Modulatable endogenous protein degradation with heterobifunctional compounds

文档序号：1660942 发布日期：2019-12-27 浏览：24次中文

阅读说明：本技术 用异双功能性化合物进行的可调节的内源性蛋白质降解 (Modulatable endogenous protein degradation with heterobifunctional compounds ) 是由 D·巴克利 G·温特 A·J·菲利普斯 T·P·赫弗南 J·布拉德纳 J·罗伯茨 B·纳于 2018-02-08 设计创作，主要内容包括：本发明提供了一种以避免与CRISPR内源性蛋白质敲除或敲入策略和提供单核苷酸校正或改变的策略相关的问题的方式调节体内基因表达的方法。本发明包含将编码异双功能性化合物靶向蛋白(dTAG)的核苷酸与编码感兴趣的内源性表达蛋白的基因的核苷酸序列框内插入基因组中,该基因在表达时产生内源性蛋白质-dTAG杂合蛋白。这允许使用异双功能性化合物的该dTAG和该融合的内源性蛋白质的靶向蛋白降解。(The present invention provides a method of modulating gene expression in vivo in a manner that avoids the problems associated with CRISPR endogenous protein knockout or knock-in strategies and strategies that provide single nucleotide correction or alteration. The invention comprises the in-frame insertion into the genome of nucleotides encoding a heterobifunctional compound targeting protein (dTAG) and a nucleotide sequence encoding a gene of interest that endogenously expresses a protein, which gene when expressed produces an endogenous protein-dTAG hybrid protein. This allows for targeted proteolytic degradation of the dTAG and the fused endogenous protein using heterobifunctional compounds.)

1. A transformed cell, comprising:

a genomically integrated nucleic acid sequence encoding a heterobifunctional compound targeting protein capable of being bound by a heterobifunctional compound;

wherein the dTAG comprises an amino acid sequence derived from EGFR, BCR-ABL, ALK, JAK2, BRAF, Src, LRRK2, PDGFR α or RET;

wherein the nucleic acid sequence encoding dTAG is integrated in-frame with the nucleic acid sequence of the gene encoding the endogenous protein in the 5 'or 3' orientation;

wherein expression of the gene encoding the endogenous protein produces an endogenous protein-dTAG hybrid protein;

wherein the heterobifunctional compound is capable of a) binding the endogenous protein-dTAG hybrid protein via the dTAG and b) binding the ubiquitin ligase in a manner that brings the endogenous protein-dTAG hybrid protein into proximity to the ubiquitin ligase;

wherein the endogenous protein-dTAG hybrid protein is ubiquitinated and then degraded by proteasome.

2. The transformed cell of claim 1, wherein the cell is a human cell.

3. The transformed cell of claim 2, wherein the human cell is a hepatocyte.

4. The transformed cell of any one of claims 1-3, wherein the heterobifunctional compound targeting protein comprises an amino acid sequence from a non-endogenous protein.

5. The transformed cell of any one of claims 1-4, wherein the heterobifunctional compound targeting protein comprises an amino acid sequence selected from SEQ ID No. 53-63.

6. The transformed cell of any one of claims 1 to 4, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from EGFR.

7. The transformed cell of claim 6, wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 53.

8. The transformed cell of claim 6, wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 54.

9. The transformed cell of claim 6, wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 55.

10. The transformed cell of claim 6, wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 56.

11. The transformed cell of any one of claims 1-4, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from BCR-ABL.

12. The transformed cell of claim 11, wherein the heterobifunctional compound targeting protein is the amino acid sequence of seq.id No. 57.

13. The transformed cell of claim 11, wherein the heterobifunctional compound targeting protein is the amino acid sequence of seq.id No. 58.

14. The transformed cell of any one of claims 1-4, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from ALK.

15. The transformed cell of claim 14, wherein the heterobifunctional compound targeting protein is an amino acid sequence of seq.id No. 59.

16. The transformed cell of any one of claims 1-4, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from JAK 2.

17. The transformed cell of claim 16, wherein the heterobifunctional compound targeting protein is the amino acid sequence of seq.id No. 60.

18. The transformed cell of any one of claims 1-4, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from BRAF.

19. The transformed cell of claim 18, wherein the heterobifunctional compound targeting protein is the amino acid sequence of seq.id No. 61.

20. The transformed cell of any one of claims 1-4, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from Src.

21. The transformed cell of claim 20, wherein the heterobifunctional compound targeting protein is the amino acid sequence of seq.id No. 62.

22. The transformed cell of claim 20, wherein the heterobifunctional compound targeting protein is the amino acid sequence of seq.id No. 63.

23. The transformed cell of any one of claims 1-4, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from LKKR 2.

24. The transformed cell of any one of claims 1 to 4, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from PDGFR α.

25. The transformed cell of any one of claims 1-4, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from RET.

26. The transformed cell of any one of claims 1-25, wherein the nucleic acid sequence encoding a heterobifunctional compound targeting protein in-frame inserts a gene encoding an endogenous protein associated with a disease that is the result of a gain-of-function mutation, amplified or increased expression, a monogenic disease, a proteinopathy, or a combination thereof.

27. The transformed cell of any one of claims 1-26, further comprising a nucleic acid sequence encoding an CRISPR RNA-directed endonuclease.

28. The transformed cell of claim 27, wherein the CRISPR RNA-guided endonuclease is selected from the group consisting of Cas1, Cas IB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Csy 9, Cse 9, Csc 9, Csa 9, Csn 9, Csm 9, Cmr 9, Csb 9, Csx 9, Csf 9, cs3672, Csx 9.

29. The transformed cell of claim 28, wherein the nucleic acid encodes a Cas9 endonuclease comprising the amino acid sequence of seq.id.no. 52.

30. The transformed cell of any one of claims 1-29, wherein the heterobifunctional compound targeting protein does not substantially interfere with the function of an endogenously expressed protein.

31. A method of modulating gene expression in a subject, comprising:

administering to the subject an effective amount of a heterobifunctional compound;

wherein the subject has one or more transformed cells that have been transformed with a nucleic acid sequence encoding a heterobifunctional compound targeting protein (dTAG);

wherein the dTAG comprises an amino acid sequence derived from EGFR, BCR-ABL, ALK, JAK2, BRAF, Src, LRRK2, PDGFR α or RET;

wherein the nucleic acid sequence encoding dTAG is integrated in-frame with the nucleic acid sequence of an endogenous protein associated with the disease in the 5 'or 3' orientation of the genome;

wherein the nucleic acid sequence encoding dTAG is inserted into a genomic sequence resulting in the production of an endogenous protein-dTAG hybrid protein upon expression;

32. The method of claim 31, wherein the cell is a human cell.

33. The method of claim 32, wherein the human cell is a hepatocyte.

34. The method of any one of claims 31-33, wherein the heterobifunctional compound targeting protein comprises an amino acid sequence from a non-endogenous protein.

35. The method of any one of claims 31-34 wherein the heterobifunctional compound targeting protein comprises an amino acid sequence selected from seq id No. 53-63.

36. The method of any one of claims 31-34, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from EGFR.

37. The method of claim 36 wherein the heterobifunctional compound targeting protein is the amino acid sequence of seq.id No. 53.

38. The method of claim 36 wherein the heterobifunctional compound targeting protein is the amino acid sequence of seq.id No. 54.

39. The method of claim 36 wherein the heterobifunctional compound targeting protein is the amino acid sequence of seq.id No. 55.

40. The method of claim 36 wherein the heterobifunctional compound targeting protein is the amino acid sequence of seq.id No. 56.

41. The method of any one of claims 31-34, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from BCR-ABL.

42. The method of claim 41 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 57.

43. The method of claim 41 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 58.

44. The method of any one of claims 31-34, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from ALK.

45. The method of claim 44 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 59.

46. The method of any one of claims 31-34, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from JAK 2.

47. The method of claim 46 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 60.

48. The method of any one of claims 31 to 34, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from BRAF.

49. The method of claim 48 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 61.

50. The method of any one of claims 31-34, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from Src.

51. The method of claim 50 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 62.

52. The method of claim 50 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 63.

53. The method of any one of claims 31-34, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from LKKR 2.

54. The method of any one of claims 31-34, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from PDGFR α.

55. The method of any one of claims 31-34, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from RET.

56. The method of any one of claims 31-55, wherein the nucleic acid sequence encoding the heterobifunctional compound targeting protein inserts in-frame a gene encoding an endogenous protein associated with a disease that is the result of a gain-of-function mutation, amplified or increased expression, a monogenic disease, a proteinopathy, or a combination thereof.

57. The method of any one of claims 31-56, further comprising a nucleic acid sequence encoding an CRISPR RNA-directed endonuclease.

58. The method of claim 57, wherein the CRISPR RNA guided endonuclease is selected from Cas1, Cas IB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Csy 9, Cse 9, Csc 9, Csa 9, Csn 9, Csm 9, Cmr 9, Csb 9, Csx 36aX, Csx 9, Csf 9, Csx 9, C.

59. The method of claim 58, wherein the nucleic acid encodes a Cas9 endonuclease comprising the amino acid sequence of SEQ ID No. 52.

60. The method of any one of claims 31-59, wherein the heterobifunctional compound targeting protein does not substantially interfere with the function of an endogenously expressed protein.

61. A method of reducing gene overexpression in a subject, comprising:

administering to the subject an effective amount of a heterobifunctional compound;

wherein the subject has one or more transformed cells that have been transformed with a nucleic acid sequence encoding a heterobifunctional compound targeting protein (dTAG);

wherein the dTAG comprises an amino acid sequence derived from EGFR, BCR-ABL, ALK, JAK2, BRAF, Src, LRRK2, PDGFR α or RET;

wherein the nucleic acid sequence encoding dTAG is integrated in-frame in the 5 'or 3' orientation with the nucleic acid sequence of an endogenous protein associated with the disease due to overexpression of the protein;

wherein the nucleic acid sequence encoding dTAG is inserted into a genomic sequence resulting in the production of an endogenous protein-dTAG hybrid protein upon expression;

62. The method of claim 61, wherein the cell is a human cell.

63. The method of claim 62, wherein the human cell is a hepatocyte.

64. The method of any one of claims 61-63, wherein the heterobifunctional compound targeting protein comprises an amino acid sequence from a non-endogenous protein.

65. The method of any one of claims 61-64 wherein the heterobifunctional compound targeting protein comprises an amino acid sequence selected from SEQ ID No. 53-63.

66. The method of any one of claims 61-64, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from EGFR.

67. The method of claim 66 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 53.

68. The method of claim 66 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 54.

69. The method of claim 66 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 55.

70. The method of claim 66 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 56.

71. The method of any one of claims 61-64, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from BCR-ABL.

72. The method of claim 71 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 57.

73. The method of claim 71 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 58.

74. The method of any one of claims 61-64, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from ALK.

75. The method of claim 74 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 59.

76. The method of any one of claims 61-64, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from JAK 2.

77. The method of claim 76 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 60.

78. The method of any one of claims 61-64, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from BRAF.

79. The method of claim 78 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 61.

80. The method of any one of claims 61-64, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from Src.

81. The method of claim 80 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 62.

82. The method of claim 80 wherein the heterobifunctional compound targeting protein is the amino acid sequence of SEQ ID No. 63.

83. The method of any one of claims 61-64, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from LKKR 2.

84. The method of any one of claims 61-64, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from PDGFR α.

85. The method of any one of claims 61-64, wherein the heterobifunctional compound targeting protein is an amino acid sequence derived from RET.

86. The method of any one of claims 61-85, wherein the nucleic acid sequence encoding the heterobifunctional compound targeting protein is inserted in-frame with a gene encoding an endogenous protein associated with a disease that is the result of a gain-of-function mutation, amplified or increased expression, a monogenic disease, a proteinopathy, or a combination thereof.

87. The method of any one of claims 31-56, further comprising a nucleic acid sequence encoding an CRISPR RNA-directed endonuclease.

88. The method of claim 87, wherein the CRISPR RNA guided endonuclease is selected from the group consisting of Cas1, Cas IB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Csy 9, Cse 9, Csc 9, Csa 9, Csn 9, Csm 9, Cmr 9, Csb 9, Csx 36x 9, Csx 9, Csf 9, Csx 9, Csf 9, Csx 9.

89. The method of claim 88, wherein the nucleic acid encodes a Cas9 endonuclease comprising the amino acid sequence of SEQ ID No. 52.

90. The method of any one of claims 61-89, wherein the heterobifunctional compound targeting protein does not substantially interfere with the function of an endogenously expressed protein.

Technical Field

Methods, compounds, and compositions for modulating endogenously expressed proteins using targeted protein degradation are described.

Background

Many tools have been developed to manipulate gene expression to interrogate the function of a gene or protein of interest. For example, techniques such as RNA interference and antisense deoxyoligonucleotides are commonly used to disrupt protein expression at the RNA and DNA levels. Homologous recombination or loss-of-function mutations can be site-specific double-stranded cleaved using zinc finger nucleases, transcription activator-like effector nucleases (TALENs) or aggregation-regulated short palindromic repeats (CRISPR) -Cas9 (Cheng, J.K. andDeler, H.S., "The genome editing toolbox: a spectra of aptamers for targeted therapy" curr.Opin. Biotechnol.,30C, (2014): 87-94; and Graham et al, Genbiol, (2015):16: 260). The CRISPR-Cas9 system has been used to modulate endogenous gene expression by incorporating specific mutations into genes of interest (see, e.g., Lo et al, Genetics, 2013; 195(2): 331-348; Yu et al, Biology Open, 2014; 3: 271-280; Park et al, PLOS One, 2013; 9(4): e 95101; Lackner et al, Nature Communications, 2015; 17(6): 1-7; U.S. Pat. Nos. 8,771,945 and 9,228,208; WO 2014/204729; and U.S. patent application 2014/0273235).

For example, the CRISPR-Cas9 system is used to mutate the human PCSK9 gene In chimeric liver-humanized mice carrying human hepatocytes (Wang, x., et al. "CRISPR-Cas9 Targeting of PCSK9 In human hepaticutes In vivo," ariioscope, Thrombosis, and Vascular Biology, (2016)). PCSK9 is successfully mutated and the CRISPR-Cas9 system has been proposed as a method for the in vivo treatment of human diseases. However, the long-term effects of permanent Genome modification are unclear and there is incomplete precision with respect to Genome editing, as well as the effect of the continuous activity of the virally-delivered CRISPR-Cas 9and the biological compensatory mechanisms that may be present in adults on direct correction (Kormann et al, "Expression of viral protein after delivery chemical modified mRNA in mice" Nat. Biotechnology, 29, (2011): 154-. Furthermore, where the expressed protein (even if not perfect) is essential for cellular function, CRISPR knockout strategies may be undesirable.

Efforts have been made to modulate gene expression in vitro using inducible degradation systems. For example, the auxin-induced degradation (AID) system in plants has been able to control protein consumption in yeast and cultured vertebrate cells. This system relies on the expression of the plant specific F-box protein TIR1, which regulates various aspects of plant growth and morphogenesis in response to the plant hormone auxin. TIR1 is a substrate recognition component of the Skp1-Cullin-F-box E3 ubiquitin ligase complex, which recognizes substrates only in the presence of auxin and targets them for degradation by the proteasome. This system has been operated and shown to produce conditional auxin-dependent protein consumption in C.elegans (Caenorhabditis elegans) as well as human HCT116 cells (see, e.g., Zhang et al, Development, 2015; 142: 4374-. However, due to the toxicity of auxin, this approach is impractical as an in vivo regulatory system.

Another approach to reversibly controlling gene expression is the use of ligand-dependent destabilizing domains and Shield-1 ligands, which allow reversible stabilization and destabilization of labeled proteins of interest in a dose-dependent manner (see, e.g., Rakhit et al, Chemistry & Biology, 2014; 21: 1238-. Fusing the destabilizing domain to the gene of interest results in the expression of a fusion protein, which is degraded by the proteasome. Shield-1 specifically binds to the destabilizing domain and inactivates protein degradation. However, this system is also not feasible as an in vivo regulatory strategy due to the need for Sield-1 to be present in the cytosol to avoid degradation. This method requires continuous administration of Shield-1 to maintain protein stability.

Accordingly, there remains an unmet need for improved systems that allow for reversible control of endogenous gene expression in vivo while providing improved treatment modalities in subjects suffering from disorders such as proteinopathies.

It is therefore an object of the present invention to provide methods, compounds and compositions to modulate gene expression in vivo in a manner that avoids the problems associated with CRISPR endogenous protein knockout or knock-in strategies.

Disclosure of Invention

The present invention provides a method of modulating gene expression in vivo in a manner that avoids the problems associated with CRISPR endogenous protein knockout or knock-in strategies and strategies that provide single nucleotide correction or alteration. The invention comprises the in-frame insertion into the genome of a nucleotide sequence encoding a heterobifunctional compound targeting protein (dTAG) and a nucleotide sequence encoding a gene of endogenously expressed protein of interest which upon expression produces an endogenous protein-dTAG hybrid protein. This allows the use of heterobifunctional compounds to target proteolytic degradation of dTAG and fused endogenous proteins in a controlled, regulated manner.

Heterobifunctional compounds as contemplated herein are compounds that bind to ubiquitin ligase in vivo via a ubiquitin ligase binding moiety and also bind to dTAG via its dTAG targeting ligand, as defined in more detail below. Heterobifunctional compounds are able to induce proteasome-mediated degradation of fused endogenous proteins via recruitment of E3 ubiquitin ligase and subsequent ubiquitination. These drug-like molecules offer the possibility of reversible, dose-responsive, adjustable, temporal control of protein levels.

Targeting endogenously expressed proteins with dTAG using heterobifunctional compounds allows reversible control of endogenously expressed proteins of interest, compared to the CRISPR-Cas9 genomic editing that incorporates irreversible transformation into the gene of interest. Thus, heterobifunctional compounds can be used as rheostats (rheostats) for protein expression, providing the ability to turn on and off endogenous protein expression upon titration of the heterobifunctional compound. Furthermore, by genomically and stably integrating the nucleic acid sequence encoding dTAG into the 5 'or 3' of the gene of the endogenous protein, side effects associated with CRISPR-Cas9, such as negative downstream consequences associated with permanently editing the gene, can be avoided.

The present invention provides a mechanism to control the degradation of endogenous proteins that mediate disease by combining genome engineering with small molecule activation/degradation modulation. The methods and compositions described herein are particularly useful for targeting endogenous proteins associated with disease due to acquired function, toxicity accumulation, overexpression or downstream enzymatic processes in which the protein may be involved. Applications of this technology include, but are not limited to, 1) targeted degradation of proteins, where pathology is the result of mutations that gain function, 2) targeted degradation of proteins, where pathology is the function of amplification or increased expression, 3) targeted degradation of proteins that manifest as monogenic diseases, 4) targeted degradation of proteins where genetic susceptibility manifests for a longer period of time and often after alternative biological compensatory mechanisms are no longer sufficient, such as, but not limited to, hypercholesterolemia and proteinopathies.

Thus, in one embodiment, a method is provided that includes at least the steps of:

(i) transforming a subject's (typically a human) relevant cells with a nucleic acid sequence encoding dTAG, wherein the nucleic acid sequence is genomically integrated in frame with a nucleic acid sequence of an endogenous protein that serves as a disease mediator, wherein the nucleic acid encoding dTAG is inserted into the genomic sequence, which upon expression produces an endogenous protein-dTAG hybrid or fusion protein; and

(ii) the method comprises administering to the subject in need thereof a heterobifunctional compound that binds a) the inserted dTAG and b) the ubiquitin ligase in such a way that the dTAG (and thus the endogenous protein-dTAG hybrid protein) is in proximity to the ubiquitin ligase, ubiquitinates the endogenous protein-dTAG hybrid protein, and is then degraded by the proteasome.

In one embodiment, the cells of the subject are transformed in vivo. In one embodiment, the cells of the subject are transformed ex vivo and administered back to the subject. In one embodiment, the cells of the subject are hepatocytes.

In one embodiment, a method is provided comprising the steps of:

administering to a subject in need thereof a heterobifunctional compound, wherein the subject has one or more cells that have been transformed with a nucleic acid sequence encoding dTAG, wherein the nucleic acid sequence is targeted to a nucleic acid sequence of an endogenous protein that serves as a disease mediator, integrates in 5 'or 3' in-frame in the genome, wherein the nucleic acid encoding dTAG inserts into the genomic sequence, producing an endogenous protein-dTAG hybrid or fusion protein upon protein expression; and wherein the heterobifunctional compound binds a) the inserted dTAG and b) the ubiquitin ligase in a manner that brings the dTAG (and thus the endogenous protein-dTAG hybrid protein) into proximity with the ubiquitin ligase such that the endogenous protein-dTAG hybrid protein is ubiquitinated and then degraded by the proteasome.

As contemplated herein, a synthetic gene encoding an endogenous protein of interest-dTAG hybrid is derived in vivo by inserting a nucleic acid encoding dTAG in a5 'or 3' targeting frame into a nucleic acid encoding a protein of interest. This results in an in-frame gene fusion that is susceptible to proteasome-mediated degradation when treated with a heterobifunctional compound capable of binding dTAG. In one main embodiment, dTAG does not substantially interfere with the function of the endogenously expressed protein. In one embodiment, dTAG is a non-endogenous peptide that allows for selectivity of the heterobifunctional compound and minimizes off-target effects when the heterobifunctional compound is administered. In one embodiment, dTAG is an amino acid sequence derived from an endogenous protein that has been modified, for example, by a "bump" strategy (see, e.g., Clackson et al, "reproducing and FKBP-ligand interface to general chemical reagents with novel specificity", PNAS 95(1998): 10437-.

Also contemplated herein is a method for in vitro allele-specific modulation of an endogenous protein by inserting a nucleic acid sequence encoding dTAG in a5 'or 3' targeting frame into a genomic sequence encoding a protein of interest, wherein the nucleic acid encoding dTAG is inserted into the genomic sequence, upon expression, producing an endogenous protein-dTAG hybrid or fusion protein, wherein the endogenous protein-dTAG is capable of being degraded by a heterobifunctional compound that binds a) the inserted dTAG and b) the ubiquitin ligase in a manner that brings the dTAG (and thus the endogenous protein-dTAG hybrid) into proximity to the ubiquitin ligase, such that the endogenous protein-dTAG hybrid is ubiquitinated and then degraded by the proteasome. By in-frame insertion of a nucleic acid encoding dTAG into a gene encoding an endogenous protein of interest using the methods described herein, expression of the resulting protein can be tightly controlled by introducing a heterobifunctional compound capable of binding dTAG, thereby resulting in degradation of the endogenous protein. Importantly, by using heterobifunctional compounds, the expression of endogenous proteins can be reversibly controlled, allowing the examination of the effect of protein expression on cells.

Thus, by modulating the expression of endogenous proteins in this manner, downstream effects that modulate protein expression can be detected in a variety of proteins and cell types as well as under a variety of physiological conditions. Because the concentration of heterobifunctional compounds within the cell can be titrated, the protein-dTAG hybrid protein concentration within the cell can be fine-tuned, allowing for the conditional alteration of the abundance of proteins within the cell and the ability to alter the phenotype within the cell as desired. In one embodiment, provided herein is a method of assessing attenuation of protein expression in a cell comprising inserting a nucleic acid sequence encoding dTAG into the 5 'or 3' framework of a genomic sequence encoding a protein of interest, wherein insertion of the nucleic acid encoding dTAG into the genomic sequence results in production of an endogenous protein-dTAG hybrid or fusion protein upon expression, wherein the endogenous protein-dTAG is capable of being degraded by a heterobifunctional compound that binds a) the inserted dTAG and b) a ubiquitin ligase in a manner that brings the dTAG (and thus the endogenous protein-dTAG hybrid protein) into proximity to the ubiquitin ligase such that the endogenous protein-dTAG hybrid protein is ubiquitinated and then degraded by the proteasome. In one embodiment, the heterobifunctional compound is administered to a cell such that the concentration of protein-dTAG hybrid protein in the cell is partially degraded. In one embodiment, the heterobifunctional compound is administered to a cell such that the concentration of the endogenous protein-dTAG hybrid protein in the cell is completely degraded.

In one embodiment, provided herein is a method of identifying a protein target associated with a disease or disorder, comprising inserting a nucleic acid sequence encoding dTAG into the 5 'or 3' frame of a genomic sequence encoding a protein of interest, wherein a nucleic acid encoding dTAG is inserted into the genomic sequence, which on expression yields an endogenous protein-dTAG hybrid or fusion protein, wherein the endogenous protein-dTAG is capable of being degraded by a heterobifunctional compound, the heterobifunctional compound binds a) the inserted dTAG and b) the ubiquitin ligase in such a way that the dTAG (and thus the endogenous protein-dTAG hybrid protein) is brought into proximity of the ubiquitin ligase, such that the endogenous protein-dTAG hybrid protein is ubiquitinated, and then degraded by the proteasome, followed by measuring the effect of protein degradation on a cellular disorder or disease state. By inserting a nucleic acid encoding dTAG in-frame into a gene encoding an endogenous protein of interest using the methods described herein, the down-regulation of various proteins can be examined and potential targets for treating disorders associated with a particular disease state can be identified. In addition, current methods can be used to validate potential proteins associated with disease states.

In a particular embodiment, the dTAG used in the present invention comprises an amino acid sequence derived from an endogenous protein kinase. In one embodiment, the endogenous protein kinase amino acid sequence comprises a mutation that inactivates the kinase. In one embodiment, the mutation in the protein kinase occurs within a conserved kinase catalytic triad amino acid sequence. In one embodiment, the conserved kinase catalytic triad amino acid sequence is TVS. In one embodiment, the conserved kinase catalytic triad amino acid sequence is HRD. In one embodiment, the conserved kinase catalytic triad amino acid sequence is DFG. In one embodiment, the conserved kinase catalytic triplet amino acid sequence is a TRD. See Kornev et al, "Surface composition of active and inactive protein ingredients activated mechanism," PNAS 2006; 103(47) 17783-17788, which are incorporated herein by reference. In one embodiment, at least one catalytic triad amino acid is substituted for alanine. In one embodiment, at least one catalytic triad amino acid replaces glycine. In one embodiment, the heterobifunctional compound comprises an allele-specific ligand capable of selectively binding to a mutein kinase sequence. In one embodiment, the mutant kinase is as described in Roskoski et al, "Classification of small molecule protein kinase inhibited up the structure of the hair drug-enzymes complex," Pharmaceutical Research http:// dx. doi.org/10.1016/j. phrs.2015.10.021, which is incorporated herein by reference and/or Roskoski et al, "A historical overview of protein kinases and the hair target small molecule inhibitors," Pharmaceutical Research (2015), http:// dx.doi.org/10.1016/j. phrs.07.10, which is incorporated herein by reference. In one embodiment, dTAG is derived from a kinase that is an analog sensitive kinase. In one embodiment, the mutant kinase is, for example, Zhang et al, "Structure-guided inhibitor design extensions of the scope of analog-sensitive kinase technology," ACS Chem biol.2013:8 (9); 1931-1938, which is incorporated herein by reference. In alternative embodiments, the dTAG used in the present invention includes, but is not limited to, amino acid sequences derived from proteins selected from EGFR, BCR-ABL, ALK, JAK2, BRAF, LRRK2, PDGFR α, and RET. In one embodiment, the protein contains one or more mutations. In one embodiment, the one or more mutations inactivate the protein.

In alternative embodiments, the dTAG used in the present invention includes, but is not limited to, amino acid sequences derived from a protein selected from the group consisting of Src, Pkd1, Kit, Jak2, Abl, Mek1, HIV integrase, and HIV reverse transcriptase.

In particular embodiments, the dTAG used in the present invention includes, but is not limited to, amino acid sequences derived from endogenously expressed proteins such as FK506 binding protein-12 (FKBP12), bromodomain-containing protein 4(BRD4), CREB binding protein (CREBBP), or transcriptional activator BRG1(SMARCA 4). In other embodiments, the dTAG used in the present invention may include, for example, hormone receptors such as estrogen receptor protein, androgen receptor protein, Retinoid X Receptor (RXR) protein, or dihydrofolate reductase (DHFR), including bacterial DHFR. In other embodiments, the dTAG may comprise, for example, an amino acid sequence derived from a bacterial dehalogenase. In other specific examples, dTAG may include proteins derived from 7, 8-dihydro-8-oxoguanine triphosphatase, AFAD, arachidonic acid 5-lipoxygenase activating protein, apolipoprotein, ASH1L, ATAD2, baculovirus IAP repeat-containing protein 2, BAZ1A, BAZ1B, BAZ2A, BAZ2B, Bcl-2, Bcl-xL, BRD1, BRD2, BRD3, BRD4, BRD5, BRD6, BRD7, BRD8, BRD9, BRD10, BRDT, BRPF1, BRPF3, BRWD3, CD209, CECR2, BBCREP 874P, E3 ligase XIAP, EP300, FALZ, fatty acid binding protein 4 from adipocytes (FA36BP), GCN5L 4, GTP-GTPase 1240, PCPAD-FAP-4, PCP-FAP-4, CREA-FAN-FAL-PGA-FAP, PGA-PGNA-FAP-4, PGA-FAN-4, PGA-FAN-L-FAN-PSD-P-4, PGA-GAP-FAN-SSP, Poly ADP-ribose polymerase 15, PRKCBP1, prostaglandin E synthase, retinal rod sensitive cGMP 3',' cyclic phosphodiesterase subunit δ, S100-a7, SMARCA2, SMARCA4, SP100, SP110, SP140, Src, Sumo-binding enzyme UBC9, superoxide dismutase, TAF1, TAF1L, tankyrase 1, tankyrase 2, TIF1a, TRIM28, TRIM33, TRIM66, WDR9, ZMYND11, or MLL 4. In still further embodiments, the dTAG may comprise, for example, an amino acid sequence derived from MDM 2.

In a particular embodiment, dTAG is derived from BRD2, BRD3, BRD4, or BRDT. In certain embodiments, dTAG is a modified or mutated BRD2, BRD3, BRD4, or BRDT protein. In certain embodiments, the one or more mutations of BRD2 include a mutation of tryptophan (W) at amino acid position 97, a mutation of valine (V) at amino acid position 103, a mutation of leucine (L) at amino acid position 110, a mutation of W at amino acid position 370, a mutation of V at amino acid position 376, or a mutation of L at amino acid position 381.

In certain embodiments, the one or more mutations of BRD3 include a mutation of W at amino acid position 57, a mutation of V at amino acid position 63, a mutation of L at amino acid position 70, a mutation of W at amino acid position 332, a mutation of V at amino acid position 338, or a mutation of L at amino acid position 345. In certain embodiments, the one or more mutations of BRD4 include a mutation of W at amino acid position 81, a mutation of V at amino acid position 87, a mutation of L at amino acid position 94, a mutation of W at amino acid position 374, a mutation of V at amino acid position 380, or a mutation of L at amino acid position 387. In certain embodiments, the one or more mutations of BRDT include a mutation of W at amino acid position 50, a mutation of V at amino acid position 56, a mutation of L at amino acid position 63, a mutation of W at amino acid position 293, a mutation of V at amino acid position 299, or a mutation of L at amino acid position 306.

In a particular embodiment, dTAG is derived from the cytoplasmic signaling protein FKBP 12. In certain embodiments, dTAG is a modified or mutated cytoplasmic signaling protein FKBP 12. In certain embodiments, the modified or mutated cytoplasmic signaling protein FKBP12 contains one or more mutations that result in an amplified binding pocket (pocket) of FKBP12 ligand. In certain embodiments, the one or more mutations comprises a mutation of phenylalanine (F) to valine (V) (F36V) (interchangeably referred to herein as FKBP or FKBP12) at amino acid position 36.

In one embodiment, the dTAG is derived from the amino acid sequence of any one of seq.id No.1 to 44 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No.1 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No.2 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 3 or a fragment thereof. In a particular embodiment, dTAG is derived from the amino acid sequence of seq.id No. 4 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 5 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 6 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 7 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 8 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 9 or a fragment thereof. In a particular embodiment, the fragment refers to the smallest amino acid sequence to which a heterobifunctional compound is required to bind. In a particular embodiment, dTAG is derived from the amino acid sequence of seq id No. 10 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 11 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 12 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 13 or a fragment thereof. In particular embodiments, the dTAG is derived from the amino acid sequence of seq.id No. 14 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 15 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 16 or a fragment thereof. In a particular embodiment, dTAG is derived from the amino acid sequence of seq.id No. 17 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 18 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 19 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 20 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 21 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 22 or a fragment thereof. In a particular embodiment, dTAG is derived from the amino acid sequence of seq.id.no. 23 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 24 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 25 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 26 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No.27 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No.28 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 29 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 30 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 31 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 32 or a fragment thereof. In a particular embodiment, dTAG is derived from the amino acid sequence of seq.id No. 33 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 34 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 35 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 36 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 37 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 38 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 39 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id.no. 40 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 41 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 42 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 43 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 44 or a fragment thereof. In particular embodiments, fragments thereof refer to the smallest amino acid sequence required to bind a heterobifunctional compound. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 62 or a fragment thereof. In a particular embodiment, the dTAG is derived from the amino acid sequence of seq.id No. 63 or a fragment thereof. In particular embodiments, fragments thereof refer to the smallest amino acid sequence required to bind a heterobifunctional compound.

In a particular embodiment, dTAG is derived from the amino acid sequence of SEQ ID No.1 or a fragment thereof, and dTAG can be bound by a heterobifunctional compound selected from any one of dFKBP-1 to dFKBP-5. dTAG is derived from the amino acid sequence of SEQ ID No.2 or a fragment thereof, and dTAG can be bound by a heterobifunctional compound selected from any one of dFKBP-6 to dFKBP-10. dTAG is derived from the amino acid sequence of SEQ ID No. 3 or a fragment thereof, and dTAG can be bound by a heterobifunctional compound selected from any one of dBET-1 to dBET-18. dTAG is derived from the amino acid sequence of SEQ ID No. 3 or a fragment thereof, and dTAG can be bound by a heterobifunctional compound selected from any one of dBromo-1 to dBromo-34. dTAG is derived from the amino acid sequence of SEQ ID No. 9 or a fragment thereof, and dTAG can be bound by a heterobifunctional compound selected from any of dHalo-1 to dHalo-2.

In one embodiment, the dTAG is derived from any of the amino acid sequences described herein or a fragment thereof, and the dTAG can be bound by a corresponding heterobifunctional compound comprising a dTAG targeting ligand capable of binding to the dTAG described herein. In one embodiment, dTAG is an amino acid sequence capable of being bound by a heterobifunctional compound as depicted in figure 29, figure 30, figure 31, figure 32, and figure 33, or any other heterobifunctional compound described herein. In one embodiment, dTAG is an amino acid sequence capable of being bound by a heterobifunctional compound comprising a dTAG targeting ligand described in table T. In a particular embodiment, the dTAG is derived from the amino acid sequence of SEQ ID No.1 or a fragment thereof, and the dTAG is capable of being bound by a heterobifunctional compound selected from any one of dFKBP-1 to dFKBP-5. In a particular embodiment, dTAG is derived from the amino acid sequence of seq.id No.1 or a fragment thereof, and dTAG can be bound by a heterobifunctional compound selected from any one of dFKBP-6 to dFKBP-13. In a particular embodiment, dTAG is derived from the amino acid sequence of seq.id No. 3 or a fragment thereof, and dTAG can be bound by a heterobifunctional compound selected from any of dBET1 through dBET 18. In a particular embodiment, dTAG is derived from the amino acid sequence of seq.id No. 3 or a fragment thereof, and dTAG can be bound by a heterobifunctional compound selected from any one of dBromo1 to dBromo 34. In a particular embodiment, dTAG is derived from the amino acid sequence of seq.id No. 9 or a fragment thereof, and dTAG can be bound by a heterobifunctional compound selected from any one of dHalo1 to dHalo 2. In a particular embodiment, the dTAG is derived from CREBBP, and the heterobifunctional compound contains a CREBBP dTAG targeting ligand selected from table T. In a particular embodiment, the dTAG is derived from SMARCA4, PB1, or SMARCA2, and the heterobifunctional compound comprises a SMARCA4/PB1/SMARCA2dTAG targeting ligand selected from table T. In particular embodiments, the dTAG is derived from TRIM24 or BRPF1, and the heterobifunctional compound comprises a TRIM24/BRPF1 dTAG targeting ligand selected from table T. In a particular embodiment, the dTAG is derived from a glucocorticoid receptor, and the heterobifunctional compound comprises a glucocorticoid dTAG targeting ligand selected from table T. In particular embodiments, the dTAG is from an estrogen or androgen receptor, and the heterobifunctional compound contains an estrogen/androgen receptor dTAG targeting ligand selected from table T. In a particular embodiment, the dTAG is derived from DOT1L, and the heterobifunctional compound comprises a DOT1L dTAG targeting ligand selected from table T. In a particular embodiment, the dTAG is derived from Ras, and the heterobifunctional compound contains a Ras dTAG targeting ligand selected from Table T. In a particular embodiment, the dTAG is derived from RasG12C, and the heterobifunctional compound comprises a RasG12C dTAG targeting ligand selected from table T. In particular embodiments, the dTAG is derived from HER3, and the heterobifunctional compound comprises a HER3 dTAG targeting ligand selected from table T. In a particular embodiment, the dTAG is derived from Bcl-2 or Bcl-XL, and the heterobifunctional compound contains a Bcl-2/Bcl-XL dTAG targeting ligand selected from Table T. In a particular embodiment, the dTAG is derived from an HDAC, and the heterobifunctional compound comprises an HDAC dTAG targeting ligand selected from table T. In particular embodiments, the dTAG is derived from a PPAR, and the heterobifunctional compound comprises a PPAR dTAG targeting ligand selected from table T. In a particular embodiment, the dTAG is derived from DHFR and the heterobifunctional compound comprises a DHFRdTAG targeting ligand selected from table T.

In one aspect, the synthetic genes of the invention include genes of interest associated with genetic diseases. As a non-limiting example, a mutant gene (e.g., encoding alpha-1 antitrypsin (A1AT)) may be inserted in-frame for dTAG targeting in a cell to produce a synthetic gene encoding a hybrid protein capable of degradation by a heterobifunctional compound of dTAG targeting the endogenous A1AT-dTAG hybrid protein. By generating an A1AT-dTAG hybrid, the function of mutated A1AT can be modulated or modulated by administration of heterobifunctional compounds, allowing the cell to maintain some function of the A1AT endogenous protein while reducing the effects of A1AT overexpression. Other non-limiting examples of proteins that can be targeted include β -catenin (CTNNB1), apolipoprotein B (APOB), angiopoietin-like protein 3(ANGPTL3), proprotein convertase subtilisin/kexin type 9 (PCSK9), apolipoprotein C3(APOC3), Low Density Lipoprotein Receptor (LDLR), C-reactive protein (CRP), apolipoprotein a (apo (a)), factor VII, factor XI, antithrombin III (SERNC PI 1), phosphatidylinositosan A species (PIG-A), C5, α -1 antitrypsin (SERPINA1), hepcidin regulation (TMPRSS6), (δ -aminoacetylpropyl synthetase 1(ALAS-1), acylCaA: diacylglycerol acyltransferase (DGAT), miR-122, miR-21, miR-155, miR-34a, prokallikrein (KLKB1), connective tissue growth factor (CCN2), intercellular adhesion molecule 1(ICAM-1), glucagon receptor (GCGR), glucocorticoid receptor (GCCR), protein tyrosine phosphatase (PTP-1B), c-Raf kinase (RAF1), fibroblast growth factor receptor 4(FGFR4), vascular adhesion molecule-1 (VCAM-1), very late antigen-4 (VLA-4), transthyretin (TTR), surviving motoneuron 2(SMN2), Growth Hormone Receptor (GHR), Dystonia Myotonic Protein Kinase (DMPK), cellular nucleic acid binding protein (CNBP or ZNF9), Clusterin (CLU), eukaryotic translation initiation factor 4E (eIF-4E), MDM2, MDM4, heat shock protein 27(HSP 27), signal transduction and transcriptional activator 3 protein (STAT3), vascular Endothelial Growth Factor (VEGF), kinesin spindle protein (KIF11), hepatitis b genome, Androgen Receptor (AR), Atonal homolog 1(ATOH1), vascular endothelial growth factor receptor 1(FLT1), retinal intoxication 1(RS1), retinal pigment epithelium specific 65kDa protein (RPE65), Rab convoy protein 1(CHM) and sodium channels, voltage gated, type X, alpha subunit (PN3 or SCN 10A). Genetic disorders include, but are not limited to, homozygous familial hypercholesterolemia, AGS1 to AGS7, PRAAS/CANDLE, SAVI, ISG15 def, SPECNCDI, hemophagocytic lymphohistiocytosis, NLRC4-MAS, CAMPS, DADA2, PLAID, tyrosinemia type I, BSEP deficiency, MRD3 gene deficiency, glycogen storage disease IV, I, Crigler-Najjar syndrome, ornithine transcarbamylase deficiency, primary hyperoxaluria, Wilson's disease, cystic fibrosis, FIC1 deficiency, citrullinemia, cystinosis, proprionics, ADA-SCID, X-linked ID, lipoprotein lipase deficiency, Leber's congenital amaurosis, and leukodystrophy.

Also contemplated herein are heterobifunctional compounds that use dTAG that can bind to the endogenous protein-dTAG hybrids of the invention and induce degradation by ubiquitination. Endogenous protein-dTAG hybrids can be modulated in subjects having a disease or disorder of target protein expression by administering to the subject a heterobifunctional compound directed to dTAG. The heterobifunctional compounds used in the present invention are small molecule antagonists that are capable of inhibiting the biological function of endogenous proteins by degradation of the endogenous protein-dTAG hybrid. They provide rapid ligand-dependent target protein degradation via chemical conjugation, e.g., derivatized phthalimide, which hijacks the function of the cereblon E3 ubiquitin ligase complex. Using this method, the endogenous protein-dTAG hybrids of the invention can be rapidly degraded with high specificity and high efficiency.

Heterobifunctional compounds useful in the present invention include those comprising a small molecule E3 ligase ligand covalently linked to a dTAG targeting ligand through linkers of different lengths and/or functions, as described in more detail below. Heterobifunctional compounds are capable of binding to dTAG and recruiting E3 ligase, for example, by binding to endogenous dTAG hybrids with cereblon protein (CRBN) containing ligase or von hippel-Lindau tumor suppressor (VHL) for ubiquitination and subsequent proteasomal degradation.

Furthermore, by combining the chemical strategy of protein degradation via the bifunctional molecules of the present application with the effectiveness of gene therapy, side effects can be modulated by turning ubiquitination on and off, as well as proteasomal degradation of the endogenous protein-dTAG hybrid, in a rapid, precise, temporal manner modulating the activity of endogenously expressed proteins.

Examples of heterobifunctional compounds useful in the present invention are further illustrated below.

In one aspect, the genomic nucleic acid sequence encodes a synthetic gene comprising an endogenous gene of interest with an in-frame 5 'or 3' insertion of a nucleic acid encoding dTAG which, when expressed, produces an endogenous protein-dTAG hybrid protein wherein the dTAG is capable of being bound by a heterobifunctional compound. Cells and animals, particularly non-human animals, carrying such genetic modifications are part of the invention.

In a particular embodiment, the genomic nucleic acid sequence encodes a synthetic gene comprising an endogenous gene of interest having a5 '-or 3' -in-frame insertion of a nucleic acid encoding dTAG, wherein dTAG is derived from the amino acid sequence of seq.id No.1 or a fragment thereof, and dTAG is capable of being bound by a heterobifunctional compound selected from any of dFKBP-1 to dFKBP-5. In a particular embodiment, the genomic nucleic acid sequence encodes a synthetic gene comprising an endogenous gene of interest having a5 '-or 3' -in-frame insertion of a nucleic acid encoding dTAG, wherein dTAG is derived from the amino acid sequence of seq.id No.2 or a fragment thereof, and dTAG is capable of being bound by a heterobifunctional compound selected from any one of dFKBP-6 to dFKBP-13. In a particular embodiment, the genomic nucleic acid sequence encodes a synthetic gene comprising an endogenous gene of interest having an in-frame insertion of 5 '-or 3' -of a nucleic acid encoding dTAG, wherein dTAG is derived from the amino acid sequence of seq.id No. 3 or a fragment thereof, and dTAG is capable of being bound by a heterobifunctional compound selected from any of dBET1 to dBET 18. In a particular embodiment, the genomic nucleic acid sequence encodes a synthetic gene comprising an endogenous gene of interest inserted in-frame 5 '-or 3' -with a nucleic acid encoding dTAG, wherein dTAG is derived from the amino acid sequence of seq.id No. 3 or a fragment thereof, and dTAG is capable of being bound by a heterobifunctional compound selected from any one of dBromo1 to dBromo 34. In a particular embodiment, the genomic nucleic acid sequence encodes a synthetic gene comprising an endogenous gene of interest having a5 '-or 3' -in-frame insertion of a nucleic acid encoding dTAG, wherein dTAG is derived from the amino acid sequence of SEQ ID No. 9 or a fragment thereof, and dTAG is capable of being bound by a heterobifunctional compound selected from dHalo 1and dHalo 2.

In one aspect, an amino acid encoded by a synthetic gene comprising an endogenous gene of interest having an in-frame 5 '-or 3' -insertion of a nucleic acid encoding dTAG is provided, wherein the amino acid is an endogenous protein-dTAG hybrid protein, wherein the dTAG is capable of being bound by a heterobifunctional compound.

In one aspect, provided herein is a transformed cell comprising a genomic nucleic acid sequence encoding a synthetic gene comprising an endogenous gene of interest with an in-frame 5 'or 3' insertion of a nucleic acid encoding dTAG that, when expressed, produces an endogenous protein-dTAG hybrid protein, wherein the dTAG is capable of being bound by a heterobifunctional compound.

In one aspect, provided herein are cells expressing a synthetic gene comprising an endogenous gene of interest having an in-frame 5 '-or 3' -insertion of a nucleic acid encoding dTAG which, when expressed, produces an endogenous protein-dTAG hybrid protein, wherein the dTAG is capable of being bound by a heterobifunctional compound.

In one particular aspect, there is provided a method of modulating the activity of an endogenous protein by genomic insertion of a nucleic acid sequence encoding dTAG which, when expressed, produces an endogenous protein-dTAG hybrid protein, wherein the dTAG is capable of binding by a heterobifunctional compound, and administering to a subject a heterobifunctional compound capable of binding to dTAG and degrading the endogenous protein-dTAG hybrid.

In a particular aspect, there is provided a method of identifying an endogenous protein associated with a disease state, wherein the activity of the endogenous protein is modulated by genomic insertion of a nucleic acid sequence encoding dTAG which, when expressed, produces an endogenous protein-dTAG hybrid protein, wherein the dTAG is capable of being bound by a heterobifunctional compound, and administering a heterobifunctional compound capable of binding to the dTAG and degrading the endogenous protein-dTAG hybrid, wherein degradation of the protein results in an alteration of the disease state.

In one embodiment, provided herein is a transformed cell comprising a nucleic acid encoding seq.id.no. 52 and a nucleic acid encoding dTAG. In one embodiment, provided herein is a transformed cell comprising a nucleic acid encoding seq.id.no. 52 and a nucleic acid encoding dTAG derived from an amino acid sequence selected from seq.id.no.1 to 44 or a fragment thereof.

In one embodiment, provided herein is a first nucleic acid ID encoding seq.id.no. 52 and a second nucleic acid encoding dTAG. In one embodiment, provided herein is a first nucleic acid encoding seq.id.no. 52 and a second nucleic acid encoding dTAG derived from an amino acid sequence selected from seq.id.no.1 to 44 or a fragment thereof.

Other aspects of the invention include polynucleotide sequences, plasmids and vectors encoding the synthetic genes of the invention, and host cells expressing the synthetic genes of the invention.

In another embodiment, the dTAG used in the present invention is an amino acid sequence derived from EGFR. In certain embodiments, dTAG is a modified or mutated EGFR protein or fragment thereof. In certain embodiments, the one or more mutations of EGFR include a substitution of leucine (L) with leucine (L) at amino acid position 858, a deletion of the amino acid sequence LREA in exon 19, an insertion of the amino acid VAIKEL in exon 19, a substitution of glycine (G) with alanine (a), cysteine (C) or serine (S) at amino acid position 719, a substitution of leucine (L) with alanine (a), cysteine (C), serine (S) at amino acid position 861, a substitution of valine (V) with alanine (a) at amino acid position 765, a substitution of threonine (T) with alanine (a) at amino acid position 783, a substitution of serine (S) with proline (P) at amino acid position 784, a substitution of threonine (T) with methionine (M) at amino acid position 790M, a substitution of threonine (T) with alanine (a) at amino acid position 854, aspartic acid (D) with tyrosine (Y) at amino acid position 761, leucine (L) with serine (S) at amino acid position 747, and cysteine (C) with serine (S) or glycine (G) at amino acid position 797. In one embodiment, dTAG is an amino acid sequence derived from seq.id.no. 53 or a fragment thereof. In one embodiment, dTAG is an amino acid sequence derived from seq.id No. 54 or a fragment thereof. In one embodiment, seq.id No. 54 has a leucine at position 163. In one embodiment, dTAG is an amino acid sequence derived from seq id No. 55 or a fragment thereof. In one embodiment, seq.id No. 55 has a leucine at position 163. In one embodiment, SEQ ID No. 55 has a threonine at position 95. In one embodiment, seq.id No. 55 has a leucine at position 163. In one embodiment, seq.id No. 55 has a threonine at position 95. In one embodiment, dTAG is an amino acid sequence derived from seq.id.no. 56 or a fragment thereof. In one embodiment, seq id No. 56 has a leucine at position 163. In one embodiment, seq.id No. 56 has a threonine at position 95. In one embodiment, seq.id No. 56 has a leucine at position 163 and a threonine at position 95.

In another embodiment, dTAG for use in the present invention is an amino acid sequence derived from BCR-ABL. In certain embodiments, dTAG is a modified or mutated BCR-ABL protein or fragment thereof. In certain embodiments, the one or more mutations of BCR-ABL comprise a substitution of tyrosine (T) with isoleucine (I) at amino acid position 315. In one embodiment, dTAG is an amino acid sequence derived from seq.id No. 57 or a fragment thereof. In one particular example, dTAG is the amino acid sequence derived from seq.id.no. 58 or a fragment thereof.

In another embodiment, the dTAG used in the present invention is an amino acid sequence derived from ALK. In certain embodiments, dTAG is a modified or mutated ALK protein or fragment thereof. In certain embodiments, the one or more mutations of ALK comprise a substitution of methionine for leucine (L) at amino acid position 1196. In one embodiment, dTAG is an amino acid sequence derived from seq.id.no. 59 or a fragment thereof.

In another embodiment, the dTAG used in the present invention is an amino acid sequence derived from JAK 2. In certain embodiments, dTAG is a modified or mutated JAK2 protein or a fragment thereof. In certain embodiments, the one or more mutations of JAK2 includes a substitution of valine (V) with valine (F) at amino acid position 617. In one embodiment, dTAG is an amino acid sequence derived from seq.id.no. 60 or a fragment thereof.

In another embodiment, dTAG for use in the present invention is an amino acid sequence derived from BRAF. In certain embodiments, dTAG is a modified or mutated BRAF protein or fragment thereof. In certain embodiments, the one or more mutations of BRAF comprise a substitution of glutamic acid (E) for valine (V) at amino acid position 600. In one embodiment, dTAG is an amino acid sequence derived from seq.id No. 61 or a fragment thereof.

In alternative embodiments, the dTAG used in the present invention includes, but is not limited to, an amino acid sequence derived from a protein selected from the group consisting of EGFR, BCR-ABL, ALK, JAK2, and BRAF. In one embodiment, the protein contains one or more mutations. In one embodiment, the one or more mutations inactivate the protein.

In another embodiment, the dTAG used in the present invention is an amino acid sequence derived from Src. In certain embodiments, dTAG is a modified or mutated Src protein or fragment thereof. In certain embodiments, the one or more mutations or modifications of Src comprises the substitution of threonine (T) with glycine (G) or alanine (a) at amino acid position 341. In one embodiment, dTAG is an amino acid sequence derived from seq.id.no. 62 or a fragment thereof. In a specific example, dTAG is an amino acid sequence derived from seq.id.no. 63 or a fragment thereof.

In another embodiment, the dTAG used in the present invention is an amino acid sequence derived from LKKR 2. In certain embodiments, dTAG is a modified or mutated LKKR2 protein or fragment thereof. In certain embodiments, the one or more mutations of LKKR2 include a substitution of arginine (R) with cysteine (C) at amino acid 1441, glycine (G) and serine (S) at amino acid 2019, and isoleucine (I) with threonine (T) at amino acid 2020.

In another embodiment, the dTAG used in the present invention is an amino acid sequence derived from PDGFR α. In certain embodiments, the dTAG is a modified or mutated PDGFR α protein or fragment thereof. In certain embodiments, the one or more mutations of PDGFR α comprise a substitution of threonine (T) with isoleucine (I) at amino acid 674.

In another embodiment, the dTAG used in the present invention is an amino acid sequence derived from RET. In certain embodiments, dTAG is a modified or mutated RET protein or fragment thereof. In certain embodiments, the one or more mutations of RET comprise a substitution of glycine (G) with serine (S) at amino acid 691. In certain embodiments, the one or more mutations of RET comprise a substitution of arginine (R) with threonine (T) at amino acid 749. In certain embodiments, the one or more mutations of RET comprise a substitution of glutamine (Q) with glutamine (Q) at amino acid 762. In certain embodiments, the one or more mutations of RET comprise a substitution of tyrosine (Y) with phenylalanine (F) at amino acid 791. In certain embodiments, the one or more mutations of RET comprise a substitution of valine (M) for valine (V) at amino acid 804. In certain embodiments, the one or more mutations of RET comprise a substitution of methionine (M) with threonine (T) at amino acid 918.

In an alternative embodiment, the dTAG used in the present invention includes, but is not limited to, an amino acid sequence derived from a protein selected from the group consisting of Kit, Jak3, Abl, Mek1, HIV reverse transcriptase and HIV integrase.

In another embodiment, the dTAG used in the present invention is an amino acid sequence derived from LKKR 2. In certain embodiments, dTAG is a modified or mutated LKKR2 protein or fragment thereof. In certain embodiments, the one or more mutations of LKKR2 include a substitution of arginine (R) with cysteine (C) at amino acid 1441, a substitution of glycine (G) with serine (S) at amino acid 2019, and a substitution of isoleucine (I) with threonine (T) at amino acid 2020.

In one embodiment, dTAG has an amino acid sequence derived from LRRK2 protein (UniProtKB-Q5S 007(LRKK2_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from amino acids 1328 to 1511 of Q5S 007. In one embodiment, dTAG is derived from amino acids 1328 to 1511 of Q5S007 where amino acid 1441 is cysteine. In one embodiment, the dTAG is derived from amino acids 1328 to 1511 of Q5S007, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-U1. In one embodiment, the dTAG is derived from amino acids 1328 to 1511 of Q5S007 (where amino acid 1441 is cysteine), and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-U1. In one embodiment, dTAG is derived from amino acids 1879 to 2138 of Q5S 007. In one particular example, dTAG is derived from amino acids 1879 to 2138 of Q5S007 where amino acid 2019 is serine. In one embodiment, dTAG is derived from amino acids 1879 to 2138 of Q5S007, wherein amino acid 2020 is threonine. In one embodiment, the dTAG is derived from amino acids 1879 to 2138 of Q5S007, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in tables T-U2 or U3. In one embodiment, the dTAG is derived from amino acids 1879 to 2138 of Q5S007 wherein amino acid 2019 is serine, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-U2. In one embodiment, the dTAG is derived from amino acids 1879 to 2138 of Q5S007 wherein amino acid 2020 is threonine and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-U3.

In one embodiment, dTAG has an amino acid sequence derived from PDGFR alpha protein (UniProtKB-P09619 (PDGFR _ HUMAN), which is incorporated herein by reference) or a variant thereof. In one embodiment, dTAG is derived from amino acids 600 to 692 of P09619. In one embodiment, dTAG is derived from amino acids 600 to 692 of P09619, where amino acid 660 is alanine. In one embodiment, the dTAG is derived from amino acids 600 to 692 of P09619, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-V1. In one embodiment, the dTAG is derived from amino acids 600 to 692 of P09619, where amino acid 660 is alanine and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-V1.

In another embodiment, the dTAG used in the present invention is an amino acid sequence derived from RET. In certain embodiments, dTAG is a modified or mutated RET protein or fragment thereof. In certain embodiments, the one or more mutations of RET comprise a substitution of glycine (G) with serine (S) at amino acid 691. In certain embodiments, the one or more mutations of RET comprise a substitution of arginine (R) with threonine (T) at amino acid 691. In certain embodiments, the one or more mutations of RET comprise a substitution of glutamine (Q) with glutamine (E) at amino acid 691. In certain embodiments, the one or more mutations of RET comprise a substitution of tyrosine (Y) with phenylalanine (F) at amino acid 691. In certain embodiments, the one or more mutations of RET comprise a substitution of valine (M) for valine (M) at amino acid 691. In certain embodiments, the one or more mutations of RET comprise a substitution of methionine (M) with threonine (T) at amino acid 691.

In one embodiment, dTAG has an amino acid sequence derived from PDGFR alpha protein (UniProtKB-P07949 (RET _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from amino acids 724 to 1016 of P07949. In one embodiment, dTAG is derived from amino acids 724 to 1016 of P07949, where amino acid 940 is isoleucine. In one embodiment, the dTAG is derived from amino acids 724 to 1016 of P07949, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W1. In one embodiment, the dTAG is derived from amino acids 724 to 1016 of P07949 (where amino acid 940 is isoleucine), and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W1. In one embodiment, the dTAG is derived from amino acids 724 to 1016 of P07949, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W2. In one embodiment, the dTAG is derived from amino acids 724 to 1016 of P07949, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W3. In one embodiment, the dTAG is derived from amino acids 724 to 1016 of P07949, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W4. In one embodiment, the dTAG is derived from amino acids 724 to 1016 of P07949, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W5. In one embodiment, the dTAG is derived from amino acids 724 to 1016 of P07949, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W6.

In a particular embodiment, dTAG is derived from the amino acid sequence of seq.id No. 53 or a fragment thereof, and dTAG is capable of being bound by a heterobifunctional compound comprising an EGFR dTAG targeting ligand selected from table T-P1. In particular embodiments, dTAG is an amino acid sequence derived from SEQ ID No. 54, or a fragment thereof, and dTAG is capable of being bound by a heterobifunctional compound comprising an EGFR dTAG targeting ligand selected from table T-P2. In particular embodiments, the dTAG is an amino acid sequence derived from SEQ ID No. 55 or a fragment thereof, and the dTAG is capable of being bound by a heterobifunctional compound comprising an EGFR dTAG targeting ligand selected from Table T-P3. In particular embodiments, the dTAG is an amino acid sequence derived from SEQ ID No. 56 or a fragment thereof, and the dTAG is capable of being bound by a heterobifunctional compound comprising an EGFR dTAG targeting ligand selected from table T-P. In particular embodiments, dTAG is an amino acid sequence derived from SEQ ID No. 57, or a fragment thereof, and dTAG is capable of being bound by a heterobifunctional compound comprising a BCR-ABL dTAG targeting ligand selected from Table T-Q1. In particular embodiments, dTAG is an amino acid sequence derived from SEQ ID No. 58 or a fragment thereof, and dTAG is capable of being bound by a heterobifunctional compound comprising a BCR-ABL dTAG targeting ligand selected from Table T-Q1. In particular embodiments, dTAG is an amino acid sequence derived from SEQ id No. 59 or a fragment thereof, and dTAG is capable of being bound by a heterobifunctional compound comprising an ALK dTAG targeting ligand selected from table T-R1. In particular embodiments, the dTAG is an amino acid sequence derived from SEQ ID No. 60, or a fragment thereof, the dTAG being capable of being bound by a heterobifunctional compound comprising a JAK 2dTAG targeting ligand selected from table T-S1. In particular embodiments, dTAG is an amino acid sequence derived from SEQ ID No. 61, or a fragment thereof, and dTAG is capable of being bound by a heterobifunctional compound comprising a BRAF dTAG targeting ligand selected from table T-T1.

In one embodiment, the dTAG is derived from LRRK2 amino acids 1328 to 1511(UnitPro-Q5S007), and the dTAG targeting ligand in the heterobifunctional compound is a ligand selected from Table T-U1. In one embodiment, the dTAG is derived from LRRK2 amino acids 1328 through 1511(UniProt-Q5S007) wherein amino acid 1441 is cysteine and the dTAG targeting ligand in the heterobifunctional compound is a ligand selected from Table T-U1. In one embodiment, dTAG is derived from LRRK2 amino acids 1879 to 2138(UniProt-Q5S 007). In one embodiment, dTAG is derived from LRRK2 amino acids 1879 to 2138(UniProt-Q5S007) wherein amino acid 2019 is serine. In one embodiment, dTAG is derived from amino acids 1879 to 2138(UniProt-Q5S007) wherein amino acid 2020 is threonine. In one embodiment, the dTAG is derived from LRRK2 amino acids 1879 to 2138(UniProt-Q5S007), and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in tables T-U2 or U3. In one embodiment, the dTAG is derived from LRRK2 amino acids 1879 to 2138(UniProt-Q5S007) wherein amino acid 2019 is serine, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-U2. In one embodiment, the dTAG is derived from LRRK2 amino acids 1879 through 2138(UniProt-Q5S007) wherein amino acid 2020 is threonine and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-U3. In one embodiment, the dTAG is derived from PDGFR amino acids 600 to 692(UniProt-P09619), and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-V1. In one embodiment, the dTAG is derived from PDGFR amino acids 600 to 692(UniProt-P09619), wherein amino acid 674 is isoleucine, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-V1. In one embodiment, dTAG is derived from RET amino acids 724 to 1016(UniProtKB-P07949), and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in tables T-W1-W6. In one embodiment, dTAG is derived from RET amino acids 724 to 1016(UniProtKB-P07949) wherein amino acid 691 is serine, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W1. In one embodiment, dTAG is derived from RET amino acids 724 to 1016(UniProtKB-P07949), wherein amino acid 749 is threonine, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W2. In one embodiment, dTAG is derived from RET amino acids 724 to 1016(UniProtKB-P07949), wherein amino acid 762 is glutamine, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W3. In one embodiment, dTAG is derived from RET amino acids 724 to 1016(UniProtKB-P07949) wherein amino acid 791 is phenylalanine, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W4. In one embodiment, dTAG is derived from RET amino acids 724 through 1016(UniProtKB-P07949) wherein amino acid 804 is methionine, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W5. In one specific example, dTAG is derived from RET amino acids 724 to 1016(UniProtKB-P07949) wherein amino acid 918 is threonine and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-W6. In one embodiment, the dTAG is derived from JAK2, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-JJJ 1. In one embodiment, the dTAG is derived from Abl, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-KKK 1. In one embodiment, the dTAG is derived from MEK1, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-LLL 1. In one embodiment, the dTAG is derived from KIT, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-MMM 1. In one embodiment, the dTAG is derived from HIV reverse transcriptase, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-NNN 1. In one embodiment, the dTAG is derived from HIV integrase, and the dTAG targeting ligand in the heterobifunctional compound is selected from the ligands in Table T-OOO 1.

In particular embodiments, dTAG is derived from a bifunctional protein selected from the group consisting of EGFR, ErbB2, ErbB4, VEGFR1, VEGFR2, VEGFR3, Kit, BCR-Abl, Src, Lyn, Hck, RET, C-Met, TrkB, Flt3, Axl, Tie2, ALK, IGF-1R, InsR, ROS1, MST1R, B-Raf, Lck, Yes, Fyn, HER 2-breast cancer, PNET, RCC, RA ML, SEGA, BTK, FGFR1/2/3/4, DDR1, PDGFR α, PDGFR β, CDK4, CDK6, Fms, Itk, T315I, Eph2A, JAK1, JAK2, JAK 9 CDK 686 8, CSF-1R, FKBP 56/mTOR, 82 1, MEK2, Brhk 68627, EphR, A-Raf, Eph B-Raf, and a targeting protein.

Heterobifunctional compounds capable of binding to the above amino acid sequences or fragments thereof can be generated using dTAG targeting ligands described in table T. In one embodiment, the genome of the nucleic acid sequence encoding dTAG derived from the above amino acid sequence or fragment thereof is inserted into a gene encoding an endogenous protein of interest which upon expression produces an endogenous protein-dTAG hybrid protein and is degraded by administering to the subject a heterobifunctional compound comprising a dTAG targeting ligand as described in table T. In one embodiment, the genome of the nucleic acid sequence encoding dTAG derived from the above amino acid sequence or fragment thereof is inserted into a gene encoding an endogenous protein of interest which, when expressed, produces an endogenous protein-dTAG hybrid protein and is degraded by administering to the subject its corresponding heterobifunctional compound capable of binding to dTAG, e.g., the heterobifunctional compounds depicted in figure 29, figure 30, figure 31, figure 32 and figure 33, or any other heterobifunctional compound described herein.

Drawings

FIG. 1 is a schematic diagram showing the "bump-well" method for selective degradation of dTAG fusion proteins. For example, a dTAG fusion may be a form of FK 506-and rapamycin binding protein, FKBP12, which forms a "pore" lumen via an amino acid mutation (F36V). This mutant FKBP12 ("raised" FKBP, also known as FKBP or FKBP12 (seq. id No.:2)) can then be selectively targeted by heterobifunctional compounds having a synthetic "bump" in the FKBP12 binding domain, i.e. a linker, and a cerebellin binding domain. The heterobifunctional compound does not target native FKBP12, thus providing selectivity for wild-type variants of the tag that naturally occur in human cells.

Figure 2 is a schematic showing genomic integration of a nucleic acid sequence encoding dTAG into the genomic locus of an endogenous gene encoding PCSK 9. Following homologous recombination, the resulting insertion produces an expression product comprising an N-terminal dTAG in-frame with the proprotein convertase subtilisin/kexin type 9 (PCSK9) protein, thereby providing a proprotein convertase subtilisin/kexin type 9 (PCSK9) -dTAG hybrid. Can be degraded by heterobifunctional compounds targeting the dTAG sequence.

FIG. 3 is a schematic representation of the genomic integration of a nucleic acid sequence encoding dTAG into the genomic locus of an endogenous gene encoding β -catenin (CTNNB 1). Following homologous recombination, the resulting insertion produces an expression product comprising an N-terminal dTAG that is in frame with the protein of β -catenin (CTNNB1), thereby providing a β -catenin (CTNNB1) -dTAG hybrid that is capable of being degraded by heterobifunctional compounds targeting the dTAG sequence.

FIG. 4 is an immunoblot of cells treated with heterobifunctional compounds of the invention. 293FT cells (CRBN-WT or CRBN-/-) expressing HA tag FKBP12WT or FKBP were treated with the indicated concentrations of dFKBP7 for 4 hours. CRBN-dependent degradation of FKBP but not FKBPWT confirmed the selective activity of dFKBP7 on mutant FKBP.

Fig. 5A and 5B are graphs measuring the activity of a panel of dFKBP heterobifunctional compounds in cells expressing FKBP fused to Nluc. Degradation of FKBP was measured as the signal ratio between NANOluc and firefly luciferase from the same polycistronic transcript (Nluc/Fluc) in wild type (fig. 7A) or CRBN-/- (fig. 7B)293FT cells after 4 hours of treatment with dFKBP at the indicated concentrations. A decrease in the signal ratio indicates FKBP (Nluc) degradation.

FIG. 6 is an immunoblot of cells treated with heterobifunctional compounds of the invention. Isogenic 293FT cells (CRBN-WT or CRBN-/-) expressing FKBP12WT or FKBP were treated with 100nM dFKBP7 or dFKBP13 for 4 hours. CRBN-dependent degradation of FKBP but not FKBP12WT or endogenous FKBP12 demonstrated the selectivity of dFKBP7 and dFKBP13 for mutant FKBP.

FIG. 7 is an immunoblot of cells treated with heterobifunctional compounds of the invention. Isogenic 293FT cells expressing HA-tagged FKBP (CRBN-WT or CRBN-/-) were treated with the indicated dose of dFKBP13 for 4 hours (example 6). These data demonstrate dose and CRBN-dependent degradation of FKBP13 on HA-labeled FKBP.

FIG. 8 is an immunoblot of cells treated with heterobifunctional compounds of the invention. 293FT cells expressing HA-tagged FKBP (CRBN-WT) were treated with 100nM dFKBP13 for the indicated time. Cells were harvested and protein lysates were immunoblotted to measure the kinetics of HA-labeled FKBP degradation induced by dFKBP 13.

FIG. 9 is an immunoblot of cells treated with heterobifunctional compounds of the invention. FKBP-expressing 293FT cells (CRBN-WT) were pretreated with 1uM Carfilzomib (proteasome inhibitor), 0.5uM MLN4924 (conjugation inhibitor) and 10uM Lenalidomide (Lenalidomide) (CRBN binding ligand) for 2 hours followed by dFKBP13 for 4 hours (example 8). The proteasome inhibitor carfilzomib rescues the degradation of FKBP13 to HA-labeled FKBP, establishing a requirement for proteasome function. Pretreatment with NAE1 inhibitor MLN4924 rescued HA-tagged FKBP, establishing dependence on CRL activity as it was expected that cullin-based ubiquitin ligase required conjugation for processive E3 ligase activity. Pretreatment with excess lenalidomide eliminated dFKBP 13-dependent FKBP degradation, confirming the requirement for CRBN to participate in degradation.

Fig. 10A and 10B are immunoblots of cells treated with heterobifunctional compounds of the present invention. Immunoblots of MV 4; the 11 protein expressing leukemia cells were fused to HA-tagged mutant FKBP. Cells were treated with indicated concentrations of FKBP-selective heterobifunctional compound, dFKBP7 or dFKBP13 for 16 hours and the abundance of the fusion protein was measured by western immunoblot analysis.

Fig. 11 is an immunoblot of NIH3T3 cells expressing KRASG12V alleles fused to FKBP at the N-or C-terminus. Cells were treated with 500nM dFKBP7 for the indicated time. Cells were harvested and immunoblotted to measure degradation of FKBP-KRASG 12V and downstream surrogates of KRAS signaling (e.g., pMEK and pAKT). The data suggests that N-terminal FKBP fusion is active and degrades upon administration of dFKBP 7.

Figure 12 is an immunoblot of NIH3T3 cells expressing FKBP fused to the N-terminus of KRASG12V treated with 1uM of the dFKBP heterobifunctional compound for 24 hours. Cells were harvested and immunoblotted to measure degradation of FKBP-KRASG 12V and downstream surrogates of KRAS signaling (e.g., pMEK and pAKT). The data suggest that dFKBP9, dFKBP12, and dFKBP13 induced efficient degradation of FKBP-KRASG 12V and inhibition of downstream signaling.

Figure 13 is an immunoblot of NIH3T3 cells expressing FKBP fused to the N-terminus of KRASG12V treated with dFKBP13 at the indicated concentration for 24 hours. Cells were harvested and immunoblotted to measure degradation of FKBP-KRASG 12V and downstream surrogates of KRAS signaling (e.g., pMEK and pAKT). The data suggest that dFKBP13 induced efficient degradation of FKBP x-KRASG 12V and effectively inhibited downstream signaling with IC 50>100 nM.

Figure 14 is an immunoblot of NIH3T3 cells expressing FKBP fused to the N-terminus of KRASG12V treated with 1uM dFKBP13 for the indicated time. Cells were harvested and immunoblotted to measure degradation of FKBP-KRASG 12V and downstream surrogates of KRAS signaling (e.g., pMEK and pAKT). These data suggest that dFKBP13 induced effective degradation of FKBP-KRASG 12V and inhibition of downstream signaling as early as 1 hour post-treatment.

FIG. 15 is an immunoblot of NIH3T3 cells expressing dTAG-KRASG12V pre-treated with 1uM carfilzomib (proteasome inhibitor), 0.5uM MLN4924 (conjugation inhibitor) and 10uM lenalidomide (CRBN binding ligand) for 2 hours prior to 4 hours of treatment with dFKBP 13.

FIG. 16 is an immunoblot of NIH3T3 cells expressing the KRAS allele, the WT or mutated form of amino acid glycine 12(G12C, G12D, and G12V) treated with 1uM dFKBP13 for 24 hours.

FIG. 17 is an immunoblot of NIH3T3 cells expressing WT or mutant KRAS alleles (G13D, Q61L and Q61R) treated with 1uM dFKBP13 for 24 hours.

Fig. 18A, 18B, 18C and 18D are graphs of phase contrast images of control NIH3T3 cells or NIH3T3 expressing FKBP fused to the N-terminus of KRASG12V treated with DMSO or dFKBP13 for 24 hours. The phase contrast images highlight the morphological changes induced upon dFKBP 13-dependent degradation of FKBP x-KRASG 12V.

Fig. 19A, 19B, 19C and 19D are proliferation profiles measuring the effect of dFKBP13 on the growth of NIH3T3 control cells expressing NIH3T3 of FKBP x-KRASG 12V. If dFKBP lasted 72 hours, cells were treated with the indicated concentrations and cell counts were measured using the ATPlite assay. The ATPlite 1step luminescence assay measures cell proliferation and cytotoxicity in cells based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin. A decrease in signal indicates a decrease in cell number.

FIG. 20 is a bar graph illustrating 48 hour treatment of NIH3T3 cells expressing dTAG-KRASG12V with dFKBP7 and dFKBP13 to induce targeted degradation of dTAG-KRASG 12V. Fixed cells were stained with propidium iodide and cell cycle analysis was performed.

Fig. 21A, 21B, 21C, 21D, 21E, 21F, 21G, 21H and 21I provide examples of degradation determining region moieties for use in the invention, wherein R is the point of attachment of a linker and X is as defined herein.

FIG. 22 provides further examples of degradation determining regions useful in the present invention, wherein R is the point of attachment of a linker and X is as defined herein.

FIG. 23 provides further examples of degradation determining regions useful in the present invention, wherein R is the point of attachment of a linker and X is as defined herein.

Fig. 24 provides an example of a connector portion for use with the present invention.

Fig. 25 provides other examples of connector portions for use with the present invention.

FIG. 26 provides an example of a heteroaliphatic linker moiety for use in the present invention.

FIG. 27 provides an example of an aromatic linker moiety useful in the present invention.

Fig. 28A, 28B, 28C, 28D, 28E, 28F and 28G provide dTAG targeting ligands for use in the invention, wherein R is the point of linker attachment.

Fig. 29A, 29B, 29C, 29D, 29E, 29F, 29G and 29H provide specific heterobifunctional compounds for use in the invention.

Fig. 30A, fig. 30B, fig. 30C, fig. 30D, fig. 30E, fig. 30F, fig. 30G, fig. 30H, fig. 30I, fig. 30J, fig. 30K, fig. 30L, fig. 30M, fig. 30N, fig. 30O, and fig. 30P provide specific heterobifunctional compounds for use in the present invention, wherein X in the above structures is a halogen selected from F, Cl, Br, and I.

Fig. 31A, fig. 31B, fig. 31C, fig. 31D, fig. 31E, fig. 31F, fig. 31G, fig. 31H, fig. 31I and fig. 31J provide specific heterobifunctional compounds for use in the present invention.

FIG. 32A, FIG. 32B, FIG. 32C, FIG. 32D, FIG. 32E, FIG. 32F, FIG. 32G, FIG. 32H, FIG. 32I, FIG. 32J, FIG. 32K, FIG. 32L, FIG. 32M, FIG. 32N, FIG. 32O, FIG. 32P, FIG. 32Q, FIG. 32R, FIG. 32S, FIG. 32T, FIG. 32U, FIG. 32V, FIG. 32W, FIG. 32X, FIG. 32Y, FIG. 32Z, FIG. 32AA, FIG. 32BB, FIG. 32CC, FIG. 32DD and FIG. 32EE provide specific heterobifunctional compounds for use in the invention, wherein R, D, and D provide specific heterobifunctional compounds of the invention^AR1And R^AR2As described herein.

Fig. 33A, 33B, 33C, 33D, 33E, 33F, 33G, 33H, 33I, 33J, 33K, 33L, 33M, 33N, 33O, 33P, 33Q, 33R, 33S, 33T, 33U, 33V, 33W provide additional heterobifunctional compounds for use in the invention.

Detailed Description

Unless otherwise indicated, the compositions disclosed herein and the implementations of making and using them in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields are within the routine skill of the art. These techniques are explained fully in the literature. See, for example, Sambrook et al, Molecular CLONING, A Laboratory Manual, Second edition, Cold Spring Harbor LABORATORY Press,1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, New York,1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTUREAND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS INDENZYMOLOGY, Vol.304, "Chromatin" (P.M.Wassarman and A.P.Wolffe, eds.), academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, "chromatography protocols" (P.B. Becker, ed.) Humana Press, Totowa, 1999.

Herein, we describe a method of using gene and protein disruption to provide a highly selective and reversible method of promoting protein degradation. The method is valuable for accurate, temporal, small molecule controlled target validation and exploration of cellular and in vivo effects of protein degradation of interest.

In this approach, a region of the target gene of interest is targeted by the guide RNA and Cas9 for insertion (knock-in) of the expression cassette of dTAG present in the Homologous Recombination (HR) targeting vector. The HR targeting vector contains homology arms at the 5 'and 3' ends of the expression cassette that are homologous to the genomic DNA surrounding the targeted gene of interest. By fusing dTAG with the target gene of interest, the resulting fusion protein after expression will be susceptible to proteasome-mediated degradation after treatment with a biologically inert small molecule heterobifunctional compound.

Genome editing in mammalian cells offers great potential for the treatment and correction of human diseases. By using short single guide rnas (sgrnas), the Cas9 endonuclease can be directed to a target genomic position, thereby inducing DNA double-strand breaks. These breaks are repaired by non-homologous end joining, which can be used to create insertions or deletions (indels) that inactivate the gene. In vivo genome editing can be accomplished by adenovirus-associated virus (AAV), lentivirus, particle, hydrodynamic injection-or electroporation-mediated methods or combinations thereof delivered with CRISPR/Cas9 (see, e.g., Kumar et al, hum. Gene ther.12, (2001): 1893-1905; Wu et al, mol. ther.18, (2010): 80-86; Ranet al, Nature 520, (2015): 186-191; Swech et al, Nat. Biotechnol.33, (2015): 102-105; Zuris et al, Nat. Biotechnol.33, (2015): 73-80; Kauffman et al, Nano. Lett.15, (2015): 7300-7306; Diverse et al, Ci. Res.115, (2014; 488; Maresch et al, Mar. 7. 10; Nature et al.: 05; Kidney et 9. 2014., 9, Nature, 2008. 10. Eq. 15; Kidney et al., 9. 2014, 9, Nature, 9. Yn. 9. multidot. 1. nat.Biotechnol.34, (2016): 328-333; and Xue et al, Nature 514, (2014):380-384, which is incorporated herein by reference), and somatic genome editing has been applied to mouse organs such as lung, liver, brain and pancreas (see, e.g., Xue et al, Nature 514, (2014): 380-384; sanchez-river et al, Nature 516, (2014): 428-; platt et al, Cell 159, (2014) 440-455; yin et al, nat. biotechnol.32, (2014) 551-553; zuckermann et al, nat. Commun.6, (2015): 7391; chiou et al, Genes Dev.29, (2015) 1576-1585; and Mazur et al, nat. Med.21, (2015): 1163-. However, the long-term effects of permanent Genome modification are unclear and there are problems with incomplete accuracy of Genome editing and the effects on direct correction of the biological compensatory mechanisms that may be present in adults (see, e.g., Fu et al, nat. Biotechnol.31(9), (2013): 822-.

Here we describe strategies for broad therapeutic use based on in vivo genomic engineering to generate knock-in fusion proteins that are produced from endogenous loci and are susceptible to degradation in a ligand-dependent, reversible, and dose-responsive manner. The fusion protein contains dTAG, which is targeted by a bi-or polyvalent hetero-bi-functional compound. Heterobifunctional compounds have the ability to bind dTAG and recruit E3 ligase, for example, the cereblon-containing CRL4A E3 ubiquitin ligase complex. This recruitment induces ubiquitination of the fusion protein (either in the dTAG domain or on the homologous protein) and subsequent degradation via UPP. By this method, the protein of interest can be targeted to rapid ubiquitin-mediated degradation with high specificity and high specificity without the need to find de novo ligand (de novo ligand) for the protein of interest. In view of the combined use of small molecules and genomic engineering, for in vivo use.

A variety of dtags may be used, including but not limited to bromodomains, e.g., the first bromodomain of BRD 4; hormone receptors, e.g., ER, AR, RXR; FKBP 12; DHFR, especially bacterial DHFR and other commonly used protein fusion tags, can be bound by ligands that can be converted into heterobifunctional compounds. In some cases, it would be advantageous to use dTAG that utilizes a "bump-hole" strategy that selectively targets the ATP binding site of protein kinases, conceptually related to the developed "bump-hole" strategy. In this case, the dTAG fusion is a form of FK 506-and rapamycin binding protein, FKBP12, which forms the lumen of the "pore" via an amino acid mutation (F36V). This mutant FKBP12 ("raised" FKBP, also known as FKBP (seq. id No.:2)) is then targeted by a heterobifunctional compound (or similar molecule) having a synthetic "bump" in the FKBP12 binding domain, a linker and a cerebellin targeting domain (e.g., an IMID derivative). this molecule does not target native FKBP12, thus providing selectivity of the heterobifunctional compound to wild-type variants of the tag naturally present in human cells. a schematic representation representing an exemplary "bump-hole" strategy is shown in fig. 1.

The invention described herein provides a mechanism to control endogenous protein degradation associated with disease by combining genome engineering with small molecule activation/de-modulation segments. Applications of this technology include, but are not limited to, 1) targeted degradation of proteins, where pathology is a function of gain of function mutations, 2) targeted degradation of proteins, where pathology is a function of amplification or increased expression, 3) targeted degradation of proteins that are manifested as monogenic diseases, 4) targeted degradation of proteins, where genetic susceptibility is manifested for a longer period of time and is often no longer sufficient after alternative biological compensation mechanisms, such as hypercholesterolemia, proteinopathies.

Definition of

The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably to refer to a polymer of deoxyribonucleotides or ribonucleotides in either a linear or circular conformation, and in either single-or double-stranded form. For the purposes of this disclosure, these terms should not be construed as limitations with respect to polymer length. The term may include known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar, and/or phosphate moiety (e.g., phosphorothioate backbone). Typically, analogs of a particular nucleotide have the same base-pairing specificity; that is, the analog of A will base pair with T.

The terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acid.

"binding" refers to a sequence-specific non-covalent interaction between a macromolecule (e.g., between a protein and a nucleic acid) or a macromolecule and a small molecule (e.g., between a protein and a drug). Not all components of a binding interaction need be sequence specific (e.g., in contact with a phosphate residue in the DNA backbone), as long as the entire interaction is sequence specific.

"recombination" refers to the process of exchanging genetic information between two polynucleotides. For the purposes of this disclosure, "homologous recombination" (HR) refers to a specialized form of this exchange that occurs during repair of double-stranded breaks in cells, e.g., via a homology-directed repair mechanism. This process requires nucleotide sequence homology, repairs the "target" molecule (i.e., the molecule undergoing double-strand cleavage) using a "donor" molecular template, and results in the transfer of genetic information from the donor to the target.

One or more targeted nucleases as described herein generate a double-stranded break in a target sequence (e.g., cellular chromatin) at a predetermined site, and a dTAG-encoding "donor" polynucleotide having homology to the nucleotide sequence in the region of the break may be introduced into the cell. The presence of double-stranded breaks has been shown to promote integration of the donor sequence. The donor sequence may be physically integrated, resulting in the introduction of all or part of the nucleotide sequence in the donor into cellular chromatin. Thus, a first sequence in cellular chromatin can be altered and converted to a sequence present in a donor polynucleotide.

In certain methods for targeted recombination and/or replacement and/or alteration of sequences in a region of interest in cellular chromatin, chromosomal sequences are altered by homologous recombination with an exogenous "donor" nucleotide sequence encoding dTAG. If sequences homologous to the break region are present, this homologous recombination is stimulated by the presence of a double-stranded break in cellular chromatin.

In any of the methods described herein, the exogenous nucleotide sequence (the "donor sequence" or "transgene") may contain a sequence that is homologous but not identical to the genomic sequence in the region of interest, thereby stimulating homologous recombination to insert a non-identical sequence, i.e., a nucleic acid sequence encoding dTAG, in the region of interest. Thus, the portion of the donor sequence that is homologous to the sequence in the region of interest exhibits about 80 to 99% (or any integer therebetween) sequence identity to the genomic sequence being replaced. In other embodiments, for example, if only 1 nucleotide differs between donor and genomic sequences that are more than 100 consecutive base pairs, the homology between the donor and genomic sequences is greater than 99%. The non-homologous portion of the donor sequence contains a nucleic acid sequence that is not present in the region of interest, such as a sequence encoding dTAG, so that a new sequence is introduced into the region of interest. In these cases, the non-homologous sequence is typically flanked by sequences that are homologous or identical to the sequence in the region of interest by 50 to 1,000 base pairs (or any integer value therebetween) or any number of base pairs greater than 1,000. In other embodiments, the donor sequence is not homologous to the first sequence and is inserted into the genome by a non-homologous recombination mechanism.

"cleavage" refers to the breaking of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods, including but not limited to enzymatic or chemical hydrolysis of the phosphodiester bond. Both single strand and double strand cleavage are possible, and double strand cleavage can occur due to two different single strand cleavage events. DNA cleavage can result in the generation of blunt or staggered ends. In certain embodiments, the fusion polypeptide is used to target double-stranded DNA cleavage.

"chromatin" is a nucleoprotein structure comprising the genome of a cell. Cellular chromatin includes nucleic acids, primarily DNA and proteins, including histone and non-histone chromosomal proteins. Most eukaryotic chromatin exists in the form of nucleosomes, in which the nucleosome core comprises DNA of about 150 base pairs associated with an octamer comprising two histones H2A, H2B, H3 and H4, respectively; and linker DNA extending between nucleosome cores (variable length depending on the organism). The molecules of histone H1 are typically associated with linker DNA. For the purposes of this disclosure, the term "chromatin" is intended to include all types of nuclear proteins, including prokaryotic and eukaryotic cells. Cellular chromatin includes both chromosomal and episomal chromatin.

An "exogenous" molecule is a molecule that is not normally present in a cell, such as certain dTAGs, but may be introduced into a cell by one or more genetic, biochemical or other methods. The foreign molecule may comprise, for example, a synthetic endogenous protein-dTAG hybrid.

An "endogenous" protein is a protein that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, the endogenous protein may be, for example, a transcription factor or enzyme or any other type of naturally expressed protein.

A "fusion" or "hybrid" protein is a protein in which two or more polypeptides are preferably covalently linked. For example, examples of fusion proteins include fusion between an endogenous protein and dTAG.

For purposes of this disclosure, "gene" includes a region of DNA encoding a gene product, as well as all regions of DNA that regulate the production of a gene product, whether or not such regulatory sequences are contiguous with the coding and/or transcribed sequences. Thus, genes include, but are not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

"Gene expression" refers to the conversion of information contained in a gene into a gene product. The gene product can be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of mRNA. Gene products also include RNA modified by methods such as capping, polyadenylation, methylation and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation and glycosylation.

"modulation" of protein expression refers to a change in the activity of a protein. Modulation of expression may include, but is not limited to, decreased protein activity or increased protein activity. For example, as contemplated herein, exposure of an endogenous protein-dTAG hybrid to a heterobifunctional compound results in degradation of the endogenous protein-dTAG hybrid, which can modulate the activity of the endogenous protein. Thus, protein inactivation may be partial or complete.

A "vector" is capable of transferring a gene sequence to a target cell. In general, "vector construct", "expression vector" and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and capable of transferring a gene sequence to a target cell. Thus, the term includes cloning and expression vectors, as well as integration vectors.

The terms "subject" and "patient" are used interchangeably and refer to mammals, such as human patients and non-human primates, as well as laboratory animals, such as rabbits, dogs, cats, rats, mice, rabbits and other animals. Thus, the term "subject" or "patient" as used herein refers to any patient or subject (e.g., a mammal) suffering from a disorder.

A. Heterobifunctional complex targeting protein (dTAG)

The present invention provides methods for making knock-in fusion proteins that are produced from endogenous gene loci and are susceptible to degradation in a ligand-dependent, reversible, and dose-responsive manner. Specifically, a nucleic acid encoding dTAG is inserted in-frame with the target gene of interest, wherein upon expression, the resulting fusion protein contains dTAG targeted by a bi-or polyvalent hetero-bifunctional compound. Heterobifunctional compounds have the ability to bind to target proteins and recruit E3 ligase, e.g., the cereblon-containing CRL4A E3 ubiquitin ligase complex. This recruitment induces ubiquitination of the fusion protein (either on dTAG or the homologous protein) and subsequent degradation via the Ubiquitin Proteasome Pathway (UPP). By this approach, proteins of interest can be targeted to rapid ubiquitin-mediated degradation with high specificity without the need to discover de novo ligands for the POI.

Heterobifunctional compound targeting proteins of synthetic genes are any amino acid sequence to which a heterobifunctional compound can bind, which when contacted with a heterobifunctional compound results in ubiquitination and degradation of the expressed endogenous protein-dTAG hybrid protein. Preferably, dTAG should not interfere with the function of the endogenously expressed protein. In one embodiment, dTAG is a non-endogenous peptide, resulting in heterobifunctional compound selectivity and allowing for the avoidance of off-target effects when administering the heterobifunctional compound. In one embodiment, dTAG is an amino acid sequence derived from an endogenous protein or fragment thereof that has been modified such that the heterobifunctional compound binds only to the modified amino acid sequence and not to the endogenously expressed protein. In one embodiment, dTAG is an endogenously expressed protein or a fragment of an endogenously expressed protein. Any amino acid sequence domain that can be used for heterobifunctional compounds through ligand binding can be used as dTAG contemplated herein. In certain embodiments, it is preferred that the smallest amino acid sequence capable of being bound by a particular heterobifunctional compound be used as dTAG.

In particular embodiments, dTAG for use in the present invention includes, but is not limited to, amino acid sequences derived from endogenously expressed proteins, such as FK506 binding protein-12 (FKBP12), bromodomain-containing protein 4(BRD4), CREB binding protein (CREBBP) and transcriptional activator BRG1(SMARCA4) or variants thereof. As contemplated herein, "variant" means any variant comprising one or several to more amino acid substitutions, deletions or additions, provided that the variant retains substantially the same function as the original sequence, in which case the ligand of the heterobifunctional compound is provided. In other embodiments, dTAG for use in the invention can include, for example, hormone receptors, e.g., estrogen receptor protein, androgen receptor protein, Retinoid X Receptor (RXR) protein, and dihydrofolate reductase (DHFR) (including bacterial DHFR, bacterial dehydrogenases, and variants).

Some embodiments of dTAG may be, but are not limited to, those derived from Hsp90 inhibitors, kinase inhibitors, MDM2 inhibitors, compounds targeting human BET bromodomain-containing proteins, compounds targeting cytoplasmic signaling protein FKBP12, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressive compounds, and compounds targeting arene receptors (AHR).

In certain embodiments, the dTAG is derived from a kinase, a BET bromodomain-containing protein, a cytoplasmic signaling protein (e.g., FKBP12), a nucleoprotein, a histone deacetylase, a lysine methyltransferase, a protein that modulates angiogenesis, a protein that modulates immune response, an Aromatic Hydrocarbon Receptor (AHR), an estrogen receptor, an androgen receptor, a glucocorticoid receptor, or a transcription factor (e.g., SMARCA4, SMARCA2, TRIM 24).

In certain embodiments, dTAG is derived from a kinase, such as, but not limited to, a tyrosine kinase (e.g., AATK, ABL, ABL, ALK, AXL, BLK, BMX, BTK, CSF1, CSK, DDR, DDR, EGFR, EPHA, EPHA, EPHA, EPHA, EPHB, EPHB, EPHB, ERBB, ERBB, FER, FES, FGFR, FGFR, FGFR, FLT, FLT, FLT, FRK, FYN, GSG, HCK, IGF1, ILK, INSR, INSRR, IRAK, KSK, JAK, JAK, JAK, KDR, KIT, LCK, LMTK, LMTK, LTK, LYN, MATK, MERK, MET, MLTK, MSNI 1, MUTK, NPR, TRK, TYRK, TRK, TRYPK, STRK, STRAK, TRK, TRYPK, TRK, STRK, TRYPK, STRAK, TRK, TRYPK, STRK, TRYPK, STRAK, TRYPK, STRK, STRAK, TRYPK, STRK, STRAK, STRK, casein kinase 2, protein kinase a, protein kinase B, protein kinase C, Raf kinase, CaM kinase, AKT, ALK, Aurora a, Aurora B, Aurora C, CHK, CLK, DAPK, DMPK, ERK, GCK, GSK, HIPK, KHS, LKB, LOK, MAPKAPK, MNK, MSSK, MST, NDR, NEK, PAK, PIM, PLK, RIP, RSK, SGK, SIK, STK, TAO, TGF- β, TLK, tsk, or PIM, e.g. cdsk, cdsk rich in cyclin-dependent kinases (e.g. Cdk).

In certain embodiments, dTAG is derived from a BET bromodomain-containing protein, such as, but not limited to, ASH1, ATAD, BAZ1, BAZ1, BAZ2, BAZ2, BRD, BRD, BRD, BRD, BRD, BRD, BRD, BRDT, BRPF, BRPF, BRWD, CECR, CREBP, EP300, FALZ, GCN5L, KIAA1240, LOC 49, MLL, PB, 933AF, PHIP, PRKCBP, SMARCA, SMARCA, SP100, SP110, SP140, TAF, TAF1, TIF1, TRIM, TRIM, TRIM, WDR, XYND, and MLL. In certain embodiments, the BET bromodomain-containing protein is BRD 4.

In certain embodiments, dTAG is derived from, but is not limited to, 7, 8-dihydro-8-oxoguanine triphosphatase, AFAD, arachidonic acid 5-lipoxygenase activating protein, apolipoprotein, baculovirus IAP repeat containing protein 2, Bcl-2, Bcl-xL, E3 ligase XIAP, fatty acid binding protein 4 from adipocytes (FABP4), GTPase k-RAS, HDAC6, hematopoietic prostaglandin D synthase, lactoglutathione lyase, Mcl-1, PA2GA, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1, poly-ADP-ribopolymerase 14, poly-ADP-ribose polymerase 15, sphingolipid activating pro-protein, prostaglandin E synthase, retinal rod rhodopsin sensitive MP 3',' 5-cyclic phosphodiesterase delta, S100-A7, Src, Sumo-conjugase UBC9, superoxide dismutase, Tankyrase (tankyrase)1 or Tankyrase 2.

In certain embodiments, dTAG is derived from nuclear proteins, including but not limited to BRD2, BRD3, BRD4, antennapedia homeodomain protein, BRCA1, BRCA2, CCAAT enhanced binding proteins, histones, Polycomb-histones, high mobility histones, telomer binding proteins, FANCA, FANCD2, FANCE, FANCF, hepatocyte nuclear factor, Mad2, NF-. kappa.B, nuclear receptor coactivators, CREB binding proteins, p55, p107, p130, Rb proteins, p53, c-fos, c-jun, c-mdm2, c-myc, and c-rel.

In particular embodiments, dTAG has the amino acid sequence derived from BRD2 ((Universal Protein Resource Knowle Base (UniProtKB) -P25440 (BRD2_ HUMAN), incorporated herein by reference), BRD3(UniProtKB-Q15059(BRD3_ HUMAN), BRD4(UniProtKB-O60885(BRD4_ HUMAN), incorporated herein by reference), or BRDT (UniProtKB-Q58F21(BRDT _ MAN), incorporated herein by reference) (see Baud HUMAN, A BUmp-and-Hole Approach to genetic control selected of BET branched chemistry Science "Science (6209: 641), and Bauch 638 and foreign gene analysis of molecular analysis, BET molecular analysis of BET molecular analysis, and analysis, C59, and molecular analysis, and analysis report No.2, C, III, C32, C, B, C, B, C, B, all incorporated herein by reference). In certain embodiments, dTAG is a modified or mutated BRD2, BRD3, BRD4, or BRDT protein (see, Baud et al, "A Bump-and-Hole Approach to engineering controlled selection of BETbrodommains chemical probes," Science 346(6209) (2014) (638) -and Baud et al, "New Synthetic Routes to Triazolo-benzodization assays," JMC 59(2016) (1492-) -1500, which is incorporated herein by reference). In certain embodiments, the one or more mutations of BRD2 include a mutation of tryptophan (W) at amino acid position 97. A mutation at amino acid position 103 of valine (V), a mutation at amino acid position 110 of leucine (L), a mutation at amino acid position 370 of W, a mutation at amino acid position 376 of V, or a mutation at amino acid position 381 of L. In certain embodiments, the one or more mutations of BRD3 include a mutation of W at amino acid position 57, a mutation of V at amino acid position 63, a mutation of L at amino acid position 70, a mutation of W at amino acid position 332, a mutation of V at amino acid position 338, or a mutation of L at amino acid position 345. In certain embodiments, the one or more mutations of BRD4 include a mutation of W at amino acid position 81, a mutation of V at amino acid position 87, a mutation of L at amino acid position 94, a mutation of W at amino acid position 37, a mutation of V at amino acid position 380, or in certain embodiments, the one or more mutations of BRDT include a mutation of W at amino acid position 50, a mutation of V at amino acid position 56, a mutation of L at amino acid position 63, a mutation of W at amino acid position 293, a mutation of V at amino acid position 299, or a mutation of L at amino acid position 306.

In certain embodiments, dTAG is derived from a kinase inhibitor, a protein inhibitor containing a BET bromodomain, a cytosolic signaling protein FKBP12 ligand, an HDAC inhibitor, a lysine methyltransferase inhibitor, an angiogenesis inhibitor, an immunosuppressive compound, and an arene receptor (AHR) inhibitor.

In particular embodiments, dTAG is derived from the cytoplasmic signaling protein FKBP 12. In certain embodiments, dTAG is a modified or mutated cytoplasmic signaling protein FKBP 12. In certain embodiments, the modified or mutated cytoplasmic signaling protein FKBP12 contains one or more mutations that result in an amplified binding pocket for FKBP12 ligand. In certain embodiments, the one or more mutations comprise a mutation (excluding the count of methionine start codons) of phenylalanine (F) to valine (V) (F36V) at amino acid position 36 (referred to as FKBP12 or FKBP, used interchangeably herein) (see Clackson et al, "identifying a FKBP-ligand interface to generated chemical processors with novel specificity," PNAS 95(1998):10437-10442, which is incorporated herein by reference).

In particular embodiments, dTAG has an amino acid sequence derived from the FKBP12 protein (UniProtKB-P62942 (FKB1A _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 1) GVQVETISPGDGRTFPKRGQTCVVHYTG MLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIP PHATLVFDVELLKLE.

In one embodiment, dTAG is an amino acid sequence derived from FKBP12, the mutation at amino acid position 36 having a phenylalanine (F) to (excluding the count of methionine start codons) valine (V) (F36V) (referred to as FKBP;) has the amino acid sequence: (SEQ. ID. NO: 2) GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKF MLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE.

In one embodiment, dTAG has an amino acid sequence derived from the BRD4 protein (UniProtKB-O60885(BRD4_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ.ID. NO. 3)

MSAESGPGTRLRNLPVMGDGLETSQMSTTQAQAQPQPANAAST NPPPPETSNPNKPKRQTNQLQYLLRVVLKTLWKHQFAWPFQQP VDAVKLNLPDYYKIIKTPMDMGTIKKRLENNYYWNAQECIQDF NTMFTNCYIYNKPGDDIVLMAEALEKLFLQKINELPTEETEIMIV QAKGRGRGRKETGTAKPGVSTVPNTTQASTPPQTQTPQPNPPPV QATPHPFPAVTPDLIVQTPVMTVVPPQPLQTPPPVPPQPQPPPAPA PQPVQSHPPIIAATPQPVKTKKGVKRKADTTTPTTIDPIHEPPSLPP EPKTTKLGQRRESSRPVKPPKKDVPDSQQHPAPEKSSKVSEQLK CCSGILKEMFAKKHAAYAWPFYKPVDVEALGLHDYCDIIKHPM DMSTIKSKLEAREYRDAQEFGADVRLMFSNCYKYNPPDHEVVA MARKLQDVFEMRFAKMPDEPEEPVVAVSSPAVPPPTKVVAPPSS SDSSSDSSSDSDSSTDDSEEERAQRLAELQEQLKAVHEQLAALSQ PQQNKPKKKEKDKKEKKKEKHKRKEEVEENKKSKAKEPPPKKT KKNNSSNSNVSKKEPAPMKSKPPPTYESEEEDKCKPMSYEEKRQ LSLDINKLPGEKLGRVVHIIQSREPSLKNSNPDEIEIDFETLKPSTL RELERYVTSCLRKKRKPQAEKVDVIAGSSKMKGFSSSESESSSES SSSDSEDSETEMAPKSKKKGHPGREQKKHHHHHHQQMQQAPAP VPQQPPPPPQQPPPPPPPQQQQQPPPPPPPPSMPQQAAPAMKSSPP PFIATQVPVLEPQLPGSVFDPIGHFTQPILHLPQPELPPHLPQPPEH STPPHLNQHAVVSPPALHNALPQQPSRPSNRAAALPPKPARPPAV SPALTQTPLLPQPPMAQPPQVLLEDEEPPAPPLTSMQMQLYLQQL QKVQPPTPLLPSVKVQSQPPPPLPPPPHPSVQQQLQQQPPPPPPPQ PQPPPQQQHQPPPRPVHLQPMQFSTHIQQPPPPQGQQPPHPPPGQ QPPPPQPAKPQQVIQHHHSPRHHKSDPYSTGHLREAPSPLMIHSP QMSQFQSLTHQSPPQQNVQPKKQELRAASVVQPQPLVVVKEEKI HSPIIRSEPFSPSLRPEPPKHPESIKAPVHLPQRPEMKPVDVGRPVI RPPEQNAPPPGAPDKDKQKQEPKTPVAPKKDLKIKN MGSWASLVQKHPTTPSSTAKSSSDSFEQFRRAAREKEEREKALK AQAEHAEKEKERLRQERMRSREDEDALEQARRAHEEARRRQEQ QQQQRQEQQQQQQQQAAAVAAAATPQAQSSQPQS MLDQQRELARKREQERRRREAMAATIDMNFQSDLLSIFEENLF。

In one embodiment, dTAG is amino acids 75 to 147 derived from seq.id.no. 3.

In one embodiment, dTAG has an amino acid sequence derived from the ASH1L protein (UniProtKB-Q9 NR48(ASH1L _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 2463 to 2533 derived from Q9NR 48.

In one embodiment, dTAG has an amino acid sequence derived from ATAD2 protein (UniProtKB-Q6 PL18(ATAD2_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 1001 to 1071 derived from Q6PL 18.

In one embodiment, dTAG has an amino acid sequence derived from the BAZ1A protein (UniProtKB-Q9 NRL2(BAZ1A _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 1446 to 1516 derived from Q9NRL 2.

In one embodiment, dTAG is a polypeptide having an amino acid sequence derived from the BAZ1B protein (UniProtKB-Q9 UIG0(BAZ1B _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 1356 to 1426 derived from Q9UIG 0.

In one embodiment, dTAG has an amino acid sequence derived from the BAZ2A protein (UniProtKB-Q9 UIF9(BAZ2A _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 1810 to 1880 derived from Q9UIF 9.

In one embodiment, dTAG has an amino acid sequence derived from the BAZ2B protein (UniProtKB-Q9 UIF8(BAZ2B _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 2077 through 2147 derived from Q9UIF 8.

In one embodiment, dTAG has an amino acid sequence derived from the BRD1 protein (UniProtKB-O95696 (BRD1_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 579 to 649 derived from O95696.

In one embodiment, dTAG has an amino acid sequence derived from BRD2 protein (UniProtKB-P25440 (BRD2_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 13)

MLQNVTPHNKLPGEGNAGLLGLGPEAAAPGKRIRKPSLLYEGFE SPTMASVPALQLTPANPPPPEVSNPKKPGRVTNQLQYLHKVVMK ALWKHQFAWPFRQPVDAVKLGLPDYHKIIKQPMDMGTIKRRLE NNYYWAASECMQDFNTMFTNCYIYNKPTDDIVLMAQTLEKIFL QKVASMPQEEQELVVTIPKNSHKKGAKLAALQGSVTSAHQVPAVSSVSHTALYTPPPEIPTTVLNIPHPSVISSPLLKSLHSAGPPLLAV TAAPPAQPLAKKKGVKRKADTTTPTPTAILAPGSPASPPGSLEPK AARLPPMRRESGRPIKPPRKDLPDSQQQHQSSKKGKLSEQLKHC NGILKELLSKKHAAYAWPFYKPVDASALGLHDYHDIIKHPMDLS TVKRKMENRDYRDAQEFAADVRLMFSNCYKYNPPDHDVVAMA RKLQDVFEFRYAKMPDEPLEPGPLPVSTAMPPGLAKSSSESSSEE SSSESSSEEEEEEDEEDEEEEESESSDSEEERAHRLAELQEQLRAV HEQLAALSQGPISKPKRKREKKEKKKKRKAEKHRGRAGADEDD KGPRAPRPPQPKKSKKASGSGGGSAALGPSGFGPSGGSGTKLPK KATKTAPPALPTGYDSEEEEESRPMSYDEKRQLSLDINKLPGEKL GRVVHIIQAREPSLRDSNPEEIEIDFETLKPSTLRELERYVLSCLRK KPRKPYTIKKPVGKTKEELALEKKRELEKRLQDVSGQLNSTKKP PKKANEKTESSSAQQVAVSRLSASSSSSDSSSSSSSSSSSDTSDSD SG。

In one embodiment, dTAG is amino acids 91 to 163 or 364 to 436 derived from seq.id No. 13.

In one embodiment, dTAG has an amino acid sequence derived from the BRD3 protein (UniProtKB-Q15059(BRD3_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 14)

MSTATTVAPAGIPATPGPVNPPPPEVSNPSKPGRKTNQLQYMQN VVVKTLWKHQFAWPFYQPVDAIKLNLPDYHKIIKNPMDMGTIK KRLENNYYWSASECMQDFNTMFTNCYIYNKPTDDIVLMAQALE KIFLQKVAQMPQEEVELLPPAPKGKGRKPAAGAQSAGTQQVAA VSSVSPATPFQSVPPTVSQTPVIAATPVPTITANVTSVPVPPAAAP PPPATPIVPVVPPTPPVVKKKGVKRKADTTTPTTSAITASRSESPP PLSDPKQAKVVARRESGGRPIKPPKKDLEDGEVPQHAGKKGKLS EHLRYCDSILREMLSKKHAAYAWPFYKPVDAEALELHDYHDIIK HPMDLSTVKRKMDGREYPDAQGFAADVRLMFSNCYKYNPPDH EVVAMARKLQDVFEMRFAKMPDEPVEAPALPAPAAPMVSKGA ESSRSSEESSSDSGSSDSEEERATRLAELQEQLKAVHEQLAALSQ APVNKPKKKKEKKEKEKKKKDKEKEKEKHKVKAEEEKKAKVA PPAKQAQQKKAPAKKANSTTTAGRQLKKGGKQASASYDSEEEE EGLPMSYDEKRQLSLDINRLPGEKLGRVVHIIQSREPSLRDSNPD EIEIDFETLKPTTLRELERYVKSCLQKKQRKPFSASGKKQAAKSK EELAQEKKKELEKRLQDVSGQLSSSKKPARKEKPGSAPSGGPSR LSSSSSSESGSSSSSGSSSDSSDSE。

In one embodiment, dTAG is amino acids 51 to 123 or 326 to 398 derived from seq.id No. 14.

In one embodiment, dTAG has an amino acid sequence derived from the BRD7 protein (UniProtKB-Q9 NPI1(BRD7_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from amino acids 148 to 218 of Q9NP 11.

In one embodiment, dTAG has an amino acid sequence derived from the BRD8 protein (UniProtKB-Q9H 0E9(BRD8_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 724 to 794 or 1120 to 1190 derived from Q9H0E 9.

In one embodiment, dTAG has an amino acid sequence derived from BRD9 protein (UniProtKB-Q9H 8M2(BRD9_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 153 to 223 derived from Q9H8M 2.

In one embodiment, dTAG has an amino acid sequence derived from a BRDT protein (UniProtKB-Q58F21(BRDT _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 15)

MSLPSRQTAIIVNPPPPEYINTKKNGRLTNQLQYLQKVVLKDLW KHSFSWPFQRPVDAVKLQLPDYYTIIKNPMDLNTIKKRLENKYY AKASECIEDFNTMFSNCYLYNKPGDDIVLMAQALEKLFMQKLSQ MPQEEQVVGVKERIKKGTQQNIAVSSAKEKSSPSATEKVFKQQE IPSVFPKTSISPLNVVQGASVNSSSQTAAQVTKGVKRKADTTTPA TSAVKASSEFSPTFTEKSVALPPIKENMPKNVLPDSQQQYNVVKT VKVTEQLRHCSEILKEMLAKKHFSYAWPFYNPVDVNALGLHNY YDVVKNPMDLGTIKEKMDNQEYKDAYKFAADVRLMFMNCYK YNPPDHEVVTMARMLQDVFETHFSKIPIEPVESMPLCYIKTDITE TTGRENTNEASSEGNSSDDSEDERVKRLAKLQEQLKAVHQQLQVLSQVPFRKLNKKKEKSKKEKKKEKVNNSNENPRKMCEQMRLK EKSKRNQPKKRKQQFIGLKSEDEDNAKPMNYDEKRQLSLNINKL PGDKLGRVVHIIQSREPSLSNSNPDEIEIDFETLKASTLRELEKYVS ACLRKRPLKPPAKKIMMSKEELHSQKKQELEKRLLDVNNQLNSR KRQTKSDKTQPSKAVENVSRLSESSSSSSSSSESESSSSDLSSSDSSDSESEMFPKFTEVKPNDSPSKENVKKMKNECIPPEGRTGVTQIGY CVQDTTSANTTLVHQTTPSHVMPPNHHQLAFNYQELEHLQTVK NISPLQILPPSGDSEQLSNGITVMHPSGDSDTTMLESECQAPVQK DIKIKNADSWKSLGKPVKPSGVMKSSDELFNQFRKAAIEKEVKA RTQELIRKHLEQNTKELKASQENQRDLGNGLTVESFSNKIQNKC SGEEQKEHQQSSEAQDKSKLWLLKDRDLARQKEQERRRREAMV GTIDMTLQSDIMTMFENNFD。

In one embodiment, the dTAG is derived from amino acids 44 to 116 or 287 to 359 of seq.id No. 15.

In one embodiment, dTAG has an amino acid sequence derived from BRPF1 protein (UniProtKB-P55201 (BRPF1_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 645 to 715 derived from P55201.

In one embodiment, dTAG has an amino acid sequence derived from BRPF3 protein (UniProtKB-Q9 ULD4(BRPF3_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 606 to 676 derived from Q9ULD 4.

In one embodiment, dTAG has an amino acid sequence derived from BRWD3 protein (UniProtKB-Q6 RI45(BRWD3_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 1158 to 1228 or 1317 to 1412 derived from Q6RI 45.

In one embodiment, dTAG has an amino acid sequence derived from the CECR2 protein (UniProtKB-Q9 BXF3(CECR2_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 451 to 521 derived from Q9BXF 3.

In one embodiment, dTAG has an amino acid sequence derived from a CREBP protein (UniProtKB-Q92793 (CBP _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from amino acids 1103 to 1175 of Q92793.

In one embodiment, dTAG has an amino acid sequence derived from the EP300 protein (UniProtKB-Q09472 (EP300_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 1067 through 1139 derived from Q09472.

In one embodiment, dTAG has an amino acid sequence derived from a FALZ protein (UniProtKB-Q12830 (BPTF _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 2944 to 3014 derived from Q12830.

In one embodiment, dTAG has an amino acid sequence derived from the GCN5L2 protein (UniProtKB-Q92830 (KAT2A _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 745 to 815 derived from Q92830.

In one embodiment, dTAG has an amino acid sequence derived from the KIAA1240 protein (UniProtKB-Q9ULI0(ATD2B _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 975 to 1045 derived from Q9ULI 0.

In one embodiment, dTAG has an amino acid sequence derived from a LOC93349 protein (UniProtKB-Q13342(SP140_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 796 to 829 derived from Q13342.

In one embodiment, dTAG has an amino acid sequence derived from an MLL protein (UniProtKB-Q03164 (KMT2A _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 1703 to 1748 derived from Q03164.

In one embodiment, dTAG has an amino acid sequence derived from PB1 protein (UniProtKB-Q86U 86(PB1_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from amino acids 63 to 134, 200 to 270, 400 to 470, 538 to 608, 676 to 746, or 792 to 862 of Q86U 86.

In one embodiment, dTAG has an amino acid sequence derived from the PCAF protein (UniProtKB-Q92831 (KAT2B _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 740 to 810 derived from Q92831.

In one embodiment, dTAG has an amino acid sequence derived from a PHIP protein (UniProtKB-Q8WWQ0(PHIP _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 1176 to 1246 or 1333 to 1403 derived from Q8WWQ 0.

In one embodiment, dTAG has a sequence derived from the PRKCBP1 protein (UniProtKB-Q9ULU4(PKCB1_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is the amino acid sequence of amino acids 165 to 235 derived from Q9ULU 4.

In one embodiment, dTAG has an amino acid sequence derived from the SMARCA2 protein (UniProtKB-P51531(SMCA2_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 1419 to 1489 derived from P51531.

In one embodiment, dTAG has an amino acid sequence derived from the SMARCA4 protein (UniProtKB-P51532(SMCA4_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 1477 to 1547 derived from P51532.

In one embodiment, dTAG has an amino acid sequence derived from the SP100 protein (UniProtKB-P23497 (SP100_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 761 to 876 derived from P23497.

In one embodiment, dTAG has an amino acid sequence derived from the SP110 protein (UniProtKB-Q9 HB58(SP110_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 581 to 676 derived from Q9HB 58.

In one embodiment, dTAG has an amino acid sequence derived from the SP140 protein (UniProtKB-Q13342(SP140_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 796 to 829 derived from Q13342.

In one embodiment, dTAG has an amino acid sequence derived from the TAF1 protein (UniProtKB-P21675 (TAF1_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is to amino acids 1397 to 1467 or 1520 to 1590 derived from P21675.

In one embodiment, dTAG has an amino acid sequence derived from the TAF1L protein (UniProtKB-Q8 IZX4(TAF1L _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 1416 to 1486 or 1539 to 1609 derived from Q8IZX 4.

In one embodiment, dTAG has an amino acid sequence derived from the TIF1A protein (UniProtKB-O15164 (TIF1A _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 932 to 987 derived from O15164.

In one embodiment, dTAG has an amino acid sequence derived from TRIM28 protein (UniProtKB-Q13263 (TIF1B _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 697 to 801 derived from Q13263.

In one embodiment, dTAG has an amino acid sequence derived from TRIM33 protein (UniProtKB-Q9 UPN9(TRI33_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 974 to 1046 derived from Q9UPN 9.

In one embodiment, dTAG has an amino acid sequence derived from TRIM66 protein (UniProtKB-O15016 (TRI66_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 1056 to 1128 derived from O15016.

In one embodiment, dTAG has an amino acid sequence derived from WDR9 protein (UniProtKB-Q9 NSI6(BRWD1_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from amino acids 1177 to 1247 or 1330 to 1400 of Q9NSI 6.

In one embodiment, dTAG has an amino acid sequence derived from ZMYND11 protein (UniProtKB-Q15326(ZMY11_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from amino acids 168 to 238 of Q15326.

In one embodiment, dTAG has an amino acid sequence derived from MLL4 protein (UniProtKB-Q9 UMN6(KMT2B _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acid 1395 to 1509 derived from Q9UMN 6.

In one embodiment, dTAG has a human amino acid sequence derived from the estrogen receptor (UniProtKB-P03372-1, which is incorporated by reference herein), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 4)

MTMTLHTKASGMALLHQIQGNELEPLNRPQLKIPLERPLGEVYL DSSKPAVYNYPEGAAYEFNAAAAANAQVYGQTGLPYGPGSEAA AFGSNGLGGFPPLNSVSPSPLMLLHPPPQLSPFLQPHGQQVPYYL ENEPSGYTVREAGPPAFYRPNSDNRRQGGRERLASTNDKGSMA MESAKETRYCAVCNDYASGYHYGVWSCEGCKAFFKRSIQGHND YMCPATNQCTIDKNRRKSCQACRLRKCYEVGMMKGGIRKDRR GGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSK KNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLT NLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIG LVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSS RFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRV LDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGM EHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEG FPATV。

In one embodiment, dTAG has an amino acid sequence derived from an estrogen receptor ligand binding domain or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 5)

SLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNL ADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRF RMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLD KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEH LYSMKCKNVVPLYDLLLE MLDAHRL。

In one embodiment, dTAG has an amino acid sequence derived from the androgen receptor (UniProtKB-P10275 (andrr _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 6)

MEVQLGLGRVYPRPPSKTYRGAFQNLFQSVREVIQNPGPRHPEA ASAAPPGASLLLLQQQQQQQQQQQQQQQQQQQQQQQETSPRQ QQQQQGEDGSPQAHRRGPTGYLVLDEEQQPSQPQSALECHPERG CVPEPGAAVAASKGLPQQLPAPPDEDDSAAPSTLSLLGPTFPGLS SCSADLKDILSEASTMQLLQQQQQEAVSEGSSSGRAREASGAPTS SKDNYLGGTSTISDNAKELCKAVSVSMGLGVEALEHLSPGEQLR GDCMYAPLLGVPPAVRPTPCAPLAECKGSLLDDSAGKSTEDTAE YSPFKGGYTKGLEGESLGCSGSAAAGSSGTLELPSTLSLYKSGAL DEAAAYQSRDYYNFPLALAGPPPPPPPPHPHARIKLENPLDYGSA WAAAAAQCRYGDLASLHGAGAAGPGSGSPSAAASSSWHTLFTA EEGQLYGPCGGGGGGGGGGGGGGGGGGGG

GGGEAGAVAPYGYTRPPQGLAGQESDFTAPDVWYPGGMVSRVP YPSPTCVKSEMGPWMDSYSGPYGDMRLETARDHVLPIDYYFPP QKTCLICGDEASGCHYGALTCGSCKVFFKRAAEGKQKYLCASR NDCTIDKFRRKNCPSCRLRKCYEAGMTLGARKLKKLGNLKLQE EGEASSTTSPTEETTQKLTVSHIEGYECQPIFLNVLEAIEPGVVCA GHDNNQPDSFAALLSSLNELGERQLVHVVKWAKALPGFRNLHV DDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNE YRMHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSII PVDGLKNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTK LLDSVQPIARELHQFTFDLLIKSHMVSVDFPEMMAEIISVQVPKILSGKVKPIYFHTQ。

In one embodiment, the dTAG has an amino acid sequence derived from the androgen receptor ligand binding domain, or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 10)

DNNQPDSFAALLSSLNELGERQLVHVVKWAKALPGFRNLHVDD QMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNEYR MHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPV DGLKNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTKLL DSVQPIARELHQFTFDLLIKSHMVSVDFPEMMAEIISVQVPKILSG KVKPIYFHT。

In one embodiment, dTAG has an amino acid sequence derived from the retinoic acid receptor (UniProtKB-P19793) (RXRA _ HUMAN), which is incorporated herein by reference, or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ.ID. NO. 7)

MDTKHFLPLDFSTQVNSSLTSPTGRGSMAAPSLHPSLGPGIGSPG QLHSPISTLSSPING MGPPFSVISSPMGPHSMSVPTTPTLGFSTGSPQLSSPMNPVSSSED IKPPLGLNGVLKVPAHPSGNMASFTKHICAICGDRSSGKHYGVY SCEGCKGFFKRTVRKDLTYTCRDNKDCLIDKRQRNRCQYCRYQ KCLAMGMKREAVQEERQRGKDRNENEVESTSSANEDMPVERIL EAELAVEPKTETYVEANMGLNPSSPNDPVTNICQAADKQLFTLV EWAKRIPHFSELPLDDQVILLRAGWNELLIASFSHRSIAVKDGILL ATGLHVHRNSAHSAGVGAIFDRVLTELVSKMRDMQMDKTELGC LRAIVLFNPDSKGLSNPAEVEALREKVYASLEAYCKHKYPEQPG RFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTFLME MLEAPHQMT。

In one embodiment, the dTAG has an amino acid sequence derived from a retinoic acid receptor ligand binding domain, or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 11)

SANEDMPVERILEAELAVEPKTETYVEANMGLNPSSPNDPVTNIC QAADKQLFTLVEWAKRIPHFSELPLDDQVILLRAGWNELLIASFS HRSIAVKDGILLATGLHVHRNSAHSAGVGAIFDRVLTELVSKMR DMQMDKTELGCLRAIVLFNPDSKGLSNPAEVEALREKVYASLEA YCKHKYPEQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPID TFLME MLEAPHQM。

In one embodiment, dTAG has an amino acid sequence derived from DHFR, E.coli, UniProtKB-Q79DQ2(Q79DQ2_ ECOLX), which is incorporated herein by reference, or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 8)

MNSESVRIYLVAAMGANRVIGNGPNIPWKIPGEQKIFRRLTEGK VVVMGRKTFESIGKPLPNRHTLVISRQANYRATGCVVVSTLSHAI ALASELGNELYVAGGAEIYTLALPHAHGVFLSEVHQTFEGDAFF PMLNETEFELVSTETIQAVIPYTHSVYARRNG。

In one embodiment, the dTAG has an amino acid sequence derived from a retinoic acid receptor ligand binding domain or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 9)

MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTS SYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFM DAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFI RPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGV VRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAV DIGPGLNLLQEDNPDLIGSEIARWLSTLEISG。

In one embodiment, dTAG has the amino acid sequence derived from the N-terminus of MDM2, or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 12)

MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQ KDTYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFG VPSFSVKEHRKIYTMIYRNLVVV。

In one embodiment, dTAG has an amino acid sequence derived from the apoptosis regulator Bcl-xL protein, UniProtKB-Q07817(B2CL1_ HUMAN), which is incorporated herein by reference, or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 16)

MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESE METPSAINGNPSWHLADSPAVNGATGHSSSLDAREVIPMAAVKQ ALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRD GVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLND HLEPWIQENGGWDTFVELYGNNAAAESRKGQERFNRWFLTGMT VAGVVLLGSLFSRK。

In one embodiment, the dTAG has an amino acid sequence derived from the CD209 antigen (UniProtKB-Q9 NNX6(CD209_ HUMAN, which is incorporated herein by reference) or a variant thereof in one embodiment, the dTAG is derived from the amino acid sequence (SEQ. ID. NO. 17)

MSDSKEPRLQQLGLLEEEQLRGLGFRQTRGYKSLAGCLGHGPLV LQLLSFTLLAGLLVQVSKVPSSISQEQSRQDAIYQNLTQLKAAVG ELSEKSKLQEIYQELTQLKAAVGELPEKSKLQEIYQELTRLKAAV GELPEKSKLQEIYQELTWLKAAVGELPEKSKMQEIYQELTRLKA AVGELPEKSKQQEIYQELTRLKAAVGELPEKSKQQEIYQELTRLK AAVGELPEKSKQQEIYQELTQLKAAVERLCHPCPWEWTFFQGN CYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQNFLQLQSSRS NRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNVG EEDCAEFSGNGWNDDKCNLAKFWICKKSAASCSRDEEQFLSPAP ATPNPPPA。

In one embodiment, dTAG has an amino acid sequence derived from the E3 ligase XIAP (UniProtKB-P98170(XIAP _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 18)

MTFNSFEGSKTCVPADINKEEEFVEEFNRLKTFANFPSGSPVSAS TLARAGFLYTGEGDTVRCFSCHAAVDRWQYGDSAVGRHRKVSP NCRFINGFYLENSATQSTNSGIQNGQYKVENYLGSRDHFALDRP SETHADYLLRTGQVVDISDTIYPRNPAMYSEEARLKSFQNWPDY AHLTPRELASAGLYYTGIGDQVQCFCCGGKLKNWEPCDRAWSE HRRHFPNCFFVLGRNLNIRSESDAVSSDRNFPNSTNLPRNPSMAD YEARIFTFGTWIYSVNKEQLARAGFYALGEGDKVKCFHCGGGLT DWKPSEDPWEQHAKWYPGCKYLLEQKGQEYINNIHLTHSLEECLVRTTEKTPSLTRRIDDTIFQNPMVQEAIR

MGFSFKDIKKIMEEKIQISGSNYKSLEVLVADLVNAQKDSMQDE SSQTSLQKEISTEEQLRRLQEEKLCKICMDRNIAIVFVPCGHLVTC KQCAEAVDKCPMCYTVITFKQKIFMS。

In one embodiment, dTAG has an amino acid sequence derived from baculovirus IAP repeat-containing protein 2(UniProtKB-Q13490(BIRC2_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 19)

MHKTASQRLFPGPSYQNIKSIMEDSTILSDWTNSNKQKMKYDFS CELYRMSTYSTFPAGVPVSERSLARAGFYYTGVNDKVKCFCCGL MLDNWKLGDSPIQKHKQLYPSCSFIQNLVSASLGSTSKNTSPMR NSFAHSLSPTLEHSSLFSGSYSSLSPNPLNSRAVEDISSSRTNPYSY AMSTEEARFLTYHMWPLTFLSPSELARAGFYYIGPGDRVACFAC GGKLSNWEPKDDAMSEHRRHFPNCPFLENSLETLRFSISNLSMQ THAARMRTFMYWPSSVPVQPEQLASAGFYYVGRNDDVKCFCC DGGLRCWESGDDPWVEHAKWFPRCEFLIRMKGQEFVDEIQGRY PHLLEQLLSTSDTTGEENADPPIIHFGPGESSSEDAVMMNTPVVK SALEMGFNRDLVKQTVQSKILTTGENYKTVNDIVSALLNAEDEK REEEKEKQAEEMASDDLSLIRKNRMALFQQLTCVLPILDNLLKA NVINKQEHDIIKQKTQIPLQARELIDTILVKGNAAANIFKNCLKEI DSTLYKNLFVDKNMKYIPTEDVSGLSLEEQLRRLQEERTCKVCM DKEVSVVFIPCGHLVVCQECAPSLRKCPICRGIIKGTVRTFLS。

In one embodiment, dTAG has an amino acid sequence derived from hematopoietic prostaglandin D synthase (UniProtKB-O60760(HPGDS _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 20)

MPNYKLTYFNMRGRAEIIRYIFAYLDIQYEDHRIEQADWPEIKST LPFGKIPILEVDGLTLHQSLAIARYLTKNTDLAGNTEMEQCHVDA IVDTLDDFMSCFPWAEKKQDVKEQMFNELLTYNAPHLMQDLDT YLGGREWLIGNSVTWADFYWEICSTTLLVFKPDLLDNHPRLVTL RKKVQAIPAVANWIKRRPQTKL。

In one embodiment, dTAG has an amino acid sequence derived from the GTPase k-RAS (UniProtKB-P01116(RASK _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 21)

MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQV VIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTK SFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQD LARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTP GCVKIKKCIIM。

In one embodiment, dTAG has an amino acid sequence derived from poly-ADP-ribose polymerase 15(UniProtKB-Q460N3(PAR15_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 22)

MAAPGPLPAAALSPGAPTPRELMHGVAGVTSRAGRDREAGSVL PAGNRGARKASRRSSSRSMSRDNKFSKKDCLSIRNVVASIQTKE GLNLKLISGDVLYIWADVIVNSVPMNLQLGGGPLSRAFLQKAGP MLQKELDDRRRETEEKVGNIFMTSGCNLDCKAVLHAVAPYWN NGAETSWQIMANIIKKCLTTVEVLSFSSITFPMIGTGSLQFPKAVF AKLILSEVFEYSSSTRPITSPLQEVHFLVYTNDDEGCQAFLDEFTN WSRINPNKARIPMAGDTQGVVGTVSKPCFTAYEMKIGAITFQVA TGDIATEQVDVIVNSTARTFNRKSGVSRAILEGAGQAVESECAVL AAQPHRDFIITPGGCLKCKIIIHVPGGKDVRKTVTSVLEECEQRK YTSVSLPAIGTGNAGKNPITVADNIIDAIVDFSSQHSTPSLKTVKV VIFQPELLNIFYDSMKKRDLSASLNFQSTFSMTTCNLPEHWTDM NHQLFCMVQLEPGQSEYNTIKDKFTRTCSSYAIEKIERIQNAFLW QSYQVKKRQMDIKNDHKNNERLLFHGTDADSVPYVNQHGFNRS CAGKNAVSYGKGTYFAVDASYSAKDTYSKPDSNGRKHMYVVR VLTGVFTKGRAGLVTPPPKNPHNPTDLFDSVTNNTRSPKLFVVFF DNQAYPEYLITFTA。

In one embodiment, dTAG has an amino acid sequence derived from poly-ADP-ribose polymerase 14(UniProtKB-Q460N5(PAR14_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 23)

MAVPGSFPLLVEGSWGPDPPKNLNTKLQMYFQSPKRSGGGECE VRQDPRSPSRFLVFFYPEDVRQKVLERKNHELVWQGKGTFKLTV QLPATPDEIDHVFEEELLTKESKTKEDVKEPDVSEELDTKLPLDG GLDKMEDIPEECENISSLVAFENLKANVTDIMLILLVENISGLSND DFQVEIIRDFDVAVVTFQKHIDTIRFVDDCTKHHSIKQLQLSPRLL EVTNTIRVENLPPGADDYSLKLFFENPYNGGGRVANVEYFPEESS ALIEFFDRKVLDTIMATKLDFNKMPLSVFPYYASLGTALYGKEK PLIKLPAPFEESLDLPLWKFLQKKNHLIEEINDEMRRCHCELTWS QLSGKVTIRPAATLVNEGRPRIKTWQADTSTTLSSIRSKYKVNPI KVDPTMWDTIKNDVKDDRILIEFDTLKEMVILAGKSEDVQSIEV QVRELIESTTQKIKREEQSLKEKMIISPGRYFLLCHSSLLDHLLTE CPEIEICYDRVTQHLCLKGPSADVYKAKCEIQEKVYTMAQKNIQ VSPEIFQFLQQVNWKEFSKCLFIAQKILALYELEGTTVLLTSCSSE ALLEAEKQMLSALNYKRIEVENKEVLHGKKWKGLTHNLLKKQN SSPNTVIINELTSETTAEVIITGCVKEVNETYKLLFNFVEQNMKIE RLVEVKPSLVIDYLKTEKKLFWPKIKKVNVQVSFNPENKQKGIL LTGSKTEVLKAVDIVKQVWDSVCVKSVHTDKPGAKQFFQDKAR FYQSEIKRLFGCYIELQENEVMKEGGSPAGQKCFSRTVLAPGVV LIVQQGDLARLPVDVVVNASNEDLKHYGGLAAALSKAAGPELQ ADCDQIVKREGRLLPGNATISKAGKLPYHHVIHAVGPRWSGYEA PRCVYLLRRAVQLSLCLAEKYKYRSIAIPAISSGVFGFPLGRCVET IVSAIKENFQFKKDGHCLKEIYLVDVSEKTVEAFAEAVKTVFKAT LPDTAAPPGLPPAAAGPGKTSWEKGSLVSPGGLQ

MLLVKEGVQNAKTDVVVNSVPLDLVLSRGPLSKSLLEKAGPEL QEELDTVGQGVAVS

MGTVLKTSSWNLDCRYVLHVVAPEWRNGSTSSLKIMEDIIRECM EITESLSLKSIAFPAIGTGNLGFPKNIFAELIISEVFKFSSKNQLKTL QEVHFLLHPSDHENIQAFSDEFARRANGNLVSDKIPKAKDTQGF YGTVSSPDSGVYEMKIGSIIFQVASGDITKEEADVIVNSTSNSFNL KAGVSKAILECAGQNVERECSQQAQQRKNDYIITGGGFLRCKNII HVIGGNDVKSSVSSVLQECEKKNYSSICLPAIGTGNAKQHPDKV AEAIIDAIEDFVQKGSAQSVKKVKVVIFLPQVLDVFYANMKKRE GTQLSSQQSVMSKLASFLGFSKQSPQKKNHLVLEKKTESATFRV CGENVTCVEYAISWLQDLIEKEQCPYTSEDECIKDFDEKEYQELN ELQKKLNINISLDHKRPLIKVLGISRDVMQARDEIEAMIKRVRLA KEQESRADCISEFIEWQYNDNNTSHCFNKMTNLKLEDARREKKK TVDVKINHRHYTVNLNTYTATDTKGHSLSVQRLTKSKVDIPAH WSDMKQQNFCVVELLPSDPEYNTVASKFNQTCSHFRIEKIERIQN PDLWNSYQAKKKTMDAKNGQTMNEKQLFHGTDAGSVPHVNRN GFNRSYAGKNAVAYGKGTYFAVNANYSANDTYSRPDANGRKH VYYVRVLTGIYTHGNHSLIVPPSKNPQNPTDLYDTVTDNVHHPS LFVAFYDYQAYPEYLITFRK。

In one embodiment, dTAG has an amino acid sequence derived from superoxide dismutase (UniProtKB-P00441(SODC _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 24)

MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLH GFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGN VTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ。

In one embodiment, dTAG has an amino acid sequence derived from the retinoid-sensitive cGMP 3',5' -cyclic phosphodiesterase subunit δ (UniProtKB-O43924 (PDE6D _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 25)

MSAKDERAREILRGFKLNWMNLRDAETGKILWQGTEDLSVPGV EHEARVPKKILKCKAVSRELNFSSTEQMEKFRLEQKVYFKGQCL EEWFFEFGFVIPNSTNTWQSLIEAAPESQMMPASVLTGNVIIETKFFDDDLLVSTSRVRLFYV。

In one embodiment, dTAG has an amino acid sequence derived from the induced myeloid leukemia cell differentiation protein Mcl-1(UniProtKB-Q07820(MCL1_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 26)

MFGLKRNAVIGLNLYCGGAGLGAGSGGATRPGGRLLATEKEAS ARREIGGGEAGAVIGGSAGASPPSTLTPDSRRVARPPPIGAEVPD VTATPARLLFFAPTRRAAPLEEMEAPAADAIMSPEEELDGYEPEP LGKRPAVLPLLELVGESGNNTSTDGSLPSTPPPAEEEEDELYRQS LEIISRYLREQATGAKDTKPMGRSGATSRKALETLRRVGDGVQR NHETAFQGMLRKLDIKNEDDVKSLSRVMIHVFSDGVTNWGRIV TLISFGAFVAKHLKTINQESCIEPLAESITDVLVRTKRDWLVKQR GWDGFVEFFHVEDLEGGIRNVLLAFAGVAGVGAGLAYLIR。

In one embodiment, dTAG has an amino acid sequence derived from the apoptosis regulator Bcl-2 (UniProtKB-Q07820(BCL2_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 27)

MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGA APAPGIFSSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALS PVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFA TVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIAL WMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHK。

In one embodiment, dTAG has an amino acid sequence derived from peptidyl-prolyl cis-trans isomerase NIMA interaction 1(UniProtKB-Q13526(PIN1_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 28)

MADEEKLPPGWEKRMSRSSGRVYYFNHITNASQWERPSGNSSS GGKNGQGEPARVRCSHLLVKHSQSRRPSSWRQEKITRTKEEALE LINGYIQKIKSGEEDFESLASQFSDCSSAKARGDLGAFSRGQMQKPFEDASFALRTGEMSGPVFTDSGIHIILRTE。

In one embodiment, dTAG has an amino acid sequence derived from tankyrase 1(UniProtKB-O95271(TNKS1_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 29)

MAASRRSQHHHHHHQQQLQPAPGASAPPPPPPPPLSPGLAPGTTP ASPTASGLAPFASPRHGLALPEGDGSRDPPDRPRSPDPVDGTSCC STTSTICTVAAAPVVPAVSTSSAAGVAPNPAGSGSNNSPSSSSSPT SSSSSSPSSPGSSLAESPEAAGVSSTAPLGPGAAGPGTGVPAVSGA LRELLEACRNGDVSRVKRLVDAANVNAKDMAGRKSSPLHFAAG FGRKDVVEHLLQ

MGANVHARDDGGLIPLHNACSFGHAEVVSLLLCQGADPNARDN WNYTPLHEAAIKGKIDVCIVLLQHGADPNIRNTDGKSALDLADP SAKAVLTGEYKKDELLEAARSGNEEKLMALLTPLNVNCHASDG RKSTPLHLAAGYNRVRIVQLLLQHGADVHAKDKGGLVPLHNAC SYGHYEVTELLLKHGACVNAMDLWQFTPLHEAASKNRVEVCSL LLSHGADPTLVNCHGKSAVDMAPTPELRERLTYEFKGHSLLQAA READLAKVKKTLALEIINFKQPQSHETALHCAVASLHPKRKQVT ELLLRKGANVNEKNKDFMTPLHVAAERAHNDVMEVLHKHGAK MNALDTLGQTALHRAALAGHLQTCRLLLSYGSDPSIISLQGFTA AQMGNEAVQQILSESTPIRTSDVDYRLLEASKAGDLETVKQLCS SQNVNCRDLEGRHSTPLHFAAGYNRVSVVEYLLHHGADVHAKD KGGLVPLHNACSYGHYEVAELLVRHGASVNVADLWKFTPLHEA AAKGKYEICKLLLKHGADPTKKNRDGNTPLDLVKEGDTDIQDLL RGDAALLDAAKKGCLARVQKLCTPENINCRDTQGRNSTPLHLA AGYNNLEVAEYLLEHGADVNAQDKGGLIPLHNAASYGHVDIAA LLIKYNTCVNATDKWAFTPLHEAAQKGRTQLCALLLAHGADPT MKNQEGQTPLDLATADDIRALLIDAMPPEALPTCFKPQATVVSASLISPASTPSCLSAASSIDNLTGPLAELAVGGASNAGDGAAGTER KEGEVAGLDMNISQFLKSLGLEHLRDIFETEQITLDVLADMGHEE LKEIGINAYGHRHKLIKGVERLLGGQQGTNPYLTFHCVNQGTILL DLAPEDKEYQSVEEEMQSTIREHRDGGNAGGIFNRYNVIRIQKV VNKKLRERFCHRQKEVSEENHNHHNERMLFHGSPFINAIIHKGFDERHAYIGGMFGAGIYFAENSSKSNQYVYGIGGGTGCPTHKDRS CYICHRQMLFCRVTLGKSFLQFSTMKMAHAPPGHHSVIGRPSVN GLAYAEYVIYRGEQAYPEYLITYQIMKPEAPSQTATAAEQKT。

In one embodiment, the dTAG has an amino acid sequence derived from tankyrase 2(UniProtKB-O9H2K2(TNKS2_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 30)

MSGRRCAGGGAACASAAAEAVEPAARELFEACRNGDVERVKRL VTPEKVNSRDTAGRKSTPLHFAAGFGRKDVVEYLLQNGANVQA RDDGGLIPLHNACSFGHAEVVNLLLRHGADPNARDNWNYTPLH EAAIKGKIDVCIVLLQHGAEPTIRNTDGRTALDLADPSAKAVLTG EYKKDELLESARSGNEEKMMALLTPLNVNCHASDGRKSTPLHLAAGYNRVKIVQLLLQHGADVHAKDKGDLVPLHNACSYGHYEV TELLVKHGACVNAMDLWQFTPLHEAASKNRVEVCSLLLSYGAD PTLLNCHNKSAIDLAPTPQLKERLAYEFKGHSLLQAAREADVTRI KKHLSLEMVNFKHPQTHETALHCAAASPYPKRKQICELLLRKGA NINEKTKEFLTPLHVASEKAHNDVVEVVVKHEAKVNALDNLGQ TSLHRAAYCGHLQTCRLLLSYGCDPNIISLQGFTALQMGNENVQ QLLQEGISLGNSEADRQLLEAAKAGDVETVKKLCTVQSVNCRDI EGRQSTPLHFAAGYNRVSVVEYLLQHGADVHAKDKGGLVPLHN ACSYGHYEVAELLVKHGAVVNVADLWKFTPLHEAAAKGKYEIC KLLLQHGADPTKKNRDGNTPLDLVKDGDTDIQDLLRGDAALLD AAKKGCLARVKKLSSPDNVNCRDTQGRHSTPLHLAAGYNNLEV AEYLLQHGADVNAQDKGGLIPLHNAASYGHVDVAALLIKYNACVNATDKWAFTPLHEAAQKGRTQLCALLLAHGADPTLKNQEGQT PLDLVSADDVSALLTAAMPPSALPSCYKPQVLNGVRSPGATADA LSSGPSSPSSLSAASSLDNLSGSFSELSSVVSSSGTEGASSLEKKEV PGVDFSITQFVRNLGLEHLMDIFEREQITLDVLVEMGHKELKEIGI NAYGHRHKLIKGVERLISGQQGLNPYLTLNTSGSGTILIDLSPDDKEFQSVEEEMQSTVREHRDGGHAGGIFNRYNILKIQKVCNKKLW ERYTHRRKEVSEENHNHANERMLFHGSPFVNAIIHKGFDERHAY IGGMFGAGIYFAENSSKSNQYVYGIGGGTGCPVHKDRSCYICHR QLLFCRVTLGKSFLQFSAMKMAHSPPGHHSVTGRPSVNGLALAE YVIYRGEQAYPEYLITYQIMRPEGMVDG。

In one embodiment, dTAG has an amino acid sequence derived from 7, 8-dihydro-8-oxoguanine triphosphatase (UniProtKB-P36639(8ODP _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 31)

MYWSNQITRRLGERVQGFMSGISPQQMGEPEGSWSGKNPGTMG ASRLYTLVLVLQPQRVLLGMKKRGFGAGRWNGFGGKVQEGETI EDGARRELQEESGLTVDALHKVGQIVFEFVGEPELMDVHVFCTD SIQGTPVESDEMRPCWFQLDQIPFKDMWPDDSYWFPLLLQKKKF HGYFKFQGQDTILDYTLREVDTV。

In one embodiment, dTAG has an amino acid sequence derived from the proto-oncogene tyrosine protein kinase Src (UniProtKB-P12931(SRC _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 32)

MGSNKSKPKDASQRRRSLEPAENVHGAGGGAFPASQTPSKPASA DGHRGPSAAFAPAAAEPKLFGGFNSSDTVTSPQRAGPLAGGVTT FVALYDYESRTETDLSFKKGERLQIVNNTEGDWWLAHSLSTGQT GYIPSNYVAPSDSIQAEEWYFGKITRRESERLLLNAENPRGTFLV RESETTKGAYCLSVSDFDNAKGLNVKHYKIRKLDSGGFYITSRT QFNSLQQLVAYYSKHADGLCHRLTTVCPTSKPQTQGLAKDAWE IPRESLRLEVKLGQGCFGEVWMGTWNGTTRVAIKTLKPGTMSPE AFLQEAQVMKKLRHEKLVQLYAVVSEEPIYIVTEYMSKGSLLDF LKGETGKYLRLPQLVDMAAQIASGMAYVERMNYVHRDLRAAN ILVGENLVCKVADFGLARLIEDNEYTARQGAKFPIKWTAPEAAL YGRFTIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGY RMPCPPECPESLHDLMCQCWRKEPEERPTFEYLQAFLEDYFTSTE PQYQPGENL。

In one embodiment, dTAG comprises the substitution of threonine (T) with glycine (G) or alanine (a) at amino acid position 341. In one embodiment, dTAG is an amino acid sequence derived from SEQ ID No. 62 or a fragment thereof.

LRLEVKLGQGCFGEVWMGTWNGTTRVAIKTLKPGTMSPEAFLQ EAQVMKKLRHEKLVQLYAVVSEEPIYIVTEYGSKGSLLDFLKGE TGKYLRLPQLVDMAAQIASGMAYVERMNYVHRDLRAANILVG ENLVCKVADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRF TIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGYRMPCPPECPESLHDLMCQCWRKEPEERPTFEYLQAFLEDYF。

In one embodiment, dTAG is an amino acid sequence derived from seq.id.no. 63 or a fragment thereof.

LRLEVKLGQGCFGEVWMGTWNGTTRVAIKTLKPGTMSPEAFLQ EAQVMKKLRHEKLVQLYAVVSEEPIYIVTEYASKGSLLDFLKGE TGKYLRLPQLVDMAAQIASGMAYVERMNYVHRDLRAANILVG ENLVCKVADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRF TIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGYRMPCPPECPESLHDLMCQCWRKEPEERPTFEYLQAFLEDYF。

In one embodiment, dTAG has an amino acid sequence derived from prostaglandin E synthase (UniProtKB-O14684(PTGES _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 33)

MPAHSLVMSSPALPAFLLCSTLLVIKMYVVAIITGQVRLRKKAFA NPEDALRHGGPQYCRSDPDVERCLRAHRNDMETIYPFLFLGFVY SFLGPNPFVAWMHFLVFLVGRVAHTVAYLGKLRAPIRSVTYTLAQLPCASMALQILWEAARHL。

In one embodiment, dTAG has an amino acid sequence derived from arachidonic acid 5-lipoxygenase activating protein (UniProtKB-P20292(AL5AP _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 34)

MDQETVGNVVLLAIVTLISVVQNGFFAHKVEHESRTQNGRSFQR TGTLAFERVYTANQNCVDAYPTFLAVLWSAGLLCSQVPAAFAG LMYLFVRQKYFVGYLGERTQSTPGYIFGKRIILFLFLMSVAGIFNYYLIFFFGSDFENYIKTISTTISPLLLIP。

In one embodiment, dTAG has the amino acid sequence of a fatty acid binding protein derived from adipocytes (UniProtKB-P15090(FABP4_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 35)

MCDAFVGTWKLVSSENFDDYMKEVGVGFATRKVAGMAKPNMI ISVNGDVITIKSESTFKNTEISFILGQEFDEVTADDRKVKSTITLDG GVLVHVQKWDGKSTTIKRKREDDKLVVECVMKGVTSTRVYER A。

In one embodiment, dTAG has an amino acid sequence derived from a PH interacting protein (UniProtKB-Q8WWQ0(PHIP _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 36)

MSCERKGLSELRSELYFLIARFLEDGPCQQAAQVLIREVAEKELL PRRTDWTGKEHPRTYQNLVKYYRHLAPDHLLQICHRLGPLLEQE IPQSVPGVQTLLGAGRQSLLRTNKSCKHVVWKGSALAALHCGR PPESPVNYGSPPSIADTLFSRKLNGKYRLERLVPTAVYQHMKMH KRILGHLSSVYCVTFDRTGRRIFTGSDDCLVKIWATDDGRLLATL RGHAAEISDMAVNYENTMIAAGSCDKMIRVWCLRTCAPLAVLQ GHSASITSLQFSPLCSGSKRYLSSTGADGTICFWLWDAGTLKINP RPAKFTERPRPGVQMICSSFSAGGMFLATGSTDHIIRVYFFGSGQ PEKISELEFHTDKVDSIQFSNTSNRFVSGSRDGTARIWQFKRREW KSILLDMATRPAGQNLQGIEDKITKMKVTMVAWDRHDNTVITA VNNMTLKVWNSYTGQLIHVLMGHEDEVFVLEPHPFDPRVLFSA GHDGNVIVWDLARGVKIRSYFNMIEGQGHGAVFDCKCSPDGQH FACTDSHGHLLIFGFGSSSKYDKIADQMFFHSDYRPLIRDANNFV LDEQTQQAPHLMPPPFLVDVDGNPHPSRYQRLVPGRENCREEQL IPQMGVTSSGLNQVLSQQANQEISPLDSMIQRLQQEQDLRRSGEAVISNTSRLSRGSISSTSEVHSPPNVGLRRSGQIEGVRQMHSNAPRS EIATERDLVAWSRRVVVPELSAGVASRQEEWRTAKGEEEIKTYR SEEKRKHLTVPKENKIPTVSKNHAHEHFLDLGESKKQQTNQHNY RTRSALEETPRPSEEIENGSSSSDEGEVVAVSGGTSEEEERAWHS DGSSSDYSSDYSDWTADAGINLQPPKKVPKNKTKKAESSSDEEEESEKQKQKQIKKEKKKVNEEKDGPISPKKKKPKERKQKRLAVGE LTENGLTLEEWLPSTWITDTIPRRCPFVPQMGDEVYYFRQGHEA YVEMARKNKIYSINPKKQPWHKMELREQELMKIVGIKYEVGLPT LCCLKLAFLDPDTGKLTGGSFTMKYHDMPDVIDFLVLRQQFDD AKYRRWNIGDRFRSVIDDAWWFGTIESQEPLQLEYPDSLFQCYN VCWDNGDTEKMSPWDMELIPNNAVFPEELGTSVPLTDGECRSLI YKPLDGEWGTNPRDEECERIVAGINQLMTLDIASAFVAPVDLQA YPMYCTVVAYPTDLSTIKQRLENRFYRRVSSLMWEVRYIEHNTR TFNEPGSPIVKSAKFVTDLLLHFIKDQTCYNIIPLYNSMKKKVLSD SEDEEKDADVPGTSTRKRKDHQPRRRLRNRAQSYDIQAWKKQC EELLNLIFQCEDSEPFRQPVDLLEYPDYRDIIDTPMDFATVRETLE AGNYESPMELCKDVRLIFSNSKAYTPSKRSRIYSMSLRLSAFFEE HISSVLSDYKSALRFHKRNTITKRRKKRNRSSSVSSSAASSPERK KRILKPQLKSESSTSAFSTPTRSIPPRHNAAQINGKTESSSVVRTRS NRVVVDPVVTEQPSTSSAAKTFITKANASAIPGKTILENSVKHSK ALNTLSSPGQSSFSHGTRNNSAKENMEKEKPVKRKMKSSVLPKA STLSKSSAVIEQGDCKNNALVPGTIQVNGHGGQPSKLVKRGPGR KPKVEVNTNSGEIIHKKRGRKPKKLQYAKPEDLEQNNVHPIRDE VLPSSTCNFLSETNNVKEDLLQKKNRGGRKPKRKMKTQKLDAD LLVPASVKVLRRSNRKKIDDPIDEEEEFEELKGSEPHMRTRNQGR RTAFYNEDDSEEEQRQLLFEDTSLTFGTSSRGRVRKLTEKAKAN LIGW。

In one embodiment, dTAG has an amino acid sequence derived from SUMO-conjugating enzyme UBC9 (UniProtKB-P63279(UBC9_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 37)

MSGIALSRLAQERKAWRKDHPFGFVAVPTKNPDGTMNLMNWE CAIPGKKGTPWEGGLFKLRMLFKDDYPSSPPKCKFEPPLFHPNV YPSGTVCLSILEEDKDWRPAITIKQILLGIQELLNEPNIQDPAQAEAYTIYCQNRVEYEKRVRAQAKKFAPS。

In one embodiment, dTAG has an amino acid sequence derived from protein S100-A7 (UniProtKB-P31151(S10A7_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 38)

MSNTQAERSIIGMIDMFHKYTRRDDKIEKPSLLTMMKENFPNFLS ACDKKGTNYLADVFEKKDKNEDKKIDFSEFLSLLGDIATDYHKQ SHGAAPCSGGSQ。

In one embodiment, dTAG has an amino acid sequence derived from phospholipase A2 (membrane-bound UniProtKB-P14555(PA2GA _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 39)

MKTLLLLAVIMIFGLLQAHGNLVNFHRMIKLTTGKEAALSYGFY GCHCGVGGRGSPKDATDRCCVTHDCCYKRLEKRGCGTKFLSYK FSNSGSRITCAKQDSCRSQLCECDKAAATCFARNKTTYNKKYQYYSNKHCRGSTPRC。

In one embodiment, dTAG has an amino acid sequence derived from histone deacetylase 6 (UniProtKB-Q9UBN7(HDAC6_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 40)

MTSTGQDSTTTRQRRSRQNPQSPPQDSSVTSKRNIKKGAVPRSIP NLAEVKKKGKMKKLGQAMEEDLIVGLQGMDLNLEAEALAGTG LVLDEQLNEFHCLWDDSFPEGPERLHAIKEQLIQEGLLDRCVSFQ ARFAEKEELMLVHSLEYIDLMETTQYMNEGELRVLADTYDSVY LHPNSYSCACLASGSVLRLVDAVLGAEIRNGMAIIRPPGHHAQH SLMDGYCMFNHVAVAARYAQQKHRIRRVLIVDWDVHHGQGTQ FTFDQDPSVLYFSIHRYEQGRFWPHLKASNWSTTGFGQGQGYTI NVPWNQVGMRDADYIAAFLHVLLPVALEFQPQLVLVAAGFDALQGDPKGEMAATPAGFAQLTHLL

MGLAGGKLILSLEGGYNLRALAEGVSASLHTLLGDPCP MLESPGAPCRSAQASVSCALEALEPFWEVLVRSTETVERDNMEE DNVEESEEEGPWEPPVLPILTWPVLQSRTGLVYDQNMMNHCNL WDSHHPEVPQRILRIMCRLEELGLAGRCLTLTPRPATEAELLTCH SAEYVGHLRATEKMKTRELHRESSNFDSIYICPSTFACAQLATGAACRLVEAVLSGEVLNGAAVVRPPGHHAEQDAACGFCFFNSVAV AARHAQTISGHALRILIVDWDVHHGNGTQHMFEDDPSVLYVSL HRYDHGTFFPMGDEGASSQIGRAAGTGFTVNVAWNGPRMGDA DYLAAWHRLVLPIAYEFNPELVLVSAGFDAARGDPLGGCQVSPE GYAHLTHLLMGLASGRIILILEGGYNLTSISESMAACTRSLLGDPP PLLTLPRPPLSGALASITETIQVHRRYWRSLRVMKVEDREGPSSS KLVTKKAPQPAKPRLAERMTTREKKVLEAG

MGKVTSASFGEESTPGQTNSETAVVALTQDQPSEAATGGATLAQ TISEAAIGGAMLGQTTSEEAVGGATPDQTTSEETVGGAILDQTTS EDAVGGATLGQTTSEEAVGGATLAQTTSEAAMEGATLDQTTSE EAPGGTELIQTPLASSTDHQTPPTSPVQGTTPQISPSTLIGSLRTLE LGSESQGASESQAPGEENLLGEAAGGQDMADSMLMQGSRGLTD QAIFYAVTPLPWCPHLVAVCPIPAAGLDVTQPCGDCGTIQENWV CLSCYQVYCGRYINGHMLQHHGNSGHPLVLSYIDLSAWCYYCQ AYVHHQALLDVKNIAHQNKFGEDMPHPH。

In one embodiment, dTAG has an amino acid sequence derived from a preproprotein (UniProtKB-P07602 (SAP _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO: 41)

MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCG AVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEI LVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEV CSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANI PLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFV QALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMQPK EICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHE VPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLP KSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPAL TVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPS AHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWN。

In one embodiment, dTAG has an amino acid sequence derived from apolipoprotein a (UniProtKB-P08519 (APOA _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ.ID. NO: 42)

MEHKEVVLLLLLFLKSAAPEQSHVVQDCYHGDGQSYRGTYSTT VTGRTCQAWSSMTPHQHNRTTENYPNAGLIMNYCRNPDAVAAP YCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQ APTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHS HSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGN GQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNY CRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVT PVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTC QAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRD PGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQR PGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPE YYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDA EGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRG TYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDA VAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLE APSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRW EYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQEC YHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAG LIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVA PPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVT GRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYC YTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAP TEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHS RTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQ CSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQ SYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCR NPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPV PSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQA WSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPG VRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPG VQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYY PNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEG TAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTY STTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAV AAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAP SEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMT PHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEY CNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECY HGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLI MNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAP PTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVT GRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYC YTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAP TEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHS RTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQ CSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQ SYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCR NPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPV PSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQA WSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPG VRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPG VQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYY PNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEG TAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTY STTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAV AAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMT PHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEY CNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECY HGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLI MNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAP PTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVT GRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYC YTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAP TEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHS RTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQ CSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQ SYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPV PSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQA WSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPG VRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPG VQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYY PNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEG TAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTY STTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDPVA APYCYTRDPSVRWEYCNLTQCSDAEGTAVAPPTITPIPSLEAPSE QAPTEQRPGVQECYHGNGQSYQGTYFITVTGRTCQAWSSMTPH SHSRTPAYYPNAGLIKNYCRNPDPVAAPWCYTTDPSVRWEYCN LTRCSDAEWTAFVPPNVILAPSLEAFFEQALTEETPGVQDCYYH YGQSYRGTYSTTVTGRTCQAWSSMTPHQHSRTPENYPNAGLTR NYCRNPDAEIRPWCYTMDPSVRWEYCNLTQCLVTESSVLATLT VVPDPSTEASSEEAPTEQSPGVQDCYHGDGQSYRGSFSTTVTGR TCQSWSSMTPHWHQRTTEYYPNGGLTRNYCRNPDAEISPWCYT MDPNVRWEYCNLTQCPVTESSVLATSTAVSEQAPTEQSPTVQDC YHGDGQSYRGSFSTTVTGRTCQSWSSMTPHWHQRTTEYYPNGG LTRNYCRNPDAEIRPWCYTMDPSVRWEYCNLTQCPVMESTLLT TPTVVPVPSTELPSEEAPTENSTGVQDCYRGDGQSYRGTLSTTIT GRTCQSWSSMTPHWHRRIPLYYPNAGLTRNYCRNPDAEIRPWC YTMDPSVRWEYCNLTRCPVTESSVLTTPTVAPVPSTEAPSEQAPP EKSPVVQDCYHGDGRSYRGISSTTVTGRTCQSWSSMIPHWHQRT PENYPNAGLTENYCRNPDSGKQPWCYTTDPCVRWEYCNLTQCS ETESGVLETPTVVPVPSMEAHSEAAPTEQTPVVRQCYHGNGQSY RGTFSTTVTGRTCQSWSSMTPHRHQRTPENYPNDGLTMNYCRN PDADTGPWCFTMDPSIRWEYCNLTRCSDTEGTVVAPPTVIQVPS LGPPSEQDCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHST FIPGTNKWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCASSSFDCGKPQVEPKKCPGSIVGGCVAHPHSWPWQVSLRTRFG KHFCGGTLISPEWVLTAAHCLKKSSRPSSYKVILGAHQEVNLESH VQEIEVSRLFLEPTQADIALLKLSRPAVITDKVMPACLPSPDYMV TARTECYITGWGETQGTFGTGLLKEAQLLVIENEVCNHYKYICA EHLARGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYARVSRFVTWIEGMMRNN。

In one embodiment, dTAG has an amino acid sequence derived from lactoglutathione lyase (UniProtKB-Q04760(LGUL _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ. ID. NO. 43)

MAEPQPPSGGLTDEAALSCCSDADPSTKDFLLQQTMLRVKDPKK SLDFYTRVLGMTLIQKCDFPIMKFSLYFLAYEDKNDIPKEKDEKI AWALSRKATLELTHNWGTEDDETQSYHNGNSDPRGFGHIGIAVP DVYSACKRFEELGVKFVKKPDDGKMKGLAFIQDPDGYWIEILNP NKMATLM。

In one embodiment, dTAG has an amino acid sequence derived from the protein afadin (UniProtKB-P55196 (AFAD _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from the amino acid sequence: (SEQ.ID. NO. 44)

MSAGGRDEERRKLADIIHHWNANRLDLFEISQPTEDLEFHGVMR FYFQDKAAGNFATKCIRVSSTATTQDVIETLAEKFRPDMRMLSSP KYSLYEVHVSGERRLDIDEKPLVVQLNWNKDDREGRFVLKNEN DAIPPKKAQSNGPEKQEKEGVIQNFKRTLSKKEKKEKKKREKEA LRQASDKDDRPFQGEDVENSRLAAEVYKDMPETSFTRTISNPEV VMKRRRQQKLEKRMQEFRSSDGRPDSGGTLRIYADSLKPNIPYK TILLSTTDPADFAVAEALEKYGLEKENPKDYCIARV MLPPGAQHSDEKGAKEIILDDDECPLQIFREWPSDKGILVFQLKR RPPDHIPKKTKKHLEGKTPKGKERADGSGYGSTLPPEKLPYLVEL SPGRRNHFAYYNYHTYEDGSDSRDKPKLYRLQLSVTEVGTEKL DDNSIQLFGPGIQPHHCDLTNMDGVVTVTPRSMDAETYVEGQRI SETTMLQSGMKVQFGASHVFKFVDPSQDHALAKRSVDGGLMV KGPRHKPGIVQETTFDLGGDIHSGTALPTSKSTTRLDSDRVSSAS STAERGMVKPMIRVEQQPDYRRQESRTQDASGPELILPASIEFRE SSEDSFLSAIINYTNSSTVHFKLSPTYVLYMACRYVLSNQYRPDIS PTERTHKVIAVVNKMVSMMEGVIQKQKNIAGALAFWMANASEL LNFIKQDRDLSRITLDAQDVLAHLVQMAFKYLVHCLQSELNNY MPAFLDDPEENSLQRPKIDDVLHTLTGAMSLLRRCRVNAALTIQ LFSQLFHFINMWLFNRLVTDPDSGLCSHYWGAIIRQQLGHIEAW AEKQGLELAADCHLSRIVQATTLLTMDKYAPDDIPNINSTCFKLN SLQLQALLQNYHCAPDEPFIPTDLIENVVTVAENTADELARSDGR EVQLEEDPDLQLPFLLPEDGYSCDVVRNIPNGLQEFLDPLCQRGF CRLIPHTRSPGTWTIYFEGADYESHLLRENTELAQPLRKEPEIITV TLKKQNGMGLSIVAAKGAGQDKLGIYVKSVVKGGAADVDGRL AAGDQLLSVDGRSLVGLSQERAAELMTRTSSVVTLEVAKQGAIY HGLATLLNQPSPMMQRISDRRGSGKPRPKSEGFELYNNSTQNGS PESPQLPWAEYSEPKKLPGDDRLMKNRADHRSSPNVANQPPSPG GKSAYASGTTAKITSVSTGNLCTEEQTPPPRPEAYPIPTQTYTREY FTFPASKSQDRMAPPQNQWPNYEEKPHMHTDSNHSSIAIQRVTR SQEELREDKAYQLERHRIEAAMDRKSDSDMWINQSSSLDSSTSS QEHLNHSSKSVTPASTLTKSGPGRWKTPAAIPATPVAVSQPIRTD LPPPPPPPPVHYAGDFDGMSMDLPLPPPPSANQIGLPSAQVAAAE RRKREEHQRWYEKEKARLEEERERKRREQERKLGQMRTQSLNP APFSPLTAQQMKPEKPSTLQRPQETVIRELQPQQQPRTIERRDLQ YITVSKEELSSGDSLSPDPWKRDAKEKLEKQQQMHIVDMLSKEI QELQSKPDRSAEESDRLRKLMLEWQFQKRLQESKQKDEDDEEE EDDDVDTMLIMQRLEAERRARLQDEERRRQQQLEEMRKREAED RARQEEERRRQEEERTKRDAEEKRRQEEGYYSRLEAERRRQHDE AARRLLEPEAPGLCRPPLPRDYEPPSPSPAPGAPPPPPQRNASYLK TQVLSPDSLFTAKFVAYNEEEEEEDCSLAGPNSYPGSTGAAVGA HDACRDAKEKRSKSQDADSPGSSGAPENLTFKERQRLFSQGQDV SNKVKASRKLTELENELNTK。

In one embodiment, dTAG has an amino acid sequence derived from epidermal growth factor receptor (EGFR, UniProtKB P00533(EGFR _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, the dTAG is derived from, includes, or is an amino acid sequence: (SEQ. ID. NO. 53): L858R)

GEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKI PVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTST VQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYL EDRRLVHRDLAARNVLVKTPQHVKITDFGRAKLLGAEEKEYHA EGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKP YDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPK FRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEE DMDDVVDADEYLIPQQG。

In one embodiment, the dTAG is derived from, includes, or is an amino acid sequence: (SEQ. ID. NO: 54): T790M)

GEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKI PVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTST VQLIMQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYL EDRRLVHRDLAARNVLVKTPQHVKITDFGRAKLLGAEEKEYHA EGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKP YDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPK FRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEE DMDDVVDADEYLIPQQG are provided. In one embodiment, SEQ ID No. 54 has a leucine at position 163.

In one embodiment, the dTAG is derived from, includes, or is an amino acid sequence: (SEQ. ID. NO. 55): C797S)

GEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKI PVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTST VQLIMQLMPFGSLLDYVREHKDNIGSQYLLNWCVQIAKGMNYL EDRRLVHRDLAARNVLVKTPQHVKITDFGRAKLLGAEEKEYHA EGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKP YDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPK FRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEE DMDDVVDADEYLIPQQG are provided. In one embodiment, SEQ id No. 55 has a leucine at position 163. In one embodiment, seq.id No. 55 has a threonine at position 95. In one embodiment, seq.id No. 55 has a leucine at position 163 and a threonine at position 95.

In one embodiment, the dTAG is derived from, includes, or is an amino acid sequence: (SEQ. ID. NO: 56): C790G)

GEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKI PVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTST VQLIMQLMPFGCGLDYVREHKDNIGSQYLLNWCVQIAKGMNYL EDRRLVHRDLAARNVLVKTPQHVKITDFGRAKLLGAEEKEYHA EGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKP YDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPK FRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEE DMDDVVDADEYLIPQQG are provided. In one embodiment, seq.id No. 56 has a leucine at position 163. In one embodiment, seq.id No. 56 has a threonine at position 95. In one embodiment, seq.id No. 56 has a leucine at position 163 and a threonine at position 95.

In one embodiment, dTAG has an amino acid sequence derived from epidermal growth factor receptor (BCR-ABL or a variant thereof) or a variant thereof. In one embodiment, the dTAG is derived from, includes, or is an amino acid sequence: (SEQ. ID. NO. 57) (T315I)

SPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLT VAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPF YIIIEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSAMEYLE KKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAG AKFPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDLSQVYELLEKDYRMERPEGCPEKVYELMRACWQWNPSDRPSF AEIHQAFETMFQES are provided. In one embodiment, seq.id No. 57 has a threonine at position 87.

In one embodiment, dTAG has an amino acid sequence derived from BCR-ABL (BCR-ABL) or a variant thereof. In one embodiment, the dTAG is derived from, includes, or is an amino acid sequence: (SEQ. ID. NO: 58):

SPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAV KTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIIT EFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSAMEYLEKKN FIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGAKF PIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDLS QVYELLEKDYRMERPEGCPEKVYELMRACWQWNPSDRPSFAEI HQAFETMFQES。

in one embodiment, dTAG has an amino acid sequence derived from ALK (ALK, UniProtKB Q9UM73(ALK _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, the dTAG is derived from, includes, or is an amino acid sequence: (seq. id No. 59) (L1196M):

ELQSPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDLKEVPR KNITLIRGLGHGAFGEVYEGQVSGMPNDPSPLQVAVKTLPEVCS EQDELDFLMEALIISKFNHQNIVRCIGVSLQSLPRFIMLELMAGG DLKSFLRETRPRPSQPSSLAMLDLLHVARDIACGCQYLEENHFIH RDIAARNCLLTCPGPGRVAKIGDFGMARDIYRAGYYRKGGCAM LPVKWMPPEAFMEGIFTSKTDTWSFGVLLWEIFSLGYMPYPSKS NQEVLEFVTSGGRMDPPKNCPGPVYRIMTQCWQHQPEDRPNFAI ILERIEYCTQDPDVINTALPIEYGPLVEEEEK are provided. In one embodiment, seq.id No. 59 has a leucine at position 136.

In one embodiment, dTAG has an amino acid sequence derived from JAK2(JAK2, UniProtKB O60674(JAK2_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, the dTAG is derived from, includes, or is an amino acid sequence: (seq. id No.:60) (V617F):

VFHKIRNEDLIFNESLGQGTFTKIFKGVRREVGDYGQLHETE VLLKVLDKAHRNYSESFFEAASMMSKLSHKHLVLNYGVCFCGD ENILVQEFVKFGSLDTYLKKNKNCINILWKLEVAKQLAWAMHF LEENTLIHGNVCAKNILLIREEDRKTGNPPFIKLSDPGISITVLPKD ILQERIPWVPPECIENPKNLNLATDKWSFGTTLWEICSGGDKPLS ALDSQRKLQFYEDRHQLPAPKAAELANLINNCMDYEPDHRPSFR AIIRDLNSLFTPD are provided. In one embodiment, seq.id No. 60 has a valine at position 82.

In one embodiment, dTAG has an amino acid sequence derived from BRAF (BRAF, UniProtKB P15056(BRAF _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, the dTAG is derived from, includes, or is an amino acid sequence: (seq. id No.:61) (V600E):

DWEIPDGQITVGQRIGSGSFGTVYKGKWHGDVAVKMLNVT APTPQQLQAFKNEVGVLRKTRHVNILLFMGYSTAPQLAIVTQWC EGSSLYHHLHASETKFEMKKLIDIARQTARGMDYLHAKSIIHRDL KSNNIFLHEDNTVKIGDFGLATEKSRWSGSHQFEQLSGSILWMA PEVIRMQDSNPYSFQSDVYAFGIVLYELMTGQLPYSNINNRDQII EMVGRGSLSPDLSKVRSNCPKRMKRLMAECLKKKRDERPSFPRI LAEIEELARE are provided. In one embodiment, seq.id No. 61 has a valine at position 152. In one embodiment, seq.id No. 61 has a tyrosine at position 153. In one embodiment, seq.id No. 61 has a valine at position 152. In one embodiment, seq.id No. 61 has lysine at position 153. In one embodiment, seq.id No. 61 has a valine at position 152 and a lysine at position 153.

In one embodiment, dTAG has an amino acid sequence derived from LRRK2 protein (UniProtKB-Q5S 007(LRRK2_ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from LRRK2 amino acids 1328 to 1511. In one embodiment, dTAG is derived from LRRK2 amino acids 1328 to 1511, where amino acid 1441 is cysteine.

In one embodiment, dTAG has an amino acid sequence derived from PDGFR alpha protein (UniProtKB-P09619 (PDGFR _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is amino acids 600 to 692 derived from P09619. In one embodiment, dTAG is amino acids 600 to 692 derived from P09619, wherein amino acid 674 is isoleucine.

In one embodiment, dTAG has an amino acid sequence derived from the RET protein (UniProtKB-P07949 (RET _ HUMAN), which is incorporated herein by reference), or a variant thereof. In one embodiment, dTAG is derived from amino acids 724 to 1016 of P07949. In one embodiment, dTAG is derived from amino acids 724 to 1016 of P07949, wherein amino acid 691 is serine. In one embodiment, dTAG is derived from amino acids 724 to 1016 of P07949, wherein amino acid 749 is threonine. In one embodiment, dTAG is derived from amino acids 724 to 1016 of P07949, where amino acid 762 is glutamine. In one embodiment, dTAG is derived from amino acids 724 to 1016 of P07949, where amino acid 791 is phenylalanine. In one embodiment, dTAG is derived from amino acids 724 to 1016 of P07949, where amino acid 804 is methionine. In one embodiment, dTAG is derived from amino acids 724 to 1016 of P07949, wherein amino acid 918 is threonine.

In one embodiment, dTAG has an amino acid sequence derived from JAK3 protein (UniProtKB-P52333 (JAK3_ HUMAN), which is incorporated herein by reference), or a variant thereof.

In one embodiment, dTAG has an amino acid sequence derived from the ABL protein (UniProtKB-P00519 (ABL _ HUMAN), which is incorporated herein by reference), or a variant thereof.

In one embodiment, dTAG has an amino acid sequence derived from MEK1 protein (UniProtKB-Q02750 (MP2K1_ HUMAN), which is incorporated herein by reference), or a variant thereof.

In one embodiment, dTAG has an amino acid sequence derived from a KIT protein (UniProtKB-P10721 (KIT _ HUMAN), which is incorporated herein by reference), or a variant thereof.

In one embodiment, dTAG has an amino acid sequence derived from the HIV reverse transcriptase protein (UniProtKB-P04585(POL _ HV1H2), which is incorporated herein by reference), or a variant thereof.

In one embodiment, dTAG has an amino acid sequence derived from the HIV integrase protein (UniProtKB-Q76353(Q76353_9HIV1), which is incorporated herein by reference), or a variant thereof.

B. Protein of interest

As contemplated herein, the dTAG strategy can be used to produce stably expressed endogenous protein-dTAG hybrids in vivo, or ex vivo or in vitro as the case may be, by inserting a dTAG nucleic acid sequence into a nucleic acid sequence encoding a protein of interest in 5 '-or 3' -in-frame genome. Following in-frame insertion of the dTAG nucleic acid sequence, the cells express the endogenous protein-dTAG hybrid, allowing the activity of the endogenous protein-dTAG hybrid to be modulated by administering a heterobifunctional compound capable of binding to dTAG and thus degrading the endogenous protein-dTAG hybrid. In one embodiment, the activity of the endogenous protein-dTAG hybrid is decreased.

In certain embodiments, the nucleic acid encoding dTAG may be inserted in-frame into the genome and encode a gene for a protein involved in the disease. Non-limiting examples of specific genes involved in disorders that can target dTAG insertion include, as non-limiting examples, alpha-1 antitrypsin (A1AT), apolipoprotein b (apob), angiopoietin-like protein 3(ANGPTL3), proprotein, invertase subtilisin/kexin type 9 (PCSK9), apolipoprotein C3(APOC3), catenin (CTNNB1), Low Density Lipoprotein Receptor (LDLR), C-reactive protein (CRP), apolipoprotein a (apo (a)), factor VII, factor XI, antithrombin III (SERPINC1), phosphatidylinositosan a species (PIG-a), C5, alpha-1 antitrypsin (SERPINA1), hepcidin regulation (TMPRSS6), (delta-aminoacetylpropionate synthase 1(ALAS-1), acylcaa: diacylglycerol acyltransferase (miR-122, miR-21, miR-155, miR-34a, prekallikrein (KLKB1), connective tissue growth factor (CCN2), intercellular adhesion molecule 1(ICAM-1), glucagon receptor (GCGR), glucocorticoid receptor (GCCR), protein tyrosine phosphatase (PTP-1B), c-Raf kinase (RAF1), fibroblast growth factor receptor 4(FGFR4), vascular adhesion molecule-1 (VCAM-1), very late antigen-4 (VLA-4), transthyretin (TTR), surviving motoneuron 2(SMN2), Growth Hormone Receptor (GHR), dystonia myotonin kinase (DMPK), cellular nucleic acid binding protein (CNBP or ZNF9), Clusterin (CLU), eukaryotic translation initiation factor 4E (eIF-4E), MDM2, MDM4, heat shock protein 27(HSP 27), signal transducer and activator of transcription 3 protein (STAT3)), Vascular Endothelial Growth Factor (VEGF), kinesin spindle protein (KIF11), hepatitis b genome, Androgen Receptor (AR), atonal homolog 1(ATOH1), vascular endothelial growth factor receptor 1(FLT1), retinal tear 1(RS1), retinal pigment epithelium-specific 65kDa protein (RPE65), Rab catenin 1(CHM), and sodium channel, voltage-gated, X-type alpha subunit (PN3 or SCN 10A). Other proteins of interest that can be targeted by dTAG insertion include proteins associated with gain-of-function mutations, such as oncogenic proteins.

In particular embodiments, the protein of interest for targeting is apoB-100, ANGPTL3, PCSK9, APOC3, CRP, ApoA, factor XI, factor VII, antithrombin III, glypican class A (PIG-A), the C5 component of complement, alpha-1-antitrypsin (A1AT), TMPRSS6, ALAS-1, DGAT-2, KLB1, CCN2, ICAM, glucagon receptor, glucocorticoid receptor, PTP-1B, FGFR4, VCAM-1, VLA-4, GCCR, TTR, SMN1, GHR, DMPK, or NAV 1.8.

In one embodiment, dTAG is 5 'or 3' in-frame genomic integration into a gene encoding an endogenous protein associated with proteinopathy. In one embodiment, dTAG is 5 'or 3' in-frame genomic integration into a gene encoding an endogenous protein associated with a disorder selected from the group consisting of in-frame genomic insertions (5 'or 3'). The gene encoding the endogenous protein is related to: alzheimer's disease (amyloid beta peptide (A β); Tau protein), brain beta-amyloid angiopathy (amyloid beta peptide (A β)), glaucomatous retinal ganglion cell degeneration (amyloid beta peptide (A β)), prion diseases (prion proteins), Parkinson's disease and other synucleinopathies (alpha-synuclein), tauopathies (microtubule-associated protein Tau (Tau protein)), frontotemporal lobar degeneration (FTLD) (Ubi +, Tau-) (TDP-43), FTLD-FUS (fused to sarcoma (FUS) protein), Amyotrophic Lateral Sclerosis (ALS) (superoxide dismutase, TDP-43, FUS), Huntington's disease and other triplet repeat diseases (protein amplified with tandem glutamine), familial British dementia (ABri), familial Danish dementia (Adan), h hereditary cerebral hemorrhage with amyloidosis (iceland) (HCHWA-I) (cystatin C), CADASIL (Notch3), Alexandria (glial fibrillary acidic protein (GFAP)), Seipin protein lesions (Seipin protein), familial amyloidosis, senile systemic amyloidosis (transthyretin protein), Serpino protein lesions (Serpin protein), AL (light chain) amyloidosis (primary systemic amyloidosis) (monoclonal immunoglobulin light chain), AH (heavy chain) amyloidosis (immunoglobulin heavy chain), AA (secondary) amyloidosis (amyloid A protein), type II diabetes mellitus (islet amyloid polypeptide (IAPP; amyrin)), medial aortic amyloidosis (Medin (mucin)), ApoAI amyloidosis (apolipoprotein), ApoAII amyloidosis (ApoAII), ApoAIV amyloidosis (apolipoprotein AIV), Finnish-type Familial Amyloidosis (FAF) (gelsolin), lysozyme amyloidosis (lysozyme), fibrinogen amyloidosis (fibrinogen), dialysis amyloidosis (β -2 microglobulin), inclusion body myositis/myopathy (amyloid β peptide (A β)), cataracts (crystallins), retinal pigment degeneration and rhodopsin mutation (rhodopsin), medullary thyroid carcinoma (calcitonin), atrial amyloidosis (atrial natriuretic factor), pituitary prolactinoma (prolactin), hereditary lattice corneal dystrophies (keratohyal), lichen amyloidosis (keratin), malachite (keratin intermediate filament protein), lactoferrin amyloidosis (lactoferrin), alveolar proteinosis (surfactant protein C (SP-C)), odontogenic (Pindborg) tumor amyloid (odontogenic ameloblast-associated protein), seminal vesiculitis amyloid (protamine I), cystic fibrosis (cystic fibrosis transmembrane conductance regulator (CFTR) protein), sickle cell disease (hemoglobin) and myasthenia gravis (CIM) (hyper-proteolytic state of myosin ubiquitination).

As contemplated herein, modulation of a protein of interest can be achieved by administering a heterobifunctional compound specific for dTAG that binds to a protein-dTAG hybrid, causing its degradation, by in-frame insertion of a nucleic acid genome encoding dTAG into the specific protein of interest. Due to the ability to modulate a particular protein of interest in this manner, this strategy can be used to treat conditions in which expression of the protein within the cell above certain threshold levels results in a diseased state. Other applications of this technology include, but are not limited to, 1.) targeted degradation of proteins, where pathology is a function of gain of function mutations, 2) targeted degradation of proteins, where pathology is a function of amplification or increased expression, 3) targeted degradation of proteins that appear as monogenic diseases, 4) targeted degradation of proteins, where genetic susceptibility appears for a longer period of time and is often no longer sufficient after alternative biological compensation mechanisms, such as, but not limited to, hypercholesterolemia and proteinopathies.

Through controlled degradation of the endogenous protein-dTAG hybrid, favorable changes in protein expression or activity kinetics can result in the prevention and/or treatment of a disorder in a subject in need thereof.

Exemplary diseases and conditions that can be treated by the presently contemplated Methods are described, for example, in U.S. application No. 20150329875 entitled "Methods and Compositions for Prevention of treatment of a Disease," which is incorporated herein by reference.

In certain embodiments, the target protein is involved in lipid metabolism. For example, hypercholesterolemia is a condition characterized by very high blood cholesterol levels, known to increase the risk of coronary artery disease. Familial hypercholesterolemia, hyperlipidemia and familial chylomicronemia are genetic disorders of the family that lead to the symptomatology observed by aberrant genes. Mutations in genes encoding LDL receptor (LDLR), apolipoprotein b (apob), angiopoietin-like protein 3(ANGPTL3) and proprotein convertase subtilisin/kexin type 9 (PCSK9) are implicated in these diseases. LDLR is used to remove LDL from plasma for internalization into cells. LDLR is a transmembrane protein, localized in clathrin-coated pits where it forms complexes with ApoB-100 (the longer gene product of ApoB) and apoE-rich lipoproteins. After endocytosis of the complex, it moves to the endosome, where lipoproteins are released from the complex and eventually degraded by lysosomes. LDLR can then be recycled back to the cell surface.

Patients deficient in apoB-100, termed "familial deficient apolipoprotein B" (FDB), frequently carry an R3500Q mutation in APOB, which results in a decreased ability of LDL to bind LDLR, decreased plasma clearance, and thus increased plasma fatty acid levels (Innerarity) et al, (1987) PNAS USA 84: 6919. FDB is generally considered an autosomal dominant disorder and occurs in approximately 1: 700 (Ginsburg and Willand (2012) genomic Personalized Medicine, volume 1and 2. Daonal Press, London, page 507). Thus, in FDB patients with apoB-100 mutant heterozygotes, specific degradation of the defective apoB-100 allele can lead to correction of the disease by inserting dTAG in-frame into the allele of hepatocytes and administering a heterobifunctional compound, thereby generating the gene product of an apo-100 deficient protein-dTAG hybrid.

Similarly, an angiopoietin-like protein 3(ANGPTL3) overexpression mutation that results in elevated levels of ANGPTL3 can cause hyperlipidemia in a subject. ANGPTL3 also acts as a dual inhibitor of lipoprotein lipase (LPL) and Endothelial Lipase (EL) and increases plasma triglycerides and high density lipoprotein cholesterol in rodents. ANGPTL3 is expressed and secreted primarily in the liver, and is commonly used to increase plasma levels of triglycerides, LDL cholesterol and HDL cholesterol, where it acts directly on the liver to regulate secretion and clearance of hepatocyte lipoproteins (musinuru et al, (2010) N Engl J Med 363:23 p.2220). Thus, the methods of the invention can be used to treat hyperlipidemia associated with ANGPTL3 overexpression by targeted degradation of proteins using the dTAG insertion strategy described herein.

PCSK9 is another gene encoding a protein that plays a major regulatory role in cholesterol homeostasis. PCSK9 binds to the epidermal growth factor-like repeat a (EGF-a) domain of LDLR and induces degradation of LDLR. Autosomal dominant, functional mutations have been described for PCSK9 (e.g., S127R, P216L, D374Y and N157K) with increased toxicity and are associated with hyperlipidemia and Familial Hypercholesterolemia (FH) due to a corresponding increase in the rate of LDLR reduction. Plasma LDL cholesterol (Abifadel et al (2003) Nat Gen 34(2): 154). Furthermore, it has been found that loss-of-function PCSK9 mutations (e.g., Y142X, C679X and R46L) result in elevated liver LDLR levels, a significant decrease in plasma LDL cholesterol levels, and an 88% reduction in the incidence of coronary heart disease (Cohen et al, (2003) New Eng J Med 354(12): 1264). Thus, the methods and compositions of the invention are useful for treating or preventing hyperlipidemia and/or FH by targeted degradation of PCSK9 protein using the dTAG insertion strategy described herein.

Familial Chylomicronemia Syndrome (FCS) is characterized by extremely high levels of plasma triglycerides and leads to a number of health problems, such as abdominal pain, liver and spleen enlargement, and recurrent acute pancreatitis. Furthermore, subjects with high triglyceride levels do not have FCS, but have similar health problems due to elevated triglycerides. The lipocalins C3 or apo-CIII encoded by the APOC3 gene are components of very low lipoproteins (VLDL), LDL, HDL and chylomicrons and generally inhibit lipolysis by inhibiting lipoprotein lipase and liver lipase. Apo-CIII inhibits hepatic uptake of triglyceride-rich particles and can be elevated in patients with hyperlipidemia (Bobik, (2008) Circulation 118:702) and is an independent cardiovascular disease risk factor. Knock-out of the APOC3 gene in mice resulted in animals with reduced plasma triglyceride levels compared to normal (Maeda et al, (1994) J Biol Chem 269(38): 23610). Thus, the methods and compositions of the present invention are useful for preventing or treating subjects suffering from lipid metabolism disorders (e.g., familial hypercholesterolemia, hyperlipidemia, and familial chylomicronemia) by targeted degradation of APOC3 protein using the dTAG insertion strategy described herein.

In other embodiments, the target protein is involved in vascular diseases, such as cardiovascular disease and coronary artery disease. Similar to the disorders of lipid metabolism discussed above, coronary artery disease can also be caused by specific genes. For example, C-reactive protein (CRP) is a protein produced in the liver that is associated with inflammatory diseases. It is an acute phase protein that binds to phosphorylcholine expressed on the surface of dead or dying cells and functions to activate the complement system to help clear the cells. In chronic inflammatory diseases, elevated CRP levels can exacerbate disease symptoms by promoting and amplifying the overall chronic inflammatory state. In addition, CRP has been shown to increase myocardial and cerebral infarct area in rat models, when translated into human patients, a more negative prognosis after heart attack can be predicted. When CRP inhibitors were introduced into these rat models, infarct size and cardiac dysfunction decreased (Pepyset al (2005) Nature 440(27): 1217). Thus, inhibition of CRP may be beneficial for both inflammatory and coronary artery disease. The methods and compositions of the invention may be used to cause modulation of CRP expression by targeted degradation of CRP protein using the dTAG insertion strategy described herein.

Plasma lipoproteins (lp (a)) are low density lipoprotein particles comprising apolipoprotein (a) (apo (a)) and are also an independent risk factor for cardiovascular disease including atherosclerosis. Apo (a) is surface-contacted with LDL via apoB-100, linked by disulfide bonds, and genetic polymorphisms associated with elevated apo (a) levels have been reported to be associated with excessive incidence of myocardial infarction (chamman et al, (2009) Atherosclerosis 203(2): 371). The lp (a) concentrations in plasma vary widely between individuals, with these concentration differences appearing to be genetically determined. The apo (a) gene contains many plasminogen kringle 4-like repeats, and the number of these kringle repeats is inversely proportional to the plasma concentration of lp (a). DNA vaccine methods designed to mount an immune response to apolipoprotein (a) and elicit antibody-mediated lp (a) clearance demonstrated lp (a) a reduction in atherosclerotic activity in mice (Kyutoku et al, (2013)) Sci Rep 3doi:10.1038/srep 1600). Thus, by targeting degradation of ApoA protein using the dTAG insertion strategy described herein, the methods and compositions of the invention can be used to reduce expression of ApoA protein, resulting in a reduction in plasma concentrations of lp (a).

Coagulation disorders, commonly referred to as thrombophilia, may produce branches in vascular disease. The complex network of biochemical events that regulate blood coagulation in mammals includes 5 proteases (factors II, VII, IX and X and protein C) that interact with 5 cofactors (tissue factor, factor V, factor VIII, thrombomodulin and surface membrane proteins) to produce fibrin, which is the main component of the clot. There is a delicate balance between strong endogenous procoagulants and thrombus resistance to ensure blood fluidity and to keep these factors ready to induce blood clotting if injury occurs. The high plasma activity of factor XI and factor VII is associated with hypercoagulable and thrombotic diseases (coronary infarction, stroke, deep vein thrombosis, pulmonary embolism) and poor patient prognosis. It has been demonstrated that people with severe factor XI deficiency are protected from ischemic brain injury and stroke (Saloman et al (2008) Blood 111: 4113). Meanwhile, high levels of FXI have been shown to be associated with higher incidence of stroke events in patients (Yang et al (2006) Am J Clin Path 126: 411). Similarly, high levels of factor VII are also associated with coronary artery disease, although this is complicated by other considerations such as how to measure factor VII and which form of protein to analyze (Chan et al (2008) Circulation 118: 2286). Thus, the methods and compositions of the invention can be used to prevent or treat a subject having a thrombotic disorder by selectively degrading coagulation factors associated with the disorder (e.g., factor VII and factor XI) by targeting degradation factor XI and/or factor VII using the dTAG insertion strategy described herein. .

As mentioned above, the balance of the coagulation cascade is crucial. Therefore, in addition to the importance of coagulation factors, inhibitors of these factors are also critical. Patients with hemophilia lack one or more components of the coagulation cascade and therefore have reduced coagulation capacity. In one of the last steps of the cascade, thrombin acts on fibrinogen to produce fibrin, which is the main component of the clot. The cascade results in the production of active thrombin allowing this to occur. To maintain systemic balance, antithrombin (also known as antithrombin III, encoded by the SERPINC1 gene) acts on thrombin to inhibit its action. In many hemophiliacs, factor deficiency is not absolute and there is some degree of clotting. Thus, an approach based on antithrombin degradation may allow the coagulation cascade to be associated with which factor is deficient and sufficient coagulation to occur when the upstream factor is restricted. This has been demonstrated using blood from hemophilia A patients (see Di Micco et al (2000) Eur J Pharmacol. March 10; 391(1-2): 1-9.). The methods and compositions of the invention are useful for treating hemophilia patients, such as hemophilia a and hemophilia B, by targeted degradation of antithrombin III protein using the dTAG insertion strategy described herein.

The target protein may also be involved in blood disorders (blood disorders). The complement system is a key participant in a variety of hematological conditions. Paroxysmal Nocturnal Hemoglobinuria (PNH) is a hemolytic disease caused by a deficiency in the PIG-A gene (see Brodsky (2008) Blood Rev 22(2): 65). The PIG-A gene product glypican A is essential for the first step in the synthesis of GPI-anchored proteins. PIG-A is found on the X chromosome and mutations in PIG-A result in erythrocytes that are sensitive to hemolysis caused by complement. Notably, these mutant cells lack the GPI-anchored proteins CD55 and CD 59. CD59 interacts directly with the complement-associated membrane attack complex (or MAC), preventing formation of lytic pores by blocking aggregation of C9, C9 being a key step in pore assembly. The function of CD55 is to accelerate the destruction of the C3 convertase, so in the absence of CD55, there is more C3 convertase, resulting in more MAC formation. Thus, the absence of both proteins results in increased lysis of the mutant erythrocytes. Complications due to increased thrombosis are the greatest problem for PNH patients (Brodsky (2008) Blood Rev 22(2): 65). 40% of PNH patients continue with thrombus formation, which can lead to stroke and acute cardiovascular disease. Thus, the methods and compositions of the invention can be used to treat and/or prevent PHN in a subject by targeted degradation of glypican class a (PIG a) using the dTAG insertion strategy described herein.

Inhibition of the C5 component of complement has been approved for the treatment of PNH and atypical hemolytic uremic syndrome (aHUS), confirming that C5 is an important therapeutic target. Hemolysis of the red blood cells associated with aHUS occurs when cells are destroyed by alternative pathways due to dysregulation of the complement system (part of innate immunity). Typically, destructive C3bBb complexes are formed on the surface of invading cells (e.g., bacteria) to accelerate their destruction as part of the alternative pathway in the complement system. The C3bBb complex can bind to another C3b to form a C3bBbC3b complex, which then acts as a C5 convertase. C5 convertase cleaves C5 into C5a and C5b, and C5b recruits C6, C7, C8, and C9 to form MACs. A group of complement regulatory proteins (e.g., CD35, CD46, CD55, and CD59) are located on the body's own cells to inhibit the activity of these proteins, thereby protecting them. However, when there is an imbalance in these regulatory proteins, the C3bBb complex may not form properly (de Jorge et al (2011) J Am Soc Nephrol 22: 137). In addition to premature destruction of red blood cells, this syndrome may also lead to kidney disease due to destruction and blockage of the glomerular filtration device. C5 negative mice were shown to be protected when crossed with mice bearing complement regulatory protein mutations, data that have been used to validate the idea of C5 as a target in aHUS (de Jorge, supra) and other diseases associated with complement dysregulation. Monoclonal antibodies specific for C5b monoclonal antibodies (eculizamab) have been successfully used to treat aHUS (Gruppo and Rother, (2009) N Engl J Med 360; 5 p544) and other complement-mediated diseases (e.g. Paroxysmal Nocturnal Hemoglobinuria (PNH) (Hillmen et al, (2013) br.j Haem 162: 62)). Thus, the methods and compositions of the invention may be used to modulate the expression of C5 to prevent or treat diseases associated with complement dysregulation by targeted degradation of C5 using the dTAG insertion strategy described herein.

Alpha-1-antitrypsin (A1AT) deficiency occurs in about 1 in 1500 to 3000 people of European descent, but is rare in individuals of Asian descent. Alpha-1-antitrypsin protein is a protease inhibitor encoded by the SERPINA1 gene and used to protect cells from the activity of proteases released by inflammatory cells, including neutrophil elastase, trypsin and protease-3 (PR-3). The deficiency is a autosomal co-dominant or recessive disease caused by a mutant SERPINA1 gene in heterozygotes, wherein reduced expression of the mutant allele or expression of the mutant A1AT protein with poor inhibitory activity results in a long-term lack of neutrophil elasticityInhibition of proteases leads to tissue damage. The most common SERPINA1 mutation comprises a Glu342Lys substitution (also known as the Z allele), which results in the formation of an ordered polymer of the protein in the endoplasmic reticulum of the patient's hepatocytes. These inclusions ultimately lead to cirrhosis, which can only be treated by liver transplantation (Yusa et al, (2011) Nature478 p.391). Aggregation within hepatocytes leads to a severe decrease in plasma A1AT levels, leading to an increased risk of this inflammatory disease. Furthermore, A1AT deficiency is associated with lung diseases, including Chronic Obstructive Pulmonary Disease (COPD), pulmonary emphysema and chronic bronchitis (Tuder et al (2010) Proc Am Thorac Soc 7(6): p.381) and may have a broader inhibition of progression of other diseases, including type 1and type 2 diabetes, acute myocardial infarction, rheumatoid arthritis, inflammatory bowel disease, cystic fibrosis, transplant rejection, graft versus host disease and multiple sclerosis (Lewis (2012) Mol Med 18(1) p.957). Population studies have shown that to avoid these diseases, the lowest ATA1 plasma threshold is about 0.5mg/mL (normal plasma levels are about 0.9 to 1.75 mg/mL in the non-inflammatory state), and for patients with severe plasma deficiency A1AT emphysema, current treatment methods alleviate symptoms mainly through the use of bronchodilators and the like, although A1AT is infused weeklyOr alternatively. The severe lung disease associated with A1AT was also ultimately treated by transplantation. Clinical trials for treatment of A1AT deficiency involve a variety of methods including delivery of concentrated A1AT protein, use of AAV constructs containing the A1AT gene by IM injection, and use of A1AT in HIV, to name a few. Thus, the compositions and methods of the invention may be used to treat or prevent diseases associated with A1AT deficiency by targeted degradation of alpha-1-antitrypsin protein using the dTAG insertion strategy described herein, thereby eliminating liver aggregates that may lead to cirrhosis.

Another liver target of interest includes any protein involved in the regulation of iron content in vivo. Iron is critical for hemoglobin production, but excess results in the production of reactive oxygen species. In patients who rely on blood transfusion (e.g. certain haemophilia, haemoglobinopathies), secondary iron overload is common. The iron-regulating hormone hepcidin and its receptors and iron channel iron transporters control dietary absorption, storage and tissue distribution of iron by promoting cellular uptake thereof. The regulation of hepcidin occurs at the transcriptional level and is sensitive to iron concentration in plasma, where increased hepcidin expression leads to decreased plasma iron concentration. The hepcidin gene is upregulated by SMAD transcription factors through a series of receptor-ligand interactions involving a receptor called hemojuvelin. Iron-related hepcidin down-regulation is in turn regulated by a protease called TMPRSS6, which cleaves hemojuvelin and prevents up-regulation of hepcidin (Ganz (2011) Blood 117: 4425). Downregulation of TMPSS6 expression by using inhibitory RNA targeting TMRSSS 6 mRNA has been shown to result in a reduction of iron overload in a mouse model (Schmidt et al, (2013) Blood 121: 1200). Thus, the methods and compositions of the invention can be used to target TMPRSS6 for degradation by using the dTAG insertion strategy described herein.

Other conditions associated with the in vivo iron utilization pathway are porphyria. These diseases are caused by a number of defects in the enzymes involved in the synthesis of hemoglobin. Acute Intermittent Porphyria (AIP) is an autosomal dominant disease, the second most common porphyria, with an incidence of about 5 to 10 of 10 thousands of people. AIP is caused by a deficiency of hydroxymethylsilane synthetase (HMBS synthase (HMBS), also known as porphobilinogen-deaminase), in which mutations in the HMBS gene are very heterogeneous, including missense and point mutations (Solis et al (1999) Mol Med 5: 664). AIP attacks, which may be life threatening, may have gastrointestinal, neuropsychiatric, cardiovascular and nervous system manifestations. Attacks have several causes, may last for several days, and often require hospitalization, and may be caused by several seemingly unrelated factors, including certain medications, infections, caloric restriction, smoking, alcohol, and hormonal fluctuations related to the menstrual cycle (Yasuda et al (2010)) Mol Ther 18(1): 17). HMB synthase is part of the heme synthesis pathway, in which glycine and succinyl-CoA are linked by a δ -aminolevulinic acid synthase 1(ALAS-1) to produce aminolevulinic acid, which is then acted upon by aminolevulinic acid dehydratase (ALAD) to produce phosphorylated fibrinogen. The phosphopropeptide is converted to hydroxymethylsilane by HMB synthase. From there, the pathway continues, ultimately producing heme (Ajioka et al (2006) Biochim Biophys Acta 1762: 723). Regardless of the trigger, all attacks lead to an increase in the enzyme delta-aminoacetylpropionic acid synthetase 1 (ALAS-1). This enzyme is the first enzyme in the hepatic heme synthesis pathway, and when induced, the deficiency of HMB synthase becomes rate-limiting and aminolevulinic acid and phosphorylated fibrinogen precursors accumulate (Yasuda, supra). Liver transplantation in AIP patients can arrest the onset, suggesting that targeting the liver may be beneficial for treatment. Furthermore, in the mouse model of AIP, the mouse had only 30% of normal HMB synthase levels, and insertion of the transgenic HMBs encoding HMB synthase resulted in a decrease in the accumulation of aminolevulinic acid and phosphorylated proprotein when administered to the mouse phenobarbital (Yasuda, supra). Double-stranded RNA designed to inhibit ALAS-1 has also been shown to reduce ALAS-1 expression in vivo and decrease phosphorylcholine accumulation in response to phenobarbital treatment in a mouse AIP model (see U.S. patent publication No. 20130281511). Thus, the methods and compositions of the invention are useful for the prevention and treatment of AIP by targeted degradation of ALAS-1 using the dTAG insertion strategy described herein.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, with a prevalence of 15% to 30% in the western population, and is caused by triglyceride accumulation in the liver. However, the prevalence increased to 58% in overweight people and 98% in obese people. Nonalcoholic steatohepatitis (NASH) is a higher form of NAFLD in which liver injury has occurred and can lead to liver failure, portal hypertension, liver cancer and cirrhosis (schwengerrand allred (2014) World J gastroenen 20(7): 1712). Evidence seems to indicate that the hepatic triglyceride accumulation observed in NALFD is closely related to hepatic insulin resistance, usually as part of type 2 diabetes and metabolic syndrome (Choi et al (2017, J Biol Chem 282(31): 22678); acyl-CaA: diacylglycerol acyltransferase (DGAT) catalyzes the last step of triglyceride synthesis by promoting the ligation of sn-1,2 Diacylglycerol (DAG) with long-chain acyl Co a. there are two major isoforms of DGAT, DGAT-1 and DGAT-2. DGAT-1, which are predominantly expressed in the small intestine, while DGAT-2 is predominantly expressed as a hepatic expression (which is an insulin response); knockout of DGAT-1 or DGAT-2 in diet-induced NALFD rats using antisense oligonucleotides is markedly improved in hepatic steatosis following knockdown, sex but not improve DGAT-1 knock-out (Choi, supra). Thus, the materials and methods of the invention can be used to alter the expression of DGAT-2 to treat NASH and NALFD, as well as to reduce hepatic insulin resistance by targeted degradation of DGAT-2 using the dTAG insertion strategy described herein.

Further vascular targets include those involved in Hereditary Angioedema (HAE). HAE is an autosomal dominant genetic disease affecting 1 out of every 50,000 people as a result of reduced levels of C1 inhibitor. Patients have recurrent episodes of swelling anywhere in the body, with tumors located in the oropharynx, larynx or abdomen, with the highest morbidity and risk of mortality (see, Tse and zuraw, (2013) Clev Clin J of Med 80(5): 297). The disease occurs by plasma extravasation into tissues due to the excessive production of bradykinin. This mechanism appears to involve cleavage of prekallikrein (also known as PKK) by activating factor XII, releasing active plasma kallikrein (which activates more factor XII). Plasma kallikrein then cleaves kininogen, releasing bradykinin. Bradykinin then binds to the B2 bradykinin receptor on endothelial cells, increasing permeability of endothelial cells. Generally, C1 inhibitors (encoded by SERPING 1) control bradykinin production by inhibiting activation of plasma kallikrein and factor XII. There are three types of HAE, i.e. type I and type II distinguished by the number and type of C1 inhibitor present and type III associated with the Thr309Lys mutation in factor XII (Prieto et al (2009) Allergy 64(2): 284). Type I HAE has low levels of C1 inhibitors, which appear to be due to expression and destruction of small amounts of C1 inhibitory protein. Type 1 accounts for approximately 85% of HAE patients. Type II patients have normal levels of C1 inhibitor, but the C1 inhibitor protein is ineffective due to mutation (Tse and zuraw, supra). More than 250 mutations in SERPING1 have been characterized that result in type I HAE, including small and large insertions and deletions as well as duplications (Rijavec et al, (2013) PLoS One 8(2): e 56712). Because of this high variability in the genetic basis of HAE, the methods and compositions of the invention can be used to prevent or treat HAE by targeting downstream participants in the expression of HAE. For example, targeting prekallikrein (KLKB1, expressed in hepatocytes) to achieve reduced expression of prekallikrein (abbreviated PKK) may result in reduced bradykinin production, regardless of the type of upstream mutation that causes HAE, resulting in reduced plasma extravasation. Thus, the methods and compositions of the invention may be used to cause a reduction in KLKB1 expression to prevent or treat HAE by targeted degradation of KLKB1 using the dTAG insertion strategy described herein.

The target may also be associated with fibrotic disease. Fibrotic diseases in various organs are the leading cause of organ dysfunction and can occur as a response to another underlying disease or as a result of a propensity for fibrosis in an individual with the disease. Fibrosis is marked by inappropriate deposition of extracellular matrix compounds such as collagen and related glycoproteins. TGF- β plays a major role in the fibrotic process, inducing fibroblasts to synthesize extracellular matrix (ECM) proteins, and it also inhibits the expression of proteins with ECM-decomposing activity (Leask (2011) J Cell Commun Signal 5: 125). There is a class of ECM regulatory proteins known as CNN proteins (the so-called first three members, CYR61 (cysteine-rich 61/CCN1), CTGF (connective tissue growth factor/CCN 2) and NOV (nephroblastoma) overexpression/CCN 3). These proteins regulate a variety of cellular functions, including cell adhesion, migration, apoptosis, survival and gene expression. TGF- β strongly upregulates CCN2 expression, acts as a cofactor for TGF- β in synergy and appears to be involved in pericyte activation, which appears to be an essential process in fibrosis (Leask supra). CCN2 is overexpressed in fibrotic tissues, including lung tissue, also found in the plasma of patients with systemic sclerosis (scleroderma). Also, CCN2 expression by use of antisense oligonucleotides (ASO) reduced chemically induced liver fibrosis, renal fibrosis due to ureteral obstruction, fibrotic scarring of skin wounds, and abscesses after partial nephrectomy of renal interstitial fibroids (Jun and Lau (2013) Nat Rev Drug Discov.10(12): 945) 963). In addition to its profibrotic role, CCN2 may be important in cancer, particularly in metastasis. It may promote tumor growth by inducing angiogenesis, and high levels of CCN2 in breast cancer cells are indicative of the potential for bone metastasis (Jun and Lau, supra). The impact of clinical progression of many of these diseases has been shown by experimental models of knockdown of CCN2 expression in various models of fibrosis, cancer, cardiovascular disease and retinopathy through the use of CCN2 modulating compounds (e.g., monoclonal antibodies or inhibitory RNAs) (Jun and Lau supra). Thus, the methods and compositions of the present invention may be used to prevent or treat fibrosis, cancer, vascular disease and retinopathy by reducing the expression of CCN2 by targeted degradation of CCN2 using the dTAG insertion strategy described herein.

In other embodiments, the target is involved in an autoimmune disease. Autoimmune diseases are common as a group, affecting over 2300 million people in the united states alone. There are several different types, with many different degrees of severity and prognosis. Generally, they are characterized by the production of autoantibodies against various self-antigens, resulting in immune responses against themselves. Autoimmune diseases of the intestine can lead to conditions such as ulcerative colitis and inflammatory/irritable bowel disease (e.g., crohn's disease). Cell surface glycoprotein intercellular adhesion molecule 1(ICAM-1) is expressed on endothelial cells and upregulated in inflammatory states and serves as a binding protein for leukocytes during translocation to tissues. Specific ICAM-1 alleles have been found to be associated with Crohn's disease (e.g., K469E allele, exon 6) or ulcerative colitis (e.g., G241R, exon 4) and may be preferentially involved in the chronic induction of inflammation found in these diseases (Braun et al, (2001) Clin Immunol.101(3): 357-60). Knock-out of ICAM in mouse models of vascular and diabetic disease has demonstrated the usefulness of this treatment (see Bourdillon et al, (2000) Ather Throm Vasc Bio 20:2630 and Okada et al, (2003) Diabetes 52: 2586). Thus, the methods and compositions of the invention can be used for a general reduction of ICAM expression in inflammatory diseases by targeted degradation of ICAM using the dTAG insertion strategy described herein.

Another common disease recently recognized as an autoimmune disease is diabetes. Glucagon is a peptide hormone released by islet alpha cells, plays a key role in regulating hepatic glucose production, and has profound hyperglycemic effects. In addition, glucagon activates a number of enzymes required for gluconeogenesis, particularly the enzyme system for converting pyruvate to phosphoenolpyruvate, which is the rate-limiting step in gluconeogenesis. Hyperglycaemia has been proposed as a causative factor in the pathogenesis of diabetes based on the following observations: 1) diabetic hyperglycemia studied from animal to human has been associated with relative or absolute hyperglycemia; 2) infusion of somatostatin inhibits endogenous glucagon release, thereby reducing blood glucose levels in diabetic patients induced by alloxan or diazoxide; 3) chronic glucagon infusion results in hepatic insulin resistance in humans (see Liang et al (2004) Diabetes 53(2): 410). Glucagon receptor (encoded by the GCGR gene) is predominantly expressed in the liver, and treatment of diabetic (db/db) mice with antisense RNA targeting glucagon receptor resulted in significant reductions in serum glucose levels, triglycerides and fatty acids compared to controls (Liang et al, supra). Similarly, Glucocorticoids (GCs) increase hepatic gluconeogenesis and play an important role in the regulation of hepatic glucose output. In db/db mice, reduction of glucocorticoid receptor (GCCR) expression by use of targeted antisense RNA results in about 40% reduction in fed and fasting glucose levels and about 50% reduction in plasma triglycerides (see Watts et al (2005) Diabetes 54(6): 1846). Thus, the methods and compositions of the present invention can be used to prevent or treat diabetes by targeting the glucagon receptor and/or glucocorticoid receptor by reducing the expression of the receptor by targeted degradation using the dTAG insertion strategy described herein.

Another potential target for insulin type 2 antidiabetics is protein tyrosine phosphatase 1B (PTP-1B). Insulin resistance is defined as a decrease in the ability of a cell to respond to insulin in terms of glucose uptake and tissue utilization. One of the most important phosphatases that regulate insulin signaling is PTP-1B, which inhibits the insulin receptor and insulin receptor substrate 1 by direct dephosphorylation. Mice that are PTP 1B-/- (mutated at both alleles) are insulin-allergic and resistant to weight gain on a high fat diet (see Fernandez-Ruiz et al, (2014) PLoS One 9(2): e 90344). Thus, the target is useful in diabetes treatment and obesity. The development of inhibitory small molecules specific for this enzyme is problematic due to the highly conserved active site pocket, but antisense oligonucleotides directed against PTP-1B have been shown to reduce PTP-1B mRNA expression in liver and adipose tissue by about 50% and to produce glucose lowering effects in hyperglycemic, insulin resistant ob/ob and db/db mice (which are repeated experiments in non-human primates) (see Swarbrick et al, (2009) Endocrin 150: 1670). Thus, the methods and compositions of the invention can be used to target PTP-1B by targeted degradation of PTP-1B using the dTAG insertion strategy described herein, resulting in increased insulin sensitivity.

A high risk factor for developing diabetic insulin against diabetes is obesity. The overweight (body mass index (BMI) of more than 25 kg/m) is estimated to be more than 10 hundred million people all over the world²More than 3 million of the people are considered to be obese (BMI ≧ 30 kg/m)²) This means that obesity is one of the biggest threats to public health today (Lagerros)(2013) The Ther Adv Gastroenterol 6 (1: 77). Obesity is closely related to complications such as insulin resistance type II diabetes, dyslipidemia, hypertension and cardiovascular disease. Obesity usually starts with changes in diet and exercise, but usually with a decrease in caloric expenditure, a parallel and mixed decrease in body energy expenditure is observed (Yu et al, (2013) PLoS One 8(7): e 66923). Fibroblast growth factor receptor 4(FGFR4) has been demonstrated to have anti-obesity effects in a mouse obesity model. FGFR4 is expressed primarily in the liver, and it and its ligand FGF19 (in humans) regulate bile acid metabolism. GFFR4/FGF19 regulates the expression of cholesterol 7 alpha-hydroxylase and its activity. In addition, FGFR4 and FGF19 appear to be involved in lipid, carbohydrate or energy metabolism. Hepatic FGFR4 expression was reduced by fasting and hepatic FGFR4 expression was increased by insulin. FGFR4 deficient mice also showed changes in lipid profile in response to different nutritional conditions compared to wild type mice. Treatment of obese mice with FGF19 increases metabolic rate and improvesObesity, hepatic steatosis, insulin sensitivity and plasma lipid levels, and also inhibits hepatic fatty acid synthesis and gluconeogenesis while increasing glycogen synthesis. Antisense reduction of FGFR4 in obese mice also resulted in reduced body weight and obesity, improved insulin sensitivity and liver steatosis, and plasma FGF15 (mouse equivalent FGF19) levels without any significant toxicity (Yu et al, supra). Thus, the methods and compositions of the invention may be used to treat obesity by reducing expression of FGFR4 through targeted degradation using the dTAG insertion strategy described herein.

Multiple Sclerosis (MS) is a chronic, disabling autoimmune disease of the central nervous system characterized by inflammation, demyelination and axonal destruction. The burst associated with relapsing MS (occurring in 85 to 95% of patients) is thought to be associated with entry of activated lymphocytes into the brain. Currently available treatments are only able to suppress recurrence rates by about 30%. The inflammatory response induces the expression of vascular adhesion molecule-1 (VCAM-1) on the vascular endothelium, and the adhesion of lymphocytes to VCAM-1 is an essential step in allowing activated cells to pass into the brain. Lymphocyte adhesion to VCAM-1 is mediated by very late binding of antigen-4 (VLA-4, also known as α 4 β 1 integrin) to the surface of activated lymphocytes (Wolf et al (2013) PLosOne 8(3): e 58438). Disruption of this interaction has been the idea behind the therapeutic use of anti-VLA-4 specific antibodies and small molecule antagonists (Wolf et al, supra). Thus, the materials and methods of the invention can be used to target VCAM-1 or VLA-4 expression by targeted degradation using the dTAG insertion strategy described herein.

Another disease of interest is cushing's disease/syndrome (CS). In this disease, the patient's serum glucocorticoid levels are elevated due to increased expression by the adrenal glands. CS is a rare disease with an incidence of between 1.8 and 2.4 patients per million per year. The most common cause of endogenous CS is pituitary adenomas that produce ACTH, which is seen in about 70% of CS patients. Cortisol-producing adrenal adenomas and ectopic ACTH-producing tumors are less common, accounting for approximately 10 to 15% of each. First line treatment of patients with pituitary-derived CS is via transsphenoid pituitary surgery (TSS) and unilateral adrenal resection of cortisol-producing adrenal adenomas. Unilateral adrenal resection is effective in almost all cortisol-producing adrenal adenoma patients, and permanent suprarenal adrenocortical insufficiency is rare. In contrast, hypopituitarism is common after TSS, ranging between 13% and 81% (see Ragnarsson and Johannsson (2013) Eur J Endocrin 169: 139). However, in some patients, surgical resection was unsuccessful, thus indicating drug treatment. One approach is to inhibit the activity of hypercortisolemia by targeting the glucocorticoid receptor (GCCR), for example, with Mifepristone (Mifepristone) (also known as RU486), which is a GCCR antagonist (see Johanssen and Allolio (2007) Eur J Endocrin 157: 561). However, RU486 has other activities (most notably inducing abortion in pregnant patients). Thus, the methods and compositions of the invention can be used to target GCCR by reducing expression through targeted degradation using the dTAG insertion strategy described herein.

Transthyretin amyloidosis (TTRA) is one of several degenerative diseases suspected of being associated with misfolded and aggregated proteins (amyloid). Transthyretin (TTR) is a tetramer produced in the liver and secreted into the bloodstream for transport of pan-retinal binding proteins. However, upon conformational change, it becomes amyloid. Partial unfolding exposes fragments of hydrophobic residues in the extended conformation, which eventually mis-assemble efficiently into substantially unstructured spherical aggregates prior to crossing the β -sheet amyloid structure (see, Johnson et al (2012) J Mol Biol421(2-3): 183). TTRA can occur in patients with sporadic and autosomal dominant forms of inheritance, including Familial Amyloid Polyneuropathy (FAP) and Familial Amyloid Cardiomyopathy (FAC). These genetic forms are usually made earlier and involve more than 100 point mutations described in the TTR gene. Generally, the more unstable a protein is to a mutation, the more likely it is to have a certain amount of amyloid pathology. The amyloid formed leads to selective destruction of cardiac tissue in FAC or peripheral and central nervous tissue in FAP. Some new therapeutic strategies for treating these diseases, such as inhibitory RNA strategies, have primarily attempted to reduce the amount of TTR to reduce the aggregation potential of proteins (Johnson et al, supra). Thus, the methods and compositions of the invention can be used to target TTR to reduce the amount of pathological forms of TTR protein and/or reduce TTR concentration, typically by targeted degradation using the dTAG insertion strategy described herein.

The methods of the invention can also be used to treat muscle disorders. Spinal muscular atrophy is an autosomal recessive genetic disease caused by mutations in the SMN1 gene, which encodes a "survival of motor neurons" (SMN) protein characterized by general muscle atrophy and dyskinesias. SMN proteins are involved in the assembly of components of the spliceosome machinery, and several defects in the SMN1 gene are associated with splice defects that result in the specific exclusion of exon 7 of the mature mRNA. These defects are particularly prevalent in spinal motor neurons and can cause spinal muscular atrophy. The severity of the SMN1 defect can be modified by bypassing SMN1, which is referred to as SMN 2. The SMN2 gene sequence differs from SMN1 by a few single nucleotide polymorphisms in exons 7 and 8 and several single nucleotide polymorphisms in intron sequences. Thus, the methods and compositions of the invention may be used to target SMN1 in an effort to reduce the amount of pathological forms of SMN1 protein and/or reduce SMN1 concentration, typically by targeted degradation using the dTAG insertion strategy described herein.

Dysregulation of Growth Hormone (GH) secretion can lead to acromegaly, a Disease of disproportionate bone, tissue and organ growth that first becomes apparent around the age of 40 (Roberts and Katznelson (2006) U.S. endocrine Disease: 71). Its incidence is about 5 cases per million, and diagnosis requires the determination of GH secretion disorders and elevated IGF1 levels. Inability to inhibit GH secretion within 2 hours after oral glucose loading is commonly used for the diagnosis of acromegaly. The normal regulation of GH secretion is performed by the pituitary gland. Hypothalamic GH-releasing hormone (GHRH), ghrelin and somatostatin regulate GH production by anterior pituitary growth hormone cells. Genes encoding GH receptors or GHRs are widely expressed and when GH molecules interact with GHR dimers, signals proceed via JAK 2-dependent and independent intracellular signal transduction pathways (see Melmed (2009) J Clin Invest 119(11): 3189). Circulating GH stimulates hepatic secretion of insulin-like growth factor-1 (IGF-1). Acromegaly occurs when benign pituitary tumors lead to increased GH secretion and thus to increased IGF-1 secretion. One GHR mutation associated with acromegaly has an in-frame deletion in exon 3, which results in a deletion of 22 amino acids in the protein. This mutant receptor, termed d 3-GHR, results in increased GH reactivity. Current therapies focus on the normalization of GH and IGF-1 levels, usually by surgical resection of pituitary tumors. Since GH induces secretion of IGF-1, targeting GHR is an attractive target for the methods and compositions of the invention. Thus, the methods and compositions of the invention can be used to target GHR by reducing expression through targeted degradation using the dTAG insertion strategy described herein.

Another disease associated with muscle atrophy is myotonic dystrophy, a chronic disease characterized by muscle atrophy, cataracts, cardiac conduction defects, endocrine changes, multiple organ damage, and myotonia (prolonged muscle contraction following voluntary contraction). Myotonic dystrophy occurs at about 13 out of every 10 million people, with two forms of disease, myotonic dystrophy type 1 (also known as Steinert's disease, MMD1 or DM1, and most common) and myotonic dystrophy type 2 (MMD2 or DM 2). Both are inherited autosomal dominant diseases caused by abnormal amplification of the 3' noncoding regions of both genes (CTG in the DMPK gene of type 1 (encoding dystrophic muscle protein kinase) and the ZNF9 gene of type 2 (encoding cellular nucleic acid-binding protein)) and DM1, the most common form of muscular dystrophy in adults. These mutations result in toxic intranuclear accumulation of mutant transcripts in the RNA content or lesions (see cailet-Boudin et al, (2014) front. Patients of type 1 have CTG copy numbers greater than 50 and have different phenotypes ranging from asymptomatic to severe. Antisense RNA technology has been used to cause specific disruption of mutant DMPK transcripts in vitro, which had no effect on the proliferation rate of DM1 myoblasts but restored their differentiation (Furling et al (2003) Gene Therapy10: 795). Thus, the methods and compositions of the invention can be used to target dystrophic myoprotein kinase or cellular nucleic acid binding proteins by targeted degradation using the dTAG insertion strategy described herein.

Chronic pain is a major health problem affecting 8000 million americans at some time during their lives, with significant associated morbidity and impact on the quality of life of the individual. Chronic pain can result from a variety of inflammatory and neurologic injury events, including cancer, infectious diseases, autoimmune related syndromes, and surgery. Voltage-gated sodium channels (VGSCs) are the basis for regulating neuronal excitability, and overexpression of these channels can produce abnormal self-discharge patterns, supporting chronic pain. There are at least nine different VGSC subtypes in the nervous system, and each subtype can be functionally classified as either sensitive to tetrodotoxin or resistant to tetrodotoxin. Neuronal sodium channel subtypes, including Nav1.3, Nav1.7, Nav1.8, and Nav1.9, are involved in the processing of nociceptive information. VGSC Nav1.8 is an anti-tetrodotoxin sodium channel, the distribution of which is limited to primary afferent neurons, most of the afferent nerves containing Nav1.8 transmitting nociceptive signals to the pain management areas of the spinal cord. Changes in the expression, transport and redistribution of nav1.8 (encoded by PN 3) following inflammation or nerve injury are thought to be the major cause of afferent nerve sensitivity and pain production (see Schuelert and McDougall (2012) artritisres Ther 14: R5). Rodent models of osteoarthritis have demonstrated that inhibition of nav1.8 channels on peripheral nerves with synaptic connections in the spinal cord is a promising treatment for nociceptive processing and may contribute to achieving more pronounced and sustained analgesia. Thus, the methods and compositions of the invention can be used to treat chronic pain by reducing local expression of NAV1.8 by targeted degradation using the dTAG insertion strategy described herein.

Cancer may also be targeted as described herein. Cancer is a general term used to describe a number of specific diseases that are combined due to a lack of regulation of cell growth. Because of the many forms involved, countless different cell types, there are also many specific gene targets associated with cancer. For example, the clusterin encoded by the CLU gene (also known as apolipoprotein J) is a heterodimeric protein that is assembled after proteolytic cleavage into two chains of a primary polypeptide CLU gene product. In recent years, two forms of clusterin have been found, the secreted and highly glycosylated form (sCLU) and the nuclear form (nCLU), where nCLU is first synthesized as the pronuclear form (pnCLU) found in the cytoplasm. The difference between the two CLU forms is related to the optional concatenation of the CLU messages and the selection of the starting ATG during message conversion. Translation of sCLU utilizes the first AUG in the full-length CLU mRNA, while translation of pnCLU begins with the splice-dependent removal of the transcribed leader segment and the full-length mRNA of exon 1 followed by the second in-frame AUG. The sCLU form appears to promote cell survival, whereas the nCLU form is associated with apoptosis. Overexpression of the protein in the form of sCLU has been found in many tumor types, including prostate, skin, pancreatic, breast, lung and colon cancers, as well as esophageal squamous cell carcinoma and neuroblastoma. Furthermore, the progression of some cancer types to high-grade and metastatic forms leads to elevated levels of sCLU (Shannan et al (2006) Cell Death Dif 13: 12). Silent sCLU expression has been induced in phase I studies of breast and prostate cancer using specific antisense oligonucleotides (ASOs) in combination with standard therapy, with increased apoptosis observed only in patients receiving ASOs and standard therapeutics (Shannon, supra). Thus, the methods and compositions of the invention may be used to treat cancers marked with increased expression of sCLU by targeted degradation using the dTAG insertion strategy described herein.

Another protein that appears to have oncogenic effects is eukaryotic translation initiation factor 4E (eIF-4E). eIF3-4E binds to the M7GpppN cap of eukaryotic mRNA (where N is any nucleotide) and is the rate-limiting member of the formation of eIF-4F complexes. eIF-4E is normally complexed with eIF-4G in the eIF-4F complex, and under normal physiological conditions, the availability of eIF-4E is negatively regulated by the binding of a family of inhibitory proteins called 4E-BPs (used to sequester eIF-4E from eIF-4G). Since eIF-4E is typically expressed at low levels, mRNA competes for available eIF-4E to be translated. Mrnas with short unstructured 5' UTRs are considered more competitive for translation because they are less dependent on unwinding activity found in the eIF-4F complex. Highly structured mRNAs are then more dependent on the translational binding of eIF-4E, and therefore these mRNAs are more readily translated when eIF3-4E is overexpressed. Growth promoting gene products such as cyclin D1, VEGF, c-myc, FGF2, heparanase, ODC and MMP9 possess these complex 5' UTRs (Mamane et al (2004) Oncogene 23:3172, Fischer (2009) Cell Cycle 8(16): 2535). In addition, eIF-4E may play a role in the modification of nuclear pore complexes and lead to an increase in the transport of these same mrnas into the cytoplasm (Culjikovic-Kraljacic et al, (2012) Cell Reports 2 p.207). eIF-4E is associated with oncogenic cell transformation and is overexpressed in several cancer types, including acute myeloid leukemia, colon cancer, breast cancer, bladder cancer, lung cancer, prostate cancer, gastrointestinal tract cancer, head and neck cancer, hodgkin's lymphoma and neuroblastoma, and elevated levels and disease levels are increasing. Targeting eIF-4E has been attempted by several different approaches, including over-expression of 4E-BP and peptides derived therefrom, development of small molecule inhibitors for preventing eIF-4E: eIFG interactions, and eIF-4E-specific antisense oligonucleotides (ASO) (Jia et al (2012) Med Res Rev 00, No.00: 1-29). ASO administration has demonstrated knockdown of eIF-4E expression in tumor cells in vitro, as well as expression in xenograft tumors in mouse models in vivo. In these mouse models, expression levels of eIF-4E were reduced by 80% without any reduction in total protein translation and without any significant toxicity, while increasing chemosensitivity to chemotherapeutic agents, increasing cancer cell apoptosis and inhibiting tumor growth (Jia, supra). Thus, the methods and compositions of the present invention can be used to treat or prevent various cancers. Expression of eIF-4F can be modulated by degradation using the dTAG insertion strategy described herein.

Vascular endothelial receptors (VEGF), which act via the receptor VEGFR, play a role in normal development and also play a role in pathological angiogenesis in cancer. In humans, there are five different VEGF family members: VEGF-A (also known as VEGF); placental growth factor (PIGF), VEGF-B, VEGF-C, and VEGF-D. VEGF-A also has three common subtypes: VEGF-121, VEGF-165 and VEGF-189. The various VEGF's have different roles in angiogenesis, with VEGF-A primarily involved in normal angiogenesis as well as tumor growth and metastasis, and VEGF-C and VEGF-D involved in normal lymphangiogenesis and malignant lymph node metastasis. Furthermore, the VEGF-A subtype may also have specific growth promoting activity in hormone-responsive tumors. Based on this knowledge, many antibodies and small molecule kinase inhibitors either directly inhibit the VEGF-VEGFR interaction or activate signal transduction pathways through the interaction. However, these therapeutic agents often have significant and potentially troublesome side effect profiles, such that active research is being conducted to develop inhibitors with increased specificity (Shibuya, (2014) Biomol Ther 11(1): 1-9). Thus, the methods and compositions of the invention can be used to prevent or treat cancer in a subject by degrading a targeted specific VEGF protein using the dTAG insertion strategy described herein.

Another protein that plays a role in several cancers is the Kinesin Spindle Protein (KSP) encoded by KIF11 gene. The most successful anticancer therapies currently in use target microtubules, with these agents having been used to treat breast, lung, ovarian, bladder and head and neck cancers. Microtubules are part of the mitotic spindle and thus targeting them successfully inhibits rapidly dividing cancer cells, but microtubules are also part of the cytoskeleton and thus treatment with these agents is also associated with serious side effects. Kinesins, particularly kinesin spindle protein, is a motor protein that binds to spindle fibers and serves to force the spindle fibers apart during chromosome segregation in cell division. Thus, targeting KSP using KSP-specific antimitotic agents will target only dividing cells and may have fewer side effects. Agents that consume KSP selectively cause cell cycle arrest in mitosis, which leads to apoptosis over a long period of time. KSP is also abundant in dividing tissues and highly expressed in tumors of the breast, colon, lung, ovary and uterus (Sarli and Gianis, (2008) Clincancer Res 14: 7583). In addition, clinical trials are underway using RNA interference that targets both KSP and VEGF in liver-affected Cancer patients (Tabernero et al, (2013) Cancer Discovery 3: 406). Thus, the methods and compositions of the invention may be used to treat or prevent cancer by targeted degradation of Kinesin Spindle Protein (KSP) using the dTAG insertion strategy described herein.

Heat shock protein 27(HSP 27, also known as heat shock protein beta-1 or HSPB1) is another cancer-associated protein. HSP 27 encoded by the HSPB1 gene is a heat shock protein initially characterized as a small chaperone protein that promotes proper refolding of damaged proteins in response to heat shock. However, ongoing investigations show that it is also involved in responses to cellular stress conditions, such as oxidative stress and chemical stress, appears to have anti-apoptotic activity, and is able to regulate actin cytoskeleton dynamics (vidyagar) et al (2012) fiber Tis Rep 5(7) under heat shock and other stress conditions. Furthermore, inhibition of HSP 27 may play a role in long-term dormancy of cancer, as studies indicate that HSP 27 is upregulated in angiogenic breast cancer cells, and that inhibition of HSP 27 in vivo results in long-term tumor dormancy (Straume et al (2012) Proc Natl Acad Sci USA 109(22): 8699-. Increased expression of heat shock proteins in tumor cells has been associated with loss of p53 function and upregulation of proto-oncogenes such as c-myc. The anti-apoptotic activity of HSP 27 protects tumor cells and has been shown to be associated with chemotherapy resistance in breast cancer and leukemia (Vidysagar, supra). Thus, HSP 27 may be a suitable target for cancer therapy, where inhibitors of proteins may be used in combination with known chemotherapies to enhance their activity. The HSP 27 inhibitor quercetin has been shown to significantly reduce tumor volume in vivo when combined with conventional chemotherapeutic agents, compared to the agent alone. In addition, HSP 27-inhibitory ASO is currently being evaluated in clinical studies of lung, ovarian, breast and pancreatic cancer (vidyagaar, supra). Thus, the methods and compositions of the invention may be used to treat cancer by inhibiting HSP 27 expression through targeted degradation of HSP 27 using the dTAG insertion strategy described herein.

Several kinases have been the target of research in anticancer therapy because they are often key regulators of cell growth. However, downstream of the signaling pathway, the role of mutant kinases is often found in the upregulation of signal transduction and activation factors of the STAT3 gene-encoded transcription 3 protein or STAT 3. In addition, it appears that both hepatitis b and hepatitis c activate Stat3, both of which are associated with the development of liver cancer. Thus, HepB and HepC viruses may disrupt the Stat3 signaling pathway and promote hepatocyte transformation (Li et al, (2006) Clin Cancer Res 12(23): 7140).

RAS proteins are a class of proteins that play a role in cell differentiation, proliferation and survival. Various members of the RAS protein family have been implicated in cancer, as aberrant RAS signaling has been found to play a role in approximately 30% of all cancers. The KRAS protein (also known as V-Ki-ras 2 Kirsten rat sarcoma virus oncogene homolog) is a GTPase that plays an essential role in normal tissue signaling. KRAS is an attractive cancer target because frequent point mutations in the KRAS gene render the protein constitutively active. Thus, KRAS may be a suitable target for cancer therapy, where small molecules targeting KRAS protein function may be used for therapeutic advantages, including in combination with known chemotherapies to enhance their activity. In one embodiment, the methods and compositions of the invention can be used to treat cancer by modulating KRAS expression by targeted degradation of KRAS using the dTAG insertion strategy described herein.

All of the various Stat proteins are transcription factors, primarily mediating signaling of cytokine and growth factor receptors. For example, IL6 and IL11 bind to their respective receptor subunits and trigger homodimerization of gp130, a transmembrane receptor that triggers Stat3 activation. Upon activation via phosphorylation of growth factor receptors, STAT3 protein dimerizes and passes through the nucleus and binds to DNA in a sequence-specific manner, upregulating many genes involved in cell proliferation. Various types of tumor cells often have kinase mutations that result in overexpression of STAT3, and thus regardless of each specific mutant kinase, a reduction in STAT3 expression may be beneficial for cancers of diverse origin (Jarnicki et al (2010) Cell Div 5: 14). Stat3 leads to malignancy through several mechanisms. It inhibits apoptosis by up-regulating pro-survival/anti-apoptotic Bcl2 protein and promotes proliferation primarily by stimulating the expression of cyclin B1, cdc2, c-myc, VEGF, H1F1 α and cyclin D1 and by inhibiting the cell cycle inhibitor p 21. Stat3 also promotes tumor metastasis by inducing extracellular matrix-degrading metalloproteinases (including MMP-2 and MMP-9). In the normal physiological state, Stat3 function is inhibited by the transcriptional inhibitor Socs3, which is normally induced by Stat3 to maintain growth balance in the cell. However, in malignant cells Stat3 overexpression can overcome the Socs3 inhibition. Thus, the methods and compositions of the invention can be used to inhibit Stat3 function and prevent or treat cancer by targeting the degradation of Stat3 using the dTAG insertion strategy described herein.

Prostate cancer (PCa) is an androgen-dependent disease that remains one of the leading causes of death in the united states and is the leading cause of cancer death in men. Although several studies have been performed, indicating that up to 42% of prostate cancer cases have genetic links (Mazaris and Tsorras (2013) Nephro Urol Mon 5(3):792-800), several types of inheritance patterns (e.g., X-linked, autosomal dominant inheritance, autosomal recessive inheritance) have been observed, indicating that no single gene or gene mutation leads to the inheritance of PCa. The cancer is dependent on the activity of the androgen receptor on growth and progression (Mahmoud et al, (2013) PLoS One 8(10): e 78479). In general, PCa can be a slowly progressive disease that can be treated using fairly conservative approaches, but in about 25 to 30% of cases, cancer can be an aggressive cancer that leads to patient death. In the case of metastatic disease, 70 to 80% of patients initially respond to androgen deprivation therapy, but at a later stage, tumors become hormone refractory and more aggressive, leading to a worsening prognosis (Mazaris and Tsiotras, supra). Hormone-refractory PCa is independent of circulating androgens, but rather is aided by AR amplification, growth factor dysregulation and AR co-amplification. In addition, mutations in the AR ligand binding domain may result in AR sensitivity to very low circulating androgen levels or to an extended group of ligands such as estrogen, progestin, epinephrine steroids, and antiandrogens. Although cancer is dependent on the activity of the AR, tumor cells that undergo these types of mutations in the AR ligand binding domain may no longer be sensitive to anti-androgen therapy. Generally, AR is present in the cytoplasm and bound by heat shock proteins to prevent its activation. Upon exposure to androgens, receptors are able to dimerize and enter the nucleus to promote expression of several growth-related genes. Thus, the methods and compositions of the present invention can be used to target the degradation of the androgen receptor by using the dTAG insertion strategy described herein to treat PCa at all stages.

Genomic in-frame insertion of dTAG

As described above, the method of the present invention is based on the in-frame insertion of a gene expressing an endogenous protein of interest into the dTAG genome. As contemplated herein, expression of a nucleic acid sequence inserted in 5 '-or 3' frame with a nucleic acid sequence encoding dTAG results in an endogenous protein-dTAG hybrid protein that is degraded by the target upon administration of a particular heterobifunctional compound.

In-frame insertion of a nucleic acid sequence encoding dTAG may be performed or effected by any known and effective genome editing method. In one aspect, the present invention utilizes the CRISPR-Cas9 system to generate knock-in endogenous protein-dTAG fusion proteins that are produced by endogenous loci and are susceptible to degradation in a ligand-dependent, reversible, and dose-responsive manner. In certain embodiments, the CRISPR-Cas9 system is used to insert an expression cassette for dTAG that is present in a Homologous Recombination (HR) "donor" sequence, wherein during homologous recombination following CRISPR-Cas internucleation, the dTAG nucleic acid sequence serves as a "donor" sequence for insertion into the genomic locus of the protein of interest. The HR targeting vector contains homology arms at the 5 'and 3' ends of the expression cassette that are homologous to the genomic DNA surrounding the targeted gene of interest. By fusing a nucleic acid sequence encoding dTAG to a target gene of interest, the resulting fusion protein contains dTAG targeted by a heterobifunctional compound.

The present invention provides an exogenous dTAG sequence (also referred to as a "donor sequence" or "donor" or "transgene") inserted into the target gene frame of interest, and the resulting fusion protein contains a dTAG targeted by a heterobifunctional compound. It will be apparent that the donor sequence need not be identical to the genomic sequence in which it is placed. The donor sequence may contain a non-homologous sequence flanking both homologous regions to allow for efficient HR at the location of interest. In addition, the donor sequence may comprise a vector molecule comprising a sequence that is not homologous to a region of interest in cellular chromatin. The donor molecule may contain several discrete regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in the region of interest, e.g., the dTAG of the invention, the sequences may be present in the donor nucleic acid molecule and flank a region of homology to the sequence of the region of interest. Alternatively, the donor molecule may be integrated into the cleaved target locus by a non-homologous end joining (NHEJ) mechanism. See, e.g., U.S.2007/0207221 and U.S.2007/0326645, which are incorporated herein by reference.

The donor dTAG coding sequence for insertion may be DNA or RNA, single and/or double stranded, and may be introduced into the cell in linear or circular form. See, e.g., U.S.2010/0047805, U.S.2007/0281361, and 2011/0207221, which are incorporated herein by reference. The donor sequence may be introduced into the cell in a circular or linear form. If introduced in a linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues are added to the 3' end of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al, Proc.Natl.Acad.Sci.84, (1987): 4959-. Other methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups and the use of modified internucleotide linkages, such as phosphorothioate, phosphoramidate, and O-methyl ribose or deoxyribose residues.

The donor polynucleotide encoding dTAG can be introduced into the cell as part of a vector molecule with additional sequences, such as CRISPR-Cas sequences, an origin of replication, a promoter and a gene encoding antibiotic resistance. In addition, the donor polynucleotide can be introduced as a naked nucleic acid, as a nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by a virus (e.g., adenovirus, AAV, herpes virus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)).

The present invention utilizes well characterized insertion strategies, such as the CRISPR-Cas9 system. Generally, a "CRISPR system" is collectively referred to as a transcript and other elements involved in expressing or directing CRISPR-associated ("Cas") gene activity, including sequences encoding the Cas gene, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portions of tracrRNA), tracr mate sequences (encompassing "direct repeats" in the context of endogenous CRISPR systems and partial direct repeats of tracrRNA processing), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and/or other sequences and transcripts from CRISPR loci. (see, e.g., Ruan, J.et., high effective CRISPR/Cas 9-mediated transgene knockin at the H11 logs In pigs. "Sci.Rep.5, (2015): 14253; and Park A, Won ST, Pentecost M, Bartkowski W, and Lee B," CRISPR/Cas9 AllowseEffecient and Complete Knock-In of a Destabilization Domain-Tagged expression Protein a Human Cell Line, Allowird Rapid Knock down of Protein function, "PLoS ONE 9(4) (2014): e 01, both associated expressed by tires reference, all incorporated herein by reference).

Cas nucleases are well known molecules. For example, the protein sequence encoded by the Cas-9 nuclease gene can be found in the SwissProt database under accession number Q99ZW 2- (seq. id No.:52):

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYP TIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEIT KAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFI ERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLF EDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPEN IVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVEN TQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSF LKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNA KLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESI LPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGK SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLP KYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYE KLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDR KRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD。

in some embodiments, a CRISPR/Cas nuclease or CRISPR/Cas nuclease system comprises a non-coding RNA molecule (guide) RNA that specifically binds DNA with sequence specificity that has nuclease function (e.g., two nuclease domains) and a Cas protein (e.g., Cas 9). Also included are donor nucleotides encoding dTAG for in-frame insertion into the genomic locus of the protein of interest.

In some embodiments, one or more elements of the CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as streptococcus pyogenes.

In some embodiments, a Cas nuclease and a gRNA (including a fusion of a crRNA specific for a target sequence and an immobilized tracrRNA) and a donor sequence encoding dTAG are introduced into a cell. Typically, a target site at the 5' end of the gRNA targets the Cas nuclease to a target site, e.g., a gene, using complementary base pairing. In some embodiments, the target site is selected based on its position 5' to a Protospacer Adjacent Motif (PAM) sequence, e.g., typically NGG or NAG. In this regard, the gRNA is targeted to a desired sequence by modifying the first 20 nucleotides of the guide RNA to correspond to the target DNA sequence.

In some embodiments, the CRISPR system induces DSBs at a target site and then homologously recombines a donor sequence encoding dTAG into the genomic locus of a protein of interest, as discussed herein. In other embodiments, Cas9 variants of "nickases" (nickases) are thought to be useful for cleaving single strands at target sites. In some aspects, pairs of nicking enzymes are used, e.g., to improve specificity, each guided by a pair of different grnas of the targeting sequence, such that when nicks are introduced simultaneously, 5' overhangs are introduced.

In general, CRISPR systems are characterized by elements that promote CRISPR complex formation at target sequence sites. Typically, in the context of forming a CRISPR complex, a "target sequence" is typically a sequence for which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes formation of the CRISPR complex, and wherein insertion of a donor sequence encoding dTAG occurs. Complete complementarity is not necessarily required if sufficient complementarity exists to cause hybridization and promote formation of the CRISPR complex.

Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., from within 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 50 or more base pairs) the target sequence. Without wishing to be bound by theory, the tracr sequence may comprise or consist of all or part of a wild-type tracr sequence (e.g., a wild-type tracr sequence of about or greater than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides) may also form part of a CRISPR complex, for example, by hybridizing along at least a portion of the tracr sequence to all or part of a tracr mate sequence operably linked to a guide sequence. In some embodiments, the tracr sequence is sufficiently complementary to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex.

As with the target sequence, in some embodiments, complete complementarity is not necessarily required. In some embodiments, the tracr sequences have at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequences when optimally aligned. In some embodiments, one or more vectors that drive expression of one or more elements of the CRISPR system are introduced into a cell such that expression of the elements of the CRISPR system directs formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence may each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed by the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not comprised in the first vector. In some embodiments, the CRISPR system elements combined in a single vector may be arranged in any suitable orientation, for example, at the 5 'position relative to the ("upstream") or at the 3' position relative to the ("downstream") second component. The coding sequence of one element may be located on the same or opposite strand and oriented in the same or opposite direction as the coding sequence of the second element. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more guide sequences, tracr mate sequences (optionally operably linked to a guide sequence) and tracr sequences embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence and tracr sequence are operably linked and expressed from the same promoter.

In some embodiments, the vector comprises a regulatory element operably linked to an enzyme coding sequence encoding an CRISPR RNA-directed endonuclease. In some embodiments, the vector comprises a regulatory element, such as a Cas protein, operably linked to an enzyme coding sequence encoding a CRISPR enzyme. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as CsnI and Csx12), Cas10, Csyl, Csy2, Csy3, csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Cmr 2, Csx 2, Csb2, Csx 2, CsaX 2, Csx 36x 2, csxf 36f 2, Csfl 36f 72, csxf 2, cspf, cscp 2, cspf 2, by way of which is incorporated herein by a homolog. These enzymes are known; for example, the amino acid sequence of the streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2 (which is incorporated herein by reference).

Cas proteins typically comprise at least one RNA recognition or binding domain. Such domains can interact with guide RNAs (grnas, described in more detail below). The Cas protein may also comprise nuclease domains, such as endonuclease domains (e.g., DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. The nuclease domain has catalytic activity for nucleic acid cleavage. Cleavage involves the breaking of covalent bonds of the nucleic acid molecule. The cleavage may produce blunt ends or staggered ends, and it may be single-stranded or double-stranded.

Examples of Cas proteins include Cas, Cas1, Cas5 (CasE d), Cas6, Cas8a, Cas8, Cas (CsnI or Csx), Cas10, CasE, Csyl, Csy, csei (CasE a), Cse (CasE b), Cse (CasE e), Cse (CasE c), Cscl, Csc, Csa, Csn, Csm, cmm, Cmr, Csbl, Csb, Csx, 2015x, Csx, Csf, and csl 966, and homologs or modified forms thereof (see WO/, which is incorporated herein by reference).

Any Cas protein that induces cleavage or double strand cleavage into the desired recognition site can be used in the methods and compositions disclosed herein.

In general, a guide sequence is any polynucleotide sequence that is sufficiently complementary to a target polynucleotide sequence to hybridize to the target sequence and specifically bind the CRISPR complex to the target sequence direct sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm.

The optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on Burrows-Wheeler transformations (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (novo technologies, ELAND (illuma, San Diego, Calif.), SOAP (available at SOAP. genetics. org. cn) and Maq (available at maq. sourceforce. net.) in some embodiments, the guide sequence has a length of about or greater than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 57, 45, 25, 35, 40, 45, 25, 45, 75, or less of the target sequence may be assessed by determining the length of the guide sequence from a suitable guide sequence The ability to bind. For example, components of the CRISPR system sufficient to form a CRISPR complex, including a guide sequence to be sequenced, can be provided to cells having the corresponding target sequence, e.g., by transfection with a vector encoding the CRISPR sequence components, and then assessing preferential cleavage within the target sequence, e.g., by the surfyor assay described herein. Similarly, cleavage of a target polynucleotide sequence in a test tube can be assessed by providing the target sequence, a component of the CRISPR complex (comprising the guide sequence to be sequenced and a control guide sequence different from the test guide sequence), and comparing the rate of binding or cleavage at the target sequence between the test and control guide sequence reactions.

The leader sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of the cell, particularly a protein of interest targeted for controlled degradation by engineering an endogenous protein-dTAG hybrid. Exemplary target sequences include target sequences unique in the target genome that provide for insertion of a dTAG donor nucleic acid in an in-frame orientation. In some embodiments, the leader sequence is selected to reduce the extent of secondary structure within the leader sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

Typically, a tracr mate sequence includes any sequence having sufficient complementarity to a tracr sequence to facilitate one or more of: (1) excision of the leader sequence flanking the tracr mate sequence in cells containing the corresponding tracr sequence; (2) forming a CRISPR complex on the target sequence, wherein the CRISPR complex comprises a tracr mate sequence that hybridizes to a tracr sequence. Typically, the degree of complementarity is referenced to the tracr mate sequence and the tracr sequence, optimally aligned along the length of the shorter of the two sequences.

As contemplated herein, the CRISPR-Cas system is used to insert a nucleic acid sequence encoding dTAG in-frame with a genomic sequence encoding a protein of interest into a eukaryote, such as a human cell. In some embodiments, the method comprises binding a CRISPR complex to a genomic sequence of a target protein of interest to effect cleavage of the genomic sequence, wherein the CRISPR complex comprises a CRISPR enzyme complexed to a guide sequence that hybridizes to an intratarget sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence that in turn hybridizes to a tracr sequence.

In some aspects, the method comprises modifying expression of the polynucleotide in the eukaryotic cell by introducing a nucleic acid encoding dTAG.

In some aspects, the polypeptide of the CRISPR-Cas system and the donor sequence are administered or introduced into a cell. The nucleic acid is typically administered in the form of an expression vector, such as a viral expression vector. In some aspects, the expression vector is a retroviral expression vector, an adenoviral expression vector, a DNA plasmid expression vector, or an AAV expression vector. In some aspects, one or more polynucleotides encoding a CRISPR-Cas system and a donor sequence delivered to a cell. In some aspects, the delivery is by delivering more than one vector.

Methods of delivering nucleic acid sequences to cells as described herein are described, for example, in U.S. patent nos. 8,586,526; 6,453,242; 6,503,717, respectively; 6,534,261; 6,599,692, respectively; 6,607,882, respectively; 6,689,558, respectively; 6,824,978, respectively; 6,933,113, respectively; 6,979,539, respectively; 7,013,219, respectively; and 7,163,824, the disclosures of all of which are incorporated herein by reference in their entirety.

Various polynucleotides as described herein can also be delivered using a vector containing sequences encoding one or more of the compositions described herein. Any vector system may be used, including but not limited to plasmid vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, poxvirus vectors; herpes viral vectors and adeno-associated viral vectors and the like are also described in U.S. Pat. nos. 6,534,261; 6,607,882, respectively; 6,824,978, respectively; 6,933,113, respectively; 6,979,539, respectively; 7,013,219, respectively; and 7,163,824, which are incorporated herein by reference in their entirety.

Methods for non-viral delivery of nucleic acids include lipofection, nuclear transfection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, artificial viral particles and agents of DNA enhance uptake. Lipofection (described, e.g., in U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) lipofection reagents are commercially available (e.g., Transfectam)^TMAnd Lipofectin^TM). Useful receptor-recognizing lipid-transfected cationic and neutral lipids for polynucleotides include those possessed by Feigner, WO 1991/17424, and WO 1991/16024. Delivery may be to a cell (e.g., in vitro or ex vivo administration) or to a target tissue (e.g., in vivo administration).

In some embodiments, delivery is by using RNA or DNA virus based system to deliver nucleic acids. The viral vectors of some aspects may be administered directly to the patient (in vivo), or they may be used to treat cells in vitro or ex vivo, and then administered to the patient. In some embodiments, virus-based systems include retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. The tropism of retroviruses can be altered by incorporation of foreign envelope proteins, thereby expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and generally producing high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with a packaging capacity of up to 6 to 10kb of exogenous sequences. The minimal cis-acting LTR is sufficient for replication and packaging of the vector, which is then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include vectors based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency Virus (SIV), Human Immunodeficiency Virus (HIV) and combinations thereof (see, e.g., Buchscher et al, J.Virol.66, (1992): 2731-.

In applications where transient expression is preferred, an adenovirus-based system may be used. Adenovirus-based vectors have very high transduction efficiency in many cell types and do not require cell division. Using such vectors, high titers and high levels of expression have been obtained. The carrier can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also used for transduction of cells with target nucleic acids, for example, in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo Gene Therapy programs (see, e.g., West et al, Virology 160 (1987): 38-47; U.S. Pat. No. 4,797,368; WO 1993/24641; Kotin, Human Gene Therapy 5, (1994): 793. Puff 801; Muzyczka, J.Clin.invest.94, (1994): 1351. construction of recombinant AAV vectors is described in numerous publications, including U.S. Pat. No.5,173,414; Tratschin et al, mol.cell. biol.5, (1985): 3251. 3260; Trtschin, et al, mol.cell.biol.4, (1984): 2082. Puff 1; Herkamon & Muzyczyc, AS 81, (1984): 3266. J.3863; and Vilski et al.3828. 2079; 1989. J.3863).

At least six viral vector methods are currently available for gene transfer in clinical trials, which utilize methods involving the production of transducible agents by gene complementation of defective vectors by insertion into helper cell lines.

pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al, Blood 85, (1995): 3048-. PA317/pLASN is the first therapeutic vector used in gene therapy trials. (Blaese et al, Science 270 (1995): 475-. Transduction efficiencies of 50% or more have been observed for MFG-S packaging carriers. (Ellem et al, Immunol immunothers.44 (1), (1997): 10-20; and Dranoff et al, hum. Gene ther.1, (1997): 111-112).

Vectors suitable for introduction of the polynucleotides described herein also include non-integrating lentiviral vectors (IDLV). See, for example, Naldini et al Proc. Natl. Acad. Sci.93, (1996): 11382-; dull et al.J.Virol.72, (1998): 8463-; zuffery et al.J.Virol.72, (1998): 9873. about.9880; follenzi et al Nature Genetics 25, (2000): 217-222; and u.s.2009/0117617.

Recombinant adeno-associated viral vectors (rAAV) can also be used to deliver the compositions described herein. All vectors are derived from plasmids that retain only the AAV inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery are key features of this vector system. (Wagner et al, Lancet 351 (1998): 91171702-3, and Kearns et al, Gene Ther.9 (1996): 748-55). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9 and AAVrh10, pseudotyped AAV such as AAV2/8, AAV2/5 and AAV2/6 and all variants thereof, can also be used in accordance with the invention.

Replication-defective recombinant adenovirus vectors (Ad) can be produced at high titers and readily infect many different cell types. Most adenoviral vectors are engineered to replace the Ad E1a, E1b and/or E3 genes with transgenes; subsequently, the replication-defective vector is propagated in human 293 cells, which provide the deleted gene function in trans. Ad vectors can transduce various types of tissues in vivo, including non-dividing, differentiated cells, such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity. An example of the use of Ad vectors in clinical trials involves polynucleotide therapy for intramuscular injection of anti-tumor immunity (Sterman et al, hum. Gene Ther.7, (1998): 1083-. Other examples of gene transfer using adenoviral vectors in clinical trials include Rosenecker et al, Infectin 24(1), (1996): 5-10; sterman et al, hum. Gene Ther.9(7), (1998): 1083-; welsh et al, hum. Gene ther.2, (1995): 205-; alvarez et al, hum. Gene Ther.5, (1997): 597-; topf et al, Gene ther.5, (1998): 507-; sterman et al, hum. Gene Ther.7, (1998): 1083-.

The packaging cells are used to form viral particles capable of infecting host cells. These cells included 293 cells packaging adenovirus, which packaged retrovirus and ψ 2 cells or PA317 cells. Viral vectors for gene therapy are typically produced by production cell lines that package nucleic acid vectors into viral particles. The vector will usually contain the minimal viral sequences required for packaging and subsequent integration into the host (if applicable), the other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are provided in trans by the packaging cell line. For example, AAV vectors for gene therapy typically have only Inverted Terminal Repeat (ITR) sequences from the AAV genome that are required for packaging and integration into the host genome. Viral DNA is packaged in cell lines that contain helper plasmids encoding other AAV genes, i.e., rep and cap, but lack ITR sequences. Cell lines are also infected with adenovirus as a helper cell. Helper viruses facilitate replication of AAV vectors and expression of AAV genes from helper plasmids. Helper plasmids are not packaged in significant amounts due to the lack of ITR sequences. Contamination with adenovirus can be reduced by, for example, heat treatment in which adenovirus is more sensitive than AAV.

The vector can be delivered with a high degree of specificity for a particular tissue type. Thus, by expressing the ligand as a fusion protein with the viral coat protein on the outer surface of the virus, the viral vector can be modified to be specific for a given cell type. The ligand is selected to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al, Proc.Natl. Acad.Sci.92, (1995):9747-9751 reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and that the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptors. This principle can be extended to other virus-target cell pairs, where the target cell expresses a receptor, and the virus expresses a fusion protein comprising a ligand for a cell-surface receptor. For example, filamentous phages may be engineered to display antibody fragments (e.g., FAB or Fv) with specific binding affinity for virtually any selected cellular receptor. Although the above description applies primarily to viral vectors, the same principles apply to non-viral vectors. These vectors can be engineered to contain specific uptake sequences that facilitate uptake by specific target cells.

The carrier can be delivered in vivo by administration to an individual subject, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, intrathecal, intratracheal, subcutaneous or intracranial infusion) or topical administration, as described below. Alternatively, the vector may be delivered to cells ex vivo, such as cells removed from an individual patient (e.g., lymphocytes, bone marrow aspirate, tissue biopsy) or universal donor hematopoietic stem cells, which are then reimplanted into the patient, typically after selection of cells that have incorporated the vector.

Vectors containing nucleases and/or donor constructs (e.g., retroviruses, adenoviruses, liposomes, etc.) can also be administered directly to an organism to transduce cells in vivo. Alternatively, naked DNA may be administered. Administration is by any route commonly used to introduce molecules into ultimate contact with blood or tissue cells, including but not limited to injection, infusion, topical administration, and electroporation. Suitable methods of administering such nucleic acids are available and well known to those skilled in the art, and while more than one route may be used to administer a particular composition, a particular route may generally provide a more direct and more effective response than another route.

In some embodiments, the polypeptide of the CRISPR-Cas system is synthesized in situ in the cell as a result of introducing a polynucleotide encoding the polypeptide into the cell. In some aspects, the polypeptide of the CRISP-Cas system can be produced extracellularly and then introduced therein. As described herein, methods for introducing a CRISPR-Cas polynucleotide construct into an animal cell are known and include, as non-limiting examples, stable transformation methods in which the polynucleotide construct is integrated into the genome of the cell, transient transformation methods in which the polynucleotide construct is not integrated into the genome of the cell and virus-mediated methods. Preferably, the CRISPR-Cas polynucleotide is transiently expressed and does not integrate into the genome of the cell. In some embodiments, the CRISPR-Cas polynucleotide can be introduced into a cell by, for example, recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, and the like. For example, in some aspects, transient transformation methods include microinjection, electroporation, or particle bombardment. In some embodiments, the CRISPR-Cas polynucleotide can be comprised in a vector, more particularly a plasmid or a virus, in view of expression in a cell.

In some embodiments, non-CRISPR-CAS viruses and non-virus-based gene transfer methods can be used to insert a nucleic acid encoding dTAG in-frame at the genomic locus of a protein of interest in a mammalian cell or target tissue. These methods can be used to administer nucleic acids encoding ZFP, ZFN, TALE and/or TALEN system components to cells in culture, or to host organisms, including donor sequences encoding dTAG for in-frame insertion of a protein of interest into a genomic locus.

Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vectors, e.g., liposomes. Viral vector delivery systems include DNA and RNA viruses that have an episomal or integrated genome upon delivery to a cell. For a review of gene therapy programs, see Anderson, Science256 (1992): 808-; nabel & Feigner, TIBTECH 11, (1993):211 + 217; mitani & Caskey, TIBTECH 11, (1993): 162-166; TIBTECH 11, (1993) 167-; miller, Nature357, (1992): 455-460; van Brunt, Biotechnology 6(10), (1988): 1149-; vigne, reactive Neurology and Neuroscience 8, (1995): 35-36; kremer & Perricaudet, British Medical Bulletin 51(1), (1995): 31-44; and Yu et al, Gene Therapy 1, (1994): 13-26.

Lipid: preparation of nucleic acid complexes, including targeted liposomes, such as immunoliposome complexes, is well known to those skilled in the art (see, e.g., Crystal, Science 270, (1995):404- & 410; Blaese et al, Cancer Gene Ther.2, (1995):291- & 297; Behr et al, Bioconjugate chem.5, (1994) & 382- & 389; Remy et al, Bioconjugate chem.5, (1994) & 647- & 654; Gao et al, Gene Therapy 2, (1995) & 710- & 722; Ahmad et al, Cancer Res.52, (1992) & 4817- & 4820; and U.S. Pat. Nos. 4,186,183,4,217,344,4,235,871,4,261,975, 4,485,054,4,501,728,4,774, 028, 028,774, 028,028, 028, 028,028, and 4,946,787).

Additional delivery methods include the use of packaging the nucleic acid to be delivered into an EnGeneIC Delivery Vehicle (EDV). These EDVs are specifically delivered to target tissues using bispecific antibodies, where one arm of the antibody is specific for the target tissue and the other arm is specific for the EDV. The antibody brings the EDV to the surface of the target cell, and then the EDV is brought into the cell by endocytosis. Once inside the cell, the contents are released (see, MacDiarmid et al Nature Biotechnology 27(7), (2009): 643).

D. Heterobifunctional compounds

The present application includes the use of heterobifunctional compounds having (i) a moiety that binds to ubiquitin ligase and (ii) a targeting moiety that binds to dTAG that has been fused to an endogenous protein intended for ubiquitination and proteasome degradation. In one embodiment, the heterobifunctional compound binds to a mutated dTAG to be selective for the corresponding endogenous protein (i.e., the dTAG targeting ligand binds to dTAG, but does not significantly bind to naturally occurring (and in some embodiments, will not significantly bind to a mutated or variant protein expressed by the host)).

Strategies for selective targeting and degradation of proteins using the Ubiquitin Proteasome Pathway (UPP) have been used for post-translational control of protein function. The heterobifunctional compounds are composed of a target protein binding ligand and an E3 ubiquitin ligase ligand. Heterobifunctional compounds are able to induce proteasome-mediated degradation of selected proteins via recruitment to E3 ubiquitin ligase and subsequent ubiquitination. These drug-like molecules offer the possibility of reversible, dose-responsive, adjustable, time-controlled response to protein levels. An early description of this compound is provided in U.S. patent No. 7,041,298 entitled "protein targeting Pharmaceutical" filed by descales et al at 9.2000 and patented at 5.2006. Sakamoto et al (PNAS 98(15) (2001):8554-8559), entitled "PROTACS: Chimeric Molecules which Target Proteins to the Skp1-Cullin F BoxComplex for inactivation and Degradation," describes a heterobifunctional compound consisting of a small molecule conjugate of MAP-AP-2 linked to a peptide capable of binding F-box protein β -TRCP, the disclosure of which is also provided in U.S. Pat. No. 3,7,041,298. The publication by Sakamoto et al (Molecular and cellular Proteomics 2(2003):1350-1358), entitled "Development of PROTACS to Target Cancer-promoting Proteins for inactivation and Degradation," describes a similar heterobifunctional compound (PROTAC2) that degrades not MAP-AP-2 but estrogen and androgen receptors. The Schneekloth et al publication (JACS 126 (2004):3748-3754), entitled "Chemical Genetic Control of protein Levels: selected in vivo Targeted differentiation", describes a similar heterobifunctional compound (PROTAC3) targeting the FK 506-binding protein (FKBP12) and shows that PROTAC2 and PROTAC3 target each with Green Fluorescent Protein (GFP) imaging. The publication by Schneekloth et al (ChemBiochem 6(2005)40-46), entitled "Chemical applications to control Intracellular protein degradation", describes the state of the art at that time using this technology. Schneekloth et al (BMCL 18(22) (2008): 5904-. WO2013/170147 to Crews et al, entitled "Compounds used for protein Degradation and Methods used Same", describes Compounds comprising a protein degrading moiety covalently bound to a linker, wherein the ClogP of the compound is equal to or higher than 1.5. A review of the above publications by Buckley et al (Angew. chem. int. Ed.53 (2014): 2312. 2330) is entitled "Small-molecular Control of intracellular Protein Levels through Modulation of the Ubiquitin Protein System". WO 2015/160845, entitled "Imide Based modules of proteins and Associated methods of Use method", assigned to Arvinas Inc., describes the Use of cerebrospinal fluid as an E3 ligase protein using the degradation determining region technique and thalidomide. The following publication by Lu et al (Chemistry and biol.22(6) (2015):755-763), entitled "Hijkaking the E3 Ubiquitin ligand peptide binding to Target BDR 4", similarly describes thalidomide-based compounds for degradation of BDR 4. Other publications describing this technology include Bondeson et al (Nature Chemical Biology 11(2015) 611-617), Gustafson et al (Angel. chem. int. Ed.54(2015):9659-9662), Buckley et al (ACSChem. Bio.10(2015):1831-1837), US 2016/0058872 assigned to Arvinas Inc., entitled "ImadeBased/models of Proteins and Association of Methods of Use", US 2016/0045607 assigned to Arvinas Inc., entitled "Etrogen-related Receptor Alpha Based Proteins and Associated Methods of Use", Yendon et al, Langeru and Cambridge engineering Inc. Biotechnology of Proteins and International publication No. 3. And.12. and International publication No. And.32. And.12. and International publication No. And.9-upright et al, (19732. And.9) and No. And.12. And.9. And.12. And.D.D.3. and U.D.D. And.D.D.D.D.D. And.3, U.D. And.D.D.D.M. And.D.D.M. And.3, and U.D.D.D.D.D.D.D. And.M. And.D.D. And.3, No. And.M. And.D.M. And.M. And.D.M. And.M. No. And.M. No. And.M. And.D. And. And.M The name is "Methods to index the targeted protein Degradation by the product of the Bifunctional Molecules".

Other descriptions of targeted Protein Degradation techniques include Itoh et al (JACS 132(16) (2010) (5820); 5826), entitled "Protein Hockdown Using Methyl Bestatin-Ligand hybrids: Design and Synthesis of indicators of inactivation-catalysis-Mediated Degradation of cellular Retinol Acid-Binding Proteins," which describes small Molecules linked to peptides that degrade Retinoic Acid Binding Proteins Using E3 ubiquitin ligase, Winter et al (Science 348(2015):1376-1381), entitled "Protein Conjugation as a Protein for in vivo Target Degradation," which describes a thalidomide-based targeted Protein Degradation technique.

The heterobifunctional compound useful in the present invention may be any heterobifunctional compound capable of binding to dTAG to induce degradation. Heterobifunctional compounds are generally known in the art, see, for example, U.S. patent 7,041,298; sakamoto et al (PNAS,2001, 98(15): 8554-8559); sakamoto et al (Molecular and cellular Proteomics 2(2003) 1350-; schneekloth et al (JACS 126 (2004): 3748-; schneekloth et al (ChemBiochem 6(2005): 40-46); schneekloth et al (BMCL 18(22) (2008: 5904-) -5908); WO 2013/170147; buckley et al (Angew. chem. int. Ed.53 (2014): 2312-2330); WO 2015/160845; lu et al (Chemistry and biol.22(6) (2015): 755-); bondeson et al (Nature Chemical Biology 11(2015): 611-617); gustafson et al (Angew. chem. int. Ed.54(2015): 9659-; buckley et al (ACS chem.Bio.10(2015): 1831-1837); U.S.2016/0058872, entitled "inside base modules of Proteins and Associated Methods of Use", assigned to U.S.2016/0045607, entitled "advanced-related Receptor Alpha Based promoters and Associated Methods of Use", assigned to Yale University, GlaxoSmithKline, and U.S.2014/0356322 Associated to Cambridge Enterprise Limited University of Cambridge, entitled "ingredients and Methods for enhanced delivery of Proteins & Associated Polypeptides E3 b.K., assigned to molecular weight analysis, assigned to molecular weight 2016 (passage 2016. express) and located in biological sample production," (III) assigned to molecular weight analysis and biological sample of Cambridge); toure et al (Angew. chem. int. Ed.55(2016): 1966-1973); itoh et al (JACS 132(16) (2010) 5820-); and Winter et al (Science 348(2015):1376-1381), each of which is incorporated herein by reference.

In general, heterobifunctional compounds suitable for use in the present application have the following general structure:

degradation determining region-linker-dTAG targeting ligand

Wherein the linker is covalently bound to a degradation determining region and a dTAG targeting ligand, the degradation determining region being a compound capable of binding to a ubiquitin ligase such as E3 ubiquitin ligase (e.g., cereblon), and the dTAG targeting ligand being capable of binding to dTAG on an endogenous protein-dTAG hybrid protein.

In certain embodiments, the application uses a compound of formula I or formula II:

wherein:

the linker is a group covalently bound to the dTAG targeting ligand and Y; and

the dTAG targeting ligand is capable of binding to or being bound by the dTAG target, thereby allowing tagging to occur.

In certain embodiments, the present application provides a compound of formula (I), or an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt thereof,

wherein:

linker (L) is a group covalently bound to dTAG targeting ligand and Y; and

the dTAG targeting ligand can bind to a dTAG targeting protein (binding to) or bind to a dTAG targeting protein (bindings to);

wherein X1, X2, Y, R₁，R₂，R₂’，R₃，R₃’，R₄，R₅And m and n are each as defined herein.

In certain embodiments, the present application provides a compound of formula (II), or an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt thereof,

wherein:

the linker is a group covalently bound to the dTAG targeting ligand and Y; and

the dTAG targeting ligand is capable of binding to the target protein (binding to) or binding to;

wherein X1, X2, Y, R₁，R₂，R₂’，R₃，R₃’，R₄，R₅And m and n are each as defined herein.

In certain embodiments, the invention employs compounds of formula III, formula IV, formula V, formula VI, formula VII, formula VIII and formula IX:

wherein:

linker (L) is a group covalently bound to dTAG targeting ligand and Z2;

the dTAG targeting ligand is capable of binding to or being bound by the target dTAG;

Z₂is a bond, alkyl, -O, -C (O) NR₂，-NR₆C (O), -NH or-NR₆；

R₆Is H, alkyl, -C (O) alkyl or-C (O) H;

X₃independently selected from O, S and CH₂；

W₂Independently selected from CH₂，CHR，C＝O，SO₂NH and N-alkyl;

Y₂independently selected from the group consisting of NH, N-alkyl, N-aryl, N-heteroaryl, N-cycloalkyl, N-heterocyclyl, O and S;

g and G 'are independently selected from H, alkyl optionally substituted with R', OH, CH₂-heterocyclyl, and benzyl optionally substituted with R';

Q₁，Q₂，Q₃and Q₄Independently selected from CH, N, CR' and N-oxide.

A₂Independently selected from alkyl, cycloalkyl, Cl and F;

R⁷selected from: -CONR 'R', -OR ', -NR' R ', -SR', -SO₂R′，—SO₂NR ' R ', -CR ' NR ' R ', -aryl, -heteroaryl, -alkyl, -cycloalkyl, -heterocyclyl, -P (O) (OR ') R ', -P(O)R′R″，—OP(O)(OR′)R″，—OP(O)R′R″，—Cl，—F， —Br，—I，—CF₃，—CN，—NR′SO₂NR′R″，—NR′CONR′ R″，—CONR′COR″，—NR′C(═N—CN)NR′R″，—C(═ N—CN)NR′R″，—NR′C(═N—CN)R″，—NR′C(═C— NO₂)NR′R″，—SO₂NR′COR″，—NO₂，—CO₂R′，—C(C═ N—OR′)R″,—CR′═CR′R″，—CCR′，—S(C═O)(C═N— R′)R″，—SF₅and-OCF₃；

R 'and R' are independently selected from the group consisting of a bond, H, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl.

Non-limiting examples of dTAG targeting ligands for use in the present invention include:

and

in some embodiments, the dTAG targeting ligand targets a mutated endogenous target or a non-endogenous target.

Degradation determining region

The degradation determining region is the moiety of a compound that links dTAG to ubiquitin ligase via a linker and a dTAG targeting ligand for proteasomal degradation. In certain embodiments, the binding to or binding to ubiquitin ligase by the binding domain is reduced. In a further embodiment, the degradation determining region is a compound that binds to E3 ubiquitin ligase. In a further embodiment, the degradation determining region is a compound that binds to cereblon. In a further embodiment, the degradation determining region is thalidomide or a derivative or analog thereof.

In certain embodiments, the degradation determining region is a moiety of formula D, formula D0, or formula D':

or an enantiomer, diastereomer, or stereoisomer thereof, wherein:

is that

Y is a bond (CH)₂)_1-6，(CH₂)_0-6-O，(CH₂)_0-6-C(O)NR₂’，(CH₂)_0-6- NR₂’C(O)，(CH₂)_0-6-NH, or (CH)₂)_0-6-NR₂；

X is C (O) or C (R)₃)₂；

X₁-X₂Is C (R)₃) N or C (R)₃)₂-C(R₃)₂；

Each R₁Independently of one another is halogen, OH, C₁-C₆Alkyl or C₁-C₆An alkoxy group;

R₂is C₁-C₆Alkyl radical C (O) -C₁-C₆Alkyl or C (O) -C₃-C₆A cycloalkyl group;

R₂' is H or C₁-C₆An alkyl group;

each R₃Independently is H or C₁-C₃An alkyl group;

each R₃' independently is C₁-C₃An alkyl group;

each R₄Independently is H or C₁-C₃An alkyl group; (ii) a Or two R₄Together with the carbon atom to which they are attached form a C (O), C3-C6 carbocyclic ring, or contain 1 or2 carbon atoms selected from N anda 4-, 5-, or 6-membered heterocyclic ring of a heteroatom of O;

R₅is H, deuterium, C₁-C₃Alkyl, F or Cl;

m is 0,1, 2or 3; and

n is 0,1 or 2;

wherein the compound is linked to another moiety (e.g., a compound or linker) throughAnd (4) covalent bonding.

In certain embodiments, the degradation determining region is a moiety of formula D, whereinIs that

In certain embodiments, the degradation determining region is a moiety of formula D, wherein X is C (O).

In certain embodiments, the degradation determining region is a moiety of formula D, wherein X is C (R)₃)₂(ii) a And each R₃Is H. In certain embodiments, X is C (R)₃)₂；R₃One of which is H and the other is C selected from methyl, ethyl and propyl₁-C₃An alkyl group. In certain embodiments, X is C (R)₃)₂(ii) a Each R₃Independently selected from methyl, ethyl and propyl.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein X₁-X₂Is C (R)₃) N. In some embodiments, X₁-X₂Is CH ═ N.In some embodiments, X₁-X₂Is C (R3) ═ N; r₃Is C selected from methyl, ethyl and propyl₁-C₃An alkyl group. In some embodiments, X₁-X₂Is C (CH3) ═ N.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein X₁-X₂Is C (R)₃)₂-C(R₃)₂(ii) a And each R₃Is H. In some embodiments, X₁-X₂Is C (R)₃)₂- C(R₃)₂(ii) a And R₃One of which is H and the other three R₃Independently C selected from methyl, ethyl and propyl₁-C₃An alkyl group. In some embodiments, and X₁-X₂Is C (R)₃)₂-C(R₃)₂； R₃Two of (A) are H, the other two R₃Independently is a C1-C3 alkyl group selected from methyl, ethyl and propyl. In some embodiments, X₁-X₂Is C (R)₃)₂-C(R₃)₂；R₃Three of which are H, the remaining R3 being C selected from methyl, ethyl and propyl₁-C₃An alkyl group.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein Y is a bond.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein Y is (CH)₂)₁，(CH₂)₂，(CH₂)₃，(CH₂)₄，(CH₂)₅Or (CH)₂)₆. In some embodiments, Y is (CH)₂)₁，(CH₂)₂Or (CH)₂)₃. In some embodiments, Y is (CH)₂)₁Or (CH)₂)₂。

In certain embodiments, the degradation determining region is a moiety of formula D, wherein Y is O, CH₂-O，(CH₂)₂-O，(CH2)₃-O，(CH₂)₄-O，(CH₂)₅-O or (CH)₂)₆-O. In some embodiments, Y is O, CH₂-O，(CH₂)₂-O or (CH)₂)₃-O. In certain embodiments, Y is O or CH₂-O. In certain embodiments, Y is O.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein Y is C (O) NR₂’，CH₂-C(O)NR₂’,(CH₂)₂-C(O)NR₂’，(CH₂)₃-C(O)NR₂’， (CH₂)₄-C(O)NR₂’，(CH₂)₅-C(O)NR₂' or (CH)₂)₆-C(O)NR₂'. In certain embodiments, Y is C (O) NR₂’，CH₂-C(O)NR₂’，(CH₂)2-C(O)NR₂' or (CH)₂)3-C(O)NR₂'. In some embodiments, Y is C (O) NR₂' or CH₂- C(O)NR₂'. In some embodiments, Y is C (O) NR₂’。

In certain embodiments, the degradation determining region is a moiety of formula D, wherein Y is NR₂’C(O)，CH₂-NR₂’C(O)，(CH₂)₂-NR₂’C(O)，(CH₂)₃-NR₂’C(O)， (CH₂)₄-NR₂’C(O)，(CH₂)₅-NR₂' C (O) or (CH)₂)₆-NR₂' C (O). In certain embodiments, Y is NR₂’C(O)，CH₂-NR₂’C(O)，(CH₂)₂-NR₂' C (O) or (CH)₂)₃-NR₂' C (O). In certain embodiments, Y is NR₂' C (O) or CH₂- NR₂' C (O). In certain embodiments, Y is NR₂’C(O)。

In certain embodiments, the degradation determining region is a moiety of formula D, wherein R₂' is H. In certain embodiments, the degradation determining region is a moiety of formula D, wherein R₂' is selected from methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, pentyl, isoPentyl and hexyl. In some embodiments, R₂' is C selected from methyl, ethyl and propyl₁-C₃An alkyl group.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein Y is NH, CH₂-NH，(CH₂)₂-NH，(CH₂)₃-NH，(CH₂)₄-NH，(CH₂)₅-NH or (CH2)₆-NH. In some embodiments, Y is NH, CH₂-NH，(CH₂)₂-NH， (CH₂)₃-NH. In certain embodiments, Y is NH or CH₂-NH. In certain embodiments, Y is NH.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein Y is NR₂， CH₂-NR₂，(CH₂)₂-NR₂，(CH₂)₃-NR₂，(CH₂)₄-NR₂，(CH₂)₅-NR₂Or (CH2)₆-NR₂. In certain embodiments, Y is NR₂、CH₂-NR₂、(CH₂)₂-NR₂Or (CH)₂)₃-NR₂. In some embodiments, Y is NR 2or CH2-NR2₂。

In certain embodiments, the degradation determining region is a moiety of formula D, wherein R₂Selected from the group consisting of methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl and hexyl. In some embodiments, R₂C selected from methyl, ethyl and propyl₁-C₃An alkyl group.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein R₂Selected from the group consisting of C (O) -methyl, C (O) -ethyl, C (O) -propyl, C (O) -butyl, C (O) -isobutyl, C (O) -tert-butyl, C (O) -pentyl, C (O) -isopentyl, and C (O) -hexyl. In certain embodiments, R₂Is C (O) -C selected from C (O) -methyl, C (O) -ethyl and C (O) -propyl₁-C₃An alkyl group.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein R₂C (O) -cyclomethyl, C (O) -cycloethyl, C (O) -cyclopropyl, C (O) -cyclobutyl, C (O) -cyclopentyl, and C (O) -cyclohexyl. In certain embodiments, R2 is c (o) -cyclohexyl.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein R₃Is H.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein R₃C selected from methyl, ethyl and propyl₁-C₃An alkyl group. In some embodiments, R₃Is methyl.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein n is 0.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein n is 1.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein n is 2.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein each R is₃Independently is a C1-C3 alkyl group selected from methyl, ethyl and propyl.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein m is 0.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein m is 1.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein m is 2.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein m is 3.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein each R is₁Independently selected from halogen (e.g., F, Cl, Br and I), OH, C₁-C₆Alkyl (e.g., methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, and hexyl) and C₁- C₆Alkoxy (e.g., methoxy, ethoxy, propoxy, butoxy, isobutoxy, t-butoxy, and pentyloxy). In a further embodiment, the degradation isThe decision region is a moiety of formula D, wherein each R₁Independently selected from the group consisting of F, Cl, OH, methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, methoxy and ethoxy.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein each R is₄Is H.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein each R is₄Is H and the other R₄Is C selected from methyl, ethyl and propyl₁-C₃An alkyl group.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein each R is₄Independently is C selected from methyl, ethyl and propyl₁-C₃An alkyl group.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein two R are₄Together with the carbon atom to which they are attached form c (o).

In certain embodiments, the degradation determining region is a moiety of formula D, wherein two R are₄Together with the carbon atom to which they are attached form a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl group.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein two R are₄Together with the carbon atom to which they are attached form a 4-, 5-or 6-membered heterocyclic ring selected from oxetane, azetidine, tetrahydrofuran, pyrrolidine, piperidine, piperazine and morpholine. In some embodiments, two R are₄Together with the carbon atom to which they are attached form an oxetane.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein R₅Is H, deuterium or C1-C3 alkyl. In a further embodiment, R₅Is in the (S) or (R) configuration. In a further embodiment, R₅Is in the (S) configuration. In certain embodiments, the degradation determining region is a moiety of formula D, wherein the compound comprises (S) -R₅And (R) -R₅A racemic mixture of (a).

In certain embodiments, the degradation determining region is a moiety of formula D, wherein R₅Is H.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein R₅Is deuterium.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein R5 is C selected from the group consisting of methyl, ethyl, and propyl₁-C₃An alkyl group. In some embodiments, R₅Is methyl.

In certain embodiments, the degradation determining region is a moiety of formula D, wherein R₅Is F or Cl. In a further embodiment, R₅Is in the (S) or (R) configuration. In a further embodiment, R₅Is in the (R) configuration. In certain embodiments, the degradation determining region is a moiety of formula D, wherein the compound comprises a racemic mixture of (S) -R5 and (R) -R5. In some embodiments, R₅Is F.

In certain embodiments, the degradation determining region is selected from the structures in FIG. 21, wherein X is H, deuterium, C₁-C₃Alkyl or halogen; and R is the point of attachment of the linker.

In some embodiments, the degradation determining region is selected from the structures in fig. 22.

In certain embodiments, the degradation determining region is selected from the structures in fig. 23.

Connector

A bond or chemical group connecting the dTAG targeting ligand to the degradation determining region. In certain embodiments, the linker is a carbon chain. In certain embodiments, the carbon chain optionally includes one, two, three, or more heteroatoms selected from N, O, and S. In certain embodiments, the carbon chain comprises only saturated chain carbon atoms. In certain embodiments, the carbon chain optionally comprises two or more unsaturated chain carbon atoms (e.g., C ═ C or C ≡ C). In certain embodiments, one or more chain carbon atoms in the carbon chain are optionally substituted with one or more substituents (e.g., oxo, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₁-C₃Alkoxy, OH, halogen, NH₂，NH(C₁-C₃Alkyl radical, N (C)₁-C₃Alkyl radical)₂，CN， C₃-C₈Cycloalkyl, heterocyclyl, phenyl, and heteroaryl).

In certain embodiments, the linker comprises at least 5 chain atoms (e.g., C, O, N, and S). In some embodiments, the linker comprises less than 20 chain atoms (e.g., C, O, N, and S). In certain embodiments, a linker comprises 5,6,7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 chain atoms (e.g., C, O, N, and S). In certain embodiments, the linker comprises 5,7, 9, 11, 13, 15, 17, or 19 chain atoms (e.g., C, O, N, and S). In certain embodiments, the linker comprises 5,7, 9, or 11 chain atoms (e.g., C, O, N, and S). In certain embodiments, the linker comprises 6,8, 10,12, 14, 16, or 18 chain atoms (e.g., C, O, N, and S). In certain embodiments, the linker comprises 6,8, 10, or 12 chain atoms (e.g., C, O, N, and S).

In certain embodiments, the linker is an optionally non-bulky substituent (e.g., oxo, C)₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₁-C₃Alkoxy, OH, halogen, NH₂，NH(C₁-C₃Alkyl radical, N (C)₁-C₃Alkyl radical)₂And CN) substituted carbon chains. In some embodiments, the non-bulky substitution is on a chain carbon atom that is close to the degradation determining region (i.e., the carbon atom to which the degradation determining region is bound is at least 3,4, or 5 chain atoms in the linker, respectively)).

In some embodiments, the linker belongs to formula L0:

or an enantiomer, diastereomer or stereoisomer thereof

P1 is an integer selected from 0 to 12;

p2 is an integer selected from 0 to 12;

p3 is an integer selected from 1 to 6;

each W is independently absent, CH₂O, S, NH or NR₅；

Z is absent, CH₂O, NH or NR₅；

Each R₅Independently is a C1-C3 alkyl group; and

q is absent or-CH₂C(O)NH-，

Wherein the linker isCovalently bonded to the degradation determining region adjacent to Q, andadjacent to Z is covalently bonded to the dTAG targeting ligand, and wherein the total number of chain atoms in the linker is less than 20.

In certain embodiments, the linker-dTAG Targeting Ligand (TL) has a structure of formula L1 or L2:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein:

p1 is an integer selected from 0 to 12;

p2 is an integer selected from 0 to 12;

p3 is an integer selected from 1 to 6;

each W is independently absent, CH₂O, S, NH or NR₅；

Z is absent, CH₂O, NH or NR₅；

Each R5 is independently C₁-C₃An alkyl group; and

TL is a dTAG targeting ligand,

wherein the linker isCovalently bonded to the degradation determining region.

In some embodiments, p1 is an integer selected from 0 to 10.

In certain embodiments, p1 is an integer selected from 2 to 10.

In certain embodiments, p1 is selected from 1,2,3,4, 5, and 6.

In certain embodiments, p1 is selected from 1,3, and 5.

In certain embodiments, p1 is selected from 1,2, and 3.

In some embodiments, p1 is 3.

In some embodiments, p2 is an integer selected from 0 to 10.

In certain embodiments, p2 is selected from 0,1, 2,3,4, 5, and 6.

In certain embodiments, p2 is an integer selected from 0 and 1.

In certain embodiments, p3 is an integer selected from 1 to 5.

In certain embodiments, p3 is selected from 2,3,4, and 5.

In certain embodiments, p3 is selected from 1,2, and 3.

In certain embodiments, p3 is selected from 2 and 3.

In some embodiments, at least one W is CH₂。

In some embodiments, at least one W is O.

In some embodiments, at least one W is S.

In some embodiments, at least one W is NH.

In some embodiments, at least one W is NR₅(ii) a And R₅Is C selected from methyl, ethyl and propyl₁-C₃An alkyl group.

In some embodiments, W is O.

In some embodiments, Z is absent.

In some embodiments, Z is CH₂。

In certain embodiments, Z is O.

In certain embodiments, Z is NH.

In certain embodiments, Z is NR₅(ii) a And R₅Is selected fromC of methyl, ethyl and propyl₁-C₃An alkyl group.

In certain embodiments, Z is part of a dTAG targeting ligand bound to a linker, i.e., Z is formed by the reaction of a functional group of the dTAG targeting ligand with the linker.

In some embodiments, W is CH₂Z is CH₂。

In certain embodiments, W is O and Z is CH₂。

In some embodiments, W is CH₂And Z is O.

In some embodiments, W is O and Z is O.

In certain embodiments, the linker-dTAG targeting ligand has a structure selected from table L:

watch L

Wherein Z, TL, and p1 are each as described above.

Any of the degradation determining regions described herein can be covalently bound to any of the linkers described herein.

In certain embodiments, the present application includes a degradation determining region-linker (DL) having the structure:

wherein each variable is as described above for formula D0 and formula L0, and dTAG targets the ligand toAdjacent to Z, is covalently bonded to DL.

In certain embodiments, the present application includes a degradation determining region-linker (DL) having the structure:

wherein each variable is as described above for formula D and formula L0, and dTAG targets the ligand toAdjacent to Z is covalently bonded to DL.

Some embodiments of the present application include bifunctional compounds having the following structure:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein each variable is as described above for formula D and formula L0, and a dTAG targeting ligand is described below.

Other embodiments of the present application relate to bifunctional compounds having the following structure:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein each variable is as described above for formula D and formula L0, and a dTAG targeting ligand is described below.

Certain embodiments of the present application relate to bifunctional compounds having one of the following structures:

in certain embodiments, the linker can be a polyethylene glycol group ranging in size from about 1 to about 12 ethylene glycol units, 1 to about 10 ethylene glycol units, about 2 to about 6 ethylene glycol units, about 2 to 5 ethylene glycol units, about 2 to 4 ethylene glycol units.

In certain embodiments, the linker is designed and optimized for the attachment position of the connector based on SAR (structure-activity relationship) and X-ray crystallography of dTAG targeting ligands.

In certain embodiments, optimal linker length and composition varies from target to target, and can be estimated based on the X-ray structure of the original dTAG targeting ligand bound to its target. Linker length and composition can also be modified to modulate metabolic stability and Pharmacokinetic (PK) and Pharmacodynamic (PD) parameters.

Where a dTAG-targeting ligand binds multiple targets, selectivity can be achieved by varying the linker length, where the ligand binds some of its targets in different binding pockets, e.g., deeper or shallower binding pockets than others.

In another embodiment, the heterobifunctional compound useful in the present invention comprises a chemical linker (L). In certain embodiments, linker group L is a group comprising one or more covalently attached a building blocks (e.g., -a1 … Aq-), wherein a1 is a group coupled to at least one of the dTAG targeting ligands or combinations thereof in the targeting region. In certain embodiments, a1 directly links a degradation determining region, dTAG targeting ligand or a combination thereof to another degradation determining region, targeting ligand or a combination thereof. In other embodiments, a1 indirectly links a degradation determining region, a dTAG targeting ligand or a combination thereof to another degradation determining region, a dTAG targeting ligand or a combination thereof through Aq.

In some embodiments, A₁To A_qEach independently being a bond, CRL₁RL₂，O， S，SO，SO₂，NR^L3，SO₂NR^L3，SONR^L3，CONR^L3，NR^L3CONR^L4， NR^L3SO₂NR^L4，CO，CR^L1＝CR^L2，C≡C，SiR^L1R^L2，P(O)R^L1， P(O)OR^L1，NR^L3C(＝NCN)NR^L4，NR^L3C(＝NCN)，NR^L3C(＝ CNO₂)NR^L4Optionally via 0 to 6R^L1And/or R^L2Radical substituted C_3-11Cycloalkyl, optionally via 0 to 6R^L1And/or R^L2Radical substituted C_3-11Heterocyclyl, optionally via 0 to 6R^L1And/or R^L2Aryl substituted by radicals, optionally with 0 to 6R^L1And/or R^L2Heteroaryl substituted by radicals, in which R is^L1Or R^L2Each independently may be linked to other a groups to form cycloalkyl and/or heterocyclyl moieties, which may be further linked through 0 to 4R^L5Substitution of radicals; wherein the content of the first and second substances,

R^L1、R^L2、R^L3、R^L4and R^L5Each independently is H, halogen, C_1-8Alkyl radical, OC_1-8Alkyl radical, SC_1-8Alkyl, NHC_1-8Alkyl radical, N (C)_1-8Alkyl radical)₂， C_3-11Cycloalkyl, aryl, heteroaryl, C_3-11Heterocyclyl radical, OC_1-8Cycloalkyl radicals, SC_1-8Cycloalkyl, NHC_1-8Cycloalkyl, N (C)_1-8Cycloalkyl radicals₂，N(C_1-8Cycloalkyl) (C)_1-8Alkyl), OH, NH₂，SH，SO₂C_1-8Alkyl, P (O) (OC)_1-8Alkyl) (C_1-8Alkyl radical, P (O) (OC)_1-8Alkyl radical)₂，CC—C_1-8Alkyl, CCH, CH ═ CH (C)_1-8Alkyl radical), C (C)_1-8Alkyl) ═ CH (C)_1-8Alkyl radical), C (C)_1-8Alkyl) ═ C (C)_1-8Alkyl radical)₂，Si(OH)₃，Si(C_1-8Alkyl radical)₃，Si(OH)(C_1-8Alkyl radical)₂，COC_1-8Alkyl radical, CO₂H, halogen, CN, CF₃，CHF₂，CH₂F， NO₂，SF₅，SO₂NHC_1-8Alkyl, SO₂N(C_1-8Alkyl radical)₂，SONHC_1-8Alkyl radical, SON (C)_1-8Alkyl radical)₂，CONHC_1-8Alkyl, CON (C)_1-8Alkyl radical)₂， N(C_1-8Alkyl) CONH (C)_1-8Alkyl radical, N (C)_1-8Alkyl) CON (C)_1-8Alkyl radical)₂， NHCONH(C_1-8Alkyl), NHCON (C)_1-8Alkyl radical)₂，NHCONH₂， N(C_1-8Alkyl) SO₂NH(C_1-8Alkyl radical, N (C)_1-8Alkyl) SO₂N(C_1-8Alkyl radical)₂， NH SO₂NH(C_1-8An alkyl group),NH SO₂N(C_1-8alkyl radical)₂，NH SO₂NH₂。

In some embodiments, q is an integer greater than or equal to 0. In some embodiments, q is an integer greater than or equal to 1.

In certain embodiments, e.g., where q is greater than 2, A_qIs a group attached to the degradation determining region, A₁And Aq are connected via a structural unit of A (the number of structural units of A: q-2).

In certain embodiments, for example, where q is 2, A_qIs a with A₁And a group to which a degradation determining region moiety is attached.

For example, where q is 1, the linker group L has the structure-A₁-, and A₁Is a group attached to a degradation determining region moiety and a dTAG targeting ligand moiety.

In further embodiments, q is an integer from 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or1 to 10.

In certain embodiments, linker (L) is selected from the structures in fig. 45.

In other embodiments, linker (L) is selected from the structures in FIG. 46, wherein

To represent

In further embodiments, the linker is an optionally substituted (poly) ethylene glycol having from 1 to about 100 ethylene glycol units, from about 1 to about 50 ethylene glycol units, from 1 to about 25 ethylene glycol units, from about 1 to 10 ethylene glycol units, from 1 to about 8 ethylene glycol units and 1 and 6 ethylene glycol units, from 2 to 4 ethylene glycol units, or an optionally substituted alkyl interspersed with optionally substituted O, N, S, P, or Si atoms. In certain embodiments, the linker is substituted with aryl, phenyl, benzyl, alkyl, alkylene, or heterocyclyl. In certain embodiments, the linker may be asymmetric or symmetric.

In any specific embodiment of a compound described herein, a linker can be any suitable moiety as described herein. In one embodiment, the linking group is a substituted or unsubstituted polyethylene glycol group having a size ranging from about 1 to about 12 ethylene glycol units, 1 to about 10 ethylene glycol units, about 2 to about 6 ethylene glycol units, about 2 to 5 ethylene glycol units, about 2 to 4 ethylene glycol units.

While the degradation determining region group and the dTAG targeting ligand group may be covalently linked to the linker group by any suitable group that is chemically stable to the linker, the linkers are independently covalently bonded to the degradation determining region group and the dTAG targeting ligand group, preferably by amide, ester, thioester, keto, carbamate (urethane), carbon or ether, wherein each group may be inserted anywhere in the degradation determining region group and the dTAG targeting ligand group to provide the degradation determining region group to the ubiquitin ligase and the dTAG targeting ligand group on the target dTAG. (it should be noted that in certain aspects where the degradation determining region group targets ubiquitin ligase, the target protein for degradation may be the ubiquitin ligase itself). The linker may be attached to an optionally substituted alkyl, alkylene, alkenyl or alkynyl, aryl or heterocyclyl group on the degradation determining region and/or on the dTAG targeting ligand group.

In some embodiments, "L" can be a straight chain having 4 to 24 straight chain atoms, the carbon atoms in the straight chain can be substituted with oxygen, nitrogen, amides, fluorinated carbons, and the like, such as the structure shown in fig. 26.

In certain embodiments, "L" can be a nonlinear chain, and can be an aliphatic or aromatic or heteroaromatic cyclic moiety, some examples of "L" include, but are not limited to, the structure of fig. 27, where X and Y are independently selected from a bond, CR^L1R^L2，O，S，SO，SO₂，NR^L3， SO₂NR^L3，SONR^L3，CONR^L3，NR^L3CONR^L4，NR^L3SO²NR^L4，CO， CR^L1＝CR^L2，C≡C，SiR^L1R^L2，P(O)R^L1，P(O)OR^L1，NR^L3C(＝ NCN)NR^L4，NR^L3C(＝NCN)，NR^L3C(＝CNO₂)NR^L4Optionally with 0 to 6R^L1And/or R^L2C3 to 11 cycloalkyl substituted by a group optionally substituted by 0 to 6R^L1Substituted C_3-11Heterocyclyl and/or R^L2Radical, optionally substituted by 0 to 6R^L1And/or R^L2Aryl substituted by radicals, optionally substituted with 0 to 6R^L1And/or R^L2A heteroaryl group substituted with R^L1Or R^L2Each independently may be linked to other A groups to form cycloalkyl and/or heterocyclyl moieties which may be further substituted with 0 to 4R^L5And (4) substituting the group.

dTAG targeting ligands

A dTAG Targeting Ligand (TL) capable of binding to or by a dTAG target, which allows ubiquitin labelling to occur;

as contemplated herein, the genome of the present invention includes a heterobifunctional compound targeting protein (dTAG) located in the cytoplasm. The heterobifunctional compound targeting protein of the genome is any amino acid sequence to which a heterobifunctional compound can bind, which when contacted with a heterobifunctional compound results in degradation of the dTAG hybrid protein. Preferably, dTAG should not interfere with the function of CAR. In one embodiment, dTAG is a non-endogenous peptide, resulting in heterobifunctional compound selectivity and allowing for the avoidance of off-target effects when administering heterobifunctional compounds. In one embodiment, dTAG is an amino acid sequence derived from an endogenous protein that has been modified such that the heterobifunctional compound binds only to the modified amino acid sequence and not to the endogenously expressed protein. In one embodiment, dTAG is an endogenously expressed protein. Any amino acid sequence domain that can be used for heterobifunctional compounds through ligand binding can be used as dTAG contemplated herein.

In particular embodiments, the dTAG used in the present invention includes, but is not limited to, amino acid sequences derived from endogenously expressed proteins, such as FK506 binding protein-12 (FKBP12), bromodomain-containing protein 4(BRD4), CREB binding protein (CREBBP) and transcriptional activator BRG1(SMARCA4) or variants thereof. As contemplated herein, "variant" means any variant, e.g., substitution, deletion, or addition of one or several to more amino acids, provided that the variant retains substantially the same function as the original sequence, which in this case provides heterobifunctional compound ligand binding. In other embodiments, the dTAG of our invention may comprise, for example, hormone receptors such as estrogen receptor protein, androgen receptor protein, Retinoid X Receptor (RXR) protein, and dihydrofolate reductase (DHFR), including bacterial DHFR, bacterial dehydrogenases, and variants.

In one embodiment, dTAG is part of any of the proteins identified herein. For example, dTAG may be the BD1 domain of BRD4 or the BD2 domain of BRD 4. In one embodiment, the targeting ligands identified herein that target the parent dTAG are used instead for the targeting moiety. In one embodiment, the BRD4 targeting ligands in table T can be used to target BD1 dTAG. In another embodiment, the BRD4 targeting ligands in table T can be used to target BD2 dTAG.

Some embodiments of the present application include dTAG-targeted TLs, including but not limited to those derived from Hsp90 inhibitors, kinase inhibitors, MDM 2inhibitors, compounds targeting proteins containing the human BET bromodomain, compounds targeting the cytoplasmic signaling protein FKBP12, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressive compounds, and compounds targeting the arene receptor (AHR).

In certain embodiments, the dTAG targeting ligand is a compound capable of binding to or binding to (bins to) a kinase-derived dTAG, a BET bromodomain-containing protein, a cytosolic signaling protein (e.g., FKBP12), a nucleoprotein, a histone deacetylase, a lysine methyltransferase, a protein that modulates angiogenesis, a protein that modulates immune response, an Aromatic Hydrocarbon Receptor (AHR), an estrogen receptor, an androgen receptor, a glucocorticoid receptor, or a transcription factor (e.g., SMARCA4, SMARCA2, TRIM 24).

In certain embodiments, dTAG is derived from a kinase to which a dTAG targeting ligand is capable of binding, including but not limited to tyrosine kinases (e.g., AATK, ABL2, ALK, AXL, BLK). BMX, BTK, CSF1, CSK, DDR, DDR, EGFR, EPHA, EPHA, EPHA, EPHA, EPHA, EPHA, EPHB, EPHB, EPHB, EPHB, ERBB, ERBB, FER, FES, FGFR, FGFR, FGFR, FGFR, FLT, FLT, FRK, FYN, GSG, HCK, IGF1, ILK, INSR, INSRR, IRK, ITK, JAK, JAK, JAK, KDR, KIT, LCR, LMTK, LMTK, LTK, LYN, MATK, MERK, MET, MLTK, MST1, MUSK, NPR, NTRK, NTRK, PDGFRAK, PDGFRB, PLK, PTK, PTNI 2, PTK, PTK, RET, RTK, TYRTR, TYRK, TRK, TRYPK, TROX, TRK, TRYPK, TROX, TRK, TRYPK, TRK, TRYPK, TRK, TRYPK, TR, ALK, Aurora a, Aurora B, Aurora C, CHK, CLK, DAPK, DMPK, ERK, GCK, GSK, HIPK, KHS, LKB, LOK, MAPKAPK, MNK, MSSK, MST, NDR, NEK, PAK, PIM, PLK, RIP, RSK, SGK, SIK, STK, TAO, TGF- β, TLK, TSSK, ULK, or ULK), a cell cycle-dependent protein kinase (e.g., Cdk to-Cdk), and a leucine-rich repeat kinase (e.g., lrk).

In certain embodiments, dTAG is derived from BET proteins containing a bromodomain to which a dTAG targeting ligand is capable of binding or binding (bins), including but not limited to ASH1, ATAD, BAZ1, BAZ2, BRD, BRPF, BRWD, CECR, CREBBP, EP300, FALZ, gc1240 n5L, KIAA, LOC93349, MLL, PB, PCAF, PHIP, PRKCBP, SMARCA, SP100, SP110, SP140, TAF1, TIF1, TRIM, WDR, ZMYND, and MLL. In certain embodiments, the BET bromodomain-containing protein is BRD 4.

In certain embodiments, dTAG is derived from a nuclear protein to which a dTAG targeting ligand is capable of binding (binding) or binding (bins), including but not limited to BRD2, BRD3, BRD4, antennapedia homeodomain protein, BRCA1, BRCA2, binding proteins that enhance CCAAT, histones, polycomb (Polymcomb) group proteins, high mobility proteins, telomere binding proteins, FANCA, FANCD2, FANCE, FANCF, hepatocyte nuclear factor, Mad2, NF-. kappa.B, nuclear receptor co-activators, CREB binding proteins, p55, p107, p130, Rb proteins, p53, c-fos, c-jun, c-mdm2, c-myc, and c-rel.

In certain embodiments, the dTAG targeting ligand is selected from the group consisting of a kinase inhibitor, a BET bromodomain containing protein inhibitor, a cytosolic signaling protein FKBP12 ligand, an HDAC inhibitor, a lysine methyltransferase inhibitor, an angiogenesis inhibitor, an immunosuppressive compound, and an arene receptor (AHR) inhibitor.

In certain embodiments, the dTAG targeting ligand is a SERM (selective estrogen receptor modulator) or a SERD (selective estrogen receptor degrader). Non-limiting examples of SERMs and SERDs are provided in WO2014/191726 assigned to Astra Zeneca, WO2013/090921, WO2014/203129, WO2014/313232 and US2013/0178445 assigned to Olema Pharmaceuticals and US patent nos. 9,078,871, 8,853,423 and 8,703,810 assigned to Seragon Pharmaceuticals, and US 2015/0005286, WO 2014/205136 and WO 2014/205138.

Additional dTAG targeting ligands include, for example, any moiety that binds to an endogenous protein (binds to the target dTAG). Illustrative dTAG targeting ligands include small molecule dTAG targeting ligands: hsp90 inhibitors, kinase inhibitors, HDM2 and MDM 2inhibitors, compounds targeting proteins containing the human BET bromodomain, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, nuclear hormone receptor compounds, immunosuppressive compounds and compounds targeting the arene receptor (AHR), and the like. Such small molecule target dTAG binding moieties also include pharmaceutically acceptable salts, enantiomers, solvates, and polymorphs of these compositions, as well as other small molecules that can target the target dTAG.

In some embodiments, the dTAG targeting ligand is a Ubc9 SUMO E2 ligase 5F6D targeting ligand, including but not limited to those described in "insight Into the interferometric Inhibition of the SUMO E2 enzymeUbc 9." by Hewitt, W.M., et al (2016) Angew.chem.int.Ed.Engl.55:5703 5707.

In another embodiment, the dTAG targeting ligand is a Tank1 targeting ligand, including but not limited to those described in "Structure of human tankyrase 1in complete with small-molecule inhibitors PJ34and XAV939," Kirby, C.A., Cheung, A., Fazal, A., Shultz, M.D., stamps, T, (2012) Actacrystallogr., Sect.F 68: 115-118; and "Structure-Efficiency Relationship of [1,2,4] Triazol-3-ylamines as Novel Nicotinamide Isospermes that is not at the inhibition Tankyrases," Shultz, M.D., et al (2013) J.Med.chem.56: 7049-.

In another embodiment, the dTAG targeting ligand is the SH2 Domain of a pp60Src targeting ligand, including but not limited to Those described in "requisitions for Specific Binding of Low Affinity Inhibitors to the SH2 Domain of pp60Src identity to third for high Affinity Binding of Full Length Inhibitors" Guundr Lange, et al, J.Med.Chem.2003,46, 5184-.

In another embodiment, The dTAG targeting ligand is a Sec7 domain targeting ligand, including but not limited to those described in "The lysomajor Protein Saponin B bonds Chloroquinone," Huta, B.P., et al, (2016) Chemedchem 11: 277.

In another embodiment, The dTAG targeting ligand is a sphingolipid-B targeting ligand, including but not limited to those described in "The structure of cytomegavirus immune modulator UL141 highlightsstructural Ig-fold versatility for receptor binding" I.M.Zajonic Acta Crystal. (2014). D70, 851).

IN another embodiment, the dTAG targeting ligand is a protein S100-A72 OWS targeting ligand, including but not limited to those described IN "2 WOS STRUCTURE OF HUMAN S100A7 IN COMPLEX WITH 2,6 ANS" DOI:10.2210/pdb2 WOS/pdb; and "Identification and Characterization of Binding Siteson S100A7," particle in Cancer and infection Pathways, "Leon, R., Murray, et al, (2009) Biochemistry 48: 10591-.

In another embodiment, the dTAG targeting ligand is a phospholipase A2 targeting ligand, including but not limited to those described in "Structure-based design of the first potential and selective inhibitor of human non-mammalian cell proliferation phospholipases A2" Schevitz, R.W., et al, Nat.Structure.biol.1995, 2, 458-465.

In another embodiment, the dTAG targeting ligand is a PHIP targeting ligand, including but not limited to those described in "A issued Fragment Library energy Rapid Synthesis Expansion stabilizing the first transported atoms of PHIP (2), an instant Bromodemian" Krojer, T.; et.chem.Sci.2016, 7, 2322-2330.

In another embodiment, the dTAG targeting ligand is a PDZ targeting ligand, including but not limited to those described in "Discovery of Low-Molecular-Weight Ligands for the AF6 PDZ Domain" MangeshJoshi, et al, Angew. chem. int. Ed. 2006,45, 3790-.

In another embodiment, the dTAG targeting ligand is a PARP15 targeting ligand, including but not limited to those described in "Structural Basis for rock of ADP-ribosyltranferase Activity in Poly (ADP-ribose) Polymerase-13/Zinc Finger scientific antibody protein," Karlberg, T.et. al., (2015) J.biol.chem.290: 7336- "7344.

In another embodiment, the dTAG targeting ligand is a PARP14 targeting ligand, including but not limited to those described in "Discovery of Ligands for ADP-Ribosyltransferases via Docking-Based virtual screening." Andersson, C.D., et al., (2012) J.Med.chem.55: 7706-; "Family-wireless profiling and structural analysis of PARP and tankyrase inhibitors," Wahlberg, E., et al (2012) Nat.Biotechnol.30: 283-; "Discovery of Ligands for ADP-Ribosyl transfer glasses vision gating-Based visual screening," Andersson, C.D., et al (2012) J.Med.Chem.55: 7706-.

In another embodiment, the dTAG targeting ligand is an MTH1 targeting ligand, including but not limited to those described in "MTH 1 inhibition ligands by prevention of the dNTP pool" Helge Gad, et al Nature, 2014,508,215- "221.

In another embodiment, the dTAG targeting ligand is an mPGES-1 targeting ligand, including but not limited to those described in "Crystal Structures of mPGES-1 Inhibitor compounds for a Basis for the rational Design of Point analytical and Anti-Inflammatory therapeutics," Luz, J.G., et al., (2015) J.Med.chem.58: 4727-.

In another embodiment, the dTAG targeting ligand is a FLAP-5-lipoxygenase-activating protein targeting ligand, including but not limited to those described in "Crystal structure of inhibitor-bound human 5-lipoxygenase-activating protein," Ferguson, A.D., McKeever, B.M., Xu, S., Wisnewski, D., Miller, D.K., Yamin, T.T., Spencer, R.H., Chu, L., Ujjainwala, F., Cunningham, B.R., Evans, J.F., Becker, J.W. (2007) Science 317: 510-512.

In another embodiment, the dTAG targeting ligand is a FA binding protein targeting ligand, including but not limited to those described in "a Real-World functional on Molecular design," Kuhn, b.; et.J.Med.chem.2016, 59, 4087-.

In another embodiment, the dTAG targeting ligand is a BCL2 targeting ligand, including but not limited to those described in "ABT-199, a potential and selective BCL-2 inhibitor, achieves inhibitor or activity while spacing platforms," Souers, A.J., et al (2013) NAT.MED. (N.Y.)19: 202-.

In another embodiment, the dTAG targeting ligand is an EGFR targeting ligand. In one embodiment, the dTAG targeting ligand is selected from erlotinib (erlotinib) (Tarceva), gefitinib (gefitinib) (Iressa), Afatinib (Afatinib) (Gilotrif), rocitinib (rocitinib) (CO-6), oximitinib (osimertinib) (Tagrisso), oximitinib (olmutinib) (Olita), naltretinib (naquotinib) (ASP8273), naltinib (nartinib) (EGF816), PF-06747775(Pfizer), ibrutinib (icotinib) (BPI-2009), neritinib (neritinib) (HKI-272; PB 272); acertinib (avitinib) (AC0010), EAI045, talotinib (tarloxotinib) (TH-4000; PR-610), PF-06459988(Pfizer), tesevatinib (tesevatinib) (XL 647; EXEL-7647; KD-019), Setinib (transtinib), WZ-3146, WZ8040, CNX-2006, and dacomitinib (dacomitinib) (PF-00299804; Pfizer). Linkers can be placed on these targeting ligands at any location that does not interfere with the binding of the ligand to EGFR. Non-limiting examples of linker binding sites are provided in table T below. In one embodiment, the EGFR-targeting ligand binds to the L858R mutant of EGFR. In another embodiment, the EGFR-targeting ligand binds to the T790M mutant of EGFR. In another embodiment, the EGFR-targeting ligand binds to a C797G or C797S mutant of EGFR. In one embodiment, the EGFR-targeting ligand is selected from erlotinib, gefitinib, afatinib, neratinib and dacatinib, and binds to the L858R mutant of EGFR. In another embodiment, the EGFR-targeting ligand is selected from the group consisting of ocitinib, rocitinib, ormotinib, naltretinib, azatinib, PF-06747775, ibrutinib, neratinib, atinib, talosotinib, PF-0645998, tixatinib, prodigitinib, WZ-3146, WZ8040, and CNX-2006. And binds to the T790M mutant of EGFR. In another embodiment, the EGFR-targeting ligand is EAI045 and binds to a C797G or C797S mutant of EGFR.

Any protein that binds to a dTAG targeting ligand group and acts on or is degraded by ubiquitin ligase is a target protein of the invention. In general, endogenous target proteins used as dTAG may comprise, for example, structural proteins, receptors, enzymes, cell surface proteins, proteins functionally associated with cell integration, including proteins involved in the following activities: catalytic activity, aromatase activity, locomotor activity, helicase activity, metabolic processes (anabolism and catabolism), antioxidant activity, proteolysis, biosynthesis, proteins with kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme modulator activity, signal transduction activity, structural molecule activity, binding activity (proteins, lipid carbohydrates), receptor activity, cell motility, membrane fusion, cell communication, bioprocess regulation, development, cell differentiation, response to stimuli, behavioral proteins, cell adhesion proteins, protein death in cells, proteins involved in trafficking (including protein transporters), nuclear transport, ion transporter activity, channel transporter activity, carrier activity, permease activity, secretion activity, electron transporter activity, pathogenesis, chaperone regulatory activity, nucleic acid binding activity, transcription regulatory activity, extracellular tissue and biogenesis activity, translation regulatory activity.

More specifically, many drug targets for human therapy represent dTAG targets, protein targets or dTAG targeting ligands can be bound and incorporated into the compounds according to the invention. These include proteins useful for restoring function in a variety of multigenic diseases, including, for example, B7.1 and B7, TINFR1m, TNFR2, NADPH oxidase, BclIBax and other pairs in the apoptotic pathway, the C5a receptor, H MG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDEI, PDEII, PDEIII, squalene cyclase inhibitors, CXCR1, CXCR2, Nitric Oxide (NO) synthetase, cyclooxygenase 1, cyclooxygenase 2,5HT receptor, dopamine receptor, G protein, i.e., Gq, histamine receptor, 5-lipoxygenase, tryptase serine protease, thymidylate synthase, purine nucleoside phosphorylase, GAPDH trypanosoma phosphorylase, carbonic anhydrase, chemokine receptor, JAW STAT, RXR and the like, HIV 1 protease, HIV 1 integrase, influenza, neuraminidase, hepatitis B reverse transcriptase, sodium channels, multidrug resistance (MDR), protein P-glycoprotein (and MRP), tyrosine kinase, CD23, CD124, tyrosine kinase P56 lck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF- α R, ICAM1, Cat + channels, VCAM, VLA-4 integrin, selectin, CD40/CD40L, novel kinases and receptors, inosine monophosphate dehydrogenase, P38 MAP kinase, RaslRaflMEWERK pathway, interleukin-1 converting enzyme, caspase, HCV, NS3 protease, HCV NS3RNA helicase, glycinamide ribosyl transferase, rhinovirus 3C protease, herpes simplex virus-1 (HSV-I), protease, cell virus (CMV) protease, poly (ADP-ribose) polymerase, cyclin kinase dependent, vascular endothelial growth factor, oxytocin receptor, microsomal transporter inhibitors, bile acid transport inhibitors, 5 alpha reductase inhibitors, angiotensin 11, glycine receptor, norepinephrine reuptake receptor, endothelin receptor, neuropeptide Y and receptor, estrogen receptor, androgen receptor, adenosine kinase and AMP deaminase, purinergic receptor (P2Y1, P2Y2, P2Y4, P2Y6, P2X1-7), farnesyl transferase, geranyl transferase, NGF a receptor of TrkA, beta-amyloid, tyrosine kinase Flk-IIKDR, vitronectin receptor, integrin receptor, Her-21neu, telomerase inhibition, cytosolic phospholipase a2, and EGF receptor tyrosine kinase. Other protein targets that can be used as dTAG include, for example, ecdysone 20-monooxygenase, ion channels of GABA-gated chloride channels, acetylcholinesterase, voltage sensitive sodium channel proteins, calcium release channels, and chloride channels. Other target proteins used as dTAG include acetyl-CoA carboxylase, adenylosuccinate synthetase, protoporphyrinogen oxidase and enolpyruvylshikimate phosphate synthase.

In one embodiment, the dTAG and dTAG targeting ligand pair is selected by screening a library of ligands. This screening was performed in Duong-Ly et al, "Kinase Inhibitor receptors advanced Opportunities to Inhibitor diseases-Associated Mutant genes"; cell Reports 14, 772-.

Haloalkane dehalogenases are another target of specific compounds according to the present invention, which can be used as dTAG. Compounds of the invention containing chloroalkane peptide binding moieties (C1-C12 are typically about C2-C10 alkyl halogen groups) are useful for inhibiting and/or degrading haloalkane dehalogenases useful for fusion proteins or related diagnostic proteins, such as PCT/US2012/063401 filed 12/6/2011, published as WO2012/078559 at 6/2012, the contents of which are incorporated herein by reference.

Non-limiting examples of dTAG targeting ligands are shown in table T below and represent dTAG targeting ligands capable of targeting protein or amino acid sequences that can be used as dTAG.

Table T:

a. BRD dTAG targeting ligand:

BRD dTAG targeting ligands, as used herein, include, but are not limited to:

and

wherein:

r is the site of linker attachment; and

r': is methyl or ethyl.

CREBP dTAG targeting ligand:

as used herein, CREBBP dTAG targeting ligands include, but are not limited to:

and

wherein:

r is the site of linker attachment;

a is N or CH; and

m is 0,1, 2,3,4, 5,6,7 or 8.

SMARCA4, PB1, and/or SMARCA2 dTAG targeting ligand:

SMARCA4, PB1, and/or SMARCA2 dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r is the site of linker attachment; and

a is N or CH; and

m is 0,1, 2,3,4, 5,6,7 or 8.

TRIM24 and/or BRPF 1dTAG targeting ligands:

TRIM24 and/or BRPF 1dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r is the site of linker attachment; and

m is 0,1, 2,3,4, 5,6,7 or 8.

E. Glucocorticoid receptor dTAG targeting ligand:

glucocorticoid dTAG targeting ligands, as used herein, include, but are not limited to:

and

wherein:

r is the site of linker attachment.

F. Estrogen and/or androgen receptor dTAG targeting ligand:

as used herein, estrogen and/or androgen dTAG targeting ligands include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Dot1l dTAG targeting ligand:

DOT1L dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r is the site of linker attachment;

a is N or CH; and

m is 0,1, 2,3,4, 5,6,7 or 8.

Ras dTAG targeting ligand:

as used herein, Ras dTAG targeting ligands include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Rasg12c dTAG targeting ligand:

RasG12C dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r is the site of linker attachment.

A her3 dTAG targeting ligand:

her3 dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r is the site of linker attachment;

r' isOr

Bcl-2or Bcl-XL dTAG targeting ligand:

bcl-2or Bcl-XL dTAG targeting ligands, as used herein, include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Hdac dTAG targeting ligand:

as used herein, HDAC dTAG targeting ligands include, but are not limited to:

wherein:

r is the site of linker attachment.

Ppar- γ dTAG targeting ligand:

PPAR- γ dTAG targeting ligands, as used herein, include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Rxr dTAG targeting ligand:

RXR dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Dhfr dTAG targeting ligand:

DHFR dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Egfr dTAG targeting ligand:

EGFR dTAG targeting ligands used herein include, but are not limited to:

1. targeting ligands targeting L858R mutant EGFR including erlotinib, gefitinib, afatinib, neratinib and dacatinib.

Wherein:

r is the site of linker attachment.

2. Ligand targeting of T790M mutant EGFR, including ocitinib, rocitinib, ormotinib, naltretinib, azatinib, PF-06747775, ibrutinib, neratinib, atintinib, tarosotinib, PF-0645998, tixatinib, setinib, WZ-3146, WZ8040 and CNX-2006:

wherein:

r is the site of linker attachment.

3. A targeting ligand targeting C797S mutant EGFR, comprising EAI 045:

wherein:

r is the site of linker attachment.

bcr-ABL dTAG targeting ligand:

as used herein, BCR-ABL dTAG targeting ligands include, but are not limited to:

1. targeting ligands targeting T315I mutant BCR-ABL (PDB #3CS9) including Nilotinib (Nilotinib) and Dasatinib (Dasatinib):

wherein:

r is the site of linker attachment.

2. Targeting ligands targeting BCR-ABL, including nilotinib, dasatinib, Ponatinib (Ponatinib) and Bosutinib:

wherein:

r is the site of linker attachment.

Alk dTAG targeting ligand:

ALK dTAG targeting ligands used herein include, but are not limited to:

1. targeting ligands targeting L1196M mutant ALK (PDB #4MKC) including Ceritinib (Ceritinib):

wherein:

r is the site of linker attachment.

S. jak2 dTAG targeting ligand:

JAK2 dTAG targeting ligands used herein include, but are not limited to:

1. ligands targeting V617F mutant JAK2, including Ruxolitinib (Ruxolitinib):

wherein:

r is the site of linker attachment.

Braf dTAG targeting ligand:

BRAF dTAG targeting ligands used herein include, but are not limited to:

1. targeting ligands targeting V600E mutant BRAF (PBD #3OG7), including Vemurafenib (Vemurafenib):

wherein:

r is the site of linker attachment.

2. Ligands directed against the target BRAF, including Dabrafenib (Dabrafenib):

wherein:

r is the site of linker attachment.

Lrrk2 dTAG targeting ligand LRRK2 dTAG targeting ligand as used herein includes, but is not limited to:

1.a targeting ligand targeting R1441C mutant LRRK2 comprising:

wherein:

r is the site of linker attachment.

2. Ligand targeting of G2019S mutant LRRK2 comprising:

wherein:

r is the site of linker attachment.

3. A targeting ligand targeting I2020T mutant LRRK2 comprising:

wherein:

r is the site of linker attachment.

Pdgfr α dTAG targeting ligand:

PDGFR α dTAG targeting ligands used herein include, but are not limited to:

1. targeting ligands targeting T674I mutant PDGFR α, including AG-1478, CHEMBL94431, dovidinib, erlotinib, gefitinib, imatinib (imatinib), Janex 1, Pazopanib (Pazopanib), PD153035, Sorafenib (Sorafenib), Sunitinib (Sunitinib), WHI-P180:

wherein:

r is the site of linker attachment.

Ret dTAG targeting ligand:

RET dTAG targeting ligands used herein include, but are not limited to:

1. targeting ligands targeting the G691S mutant RET, including tozasertib

Wherein:

r is the site of linker attachment.

2. Targeting ligands targeting the R749T mutant RET, including tazarotene

Wherein:

r is the site of linker attachment.

3. Targeting ligands targeting E762Q mutant RET, including tozasertib

Wherein:

r is the site of linker attachment.

4. Targeting ligands targeting the Y791F mutant RET, including tozasertib

Wherein:

r is the site of linker attachment.

5. Targeting ligands targeting the V804M mutant RET, including tozasertib

Wherein:

r is the site of linker attachment.

6. Targeting ligands targeting M918T mutant RET, including tazarotene

Wherein:

r is the site of linker attachment.

Heat shock protein 90(HSP90) dTAG targeting ligand:

heat shock protein 90(HSP90) dTAG targeting ligands, as used herein, include but are not limited to:

1. in Vallee et al, "Tricyclic Series of Heat Shock Protein 90(HSP90) Inhibitors Part I: HSP90 Inhibitors identified in Discovery of Tricyclic Imidazo [4,5-C ] pyridines potential Inhibitors of the HSP90 molecular Chaperone (2011) J.Med.chem.54:7206, comprising (N- [4- (3H-Imidazo [4,5-C ] pyridin-2-yl) -9H-fluoren-9-yl ] -succinamide):

wherein the linker group L or- (L-degradation determining region) group is derivatized, for example, via a terminal amide group linkage;

HSP90 inhibitor p54 (modified) (8- [ (2, 4-dimethylphenyl) sulfanyl ] -3] pent-4-yn-1-yl-3H-purin-6-amine):

wherein the linker group L or- (L-degradation determining region) group is derivatized, for example, via a terminal ethynyl linkage;

3. HSP90 Inhibitors (modified) identified in Brough et al, "4, 5-Diarylisoxazole HSP90 Chaperone Inhibitors: functional Therapeutic Agents for the treatment of Cancer," J.Med. CHEM.Vol:51, page:196(2008), including the compound 2GJ (5- [2, 4-dihydroxy-5- (1-methylethyl) phenyl ] -n-ethyl-4- [4- (morpholin-4-ylmethyl) phenyl ] isoxazole-3-carboxamide) having the structure shown below:

wherein the linker group L or the- (L-degradation determining region) group is derivatized via, for example, an amide group linkage (on the amine or on the alkyl group on the amine);

4. in Wright et al, "Structure-Activity Relationships in pure-Based Inhibitor Binding to HSP90 Isoform," Chem biol.2004 June; 11(6) HSP90 inhibitors identified in 775-85 (modified), including the HSP90 inhibitor PU3 having the structure shown below:

wherein the linker group L or- (L-degradation determining region) is derivatized, for example, via a butyl linkage; and

HSP90 inhibitor geldanamycin ((4E, 6Z, 8S, 9S, 10E, 12S, 13R, 14S, 16R) -13-hydroxy-8, 14, 19-trimethoxy-4, 10,12, 16-tetramethyl-3, 20, 22-trioxo-2-azabicyclo [16.3.1] (derivatized) or any derivative thereof (such as 17-alkylamino-17-demethoxygeldanamycin ("17-AAG") or 17- (2-dimethylaminoethyl) amino-17) -demethoxygeldanamycin ("17-DMAG") (derivatized where linker groups L or- (L-degradation determining region) groups are linked via, for example, an amide group).

Kinase and phosphatase dTAG targeting ligands:

kinase and phosphatase dTAG targeting ligands used herein include, but are not limited to:

1. erlotinib derivative tyrosine kinase inhibitors:

wherein R is a linker group L or a- (L-degradation determining region) group connected via, for example, an ether group;

2. kinase inhibitor sunitinib (derivatised):

wherein R is a linker group L or a- (L-degradation determining region) group is derivatized, for example, with an azole moiety attached;

3. kinase inhibitor sorafenib (derivatization):

wherein R is a linker group L or a- (L-degradation determining region) group is derivatized, for example, with an amide moiety attached;

4. kinase inhibitor dasatinib (derivatization):

wherein R is a linker group L or is derivatized with a- (L-degradation determining region) linkage such as a pyrimidine;

5. kinase inhibitor lapatinib (derivatization):

wherein the linker group L or the- (L-degradation determining region) group is derivatized via a terminal methyl linkage such as a sulfonylmethyl group;

6. kinase inhibitor U09-CX-5279 (derivatized):

wherein the L or- (L-degradation determining region) group is derivatized via, for example, an amine (aniline), carboxylic acid or amine α linkage to a cyclopropyl or cyclopropyl group;

7. kinase Inhibitors identified in Millan et al, Design and Synthesis of interferometric P38 Inhibitors for the treatment of Chronic Obstructive Disease, J.MED. CHEM.Vol:54, page:7797(2011) include kinase Inhibitors Y1W and Y1X (derivations) having the structures shown below:

YIX (1-ethyl-3- (2- { [3- (1-methylethyl) [1,2,4] triazolo [4,3-a ] pyridin-6-yl ] sulfanyl } benzyl) urea in which the linker group L or the- (L-degradation determining region) group is derivatized, for example, via an isopropyl linkage;

1- (3-tert-butyl-1-phenyl-1H-pyrazol-5-yl) -3- (2- { [3- (1-methylethyl) - [1,2,4] triazolo [4,3-a ] pyridin-6-yl ] sulfanyl } benzyl) urea, wherein the linker group L or the- (L-delocalized) group is derivatized, for example, preferably via an isopropyl or tert-butyl linkage;

8. kinase Inhibitors identified in Schenkel et al, Discovery of patent and high Selective Thienopyridine Janus 2Inhibitors j.med.chem.,2011, 54(24), pp 8440-:

4-amino-2- [4- (tert-butylsulfamoyl) phenyl ] -N-methylthieno [3,2-c ] pyridine-7-carboxamidothiopyridine 19, in which the linker group L or the- (L-degradation determining region) group is derivatized, for example, via a terminal methyl linkage to the amide moiety:

4-amino-N-methyl-2- [4- (morpholin-4-yl) phenyl ] thieno [3,2-c ] pyridine-7-carboxamide thienopyridine 8, in which the linker group L or the- (L-degradation determining region) group is derivatized, for example, via a terminal methyl linkage to the amide moiety;

9. in Van Eis et al, "2, 6-Naphthyridines as potential and selective inhibitors of the novel protein kinase C isozymes," Bio rg. Med. chem. Lett.2011Dec.15; 21(24) 7367-72, comprising kinase inhibitor 07U having the structure shown below:

2-methyl N.sub.1- [3- (pyridin-4-yl) -2, 6-naphthyridin-1-yl ] propane-1, 2-diamine in which the linker group L or the- (L-degradation determining region) group is derivatized, for example, via a secondary amine or a terminal amino linkage;

lountos et al, "Structural Characterization of Inhibitor compounds with Checkpoint Kinase 2(Chk2), Drug Target for Cancer Therapy," J.STRUCT.BIOL.Vol:176, pag:292(2011) comprising the kinase inhibitor YCF having the structure shown below:

wherein the linker group L or- (L-degradation determining region) group is derivatized, for example, via attachment of any of the terminal hydroxyl groups;

kinase inhibitors identified in "Structural Characterization of Inhibitor compounds with Checkpoint Kinase 2(Chk2), a Drug Target for Cancer Therapy," J.STRUCT.BIOL.Vol:176, pag:292(2011) include Kinase inhibitors XK9 and NXP (derivatives) having the structures shown below:

n- {4- [ (1E) -N- (N-hydroxycarbamimidoyl) etha-zino ] phenyl } -7-nitro-1H-indole-2-carboxamide

N- {4- [ (1E) -N-carbamimidoylethyldiazoxy ] phenyl } -1H-indole-3-carboxamide, wherein the linker group L or the- (L-degradation determining region) group is derivatized, for example, via a terminal hydroxy (XK9) or hydrazone group (NXP) linkage;

12. the kinase inhibitor afatinib (derivatised) (N- [4- [ (3-chloro-4-fluorophenyl) amino ] -7- [ [ (3S) -tetrahydro-3-furanyl ] oxy ] -6-quinazolinyl ] -4 (dimethylamino) -2-butenamide) (wherein the linker group L or the- (L-degradation determining region) group is derivatised, e.g. via an aliphatic amine linkage);

13. kinase inhibitor fostamatinib (derivatization) ([6- ({ 5-fluoro-2- [ (3,4, 5-trimethoxyphenyl) amino ] pyrimidin-4-yl } amino) -2, 2-dimethyl-3-oxo-2, 3-dihydro-4H-pyrido [3,2-b ] -1, 4-oxazin-4-yl ] methyl disodium phosphate hexahydrate) (where the linker group L or the- (L-degradation determining region) group is derivatized, e.g., via methoxy linkage);

14. kinase inhibitor gefitinib (derivatized) (N- (3-chloro-4-fluoro-phenyl) -7-methoxy-6- (3-morpholin-4-ylpropoxy) quinazolin-4-amine):

wherein the linker group L or- (L-degradation determining region) group is derivatized, for example, via a methoxy or ether linkage;

15. kinase inhibitor lenvatinib (derivatised) (4- [ 3-chloro-4- (cyclopropylcarbamoylamino) phenoxy ] -7-methoxy-quinoline-6-carboxamide) (wherein the linker group L or- (L-degradation determining region)) is derivatised e.g. via a cyclopropyl linkage);

16. the kinase inhibitor vandetanib (derivatised) (N- (4-bromo-2-fluorophenyl) -6-methoxy-7- [ (1-methylpiperidin-4-yl) methoxy ] quinazolin-4-amine) (wherein the linker group L or the- (L-degradation determining region) group is derivatised, e.g. via a methoxy or hydroxy linkage);

17. the kinase inhibitor vemurafenib (derivatized) (propane-1-sulfonic acid {3- [5- (4-chlorophenyl) -1H-pyrrolo [2,3-b ] pyridine-3-carbonyl ] -2, 4-difluoro-phenyl } -amide) wherein the linker group L or- (L-degradation determining region) group is derivatized, e.g., via a sulfonyl linkage;

18. kinase inhibitor Gleevec (derivatization)

Wherein R is derivatized as a linker group L or a- (L-degradation determining region) group, for example via an amide group or via an aniline group linkage;

19. kinase inhibitor pazopanib (derivatised) (VEGFR3 inhibitor):

wherein R is a linker group L or a- (L-degradation determining region) group, e.g. attached to a phenyl moiety or derivatized via an aniline group;

20. kinase inhibitor AT-9283 (derivatized) Aurora kinase inhibitors

Wherein R is, for example, a linker group L or a- (L-degradation determining region) group attached to the phenyl moiety);

21. kinase inhibitor TAE684 (derivatized) ALK inhibitors

Wherein R is, for example, a linker group L or a- (L-degradation determining region) group attached to the phenyl moiety);

22. kinase inhibitor nilotanib (derivatized) Abl inhibitor:

wherein R is a linker group L or a- (L-degradation determining region) group, e.g., derivatized with a phenyl moiety or an aniline group;

23. kinase inhibitor NVP-BSK805 (derivatized) JAK 2inhibitor

Wherein R is a linker group L or a- (L-degradation determining region) group, e.g., derivatized with a phenyl moiety or a oxadiazolyl group;

24. kinase inhibitor crizotinib-derived Alk inhibitors

Wherein R is a linker group L or a- (L-degradation determining region) group such as a linker phenyl moiety or diazolyl derivatisation;

25. kinase inhibitor JNJ FMS (derivatised) inhibitors

Derivatization, wherein R is- (L-degradation determining region) e.g. a linker group L attached to the phenyl moiety or a- (L-degradation determining region) group;

26. kinase inhibitor fornicinib (derivatised) Met inhibitors

Derivatization, wherein R is- (L-degradation determining region) e.g. a linker group L or a- (L-degradation determining region) group linking a hydroxyl or ether group on a phenyl moiety or a quinoline moiety;

27. allosteric protein tyrosine phosphatase inhibitor PTP1B (derivatized):

wherein the linker group L or- (L-degradation determining region) group is derivatized such as shown at R;

an inhibitor of the SHP-2 tyrosine phosphatase domain (derivatisation):

wherein for example at R a linker group L or a- (L-degradation determining region) group is attached;

an inhibitor of BRAF (BRAFV600E)/MEK (derivatized):

wherein, for example, R is derivatized by attachment of a linker group L or a- (L-degradation determining region) group;

30. inhibitors of tyrosine kinase ABL (derivatized)

Wherein, for example, R is derivatized by attachment of a linker group L or a- (L-degradation determining region) group;

31. kinase inhibitor OSI-027 (derivatized) mTORC1/2 inhibitors

Wherein, for example, R is derivatized by attachment of a linker group L or a- (L-degradation determining region) group;

32. kinase inhibitor OSI-930 (derivatized) c-Kit/KDR inhibitors

Wherein, for example, R is derivatized by attachment of a linker group L or a- (L-degradation determining region) group; and

33. kinase inhibitor OSI-906 (derivatized) IGF1R/IR inhibitor

Wherein, for example, the linker group L or the- (L-degradation determining region) group is attached at R.

Wherein, in any of the embodiments described in chapters I through XVII, "R" represents a site for attachment of a linker group L or a- (L-degradation determining region) group on the piperazine moiety.

Hdm2 and/or MDM2 dTAG targeting ligands:

HDM2 and/or MDM2 dTAG targeting ligands used herein include, but are not limited to:

the HDM2/MDM 2inhibitors identified In Vassilev, et al, In vivo activation of the p53 pathway by small-molecular antagonists of 2, SCIENCE vol:303, pag: 844-:

(wherein, for example, a methoxy group or a hydroxyl group is attached to the linker group L or the- (L-degradation determining region) group for derivatization);

(wherein the attachment at the linker group L or- (L-degradation determining region) group is derivatised e.g. at methoxy or hydroxy);

(wherein at the linker group L or- (L-degradation determining region) the group is derivatised, e.g.via a methoxy or as a hydroxyl linkage); and

2. trans-4-iodo-4' -boryl-chalcones

(derivatisation, wherein linker group L or- (L-degradation determining region) group is attached, for example, via a hydroxyl group.

Human BET bromodomain-containing protein dTAG targeting ligand:

in certain embodiments, a "dTAG targeting ligand" can be a ligand that binds to the bromo-and extra terminal domain (BET) proteins BRD2, BRD3, and BRD 4. Compounds targeting BET bromodomain-containing proteins in humans include, but are not limited to, compounds related to the targets described below, wherein "R" or "linker" represents the site of attachment of a linker group L or a- (L-degradation determining region) group, for example:

1.JQ1,Filippakopoulos et al.Selective inhibition of BET bromodomains.Nature(2010):

2.I-BET,Nicodeme et al.Suppression of Inflammation by a Synthetic Histone Mimic.Nature(2010).Chung et al.Discovery and Characterization ofSmall Molecule Inhibitors of the BET Family Bromodomains.J.Med Chem.(2011):

3. compounds described in Hewings et al.3,5-Dimethylisoxazoles Act as Acetyl-lysine Bromodomain ligands.J.Med.chem. (2011) 546761-6770.

4.I-BET151,Dawson et al.Inhibition of BET Recruitment to Chromatin as an Effective Treatment for MLL-fusion Leukemia. Nature(2011):

5. Carbazole type (US 2015/0256700)

6. Pyrrolopyridine types (US 2015/0148342)

7. Tetrahydroquinoline type (WO 2015/074064)

8. Triazole pyrazine type (WO 2015/067770)

9. Pyridones (WO 2015/022332)

10. Quinazolinones (WO 2015/015318)

11. Dihydropyridinazinones (WO 2015/011084)

(wherein, in each case, R or L or a linker designates a linking site for e.g.linking a linker group L or a- (L-degradation determining region) group).

Hdac dTAG targeting ligand:

HDAC dTAG targeting ligands as used herein include, but are not limited to:

1.Finnin,M.S.et al.Structures of Histone Deacetylase Homologue Bound to the TSA and SAHA Inhibitors.Nature 40,188-193(1999)。

(wherein "R" represents a linking site derivatized, for example, with a linker group L or a- (L-degradation determining region) group); and

2. a compound defined by formula (I) of PCT WO0222577 ("deacylase inhibitor") (wherein the linker group L or the- (L-degradation determining region) group is derivatized, for example, via a hydroxyl group linkage);

human lysine methyltransferase dTAG targeting ligand:

human lysine methyltransferase dTAG targeting ligands used herein include, but are not limited to:

1.Chang et al.Structural Basis for G9a-Like protein Lysine Methyltransferase Inhibition by BIX-1294.Nat.Struct.Biol.(2009) 16(3)312。

(wherein "R" is derivatized designating a site for attachment of, for example, a linker group L or a- (L-degradation determining region) group);

2.Liu,F.et al Discovery of a 2,4-Diamino-7- aminoalkoxyquinazoline as a Potent and Selective Inhibitor of Histone Methyltransferase G9a.J.Med.Chem.(2009)52(24)7950。

(wherein "R" is derivatized designating potential sites for attachment of, for example, a linker group L or a- (L-degradation determining region) group);

3. azacitidine (derivatized) (4-amino-1- (3-D-ribofuranosyl-1, 3, 5-triazin-2 (1H) -one) in which the linker group L or- (L-degradation determining region) is derivatized, for example, via a hydroxyl or amino linker group), and

4. decitabine (derivatized) (4-amino-1- (2-deoxy-b-D-erythro-pentofuranosyl) -1,3, 5-triazin-2 (1H) -one) (in which the linker group L or the- (L-degradation determining region) group is derivatized, for example, via a hydroxyl or amino linkage).

DD. targeting ligands by dTAG of functional tissues

Angiogenesis inhibitors:

angiogenesis inhibitors, including but not limited to:

sakamoto, et al, Development of procedure to target cancer-promoting proteins for solubilization and differentiation, Mol Cell Proteomics 2003 December; 2(12) GA-1 (derivatized) and derivatives and analogs thereof as described in 1350-8, having structures and binding to conjugates;

Rodriguez-Gonzalez, et al, Targeting stereo hormone receptors for solubilization and differentiation in break and state cancer, Oncogene (2008)27, 7201-;

sakamoto, et al, Development of procedure to target cancer-promoting proteins for solubilization and differentiation, Mol Cell Proteomics 2003 December; 1350-estradiol, testosterone (derivatized) and related derivatives including but not limited to DHT and its derivatives and analogs; and

sakamoto, et al, Protacs, molecular molecules which target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation Proc NatlAcad Sci USA.2001 Jul.17; 98(15) 8554-9 and the oophorin, fumagillin (derivatization) and derivatives and analogs thereof described in U.S. patent No. 7,208,157, having structure and binding to a linker group L or a- (L-degradation determining region) group.

Immunosuppressive compounds:

immunosuppressive compounds, including but not limited to:

AP21998 (derivatization) described in Chemical Genetic Control of Protein Levels: selected in Vivo Targeted Degradation, J.AM. CHEM.SOC.2004, 126, 3748-containing 3754, having the structure and binding to linker groups L or- (L-Degradation determining region) groups, such as Schnekloth et al, Chemical Genetic Control of Protein Levels: alternative in vivo is generally described. Targeted degeneration, J. And (6) AM. CHEM. And (4) SOC.2004, 126, 3748-3754;

2. glucocorticoids (e.g., hydrocortisone, prednisone, prednisolone and methylprednisolone), wherein the linker group L or- (L-degradation determining region) group is derivatized, e.g., with any hydroxyl group, and beclomethasone dipropionate (derived from the linker group) group or- (L-degradation determining region) group is bound, e.g., to the propionate;

3. methotrexate (where the linker group or- (L-degradation determining region) group can be derivatized, for example, in combination with any of the terminal hydroxyl groups);

4. cyclosporine (derivatised, wherein the linker group or- (L-degradation determining region) group may be derivatised in combination with any of the e.g. butyl groups);

5. tacrolimus (FK-506) and rapamycin (derivatised, where linker group L or- (L-degradation determining region) group may be attached, e.g. at either of the methoxy groups); and

6. actinomycin (derivatisation in which the linker group L or the- (L-degradation determining region) group may be bound to either of the isopropyl groups, for example).

Aryl Hydrocarbon Receptor (AHR) dTAG targeting ligand:

AHR dTAG targeting ligands used herein include, but are not limited to:

apigenin (derivatized in a manner that binds to a linker group L or a- (L-Degradation determining region) group as generally described in Lee, et al, Targeted differentiation of the Aryl Hydrocarbon Receptor by the PROTAC Approach: A Useful Chemical Genetic Tool, Chem Bio Chem Volume 8, Issue 17, pages 2058-; and

boitano, et al, Aryl Hydrocarbon receptors promoter products of the Expansion of Human hepatogenic Stem Cells, Science 10 Sep, 2010: Vol.329no.5997 pp.1345-1348 (derivatized by allowing the linker group L or- (L-degradation determining region) to bind).

Raf dTAG targeting ligand:

RAF dTAG targeting ligands used herein include, but are not limited to:

(wherein, for example, "R" designates the site at which the linker group L or the- (L-degradation determining region) group is attached and is derivatized).

FKBP dTAG targeting ligand:

FKBP dTAG targeting ligands used herein include, but are not limited to:

(wherein "R" designates the site at which the linker group L or the- (L-degradation determining region) group is attached and is derivatized).

HH. Androgen Receptor (AR) dTAG targeting ligand:

AR dTAG targeting ligands, as used herein, include, but are not limited to:

ligands of RU59063 androgen receptor (derivatization)

(wherein "R" designates the site at which the linker group L or the- (L-degradation determining region) group is attached and is derivatized).

2. SARM ligands (derivatisation) of the androgen receptor

(derivatization, wherein "R" represents the site of attachment of the linker group L or the- (L-degradation determining region) group).

3. Androgen receptor ligand DHT (derivatization)

(wherein "R" designates the site at which the linker group L or the- (L-degradation determining region) group is attached and is derivatized).

MDV3100 ligand (derivatization)

ARN-509 ligands (derivatised)

6. Hexahydrobenzisoxazole

7. Tetramethylcyclobutane

Estrogen Receptor (ER) dTAG targeting ligand:

ER dTAG targeting ligands as used herein include, but are not limited to:

1. estrogen receptor ligands

(wherein "R" designates the site at which the linker group L or the- (L-degradation determining region) group is attached and is derivatized).

Thyroid hormone receptor (TR) dTAG targeting ligand:

TR dTAG targeting ligands used herein include, but are not limited to:

1. thyroid hormone receptor ligands (derivatisation)

(wherein "R" designates the site at which the linker group L or the- (L-degradation determining region) group is attached, and MOMO is derivatized representing methoxymethoxy).

Hiv protease dTAG targeting ligand:

HIV protease dTAG targeting ligands used herein include, but are not limited to:

HIV protease inhibitors (derivatization)

(wherein "R" designates the site at which the linker group L or the- (L-degradation determining region) group is attached and is derivatized). See J.Med.chem.2010,53, 521-reservoir 538.

HIV protease inhibitors

(wherein "R" designates a potential site for attachment of a linker group L or a- (L-degradation determining region) group for derivatization). See J.Med.chem.2010,53, 521-reservoir 538.

Hiv integrase dTAG targeting ligand:

HIV integrase dTAG targeting ligands used herein include, but are not limited to:

HIV integrase inhibitors (derivatization)

(wherein "R" designates the site at which the linker group L or the- (L-degradation determining region) group is attached and is derivatized). See j.med.chem.2010,53,6466.

HIV integrase inhibitors (derivatization)

(wherein "R" designates the site at which the linker group L or the- (L-degradation determining region) group is attached and is derivatized). See j.med.chem.2010,53,6466.

Hcv protease dTAG targeting ligand:

HCV protease dTAG targeting ligands used herein include, but are not limited to:

HCV protease inhibitors (derivatization)

(wherein "R" designates the site at which the linker group L or the- (L-degradation determining region) group is attached and is derivatized).

NN. acyl-protein thioesterases-1 and-2 (APT1 and APT2) dTAG targeting ligand: acyl-protein thioesterase-1 and-2 (APT1 and APT2) dTAG targeting ligands used herein include, but are not limited to:

inhibitors of APT1 and APT2 (derivatised)

(wherein "R" designates the site at which the linker group L or the- (L-degradation determining region) group is attached and is derivatized). See, angelw. chem. int. ed.2011,50, 9838-.

Bcl2 dTAG targeting ligand:

BCL2 dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r is the site of linker attachment.

BCL-XL dTAG targeting ligand:

as used herein, BCL-XL dTAG targeting ligands include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Fa binding protein dTAG targeting ligand:

FA dTAG targeting ligands used herein include, but are not limited to:and

wherein:

r is the site of linker attachment.

Flap-5-lipoxygenase activating protein dTAG targeting ligand:

as used herein, FLAP-5-lipoxygenase activating protein dTAG targeting ligands include, but are not limited to:

wherein:

r is the site of linker attachment.

Hdac6 Zn finger domain dTAG targeting ligand:

HDAC6 Zn finger domain dTAG targeting ligands used herein include, but are not limited to:

wherein:

r is the site of linker attachment.

TT. tricyclic domain V4 BVV dTAG targeting ligand:

as used herein, a tricyclic domain V4 BVV dTAG targeting ligand includes, but is not limited to:

wherein:

r is the site of linker attachment.

Uu, lactoyl glutathione lyase dTAG targeting ligand:

as used herein, lactoyl glutathione lyase dTAG targeting ligands include, but are not limited to:

wherein:

r is the site of linker attachment.

Mpges-1 dTAG targeting ligand:

mPGES-1 dTAG targeting ligands, as used herein, include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Mthh 1dTAG targeting ligand:

as used herein, MTH 1dTAG targeting ligands include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Parp14 dTAG targeting ligand:

as used herein, PARP14 dTAG targeting ligands include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Yy. parp15 dTAG targeting ligand:

as used herein, PARP15 dTAG targeting ligands include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Pdz domain dTAG targeting ligand:

PDZ domain dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r and R' are the sites to which the linkers are attached.

Phip dTAG targeting ligand:

as used herein, a PHIP dTAG targeting ligand includes, but is not limited to:

wherein:

r is the site of linker attachment.

Phospholipase a2 domain dTAG targeting ligand:

as used herein, phospholipase a2 domain dTAG targeting ligands include, but are not limited to:and

wherein:

r is the site of linker attachment.

Ccc. protein S100-a 72 WOS dTAG targeting ligand:

protein S100-a 72 WOS dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Ddd, sphingolipid activator-B dTAG targeting ligand:

as used herein, a sphingolipid activator-B dTAG targeting ligand includes, but is not limited to:

and

wherein:

r is the site of linker attachment.

Sec7 dTAG targeting ligand:

sec7 dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Pp60Src dTAG targeting ligand SH2 domain:

the pp60Src dTAG targeting ligand SH2 domain used herein includes, but is not limited to:

and

wherein:

r is the site of linker attachment.

Tnk 1dTAG targeting ligand:

the Tank 1dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Hhh. ubch 9 SUMO E2 ligase SF6D dTAG targeting ligand:

ubc9 SUMO E2 ligase SF6D dTAG targeting ligands used herein include, but are not limited to:

and

wherein:

r is the site of linker attachment.

Src (c-Src) dTAG targeting ligand:

src dTAG targeting ligands, as used herein, include, but are not limited to:

a Src targeting ligand comprising AP 23464:

wherein:

r is the site of linker attachment.

Src-AS1 and/or Src AS2 targeting ligands include:

wherein:

r is the site of linker attachment.

Jjj. jak3 dTAG targeting ligand:

JAK3 dTAG targeting ligands used herein include, but are not limited to:

1. for ligands targeting JAK3, including Tofacitinib (Tofacitinib):

wherein:

r is the site of linker attachment.

Abl dTAG against ligand:

abl dTAG targeting ligands, as used herein, include, but are not limited to:

1. for Abl-targeting ligands, including tofacitinib and ponatinib:

wherein:

r is the site of linker attachment.

Mek 1dTAG targeting ligand:

MEK 1dTAG targeting ligands, as used herein, include, but are not limited to:

1. ligands directed against MEK1, including PD318088, Trametinib (Trametinib) and G-573:

wherein:

r is the site of linker attachment.

Kit dTAG targeting ligand:

KIT dTAG targeting ligands used herein include, but are not limited to:

1. ligands directed to KIT targeting include Regorafenib:

wherein:

r is the site of linker attachment.

Hiv reverse transcriptase dTAG targeting ligand:

HIV reverse transcriptase dTAG targeting ligands used herein include, but are not limited to:

1. targeting ligands targeting HIV reverse transcriptase, including efavirenz, tenofovir, Emtricitabine (Emtricitabine), ritonavir, latiravir (Raltegravir) and ritatavir (Atazanavir):

wherein:

r is the site of linker attachment.

Hiv protease Dtag targeting ligand:

HIV protease dTAG targeting ligands used herein include, but are not limited to:

targeting ligands for HIV protease, including ritonavir, raltavir and rituximab:

wherein:

r is the site of linker attachment.

In one embodiment, any of the aforementioned dTAG targeting ligands of table T is used to target any of the dtags described herein.

Many dTag targeting ligands are capable of binding more than one dTag, e.g., afatinib binds to EGFR, ErbB2 and ErbB4 proteins. This allowed for a double attack of many proteins as shown in table Z. In one embodiment, the dTAG targeting ligand is selected from the "dTAG targeting ligand" columns in table Z and the dTAG is selected from the corresponding "dTAG" row.

Table z.dtag targeting ligands and corresponding dtags

In certain embodiments, the present application includes compounds comprising the dTAG targeting ligands shown in table 1.

TABLE 1.dTAG targeting ligands 1-6

In certain embodiments, the dTAG targeting ligand is a compound of formula TL-I:

or a pharmaceutically acceptable salt thereof, wherein:is that

A¹Is S or C ═ C;

A²is NRa5 or O;

nn1 is 0,1 or 2;

each Ra¹Independently is C₁-C₃Alkyl radical (CH)₂)_0-3-CN，(CH₂)_0-3-halogen, (CH)₂)_0-3-OH，(CH₂)_0-3-C₁-C₃Alkoxy radical, C (O) NRa⁵L，OL，NRa⁵L or L;

Ra²is H, C₁-C₆Alkyl radical (CH)₂)_0-3-heterocyclyl (CH)₂)_0-3-phenyl or L, wherein the heterocyclyl comprises a saturated 5-or 6-membered ring and 1 to 2 heteroatoms selected from N, O and S, and optionally via C₁-C₃Alkyl, L or C (O) L substituted, and wherein phenyl is optionallyWarp C₁-C₃Alkyl, CN, halogen, OH, C₁-C₃Or L alkoxy;

nn2 is 0,1, 2or 3;

each Ra³Independently is C₁-C₃Alkyl radical (CH)₂)_0-3-CN，(CH₂)_0-3Halogen, L or C (O) NRa⁵L；

Ra⁴Is C₁-C₃An alkyl group;

Ra⁵is H or C₁-C₃An alkyl group; and

l is a linker which is a linker of,

provided that the compound of formula TL-I is substituted with only one L.

In some embodiments of the present invention, the,is that

In some embodiments, A¹Is S.

In some embodiments, A¹Is C ═ C.

In some embodiments, A²Is NRa⁵. In a further embodiment, Ra⁵Is H. In other embodiments, Ra⁵Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl). In a further embodiment, Ra⁵Is methyl.

In some embodiments, A²Is O.

In some embodiments, nn1 is 0.

In certain embodiments, nn1 is 1.

In certain embodiments, nn1 is 2.

In some embodiments, at least one Ra¹Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl). In a further embodiment, at least one Ra¹Is methyl. In a further embodiment, two Ra¹Is methyl.

In some embodiments, at least one Ra¹Is CN, (CH)₂)-CN，(CH₂)₂-CN or (CH)₂₎₃-CN. In a further embodiment, at least one Ra¹Is (CH)₂)-CN。

In some embodiments, at least one Ra¹Is halogen (e.g. F, Cl or Br), (CH)₂) -halogen, (CH)₂)₂-halogen or (CH)₂)₃-a halogen. In a further embodiment, at least one Ra¹Is Cl, (CH)₂)-Cl，(CH₂)₂-Cl or (CH)₂)₃-Cl。

In some embodiments, at least one Ra¹Is OH, (CH)₂)-OH，(CH₂)₂-OH or (CH)₂)₃-OH。

In some embodiments, at least one Ra¹Is C₁-C₃Alkoxy (e.g., methoxy, ethoxy or propoxy), (CH)₂)-C₁-C₃Alkoxy (CH)₂)₂-C₁-C₃Alkoxy or (CH)₂)₃-C₁-C₃An alkoxy group. In some embodiments, at least one Ra¹Is methoxy.

In some embodiments, an Ra¹Is C (O) NRa⁵And L. In a further embodiment, Ra⁵Is H. In other embodiments, Ra⁵Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl).

In some embodiments, an Ra¹Is OL.

In some toolsIn the example, one Ra¹Is NRa⁵And L. In a further embodiment, Ra⁵Is H. In other embodiments, Ra⁵Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl). In other embodiments, Ra⁵Is methyl.

In some embodiments, an Ra¹Is L.

In some embodiments, Ra²Is H.

In some embodiments, R^a2Is straight chain C₁-C₆Or branched C₃-C₆Alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, or hexyl). In a further embodiment, Ra²Is methyl, ethyl or tert-butyl.

In some embodiments, Ra²Is a heterocyclic radical, (CH)₂) -heterocyclyl (CH)₂)₂-a heterocyclic radical or (CH)₂)₃-a heterocyclic group. In a further embodiment, Ra²Is (CH)₂)₃-a heterocyclic group. In a further embodiment, heterocyclyl is selected from pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidyl, morpholinyl, and thiomorpholinyl. In further embodiments, heterocyclyl is piperazinyl.

In some embodiments, the heterocyclic group is through C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl) substituted.

In certain embodiments, heterocyclyl is substituted with C (O) L.

In certain embodiments, heterocyclyl is substituted with L.

In some embodiments, Ra²Is phenyl, (CH)₂) -phenyl, (CH)₂)₂-phenyl or (CH)₂)₃-phenyl. In a further embodiment, Ra²Is phenyl.

In some embodiments, the phenyl group is substituted with C₁-C₃Alkyl (e.g. methyl, ethyl)Propyl or isopropyl). In certain embodiments, the phenyl group is substituted with CN. In certain embodiments, the phenyl group is substituted with a halogen (e.g., F, Cl, or Br). In certain embodiments, the phenyl group is substituted with OH. In some embodiments, the phenyl group is substituted with C₁-C₃Alkoxy (e.g., methoxy, ethoxy, or propoxy) substituted.

In certain embodiments, the phenyl is substituted with L.

In some embodiments, Ra²Is L.

In some embodiments, nn2 is 0.

In certain embodiments, nn2 is 1.

In certain embodiments, nn2 is 2. In certain embodiments, nn2 is 3.

In some embodiments, at least one Ra³Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl). In a further embodiment, at least one Ra 3 is methyl.

In some embodiments, at least one Ra³Is CN, (CH)₂)-CN，(CH₂)₂-CN or (CH)₂)₃-CN. In a further embodiment, at least one Ra³Is CN.

In some embodiments, at least one Ra³Is halogen (e.g. F, Cl or Br), (CH)₂) -halogen, (CH)₂)₂-halogen or (CH2) 3-halogen. In a further embodiment, at least one Ra³Is Cl, (CH)₂)-Cl，(CH₂)₂-Cl or (CH)₂)₃-Cl. In further specific examples, at least one Ra³Is Cl.

In some embodiments, an Ra³Is L.

In some embodiments, an Ra³Is C (O) NRa⁵And L. In a further embodiment, Ra⁵Is H. In other embodiments, Ra⁵Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl).

In some embodiments, Ra⁴Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl). In a further embodiment, Ra⁴Is methyl.

In some embodiments, Ra⁵Is H.

In some embodiments, Ra⁵Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl). In a further embodiment, Ra⁵Is methyl.

In some embodiments, the first and second substrates are,is thatAnd A¹Is S.

In some embodiments, the first and second substrates are,is thatAnd A¹Is C ═ C.

In some embodiments of the present invention, the,is thatAnd A¹Is C ═ C.

In some embodiments, A²Is NH, and Ra²Is (CH2)_0-3-a heterocyclic group. In a further embodiment, Ra²Is (CH)₂)₃-a heterocyclic group. In further embodiments, heterocyclyl is piperazinyl. In a further embodiment, the heterocyclic group is substituted with C₁-C₃Alkyl, L or C (O) L.

In some embodiments, A²Is NH, and Ra²Is (CH)₂)_0-3-phenyl. In a further embodiment, Ra²Is phenyl. In further embodiments, the phenyl group is substituted with OH or L.

In some embodiments, A²Is NH, Ra²Is L.

In some embodiments, A²Is NH, and Ra²Is H or C₁-C₆An alkyl group. In a further embodiment, Ra²Is C₁-C₄An alkyl group.

In some embodiments, A²Is O, and Ra²Is H or C₁-C₆An alkyl group. In a further embodiment, Ra²Is C₁-C₄An alkyl group.

In certain embodiments, the dTAG targeting ligand is a compound of formula TL-I1:

or a pharmaceutically acceptable salt thereof, wherein A²，Ra¹，Ra²，Ra³，Ra⁴，Ra⁵And nn1 and nn2 are each as defined above for formula TL-1.

A²，Ra¹，Ra²，Ra³，Ra⁴，Ra⁵Each of nn1 and nn2 may be selected from moieties described in formula TL-1 above. A. the²，Ra¹，Ra²，Ra³，Ra⁴，Ra⁵，nn¹And nn²Each part of a definition of (a) may be related to a²，Ra¹，Ra²，Ra³，Ra⁴，Ra⁵Any of the moieties defined by the other moieties in nn1 and nn25 are combined as described above for formula TL-1.

In certain embodiments, the dTAG targeting ligand is a compound of the formula TL-I1a through TL-I1 d:

or a pharmaceutically acceptable salt thereof, wherein:

each Ra⁶Independently is C₁-C₃Alkyl radical (CH)₂)_0-3-CN，(CH₂)_0-3-halogen, (CH)₂)_0-3-OH or (CH)₂)_0-3-C₁-C₃An alkoxy group;

Ra⁷is (CH)₂)_0-3-heterocyclyl (CH)₂)_0-3-phenyl or L, wherein heterocyclyl comprises a saturated 5-or 6-membered ring and 1 to 2 heteroatoms selected from N, O and S, and is substituted with L or c (O) L, and wherein the phenyl is substituted with L;

Ra⁸is H, C₁-C₆Alkyl radical (CH)₂)_0-3-heterocyclyl or (CH)₂)_0-3-phenyl, wherein the heterocyclyl comprises a saturated 5-or 6-membered ring and 1 to 2 heteroatoms selected from N, O and S, and optionally via C₁-C₃Alkyl substituted, and wherein the phenyl is optionally C₁-C₃Alkyl, CN, halogen, OH or C₁-C₃Alkoxy substitution;

Ra¹⁰is C₁-C₃Alkyl radical (CH)₂)_0-3-CN or (CH)₂)_0-3-a halogen; and

A2，Ra⁴，Ra⁵and nn1 and L are each as defined above for formula TL-1.

In certain embodiments, nn1 is 0.

In certain embodiments, nn1 is 1.

In certain embodiments, nn1 is 2.

In some embodiments, at least one Ra⁶Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl). In a further embodiment, at least one Ra⁶Is methyl. In a further embodiment, two Ra⁶Is methyl.

In some embodiments, at least one Ra⁶Is CN, (CH)₂)-CN，(CH₂)₂-CN or (CH)₂)₃-CN. In further detailIn the example, at least one Ra⁶Is (CH)₂)-CN。

In some embodiments, at least one Ra⁶Is halogen (e.g. F, Cl or Br), (CH)₂) -halogen, (CH)₂)₂-halogen or (CH)₂)₃-a halogen. In a further embodiment, at least one Ra⁶Is Cl, (CH)₂)-Cl，(CH₂)₂-Cl or (CH)₂)₃-Cl。

In some embodiments, at least one Ra⁶Is OH, (CH)₂)-OH，(CH₂)₂-OH or (CH)₂)₃-OH。

In some embodiments, at least one Ra⁶Is C₁-C₃Alkoxy (e.g., methoxy, ethoxy or propoxy), (CH)₂)-C₁-C₃Alkoxy (CH)₂)₂-C₁-C₃Alkoxy or (CH)₂)₃-C₁-C₃An alkoxy group. In some embodiments, at least one Ra⁶Is methoxy.

In some embodiments, Ra⁷Is a heterocyclic radical, (CH)₂) -heterocyclyl (CH)₂)₂-a heterocyclic radical or (CH)₂)₃-a heterocyclic group. In a further embodiment, Ra⁷Is (CH)₂)₃-a heterocyclic group. In a further embodiment, heterocyclyl is selected from pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidyl, morpholinyl, and thiomorpholinyl. In further embodiments, heterocyclyl is piperazinyl.

In certain embodiments, heterocyclyl is substituted with C (O) L.

In certain embodiments, heterocyclyl is substituted with L.

In some embodiments, Ra⁷Is phenyl, (CH)₂) -phenyl, (CH)₂)₂-phenyl or (CH)₂)₃-phenyl. In a further embodiment, Ra⁷Is phenyl.

In some embodiments, Ra⁷Is L.

In some embodiments, Ra⁸Is H.

In some embodiments, Ra⁸Is straight chain C₁-C₆Or branched C₃-C₆Alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, or hexyl). In a further embodiment, Ra⁸Is methyl, ethyl or tert-butyl.

In some embodiments, Ra⁸Is a heterocyclic radical, (CH)₂) -heterocyclyl (CH)₂)₂-a heterocyclic radical or (CH)₂)₃-a heterocyclic group. In a further embodiment, Ra⁸Is (CH)₂)₃-a heterocyclic group. In a further embodiment, heterocyclyl is selected from pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidyl, morpholinyl, and thiomorpholinyl. In further embodiments, heterocyclyl is piperazinyl.

In some embodiments, the heterocyclic group is through C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl) substituted.

In some embodiments, Ra⁸Is phenyl, (CH)₂) -phenyl, (CH)₂)₂-phenyl or (CH)₂)₃-phenyl. In a further embodiment, Ra⁸Is phenyl.

In some embodiments, the phenyl group is substituted with C¹-C³Alkyl (e.g., methyl, ethyl, propyl, or isopropyl) substituted. In certain embodiments, the phenyl group is substituted with CN. In certain embodiments, the phenyl group is substituted with a halogen (e.g., F, Cl, or Br). In certain embodiments, the phenyl group is substituted with OH. In some embodiments, the phenyl group is substituted with C₁-C₃Alkoxy (e.g., methoxy, ethoxy, or propoxy) substituted.

In some embodiments, Ra¹⁰Is C₁-C₃Alkyl (e.g. methyl, ethyl, propyl or isopropyl)A base).

In some embodiments, Ra¹⁰Is CN, (CH)₂)-CN，(CH₂)₂-CN or (CH)₂)₃-CN。

In some embodiments, Ra¹⁰Is halogen (e.g., F, Cl or Br), (CH)₂) -halogen, (CH)₂)₂-halogen or (CH)₂)₃-a halogen. In a further embodiment, Ra¹⁰Is Cl, (CH)₂)-Cl，(CH₂)₂-Cl or (CH)₂)₃-Cl. In a further embodiment, Ra¹⁰Is Cl.

A²，Ra⁴，Ra⁵And nn1 may each be selected from moieties described in formula TL-1 above. A. the²，Ra⁴，Ra⁵，Ra⁶，Ra⁷，Ra⁸，Ra¹⁰And each of the parts defined as one of nn1 may be associated with a²，Ra⁴，Ra⁵，Ra⁶，Ra⁷，Ra⁸，Ra¹⁰And any partial combination defined by the other of nn1, as described above and in formula TL-1.

In certain embodiments, the dTAG targeting ligand is a compound of formula TL-I2:

or a pharmaceutically acceptable salt thereof, wherein A²，Ra¹，Ra²，Ra³，Ra⁴，Ra⁵And nn1 and nn2 are each as defined above for formula TL-1.

A²，Ra¹，Ra²，Ra³，Ra⁴，Ra⁵Each of nn1 and nn2 may be selected from moieties described in formula TL-1 above. A. the²，Ra¹，Ra²，Ra³，Ra⁴，Ra⁵Each moiety of the definition of one of nn1 and nn2 may be related to be a²，Ra¹，Ra²，Ra³， Ra⁴，Ra⁵Any combination of moieties defined elsewhere in nn1 and nn2, as described above for formula TL-1.

In certain embodiments, the dTAG targeting ligand is a compound of the formulae TL-I2a through TL-I2 c:

or a pharmaceutically acceptable salt thereof, wherein A²，Ra⁴，Ra⁵Each of nn1 and L is as defined above for formula TL-1, and Ra⁶，Ra⁷，Ra⁸And Ra¹⁰Each as defined above in formulas TL-I1a through TL-I1 d.

A²，Ra⁴，Ra⁵And each of nn1 may be selected from moieties described in formula TL-1 above, and Ra⁶，Ra⁷，Ra⁸And Ra¹⁰Each of which may be selected from moieties described above in formulas TL-I1a through TL-I1 d. A. the²，Ra⁴，Ra⁵，Ra⁶，Ra⁷，Ra⁸And Ra¹⁰And each of the parts defined as one of nn1 may be associated with a²，Ra⁴，Ra⁵，Ra⁶， Ra⁷，Ra⁸And Ra¹⁰And any combination of moieties defined for other moieties in nn1, as described above for formulas TL-1 and TL-I1a through TL-I1 d.

In certain embodiments, the dTAG targeting ligand is a compound of formula TL-I3:

or a pharmaceutically acceptable salt thereof.

A²，Ra¹，Ra²，Ra³，Ra⁴，Ra⁵And nn1 and nn2 are each as defined above for formula TL-1. A. the²，Ra¹，Ra²，Ra³，Ra⁴，Ra⁵Each of nn1 and nn2 may be selected from moieties described in formula TL-1 aboveAnd (4) dividing. A. the²，Ra¹，Ra²，Ra³，Ra⁴， Ra⁵Each moiety defined by one of nn1 and nn2 may be related to a²，Ra¹，Ra²， Ra³，Ra⁴，Ra⁵Any of the moieties defined elsewhere in nn1 and nn2 are combined as described above for formula TL-1.

In certain embodiments, the dTAG targeting ligand is a compound of the formulae TL-I3a through TL-I3 c:

or a pharmaceutically acceptable salt thereof, wherein:

Ra⁹is C (O) NRa⁵L，OL，NRa⁵L or L;

A²，Ra⁴，Ra⁵and each of nn1 and L is as defined above for formula TL-1; and

Ra⁶，Ra⁷，Ra⁸and Ra¹⁰Each as defined above in formula TL-I1a-TL-I1 d.

In some embodiments, Ra⁹Is C (O) NRa⁵And L. In a further embodiment, Ra⁵Is H. In other embodiments, Ra⁵Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl).

In some embodiments, Ra⁹Is OL.

In some embodiments, Ra⁹Is NRa⁵And L. In a further embodiment, Ra⁵Is H. In other embodiments, Ra⁵Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl). In other embodiments, Ra⁵Is methyl.

In some embodiments, Ra⁹Is L.

A²，Ra⁴，Ra⁵And each of nn1 may be selected from moieties described in formula TL-1 above, and Ra⁶，Ra⁷，Ra⁸And Ra¹⁰Each of which may be selected from moieties described above in formulas TL-I1a through TL-I1 d. A. the²，Ra⁴，Ra⁵，Ra⁶，Ra⁷，Ra⁸， Ra⁹，Ra¹⁰And each of the parts defined as one of nn1 may be associated with a²，Ra⁴，Ra⁵， Ra6，Ra⁷，Ra⁸，Ra⁹，Ra¹⁰And any portion defined by nn1, as described above and in formulas TL-I and TL-I1a through TL-I1 d.

In certain embodiments, the dTAG targeting ligand is a compound of the formula TL-VI:

or a pharmaceutically acceptable salt thereof, wherein:

Rf¹is C (O) NRf²L，OL，NRf²L or L;

Rf²independently is H or C₁-C₃An alkyl group; and

l is a linker.

In certain embodiments, Rf¹Is C (O) NRf²And L. In a further embodiment, Rf²Is H. In other embodiments, Rf²Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl).

In certain embodiments, Rf¹Is OL.

In certain embodiments, Rf¹Is NRe⁴And L. In a further embodiment, Rf²Is H. In other embodiments, Rf²Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl). In other embodiments, Rf²Is methyl.

In certain embodiments, Rf¹Is L.

In certain embodiments, the dTAG targeting ligand is a compound of formula TL-VII:

or a pharmaceutically acceptable salt thereof, wherein:

T₇is CH₂Or CH₂CH₂；

Rg¹Is C (O) Rg⁵Or (CH2)1-3Rg⁶；

nn10 is 0,1, 2or 3;

nn11 is 0,1, 2or 3;

each Rg²Independently is C₁-C₃Alkyl radical, C₁-C3 alkoxy, CN or halogen;

Rg³is C (O) NRg⁴L，OL，NRg⁴L，L，O-(CH₂)_1-3-C(O)NRg⁴L, or NHC (O) - (CH)₂)_1-3-C(O)NRg⁴L；

Rg⁴Is H or C₁-C₃An alkyl group;

Rg⁵is C₁-C₆An alkyl group;

Rg⁶is optionally passed through C₁-C₃Alkyl radical, C₁-C₃Alkoxy, CN or halogen substituted phenyl; and

l is a linker.

In some embodiments, T⁷Is CH₂。

In some embodiments, T⁷Is CH₂CH₂。

In certain embodiments, Rg¹Is C (O) Rg⁵。

In certain embodiments, Rg¹Is (CH)₂)-Rg⁶，(CH₂)₂-Rg⁶Or (CH)₂)₃- Rg⁶。

In certain embodiments, Rg⁵Is straight chain C₁-C₆Or branched C₃-C₆Alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, or hexyl).

In certain embodiments, Rg⁶Is unsubstituted phenyl.

In certain embodiments, Rg⁶Is phenyl substituted with one, two, three or more substituents independently selected from C₁-C₃Alkyl (e.g. methyl, ethyl, propyl or isopropyl), C₁-C₃Alkoxy (e.g., methoxy, ethoxy or propoxy), CN and halogen (e.g., F, Cl or Br).

In certain embodiments, nn10 is 0.

In certain embodiments, nn10 is 1.

In certain embodiments, nn10 is 2.

In some embodiments, nn10 is 3.

In certain embodiments, nn11 is 0.

In certain embodiments, nn11 is 1.

In certain embodiments, nn11 is 2.

In some embodiments, nn11 is 3.

In certain embodiments, at least one Rg²Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl). In further embodiments, at least one Rg²Is methyl.

In certain embodiments, at least one Rg²Is C₁-C₃Alkoxy (e.g., methoxy, ethoxy, or propoxy). In further embodiments, at least one Rg²Is methoxy.

In certain embodiments, at least one Rg²Is CN.

In certain embodiments, at least one Rg²Is halogen (e.g., F, Cl or Br).

In certain embodiments, Rg³Is C (O) NRg⁴And L. In a further embodiment, Rg⁴Is H, in other embodiments, Rg⁴Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl).

In certain embodiments, Rg³Is OL.

In certain embodiments, Rg³Is NRg⁴And L. In a further embodiment, Rg⁴Is H. In other embodiments, Rg⁴Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl). In other embodiments, Rg⁴Is methyl.

In certain embodiments, Rg³Is L.

In certain embodiments, Rg³Is O- (CH)₂)-C(O)NRg⁴L，O-(CH₂)₂-C (O) NRg4L or O- (CH)₂)₃-C(O)NRg⁴And L. In a further embodiment, Rg³Is O- (CH)₂)-C(O)NRg⁴And L. In a further embodiment, Rg⁴Is H. In other embodiments, Rg⁴Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl).

In certain embodiments, Rg³Is NHC (O) - (CH)₂)-C(O)NRg⁴L， NHC(O)-(CH₂)₂-C(O)NRg⁴L, or NHC (O) - (CH)₂)₃-C(O)NRg⁴And L. In a further embodiment, Rg³Is NHC (O) - (CH)₂)-C(O)NRg⁴L，NHC(O)- (CH₂)₂-C(O)NRg⁴And L. In a further embodiment, Rg³Is NHC (O) - (CH)₂)₂- C(O)NRg⁴And L. In a further embodiment, Rg⁴Is H. In other embodiments, Rg⁴Is C₁-C₃Alkyl (e.g., methyl, ethyl, propyl, or isopropyl).

In certain embodiments, the dTAG targeting ligand is selected from the structures shown in fig. 28, wherein R is the site of attachment of a linker.

In certain embodiments, a dTAG-targeting ligand or target is selected based on the presence (known dTAG binding moiety) and the ability to develop potent and selective ligands with functional sites that can accommodate the linker. Some embodiments relate to dTAG targeting ligands with lower selectivity that can benefit from degradation associated with proteomics as a measure of compound selectivity or target ID.

Some embodiments of the application relate to 30% to 100% degradation or loss of the CAR. Certain embodiments involve a 50 to 100% loss of CAR. Other embodiments involve a loss of CAR of 75 to 95%.

Non-limiting examples of heterobifunctional compounds useful in the present invention include those shown in FIGS. 29, 30, 31, and 32.

Figure 29 provides specific heterobifunctional compounds useful in the present invention.

FIG. 30 provides specific heterobifunctional compounds useful in the present invention, wherein X in the above structures is a halogen selected from F, Cl, Br and I.

Figure 31 provides specific heterobifunctional compounds useful in the present invention.

Figure 32 provides specific heterobifunctional compounds useful in the present invention,

wherein: r^AR1Is selected from: andand

R^AR2is selected from: and

other compounds useful in the invention include the structures of fig. 33.

Some of the aforementioned heterobifunctional compounds comprise one or more asymmetric centers and thus may exist in various isomeric forms, such as stereoisomers and/or diastereomers. Thus, the compounds and pharmaceutical compositions thereof may be in the form of individual enantiomers, diastereomers or geometric isomers, or may be in the form of a mixture of stereoisomers. In certain embodiments, the compounds of the present application are enantiomerically pure compounds. In certain other embodiments, a mixture of stereoisomers or diastereomers is provided.

Furthermore, unless otherwise specified, certain heterobifunctional compounds as described herein may have one or more double bonds, which may exist as either Z or E isomers. The present application also includes compounds as individual isomers substantially free of other isomers, or as mixtures of various isomers, such as racemic mixtures of stereoisomers. In addition to the above compounds themselves, the present application also includes pharmaceutically acceptable derivatives of these heterobifunctional compounds and compositions comprising one or more of the compounds of the present application and one or more pharmaceutically acceptable excipients or additives.

The heterobifunctional compounds of the present application can be prepared by crystallizing the compounds under different conditions, and can exist as one of the polymorphs of the compounds forming part of the present application, or a combination thereof. For example, recrystallization can be performed using different solvents or different solvent mixtures to identify and/or prepare different polymorphs; by crystallization at different temperatures; or from very fast to very slow cooling during crystallization by using various cooling modes. Polymorphs can also be obtained by heating or melting the compound followed by gradual or rapid cooling. The presence of polymorphs can be determined by solid probe NMR spectroscopy, IR spectroscopy, differential scanning calorimetry, powder X-ray diffraction patterns and/or other techniques. Thus, the present application includes heterobifunctional compounds, derivatives thereof, tautomeric forms thereof, stereoisomers thereof, polymorphs thereof, pharmaceutically acceptable salts thereof, pharmaceutically acceptable solvates thereof and pharmaceutically acceptable compositions containing the compounds.

General Synthesis of heterobifunctional Compounds

The heterobifunctional compounds described herein can be prepared by methods known to those skilled in the art. In one non-limiting example, the disclosed heterobifunctional compounds can be prepared by the following scheme.

Reaction scheme 1

Reaction scheme 2

As shown in reaction scheme 1, heterobifunctional compounds for use in the present invention can be prepared by chemically binding the degradation determining region and linker, followed by the addition of a dTAG targeting ligand. Similarly, in scheme 2, heterobifunctional compounds useful in the present invention are prepared by first chemically combining a dTAG targeting ligand and a linker, and then adding a degradation determining region.

As shown above and in the following reaction schemes, one skilled in the art can readily synthesize heterobifunctional compounds for use in the present invention by a variety of methods and chemical reactions.

Reaction scheme 3

Reaction scheme 3: in step 1, the nucleophilic degradation determining region displaces a leaving group on the linker to produce a degradation determining region linker fragment. In step 2, the protecting group is removed by methods known in the art to release the nucleophilic site on the linker. In step 3, the nucleophilic degradation determining region linker fragment displaces a leaving group on the dTAG targeting ligand to form a compound for use in the present invention. In another embodiment, step 1 and/or step 2 is accomplished by a coupling reaction rather than nucleophilic attack.

Reaction scheme 4

Reaction scheme 4: in step 1, a nucleophilic dTAG-targeting ligand displaces a leaving group on the linker to prepare a dTAG-targeting ligand linker fragment. In step 2, the protecting group is removed by methods known in the art to release the nucleophilic site on the linker. In step 3, the nucleophilic dTAG-targeting ligand linker fragment displaces the leaving group on the degradation determining region to form a compound for use in the present invention. In another embodiment, step 1 and/or step 2 is accomplished by a coupling reaction rather than nucleophilic attack.

Reaction scheme 5

Reaction scheme 6

Reaction scheme 5 and reaction scheme 6: in step 1, the nucleophilic degradation determining region displaces a leaving group on the linker to produce a degradation determining region linker fragment. In step 2, the protecting group is removed by methods known in the art to release the nucleophilic site on the linker. In step 3, the nucleophilic degradation determining region linker fragment displaces a leaving group on the dTAG targeting ligand to form the compound of formula I or formula II. In another embodiment, step 1 and/or step 2 is accomplished by a coupling reaction rather than nucleophilic attack.

a) Reacting tert-butyl (2-aminoethyl) carbamate or an analog thereof (e.g., n ═ 1 to 20) (1) or an analog thereof (e.g., n ═ 1 to 20) with chloroacetyl chloride under suitable conditions to produce tert-butyl (2- (2-chloroacetamido) ethyl) carbamate or an analog thereof (e.g., n ═ 1 to 20) (2);

b) reacting tert-butyl (2- (2-chloroacetamido) ethyl) carbamate or analog thereof (2) with dimethyl 3-hydroxyphthalite under suitable conditions to provide 3- (2- ((2- ((tert-butoxycarbonyl) amino)) ethyl) amino) -2-oxoethoxy) phthalate or analog thereof (3);

c) reacting dimethyl 3- (2- ((2- ((tert-butoxycarbonyl) amino) ethyl) amino) -2-oxoethoxy) phthalate or an analogue thereof (3) with a strong base and then with 3-aminopiperidin-2, 6-reactive-dione hydrochloride to produce tert-butyl (2- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamido) ethyl) carbamate or an analogue thereof (4);

d) deprotecting compound (4) to provide diaminoacetyl-O-thalidomide trifluoroacetate or analog thereof (5);

e) compound (5) is reacted with an acid derivative of a dTAG targeting ligand (compound (6)) under suitable conditions to give bifunctional compound (7).

In certain embodiments, the above process is carried out in a solution phase. In certain other embodiments, the above method is performed on a solid phase. In certain embodiments, the synthetic methods are applicable to high throughput techniques or techniques commonly used in combinatorial chemistry.

Representative Synthesis of heterobifunctional Compounds

Unless otherwise indicated, starting materials are commercially available or readily available by laboratory synthesis by anyone familiar with the art. Generally described below are methods and general guidelines for the synthesis of the compounds generally described and the subclasses and classes herein.

Synthesis example 1': synthesis of IMiD derivatives and degradation determining regions

General procedure I: IMiD condensation

2- (2, 6-dioxopiperidin-3-yl) -4-hydroxyisoindoline-1, 3-dione (D-1)

In a 20mL glass vial, a mixture of 3-hydroxyphthalic anhydride (500mg, 3.05 mmol, 1 equiv.), potassium acetate (927mg, 9.44mmol, 3.1 equiv.), and 3-aminopiperidine-2, 6-dione hydrochloride (552mg, 3.35mmol, 1.1 equiv.) in acetic acid (10.2 mL, 0.3M) was heated to 90 ℃ overnight. The black reaction mixture was cooled to room temperature and diluted to 20mL with water, followed by cooling on ice for 30 minutes. The resulting slurry was transferred to a 50mL Falcon tube and centrifuged at 3500rpm for 5 minutes. The supernatant was discarded and the black solid was transferred to 250mL RBF containing methanol and concentrated in vacuo. By flash column Chromatography (CH)₂Cl₂: MeOH (9: 1)) on silica gel to give the title compound as a white solid (619mg, 74%).¹H NMR(400MHz,DMSO-d₆) δ 11.07(s,1H),7.65(dd, J ═ 8.4,6.8Hz,1H),7.31(d, J ═ 6.8Hz,1H), 7.24(d, J ═ 8.4Hz,1H),5.06(dd, J ═ 12.8,5.4Hz,1H), 2.94-2.82 (m,1H), 2.64-2.43 (m,2H), 2.08-1.97 (m, 1H); calculating C₁₃H₁₁N₂O₅[M+H]⁺275.07, ms (esi), found to be 275.26.

2- (2, 6-dioxopiperidin-3-yl) -4-nitroisoindoline-1, 3-dione (D-10)

Flash column Chromatography (CH) using 3-nitrophthalic anhydride (300mg, 1.55mmol, 1 eq), potassium acetate (473mg, 4.82mmol, 3.1 eq) and 3-aminopiperidine-2, 6-dione hydrochloride (281mg, 1.71mmol) according to general procedure I₂Cl₂: MeOH (9: 1)) on silica gel to give the title compound as a pale yellow solid (280mg, 59%).¹H NMR(500MHz,DMSO-d₆) δ 11.17(s,1H),8.35(d, J ═ 8.1Hz,1H), 8.24(d, J ═ 7.5Hz,1H), 8.14-8.10 (m,1H),5.20(dd, J ═ 12.9, 5.5Hz,1H), 2.93-2.84 (m,1H), 2.64-2.45 (m,2H), 2.11-2.04 (m, 1H); calculating C₁₃H₁₀N₃O₆[M+H]⁺304.06, ms (esi), found to be 304.19.

2- (2, 6-dioxopiperidin-3-yl) -5-nitroisoindoline-1, 3-dione (D-2)

Following general procedure I, 4-nitrophthalic anhydride (300mg, 1.55 mmol), potassium acetate (473mg, 4.82 mmol) were usedmmol) and 3-aminopiperidine-2, 6-dione hydrochloride (281mg, 1.71mmol) by flash column Chromatography (CH)₂Cl₂: MeOH (30: 1)) on silica gel gave the title compound as a white solid (409mg, 87%).¹H NMR(500MHz,DMSO-d₆) δ 11.18(s,1H),8.68(dd, J ═ 8.1,1.9 Hz,1H),8.56(d, J ═ 1.9Hz,1H),8.19(d, J ═ 8.1Hz,1H),5.24(dd, J ═ 12.9,5.4Hz,1H),2.90(ddd, J ═ 17.2,13.9,5.5Hz,1H), 2.69-2.48 (m,2H), 2.14-2.05 (m, 1H); calculating C₁₃H₁₀N₃O₆[M+H]⁺304.06, ms (esi), found to be 304.19.

2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-6)

According to general method I, using phthalic anhydride (155mg, 1.05mmol), potassium acetate (318mg, 3.24mmol) and 3-aminopiperidine-2, 6-dione hydrochloride (189mg, 1.15mmol), by flash column chromatography (CH2 Cl)₂: MeOH (15: 1)) on silica gel gave the title compound as a white solid (235mg, 87%).¹H NMR (500MHz,DMSO-d₆) δ 11.13(s,1H), 8.00-7.76 (m,4H),5.16(dd, J ═ 12.8,5.4Hz,1H),2.89(ddd, J ═ 16.8,13.7,5.4Hz,1H), 2.65-2.42 (m,2H), 2.12-1.99 (m, 1H); calculation of C₁₃H₁₁N₂O₄[M+H]⁺259.07, ms (esi), found to be 259.23.

2- (2, 5-dioxopyrrolidin-3-yl) isoindoline-1, 3-dione (D-7)

According to general method I, using phthalic anhydride (90mg, 0.608mmol), potassium acetate (185mg, 1.88mmol) and 3-aminopyrrolidine-2, 5-dione hydrochloride (101mg, 0.668mmol), by flash column Chromatography (CH)₂Cl₂: MeOH (14: 1)) on silica gel gave the title compound as a white solid (95mg, 64%). Calculating C₁₂H₉N₂O₄[M+H]⁺245.06, ms (esi), found to be 45.26.

2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindoline-5-carboxylic acid (D-13)

Following general procedure I, using 1,2, 4-benzenetricarboxylic anhydride (200mg, 1.04mmol), ethyl acetatePotassium (317mg, 3.23mmol) and 3-aminopiperidine-2, 6-dione hydrochloride (188mg, 1.15 mmol). By flash column Chromatography (CH)₂Cl₂: MeOH (9: 1)) was purified on silica gel to give the title compound as a white solid (178mg, 57%). Calculating C₁₄H₁₁N₂O₆[M+H]⁺303.06, ms (esi), found to be 303.24.

2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (D-14)

After purification by flash column chromatography (CH2Cl 2: MeOH (50: 1)) on silica gel using 3-fluorophthalic anhydride (200mg, 1.20mmol), potassium acetate (366mg, 3.73mmol) and 3-aminopiperidine-2, 6-dione hydrochloride (218mg, 1.32mmol) according to general method I, the title compound was obtained as a white solid (288mg, 86%).¹H NMR (500MHz,DMSO-d₆) δ 11.15(s,1H),7.96(ddd, J ═ 8.3,7.3,4.5Hz, 1H), 7.82-7.71 (m,2H),5.17(dd, J ═ 13.0,5.4Hz,1H),2.90(ddd, J ═ 17.1,13.9,5.4Hz,1H), 2.65-2.47 (m,2H), 2.10-2.04 (m,1H), calculate C₁₃H₁₀FN₂O₄[M+H]⁺277.06, ms (esi), found to be 277.25.

2- (2, 6-dioxopiperidin-3-yl) -4-methylisoindoline-1, 3-dione (D-19)

According to general method I, using 3-methylphthalic anhydride (150mg, 0.925 mmol), potassium acetate (281mg, 2.87mmol) and 3-aminopiperidine-2, 6-dione hydrochloride (167mg, 1.02mmol), by flash column Chromatography (CH)₂Cl₂: MeOH (15: 1)) on silica gel gave the title compound as a white solid (168mg, 67%). Calculating C₁₄H₁₃N₂O₄[M+H]⁺273.09, ms (esi), found to be 273.24.

2- (2, 6-dioxopiperidin-3-yl) -5-fluoroisoindoline-1, 3-dione (D-24)

Following general procedure I, using 4-fluorophthalic anhydride (200mg, 1.20mmol), potassium acetate (366mg, 3.73mmol) and 3-aminopiperidine-2, 6-dione hydrochloride (218mg, 1.32mmol), by flash column Chromatography (CH)₂Cl₂: MeOH (15: 1)) on silica gelAfter workup, the title compound was obtained as a white solid (254mg, 76%). Calculating C₁₃H₁₀FN₂O₄[M+H]⁺277.06, ms (esi), found to be 277.24.

2- (2, 6-dioxopiperidin-4-yl) isoindoline-1, 3-dione (D-43)

After purification by flash column chromatography (CH2Cl 2: MeOH (9: 1)) on silica gel using phthalic anhydride (60mg, 0.311mmol), potassium acetate (95mg, 0.963mmol) and 4-aminopiperidine-2, 6-dione hydrochloride (56mg, 0.342mmol) according to general method I, the title compound was obtained as a white solid (40mg, 43%). Calculating C₁₃H₁₁N₂O₄[M+H]⁺259.07, ms (esi), found to be 259.18.

General procedure II: reduction of aromatic nitro groups

4-amino-2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-4)

2- (2, 6-dioxopiperidin-3-yl) -4-nitroisoindoline-1, 3-dione (173mg, 0.854mmol), Pd (OAc)₂(12.8mg, 0.0854mmol, 10 mol%) potassium fluoride (66mg, 1.71mmol, 2 equiv.) in THF: a solution of water (8: 1) (5.7mL, 0.1M) was stirred at room temperature. Triethylsilane (365 μ L, 3.41mmol, 4 equiv.) was added slowly and the resulting black solution was stirred at room temperature for 1 hour. The reaction mixture was filtered through a pad of celite, washing with excess ethyl acetate. The filtrate was concentrated in vacuo and the residue was purified by flash column Chromatography (CH)₂Cl₂: MeOH (7: 1)) was purified on silica gel to give the title compound as a yellow powder (72mg, 46%).¹H NMR(500MHz,DMSO-d₆) δ 11.08(s, 1H),7.47(dd, J ═ 8.5,7.0Hz,1H), 7.06-6.95 (m,1H), 6.59-6.44 (m,1H),5.04(dd, J ═ 12.7,5.4Hz,1H), 2.93-2.82 (m,1H), 2.64-2.45 (m,2H), 2.05-1.98 (m, 1H); calculating C₁₃H₁₁N₃O₄[M+H]⁺274.08, ms (esi), found to be 274.23.

2- (2, 6-dioxopiperidin-3-yl) -5-nitroisoindole-1, 3-dione (D-8)

Following general procedure II, using 2- (2, 6-dioxopiperidin-3-yl) -5-nitroisoindoline-1, 3-dione (100mg, 0.330mmol), Pd (OAc)₂(7.4mg, 0.033mmol) potassium fluoride (38mg, 0.660mmol) and triethylsilane (211. mu.L, 1.32mmol) were purified by flash column chromatography (CH2Cl 2: MeOH (9: 1))) on silica gel to give the title compound as a yellow solid (33mg, 37%).¹H NMR(500MHz,DMSO- d₆) δ 11.05(s,1H),7.52(d, J ═ 8.2Hz,1H),6.94(d, J ═ 2.0Hz,1H), 6.83(dd, J ═ 8.2,2.0Hz,1H),6.55(s,2H),5.01(dd, J ═ 12.8,5.4Hz,1H), 2.86(ddd, J ═ 16.9,13.9,5.5Hz,1H), 2.68-2.43 (m,2H), 2.03-1.93 (m, 1H); calculating C₁₃H₁₂N₃O₄[M+H]⁺274.08, ms (esi), found to be 274.59.

4-amino-2- (1-benzyl-2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-12)

Following general procedure II, using 2- (1-benzyl-2, 6-dioxopiperidin-3-yl) -4-nitroisoindoline-1, 3-dione (48mg, 0.122mmol), Pd (OAc)₂(2.7mg, 0.0122 mmol), potassium fluoride (14mg, 0.244mmol) and triethylsilane (78 μ L, 0.488 mmol) gave, after purification by flash column chromatography (0 to 100% hexanes in EtOAc), the title compound as a yellow solid (7mg, 16%). Calculations C₂₀H₁₈N₃O₄ [M+H]⁺364.13, ms (esi), found to be 364.34.

3- (5-amino-2-methyl-4-oxoquinazolin-3 (4H) -yl) piperidine-2, 6-dione (D-17)

Following general procedure II, using 3- (2-methyl-5-nitro-4-oxoquinazolin-3 (4H) -yl) piperidine-2, 6-dione (21mg, 0.0664mmol), Pd (OAc)₂(1.5mg, 0.0066 mmol), potassium fluoride (7.7mg, 0.133mmol) and triethylsilane (42 μ L, 0.266 mmol) were purified by preparative HPLC to give the title compound as a white solid (7mg, 37%). Calculating C₁₄H₁₅N₄O₃[M+H]⁺287.11, ms (esi), found to be 287.30.

3- (7-amino-1-oxoisoindolin-2-yl) piperidine-2, 6-dione (D-41)

Following general procedure II, using 3- (7-nitro-1-oxoisoindolin-2-yl) piperidine-2, 6-dione (11mg, 0.038mmol), Pd (OAc)₂(0.9mg, 0.0038mmol), potassium fluoride (4.4mg, 0.076mmol) and triethylsilane (24 μ L, 0.152 mmol) by flash column chromatography (0 to 10% MeOH in CH)₂Cl₂Solution) on silica gel to give the title compound as a yellow solid (2mg, 21%). Calculating C₁₃H₁₄N₃O₃ [M+H]⁺260.10, ms (esi), found to be 260.52.

General procedure III: acylation of anilines

N- (2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) acetamide (D-5)

In a 4mL glass vial, a mixture of 5-amino-2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (30mg, 0.110mmol, 1 eq) and acetyl chloride (26 μ L, 0.220 mmol, 2 eq) in THF (1.8mL, 0.1M) was heated to reflux overnight. The reaction mixture was filtered and the filter cake was taken up in Et₂O wash to afford the title compound as a white solid (27mg, 47%) which was used without further purification.¹H NMR(500MHz, DMSO-d₆) δ 11.11(s,1H),10.63(s,1H),8.24(d, J ═ 1.5Hz,1H), 7.91-7.83 (m,2H),5.11(dd, J ═ 12.8,5.4Hz,1H),2.88(ddd, J ═ 17.0,13.8,5.4Hz,1H), 2.63-2.46 (m,2H),2.13(s,3H), 2.09-2.00 (m, 1H); calculating C₁₅H₁₄N₃O₅[M+H]⁺316.09, ms (esi), found to be 316.23.

N- (2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) acetamide (D-3)

Following general procedure III, using 4-amino-2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (50mg, 0.183mmol) and acetyl chloride (26 μ L, 0.366mmol), the title compound was obtained as a white solid (10mg, 17%).¹H NMR(500MHz, DMSO-d₆) δ 11.14(s,1H),9.73(s,1H),8.44(d, J ═ 8.4Hz,1H), 7.83(dd, J ═ 8.4,7.3Hz,1H),7.62(d, J ═ 7.2Hz,1H),5.14(dd, J ═ 12.9,5.4Hz,1H),2.90(ddd, J ═ 17.1,13.9,5.4Hz,1H), 2.66-2.45 (m,2H),2.19(s,3H), 2.14-2.00 (m, 1H); calculating C₁₅H₁₄N₃O₅ [M+H]⁺316.09, ms (esi), found to be 316.27.

2-chloro-N- (2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) acetamide (D-32)

Following general procedure III, using 5-amino-2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (10mg, 0.0366mmol) and chloroacetyl chloride (6 μ L, 0.0732mmol), the title compound was obtained as a white solid (7.1mg, 55%). Calculating C₁₅H₁₃ClN₃O₅ [M+H]⁺350.05, ms (esi), found to be 350.23.

2-chloro-N- (2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindol-4-yl) acetamide (D-34)

Following general procedure III, using 3- (4-amino-1-oxoisoindolin-2-yl) piperidine-2, 6-dione (20mg, 0.0771mmol) and chloroacetyl chloride (12 μ L, 0.154mmol), the title compound was obtained as a white solid (14.9mg, 56%).¹H NMR(500MHz, DMSO-d₆) δ 11.02(s,1H),10.20(s,1H),7.81(dd, J ═ 7.7,1.3Hz, 1H), 7.65-7.47 (m,2H),5.16(dd, J ═ 13.3,5.1Hz,1H), 4.45-4.34 (m,2H),4.33(s,2H), 3.00-2.85 (m,1H), 2.68-2.56 (m,1H), 2.41-2.28 (m,1H), 2.09-1.97 (m, 1H); calculating C₁₅H₁₅ClN₃O₄[M+H]⁺336.07, ms (esi), found to be 336.31.

N- (2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindol-4-yl) acrylamide (D-35)

Following general procedure III, using 3- (4-amino-1-oxoisoindolin-2-yl) piperidine-2, 6-dione (20mg, 0.0771mmol) and acryloyl chloride (13 μ L, 0.154mmol), the title compound was obtained as a white solid (18mg, 76%).¹H NMR(500MHz, DMSO-d₆)δ15.77(s,1H),14.81(s,1H),12.65(dd,J＝7.4,1.6Hz, 1H),12.37–12.18(m,2H),11.28(dd,J＝17.0,10.2Hz,1H),11.06(dd,J ═ 17.0,1.9Hz,1H),10.57(dd, J ═ 10.2,1.9Hz,1H),9.91(dd, J ═ 13.3,5.1Hz,1H), 9.24-9.05 (m,2H),7.67(ddd, J ═ 17.2,13.7, 5.5Hz,1H),7.36(dt, J ═ 17.3,3.8Hz,1H), 7.20-7.03 (m,1H), 6.83-6.72 (m, 1H); calculating C₁₆H₁₆N₃O₄[M+H]⁺314.11, ms (esi), found to be 314.24.

N- (2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) acrylamide (D-36)

Following general procedure III, using 5-amino-2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (10mg, 0.0366mmol) and acryloyl chloride (6 μ L, 0.0732mmol), the title compound was obtained as a white solid (8.8mg, 73%).¹H NMR(500MHz, DMSO-d₆) δ 11.12(s,1H),10.83(s,1H),8.33(d, J ═ 1.8Hz,1H), 7.99(dd, J ═ 8.2,1.9Hz,1H),7.90(d, J ═ 8.2Hz,1H),6.48(dd, J ═ 17.0,10.1Hz,1H),6.36(dd, J ═ 17.0,1.9Hz,1H),5.88(dd, J ═ 10.0,1.9Hz,1H),5.13(dd, J ═ 12.8,5.5Hz,1H), 2.95-2.84 (m,1H), 2.67-2.46 (m,2H), 2.09-2.01 (m, 1H); calculating C₁₆H₁₄N₃O₅ [M+H]⁺328.09, ms (esi), found to be 328.23.

N- (2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindol-4-yl) acetamide (D-37)

Following general procedure III, using 3- (4-amino-1-oxoisoindolin-2-yl) piperidine-2, 6-dione (20mg, 0.0771mmol) and acetyl chloride (11 μ L, 0.154mmol), the title compound was obtained. White solid (17 mg, 71%). MS (ESI) calculation of C₁₅H₁₆N₃O₄ [M+H]⁺302.11, found to be 301.99.

N- (2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindol-4-yl) cyclopropanecarboxamide (D-38)

Following general procedure III, using 3- (4-amino-1-oxoisoindolin-2-yl) piperidine-2, 6-dione (20mg, 0.0771mmol) and cyclopropanecarbonyl chloride (11 μ L, 0.154mmol), the title compound was obtained as a white solid (19mg, 75%).¹H NMR (500MHz,DMSO-d₆)δ11.01(s,1H),10.06(s,1H),7.84(dd,J＝ 7.2,1.9Hz,1H),7.66–7.38(m,2H),5.14(dd,J＝13.3,5.1Hz,1H), 4.52-4.30 (m,2H),2.92(ddd, J ═ 17.3,13.6,5.4Hz,1H), 2.64-2.54 (m,1H), 2.45-2.27 (m,1H), 2.08-1.95 (m,1H), 1.93-1.83 (m,1H), 0.90-0.75 (m, 4H); calculating C₁₇H₁₈N₃O₄[M+H]⁺328.13, ms (esi), found to be 328.00.

General procedure IV: quinazolinone condensation

3- (2-methyl-4-oxoquinazolin-3 (4H) -yl) piperidine-2, 6-dione (D-9)

In a 20mL glass vial, anthranilic acid (100mg, 0.729mmol, 1 eq.), acetic acid (42 μ L, 0.729mmol, 1 eq.) and P (OPh)₃(479. mu.L, 1.82mmol, 2.5 equiv.) in pyridine (1.0uL, 0.7M) heated to 90 ℃. After 4h, the reaction mixture was cooled to room temperature and 3-aminopiperidine-2, 6-dione hydrochloride (144mg, 0.875mmol, 1.2 equiv.) was added. The reaction mixture was heated to 90 ℃ for 1.5 hours and then stirred at room temperature overnight. The reaction mixture was dissolved in EtOAc (15mL) and water (15 mL). The organic layer was washed with brine (2X 25mL) and Na₂SO₄Dried and concentrated in vacuo. Flash column chromatography (0-5% MeOH in CH)₂Cl₂Solution) the residue was purified on silica gel to give the title compound as a white solid (79mg, 40%).¹H NMR(500MHz,DMSO-d₆) δ 11.03(s,1H),8.03(dd, J ═ 7.9,1.5Hz,1H),7.82(ddd, J ═ 8.5,7.1,1.6Hz,1H),7.62(dd, J ═ 8.3,1.1Hz,1H),7.50(ddd, J ═ 8.1,7.1,1.1Hz,1H),5.27(dd, J ═ 11.5,5.7Hz,1H), 2.92-2.78 (m,1H), 2.73-2.56 (m,5H), 2.26-2.06 (m, 1H); calculating C₁₄H₁₄N₃O₃[M+H]⁺272.10, ms (esi), found to be 272.33.

3- (2-methyl-4-oxoquinazolin-3 (4H) -yl) pyrrolidine-2, 5-dione (D-11)

Following general procedure IV, anthranilic acid (200mg, 1.46mmol), acetic acid (84. mu.L, 1.46mmol), P (OPh)₃(959 μ L, 3.65mmol) and 3-aminopyrrolidine-2, 5-dione hydrochloric acidSalt (263mg, 1.75mmol) by flash column Chromatography (CH)₂Cl₂: MeOH (15: 1)) on silica gel to give the title compound as a white solid (25mg, 7%). Calculating C₁₃H₁₂N₃O₃[M+H]⁺258.09, ms (esi), found to be 258.22.

3- (5-fluoro-2-methyl-4-oxoquinazolin-3 (4H) -yl) piperidine-2, 6-dione (D-66)

Following general procedure IV, using 6-fluoroanthranilic acid (100mg, 0.645 mmol), acetic acid (37. mu.L, 0.644mmol), P (OPh)₃(424. mu.L, 1.61mmol) and 3-aminopiperidine-2, 6-dione hydrochloride (127mg, 0.774mmol) by flash column chromatography (0 to 10% MeOH in CH)₂Cl₂) Purification on silica gel gave the title compound as a white solid (70mg, 38%).¹H NMR(500MHz,DMSO-d₆) δ 11.03(s,1H), 7.84-7.76 (m,1H),7.44(dd, J ═ 8.2,1.0Hz,1H),7.25(ddd, J ═ 11.1,8.2,1.0Hz,1H),5.24(dd, J ═ 11.3,5.7Hz,1H), 2.90-2.75 (m,1H),2.62(s,3H), 2.61-2.56 (m,2H), 2.20-2.12 (m, 1H); calculating C₁₄H₁₃FN₃O₃[M+H]⁺290.09, ms (esi), found to be 290.27.

3- (2-methyl-5-nitro-4-oxoquinazolin-3 (4H) -yl) piperidine-2, 6-dione (D-67)

Following general procedure IV, using 6-nitroanthranilic acid (100mg, 0.549mmol), acetic acid (31 μ L, 0.549mmol), P (OPh)₃(361. mu.L, 1.37mmol) and 3-aminopiperidine-2, 6-dione hydrochloride (108mg, 0.659mmol) by flash column chromatography (0 to 10% MeOH in CH)₂Cl₂) Purification on silica gel gave the title compound as a white solid (29mg, 17%). Calculating C₁₄H₁₃N₄O₅[M+H]⁺317.09, ms (esi), found to be 317.58.

General procedure V: amide coupling

N-benzyl-2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindoline-5-carboxamide (D-15)

In a 4mL glass vial, a solution of 2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindoline-5-carboxylic acid (10mg, 0.033mmol, 1 eq.), HATU (13mg, 0.033mmol, 1 eq.), DIPEA (17. mu.L, 0.099mmol, 3 eq.) and benzylamine (4. mu.L, 0.036mmol, 1.1 eq.) in DMF (331. mu.L, 0.1M) was stirred at room temperature overnight. The reaction mixture was diluted to 4mL with MeOH, filtered, and then purified by preparative HPLC to give the title compound as a white solid (6mg, 46%). Calculating C₂₁H₁₈N₃O₅[M+H]⁺392.12, ms (esi), found to be 392.33.

General procedure VI: nucleophilic aromatic substitution

4- (benzylamino) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-16)

In a 4mL glass vial, 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (10mg, 0.036mmol, 1 equiv.), benzylamine (4.4. mu.L, 0.040 mmol, 1.1 equiv.) and DIPEA (13. mu.L, 0.072mmol, 2 equiv.) were heated to 90 ℃ overnight in NMP (362. mu.L, 0.1M). The reaction mixture was cooled to room temperature and dissolved in EtOAc (15 mL). The organic layer was washed with NaHCO₃(aqueous) (15mL), water (15mL) and brine (3X 15mL), followed by Na₂SO₄Dried and concentrated in vacuo. The residue was purified by flash column chromatography (0 to 100% EtOAc in hexanes) on silica gel to give the title compound as a yellow film (5mg, 38%).¹H NMR (500MHz, chloroform-d) δ 8.10(s,1H),7.44(dd, J ═ 8.5,7.1Hz,1H), 7.40-7.25 (m,5H), 7.12(d, J ═ 7.1Hz,1H),6.84(d, J ═ 8.5Hz,1H),6.71(t, J ═ 5.9Hz,1H), 4.93(dd, J ═ 12.3,5.3Hz,1H),4.51(d, J ═ 5.9Hz,2H), 2.93-2.66 (m,3H), 2.21-2.07 (m, 1H); calculating C₂₀H₁₈N₃O₄[M+H]⁺364.13, ms (esi), found to be 364.31.

2- (2, 6-dioxopiperidin-3-yl) -4- (isopropylamino) isoindoline-1, 3-dione (D-18)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (30mg, 0.109mmol), isopropylamine (10. mu.L, 0.119mmol) and DIPEA (21. mu.L, 0.119mmol), purification (`) by flash column chromatography on silica gel gave the title compound as a yellow film (11mg, 32%) (0 to 100% EtOAc in hexane). Calculating C₁₆H₁₈N₃O₄[M+H]⁺316.13, ms (esi), found to be 316.65.

4- (diethylamino) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-21)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (30mg, 0.109mmol), diethylamine (11. mu.L, 0.130mmol) and DIPEA (32. mu.L), purification by flash column chromatography (0-100% EtOAc in hexane) on silica gel afforded the title compound as a yellow film (28mg, 97%). Calculating C₁₇H₂₀N₃O₄[M+H]⁺330.14, ms (esi), found to be 330.62.

5- (benzylamino) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-25)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -5-fluoroisoindoline-1, 3-dione (30mg, 0.109mmol), benzylamine (13 μ L, 0.119mmol) and DIPEA (38 μ L, 0.217mmol), purification by flash column chromatography (0 to 100% EtOAc in hexanes) gave the title compound as a yellow film (6mg, 15%). Calculating C₂₀H₁₈N₃O₄[M+H]⁺364.13, ms (esi), found to be 364.34.

2- (2, 6-dioxopiperidin-3-yl) -5- (isopropylamino) isoindoline-1, 3-dione (D-26)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -5-fluoroisoindoline-1, 3-dione (30mg, 0.109mmol), isopropylamine (11 μ L, 0.130mmol) and DIPEA (38 μ L, 0.127mmol), purification by flash column chromatography (0 to 100% EtOAc in hexanes) on silica gel afforded the title compound as a yellow film (6mg, 17%).¹H NMR (500MHz, chloroform-d)δ 8.00(s,1H),7.53(d, J ═ 8.3Hz,1H), 6.87(d, J ═ 2.1Hz,1H),6.64(dd, J ═ 8.3,2.2Hz,1H),4.86(dd, J ═ 12.3,5.4Hz,1H),4.30(d, J ═ 7.8Hz,1H), 2.86-2.58 (m,3H), 2.12-2.01 (m,1H), 1.26-1.15 (m, 6H); calculating C₁₆H₁₈N₃O₄[M+H]⁺316.13, ms (esi), found to be 316.30.

5- (diethylamino) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-27)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -5-fluoroisoindoline-1, 3-dione (30mg, 0.109mmol), diethylamine (14. mu.L, 0.130mmol) and DIPEA (38. mu.L, 0.127mmol), flash column chromatography (0 to 100% EtOAc in hexane) on purified silica gel afforded the title compound as a yellow film (6mg, 31%).¹H NMR (500MHz, chloroform-d) δ 8.08(s,1H),7.57(d, J ═ 8.6 Hz,1H),6.98(d, J ═ 2.4Hz,1H),6.72(dd, J ═ 8.7,2.4Hz,1H), 4.90-4.80 (m,1H),3.40(q, J ═ 7.1Hz,4H), 2.89-2.61 (m,3H), 2.11-2.01 (m,1H),1.16(t, J ═ 7.1Hz, 6H); calculating C₁₇H₂₀N₃O₄[M+H]⁺330.14, ms (esi), found to be 330.69.

2- (2, 6-dioxopiperidin-3-yl) -5- ((furan-2-ylmethyl) amino) isoindoline-1, 3-dione (D-28)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -5-fluoroisoindoline-1, 3-dione (50mg, 0.181mmol), furfuryl amine (18. mu.L, 0.199mmol) and DIPEA (63. mu.L, 0.362mmol), by flash column chromatography (0 to 5% MeOH in CH)₂Cl₂) Purification on silica gel gave the title compound as a yellow film (8mg, 13%). Calculating C₁₈H₁₆N₃O₄[M+H]⁺354.11, ms (esi), found to be 354.25.

Tert-butyl (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) amino) ethyl) carbamate (D-29)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (50mg, 0.181mmol), 1-Boc-ethylenediamine (32mg, 0.199mmol) and DIPEA (63. mu.L, 0.362mmol), by flash column chromatography (0.L)To 10% MeOH in CH2Cl₂) After purification on silica gel, the title compound was obtained as a yellow film (31mg, 41%).¹H NMR(500MHz,CDCl₃) δ 8.08(bs,1H),7.50 (dd, J ═ 8.5,7.1Hz,1H),7.12(d, J ═ 7.1Hz,1H),6.98(d, J ═ 8.5Hz,1H), 6.39(t, J ═ 6.1Hz,1H), 4.96-4.87 (m,1H),4.83(bs,1H), 3.50-3.41 (m,2H), 3.41-3.35 (m,2H), 2.92-2.66 (m,3H), 2.16-2.09 (m,1H),1.45(s, 9H); calculating C₂₀H₂₅N₄O₆[M+H]⁺417.18, ms (esi), found to be 417.58.

(2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-5-yl) amino) ethyl) carbamic acid tert-butyl ester (D-30)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -5-fluoroisoindoline-1, 3-dione (50mg, 0.181mmol), 1-Boc-ethylenediamine (32mg, 0.199mmol) and DIPEA (63 μ L, 0.362mmol), by flash column chromatography (0 to 10% MeOH in CH)₂Cl₂) Purification on silica gel gave the title compound as a yellow film (22mg, 29%). Calculating C₂₀H₂₅N₄O₆[M+H]⁺417.18, ms (esi), found to be 417.32.

2- (2, 6-dioxopiperidin-3-yl) -4- ((furan-2-ylmethyl) amino) isoindoline-1, 3-dione (D-31)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (19.5mg, 0.0706mmol), furfuryl amine (7. mu.L, 0.078mmol) and DIPEA (25. mu.L, 0.141mmol), by flash column chromatography (0 to 2.5% MeOH in CH)₂Cl₂) Purification on silica gel afforded the title compound as a yellow film (19mg, 76%). Calculating C₁₈H₁₆N₃O₄[M+H]⁺354.11, ms (esi), found to be 354.27.

3- (5- (benzylamino) -2-methyl-4-oxoquinazolin-3 (4H) -yl) piperidine-2, 6-dione (D-39)

In accordance with general method VI, the reaction mixture was heated to 170 ℃ instead of 90 ℃, using 3- (5-fluoro-2-methyl-4-oxoquinazolin-3 (4H) -yl) piperidine-2, 6-dione (30mg,0.104mmol), benzylamine (13. mu.L, 0.114mmol) and DIPEA (36. mu.L, 0.207mmol) by flash column chromatography (0 to 10% MeOH in CH)₂Cl₂) Purification on silica gel gave the title compound as a white solid (15mg, 38%).¹H NMR (500MHz, chloroform-d) δ 8.73(t, J ═ 5.7Hz,1H),8.39(s,1H),7.41(t, J ═ 8.1Hz,1H), 7.39-7.19 (m,5H),6.77(d, J ═ 7.7Hz,1H),6.41(d, J ═ 8.3Hz,1H),4.67(dd, J ═ 11.5,5.9Hz,1H),4.43(d, J ═ 5.7Hz,2H), 3.03-2.79 (m,2H), 2.72-2.61 (m,1H),2.60(s,3H), 2.15-2.07 (m, 1H); calculating C₂₁H₂₁N₄O₃[M+H]⁺377.16, ms (esi), found to be 377.02.

3- (5- (isopropylamino) -2-methyl-4-oxoquinazolin-3 (4H) -yl) piperidine-2, 6-dione (D-40)

In accordance with general method VI, except that the reaction mixture was heated to 170 ℃ instead of 90 ℃, using 3- (5-fluoro-2-methyl-4-oxoquinazolin-3 (4H) -yl) piperidin-2, 6-one (30mg, 0.104mmol), isopropylamine (10 μ L, 0.114mmol) and DIPEA (36 μ L, 0.207mmol), by flash column chromatography on silica gel (0 to 10% MeOH in CH)₂Cl₂) Purification afforded the title compound as a white solid (5mg, 15%).¹H NMR (500MHz, chloroform-d) δ 8.31(s,1H),8.21(d, J ═ 7.2Hz,1H), 7.50-7.37 (m,1H),6.70(dd, J ═ 7.9,0.9Hz,1H),6.47(d, J ═ 8.4Hz,1H), 4.65(dd, J ═ 11.4,5.9Hz,1H), 3.69-3.56 (m,1H), 3.03-2.80 (m,3H),2.58(s,3H), 2.14-2.03 (m,1H),1.27(d, J ═ 2.7Hz,3H), 1.26(d, J ═ 2.7Hz, 3H); calculating C₁₇H₂₁N₄O₃[M+H]⁺329.16, ms (esi), found to be 329.97.

2- (2, 6-dioxopiperidin-3-yl) -4- ((2-hydroxyethyl) amino) isoindoline-1, 3-dione (D-68)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (30mg, 0.109mmol), aminoethanol (7. mu.L, 0.119mmol) and DIPEA (38. mu.L, 0.217mmol), by flash column chromatography (0 to 5% MeOH in CH)₂Cl₂) Purification on silica gel gave the title compound as a yellow film (6mg, 18%).¹H NMR (500MHz, chloroform-d) delta 8.26(s,1H),7.50(dd, J ═ 8.5,7.1Hz,1H),7.12(d, J ═ 7.0Hz,1H),6.95(d, J ═ 8.5Hz,1H),6.50(t, J ═ 5.9Hz,1H), 4.97-4.85 (m,1H), 3.94-3.79 (m,2H),3.47(q, J ═ 5.5Hz,2H), 3.03-2.68 (m,3H), 2.19-2.04 (m, 1H); calculating C₁₅H₁₆N₃O₅[M+H]⁺318.11, ms (esi), found to be 318.22.

4- (cyclopropylamino) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D47)

According to general method VI, using 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (20mg, 0.0724mmol), cyclopropylamine (6. mu.L, 0.080mmol) and DIPEA (25. mu.L, 0.141mmol), by flash column chromatography (0 to 5% MeOH in CH)₂Cl₂) Purification on silica gel afforded the title compound as a yellow film (16mg, 70%).¹H NMR (500MHz, chloroform-d) δ 8.05(s,1H),7.53(dd, J ═ 8.5,7.1Hz,1H), 7.33-7.21 (m,1H),7.15(dd, J ═ 7.1,0.7Hz,1H),6.44(bs, 1H), 4.95-4.85 (m,1H), 2.98-2.66 (m,3H), 2.62-2.50 (m,1H), 2.19-2.06 (m,1H), 0.92-0.78 (m,2H), 0.67-0.56 (m, 2H); calculating C₁₆H₁₆N₃O₄[M+H]⁺314.11, ms (esi), found to be 314.54.

4- ((2- (1H-indol-3-yl) ethyl) amino) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-48)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (20mg, 0.0724mmol), tryptamine (13mg, 0.080mmol) and DIPEA (25. mu.L, 0.144mmol), flash column chromatography on silica gel (0 to 10% MeOH in CH)₂Cl₂) Purification gave the title compound as a yellow film (10mg, 33%).¹H NMR (500MHz, chloroform-d) δ 8.14(s,1H),8.11(s,1H), 7.65-7.55 (m,1H), 7.45(dd, J ═ 8.6,7.1Hz,1H),7.37(dt, J ═ 8.2,0.9Hz,1H), 7.21(ddd, J ═ 8.2,7.0,1.2Hz,1H), 7.16-7.04 (m,3H),6.88(d, J ═ 8.5Hz,1H),6.34(t, J ═ 5.6Hz,1H),4.89(dd, J ═ 12.4,5.4Hz,1H), 3.59(td, J ═ 6.8,5.5, 2H), 3.19-3.03 (m,2H),2.93, 2H (m,2H), 3.04 (m,2H, 14H, 1H); calculating C₂₃H₂₁N₄O₄[M+H]⁺417.16, ms (esi), found to be 417.26.

2- (2, 6-dioxopiperidin-3-yl) -4- ((4-hydroxyphenylethyl) amino) isoindoline-1, 3-dione (D-49)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (20mg, 0.0724mmol), tyramine (11mg, 0.080mmol) and DIPEA (25. mu.L, 0.144mmol), by flash column chromatography (0 to 5% MeOH in CH)₂Cl₂) Purification on silica gel gave the title compound as a yellow film (15mg, 54%).¹H NMR (500MHz, chloroform-d) δ 8.20(s,1H),7.51(dd, J ═ 8.5,7.1Hz,1H), 7.17-7.08 (m,2H),6.90(d, J ═ 8.5Hz,1H), 6.85-6.72 (m,2H), 4.95-4.90 (m,1H), 3.52-3.46 (m,2H), 2.97-2.87 (m,2H), 2.86-2.72 (m,2H), 2.21-2.09 (m, 1H); calculating C₂₁H₂₀N₃O₅[M+H]⁺394.14, ms (esi), found to be 394.25.

4- ((2- (1H-imidazol-2-yl) ethyl) amino) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-50)

Following general procedure VI, using 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (20mg, 0.0724mmol), histamine (15mg, 0.080mmol) and DIPEA (25. mu.L, 0.144mmol), by flash column chromatography (0 to 10% MeOH in CH)₂Cl₂) Purification on silica gel gave the title compound as a yellow film (5mg, 19%).¹H NMR (500MHz, chloroform-d) δ 8.19(s,1H),7.61(d, J ═ 1.2Hz,1H), 7.47(dd, J ═ 8.5,7.1Hz,1H),7.07(d, J ═ 6.9Hz,1H), 6.96-6.83 (m,2H),6.39(t, J ═ 5.7Hz,1H), 4.97-4.79 (m,1H),3.59(q, J ═ 6.5Hz,2H),2.95(t, J ═ 6.6Hz,2H), 2.92-2.62 (m,2H), 2.16-2.04 (m, 1H); (ii) a Calculating C₁₈H₁₈N₅O₄[M+H]⁺368.14, ms (esi), found to be 368.47.

General procedure VII: acylation of primary amines

N- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) methyl) cyclopropanecarboxamide (D-22)

In a 4mL glass vial, 4- (aminomethyl) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (25mg, 0.087mmol, 1 equiv.) and DIPEA (30. mu.L, 0.174mmol, 2 equiv.) in MeCN (250. mu.L, 0.35M) were cooled to 0 ℃. Cyclopropanecarbonyl chlorocyclopropane pseudoacid chloride (8.7 μ L, 0.096mmol) was added slowly and the reaction mixture was stirred at room temperature overnight. The product was isolated by filtration to give the title compound as a white solid (4.8mg, 15%) which was used without further purification. Calculating C₁₈H₁₈N₃O₅ [M+H]⁺356.12, ms (esi), found to be 356.32.

N- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) methyl) acetamide (D-23)

Following general procedure VII, using 4- (aminomethyl) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (25mg, 0.087mmol), DIPEA (30 μ L, 0.174 mmol) and acetyl chloride (7 μ L, 0.096mmol), the title compound was obtained as a white solid (4.5mg, 16%).¹H NMR(500MHz,DMSO-d₆) δ 11.13(s,1H), 8.47(t, J ═ 6.0Hz,1H), 7.88-7.76 (m,2H),7.70(dt, J ═ 7.3, 1.1Hz,1H),5.15(dd, J ═ 12.7,5.4Hz,1H),4.69(d, J ═ 6.0Hz,2H), 2.90(ddd, J ═ 16.8,13.8,5.4Hz,1H), 2.64-2.44 (m,2H), 2.15-2.01 (m,1H),1.92(s, 3H); calculating C₁₆H₁₆N₃O₅[M+H]⁺330.11, ms (esi), found to be 330.05.

2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) amino) ethan-1-am-monium-2, 2, 2-trifluoroacetate (D-33)

To a stirred solution of tert-butyl (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) amino) ethyl) carbamate (205mg, 0.492mmol, 1 eq) in dichloromethane (2.25mL) was added trifluoroacetic acid (0.250 mL). The reaction mixture was stirred at room temperature for 4 hours, then the volatiles were removed in vacuo. The title compound was obtained as a yellow solid (226mg,>95%) which is different fromFurther purifying for use.¹H NMR (500MHz, MeOD) δ 7.64(d, J ═ 1.4Hz,1H), 7.27-7.05 (m,2H),5.10 (dd, J ═ 12.5,5.5Hz,1H),3.70(t, J ═ 6.0Hz,2H), 3.50-3.42 (m,2H), 3.22(t, J ═ 6.0Hz,1H), 2.93-2.85 (m,1H), 2.80-2.69 (m,2H), 2.17-2.10 (m, 1H); calculating C₁₅H₁₇N₄O₄[M+H]⁺317.12, ms (esi), found to be 317.53.

General procedure VIII: phenol alkylation

2- (2, 6-dioxopiperidin-3-yl) -4- ((4- (morpholinomethyl) benzyl) oxy) isoindoline-1, 3-dione (D-45)

In a 4mL glass vial, 2- (2, 6-dioxopiperidin-3-yl) -4-hydroxyisoindoline-1, 3-dione (30mg, 0.109mmol, 1 eq.) and K₂CO₃(15mg, 0.109mmol, 1 equiv.) in DMF (365. mu.L, 0.3M) was stirred at room temperature. 4- (4- (bromomethyl) benzyl) morpholine (30mg, 0.109mmol, 1 eq) in DMF (200 μ L) was added and the reaction mixture was stirred at room temperature for 4 days. The reaction mixture was dissolved in water (15mL) and EtOAc (15mL), the organic layer was washed with brine (3X 15mL), Na₂SO₄Dried and concentrated in vacuo. Flash column chromatography (0 to 10% MeOH in CH)₂Cl₂) The residue was purified on silica gel to give the title compound as a white solid (20mg, 40%).¹H NMR(500MHz,DMSO-d₆) δ 11.10(s,1H),7.82(dd, J ═ 8.5,7.2Hz, 1H),7.60(d, J ═ 8.5Hz,1H), 7.50-7.42 (m,3H),7.35(d, J ═ 8.1Hz, 2H),5.35(s,2H),5.09(dd, J ═ 12.8,5.5Hz,1H), 3.64-3.51 (m,4H), 3.46(s,2H),2.88(ddd, J ═ 17.0,14.1,5.4Hz,1H), 2.63-2.47 (m,2H), 2.38-2.31 (m,4H), 2.07-1.99 (m, 1H); calculating C₂₅H₂₆N₃O₆ [M+H]⁺464.18, ms (esi), found to be 464.00.

4- (benzyloxy) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-46)

According to general method VIII, using 2- (2, 6-dioxopiperidin-3-yl) -4-hydroxyisoindoleIndole-1, 3-dione (30mg, 0.109mmol), K₂CO₃(15mg, 0.109mmol) and benzyl bromide (8. mu.L, 0.109mmol) by flash column chromatography (0 to 10% MeOH in CH)₂Cl₂Solution) on silica gel to give the title compound as a white solid (8mg, 20%).¹H NMR(500MHz,DMSO-d₆) δ 11.10(s,1H),7.83(dd, J ═ 8.5,7.3Hz,1H),7.60(d, J ═ 8.5Hz,1H), 7.53-7.50 (m,2H),7.47 (d, J ═ 7.2Hz,1H), 7.45-7.39 (m,2H), 7.38-7.32 (m,1H),5.38(s, 2H),5.09(dd, J ═ 12.8,5.5Hz,1H),2.88(ddd, J ═ 16.9,13.8,5.5 Hz,1H), 2.64-2.46 (m,2H), 2.07-1.99 (m, 1H); calculating C₂₀H₁₇N₂O₅ [M+H]⁺365.11, ms (esi), found to be 365.21.

2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) amino) ethyl 4-methylbenzenesulfonate (D-44)

In a 4mL glass vial, 2- (2, 6-dioxopiperidin-3-yl) -4- ((2-hydroxyethyl) amino) isoindoline-1, 3-dione (7mg, 0.0221mmol, 1 eq.) and Et₃N (3. mu.L, 0.033mmol, 1.5 eq) in CH₂Cl₂(200. mu.L) was stirred at room temperature. Tosyl chloride (6mg, 0.026mmol, 1.2 equiv.) in CH was added₂Cl₂(100. mu.L) and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and the residue was purified by flash column chromatography (0 to 10% MeOH in CH)₂Cl₂) Purification on silica gel gave the title compound as a white solid (4mg, 40%).¹H NMR(500MHz,DMSO-d₆) δ 11.13(s,1H), 7.64-7.59 (m,2H),7.46(dd, J ═ 8.6,7.1Hz,1H), 7.33-7.27 (m,2H), 7.04-6.93 (m,2H),6.58(t, J ═ 6.4Hz,1H), 5.09(dd, J ═ 12.7,5.4Hz,1H),4.15(t, J ═ 5.1Hz,2H), 3.65-3.52 (m,2H), 2.97-2.83 (m,1H), 2.67-2.46 (m,2H),2.27(s,3H), 2.12-2.02 (m, 1H); calculating C₂₂H₂₂N₃O₇S[M+H]⁺472.12, ms (esi), found 472.39.

(R) -4-hydroxy-2- (3-methyl-2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-52)

Hydroxyisobenzofuran-1, 3-dione (147.08mg, 0.896mmol, 1 eq.) was added to (R) -3-amino-3-methylpiperidine-2, 6-dione hydrochloric acid (127.32mg, 0.896mmol, 1 eq.). Pyridine (3.584mL, 0.25M) was then added to the mixture and stirred at 110 ℃ for 17 hours. The mixture was diluted with methanol and concentrated under reduced pressure. The crude material was purified by column chromatography (ISCO, 24g silica column, 0 to 10% MeOH/DCM, 25 min gradient) to give a white oil (110.9mg, 42.63% yield).¹H NMR(400MHz,DMSO-d₆)δ10.95(s,1H),7.61(dd,J＝8.4,7.2Hz, 1H),7.27–7.14(m,2H),2.73–2.63(m,1H),2.57–2.51(m,1H), 2.04–1.97(m,1H),1.86(s,3H)。

LCMS 289(M+H)。

(S) -4-hydroxy-2- (3-methyl-2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-53)

4-hydroxyisobenzofuran-1, 3-dione (148.99mg, 0.907mmol, 1 equivalent) was added to (S) -3-amino-3-methylpiperidine-2, 6-dione hydrochloric acid (128.97mg, 0.907mmol, 1 equivalent). Pyridine (3.628mL, 0.25M) was then added to the mixture and stirred at 110 ℃ for 17 hours. The mixture was diluted with methanol and concentrated under reduced pressure. The crude material was purified by column chromatography (ISCO, 24g silica column, 0 to 10% MeOH/DCM, 25 min gradient) to give a white oil (150mg, 57.4% yield).¹H NMR(400MHz,DMSO-d₆)δ10.95(s,1H),7.62(dd,J＝8.4,7.2Hz, 1H),7.27–7.16(m,2H),2.75–2.62(m,1H),2.55(dd,J＝14.0,4.3 Hz,1H),2.05–1.96(m,1H),1.86(s,3H).LCMS 289(M+H)。

(S) -2- ((2- (3-methyl-2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetic acid (D-55)

To TFA (0.63mL, 0.1M) was added tert-butyl (S) -2- ((2- (3-methyl-2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetate (25.4mg, 0.063 mmol, 1 equiv.) and the mixture was stirred at 50 ℃ for 1 hour. The mixture was then diluted with methanol and concentrated under reduced pressure to give a white powder (20.5mg, 93.9% yield)) It was used without further purification.¹H NMR (500MHz, methanol-d)₄)δ7.81 –7.75(m,1H),7.50(d,J＝7.3Hz,1H),7.45(d,J＝8.6Hz,2H), 7.43–7.37(m,3H),5.09(dd,J＝12.8,5.5Hz,1H),4.76(s,2H), 4.63(dd,J＝9.1,5.2Hz,1H),3.66–3.55(m,30H),3.51–3.41(m, 5H),2.90–2.83(m,1H),2.79–2.71(m,2H),2.69(s,3H),2.43(s, 3H),2.14(ddt,J＝10.5,5.5,3.2Hz,1H),1.69(s,3H).LCMS 347 (M+H)。

(R) -2- ((2- (3-methyl-2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetic acid (D-54)

To TFA (1.78mL, 0.1M) was added tert-butyl (R) -2- ((2- (3-methyl-2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetate (71.3mg, 0.178 mmol, 1 eq) and the mixture was stirred at 50 ℃ for 1 hour. The mixture was then diluted with methanol and concentrated under reduced pressure to give a white powder (47.2mg, 76.63% yield), which was used on without further purification.¹H NMR (400MHz, methanol-d)₄)δ7.72 (ddd,J＝8.5,7.3,5.0Hz,1H),7.46–7.42(m,1H),7.30(dd,J＝8.6, 4.5Hz,1H),4.94(d,J＝5.3Hz,2H),2.81–2.56(m,2H),2.24– 2.07(m,1H),2.00(s,2H),0.90(t,J＝6.5Hz,2H).LCMS 347 (M+H)。

4, 7-dichloro-2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (D-51)

4, 7-Dichloroisobenzofuran-1, 3-dione (434.6mg, 2.002mmol, 1 equivalent) was added to 3-aminopiperidine-2, 6-dione hydrochloric acid (362.6mg, 2.203mmol, 1.1 equivalents). Potassium acetate (609.07mg, 6.206mmol, 3.1 equiv.) and acetic acid (6.67mL, 0.3M) were then added to the mixture and stirred at 90 ℃ for 18 h. The mixture was cooled to room temperature, diluted with deionized water and centrifuged for 5 minutes. The precipitate was diluted with methanol and concentrated under reduced pressure. The crude material was purified by column chromatography (ISCO, 12g silica column, 0 to 10% MeOH/DCM, 25 min gradient) to give a white powder (160.4mg, 24.5% yield).¹H NMR(500MHz,DMSO-d₆)δ11.15(s,1H),7.91 (s,2H),5.17(dd,J＝12.9,5.4Hz,1H),2.88(ddd,J＝17.2,13.9,5.4 Hz,1H),2.68–2.54(m,1H),2.05(ddd,J＝10.5,5.4,2.7Hz,1H). LCMS 328(M+H)。

Synthesis example 1: synthesis of dBET1

(1) Synthesis of JQ-acid

JQ1(1.0g, 2.19mmol, 1 eq.) was dissolved in formic acid (11mL, 0.2M) and stirred at room temperature for 75 h. The mixture was concentrated under reduced pressure to give a yellow solid (0.99g, quantitative yield), which was used without purification.¹H NMR (400MHz, methanol-d)₄)δ7.50 –7.36(m,4H),4.59(t,J＝7.1Hz,1H),3.51(d,J＝7.1Hz,2H), 2.70(s,3H),2.45(s,3H),1.71(s,3H).LCMS 401.33(M+H)。

N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate was synthesized according to a previously published method (Fischer et al, Nature 512(2014): 49).

(2) Synthesis of dBET1

JQ-acid (11.3mg, 0.0281mmol, 1 eq) and N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate (14.5mg, 0.0281mmol, 1 eq) were dissolved in DMF (0.28mL, 0.1M) at room temperature. DIPEA (14.7. mu.l, 0.0843mmol, 3 equiv.) and HATU (10.7mg, 0.0281mmol, 1 equiv.) were then added and the mixture stirred for 19 h. The mixture was then purified by preparative HPLC to give dBET1 as a yellow solid (15.90mg, 0.0202mmol, 72%).¹H NMR (400MHz, methanol-d)₄)δ7.77(dd,J＝8.3,7.5Hz,1H),7.49(d,J＝7.3 Hz,1H),7.47–7.37(m,5H),5.07(dd,J＝12.5,5.4Hz,1H),4.74(s, 2H),4.69(dd,J＝8.7,5.5Hz,1H),3.43–3.32(m,3H),3.29–3.25 (m,2H),2.87–2.62(m,7H),2.43(s,3H),2.13–2.04(m,1H),1.72 –1.58(m,7H)。¹³C NMR(100MHz,cd₃od)δ174.41,172.33, 171.27,171.25,169.87,168.22,167.76,166.73,166.70,156.26, 138.40,138.23,137.44,134.83,133.92,133.40,132.30,132.28, 131.97,131.50,129.87,121.85,119.31,118.00,69.53,54.90,50.54,40.09,39.83,38.40,32.12,27.74,27.65,23.61,14.42,12.97,11.57. LCMS 785.44(M+H)。

Synthesis example 2: synthesis of dBET4

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.438 mL) was added to (R) -JQ-acid (prepared in analogy to JQ-acid from (R) -JQ 1) (14.63mg, 0.0365mmol, 1 eq.) 0.0438mmol 1.2 eq) at room temperature. DIPEA (19.1. mu.L, 0.1095mmol, 3 equiv.) and HATU (15.3mg, 0.0402mmol, 1.1 equiv.) were added and the mixture was stirred for 24 h, then diluted with MeOH and concentrated under reduced pressure. The crude material was purified by preparative HPLC to give a yellow solid (20.64mg, 0.0263mmol, 72%).¹H NMR (400MHz, methanol-d)₄)δ 7.79(dd,J＝8.4,7.4Hz,1H),7.51(d,J＝7.3Hz,1H),7.47–7.39 (m,5H),5.11–5.06(m,1H),4.75(s,2H),4.68(dd,J＝8.8,5.5Hz, 1H),3.47–3.31(m,5H),2.83–2.65(m,7H),2.44(s,3H),2.13– 2.06(m,1H),1.68(s,3H),1.67–1.60(m,4H).¹³C NMR(100MHz, cd₃od)δ174.43,172.40,171.29,169.92,168.24,167.82,166.71, 156.31,153.14,138.38,138.24,137.54,134.88,133.86,133.44, 132.29,132.00,131.49,129.88,122.46,121.90,119.38,118.02, 69.59,54.96,50.55,40.09,39.84,38.45,32.14,27.75,27.65,23.62, 14.41,12.96,11.56.MS 785.48(M+H)。

Synthesis example 3: synthesis of dBET3

A solution of 0.1M N- (2-aminoethyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.475 mL, 0.0475mmol, 1.2 equiv) was added Q-acid (15.86mg, 0.0396 mmol, 1 equiv) at room temperature. DIPEA (20.7. mu.l, 0.1188mmol, 3 equivalents) and HATU (16.5mg, 0.0435mmol, 1.1 equivalents) were then added and the mixture was stirred for 24 hours and then purified by preparative HPLC to give a yellow solid (22.14mg, 0.1 equivalent).0292 mmol，74％)。¹H NMR (400MHz, methanol-d)₄)δ7.82–7.75(m,1H), 7.52–7.32(m,6H),5.04(dd,J＝11.6,5.5Hz,1H),4.76(d,J＝3.2 Hz,2H),4.66(d,J＝6.6Hz,1H),3.58–3.35(m,6H),2.78–2.58 (m,6H),2.48–2.41(m,3H),2.11–2.02(m,1H),1.70(d,J＝11.8 Hz,3H)。¹³C NMR(100MHz,cd₃od)δ174.38,171.26,171.19, 170.26,168.86,168.21,167.76,166.72,156.27,153.14,138.44, 138.36,138.19,134.87,133.71,132.31,131.57,131.51,129.90, 129.86,121.81,119.36,117.95,69.48,54.83,50.52,40.09,39.76, 38.30,32.09,23.63,14.40,11.61.LCMS 757.41(M+H)。

Synthesis example 4: synthesis of dBET5

A solution of 0.1M N- (6-aminohexyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.247mL, 0.0247mmol, 1 eq) was added to JQ-acid (9.9mg, 0.0247mmol, 1 eq) at room temperature. DIPEA (12.9. mu.l, 0.0741mmol, 3 equivalents) and HATU (9.4mg, 0.0247mmol, 1 equivalent) were then added. The mixture was stirred for 21 hours, then diluted with MeOH and concentrated under reduced pressure. The crude material was purified by preparative HPLC to give a yellow solid (13.56mg, 0.0167mmol, 67%).¹H NMR (400MHz, methanol-d)₄)δ7.82–7.78(m,1H),7.53(dd,J＝7.3,2.0Hz,1H), 7.49–7.37(m,5H),5.10(dt,J＝12.4,5.3Hz,1H),4.76(s,2H), 4.70(dd,J＝8.7,5.5Hz,1H),3.42–3.33(m,2H),3.25(dt,J＝12.3, 6.0Hz,3H),2.87–2.67(m,7H),2.48–2.42(m,3H),2.14–2.09(m, 1H),1.69(d,J＝4.8Hz,3H),1.58(s,4H),1.42(d,J＝5.2Hz,4H)。¹³C NMR(100MHz,cd₃od)δ174.51,171.31,171.26,169.82,168.27, 168.26,167.75,156.26,150.46,138.20,134.92,133.92,133.47, 132.34,132.01,131.52,129.88,121.69,119.34,117.95,111.42, 69.39,54.97,50.56,40.39,40.00,38.40,32.15,30.46,30.16,27.58, 27.48,23.64,14.41,12.96,11.55.LCMS 813.38。

Synthesis example 5: synthesis of dBET6

0.1M N- (8-Aminooctyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate was added to JQ-acid (7.66mg, 0.0191mmol, 1 eq) under a solution of DMF (0.191 mL, 0.0191mmol, 1 eq) at room temperature. DIPEA (10. mu.L, 0.0574mmol, 3 equivalents) and HATU (7.3mg, 0.0191mmol, 1 equivalent) were added and the mixture was stirred for 22 h, diluted with MeOH and concentrated under reduced pressure. The crude material was purified by preparative HPLC to give a cream solid. (8.53mg, 0.0101mmol, 53%).¹H NMR (400MHz, methanol-d)₄)δ7.80(dd,J＝8.4,7.4Hz,1H),7.53(d,J＝7.4Hz, 1H),7.49–7.36(m,5H),5.10(dt,J＝12.3,5.3Hz,1H),4.75(s,2H), 4.69(dd,J＝8.8,5.3Hz,1H),3.42(dd,J＝15.0,8.9Hz,1H),3.30– 3.18(m,4H),2.90–2.64(m,7H),2.45(s,3H),2.13(dtt,J＝10.8, 5.2,2.6Hz,1H),1.71(d,J＝4.4Hz,3H),1.56(d,J＝6.2Hz,4H), 1.33(d,J＝17.1Hz,8H)。¹³C NMR(100MHz,cd₃od)δ174.50, 172.38,171.30,169.81,168.28,167.74,166.64,156.25,138.38, 138.20,137.55,134.92,133.88,133.42,132.27,132.02,131.50, 129.85,121.66,119.30,117.95,69.37,55.01,50.58,40.51,40.12, 38.44,32.18,30.46,30.33,30.27,30.21,27.91,27.81,23.63,14.42, 12.96,11.55.LCMS 841.64(M+H)。

Synthesis example 6: synthesis of dBET9

A solution of 0.1M N- (3- (2- (2- (3-aminopropoxy) ethoxy) propyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.321mL, 0.0321mmol, 1 eq) was added JQ-acid (12.87mg, 0.0321mmol, 1 eq) at room temperature. DIPEA (16.8 μ l, 0.0963mmol, 3 equiv.) and HATU (12.2mg, 0.0321mmol, 1 equiv.) were added and the mixture was stirred for 24 h, diluted with MeOH and concentrated under reduced pressure. The crude material was purified by preparative HPLC to give a yellow oil. (16.11mg, 0.0176mmol, 55%).

¹H NMR (400MHz, methanol-d)₄)δ7.79(dd,J＝8.4,7.4Hz,1H),7.52(d, J＝7.2Hz,1H),7.49–7.36(m,5H),5.10(dd,J＝12.5,5.5Hz,1H), 4.78–4.67(m,3H),3.64–3.52(m,11H),3.48–3.32(m,6H),2.94 –2.64(m,7H),2.52–2.43(m,3H),2.18–2.08(m,1H),1.81(p,J＝6.3Hz,4H),1.73–1.67(m,3H).LCMS 918.45(M+H)。

Synthesis example 7: synthesis of dBET17

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.281mL, 0.0281mmol, 1 eq) was added to (S) -2- (4- (4-cyanophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetic acid (11mg, 0.0281mmol, 1 eq) at room temperature. DIPEA (14.7. mu.L, 0.0843mmol, 3 equiv.) and HATU (10.7mg, 0.0281mmol, 1 equiv.) were added and the mixture stirred for 24 h, diluted with EtOAc, washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated. Purification by column chromatography (ISCO, 4g silica gel column 0 to 10% MeOH/DCM) gave a white solid (14.12mg, 0.0182mmol, 65%).

¹H NMR (400MHz, methanol-d)₄)δ7.82–7.72(m,3H),7.61(dd,J＝8.5, 2.0Hz,2H),7.51(d,J＝7.9Hz,1H),7.44–7.40(m,1H),5.11– 5.05(m,1H),4.76(s,2H),4.66(dd,J＝9.0,5.1Hz,1H),3.48–3.32 (m,4H),3.30–3.23(m,1H),2.87–2.61(m,7H),2.43(s,3H),2.10(dt,J＝10.7,5.2Hz,1H),1.70–1.59(m,7H).¹³C NMR(100MHz, cd₃od)δ174.42,172.65,171.27,169.92,168.25,167.80,165.88, 156.31,143.55,138.24,134.88,133.92,133.50,133.39,131.72, 131.46,130.55,121.93,119.39,119.21,118.02,115.17,69.59,55.50, 50.55,40.10,39.83,38.86,32.11,27.78,27.67,23.62,14.41,12.91,11.64.LCMS 776.39(M+H)。

Synthesis example 8: synthesis of dBET15

N- (6-Aminohexyl) -2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindoline-5-carboxamide trifluoroacetate (13.29mg, 0.258mmol, 1 eq.) and JQ-acid (10.3mg, 0.0258mmol, 1 eq.) are dissolved in DMF (0.26 mL). DIPEA (13.5 μ l, 0.0775mmol, 3 eq) was added followed by HATU (9.8mg, 0.0258mmol, 1 eq) and the mixture was stirred at room temperature. After 24 h, the material was diluted with DCM and purified by column chromatography (ISCO, 0 to 15% MeOH/DCM) followed by preparative HPLC to give a pale yellow solid (11.44mg, 0.0146 mmol, 57%).

¹H NMR (400MHz, methanol-d)₄)δ8.29–8.23(m,2H),7.93(dd,J＝8.1, 4.2Hz,1H),7.50–7.34(m,4H),5.17–5.11(m,1H),4.75–4.69(m, 1H),3.53–3.32(m,6H),3.25(dd,J＝13.8,6.7Hz,1H),2.90–2.67 (m,6H),2.49–2.38(m,3H),2.18–2.10(m,1H),1.64(d,J＝22.4 Hz,6H),1.47(s,4H).¹³C NMR(100MHz,cd₃od)δ174.48,171.17, 168.05,168.03,167.99,167.70,166.63,141.81,138.40,137.47, 135.09,134.77,134.74,133.96,133.94,133.38,132.24,132.05, 131.44,129.85,124.57,123.12,123.09,54.98,50.78,40.88,40.08, 38.37,32.13,30.40,30.23,27.34,27.26,23.58,14.40,12.96,11.54. LCMS783.43(M+H).

Synthesis example 9: synthesis of dBET2

(1) Synthesis of ethyl (R) -4- ((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5, 6,7, 8-tetrahydropteridin-2-yl) amino) -3-methoxybenzoate

The reaction mixture was washed with (R) -2-chloro-8-cyclopentyl-7-ethyl-5-methyl-7, 8-dihydropteridin-6 (5H) -one (44.2mg,0.15mmol, 1 eq), ethyl 4-amino-3-methoxybenzoate (35.1 mg, 0.18mmol, 1.2 eq), Pd₂dba₃(6.9mg, 0.0075mmol, 5 mol%), XPhos (10.7mg, 0.0225mmol, 15 mol%) and potassium carbonate (82.9 mg, 0.60mmol, 4 equiv.) were dissolved in tBuOH (1.5mL, 0.1M) and heated to 100 ℃. After 21 h, the mixture was cooled to room temperature, filtered through celite, washed with DCM and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 100% EtOAc/hexanes gradient over 18 min) afforded a yellow oil (52.3mg, 0.115mmol, 77%).¹H NMR (400MHz, chloroform-d) δ 8.57(d, J ═ 8.5Hz,1H), 7.69(td, J ═ 6.2,2.9Hz,2H),7.54(d, J ═ 1.8Hz,1H),4.52 (t, J ═ 7.9Hz,1H),4.37(q, J ═ 7.1Hz,2H),4.23(dd, J ═ 7.9,3.7Hz, 1H),3.97(s,3H),3.33(s,3H), 2.20-2.12 (M,1H), 2.03-1.97 (M,1H), 1.86(ddd, J ═ 13.9,7.6,3.6Hz,4H), 1.78-1.65 (M,4H),1.40 (t, 7.88H), 3.26H (t, 3H + 454.32H).

(2) Synthesis of (R) -4- ((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5, 6,7, 8-tetrahydropteridin-2-yl) amino) -3-methoxybenzoic acid

Ethyl (R) -4- ((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5, 6,7, 8-tetrahydropteridin-2-yl) amino) -3-methoxybenzoate (73.8mg, 0.163mmol, 1 eq) and LiOH (11.7mg, 0.489mmol, 3 eq) were dissolved in MeOH (0.82mL), THF (1.63mL) and water (0.82 mL). After 20 h, an additional 0.82mL of water was added and the mixture was stirred for an additional 24 h and then purified by preparative HPLC to give a cream-colored solid (53mg, 0.125mmol, 76%).¹H NMR (400MHz, methanol-d)₄)δ 7.97(d,J＝8.4Hz,1H),7.67(dd,J＝8.3,1.6Hz,1H),7.64–7.59 (m,2H),4.38(dd,J＝7.0,3.2Hz,1H),4.36–4.29(m,1H),3.94(s, 3H),3.30(s,3H),2.13–1.98(m,2H),1.95–1.87(m,2H),1.87– 1.76(m,2H),1.73–1.57(m,4H),0.86(t,J＝7.5Hz,3H).¹³C NMR (100MHz,cd₃od)δ168.67,163.72,153.59,150.74,150.60,130.95, 127.88,125.97,123.14,121.68,116.75,112.35,61.76,61.66,56.31,29.40,29.00,28.68,28.21,23.57,23.41,8.69.LCMS 426.45(M+H)。

(3) Synthesis of dBET2

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.183 mL, 0.0183mmol, 1.2 equivalents) was added to (R) -4- ((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5, 6,7, 8-tetrahydropteridin-2-yl) amino) 3-methoxybenzoic acid (6.48mg, 0.0152mmol, 1 equivalent) at room temperature. DIPEA (7.9 μ l, 0.0456mmol, 3 equivalents) and HATU (6.4mg, 0.0168mmol, 1.1 equivalents) were added and the mixture was stirred for 23 hours and then purified by preparative HPLC to give a yellow solid (9.44mg, 0.0102 mmol, 67%).¹H NMR (400MHz, methanol-d)₄)δ7.84–7.77(m,2H), 7.58(d,J＝1.8Hz,2H),7.53–7.46(m,2H),7.42(d,J＝8.4Hz, 1H),5.11–5.05(m,1H),4.76(s,2H),4.48(dd,J＝6.5,3.1Hz,1H), 4.33–4.24(m,1H),3.95(s,3H),3.49–3.35(m,4H),2.97(d,J＝ 10.5Hz,3H),2.89–2.65(m,5H),2.17–1.99(m,4H),1.89(dd,J＝ 14.5,7.3Hz,2H),1.69–1.54(m,6H),1.36(dt,J＝7.6,3.9Hz,1H), 0.85(t,J＝7.5Hz,3H)。¹³C NMR(100MHz,cd₃od)δ176.52, 174.48,173.05,171.34,169.99,168.91,168.25,167.80,164.58, 156.34,154.48,153.10,150.63,138.22,134.89,133.96,129.53, 123.93,121.87,120.78,119.36,117.99,111.54,69.55,63.29,63.10, 56.68,50.55,40.71,39.86,32.15,29.43,29.26,28.73,28.63,27.81, 27.77,24.25,23.63,8.47.LCMS 810.58(M+H)。

Synthesis example 10: synthesis of dBET7

A solution of 0.1M N- (6-aminohexyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.186mL, 0.0186mmol 1 eq) was added to (R) -4- ((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5, 6,7, 8-tetrahydropteridin-2-yl) amino) -3-methoxybenzoic acid (7.9mg, 0.0186mmol, 1 eq) at room temperature. DIPEA (9.7 μ l, 0.0557mmol, 3 equivalents) and HATU (7.1mg, 0.0186mmol, 1 equivalent) were added and the mixture was stirred for 19 h and then purified by preparative HPLC to give the desired trifluoroacetate salt as a yellow solid (13.62mg, 0.0143mmol, 77%).

¹H NMR (400MHz, methanol-d)₄)δ7.80(t,J＝8.3Hz,2H),7.61–7.57 (m,2H),7.55–7.49(m,2H),7.42(d,J＝8.4Hz,1H),5.13(dd,J＝ 12.6,5.5Hz,1H),4.75(s,2H),4.48(dd,J＝6.5,3.2Hz,1H),4.33–4.24(m,1H),3.97(s,3H),3.40(t,J＝7.1Hz,2H),3.34(d,J＝6.7Hz,2H),3.30(s,3H),2.98(d,J＝8.5Hz,1H),2.89–2.82(m,1H), 2.79–2.63(m,3H),2.17–2.00(m,4H),1.91(dt,J＝14.4,7.1Hz, 3H),1.61(dt,J＝13.4,6.6Hz,7H),1.47–1.41(m,3H),0.86(t,J＝ 7.5Hz,3H).¹³C NMR(100MHz,cd₃od)δ174.54,171.37,169.84, 168.84,168.27,167.74,164.59,156.26,154.47,153.18,150.69, 138.19,134.91,134.05,129.47,124.78,124.01,121.65,120.77, 119.29,117.92,117.86,111.55,69.34,63.31,63.13,56.67,50.53, 40.97,39.96,32.16,30.42,30.19,29.42,29.26,28.72,28.62,27.65, 27.46,24.26,23.65,8.47.LCMS 838.60(M+H).

Synthesis example 11: synthesis of dBET8

A solution of 0.1M N- (8-aminooctyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.186mL, 0.0186mmo, 1 eq) was added to (R) -4- ((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5, 6,7, 8-tetrahydropteridin-2-yl) amino) -3-methoxybenzoic acid (7.9mg, 0.0186mmol, 1 eq) at room temperature. DIPEA (9.7 μ l, 0.0557mmol, 3 equivalents) and HATU (7.1mg, 0.0186mmol, 1 equivalent) were added and the mixture was stirred for 16 h and then purified by preparative HPLC to give the desired trifluoroacetate as an off-white solid (7.15mg, 0.007296mmol, 39%).

¹H NMR (400MHz, methanol-d)₄)δ7.83–7.77(m,2H),7.61–7.56(m, 2H),7.55–7.50(m,2H),7.42(d,J＝8.5Hz,1H),5.13(dd,J＝12.6, 5.5Hz,1H),4.75(s,2H),4.49(dd,J＝6.6,3.3Hz,1H),4.33–4.24 (m,1H),3.97(s,3H),3.39(t,J＝7.1Hz,2H),3.34–3.32(m,2H),3.30(s,3H),3.01–2.83(m,2H),2.82–2.65(m,3H),2.17–2.01 (m,4H),1.91(dt,J＝14.2,7.4Hz,1H),1.68–1.54(m,7H),1.37(s, 7H),0.86(t,J＝7.5Hz,3H)。¹³C NMR(100MHz,cd₃od)δ174.52, 171.35,169.81,168.85,168.28,167.74,164.58,156.27,154.47, 153.89,150.64,138.19,134.93,134.18,129.52,129.41,124.91, 123.83,121.67,120.76,119.31,117.95,117.89,111.57,69.37,63.37, 63.17,56.67,50.58,41.12,40.12,32.19,30.43,30.28,30.22,30.19, 29.40,29.25,28.71,28.62,27.94,27.75,24.29,23.65,8.46.LCMS 866.56(M+H)。

Synthesis example 12: synthesis of dBET10

At room temperature, a solution of 0.1M N- (3- (2- (2- (3-aminopropoxy) ethoxy) propyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-methyl-1-oxy) acetamide trifluoroacetate in DMF (0.172mL, 0.0172mmol, 1 eq) was added (R) -4- ((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5, 6,7, 8-tetrahydropteridin-2-yl) amino) -3-methoxybenzoic acid (7.3mg, 0.72 mmol, 1 eq.) DIPEA (9.0 was added, 0.0515mmol, 3 equivalents) and HATU (6.5mg, 0.0172mmol, 1 equivalent), the mixture was stirred for 23 hours and then purified by preparative HPLC to give the desired trifluoroacetate salt as a shutdown white oil (10.7mg, 0.0101mmol, 59%).

¹H NMR (400MHz, methanol-d)₄)δ7.78(d,J＝8.3Hz,1H),7.75(dd,J＝ 8.4,7.4Hz,1H),7.56–7.51(m,2H),7.49–7.44(m,2H),7.36(d,J ＝8.4Hz,1H),5.08(dd,J＝12.4,5.4Hz,1H),4.69(s,2H),4.44(dd, J＝6.7,3.2Hz,1H),4.30–4.21(m,1H),3.92(s,3H),3.59–3.42(m,12H),3.35(t,J＝6.7Hz,2H),3.25(s,3H),2.95–2.64(m,5H), 2.13–1.95(m,4H),1.91–1.71(m,7H),1.65–1.48(m,4H),0.81(t, J＝7.5Hz,3H)。¹³C NMR(100MHz,cd₃od)δ174.50,171.35, 169.83,168.77,168.25,167.68,164.57,156.26,154.47,153.05, 150.59,138.19,134.92,133.89,129.53,124.57,123.98,121.72, 120.75,119.26,117.95,117.86,111.54,71.51,71.46,71.28,71.20, 70.18,69.65,69.41,63.27,63.07,56.71,50.57,38.84,37.59,32.17, 30.41,30.32,29.46,29.26,28.73,28.64,24.27,23.65,8.49.LCMS 942.62(M+H)。

Synthesis example 13: synthesis of dBET16

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.402mL, 0.0402mmol, 1 eq) was added to (R) -4- ((4-cyclopentyl-1, 3-dimethyl-2-oxo-1, 2,3, 4-tetrahydropyrido [2,3-b ] at room temperature]Pyrazin-6-yl) amino) -3-methoxybenzoic acid (16.55 mg, 0.0402mmol, 1 eq). DIPEA (21. mu.l, 0.1206mmol, 3 equiv.) and HATU (15.3mg, 0.0402mmol, 1 equiv.) were added and the mixture was stirred for 21 h, then purified by preparative HPLC, followed by column chromatography (ISCO, 12g) NH₂Silica column, 0 to 15% MeOH/DCM, 20 min gradient), gave HPLC to give a brown solid (10.63mg, 0.0134mmol, 33%).

¹H NMR (400MHz, methanol-d)₄)δ8.22(d,J＝8.4Hz,1H),7.78(dd,J＝ 8.4,7.4Hz,1H),7.73–7.68(m,1H),7.49(d,J＝7.4Hz,2H),7.46 –7.39(m,2H),6.98(d,J＝8.8Hz,1H),5.97–5.87(m,1H),5.06 (dd,J＝12.6,5.4Hz,1H),4.76(s,2H),3.98(s,3H),3.61(s,2H),3.44–3.36(m,4H),2.92(s,1H),2.78(dd,J＝14.3,5.2Hz,1H), 2.68(ddd,J＝17.7,8.2,4.5Hz,2H),2.36–2.26(m,2H),2.10– 1.90(m,5H),1.76–1.62(m,6H),1.31(d,J＝16.0Hz,4H).LCMS 795.38(M+H)。

Synthesis example 14: synthesis of dBET11

(1) Synthesis of ethyl 4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimido [5,4-b ] [1,4] diazepan-2-yl) amino) -3-methoxybenzoate

2-chloro-5, 11-dimethyl-5H-benzo [ e]Pyrimido [5,4-b ] s][1,4]Diazepatrien-6 (11H) -one (82.4mg, 0.30mmol, 1 eq), ethyl 4-amino-3-methoxybenzoate (70.3mg, 0.36mmol, 1.2 eq) Pd₂dba₃(13.7mg, 0.015 mmol, 5 mol%), XPhos (21.5mg, 0.045mmol, 15 mol%) and potassium carbonate (166mg, 1.2mmol, 4 equiv.) were dissolved in tBuOH (3.0mL) and heated to 100 ℃. After 17 hours, the mixture was cooled to room temperature and filtered through celite. The mixture was purified by column chromatography (ISCO, 12g silica gel column, 0 to 100% EtOAc/hexanes, 19 min gradient) to give an off-white solid (64.3mg, 0.148mmol, 49%).

¹H NMR(400MHz,50％cd₃od/cdcl₃)δ8.51(d,J＝8.5Hz,1H), 8.17(s,1H),7.73(ddd,J＝18.7,8.1,1.7Hz,2H),7.52(d,J＝1.8 Hz,1H),7.46–7.41(m,1H),7.15–7.10(m,2H),4.34(q,J＝7.1 Hz,4H),3.95(s,3H),3.47(s,3H),3.43(s,3H),1.38(t,J＝7.1Hz,3H).¹³C NMR(100MHz,50％cd₃od/cdcl₃)δ169.28,167.39,164.29, 155.64,151.75,149.73,147.45,146.22,133.88,133.18,132.37, 126.44,124.29,123.70,123.36,122.26,120.58,118.05,116.83, 110.82,61.34,56.20,38.62,36.25,14.51.LCMS 434.33(M+H).

(2) Synthesis of 4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimido [5,4-b ] [1,4] diazepan-2-yl) amino) -3-methoxybenzoic acid

Ethyl 4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimido [5,4-b ] [1,4] diazepan-2-yl) amino) -3-methoxybenzoate (108.9mg, 0.251mmol, 1 eq) and LiOH (18mg) were dissolved in THF (2.5mL) and water (1.25 mL). After 24 h MeOH (0.63mL) was added to improve solubility) and stirred for a further 24 h, then diluted with MeOH and purified by preparative HPLC to give a light yellow solid (41.31 mg).

¹H NMR (400MHz, methanol-d)₄)δ8.51(d,J＝8.5Hz,1H),8.22(s,1H), 7.73(ddd,J＝11.8,8.1,1.7Hz,2H),7.57(d,J＝1.8Hz,1H),7.49– 7.44(m,1H),7.19–7.11(m,2H),3.97(s,3H),3.48(s,3H),3.45(s, 3H)。LCMS 406.32(M+H)。

(3) Synthesis of dBET11

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.190mL, 0.0190mmol, 1 eq) was added to 4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimido [5,4-b ] [1,4] diazepan-2-yl) amino) -3-methoxybenzoic acid (7.71mg, 0.0190mmol, 1 eq) at room temperature. DIPEA (9.9 μ l, 0.0571mmol, 3 equivalents) and HATU (7.2mg, 0.0190mmol, 1 equivalent) were added and the mixture was stirred for 22 h and then purified by preparative HPLC to give the desired trifluoroacetate salt as a cream solid (6.72mg, 0.00744mmol, 39%).

¹H NMR (400MHz, methanol-d)₄)δ8.46(d,J＝8.3Hz,1H),8.21(s,1H), 7.79–7.73(m,2H),7.52(d,J＝7.1Hz,1H),7.50–7.43(m,3H), 7.33(d,J＝8.2Hz,1H),7.15(dd,J＝7.7,5.9Hz,2H),4.98(dd,J＝ 12.0,5.5Hz,1H),4.69(s,2H),3.97(s,3H),3.49(s,3H),3.46–3.34 (m,7H),2.81–2.67(m,3H),2.13–2.08(m,1H),1.69(dt,J＝6.6, 3.5Hz,4H).¹³C NMR(100MHz,cd₃od)δ173.40,170.10,169.68, 169.00,168.85,167.60,167.15,164.77,156.01,155.42,151.83, 150.03,148.21,137.82,134.12,133.48,132.58,132.52,128.11, 126.72,124.54,122.33,121.06,120.63,118.77,118.38,117.94, 117.62,109.67,68.90,56.33,49.96,40.16,39.48,38.72,36.34, 31.82,27.24,23.16.LCMS790.48(M+H)。

Synthesis example 15: synthesis of dBET12

A solution of 0.1M N- (3- (2- (2- (3-aminopropoxy) ethoxy) propyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindoline) 4-methyl-1-oxy) acetamide trifluoroacetate in DMF (0.186mL, 0.0186mmol, 1 eq) was added to 4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimido [5,4-b ] [1,4] diazepan-2-yl) amino) -3-methoxybenzoic acid (7.53mg, 0.0186mmol, 1 eq) at room temperature. DIPEA (9.7 μ l, 0.0557mmol, 3 equiv.) and HATU (7.1mg, 0.0186mmol, 1 equiv.) were added and the mixture was stirred for 22 h then purified by preparative HPLC to afford HPLC the desired trifluoroacetate salt as a cream solid (7.50mg, 0.00724mmol, 39%).

¹H NMR (400MHz, methanol-d)₄)δ8.46(d,J＝8.9Hz,1H),8.21(s,1H), 7.73(dd,J＝15.2,7.8Hz,2H),7.50–7.42(m,3H),7.28(d,J＝8.5 Hz,1H),7.15(t,J＝7.7Hz,2H),5.01(dd,J＝11.8,5.8Hz,1H), 4.68(s,2H),3.97(s,3H),3.67–3.58(m,7H),3.58–3.43(m,10H), 3.39(t,J＝6.8Hz,2H),3.35(s,2H),2.97(s,1H),2.84–2.70(m, 3H),2.16–2.07(m,1H),1.93–1.76(m,4H).LCMS 922.57(M+H).

Synthesis example 16: synthesis of dBET13

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.501mL, 0.0501mmol 1 equiv) was added 2- ((2- (4- (3, 5-dimethylisoxazol-4-yl) phenyl) imidazo [1,2-a ] o]Pyrazin-3-yl) amino) acetic acid (as synthesized in McKeown et al, j.med.chem,2014,57, 9019) (18.22mg, 0.0501mmol, 1 equivalent) was synthesized at room temperature. DIPEA (26.3 μ l, 0.150mmol, 3 equivalents) and HATU (19.0mg, 0.0501mmol, 1 equivalent) were added and the mixture was stirred for 21 h and then purified by preparative HPLC to give the desired trifluoroacetate salt as a dark yellow oil (29.66mg, 0.0344mmol, 69%).¹H NMR (400MHz, methanol-d)₄)δ9.09(s,1H),8.65(d,J＝5.2Hz,1H),8.14–8.06(m,2H), 7.94–7.88(m,1H),7.80–7.74(m,1H),7.59–7.47(m,3H),7.40 (dd,J＝8.4,4.7Hz,1H),5.11–5.06(m,1H),4.72(d,J＝9.8Hz, 2H),3.90(s,2H),3.25–3.22(m,1H),3.12(t,J＝6.4Hz,1H),2.96 (s,2H),2.89–2.79(m,1H),2.76–2.62(m,2H),2.48–2.42(m, 3H),2.29(s,3H),2.10(ddq,J＝10.2,5.3,2.7Hz,1H),1.49–1.45 (m,2H),1.37(dd,J＝6.7,3.6Hz,2H).¹³C NMR(100MHz,cd₃od) δ174.45,171.98,171.35,169.88,168.17,167.85,167.40,159.88, 156.28,141.82,138.26,135.85,134.82,133.09,132.06,130.75, 129.67,122.07,121.94,119.30,118.98,118.06,117.24,69.56,50.56, 40.05,39.73,32.13,27.53,23.62,18.71,17.28,11.64,10.85.LCMS 748.49(M+H)。

Synthesis example 17: synthesis of dBET14

A solution of 0.1M N- (3- (2- (2- (3-aminopropoxy) ethoxy) propyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.510mL, 0.0510mmol, 1 eq) was added to 2- ((2- (4- (3, 5-dimethylisoxazol-4-yl) phenyl) imidazo [1,2- α ] pyrazin-3-yl) amino) acetic acid (as synthesized in McKeown et al, J.Med.chem,2014,57, 9019) (18.52mg, 0.0510mmol, 1 eq) at room temperature. DIPEA (26.6 μ l, 0.153mmol, 3 equivalents) and HATU (19.4mg, 0.0510mmol, 1 equivalent) were added and the mixture was stirred for 22 h, then purified by preparative HPLC to give the desired trifluoroacetate salt as a dark yellow oil (32.63mg, 0.0328mmol, 64%).

¹H NMR (400MHz, methanol-d)₄)δ9.09(s,1H),8.66(d,J＝5.4Hz,1H), 8.17–8.08(m,2H),7.92(d,J＝5.6Hz,1H),7.77(dd,J＝8.4,7.4 Hz,1H),7.60–7.47(m,3H),7.39(d,J＝8.4Hz,1H),5.09(dd,J＝ 12.4,5.5Hz,1H),4.71(s,2H),3.91(s,2H),3.62–3.46(m,10H),3.38(dt,J＝16.0,6.4Hz,3H),3.18(t,J＝6.8Hz,2H),2.97(s,1H), 2.89–2.81(m,1H),2.78–2.66(m,2H),2.47(s,3H),2.31(s,3H), 2.16–2.08(m,1H),1.79(dt,J＝12.8,6.5Hz,2H),1.64(t,J＝6.3 Hz,2H).¹³C NMR(100MHz,cd₃od)δ174.48,171.88,171.34, 169.80,168.22,167.69,167.42,159.87,156.24,141.87,138.21, 135.89,134.88,133.13,132.04,130.76,129.67,122.08,121.69, 119.20,117.94,117.23,71.44,71.22,71.10,69.92,69.62,69.38, 50.57,49.64,38.11,37.55,32.16,30.30,30.20,23.63,11.67,10.88. LCMS 880.46(M+H)。

Synthesis example 18: synthesis of dBET18

(1) Synthesis of tert-butyl (S) -4- (3- (2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetamido) propyl) piperazine-1-carboxylate

JQ-acid (176.6mg, 0.441mmol, 1 eq.) was dissolved in DMF (4.4mL) at room temperature. HATU (176mg, 0.463mmol, 1.05 equiv) was added followed by DIPEA (0.23mL), 1.32mmol, 3 equiv). After 10 min, a solution of tert-butyl 4- (3-aminopropyl) piperazine-1-carboxylate (118mg, 0.485mmol, 1.1 eq) in DMF (0.44mL) was added. After 24 h, the mixture was diluted with half-saturated sodium bicarbonate, extracted twice with DCM and once with EtOAc. The combined organic layers were dried over sodium sulfate, filtered and concentrated. Purification by column chromatography (ISCO, 24g silica gel column, 0-15% MeOH/DCM, 23 min gradient) gave a yellow oil (325.5mg, quantitative yield).¹H NMR (400MHz, chloroform-d) δ 7.67(t, J ═ 5.3Hz,1H),7.41 to 7.28(m,4H), 4.58(dd, J ═ 7.5,5.9Hz,1H),3.52 to 3.23(m,8H),2.63(s, 9H),2.37(s,3H),1.80 to 1.69(m,2H),1.64(s,3H),1.42(s,9H).¹³C NMR(100MHz,cdcl₃)δ171.41,164.35,155.62,154.45,150.20, 136.92,136.64,132.19,131.14,130.98,130.42,129.98,128.80, 80.24,56.11,54.32,52.70,38.96,37.85,28.42,25.17,14.43,13.16, 11.82.LCMS 626.36(M+H)。

(2) Synthesis of (S) -2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) -N- (3- (piperazin-1-yl) propyl) acetamide

Tert-butyl (S) -4- (3- (2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetamido) propyl) piperazine-1-carboxylate (325.5mg) was dissolved in DCM (5mL) and MeOH (0.5 mL). 4M HCl in dioxane (1mL) was added and the mixture was stirred for 16 h and then concentrated under a stream of nitrogen to give a yellow solid (231.8mg), which was used without further purification.

¹H NMR (400MHz, methanol-d)₄)δ7.64–7.53(m,4H),5.05(t,J＝7.1 Hz,1H),3.81–3.66(m,6H),3.62–3.33(m,9H),3.30(p,J＝1.6 Hz,1H),2.94(s,3H),2.51(s,3H),2.09(dq,J＝11.8,6.1Hz,2H), 1.72(s,3H).¹³C NMR(100MHz,cd₃od)δ171.78,169.38,155.83, 154.03,152.14,140.55,136.33,134.58,134.53,133.33,132.73, 130.89,130.38,56.07,53.54,41.96,37.22,36.23,25.11,14.48, 13.14,11.68.LCMS 526.29(M+H)。

(3) Synthesis of tert-butyl (S) - (6- (4- (3- (2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetamido) propyl) piperazin-1-yl) -6-oxohexyl) carbamate

(S) -2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) -N- (3- (piperazin-1-yl) propyl) acetamide (62.1mg) and 6- ((tert-butoxycarbonyl) amino) hexanoic acid (24.0mg, 0.1037mmol, 1 equivalent) were dissolved in DMF (1 mL). DIPEA (72.2. mu.l, 0.4147mmol, 4 equiv.) was added followed by HATU (39.4mg, 0.1037mmol, 1 equiv.) and the mixture stirred for 25 h. The mixture was diluted with half-saturated sodium bicarbonate and extracted three times with DCM. The combined organic layers were dried over sodium sulfate, filtered and concentrated. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 15% MeOH/DCM, 15 min gradient) afforded a yellow oil (71.75mg, 0.0970mmol, 94%).

¹H NMR (400MHz, chloroform-d) δ 7.61(s,1H), 7.43-7.28 (m,4H),4.63 (s,1H), 4.61-4.56 (m,1H), 3.82-3.21 (m,10H), 3.11-3.01 (m,2H), 2.61(d, J ═ 24.3Hz,9H),2.38(s,3H),2.28(t, J ═ 7.4Hz,2H), 1.73(dq, J ═ 13.8,7.4Hz,2H), 1.63-1.55 (m,2H), 1.53-1.24 (m,14H).¹³C NMR(100MHz,cdcl₃)δ171.63,171.11,164.34,156.17, 155.66,150.21,136.96,136.72,132.25,131.14,131.01,130.47, 130.00,128.85,79.11,56.42,54.46,53.06,52.82,45.04,41.02, 40.47,39.29,38.33,33.00,29.90,28.54,26.60,25.29,24.86,14.47, 13.20,11.86.LCMS 739.37(M+H)。

(4) Synthesis of (S) -N- (3- (4- (6-aminocaproyl) piperazin-1-yl) propyl) -2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetamide

Tert-butyl (S) - (6- (4- (3- (2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetamido) propyl) piperazin-1-yl) -6-oxohexyl) carbamate (71.75mg, 0.0970mmol, 1 equivalent) was dissolved in DCM (2mL) and MeOH (0.2 mL). A solution of 4M HCl in dioxane (0.49 mL) was added and the mixture was stirred for 2 hours then concentrated under a stream of nitrogen and then in vacuo to give a yellow foam (59.8mg, 0.0840mmol, 87%).

¹H NMR (400MHz, methanol-d)₄)δ7.68–7.53(m,4H),5.04(d,J＝6.6 Hz,1H),4.66(d,J＝13.6Hz,1H),4.23(d,J＝13.6Hz,1H),3.63– 3.34(m,7H),3.29–3.00(m,5H),2.95(d,J＝6.0Hz,5H),2.51(d, J＝9.2Hz,5H),2.08(s,2H),1.77–1.62(m,7H),1.45(dt,J＝15.3,8.6Hz,2H).¹³C NMR(100MHz,cd₃od)δ173.77,171.84,169.35, 155.85,153.99,140.56,136.40,134.58,133.35,132.70,130.39, 55.83,53.57,52.92,52.70,43.57,40.55,39.67,37.33,36.25,33.17, 28.26,26.94,25.33,25.26,14.49,13.15,11.65.LCMS639.35(M+H)。

(5) Synthesis of dBET18

(S) -N- (3- (4- (6-aminocaproyl) piperazin-1-yl) propyl) -2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetamide dihydrochloride (20.0mg, 0.0281mmol, 1 eq) and 2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetic acid (9.32mg, 0.0281mmol, 1 eq) were dissolved in DMF (0.281 mL). DIPEA (19.6. mu.l, 0.1124mmol, 4 equiv.) was added followed by HATU (10.7mg, 0.0281mmol, 1 equiv.). After 24 hours, the mixture was diluted with MeOH and purified by preparative HPLC to give the desired trifluoroacetate salt.

¹H NMR (400MHz, methanol-d)₄)δ7.83–7.79(m,1H),7.54(d,J＝7.1 Hz,1H),7.45(q,J＝8.8Hz,5H),5.12(dd,J＝12.5,5.4Hz,1H), 4.76(s,2H),4.68(t,J＝7.3Hz,1H),3.59–3.32(m,8H),3.28– 3.18(m,4H),2.87(ddd,J＝19.0,14.7,5.3Hz,2H),2.80–2.65(m, 6H),2.44(d,J＝6.8Hz,5H),2.33–2.25(m,1H),2.14(dd,J＝9.8, 4.9Hz,1H),2.06–1.89(m,3H),1.70(s,3H),1.61(dq,J＝14.4,7.3, 6.9Hz,4H),1.45–1.37(m,2H).¹³C NMR(100MHz,cd₃od)δ 174.52,173.97,173.69,171.44,169.88,168.26,167.83,166.72, 156.36,138.28,137.84,134.89,133.52,132.12,131.83,131.38, 129.89,121.87,119.32,118.01,69.52,55.64,55.03,52.79,50.58, 43.69,39.77,38.57,36.89,33.47,32.16,29.93,27.34,25.76,25.45, 23.63,14.39,12.94,11.66.LCMS 953.43(M+H)。

Synthesis example 19: synthesis of dBET19

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (235. mu.l, 0.0235mmol, 1 eq) was added to (S) -2- (4- (4-chlorophenyl) -2- (cyanomethyl) -3, 9-dimethyl-6H-thieno [3,2-f ] thiophene at room temperature][1,2,4]Triazolo [4,3-a][1,4]Diazepan-6-yl) acetic acid (10mg, 0.0235mmol, 1 equiv.). DIPEA (12.3. mu.L, 0.0704mmol, 3 equiv.) and HATU (8.9mg, 0.0235mmol, 1 equiv.) were added and the mixture was stirred for 18.5 h. The mixture was then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as a white solid (12.96mg, 0.0160mmol, 68%).¹H NMR (400MHz, chloroform-d) δ 7.80(dd, J ═ 8.4,7.4Hz,1H), 7.55-7.37 (M,6H), 5.14-5.06 (M,1H),4.77(d, J ═ 1.5Hz,2H),4.64(dd, J ═ 8.0,5.6Hz,1H), 3.45-3.32 (M,5H), 3.29-3.21 (M,2H), 2.83-2.66 (M,6H),2.58(s,3H), 2.14-2.06 (M,1H), 1.71-1.57 (M,4H), LCMS 810.30, M + H).

Synthesis example 20: synthesis of dBET20

At room temperature, 3- ((2- ((4- (4- (4-Ammonia))Phenylbutyryl) piperazin-1-yl) phenyl) amino) -5-methylpyrimidin-4-yl) amino) -N- (tert-butyl) benzenesulfonamide trifluoroacetate (7.41mg, 0.0107mmol, 1 eq) and 2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetic acid (3.6mg, 0.0107mmol, 1 eq) are dissolved in DMF (214 μ l, 0.05M). DIPEA (5.6. mu.l, 0.0321mmol, 3 equiv.) and HATU (4.1mg, 0.0107mmol, 1 equiv.) were added. After 22.5 h, the mixture was diluted with MeOH and purified by preparative HPLC to give the desired product as a brown residue (6.27mg, 0.00701mmol, 65%).¹H NMR (500MHz, methanol-d)₄) δ8.06(s,1H),7.84–7.75(m,3H),7.65(s,1H),7.55(t,J＝7.8Hz, 2H),7.45(d,J＝8.4Hz,1H),7.25–7.20(m,2H),6.99(d,J＝8.8 Hz,2H),5.11(dd,J＝12.5,5.4Hz,1H),4.78(s,2H),3.79–3.66(m, 4H),3.40(t,J＝6.6Hz,2H),3.24–3.13(m,4H),2.82–2.68(m, 3H),2.52(t,J＝7.4Hz,2H),2.24–2.19(m,3H),2.12(dd,J＝10.2, 5.1Hz,1H),1.92(dd,J＝13.4,6.4Hz,2H),1.18(s,9H).LCMS 895.63(M+H)。

Synthesis example 21: synthesis of dBET21

A solution of 0.1M 4- ((10-aminodecyl) oxy) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione trifluoroacetate in DMF (232. mu.l, 0.0232mmol, 1 eq) was added JQ-acid (9.3mg, 0.0232mmol, 1 eq) at room temperature. DIPEA (12.1. mu.l, 0.0696mmol, 3 equiv.) and HATU (8.8mg, 0.0232mmol, 1 equiv.) were added and the mixture was stirred for 18 h. The mixture was then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by preparative HPLC followed by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) gave the desired product as an off-white residue (1.84mg, 0.00235mmol, 10%).¹H NMR (500MHz, methanol-d)₄)δ7.77–7.73(m,1H),7.50–7.33(m,6H), 5.09(dd,J＝12.5,5.5Hz,1H),4.62(s,1H),4.21(t,J＝6.4Hz,2H), 3.36(s,2H),2.87–2.67(m,6H),2.44(s,3H),1.88–1.82(m,2H), 1.70(s,3H),1.58(s,4H),1.29(s,8H).LCMS 784.51(M+H)。

Synthesis example 22: synthesis of dBET22

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (247. mu.l, 0.0247mmol, 1 eq) was added to (S) -4- (4-chlorophenyl) -6- (2-methoxy-2-oxoethyl) -3, 9-dimethyl-6H-thieno [3,2-f ] at room temperature][1,2,4]Triazolo [4,3-a][1,4]Diazacycloheptatriene-2-carboxylic acid (10.98mg, 0.0247mmol, 1 eq). DIPEA (12.9. mu.L, 0.0740mmol, 3 equiv.) and HATU (9.4mg, 0.0247mmol, 1 equiv.) were added. The mixture was then stirred for 21 hours, then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as a white solid (9.79mg, 0.0118mmol, 48%).¹H NMR (400MHz, methanol-d)₄)δ7.80(dd,J＝ 8.4,7.4Hz,1H),7.51(dd,J＝7.1,1.5Hz,1H),7.48–7.34(m,5H), 5.11(ddd,J＝12.4,5.4,3.5Hz,1H),4.76(s,2H),4.69(td,J＝7.2, 1.4Hz,1H),3.76(s,3H),3.55(d,J＝7.2Hz,2H),3.48–3.33(m, 4H),2.93–2.82(m,1H),2.78–2.64(m,5H),2.14–2.07(m,1H),1.96(d,J＝0.9Hz,3H),1.66(s,4H).LCMS 829.39(M+H)。

Synthesis example 23: synthesis of dBET23

At room temperature, a solution of 0.1M N- (8-aminooctyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (220. mu.l, 0.0220mmol, 1 eq) was added (S) -4- (4-chlorophenyl) -6- (2-methoxy-2-oxoethyl) -3, 9-dimethyl-6H-thieno [3,2-f ] thiophene])[1,2,4]Triazolo [4,3-a][1,4]Diazo compoundsCycloheptatriene-2-carboxylic acid (9.87mg, 0.0220mmol, 1 eq). DIPEA (11.5. mu.l, 0.0660mmol, 3 equiv.) and HATU (8.4mg, 0.0220mmol, 1 equiv.) were added. The mixture was then stirred for 21 hours, then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as a white solid (8.84mg, 0.00998 mmol, 45%).¹H NMR (400MHz, methanol-d)₄)δ7.81(dd,J＝8.4,7.4 Hz,1H),7.53(d,J＝7.3Hz,1H),7.50–7.39(m,5H),5.12(dd,J＝ 12.6,5.4Hz,1H),4.75(s,2H),4.68(t,J＝7.2Hz,1H),3.76(s,3H), 3.54(d,J＝7.2Hz,2H),3.39–3.32(m,3H),3.29(s,1H),2.90– 2.83(m,1H),2.79–2.68(m,5H),2.14(dd,J＝8.9,3.7Hz,1H),1.99(s,3H),1.65–1.53(m,4H),1.36(d,J＝6.5Hz,8H).LCMS 885.47(M+H)。

Synthesis example 24: synthesis of dBET24

Step 1: synthesis of tert-butyl (2- (2- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamido) ethoxy) ethyl) carbamate

2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetic acid (200mg, 0.602mmol, 1 eq.) was dissolved in DMF (6.0mL, 0.1M). HATU (228.9mg, 0.602mmol, 1 equiv.), DIPEA (0.315mL, 1.81mmol, 3 equiv.) and N-Boc-2,2' - (ethylenedioxy) diethylamine (0.143mL, 0.602mmol, 1 equiv.) were added sequentially. After 6h, additional HATU (114mg, 0.30mmol, 0.5 equiv.) was added to ensure completion of the reaction. After an additional 24 hours, the mixture was diluted with EtOAc and washed twice with saturated sodium bicarbonate, water and brine. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 12g silica gel column, 0 to 15% MeOH/DCM, 15 min gradient) afforded the desired product as a yellow oil (0.25g, 0.44mmol, 74%).¹H NMR (400MHz, methanol-d)₄)δ7.82–7.75(m,1H),7.51(d,J＝7.4Hz,1H),7.41(d,J＝8.5 Hz,1H),5.13(dd,J＝12.4,5.5Hz,1H),4.76(s,2H),3.66–3.58(m, 6H),3.53–3.45(m,4H),3.19(t,J＝5.6Hz,2H),2.95–2.83(m, 1H),2.80–2.67(m,2H),2.19–2.12(m,1H),1.41(s,9H).LCMS 563.34(M+H)。

Step 2: synthesis of N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate

Tert-butyl (2- (2- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamido) ethoxy) ethyl) carbamate (0.25g, 0.44mmol, 1 eq) was dissolved in TFA (4.5mL) and heated to 50 ℃. After 3 hours, the mixture was cooled to room temperature, diluted with MeOH, and concentrated under reduced pressure. Purification by preparative HPLC gave the desired product as a brown solid (0.197g, 0.342mmol, 77%).¹H NMR (400MHz, methanol-d)₄)δ7.81(ddd,J＝8.4,7.4,1.1 Hz,1H),7.55–7.50(m,1H),7.43(d,J＝8.5Hz,1H),5.13(dd,J＝ 12.7,5.5Hz,1H),4.78(s,2H),3.74–3.66(m,6H),3.64(t,J＝5.4 Hz,2H),3.52(t,J＝5.3Hz,2H),3.14–3.08(m,2H),2.89(ddd,J＝ 17.5,13.9,5.2Hz,1H),2.80–2.66(m,2H),2.16(dtd,J＝13.0,5.7, 2.7Hz,1H).LCMS 463.36(M+H)。

Step 2 Synthesis of dBET24

A solution of 0.1M N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) acetamide trifluoroacetate in DMF (0.324mL, 0.0324mmol, 1 eq) was added JQ-acid (13.0mg, 0.324mmol, 1 eq.) followed by DIPEA (16.9. mu.L, 0.0972mmol, 3 eq.) and HATU (12.3mg, 0.0324mmol, 1 eq.) and the mixture stirred at room temperature for 18 h, then the mixture was diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine, then the organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure, column chromatography (ISCO, 4g silica gel, 0 to 10% MeOH/DCM, gradient 25 min) to afford the desired product as an off-white solid (20.0mg, 0.0236mmol, 73%).¹H NMR (400MHz, methanol-d)₄)δ7.77–7.72(m,1H),7.49(d, J＝7.4Hz,1H),7.45–7.35(m,5H),5.09(ddd,J＝12.3,5.4,3.7Hz, 1H),4.76(s,2H),4.60(dd,J＝8.9,5.3Hz,1H),3.68–3.62(m,6H), 3.59(t,J＝5.6Hz,2H),3.54–3.48(m,2H),3.47–3.35(m,4H), 2.84(ddd,J＝19.4,9.9,4.6Hz,1H),2.77–2.69(m,2H),2.68(d,J ＝1.8Hz,3H),2.43(s,3H),2.12(dt,J＝9.8,5.3Hz,1H),1.68(s, 3H).LCMS845.39(M+H)。

Synthesis example 25: synthesis of dBET25

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (183. mu.l, 0.0183mmol, 1 eq) was added to (S) -4- (4-chlorophenyl) -6- (2-methoxy-2-oxoethyl) -2, 9-dimethyl-6H-thieno [3,2-f ] thiophene at room temperature])[1,2,4]Triazolo [4,3-a][1,4]Diazacycloheptatriene-3-carboxylic acid (8.16mg, 0.0183mmol, 1 equivalent). DIPEA (9.6. mu.l, 0.0550mmol, 3 equiv.) and HATU (7.0mg, 0.0183mmol, 1 equiv.) were added. The mixture was then stirred for 23 hours, then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as a yellow solid (4.39mg, 0.00529mmol, 29%). ¹H NMR (400MHz, methanol-d)₄)δ7.82(dd,J＝8.4,7.4Hz,1H), 7.55(d,J＝7.3Hz,1H),7.45(d,J＝8.2Hz,1H),7.43–7.31(m, 4H),5.16–5.10(m,1H),4.77(d,J＝1.5Hz,2H),4.56(s,1H),3.74 (d,J＝1.8Hz,3H),3.66–3.60(m,1H),3.50(dd,J＝16.5,7.3Hz, 1H),3.37–3.32(m,1H),3.28(s,3H),2.85(t,J＝7.2Hz,2H),2.75 (d,J＝7.8Hz,1H),2.71(d,J＝0.9Hz,3H),2.59(d,J＝1.0Hz,3H), 2.18–2.10(m,1H),1.36–1.24(m,4H).LCMS 829.38(M+H)。

Synthesis example 26: synthesis of dBET26

0.1M N- (8-Aminooctyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (186. mu.M) at room temperatureLiter, 0.0186mmol, 1eq) of solution was added (S) -4- (4-chlorophenyl) -6- (2-methoxy-2-oxoethyl) -2, 9-dimethyl-6H-thieno [3, 2-f)])[1,2,4]Triazolo [4,3-a][1,4]Diazacycloheptatriene-3-carboxylic acid (8.26mg, 0.0186mmol, 1 equivalent). DIPEA (9.7. mu.l, 0.0557mmol, 3 equiv.) and HATU (7.1mg, 0.0186mmol, 1 equiv.) were added. The mixture was then stirred for 23 hours, then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as a cream colored solid (6.34mg, 0.00716mmol, 38%).¹H NMR (400MHz, methanol-d)₄)δ 7.83–7.78(m,1H),7.53(dd,J＝7.3,2.2Hz,1H),7.45–7.38(m, 3H),7.32(dd,J＝8.5,1.3Hz,2H),5.16–5.08(m,1H),4.76(s,2H), 4.56(s,1H),3.75(s,3H),3.66(dd,J＝15.9,8.7Hz,1H),3.50(dd,J ＝16.9,6.9Hz,1H),3.32(d,J＝2.8Hz,4H),2.84–2.74(m,3H), 2.70(d,J＝1.1Hz,3H),2.66–2.54(m,3H),2.14(d,J＝5.3Hz, 1H),1.62–1.22(m,12H).LCMS 885.48(M+H)。

Synthesis example 27: synthesis of dBET27

A solution of 0.1M 4- (2- (2-aminoethoxy) ethoxy) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione trifluoroacetate in DMF (257 microliters, 0.0257mmol, 1eq) was added JQ-acid (10.3mg, 0.0257mmol, 1 eq). DIPEA (13.4 μ l, 0.0771mmol, 3 equiv.) and HATU (9.8mg, 0.0257mmol, 1 equiv.) were then added and the mixture was stirred at room temperature for 18 h. The mixture was then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as a white solid (14.53mg, 0.0195mmol, 76%).¹H NMR (400MHz, methanol-d)₄) δ7.75(ddd,J＝8.5,7.3,1.3Hz,1H),7.47–7.30(m,6H),5.00(ddd, J＝25.4,12.2,5.2Hz,1H),4.61(td,J＝9.4,5.0Hz,1H),4.36(q,J ＝4.8Hz,2H),3.96–3.89(m,2H),3.74(q,J＝5.6Hz,2H),3.53–3.41(m,3H),3.30–3.24(m,1H),2.78–2.53(m,6H),2.41(d,J＝ 3.9Hz,3H),2.09–1.98(m,1H),1.67(d,J＝5.0Hz,3H)。

Synthesis example 28: synthesis of dBET28

A solution of 0.1M 4- (4-aminobutoxy) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione trifluoroacetate in DMF (202. mu.l, 0.0202mmol, 1eq) was added to JQ meso-acid (8.1mg, 0.0202mmol, 1 eq). DIPEA (10.6. mu.l, 0.0606mmol, 3 equiv.) and HATU (7.7mg, 0.0202mmol, 1 equiv.) were then added and the mixture was stirred at room temperature for 18.5 h. The mixture was then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as a cream colored solid (10.46mg, 0.0144mmol, 71%).¹H NMR (400MHz, methanol-d)₄)δ7.76(t,J＝7.5Hz,1H),7.43(td,J＝6.5,2.5Hz,4H), 7.34(t,J＝8.8Hz,2H),5.08–4.98(m,1H),4.64(td,J＝9.1,5.0Hz, 1H),4.26(t,J＝5.3Hz,2H),3.57–3.32(m,4H),2.84–2.59(m, 6H),2.45–2.37(m,3H),2.08–2.01(m,1H),2.00–1.91(m,2H), 1.82(dq,J＝13.8,6.9Hz,2H),1.68(d,J＝11.7Hz,3H).LCMS728.38(M+H)。

Synthesis example 29: synthesis of dBET29

A solution of 0.1M 4- ((6-aminohexyl) oxy) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione in DMF (205. mu.l, 0.0205mmol, 1eq) was added JQ-acid (8.2mg, 0.0205mmol, 1 eq). DIPEA (10.7. mu.l, 0.0614mmol, 3 equiv.) and HATU (7.8mg, 0.0205mmol, 1 equiv.) were then added and the mixture was cooled at room temperatureStirred for 19 hours. The mixture was then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as a white solid (8.04mg, 0.0106mmol, 52%).¹H NMR (400MHz, methanol-d)₄)δ7.75–7.71(m,1H),7.51– 7.34(m,6H),5.07(ddd,J＝12.1,5.4,2.4Hz,1H),4.62(dd,J＝9.0, 5.2Hz,1H),4.22(t,J＝6.4Hz,2H),3.44–3.32(m,2H),3.29–3.21(m,2H),2.88–2.65(m,6H),2.43(s,3H),2.13–2.06(m,1H), 1.86(dt,J＝13.9,6.7Hz,2H),1.68(s,3H),1.59(dq,J＝14.2,7.0 Hz,4H),1.54–1.45(m,2H).LCMS 756.40(M+H).

Synthesis example 30: synthesis of dBET30

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (163. mu.l, 0.0163mmol, 1eq) was added to (S) -4- (4-chlorophenyl) -3, 9-dimethyl-6- (2- ((3- (4-methylpiperazin-1-yl) propyl) amino)) -2-oxoethyl) -6H-thieno [3,2-f ] at room temperature][1,2,4]Triazolo [4,3-a][1,4]Diazepine-2-carboxylic acid (9.31mg, 0.0163mmol, 1 eq). DIPEA (8.5. mu.l, 0.0490mmol, 3 equiv.) and HATU (6.2mg, 0.0163mmol, 1 equiv.) were added. The mixture was then stirred for 23.5 hours, then purified by preparative HPLC to give the desired product as a yellow oil (11.48mg, 0.0107mmol, 66%).¹H NMR (400MHz, methanol-d)₄)δ 7.82–7.78(m,1H),7.54–7.35(m,6H),5.09(td,J＝12.7,5.4Hz, 1H),4.77–4.70(m,3H),3.56–3.31(m,12H),3.23(dd,J＝8.0,6.0 Hz,3H),3.05(d,J＝3.2Hz,2H),2.93–2.81(m,5H),2.78–2.63 (m,5H),2.15–2.05(m,2H),1.96–1.86(m,4H),1.68(s,4H). LCMS 954.55(M+H)。

Synthesis example 31: synthesis of dBET31

A solution of 0.1M N- (8-aminooctyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (153. mu.l, 0.0153mmol, 1eq) was added to (S) -4- (4-chlorophenyl) -3, 9-dimethyl-6- (2- ((3- (4-methylpiperazin-1-yl) propyl) amino)) -2-oxoethyl) -6H-thieno [3,2-f ] at room temperature][1,2,4]Triazolo [4,3-a][1,4]Diazepine-2-carboxylic acid (8.7mg, 0.0153mmol, 1 equivalent). DIPEA (7.9. mu.l, 0.0458mmol, 3 equiv.) and HATU (5.8mg, 0.0153mmol, 1 equiv.) were added. The mixture was then stirred for 25 hours and then purified by preparative HPLC to give the desired product as a brown oil (not brown in stool but somewhat brick-like in color) (9.52mg, 0.00847mmol, 55%).¹H NMR (400MHz, methanol-d)₄)δ7.81(dd,J＝8.4,7.4Hz,1H),7.59–7.40(m, 6H),5.12(dd,J＝12.5,5.4Hz,1H),4.75(s,2H),4.71(t,J＝7.4Hz, 1H),3.53–3.34(m,8H),3.29–3.11(m,6H),3.03–2.61(m,13H), 2.15(s,1H),2.01–1.84(m,5H),1.59(s,4H),1.37(s,8H).LCMS1010.62(M+H)。

Synthesis example 32: synthesis of dBET32

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (180 μ l, 0.0180mmol, 1eq) was added 4- (4- (4- ((4- ((3- (N- (tert-butyl) sulfamoyl) phenyl) amino) -5-methylpyrimidin-2-yl) amino) phenyl) piperazin-1-yl) -4-oxobutanoic acid (10.7mg, 0.0180mmol, 1eq) at room temperature. DIPEA (9.4. mu.l, 0.0539 mmol, 3 equiv.) and HATU (6.8mg, 0.0180mmol, 1 equiv.) were added and the mixture was stirred for 19 h. The mixture was then diluted with methanol and purified by preparative HPLC to give the desired product as a brown oil (4.40mg, 0.00449mmol, 25%).¹H NMR (500MHz, methanol-d)₄)δ8.08(d,J＝13.6Hz,1H),7.84–7.76 (m,3H),7.63(s,1H),7.57–7.51(m,2H),7.41(d,J＝8.4Hz,1H), 7.22(td,J＝6.7,2.2Hz,2H),7.03–6.97(m,2H),5.14(dd,J＝12.5, 5.5Hz,1H),4.76(d,J＝16.8Hz,2H),3.72(dt,J＝10.0,5.2Hz,4H),3.34–3.33(m,1H),3.23–3.12(m,5H),2.97(dd,J＝8.8,4.0 Hz,3H),2.80–2.69(m,4H),2.64(dd,J＝7.6,5.5Hz,1H),2.50(t, J＝6.8Hz,1H),2.22(dd,J＝2.4,0.9Hz,3H),2.17–2.11(m,1H), 1.67–1.52(m,4H),1.18(d,J＝0.8Hz,9H).LCMS 980.64(M+H)。

Synthesis example 33: synthesis of dBET33

A solution of 0.1M N- (8-aminooctyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (188 microliters, 0.0188mmol, 1eq) was added to 4- (4- (4- ((4- ((3- (N- (tert-butyl) sulfamoyl) phenyl) amino) -5-methylpyrimidin-2-yl) amino) phenyl) piperazin-1-yl) -4-oxobutanoic acid (10.8mg, 0.0188mmol, 1eq) at room temperature. DIPEA (9.8. mu.l, 0.0564 mmol, 3 equiv.) and HATU (7.1mg, 0.0188mmol, 1 equiv.) were added and the mixture was stirred for 23 h. The mixture was then diluted with methanol and purified by preparative HPLC to give the desired product as a brown residue (7.41mg, 0.00715mmol, 38%).¹H NMR (500MHz, methanol-d)₄)δ8.06(s,1H),7.80(ddd,J＝10.5,7.6,3.2 Hz,3H),7.65(d,J＝4.5Hz,1H),7.57–7.51(m,2H),7.41(dd,J＝ 8.4,2.9Hz,1H),7.25(td,J＝6.7,2.9Hz,2H),7.02(t,J＝8.0Hz, 2H),5.16–5.09(m,1H),4.75(d,J＝9.5Hz,2H),3.76(dq,J＝16.0,5.3Hz,4H),3.29–3.12(m,7H),3.00–2.67(m,7H),2.51(t,J＝ 6.8Hz,1H),2.22(d,J＝3.1Hz,3H),2.13(dtd,J＝10.4,5.7,3.1Hz, 1H),1.59–1.52(m,2H),1.51–1.43(m,2H),1.32(t,J＝16.6Hz, 8H),1.18(d,J＝1.3Hz,9H).LCMS 1036.69(M+H)。

Synthesis example 34: synthesis of dBET34

A solution of 0.1M N- (3- (2- (2- (3-aminopropoxy) ethoxy) propyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxaindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (173. mu.l, 0.0173mmol, 1eq) was added 4- (4-(4- ((4- ((3- (N- (tert-butyl) sulfamoyl) phenyl) amino) -5-methylpyrimidin-2-yl) amino) phenyl) piperazin-1-yl) -4-oxobutanoic acid (10.3mg, 0.0173mmol, 1 eq). DIPEA (9.0. mu.l, 0.0519mmol, 3 equiv.) and HATU (6.6mg, 0.0173mmol, 1 equiv.) were added and the mixture was stirred for 25 h. The mixture was then diluted with methanol and purified by preparative HPLC to give the desired product as a brown residue (7.99 mg, 0.00718mmol, 42%).¹H NMR (500MHz, methanol-d)₄)δ8.06(s, 1H),7.83–7.76(m,3H),7.65(s,1H),7.58–7.50(m,2H),7.43(dd, J＝17.7,8.4Hz,1H),7.27–7.21(m,2H),7.02(t,J＝8.0Hz,2H), 5.13(dt,J＝12.7,5.2Hz,1H),4.76(d,J＝12.4Hz,2H),3.73(q,J＝6.3Hz,4H),3.63–3.49(m,10H),3.41(q,J＝6.6Hz,2H),3.27– 3.15(m,5H),3.01–2.81(m,4H),2.79–2.63(m,5H),2.50(t,J＝ 6.8Hz,1H),2.22(d,J＝2.3Hz,3H),2.17–2.11(m,1H),1.88– 1.70(m,4H),1.18(d,J＝1.2Hz,9H).LCMS 1112.74(M+H)。

Synthesis example 35: synthesis of dBET35

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindol-4-yl) amino) acetamide trifluoroacetate in DMF (185. mu.l, 0.0185mmol, 1eq) was added JQ-acid (7.4mg, 0.0185mmol, 1 eq). DIPEA (9.6 microliters, 0.0554mmol, 3 equivalents) and HATU (7.0mg, 0.0185mmol, 1 equivalent) were then added, and the mixture was stirred at room temperature for 17 hours. The mixture was then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 15% MeOH/DCM, 25 min gradient) afforded the desired product as a white solid (2.71mg, 0.00351mmol, 19%).¹H NMR (500MHz, methanol-d)₄)δ7.48–7.37(m,4H),7.34(t,J＝7.8Hz,1H),7.14(dd,J＝ 7.4,2.4Hz,1H),6.67(d,J＝8.1Hz,1H),5.14(td,J＝13.5,5.2Hz, 1H),4.66–4.60(m,1H),4.59(d,J＝8.3Hz,2H),4.43–4.31(m, 2H),3.88(s,2H),3.25(dd,J＝14.8,7.1Hz,4H),2.94–2.72(m, 3H),2.68(d,J＝4.9Hz,3H),2.49–2.40(m,4H),2.21–2.12(m, 1H),1.68(s,3H),1.53(s,4H).LCMS 770.51(M+H)。

Synthesis example 36: synthesis of dBET36

A solution of 0.1M N- (4-aminobutyl) -2- (2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) acetamide trifluoroacetate in DMF (222. mu.l, 0.0222mmol, 1eq) was added JQ-acid (8.9mg, 0.0222mmol, 1 eq). DIPEA (11.6 μ l, 0.0666mmol, 3 equiv.) and HATU (8.4mg, 0.0222mmol, 1 equiv.) were then added and the mixture was stirred at room temperature for 17.5 h. The mixture was then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 15% MeOH/DCM, 25 min gradient) afforded the desired product as a white solid (12.42mg, 0.0156mmol, 70%).¹H NMR (500MHz, methanol-d)₄)δ7.80–7.74(m,2H),7.68(d,J＝6.8Hz,1H),7.42(q,J＝ 8.7Hz,4H),5.11(dt,J＝12.3,4.6Hz,1H),4.63(dd,J＝8.8,5.5Hz, 1H),4.10–4.00(m,2H),3.39(ddd,J＝14.9,8.8,2.5Hz,1H),3.30 –3.21(m,5H),2.88–2.76(m,1H),2.74–2.65(m,5H),2.44(s, 3H),2.15–2.08(m,1H),1.69(s,3H),1.63–1.55(m,4H).LCMS 769.49(M+H)。

Synthesis example 37: synthesis of dBET37

A solution of 0.1M 6-amino-N- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) methyl) hexanamide trifluoroacetate in DMF (195. mu.l, 0.0195mmol, 1eq) was added to JQ-acid (7.8mg, 0.0195mmol, 1 eq). DIPEA (10.2. mu.l, 0.0584mmol, 3 equiv.) and HATU (7.4mg, 0.0195mmol, 1 equiv.) were then added and the mixture was stirred at room temperature for 18 h. The mixture was then diluted with EtOAc and saturated carbonic acidSodium hydrogen, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 15% MeOH/DCM, 25 min gradient) afforded the desired product as a white solid (11.83mg, 0.0151mmol, 77%).¹H NMR (500MHz, methanol-d)₄)δ7.78–7.74(m,2H),7.71(dd,J＝5.3,3.5Hz,1H),7.42 (q,J＝8.5Hz,4H),5.13(dd,J＝12.6,5.5Hz,1H),4.82(s,2H), 4.63(dd,J＝8.8,5.5Hz,1H),3.40(ddd,J＝15.0,8.8,1.6Hz,1H), 3.30–3.21(m,3H),2.86(ddd,J＝18.4,14.6,4.8Hz,1H),2.74(ddd, J＝13.8,10.1,2.8Hz,2H),2.69(s,3H),2.44(s,3H),2.30(t,J＝7.4 Hz,2H),2.13(dtd,J＝12.9,4.9,2.3Hz,1H),1.74–1.64(m,5H),1.59(p,J＝7.0Hz,2H),1.46–1.38(m,2H).LCMS 783.47(M+H)。

Synthesis example 38: synthesis of dBET38

Step 1: (Synthesis of 3- (3- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetylamino) propoxy) propyl tert-butyl ester) Carbamate

Tert-butyl (3- (3-aminopropoxy) propyl) carbamate (134.5mg, 0.579mmol, 1eq) was dissolved in DMF (5.79mL, 0.05M) and 2- ((2- (2, 6-dioxopiperidine) -3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetic acid (192.38 mg, 0.579mmol, 1eq) was added. DIPEA (0.28mL, 1.74mmol, 3 equiv.) and HATU (153.61mg, 0.579mmol, 1 equiv.) were added and the mixture was stirred at room temperature for 18 h. The mixture was then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give a yellow oil (157.1 mg). The crude material was purified by column chromatography (ISCO, 12g silica column, 0 to 15% MeOH/DCM, 25 min gradient) to give a yellow oil (121.3mg, 0.222mmol, 38.27%).¹H NMR (400MHz, methanol-d)₄)δ7.78(dd,J ＝8.4,7.4Hz,1H),7.50(d,J＝7.3Hz,1H),7.41(d,J＝8.5Hz,1H), 5.13(dd,J＝12.4,5.5Hz,1H),4.75(s,2H),3.53–3.37(m,6H), 3.14–3.07(m,2H),2.94–2.88(m,1H),2.79–2.68(m,2H),2.16 (ddd,J＝12.8,6.6,2.7Hz,1H),1.81(p,J＝6.4Hz,2H),1.73–1.65(m,2H),1.40(s,9H).LCMS 547.6(M+H).

Step 2: synthesis of N- (3- (3-aminopropoxy) propyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamide trifluoroacetate

TFA (2.22ml, 0.1M) was added to tert-butyl (3- (3- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxyacetamido) propoxy) propyl) carbamate (121.3mg, 0.222mmol, 1eq) and the mixture was stirred at 50 ℃ for 2h then dissolved in MeOH and concentrated under reduced pressure to give a brown oil (114.1mg) which was used further without further purification.¹H NMR (400MHz, methanol-d)₄)δ7.81–7.74(m,1H),7.50(d,J＝7.3Hz,1H),7.41(d,J＝ 8.5Hz,1H),5.12(dd,J＝12.7,5.5Hz,1H),4.76(s,2H),3.57–3.52 (m,2H),3.48(t,J＝5.9Hz,2H),3.40(t,J＝6.6Hz,2H),3.06(t,J＝ 6.5Hz,2H),2.87(ddd,J＝14.1,10.1,7.0Hz,1H),2.79–2.65(m, 2H),2.15(dtd,J＝12.8,5.5,2.6Hz,1H),1.92(dt,J＝11.7,5.9Hz, 2H),1.81(p,J＝6.3Hz,2H).LCMS 447.2(M+H)。

And step 3: synthesis of dBET38

A solution of 0.1M N- (3- (3-aminopropoxy) propyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.215mL, 0.0215mmol, 1eq) was added JQ-acid (8.6mg, 0.0215mmol, 1eq) at room temperature. DIPEA (11.2. mu.l, 0.0644mmol, 3 equiv.) and HATU (8.2mg, 0.0215mmol, 1 equiv.) were added. After 19 h, the mixture was diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 15% MeOH/DCM, 25 min gradient) afforded the desired product as a cream colored solid (10.6mg, 0.0127mmol, 59%).¹H NMR (500MHz, methanol-d)₄)δ7.79–7.74(m,1H),7.50(d,J＝8.1Hz,1H),7.46–7.36 (m,5H),5.11(ddd,J＝12.4,5.5,1.7Hz,1H),4.73(s,2H),4.62(ddd, J＝8.7,5.4,1.4Hz,1H),3.50(q,J＝6.3Hz,4H),3.43(t,J＝6.5Hz, 2H),3.41–3.32(m,3H),3.29–3.24(m,1H),2.85(ddd,J＝18.3, 14.6,4.2Hz,1H),2.77–2.65(m,5H),2.43(s,3H),2.17–2.09(m, 1H),1.80(h,J＝6.4Hz,4H),1.68(s,3H).LCMS 829.32(M+H)。

Synthesis example 39: synthesis of dBET39

A solution of 0.1M 4- ((10-aminodecyl) oxy) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione trifluoroacetate in DMF (0.212mL, 0.0212mmol, 1eq) is added to JQ-acid (8.5mg, 0.0212mmol, 1eq) at room temperature. DIPEA (11.1. mu.l, 0.0636mmol, 3 equiv.) and HATU (8.1mg, 0.0212mmol, 1 equiv.) were added. After 19 h, the mixture was diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 15% MeOH/DCM, 25 min gradient) and preparative HPLC afforded the desired product (0.39mg, 0.00048 mmol, 2.3%).¹H NMR (500MHz, methanol-d)₄)δ7.77–7.73(m,1H), 7.56–7.31(m,6H),5.11–5.06(m,1H),4.62(dd,J＝9.2,5.0Hz, 1H),4.58(s,2H),4.21(t,J＝6.3Hz,2H),3.42–3.38(m,1H),3.24 –3.20(m,1H),2.90–2.68(m,6H),2.45(d,J＝6.7Hz,3H),2.11(s, 1H),1.83(dd,J＝14.7,6.6Hz,2H),1.70(s,3H),1.61–1.49(m, 4H),1.32(d,J＝23.2Hz,10H).LCMS 812.60(M+H)。

Synthesis example 40: synthesis of dBET40

0.1M 4- (2- (2- (2-aminoethoxy) ethoxy) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione trifluoroacetate in DMF (0.242mL, 0.0242mmol, 1eq) was added JQ-acid (9.7mg, 0.0242mmol, 1eq) at room temperature. DIPEA (12.6. mu.l, 0.0726mmol, 3 equiv.) and HATU (9.2mg, 0.0242mmol, 1 equiv.) were added. After 22 h, the mixture was diluted with EtOAc and saturated carbonic acidSodium hydrogen, water and brine. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) and preparative HPLC gave the desired product as a brown oil (4.74mg, 0.00601mmol, 25%).¹H NMR (500MHz, methanol-d)₄)δ7.77–7.67(m,1H),7.52–7.36(m,5H),5.09 –5.03(m,1H),4.64(d,J＝4.8Hz,1H),4.40–4.32(m,2H),3.97– 3.88(m,2H),3.81–3.74(m,2H),3.69–3.60(m,5H),3.55–3.38 (m,4H),2.89–2.54(m,6H),2.45(d,J＝5.9Hz,3H),2.11(s,1H), 1.70(d,J＝8.6Hz,3H).LCMS 788.42(M+H)。

Synthesis example 41: synthesis of dBET41

Step 1: synthesis of tert-butyl (4- ((2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamido) methyl) benzyl) carbamate

Tert-butyl (4- (aminomethyl) benzyl) carbamate (183.14mg, 0.755mmol, 1eq) was dissolved in DMF (15.1mL, 0.05M) and 2- ((2- (2, 6-dioxopiperidin-3-one- (. alpha. -3, 3-dioxoisoindolin-4-yl) oxy) acetic acid (250.90mg, 0.755mmol, 1eq) was added. DIPEA (0.374mL, 2.265mmol, 3 equiv.) and HATU (296.67mg, 0.755mmol, 1 equiv.) were added and the mixture was stirred at room temperature for 20 h. The mixture was then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give a light brown oil. The crude material was purified by column chromatography (ISCO, 12g silica column, 0 to 15% MeOH/DCM, 25 min. gradient) to give a light brown oil (373.1mg, 0.678mmol, 89.8%).¹H NMR(500MHz,DMSO-d₆)δ11.10(s,2H),8.48(t,J＝ 5.8Hz,1H),7.80(dd,J＝8.4,7.3Hz,1H),7.49(d,J＝7.2Hz,1H), 7.40(d,J＝8.6Hz,1H),7.26–7.08(m,4H),5.11(dd,J＝12.9,5.4 Hz,1H),4.86(s,2H),4.33(d,J＝3.9Hz,2H),4.09(d,J＝5.3Hz, 2H),2.65–2.51(m,3H),2.07–1.99(m,1H),1.38(s,9H).LCMS 551.5(M+H)。

Step 2: synthesis of N- (4- (aminomethyl) benzyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate

TFA (6.77mL, 0.1M) was added to tert-butyl (4- ((2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamido) methyl) benzyl) carbamate (373.1mg, 0.677mmol, 1eq) ester. The mixture was stirred at 50 ℃ for 1.5 hours. The mixture was then dissolved in MeOH and concentrated under reduced pressure to give a brown oil (270.29mg), which was used without further purification.¹H NMR(500 MHz,DMSO-d₆)δ11.11(s,1H),8.55(t,J＝6.2Hz,1H),8.07(s, 3H),7.81(dd,J＝8.5,7.3Hz,1H),7.51(d,J＝7.2Hz,1H),7.40(dd, J＝14.9,8.3Hz,3H),7.31(d,J＝8.2Hz,2H),5.11(dd,J＝12.9,5.4Hz,1H),4.87(s,2H),4.37(d,J＝6.1Hz,2H),4.01(q,J＝5.8 Hz,2H),2.66–2.51(m,3H),2.07–1.99(m,1H).LCMS 451.3 (M+H)。

And step 3: synthesis of dBET41

A solution of 0.1M N- (4- (aminomethyl) benzyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (0.237mL, 0.0237mmol, 1eq) was added to JQ-acid (9.5mg, 0.0237mmol, 1eq) at room temperature. After 23 hours, the mixture was diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as a cream colored solid (11.8mg, 0.0142 mmol, 60%).¹H NMR (500MHz, methanol-d)₄)δ7.80–7.75(m,1H), 7.51(dd,J＝7.3,1.5Hz,1H),7.41(d,J＝8.4Hz,1H),7.36(d,J＝2.2Hz,4H),7.34–7.28(m,4H),5.10–5.00(m,1H),4.82(s,2H), 4.67–4.64(m,1H),4.61–4.42(m,4H),4.34(dd,J＝14.9,12.8Hz, 1H),3.49(ddd,J＝14.8,9.5,5.2Hz,1H),2.83–2.75(m,1H),2.73 –2.61(m,5H),2.44–2.39(m,3H),2.06(ddq,J＝9.8,4.7,2.6Hz, 1H),1.66(d,J＝4.2Hz,3H).LCMS832.92(M+H)。

Synthesis example 42: synthesis of dBET42

A solution of 0.1M 5-amino-N- (2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) pentanamide trifluoroacetate in DMF (222. mu.l, 0.0222mmol, 1eq) was added JQ-acid (8.9mg, 0.0222mmol, 1 eq). DIPEA (11.6 μ l, 0.0666mmol, 3 equiv.) and HATU (8.4mg, 0.0222mmol, 1 equiv.) were then added and the mixture was stirred at room temperature for 24 h. The mixture was then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as a white solid (12.23mg, 0.0165mmol, 74%).¹H NMR (500MHz, methanol-d)₄)δ 7.76–7.71(m,1H),7.66–7.62(m,1H),7.51(td,J＝7.8,2.5Hz, 1H),7.45–7.35(m,4H),5.11(ddd,J＝13.2,11.3,5.2Hz,1H),4.63 (ddd,J＝8.8,5.7,3.2Hz,1H),4.47(s,2H),3.45–3.32(m,3H), 3.30–3.27(m,1H),2.90–2.80(m,1H),2.73–2.63(m,4H),2.49(t, J＝7.4Hz,2H),2.46–2.38(m,4H),2.11(ddtd,J＝12.8,10.5,5.3, 2.3Hz,1H),1.84–1.75(m,2H),1.66(dd,J＝16.2,7.6Hz,5H). LCMS 741.46(M+H)。

Synthesis example 43: synthesis of dBET43

A solution of 0.1M 7-amino-N- (2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindol-4-yl) heptanamide trifluoroacetate in DMF (227. mu.l, 0.0227mmol, 1eq) was added to JQ-acid (9.1mg, 0.0227mmol, 1 eq). DIPEA (11.9. mu.l, 0.0681mmol, 3 equiv.) and HATU (8.6mg, 0.0227mmol, 1 equiv.) were then added and the mixture was stirred at room temperature for 25.5 h. The mixture was then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) affordedTo the desired product as an off-white solid (12.58mg, 0.0164mmol, 72%).¹H NMR (500MHz, methanol-d)₄)δ 7.71(d,J＝7.9Hz,1H),7.64(d,J＝7.4Hz,1H),7.51(t,J＝7.8Hz, 1H),7.46–7.38(m,4H),5.14(ddd,J＝13.3,5.2,2.2Hz,1H),4.62(ddd,J＝8.6,5.6,2.1Hz,1H),4.49–4.45(m,2H),3.39(ddd,J＝ 14.9,8.7,1.3Hz,1H),3.30–3.24(m,3H),2.93–2.83(m,1H),2.79 –2.65(m,4H),2.50–2.40(m,6H),2.16(ddq,J＝9.9,5.2,2.6Hz, 1H),1.78–1.70(m,2H),1.68(d,J＝2.1Hz,3H),1.63–1.57(m, 2H),1.50–1.42(m,4H).LCMS 769.55(M+H)。

Synthesis example 44: synthesis of dBET44

A solution of 0.1M 8-amino-N- (2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) octanoyl amide trifluoroacetate in DMF (217. mu.l, 0.0217mmol, 1eq) was added JQ-acid (8.7mg, 0.0217mmol, 1 eq). DIPEA (11.3. mu.l, 0.0651mmol, 3 equiv.) and HATU (8.3mg, 0.0217mmol, 1 equiv.) were then added and the mixture was stirred at room temperature for 20.5 h. The mixture was then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0-10% MeOH/DCM, 25 min gradient) afforded the desired product as a cream colored solid (14.28mg, 0.0182mmol, 84%).¹H NMR (500MHz, methanol-d)₄) δ7.72–7.68(m,1H),7.64(d,J＝7.5Hz,1H),7.51(t,J＝7.7Hz, 1H),7.46–7.39(m,4H),5.14(dt,J＝13.3,5.0Hz,1H),4.62(dd,J ＝8.8,5.4Hz,1H),4.48–4.44(m,2H),3.40(ddd,J＝14.9,8.8,0.9 Hz,1H),3.26(dt,J＝13.2,6.9Hz,3H),2.88(ddd,J＝18.7,13.5, 5.4Hz,1H),2.75(dddd,J＝17.6,7.1,4.5,2.4Hz,1H),2.68(d,J＝ 2.2Hz,3H),2.49–2.39(m,6H),2.17(ddt,J＝9.8,5.3,2.3Hz,1H),1.76–1.70(m,2H),1.70–1.67(m,3H),1.61–1.54(m,2H),1.42 (s,6H).LCMS 783.53(M+H)。

Synthesis example 45: synthesis of dBET45

A solution of 0.1M N- (8-aminooctyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (268. mu.l, 0.0268mmol, 1eq) was added to (R) -4- ((4-cyclopentyl-1, 3-dimethyl-2-oxo-1, 2,3, 4-tetrahydropyrido [2,3-b ] at room temperature]Pyrazine-) 6-yl) amino) -3-methoxybenzoic acid (11.0 mg, 0.0268mmol, 1 eq). DIPEA (14.0. mu.L, 0.0804mmol, 3 equiv.) and HATU (10.2mg, 0.0268mmol, 1 equiv.) were then added and the mixture stirred for 18.5 h. The mixture was then diluted with methanol and purified by preparative HPLC to give the desired product as a dark brown solid (10.44mg, 0.0108mmol, 40%).¹H NMR (500MHz, methanol-d)₄)δ8.38(d,J＝8.4Hz,1H), 7.80–7.75(m,1H),7.55–7.48(m,1H),7.48–7.35(m,3H),7.27 (d,J＝8.3Hz,1H),6.45(d,J＝8.2Hz,1H),5.12(dd,J＝12.5,5.5 Hz,1H),4.72(d,J＝5.1Hz,2H),4.53(s,1H),4.28(d,J＝6.8Hz, 1H),3.98(d,J＝4.1Hz,3H),3.48–3.33(m,4H),2.90–2.82(m, 1H),2.80–2.69(m,2H),2.18–2.01(m,4H),1.88–1.52(m,10H), 1.34(d,J＝42.9Hz,10H),1.17(d,J＝6.8Hz,3H).LCMS851.67 (M+H)。

Synthesis example 46: synthesis of dBET46

A solution of 0.1M N- (3- (2- (2- (3-aminopropoxy) ethoxy) propyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-diisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (256 microliters, 0.0256mmol, 1 equivalent) was added to (R) -4- ((4-cyclopentyl-1, 3-dimethyl-2-oxo-1, 2,3, 4-tetrahydropyrido [2,3-b ] at room temperature]Pyrazin-6-yl) amino) -3-methoxybenzoic acid (10.5mg, 0.0256mmol, 1 eq). DIPEA (13.4. mu.l, 0.0767mmol, 3 equiv.) and HATU (9.7mg, 0.0256mmol, 1 equiv.) were then added and the mixture stirred for 20 h. The mixture was then diluted with methanol and purified by preparative HPLC to give the desired product as a dark brown solid (13).69mg，0.0132mmol，51％)。¹H NMR (500MHz, methanol-d)₄)δ 8.28–8.24(m,1H),7.74–7.71(m,1H),7.49(dd,J＝7.3,3.7Hz, 1H),7.39–7.34(m,2H),7.28–7.25(m,1H),7.14–7.10(m,1H), 6.34(d,J＝8.3Hz,1H),5.01–4.97(m,1H),4.62(s,2H),4.25(q,J ＝6.7Hz,1H),3.95(d,J＝5.4Hz,3H),3.60(ddd,J＝9.0,6.1,1.6 Hz,8H),3.53–3.46(m,6H),3.40–3.37(m,2H),2.78(td,J＝11.1, 6.6Hz,3H),2.16–2.00(m,4H),1.84(ddt,J＝33.5,13.0,6.4Hz,7H),1.75–1.60(m,6H),1.17(d,J＝6.8Hz,3H).LCMS 927.74 (M+H)。

Synthesis example 47: synthesis of dBET50

A solution of 0.1M N- (3- (2- (2- (3-aminopropoxy) ethoxy) propyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (200. mu.l, 0.0200mmol, 1eq) was added to (S) -4- (4-chlorophenyl) -6- (2-methoxy-2-oxoethyl) -3, 9-dimethyl-6H-thieno [3,2-f ] at room temperature][1,2,4]Triazolo [4,3-a][1,4]Diazepatriene-2-carboxylic acid (8.9mg, 0.020mmol, 1 equivalent). DIPEA (10.5. mu.l, 0.060mmol, 3 equivalents) and HATU (7.6mg, 0.020mmol, 1 equivalent) were added. The mixture was then stirred for 17 hours, then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as a cream colored solid (9.31mg, 0.00968mmol, 48%).¹H NMR (500MHz, methanol-d)₄)δ7.82–7.78(m,1H),7.52(dd,J＝7.1,1.6Hz,1H), 7.49–7.40(m,5H),5.10(ddd,J＝12.8,5.5,2.9Hz,1H),4.74(s, 2H),4.67(t,J＝7.1Hz,1H),3.76(s,3H),3.62–3.50(m,14H),3.49 –3.43(m,2H),3.40(q,J＝6.5Hz,2H),2.87(ddd,J＝17.6,14.0, 5.3Hz,1H),2.79–2.67(m,5H),2.12(ddq,J＝10.3,5.4,2.9Hz, 1H),2.00(s,3H),1.86(q,J＝6.3Hz,2H),1.80(p,J＝6.4Hz,2H)。 LCMS 961.67(M+H)。

Synthesis example 48: synthesis of dBET51

A solution of 0.1M N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (200. mu.l, 0.0200mmol, 1eq) was added to (S) -4- (4-chlorophenyl) -6- (2-methoxy-2-oxoethyl) -3, 9-dimethyl-6H-thieno [3,2-f ] at room temperature][1,2,4]Triazolo [4,3-a][1,4]Diazepatriene-2-carboxylic acid (8.9mg, 0.020mmol, 1 equivalent). DIPEA (10.5. mu.l, 0.060mmol, 3 equiv.) and HATU (7.6mg, 0.020mmol, 1 equiv.) were added. The mixture was then stirred for 17 hours, then diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as an off-white solid (8.38mg, 0.00942mmol, 47%).¹H NMR (500MHz, methanol-d)₄)δ 7.80(dd,J＝8.4,7.4Hz,1H),7.52(dd,J＝7.2,1.3Hz,1H),7.48– 7.38(m,5H),5.08(ddd,J＝12.7,5.5,1.6Hz,1H),4.74(d,J＝2.7 Hz,2H),4.66(t,J＝7.1Hz,1H),3.75(d,J＝3.0Hz,3H),3.65(t,J ＝4.1Hz,6H),3.59(t,J＝5.3Hz,2H),3.57–3.49(m,4H),3.49– 3.40(m,2H),2.93–2.84(m,1H),2.78–2.64(m,5H),2.15–2.09 (m,1H),2.00(d,J＝0.9Hz,3H).LCMS 889.58(M+H)。

Synthesis example 49: synthesis of dBET52

A solution of 0.1M N- (2- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamido trifluoroacetate in DMF (200. mu.l, 0.020mmol, 1eq) was added JQ-acid (8.0mg, 0.020mmol, 1eq) at room temperature. DIPEA (10.5. mu.l, 0.060mmol, 3 equiv.) and HATU (7.6mg, 0.020mmol, 1 equiv.) were added. After 17.5 hours, mixThe mixture was diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product as a colorless residue (9.12mg, 0.01025mmol, 51%).¹H NMR (500MHz, methanol-d)₄)δ7.77(t,J＝7.9Hz,1H),7.50(dd,J＝ 7.3,1.5Hz,1H),7.47–7.36(m,5H),5.09(ddd,J＝13.0,7.6,5.5 Hz,1H),4.76(s,2H),4.62(dd,J＝9.1,5.1Hz,1H),3.62(ddt,J＝ 17.3,11.2,6.5Hz,12H),3.52–3.41(m,5H),3.28(d,J＝5.1Hz, 1H),2.90–2.81(m,1H),2.79–2.66(m,5H),2.44(s,3H),2.16– 2.09(m,1H),1.69(s,3H).LCMS 889.38(M+H)。

Synthesis example 50: synthesis of dBET53

A solution of 0.1M N- (14-amino-3, 6,9, 12-tetraoxatetradecyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (200. mu.l, 0.020mmol, 1eq) was added JQ-acid (8.0mg, 0.020mmol, 1eq) at room temperature. DIPEA (10.5. mu.l, 0.060mmol, 3 equiv.) and HATU (7.6mg, 0.020mmol, 1 equiv.) were added. After 17.5 h, HATU (7.6mg) and DIPEA (10.5. mu.l) were added and the mixture was stirred for an additional 5 h. The mixture was diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product (3.66 mg).¹H NMR (500MHz, methanol-d)₄)δ7.79(dd,J＝8.4,7.4Hz, 1H),7.51(d,J＝7.3Hz,1H),7.45(d,J＝7.7Hz,2H),7.43–7.36 (m,3H),5.08(ddd,J＝12.7,5.5,2.2Hz,1H),4.78–4.74(m,2H), 4.62(dd,J＝9.1,5.1Hz,1H),3.70–3.51(m,16H),3.50–3.41(m, 5H),3.27(dd,J＝5.1,2.3Hz,1H),2.87(ddt,J＝18.2,9.5,4.9Hz, 1H),2.78–2.66(m,5H),2.44(s,3H),2.16–2.09(m,1H),1.69(s, 3H).LCMS 933.43(M+H)。

Synthesis example 51: synthesis of dBET54

A solution of 0.1M N- (17-amino-3, 6,9,12, 15-pentaoxaheptadecyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (200. mu.l, 0.020mmol, 1eq) was added JQ-acid (8.0mg, 0.020mmol, 1eq) at room temperature. DIPEA (10.5. mu.l, 0.060mmol, 3 equiv.) and HATU (7.6mg, 0.020mmol, 1 equiv.) were added. After 16 h, the mixture was diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) afforded the desired product (6.27mg, 0.00641mmol, 32%).¹H NMR (500MHz, methanol-d)₄) δ7.81–7.76(m,1H),7.51(d,J＝7.1Hz,1H),7.47–7.38(m,5H), 5.09(dd,J＝12.6,5.5Hz,1H),4.77(s,2H),4.62(dd,J＝8.8,5.0 Hz,1H),3.67–3.55(m,20H),3.46(ddd,J＝20.1,10.2,4.7Hz,5H), 3.28(d,J＝5.1Hz,1H),2.91–2.83(m,1H),2.78–2.68(m,5H), 2.44(s,3H),2.16–2.10(m,1H),1.72–1.66(m,3H).LCMS 977.50 (M+H)。

Synthesis example 52: synthesis of dBET55

To a solution of 0.1M N- (29-amino-3, 6,9,12,15,18,21,24, 27-nonanoyloxy-dioctadecyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (200. mu.l, 0.020mmol, 1eq) at room temperature was added JQ-acid (8.0mg, 0.020mmol, 1 eq). DIPEA (10.5. mu.L, 0.060mmol, 3 equiv.) and HATU (7.6mg, 0.020mmol, 1 equiv.) were added. After 18 h, the mixture was diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. Column chromatography (ISCO, 4g silica gel column, 0 to 10% M)eOH/DCM, 25 min gradient) to give the desired product (10.55mg, 0.00914mmol, 46%).¹H NMR (500MHz, methanol-d)₄)δ7.82(dd,J＝8.4,7.4Hz,1H),7.55(d,J＝7.0Hz, 1H),7.49–7.41(m,5H),5.13(dd,J＝12.6,5.5Hz,1H),4.80(s, 2H),4.65(dd,J＝9.1,5.1Hz,1H),3.68–3.58(m,36H),3.53–3.44 (m,5H),2.94–2.86(m,1H),2.81–2.70(m,5H),2.46(s,3H),2.19 –2.13(m,1H),1.74–1.69(m,3H).LCMS1153.59(M+H)。

Synthesis example 53: synthesis of dBET56

A solution of 0.1M N- (35-amino-3, 6,9,12,15,18,21,24,27,30, 33-decapentaene triacontyl) -2- ((2- (2, 6-dioxopiperidine) -3-methyl) -1, 3-oxoisoindolin-4-yl) oxy) acetamide trifluoroacetate in DMF (200. mu.l, 0.020mmol, 1eq) was added to JQ-acid (8.0mg, 0.020mmol, 1eq) at room temperature. DIPEA (10.5. mu.l, 0.060mmol, 3 equiv.) and HATU (7.6mg, 0.020mmol, 1 equiv.) were added. After 20 h, the mixture was diluted with EtOAc and washed with saturated sodium bicarbonate, water and brine. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification by column chromatography (ISCO, 4g silica gel column, 0 to 10% MeOH/DCM, 25 min gradient) gave the desired product as an oily residue (9.03mg, 0.00727 mmol, 36%).¹H NMR (500MHz, methanol-d)₄)δ7.81(dd,J＝8.4,7.4 Hz,1H),7.53(d,J＝7.1Hz,1H),7.50–7.40(m,5H),5.11(dd,J＝ 12.6,5.5Hz,1H),4.78(s,2H),4.68(dd,J＝8.6,5.0Hz,1H),3.69– 3.56(m,44H),3.52–3.43(m,5H),3.34(dd,J＝7.9,3.5Hz,1H), 2.88(ddd,J＝18.0,14.0,5.2Hz,1H),2.79–2.68(m,5H),2.46(s, 3H),2.17–2.12(m,1H),1.71(s,3H).LCMS 1241.60(M+H)。

Synthesis example 54: synthesis of dBET57

Step 1: synthesis of 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione

To a solution of 4-fluoroisobenzofuran-1, 3-dione (200mg, 1.20mmol, 1 equiv.) in AcOH (4.0mL, 0.3M) was added 2, 6-dioxopiperidine-3-amine hydrochloride (218 mg, 1.32mmol, 1.1 equiv.) and potassium acetate (366mg, 3.73mmol, 3.1 equiv.). The reaction mixture was heated to 90 ℃ overnight, then diluted to 20mL with water and cooled on ice for 30 min. The resulting slurry was filtered and purified by flash column chromatography (2% MeOH in CH)₂Cl₂Solution of R_f0.3) on silica gel to give the title compound as a white solid (288mg, 86%).¹H NMR(500MHz,DMSO- d₆) δ 11.15(s,1H),7.96(ddd, J ═ 8.3,7.3,4.5Hz,1H), 7.82-7.71 (m,2H),5.17(dd, J ═ 13.0,5.4Hz,1H),2.90(ddd, J ═ 17.1,13.9, 5.4Hz,1H), 2.65-2.47 (m,2H), 2.10-2.04 (m,1H), calculate C₁₃H₁₀FN₂O₄[M+H]⁺277.06, ms (esi), found to be 277.25.

Step 2: synthesis of tert-butyl (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) amino) ethyl) carbamate

To a stirred solution of 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (174mg, 0.630mmol, 1 equiv.) in DMF (6.3mL, 0.1M) was added DIPEA (220. mu.L, 1.26mmol, 2 equiv.) and 1-Boc-ethylenediamine (110. mu.L, 0.693mmol, 1.1 equiv.). The reaction mixture was heated to 90 ℃ overnight, then cooled to room temperature and dissolved in EtOAc (30mL) and water (30 mL). The organic layer was washed with brine (3X 20mL) and Na₂SO₄Dried and concentrated in vacuo. The residue was purified by flash column chromatography (0 → 10% MeOH in CH2Cl 2) on silica gel to give the title compound as a yellow solid (205mg, 79%).¹H NMR(500MHz,CDCl₃)δ8.08(bs, 1H),7.50(dd,J＝8.5,7.1Hz,1H),7.12(d,J＝7.1Hz,1H),6.98(d, J＝8.5Hz,1H),6.39(t,J＝6.1Hz,1H),4.96–4.87(m,1H),4.83 (bs,1H),3.50–3.41(m,2H),3.41–3.35(m,2H), 2.92-2.66 (m,3H), 2.16-2.09 (m,1H),1.45(s, 9H); calculating C₂₀H₂₅N₄O₆[M+H]⁺417.18, ms (esi), found to be 417.58.

And step 3: synthesis of 2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) ethan-1-ammonium 2,2, 2-trifluoroacetate

To a stirred solution of tert-butyl (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) amino) ethyl) carbamate (205mg, 0.492mmol, 1eq) in dichloromethane (2.25mL) was added trifluoroacetic acid (0.250 mL). The reaction mixture was stirred at room temperature for 4 hours, then the volatiles were removed in vacuo. The title compound was obtained as a yellow solid (226mg,>95%) which was used without further purification.¹H NMR (500MHz, MeOD) δ 7.64(d, J ═ 1.4Hz,1H), 7.27-7.05 (m,2H),5.10 (dd, J ═ 12.5,5.5Hz,1H),3.70(t, J ═ 6.0Hz,2H), 3.50-3.42 (m,2H), 3.22(t, J ═ 6.0Hz,1H), 2.93-2.85 (m,1H), 2.80-2.69 (m,2H), 2.17-2.10 (m, 1H); calculating C₁₅H₁₇N₄O₄[M+H]⁺317.12, ms (esi), found to be 317.53.

Step 2: synthesis of dBET57

JQ-acid (8.0mg, 0.0200mmol, 1 equiv.) and 2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) amino) ethan-1-aminium 2,2, 2-trifluoroacetate (8.6mg, 0.0200mmol, 1 equiv.) were dissolved in DMF (0.200mL, 0.1M) at room temperature. DIPEA (17.4. mu.L, 0.100mmol, 5 equiv.) and HATU (7.59mg, 0.0200mmol, 1 equiv.) were then added and the mixture was stirred at room temperature overnight. The reaction mixture was dissolved in EtOAc (15mL) and saturated NaHCO₃(aqueous) (15mL), water (15mL) and brine (3 × 15 mL). The organic layer was washed with Na₂SO₄Drying and vacuum concentrating. Flash column chromatography (0 → 10% MeOH in CH2Cl2, R)_f0.3 (10% MeOH in CH)₂Cl₂Solution)) on silica gel to give the title compound as a bright yellow solid (11.2mg, 80%).¹H NMR(400MHz,CDCl₃) δ 8.49(bs,0.6H),8.39(bs,0.4H), 7.51-7.43 (m,1H),7.38(d, J ═ 7.8Hz,2H),7.29(dd, J ═ 8.8,1.7Hz,2H),7.07(dd, J ═ 7.1,4.9Hz, 1H),6.97(dd, J ═ 8.6,4.9Hz,1H),6.48(t, J ═ 5.9Hz,1H),6.40(t, J ═ 5.8Hz,0.6H), 4.91-4.82 (m,0.4H), 4.65-4.60 (m,1H), 3.62-3.38 (m,6H), 2.87-2.64 (m,3H),2.63, 3.40(m, 3H), 2.04 (s, 2.6H), 2.04 (r, 3.6H), 2.6.6H, 2.6 hrs (m,12 r, 2.04, 2.6H); calculating C₃₄H₃₂ClN₈O₅S[M+H]⁺700.19, ms (esi), found to be 700.34.

Synthesis example 55: dGR1 Synthesis

Synthesis example 56: dGR2 Synthesis:

synthesis example 57: dGR3 Synthesis:

synthesis example 58: synthesis of dFKBP-1

(1) Synthesis of SLF-succinate

SLF (25mg, 2.5mL of a 10mg/mL MeOAc solution, 0.0477mmol, 1 equiv.) was mixed with DMF (0.48mL, 0.1M) and succinic anhydride (7.2mg, 0.0715 mmol, 1.5 equiv.)) Combine and stir at room temperature for 24 hours. Low conversion was observed and the mixture was placed in N₂Air flow to remove MeOAc. An additional 0.48mL of DMF was added, along with an additional 7.2mg of succinic anhydride and DMAP (5.8mg, 0.0477mmol, 1 equivalent). The mixture was then stirred for an additional 24 hours and then purified by preparative HPLC to give SLF-succinate as a yellow oil (24.06mg, 0.0385mmol, 81%).¹H NMR (400MHz, methanol-d 4) δ 7.62(d, J ═ 10.7Hz,1H),7.44(d, J ═ 8.0Hz,1H),7.26(td, J ═ 7.9,2.7Hz,1H), 7.07-6.97 (m,1H), 6.80(dd, J ═ 8.1,2.1Hz,1H), 6.74-6.66 (m,2H),5.73(dd, J ═ 8.1, 5.5Hz,1H),5.23(d, J ═ 4.8Hz,1H),3.83(s,3H),3.81(s,3H), 3.39-3.29 (m,4H),3.21(td, J ═ 13.2,3.0, 1H),2.68, 2.2H, 2.19(m,2H), 1.19.19-2H, 1H), 3.19(m, 2H), 1.27.27.27.27H, 1H, 5H, 1. LCMS 624.72(M + H).

(2) Synthesis of dFKBP-1

N- (4-Aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamide trifluoroacetate (9.9mg, 0.0192mmol, 1eq) was added to a solution of SLF succinate (11.98mg, 0.0192mmol, 1eq) in 0.192mL DMF (0.1M). DIPEA (10.0. mu.l, 0.0575mmol, 3 equiv.) was added followed by HATU (7.3mg, 0.0192mmol, 1 equiv.). The mixture was stirred for 17 hours, then diluted with MeOH and purified by preparative HPLC to give dFKBP-1 (7.7mg, 0.00763mmol, 40%) as a yellow solid.

¹H NMR (400MHz, methanol-d 4) δ 7.81(s,1H), 7.77-7.70 (m,1H), 7.55-7.49 (m,2H),7.26(dd, J ═ 8.0,5.3Hz,2H), 7.05-6.99 (m,1H), 6.77(d, J ═ 8.8Hz,1H),6.66(d, J ═ 6.8Hz,2H), 5.77-5.72 (m,1H),5.24(d, J ═ 4.8Hz,1H),4.99(dd, J ═ 12.3,5.7Hz,1H), 4.68-4.59 (m,2H),3.82(s,3H),3.81(s,3H),3.32 (J, 3.32 ═ 3.3, 6, 4.3, 3.9H), 3.9, 14H, 14.9, 14H, 9H, 14H, 9H, 14H, 2H, 9H, 1.43-1.30 (m,2H), 1.20-1.04 (m,6H), 0.90-0.76 (m, 3H). 13C NMR (100MHz, cd3od) delta 208.51,173.27,172.64,171.63,169.93,169.51,168.04,167.69, 167.09,166.71,154.92,149.05,147.48,140.76,138.89,137.48, 133.91,133.67,129.36,122.19,120.61,120.54,119.82,118.41,118.12,117.79,112.12,111.76,68.54,56.10,55.98,51.67,46.94, 44.57,39.32,39.01,38.23,32.64,31.55,31.43,26.68,26.64,25.08, 23.52,23.21,22.85,21.27,8.76.LCMS1009.66(M+H)。

Synthesis example 59: synthesis of dFKBP-2

(1) Synthesis of tert-butyl (1-chloro-2-oxo-7, 10, 13-trioxa-3-azahexadecan-16-yl) carbamate

Tert-butyl (3- (2- (2- (3-aminopropoxy) ethoxy) propyl) carbamate (1.0g, 3.12mmol, 1eq) was dissolved in THF (31mL, 0.1M). DIPEA (0.543mL, 3.12mmol, 1 equiv.) was added and the solution was cooled to 0 ℃. Chloroacetyl chloride (0.273mL, 3.43mmol, 1.1 equiv.) was added and the mixture was allowed to warm slowly to room temperature. After 24 h, the mixture was diluted with EtOAc, washed with saturated sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give a yellow oil (1.416g), which was used without further purification.

¹H NMR (400MHz, chloroform-d) δ 7.24(s,1H),5.00(s,1H), 3.98-3.89 (m,2H),3.54(dddt, J ═ 17.0,11.2,5.9,2.2Hz,10H), 3.47-3.40 (m,2H), 3.37-3.31 (m,2H), 3.17-3.07 (m,2H), 1.79-1.70 (m,2H), 1.67(p, J ═ 6.1Hz,2H),1.35(s,9H).¹³C NMR(100MHz,cdcl3)δ 165.83,155.97,78.75,70.49,70.47,70.38,70.30,70.14,69.48, 42.61,38.62,38.44,29.62,28.59,28.40.LCMS 397.37(M+H)。

(2) Synthesis of dimethyl 3- ((2, 2-dimethyl-4, 20-dioxo-3, 9,12, 15-tetraoxa-5, 19-diazadidodec-21-yl) oxy) phthalate

Tert-butyl (1-chloro-2-oxo-7, 10, 13-trioxa-3-azahexadecan-16-yl) carbamate (1.41g, 3.12mmol, 1eq) was dissolved in MeCN (32mL, 0.1M). Dimethyl 3-hydroxyphthalite (0.721g, 3.43mmol, 1.1 equiv.) and cesium carbonate (2.80g, 8.58mmol, 2.75 equiv.) were added. The flask was fitted with a reflux condenser and heated to 80 ℃ for 19 hours. The mixture was cooled to room temperature and diluted with water, extracted once with chloroform and twice with EtOAc. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by column chromatography (ISCO, 24g silica column, 0 to 15% MeOH/DCM, 22 min gradient) to give a yellow oil (1.5892g, 2.78mmol, 89%, two steps).

¹H NMR (400MHz, chloroform-d) δ 7.52(d, J ═ 7.8Hz,1H),7.35(t, J ═ 8.1Hz,1H),7.04(d, J ═ 8.3Hz,1H),7.00(t, J ═ 5.3Hz,1H),5.06(s,1H),4.46(s,2H),3.83(s,3H),3.78(s,3H), 3.47(ddd, J ═ 14.9,5.5,2.8Hz,8H),3.39(dt, J ═ 9.4,6.0Hz,4H),3.29(q, J ═ 6.5Hz,2H),3.09(d, J ═ 6.0Hz,2H),1.70(p, J ═ 6.5, 2H),1.63(p, 1.31H), 1.31H, 1H, 31H).¹³C NMR(100MHz,cdcl3)δ167.68, 167.36,165.45,155.93,154.41,130.87,129.60,125.01,123.20, 117.06,78.60,70.40,70.17,70.06,69.39,68.67,68.25,52.77,52.57,38.38,36.58,29.55,29.20,28.34.LCMS 571.47(M+H)。

(3) Synthesis of N- (3- (2- (2- (3-aminopropoxy) ethoxy) propyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamide trifluoroacetate dimethyl 3- ((2, 2-dimethyl-4, 20-dioxo-3, 9,12, 15-tetraoxa-5, 19-diazadidodec-21-yl) oxy) phthalate (1.589g, 2.78mmol, 1eq) was dissolved in EtOH (14mL, 0.2M). Aqueous 3M NaOH (2.8mL, 8.34mmol, 3 equiv.) was added and the mixture was heated to 80 ℃ for 22 hours. The mixture was then cooled to room temperature and diluted with 50mL of DCM and 20mL of 0.5M HCl. The layers were separated and the organic layer was washed with 25mL of water. The aqueous layers were combined and extracted three times with 50mL of chloroform. The combined organic layers were dried over sodium sulfate, filtered and concentrated to give 1.53g of material which was used without further purification. LCMS 553.44.

The resulting material (1.53g) and 3-aminopiperidine-2, 6-dione hydrochloride (0.480g, 2.92mmol, 1eq) were dissolved in pyridine (11.7mL, 0.25M) and heated to 110 ℃ for 17 hours. The mixture was cooled to room temperature and concentrated under reduced pressure to give crude tert-butyl (1- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-oxoisoindolin-4-yl) oxy) -2-oxo-7, 10, 13-trioxa-3-azahexadecan-16-yl) carbamate as a black sludge (3.1491g), which was used on without further purification. LCMS 635.47.

Crude tert-butyl (1- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) -2-oxo-7, 10, 13-trioxa-3-azahexadecan-16-yl) carbamate (3.15g) was dissolved in TFA (20mL) and heated to 50 ℃ for 2.5 hours. The mixture was cooled to room temperature, diluted with MeOH and concentrated under reduced pressure. This material was purified by preparative HPLC to give N- (3- (2- (2- (3-aminopropoxy) ethoxy) propyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamide trifluoroacetate (1.2438g, 1.9598mmol, 71%, 3 steps) as a dark red oil.

¹H NMR (400MHz, methanol-d 4) δ 7.77(dd, J ═ 8.3,7.5Hz,1H),7.49 (d, J ═ 7.3Hz,1H),7.40(d, J ═ 8.5Hz,1H),5.12(dd, J ═ 12.8,5.5 Hz,1H),4.75(s,2H), 3.68-3.51 (m,12H),3.40(t, J ═ 6.8Hz,2H), 3.10(t, J ═ 6.4Hz,2H), 2.94-2.68 (m,3H),2.16(dtd, J ═ 12.6,5.4, 2.5Hz,1H),1.92(p, J ═ 6.1, 2H), 1.86-1.77 (m,2H).¹³C NMR (100MHz,cd3od)δ173.17,169.97,168.48,166.87,166.30,154.82, 136.89,133.41,120.29,117.67,116.58,69.96,69.68,69.60,68.87, 68.12,67.92,49.19,38.62,36.14,30.80,28.92,26.63,22.22.LCMS 536.41(M+H).

(4) Synthesis of dFKBP-2

N- (3- (2- (2- (3-aminopropoxy) ethoxy) propyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) acetamide trifluoroacetate (12.5mg, 0.0193mmol, 1eq) was added to a solution of SLF-succinate (12.08mg, 0.0193mmol, 1eq) in 0.193mL of DMF (0.1M) DIPEA (10.1. mu.L, 0.0580mmol, 3 eq) and HATU (7.3mg, 0.0193mmol, 1eq) and the mixture was stirred for 19 h before the mixture was diluted with MeOH and purified by preparative HPLC to give dFKBP-2(9.34mg, 0.00818mmol, 42%) as a yellow oil.

¹H NMR (400MHz, 50% MeOD/chloroform-d) delta 7.76-7.70 (m,1H), 7.58-7.45 (m,3H),7.26(t, J ═ 8.2Hz,2H), 7.05-6.98(m,1H),6.77(d, J＝7.9Hz,1H),6.71–6.63(m,2H),5.73(dd,J＝8.1,5.6Hz,1H), 5.23(d,J＝5.4Hz,1H),5.03–4.95(m,1H),4.64(s,2H),3.82(s, 3H),3.80(s,3H),3.62–3.52(m,8H),3.47(t,J＝6.1Hz,2H),3.44 –3.33(m,3H),3.27–3.14(m,3H),2.84–2.70(m,3H),2.64–2.47 (m,6H),2.34(d,J＝14.1Hz,1H),2.24(dd,J＝14.3,9.3Hz,2H), 2.13–2.00(m,2H),1.83(p,J＝6.3Hz,2H),1.67(dtd,J＝38.4, 16.8,14.8,7.0Hz,7H),1.51–1.26(m,3H),1.22–1.05(m,6H), 0.80(dt,J＝39.8,7.5Hz,3H).¹³C NMR(100MHz,cdcl3)δ208.64, 173.39,173.01,171.76,170.11,169.62,168.24,167.92,167.36, 166.69,155.02,149.23,147.66,140.94,139.18,137.57,134.09, 133.91,129.49,122.32,120.75,120.52,119.93,118.42,117.75, 112.33,111.98,70.77,70.51,70.40,69.45,69.04,68.48,56.20, 56.10,51.88,47.09,44.78,38.40,37.48,36.91,32.80,32.71,31.70, 31.59,31.55,29.53,29.30,26.77,25.22,23.63,23.33,22.98,21.43. LCMS 1141.71(M+H).

Synthesis example 60: synthesis of dFKBP-3

A solution of 0.1M N- (4-aminobutyl) -2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) oxy) acetamide trifluoroacetate (0.233mL, 0.0233mmol, 1 equiv.) is added to 2- (3((R) -3- (3, 4-dimethoxyphenyl) -1- (((S) -1- (3, 3-dimethyl-2-oxopentanoyl) pyrrolidine-2-carbonyl) oxy) propyl) phenoxy) acetic acid (13.3mg, 0.0233mmol, 1 equiv.). DIPEA (12.2. mu.l, 0.0700mmol, 3 equiv.) was added followed by HATU (8.9mg, 0.0233mmol, 1 equiv.). The mixture was stirred for 23 h, then diluted with MeOH and purified by preparative HPLC to give a white solid (10.72mg, 0.0112mmol, 48%).

¹H NMR (400MHz, methanol-d)₄)δ7.79–7.74(m,1H),7.52(d,J＝7.4 Hz,1H),7.33(d,J＝8.4Hz,1H),7.26(t,J＝8.1Hz,1H),6.97– 6.90(m,2H),6.89–6.84(m,1H),6.79(dd,J＝8.2,1.9Hz,1H), 6.73–6.64(m,2H),5.73–5.65(m,1H),5.07–4.99(m,1H),4.67 (s,2H),4.57–4.51(m,1H),4.48(dd,J＝5.7,2.5Hz,2H),3.82(d,J ＝1.9Hz,3H),3.80(s,3H),3.66–3.39(m,3H),2.88–2.48(m,6H), 2.42–1.87(m,9H),1.73–1.51(m,6H),1.19–0.92(m,6H),0.75 (dt,J＝56.7,7.5Hz,3H).LCMS 954.52(M+H)。

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