ENPP1 inhibitors and methods of modulating immune responses
阅读说明：本技术 Enpp1抑制剂和调节免疫反应的方法 (ENPP1 inhibitors and methods of modulating immune responses ) 是由 L.李 M.史密斯 J.A.卡罗扎 V.博纳特 于 2020-01-30 设计创作，主要内容包括：本申请提供了用于抑制ENPP1的化合物、组合物和方法。主题方法的方面包括将样品与ENPP1抑制剂化合物接触以抑制ENPP1的cGAMP水解活性。在一些情况下,该ENPP1抑制剂化合物是细胞不可渗透的。ENPP1抑制剂化合物可以在细胞外发挥作用,阻断cGAMP的降解。还提供了用于治疗癌症的药物组合物和方法。该方法的方面包括向受试者给药治疗有效量的ENPP1抑制剂以治疗受试者的癌症。在某些情况下,该癌症是实体瘤癌症。还提供了向受试者施用放射疗法联合向受试者给药ENPP1抑制剂的方法。该放射疗法可以在主题方法中以有效降低对受试者造成的放射损伤,但是仍然能激发免疫响应的剂量和/或频率来施用。(The present application provides compounds, compositions and methods for inhibiting ENPP 1. Aspects of the subject methods include contacting a sample with an ENPP1 inhibitor compound to inhibit cGAMP hydrolytic activity of ENPP 1. In some cases, the ENPP1 inhibitor compound is cell impermeable. ENPP1 inhibitor compounds can act extracellularly, blocking the degradation of cGAMP. Pharmaceutical compositions and methods for treating cancer are also provided. Aspects of the methods include administering to the subject a therapeutically effective amount of an ENPP1 inhibitor to treat cancer in the subject. In some cases, the cancer is a solid tumor cancer. Methods of administering radiation therapy to a subject in conjunction with administering an ENPP1 inhibitor to the subject are also provided. The radiation therapy can be administered in the subject methods at a dose and/or frequency effective to reduce radiation damage to the subject, but still elicit an immune response.)
1. A compound of formula (I), or a prodrug, pharmaceutically acceptable salt, or solvate thereof:
wherein the content of the first and second substances,
X1is a hydrophilic head group (e.g., a phosphorus-containing group capable of binding zinc ions);
a is a ring system selected from: aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocycle, and substituted heterocycle;
L1and L2Independently a covalent bond or a linker;
Z3is absent or selected from NR22O and S;
Z2is CR12Or N;
Z1is CR11Or N;
R1selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkylaryl, substituted alkylaryl, alkylheteroaryl, substituted alkylheteroaryl, alkenylaryl (e.g., vinylaryl), substituted alkenylaryl, alkenylheteroaryl (e.g., vinylheteroaryl), substituted alkenylheteroaryl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic;
R11and R12Independently selected from the group consisting of H, cyano, trifluoromethyl, halogen, alkyl, and substituted alkyl;
R22selected from the group consisting of H, alkyl, and substituted alkyl; and
R2to R5Independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF 3Halogen, cyano, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or wherein R is2And R3、R3And R4Or R4And R5Together with the carbon atoms to which they are attached provide a fused ring (e.g., a 5-or 6-membered monocyclic ring) selected from the group consisting of heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.
2. The compound according to claim 1, wherein the compound is of formula (II):
wherein Z31Selected from NR22O and S.
3. A compound according to claim 2, wherein:
Z31is NR22(ii) a And
R22selected from H, C(1-6)Alkyl and substituted C(1-6)An alkyl group.
4. A compound according to claim 2 or 3, wherein the compound is of formula (III):
wherein R is31To R34Each independently selected from H, halogen, alkyl and substituted alkyl, or R31And R32Or R33And R34Are cyclic and together with the carbon atom to which they are attached provide a cycloalkyl, substituted cycloalkyl, heterocyclyl or substituted heterocyclyl ring; and
n and m are each independently an integer from 0 to 6 (e.g., 0-3).
5. The compound according to claim 4, wherein n + m is 0 to 3 (e.g., 0, 1, 2, or 3).
6. A compound according to any one of claims 1-5, wherein the ring system A is selected from phenyl, substituted phenyl, pyridyl, substituted pyridyl, pyrimidine, substituted pyrimidine, piperidine, substituted piperidine, piperazine, substituted piperazine, pyridazine, substituted pyridazine, cyclohexyl and substituted cyclohexyl.
7. The compound according to claim 6, wherein the ring system A is selected from:
wherein:
Z5selected from N and CR6；
R6Each selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide;
p is an integer of 0 to 4; and
q is an integer of 0 to 2.
8. The compound according to any one of claims 4-7, wherein the compound is of formula (IV):
wherein:
Z11and Z21Independently selected from N and c (cn);
each R is6Independently selected from the group consisting of H, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, and halogen; and
p is an integer of 0 to 4.
9. The compound according to claim 8, wherein the compound is of formula (V):
wherein:
R41to R44Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide.
10. The compound according to claim 9, wherein the compound is a compound of one of formulae (VIa) - (VIb):
11. The compound according to any one of claims 1-10, wherein R1Selected from the group consisting of H, alkylaryl, substituted alkylaryl, alkylheteroaryl, substituted alkylheteroaryl, alkenylaryl (e.g., vinylaryl), substituted alkenylaryl, alkenylheteroaryl (e.g., vinylheteroaryl), substituted alkenylheteroaryl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.
12. A compound according to claim 11, wherein R1Selected from the group consisting of H, vinyl aryl, substituted vinyl aryl, vinyl heteroaryl, and substituted vinyl heteroaryl.
13. The compound according to claim 10, wherein the compound is a compound of one of formulas (VIIa) - (VIIb):
14. the compound according to any one of claims 1-13, wherein R2To R5Independently selected from H, OH, alkyl, substituted alkyl, alkoxy, substituted alkoxy, -OCF3Halogen, halogenCyano, amines, substituted amines, amides, heterocycles and substituted heterocycles.
15. A compound according to claim 14, wherein R2To R5Independently selected from H, OH, C(1-6)Alkoxy, -OCF3、C(1-6)Alkylamino radical, di-C(1-6)Alkylamino, F, Cl, Br and CN.
16. A compound according to claim 14, wherein:
R3And R4Independently is an alkoxy group; and
R2and R5Is hydrogen.
17. A compound according to claim 14, wherein:
R3is an alkoxy group; and
R2、R4and R5Is hydrogen.
18. A compound according to claim 14, wherein:
R4is an alkoxy group; and
R2、R3and R5Is hydrogen.
19. A compound according to claim 14, wherein:
R2、R3and R4Is H; and
R5is an alkoxy group.
20. A compound according to any one of claims 4 to 19, wherein n is 0 and m is 1.
21. A compound according to any one of claims 4 to 19, wherein n is 1 and m is 0.
22. A compound according to any one of claims 4 to 19 wherein n and m are both 1.
23. A compound according to any one of claims 4 to 19, wherein n and m are both 0.
24. A compound according to claim 1, wherein Z3Is absent, and Z2Is CR12Wherein R is12Is cyano, wherein the compound is a compound of formula (X):
wherein L is11And L12Independently a covalent bond or a linker.
25. The compound according to claim 24, wherein said ring system a is selected from the group consisting of phenyl, substituted phenyl, pyridyl, substituted pyridyl, pyrimidine, substituted pyrimidine, piperidine, substituted piperidine, piperazine, substituted piperazine, pyridazine, substituted pyridazine, cyclohexyl, and substituted cyclohexyl.
26. The compound according to claim 24 or 25, wherein the compound is of formula (XI):
wherein:
each Z5Independently selected from N and CR16；
Each R is16Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and
r is an integer of 0 to 8.
27. The compound according to claim 26, wherein the compound is of formula (XII):
28. the compound according to claim 27, wherein Z5Is N.
29. The compound according to claim 28, wherein the compound is of formula (XIII):
where s is an integer from 0 to 6 (e.g., 0 to 3).
30. The compound according to claim 29, wherein the compound is of formula (XIV):
31. the compound according to any one of claims 24-30, wherein R2To R5Independently selected from H, OH, alkyl, substituted alkyl, alkoxy, substituted alkoxy, -OCF3Halogen, cyano, amine, substituted amine, amide, heterocycle, and substituted heterocycle.
32. The compound according to claim 31, wherein R 2To R5Independently selected from H, OH, C(1-6)Alkoxy, -OCF3、C(1-6)Alkylamino radical, di-C(1-6)Alkylamino, F, Cl, Br and CN.
33. A compound according to claim 31, wherein:
R3and R4Independently is an alkoxy group; and
R2and R5Is hydrogen.
34. A compound according to claim 31, wherein:
R3is an alkoxy group; and
R2、R4and R5Is hydrogen.
35. A compound according to claim 31, wherein:
R4is an alkoxy group; and
R2、R3and R5Is hydrogen.
36. A compound according to claim 31, wherein:
R2、R3and R4Is H; and
R5is an alkoxy group.
37. The compound according to any one of claims 29-36, wherein s is 1.
38. The compound according to any one of claims 29-36, wherein s is 2.
39. The compound according to any one of claims 29-36, wherein s is 3.
40. The compound according to any one of claims 1-39, wherein X1Including a precursor moiety that masks the charged phosphorus-containing groups capable of binding zinc ions.
41. The compound according to any one of claims 41-40, wherein X1Selected from the group consisting of phosphonic acids, phosphonates, phosphates, thiophosphates, phosphoramidates, and thiophosphates.
42. The compound according to any one of claims 1-41, wherein X 1Is of formula (XV):
wherein:
Z6is absent or selected from O and CH2；
Z7And Z9Each independently selected from O and NR10Wherein R is10Is H, alkyl or substituted alkyl;
Z8selected from O and S; and
R8and R9Each independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, non-aromatic heterocycle, substituted non-aromatic heterocycle, cycloalkyl, substituted cycloalkyl, and a precursor moiety.
43. A compound according to claim 42, wherein X1Selected from one of formulae (XVa) to (XVf):
wherein:
R10and R11Each independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, acyl, substituted acyl, and a precursor moiety.
44. A compound according to claim 43, wherein X1Selected from:
or a pharmaceutically acceptable salt thereof.
45. The compound according to claim 13, wherein the compound is selected from the structures:
46. the compound according to claim 30, wherein the compound is selected from the structures:
47. a pharmaceutical composition comprising:
means for inhibiting the function of ENPP 1; and
a pharmaceutically acceptable excipient.
48. The pharmaceutical composition according to claim 47, wherein the means for inhibiting the function of ENPP1 is an inhibitor of ENPP1 according to any one of claims 1 to 46.
49. A pharmaceutical composition for treating cancer, comprising:
an ENPP1 inhibitor according to any one of claims 1-46; and
a pharmaceutically acceptable excipient.
50. A method of inhibiting ENPP1, the method comprising:
contacting a sample comprising ENPP1 with an ENPP1 inhibitor according to any one of claims 1 to 46 to inhibit cGAMP hydrolysis activity of ENPP 1.
51. The method of claim 50, wherein the ENPP1 inhibitor is cell impermeable.
52. The method of claim 50, wherein the ENPP1 inhibitor is cell permeable.
53. The method according to any one of claims 50-52, wherein the sample is a cell sample.
54. The method of claim 53, wherein the sample comprises cGAMP.
55. The method of claim 54, wherein the level of cGAMP is elevated in the cell sample (e.g., relative to a control sample not contacted with the ENPP1 inhibitor).
56. A method of treating cancer, the method comprising:
administering to a subject having cancer a therapeutically effective amount of an ENPP1 inhibitor according to any one of claims 1-46 to treat the cancer in the subject.
57. A method of treating cancer, the method comprising:
administering to a subject having cancer a therapeutically effective amount of a means for inhibiting ENPP1 function to treat the cancer in the subject.
58. The method according to claim 56 or 57, wherein the cancer is a solid tumor cancer.
59. The method according to any one of claims 56-58, wherein the cancer is selected from the group consisting of adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, colon cancer, stomach cancer, ovarian cancer, cervical cancer, uterine cancer, esophageal cancer, colorectal cancer, prostate cancer, pancreatic cancer, lung cancer (both small cell and non-small cell), thyroid cancer, malignant epithelial tumors, sarcomas, glioblastomas, melanomas, and various head and neck tumors.
60. A method according to claim 59, wherein the cancer is breast cancer.
61. The method according to claim 56 or 57, wherein the cancer is lymphoma.
62. The method of claim 59, wherein the cancer is glioblastoma.
63. The method of any one of claims 56-62, further comprising administering to the subject an effective amount of one or more additional active agents.
64. The method according to claim 63, wherein the one or more additional active agents is a chemotherapeutic agent or an immunotherapeutic agent.
65. The method according to claim 63 or 64, wherein the one or more additional active agents is a small molecule, an antibody fragment, an antibody-drug conjugate, an aptamer, or a protein.
66. The method according to any one of claims 63-65, wherein the one or more additional active agents comprises a checkpoint inhibitor.
67. The method according to claim 66, wherein the checkpoint inhibitor is selected from the group consisting of a cytotoxic T-lymphocyte-associated antigen 4(CTLA-4) inhibitor, a programmed death 1(PD-1) inhibitor and a PD-L1 inhibitor.
68. The method according to any one of claims 63-65, wherein the one or more additional active agents comprise a chemotherapeutic agent.
69. The method according to claim 68, wherein the chemotherapeutic agent is a cGAMP-inducing chemotherapeutic agent.
70. The method according to claim 69, wherein the cGAMP-inducing chemotherapeutic agent is an anti-mitotic or antineoplastic agent administered in an amount effective to induce cGAMP production in the subject.
71. The method of any one of claims 56-70, further comprising administering radiation therapy to the subject.
72. The method of claim 71, wherein the inhibitor is administered to the subject prior to radiation therapy.
73. The method according to claim 71, wherein the inhibitor is administered after the subject is exposed to radiation therapy.
74. The method according to claim 72 or 73, wherein the radiation therapy induces cGAMP production in the subject.
75. The method of any one of claims 71-74 wherein the radiation therapy is administered at a dose and/or frequency effective to reduce radiation damage to the subject.
76. The method according to any one of claims 56-75, wherein the ENPP1 inhibitor is cell impermeable.
77. The method of any one of claims 56-75, wherein the ENPP1 inhibitor is cell permeable.
78. An ENPP1 inhibitor according to any one of claims 1-46 for use in the treatment of cancer.
79. Use of an ENPP1 inhibitor according to any one of claims 1-46 in the manufacture of a medicament for the treatment of cancer.
80. A method of modulating an immune response in a subject, the method comprising:
administering to the subject a therapeutically effective amount of an ENPP1 inhibitor according to any one of claims 1-46 to treat an inflammatory disorder in the subject.
81. A method of modulating an immune response in a subject, the method comprising:
administering to the subject a therapeutically effective amount of a means for inhibiting ENPP1 function to treat the inflammatory disorder in the subject.
82. A compound according to claim 1, wherein Z3Is absent, and Z2Is CR12Wherein R is12Is cyano, halogen or NH, wherein the compound is a compound of formula (X):
Wherein L is11And L12Independently a covalent bond or a linker.
83. The compound according to claim 82, wherein said ring system A is selected from the group consisting of phenyl, substituted phenyl, pyridyl, substituted pyridyl, pyrimidine, substituted pyrimidine, piperidine, substituted piperidine, piperazine, substituted piperazine, pyridazine, substituted pyridazine, cyclohexyl, and substituted cyclohexyl.
84. The compound according to claim 82 or 83, wherein the compound is of formula (XI):
wherein:
each Z5Independently selected from N and CR16；
Each R is16Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and
r is an integer of 0 to 8.
85. The compound according to claim 82, wherein the compound is of formula (XII):
86. the compound according to claim 85, wherein Z5Is N.
87. The compound according to claim 84, wherein the compound is of formula (XIII):
where s is an integer from 0 to 6 (e.g., 0 to 3).
88. The compound according to claim 84, wherein the compound is of formula (XIV):
89. The compound according to any one of claims 82-87, wherein R2To R5Independently selected from H, OH, alkyl, substituted alkyl, alkoxy, substituted alkoxy, -OCF3Halogen, cyano, amine, substituted amine, amide, heterocycle, and substituted heterocycle.
90. The compound according to claim 89, wherein R2To R5Independently selected from H, OH, C(1-6)Alkoxy, -OCF3、C(1-6)Alkylamino radical, di-C(1-6)Alkylamino, F, Cl, Br and CN.
Background
Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) activates the interferon gene stimulating factor (STING) pathway, which is an important anticancer innate immune pathway. The cGAS-cGAMP-STING pathway is activated in the presence of cytoplasmic DNA, probably due to microbial infection or pathophysiological conditions including cancer and autoimmune diseases. Cyclic GMP-AMP synthase (cGAS) belongs to the family of nucleotidyl transferases and is a versatile DNA sensor that is activated upon binding to cytoplasmic dsDNA to produce the signaling molecule (2 '-5', 3 '-5') cyclic GMP-AMP (or 2 ', 3' -cGAMP or cyclic guanosine monophosphate adenosine monophosphate, cGAMP). 2 ', 3' -cGAMP acts as a second messenger during microbial infection, binding and activating STING, leading to the production of type I Interferons (IFNs) and other co-stimulatory molecules that trigger an immune response. In addition to its role in infectious diseases, the STING pathway has become a target for cancer immunotherapy and autoimmune diseases.
Ectonucleotide pyrophosphatase/phosphodiesterase 1(ENPP1) is the major cGAMP hydrolase that degrades cGAMP. ENPP1 is a member of the ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family and is a type II transmembrane glycoprotein comprising two identical disulfide-bonded subunits. ENPP1 has broad specificity to cleave a variety of substrates including the phosphodiester bonds of nucleotides and nucleotide sugars, and the pyrophosphate bonds of nucleotides and nucleotide sugars. ENPP1 may function to hydrolyze nucleoside 5' triphosphates to their corresponding monophosphates and may also hydrolyze polyadenylic acid polyphosphates.
Disclosure of Invention
Compounds, compositions and methods for inhibiting ENPP1 are provided. Aspects of the subject methods include contacting a sample with an ENPP1 inhibitor compound to inhibit cGAMP hydrolytic activity of ENPP 1. In some cases, the ENPP1 inhibitor compound is cell impermeable. ENPP1 inhibitor compounds can act extracellularly to block the degradation of cGAMP. Pharmaceutical compositions and methods for treating cancer are also provided. Aspects of the methods include administering to the subject a therapeutically effective amount of an ENPP1 inhibitor to treat cancer in the subject. In some cases, the cancer is a solid tumor cancer. Methods of administering radiation therapy to a subject in conjunction with administering an ENPP1 inhibitor to the subject are also provided. The radiation therapy can be administered in the subject methods at a dose and/or frequency effective to reduce radiation damage to the subject, but still elicit an immune response.
These and other advantages and features of the disclosure will become apparent to those skilled in the art upon reading the details of the compositions and methods of use described more fully below.
Drawings
The invention is best understood from the following detailed description when read with the accompanying drawing figures. The patent or application document contains at least one color drawing. It is emphasized that, according to common practice, the various features of the drawings are not to scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawing are the following figures. It should be understood that the drawings described below are for illustration purposes only. The figures are not intended to limit the scope of the present teachings in any way.
FIG. 1, panels A through J, shows demonstration of cGAMP as a soluble factor from 293T cGAS ENPP1-/-Experimental results of cell output.
Fig. 2, panels a through C, show experimental results demonstrating that ENPP1 can modulate extracellular cGAMP.
Figure 3, panels a through F, illustrate the structure and activity of an exemplary ENPP1 inhibitor (compound 1) in various cellular assays.
Fig. 4, panels a through E, show experimental results demonstrating that cancer cells express cGAS and continue to export cGAMP in culture.
Figure 5, panels a through I, show experimental results demonstrating that the sequestration of extracellular cGAMP reduces tumor-associated dendritic cells in a tumor cGAS and host STING-dependent manner.
FIG. 6, panels A through D, shows a schematic ENPP1-/-Tumor recruitment innate immune infiltration, less aggressive, and more susceptible to IR and anti-CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) therapy.
FIG. 7, panels A through C, show experimental results demonstrating that ENPP1 inhibition exerts an anti-tumor effect in conjunction with IR therapy and anti-CTLA-4.
FIG. 8, panels A through D, illustrates the use of the LC-MS/MS method and 293T cGAS ENPP1Is low inAnd 293T cGAS ENPP1-/-Cell lines were evaluated for ENPP1 hydrolytic activity and cGAMP levels.
FIG. 9, panels A through B, shows an explanatory CD14+Experimental schematic and results of primary human Peripheral Blood Mononuclear Cells (PBMCs) responsive to extracellular cGAMP.
Figure 10, panels a to B, shows the results of an experiment comparing ENPP1 inhibitory activity of compound 1 and compound QS1 and shows the activity of QS1 in a cellular assay.
Figure 11, panels a through F, shows experimental results demonstrating that an exemplary ENPP1 inhibitor, compound 1(STF-1084), is cell impermeable, specific for ENPP1, and non-toxic.
Fig. 12, panels a through E, show experimental results demonstrating that cancer cells continually export cGAMP in culture.
Fig. 13, panels a through D, show experimental results demonstrating that the sequestration of extracellular cGAMP reduces tumor-associated dendritic cells in a tumor cGAS and host STING-dependent manner.
FIG. 14, panels A through F, shows a graph showing the established ENPP1-/-The experimental results that tumors cause an increase in tumor-associated dendritic cells, are less aggressive, and are more susceptible to IR and anti-CTLA-4 therapy.
FIG. 15 shows a graph of data demonstrating that ENPP1 inhibition (e.g., using Compound 1; STF-1084) synergistically increased tumor-associated dendritic cells with IR treatment.
Fig. 16 shows a schematic illustrating different patterns of cGAMP delivery from synthetic cells to target cells.
Fig. 17 shows a schematic diagram illustrating that cGAMP is a cancer risk signal secreted by cancer cells in vivo.
Fig. 18A-18C show data illustrating that an exemplary ENPP1 inhibitor (compound 1) can increase the amount of extracellular cGAMP present in a cellular system.
Fig. 19A-19B show experimental schematic and results illustrating that an exemplary ENPP1 inhibitor (compound 1) can increase cGAMP-stimulated interferon transcription.
Figures 20A-20B show data illustrating that an exemplary ENPP1 inhibitor (compound 1) can increase the number of tumor-associated dendritic cells in a mouse tumor model.
FIGS. 21A-21C show experimental results illustrating the synergy of ENPP1 inhibition with IR therapy and anti-CTLA-4 to exert an anti-tumor effect.
Fig. 22 shows a schematic illustrating ENPP1 as an innate immune checkpoint that regulates the neurotransmitter cGAMP.
Definition of
Before the embodiments of the disclosure are further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of embodiments of the present disclosure.
It must be noted that, as used herein and in the appended claims, the singular forms "a," an, "and" the "include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes not only a single compound, but also a combination of two or more compounds, reference to "a substituent" includes a single substituent as well as two or more substituents, and so forth.
In describing and claiming the present invention, certain terminology will be used in accordance with the definitions set forth below. It should be understood that the definitions provided herein are not intended to be mutually exclusive. Thus, some chemical moieties may fall within the definition of more than one term.
The phrases "for example", "for instance", "such as" or "including" are intended to introduce examples that further clarify more general subject matter. These examples are provided solely to aid in the understanding of the present disclosure and are not meant to be limiting in any way.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. In addition, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.
The terms "active agent", "antagonist", "inhibitor", "drug" and "pharmacologically active agent" are used interchangeably herein to refer to a chemical material or compound that, when administered to an organism (human or animal), induces a desired pharmacological and/or physiological effect through a local and/or systemic effect.
The terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and/or physiological effect, such as reducing tumor burden. In the case of total or partial prevention of the disease or its symptoms, the effect may be prophylactic; and/or the effect may be therapeutic in terms of a partial or complete cure for the disease and/or side effects due to the disease. As used herein, "treatment" is intended to encompass any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing a disease or symptoms of a disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease (e.g., including diseases that may be associated with or caused by a primary disease (as in the case of liver fibrosis, which may lead to chronic HCV infection), (b) inhibiting a disease, i.e., arresting its development, and (c) alleviating a disease, i.e., regression of the disease (e.g., reduction in tumor burden).
The term "pharmaceutically acceptable salt" means a salt (a salt with a counterion that has acceptable mammalian safety for a given dosage regimen) that is acceptable for administration to a patient such as a mammal. Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and may be derived from pharmaceutically acceptable inorganic or organic acids. "pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of compounds derived from a variety of organic and inorganic counterions well known in the art, and includes, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, benzenesulfonate, methanesulfonate, acetate, maleate, oxalate, and the like.
The terms "individual", "host", "subject" and "patient" are used interchangeably herein and refer to animals, including but not limited to humans and non-human primates, including simians and humans; rodents, including rats and mice; (ii) a bovine animal; an equine animal; a ovine animal; a feline; a canine; and the like. "mammal" means one or more members of any mammalian species and includes, for example, canines; a feline; an equine animal; (ii) a bovine animal; a ovine animal; rodents, and the like, as well as primates, such as non-human primates and humans. Non-human animal models, such as mammals, e.g., non-human primates, murine animals, lagomorphs, and the like, can be used for experimental studies.
The terms "determining," "measuring," "evaluating," and "determining" are used interchangeably and include both quantitative and qualitative determinations.
The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions containing heterologous and native leader sequences with or without an N-terminal methionine residue; an immunologically labeled protein; fusion proteins with detectable fusion partners, such as fusion proteins comprising fluorescent protein, beta-galactosidase, luciferase, etc. as fusion partners; and the like.
The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mrna), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.
By "therapeutically effective amount" or "effective amount" is meant the amount of a compound that, when administered to a mammal or other subject to treat a disease, disorder, or condition, is sufficient to effect such treatment for the disease, disorder, or condition. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity, as well as the age, weight, etc., of the subject to be treated.
The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound (e.g., an aminopyrimidine compound as described herein) calculated to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage form depend on the particular compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.
The terms "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" refer to excipients, diluents, carriers, or adjuvants that may be used to prepare pharmaceutical compositions that are generally safe, non-toxic, and biologically or otherwise undesirable, and include excipients, diluents, carriers, and adjuvants that are acceptable for veterinary use as well as human pharmaceutical use. As used in the specification and claims, "pharmaceutically acceptable excipients, diluents, carriers and adjuvants" includes one or more than one such excipient, diluent, carrier and adjuvant.
The term "pharmaceutical composition" is intended to encompass compositions suitable for administration to a subject such as a mammal, particularly a human. Generally, a "pharmaceutical composition" is sterile and preferably free of contaminants capable of eliciting an undesirable response in a subject (e.g., one or more compounds in the pharmaceutical composition are pharmaceutical grade). The pharmaceutical compositions can be designed to be administered to a subject or patient in need thereof via a number of different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, and the like.
The phrases "having" or "having a structure" are not intended to be limiting and are used in the same manner that the term "comprising" is commonly used. The term "independently selected from" is used herein to indicate that the listed elements, e.g., R groups, etc., may be the same or different.
The terms "may", "optionally" or "may optionally" mean that the subsequently described circumstance may or may not occur, and so the description includes instances where said circumstance occurs and instances where it does not. For example, the phrase "optionally substituted/optionally … … substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and thus, the description includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.
"acyl" refers to the groups H-C (O) -, alkyl-C (O) -, substituted alkyl-C (O) -, alkenyl-C (O) -, substituted alkenyl-C (O) -, alkynyl-C (O) -, substituted alkynyl-C (O) -, cycloalkyl-C (O) -, substituted cycloalkyl-C (O) -, cycloalkenyl-C (O) -, substituted cycloalkenyl-C (O) -, aryl-C (O) -, substituted aryl-C (O) -, heteroaryl-C (O) -, substituted heteroaryl-C (O) -, heterocyclyl-C (O) -, and substituted heterocyclyl-C (O) -, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein. For example, acyl includes the "acetyl" group CH 3C(O)-。
The term "alkyl" generally refers to a branched or unbranched saturated hydrocarbyl group (i.e., a mono-radical hydrocarbyl group), although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl, and the like. Typically, although not necessarily, alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms. The term "lower alkyl" means an alkyl group of 1 to 6 carbon atoms. "substituted alkyl" refers to an alkyl group substituted with one or more substituent groups, and this includes examples in which two hydrogen atoms from the same carbon atom in the alkyl substituent group are replaced, such as in the case of a carbonyl group (i.e., a substituted alkyl group may include a-C (═ O) -moiety). The terms "heteroatom-containing alkyl" and "heteroalkyl" refer to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom, as described in further detail below. The terms "alkyl" and "lower alkyl" if not otherwise stated, include straight-chain, branched, cyclic, unsubstituted, substituted and/or heteroatom-containing alkyl or lower alkyl, respectively.
The term "substituted alkyl" is intended to include alkyl groups as defined herein wherein one or more carbon atoms in the alkyl chain are optionally replaced by a heteroatom such as-O-, -N-, -S (O) N- (wherein N is 0 to 2), -NR- (wherein R is hydrogen or alkyl) having 1 to 5 substituents selected from: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO 2-alkyl, -SO 2-aryl, -SO 2-heteroaryl, and-NRaRb, where R' and R "may be the same or different and are selected from hydrogen, halogen, amino, hydroxy, amino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, and-NRaRb, Optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocycle.
The term "alkenyl" refers to a straight, branched, or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like. Typically, although again not necessarily, alkenyl groups herein may contain from 2 to about 18 carbon atoms, and may contain, for example, from 2 to 12 carbon atoms. The term "lower alkenyl" means an alkenyl group of 2 to 6 carbon atoms. The term "substituted alkenyl" refers to alkenyl substituted with one or more substituent groups, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to alkenyl in which at least one carbon atom is replaced with a heteroatom. The terms "alkenyl" and "lower alkenyl", if not otherwise stated, include straight-chain, branched, cyclic, unsubstituted, substituted and/or heteroatom-containing alkenyl and lower alkenyl, respectively.
The term "alkynyl" refers to a straight or branched chain hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Typically, although again not necessarily, alkynyl groups herein may contain 2 to about 18 carbon atoms, and such groups may also contain 2 to 12 carbon atoms. The term "lower alkynyl" means an alkynyl group of 2 to 6 carbon atoms. The term "substituted alkynyl" refers to alkynyl groups substituted with one or more substituent groups, and the terms "heteroatom-containing alkynyl" and "heteroalkynyl" refer to alkynyl groups in which at least one carbon atom is replaced with a heteroatom. The terms "alkynyl" and "lower alkynyl" include straight-chain, branched-chain, unsubstituted, substituted and/or heteroatom-containing alkynyl and lower alkynyl groups, respectively, if not otherwise specified.
The term "alkoxy" refers to an alkyl group bonded through a single terminal ether linkage; that is, an "alkoxy" group may be represented as-O-alkyl, where alkyl is as defined above. "lower alkoxy" group means an alkoxy group containing 1 to 6 carbon atoms and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy and the like. Substituents denoted herein as "C1-C6 alkoxy" or "lower alkoxy" may, for example, contain 1 to 3 carbon atoms, and as a further example, such substituents may contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy). The designations "-OMe" and "MeO-" refer to methoxy.
The term "substituted alkoxy" refers to the group, substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, wherein substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.
The term "aryl", unless otherwise specified, refers to an aromatic substituent which typically, but not necessarily, contains from 5 to 30 carbon atoms and contains a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group, such as a methylene or ethylene moiety). The aryl group may, for example, contain 5 to 20 carbon atoms, and as a further example, the aryl group may contain 5 to 12 carbon atoms. For example, an aryl group can contain one aromatic ring or two or more fused or linked aromatic rings (i.e., biaryl, aryl-substituted aryl, etc.). Examples include phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, benzophenone, and the like. "substituted aryl" refers to an aryl moiety substituted with one or more substituent groups, and the terms "heteroatom-containing aryl" and "heteroaryl" refer to aryl substituents in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail below. Aryl is intended to include stable cyclic, heterocyclic, polycyclic and polyheterocyclic unsaturated C 3-C14Moieties (such as, but not limited to, phenyl, biphenyl, naphthyl, pyridyl, furyl, thienyl, imidazolyl, pyrimidinyl, and oxazolyl), which can also be substituted with 1-5 members selected from the group consisting of: hydroxy, C1-C8Alkoxy radical, C1-C8Branched or straight chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halo, trifluoromethyl, cyano and carboxy (see, e.g., Katritzky, Handbook of Heterocyclic Chemistry). The term "aryl" includes unsubstituted, substituted and/or heteroatom-containing aromatic substituents, if not otherwise specified.
The term "aralkyl" refers to an alkyl group having an aryl substituent, and the term "alkaryl" refers to an aryl group having an alkyl substituent, wherein "alkyl" and "aryl" are defined above. Typically, the aralkyl and alkaryl groups herein contain from 6 to 30 carbon atoms. Aralkyl and alkaryl groups may, for example, contain from 6 to 20 carbon atoms, and as a further example, such groups may contain from 6 to 12 carbon atoms.
The term "alkylene" refers to a diradical alkyl group. Unless otherwise specified, such groups include saturated hydrocarbon chains containing from 1 to 24 carbon atoms, which may or may not be substituted, which may contain one or more cycloaliphatic groups, and which may contain heteroatoms. "lower alkylene" refers to an alkylene linkage containing 1 to 6 carbon atoms. Examples include methylene (- -CH) 2- - - - - -, ethylene (- -CH)2CH2- - - - -, propylene (- -CH)2CH2CH2- - - - - -, 2-methylpropylidene (- -CH)2--CH(CH3)--CH2- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -2)6- - -) and the like.
Similarly, the terms "alkenylene," "alkynylene," "arylene," "aralkylene," and "alkarylene" refer to di-alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.
The term "amino" refers to the group-NRR ', where R and R' are independently hydrogen or non-hydrogen substituents, where non-hydrogen substituents include, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and/or heteroatom-containing variants thereof.
The terms "halo" and "halogen" are used in the conventional sense to refer to a chloro, bromo, fluoro, or iodo substituent.
"carboxy", "carboxyl" or "carboxylic acid (carboxylate)" means-CO2H or a salt thereof.
"cycloalkyl" refers to a cycloalkyl group of 3 to 10 carbon atoms having single or multiple cyclic rings, including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or polycyclic structures such as adamantyl, and the like.
The term "substituted cycloalkyl" refers to a cycloalkyl group having 1 to 5 substituents or 1 to 3 substituents selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkylCycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl2-alkyl, -SO2-substituted alkyl, -SO2-aryl and-SO2-a heteroaryl group.
The term "heteroatom-containing" as in "heteroatom-containing alkyl" (also referred to as "heteroalkyl" group) or "heteroatom-containing aryl" (also referred to as "heteroaryl" group) refers to a molecule, linkage, or substituent in which one or more carbon atoms are replaced by an atom other than carbon, such as nitrogen, oxygen, sulfur, phosphorus, or silicon, typically nitrogen, oxygen, or sulfur. Similarly, the term "heteroalkyl" refers to a heteroatom-containing alkyl substituent, the term "heterocycloalkyl" refers to a heteroatom-containing cycloalkyl substituent, the term "heterocyclic" or "heterocycle" refers to a heteroatom-containing cyclic substituent, and the terms "heteroaryl" and "heteroaromatic" refer to a heteroatom-containing "aryl" and "aromatic" substituent, respectively, and the like. Examples of heteroalkyl groups include alkoxyaryl, alkylthio-substituted alkyl, N-alkylated aminoalkyl, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridyl, quinolyl, indolyl, furyl, pyrimidinyl, imidazolyl, 1,2, 4-triazolyl, tetrazolyl, and the like, and examples of heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl, and the like.
"heteroaryl" refers to an aromatic group having from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and from 1 to 10 heteroatoms selected from oxygen, nitrogen and sulfur, within the ring. Such heteroaryl groups may have a single ring (such as pyridyl, imidazolyl or furyl) or multiple condensed rings in a ring system (e.g., as in groups such as indolizinyl, quinolinyl, benzofuran, benzofuranylAs is the case with imidazolyl or benzothienyl groups), wherein at least one ring of the ring system is aromatic, provided that the point of attachment is through an atom of the aromatic ring. In certain embodiments, one or more nitrogen and/or sulfur ring atoms of the heteroaryl group are optionally oxidized to provide an N-oxide (N → O), sulfinyl, or sulfonyl moiety. The term includes, for example, pyridyl, pyrrolyl, indolyl, thienyl and furyl. Unless otherwise limited by the definition for the heteroaryl substituent, such heteroaryl groups may be optionally substituted with 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxyl, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkylaryl, aryl, aryloxy, azido, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, heteroaryl, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, substituted aryl, substituted heteroaryl, and optionally substituted heteroaryl, -SO-aryl, -SO-heteroaryl, -SO 2-alkyl, -SO2-substituted alkyl, -SO2-aryl and-SO2-heteroaryl, and trihalomethyl.
The terms "heterocycle", "heterocyclic" and "heterocyclyl" refer to a saturated or unsaturated group having a single ring or multiple fused rings (including fused bridged ring systems and spiro ring systems) and having from 3 to 15 ring atoms (including 1-4 heteroatoms). These ring heteroatoms are selected from nitrogen, sulfur and oxygen, where in the fused ring system one or more of the rings may be cycloalkyl, heterocycloalkyl, aryl or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, one or more nitrogen and/or sulfur atoms of the heterocyclic group is optionally oxidized to provide an N-oxide, -S (O) -or-SO2-a moiety.
Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indoline, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3, 4-tetrahydroisoquinoline, 4,5,6, 7-tetrahydrobenzo [ b ] thiophene, thiazole, thiazolidine, thiophene, benzo [ b ] thiophene, morpholinyl, thiomorpholinyl (also referred to as thiomorpholinyl), 1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl and the like.
Unless otherwise limited by the definition for the heterocyclic substituent, such heterocyclic groups may be optionally substituted with 1 to 5 or 1 to 3 substituents selected from: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocycloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocycloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2Heteroaryl and fused heterocycles.
"hydrocarbyl" refers to monovalent hydrocarbyl groups containing from 1 to about 30 carbon atoms (including from 1 to about 24 carbon atoms, further including from 1 to about 18 carbon atoms, and further including from about 1 to 12 carbon atoms), including straight, branched, cyclic, saturated and unsaturated species such as alkyl groups, alkenyl groups, aryl groups, and the like. The hydrocarbyl group may be substituted with one or more substituent groups. The term "heteroatom-containing hydrocarbyl" refers to hydrocarbyl in which at least one carbon atom is replaced with a heteroatom. Unless otherwise indicated, the term "hydrocarbyl" should be construed to include substituted and/or heteroatom-containing hydrocarbyl moieties.
As referred to in some of the foregoing definitions, "substituted" as in "substituted hydrocarbyl," "substituted alkyl," "substituted aryl," and the like means that in the hydrocarbyl, alkyl, aryl, or other moiety, at least one hydrogen atom bonded to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, but are not limited to, functional groups and hydrocarbyl moieties C1-C24 alkyl (including C1-C18 alkyl, further including C1-C12 alkyl, and further including C1-C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, and further including C2-C6 alkynyl), C5-C30 aryl (including C5-C20 aryl, and further including C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, and further including C6-C12 aralkyl). The above hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any group described herein should be construed to include substituted and/or heteroatom-containing moieties in addition to unsubstituted groups.
"Sulfonyl" refers to the group SO2Alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-cycloalkyl, SO2-substituted cycloalkyl, SO2Cycloalkenyl radical, SO2-substituted cycloalkenyl, SO2Aryl, SO2Substituted aryl, SO2Heteroaryl, SO2-substituted heteroaryl, SO2-heterocycle and SO2-substituted heterocycle, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein. Sulfonyl radicals include, for example, methyl-SO2-, phenyl-SO2And 4-methylphenyl-SO2-。
The term "functional group" means a chemical group such as halogen, hydroxy, mercapto, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (- (CO) -O-alkyl), C6-C20 aryloxycarbonyl (- (CO) -O-aryl), halocarbonyl (-CO) -X, where X is halogen), C2-C24 alkylcarbonate (-carbonate) C (carbonate O- (CO) -O-alkyl), C6-C20 arylcarboxylate (-O- (CO) -O-aryl), Carboxyl (-COOH), carboxylate (-COO-), carbamoyl (- (CO) -NH) 2) Monosubstituted C1-C24 alkylcarbamoyl (- (CO) -NH (C1-C24 alkyl)), disubstituted alkylcarbamoyl (- (CO) -N (C1-C24 alkyl)2) Monosubstituted arylcarbamoyl (- (CO) -NH-aryl), thiocarbamoyl (- (CS) -NH2) Ureido (-NH- (CO) -NH)2) Cyano (-C.ident.N), isocyano (-N +. ident.C-), cyanato (-O-C.ident.N), isocyanato (-O-N +. ident.C-), isothiocyanato (-S-C.ident.N), azido (-N +. ident.N-), formyl (- (CO) -H), thiocarbonyl (- (CS) -H), amino (-NH-C.ident.N-), cyano (-C.2) Mono-and di- (C1-C24 alkyl) substituted amino, mono-and di- (C5-C20 aryl) substituted amino, C2-C24 alkylamido (-NH- (CO) -alkyl), C5-C20 arylamido (-NH- (CO) -aryl), imino (-CR ═ NH, wherein R ═ hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C20 alkylaryl, C6-C20 arylalkyl, etc.), alkylimino (-CR ═ N (alkyl), wherein R ═ hydrogen, alkyl, aryl, alkylaryl, etc., arylimino (-CR ═ N (aryl), wherein R ═ hydrogen, alkyl, aryl, alkylaryl, etc.), nitro (-NO (-NO (NO), amino groups (-N (aryl), and the like2) Nitroso group (-NO), sulfo group (-SO)2-OH), sulfonato (-SO)2-O-), C1-C24 alkylthio (-S-alkyl; also known as "alkylthio"), arylthio (-S-aryl); also known as "arylthio"), C1-C24 alkylsulfinyl (- (SO) -alkyl), C5-C20 arylsulfinyl (- (SO) -aryl), C1-C24 alkylsulfonyl (-SO) 2-alkyl), C5-C20 arylsulfonyl (-SO)2Aryl), phosphono (-P (O) (OH)2) Phosphonate (-P (O) (-))2) Phosphonous acid (-P (O-), phosphoric acid (-PO)2) And phosphino (-PH)2) Mono-and di- (C1-C24 alkyl) substituted phosphino, mono-and di- (C5-C20 aryl) substituted phosphines. In addition, the foregoing functional groups may be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above, if the particular group so permits.
"linking" or "linker" as in "linker", "linker moiety", and the like, means a linking moiety that connects two groups via a covalent bond. The linking group may be linear, branched, cyclic, or a single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups including, but not limited to: amido (-NH-CO-), ureidoidene (-NH-CO-NH-), imide (-CO-NH-CO-), oxo (-O-), thiobridging (-S-), epidioxy (-O-), carbonyldioxy (-O-CO-O-), alkyldioxy (-O- (CH2) n-O-), epidioxyimino (-O-NH-), epimino (-NH-), carbonyl (-CO-), and the like. In some cases, one, two, three, four, or five or more carbon atoms of the linker backbone may be optionally substituted with sulfur, nitrogen, or oxygen heteroatoms. The bonds between the backbone atoms may be saturated or unsaturated, and typically no more than one, two or three unsaturated bonds are present in the linker backbone. The linking group may comprise one or more substituents, for example with alkyl, aryl or alkenyl groups. The linking group may include, but is not limited to, one or more poly (ethylene glycol) units (e.g., - (CH) 2-CH2-O) -); an ether; a thioether; an amine; alkyl (e.g., (C)1-C12) Alkyl) which may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1-dimethylethyl (tert-butyl), and the like. The linker backbone may comprise a cyclic group, for example an aryl, heterocyclic or cycloalkyl group, wherein 2 or more atoms, such as 2, 3 or 4 atoms, of the cyclic group are comprised in the backbone. The linker may be cleavable or non-cleavable. Any convenient orientation and/or attachment of the linking group to the linked group may be used.
When the term "substituted" appears before the list of possible substituted groups, it means that the term applies to each member of the group. For example, the phrase "substituted alkyl and aryl" should be interpreted as "substituted alkyl and substituted aryl".
In addition to the disclosure herein, the term "substituted" when used to modify a specified group or radical can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of the others, replaced with the same or different substituent as defined below.
In addition to the groups disclosed for each term herein, for substitution of one or more hydrogens on saturated carbon atoms in the indicated group or radicals (any two hydrogens on a single carbon may be replaced with ═ O, ═ NR 70、＝N-OR70、＝N2Or ═ S substitution) is-R unless otherwise specified60Halogen, ═ O, -OR70、-SR70、-NR80R80Trihalomethyl, -CN, -OCN, -SCN, -NO2、＝N2、-N3、-SO2R70、-SO2O–M+、-SO2OR70、-OSO2R70、-OSO2O–M+、-OSO2OR70、-P(O)(O–)2(M+)2、-P(O)(OR70)O–M+、-P(O)(OR70)2、-C(O)R70、-C(S)R70、-C(NR70)R70、-C(O)O–M+、-C(O)OR70、-C(S)OR70、-C(O)NR80R80、-C(NR70)NR80R80、-OC(O)R70、-OC(S)R70、-OC(O)O-M+、-OC(O)OR70、-OC(S)OR70、-NR70C(O)R70、-N R70C(S)R70、-NR70CO2 –M+、-NR70CO2R70、-NR70C(S)OR70、-NR70C(O)NR80R80、-NR70C(NR70)R70and-NR70C(NR70)NR80R80Wherein R is60Selected from: optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70Independently is hydrogen or R60(ii) a Each R80Independently is R70Or alternatively, two R80’Together with the nitrogen atom to which they are bound form a 5-, 6-or 7-membered heterocycloalkyl which may optionally contain 1 to 4 additional heteroatoms, which may be the same or different, selected from O, N and S, wherein N may have-H or C1-C3Alkyl substitution; and each M+Are counterions having a net single positive charge. Each M+May independently be, for example, an alkali metal ion, such as K+、Na+、Li+(ii) a Ammonium ions, such as+N(R60)4(ii) a Or alkaline earth metal ions, such as [ Ca ]2+]0.5、[Mg2+]0.5Or [ Ba ]2+]0.5("subscript 0.5 means that one counterion to the divalent alkaline earth metal ion can be the ionized form of the compound of the invention and the other is a typical counterion such as chloride, or two ionized compounds disclosed herein can be used as the counterion for such divalent alkaline earth metal ion, or a secondary ionized compound of the invention can be used as the counterion for such divalent alkaline earth ion). As specific examples, -NR 80R80Is intended to include-NH2-NH-alkyl, -N-pyrrolidinyl, -N-piperazinyl, -4N-methyl-piperazin-1-yl, and-N-morpholinyl.
In addition to the disclosure herein, a substituent for a hydrogen on an unsaturated carbon atom in a "substituted" alkene, alkyne, aryl and heteroaryl group is-R unless otherwise specified60Halogen, -O-M+、-OR70、-SR70、-S–M+、-NR80R80Trihalomethyl, -CF3、-CN、-OCN、-SCN、-NO、-NO2、-N3、-SO2R70、-SO3 –M+、-SO3R70、-OSO2R70、-OSO3 –M+、-OSO3R70、-PO3 -2(M+)2、-P(O)(OR70)O–M+、-P(O)(OR70)2、-C(O)R70、-C(S)R70、-C(NR70)R70、-CO2 –M+、-CO2R70、-C(S)OR70、-C(O)NR80R80、-C(NR70)NR80R80、-OC(O)R70、-OC(S)R70、-OCO2 –M+、-OCO2R70、-OC(S)OR70、-NR70C(O)R70、-NR70C(S)R70、-NR70CO2 –M+、-NR70CO2R70、-NR70C(S)OR70、-NR70C(O)NR80R80、-NR70C(NR70)R70and-NR70C(NR70)NR80R80Wherein R is60、R70、R80And M+As previously defined, provided that in the case of a substituted alkene or alkyne, the substituent is not-O-M+、-OR70、-SR70or-S–M+。
In addition to the groups disclosed for each term herein, the substituent group for a hydrogen on a nitrogen atom in "substituted" heteroalkyl and cycloheteroalkyl groups is-R unless otherwise specified60、-O-M+、-OR70、-SR70、-S-M+、-NR80R80Trihalomethyl, -CF3、-CN、-NO、-NO2、-S(O)2R70、-S(O)2O-M+、-S(O)2OR70、-OS(O)2R70、-OS(O)2O-M+、-OS(O)2OR70、-P(O)(O-)2(M+)2、-P(O)(OR70)O-M+、-P(O)(OR70)(OR70)、-C(O)R70、-C(S)R70、-C(NR70)R70、-C(O)OR70、-C(S)OR70、-C(O)NR80R80、-C(NR70)NR80R80、-OC(O)R70、-OC(S)R70、-OC(O)OR70、-OC(S)OR70、-NR70C(O)R70、-NR70C(S)R70、-NR70C(O)OR70、-NR70C(S)OR70、-NR70C(O)NR80R80、-NR70C(NR70)R70and-NR70C(NR70)NR80R80Wherein R is60、R70、R80And M+As previously defined.
In addition to the disclosure herein, in a certain embodiment, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
Unless otherwise specified, the nomenclature of substituents not explicitly defined herein is obtained by: the terminal portions of the functional groups are named first, and then the adjacent functional groups are named toward the point of attachment. For example, the substituent "aralkyloxycarbonyl" refers to the group (aryl) - (alkyl) -O-C (O) -.
For any group disclosed herein that contains one or more substituents, it is, of course, understood that such groups do not contain any substitutions or substitution patterns that are sterically impractical and/or synthetically impractical. In addition, the subject compounds include all stereochemically isomeric forms resulting from substitution of such compounds.
In certain embodiments, substituents may contribute to the optical and/or stereoisomerism of a compound. Salts, solvates, hydrates and prodrug forms of the compounds are also of interest. The present disclosure encompasses all such forms. Accordingly, the compounds described herein include salts, solvates, hydrates, prodrugs and isomeric forms thereof, including pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof. In certain embodiments, the compounds may be metabolized to a pharmaceutically active derivative.
Unless otherwise specified, reference to an atom is intended to include an isotope of that atom. For example, reference to H is intended to include1H、2H (i.e., D) and3h (i.e., T), and reference to C is intended to include12All isotopes of C and carbon (such as13C)。
It will be apparent to those skilled in the art upon reading this disclosure that each of the various embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with any of the other several embodiments without departing from the scope or spirit of the present invention. Any described method may be performed in the order of events recited or in any other order that is logically possible.
While the apparatus and method have or will be described for the sake of grammatical fluidity and functional explanation, it is to be expressly understood that the claims are not to be construed as necessarily limited in any way by the interpretation of "method" or "step" unless expressly stated under 35u.s.c. § 112, but according to the doctrine of equivalents, the full meaning and range of equivalents of the definitions provided by the claims are to be accorded, and in the event that the claims are expressly stated under 35u.s.c. § 112, full legal equivalents are to be accorded, according to 35u.s.c. § 112.
Definitions for other terms and concepts appear throughout the detailed description.
Detailed Description
As summarized above, aspects of the present disclosure include compounds, compositions, and methods for inhibiting ENPP 1. Aspects of the methods include contacting the sample with an ENPP1 inhibitor compound to inhibit cGAMP hydrolytic activity of ENPP 1. These compounds, compositions and methods find use in a variety of applications where inhibition of ENPP1 is desired.
Also provided are pharmaceutical compositions and methods of treating cancer using the subject ENPP1 inhibitor compounds. Aspects of the methods include administering to the subject a therapeutically effective amount of an ENPP1 inhibitor compound to inhibit hydrolysis of cGAMP and treat cancer in the subject.
ENPP 1-inhibitor compounds
The subject ENPP1 inhibitor compounds may include a core structure based on an aryl or heteroaryl ring system, such as a quinazoline or quinoline group, attached to a hydrophilic head group. The linker between the aryl or heteroaryl ring system and the hydrophilic head group may include a monocyclic aryl, heteroaryl, carbocyclic or heterocyclic ring and one or more acyclic linking moieties. The quinazoline or quinoline core structure may be substituted at the 4-position with a linker. The aryl or heteroaryl ring system is optionally further substituted. The present disclosure includes compounds having a quinoline core structure substituted at the 4-position with a linker and at the 3-position with a cyano group. In some cases, the linker includes a 1, 4-disubstituted 6-membered aryl or heteroaryl cyclic group, such as phenyl or substituted phenyl. In some cases, the linker includes a 1, 4-disubstituted 6-membered saturated heterocyclic or carbocyclic ring, such as an N1, 4-disubstituted piperidine ring or an N1, N4-disubstituted piperazine ring. Other aspects of the subject ENPP1 inhibitor compounds are described below and in PCT application number PCT/US2018/050018 filed by Li et al on 7/9/2018, the disclosure of which is incorporated herein by reference in its entirety.
The term "hydrophilic head group" refers to a group attached to a core aryl or heteroaryl ring system that is hydrophilic and well solvated in aqueous environments, such as under physiological conditions, and has low permeability to cell membranes. In some cases, low permeability to cell membranes means a permeability coefficient of 10-4cm/s or less, such as 10-5cm/s or less, 10- 6cm/s or less, 10-7cm/s or less, 10-8cm/s or less, 10-9cm/s or less, or even less, as measured by any convenient method of passive diffusion of the isolated hydrophilic head group through a membrane (e.g., a cell monolayer, such as a colorectal Caco-2 or renal MDCK cell line). See, e.g., Yang and Hinner, Methods Mol biol.2015; 1266: 29-53. hydrophilic head groups can impart improved water to molecules attached theretoSolubility and reduced cell permeability. The hydrophilic head group can be any convenient hydrophilic group that is well solvated in an aqueous environment and has low permeability to the membrane. In some cases, the hydrophilic groups are discrete functional groups (e.g., as described herein) or substituted versions thereof. In general, the permeability of charged groups or larger groups without electrical polarity is low. In some cases, the hydrophilic head group is charged, such as positively or negatively charged. In some embodiments, the hydrophilic head group is not itself cell permeable and renders the subject compounds cell impermeable. It will be appreciated that the hydrophilic head group or prodrug form thereof may be selected to provide the desired cell permeability of the subject compounds. In some cases, the hydrophilic head group is a neutral hydrophilic group. In some cases, the hydrophilic head group is contained in a prodrug form, and thus comprises a precursor moiety that is removable in vivo. In certain instances, the subject compounds are cell permeable.
The hydrophilic head group may be any convenient group capable of binding or chelating a zinc ion, or a prodrug form thereof. In some cases, the hydrophilic head group is a phosphorous-containing group. Target phosphorus-containing groups that may be used in the subject ENPP1 inhibitors include, but are not limited to, phosphonic acids or phosphonates, phosphates, thiophosphates, phosphoramidates, and thiophosphamides, or salts thereof, or prodrug forms thereof (e.g., as described herein).
Exemplary target ENPP1 inhibitor compounds that include quinazoline and isoquinoline ring systems are listed in the structures of compounds of formulae (I) - (XVb) and tables 1-2.
In some cases, the subject ENPP1 inhibitor compounds are compounds of formula (I), or a prodrug, pharmaceutically acceptable salt, or solvate thereof:
wherein the content of the first and second substances,
X1is a hydrophilic head group (e.g., as described herein);
a is a ring system selected from: aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocycle, and substituted heterocycle;
L1and L2Independently a covalent bond or a linker;
Z3Is absent or selected from NR22O and S;
Z2is CR12Or N;
Z1is CR11Or N;
R1selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkylaryl, substituted alkylaryl, alkylheteroaryl, substituted alkylheteroaryl, alkenylaryl (e.g., vinylaryl), substituted alkenylaryl, alkenylheteroaryl (e.g., vinylheteroaryl), substituted alkenylheteroaryl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic;
R11and R12Independently selected from the group consisting of H, cyano, trifluoromethyl, halogen, alkyl, and substituted alkyl;
R22selected from the group consisting of H, alkyl, and substituted alkyl; and
R2to R5Independently selected from H, OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, -OCF3Halogen, cyano, amine, substituted amine, amide, heterocycle, and substituted heterocycle; or wherein R is2And R3、R3And R4Or R4And R5Together with the carbon atoms to which they are attached provide a fused ring (e.g., a 5-or 6-membered monocyclic ring) selected from the group consisting of heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl.
In certain embodiments of formula (I), Z3Is absent. In certain embodiments of formula (I), Z 3Is NR22Wherein R is22Selected from H, C(1-6)Alkyl and substituted C(1-6)An alkyl group. In some cases, Z3Is NH. In some cases, Z3Is NR22And R is22Is C(1-6)Alkyl groups, for example, methyl, ethyl, propyl, pentyl or hexyl. In some cases, Z3Is NR22And R is22Is substituted C(1-6)An alkyl group. In some cases of formula (I), Z3Is O. In some cases of formula (I), Z3Is S.
In some cases of formula (I), Z1Is CR11And R is11Selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogens. In some cases, the alkyl or substituted alkyl is C1-5An alkyl group. In some cases of formula (I), Z1Is CR11And R is11Is hydrogen. In some cases, R11Is cyano.
In some cases, R11Is trifluoromethyl. In some cases, R11Is halogen, such as Br, I, Cl or F. In some cases, R11Is alkyl, e.g. C1-5An alkyl group. In some cases, R11Being substituted alkyl, e.g. substituted C1-5An alkyl group.
In some cases of formula (I), Z2Is CR12And R is12Selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogens. In some cases, the alkyl or substituted alkyl is C1-5An alkyl group. In some cases of formula (I), Z 2Is CR12And R is12Is hydrogen. In some cases, R12Is cyano. In some cases, R12Is trifluoromethyl. In some cases, R12Is halogen, such as Br, I, Cl or F. In some cases, R12Is alkyl, e.g. C1-5An alkyl group. In some cases, R12Being substituted alkyl, e.g. substituted C1-5An alkyl group.
In certain embodiments of formula (I), Z1And Z2Is N. In certain embodiments of formula (I), Z1Is CR11And Z is2Is N. In some cases of formula (I), Z1Is N, and Z2Is CR12. In some cases of formula (I), Z1Is CR11And Z is2Is CR12. In some cases of formula (I), Z1Is N, and Z2Is N.
In certain embodiments of formula (I), L1And L2Each is a covalent bond. In some cases, L1And L2Each is a linking group. In some cases, L1Is a covalent bond, and L2Is a linking group. In some cases, L1Is a linking group, and L2Is a covalent bond. Any convenient linker may be used to attach A to X and/or A to Z3(e.g., as described herein). In some cases, a is linked to X by a covalent bond. In some cases, a is linked to X through a linear linker of 1 to 12 atoms in length, e.g., 1 to 10, 1 to 8, or 1 to 6 atoms in length, e.g., 1, 2, 3, 4, 5, or 6 atoms in length. The linking group L 2Can be (C)1-6) Alkyl linking group or substituted (C)1-6) Alkyl linking group, optionally interrupted by a heteroatom or linking functional group such as an ester (-CO)2-), amido (CONH), carbamato (carbamate) (OCONH), ether (-O-), thioether (-S-) and/or an amino group (-NR-, wherein R is H or alkyl). In some cases, a is linked to Z by a covalent bond3. In some cases, A is linked to Z through a straight chain linker of 1 to 12 atoms in length, e.g., 1 to 10, 1 to 8, or 1 to 6 atoms in length, e.g., 1, 2, 3, 4, 5, or 6 atoms in length3. The linking group L1Can be (C)1-6) Alkyl linking group or substituted (C)1-6) Alkyl linking groups, optionally interrupted by hetero atoms or linking functional groups such as keto (CO), ester (-CO)2-), amido (CONH), carbamato (OCONH), ether (-O-), thioether (-S-) and/or an amino group (-NR-, wherein R is H or alkyl). When Z is3Is NR22When the linking group L is present1May include a terminal keto group (C ═ O), with Z3Together provide an amido group (NR)22CO) connection. When Z is31When O or S is present, the linking group L1May include a terminal keto group (C ═ O), with Z31Together providing an ester or thioester group linkage.
In certain embodiments of formula (I), Z3A phosphorus-containing group capable of binding zinc ions, or a prodrug form thereof.
In some cases of formula (I), Z3Selected from NR22O and S. Thus, the subject ENPP1 inhibitor compounds of formula (I) can be described by formula (II):
wherein Z31Selected from NR22O and S.
In certain embodiments of formula (II), Z31Is NR22Wherein R is22Selected from H, C(1-6)Alkyl and substituted C(1-6)An alkyl group. In some cases, Z31Is NH. In some cases, Z31Is NR22And R is22Is C(1-6)Alkyl groups, for example, methyl, ethyl, propyl, pentyl or hexyl. In some cases, Z31Is NR22And R is22Is substituted C(1-6)An alkyl group. In some cases of formula (I), Z31Is O. In some cases of formula (I), Z31Is S.
In some cases of formula (II), Z1Is CR11And R is11Selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogens. In some cases, the alkyl or substituted alkyl is C1-5An alkyl group. In some cases of formula (II), Z1Is CR11And R is11Is hydrogen. In some cases, R11Is cyano.
In some cases, R11Is trifluoromethyl. In some cases, R11Is halogen, such as Br, I, Cl or F. In some cases, R11Is alkyl, e.g. C1-5An alkyl group. In some cases, R11Is a substituted alkaneRadicals, e.g. substituted C1-5An alkyl group.
In some cases of formula (II), Z2Is CR12And R is12Selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogens. In some cases, the alkyl or substituted alkyl is C1-5An alkyl group. In some cases of formula (II), Z2Is CR12And R is12Is hydrogen. In some cases, R12Is cyano. In some cases, R12Is trifluoromethyl. In some cases, R12Is halogen, such as Br, I, Cl or F. In some cases, R12Is alkyl, e.g. C1-5An alkyl group. In some cases, R12Being substituted alkyl, e.g. substituted C1-5An alkyl group.
In certain embodiments of formula (II), Z1And Z2Is N. In certain embodiments of formula (I), Z1Is CR11And Z is2Is N. In some cases of formula (I), Z1Is N, and Z2Is CR12. In some cases of formula (I), Z1Is CR11And Z is2Is CR12. In some cases of formula (I), Z1Is N, and Z2Is N.
In certain embodiments of formula (II), L1And L2Each is a covalent bond. In some cases, L1And L2Each is a linking group. In some cases, L1Is a covalent bond, and L2Is a linking group. In some cases, L1Is a linking group, and L2Is a covalent bond. Any convenient linker may be used to attach A to X and/or A to Z 3(e.g., as described herein). In some cases, a is linked to X by a covalent bond. In some cases, a is linked to X through a linear linker of 1 to 12 atoms in length, e.g., 1 to 10, 1 to 8, or 1 to 6 atoms in length, e.g., 1, 2, 3, 4, 5, or 6 atoms in length. The linking group L2Can be (C)1-6) Alkyl linking group or substituted (C)1-6) Alkyl linking group, optionally interrupted by hetero atoms or attached theretoFunctional groups such as keto (CO), ester (-CO)2-), amido (CONH), carbamato (OCONH), ether (-O-), thioether (-S-) and/or an amino group (-NR-, wherein R is H or alkyl). In some cases, a is linked to Z by a covalent bond3. In some cases, A is linked to Z through a straight chain linker of 1 to 12 atoms in length, e.g., 1 to 10, 1 to 8, or 1 to 6 atoms in length, e.g., 1, 2, 3, 4, 5, or 6 atoms in length3. The linking group L1Can be (C)1-6) Alkyl linking group or substituted (C)1-6) Alkyl linking groups, optionally interrupted by hetero atoms or linking functional groups such as keto (CO), ester (-CO)2-), amido (CONH), carbamato (OCONH), ether (-O-), thioether (-S-) and/or an amino group (-NR-, wherein R is H or alkyl). When Z is31Is NR22When the linking group L is present 1May include a terminal keto group (C ═ O), with Z31Together provide an amido group (NR)22CO) connection. When Z is31When O or S is present, the linking group L1May include a terminal keto group (C ═ O), with Z31Together providing an ester or thioester group linkage.
In some cases of formula (II), the subject ENPP1 inhibitor compound is a compound of formula (III):
wherein:
R31to R34Each independently selected from H, halogen, alkyl and substituted alkyl, or R31And R32Or R33And R34Are cyclic and together with the carbon atom to which they are attached provide a cycloalkyl, substituted cycloalkyl, heterocyclyl or substituted heterocyclyl ring; and
n and m are each independently an integer from 0 to 6 (e.g., 0-3).
In certain embodiments of formula (III), Z31Is NR22Wherein R is22Selected from H, C(1-6)Alkyl and substituted C(1-6)An alkyl group. In some casesLower, Z31Is NH. In some cases, Z31Is NR22And R is22Is C(1-6)Alkyl groups, for example, methyl, ethyl, propyl, pentyl or hexyl. In some cases, Z31Is NR22And R is22Is substituted C(1-6)An alkyl group. In some cases of formula (III), Z31Is O. In some cases of formula (III), Z31Is S.
In the formula (II), when Z31Is NR22When the linking group L is present1May include a terminal keto group (C ═ O), with Z 31Together provide an amido group (NR)22CO) connection. Thus, in some cases of formula (II), the subject ENPP1 inhibitor compound is a compound of formula (IIIa):
wherein:
Z41is-NR22C(＝O)-；
R31To R34Each independently selected from H, halogen, alkyl and substituted alkyl, or R31And R32Or R33And R34Are cyclic and together with the carbon atom to which they are attached provide a cycloalkyl, substituted cycloalkyl, heterocyclyl or substituted heterocyclyl ring; and
n and m are each independently an integer from 0 to 6 (e.g., 0-3).
In some cases of formulae (III) - (IIIa), Z1Is CR11And R is11Selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogens. In some cases, the alkyl or substituted alkyl is C1-5An alkyl group. In some cases of formulae (III) - (IIIa), Z1Is CR11And R is11Is hydrogen. In some cases, R11Is cyano. In some cases, R11Is trifluoromethyl. In some cases, R11Is halogen, such as Br, I, Cl or F. In some cases, R11Is alkyl, e.g. C1-5An alkyl group. In some cases, R11Being substituted alkyl, e.g. substituted C1-5An alkyl group.
In some cases of formulae (III) - (IIIa), Z2Is CR12And R is12Selected from hydrogen, cyano, trifluoromethyl, halogen, alkyl and substituted alkyl hydrogens. In some cases, the alkyl or substituted alkyl is C 1-5An alkyl group. In some cases of formulae (III) - (IIIa), Z2Is CR12And R is12Is hydrogen. In some cases, R12Is cyano. In some cases, R12Is trifluoromethyl. In some cases, R12Is halogen, such as Br, I, Cl or F. In some cases, R12Is alkyl, e.g. C1-5An alkyl group. In some cases, R12Being substituted alkyl, e.g. substituted C1-5An alkyl group.
In certain embodiments of formulas (III) - (IIIa), Z1And Z2Is N. In certain embodiments of formulas (III) - (IIIa), Z1Is CR11And Z is2Is N. In some cases of formulae (III) - (IIIa), Z1Is N, and Z2Is CR12. In some cases of formulae (III) - (IIIa), Z1Is CR11And Z is2Is CR12. In some cases of formulae (III) - (IIIa), Z1Is N, and Z2Is N.
In certain embodiments of formulas (III) - (IIIa), R31To R34Each is hydrogen. In certain embodiments, R31To R34At least one of which is halogen. In certain embodiments, R31To R34At least one of which is an alkyl group. In certain embodiments, R31To R34At least one of which is a substituted alkyl group. In some cases, R31To R34One is halogen and the others are selected from hydrogen, halogen, alkyl and substituted alkyl. In some cases, R 31To R34One is alkyl and the others are selected from hydrogen, halogen, alkyl and substituted alkyl. In some casesUnder the condition of R31To R34One is a substituted alkyl group and the others are selected from the group consisting of hydrogen, halogen, alkyl and substituted alkyl. In some cases, R31To R34One is halogen and the remainder are hydrogen. In some cases, R31To R34One is alkyl and the remainder are hydrogen. In some cases, R31To R34One is substituted alkyl and the remainder are hydrogen.
In certain embodiments of formulas (III) - (IIIa), n is an integer from 0 to 3. In some cases, n is 0. In some cases, n is 1. In some cases, n is 2. In some cases, n is 3. In certain embodiments of formulas (III) - (IIIa), m is an integer from 0 to 3. In some cases, m is 0. In some cases, m is 1. In some cases, m is 2. In some cases, m is 3. In some cases, n is 0 and m is 1. In some cases, n is 0 and m is 2. In some cases, n is 0 and m is 3. In some cases, n is 1 and m is 0. In some cases, n is 1, and m is 1. In some cases, n is 1 and m is 2. In some cases, n is 1 and m is 3. In some cases, n is 2, and m is 0. In some cases, n is 2 and m is 1. In some cases, n is 2 and m is 2. In some cases, n is 2 and m is 3. In some cases, n is 3 and m is 0. In some cases, n is 3 and m is 1. In some cases, n is 3 and m is 2. In some cases, n is 3 and m is 3. In some cases, n + m is an integer from 0 to 3. In some cases, n + m is 0. In some cases, n + m is 1. In some cases, n + m is 2. In some cases, n + m is 3.
In some embodiments of any of formulas (I) through (IIIa), the ring system a is selected from the group consisting of phenyl, substituted phenyl, pyridyl, substituted pyridyl, pyrimidine, substituted pyrimidine, piperidine, substituted piperidine, piperazine, substituted piperazine, pyridazine, substituted pyridazine, cyclohexyl, and substituted cyclohexyl. In some cases, the ring system a is phenyl or substituted phenyl. In some cases, the ring system a is pyridyl or substituted pyridyl. In some cases, the ring system a is a pyrimidine or substituted pyrimidine. In some cases, the ring system a is piperidine or substituted piperidine. In some cases, the ring system a is piperazine or substituted piperazine. In some cases, the ring system a is cyclohexyl or substituted cyclohexyl.
In some embodiments, the ring system a is described by formula (a 1):
wherein:
R6each selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and
p is an integer of 0 to 4.
In some cases, a1 is phenylene. In some cases, a1 is monosubstituted phenylene. In some cases, a1 is a disubstituted phenylene group. In some cases, a1 is a trisubstituted phenylene group. In some cases, a1 is a tetrasubstituted phenylene. In some cases, the substituents of the phenylene group are selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, and hexyl) and halogen (e.g., F, Cl, I, or Br).
In some embodiments, the A1 ring is described by formula (A1 a):
in some embodiments, the ring system a is described by formula (a 2):
wherein:
Z5selected from N and CR6；
R6Each selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and
q is an integer of 0 to 2.
In some cases, a2 is pyridyl. In some instances, a2 is a substituted pyridyl group. In some cases, the pyridyl group is monosubstituted pyridyl. In other cases, the pyridyl is disubstituted pyridyl. In other cases, the pyridyl is a trisubstituted pyridyl. In some cases, Z5Is N, such that a2 is pyrimidinyl. In some cases, a2 is a substituted pyrimidinyl group. In some cases, the pyrimidinyl group is monosubstituted. In some cases, the pyrimidinyl group is disubstituted. In certain embodiments of a2, the substituents are selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, and hexyl), trifluoromethyl, and halogen (e.g., F, Cl, I, or Br).
In some embodiments, the ring system a is described by formula (a 3):
wherein:
Z5selected from N and CR6；
R6Each selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and
q is an integer of 0 to 2.
In some cases, a3 is pyridyl. In some instances, a3 is a substituted pyridyl group. In some cases, the pyridyl group is monosubstituted pyridyl. In other cases, the pyridyl is disubstituted pyridyl. In other situationsIn the case, the pyridyl group is a trisubstituted pyridyl group. In some cases, Z5Is N, such that a3 is pyrimidinyl. In some cases, a3 is a substituted pyrimidinyl group. In some cases, the pyrimidinyl group is monosubstituted. In some cases, the pyrimidinyl group is disubstituted. In certain embodiments of a3, the substituents are selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, and hexyl), trifluoromethyl, and halogen (e.g., F, Cl, I, or Br).
In some embodiments, the ring system a is described by formula (a 4):
Wherein:
Z5is N;
R6each selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and
q is an integer of 0 to 2.
In some cases, a4 is a substituted pyrimidinyl group. In some cases, the pyrimidinyl group is monosubstituted. In some cases, the pyrimidinyl group is disubstituted. In certain embodiments of a4, the substituents are selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, and hexyl), trifluoromethyl, and halogen (e.g., F, Cl, I, or Br).
In some cases of formulae (III) - (IIIa), the ENPP1 inhibitor compound is a compound of formulae (IV) - (IVa):
wherein:
Z31selected from NR22O and S;
Z41is-NR22C(＝O)-；
Z11And Z21Independently selected from N and c (cn);
R31to R34Each independently selected from H, halogen, alkyl and substituted alkyl, or R31And R32Or R33And R34Are cyclic and together with the carbon atom to which they are attached provide a cycloalkyl, substituted cycloalkyl, heterocyclyl or substituted heterocyclyl ring;
R6each independently selected from the group consisting of H, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, and halogen;
p is an integer of 0 to 4; and
n and m are each independently an integer from 0 to 6 (e.g., 0-3).
In certain embodiments of formulae (IV) - (IVa), Z31Is NR22Wherein R is22Selected from H, C(1-6)Alkyl and substituted C(1-6)An alkyl group. In some cases, Z31Is NH. In some cases, Z31Is NR22And R is22Is C(1-6)Alkyl groups, for example, methyl, ethyl, propyl, pentyl or hexyl. In some cases, Z31Is NR22And R is22Is substituted C(1-6)An alkyl group. In some cases of formulae (IV) - (IVa), Z31Is O. In some cases of formulae (IV) - (IVa), Z31Is S.
In certain embodiments of formulae (IV) - (IVa), Z11And Z21Is N. In certain embodiments of formulae (IV) - (IVa), Z11Is C (CN), and Z21Is N. In some cases of formulae (IV) - (IVa), Z11Is N, and Z21Is C (CN). In some cases of formulae (IV) - (IVa), Z11Is C (CN), and Z21Is C (CN). In some cases of formulae (IV) - (IVa), Z11Is N, and Z21Is N.
In certain embodiments of formulas (IV) - (IVa), R31To R34Each is hydrogen. In certain embodiments, R31To R34At least one of which is halogen. In certain embodiments, R31To R34At least one of which is an alkyl group. In certain embodiments, R31To R 34At least one of which is a substituted alkyl group. In some cases, R31To R34One is halogen and the others are selected from hydrogen, halogen, alkyl and substituted alkyl. In some cases, R31To R34One is alkyl and the others are selected from hydrogen, halogen, alkyl and substituted alkyl. In some cases, R31To R34One is a substituted alkyl group and the others are selected from the group consisting of hydrogen, halogen, alkyl and substituted alkyl. In some cases, R31To R34One is halogen and the remainder are hydrogen. In some cases, R31To R34One is alkyl and the remainder are hydrogen. In some cases, R31To R34One is substituted alkyl and the remainder are hydrogen.
In certain embodiments of formulas (IV) - (IVa), n is an integer from 0 to 3. In some cases, n is 0. In some cases, n is 1. In some cases, n is 2. In some cases, n is 3. In certain embodiments of formulas (IV) - (IVa), m is an integer from 0 to 3. In some cases, m is 0. In some cases, m is 1. In some cases, m is 2. In some cases, m is 3. In some cases, n is 0 and m is 1. In some cases, n is 0 and m is 2. In some cases, n is 0 and m is 3. In some cases, n is 1 and m is 0. In some cases, n is 1, and m is 1. In some cases, n is 1 and m is 2. In some cases, n is 1 and m is 3. In some cases, n is 2, and m is 0. In some cases, n is 2 and m is 1. In some cases, n is 2 and m is 2. In some cases, n is 2 and m is 3. In some cases, n is 3 and m is 0. In some cases, n is 3 and m is 1. In some cases, n is 3 and m is 2. In some cases, n is 3 and m is 3. In some cases, n + m is an integer from 0 to 3. In some cases, n + m is 0. In some cases, n + m is 1. In some cases, n + m is 2. In some cases, n + m is 3.
In some cases of formula (IVa), n is 0, and m is 0-2, e.g., m is 1 or 2.
In some cases of formulas (IV) - (IVa), the ENPP1 inhibitor compound is a compound of formulas (V) - (Va):
wherein:
R41to R44Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide.
In certain embodiments of formulas (V) - (Va), Z11And Z21Is N. In certain embodiments of formulas (V) - (Va), Z11Is C (CN), and Z21Is N. In some cases of formulas (V) - (Va), Z11Is N, and Z21Is C (CN). In some cases of formulas (V) - (Va), Z11Is C (CN), and Z21Is C (CN). In some cases of formulas (V) - (Va), Z11Is N, and Z21Is N.
In some cases of formulas (V) - (Va), the subject ENPP1 inhibitor compound is a compound of one of formulas (VIa) - (VId):
in certain embodiments of formulae (VIa) - (VId), R41To R44Each is hydrogen. In certain embodiments, R41To R44At least one of which is an alkyl group or a substituted alkyl group. In some instancesIn the embodiment, R 41To R44At least one of which is a hydroxyl group. In certain embodiments, R41To R44At least one of which is alkoxy or substituted alkoxy. In some cases, R41To R44At least one of which is trifluoromethyl. In some cases, R41To R44At least one of which is halogen. In some cases, R41To R44At least one of which is an acyl group or a substituted acyl group. In some cases, R41To R44At least one of which is a carboxyl group. In some cases, R41To R44At least one of which is formamide or a substituted formamide. In some cases, R41To R44At least one of which is a sulfonyl group or a substituted sulfonyl group. In some cases, R41To R44Is a sulfonamide or a substituted sulfonamide. In some cases, R31To R34One is hydrogen and the others are selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide and substituted sulfonamide. In some cases, R31To R34Two of which are hydrogen and the others are selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide and substituted sulfonamide. In some cases, R 31To R34Three of which are hydrogen and the others are selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide and substituted sulfonamide.
In certain embodiments of formulas (VIa) - (VId), n is an integer from 0 to 3. In some cases, n is 0. In some cases, n is 1. In some cases, n is 2. In some cases, n is 3. In certain embodiments of formulas (VIa) - (VId), m is an integer from 0 to 3. In some cases, m is 0. In some cases, m is 1. In some cases, m is 2. In some cases, m is 3. In some cases, n is 0 and m is 1. In some cases, n is 0 and m is 2. In some cases, n is 0 and m is 3. In some cases, n is 1 and m is 0. In some cases, n is 1, and m is 1. In some cases, n is 1 and m is 2. In some cases, n is 1 and m is 3. In some cases, n is 2, and m is 0. In some cases, n is 2 and m is 1. In some cases, n is 2 and m is 2. In some cases, n is 2 and m is 3. In some cases, n is 3 and m is 0. In some cases, n is 3 and m is 1. In some cases, n is 3 and m is 2. In some cases, n is 3 and m is 3. In some cases, n + m is an integer from 0 to 3. In some cases, n + m is 0. In some cases, n + m is 1. In some cases, n + m is 2. In some cases, n + m is 3.
In certain embodiments of formulae (VIa) - (VId), R22Is hydrogen. In some cases, R22Is an alkyl group. In some cases, R22Is a substituted alkyl group. In some cases, the alkyl or substituted alkyl is C(1-6)An alkyl group.
In certain embodiments of any of formulas (I) - (VId), R1Selected from the group consisting of hydrogen, alkylaryl, substituted alkylaryl, alkylheteroaryl, substituted alkylheteroaryl, alkenylaryl (e.g., vinylaryl), substituted alkenylaryl, alkenylheteroaryl (e.g., vinylheteroaryl), substituted alkenylheteroaryl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.
In some cases of formulae (I) - (VId), R1Is hydrogen. In some cases, R1Is aryl or substituted aryl. In some cases, R1Is heteroaryl or substituted heteroaryl. In some cases, R1Is alkylaryl or substituted alkylaryl. In some cases, R1Is an alkylheteroaryl or substituted alkylheteroaryl. In some cases, R1Is alkeneAn aryl group or a substituted alkenylaryl group. In some cases, R1Is a vinyl aryl group. In some cases, R1Is a substituted vinyl aryl group. In some cases, R 1Is a vinyl heteroaryl group. In some cases, R1Is alkenylheteroaryl or substituted alkenylheteroaryl. In some cases, R1Is a substituted vinyl heteroaryl.
In some cases of formulas (VIa) - (VId), the ENPP1 inhibitor compound is a compound of one of formulas (VIIa) - (VIIb):
in certain embodiments of any of formulas (I) - (VIIb), R2To R5Independently selected from H, OH, alkyl, substituted alkyl, alkoxy, substituted alkoxy, -OCF3Halogen, cyano, amine, substituted amine, amide, heterocycle, and substituted heterocycle.
In certain embodiments of any of formulas (I) - (VIIb), R2To R5Independently selected from hydrogen, OH, C(1-6)Alkoxy, -OCF3、C(1-6)Alkylamino radical, di-C(1-6)Alkylamino, F, Cl, Br and CN.
In some cases, R2To R5At least one of which is hydrogen. In some cases, R2To R5At least two of which are hydrogen. In some cases, R2To R5Each is hydrogen. In some cases, R2To R5At least one of which is a hydroxyl group. In some cases, R2To R5At least one of which is an alkyl group or a substituted alkyl group. In some cases, R2To R5At least one of which is alkoxy or substituted alkoxy.
In some cases, the alkoxy or substituted alkoxy is C (1-6)Alkoxy, for example, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy. In some cases, R2To R5At least one of which is methoxy. In some cases, R2To R5At least one of which is-OCF3. In some cases, R2To R5At least one of which is halogen. In some cases, the halogen is fluorine. In some cases, the halogen is chlorine. In some cases, the halogen is bromine. In some cases, R2To R5At least one of which is cyano. In some cases, R2To R5At least one of which is an amine or a substituted amine. In some cases, R2To R5At least one of them is C(1-6)An alkylamino group. In some cases, R2To R5At least one of which is di-C(1-6)An alkylamino group. In some cases, R2To R5At least one of which is an amide. In some cases, R2To R5At least one of which is a heterocycle or a substituted heterocycle.
In some cases of formulas (I) - (VIIb), R3And R4Independently is an alkoxy group; and R is2And R5Are both hydrogen. In some cases, the alkoxy group is methoxy. In some cases, R3Is an alkoxy group; and R is2、R4And R5Is hydrogen. In some cases, R4Is an alkoxy group; and R is2、R3And R 5Each is hydrogen. In some cases, R2、R3And R4Is hydrogen, and R5Is an alkoxy group. In some cases, the alkoxy group is C(1-6)An alkoxy group. In some cases, the alkoxy group is methoxy. In some cases, the alkoxy group is ethoxy. In some cases, the alkoxy group is propoxy. In some cases, the alkoxy group is butoxy. In some cases, the alkoxy group is pentyloxy. In some cases, the alkoxy group is a hexyloxy group.
In some cases of formulae (VIc) - (VId), R41-R44Each independently of the other being H, halogen, C(1-6)Alkyl or C(1-6)An alkoxy group. In some cases of formulae (VIc) - (VId), m is 1 or 2. In formulae (VIc) - (VId)In some cases, R2Is H, and R3To R5Independently selected from hydrogen, C(1-6)Alkoxy, F, Cl and C(1-6)An alkyl group.
In some cases of formulas (VIIa) - (VIIb), the subject ENPP1 inhibitor compound is a compound of one of formulas (VIIc) - (VIIl):
in some cases of formula (VIa), the ENPP1 inhibitor compound is a compound of formula (VIIm):
in certain embodiments of formula (VIIm), R2To R5Independently selected from H, OH, alkyl, substituted alkyl, alkoxy, substituted alkoxy, -OCF3Halogen, cyano, amine, substituted amine, amide, heterocycle, and substituted heterocycle. In certain embodiments of formula (VIIm), R 2To R5Independently selected from hydrogen, OH, C(1-6)Alkoxy, -OCF3、C(1-6)Alkylamino radical, di-C(1-6)Alkylamino, F, Cl, Br and CN. In certain embodiments of formula (VIIm), n + m ═ 1. In certain embodiments of formula (VIIm), n + m is 2. In certain embodiments of formula (VIIm), n is 1 and m is 0.
In some cases of formula (VIIm), R3To R5At least one of which is hydrogen. In some cases, R3To R5At least two of which are hydrogen. In some cases, R3To R5Each is hydrogen. In some cases, R3To R5At least one of which is a hydroxyl group. In some cases, R3To R5At least one of which is an alkyl group or a substituted alkyl group. In some cases, R3To R5At least one of which is alkoxy or substituted alkoxy. In some cases of formula (VIIm), the alkoxy or substituted alkoxy is C(1-6)Alkoxy, for example, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy. In some cases, R3To R5At least one of which is methoxy. In some cases of formula (VIIm), R3To R5At least one of which is-OCF3. In some cases, R3To R5At least one of which is halogen. In some cases, the halogen is fluorine. In some cases, the halogen is chlorine. In some cases, the halogen is bromine. In some cases, R 3To R5At least one of which is cyano. In some cases, R3To R5At least one of which is an amine or a substituted amine. In some cases, R3To R5At least one of them is C(1-6)An alkylamino group. In some cases, R3To R5At least one of which is di-C(1-6)An alkylamino group. In some cases of formula (VIIm), R3To R5At least one of which is an amide. In some cases, R3To R5At least one of which is a heterocycle or a substituted heterocycle.
In some cases of formula (VIIm), R3And R4Independently is an alkoxy group; and R is2And R5Are both hydrogen. In some cases, the alkoxy group is methoxy. In some cases, R3Is an alkoxy group; and R is2、R4And R5Is hydrogen. In some cases, R4Is an alkoxy group; and R is2、R3And R5Each is hydrogen. In some cases of formula (VIIm), R2、R3And R4Is hydrogen, and R5Is an alkoxy group. In some cases, the alkoxy group is C(1-6)An alkoxy group. In some cases, the alkoxy group is methoxy. In some cases, the alkoxy group is ethoxy. In some cases, the alkoxy group is propoxyAnd (4) a base. In some cases, the alkoxy group is butoxy. In some cases, the alkoxy group is pentyloxy. In some cases, the alkoxy group is a hexyloxy group.
In certain embodiments of formula (VIIm), n is 0 to 3, and m is 0 to 3. In some cases of formula (VIIm), m is 0. In some cases, m is 1. In some cases, m is 2. In some cases, m is 3. In some cases, n is 0 and m is 1. In some cases, n is 0 and m is 2. In some cases, n is 0 and m is 3. In some cases, n is 1 and m is 0. In some cases, n is 1, and m is 1. In some cases, n is 1 and m is 2. In some cases, n is 1 and m is 3. In some cases, n is 2, and m is 0. In some cases, n is 2 and m is 1. In some cases, n is 2 and m is 2. In some cases, n is 2 and m is 3. In some cases, n is 3 and m is 0. In some cases, n is 3 and m is 1. In some cases, n is 3 and m is 2. In some cases, n is 3 and m is 3. In some cases, n + m is an integer from 0 to 3. In some cases, n + m is 0. In some cases, n + m is 1. In some cases, n + m is 2. In some cases, n + m is 3.
In certain instances of the ENPP1 inhibitor compounds of formula (I), Z3Is absent. In certain embodiments of formula (I), Z 3Is absent, Z2Is CR12，R12Is cyano and the compound is described by formula (X):
wherein L is11And L12Independently a covalent bond or a linker. In some cases of formula (X), L11Is a covalent bond.
In some embodiments of formula (X), the ring system a is selected from the group consisting of phenyl, substituted phenyl, pyridyl, substituted pyridyl, pyrimidine, substituted pyrimidine, piperidine, substituted piperidine, piperazine, substituted piperazine, pyridazine, substituted pyridazine, cyclohexyl, and substituted cyclohexyl. In some cases, the ring system a is phenyl or substituted phenyl. In some cases, the ring system a is pyridyl or substituted pyridyl. In some cases, the ring system a is a pyrimidine or substituted pyrimidine. In some cases, the ring system a is piperidine or substituted piperidine. In some cases, the ring system a is piperazine or substituted piperazine. In some cases, the ring system a is cyclohexyl or substituted cyclohexyl.
In some embodiments, the ring system a is described by one of formulae (a1) - (a4) (e.g., as described herein):
wherein:
Z5selected from N and CR6；
R6Each selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide;
p is an integer of 0 to 4; and
q is an integer of 0 to 2.
In some embodiments, the a ring is described by formula (a 5):
wherein:
Z5each independently selected from N and CR16；
R16Each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide; and
r is an integer of 0 to 8.
In some cases, a5 is piperidine or substituted piperidine. In some cases, a5 is piperazine or substituted piperazine. In some cases, a5 is cyclohexyl or substituted cyclohexyl. In certain embodiments of a5, r is greater than 0, e.g., 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, a5 includes one R16A group. In some cases, a5 includes two R16A group. In some cases, a5 includes three R16A group. In some cases, a5 includes four R16A group. In certain embodiments, the substituents are selected from lower alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, and hexyl), trifluoromethyl, and halogen (e.g., F, Cl, I, or Br).
In certain embodiments, the a ring has any one of formulas (A5a) - (A5 c):
In certain embodiments, the a ring is cyclohexyl having the relative configuration of formula (A5d) or (A5 e):
in certain instances of formula (X), the subject ENPP1 inhibitor compounds are compounds of formula (XI):
wherein:
Z5each independently selected from N and CR16；
R16Each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonylAn amine; and
r is an integer of 0 to 8.
In certain embodiments of formula (XI), at least one Z5Is N. In certain embodiments of formula (XI), one Z5Is N, and another Z5Is CR16. In some cases of formula (XI), two Z5The radicals are all CR16. In some cases of formula (XI), two Z5The radicals are all N.
In certain embodiments of the compounds of any one of formulas (X) - (XI), L11And L12Each is a covalent bond. In some cases, L11And L12Each is a linking group. In some cases, L11Is a covalent bond, and L12Is a linking group. In some cases, L11Is a linking group, and L12Is a covalent bond. Any convenient linker may be used as L 11And L12. In some cases, L11Is a covalent bond. In some cases, L11Is a straight chain linker of 1 to 12 atoms in length, for example 1 to 10, 1 to 8 or 1 to 6 atoms in length, for example 1, 2, 3, 4, 5 or 6 atoms in length. The linking group L11Can be (C)1-6) Alkyl linking group or substituted (C)1-6) Alkyl linking group, optionally interrupted by a heteroatom or linking functional group such as an ester (-CO)2-), amido (CONH), carbamato (OCONH), ether (-O-), thioether (-S-) and/or an amino group (-NR-, wherein R is H or alkyl). In some cases, L12Is a covalent bond. In some cases, L12Are linkers of 1 to 12 atoms in length, for example 1 to 10, 1 to 8 or 1 to 6 atoms in length, for example 1, 2, 3, 4, 5 or 6 atoms in length. The linking group L12Can be (C)1-6) Alkyl linking group or substituted (C)1-6) Alkyl linking group, optionally interrupted by a heteroatom or linking functional group such as an ester (-CO)2-), amido (CONH), carbamato (OCONH), ether (-O-), thioether (-S-) and/or an amino group (-NR-, wherein R is H or alkyl).
In some cases of formula (XI), the subject ENPP1 inhibitor compound is a compound of formula (XII):
in certain embodiments of the compounds of formula (XII), Z 5Is CR16Wherein R is16Selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, trifluoromethyl, halogen, acyl, substituted acyl, carboxy, carboxamide, substituted carboxamide, sulfonyl, substituted sulfonyl, sulfonamide, and substituted sulfonamide. In some cases of the compounds of the formula (XII), Z5Is N.
In certain embodiments of the compounds of formula (XII), L12Is a covalent bond. In some cases, L12Is a linking group. Any convenient linker may be used as L12. In some cases, L12Is a straight chain linker of 1 to 12 atoms in length, for example 1 to 10, 1 to 8 or 1 to 6 atoms in length, for example 1, 2, 3, 4, 5 or 6 atoms in length. The linking group L12Can be (C)1-6) Alkyl linking group or substituted (C)1-6) Alkyl linking group, optionally interrupted by a heteroatom or linking functional group such as an ester (-CO)2-), amido (CONH), carbamato (OCONH), ether (-O-), thioether (-S-) and/or an amino group (-NR-, wherein R is H or alkyl).
In some cases of formula (XII), the subject ENPP1 inhibitor compounds are compounds of formula (XIII):
wherein:
R35and R36Each independently selected from H, halogen, alkyl and substituted alkyl, or R 35And R36Are cyclic and together with the carbon atom to which they are attached provide a cycloalkyl, substituted cycloalkyl, heterocyclyl or substituted heterocyclyl ring; and
s is an integer from 0 to 6 (e.g., 0 to 3).
In certain embodiments of formula (XIII), R35And R36Each is hydrogen. In certain embodiments, R35Or R36At least one of which is halogen. In certain embodiments, R35Or R36At least one of which is an alkyl group. In certain embodiments, R35Or R36At least one of which is a substituted alkyl group. In some cases, R35Is halogen, and R36Selected from the group consisting of hydrogen, halogen, alkyl, and substituted alkyl. In some cases, R35Is alkyl, and R36Selected from the group consisting of hydrogen, halogen, alkyl, and substituted alkyl. In some cases, R35Is a substituted alkyl group, and R36Selected from the group consisting of hydrogen, halogen, alkyl, and substituted alkyl. In some cases, R35Is halogen, and R36Is hydrogen. In some cases, R35Is alkyl, and R36Is hydrogen. In some cases, R35Is a substituted alkyl group, and R36Is hydrogen.
In certain embodiments of formula (XIII), s is an integer from 0 to 3. In some cases, s is 0. In some cases, s is 1. In some cases, s is 2. In some cases, s is 3.
In some cases of formula (XIII), the subject ENPP1 inhibitor compounds are of formula (XIV):
where s is an integer from 0 to 6 (e.g., 0 to 3).
In certain embodiments of formula (XIII), s is an integer from 0 to 3. In some cases, s is 0. In some cases, s is 1. In some cases, s is 2. In some cases, s is 3.
In certain embodiments of any of formulas (X) - (XIV), R2To R5Independently selected from H, OH, alkyl, substituted alkyl, alkoxy, substituted alkoxy, -OCF3Halogen, cyano, amine, substituted amine, amideHeterocyclic and substituted heterocyclic.
In certain embodiments of any of formulas (X) - (XIV), R2To R5Independently selected from hydrogen, OH, C(1-6)Alkoxy, -OCF3、C(1-6)Alkylamino radical, di-C(1-6)Alkylamino, F, Cl, Br and CN.
In some cases of any of formulas (X) - (XIV), R2To R5At least one of which is hydrogen. In some cases, R2To R5At least two of which are hydrogen. In some cases, R2To R5At least three of which are hydrogen. In some cases, R2To R5Each is hydrogen. In some cases, R2To R5At least one of which is a hydroxyl group. In some cases, R2To R5At least one of which is an alkyl group or a substituted alkyl group. In some cases, R 2To R5At least one of which is alkoxy or substituted alkoxy. In some cases, the alkoxy or substituted alkoxy is C(1-6)Alkoxy, for example, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy. In some cases, R2To R5At least one of which is methoxy. In some cases, R2To R5At least one of which is-OCF3. In some cases, R2To R5At least one of which is halogen. In some cases, the halogen is fluorine. In some cases, the halogen is chlorine. In some cases, the halogen is bromine. In some cases, R2To R5At least one of which is cyano. In some cases, R2To R5At least one of which is an amine or a substituted amine. In some cases, R2To R5At least one of them is C(1-6)An alkylamino group. In some cases, R2To R5At least one of which is di-C(1-6)An alkylamino group. In some cases, R2To R5At least one of which is an amide. In some cases, R2To R5At least one of which is a heterocycle or a substituted heterocycle.
In some cases of any of formulas (X) - (XIV), R3And R4Independently is an alkoxy group; and R is2And R5Are both hydrogen. In some cases, R 3Is an alkoxy group; and R is2、R4And R5Is hydrogen. In some cases, R4Is an alkoxy group; and R is2、R3And R5Each is hydrogen. In some cases, R2、R3And R4Is hydrogen, and R5Is an alkoxy group. In some cases, the alkoxy group is C(1-6)An alkoxy group. In some cases, the alkoxy group is methoxy. In some cases, the alkoxy group is ethoxy. In some cases, the alkoxy group is propoxy. In some cases, the alkoxy group is butoxy. In some cases, the alkoxy group is pentyloxy. In some cases, the alkoxy group is a hexyloxy group.
In some cases of formula (XIV), the subject ENPP1 inhibitor compound is a compound of one of formulae (XIVa) - (XIVe):
where s is an integer from 0 to 6 (e.g., 0 to 3).
In some cases of formula (I), the subject ENPP1 inhibitor compound is a compound of formula (XVa) or (XVb):
wherein:
s is 0 to 3;
R21is C(1-6)Alkyl or substituted C(1-6)An alkyl group; and
R3and R4Selected from Cl and F.
In some cases of formulas (XVa) - (XVb), R21Selected from methyl, ethyl, n-propyl and isopropyl. In some cases, R21Is methyl. In some cases of formulas (XVa) - (XVb), R3And R4Is Cl.
In some cases, R3And R4Is F. In some cases of formulas (XVa) - (XVb), s is 2. In some cases, s is 1. In some embodiments of formulae (XVa) - (XVb), s is 2; r 21Is methyl or isopropyl; and R is3And R4Selected from Cl and F.
In some cases of formulae (XVa) - (XVb), the subject ENPP1 inhibitor compound is a compound of one of the following structures or a prodrug thereof (e.g., as described herein):
as described above, X1In the form of a hydrophilic head group or prodrug thereof. Any embodiment of the hydrophilic head group described herein can be incorporated into any embodiment of formulae (I) - (XVb) described herein. In some embodiments of formulas (I) - (XVb), X1A hydrophilic head group comprising a charged group capable of binding zinc ions, or a prodrug form thereof. In some cases, the hydrophilic head group capable of binding zinc ions is a phosphorous-containing functional group (e.g., as described herein).
In some embodiments of formulas (I) - (XVb), the hydrophilic head group (X)1) Selected from the group consisting of phosphonic or phosphonic acid, phosphonate, phosphate ester, thiophosphate, phosphoramidate, thiophosphate, sulfonate, sulfonic acid, sulfate, hydroxamic acid, keto acid, amide, and carboxylic acid. In some embodiments of any of formulas (I) - (XVb), the hydrophilic head group is a phosphonic acid, a phosphonate, or a salt thereof. In some embodiments of any of formulas (I) - (XVb), the hydrophilic head group is a phosphate, or a salt thereof. In some embodiments of any of formulas (I) - (XVb), the hydrophilic head group is a phosphonate or a phosphate. In some embodiments of any of formulas (I) - (XVb), the hydrophilic head group is a thiophosphate. Some embodiments of any of formulas (I) - (XVb) In embodiments, the hydrophilic head group is a phosphorothioate. In some embodiments of any of formulas (I) - (XVb), the hydrophilic head group is an amino phosphate. In some embodiments of any of formulas (I) - (XVb), the hydrophilic head group is a phosphoroamidothioate.
Specific examples of targeted hydrophilic head groups that can be incorporated into any of the embodiments of formulae (I) - (XVb) described herein include, but are not limited to, head groups comprising a first moiety selected from the group consisting of: phosphate Radical (RPO)4H-) Phosphonic acid (RPO)3H-) Boric acid (RBO)2H2) Carboxylate Radical (RCO)2 -) Sulfate Radical (RSO)4 -) Sulfonate Radical (RSO)3 -) Amine (RNH)3 +) Glycerol, a sugar such as lactose or derived from hyaluronic acid, polar amino acids, polyethylene oxide and oligoethylene glycol, said first moiety being optionally conjugated to a residue of a second moiety selected from: choline, ethanolamine, glycerol, nucleic acids, sugars, inositol, amino acids or amino acid esters (e.g., serine), and lipids (e.g., fatty acids or hydrocarbon chains, such as C8-C30 saturated or unsaturated hydrocarbons). The head group may contain various other modifications, for example in the case of oligo-and polyethylene oxides (PEG) containing head groups, such PEG chains may be terminated with methyl groups or have a distal functional group for further modification. Examples of hydrophilic head groups also include, but are not limited to, thiophosphates, phosphocholines, phosphoglycerides, phosphoethanolamines, phosphoserines, phosphoinositides, ethylphosphorylcholines (ethylphosphorylcholines), polyethylene glycols, polyglycerols, melamines, glycosamines, trimethylamine, spermine, spermidine, and conjugated carboxylates, sulfates, boronic acids, sulfonates, sulfates, and carbohydrates.
In some cases of any of formulas (I) - (XVb), the hydrophilic head group X1Is of formula (XVI):
wherein:
Z6is absent or selected from O and CH2；
Z7And Z9Each independently selected from O and NR10Wherein R is10Is H, alkyl or substituted alkyl;
Z8selected from O and S; and
R8and R9Each independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, non-aromatic heterocycle, substituted non-aromatic heterocycle, cycloalkyl, substituted cycloalkyl, and a precursor moiety.
In some embodiments of formula (XVI), Z6Is absent. In other cases, Z6Is CH2. In other cases, Z6Is oxygen. In some embodiments of formula (XVI), Z7Is oxygen, and Z9Is NR10. In some cases, Z7Is NR10And Z is9Is oxygen. In some cases, Z7And Z9Are all oxygen. In other cases, Z7And Z9Are all NR10. In some cases, Z8Is oxygen. In other cases, Z8Is sulfur.
In some embodiments of formula (XVI), Z7、Z8And Z9Are all oxygen atoms, and Z6Is absent or is CH2. In other cases, Z8Is a sulfur atom, Z7And Z9Are all oxygen atoms, and Z6Is absent or is CH 2. In other cases, Z8Is a sulfur atom, Z6、Z7And Z9Are all oxygen atoms. In some cases, Z8Is an oxygen atom, Z7Is NR10，Z9Is an oxygen atom, and Z6Is absent or is CH2. In other cases, Z8Is an oxygen atom, Z7Is NR10，Z6And Z9Are all oxygen atoms. In other cases, Z8Is an oxygen atom, Z7And Z9Each independently is NR10And Z is6Is an oxygen atom. In other cases, Z8Is an oxygen atom, Z7And Z9Each independently is NR10And Z is6Is absent or is CH2. In some cases, Z7And Z9Each being identical. In other cases, Z7And Z9Is different. It is to be understood that the group of formula (XVI) may include one or more tautomeric forms of the structure depicted, and is intended to include all such forms and salts thereof.
In some embodiments of formula (XVI), Z7And Z9At least one of which is NR10. In some cases, R10Is hydrogen. In some cases, R10Is an alkyl group. In some other cases, R10Is a substituted alkyl group. In some cases, Z7And Z9Are all NR10. In some cases, Z7And Z9Are all NR10And R is10、R8And R9Each independently hydrogen. In some cases, Z7And Z9Are all NR10，R10Each is alkyl, and R8And R9Each is hydrogen. In some cases, Z 7And Z9Are all NR10，R10Each is a substituted alkyl group (e.g., an alkyl group substituted with an ester or carboxyl group), and R8And R9Each is hydrogen.
In some embodiments of formula (XVI), R8And R9Are all hydrogen atoms. In some cases, R8And R9At least one of which is a substituent other than hydrogen. In other cases, R8And R9Are all substituents other than hydrogen. In some cases, R8And R9At least one of which is an alkyl group or a substituted alkyl group. In some cases, R8And R9At least one of which is alkenyl or substituted alkenyl. In some other cases, R8And R9At least one of which is aryl or substituted aryl. In some cases, R8And R9At least one of which is an acyl group or a substituted acyl group. In some cases, R8And R9At least one of which is heteroaryl or substituted heteroaryl. In some cases, R8And R9At least one of which is cycloalkyl or substituted cycloalkyl. In some cases, R8And R9Are both alkyl (e.g., lower alkyl). In some cases, R8And R9Are all substituted alkyl (e.g. C)(1-6)Alkyl substituted with alkoxy, substituted alkoxy, ester, or carboxyl). In some cases, R8And R9Comprises a precursor moiety. In some cases, R 8And R9Are both phenyl groups. In some cases, R8And R9Are the same. In other cases, R8And R9Is different.
In some cases of any of formulas (I) - (XVb), the hydrophilic head group X1Selected from any one of formulas (XVIa) to (XVIf):
wherein:
R10and R11Each independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, carboxyl, substituted carboxyl, and a precursor moiety (promoity) (e.g., as described herein).
In some embodiments of formulas (XVIa) to (XVif), R10And R11Are all hydrogen atoms. In some cases, R10And R11At least one of which is a substituent other than hydrogen. In other cases, R10And R11Are all substituents other than hydrogen. In some cases, R10And R11Are the same. In other cases, R10And R11Is different. In some cases, R10And R11At least one ofIs alkyl or substituted alkyl. In some cases, R10And R11At least one of which is aryl or substituted aryl. In some cases, R10And R11Are both alkyl or substituted alkyl. In some cases, R 10And R11Are both aryl or substituted aryl. In some cases, R10And R11Are both acyl or substituted acyl. In some cases, R10And R11Are both lower alkyl groups. In some cases, R10And R11Are all substituted alkyl (e.g. C)(1-6)Alkyl substituted with alkoxy, substituted alkoxy, ester, or carboxyl). In some cases, R10And R11Comprises a precursor moiety. In some cases, R10And R11Are both phenyl groups.
In some cases of formulae (XVIa) to (XVId), R10And R11Comprises a cleavable group or a self-immolative (self-immolative) precursor moiety. The self-eliminating group may be a disulfide-linked precursor moiety or a self-eliminating ester containing a precursor moiety. In some cases, R10And/or R11Comprising the formula-CH2CH2-SS-R12A disulfide-linked precursor moiety of (a), wherein R12Is alkyl or substituted alkyl. In some cases, R12Is a C8-C30 saturated or unsaturated hydrocarbon chain. In some cases, R10And/or R11Comprising the formula-CH2OCOR13Wherein R is13Is H, alkyl or substituted alkyl. In some cases, R10And/or R11Comprising the formula-CH2C(R14)2CO2R14Wherein R is14Each independently is H, alkyl or substituted alkyl.
In some cases of any of formulas (I) - (XVb), the hydrophilic head group X1Or a prodrug form thereof is selected from:
or a pharmaceutically acceptable salt thereof.
In some cases of any of formulas (I) - (XVb), the hydrophilic head group X1Is a radical of formula (XVI):
wherein:
R81and R91Each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, ester, amide, heterocycle, substituted heterocycle, cycloalkyl and substituted cycloalkyl, or R81And R91Together with the atoms to which they are attached form a group selected from heterocycles and substituted heterocycles.
In some embodiments of formula (XVI), R81And R91Are all hydrogen atoms. In other cases, R81And R91Are all substituents other than hydrogen.
In some cases of any of formulas (I) - (XVb), the hydrophilic head group X1Is a radical of formula (XVII):
in some cases of any of formulas (I) - (XVb), the hydrophilic head group X1Is a radical of formula (XVIII):
wherein:
Z61is absent or selected from O and CH2。
In some embodiments of formula (XVIII), the hydrophilic head group is selected from one of the following groups:
in some cases of any of formulas (I) - (XVb), the hydrophilic head group X 1Is a group of formula (XIX):
in some cases of any of formulas (I) - (XVb), the hydrophilic head group X1Is a group of formula (XX):
wherein:
R92selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, ester, amide, heterocycle, substituted heterocycle, cycloalkyl, and substituted cycloalkyl.
In some embodiments of formula (XX), R92Is hydrogen. In other cases, R92Are substituents other than hydrogen. In certain embodiments, R92Is alkyl or substituted alkyl. In certain embodiments of formula (XX), the hydrophilic head group is a group of the following structure:
in some cases of any of formulas (I) - (XVb), the hydrophilic head group X1Is a group of formula (XXI):
it is understood that the formulae (I) - (XVb)) Group X of any one of1Any of the hydroxyl and amine groups in (a) may optionally be further substituted with any convenient group (e.g., alkyl, substituted alkyl, phenyl, substituted phenyl, ester group, etc.). It will be appreciated that any convenient alternative hydrophilic group may be used as the group X in the compounds of any of formulae (I) - (XVb)1。
In certain embodiments, the ENPP1 inhibitor compound is described by the structure of table 1 or a prodrug thereof (e.g., as described herein), or one of its pharmaceutically acceptable salts.
Table 1: ENPP1 inhibitor compounds
In certain embodiments, the ENPP1 inhibitor compound is described by the structure of table 2 or a prodrug thereof (e.g., as described herein), or one of its pharmaceutically acceptable salts.
Table 2: ENPP1 inhibitor compounds
In certain embodiments, the ENPP1 inhibitor compound is described by the structure of table 3 or a prodrug thereof (e.g., as described herein), or one of its pharmaceutically acceptable salts.
Table 3: ENPP1 inhibitor compounds
In certain embodiments, the compound is described by the structure of one of the compounds of tables 1-3 (herein, reference to tables 1-3 includes table 3 a). It is understood that any of the compounds shown in tables 1-3 may exist in the form of a salt. In some cases, the salt form of the compound is a pharmaceutically acceptable salt. It is understood that any of the compounds shown in tables 1-3 may exist in prodrug form.
In some embodiments, the compound is described by the structure of one of the compounds of table 3 a.
TABLE 3a
Aspects of the present disclosure include ENPP1 inhibitor compounds (e.g., as described herein), salts (e.g., pharmaceutically acceptable salts) thereof, and/or solvate, hydrate, and/or prodrug forms thereof. In addition, it is to be understood that in any compound described herein having one or more chiral centers, each center can independently have the R-configuration or the S-configuration, or mixtures thereof, if absolute stereochemistry is not explicitly indicated. It is understood that all permutations of salts, solvates, hydrates, prodrugs and stereoisomers are intended to be encompassed by the present disclosure.
In some embodiments, the subject ENPP1 inhibitor compound or prodrug form thereof is provided in the form of a pharmaceutically acceptable salt. Compounds containing amine-or nitrogen-containing heteroaryl groups can be basic in nature and, therefore, can react with a wide variety of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Acids commonly used to form such salts include inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acids, and organic acids such as p-toluenesulfonic, methanesulfonic, oxalic, p-bromophenylsulfonic, carbonic, succinic, citric, benzoic and acetic acids, and related inorganic and organic acids. Thus, such pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, decanoate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, decanoate, octanoate, hexanoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, nitrobenzoate, and mixtures thereof, Phenylbutyrate, citrate, lactate, beta-hydroxybutyrate, glycolate, maleate, tartrate, mesylate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, hippurate, gluconate, lactobionate and the like. In certain particular embodiments, pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid and hydrobromic acid, as well as those formed with organic acids such as fumaric acid and maleic acid.
In some embodiments, the subject compounds are provided in prodrug form. "prodrug" refers to a derivative of an active agent that requires conversion in vivo to release the active agent. In certain embodiments, the transformation is an enzymatic transformation. Prodrugs are often, although not necessarily, pharmacologically inactive until converted to the active agent. A "precursor moiety" is a form of protecting group that, when used to mask a functional group within an active agent, converts the active agent into a prodrug. In some cases, the precursor moiety is attached to the drug via one or more bonds that are cleaved in vivo by enzymatic or non-enzymatic means. Any convenient prodrug form of the subject compounds may be prepared, for example, according to the strategies and methods described by Rautio et al ("Prodrugs: design and clinical applications", Nature Reviews Drug Discovery 7, 255-. In some cases, the precursor moiety is attached to a hydrophilic head group of the subject compound. In some cases, the precursor moiety is attached to a hydroxyl or carboxylic acid group of the subject compound. In some cases, the precursor moiety is an acyl group or a substituted acyl group. In some cases, the precursor moiety is an alkyl or substituted alkyl group that forms an ester functional group (e.g., phosphonate, phosphate, etc.) when attached to the hydrophilic head group of the subject compound, for example.
In some embodiments, the subject compounds are phosphonate esters or phosphate ester prodrugs, which can be converted to compounds that include a phosphonic acid or phosphonate or phosphate head group.
In some embodiments, the subject compounds, prodrugs, stereoisomers, or salts thereof are provided in the form of solvates (e.g., hydrates). The term "solvate" as used herein refers to a complex or aggregate formed by molecules of one or more solutes (e.g., prodrugs or pharmaceutically acceptable salts thereof) and one or more solvent molecules. Such solvates are typically crystalline solids having a substantially fixed solute to solvent molar ratio. Representative solvents include, for example, water, methanol, ethanol, isopropanol, acetic acid, and the like. When the solvent is water, the solvate formed is a hydrate.
In some embodiments, the subject compounds are provided by oral administration and absorbed into the bloodstream. In some embodiments, the subject compound has an oral bioavailability of 30% or greater. The subject compounds or formulations thereof may be modified using any convenient method to increase absorption through the intestinal lumen or their bioavailability.
In some embodiments, the subject compounds are metabolically stable (e.g., remain substantially intact in vivo during the half-life of the compound). In certain embodiments, the half-life (e.g., in vivo half-life) of a compound is 5 minutes or more, such as 10 minutes or more, 12 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 60 minutes or more, 2 hours or more, 6 hours or more, 12 hours or more, 24 hours or more, or even more.
Methods of inhibiting ENPP1
As summarized above, aspects of the present disclosure include ENPP1 inhibitors, and methods of inhibition using the ENPP1 inhibitors. ENPP1 is a member of the ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family. Thus, aspects of the subject methods include inhibiting the hydrolase activity of ENPP1 against cGAMP. The present inventors have discovered that cGAMP may have an important extracellular biological function that may be enhanced by preventing extracellular degradation of cGAMP, such as hydrolysis by its degradative enzyme ENPP 1. In certain examples, ENPP1 inhibits the target to be located extracellularly, and the subject ENPP1 inhibitory compounds are cell impermeable and therefore unable to diffuse into the cell. Thus, the subject methods can provide selective extracellular inhibition of ENPP1 hydrolase activity and increase extracellular levels of cGAMP. Thus, in some instances, an ENPP1 inhibitory compound is a compound that inhibits the activity of ENPP1 extracellularly. Experiments conducted by the present inventors have shown that inhibition of ENPP1 activity increases extracellular cGAMP and may therefore enhance the STING pathway.
By inhibiting ENPP1 is meant that the activity of the enzyme (e.g., relative to a control in any convenient in vitro inhibition assay) is reduced by 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more. In some cases, inhibiting ENPP1 means decreasing the activity of the enzyme by one 2 or less, such as by one 3 or less, by one 5 or less, by one 10 or less, by one 100 or less, or by one 1000 or less, relative to its normal activity (e.g., relative to a control, as measured by any convenient assay).
In some cases, the method is a method of inhibiting ENPP1 in a sample. The term "sample" as used herein relates to a material or mixture of materials containing one or more target components, typically but not necessarily in fluid form.
In some embodiments, methods of inhibiting ENPP1 are provided, the methods comprising contacting a sample with a cell-impermeable ENPP1 inhibitor to inhibit cGAMP hydrolysis activity of ENPP 1. In some cases, the sample is a cell sample. In some cases, the sample comprises cGAMP. In certain instances, cGAMP levels are elevated in the cell sample (e.g., relative to a control sample that has not been contacted with the inhibitor). The subject methods can provide increased levels of cGAMP. By "increased cGAMP level" is meant a level of cGAMP in a sample of cells contacted with a subject compound, wherein the cGAMP level in the sample is increased by 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, or even more, relative to a control sample not contacted with the agent.
In certain embodiments, the ENPP1 inhibitor is an inhibitor as defined herein. In some embodiments, the ENPP1 inhibitor is an inhibitor according to any one of formulas (I) - (XVb) (e.g., as described herein). In some cases, the ENPP1 inhibitor is a compound of any one of tables 1-3 (e.g., as described herein). In some cases, the ENPP1 inhibitor is cell impermeable.
In some embodiments, the ENPP1 inhibitor is configured to be cell permeable. In some embodiments, methods of inhibiting ENPP1 are provided, the methods comprising contacting a sample with a cell permeable ENPP1 inhibitor to inhibit ENPP 1.
In some embodiments, the subject compounds have ENPP1 inhibitory properties that reflect activity against additional enzymes. In some embodiments, the subject compounds specifically inhibit ENPP1 without undesirably inhibiting one or more other enzymes.
In some embodiments, the compounds of the present disclosure interfere with the interaction of cGAMP and ENPP 1. For example, the subject compounds can function to increase extracellular cGAMP by inhibiting the hydrolase activity of ENPP1 against cGAMP. Without being bound by any particular theory, it is believed that increasing extracellular cGAMP activates the STING pathway.
In some embodiments, the subject compound inhibits ENPP1, as determined by an inhibition test, e.g., by an assay that determines the level of activity of an enzyme in a cell-free system or in a cell after treatment with the subject compound relative to a control, by measuring IC, respectively50Or EC50The value is obtained. In certain embodiments, a subject compound has an IC of 10 μ Μ or less, such as 3 μ Μ or less, 1 μ Μ or less, 500nM or less, 300nM or less, 200nM or less, 100nM or less, 50nM or less, 30nM or less, 10nM or less, 5nM or less, 3nM or less, 1nM or less, or even less50Value (or EC)50Value).
As summarized above, aspects of the present disclosure include methods of inhibiting ENPP 1. The subject compounds (e.g., as described herein) can inhibit the activity of ENPP1 in a range of 10% to 100%, such as 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In certain assays, the subject compounds may be present at 1x 10-6M or less (e.g., 1x 10)-6M or less, 1x 10-7M or less, 1x 10-8M or less, 1x 10-9M or less, 1x 10-10M or less, or 1x 10 -11M or less) IC50Inhibiting its target.
There are many protocols that can be used to determine ENPP1 activity, and include, but are not limited to, cell-free assays, such as binding assays; assays using purified enzymes, cellular assays in which cellular phenotype is measured, such as gene expression assays; and in vivo assays involving specific animals (which in certain embodiments may be animal models of disorders associated with the pathogen of interest).
In some embodiments, the subject methods are in vitro methods comprising contacting a sample with a subject compound that specifically inhibits ENPP 1. In certain embodiments, the sample is suspected of containing ENPP1, and the subject methods further comprise assessing whether the compound inhibits ENPP 1.
In certain embodiments, the subject compounds are modified compounds that include a label, e.g., a fluorescent label, and the subject methods further include detecting the label (if present) in the sample, e.g., using optical detection.
In certain embodiments, the compound is modified with a support or with an affinity group that is bound to a support (e.g., biotin) such that any sample that is not bound to the compound can be removed (e.g., by washing). The specifically bound ENPP1, if present, can be detected using any convenient method, such as binding using a labeled target-specific probe or using a fluorescent protein reagent.
In another embodiment of the subject method, the known sample contains ENPP 1.
In some embodiments, the method is a method of reducing cancer cell proliferation, wherein the method comprises contacting the cell with an effective amount of the subject ENPP1 inhibitor compound (e.g., as described herein) to reduce cancer cell proliferation. In certain instances, the subject ENPP1 inhibitor compounds may act intracellularly. The method can be performed in conjunction with a chemotherapeutic agent (e.g., as described herein). Cancer cells can be in vitro or in vivo. In certain examples, the method comprises contacting the cell with an ENPP1 inhibitor compound (e.g., as described herein), and contacting the cell with a chemotherapeutic agent. Any convenient cancer cell can be targeted.
Method of treatment
Aspects of the present disclosure include methods for inhibiting the hydrolase activity of ENPP1 against cGAMP, to provide increased levels of cGAMP and/or downstream regulation (e.g., activation) of the STING pathway. The inventors have discovered that cGAMP can be present in the extracellular space, and ENPP1 can control extracellular levels of cGAMP. The inventors have also discovered that cGAMP can have important extracellular biological functions in vivo. The results described and demonstrated herein demonstrate that ENPP1 inhibition according to the subject methods can modulate STING activity in vivo and thus can be used to treat a variety of diseases, such as targets for cancer immunotherapy. Thus, the subject methods can provide selective extracellular inhibition of ENPP1 activity (e.g., cGAMP hydrolase activity) to increase extracellular levels of cGAMP and activate the interferon gene stimulating factor (STING) pathway. In some examples, the subject methods are methods for enhancing STING-mediated responses in a subject. In some examples, the subject methods are methods for modulating an immune response in a subject.
By "STING-mediated response" is meant any response mediated by STING, including but not limited to, for example, immune responses to bacterial, viral and eukaryotic pathogens. See, e.g., Ishikawa et al, Immunity 29:538-550 (2008); ishikawa et al Nature 461:788-792 (2009); and Immunity 35:194-207(2011) by Sharma et al. STING may also play a role in certain autoimmune diseases that arise as a result of inappropriate recognition of self-DNA (see, e.g., Gall et al Immunity 36:120-131(2012), and may also play a role in inducing adaptive Immunity in response to DNA vaccines (see, e.g., Ishikawa et al Nature 461:788-792 (2009). increasing STING-mediated response in a subject means that STING-mediated response in a subject is increased compared to a control subject (e.g., a subject not administered the subject compound).
In some cases, a STING-mediated response comprises increasing production of an interferon (e.g., type I Interferon (IFN), type III Interferon (IFN)) in the subject. Interferons (IFNs) are proteins with a variety of biological activities, such as anti-viral, immunomodulatory and anti-proliferative. IFNs are relatively small, species-specific single-chain polypeptides produced by mammalian cells in response to exposure to various inducers (such as viruses, polypeptides, mitogens, and the like). Interferons protect animal tissues and cells from virus attack and are important host defense mechanisms. Interferons can be classified into type I, type II and type III interferons. Mammalian type I interferons of interest include IFN- α (alpha), IFN- β (beta), IFN- κ (kappa), IFN- δ (delta), IFN- ε (epsilon), IFN- τ (tau), IFN- ω (omega), and IFN- ζ (zeta, also known as a limiter (limitin)).
Interferons are useful in the treatment of a variety of cancers because these molecules have anti-cancer activity that acts at various levels. The interferon protein can directly inhibit the proliferation of human tumor cells. In some cases, the antiproliferative activity is also synergistic with various approved chemotherapeutic agents such as cisplatin, 5FU, and paclitaxel. Immunomodulatory activity of interferon proteins may also lead to the induction of anti-tumor immune responses. This response includes activation of NK cells, stimulation of macrophage activity and induction of MHC class I surface expression, resulting in induction of anti-tumor cytotoxic T lymphocyte activity. In addition, interferons play a role in the cross presentation of antigens in the immune system. In addition, several studies have further shown that IFN- β proteins may have anti-angiogenic activity. Angiogenesis (neovascularization) is critical to the growth of solid tumors. IFN- β can inhibit angiogenesis by inhibiting the expression of pro-angiogenic factors such as bFGF and VEGF. Interferon proteins may also inhibit tumor invasion by modulating the expression of enzymes important in tissue remodeling, such as collagenase and elastase.
Aspects of the methods include administering to a subject having cancer a therapeutically effective amount of an ENPP1 inhibitor to treat the cancer in the subject. In some examples, the subject is a subject diagnosed with or suspected of having cancer. Any convenient ENPP1 inhibitor can be used in the subject methods of treating cancer. In certain instances, the ENPP1 inhibitor compound is a compound as described herein. In certain instances, the ENPP1 inhibitor is a cell impermeable compound. In certain instances, the ENPP1 inhibitor is a cell permeable compound. In some cases, the cancer is a solid tumor cancer. In certain embodiments, the cancer is selected from adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, colon cancer, stomach cancer, ovarian cancer, cervical cancer, uterine cancer, esophageal cancer, colorectal cancer, prostate cancer, pancreatic cancer, lung cancer (both small cell and non-small cell), thyroid cancer, malignant epithelial tumors, sarcomas, glioblastomas, melanomas, and various head and neck tumors. In some cases, the cancer is breast cancer. In certain embodiments, the cancer is lymphoma.
Aspects of the methods include administering to the subject a therapeutically effective amount of a cell impermeable ENPP1 inhibitor to inhibit hydrolysis of cGAMP and treat cancer in the subject. In some cases, the cancer is a solid tumor cancer. In certain embodiments, the cancer is selected from adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, colon cancer, stomach cancer, ovarian cancer, cervical cancer, uterine cancer, esophageal cancer, colorectal cancer, prostate cancer, pancreatic cancer, lung cancer (both small cell and non-small cell), thyroid cancer, malignant epithelial tumors, sarcomas, glioblastomas, melanomas, and various head and neck tumors. In certain embodiments, the cancer is breast cancer. In some cases, the cancer is lymphoma.
In some embodiments of the methods disclosed herein, the cell-impermeable ENPP1 inhibitor is an inhibitor of any one of formulas (I) - (XVb) (e.g., as described herein). In some cases, the ENPP1 inhibitor is a compound in tables 1-3 or a prodrug form thereof (e.g., as described herein).
In some embodiments of the methods disclosed herein, the ENPP1 inhibitor is cell permeable.
Accordingly, aspects of the methods include contacting a sample with a subject compound (e.g., as described above) under conditions in which the compound inhibits ENPP 1. Any convenient protocol for contacting the compound with the sample may be employed. The particular protocol employed may vary, e.g., depending on whether the sample is in vitro or in vivo. For in vitro protocols, contacting the sample with the compound can be accomplished using any convenient protocol. In some examples, the sample comprises cells maintained in a suitable culture medium, and the complex is introduced into the culture medium. For in vivo protocols, any convenient administration protocol may be employed. Depending on the potency of the compound, the cells of interest, the mode of administration, the number of cells present, various protocols can be employed.
In some embodiments, the subject methods are methods of treating cancer in a subject. In some embodiments, the subject methods comprise administering to the subject an effective amount of a subject compound (e.g., as described herein), or a pharmaceutically acceptable salt thereof. The subject compounds can be administered as part of a pharmaceutical composition (e.g., as described herein). In certain examples of this method, the compound administered is a compound of one of formulas (I) - (XVb) (e.g., as described herein). In certain examples of the method, the compound administered is described by one of the compounds in tables 1-3.
In some embodiments, an "effective amount" is an amount of a subject compound that, when administered to a subject in a single dose or multiple doses, in monotherapy or in combination therapy, is effective to inhibit ENPP1 by about 20% (20% inhibition), at least about 30% (30% inhibition), at least about 40% (40% inhibition), at least about 50% (50% inhibition), at least about 60% (60% inhibition), at least about 70% (70% inhibition), at least about 80% (80% inhibition), or at least about 90% (90% inhibition), as compared to ENPP1 activity in a subject treated in the absence of the compound, or alternatively as compared to ENPP1 activity in a subject before or after treatment with the compound.
In some embodiments, a "therapeutically effective amount" is an amount of a subject compound that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to reduce tumor burden in the subject by about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as compared to tumor burden in an individual in the absence of treatment with the compound, or alternatively as compared to tumor burden in the subject before or after treatment with the compound. As used herein, the term "tumor burden" refers to the total mass of tumor tissue carried by a subject with cancer.
In some embodiments, a "therapeutically effective amount" is an amount of a subject compound effective to reduce the dose of radiation therapy required to observe tumor shrinkage in a subject by about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% when administered to an individual in one or more doses, in monotherapy or in combination therapy, as compared to the dose of radiation therapy required to observe tumor shrinkage in an individual in the absence of treatment with the compound.
In some embodiments, a "therapeutically effective amount" of a compound is an amount effective to achieve a 1.5-log, 2-log, 2.5-log, 3-log, 3.5-log, 4-log, 4.5-log, or 5-log reduction in tumor size when administered in one dose or multiple doses to an individual having cancer.
In some embodiments, an effective amount of a compound is in the range of about 50ng/ml to about 50 μ g/ml (e.g., about 50ng/ml to about 40 μ g/ml, about 30ng/ml to about 20 μ g/ml, about 50ng/ml to about 10 μ g/ml, about 50ng/ml to about 1 μ g/ml, about 50ng/ml to about 800ng/ml, about 50ng/ml to about 700ng/ml, about 50ng/ml to about 600ng/ml, about 50ng/ml to about 500ng/ml, about 50ng/ml to about 400ng/ml, about 60ng/ml to about 400ng/ml, about 70ng/ml to about 300ng/ml, about 60ng/ml to about 100ng/ml, about 65ng/ml to about 85ng/ml, about 70ng/ml to about 90ng/ml, or, About 200ng/ml to about 900ng/ml, about 200ng/ml to about 800ng/ml, about 200ng/ml to about 700ng/ml, about 200ng/ml to about 600ng/ml, about 200ng/ml to about 500ng/ml, about 200ng/ml to about 400ng/ml, or about 200ng/ml to about 300 ng/ml).
In some embodiments, an effective amount of a compound is an amount in the range of about 10pg to about 100mg, e.g., about 10pg to about 50pg, about 50pg to about 150pg, about 150pg to about 250pg, about 250pg to about 500pg, about 500pg to about 750pg, about 750pg to about 1ng, about 1ng to about 10ng, about 10ng to about 50ng, about 50ng to about 150ng, about 150ng to about 250ng, about 250ng to about 500ng, about 500ng to about 750ng, about 750ng to about 1 μ g, about 1 μ g to about 10 μ g, about 10 μ g to about 50 μ g, about 50 μ g to about 150 μ g, about 150 μ g to about 250 μ g, about 250 μ g to about 500 μ g, about 500 μ g to about 750 μ g, about 750 μ g to about 1mg, about 1mg to about 50mg, about 1mg to about 100mg, or about 100 mg. The amount may be that of a single dose or may be the total daily amount. The total daily amount may be in the range of 10pg to 100mg, or may be in the range of 100mg to about 500mg, or may be in the range of 500mg to about 1000 mg.
In some embodiments, a single dose of the compound is administered. In other embodiments, multiple doses are administered. In the case of multiple doses administered over a period of time, the compound may be administered twice daily (qid), daily (qd), every other day (qod), every third day, three times weekly (tiw), or twice weekly (biw) over a period of time. For example, the compound is administered qid, qd, qod, tiw or biw over a period of from one day to about 2 years or more. For example, depending on various factors, the compound is administered at any of the above frequencies for one week, two weeks, one month, two months, six months, one year, or two years or more.
Administration of a therapeutically effective amount of a subject compound to an individual having cancer may result in one or more of the following: 1) a reduction in tumor burden; 2) a reduction in the radiation therapy dose required to achieve tumor reduction; 3) a reduction in the spread of cancer from one cell to another in an individual; 4) decreased morbidity or mortality with clinical outcome; 5) the overall time of treatment is reduced when used in combination with other anti-cancer agents; and 6) improvement in disease response indicators (e.g., reduction in one or more symptoms of cancer). Any of a variety of methods may be used to determine whether a treatment is effective. For example, assays can be performed on biological samples taken from individuals who have been treated with the subject methods.
Any of the compounds described herein can be used in the subject methods of treatment. In certain examples, the compound is one of formulas (I) - (XVb) (e.g., as described herein). In certain instances, the compound is one of the compounds in tables 1-3 or a prodrug form thereof. In some cases, the compounds used in the subject methods are not cell permeable. In some cases, the compounds used in the subject methods have poor cell permeability.
In some embodiments, the compound specifically inhibits ENPP 1. In some embodiments, the compound modulates the activity of cGAMP. In some embodiments, the compound interferes with the interaction of ENPP1 and cGAMP. In some embodiments, the compound causes activation of the STING pathway.
In some embodiments, the subject is a mammal. In certain examples, the subject is a human. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal disease models), and non-human primates (e.g., chimpanzees and monkeys). The subject may be in need of treatment for cancer. In some examples, the subject methods include diagnosing cancer, including any of the cancers described herein. In some embodiments, the compound is administered as a pharmaceutical product.
In certain embodiments, the ENPP1 inhibitor compound is a modified compound comprising a label, and the method further comprises detecting the label in the subject. The choice of label depends on the detection means. Any convenient labeling and detection system may be used in The subject methods, see, e.g., Baker, "The book picture," Nature,463,2010, pages 977-. In certain embodiments, the compound comprises a fluorescent label suitable for optical detection. In certain embodiments, the compound comprises a radioactive label for detection using Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT). In some cases, the compound comprises a paramagnetic label suitable for tomographic detection. As described above, the subject compounds may be labeled, but in some methods, the compound is not labeled and a second labeling agent is used for imaging.
Combination therapy
The subject compounds can be administered to a subject alone or in combination with an additional, i.e., second, active agent. Combination therapy methods wherein the subject ENPP1 inhibitor compounds can be used in combination with a second active agent or additional therapy (e.g., radiation therapy). The terms "agent," "compound," and "drug" are used interchangeably herein. For example, the ENPP1 inhibitor compound may be administered alone or in combination with one or more other drugs, such as drugs for treating diseases of interest (including but not limited to immunomodulatory diseases and disorders and cancer). In some embodiments, the subject methods further comprise co-administering, concomitantly or sequentially, a second agent, such as a small molecule, chemotherapeutic agent, antibody fragment, antibody-drug conjugate, aptamer, protein, or checkpoint inhibitor. In some embodiments, the method further comprises administering radiation therapy to the subject.
The terms "co-administration" and "in combination with. In one embodiment, the agents are present in the cell or in the body of the subject at the same time, or exert their biological or therapeutic effects at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agent is in a separate composition or unit dosage form. In certain embodiments, the second therapeutic agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before administration of the second therapeutic agent), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after administration of the second therapeutic agent) administration of the second therapeutic agent.
By "concomitant administration" of a known therapeutic drug or additional therapy with a pharmaceutical composition of the present disclosure is meant that the compound and second agent or additional therapy are administered at a time such that both the known drug and the composition of the present invention will have a therapeutic effect. With respect to administration of the subject compounds, such concomitant administration may involve administration of the drug at the same time (i.e., at the same time), before, or after. The route of administration of the two agents may vary, with representative routes of administration being described in more detail below. For particular drugs or therapies and compounds of the present disclosure, one of ordinary skill in the art will readily determine the appropriate timing of administration, order of administration, and dosage of administration.
In some embodiments, the compounds (e.g., the subject compound and at least one additional compound or therapy) are administered to the subject within twenty-four hours of each other, such as within 12 hours of each other, within 6 hours of each other, within 3 hours of each other, or within 1 hour of each other. In certain embodiments, the compounds are administered within 1 hour of each other. In certain embodiments, the compounds are administered substantially simultaneously. By substantially simultaneous administration is meant that the compounds are administered to the subject within about 10 minutes or less of each other, such as 5 minutes or less of each other, or 1 minute or less.
Pharmaceutical preparations of the subject compounds and a second active agent are also provided. In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts, or they may be used alone or in appropriate combination with other pharmaceutically active compounds, or in combination with other pharmaceutically active compounds.
In conjunction with any of the subject methods, an ENPP1 inhibitor compound (e.g., as described herein) (or a pharmaceutical composition comprising such a compound) can be administered in combination with another drug designed to reduce or prevent inflammation, treat or prevent chronic inflammation or fibrosis, or treat cancer. In each case, the ENPP1 inhibitor compound may be administered prior to, concurrently with, or subsequent to the administration of the other drug. In certain instances, the cancer is selected from adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, colon cancer, stomach cancer, ovarian cancer, cervical cancer, uterine cancer, esophageal cancer, colorectal cancer, prostate cancer, pancreatic cancer, lung cancer (both small cell and non-small cell), thyroid cancer, malignant epithelial tumors (carcinomas), sarcomas, gliomas, glioblastomas, melanomas, and various head and neck tumors.
For the treatment of cancer, the ENPP1 inhibitor compound may be administered in combination with a chemotherapeutic agent selected from the group consisting of: alkylating agents, nitrosoureas, antimetabolites, tumor antibiotics, plant (vinca) alkaloids, steroid hormones, taxanes, nucleoside analogs, steroids, anthracyclines, thyroid hormone replacement drugs, thymidylate targeting drugs, chimeric antigen receptor/T cell therapy, chimeric antigen receptor/NK cell therapy, apoptosis modulator inhibitors (e.g., B-cell CLL/lymphoma 2(BCL-2) BCL-2-like 1(BCL-XL) inhibitors), CARP-1/CCAR1 (cell division cycle and apoptosis modulator 1) inhibitors, colony stimulating factor-1 receptor (CSF1R) inhibitors, CD47 inhibitors, cancer vaccines (e.g., Th 17-induced dendritic cell vaccines or genetically modified tyrosinases such as) And other cell therapies.
Specific chemotherapeutic agents of interest include, but are not limited to, gemcitabine, docetaxel, bleomycin, erlotinib, gefitinib, lapatinib, imatinib, dasatinib, nilotinib, bosutinib, crizotinib, ceritinib, tremetinib, bevacizumab, sunitinib, sorafenib, trastuzumab, Ado-trastuzumab emtansine (Ado-trastuzumab emtansine), rituximab, ipilimu, rapamycin, sirolimus, everolimus, methotrexate, doxorubicin, Abraxane, flucillin (Folfirinox), cisplatin, carboplatin, 5-fluorouracil, Teysumo, paclitaxel, prednisone, levothyroxine, pemetrexed, naltrexatux (navitoclax), and ABT-199. Peptide compounds may also be used. Targeted cancer chemotherapeutic agents include, but are not limited to dolastatin (dolastatin) and its active analogs and derivatives; and auristatins (auristatins) and their active analogs and derivatives (e.g., monomethyl auristatin d (mmad), monomethyl auristatin e (mmae), monomethyl auristatin f (mmaf), etc.). See, e.g., WO 96/33212, WO 96/14856, and u.s.6,323,315. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and analogs thereof Derivatives (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA93: 8618-8623); duocarmycins and active analogs and derivatives thereof (including, for example, synthetic analogs, KW-2189 and CB 1-TM 1); and benzodiazepinesAnd active analogs and derivatives thereof (e.g. pyrrolobenzodiazepines)(PBD)。
In some embodiments, the ENPP1 inhibitor compound may be administered in combination with a chemotherapeutic agent to treat cancer. In certain instances, the chemotherapeutic agent is gemcitabine. In some cases, the chemotherapeutic agent is docetaxel. In some cases, the chemotherapeutic agent is Abraxane.
For the treatment of cancer (e.g., solid tumor cancer), an ENPP1 inhibitor compound can be administered in combination with an immunotherapeutic agent. An immunotherapeutic is any convenient agent used to treat a disease by inducing, enhancing or suppressing an immune response. In some cases, the immunotherapeutic agent is an immune checkpoint inhibitor. For example, figures 21A-4C show that an exemplary ENPP1 inhibitor can act synergistically with immune checkpoint inhibitors in a mouse model. Any convenient checkpoint inhibitor may be used, including but not limited to cytotoxic T-lymphocyte-associated antigen 4(CTLA-4) inhibitors, programmed death 1(PD-1) inhibitors, and PD-L1 inhibitors. In certain examples, the checkpoint inhibitor is selected from a cytotoxic T-lymphocyte-associated antigen 4(CTLA-4) inhibitor, a programmed death 1(PD-1) inhibitor, and a PD-L1 inhibitor. Exemplary target checkpoint inhibitors include, but are not limited to, ipilimumab, pembrolizumab, and nivolumab. In certain embodiments, one or more immunomodulatory polypeptides may be administered in combination with a colony stimulating factor-1 receptor (CSF1R) inhibitor for the treatment of cancer and/or inflammatory diseases. Targeted CSF1R inhibitors include, but are not limited to, inflixtuzumab (emactuzumab).
Any convenient cancer vaccine therapy and agents can be used in combination with the subject ENPP1 inhibitor compounds, compositions, and methods. For the treatment of cancer, such as ovarian cancer, the ENPP1 inhibitor compound may be administered in combination with a vaccine therapy, such as a Dendritic Cell (DC) vaccination agent that promotes Th1/Th17 immunization. Th17 cell infiltration was associated with a significant prolongation of overall survival in ovarian cancer patients. In some cases, ENPP1 inhibitor compounds may be used as adjuvant therapy in combination with Th 17-induced vaccination.
Also of interest are agents that are CARP-1/CCARI (regulator of cell division cycle and apoptosis 1) inhibitors, including but not limited to those described by Rishi et al, Journal of biological Nanotechnology, Vol.11, 9 th 2015, 9 th, p.1608-1627 (20) and CD47 inhibitors (including but not limited to anti-CD 47 antibody agents such as Hu5F 9-G4).
In certain instances, the combination provides an enhanced effect relative to either component alone; in some cases, the combination provides a superadditive or synergistic effect with respect to the combined or additive effect of the components. Various combinations of the subject compounds and chemotherapeutic agents, used sequentially or simultaneously, may be employed. For multiple administrations, for example, the two agents may be directly alternated, or two or more doses of one agent may be alternated with a single dose of the other agent. The simultaneous administration of the two agents may also be alternated or otherwise interspersed with the administration of the single agent. In some cases, the time between administrations can be a period of from about 1-6 hours to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 weeks or more after initiation of treatment.
Combination with cGAMP-inducing chemotherapy
Aspects of the present disclosure include methods of treating cancer, wherein an ENPP1 inhibitor compound (or a pharmaceutical composition comprising such a compound) can be administered in combination with a chemotherapeutic agent capable of inducing cGAMP production in vivo. The production of 2 '3' -cGAMP in a subject can be induced when the subject is exposed to an effective amount of a particular chemotherapeutic agent. When the subject ENPP1 inhibitor compounds are co-administered to prevent cGAMP degradation, induced levels of cGAMP can be maintained and/or enhanced, e.g., enhanced, compared to levels achieved with either agent alone. Any convenient chemotherapeutic agent that can cause DNA damage and induce cGAMP production by dying cells due to overwhelming repair or degradation mechanisms can be used in the subject combination therapy methods, such as alkylating agents, nucleic acid analogs, and intercalating agents. In some cases, the cGAMP-inducing chemotherapeutic agent is an anti-mitotic agent. Anti-mitotic agents are agents that act by damaging DNA or binding to microtubules. In some cases, the cGAMP-inducing chemotherapeutic agent is an antineoplastic agent.
Target cancers that may be treated using the subject combination therapies include, but are not limited to, adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, colon cancer, stomach cancer, ovarian cancer, cervical cancer, uterine cancer, esophageal cancer, colorectal cancer, prostate cancer, pancreatic cancer, lung cancer (both small cell and non-small cell), thyroid cancer, malignant epithelial tumors, sarcomas, gliomas, glioblastomas, melanomas, and various head and neck tumors. In some cases, the cancer is breast cancer. In certain examples, the cancer is a glioma or a glioblastoma.
Chemotherapeutic agents of interest include, but are not limited to, uracil analogs, fluorouracil prodrugs, thymidylate synthase inhibitors, deoxycytidine analogs, DNA synthesis inhibitors (e.g., causing S phase apoptosis), folic acid analogs, dehydrofolate reductase inhibitors, anthracyclines, intercalators (e.g., causing double strand breaks), topoisomerase IIa inhibitors, taxanes, microtubule disassembly inhibitors (e.g., causing G2/M phase block/apoptosis), microtubule assembly inhibitors, microtubule function stabilizers (e.g., causing G2/M-phase apoptosis), tubulin polymerization promoters, tubulin binders (e.g., causing apoptosis due to M-phase block), epothilone B analogs, vinca alkaloids, nitrogen mustards, nitrosoureas, DNA alkylating agents (e.g., causing interchain cross-linking, apoptosis via p 53), VEGF inhibitors, anti-angiogenic antibodies, HER2 inhibitors, quinazoline HER2 inhibitors, EGFR inhibitors, tyrosine kinase inhibitors, sirolimus analogs, mTORC1 inhibitors (as combined with exemestane ═ aromatase inhibitors that inhibit estrogen production in breast cancer), triazenes, dacarbazine prodrugs, methylhydrazine.
Exemplary targeted breast cancer chemotherapeutic agents include, but are not limited to, capecitabine, carmofur, fluorouracil, tegafur, gemcitabine, methotrexate, doxorubicin, epirubicin, docetaxel, ixabepilone, vindesine, vinorelbine, cyclophosphamide, bevacizumab, pertuzumab, trastuzumab, lapatinib, and everolimus. Exemplary glioma/glioblastoma-associated antineoplastic agents include, but are not limited to, carmustine, lomustine, temozolomide, procarbazine, vincristine, and bevacizumab. Exemplary targeted DNA damage chemotherapeutic agents include, but are not limited to, melphalan, cisplatin, and etoposide, fluorouracil, gemcitabine.
Combination radiation therapy
Alternatively, for methods of treating cancer, an ENPP1 inhibitor compound (or a pharmaceutical composition comprising such a compound) may be administered in combination with radiation therapy. In certain embodiments, the method comprises administering radiation therapy to the subject. Likewise, the ENPP1 inhibitor compound may be administered before or after the administration of radiation therapy. Thus, the subject methods may further comprise administering radiation therapy to the subject. The combination of radiation therapy and administration of the subject compounds can provide a synergistic therapeutic effect. Production of 2 '3' -cGAMP can be induced in a subject when the subject is exposed to a suitable dose and/or frequency of radiation during Radiation Therapy (RT). These induced cGAMP levels can be maintained and/or enhanced, e.g., enhanced, when the subject ENPP1 inhibitor compound is co-administered to prevent cGAMP degradation, e.g., compared to levels achieved using RT alone. For example, fig. 21A shows that an exemplary ENPP1 inhibitor can act synergistically with Radiation Therapy (RT) to reduce tumor burden in a mouse model. Accordingly, aspects of the subject methods include administering a reduced dose and/or frequency/course of radiation therapy as compared to a therapeutically effective dose and/or frequency/course of radiation therapy (regimen) alone. In some cases, radiation therapy is administered in combination with the subject compounds at a dose and/or frequency effective to reduce radiation damage to the subject (e.g., radiation damage that would be expected to occur at a therapeutically effective dose and/or frequency/course of radiation therapy alone).
In some cases, the method comprises administering an ENPP1 inhibitor to the subject prior to radiation therapy. In some cases, the method comprises administering an ENPP1 inhibitor to the subject after exposing the subject to radiation therapy. In certain instances, the methods comprise sequentially administering radiation therapy, followed by administration of an ENPP1 inhibitor, followed by administration of a checkpoint inhibitor to a subject in need thereof.
Practicality of use
For example, the compounds and methods of the invention as described herein can be used in a variety of applications. Target applications include, but are not limited to: research applications and therapeutic applications. The method of the present invention may be used in a variety of different applications, including any convenient application where it is desirable to inhibit ENPP 1.
The subject compounds and methods find use in a variety of research applications. The subject compounds and methods are useful for optimizing the bioavailability and metabolic stability of a compound.
The subject compounds and methods find use in a variety of therapeutic applications. Targeted therapeutic applications include those in the treatment of cancer. Thus, the subject compounds are useful in treating a variety of different conditions in which inhibition and/or treatment of cancer in a host is desired. For example, the subject compounds and methods can be used to treat solid tumor cancers (e.g., as described herein).
Pharmaceutical composition
The compounds discussed herein may be formulated using any convenient excipients, reagents and methods. Compositions formulated with one or more pharmaceutically acceptable excipients are provided. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been fully described in a number of publications including, for example, A.Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20 th edition, Lippincott, Williams, & Wilkins; pharmaceutical document Forms and Drug Delivery Systems (1999) edited by h.c. ansel et al, 7 th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) edited by A.H.Kibbe et al, 3 rd edition of Amerer. Pharmaceutical Assoc.
Pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents are readily available to the public. In addition, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizing agents, wetting agents and the like are readily available to the public.
In some embodiments, the subject compounds are formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers, in an intensity range of 5mM to 100 mM. In some embodiments, the aqueous buffer includes reagents that provide an isotonic solution. Such agents include, but are not limited to, sodium chloride; and sugars such as mannitol, dextrose, sucrose, and the like. In some embodiments, the aqueous buffer further comprises a non-ionic surfactant such as polysorbate 20 or 80. Optionally, the formulation may further comprise a preservative. Suitable preservatives include, but are not limited to, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the formulation is stored at about 4 ℃. The formulations may also be lyophilized, in which case they typically comprise a cryoprotectant, such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored for long periods of time even at ambient temperature. In some embodiments, the subject compounds are formulated for sustained release.
In some embodiments, the subject compound and a second active agent (e.g., as described herein), such as a small molecule, chemotherapeutic agent, antibody fragment, antibody-drug conjugate, aptamer, or protein, etc., are administered to an individual in a formulation (e.g., in the same formulation or in separate formulations) containing one or more pharmaceutically acceptable excipients. In some embodiments, the second active agent is a checkpoint inhibitor, e.g., a cytotoxic T lymphocyte-associated antigen 4(CTLA-4) inhibitor, a programmed death 1(PD-1) inhibitor, or a PD-L1 inhibitor.
In another aspect of the invention, there is provided a pharmaceutical composition comprising or consisting essentially of: the compounds of the present invention, or pharmaceutically acceptable salts, isomers, tautomers or prodrugs thereof, further comprise one or more additional active agents of interest. Any convenient active agent can be used in combination with the subject compounds in the subject methods. In some examples, the additional agent is a checkpoint inhibitor. The subject compounds and checkpoint inhibitors, as well as additional therapeutic agents for combination therapy as described herein, may be administered orally, subcutaneously, intramuscularly, intranasally, parenterally, or by other routes. The subject compound and the second active agent (if present) can be administered by the same route of administration or by different routes of administration. The therapeutic agent may be administered by any suitable means including, but not limited to, for example, oral administration, rectal administration, nasal administration, topical (including transdermal, aerosol, buccal and sublingual) administration, vaginal administration, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, intravesical administration or injection into the affected organ. In some cases, the therapeutic agent may be administered intranasally. In some cases, the therapeutic agent may be administered intratumorally.
In some embodiments, the subject compound and chemotherapeutic agent are administered to the individual in a formulation (e.g., in the same formulation or in separate formulations) with one or more pharmaceutically acceptable excipients. Chemotherapeutic agents include, but are not limited to, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptide compounds may also be used. Suitable cancer chemotherapeutic agents include dolastatin and its active analogs and derivatives; and auristatins and their active analogs and derivatives (e.g., monomethyl auristatin d (mmad), monomethyl auristatin e (mmae), monomethyl auristatin f (mmaf), etc.). See, for example, WO 96/33212, WO 96/14856, and u.s.6,323,315. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623); duocarmycin and its preparation methodActive analogs and derivatives (e.g., including synthetic analogs, KW-2189 and CB 1-TM 1); and benzodiazepinesAnd active analogs and derivatives thereof (e.g., pyrrolobenzodiazepines) (PBD)。
The subject compound and the second chemotherapeutic agent, as well as additional therapeutic agents for use in combination therapy as described herein, may be administered orally, subcutaneously, intramuscularly, parenterally or by other routes. The subject compound and the second chemotherapeutic agent may be administered by the same route of administration or by different routes of administration. The therapeutic agent may be administered by any suitable means including, but not limited to, for example, oral administration, rectal administration, nasal administration, topical (including transdermal, aerosol, buccal and sublingual) administration, vaginal administration, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, intravesical administration or injection into the affected organ.
The subject compounds may be administered in unit dosage form and may be prepared by any method known in the art. Such methods comprise bringing the subject compound into association with a pharmaceutically acceptable carrier or diluent which constitutes one or more accessory ingredients. The pharmaceutically acceptable carrier is selected according to the chosen route of administration and standard pharmaceutical practice. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. The carrier may be a solid or a liquid, and is generally selected based on the type of administration used.
Examples of suitable solid carriers include lactose, sucrose, gelatin, agar and loose powders. Examples of suitable liquid carriers include water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions, and solutions or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules. Such liquid carriers can contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners, and melting agents. Preferred carriers are edible oils such as corn oil or canola oil. Polyethylene glycols, such as PEG, are also good carriers.
Any drug delivery device or system that provides the dosing regimen of the present disclosure may be used. A variety of delivery devices and systems are known to those skilled in the art.
Examples
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments of the present disclosure, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric.
While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to fall within the scope of the appended claims.
Example 1: synthesis of Compounds
The compounds may be synthesized using any convenient method. Methods that may be useful for preparing the compounds of the present disclosure include the exemplary synthetic methods described in examples 1a-1c, as well as those described in PCT application number PCT/US2018/050018 filed by Li et al on 7/9/2018, the disclosure of which is incorporated herein by reference in its entirety. Numerous methods of providing well-known chemical synthesis schemes and conditions useful for the synthesis of the disclosed compoundsGeneral references are also available (see, e.g., Smith and March, March's Advanced Organic Chemistry: Reactions, mechanics, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Inc. Qualitative analytical Analysis, Fourth Edition, New York: Longman, 1978). The reaction can be carried out by Thin Layer Chromatography (TLC), LC/MS and by LC/MS and 1H NMR-characterized reaction products were monitored. The intermediates and the final product can be purified by silica gel chromatography or by HPLC.
Example 1 a: exemplary synthetic scheme for Compound 1
The synthesis of compound 1 (which may be useful in preparing the compounds of the present disclosure) is shown below:
preparation of dimethyl (2- (piperidin-4-yl) ethyl) phosphonate
Sodium hydride (2.16g,54.11mmol) was added carefully to a stirred solution of bis (dimethoxyphosphoryl) methane (11.42g,49.19mmol) in toluene (100mL) at room temperature. The reaction mixture was then placed under a nitrogen atmosphere and a solution of 1-benzylpiperidine-4-carbaldehyde (10g,49.19mmol) in toluene (50mL) was added slowly, maintaining the temperature below 40 ℃. The resulting mixture was stirred at room temperature for 16 hours and then quenched by addition of saturated aqueous ammonium chloride solution. The organic phase was separated, washed with brine and dried (MgSO)4) And evaporated to dryness. Chromatography (120g SiO2(ii) a 5 to 100% gradient EtOAc in hexanes) to give dimethyl (E) - (2- (1-benzylpiperidin-4-yl) vinyl) phosphonate (6.2g, 16%) as a colorless oil.
To a mixture of dimethyl (E) - (2- (1-benzylpiperidin-4-yl) vinyl) phosphonate (3.7g,12.0mmol) in ethanol (40mL) was added Pd/C (1.1g,10.3 mmol). The mixture was placed under an atmosphere of hydrogen and stirred at room temperature for 12 hours, filtered and evaporated to dryness under reduced pressure to give dimethyl (2- (piperidin-4-yl) ethyl) phosphonate (2.7g, 100%) as a colorless oil.
Preparation of dimethyl (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonate
Diisopropylethylamine (0.6g,8.9mmol) was added to a mixture of dimethyl (2- (piperidin-4-yl) ethyl) phosphonate (1.1g,4.9mmol) and 4-chloro-6, 7-dimethoxyquinazoline (1.0g,4.5mmol) in isopropanol (20 mL). After stirring for 3 hours at 90 ℃, the reaction mixture was cooled and evaporated to dryness. Purification on silica gel (5% MeOH in dichloromethane) afforded dimethyl (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonate (755mg, 37%) as an oil.
LC-MS：m/z＝410.25[M+H]+
1H NMR(500MHz,CDCl3)δ8.65(s,1H),7.23(s,1H),7.09(s,1H),4.19(dq,J＝14.0,2.9,2.4Hz,2H),4.02(s,3H),3.99(s,3H),3.77(s,3H),3.75(s,3H),3.05(td,J＝12.8,2.3Hz,2H),1.93–1.77(m,4H),1.67(ddd,J＝14.1,9.5,5.9Hz,3H),1.46(qd,J＝12.2,3.7Hz,2H).
Preparation of dimethyl (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid (compound 1) Prepare for
Bromotrimethylsilane (3.67g,24mmol) was added to a cooled solution of dimethyl (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonate (3.25g,7.94mmol) in chloroform (60mL), which was cooled by an ice bath. The reaction mixture was allowed to warm to room temperature and quenched after 90 minutes by the addition of methanol (20 mL). The mixture was evaporated to dryness under reduced pressure and then solvated in methanol (100 mL). The reaction mixture was concentrated to half volume, filtered to remove the precipitate and evaporated to dryness. The residue was crystallized from dichloromethane, filtered and dried in vacuo to give dimethyl (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid (2.1g, 69%).
LC-MS：m/z＝381.8[M+H]+
1H NMR(500MHz,DMSO-d6)δ8.77(s,1H),7.34(s,1H),7.23(s,1H),4.71(d,J＝13.1Hz,2H),3.99(s,3H),3.97(s,3H),3.48(t,J＝12.7Hz,2H),3.18(s,1H),1.97–1.90(m,2H),1.62–1.43(m,4H),1.40–1.27(m,2H).
Example 1 b: synthesis of Compound 5 (Table 1)
The synthetic scheme set forth below was used to prepare compound 5:
example 1 c: synthesis of Compound 6 (Table 1)
The synthetic scheme set forth below was used to prepare compound 6:
chemical synthesis: unless otherwise indicated, the reactions were carried out under ambient atmosphere. For the thickness of 250mm,Glass backed F254 silica (silica, Quebec City, Canada) was subjected to qualitative TLC analysis. Visualization was by ultraviolet light and exposure to p-anisaldehyde or KMnO4The staining solution is then heated to complete. Make itAll solvents used were of ACS grade Sure-Seal, all other reagents were used as received unless otherwise stated. The synthesis of non-commercial 4-chloroquinazolines and 4-chloro-3-quinolinecarbonitriles and amine building blocks is described in supplementary information. Using a silica gel flash column (SiliaSepTM40-63mm,) Flash chromatography was performed on a Teledyne Isco purification system. HPLC was performed on an Agilent 1260Infinity preparative scale purification system using an Agilent PrepHT Zorbax Eclipse XDB-C18 reverse phase chromatography column (21.2X 250 mm). Using recording on a Bruker AV-500 spectrometer1The H spectra and low resolution mass spectra (ESI-MS) collected on a Shimadzu 20-20ESI LCMS instrument were used for structure determination. Using a recording on a Bruker AV-500 or AV-400 spectrometer 1The H spectra and low resolution mass spectra (ESI-MS) collected on a Shimadzu 20-20ESI LCMS instrument were used for structure determination. Determination of the final Compound purity according to HPLC-MS>95 percent. All final compounds1The H spectrum was consistent with the expected structure.
Synthesis of ureas 4 and 5.
a)DIPEA，i-PrOH.
Preparation of 1- (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) urea 4 (in Table 3 a)
To a solution of 1- (2- (piperidin-4-yl) ethyl) urea 64(173mg,1.01mmol) in isopropanol (5mL) under a nitrogen atmosphere was added 4-chloro-6, 7-dimethoxyquinazoline 63(181mg,0.81mmol) and N, N-diisopropylethylamine (391mg,3.03 mmol). The mixture was stirred at room temperature for 2 hours and then evaporated to dryness under reduced pressure. Purification (preparative HPLC) gave the title compound 4(172mg, 47%) as light yellow crystals.
LCMS：[M+H]+m/z 360.1H NMR(400MHz,DMSO-d6) Δ 8.49(s,1H),7.17(s,1H),7.07(s,1H), 5.92-5.90 (m,1H),5.36(br s,2H), 4.13-4.09 (m,2H),3.90(s,3H),3.88(s,3H), 3.04-2.94 (m,4H), 1.81-1.78 (m,2H), 1.62-1.56 (m,1H) and 1.38-1.33 (m,4H).
Preparation of 1- ((1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) methyl) urea 5 (in Table 3 a)
To a solution of 1- (piperidin-4-ylmethyl) urea 65(155mg,0.97mmol) in isopropanol (10mL) under a nitrogen atmosphere were added 4-chloro-6, 7-dimethoxyquinazoline 63(174mg,0.78mmol) and N, N-diisopropylethylamine (394mg,2.9 mmol). The mixture was stirred at 10 ℃ for 3 hours and then evaporated to dryness under reduced pressure. Chromatography (SiO) 2: 0 to 6% MeOH in dichloromethane) to afford the desired product 5(150mg, 44%) as a white solid.
LCMS：[M+H]+m/z 346.0 1H NMR (400MHz, methanol-d)4) δ 8.44(s,1H),7.14(s,1H),7.12(s,1H), 4.28-4.24 (m,2H),3.96(s,3H),3.94(s,3H), 3.13-3.07 (m,4H), 1.94-1.87 (m,3H) and 1.50-1.41 (m,2H).
Preparation of 3- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) propionic acid 6 (in Table 3 a)
4-chloro-6, 7-dimethoxy-quinazoline 63(3.14g,13.98mmol) and 3- (4-piperidinyl) propionic acid (2.0g,12.72mmol) were suspended in isopropanol (100mL) and stirred at 90 ℃ for 3 hours. Once cooled, the mixture was evaporated to dryness under reduced pressure. Then the residue is reacted with CH2Cl2(20mL) were triturated together to give the title compound 6(1.87g, 42%) as a white solid.
1H NMR (400MHz, methanol-d)4)δ
Preparation of (2- (1- (6, 7-dimethoxyquinolin-4-yl) piperidin-4-yl) ethyl) boronic acid 7 (in Table 3 a)
a)Cp2ZrCl2，PhMe60℃；b)Pd/C，H2MeOH; c) HCI (aq), MeOH/hexanes; (ii) a d)63, DIEA, THF80 ℃.
A solution of tert-butyl 4-ethynylpiperidine-1-carboxylate 66(2.92g,13.95mmol), bis (cyclopentadienyl) zirconium chloride hydride (150mg,0.518mmol) and 4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane 67(1.49g,11.63mmol) in solvent was stirred at 60 ℃ for 16 h, then diluted with ether and evaporated to dryness under reduced pressure. Chromatography (SiO) 2(ii) a 2-5% ethyl acetate in petroleum ether) to yield 68(4.2g, 89%). A mixture of tert-butyl (E) -4- (2- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) vinyl) piperidine-1-carboxylate 68(4.2g,12.46mmol) and palladium on carbon (840mg, 20% w/w) in MeOH (500mL) was placed under a hydrogen atmosphere and stirred at room temperature for 16 h. Then the mixture is passed throughThe pad was filtered and then evaporated to dryness under reduced pressure to give 69(4.2g, 92%). 1M aqueous HCl (4mL) was added to a cooled (0 ℃ C.) mixture of 73(460mg,1.36mmol) in MeOH/hexane (5mL/5 mL). The mixture was warmed to room temperature and stirred for 3 hours, then evaporated to dryness under reduced pressure to give (2- (piperidin-4-yl) ethyl) boronic acid 70(180mg, 68%) as the hydrochloride salt. To a solution of 70(140mg,1.04mmol) in THF (5mL) was added 4-chloro-6, 7-dimethoxyquinazoline 63(180mg,0.935mmol) followed by N, N-diisopropylethylamine (360mg,1.87 mmol). The mixture was stirred at 80 ℃ for 16 hours and then evaporated to dryness under reduced pressure. Purification (preparative HPLC) gave the title compound as a pale yellow solid (105 mg; 37%).
LCMS：[M+H]+m/z 346.3.1H NMR(400MHz,DMSO-d6) δ 8.67(s,1H),7.26(s,1H),7.25(s,1H), 4.62-4.59 (m,2H),3.92(s,3H),3.90(s,3H), 3.42-3.36 (m,4H),2.46(s,1H), 1.88-1.86 (m,2H), 1.29-1.14 (m,3H) and 0.60-0.56 (m,2H).
Preparation of hydroxamic acids 8 and 9
a)63,i-PrOH,100℃；b)NaOH,THF,H20；c)NH2OH.HCI,DIPEA,BOP,THF.
Preparation of 2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) -N-hydroxyacetamide 8 (in Table 3 a)
A mixture of 4-chloro-6, 7-dimethoxyquinazoline 63(600mg,2.68mmol) and ethyl 2- (piperidin-4-yl) acetate 71(504mg,2.95mmol) in i-PrOH (6mL) was stirred in a sealed tube at 100 ℃ for 16 hours. The reaction mixture was then concentrated under reduced pressure, and the residue was purified by silica gel chromatography to give ethyl 2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) acetate (750mg, 77%). 2M NaOH in H2A solution of O (1mL) was added to a mixture of ethyl 2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) acetate (250mg,0.696mmol) in THF (10 mL). The mixture was stirred at room temperature for 16 hours and then quenched by the addition of 1M HCl solution. The organic phase was extracted with ethyl acetate, washed with brine and dried (Na)2SO4) And evaporated to dryness under reduced pressure to give acid 72(200mg, 86%) as a white solid.
To a mixture of acid 72(300mg, 0.906mmol) in THF (10mL) was added NH2OH HCl (76mg, 1.09mmol), DIEA (468mg, 3.63mmol) and (benzotriazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate (BOP) (481mg, 1.09 mmol). The mixture was stirred at room temperature for 16 hours and then diluted with water, extracted with ethyl acetate, washed with brine solution, dried (Na) 2SO4) And evaporated to dryness under reduced pressure. Chromatography (SiO)2Solvent) to afford the title product 8(180mg, 77%) as a white solid. LCMS: [ M + H ]]+m/z 347.10.1H NMR(400MHz，D2O) delta 8.42(s, 1H), 7.13(s, 1H), 7.00(s, 1H), 4.68-4.62(m, 2H), 3.95(s, 3H), 3.91(s, 3H), 3.51-3.45(m, 2H), 2.21-2.15(m, 3H), 1.93-1.0(m, 2H) and 1.45-1.36(m, 2H).
Preparation of 1- (6, 7-dimethoxyquinazolin-4-yl) -N-hydroxypiperidine-4-carboxamide 9 (in Table 3 a).
Synthesized according to the procedure of 8 but using ethyl piperidine-4-carboxylate 73.
LCMS：[M+H]+m/z 333.25.1H NMR(400MHz，D2O) delta 8.39(s, 1H), 7.04(s, 1H), 6.94(s, 1H), 4.62-4.58(m, 2H), 3.91(s, 3H), 3.86(s, 3H), 3.47-3.41(m, 2H), 2.65-2.60(m, 1H), 1.97-1.94(m, 2H) and 1.82-1.77(m, 2H).
For the general procedure of compounds 10, 11, 12, 13 and 16 (in table 3 a).
a)i-PrOH，100℃；b)Pyridine compound，PSCl3Then H2O; or c)Pyridine compound，POCl3，Then theH2O；d)PPh3，I2，Imidazole，CH2Cl2；e)PO(OBn)2，
DBU，MeCN；f)Pd/C，H2，MeOH.
Preparation of 2- (1- (6, 7-dimethoxyquinolin-4-yl) piperidin-4-yl) ethan-1-ol 77
A mixture of 4-chloro-6, 7-dimethoxyquinazoline 63(1.0g, 4.46mmol) and piperidin-4-ylethanol 79(633mg, 4.91mmol) in isopropanol (10mL) was stirred in a sealed tube at 100 ℃ for 16 hours. After cooling, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (SiO) 2(ii) a EtOAc in petroleum ether) to give 2- (1- (6, 7-dimethoxyquinolin-4-yl) piperidin-4-yl) ethan-1-ol 75(1.3g, 91%).
Preparation of (1- (6, 7-dimethoxyquinolin-4-yl) piperidin-4-yl) methanol 78
A mixture of 4-chloro-6, 7-dimethoxyquinazoline 63(900mg,4.02mmol) and piperidin-4-ylmethanol 76(508mg,4.42mmol) in i-PrOH (10mL) was stirred in a sealed tube at 100 ℃ for 16 hours. After cooling, the reaction mixture was evaporated to dryness under reduced pressure. By chromatography (SiO)2(ii) a 10 to 80% ethyl acetate in petroleum ether) to give (1- (6, 7-dimethoxy)Quinolin-4-yl) piperidin-4-yl) methanol 78(1g, 82%).
Preparation of 2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl dihydrogen phosphate 10 (in Table 3 a)
2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethan-1-ol 77(340mg,1.07mmol) was dissolved in 10mL anhydrous pyridine, which was then cooled to-15 ℃ and stirred for 10 min. In N2Dropwise adding POCl under atmosphere3(821mg,5.4mmol), the reaction temperature was slowly raised to 0 ℃ and then stirred for a further 30 minutes. The mixture was poured into sodium bicarbonate solution (800mg in 250mL water) at 0 ℃. The desired compound was extracted with dichloromethane. The organic phase was concentrated and purified by preparative HPLC to give 2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl dihydrogen phosphate 10(52mg, 12%) as a white solid.
LCMS：[M+H]+m/z 398.1H NMR(400MHz,DMSO-d6) δ 8.54(s,1H),7.17(s,1H),7.15(s,1H), 4.28-4.16 (m,2H),3.93(s,8H), 3.13-3.04 (m,2H), 1.90-1.80 (m,2H),1.75(s,1H),1.59(d, J ═ 6.4Hz,2H) and 1.44-1.32 (m,2H).
Preparation of dihydro- (1- (6, 7-dimethoxyquinolin-4-yl) piperidin-4-yl) methyl phosphate 11 (in Table 3 a)
(1- (6, 7-Dimethoxyquinolin-4-yl) piperidin-4-yl) methanol 78(100mg,0.33mmol) was dissolved in anhydrous pyridine (3mL), which was then cooled to-15 ℃ and stirred for 10 minutes. Dropping POCl in nitrogen atmosphere3(253mg,1.65 mmol). The reaction temperature was slowly raised to 0 ℃ and then stirred for another 30 minutes. The mixture was poured into NaHCO at 0 deg.C3Aqueous solution (160mg in 50mL water). The desired compound was extracted with dichloromethane and then evaporated to dryness under reduced pressure. Purification by preparative HPLC after lyophilization afforded (1- (6, 7-dimethoxyquinolin-4-yl) piperidin-4-yl) methyl phosphate 11(70mg, 55%) as a white powder. LCMS: [ M + H ]]+m/z 384.20.1H NMR(400MHz,DMSO-d6)δ8.74(d,J＝1.7Hz,1H),7.31(s,1H),7.20(s,1H),4.66(d,J＝13.0Hz,1H),3.97(m,J＝12.6,1.6Hz,8H),3.76(t,J＝6.6Hz,3H),2.19–2.00(m,1H),1.92(d,J＝13.5Hz,2H),1.45(dd,J＝14.2,10.7Hz,1H).
Preparation of O- (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) O, O-dihydrophosphorothioate 12 (in Table 3 a)
To a solution of 2- (1- (6, 7-dimethoxyquinolin-4-yl) piperidin-4-yl) ethan-1-ol 77(150mg,0.473mmol) in anhydrous pyridine (5mL) at-15 deg.C was added P (S) Cl 3(477mg,2.84 mmol). After stirring at 0 ℃ for 0.5 h, the mixture is poured into NaHCO3(238mg,2.84mmol) of H2O (50mL) solution. The mixture was stirred at 0 ℃ for 2 hours. The progress of the reaction mixture was monitored by LCMS. The mixture was then concentrated under reduced pressure, and the residue was purified by preparative HPLC to give compound 12(16mg, 8%) as a pale yellow solid. LCMS: [ M + H ]]+m/z414.05.1H NMR(400MHz,DMSO-d6)δ8.62(s,1H),7.19(d,J＝7.7Hz,2H),4.45(d,J＝12.3Hz,2H),3.91(d,J＝11.3Hz,10H),1.86(d,J＝12.2Hz,3H),1.56(d,J＝6.4Hz,2H),1.34(d,J＝10.7Hz,2H).
Preparation of O- ((1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) methyl) O, O-dihydrophosphorothioate 13 (in Table 3 a)
To a solution of (1- (6, 7-dimethoxyquinolin-4-yl) piperidin-4-yl) methanol 78(100mg,0.330mmol) in anhydrous pyridine (5mL) at-15 deg.C was added P (S) Cl3(280mg,1.98 mmol). After stirring at 0 ℃ for 0.5 h, the mixture is poured into NaHCO3(116mg,1.98mmol) of H2O (50mL) solution. The mixture was stirred at 0 ℃ for 2 hours. The mixture was evaporated to dryness under reduced pressure and the residue was purified by preparative HPLC to give compound 13(10mg, 7.6%) as a yellow solid. LCMS: [ M + H ]]+m/z 400.15.1H NMR(400MHz,DMSO-d6)δ8.54(s,1H),7.18(s,1H),7.11(s,1H),4.25(d,J＝13.4Hz,2H),3.89(d,J＝9.1Hz,6H),3.76(s,2H),3.10(d,J＝11.8Hz,3H),1.94(s,1H),1.81(d,J＝12.7Hz,2H),1.39(d,J＝11.4Hz,1H).
Preparation of ((1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) methyl) phosphonic acid 16
Mixing PPh3(3.39g,15mmol) and imidazole (1.02g,15mmol) in anhydrous CH2Cl2(40mL) was stirred at 0 ℃ for 10 minutes, and then I was added 2(3.8g,15 mmol). The crude reaction mixture was placed under a nitrogen atmosphere and stirred for a further 10 minutes and then (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) methanol 78(3.03g,10mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction is carried out by adding Na2S2O3The aqueous solution is terminated. The crude mixture is treated with CH2Cl2Extraction, washing with water, brine and drying (Na)2SO4) And evaporated to dryness under reduced pressure. Recrystallization from methanol gave 4- (4- (iodomethyl) piperidin-1-yl) -6, 7-dimethoxyquinazoline (2.28g, 56%) as a pale yellow solid. LCMS: [ M + H ]]+m/z 414.3.1H NMR(400MHz,CDCl3)δ8.63(d,J＝1.3Hz,1H),7.28(s,1H),7.07(s,1H),4.23(s,2H),4.00(s,6H),3.19(d,J＝6.5Hz,2H),3.08(s,2H),2.11–2.00(m,2H),1.82(s,1H),1.49(s,2H),1.29–1.20(m,1H).
1, 8-diazabicyclo (5.4.0) undec-7-ene (DBU) (9.2g,60.5mmol) was added to a cooled (0 ℃ C.) solution of bis (benzyloxy) (oxo) -lambda 4-phosphine (9.5g,36.3mmol) in anhydrous MeCN (40 mL). After 10 min, 4- (4- (iodomethyl) piperidin-1-yl) -6, 7-dimethoxyquinazoline (5.0g,12.1mmol) was added. The resulting mixture was stirred overnight and then evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate, washed with water, brine and dried (MgSO)4) And evaporated to dryness under reduced pressure. By FCC [ CH2Cl2：MeOH(50：1)]Purification gave dibenzyl ((1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) methyl) phosphonate (1.1g, 18%) as a colorless viscous oil. LCMS: [ M + H ] ]+m/z 548.20.1H NMR(400MHz,CDCl3)δ8.57(s,1H),7.89(s,1H),7.39–7.33(m,10H),6.99(s,1H),5.08(m,3H),4.96(m,2H),4.64(d,J＝13.5Hz,2H),4.09(s,3H),3.93(s,3H),3.27(d,J＝12.9Hz,2H),2.05(d,J＝13.9Hz,5H),1.76(m,4H),1.42(d,J＝12.5Hz,2H).
A mixture containing dibenzyl ((1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) methyl) phosphonate (660mg,1.2mmol) and Pd/C (132mg, 20% w/w) in MeOH (20mL) was placed in H2Under an atmosphere, and stirred at room temperature. After 4 hours, the crude product is washedThe mixture is passed throughThe pad was filtered and the filtrate was evaporated to dryness under reduced pressure. Purification by preparative HPLC gave ((1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) methyl) phosphonic acid 16(125mg, 28%) as a pale yellow solid. LCMS: [ M + H ]]+m/z 368.10.1H NMR(400MHz,DMSO-d6)δ8.72(s,1H),7.29(s,2H),4.60(d,J＝12.8Hz,2H),3.95(d,J＝11.2Hz,6H),3.46(s,2H),2.09(s,3H),1.61(s,2H),1.42(s,2H).
Preparation of (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) propyl) phosphonic acid 14 (in Table 3 a)
a)PPh3，I2Imidazole, CH2Cl2；b)P(O)H(OEt)2，Cs2CO3，DMF；c)TFA，CH2Cl2；d)63，DIPEA，
CH2Cl2；e)TMSBr,MeCN,60℃
Iodine (1.35g,5.34mmol) was added to PPh3(1.4g,5.34mmol) and imidazole (0.36g,5.34mmol) in CH2Cl2(20mL) in solution. The mixture was stirred at room temperature for 0.5 h, and then 79(1.0g,4.11mmol) of CH was added dropwise2Cl2(5mL) of the solution. The reaction mixture was stirred at room temperature for 4 hours, and then saturated Na was used2SO3Stopping the solution with CH2Cl2And (4) extracting. The organic phase was washed with water, brine and dried (Na)2SO4) And evaporated to dryness under reduced pressure. Chromatography (SiO)2(ii) a 5% EtOAc in petroleum ether) to give tert-butyl 4- (3-iodopropyl) piperidine-1-carboxylate (1.0g, 68% yield) as a pale yellow oil.
To a mixture of tert-butyl 4- (3-iodopropyl) piperidine-1-carboxylate (1.0g,2.83mmol) in DMF (50mL) was added diethyl phosphonate (0.58g,4.24mmol) and Cs2CO3(1.84g,5.66mmol). The reaction mixture was stirred at room temperature under nitrogen atmosphere overnight and then quenched by the addition of water. The organic phase was washed with water, brine and dried (Na)2SO4) And evaporated to dryness under reduced pressure. Chromatography (SiO)2(ii) a 20% EtOAc in petroleum ether) to give 80(0.78g, 76%) as a light yellow oil. LCMS: [ M + H ]]+m/z 364.30.
To 80(0.78g,2.14mmol) of CH2Cl2To the solution (8mL) was added TFA (1.5mL,21.4 mmol). The mixture was stirred at room temperature for 4 hours and then evaporated to dryness under reduced pressure to give crude 81 as an oil which was used in the next step without further purification. LCMS: [ M + H ]]+m/z 264.25。
DIPEA (1.37g,10.63mmol) was added to diethyl phosphonate (597mg,2.65mmol) and crude 81 in CH2Cl2(10 mL). The mixture was stirred at room temperature overnight and then saturated NH4Stopping with aqueous Cl solution and using CH2Cl2And (4) extracting. The organic phase was washed with water, brine and dried (Na)2SO4) And evaporated to dryness under reduced pressure. Chromatography (SiO)2CH of 5% MeOH2Cl2Solution) to give the intermediate diethyl phosphonate (0.5g, 39%) as a yellow oil. It was solvated in MeCN (10mL) and TMSBr (1.46mL,11.07mmol) was added. The resulting mixture was stirred at 60 ℃ for 6 hours and then cooled to room temperature and evaporated to dryness under reduced pressure. Chromatography (preparative HPLC) afforded 14 as a white solid (220mg, 50%). LCMS: [ M + H ] ]+m/z396.20.1H NMR (400MHz, methanol-d)4) δ 8.51(s,1H),7.33(s,1H),7.14(s,1H),4.02(s,3H),3.97(s,3H),3.49(t, J ═ 12Hz,2H), 2.00-1.97 (m,3H), 1.75-1.66 (m,5H) and 1.45-1.37 (m,5H).
Preparation of (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) thiophosphine O, O-acid 17
(2- (piperidine-4) under stirring-yl) ethyl) thiophosphonic acid O, O-diethyl ester (400mg,1.51mmol) and DIPEA (927mg,7.19mmol) in DMSO (10mL) was added 4-chloro-6, 7-dimethoxy-quinazoline 66(403mg,1.80 mmol). The reaction mixture was placed under a nitrogen atmosphere and then stirred at 80 ℃ for 16 hours. The reaction mixture was cooled to room temperature, diluted with water, and extracted with ethyl acetate. The organic phase was dried (Na)2SO4) And evaporated to dryness under reduced pressure. Purification (SiO)20-100% EtOAc in hexane) to give (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) thiophosphonic acid O, O-diethyl ester (380mg, 46%). A stirred solution of (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) thiophosphonic acid O, O-diethyl ester (45mg,0.099mmol) in TMSI (7mL) was stirred at 60 ℃ for 16 hours and then cooled to room temperature. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was dried (Na) 2SO4) And then evaporated to dryness under reduced pressure. Purification by preparative HPLC gave (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) thiophosphine O, O-acid 17(13mg, 32%) as a white solid. LCMS: [ M + H ]]+m/z 396.25.1H NMR(400MHz,DMSO-d6)δ8.51(s,1H),7.33(s,1H),7.16(s,1H),4.02(s,3H),3.97(s,3H),3.58(d,J＝10.4Hz,3H),3.48(t,J＝12.0Hz,2H),2.00(d,J＝11.7Hz,2H),1.81(s,1H),1.64(d,J＝17.9Hz,2H),1.61–1.51(m,2H),1.45–1.32(m,2H).
(2- (4- (6, 7-Dimethoxyquinazolin-4-yl) piperidin-1-yl) ethyl) phosphonic acid 18 (in Table 3 a)
LCMS：[M+H]+m/z 382.251H NMR(400MHz,D2O) delta 8.65(s,1H),6.93(s,1H),6.76(s,1H),3.86(s,3H),3.84(s,3H), 3.30-3.22 (m,1H), 3.18-3.15 (m,2H), 2.73-2.66 (m,2H), 2.37-2.32 (m,2H) and 1.79-1.63 (m,6H).
(2- (4- (6, 7-dimethoxyquinazolin-4-yl) piperazin-1-yl) ethyl) phosphonic acid hydrobromide 19 (in Table 3 a).
a)iPrOH,Δ；b)H2O,50℃；c)TMSBr,CHCIs，DMF
The mixture containing pyrazine 82 and compound 63 in propan-2-ol was heated to reflux for 30 minutes and then cooled to room temperature. The reaction mixture was quenched with water and extracted into chloroform. The organic phase was separated, washed with water, brine and dried (Na)2SO4) And evaporated to dryness under reduced pressure. Recrystallization from ether gave 83(1.65g, 84%) as a white solid. Piperazine 83(0.31g,1.1mmol) was dissolved in water (20mL) and phosphonic acid vinyl ester 84(0.19g,1.2mmol) was added. The resulting mixture was heated at 50 ℃ for 1 hour, and then cooled to room temperature. Extracting with chloroform, and drying (Na)2SO4) And evaporated to dryness under reduced pressure. Chromatography (SiO) 212g of a mixture; CH of 15% MeOH2Cl2Solution) followed by recrystallization from diethyl ether gave ethyl ester 85(0.23 g; 49%) as a white solid. LCMS: [ M + H ]]+m/z 410.101H NMR (500MHz, chloroform-d) δ 8.67(s,1H),7.25(s,1H),7.09(s,1H),4.01(d, J ═ 18.8Hz,6H),3.77(d, J ═ 10.9Hz,6H),3.68(t, J ═ 4.9Hz,4H),3.49(s,2H), 2.81-2.66 (m,6H), 2.11-2.00 (m, 2H). Trimethylsilyl bromide (198mg,1.3mmol) was added to 4- [4- (2-diethoxyphosphorylethyl) piperazin-1-yl]-6, 7-dimethoxy-quinazoline 85(600mg,3.8mmol) in chloroform (20mL) and DMF (5 mL). The resulting solution was stirred at room temperature for 3 hours and then quenched by the addition of methanol. The mixture was evaporated to dryness under reduced pressure and recrystallized from methanol-ether to give the desired product 19(0.23g, 89%) as HBr salt. LCMS: [ M + H ]]+m/z 382.8.1H NMR(500MHz,DMSO-d6)δ8.97(s,1H),7.96(s,1H),7.42(s,1H),7.35(s,1H),4.02(s,3H),4.00(s,3H),3.34(t,J＝8.6Hz,2H),3.17(s,2H),2.90(s,2H),2.74(s,2H),2.20–2.08(m,2H).
Preparation of (4- (6, 7-dimethoxyquinazolin-4-yl) phenethyl) phosphonic acid 20 (in Table 3 a)
a) nBuli, triisopropyl borate, THF; b) pd (PPh)3)4，K2CO3，H2O，THF，65℃；c)PPh3Imidazole, I2, CH2Cl2；d)(BnO)2P(O)H，Cs2CO3，DMF；θ)Pd/C，H2，MeOH.
2.5M n-butyllithium (24mL) was added to a solution of 2- (4-bromophenyl) ethan-1-ol 86(5.0g,24.8mmol) in anhydrous THF (100mL) at-78 deg.C under a nitrogen atmosphere. After stirring for 1 hour, triisopropyl borate (8.6mL) was added to the mixture. The reaction mixture was stirred at room temperature for 1 hour and then quenched by the addition of 2M HCl solution (100mL) and stirred for 1 hour. The mixture was extracted with dichloromethane (3X 100mL) and dried (Na) 2SO4) And evaporated to dryness under reduced pressure. Chromatography (SiO)2(ii) a Dichloromethane: methanol, 1: 0 to 20: 0) work-up gave 87(1.34g, 33%) boronic acid as a pale yellow solid. The material was then dissolved in a solution of THF (30mL) and water (10 mL). 4-chloro-6, 7-dimethoxyquinazoline 63(2.24g,10.0mmol) and potassium carbonate (2.76g,20.0mmol) were added to the solution followed by tetrakis (triphenylphosphine) palladium (0.5g,0.43 mmol). The resulting mixture was stirred at 65 ℃ for 16 h and then diluted with ethyl acetate, washed with brine and dried (Na)2SO4) And evaporated to dryness under reduced pressure. Chromatography (SiO)2: (0 to 10% methanol in dichloromethane) to give 88(1.43g, 60%) as a pale yellow solid.
To a solution of triphenylphosphine (2.36g,9.0mmol) in dichloromethane (24mL) at 0 deg.C was added imidazole (700mg,10.28 mmol). After stirring for 10 minutes, I is added2(2.3g,9.0 mmol). After stirring for a further 10 minutes, a solution of compound 89(1.5g,4.8mmol) in dichloromethane (12mL) was added. The mixture was warmed to room temperature and stirred for 5 hours. The mixture was then diluted with dichloromethane (36mL), washed with brine, and dried (Na)2SO4) And evaporated to dryness under reduced pressure. Chromatography (SiO)2: petroleum ether: acetic acid Ethyl ester 10: 1) work up gave 89(4.0g) as a colorless oil.
Cesium carbonate (1.426g,4.4mmol) was added to a mixture of crude 89(930mg,2.2mmol) and dibenzyl phosphonate (884mg,3.37mmol) in DMF (20 mL). The mixture was placed under a nitrogen atmosphere and stirred at room temperature for 3 hours. Once complete, the reaction mixture was filtered and evaporated to dryness under reduced pressure. Chromatography (C18 column: water: acetonitrile, 1: 0 to 80: 1) followed by lyophilization afforded the dibenzyl intermediate (750mg, 79%) as an off-white solid. Dibenzyl (4- (6, 7-dimethoxyquinazolin-4-yl) phenethyl) phosphonate (230mg,0.41mmol) was dissolved in MeOH (20 mL). Pd/C (46mg, 20% w/w) was added and the mixture was stirred at room temperature under an atmosphere of hydrogen for 24 hours and then passedAnd (5) filtering. Chromatography (preparative HPLC under acidic conditions) afforded compound 20(55.4mg, 36%) as a yellow solid. LCMS: [ M + H ]]+m/z 375.0.1H NMR(400MHz,DMSO-d6)): δ 9.09(s,1H),7.75-7.73(d, J ═ 8.0Hz,2H), 7.45-7.43 (m,2H),7.41(s,1H),7.32(s,1H),4.08(s,1H),3.98(s,3H),3.91(s,1H),3.81(s,3H), 2.89-2.87 (m,3H) and 1.89(m,2H).
Synthesis of (4- ((6, 7-dimethoxyquinolin-4-yl) amino) phenyl) phosphonic acid, sodium salt 21 (in Table 3 a)
A mixture of 4-chloro-6, 7-dimethoxy-quinazoline 67(0.67g,3.0mmol) and diethyl (4-aminophenyl) phosphonate (0.69g,3.0mmol) in iPrOH (10mL) was heated to reflux overnight. The solid precipitate was filtered, washed with EtOAc and dried to give diethyl (4- ((6, 7-dimethoxyquinazolin-4-yl) amino) phenyl) phosphonate (0.92g, 73% yield) as a white solid. The product was dissolved in MeCN (20mL) and trimethylsilyl bromide (2.8mL,22mmol) was added to it. The resulting mixture was stirred at 60 ℃ for 6 hours, cooled, and then evaporated to dryness under reduced pressure. Passing the crude residue throughSaturated NaHCO was added3The aqueous solution (adjusted to pH 8) was terminated. The resulting mixture was purified by preparative HPLC (under neutral conditions) and then lyophilized to give the desired product 21(300mg, 35% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 362.10.1H NMR(400MHz,D2O) δ 7.73(s,1H),7.53(t, J ═ 9.8Hz,2H),7.22(d, J ═ 7.4Hz,2H),6.39(s,1H),6.16(s,1H) and 3.39(s,6H).
Synthesis of (4- ((6, 7-dimethoxyquinolin-4-yl) amino) benzyl) phosphonic acid, sodium salt 22 (in Table 3 a)
A mixture of 4-chloro-6, 7-dimethoxy-quinazoline 67(0.34g,1.5mmol) and diethyl (4-aminobenzyl) phosphonate (0.36g,3.0mmol) in iPrOH (10mL) was heated to reflux overnight. The precipitate was filtered, washed with EtOAc, and evaporated to dryness under reduced pressure, and then dissolved in acetonitrile (20 mL). To this was added trimethylsilyl bromide (0.58mL,4.6 mmol). The mixture was stirred at 60 ℃ for 6 hours. After concentration, the residue was taken up with saturated NaHCO 3The aqueous solution was treated until the solution reached a pH of 8. The mixture was purified by preparative HPLC (neutral) to give 22(104mg, 57%) as an off-white solid. LCMS: [ M + H ]]+m/z 376.10.1H NMR(400MHz,D2O) δ 7.77(s,1H),7.15(s,4H),6.49(s,1H),6.21(s,1H),3.47(s,6H) and 2.70(d, J ═ 19.5Hz,2H).
Sodium (4- (((6, 7-dimethoxyquinolin-4-yl) amino) methyl) phenyl) phosphonate 23 (also referred to as 4 in table 1).
a)iPrOH，Δ；b)Et3N，KOAc，Pd(OAc)2，dppf，(EtO)2PH，THF；c)TMSBr，MeCN，60℃.
4-chloro-6, 7-dimethoxy-quinazoline 63(0.93g,4.14mmol) and (4-bromophenyl) methylamine 90(0.77g,4.14mmol) were placed in iPrOH (10)mL) was heated to reflux overnight. Filtering the solid precipitate; washed with ethyl acetate and evaporated to dryness under reduced pressure to give N- (4-bromobenzyl) -6, 7-dimethoxyquinazolin-4-amine hydrochloride 91(1.5g, 88%) as a white solid. Triethylamine (0.37mL,2.68mmol) was added to KOAc (11mg,0.112mmol), Pd (OAc)2A mixture of (5.5mg,0.025mmol), dppf (27mg,0.049mmol) in THF (10mL) and purged with nitrogen. Triethylamine (0.37mL,2.68mmol) was added. After stirring at 70 ℃ for 15 minutes, a solution of N- (4-bromobenzyl) -6, 7-dimethoxyquinazolin-4-amine hydrochloride (0.5g,1.22mmol) and diethyl phosphonate (0.16g,1.22mmol) in THF (10mL) was added. The reaction was stirred at reflux for 6h, and then partitioned between EtOAc (30mL) and water (20 mL). The organic phase was separated, washed with water, brine and dried (Na) 2SO4) And evaporated to dryness under reduced pressure. By column chromatography (SiO)2(ii) a 50% petroleum ether in ethyl acetate) to give diethyl (4- (((6, 7-dimethoxyquinazolin-4-yl) amino) methyl) phenyl) phosphonate (0.2g, 38%) as a yellow solid.
To a solution of diethyl (4- (((6, 7-dimethoxyquinazolin-4-yl) amino) methyl) phenyl) phosphonate (0.5g,1.16mmol) in MeCN (20mL) was added TMSBr (1.45mL,11.5 mmol). The mixture was stirred at 60 ℃ for 6 hours, cooled to room temperature, and then evaporated under reduced pressure. The residue was taken up with saturated NaHCO3The aqueous solution (pH 9) was stopped and the resulting mixture was purified by preparative HPLC (neutral) to give the title product 23 as an off-white solid (102mg, 22%). LCMS: [ M-H ]]+m/z：374.00.1H NMR(400MHz,D2O)δ8.00(s,1H),7.61(s,2H),7.29(d,J＝7.6Hz,2H),6.70(s,1H),6.55(s,1H),4.62(s,2H),3.75(d,J＝18.2Hz,6H).
General procedure for the Synthesis of dimethyl (2- (piperidin-4-yl) ethyl) phosphonate 92 and diethyl (2- (piperidin-4-yl) ethyl) phosphonate 93
a)NaH，PhMe；b)Pd/C，H2，EtOH.
Sodium hydride (1.1mol. eq) was carefully added to a stirred solution of bis (dimethoxyphosphoryl) methane 92 or bis (diethoxyphosphoryl) methane 93(1mol. eq) in toluene at room temperature. The reaction mixture was then placed under a nitrogen atmosphere and a solution of 1-benzylpiperidine-4-carbaldehyde 94(1mol. eq) in toluene was added slowly, maintaining the temperature below 40 ℃. The resulting mixture was stirred at room temperature for 16 hours and then saturated NH was added 4And stopping with an aqueous solution of Cl. The organic phase was separated, washed with brine and dried (MgSO)4) And evaporated to dryness. Chromatography (120g SiO2(ii) a A gradient of 5 to 100% EtOAc in hexanes) to afford either (E) - (2- (1-benzylpiperidin-4-yl) vinyl) phosphonic acid dimethyl ester or (E) - (2- (1-benzylpiperidin-4-yl) vinyl) phosphonic acid diethyl ester as a colorless oil. To a mixture of dimethyl (E) - (2- (1-benzylpiperidin-4-yl) vinyl) phosphonate or diethyl (E) - (2- (1-benzylpiperidin-4-yl) vinyl) phosphonate (1mol. eq) in ethanol was added a catalytic amount of Pd/C. The mixture was placed under an atmosphere of hydrogen and stirred at room temperature for 12 hours, filtered and evaporated to dryness under reduced pressure to give dimethyl (2- (piperidin-4-yl) ethyl) phosphonate 95 and diethyl (2- (piperidin-4-yl) ethyl) phosphonate 96 as a colorless oil.
General procedure for the Synthesis of dibenzyl (2- (piperidin-4-yl) ethyl) phosphonate 100
a)PPh3，I2Imidazole, CH2Cl2；b)P(O)OBn2，DBU，MeCN；c)TFA，CH2Cl2
Iodine (1.5mol. eq.) was added to PPh3CH of (1.5mol. eq.) and imidazole (1.5mol. eq.)2Cl2In solution. After stirring for 10 minutes, 97(1.0mol. eq.) of CH was added dropwise2Cl2And (3) solution. The mixture was stirred at room temperature for 2 hours byPad filtrationAnd treated with 5% sodium thiosulfate solution. The mixture was extracted with ethyl acetate, washed with brine and dried (Na) 2SO4) And evaporated to dryness under reduced pressure. Chromatography gave 98 as an oil.
DBU (5.0mol. eq.) was added to a solution of compound 98(3.0mol. eq.) in MeCN at 40 ℃. After stirring for 10 minutes, a solution of dibenzyl phosphonate (1.0mol equivalent) in MeCN was added dropwise. After stirring for 2 hours, the reaction mixture was evaporated to dryness under reduced pressure and purified by chromatography to give 99.
A solution of compound 99(1.0mol. eq) in TFA/DCM was stirred at room temperature for 1 hour and then evaporated to dryness under reduced pressure to give 100 as an oil.
General procedure for the Synthesis of (2- (1- (quinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid, (2- (1- (quinolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid, and (2- (1- (isoquinolin-1-yl) piperidin-4-yl) ethyl) phosphonic acid
The method A comprises the following steps:
diisopropylethylamine (2mol. eq) was added to a mixture of dimethyl (2- (piperidin-4-yl) ethyl) phosphonate 95 or diethyl (2- (piperidin-4-yl) ethyl) phosphonate 96(1.1mol. eq) and 4-chloroquinazoline, 4-chloroquinoline or 1-chloroisoquinoline (1mol. eq) in isopropanol (0.1M reaction concentration). After stirring for 3 hours at 90 ℃, the reaction mixture was cooled and evaporated to dryness. Purification on silica gel (5% MeOH in dichloromethane) afforded dimethyl phosphonate or diethyl phosphonate. To a cooled (0 ℃) solution of phosphonate (1mol. eq) in chloroform or dichloromethane (0.5M reaction concentration) was added trimethylsilyl bromide (3mol. eq). The reaction mixture was warmed to room temperature and after 90 minutes was quenched by the addition of methanol. The mixture was evaporated to dryness under reduced pressure and then solvated in methanol. The reaction mixture was concentrated to half volume, filtered to remove the precipitate, and then evaporated to dryness. The residue was crystallized from dichloromethane, filtered and dried under reduced pressure to give the desired hydrobromide salt of phosphonic acid.
The method B comprises the following steps:
diisopropylethylamine (3mol. eq) was added to dimethyl (2- (piperidin-4-yl) ethyl) phosphonate 95 or diethyl (2- (piperidin-4-yl) ethyl) phosphonate96(1.1mol. eq.) and 4-chloroquinazoline, 4-chloroquinoline or 1-chloroisoquinoline (1mol. eq.) in dichloromethane (0.1M reaction concentration). After stirring overnight at room temperature, the reaction mixture was purified by addition of saturated NH4And stopping with an aqueous solution of Cl. The organic phase was separated, washed with water and brine, and dried (Na)2SO4) And evaporated to dryness under reduced pressure. Purification on silica gel (5% MeOH in dichloromethane) afforded dimethyl phosphonate or diethyl phosphonate. To a cooled (0 ℃) solution of dimethyl phosphonate or diethyl phosphonate (7mol. eq) in acetonitrile (0.1M reaction concentration) was added trimethylsilyl bromide (3mol. eq). The reaction mixture was stirred at 60 ℃ for 6 hours, cooled and evaporated to dryness under reduced pressure, and the crude residue was purified by addition of saturated NaHCO3The aqueous solution was stopped (until a pH of 8-9 was observed). The crude residue was purified by preparative HPLC (neutral) to give the sodium salt of phosphonic acid.
The method C comprises the following steps:
diisopropylethylamine (3mol. eq) was added to a mixture of dibenzyl (2- (piperidin-4-yl) ethyl) phosphonate 100(1.1mol. eq) and 4-chloroquinazoline, 4-chloroquinoline or 1-chloroisoquinoline (1mol. eq) in dichloromethane (0.1M reaction concentration). After stirring overnight at room temperature, the reaction mixture was purified by addition of saturated NH 4And stopping with an aqueous solution of Cl. The organic phase was separated, washed with water and brine, and dried (Na)2SO4) And evaporated to dryness under reduced pressure. Purification on silica gel (5% MeOH in dichloromethane) afforded dibenzyl phosphonate. A mixture of dibenzyl phosphonate (1mol. equivalent) and Pd/C in MeOH was placed under a hydrogen atmosphere and stirred at room temperature for 2 hours. Then the mixture is passed throughFiltration and evaporation to dryness under reduced pressure gave the phosphonic acid.
Preparation of (2- (1- (6, 7-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid (or Compound 1)
Prepared according to method a to give 15(2.1g, 69%) as an off-white solid. LCMS: [ M + H ]]+m/z 381.8.1H NMR(500MHz,DMSO-d6)δ8.77(s,1H),7.34(s,1H),7.23(s,1H),4.71(d,J＝13.1Hz,2H),3.99(s,3H),3.97(s,3H),3.48(t,J＝12.7Hz,2H),3.18(s,1H),1.97–1.90(m,2H),1.62–1.43(m,4H),1.40–1.27(m,2H).
Preparation of (4- (((6, 7-dimethoxyquinazolin-4-yl) amino) methyl) benzyl) phosphonic acid 24 (also referred to as 5 in the tables herein)
Prepared according to procedure B. The product was isolated by preparative HPLC (9% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 390.15.1H NMR(400MHz,D2O)δ8.12(s,1H),7.22(s,4H),7.11(s,1H),6.91(s,1H),4.79(s,2H),4.76(s,2H),3.98(s,3H),3.91(s,3H),2.79(s,1H),2.74(s,1H)
Preparation of (2- (1- (6-methoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 25
Prepared according to method a to give 25 (50% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 352.10.1H NMR (400MHz, methanol-d)4) Δ 8.57(s,1H), 7.74-7.73 (m,1H), 7.68-7.66 (m,1H),7.46(d,1H),4.96(br s,2H),3.98 (s,3H),3.57(br s,2H),2.65(s,2H), 2.07-2.04 (m,2H),1.81(m,1H), 1.79-1.75 (m,2H), 1.66-1.63 (m,2H) and 1.46-1.44 (m,2H).
Preparation of (2- (1- (6-hydroxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 26
Prepared according to method C26 (7% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 338.15.1H NMR(400MHz,DMSO-d6) δ 8.48(s,1H),7.65(d, J ═ 8.8Hz,1H),7.32(d, J ═ 8.8Hz,1H),7.18(s,1H), 4.21-4.17 (m,2H), 2.99-2.95 (m,2H), 1.82-1.79 (m,2H), 1.53-1.49 (m,5H) and 1.30-1.19 (m,2H).
Preparation of (2- (1- (7-methoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 27
Prepared according to method a to give 27 (95% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 352.0 1H NMR (500MHz, methanol-d)4) δ 8.55(s,1H),8.10(d, J ═ 10Hz,1H),7.30(dd, J ═ 10Hz and 5Hz,1H),7.10(d, J ═ 5Hz,1H),4.01(s,3H), 3.57-3.48 (m,2H),2.65(s,1H), 2.05-2.02 (m,2H), 1.94-1.90 (m,1H), 1.80-1.74 (m,2H), 1.65-1.60 (m,2H) and 1.46-1.41 (m,2H).
Preparation of (2- (1- (7-ethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 28
Prepared according to method B to give 28 as an off-white solid.
Preparation of (2- (1- (7-hydroxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 29
Prepared according to procedure B to give 29 (4% yield) as a pale yellow solid. LCMS: [ M + H ]]+m/z 338.25.1H NMR(400MHz,DMSO-d6) δ 11.49(s,1H),8.64(s,1H),7.96(d, J ═ 9.2Hz,1H),7.10(dd, J ═ 9.2and 2.2Hz,1H),7.00(d, J ═ 2.2Hz,1H),4.62(br s,2H),3.38(br s,2H),1.87(d, J ═ 12.7Hz,2H),1.72(br s,1H), 1.58-1.38 (m,4H) and 1.28-1.22 (m,2H).
Preparation of (2- (1- (7-aminoquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 30
Prepared according to procedure C to give 30 (32% yield) as a light yellow solid. LCMS: [ M + H ]]+m/z 337.10.1H NMR(400MHz,DMSO-d6) δ 8.35(s,1H),7.62(d, J ═ 8.8Hz,1H),6.81(d, J ═ 8.8Hz,1H),6.61(br s,1H),6.30(br s,2H), 4.26-4.20 (m,2H), 3.10-2.90 (m,2H), 1.79-1.76 (m,2H), 1.60-1.30 (m,5H) and 1.25-1.20 (m,2H).
Preparation of (2- (1- (7-isopropoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 31
Prepared according to method B to give 31 as a light yellow solid. LCMS: [ M + H ]]+m/z 337.10.1H NMR(400MHz,DMSO-d6) δ 8.35(s,1H),7.62(d, J ═ 8.8Hz,1H),6.81(d, J ═ 9.2Hz,1H),6.66(s,1H),6.30(br s,2H), 4.25-4.21 (m,2H), 3.08-2.96 (m,2H), 1.81-1.75 (m,2H), 1.65-1.31 (m,5H) and 1.27-1.18 (m,2H).
Preparation of (2- (1- (8-methoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 32
Prepared according to method B to give 32 (32% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 352.15.1H NMR(400MHz,DMSO-d6) δ 7.92(s,1H), 6.92-6.88 (m,1H),6.80(d, J ═ 7.6Hz,1H),6.71(d, J ═ 8.4Hz,1H), 3.76-3.70 (m,2H),3.71(s,3H),2.72(t, J ═ 12Hz,2H),1.64(d, J ═ 12Hz,2H), 1.51-1.28 (m,5H) and 1.02-0.94 (m,2H).
Preparation of (2- (1- (8-ethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 33 (also referred to in the tables herein as 226)
Prepared according to method B to give 33 as a white solid. LCMS: [ M + H ]]+m/z 366.20.1H NMR(400MHz,DMSO-d6)δ1H NMR(400MHz,D2O)δ8.17(s,1H),7.21–7.09(m,2H),4.13–3.98(m,4H),2.97(t,J＝12.4Hz,2H),1.82(d,J＝13.0Hz,2H),1.56–1.35(m,8H),1.26(q,J＝11.4Hz,2H).
Preparation of (2- (1- (8-isopropoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 34 (in Table 3 a)
Prepared according to method B to give 34 (43% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 1H NMR(400MHz,DMSO-d6)δ1H NMR(400MHz,D2O)δ8.10(s,1H),7.11(d,J＝7.5Hz,2H),7.03(d,J＝7.1Hz,1H),3.94(d,J＝13.0Hz,2H),2.86(t,J＝12.6Hz,2H),1.67(d,J＝13.1Hz,2H),1.40–1.07(m,13H).
Preparation of (2- (1- (8-hydroxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 36 (in Table 3 a)
Prepared according to method B to give 35 as an off-white solid.
LCMS：[M+H]+m/z 1H NMR(400MHz,DMSO-d6)δ1H NMR(400MHz,D2O)
Preparation of (2- (1- (5, 8-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 36
Prepared according to method B to give 36 as an off-white solid. LCMS: [ M + H ]]+m/z 382.15.1H NMR(400MHz,DMSO-d6)δ8.47(s,1H),7.54(d,J＝8.9Hz,1H),7.15(d,J＝8.8Hz,1H),3.95(d,J＝12.0Hz,8H),1.82(s,2H),1.67(s,1H),1.59–1.30(m,6H),1.21(s,2H).
Preparation of (2- (1- (6, 8-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 37
Prepared according to method C to give 37 (15% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 382.151H NMR(400MHz,DMSO-d6) δ 8.54(s,1H),7.11(s,1H),6.86-6.82(m,1H),4.50(d, J ═ 12.4Hz,1H),4.27(m,1H),3.85(m,6H),3.74(m,2H),3.27(m,2H), 1.88-1.85 (m,2H),1.66(m,1H), 1.54-1.48 (m,4H) and 1.29(m,2H).
Preparation of (2- (1- (7, 8-dimethoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 38
Prepared according to method C to give 38 (16% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 382.15 1H NMR(400MHz,DMSO-d6) δ 8.60(s,1H),7.90(d, J ═ 9.6Hz,1H),7.49(d, J ═ 9.2Hz,1H),4.69(m,2H),4.02(s,3H),3.89(s,3H),3.46(m,2H),1.90(d, J ═ 12.8Hz,2H),1.75(m,1H), 1.53-1.49 (m,4H) and 1.31-1.28 (m,2H).
Preparation of (2- (1- (7, 8-dihydro- [1,4] dioxadieno [2,3-g ] quinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 39 (in Table 3 a)
Prepared according to method C to give 39 (7% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 380.15.1H NMR(400MHz,DMSO-d6)δ8.64(s,1H),7.48(s,1H),7.19(s,1H),4.56(d,J＝11.8Hz,2H),4.45(d,J＝3.0Hz,2H),4.38(d,J＝3.3Hz,2H),3.39(s,1H),3.33(s,1H),1.87(d,J＝12.2Hz,2H),1.71(s,1H),1.58–1.38(m,4H),1.26(d,J＝10.2Hz,2H).
Preparation of (2- (1- (5-fluoro-8-methoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 40 (in Table 3 a)
Prepared according to procedure C to give 40 (7% yield) as a light yellow solid. LCMS: [ M + H ]]+m/z 370.10.1H NMR(400MHz,DMSO-d6) δ 8.55(s,1H), 7.44-7.38 (m,2H), 4.28-4.23 (m,2H),3.94(s,3H), 3.28-3.18 (m,2H), 1.82-1.78 (m,2H), 1.70-1.66 (m,1H), 1.49-1.23 (m,4H) and 1.26-1.09 (m,2H).
Preparation of (2- (1- (6-fluoro-8-methoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 41 (in Table 3 a)
Prepared according to method B to give 41 (44% yield) as a white solid. LCMS: [ M + H ]]+m/z 366.15.1H NMR(400MHz,DMSO-d6) δ 8.02(d, J ═ 9.2Hz,1H),7.21(d, J ═ 9.2Hz,1H),7.04(s,1H),3.93(s,3H),2.49(s,3H),1.91-1.88(m,2H),1.74(m,1H),1.53-1.49(m,5H) and 1.29-1.27(m,3H).
Preparation of (2- (1- (6-chloro-8-methoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 42 (in Table 3 a)
Prepared according to method B to give 42 (42% yield) as a light yellow solid. LCMS: [ M + H ]]+m/z 386.10.1H NMR(400MHz,D2O)δ8.03(s,1H),6.91–6.88(m,2H), 3.92-3.89 (m,2H),3.77(s,3H), 2.93-2.87 (m,2H), 1.70-1.67 (m,2H) and 1.41-1.12 (m,7H).
Preparation of (2- (1- (7-chloro-8-methoxyquinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 43 (in Table 3 a)
Prepared according to procedure B to give 43 (50% yield) as a white solid. LCMS: [ M + H ]]+m/z 386.05.1H NMR(400MHz,D2O) delta 8.07(s,1H), 7.10-7.06 (m,2H), 3.95-3.91 (m,2H),3.71(s,3H), 2.96-2.90 (m,2H), 1.71-1.68 (m,2H) and 1.42-1.01 (m,7H).
Preparation of 2- [1- [6, 7-dimethoxy-2- [ (E) -2- (3-pyridyl) ethenyl ] quinazolin-4-yl ] -4-piperidyl ] ethyl-hydroxy-phosphinic acid 44
Prepared according to procedure B to give 44 (49% yield) as a light yellow solid. LCMS: [ M + H ]]+m/z 485.25.1H NMR(400MHz,D2O) δ 8.09(s,1H),7.94(s,1H),7.36(d, J ═ 8Hz,1H),7.06(s,1H),6.74(d, J ═ 16.8Hz 1H),6.58(d, J ═ 3.2Hz,1H),6.40(d, J ═ 3.2Hz,1H),6.24(d, J ═ 16.8Hz,1H), 3.94-3.91 (m,2H),3.84(s,3H),3.67(s,3H), 2.96-2.90 (m,2H),1.96-1.93(m,2H) and 1.56-1.32 (m,7H).
(E) Preparation of (2- (1- (8-methoxy-2- (2- (pyridin-3-yl) vinyl) quinazolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 45
Prepared according to procedure B to give 45 (79% yield) as a yellow solid. LCMS: [ M + H ]]+m/z 455.20.1H NMR (400MHz, methanol-d)4)δ9.07(br s,1H),8.70(br s,1H),8.62–8.60(m,1H),8.31(d,1H),7.89–7.88(m,1H),7.70–7.54(m,4H), 5.20-5.00 (m,2H)4.13(s,3H), 3.58-3.50 (m,2H), 2.07-2.02 (m,2H), 1.88-1.82 (m,1H), 1.78-1.64 (m,4H) and 1.51-1.46 (m,2H).
Preparation of (2- (1- (6, 7-dimethoxyquinolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 46
Prepared according to procedure C to give 46 (22% yield) as a white solid. LCMS: [ M + H ]]+m/z 381.30.1H NMR (400MHz, methanol-d)4) δ 8.35(d, J ═ 6.8Hz,1H), 7.29-7.27 (m,2H),7.11(d, J ═ 6.8Hz,1H), 4.27-4.23 (m,2H),4.03(s,3H),4.02(s,3H), 3.40-3.32 (m,2H), 2.06-2.03 (m,4H) 1.82-1.79 (m,3H) and 1.62-1.48 (m,2H).
Preparation of (2- (1- (3-cyano-6, 7-dimethoxyquinolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 47 (also referred to as 42 in Table 2)
Prepared according to method B to give 47 (47% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 406.20.1H NMR(400MHz,D2O)δ8.00(s,1H),6.62(s,1H),6.40(s,1H),3.75(s,3H),3.66(s,3H),3.18(d,J＝12.3Hz,2H),2.94(t,J＝12.2Hz,2H),1.72(d,J＝12.7Hz,2H),1.43–1.30(m,6H),1.17–1.04(m,2H).
Preparation of (2- (1- (3-cyano-6-methoxyquinolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 48 (also referred to as 44 in Table 2)
Preparation according to method B gave 48 (16% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 376.20.1H NMR(400MHz,D2O)δ8.15(s,1H),7.51(s,1H),7.18(s,1H),6.88(s,1H),3.71(s,3H),3.60–3.51(m,2H),3.15–3.08(m,2H),1.81–174(m,2H) and 1.41-1.15 (m,7H).
Preparation of (2- (1- (3-cyano-7-methoxyquinolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 49 (also referred to as 43 in Table 2)
Prepared according to method B to give 49 (23% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 376.20.1H NMR(400MHz,D2O) δ 7.94(s,1H),7.39(d, J ═ 9.4Hz,1H),6.84 to 6.64(m,2H),3.90(s,3H),3.59(d, J ═ 12.4Hz,2H),3.22(t, J ═ 12Hz,2H),1.89(d, J ═ 12.8Hz,2H),1.62 to 1.45(m,5H) and 1.33 to 1.25(m,2H).
Preparation of (2- (1- (3-cyano-8-methoxyquinolin-4-yl) piperidin-4-yl) ethyl) phosphonic acid 50 (also referred to as 45 in Table 1)
Prepared according to method B to give 50 as an off-white solid. LCMS: [ M + H ]]+m/z376.20.1H NMR(400MHz,D2O) δ 8.03(s,1H), 7.27-7.23 (m,1H), 7.11-7.09 (m,1H), 7.04-7.02 (m,1H),3.90(s,3H),3.43(br d, J ═ 12.4Hz,2H),3.06(br t, J ═ 12Hz,2H),1.80(br d, J ═ 12.8Hz,2H), 1.50-1.47 (m,5H) and 1.31-1.24 (m,2H).
Preparation of (2- (1- (6, 7-Dimethoxyisoquinolin-1-yl) piperidin-4-yl) ethyl) phosphonic acid 51
Prepared according to method B to give 51 (30% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 381.10.1H NMR(400MHz,D2O)δ1H NMR(400MHz,DMSO-d6): δ 7.79(d, J ═ 6.4Hz,1H), 7.51-7.44 (m,2H),7.31(s,1H),3.96(d, J ═ 4.8Hz,6H),3.23(m,4H),1.88(m,2H),1.55(m,2H),1.48(m,1H) and 1.45(m,4H).
Preparation of (2- (1- (4-cyano-6, 7-dimethoxyisoquinolin-1-yl) piperidin-4-yl) ethyl) phosphonic acid 52 (in Table 3 a)
Prepared according to method B to give 52 (50% yield) as an off-white solid. LCMS: [ M + H ]]+m/z 1H NMR(400MHz,D2O)δ
O- ((1- (8-Methoxyquinazolin-4-yl) piperidin-4-yl) methyl) O, O-dihydrothiophosphate 53 (in)
Preparation of table 3 a)
To a solution of (1- (8-methoxyquinazolin-4-yl) piperidin-4-yl) methanol (prepared using the same method as compound 80) (500mg,1.83mmol) in pyridine (5mL) was added dropwise thiophosphoryl chloride (1.6g,9.45mmol) at-15 ℃. After stirring at 0 ℃ for 1 hour, the reaction mixture was added to a solution of sodium bicarbonate (923mg,10.98mmol) in water (20mL) at 0 ℃. The resulting mixture was stirred at 0 ℃ for 2 hours and then evaporated to dryness under reduced pressure. Purification (preparative HPLC) gave 53(83mg, 12%) as a white solid. LCMS: [ M + H ] ]+m/z 354.10 1H NMR(400MHz,,DMSO-d6)δ8.59(s,1H),7.59–7.50(m,2H),7.45(dd,J＝6.5,2.4Hz,1H),4.53(d,J＝12.7Hz,2H),3.97(s,3H),3.80–3.74(m,4H),2.03(s,1H),1.86(d,J＝13.5Hz,2H),1.41(q,J＝11.8Hz,2H).
Preparation of ((1- (8-methoxyquinazolin-4-yl) piperidin-4-yl) oxy) methyl) phosphonic acid 54 (in Table 3 a)
Prepared using the same method as compounds 10 and 11. The mixture was purified (preparative HPLC 0.1% TFA) to give 54 as an off-white solid.
LCMS：[M+H]+m/z 353.3.1H NMR(400MHz,D2O)δ8.40(s,1H),7.54(s,2H),7.40(d,J＝7.0Hz,1H),4.37(s,3H),4.01–3.88(m,9H),3.71(d,J＝9.3Hz,4H),3.63(s,1H),2.11(s,2H),1.81(s,2H).
Preparation of (4- (8-Methoxyquinazolin-4-yl) phenethyl) phosphonic acid 55 (in Table 3 a)
Prepared using the same method as compound 20 as a white solid.
LCMS：[M+H]+m/z 345.10.1H NMR(400MHz,D2O)δ
Preparation of (4- (((8-methoxyquinazolin-4-yl) amino) methyl) phenyl) phosphonic acid 56
Prepared according to the same method as compound 23. LCMS: [ M + H ]]+m/z 346.10.1H NMR(400MHz,D2O) delta 8.20(s,1H), 7.62-7.57 (m,2H), 7.43-7.41 (m,2H), 7.31-7.28 (m,2H), 7.23-7.21 (m,1H) and 2.93(s,3H).
Preparation of (4- (((3-cyano-8-methoxyquinolin-4-yl) amino) methyl) phenyl) phosphonic acid 57 (also referred to as 52 in Table 1)
Prepared according to the same procedure as compound 23 to give 57 as an off-white solid. LCMS: [ M + H ]]+m/z 370.10.1H NMR(400MHz,D2O) delta 8.02(s,1H), 7.48-7.43 (m,2H, 7.36-7.30 (m,2H), 7.15-7.09 (m,3H),4.77(s,2H) and 3.81(s,3H).
Preparation of (4- (((3-cyano-8-methoxyquinolin-4-yl) amino) methyl) benzyl) phosphonic acid 58 (also referred to as 211 in Table 2)
Prepared according to the same method as compound 23 to give 58 as a white solid. LCMS: [ M + H ] ]+m/z 384.15.1H NMR (400MHz, methanol-d)4) δ 8.32(s,1H),7.77(d, J ═ 8.4Hz,1H),7.50(t, J ═ 8.4Hz,1H), 7.35-7.33 (m,2H),7.26(d, J ═ 7.6Hz,1H),7.20(d, J ═ 8.4Hz,2H),5.02(s,2H),3.99(s,3H) and 2.85(d, J ═ 20Hz,2H).
Preparation of (3- (1- (3-cyano-8-methoxyquinolin-4-yl) piperidin-4-yl) propyl) phosphonic acid 59 (also referred to as 210 in Table 2)
Prepared according to the same method as compound 14 to give 59 as a white solid. LCMS: [ M + H ]]+m/z 390.20.1H NMR(400MHz,D2O) δ 8.30(s,1H),7.39(br s,2H),7.19(br s,1H),3.98(s,3H), 3.73-3.70 (m,2H),3.30(t, J ═ 12Hz,2H), 1.92-1.88 (m,2H), 1.70-1.45 (m,3H) and 1.40-1.27 (m,6H).
Preparation of (4- (((3-cyano-8-methoxyquinolin-4-yl) amino) methyl) phenyl) boronic acid 60 (also referred to as 214 in Table 2)
a)Et3N，CH3OCH2CH2OH；b)PdCl2(dppf)，KOAc，B2Pin2，DMSO，80℃；c)HCl，EtOAc
To a solution of compound 101(0.97g,5.0mmol) in 2-methoxyethanol (10mL) was added (4-bromophenyl) methylamine 90(1.74g,10.0mmol) and Et3N (1.51g,15 mmol). The mixture was heated at 100 ℃ overnight, cooled to room temperature, and then evaporated to dryness under reduced pressure. Chromatography (35% EtOAc in petroleum ether) afforded 102(1.5g, 88%),as a white solid. To a solution of compound 102(69mg,0.2mmol) in DMSO (3mL) was added bis (pinacolato) diboron (61.0mg,0.24mmol), potassium acetate (58.8mg,3.0mmol), Pd (dppf) Cl 2(7.4mg,0.05 mmol). The reaction was degassed by purging with nitrogen and then heated at 80 ℃ for 48 hours. The mixture was cooled to room temperature and diluted with ethyl acetate and then passed throughThe pad is filtered. The filtrate was evaporated to dryness under reduced pressure. The residue was dissolved in EtOAc (10mL) and a solution of HCl (4M,0.2mL,4.0mmol) in EtOAc was added. The mixture was stirred at room temperature overnight and then evaporated to dryness under reduced pressure. Chromatography (preparative hplc (tfa)) afforded 60(40.5mg, 65%, two steps) as a white solid. LCMS: [ M + H ]]+m/z 334.15.1H NMR (400MHz, methanol-d)4)δ8.69(s,1H),7.90(d,J＝8.4Hz,1H),7.73(t,J＝8.3Hz,2H),7.61(d,J＝8.1Hz,2H),7.41(dd,J＝17.3,7.8Hz,2H),5.05(s,2H),4.13(s,3H).
Preparation of (2- (1- (3-cyano-8-methoxyquinolin-4-yl) piperidin-4-yl) ethyl) boronic acid 61 (also referred to as 216 in Table 2)
Prepared according to the same procedure as compound 7. Compound 61 was isolated as a yellow solid. LCMS: [ M + H ]]+m/z 340.20.1H NMR (400MHz, methanol-d)4) δ 8.81(s,1H),7.83(d, J ═ 12Hz,1H),7.72(t, J ═ 8Hz,1H),7.60(d, J ═ 8Hz,1H),4.51(d, J ═ 12Hz,2H),3.81(t, J ═ 12Hz,2H),3.31(s,3H),2.66(s,1H), 2.05(br d, J ═ 12Hz,2H), 1.72-1.30 (m,4H) and 0.91-0.85 (m,2H).
Preparation of 4- (((3-cyano-8-methoxyquinolin-4-yl) amino) methyl) -N-hydroxybenzamide 62 (also referred to as 220 in Table 2)
a)CH3OCH2CH2O；b)NaOH，THF/H2O；c)NH2OH.HCl，BOP，DIPEA，THF
A solution of 101(2.0g,8.9mmol) and 103(1.5g, 8.9mmol) in 2-methoxyethanol (40mL) was heated to reflux overnight and then cooled to room temperature. The reaction mixture was evaporated to dryness under reduced pressure and then triturated with EtOAc, filtered and dried to give crude compound 104(1.7g) as a light yellow solid.
To a solution of compound 104(0.5g,1.55mmol) in THF (20mL) was added NaOH (0.17g,4.65mmol, dissolved in 2mL of water). The mixture was heated at 45 ℃ overnight. The cooled solution was concentrated under reduced pressure and the residue was treated with aqueous HCl (2N) until a pH of 5.5 was reached. The resulting precipitate was filtered and dried to give the crude acid intermediate (0.3g, 62% yield) as a pale yellow solid. The crude acid was dissolved in DMF (10mL) and then cooled to 0 ℃ and placed under nitrogen. BOP (0.48g,1.06mmol) and DIPEA (0.50g,3.88mmol) were added followed by HONH2-HCl (0.09g,1.26 mmol). The mixture was stirred at room temperature overnight, quenched with water (50mL), and extracted with EtOAc. The organic phase was washed with water and brine and dried (Na)2SO3) And evaporated to dryness under reduced pressure. Chromatography (5% MeOH in CH)2Cl2Solution) and then subjected to preparative HPLC (H)+0.1% TFA) to give 62(34mg, 10%) as an off-white solid. LCMS: [ M + H ]]+ 1H NMR(400MHz,DMSO-d6)δ11.19(s,1H),9.06(s,1H),8.55(s,1H),8.45(d,J＝8.7Hz,1H),7.72(d,J＝7.4Hz,2H),7.39(dd,J＝4.6,2.9Hz,2H),7.29(dd,J＝12.3,5.0Hz,2H),5.11(s,2H),3.93(s,3H).
Example 2: evaluation of Compound Activity
Selected compounds of tables 1-3 and other derivatives were prepared and evaluated in an ENPP1 activity assay using p-nitrophenol thymidine monophosphate (TMP-pNP) as a substrate. At room temperature, the purified recombinant mouse ENPP1(0.5nM) was diluted 5-fold with TMP-pNP (2. mu.M), ENPP1 inhibitor in 100mM Tris, 150mM NaCl, 2mM CaCl 2、200μM ZnCl2pH 7.5 on enzymeThe reaction is ready for use. The progress of the reaction was monitored for 20 minutes by measuring the absorbance at 400nm of the p-nitrophenol ester produced by the reaction. The slope of product formation was determined, plotted using Graphpad Prism 7.03 and fitted to obtain IC50The value is obtained.
Compounds were also evaluated in ENPP1 enzyme activity assays using cGAMP as a substrate. Methods that can be used to evaluate the subject compounds include those described by Li et al, PCT application number PCT/US2018/050018 filed on 7/9/2018. An exemplary method is set forth below.
Materials:
mouse ENPP 1: according to Kato et al PNAS (2012)109 (42): 16876-8.cGAMP expression and purification: according to Li et al nat. chem. biol. (2014) 10: 1043-8, and purifying. Polyphosphate AMP Phosphotransferase (PAP): the PAP gene (GenBank: AB092983.1) was synthesized (Integrated DNA Technologies) and cloned into the pTB146 vector with His-SUMO C-terminal tag. BL21(DE3) cells transformed with the plasmid were grown and induced overnight at 16 ℃ with 0.75mM IPTG at OD600 ═ 1. Cells were resuspended in buffer containing 50mM Tris pH 7.5, 400mM NaCl, 10mM imidazole, 2mM DTT, protease inhibitor (Roche) and lysed using two freeze-thaw cycles and sonication. All subsequent steps were carried out at 4 ℃. Lysates were cleared by centrifugation at 40,000rcf for 1 hour, and supernatants were incubated with HisPur cobalt resin (Thermo Fisher Scientific) for 2 hours. The resin was washed twice with 30mL buffer containing 50mM Tris pH 7.5, 150mM NaCl and the protein was eluted with 50mM Tris pH 7.5, 150mM NaCl, 600mM imidazole. Anion exchange chromatography (HiTrap Q HP) was performed. Muscle kinase (Millipore Sigma), CellTiterGlo (Promega)
Exemplary procedure for ENPP1 enzyme activity assay:
at room temperature, 3nM mouse ENPP1 was diluted 5-fold serially with 5uM cGAMP and compound in 50mM Tris pH 7.6, 250nM NaCl, 500uM CaCl2And 1uM ZnCl2Was incubated in the buffer (total reaction volume ═ 10mL) for 3 hours, and then the reaction was heat inactivated at 95 ℃ for 10 minutes. AMP degradation products are converted to ATP, which is detected using luciferase. To this end, according to Goueli et al in EP2771480, enzyme mixtures of Polyphosphate AMP Phosphotransferase (PAP) and myokinase are prepared. Briefly, PAP was diluted to 2mg/mL in a buffer containing 50mM Tris, pH 7.5, 0.1% NP-40. The myokinase was diluted to 2KU/mL in a buffer containing 3.2mM ammonium sulfate, pH 6.0, 1mM EDTA and 4mM polyphosphate. Heat-inactivated ENPP1 reaction was reacted with PAP (0.01mg/mL) and myokinase (0.0075U/mL) in the presence of 40mM Tris pH 7.5, 0.05mg/mL Prionix, 5mM MgCl220mM polyphosphate and 0.15g/L phenol red (for pipetting) in buffer for 3 hours (total reaction volume 20 mL). CellTiterGlo (20uL) was added to the reaction and luminescence was measured according to the manufacturer's protocol. After fitting into a function 100/(1+ ([ Compound ]]/IC50)), the data were normalized to 100% enzyme activity (no compound) and 0% enzyme activity (no enzyme).
IC50 values fall within the range indicated by the letters a-C, where a represents IC50 values of less than 50nM, B represents IC50 values between 50nM and 100nM, and C represents IC50 values of greater than 100 nM.
Table 4: ENPP1 enzyme activity. IC50 value: a (<50 nM); b (50 nM-100 nM); c (>100nM).
TABLE 2 Compounds
Example 3: display of extracellular ENPP1 and inhibition of extracellular ENPP1
Referring to fig. 18A to 18C, it was observed that ENPP1 controls extracellular cGAMP levels and that cGAMP levels can be restored by treating cells with an ENPP1 inhibitor (e.g., compound 1).
293T cGAS ENPP1-/-Cells were transfected with the human ENPP1 expression plasmid and cGAMP hydrolase activity was confirmed in whole cell lysates (fig. 18A). 293T cells were purchased from ATCC and transfected with virus to stably express mouse cGAS. 293T mcGAS ENPP1-/-Is produced by viral transfection of CRISPR sgRNA (5 'CACCGCTGGTTCTATGCACGTCTCC-3') (SEQ ID NO: 1) targeting human ENPP1In (1). 293T mcGAS ENPP1-/-Cells were plated in tissue culture-treated plates coated with PurCol (advanced BioMatrix) in DMEM (Corning Cellgro) supplemented with 10% FBS (Atlanta biologics) (v/v) and 100U/mL penicillin-streptomycin (ThermoFisher). 12-24 hours after plating, cells were transfected with Fugene 6(Promega) according to the manufacturer's instructions plus pcDNA3 plasmid DNA (empty or containing human ENPP1) at the indicated concentration. 24 hours after transfection, cells were lysed to analyze ENPP1 expression by western blotting (using antibodies rabbit anti-ENPP 1(L520,1:1000) and mouse anti-tubulin (DM1A,1:2,000), Cell Signaling Technologies). By reaction in 10mM Tris,150mM NaCl,1.5mM MgCl 21% NP-40, cleaved 1X10 in pH 9.06Individual cells to produce whole cell lysates. Will be provided with32P-cGAMP (5 μ M) was incubated with whole cell lysate and degradation was monitored as described in example 2 (fig. 18A) above.
In intact cells, ENPP1 expression depleted extracellular cGAMP, but did not affect intracellular cGAMP concentrations (fig. 18B). In the transfection of 293T mcGAS ENPP1 with pcDNA3 (empty or human ENPP1)-/-After 24 hours, the medium was removed and replaced with serum-free DMEM supplemented with 1% insulin-transferrin-selenium-sodium pyruvate (ThermoFisher) and 100U/mL penicillin-streptomycin. 12-24 hours after medium change, the medium was removed and the cells were washed off the plate with cold PBS. Both the media and cells were centrifuged at 1000rcf for 10 min at 4 ℃ and prepared for cGAMP concentration measurement by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cells were supplemented with 500nM cyclic GMP-one as internal standard at 30 to 100. mu.L13C10,15N5-AMP in 50:50 acetonitrile: water and centrifuged at 15,000rcf for 20 min at 4 ℃ to remove insoluble fractions. Removal of 500nM cyclic GMP-supplemented as internal standard13C10,15N5-AMP and 20% formic acid medium. Samples were analyzed for cGAMP, ATP, and GTP content on Shimadzu HPLC (San Francisco, CA) with an autosampler set at 4 ℃ and linked to AB Sciex 4000QTRAP (Foster City, CA). A volume of 10. mu.L was injected onto a Biobasic AX LC column, 5 μm,50X 3mm (thermo scientific). Mobile phase of Acetonitrile solution (B) containing 100mM ammonium carbonate (A) and 0.1% formic acid. The initial condition was 90% B, and was maintained for 0.5 min. The mobile phase was ramped up to 30% a from 0.5min to 2.0min, held at 30% a from 2.0min to 3.5min, ramped up to 90% B from 3.5min to 3.6min, and maintained at 90% B from 3.6min to 5 min. The flow rate was set to 0.6 mL/min. The mass spectrometer was operated in electrode spray positive ion mode with the source temperature set at 500 ℃. Declustering and collision-induced dissociation was achieved using nitrogen. Declustering potential and collision energy were optimized by direct infusion of standards. For each molecule, MRM transition(s) (m/z), DP (V) and CE (V) are as follows: ATP (508)>136,341,55)，GTP(524>152,236,43)，cGAMP(675>136,121,97；675>312,121,59；675>152,121,73), internal standard cyclic GMP-13C10,15N5-AMP(690>146,111,101；690>152,111,45；690>327,111,47), extraction standard circularity13C10,15N5-GMP-13C10,15N5-AMP(705>156,66,93；705>162,66,73)。
Inhibition of ENPP1 blocked the degradation of extracellular cGAMP (fig. 18C). The same experiment was performed as above, this time also including 50 μ M of ENPP1 inhibitor (compound 1) when the medium was changed. In the presence of the inhibitor, the extracellular cGAMP concentration in the medium was restored to the previous level.
FIG. 18A shows transfection with empty vector and vector containing human ENPP1 and analysis of ENPP1 protein expression using Western blotting (Top) and ENPP1 using Thin Layer Chromatography (TLC) after 24 hours 32293T cGAS ENPP1 with P-cGAMP hydrolytic Activity (bottom)-/-A cell. FIG. 18B shows intracellular and extracellular cGAMP concentrations using LC-MS/MS. BQL ═ below the limit of quantitation. Mean ± SEM (n ═ 2). P ═ 0.005 (student's t test). FIG. 18C shows 293T cGAS ENPP1 transfected with an empty vector or a vector containing human ENPP1 in the presence or absence of 50. mu.M Compound 1-/-Intracellular and extracellular cGAMP concentrations of cells. BQL ═ below the limit of quantitation. Mean ± SEM (n ═ 2). P ═ 0.0013 (student's t test).
Example 4: ENPP1 inhibition increases cG in primary CD14+ monocytesAMP activation
Using an ENPP1 inhibitor (Compound 1), a test was performed by 293T cGAS ENPP1Is low inWhether cGAMP exported by cell lines can be expressed by human CD14+Monocyte Antigen Presenting Cells (APC) were detected (fig. 19A). 293T cGAS ENPP1Is low inCells were transfected with pcDNA (empty or pcDNA containing human ENPP 1). Primary human Peripheral Blood Mononuclear Cells (PBMCs) were isolated by subjecting enriched buffy coats from whole blood to a Percoll density gradient. Using CD14+MicroBeads (Miltenyi) isolation of CD14+A monocyte. Culturing of CD14 in RMPI supplemented with 2% human serum and 100U/mL penicillin-streptomycin +A monocyte. In the transfection of 293T cGAS ENPP1Is low in8 hours after the cells, the medium was changed to RMPI supplemented with 2% human serum and 100U/mL penicillin-streptomycin, with or without the addition of the exemplary ENPP1 inhibitor Compound 1. 24 hours after medium replacement, 293T cGAS ENPP1Is low inCell supernatants were transferred to CD14+Monocytes (fig. 19A). 24-26 hours after transfer of the supernatant, total RNA was extracted using Trizol (Thermo Fisher Scientific) and reverse transcribed with Maxima H Minus reverse transcriptase (Thermo Fisher Scientific). Real-time RT-PCR was performed on a 7900HT fast real-time PCR system (Applied Biosystems) in duplicate with an AccuPower 2X Greenstar qPCR Master Mix (Bioneer). For each sample, the data were normalized to CD14 expression. Fold induction was calculated using Δ Δ Ct. Primers for human IFNB 1: fwd (5'-AAACTCATGAGCAGTCTGCA-3') (SEQ ID NO:2), rev (5'-AGGAGATCTTCAGTTTCGGAGG-3') (SEQ ID NO: 3); primers for human CD 14: fwd (5'-GCCTTCCGTGTCCCCACTGC-3') (SEQ ID NO:4), rev (5'-TGAGGGGGCCCTCGACG-3') (SEQ ID NO: 5).
293T cGAS ENPP1 from cGAS expressingIs low inCell supernatants induced CD14+ IFNB1 expression, while supernatants from cGAS-null 293T cells failed, suggesting that extracellular cGAMP exported by cancer cells can be encoded by CD14 +Cells were detected as signaling factors (fig. 19B). ENPP1 at 293T cGAS ENPP1Is low inTransient overexpression on cells leads to extracellular cGAMP degradation and CD14+IFNB1 expression decreased, but addition of compound 1 rescued extracellular cGAMP levels and induced CD14+IFNB1 expression (fig. 19B).
Referring to fig. 19A, a schematic of a supernatant transfer experiment is shown. FIG. 19B shows cGAS-free 293T cells or 293T cGAS ENPP1 transfected with DNA and incubated in the presence or absence of Compound 1Is low inA cell. Supernatants from these cells were transferred to primary CD14+Human PBMC. IFNB1 mRNA levels were normalized to CD14 and compared to untreated CD14+Cells were counted for fold induction. Mean ± SEM (n ═ 2). P<0.05,***P<0.001 (one-way ANOVA).
Example 5: ENPP1 inhibition synergizes with Ionizing Radiation (IR) treatment to increase tumor-associated dendritic cells Cell
Whether cancer cell lines export cGAMP and whether Ionizing Radiation (IR) affects the levels of extracellular cGAMP produced has been tested. Ionizing Radiation (IR) has been shown to increase cytoplasmic DNA in tumor cells and activate cGAS-dependent IFN- β production in tumor cells (Bakhoum et al nat. commun. (2015)6: 1-10; and vanpoule nat. commun. (2017)8: 15618). At 24 hours after plating, 4T1 cells were treated with 20Gy IR using a cesium source and the media was changed and supplemented with 50uM of ENPP1 inhibitor (compound 1) to inhibit ENPP1 present in the cell culture. The medium was harvested at the indicated times, centrifuged at 1000x g to remove residual cells, acidified with 0.5% acetic acid, and supplemented with cyclic-acetate as extraction standard 13C10,15 5-GMP-13C10,15N5AMP (appropriate amount at final concentration of 2. mu.M in 100. mu.L). The media was applied to a HyperSep Aminopropyl SPE column (ThermoFisher Scientific) to enrich for cGAMP as previously described (Gao et al, Proc. Natl. Acad. Sci. U.S.A. (2015)112: E5699-705). The eluate was evaporated to dryness and redissolved in 50:50 acetonitrile: water supplemented with 500nM internal standard. The media was submitted for mass spectrometric quantification of cGAMP.
Continuous cGAMP output was detected in 4T1 cells within 48 hours. At 48 hours, cells treated with IR had significantly higher extracellular cGAMP levels compared to untreated cells.
Next, the effect of IR in combination with the exemplary ENPP1 inhibitor compound 1 on the number of tumor-associated dendritic cells in the mouse 4T1 tumor model was investigated (fig. 20B). In 7 to 9 week old female Balb/c mice (Jackson Laboratories), suspend in 50L PBS 1x 106A 4T 1-luciferase tumor cell was inoculated into the mammary fat pad. Two days after injection, tumors were irradiated with 20Gy using a 225kVp cabinet X-ray irradiator (IC 250, Kimtron inc., CT) filtered with 0.5mm Cu. Anesthetized animals were shielded with a 3.2mm lead shield with a 15x 20mm hole for tumor placement. Mice were injected intratumorally with 100 μ L of 1mM compound 1 in PBS or PBS alone. The next day, tumors were extracted and incubated in RPMI + 10% FBS containing 20. mu.g/mL DNAse type IV (Sigma-Aldrich) and 1mg/mL collagenase from Clostridium histolyticum (Sigma-Aldrich) for 30min at 37 ℃. Tumors were passed through a 100 μm cell filter (Sigma-Aldrich) and red blood cell lysis buffer (155mM NH) was used at room temperature 4Cl,12mM NaHCO30.1mM EDTA) to lyse the red blood cells for 5 minutes. Cells were stained with live/dead immortable near-infrared dead cell staining kit (Thermo Fisher Scientific), Fc blocked using TruStain fcX for 10min, and then antibody stained with CD11c, CD45, and I-a/I-E (all from Biolegend). Cells were analyzed using SH800S cell sorter (Sony) or LSR II (BD Biosciences). Data were analyzed by statistical analysis using FlowJo V10 software (Treestar) and Prism 7.04 software (Graphpad), and statistical significance was assessed using unpaired t-test and Welch correction.
Intratumoral injection of compound 1 did not alter the tumor-associated leukocyte composition compared to the PBS control (fig. 20B), indicating that ENPP1 had no significant effect in clearing basal levels of extracellular cGAMP in this tumor model. However, when tumors were pretreated with IR, compound 1 was observed to increase tumor-associated CD11c+Group (fig. 20B).
The results are shown in fig. 20A and 20B. Fig. 20A shows extracellular cGAMP produced by 4T1 cells within 48 hours. At time 0, the cells were left outMedium supplemented with 50 μ M compound 1 was either treated and refreshed with 20Gy IR. Mean ± SEM (n ═ 2). P ═ 0.004 (student's t test). FIG. 20B shows 4T1 cells (1X 10) injected in situ into BALB/cJ mice on day 0 6). Tumors were left untreated or treated with 20Gy IR and injected intratumorally with PBS (n-5 for IR (0 Gy; n-4 for IR (20 Gy)) or compound 1 (n-5) on day 2. Tumors were harvested on day 3 and analyzed by FACS. P ═ 0.047(Welch t test).
Example 6: ENPP1 inhibition exerts an anti-tumor effect synergistically with IR treatment and anti-CTLA-4
It has been investigated whether further increases in vitro cGAMP by using Ionizing Radiation (IR) and an exemplary ENPP1 inhibitor such as compound 1 could improve the immunodetection and clearance of tumors in vivo.
In 7 to 9 week old female Balb/c mice (Jackson Laboratories), 5X 10 suspended in 50. mu.L PBS4Individual 4T 1-luciferase cells were seeded into mammary fat pads. When tumor volume (determine length)2x width/2) up to 80mm3To 120mm3When the tumor was irradiated at 20Gy using a 225kVp cabinet X-ray irradiator (IC 250, Kimtron inc., CT) filtered with 0.5mm Cu. Anesthetized animals were shielded with a 3.2mm lead shield with a 15x 20mm hole for tumor placement. On days 2, 4 and 7 after IR, 100 μ L of 100 μ M Compound 1 and/or 10 μ g cGAMP in PBS, or PBS alone, was injected intratumorally. Alternatively, on days 2, 5 and 7 post-IR, compound 1 or PBS alone was injected intratumorally at 1mM in PBS and intraperitoneally at 200 μ g anti-CTLA-4 antibody or syrian hamster IgG antibody (both from BioXCell). Mice from different treatment groups were co-housed in each cage to eliminate the cage effect. The experimenters were blinded throughout the study. Tumor volumes were recorded every other day. Tumor volumes were analyzed using the generalized estimation equation to explain the correlation in mice. Treatment groups were compared pairwise at each time point using post hoc testing, and multiple comparisons were made using Tukey adjustments. Animal deaths were plotted in a Kaplan Meier curve using Graphpad prism7.03, and Statistical significance was assessed using the Logrank Mantel-Cox test. All animal procedures were approved by the laboratory animal care management panel.
Administration of compound 1 enhanced the tumor shrinkage effect of IR treatment, but was not significant (fig. 21A). Although intratumoral cGAMP injection had no effect on IR treatment, compound 1 injection in addition to cGAMP synergistically reduced tumor size, prolonged survival, and achieved a 10% cure rate (fig. 21A and 21B).
Synergy with the adaptive immune checkpoint blockade anti-CTLA-4 was also tested. In the absence of IR, anti-CTLA-4 and compound 1 treatment had no effect on prolongation of survival (figure 21C). However, combining IR pretreatment with compound 1 and anti-CTLA-4 exerted a significant synergistic effect and achieved a 10% cure rate. Taken together, these results indicate that enhancing extracellular cGAMP by combining IR treatment with ENPP1 inhibition can increase tumor immunogenicity and exert antitumor effects.
The results are shown in fig. 21A, which shows the tumor shrinkage effect of compound 1 in combination with IR. The established tumors (100. + -. 20 mm)3) Treatment was once with 20Gy IR, followed by 3 intratumoral injections of PBS or therapeutic agent on days 2, 4 and 7 post IR (n-9/treatment group). Mice from different treatment groups were housed together and blinded to the experimenters. Tumor volumes were analyzed in a generalized estimation equation to account for correlations in mice. Treatment groups were compared pairwise at each time point using post hoc testing, and multiple comparisons were made using Tukey adjustments. FIG. 21B shows the Kaplan Meier curve of FIG. 21A with P values determined by the log rank Mantel-Cox test. Figure 21C shows anti-CTLA 4 or IgG isotype control antibody injected intraperitoneally on days 2, 5, and 7 post-IR (n-8 for IR (0) + compound 1+ CTLA-4 treatment group; n-17-19 for all other treatment groups), in addition to the same procedure as figure 21B. Statistical analysis was performed as shown in fig. 21B.
Taken together, these results indicate that cGAMP is present extracellularly, and that the subject ENPP1 inhibitor can act extracellularly; thus, extracellular inhibition of ENPP1 was shown to be sufficient for therapeutic effect. ENPP1 qualifies as an innate immune checkpoint. These experiments showed that extracellular inhibition of ENPP1 enabled cGAMP to enhance anticancer immunity and to be synergistically combined with immune checkpoint blockade drugs that have been available as therapeutics (fig. 22).
Example 7: 2 '3' -cGAMP is an neurotransmitter produced by cancer cells and regulated by ENPP1
Introduction to
2 '3' -cyclic GMP-AMP (cGAMP) is characterized as an intracellular second messenger that responds to cytosolic dsDNA synthesis and activates the innate immune STING pathway. Its extracellular hydrolase ENPP1 suggests the presence of extracellular cGAMP. Using mass spectrometry, cGAMP was detected as a soluble factor that was continuously exported by the engineered cell line, but was subsequently efficiently cleared by ENPP 1. cGAMP export was detected in cancer cell lines commonly used in mouse tumor models by developing a potent, specific and cell-impermeable inhibitor of ENPP 1. In tumors, depletion of extracellular cGAMP using neutralizing proteins would decrease tumor-associated dendritic cells. Promotion of extracellular cGAMP by gene knockout and pharmacological inhibition of ENPP1 increased tumor-associated dendritic cells, narrowed the tumor, and treated tumors in concert with ionizing radiation and anti-CTLA-4. In summary, cGAMP is an anticancer neurotransmitter that is released by tumors and detected by the host's innate immunity.
The second messenger, 2 '3' -cyclic GMP-AMP (cGAMP), plays a key role in antiviral and anticancer innate immunity. It is synthesized by the enzyme cyclic-GMP-AMP synthase (cGAS), in response to double stranded DNA (dsDNA) in the cytosol, which is a danger signal for intracellular pathogens and damaged or cancerous cells. cGAMP binds to and activates Endoplasmic Reticulum (ER) surface receptor stimulators of its interferon gene (STING) to activate the production of type 1 Interferon (IFN). These potent cytokines trigger downstream innate and adaptive immune responses to clear threats.
In addition to activating STING within its cells of origin, cGAMP can also diffuse to bystander cells through gap junctions in epithelial cells. This mechanism of intercellular communication reminds the neighboring cells of the damaged cell, which is unfortunately also responsible for drug-induced hepatotoxic spread and brain metastases. In addition, cytosolic cGAMP can be packaged into budding viral particles and spread during the next round of infection. In both modes of transmission, cGAMP is never exposed to the extracellular space.
The only enzyme responsible for the detectable cGAMP hydrolase activity is the ectonucleotide pyrophosphatase phosphodiesterase 1(ENPP1) (see, e.g., Li, L., et al, Hydrolysis of 2 '3' -cGAMP by ENPP1 and design of nonhydrolytic enzymes and. Nat. chem. biol.10, 1043-8 (2014)). This is surprising because ENPP1 was annotated as an extracellular enzyme, both as a membrane-bound form anchored by a single-pass transmembrane domain, and as a soluble protein that was cleaved in serum. cGAMP (which has two negative charges and is presumed not to passively cross the cell membrane) can enter the cell to activate STING (see, e.g., Gao, P. et al Structure-function analysis of STING Activation by c [ G (2 ', 5') pA (3 ', 5') p ] and targeting by anti-viral DMXAA. cell 154, 748-762 (2013); and Corrales, L. et al Direct Activation of STING in the Tumor microorganism gains to position and systematic Tumor Regression and immunity. cell 11, 1018-1030 (2015)), indicating the presence of a transport channel for cGAMP. Because it can enter cells, cGAMP analogs are currently being tested in clinical trials to treat metastatic solid tumors by intratumoral injection. Extracellular cGAMP is known to be introduced and has anticancer effects, and the dominant cGAMP hydrolase is extracellular, so it is assumed that cGAMP is exported to the extracellular space to signal other cells and is regulated by extracellular degradation.
The role of cancer export cGAMP as well as extracellular cGAMP in anticancer immunoassays is demonstrated herein. Using gene knock-out and pharmacological inhibition, the role of ENPP1 in controlling extracellular cGAMP concentration, immune infiltration, and tumor progression was also investigated. In summary, cGAMP is characterized as an neurotransmitter that is regulated by ENPP 1.
Materials and methods
Reagents, antibodies and cell lines
[α-32P]ATP (800Ci/mmol,10mCi/mL, 250. mu. Ci) and [ 2 ]35S]ATP. alpha.S (1250Ci/mmol,12.5mCi/mL, 250. mu. Ci)From Perkin Elmer. Adenosine triphosphate, guanosine triphosphate, adenosine-13C10,15N5Guanosine 5' -triphosphate13C10,15N5Triphosphoric acid, 4-nitrophenyl phosphate and bis (4-nitrophenyl) phosphate were purchased from Sigma-Aldrich and were>98% atomic purity. 2 '3' -cGAMP was purchased from Invivogen. The Caco-2 kit was purchased from Cyprotex. Kinomie screening was performed by Eurofins. PAMPA and MDCK permeability assays were performed by Quintara Discovery. Total protein content was quantified using the BCA assay (ThermoFisher). Cell viability was quantified using the CellTiterGlo assay (Promega). Full-length human ENPP1 was cloned into pcDNA3 vector. A set of 4 ON-TARGETplus ENPP1 siRNAs (LQ-003809-00-0002) were purchased from Dharmacon. Synthesis of QS1 is as previously described25. Immunoblotting the following monoclonal antibodies were used: rabbit anti-cGAS (D1D3G Cell Signaling, 1: 1,000), rabbit anti-mouse cGAS (D2O8O Cell Signaling, 1: 1,000), mouse anti-tubulin (DM1A Cell Signaling, 1: 2,000) and rabbit anti-STING (D2P2F Cell Signaling, 1: 1,000), IRDye 800CW goat anti-rabbit (LI-COR, 1: 15,000) and IRDye 680RD goat anti-mouse (LI-COR, 1: 15,000).
293T cells were purchased from ATCC and virus transfected to stably express mouse cGAS. 293T cGAS ENPP1lowCells were generated by viral transfection of CRISPR sgrnas targeting human ENPP1 (5'-CACCGCTGGTTCTATGCACGTCTCC-3'), and 293T mcGAS ENPP1 was selected after cloning single cells from this pool-/-A cell. 4T1 and E0771 cGAS-/-Cells were generated by viral transfection of CRISPR sgrnas (using lenticrispr rv2-blast, addge plasmid #83480) targeting mouse Mb21d1 (5'-CACCGGAAGGGGCGCGCGCTCCACC-3'). Cells were selected after single cell cloning. 4T1-Luc ENPP1-/-Cells were generated by viral transfection of CRISPR sgrnas targeting mouse Enpp1 (5'-GCTCGCGCCCATGGACCT-3' and 5'-ATATGACTGTACCCTACGGG-3') or scrambled sequences (using lenticrisprrv 2-blast) (Sanjana, n.e., Shalem, O.&Zhang, F.improved vectors and genes-wide libraries for CRISPR screening. Nat.methods 11, 783-784 (2014)). 4T1-Luc shcGAS cells were transfected with shRNA (5' -CAGGATTGAGCTACAA) by virus using plasmid pGH188GAATAT-3'). Cells with shRNA were selected with blasticidin and classified for GFP expression and used as the experimental pool. MDA-MB-231 was purchased from ATCC, E0771 was purchased from CH3 BioSystems, and 4T 1-luciferase and HEK293S GnT1 expressing secreted mENPP1 were obtained -A cell.
Cell culture
The cell lines were maintained in DMEM (Corning Cellgro) (293T, MC38) or RPMI (Corning Cellgro) (4T1-Luc, E0771, MDA-MD-231) supplemented with 10% FBS (Atlanta biologics) (v/v) and 100U/mL penicillin-streptomycin (ThermoFisher). Primary human Peripheral Blood Mononuclear Cells (PBMCs) were isolated by subjecting enriched buffy coats from whole blood to a Percoll density gradient. Using CD14+MicroBeads (Miltenyi) isolation of CD14+PBMC. Culturing of CD14 in RMPI supplemented with 2% human serum and 100U/mL penicillin-streptomycin+PBMC。
Expression and purification of recombinant proteins
sscGAS: using primer pair fwd: (5'-CTGGAAGTTCTGTTCCAGGGGCCCCATATGGGCGCCTGGAAGCTCCAG AC-3') and rev: (5'-GATCTCAGTGGTGGTGGTGGTGGTGCTCGAGCCAAAAAACTGGAAAT CCATTGT-3') the DNA sequence encoding porcine cGAS (residue 135-497) was amplified from a porcine cDNA library. The PCR product was inserted into pDB-His-MBP by Gibson assembly and expressed in Rosetta cells. Cells were grown in 2XYT medium containing kanamycin (100. mu.g/ml) when OD was applied600Induced with 0.5mM IPTG when 1 was reached and grown overnight at 16 ℃. All the following procedures involving proteins and cell lysates were performed at 4 ℃. Cells were pelleted and lysed in 20mM HEPES pH 7.5, 400mM NaCl, 10% glycerol, 10mM imidazole, 1mM DTT and protease inhibitor cocktail (cOmplexlite, EDTA-free tablets, Roche). Cell extracts were cleared by ultracentrifugation at 50,000x g for 1 hour. The clear supernatant was incubated with HisPur cobalt resin (ThermoFisher Scientific; 1mL resin per liter of bacterial culture). The cobalt resin was washed with 20mM HEPES pH 7.5, 1M NaCl, 10% glycerol, 10mM imidazole, 1mM DTT. The proteins were eluted from the resin with 20mM HEPES pH 7.5, 1M NaCl, 10% glycerol and 300mM imidazole in 1mM DTT And (6) next. The His-MBP-sscGAS containing fractions were combined, concentrated, and dialyzed against 20mM HEPES pH7.5, 400mM NaCl, 1mM DTT. The protein is flash frozen into aliquots for future use.
STING: mouse STING (residue 139-378) was inserted into the pTB146 His-SUMO vector and expressed in Rosetta cells. Cells were grown in 2XYT medium containing 100. mu.g/mL ampicillin, when OD600Induction was carried out overnight at 16 ℃ with 0.75mM IPTG when 1 was reached. All subsequent manipulations with protein and cell lysates were performed at 4 ℃. Cells were pelleted and lysed in 50mM Tris pH7.5, 400mM NaCl, 10mM imidazole, 2mM DTT and protease inhibitor (cOmplexate, EDTA-free protease inhibitor cocktail, Roche). Cells were lysed by sonication, and lysates were cleared by ultracentrifugation at 50,000rcf for 1 hour. The clear supernatant was incubated with HisPur cobalt resin (ThermoFisher Scientific; 1mL resin per 1L bacterial culture) for 30 minutes. The resin bound proteins were washed with 50 column volumes of 50mM Tris pH7.5, 150mM NaCl, 2% triton X-114, 50CV of 50mM Tris pH7.5, 1M NaCl (titration rate per wash set at 1 drop/2-3 seconds, taking 2-3 hours) and 20CV of 50mM Tris pH7.5, 150mM NaCl. The protein was eluted from the resin with 600mM imidazole in 50mM Tris pH7.5, 150mM NaCl. The His-SUMO-STING containing fractions were combined, concentrated, and dialyzed against 50mM Tris pH7.5, 150mM NaCl while incubated overnight with SUMOLASE enzyme His-ULP1 to remove the His-SUMO tag. The solution was again incubated with hispu cobalt resin to remove the His-SUMO tag and STING was collected from the effluent. The protein was dialyzed against 50mM Tris pH7.5 and used Fplc (GE Healthcare) was loaded onto a HitrapQ anion exchange column (GE Healthcare) and eluted with a NaCl gradient. The STING-containing fractions were pooled and the buffer was changed to PBS and stored at 4 ℃ until use.
ENPP 1: mENPP1 was prepared as described in Kato, K. et al (Expression, publication, crystallization and prediction X-ray crystallization analysis of Enpp1.acta crystallization. Sect.F. Structure.Biol.Crystal.Commun.68, 778-782 (2012), and Crystal structure of Enpp1, an extracellular fibrous in volatile in bone crystallization and insulin signaling. Proc.Natl.Acad.Sci.U.S. A.109, 16876-81 (2012)).
Liquid chromatography-tandem mass spectrometry
Cyclic GMP-13C10,15N5-AMP is used as internal standard and is cyclic13C10,15 5-GMP-13C10,15N5-AMP was used as extraction standard. Isotope-labeled cGAMP standards were prepared by incubating 1mM ATP (isotope-labeled), 1mM GTP (isotope-labeled), 20mM MgCl in 100mM Tris, pH 7.520.1mg/mL herring testis DNA (Sigma) and 2. mu.M sscGAS overnight. The reaction was heated at 95 ℃ and filtered through a 3kDa centrifugal filter. The water was removed on a rotary evaporator. PLRP-S Polymer reverse phase preparative columns (Per HPLC) were used on a preparative HPLC (1260Infinity LC System; Agilent Technologies) connected to a UV-vis detector (ProStar; Agilent Technologies) and a fraction collector (440-LC; Agilent Technologies) 8 μm,300 × 25 mm; agilent Technologies) purified cGAMP from the crude reaction mixture. The flow rate was set at 25 mL/min. The mobile phase consisted of 10mM triethylammonium acetate in water and acetonitrile. The first 5 minutes of mobile phase was 2% acetonitrile. Acetonitrile then rose from 5-20 minutes to 30%, from 20-22 minutes to 90%, from 22-25 minutes to 90%, and then from 25-28 minutes to 2%. The cGAMP-containing fraction was lyophilized and resuspended in water. The concentration was determined by measuring the absorbance at 280 nm. Samples were analyzed for cGAMP, ATP and GTP content on Shimadzu HPLC (San Francisco, CA), autosampler set at 4 ℃ and attached to AB Sciex 4000QTRAP (Foster City, CA). A10. mu.L volume was injected onto a Biobasic AX LC column, 5 μm,50X 3mm (thermo scientific). The mobile phase consisted of 100mM ammonium carbonate (A) and 0.1% formic acid in acetonitrile (B). Initial conditions were 90% B, hold for 0.5 min. The mobile phase rose to 30% a from 0.5 min to 2.0 min, remained at 30% a from 2.0 min to 3.5 min, rose to 90% B from 3.5 min to 3.6 min, and remained at 90% B from 3.6 min to 5 min. The flow rate was set to 0.6 mL/min. The mass spectrometer was operated in the electrode spray positive ion mode with the source temperature set at 500 ℃. Declustering and collision induced dissociation was achieved using nitrogen. De-clustering potential and collision energy were optimized by direct injection of standards. For each molecule, MRM transitions (m/z), DP (V), and CE (V) are as follows: ATP (508) >136,341,55)、GTP(524>152,236,43)、cGAMP(675>136,121,97；675>312,121,59；675>152,121,73), internal standard cyclic GMP-13C10,15N5-AMP(690>146,111,101；690>152,111,45；690>327,111,47), extraction standard Ring13C10,15N5-GMP-13C10,15N5-AMP(705>156,66,93；705>162,66,73)。
-/-Output assay in 293TcGAS ENPP1 cells
293T cGAS ENPP1-/-Cells were seeded in tissue culture treated plates coated with purcol (advanced biometrix). After 24 hours, the medium was gently removed and replaced with serum-free DMEM supplemented with 1% insulin-transferrin-selenium-sodium pyruvate (ThermoFisher) and 100U/mL penicillin-streptomycin. At the indicated times, the medium was removed and the cells were washed off the plate with cold PBS. Both the medium and the cells were centrifuged at 1000rcf for 10 min at 4 ℃. Cells were lysed in 30 to 100 μ L of 50:50 acetonitrile: water supplemented with 500nM internal standard and centrifuged at 15,000rcf for 20 min at 4 ℃ to remove insoluble fractions. If concentration is not required, an aliquot of the medium is removed and supplemented with 500nM internal standard and 20% formic acid. If concentration is required, the medium is acidified with 0.5% acetic acid and supplemented with extraction standards (appropriate amount to a final concentration of 2. mu.M in 100. mu.L). Culture medium was applied to a HyperSep Aminopropyl SPE column (ThermoFisher Scientific) as described in Gao, D.et al (Activation of cyclic GMP-AMP synthsase by self-DNA mice, Proc. Natl. Acad. Sci. U.S.A.112, E5699-705 (2015))) To enrich for cGAMP. The eluate was evaporated to dryness and reconstituted in 50:50 acetonitrile: water supplemented with 500nM internal standard. Media and cell extracts were provided for mass spectrometric quantification of cGAMP, ATP and GTP.
293T cGAS -/-Transfection stimulation of ENPP1 cells
293T cGAS ENPP1 was transfected with Fugene 6(Promega) according to the manufacturer's instructions plus pcDNA3 plasmid DNA (empty or containing human ENPP1) at the indicated concentration-/-A cell. Output assays were performed as described above 24 hours after transfection.
Conditioned Medium transfer
293T cGAS ENPP1, as described aboveIs low inCells were plated and transfected with plasmid DNA. 24 hours after transfection, the medium was changed to RPMI + 2% human serum + 1% penicillin-streptomycin, +/-2. mu.M cGAMP, +/-20nM recombinant mENPP1 or +/-50uM Compound 1. 24 hours after medium change, 293T cGAS ENPP1Is low inThe conditioned medium was removed from the cells and separated from freshly isolated CD14+PBMC are incubated. Analysis of CD14 after 14-16 hours+Gene expression of PBMC.
RT-PCR analysis
Total RNA was extracted using Trizol (Thermo Fisher Scientific) and reverse transcribed using Maxima H Minus reverse transcriptase (Thermo Fisher Scientific). Real-time RT-PCR was performed repeatedly on a 7900HT fast real-time PCR system (Applied Biosystems) using an AccuPower 2X Greenstar qPCR Master Mix (Bioneer). Data for each sample were normalized to CD14, ACTB, or GAPDH expression. Fold induction was calculated using Δ Δ Ct. Primers for human IFNB 1: fwd (5'-AAACTCATGAGCAGTCTGCA-3'), rev (5'-AGGAGATCTTCAGTTTCGGAGG-3'); primers for human CD 14: fwd (5'-GCCTTCCGTGTCCCCACTGC-3'), rev (5'-TGAGGGGGCCCTCGACG-3'); primers for human ACTB: fwd (5'-GGCATCCTCACCCTGAAGTA-3'), rev (5'-AGAGGCGTACAGGGATAGCA-3'); primers for human GAPDH: fwd (5'-CCAAGGTCATCCATGACAAC-3'); rev (5'-CAGTGAGCTTCCCGTTCAG-3').
32TLC assay for P-cGAMP degradationStator
By mixing unlabeled ATP (1mM) with a reagent containing ATP32GTP of P-ATP (1mM) together with 2. mu.M purified recombinant porcine cGAS in 20mM Tris pH 7.5,2mM MgCl2Synthesis of radiolabeled DNA from herring testis DNA 100. mu.g/mL incubated overnight at room temperature32P cGAMP, and the remaining nucleotide starting material was degraded with alkaline phosphatase at 37 ℃ for 4 hours. Cell lysates were prepared by washing 100. mu.L of 10mM Tris, 150mM NaCl, 1.5mM MgCl21% NP-40, pH 9.0 scraping and cleavage 1X106Individual cell (293T) or 10x106Produced by individual cells (4T1-Luc, E0771 and MDA-MB-231). For 4T1-Luc, E0771, and MDA-MB-231, the total protein concentration of the lysate was measured using the BCA assay (Pierce, Thermo Fisher) and the samples were normalized so that the same amount of protein was used for each lysate reaction. The probe is used for detecting the position of the probe32P-cGAMP (5. mu.M) with mENPP1(20nM) or whole cell lysate in 100mM Tris, 150mM NaCl, 2mM CaCl2、200μM ZnCl2Incubate at pH 7.5 or pH 9.0 for the indicated time. To generate an inhibition curve, 5-fold dilutions of ENPP1 inhibitor were included in the reaction. Degradation was assessed by TLC (see, e.g., Li, L. et al Hydrolysis of 2 '3' -cGAMP by ENPP1 and design of nonhydrolyzable analytes Nat. chem. biol.10, 1043-8 (2014)). Plates were exposed on fluorescent screens (Molecular Dynamics) and imaged on Typhoon 9400, and ImageJ pairs were used 32P signal was quantified. Inhibition curves were fitted using Graphpad Prism 7.03 to obtain IC50The value is obtained. Using Cheng-Prusoff equation Ki,app＝IC50/(1+[S]/Km) Will IC50Conversion of value to Ki,appThe value is obtained.
ALPL and ENPP2 inhibition assays
Inhibition assays for other ectonucleotidases were performed by incubating reaction components in a 96-well plate format at room temperature and monitoring the production of 4-nitrophenol by measuring absorbance at 400nM in a plate reader (Tecan). ALPL: at room temperature, in a buffer pH 9.0 (containing 50mM Tris, 20. mu.M ZnCl)2、1mM MgCl2) 0.1nM ALPL, 2. mu.M 4-nitrophenyl phosphate and various concentrationsAnd (3) an inhibitor. ENPP 2: at buffer pH 9.0 (containing 100mM Tris, 150mM NaCl, 200. mu.M ZnCl)2、2mM CaCl2) 2nM ENPP2, 500. mu.M bis (4-nitrophenyl) phosphate and different concentrations of inhibitor.
Output assay in cancer cell lines
4T1-Luc, E0771 and MC38 cells were replaced with new medium supplemented with 50. mu.M Compound 1. Collecting the culture medium at a designated time; cells were washed off the plate with PBS, pelleted at 1000rcf, and pelleted with 4mL 50: 50 parts of acetonitrile: water was lysed and centrifuged at 15,000 rcf. cGAMP was enriched from culture media and cell supernatants using a HyperSep Aminopropyl SPE column as described above and submitted for mass spectrometric quantitation.
Mouse model of 4T1-Luc tumor
In 7 to 9 weeks old female BALB/c mice (Jackson Laboratories), 5X 10 suspended in 50. mu.L PBS4Or 5x 1054T 1-Luc-luciferase cells were seeded into mammary fat pads. When tumor volume (determine length)2x width/2) up to 80mm3To 120mm3Tumors were irradiated to 20Gy using a 225kVp cabinet X-ray irradiator (IC-250, Kimtron inc., CT) filtered with 0.5mm Cu. Anesthetized animals were shielded with a 3.2mm lead shield with a 15x 20mm hole for tumor placement. On days 2, 4 and 7 after IR, 100 μ L of 100 μ M compound 1 and/or 10 μ g cGAMP in PBS, or PBS alone, was injected intratumorally. Alternatively, on days 2, 5 and 7 post-IR, compound 1 or PBS alone was injected intratumorally at 1mM in PBS and intraperitoneally at 200 μ g anti-CTLA-4 antibody or syrian hamster IgG antibody (both from BioXCell). Mice from different treatment groups were co-housed in each cage to eliminate the cage effect. The experimenters were blinded throughout the study. Tumor volumes were recorded every other day. Tumor volumes were analyzed using the generalized estimation equation to explain the correlation in mice. Treatment groups were compared pairwise at each time point using post hoc testing, and multiple comparisons were made using Tukey adjustments. Animal deaths were plotted in a Kaplan Meier curve using Graphpad Prism 7.03 And statistical significance was assessed using the Log-rank Mantel-Cox test. All mice were housed at the university of stanford according to the institutional animal care and use committee at the university of stanford, and the procedures were approved by the university of stanford laboratory animal care management group.
FACS analysis of tumors
Female BALB/C WT (4T1-Luc tumor) or C57BL/6(E0771 tumor) WT, cGAS at 7 to 9 weeks of age-/-Or STINGgt/gt(referred to as STING)-/-) In mice (Jackson Laboratories), 1X 10 suspended in 50. mu.L PBS6Tumor cells were seeded into the mammary fat pad. Two days after injection, the tumors were irradiated as described and 100 μ L of 1mM compound 1 in PBS, or PBS alone, was injected intratumorally. For experiments using STING and mENPP1, 100 μ L of 100 μ M neutralizing STING or non-binding STING (R237A) or 700nM mENPP1 or PBS was injected intratumorally. The next day, tumors were extracted and incubated in RPMI + 10% FBS containing 20. mu.g/mL DNase type I IV (Sigma-Aldrich) and 1mg/mL collagenase from Clostridium histolyticum (Sigma-Aldrich) for 30min at 37 ℃. Tumors were passed through a 100 μm cell filter (Sigma-Aldrich) and red blood cell lysis buffer (155mM NH) was used at room temperature 4Cl、12mM NaHCO30.1mM EDTA) lysed erythrocytes for 5 minutes. Cells were stained with live/dead immortable near-infrared dead cell staining kit (Thermo Fisher Scientific), Fc blocked using TruStain fcX for 10min, and then antibody stained with CD11c, CD45, and I-a/I-E (all from Biolegend). Cells were analyzed using SH800S cell sorter (Sony) or LSR II (BD Biosciences). Data were analyzed by statistical analysis using FlowJo V10 software (Treestar) and Prism 7.04 software (Graphpad), and statistical significance was assessed using unpaired t-test and Welch correction.
In vivo imaging
Mice were injected intraperitoneally with 3mg of XenoLight D-fluorescein (Perkin-Elmer) in 200. mu.l of water and imaged using a Lago X in vivo Imaging system (Spectral Instruments Imaging). The target height was set to 1.5cm, binning was 4, FStop was 1.2, and exposure time was 120 s. Images were analyzed using aura 2.0.1 software (Spectral Instruments Imaging).
Results
cGAMP as a soluble factor from 293T cGAS -/-Export in ENPP1 cells
To test the hypothesis that cGAMP is present extracellularly, we first developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect cGAMP in complex mixtures. Using two isotope-labeled cGAMP standards (fig. 1, panel a), we can quantify cGAMP concentrations as low as 0.5nM in basal cell culture medium and serum-containing medium, and we can quantify intracellular cGAMP concentrations in cell extracts in the same experiment (fig. 1, panel B and fig. 8, panels a and B). We chose to use 293T cells that do not express cGAS or STING. By stably expressing mouse cGAS and using CRISPR knock-out ENPP1, we created 293T cGAS ENPP1 Is low inCell line (fig. 8, panel C). We then isolated a clone to create 293T cGAS ENPP1-/-Cell line (fig. 8, panel C). We also used serum-free medium, since the serum contained the proteolytically soluble form of ENPP 1. Using this cell culture system without ENPP1, we performed 293T cGAS ENPP1 without any stimulation-/-Constant low micromolar basal intracellular cGAMP concentrations were detected in the cells (fig. 1, panel C). This is not surprising, since erroneous DNA isolation leads to the presence of abundant cytoplasmic dsDNA in cancer cells (see, e.g., Mackenzie, K.J. et al, cGAS fundamental of microcatheter linkage gene importance.Nature 548, 461-465 (2017); Harding, S.M. Mitic progression yielding DNA database genes expression protocol with genetic DNA nature 548, 466-470 (2017); and Bakhoum, S.F. et al, Chromosomal architecture genes genome of a cytoponic DNA response. Nature.467-472 (2018)). After replenishing the cells with fresh medium, we measured a linear increase in extracellular cGAMP concentration to 100nM after 30 hours (fig. 1, panel D). At 30 hours, the number of extracellular cGAMP molecules was equal to the number of intracellular cGAMP molecules (graph) 1, panel E). We detected negligible amounts of cell death based on extracellular Lactose Dehydrogenate (LDH) activity, indicating that cGAMP in the culture medium was exported by living cells (fig. 1, panel E). We calculate the output ratio (v)export) Is 220 molecular cells-1s-1(FIG. 1, panel F). Finally, cGAMP in the culture medium can pass through the 10kDa filter without any retention (which should retain extracellular vesicles and proteins), suggesting that cGAMP is exported as a freely soluble molecule (fig. 1, panel H).
To further confirm that extracellular cGAMP secreted by 293T cells exists predominantly in soluble form, but not in extracellular vesicles, we used CD14+Human Peripheral Blood Mononuclear Cells (PBMCs) were used as the reporter. These cells have previously been shown to take up soluble cGAMP, leading to the production of IFN- β17. We observed CD14+PBMC responded to submicromolar concentrations of soluble cGAMP by up-regulating IFNB1 (fig. 9). Conditioned Medium (obtained from DNA-transfected cGAS-expressing 293T cGAS ENPP1Is low inCells other than cGAS-null 293T cells from DNA transfection) in CD14+IFNB1 expression was induced in the cells, indicating that this activity is the result of extracellular cGAMP produced by 293T cells (fig. 1, panels H and I). Addition of purified soluble recombinant mouse ENPP1(mENPP1) (fig. 8, panel D) depleted detectable cGAMP in conditioned media and also abolished this activity (fig. 1, panels H and J). Since soluble ENPP1(MW ═ 100kDa) is not permeable to membranes and therefore only accessible to soluble extracellular cGAMP, we concluded that 293T cells secrete soluble cGAMP. In summary, our data suggest that this artificial cancer cell line maintains its intracellular cGAMP in a stable state by exporting it as a soluble factor into the extracellular medium.
Fig. 1, panels a to J: cGAMP as a soluble factor from 293T cGAS ENPP1-/-And (4) outputting in the cells. a, chemical structure of cGAMP and single isotope labeled cGAMP. cGAMP was detected by LC-MS/MS with a lower limit of quantitation of 4 nM. (left) liquid chromatograms of 0, 4 and 10nM cGAMP and 500nM monoisotopically labeled cGAMP (heavy 15Da) as internal standards; (right) external standard curve of cGAMP, R20.996. Data representation>10 independent experiments. c-d, measured using LC-MS/MS from 293T cGAS ENPP1-/-Intracellular and extracellular concentrations of cGAMP in cells (without exogenous stimulation). At time 0, cells were supplemented with serum-free media. Mean ± SEM (n ═ 2), some error bars were too small to visualize. Data are representative of three independent experiments. e, the ratio of extracellular cGAMP molecules/total cGAMP molecules calculated from the data in (c) and (d) (left y-axis) is compared to the ratio of extracellular lactose dehydrogen/total Lactose Dehydrogen (LDH) activity (right y-axis). f, the amount of cGAMP output per cell over time calculated from the data in (d). The output rate was found using linear regression. g, 293T cGAS ENPP1 measured before and after passing the culture medium through a 10kDa filter-/-Intracellular and extracellular cGAMP concentrations produced by the cells. Mean ± SEM (n ═ 2). Data are representative of two independent experiments. Schematic representation of conditioned medium transfer experiments for h, (i) and (j). cGAS-null293T or 293T cGAS ENPP1 Is low inCells were transfected with empty pcDNA vector and treated with +/-20nM recombinant mouse ENPP1(mENPP 1). Transfer of conditioned Medium from these cells to Primary CD14+Human PBMC. i, IFNB1 mRNA levels were normalized to CD14 and compared to untreated CD14+Cells were counted for fold induction. Mean ± SEM (n ═ 4). P ═ 0.0003 (one-way ANOVA). cGAMP concentrations were measured in conditioned media. Mean ± SEM (n ═ 2). P ═ 0.0002 (one-way ANOVA). Data are representative of two independent experiments. j, IFNB1 mRNA levels were normalized to CD14 and compared to untreated CD14+Cells were counted for fold induction. Mean ± SEM (n ═ 2). P ═ 0.04 (one-way ANOVA). cGAMP concentrations were measured in conditioned media. Mean ± SEM (n ═ 2). P ═ 0.002 (one-way ANOVA). Data are representative of two independent experiments.
Fig. 8, panels a to D: development of LC-MS/MS method and construction of 293T cGAS ENPP1Is low inAnd 293T cGAS ENPP1-/-A cell line. a liquid chromatogram of cGAMP at 0, 20 and 80 nM; liquid chromatogram of monoisotopically labeled internal standard cGAMP (weight 15Da) at 500 nM; and liquid chromatogram of the two-isotope labeled extraction standard cGAMP (weight 30Da) at 2. mu.M. What is needed is There is the chemical structure of the analyte. b, cell number was calibrated to ATP concentration measured by LC-MS/MS. Mean ± SEM (n ═ 2). c, analysis by western blot of 293T, 293T cGAS ENPP1-/-And 293T cGAS ENPP1Is low incGAS expression of cell lines (left). 293T cGAS, 293T cGAS ENPP1-/-And 293T cGAS ENPP1Is low inIn a whole cell lysate of 100 ten thousand cells each32The ENPP1 hydrolytic activity of P-cGAMP was measured by TLC and autoradiography (right). Lysate data are representative of two independent experiments. d, Coomassie gel (Coomassie gel) of recombinant mouse ENPP1 purified from the culture medium; the eluted fractions were pooled before use (left). TLC analysis of mouse ENPP1 pairs32Degradation of P-cGAMP (right).
Fig. 9, panel a: CD14+PBMC respond to extracellular cGAMP. Stimulation of CD14 with extracellular cGAMP+Schematic representation of PBMC. b, human CD14 measured by RT-qPCR+IFNB1 induction of PMBC, human CD14+PMBC were stimulated with increasing concentrations of extracellular cGAMP for 16 hours. Mean ± SEM (n ═ 2 technical qPCR replicates).
ENPP1 regulates extracellular cGAMP only
Since we were able to observe extracellular cGAMP for the first time when we knocked ENPP1 out of 293T cells and cultured them in media without ENPP1, we investigated whether only extracellular cGAMP is regulated by ENPP 1. Despite extracellular annotation (annotation), ENPP1 may flip direction on membranes, such as the enzyme CD38 (see, e.g., Zhao, y.j., Lam, c.m.c.). &Lee, h.c. the membrane-bound enzyme CD38 exists in two-openingorientations, sci.signal.5, ra67(2012)), or it may be active when synthesized in the ER lumen, and cGAMP may pass through the ER membrane (fig. 2, panel a). To investigate the localization of ENPP1 activity, we transfected 293T cGAS ENPP1 with the human ENPP1 expression plasmid-/-Cells and their activity in whole cell lysates was confirmed (fig. 2, panel B). In intact cells, ENPP1 expression consumed extracellular cGAMP, but did not affect intracellular cGAMP concentrations (fig. 2, panel C). Thus, in these cells, extracellular, but not intracellular, cGAMP is regulated by ENPP 1.
Fig. 2, panels a to C: ENPP1 regulates only extracellular cGAMP. a, three possible cellular locations for ENPP1 activity. b,293T cGAS ENPP1-/-Cells were transfected with either the empty vector or the vector containing human ENPP1 and after 24 hours ENPP1 protein expression was analyzed using western blot (top) and ENPP1 was analyzed using Thin Layer Chromatography (TLC)32P-cGAMP hydrolytic activity (bottom). Data are representative of two independent experiments. c, intracellular and extracellular cGAMP concentrations were measured using LC-MS/MS. BQL ═ below the limit of quantitation. Mean ± SEM (n ═ 2). P ═ 0.002 (student's t test). Data are representative of three independent experiments.
Development of cell impermeable ENPP 1inhibitors
To investigate the physiological relevance of extracellular cGAMP and why it needs to be regulated by specific hydrolases, we attempted to modulate its concentration by pharmacological inhibition of ENPP 1. We first tested the nonspecific ENPP 1inhibitor QS1 (FIG. 10, panel A) (Patel, S.D. et al, Quinazolin-4-piperidine-4-methyl amides PC-1 inhibitors: adapting hERG intermediates through structured base design, Bioorganic Chem. chem.Lett.19, 3339-3343 (2009), and Shayidin, E.E. et al, Quinazolin-4-piperidine amides specific inhibitors of human NPP1 and predictive Pathology of functional cells Br. J.172, 4189-4199). Although QS1 can inhibit extracellular cGAMP degradation in cells overexpressing ENPP1, it also partially blocked cGAMP export in ENPP1 knockout cells (fig. 10, panel B). QS1 treated cells have elevated intracellular cGAMP, again demonstrating that output is an important mechanism for maintaining cGAMP homeostasis in cancer cells. In our export studies, export blocking activity precluded QS1 as a tool for studying extracellular cGAMP. Phosphonic acid (Phosphonate) analogues (compound 1) are intended to chelate Zn at the catalytic site of ENPP1 2+And minimizes cell permeability and avoids intracellular miss-targeting (fig. 3, panel a). K of Compound 1i,appAt 110 ± 10nM (fig. 3, panel B), which is about 60 times more potent than QS1 (fig. 10, panel a).
We confirmed the compounds by performing three independent permeability assays1 is cell impermeable: parallel Artificial Membrane Permeability Assay (PAMPA) (fig. 11, panel a); intestinal cell Caco-2 permeability assay (fig. 11, panel B); and epithelial cell MDCK permeability assay (fig. 11, panel C). Compound 1 belongs to the category of impermeable compounds in all three assays, compared to a control compound with high and low cell permeability. In addition, it is directed against the closely related ectonucleotidase alkaline phosphatase (K)i,app>100 μ M) and ENPP2 (K)i,app5.5 μ M) was lower (fig. 11, panel D). Although we do not want compound 1 to be off-target inside the cell due to its low cell permeability, we tested its binding to a panel of 468 kinases to further determine its specificity. Although structurally similar to AMP, compound 1 bound only two kinases at 1 μ M (fig. 11, panel E). Compound 1 also shows high stability in human and mouse liver microsomes (t) 1/2>159 min). In summary, we demonstrate that compound 1 is a potent, cell-impermeable, specific, stable ENPP1 inhibitor.
Then, we measured the efficacy of compound 1 in maintaining the extracellular cGAMP concentration in 293T cGAS cells overexpressing ENPP1 and obtained an IC of 340 ± 160nM50Values (fig. 3, panel C) where 10 μ M was sufficient to completely block extracellular cGAMP degradation (fig. 3, panel D). Unlike QS1, compound 1 had no effect on intracellular cGAMP, suggesting that it did not affect cGAMP output (fig. 3, panel D). Thus, compound 1 is an excellent tool compound for ENPP1 inhibitors, specifically increasing extracellular cGAMP concentrations.
Finally, we tested compound 1 for enhancement of CD14+The potency of PBMC in terms of extracellular cGAMP signaling that could be detected. We first demonstrated that compound 1 was not toxic to PBMC at the concentrations used (fig. 11, panel F). Conditioned media from 293T cGAS cells overexpressing ENPP1 failed in CD14+IFNB1 expression was induced in cells (fig. 3, panels E and F). However, compound 1 restored extracellular cGAMP levels and CD14 in the culture medium+Induction of IFNB1 expression in cells (fig. 3, panel F). These results indicate that the enzymatic activity of ENPP1 (rather than a potential scaffold effect as a transmembrane protein) is inhibited Manufacture CD14+PBMC response to extracellular cGAMP. Taken together, our data indicate that extracellular cGAMP levels can be reduced by ENPP1 expression and increased by ENPP1 inhibition, which affects CD14 in vitro+Activation of PBMC.
Fig. 3, panels a to F: activity of cell impermeable ENPP1 inhibitor. a, chemical structure of compound 1. b, in order to32Inhibitory Activity of Compound 1 on purified mouse ENPP1 at pH 7.5 with P-cGAMP as substrate (K)i,app110 ± 10 nM). Mean ± SEM (n ═ 3 independent experiments), some error bars were too small to visualize. c, Compound 1 vs 293T cGAS ENPP1-/-Inhibitory Activity (IC) of human ENPP1 transiently expressed in cells50340 ± 160 nM). Mean ± SEM (n ═ 2). d 293T cGAS ENPP1 transfected with empty pcDNA vector or vector containing human ENPP1 in the presence or absence of 10. mu.M Compound 1-/-Intracellular and extracellular cGAMP concentrations of cells. BQL ═ below the limit of quantitation. Mean ± SEM (n ═ 3). P<0.0001 (one-way ANOVA). Data are representative of two independent experiments. e, schematic representation of the conditioned medium experiments. 293T cGAS ENPP1Is low inCells were transfected with vectors containing human ENPP1 and incubated in the presence or absence of compound 1. Transfer of conditioned media from these cells to primary CD14 +Human PBMC. f, IFNB1 mRNA levels were normalized to CD14 and compared to untreated CD14+Cells were counted for fold induction. Mean ± SEM (n ═ 2). P ═ 0.007 (one-way ANOVA). cGAMP concentrations were measured in conditioned media. Mean ± SEM (n ═ 2). P ═ 0.006 (one-way ANOVA). Data are representative of two independent experiments.
Fig. 10, panels a to B: improvement of Compound 1 over QS1
a, structure of QS1 and its use at pH 7.532Inhibitory Activity of P-cGAMP as substrate on purified mouse ENPP1 (compared with Compound 1) (QS 1Ki,app6.4 ± 3.2 μ M). Mean ± SEM (n ═ 2 independent experiments). b 293T cGAS ENPP1 transfected with an empty vector or a vector containing human ENPP1 in the presence or absence of QS1-/-Intracellular, extracellular and total cGAMP of cells. Mean value ± SEM (n ═ 2)).*P<0.05.**P<0.01 (one-way ANOVA).
Fig. 11, panels a to F: compound 1 is cell impermeable, specific for ENPP1, and non-toxic. a, permeability of compound 1 in artificial membrane permeability assay (PAMPA).
b, permeability of Compound 1 in intestinal cell Caco-2 assay. PA as peak area and IS as internal standard. Compounds (including compound 1), atenolol (low passive permeability negative control) and propranolol (high passive permeability positive control) were incubated on top of Caco-2 monolayers for 2 hours. The compound concentration outside the substrate was monitored by LC-MS/MS. Apparent permeability (P) was calculated from the slope app). Data are representative of two independent experiments. c, permeability of compound 1 in the epithelial cell MDCK permeability assay. d, inhibitory activity of compound 1 on alkaline phosphatase (ALPL) and ENPP 2. Mean ± SEM (n ═ 2). e, kinase panel interaction plot for compound 1 (468 kinases tested), kinase inhibition is described as a percentage of control. Using TREETMSoftware tool generates images, via KINOME(a division of the Discovex company) permit reprinting.DiscoverX corporation 2010. f, cell viability as determined by CellTiterGlo. Total PBMC and CD14+PBMCs were incubated with compound 1 for 16 hours and then ATP levels were detected using CellTiterGlo. Data were normalized to no compound 1 to calculate percent cell viability.
Cancer cells express cGAS and continuously export cGAMP in culture
To determine whether extracellular cGAMP can serve as a danger signal for secretion by cancer cells in vivo, we first attempted to determine tumor models that export cGAMP. We tested one human cancer cell line (MDA-MB-231) and three mouse cancer cell lines (E0771, MC38 and 4T1-Luc, which are luciferase-expressing 4T1 cell lines for in vivo imaging) in culture, all of which express cGAS (FIG. 4, Small Fig. a). Intracellular cGAMP concentrations in these cells are difficult to detect. However, we were able to detect 5.8x 10 in 4T1-Luc cells by additional concentration and purification steps-10nmol/cell (. about.150 nM) intracellular cGAMP (FIG. 4, panel B). Knockdown of cGAS using shRNA resulted in decreased cGAS protein levels and decreased intracellular cGAMP levels, demonstrating that cGAS expression controls the amount of cGAMP present in 4T1-Luc cells (fig. 12, panels a and B). Using compound 1 to inhibit cell surface and soluble ENPP1 in cell culture medium, we detected sustained cGAMP output in all these cell lines and extracellular cGAMP levels reached approximately 6x 10 within 48 hours-9nmol/cell (about 10nM when diluted into the medium) (fig. 4, panels C and D and fig. 12, panels C and D). Notably, it is approximately 10 times the amount of cGAMP present in cells, suggesting that cancer cells effectively eliminate their cGAMP by export. Ionizing Radiation (IR) can increase cytoplasmic DNA and activate cGAS-dependent IFN- β production in tumor cells (see, e.g., Bakhoum, S.F. et al, digital chromosomal instability peptides with specificity to radiation therapy. Nat. Commun.6, 1-10 (2015), and Vanpoulle-Box, C. et al, DNA exoenzyme Trex1 regulation of radiotherapy-induced expression immunogenicity. Nat. Commun.8,15618 (2017)). Indeed, IR treatment also increased extracellular cGAMP production in 4T1-Luc cells after 2 days (fig. 4, panel E and fig. 12, panel E). Taken together, our data indicate that these cancer cell lines are constantly producing and efficiently exporting cGAMP, and can be stimulated with IR to produce more extracellular cGAMP.
Fig. 4, panels a to E: cancer cells express cGAS and continually export cGAMP in culture. a, cGAS expression of 4T1-Luc, E0771, MDA-MB-231 and MC38 was analyzed by Western blot. b, estimation of intracellular cGAMP concentration in 4T1-Luc cells without exogenous stimulation. Mean ± SEM (n ═ 2). c, extracellular cGAMP produced by MC38 cells within 48 hours. At time 0, cells were refreshed with medium supplemented with 50 μ M compound 1. Mean ± SEM (n ═ 2). Data are representative of two independent experiments. d, extracellular cGAMP produced by 4T1-Luc, E0771 and MDA-MB-231 cells was measured after 48 hours in the presence of 50. mu.M Compound 1. BQL ═ below the limit of quantitation. Mean ± SEM (n ═ 2). e, extracellular cGAMP produced by 4T1-Luc cells within 48 hours. At time 0, cells were untreated or treated with 20Gy IR and refreshed with medium supplemented with 50 μ M compound 1. Mean ± SEM (n ═ 2). P ═ 0.04 (student's t test).
Fig. 12, panels a to E: cancer cells continuously export cGAMP in culture. a, cGAS expression was analyzed by western blot for 4T1-Luc WT and 4T1-Luc shcGAS cell lines. b, intracellular cGAMP from 4T1-Luc WT and 4T1-Luc shcGAS cell lines without exogenous stimulation. Mean ± SEM (n ═ 2). 4T1-Luc WT data are reproduced from FIG. 4, panel b for comparison. c, fig. 4, panel c shows the extracellular cGAMP (expressed in media concentration units) of the experiment. d, fig. 4, panel d shows the extracellular cGAMP (expressed in media concentration units) of the experiment. BQL ═ below the limit of quantitation. Mean ± SEM (n ═ 2). e, fig. 4, panel c, extracellular cGAMP (expressed in media concentration units) for the experiments shown. Mean ± SEM (n ═ 2). P ═ 0.004 (student's t test).
Isolation (Sequestration) of extracellular cGAMP reduces tumor-dependent cGAS and host STING Related dendritic cells
In tumors, the extracellular space is estimated to be 0.3-0.8 times the volume of the intracellular space28. However, in cell culture, the volume of the extracellular space is approximately 250-fold and 1000-fold the volume of the intracellular space. We are every 1x 1061mL of medium was used for each cell, and it was estimated that a cell volume of approximately 1-4pL was used for this calculation. Thus, our cell culture system diluted the extracellular space by 300-3000 fold compared to the tumor microenvironment. Given this dilution factor and our measurement of nanomolar extracellular cGAMP exported by cancer cells in vitro, we predicted that extracellular cGAMP in the tumor microenvironment could reach the micromolar range, which could lead to innate immune recognition of tumor cells. Recognizing the limitations of our in vitro cell experiments, we turned to in vivo experiments to study the effects of extracellular cGAMP (fig. 5, panel a). First, we attempted to exploit cGAS in the C57BL/6 background by knocking out cGAS in tumor cells (fig. 13, panel a)-/-And STING-/-Mouse come trueThe importance of the tumor versus the host cGAMP. We also developed a neutralizing agent as a tool to specifically sequester extracellular cGAMP (fig. 5, panel a). We used the soluble cytoplasmic domain of STING (FIG. 5, panel B) at a K of 73. + -. 14nM dcGAMP was bound (fig. 5, panel C). We also generated the R237A mutant STING (see, e.g., Gao, P. et al, Cell 154, 748-762 (2013)) as a non-binding STING control (FIG. 5, panels B-D). To test the neutralizing efficacy of these proteins in cell culture, we used CD14+PBMC. Wild-type (WT) STING (neutralization STING) can be predicted at 2: 1 stoichiometric ratio neutralized extracellular cGAMP, whereas unbound STING had no effect even at 200-fold higher concentrations (fig. 5, panel E).
We established an E0771 orthotopic tumor in mice, followed by intratumoral injection of neutralizing STING to deplete extracellular cGAMP, and resection of the tumor to stain tumor-associated leukocytes. Neutralization of STING significantly reduced total CD45 in WT E0771 tumors+/MHC-II+CD11c in a population of tumor associated Antigen Presenting Cells (APCs)+Dendritic cell population, indicating that extracellular cGAMP can be detected by the immune system (fig. 5, panels F and G and fig. 13, panel B). When tumors were in cGAS-/-Extracellular cGAMP depletion also decreased CD11c upon growth in mice+Population, indicating that the host cells did not significantly contribute to extracellular cGAMP production (fig. 5, panel G and fig. 13, panel B). In contrast, when cGAS is used-/-E0771 cells (multiple clones pooled to achieve clean knockdown, but with minimal cloning effect) or STING -/-In mice, extracellular cGAMP depletion did not affect CD11c+And (4) clustering. This suggests that tumor cells, but not host cells, are the major producers of extracellular cGAMP, which is then sensed by the host STING (fig. 5, panel G and fig. 13, panel B). We also tested the 4T1-Luc tumor model in situ against a BALB/c background. Although cGAS and STING knockout strains have not been established in this context, we knocked out cGAS in 4T1-Luc tumors. Intratumoral injection of neutralizing STING into WT 4T1-Luc tumors significantly reduced CD45+/MHC II+Tumor-associated CD11c in the population+Group (fig. 5, panel H, fig. 13, panel C). In contrast, extracellularcGAMP depletion vs cGAS-/-4T1-Luc tumors had no effect (FIG. 5, panel H and FIG. 13, panel C). We also consumed extracellular cGAMP by intratumoral injection of mENPP1 protein (fig. 8, panel D), and again observed CD45+/MHC II+CD11c in the group+Cell depletion (fig. 5, panel I and fig. 13, panel D). Together, our results for the E0771 and 4T1-Luc models demonstrate that extracellular cGAMP produced by tumor cells activates the innate immune response in a host STING-dependent but host cGAS-independent manner. Taken together, our data indicate that extracellular cGAMP produced by cancer cells is a danger signal for triggering innate immune responses.
Fig. 5, panels a to I: the sequestration of extracellular cGAMP reduces tumor-associated dendritic cells in a tumor cGAS and host STING-dependent manner. a, experimental setup to evaluate the effect of extracellular cGAMP in vivo. b, Coomasie gel of recombinant mouse WT STING and R237A STING. c, binding curves of mouse WT STING (neutralizing) and R237A STING (non-binding) cytoplasmic domains by using radiolabels35Membrane binding assay determination of S-cGAMP as probe. Mean ± SEM (n ═ 2, from two independent experiments). d, crystal structure of mouse WT STING complexed with cGAMP, with R237 highlighted in pink (PDB ID 4 LOJ). e, treatment of CD14 with 2. mu.M cGAMP in the presence of neutralized or unbound STING (2. mu.M to 100. mu.M, 2.5 fold dilution)+IFNB1 mRNA fold induction in PBMC. Mean ± SEM (n ═ 2 qPCR technique replicates). f, WT or cGAS on day 0-/-E0771 cells (1X 10)6) In situ injection into WT, cGAS-/-Or STING-/-C57BL/6J mice. Intratumoral injection of neutralized STING (WT mice n-5; cGAS) on day 2-/-Mouse n-5; STING-/-Mouse n-4) or unbound STING (WT mouse n-5; cGAS-/-Mouse n-4; STING-/-Mouse n-5). Tumors were harvested on day 3 and analyzed by FACS. The samples were tested in FSC-A/SSC-A, singlet (FSC-W), viable cells, CD45 +、MHC II+、CD11c+Gating is performed on cells in the population. g, CD11c of Total APC+Percentage of cells. Mean. + -. SD. P ═ 0.015 · P ═ 0.008(Welch t test). h, use WT (neutralization S)TING n is 3; unbound STING n ═ 2) or cGAS-/-4T1-Luc cells (n ═ 5) the same procedures as in (f) and (g) were performed in WT BALB/cJ mice. Mean. + -. SD. P is 0.011. I.e., 4T1-Luc cells (1X 10)6) WT BALB/cJ mice were injected in situ on day 0. Day 2 intratumoral injections of PBS (n-5) or recombinant mouse ENPP1(mENPP1) (n-6). Tumors were harvested on day 3 and analyzed by FACS. Mean. + -. SD. P ═ 0.033 (Welch t test).
Fig. 13, panels a to D: the sequestration of extracellular cGAMP reduces tumor-associated dendritic cells in a tumor cGAS and host STING-dependent manner. a, subcloned E0771 (left) and 4T1-Luc (right) cGAS from CRISPR knock-out pools-/-A cell. E0771 cGAS-/-Subclones 1, 2, 4, 6, 8, and 9 were mixed prior to injection into mice. 4T1-Luc cGAS-/-Subclones 4, 7 and 8 were mixed prior to injection into mice. b, geometric mean of experiments as shown in fig. 5 g. Mean ± SD ═ P0.049 (WT tumor/WT host). P ═ 0.015(WT tumor/cGAS)-/-Host) ((Welch t test). c, geometric mean of the experiment as shown in fig. 5 h. Mean ± SD. × P ═ 0.009(Welch t test). d, geometric mean of the experiments as shown in fig. 5 i. Mean ± SD <0.015(Welch t test).
Increasing extracellular cGAMP by decreasing ENPP1 activity increases dendritic cell infiltration and makes breast tumors more amenable to treatment Therapy
ENPP1 is highly expressed in some Breast cancers, and its levels correlate with poor prognosis (see, e.g., Lau, W.M. et al Enpp 1: A Potential facility of Breast Cancer Bone Metastasis. PLoS One 8, 1-5 (2013); Takahashi, R.U. et al Loss of microRNA-27b controls to break Cancer cell generation by activity ENPP1.Nat. Commin. 6, 1-15 (2015); and Umar, A. et al Identification of a reactive Protein Profile Association with Tamoxin therapeutic treatment in Cancer. cell. Protics 8,1278 (1294)). High ENPP1 expression may be a mechanism by which breast cancer depletes extracellular cGAMP and inhibits immunodetection. We measured ENPP1 activity in three triple negative breast cancer cells, 4T1-Luc, E0771 and MDA-MB-231, wherein MDA-MB-231 and 4T1-Luc showed high ENPP1 activity (FIG. 14, panel A). Therefore, we selected a triple negative, metastatic and in situ 4T1-Luc mouse model to investigate the effect of ENPP1 on tumor immunodetection, growth and therapeutic response.
We first tested the effect of ENPP1 on tumor infiltrating dendritic cells. We knocked out ENPP1 in 4T1-Luc cells, verified clones by lack of enzymatic activity (commercial ENPP1 antibody is not sensitive enough to verify knock-out), and merged multiple clones to minimize clonal effects (figure 14, panel B). After 4T1-Luc implantation, we treated the tumors with a 20Gy IR dose to induce cGAMP production, and resected the tumors 24 hours later to analyze their tumor-associated leukocyte composition. ENPP1-/-Tumors have larger tumor associated CD11c than WT tumors+Clusters (fig. 6, panel a, fig. 14, panel C). We then tested the effect of ENPP1 on 4T1-Luc tumor immune rejection. This is an invasive tumor model, usually metastasized to the lungs within two weeks after tumor implantation (pulski, B.A).&Ostrand-Rosenberg,S.Reduction of Established Spontaneous Mammary Carcinoma Metastases following Immunotherapy with Major Histocompatibility Complex Class II and B7.1 Cell-based Tumor Vaccines.Cancer Res.58,1486–1493(1998))。ENPP1-/-The tumor reaches 100mm3The previous initial tumor growth rate was the same as the WT tumor, indicating that we did not select a clone that grew slowly (fig. 14, panel D). However, the established ENPP1-/-Tumors were less invasive (fig. 6, panel B) and more sensitive to IR response (fig. 6, panel B). Without IR, the adaptive immune checkpoint blocker anti-CTLA-4 did not interact with ENPP1-/-Tumors were reduced synergistically (fig. 14, panels E and F). Strikingly, anti-CTLA-4 healed 40% of ENPP1 when we induced cGAMP production using IR -/-Tumors, but no cure for either WT tumor (fig. 6, panel C). Direct intratumoral injection of extracellular cGAMP in ENPP1-/-Tumors were more potent than WT tumors and 30% of mice were cured in synergy with IR (figure 6, panel D) and no anti-CTLA-4 was present. In conclusion, ENPP1 inhibits extracellular cGAMP, 4T1-Luc tumorsInnate immunity detects and negatively impacts their response to IR and adaptive immune checkpoint blockade.
Fig. 6, panels a to D: ENPP1-/-Tumor recruitment is innate immune-infiltration, less aggressive and more susceptible to IR and anti-CTLA-4 therapy. a, WT or ENPP1 on day 0-/-4T1-Luc cell (1X 10)6) Injected in situ into WT BALB/cJ mice (n-5 per group). Tumors were treated with 20Gy of IR on day 2. Tumors were harvested on day 3 and analyzed by FACS. Combining multiple ENPP1 prior to injection-/-4T1-Luc cell clones to minimize cloning effects. P ═ 0.008(Welch t test). b, the determined WT or ENPP1-/-4T1-Luc tumor (100 + -20 mm)3) Treatment with 0Gy or 20Gy IR once, followed by 3 intraperitoneal injections of IgG (n-9 for WT 4T1-Luc, and ENPP1 for ENPP 1) on days 2, 5, and 7 post-IR-/-4T1-Luc, n ═ 10). Tumor volume and Kaplan Meier curves are shown. P-values were determined by pairwise comparisons using day 20 post-hoc testing and Tukey adjustment (tumor volume) and the log rank Mantel-Cox test (Kaplan Meier). P <0.0001.c, WT or ENPP1 determined-/-4T1-Luc tumor (100 + -20 mm)3) Treatment was once with 0Gy or 20Gy IR followed by 3 intraperitoneal injections of anti-CTLA-4 (n ═ 10 for all groups) on days 2, 5 and 7 post IR. Tumor volume and Kaplan Meier curves are shown. P-values were determined by pairwise comparisons using day 20 post-hoc testing and Tukey adjustment (tumor volume) and the log rank Mantel-Cox test (Kaplan Meier). P<0.0001. In ENPP1-/-In the 4T1-Luc + IR (20) + anti-CTLA-4 treatment group, 4/10 (40%) mice were tumor-free survivors verified by bioluminescence imaging. d, established infection with 4T1-Luc tumor or ENPP1 scrambled with sgRNA sequence-/-4T1-Luc tumor (100 + -20 mm)3) Treatment was once with 20Gy IR, followed by 3 intratumoral injections of 10 μ g cGAMP (n ═ 10, two groups) on days 2, 4, and 7 post-IR. Tumor volume and Kaplan Meier curves are shown. P-values were determined by pairwise comparisons using day 20 post-hoc testing and Tukey adjustment (tumor volume) and the log rank Mantel-Cox test (Kaplan Meier). P<0.05,****P<0.0001. In ENPP1-/-4T1-Luc + IR (20) cGAMP treatmentIn the group, 3/10 (30%) mice were tumor-free survivors verified by bioluminescence imaging. Mice from different treatment groups in b-d were co-housed and the experimenter was blinded.
Fig. 14, panels a to F: established ENPP1-/-Tumors cause an increase in tumor-associated dendritic cells, are less aggressive, and are more susceptible to IR and anti-CTLA-4 therapy. a, use of32P-cGAMP degradation activity of ENPP1 in 4T1-Luc, E0771 and MDA-MB231 cells was determined. Data are representative of three independent experiments. b, use of32P-cGAMP degradation assay validation ENPP1-/-4T1-Luc clone. Lysates from different clones were normalized by protein concentration. ENPP1 was injected into mice-/-4T1-Luc clones 2-6 and 13-18 were pooled. c, geometric mean of the experiments shown in fig. 6 a. Mean ± SD ═ P0.012 (Welch t test). d, (left) WT (n 55) and ENPP1-/-(n-55) tumor volume of 4T1-Luc cells on the day of treatment; (Right) initial tumor growth rate expressed as up to 100mm3±20mm3Tumor volume required for size/day. Mean ± SD (Welch t test). e, bioluminescence image of tumor-bearing mice. f, the data shown in FIGS. 6a, b are re-plotted to highlight the comparison between the IgG and anti-CTLA-4 treatment groups. Established WT or ENPP1 was treated with 3 intraperitoneal injections of IgG or anti-CTLA-4 on days 2, 5, and 7 after tumors reached desired size-/-4T1-Luc tumor (100 + -20 mm)3) (n-9 for WT 4T1-Luc + IgG, and n-10 for all other groups). Tumor volume and Kaplan Meier curves are shown. P-values were determined by pairwise comparisons using day 20 post-hoc testing and Tukey adjustment (tumor volume) and the log rank Mantel-Cox test (Kaplan Meier).
Our genetic results indicate that ENPP1 is a potential target for pharmacological inhibition. Our developed ENPP1 inhibitor (compound 1) showed rapid clearance upon intratumoral injection. Without extensive research by pharmaceutical companies on the route of administration and corresponding formulation optimization, which is usually done later in drug development, we asked whether compound 1 has an effect in vivo. We injected tumors with compound 1 immediately after IR treatment and observed tumor-associated CD11c+Population increased after 24 hours (fig. 7, panel a and fig. 15). Notably, compound 1 synergized with IR and anti-CTLA-4 to achieve a cure rate of 10% (figure 7, panel B). Finally, compound 1 was observed to shrink tumors synergistically with IR and cGAMP, prolong survival, and achieve a cure rate of 10% (fig. 7, panel C). Taken together, these results indicate that ENPP1 can be pharmacologically targeted to enhance innate immune recognition of cancer.
Fig. 7, panels a to C: ENPP1 inhibition exerts an anti-tumor effect in synergy with IR therapy and anti-CTLA-4. a, 4T1-Luc cells (1x 10)6) WT BALB/cJ mice were injected in situ on day 0. Tumors were treated with 20Gy IR and intratumorally injected on day 2 with either PBS (n-4) or compound 1 (n-5). Tumors were harvested on day 3 and analyzed by FACS. P ═ 0.047(Welch t test). b, the determined 4T1-Luc tumor (100 +/-20 mm) 3) Treatment with 20Gy IR once followed by 3 intratumoral injections of PBS or compound 1 on days 2, 4 and 7 and intraperitoneal injection of anti-CTLA-4 on days 2, 5 and 7 (n ═ 17-19 for all treatment groups). Tumor volume and Kaplan Meier curves are shown. P-values were determined by pairwise comparisons using day 40 post-hoc testing and Tukey adjustment (tumor volume) and the log rank Mantel-Cox test (Kaplan Meier). c, the determined 4T1-Luc tumor (100 +/-20 mm)3) Treatment was once with 20Gy IR, followed by 3 separate intratumoral injections of cGAMP or cGAMP + compound 1 on days 2, 4, and 7 post-IR (n ═ 9 per treatment group). Tumor volume and Kaplan Meier curves are shown. P-values were determined by pairwise comparisons using day 40 post-hoc testing and Tukey adjustment (tumor volume) and the log rank Mantel-Cox test (Kaplan Meier).
Figure 15 shows ENPP1 inhibition increases tumor-associated dendritic cells synergistically with IR treatment. The geometric mean of the experiment is shown in figure 7, panel a. Mean. + -. SD. P <0.05(Welch t test).
FIG. 16: different patterns of cGAMP delivery from synthetic cells to target cells. (1) Propagating through gap junctions; (2) packaging into a budding virus particle and transmitting in the next round of infection; and (3) export to the extracellular space.
FIG. 17: cGAMP is a cancer risk signal. APC can sense tumor cells through different cGAS-dependent mechanisms: (1) activation of APC cGAS by tumor-derived dsDNA, (2) APC sensing of type I IFN secreted by tumor cells, and (3) APC sensing of cGAMP is constitutively produced and exported by tumor cells.
Discussion of the related Art
The results of the present disclosure provide evidence of cGAMP output. Intercellular cGAMP transfer can occur through gap junctions and viral particles. The present disclosure provides in vitro and in vivo evidence that cGAMP can cross the extracellular space (see, e.g., fig. 16). cGAMP output is a marker for cancer cells, as all cell lines we tested can synthesize and export cGAMP without external stimuli. Since Chromosomal instability and abnormal cytoplasmic dsDNA are considered intrinsic to tumors, tumor cells rarely inactivate cGAS (see, e.g., Bakhoum, s.f., et al, chromosome activity drive across tumor a cytopolic DNA response, nature 553, 467-. Since no cytosolic cGAMP hydrolase has been identified and ENPP1 is unable to degrade intracellular cGAMP, export is currently the only mechanism for cGAMP removal from the cytosol and represents another approach to turn off intracellular STING signaling in addition to ubiquitin-mediated STING degradation (see, e.g., Konno, h., Konno, K. & Barber, g.n. cyclic nucleotides trigger ULK1(ATG1) phosphorylation of STING to predicted contained in animal signaling. cell 155,688 + 698 (2013)). However, this clearance mechanism exposes the cancer cells to immunoassay.
Indeed, our results indicate that cGAMP exported by cancer cells is a danger signal detected by the immune system. New antigens from cancer cells are presented by APC to cross (cross) primary cytotoxic CD8+T cells, ultimately performing cancer-specific killing. However, there is little understanding of how APCs initially detect cancer cells. Immunogenic tumors release dsDNA as a vaccine to CD11c+Danger signal of Dendritic Cells, an important APC type (see, e.g., Xu, M.M. et al, Dendritic Cells but Not Macrophages Sense Tumor Mitochondrial DNA for Cross-prim)Immunity 47, 363-373 (2017)). In addition, cancer cells respond to radiation-induced autoplasmatic dsDNA and produce IFN as a danger signal (vanpoulle-Box, c. et al DNA exoenzyme Trex1 regulation radiotherapeutics-induced tumor immunity. nat. commun.8,15618 (2017)). The catalytic activity of Tumor cGAS correlates in a host STING-dependent manner with Tumor immunity in the B16 melanoma model (see, e.g., Marcus, a. et al, Tumor-Derived cGAMP triggerrs a STING-Mediated Interferon Response in Non-Tumor Cells to activity the NK Cell Response, immunity 49,754-763. e4(2018)), suggesting that cGAMP can metastasize from Tumor Cells to host Cells, but the mechanism is unknown. Here we provide direct evidence that cancer cells produce soluble extracellular cGAMP as a danger signal, leading to an increase in the number of dendritic cells in the tumor microenvironment (fig. 17). cGAMP output is an important mode of cGAMP communication between cells that are not physically connected but are in close proximity. Unlike cytokines, cGAMP cannot travel long distances in the extracellular space without being degraded and/or diluted below its effective concentration. This property is shared with neurotransmitters, making cGAMP the first defined neurotransmitter.
Therefore, if cGAMP cannot be rapidly cleared, release of cGAMP into the extracellular space is a fatal problem for cancer. We demonstrate that ENPP1 down-regulates extracellular cGAMP signaling and its downstream anti-cancer immune activation in mice in vitro. Since tumor-derived soluble cGAMP is freely diffusible, overexpression of ENPP1 on one cell surface certainly scavenges cGAMP in the nearby microenvironment and provides adaptability to its neighborhood. In humans, the level of ENPP1 expression in breast cancer is associated with drug resistance (see, e.g., Umar, a. et al mol. cell. proteomics 8, 1278-1294 (2009)), bone metastasis (see, e.g., Lau, w.m. et al PLoS One 8, 1-5 (2013)), and poor prognosis (Takahashi, r.u. et al nat. commu.6, 1-15 (2015)). ENPP1 can be targeted for inhibition as an innate immune checkpoint for cancer immunotherapy.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Accordingly, the foregoing merely illustrates the principles of the invention. It will thus be appreciated that those skilled in the art will be able to devise various arrangements that, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention is embodied by the appended claims.
Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention is embodied by the appended claims. In the claims, 35u.s.c. § 112(f) or 35u.s.c. § 112(6) being expressly defined only when the precise phrase "method for … …" or the precise phrase "step for … …" is referred to at the beginning of such limitations in the claims in order to limit the limitations in the claims; 35u.s.c. § 112(f) or 35u.s.c. § 112(6) are not cited if no such accurate phrases are used in the limitations of the claims.
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