Novel AUTOTAC chimeric compounds and compositions comprising the same for preventing, ameliorating or treating diseases through targeted protein degradation
阅读说明：本技术 新autotac嵌合化合物及包含其的通过靶向的蛋白质降解来预防、改善或治疗疾病的组合物 (Novel AUTOTAC chimeric compounds and compositions comprising the same for preventing, ameliorating or treating diseases through targeted protein degradation ) 是由 权容兑 池昌勋 斯利尼瓦斯劳·甘尼皮瑟蒂 金希妍 文修兰 郑灿勋 郑宜静 成基云 于 2019-07-24 设计创作，主要内容包括：本发明涉及一种自噬靶向性嵌合(AUTOTAC)化合物,其中新颖的p62配体和靶结合配体通过连接子连接,或者该AUTOTAC化合物的立体异构物、溶剂化物、水合物或前药；以及包含其作为活性成分的用于通过靶向蛋白降解来预防或治疗疾病的药物组合物或食品组合物。本发明不仅允许采用靶向的特定蛋白质的浓度调节,而且允许将药物和其它低分子量化合物递送至溶酶体。根据本发明的AUTOTAC化合物选择性移除特定蛋白质且因此可有用地用作预防、改善或治疗各种疾病的药物组合物。(The present invention relates to an autophagy-targeting chimeric (AUTOTAC) compound, wherein a novel p62 ligand and a target binding ligand are linked by a linker, or a stereoisomer, solvate, hydrate or prodrug of the AUTOTAC compound; and a pharmaceutical composition or food composition for preventing or treating diseases by targeted protein degradation comprising the same as an active ingredient. The present invention not only allows for concentration modulation of specific proteins that are targeted, but also allows for the delivery of drugs and other low molecular weight compounds to lysosomes. The AUTOTAC compound according to the present invention selectively removes a specific protein and thus may be usefully used as a pharmaceutical composition for preventing, improving or treating various diseases.)
1. A chimeric compound comprising a p62 ligand, a Target Binding Ligand (TBL), and a linker.
2. The chimeric compound of claim 1, wherein the chimeric compound has the structure of the following chemical formula 1,
[ chemical formula 1]
Connector
Wherein the content of the first and second substances,
a represents a target binding ligand, B represents a p62 ligand, and A and B are linked to a linker.
3. The chimeric compound of claim 1, wherein the target-binding ligand is one or more selected from the group consisting of the following compounds, or has a derivative structure derived from these structures:
structure of target binding ligand:
4. the chimeric compound of claim 1, wherein the p62 ligand has the structure of formula 2 below or a derivative thereof:
[ chemical formula 2]
Wherein the content of the first and second substances,
w is a C6-C10 aryl group;
l is- (CH)2)n1-or-O- (CH)2)n2-CH (OH) -, with the proviso that-O- (CH)2)n2O in-CH (OH) -is bonded to a benzene ring, wherein n1 is an integer of 1 to 4;
n2 is an integer from 1 to 4;
m is an integer of 0 to 2;
Rais R1OR-OR1，
Wherein R is1Is hydrogen or- (CH)2)n3-R'1，
R'1Is unsubstituted phenyl or is substituted by hydroxy, halogen, C1-4Alkyl radical, C1-4Alkoxy, nitro, amino, (C)1-4Alkyl) amino or di (C)1-4Alkyl) amino-substituted phenyl groups,
n3 is an integer from 1 to 6;
Rbis-OR2，
Wherein R is2Is hydrogen or- (CH)2)n4-R'2，
R'2Is unsubstituted phenyl or is substituted by hydroxy, halogen, C1-4Alkyl radical, C1-4Alkoxy, nitro, amino, (C)1-4Alkyl) amino or di (C)1-4Alkyl) amino-substituted phenyl groups,
n4 is an integer from 1 to 6;
Rcis- (CH)2)n5-OH、-(CH2)n5-NH-C(＝NH)NH2、-C(＝NH)NH2、-CH(R3) -COOH or-CH (COO-R)4)-CH2CH2CH2-NH-C(＝NH)NH2、-(CH2)n5-O-(CH2)n5-OH、-CONH(CH2)n5-OH、-CO(CH2)n6-OH、-(CH2)n6-CH(NH2)-COOH、-(CH2)n6-CONH2，
n5 is an integer from 2 to 4,
n6 is an integer from 1 to 4,
R3is hydrogen or C1-4An alkyl group, a carboxyl group,
R4is C1-4Alkyl radical, and
Rdis hydrogen, halogen, C1-4Alkoxy or C1-4An alkyl group.
5. The chimeric compound of claim 1, wherein the linker has a-Q- (CH)2CH2O)x-(CH2)y-P-or-Q- (CH)2CH2CH2O)x-(CH2)y-P-or-Q- (CH)2CH2NH)x-(CH2)y-P-or-Q- (CH)2CH2CONH)x-(CH2)y-the structure of the P-group,
wherein Q comprises-NH-, -O-, ═ N-N (CH)3) And is a moiety that is modified by binding to the target binding ligand;
p comprises-NH-, -O-, -CH2-, -C (═ O) -, and is a moiety modified by binding to a P62 ligand;
x is an integer from 0 to 4; and is
y is an integer of 0 to 3.
6. The chimeric compound of claim 4,
w is phenyl;
l is- (CH)2)n1-or-O- (CH)2)n2-CH (OH) -, with the proviso that-O- (CH)2)n2O in-CH (OH) -is bonded to a benzene ring, wherein n1 is an integer of 0 to 1;
n2 is an integer from 1 to 2;
m is a whole of 0 to 2;
Rais hydrogen or-O- (CH)2)n3-R'1，
R'1Is unsubstituted phenyl or phenyl substituted by hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino or dimethylamino,
n3 is an integer from 1 to 4;
Rbis hydroxy or-O- (CH)2)n4-R'2，
R'2Is unsubstituted phenyl or phenyl substituted by hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino or dimethylamino,
n4 is an integer from 1 to 4;
Rcis- (CH)2)n5-OH、-(CH2)n5-NH-C(＝NH)NH2、-C(＝NH)NH2、-(CH2)n5-O-(CH2)n5-OH、-CONH(CH2)n5-OH、-CO(CH2)n6-OH、-(CH2)n6-CH(NH2) -COOH or- (CH)2)n6-CONH2，
n5 is an integer from 2 to 3,
n6 is an integer of 1 to 2, and
Rdis hydrogen, halogen, C1-2Alkoxy or C1-2An alkyl group.
7. The chimeric compound of claim 5, wherein the bond between P and the P62 ligand is-CONH-, -O-, -NH-, -NHCO-, or-COO-.
8. The chimeric compound of claim 1, represented by a compound selected from the group consisting of compounds 1 to 13:
1) (2E,4E,6E,8E) -N- (2- (2- (2- (((R) -3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) amino) ethoxy) ethyl) -3, 7-dimethyl-9- (2,6, 6-trimethylcyclohex-1-en-1-yl) non-2, 4,6, 8-tetraacrylamide;
2) (2E,4E,6E,8E) -N- (2- (2- (2- ((3, 4-bis (benzyloxy) benzyl) amino) ethoxy) ethyl) -3, 7-dimethyl-9- (2,6, 6-trimethylcyclohex-1-en-1-yl) non-2, 4,6, 8-tetraalkenamide;
3) (R) -N- (15- (3, 4-bis (benzyloxy) phenoxy) -14-hydroxy-3, 6, 9-trioxa-12-azepentadecyl) -4-phenylbutanamide;
4) n- (1- (3, 4-bis (benzyloxy) phenyl) -5,8, 11-trioxa-2-azatridecan-13-yl) -4-phenylbutanamide;
5)3- (3- (benzo [ d ] [1,3] dioxol-5-yl) -1H-pyrazol-5-yl) -N- (2- (2- (2- ((3- ((4-fluorobenzyl) oxy)) benzyl) amino) ethoxy) ethyl) aniline;
6) (3R,4S,5S,6R) -5-methoxy-4- ((2R,3R) -2-methyl-3- (3-methylbut-2-en-1-yl) oxiran-2-yl) -1-oxaspiro [2.5] octan-6-yl (13E,15E,17E,19E) -1- (3- (phenylmethoxy) phenyl) -12-oxo-5, 8-dioxa-2, 11-diaza-heneicosyl-13, 15,17, 19-tetraen-21-oic acid ester;
7)3- (3, 5-dichlorophenyl) -5- ((R) -15- (3, 4-diphenylethoxyphenoxy) -14-hydroxy-6, 9-dioxa-3, 12-diazadecanyl) -5-methyloxazolidine-2, 4-dione;
8) (R) -1- (4- (benzyloxy) -3- (3-phenylpropyloxy) phenoxy) -3- ((2- (2- (2- (4- (2-phenyl-5, 7-bis (trifluoromethyl) pyrazolo [1,5-a ] pyrimidin-3-yl) phenoxy) ethoxy) ethyl) amino) propan-2-ol;
9) (R, Z) -4- ((2- (2- (2- ((3- (3, 4-diphenylethoxyphenoxy) -2-hydroxypropyl) amino) ethoxy) ethyl) imino) -2-phenyl-4H-chromene-5, 6, 7-triol;
10) (E) -5- (4- (2- (2- (2- ((3, 4-bis (benzyloxy) benzyl) amino) ethoxy) styryl) benzene-1, 3-diol;
11) (R) -2- (4- (benzo [ d ] thiazol-2-yl) phenyl) -14- (3, 4-bis (benzyloxy) phenoxy) -5, 8-dioxa-2, 11-diazatetram-13-ol;
12) (1E,6E) -1- (4- (2- (2- (2- (((R) -3- (3- (benzyloxy) -4-phenylethoxyphenoxy) -2-hydroxypropyl) amino) ethoxy) -3-methoxyphenyl) -7- (4-hydroxy-3-methoxyphenyl) hepta-1, 6-diene-3, 5-dione; and
13) (R) -1- (3-phenylethoxyphenoxy) -3- ((2- (2- (2- ((6- (trifluoromethoxy)) benzo [ d ] thiazol-2-yl) amino) ethoxy) ethyl) amino) propan-2-ol.
9. A pharmaceutical composition for preventing, improving or treating diseases, which comprises the compound of chemical formula 1, pharmaceutically acceptable salt, stereoisomer, solvate, hydrate or prodrug thereof according to any one of claims 1 to 8.
10. The pharmaceutical composition of claim 9, wherein the disease is cancer, a protein conformation disease, a rare disease, or a refractory disease.
11. The pharmaceutical composition of claim 9, which directly eliminates pathogenic proteins causing said disease.
12. The pharmaceutical composition of claim 10, wherein the protein conformation disease is a neurodegenerative disease, alpha-1 antitrypsin deficiency, keratopathy, retinitis pigmentosa, type 2 diabetes, or cystic fibrosis.
13. The pharmaceutical composition of claim 12, wherein the neurodegenerative disease is selected from the group consisting of: lyme borreliosis, fatal familial insomnia, creutzfeldt-jakob disease (CJD), Multiple Sclerosis (MS), dementia, alzheimer's disease, epilepsy, parkinson's disease, stroke, huntington's disease, pick's disease, Amyotrophic Lateral Sclerosis (ALS), spinocerebellar ataxia, other poly-Q diseases, hereditary cerebral amyloid angiopathy, familial amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), injection local amyloidosis, beta-2 microglobulin amyloidosis, hereditary non-neuropathic amyloidosis, alexander-law disease, and hereditary systemic amyloidosis of the finland type.
14. A pharmaceutical composition for autophagy delivery or degradation of pathogenic pathological proteins and misfolded proteins, comprising a compound of chemical formula 1, a pharmaceutically acceptable salt, stereoisomer, solvate, hydrate, or prodrug thereof of any one of claims 1 to 8.
15. A method of increasing degradation of a target protein comprising treating a cell or a p62 protein with the chimeric compound of any one of claims 1 to 8.
16. A method of increasing the degradation of organelles and structures comprising treating a cell or a p62 protein with the chimeric compound of any one of claims 1 to 8.
17. A method of increasing the degradation of viruses and bacteria that invade cells, comprising treating the cells or the p62 protein with the chimeric compound of any one of claims 1 to 8.
18. A method of delivering drugs or small molecule compounds to autophagy and lysosomes comprising treating cells or p62 protein with the chimeric compound of any one of claims 1 to 8.
19. A method of self-oligomerization and autophagy activation of p62 comprising treating a cell or p62 protein with the chimeric compound of any one of claims 1 to 8.
20. A method of increasing degradation by lysosomes by attaching specific proteins in cells to p62 and delivering them to autophagy, comprising treating the cells or the p62 protein with the chimeric compound of any one of claims 1 to 8.
21. A method of using a drug in which the chimeric compound of any one of claims 1 to 8 is linked to a therapeutic antibody that specifically binds to a protein exposed on a cell membrane and delivering the drug to a lysosome via an endosome.
Technical Field
The present invention relates to a novel chimeric compound, in particular a chimeric compound in which a p62 ligand and a target binding ligand are linked by a linker; and a pharmaceutical composition or food composition for preventing or treating diseases by targeted protein degradation comprising the same.
Background
The N-terminal canonical pathway is a proteolytic system in which a particular N-terminal residue of the protein serves as a degradation signal. The N-terminal regular degradation signal is through the type I basic residues including N-terminal arginine (Nt-Arg), lysine (Nt-Lys) and histidine (Nt-His); and type II hydrophobic residues including phenylalanine (Nt-Phe), leucine (Nt-Leu), tryptophan (Nt-Trp), tyrosine (Nt-Tyr), and isoleucine (Nt-Ile). These N-terminal residues bind to a specific N-recognizer (hereinafter referred to as N-ligand). The present inventors have for the first time discovered or cloned previously known N recognizers, namely UBR1, UBR2, UBR4 and UBR5, and found that they utilize the UBR cassette as a substrate recognition domain (Tasaki, T., et al, Mol Cell Biol 25,7120-36 (2005)). The inventors have also found that the UBR cassette binds to type I N-terminal canonical ligands (Nt-Arg, Nt-Lys, Nt-His) such as the N-terminal arginine residue to recognize the substrate and link the ubiquitin chain to this substrate. It was further found that UBR1 and UBR2 have N domains (Sriram, S.M., Kim, B.Y. & Kwon, Y.T., Nat Rev Mol Cell Biol 12,735-47(2011)) that play an important role in the binding of type 2N-terminal canonical ligands (Nt-Trp, Nt-Phe, Nt-Tyr, Nt-Leu, and Nt-Ile). The ubiquitinated substrate resulting from the binding between the N-recognizer and the N-terminal canonical ligand is delivered to the proteasome where it is degraded into short peptides. In this process, specific N-terminal residues (Nt-Arg, Nt-His, Nt-Lys, Nt-Trp, Nt-Phe, Nt-Tyr, Nt-Leu) are the primary determinants of binding, as the N-recognizer provides the majority of the hydrogen bonds required to target the N-terminal regular substrate (Sriram, S.M. & Kwon, y.t., Nat Struct Mol Biol 17,1164-5 (2010)).
Intracellular protein degradation is mainly carried out by the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system. In general, UPS can modulate the intracellular concentration of normally folded regulators or degrade misfolded and proteins that have lost their function. At this time, the substrate is estimated to be recognized and ubiquitinated directly or indirectly by about 500 to 1,000E 3 ligases, and then unfolded into a polypeptide helix and degraded by proteasomes (Ji and Kwon, Mol Cells 40,441-449 (2017)). In normal cells, the processes of the ubiquitin-proteasome system are smooth, but disease-related proteins become misfolded proteins that fold incorrectly, or aggregates produced by accumulation of misfolded proteins block proteasome function, or proteasome function declines during aging, or degradation of disease-related proteins does not proceed smoothly due to reprogramming of protein transcription and translation (Ciechanover, a. & Kwon, y.t., Exp Mol Med 47, e147 (2015)). In representative examples, the respective major pathological proteins of protein conformational diseases (alzheimer's disease, huntington's disease, parkinson's disease, human mad cow disease, greick's disease/amyotrophic lateral sclerosis, alpha-1 antitrypsin deficiency, keratopathy, type 2 diabetes, etc.) are misfolded, ubiquitinated and accumulated, while these excess protein wastes are converted back into aggregates (Aguzzi and O ' Connor, Nat Rev Drug Discov 9,237-48 (2010)). These particular muteins have the strong property of being converted into aggregates and therefore not being degraded into the proteasome as described above. The reason for this is that, because the proteasome has a narrow internal diameter of about 13 angstroms, a misfolded protein must be unfolded, but it cannot be unfolded when the protein is aggregated. In another representative example, cancer cells are known to increase transcription and translation of oncogenic proteins while inhibiting degradation by reprogramming intracellular transcription and translation (Xiong et al, J Cell Physiol 234, 14031-. Furthermore, due to the ubiquitin-proteasome system, the degradation of the subunit proteins and transmembrane proteins forming the complex is also limited.
Autophagy (autophagy) is the major intracellular protein degradation system in addition to UPS (ubiquitin-proteasome system). Autophagy is a protein degradation process that substantially maintains cell homeostasis and gene stability by degrading organelles of aging or damaged Cells or damaged or abnormally folded proteins and aggregates thereof (Ji and Kwon, Mol Cells 40,441-449 (2017)). In particular, when the case protein and its aggregates accumulate in the cytoplasm, it may become a cytotoxic substance, and thus should be received and degraded by autophagy. The mechanisms of autophagy are mainly divided into major autophagy (macroautophagy), minor autophagy (microaautophagy) and concomitant protein-mediated autophagy, and they are divided into major autophagy and selective autophagy depending on the purpose of degrading intracellular substrates (Dikic, I. & Elazar, z., Nat Rev Mol Cell Biol 19, 349-. Among these, selective autophagy and the accompanying protein-mediated autophagy cause the selective degradation of undesirable intracellular proteins and dysfunctional organelles. The development of new therapies based on diseases that accumulate pathologically misfolded proteins and dysfunctional organelles by inducing selective autophagy is currently establishing a new paradigm. The p62/SQSTM1/Sequestosome-1 protein is crucial for initiating the formation of autophagosomes as mediators in the mechanism of selective autophagy and for delivering the contents. At this point, the p62 protein binds to pathological proteins and their aggregates, which are then delivered to autophagosomes. p62 undergoes self-oligomerization ((Dikic, I. & Elazar, z., Nat Rev Mol Cell Biol 19,349-364(2018)) as a key process in delivering pathological proteins to autophagosomes at which time the pathological proteins are concentrated together to reduce volume, thereby facilitating degradation using autophagy PB1 region regulates self-oligomerization of p62, but its regulatory mechanism is not well known.
By the mechanisms described above, autophagy is critical for maintaining cell homeostasis by modulating intracellular changes in damaged proteins and cellular organelles. When autophagy function is diminished, it leads to accumulation and aggregation of misfolded proteins, resulting in protein conformational diseases or cancer. Studies on the activation of global autophagy to treat these diseases continue to be actively carried out (Ciechanover, a. & Kwon, y.t., Exp Mol Med 47, e147 (2015)). A typical modulator that inhibits whole body autophagy is mTOR. The method of activating autophagy using mTOR inhibitors is most widely used (Jung, c.h., Ro, s.h., Cao, j., Otto, N.M. & Kim, d.h., FEBS Lett 584,1287-95 (2010)). Specifically, amyloid β (Ab) and tau can be eliminated while improving cognitive ability in AD animal models overexpressing APP by treatment with rapamycin (rapamycin) (Caccamo, a., Majumder, s., Richardson, a., Strong, R. & oldo, s., J Biol Chem 285,13107-20 (2010)); tau protein can be eliminated in animal models of AD that overexpress tau protein (Rodriguez-Navarro, j.a.et al, Neurobiol Dis 39,423-38 (2010)); and over-expressed mutant alpha-synuclein aggregates can be eliminated in a PD mouse model (Webb, j.l., Ravikumar, b., Atkins, J., Skepper, J.N. & Rubinsztein, d.c., J Biol Chem 278,25009-13 (2003)). It was demonstrated in HD mice that the use of CCI-779, a rapamycin-like substance, effectively eliminated Huntington protein aggregates and improved animal performance and cognition (Ravikumar, B., Duden, R. & Rubinsztein, D.C., Hum Mol Genet 11,1107-17 (2002)). However, mTOR plays a very important role in various intracellular pathways including NF-kB. Therefore, although it exhibits excellent activity in eliminating misfolded protein aggregates of protein conformational diseases, there is a limitation in using these monolithic autophagy activators, for which mTOR is known as a drug target, as therapeutic agents. Furthermore, there is no effective autophagosome-targeted therapy and therapeutic agents that target disease-inducing proteins.
In the central dogma, genetic information stored in DNA is transcribed into RNA and translated into protein to regulate cellular function. For DNA, target cleavage can be performed using DNA editing techniques such as CRISPR; for RNA, siRNA can be used for target degradation. However, for proteins, targeted degradation techniques are relatively limited. If a disease-inducing protein can be degraded by targeting it, it can serve as a platform technology for drug development in the pharmaceutical industry. ProTAC (proteolytic targeting chimera) is a technology developed for the production of intracellular degradation target proteins. ProTAC uses chimeric compounds with a ligand recognizing the target protein and a ligand recognizing the E3 ubiquitin enzyme (An and Fu, EBiomedicine 36, 553-562 (2018)). When the target binding binds to the disease-inducing protein and brings it close to E3, E3 recognizes as a substrate and undergoes ubiquitination to induce proteasomal degradation. Because the current paradigm for disease treatment is protease inhibition, it is important to develop new therapies for proteins that cannot be targeted by existing therapeutics. In these respects, PROTAC, which is capable of selectively degrading proteins under the ubiquitin-proteasome system, is an attractive new therapy development approach for proteins that cannot be targeted by traditional enzyme inhibition methods. However, PROTAC causes proteasomal degradation using only ligands that recognize E3 ubiquitinase, and thus target proteins are difficult to degrade when they are misfolded to form aggregates, or to form complexes or bind to membrane structures (boneson et al, Cell Chem Biol 25,78-87.E5 (2019)). In addition, ProTAC cannot degrade organelles such as the endoplasmic reticulum and mitochondria, as well as pathogens such as viruses and bacteria. Therefore, there is a need to develop a method that can target pathological proteins, organelles and aggregates and deliver them to selective autophagy.
In order to regulate cellular functions, methods of indirectly regulating protein activity and concentration by editing DNA or degrading RNA are widely used. In particular, the use of siRNA enables target degradation of RNA, but siRNA has low cell permeability and is delivered into cells using transfection reagents and the like. Therefore, the method is complicated and expensive. Furthermore, if the target protein is stable, it is difficult to reduce the concentration of the protein even if the RNA is degraded. Therefore, it is necessary to develop a method or a substance (i.e., a protein degradation agent) that can be directly degraded by targeting a specific protein.
Disclosure of Invention
Traditional drugs such as small molecule synthetic compounds, antibodies, proteins, peptides, etc. are different from each other, but the basic principle is to bind to the active site of a specific protein, inhibiting the activity of pathogenic proteins, thereby exhibiting drug efficacy. These traditional drug development methods have certain limitations. First, high drug concentrations are required, which may cause side effects of the drug. Because the drug-protein binding is not a stable covalent bond, the binding may be broken. That is, a drug exhibits drug efficacy when bound to a target protein, but does not exhibit drug efficacy when separated from the target protein. Therefore, to maintain drug efficacy, high drug concentrations must be maintained throughout the body. However, increasing the concentration of the drug for drug efficacy allows the drug to bind to other unintended proteins, which may lead to drug side effects. Second, drug target proteins are limited. To date, approximately 400 drug target proteins have been licensed by the U.S. Food and Drug Administration (FDA). These are more than 90% enzymes, transport proteins, channel/membrane proteins, etc. Because these drug target proteins have an active site and a binding site, they are relatively easy to find with relatively traditional drug development methods. However, it is estimated that there are 3,000 disease-associated proteins, and currently only about 13% of licensed drugs are used for this target. Therefore, there is a need to transfer a new drug development paradigm, and when using target protein degradation technology, it has the following advantages over existing therapeutics. First, it can regulate an undeliverable protein that is difficult to target with conventional drugs because it can degrade transcription factors, bind proteins involved in signal transduction through proteins, and aggregate and accumulate proteins such as tau protein. Secondly, it can overcome the phenomenon in which the target protein is overexpressed and overactivated or drug-resistant or drug efficacy is reduced by activation of other signaling systems. Third, since the target protein-degrading material is repeatedly used after degrading the target protein, it can be administered at a low dose, and also can reduce side effects associated therewith. Fourth, since a specific time is required for degradation and regeneration of the target protein, the administration period is increased and economic efficiency is high.
In order to solve the problems of the above-mentioned existing therapeutic agents, it is an object of the present invention to provide a novel AUTOTAC (autophagy targeting chimera) compound in which a novel p62 ligand compound inducing activation and oligomerization of p62 protein is linked to a target binding ligand via a linker (FIG. 1).
It is another object of the present invention to provide a pharmaceutical or food composition for eliminating disease-inducing proteins comprising the aforementioned AUTOTAC compound as an active ingredient.
It is yet another object of the present invention to provide a biochemical screening method and technique for achieving degradation of intracellular target proteins.
To achieve the above objects, one embodiment of the present invention provides a novel AUTOTAC chimeric compound in which a ligand that binds the ZZ domain of p62 is linked to a target-binding ligand by a linker.
Another embodiment of the present invention provides (1) a method of inducing p62 oligomerization and structural activation; (2) methods of increasing p62-LC3 binding; (3) methods of delivering p62 and a target protein to autophagosomes; (4) a method of activating the biosynthesis of autophagosomes; (5) methods of targeting and delivering a target protein to autophagy; (6) methods of targeting and inactivating a target protein; and (7) methods of degrading a target protein by lysosomes.
In another embodiment, the present invention provides a pharmaceutical composition or health functional food for preventing or treating diseases, which contains the aforementioned chimeric compound of AUTOTAC as an active ingredient. Preferably, the disease includes not only cancer or degenerative brain diseases, but also other diseases where targeted degradation of certain proteins is expected to have a therapeutic effect, such as rare refractory diseases.
The core technology of the invention is as follows: (1) linking a target protein to p62 using an AUTOTAC material, (2) inducing self-oligomerization of p62, (3) complexing the target protein with p62 to biologically inactivate the target protein, (4) subsequently delivering the target protein-p 62 complex to autophagy membranes such as phagophores (phagophores), and (5) degrading the target protein-p 62 complex in lysosomes.
The pharmacokinetics and core technology of the present invention are summarized in figure 1.
Specifically, as shown in fig. 1, the autosac chimeric compound has a structure in which a Target Binding Ligand (TBL) and a p62 ZZ domain ligand (i.e., an Autophagy Targeting Ligand (ATL)) are linked by a linker. It binds to the target protein through this target ligand, whereas the autophagy-targeting ligand oligomerizes after binding to the p62 ZZ domain (which is the autophagy receptor). Subsequently, p62 was targeted by autophagy by binding to LC3 protruding from the autophagosome membrane, and the target protein was then degraded in lysosomes. In the present invention, it was attempted to effectively eliminate the target protein by activating p62 using a small molecule ligand that binds to the p62 ZZ domain. To date, PROTAC compounds exist that use the ubiquitin-proteasome system (UPS) to target protein degradation, but there have been no reports on the use of autophagy to degrade low molecular weight compounds of a target protein. When autophagy other than PROTAC is used, it is possible to degrade not only proteins that cannot be degraded by PROTAC, such as misfolded protein aggregates, membrane-bound proteins, and subunits of complexes, but also intracellular structures (inflammatories, stress granules, etc.), organelles (endoplasmic reticulum, mitochondria, peroxisomes, etc.), and pathogens (viruses, bacteria, etc.) that invade cells. By such target degradation, preventive and therapeutic effects in various diseases can be expected.
The p62 protein is important for initiating the formation of autophagosomes, which are mediators in selective autophagy machinery, and the delivery of the contents, i.e., the target protein. It has been observed that the novel AUTOTAC chimeric compounds according to the present invention induce self-oligomerization of p62 by p62 activation. Furthermore, it was confirmed that the target protein was delivered to autophagosome by this self-oligomerization, whereby the target protein was degraded in lysosomes.
Since the AUTOTAC chimeric compound according to the present invention not only causes intracellular autophagy but also causes autophagosomes to target a target protein, it is able to selectively degrade proteins that cannot be targeted by conventional enzyme inhibition methods under the autophagy mechanism, which can provide novel therapeutic agents.
The AUTOTAC chimeric compound according to the present invention has a structure in which a Target Binding Ligand (TBL) is linked to a ligand that binds to the ZZ domain of p62 protein, i.e., an Autophagy Targeting Ligand (ATL), via a linker. Autophagy is activated by an autophagy targeting ligand, bringing the target protein to autophagosomes and degrading it in lysosomes.
The present invention is a platform technology capable of degrading a desired target protein when a ligand of a protein to be degraded is linked to an autophagy-targeting ligand-linker, and thus can be used as a drug for preventing, ameliorating and treating various diseases.
Drawings
Figure 1 shows a schematic diagram showing the construction of the novel AUTOTAC chimeric compound according to the present invention and showing that the compound binds to p62 protein and simultaneously to target proteins, organelles and aggregates, which are delivered to autophagy bodies (which are mediators of large autophagy through p62 protein) and finally degraded by lysosomes.
FIGS. 2a, 2b and 2c are immunoblot assays showing the efficacy of oligomerization and high molecular weight aggregation of the corresponding p62 protein from the AUTOTAC compound. This shows that oligomerization and high molecular weight aggregation of p62 protein increases with treatment of the compound. Immunoblots show representative results from three or more independent experiments.
FIG. 3 is the result of an immunoblot assay showing the efficacy of degradation of the corresponding target protein by the AUTOTAC compound according to the present invention. This shows that the target protein level decreases with treatment of the compound. Immunoblots show representative results from three or more independent experiments.
FIGS. 4a and 4b are immunoblot assays showing the mechanism of efficacy of degradation of the corresponding target proteins mediated by the AUTOTAC compounds according to the invention via the autophagy-lysosomal pathway. This shows that target protein levels are reduced by treatment with the compound, whereas target protein levels are increased again when treated with Hydroxychloroquine (HCQ) which is an inhibitor of the autophagy-lysosomal pathway. Immunoblots show representative results from three or more independent experiments.
FIGS. 5a, 5b and 5c are immunoblot assay results showing that the target protein degradation efficacy of the AUTOTAC chimeric compounds of the invention is superior to that of either p62 ligand alone or the target binding ligand, respectively. This shows that the level of target protein after treatment with the AUTOTAC chimeric compound is significantly reduced compared to after treatment with either p62 ligand or the target protein ligand. Immunoblots show representative results from three or more independent experiments.
FIGS. 6a, 6b and 6c are results of immunofluorescent staining assays showing the efficacy of delivering a target protein together with p62 protein to autophagy by an AUTOTAC chimeric compound according to the present invention. It can be confirmed that intracellular fluorescent spots (puncta) and coexistence of the target protein and p62 protein with the AUTOTAC compound are gradually increased after the treatment with the compound.
Detailed Description
The present invention will be described in more detail below.
The present invention provides a chimeric compound comprising a p62 ligand (autophagy targeting ligand, ATL), a Target Binding Ligand (TBL) and a linker; and a method of using the aforementioned compounds to deliver target proteins, organelles and aggregates to autophagosomes (which are mediators of macroautophagy) together with p62 and to be degraded in lysosomes. By this method, the present invention provides a method for preventing, ameliorating or treating a disease by delivering pathogenic proteins, organelles and aggregates to autophagy and degrading them, and a composition for autophagy activation of pathological proteins, organelles and aggregates associated with a disease and for preventing, ameliorating or treating the disease, comprising the above complex (hereinafter also referred to as "chimeric compound").
The present inventors have found that by using a p62 ligand, the p62 ligand can activate autophagy, effectively delivering target proteins, organelles and aggregates to autophagosomes and eliminating them. The inventors also found that when the ligand is used in combination with a ligand capable of binding the above pathological protein, it exhibits very excellent pathological protein and aggregate elimination efficacy, and does not need to optimize the length of the linker in order to form a ternary complex (target protein-linker-E3 ligase ligand) for pathological protein folding, E3 ligase selectivity, and efficient degradation, unlike conventional PROTAC compounds. The novel chimeric compound in which a p62 ligand is linked to a target binding ligand that binds to a target protein via a linker, which was developed by the present inventors, was named AUTOTAC (AUTOphagy Targeting Chimera).
The present invention features a technique capable of degrading a desired protein by linking a ligand of a protein to be degraded to a linker linked to a p62 ligand. That is, a chimeric compound in which a p62 ligand and a target binding ligand are linked by a linker is a bifunctional small molecule, and the target protein (which will degrade near p62 associated with autophagy of the protein) binds to the ligands that target them, forming a structure that can readily degrade the target protein. The gist of the present invention resides in that a therapeutic effect is expected by linking a protein associated with various diseases to be prevented or treated to p62 and degrading a target protein of interest.
In a preferred embodiment, the chimeric compound comprising a p62 ligand, a target-binding ligand and a linker according to the present invention has the form of a chimeric compound in which the p62 ligand and the target-binding ligand are linked through a linker, and more preferably, it may have the structure of the following chemical formula 1.
[ chemical formula 1]
Wherein A represents a target binding ligand and B represents a p62 ligand. In chemical formula 1, a and B may be respectively connected to at least one linker.
In the present invention, a target-binding ligand is a ligand that binds to a specific target protein in the body (more specifically, a pathological protein causing a disease to be targeted) or an organelle or aggregate. These target proteins include, but are not limited to, proteins preferably associated with cancer and proteins associated with various protein conformational diseases. These target binding ligands are not particularly limited in use as long as they are those that can bind to a protein associated with a disease to be prevented, improved and treated, preferably a target protein, organelle or aggregate associated with a disease caused by a pathological protein, cancer, protein conformational disease, intractable disease or genetic disease. Specific embodiments may include, but are not limited to, one or more selected from the group consisting of the compounds shown in table 1 below, or derivative structures derived from these structures.
The "derivative" structure refers to a structure in which a part of the structure of the target-binding ligand is modified by binding to a linker (for example, the linking portion of the target-binding ligand to the linker is changed by an amide group generated by binding a carboxyl group in a substituent to the linker having an amino group).
[ Table 1]
The linker connecting a and B in chemical formula 1 is not particularly limited in use as long as it has a structure in which both a and B are structurally connected. For example, the linker may be-Q- (CH)2CH2O)x-(CH2)y-P-or-Q- (CH)2CH2CH2O)x-(CH2)y-P-or-Q- (CH)2CH2NH)x-(CH2)y-P-or-Q- (CH)2CH2CONH)x-(CH2)y-P- (wherein Q comprises-NH-, -O-, ═ N-N (CH)3) -which is a moiety modified by binding to a target binding ligand; p includes-NH-, -O-, -CH2-, -C (═ O) -, which is a moiety modified by binding to a p62 ligand; x is an integer from 0 to 4; and y is an integer of 0 to 3), but is not limited thereto. Preferably, the bond between P and the P62 ligand can be-CONH-, -O-, -NH-, -NHCO-, or-COO-, and to form this bond, a portion of the P62 ligand (e.g., the Rc moiety and the portion of the target-binding ligand) can be structurally modified, as is well known in the art.
In the present invention, p62 ligand refers to p62, more specifically, the material that binds to the ZZ domain of p 62. These p62 ligands increase oligomerization of p62 and activate autophagy, particularly macroautophagy, due to binding to the p62 ZZ domain. In a preferred embodiment, the p62 ligand may have the structure of chemical formula 2 below.
[ chemical formula 2]
Wherein the content of the first and second substances,
w is a C6-C10 aryl group;
l is- (CH)2)n1-or-O- (CH)2)n2-CH (OH) -, with the proviso that-O- (CH)2)n2O in-CH (OH) -is bonded to a benzene ring, wherein n1 is an integer of 1 to 4;
n2 is an integer from 1 to 4;
m is an integer of 0 to 2;
Rais R1OR-OR1，
Wherein R is1Is hydrogen or- (CH)2)n3-R'1，
R'1Is unsubstituted phenyl or is substituted by hydroxy, halogen, C1-4Alkyl radical, C1-4Alkoxy, nitro, amino, (C)1-4Alkyl) amino or di (C)1-4Alkyl) amino-substituted phenyl groups,
n3 is an integer from 1 to 6;
Rbis-OR2，
Wherein R is2Is hydrogen or- (CH)2)n4-R'2，
R'2Is unsubstituted phenyl or is substituted by hydroxy, halogen, C1-4Alkyl radical, C1-4Alkoxy, nitro, amino, (C)1-4Alkyl) amino or di (C)1-4Alkyl) amino-substituted phenyl groups,
n4 is an integer from 1 to 6;
Rcis- (CH)2)n5-OH、-(CH2)n5-NH-C(＝NH)NH2、-C(＝NH)NH2、-CH(R3) -COOH or-CH (COO-R)4)-CH2CH2CH2-NH-C(＝NH)NH2、-(CH2)n5-O-(CH2)n5-OH、-CONH(CH2)n5-OH、-CO(CH2)n6-OH、-(CH2)n6-CH(NH2)-COOH、-(CH2)n6-CONH2，
n5 is an integer from 2 to 4,
n6 is an integer from 1 to 4,
R3is hydrogen or C1-4An alkyl group, a carboxyl group,
R4is C1-4Alkyl radical, and
Rdis hydrogen, halogen, C1-4Alkoxy or C1-4An alkyl group.
Preferably, W may be phenyl.
Preferably, L is- (CH)2)n1-or-O- (CH)2)n2-CH (OH) -, with the proviso that-O- (CH)2)n2O in-CH (OH) -is bonded to a benzene ring.
Preferably, n1 may be an integer from 0 to 1.
Preferably, n2 may be an integer from 1 to 2.
Preferably, RaCan be hydrogen or-O- (CH)2)n3-R'1。
Preferably, R'1Can be unsubstituted phenyl or phenyl substituted with hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino or dimethylamino.
Preferably, n3 may be an integer from 1 to 4.
Preferably, RbCan be hydroxyl or-O- (CH)2)n4-R'2。
Preferably, R'2Can be unsubstituted phenyl or phenyl substituted with hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino or dimethylamino.
Preferably, n4 may be an integer from 1 to 4.
Preferably, RcCan be- (CH)2)n5-OH、-(CH2)n5-NH-C(＝NH)NH2、-C(＝NH)NH2、-(CH2)n5-O-(CH2)n5-OH、-CONH(CH2)n5-OH、-CO(CH2)n6-OH、-(CH2)n6-CH(NH2) -COOH or- (CH)2)n6-CONH2。
Preferably, n5 may be an integer from 2 to 3.
Preferably, n6 may be an integer from 1 to 2.
Preferably, RdCan be hydrogen, halogen, C1-2Alkoxy or C1-2An alkyl group.
For binding to a linker, the p62 ligand may be attached to the linker in the form of a derivative (in which some groups have been modified to facilitate binding to the linker). This may be varied by those skilled in the art using known techniques as appropriate depending on the type of p62 ligand, the type of linker and their binding format, and these modified derivative forms are also included in the p62 ligand of the present invention.
In one embodiment, the AUTOTAC chimeric compound according to the present invention may be a compound shown in the following Table 2, but is not limited thereto.
[ Table 2]
Also, the compounds of the present invention may exist in the form of pharmaceutically acceptable salts. As salts, addition salts formed by pharmaceutically acceptable free acids can be used. The term "pharmaceutically acceptable salt" as used herein refers to any organic or inorganic addition salt of the compounds represented by chemical formulas 1 to 3, wherein the adverse effects caused by the salt do not impair the beneficial effects of the compounds at concentrations that exhibit relatively non-toxic and non-harmful effective activity to patients.
Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess of aqueous acid and precipitating the resulting salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Alternatively, water or an alcohol containing equimolar amounts of the compound and the acid (e.g., ethylene glycol monomethyl ether) may be heated, and then the resulting mixture may be dried by evaporation or the precipitated salt may be filtered under suction.
In this case, the free acid may be an inorganic acid or an organic acid. Examples of inorganic acids include, but are not limited to, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and stannic acid. Examples of organic acids include, but are not limited to, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid.
In addition, bases can be used to prepare pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt and subsequently evaporating the filtrate until drying. At this time, as the metal salt, sodium, potassium or calcium salt is particularly suitable in the pharmaceutical field, but the present invention is not limited thereto. Furthermore, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).
Unless otherwise indicated herein, pharmaceutically acceptable salts of the compounds of the present invention include salts of acidic or basic groups, which may be present in the compounds of formula 1. For example, pharmaceutically acceptable salts include sodium, calcium, and potassium salts of hydroxyl groups; and other pharmaceutically acceptable salts of the amino group including hydrobromide, sulphate, bisulphate, phosphate, hydrogenphosphate, dihydrogenphosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulphonate (methanesulphonate/mesylate) and p-toluenesulphonate (p-tolenesulphonate/tosylate). Salts can be prepared using salt preparation methods known in the art.
The salt of the compound of chemical formula 1 of the present invention is a pharmaceutically acceptable salt, and may be used without particular limitation so long as it is a salt exhibiting pharmacological activity equivalent to that of the compound of chemical formula 1.
In addition, the compound represented by chemical formula 1 according to the present invention includes, but is not limited to, not only pharmaceutically acceptable salts thereof, but also all solvates or hydrates and all possible stereoisomers that may be prepared therefrom. All stereoisomers of the compounds of the present invention (e.g., those that may exist due to asymmetric carbons on various substituents), including enantiomeric forms and diastereomeric forms, are contemplated within the scope of the invention. Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers (e.g., as pure or substantially pure optical isomers with the indicated activity) or may, for example, be as racemates or blended with all other stereoisomers or other selected stereoisomers. The chiral center of the compounds of the invention may have an S or R configuration as defined by the IUPAC 1974 recommendation. The racemic forms can be analyzed by physical methods such as fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. The individual optical isomers may be obtained from the racemates by any suitable method, including but not limited to salt formation with an optically active acid followed by crystallization.
Solvates and stereoisomers of the compound represented by chemical formula 1 may be prepared from the compound represented by chemical formula 1 using methods known in the art.
In addition, the compound represented by chemical formula 1 according to the present invention may be prepared in a crystalline form or an amorphous form, and when the compound is prepared in a crystalline form, it may be optionally hydrated or solvated. In the present invention, the compound of chemical formula 1 may include not only a stoichiometric hydrate but also a compound containing various amounts of water. The solvate of the compound of chemical formula 1 according to the present invention includes both stoichiometric and non-stoichiometric solvates.
The compound of chemical formula 1 according to the present invention may be prepared by the following exemplary method, specific examples of which are the same as in the reaction formulae described in the following examples.
In the preparation method of the present invention, the reactants used in the reaction formula may be commercially available compounds, or may be synthesized by directly conducting one or more reactions known in the art or by appropriately modifying the reaction. For example, the reactants are synthesized by performing one or more reactions in a series of orders, considering the presence, type and/or position of the reactive functional group and/or hetero element contained in the skeleton structure, but is not limited thereto.
The compound of chemical formula 1 according to the present invention acts as a chimeric ligand binding to the ZZ domain of p62 and simultaneously binding to target proteins, organelles, and aggregates, thereby activating the function of p62 and delivering target proteins, organelles (endoplasmic reticulum, mitochondria, peroxisomes, etc.), intracellular structures (inflammatories, stress particles, etc.), pathogens (viruses, bacteria, etc.), and aggregates invading cells to autophagy for degradation, similar to p 62.
Accordingly, in another aspect, the present invention provides a pharmaceutical composition for autophagy activation, comprising a compound of formula 1, a pharmaceutically acceptable salt, a stereoisomer, a hydrate, a solvate, or a prodrug thereof.
The compound of chemical formula 1 according to the present invention can eliminate pathological proteins, organelles and aggregates of pathological protein-related diseases by delivering target proteins, organelles and aggregates to autophagy and degrading them. In addition, the compound of chemical formula 1 is a p62 ligand, which binds to the p62 ZZ domain and activates the PB1 domain and the LIR domain of the p62 protein, so that p62 oligomerizes and forms aggregates, while increasing the delivery of target proteins, organelles and aggregates, and p62 protein to autophagosomes. By the above method, the target protein, organelles and aggregates are effectively eliminated (see FIG. 1). The target protein may be a major protein of a pathological protein-related disease, more particularly at least one selected from the group consisting of: prion protein (prion protein), Amyloid Precursor Protein (APP), alpha-synuclein, superoxide dismutase 1, tau protein (tau), immunoglobulin, amyloid A, transthyretin, beta 2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, islet amyloid polypeptide, ANF, peptin, insulin, lysozyme, fibrinogen, Huntington protein, alpha-1-antitrypsin Z, phacoelenteranin, C9 open reading frame 72(C9orf72), gliobulinic protein, cystic fibrosis transmembrane conductance regulator protein, rhodopsin (rhodosin) and spinocerebellar ataxia protein (ataxin), as well as other proteins with poly-Q extensions.
Accordingly, in yet another aspect, the present invention provides a pharmaceutical composition comprising the AUTOTAC chimeric compound of chemical formula 1, a pharmaceutically acceptable salt, a stereoisomer, a hydrate, a solvate, or a prodrug thereof. The pharmaceutical composition is useful for preventing or treating a disease targeted by a target-binding ligand. Without limitation, such a disease may be a target as long as it can bind to a target binding ligand, preferably it may be a cancer or a protein conformation disease, more preferably it includes various diseases that can be expected to have a therapeutic effect when targeting and degrading a particular protein, such as rare or refractory diseases or genetic diseases. The pharmaceutical composition according to the present invention is characterized by directly eliminating pathogenic proteins inducing the above-mentioned diseases.
In another aspect, the present invention provides a pharmaceutical composition for autophagy delivery or degradation of pathologically pathogenic proteins and misfolded proteins, comprising the compound of chemical formula 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate, or prodrug thereof.
The term "protein conformation disease" or "diseases associated with protein aggregation" as used herein refers to those diseases characterized by the presence of misfolded protein aggregates, examples of which include, but are not limited to, neurodegenerative diseases, alpha-1 antitrypsin deficiency, keratopathy, retinitis pigmentosa, type 2 diabetes, cystic fibrosis, and the like.
The term "aggregation" according to the present invention refers to the formation of oligomeric or multimeric complexes of typically one or more types of proteins, which may be accompanied by the integration of additional biomolecules, such as carbohydrates, nucleic acids and lipids, into the complex. These aggregated proteins can form deposits in specific tissues, more preferably in neural tissues or brain tissues. The degree of aggregation depends on the particular disease.
The neurodegenerative disease herein is preferably selected from the group consisting of: lyme borreliosis (Lyme borreliosis), fatal familial insomnia, Curie's Disease (Creutzfeldt-Jakob Disease; CJD), Multiple Sclerosis (MS), dementia, Alzheimer's Disease, epilepsy, Parkinson's Disease, stroke, Huntington's Disease, pick's Disease, Amyotrophic Lateral Sclerosis (ALS), spinocerebellar ataxia, other poly-Q diseases, hereditary cerebral amyloid angiopathy, familial amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), injection of local amyloidosis, beta-2 microglobulin amyloidosis, hereditary non-neuropathic amyloidosis, Alexander Disease, and Finland-type hereditary systemic amyloidosis.
The dosage of the pharmaceutical composition of the present invention can vary widely depending on the body weight, age, sex or health condition of the patient, diet, administration period, administration method, excreta and severity of disease. However, effective doses are typically about 1ng to 10 mg/day, and especially about 1ng to 1 mg/day for an adult (60 kg). Since the dosage may vary depending on various conditions, it will be apparent to one skilled in the relevant art that the dosage may be increased or decreased. Thus, the scope of the invention is not limited in any way by the foregoing dosages. With respect to the number of administrations, the administration may be performed once per day or divided into several administrations within a desired range, and the administration period is not particularly limited or not.
As used herein, the term "treatment" refers to all measures to alleviate or advantageously alter the symptoms of various diseases associated with misfolded protein aggregation as well as diseases targeted for protein degradation by administering the pharmaceutical composition of the invention.
As described above, the compounds of the present invention exhibit the following effects: (1) inducing p62 oligomerization and structural activation; (2) enhancing p62-LC3 binding, and (3) enhancing the delivery of p62 to the autophagosome; (4) activating autophagy; and finally (5) elimination of the targeted protein. Therefore, the pharmaceutical composition containing the compound as an active ingredient can be used for the prevention, amelioration or treatment of various desired diseases, preferably cancer or protein conformational diseases.
For example, the compositions of the present invention may further comprise a pharmaceutically acceptable carrier, diluent or excipient. The composition may be used in various forms such as oral dosage forms of powder, granule, troche, capsule, suspension, emulsion, syrup, spray, and injection of sterile injectable solution, which are formulated by conventional methods according to the purpose of each of the intended uses. The compositions can be administered via a variety of routes, including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. Examples of suitable carriers, excipients, or diluents that may be included in such compositions may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition, the composition of the present invention may further include fillers, anticoagulants, lubricants, humectants, fragrances, emulsifiers, preservatives and the like.
Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and these solid dosage forms are formulated by mixing the composition of the present invention with one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin and the like. In addition, lubricants other than simple excipients, such as magnesium stearate and talc, may be used.
Liquid preparations for oral administration may be illustrated as suspensions, solutions, emulsions, syrups and the like, and may include various excipients such as moisturizers, sweeteners, aromatics, preservatives and the like, and water and liquid paraffin as common diluents.
Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsion reagents, lyophilisates and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, polyethylene glycol, Tween 61, cacao butter, lauryl ester, glycerogelatin or the like can be used. In another aspect, the injectable formulation may include conventional additives such as solubilizers, isotonics agents, suspending agents, emulsifying agents, stabilizing agents, or preservatives.
The formulations can be prepared according to conventional mixing, granulating or coating methods and contain an amount of the active ingredient effective for medical treatment, particularly for preventing, ameliorating or treating diseases associated with protein aggregation.
In this case, the composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein refers to an amount sufficient to treat a disease and insufficient to cause side effects at a reasonable benefit/risk ratio applicable to any medical treatment. The level of an effective amount may be determined depending on the patient's health, type of disease, severity of disease, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, rate of excretion, duration of treatment, combination, factors including concurrent use of other drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and it may be administered simultaneously or sequentially with conventional therapeutic agents, and administered one or more times. Taking all of the above factors into account, it is crucial that the minimum amount that can provide the maximum effect without side effects is administered, which can be readily determined by the person skilled in the art.
For example, the dose may be increased or decreased depending on the administration route, severity of disease, sex, body weight, age, and the like, and the scope of the present invention is not limited in any way by the aforementioned dose.
The preferred dosage of the compound according to the present invention may vary depending on the condition and body weight of the patient, the severity of the disease, the type of the drug, and the route and duration of administration, but it may be appropriately selected by those skilled in the art.
In another aspect, the present invention provides a method for preventing, ameliorating or treating a disease, preferably cancer, a pathological protein-related disease, a disease associated with protein aggregation, comprising administering to a subject in need thereof a pharmaceutical composition of the present invention.
In yet another aspect, the invention provides (i) methods of increasing the degradation of a target protein, (ii) methods of increasing the degradation of organelles and structures; (iii) methods of increasing the degradation of various viruses and bacteria that invade cells, (iv) methods of delivering drugs or small molecule compounds to autophagy and lysosomes, (v) methods of self-oligomerization and autophagy activation of p62, and (vi) methods of increasing degradation by lysosomes by attaching specific proteins in cells to p62 and delivering them to autophagy, including compounds of formula 1, pharmaceutically acceptable salts, stereoisomers, solvates, hydrates, or prodrugs thereof.
In another aspect, the invention provides a method of using a drug in which a chimeric compound according to the invention is linked to a therapeutic antibody that specifically binds to a protein exposed on the cell membrane and delivering this drug to the lysosome by endosome. The therapeutic antibody may be used without limitation so long as it exhibits pharmaceutical activity against a disease requiring treatment.
As used herein, the term "subject" refers to all animals including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs, which have the target disease to be prevented, ameliorated, or treated. The target disease, preferably cancer, a disease associated with a pathological protein, or a disease associated with misfolded protein aggregation, may be effectively prevented, ameliorated or treated by administering the pharmaceutical composition of the invention to a subject. In addition, since the pharmaceutical composition of the present invention serves as a p62 ligand to activate autophagy, aggregates of misfolded proteins are eliminated due to autophagy activation, and thus, a prophylactic or therapeutic effect of diseases associated with these aggregated proteins is exhibited, which can exhibit a synergistic effect by being administered in combination with existing therapeutic agents.
As used herein, the term "administering" refers to introducing a prescribed amount of a substance into a patient in some appropriate way, and the composition of the invention can be administered via any of the general routes as long as it can reach the target tissue. For example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration may be performed, but the present invention is not limited to these exemplified modes of administration. Furthermore, the pharmaceutical compositions of the present invention may be administered using any device capable of delivering the active ingredient to the target cells. Preferred modes of administration and formulations are intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intravenous drip, and the like. Injectable formulations can be prepared using saline, aqueous solutions such as ringer's solution, and non-aqueous solutions such as vegetable oils, higher fatty acid esters (e.g., ethyl oleate, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Injectable formulations may include a pharmaceutical carrier, including stabilizers for preventing degenerative disorders (e.g., ascorbic acid, sodium bisulfite, sodium metabisulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, and preservatives for inhibiting microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).
In another aspect, the present invention provides a food composition for preventing or improving a protein conformational disease, comprising the AUTOTAC chimeric compound of chemical formula 1, a pharmaceutically acceptable salt, a stereoisomer, a hydrate, a solvate, or a prodrug thereof. The food composition is a health functional food and it can be used by itself via the preparation or included in other health functional foods as an additive to the health functional foods. The health functional food means a food having a body regulation function such as prevention or improvement of diseases, bio-defense, immunity, recovery of rehabilitation, aging inhibition, and the like, and it should not be harmful to the human body when taken for a long period of time. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (preventive, health care or therapeutic treatment).
The kind of food is not particularly limited. Examples of foods to which the above substances can be added are meat, sausage, bread, chocolate, candy, snack, cookie, pizza, stretched noodles, other noodles, jelly, dairy products including ice cream, soup, beverage, tea, drink, alcoholic drink, vitamin complex, etc., and they include all health functional foods in common knowledge.
The food composition of the present invention may comprise ingredients commonly used in the preparation of foods or food additives, in particular, a flavoring agent; natural sweeteners such as monosaccharides such as glucose, fructose and the like, disaccharides such as maltose, sucrose and the like, and dextrins, cyclodextrins as natural carbohydrates, or synthetic sweeteners such as saccharin, aspartame; nutrients; a vitamin; an electrolyte; a colorant; an organic acid; a protective colloid binder; a pH adjusting agent; a stabilizer; a preservative; glycerol; alcohol; carbonating agents used for carbonated drinks, and the like.
In a specific embodiment of the present invention, the novel AUTOTAC chimeric compounds 1 to 13 represented by chemical formula 1 are newly synthesized. Furthermore, in order to evaluate whether the novel AUTOTAC chimeric compound according to the present invention can increase autophagy phenomenon in cultured cells, cell lines mainly expressing a target protein (MCF7, NTERA-2, ACHN, U87-MG, LNCaP, HEK293T) or gene recombinant cell lines (SH-SY5Y-tau, HeLa-HttQ97, PC12-a-synA30P) were treated with the AUTOTAC chimeric compound according to the present invention and cultured, and then the target protein degrading activity in cultured cells was confirmed using immunoblotting. As a result, not only degradation of each target protein mediated by autophagy treated with the AUTOTAC compound according to the present invention at a concentration but also degradation efficacy superior to that of the p62 ligand or the target protein constituting the chimeric compound was gradually confirmed. Therefore, it was confirmed that the AUTOTAC compound according to the present invention activates and oligomerizes p62 protein, thereby delivering to autophagosomes, while selectively and effectively eliminating proteins and aggregates thereof associated with various target diseases such as cancer-associated proteins or protein conformational diseases.
Examples
The present invention will be described in detail below with reference to examples. However, these examples are for illustrative purposes only, and the present invention is not limited by these examples.
The compounds of formulae 1-13 according to the present invention are prepared according to the methods of examples 1 to 13 below.
[ Table 3]
Various synthetic methods are known in terms of starting materials for synthesizing the compounds of the present invention, and if commercially available, are available from commercial suppliers. Examples of reagent suppliers include, but are not limited to, Sigma-Aldrich, TCI, Wako, Kanto, Fluorchem, Acros, Alfa, and Fluka, among others.
The compounds of the present invention can be prepared from readily available starting materials using the following general methods and procedures. As to typical or preferred process conditions (i.e., reaction temperature, time, molar ratios of reactants, solvents, pressures), and the like, other process conditions may also be used unless otherwise specified. The optimum reaction conditions may vary with the particular reactants or solvents used. These conditions can be determined by one skilled in the art through routine optimization procedures.
The production methods of examples 1 to 13 are described below.
Example 1: preparation of (2E,4E,6E,8E) -N- (2- (2- (2- (((R) -3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) amino) ethoxy) ethyl) -3, 7-dimethyl-9- (2,6, 6-trimethylcyclohex-1-en-1-yl) non-2, 4,6, 8-tetraacrylamide (RTEG-1104)
Step 1) preparation of 3, 4-bis (benzyloxy) benzaldehyde (1101)
After 3, 4-dihydroxybenzaldehyde (0.50g, 3.62mmol) was dissolved in anhydrous DMF (5ml), potassium carbonate (K) was added2CO31.50g, 10.86mmol) and then bromotoluene (0.92mL, 7.96mmol) is slowly added to the reaction and stirred at 60 ℃ for 4 hours. When the reaction was complete, the reaction mixture was cooled to room temperature, diluted with purified water and extracted twice with diethyl ether (50 ml). The organic layer was washed twice with purified water (50ml) and then once with a saturated aqueous sodium chloride solution (50 ml). Then, anhydrous sodium sulfate was added to the organic layer and stirred, followed by filtration under reduced pressure. The filtered solution was concentrated and then purified by column chromatography to obtain 3, 4-bis (benzyloxy) benzaldehyde (1101, 1.04g, yield: 90%).1H NMR(CDCl3，300MHz)δ9.81(s,1H),7.49-7.31(m,12H),7.04(d,J＝8.3Hz,1H),5.27(s,2H),5.22(s,2H)；ESIMS m/z：319.33[M+H]+。
Step 2) preparation of 3, 4-bis (benzyloxy) phenol (1102)
Dichloromethane (15ml) was added to and dissolved in 3, 4-bis (benzyloxy) benzaldehyde (1101, 1.00g, 3.0mmol, 1eq.) and mCPBA (0.78g, 4.5mmol, 1.5eq.) was added to the reaction and stirred at room temperature for 4 hours. The reaction mixture was diluted with ethyl acetate, washed with saturated aqueous sodium carbonate solution, and the organic layer was separated. The organic layer was washed with an aqueous solution of sodium chloride, then dehydrated with anhydrous sodium sulfate and filtered under reduced pressure. The filtered solution was concentrated and then redissolved in methanol (10 ml). 6N NaOH was added thereto, and the mixture was stirred at room temperature for 30 minutes. To the reaction was added 4N HCl solution and stirred for further 30 minutes. The reaction mixture was diluted with ethyl acetate (50ml), washed with brine, dried over anhydrous sodium sulfate and filtered under reduced pressure. The filtered solution was concentrated and then purified by column chromatography (hexane/ethyl acetate ratio: 7/3) to obtain 3, 4-bis (benzyloxy) phenol (1102, 0.87g, yield: 90%).1H NMR(CDCl3300 MHz): δ 7.25-7.42 (m,10H),6.80(d,1H, J ═ 9.0Hz),6.48(d,1H, J ═ 3.0Hz),6.29(dd,1H, J ═ 3.0 and 9.0Hz),5.08(d,4H, J ═ 15Hz),4.55(s, 1H); ESIMS M/z 307.25[ M + H ]]+。
Step 3) preparation of R-2- ((3, 4-bis (benzyloxy) phenoxy) methyl) oxirane (1103)
Diluting 3, 4-Diphenylmethoxy with ethanol (10ml)Phenol (1102, 306mg, 1.0mmol) was added followed by aqueous KOH (66 mg, 1.2mmol, 1ml) and (R) -2- (chloromethyl) oxirane (410ul, 5.0 mmol). The reaction mixture was stirred at room temperature for 5 hours, and then the organic solvent was removed under reduced pressure. The concentrated reaction mixture was again diluted with ethyl acetate, washed with water and then brine. The extracted organic layer was dehydrated with anhydrous sodium sulfate, and then filtered under reduced pressure. The filtered organic layer was concentrated and purified by column chromatography to obtain pure R-2- ((3, 4-bis (benzyloxy) phenoxy) methyl) oxirane (1103, 297mg, yield: 82%). ESIMS m/z: 363.5[ M + H]+。
Step 4) preparation of (R) -1- ((2- (2- (2-Aminoethoxy) ethoxy) ethyl) amino-3- (3, 4-bis (benzyloxy) phenoxy) propan-2-ol (AL-1)
R-2- ((3, 4-bis (benzyloxy) phenoxy) methyl) oxirane (1103, 270mg, 0.75mmol) was dissolved in absolute ethanol (5ml), and 2,2' - (ethane-1, 2-diylbis (oxy)) bis (ethane-1-amine) (880mg, 5.9mmol) was added and stirred at room temperature for 8 hours. After confirming the reaction by TLC, the reaction solvent was concentrated under reduced pressure. Water was added to the concentrated reaction, which was extracted with dichloromethane (3X 5 mL). The extracted organic layer was dehydrated with anhydrous sodium sulfate, and then filtered under reduced pressure. The filtered organic layer was concentrated and purified by column chromatography (dichloromethane: methanol ═ 19: 1) to give (R) -1- ((2- (2- (2-aminoethoxy) ethoxy) ethyl) amino-3- (3, 4-bis (benzyloxy) phenoxy) propan-2-ol (AL-1, 267mg, yield: 70%). ESIMS M/z: 511.5[ M + H ] +.
Step 5) 1- ((2- (2- (2-aminoethoxy) ethoxy) ethyl) amino-3- (3, 4-bis (benzyloxy) phenoxy) propan-2-ol (AL-1, 100mg, 0.19mmol) was dissolved in DMF (4ml) followed by the addition of 1-ethyl-3- (3-trimethylaminopropyl) carbodiimide (EDCI, 44mg, 0.285mmol), hydroxybenzotriazole (HOBt, 38.5mg, 0.285mmol) and retinoic acid (60mg, 0.2mol) in that order. Then, N-diisopropylethylamine (DIPEA, 0.6ml) was added thereto, and stirred at room temperature for 12 hours. Water was added to the reaction, followed by extraction twice with ethyl acetate, and then washing the organic layer once with brine. Feeding the organic layer with anhydrous sodium sulfateThe mixture was dehydrated and filtered under reduced pressure. The filtrate was concentrated under reduced pressure and purified by high-resolution liquid chromatography to obtain a white solid, (2E,4E,6E,8E) -N- (2- (2- (2- (((R) -3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) amino) ethoxy) ethyl) -3, 7-dimethyl-9- (2,6, 6-trimethylcyclohex-1-en-1-yl) non-2, 4,6, 8-tetraacrylamide (RTEG-1104, 60mg, yield: 41%).1H NMR(400MHz,DMSO-d6) δ (ppm)1.01(s,6H),1.53(m,2H),1.74-1.79(m,5H),1.96(m,2H),2.12(s,3H),2.42(s,3H),2.56-2.81(m,4H),3.04(m,2H),3.52-3.54(m,6H),3.67(m,2H),3.95-4.05(m,3H),5.16(s,4H),5.37(br s,1H),5.91(br s,1H),6.22(s,2H),6.51(s,4H),6.57-6.61(m,2H),6.98(d,1H),7.32-7.48(m,10H),8.51(br s, 1H); ESI-MS calculation of m/z C49H64N2O7[M+H]+794.35 measurement 793.06
Example 2: preparation of (2E,4E,6E,8E) -N- (2- (2- (2- ((3, 4-bis (benzyloxy) benzyl) amino) ethoxy) ethyl) -3, 7-dimethyl-9- (2,6, 6-trimethylcyclohex-1-en-1-yl) non-2, 4,6, 8-tetraenamide (RTEG-1105)
Step 1) preparation of 2- (2- (2-aminoethoxy) ethoxy) -N- (3, 4-bis (benzyloxy) benzyl) ethan-1-amine (AL-2)
3, 4-bis (benzyloxy) benzaldehyde (1101, 0.5g, 1.57mmol) was dissolved in acetonitrile (ACN, 10ml), and tert-butyl (2- (2- (2-aminoethoxy) ethoxy) ethyl) carbamate (468mg, 1.88mmol) was added thereto and stirred at 60 to 70 ℃ for 5 hours. After cooling to room temperature, sodium borohydride (NaBH4, 106mg, 2.82mmol) was slowly added to the reaction, followed by stirring at room temperature for about 5 hours. To the reaction solution was added water to complete the reaction, and the compound was extracted with ethyl acetate (50 mL. times.3). The extracted organic solvent layer was washed with brine and dehydrated using sodium sulfate. The filtered solvent was concentrated and then dissolved again in dichloromethane (6 ml). Trifluoroacetic acid (TFA, 2ml) was added thereto, and the mixture was stirred at room temperature for 2 hours and then reducedThe solvent was concentrated under reduced pressure. The concentrate was purified by column chromatography (dichloromethane/methanol ═ 15: 1) to give 2- (2- (2-aminoethoxy) ethoxy) -N- (3, 4-bis (benzyloxy) benzyl) ethan-1-amine (AL-2, 590mg, yield: 84%) as a pale yellow liquid.1H NMR(400MHz,DMSO-d6)δ(ppm)2.59(t,2H),3.32-3.61(m,12H),4.62(br s,1H),5.10(s,4H),6.82(d,1H),6.97(d,1H,7.06(s,1H),7.30-7.46(m,10H)。
Step 2) in the same manner as in step 5 of example 1, (2E,4E,6E,8E) -N- (2- (2- (2- ((3, 4-bis (benzyloxy) benzyl) amino) ethoxy) ethyl) -3, 7-dimethyl-9- (2,6, 6-trimethylcyclohex-1-en-1-yl) non-2, 4,6, 8-tetraalkenamide (RTEG-1105) was synthesized.1H NMR(400MHz,DMSO-d6) δ (ppm)1.01(s,6H),1.54(m,2H),1.75-1.80(m,5H),1.98(m,2H),2.12(s,3H),2.42(s,3H),2.72(t,2H),3.04(t,2H),3.51-3.54(m,6H),3.67(t,2H),3.76(s,2H),5.14(s,4H),6.22(m,2H),6.37(br s,1H),6.51(s,4H),6.80(d,1H),6.87(d,1H),6.99(s,1H),7.31-7.46(m,10H),8.41(br s, 1H); ESI-MS calculation of M/z C47H60N2O5[ M + H ]]+734.3 measurement 733.01
Example 3: preparation of (R) -N- (15- (3, 4-bis (benzyloxy) phenoxy) -14-hydroxy-3, 6, 9-trioxa-12-azepinyl) -4-phenylbutanamide (PBA-1104)
Step 1) 2,2'- (2,2' -oxybis (ethane-2, 1-diyl) bis (oxy)) diethylamine (21g, 73.1mmol) was dissolved in dichloromethane (400ml), and then hydroxybenzotriazole (HOBt, 12.3g, 91.5mmol) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI, 17.6g, 91.5mmol) were added in that order. To this was added triethylamine (Et3N, 18.5g, 180mmol) and cooled to 0 ℃. 4-phenylbutyric acid (15g, 91.5mmol) was dissolved in dichloromethane (200ml), which was then added to the reaction, followed by stirring at room temperature for 12 hours. The reaction mixture was diluted with water, followed by extraction with dichloromethane (50ml × 3 times), and the organic layer was dehydrated with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and then purified by silica gel column chromatography to give N- (2- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -4-phenylbutylamide (TL-1, 12g) as a yellow liquid. ESIMS m/z: 339.1[ M + H ] +.
Step 2) N- (2- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -4-phenylbutylamide (TL-1, 100mg, 0.295mmol) was dissolved in methanol (6ml), to which R-2- ((3, 4-bis (benzyloxy) phenoxy) methyl) oxirane (1103, 128mg, 0.354mmol) was added and stirred at 50 ℃ for 10 hours. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography to give (R) -N- (15- (3, 4-bis (benzyloxy) phenoxy) -14-hydroxy-3, 6, 9-trioxa-12-azepentadecyl) -4-phenylbutanamide (PBA-1104, 2013mg, yield: 50%) as a white solid.1H NMR(400MHz,DMSO-d6) δ (ppm)1.25(s,1H),1.92(m,2H),2.31(t,2H),2.56-2.81(m,4H),3.28(m,2H),3.51-3.53(m,10H),3.67(t,2H),3.95-4.20(m,3H),5.14(s,4H),5.37(br s,1H),5.90(br s,1H),6.57(s,1H),6.67(d,1H),6.96(d,1H),7.16(m,3H),7.30(mk 8H),7.45(m, 4H); ESI-MS calculation of M/z C41H52N2O8[ M + H ]]+701.5 measurement 700.87
Example 4: preparation of N- (1- (3, 4-bis (benzyloxy) phenyl) -5,8, 11-trioxa-2-azatridecan-13-yl) -4-phenylbutanamide (PBA-1105)
Step 1) preparation of N- (1- (3, 4-bis (benzyloxy) phenyl) -5,8, 11-trioxa-2-azatridecan-13-yl) -4-phenylbutanamide (PBA-1105)
N- (2- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -4-phenylbutylamide (TL-1) (10g, 29.6mmol) was dissolved in methanol (MeOH, 150mL) and 3, 4-dihydroxybenzaldehyde (10.3g, 32.3mmol) was added thereto. The mixture was then stirred at 65 ℃ for about 5 hours. After cooling to room temperature, sodium borohydride (NaBH) was added42.2g, 57.9mmol) and stirred at room temperature for about 5 hours. To the reaction solution was added water to complete the reaction, and the compound was extracted with ethyl acetate (50 mL. times.3). The extracted organic solvent layer was washed with brine, and water was removed using sodium sulfate. The filtered solvent is concentrated and then diluted with waterPurification was performed by column chromatography using silica gel (dichloromethane/methanol ═ 15: 1). Thus, a yellow liquid, N- (1- (3, 4-bis (benzyloxy) phenyl) -5,8, 11-trioxa-2-azatridecan-13-yl) -4-phenylbutanamide (PBA-1105) (10.3g) was obtained.1H NMR(CDCl3400MHz) delta (ppm)7.45-7.42(m,4H),7.36-7.29(m,7H),7.27-7.26(m,1H),7.18-7.15(m,3H),6.99(d,1H),6.83(d,1H),6.41(brs,1H),5.15(d,4H),3.71(s,2H),3.60-3.55(m,10H),3.55-3.49(m,2H),3.42-3.39(m,2H),2.75-2.73(m,2H),2.65-2.61(m,2H),2.18-2.15(m,2H),1.97-1.93(m,2H),1.25(brs, 1H); ESI-MS calculation of M/z C39H48N2O6[ M + H ]]+641.00 measurement 640.82
Example 5: preparation of 3- (3- (benzo [ d ] [1,3] dioxol-5-yl) -1H-pyrazol-5-yl) -N- (2- (2- (2- ((3- ((4-fluorobenzyl) oxy)) benzyl) amino) ethoxy) ethyl) aniline (Angle 138b-F105)
Step 1) preparation of 3- ((4-fluorobenzyl) oxy) benzaldehyde (YT-102)
Adding potassium carbonate (K)2CO3112.5g, 0.81mol) and 1- (bromomethyl) -4-fluorobenzene (95g, 0.50mol) were added to a solution of 3-hydroxybenzaldehyde (50g, 0.41mol) in acetonitrile (ACN, 500 mL). The mixture was allowed to stir at 60 ℃ for about 10 hours. After completion of the reaction, the reaction solution was filtered and concentrated. To this was added petroleum ether/ethyl acetate (PE/EA, 20: 1, 20mL), stirred for about 1 hour, and then concentrated through a filter. Thus was obtained an off-white solid, 3- ((4-fluorobenzyl) oxy) benzaldehyde (YT-102, 25 g).1H NMR(DMSO-d6，400MHz)δ(ppm)9.98(s,1H),7.54-7.50(m,5H),7.36(m,1H),7.23(m,2H),5.18(s,2H)
Step 2) preparation of tert-butyl 3- (benzo [ d ] [1,3] dioxol-5-yl) -5- (3-bromophenyl) -1H-pyrazole-1-carboxylate (T-1)
Reacting 3- (benzo [ d ]][1,3]Dioxolen-5-yl) -5- (3-bromophenyl) -1H-pyrazole (200mg, 0.58mmol) was dissolved in dichloromethane (DCM, 4mL) and triethylamine (TEA, 88mg, 0.87mmol) and bisDi-tert-butyl carbonate (Boc2O, 153mg, 0.70 mmol). The mixture was stirred at room temperature for about 4 hours. After completion of the reaction, the filtered solution was concentrated and purified by column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 15 to 1: 5). Thus, a white solid, tert-butyl 3- (benzo [ d ]][1,3]Dioxol-5-yl) -5- (3-bromophenyl) -1H-pyrazole-1-carboxylate (T-1, 200 mg).1H NMR(DMSO-d6，400MHz)δ(ppm)8.09-7.43(m,5H),7.11-6.94(m,3H),6.08(s,2H),1.34(d,J＝20Hz,9H)
Step 3) preparation of 3- (benzo [ d ] [1,3] dioxol-5-yl) -5- (3- ((2, 2-dimethyl-4-oxo-3, 8, 11-trioxa-5-azatridecan-13-yl) amino) phenyl) -1H-pyrazole-1-carboxylic acid tert-butyl ester (TL-2)
Tert-butyl 3- (benzo [ d ] [1,3] dioxol-5-yl) -5- (3-bromophenyl) -1H-pyrazole-1-carboxylate (T-1, 200mg, 0.45mmol) was dissolved in dimethyl sulfoxide (DMSO, 4mL), to which was then added tert-butyl (2- (2- (2-aminoethoxy) ethoxy) ethyl) carbamate (L-1, 168mg, 0.68mmol) and potassium tert-butoxide (T-BuOK, 101mg, 0.90 mmol). The mixture was stirred at 120 ℃ for about 16 hours. The reaction mixture was filtered and concentrated, then purified by preparative high performance liquid chromatography (Prep-HPLC). Thus, a pale yellow solid, tert-butyl 3- (benzo [ d ] [1,3] dioxol-5-yl) -5- (3- ((2, 2-dimethyl-4-oxo-3, 8, 11-trioxa-5-azatridecan-13-yl) amino) phenyl) -1H-pyrazole-1-carboxylate (TL-2, 180mg) was obtained.
Step 4) preparation of N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -3- (3- (benzo [ d ] [1,3] dioxol-5-yl) -1H-pyrazol-5-yl) aniline (TL-3)
Tert-butyl 3- (benzo [ d ] [1,3] dioxol-5-yl) -5- (3- ((2, 2-dimethyl-4-oxo-3, 8, 11-trioxa-5-azatridecan-13-yl) amino) phenyl) -1H-pyrazole-1-carboxylate (TL-2, 180mg, 0.29mmol) was dissolved in ethyl acetate (EA, 4mL), to which was then added a hydrochloric acid/ethyl acetate (1mL, 3N) solution. The mixture was stirred at room temperature for about 2 hours. The reaction mixture was filtered and concentrated. Thus, white solid, N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -3- (3- (benzo [ d ] [1,3] dioxol-5-yl) -1H-pyrazol-5-yl) aniline (TL-3, 100mg) was obtained.
Step 5) preparation of 3- (3- (benzo [ d ] [1,3] dioxol-5-yl) -1H-pyrazol-5-yl) -N- (2- (2- (2- ((3- ((4-fluorobenzyl) oxy)) benzyl) amino) ethoxy) ethyl) propylamine (Angle 138b-F105)
Reacting N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -3- (3- (benzo [ d ]][1,3]Dioxolen-5-yl) -1H-pyrazol-5-yl propylamine (TL-3, 90mg, 0.22mmol) was dissolved in methanol (MeOH, 2mL), followed by addition of 3- ((4-fluorobenzyl) oxy) benzaldehyde (YT-102, 50mg, 0.22mmol), and stirring at 65 ℃. Then, sodium borohydride (NaBH) was added thereto at 5 ℃416mg, 0.44mmol) and then further stirred for about 1 hour. The reaction mixture was concentrated and then purified by preparative high performance liquid chromatography (Prep-HPLC). Thus obtaining a colorless liquid, 3- (3- (benzo [ d ]][1,3]Dioxol-5-yl) -1H-pyrazol-5-yl) -N- (2- (2- (2- ((3- ((4-fluorobenzyl) oxy)) benzyl) amino) ethoxy) ethyl) propylamine (Anle138b-F105, 15 mg).1H NMR(DMSO-d6400MHz) δ (ppm)13.08(brs,1H),7.47(q, J ═ 6Hz,2H),7.37-7.31(m,2H),7.21-7.17(m,3H),7.11(t, J ═ 8Hz,1H),7.00-6.96(m,5H),6.88-6.82(m,2H),6.56(d, J ═ 8Hz,1H),6.04(s,2H),5.59(brs,1H),5.04(s,2H),3.66(s,2H),3.60-3.52(m,6H),3.47(t, J ═ 6Hz,2H),3.26-3.22(m,2H),2.61(t, J ═ 5.6Hz, 2H); ESI-MS calculation of m/z C36H37FN4O5[M+H]+625.10 measurement 624.71
Example 6: preparation of (3R,4S,5S,6R) -5-methoxy-4- ((2R,3R) -2-methyl-3- (3-methylbut-2-en-1-yl) oxiran-2-yl) -1-oxaspiro [2.5] octan-6-yl (13E,15E,17E,19E) -1- (3- (phenylmethoxy) phenyl) -12-oxo-5, 8-dioxa-2, 11-diaza-heneico-13, 15,17, 19-tetraen-21-oic acid ester (fumagillin-105)
Step 1) preparation of 3- (Phenylmethoxy) benzaldehyde (201)
Adding potassium carbonate (K)2CO3112.5g, 0.81mol) and bromomethylbenzene (85g, 0.50mol) were added to a solution of 3-hydroxybenzaldehyde (50g, 0.41mol) in acetonitrile (ACN, 500 mL). The mixture was stirred at 60 ℃ for about 10 hours. After completion of the reaction, the reaction solution was filtered, and then concentrated. Then, petroleum ether/ethyl acetate (PE/EA, 20: 1, 20mL) was added thereto, and further stirred for about 1 hour, followed by concentration through a filter. Thus, an off-white solid, 3- (benzyloxy) benzaldehyde (201, 25g) was obtained.1H NMR(DMSO-d6，400MHz)δ(ppm)9.97(s,1H),7.54-7.51(m,3H),7.47(d,J＝7.2Hz,2H),7.42-7.34(m,4H),5.19(s,2H)
Step 2) preparation of 2- (2- (2-aminoethoxy) ethoxy) -N- (3- (phenylmethoxy) phenylmethyl) ethane-1-amine (AL-3)
3- (Phenylmethoxy) benzaldehyde (201, 50g, 235.8mmol) was dissolved in methanol (MeOH, 500mL), to which was then added 2,2' - (ethane-1, 2-diylbis (oxy)) diethylamine (L-2, 34.9g, 235.8 mmol). The mixture was stirred at 65 ℃ for about 6 hours. After cooling to room temperature, sodium borohydride (NaBH) was added48.95g, 235.8mmol), stirred at 50 ℃ overnight. Water was added to complete the reaction and the compound was extracted with ethyl acetate (EtOAc, 50 mL. times.3). The extracted organic layer was washed with brine, then washed with sodium sulfate (Na)2SO4) The water is removed. The solution was concentrated and then purified by column chromatography using silica gel (dichloromethane/methanol ═ 12: 1). Thus was obtained a yellow liquid, 2- (2- (2-aminoethoxy) ethoxy) -N- (3- (phenylmethoxy) benzyl) ethan-1-amine (AL-3, 20 g). 1H NMR (DMSO + D2O,400MHz) δ (ppm)7.41-7.29(m,5H),7.18(t, J ═ 8Hz,1H),6.94(s,1H),6.86-6.81(m,2H),5.03(s,2H),3.60(s,2H),3.45-3.42(m,6H),3.33(t, J ═ 5.6Hz,2H),2.57-2.52(m, 4H); ESI-MS calculation of m/z C20H28N2O3[M+H]+345.10 measurement 344.46
Step 3) preparation of (3R,4S,5S,6R) -5-methoxy-4- ((2R,3R) -2-methyl-3- (3-methylbut-2-en-1-yl) oxiran-2-yl) -1-oxaspiro [2.5] octan-6-yl (13E,15E,17E,19E) -1- (3- (phenylmethoxy) phenyl) -12-oxo-5, 8-dioxa-2, 11-diaza-heneico-13, 15,17, 19-tetraene-21-oic acid ester (fumagillin-105)
2- (2- (2-Aminoethoxy) ethoxy) -N- (3- (benzyloxy) benzyl) ethan-1-amine (AL-3, 70mg, 0.22mmol) was dissolved in dichloromethane (DCM, 2mL) and then Fumagillin (Fumagillin) (100mg, 0.22mmol), hydroxybenzotriazole (HOBT, 34mg, 0.25mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI, 48mg, 0.25mmol) and triethylamine (Et3N, 44mg, 0.44mmol) were added thereto. The mixture was stirred at 30 ℃ for about 10 hours. After completion of the reaction, the compound was extracted with dichloromethane (DCM, 50mL × 3). The extracted organic solvent layer was washed with brine, and water was removed using sodium sulfate. The filtered solvent was concentrated and then purified using preparative high performance liquid chromatography (Prep-HPLC). Thus, a yellow solid, (3R,4S,5S,6R) -5-methoxy-4- ((2R,3R) -2-methyl-3- (3-methylbut-2-en-1-yl) oxiran-2-yl) -1-oxaspiro [2.5] was obtained]Octane-6-yl (13E,15E,17E,19E) -1- (3- (phenylmethoxy) phenyl) -12-oxo-5, 8-dioxa-2, 11-diaza-heneicosyl-13, 15,17, 19-tetraen-21-oic acid ester (fumagillin-105, 3 mg). ESI-MS calculation of m/z C46H60N2O9[M]+784.90 measurement 784.99
Example 7: preparation of 3- (3, 5-dichlorophenyl) -5- ((R) -15- (3, 4-diphenylethoxyphenoxy) -14-hydroxy-6, 9-dioxa-3, 12-diazepidecyl) -5-methyloxazolidine-2, 4-dione (Endocenone-2204)
Step 1) preparation of 3-hydroxy-4-phenethyloxybenzaldehyde (A-1)
3, 4-dihydroxybenzaldehyde (200g, 1.4mol) was dissolved in acetonitrile (ACN, 2L), to which potassium carbonate (K) was added2CO3260g, 1.9mol) and 1- (2-bromoethyl) benzene (267g, 1.4 mol). The mixture was stirred at 80 ℃ for about 16 hours. After completion of the reaction with hydrochloric acid (1.5L, 1N), the compound was extracted with ethyl acetate (EA, 1L × 3). The extracted organic solvent layer was washed with brine, water was removed with sodium sulfate, and then filtered. By column chromatography using silica gel (ethyl acetate/petroleum ether ═ 20: 1 to10: 1) the concentrated solution was purified. Thus, 3-hydroxy-4-phenethyloxybenzaldehyde (A-1, 320g) was obtained as a white solid. ESI-MS calculation of m/z C15H14O3[M+H]+243.0 measurement 242.27
Step 2) preparation of 3, 4-diphenylethoxybenzaldehyde (2201)
3-hydroxy-4-phenylethoxybenzaldehyde (A-1, 320g, 1.32mol) was dissolved in tetrahydrofuran (THF, 5L) and then 2-phenylethyl alcohol (193.5g, 1.59mol), triphenylphosphine (PPh3, 520g, 1.98mol) and diisopropyl azodicarboxylate (DIAD, 400g, 1.98mol) were added thereto. The mixture was stirred at 65 ℃ for about 16 hours. Water (100mL) was added to the reaction solution to complete the reaction, and the mixture was extracted with ethyl acetate (100mLx 2). Sodium sulfate was added to the extracted organic solvent layer to remove water, filtered and concentrated. The concentrated compound was purified by column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 15 to 1: 10). Thus, 3, 4-diphenylethoxybenzaldehyde (2201, 160g) was obtained as a white solid.1H NMR(DMSO-d6400MHz) δ (ppm)9.81(s,1H),7.51(dd, J ═ 8Hz and 1.6Hz,1H),7.40-7.17(m,12H),4.27(t, J ═ 6.8Hz,2H),4.22(t, J ═ 6.8Hz,2H),3.06(q, J ═ 6.4Hz, 4H); ESI-MS calculation of m/z C23H22O3[M+H]+346.90 measurement 346.43
Step 3) preparation of 3, 4-Diphenyl Ethoxyphenol (2202)
3, 4-Benzyloxybenzaldehyde (2201, 160g, 0.46mmol) was dissolved in dichloromethane (DCM, 2L) and m-chloroperoxybenzoic acid (m-CPBA, 119g, 069mmol) was added thereto. The mixture was stirred at room temperature for about 16 hours. After completion of the reaction, the solution was filtered and then concentrated. The concentrated solution was purified by column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 15 to 1: 5). Thus, 3, 4-diphenylethoxyphenol (2202, 100g) was obtained as an off-white solid. ESI-MS calculation of m/z C22H22O3[M+H]+335.00 measurement 334.42
Step 4) preparation of (R) -2- ((3, 4-Diphenyl-ethoxy-phenoxy) methyl) oxirane (A-2)
3, 4-Diphenylethoxyphenol (2202) (5g, 15mmol) was dissolved in ethanol (EtOH, 50 m)L), then water (2.5mL) and potassium hydroxide (KOH, 1.93g, 34.5mmol) were added. Then, (R) -2- (chloromethyl) oxirane (8g, 87mmol) was added and stirred at room temperature for about 16 hours. To the reaction solution was added water (50mL) to complete the reaction, and the compound was extracted with ethyl acetate (EA, 20 mL. times.3). The extracted organic solvent was washed with brine, water was removed with sodium sulfate, filtered and concentrated. The concentrated solution was extracted using column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 15 to 1: 10). Thus, white solid, (R) -2- ((3, 4-diphenylethoxyphenoxy) methyl) oxirane (A-2, 2.5g) was obtained. ESI-MS calculation of m/z C25H26O4[M+H]+391.00 measurement 390.48
Step 5) preparation of (R) -1- ((2- (2- (2-aminoethoxy) ethoxy) ethyl) amino) -3- (3, 4-diphenylethoxyphenoxy) propan-2-ol (AL-4)
(R) -2- ((3, 4-Diphenylethoxyphenoxy) methyl) oxirane (A-2, 20g, 51.2mmol) was dissolved in acetonitrile (ACN, 200mL), to which was then added 2- (2- (2-aminoethoxy) ethoxy) ethylamine (L-2, 15.2g, 102.5 mmol). The mixture was stirred at 70 ℃ for about 40 hours. When the reaction was complete, the reaction solution was filtered and concentrated. The concentrated solution was purified by column chromatography using silica gel (dichloromethane/methanol ═ 100: 1 to 30: 1). Thus, a yellow liquid, (R) -1- ((2- (2- (2-aminoethoxy) ethoxy) ethyl) amino) -3- (3, 4-diphenylethoxyphenoxy) propan-2-ol (AL-4, 12g) was obtained.1H NMR(CD3OD, 400MHz) δ (ppm)8.52(s,3H),7.30-7.20(m,10H),6.85(d, J ═ 8Hz,1H),6.60(d, J ═ 2.8Hz,1H),6.47(dd, J ═ 8Hz and 2Hz,1H),4.16(t, J ═ 6.8Hz,3H),4.08(t, J ═ 6.8Hz,2H),3.96(q, J ═ 4Hz,2H),3.79(t, J ═ 5.2Hz,2H),3.70(m,7H),3.32-3.25(m,3H),3.17-2.98(m, 8H); ESI-MS calculation of m/z C31H42N2O6[M+H]+539.20 measurement 538.69
Step 6) preparation of liquid 3- (3, 5-dichlorophenyl) -5- ((R) -15- (3, 4-diphenylethoxyphenoxy) -14-hydroxy-6, 9-dioxa-3, 12-diazepidecyl) -5-methyloxazolidine-2, 4-dione (Endocenone-2204)
Reacting (R) -1- ((2- (2- (2-aminoethoxy) ethoxy) ethyl)Amino) -3- (3, 4-diphenylethoxyphenoxy) propan-2-ol (AL-4, 120mg, 0.22mmol) was dissolved in methanol (MeOH, 2mL) and then incorporated with enoxalone (Vinclozolin) (117mg, 0.33 mmol). The mixture was stirred at 65 ℃ for about 16 hours. Upon completion of the reaction, the solution was filtered and concentrated. The concentrated solution was purified by preparative high performance liquid chromatography (Prep-HPLC). Thus, a colorless liquid, 3- (3, 5-dichlorophenyl) -5- ((R) -15- (3, 4-diphenylethoxyphenoxy) -14-hydroxy-6, 9-dioxa-3, 12-diazapentanyl) -5-methyloxazolidine-2, 4-dione (cycloheximide-2204, 15mg) was obtained.1H NMR(DMSO-d6400MHz) δ (ppm)1.65(m,3H),2.65(m,4H),2.97(m,4H),3.17(m,2H),3.42(m,8H),3.57(d, J ═ 8Hz,1H),3.80(m,3H),4.02(t, J ═ 8Hz,2H),4.13(t, J ═ 8Hz,2H),4.90(b,1H),5.33(m,2H),6.20(m,1H),6.38(d, J ═ 8Hz,1H),6.54(m,1H),6.82(d, J ═ 8Hz,1H),7.25(m,11H),7.49(d, J ═ 1Hz,1H),7.76(s,1H),9.70(s, 1H); ESI-MS calculation of m/z C43H51Cl2N3O9[M+H]+825.00 measurement 824.79
Example 8: preparation of (R) -1- (4- (benzyloxy) -3- (3-phenylpropyloxy) phenoxy) -3- ((2- (2- (2- (4- (2-phenyl-5, 7-bis (trifluoromethyl) pyrazolo [1,5-a ] pyrimidin-3-yl) phenoxy) ethoxy) ethyl) amino) propan-2-ol (PHTPP-1304)
Step 1) preparation of 4- (Phenylmethoxy) -3-hydroxybenzaldehyde (A-3)
3, 4-Dihydroxybenzaldehyde (500g, 3.62mol) was dissolved in acetonitrile (ACN, 7L) and then sodium bicarbonate (NaHCO3, 395g, 4.71mol) and benzyl bromide (BnBr, 619g, 3.62mol) were added thereto. The mixture was stirred at 80 ℃ for about 16 hours. The reaction was completed using hydrochloric acid (3L, 1N), and the compound was extracted with ethyl acetate (3L × 3). The extracted organic solvent layer was washed with brine, the remaining water was removed with sodium sulfate, filtered to remove impurities and concentrated. The concentrated solution was purified by column chromatography using silica gel (EA/PE ═ 20: 1 to 10: 1). Thus, 4- (benzyloxy) -3-hydroxybenzaldehyde was obtained as a white solid(A-3，250g)。1H NMR(DMSO-d6，400MHz)δ(ppm)9.76(s,1H),9.65(s,1H),7.49(d,J＝6.8Hz,2H),7.42-7.34(m,4H),7.29(d,J＝2Hz,1H),7.19(d,J＝8Hz,1H),5.23(s,2H)
Step 2) preparation of 4- (benzyloxy) -3- (3-phenylpropyloxy) benzaldehyde (1301)
4- (Phenylmethoxy) -3-hydroxybenzaldehyde (A-3, 120g, 526mmol) was dissolved in tetrahydrofuran (THF, 2L), to which was then added 3-phenylprop-1-ol (85.9g, 631mmol), triphenylphosphine (PPh3, 206.9g, 789mmol) and diisopropyl azodicarboxylate (DIAD, 159.5g, 789 mmol). The mixture was stirred at 65 ℃ for about 16 hours. Upon completion of the reaction, the reaction mixture was filtered and concentrated. The concentrated solution was purified by column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 15 to 1: 10). Thus, 4- (benzyloxy) -3- (3-phenylpropyloxy) benzaldehyde (1301, 100g) was obtained as a white solid.1H NMR(DMSO-d6，400MHz)δ(ppm)9.82(s,1H),7.55-7.50(m,3H),7.38(t,J＝7.2Hz,3H),7.36-7.34(m,1H),7.29-7.25(m,3H),7.21-7.16(m,3H),5.26(s,2H),4.04(t,J＝6.4Hz,2H),2.76(t,J＝8Hz,2H),2.04(t,J＝7.2Hz,2H)
Step 3) preparation of 4- (benzyloxy) -3- (3-phenylpropyloxy) phenol (1302)
4- (Phenylmethoxy) -3- (3-phenylpropyloxy) benzaldehyde (1301, 100g, 289mmol) was dissolved in dichloromethane (DCM, 900mL) and m-chloroperoxybenzoic acid (m-CPBA, 74.7g, 433mmol) was added thereto. The mixture was stirred at room temperature for about 16 hours. Upon completion of the reaction, the reaction mixture was filtered and then concentrated. The concentrated solution was purified by column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 15 to 1: 5). Thus, 4- (benzyloxy) -3- (3-phenylpropyloxy) phenol (1302, 66g) was obtained as an off-white solid.1H NMR(DMSO-d6400MHz) δ (ppm)9.00(s,1H),7.44-7.18(m,10H),6.81(d, J ═ 8Hz,1H),6.39(d, J ═ 2.8Hz,1H),6.22(dd, J ═ 8.8Hz and 2.8Hz,1H),4.96(s,2H),3.90(t, J ═ 6Hz,2H),2.74(t, J ═ 8Hz,2H),2.01(q, J ═ 5.6Hz,2H)
Step 4) preparation of (R) -2- ((4- (benzyloxy) -3- (3-phenylpropyloxy) phenoxy) methyl) oxirane (A-4)
4- (benzyloxy) benzeneThe yl) -3- (3-phenylpropyloxy) phenol (1302, 40g, 60mmol) was dissolved in ethanol (EtOH, 800mL) and then water (40mL) and potassium hydroxide (KOH, 8.0g, 143mmol) were added thereto. Then, (R) -2- (chloromethyl) oxirane (33.2g, 359mmol) was added, followed by stirring at room temperature for about 16 hours. When the reaction was completed, water (1600mL) was added to the reaction solution to complete the reaction, and the compound was extracted with ethyl acetate (1600 mL. times.3). The extracted organic solvent layer was washed with brine, and then the remaining water was removed with sodium sulfate. Impurities were removed using a filter, and then concentrated, and the concentrated solution was purified using column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 15 to 1: 10). Thus, white solid, (R) -2- ((4- (benzyloxy) -3- (3-phenylpropyloxy) phenoxy) methyl) oxirane (A-4, 30g) was obtained.1H NMR(DMSO-d6400MHz) δ (ppm)7.46-7.17(m,10H),6.93(d, J ═ 8.8Hz,1H),6.61(d, J ═ 3.2Hz,1H),6.43(dd, J ═ 8.8Hz and 2.8Hz,1H),5.02(s,2H),4.23(dd, J ═ 7.6Hz and 2.8Hz,1H),3.97(t, J ═ 6.4Hz,2H),3.75(dd, J ═ 7.2Hz and 1.6Hz,1H),3.28(m,1H),2.82(dd, J ═ 5.2Hz and 4Hz,1H),2.75(t, J ═ 7.6Hz,2H),2.73-2.67(m,1H),2.01(m,2H)
Step 5) preparation of tert-butyl (2- (2- (2-hydroxyethoxy) ethoxy) ethyl) carbamate (L-4)
2- (2- (2-Aminoethoxy) ethoxy) ethan-1-ol (L-3, 5g, 33mmol) was dissolved in dichloromethane (DCM, 100mL) to which was added triethylamine (TEA, 4.1g, 40mmol) and di-tert-butyl dicarbonate (Boc2O, 8.1g, 37 mmol). The mixture was stirred at room temperature for about 16 hours. Upon completion of the reaction, the reaction mixture was filtered and then concentrated. The concentrated solution was purified by column chromatography using silica gel (EA/PE ═ 1: 3 to 1: 1). Thus, colorless liquid, (tert-butyl 2- (2- (2-hydroxyethoxy) ethoxy) ethyl) carbamate (L-4, 4.2g) was obtained.1H NMR(DMSO-d6，400MHz)δ(ppm)6.74(m,1H),4.56(t,J＝5.6Hz,1H),3.51-3.46(m,6H),3.42-3.37(m,4H),3.07-3.03(m,2H),1.37(s,9H)
Step 6) preparation of tert-butyl (2- (2- (2- (4- (2-phenyl-5, 7-bis (trifluoromethyl) pyrazolo [1,5-a ] pyrimidin-3-yl) phenoxy) ethoxy) ethyl) carbamate (TL-4)
(2- (2- (2-hydroxyethoxy) ethoxy) ethyl) carbamic acid tert-butyl ester (L-4, 300mg, 1.2mmol) was dissolved in tetrahydrofuran (THF, 5mL) and 4- [ 2-phenyl-5, 7-bis (trifluoromethyl) pyrazolo [1,5-a ] pyrimidin-3-yl ] phenol (PHTPP, 509mg, 1.2mmol), triphenylphosphine (PPh3, 377mg, 1.44mmol) and diisopropyl azodicarboxylate (DIAD, 291mg, 1.44mmol) were added thereto. The mixture was stirred at 65 ℃ for about 16 hours. After completion of the reaction, the reaction was terminated, and the solution was filtered and concentrated. The concentrated solution was purified by column chromatography using silica gel (DCM/MeOH ═ 100: 1 to 50: 1). Thus, tert-butyl (2- (2- (2- (4- (2-phenyl-5, 7-bis (trifluoromethyl) pyrazolo [1,5-a ] pyrimidin-3-yl) phenoxy) ethoxy) ethyl) carbamate (TL-4, 200mg) was obtained as a yellow solid.
Step 7) preparation of 2- (2- (2- (4- (2-phenyl-5, 7-bis (trifluoromethyl) pyrazolo [1,5-a ] pyrimidin-3-yl) phenoxy) ethoxy) ethan-1-amine (TL-5)
Tert-butyl (2- (2- (2- (4- (2-phenyl-5, 7-bis (trifluoromethyl) pyrazolo [1,5-a ] pyrimidin-3-yl) phenoxy) ethoxy) ethyl) carbamate (TL-4, 200mg, 0.31mmol) was dissolved in ethyl acetate (EA, 4mL), to which was then added hydrochloric acid (g)/ethyl acetate (1 mL). The mixture was stirred at room temperature for about 2 hours. After completion of the reaction, the reaction was terminated, and the solution was filtered and concentrated. The concentrated solution was subjected to a purification treatment to obtain 2- (2- (2- (4- (2-phenyl-5, 7-bis (trifluoromethyl) pyrazolo [1,5-a ] pyrimidin-3-yl) phenoxy) ethoxy) ethan-1-amine (TL-5, 120mg) as a yellow solid.
Step 8) preparation of (R) -1- (4- (benzyloxy) -3- (3-phenylpropyloxy) phenoxy) -3- ((2- (2- (2- (4- (2-phenyl-5, 7-bis (trifluoromethyl) pyrazolo [1,5-a ] pyrimidin-3-yl) phenoxy) ethoxy) ethyl) amino) propan-2-ol (PHTPP-1304)
2- (2- (2- (4- (2-phenyl-5, 7-bis (trifluoromethyl)) pyrazolo [1, 5-a)]Pyrimidin-3-yl) phenoxy) ethoxy) ethan-1-amine (TL-5, 120mg, 0.22mmol) was dissolved in methanol (MeOH, 2mL) and (R) -2- ((4- (benzyloxy) -3- (3-phenylpropoxy) phenoxy) methyl) oxirane (a-4, 117mg, 0.33mmol) was added thereto. The mixture was stirred at 65 ℃ for about 16And (4) hours. After completion of the reaction, the reaction was terminated, the solution was filtered and concentrated, and the concentrated solution was purified by preparative high performance liquid chromatography (Prep-HPLC). Thus, a yellow solid, (R) -1- (4- (benzyloxy) -3- (3-phenylpropyloxy) phenoxy) -3- ((2- (2- (2- (4- (2-phenyl-5, 7-bis (trifluoromethyl) pyrazolo [1, 5-a) was obtained]Pyrimidin-3-yl) phenoxy) ethoxy) ethyl) amino) propan-2-ol (PHTPP-1304, 15 mg).1H NMR(DMSO-d6400MHz) δ (ppm)1.99(m,2H),2.73(m,6H),3.55(m,8H),3.80(m,5H),3.94(t, J ═ 8Hz,2H),4.13(t, J ═ 4Hz,2H),4.99(s,2H),6.40(d, J ═ 8Hz,1H),6.55(d, J ═ 4Hz,1H),6.89(d, J ═ 8Hz,1H),7.04(d, J ═ 8Hz,2H),7.18(t, J ═ 4Hz,3H),7.27(dd, J ═ 4Hz and 4Hz,3H),7.35(m,4H),7.44(m,5H),7.61(m,2H),8.07(s, 8H), 1.42 (s, 1H); ESI-MS calculation of M/z C51H50F6N4O7[ M + H ]]+945.10 measurement 944.97
Example 9: preparation of (R, Z) -4- ((2- (2- (2- ((3- (3, 4-diphenylethoxyphenoxy) -2-hydroxypropyl) amino) ethoxy) ethyl) imino) -2-phenyl-4H-chromene-5, 6, 7-triol (Begatran-2204)
Step 1) preparation of tert-butyl (2- (2- (2-aminoethoxy) ethoxy) ethyl) carbamate (L-1)
2,2' - (ethane-1, 2-diylbis (oxy)) diethylamine (L-2, 5g, 33.7mmol) was dissolved in dichloromethane (DCM, 100mL) and di-tert-butyl dicarbonate (Boc2O, 7.36g, 33.7mmol) was added thereto. The mixture was stirred at room temperature for about 16 hours. After completion of the reaction, the reaction solution was filtered and concentrated. The concentrated solution was purified by column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 3 to 1: 1). Thus, colorless liquid, (tert-butyl 2- (2- (2-aminoethoxy) ethoxy) ethyl) carbamate (L-2, 2.2g) was obtained.
Step 2) preparation of tert-butyl (Z) - (2- (2- (2- ((5,6, 7-trihydroxy-2-phenyl-4H-chromen-4-ylidene) amino) ethoxy) ethyl) carbamate (TL-6)
Tert-butyl (2- (2- (2-aminoethoxy) ethoxy) ethyl) carbamate (TL-6, 300mg, 1.21mmol) was dissolved in methanol (MeOH, 5mL) and 5,6, 7-trihydroxyflavone (Begazine, Baicalein, 326mg, 1.21mmol) was added thereto. The mixture was stirred at 65 ℃ for about 16 hours. When the reaction was complete, the reaction solution was filtered and concentrated. The concentrated solution was purified by preparative high performance liquid chromatography (Prep-HPLC). Thus, tert-butyl (Z) - (2- (2- (2- ((5,6, 7-trihydroxy-2-phenyl-4H-chromen-4-ylidene) amino) ethoxy) ethyl) carbamate (TL-6, 120mg) was obtained as a yellow solid.
Step 3) preparation of (Z) -4- ((2- (2- (2-aminoethoxy) ethoxy) ethyl) imino) -2-phenyl-4H-chromene-5, 6, 7-triol (TL-7)
Tert-butyl (Z) - (2- (2- (2- ((5,6, 7-trihydroxy-2-phenyl-4H-chromen-4-ylidene) amino) ethoxy) ethyl) carbamate (TL-6, 120mg, 0.24mmol) was dissolved in ethyl acetate (EA, 2mL) to which was then added hydrochloric acid (g)/ethyl acetate (1 mL). The mixture was stirred at room temperature for about 2 hours. Upon completion of the reaction, the reaction mixture was extracted with ethyl acetate, filtered and concentrated. Thus, a yellow solid, (Z) -4- ((2- (2- (2-aminoethoxy) ethoxy) ethyl) imino) -2-phenyl-4H-chromene-5, 6, 7-triol (TL-7, 90mg) was obtained.
Step 4) preparation of (R, Z) -4- ((2- (2- (2- ((3- (3, 4-diphenylethoxyphenoxy) -2-hydroxypropyl) amino) ethoxy) ethyl) imino) -2-phenyl-4H-chromene-5, 6, 7-triol (Baicalein-2204)
(Z) -4- ((2- (2- (2-aminoethoxy) ethoxy) ethyl) imino) -2-phenyl-4H-chromene-5, 6, 7-triol (TL-7, 90mg, 0.22mmol) was dissolved in methanol (MeOH, 2mL) and (R) -2- ((3, 4-diphenylethoxyphenoxy) methyl) oxirane (A-2, 87mg, 0.22mmol) was added thereto. The mixture was stirred at 65 ℃ for about 16 hours. When the reaction was complete, the reaction solution was filtered and concentrated. The concentrated solution was purified by preparative high performance liquid chromatography (Prep-HPLC). Thus, a yellow solid, (R, Z) -4- ((2- (2- (2- ((3- (3, 4-diphenylethoxyphenoxy) -2-hydroxypropyl) amino) ethoxy) ethyl) imino) -2-phenyl-4H-chromene-5, 6, 7-triol (bexain-2204, 12mg) was obtained.1H NMR(DMSO-d6，400MHz) δ (ppm)2.91(m,7H),3.35(m,11H),3.80(m,3H),3.96(t, J ═ 8Hz,2H),4.08(t, J ═ 8Hz,2H),6.32(dd, J ═ 4Hz and 4Hz,1H),6.37(s,1H),6.47(s,1H),6.76(d, J ═ 12Hz,2H),7.25(m,10H),7.58(t, J ═ 4Hz,3H),7.99(d, J ═ 8Hz,2H),8.42(s, 1H); ESI-MS calculation of m/z C46H50N2O10[M+H]+791.20 measurement 790.91
Example 10: (E) preparation of (E) -5- (4- (2- (2- (2- ((3, 4-bis (benzyloxy) benzyl) amino) ethoxy) styryl) benzene-1, 3-diol (resveratrol-1105)
Step 1) preparation of 2- (2- (2- ((3, 4-bis (benzyloxy) benzyl) amino) ethoxy) ethan-1-ol (AL-5)
3, 4-bis (benzyloxy) benzaldehyde (1101, 25g, 78.6mmol) was dissolved in methanol (MeOH, 250mL), to which was then added 2- (2- (2-aminoethoxy) ethoxy) ethanol (L-3, 11.7g, 78.6 mmol). The mixture was stirred at 65 ℃ for about 6 hours. After cooling to room temperature, sodium borohydride (NaBH) was further added43g, 78.6mmol) and then stirred at 50 ℃ overnight. To the reaction solution was added water to complete the reaction, and the compound was extracted with ethyl acetate (EtOAc, 50 mL. times.3). The extracted compound was washed with brine and the remaining water was removed with sodium sulfate. After filtration, the solution was concentrated and the concentrated solution was purified by column chromatography using silica gel (dichloromethane/methanol ═ 20: 1). Thus, 2- (2- (2- ((3, 4-bis (benzyloxy) benzyl) amino) ethoxy) ethan-1-ol (AL-5, 13g) was obtained as a yellow liquid.1H NMR(DMSO-d6400MHz) δ (ppm)7.46-7.30(m,10H),7.06(d, J ═ 1.6Hz,1H),6.97(d, J ═ 8Hz,1H),6.82(d, J ═ 1.2Hz,1H),5.10(d, J ═ 4Hz,4H),3.61(s,2H),3.50-3.39(m,11H),2.58(t, J ═ 6Hz,2H) ESI-MS calculation m/z C27H33N5O5[M+H]+452.10 measurement 451.56
Step 2) preparation of tert-butyl (3, 4-bis (benzyloxy) benzyl) (2- (2- (2-hydroxyethoxy) ethoxy) ethyl) carbamate (AL-6)
2- (2- (2- ((3, 4-bis (benzyloxy) benzyl) amino) ethoxy) ethan-1-ol (AL-5, 500mg, 1.11mmol) was dissolved in dichloromethane (DCM, 6mL) to which was then added triethylamine (TEA, 168mg, 1.66mmol) and di-tert-butyl dicarbonate (Boc2O, 290mg, 1.33 mmol). The mixture was stirred at room temperature for about 4 hours. When the reaction was complete, the reaction solution was filtered and concentrated. The concentrated solution was purified by column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 5 to 1: 1). Thus, colorless tert-butyl (3, 4-bis (benzyloxy) benzyl) (2- (2- (2-hydroxyethoxy) ethoxy) ethyl) carbamate (AL-6, 520mg) was obtained.
Step 3) preparation of tert-butyl (E) - (3, 4-bis (benzyloxy) benzyl) (2- (2- (2- (4- (3, 5-dihydroxystyryl) phenoxy) ethoxy) ethyl) carbamate (ATL-1)
Tert-butyl (3, 4-bis (benzyloxy) benzyl) (2- (2- (2-hydroxyethoxy) ethoxy) ethyl) carbamate (AL-6, 300mg, 0.54g) was dissolved in tetrahydrofuran (THF, 5mL) and Resveratrol (Resveratrol) (149mg, 0.65mmol), triphenylphosphine (PPh3, 214mg, 0.82mmol) and diisopropyl azodicarboxylate DIAD (165mg, 0.82mmol) were added thereto. The mixture was stirred at 65 ℃ for about 16 hours. After completion of the reaction, the reaction solution was filtered and concentrated. The concentrated solution was purified by column chromatography using silica gel (dichloromethane/methanol ═ 100: 1 to 50: 1). Thus, tert-butyl (E) - (3, 4-bis (benzyloxy) benzyl) (2- (2- (2- (4- (3, 5-dihydroxystyryl) phenoxy) ethoxy) ethyl) carbamate (ATL-1, 200mg) was obtained as a yellow solid.
Step 4) preparation of (E) -5- (4- (2- (2- (2- ((3, 4-bis (benzyloxy) benzyl) amino) ethoxy) styryl) benzene-1, 3-diol (resveratrol-1105)
Tert-butyl (E) - (3, 4-bis (benzyloxy) benzyl) (2- (2- (2- (4- (3, 5-dihydroxystyryl) phenoxy) ethoxy) ethyl) carbamate (ATL-1, 200mg) was dissolved in ethyl acetate (EA, 4mL), to which was then added hydrochloric acid (g)/ethyl acetate (1 mL). The mixture was stirred at room temperature for about 2 hours. Reaction ofAfter completion, the reaction solution was filtered and then concentrated. The concentrated solution was purified by high resolution liquid chromatography (Prep-HPLC). Thus, a white solid, (E) -5- (4- (2- (2- (2- ((3, 4-bis (benzyloxy) benzyl) amino) ethoxy) styryl) benzene-1, 3-diol (resveratrol-1105, 15mg) was obtained.1H NMR(DMSO-d6400MHz) δ (ppm)2.58(t, J ═ 4Hz,2H),3.45(t, J ═ 4Hz,2H),3.51(m,2H),3.57(m,4H),3.73(m,2H),4.06(m,2H),5.07(m,4H),6.12(m,1H),6.40(m,2H),6.90(m,7H),7.39(m,12H),9.20(s, 2H); ESI-MS calculation of m/z C41H43NO7[M+H]+662.10 measurement 661.80
Example 11: preparation of (R) -2- (4- (benzo [ d ] thiazol-2-yl) phenyl) -14- (3, 4-bis (benzyloxy) phenoxy) -5, 8-dioxa-2, 11-diazatetram-13-ol (BTA-1-1104)
Step 1) preparation of 2, 2-dimethyl-4-oxo-3, 8, 11-trioxa-5-azatridecan-13-yl-methanesulfonate (L-5)
(2- (2- (2-hydroxyethoxy) ethoxy) ethyl) carbamic acid tert-butyl ester (L-4, 1g, 4.0mmol) was dissolved in dichloromethane (DCM, 15mL) and triethylamine (TEA, 0.486g, 4.8mmol) and methanesulfonyl chloride (MsCl, 0.504g, 4.4mmol) were added thereto. The mixture was stirred at room temperature for about 4 hours. Upon completion of the reaction, the compound was extracted, filtered, and then concentrated. The concentrated solution was purified by column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 5 to 1: 1). Thus, colorless liquid, 2, 2-dimethyl-4-oxo-3, 8, 11-trioxa-5 azatridecan-13-yl-methanesulfonate (L-5, 1g) was obtained.
Step 2) preparation of tert-butyl (2- (2- (2- ((4- (benzo [ d ] thiazol-2-yl) phenyl) (methyl) amino) ethoxy) ethyl) carbamate (TL-8)
2, 2-dimethyl-4-oxo-3, 8, 11-trioxa-5-azatridecan-13-yl-methanesulfonate (L-5, 300mg, 0.92mmol) was dissolved in dimethyl sulfoxide (DMSO, 5mL), to which was then added 2- (4' -methylaminophenyl) benzothiazole (BTA-1, 220mg, 0.92mmol) and potassium tert-butoxide (t-BuOK, 154mg, 1.37 mmol). The mixture was stirred at 120 ℃ for about 16 hours. When the reaction was completed, the reaction solution was filtered and then concentrated. The concentrated solution was purified by preparative high performance liquid chromatography (Prep-HPLC). Thus, tert-butyl (2- (2- (2- ((4- (benzo [ d ] thiazol-2-yl) phenyl) (methyl) amino) ethoxy) ethyl) carbamate (TL-8, 180mg) was obtained as a yellow liquid.
Step 3) preparation of N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -4- (benzo [ d ] thiazol-2-yl) -N-toluidine (TL-9)
Tert-butyl (2- (2- (2- ((4- (benzo [ d ] thiazol-2-yl) phenyl) (methyl) amino) ethoxy) ethyl) carbamate (TL-8, 180mg, 0.38mmol) was dissolved in ethyl acetate (EA, 4mL), to which was then added hydrochloric acid (g)/ethyl acetate (1 mL). The mixture was stirred at room temperature for 2 hours. Upon completion of the reaction, the compound was extracted, filtered, and then concentrated. Thus was obtained a yellow solid, N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -4- (benzo [ d ] thiazol-2-yl) -N-toluidine (TL-9, 120 mg).
Step 4) preparation of (R) -2- (4- (benzo [ d ] thiazol-2-yl) phenyl) -14- (3, 4-bis (benzyloxy) phenoxy) -5, 8-dioxan-2, 11-diazatetradecane-13-ol (BTA-1-1104)
Reacting N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -4- (benzo [ d ]]Thiazol-2-yl) -N-toluidine (TL-9, 120mg, 0.33mmol) was dissolved in methanol (MeOH, 2mL) and (R) -2- ((3, 4-bis (benzyloxy) phenoxy) methyl) oxirane (1103, 117mg, 0.33mmol) was added thereto. The mixture was stirred at 65 ℃ for about 16 hours. Upon completion of the reaction, the compound was extracted, filtered, and then concentrated. The concentrated solution was purified by preparative high performance liquid chromatography (Prep-HPLC). Thus, a yellow solid, (R) -2- (4- (benzo [ d ] is obtained]Thiazol-2-yl) phenyl) -14- (3, 4-bis (benzyloxy) phenoxy) -5, 8-dioxa-2, 11-diazatetran-13-ol (BTA-1-1104, 8 mg).1H NMR(DMSO-d6，400MHz)δ(ppm)1.15(d,J＝8Hz,3H),1.23(s,1H),2.67(m,4H),3.50(m,11H),3.81(m,4H),5.01(s,2H),5.10(s,2H),6.27(d,J＝8Hz,1H),6.41(d,J＝8Hz,1H),6.69(m,3H),6.91(d,J＝12Hz,1H),7.38(m,12H),7.79(d, J ═ 8Hz,2H),7.89(d, J ═ 8Hz,1H),7.90(d, J ═ 8Hz,1H),8.40(s, 1H); ESI-MS calculation of m/z C43H47N3O6S[M+H]+734.10 measurement 733.92
Example 12: preparation of (1E,6E) -1- (4- (2- (2- (((R) -3- (3- (benzyloxy) -4-phenylethoxyphenoxy) -2-hydroxypropyl) amino) ethoxy) -3-methoxyphenyl) -7- (4-hydroxy-3-methoxyphenyl) hepta-1, 6-diene-3, 5-dione (curcumin-1204)
Step 1) preparation of 4- (Phenylmethoxy) -3-Phenylethoxybenzaldehyde (1201)
4- (Phenylmethoxy) -3-hydroxybenzaldehyde (A-3, 50g, 219mmol) was dissolved in tetrahydrofuran (THF, 1L) and 2-phenylethyl alcohol (32.1g, 263mmol), triphenylphosphine (PPh3, 86.2g, 329mmol) and diisopropyl azodicarboxylate (DIAD, 66.4g, 329mmol) were added thereto. The mixture was stirred at 65 ℃ for about 16 hours. Upon completion of the reaction, the compound was extracted, filtered and concentrated. The concentrated solution was purified by column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 15 to 1: 10). Thus, 4- (benzyloxy) -3-phenethyloxybenzaldehyde (1201, 25g) was obtained as a white solid.
1H NMR(DMSO-d6400MHz) δ (ppm)9.82(s,1H),7.52(dd, J ═ 8Hz and 2Hz,1H),7.43-7.40(m,5H),7.37-7.32(m,3H),7.28-7.21(m,4H),5.21(s,2H),4.26(t, J ═ 6.4Hz,2H),3.05(t, J ═ 6.4Hz,2H)
Step 2) preparation of 4- (Phenylmethoxy) -3-Phenoxyphenol (1202)
4- (Phenylmethoxy) -3-phenylethoxybenzaldehyde (1201, 50g, 150mmol) was dissolved in dichloromethane (DCM, 500mL) and m-chloroperoxybenzoic acid (m-CPBA, 39g, 225mmol) was added thereto. The mixture was stirred at room temperature for about 16 hours. Upon completion of the reaction, the compound was extracted, filtered and concentrated. The concentrated solution was purified by column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 15 to 1: 5). So as to obtain a white solid which is,4- (Phenylmethoxy) -3-phenethyloxyphenol (1202, 32 mg).1H NMR(DMSO-d6400MHz) δ (ppm)9.00(s,1H),7.36-7.20(m,10H),6.78(d, J ═ 8Hz,1H),6.43(d, J ═ 2.8Hz,1H),6.22(dd, J ═ 8Hz and 2.8Hz,1H),4.86(s,2H),4.13(t, J ═ 6.8Hz,2H),3.02(t, J ═ 6.4Hz,2H)
Step 3) preparation of (R) -2- ((4- (benzyloxy) -3-phenethyloxyphenoxy) methyl) oxirane (1203)
4- (Phenylmethoxy) -3-phenethyloxyphenol (1203, 40g, 64mmol) was dissolved in ethanol (EtOH, 800mL), to which was then added water (40mL) and potassium hydroxide (KOH, 8.2g, 146 mmol). Then, (R) -2- (chloromethyl) oxirane (34.6g, 374mmol) was added, and the mixture was further stirred at room temperature for 16 hours. Water (1600mL) was added to the reaction solution to complete the reaction, and the mixture was extracted with ethyl acetate (1600 mL. times.3). The extracted organic solvent layer was washed with brine and the remaining water was removed with sodium sulfate. The resulting material was filtered to remove impurities and concentrated. The concentrated solution was purified by column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 15 to 1: 10). Thus, white solid, (R) -2- ((4- (benzyloxy) -3-phenethyloxyphenoxy) methyl) oxirane (1203, 34g) was obtained.1H NMR(DMSO-d6400MHz) δ (ppm)7.38-7.20(m,10H),6.90(d, J ═ 8.8Hz,1H),6.64(d, J ═ 2Hz,1H),6.41(dd, J ═ 8.8Hz and 2.8Hz,1H),4.93(s,2H),4.25-4.17(m,3H),3.75(dd, J ═ 11.2Hz and 6.4Hz,1H),3.28(m,1H),3.03(t, J ═ 6.8Hz,2H),2.81(t, J ═ 4.4Hz,1H),2.67 (t, J ═ 5.2Hz, 1H),2.8 Hz,1H)
Step 4) preparation of tert-butyl (2- (2- (2- (4- ((1E,6E) -7- (4-hydroxy-3-methoxyphenyl) -3, 5-dioxohept-1, 6-dien-1-yl) -2-methoxyphenoxy) ethoxy) ethyl) carbamate (TL-10)
(2- (2- (2-hydroxyethoxy) ethoxy) ethyl) carbamic acid tert-butyl ester (L-4, 300mg, 1.2mmol) was dissolved in tetrahydrofuran (THF, 5mL), to which curcumin (curcumin) (442mg, 1.2mmol), triphenylphosphine (PPh3, 377mg, 1.44mmol) and diisopropyl azodicarboxylate (DIAD, 291mg, 1.44mmol) were then added. The mixture was stirred at 65 ℃ for about 16 hours. Upon completion of the reaction, the compound was extracted, filtered and concentrated. By column chromatography using silica gel (dichloromethane)Methanol 100: 1 to 50: 1) the concentrated solution was purified. Thus, tert-butyl (2- (2- (2- (4- ((1E,6E) -7- (4-hydroxy-3-methoxyphenyl) -3, 5-dioxohept-1, 6-dien-1-yl) -2-methoxyphenoxy) ethoxy) ethyl) carbamate (TL-10, 200mg) was obtained as a yellow solid.1H NMR(DMSO-d6400MHz) δ (ppm)9.87(brs,1H),7.57(dd, J ═ 16Hz and 4Hz,2H),3.34(dd, J ═ 12Hz and 1.6Hz,2H),7.24(dd, J ═ 8Hz and 1.2Hz,1H),7.16(dd, J ═ 8Hz and 1.2Hz,1H),7.02(d, J ═ 8Hz,1H),6.85-6.74(m,4H),6.08(s,1H),4.13(t, J ═ 4Hz,2H),3.83(m,6H),3.75(t, J ═ 4Hz,2H),3.59(q, J ═ 4Hz,2H),3.52(q, J ═ 4, 2H),3.40 (m, 3.38H), 3.06H, 3.06(q, J ═ 4H), 3.06H, 3.6H, 1H)
Step 5) preparation of (1E,6E) -1- (4- (2- (2- (2-aminoethoxy) ethoxy) -3-methoxyphenyl) -7- (4-hydroxy-3-methoxyphenyl) hepta-1, 6-diene-3, 5-dione (TL-11)
Tert-butyl (2- (2- (2- (4- ((1E,6E) -7- (4-hydroxy-3-methoxyphenyl) -3, 5-dioxohept-1, 6-dien-1-yl) -2-methoxyphenoxy) ethoxy) ethyl) carbamate (TL-10, 200mg, 0.33mmol) was dissolved in ethyl acetate (EA, 4mL), to which was then added hydrochloric acid (g)/ethyl acetate (1 mL). The mixture was stirred at room temperature for about 2 hours. After completion of the reaction, the compound was extracted, filtered, and then concentrated. Thus, a yellow solid, (1E,6E) -1- (4- (2- (2- (2-aminoethoxy) ethoxy) -3-methoxyphenyl) -7- (4-hydroxy-3-methoxyphenyl) hepta-1, 6-diene-3, 5-dione (TL-11, 120mg) was obtained.
Step 6) preparation of (1E,6E) -1- (4- (2- (2- (2- (((R) -3- (3- (benzyloxy) -4-phenethyloxyphenoxy) -2-hydroxypropyl) amino) ethoxy) -3-methoxyphenyl) -7- (4-hydroxy-3-methoxyphenyl) hepta-1, 6-diene-3, 5-dione (curcumin-1204)
(1E,6E) -1- (4- (2- (2- (2-aminoethoxy) ethoxy) -3-methoxyphenyl) -7- (4-hydroxy-3-methoxyphenyl) hepta-1, 6-diene-3, 5-dione (TL-11, 120mg, 0.24mmol) was dissolved in methanol (MeOH, 2mL) and (R) -2- ((4- (benzyloxy) -3-phenethyloxyphenoxy) methyl) oxirane (1203, 90mg, 0.24mmol) was added thereto. The mixture was stirred at 65 ℃ for about 16 hours. On completion of the reaction, the compound is extractedFiltered and then concentrated. The concentrated solution was purified by preparative high performance liquid chromatography (Prep-HPLC). Thus, a yellow solid, (1E,6E) -1- (4- (2- (2- (2- (((R) -3- (3- (benzyloxy) -4-phenethyloxyphenoxy) -2-hydroxypropyl) amino) ethoxy) -3-methoxyphenyl) -7- (4-hydroxy-3-methoxyphenyl) hepta-1, 6-diene-3, 5-dione (curcumin-1204, 12mg) was obtained.1H NMR(DMSO-d6+ D2O,400MHz) δ (ppm)2.81(m,1H),2.92(m,3H),3.00(t, J ═ 6.4Hz,2H),3.56(m,9H),3.80(m,9H),3.97(b,2H),4.09(m,2H),4.14(t, J ═ 6.4Hz,2H),4.88(s,2H),6.36(dd, J ═ 8.8Hz and 2.8Hz,1H),6.56(D, J ═ 2.8Hz,1H),6.79(m,4H),6.97(D, J ═ 8Hz,1H),7.23(m,14H),7.53(D, J ═ 16Hz,2H),8.33(s, 1H); ESI-MS calculation of m/z C51H57NO12[M]+876.10 measurement 876.01
Example 13: preparation of (R) -1- (3-Phenoxyphenoxy) -3- ((2- (2- (2- ((6- (trifluoromethoxy)) benzo [ d ] thiazol-2-yl) amino) ethoxy) ethyl) amino) propan-2-ol (riluzole-204)
Step 1) preparation of 3-Phenoxyphenol (A-5)
Resorcinol (50g, 0.45mol) was dissolved in acetonitrile (ACN, 500mL), to which potassium carbonate (K) was then added2CO3112.5g, 0.81mol) and (2-bromoethyl) benzene (83.2g, 0.45 mol). The mixture was stirred at 60 ℃ for about 10 hours. After completion of the reaction, the compound was extracted, filtered and concentrated. The concentrated solution was purified by column chromatography using silica gel (petroleum ether/ethyl acetate ═ 5: 1). Thus, 3-phenethyloxyphenol (A-5, 25g) was obtained as a yellow liquid.1H NMR(CDCl3，400MHz)δ(ppm)7.31-7.23(m,5H),7.11(t,J＝8Hz,1H),6.50-6.39(m,3H),4.74(s,1H),4.14(m,2H),3.08(t,J＝7.2Hz,2H)
Step 2) preparation of (R) -2- ((3-Phenoxyphenoxy) methyl) oxirane (A-6)
3-Phenoxyphenol (A-5, 25g, 116.8mmol) was dissolved in ethanol (EtOH, 500mL), to which was then added water(25mL) and potassium hydroxide (KOH, 11.1g, 278.3 mmol). Then, (R) -2- (chloromethyl) oxirane (64.6g, 698.8mmol) was added, followed by stirring at room temperature for about 16 hours. Water (1000mL) was added to the reaction solution to complete the reaction, and the compound was extracted with ethyl acetate (500 mL. times.3). The extracted organic solvent layer was washed with brine, and the remaining water was removed with sodium sulfate. The resulting material was filtered to remove impurities and concentrated. The concentrated solution was purified by column chromatography using silica gel (ethyl acetate/petroleum ether ═ 1: 15 to 1: 10). Thus, a yellow liquid (R) -2- ((3-phenethyloxyphenoxy) methyl) oxirane (A-6, 18g) was obtained.1H NMR(DMSO-d6400MHz) δ (ppm)7.32-7.28(m,4H),7.24-7.14(m,2H),6.52(m,3H),4.29(dd, J ═ 11.2Hz and 2Hz,1H),4.16(t, J ═ 6.8Hz,2H),3.79(m,1H),3.34(s,1H),3.01(t, J ═ 6Hz,2H),2.82(t, J ═ 4Hz,1H),2.69-2.67(m,1H)
Step 3) preparation of tert-butyl (2- (2- (2- ((6- (trifluoromethoxy) benzo) [ d ] thiazol-2-yl) amino) ethoxy) ethyl) carbamate (TL-12)
2, 2-dimethyl-4-oxo-3, 8, 11-trioxa-5 azatridecan-13-yl-methanesulfonate (L-5, 300mg, 0.92mmol) was dissolved in dimethyl sulfoxide (DMSO, 5mL), to which Riluzole (Riluzole) (214mg, 0.92mmol) and potassium tert-butoxide (t-BuOK, 154mg, 1.37mmol) were then added. The mixture was stirred at 120 ℃ for about 16 hours. After completion of the reaction, the compound was extracted, filtered, and then concentrated. The concentrated solution was purified by preparative high performance liquid chromatography (Prep-HPLC). Thus, a pale yellow liquid, (2- (2- (2- ((6- (trifluoromethoxy) benzo [ d)]Thiazol-2-yl) amino) ethoxy) ethyl) carbamic acid tert-butyl ester (TL-12, 180 mg).1H NMR(DMSO-d6400MHz) δ (ppm)8.27-8.25(m,1H),7.78(d, J ═ 1.6Hz,1H),7.41(d, J ═ 8Hz,1H),7.18(dd, J ═ 8Hz and 1.6Hz,1H),6.74(m,1H),3.63-3.51(m,12H),3.05(q, J ═ 6Hz,2H),1.36(s,11H)
Step 4) preparation of N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -6- (trifluoromethoxy) benzo [ d ] thiazol-2-amine (TL-13)
Tert-butyl (2- (2- (2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) ethoxy) ethyl) carbamate (TL-12, 180mg, 0.39mmol) was dissolved in ethyl acetate (EA, 4mL), to which was then added hydrochloric acid (g)/ethyl acetate (1 mL). The mixture was stirred at room temperature for about 2 hours. Upon completion of the reaction, the compound was extracted, filtered and concentrated. Thus was obtained a yellow solid, N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -6- (trifluoromethoxy) benzo [ d ] thiazol-2-amine (TL-13, 120 mg).
Step 5) preparation of (R) -1- (3-Phenoxyphenoxy) -3- ((2- (2- (2- ((6- (trifluoromethoxy)) benzo [ d ] thiazol-2-yl) amino) ethoxy) ethyl) amino) propan-2-ol (riluzole-204)
Reacting N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -6- (trifluoromethoxy) benzo [ d]Thiazol-2-amine (TL-13, 120mg, 0.33mmol) was dissolved in methanol (MeOH, 2mL) and (R) -2- ((3-phenethyloxyphenoxy) methyl) oxirane (A-6, 89mg, 0.33mmol) was added thereto. The mixture was stirred at 65 ℃ for about 16 hours. After completion of the reaction, the compound was extracted, filtered and concentrated. The concentrated solution was purified by preparative high performance liquid chromatography (Prep-HPLC). Thus, a colorless liquid was obtained, (R) -1- (3-phenethyloxyphenoxy) -3- ((2- (2- (2- ((6- (trifluoromethoxy)) benzo [ d)]Thiazol-2-yl) amino) ethoxy) ethyl) amino) propan-2-ol (riluzole-204, 15 mg). 1H NMR (400MHz, DMSO-d6) δ (ppm)2.60(m,4H),3.00(t, J ═ 8Hz,2H),3.52(m,10H),3.85(m,3H),4.15(t, J ═ 8Hz,2H),6.48(dd, J ═ 12Hz and 8Hz,3H),7.18(m,3H),7.31(d, J ═ 4Hz,4H),7.43(d, J ═ 12Hz,1H),7.75(s, 1H); ESI-MS calculation of m/z C31H36F3N3O6S[M+H]+636.10 measurement 635.70
EXAMPLE 1 evaluation of oligomerization Activity of p62 protein in cultured cells by immunoblotting
To evaluate the efficacy of p62 protein oligomerization activity of compounds 1 to 13 (examples 1 to 13), HEK293 cell lines were collected, which were human embryonic kidney derived cells. Cells were treated with the compound of the example and the p62 ligand compound YTK-1105 and DMSO-treated cells were used as a control.
To measure intracellular p62 protein activation and oligomerization in cells treated with these compounds, each cell was dispensed into 100pi dishes. After further incubation for 24 hours, the cells were collected so that the cells were completely attached to the surface of the plate. 100ul of lysis buffer (20mM Tris (pH 7.4), 150mM NaCl, 1% Triton X-100, 2mM NaF, 2mM EDTA, 2mM beta-glycerophosphate, 5mM sodium orthovanadate, 1mM PMSF, leupeptin, aprotinin) was injected into each sample to lyse the cells. Based on the total protein concentration measured, each sample was treated with the test compound at room temperature for 2 hours, and then a sample buffer was added to allow it to react at 95 ℃ for 10 minutes. After the reaction, 25ul of the sample was taken and distributed in each well of the acrylamide gel, followed by immunoblotting. Immunoblots show representative results from three or more independent experiments. The results are shown in FIGS. 2a to 2 c.
As shown in fig. 2a to 2c, it was confirmed that treatment with the AUTOTAC chimeric compound according to the present invention and the p62 ligand compound YTK-1105 results in a decrease in monomers of p62 protein, and an increase in oligomers and polymer aggregates, unlike that when treated with DMSO as a control group.
EXAMPLE 2 evaluation of target protein degradation in cultured cells by immunoblotting
To evaluate the target protein degradation efficacy of the compounds (examples 1 to 13), cell lines predominantly expressing the target protein (MCF7, NTERA-2, ACHN, U87-MG, LNCaP, HEK293T) or recombinant cell lines (SH-SY5Y-tau, HeLa-HttQ97, PC12-a-synA30P) were cultured in 12-well plates and treated with the corresponding AUTOTAC chimeric compounds at concentrations, followed by immunoblotting as in Experimental example 1. Immunoblots show representative results from three or more independent experiments. The results are shown in FIGS. 3a, 3b and 3 c.
As shown in fig. 3a, 3b and 3c, it was confirmed that the level of the target protein gradually increased with the concentration of the compound when treated with the AUTOTAC chimeric compound according to the present invention.
EXAMPLE 3 evaluation of target protein degradation mechanism in cultured cells by immunoblotting
To assess whether the target protein degradation mechanism of the aforementioned compounds (examples 1-13) was mediated by autophagy, cell lines or genetically recombinant cell lines expressing mainly the target protein were cultured in 12-well plates and treated with 2.5 μ M of the corresponding AUTOTAC chimeric compound alone or in combination with 10 μ M of Hydroxychloroquine (HCQ), an inhibitor of the autophagy-lysosomal pathway, followed by immunoblotting as in Experimental example 1. Immunoblots show representative results from three or more independent experiments. The results are shown in FIGS. 4a and 4 b.
As shown in fig. 4a and 4b, it was confirmed that the level of the target protein was decreased when treated with the autosac chimeric compound according to the present invention alone, and the decreased level of the target protein was increased again when treated with binding HCQ (inhibitor of autophagy-lysosomal pathway).
EXAMPLE 4 comparative evaluation of target protein degradation efficacy in cultured cells Using immunoblotting
To evaluate whether the target protein degradation efficacy of the compounds (examples 1 to 13) was superior to that of the p62 ligand or the target protein ligand, which is a chimeric component, cell lines or gene recombinant cell lines mainly expressing the target protein were cultured in 12-well plates and treated with the corresponding AUTOTAC chimeric compound, p62 ligand or target protein ligand, followed by immunoblotting as in Experimental example 1. Immunoblots show representative results from three or more independent experiments. The results are shown in fig. 5a to 5 c.
As shown in fig. 5a to 5c, it was confirmed that the level of the target protein after the treatment with the autoctac chimeric compound according to the present invention was significantly reduced compared to that after the treatment with the p62 ligand or the target protein ligand.
EXAMPLE 5 evaluation of P62-mediated Activity of delivering target protein in cultured cells Using immunofluorescence staining and confocal microscopy
To demonstrate the efficacy of p62 mediated delivery of target proteins to autophagy following treatment with compounds (examples 1-13), immunofluorescent staining was performed using p62 and each target protein as markers. The glass cover is placed on a 24-well plate for immunofluorescence staining, and a cell line or a gene recombinant cell line mainly expressing the target protein is cultured. Cells were distributed and cultured for 24 hours, and then treated with 2.5. mu.M of the novel AUTOTAC chimeric ligand according to the present invention. The cells were further cultured for 24 hours to exert the effect of the compound, the medium was removed, and the cells were fixed with formaldehyde at room temperature. To prevent non-specific staining, cells were allowed to react with a blocking solution at room temperature for 1 hour, and then the p62 antibody and the target protein antibody diluted to a specific ratio were treated with the blocking solution and then reacted at room temperature for 1 hour. After washing the antibody-treated cells three times with PBS, the goat-derived secondary antibody was diluted to a specific ratio using a blocking solution, and then reacted at room temperature for 30 minutes. The cells were washed three times again with PBS, and were subjected to DAPI staining treatment for intracellular core staining, and then the expression levels of p62 and LC3, intracellular fluorescent spot formation, and intracellular coexistence levels were observed using a confocal microscope. The results are shown in fig. 6a to 6 c. Immunofluorescent staining showed representative results from three or more independent experiments.
As shown in FIGS. 6a to 6c, it was confirmed that the formation of a fluorescent spot of intracellular p62 protein, the formation of a fluorescent spot of intracellular target protein and local coexistence thereof were increased after the treatment with the AUTOTAC chimeric compound according to the present invention.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：作为LSD1抑制剂的杂螺环类化合物及其应用