CN117447466A - Oxazole compound and preparation method and application thereof - Google Patents

Oxazole compound and preparation method and application thereof Download PDF

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CN117447466A
CN117447466A CN202210851738.3A CN202210851738A CN117447466A CN 117447466 A CN117447466 A CN 117447466A CN 202210851738 A CN202210851738 A CN 202210851738A CN 117447466 A CN117447466 A CN 117447466A
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罗成
林华
李佳城
刘丽萍
王明宇
陈凯先
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Shanghai Institute of Materia Medica of CAS
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract

The invention relates to an oxazole compound and a preparation method and application thereof, and in particular discloses an oxazole compound represented by a general formula I, and pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, atropisomers, polymorphs, solvates or isotopically labeled compounds thereof. The compound shown in the formula (I) has high-efficiency and selective CDK9-cyclin T1 complex degradation, and can be used for preparing medicines for preventing and/or treating diseases or symptoms mediated by CDK9-cyclin T1 complex, especially tumors.

Description

Oxazole compound and preparation method and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a compound for degrading CDK9-cyclin T1 complex, a preparation method, a pharmaceutical composition and application thereof.
Background
Cyclin-dependent kinases (CDKs) are important regulatory proteins in the life cycle activities of mammalian cells, belonging to the conserved serine/threonine protein kinase family. CDKs consist of a dimeric complex of a cell cycle catalytic subunit and a regulatory subunit, which bind to cyclin (cyclin) and bind to a cell cycle (including the prophase of DNA synthesis (G) 1 Stage), DNA synthesis stage (S stage), DNA synthesis late stage (G) 2 Phase), mitosis phase (M phase), and G in a dormant state of cells 0 Phase), transcription of cells, DNA damage and repair, stem cells, metabolism, epigenetic and angiogenic processes. It has now been found that the human genome encodes 21 CDKs and 29 cyclin proteins, which are divided into two classes according to their primary functions: one is a subfamily that regulates cell cycle, including regulation of G 1 CDKs (CDK 2-cyclin E, CDK4-cyclin D, CDK6-cyclin D, etc.) and modulation of S phase/G 2 CDKs (CDK 1-cyclin A, CDK1-cyclin B, CDK2-cyclin A, etc.) of phase/M; one class is CDK7-cyclin H, CDK8-cyclin C, CDK9-cyclin T, CDK11-cyclin L, etc. that are involved in cell transcriptional regulation. The CDKs subfamilies which are divided according to functions act on each regulation site in a cooperativity and periodicity mode in the whole cell growth and proliferation process, so that the normal operation of the whole cell life cycle is ensured. Since tumors and various proliferative diseases are caused by abnormal changes in the cell cycle, CDKs are important targets for the treatment of these diseases.
Cyclin-dependent kinase 9 (CDK 9) forms a complex with cyclin T or cyclin K as a CDKs family member, forward transcriptionally-extending factor b (P-TEFb), which regulates cellular transcription by phosphorylating the carboxy-terminal domain (CTD) of the largest subunit (RPB 1) of RNA polymerase II (RNAPII) to facilitate transcriptional extension. Given that P-TEFb is a universal transcription factor for the efficient expression of most genes, CDK9 is widely found in the transcription process of normal cells and thus does not appear to be an ideal target for the treatment of any disease. However, a great deal of research shows that CDK9 is closely related to pathogenesis of tumors (leukemia, breast cancer and liver cancer), heart diseases and AIDS, and is a key target of the related drugs for the diseases. Through studies of the CDK9 structure, it was found that it has a folding region of the protein kinase consisting of C-and N-terminal kinase domains and a short C-terminal extension, the cleft between the N-and C-terminal kinase leaves being an ATP binding site and being highly conserved, many fragments in the ATP binding pocket, such as the α -helix, hinge and G-loop regions, have better flexibility than other CDKs family members, creating ideal conditions for the development of specific CDK9 inhibitors.
In recent years, this field has reported a variety of highly selective CDK9 inhibitors, some of which have entered the clinical trial phase. Bayer in 2017 developed the first highly specific CDK9 inhibitor Atuveciclib (BAY-1143572), which is an aminotriazine derivative, and is currently in phase I clinical trials for the treatment of advanced acute leukemia. BAY-1143572 has been reported to have synergistic anti-esophageal adenocarcinoma effects with 5-fluorouracil and also significantly reduce invasion of ATL tumors in the liver and bone marrow of mice. After this, bayer company has further developed a second highly selective CDK9 inhibitor, BAY-1251152 with an aminopyridine structure, which is in phase i clinical trials for the treatment of acute leukemia and the treatment of advanced solid tumors with single agents. In addition, the novel high-efficiency aminopyridine structure CDK9 inhibitor AZD-4573 is also developed by the aspartame, and the drug is in the phase I clinical trial stage of the malignant tumor of the blood system. Research shows that AZD-4573 has good activity in xenograft tumor models of multiple blood tumors, and the combination of the AZD-4573 and a tyrosine kinase (BTK) inhibitor, namely Acalabarutinib, improves the anti-tumor activity.
In addition to the large number of reported specific inhibitors of CDK9, more work has focused on the study of specific degradants of CDK 9. The sandep Rana topic group in 2017 reported the first selective degradants of CDK9 based on proteolytically targeted chimeras (Proteolysis targeting chimera, PROTAC). The structure of the molecule is a connector of aminopyrazole derivative and CRBN ligand thalid, and western blots experiments show that the concentration of the degradation agent can reduce CDK9 protein expression by 56% and 65% when 10 mu M and 20 mu M. In 2018, the Nathanael S.Gray team developed a CDK9 selective degradation agent, THAL-SNS-032, based on the inhibitor SNS-032 of CDK9 and the CRBN ligand, thaldimide, which induced rapid degradation of 99% CDK9 at 50nM and did not affect the expression of proteins at other SNS-032 targets, and furthermore had a more durable pharmacokinetic effect compared to other inhibitors. The Zhiyu Li team 2018 also reported a selective degradation agent for CDK9 based on the natural product wogonin and CRBN ligand pomalidomide, which compound showed good cell proliferation inhibitory activity (ic50=17±1.9 μm) and low activity against CDK 9-low expressing cell lines with good selectivity profile.
Although many specific inhibitors targeting CDK9 and selective degradants for proac-based CDK9 are reported today, the currently reported CDK9 degradants are designed based on CDK9 and mainly cause degradation of monomeric CDK9 with little impact on cyclin T1. Considering that cyclin T1 performs many non-catalytic functions, a small molecule degradation agent that degrades CDK9-cyclin T1 complex is an urgent object to be developed.
Disclosure of Invention
Based on the above problems in the prior art, it is an object of the present invention to provide a pharmaceutical composition comprising a 5- (tert-butyl) -2- ((thiazol-5-ylthio) methyl) oxazole compound or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate or isotopically labeled compound thereof.
It is another object of the present invention to provide a process for the preparation of the above compounds.
It is a further object of the present invention to provide a pharmaceutical composition comprising a therapeutically effective amount of one or more of the above compounds, or pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, atropisomers, polymorphs, solvates or isotopically-labeled compounds thereof.
It is a further object of the present invention to provide the use of a compound as defined above, or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate or isotopically-labelled compound thereof, in the manufacture of a medicament for the treatment of a disease or condition mediated by CDK9 and/or cyclin T1 dysfunction.
It is a further object of the present invention to provide a method of treating a disease or condition mediated by CDK9 and/or cyclin T1 dysfunction, comprising administering to a subject a therapeutically effective amount of one or more of the above compounds, or pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, atropisomers, polymorphs, solvates or isotopically labeled compounds thereof.
In a first aspect of the present invention, there is provided an oxazole compound of the general formula I, a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotopically labeled compound thereof:
wherein A is selected from-C (=O) (CH 2 ) x -、-C(＝O)(CH 2 ) x NHC(＝O)-、-(CH 2 ) x NHC(＝O)(CH 2 ) y -、-(CH 2 ) x -、-(CH 2 CH 2 O) x (CH 2 ) y NHC(＝O)CH 2 -、-(CH 2 ) x -B-C(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)NH-；
x is independently selected from 0, 1, 2, 3, 4, 5, 6;
y is independently selected from 0, 1, 2;
b is a 4-8 membered heterocyclic ring containing 1, 2 or 3 heteroatoms selected from N, O, S; preferably a 4-6 membered heterocyclic ring, the heteroatom being N; more preferably the heterocycle is azetidine, azacyclopentane, azacyclohexane; more preferably B is Wherein z is independently selected from 1, 2, 3; particularly preferred is +.>
R is selected from C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 10 Cycloalkyl oxy, C 6 -C 12 Aryl, 5-7 membered heteroaryl, the foregoing C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 10 Cycloalkyl oxy, C 6 -C 12 Aryl, 5-7 membered heteroaryl groups may be substituted with one or more of the following groups: c (C) 1 -C 6 Alkyl, deuterium, halogen, cyano, nitro, amino, hydroxy, halogen substituted C 1 -C 6 Alkyl, hydroxy substituted C 1 -C 6 Alkyl, C 1 -C 6 Alkyloxy, halogen substituted C 1 -C 6 Alkyloxy, phenyl, -NH-Boc-, - (CH) 2 ) z -N＝C(NH-Boc) 2 The heteroaryl contains 1, 2 or 3 heteroatoms selected from N, O, S, and z is 0, 1, 2 or 3; r is preferably selected from C 1 -C 6 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 8 Cycloalkyl oxy, C 1 -C 6 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 8 The cycloalkyloxy group may be substituted with one or more of the following groups: c (C) 1 -C 6 Alkyl, deuterium, halogen, cyano, nitro, amino, hydroxy, halogen substituted C 1 -C 6 Alkyl, hydroxy substituted C 1 -C 6 Alkyl, C 1 -C 6 Alkyloxy, halogen substituted C 1 -C 6 Alkyloxy, phenyl, -NH-Boc-, - (CH) 2 ) z -N＝C(NH-Boc) 2 Z is 0, 1, 2 or 3; r is more preferably selected from C 1 -C 4 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 6 Cycloalkyl oxy, C 1 -C 4 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 6 The cycloalkyloxy group may be substituted with one or more of the following groups: c (C) 1 -C 4 Alkyl, deuterium, halogen, cyano, nitro, amino, hydroxy, halogen substituted C 1 -C 4 Alkyl, hydroxy substituted C 1 -C 4 Alkyl, C 1 -C 4 Alkyloxy, halogen substituted C 1 -C 4 Alkyloxy, phenyl, -NH-Boc-, - (CH) 2 ) 3 -N＝C(NH-Boc) 2 The method comprises the steps of carrying out a first treatment on the surface of the R is more preferably selected from the group consisting of methyl, ethyl, n-propyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy, the foregoing methyl, ethyl, propyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy being substituted with one or more of the following groups: methyl, ethyl, n-propyl, isopropyl, deuterium, halogen, cyano, nitro, amino, hydroxy, phenyl, -NH-Boc-, - (CH) 2 ) 3 -N＝C(NH-Boc) 2 The method comprises the steps of carrying out a first treatment on the surface of the More preferably, R is selected from adamantyl, cyclohexyloxy, triphenylmethyl, 2-triphenylethyl, unsubstituted or substituted with one or two members selected from the group consisting of methyl, isopropyl,further preferred, R is selected from adamantyl,/->Triphenylmethyl, 2-triphenylethyl, -, and->
In some preferred embodiments, according to the invention, in the definition of substituents, a is selected from-C (=o) (CH 2 ) x -、-C(＝O)(CH 2 ) x NHC(＝O)-、-(CH 2 ) x NHC(＝O)(CH 2 ) y -、-(CH 2 ) x -、-(CH 2 CH 2 O) x (CH 2 ) y NHC(＝O)CH 2 -、-(CH 2 ) x -B-C(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)NH-；
x is independently selected from 0, 1, 2, 3, 4, 5, 6;
y is independently selected from 0, 1, 2;
b is 4-8 membered nitrogen heterocycle;
r is selected from C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 10 Cycloalkyl oxy, C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 10 The cycloalkyloxy group may be substituted with one or more of the following groups: c (C) 1 -C 6 Alkyl, deuterium, halogen, cyano, nitro, amino, hydroxy, halogen substituted C 1 -C 6 Alkyl, hydroxy substituted C 1 -C 6 Alkyl, C 1 -C 6 Alkyloxy, halogen substituted C 1 -C 6 Alkyloxy, phenyl, -NH-Boc, - (CH) 2 ) 3 -N＝C(NH-Boc) 2 。
In some preferred embodiments, a is selected from-C (=o) (CH 2 ) x -、-C(＝O)(CH 2 ) x NHC(＝O)-、-(CH 2 ) x NHC(＝O)(CH 2 ) y -、-(CH 2 ) x -、-(CH 2 CH 2 O) x (CH 2 ) y NHC(＝O)CH 2 -、-(CH 2 ) x -B-C(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)NH-；
x is independently selected from 0, 1, 2, 3, 4, 6;
y is independently selected from 0, 1, 2;
b is 4-8 membered nitrogen heterocycle;
r is selected from C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 8 Cycloalkyl oxy, C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 8 The cycloalkyloxy group may be substituted with one or more (e.g., 1, 2, or 3) of the following groups: c (C) 1 -C 6 Alkyl, phenyl, -NH-Boc, - (CH) 2 ) 3 -N＝C(NH-Boc) 2 The method comprises the steps of carrying out a first treatment on the surface of the Preferably selected from C 1 -C 4 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 8 Cycloalkyl oxy, C 1 -C 4 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 8 The cycloalkyloxy group may be substituted with one or more of the following groups: c (C) 1 -C 4 Alkyl, phenyl, -NH-Boc- (CH) 2 ) 3 -N＝C(NH-Boc) 2 。
In some preferred embodiments, a is selected from-C (=o) (CH 2 ) x -、-C(＝O)(CH 2 ) x NHC(＝O)-、-(CH 2 ) x NHC(＝O)(CH 2 ) y -、-(CH 2 ) x -、-(CH 2 CH 2 O) x (CH 2 ) y NHC(＝O)CH 2 -、-(CH 2 ) x -B-C(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)NH-；
x is independently selected from 0, 1, 2, 3, 4, 5, 6;
y is independently selected from 0, 1, 2;
b is 6-membered nitrogen heterocyclyl;
R is selected from methyl, ethyl, n-propyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy, the foregoing methyl, ethyl, n-propyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy may be substituted with one or more of the following groups: methyl, ethyl, n-propyl, isopropyl, deuterium, halogen, cyano, nitro, amino, hydroxy, phenyl, -NH-Boc-, - (CH) 2 ) 3 -N＝C(NH-Boc) 2 。
In one embodiment, a is preferably selected from-C (=o) CH 2 -、-C(＝O)(CH 2 ) 2 -、-C(＝O)CH 2 NHC(＝O)-、-(CH 2 ) 2 NHC(＝O)-、-(CH 2 ) 4 NHC(＝O)CH 2 -、-(CH 2 ) 6 NHC(＝O)-CH 2 -、-(CH 2 ) 2 NHC(＝O)CH 2 -、-CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、-CH 2 CH 2 O-(CH 2 ) 2 NHC(＝O)CH 2 -、-(CH 2 CH 2 O) 2 (CH 2 ) 2 NHC(＝O)CH 2 -、-CH 2 -B-C(＝O)CH 2 -、-S(＝O) 2 (CH 2 ) 2 NHC(＝O)CH 2 -、-S(＝O) 2 (CH 2 ) 2 NHC(＝O)NH-、-S(＝O) 2 (CH 2 ) 2 NHC (=o) -; the other substituents are as defined above.
More preferably, the process is carried out,
a is preferably selected from-C (=O) CH 2 -、-C(＝O)(CH 2 ) 2 -、-C(＝O)CH 2 NHC(＝O)-、-(CH 2 ) 2 NHC(＝O)-、-(CH 2 ) 4 NHC(＝O)CH 2 -、-(CH 2 ) 6 NHC(＝O)CH 2 -、-(CH 2 ) 2 NHC(＝O)CH 2 -、-CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、-CH 2 CH 2 O-(CH 2 ) 2 NHC(＝O)CH 2 -、-(CH 2 CH 2 O) 2 (CH 2 ) 2 NHC(＝O)CH 2 -、-CH 2 -B-C(＝O)CH 2 -、-S(＝O) 2 (CH 2 ) 2 NHC(＝O)CH 2 -、-S(＝O) 2 (CH 2 ) 2 NHC(＝O)NH-、-S(＝O) 2 (CH 2 ) 2 NHC(＝O)-；
B isWherein the method comprises the steps ofz is independently selected from 1, 2, 3; particularly preferred is +.>
R is selected from adamantyl, cyclohexyloxy, triphenylmethyl, 2-triphenylethyl, unsubstituted or substituted by one or two selected from methyl, isopropyl,further preferred, R is selected from adamantyl,/->Triphenylmethyl, 2-triphenylethyl, -, and->
In some preferred embodiments, the compound of formula (I) is specifically selected from the following structures:
wherein x, y, R are as defined previously and n is 1, 2, 3 or 4.
According to the invention, particularly preferably, the compounds of formula (I) above are:
In a second aspect of the present invention, there is provided a process for the preparation of a compound of formula (I) as defined above. All final compounds of the invention are prepared by the methods described in or analogous to these schemes, which are well known to those of ordinary skill in the art of organic chemistry, and all variables employed in these schemes are defined below.
The method is selected from one of the following methods:
the synthesis method comprises the following steps:
wherein R is as previously defined;
step 1-1: the compounds 1A and 1B are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate a compound 1C through amidation reaction;
or alternatively, the first and second heat exchangers may be,
wherein R is as previously defined;
step 1-2: reacting the compound 1D with trimethyl phosphorylacetate in tetrahydrofuran solution at 0-5 ℃ in the presence of sodium hydride to generate a compound 1E;
step 1-3: dissolving the compound 1E in methanol, adding sodium hydroxide aqueous solution, and hydrolyzing at 70 ℃ to generate a compound 1F;
step 1-4: the compound 1F is hydrogenated in methanol solution by Pd/C catalyst to obtain compound 1G;
step 1-5: the compound 1G and the compound 1A are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate a compound 1H through amidation reaction;
The synthesis method II comprises the following steps:
wherein R is as previously defined;
step 2-1: the compound 2A, glycine methyl ester hydrochloride and triethylamine are activated in dimethylformamide solution at room temperature, and then are reacted to generate a compound 2B through EDCI and HOBt;
step 2-2: dissolving the compound 2B in methanol, adding sodium hydroxide aqueous solution, and hydrolyzing at 70 ℃ to generate a compound 2C;
step 2-3: the compound 2C and the compound 1A are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate a compound 2D through amidation reaction;
and a synthesis method III:
wherein R is as previously defined;
step 3-1: the compound 3A and 2, 2-diethoxyethyl-1-amine are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate a compound 3B through amidation reaction;
step 3-2: dissolving the compound 3B in acetonitrile, adding a 1N hydrochloric acid solution, and reacting at room temperature to obtain a compound 3C;
step 3-3: reacting the compound 3C with the compound 1A and triethylamine in a dichloromethane solution at room temperature in the presence of sodium triacetoxyborohydride to generate a compound 3D;
and a synthesis method:
wherein R is as previously defined; n is taken from 0,1,2,3,4,5;
Step 4-1: reducing the compound 4A into a compound 4B by lithium aluminum hydride in tetrahydrofuran solution at 0 ℃;
step 4-2: dissolving oxalyl chloride in dichloromethane solution, cooling to-78deg.C, adding DMSO, reacting for a period of time, adding compound 4B, reacting, adding triethylamine, heating to room temperature, and reacting to obtain compound 4C;
step 4-3: reacting compound 4C with compound 1A and triethylamine in a dichloromethane solution in the presence of sodium triacetoxyborohydride at room temperature to produce 4D;
or alternatively, the first and second heat exchangers may be,
wherein R is as previously defined; n is taken from 0,1,2,3,4,5;
step 4-4: adding concentrated sulfuric acid into a methanol solution of the compound 4A, and reacting at 80 ℃ to obtain a compound 4E;
step 4-5: compound 4E was reduced to compound 4B by lithium aluminum hydride in tetrahydrofuran solution at 0 ℃;
step 4-6: the compound 4B reacts with pyridine chlorochromate in dichloromethane solution at room temperature to obtain a compound 4C;
step 4-7: reacting compound 4C with compound 1A and triethylamine in a dichloromethane solution in the presence of sodium triacetoxyborohydride at room temperature to produce 4D;
the synthesis method is as follows:
/>
wherein X is- (CH) 2 ) x -，-(CH 2 CH 2 O) x (CH 2 ) y -x, y, R are as previously defined;
Step 5-1: the compound 5A and the compound 5B are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate a compound 5C through amidation reaction;
step 5-2: adding triethylamine into a dichloromethane solution of the compound 5C, cooling to 0 ℃, dropwise adding a dichloromethane solution of methanesulfonic anhydride, and reacting at room temperature to generate a compound 5D;
step 5-3: reacting the compound 5D with the compound 1A in a dimethylformamide solution at 80 ℃ in the presence of triethylamine to generate a compound 5E;
or alternatively, the first and second heat exchangers may be,
r is as defined above, n is selected from 0,1,2,3;
step 5-4: the compound 5A and the compound 5F are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate a compound 5G through amidation reaction;
step 5-5: adding triethylamine into a dichloromethane solution of the compound 5G, cooling to 0 ℃, dropwise adding a dichloromethane solution of methanesulfonic anhydride, and reacting at room temperature to generate a compound 5H;
step 5-6: reacting the compound 5H with the compound 1A in dimethylformamide solution at 80 ℃ in the presence of triethylamine to generate a compound 5I;
the synthesis method is six:
wherein R is as previously defined;
step 6-1: the compound 1A and chloroethane-1-sulfonyl chloride react in methylene dichloride solution at the temperature of minus 40 ℃ in the presence of triethylamine to generate a compound 6A;
Step 6-2: compound 6A is reacted in acetonitrile solution at room temperature in the presence of cesium carbonate and ammonium hydroxide to produce compound 6B;
step 6-3: compound 6B and compound 6C are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate compound 6D through amidation reaction; or the compound 6B and the compound 6C are subjected to amidation reaction in acetonitrile solution in the presence of N-methylimidazole and tetramethyl chlorourea hexafluorophosphate to generate a compound 6D;
step 6-4: compound 6B and compound 6E were reacted in dichloromethane solution at room temperature to yield compound 6F.
In a third aspect of the present application, there is provided a pharmaceutical composition comprising a compound of the first aspect, one or more of its pharmaceutically acceptable salts, enantiomers, diastereomers, atropisomers, racemates, polymorphs, solvates, or isotopically-labeled compounds, and a pharmaceutically acceptable carrier, diluent or excipient.
In another preferred embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulating agent, an anti-inflammatory agent, an anti-alzheimer agent and combinations thereof.
In a fourth aspect of the invention there is provided the use of a compound according to the first aspect, a pharmaceutically acceptable salt, enantiomer, diastereomer, atropisomer, polymorph, solvate, isotopically-labelled compound or racemate thereof, or a pharmaceutical composition according to the second aspect, for the manufacture of a medicament for the prophylaxis and/or treatment of a disease or condition mediated by the CDK9-cyclin T1 complex.
In another preferred embodiment, the disease or disorder mediated by the CDK9-cyclin T1 complex is selected from the group consisting of: breast cancer, osteosarcoma, endometrial tumor, leukemia, lung cancer, prostate cancer, melanoma, ovarian cancer, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdoid tumor, hepatocellular carcinoma, biliary tract cancer, bladder cancer, brain tumor, neuroblastoma, schwannoma, glioma, glioblastoma, astrocytoma, endometrial cancer, esophageal cancer, head and neck cancer, pancreatic cancer, renal cell carcinoma.
In a fifth aspect of the invention there is provided a method of treating a disease or condition mediated by the CDK9-cyclin T1 complex, particularly a tumour, comprising administering to a subject a therapeutically effective amount of one or more compounds described above or pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, atropisomers, polymorphs, solvates or isotopically labelled compounds thereof. In some embodiments, the method further comprises administering to a subject in need thereof an effective dose of an additional therapeutic agent.
The beneficial effects are that:
the invention synthesizes a series of high-efficiency and selective CDK9-cyclin T1 complex small molecule degradation agent derivatives. The previously reported CDK9 small molecule degrading agent mainly aims at CDK9 monomeric protein, and the compound disclosed by the invention can selectively degrade CDK9 and cyclin T1 simultaneously to degrade CDK9-cyclin T1 complex.
At the cellular level, the compound of the invention has proved to obviously inhibit the proliferation of prostate cancer cells, block the cell cycle, induce apoptosis and have stronger effect than the inhibitor SNS032. At the protein level, compound LL-K9-015 significantly inhibited the phosphorylation level of RNA pol II, significantly down-regulated the protein levels of MCL1, cMyc, AR, ARv7, KLK3, NKX3-1, and the inhibition was more complete than SNS032. At the transcriptome level MYC and AR mediated carcinomatous aberrant transcription is inhibited. This is the first case study using small molecule degradants to explore the therapeutic potential against the CDK9-cyclin T1 complex.
The CDK9-cyclin T1 complex small molecule degradation agent, such as LL-K9-015, is a powerful and selective CDK9-cyclin T1 complex small molecule degradation agent, and is a valuable chemical probe for non-catalytic related functional research of CDK9-cyclin T1 complexes.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. Each feature disclosed in the description may be replaced by alternative features serving the same, equivalent or similar purpose. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows the measurement results of the activity of the compound of the invention in degrading CDK9-cyclin T1 complex.
FIG. 2 shows degradation potency DC of LL-K9-015 50 Wherein A is the result of immunoblot analysis of LL-K9-015 degradation of CDK9-cyclin T1 complex; b is a degradation efficacy curve of LL-K9-015 fitted according to three independent replicates.
FIG. 3 is a graph showing the results of proteomic assays of the kinetics of degradation of the CDK9-cyclin T1 complex by LL-K9-015, wherein A is the relative protein level change of CDK9 and cyclin T1 over time in 22RV1 cells treated with LL-K9-015; b is the relative protein levels of CDK9 and cyclin T1 in 22RV1 cells treated with SNS032 over time.
FIG. 4 shows the results of immunoblotting for determining the kinetics of degradation of LL-K9-015 to CDK9-cyclin T1 complex.
FIG. 5 shows the results of degradation selectivity, wherein A is the CDK protein content of 22RV1 cells treated with LL-K9-015 for 24 hours; b is the amount of cyclin proteins binding CDK2, CDK7 and CDK9 after treatment of 22RV1 cells for 24 hours with LL-K9-015; c is the proteome change caused by LL-K9-015 group relative to control group (DMSO); d is the proteome change caused by SNS032 relative to the control (DMSO).
FIG. 6 shows the results of in vitro antitumor activity, wherein A is the effect of SNS032 and LL-K9-015 treatment of 22RV1 cells on cell viability for 5 days; b is the effect of SNS032 and LL-K9-015 on clone formation.
FIG. 7 is a graph showing the cell cycle and apoptosis results, wherein A is the distribution of cell cycles 24 hours after treatment of cells with LL-K9-015 and SNS032, and bar graphs represent mean.+ -. Standard deviation of two independent experiments; b is the result of detecting apoptosis after 24 hours of cell action of LL-K9-015 and SNS032, each point represents the result of an independent experiment, and the bar graph represents mean.+ -. Standard deviation, n=3.
FIG. 8 is an immunoblot analysis of CDK 9-associated pathways and proteins of target proteins, wherein A is the result of the immunoblot analysis of CTD phosphorylation and CDK9 target protein levels; b is the result of immunoblot analysis of AR and AR downstream signaling pathways.
FIG. 9 is a thermal graph of the effect of degradants on transcriptomes, wherein A is the heat map of each set of differential genes; b is the transcription factor network analysis of the genes specifically affected by LL-K9-015, and the name of the hinge transcription factor is shown in the figure; c is a heat map of a representative MYC target gene; d is a heat map of a representative AR target gene.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions (e.g.those described in Sambrook et al, molecular cloning: A laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989)) or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
Through extensive and intensive studies, the inventor of the application synthesizes a series of high-efficiency and selective small molecule degradation agent derivatives of CDK9-cyclin T1 complex with a new oxazole skeleton structure. At the cellular level, the compounds can obviously inhibit the proliferation of prostate cancer cells, block the cell cycle, induce apoptosis, and down regulate the level of regulated proteins of CDK9-cyclin T1 complex, including cMyc, MCL-1, AR and the like. At the transcriptome level MYC and AR mediated carcinomatous aberrant transcription is inhibited.
Terminology
In the present invention, unless otherwise indicated, terms used have the ordinary meanings known to those skilled in the art.
In the present invention, the term "C 1 -C 10 "means having 1, 2, 3, 4, 5,6. 7, 8, 9 or 10 carbon atoms, "C 3 -C 10 "means having 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms, the term" C 1 -C 4 ”、“C 1 -C 6 ”、“C 3 -C 8 ”、“C 6 -C 12 "has a similar meaning.
In the present invention, the term "4-8 membered" means having 4-8 ring atoms, the term "5-7 membered" means having 5-7 ring atoms, and so on.
In the present invention, the term "alkyl" means a saturated straight or branched chain linear hydrocarbon moiety, e.g., the term "C 1 -C 10 Alkyl "refers to a straight or branched alkyl group having 1 to 10 carbon atoms and includes, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and the like; ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl are preferred; the term "C 1 -C 6 Alkyl ", C 1 -C 4 Alkyl has similar meaning.
In the present invention, the term "cycloalkyl" means a saturated cyclic hydrocarbyl moiety, including monocyclic, bicyclic, and tricyclic, e.g., the term "C 3 -C 10 Cycloalkyl "refers to a cyclic alkyl group having 3 to 10 carbon atoms in the ring and includes, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, adamantyl (exemplary tricyclic cycloalkyl groups), and the like.
In the present invention, the term "C 3 -C 10 Cycloalkyloxy "refers to a cyclic alkyl-oxy group having 3 to 10 carbon atoms in the ring, including, without limitation, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy, cyclooctyloxy, cyclononanyloxy, cyclodecyloxy. The term "C 3 -C 8 Cycloalkyloxy "has similar meaning.
In the present invention, the term "aryl" means a hydrocarbyl moiety comprising one or more aromatic rings. For example, the term "C 6 -C 12 Aryl radicals "Refers to an aromatic cyclic group having 6 to 12 carbon atoms, such as phenyl, naphthyl, etc., which does not contain a heteroatom in the ring. Examples of aryl groups include, but are not limited to, phenyl (Ph), naphthyl, pyrenyl, anthryl, and phenanthryl.
The term "heterocycle" refers to a saturated or partially unsaturated, non-aromatic heterocycle of three to fifteen members, preferably three to ten members, comprising one to four heteroatoms selected from oxygen, nitrogen and sulfur: mono-, bi-or tricyclic heterocycles containing one to three nitrogen atoms and/or one oxygen or sulfur atom or one or two oxygen and/or sulfur atoms in addition to the carbocycle members; if the ring contains more than one oxygen atom, they are not directly adjacent; such as, but not limited to, oxiranyl, oxetanyl, aziridinyl, azetidinyl, and the like.
The term "heteroaryl" refers to a monocyclic or bicyclic aromatic ring radical containing at least one heteroatom selected from nitrogen, oxygen or sulfur as ring member; "5-7 membered heteroaryl" refers to heteroaryl groups containing 5 to 7 ring atoms. Examples of heteroaryl groups include, but are not limited to, the following groups: pyrrolyl, imidazolyl, pyrazolyl, 1,2, 3-triazolyl, pyridinyl, pyridonyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, quinolinyl, and the like.
In the present invention, the term Boc means t-butoxycarbonyl (t-Butyloxy carbonyl).
Unless otherwise indicated, alkyl, cycloalkyl, cycloalkyloxy, aryl, and heteroaryl groups described herein are substituted and unsubstituted groups. Possible substituents on alkyl, cycloalkyl, cycloalkyloxy, aryl and heteroaryl groups include, but are not limited to: hydroxy, amino, nitro, nitrile, halogen, C1-C6 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C1-C20 heterocycloalkyl, C1-C20 heterocycloalkenyl, C1-C6 alkoxy, aryl, heteroaryl, heteroaryloxy, C1-C10 alkylamino, C1-C20 dialkylamino, arylamino, diarylamino, C1-C10 alkylsulfinyl, arylsulfinyl, C1-C10 alkylimino, C1-C10 alkylsulfonimino, arylsulfonyl imino, mercapto, C1-C10 alkylthio, C1-C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, guanidino, ureyl, cyano, acyl, thio acyl, acyloxy, carboxyl and carboxylate groups. On the other hand, cycloalkyl, heterocycloalkyl, heterocycloalkenyl, aryl and heteroaryl may also be fused to each other.
In the present invention, the substitution is mono-substitution or poly-substitution, and the poly-substitution is di-substitution, tri-substitution, tetra-substitution, or penta-substitution. The disubstitution means having two substituents and so on.
The pharmaceutically acceptable salts of the present invention may be salts of anions with positively charged groups on the compounds of formula I. Suitable anions are chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methylsulfonate, trifluoroacetate, acetate, malate, tosylate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate or maleate. Similarly, salts may be formed from cations with negatively charged groups on the compounds of formula I. Suitable cations include sodium, potassium, magnesium, calcium and ammonium ions, such as tetramethylammonium.
In another preferred embodiment, "pharmaceutically acceptable salt" refers to the salt of a compound of formula I with an acid selected from the group consisting of: hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, sulfamic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalacetic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid, and the like; or a sodium, potassium, calcium, aluminum or ammonium salt of a compound of formula I with an inorganic base; or the methylamine, ethylamine or ethanolamine salt of the compounds of the formula I with organic bases.
Pharmaceutical composition
The invention also provides a pharmaceutical composition comprising an active ingredient in a safe and effective amount, and a pharmaceutically acceptable carrier.
The "active ingredient" described herein refers to the compound of formula I described herein.
The "active ingredient" and the pharmaceutical composition of the invention are used for preparing medicines for treating diseases or symptoms mediated by CDK9-cyclin T1 complex. The "active ingredients" and pharmaceutical compositions of the invention are useful as inhibitors and degradants of the CDK9-cyclin T1 complex.
"safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 1-2000mg of active ingredient per dose, more preferably 10-200mg of active ingredient per dose. Preferably, the "one dose" is a tablet.
"pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatibility" as used herein means that the components of the composition are capable of blending with and between the active ingredients of the present invention without significantly reducing the efficacy of the active ingredients. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulphate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water, and the like.
The mode of administration of the active ingredient or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and the like.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like. In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Suspensions, in addition to the active ingredient, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances, and the like.
Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
The compounds of the invention may be administered alone or in combination with other therapeutic agents, such as antineoplastic agents.
When a pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective dose, and the daily dose is usually 1 to 2000mg, preferably 20 to 500mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Example 1: preparation of Compound SNS-032
Step 1:
1-amino-3, 3-dimethylbut-2-one hydrochloride (6.0 g,40mmol,1 eq.) was dissolved in DCM (200 mL), cooled to-5deg.C and triethylamine (22 mL,160mmol,4 eq.) was slowly added. The resulting mixture was cooled to-10 ℃. Chloroacetyl chloride (3.5 mL,44mmol,1.1 eq.) was dissolved in ice DCM (20 mL) and the above mixture was added dropwise over 15 minutes while maintaining the reaction temperature below-5 ℃. The reaction mixture was stirred at-5℃for 2 hours and then quenched with 1N aqueous HCl (100 mL). The phases were separated and the organic phase was washed sequentially with 1N aqueous HCl (100 mL) and water (30 mL), dried (Na 2 SO 4 ) Concentration in vacuo afforded 2-chloro-N- (3, 3-dimethyl-2-oxobutyl) acetamide (2) as a yellow solid (5.5 g, 72%). MS (ESI) 191.97[ M+H ]] + .1H NMR(400MHz,CDCl 3 )δ7.40(brs,1H),4.30(d,J＝4.4Hz,2H),4.07(s,2H),1.20(s,9H).
Step 2:
into a 100mL thick-walled pressure-resistant bottle were charged 2-chloro-N- (3, 3-dimethyl-2-oxobutyl) acetamide (2) (8.0 g,41.7mmol,1 eq.) and POCl 3 (16 mL,171.0mmol,4.1 eq.). The reaction mixture was heated to 110 ℃ and stirred overnight. After cooling to room temperature, the reaction mixture was carefully poured into ice water (200 mL). The mixture was extracted with methyl tert-butyl ether (2X 200 mL). The organic extracts were combined and neutralized with saturated sodium bicarbonate to ph=7-8. The organic phase was separated, washed successively with saturated sodium bicarbonate (200 mL), water (200 mL) and NaCl brine (100 mL), dried (Na 2 SO 4 ) Concentrated in vacuo to give 5- (tert-butyl) -2- (chloromethyl) oxazole (3) as a black oil (5.3 g, 73%). MS (ESI) 173.98[ M+H ]] + . 1 H NMR(400MHz,CDCl 3 )δ6.68(s,1H),4.57(s,2H),1.29(s,9H).
Step 3:
5-Bromothiazol-2-amine (4) (3.56 g,20mmol,1 eq.) and potassium thiocyanate (19.4 g,200mmol,10 eq.) are dissolved in methanol (150 mL), heated to 60℃and stirred overnight. The reaction mixture was concentrated in vacuo, then extracted with ethyl acetate (EA, 3×100 mL), washed with NaCl brine, dried (Na 2 SO 4 ) 5-thiocyanothiazole-2-amine (5) was obtained as a yellow solid (2.0 g, 64%). MS (ESI) 157.90[ M+H ]] + .1H NMR(400MHz,CD 3 OD)δ7.33(s,1H).
Step 4:
to a 250mL three-necked round bottom flask was added 5-thiocyanothiazole-2-amine (5) (3.14 g,20mmol,1 eq.) and absolute ethanol (120 mL). NaBH was added in portions under nitrogen 4 (1.5 g,40mmol,2 eq.). The mixture was stirred at room temperature for 1 hour, then acetone (100 mL) was slowly added. After 1 hour, 5- (tert-butyl) -2- (chloromethyl) oxazole (3) (3.8 g,22mmol,1.1 eq.) was dissolved in EtOH (10 mL) and slowly added to the reaction mixture which was heated to 80℃and refluxed for 2 hours. After the reaction was completed, it was cooled to room temperature, concentrated in vacuo, and then extracted with ethyl acetate (3X 100 mL) and NaCl brine (3X 200 mL). The organic phase was separated, dried (Na 2 SO 4 ) Concentrating in vacuum to obtain crude product solid. The crude product was purified by flash chromatography on silica gel (PE: ea=1:1) to give 5- (((5- (tert-butyl) oxazol-2-yl) methyl) thio) thiazol-2-amine (6) as a yellow solid (3.6 g, 67%). MS (ESI) 270.00[ M+H ]] + .1H NMR(400MHz,CDCl 3 )δ6.97(s,1H),6.58(s,1H),5.26(brs,2H),3.88(s,2H),1.26(s,9H).
Step 5:
1- (tert-Butoxycarbonyl) piperidine-4-carboxylic acid (4.58 g,20mmol,2 eq.) was dissolved in DMF (100 mL), HATU (7.6 g,20mmol,2 eq.) and DIPEA (6.95 mL,40mmol,4 eq.) were added and the mixture stirred for 10 min before 5- (((5- (tert-butyl) oxazol-2-yl) methyl) thio) thiazol-2-amine (6) (2.69 g,10mmol,1 eq.) was added. The mixture was stirred at room temperature overnight, after the reaction was completed, water (100 mL) was added to give a large amount of a white solid precipitate, which was washed with water and dried to give tert-butyl 4- ((5- (((5- (tert-butyl) oxazol-2-yl) methyl) thio) thiazol-2-yl) carbamoyl) piperidine-1-carboxylate (7) as a white solid (3.7 g, 77%). MS (ESI) 481.11[ M+H ]] + .1H NMR(400MHz,CDCl 3 )δ7.28(s,1H),6.58(s,1H),4.21–4.08(m,2H),3.95(s,2H),2.89–2.80(m,2H),2.58–2.51(m,1H),1.89–1.83(m,2H),1.80–1.73(m,2H),1.46(s,9H),1.24(s,9H).
Step 6:
/>
tert-butyl 4- ((5- (((5- (tert-butyl) oxazol-2-yl) methyl) thio) thiazol-2-yl) carbamoyl) piperidine-1-carboxylate (7) (2.4 g) was dissolved in EtOH (20 mL), HCl in EtOH (2M, 20 mL) was slowly added and the reaction was stirred at room temperature overnight. The solution was concentrated in vacuo to give the crude product, saturated sodium bicarbonate adjusted ph=7-8, extracted with DCM, concentrated in vacuo and purified by flash chromatography (DCM: meoh=10:1) to give compound SNS-032 as a white solid (1.8 g, 95%). MS (ESI) 381.14[ M+H ] ] + .1H NMR(400MHz,CD 3 OD)δ7.45(s,1H),7.15(s,1H),4.21(s,2H),3.48(dt,J＝13.0,3.7Hz,2H),3.12(td,J＝12.4,3.0Hz,2H),2.90–2.84(m,1H),2.15–2.09(m,2H),2.00–1.92(m,2H),1.29(s,9H).
Example 2: preparation of Compound LL-K9-001
Step 1:
1-adamantaneacetic acid (48.5 mg,0.25mmol,1 eq.) HATU (114 mg,0.3mmol,1.2 eq.) and DIPEA (173.8. Mu.L, 1mmol,4 eq.) were dissolved in DMF (5 mL), the mixture was stirred for 10 min, then SNS-032 (95 mg,0.25mmol,1 eq.) was added and stirred overnight at room temperature. The reaction solution was extracted with EA and water. The organic phase was separated, washed with NaCl brine and concentrated in vacuo to give a crude product as a solid. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) and the pure fractions were concentrated to give LL-K9-001 (65 mg, 47%) as a white solid. HRMS [ M+H ]] + C 29 H 41 N 4 O 3 S 2 + Calculated as 557.2620, found 577.2664. 1 H NMR(400MHz,CD 3 OD)δ7.32(s,1H),6.68(s,1H),4.62–4.56(m,1H),4.18–4.12(m,1H),3.99(s,2H),3.24–3.16(m,1H),2.79–2.75(m,1H),2.74–2.70(m,1H),2.27(d,J＝13.2Hz,1H),2.19(d,J＝13.2Hz,1H),2.01–1.86(m,6H),1.74-1.67(m,13H),1.24(s,9H).
Example 3: preparation of Compound LL-K9-002
Prepared from SNS-032 (95 mg,0.25mmol,1 eq.) and (-) -menthoxyacetic acid (53.5 mg,0.25mmol,1 eq.) in the same or similar manner as in example 2. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) followed by semi-preparative HPLC (flow rate, 10mL/min; solvent a MeOH: mecn=1:1; solvent B is 0.1% tfa in water; gradient: eluting from 5% a to 95% a over 30min, 30-45min 95% a) to give LL-K9-002 (95 mg, 66%) as a white solid. HRMS [ M+H ] ] + C 29 H 45 N 4 O 4 S 2 + Calculated as 577.2882, found 577.2931. 1 H NMR(600MHz,DMSO-d 6 )δ12.32(s,1H),7.40(s,1H),6.71(s,1H),4.35–4.27(m,1H),4.18(dd,J＝20.5,12.9Hz,1H),4.05(s,2H),4.04–3.97(m,1H),3.95–3.84(m,1H),3.11(qd,J＝11.0,3.6Hz,1H),3.05–2.97(m,1H),2.75–2.70(m,1H),2.65–2.56(m,1H),2.21–2.11(m,2H),1.82–1.78(m,2H),1.63–1.54(m,3H),1.44–1.38(m,1H),1.33–1.28(m,1H),1.25–1.22(m,1H),1.16(s,9H),0.97–0.92(m,1H),0.87(dd,J＝19.6,6.8Hz,6H),0.83–0.72(m,5H).
Example 4: preparation of Compound LL-K9-003
Step 1:
1-adamantaneacetic acid (1.94 g,10mmol,1 eq.) HATU (4.56 g,12mmol,1.2 eq.) and DIPEA (6.95 mL,40mmol,4 eq.) were dissolved in DMF (50 mL) and the mixture stirred for 10 min before 2, 2-diethoxyethyl-1-amine (1.33 g,10mmol,1 eq.) was added. The mixture was stirred at room temperature overnight and then extracted with EA and water. The organic phase was separated, washed with NaCl brine and concentrated in vacuo to give a crude product as a solid. The crude product was purified by flash chromatography on silica gel (PE: ea=2:1) and the pure fractions were concentrated in vacuo to give 2- ((3 r,5r,7 r) -adamantan-1-yl) -N- (2, 2-diethoxyethyl) acetamide as a white solid (2.2 g, 71%). HRMS [ M+H ]] + C 18 H 32 NO 3 + Calculated as 310.2382, found 310.2390. 1 H NMR(400MHz,CDCl 3 )δ5.61(s,1H),4.47(t,J＝5.3Hz,1H),3.73–3.64(m,2H),3.56–3.47(m,2H),3.36(t,J＝5.6Hz,2H),1.96–1.91(m,5H),1.70–1.59(m,12H),1.19(t,J＝7.1Hz,6H).
Step 2:
2- ((3R, 5R, 7R) -adamantan-1-yl) -N- (2, 2-diethoxyethyl) acetamide (3.1 g,10mmol,1 eq.) was dissolved in MeCN (50 mL), HCl (1N, 50 mL) was added and the mixture stirred overnight at room temperature, then neutralized to pH 7-8 with saturated sodium bicarbonate, extracted with EA and water, na 2 SO 4 Drying and concentration in vacuo afforded 2- ((3R, 5R, 7R) -adamantan-1-yl) -N- (2-oxoethyl) acetamide, which was used without further purification In the next step.
Step 3:
SNS-032 (95 mg,0.25mmol,1 eq.) was dissolved in DCM (10 mL), triethylamine (69. Mu.L, 0.5mmol,2 eq.) and compound 2- ((3R, 5R, 7R) -adamantan-1-yl) -N- (2-oxoethyl) acetamide (117.5 mg,0.5mmol,2 eq.) were added and the mixture stirred for 15min, followed by sodium triacetoxyborohydride (106 mg,0.5mmol,2 eq.). The mixture was stirred at room temperature overnight. The organic phase was then separated by extraction with DCM and water, washed with NaCl brine and concentrated in vacuo to give the crude product. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) followed by semi-preparative HPLC (flow rate, 10mL/min; solvent a MeOH: mecn=1:1; solvent B is 0.1% tfa in water; gradient: eluting from 5% a to 95% a over 30min, 30-45min 95% a) to give LL-K9-003 (99 mg, 66%) as a white solid. HRMS [ M+H ]] + C 31 H 46 N 5 O 3 S 2 + Calculated as 600.3042, found 600.3089. 1 H NMR(400MHz,CD 3 OD)δ7.33(s,1H),6.67(s,1H),3.99(s,2H),3.85–3.72(m,2H),3.56(t,J＝6.1Hz,2H),3.28–2.23(m,2H),3.14–3.02(m,2H),2.84–2.74(m,1H),2.22–2.15(m,2H),2.02–1.95(m,6H),1.80–1.61(m,13H),1.25(s,9H).
Example 5: preparation of Compound LL-K9-004
Prepared by the same or similar method as example 4, the structure and characterization data are as follows:
HRMS[M+H] + C 31 H 50 N 5 O 4 S 2 + calculated as 620.3304, found 620.3355. 1 H NMR(600MHz,DMSO-d 6 )δ12.46(s,1H),7.89(t,J＝5.6Hz,1H),7.41(s,1H),6.72(s,1H),4.06(s,2H),3.97(d,J＝15.0Hz,1H),3.83(d,J＝15.0Hz,1H),3.66–3.58(m,2H),3.49–3.46(m,2H),3.19–3.11(m,3H),3.05–2.93(m,2H),2.78–2.71(m,1H),2.22–2.16(m,1H),2.06–2.01(m,3H),1.87–1.78(m,2H),1.64–1.55(m,2H),1.27–1.21(m,2H),1.17(s,9H),0.98–0.90(m,1H),0.88(dd,J＝9.4,6.9Hz,6H),0.84–0.78(m,2H),0.74(d,J＝6.9Hz,3H).
Example 6: preparation of Compound LL-K9-005
Step 1:
(-) -Bengalenical acid (1.07 g,5mmol,1 eq.) HATU (2.28 g,6mmol,1.2 eq.) and DIPEA (3.5 mL,20mmol,4 eq.) were dissolved in DMF (20 mL) and the mixture stirred for 10 min. 4-aminobutan-1-ol (445 mg,5mmol,1 eq.) was added and the mixture stirred at room temperature overnight. The organic phase was then separated by EA and water extraction, washed with NaCl brine and concentrated in vacuo to give the crude product. The crude product was purified by flash chromatography on silica gel (PE: ea=1:4) and the pure fractions were concentrated in vacuo to give N- (4-hydroxybutyl) -2- (((1 r,2s,5 r) -2-isopropyl-5-methylcyclohexyl) oxy) acetamide as a yellow oil (1.03 g, 72%). HRMS [ M+H ]] + C 16 H 32 NO 3 + Calculated as 286.2382, found 286.2386. 1 H NMR(400MHz,CDCl 3 )δ6.72(s,1H),4.05(d,J＝15.1Hz,1H),3.84(d,J＝15.1Hz,1H),3.67(t,J＝5.7Hz,2H),3.33(q,J＝6.4Hz,2H),3.13(td,J＝10.6,4.1Hz,1H),2.11–2.00(m,2H),1.69–1.56(m,6H),1.41–1.27(m,2H),1.02–0.94(m,1H),0.92–0.81(m,8H),0.77(d,J＝7.0Hz,3H).
Step 2:
n- (4-hydroxybutyl) -2- (((1R, 2S, 5R) -2-isopropyl-5-methylcyclohexyl) oxy) acetamide (560 mg,2mmol,1 eq.) was dissolved in DCM (10 mL) and addedNEt 3 (554. Mu.L, 4mmol,2 eq.). The mixture was cooled to 0deg.C, and methanesulfonic anhydride (697 mg,4mmol,2 eq.) was dissolved in DCM (4 mL) and added dropwise to the reaction. The mixture was stirred at room temperature overnight, then extracted with DCM and water, washed with NaCl brine, na 2 SO 4 Drying and concentration in vacuo afforded butyl 4- (2- (((1R, 2S, 5R) -2-isopropyl-5-methylcyclohexyl) oxy) acetamido) methanesulfonate as a yellow oil (640 mg, 88%) which was used in the next step without further purification. HRMS [ M+H ] ] + C 17 H 34 NO 5 S + Calculated as 364.2158, found 364.2166. 1 H NMR(400MHz,CDCl 3 )δ6.65(s,1H),4.22(t,J＝6.2Hz,2H),4.02(d,J＝15.1Hz,1H),3.81(d,J＝15.1Hz,1H),3.30(dd,J＝13.4,6.8Hz,2H),3.11(td,J＝10.5,4.0Hz,1H),2.98(s,3H),2.11–2.04(m,1H),2.03–1.98(m,1H),1.80–1.71(m,2H),1.67–1.57(m,4H),1.37–1.23(m,2H),1.00–0.92(m,1H),0.88(d,J＝6.8Hz,6H),0.85–0.79(m,2H),0.74(d,J＝7.0Hz,3H).
Step 3:
SNS-032 (95 mg,0.25mmol,1 eq.) was dissolved in DMF (3 mL) and NEt was added 3 (69. Mu.L, 0.5mmol,2 eq.) and butyl 4- (2- (((1R, 2S, 5R) -2-isopropyl-5-methylcyclohexyl) oxy) acetamido) methanesulfonate (181.5 mg,0.5mmol,2 eq.). The mixture was heated to 80 ℃, stirred overnight, then extracted with EA and water, the organic phase separated, washed with NaCl brine and concentrated in vacuo to afford a crude product as a solid. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) and the pure fractions were concentrated in vacuo to afford LL-K9-005 (68 mg, 42%) as a yellow solid. HRMS [ M+H ]] + C 33 H 54 N 5 O 4 S 2 + Calculated as 648.3617, found 648.3671. 1 H NMR(600MHz,DMSO-d 6 )δ12.27(s,1H),7.50(t,J＝5.9Hz,1H),7.37(s,1H),6.71(s,1H),4.04(s,2H),3.90(d,J＝14.7Hz,1H),3.77(d,J＝14.7Hz,1H),3.13–3.09(m,3H),2.97–2.84(m,2H),2.49–2.43(m,1H),2.42–2.21(m,2H),2.20–2.14(m,1H),2.04–2.01(m,1H),1.98–1.91(m,1H),1.79–1.73(m,2H),1.65–1.58(m,3H),1.57–1.53(m,1H),1.43–1.39(m,4H),1.34–1.29(m,1H),1.27–1.19(m,2H),1.16(s,9H),0.96–0.89(m,1H),0.86(dd,J＝8.6,6.9Hz,6H),0.82–0.77(m,2H),0.73(d,J＝6.9Hz,3H).
Example 7: preparation of LL-K9-006
Prepared by the same or similar methods as example 6, intermediate and final product structures and characterization data are as follows:
alcohol intermediates
N- (6-hydroxyhexyl) -2- (((1R, 2S, 5R) -2-isopropyl-5-methylcyclohexyl) oxy) acetamide. HRMS [ M+H ]] + C 18 H 36 NO 3 + Calculated as 314.2695, found 314.2701. 1 H NMR(400MHz,CDCl 3 )δ6.62(s,1H),4.05(d,J＝15.1Hz,1H),3.83(d,J＝15.1Hz,1H),3.62(t,J＝6.5Hz,2H),3.28(dd,J＝13.4,6.7Hz,2H),3.13(td,J＝10.6,4.0Hz,1H),2.14–1.99(m,2H),1.68–1.48(m,6H),1.43–1.26(m,6H),1.03–0.93(m,1H),0.92–0.80(m,8H),0.77(d,J＝7.0Hz,3H).
Methanesulfonyl intermediates/>
6- (2- ((1R, 2S, 5R) -2-isopropyl-5-methylcyclohexyl) oxy) acetamido methyl sulfonate. HRMS [ M+H ]] + C 19 H 38 NO 5 S + Calculated as 392.2471, found 392.2484. 1 H NMR(400MHz,CDCl 3 )δ6.66(s,1H),4.22(t,J＝6.5Hz,2H),4.07(d,J＝15.2Hz,1H),3.86(d,J＝15.2Hz,1H),3.29(dd,J＝13.4,6.8Hz,2H),3.14(td,J＝10.5,4.0Hz,1H),3.00(s,3H),2.15–1.99(m,2H),1.80–1.71(m,2H),1.69–1.61(m,2H),1.58–1.50(m,2H),1.48–1.30(m,6H),1.03–0.95(m,1H),0.92(d,J＝6.8Hz,6H),0.88–0.82(m,2H),0.78(d,J＝7.0Hz,3H).
LL-K9-06
HRMS[M+H] + C 35 H 58 N 5 O 4 S 2 + Calculated as 676.3930, found 676.3984. 1 H NMR(600MHz,DMSO-d 6 )δ12.46(s,1H),7.50(t,J＝5.8Hz,1H),7.41(s,1H),6.72(s,1H),4.06(s,2H),3.90(d,J＝14.7Hz,1H),3.78(d,J＝14.7Hz,1H),3.57–3.53(m,2H),3.13(dd,J＝10.7,4.1Hz,1H),3.09(d,J＝6.6Hz,2H),3.05–3.01(m,2H),2.96–2.88(m,2H),2.74(tt,J＝12.1,3.4Hz,1H),2.20–2.15(m,1H),2.06–2.00(m,3H),1.87–1.77(m,2H),1.63–1.55(m,4H),1.44–1.40(m,2H),1.28–1.22(m,6H),1.17(s,9H),0.93(qd,J＝13.0,3.3Hz,1H),0.89–0.86(m,6H),0.83–0.78(m,2H),0.74(d,J＝7.0Hz,3H).
Example 8: preparation of LL-K9-007
Prepared by the same or similar methods as example 6, intermediate and final product structures and characterization data are as follows:
alcohol intermediates
N- (2- (2-hydroxyethoxy) ethoxy) ethyl) -2- ((1 r,2s,5 r) -2-isopropyl-5-methylcyclohexyl) acetamide. HRMS [ M+H ]] + C 18 H 36 NO 5 + Calculated as 346.2594, found 346.2602. 1 H NMR(400MHz,CDCl 3 )δ7.00(s,1H),4.07(d,J＝15.2Hz,1H),3.86(d,J＝15.2Hz,1H),3.76–3.72(m,2H),3.65–3.57(m,8H),3.54–3.45(m,2H),3.14(td,J＝10.6,4.0Hz,1H),2.16–2.09(m,1H),2.06–2.01(m,1H),1.69–1.60(m,2H),1.40–1.26(m,2H),1.03–0.94(m,1H),0.93–0.81(m,8H),0.77(d,J＝7.0Hz,3H).
Methanesulfonyl intermediates
2- (2- (2- ((1R, 2S, 5R) -2-isopropyl-5-A)Methylcyclohexyl) oxy) acetamido) ethoxy) ethyl methanesulfonate. HRMS [ M+H ]] + C 19 H 38 NO 7 S + Calculated as 424.2369, found 424.2382. 1 H NMR(400MHz,CDCl 3 )δ6.95(s,1H),4.40–4.33(m,2H),4.06(d,J＝15.2Hz,1H),3.84(d,J＝15.2Hz,1H),3.78–3.73(m,2H),3.67–3.59(m,4H),3.58–3.52(m,2H),3.51–3.45(m,2H),3.13(td,J＝10.6,4.1Hz,1H),3.05(s,3H),2.16–2.06(m,1H),2.06–2.00(m,1H),1.69–1.59(m,2H),1.35–1.26(m,2H),1.02–0.93(m,1H),0.92–0.88(m,6H),0.88–0.81(m,2H),0.76(d,J＝7.0Hz,3H).
LL-K9-007
HRMS[M+H] + C 35 H 58 N 5 O 6 S 2 + Calculated as 708.3829, found 708.3886. 1 H NMR(600MHz,DMSO-d 6 )δ12.48(s,1H),7.49(t,J＝5.8Hz,1H),7.40(s,1H),6.71(s,1H),4.05(s,2H),3.92(d,J＝14.8Hz,1H),3.79(d,J＝14.8Hz,1H),3.76–3.74(m,2H),3.58–3.53(m,6H),3.44(t,J＝6.0Hz,2H),3.30–3.25(m,4H),3.11(td,J＝10.6,4.0Hz,1H),3.05–2.95(m,2H),2.73(tt,J＝12.1,3.4Hz,1H),2.18–2.13(m,1H),2.04–2.00(m,3H),1.92–1.82(m,2H),1.62–1.58(m,1H),1.57–1.53(m,1H),1.33–1.28(m,1H),1.23–1.20(m,1H),1.16(s,9H),0.94–0.88(m,1H),0.87–0.84(m,6H),0.81–0.76(m,2H),0.72(d,J＝6.9Hz,3H).
Example 9: preparation of LL-K9-008
Prepared by the same or similar methods as example 6, intermediate and final product structures and characterization data are as follows:
alcohol intermediates
1- (4- (hydroxymethyl) piperidin-1-yl) -2- ((1 r,2s,5 r) -2-isopropyl-5-methylcyclohexyl) oxy) ethan-1-one. HRMS [ M+H ]] + C 18 H 34 NO 3 + Calculated as 312.2539, found 312.2547. 1 H NMR(400MHz,CDCl 3 )δ4.62–4.50(m,1H),4.19(dd,J＝12.6,3.1Hz,1H),4.09–3.95(m,2H),3.54–3.43(m,2H),3.20–3.10(m,1H),3.05–2.95(m,1H),2.62–2.50(m,1H),2.26–2.06(m,2H),1.83–1.70(m,3H),1.66–1.56(m,2H),1.38–1.28(m,1H),1.25–1.13(m,3H),1.00–0.92(m,1H),0.88(dd,J＝8.0,7.0Hz,6H),0.85–0.77(m,2H),0.74(dd,J＝9.8,7.0Hz,3H).
Methanesulfonyl intermediates
(1- (2- ((1 r,2s,5 r) -2-isopropyl-5-methylcyclohexyl) oxy) acetyl) piperidin-4-yl) methyl methanesulfonate. HRMS [ M+H ]] + C 19 H 36 NO 5 S + Calculated as 390.2314, found 390.2326. 1 H NMR(400MHz,CDCl 3 )δ4.63–4.54(m,1H),4.19(dd,J＝12.7,2.8Hz,1H),4.11–3.99(m,4H),3.17–3.03(m,1H),3.00(s,3H),2.62–2.51(m,1H),2.21–1.96(m,3H),1.84–1.74(m,2H),1.64–1.56(m,2H),1.35–1.20(m,4H),1.00–0.91(m,1H),0.90–0.85(m,6H),0.83–0.77(m,2H),0.76–0.70(m,3H).
LL-K9-008：
HRMS[M+H] + C 35 H 56 N 5 O 4 S 2 + Calculated as 674.3774, found 674.3827. 1 H NMR(600MHz,DMSO-d 6 )δ12.46(s,1H),7.41(s,1H),6.72(s,1H),4.30(t,J＝14.3Hz,1H),4.17(d,J＝12.8Hz,1H),4.06(s,2H),3.99(dd,J＝12.8,4.9Hz,1H),3.91–3.81(m,2H),3.62–3.58(m,2H),3.14–3.07(m,1H),3.00–2.96(m,3H),2.96–2.90(m,2H),2.74(tt,J＝12.0,3.6Hz,1H),2.60–2.53(m,1H),2.20–2.00(m,6H),1.95–1.87(m,2H),1.77–1.73(m,2H),1.63–1.59(m,1H),1.57–1.54(m,1H),1.33–1.29(m,1H),1.24–1.22(m,1H),1.17(s,9H),0.96–0.91(m,1H),0.88(d,J＝6.5Hz,3H),085 (d, j=7.0 hz, 3H), 0.83-0.78 (m, 1H), 0.75-0.70 (m, 4H). Example 10: preparation of LL-K9-009
Prepared by the same or similar methods as example 6, intermediate and final product structures and characterization data are as follows:
alcohol intermediates
N- (2- (2-hydroxyethoxy) ethyl) -2- ((1R, 2S, 5R) -2-isopropyl-5-methylcyclohexyl) oxy) acetamide. HRMS [ M+H ]] + C 16 H 32 NO 4 + Calculated as 302.2331, found 302.2339. 1 H NMR(400MHz,CDCl 3 )δ6.97(s,1H),4.07(d,J＝15.2Hz,1H),3.85(d,J＝15.2Hz,1H),3.76–3.70(m,2H),3.61–3.54(m,4H),3.53–3.47(m,2H),3.14(td,J＝10.6,4.0Hz,1H),2.13–2.08(m,1H),2.06–2.01(m,1H),1.68–1.60(m,2H),1.38–1.26(m,2H),1.03–0.94(m,1H),0.91(d,J＝6.7Hz,6H),0.88–0.81(m,2H),0.77(d,J＝7.0Hz,3H).
Methanesulfonyl intermediates
Ethyl 2- (2- (2- ((1 r,2s,5 r) -2-isopropyl-5-methylcyclohexyl) oxy) acetamido) ethoxy) methylsulfonate. HRMS [ M+H ]] + C 17 H 34 NO 6 S + Calculated as 380.2107, found 380.2114. 1 H NMR(400MHz,CDCl 3 )δ6.92(s,1H),4.38–4.33(m,2H),4.06(d,J＝15.2Hz,1H),3.85(d,J＝15.2Hz,1H),3.77–3.71(m,2H),3.63–3.56(m,2H),3.55–3.45(m,2H),3.14(td,J＝10.7,4.1Hz,1H),3.05(s,3H),2.13–2.08(m,1H),2.06–2.01(m,1H),1.68–1.60(m,2H),1.40–1.30(m,2H),1.00–0.94(m,1H),0.91(d,J＝6.8Hz,6H),0.86–0.82(m,2H),0.77(d,J＝7.0Hz,3H).
LL-K9-009
HRMS[M+H] + C 33 H 54 N 5 O 5 S 2 + Calculated as 664.3566, found 664.3618. 1 H NMR(600MHz,DMSO-d 6 )δ12.24(s,1H),7.37(s,1H),6.71(s,1H),4.04(s,2H),3.92(d,J＝14.8Hz,1H),3.79(d,J＝14.9Hz,1H),3.49(t,J＝5.9Hz,2H),3.42(t,J＝5.8Hz,2H),3.29–3.24(m,2H),3.12(td,J＝10.6,4.0Hz,1H),2.94–2.87(m,2H),2.48–2.44(m,2H),2.44–2.39(m,1H),2.19–2.13(m,1H),2.05–2.01(m,1H),2.01–1.92(m,2H),1.76–1.70(m,2H),1.62–1.54(m,4H),1.35–1.28(m,1H),1.23–1.21(m,1H),1.16(s,9H),0.97–0.89(m,1H),0.87–0.85(m,6H),0.82–0.77(m,2H),0.73(d,J＝7.0Hz,3H).
Example 11: preparation of LL-K9-010
Step 1:
1-adamantaneacetic acid (0.97 g,5mmol,1 eq.) HATU (2.28 g,6mmol,1.2 eq.) and DIPEA (3.5 mL,20mmol,4 eq.) were dissolved in DMF (20 mL) and the mixture stirred for 10 min. 4-aminobutan-1-ol (445 mg,5mmol,1 eq.) was added and the mixture stirred at room temperature overnight. The organic phase was then separated by EA and water extraction, washed with NaCl brine and concentrated in vacuo to give the crude product. The crude product was purified by flash chromatography on silica gel (PE: ea=1:4) and the pure fractions were concentrated in vacuo to give 2- ((3 r,5r,7 r) -adamantan-1-yl) -N- (4-hydroxybutyl) acetamide as a yellow oil (1.1 g, 83%). MS (ESI) 266.23[ M+H ] ] + .1H NMR(600MHz,CDCl 3 )δ5.73(s,1H),3.68–3.65(m,2H),3.28–3.24(m,2H),1.96–1.89(m,5H),1.70–1.66(m,3H),1.63–1.58(m,13H).
Step 2:
2- ((3R, 5R, 7R) -adamantan-1-yl) -N- (4-hydroxybutyl) acetamide (530 mg,2mmol,1 eq.) was dissolved in DCM (10 mL) and addedNEt 3 (554. Mu.L, 4mmol,2 eq.). The mixture was cooled to 0deg.C, and methanesulfonic anhydride (697 mg,4mmol,2 eq.) was dissolved in DCM (4 mL) and added dropwise to the reaction. The mixture was stirred at room temperature overnight, then extracted with DCM and water, washed with NaCl brine, na 2 SO 4 Drying and concentration in vacuo afforded butyl 4- (2- ((3 r,5r,7 r) -adamantan-1-yl) acetamido) methylsulfonate as a yellow oil (560 mg, 86%) which was used in the next step without further purification. MS (ESI) 344.45[ M+H ]] + .1H NMR(600MHz,CDCl 3 )δ5.64(s,1H),4.24(t,J＝6.3Hz,2H),3.26(dd,J＝13.1,6.9Hz,2H),3.00(s,3H),1.95–1.89(m,5H),1.79–1.75(m,2H),1.69–1.65(m,3H),1.62–1.58(m,11H).
Step 3:
SNS-032 (95 mg,0.25mmol,1 eq.) was dissolved in DMF (3 mL) and NEt was added 3 (69. Mu.L, 0.5mmol,2 eq.) and butyl 4- (2- ((3R, 5R, 7R) -adamantan-1-yl) acetamido) methylsulfonate (171.5 mg,0.5mmol,2 eq.). The mixture was heated to 80 ℃, stirred overnight, then extracted with EA and water, the organic phase separated, washed with NaCl brine and concentrated in vacuo to afford a crude product as a solid. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) and concentrated in vacuo to afford LL-K9-010 (64 mg, 41%) as a yellow solid. HRMS [ M+H ] ] + C 33 H 50 N 5 O 3 S 2 + Calculated as 628.3355, found 628.3408. 1 H NMR(400MHz,DMSO-d 6 )δ12.24(s,1H),7.67(t,J＝5.5Hz,1H),7.38(s,1H),6.72(s,1H),4.04(s,2H),3.04–2.99(m,2H),2.92–2.85(m,2H),2.46–2.41(m,1H),2.31–2.21(m,2H),1.91–1.80(m,7H),1.77–1.72(m,2H),1.67–1.54(m,14H),1.43–1.35(m,4H),1.16(s,9H).
Example 12: preparation of LL-K9-011
Prepared by the same or similar methods as example 11, intermediate and final product structures and characterization data are as follows:
alcohol intermediates
2- ((3 r,5r,7 r) -adamantan-1-yl) -N- (6-hydroxyhexyl) acetamide. MS (ESI) 294.45[ M+H ]] + .1H NMR(600MHz,CDCl 3 )δ5.49(s,1H),3.63–3.60(m,2H),3.24–3.20(m,2H),1.96–1.89(m,5H),1.70–1.66(m,3H),1.63–1.59(m,9H),1.56–1.48(m,4H),1.40–1.32(m,4H).
Methanesulfonyl intermediates
6- (2- ((3R, 5R, 7R) -adamantan-1-yl) acetamido) methylsulfonic acid hexyl ester
MS(ESI)372.31[M+H] + .1H NMR(600MHz,CDCl 3 )δ5.47(s,1H),4.21(t,J＝6.5Hz,2H),3.24–3.19(m,2H),2.99(s,3H),1.96–1.89(m,5H),1.75–1.72(m,2H),1.69–1.66(m,3H),1.63–1.59(m,9H),1.52–1.47(m,2H),1.43–1.40(m,2H),1.37–1.33(m,2H).
LL-K9-011
HRMS[M+H] + C 35 H 54 N 5 O 3 S 2 + Calculated as 656.3668, found 656.3720. 1 H NMR(400MHz,DMSO-d 6 )δ12.25(s,1H),7.65(t,J＝5.5Hz,1H),7.38(s,1H),6.72(s,1H),4.04(s,2H),3.02–2.89(m,4H),2.47–2.41(m,1H),2.35–2.23(m,2H),1.92–1.79(m,7H),1.78–1.72(m,2H),1.66–1.54(m,14H),1.41–1.35(m,4H),1.27–1.23(m,4H),1.17(s,9H).
Example 13: preparation of LL-K9-012
Prepared by the same or similar methods as example 11, intermediate and final product structures and characterization data are as follows:
alcohol intermediates
2- ((3R, 5R, 7R) -adamantan-1-yl) -N- (2- (2- (2-hydroxyethoxy) ethoxy) acetamide MS (ESI) 326.23[ M+H)] + . 1 H NMR(600MHz,CDCl 3 )δ6.30(s,1H),3.69–3.67(m,2H),3.61–3.59(m,2H),3.58–3.54(m,4H),3.51–3.48(m,2H),3.39–3.36(m,2H),1.91–1.87(m,5H),1.65–1.61(m,3H),1.58–1.54(m,9H).
Methanesulfonyl intermediates
Ethyl 2- (2- (2- (2- ((3R, 5R, 7R) adamantan-1-yl) acetamido) ethoxy) methanesulfonate MS (ESI) 404.20[ M+H)] + . 1 H NMR(600MHz,CDCl 3 )δ5.88(s,1H),4.38–4.36(m,2H),3.77–3.75(m,2H),3.66–3.64(m,2H),3.61–3.59(m,2H),3.54–3.51(m,2H),3.44–3.41(m,2H),3.06(s,3H),1.95–1.92(m,5H),1.69–1.67(m,3H),1.62–1.59(m,9H).
LL-K9-012
HRMS[M+H] + C 35 H 54 N 5 O 5 S 2 + Calculated as 688.3566, found 688.3622. 1 H NMR(600MHz,DMSO-d 6 )δ12.46(s,1H),7.75(t,J＝5.6Hz,1H),7.41(s,1H),6.72(s,1H),4.06(s,2H),3.75–3.73(m,2H),3.59–3.56(m,4H),3.55–3.53(m,2H),3.40–3.38(m,2H),3.30–3.26(m,2H),3.19–3.16(m,2H),3.04–2.96(m,2H),2.73(tt,J＝12.2,3.5Hz,1H),2.07–2.01(m,2H),1.90–1.81(m,7H),1.65–1.62(m,3H),1.56–1.52(m,9H),1.17(s,9H).
Example 14: preparation of LL-K9-013
Prepared by the same or similar methods as example 11, intermediate and final product structures and characterization data are as follows:
alcohol intermediates
2- ((3 s) -adamantan-1-yl) -1- (4- (hydroxymethyl) piperidin-1-yl) ethan-1-one. MS (ESI) 292.43[ M+H ] ] + . 1 H NMR(600MHz,CDCl 3 )δ4.66–4.61(m,1H),3.96–3.91(m,1H),3.47–3.90(m,2H),2.96(td,J＝13.2,2.7Hz,1H),2.48(td,J＝12.9,2.6Hz,1H),2.14(d,J＝13.3Hz,1H),2.07(d,J＝13.3Hz,1H),1.92–1.89(m,3H),1.79–1.75(m,1H),1.74–1.66(m,2H),1.65–1.62(m,3H),1.60–1.56(m,9H),1.15–1.03(m,2H).
Methanesulfonyl intermediates/>
(1- (2- ((3 s) -adamantan-1-yl) acetyl) piperidin-4-yl) methyl methanesulfonate. MS (ESI) 370.52[ M+H ]] + . 1 H NMR(600MHz,CDCl 3 )δ4.72–4.69(m,1H),4.06–4.02(m,2H),4.00-3.96(m,1H),2.98(s,3H),2.58–2.45(m,2H),2.14(d,J＝13.4Hz,1H),2.08(d,J＝13.4Hz,1H),1.99–1.94(m,1H),1.93–1.91(m,3H),1.82–1.78(m,1H),1.76–1.73(m,1H),1.66–1.63(m,3H),1.61–1.58(m,9H),1.20–1.12(m,2H).
LL-K9-013
HRMS[M+H] + C 35 H 52 N 5 O 3 S 2 + Calculated as 654.3512, found 654.3564. 1 H NMR(400MHz,DMSO-d 6 )δ12.45(s,1H),7.41(s,1H),6.72(s,1H),4.47–4.42(m,1H),4.06(s,2H),4.02–3.96(m,1H),3.63–3.55(m,2H),3.03–2.91(m,4H),2.76–2.69(m,1H),2.10–2.08(m,1H),2.05–1.98(m,3H),1.93–1.88(m,4H),1.77–1.70(m,2H),1.68–1.55(m,14H),1.27–1.22(m,4H),1.18(s,9H).
Example 15: preparation of LL-K9-014
Prepared by the same or similar methods as example 11, intermediate and final product structures and characterization data are as follows:
alcohol intermediates
2- ((3 r,5r,7 r) -adamantan-1-yl) -N- (2- (2-hydroxyethoxy) ethyl) acetamide. MS (ESI) 282.20[ M+H ]] + . 1 H NMR(600MHz,CDCl 3 )δ6.42(t,J＝5.6Hz,1H),3.69–3.67(m,2H),3.52–3.49(m,4H),3.41–3.37(m,2H),1.92–1.89(m,5H),1.65–1.62(m,3H),1.58–1.55(m,9H).
Methanesulfonyl intermediates
Ethyl 2- (2- (2- ((3 r,5r,7 r) -adamantan-1-yl) acetamido) ethoxy) methylsulfonate. MS (ESI) 360.18[ M+H ]] + . 1 H NMR(600MHz,CDCl 3 )δ6.07(t,J＝4.9Hz,1H),4.33–4.31(m,2H),3.68–3.66(m,2H),3.52(t,J＝5.2Hz,2H),3.40–3.36(m,2H),3.01(s,3H),1.91–1.88(m,5H),1.65–1.62(m,3H),1.58–1.55(m,9H).
LL-K9-014
HRMS[M+H] + C 33 H 50 N 5 O 4 S 2 + Calculated as 644.3304, found 644.3353. 1 H NMR(400MHz,DMSO-d 6 )δ12.24(s,1H),7.72(t,J＝5.5Hz,1H),7.38(s,1H),6.71(s,1H),4.04(s,2H),3.48(t,J＝5.5Hz,2H),3.20–3.15(m,2H),2.94–2.87(m,2H),2.48–2.39(m,3H),1.98–1.80(m,9H),1.76–1.71(m,2H),1.66–1.54(m,14H),1.16(s,9H).
Example 16: preparation of LL-K9-015
Step 1:
SNS-032 (3.8 g,10mmol,1 eq.) was dissolved in DCM (40 mL) and NEt was added 3 (5.5 mL,40mmol,4 eq.) the mixture was cooled to-40 ℃. Ethyl chloride-1-sulfonyl chloride (2.1 mL,20mmol,2 eq.) was dissolved in ice DCM (4 mL) and added dropwise to the reaction solution. The mixture was stirred at-40 ℃ for 2 hours, then quenched with water, extracted with DCM and water, washed with NaCl brine and concentrated in vacuo to give the crude product as a solid. The crude product was purified by flash chromatography on silica gel (DCM: meoh=40:1) and the pure fractions were concentrated to give N- (5- (((5- (tert-butyl) oxazol-2-yl) methyl) thio) thiazol-2-yl) -1- (vinylsulfonyl) piperidine-4-carboxamide as a yellow solid (2.2 g, 47%). HRMS [ M+H ] ] + C 19 H 27 N 4 O 4 S 3 + Calculated as 471.1194, found 471.1210. 1 H NMR(400MHz,CD 3 OD)δ7.32(s,1H),6.69–6.62(m,2H),6.19(d,J＝16.6Hz,1H),6.12(d,J＝10.0Hz,1H),3.99(s,2H),3.72(dt,J＝12.8,3.6Hz,2H),2.76(td,J＝12.1,2.8Hz,2H),2.58(tt,J＝11.2,3.9Hz,1H),1.98–1.92(m,2H),1.86–1.77(m,2H),1.25(s,9H).
Step 2:
n- (5- (((5- (tert-butyl) oxazol-2-yl) methyl) thio) thiazol-2-yl) -1- (vinylsulfonyl) piperidine-4-carboxamide (2.35 g,5mmol,1 eq.) was dissolved in MeCN (100 mL) and Cs was added 2 CO 3 (3.25 g,10mmol,2 eq.) and NH 4 OH (48%, 40 mL). The reaction was stirred at room temperature overnight and then concentrated in vacuo. The mixture was extracted with DCM and water, washed with NaCl brine and concentrated in vacuo to give the crude product as a solid. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) and the pure fractions were concentrated in vacuo to give 1- ((2-aminoethyl) sulfonyl) -N- (5- (((5- (tert-butyl)) oxazol-2-yl) methyl) thio) thiazol-2-yl) piperidine-4-carboxamide as a yellow solid (1.8 g, 75%). HRMS [ M+H ]] + C 19 H 30 N 5 O 4 S 3 + Calculated as 488.1460, found 488.1479. 1 H NMR(400MHz,DMSO-d 6 )δ7.38(s,1H),6.71(s,1H),4.04(s,2H),3.66–3.58(m,2H),3.12(t,J＝6.9Hz,2H),2.91(t,J＝6.9Hz,2H),2.87–2.79(m,2H),2.66–2.58(m,1H),1.91–1.86(m,2H),1.64–1.54(m,2H),1.17(s,9H).
Step 3:
(-) -Bengalenical acid (53.5 mg,0.25mmol,1 eq.) is dissolved in DMF (5 mL), HATU (114 mg,0.3mmol,1.2 eq.) and DIPEA (173.8. Mu.L, 1mmol,4 eq.) are added, the mixture is stirred for 10 min, then 1- ((2-aminoethyl) sulfonyl) -N- (5- (((5- (tert-butyl)) oxazol-2-yl) methyl) thio) thiazol-2-yl) piperidine-4-carboxamide (122 mg,0.25mmol,1 eq.) is added and the mixture is stirred overnight at room temperature, then extracted with EA and water, washed with NaCl brine and concentrated in vacuo to give the crude product as a solid. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) followed by semi-preparative HPLC (flow rate, 10mL/min; solvent a MeOH: mecn=1:1; solvent B is 0.1% tfa in water; gradient: eluting from 5% a to 95% a over 30min, 30-45min 95% a) to give LL-K9-015 (102 mg, 60%) as a white solid. HRMS [ M+H ] ] + C 31 H 50 N 5 O 6 S 3 + Calculated as 684.2923, found 684.2989. 1 H NMR(400MHz,CD 3 OD)δ7.32(s,1H),6.68(s,1H),4.08(d,J＝15.3Hz,1H),3.99(s,2H),3.90(d,J＝15.2Hz,1H),3.84–3.78(m,2H),3.69(td,J＝6.5,1.5Hz,2H),3.25(t,J＝6.6Hz,2H),3.21(dd,J＝10.7,4.2Hz,1H),2.95(td,J＝12.2,2.5Hz,2H),2.63(tt,J＝11.2,3.7Hz,1H),2.29–2.21(m,1H),2.13–2.07(m,1H),1.99–1.92(m,2H),1.86–1.75(m,2H),1.72–1.63(m,2H),1.36–1.28(m,2H),1.25(s,9H),1.09–0.98(m,1H),0.96–0.87(m,8H),0.80(d,J＝7.0Hz,3H).
Example 17: preparation of LL-K9-016
1-adamantaneacetic acid (48.5 mg,0.25mmol,1 eq) was dissolved in DMF (5 mL), HATU (114 mg,0.3mmol,1.2 eq) and DIPEA (173.8. Mu.L, 1mmol,4 eq) were added, the mixture was stirred for 10 min, then the compound 1- ((2-aminoethyl) sulfonyl) -N- (5- (((5- (tert-butyl)) oxazol-2-yl) methyl) thio) thiazol-2-yl) piperidine-4-carboxamide (122 mg,0.25mmol,1 eq) was added and the mixture stirred overnight at room temperature, then extracted with EA and water, washed with NaCl brine and concentrated in vacuo to give the crude product as a solid. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) and the pure fractions were concentrated to give LL-K9-016 as a white solid (107 mg, 66%). HRMS [ M+H ]] + C 31 H 46 N 5 O 5 S 3 + Calculated as 664.2661, found 664.2721. 1 H NMR(400MHz,CD 3 OD)δ7.32(s,1H),6.68(s,1H),3.99(s,2H),3.80(dt,J＝12.2,3.0Hz,2H),3.57(t,J＝6.8Hz,2H),3.21(t,J＝6.8Hz,2H),2.95(td,J＝12.2,2.6Hz,2H),2.63(tt,J＝11.2,3.7Hz,1H),1.99–1.91(m,7H),1.85–1.63(m,14H),1.25(s,9H).
Example 18: preparation of LL-K9-017
1- ((2-aminoethyl) sulfonyl) -N- (5- (((5- (tert-butyl)) oxazol-2-yl) methyl) thio) thiazol-2-yl) piperidine-4-carboxamide (122 mg,0.25mmol,1 eq) and 2-isocyanatoadamantane (44 mg,0.25mmol,1 eq) were dissolved in DCM (10 mL) and the mixture stirred overnight at room temperature then extracted with DCM and water, washed with NaCl brine and concentrated in vacuo to give the crude product as a solid. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) and the pure fractions were concentrated in vacuo to give LL-K9-017 as a white solid (98 mg, 59%). HRMS [ M+H ] ] + C 30 H 45 N 6 O 5 S 3 + Calculated as 665.2614, found 665.2670. 1 H NMR(400MHz,CD 3 OD)δ7.32(s,1H),6.68(s,1H),3.99(s,2H),3.82–3.76(m,2H),3.49(t,J＝6.4Hz,2H),3.17(t,J＝6.4Hz,2H),2.96–2.89(m,2H),2.62(tt,J＝11.2,3.8Hz,1H),2.03–1.92(m,10H),1.85–1.69(m,9H),1.25(s,9H).
Example 19: preparation of LL-K9-018
1- ((2-aminoethyl) sulfonyl) -N- (5- (((5- (tert-butyl)) oxazol-2-yl) methyl) thio) thiazol-2-yl) piperidine-4-carboxamide (122 mg,0.25mmol,1 eq.) N5- [ bis [ [ (1, 1-dimethylethoxy) carbonyl []Amino group]Methylene group]-N2- [ (1, 1-dimethylethoxy) carbonyl ]]L-ornithine (119 mg,0.25mmol,1 eq.) N-methylimidazole (NMI) (60. Mu.L) was dissolved in MeCN (10 mL), stirred at room temperature for 10min, then tetramethyl chlorourea hexafluorophosphate (TCFH) (77 mg,0.28mmol,1.1 eq.) was added and the mixture stirred at room temperature overnight, then extracted with DCM and water, washed with NaCl brine and concentrated in vacuo to give the crude product. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) and the pure fractions were concentrated in vacuo to give LL-K9-018 as a white solid (156 mg, 66%). HRMS [ M+H ]] + C 40 H 66 N 9 O 11 S 3 + Calculated as 944.4044, found 944.4125. 1 H NMR(400MHz,CD 3 OD)δ7.32(s,1H),6.68(s,1H),4.04–4.00(m,1H),3.99(s,2H),3.83–3.78(m,2H),3.37(t,J＝6.4Hz,2H),3.21(t,J＝6.7Hz,2H),2.98–2.90(m,2H),2.66–2.59(m,1H),1.97–1.91(m,2H),1.88–1.73(m,4H),1.68–1.59(m,4H),1.52(s,9H),1.47(s,9H),1.45(s,9H),1.24(s,9H).
Example 20: preparation of Compound LL-K9-019
Step 1:
1-adamantanecarboxylic acid (0.5 g,2.8 mmol) was dissolved in DMF (10 mL), EDCI (1.07 g,5.5 mmol), HOBt (0.375 g,2.8 mmol), glycine methyl ester hydrochloride (0.35 g,22.8 mmol) was added followed by triethylamine (1.24)mL,8.9 mmol). The mixture was stirred at room temperature for 3 hours, and the reaction was quenched with water (50 mL). The aqueous phase was extracted with EA (2X 150 mL), the combined organic phases were washed with water (2X 80 mL), saturated NaCl brine (50 mL), and dried (Na 2 SO 4 ) Concentrating in vacuum. A white solid (0.59 g, 84%) was obtained and used in the next step without further purification.
Step 2:
1- ((3 r,5r,7 r) -adamantyl-1-carbonyl) -glycine methyl ester (0.59 g,2.3 mmol) was dissolved in methanol (10 mL), sodium hydroxide (188 mg,4.6 mmol) was dissolved in water (1 mL), and aqueous sodium hydroxide solution was added to a methanol solution of 1- ((3 r,5r,7 r) -adamantyl-1-carbonyl) -glycine methyl ester. The mixture was reacted at 70℃for 2 hours, after which time the system was cooled to room temperature and water (100 mL) was added. The aqueous phase was adjusted to ph=1 with dilute hydrochloric acid (6M) and extracted with EA (2×70 mL), dried (Na 2 SO 4 ) Concentrated in vacuo to give a white solid (0.4 g, 71%) which was used in the next step without further purification.
Step 3:
/>
1- ((3 r,5r,7 r) -adamantyl-1-carbonyl) -glycine (59.25 mg,0.25mmol,1 eq.) HATU (68.5 mg,0.18mmol,1.2 eq.) and DIPEA (105. Mu.L, 0.6mmol,4 eq.) were dissolved in DMF (2 mL), the mixture stirred for 10 min, then SNS-032 (57 mg,0.15mmol,1 eq.) was added. The mixture was stirred at room temperature overnight and extracted with DCM and water. The organic phase was separated, washed with NaCl brine and concentrated in vacuo to give a crude product as a solid. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) and the pure fractions were concentrated to give LL-K9-019 (58.40 mg, 65%) as a white solid. 1 H NMR(600MHz,Chloroform-D)δ7.26(s,1H),6.89(t,J＝3.9Hz,1H),6.56(s,1H),4.47(dd,J＝9.7,3.6Hz,1H),4.06(qd,J＝17.5,3.9Hz,2H),3.94(s,2H),3.84–3.78(m,1H),3.14–3.09(m,1H),2.94–2.87(m,1H),2.76–2.70(m,1H),2.02(s,3H),1.95–1.89(m,2H),1.86(d,J＝2.4Hz,6H),1.80(ddd,J＝23.9,14.3,3.9Hz,2H),1.69(dd,J＝31.1,11.9Hz,6H),1.23(s,9H)。
Example 21: preparation of Compound LL-K9-020
Step 1:
1-adamantanecarboxylic acid (25 g,138 mmol) was dissolved in methanol (150 mL), concentrated sulfuric acid (4 mL) was added with stirring, and the mixture was refluxed at 80℃for 3 hours. The organic phase was concentrated in vacuo, the remaining oil diluted with water (250 mL), extracted with EA (2X 250 mL), the organic phase washed with water (2X 150 mL), naCl brine (100 mL), and dried (Na) 2 SO 4 ) Concentrated in vacuo and the crude product was passed on a silica gel column (mobile phase 2% (EA: PE)) to give methyl 1-adamantane carboxylate as a white solid (21.8 g, 81%). 1 H NMR(600MHz,Chloroform-D)δ3.61(s,3H),1.97(s,3H),1.85(d,J＝2.7Hz,6H),1.67(q,J＝12.2Hz,6H).
Step 2:
lithium Aluminum Hydride (LAH) (6.41 g,168 mmol) was suspended in THF (250 mL) under nitrogen and cooled to 0deg.C. Methyl 1-adamantanecarboxylate (21.8 g,112 mmol) was dissolved in THF (50 mL), and the reaction solution was slowly dropped at 0 ℃. After the completion of the dropwise addition, the mixture was stirred at room temperature for 1 hour, and then EA (100 mL) and water (20 mL) were added dropwise at 0 ℃. All liquids were filtered through celite, the solvent was removed in vacuo, and dried (Na 2 SO 4 ) A colorless oil (17 g, 91%) was obtained. 1 H NMR(600MHz,Chloroform-D)δ3.17(s,2H),1.96(s,3H),1.73–1.60(m,6H),1.48(d,J＝2.5Hz,6H).
Step 3:
chromium chloridePyridine (PCC) (23.17 g,107.5 mmol) was suspended in DCM (150 mL) and DCM (100 mL) dissolved in 1-adamantanemethanol (10.5 g,63.2 mmol) was added with stirring at room temperature. After 1 hour, it was diluted with diisopropyl ether (750 mL) and filtered through celite. The filtrate was washed with 1N NaOH (250 mL), water (2X 150 mL), and the organic phase was dried (Na 2 SO 4 ) White solid (8.94 g, 86%) was obtained. 1 H NMR(600MHz,Chloroform-D)δ9.30(s,1H),2.05(s,3H),1.76(d,J＝12.4Hz,3H),1.72–1.66(m,9H)。
Step 4:
SNS-032 (57 mg,0.15mmol,1 eq.) was dissolved in DCM (2 mL), triethylamine (41.6. Mu.L, 0.3mmol,2 eq.) and the compound adamantane-formaldehyde (61.5 mg,0.375mmol,2.5 eq.) were added and the mixture stirred for 45min before sodium triacetoxyborohydride (32.0 mg,0.3mmol,2 eq.) was added. The mixture was stirred at room temperature overnight, then extracted with DCM and water, the organic phase separated, washed with NaCl brine and concentrated in vacuo to give the crude product. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) to give LL-K9-020 (41.2 mg, 52%) as a white solid. 1 H NMR(600MHz,Chloroform-D)δ7.30(s,1H),6.59(s,1H),3.95(s,2H),2.82(d,J＝11.1Hz,2H),2.31(d,J＝11.4Hz,1H),2.26(t,J＝10.9Hz,2H),1.90(dd,J＝29.9,9.4Hz,5H),1.79–1.66(m,7H),1.62(d,J＝11.7Hz,3H),1.47(s,6H),1.24(s,9H)。
Example 22: preparation of Compound LL-K9-021
Step 1:
sodium hydride (2.38 g,99 mmol) was suspended in THF (200 mL), trimethyl phosphorylacetate (TMPA) (12.89 g,70.8 mmol) was slowly added at 0deg.C, stirred for 15 min, then 1-adamantane-formaldehyde in THF was added dropwise at 0deg.C, stirred for 1.5 h at 5deg.C, quenched with water (50 mL), and THF was removed in vacuo. The mixture was added with water (150 mL) and extracted with EA (2X 150 mL). The organic phase was desolventized in vacuo and dried to give a white solid mixture (10.75 g, 91%) which was used in the next step without further purification.
Step 2:
methyl 1-adamantyl propionate (12.75 g,57.9 mmol) was dissolved in methanol (150 mL), sodium hydroxide (6.95 g,173 mmol) was dissolved in water (10 mL), sodium hydroxide solution was added to the methyl 1-adamantyl propionate in methanol, and after stirring at 70℃for 2 hours, the mixture was desolvated in vacuo, water (150 mL) was added, and extracted with EA (150 mL). The organic phase was adjusted to ph=2 with hydrochloric acid and then cooled to 5 ℃ for 30 minutes, and the precipitate was filtered and dried in vacuo to give a white solid (7.4 g, 62%). 1 H NMR(600MHz,Chloroform-D)δ6.92(d,J＝15.9Hz,1H),5.67(d,J＝15.9Hz,1H),2.02(s,3H),1.75(d,J＝12.3Hz,3H),1.66(d,J＝11.3Hz,3H),1.63(d,J＝2.5Hz,6H)。
Step 3:
1-adamantyl-allylic acid (7.4 g,33.9 mmol) was dissolved in 200mL of methanol, and 10% Pd/C (10%, 1.48 g) was added. The mixture was bubbled with nitrogen for 15 minutes, and then reacted at room temperature under a hydrogen atmosphere for 1 day. The reaction mixture was filtered through celite and the solvent was removed in vacuo to give a white solid (7.31 g, 97%) which was used in the next step without further purification.
Step 4:
1-adamantylpropionic acid (31 mg,0.25mmol,1 eq.) HATU (68.5 mg,0.18mmol,1.2 eq.) and DIPEA (105. Mu.L, 0.6mmol,4 eq.) were dissolved in DMF (2 mL), the mixture was stirred for 10 min, then SNS-032 @ was added57mg,0.15mmol,1 eq). The mixture was stirred at room temperature overnight and extracted with DCM and water. The organic phase was separated, washed with NaCl brine and concentrated in vacuo to give a crude product as a solid. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) and the purified fractions were concentrated to give LL-K9-021 (50.44 mg, 59%) as a white solid. 1 H NMR(600MHz,Chloroform-D)δ7.26(s,1H),6.55(s,1H),4.53(d,J＝13.3Hz,1H),3.93(s,2H),3.90(d,J＝13.6Hz,1H),3.12(d,J＝11.0Hz,1H),2.74(dd,J＝18.2,6.9Hz,1H),2.67(ddd,J＝14.6,10.7,3.7Hz,1H),2.30–2.25(m,2H),1.95–1.86(m,5H),1.84–1.77(m,1H),1.69(dd,J＝28.0,11.7Hz,4H),1.58(d,J＝11.7Hz,3H),1.44(s,6H),1.40(d,J＝7.5Hz,2H),1.21(s,9H)。
Example 23: preparation of Compound LL-K9-022
Step 1:
1-adamantylpropionic acid (7.2 g,34.6 mmol) was added to 150mL of methanol with stirring, concentrated sulfuric acid (2 mL) was added, the mixture was reacted at 80℃for 3 hours, the reaction was desolventized in vacuo, the residue was diluted with water (250 mL), extracted with EA (2X 250 mL), the organic phase was washed with saturated NaCl brine (1X 100 mL), and dried (Na) 2 SO 4 ) The solvent was removed in vacuo to give a white solid (7.34 g, 95%) which was used in the next step without further purification.
Step 2:
lithium aluminum hydride (LAH, 2.51g,66 mmol) was dissolved in THF (100 mL), a solution of 1-adamantyl methyl propionate (7.34 g,33 mmol) in THF (50 mL) was added dropwise at 0deg.C, and after stirring the mixture at room temperature for 1 hour, the reaction solution was cooled to 0deg.C again, and EA (20 mL) and water (5 mL) were added dropwise slowly. The mixture was filtered through celite, dried (Na 2 SO 4 ) Concentrating in vacuum. The crude product was passed through a silica gel column (EA: pe=5%) to give an oil (5.2 g, 81%) which was purified without further purificationFor the next step.
Step 3:
PCC (7.55 g,36 mmol) was suspended in DCM (50 mL) and DCM (50 mL) dissolved in 1-adamantylpropanol (3.5 g,18 mmol) was added. After the mixture was stirred at room temperature for 1 hour, diisopropyl ether (150 mL) was added and filtered through celite. Then washed with 1N sodium hydroxide (2X 100 mL) and water (2X 100 mL), dried (Na 2 SO 4 ) Concentrated in vacuo to give a white oil (3.13 g, 90%) which was used in the next step without further purification.
Step 4:
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SNS-032 (57 mg,0.15mmol,1 eq.) was dissolved in DCM (2 mL), triethylamine (41.6. Mu.L, 0.3mmol,2 eq.) and the compound adamantane propionaldehyde (72.0 mg,0.375mmol,2.5 eq.) were added and the mixture stirred for 45min, then sodium triacetoxyborohydride (32 mg,0.3mmol,2 eq.) was added. The mixture was stirred at room temperature overnight, then extracted with DCM and water, the organic phase separated, washed with NaCl brine and concentrated in vacuo to give the crude product. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) to give LL-K9-022 as a white solid (37.6 mg, 45%). 1 H NMR(600MHz,Chloroform-D)δ7.31(s,1H),6.58(s,1H),3.95(s,2H),3.06(d,J＝11.5Hz,2H),2.46(d,J＝6.4Hz,1H),2.41–2.33(m,2H),2.15(s,2H),1.93(d,J＝20.3Hz,7H),1.67(d,J＝11.9Hz,3H),1.59(d,J＝11.6Hz,3H),1.49(dd,J＝13.0,8.2Hz,2H),1.44(s,6H),1.23(s,9H),1.03–0.98(m,2H)。
Example 24: preparation of Compound LL-K9-023
Step 1:
1-adamantaneacetic acid (2.00 g,10.3mmol,1 eq.) was dissolved in THF (34 mL) under nitrogen, cooled to 0deg.C, and LAH (1M solution in THF, 15.5mL,15.4mmol,1.5 eq.) was slowly added dropwise. After the completion of the dropwise addition, the reaction mixture was warmed to room temperature and reacted for 18 hours. After 0℃water (50 mL) was added and quenched, the mixture was extracted with EA (2X 50 mL). The organic phases were combined, washed with 3N hydrochloric acid (20 mL), saturated NaCl brine, dried (Na 2 SO 4 ) Concentrated in vacuo to give a colorless oil (1.78 g, 96%). 1 H NMR(600MHz,Chloroform-D)δ3.72–3.68(m,2H),1.93(s,3H),1.69(d,J＝12.1Hz,3H),1.65–1.59(m,3H),1.51(d,J＝2.2Hz,6H),1.40–1.35(m,2H)。
Step 2:
oxalyl chloride (1 mL,11.7mmol,2 eq.) was dissolved in DCM (80 mL) and cooled to-78deg.C. DMSO (1.65 mL,23.3mmol,2.4 eq.) was added dropwise, followed by stirring for 15 min and 1-adamantaneethanol (1.75 g,9.71mmol,1 eq.) was added dropwise to DCM (17 mL). After the completion of the dropwise addition, stirring was carried out at the same temperature for 30 minutes. Triethylamine (8.2 mL,58.2mmol,6 eq) was slowly added dropwise over 20 min after the reaction, then the reaction was slowly warmed to room temperature and stirred at room temperature for 40 min, saturated sodium bicarbonate (50 mL) was added and stirred for 30 min before extraction with DCM (3×50 mL). The combined organic phases were washed with water (50 mL), saturated NaCl brine (50 mL), dried (Na 2 SO 4 ) Concentrating in vacuum. Column over silica gel (EA: pe=5%). A colorless oil (1.43 g,8.02mmol, 83%) was obtained. 1 H NMR(600MHz,Chloroform-D)δ9.87(t,J＝3.3Hz,1H),2.12(d,J＝3.3Hz,2H),1.98(s,3H),1.72(d,J＝12.1Hz,3H),1.65(dd,J＝13.4,2.0Hz,9H)。
Step 3:
SNS-032 (57 mg,0.15mmol,1 eq.) was dissolved in DCM (2 mL) and triethylamine (41.6. Mu.L, 0.3mmol,2 eq.) and the compound adamantane acetaldehyde (66)75mg,0.375mmol,2.5 eq.) and the mixture stirred for 45min, then sodium triacetoxyborohydride (32 mg,0.3mmol,2 eq.) was added. The mixture was stirred at room temperature overnight. The organic phase was then separated by extraction with DCM and water, washed with NaCl brine and concentrated in vacuo to give the crude product. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) to give LL-K9-023 (36.5 mg, 60%) as a white solid. 1 H NMR(600MHz,Chloroform-D)δ7.32(s,1H),6.58(s,1H),3.95(s,2H),3.03(d,J＝11.5Hz,2H),2.42(d,J＝5.2Hz,1H),2.40–2.35(m,2H),2.05(t,J＝14.7Hz,2H),1.91(s,7H),1.67(t,J＝8.9Hz,3H),1.60(d,J＝11.5Hz,3H),1.47(d,J＝2.1Hz,6H),1.31–1.27(m,2H),1.22(s,9H)。
Example 25: preparation of Compound LL-K9-024
Step 1:
3, 3-triphenylacetic acid (2.88 g,10mmol,1 eq.) HATU (4.56 g,12mmol,1.2 eq.) and DIPEA (6.95 mL,40mmol,4 eq.) were dissolved in DMF (20 mL), the mixture stirred for 10 min, then 2, 2-diethoxyethyl-1-amine (1.33 g,10mmol,1 eq.) was added. The mixture was stirred at room temperature overnight and then extracted with EA and water. The organic phase was separated, washed with NaCl brine and concentrated in vacuo to give a crude product as a solid. The crude product was purified by flash chromatography on silica gel (PE: ea=2:1) and concentrated in vacuo to give N- (2, 2-diethoxyethyl) -2, 2-triphenylacetamide as a white solid (2.86 g, 71%). 1 H NMR(400MHz,CDCl 3 )δ7.30–7.22(m,15H),6.06(s,1H),4.46(t,J＝5.5Hz,1H),3.62(dq,J＝9.3,7.1Hz,2H),3.48–3.39(m,4H),1.11(t,J＝7.0Hz,6H)。
Step 2:
n- (2, 2-Diethoxyethyl) -2, 2-triphenylacetamide (302 mg,0.75mmol,1 eq.) was dissolved in MeCN (1 mL), addedHCl (1N, 1 mL) was added, the mixture was stirred at room temperature overnight, extracted with DCM and water, the post organic phase washed with water, saturated NaCl brine, na 2 SO 4 Dried, concentrated in vacuo, and N- (2-oxyethyl) -2, 2-triphenylacetamide was used in the next step without further purification.
Step 3:
SNS-032 (57 mg,0.15mmol,1 eq.) was dissolved in DCM (2 mL), triethylamine (41.6. Mu.L, 0.3mmol,2 eq.) and the compound N- (2, 2-diethoxyethyl) -2, 2-triphenylacetamide (123.0 mg,0.375mmol,2.5 eq.) were added and the mixture stirred for 45min, followed by sodium triacetoxyborohydride (32 mg,0.3mmol,2 eq.). The mixture was stirred at room temperature overnight. The organic phase was then separated by extraction with DCM and water, washed with NaCl brine and concentrated in vacuo to give the crude product. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) to give LL-K9-024 (35.4 mg, 34%) as a white solid. 1 H NMR(600MHz,Chloroform-D)δ7.30–7.25(m,15H),6.58(s,1H),6.53(t,J＝4.4Hz,1H),3.95(s,2H),3.42(d,J＝5.2Hz,2H),2.73(d,J＝11.2Hz,2H),2.41(s,2H),2.27(t,J＝10.7Hz,1H),1.92(s,2H),1.70(d,J＝11.5Hz,2H),1.60–1.52(m,2H),1.24(s,9H)。
Example 26: preparation of Compound LL-K9-025
Step 1:
to 3, 3-triphenylpropionic acid (3.02 g,10mmol,1 eq.) HATU (4.56 g,12mmol,1.2 eq.) and DIPEA (6.95 mL,40mmol,4 eq.) were dissolved in DMF (20 mL), the mixture was stirred for 10 min, then 2, 2-diethoxyethyl-1-amine (1.33 g,10mmol,1 eq.) was added and the mixture stirred at room temperature overnight, then extracted with EA and water. The organic phase was separated, washed with NaCl brine and concentrated in vacuo to give a crude product as a solid. By rapid silica gelThe crude product was purified by chromatography (PE: ea=2:1) and concentrated in vacuo to give N- (2, 2-diethoxyethyl) -3, 3-triphenylpropionamide as a white solid (3.54 g, 85%). 1 H NMR(400MHz,CDCl 3 )δ7.31–7.27(m,12H),7.24–7.18(m,3H),5.12(s,1H),4.12(t,J＝5.4Hz,1H),3.58(s,2H),3.55–3.47(m,2H),3.41–3.29(m,2H),3.07(t,J＝5.6Hz,2H),1.11(t,J＝7.0Hz,6H)。
Step 2:
n- (2, 2-Diethoxyethyl) -2, 2-triphenylacetamide (312 mg,0.75mmol,1 eq.) was dissolved in MeCN (1 mL), HCl (1N, 1 mL) was added, the mixture was stirred at room temperature overnight, extracted with DCM and water, and the organic phase was washed with water, saturated NaCl brine, na 2 SO 4 Drying and vacuum concentration to obtain N- (2-oxyethyl) -3, 4-triphenyl propionamide, which can be used for the next step without further purification.
Step 3:
SNS-032 (57 mg,0.15mmol,1 eq.) was dissolved in DCM (2 mL), triethylamine (41.6. Mu.L, 0.3mmol,2 eq.) and the compound N- (2-oxyethyl) -3, 3-triphenylpropionamide (257.0 mg,0.375mmol,2.5 eq.) were added and the mixture stirred for 45min, then sodium triacetoxyborohydride (32.0 mg,0.3mmol,2 eq.) was added. The mixture was stirred at room temperature overnight, then extracted with DCM and water, the organic phase separated, washed with NaCl brine and concentrated in vacuo to give the crude product. The crude product was purified by flash chromatography on silica gel (DCM: meoh=20:1) followed by semi-preparative HPLC (flow rate, 10mL/min; solvent a MeOH: mecn=1:1; solvent B is 0.1% tfa in water; gradient: eluting from 5% a to 95% a over 30min, 30-45min 95% a) to give LL-K9-025 (17 mg, 16%) as a white solid. 1 H NMR(600MHz,Chloroform-D)δ7.29–7.21(m,15H),7.09(s,1H),6.62(s,1H),3.99(s,2H),3.59(s,2H),3.30-3.48(m,4H),2.94-3.03(m,1H),2.84(s,2H),2.63-2.71(m,1H),2.28–2.04(m,1H),1.27(s,9H)。
EXAMPLE 27 determination of the Activity of the Compounds of the invention to degrade CDK9-cyclin T1 Complex
The ability of each of the compounds in the above examples to degrade CDK9-cyclin T1 in vitro was measured and the cell line selected as prostate cancer cell 22RV1 was as follows: cells were cultured using RPMI1640 medium (Gibco, available from Life Technologies, 22400-089) to which 10% fetal bovine serum (Gibco, available from Life Technologies, 10099-141) and 1% antibiotics (penicillin and streptomycin, available from Life Technologies, 10378016) were added. Before testing, cells in logarithmic growth phase were selected at 5X 10 5 Individual cells/well were seeded in 12-well plates. After 24 hours, the compounds of each example were diluted to 3. Mu.M, 1. Mu.M and 0.3. Mu.M with medium, and the medium for each well of cells was changed to medium liquid containing the compound of the example at the above concentration. After 24 hours of treatment, the medium was discarded, and the cells were washed with pre-chilled PBS, and after the liquid was removed, SDS cell lysis buffer was added, and the cells were lysed by leaving on ice for 15 minutes, and then the sample was heated on a 99℃metal bath heater for 10 minutes. Lysates were separated by SDS-PAGE gel electrophoresis, total protein was transferred to NC membrane, and after blocking with TBST buffer containing 5% skim milk for one hour, target protein was identified using antibodies against CDK9 (Cell Signaling Technology, cat# 2316) and against cyclin T1 (Abcam, cat# ab 184703), and internal reference GAPDH was identified using antibodies against GAPDH (Cell Signaling Technology, cat# 5174). After the above primary antibodies were incubated overnight with the membrane, the membrane was washed with TBST buffer, and the remaining primary antibodies were washed off, and incubated on a shaker for 10 minutes each time, 3 times in total. After 1 hour incubation with the secondary antibody with the membrane, the membrane was washed with TBST buffer and the remaining primary antibody was washed off, and incubated on a shaker for 10 minutes each time for a total of 3 washes. The developer was SuperSignal WestDura (available from Thermo Scientific, cat# 34076) and the developer was GE ImageQuant LAS 4000 systems. The specific degradation activity of the above compounds is shown in FIG. 1.
The result shows that the compound disclosed by the invention can degrade CDK9 and cyclin T1 proteins, and the compound disclosed by the invention can realize the degradation of CDK9-cyclin T1 complex, especially LL-K9-015 in the compound, and has strong degradation activity. The degradation activity of compound LL-K9-015 on CDK9 and cyclin T1 in the concentration range of 12. Mu.M-0. Mu.M (2-fold dilution, initial concentration 12. Mu.M, 9 concentration points) was further analyzed and Western immunoblots were analyzed for gray scale using ImageJ, graphPad Prism9 fitted with a concentration-relative protein abundance curve to give LL-K9-015 DCs for CDK9 and cyclin T1 50 0.662 μm and 0.589 μm, respectively (fig. 2).
Example 28 determination of degradation kinetics
The kinetics of degradation of compound LL-K9-015 in 22RV1 cells was determined in this example using DIA-MS based proteomics and Western immunoblotting.
Proteomics method based on DIA-MS the procedure for determining the degradation kinetics of LL-K9-015 is as follows: 22RV1 cells were used in 1.2X10 manner 6 A density of/ml was applied to a 6-well plate. After cell attachment, LL-K9-015 was diluted to a final concentration of 3. Mu.M in culture medium, SNS032 was used as control inhibitor, and also to 3. Mu.M in culture medium containing DMSO at the same concentration as a blank. The compound groups were dosed simultaneously, each group being set for 5 incubation times, 3 hours, 9 hours, 12 hours, 24 hours, 36 hours, respectively. Three duplicate samples were set for each group. After reaching the incubation time, each group of samples was washed 3 times with pre-chilled PBS buffer, 200. Mu.L of cell lysate (4% SDS,100mM Tris-HCl, pH 7.6,0.1M DTT) was added, and the cells were scraped off with a disposable cell scraper and placed in the EP tube. Heating at 100deg.C for 10 min, then sonicating for 30s, collecting supernatant, determining protein concentration by tryptophan method, and performing quality detection on cell sample by SDS-PAGE electrophoresis. And (5) after the quality is qualified, performing enzymolysis by using a FASP method. Desalting the obtained peptide fragment with C18 membrane, lyophilizing, and storing in-80deg.C refrigerator. Dissolving the dried peptide sample with 0.1% formic acid water, quantifying nanodrop, taking 1ug peptide, and separating by EASY-nLC 1000 nano-flow ultra-high performance liquid chromatography with flow rate of 300nl min-1. The column was a self-contained nanofiltration C18 column (3 um,120a,75um x 15 cm). Mobile phase A was 0.1% Formic acid-water solution, mobile phase B was 0.1% formic acid-acetonitrile solution. The analysis gradient is 0-72 min,3% -27% B; 72-82 min, 27-35% B; 82-84 min, 35-100% B;84-90min,100% B. The sample was separated by liquid chromatography and mass spectrometry was performed using a Q exact HF mass spectrometer. The Full MS-DIA method has the primary resolution of 120K, the scanning range of 350-1500 m/z, the AGC of 3e6 and the maximum ion implantation time of 100MS. The fragmentation energy of the parent ion was 27%. The resolution of the secondary scan was 30K, AGC to 5e5 and the maximum ion implantation time was 50ms. The obtained data refers to Uniprot human protein database, DIA original data is searched by using DIA-NN1.7.16 software built-in parameters. The GraphPad Prism 9.3 draws a degradation curve of key proteins, and the result shows that the compound LL-K9-015 can realize synchronous degradation of CDK9 and cyclin T1, while the prototype compound SNS032 does not have the capacity of degrading CDK9 and cyclin T1 (figure 3).
The procedure for determining the degradation kinetics of LL-K9-015 based on Western blotting was as follows: 22RV1 cells were used in an amount of 0.5X10 6 The density of individual cells/wells was plated on 12-well plates, after which cells were attached, the cells were treated with medium containing 3. Mu.M LL-K9-015 for 0, 1, 3, 5, 7, 9, 11, 16, 24, 36, 48 hours, lysed with SDS, and denatured at 99℃for 10 minutes. A western blot experiment was then performed to detect the abundance of the target protein. The results show that the compound LL-K9-015 can realize the synchronous degradation of CDK9 and cyclin T1, the degradation of CDK9 and cyclin T1 begins to occur in 9-11 hours, and two proteins are significantly degraded in 24 hours. This suggests that LL-K9-015 induced degradation of CDK9-cyclin T1 complex (FIG. 4).
EXAMPLE 29 evaluation of degradation Selectivity within the CDK, cyclin family
In this example, human prostate cancer cells 22RV1 were measured at a ratio of 0.5X10 6 The density of individual cells/wells was plated on 12-well plates, after cell attachment, the cell growth medium was changed to that containing 6. Mu.M-0.38. Mu.M LL-K9-015, treated for 24 hours, lysed with SDS, and denatured at 99℃for 10 minutes. Western blot experiments were then performed to detect the abundance of CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK9, cyclin E1, cyclin T1, cyclin H, and cyclin T2. ExperimentThe results indicate that LL-K9-015 selectively degrades CDK9-cyclin T1 complex without significant effect on the remaining CDK family of proteins. Although prototype compound SNS032 also inhibited CDK2 and CDK7, LL-K9-015 had only a weak down-regulation of the regulatory subunit cyclin E1 of CDK2 at high concentrations, no significant effect on the regulatory subunit cyclin H of CDK7, and only a partial degradation of the minor binding subunit cyclin T2 of CDK9 at high concentrations (fig. 5A-5B).
Example 30 proteome evaluation of degradation selectivity
This example evaluates the effect of LL-K9-015 on whole proteome according to the method of the proteome of DIA-MS in example 28. In example 28, the CDK9-cyclin T1 complex was significantly degraded 24 hours after treatment of the cells with LL-K9-015, and 24 hours was chosen as the time point for analysis of proteome selectivity. The LL-K9-015 and SNS032 group differential expression proteins are screened by using R4.1.1 according to the standard that the fold change is more than or equal to 1.5 or 0.67 and the P-value is less than or equal to 0.05. Volcanic plot shows that SNS032 has slight up-regulation effect on cyclin T1, significant down-regulation effect on cyclin D1, and no effect on the rest CDK and cyclin proteins; LL-K9-015 significantly down-regulates CDK9 and cyclin T1. Since CDK9 regulates transcription of cycle-associated cyclin proteins, cycle-associated cyclin A2, cyclin B1 and cyclin D1 are also significantly down-regulated. Other binding targets for the prototype compound SNS032 of LL-K9-015 were not identified as differential proteins, indicating selective degradation of CDK9-cyclin T1 complex by LL-K9-015 (FIGS. 5C-5D).
Example 31 determination of in vitro anti-tumor Activity of some Compounds
In this example, the inhibitory activity of a compound against cell proliferation was examined using human prostate cancer cell line 22RV1 cells as a model. When the cells are in exponential growth phase, the cells are harvested, counted and inoculated. In 96-well plates, 100 μM cells per well was made 5000 cells per well, and a blank was added with 100 μM complete medium; compound treatment was given simultaneously, with a concentration gradient of 100 μm as starting concentration, three-fold gradient dilution, 9 concentration points total. The change in cell proliferation after 5 days of administration was detected by CellTiter-Glo method (Promega, G7572). The checking method comprises the following steps: the prepared CellTiter-Glo was added to 100. Mu.M to 96 well plates and the cell plates were placedShaking on a shaking table for 10min, and standing at normal temperature for 5min. The treated cells were transferred to a white 384 well plate (OptiPlate-384, available from Perkinelmer, inc. 6007299) at 60. Mu.L/well and cold-luminescence signals were detected at 400-700 nm using a multifunctional microplate reader EnVision (available from Perkinelmer, inc.). Analysis of data using GraphPad Prism 9.3 software, mapping of cell viability on ordinate and drug concentration on cross-arm, and calculation of IC for inhibition of proliferation of each cell line by the compound 50 。
The cell viability (%) was calculated as:
survival (%) = (dosing well OD-blank well OD)/(control well OD-blank well OD) ×100%
Table 1 partial example compounds antiproliferative activity data on prostate cancer cells 22RV1
Compounds of formula (I) IC 50 (μM)
LL-K9-003 0.068
LL-K9-016 0.255
LL-K9-017 0.564
LL-K9-015 0.120
LL-K9-023 0.236
LL-K9-024 0.350
SNS032 (prototype Compound control) 0.384
The in vitro antiproliferative activity of the compounds with superior degradation activity is shown in Table 1, which shows that several compounds have stronger antiproliferative activity, especially LL-K9-015 and LL-K9-003, compared to the prototype compound SNS 032. Therefore, the compound can be used for preparing anticancer drugs. The compound provided by the invention has remarkable capability of degrading CDK9-cyclin T1 complex, and can be used in medicines for treating diseases related to CDK9-cyclin T1, especially in treatment of tumors, because CDK9-cyclin T1 complex has a critical role in tumor cell growth and proliferation.
EXAMPLE 32 cloning experiments
In this example, human prostate cancer cell line 22RV1 cells in logarithmic growth phase were inoculated into 12-well transparent cell culture plates at 800 cells/well, after 24 hours, the medium per well was changed to medium containing 400nM, 200nM, 100nM of LL-K9-015 or SNS032 of the same concentration, and the culture was continued with medium containing 0.1% (v/v) DMSO as a control for 14 days, during which the medium containing the corresponding compound or 0.1% (v/v) DMSO was changed to freshly prepared medium containing the compound or DMSO every 2 days. After 14 days of incubation, the medium was discarded, the cells were washed twice with PBS buffer, fixed with 4% paraformaldehyde for 10 min, washed twice with PBS, then stained with crystal violet staining solution (Biyundian, cat# C0121) for 20 min, washed with PBS to remove excess staining solution, dried at room temperature, and photographed. The results indicate that LL-K9-015 dose-dependently inhibited 22RV1 cell clonogenic and was significantly stronger than SNS032 (FIG. 6B). This indicates that the degradation of CDK9-cyclin T1 has a stronger ability to inhibit tumor cell proliferation.
Example 33 cell cycle and apoptosis experiments
In this example, human prostate cancer cell line 22RV1 cells in logarithmic growth phase were cultured in a 6X 10 manner 5 After 24 hours of seeding of the cells/well into 6-well transparent cell culture plates, 1. Mu.M or 0.3. Mu.M compound was added and incubation was continued in a cell incubator at 37℃with 5% CO2 for 24 hours, with 0.1% (v/v) DMSO as a control. Cells were collected by centrifugation at 1000rpm and washed once with PBS to remove the medium. Cells were finally resuspended in pre-chilled PBS containing 70% (v/v) ethanol and fixed overnight at 4 ℃. The ethanol was removed by centrifugation and resuspended after one wash with FACS buffer. Finally, RNase and Propidium Iodide (PI) were added at the recommended concentrations (BD Pharmingen), stained at room temperature for 20min, and examined on a flow cytometer (BioRad ZE 5). Propidium Iodide dye can be combined with RNA and DNA, and DNA can be detected only by degrading RNA with RNase, the number of DNA in G1 phase of a cell is half of that in G2 phase, and the number of DNA in S phase is between the two, so that the cycle state of the cell can be judged by detecting the fluorescent signal value of the dye.
In this example, the effect of compounds LL-K9-015 and SNS032 on apoptosis of 22RV1 was examined by apoptosis experiments, by first dividing 22RV1 cells by 6X 10 5 Individual cell densities were seeded in 12-well plates (Corning). After 24 hours of compound treatment, cell samples were collected. Samples were then treated with an Annexin V-FITC apoptosis detection kit (nanking biosciences, a 211-01) and the apoptotic state was detected with a BioRad ZE5 flow meter, and data analysis was done using Modfit and FlowJo software (version 7.6.1). The results showed that after 24 hours of treatment with LL-K9-015 and SNS032, a portion of 22RV1 cells were arrested in the G1/S phase and a portion of cells were arrested in the sub-G1 phase (apoptotic phase), with LL-K9-015 having a greater arrest capacity. Meanwhile, detection of apoptosis levels by flow cytometry showed that LL-K9-015 caused more apoptosis than SNS032 (FIG. 7).
EXAMPLE 34 Western blot experiments of CDK 9-associated pathway and target protein proteins
In this example, human prostate cancer cells 22RV1 were measured at a ratio of 0.5X10 6 The density of individual cells/well was spread on a 12-well plate, and after the cells had adhered to the wall, the cell growth medium was changed to contain 3. Mu.M, 1. Mu.M, 0.3mu.M of LL-K9-015 or SNS032 medium, treated for 24 hours, lysed with SDS and denatured at 99℃for 10 minutes. Western blot experiments were then performed to detect the abundance of MCL1, cMyc, AR, ARv, KLK3, NKX3-1, phospho-Rpb1 CTD (Ser 5), phospho-Rpb1 CTD (Ser 2), and RNA pol II antibody. Experimental results show that LL-K9-015 significantly inhibited RNA pol II phosphorylation, significantly down-regulated protein levels of MCL1, cMyc, AR, ARv7, KLK3, NKX3-1, and inhibited more completely than SNS032 (FIG. 8).
Example 35: transcriptome analysis
In this example, prostate cancer cells 22RV1 were treated with 2. Mu.M LL-K9-015, 2. Mu.M Thal-SNS032, 2. Mu.M MSNS032 and 0.1% DMSO for 24 hours, and cell samples were collected and were phenol/chloroform extracted for total RNA, purity was checked by NanoDrop 2000&8000 micro-spectrophotometry, concentration was determined by Agilent 2100Bioanalyzer, and integrity was checked by Agilent RNA 6000Nano Kit. Only samples that passed the purity and integrity tests were subjected to the next library analysis. The pooled samples were high-throughput sequenced by Illumina NovaSeq 6000, sequencing read length PE150. The washed data were aligned by STAR software and the reference genome was UCSC hg19. And submitting the compared bam file to the featuresource software for quantitative analysis. Differential gene analysis was performed by R package Deseq2, with differential gene threshold of logFC >1.5, corrected p value less than 0.05. The clustering analysis is completed by R packets Clusterfile, the p value of the clustering is corrected by adopting an FDR method, and the threshold value of the significance is that the corrected p value is smaller than 0.05. The results indicate that the changes in the transcriptome caused by LL-K9-015 are significantly different from those caused by SNS032 and Thal-SNS032, and that LL-K9-015 inhibits MYC and AR mediated oncogenes and down regulates MYC and AR target genes (FIG. 9).
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (10)

1. An oxazole compound of the general formula I, a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotopically labeled compound thereof:
wherein A is selected from-C (=O) (CH 2 ) x -、-C(＝O)(CH 2 ) x NHC(＝O)-、-(CH 2 ) x NHC(＝O)(CH 2 ) y -、-(CH 2 ) x -、-(CH 2 CH 2 O) x (CH 2 ) y NHC(＝O)CH 2 -、-(CH 2 ) x -B-C(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)NH-；
x is independently selected from 0, 1, 2, 3, 4, 5, 6;
y is independently selected from 0, 1, 2;
b is a 4-8 membered heterocyclic ring containing 1, 2 or 3 heteroatoms selected from N, O, S; preferably a 4-6 membered heterocyclic ring, the heteroatom being N; more preferably the heterocycle is azetidine, azacyclopentane, azacyclohexane; more preferably B isWherein z is independently selected from 1, 2, 3; particularly preferred +.>
R is selected from C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 10 Cycloalkyl oxy, C 6 -C 12 Aryl, 5-7 membered heteroaryl, the foregoing C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 10 Cycloalkyl oxy, C 6 -C 12 Aryl, 5-7 membered heteroaryl groups may be substituted with one or more of the following groups: c (C) 1 -C 6 Alkyl, deuterium, halogen, cyano, nitro, amino, hydroxy, halogen substituted C 1 -C 6 Alkyl, hydroxy substituted C 1 -C 6 Alkyl, C 1 -C 6 Alkyloxy, halogen substituted C 1 -C 6 Alkyloxy, phenyl, -NH-Boc-, - (CH) 2 ) z -N＝C(NH-Boc) 2 The heteroaryl contains 1, 2 or 3 heteroatoms selected from N, O, S, and z is 0, 1, 2 or 3; r is preferably selected from C 1 -C 6 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 8 Cycloalkyl oxy, C 1 -C 6 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 8 The cycloalkyloxy group may be substituted with one or more of the following groups: c (C) 1 -C 6 Alkyl, deuterium, halogen, cyano, nitro, amino, hydroxy, halogen substituted C 1 -C 6 Alkyl, hydroxy substituted C 1 -C 6 Alkyl, C 1 -C 6 Alkyloxy, halogen substituted C 1 -C 6 Alkyloxy, phenyl, -NH-Boc-, - (CH) 2 ) z -N＝C(NH-Boc) 2 Z is 0, 1, 2 or 3; r is more preferably selected from C 1 -C 4 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 6 Cycloalkyl oxy, C 1 -C 4 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 6 The cycloalkyloxy group may be substituted with one or more of the following groups: c (C) 1 -C 4 Alkyl, deuterium, halogen, cyano, nitro, amino, hydroxy, halogen substituted C 1 -C 4 Alkyl, hydroxy substituted C 1 -C 4 Alkyl, C 1 -C 4 Alkyloxy, halogen substituted C 1 -C 4 Alkyloxy, phenyl, -NH-Boc-, - (CH) 2 ) 3 -N＝C(NH-Boc) 2 The method comprises the steps of carrying out a first treatment on the surface of the R is more preferably selected from methyl, ethyl, n-methylPropyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy, the foregoing methyl, ethyl, propyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy may be substituted with one or more of the following groups: methyl, ethyl, n-propyl, isopropyl, deuterium, halogen, cyano, nitro, amino, hydroxy, phenyl, -NH-Boc-, - (CH) 2 ) 3 -N＝C(NH-Boc) 2 The method comprises the steps of carrying out a first treatment on the surface of the More preferably, R is selected from adamantyl, cyclohexyloxy, triphenylmethyl, 2-triphenylethyl, unsubstituted or substituted with one or two members selected from the group consisting of methyl, isopropyl,further preferred, R is selected from adamantyl,/->Triphenylmethyl, 2-triphenylethyl, -, and->
2. The method of claim 1, wherein the oxazole compound, pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate or isotopically labeled compound,
a is selected from-C (=O) (CH 2 ) x -、-C(＝O)(CH 2 ) x NHC(＝O)-、-(CH 2 ) x NHC(＝O)(CH 2 ) y -、-(CH 2 ) x -、-(CH 2 CH 2 O) x (CH 2 ) y NHC(＝O)CH 2 -、-(CH 2 ) x -B-C(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)NH-；
x is independently selected from 0, 1, 2, 3, 4, 5, 6;
y is independently selected from 0, 1, 2;
b is 4-8 membered nitrogen heterocycle;
r is selected from C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 10 Cycloalkyl oxy, C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 10 The cycloalkyloxy group may be substituted with one or more of the following groups: c (C) 1 -C 6 Alkyl, deuterium, halogen, cyano, nitro, amino, hydroxy, halogen substituted C 1 -C 6 Alkyl, hydroxy substituted C 1 -C 6 Alkyl, C 1 -C 6 Alkyloxy, halogen substituted C 1 -C 6 Alkyloxy, phenyl, -NH-Boc, - (CH) 2 ) 3 -N＝C(NH-Boc) 2 。
3. The method according to claim 1 or 2, wherein the oxazole compound, pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate or isotopically labeled compound,
a is selected from-C (=O) (CH 2 ) x -、-C(＝O)(CH 2 ) x NHC(＝O)-、-(CH 2 ) x NHC(＝O)(CH 2 ) y -、-(CH 2 ) x -、-(CH 2 CH 2 O) x (CH 2 ) y NHC(＝O)CH 2 -、-(CH 2 ) x -B-C(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)NH-；
x is independently selected from 0, 1, 2, 3, 4, 6;
y is independently selected from 0, 1, 2;
b is 4-8 membered nitrogen heterocycle;
r is selected from C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 8 Cycloalkyl oxy, C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 8 The cycloalkyloxy group may be substituted with one or more (e.g., 1, 2, or 3) of the following groups: c (C) 1 -C 6 Alkyl, phenyl, -NH-Boc, - (CH) 2 ) 3 -N＝C(NH-Boc) 2 The method comprises the steps of carrying out a first treatment on the surface of the Preferably selected from C 1 -C 4 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 8 Cycloalkyl oxy, C 1 -C 4 Alkyl, C 3 -C 10 Cycloalkyl, C 3 -C 8 The cycloalkyloxy group may be substituted with one or more of the following groups: c (C) 1 -C 4 Alkyl, phenyl, -NH-Boc- (CH) 2 ) 3 -N＝C(NH-Boc) 2 。
4. The method according to claim 1 to 3, wherein the oxazole compound, pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate or isotopically labeled compound,
a is selected from-C (=O) (CH 2 ) x -、-C(＝O)(CH 2 ) x NHC(＝O)-、-(CH 2 ) x NHC(＝O)(CH 2 ) y -、-(CH 2 ) x -、-(CH 2 CH 2 O) x (CH 2 ) y NHC(＝O)CH 2 -、-(CH 2 ) x -B-C(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)(CH 2 ) y -、-S(＝O) 2 (CH 2 ) x NHC(＝O)NH-；
x is independently selected from 0, 1, 2, 3, 4, 5, 6;
y is independently selected from 0, 1, 2;
b is 6-membered nitrogen heterocyclyl;
r is selected from methyl, ethyl, n-propyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy, the foregoing methyl groupsThe group, ethyl, n-propyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy may be substituted with one or more of the following groups: methyl, ethyl, n-propyl, isopropyl, deuterium, halogen, cyano, nitro, amino, hydroxy, phenyl, -NH-Boc-, - (CH) 2 ) 3 -N＝C(NH-Boc) 2 。
5. The method according to claim 1 to 4, wherein the oxazole compound, pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate or isotopically labeled compound,
A is selected from-C (=O) CH 2 -、-C(＝O)(CH 2 ) 2 -、-C(＝O)CH 2 NHC(＝O)-、-(CH 2 ) 2 NHC(＝O)-、-(CH 2 ) 4 NHC(＝O)CH 2 -、-(CH 2 ) 6 NHC(＝O)-CH 2 -、-(CH 2 ) 2 NHC(＝O)CH 2 -、-CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、-CH 2 CH 2 O-(CH 2 ) 2 NHC(＝O)CH 2 -、-(CH 2 CH 2 O) 2 (CH 2 ) 2 NHC(＝O)CH 2 -、-CH 2 -B-C(＝O)CH 2 -、-S(＝O) 2 (CH 2 ) 2 NHC(＝O)CH 2 -、-S(＝O) 2 (CH 2 ) 2 NHC(＝O)NH-、-S(＝O) 2 (CH 2 ) 2 NHC (=o) -; the other substituents are as defined above;
preferably, the method comprises the steps of,
a is selected from-C (=O) CH 2 -、-C(＝O)(CH 2 ) 2 -、-C(＝O)CH 2 NHC(＝O)-、-(CH 2 ) 2 NHC(＝O)-、-(CH 2 ) 4 NHC(＝O)CH 2 -、-(CH 2 ) 6 NHC(＝O)CH 2 -、-(CH 2 ) 2 NHC(＝O)CH 2 -、-CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、-CH 2 CH 2 O-(CH 2 ) 2 NHC(＝O)CH 2 -、-(CH 2 CH 2 O) 2 (CH 2 ) 2 NHC(＝O)CH 2 -、-CH 2 -B-C(＝O)CH 2 -、-S(＝O) 2 (CH 2 ) 2 NHC(＝O)CH 2 -、-S(＝O) 2 (CH 2 ) 2 NHC(＝O)NH-、-S(＝O) 2 (CH 2 ) 2 NHC(＝O)-；
B isWherein z is independently selected from 1, 2, 3; particularly preferred is +.>
R is selected from adamantyl, cyclohexyloxy, triphenylmethyl, 2-triphenylethyl, unsubstituted or substituted by one or two selected from methyl, isopropyl,further preferably, R is selected from adamantyl,triphenylmethyl, 2-triphenylethyl, -, and->
6. The oxazole compound of any of claims 1 to 5, pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, atropisomers, polymorphs, solvates or isotopically labeled compounds thereof, wherein the oxazole compound is selected from the following structures:
wherein x, y, R are as defined previously and n is 1, 2, 3 or 4.
7. The oxazole compound of any of claims 1 to 6, pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, atropisomers, polymorphs, solvates or isotopically labeled compounds thereof, wherein the oxazole compound is selected from the following structures:
8. A process for the preparation of an oxazole compound as claimed in any of claims 1 to 7 wherein the process is selected from one of the following:
the synthesis method comprises the following steps:
wherein R is defined in the respective claims;
step 1-1: the compounds 1A and 1B are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate a compound 1C through amidation reaction;
or alternatively, the first and second heat exchangers may be,
wherein R is defined in the respective claims;
step 1-2: reacting the compound 1D with trimethyl phosphorylacetate in tetrahydrofuran solution at 0-5 ℃ in the presence of sodium hydride to generate a compound 1E;
step 1-3: dissolving the compound 1E in methanol, adding sodium hydroxide aqueous solution, and hydrolyzing at 70 ℃ to generate a compound 1F;
step 1-4: the compound 1F is hydrogenated in methanol solution by Pd/C catalyst to obtain compound 1G;
step 1-5: the compound 1G and the compound 1A are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate a compound 1H through amidation reaction;
the synthesis method II comprises the following steps:
wherein R is defined in the respective claims;
step 2-1: the compound 2A, glycine methyl ester hydrochloride and triethylamine are activated in dimethylformamide solution at room temperature, and then are reacted to generate a compound 2B through EDCI and HOBt;
Step 2-2: dissolving the compound 2B in methanol, adding sodium hydroxide aqueous solution, and hydrolyzing at 70 ℃ to generate a compound 2C;
step 2-3: the compound 2C and the compound 1A are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate a compound 2D through amidation reaction;
and a synthesis method III:
wherein R is defined in the respective claims;
step 3-1: the compound 3A and 2, 2-diethoxyethyl-1-amine are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate a compound 3B through amidation reaction;
step 3-2: dissolving the compound 3B in acetonitrile, adding a 1N hydrochloric acid solution, and reacting at room temperature to obtain a compound 3C;
step 3-3: reacting the compound 3C with the compound 1A and triethylamine in a dichloromethane solution at room temperature in the presence of sodium triacetoxyborohydride to generate a compound 3D;
and a synthesis method:
wherein R is defined in the respective claims; n is taken from 0,1,2,3,4,5;
step 4-1: reducing the compound 4A into a compound 4B by lithium aluminum hydride in tetrahydrofuran solution at 0 ℃;
step 4-2: dissolving oxalyl chloride in tetrahydrofuran solution, cooling to-78 ℃, adding DMSO, reacting for a period of time, adding compound 4B, continuing to react, adding triethylamine in the later period of reaction, and heating to room temperature, and continuing to react to obtain compound 4C;
Step 4-3: reacting compound 4C with compound 1A and triethylamine in a dichloromethane solution in the presence of sodium triacetoxyborohydride at room temperature to produce 4D;
or alternatively, the first and second heat exchangers may be,
wherein R is defined in the respective claims; n is taken from 0,1,2,3,4,5;
step 4-4: adding concentrated sulfuric acid into a methanol solution of the compound 4A, and reacting at 80 ℃ to obtain a compound 4E;
step 4-5: compound 4E was reduced to compound 4B by lithium aluminum hydride in tetrahydrofuran solution at 0 ℃;
step 4-6: the compound 4B reacts with pyridine chlorochromate in dichloromethane solution at room temperature to obtain a compound 4C;
step 4-7: reacting compound 4C with compound 1A and triethylamine in a dichloromethane solution in the presence of sodium triacetoxyborohydride at room temperature to produce 4D;
the synthesis method is as follows:
wherein X is- (CH) 2 ) x -，-(CH 2 CH 2 O) x (CH 2 ) y -x, y, R are each as defined in the respective claims;
step 5-1: the compound 5A and the compound 5B are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate a compound 5C through amidation reaction;
step 5-2: adding triethylamine into a dichloromethane solution of the compound 5C, cooling to 0 ℃, dropwise adding a dichloromethane solution of methanesulfonic anhydride, and reacting at room temperature to generate a compound 5D;
Step 5-3: reacting the compound 5D with the compound 1A in a dimethylformamide solution at 80 ℃ in the presence of triethylamine to generate a compound 5E;
or alternatively, the first and second heat exchangers may be,
r is defined in the corresponding claims, n is selected from 0,1,2,3;
step 5-4: the compound 5A and the compound 5F are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate a compound 5G through amidation reaction;
step 5-5: adding triethylamine into a dichloromethane solution of the compound 5G, cooling to 0 ℃, dropwise adding a dichloromethane solution of methanesulfonic anhydride, and reacting at room temperature to generate a compound 5H;
step 5-6: reacting the compound 5H with the compound 1A in dimethylformamide solution at 80 ℃ in the presence of triethylamine to generate a compound 5I;
the synthesis method is six:
wherein R is defined in the respective claims;
step 6-1: the compound 1A and chloroethane-1-sulfonyl chloride react in methylene dichloride solution at the temperature of minus 40 ℃ in the presence of triethylamine to generate a compound 6A;
step 6-2: compound 6A is reacted in acetonitrile solution at room temperature in the presence of cesium carbonate and ammonium hydroxide to produce compound 6B;
Step 6-3: compound 6B and compound 6C are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate compound 6D through amidation reaction; or the compound 6B and the compound 6C are subjected to amidation reaction in acetonitrile solution in the presence of N-methylimidazole and tetramethyl chlorourea hexafluorophosphate to generate a compound 6D;
step 6-4: compound 6B and compound 6E were reacted in dichloromethane solution at room temperature to yield compound 6F.
9. A pharmaceutical composition comprising a compound of any one of claims 1-7, one or more of its pharmaceutically acceptable salts, enantiomers, diastereomers, atropisomers, racemates, polymorphs, solvates, or isotopically labeled compounds, and a pharmaceutically acceptable carrier, diluent, or excipient;
preferably, the pharmaceutical composition further comprises at least one additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulator, an anti-inflammatory agent, an anti-alzheimer agent and combinations thereof.
10. Use of a compound according to any one of claims 1 to 8, a pharmaceutically acceptable salt, enantiomer, diastereomer, atropisomer, polymorph, solvate, isotopically-labeled compound or racemate thereof, or a pharmaceutical composition according to claim 9, for the manufacture of a medicament for the prevention and/or treatment of a disease or condition mediated by the CDK9-cyclin T1 complex;
Preferably, the disease or disorder mediated by the CDK9-cyclin T1 complex is selected from: breast cancer, osteosarcoma, endometrial tumor, leukemia, lung cancer, prostate cancer, melanoma, ovarian cancer, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdoid tumor, hepatocellular carcinoma, biliary tract cancer, bladder cancer, brain tumor, neuroblastoma, schwannoma, glioma, glioblastoma, astrocytoma, endometrial cancer, esophageal cancer, head and neck cancer, pancreatic cancer, renal cell carcinoma.
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