Oxazoles, processes for their preparation and uses thereof

Abstract

The present application relates to a kind of oxazole compounds and preparation method and purposes thereof, specifically, the present application discloses oxazole compounds represented by general formula I, and its pharmaceutically acceptable salt, enantiomer, diastereoisomer, racemate, atropisomer, polymorph, solvate or isotopically labeled compound.The compound represented by the formula (I) has high-efficiency, selective CDK9-cyclin T1 complex degradation effect, and can be used for preparing the drug for preventing and / or treating the disease or disorder mediated by CDK9-cyclin T1 complex, especially tumor.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a compound that degrades the CDK9-cyclin T1 complex, its preparation method, pharmaceutical composition, and its uses. Background Technology

[0002] Cyclin-dependent kinases (CDKs) are important regulatory proteins in the life cycle of mammalian cells, belonging to the conserved serine / threonine protein kinase family. CDKs consist of a dimeric complex composed of catalytic and regulatory subunits of the cell cycle. By binding to cyclins, they play an irreplaceable role in various biological processes, including the cell cycle (including pre-DNA synthesis (G1 phase), DNA synthesis (S phase), post-DNA synthesis (G2 phase), mitosis (M phase), and the G0 phase of cell dormancy), transcription, DNA damage and repair, stem cells, metabolism, epigenetics, and angiogenesis. Currently, the human genome has been identified as encoding 21 cyclin-dependent kinase inhibitors (CDKs) and 29 cyclin proteins. Based on their primary functions, these are divided into two subfamilies: one family regulates the cell cycle, including CDKs that regulate G1 / S phase (CDK2-cyclin E, CDK4-cyclin D, CDK6-cyclin D, etc.) and CDKs that regulate S / G2 / M phase (CDK1-cyclin A, CDK1-cyclin B, CDK2-cyclin A, etc.); the other family participates in transcriptional regulation, including CDK7-cyclin H, CDK8-cyclin C, CDK9-cyclin T, and CDK11-cyclin L. These functionally distinct CDK subfamilies act synergistically and cyclically at various regulatory sites throughout cell growth and proliferation, ensuring the normal functioning of the entire cell life cycle. Since tumors and many proliferative diseases are caused by abnormal changes in the cell cycle, CDKs have become important targets for the treatment of these diseases.

[0003] As a member of the CDKs family, cyclin-dependent kinase 9 (CDK9) forms a complex with cyclin T or cyclin K called positive transcription elongation factor b (P-TEFb). P-TEFb promotes transcriptional elongation by phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) (RPB1), thereby regulating cellular transcription. Given that P-TEFb is a universal transcription factor for the efficient expression of most genes, and CDK9 is widely present in the transcriptional process of normal cells, it might seem that it is not an ideal target for treating any disease. However, numerous studies have shown that CDK9 is closely related to the pathogenesis of tumors (leukemia, breast cancer, and liver cancer), heart disease, and AIDS, making it a key target for drugs related to these diseases. Studies of the CDK9 structure have revealed that it has a protein kinase fold region consisting of C-terminal and N-terminal kinase domains and a short C-terminal extension region. The cleavage between the N-terminal and C-terminal kinase lobes is an ATP-binding site and is highly conserved. Many segments in the ATP-binding pocket, such as the α-helix, hinge region, and G-loop region, have better flexibility than other CDKs family members, which creates ideal conditions for developing specific CDK9 inhibitors.

[0004] In recent years, several highly selective CDK9 inhibitors have been reported in this field, with some compounds already in clinical trials. In 2017, Bayer developed the first highly specific CDK9 inhibitor, Atuveciclib (BAY-1143572), an aminotriazine derivative, which is currently in Phase I clinical trials for the treatment of advanced acute leukemia. Reports indicate that BAY-1143572 exhibits synergistic anti-esophageal adenocarcinoma activity with 5-fluorouracil and significantly reduces the invasion of ATL tumors in the liver and bone marrow of mice. Following this, Bayer further developed a second highly selective CDK9 inhibitor, BAY-1251152, with an aminopyridine structure, which is currently in Phase I clinical trials for the treatment of acute leukemia and as a monotherapy for advanced solid tumors. Additionally, AstraZeneca has developed a novel and highly effective aminopyridine CDK9 inhibitor, AZD-4573, which is in Phase I clinical trials for hematologic malignancies. Studies have shown that AZD-4573 has good activity in xenograft models of multiple hematologic malignancies, and its combined use with the tyrosine kinase (BTK) inhibitor acalabrutinib enhances its antitumor activity.

[0005] Beyond the numerous reports on specific CDK9 inhibitors, much more research has focused on CDK9-specific degraders. In 2017, Sandeep Rana's group reported the first selective CDK9 degrader based on a protein hydrolysis-targeting chimera (PROTAC). This molecule is a linker between an aminopyrazole derivative and the CRBN ligand thalidomide. Western blot experiments showed that this degrader reduced CDK9 protein expression by 56% and 65% at concentrations of 10 μM and 20 μM, respectively. In 2018, Nathanael S. Gray's team developed the CDK9 inhibitor SNS-032 and the CRBN ligand thalidomide-based selective CDK9 degrader THAL-SNS-032. At 50 nM, it induced rapid degradation of 99% of CDK9 without affecting the expression of other SNS-032 target proteins. Furthermore, compared to other inhibitors, this degrader exhibited a more durable pharmacokinetic effect. In 2018, Zhiyu Li's team also reported a selective degrader of CDK9 based on the natural product baicalin and the CRBN ligand pomalidomide. This compound exhibited good cell proliferation inhibitory activity (IC50 = 17 ± 1.9 μM) and low activity against cell lines with low CDK9 expression, demonstrating good selectivity.

[0006] Although many specific inhibitors targeting CDK9 and selective degraders of CDK9 based on PROTAC have been reported, these CDK9 degraders are all designed based on CDK9 and mainly cause the degradation of monomeric CDK9, with little impact on cyclin T1. Considering that cyclin T1 performs many non-catalytic functions, small molecule degraders that degrade the CDK9-cyclin T1 complex are urgently needed for development. Summary of the Invention

[0007] In view of the above-mentioned problems existing in the prior art, one object of the present invention is to provide a compound containing 5-(tert-butyl)-2-((thiazolyl-5-ylthio)methyl)oxazole or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, transisomer, polymorph, solvate or isotopically labeled compound.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned compound.

[0009] Another object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of one or more of the above-mentioned compounds or their pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, transisomers, polymorphs, solvates or isotopically labeled compounds.

[0010] Another object of the present invention is to provide the use of the above-described compounds or pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, transisomers, polymorphs, solvates or isotopically labeled compounds in the preparation of medicaments for treating diseases or conditions mediated by CDK9 and / or cyclin T1 dysfunction.

[0011] Another object of the present invention is to provide a method for treating a disease or condition mediated by CDK9 and / or cyclin T1 dysfunction, by administering to a subject a therapeutically effective amount of one or more of the above-mentioned compounds or their pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, transisomers, polymorphs, solvates or isotopically labeled compounds.

[0012] In a first aspect, the present invention provides an oxazole compound represented by general formula I, a pharmaceutically acceptable salt thereof, an enantiomer, a diastereomer, a racemic mixture, a transisomer, a polymorph, a solvate, or an isotopically labeled compound:

[0013]

[0014] Where A is selected from -C(=O)(CH2). x -、-C(=O)(CH2) x NHC(=O)-、-(CH2) x NHC(=O)(CH2) y -、-(CH2) x -、-(CH2CH2O) x (CH2) y NHC(=O)CH2-、-(CH2) x -BC(=O)(CH2) y -、-S(=O)2(CH2) x NHC(=O)(CH2) y -、-S(=O)2(CH2) x NHC(=O)NH-;

[0015] x is independently selected from 0, 1, 2, 3, 4, 5, 6;

[0016] y is independently selected from 0, 1, and 2;

[0017] B is a 4-8 membered heterocycle, wherein the heterocycle contains 1, 2, or 3 heteroatoms selected from N, O, and S; preferably a 4-6 membered heterocycle, wherein the heteroatom is N; more preferably, the heterocycle is azirrobutane, azirropentane, or azirrohexane; even more preferably, B is... Where z is independently selected from 1, 2, and 3; particularly preferred is...

[0018] R is selected from C1-C 10 Alkyl, C3-C 10 cycloalkyl, C3-C 10 Cycloalkyloxy, C6-C 12 Aryl, 5-7 membered heteroaryl, the aforementioned C1-C 10 Alkyl, C3-C 10 cycloalkyl, C3-C 10 Cycloalkyloxy, C6-C 12 The aryl and 5-7 heteroaryl groups can be substituted by one or more of the following groups: C1-C6 alkyl, deuterium, halogen, cyano, nitro, amino, hydroxyl, halogen-substituted C1-C6 alkyl, hydroxyl-substituted C1-C6 alkyl, C1-C6 alkyloxy, halogen-substituted C1-C6 alkyloxy, phenyl, -NH-Boc-, -(CH2). z -N=C(NH-Boc)2, wherein the heteroaryl group contains 1, 2, or 3 heteroatoms selected from N, O, and S, and z is 0, 1, 2, or 3; R is preferably selected from C1-C6 alkyl groups, C3-C6 alkyl groups, and C4-C6 alkyl groups. 10 Cycloalkyl, C3-C8 cycloalkyloxy, the aforementioned C1-C6 alkyl, C3-C 10 Cycloalkyl and C3-C8 cycloalkyloxy groups can be substituted with one or more of the following groups: C1-C6 alkyl, deuterium, halogen, cyano, nitro, amino, hydroxyl, halogen-substituted C1-C6 alkyl, hydroxyl-substituted C1-C6 alkyl, C1-C6 alkyloxy, halogen-substituted C1-C6 alkyloxy, phenyl, -NH-Boc-, -(CH2) z -N=C(NH-Boc)2, z is 0, 1, 2 or 3; R is more preferably selected from C1-C4 alkyl, C3-C 10 Cycloalkyl, C3-C6 cycloalkyloxy, the aforementioned C1-C4 alkyl, C3-C 10The cycloalkyl and C3-C6 cycloalkyloxy groups may be substituted with one or more of the following groups: C1-C4 alkyl, deuterium, halogen, cyano, nitro, amino, hydroxyl, halogen-substituted C1-C4 alkyl, hydroxyl-substituted C1-C4 alkyl, C1-C4 alkyloxy, halogen-substituted C1-C4 alkyloxy, phenyl, -NH-Boc-, -(CH2)3-N=C(NH-Boc)2; R is more preferably selected from methyl, ethyl, n-propyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy The aforementioned methyl, ethyl, propyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclopentyloxy, cyclohexyloxy, and cycloheptyloxy groups may be substituted with one or more of the following groups: methyl, ethyl, n-propyl, isopropyl, deuterium, halogen, cyano, nitro, amino, hydroxyl, phenyl, -NH-Boc-, -(CH2)3-N=C(NH-Boc)2; more preferably, R is selected from adamantyl, an unsubstituted or substituted cyclohexyloxy group selected from one or both of methyl and isopropyl, triphenylmethyl, 2,2,2-triphenylethyl, More preferably, R is selected from adamantyl alkyl groups. Triphenylmethyl, 2,2,2-triphenylethyl

[0019] In some preferred embodiments, according to the invention, A is selected from -C(=O)(CH2) as defined in the substituents. x -、-C(=O)(CH2) x NHC(=O)-、-(CH2) x NHC(=O)(CH2) y -、-(CH2) x -、-(CH2CH2O) x (CH2) y NHC(=O)CH2-、-(CH2) x -BC(=O)(CH2) y -、-S(=O)2(CH2) x NHC(=O)(CH2) y -、-S(=O)2(CH2) x NHC(=O)NH-;

[0020] x is independently selected from 0, 1, 2, 3, 4, 5, 6;

[0021] y is independently selected from 0, 1, and 2;

[0022] B is a 4-8 membered nitrogen heterocycle;

[0023] R is selected from C1-C 10 Alkyl, C3-C 10cycloalkyl, C3-C 10 Cycloalkyloxy, the aforementioned C1-C 10 Alkyl, C3-C 10 cycloalkyl, C3-C 10 Cycloalkyloxy groups may be substituted by one or more of the following groups: C1-C6 alkyl, deuterium, halogen, cyano, nitro, amino, hydroxyl, halogen-substituted C1-C6 alkyl, hydroxyl-substituted C1-C6 alkyl, C1-C6 alkyloxy, halogen-substituted C1-C6 alkyloxy, phenyl, -NH-Boc, -(CH2)3-N=C(NH-Boc)2.

[0024] In some preferred examples, A is selected from -C(=O)(CH2). x -、-C(=O)(CH2) x NHC(=O)-、-(CH2) x NHC(=O)(CH2) y -、-(CH2) x -、-(CH2CH2O) x (CH2) y NHC(=O)CH2-、-(CH2) x -BC(=O)(CH2) y -、-S(=O)2(CH2) x NHC(=O)(CH2) y -、-S(=O)2(CH2) x NHC(=O)NH-;

[0025] x is independently selected from 0, 1, 2, 3, 4, and 6;

[0026] y is independently selected from 0, 1, and 2;

[0027] B is a 4-8 membered nitrogen heterocycle;

[0028] R is selected from C1-C 10 Alkyl, C3-C 10 Cycloalkyl, C3-C8 cycloalkyloxy, the aforementioned C1-C 10 Alkyl, C3-C 10 The cycloalkyl group or C3-C8 cycloalkyloxy group may be substituted by one or more (e.g., 1, 2, or 3) of the following groups: C1-C6 alkyl, phenyl, -NH-Boc, -(CH2)3-N=C(NH-Boc)2; preferably selected from C1-C4 alkyl, C3-C 10 Cycloalkyl, C3-C8 cycloalkyloxy, the aforementioned C1-C4 alkyl, C3-C 10Cycloalkyl and C3-C8 cycloalkyloxy groups can be substituted by one or more of the following groups: C1-C4 alkyl, phenyl, -NH-Boc-, -(CH2)3-N=C(NH-Boc)2.

[0029] In some preferred examples, A is selected from -C(=O)(CH2). x -、-C(=O)(CH2) x NHC(=O)-、-(CH2) x NHC(=O)(CH2) y -、-(CH2) x -、-(CH2CH2O) x (CH2) y NHC(=O)CH2-、-(CH2) x -BC(=O)(CH2) y -、-S(=O)2(CH2) x NHC(=O)(CH2) y -、-S(=O)2(CH2) x NHC(=O)NH-;

[0030] x is independently selected from 0, 1, 2, 3, 4, 5, 6;

[0031] y is independently selected from 0, 1, and 2;

[0032] B is a 6-membered nitrogen heterocyclic group;

[0033] R is selected from methyl, ethyl, n-propyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclopentyloxy, cyclohexyloxy, and cycloheptyloxy. The aforementioned methyl, ethyl, n-propyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclopentyloxy, cyclohexyloxy, and cycloheptyloxy groups may be substituted by one or more of the following groups: methyl, ethyl, n-propyl, isopropyl, deuterium, halogen, cyano, nitro, amino, hydroxyl, phenyl, -NH-Boc-, and -(CH2)3-N=C(NH-Boc)2.

[0034] In one embodiment, A is preferably selected from -C(=O)CH2-, -C(=O)(CH2)2-, -C(=O)CH2NHC(=O)-, -(CH2)2NHC(=O)-, -(CH2)4NHC(=O)CH2-, -(CH2)6NHC(=O)-CH2-, -(CH2)2NHC(=O)CH2-, -CH2-, -(CH2)2-, -(CH2)3-, -CH2CH2O-(CH2)2NHC(=O)CH2-, -(CH2CH2O)2(CH2)2NHC(=O)CH2-, -CH2-BC(=O)CH2-, -S(=O)2(CH2)2NHC(=O)CH2-, -S(=O)2(CH2)2NHC(=O)NH-, -S(=O)2(CH2)2NHC(=O)-; other substituents are as defined above.

[0035] More preferably,

[0036] A is preferably selected from -C(=O)CH2-, -C(=O)(CH2)2-, -C(=O)CH2NHC(=O)-, -(CH2)2NHC(=O)-, -(CH2)4NHC(=O)CH2-, -(CH2)6NHC(=O)CH2-, -(CH2)2NHC(=O)CH2-, -CH2-, -(CH2)2-, -(CH2)3-, -CH2CH2O-(CH2)2NHC(=O)CH2-, -(CH2CH2O)2(CH2)2NHC(=O)CH2-, -CH2-BC(=O)CH2-, -S(=O)2(CH2)2NHC(=O)CH2-, -S(=O)2(CH2)2NHC(=O)NH-, -S(=O)2(CH2)2NHC(=O)-;

[0037] B is Where z is independently selected from 1, 2, and 3; particularly preferred is...

[0038] R is selected from adamantyl, unsubstituted or substituted with one or both of methyl and isopropyl cyclohexyloxy, triphenylmethyl, 2,2,2-triphenylethyl. More preferably, R is selected from adamantyl alkyl groups. Triphenylmethyl, 2,2,2-triphenylethyl

[0039] In some preferred embodiments, the compound of formula (I) is specifically selected from the following structures:

[0040]

[0041]

[0042] Where x, y, and R are as defined above, and n is 1, 2, 3, or 4.

[0043] According to the present invention, particularly preferably, the compound of formula (I) above is:

[0044]

[0045]

[0046]

[0047]

[0048] A second aspect of the invention provides a method for preparing compounds of formula (I) above. All final compounds of the invention are prepared by the methods described in these illustrative reaction formulas or by similar methods, methods well known to those skilled in the art of organic chemistry, all of which are variable factors applied in these illustrative reaction formulas as defined below.

[0049] The method is selected from one of the following methods:

[0050] Synthesis Method 1:

[0051]

[0052] Where R is as defined above;

[0053] Step 1-1: Compounds 1A and 1B are activated by HATU and DIPEA in dimethylformamide solution at room temperature, and undergo an amidation reaction to generate compound 1C;

[0054] or,

[0055]

[0056] Where R is as defined above;

[0057] Steps 1-2: Compound 1D reacts with trimethyl phosphoroacetate in tetrahydrofuran solution at 0-5°C in the presence of sodium hydride to generate compound 1E;

[0058] Steps 1-3: Compound 1E is dissolved in methanol, and an aqueous sodium hydroxide solution is added. The mixture is then hydrolyzed at 70°C to generate compound 1F.

[0059] Steps 1-4: Compound 1F is hydrogenated in methanol solution by a Pd / C catalyst to obtain compound 1G;

[0060] Steps 1-5: Compound 1G and Compound 1A are reacted in dimethylformamide solution at room temperature by activation with HATU and DIPEA to generate Compound 1H;

[0061] Synthesis Method Two:

[0062]

[0063] Where R is as defined above;

[0064] Step 2-1: Compound 2A, glycine methyl ester hydrochloride, and triethylamine react in dimethylformamide solution at room temperature with EDCI and HOBt to generate compound 2B;

[0065] Step 2-2: Compound 2B is dissolved in methanol, and an aqueous solution of sodium hydroxide is added. The solution is then hydrolyzed at 70°C to generate compound 2C.

[0066] Steps 2-3: Compound 2C and compound 1A are reacted in dimethylformamide solution at room temperature by activation with HATU and DIPEA to generate compound 2D;

[0067] Synthesis Method 3:

[0068]

[0069] Where R is as defined above;

[0070] Step 3-1: Compound 3A reacts with 2,2-diethoxyethyl-1-amine in a dimethylformamide solution at room temperature, and is activated by HATU and DIPEA to undergo an amidation reaction to generate compound 3B;

[0071] Step 3-2: Compound 3B is dissolved in acetonitrile, 1N hydrochloric acid solution is added, and the reaction is carried out at room temperature to obtain compound 3C;

[0072] Step 3-3: Compound 3C reacts with compound 1A and triethylamine in a dichloromethane solution in the presence of sodium triacetoxyborohydride at room temperature to generate compound 3D;

[0073] Synthesis Method Four:

[0074]

[0075] Where R is as defined above; n is taken from 0, 1, 2, 3, 4, 5;

[0076] Step 4-1: Compound 4A is reduced to compound 4B by lithium aluminum hydride in tetrahydrofuran solution at 0°C;

[0077] Step 4-2: Oxaloyl chloride was dissolved in dichloromethane solution, cooled to -78°C, DMSO was added and reacted for a period of time, then compound 4B was added and the reaction continued. Triethylamine was added in the later stage of the reaction, and the temperature was raised to room temperature to continue the reaction, yielding compound 4C.

[0078] Step 4-3: Compound 4C reacts with compound 1A and triethylamine in a dichloromethane solution in the presence of sodium triacetoxyborohydride at room temperature to generate 4D;

[0079] or,

[0080]

[0081] Where R is as defined above; n is taken from 0, 1, 2, 3, 4, 5;

[0082] Step 4-4: Compound 4A is reacted with concentrated sulfuric acid in methanol solution at 80°C to obtain compound 4E;

[0083] Steps 4-5: Compound 4E is reduced to compound 4B by lithium aluminum hydride in tetrahydrofuran solution at 0°C;

[0084] Steps 4-6: Compound 4B reacts with pyridine chlorochromate in a dichloromethane solution at room temperature to give compound 4C;

[0085] Steps 4-7: Compound 4C reacts with compound 1A and triethylamine in a dichloromethane solution in the presence of sodium triacetoxyborohydride at room temperature to generate 4D;

[0086] Synthesis Method 5:

[0087]

[0088] Where X is -(CH2) x -,-(CH2CH2O) x (CH2) y - x, y, and R are as defined previously;

[0089] Step 5-1: Compound 5A and compound 5B are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate compound 5C;

[0090] Step 5-2: In a dichloromethane solution, triethylamine is added to compound 5C, the mixture is cooled to 0°C, and a dichloromethane solution of methanesulfonic anhydride is added dropwise. The mixture is allowed to react at room temperature to produce compound 5D.

[0091] Step 5-3: Compound 5D and compound 1A react in dimethylformamide solution at 80°C in the presence of triethylamine to generate compound 5E;

[0092] or,

[0093]

[0094] R is as defined above, and n is selected from 0, 1, 2, 3;

[0095] Step 5-4: Compound 5A and compound 5F are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate compound 5G;

[0096] Step 5-5: In a dichloromethane solution, triethylamine is added to compound 5G, the mixture is cooled to 0°C, and a dichloromethane solution of methanesulfonic anhydride is added dropwise. The mixture is allowed to react at room temperature to produce compound 5H.

[0097] Steps 5-6: Compound 5H and compound 1A react in dimethylformamide solution at 80°C in the presence of triethylamine to generate compound 5I;

[0098] Synthesis Method Six:

[0099]

[0100] Where R is as defined above;

[0101] Step 6-1: Compound 1A reacts with chloroethane-1-sulfonyl chloride in a dichloromethane solution at -40°C in the presence of triethylamine to produce compound 6A;

[0102] Step 6-2: Compound 6A reacts in acetonitrile solution at room temperature in the presence of cesium carbonate and ammonium hydroxide to form compound 6B;

[0103] Step 6-3: Compound 6B and Compound 6C undergo an amidation reaction in dimethylformamide solution at room temperature, activated by HATU and DIPEA, to generate Compound 6D; or Compound 6B and Compound 6C undergo an amidation reaction in acetonitrile solution in the presence of N-methylimidazole and tetramethylchlorourea hexafluorophosphate to generate Compound 6D.

[0104] Step 6-4: Compound 6B and compound 6E react in a dichloromethane solution at room temperature to produce compound 6F.

[0105] In a third aspect, this application provides a pharmaceutical composition comprising one or more of the compounds described in the first aspect, pharmaceutically acceptable salts, enantiomers, diastereomers, transisomers, racemates, polymorphs, solvates, or isotopically labeled compounds, and pharmaceutically acceptable carriers, diluents, or excipients.

[0106] In another preferred embodiment, the pharmaceutical composition further comprises at least one other therapeutic agent selected from anticancer agents, immunomodulators, anti-inflammatory agents, anti-Alzheimer's agents, and combinations thereof.

[0107] A fourth aspect of the invention provides the use of the compounds of the first aspect, pharmaceutically acceptable salts thereof, enantiomers, diastereomers, transisomers, polymorphs, solvates, isotopically labeled compounds or racemates, or pharmaceutical compositions of the second aspect, for the preparation of medicaments for the prevention and / or treatment of diseases or conditions mediated by the CDK9-cyclin T1 complex.

[0108] In another preferred embodiment, the disease or condition 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, bile duct and gallbladder cancer, bladder cancer, brain tumor, neuroblastoma, schwannoma, glioma, glioblastoma and astrocytoma, endometrial cancer, esophageal cancer, head and neck cancer, pancreatic cancer, and renal cell carcinoma.

[0109] A fifth aspect of the invention provides a method for treating a disease or condition mediated by the CDK9-cyclin T1 complex, particularly a tumor, the method comprising administering to a subject a therapeutically effective amount of one or more of the above-described compounds or pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, transisomers, polymorphs, solvates, or isotopically labeled compounds thereof. In some embodiments, the method further comprises administering to a subject in need an effective dose of other therapeutic agents.

[0110] Beneficial effects:

[0111] This invention synthesizes a series of highly efficient and selective small-molecule degradative derivatives of the CDK9-cyclin T1 complex. Previously reported CDK9 small-molecule degradatives primarily target the CDK9 monomeric protein; the compounds in this invention can selectively degrade both CDK9 and cyclin T1 simultaneously, achieving the degradation of the CDK9-cyclin T1 complex.

[0112] At the cellular level, the compounds of this invention were shown to significantly inhibit the proliferation of prostate cancer cells, block the cell cycle, and induce apoptosis, with effects stronger than the inhibitor SNS032. At the protein level, compound LL-K9-015 significantly inhibited RNA polymerase II phosphorylation and significantly downregulated the protein levels of MCL1, cMyc, AR, ARv7, KLK3, and NKX3-1, with more thorough inhibition than SNS032. At the transcriptomic level, MYC and AR-mediated oncogenic aberrations were inhibited. This is the first case study to explore the therapeutic potential of the CDK9-cyclin T1 complex using small molecule degraders.

[0113] The CDK9-cyclin T1 complex small molecule degrader of this invention, such as LL-K9-015, is a potent and selective CDK9-cyclin T1 complex small molecule degrader, and will be a valuable chemical probe for the study of non-catalytic related functions of the CDK9-cyclin T1 complex.

[0114] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0115] Figure 1 The results show the activity of the compound of this invention in degrading the CDK9-cyclin T1 complex.

[0116] Figure 2 The degradation efficiency of LL-K9-015 is DC 50 The results of the assay are shown in Figure A, where A represents the immunoblotting analysis results of LL-K9-015 degrading the CDK9-cyclin T1 complex; and B represents the degradation efficiency curve of LL-K9-015 fitted based on three independent replicate experiments.

[0117] Figure 3 The results of proteomic determination of the degradation kinetics of CDK9-cyclin T1 complex by LL-K9-015 were presented. In the figure, A represents the change in the relative protein levels of CDK9 and cyclin T1 over time in 22RV1 cells treated with LL-K9-015; and B represents the change in the relative protein levels of CDK9 and cyclin T1 over time in 22RV1 cells treated with SNS032.

[0118] Figure 4The results of the degradation kinetics of LL-K9-015 on CDK9-cyclin T1 complex were determined by immunoblotting.

[0119] Figure 5 To show the results of degradation selectivity, A represents the CDK protein content of 22RV1 cells after 24 hours of LL-K9-015 treatment; B represents the content of cyclin proteins binding CDK2, CDK7, and CDK9 after 24 hours of LL-K9-015 treatment; C represents the proteomic changes in the LL-K9-015 group compared to the control group (DMSO); and D represents the proteomic changes in the SNS032 group compared to the control group (DMSO).

[0120] Figure 6 The results represent the in vitro antitumor activity, where A shows the effect of SNS032 and LL-K9-015 treatment on 22RV1 cells for 5 days on cell viability; and B shows the effect of SNS032 and LL-K9-015 on colony formation.

[0121] Figure 7 The results of cell cycle and apoptosis experiments are shown. In A, the distribution of cell cycle is shown after 24 hours of treatment with LL-K9-015 and SNS032. The bar chart represents the mean ± standard deviation of two independent experiments. In B, the detection results of cell apoptosis are shown after 24 hours of treatment with LL-K9-015 and SNS032. Each point represents the result of an independent experiment. The bar chart represents the mean ± standard deviation. n = 3.

[0122] Figure 8 Immunoblotting analysis of CDK9-related pathway and target proteins, where A represents the immunoblotting results of CTD phosphorylation and CDK9 target protein levels; B represents the immunoblotting results of AR and AR downstream signaling pathways.

[0123] Figure 9 The effects of the degrading agent on the transcriptome are shown in Figure 1. A is a heatmap of differentially expressed genes in each group; B is a transcription factor network analysis of genes specifically affected by LL-K9-015, showing the names of pivot transcription factors; C is a heatmap of representative MYC target genes; and D is a heatmap of representative AR target genes. Detailed Implementation

[0124] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions (such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0126] Through extensive and in-depth research, the inventors of this application have synthesized a series of highly efficient and selective small-molecule degradative derivatives of the CDK9-cyclin T1 complex with novel oxazole backbone structures. At the cellular level, the compounds significantly inhibit the proliferation of prostate cancer cells, block the cell cycle, induce apoptosis, and downregulate the levels of proteins regulated by the CDK9-cyclin T1 complex, including cMyc, MCL-1, and AR. At the transcriptomic level, MYC and AR-mediated oncogenic aberrations are suppressed.

[0127] the term

[0128] In this invention, unless otherwise specified, the terms used have the general meanings known to those skilled in the art.

[0129] In this invention, the term "C1-C" 10 "C3-C" refers to a carbon atom with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 atoms. 10 "C1-C4" refers to carbon atoms with 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The terms "C1-C4", "C1-C6", "C3-C8", and "C6-C9" are also used. 12 "has a similar meaning."

[0130] In this invention, the term "4-8-element" refers to having 4-8 ring atoms, the term "5-7-element" refers to having 5-7 ring atoms, and so on.

[0131] In this invention, the term "alkyl" refers to a saturated straight-chain or branched linear hydrocarbon moiety, such as the term "C1-C". 10"Alkyl" refers to a straight-chain 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, etc.; preferably ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl; the terms "C1-C6 alkyl" and "C1-C4 alkyl" have similar meanings.

[0132] In this invention, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon moiety, including monocyclic, bicyclic, and tricyclic, for example, the term "C3-C". 10 "Cycloalkyl" refers to a cyclic alkyl group having 3 to 10 carbon atoms on a ring, and includes, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, adamantyl (exemplary tricyclic cycloalkyl), etc.

[0133] In this invention, the term "C3-C" 10 "Cycloalkyloxy" refers to a cyclic alkyl-oxy group having 3 to 10 carbon atoms on the ring, including, but not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy, cyclooctyloxy, cyclononyloxy, and cyclodecyloxy. The term "C3-C8 cycloalkyloxy" has a similar meaning.

[0134] In this invention, the term "aryl" refers to a hydrocarbon moiety comprising one or more aromatic rings. For example, the term "C6-C..." 12 "Aryl" refers to an aromatic cyclic group with 6 to 12 carbon atoms that does not contain heteroatoms on the ring, such as phenyl and naphthyl. Examples of aryl groups include, but are not limited to, phenyl (Ph), naphthyl, pyrene, anthracene, and phenanthrene.

[0135] The term "heterocycle" refers to a ternary to 15-membered, preferably ternary to 12-membered, saturated or partially unsaturated non-aromatic heterocycle containing one to four heteroatoms selected from oxygen, nitrogen, and sulfur: monocyclic, bicyclic, 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 carbocyclic member; if the ring contains more than one oxygen atom, they are not directly adjacent; for example (but not limited to) ethylene oxide, oxobutyryl, aziridinyl, aziridine, aziridine, aziridinehexyl, etc.

[0136] The term "heteroaryl" refers to a monocyclic or bicyclic aromatic ring group containing at least one heteroatom selected from nitrogen, oxygen, or sulfur as a ring member; "5-7 membered heteroaryl" refers to a heteroaryl group containing 5 to 7 ring atoms. Examples of heteroaryl groups include, but are not limited to, the following groups: pyrrole, imidazolyl, pyrazolyl, 1,2,3-triazolyl, pyridinyl, pyridonel, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, quinolinyl, etc.

[0137] In this invention, the term Boc refers to t-Butyloxycarbonyl.

[0138] Unless otherwise stated, the alkyl, cycloalkyl, cycloalkyloxy, aryl, and heteroaryl groups mentioned herein are substituted and unsubstituted groups. Possible substituents on alkyl, cycloalkyl, cycloalkyloxy, aryl, and heteroaryl groups include, but are not limited to: hydroxyl, 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 alkylaminosulfonyl, arylaminosulfonyl, C1-C10 alkylimino, C1-C10 alkylsulfonylimino, arylsulfonylimino, mercapto, C1-C10 alkylthio, C1-C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, guanidine, urea, cyano, acyl, thioacyl, acyloxy, carboxyl, and carboxylic acid ester groups. On the other hand, cycloalkyl, heterocycloalkyl, heterocycloalkenyl, aryl, and heteroaryl groups can also be fused together.

[0139] In this invention, the substitution can be monosubstituted or polysubstituted, and the polysubstituted can be disubstituted, trisubstituted, tetrasubstituted, or pentasubstituted. Disubstituted means having two substituents, and so on.

[0140] The pharmaceutically acceptable salts described in this invention can be salts formed by anion and a positively charged group on a compound of Formula I. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate, or maleate. Similarly, salts can be formed by cations and negatively charged groups on a compound of Formula I. Suitable cations include sodium, potassium, magnesium, calcium, and ammonium ions, such as tetramethylammonium ions.

[0141] In another preferred embodiment, "pharmaceutically acceptable salt" refers to a salt formed by 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, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic 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, pyric acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, and hydroxyethanesulfonic acid; or a sodium, potassium, calcium, aluminum, or ammonium salt formed by a compound of Formula I with an inorganic base; or a methylamine, ethylamine, or ethanolamine salt formed by a compound of general Formula I with an organic base.

[0142] Pharmaceutical Composition

[0143] The present invention also provides a pharmaceutical composition comprising an active ingredient within a safe and effective range, and a pharmaceutically acceptable carrier.

[0144] The "active ingredient" mentioned in this invention refers to the compound of general formula I described in this invention.

[0145] The "active ingredient" and pharmaceutical composition described in this invention are used to prepare remedies for treating diseases or conditions mediated by the CDK9-cyclin T1 complex. The "active ingredient" and pharmaceutical composition described in this invention can also be used as CDK9-cyclin T1 complex inhibitors and degrading agents.

[0146] "Safe and effective dose" refers to an amount of active ingredient sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of active ingredient per dose, more preferably 10-200 mg of active ingredient per dose. Preferably, "one dose" refers to one tablet.

[0147] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredient of the invention without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers. Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0148] There are no particular limitations on the administration of the active ingredients or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0149] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0150] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0151] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0152] 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 non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0153] The compounds of this invention can be administered alone or in combination with other therapeutic agents (such as antitumor drugs).

[0154] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 20–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0155] Example 1: Preparation of compound SNS-032

[0156] Step 1:

[0157]

[0158] 1-Amino-3,3-dimethylbutanone hydrochloride (6.0 g, 40 mmol, 1 equivalent) was dissolved in DCM (200 mL), cooled to -5 °C, and triethylamine (22 mL, 160 mmol, 4 equivalent) was slowly added. The resulting mixture was cooled to -10 °C. Chloroacetyl chloride (3.5 mL, 44 mmol, 1.1 equivalent) was dissolved in ice-cold DCM (20 mL), and the mixture was added dropwise over 15 minutes while maintaining the reaction temperature below -5 °C. The reaction mixture was stirred at -5 °C for 2 hours, and then quenched with 1N HCl aqueous solution (100 mL). The phases were separated, and the organic phase was washed successively with 1N HCl aqueous solution (100 mL) and water (30 mL), dried (Na2SO4), and concentrated under vacuum to give 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, CDCl3) δ7.40 (brs, 1H), 4.30 (d, J = 4.4Hz, 2H), 4.07 (s, 2H), 1.20 (s, 9H).

[0159] Step 2:

[0160]

[0161] Add 2-chloro-N-(3,3-dimethyl-2-oxobutyl)acetamide (2) (8.0 g, 41.7 mmol, 1 equivalent) and POCl3 (16 mL, 171.0 mmol, 4.1 equivalent) to a 100 mL thick-walled pressure-resistant flask. Heat the reaction mixture to 110 °C and stir overnight. After cooling to room temperature, carefully pour the reaction mixture into ice water (200 mL). Extract the mixture with methyl tert-butyl ether (2 × 200 mL). Combine the organic extracts and neutralize with saturated sodium bicarbonate to pH 7-8. Separate the organic phase, wash successively with saturated sodium bicarbonate (200 mL), water (200 mL), and NaCl brine (100 mL), dry (Na2SO4), and concentrate under vacuum 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, CDCl3) δ6.68(s,1H),4.57(s,2H),1.29(s,9H).

[0162] Step 3:

[0163]

[0164] 5-Bromothiazol-2-amine (4) (3.56 g, 20 mmol, 1 equivalent) and potassium thiocyanate (19.4 g, 200 mmol, 10 equivalent) were dissolved in methanol (150 mL) and heated to 60 °C with stirring overnight. The reaction mixture was concentrated under vacuum, then extracted with ethyl acetate (EA, 3 × 100 mL), washed with NaCl brine, and dried over Na₂SO₄ to give 5-thiocyanothiazol-2-amine (5) as a yellow solid (2.0 g, 64%). MS (ESI) 157.90 [M+H] + .1H NMR(400MHz,CD3OD)δ7.33(s,1H).

[0165] Step 4:

[0166]

[0167] Add 3.14 g (20 mmol, 1 equivalent) of 5-thiocyanothiazol-2-amine (5) and 120 mL of anhydrous ethanol to a 250 mL three-necked round-bottom flask. Add 1.5 g (40 mmol, 2 equivalent) of NaBH4 in portions under nitrogen atmosphere. Stir the mixture at room temperature for 1 hour, then slowly add 100 mL of acetone. After 1 hour, dissolve 3-(tert-butyl)-2-(chloromethyl)oxazole (3) (3.8 g, 22 mmol, 1.1 equivalent) in EtOH (10 mL) and slowly add it to the above reaction mixture. Heat the reaction mixture to 80 °C and reflux for 2 hours. After the reaction is complete, cool to room temperature, concentrate under vacuum, and then extract with ethyl acetate (3 × 100 mL) and NaCl brine (3 × 200 mL). Separate the organic phase, dry (Na2SO4), concentrate under vacuum, and give the crude product solid. The crude product was purified by rapid silica gel chromatography (PE:EA = 1:1) to give 5-(((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazole-2-amine (6), as a yellow solid (3.6 g, 67%). MS (ESI) 270.00 [M+H] + .1H NMR (400MHz, CDCl3) δ6.97(s,1H),6.58(s,1H),5.26(brs,2H),3.88(s,2H),1.26(s,9H).

[0168] Step 5:

[0169]

[0170] 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (4.58 g, 20 mmol, 2 equivalents) was dissolved in DMF (100 mL), and HATU (7.6 g, 20 mmol, 2 equivalents) and DIPEA (6.95 mL, 40 mmol, 4 equivalents) were added. The mixture was stirred for 10 minutes, and then 5-(((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazole-2-amine (6) (2.69 g, 10 mmol, 1 equivalent) was added. The mixture was stirred overnight at room temperature. After the reaction was complete, water (100 mL) was added, and a large amount of white solid precipitate was formed. The precipitate was washed with water and dried to give tert-butyl 4-((5-(((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazole-2-yl)carbamoyl)piperidine-1-carboxylic acid ester (7), which was a white solid (3.7 g, 77%). MS(ESI)481.11[M+H] + .1H NMR(400MHz, CDCl3)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).

[0171] Step 6:

[0172]

[0173] 2.4 g of tert-butyl 4-((5-(((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazolyl-2-yl)carbamoyl)piperidin-1-carboxylic acid ester (7) was dissolved in 20 mL of EtOH. A 2 M, 20 mL solution of HCl in EtOH was slowly added, and the reaction mixture was stirred overnight at room temperature. The solution was concentrated under vacuum to obtain a crude product. The pH was adjusted to 7-8 with saturated sodium bicarbonate, extracted with DCM, concentrated under vacuum, and purified by rapid silica gel 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, CD3OD) 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).

[0174] Example 2: Preparation of compound LL-K9-001

[0175] Step 1:

[0176]

[0177] 1-Adamantaneacetic acid (48.5 mg, 0.25 mmol, 1 equivalent), HATU (114 mg, 0.3 mmol, 1.2 equivalent), and DIPEA (173.8 μL, 1 mmol, 4 equivalent) were dissolved in DMF (5 mL). The mixture was stirred for 10 minutes, and then SNS-032 (95 mg, 0.25 mmol, 1 equivalent) was added. The mixture was 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 under vacuum to give a crude product solid. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), and the pure fraction was concentrated to give LL-K9-001 (65 mg, 47%) as a white solid. HRMS[M+H] + C 29 H 41 N4O3S2 + The calculated value is 557.2620, and the result is 577.2664. 1 H NMR (400MHz, CD3OD) 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),2.79–2.7 5(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).

[0178] Example 3: Preparation of compound LL-K9-002

[0179]

[0180] The product was prepared using the same or similar method as in Example 2, from SNS-032 (95 mg, 0.25 mmol, 1 equivalent) and (-)-monthoxyacetic acid (53.5 mg, 0.25 mmol, 1 equivalent). The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), followed by semi-preparative HPLC (flow rate, 10 mL / min; solvent A: MeOH:MeCN = 1:1; solvent B: 0.1% TFA aqueous solution; gradient: elution from 5% A to 95% A over 30 min, followed by elution with 95% A over 30-45 min) to obtain a white solid LL-K9-002 (95 mg, 66%). HRMS [M+H] + C 29 H 45 N4O4S2 + The calculated value is 577.2882, and the value found is 577.2931. 1 H NMR (600MHz, DMSO-d6) 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),2.75–2.7 0(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).

[0181] Example 4: Preparation of compound LL-K9-003

[0182] Step 1:

[0183]

[0184] 1-Adamantaneacetic acid (1.94 g, 10 mmol, 1 equivalent), HATU (4.56 g, 12 mmol, 1.2 equivalent), and DIPEA (6.95 mL, 40 mmol, 4 equivalent) were dissolved in DMF (50 mL). The mixture was stirred for 10 minutes, and then 2,2-diethoxyethyl-1-amine (1.33 g, 10 mmol, 1 equivalent) was added. The mixture was stirred overnight at room temperature and then extracted with EA and water. The organic phase was separated, washed with NaCl brine, and concentrated under vacuum to give a crude product solid. The crude product was purified by rapid silica gel chromatography (PE:EA = 2:1), and the pure fraction was concentrated under vacuum to give 2-((3R,5R,7R)-adamantane-1-yl)-N-(2,2-diethoxyethyl)acetamide as a white solid (2.2 g, 71%). HRMS[M+H] + C 18 H 32 NO3 + The calculated value is 310.2382, but we found 310.2390. 1 HNMR(400MHz, CDCl3)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).

[0185] Step 2:

[0186]

[0187] 3.1 g, 10 mmol, 1 equivalent of 2-((3R,5R,7R)-adamantane-1-yl)-N-(2,2-diethoxyethyl)acetamide was dissolved in MeCN (50 mL), and HCl (1 N, 50 mL) was added. The mixture was stirred overnight at room temperature, then neutralized to pH 7-8 with saturated sodium bicarbonate, extracted with EA and water, dried over Na2SO4, and concentrated under vacuum to give 2-((3R,5R,7R)-adamantane-1-yl)-N-(2-oxyethyl)acetamide, which can be used in the next step without further purification.

[0188] Step 3:

[0189]

[0190] SNS-032 (95 mg, 0.25 mmol, 1 equivalent) was dissolved in DCM (10 mL), and triethylamine (69 μL, 0.5 mmol, 2 equivalents) and compound 2-((3R,5R,7R)-adamantane-1-yl)-N-(2-oxoethyl)acetamide (117.5 mg, 0.5 mmol, 2 equivalents) were added. The mixture was stirred for 15 min, and then sodium triacetoxyborohydride (106 mg, 0.5 mmol, 2 equivalents) was added. The mixture was stirred overnight at room temperature. Then, it was extracted with DCM and water, the organic phase was separated, washed with NaCl brine, and concentrated under vacuum to give the crude product. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), followed by semi-preparative HPLC (flow rate, 10 mL / min; solvent A: MeOH:MeCN = 1:1; solvent B: 0.1% TFA aqueous solution; gradient: elution from 5% A to 95% A over 30 min, then elution with 95% A over 30-45 min) to obtain a white solid LL-K9-003 (99 mg, 66%). HRMS [M+H] + C 31 H 46 N5O3S2 + The calculated value is 600.3042, but the result is 600.3089. 1 H NMR (400MHz, CD3OD) 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).

[0191] Example 5: Preparation of compound LL-K9-004

[0192] The compound was prepared using the same or similar method as in Example 4. The compound structure and characterization data are as follows:

[0193]

[0194] HRMS[M+H] + C 31 H 50 N5O4S2 + The calculated value is 620.3304, but the value found is 620.3355. 1H NMR (600MHz, DMSO-d6) 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,2 H),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).

[0195] Example 6: Preparation of compound LL-K9-005

[0196] Step 1:

[0197]

[0198] (-)-Monthoxyacetic acid (1.07 g, 5 mmol, 1 equivalent), HATU (2.28 g, 6 mmol, 1.2 equivalent), and DIPEA (3.5 mL, 20 mmol, 4 equivalent) were dissolved in DMF (20 mL), and the mixture was stirred for 10 minutes. 4-Aminobut-1-ol (445 mg, 5 mmol, 1 equivalent) was added, and the mixture was stirred overnight at room temperature. The mixture was then extracted with EA and water, the organic phase was separated, washed with NaCl brine, and concentrated under vacuum to obtain the crude product. The crude product was purified by rapid silica gel chromatography (PE:EA = 1:4), and the pure fraction was concentrated under vacuum to give N-(4-hydroxybutyl)-2-(((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamide, as a yellow oil (1.03 g, 72%). HRMS[M+H] + C 16 H 32 NO3 + The calculated value is 286.2382, and the value is found to be 286.2386. 1H NMR (400MHz, CDCl3) 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).

[0199] Step 2:

[0200]

[0201] N-(4-hydroxybutyl)-2-(((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamide (570 mg, 2 mmol, 1 equivalent) was dissolved in DCM (10 mL), and NEt3 (554 μL, 4 mmol, 2 equivalents) was added. The mixture was cooled to 0 °C, and methanesulfonic anhydride (697 mg, 4 mmol, 2 equivalents) was dissolved in DCM (4 mL) and added dropwise to the reaction mixture. The mixture was stirred overnight at room temperature, then extracted with DCM and water, washed with NaCl brine, dried over Na2SO4, and concentrated under vacuum to give 4-(2-(((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamido)methanesulfonate as a yellow oil (640 mg, 88%), which could be used in the next step without further purification. HRMS[M+H] + C 17 H 34 NO5S + The calculated value is 364.2158, but we found 364.2166. 1H NMR (400MHz, CDCl3) 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).

[0202] Step 3:

[0203]

[0204] SNS-032 (95 mg, 0.25 mmol, 1 equivalent) was dissolved in DMF (3 mL), and NEt3 (69 μL, 0.5 mmol, 2 equivalents) and 4-(2-(((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamido)butyl methanesulfonate (181.5 mg, 0.5 mmol, 2 equivalents) were added. The mixture was heated to 80 °C and stirred overnight, then extracted with EA and water. The organic phase was separated, washed with NaCl brine, and concentrated under vacuum to give a crude product solid. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), and the pure fraction was concentrated under vacuum to give a yellow solid LL-K9-005 (68 mg, 42%). HRMS[M+H] + C 33 H 54 N5O4S2 + The calculated value is 648.3617, but the result is 648.3671. 1H NMR (600MHz, DMSO-d6) 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,1 H),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).

[0205] Example 7: Preparation of LL-K9-006

[0206] The intermediate and final products were prepared using the same or similar methods as in Example 6. The structures and characterization data of the intermediates and final products are as follows:

[0207] alcohol intermediates

[0208] N-(6-hydroxyhexyl)-2-(((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamide. HRMS[M+H] + C 18 H 36 NO3 + The calculated value is 314.2695, and the value found is 314.2701. 1 H NMR (400MHz, CDCl3) 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).

[0209] methanesulfonyl intermediate

[0210] 6-(2-((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamimide hexyl methanesulfonate. HRMS[M+H] + C 19 H 38 NO5S + The calculated value is 392.2471, but the value is 392.2484. 1 H NMR (400MHz, CDCl3) 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).

[0211] LL-K9-06

[0212]

[0213] HRMS[M+H] + C 35 H 58 N5O4S2 + The calculated value is 676.3930, and the value found is 676.3984. 1H NMR (600MHz, DMSO-d6) 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,2 H),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).

[0214] Example 8: Preparation of LL-K9-007

[0215] The intermediate and final products were prepared using the same or similar methods as in Example 6. The structures and characterization data of the intermediates and final products are as follows:

[0216] alcohol intermediates

[0217] N-(2-(2-hydroxyethoxy)ethoxy)ethyl)-2-((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)acetamide. HRMS[M+H] + C 18 H 36 NO5 + The calculated value is 346.2594, but the actual value is 346.2602. 1H NMR (400MHz, CDCl3) 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=1 0.6,4.0Hz,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).

[0218] methanesulfonyl intermediate

[0219] 2-(2-(2-(2-((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamimide)ethoxy)ethyl methanesulfonate. HRMS[M+H] + C 19 H 38 NO7S + The calculated value is 424.2369, but the value found is 424.2382. 1 H NMR(400MHz, CDCl3) δ6.95(s,1H),4.40–4.33(m,2H),4.06(d,J=15.2Hz,1H),3.84(d,J=15.2H z,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).

[0220] LL-K9-007

[0221]

[0222] HRMS[M+H] + C 35 H 58 N5O6S2 + The calculated value is 708.3829, and the actual value is 708.3886.1 H NMR (600MHz, DMSO-d6) 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).

[0223] Example 9: Preparation of LL-K9-008

[0224] The intermediate and final products were prepared using the same or similar methods as in Example 6. The structures and characterization data of the intermediates and final products are as follows:

[0225] alcohol intermediates

[0226] 1-(4-(hydroxymethyl)piperidin-1-yl)-2-((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)ethane-1-one. HRMS[M+H] + C 18 H 34 NO3 + The calculated value is 312.2539, but the value found is 312.2547. 1H NMR(400MHz, CDCl3)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).

[0227] methanesulfonyl intermediate

[0228] (1-(2-((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetyl)piperidin-4-yl)methylmethanesulfonate. HRMS[M+H] + C 19 H 36 NO5S + The calculated value is 390.2314, but the value found is 390.2326. 1 H NMR (400MHz, CDCl3) 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).

[0229] LL-K9-008:

[0230]

[0231] HRMS[M+H] + C 35 H 56 N5O4S2 + The calculated value is 674.3774, and the value found is 674.3827. 1H NMR (600MHz, DMSO-d6) 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), 0.85(d,J=7.0Hz,3H), 0.83–0.78(m,1H), 0.75–0.70(m,4H). Example 10: Preparation of LL-K9-009

[0232] The intermediate and final products were prepared using the same or similar methods as in Example 6. The structures and characterization data of the intermediates and final products are as follows:

[0233] alcohol intermediates

[0234] N-(2-(2-hydroxyethoxy)ethyl)-2-((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamide. HRMS[M+H] + C 16 H 32 NO4 + The calculated value is 302.2331, but we found 302.2339. 1H NMR (400MHz, CDCl3) 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).

[0235] methanesulfonyl intermediate

[0236] 2-(2-(2-((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamimide)ethoxy)methylsulfonate ethyl ester. HRMS[M+H] + C 17 H 34 NO6S + The calculated value is 380.2107, but the result is 380.2114. 1 H NMR (400MHz, CDCl3) δ6.92 (s, 1H), 4.38–4.33 (m, 2H), 4.06 (d, J = 15.2Hz, 1H), 3.85 (d, J = 1 5.2Hz,1H),3.77–3.71(m,2H),3.63–3.56(m,2H),3.55–3.45(m,2H),3.14(td,J=10.7,4.1 Hz,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).

[0237] LL-K9-009

[0238]

[0239] HRMS[M+H] + C 33 H 54 N5O5S2 +The calculated value is 664.3566, and the value found is 664.3618. 1 H NMR (600MHz, DMSO-d6) 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).

[0240] Example 11: Preparation of LL-K9-010

[0241] Step 1:

[0242]

[0243] 1-Adamantaneacetic acid (0.97 g, 5 mmol, 1 equivalent), HATU (2.28 g, 6 mmol, 1.2 equivalent), and DIPEA (3.5 mL, 20 mmol, 4 equivalent) were dissolved in DMF (20 mL), and the mixture was stirred for 10 minutes. 4-Aminobut-1-ol (445 mg, 5 mmol, 1 equivalent) was added, and the mixture was stirred overnight at room temperature. The mixture was then extracted with EA and water, the organic phase was separated, washed with NaCl brine, and concentrated under vacuum to obtain the crude product. The crude product was purified by rapid silica gel chromatography (PE:EA = 1:4), and the pure fraction was concentrated under vacuum to give 2-((3R,5R,7R)-adamantane-1-yl)-N-(4-hydroxybutyl)acetamide as a yellow oil (1.1 g, 83%). MS (ESI) 266.23 [M+H] +.1H NMR (600MHz, CDCl3) 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).

[0244] Step 2:

[0245]

[0246] 2-((3R,5R,7R)-adamantane-1-yl)-N-(4-hydroxybutyl)acetamide (530 mg, 2 mmol, 1 equivalent) was dissolved in DCM (10 mL), and NEt3 (554 μL, 4 mmol, 2 equivalents) was added. The mixture was cooled to 0 °C, and methanesulfonic anhydride (697 mg, 4 mmol, 2 equivalents) was dissolved in DCM (4 mL) and added dropwise to the reaction mixture. The mixture was stirred overnight at room temperature, then extracted with DCM and water, washed with NaCl brine, dried over Na2SO4, and concentrated under vacuum to give 4-(2-((3R,5R,7R)-adamantane-1-yl)acetamamido)methylsulfonate butyl ester, a yellow oil (590 mg, 86%), which could be used in the next step without further purification. MS (ESI) 344.45 [M+H] + .1H NMR (600MHz, CDCl3) 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).

[0247] Step 3:

[0248]

[0249] SNS-032 (95 mg, 0.25 mmol, 1 equivalent) was dissolved in DMF (3 mL), and NEt3 (69 μL, 0.5 mmol, 2 equivalents) and butyl 4-(2-((3R,5R,7R)-adamantane-1-yl)acetamido)methylsulfonate (171.5 mg, 0.5 mmol, 2 equivalents) were added. The mixture was heated to 80 °C and stirred overnight, then extracted with EA and water. The organic phase was separated, washed with NaCl brine, and concentrated under vacuum to give a crude product solid. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), and the purified fraction was concentrated under vacuum to give a yellow solid LL-K9-010 (64 mg, 41%). HRMS[M+H] + C 33 H 50 N5O3S2 + The calculated value is 628.3355, but the actual value is 628.3408. 1 H NMR (400MHz, DMSO-d6) 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).

[0250] Example 12: Preparation of LL-K9-011

[0251] The intermediate and final product were prepared using the same or similar methods as in Example 11. The structures and characterization data of the intermediate and final product are as follows:

[0252] alcohol intermediates

[0253] 2-((3R,5R,7R)-adamantane-1-yl)-N-(6-hydroxyhexyl)acetamide. MS (ESI) 294.45 [M+H] + .1H NMR(600MHz, CDCl3)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).

[0254] methanesulfonyl intermediate

[0255] 6-(2-((3R,5R,7R)-adamantane-1-yl)acetamyl)hexyl methanesulfonate

[0256] MS(ESI) 372.31 [M+H] + .1H NMR(600MHz, CDCl3)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).

[0257] LL-K9-011

[0258]

[0259] HRMS[M+H] + C 35 H 54 N5O3S2 + The calculated value is 656.3668, and the value found is 656.3720. 1 H NMR (400MHz, DMSO-d6) 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).

[0260] Example 13: Preparation of LL-K9-012

[0261] The intermediate and final product were prepared using the same or similar methods as in Example 11. The structures and characterization data of the intermediate and final product are as follows:

[0262] alcohol intermediates

[0263] 2-((3R,5R,7R)-adamantane-1-yl)-N-(2-(2-(2-hydroxyethoxy)ethoxy)acetamide. MS (ESI) 326.23 [M+H]+ . 1 H NMR(600MHz, CDCl3)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).

[0264] methanesulfonyl intermediate

[0265] 2-(2-(2-(2-((3R,5R,7R)adamantane-1-yl)acetamamido)ethoxy)ethyl methanesulfonate. MS(ESI) 404.20 [M+H] + . 1 H NMR(600MHz, CDCl3)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).

[0266] LL-K9-012

[0267]

[0268] HRMS[M+H] + C 35 H 54 N5O5S2 + The calculated value is 688.3566, and the result is 688.3622. 1H NMR(600MHz,DMSO-d6)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).

[0269] Example 14: Preparation of LL-K9-013

[0270] The intermediate and final product were prepared using the same or similar methods as in Example 11. The structures and characterization data of the intermediate and final product are as follows:

[0271] alcohol intermediates

[0272] 2-((3s)-adamantane-1-yl)-1-(4-(hydroxymethyl)piperidin-1-yl)ethane-1-one. MS (ESI) 292.43 [M+H] + . 1 H NMR (600MHz, CDCl3) δ4.66–4.61(m,1H),3.96–3.91(m,1H),3.47–3.90(m,2 H),2.96(td,J=13.2,2.7Hz,1H),2.48(td,J=12.9,2.6Hz,1H),2.14(d,J=1 3.3Hz,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).

[0273] methanesulfonyl intermediate

[0274] Methyl (1-(2-((3s)-adamantane-1-yl)acetyl)piperidin-4-yl)methyl methanesulfonate. MS(ESI) 370.52 [M+H] + . 1H NMR (600MHz, CDCl3) 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).

[0275] LL-K9-013

[0276]

[0277] HRMS[M+H] + C 35 H 52 N5O3S2 + The calculated value is 654.3512, but the result is 654.3564. 1 H NMR(400MHz,DMSO-d6)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,4 H),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).

[0278] Example 15: Preparation of LL-K9-014

[0279] The intermediate and final product were prepared using the same or similar methods as in Example 11. The structures and characterization data of the intermediate and final product are as follows:

[0280] alcohol intermediates

[0281] 2-((3R,5R,7R)-adamantane-1-yl)-N-(2-(2-hydroxyethoxy)ethyl)acetamide. MS(ESI) 282.20 [M+H] + . 1H NMR(600MHz, CDCl3)δ6.42(t,J=5.6Hz,1H),3.69–3.67(m,2H),3.52–3.49(m,4 H),3.41–3.37(m,2H),1.92–1.89(m,5H),1.65–1.62(m,3H),1.58–1.55(m,9H).

[0282] methanesulfonyl intermediate

[0283] 2-(2-(2-((3R,5R,7R)-adamantane-1-yl)acetamamido)ethoxy)methylsulfonate. MS(ESI) 360.18 [M+H] + . 1 H NMR (600MHz, CDCl3) δ6.07(t,J=4.9Hz,1H),4.33–4.31(m,2H),3.68–3.66(m,2H),3.52(t,J=5.2 Hz,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).

[0284] LL-K9-014

[0285]

[0286] HRMS[M+H] + C 33 H 50 N5O4S2 + The calculated value is 644.3304, but the result is 644.3353. 1 H NMR (400MHz, DMSO-d6) 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).

[0287] Example 16: Preparation of LL-K9-015

[0288] Step 1:

[0289]

[0290] SNS-032 (3.8 g, 10 mmol, 1 equivalent) was dissolved in DCM (40 mL), and NEt3 (5.5 mL, 40 mmol, 4 equivalent) was added. The mixture was cooled to -40 °C. Ethyl chloride-1-sulfonyl chloride (2.1 mL, 20 mmol, 2 equivalent) was dissolved in ice-cold DCM (4 mL) and added dropwise to the reaction mixture. The mixture was stirred at -40 °C for 2 hours, then quenched with water, extracted with DCM and water, washed with NaCl brine, and concentrated under vacuum to obtain a crude solid. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 40:1), and the pure fraction was concentrated to give N-(5-(((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazolyl-2-yl)-1-(vinylsulfonyl)piperidine-4-carboxamide, a yellow solid (2.2 g, 47%). HRMS[M+H] + C 19 H 27 N4O4S3 + The calculated value is 471.1194, but the value found is 471.1210. 1 H NMR (400MHz, CD3OD) 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=1 2.8,3.6Hz,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).

[0291] Step 2:

[0292]

[0293] N-(5-(((5-(tert-butyl))oxazol-2-yl)methyl)thio)thiazol-2-yl)-1-(vinylsulfonyl)piperidine-4-carboxamide (2.35 g, 5 mmol, 1 equivalent) was dissolved in MeCN (100 mL), and Cs₂CO₃ (3.25 g, 10 mmol, 2 equivalent) and NH₄OH (48%, 40 mL) were added. The reaction was stirred overnight at room temperature and then concentrated under vacuum. The mixture was extracted with DCM and water, washed with NaCl brine, and concentrated under vacuum to give a crude solid. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), and the pure fraction was concentrated under vacuum 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 N5O4S3 + The calculated value is 488.1460, but the value found is 488.1479. 1 H NMR (400MHz, DMSO-d6) 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),2.9 1(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).

[0294] Step 3:

[0295]

[0296] Dissolve (-)-monthoxyacetic acid (53.5 mg, 0.25 mmol, 1 equivalent) in DMF (5 mL), add HATU (114 mg, 0.3 mmol, 1.2 equivalent) and DIPEA (173.8 μL, 1 mmol, 4 equivalent), stir the mixture for 10 minutes, then add 1-((2-aminoethyl)sulfonyl)-N-(5-(((5-(tert-butyl))oxazol-2-yl)methyl)thio)thiazolyl-2-yl)piperidine-4-carboxamide (122 mg, 0.25 mmol, 1 equivalent), stir the mixture overnight at room temperature, then extract with EA and water, wash with NaCl brine and concentrate under vacuum to give crude product solid. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), followed by semi-preparative HPLC (flow rate, 10 mL / min; solvent A: MeOH:MeCN = 1:1; solvent B: 0.1% TFA aqueous solution; gradient: elution from 5% A to 95% A over 30 min, then elution with 95% A over 30-45 min) to obtain a white solid LL-K9-015 (102 mg, 60%). HRMS [M+H] + C 31 H 50 N5O6S3 + The calculated value is 684.2923, but the result is 684.2989. 1 HNMR(400MHz,CD3OD)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).

[0297] Example 17: Preparation of LL-K9-016

[0298]

[0299] 1-Adamantaneacetic acid (48.5 mg, 0.25 mmol, 1 equivalent) was dissolved in DMF (5 mL), and HATU (114 mg, 0.3 mmol, 1.2 equivalent) and DIPEA (173.8 μL, 1 mmol, 4 equivalent) were added. The mixture was stirred for 10 minutes, and then compound 1-((2-aminoethyl)sulfonyl)-N-(5-(((5-(tert-butyl))oxazol-2-yl)methyl)thio)thiazolyl-2-yl)piperidine-4-carboxamide (122 mg, 0.25 mmol, 1 equivalent) was added. The mixture was stirred overnight at room temperature, then extracted with EA and water, washed with NaCl brine, and concentrated under vacuum to give a crude product solid. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), and the pure fraction was concentrated to give LL-K9-016 as a white solid (107 mg, 66%). HRMS[M+H] + C 31 H 46 N5O5S3 + The calculated value is 664.2661, but the result is 664.2721. 1 H NMR (400MHz, CD3OD) 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).

[0300] Example 18: Preparation of LL-K9-017

[0301]

[0302] 1-((2-aminoethyl)sulfonyl)-N-(5-(((5-(tert-butyl))oxazol-2-yl)methyl)thio)thiazo-2-yl)piperidin-4-carboxamide (122 mg, 0.25 mmol, 1 equivalent) and 2-isocyanate adamantane (44 mg, 0.25 mmol, 1 equivalent) were dissolved in DCM (10 mL). The mixture was stirred overnight at room temperature, then extracted with DCM and water, washed with NaCl brine, and concentrated under vacuum to give a crude product solid. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), and the pure fraction was concentrated under vacuum to give LL-K9-017 as a white solid (98 mg, 59%). HRMS[M+H] + C 30 H45 N6O5S3 + The calculated value is 665.2614, but the result is 665.2670. 1 H NMR (400MHz, CD3OD) 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).

[0303] Example 19: Preparation of LL-K9-018

[0304]

[0305] 1-((2-aminoethyl)sulfonyl)-N-(5-(((5-(tert-butyl))oxazol-2-yl)methyl)thio)thiazolyl-2-yl)piperidine-4-carboxamide (122 mg, 0.25 mmol, 1 equivalent), N5-[bis[[(1,1-dimethylethoxy)carbonyl]amino]methylene]-N2-[(1,1-dimethylethoxy)carbonyl]-L-ornithine (119 mg, 0.25 mmol, 1 equivalent), and N-methylimidazolium (NMI) (60 μL) were dissolved in MeCN (10 mL) and stirred at room temperature for 10 min. Then, tetramethylchlorourea hexafluorophosphate (TCFH) (77 mg, 0.28 mmol, 1.1 equivalent) was added. The mixture was stirred at room temperature overnight, then extracted with DCM and water, washed with NaCl brine, and concentrated under vacuum to obtain the crude product. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), and the pure fraction was concentrated under vacuum to give LL-K9-018 as a white solid (156 mg, 66%). HRMS [M+H] + C 40 H 66 N9O 11 S3 + The calculated value is 944.4044, but the actual value is 944.4125. 1H NMR (400MHz, CD3OD) 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).

[0306] Example 20: Preparation of compound LL-K9-019

[0307] Step 1:

[0308]

[0309] 1-Adamantanecarboxylic acid (0.5 g, 2.8 mmol) was dissolved in DMF (10 mL), and EDCI (1.07 g, 5.5 mmol), HOBt (0.375 g, 2.8 mmol), and glycine methyl ester hydrochloride (0.35 g, 22.8 mmol) were 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 (2 × 150 mL), and the combined organic phases were washed with water (2 × 80 mL) and saturated NaCl brine (50 mL), dried (Na₂SO₄), and concentrated under vacuum. A white solid (0.59 g, 84%) was obtained, which could be used in the next step without further purification.

[0310] Step 2:

[0311]

[0312] 1-((3r,5r,7r)-adamantyl-1-carbonyl)-glycine methyl ester (0.59 g, 2.3 mmol) was dissolved in methanol (10 mL), and sodium hydroxide (188 mg, 4.6 mmol) was dissolved in water (1 mL). The sodium hydroxide aqueous solution was added to the methanol solution of 1-((3r,5r,7r)-adamantyl-1-carbonyl)-glycine methyl ester. The mixture was reacted at 70 °C for 2 hours, then cooled to room temperature, and water (100 mL) was added. The aqueous phase was adjusted to pH 1 with dilute hydrochloric acid (6 M), extracted with EA (2 × 70 mL), dried (Na₂SO₄), and concentrated under vacuum to give a white solid (0.4 g, 71%), which could be used in the next step without further purification.

[0313] Step 3:

[0314]

[0315] 1-((3r,5r,7r)-adamantyl-1-carbonyl)-glycine (59.25 mg, 0.25 mmol, 1 equivalent), HATU (68.5 mg, 0.18 mmol, 1.2 equivalent), and DIPEA (105 μL, 0.6 mmol, 4 equivalent) were dissolved in DMF (2 mL). The mixture was stirred for 10 minutes, and then SNS-032 (57 mg, 0.15 mmol, 1 equivalent) was added. The mixture was stirred overnight at room temperature and extracted with DCM and water. The organic phase was separated, washed with NaCl brine, and concentrated under vacuum to give a crude product solid. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), and the pure fraction was 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).

[0316] Example 21: Preparation of compound LL-K9-020

[0317] Step 1:

[0318]

[0319] 1-Adamantanecarboxylic acid (25 g, 138 mmol) was dissolved in methanol (150 mL), and concentrated sulfuric acid (4 mL) was added with stirring. The mixture was refluxed at 80 °C for 3 hours. The organic phase was concentrated under vacuum, and the remaining oily substance was diluted with water (250 mL). It was extracted with EA (2 × 250 mL), and the organic phase was washed with water (2 × 150 mL) and NaCl brine (100 mL). The mixture was dried (Na2SO4), concentrated under vacuum, and the crude product was passed through a silica gel column (mobile phase 2% (EA:PE)) to give methyl 1-adamantanecarboxylate 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).

[0320] Step 2:

[0321]

[0322] Under a nitrogen atmosphere, lithium aluminum hydride (LAH) (6.41 g, 168 mmol) was suspended in THF (250 mL) and cooled to 0 °C. Separately, methyl 1-adamantane carboxylate (21.8 g, 112 mmol) was dissolved in THF (50 mL) and slowly added dropwise to the reaction mixture at 0 °C. After the addition was complete, the mixture was stirred at room temperature for 1 hour, and then EA (100 mL) and water (20 mL) were added dropwise at 0 °C. All liquids were filtered through diatomaceous earth, solvent removed under vacuum, and dried (using Na₂SO₄) to obtain a colorless oily substance (17 g, 91%). 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).

[0323] Step 3:

[0324]

[0325] Pyridine chlorochromate (PCC) (23.17 g, 107.5 mmol) was suspended in DCM (150 mL), and DCM (100 mL) containing 1-adamantyl methanol (10.5 g, 63.2 mmol) was added with stirring at room temperature. After 1 hour, the mixture was diluted with diisopropyl ether (750 mL) and filtered through diatomaceous earth. The filtrate was washed with 1N NaOH (250 mL) and water (2 × 150 mL), and the organic phase was dried over Na₂SO₄ to give a white solid (8.94 g, 86%). 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).

[0326] Step 4:

[0327]

[0328] SNS-032 (57 mg, 0.15 mmol, 1 equivalent) was dissolved in DCM (2 mL), and triethylamine (41.6 μL, 0.3 mmol, 2 equivalents) and the compound adamantane formaldehyde (61.5 mg, 0.375 mmol, 2.5 equivalents) were added. The mixture was stirred for 45 min, and then sodium triacetoxyborohydride (32.0 mg, 0.3 mmol, 2 equivalents) was added. The mixture was stirred overnight at room temperature, then extracted with DCM and water. The organic phase was separated, washed with NaCl brine, and concentrated under vacuum to obtain the crude product. The crude product was purified by rapid silica gel chromatography (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).

[0329] Example 22: Preparation of compound LL-K9-021

[0330] Step 1:

[0331]

[0332] Sodium hydride (2.38 g, 99 mmol) was suspended in THF (200 mL). Trimethyl phosphoroacetate (TMPA) (12.89 g, 70.8 mmol) was slowly added at 0 °C. After stirring for 15 minutes, a THF solution of 1-adamantaneformaldehyde was added dropwise at 0 °C. The mixture was then stirred at 5 °C for 1.5 hours. The reaction was quenched with water (50 mL), and THF was removed under vacuum. The mixture was then added to water (150 mL) and extracted with EA (2 × 150 mL). The organic phase was desolventized under vacuum and dried to give a white solid mixture (10.75 g, 91%), which could be used for the next step without further purification.

[0333] Step 2:

[0334]

[0335] 1-Adamantyl allyl methyl ester (12.75 g, 57.9 mmol) was dissolved in methanol (150 mL), and sodium hydroxide (6.95 g, 173 mmol) was dissolved in water (10 mL). The sodium hydroxide solution was added to the methanol solution of 1-adamantyl allyl methyl ester, and the mixture was stirred at 70 °C for 2 hours. The solvent was removed from the mixture under vacuum, and water (150 mL) was added. The mixture was extracted with EA (150 mL). The organic phase was adjusted to pH 2 with hydrochloric acid, cooled to 5 °C, and allowed to stand for 30 minutes. The precipitate was filtered and dried under vacuum to give a white solid (7.4 g, 62%). 1 HNMR(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).

[0336] Step 3:

[0337]

[0338] 1-Adamantylallyl acid (7.4 g, 33.9 mmol) was dissolved in 200 mL of methanol, and 10% Pd / C (10%, 1.48 g) was added. The mixture was bubbled under nitrogen for 15 minutes, and then reacted at room temperature for 1 day under a hydrogen atmosphere. The reaction mixture was filtered through diatomaceous earth and the solvent was removed under vacuum to give a white solid (7.31 g, 97%), which could be used for the next step without further purification.

[0339] Step 4:

[0340]

[0341] 1-Adamantane propionic acid (31 mg, 0.25 mmol, 1 equivalent), HATU (68.5 mg, 0.18 mmol, 1.2 equivalent), and DIPEA (105 μL, 0.6 mmol, 4 equivalent) were dissolved in DMF (2 mL). The mixture was stirred for 10 minutes, and then SNS-032 (57 mg, 0.15 mmol, 1 equivalent) was added. The mixture was stirred overnight at room temperature and extracted with DCM and water. The organic phase was separated, washed with NaCl brine, and concentrated under vacuum to give a crude product solid. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), and the purified fraction was concentrated to give LL-K9-021 (50.44 mg, 59%) as a white solid. 1H 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).

[0342] Example 23: Preparation of compound LL-K9-022

[0343] Step 1:

[0344]

[0345] 1-Adamantylpropionic acid (7.2 g, 34.6 mmol) was dissolved in 150 mL of methanol, and concentrated sulfuric acid (2 mL) was added with stirring. The mixture was reacted at 80 °C for 3 hours. The solvent was removed from the reactants under vacuum, and the residue was diluted with water (250 mL) and extracted with EA (2 × 250 mL). The organic phase was washed with saturated NaCl brine (1 × 100 mL) and dried (Na2SO4). The solvent was removed under vacuum to give a white solid (7.34 g, 95%), which could be used in the next step without further purification.

[0346] Step 2:

[0347]

[0348] Lithium aluminum hydride (LAH, 2.51 g, 66 mmol) was dissolved in THF (100 mL). A THF solution containing methyl 1-adamantylpropionate (7.34 g, 33 mmol) was added dropwise at 0 °C in 50 mL of THF. After stirring the mixture at room temperature for 1 hour, the reaction mixture was cooled to 0 °C, and EA (20 mL) and water (5 mL) were slowly added dropwise. The mixture was filtered through diatomaceous earth, dried (Na₂SO₄), and concentrated under vacuum. The crude product was passed through a silica gel column (EA:PE = 5%) to obtain an oily substance (5.2 g, 81%), which could be used in the next step without further purification.

[0349] Step 3:

[0350]

[0351] PCC (7.55 g, 36 mmol) was suspended in DCM (50 mL), and DCM (50 mL) containing 1-adamantylpropanol (3.5 g, 18 mmol) was added. The mixture was stirred at room temperature for 1 hour, then diisopropyl ether (150 mL) was added, and the mixture was filtered through diatomaceous earth. It was then washed with 1N sodium hydroxide (2 × 100 mL) and water (2 × 100 mL), dried (Na₂SO₄), and concentrated under vacuum to give a white oily substance (3.13 g, 90%), which could be used in the next step without further purification.

[0352] Step 4:

[0353]

[0354] SNS-032 (57 mg, 0.15 mmol, 1 equivalent) was dissolved in DCM (2 mL), and triethylamine (41.6 μL, 0.3 mmol, 2 equivalents) and the compound adamantanepropionaldehyde (72.0 mg, 0.375 mmol, 2.5 equivalents) were added. The mixture was stirred for 45 min, and then sodium triacetoxyborohydride (32 mg, 0.3 mmol, 2 equivalents) was added. The mixture was stirred overnight at room temperature, then extracted with DCM and water. The organic phase was separated, washed with NaCl brine, and concentrated under vacuum to obtain the crude product. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1) to give LL-K9-022 (37.6 mg, 45%) as a white solid. 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).

[0355] Example 24: Preparation of compound LL-K9-023

[0356] Step 1:

[0357]

[0358] Under a nitrogen atmosphere, 1-adamantaneacetic acid (2.00 g, 10.3 mmol, 1 equivalent) was dissolved in THF (34 mL), cooled to 0 °C, and LAH (1 M THF solution, 15.5 mL, 15.4 mmol, 1.5 equivalent) was slowly added dropwise. After the addition was complete, the mixture was brought to room temperature and reacted for 18 hours. The reaction was then quenched with water (50 mL) at 0 °C, and the mixture was extracted with EA (2 × 50 mL). The combined organic phases were washed with 3N hydrochloric acid (20 mL) and saturated NaCl brine, dried (Na₂SO₄), and concentrated under vacuum 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).

[0359] Step 2:

[0360]

[0361] Oxaloyl chloride (1 mL, 11.7 mmol, 2 equivalents) was dissolved in DCM (80 mL) and cooled to -78 °C. DMSO (1.65 mL, 23.3 mmol, 2.4 equivalents) was added dropwise, and the mixture was stirred for 15 minutes. Then, 1-adamantaneethanol (1.75 g, 9.71 mmol, 1 equivalent) was added dropwise to DCM (17 mL). After the addition was complete, the mixture was stirred at the same temperature for 30 minutes. Triethylamine (8.2 mL, 58.2 mmol, 6 equivalents) was slowly added dropwise over the next 20 minutes. The reaction mixture was then slowly heated to room temperature and stirred at room temperature for 40 minutes. Saturated sodium bicarbonate (50 mL) was added and the mixture was stirred for 30 minutes. The mixture was then extracted with DCM (3 × 50 mL). The combined organic phases were washed with water (50 mL) and saturated NaCl brine (50 mL), dried (Na2SO4), and concentrated under vacuum. The solution was then passed through a silica gel column (EA:PE = 5%). A colorless oily substance was obtained (1.43 g, 8.02 mmol, 83%). 1 H NMR (600MHz, Chloroform-D) δ9.87 (t, J = 3.3 Hz, 1H), 2.12 (d, J = 3.3 Hz, 2H), 1.98 (s, 3H), 1.72 (d, J = 12.1 Hz, 3H), 1.65 (dd, J = 13.4, 2.0 Hz, 9H).

[0362] Step 3:

[0363]

[0364] SNS-032 (57 mg, 0.15 mmol, 1 equivalent) was dissolved in DCM (2 mL), and triethylamine (41.6 μL, 0.3 mmol, 2 equivalents) and the compound adamantaneacetaldehyde (66.75 mg, 0.375 mmol, 2.5 equivalents) were added. The mixture was stirred for 45 min, and then sodium triacetoxyborohydride (32 mg, 0.3 mmol, 2 equivalents) was added. The mixture was stirred overnight at room temperature. Then, it was extracted with DCM and water, the organic phase was separated, washed with NaCl brine and concentrated under vacuum to obtain the crude product. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1) to give LL-K9-023 (36.5 mg, 60%) as a white solid. 1 HNMR(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).

[0365] Example 25: Preparation of compound LL-K9-024

[0366] Step 1:

[0367]

[0368] 3,3,3-Triphenylacetic acid (2.88 g, 10 mmol, 1 equivalent), HATU (4.56 g, 12 mmol, 1.2 equivalent), and DIPEA (6.95 mL, 40 mmol, 4 equivalent) were dissolved in DMF (20 mL). The mixture was stirred for 10 minutes, and then 2,2-diethoxyethyl-1-amine (1.33 g, 10 mmol, 1 equivalent) was added. The mixture was stirred overnight at room temperature and then extracted with EA and water. The organic phase was separated, washed with NaCl brine, and concentrated under vacuum to give a crude product solid. The crude product was purified by rapid silica gel chromatography (PE:EA = 2:1) and concentrated under vacuum to give N-(2,2-diethoxyethyl)-2,2,2-triphenylacetamide as a white solid (2.86 g, 71%). 1H NMR (400MHz, CDCl3) 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).

[0369] Step 2:

[0370]

[0371] N-(2,2-diethoxyethyl)-2,2,2-triphenylacetamide (302 mg, 0.75 mmol, 1 equivalent) was dissolved in MeCN (1 mL), and HCl (1 N, 1 mL) was added. The mixture was stirred overnight at room temperature, extracted with DCM and water, and the organic phase was washed with water, then washed with saturated NaCl brine, dried over Na2SO4, and concentrated under vacuum. N-(2-oxyethyl)-2,2,2-triphenylacetamide can be used in the next step without further purification.

[0372] Step 3:

[0373]

[0374] SNS-032 (57 mg, 0.15 mmol, 1 equivalent) was dissolved in DCM (2 mL), and triethylamine (41.6 μL, 0.3 mmol, 2 equivalents) and compound N-(2,2-diethoxyethyl)-2,2,2-triphenylacetamide (123.0 mg, 0.375 mmol, 2.5 equivalents) were added. The mixture was stirred for 45 min, and then sodium triacetoxyborohydride (32 mg, 0.3 mmol, 2 equivalents) was added. The mixture was stirred overnight at room temperature. Then, it was extracted with DCM and water, the organic phase was separated, washed with NaCl brine and concentrated under vacuum to give the crude product. The crude product was purified by rapid silica gel chromatography (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),2.7 3(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).

[0375] Example 26: Preparation of compound LL-K9-025

[0376] Step 1:

[0377]

[0378] 3,3,3-Triphenylpropionic acid (3.02 g, 10 mmol, 1 equivalent), HATU (4.56 g, 12 mmol, 1.2 equivalent), and DIPEA (6.95 mL, 40 mmol, 4 equivalent) were dissolved in DMF (20 mL). The mixture was stirred for 10 minutes, and then 2,2-diethoxyethyl-1-amine (1.33 g, 10 mmol, 1 equivalent) was added. The mixture was stirred overnight at room temperature and then extracted with EA and water. The organic phase was separated, washed with NaCl brine, and concentrated under vacuum to give a crude product solid. The crude product was purified by rapid silica gel chromatography (PE:EA = 2:1) and concentrated under vacuum to give N-(2,2-diethoxyethyl)-3,3,3-triphenylpropionamide as a white solid (3.54 g, 85%). 1 H NMR (400MHz, CDCl3) 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).

[0379] Step 2:

[0380]

[0381] N-(2,2-diethoxyethyl)-2,2,2-triphenylacetamide (312 mg, 0.75 mmol, 1 equivalent) was dissolved in MeCN (1 mL), and HCl (1 N, 1 mL) was added. The mixture was stirred overnight at room temperature, extracted with DCM and water, and the organic phase was washed with water and saturated NaCl brine, dried over Na2SO4, and concentrated under vacuum to obtain N-(2-oxyethyl)-3,3,4-triphenylpropionamide, which can be used in the next step without further purification.

[0382] Step 3:

[0383]

[0384] SNS-032 (57 mg, 0.15 mmol, 1 equivalent) was dissolved in DCM (2 mL), and triethylamine (41.6 μL, 0.3 mmol, 2 equivalents) and compound N-(2-oxoethyl)-3,3,3-triphenylpropionamide (257.0 mg, 0.375 mmol, 2.5 equivalents) were added. The mixture was stirred for 45 min, and then sodium triacetoxyborohydride (32.0 mg, 0.3 mmol, 2 equivalents) was added. The mixture was stirred overnight at room temperature, then extracted with DCM and water. The organic phase was separated, washed with NaCl brine, and concentrated under vacuum to obtain the crude product. The crude product was purified by rapid silica gel chromatography (DCM:MeOH = 20:1), and then purified by semi-preparative HPLC (flow rate, 10 mL / min; solvent A: MeOH:MeCN = 1:1; solvent B: 0.1% TFA aqueous solution; gradient: elution from 5% A to 95% A over 30 min, followed by elution with 95% A over 30-45 min) to obtain a white solid LL-K9-025 (17 mg, 16%). 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).

[0385] Example 27: Determination of the activity of the compounds of the present invention in degrading the CDK9-cyclin T1 complex.

[0386] The ability of each compound in the above examples to degrade CDK9-cyclin T1 in vitro was determined. The selected cell line was prostate cancer cell line 22RV1. The specific operation was as follows: Cells were cultured in RPMI 1640 medium (Gibco, purchased from Life Technologies, 22400-089), with 10% fetal bovine serum (Gibco, purchased from Life Technologies, 10099-141) and 1% antibiotics (penicillin and streptomycin, purchased from Life Technologies, 10378016) added to the medium. Before the test, cells in the logarithmic growth phase were selected and cultured at a density of 5 × 10⁶ cells / year. 5Cells / well were seeded in 12-well plates. After 24 hours, the compounds in each example were diluted to 3 μM, 1 μM, and 0.3 μM with culture medium, and the culture medium for each well was replaced with culture medium containing the compounds of the examples at the above concentrations. After 24 hours of treatment, the culture medium was discarded, and the cells were washed with pre-cooled PBS. After aspirating the liquid, SDS cell lysis buffer was added, and the cells were incubated on ice for 15 minutes to lyse. The samples were then heated on a 99°C metal bath heater for 10 minutes. The lysate was separated by SDS-PAGE gel electrophoresis, and the total protein was transferred to an NC membrane. After blocking with TBST buffer containing 5% skim milk for one hour, the target protein was recognized using antibodies against CDK9 (Cell Signaling Technology, catalog number: 2316) and cyclin T1 (Abcam, catalog number: ab184703), and the internal control GAPDH was recognized using an antibody against GAPDH (Cell Signaling Technology, catalog number: 5174). After incubating the primary antibody with the membrane overnight, the membrane was washed with TBST buffer to remove residual primary antibody, incubating for 10 minutes on a shaker for a total of 3 washes. After incubating the membrane with the secondary antibody for 1 hour, the membrane was washed again with TBST buffer to remove residual primary antibody, incubating for 10 minutes on a shaker for a total of 3 washes. The developing solution was SuperSignal WestDura (purchased from ThermoScientific, catalog number: 34076), and the developing system was a GE ImageQuant LAS 4000 system. The specific degradation activities of the above compounds are as follows: Figure 1 As shown.

[0387] The results showed that the compounds disclosed in this invention can degrade CDK9 and cyclin T1 proteins, indicating that the degradation of the CDK9-cyclin T1 complex can be achieved, especially LL-K9-015, which has strong degradative activity. Further analysis of the degradative activity of compound LL-K9-015 against CDK9 and cyclin T1 in the concentration range of 12 μM-0 μM (2-fold dilution, starting concentration 12 μM, 9 concentration points) was performed. ImageJ was used to analyze the grayscale of the protein immunoblot, and GraphPad Prism9 was used to fit the concentration-relative protein abundance curve to obtain the DC values ​​of LL-K9-015 against CDK9 and cyclin T1. 50 The values ​​are 0.662 μM and 0.589 μM, respectively. Figure 2 ).

[0388] Example 28: Determination of Degradation Kinetics

[0389] In this embodiment, the degradation kinetics of compound LL-K9-015 in 22RV1 cells were determined using DIA-MS-based proteomics and Western blotting.

[0390] The procedure for determining the degradation kinetics of LL-K9-015 using DIA-MS proteomics is as follows: 22RV1 cells were injected at 1.2 × 10⁻⁶ cells per cell line. 6Cells were seeded at a density of 1 / ml in 6-well plates. After cell attachment, LL-K9-015 was diluted in the culture medium to a final concentration of 3 μM, and SNS032, as a control inhibitor, was also diluted to 3 μM. Culture medium containing the same concentration of DMSO served as a blank control. All compound groups were administered simultaneously, with five incubation times set for each group: 3 hours, 9 hours, 12 hours, 24 hours, and 36 hours. Three replicates were prepared for each group. After reaching the incubation time, each sample was washed three times with pre-cooled PBS buffer, and then 200 μL of cell lysis buffer (4% SDS, 100 mM Tris-HCl, pH 7.6, 0.1 M DTT) was added. Cells were scraped off using a disposable cell scraper and placed in EP tubes. The tubes were heated at 100°C for 10 minutes, followed by sonication for 30 seconds. The supernatant was collected, and protein concentration was determined using the tryptophan method. SDS-PAGE electrophoresis was used to assess the quality of the cell samples. After passing quality checks, the samples were digested using the FASP method. The obtained peptides were desalted using a C18 membrane, lyophilized, and stored at -80°C for later use. The dried peptide sample was dissolved in 0.1% formic acid solution, quantified using nanodrop, and 1 μg of peptide was taken for separation using EASY-nLC 1000 nanoflow ultra-high performance liquid chromatography at a flow rate of 300 nmol / min. The chromatographic column was a self-made nanoflow C18 column (3 μm, 120A, 75 μm * 15 cm). Mobile phase A was 0.1% formic acid-water solution, and mobile phase B was 0.1% formic acid-acetonitrile solution. The analytical gradient was: 0–72 min, 3%–27% B; 72–82 min, 27%–35% B; 82–84 min, 35%–100% B; 84–90 min, 100% B. After HPLC separation, the sample was analyzed by Q Exactive HF mass spectrometry. The full MS-DIA method had a first-level resolution of 120K, a scan range of m / z 350–1500, an AGC of 3e6, and a maximum ion implantation time of 100 ms. The fragmentation energy of the precursor ion was 27%. The second-level scan had a resolution of 30K, an AGC of 5e5, and a maximum ion implantation time of 50 ms. Data were obtained from the Uniprot human protein database, and the raw DIA data was searched using the built-in parameters of DIA-NN 1.7.16 software. Degradation curves of key proteins were plotted using GraphPad Prism 9.3. The results showed that the compound LL-K9-015 of this invention could simultaneously degrade CDK9 and cyclin T1, while the prototype compound SNS032 did not have the ability to degrade CDK9 and cyclin T1. Figure 3 ).

[0391] The procedure for determining the degradation kinetics of LL-K9-015 based on Western blotting is as follows: 22RV1 cells were injected with 0.5 × 10⁻⁶ cells. 6Cells were seeded at a density of 100 cells / well in 12-well plates. After cell adhesion, cells were treated with medium containing 3 μM LL-K9-015 for 0, 1, 3, 5, 7, 9, 11, 16, 24, 36, and 48 hours. Cells were then lysed with SDS and denatured at 99°C for 10 minutes. Western blotting was subsequently performed to detect the abundance of the target proteins. The results showed that the compound LL-K9-015 of this invention can achieve simultaneous degradation of CDK9 and cyclin T1. Degradation of CDK9 and cyclin T1 began to appear after 9-11 hours, and both proteins were significantly degraded after 24 hours. This indicates that LL-K9-015 induces the degradation of the CDK9-cyclin T1 complex. Figure 4 ).

[0392] Example 29: Evaluation of degradation selectivity within the CDK and cyclin families

[0393] In this embodiment, human prostate cancer cells 22RV1 were administered at a concentration of 0.5 × 10⁻⁶. 6 Cells were seeded at a density of 100 cells / well in 12-well plates. After cell attachment, the cell growth medium was replaced with medium containing 6 μM-0.38 μM LL-K9-015, and the cells were treated for 24 hours. Cells were then lysed with SDS and denatured at 99°C for 10 minutes. Western blotting was subsequently performed to detect the abundance of CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK9, cyclin E1, cyclin T1, cyclin H, and cyclin T2. ​​The results showed that LL-K9-015 selectively degrades the CDK9-cyclin T1 complex without significantly affecting other CDK family proteins. Although the prototype compound SNS032 can also inhibit CDK2 and CDK7, LL-K9-015 only slightly downregulated the CDK2 regulatory subunit cyclin E1 at high concentrations, had no significant effect on the CDK7 regulatory subunit cyclin H, and only partially degraded the CDK9 minor binding subunit cyclin T2 at high concentrations. Figure 5 A- Figure 5 B).

[0394] Example 30: Degradation-selective proteomic assessment

[0395] This example evaluates the effect of LL-K9-015 on the whole proteome using the DIA-MS proteomics method described in Example 28. In Example 28, after LL-K9-015 treatment of cells for 24 hours, the CDK9-cyclin T1 complex was significantly degraded, and 24 hours was selected as the time point for analyzing proteomic selectivity. Differentially expressed proteins in the LL-K9-015 and SNS032 groups were screened using R 4.1.1 with a fold change ≥1.5 or 0.67 and a P-value ≤0.05. Volcano plots showed that SNS032 had a slight upregulation of cyclin T1 and a significant downregulation of cyclin D1, but no effect on other CDK and cyclin proteins; LL-K9-015 significantly downregulated CDK9 and cyclin T1. Since CDK9 regulates the transcription of cycle-related cyclin proteins, cycle-related cyclin A2, cyclin B1, and cyclin D1 were also significantly downregulated. Other binding targets of the prototype compound SNS032 of LL-K9-015 were not identified as differentially expressed proteins, indicating that LL-K9-015 selectively degrades the CDK9-cyclin T1 complex. Figure 5 C- Figure 5 D).

[0396] Example 31: Determination of the in vitro antitumor activity of some compounds

[0397] This embodiment uses the human prostate cancer cell line 22RV1 as a model to investigate the inhibitory activity of the compound on cell proliferation. When the cells are in the exponential growth phase, they are harvested, counted, and seeded. In 96-well plates, 100 μM of the compound is added to each well to achieve a cell count of 5000 cells per well. A blank control is added to 100 μM of complete culture medium. Simultaneously, the compound is administered at a concentration gradient starting at 100 μM and then serially diluted three-fold to a total of nine concentration points. Changes in cell proliferation after 5 days of treatment are detected using the CellTiter-Glo method (Promega, G7572). The method is as follows: 100 μM of the prepared CellTiter-Glo is added to a 96-well plate, the plate is shaken on a shaker for 10 min, and then incubated at room temperature for 5 min. The treated cells were transferred at 60 μL / well to white 384-well plates (OptiPlate-384, PerkinElmer, 6007299). The cryoluminescence signal was detected using an EnVision microplate reader (PerkinElmer) at wavelengths of 400–700 nm. Data were analyzed using GraphPad Prism 9.3 software, with cell viability on the ordinate and drug concentration on the x-axis. The IC50 of the compound for inhibiting cell proliferation in each cell line was calculated. 50 .

[0398] The cell viability (%) is calculated as follows:

[0399] Survival rate (%) = (OD of drug delivery well - OD of blank well) / (OD of control well - OD of blank well) × 100%

[0400] Table 1 shows the antiproliferative activity data of some of the compounds in the examples against prostate cancer cells 22RV1.

[0401] 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

[0402] Table 1 shows the in vitro antiproliferative activity of compounds with superior degradation activity. The results indicate that several compounds exhibit stronger antiproliferative activity compared to the prototype compound SNS032, particularly LL-K9-015 and LL-K9-003. Therefore, the compounds of this invention can be used in the preparation of anticancer drugs. The compounds provided by this invention have a significant ability to degrade the CDK9-cyclin T1 complex. Since the CDK9-cyclin T1 complex plays a crucial role in the growth and proliferation of tumor cells, the compounds provided by this invention can be used in drugs for treating diseases related to CDK9-cyclin T1, especially in the treatment of tumors.

[0403] Example 32: Cloning Experiment

[0404] In this embodiment, 22RV1 human prostate cancer cell line in logarithmic growth phase was seeded at 800 cells / well into 12-well clear cell culture plates. After 24 hours, the culture medium in each well was replaced with medium containing 400 nM, 200 nM, or 100 nM LL-K9-015, or the same concentration of SNS032. Medium containing 0.1% (v / v) DMSO was used as a control. The cells were cultured for another 14 days, during which the medium containing the corresponding compound or 0.1% (v / v) DMSO was replaced with freshly prepared medium containing the compound or DMSO every 2 days. After 14 days of culture, the culture medium was discarded, and the cells were washed twice with PBS buffer. The cells were then fixed with 4% paraformaldehyde for 10 minutes, washed twice with PBS, and then stained with crystal violet staining solution (Beyotime, catalog number: C0121) for 20 minutes. After washing away excess staining solution with PBS, the cells were air-dried at room temperature and photographed. The results showed that LL-K9-015 dose-dependently inhibited colony formation in 22RV1 cells, and was significantly stronger than SNS032 (e.g., Figure 6 B). This indicates that degrading CDK9-cyclin T1 has a stronger ability to inhibit tumor cell proliferation.

[0405] Example 33: Cell cycle and apoptosis experiments

[0406] In this embodiment, human prostate cancer cell line 22RV1 cells in the logarithmic growth phase were divided into 6×10 5 Cells were seeded per well into 6-well clear cell culture plates. After 24 hours, 1 μM or 0.3 μM of the compound was added, with 0.1% (v / v) DMSO as a control, and the cells were cultured for another 24 hours in a cell culture incubator at 37°C and 5% CO2. Cells were collected by centrifugation at 1000 rpm and washed once with PBS to remove the culture medium. The cells were then resuspended in pre-chilled PBS containing 70% (v / v) ethanol and fixed overnight at 4°C. After centrifugation to remove the ethanol, the cells were washed once with FACS buffer and resuspended. Finally, the recommended concentration of RNase and propidium iodide (PI) (BD Pharmingen) was added, and the cells were stained at room temperature in the dark for 20 minutes. The cells were then analyzed using a flow cytometer (BioRad ZE5). Propidium Iodide dye can bind to both RNA and DNA. By degrading RNA with RNase, only DNA can be detected. The amount of DNA in cells during G1 phase is half that in G2 phase, and the amount of DNA in S phase is between the two. Therefore, the cell cycle state can be determined by detecting the fluorescence signal value of the dye.

[0407] This embodiment utilizes an apoptosis assay to detect the effects of compounds LL-K9-015 and SNS032 on apoptosis in 22RV1 cells. Firstly, 22RV1 cells were cultured at 6 × 10⁶ cells per cell line. 5 Cells were seeded at a density of 1000 cells / well in 12-well plates (Corning). Cell samples were collected 24 hours after compound treatment. Samples were then treated with the Annexin V-FITC apoptosis detection kit (Nanjing Novizan Biotechnology Co., Ltd., A211-01), and apoptosis status was detected using a BioRad ZE5 flow cytometer. Data analysis was performed using Modfit and FlowJo software (version 7.6.1). Results showed that after 24 hours of treatment with LL-K9-015 and SNS032, some 22RV1 cells were arrested in the G1 / S phase, and some were arrested in the sub-G1 phase (apoptotic phase), with LL-K9-015 exhibiting stronger arrest ability. Simultaneously, flow cytometry analysis of apoptosis levels showed that LL-K9-015 induced more apoptosis than SNS032 (e.g., ...). Figure 7 ).

[0408] Example 34: Immunoblotting assay of CDK9-related pathway and target proteins

[0409] In this embodiment, human prostate cancer cells 22RV1 were administered at a concentration of 0.5 × 10⁻⁶. 6Cells were seeded at a density of 100 cells / well in 12-well plates. After cell attachment, the cell growth medium was replaced with medium containing 3 μM, 1 μM, or 0.3 μM LL-K9-015 or SNS032. Cells were treated for 24 hours, then lysed with SDS and denatured at 99°C for 10 minutes. Western blotting was then performed to detect the abundance of MCL1, cMyc, AR, ARv7, KLK3, NKX3-1, Phospho-Rpb1 CTD (Ser5), Phospho-Rpb1 CTD (Ser2), and RNA pol II antibody. The results showed that LL-K9-015 significantly inhibited RNA pol II phosphorylation and significantly downregulated the protein levels of MCL1, cMyc, AR, ARv7, KLK3, and NKX3-1, with a more thorough inhibitory effect than SNS032 (e.g., ...). Figure 8 ).

[0410] Example 35: Transcriptome Analysis

[0411] In this embodiment, prostate cancer cells 22RV1 were treated with 2 μM LL-K9-015, 2 μM Thal-SNS032, 2 μM MSNS032, and 0.1% DMSO. Cell samples were collected after 24 hours, and total RNA was extracted from the samples using the phenol / chloroform method. Before library construction, purity was tested using a NanoDrop 2000 & 8000 micro spectrophotometer, concentration was determined using an Agilent 2100 Bioanalyzer, and integrity was tested using an Agilent RNA 6000 Nano Kit. Only samples that passed the purity and integrity tests were used for the next step of library construction and analysis. After library construction, the samples were sequenced using an Illumina NovaSeq 6000 with a read length of PE150. The cleaned data were aligned using STAR software with the UCSC hg19 genome as the reference genome. The aligned BAM file was then submitted to featureCounts software for quantitative analysis. Differential gene analysis was performed using the R package Deseq2, with a threshold of logFC > 1.5 and a corrected p-value < 0.05. Cluster analysis was performed using the R package Clusterfile, with the p-value for clustering corrected using the FDR method, and a significance threshold of a corrected p-value < 0.05. The results showed that the transcriptomic changes induced by LL-K9-015 were significantly different from those induced by SNS032 and Thal-SNS032. LL-K9-015 inhibited MYC and AR-mediated tumor-promoting abnormal transcription and downregulated MYC and AR target genes (such as...). Figure 9 ).

[0412] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An oxazole compound represented by general formula I, its pharmaceutically acceptable salt or racemate: (I) in, A is selected from -C(=O)(CH2) x -, -C(=O)(CH2) x NHC(=O)-, -(CH2) x NHC(=O)(CH2) y -, -(CH2) x -, -(CH2CH2O) x (CH2) y NHC(=O)CH2-, -(CH2) x -B-C(=O)(CH2) y -, -S(=O)2(CH2) x NHC(=O)(CH2) y -, -S(=O)2(CH2) x NHC(=O)NH-; x is independently selected from 0, 1, 2, 3, 4, 5, 6; y is independently selected from 0, 1, and 2; B is , where z is independently selected from 1, 2, and 3; R is selected from adamantyl, unsubstituted or substituted with one or both of methyl and isopropyl cyclohexyloxy, triphenylmethyl, 2,2,2-triphenylethyl. .

2. The oxazole compound, its pharmaceutically acceptable salt or racemic mixture according to claim 1, characterized in that, A is selected from -C(=O)CH2-, -C(=O)(CH2)2-, -C(=O)CH2NHC(=O)-, -(CH2)2NHC(=O)-, -(CH2)4NHC(=O)CH2-, -(CH2)6NHC(=O)-CH2-, -(CH2)2NHC(=O)CH2-, -CH2-, -(CH2)2-, -(CH2)3-, - CH2CH2O-(CH2)2NHC(=O)CH2-, -(CH2CH2O)2(CH2)2NHC(=O)CH2-, -CH2-BC(=O)CH2-, -S(=O)2(CH2)2NHC(=O)CH2-, -S(=O)2(CH2)2NHC(=O)NH-, -S(=O)2(CH2)2NHC(=O)-; and / or B is .

3. The oxazole compound, its pharmaceutically acceptable salt or racemic mixture according to claim 1, characterized in that, A is selected from -C(=O)CH2-, -C(=O)(CH2)2-, -C(=O)CH2NHC(=O)-, -(CH2)2NHC(=O)-, -(CH2)4NH C(=O)CH2-, -(CH2)6NHC(=O)CH2-, -(CH2)2NHC(=O)CH2-, -CH2-, -(CH2)2-, -(CH2)3-, -CH2CH2O-(CH2)2NHC(=O)CH2-, -(CH2CH2O)2(CH2)2NHC(=O)CH2-, -CH2-BC(=O)CH2- , -S(=O)2(CH2)2NHC(=O)CH2-, -S(=O)2(CH2)2NHC(=O)NH-, -S(=O)2(CH2)2NHC(=O)-; B is , where z is independently selected from 1, 2, and 3.

4. The oxazole compound, its pharmaceutically acceptable salt or racemic mixture according to claim 1, characterized in that, A is selected from -C(=O)CH2-, -C(=O)(CH2)2-, -C(=O)CH2NHC(=O)-, -(CH2)2NHC(=O)-, -(CH2)4NH C(=O)CH2-, -(CH2)6NHC(=O)CH2-, -(CH2)2NHC(=O)CH2-, -CH2-, -(CH2)2-, -(CH2)3-, -CH2CH2O-(CH2)2NHC(=O)CH2-, -(CH2CH2O)2(CH2)2NHC(=O)CH2-, -CH2-BC(=O)CH2- , -S(=O)2(CH2)2NHC(=O)CH2-, -S(=O)2(CH2)2NHC(=O)NH-, -S(=O)2(CH2)2NHC(=O)-; B is ; R is selected from adamantyl alkyl group. Triphenylmethyl, 2,2,2-triphenylethyl .

5. The oxazole compound, its pharmaceutically acceptable salt or racemic mixture according to any one of claims 1-4, characterized in that, The oxazole compounds are selected from the following structures: Where x, y, and R are as defined in the corresponding claims, and n is 1, 2, 3, or 4.

6. The oxazole compound, its pharmaceutically acceptable salt or racemic mixture according to claim 1, characterized in that, The oxazole compounds are selected from the following structures: 。 7. A method for preparing the oxazole compound according to any one of claims 1-6, characterized in that, The method is selected from one of the following methods: Synthesis Method 1: Wherein, R is as defined in the respective claims; Step 1-1: Compounds 1A and 1B are activated by HATU and DIPEA in dimethylformamide solution at room temperature, and undergo an amidation reaction to generate compound 1C; or, Wherein, R is as defined in the respective claims; Steps 1-2: Compound 1D reacts with trimethyl phosphoroacetate in tetrahydrofuran solution at 0-5°C in the presence of sodium hydride to generate compound 1E; Steps 1-3: Compound 1E is dissolved in methanol, and an aqueous sodium hydroxide solution is added. The mixture is then hydrolyzed at 70°C to generate compound 1F. Steps 1-4: Compound 1F was hydrogenated in methanol solution by a 10% Pd / C catalyst to obtain compound 1G; Steps 1-5: Compound 1G and Compound 1A are reacted in dimethylformamide solution at room temperature by activation with HATU and DIPEA to generate Compound 1H; Synthesis Method Two: Wherein, R is as defined in the respective claims; Step 2-1: Compound 2A, glycine methyl ester hydrochloride, and triethylamine react in dimethylformamide solution at room temperature with EDCI and HOBt to generate compound 2B; Step 2-2: Compound 2B is dissolved in methanol, and an aqueous solution of sodium hydroxide is added. The solution is then hydrolyzed at 70°C to generate compound 2C. Steps 2-3: Compound 2C and compound 1A are reacted in dimethylformamide solution at room temperature by activation with HATU and DIPEA to generate compound 2D; Synthesis Method 3: Wherein, R is as defined in the respective claims; Step 3-1: Compound 3A reacts with 2,2-diethoxyethyl-1-amine in a dimethylformamide solution at room temperature, and is activated by HATU and DIPEA to undergo an amidation reaction to generate compound 3B; Step 3-2: Compound 3B is dissolved in acetonitrile, 1N hydrochloric acid solution is added, and the reaction is carried out at room temperature to obtain compound 3C; Step 3-3: Compound 3C reacts with compound 1A and triethylamine in a dichloromethane solution in the presence of sodium triacetoxyborohydride at room temperature to generate compound 3D; Synthesis Method Four: Wherein, R is as defined in the corresponding claims; n is taken from 0, 1, 2, 3, 4, 5; Step 4-1: Compound 4A is reduced to compound 4B by lithium aluminum hydride in tetrahydrofuran solution at 0°C; Step 4-2: Oxaloyl chloride was dissolved in tetrahydrofuran solution, cooled to -78°C, DMSO was added and reacted for a period of time, then compound 4B was added and the reaction continued. Triethylamine was added in the later stage of the reaction, and the temperature was raised to room temperature to continue the reaction, yielding compound 4C. Step 4-3: Compound 4C reacts with compound 1A and triethylamine in a dichloromethane solution in the presence of sodium triacetoxyborohydride at room temperature to generate 4D; or, Wherein, R is as defined in the corresponding claims; n is taken from 0, 1, 2, 3, 4, 5; Step 4-4: Compound 4A is reacted with concentrated sulfuric acid in methanol solution at 80°C to obtain compound 4E; Steps 4-5: Compound 4E is reduced to compound 4B by lithium aluminum hydride in tetrahydrofuran solution at 0°C; Steps 4-6: Compound 4B reacts with pyridine chlorochromate in a dichloromethane solution at room temperature to give compound 4C; Steps 4-7: Compound 4C reacts with compound 1A and triethylamine in a dichloromethane solution in the presence of sodium triacetoxyborohydride at room temperature to generate 4D; Synthesis Method 5: Where X is -(CH2) x -,-(CH2CH2O) x (CH2) y -, x, y, and R are as defined in the respective claims; Step 5-1: Compound 5A and compound 5B are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate compound 5C; Step 5-2: In a dichloromethane solution, triethylamine is added to compound 5C, the mixture is cooled to 0°C, and a dichloromethane solution of methanesulfonic anhydride is added dropwise. The mixture is allowed to react at room temperature to produce compound 5D. Step 5-3: Compound 5D and compound 1A react in dimethylformamide solution at 80°C in the presence of triethylamine to generate compound 5E; or, R are as defined in the respective claims, and n is selected from 0, 1, 2, 3; Step 5-4: Compound 5A and compound 5F are activated by HATU and DIPEA in dimethylformamide solution at room temperature to generate compound 5G; Step 5-5: In a dichloromethane solution, triethylamine is added to compound 5G, the mixture is cooled to 0°C, and a dichloromethane solution of methanesulfonic anhydride is added dropwise. The mixture is allowed to react at room temperature to produce compound 5H. Steps 5-6: Compound 5H and compound 1A react in dimethylformamide solution at 80°C in the presence of triethylamine to generate compound 5I; Synthesis Method Six: Wherein, R is as defined in the respective claims; Step 6-1: Compound 1A reacts with chloroethane-1-sulfonyl chloride in a dichloromethane solution at -40°C in the presence of triethylamine to generate compound 6A; Step 6-2: Compound 6A reacts in acetonitrile solution at room temperature in the presence of cesium carbonate and ammonium hydroxide to form compound 6B; Step 6-3: Compound 6B and Compound 6C undergo an amidation reaction in dimethylformamide solution at room temperature, activated by HATU and DIPEA, to generate Compound 6D; or Compound 6B and Compound 6C undergo an amidation reaction in acetonitrile solution in the presence of N-methylimidazole and tetramethylchlorourea hexafluorophosphate to generate Compound 6D. Step 6-4: Compound 6B and compound 6E react in a dichloromethane solution at room temperature to produce compound 6F.

8. A pharmaceutical composition comprising one or more of the compounds of any one of claims 1-6, a pharmaceutically acceptable salt, a racemic mixture, and a pharmaceutically acceptable carrier or excipient.

9. The pharmaceutical composition according to claim 8, wherein, The pharmaceutical composition comprises at least one other therapeutic agent selected from anticancer agents, immunomodulators, anti-inflammatory agents, anti-Alzheimer's agents, and combinations thereof.

10. Use of the compound of any one of claims 1-6, its pharmaceutically acceptable salt or racemate, or the pharmaceutical composition of claim 8 or 9 for the preparation of a medicament for the prevention and / or treatment of diseases or conditions mediated by the CDK9-cyclin T1 complex.

11. The use according to claim 10, wherein, The diseases or conditions mediated by the CDK9-cyclin T1 complex are 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, bile duct and gallbladder cancer, bladder cancer, brain tumor, neuroblastoma, schwannoma, glioma, glioblastoma and astrocytoma, endometrial cancer, esophageal cancer, head and neck cancer, pancreatic cancer, and renal cell carcinoma.

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