A cdk9-cyclin t1 protein interaction inhibitor and uses thereof

Abstract

A CDK9-Cyclin T1 protein interaction inhibitor and application thereof are used for preparing a medicine for treating diseases related to CDK9, CDK9 / Cyclin T1 activity or expression amount. By targeting binding of CDK9, the protein interaction of CDK9 and Cyclin T1 is destroyed, and then the related signal path is regulated, tumor cell apoptosis is induced, and the effect of inhibiting tumor cell proliferation is achieved. Therefore, the CDK9-Cyclin T1 protein interaction inhibitor disclosed in the application can be used for treating cancer or related diseases.

Description

Technical Field

[0001] This invention relates to the field of chemical drug technology, and in particular to a CDK9-Cyclin T1 protein interaction inhibitor and its application. Background Technology

[0002] Cyclin-dependent kinase 9 (CDK9) is an important member of the CDK family, playing a crucial role in transcriptional regulation. The CDK9 / cyclin complex is involved in various cellular functions, while the CDK9-Cyclin T1 complex participates in the formation of positive transcription elongation factor b (P-TEFb), playing a vital role in regulating transcriptional elongation. Increasing research indicates that aberrant expression and dysfunction of CDK9 are closely related to tumorigenesis and development, making it an important drug target for cancer treatment. Existing studies have shown that CDK9-Cyclin T1 protein-protein interaction inhibitors can significantly inhibit the proliferation and migration of triple-negative breast cancer (TNBC) cells in vitro and in vivo. Therefore, developing novel CDK9 inhibitors targeting CDK9-Cyclin T1 interaction is an effective strategy for cancer therapy. Summary of the Invention

[0003] The purpose of this invention is to solve the above-mentioned problems in the prior art and to provide a CDK9-Cyclin T1 protein interaction inhibitor and its application.

[0004] The primary objective of this invention is to provide a compound of general formula (I) or its stereoisomers, its hydrates, its solvates, its deuterated derivatives, its prodrugs, its metabolites, its intermediates, its pharmaceutically acceptable salts or cocrystals, and its pharmaceutically acceptable carriers.

[0005]

[0006] Among them, X is preferably Cl or H; Linker is preferably -CH2- or carbonyl. R1 is a piperidine group, 2-methylaminoethanol, or a piperazine group, preferably 4-hydroxypiperidine, N-methylpiperazine, N-ethylpiperazine, N-isopropylpiperazine, 4-methylpiperidine, 3-methylpiperidine, 2-methylaminoethanol, 4-aminopiperidine, 4-tert-butoxycarbonylaminopiperidine, 4-methanolpiperidine, or 3-methanolpiperidine; R2 is H, halogen, alkyl, or alkoxy, preferably H, 4-F, 4-CH3, 4-OCH3, 4-CF3, 4-OCH2CH3, 2-CH3, 3-CH3, or 3-OCH3.

[0007] A pharmaceutical composition comprising a compound of formula (I) of the present invention or its stereoisomer, its hydrate, its solvate, its deuterated product, its prodrug, its metabolite, its intermediate, its pharmaceutically acceptable salt or cocrystal, and a pharmaceutically acceptable carrier.

[0008] The pharmaceutically acceptable salt is a pharmaceutically acceptable addition salt formed by a compound of general formula (I) and an acid. The acid used for salt formation includes inorganic and organic acids, with preferred inorganic and organic acids being hydrochloric acid, sulfuric acid, phosphoric acid, and methanesulfonic acid. Organic acids include acetic acid, trichloroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, digluconic acid, aspartic acid, and tartaric acid.

[0009] The pharmaceutically acceptable carrier refers to an excipient or diluent that does not cause significant irritation to the organism and does not interfere with the biological activity and properties of the compound given.

[0010] Any compound or its stereoisomers, hydrates, solvates, deuterated derivatives, prodrugs, metabolites, intermediates, pharmaceutically acceptable salts or cocrystals, and pharmaceutically acceptable carriers described herein are used to treat CDK9 and CDK9 / Cyclin T1-related diseases, such as tumors, including breast cancer, lung cancer, liver cancer, kidney cancer, cervical cancer, etc.

[0011] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0012] The CDK9-Cyclin T1 protein interaction inhibitor disclosed in this invention targets and binds to CDK9, disrupting the protein interaction between CDK9 and Cyclin T1, thereby regulating related signaling pathways and inducing tumor cell apoptosis, thus inhibiting tumor cell proliferation. Therefore, the CDK9-Cyclin T1 protein interaction inhibitor disclosed in this invention can be used for the treatment of cancer or related diseases. Attached Figure Description

[0013] Figure 1 The results show the detection of the inhibitory effect of some of the disclosed compounds in Example 3 on CDK9 phosphorylated RNApol II (S2).

[0014] Figure 2 The results show the detection of the selective inhibition of the interaction between CDK9 and Cyclin T1 by compound B19 in Example 4.

[0015] Figure 3 The results show the detection of the specific binding of compound B19 to CDK9 in Example 5.

[0016] Figure 4The results show the detection of CDK9-dependent biological functions of compound B19 in Example 6.

[0017] Figure 5 The results show the effects of different concentrations of B19 on the levels of downstream factors of CDK9 in Example 8.

[0018] Figure 6 The results show the effects of compound B19 in Example 9 on the proliferation and apoptosis of MDA-MB-231 cells.

[0019] Figure 7 The results show the effect of compound B19 on the migration of MDA-MB-231 cells in Example 10.

[0020] Figure 8 The results show the detection of the synergistic killing effect of compound B19 in combination with olaparib on MDA-MB-231 tumor cells in Example 11.

[0021] Figure 9 The results show the changes in body weight in mice during the acute toxicity study of compound B19 in Example 12.

[0022] Figure 10 The blood concentration-time curve (drug-time curve) of rats after intraperitoneal injection of 50 mg / kg B19 in Example 13 is shown.

[0023] Figure 11 The results show that compound B19 in Example 14 significantly inhibited the growth of 4T1 xenografts in nude mice. Detailed Implementation

[0024] To make the technical problems, technical solutions, and beneficial effects of this invention clearer and more understandable, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The scope of protection of this invention includes, but is not limited to, these embodiments.

[0025] The specific synthetic route of the CDK9-Cyclin T1 protein interaction inhibitor provided by this invention is as follows:

[0026]

[0027] In a preferred embodiment of the present invention, X is preferably Cl or H; Linker is preferably -CH2-, -C=O-, -C=OCH2-, or -C=OCH2CH2-; R1 is a piperidine group or 2-methylaminoethanol or piperazine group, preferably 4-hydroxypiperidine, N-methylpiperazine, N-ethylpiperazine, N-isopropylpiperazine, 4-methylpiperidine, 3-methylpiperidine, 2-methylaminoethanol, 4-aminopiperidine, 4-tert-butoxycarbonylaminopiperidine, 4-methanolpiperidine, or 3-methanolpiperidine; R2 is H, halogen, alkyl, or alkoxy, preferably H, 4-F, 4-CH3, 4-OCH3, 4-CF3, 4-OCH2CH3, 2-CH3, 3-CH3, or 3-OCH3.

[0028] The preparation method of the CDK9-Cyclin T1 protein interaction inhibitor provided by this invention is as follows:

[0029] The raw material (1) 4-bromo-2-fluoropyridine or 2-fluoro-4-iodo-5-chloropyridine was reacted with different amino groups in DMF as solvent and anhydrous potassium carbonate as base, and the mixture was heated to reflux to carry out nucleophilic substitution reaction to obtain substituted or unsubstituted N-benzyl-4-bromopyridine-2-amine, 4-(4-bromopyridine-2-yl)amine derivative, 1-(5-chloro-4-iodopyridine-2-yl)piperidin-4-ol, (1-(5-chloro-4-iodopyridine-2-yl)piperidin-4-yl)methanol and other intermediates. (2) The intermediate boronic acid ester was prepared by reacting 4-(4-bromopyridine-2-yl)amine derivative, 1-(5-chloro-4-iodopyridine-2-yl)piperidin-4-ol, (1-(5-chloro-4-iodopyridine-2-yl)piperidin-4-yl)methanol and other intermediates with pinacol diboronic acid ester. (3) Using raw material (4) 4-methoxyphenylacetic acid or 4-methoxyphenylpropionic acid as a solvent, thionyl chloride is stirred and heated to reflux to generate 4-methoxyphenylacetyl chloride or 4-methoxyphenylpropionyl chloride. Raw material (5) 2-amino-4-bromopyridine or 5-chloro-4-iodopyridine-2-amine is added dropwise with tetrahydrofuran as a solvent and pyridine as a base under ice bath conditions and stirring to carry out an amide condensation reaction to obtain substituted or unsubstituted N-(4-bromopyridin-2-yl)-4-fluorobenzamide, N-(4-bromopyridin-2-yl)-3-chloro4-fluorobenzamide, N-(4-bromopyridin-2-yl)-4-methylbenzamide, N-(4-bromopyridin-2-yl)-3,5-dichlorobenzamide, and N-(4-bromopyridin-2-yl)-3,5-dichlorobenzamide. Intermediates such as pyridin-2-yl)-4-methoxybenzamide, N-(4-bromopyridin-2-yl)-4-trifluoromethylbenzamide, N-(4-bromopyridin-2-yl)-3,4,5-trimethoxybenzamide, N-(4-bromopyridin-2-yl)-2-methylbenzamide, N-(4-bromopyridin-2-yl)-3-methylbenzamide, N-(4-bromopyridin-2-yl)-3-methoxybenzamide, N-(4-bromopyridin-2-yl)-4-ethoxybenzamide, N-(4-bromopyridin-2-yl)-2-(4-methoxyphenyl)acetamide, N-(4-bromopyridin-2-yl)-3-(4-methoxyphenyl)propionamide, and N-(5-chloro-4-iodopyridin-2-yl)-4-methoxybenzamide (7). The intermediate borate ester (3) was subjected to Suzuki coupling reaction with the intermediate substituted / unsubstituted N-benzyl-4-bromopyridin-2-amine or the intermediate (7) to obtain the target compound (8).

[0030] Table 1. Structure and proton spectrum of the compounds described in this invention 1 Characterization data from H NMR and high-resolution mass spectrometry (HRMS)

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] Example 1: Preparation of N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridine]-2-yl)-4-methoxybenzamide (B19)

[0041] This embodiment uses the synthesis of B19 as an example to illustrate the synthesis of the compounds disclosed in this invention. The specific steps are as follows:

[0042] (1) Synthesis of intermediate N-(4-bromopyridin-2-yl)-4-methoxybenzamide: In a dry 50 mL reaction flask, under ice bath conditions, 2-amino-4-bromopyridine (500 mg, 2.89 mmol), pyridine (684.93 mg, 8.67 mmol), and THF (10 mL) were added sequentially, and stirring was continued for 0.5 h; then, a THF mixture of 4-methoxybenzoyl chloride (591.60 mg, 3.74 mmol) was slowly added dropwise while stirring, and the reaction was carried out under ice bath conditions for 1 h. The reaction was stopped after TLC monitoring showed that the reaction was complete, the solvent was concentrated under vacuum and then ice water was added and stirred, a solid was precipitated, and filtered to obtain a filter cake (crude product). The crude product was separated by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 5:1, v / v) to give 649 mg of white N-(4-bromopyridin-2-yl)-4-methoxybenzamide, with a yield of 73%.

[0043] (2) Synthesis of intermediate 1-(4-bromopyridin-2-yl)piperidin-4-ol: In a dry 50 mL reaction flask, 2-fluoro-4-bromopyridine (1.00 g, 11.36 mmol) and 4-hydroxypiperidine (1.41 g, 13.63 mmol) were dissolved in 25 mL of DMF. Anhydrous potassium carbonate (4.71 g, 34.09 mmol) was added. After purging the reaction system with nitrogen, the temperature was raised to 90 °C and reacted for 3 h. After the reaction was confirmed to be complete by TLC, heating was stopped, and the reaction solution was poured into 100 mL of ice water with stirring. A solid precipitated, which was filtered, and the filter cake was dried to obtain 2.54 g of white solid 1-(4-bromopyridin-2-yl)piperidin-4-ol, with a yield of 83%.

[0044] (3) Synthesis of 1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)pyridin-2-yl)piperidine-4-ol: In a dry 100 mL reaction flask, 2.54 g (9.88 mmol) of 1-(4-bromopyridin-2-yl)piperidine-4-ol obtained in the previous step, 3.76 g (14.82 mmol) of pinacol diborate, 0.1 mmol (1,1'-bis(diphenylphosphino)ferrocene)palladium dichloride, and 2.91 g (29.64 mmol) of dry anhydrous potassium acetate were added to 30 mL of dry 1,4-dioxane. Nitrogen gas was purged, and the mixture was heated to 11 °C under nitrogen protection. The reaction was carried out at 0℃ for 4 h. After the reaction of 1-(4-bromopyridin-2-yl)piperidin-4-ol was complete, the solvent was removed by concentration under reduced pressure. The mixture was extracted three times with ethyl acetate and water (3 x 100 mL). The organic phases were combined, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered to remove the drying agent. The residue after concentration under reduced pressure was mixed with silica gel and purified by column chromatography (isocratic elution method, dichloromethane:methanol = 20:1, v / v) to obtain 2.23 g of 1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)pyridin-2-yl)piperidin-4-ol, a white powder intermediate, with a yield of 74%.

[0045] (4) N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-methoxybenzamide (B19): Take a 50 mL round-bottom flask and weigh the intermediate N-(4-bromopyridin-2-yl)-4-methoxybenzamide (300 mg, 0.97 mmol), the intermediate 1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)pyridin-2-yl)piperidin-4-ol (444.20 mg, 1.47 mmol), anhydrous potassium carbonate (414 mg, 3 mmol), and 1,1 '-Bis(diphenylphosphino)ferrocene]palladium dichloride (30 mg) was dissolved in 5 mL of solvent (ethylene glycol dimethyl ether: water = 4:1), purged with nitrogen three times, heated to 80 °C and reacted overnight. TLC showed that the reaction had reached equilibrium, the reaction was stopped, the solvent was removed by concentration under reduced pressure, the residue was mixed with silica gel, and purified by column chromatography (isocratic elution method, petroleum ether / ethyl acetate = 1:1, v / v) to give compound N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridine]-2-yl)-4-methoxybenzamide, 125 mg white solid, yield 32%.

[0046] Other compounds listed in Table 1:

[0047] 4-Fluoro-N-(2'-(4-methylpiperidin-1-yl)-[4,4'-bipyridin]-2-yl)benzamide (B1), 4-methyl-N-(2'-(4-methylpiperidin-1-yl)-[4,4'-bipyridin]-2-yl)benzamide (B2), 3,4-dichloro-N-(2'-(4-methylpiperidin-1-yl)-[4,4'-bipyridin]-2-yl)benzamide (B3), N-(2'-(4-methylpiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-(trifluoromethyl)benzamide (B4), 4-methoxy-N-(2'-(4-methylpiperidin-1-yl)-[4,4'-bipyridin]-2-yl)benzamide (B5) 4-Fluoro-N-(2'-(3-methylpiperidin-1-yl)-[4,4'-bipyridin]-2-yl)benzamide (B6), 4-methoxy-N-(2'-(4-methylpiperazin-1-yl)-[4,4'-bipyridin]-2-yl)benzamide (B7), N-(2'-(3-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-methylbenzamide (B8), N-(5-chloro-2'-(3-(hydroxymethyl)piperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-methylbenzamide (B9), N-(2'-(3-(hydroxymethyl)piperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-methoxybenzamide (B10), Tertiary butyl (1-(2'-(4-methoxybenzamide)-[4,4'-bipyridin]-2-yl)piperidin-4-yl)carbamate (B11), N-(2'-(4-aminopiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-methoxybenzamide (B12), N-(2'-((2-hydroxyethyl)(methyl)amino)-[4,4'-bipyridin]-2-yl)-4-methoxybenzamide (B13), N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-2-methylbenzamide (B14), 2-chloro-N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-[4,4'-bipyridin] N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-3-methylbenzamide (B16), N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-methylbenzamide (B17), N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-(trifluoromethyl)benzamide (B18), N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-methoxybenzamide (B19), 3,5-dichloro-N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-Bipyridin-2-yl)benzamide (B20), N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-3,4,5-trimethoxybenzamide (B21), 4-ethoxy-N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)benzamide (B22), N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-3-methoxybenzamide (B23), N-(5-chloro-2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-methylbenzamide (B25), 4-fluoro-N-(2'-(4-) 4-(5'-chloro-2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)benzamide (B24), N-(5'-chloro-2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-methoxybenzamide (B26), 4-fluoro-N-(2'-(4-(hydroxymethyl)piperidin-1-yl)-[4,4'-bipyridin]-2-yl)benzamide (B27), N-(2'-(4-(hydroxymethyl)piperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-methoxybenzamide (B28), N-(5'-chloro-2'-(4-(hydroxymethyl)piperidin-1-yl)-[4,4'-bipyridin]-2-yl)-4-methoxybenzamide Amide (B29), 3-chloro-4-fluoro-N-(2'-(4-methylpiperidin-1-yl)-[4,4'-bipyridin]-2-yl)benzamide (B30), 3,5-dichloro-N-(2'-(4-methylpiperidin-1-yl)-[4,4'-bipyridin]-2-yl)benzamide (B31), N-(2'-(4-ethylpiperazin-1-yl)-[4,4'-bipyridin]-2-yl)-4-fluorobenzamide (B32), N-(2'-(4-ethylpiperazin-1-yl)-[4,4'-bipyridin]-2-yl)-4-methoxybenzamide (B33), N-(2'-(4-isopropylpiperazin-1-yl)-[4,4'-bipyridin]-2-yl) 4-Methoxybenzamide (B34), 4-fluoro-N-(2'-((2-hydroxyethyl)(methyl)amino)-[4,4'-bipyridin]-2-yl)benzamide (B35), 3-chloro-4-fluoro-N-(2'-((2-hydroxyethyl)(methyl)amino)-[4,4'-bipyridin]-2-yl)benzamide (B36), 1-(2'-((4-methoxybenzyl)amino)-[4,4'-bipyridin]-2-yl)piperidin-4-ol (C1), N-(2'-(4-hydroxypiperidin-1-yl)-[4,4'-bipyridin]-2-yl)-2-(4-methoxyphenyl)acetamide (C2), N-(2'-(4-hydroxypiperidin-1-yl)-[4,The synthetic methods for compounds B19 are similar to those for compound B19.

[0048] Example 2: Evaluation of the antitumor cell proliferation activity of the compounds in this invention (IC50) 50 )

[0049] Log-phase MDA-MB-231, A549, HeLa, and LO2 cells were seeded into 96-well plates and incubated overnight at 37°C. The test compound solution was initially diluted to 20 μM using a three-fold dilution method and added to each well, with three replicates per group. A proportionally diluted DMSO solution was used as a control. After culturing for 72 h, 0.5 mg / mL MTT was added, and the plates were incubated at 37°C for 3-4 h. The supernatant was discarded, and 100 μL of DMSO was added to each well. The plates were shaken for 10 min to fully dissolve the formazan. The absorbance at 492 nm was then measured using a microplate reader. Experimental data are expressed as follows: Calculate the inhibition rate, and then calculate the half-inhibitory concentration (IC50) based on the inhibition rate at each concentration. 50 The experimental results are shown in Table 2. The results show that the compound in this invention has a significant inhibitory effect on tumor cell proliferation, and the inhibitory activity of the compound on LO2 (normal human hepatocytes) is generally lower than that on cancer cells, indicating good safety.

[0050] Table 2. Evaluation of the antitumor cell proliferation activity of some compounds in this invention

[0051]

[0052]

[0053] Example 3: Evaluation of the inhibitory activity of the compounds of the present invention against CDK9 phosphorylated RNApol II (S2)

[0054] Whether the kinase activity of CDK9 is inhibited or the interaction between CDK9 and Cyclin T1 is disrupted, the phosphorylation of serine 2 of RNA polymerase in vivo is inhibited. Based on the MTT results, the candidate compound showed significantly better inhibitory activity against MDA-MB-231 than A549 and Hela in human non-small cell lung cancer cell line A549, human cervical cancer cell line Hela, and triple-negative breast cancer cell line MDA-MB-231. Therefore, this invention evaluated the expression level of phosphorylated protein at serine 2 of RNA polymerase by the preferred compound in the MDA-MB-231 cell line.

[0055] Western Blot Experiment: Logarithmic growth phase MDA-MB-231 cells were seeded in 12-well plates and cultured overnight at 37°C. A 1 μM ICDK9 solution was used as a positive control. After culturing for another 24 hours in medium containing 1 μM of the compound, the medium was discarded, and the cells were washed three times with pre-cooled 1×PBS. Cell lysis buffer was added, and the cells were lysed on ice for 30 min. The cells were then centrifuged at 12000g at 4°C for 15 min. The supernatant was collected, loaded with loading buffer, and boiled at 100°C for 10 min to obtain protein samples. Protein samples were then analyzed by Western blot. The experimental results and their quantitative analysis are as follows: Figure 1 As shown in the figure. The results indicate that the preferred compounds can inhibit the protein expression of CDK9 phosphorylated RNApol II (S2) at a certain level, with compound B19 significantly inhibiting the phosphorylation level of RNApol II (S2).

[0056] Figure 1 A is a Western blot analysis of the effect of the compound on the phosphorylation level (P-ser2) of RNA polⅡ(S2).

[0057] Figure 1 B and 1C are Figure 1 Quantitative analysis of P-ser2 bands in a Western blot.

[0058] The above indicates that compound B19 can inhibit the phosphorylation level of RNA polⅡ(S2).

[0059] Example 4: Compound B19 exerts its biological function by disrupting the interaction between CDK9 and Cyclin T1.

[0060] Exogenous CO-IP experiment: HEK293t cells were transfected with the flag-CDK9 plasmid. 24 h after transfection, the supernatant was discarded, and the cells were washed twice with pre-chilled 1×PBS. Pre-chilled cell lysis buffer was then added, and the cells were lysed on ice for 30 min. All cells were scraped off with a spatula and transferred to pre-chilled 1.5 mL ep lysate tubes. The cell lysate was centrifuged at 12000 g for 15 min at 4 °C. The supernatant was collected, and the lysis buffer was treated with 1 μM and 3 μM B19 for 8 h. 10% of the protein supernatant was taken, loaded with loading buffer, and boiled at 100 °C for 10 min as input. 1 μg of Flag antibody stock solution was added to the remaining protein supernatant, and the mixture was then incubated overnight at 4 °C using a vertical mixer. Add 15 μL of protein A / G agarose beads to each sample (wash protein A / G agarose beads three times with 1×PBS), then incubate in a vertical mixer at 4°C for 3-4 h to allow antibody conjugation to the agarose beads. After immunoprecipitation, centrifuge at 3000 rpm for 5 min at 4°C, carefully aspirate the supernatant, and wash the remaining agarose beads at the bottom of the tube 3-4 times with lysis buffer, aspirating as much residual supernatant as possible on the last wash. Add 20 μL of 2× loading buffer to the precipitated agarose beads and boil at 100°C for 10 min. Protein samples are detected by Western blot. Transfect CDK7 plasmid into HEK293t cells, following the same steps as above.

[0061] Endogenous CO-IP: MDA-MB-231 cells were cultured in 15cm dishes, with 3μM ICDK9 as a positive control. MDA-MB-231 cells were treated with 1μM and 3μM compound B19 for 8 hours, respectively. The supernatant was discarded, and other steps were the same as above. Experimental results and quantitative analysis are as follows: Figure 2 As shown.

[0062] Figure 2 A is the concentration-dependent inhibition of the interaction between CDK9 and Cyclin T1 in exogenous co-ip.

[0063] Figure 2 B is Figure 2 Quantitative analysis of Cyclin T1 and Cyclin K bands in a Western blot.

[0064] Figure 2 In exogenous co-ip, compound B19 does not affect the interaction between CDK7 and Cyclin H.

[0065] Figure 2 D is Figure 2 Quantitative analysis of Cyclin H bands in C-Western blot.

[0066] Figure 2 E is the effect of treating endogenous co-ip cells in MDA-MB-231 cells with the drug, showing that compound B19 inhibits the interaction between CDK9 and Cyclin T1 in a concentration-dependent manner, while the positive control ICDK9 does not affect the interaction between CDK9 and Cyclin T1.

[0067] Figure 2 F is Figure 2 Quantitative analysis of Cyclin T1 bands in E Western blot.

[0068] This indicates that compound B19 can exert its biological function by inhibiting the CDK9-CyclinT1 interaction.

[0069] Example 5: Compound B19 specifically binds to CDK9

[0070] CETSA (Cellular Thermal Shift Assay) is an experiment to detect the binding efficiency of intracellular drugs to target proteins. The principle is that target proteins typically become stable when bound to drug molecules. That is, as temperature increases, proteins degrade; when a protein binds to a drug, at the same temperature, the amount of undegraded protein increases, and the thermal melting curve of the complex protein shifts to the right.

[0071] CETSA Experiment: HEK293t cells were transfected with the flag-CDK9 plasmid. 24 h after transfection, the supernatant was discarded, and the cells were washed twice with pre-chilled 1×PBS. Pre-chilled cell lysis buffer was then added, and the cells were lysed on ice for 30 min. All cells were scraped off with a spatula and transferred to EP tubes. The cells were centrifuged at 12000g for 15 min at 4°C. The supernatant was collected, and 1 μM B19 was added. The cells were incubated at 4°C for 6 h. The supernatant was then evenly divided into 6 PCR tubes. The PCR tubes were placed in a PCR instrument with gradient heating at 45°C, 46.4°C, 50.2°C, 56.8°C, 62°C, 63.6°C, and 65°C for 30 s, incubated at room temperature for 2 min, and then centrifuged at 12000g for 15 min at 4°C. The supernatant was collected, and 4× loading buffer was added in equal proportions. The cells were then boiled in a metal bath for denaturation. Western blotting was then performed to detect the binding ability of the compounds to proteins.

[0072] MDA-MB-231 cells were cultured in 15cm dishes and treated with 1μM B19 for 8 hours. The supernatant was discarded, and the cells were washed twice with pre-chilled 1×PBS. Pre-chilled cell lysis buffer was then added, and the cells were lysed on ice for 30 minutes. All cells were scraped off with a spatula and transferred to EP tubes. The cells were centrifuged at 12000g for 15 minutes at 4°C. The supernatant was collected and evenly divided into six PCR tubes. The PCR tubes were placed in a PCR instrument with gradient heating at 45°C, 46.4°C, 50.2°C, 56.8°C, 62°C, 63.6°C, and 65°C for 30 seconds each, incubated at room temperature for 2 minutes, and then centrifuged at 12000g for 15 minutes at 4°C. The supernatant was collected, and 4× loading buffer was added in equal proportions. The cells were then boiled in a metal bath for denaturation. Western blotting was then performed to detect the binding ability of the compounds to the proteins. The experimental results are as follows: Figure 3 As shown.

[0073] Figure 3 A is a Western blotting analysis of the GETSA experiment performed on 293t cell lysates treated with the drug, showing that B19 can bind to CDK9 and stabilize the CDK9 protein.

[0074] Figure 3 B is Figure 3 A quantitative analysis of CDK9 bands in a Western blot.

[0075] Figure 3 C involves drug treatment in MDA-MB-231 cells, and Western blotting shows that B19 specifically binds to CDK9 within the CDK family.

[0076] Figure 3 D is Figure 3 Quantitative analysis of CDK9 bands in C-ray Western blot.

[0077] The above indicates that compound B19 can specifically bind to CDK9 to exert its biological function.

[0078] Example 6: Compound B19 exerts its biological function in dependence on CDK9

[0079] CDK9 was knocked down in the MDA-MB-231 cell line using sh-RNA technology to obtain the MDA-MB-231-shCDK9 cell line. Logarithmically growing MDA-MB-231-WT and MDA-MB-231-shCDK9 cells were seeded in six-well plates, and 1 μM B19 was added to each well. Western blotting was used to detect downstream protein expression. MTT assays were performed to assess the cell viability of wild-type MDA-MB-231 cells and MDA-MB-231-shCDK9 cells after treatment with different concentrations of compound B19 for 72 h. Results are as follows: Figure 4 As shown.

[0080] Figure 4 A shows the expression of CDK9 downstream factors in MDA-MB-231 cells (DMSO or B19) after drug treatment with wild-type MDA-MB-231 and sh-CDK9, as detected by Western Blot, indicating that B19 depends on CDK9 to perform its biological functions.

[0081] Figure 4 B, 4C, 4D, 4E, 4F, 4G, and 4H are respectively Figure 4 Quantitative analysis of CDK9, Cyclin T1, p-ser2, C-myc, Mcl-1, Bcl-2, and Cleaved-parp bands in a Western blot.

[0082] Figure 4 I. Different concentrations of B19 were used to treat wild-type MDA-MB-231 and sh-CDK9 MDA-MB-231 cells for 72 h, and the IC50 of the compounds in the two cell lines was detected using the MTT assay. 50 The results showed that B19's anti-proliferative ability was weakened in sh-CDK9 MDA-MB-231 cells compared to wild-type MDA-MB-231 cells.

[0083] Figure 4 J is Figure 4 I-cell IC 50 The numerical comparison histogram.

[0084] The above indicates that compound B19 depends on CDK9 to exert its biological functions.

[0085] Example 7: B19 significantly inhibits the proliferation of triple-negative breast cancer cell lines

[0086] Log-growing cells MDA-MB-231, BT549, Hs578t, 4T1, A498, HepG2, A549, HeLa, LO2, HK2, and MCF-10A were seeded in 96-well plates and subjected to MTT assays to detect the anti-cancer cell proliferation activity (IC50) of the compounds.50 (), used to evaluate the anticancer activity of compound B19.

[0087] The results are shown in Table 3. Compound B19 significantly inhibited the proliferation of triple-negative breast cancer cell lines (MDA-MB-231, BT549, Hs578t, 4T1) better than other cancer cell lines such as cervical cancer HeLa, lung cancer A549, renal cancer A498, and liver cancer HepG2, as well as normal cell lines (MCF-10A, LO2, HK2), indicating that compound B19 has a certain selectivity for triple-negative breast cancer cell lines.

[0088] Table 3. Compound B19 significantly inhibited the proliferation of triple-negative breast cancer cell lines.

[0089]

[0090] Example 8: B19 concentration-dependent inhibition of CDK9 downstream proteins

[0091] The main downstream regulatory proteins of CDK9 are Mcl-1, C-myc, etc. These proteins are involved in cell growth and cell cycle processes, and control the proliferation and survival of tumor cells.

[0092] Logarithmically growing MDA-MB-231 cells were seeded into 6-well plates. After cell adhesion, the medium was replaced with culture medium containing different concentrations of compound B19. After 24 hours, the supernatant was discarded, and the cells were washed three times with chilled 1×PBS. Pre-chilled cell lysis buffer was added, and the procedure was repeated as above. Western blotting was then performed to detect protein expression results. Figure 5 As shown.

[0093] Figure 5 A is a Western blotting study of CDK9 downstream proteins and apoptosis-related proteins, indicating that B19 concentration-dependent inhibition of CDK9 downstream proteins induces apoptosis.

[0094] Figure 5 B, 5C, 5D, 5E, 5F, and 5G are respectively Figure 5 Quantitative analysis of p-ser2, p-ser5, C-myc, Mcl-1, Bcl-2, and Cleaved-parp bands in a Western blot.

[0095] In summary: B19 concentration-dependent inhibition of CDK9 downstream proteins.

[0096] Example 9: B19 concentration-dependent inhibition of MDA-MB-231 cell proliferation and induction of apoptosis

[0097] (1) Cell colony formation assay: The ability of compound B19 to inhibit cell proliferation was determined by cell colony formation. Log-phase MDA-MB-231 cells were seeded in 6-well plates and cultured at 37℃ for 48 h. After culturing for another ten days, culture medium containing different concentrations (12, 37, 111, 333, 1000 nM) of B19 was added. The culture medium was discarded, and the cells were washed three times with 1×PBS. The cells were fixed with methanol at room temperature for 15 min, the methanol was discarded, and the cells were washed three times with 1×PBS. After drying, 0.5% crystal violet solution (diluted with PBS) was added for staining at room temperature for 10-15 min. The staining solution was removed, and the cells were washed with ultrapure water until no background color was visible. Colony counts were observed under a microscope, and photographs were taken and recorded. Figure 6 A) and perform quantitative analysis ( Figure 6 B).

[0098] (2) Western Blot experiment: MDA-MB-231 cells in logarithmic growth phase were seeded into six-well plates. After cell adhesion, the medium was replaced with a medium containing different concentrations of compound B19. After 24 hours, the supernatant was discarded, and the cells were washed three times with pre-cooled 1×PBS. Chilled cell lysis buffer was added, and the Western Blot experiment was performed as above. Figure 6 C) After apoptosis occurs, the expression of related proteins changes significantly. PARP protein is cleaved during early apoptosis; therefore, PARP cleavage can serve as a marker of early apoptosis. Its bands were quantified using grayscale analysis. Figure 6 D).

[0099] (3) Annexin V-FITC Apoptosis Detection Assay: The effect of compound B19 on cell apoptosis was detected by flow cytometry. Log-phase MDA-MB-231 cells were seeded in 6-well plates and cultured overnight at 37°C. ICDK9 was used as a positive control. Cells were cultured for 72 hours with different concentrations of B19 (37, 111, 333, and 1000 nM). The culture medium was discarded, and the cells were washed with pre-cooled 1×PBS, digested with trypsin, and collected into pre-cooled 1.5 mL ep tubes. After centrifugation at 2500 rpm for 5 min, the supernatant was removed. The cells were washed twice with pre-cooled PBS, centrifuged again, and the supernatant was removed. The tube walls were gently tapped to prevent cell clumping. The cells were stained with Annexin V-FITC apoptosis detection kit at room temperature in the dark for 20 min, and the apoptosis was detected by flow cytometry. The experimental results are as follows: Figure 6 As shown in E and 6F.

[0100] Figure 6 A shows the cell colony formation assay of MDA-MB-231 cells treated with different concentrations of B19, indicating that B19 inhibits MDA-MB-231 proliferation in a concentration-dependent manner.

[0101] Figure 6 B is Figure 6 A. Quantitative analysis of the dissolution of crystal violet.

[0102] Figure 6 C represents the detection of apoptosis markers parp and cleaved-parp expression by Western blotting at different concentrations of B19 treatment, indicating that B19 concentration-dependent induction of parp cleavage.

[0103] Figure 6 D is Figure 6 Quantitative analysis of Cleaved-Parp in C Western Blot.

[0104] Figure 6 E represents the flow cytometry detection of apoptosis rates at different concentrations of B19, indicating that B19 induces apoptosis in a concentration-dependent manner.

[0105] Figure 6 F is Figure 6 Quantitative analysis of apoptotic cells in E.

[0106] The above indicates that B19 concentration-dependent inhibition of MDA-MB-231 cell proliferation and induction of apoptosis.

[0107] Example 10: B19 concentration- and time-dependent inhibition of MDA-MB-231 migration

[0108] (1) Cell scratch assay: MDA-MB-231 cells in the logarithmic growth phase were seeded in a six-well plate. After a monolayer of dense cells was formed, straight lines were drawn with a 200 μl pipette tip. Cell debris was washed with PBS and cultured in serum-free medium containing 0.1 μM of compound B19. The scratch healing was observed at four time points: 0 h, 6 h, 12 h, and 24 h under an inverted microscope, and quantified using ImageJ.

[0109] (2) Transwell assay: Medium containing 10% fetal bovine serum was added to the lower chamber of the Transwell. Logarithmically growing MDA-MB-231 cells were seeded into the upper chamber, which was then replaced with serum-free medium containing different concentrations of compound B19. 1 μM ICDK9 was used as a positive control. After 24 h of culture, the Transwell chamber was removed, gently rinsed with PBS, air-dried, fixed with paraformaldehyde, stained with 0.1% crystal violet, washed with deionized water, and air-dried. Cell migration and condition were observed under an inverted microscope, and quantification was performed using ImageJ. The experimental results are as follows: Figure 7 As shown.

[0110] Figure 7 A was a scratch experiment performed on a 0.1 μM B19 treatment. Photos were taken at different time points to detect its migration level, indicating that B19 inhibited the migration of MDA-MB-231 in a time-dependent manner.

[0111] Figure 7 B is Figure 7 Quantitative analysis of A's migration ability.

[0112] Figure 7 C represents the Transwell assay performed on MDA-MB-231 cells treated with different concentrations of B19 for 24 hours to assess their invasive ability. This indicates that B19 concentration-dependently inhibits MDA-MB-231 invasion.

[0113] Figure 7 D is Figure 7 Quantitative analysis of the dissolution of crystal violet C.

[0114] The above indicates that B19 inhibits MDA-MB-231 migration and invasion in a concentration- and time-dependent manner.

[0115] Example 11: Compound B19 combined with olaparib synergistically kills tumor cells

[0116] CI (Combined Index Analysis): CompuSyn is an application software based on the Chou-Talalay mathematical model that can directly calculate CI values ​​from imported data, making it convenient and fast. The magnitude of the CI value determines the combined effect of two drugs: when CI < 1, the two drugs have a synergistic effect; when CI = 1, the two drugs have an additive effect; when CI > 1, the two drugs have an antagonistic effect. Simultaneously, CompuSyn software will draw an Fa-CI diagram of the equivalent dose-response ratio based on the obtained data.

[0117] Log-phase MDA-MB-231 cells were seeded in 96-well plates. B19 was initially diluted three times at 1.33 μM, and olaparib was initially diluted three times at 16.67 μM. The cells were treated with a combination of B19 and olaparib for 48 h. An MTT assay was performed to detect the cell proliferation inhibitory activity of the compounds. The combination index was calculated using CompuSyn. The experimental results are as follows: Figure 8 As shown. Figure 8 The results showed that the combined use of B19 and Olaparib had a synergistic effect, providing a theoretical basis for the clinical treatment of TNBC.

[0118] Example 12: Acute toxicity study of compound B19 in mice

[0119] In accordance with the guidelines of the Animal Care and Use Committee of Xiamen University, an acute toxicity study protocol for mice was approved. The preferred compound B19 was evaluated for acute toxicity in 4-6 week old (20 g) mice. The compound was prepared in 10% DMSO, 2% T-80, and 88% ultrapure water, and administered intraperitoneally at doses of 62.5 mg / kg, 125 mg / kg, 250 mg / kg, 500 mg / kg, 750 mg / kg, and 1000 mg / kg (n=5 per group), with observation for two weeks.

[0120] Table 4 shows the acute toxicity study of compound B19 in mice. All mice given the highest dose (1000 mg / kg) died, two mice gave the higher dose (750 mg / kg) and no mice died in the other dose groups. The median lethal dose (LD50) of compound B19 was calculated using the Probit method. 50 It is 537 mg / kg.

[0121] Figure 9 The graph shows the weight changes of mice in the acute toxicity study of compound B19. The higher dose group (750 mg / kg) showed a decline in mental state within 5 days after administration. The 3 mice that did not die recovered well after 5 days and had a slower weight gain. In contrast, the mice in other dose groups showed more significant weight gain and good mental state.

[0122] The above indicates that compound B19 has good safety.

[0123] Table 4. Acute toxicity evaluation of compound B19 in mice

[0124]

[0125] Example 13: Pharmacokinetic Study of Compound B19 in Rats

[0126] The pharmacokinetic study protocol was approved according to the guidelines of the Animal Care and Use Committee of Xiamen University. The preferred compound B19 was pharmacokinetically evaluated in 10-14 week old (200-220g) Sprague-Dawley rats. The compound was formulated in 10% DMSO, 2% T-80, and 88% ultrapure water and administered intraperitoneally (ip) at a dose of 25 mg / kg. Female SD rats were randomly divided into groups of three. Blood samples were collected in tubes containing heparin sodium (10 mg / mL, 30 μL) at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after administration. Plasma was separated from blood after centrifugation at 4000 rpm for 10 min at 4°C (100 μL). The levels of compound B19 in plasma samples were determined by LC / MS / MS using an ABI 3200QTRAP triple quadrupole system operating in positive electrospray mode. Pharmacokinetic parameters of compound B19 were fitted using DAS 3.0 software.

[0127] Table 5 shows the pharmacokinetic properties of compound B19; Figure 10 The plasma concentration-time curve (drug-time curve) of 50 mg / kg B19 administered intraperitoneally to rats is shown. The results indicate that the peak concentration of compound B19 in rats after intraperitoneal injection was 0.5 h, Cmax was 15471 ± 952.84 μg / L, half-life was 1.67 ± 0.02 h, AUC(0-t) was 17042.05 ± 1878.12 μg / L*h, and AUMC(0-t) was 21387.42 ± 2260.07 h*h*μg / L.

[0128] Table 5. Pharmacokinetic parameters of compound B19 in rats

[0129]

[0130] Example 14: Compound B19 can inhibit the growth of triple-negative breast cancer xenografts in nude mice.

[0131] 4T1 cell suspension was subcutaneously injected into the right anterior axilla of nude mice. A control group (solvent), a low-dose group (12.5 mg / kg), and a high-dose group (25.0 mg / kg) were established, and administration was repeated for 14 days. After administration, the mice were euthanized by cervical dislocation, the tumor was removed, photographed, and weighed. Results are as follows: Figure 11 As shown in A-11E.

[0132] Figure 11 The changes in body weight of mice in each group during the experimental period A indicated that compound B19 did not cause a significant reduction in body weight in nude mice during administration and had no obvious toxicity.

[0133] Figure 11 B shows the changes in tumor volume in each group of mice during the experimental period, indicating that compound B19 can significantly inhibit the growth of 4T1 cell xenografts.

[0134] Figure 11 C shows the tumor weight of each group of mice on day 14. Compound B19 significantly inhibited the weight of 4T1 cell xenografts. After treatment with compound B19, tumor growth in nude mice was significantly inhibited. The average tumor weight in the 12.5 mg / kg group was 0.453 ± 0.08 g, and the average tumor weight in the 25 mg / kg group was 0.174 ± 0.08 g, which were significantly lower than the average tumor weight of 1.249 ± 0.29 g in the control group.

[0135] Figure 11 D shows the size of xenografts in each group of mice on day 14, indicating that compound B19 can significantly inhibit the growth of 4T1 cell xenografts.

[0136] Figure 11 E shows the H&E staining of heart, liver, spleen, lung, kidney and tumor tissues in control and treatment mice. It shows that compound B19 has a significant killing effect on tumor tissue, but no obvious damage in organs. This indicates that compound B19 can effectively inhibit the growth of tumors in nude mice, while not producing obvious toxicity to tissues and organs.

[0137] In summary, compound B19 significantly inhibited the growth of 4T1 cell xenografts at doses of 12.5 mg / kg and 25 mg / kg, with inhibition rates (TGI) of 63.1% and 86.1%, respectively; and it also showed certain in vivo safety.

Claims

1. A CDK9-Cyclin T1 protein interaction inhibitor, characterized in that: Compounds with the following structures: 。 2. A pharmaceutical composition, characterized in that: A compound containing the structure shown in claim 1.

3. The application of the CDK9-Cyclin T1 protein interaction inhibitor of claim 1 or the pharmaceutical composition of claim 2, characterized in that: Application in the preparation of drugs for treating diseases related to CDK9, CDK9 / Cyclin T1 activity or expression levels.

4. The application of the CDK9-Cyclin T1 protein interaction inhibitor as described in claim 1 or the pharmaceutical composition as described in claim 2, characterized in that: Applications in the preparation of drugs for the treatment of, inhibition of or degradation of CDK9-related diseases.

5. The application as described in claim 3 or 4, characterized in that: The diseases mentioned include breast cancer, lung cancer, liver cancer, cervical cancer, and kidney cancer.

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