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CN111253371B - Small molecule regulating agent targeting CDK9, and synthesis method and application thereof - Google Patents

Small molecule regulating agent targeting CDK9, and synthesis method and application thereof Download PDF

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CN111253371B
CN111253371B CN201911196786.8A CN201911196786A CN111253371B CN 111253371 B CN111253371 B CN 111253371B CN 201911196786 A CN201911196786 A CN 201911196786A CN 111253371 B CN111253371 B CN 111253371B
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卞金磊
李志裕
仇夏秋
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China Pharmaceutical University
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Abstract

The invention discloses a targeted CDK9 small molecule regulator, which has a structure shown in a formula (I) and comprises a CDK9 inhibitor BAY-1143572, a connecting chain and pomalidomide, wherein the connecting chain is connected with BAY-1143572 and pomalidomide through a covalent bond. The preparation method is simple to operate and mild in condition, and the obtained chimera molecule has CDK9 degradation activity and an obvious anti-tumor effect.

Description

Small molecule regulating agent targeting CDK9, and synthesis method and application thereof
Technical Field
The invention relates to the field of chemical medicine, in particular to a small molecule regulator targeting CDK9 based on a protein degradation strategy, or a stereoisomer, a pharmaceutically acceptable salt, a hydrate, a prodrug or a solvate thereof, a synthetic method of the small molecule regulator, and a use method of the small molecule regulator in the field of medicine.
Technical Field
The ubiquitin-proteasome pathway (UPP) is a key pathway for regulating intracellular protein levels by degrading proteins, and is involved in the degradation of more than 80% of proteins in cells. The UPP pathway consists of ubiquitin, ubiquitin activating enzyme E1, ubiquitin transferase E2s, ubiquitin ligase E3s and proteasome. The ubiquitin-proteasome degradation pathway involves two major phases. The first stage is the interaction of ubiquitin with protein substrates: forming a high-energy thioester bond E1-ubiquitin complex, transferring activated ubiquitin (E1-ubiquitin complex) to E2s, releasing E1 to form the high-energy bond E2-ubiquitin complex, then identifying and combining a substrate by E3s, transferring ubiquitin on the E2-ubiquitin complex to E3s to form the high-energy bond complex, then forming an amido bond by the substrate through epsilon-amino of lysine and connecting the substrate with the ubiquitin, and adding ubiquitin molecules one by one to form a chain structure. The second stage is degradation of the substrate by the proteasome: the substrate ubiquitin chain interacts with the ubiquitin receptor of proteasome 19S, is gradually degraded, and under the action of ubiquitin C-terminal hydrolase, deubiquitinase and oligopeptidase, ubiquitin molecules are released (can participate in the circulation again).
CDK9 is one of the most critical molecules in the development of tumorigenesis, and is capable of forming complexes with the corresponding cyclin T/K, phosphorylating RNA polymerase II (RNAPII) and several negative transcription elongation factors (NELF and N-TEFs), thereby extending transcription from the initiation site. Meanwhile, the abnormal expression level and kinase activity of CDK9 can induce the up-regulation of a large number of pro-survival factors through phosphorylation RNAPII, so that the expression of various proteins (such as anti-apoptosis proteins Bcl-2, mcl-1 cyclin D1, p53 pathway-related proteins; NF-kB pathway-related proteins, VEGF related to tumor microenvironment, and the like) in tumor cells or the mRNA level of the proteins is abnormal. Blocking the phosphorylation of CDK9 on RNAPII and negative transcription elongation factor, reducing RNA synthesis, down-regulating the expression of short anti-apoptosis protein and related protein, inducing tumor cell apoptosis, and playing the role of anti-tumor.
Figure RE-GDA0002465180210000021
Research has shown that compound a (pomalidomide) analogues can bind to the E3 ubiquitin ligase cereblon, and ubiquitin can be used to label specific proteins, which are then grayed.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a small molecule regulator for targeted ubiquitination degradation of CDK9 protein.
The invention also aims to solve the technical problem of providing the application of the small molecule regulator for targeted ubiquitination degradation of CDK9 protein in prevention and treatment of tumors.
The technical scheme is as follows: in order to solve the technical problems, the invention provides the following technical scheme:
the structure of the small molecule regulator is shown as a formula (I), and the small molecule regulator comprises a CDK9 inhibitor BAY-1143572, a connecting chain and pomalidomide, wherein the connecting chain is connected with BAY-1143572 and pomalidomide through covalent bonds;
Figure RE-GDA0002465180210000022
wherein the CDK9 inhibitor BAY-1143572 binds CDK9;
pomalidomide binds to ubiquitin ligase (CRBN).
Further, the structure of the small molecule regulator is shown as a formula (II), wherein a sulfinyl imino group of a CDK9 inhibitor BAY-1143572 is covalently combined with a connecting chain;
Figure RE-GDA0002465180210000031
further, the structure of the small molecule regulator is shown in a formula (III), wherein the fluorine atom end of the BAY-1143572 inhibitor CDK9 is replaced by piperazine and then is covalently combined with a connecting chain;
Figure RE-GDA0002465180210000032
wherein the structure of the connecting chain is shown as a formula (IV), or a stereoisomer, a pharmaceutically acceptable salt, a hydrate, a prodrug or a solvate thereof,
Figure RE-GDA0002465180210000033
wherein,
x is an integer selected from 0 to 14;
m is an integer selected from 0 to 14;
n is an integer selected from 0 to 14;
z is an integer selected from 0 to 4;
a is CH 2 Or C (O);
b is CH 2 Or
Figure RE-GDA0002465180210000034
Wherein the connecting chain passes beside A
Figure RE-GDA0002465180210000035
Covalently bound to the CDK9 inhibitor BAY-1143572 and through CH 2 Beside the table
Figure RE-GDA0002465180210000036
Covalently bound to the ubiquitin ligase (CRBN) ligand pomalidomide.
Preferably, the structure of the small molecule regulator is shown as formulas (A01), (B01), (C01), (D01) and (E01);
Figure RE-GDA0002465180210000041
a pharmaceutical composition comprising one or more of any of the small molecule modulators, stereoisomers, and pharmaceutically acceptable salts thereof.
Wherein the dosage form of the pharmaceutical composition comprises: such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, and ampoules (solutions or suspensions), and the like, preferably tablets, capsules, liquids, suspensions, and ampoules (solutions or suspensions).
The application of the targeted CDK9 small molecule regulating agent in preparing antitumor drugs. Through screening in enzymatic degradation experiments, it was found that some embodiments involve 80% to 100% CDK9 target protein degradation, and some embodiments involve 5% to 60% CDK9 target protein degradation. Accordingly, the compounds of the invention are useful in diseases associated with aberrant expression of CDK9 activity, such as various cancers.
The application of the targeted CDK9 small molecule regulator in preparing a medicament for preventing, treating or prognosing diseases related to abnormal expression of CDK 9.
Wherein the disease comprises: breast cancer, non-small cell lung cancer, acute lymphocytic leukemia, acute myelogenous leukemia, hypoplastic acute leukemia, adult T-lymphocytic leukemia, plasma cell leukemia, mast cell leukemia, eosinophilic leukemia, basophilic leukemia, mixed cell leukemia, central nervous system leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome.
Has the advantages that:
the invention discloses a small molecule regulator targeting CDK9 based on a protein degradation strategy, which can be used as a unique anticancer drug or be used together with one or more other anticancer drugs. The preparation method is simple to operate and mild in condition, and the obtained chimera molecule has CDK9 degradation activity and an obvious anti-tumor effect.
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FIG. 1 inhibitory Effect of some of the compounds of the present invention on the proliferation of MV4-11 cells.
FIG. 2 potency of a partial compound of the invention to degrade CDK9 at an administration concentration of 500 nM.
FIG. 3 shows the ability of a partial compound of the invention to degrade CDK9 and the like at a dose of 500 nM.
FIG. 4 shows the degradation ability of compound A03 of the present invention on various anti-apoptotic proteins.
Figure 5 the protein degradation effect of compound a03 of the invention fades away after removal of the drug.
Detailed Description
Example 1
Figure RE-GDA0002465180210000061
Preparation of 5- ((3- ((4- (4-fluoro-2-methoxyphenyl) -1,3, 5-triazin-2-yl) amino) benzyl) (methyl) (oxo) -sulfinamino) amino) -5-oxoglutaric acid (1-3)
3ml of DMF was measured to dissolve Compound 1-1 (BAY-1143572) (200mg, 0.52mmol), 100mg of Compound 1-2 (136mg, 1.03mmol) and DMTMM (171mg, 0.62mmol) were added, and stirring was carried out at 35 ℃ for 2 hours. Diluting with water, extracting with ethyl acetate, washing the organic layer with saturated brine, drying over anhydrous sodium sulfate, filtering, and concentrating under reduced pressure. The crude product was purified by silica gel column chromatography (0-10% MeOH/DCM) to give a pale white solid (123mg, 48%). HRMS (ESI) M/z [ M + H ]] + .Calcd for C 23 H 24 FN 5 O 5 S 501.1482,found 501.1478。
N 1 - (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-phthalimido-4-yl) amino) ethyl) -N 3 Preparation of (- ((3- ((4- (4-fluoro-2-methoxyphenyl) -1,3, 5-triazin-2-yl) amino) benzyl) (methyl) (oxo) sulfonimidyl) propanediamide (A01)
2ml of DCM was measured and added with compound 1-3 (123mg, 0.25mmol), compound 1-4 (77.59 mg,0.25 mmol), HATU (186.31mg, 0.49mmol) and DIPEA (158.03mg, 1. Mu.l)225 mmol), stirring at room temperature for 30min. Diluted with water, extracted with DCM, the organic layer washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (0-10% MeOH/DCM) to give a pale yellow solid (37mg, 19%). 1 H NMR(300MHz,DMSO),δ:1.83-1.87(m,2H,CH 2 ), 2.05-2.09(m,2H,CH 2 ),2.14-2.25(m,4H,CH 2 ×2),2.34(m,2H,CH 2 ),2.38(m,2H, CH 2 ),2.50(s,3H,CH 3 ),3.35(t,2H,CH 2 ),3.42(t,2H,CH 2 ),3.79(s,3H,CH 3 ),3.83 (s,2H,CH 2 ),4.44(t,1H,CH),6.79(s,1H,NH),6.97-7.59(m,10H,ArH×10),7.70(s, 1H,NH),9.09(s,1H,triazine-H),9.43(s,1H,NH),11.06(s,1H,NH)。HRMS(ESI): m/z[M+H] + .Calcd for C 38 H 38 FN 9 O 8 S 799.2548,found 799.2539。
Example 2
Figure RE-GDA0002465180210000071
Preparation of imino (3- ((4- (2-methoxy-4- (piperazin-1-yl) phenyl) -1,3, 5-triazin-2-yl) amino) benzyl) (methyl) -sulfonamidone (1-6)
Compound 1-1 (BAY-1143572) (1.5g, 3.88mmol) was dissolved in 25ml of DMSO, and compound 1-5 (1.66g, 19.28mmol) and DIPEA (2.49g, 19.28mmol) were added and stirred at 80 ℃ for 4 hours. The mixture was filtered and passed through reverse phase HPLC (0-100% MeOH/H) 2 O) to yield a yellow solid (1.35 g, 77%). HRMS (ESI) M/z [ M + H ]] + .Calcd for C 22 H 27 N 7 O 2 S 453.1947,found 453.1953。
Example 3
Figure RE-GDA0002465180210000072
Preparation of 2-bromo-N- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-phthalimido-4-yl) amino) ethyl) acetamide (1-8)
15ml of DCM lysis compound was metered inTo 1-4 (200mg, 0.63mmol) of substance were added compounds 1-7 (87 mg, 0.63mmol), HATU (479mg, 1.26mmol) and DIPEA (406 mg, 3.15mmol), and the mixture was stirred at room temperature for 30min. Diluted with water, extracted with DCM, the organic layer washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (0-10% MeOH/DCM) to give a yellow solid (124mg, 45%). HRMS (ESI) M/z [ M + H ]] + .Calcd for C 17 H 17 BrN 4 O 5 436.0382,found 436.0377。
Preparation of N- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-phthalimido-4-yl) amino) ethyl) -2- (4- (3-methoxy-4- (4- ((3- ((S-methylsulfonimidoyl) methyl) phenyl) amino) -1,3, 5-triazin-2-yl) phenyl) piperazin-1-yl) acetamide (B01)
Compound 1-8 (124mg, 0.28mmol) was dissolved in 5ml of DMSO, compound 1-6 (127 mg, 0.28mmol) and DIPEA (181mg, 1.4 mmol) were added, and the mixture was stirred at 78 ℃ for 4 hours. The mixture was filtered and passed through reverse phase HPLC (0-100% MeOH/H) 2 O) to yield a yellow solid (14mg, 7%). 1 H NMR(300MHz,DMSO),δ:2.04-2.27(m,4H,CH 2 ×2),2.50(s,3H,CH 3 ),2.72(t,4H, CH 2 ×2),3.18(s,2H,CH 2 ),3.38(t,4H,CH 2 ×2),3.41(t,2H,CH 2 ),3.63(t,2H,CH 2 ), 3.79(s,3H,CH 3 ),3.86(s,2H,CH 2 ),4.46(t,1H,CH),6.50-6.61(m,2H,ArH),6.82 (s,1H,NH),6.98-7.53(m,8H,ArH×8),7.83(s,1H,NH),9.11(s,1H,triazine-H), 9.45(s,1H,NH),11.08(s,1H,NH)。HRMS(ESI):m/z[M+H] + .Calcd for C 39 H 43 N 11 O 7 S 809.3068,found 809.3058。
Example 4
Figure RE-GDA0002465180210000081
Preparation of N- ((3- ((4- (4-fluoro-2-methoxyphenyl) -1,3, 5-triazin-2-yl) amino) benzyl) (methyl) (oxo) -sulfinamino) pent-4-ynylamide (1-10)
3ml of DMF was measured and the compound 1-1 (BAY-1143572) (200mg, 0.52mmol) was dissolved, and 100mg was addedCompound 1-9 (101mg, 1.03mmol) and DMTMM (171mg, 0.62mmol) were stirred at 35 ℃ for 2h. Diluting with water, extracting with ethyl acetate, washing the organic layer with saturated brine, drying over anhydrous sodium sulfate, filtering, and concentrating under reduced pressure. The crude product was purified by silica gel column chromatography (0-10% MeOH/DCM) to give a white solid (123mg, 48%). HRMS (ESI) M/z [ M + H ]] + .Calcd for C 23 H 22 FN 5 O 3 S 467.1427,found 467.1438。
Example 5
Figure RE-GDA0002465180210000091
Preparation of 4-bromo-N- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-phthaloyl-4-yl) amino) ethyl) butanamide (1-12)
Compound 1-4 (200mg, 0.63mmol) was dissolved in 15ml of DCM, and compound 1-11 (105 mg, 0.63mmol), HATU (479mg, 1.26mmol) and DIPEA (406mg, 3.15mmol) were added and stirred at room temperature for 30min. Diluted with water, extracted with DCM, and the organic layer washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (0-10% MeOH/DCM) to give a yellow solid (132mg, 45%). HRMS (ESI) M/z [ M + H ]] + .Calcd for C 17 H 17 BrN 4 O 5 464.0695,found 464.0692。
Preparation of 4-azido-N- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-phthalimido-4-yl) amino) ethyl) butanamide (1-14)
Compound 1-12 (132mg, 0.28mmol) was dissolved in 3ml of DMSO, and compound 1-13 (37mg, 0.57mmol) was added thereto, followed by stirring overnight at room temperature. Diluted with water, extracted with DCM, the organic layer washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. A yellow solid (112mg, 94%) was obtained. HRMS (ESI) M/z [ M + H ]] + .Calcd for C 19 H 21 N 7 O 5 427.1604,found 427.1601。
Preparation of N- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-phthalimido-4-yl) amino) ethyl) -4- (4- (3- (((3- ((4- (4-fluoro-2-methoxyphenyl) -1,3, 5-triazin-2-yl) amino) benzyl) (methyl) (oxo) -sulfonylimino) amino) -3-oxopropyl) -1H-1,2, 3-triazol-1-yl) butanamide (C01)
2ml of HMPA was measured to dissolve Compound 1-10 (122mg, 0.26mmol), and Compound 1-14 (112mg, 0.26mmol), cuI (5.69mg, 0.03mmol), DIPEA (1.3ml, 13mmol) were added and stirred at room temperature overnight. Diluted with water, extracted with DCM, the organic layer washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (0-10% MeOH/DCM) to give a yellow solid (32mg, 14%). 1 H NMR(300MHz,DMSO),δ:2.02-2.34(m,8H,CH 2 ×2),2.50(s,3H, CH 3 ),2.73(t,2H,CH 2 ),2.88(t,2H,CH 2 )3.41(t,2H,CH 2 ),3.63(t,2H,CH 2 ),3.69(s, 3H,CH 3 ),3.75(s,2H,CH 2 ),4.48(t,1H,CH),4.56(t,2H,CH 2 ),6.75(s,1H,NH), 6.97-7.59(m,10H,ArH×10),7.85(s,1H,1,2,3-triazol-H),8.03(s,1H,NH),9.15(s, 1H,triazine-H),9.50(s,1H,NH),11.07(s,1H,NH)。HRMS(ESI):m/z[M+H] + .Calcd for C 42 H 43 FN 12 O 8 S 894.3032,found 894.3030。
Example 6
Figure RE-GDA0002465180210000101
Preparation of (3- ((4- (4- (4- (but-3-yn-1-yl) piperazin-1-yl) -2-methoxyphenyl) -1,3, 5-triazin-2-yl) amino) benzyl) (imino) (methyl) -sulfonamidone (1-16)
Compound 1-6 (200mg, 0.44mmol) was dissolved in 5ml of DMSO, and compound 1-6 (58 mg, 0.44mmol) and DIPEA (285mg, 2.2mmol) were added and stirred at 78 ℃ for 4 hours. Diluted with water, extracted with DCM, the organic layer washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (0-10% MeOH/DCM) to give a yellow solid (192mg, 95%). HRMS (ESI) M/z [ M + H ]] + .Calcd for C 26 H 21 N 7 O 2 S 505.2260,found 505.2255。
Preparation of N- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-phthalimido-4-yl) amino) ethyl) -4- (4- (2- (4- (3-methoxy-4- (4- ((3- ((S-methylsulfonimidoyl) methyl) phenyl) amino) -1,3, 5-triazin-2-yl) phenyl) piperazin-1-yl) ethyl) -1H-1,2, 3-triazol-1-yl) butanamide (D01)
Compound 1-16 (192mg, 0.38mmol) was dissolved in 2ml of HMPA, and compound 1-14 (162mg, 0.38mmol), cuI (7.59mg, 0.04mmol) and DIPEA (1.9ml, 19mmol) were added thereto, and the mixture was stirred at room temperature overnight. Diluted with water, extracted with DCM, the organic layer washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (0-10% MeOH/DCM) to give a yellow solid (46mg, 13%). 1 H NMR(300MHz,DMSO),δ:2.00-2.38(m,8H,CH 2 ×4),2.50(s,3H, CH 3 ),2.65-2.69(t,4H,CH 2 ×2),3.41-3.44(m,10H,CH 2 ×5),3.67(t,2H,CH 2 ),3.75 (s,3H,CH 3 ),3.88(s,2H,CH 2 ),4.42(t,2H,CH 2 ),4.48(t,1H,CH),6.75(s,1H,NH), 6.96-7.58(m,10H,ArH×10),7.81(s,1H,1,2,3-triazol-H),8.02(s,1H,NH),9.14(s, 1H,triazine-H),9.40(s,1H,NH),11.04(s,1H,NH)。HRMS(ESI):m/z[M+H] + .Calcd for C 45 H 52 N 14 O 7 S 932.3864,found 932.3862。
Example 7
Figure RE-GDA0002465180210000111
N 1 - (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-phthalimido-4-yl) amino) ethoxy) ethyl) -N 5 Preparation of (- ((3- ((4- (4-fluoro-2-methoxyphenyl) -1,3, 5-triazin-2-yl) amino) benzyl) (methyl) (oxo) sulfonimidyl) propanediamide (E01)
Compound 1-3 (123mg, 0.25mmol), compound 1-15 (112mg, 0.25 mmol), HATU (186.31mg, 0.49mmol), and DIPEA (158.03mg, 1.225mmol) were added to 2ml of DCM, and stirred at room temperature for 30min. Diluted with water, extracted with DCM, and the organic layer washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (0-10% MeOH/DCM) to give a light yellow solid (44mg, 19%). 1 H NMR(300MHz,DMSO),δ:2.01-2.38(m,10H,CH 2 ×5),2.48(s, 3H,CH 3 ),3.28(t,2H,CH 2 ),3.46-3.67(m,14H,CH 2 ×7),3.77(s,3H,CH 3 ),3.81(s, 2H,CH 2 ),4.46(t,1H,CH),6.83(s,1H,NH),6.95-7.54(m,10H,ArH×10),7.93(s, 1H,NH),9.18(s,1H,triazine-H),9.50(s,1H,NH),11.21(s,1H,NH)。HRMS(ESI): m/z[M+H] + .Calcd for C 38 H 38 FN 9 O 8 S 932.3378,found 932.3367。
The following are the pharmacological tests and results of some of the compounds of the invention:
example 8: determination of protein degradation Activity of partial Compounds of the present invention
The experimental method comprises the following steps:
MV4-11 cells were treated with DMSO as control, and after 6 hours with 500nM of BAY-1143572 (BAY in fig. 2), 500nM of pomalidomide (p.m. in fig. 2), 250nM of THAL-SNS-032 (Olson cm. Et al. Nat. Chem.biol.,2018, 14) and 500nM of the partial chimera compound of the invention (i.e., compounds a01, a02, a03, a04, B01, B02, B03, B04), the cells were washed and resuspended in RIPA lysis buffer (formulated from RIPA lysate (strong): protease inhibitor PMSF =99: 1) and lysed on ice for 30min. The lysate was centrifuged at 12500rpm for 30min and the supernatant was taken. Carrying out protein quantification by using a BCA kit, adding a sample buffer solution, and then placing the sample in a metal bath at 100 ℃ for 8min to denature the protein; equal amounts of protein were loaded onto the gel, the proteins on the gel were transferred to a PVDF membrane (polyvinylidene difluoride membrane), coated with antibodies to CDK9 and β -actin, and developed.
As can be seen from figure 2, compounds a02, a03 of the invention showed complete degradation of CDK9 at 500nM, and a04, B01, a01 showed partial degradation of CDK9 at 500nM, whereas no degradation was observed with the compounds BAY-1143572 (BAY in figure 2) and pomalidomide (p.m. in figure 2).
Alternatively, MV4-11 cells were controlled with DMSO, and 500nM of partial chimeric compound a03 of the invention was administered after 6 hours, the cells were washed and resuspended in RIPA lysis buffer (formulated from RIPA lysate (strong): protease inhibitor PMSF = 99) and lysed on ice for 30min. The lysate was centrifuged at 12500rpm for 30min and the supernatant was taken. Protein quantification is carried out by using a BCA kit, and after a sample is added with a loading buffer solution, the sample is placed in a metal bath at 100 ℃ for 8min to denature the protein; equal amounts of protein were loaded onto the gel, proteins on the gel were blotted onto a PVDF membrane (polyvinylidene difluoride membrane), coated with antibodies to CDK9, CDK1/2, CDK5, CDK6, CDK7, CDK8, and β -actin, and developed.
As can be seen in fig. 3, compound a03 of the present invention selectively degrades CDK9 at a dosing concentration of 500 nM.
Alternatively, MV4-11 cells were controlled with DMSO, dosed with 1, 10, 30, 100, 250, 500nM of the partial chimera compound a03 of the invention, and after 6 hours, the cells were washed and resuspended in RIPA lysis buffer (prepared from RIPA lysate (strong): protease inhibitor PMSF = 99. The lysate was centrifuged at 12500rpm for 30min and the supernatant was taken. Protein quantification is carried out by using a BCA kit, and after a sample is added with a loading buffer solution, the sample is placed in a metal bath at 100 ℃ for 8min to denature the protein; equivalent amount of protein was loaded on the gel, the protein on the gel was transferred to PVDF membrane (polyvinylidene fluoride membrane), and anti-apoptotic proteins MCL-1, PARP-1, BCL-2, XIAP, caspase-3 antibody and β -actin antibody of CDK9 pathway were applied for visualization.
As can be seen from fig. 4, compound a03 of the present invention has concentration-dependent degradation of various anti-apoptotic proteins.
Alternatively, MV4-11 cells were controlled with DMSO, and 250nM of the partial chimera compound a03 of the present invention was administered after 6 hours, the cells were centrifuged and fresh medium was added again, the cells were washed at 0, 1,3, 6, 12 hours and resuspended in RIPA lysis buffer (prepared from RIPA lysate (strong): protease inhibitor PMSF = 99). The lysate was centrifuged at 12500rpm for 30min and the supernatant was taken. Protein quantification is carried out by using a BCA kit, and after a sample is added with a loading buffer solution, the sample is placed in a metal bath at 100 ℃ for 8min to denature the protein; equal amounts of protein were loaded onto the gel, the proteins on the gel were transferred to a PVDF membrane (polyvinylidene difluoride membrane), coated with antibodies to CDK9 and β -actin, and developed.
As can be seen from fig. 5, the protein degradation effect of compound a03 of the present invention is gradually lost after the removal of the drug, and the target protein CDK9 is quickly restored to the normal level.
Example 9: cell proliferation inhibitory Activity of some Compounds of the present application
The experimental method comprises the following steps:
MTT powder was prepared into 5mg/ml stock solution with PBS, sterilized by filtration through a small filter tip of 0.22 μm, and then dispensed into sterilized EP tubes and stored at-20 ℃ in the dark. Selecting cells in a nearly logarithmic growth phase, centrifuging the cells to precipitate cells and removing supernatant, and re-suspending the cells in a normal culture medium; the cell counting plate counts, for MV4-11 cells, the cell concentration was adjusted to 40000 cells/ml using culture medium, then 100. Mu.l of cell suspension was randomly added to a 96-well plate, at this time the number of cells per well was about 4000, the drug was configured to 6 concentrations from high to low, 3 replicates per experimental group were set, and 100. Mu.l of drug was added per well. The group with no cells and medium was a blank group, and the group with cells and medium but no medium was a negative group. After incubation in an incubator for 72h, 20. Mu.l of MTT stock solution per well under dark conditions, 37 ℃,5% of the cells were incubated for further 4h under CO2 conditions to allow the formation of crystals. After the crystals are generated, slightly sucking out the original culture medium, adding 150 mu l of DMSO for dissolving, and shaking at room temperature for 5min to ensure that the crystals are completely dissolved; the 96-well plate was quickly removed and the absorbance at 492nm was measured in an ELISA. Analysis of data by nonlinear regression (Graphpad prism) to generate IC 50 The value is obtained. Measuring the proliferation inhibitory activity of the compound on cells using the method described above, wherein IC 50 The CDK9 inhibitor BAY-1143572, currently in phase I clinical trials, was used as a control as follows.
The results of the experiment are shown in the table.
TABLE 1 proliferation inhibitory Activity of partial Compounds of the invention on MV4-11 cells IC 50
Figure RE-GDA0002465180210000141
As can be seen from Table 1 and FIG. 1, some of the compounds of the present invention have excellent inhibitory activity against the proliferation of MV4-11 cells.

Claims (8)

1. The targeted CDK9 small molecule regulator is characterized by having a structure shown in a formula (II), wherein a sulfinyl imino group of a CDK9 inhibitor BAY-1143572 is covalently bonded with a connecting chain;
Figure DEST_PATH_IMAGE001
the structure of the connecting chain is shown as a formula (IV),
Figure 650540DEST_PATH_IMAGE002
wherein,
x is an integer selected from 0 to 14;
m is an integer selected from 0 to 14;
n is an integer selected from 0 to 14;
z is an integer selected from 0 to 4;
a is CH 2 Or C (O);
b is CH 2 Or
Figure DEST_PATH_IMAGE003
；
Wherein the linker is covalently bound to the CDK9 inhibitor BAY-1143572 through the side of A and through CH 2 The next one covalently binds to the ubiquitin ligase (CRBN) ligand pomalidomide.
2. The targeted CDK9 small molecule modulator according to claim 1, which has a structure shown in formula (III), wherein the fluorine atom end of the CDK9 inhibitor BAY-1143572 is replaced by piperazine and then is covalently bonded with a connecting chain;
Figure 932792DEST_PATH_IMAGE004
。
3. the targeted CDK9 small molecule modulator according to claim 1, wherein the structure of the small molecule modulator is shown as formula (A01), (B01), (C01), (D01), (E01);
Figure 869787DEST_PATH_IMAGE006
（A01）
Figure DEST_PATH_IMAGE007
（B01）
Figure DEST_PATH_IMAGE008
（C01）
Figure DEST_PATH_IMAGE009
（D01）
Figure DEST_PATH_IMAGE010
（E01）。
4. a pharmaceutical composition characterized by: comprising the small molecule modulator of any one of claims 1-3.
5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is in a dosage form comprising: tablets, pills, powders, liquids, suspensions, emulsions, granules.
6. The use of the targeted CDK9 small-molecule regulator as defined in any one of claims 1 to 3 in the preparation of an antitumor medicament.
7. Use of the targeted CDK9 small molecule modulator of any one of claims 1 to 3 in the preparation of a medicament for preventing, treating or prognosing a disease associated with abnormal CDK9 expression.
8. The use according to claim 7, wherein said disease comprises: breast cancer, non-small cell lung cancer, acute lymphocytic leukemia, acute myelogenous leukemia, hypoplastic acute leukemia, adult T-lymphocytic leukemia, plasma cell leukemia, mast cell leukemia, eosinophilic leukemia, basophilic leukemia, mixed cell leukemia, central nervous system leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome.
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