A compound for targeted degradation of CDK6 and its application

Abstract

The present invention belongs to the field of medicine, and specifically relates to a compound and application for targeted degradation of CDK6. The present invention first provides a compound or a pharmaceutically acceptable salt thereof for targeted degradation of CDK6 as shown in formula (I), wherein the compound inhibits the expression of CDK6 downstream proteins, blocks the cell cycle in the G0 / G1 phase, induces cell apoptosis, and can be used as an inhibitor of CDK6 for the preparation of anti-tumor drugs; secondly, the present invention also performs PROTAC modification on the above-mentioned compound to obtain a PROTAC compound, which is used as a CDK6 degrader, further improves the degradation activity of CDK6, and then promotes the apoptosis of tumor cells, and has the potential to become an effective treatment for tumors.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a compound for targeted degradation of CDK6 and its application. Background Art

[0002] At present, most of the anticancer drugs widely used in clinical practice are small molecule inhibitor compounds of various types. Small molecule inhibitors used in clinical practice mainly include platinum drugs, fluorouracil, irinotecan, paclitaxel and other drugs. However, the mechanism of action of small molecule inhibitors is mostly to occupy or block the "active site" of the protein (such as competitive inhibition and metabolic inhibition of the ATP binding site of protein kinase). Usually, only when more than 90% of the target protein binds to the inhibitor can the drug-related inhibitory effect be achieved, so large doses must be administered to achieve efficacy. During this period, it is difficult for small molecule inhibitors to distinguish between tumor cells and rapidly dividing non-malignant cells, resulting in serious toxic side effects. In addition, although many therapeutic targets have been identified in the human proteome, more than 80% of proteins cannot be used as small molecule targets (such as transcription factors, scaffold proteins, etc.), which are called "undruggable" proteins. The most common reason is that many proteins do not have "active sites", so small molecule drugs are less likely to exert inhibitory effects, which is one of the reasons why the development of small molecule inhibitors is increasingly restricted. However, protease-targeted chimeras (PROTACs) can better avoid these problems.

[0003] PROTACs are composed of three parts: ① target protein (POI) binding ligand, ② E3 ubiquitin ligase recruitment ligand, and ③ Linker connecting the first two, recruiting E3 ligase to the surface of the target protein, and using the inherent ubiquitination process of the organism to target the protein for degradation. Since PROTACs recruit E3 ubiquitin ligases, and E3 ubiquitin ligases ubiquitinate the target protein, the degradation of the target protein is a "transient process", so it can be used as a catalyst to repeatedly degrade the target protein at low concentrations, thereby achieving the cycle and efficient degradation of the target protein at low concentrations. Moreover, PROTACs can greatly expand the range of target proteins, and do not need to have "active sites" like small molecule inhibitors in the past, as long as the small molecule can bind to the appropriate "crack" or specific structure in the target protein, regardless of whether it is an "active site". Small molecules can recruit E3 ubiquitin ligands, regardless of whether it is an "active site". E3 ubiquitin ligands can be recruited to degrade target proteins using the ubiquitin-proteosome system. PROTACs technology has become an increasingly popular research activity due to its unique advantages of targeting non-drug-forming proteins, low-dose catalysis, and high performance. However, the discovery of existing PROTACs is mainly achieved through the modification of existing targeted inhibitors. The exploration of a large number of "undruggable" proteins and proteins without clear targeted inhibitors is limited, so a suitable method is needed to try to explore them. Virtual screening of target proteins using virtual combinatorial chemistry databases is one of the methods worth considering.

[0004] Using virtual combinatorial chemistry to establish a "REAL" database to discover targeted small molecule inhibitors is an emerging and promising method that uses computers to establish a very large and realistic virtual combinatorial chemistry database for virtual screening of targeted proteins. This method has led to the discovery of active small molecules at the pM level. This method is more realistic and reasonable than virtual screening methods using public databases because: First, the molecules in the database are often built from existing small molecule blocks through fixed chemical reaction lines. Second, the number of different molecular blocks that can be virtually combined into new molecular combinations is very large, which has a wider potential than existing databases. Therefore, it may be valuable to apply this method to the discovery of PROTACs.

[0005] The present invention provides a potential cyclin-dependent kinase 6 (CDK6) targeted inhibitor, which inhibits the expression of CDK6 downstream proteins, blocks the cell cycle in the G0 / G1 phase, and induces cell apoptosis; and compound 10 is modified with PROTAC to obtain a targeted CDK6 degrader PROTAC compound, which further improves the degradation activity of CDK6, thereby promoting the apoptosis of tumor cells, and has the potential to become an effective treatment for tumors. Summary of the invention

[0006] In view of the above technical problems, the purpose of the present invention is to provide a potential cycloprotein-dependent kinase 6 (CDK6) targeted inhibitor and application thereof, which specifically includes the following contents:

[0007] In a first aspect, the present invention provides a compound for targeted inhibition of CDK6 or a pharmaceutically acceptable salt thereof, wherein the structural formula of the compound is shown in the following formula (I):

[0008]

[0009] Wherein, the R1 is selected from

[0010] The R2 is selected from

[0011] The R3 is selected from -CH2CCH, -CH2CHCH2, -CH2CH2CH3, -CH2CH2SCH3, -CH2CH(CH3)2, -CH2CH2CH(CH3)2, or R3 represents -CH2CH2- which forms a three-membered ring with C at the position where R3 is substituted in formula (I).

[0012] Preferably, the structural formula of the compound is any one of the following:

[0013]

[0014] In a second aspect, the present invention provides use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a CDK6 inhibitor.

[0015] In a third aspect, the present invention provides use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

[0016] In a fourth aspect, the present invention provides a PROTAC compound or a pharmaceutically acceptable salt thereof for targeting the degradation of CDK6, wherein the PROTAC compound is obtained by modifying the compound described in the first aspect above with a ligand molecule of an E3 ligase.

[0017] Preferably, the ligand molecule of the E3 ligase is obtained by connecting the compound described in the first aspect via a Linker.

[0018] Preferably, the ligand molecule of the E3 ligase comprises the following structure:

[0019]

[0020] Preferably, the structural formula of the PROTAC compound is as shown in the following formula (II):

[0021]

[0022] Wherein, R4 is -CH2CCH or -CH2CH2CH(CH3)2;

[0023] The Linker R n Includes the following structure:

[0024]

[0025] Preferably, the structural formula of the PROTAC compound is any one of the following:

[0026]

[0027] In a fifth aspect, the present invention provides a use of the PROTAC compound or a pharmaceutically acceptable salt thereof described in the fourth aspect in the preparation of a CDK6 degrader.

[0028] In a sixth aspect, the present invention provides a use of the PROTAC compound or a pharmaceutically acceptable salt thereof described in the fourth aspect in the preparation of an anti-tumor drug.

[0029] In a seventh aspect, the present invention provides a pharmaceutical composition, comprising the compound described in the first aspect above or a pharmaceutically acceptable salt thereof, and / or the PROTAC compound described in the third aspect above or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or a combination thereof.

[0030] The beneficial effects of the present invention are as follows: first, the present invention provides a potential cyclin-dependent kinase 6 (CDK6) targeted inhibitor compound, which inhibits the expression of CDK6 downstream proteins, blocks the cell cycle in the G0 / G1 phase, and induces cell apoptosis, and can be used as a CDK6 inhibitor for the preparation of anti-tumor drugs; secondly, the present invention modifies the above compound with PROTAC to obtain a PROTAC compound, which targets and degrades CDK6, and as a CDK6 degrader, further improves the degradation activity of CDK6, thereby promoting the apoptosis of tumor cells, and has the potential to become an effective treatment for tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 Experimental research process; A is the main construction process of CCSMD; B is the process of using CCSMD to virtually screen CDK6 and obtain the molecules to be synthesized; C is the process of structural optimization and PROTAC modification based on the best active compound 10 in the virtual screening results;

[0033] Figure 2 Evaluation of the biological activity of compound 10; A is the result of cell cycle detection; B is the result of protein level detection; C is the result of Hoechst / PI double staining to detect cell apoptosis or necrosis; D is the result of FITC-Annexin V and PI double staining to detect cell apoptosis;

[0034] Figure 3 Based on the structural optimization of compound 10; wherein A is the compound divided into three parts I, II and III for structural optimization; B is the structure and synthesis process of each compound;

[0035] Figure 4 Evaluation of the biological activity of compound 2f; A is the cytotoxicity test of compound 2f on gastric cancer AGS and normal cells GES-1; B is the cell cycle detection result; C is the protein level detection result; D is the cell apoptosis or necrosis detection result; E is the apoptosis detection result using FITC-Annexin V and PI double staining;

[0036] Figure 5 PROTAC modification of compound 10; wherein A is the docking of compound 10 with CDK6 protein; B is the PROTAC modification process of compound 10;

[0037] Figure 6 Evaluation of the biological activity of 10-PROTAC; A is the cytotoxicity experiment of 10-PROTAC on gastric cancer AGS and normal cells GES-1; B is the cell cycle detection result; C is the protein level detection result;

[0038] Figure 7PROTAC modification and bioactivity evaluation of compound 2f; A is the modification of 2f with PROTAC and replacement with linkers of different lengths, and the IC50s of each compound on AGS cells are detected; B is the cytotoxicity test of 2f-PROTAC on gastric cancer AGS and normal cells GES-1; C is the cell cycle test result; D is the protein level test result;

[0039] Figure 8 Acute toxicity experiment of 2f-PROTAC in mice; A shows the changes in body weight gain of KM mice during the experiment; B shows the HE-stained sections of the heart, liver, spleen, lungs and kidneys of KM mice. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. Specific conditions are not specified in the embodiments, and the conditions recommended by the manufacturer are carried out according to conventional conditions or manufacturers. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0041] The experimental methods described in the following examples are as follows:

[0042] (1) Cell culture

[0043] AGS gastric cancer cells were purchased from ATCC (LOT: 70012225, Manassas, VA), and GES-1 normal gastric mucosal cells were laboratory owned cells. AGS and GES-1 were grown in RPMI medium 1640 from HyClone. All culture media were supplemented with 10% heat-inactivated fetal bovine serum (Biological Industries, USA) and 100 units / ml penicillin and 100 μg / ml streptomycin (Solarbio, China).

[0044] (2) Cell antiproliferation test

[0045] 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) was used to determine the growth inhibitory effect of the screened molecules on AGS cells. The cells were plated in 96-well plates at 10,000 cells / 100 μL / well. After 24 hours, the cells were treated with compounds in fresh medium. After 48 hours, 10 μL of MTT solution (5 mg / mL) was added to each well, the medium was removed, 100 μL of DMSO was added as a lysis solution, and the absorbance was measured at 490 nm after shaking the wells.

[0046] (3) Hoechst / PI fluorescence staining experiment

[0047] Cultured AGS cells were inoculated in 12-well plates, with 50,000 cells per well. After 24 hours of culture, the original culture medium was aspirated, and culture medium containing appropriate concentrations of compounds was added for 48 hours of culture, digested with trypsin, and collected by centrifugation. 5 μL Hoechst staining solution and 5 μL PI staining solution were added to each sample, mixed well, and incubated in an ice bath for 30 minutes. The corresponding parameters of the flow cytometer (NovoCyte Quanteon) were adjusted, 10,000 cells were collected and the detection was terminated. The data were recorded and analyzed using NovoExpress software.

[0048] (4) Cell apoptosis experiment

[0049] AGS cells were seeded in six-well plates at 3 × 10 5 cells and cultured for 24 hours. Aspirate the original culture medium and add the culture medium containing the corresponding concentration of compound for 48 hours of culture. After the culture is completed, add 1mL of EDTA-free trypsin to each well to digest the cells. Collect the cells in PBS buffer, centrifuge at 1200rpm for 5 minutes, and discard the supernatant. Resuspend the cells (200μL) with Binding buffer in the cell apoptosis detection kit, then aspirate 100μL of cell suspension into the flow tube, add 5μL FITC-Annexin V staining solution, incubate at room temperature in the dark for 10 minutes, and add 10μL PI staining solution and 400μL PBS buffer to each tube before loading. Adjust the corresponding parameters of the flow cytometer (NovoCyte Quanteon), collect 10,000 cells and end the detection, record the data and analyze it using NovoExpress software.

[0050] (5) Cell cycle analysis

[0051] AGS cells were seeded in six-well plates at 6 × 10 cells per well. 5 cells and cultured for 24 h. The original culture medium was aspirated, and the culture medium containing the corresponding concentration of the compound was added and cultured for 48 h. The AGS cells were collected by trypsin digestion, washed with pre-cooled PBS solution, and left in a centrifuge tube. 300 μL of PBS was added, mixed with 350 μL of anhydrous ethanol twice, and fixed at 4 ° C overnight. After fixation, centrifuged at 2000 rpm for 8 min, the supernatant was discarded, the cells were resuspended in pre-cooled PBS solution, and washed to remove the residual fixative. The cells were resuspended in 100 μL of RNase and incubated at 37 ° C for 30 min. The suspension was transferred to a centrifuge tube and stained with 400 μL of propidium iodide staining solution. Finally, the corresponding parameters of the flow cytometer (NovoCyte Quanteon) were adjusted, 20,000 cells were collected and the detection was ended, and the data were recorded and analyzed using NovoExpress software.

[0052] (6) Immunoblotting

[0053] Cell culture: AGS cells in the logarithmic growth phase were cultured at 6×10 5 The cells were seeded at a density of 10 cells / well in a six-well plate, with a negative control group (DMSO) and a drug-treated group (compounds described in the present application, including 2 μM compound 10 group, 1 μM compound 2f group, etc.). The cells were cultured in an incubator for 24 hours to allow them to adhere to the wall.

[0054] Drug administration: According to the experimental plan, a drug (compound described in the present application, such as compound 10) of appropriate concentration is prepared in RPMI medium, and culture is continued for 48 h.

[0055] Protein extraction: The cells in the wells digested with trypsin were collected with pre-cooled medium, centrifuged at 1500rpm for 5min, and the supernatant was discarded. The cells were mixed and suspended with pre-cooled RIPA lysis buffer containing 1% protein phosphatase and 1% PMSF to disperse the cells and make them fully contact with the lysis buffer. The cell suspension was transferred to a 1.5mL centrifuge tube and lysed on ice for 40min, and the centrifuge tube was flipped every ten minutes to resuspend the cells. After lysis, centrifuged at 12,000rpm for 15min, the tannin in the tube was carefully removed with a pipette tip, and 2μL of the total protein suspension in PBS diluted ten times was aspirated and added to a 96-well plate for protein quantitative detection according to the procedure of the BCA quantitative kit. An appropriate amount of protein loading buffer was added to the remaining total protein suspension, and the protein was denatured in a metal bath (100℃, 10 minutes). The denatured protein sample was stored in a refrigerator at -80℃ for subsequent experiments.

[0056] 30 μg of total protein was added to each lane, and the volume of each group was adjusted to the same level with diluted protein loading buffer. The protein distribution was indicated by rainbow 245 broad-spectrum protein marker. Electrophoresis was performed for 45 min at a constant pressure of 60 V to allow the leading edge indicator to run to the boundary of the concentrated gel and the separation gel, and then electrophoresis was performed for 90 min at a constant pressure of 12 V, using a wet transfer membrane, 300 mA, 120 min. The membrane was transferred at a constant current for 120 min, and an ice bath was maintained during the transfer process. The cleaned PVDF membrane was cut according to the size of the target protein and soaked in the primary antibody prepared by antibody dilution (dilution ratio 1:1000). Incubate in a refrigerator at 4 ° C overnight. After incubation, the PVDF membrane was washed with TBST (3×10 min), and then incubated with the secondary antibody prepared by antibody dilution (dilution ratio 1:5000) for 120 min. After incubation, the secondary antibody remaining on the surface of the PVDF membrane was washed with TBST (3×10 min). After draining the TBST solution on the PVDF surface, add the prepared ECL luminescent liquid dropwise to fully cover the membrane, put the membrane into an exposure instrument for exposure and export for photography, and use Image J software to perform grayscale analysis of the target band.

[0057] (7) Virtual screening and docking

[0058] The crystal structures of human cyclophosphamide-dependent kinase 6 (CDK6) and palbociclib (PDB: 5L2I) were used as target protein structures, and the box that completely enclosed the ligand was selected as the docking site. The open source molecular docking software Autodockvina was selected for docking. The docking results were visually inspected by the Chimera 1.14 visualization tool.

[0059] (8) Statistical analysis

[0060] All results are expressed as mean ± standard deviation, and the data were analyzed by SPSS 22.0.

[0061] (9) Acute toxicity test

[0062] Twelve female KM mice within the standard weight range of mice were selected and divided into two groups, with six mice in each group. All mice were weighed and their weights were recorded after fasting for 24 hours without water restriction. The drug was administered by gavage at a dose of 1000 mg / kg according to their body weight. After administration, food supply was resumed, and the activity status, weight, and appearance of all mice were observed and recorded every day. After 14 days, all mice were killed, and the heart, liver, spleen, lungs, and kidneys were taken. After washing with sterile saline, they were placed in 4% paraformaldehyde and treated for 48 hours. After that, HE staining sections were performed, and photos were taken using a microscope to observe whether there was obvious damage to the mouse organs.

[0063] The specific structures of the compounds involved in the examples of this application are shown below:

[0064]

[0065]

[0066] Example 1 Activity Study of Compound 10

[0067] Compound 10 was obtained by virtual screening using a self-built database and served as the basis for targeting CDK6 degradation. In an earlier study (http: / / dx.doi.org / 10.2139 / ssrn.4239455), we obtained a combinatorial chemical small molecule database (CCSMD) by searching for various types of molecular blocks, using Rkit for virtual combination based on simple chemical reactions, and removing unreliable molecules ( Figure 1 Afterwards, Autodock vina was used on a Linux platform to perform virtual screening of CCSMD and CDK6, and all scored molecules were ranked, the top 100 molecules were selected for manual verification, and finally 13 molecules were selected for synthesis ( Figure 1 Compound 10 is the most active molecule. Based on this, we modified compound 10 with PROTAC, optimized its structure, and optimized its structure with PROTAC ( Figure 1 (as shown in C).

[0068] 1. Synthesis of Compound 10

[0069] The raw material (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)pent-4-ynoic acid (50.0 mg, 0.149 mmol) was added to 5 ml of thionyl chloride and stirred at 75 ° C for 2 hours. The system was then concentrated under reduced pressure until a white solid was produced to obtain the intermediate (9H-fluorene-9-yl)methyl (S)-(1-chloro-1-oxo-4-alkyn-2-yl)carbamate, which was added to THF, and then the raw materials 4-chloro-2,5-dimethylaniline (23.1 mg, 0.148 mmol) and DIPEA (77.0 mg, 0.596 mmol) were added to THF and stirred for 3 hours under ice bath conditions. Afterwards, the system was concentrated under reduced pressure, the residue was purified by silica gel column chromatography, and the product was purified with petroleum ether / ethyl acetate (4:1) to obtain white solid compound 10 (46.5 mg, yield 66%).

[0070] Compound 10: yield, 66%; white solid; 1H NMR(400MHz,DMSO-d6)δ9.47(s,1H),7.85(d,J=7.5Hz,2H),7.76(d,J=8.0Hz,1 H),7.70(d,J=7.4Hz,2H),7.37(t,J=7.4Hz,2H),7.31(s,1H),7.30–7.24(m,2H ),7.23(s,1H),4.36(q,J=7.4Hz,1H),4.28(t,J=7.1Hz,2H),4.20(t,J=6.9Hz, 1H), 2.90 (s, 1H), 2.58 (qd, J = 17.6, 16.4, 7.0Hz, 2H), 2.22 (s, 3H), 2.10 (s, 3H).

[0071] 2. Compound 10 induces apoptosis, arrests cells in the G0 / G1 phase, and causes downregulation of Phospho-Rb

[0072] After AGS was treated with the above-synthesized compound 10 for 48 hours, cell cycle detection and detection of CDK6 and CDK6 downstream proteins were performed. The results showed that compound 10 blocked AGS in the G0 / G1 phase and downregulated the S phase ( Figure 2 It has no significant effect on the expression of CDK6 protein, but can significantly inhibit the phosphorylation of the downstream protein Rb mediated by CDK6 ( Figure 2 This is consistent with the performance of CDK6 inhibitors; in addition, compound 10 can also induce cell apoptosis, and mainly manifests in the late stage of cell apoptosis ( Figure 2 C and D).

[0073] According to the above experimental results, the above synthesized compound 10 may be an effective CDK6 inhibitor.

[0074] Example 2 Structural optimization and activity detection of compound 10

[0075] Compound 10 was divided into three parts according to its structure, namely Ⅰ, Ⅱ and Ⅲ ( Figure 3 By replacing these three parts with different structures, the structure is optimized; the synthesis process is as follows Figure 3 As shown in B.

[0076] The synthesis process of compounds 1a-1e and 2a-2f is similar (the difference is that the structures of parts Ⅰ and Ⅱ are different, see Figure 3 In this example, compound 2f is taken as an example and synthesized as follows:

[0077] N-[(9H-fluoren-9-ylmethoxy)carbonyl]-5-methyl-L-norleucine (135.2 mg, 0.368 mmol) and HATU (154.0 mg, 0.405 mmol) were placed in a 50 mL round-bottom flask, and 15 mL of dichloromethane and 1 mL of DIPEA were added. After stirring for one hour, the system became clear, and 6-bromo-2-aminonaphthalene (85.9 mg, 0.405 mmol) was added. The mixture was stirred overnight. As the reaction was completed, the product was slowly precipitated. After the reaction was completed, the system was evaporated to dryness using a rotary evaporator, and 30 mL of ethyl acetate was added to dissolve the system. The organic phase was extracted three times with a saturated ammonium chloride solution, and then the organic phase was extracted three times with a saturated sodium chloride solution. The organic phase was then dried over anhydrous sodium sulfate, the ethyl acetate was evaporated to dryness and the crude product was collected. The crude product was purified by column chromatography using a developing solvent ratio of petroleum ether: ethyl acetate = 3:1. All the pure products were collected and weighed to obtain 140.8 mg of a light yellow solid with a yield of 63.8%. The product was stored at 4°C.

[0078]

[0079] Compound 2f; yield 63.8%; white solid; 1 H NMR (400MHz, DMSO-d6) δ10.31(s,1H),8.31(d,J=2.0Hz,1H),8.07(d,J=2.0Hz,1H),7.83(t,J=8.8H z,3H),7.76(d,J=8.9Hz,1H),7.74–7.67(m,2H),7.62(dd,J=8.9,2.1Hz,1H),7.53(dd,J=8.8,2.1H z,1H),7.37(tdd,J=7.5,3.4,1.2Hz,2H),7.28(tt,J=7.4,1.4Hz,2H),4.23(tq,J=14.1,7.2Hz,3H) ,4.12(td,J=8.6,8.1,4.0Hz,1H),2.64(s,1H),1.17(d,J=6.7Hz,5H),0.82(dd,J=6.6,1.7Hz,6H). 13C NMR (101MHz, DMSO-D6) δ172.21,156.68,144.42,144.32,141.25,137.64,132.50,131.39,130.11,129.87,128.19 ,127.59,125.89,121.58,120.66,118.02,115.69,66.18,56.31,47.17,35.27,30.32,27.90,23.10,22.90.MS-ESI m / z:calcd for C 32 H 31 BrN2O3[M+Na] + :593.1416; found:593.1387.

[0080] The synthesis process of compounds 3a-3c is similar (the difference is that the structure of part III is different, see Figure 3 In this embodiment, compound 3a is taken as an example and synthesized as follows:

[0081] Synthesis of 3-(9H-carbazole-9-yl)propionic acid: Carbazole (1.3264 g, 7.939 mmol), sodium hydroxide (0.6351 g, 15.878 mmol), ethyl 3-bromopropionate (1.6416 g, 8.733 mmol) and 10 mL of DMF were placed in a 50 mL beaker and reacted at high heat for 5 min in a Grids microwave oven. After the reaction, 100 mL of water was added to dissolve the reaction system. The pH was adjusted to pH = 1 with hydrochloric acid. The product gradually precipitated and the system was filtered to obtain 1.3534 g of the product with a yield of 71.3%.

[0082]

[0083] Synthesis of (S)-2-amino-N-(6-bromonaphthalene-2-yl)pent-4-ynamide: Compound 10 (129.1 mg, 0.240 mmol) was dissolved in 10 mL of tetrahydrofuran, 1 mL of diethylamine was added, and the reaction system was stirred at room temperature for 3 hours, then evaporated to dryness, and the crude product was purified by column chromatography using a developing solvent ratio of petroleum ether: ethyl acetate = 1:2. All the pure products were collected and weighed to obtain 44.8 mg of a light yellow solid with a yield of 59.1%. The product was stored in a refrigerator at 4°C.

[0084]

[0085] Synthesis of compound 3a: 3-(9H-carbazole-9-yl)propionic acid (33.9 mg, 0.142 mmol) and HATU (59.3 mg, 0.155 mmol) were placed in a 50 mL round-bottom flask, 15 mL of dichloromethane and 1 mL of DIPEA were added, and the system was clarified after stirring for one hour. Then (S)-2-amino-N-(6-bromonaphthalene-2-yl)pent-4-ynamide (44.8 mg, 0.142 mmol) was added and stirred overnight. After the reaction was completed, the system was evaporated to dryness using a rotary evaporator, 30 mL of ethyl acetate was added to dissolve the system, and the organic phase was extracted three times with a saturated ammonium chloride solution, and then the organic phase was extracted three times with a saturated sodium chloride solution. The organic phase was then dried over anhydrous sodium sulfate, the ethyl acetate was evaporated to dryness and the crude product was collected. The crude product was purified by column chromatography using a developing solvent ratio of petroleum ether: ethyl acetate = 2:1. All the pure products were collected and weighed to obtain 55.4 mg of a light yellow solid with a yield of 72.7%. The product was stored in a refrigerator at 4°C.

[0086]

[0087] Compound 3a; final yield 42.9%; light yellow solid; 1 H NMR (400MHz, DMSO-d6) δ10.41 (s, 1H), 8.51 (d, J = 7.8Hz, 1H), 8.25 (d, J = 2.0Hz, 1H),8.13–8.05(m,3H),7.83(d,J=8.9Hz,1H),7.76(d,J=8.8Hz,1H),7.56(qd,J =8.8,8.0,2.0Hz,4H),7.38(ddd,J=8.3,7.1,1.2Hz,2H),7.13(t,J=7.4Hz,2H), 4.58(td,J=7.2,3.3Hz,3H),3.28(s,1H),2.81–2.76(m,1H),2.73–2.59(m,3H). 13 C NMR (101MHz, DMSO-D6) δ170.82,169.54,140.24,137.30,132.40,131.51,130.15,129.91,129.88,128.25,126. 18,122.68,121.66,120.75,119.33,118.17,116.01,109.89,80.62,73.85,53.03,40.68,35.12,22.29.MS-ESI m / z:calcd forC 30 H 24 BrN3O2[M+Na] +:560.0590; found:560.0924.

[0088] Although the present embodiment used compound 2f and compound 3a as examples for synthesis, the structures of the synthetic compounds of compounds 1a-1e, 2a-2f and 3a-3c described in the present application were verified, and the inhibitory effects of all compounds on AGS were studied. The verification results are shown in Table 1.

[0089] Table 1 IC values ​​of all compounds for AGS 50

[0090] Compound <![CDATA[IC 50 (μM)]]> Compound <![CDATA[IC 50 (μM)]]> Compound <![CDATA[IC 50 (μM)]]> 10 1.16±0.14 1a 13.62±0.48 1b 9.12±1.65 1c 19.21±1.67 1d 2.03±0.18 1e 16.51±0.46 2a 1.46±0.47 2b 0.73±0.01 2c 3.83±0.01 2d 0.57±0.08 2e 1.37±0.19 2f 0.56±0.06 3a 0.76±0.12 3b 0.92±0.29 3c 1.85±0.03

[0091] Summarizing the above experimental results, it was found that the substitution of groups from small to large in zone I showed that groups that were too large may reduce activity, adding N atoms did not significantly increase activity, adding halogens had a higher increase in activity, and non-polar groups led to a decrease in activity. Adding larger non-polar groups in zone II significantly increased activity, while polar groups showed a decrease in activity; for the modification of the groups in zone III, it was found that larger groups were more active. Compounds 1a-1e, 2a-2f, and 3a-3c described in the present application all had good inhibitory activity against AGS, among which compound 2f had the best anti-tumor activity.

[0092] Example 3 Compound 2f downregulates phosphorylated Rb, organizes cells in the G0 / G1 phase, and induces apoptosis in the late apoptotic phase

[0093] The cytotoxicity inhibition of compound 2f obtained in Example 2 was evaluated, and it was found that at the same concentration, compound 2f had a significant inhibitory effect on AGS, but had no inhibitory effect on GES-1 ( Figure 4 Compound 2f at a concentration of 8 μM can inhibit the arrest of the cell cycle at the G0 / G1 phase ( Figure 4 At the protein level, it was found that compound 2f at a concentration of 1 μM could significantly downregulate the phosphorylation of CDK6 downstream protein Rb, but had no significant effect on the protein concentration of Rb and CDK6 concentration ( Figure 4 These results are consistent with the performance of CDK6 inhibitors. In addition, it was found that compound 2f can induce cell apoptosis, and apoptosis is mainly manifested in the late stage of cell apoptosis ( Figure 4 D and E).

[0094] Through the above experiments, it was found that the biological activity of compound 2f described in the present application is consistent with that of CDK6 inhibitors and is a CDK6 inhibitor.

[0095] Example 4 PROTACs modification of compound 10

[0096] From the docking results of compound 10 and CDK6, we can see that ( Figure 5 As shown in Figure A, the naphthalene ring of compound 10 is exposed outside the protein pocket. Therefore, this part is modified by replacing the bromine atom with a carboxyl group, and then amide condensation is performed on this carboxyl group to connect the E3 ubiquitin ligand. The main synthetic route after modification is shown in Figure 5 As shown in B. After the synthesis route was completed, the biological activity was evaluated and it was found to have certain activity, so this molecule was used for the next step of biological activity evaluation.

[0097] 1.10-PROTAC synthesis

[0098] Synthesis of 10-acid: Pour the raw material (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pent-4-ynoic acid (167.2 mg, 0.499 mmol) into a 50 mL round-bottom flask, add 5 mL of thionyl chloride and then 20 μL of DMF (catalytic amount), stir at room temperature for one hour, use rotary evaporation to remove thionyl chloride to obtain a light yellow solid, and then add 10 mL of THF dissolved 6-amino-2-naphthoic acid (88.6 mg, 0.474 mmol), stir in an ice bath for two hours, add 1 mL of methanol to quench the reaction, then evaporate the system to dryness, add 50 ml of ethyl acetate to dissolve the system, extract the organic phase three times with a saturated ammonium chloride solution, and then extract the organic phase three times with a saturated sodium chloride solution. The organic phase was then dried over anhydrous sodium sulfate, the ethyl acetate was evaporated to dryness and the crude product was collected. The crude product was purified by column chromatography. The developing solvent ratio used was petroleum ether: ethyl acetate = 1000:200. 0.1 mL of acetic acid was added to every 100 mL of the developing solvent. All the pure products were collected and weighed to obtain 134.3 mg of a light yellow solid with a yield of 50.1%. The product was stored in a refrigerator at 4°C.

[0099]

[0100] Synthesis of ELB8: N-Boc-1,6-hexanediamine (375.0 mg, 1.528 mmoL), 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (383.8 mg, 1.389 mmoL) and 1 mL of DIPEA were mixed in a 50 mL round-bottom flask, 15 mL of 1,4-dioxane was added to dissolve, and the mixture was stirred at 90 degrees Celsius for 12 hours. During the stirring process, the system gradually produced a yellow-green fluorescent product. After the reaction was completed, the system was evaporated to dryness using a rotary evaporator, 50 mL of ethyl acetate was added to dissolve, and the organic phase was extracted three times using a saturated ammonium chloride solution and a saturated sodium chloride solution, respectively. The crude product was then purified by column chromatography, and the developing solvent ratio used was petroleum ether: ethyl acetate = 1:1. After all the pure products were collected, they were evaporated to dryness using a rotary evaporator to obtain 434.3 mg of pure product with a yield of 66.2%. The pure product is a yellow-green oily liquid, showing yellow-green fluorescence at a wavelength of 365nm.

[0101]

[0102] Synthesis of ELN8: ELB8 (434.3 mg, 0.919 mmol) was dissolved in 10 mL of dichloromethane and 1 mL of trifluoroacetic acid, stirred at room temperature for two hours, and the system was evaporated to dryness using a rotary evaporator. The crude product was then directly purified by column chromatography, using a developing solvent ratio of dichloromethane: methanol = 10:1, and 1 mL of triethylamine was added to each 100 mL of developing solvent. After all the pure products were collected and concentrated, 253.7 mg of the pure product was obtained, with a yield of 66.2%. The pure product was a viscous liquid, and the pure product was stored at 4°C.

[0103]

[0104] Synthesis of compound 10PL8: 10-acid (80.3 mg, 0.159 mmoL), ELN8 (65.2 mg, 0.175 mmoL), HATU (66.6 mg, 0.175 mmoL) and 1 mL of DIPEA were mixed in a 50 mL round-bottom flask, and 2 mL of DMF and 15 mL of THF were added. Stir overnight at room temperature. After the reaction was completed, THF was evaporated using a rotary evaporator, and then 50 ml of ethyl acetate was added to dissolve the system. The system was extracted with a saturated ammonium chloride solution and a saturated sodium chloride solution three times. The organic phase was then dried with anhydrous sodium sulfate, and the crude product was collected after evaporating ethyl acetate. The crude product was purified by column chromatography. The developing solvent ratio used was petroleum ether: ethyl acetate = 1:3. All the pure products were collected and weighed to obtain 55.9 mg of a yellow-green solid with a yield of 39.4%. The product was stored at 4 °C.

[0105]

[0106] Compound 10PL8; final yield: 19.73%; yellow-green solid; 1 H NMR (400MHz, DMSO-d6) δ11.04(s,1H),10.43(s,1H),8.50(q,J=5.1,4.6Hz,1H),8.32(d,J=2.8Hz,2H),7.92(d,J=8.8Hz,1H),7.86(d,J=3.6Hz,2 H),7.84(s,3H),7.71(d,J=7.4Hz,2H),7.64(td,J=9.6,8.9,7.3Hz,2H),7.52(dd,J=8.5,7.0Hz,1H),7.37(td,J=7.5,2.5Hz,2H),7.29(td,J=7. 4,3.6Hz,2H),6.97(d,J=7.1Hz,1H),6.49(t,J=5.9Hz,1H),5.00(dd,J=13.0,5.3Hz,2H),4.43–4.31(m,1H),4.31–4.17(m,3H),3.49(t,J=7.1Hz ,1H),3.27–3.22(m,4H),2.89(d,J=2.8Hz,1H),1.72(s,1H),1.56–1.51 (m,3H),1.37–1.33(m,4H),1.23(d,J=5.6Hz,2H),1.19(d,J=4.4Hz,2H). 13 C NMR (101MHz, DMSO-D6) δ173.35,170.65,170.09,169.49,167.85,166.66,156.45,146.97,144 .32,141.26,136.81,135.11,132.74,131.37,130.08,129.41,128.19,127.82,127.64,127.60 ,125.87,125.84,125.27,120.66,117.71,115.64,110.90,109.56,103.58,73.76,73.74,66.5 7,66.36,55.02,49.07,47.15,42.34,32.37,31.52,29.66,29.20,26.80,26.64,22.69.MS-ESI m / z:calcd for C 50 H 46 N6O8[M+Na] + :811.3275; found:811.3251.

[0107] For PROTAC modification of compound 10, since both carbonyl and bromine atoms are highly polar groups, carboxylic acid groups are used for substitution, which will not cause much change in polarity. Compound 10PL8 is the best active PROTAC molecule, and its optimal linker length is close to the optimal length studied previously. Compound 10PL8 can degrade CDK6, downregulate phosphorylated Rb, and block cells in the G0 / G1 phase.

[0108] The cell cycle assay was performed on compound 10PL8, and it was found that the compound could significantly block AGS cells in the G0 / G1 phase at a concentration of 50 μM in WB, but had a weak inhibitory effect on GES-1 ( Figure 6 Its IC for AGS cells 50 The compound 10PL8 can significantly degrade CDK6 and significantly downregulate phosphorylated Rb. The compound 10PL8 has an inhibitory effect on the DC of CDK6. 50 26.67±5.09μM( Figure 6 In order to verify whether the downregulation of CDK6 by compound 10PL8 was affected by compound 10PL8, rather than the interference of compound 10 or E3 ubiquitin ligase. The results showed that neither compound 10 alone nor E3 ubiquitin ligase ligase alone caused the downregulation of CDK6, further confirming that compound 10PL8 degraded CDK6 ( Figure 6 Compound 10PL8 blocks cells in the G0 / G1 phase, and compound 10PL8 significantly downregulates CDK6, and the above results are consistent with the performance of CDK6 degraders. Therefore, it can be determined that compound 10PL8 described in the present application is a CDK6 degrader.

[0109] Example 5 PROTACs modification of compound 2f

[0110] 1.2f-PROTAC synthesis

[0111] Using the same synthetic route as 10-PROTAC, three PROTAC molecules (compounds 2fPL6, 2fPL8, and 2fPL10) were synthesized, and the IC values ​​of the three PROTAC molecules against AGS were measured. 50 (like Figure 7 The results show that the compounds 2fPL6, 2fPL8 and 2fPL10 described in the present application have good inhibitory activity against AGS.

[0112] Among them, the synthesis process of compounds 2fPL6, 2fPL8 and 2fPL10 is similar to the synthesis process of compound 10-PROTAC, so part of the synthesis process mainly refers to the synthesis of 10-PROTAC.

[0113]

[0114] Compound 2fPL6; yield 33.8%; yellow-green solid; 1 H NMR (400MHz, DMSO-d6) δ11.04(s,1H),10.32(s,1H),8.57(d,J=5.6Hz,1H),8.31(d,J=5.0Hz,1H),7.99–7.78(m,5H),7.68(dt ,J=23.1,8.0Hz,4H),7.51(t,J=7.9Hz,1H),7.36(td,J=7.5,4.2Hz,2H),7.29(d,J=7.7Hz,2H),7.07(d,J=8.6Hz,1H),6.96(d, J=6.9Hz,1H),6.53(s,1H),5.00(dd,J=12.8,5.2Hz,1H),4.37–4.04(m,4H),3.50(t,J=6.9Hz,2H),3.05–2.69(m,2H),2.52(d, J=25.7Hz,2H),2.07–1.90(m,1H),1.72(s,1H),1.61(s,5H),1.51(dt,J=13.1,6.6Hz,2H),1.18(s,3H),0.83(d,J=6.5Hz,6H). 13 C NMR(101MHz,DMSO-D6)δ173.35,172.26,170.65,169.47,167.84,166.75,156.68,146.96,14 4.43,144.33,141.27,138.48,136.79,135.20,132.76,131.20,130.07,129.30,128.18,127 .79,127.59,125.88,125.23,121.11,120.65,117.78,115.41,110.92,109.57,66.19,56.36 ,49.07,47.20,42.11,35.26,31.51,30.35,27.90,27.12,26.82,23.10,22.90,22.69.MS-ESI m / z:calcd for C 50 H 50 N6O8[M+Na] +:885.3588; found:885.3526.

[0115]

[0116] Compound 2fPL8; yield 37.4%; yellow-green solid; 1 H NMR (400MHz, DMSO-d6) δ11.04(s,1H),10.29(s,1H),8.50(t,J=5.4Hz,1H),8.31(d,J=3.5Hz,2H),7.91(d,J=9.0Hz,1H),7.86(s,1H),7.8 4(d,J=4.6Hz,3H),7.74–7.66(m,3H),7.64(d,J=8.9Hz,1H),7.55–7.49(m,1H),7.37(td,J=7.5,3.7Hz,2H),7.28(t,J=7.5Hz,2H),7.05( d,J=8.6Hz,1H),6.97(d,J=7.0Hz,1H),6.50(t,J=6.0Hz,1H),5.00(dd,J=12.9,5.4Hz,1H),4.30–4.06(m,3H),3.25(t,J=6.6Hz,3H),2.8 9–2.78(m,1H),2.59–2.47(m,1H),2.02–1.90(m,2H),1.51(dt,J=13.2,6.6Hz,4H),1.39–1.32(m,4H),1.19(s,5H),0.83(d,J=6.6Hz,6H). 13 C NMR (101MHz, DMSO-D6) δ173.36,172.26,170.65,169.50,167.85,166.68,156.68,146.96,144.4 1,144.32,141.26,138.47,135.18,132.73,131.26,129.31,127.98,127.79,127.39,125.86,12 5.31,121.11,120.71,117.96,117.41,115.56,115.26,111.18,109.55,55.44,48.80,47.31,42 .33,40.57,38.91,38.61,35.22,29.66,29.20,28.01,27.82,26.79,26.64,23.13,22.89.MS-ESI m / z:calcd for C 52 H 54 N6O8[M+Na] +:913.3901; found:913.3827.

[0117]

[0118] Compound 2fPL10; yield 30.1%; yellow-green solid; 1 H NMR (400MHz, DMSO-d6) δ11.05(s,1H),10.33(s,1H),8.56(t,J=5.6Hz,1H),8.32( d,J=2.4Hz,2H),7.91(d,J=8.9Hz,1H),7.88–7.79(m,4H),7.74–7.67(m,3H),7.64 (dd,J=8.9,2.0Hz,1H),7.49(dd,J=8.6,7.1Hz,1H),7.37(td,J=7.4,4.1Hz,2H), 7.31–7.25(m,2H),7.02(d,J=8.6Hz,1H),6.97(d,J=7.0Hz,1H),6.54(s,1H),5.01 (dd,J=12.9,5.4Hz,1H),4.36–4.09(m,5H),3.58(d,J=5.5Hz,2H),3.52(s,2H),3 .43(t,J=5.7Hz,2H),3.37(t,J=5.5Hz,2H),2.94(s,1H),2.83(ddd,J=17.3,14.0, 5.4Hz,1H),2.58–2.48(m,2H),1.97(ddd,J=11.8,6.1,3.8Hz,1H),1.78–1.57(m, 2H), 1.51 (dq, J=13.3, 6.7Hz, 1H), 1.24–1.09 (m, 2H), 0.83 (dd, J=6.7, 1.6Hz, 6H). 13CNMR(101MHz,DMSO-D6)δ173.35,172.27,170.65,169.47,167.82,166.83,156.68,146.88,14 4.43,144.32,141.26,138.53,135.24,132.60,130.92,130.23,129.27,128.36,127.86,127.5 6,127.39,125.89,121.11,120.71,120.57,111.49,110.92,109.77,70.20,69.51,69.40,56.3 9,55.45,49.36,48.79,42.20,40.58,35.41,35.23,27.97,23.13,23.05,22.95,22.86.MS-ESI m / z:calcd for C 52 H 54 N6O 10 [M+Na] + :945.3799; found:945.3741.

[0119] 2.2f-PROTAC significantly degraded CDK6 and downregulated phosphorylated Rb, and blocked cells in the G0 / G1 phase

[0120] The results of the activity test in this embodiment showed that compound 2fPL8 had a more obvious inhibitory effect on AGS and a weaker inhibitory effect on GES-1 ( Figure 7 Compound 2fPL8 can block AGS in the G0 / G1 phase at a concentration of 8 μM ( Figure 7 Compound 2fPL8 can also cause significant degradation of CDK6 and significantly downregulate phosphorylated Rb, but has no significant effect on Rb ( Figure 7 (as shown in D in the figure);

[0121] From the above experiments, it can be seen that compound 2fPL8 can significantly downregulate CDK6 and phosphorylated Rb, blocking cells in the G0 / G1 phase, which is consistent with the performance of CDK6 degraders. Therefore, it can be judged that compound 2fPL8 can be a CDK6 degrader, targeting the degradation of CDK6, and its DC 50 It is 4.80±0.79μM.

[0122] 3. Acute toxicity test of compound 2fPL8 in mice

[0123] Two groups of KM female mice with similar body weight were used for acute toxicity experiments. After fasting for 24 hours, one group was intragastrically administered with 1000 mg / kg of compound 2fPL8 and observed for two weeks. The body weight was recorded every day ( Figure 8 (As shown in A), it was found that the weight gain of the drug-administered group in the first four days was significantly lower than that of the control group, but after the fifth day, the weight gains of the two groups began to approach, and after the tenth day, the weight gains of the two groups began to be consistent. No mice died during the experiment. The mice in the experimental group were obviously listless, moved less, and shrank into a ball one to two days after gavage, and were obviously normal after the third day.

[0124] After 14 days, all mice were treated, and the heart, liver, spleen, lung and kidney were taken for tissue sectioning and HE staining. Then, the organ sections of mice in the control group and the treatment group were photographed. The two groups of photos were analyzed ( Figure 8 It was determined that compound 2fPL8 had certain biosafety for mice at a dose of 1000 mg / kg.

[0125] The present invention uses Python's RDKit package to intelligently combine small molecule building blocks through simple synthetic reaction conditions, forming an online virtual database with simple synthesis features. The total number of molecules reached 12,904. The database was used to virtually screen CDK6 as a target protein, and a total of 13 compounds were screened and synthesized. Among them, 76.92% of the molecules were active at 10μM. The IC of the most active molecule 10 50 The results showed that the amide reaction can effectively inhibit the expression of CDK6 in the presence of amides, which is 1.16±0.14μM, respectively, which is superior to CIS at the cellular level. The above experiments demonstrated the feasibility of using various small molecule building blocks to establish a virtual database through combinatorial chemistry, and to screen and discover potential CDK6 inhibitors. This work explored the application of the molecular library constructed by virtual amide reaction in virtual screening and verified that the amide reaction has a realistic hit rate in actual virtual screening.

[0126] Further research was conducted on the active compounds of the potential inhibitors that have been discovered, and compound 10 was found to effectively inhibit CDK6. This compound has a significant inhibitory effect on the gastric cancer cell line AGS with abnormally high expression of CDK6, and the compound has no significant inhibitory effect on normal gastric epithelial cells GES-1. This result shows that the compound has the potential for further transformation. The cells treated with compound 10 were stained with Hoechst / PI fluorescent dyes and then detected by flow cytometry. It was found that the compound can induce cell apoptosis rather than necrosis. After staining with FITC / PI dyes, it was found that the compound caused cell apoptosis and manifested in the late stage of cell apoptosis. At the same time, the cell cycle of cells treated with this compound was detected, and it was found that compound 10 could block cells in the G0 / G1 phase, which is also consistent with the performance of CDK6 inhibitors. Using WB to detect at the protein level, cells treated with compound 10 can significantly downregulate the downstream protein phosphorylation Rb of CDK6, but have no obvious effect on the expression of CDK6 and Rb.

[0127] After verifying that compound 10 is a CDK6 inhibitor, compound 10 was modified with PROTAC. In related studies, the best linker length of PROTAC molecules that degrade CDK6 was about 9 atoms. Based on this, compound 10 was modified with PROTAC to obtain a PROTAC molecule 10-PROTAC with a linker length of about 9 atoms. 10-PROTAC was tested for anti-proliferation of AGS, and it was found that the compound had limited inhibitory effect on AGS. Cell cycle detection was performed on it, and it was found that the compound could effectively block cells in the G0 / G1 phase at a concentration of 50μM, and WB was used to find that CDK6 protein can be effectively degraded at the protein level. Experiments have shown that 10-PROTAC is a new type of CDK6 degrader, but its degradation effect is limited, so further structural optimization is needed in the hope of discovering PROTAC molecules with better activity.

[0128] Compound 10 was split into I, II, and III with structural replacement for each part. All the replaced molecules were tested for cell anti-proliferation, and compound 2f with better activity was found. Compound 2f was also tested for anti-proliferation of gastric cancer cells AGS and normal gastric epithelial cells GES-1, and it was found that compound 2f could effectively inhibit AGS but had no obvious inhibitory effect on GES-1. After staining with Hoechst / PI fluorescent dye and FITC / PI dye, it was found that the compound caused cell apoptosis and manifested in the late stage of cell apoptosis. Cell cycle detection was performed on cells treated with the compound, and it was found that compound 2f also made AGS block cells in the G0 / G1 phase. At the protein level, compound 2f could also downregulate the downstream protein phosphorylation Rb of CDK6, but had no obvious effect on Rb and CDK6, and was a CDK6 inhibitor.

[0129] Therefore, the CDK6 inhibitor 2f was further modified by PROTAC, and the anti-proliferation test of AGS was performed on the modified PROTAC molecule. It was found that 2f-PROTAC had a significant increase in activity compared with 10-PROTAC. Further testing of the 2f-PROTAC with improved activity found that 2f-PROTAC had a poor inhibitory effect on GES-1, and the inhibitory effect at the maximum concentration was not high, which is conducive to further research. Cell cycle detection was performed on AGS treated with 2f-PROTAC, and it was found that under high concentration treatment, the compound could block cells in the G0 / G1 phase. Using WB to detect at the protein level, it was found that 2f-PROTAC can significantly downregulate CDK6 expression, and significantly downregulate the downstream protein phosphorylation Rb of CDK6, which proves that 2f-PROTAC is a CDK6 degrader.

[0130] An acute toxicity experiment on 2f-PROTAC in mice revealed that 2f-PROTAC was less toxic to mice, with no mouse deaths at a dose of 1000 mg / kg. After the acute toxicity experiment, samples were collected from the mice and the heart, liver, spleen, lungs, and kidneys were sectioned with HE staining, revealing that the organs of the mice fed 2f-PROTAC showed no obvious damage, indicating that 2f-PROTAC has good biosafety.

Claims

1. A compound for targeted inhibition of CDK6 or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula of the compound is shown in the following formula (I): Wherein, the R1 is selected from The R2 is selected from The R3 is selected from -CH2CCH, -CH2CHCH2, -CH2CH2CH3, -CH2CH2SCH3, -CH2CH(CH3)2, -CH2CH2 CH(CH3)2, or R3 represents -CH2CH2- which forms a three-membered ring with C at the position where R3 is substituted in formula (I).

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that The structural formula of the compound is shown as any one of the following:

3. Use of the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof in the preparation of a CDK6 inhibitor.

4. The use according to claim 3, characterized in that The CDK6 inhibitor is an anti-gastric cancer drug.

5. A PROTAC compound or a pharmaceutically acceptable salt thereof that targets and degrades CDK6, characterized in that: The PROTAC compound is obtained by modifying the compound according to claim 1 or 2 with a ligand molecule of an E3 ligase.

6. The PROTAC compound or a pharmaceutically acceptable salt thereof according to claim 5, wherein The ligand molecule of the E3 ligase is modified after being connected with the compound according to claim 1 or 2 by a linker.

7. The PROTAC compound or a pharmaceutically acceptable salt thereof according to claim 6, wherein The ligand molecule of the E3 ligase comprises the following structure:

8. The PROTAC compound or a pharmaceutically acceptable salt thereof according to claim 7, wherein The structural formula of the PROTAC compound is shown in the following formula (II): Wherein, R4 is -CH2CCH or -CH2CH2 CH(CH3)2; The Linker R n Includes the following structure:

9. The PROTAC compound or a pharmaceutically acceptable salt thereof according to claim 8, wherein The structural formula of the PROTAC compound is shown as any one of the following:

10. Use of a PROTAC compound or a pharmaceutically acceptable salt thereof according to any one of claims 5 to 9 in the preparation of a CDK6 degrader.

11. The use according to claim 10, characterized in that The CDK6 degrader is an anti-gastric cancer drug.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, and / or the PROTAC compound according to any one of claims 5 to 9 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

13. The pharmaceutical composition according to claim 12, characterized in that The auxiliary agent is a carrier, a diluent, a vehicle or a combination thereof.

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