A CDK4 protein hydrolysis targeting chimera and its preparation method, pharmaceutical composition and application

Abstract

This invention belongs to the field of chemical pharmaceutical technology and specifically relates to a CDK4 proteolysis-targeted chimera, its preparation method, pharmaceutical composition, and application. The structure of this proteolysis-targeted chimera is shown in Formula I, where the linker is any chemically feasible linker structure. The preparation process is simple and easy. The resulting proteolysis-targeted chimera or its pharmaceutically acceptable salt exhibits highly effective targeted degradation of CDK4, inhibits tumor cell proliferation, and exhibits potent anti-tumor activity, making it suitable for the development of anti-tumor drugs. #imgabs0# Formula I.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical medicines, and specifically relates to a CDK4 protein hydrolysis-targeting chimera, a preparation method thereof, a pharmaceutical composition and applications thereof. Background Art

[0002] Proteolysis-targeting chimeras (PROTACs), which use bifunctional small molecules to induce target protein degradation, are one of the most exciting new drug modalities pioneered in recent years. PROTACs consist of a target protein ligand, an E3 ubiquitin ligase ligand, and an appropriate linker. They form a ternary complex called "target protein-PROTAC-E3 ligase" and degrade the target protein through the ubiquitin-proteasome system (UPS). Compared to traditional small molecule inhibitors, PROTACs offer unique advantages, including catalytic properties, reduced dosing and frequency, more potent and longer-lasting effects, increased selectivity, reduced potential toxicity, overcoming drug resistance, and expanding the target space. PROTAC design requires a known target protein ligand and an E3 ligase ligand as a protein decoy, and their development relies on the discovery and optimization of these ligands. The lack of available E3 ligase ligands and the low drugability of existing major E3 ligase ligands have limited PROTAC development.

[0003] Molecular glue degraders are small molecules that can induce interactions between E3 ubiquitin ligase substrate receptors and target proteins, leading to their degradation via ubiquitination. They possess dual-ligand structural characteristics with both E3 ubiquitin ligases and target proteins. This property of molecular glues binding to E3 ligases makes them an effective tool for discovering novel E3 ligase ligands.

[0004] Cyclin-dependent kinases (CDKs) are key cellular enzymes that regulate eukaryotic cell division and proliferation. The catalytic units of CDKs are activated by cyclins. Several mammalian cyclins have been identified, among which Cyclin B / CDK1, Cyclin A / CDK2, Cyclin E / CDK2, Cyclin D / CDK4, and Cyclin D / CDK6 play important roles in regulating cell cycle progression. Other functions of cyclins / CDKs, such as transcriptional regulation, DNA repair, differentiation, and apoptosis, have also been reported. Increased activity or transient aberrant activation of CDKs can contribute to the development of various tumors. Therefore, cyclin-dependent kinase inhibitors are considered promising therapeutic agents for the treatment of cancer. Specifically, CDK4 / 6 inhibitors have demonstrated remarkable clinical efficacy in breast cancer and other cancers, either as monotherapy or in combination with other therapeutic agents. For example, the CDK4 / 6 inhibitors palbociclib and ribociclib have been approved for marketing. However, the development of both primary and acquired drug resistance has limited the use of CDK4 / 6 inhibitors. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and provide a CDK4 proteolysis-targeting chimera, a preparation method, a pharmaceutical composition, and applications thereof, specifically employing the following technical solutions:

[0006] A CDK4 proteolysis targeting chimera, the structural formula of which is shown in Formula I:

[0007] Formula I, wherein Linker is any chemically feasible linking structure.

[0008] Previously, the inventors serendipitously discovered that CP0371, a highly permeable and druggable anti-tumor lead compound derived from the semi-synthetic modification of the natural triterpenoid oleanolic acid (OA), can act as a molecular glue to bind to DNA damage binding protein 1 (DDB1), thereby mediating the degradation of phosphoglycerate dehydrogenase (PHGDH). Further research revealed that DDB1 is an important component of multiple E3 ligases. Developing E3 ligase ligase ligands that bind to DDB1 may help expand the PROTAC target protein library and potentially target more undruggable proteins. Leveraging CP0371's ability to bind to DDB1 and its good permeability, CP0371 has the potential to be used as a novel E3 ligase ligand in PROTAC molecular design and development.

[0009] Therefore, the present invention is based on the molecular glue mechanism of the pentacyclic triterpenoid natural product oleanolic acid derivative, which is used as an E3 ligase ligand for the design of protein hydrolysis targeting chimeras, and a new EGFR-PROTAC degrader is developed based on the classic CDK4 inhibitor as the target protein ligand. The inventors of the present invention have found in a large number of studies that the above-mentioned protein hydrolysis targeting chimera or its pharmaceutically acceptable salt can be used in the preparation of a drug for treating and / or preventing cancer, and can be used as an active ingredient in a pharmaceutical composition for treating cancer. It can effectively degrade CDK4 in various malignant tumor cells at the protein level and significantly inhibit the proliferation of malignant tumor cells at the cellular level. The pharmaceutical composition contains one or more pharmaceutically acceptable excipients, and the dosage form of the above-mentioned pharmaceutical composition is any pharmaceutically acceptable dosage form, and can also be used as a drug for targeted degradation of CDK4.

[0010] The above cancers include: gynecological cancers, such as ovarian cancer, cervical cancer, vaginal cancer, pudendal cancer, uterine / endometrial cancer, gestational trophoblastic tumor, fallopian tube cancer, uterine sarcoma; endocrine cancers, such as adrenal cortical cancer, pituitary cancer, pancreatic cancer, thyroid cancer, parathyroid cancer, thymic cancer, multiple endocrine neoplasia; bone cancers, such as osteosarcoma, Ewing's sarcoma, chondrosarcoma, etc.; lung cancers, such as small cell lung cancer, non-small cell lung cancer; brain and CN S tumors, such as neuroblastoma, acoustic neuroma, glioma and other brain tumors, spinal cord tumors, breast cancer, colorectal cancer, advanced colorectal adenocarcinoma; gastrointestinal cancers, such as liver cancer, extrahepatic bile duct cancer, gastrointestinal carcinoid tumors, gallbladder cancer, stomach cancer, esophageal cancer, small intestine cancer; genitourinary cancers, such as penile cancer, testicular cancer, prostate cancer; head and neck tumors, such as nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, oral cancer, lip cancer, salivary gland cancer, laryngeal cancer, lower limb cancer, etc. Pharyngeal cancer, pharyngeal cancer; blood cancers, such as acute myeloid leukemia, acute lymphocytic leukemia, childhood leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, acute promyelocytic leukemia, plasma cell leukemia; bone marrow cancer blood diseases, such as myelodysplastic syndrome, myeloproliferative disorders, Fanconi anemia, aplastic anemia, essential macroglobulinemia; lymphoma, such as Hodgkin's disease, non-Hodgkin's disease The invention also includes the following: sarcoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma; eye cancers, including retinoblastoma and uveal melanoma; skin cancers, such as melanoma, non-melanoma skin cancer, and Merkel cell carcinoma; soft tissue sarcomas, such as Kaposi's sarcoma, childhood soft tissue sarcoma, and adult soft tissue sarcoma; urinary system cancers, such as renal cell carcinoma, Wilms' tumor, bladder cancer, urethral cancer, and metastatic cell carcinoma. It is preferably used to treat colorectal cancer.

[0011] As a further preferred embodiment, the linker is a saturated fatty chain, an unsaturated fatty chain or a fatty acid chain.

[0012] As a further preferred embodiment, the molecular structure of the CDK4 proteolysis targeting chimera is any one of Formula II to Formula IV:

[0013] Formula II,

[0014] Formula III,

[0015] Formula IV;

[0016] Wherein, in formula II, n is selected from any positive integer from 1 to 12; in formula III, n is selected from any integer from 0 to 8;

[0017] In formula IV, n is any positive integer selected from 1-9.

[0018] As a further preferred embodiment, the molecular structure of the above-mentioned proteolysis targeting chimera is as follows:

[0019] . The compounds prepared by the present invention exhibit good anti-proliferative activity in several different cell lines, and these compounds generally perform better in colorectal cancer cells, especially in the HCT-116 cell line; in addition, compound M1 exhibits high anti-proliferative activity in multiple tumor cell lines.

[0020] The present invention also provides a method for preparing the above-mentioned CDK4 protein hydrolysis targeting chimera or a pharmaceutically acceptable salt thereof. The route is simple, the raw materials used in the route are cheap and readily available, and the overall reaction yield is high. The preparation route is route 1, route 2 or route 3:

[0021] Route 1:

[0022] ;

[0023] Route 2:

[0024] ;

[0025] Route 3:

[0026] .

[0027] In the online route one, oleanolic acid A is reacted with N-Boc ethylenediamine under the action of HATU to undergo amide condensation to obtain intermediate C. After removing Boc, it reacts with 4-methoxyisocyanate to obtain urea intermediate D. D undergoes monoesterification with diacids of different lengths to obtain E1-E2, which is then condensed with ribociclib F to obtain the final products G1-G2. In route two, 2-azidoethanol is reacted with p-toluenesulfonyl chloride under the action of triethylamine (TEA) to obtain intermediate J, which is then substituted with ribociclib under the action of Cs2CO3 and KI to obtain intermediate K. Intermediate D undergoes esterification with alkyne acids of different lengths to obtain L1-L3, which is then subjected to a click reaction with intermediate K to obtain the final products M1-M3. In route three, intermediate D undergoes a substitution reaction with alkyne bromides of different lengths under the action of sodium hydride to obtain N1-N3, which is then subjected to a click reaction with intermediate K to obtain the final products O1-O3.

[0028] The present invention also provides a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, wherein the excipients include at least one of carbohydrates, polymers, lipids or minerals.

[0029] The present invention has the following beneficial effects: the preparation process is simple and easy, and the prepared proteolysis-targeted chimera or its pharmaceutically acceptable salt has the effect of efficiently targeting and degrading CDK4 with high selectivity. Specifically, CP0371, which has a "molecular glue" function, is used as a novel E3 ligase ligand, and ribociclib is used as a target protein ligand. Linkers of different types and lengths are designed to synthesize and screen a new compound. Compound M1 can effectively induce the degradation of CDK4 in the human colorectal cancer cell line HCT-116 in a dose-dependent manner, has considerable bioavailability, and exhibits excellent in vivo anti-colon cancer effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure shows the concentration-dependent degradation of CDK4 protein by compound M1 in HCT-116 cell line. DETAILED DESCRIPTION

[0031] The following will provide a clear and complete description of the concept, specific structure and technical effects of the present invention in conjunction with the embodiments and drawings to fully understand the purpose, scheme and effects of the present invention.

[0032] Example 1

[0033] Preparation of CDK4 proteolysis-targeting chimeras

[0034] Preparation of compound G1:

[0035] The structure of compound G1 is as follows:

[0036] , The preparation process is as follows:

[0037] Step 1: Preparation of compound E1:

[0038] The structure of compound E1 is as follows:

[0039] ;

[0040] The specific preparation method is as follows: under an ice bath, compound D (648 mg, 1.00 mmol) was dissolved in dichloromethane (10 mL), and N,N'-dicyclohexylcarbodiimide (413 mg, 2.00 mmol), 4-dimethylaminopyridine (61.3 mg, 0.500 mmol) and pimelic acid (192 mg, 1.20 mmol) were added respectively. The ice bath was removed after 5 minutes, and the mixture was stirred at room temperature overnight. The liquid in the bottle was concentrated using a rotary evaporator, then washed with water (20 mL), extracted with ethyl acetate (3 × 20 mL), and the organic phase was dried over anhydrous sodium sulfate. After concentration, compound E1 was obtained by flash column chromatography on silica gel (petroleum ether: ethyl acetate = 3:1).

[0041] Compound E1 was tested and the test results were as follows: HRMS (ESI) calculated for C 47 H 70 N3O7 - [MH] - :788.5219, found. 788.5217.

[0042] The chemical formula of compound D in the above steps is as follows:

[0043]

[0044] Step 2: Preparation of compound G1:

[0045] The specific preparation method is as follows: E1 (158 mg, 0.200 mmol) and compound F (86.9 mg, 0.0.200 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N,N`,N`-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (152 mg, 0.400 mmol) and diisopropylethylamine (64.6 mg, 0.500 mmol) were added. The reaction was allowed to react at room temperature overnight. After the reaction was completed, the reaction solution was washed with 1N HCl solution (10 mL), saturated sodium bicarbonate solution (10 mL) and saturated NaCl solution (10 mL), respectively, and extracted with ethyl acetate (3×10 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1 to 2:1) to obtain compound G1 (white solid, 101 mg, 42%).

[0046] Compound G1 was tested, and the test results were as follows: 1 H NMR (400 MHz, CDCl3)δ 9.49 (s,1H), 9.00 (s, 1H), 8.11 (d,J = 1.3 Hz, 1H), 7.56 (d, J = 3.2 Hz, 2H), 7.36 –7.24 (m, 2H), 7.16 – 6.97 (m, 2H), 6.93 – 6.76 (m, 2H),6.52 (d, J = 0.7 Hz,1H), 6.18 (d, J = 0.7 Hz, 1H), 5.49 (s, 1H),4.71 (s, 1H), 4.41 (s, 1H), 3.80(s, 3H), 3.62 (d, J = 20.7 Hz, 4H),3.40 (d, J = 1.1 Hz, 2H), 3.38 – 3.28 (m, 2H),3.24 (d, J = 1.8Hz, 4H), 3.05 (s, 6H), 2.33 (s, 2H), 2.31 – 2.20 (m, 3H), 2.17– 2.07 (m, 3H),2.04 – 1.89 (m, 6H), 1.86 – 1.77 (m, 4H), 1.77 – 1.60 (m, 6H),1.60 – 1.56 (m,4H), 1.53 (dd, J = 13.0, 5.7 Hz, 2H), 1.46 – 1.39 (m, 3H), 1.38– 1.34(m, 4H), 1.34 – 1.28 (m, 2H), 1.24 (s, 1H), 1.07 – 1.01 (m, 7H), 1.00(s, 3H),0.96 (s, 3H), 0.91 (s, 3H), 0.86 (d, J = 3.1 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 177.7, 174.5, 174.8,166.6, 159.9, 159.3, 159.2, 149.2, 148.5, 145.1,138.8, 138.3, 137.8, 134.0,124.3, 123.0, 121.2, 118.9, 117.0, 116.4, 118.6,110.8, 83.5, 71.2, 62.3, 56.6,55.6, 55.1, 49.9, 48.2, 48.1, 47.8, 43.6, 42.0,41.7, 40.1, 40.0, 38.4, 38.1,38.0, 37.8, 37.2, 37.0, 36.4, 35.6, 34.5, 33.0,32.8, 32.3, 31.0, 29.7, 29.3,29.0, 27.3, 25.8, 25.7, 25.2, 23.4, 22.5, 19.9,17.5. HRMS (ESI): m / z calcd for C 70 H 100 N 11 O7 + [M+H] + : 1206.7802; found 1206.7805.

[0047] The chemical formula of compound F in the above steps is as follows:

[0048]

[0049] Example 2

[0050] Preparation of CDK4 proteolysis-targeting chimeras

[0051] Preparation of compound G2

[0052] The structure of compound G2 is as follows:

[0053] , its preparation process is as follows:

[0054] Compound G2 was obtained by following the synthetic steps of compound G1 described in Example 1, except that pimelic acid was replaced by suberic acid, while other experimental conditions remained unchanged.

[0055] Compound G2 was tested, and the test results were as follows: 1 H NMR (400 MHz, CDCl3)δ 9.49 (s,1H), 9.00 (s, 1H), 8.11 (d,J = 1.3 Hz, 1H), 7.56 (d, J = 3.2 Hz, 2H), 7.40 –7.25 (m, 2H), 7.17 – 7.00 (m, 2H), 6.91 – 6.80 (m, 2H),6.52 (d, J = 0.7 Hz,1H), 6.18 (d, J = 0.7 Hz, 1H), 5.49 (s, 1H),4.71 (s, 1H), 4.41 (s, 1H), 3.80(s, 3H), 3.62 (d, J = 20.7 Hz, 4H),3.40 (d, J = 1.1 Hz, 2H), 3.38 – 3.28 (m, 2H),3.24 (s, 4H), 3.05 (s,6H), 2.33 (s, 2H), 2.31 – 2.21 (m, 3H), 2.16 – 2.06 (m,3H), 2.03 – 1.88 (m,6H), 1.85 (s, 1H), 1.83 – 1.60 (m, 9H), 1.58 (s, 2H),1.56 – 1.48 (m, 4H), 1.44– 1.39 (m, 3H), 1.38 – 1.27 (m, 8H), 1.24 (s, 1H),1.07 – 1.01 (m, 7H), 1.00(s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.86 (d, J = 3.1Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 177.1, 174.8, 174.0,166.6, 159.3, 159.1,159.0, 149.0, 148.5, 145.1, 138.3, 138.0, 137.8, 134.7,124.3, 123.0, 121.5,118.9, 117.0, 116.4, 113.2, 110.6, 83.5, 65.2, 56.3, 55.8,55.1, 49.9, 48.2,48.1, 47.8, 43.1, 42.8, 42.0, 41.4, 40.5, 40.9, 38.9, 38.7,38.0, 37.8, 37.2,37.1, 36.4, 35.3, 33.0, 32.8, 32.3, 31.7, 31.0, 29.9, 29.7,29.3, 29.0, 27.5,26.0, 25.8, 25.2, 23.4, 22.5, 19.9, 17.9. HRMS (ESI): m / z calcd forC 71 H 102 N 11 O7 + [M+H] + :1220.7958; found 1220.7953.

[0056] Example 3

[0057] Preparation of CDK4 proteolysis-targeting chimeras

[0058] Preparation of compound M1

[0059] The structure of compound M1 is as follows:

[0060] , its preparation process is as follows:

[0061] Step 1: Preparation of compound L1:

[0062] The structure of compound L1 is as follows:

[0063] ;

[0064] The specific preparation method is as follows: under an ice bath, compound D (648 mg, 1.00 mmol) was dissolved in dichloromethane (10 mL), and N,N'-dicyclohexylcarbodiimide (413 mg, 2.00 mmol), 4-dimethylaminopyridine (61.3 mg, 0.500 mmol) and propiolic acid (84.1 mg, 1.20 mmol) were added respectively. The ice bath was removed after 5 minutes, and the mixture was stirred at room temperature overnight. The liquid in the bottle was concentrated using a rotary evaporator, then washed with water (20 mL), extracted with ethyl acetate (3 × 20 mL), and the organic phase was dried over anhydrous sodium sulfate. After concentration, compound L1 was obtained by flash column chromatography on silica gel (petroleum ether: ethyl acetate = 3:1).

[0065] Compound L1 was tested and the test results were as follows: HRMS (ESI) calculated for C 43 H 62 N3O5 + [M+H] + :700.4684, found. 700.4687.

[0066] Step 2: Preparation of compound M1:

[0067] The specific preparation method is as follows: L1 (140 mg, 0.200 mmol) and compound K (77.8 mg, 0.0.200 mmol) are dissolved in t CuSO4•5H2O (25.0 mg, 0.100 mmol) and sodium L-ascorbate (35.6 mg, 0.180 mmol) were added to a BuOH / H2O (6.4 mL, 1:1) mixed solvent and reacted at room temperature overnight. After the reaction was completed, CuSO4•5H2O was filtered out and the filtrate was concentrated in vacuo and separated by silica gel flash column chromatography (dichloromethane: methanol = 8:1) to obtain compound M1 (white solid, 74.1 mg, 34%).

[0068] Compound M1 was tested, and the test results are as follows:

[0069] 1 H NMR (400 MHz, CDCl3) δ8.71 (s, 1H), 7.99 (s, 2H), 7.21 (d, J = 8.4Hz, 2H), 6.82 (d, J = 8.5 Hz, 2H), 6.55 (t, J= 5.4 Hz, 1H), 6.45 (s, 1H), 5.64 (s, 1H), 5.39 (d, J = 3.7 Hz, 1H), 4.78 (t, J = 8.8 Hz, 1H), 4.32 (t, J = 4.9 Hz,2H), 3.77 (s, 3H), 3.69 (t, J = 5.1 Hz, 4H), 3.48 (t, J = 5.0 Hz, 2H), 3.42 (dd, J = 9.8, 5.4 Hz, 1H), 3.39 – 3.34 (m, 1H), 3.30(q, J = 5.9, 5.5 Hz, 1H), 3.20(dt, J = 7.6, 4.1 Hz, 2H), 3.15(s, 6H), 2.60 – 2.53 (m, 2H), 2.05 (dt, J = 16.4,8.1 Hz, 5H), 1.90 –1.85 (m, 2H), 1.76 – 1.69 (m, 4H), 1.58 (d, J = 13.0 Hz,5H), 1.52 (d, J = 15.1 Hz, 4H), 1.46 (d, J = 1.9 Hz, 1H), 1.43 (s, 1H), 1.39 (d, J = 4.7 Hz, 1H), 1.35 (s, 1H), 1.32 (d, J = 2.9 Hz, 1H), 1.25 (s, 6H),1.13 (s,3H), 0.98 (s, 3H), 0.93 – 0.80 (m, 13H), 0.77 (s, 3H), 0.72 (s, 3H).HRMS(ESI): m / z calcd for C 68 H 95 N 14 O6 + [M+H] + : 1203.7554; found 1203.7559.

[0070] The chemical formula of compound K in the above steps is as follows:

[0071]

[0072] Example 4

[0073] Preparation of CDK4 proteolysis-targeting chimeras

[0074] Preparation of compound M2

[0075] The structure of compound M2 is as follows:

[0076] , its preparation process is as follows:

[0077] The specific preparation method is: Compound M2 is obtained by following the synthetic steps of Compound M1 described in Example 3, except that propiolic acid is replaced by butynic acid, and other experimental conditions remain unchanged.

[0078] Compound M2 was tested, and the test results are as follows:

[0079] 1 H NMR (400 MHz, CDCl3)δ 8.72 (s, 2H), 8.44 – 8.28 (m, 4H), 8.02 (d, J =2.9 Hz, 2H), 7.74 (s,2H), 7.30 (dd, J = 9.1, 3.0 Hz, 2H), 6.43 (s, 2H), 4.78(t, J =8.9 Hz, 2H), 4.49 (t, J = 6.3 Hz, 4H), 3.83 (s, 4H), 3.72 (s, 6H), 3.16(d, J = 4.9 Hz, 6H), 3.14 – 3.16 (m, 13H), 2.90 (t, J = 6.3 Hz,4H), 2.74 – 2.65 (m,8H), 2.22 – 1.89 (m, 10H), 1.82 – 1.58 (m, 6H), 1.18 –1.34 (m, 10H), 0.91 –0.75 (m, 6H). HRMS (ESI): m / z calcd for C 69 H 97 N 14 O6 + [M+H] + : 1217.7710; found1217.7706.

[0080] Example 5

[0081] Preparation of CDK4 proteolysis-targeting chimeras

[0082] Preparation of compound M3

[0083] The structure of compound M3 is as follows:

[0084] ;

[0085] The specific preparation method is: Compound M3 is obtained by following the synthetic steps of Compound M1 described in Example 3, except that propiolic acid is replaced by pentynoic acid, and other experimental conditions remain unchanged.

[0086] Compound M3 was tested, and the test results are as follows: 1 H NMR (400 MHz, CDCl3)δ 9.49 (s,1H), 9.00 (s, 1H), 7.96 (d, J = 1.3 Hz, 1H), 7.56 (d, J = 3.2 Hz, 2H), 7.50 (s,1H), 7.35 – 7.26 (m, 2H), 7.16 – 6.97 (m, 2H), 6.89 –6.80 (m, 2H), 6.52 (d, J =0.7 Hz, 1H), 6.18 (d, J = 0.7 Hz, 1H),5.49 (s, 1H), 4.73 (s, 1H), 4.41 (s, 1H),4.21 (d, J = 1.3 Hz, 2H), 3.80(s, 3H), 3.40 (d, J = 1.1 Hz, 2H), 3.37 – 3.28 (m,2H), 3.23 (s, 3H), 3.05 (s, 6H), 3.03 – 2.90 (m, 2H), 2.87 – 2.74 (m, 2H), 2.73 (d, J = 1.9Hz, 2H), 2.65 (s, 2H), 2.59 (s, 2H), 2.28 (s, 1H), 2.17 – 2.06(m, 3H), 2.02 –1.91 (m, 5H), 1.90 (d, J = 5.0 Hz, 1H), 1.85 (s, 1H), 1.82 (d, J =0.9 Hz, 2H), 1.78 (s, 1H), 1.75 (d, J= 13.0 Hz, 1H), 1.72 – 1.59 (m,5H), 1.54(d, J = 5.7 Hz, 1H), 1.51 (d, J = 5.7 Hz, 1H), 1.45 –1.39 (m, 3H), 1.38 – 1.28(m, 4H), 1.24 (s, 1H), 1.08 – 1.01 (m, 7H), 1.00 (s,3H), 0.96 (s, 3H), 0.91(s, 3H), 0.86 (d, J = 3.1 Hz, 6H). 13 C NMR (100 MHz, CDCl3) δ 177.1, 174.1,166.6,159.3, 159.1, 159.0, 149.0, 148.5, 146.3, 145.4, 138.3, 138.0, 137.4,134.0,128.9, 124.3, 123.0, 121.2, 118.9, 117.0, 116.4, 114.2, 110.8, 83.0,63.2,59.9, 56.7, 56.3, 55.6, 55.1, 54.8, 54.3, 49.9, 48.2, 47.8, 43.1, 42.0,41.7,40.5, 40.2, 38.9, 38.7, 38.0, 37.9, 37.2, 37.0, 33.0, 32.8, 32.3, 32.2,31.0,30.5, 29.7, 29.0, 25.8, 25.2, 24.1, 23.3, 22.5, 19.9, 17.5. HRMS (ESI):m / z calcd forC 70 H 99 N 14 O6 + [M+H] + :1231.7867; found 1231.7866.

[0087] Example 6

[0088] Preparation of CDK4 proteolysis-targeting chimeras

[0089] Preparation of compound O1

[0090] The structure of compound O1 is as follows:

[0091] , its preparation process is as follows:

[0092] Step 1: Preparation of compound N1:

[0093] The structure of compound N1 is as follows:

[0094] ;

[0095] The specific preparation method is as follows: under an ice bath, compound D (648 mg, 1.00 mmol) was dissolved in tetrahydrofuran (10 mL), sodium hydride (40 mg, 1.00 mmol) was slowly added, and 3-bromopropyne (143 mg, 1.20 mmol) was slowly added after 10 minutes. The ice bath was removed after 10 minutes, and the mixture was stirred at room temperature overnight. The liquid in the bottle was concentrated using a rotary evaporator, then washed with water (20 mL), extracted with ethyl acetate (3 × 20 mL), and the organic phase was dried over anhydrous sodium sulfate. After concentration, compound N1 was obtained by flash column chromatography on silica gel (petroleum ether: ethyl acetate = 3:1).

[0096] Compound N1 was tested and the test results were as follows: HRMS (ESI) calculated for C 43 H 64 N3O4 + [M+H] + :686.4891, found. 686.4894.

[0097] The chemical formula of compound D in the above steps is as follows:

[0098] ;

[0099] Step 2: Preparation of compound O1:

[0100] The specific preparation method is as follows: N1 (137 mg, 0.200 mmol) and compound K (77.8 mg, 0.0.200 mmol) are dissolved in t CuSO4•5H2O (25.0 mg, 0.100 mmol) and sodium L-ascorbate (35.6 mg, 0.180 mmol) were added to a BuOH / H2O (6.4 mL, 1:1) mixed solvent and reacted at room temperature overnight. After the reaction was completed, CuSO4•5H2O was filtered out and the filtrate was concentrated in vacuo and separated by silica gel flash column chromatography (dichloromethane: methanol = 8:1) to obtain compound O1 (white solid, 98.9 mg, 44%).

[0101] Compound O1 was tested, and the test results were as follows: 1H NMR (400 MHz, CDCl3)δ 9.49 (s,1H), 9.00 (s, 1H), 7.96 (d, J = 1.3 Hz, 1H), 7.69 (s, 1H),7.56 (d, J = 3.2 Hz,2H), 7.37 – 7.23 (m, 2H), 7.14 – 6.96 (m, 2H), 6.85(d, J = 7.6 Hz, 2H), 6.52(d, J = 0.7 Hz, 1H), 6.18 (d, J = 0.7 Hz, 1H), 5.49 (s, 1H), 4.85 – 4.59 (m, 2H),4.41 (s, 1H), 4.32 – 4.17 (m,2H), 3.80 (s, 3H), 3.56 (s, 1H), 3.40 (d, J = 1.1Hz, 2H), 3.37 – 3.27(m, 2H), 3.23 (d, J = 5.6 Hz, 4H), 3.05 (s, 6H), 2.90 –2.74 (m, 2H),2.62 (d, J = 21.8 Hz, 4H), 2.28 (t, J = 1.0 Hz, 1H), 2.18 – 2.07(m,3H), 2.03 – 1.96 (m, 2H), 1.96 – 1.88 (m, 4H), 1.85 (s, 1H), 1.82 (d, J = 0.9Hz, 2H), 1.79 – 1.72 (m, 2H), 1.71 – 1.60 (m, 5H), 1.53 (dd, J =13.0, 5.9 Hz,3H), 1.42 (dd, J = 13.1, 1.2 Hz, 2H), 1.39 – 1.27 (m, 4H),1.20 (s, 1H), 1.03(dd, J = 13.5, 5.1 Hz, 7H), 0.96 (d, J = 1.8Hz, 6H), 0.91 (s, 3H), 0.86 (d, J = 2.8Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 177.7, 166.6, 159.9,159.1, 159.1, 149.2,148.5, 145.4, 143.9, 138.3, 138.0, 137.8, 134.7, 127.2,124.3, 123.0, 121.5,118.9, 117.0, 116.9, 114.4, 110.6, 87.6, 77.8, 69.4, 63.2,56.1, 56.0, 55.8,54.8, 54.7, 54.6, 49.9, 48.6, 47.4, 43.6, 42.8, 41.5, 40.1,39.3, 39.1, 38.8,38.1, 38.0, 37.8, 37.4, 37.2, 33.0, 32.5, 32.3, 30.8, 30.0,29.7, 25.8, 25.2,23.2, 22.8, 19.9, 17.5. HRMS (ESI): m / z calcd for C 68 H 97 N 14 O5 + [M+H] + : 1189.7761;found 1189.7766.

[0102] The chemical formula of compound K in the above steps is as follows:

[0103] ;

[0104] Example 7

[0105] Preparation of CDK4 proteolysis-targeting chimeras

[0106] Preparation of compound O2

[0107] The structure of compound O2 is as follows:

[0108] , its preparation process is as follows:

[0109] Compound O2 was obtained by following the synthetic steps of compound O1 described in Example 6 except that 3-bromopropyne was replaced by 4-bromo-n-butyne, while other experimental conditions remained unchanged.

[0110] Compound O2 was tested, and the test results are as follows: 1 H NMR (400 MHz, CDCl3)δ 9.49 (s,1H), 9.00 (s, 1H), 7.96 (d, J<h2 style=";text-align:left;direction:ltr">= 1.3 Hz, 1H), 7.69 – 7.50 (m, 3H), 7.41 – 7.22 (m, 2H), 7.18 – 6.96 (m, 2H), 6.91 – 6.80 (m, 2H), 6.52 (d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 0.7 Hz, 1H),6.18 (d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 0.7 Hz, 1H), 5.49 (s, 1H), 4.41 (s, 1H),4.21 (d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 1.3 Hz, 2H), 3.80 (s, 3H), 3.72 – 3.49 (m, 2H), 3.40 (d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 1.1 Hz, 2H), 3.38 – 3.28 (m, 3H), 3.23 (d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 5.6 Hz, 4H), 3.05 (s,6H), 2.96 – 2.73 (m, 4H), 2.65 (s, 2H),2.59 (s, 2H), 2.28 (t,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 1.0Hz, 1H), 2.21 – 2.06 (m, 3H), 2.04 – 1.96 (m,2H), 1.95 – 1.68 (m, 5H), 1.68 –1.58 (m, 5H), 1.53 (dd,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 13.0, 5.9 Hz, 4H), 1.42 (dd,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =13.1, 1.2 Hz, 3H), 1.39 – 1.24(m, 6H), 1.20(s, 1H), 1.07 – 0.99(m, 7H), 0.96(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 2.8 Hz, 6H), 0.91 (s, 3H), 0.86 (d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 2.8 Hz, 6H).<h2 style=";text-align:left;direction:ltr"> 13C NMR (100 MHz, CDCl3) δ 177.1, 166.1, 159.3,159.1, 159.0, 149.0, 148.5, 145.4,142.6, 138.8, 138.0, 137.8, 134.7, 128.8,124.3, 123.0, 121.2, 118.3, 117.0,116.4, 113.3, 110.8, 87.2, 71.0, 69.9, 63.2,56.7, 56.6, 55.6, 54.2, 54.5,54.6, 49.9, 49.1, 48.6, 47.8, 43.1, 42.8, 41.5,40.1, 39.1, 39.0, 38.7, 38.2,38.0, 37.8, 37.6, 33.0, 32.8, 32.3, 30.8, 29.9,29.7, 27.4, 25.0, 25.2, 23.6,22.8, 19.5, 17.2. HRMS (ESI): m / z calcd for C 69 H 99 N 14 O5 + [M+H] + : 1203.7917; found1203.7912.

[0111] Example 8

[0112] Preparation of CDK4 proteolysis-targeting chimeras

[0113] Preparation of compound O3

[0114] The structure of compound O3 is as follows:

[0115] The preparation process is as follows

[0116] Compound O3 was obtained by following the synthetic steps of compound O1 described in Example 6 except that 3-bromopropyne was replaced by 5-bromo-n-pentyne, while other experimental conditions remained unchanged.

[0117] Compound O3 was tested, and the test results were as follows: 1 H NMR (400 MHz, CDCl3)δ 9.49 (s,1H), 9.00 (s, 1H), 7.96 (d, J= 1.2 Hz, 1H), 7.65 – 7.44 (m,3H), 7.38 – 7.19(m, 2H), 7.14 – 6.97 (m, 2H), 6.89 – 6.76 (m, 2H), 6.52 (d, J = 0.7 Hz, 1H),6.18 (d, J = 0.6 Hz, 1H), 5.49 (s, 1H), 4.41 (s, 1H),4.21 (d, J = 1.3 Hz, 2H),3.80 (s, 3H), 3.63 (d, J = 12.5 Hz, 1H),3.49 (d, J = 12.5 Hz, 1H), 3.44 – 3.28(m, 5H), 3.23 (s, 4H), 3.05 (s,6H), 2.90 – 2.71 (m, 4H), 2.62 (d, J = 21.8 Hz,4H), 2.28 (t, J =1.0 Hz, 1H), 2.18 – 2.07 (m, 3H), 2.03 – 1.92 (m, 5H), 1.91 –1.79 (m, 6H),1.79 – 1.71 (m, 2H), 1.71 – 1.61 (m, 4H), 1.58 (d, J = 12.9 Hz,1H),1.53 (dd, J = 13.0, 5.9 Hz, 3H), 1.42 (dd, J = 13.1, 1.2 Hz, 2H),1.39 – 1.28(m, 3H), 1.20 (s, 1H), 1.08 – 0.98 (m, 7H), 0.96 (d, J = 2.8Hz, 6H), 0.91 (s,3H), 0.86 (d, J = 2.8 Hz, 6H). 1313C NMR (100 MHz, CDCl3) δ 177.7, 166.6, 159.9, 159.3, 159.2, 149.2, 148.5, 145.4, 144.7, 138.8, 138.0, 137.4, 134.7, 131.2, 124.3, 123.0, 121.5, 118.3, 117.0, 116.4, 113.3, 110.8, 87.2, 69.9, 64.5, 62.7, 56.8, 56.3, 55.8, 54.8, 54.6, 54.3, 49.9, 48.2, 47.8, 43.1, 42.0, 41.8, 40.1, 39.7, 39.6, 39.0, 38.1, 38.0, 37.9, 37.6, 33.0, 32.5, 32.3, 31.6, 30.9, 30.2, 29.1, 29.0, 26.4, 25.8, 25.8, 23.2, 22.7, 19.5, 17.2. HRMS (ESI): m / z calcd for C 70 H 101 N 14 O5 + [M+H] + : 1217.8074; found 1217.8077。

[0118] Example 9

[0119] Screening of the anti-tumor cell proliferation effect of compounds

[0120] The above-mentioned PROTAC molecules based on the pentacyclic triterpenoid molecular glue CP0371 (the proteolysis-targeting chimera prepared in Examples 1-8) as the E3 ligase ligand and Ribociclib as the target protein ligand were screened for anti-tumor cell proliferation experiments. MTT experiments were carried out in three different malignant tumor cell lines (human colorectal cancer cells HCT-116, HCT-15, human lung cancer cells HCC-827). The results are shown in the following table. In order to compare the activity differences of the compounds more intuitively, the IC 50 was divided into four categories: 1 μM < IC 50 < 3 μM (A), 3 μM < IC50 < 10 μM (B), 10 μM < IC50 (C). The test results are shown in the following table: The specific test results are shown as follows.

[0121]

[0122] From the above results, it can be seen that the above compounds exhibited good anti-proliferative activity in several different cell lines, and these compounds generally performed better in colorectal cancer cells, especially in the HCT-116 cell line; in addition, compound M1 exhibited high anti-proliferative activity in multiple tumor cell lines. Therefore, subsequent evaluations will select compound M1 for further evaluation in the HCT-116 cell line.

[0123] Example 10

[0124] Compound M1 degrades CDK4 in a dose-dependent manner

[0125] The preferred compound M1 in Example 9 was evaluated for its ability to degrade EGFR in human colorectal cancer cell line HCT-116 using Western blotting. The results are shown in FIG. Figure 1 The results showed that with the increase of compound M1 concentration, the amount of CDK4 degradation in HCT-116 cells was significantly increased, indicating that the degradation of EGFR by compound M1 was concentration-dependent.

[0126] Example 11

[0127] Evaluation of the bioavailability of compound M1

[0128] The low bioavailability of PROTAC molecules usually limits their in vivo efficacy. One of the key factors limiting the bioavailability of PROTAC molecules is the poor solubility of conventional E3 ligase ligands. The novel E3 ligase ligand CP0371 selected in this invention has the characteristics of good solubility and is expected to exert higher in vivo bioavailability. The preferred compound, M1, was evaluated in rats for pharmacokinetic analysis. After overnight fasting, three rats were gavaged (20 mg / kg) and three rats were administered via tail vein (5 mg / kg). Blood was then collected at 5, 15, 30, 1, 2, 4, 6, and 8 hours. The blood was then placed in a pre-heparinized tube and gently tapped to thoroughly mix the blood with the heparin. Plasma was then obtained by centrifugation (4°C, 3000 rpm, 10 min). Fifty microliters of plasma was collected, 50 microliters of diluent (50% methanol / water) and 250 microliters of methanol precipitant were added, and the mixture was vortexed and centrifuged (4°C, 12000 rpm, 10 min). The supernatant was sealed and analyzed by LC-MS / MS. Parameters were analyzed using WinNonlin software. Results showed that compound M1 had a good oral bioavailability of 32.7%, a significant improvement compared to other PROTAC molecules.

[0129] Example 12

[0130] Safety evaluation of compound M1

[0131] The hERG cardiotoxicity of the preferred compound M1 was evaluated. HEK293 cells stably transfected and expressing human myocardial HERG ion channels were used. The cultured cells were placed under an inverted microscope, and the recording electrodes were brought into contact with the cell surface using a micromanipulator. Membrane capacitance compensation and series resistance compensation were then performed to smooth the current line for subsequent testing. An 8-channel perfusion drug delivery system was controlled by a magnetic valve, and drug delivery was performed through the drug delivery electrode. The prepared compound M1 stock solution was diluted to 30 μM, 10 μM, 3 μM, 1 μM, and 0.3 μM in proportion and added to the perfusion drug delivery system. The flow rate was controlled by gravity to continuously perfuse the cells. The drug delivery electrode was connected and adjusted to the upper left of the cell. After the cell current was recorded, it was perfused with the control group (without compound M1). After the current stabilized, the compound M1 was perfused and its effect was observed. According to Clampfit software analysis, the compound M1 cardiotoxicity IC 50 The concentration of 15.89 μM showed high safety.

[0132] Example 13

[0133] In vivo efficacy evaluation of compound M1

[0134] The in vivo efficacy of compound M1 was evaluated, and HCT-116 cells were used to establish a xenograft model. HCT-116 cells were inoculated subcutaneously in the axilla of nude mice, and the tumors grew to 70-110 mm. 3 The mice were randomly divided into a control group (5 mice) and a Compound M1-treated group (40 mpk, 5 mice) for 15 days. There was no significant weight fluctuation or abnormality in either the treated or control groups, indicating the safety of Compound M1. Tumor weight and volume in the treated group were significantly lower than those in the control group, with a tumor inhibition rate ((1 - tumor weight in the treated group / tumor weight in the control group) * 100%) of 66.7%. This demonstrates that Compound M1 has strong in vivo efficacy.

[0135] Although the present invention has been described in considerable detail and with particularity with respect to several described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but rather should be construed as providing a broad possible interpretation of these claims in view of the prior art by reference to the appended claims, thereby effectively encompassing the intended scope of the invention. In addition, the invention has been described above in terms of embodiments foreseen by the inventors for the purpose of providing a useful description, and those insubstantial modifications of the invention that are not currently foreseen may still represent equivalent modifications of the invention.

Claims

1. A CDK4 protein hydrolysis targeting chimera, wherein the molecular structure of the CDK4 protein hydrolysis targeting chimera is any one of Formula II to Formula IV: Formula II, Formula III, Formula IV; in, In Formula II, n is selected from any positive integer from 1 to 12; in Formula III, n is selected from any integer from 0 to 8; In formula IV, n is any positive integer selected from 1-9.

2. The CDK4 proteolysis targeting chimera according to claim 1, characterized in that The molecular structure of the CDK4 proteolysis targeting chimera is shown below: 。 3. A method for preparing the CDK4 proteolysis targeting chimera according to claim 1, characterized in that: Its preparation route is route 1, route 2 or route 3: Route 1: ； Route 2: ； Route 3: 。 4. Use of the CDK4 proteolysis targeting chimera or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2 in the preparation of a drug for treating and / or preventing cancer, characterized in that: The cancers were colorectal cancer and lung cancer.

5. A pharmaceutical composition, characterized in that The main active ingredient is the CDK4 protein hydrolysis targeting chimera or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

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