PROTAC compound targeting CMYC as well as preparation method and application of PROTAC compound

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a PROTAC compound targeting CMYC as well as a preparation method and application of the PROTAC compound. Compared with an existing CMYC inhibitor, the PROTAC compound capable of precisely targeting CMYC provided by the invention can thoroughly degrade CMYC protein in cells through a ubiquitin-proteasome system, realizes efficient degradation of CMYC in tumor cells, has a relatively good curative effect in various tumor models, and has a good application prospect. The compound can effectively kill colorectal cancer cells and has small influence on normal cells; moreover, the preparation method of the PROTAC compound targeting CMYC is designed according to the conformational relationship between the inhibitor and CMYC protein, and molecular sites are more suitable for Euclidean reaction; meanwhile, by protecting the E3 ubiquitin ligase ligand, the selectivity of the reaction process is ensured, the reaction conditions are mild, and the method is simple and convenient.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and particularly discloses a PROTAC compound targeting CMYC as well as a preparation method and application thereof. BACKGROUND

[0002] CMYC (often written as CMYC or MYC) is a kind of "super" transcription factor in cells, which can form a dimer with its partner protein MAX and bind to the E-box sequence, thereby widely amplifying the transcription output of active promoters in cells, regulating ribosome biogenesis, protein synthesis, cell cycle progression, energy metabolism and cell growth and other basic biological processes, and thus is regarded as one of the core pivots driving tumor occurrence and maintenance (the influence range can reach a considerable part of the cell genome) (document 1. Chen H, Liu H, Qing G. Targeting oncogenic Myc as a strategy for cancer treatment. Signal Transduction and Targeted Therapy. 2018;3:5. doi:10.1038 / s41392-018-0008-7.).

[0003] In various solid tumors and hematological malignancies, MYC realizes persistent high expression / high activity through (1) gene amplification, (2) chromosomal translocation, (3) persistent activation of upstream signals (such as RAS / MAPK, WNT / β-catenin, etc.) or (4) abnormal protein stability, and the abnormal expression of MYC is often related to invasiveness, accelerated proliferation speed, treatment resistance and poor prognosis (document 2. Stine ZE, Walton ZE, Altman BJ, Hsieh AL, Dang CV. MYC, Metabolism, and Cancer. Cancer Discovery. 2015;5(10):1024-1039. doi:10.1158 / 2159-8290.CD-15-0507.).

[0004] The oncogenic activity of CMYC is not only reflected in driving cell cycle and proliferation, but also through reprogramming cellular metabolism (increasing glucose metabolism, glutamate transport and nucleotide biosynthesis, etc.), maintaining the high demand of nutrients / energy for cancer cells and supporting rapid growth; in addition, MYC can also affect the stemness characteristics, escape immune surveillance and induce the overall "amplification" effect of gene expression, thus playing a multi-faceted role in tumor occurrence, metastasis and tumor microenvironment regulation (literature 3. Gregory MA, Hann SR. Phosphorylation by glycogen synthase kinase-3 controls CMYC proteolysis and subnuclear localization. Journal of Biological Chemistry. 2000s.).

[0005] The CMYC protein level is strictly regulated by post-translational modification: the phosphorylation-dephosphorylation cascade at S62, T58 and other sites determines the stability of MYC - S62 phosphorylation temporarily stabilizes MYC, while subsequent T58 phosphorylation can guide it to be recognized by E3 ubiquitin ligase such as Fbw7 and degraded by proteasome; therefore, dysfunction of these modification enzymes (such as GSK-3, ERK, etc.) or Fbw7 will lead to abnormal stability of MYC in tumors and promote oncogenicity (literature 4. Welcker M, Orian A, Jin J, et al. The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent CMYC degradation. Proceedings of the National Academy of Sciences (PNAS). 2004;101(24):9085-9090. doi:10.1073 / pnas.0402770101.).

[0006] Given the "central hub" role of MYC in multiple tumors, it has long been considered an extremely attractive but "difficult to drug" target (due to the protein's large amount of no secondary structure, functional dependence on protein-protein and protein-DNA interactions). In recent years, direct and indirect inhibition strategies against MYC are rapidly developing - including small molecules or peptides / microproteins that interfere with MYC-MAX dimerization / DNA binding (e.g. Omomyc-derived agents), strategies to inhibit MYC transcription / translation, promote MYC degradation, and synthetic lethal combination therapies targeting MYC overexpression (5. Garralda E, Beaulieu M-E, Moreno V, et al. **MYC targeting by OMO-103 in solid tumors: a phase 1 trial. Nature Medicine. 2024; 30: 762-771. doi: 10.1038 / s41591-024-02805-1.).

[0007] Recent studies have also found that in rheumatoid arthritis (RA), CMYC is involved in the apoptosis and destruction of articular chondrocytes (Document 6. Yatsugi, N., Tsukazaki, T., Osaki, M. et al. Apoptosis of articular chondrocytes in rheumatoid arthritis and osteoarthritis: correlation of apoptosis with degree of cartilage destruction and expression of apoptosis-related proteins of p53 and CMYC. J Orthop Sci 5, 150-156 (2000). https: / / doi.org / 10.1007 / s007760050142). At the same time, in a classic early study, it has been found that the expression of CMYC in the mucosa of ulcerative colitis and Crohn's disease patients biopsy is increased, which proves that CMYC is also highly related to chronic inflammation of the intestine (Document 7. Macpherson AJ, Chester KA, Robson L, et al Increased expression of CMYC proto-oncogene in biopsies of ulcerative colitis and Crohn's colitis. Gut 1992; 33: 651-656. https: / / doi.org / 10.1136 / gut.33.5.651). Therefore, the CMYC degradation PROTAC drug developed by the present application also has good application potential for important chronic refractory diseases other than tumor diseases.

[0008] Nevertheless, how to efficiently and selectively inhibit MYC activity (or destroy its downstream key metabolic / transcriptional programs) in tumor cells while ensuring normal tissue tolerance is still the core problem of current research and drug development, and the heterogeneity of MYC carcinogenic mechanism in different tumor types and its interaction with signal pathways / microenvironment also greatly limits the efficacy of existing small molecule inhibitors in different tumors. Therefore, the MYC-PROTAC protein degradation drug developed by the present application will provide a more thorough and effective treatment plan for the clinical treatment of various types of tumors. SUMMARY

[0009] The purpose of this application is to overcome the shortcomings of the prior art and provide a PROTAC compound targeting CMYC, its preparation method, and its application. This application provides a PROTAC compound that precisely targets CMYC, achieving efficient degradation of CMYC within tumor cells and demonstrating good therapeutic effects in various tumor models.

[0010] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a PROTAC compound targeting CMYC, the PROTAC compound comprising a target protein ligand, a linker and an E3 ubiquitin ligase ligand, wherein the target protein ligand is linked by the linker and the E3 ubiquitin ligase ligand; The target protein ligands include MYCi361 or KDJ-Pyr-9; When the target protein ligand is MYCi361, the structural formula of the PROTAC compound is shown in formula (I); Formula (I); The target protein ligand is KDJ-Pyr-9, and the structural formula of the PROTAC compound is shown in formula (II); Formula (II); In equation (I), n is a natural number from 2 to 8; In equation (II), m is a natural number from 2 to 8.

[0011] The PROTAC compound (PROteolysis Targeting Chimera) provided in this application differs from traditional small molecule inhibitors. It utilizes a naturally occurring ubiquitin-protease system in cells to induce the degradation of targeted proteins. The PROTAC compound consists of three parts: a target protein ligand, a linker, and an E3 ubiquitin ligase ligand.

[0012] Compared to traditional small molecule inhibitors used in cancer treatment, PROTAC compounds have several potential advantages: they are event-driven (rather than site-driven - directly inhibiting the functional activity of target proteins), so they can achieve good efficacy at very low doses; after initiating protein degradation, PROTACs are separated from the complex and enter the next catalytic cycle, resulting in high drug action efficiency; each PROTAC can degrade many protein molecules, and has the potential to overcome untreatable targets.

[0013] In this application, the target protein ligands used include MYCi361 or KDJ-Pyr-9. Choosing this ligand has advantages in substrate specificity, safety and optimization of physicochemical properties.

[0014] As a preferred embodiment of the targeting CMYC PROTAC compound described in the present application, in the formula (I), the carbon chain of -(C)n- is connected to the amide group in VHL of the E3 ligase ligand; and / or, in the formula (II), the carbon chain of -(C)m- is connected to the amide group in VHL of the E3 ligase ligand.

[0015] The present application selects the Cl atom site in the benzene ring of the MYCi361 molecule as the Linker linker site: this site is a non-active region site on the precursor CMYC ligand MYCi361 molecule of the targeting CMYC PROTAC compound. The full name of the MYCi361 molecule is 6-((4-chlorobenzyl)oxy)-3-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)-3',5'-bis(trifluoromethyl)-[1,1'-biphenyl]-2-ol. Based on the structure-activity relationship between the MYCi361 molecule and CMYC, the PROTAC formed by connecting the linker and the E3 ligase ligand at this site better targets CMYC, and this site is easy to couple, so it is the best connection site for the Linker linker. The carbon chain of -(C) n - is connected to the amide group of the VHL ligand. By exchanging the terminal acetyl group of VHL with a suitable linker, the carbon chain of Linker can be easily connected to VHL through an amide bond.

[0016] The present application selects the amide group site on the benzene ring of the KDJ-Pyr-9 molecule as the Linker linker site: this site is a non-active region site on the precursor CMYC ligand KDJ-Pyr-9 molecule of the targeting CMYC PROTAC compound. The full name of the KDJ-Pyr-9 molecule is 4-(2-(furan-2-yl)-6-(4-nitrophenyl)pyridin-4-yl)benzamide. Based on the structure-activity relationship between the KDJ-Pyr-9 molecule and CMYC, the PROTAC formed by connecting the linker and the E3 ligase ligand at this site better targets CMYC, and this site is easy to couple, so it is the best connection site for the Linker linker. The carbon chain of -(C) m - is connected to the amide group of the VHL ligand. By exchanging the terminal acetyl group of VHL with a suitable linker, the carbon chain of Linker can be easily connected to VHL through an amide bond.

[0017] As a preferred embodiment of the PROTAC compound targeting CMYC described in the present application, the hydroxyl on the VHL of the E3 ligase ligand is modified by TBSCl.

[0018] The hydroxyl on the VHL is modified with TBSCl. By protecting the hydroxyl of VHL in advance with TBS, the E3 ligase ligand is prevented from competing with the carboxyl of the linker and from side reactions in the amide bond formation reaction, ensuring the selectivity of the reaction. After the synthesis of the PROTAC is completed, the TBS protecting group is removed by TBAF.

[0019] As a preferred embodiment of the PROTAC compound targeting CMYC described in the present application, the PROTAC compound is P-MYC1-4 or P-MYC1-8; The structural formula of P-MYC1-4 and P-MYC1-8 is shown in formula (III); Formula (III).

[0020] In the technical solution of the present application, compared with the current CMYC inhibitor MYCi361, the P-MYC1-4 and P-MYC1-8 compounds can degrade CMYC in cells, further control and kill tumors, especially colorectal cancer cells. Compared with MYCi361, the P-MYC1-4 and P-MYC1-8 compounds have obvious killing effect on human CRC cells and weak killing effect on human normal cells.

[0021] As a preferred embodiment of the PROTAC compound targeting CMYC described in the present application, the PROTAC compound is P-MYC2-6; The structural formula of P-MYC2-6 is shown in formula (IV); Formula (IV).

[0022] In the present application, compared with the current CMYC inhibitor KDJ-Pyr-9, the P-MYC2-6 compound can degrade CMYC in cells, further control and kill tumors, especially colorectal cancer cells. Compared with KDJ-Pyr-9, the P-MYC2-6 compound has obvious killing effect on human CRC cells and weak killing effect on human normal cells.

[0023] The application provides three PROTAC compounds targeting CMYC, P-MYC1-4, P-MYC1-8 and P-MYC2-6, which can degrade CMYC protein in cells through the ubiquitin-proteasome system, compared with existing CMYC inhibitors. The three compounds can effectively kill cancer cells (such as colorectal cancer cells) while having little effect on normal cells.

[0024] The application also provides a ternary complex comprising a target protein, the PROTAC compound and an E3 ligase, which ubiquitinates the target protein.

[0025] In the technical solution of the application, the PROTAC compound specifically recognizes and binds to the target through the target protein ligand at one end, and specifically recognizes and binds to the E3 ligase through the E3 ligase ligand at the other end, forming a ternary complex of target protein-PROTAC-E3 ligase. In this complex, the target protein is ubiquitinated by the E3 ligase, and the ubiquitinated target protein is recognized and degraded by the proteasome, thereby inhibiting the function of the target protein.

[0026] The application also provides a preparation method of the above-mentioned PROTAC compound targeting CMYC, comprising the following steps: (1) dissolve the substrate (2R, 4R)-1-[(S)-2-amino-3, 3-dimethylbutanoyl]-4-hydroxy-N-[4-(4-methylthiazol-5-yl) benzyl] pyrrolidine-2-carboxamide in DMF, add imidazole and TBS-Cl for reaction to obtain a reaction solution; add ethyl acetate to the reaction solution, separate organic phase I, and extract the aqueous phase with ethyl acetate; wash organic phase I, dry, filter and concentrate under reduced pressure to obtain a crude product; dissolve the crude product in DMF, add a Fmoc-protected amino acid linker, 1-hydroxybenzotriazole and a DMF solution of N, N'-diisopropyl carbodiimide for reaction and stirring, and then add a DMF solution to obtain a reaction mixture; quench the reaction mixture with water, extract with dichloromethane to obtain organic phase II, wash organic phase II, dry, filter and concentrate under reduced pressure to obtain a VHL ligand linker conjugate; (2) dissolve the VHL ligand linker conjugate obtained in step (1) in toluene, add Pd(OAc)2, MYCi361 and NaOtBu, and then add aryl chloride ArCl for stirring reaction to obtain a reaction solution; add TBAF dissolved in THF to the reaction solution for stirring to obtain a reaction mixture; quench the reaction mixture with water, extract the aqueous phase with ethyl acetate to obtain organic phase III, wash organic phase I, dry, filter and concentrate under reduced pressure, purify to obtain a PROTAC compound; or, (2) The VHL ligand-linker conjugate obtained in step (1) is dissolved in toluene, a DMF solution of KDJ-Pyr-9, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide is added, then a DMF solution is added for stirring to obtain a reaction mixture; the reaction mixture is quenched with water and extracted with dichloromethane to obtain an extraction product, which is concentrated, THF-dissolved TBAF is added for continuous stirring, then quenched with water, the aqueous phase is extracted with ethyl acetate to obtain an organic phase IV, which is washed, dried, filtered and concentrated under reduced pressure, purified and PROTAC compounds are obtained.

[0027] The application provides three preparation methods of the CMYC-targeted PROTAC compounds, which are designed by the conformational relationship between the inhibitor and the CMYC protein, and the molecular sites are more suitable for the Euclidean reaction; meanwhile, the selectivity of the reaction process is ensured by protecting the E3 ubiquitin ligase ligand, and the reaction conditions are mild and the method is simple.

[0028] The application also provides application of the CMYC-targeted PROTAC compounds in preparation of tumor treatment drugs.

[0029] The application provides application of the CMYC-targeted PROTAC compounds, and the drugs have good anticancer effects in various cells such as colorectal cancer and breast cancer, and have no obvious toxic side effects on non-cancer cell lines.

[0030] As a preferred embodiment of the application, the tumor comprises at least one of colorectal cancer, gastric cancer and breast cancer.

[0031] The application also provides a tumor drug, which comprises the CMYC-targeted PROTAC compound or the ternary complex.

[0032] The CMYC-targeted PROTAC compounds (including P-MYC1-4, P-MYC1-8 and P-MYC2-6) described in the application have the following core mechanism: the CMYC-targeting ligand (MYCi361 or KDJ-Pyr-9 derivative structure) in the compound molecule specifically binds to the inactive region of the CMYC protein, and the linker mediates the recruitment of the E3 ubiquitin ligase VHL to form a “CMYC-PROTAC-VHL” ternary complex; the E3 ligase in the complex can catalyze the ubiquitination modification of the CMYC protein, and the ubiquitinated CMYC protein is recognized and degraded by the 26S proteasome, so as to completely block the oncogenic pathways such as cell proliferation, metabolic reprogramming and stemness maintenance regulated by CMYC, and finally realize tumor killing. Based on this mechanism, the compounds can be used as active ingredients in the preparation of various CMYC-dependent tumor treatment drugs, and the specific preparation methods are as follows: The PROTAC compound described in the present application has significant therapeutic potential for tumors caused by CMYC gene amplification, abnormal protein stability, or persistent activation of upstream signals leading to high expression of CMYC, and is preferably applied to the following tumor types: Colorectal cancer: for colorectal cancer cells with abnormally high expression of CMYC (such as the HCT116 cell line), the in vitro proliferation and growth can be effectively inhibited, and the toxicity to normal colon epithelial cells (such as NCM460) is extremely low; Gastric cancer: for diffuse and intestinal type gastric cancer cells (such as HGC27, MGC803, and SMU719 cell lines), the abnormally accumulated CMYC protein can be targeted for degradation; Breast cancer: focusing on triple-negative breast cancer (limited clinical treatment methods and high proportion of CMYC high expression), the triple-negative breast cancer cell lines MDA-MB-231 and SUM-159T have strong killing effects. The PROTAC compound of the present application can efficiently degrade CMYC protein, inhibit cell colony formation, and reduce the colony formation rate to less than 20%.

[0033] The PROTAC compound has the best application effect in the treatment of triple-negative breast cancer (MDA-MB-231 and SUM-159T cell lines) and colorectal cancer (HCT116 cell line).

[0034] Compared with the prior art, the present application has the following beneficial effects: The present application provides a PROTAC compound targeting CMYC and its preparation method and application. The present application provides a PROTAC compound (P-MYC1-4, P-MYC1-8, P-MYC2-6) precisely targeting CMYC. Through the ubiquitin-proteasome system, compared with existing CMYC inhibitors, the CMYC protein in cells can be completely degraded, achieving efficient degradation of CMYC in tumor cells. The above compounds have good efficacy in various tumor models, can effectively kill colorectal cancer cells while having little effect on normal cells; and the preparation method of the PROTAC compound targeting CMYC is designed based on the conformational relationship between the inhibitor and the CMYC protein, and the molecular site is more suitable for the E3 ubiquitin ligase reaction. At the same time, the E3 ubiquitin ligase ligand is protected to ensure the selectivity of the reaction process, the reaction conditions are mild, and the method is simple. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the P-MYC1-4 compound is shown in Figure 1; Figure 2 The nuclear magnetic resonance hydrogen spectrum of the P-MYC1-8 compound is shown in Figure 2; Figure 3 The nuclear magnetic resonance hydrogen spectrum of the P-MYC2-6 compound is shown in Figure 3; Figure 4 The half-killing concentration (IC 50 ) of P-MYC1-8 to different cells. Figure 5 The protein degradation WB diagram of CMYC of the PROTAC compound to different cells. DETAILED DESCRIPTION

[0036] For the purpose, technical scheme and advantages of the present application, the present application will be further described below in conjunction with the drawings and specific examples.

[0037] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified, and the components used in each parallel experiment are the same.

[0038] The present application provides a PROTAC compound targeting CMYC, whose general formula is shown in formula (I) or (II).

[0039] Formula (I) Formula (II); In formula (I), n is 2 and 6, R is -(C)2 and -(C)6, and the structural formula of the PROTAC compound (P-MYC1-4, P-MYC1-8) is shown in formula (III). Formula (III). In formula (II), m is 5, and R is -(C)5. The structural formula of the PROTAC compound (P-MYC2-6) is shown in formula (IV). Formula (IV).

[0040] Example 1, a PROTAC compound targeting CMYC and a preparation method thereof The present embodiment provides a preparation method of a PROTAC compound (P-MYC1-4, P-MYC1-8) targeting CMYC, comprising the following steps: (1) Synthesis of VHL ligand-linker conjugate: The substrate (2R,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl) pyrrolidine-2-carboxamide is dissolved in DMF, and imidazole and TBS-Cl are added, and the reaction mixture is obtained by reacting at 0 ℃ and then warming to room temperature; after stirring for 24 hours, the reaction is quenched with dilute hydrochloric acid to obtain a reaction solution; ethyl acetate (EA) is added to the reaction solution, and the organic phase I is separated, and the aqueous phase is extracted with ethyl acetate; the combined organic phase I is washed with water and then saturated brine, and then dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude product; the obtained crude product is directly used in the next step reaction without further purification. The crude product (TBS-protected compound) is dissolved in DMF, and a DMF solution of Fmoc-protected amino acid linker, 1-hydroxybenzotriazole (HOBt) and N,N'-diisopropylcarbodiimide (DIC) is added, and stirred at room temperature for 1 hour. After the crude product is completely reacted, piperidine (DMF solution, 5% w / v) is added, and stirring is continued at room temperature for 1 hour to obtain a reaction mixture; the reaction mixture is quenched with water, and extracted with dichloromethane (DCM) to obtain an organic phase II; the organic phase II is washed with water and saturated brine, dried over Na2SO4, filtered and concentrated under reduced pressure, and the obtained crude product is directly used in the next step reaction without further purification to obtain the VHL ligand linker conjugate.

[0041] The synthetic route is as follows:

[0042] (2) Synthesis of P-MYC1-4 and P-MYC1-8: The VHL ligand linker conjugate synthesized in the above step is dissolved in toluene, Pd(OAc)2, ligand and NaOtBu are added under argon protection, and then aryl chloride (ArCl) is added, and the reaction is stirred at room temperature. After the reaction is completed, TBAF dissolved in THF is added to the reaction system, and stirring is continued for 24 hours. The reaction mixture is quenched with water, the aqueous phase is extracted with ethyl acetate (EA), and the combined organic phase is washed with saturated brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The obtained crude product is purified by silica gel column chromatography to obtain the target compound.

[0043] The synthetic route is as follows:

[0044] wherein the nuclear magnetic resonance hydrogen spectrum (d 6 -DMSO, 300 MHz): 11.10 (s, 1H), 9.35(s, 1H), 8.65 (s, 1H), 8.20–7.15 (m, 12H), 6.45 (s, 1H), 4.52 (t, 2H), 3.78(s, 3H,), 2.45 (t, 2H), 1.90–1.55 (m, 4H), 0.94 (s, 9H), confirming that the structure is correct, as Figure 1 shown.

[0045] wherein the nuclear magnetic resonance hydrogen spectrum (d 6 -DMSO, 300 MHz): 0.95 (s, 9H), 1.20–1.40 (m, 10H), 1.62 (m, 2H), 2.05 (m, 2H), 2.85 (t, 2H), 3.35–3.65 (m, 4H), 3.85(dd, 1H), 4.20–4.45 (m, 2H), 5.10 (s, 1H), 6.80–7.60 (m, 8–10H), 7.90–8.40 (m, 2–3H), 8.50 (br s, 1H), confirming that the structure is correct, as Figure 2 shown.

[0046] Example 2, a PROTAC compound targeting CMYC and a preparation method thereof The present embodiment provides a preparation method of a PROTAC compound (P-MYC2-6) targeting CMYC, comprising the following steps: (1) Synthesis of VHL ligand linker conjugate: The substrate (2R,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl) pyrrolidine-2-carboxamide is dissolved in DMF, and imidazole and TBS-Cl are added, and the reaction mixture is reacted at 0°C, and then the reaction mixture is warmed to room temperature; after stirring for 24 hours, the reaction is quenched with dilute hydrochloric acid to obtain a reaction solution; ethyl acetate (EA) is added to the reaction solution, and the organic phase I is separated, and the aqueous phase is extracted with ethyl acetate; the combined organic phase I is sequentially washed with water and saturated brine, and then dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude product; the obtained crude product is directly used in the next step reaction without further purification. The crude product (TBS-protected compound) is dissolved in DMF, and a DMF solution of an Fmoc-protected amino acid linker, 1-hydroxybenzotriazole (HOBt), and N,N'-diisopropylcarbodiimide (DIC) is added, and stirred at room temperature for 1 hour. After the crude product (TBS-protected compound) is completely reacted, piperidine (DMF solution, 5% w / v) is added, and stirring is continued at room temperature for 1 hour to obtain a reaction mixture; the reaction mixture is quenched with water, and extracted with dichloromethane (DCM) to obtain an organic phase II; the organic phase II is washed with water and saturated brine, dried over Na2SO4, filtered, and concentrated under reduced pressure, and the obtained crude product is directly used in the next step reaction without further purification to obtain a VHL ligand linker conjugate.

[0047] The synthetic route is as follows:

[0048] (2) Synthesis of P-MYC2-6: The VHL ligand linker conjugate synthesized in the above step is dissolved in toluene, and a DMF solution of KDJ-Pyr-9, HOBt, and DIC is added under argon protection, and stirred at room temperature for 1 hour. After the reaction is completed, piperidine (DMF solution, 5% w / v) is added, and stirring is continued at room temperature for 1 hour. The reaction mixture is quenched with water, and extracted with dichloromethane (DCM). After the extracted product is concentrated, THF-dissolved TBAF is added to a round-bottom flask, and stirring is continued for 24 hours. The reaction mixture is quenched with water, and the aqueous phase is extracted with ethyl acetate (EA), and the combined organic phase is washed with saturated brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude product is purified by silica gel column chromatography to obtain the target compound.

[0049] The synthesis route is as follows:

[0050] Among them, the 1H NMR spectrum (d) of the P-MYC2-6 compound 6 -DMSO, 300MHz): 0.94 (s, 9H), 1.33(m, 3H), 1.53 (m, 1H), 2.05 (t, 7H), 2.24 (m, 22H), 2.48(m, 2H), 3.30 (t, 1H), 3.73(m, 1H), 4.27 (m, 1H), 4.40 (t, 2H), 5.37 (s, 1H), 6.90 (m, 1H), 7.47(d,2H), 7.59(dd, 1H), 7.73-7.82 (m, 4H), 7.95(d, 1H),8.05(d,1H),8.33-8.43(m, 2H),8.87(s, 1H), 9.08 (s, 1H), confirming its correct structure, such as Figure 3 As shown.

[0051] Example 3, comparison of drug tumor killing activity of P-MYC1-4, P-MYC1-8, P-MYC2-6 1. Detection of cell proliferation inhibition activity (CCK8 assay): (1) Preparation before cell seeding: Take each tumor cell line in logarithmic growth phase (HCT116, MGC803, HGC27, MDA-MB-231, SUM-159T), discard the old culture medium, and gently wash twice with PBS buffer; add 0.25% trypsin-EDTA digestion solution, incubate at 37℃ for 2-3 min until the cells detach from the cell wall, add the corresponding culture medium containing serum to stop digestion, and gently pipette to form a single cell suspension.

[0052] (2) Cell counting and seeding: Single-cell suspensions were counted using a cell counting chamber. The seeding concentration was adjusted according to the cell growth rate: 8 × 10³ cells per well for HCT116 and 1 × 10³ cells per well for HGC27 / MGC803. 4 10 cells, A-MB-231 / SUM-159T per well, 1×10⁶ cells / well 4 100 μL of single-cell suspension was added to each well of a 96-well plate, and 100 μL of the corresponding culture medium was added to the edge wells (columns 1 and 12) as a blank control. The 96-well plate was placed in a 37°C, 5% CO2 incubator and incubated for 24 h to allow the cells to adhere and resume logarithmic growth.

[0053] (3) Drug gradient treatment: After the cells adhere, discard the old culture medium in each experimental well of the 96-well plate, and add 100 μL of fresh culture medium containing different concentrations of drugs to each experimental well. The final concentration gradient of the drug is set as: 0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, and the final volume of each well is ensured to be 200 μL, and the final DMSO concentration is ≤0.1%. Each drug concentration is set with 3 replicate wells, and the solvent control group (0 μM drug) is added with corresponding culture medium containing 0.1% DMSO. After the treatment is completed, the 96-well plate is placed back into the incubator and incubated for 48 h.

[0054] (4) CCK8 coloration and absorbance detection: After the drug incubation is completed, the 96-well plate is taken out, and 100 μL of supernatant is gently aspirated from each well. In a dark environment, 20 μL of CCK8 reagent is added to each well. The 96-well plate is gently shaken, and then placed back into the 37°C, 5% CO2 incubator for dark incubation for 1 h. After the incubation is completed, the absorbance of each well at 450 nm is detected by an enzyme marker.

[0055] (5) Cell survival rate calculation: The cell survival rate under each drug concentration is calculated according to the following formula: cell survival rate (%) = [(net OD value of drug group - net OD value of blank group) / (net OD value of solvent control group - net OD value of blank group)] x 100%, wherein the net OD value of the blank group is that of the edge well containing only the culture medium, and the net OD value of the solvent control group is that of the 0 μM drug treatment well.

[0056] The results are shown in Table 1 and Figure 4 .

[0057] Table 1 2, CMYC protein degradation efficiency detection (Western blot method): (1) Drug treatment and protein extraction: Take a 6-well plate, inoculate 2 x 10 5 cells of tumor cells (HCT116) in the logarithmic growth phase in each well, and incubate for 24 h until the cells adhere. Add P-MYC2-6 with a final concentration of 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, and 2 μM to each well, and add 0.1% DMSO to the solvent control group (ctrl). After incubation for 24 h, discard the culture medium, wash twice with PBS, add 200 μL of RIPA lysis buffer containing protease inhibitors to each well, and lyse on ice for 30 min. Scrape the cell lysate with a cell scraper, transfer it to a 1.5 mL EP tube, centrifuge at 12000 rpm for 15 min at 4°C, and take the supernatant.

[0058] (2) Protein quantification and electrophoresis: The protein concentration of the protein extract was detected by BCA protein quantification kit, and the protein loading amount was adjusted according to the quantification result (10-15 μg protein per well); 5x SDS loading buffer was added, and the protein was denatured by boiling at 100℃ for 5 min; 10% SDS-PAGE gel was prepared, and protein electrophoresis was carried out (concentrated gel 80V, separation gel 120V, until the bromophenol blue indicator reached the bottom of the gel).

[0059] (3) Transferring membrane and immunohybridization: After electrophoresis, the protein was transferred to PVDF membrane (300mA, 60min); after transferring membrane, the PVDF membrane was blocked with 5% skim milk (prepared with TBST) for 1h (room temperature, shaking bed oscillation); the blocking solution was discarded, and the primary antibody diluent (CMYC antibody 1:1000, GAPDH antibody 1:1000, prepared with TBST) was added, and the shaking bed was incubated at 4℃ overnight; the next day, the membrane was washed with TBST for 3 times (10min each time), and the HRP labeled secondary antibody diluent (anti-rabbit secondary antibody 1:1000, prepared with TBST) was added, and the room temperature was incubated for 1h; the membrane was washed with TBST for 3 times again (10min each time), and ECL chemiluminescence solution was added, and the image was collected by chemiluminescence imaging system. The results are shown in Figure 5

[0060] The PROTAC compound (P-MYC1-4, P-MYC1-8, P-MYC2-6) provided by the application can completely degrade the CMYC protein in the cell through the ubiquitin-proteasome system, compared with the existing CMYC inhibitor, can realize the efficient degradation of CMYC in tumor cells, has good curative effect in various tumor models, and the above compounds can effectively kill colorectal cancer cells while having less effect on normal cells.

[0061] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the application and not to limit the protection scope of the application. Although the application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the application can be modified or replaced equivalently without departing from the essence and scope of the technical solutions of the application.​

Claims

1. A PROTAC compound targeting CMYC, characterized in that, The PROTAC compound includes a target protein ligand, a linker, and an E3 ubiquitin ligase ligand, wherein the target protein ligand is linked by the linker and the E3 ubiquitin ligase ligand. The target protein ligands include MYCi361 or KDJ-Pyr-9; When the target protein ligand is MYCi361, the structural formula of the PROTAC compound is shown in formula (I); Formula (I); The target protein ligand is KDJ-Pyr-9, and the structural formula of the PROTAC compound is shown in formula (II); Formula (II); In equation (I), n is a natural number from 2 to 8; In equation (II), m is a natural number from 2 to 8.

2. The PROTAC compound targeting CMYC as described in claim 1, characterized in that, In formula (I), the carbon chain of -(C)n- is linked to the acetamide group in the VHL of the E3 ligase ligand; And / or, in formula (II), the carbon chain of -(C)m- is linked to the acetamide group in the VHL of the E3 ligase ligand.

3. The PROTAC compound targeting CMYC as described in claim 2, characterized in that, The hydroxyl group on the VHL of the E3 ligase ligand is modified with TBSCl.

4. The PROTAC compound targeting CMYC as described in claim 1, characterized in that, The PROTAC compound is P-MYC1-4 or P-MYC1-8; The structural formulas of P-MYC1-4 and P-MYC1-8 are shown in formula (III); Equation (III).

5. The PROTAC compound targeting CMYC as described in claim 1, characterized in that, The PROTAC compound is P-MYC2-6; The structural formula of P-MYC2-6 is shown in formula (IV); Formula (IV).

6. A ternary composite, characterized in that, The complex comprises a target protein, a PROTAC compound as described in any one of claims 1 to 5, and an E3 ligase, wherein the E3 ligase ubiquitinates and modifies the target protein.

7. The method for preparing the PROTAC compound targeting CMYC according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) The substrate (2R,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[4-(4-methylthiazolyl-5-yl)benzyl]pyrrolidine-2-carboxamide was dissolved in DMF, and imidazole and TBS-Cl were added to react and obtain a reaction solution. Ethyl acetate was added to the reaction solution to separate organic phase I, and the aqueous phase was extracted with ethyl acetate. Organic phase I was washed, dried, filtered and concentrated under reduced pressure to obtain crude product. The crude product was dissolved in DMF, and a DMF solution of Fmoc-protected amino acid linker, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide was added and stirred. DMF solution was added again to obtain a reaction mixture. The reaction mixture was quenched with water and extracted with dichloromethane to obtain organic phase II. Organic phase II was washed, dried, filtered and concentrated under reduced pressure to obtain VHL ligand linker conjugate. (2) The VHL ligand linker conjugate obtained in step (1) was dissolved in toluene, Pd(OAc)2, MYCi361 and NaOtBu were added, and then Arl chloride ArCl was added and stirred to obtain a reaction solution; TBAF dissolved in THF was added to the reaction solution and stirred to obtain a reaction mixture; the reaction mixture was quenched with water, the aqueous phase was extracted with ethyl acetate to obtain organic phase III, organic phase I was washed, dried, filtered and concentrated under reduced pressure, purified to obtain PROTAC compound; or, (2) The VHL ligand linker conjugate obtained in step (1) was dissolved in toluene, and a DMF solution of KDJ-Pyr-9, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide was added. Then, the DMF solution was added and stirred to obtain a reaction mixture. The reaction mixture was quenched with water and extracted with dichloromethane to obtain an extract. The extract was concentrated, and TBAF dissolved in THF was added and stirred. Then, the mixture was quenched with water. The aqueous phase was extracted with ethyl acetate to obtain organic phase IV. Organic phase IV was washed, dried, filtered and concentrated under reduced pressure to obtain the PROTAC compound.

8. The use of the PROTAC compound targeting CMYC as described in any one of claims 1 to 5 in the preparation of a tumor therapeutic agent.

9. The application as described in claim 8, characterized in that, The tumor includes at least one of colorectal cancer, gastric cancer, and breast cancer.

10. A tumor drug, characterized in that, The tumor drug includes a PROTAC compound targeting CMYC as described in any one of claims 1 to 5, or a ternary complex as described in claim 6.

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