Ezh2 and brd4 dual-targeted inhibitors and uses thereof

Abstract

The application discloses an EZH2 and BRD4 double-target inhibitor and application thereof, wherein the double-target inhibitor is obtained by coupling a structural fragment for inhibiting a BRD4 protein and a structural fragment for inhibiting an EZH2 protein through a linker. The inventors find that the structural fragment for inhibiting the EZH2 protein and the structural fragment for inhibiting the BRD4 protein are restructured, coupled together through the linker, and the length of the linker is adjusted, so that the obtained EZH2 and BRD4 double-target inhibitor has an unexpected anti-tumor effect, the anti-tumor effect is better than that of an individual EZH2 inhibitor or BRD4 inhibitor, and is also better than that of a combination of the two under the same conditions.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to an EZH2 and BRD4 dual-target inhibitor and application thereof. BACKGROUND

[0002] In recent decades, epigenetic regulation has been intensively studied. Abnormal changes in epigenetics observed in cancer cells, particularly abnormal histone modifications, have been extensively studied for their roles in cancer. A series of small molecule compounds that specifically inhibit histone modification enzymes have also been developed, and have achieved certain therapeutic effects. Epigenetic drugs have been shown to improve the efficiency of various major cancer treatment methods, such as chemotherapy, radiotherapy, targeted therapy and immunotherapy. In addition, due to the interaction between multiple epigenetic processes, combining multiple epigenetic drugs has also become an effective method for targeting tumors.

[0003] The protein expressed by the EZH2 gene is called histone lysine N-methyltransferase EZH2 (Enhancer of zeste homolog 2, zeste homolog enhancer 2), which is a member of the polycomb family. EZH2 can promote the mono-, di- and tri-methylation of lysine 27 (H3K27) of histone H3, and is the enzymatic subunit protein of polycomb repressive complex 2 (PRC2). EZH2 is associated with a variety of biological functions, including transcriptional regulation in hematopoiesis, development and cell differentiation. EZH2 mainly silences genes by tri-methylating H3K27, a histone modification that plays a unique role in epigenetic regulation of gene transcription. Tri-methylated H3K27 is an important modification that keeps chromosomes in a tight state, and can promote chromatin condensation and heterochromatin formation. The polygene complex 2 (PRC2) containing EZH2 can also be involved in the recruitment of DNA methyltransferase, which leads to increased DNA methylation. Specific genes identified as targets of EZH2-mediated transcriptional repression include HOXA9, HOXC8, MYT1, CDKN2A and retinoic acid target genes, etc.

[0004] EZH2 contributes to cancer cell division and proliferation, and is overexpressed in many cancers such as leukemia, bladder cancer, kidney cancer, uterine cancer and prostate cancer, as well as melanoma and lymphoma, and is recognized as an anticancer target protein. The development of EZH2 inhibitors as cancer treatment drugs has been widely studied and focused on. So far, some EZH2 inhibitors, including GSK126, EPZ6438, PF-06821497 and CPI-1205, have entered clinical trials. Among them, EPZ6438 as an EZH2 specific small molecule inhibitor has excellent efficacy and drug-like properties, including good oral bioavailability. EPZ6438 (tazemetostat) has been approved for the treatment of locally advanced or metastatic epithelioid sarcoma and follicular lymphoma, and has also entered clinical trials for the treatment of diffuse large B-cell lymphoma. However, there are limitations in the tumor species of EZH2 inhibitors, which mainly show good efficacy in some hematological tumors, especially diffuse large B-cell lymphoma; while in solid tumors, only solid tumors with mutations in SWI / SNF members and BAP1 mutations have certain inhibitory effects. EZH2 inhibitors have weak killing effects on most solid tumors with overexpression of wild-type EZH2 and increased H3K27 trimethylation. Based on the molecular mechanism of tumor cells not being sensitive or resistant to EZH2 inhibitors, it has good scientific value to continue to develop anti-tumor drug molecules targeting the EZH2 target protein to expand the clinical application of EZH2 inhibitors in solid tumors.

[0005] In most solid tumors, while EZH2 inhibition reduces H3K27me3 levels, it also increases H3K27 acetylation (H3K27ac), leading to resistance to EZH2 inhibitors. The increased H3K27ac and oncogenic transcriptional reprogramming caused by EZH2 inhibition may limit the efficacy of EZH2 inhibitors in solid tumors. BRD4, a member of the Bromo and extra-C terminal domain (BET) protein family, can read acetylated lysine residues from histones, promoting gene transcription. (+)-JQ1 is the first reported selective BRD4 inhibitor that mimics the interaction between BET protein and acetyl-lysine residues on histones, exhibiting effective anticancer activity. Several BRD4 inhibitors, including I-BET762, OTX-015, and ABBV-075, have been reported, some of which are in clinical trials. BRD4 inhibitors can reduce EZH2 inhibitor resistance caused by H3K27ac upregulation, thus improving the efficacy of EZH2 inhibitors. The results indicate that the combined use of BRD4 inhibitors and EZH2 inhibitors can induce the sensitivity of solid tumor cells to EZH2 inhibition, exhibiting a good synergistic anti-cancer effect. However, EZH2 and BRD4 are independent proteins, requiring the simultaneous use of both BRD4 and EZH2 inhibitors. Summary of the Invention

[0006] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a dual-target inhibitor of EZH2 and BRD4 and its application.

[0007] The technical solution adopted in this invention is:

[0008] The first aspect of the present invention provides:

[0009] A dual-target inhibitor of EZH2 and BRD4, wherein the dual-target inhibitor is obtained by linking a structural fragment that inhibits BRD4 protein and a structural fragment that inhibits EZH2 protein through a linker, wherein the main chain of the linker contains 1 to 14 atoms.

[0010] In some examples of dual-target inhibitors, the linker's main chain contains 2 to 7 atoms, preferably 5 to 7 atoms. Further, the linker's main chain contains 6 or 7 atoms. Experimental data show that the length of the main chain has a significant impact on the activity of the dual-target inhibitor.

[0011] In some examples of dual-target inhibitors, the structural fragment that inhibits the EZH2 protein may be selected from known EZH2 protein inhibitors such as EPZ6438, GSK126, PF-06821497, or CPI-1205, or structural fragments with EZH2 protein inhibitory activity.

[0012] In some examples of dual-target inhibitors, the structural fragment that inhibits the BRD4 protein may be selected from known BRD4 protein inhibitors such as (+)-JQ1, I-BET762, OTX-015, CPI-0610 or TEN-010, or structural fragments with BRD4 protein inhibitory activity.

[0013] In some examples of dual-target inhibitors, the linker's backbone is an alkyl chain, a polyethylene glycol chain, or an ether-containing alkyl chain. These backbones exhibit good stability while also possessing good flexibility.

[0014] In some examples of dual-target inhibitors, at least one end of the main chain is coupled via -NH- to a structural fragment that inhibits the BRD4 protein or the EZH2 protein.

[0015] In some examples of dual-target inhibitors, the general structural formula of the dual-target inhibitor is shown in Formula I:

[0016] In some examples of dual-target inhibitors, the linker is selected from one of the formulas X1 to X15, where n or m is an integer:

[0017]

[0018]

[0019] In some examples of dual-target inhibitors, the structural formula is one of formulas D1 to D12:

[0020] In equations D1 to D12,

[0021] A second aspect of the present invention provides:

[0022] A pharmaceutical composition comprising an active ingredient and excipients, wherein the active ingredient comprises the dual-target inhibitor of EZH2 and BRD4 as described in the first aspect of the present invention, or one of its pharmaceutical salts, crystals, or solvates.

[0023] In some instances, the pharmaceutical composition is used to treat tumors.

[0024] In some examples of pharmaceutical compositions, the tumor is a solid tumor or a hematoma.

[0025] In some examples of pharmaceutical compositions, the solid tumor is selected from lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, kidney cancer, uterine cancer, prostate cancer, or melanoma; the hematologic malignancy is selected from leukemia or lymphoma.

[0026] A third aspect of the present invention provides:

[0027] The application of the EZH2 and BRD4 dual-target inhibitor described in the first aspect of this invention in the preparation of a drug for treating tumors.

[0028] In some applications, the tumor is a solid tumor or a hematoma.

[0029] In some application examples, the solid tumor is selected from lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, kidney cancer, uterine cancer, prostate cancer, or melanoma; the hematologic malignancy is selected from leukemia or lymphoma.

[0030] The beneficial effects of this invention are:

[0031] EZH2 and BRD4 are independent proteins, physically isolated within the cell. Theoretically, coupling a BRD4 inhibitor and an EZH2 inhibitor together to form a dual-target inhibitor would only react with and inhibit one of EZH2 or BRD4. Compared to traditional single-target inhibitors, this dual-target inhibitor theoretically does not offer an advantage in inhibiting the activity of a single target protein. However, the inventors discovered that by structurally modifying the structural fragments that inhibit EZH2 and BRD4 proteins and coupling them together via a linker, and adjusting the linker length, the resulting dual-target inhibitor of EZH2 and BRD4 exhibited unexpected anti-tumor effects. Its anti-tumor efficacy was superior to that of either EZH2 or BRD4 inhibitors alone, and also superior to the combination of both under the same conditions. Attached Figure Description

[0032] Figure 1 The effects of the compounds of this invention on the levels of H3K27me3 downstream of EZH2 and c-Myc downstream of BRD4;

[0033] Figure 2 The effects of different concentrations of compound D7 of this invention on the levels of H3K27me3 downstream of EZH2 and c-Myc downstream of BRD4;

[0034] Figure 3 The effects of compound D7 of this invention and control compounds EPZ6438 and JQ1 on the viability of multiple solid tumor cell lines are investigated.

[0035] Figure 4 The antitumor effects of compound D7 of the present invention and control compounds EPZ6438 and JQ1 in the A549 nude mouse subcutaneous xenograft model are presented.

[0036] Figure 5 It refers to the anti-tumor effects of different compounds on different tumor cell lines. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings. It should be noted that the following embodiments are based on the present technical solution and provide detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to these embodiments.

[0038] In the following examples, the intermediates have the same number, meaning that intermediates with the same number have the same structure.

[0039] Example 1: Synthesis of compound D7

[0040] Synthesis of intermediate 6:

[0041]

[0042] Reagents and reaction conditions: (a) Fe, NH4Cl, MeOH, 90℃; (b) tetrahydro-4H-pyran-4-one, AcOH, Na(AcO)3BH,1,2-dichloroethane; (c) acetaldehyde, A cOH, Na(AcO)3BH, 1,2-dichloroethane; (d) Boc2O, H2, Raney-Ni, MeOH; (e) HCl, MeOH; (f) NaOH, EtOH, 60°C; (g) HOBt, EDCI, NMM, DMSO.

[0043] As shown in the reaction process above, the synthesis was carried out using commercially available compound 1 (900 mg, 3.28 mmol) as the starting material. Iron powder and ammonium chloride were added, and the mixture was refluxed overnight at 90 °C in methanol solution. After the reaction was confirmed to be complete by TLC, the reaction system was filtered, and the filtrate was collected and distilled under reduced pressure. The concentrated product was purified by silica gel column chromatography using an ethyl acetate / petroleum ether system to obtain a pale yellow liquid intermediate 2 (723 mg, 2.96 mmol), with a yield of approximately 90%.

[0044] Intermediate 2 (700 mg, 2.87 mmol), tetrahydropyranone, and acetic acid were added to a round-bottom flask containing dry anhydrous 1,2-dichloroethane (DCE) and stirred at room temperature for 30 minutes. Then, sodium triacetoxyborohydride was added in an ice bath, and the reaction was allowed to proceed for 24 hours at room temperature after removing the ice bath. After TLC analysis to confirm complete reaction, the solvent was removed by vacuum distillation. The concentrated crude product was extracted using an ethyl acetate / water system, the organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The purified white solid was dissolved in an appropriate amount of anhydrous DCE, acetic acid was added, and acetaldehyde was injected into the reaction system. The mixture was stirred at room temperature for 30 minutes, and then sodium triacetoxyborohydride was added in an ice bath, and the reaction was allowed to proceed for 24 hours at room temperature. The post-treatment was the same as for the white solid, yielding a pale yellow liquid intermediate 3 (890 mg, 2.5 mmol), with a yield of approximately 87%.

[0045] Using commercially purchased compound 4 as a starting material (500 mg, 3.37 mmol), a methanol solution was added with appropriate amounts of Raney nickel and Boc anhydride. The mixture was purged three times with hydrogen and reacted overnight under hydrogen protection. After TLC detection of complete reaction, the reaction system was filtered, the filtrate was collected and subjected to vacuum distillation, and the concentrated product was purified to obtain a white solid. This solid was added to the reaction system of acetyl chloride and methanol solution under ice bath conditions and reacted at room temperature for 3 hours. After TLC detection of complete reaction, extraction was performed. First, extraction was performed with dichloromethane / water, the aqueous phase was collected, the pH of the aqueous phase was adjusted to 3-4 with 1M hydrochloric acid solution, and extraction was performed with ethyl acetate. The organic phase was collected and subjected to vacuum distillation to obtain the hydrochloride salt of product intermediate 5.

[0046] Intermediates 3 (787 mg, 210 mmol), 5 (403.5 mg, 265 mmol), HOBT, and EDCI were added to a DMSO solution, and N-methylmorpholine was injected into the reaction system. The mixture was stirred overnight at room temperature. After the reaction was confirmed to be complete by TLC, the mixture was extracted with an ethyl acetate / water system, the organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The concentrated product was purified by silica gel column chromatography using a methanol / dichloromethane system to obtain a pale yellow solid intermediate 6 (1 g, 210 mmol), with a yield of approximately 95%. 1H NMR (400MHz, CDCl3) δ11.12(s,1H),7.22(d,J=1.8Hz,1H),7.18(d,J=1.8Hz,1H),7.12(t,J=5.6Hz,1H),5.95(s,1H),4.52(d,J=5.9Hz,2H),3.95 (d,J=11.4Hz,2H),3.35-3.27(m,2H),3.02(q,J=6.9Hz,2H),2.93(m,1H) ,2.39(s,3H),2.24(d,J=2.3Hz,6H),1.66(m,4H),0.85(t,J=7.0Hz,3H).

[0047] Synthesis of intermediate 13:

[0048]

[0049] Reagents and reaction conditions: (a) 2-butanone, 7-morpholine, EtOH, 70℃; (b) Fmoc-Asp(Ot-Bu)-OH, EDCI, HOBT, DMF, 40℃; (c) piperidine, CH2Cl2; (d) AcOH, EtOH, 80℃; (e) KOt-Bu, THF, PO(OMe)2Cl, acethydrazide, n-BuOH; (f) 20% TFA, CH2Cl2.

[0050] As shown in the reaction procedure above, sulfur (2.79 g, 86.86 mmol) was added to an ethanolic solution of compound 7 (4-chlorobenzoylacetonitrile) (1.24 g, 6.9 mmol), 2-butanone, and morpholine. The mixture was heated to 70 °C. After 12 hours, the reaction mixture was cooled to room temperature and poured into brine. The aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product of the residue was purified by silica gel column chromatography using an ethyl acetate / petroleum ether system to give intermediate 8 (17.5 g, 98.6%) as a yellow solid. Fmoc-Asp(Ot-Bu)-OH (3.72 g, 9.03 mmol), EDCI, and HOBt were added to an N,N-dimethylformamide (DMF) solution. The mixture was stirred at room temperature for 5 minutes, and then intermediate 8 (2.00 g, 7.53 mmol) was added. The resulting mixture was stirred at 40 °C for 24 hours, then extracted with an ethyl acetate / water system. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography using an ethyl acetate / petroleum ether system to give intermediate 9 (2.01 g, 54.0%), which was a brown oil. Intermediate 9 (1.90 g, 3.03 mmol) and piperidine (0.56 mL, 6.06 mmol) were added to a dichloromethane solution. The mixture was stirred at room temperature for 30 minutes, then extracted with an ethyl acetate / water system. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using an ethyl acetate / petroleum ether system to give a yellow solid free amine intermediate 10 (1.11 g, 86.3%).

[0051] Intermediate 10 (1.02 g, 2.33 mmol) was dissolved in 10 mL of ethanol, followed by the addition of 1.5 mL of acetic acid. After 2 hours, all solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography using an ethyl acetate / petroleum ether system to give compound 11 (0.907 g, 93.4%) as a white solid. At -78 °C, a solution of potassium tert-butoxide in tetrahydrofuran (1.0 M, 0.859 mL, 0.859 mmol) was added to a solution of intermediate 11 (300 mg, 0.716 mmol) in tetrahydrofuran (2 mL). The reaction mixture was heated to -10 °C and stirred at room temperature for 30 minutes. Then, the reaction mixture was cooled to -78 °C, and PO(OMe)₂Cl (0.154 mL, 0.32 mmol) was added. The resulting mixture was heated to -10 °C over 1 hour. After the addition of acetic acid hydrazide (117 mg, 1.575 mmol), the reaction mixture was stirred at room temperature for 1 hour. Add 6 ml of 1-butanol and heat the reaction mixture to 90 °C. After 1 hour, remove all solvent under reduced pressure. The residue was purified by silica gel column chromatography using an ethyl acetate / petroleum ether system to give JQ1 (180 mg, 88.1%) as a white solid. 1 HNMR(400MHz, CDCl3), δ7.40(d,J=8.4Hz,2H),7.32(d,J=8.3Hz,2H),4.55(t,J=7 .0Hz,1H),3.62-3.46(m,2H),2.66(s,3H),2.40(s,3H),1.68(s,3H),1.49(s,9H).

[0052] Finally, trifluoroacetic acid (0.4 mL) was added to a solution of JQ1 (180 mg, 408 μmol) in ethyl acetate (2 mL). The reaction mixture was stirred at room temperature for 2 hours. Then, all solvent was removed under reduced pressure to obtain 13 without further purification.

[0053] Synthesis of compound D7

[0054]

[0055] Reagents and reaction conditions: (a) TsCl, TEA, DMAP, CH2Cl2; (b) 4-hydroxyphenylboronic acid pinacol ester, K2CO3, DMF, 70℃; (c) Intermediate 6, Pd(PPh3)4, K2CO3, DMF, 90℃; (d) 20% TFA, CH2Cl2; (e) Intermediate 13, HATU, DIPEA, DMF.

[0056] To a dichloromethane solution of compound 14d (950 mg, 5.96 mmol), 4-toluenesulfonyl chloride, TEA, and DMAP were added. The reaction mixture was stirred at room temperature for 3 hours, then diluted with 100 mL of dichloromethane. The organic phase was washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using an ethyl acetate / petroleum ether system to give a colorless oil (1.04 g, 46.0%). To a DMF (5 mL) solution of the above colorless oil (1.04 g, 2.80 mmol), pinacol 4-hydroxyphenylboronic acid and potassium carbonate were added. The reaction mixture was stirred at 70 °C for 5 hours, then cooled to room temperature, extracted with an ethyl acetate / water system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using an ethyl acetate / petroleum ether system to give a yellow oily intermediate 15d (892 mg, 78.6%). Intermediate 15d (102 mg, 251 μmol), potassium carbonate, and Pd(PPh3)4 were added to a DMF (2 mL) solution of intermediate 6 (100 mg, 210 μmol). The mixture was purged and refilled three times with argon. The mixture was stirred at 90 °C for 9 hours, then extracted with an ethyl acetate / water system. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using an ethanol / dichloromethane system to give 16d (120 mg, 84.7%) as a yellow solid. Then, intermediate 16d (70 mg, 82.9 μmol) was dissolved in dichloromethane, and TFA (0.2 μL) was added. The resulting mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure to provide the free amine, which required no further purification. Intermediate 13 (43.0 mg, 107 μmol), DIPEA, and HATU were added sequentially to the DMF solution of the amine. The resulting mixture was stirred at room temperature for 10 hours, then extracted with an ethyl acetate / water system. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using an ethanol / dichloromethane system to give compound D7 (78 mg, 75.9%) as a yellow powder. 1H NMR (400MHz, d6-DMSO) δ11.46(s,1H),8.21(t,J=5.6Hz,1H),8.17(t,J=4.8Hz,1H),7.53(d,J=8.7Hz,2H),7.47(d,J=8.8Hz,2H),7.42(d, J=8.6Hz,2H),7.35(s,1H),7.18(s,1H),6.98(d,J=8.7Hz,2H),5.86(s,1H),4.52(dd,J=8.1,6.0Hz,1H),4.30(d,J=4.8Hz,2H),3.98(t,J= 6.4Hz,2H),3.83(d,J=10.6Hz,2H),3.30-3.25(m,6H),3.17(m,J=6.2Hz,2H),3.08(m,J=7.0Hz,2H),3.02(m,1H),2.59(s,3H),2.40(s,3H) ,2.24(s,3H),2.21(s,3H),2.11(s,3H),1.78–1.72(m,2H),1.67(d,J=11.7Hz,2H),1.61(s,3H),1.53–1.47(m,4H),0.83(t,J=6.9Hz,3H). 13 C NMR(101MHz,d6-DMSO)δ169.4,169.1,163.0,162.9,158.2,155.1,149.8,149.5,148.8,1 42.7,139.5,136.9,136.7,135.2,132.2,132.1,131.9,130.7,130.1,129.8,129.6,128. 4,127.6,122.5,121.6,120.4,114.8,107.3,67.5,66.3,57.8,53.9,41.2,38.3,37.7,34 .9,30.3,29.0,28.4,22.9,18.9,18.2,14.5,14.0,12.7,12.7,11.3.HRMS(ESI,m / z)calcd forC 53 H 61 ClN8O5S[M+H] + :957.4247,found:957.4203.Purity:97.2%.

[0057] Example 2: Synthesis of compound D2

[0058]

[0059] Reagents and reaction conditions: (a) Boc2O, NaOH, THF, H2O; (b) Intermediate 6, Pd(PPh3)4, K2CO3, DMF, 90℃; (c) 20% TFA, CH2Cl2; (d) Intermediate 13, HATU, DIPEA, DMF.

[0060] An aqueous solution of Boc₂O and sodium hydroxide was added to a THF (4 mL) solution of compound 17b (0.2 g, 857 μmol). The mixture was stirred overnight at room temperature. Extraction was then performed using an ethyl acetate / water system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using an ethyl acetate / petroleum ether system to give a Boc-protected intermediate (115 mg, 40.2%) as a yellow solid. Freshly prepared intermediate (83.9 mg, 251 μmol), K₃PO₄, and Pd(PPh₃)₄ were added to a DMF solution containing intermediate 6 (100 mg, 209 μmol). The mixture was purged and refilled three times with argon. The mixture was stirred at 90 °C for 9 hours, then extracted using an ethyl acetate / water system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a methanol / dichloromethane system to give intermediate 18b (109 mg, 86.2%) as a yellow solid. Intermediate 18b (50 mg, 82.9 μmol) was then dissolved in dichloromethane (1 mL), and TFA (0.2 μL) was added. The mixture was stirred at room temperature for 3 hours. Concentration of the solvent under reduced pressure provided the free amine, which could be used for the next step without further purification. Intermediate 13 (35.0 mg, 97.3 μmol), DIPEA, and HATU were added sequentially to a DMF (2 mL) solution containing the free amine (43.9 mg, 87.3 μmol). The resulting mixture was stirred at room temperature for 10 hours, then extracted with ethyl acetate / water. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a methanol / dichloromethane system to give compound D2 (51 mg, 66.0%) as a yellow powder. 1H NMR (400MHz, d6-DMSO) δ11.48(s,1H),8.79(t,J=5.9Hz,1H),8.19(t,J=4.9Hz,1H),7.57(d,J=8.2Hz,2H),7.44(d,J=8.8Hz,2H),7.42–7 .37(m,4H),7.34(d,J=1.2Hz,1H),7.23(d,J=1.4Hz,1H),5.87(s,1H),4.56(dd,J=8.7,5.5Hz,1H),4.42(d,J=6.0Hz,1H),4.35(d,J=5.7 Hz,1H),4.31(d,J=4.8Hz,2H),3.83(d,J=10.6Hz,2H),3.27(d,J=5.1Hz,2H),3.28–3.22(m,2H),3.10–3.06(m,2H),3.01(s,1H),2.61(s ,3H),2.41(s,3H),2.25(s,3H),2.22(s,3H),2.11(s,3H),1.65(d,J=11.9Hz,2H),1.61(s,3H),1.55–1.51(m,2H),0.83(t,J=6.9Hz,3H). 13 CNMR(101MHz,d6-DMSO)δ169.7,169.1,163.1,163.0,155.1,149.9,149.5,148.9,1 42.8,139.7,138.8,138.4,137.0,136.7,135.3,132.6,132.3,130.8,130.1,129.8, 129.6,128.4,127.8,126.4,122.8,121.6,120.8,107.4,69.8,66.3,57.9,54.1,41 .8,41.2,37.8,34.9,30.3,19.0,18.2,14.6,14.0,12.7,11.3.HRMS(ESI,m / z)calcd for C 49 H 53 ClN8O4S[MH] - :883.3526,found:883.3556.Purity:97.7%.

[0061] Example 3: Synthesis of Compound D1

[0062]

[0063] Reagents and reaction conditions: (a) Boc2O, NaOH, THF, H2O; (b) Intermediate 6, Pd(PPh3)4, K2CO3, DMF, 90℃; (c) 20% TFA, CH2Cl2; (d) 13, HATU, DIPEA, DMF.

[0064] As shown in the reaction process above, the synthesis of compound D1 is described in the synthesis process of compound D2. Compound D1: 1 H NMR (400MHz, d6-DMSO) δ11.46(s,1H),10.43(s,1H),8.18(t,J=4.9Hz,1H),7.73(d,J=8.6Hz,2H),7.60(d,J=8.7Hz,2H),7.4 9(d,J=8.7Hz,2H),7.43(d,J=8.6Hz,2H),7.40(s,1H),7.23(s,1H),5.86(s,1H),4.62(t,J=7.1Hz,1H),4.30(d,J=4.8Hz,2H ),3.83(d,J=10.5Hz,2H),3.54(d,J=7.0Hz,2H),3.26(t,2H),3.09(dd,J=13.9,7.0Hz,2H),3.01(d,J=10.8Hz,1H),2.61(s, 3H),2.43(s,3H),2.24(s,3H),2.22(s,3H),2.11(s,3H),1.69(s,2H),1.64(s,3H),1.55–1.51(m,2H),0.84(t,J=6.9Hz,3H). 13 C NMR(101MHz,d6-DMSO)δ169.1,168.7,163.3,163.1,155.1,149.9,149.5,148.9 ,142.7,139.6,138.6,136.8,136.7,135.3,134.5,132.3,130.8,130.1,129.9,1 29.6,128.5,126.8,122.6,121.6,120.5,119.5,107.4,66.3,57.9,53.8,41.3, 38.7,34.9,30.3,19.0,18.2,14.5,14.1,12.8,12.7,11.3.HRMS(ESI,m / z)calcd for C 48 H 51 ClN8O4S[M+H] + :871.3515,found:871.3481.Purity:96.5%.

[0065] Example 4: Synthesis of compound D3

[0066]

[0067] Reagents and reaction conditions: a) 4-methoxycarbonylphenylboronic acid, K2CO3, Pd(PPh3)4, DMF, 100℃; b) NaOH, EtOH, 60℃.

[0068] As shown in the reaction process above, the synthesis of compound D3 is described in the synthesis process of compound D2. Compound D3: 1 H NMR (400MHz, d6-DMSO) δ11.46(s,1H),8.32(t,J=5.4Hz,1H),8.17(t,J=4.8Hz,1H),7.55(d,J=8.1Hz,2H),7.44(d,J=8.7Hz,2 H),7.40(d,J=8.8Hz,2H),7.38(s,1H),7.32(d,J=8.1Hz,2H),7.22(s,1H),5.86(s,1H),4.52(t,J=7.0Hz,1H),4.30(d,J=4.8H z,2H),3.82(d,J=9.9Hz,2H),3.51(s,4H),3.28–3.22(m,2H),3.09(dd,J=6.9Hz,2H),3.03–3.01(m,1H),2.80(s,2H),2.60(s, 3H),2.41(s,3H),2.25(s,3H),2.21(s,3H),2.11(s,3H),1.65(d,J=13.8Hz,2H),1.62(s,3H),1.56–1.49(m,2H),0.83(s,3H). 13 C NMR (101MHz, d6-DMSO) δ169.4,169.0,163.0,155.1,149.8,149.4,148.8,142.7,139. 6,138.7,137.7,137.1,136.7,135.2,132.5,132.2,130.7,130.1,129.8,129.6,129.2 ,128.4,126.5,122.8,121.6,120.7,107.3,99.5,69.8,66.3,57.8,53.8,41.2,37.6, 34.9,34.8,30.3,29.0,18.9,18.2,14.5,14.0,12.7,12.6,11.3.HRMS(ESI,m / z)calcd for C 50 H 55ClN8O4S[M–H] - :897.3683,found:897.3662.Purity:97.5%.

[0069] Example 5: Synthesis of compound D4

[0070]

[0071] Reagents and reaction conditions: (a) TsCl, TEA, DMAP, CH2Cl2; (b) 4-hydroxyphenylboronic acid pinacol ester, K2CO3, DMF, 70℃; (c) Intermediate 6, Pd(PPh3)4, K2CO3, DMF, 90℃; (d) 20% TFA, CH2Cl2; (e) Intermediate 13, HATU, DIPEA, DMF.

[0072] As shown in the reaction process above, the synthesis of compound D4 is described in the synthesis process of compound D7. Compound D4: 1 H NMR (400MHz, d6-DMSO) δ11.47(s,1H),8.58(t,J=5.5Hz,1H),8.17(t,J=4.9Hz,1H),7.58(d,J=8.7Hz,2H),7.36(d,J=8.7Hz,3H),7.26(d,J=8 .7Hz,2H),7.21(d,J=1.4Hz,1H),7.06(d,J=8.8Hz,2H),5.86(s,1H),4.53(dd,J=8.8,5.4Hz,1H),4.30(d,J=4.8Hz,2H),4.07(d,J=5.0Hz,2H ),3.83(d,J=11.0Hz,2H),3.49–3.37(m,2H),3.27(t,J=11.1Hz,2H),3.18(dd,J=14.9,5.3Hz,2H),3.11–3.06(m,2H),3.03–3.01(m,1H),2.5 9(s,3H),2.40(s,3H),2.25(s,3H),2.21(s,3H),2.11(s,3H),1.67(d, J=11.6Hz,2H),1.60(s,3H),1.53–1.51(m,2H),0.84(t,J=7.0Hz,3H). 13C NMR(101MHz,d6-DMSO)δ170.0,169.1,167.0,163.0,158.1,155.1,149.8,149.5,148.8,142.7,139.5,136.9 ,136.7,135.1,132.5,132.3,132.1,131.7,131.6,130.7,130.1,129.8,129.5,129.5,128.6,128.3,127.8, 122.6,121.6,120.6,114.9,107.3,67.4,66.8,66.3,57.9,53.9,41.3,38.4,38.1,37.5,34.9,30.3,29.9,2 9.8,28.4,23.4,23.2,22.4,18.9,18.2,14.5,14.0,13.9,12.7,12.7,11.3,10.9,10.8.HRMS(ESI,m / z)calcd for C 50 H 55 ClN8O5S[MH] - :913.3632,found:913.3611.Purity:100%.

[0073] Example 6: Synthesis of compound D5

[0074]

[0075] Reagents and reaction conditions: (a) TsCl, TEA, DMAP, CH2Cl2; (b) 4-hydroxyphenylboronic acid pinacol ester, K2CO3, DMF, 70℃; (c) Intermediate 6, Pd(PPh3)4, K2CO3, DMF, 90℃; (d) 20% TFA, CH2Cl2; (e) Intermediate 13, HATU, DIPEA, DMF.

[0076] As shown in the reaction process above, the synthesis of compound D5 is described in the synthesis process of compound D7. Compound D7: 1H NMR(400MHz,d6-DMSO)δ11.46(s,1H),8.32(t,J=5.6Hz,1H),8.17(t,J=4.9Hz,1H),7.53(d,J=8.7Hz,2H),7.41(s,4H),7.34(s,1H), 7.18(s,1H),6.99(d,J=8.8Hz,2H),5.86(s,1H),4.52(dd,J=8.0,6.2Hz,1H),4.30(d,J=4.9Hz,2H),4.09–4.02(m,2H),3.84(d,J=10. 5Hz,2H),3.29–3.22(m,4H),3.23(d,J=5.8Hz,2H),3.12–3.05(m,2H),3.02–2.99(m,J=11.2Hz,1H),2.59(s,3H),2.40(s,3H),2.24(s ,3H),2.21(s,3H),2.11(s,3H),1.97–1.89(m,2H),1.67(d,J=11.3Hz,2H),1.60(s,3H),1.53(d,J=8.6Hz,2H),0.84(t,J=7.0Hz,3H). 13 C NMR(101MHz,d6-DMSO)δ169.6,169.1,163.0,162.9,158.1,155.1,149.8,149.4,148 .8,142.7,139.5,136.9,136.7,135.2,132.2,131.9,130.7,130.0,129.8,129.6,12 8.4,127.6,122.5,121.6,120.5,114.9,107.3,66.3,65.3,57.9,53.9,41.2,37.7,3 5.4,34.9,30.3,29.1,18.9,18.2,14.5,14.0,12.7,12.6,11.3.HRMS(ESI,m / z)calcd forC 51 H 57 ClN8O5S[M–H] - :927.3788,found:927.3778.Purity:99.5%.

[0077] Example 7: Synthesis of Compound D6

[0078]

[0079] Reagents and reaction conditions: (a) TsCl, TEA, DMAP, CH2Cl2; (b) 4-hydroxyphenylboronic acid pinacol ester, K2CO3, DMF, 70℃; (c) Intermediate 6, Pd(PPh3)4, K2CO3, DMF, 90℃; (d) 20% TFA, CH2Cl2; (e) Intermediate 13, HATU, DIPEA, DMF.

[0080] As shown in the reaction process above, the synthesis of compound D6 is described in the synthesis process of compound D7. Compound D6: 1 H NMR (400MHz, d6-DMSO) δ11.47(s,1H),8.26(t,J=5.5Hz,1H),8.17(t,J=4.9Hz,1H),7.53(d,J=8.6Hz,2H),7.42(s,4H),7.34(s,1H),7. 18(s,1H),6.98(d,J=8.6Hz,2H),5.86(s,1H),4.52(dd,J=8.3,5.8Hz,1H),4.29(d,J=4.8Hz,2H),4.02(t,J=6.4Hz,2H),3.82(d,J=10. 2Hz,2H),3.23(dd,J=21.7,8.7Hz,6H),3.18–3.15(m,2H),3.07(dd,J=13.7,6.8Hz,2H),3.01–2.98(m,1H),2.59(s,3H),2.40(s,3H),2 .23(s,3H),2.21(s,3H),2.10(s,3H),1.80–1.74(m,2H),1.65(d,J=12.0Hz,2H),1.61(s,3H),1.53–1.50(m,2H),0.82(t,J=6.9Hz,3H). 13C NMR (101MHz, d6-DMSO) δ169.5,169.2,163.0,158.2,155.1,149.8,149.5,148.8,142. 7,139.5,136.9,136.8,135.2,132.3,132.2,131.9,130.7,130.1,129.8,129.6,128.4 ,127.7,122.6,121.6,120.5,114.8,107.4,67.3,66.3,57.9,54.0,41.3,38.2,37.7, 34.9,30.3,26.2,26.0,19.0,18.2,14.5,14.0,12.7,12.7,11.3.HRMS(ESI,m / z)calcd for C52H59ClN8O5S[M+H] + :943.4090,found:943.4098.Purity:97.5%.

[0081] Example 8: Synthesis of compound D8

[0082]

[0083] Reagents and reaction conditions: (a) 4-methoxycarbonylphenylboronic acid, K2CO3, Pd(PPh3)4, DMF, 90℃; (b) NaOH, EtOH, 60℃; (c) tert-butyl(2-(2-aminoethoxy)ethyl)carbamate, HATU, DIPEA, DMF; (d) 20% TFA, CH2Cl2; (e) Intermediate 13, HATU, DIPEA, DMF; (f) Boc2O, NaOH, THF, H2O.

[0084] 4-Methoxycarbonylphenylboronic acid, K₂CO₃, and Pd(PPh₃)₄ were added to a DMF (20 mL) solution of intermediate 6 (1 g, 2.10 mmol). The solution was purged and refilled three times with argon. The mixture was stirred at 90 °C for 9 hours, then extracted with an ethyl acetate / water system. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a methanol / dichloromethane system to give the coupling product as a yellow solid (0.82 g, 73.5%). The obtained product was hydrolyzed in a sodium hydroxide solution containing ethanol at 60 °C to give intermediate 19. Intermediate 19 (50.7 mg, 97.9 μmol), DIPEA, and HATU were added sequentially to a DMF (2 mL) solution containing tert-butyl (2-(2-aminoethoxy)ethyl)carbamate. The resulting mixture was stirred at room temperature for 10 hours, then extracted with an ethyl acetate / water system. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a methanol / dichloromethane system to give a yellow powder (59 mg, 85.6%). The yellow powder (59 mg, 97.9 μmol) was then dissolved in dichloromethane (1 mL), and TFA (0.2 μL) was added. The mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure to provide the free amine, which could be used for the next step without further purification. The amine (26.0 mg, 43.1 μmol) was dissolved in DMF (2 mL), and intermediate 13 (17.3 mg, 43.1 μmol), DIPEA, and HATU were added sequentially. The resulting mixture was stirred at room temperature for 10 hours, then extracted with an ethyl acetate / water system. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a methanol / dichloromethane system to obtain D8 (27.0 mg, 65.6%) as a yellow powder. 1H NMR (400MHz, d6-DMSO) δ11.46(s,1H),8.55(t,J=5.7Hz,1H),8.31(t,J=5.3Hz,1H),8.22(t,J=4.7Hz,1H),7.94(d,J=8.3Hz,2H),7.70(d,J=8.4 Hz,2H),7.47(d,J=8.7Hz,2H),7.44(s,1H),7.41(d,J=8.6Hz,2H),7.28 (s,1H),5.86(s,1H),4.54–4.48(m,1H),4.30(d,J=4.8Hz,2H),3.83(d,J =10.4Hz,2H),3.60(t,J=6.2Hz,2H),3.53–3.49(m,2H),3.47(d,J=5.9H z,2H),3.28–3.21(m,6H),3.10(dd,J=13.8,6.8Hz,2H),3.01(m,1H),2.5 8(s,3H),2.39(s,3H),2.26(s,3H),2.21(s,3H),2.11(s,3H),1.66(d,J =12.9Hz,2H),1.59(s,3H),1.53(d,J=8.6Hz,2H),0.83(t,J=6.9Hz,3H). 13 C NMR(101MHz,d6-DMSO)δ169.8,168.9,166.0,163.0,155.1,149.8,149.5 ,149.0,142.3,139.7,136.7,136.2,135.2,132.2,130.7,130.1,129.8, 128.4,127.8,126.3,123.0,69.0,68.8,66.3,57.9,53.8,41.2,37.5,34 .9,30.3,18.9,18.2,14.6,14.0,12.8,12.6,11.2.HRMS(ESI,m / z)calcd for C 53 H60ClN9O6S[M–H]-:984.4003,found:984.4014.Purity:97.3%.

[0085] Example 9: Synthesis of compound D9

[0086]

[0087] Reagents and reaction conditions: (a) Boc2O, NaOH, THF, H2O; (b) Intermediate 18a, HATU, DIPEA, DMF; (c) 20% TFA, CH2Cl2; (d) Intermediate 13, HATU, DIPEA, DMF.

[0088] An aqueous solution of Boc₂O and sodium hydroxide was added to a THF (4 mL) solution containing compound 20a (0.2 g, 1.38 mmol). The mixture was stirred overnight at room temperature, then extracted with an ethyl acetate / water system. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using an ethyl acetate / petroleum ether system to give intermediate 21a (378 mg, 100%) as a yellow solid. The Boc group of intermediate 18a (30 mg, 122 μmol) was removed under acidic conditions to give a free amine. Compound 21a (59.8 mg, 122 μmol), HATU, and DIPEA were added sequentially to a DMF solution containing the free amine. The resulting mixture was stirred at room temperature for 10 hours, then extracted with an ethyl acetate / water system. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a methanol / dichloromethane system to obtain a yellow powder intermediate 22a (41.0 mg, 46.8%). Intermediate 22a (41.0 mg, 122 μmol) was then dissolved in dichloromethane (1 mL), and TFA (0.2 μL) was added. The mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure to obtain the free amine, which could be used for the next step without further purification. The amine (9.0 mg, 22.5 μmol) was dissolved in DMF (2 mL), and intermediate 13 (13.0 mg, 22.5 μmol), DIPEA, and HATU were added sequentially. The resulting mixture was stirred at room temperature for 10 hours, then extracted with an ethyl acetate / water system. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a methanol / dichloromethane system to obtain a yellow powder compound D9 (13.0 mg, 58.0%). 1H NMR (400MHz, d6-DMSO) δ11.45(s,1H),9.97(s,1H),8.17(t,J=5.0Hz,2H),7.67(d,J=8.6Hz,2H),7.55(d,J=8.6Hz,2H),7.4 9(d,J=8.7Hz,2H),7.42(d,J=8.6Hz,2H),7.38(s,1H),7.20(s,1H),5.86(s,1H),4.54–4.47(m,1H),4.29(d,J=4.8Hz,2H),4 .22(t,J=6.6Hz,1H),3.83(d,J=9.4Hz,2H),3.27–3.16(m,8H),3.08–3.02(m,3H),2.59(s,3H),2.40(s,3H),2.31(t,J=7.3H z,2H),2.23(s,3H),2.21(s,3H),2.11(s,3H),1.64(d,J=2.8Hz,2H),1.61(s,3H),1.53–1.33(m,9H),0.82(d,J=7.0Hz,3H). 13 C NMR(101MHz,d6-DMSO)δ169.3,169.1,163.0,155.1,154.4,149.8,149.5,148.8,148.8,148.4,139.6, 138.7,136.8,134.3,132.2,132.2,131.5,130.7,130.1,129.6,128.6,128.5,126.7,120.5,119.4,10 9.4,107.4,73.8,66.3,57.9,53.9,41.2,40.8,38.4,37.7,36.4,34.9,34.5,31.3,31.2,30.3,30.0,2 9.1,29.0,28.4,26.1,25.0,18.9,18.6,18.2,14.5,14.0,13.5,12.7,12.6,11.3.HRMS(ESI,m / z)calcd for C 55 H 64 ClN9O5S[M–]:997.4445,found:997.4487.Purity:95.0%.

[0089] Example 10: Synthesis of compound D10

[0090]

[0091] Reagents and reaction conditions: (a) Boc2O, NaOH, THF, H2O; (b) Intermediate 18a, HATU, DIPEA, DMF; (c) 20% TFA, CH2Cl2; (d) Intermediate 13, HATU, DIPEA, DMF.

[0092] Compound D10 was obtained by referring to the synthetic method of compound D9. Compound D10: 1 H NMR(400MHz,d6-DMSO)δ11.46(s,1H),9.97(s,1H),8.17(s,1H),7.75–7.70(m,1H) ,7.67(d,J=8.3Hz,2H),7.56(d,J=8.3Hz,2H),7.48(d,J=8.2Hz,2H),7.42(d,J=8. 2Hz,2H),7.38(s,1H),7.21(s,1H),5.86(s,1H),4.55–4.47(m,1H),4.30(d,J=4.3 Hz,2H),4.16–4.10(m,1H),4.02(d,J=6.4Hz,1H),3.83(d,J=10.3Hz,2H),3.28–3.2 0(m,J=9.4,4H),3.11–3.06(m,2H),3.03–2.97(m,1H),2.94–2.89(m,1H),2.59(s, 3H),2.40(s,3H),2.34–2.28(m,2H),2.24(s,3H),2.21(s,3H),2.11(s,3H),1.64( s,2H),1.62(s,3H),1.52(d,J=13.4Hz,2H),1.47–1.43(m,2H),1.37–1.29(m,6H), 1.20–1.15(m,2H),0.93(d,J=6.6Hz,2H),0.90–0.86(m,2H),0.82(d,J=6.7Hz,3H). 13C NMR(101MHz,d6-DMSO)δ171.3,169.1,167.0,163.0,163.0,155.1,149.8,149.5,148.8,142.7,139.6,138.8, 136.7,135.2,132.2,131.7,131.6,130.7,130.1,129.8,129.6,128.7,128.4,126.7,121.6,119.4,107.4,71. 2,67.4,66.3,57.9,53.9,41.3,38.5,38.1,37.7,36.4,34.9,34.3,30.3,29.8,29.2,28.7,28.6,28.4,27.2,2 6.3,25.1,23.3,22.4,19.0,18.9,18.2,14.5,14.0,13.9,12.7,12.7,11.3,11.00,10.8.HRMS(ESI,m / z)calcd for C 56 H 66 ClN9O5S[MH] - :1010.4523,found:1010.4562.Purity:95.0%.

[0093] Example 11: Synthesis of compound D11

[0094]

[0095] Reagents and reaction conditions: (a) Boc2O, NaOH, THF, H2O; (b) Intermediate 18a, HATU, DIPEA, DMF; (c) 20% TFA, CH2Cl2; (d) Intermediate 13, HATU, DIPEA, DMF.

[0096] Compound D11 was synthesized using the same method as compound D9. Compound D11: 1H NMR (400MHz, d6-DMSO) δ11.47(s,1H),10.04(s,1H),8.27(t,J=5.5Hz,1H),8.19(t,J=4.9Hz,1H),7.67(d,J=8.7Hz,2H),7.56(d,J=8.7Hz,2H),7 .47(d,J=8.8Hz,2H),7.41(d,J=8.6Hz,2H),7.38(s,1H),7.20(s,1H),5 .86(s,1H),4.53–4.47(m,1H),4.29(d,J=4.9Hz,2H),3.81(d,J=9.8Hz,2 H),3.70(t,J=6.2Hz,2H),3.52(d,J=2.2Hz,10H),3.34–3.15(m,6H),3.07(dd,J=14.1,7.0Hz,2H),2.99(d,J=10.9Hz,1H),2.58(s,3H),2.55(d, J=6.2Hz,2H),2.39(s,3H),2.22(s,3H),2.20(s,3H),2.10(s,3H),1.65( d,J=11.4Hz,2H),1.60(s,3H),1.55–1.45(m,2H),0.81(t,J=6.9Hz,3H). 13 C NMR(126MHz,d6-DMSO)δ169.8,169.3,169.2,163.1,155.2,149.9,149.7,148.9,142.8,139.6,1 38.7,136.8,136.8,135.3,134.5,132.3,130.8,130.2,129.9,129.6,128.5,126.8,122.6,121. 7,120.5,119.4,107.5,69.8,69.8,69.7,69.7,69.3,66.7,66.4,59.8,57.9,53.9,41.3,38.7,3 7.6,37.2,34.9,30.4,20.8,19.0,18.2,14.6,14.1,14.0,12.8,12.7,11.3.HRMS(ESI,m / z)calcd forC 56 H 66 ClN9O5S[MH] - :1072.4527,found:1072.4544.Purity:95.0%.

[0097] Example 12: Synthesis of compound D12

[0098]

[0099] Reagents and reaction conditions: (a) TsCl, TEA, DMAP, CH2Cl2; (b) 4-hydroxyphenylboronic acid pinacol ester, K2CO3, DMF, 70℃; (c) Intermediate 6, Pd(PPh3)4, K2CO3, DMF, 90℃; (d) 20% TFA, CH2Cl2; (e) Intermediate 13, HATU, DIPEA, DMF.

[0100] Compound D12 was synthesized using the same method as compound D7. LRMS(ESI, m / z)calcd for C 52 H 60 ClN8O6S[M+H] + :959.40,found:959.50.

[0101] Example 13: Western Blot Detection of the Inhibitory Effects of Compounds on EZH2 and BRD4

[0102] (1) Extraction of total protein: Transfer cells treated with DMSO or other drugs to centrifuge tubes, collect cells by centrifugation, wash twice with PBS, and transfer to 1.5 mL EP tubes. Add 100-150 μL of lysis buffer to each EP tube according to the number of cells, mix well, and incubate at room temperature for 10 minutes. Then place the EP tubes in a constant temperature metal bath and boil at 95°C for 15 minutes.

[0103] (2) Protein content determination: Protein content determination was performed in accordance with the instructions of the Pierce BCA Protein Quantitative Kit.

[0104] (3) SDS-PAGE gel electrophoresis preparation: After cleaning the glass plates, let them air dry. Prepare a 12% separating gel and a 5% stacking gel according to the formula. Sample loading: Add 4x SDS loading buffer to the protein sample according to the ratio to make the final SDS concentration 1x. After mixing well, place the sample in a constant temperature metal bath at 95°C for 10 minutes. Calculate and add 10ug of protein sample to each well. Electrophoresis: Set the voltage of the stacking gel to 80V at the beginning. After the band enters the separating gel, adjust the constant voltage to 120V until the bromophenol blue band reaches the bottom.

[0105] (4) Transfer: Prepare a methanol-soaked PVDF membrane, filter paper soaked in transfer solution, a black sponge, separating gel, transfer clamp, and filter paper, placing them in a tray containing transfer solution. Place the transfer clamp with the black side down. On the transfer clamp, place the sponge, filter paper, separating gel, PVDF membrane, another sheet of filter paper, and the sponge in sequence, carefully removing air bubbles with a glass rod during the process. Secure the membrane in the transfer tank. Use the wet transfer method with a constant current of 0.25A, transferring for 1-3 hours depending on the molecular weight.

[0106] (5) Immunoblot blocking: Place the transferred PVDF membrane in a plastic box containing 5% skim milk powder and block at room temperature for 1 hour. Wash the membrane with TBST for 10 minutes. Primary antibody incubation: Dilute the primary antibody with Beyotime's primary antibody diluent according to the instructions. Place the transfer membrane in the diluted primary antibody and incubate overnight at 4°C on a shaker. Secondary antibody incubation: Wash the membrane with TBST for 10 minutes × 3 times the next day. Transfer the transfer membrane to the diluted secondary antibody and incubate at room temperature for 1 hour. Then wash again with TBST for 10 minutes × 3 times. ECL development: Apply the prepared ECL luminescence solution evenly to the band to be detected. After placing in the dark for one minute, develop and photograph using a chemiluminescence imaging system.

[0107] Example 14: MTS detection of the killing effect of compounds on tumor cells

[0108] Using pancreatic cancer cell line AsPC-1, lung cancer cell line A549, and colorectal cancer cell line HCT116 as examples, cells in the logarithmic growth phase were centrifuged, the supernatant was discarded, washed twice with PBS, resuspended, and counted. Cells were seeded evenly in 96-well plates at a density of 500-1500 cells / 100 μL. Replica wells and blank controls were set up. The drug was diluted with fresh culture medium to the required concentration, and 100 μL was added to each well of the seeded 96-well plate. The plates were incubated at 37°C with CO2 for 6 days. 20 μL of MTS solution was added to each well, and the reaction continued for 4 hours. The absorbance (OD) of different wells was measured at 490 nm using a microplate reader.

[0109] Example 15: Experimental Examination of the Antitumor Effect of Compound D7 in a Human Lung Cancer Subcutaneous Xenograft Tumor Model in Nude Mice

[0110] (1) Collect tumor cells in the logarithmic growth phase, wash twice with PBS solution, resuspend the cells in serum-free RPMI-1640 medium, and calculate the required number of cells for seeding. A549 model cells: 2.5 × 10⁻⁶ 6 / 100μL;

[0111] (2) 100 μL of cell suspension was injected subcutaneously into the right axilla of 8-week-old nude mice;

[0112] (3) When the tumor volume reaches 50-100 mm 3Mice were randomly divided into four groups: a solvent control group and three drug treatment groups.

[0113] (4) Drug administration: JQ1 (30mg / kg), EPZ6438 (30mg / kg) and D7 (60mg / kg) were given in the drug treatment group, and DMSO (10%, DMSO:castor oil:PBS buffer = 1:1:8) was given in the solvent control group. 200μL of drug or solvent was injected into each animal intraperitoneally every day.

[0114] (5) When a subcutaneous tumor is visible to the naked eye, tumor growth measurements should be performed every two days. The length (L) and width (W) of the tumor should be measured and recorded using calipers. The tumor volume is calculated using the formula V (mm²). 3 )=(L×W 2 ) / 2, plot the tumor growth curve based on the tumor size;

[0115] (6) When the tumor volume in the control group reached 1000-2000 mm 3 The experiment was terminated at the appropriate time. Nude mice were euthanized by dislocation, tumor tissue was collected, photographed, and weighed.

[0116] To further verify the effect of the compounds of this invention on EZH2 protein levels, AsPC-1 cells were treated with 1 μM of compound D1-D11 or DMSO for 48 h. The levels of H3K27me3 and c-Myc were then measured using Western blot, with histone H3 and actin as controls. Figure 1 As shown, the results indicate that compound D7 most significantly reduced H3K27me3 protein levels and also reduced c-Myc levels.

[0117] Furthermore, such as Figure 2 The effect of different concentrations of compound D7 of this invention on the protein levels of EZH2 and BRD4 is shown in the figure. AsPC-1 cells were treated with the specified concentration of the compound for 48 hours, and the levels of H3K27me3 and c-Myc were measured using Western blot, with histone H3 and actin as controls. The results showed that D7 could inhibit the enzyme function of EZH2 and BRD4 in a concentration-dependent manner.

[0118] Figure 3This study demonstrates the effects of compound D7 of the present invention and existing EZH2 inhibitors EPZ6438 and BRD4 inhibitor JQ1 on the viability of different solid tumor cells. The present invention treated pancreatic cancer cell line AsPC-1, lung cancer cell line A549, and colorectal cancer cell line HCT116 at specified concentrations of the compound for six days, and then measured cell viability using MTS. The results showed that compound D7 of the present invention had a stronger killing effect compared to EPZ6438 and JQ1.

[0119] like Figure 4 As shown, in the A549 nude mouse subcutaneous xenograft model, compound D7 of the present invention exhibits a more effective antitumor effect compared to EPZ6438, JQ1, or a combination of both.

[0120] Figure 5 The results show that different compounds have antitumor effects in different tumor cell lines. It can be seen that compound D7 of the present invention has a more effective antitumor effect compared with EPZ6438, JQ1 or a combination of the two.

[0121] Table 1 shows the IC50 values ​​of different compounds D1-D11, EPZ5438, and JQ1 in AsPC-1 and H460 cells. 50 (μm).

[0122] Table 1. IC50 values ​​of different compounds in AsPC-1 and H460 cells. 50

[0123]

[0124] The data in Table 1 shows that:

[0125] 1) When the main chain length is 1 to 14 atoms, the activity of the dual-target inhibitor is significantly improved compared with EPZ6438;

[0126] 2) When the main chain length is 3 to 7 atoms, the activity of the dual-target inhibitor is significantly improved compared with JQ1;

[0127] 3) Linker length significantly affects the antitumor activity of compounds. Specifically, when the main chain length is 1 to 7 atoms, the activity of dual-target inhibitors increases with the increase of main chain length. When the main chain length is 3 to 7 atoms, the activity of dual-target inhibitors is significantly improved compared with JQ1 and EPZ6438. When the length is 5 to 7 atoms, it has better activity, and when it is 6 to 7 atoms, it is expected to have the best activity.

[0128] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A dual-target inhibitor of EZH2 and BRD4, characterized in that: The dual-target inhibitor can simultaneously inhibit both EZH2 and BRD4 proteins. It is obtained by linking a structural fragment that inhibits BRD4 protein and a structural fragment that inhibits EZH2 protein through a linker, and its structural formula is one of formulas D3-D7 and D12: , , , , , In equations D3 to D7 and D12, R1 = R2= .

2. A pharmaceutical composition comprising an active ingredient and excipients, characterized in that: Its active ingredients include the dual-target inhibitor of EZH2 and BRD4 as described in claim 1, or one of its pharmaceutical salts, crystals, or solvates.

3. The pharmaceutical composition according to claim 2, characterized in that: It is used to treat tumors.

4. The use of a dual-target inhibitor of EZH2 and BRD4 in the preparation of a drug for treating tumors, wherein the dual-target inhibitor of EZH2 and BRD4 is as described in claim 1, and the tumor is lung cancer, colorectal cancer, or pancreatic cancer.

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