CN115920073A - EZH2 and BRD4 double-target-point inhibitor and application thereof - Google Patents

Abstract

The invention 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 BRD4 protein and a structural fragment for inhibiting EZH2 protein through a linker. The inventor researches and discovers that the structural fragment for inhibiting the EZH2 protein and the structural fragment for inhibiting the BRD4 protein are structurally modified and coupled together through a linker, and the length of the linker is adjusted, so that the obtained EZH2 and BRD4 dual-target inhibitor has an unexpected anti-tumor effect, and the anti-tumor effect is superior to that of a single EZH2 inhibitor or a single BRD4 inhibitor and is also superior to that of the combination of the two inhibitors under the same condition.

Description

EZH2 and BRD4 double-target-point inhibitor and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to an EZH2 and BRD4 double-target inhibitor and application thereof.

Background

In recent decades, the epigenetic regulation of the surface has been studied intensively. The role of the abnormal epigenetic changes observed in cancer cells, and in particular abnormal histone modifications, in cancer has been extensively studied. At present, people also develop a series of small molecular compounds for specifically inhibiting histone modification enzyme, and obtain certain therapeutic effect. Epigenetic drugs have been shown to improve the efficacy of a variety of major cancer treatment modalities, such as chemotherapy, radiation therapy, targeted therapies, and immunotherapy. In addition, due to the interactions between multiple epigenetic processes, combining multiple epigenetic drugs is also an effective method to target tumors.

The protein expressed by the EZH2 gene, known as histone lysine N-methyltransferase EZH2 (Enhancer of zeste homolog 2), is a member of the polycomb family. EZH2 is capable of promoting mono-, di-and trimethylation of lysine 27 (H3K 27) of histone H3, and is an enzymatic subunit protein of polycomb inhibitory complex 2 (PRC 2). EZH2 is associated with a variety of biological functions, including transcriptional regulation in hematopoiesis, development, and cell differentiation. EZH2 silences genes primarily by trimethylating H3K27, a histone modification that plays a unique role in epigenetic regulation of gene transcription. Trimethylated H3K27 is an important modification of chromosomes to maintain a compact state, promoting chromatin condensation and heterochromatin formation. Polygene complex 2 (PRC 2) containing EZH2 may also be involved in the recruitment of DNA methyltransferases, which leads to increased DNA methylation. Specific genes currently identified as targets for EZH 2-mediated transcriptional repression include HOXA9, HOXC8, MYT1, CDKN2A, and retinoic acid target genes, among others.

EZH2, which is implicated in cancer cell division and proliferation, is overexpressed in many cancers, including leukemia, bladder, kidney, uterus and prostate cancers, as well as melanoma and lymphoma, and is recognized as a target protein for cancer therapy. The development of EZH2 inhibitors as cancer therapeutic drugs has been extensively studied and focused on. To date, several EZH2 inhibitors, including GSK126, EPZ6438, PF-06821497 and CPI-1205, have been entered into clinical trials. Among them, EPZ6438 has excellent drug efficacy and drug-like properties, including good oral bioavailability, as a specific small molecule inhibitor of EZH 2. EPZ6438 (tazemetostat) is currently approved for the treatment of locally advanced or metastatic epithelioid sarcoma and follicular lymphoma, while also entering clinical trials for the treatment of diffuse large B-cell lymphoma. However, EZH2 inhibitors have tumor species limitations, and show good efficacy mainly in some hematological tumors, especially diffuse large B-cell lymphoma; on the other hand, the inhibitor has certain inhibiting effect on the solid tumors with SWI/SNF member gene mutation and BAP1 mutation. EZH2 inhibitors are less effective in killing most solid tumors with over-expression of wild-type EZH2 and increased degree of H3K27 trimethylation. Based on the molecular mechanism that tumor cells are insensitive or resistant to the EZH2 inhibitor, the clinical application of the EZH2 inhibitor in solid tumors is expanded by continuously developing anti-tumor drug molecules aiming at the EZH2 target protein.

In most solid tumors, EZH2 inhibition, although reducing H3K27me3 levels, also increases the degree of H3K27 acetylation (H3K 27 ac), which in turn leads to solid tumors resistant to EZH2 inhibitors. The increase in H3K27ac and oncogenic transcriptional reprogramming caused by EZH2 inhibition may limit the effect of EZH2 inhibitors in solid tumors. BRD4 is a member of the bromodomain-containing and extra-terminal domain (BET) protein family, which is capable of reading acetylated lysine residues in histones, facilitating gene transcription. (+) -JQ1 was the first reported selective inhibitor of BRD4, which mimics the interaction between BET protein and acetyl-lysine on histones and has potent anti-cancer effects. Several BRD4 inhibitors including I-BET762, OTX-015 and ABBV-075 are reported in succession, some of which have entered clinical trials. The BRD4 inhibitor can reduce the drug resistance of the EZH2 inhibitor caused by the up-regulation of H3K27ac and improve the curative effect of the EZH2 inhibitor. Research results show that the combined use of the BRD4 inhibitor and the EZH2 inhibitor can induce the sensitivity of solid tumor cells to the EZH2 inhibition, and has better synergistic anticancer effect. However, EZH2 and BRD4 are separate proteins, and it is desirable to use both a BRD4 inhibitor and an EZH2 inhibitor.

Disclosure of Invention

The present invention aims to overcome at least one of the deficiencies of the prior art and to provide an EZH2 and BRD4 dual-target inhibitor and uses thereof.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

the double-target-point inhibitor for the EZH2 and the BRD4 is obtained by coupling a structural fragment for inhibiting the BRD4 protein and a structural fragment for inhibiting the EZH2 protein through a linker, wherein the main chain of the linker contains 1-14 atoms.

In some examples of dual-target inhibitors, the linker backbone is 2 to 7 atoms, preferably 5 to 7 atoms. Further, the main chain of the linker contains 6 or 7 atoms. Experimental data show that the length of the backbone has a significant effect on the activity of the dual-target inhibitor.

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 a structural fragment that has an EZH2 protein inhibitory effect.

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 a structural fragment that inhibits the BRD4 protein.

In some examples of dual-target inhibitors, the linker backbone is an alkyl chain, a polyethylene glycol chain, or an ether-containing alkyl chain. These backbones have better stability and at the same time better flexibility.

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

In some examples of dual-target inhibitors, the dual-target inhibitor has the general structural formula shown in formula I:

in some examples of dual-target inhibitors, the linker is selected from one of the formulas X1 to X15, wherein n or m is an integer:

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in some examples of dual-target inhibitors, the structural formula is one of formulas D1-D12:

in formulas D1-D12, is selected>

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In a second aspect of the present invention, there is provided:

a pharmaceutical composition comprises an active ingredient and an excipient, wherein the active ingredient comprises the EZH2 and BRD4 double-target inhibitor of the first aspect of the invention, or one of pharmaceutically acceptable salts, crystals and solvates thereof.

In some examples of pharmaceutical compositions, it is used to treat tumors.

In some examples of the pharmaceutical composition, the tumor is a solid tumor or a hematologic tumor.

In some examples of pharmaceutical compositions, the solid tumor is selected from lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, renal cancer, uterine cancer, prostate cancer, or melanoma; the hematological tumor is selected from leukemia or lymphoma.

In a third aspect of the present invention, there is provided:

the invention relates to the use of a double-target EZH2 and BRD4 inhibitor according to the first aspect in the manufacture of a medicament for the treatment of a tumour.

In some examples of use, the tumor is a solid tumor or a hematological tumor.

In some examples of use, the solid tumor is selected from lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, renal cancer, uterine cancer, prostate cancer, or melanoma; the hematological tumor is selected from leukemia or lymphoma.

The invention has the beneficial effects that:

EZH2 and BRD4 are separate proteins, physically isolated within the cell, and theoretically coupling a BRD4 inhibitor and an EZH2 inhibitor together forms a dual-target inhibitor that reacts with and inhibits only one of EZH2 and BRD4, which does not have an advantage in inhibiting the theoretical activity of a single target protein over a traditional single-target inhibitor. However, the inventor researches and discovers that the structural fragments for inhibiting the EZH2 protein and the structural fragments for inhibiting the BRD4 protein are structurally modified and coupled together through a linker, and the length of the linker is adjusted, so that the obtained double-target inhibitor of the EZH2 and the BRD4 has an unexpected anti-tumor effect, and the anti-tumor effect is superior to that of a single EZH2 inhibitor or a single BRD4 inhibitor and is also superior to that of the combination of the two inhibitors under the same condition.

Drawings

FIG. 1 is a graph of the effect of compounds of the invention on the levels of H3K27me3 and C-Myc protein downstream of BRD4 in EZH 2;

FIG. 2 is a graph showing the effect of different concentrations of compound D7 on the levels of H3K27me3 and c-Myc protein downstream of BRD4 in the presence of EZH 2;

FIG. 3 shows the effect of inventive compound D7 and control compounds EPZ6438, JQ1 on the viability of multiple solid tumor cells.

FIG. 4 shows the antitumor effect of compound D7 of the present invention and control compounds EPZ6438, JQ1 in a nude mouse subcutaneous transplantation tumor model A549.

FIG. 5 is a graph of the antitumor effect of different compounds on different tumor cell lines.

Detailed Description

The present invention will be further described with reference to the accompanying drawings, and it should be noted that the following examples are provided to give detailed embodiments and specific operation procedures on the premise of the technical solution, but the protection scope of the present invention is not limited to the examples.

In the following examples, the numbering of the intermediates is the same, i.e., intermediates having the same numbering have the same structure.

Example 1: synthesis of Compound D7

Synthesis of intermediate 6:

reagents and reaction conditions: (a) Fe, NH ₄ Cl,MeOH,90℃；(b)tetrahydro-4H-pyran-4-one,AcOH,Na(AcO) ₃ BH,1,2-dichloroethane；(c)acetaldehyde,AcOH,Na(AcO) ₃ BH,1,2-dichloroethane；(d)Boc ₂ O,H ₂ ,Raney-Ni,MeOH；(e)HCl,MeOH；(f)NaOH,EtOH,60℃；(g)HOBt,EDCI,NMM,DMSO。

As shown in the above reaction procedure, the synthesis was carried out starting from commercially available Compound 1 (900mg, 3.28mmol), iron powder and ammonium chloride were added, and the reaction was refluxed at 90 ℃ overnight in a methanol solution. After TLC detection reaction is completed, the reaction system is filtered, the filtrate is collected and distilled under reduced pressure, and the product after concentration is separated and purified by silica gel column chromatography using ethyl acetate/petroleum ether system to obtain light yellow liquid product intermediate 2 (723mg, 2.96mmol), with the yield of about 90%.

Intermediate 2 (700mg, 2.87mmol), 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 to the ice bath, and the ice bath was removed to react at room temperature for 24h. After TLC detection reaction is completed, carrying out reduced pressure distillation to remove the solvent, extracting the concentrated crude product by using an ethyl acetate/water system, collecting an organic phase, drying the organic phase by using anhydrous sodium sulfate, carrying out reduced pressure distillation to remove the solvent, dissolving a white solid obtained by purifying the concentrated product in a proper amount of anhydrous DCE, adding acetic acid, injecting acetaldehyde into a reaction system, stirring the mixture at room temperature for reaction for 30 minutes, adding sodium triacetoxyborohydride into an ice bath, and reacting at room temperature for 24 hours. Work-up was carried out as described above for the white solid to give intermediate 3 (890mg, 2.5mmol) as a pale yellow liquid in about 87% yield.

A commercially available compound 4 as a raw material (500mg, 3.37mmol) in methanol was added with an appropriate amount of Raney nickel and Boc anhydride, and reacted overnight under hydrogen protection with hydrogen displacement gas three times. And after the TLC detection reaction is completed, filtering the reaction system, collecting filtrate, carrying out reduced pressure distillation, purifying the concentrated product to obtain a white solid, adding the white solid into the reaction system of acetyl chloride and methanol solution under the ice bath condition, and reacting for 3 hours at room temperature. After the TLC detects that the reaction is complete, the extraction operation is carried out. Firstly, extracting by using dichloromethane/water, collecting an aqueous phase, adjusting the pH value of the aqueous phase to 3-4 by using 1M hydrochloric acid solution, extracting by using ethyl acetate, collecting an organic phase, and distilling under reduced pressure to obtain a hydrochloride of a product intermediate 5.

Intermediate 3 (787 mg, 210mmol), 5 (403.5 mg, 265mmol), HOBT and EDCI were added to the DMSO solution, and N-methylmorpholine was injected into the reaction system and stirred at room temperature overnight. After TLC detection reaction is completed, extraction is carried out by using ethyl acetate/water system, an organic phase is collected and dried by using anhydrous sodium sulfate, the solvent is removed by reduced pressure distillation, and the concentrated product is subjected to silica gel column chromatography separation and purification by using a methanol/dichloromethane system to obtain a light yellow solid intermediate 6 (1g, 210mmol), wherein the yield is about 95%. ¹ H 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)。

Synthesis of intermediate 13:

reagents and reaction conditions: (a) 2-butanone,7, morphholine, etOH,70 ℃; (b) Fmoc-Asp (Ot-Bu) -OH, EDCI, HOBT, DMF,40 ℃; (c) piperidine, CH ₂ Cl ₂ ；(d)AcOH,EtOH,80℃；(e)KOt-Bu,THF,PO(OMe) ₂ Cl,acethydrazide,n-BuOH；(f)20％TFA,CH ₂ Cl ₂ 。

As shown in the above reaction procedure, sulfur (2.79g, 86.86mmol) was added to a solution of compound 7 (4-chlorobenzoylacetonitrile) (1.24g, 6.9 mmol), 2-butanone and morpholine in ethanol, and the mixture was heated to 70 ℃. 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 subjected to silica gel column chromatography using ethyl acetate/petroleum ether system to obtain intermediate 8 (17.5g, 98.6%) as a yellow solid. To a solution of N, N-Dimethylformamide (DMF) were added Fmoc-Asp (Ot-Bu) -OH (3.72g, 9.03mmol), EDCI, and HOBt. The mixture was stirred at room temperature for 5 minutes, then intermediate 8 (2.00g, 7.53mmol) was added. The resulting mixture was stirred at 40 ℃ for 24 hours, followed by extraction with an ethyl acetate/water system, collection of the organic phase, drying with anhydrous sodium sulfate, removal of the solvent by distillation under reduced pressure, and separation and purification of the residue by silica gel column chromatography using an ethyl acetate/petroleum ether system to give intermediate 9 (2.01g, 54.0%) as a brown oil. Intermediate 9 (1.90g, 3.03mmol) and piperidine (0.56mL, 6.06mmol) were added to the dichloromethane solution. The mixture was stirred at room temperature for 30 minutes, then extracted with ethyl acetate/water system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography using ethyl acetate/petroleum ether system to give the free amine intermediate 10 (1.11g, 86.3%) as a yellow solid.

Intermediate 10 (1.02g, 2.33mmol) was dissolved in 10mL of ethanol solution, followed by addition of 1.5mL of acetic acid. After 2 hours, all solvents were removed under reduced pressure. The residue was subjected to silica gel column using ethyl acetate/petroleum ether systemChromatography separation and purification gave compound 11 (0.907g, 93.4%) as a white solid. A solution of potassium tert-butoxide in tetrahydrofuran (1.0M, 0.859mL, 0.859mmol) was added to a solution of intermediate 11 (300mg, 0.716mmol) in tetrahydrofuran (2 mL) at-78 deg.C. The reaction mixture was warmed to-10 ℃ and stirred at room temperature for 30 minutes. The reaction mixture was then cooled to-78 deg.C, then PO (OMe) 2Cl (0.154mL, 0.32mmol) was added. The resulting mixture was warmed to-10 ℃ over 1 hour. After addition of acetic acid hydrazide (117mg, 1.575mmol), the reaction mixture was stirred at room temperature for 1 hour. 1-Butanol (6 ml) was added and the reaction mixture was heated to 90 ℃. After 1 hour, all solvents were removed under reduced pressure. The residue was subjected to silica gel column chromatography using an ethyl acetate/petroleum ether system to give JQ1 (180mg, 88.1%) as a white solid. ¹ HNMR(400MHz,CDCl ₃ )，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)。

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

Synthesis of Compound D7

Reagents and reaction conditions: (a) TsCl, TEA, DMAP, CH ₂ Cl ₂ ；(b)4-hydroxyphenylboronic acid pinacol ester,K ₂ CO ₃ DMF,70 ℃; (c) Intermediate 6,Pd (PPh) ₃ ) ₄ ,K ₂ CO ₃ ,DMF,90℃；(d)20％TFA,CH ₂ Cl ₂ (ii) a (e) intermediate 13, HATU, DIPEA, DMF.

Add 4-Toluenesulfonylchloride, TEA and DMAP to a solution of Compound 14d (950mg, 5.96mmol) in dichloromethane. The reaction mixture was stirred at room temperature for 3 hours and then diluted with 100mL of dichloromethane. The organic phase was washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure.The residue was subjected to silica gel column chromatography using an ethyl acetate/petroleum ether system to give a colorless oil (1.04g, 46.0%). To a solution of the above colorless oil (1.04g, 2.80mmol) in DMF (5 mL) was added 4-hydroxyphenylboronic acid pinacol ester and potassium carbonate. The reaction mixture was stirred at 70 ℃ for 5 hours, then cooled to room temperature, extracted with ethyl acetate/water system, the organic phase collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography using ethyl acetate/petroleum ether system to give intermediate 15d (892mg, 78.6%) as a yellow oil. To a solution of intermediate 6 (100mg, 210. Mu. Mol) in DMF (2 mL) was added intermediate 15d (102mg, 251. Mu. Mol), potassium carbonate and Pd (PPh) ₃ ) ₄ . The mixture was purged and refilled with argon three times. The mixture was stirred at 90 ℃ for 9 hours, then extracted with ethyl acetate/water system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography using an ethanol/dichloromethane system to give 16d (120mg, 84.7%) as a yellow solid. Then, intermediate 16d (70mg, 82.9. Mu. Mol) was dissolved in dichloromethane, TFA (0.2. Mu.L) was added, and the resulting mixture was stirred at room temperature for 3 hours. Removal of the solvent under reduced pressure afforded the free amine without further purification. Intermediate 13 (43.0 mg, 107. Mu. Mol), DIPEA and HATU were added sequentially to the amine in DMF. The resulting mixture was stirred at room temperature for 10 hours, then extracted with 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 (78mg, 75.9%) as a yellow powder. ¹ H 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)δ169.4,169.1,163.0,162.9,158.2,155.1,149.8,149.5,148.8,142.7,139.5,136.9,136.7,135.2,132.2,132.1,131.9,130.7,130.1,129.8,129.6,128.4,127.6,122.5,121.6,120.4,114.8,107.3,67.5,66.3,57.8,53.9,41.2,38.3,37.7,34.9,30.3,29.0,28.4,22.9,18.9,18.2,14.5,14.0,12.7,12.7,11.3.HRMS(ESI,m/z)calcd for C ₅₃ H ₆₁ ClN ₈ O ₅ S[M+H] ⁺ :957.4247,found:957.4203.Purity:97.2％。

Example 2: synthesis of Compound D2

Reagents and reaction conditions: (a) Boc ₂ O,NaOH,THF,H ₂ O; (b) Intermediate 6,Pd (PPh) ₃ ) ₄ ,K ₂ CO ₃ ,DMF,90℃；(c)20％TFA,CH ₂ Cl ₂ (ii) a (d) intermediate 13, HATU, DIPEA, DMF.

To a solution of compound 17b (0.2g, 857. Mu. Mol) in THF (4 mL) was added Boc ₂ O and aqueous sodium hydroxide solution. The mixture was stirred at room temperature overnight. Extraction was then performed with an ethyl acetate/water system, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography purification using ethyl acetate/petroleum ether system to give the Boc protected intermediate (115mg, 40.2%) as a yellow solid. To a solution of intermediate 6 (100mg, 209. Mu. Mol) in DMF was added the newly prepared intermediate (83.9 mg, 251. Mu. Mol), K ₃ PO ₄ And Pd (PPh) ₃ ) ₄ . The mixture was purged and refilled with argon three times. The mixture was stirred at 90 ℃ for 9 hours, then extracted with ethyl acetate/water system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography using a methanol/dichloromethane system to give intermediate 18b (109mg, 86.2%) as a yellow solid. Intermediate 18b (50mg, 82.9. Mu. Mol) was then dissolved in dichloromethaneTo (1 mL) was added TFA (0.2. Mu.L), and the mixture was stirred at room temperature for 3 hours. Concentration of the solvent under reduced pressure afforded the free amine, which was used in the next step without further purification. To a solution of free amine (43.9mg, 87.3. Mu. Mol) in DMF (2 mL) was added intermediate 13 (35.0 mg, 97.3. Mu. Mol), DIPEA and HATU in that order. The resulting mixture was stirred at room temperature for 10 hours, then extracted with ethyl acetate/water system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography using a methanol/dichloromethane system to give Compound D2 (51mg, 66.0%) as a yellow powder. ¹ H 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＝5.7Hz,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 C NMR(101MHz,d6-DMSO)δ169.7,169.1,163.1,163.0,155.1,149.9,149.5,148.9,142.8,139.7,138.8,138.4,137.0,136.7,135.3,132.6,132.3,130.8,130.1,129.8,129.6,128.4,127.8,126.4,122.8,121.6,120.8,107.4,69.8,66.3,57.9,54.1,41.8,41.2,37.8,34.9,30.3,19.0,18.2,14.6,14.0,12.7,11.3.HRMS(ESI,m/z)calcd for C ₄₉ H ₅₃ ClN ₈ O ₄ S[M-H] ^- :883.3526,found:883.3556.Purity:97.7％。

Example 3: synthesis of Compound D1

Reagents and reaction conditions: (a) Boc ₂ O,NaOH,THF,H ₂ O; (b) Intermediate 6,Pd (PPh) ₃ ) ₄ ,K ₂ CO ₃ ,DMF,90℃；(c)20％TFA,CH ₂ Cl ₂ ；(d)13,HATU,DIPEA,DMF。

As shown in the above reaction process, the synthesis of compound D1 is shown 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),7.49(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)δ169.1,168.7,163.3,163.1,155.1,149.9,149.5,148.9,142.7,139.6,138.6,136.8,136.7,135.3,134.5,132.3,130.8,130.1,129.9,129.6,128.5,126.8,122.6,121.6,120.5,119.5,107.4,66.3,57.9,53.8,41.3,38.7,34.9,30.3,19.0,18.2,14.5,14.1,12.8,12.7,11.3.HRMS(ESI,m/z)calcd for C ₄₈ H ₅₁ ClN ₈ O ₄ S[M+H] ⁺ :871.3515,found:871.3481.Purity:96.5％。

example 4: synthesis of Compound D3

Reagents and reaction conditions: a) 4-methoxyarylphenylboronic acid, K ₂ CO ₃ ,Pd(PPh ₃ ) ₄ ,DMF,100℃；b)NaOH,EtOH,60℃。

As shown in the above reaction process, the synthesis of compound D3 is shown 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,2H),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＝4.8Hz,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 ₅₅ ClN ₈ O ₄ S[M–H] ^- :897.3683,found:897.3662.Purity:97.5％。

example 5: synthesis of Compound D4

Reagents and reaction conditions: (a) TsCl, TEA, DMAP, CH ₂ Cl ₂ ；(b)4-hydroxyphenylboronic acid pinacol ester,K ₂ CO ₃ DMF,70 ℃; (c) Intermediate 6,Pd (PPh) ₃ ) ₄ ,K ₂ CO ₃ ,DMF,90℃；(d)20％TFA,CH ₂ Cl ₂ (ii) a (e) intermediate 13, HATU, DIPEA, DMF.

As shown in the above reaction procedure, the synthesis of compound D4 is described in the synthesis 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),2.59(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). ¹³ C NMR(101MHz,d6-DMSO)δ170.0,169.1,167.0,163.0,158.1,155.1,149.8,149.5,148.8,142.7,139.5,136.9,136.7,135.1,132.5,132.3,132.1,131.7,131.6,130.7,130.1,129.8,129.5,129.5,128.6,128.3,127.8,122.6,121.6,120.6,114.9,107.3,67.4,66.8,66.3,57.9,53.9,41.3,38.4,38.1,37.5,34.9,30.3,29.9,29.8,28.4,23.4,23.2,22.4,18.9,18.2,14.5,14.0,13.9,12.7,12.7,11.3,10.9,10.8.HRMS(ESI,m/z)calcd for C ₅₀ H ₅₅ ClN ₈ O ₅ S[M-H] ^- :913.3632,found:913.3611.Purity:100％。

example 6: synthesis of Compound D5

Reagents and reaction conditions: (a) TsCl, TEA, DMAP, CH ₂ Cl ₂ ；(b)4-hydroxyphenylboronic acid pinacol ester,K ₂ CO ₃ DMF,70 ℃; (c) Intermediate 6,Pd (PPh) ₃ ) ₄ ,K ₂ CO ₃ ,DMF,90℃；(d)20％TFA,CH ₂ Cl ₂ (ii) a (e) intermediate 13, HATU, DIPEA, DMF.

As shown in the above reaction procedure, the synthesis of compound D5 is described in the synthesis of compound D7. Compound D7: ¹ H 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)δ169.6,169.1,163.0,162.9,158.1,155.1,149.8,149.4,148.8,142.7,139.5,136.9,136.7,135.2,132.2,131.9,130.7,130.0,129.8,129.6,128.4,127.6,122.5,121.6,120.5,114.9,107.3,66.3,65.3,57.9,53.9,41.2,37.7,35.4,34.9,30.3,29.1,18.9,18.2,14.5,14.0,12.7,12.6,11.3.HRMS(ESI,m/z)calcd for C ₅₁ H ₅₇ ClN ₈ O ₅ S[M–H] ^- :927.3788,found:927.3778.Purity:99.5％。

example 7: synthesis of Compound D6

Reagents and reaction conditions: (a) TsCl, TEA, DMAP, CH ₂ Cl ₂ ；(b)4-hydroxyphenylboronic acid pinacol ester,K ₂ CO ₃ DMF,70 ℃; (c) Intermediate 6,Pd (PPh) ₃ ) ₄ ,K ₂ CO ₃ ,DMF,90℃；(d)20％TFA,CH ₂ Cl ₂ (ii) a (e) intermediate 13, HATU, DIPEA, DMF.

As shown in the above reaction procedure, the synthesis of compound D6 is described in the synthesis 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). ¹³ C 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％。

example 8: synthesis of Compound D8

Reagents and reaction conditions: (a) 4-methoxyarylphenylboronic acid, K ₂ CO ₃ ,Pd(PPh ₃ ) ₄ ,DMF,90℃；(b)NaOH,EtOH,60℃；(c)tert-butyl(2-(2-aminoethoxy)ethyl)carbamate,HATU,DIPEA,DMF；(d)20％TFA,CH ₂ Cl ₂ (ii) a (e) intermediate 13, HATU, DIPEA, DMF; (f) Boc ₂ O,NaOH,THF,H ₂ O。

To intermediate 6 (1g, 2)10 mmol) of DMF (20 mL) was added 4-methoxycarbonylphenylboronic acid, K ₂ CO ₃ And Pd (PPh) ₃ ) ₄ . The solution was purged and refilled 3 times with argon. After stirring at 90 ℃ for 9 hours, extraction was carried out with ethyl acetate/water system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography using a methanol/dichloromethane system to give the coupled product as a yellow solid (0.82g, 73.5%). The product obtained is hydrolyzed in sodium hydroxide solution containing ethanol at 60 ℃ to give intermediate 19. To a solution of tert-butyl (2- (2-aminoethoxy) ethyl) carbamate in DMF (2 mL) was added intermediate 19 (50.7 mg, 97.9. Mu. Mol), DIPEA and HATU in that order. The resulting mixture was stirred at room temperature for 10 hours, then extracted with ethyl acetate/water system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography using a methanol/dichloromethane system to give a yellow powder (59mg, 85.6%). Then, the yellow powder (59mg, 97.9. Mu. Mol) was dissolved in dichloromethane (1 mL), TFA (0.2. Mu.L) was added, and the mixture was stirred at room temperature for 3 hours. Removal of the solvent under reduced pressure afforded the free amine, which was used in the next step without further purification. The above amine (26.0 mg, 43.1. Mu. Mol) was dissolved in DMF (2 mL) solution, and intermediate 13 (17.3 mg, 43.1. Mu. Mol), DIPEA and HATU were added in that order. The resulting mixture was stirred at room temperature for 10 hours, then extracted with ethyl acetate/water system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography using a methanol/methylene chloride system to give D8 (27.0 mg, 65.6%) as a yellow powder. ¹ H 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＝8.4Hz,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＝5.9Hz,2H),3.28–3.21(m,6H),3.10(dd,J＝13.8,6.8Hz,2H),3.01(m,1H),2.58(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 H ₆ 0ClN ₉ O ₆ S[M–H]-:984.4003,found:984.4014.Purity:97.3％。

Example 9: synthesis of Compound D9

Reagents and reaction conditions: (a) Boc ₂ O,NaOH,THF,H ₂ O; (b) intermediate 18a, hatu, dipea, dmf; (c) 20% of TFA ₂ Cl ₂ (ii) a (d) intermediate 13, HATU, DIPEA, DMF.

To a solution of compound 20a (0.2g, 1.38mmol) in THF (4 mL) was added Boc ₂ O and aqueous sodium hydroxide solution. The mixture was stirred at room temperature overnight, then extracted with 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 ethyl acetate/petroleum ether system to give intermediate 21a (378mg, 100%) as a yellow solid. The Boc group of intermediate 18a (30mg, 122. Mu. Mol) was removed under acidic conditions to give the free amine. Compound 21a (59.8mg, 122 μmol), HATU and DIPEA were added successively to a DMF solution containing the free amine, and the resulting mixture was stirred at room temperature for 10 hours, followed by extraction with an ethyl acetate/water system, collection of the organic phase, drying over anhydrous sodium sulfate, filtration and concentration under reduced pressure. The residue was subjected to silica gel column chromatography purification using a methanol/dichloromethane system to give intermediate 22a (41.0 mg, 46.8%) as a yellow powder. Then, intermediate 22a (41.0 mg, 122. Mu. Mol) was dissolved in dichloromethane (1 mL), TFA (0.2. Mu.L) was added, and the mixture was stirred at room temperature for 3 hours. Removal of the solvent under reduced pressure gave the free amine, which was used without further purificationAnd then the next operation is carried out. The above amine (9.0 mg, 22.5. Mu. Mol) was dissolved in DMF (2 mL), and intermediate 13 (13.0 mg, 22.5. Mu. Mol), DIPEA and HATU were added in that order. The resulting mixture was stirred at room temperature for 10 hours, then extracted with ethyl acetate/water system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography purification using a methanol/dichloromethane system to give Compound D9 (13.0 mg, 58.0%) as a yellow powder. ¹ H 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),7.49(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＝7.3Hz,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)δ169.3,169.1,163.0,155.1,154.4,149.8,149.5,148.8,148.8,148.4,139.6,138.7,136.8,134.3,132.2,132.2,131.5,130.7,130.1,129.6,128.6,128.5,126.7,120.5,119.4,109.4,107.4,73.8,66.3,57.9,53.9,41.2,40.8,38.4,37.7,36.4,34.9,34.5,31.3,31.2,30.3,30.0,29.1,29.0,28.4,26.1,25.0,18.9,18.6,18.2,14.5,14.0,13.5,12.7,12.6,11.3.HRMS(ESI,m/z)calcd for C ₅₅ H ₆₄ ClN ₉ O ₅ S[M–]:997.4445,found:997.4487.Purity:95.0％。

Example 10: synthesis of Compound D10

Reagents and reaction conditions: (a) Boc ₂ O,NaOH,THF,H ₂ O; (b) intermediate 18a, hatu, dipea, dmf; (c) 20% of TFA ₂ Cl ₂ (ii) a (d) intermediate 13, HATU, DIPEA, DMF.

The synthesis of reference compound D9 gives compound D10. 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＝4.3Hz,2H),4.16–4.10(m,1H),4.02(d,J＝6.4Hz,1H),3.83(d,J＝10.3Hz,2H),3.28–3.20(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). ¹³ C NMR(101MHz,d6-DMSO)δ171.3,169.1,167.0,163.0,163.0,155.1,149.8,149.5,148.8,142.7,139.6,138.8,136.7,135.2,132.2,131.7,131.6,130.7,130.1,129.8,129.6,128.7,128.4,126.7,121.6,119.4,107.4,71.2,67.4,66.3,57.9,53.9,41.3,38.5,38.1,37.7,36.4,34.9,34.3,30.3,29.8,29.2,28.7,28.6,28.4,27.2,26.3,25.1,23.3,22.4,19.0,18.9,18.2,14.5,14.0,13.9,12.7,12.7,11.3,11.00,10.8.HRMS(ESI,m/z)calcd for C ₅₆ H ₆₆ ClN ₉ O ₅ S[M-H] ^- :1010.4523,found:1010.4562.Purity:95.0％。

example 11: synthesis of Compound D11

The reagent and the reaction condition are (a) Boc ₂ O,NaOH,THF,H ₂ O; (b) intermediate 18a, HATU, DIPEA, DMF; (c) 20% of TFA ₂ Cl ₂ (ii) a (d) intermediate 13, HATU, DIPEA, DMF.

Reference synthesis of compound D9 compound D11 was synthesized. Compound D11: ¹ H 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,2H),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)δ169.8,169.3,169.2,163.1,155.2,149.9,149.7,148.9,142.8,139.6,138.7,136.8,136.8,135.3,134.5,132.3,130.8,130.2,129.9,129.6,128.5,126.8,122.6,121.7,120.5,119.4,107.5,69.8,69.8,69.7,69.7,69.3,66.7,66.4,59.8,57.9,53.9,41.3,38.7,37.6,37.2,34.9,30.4,20.8,19.0,18.2,14.6,14.1,14.0,12.8,12.7,11.3.HRMS(ESI,m/z)calcd for C ₅₆ H ₆₆ ClN ₉ O ₅ S[M-H] ^- :1072.4527,found:1072.4544.Purity:95.0％。

example 12: synthesis of Compound D12

Reagents and reaction conditions: (a) TsCl, TEA, DMAP, CH ₂ Cl ₂ ；(b)4-hydroxyphenylboronic acid pinacol ester,K ₂ CO ₃ DMF,70 ℃; (c) Intermediate 6,Pd (PPh) ₃ ) ₄ ,K ₂ CO ₃ ,DMF,90℃；(d)20％TFA,CH ₂ Cl ₂ (ii) a (e) intermediate 13, HATU, DIPEA, DMF.

Reference synthesis of compound D7 compound D12 was synthesized. LRMS (ESI, m/z) calcd for C ₅₂ H ₆₀ ClN ₈ O ₆ S[M+H] ⁺ :959.40,found:959.50。

Example 13: protein immunoblotting (Western Blot) to detect the inhibitory effect of compounds on EZH2 and BRD4

(1) Extraction of total protein: the cells treated with DMSO or drug were transferred to a centrifuge tube, harvested by centrifugation and washed twice with PBS, and transferred to a 1.5mL EP tube. 100-150uL of lysis buffer was added to each EP tube in terms of cell number, and the mixture was mixed and left at room temperature for 10 minutes. The EP was then placed on a thermostatted metal bath and cooked for 15 minutes at 95 ℃.

(2) Protein content determination: protein content determination was performed with reference to Pierce BCA protein quantification kit instructions.

(3) And (3) preparing gel by SDS-PAGE gel electrophoresis: and (3) placing and airing the cleaned glass plate, and preparing 12% of separation glue and 5% of concentrated glue according to the formula in sequence. And (3) adding 4xSDS loading buffer to the protein sample in proportion to make the final concentration of SDS be 1x. After mixing uniformly, the sample is put into a constant temperature metal bath and boiled for 10 minutes at 95 ℃. After calculation, 10ug of protein sample was added to each well in equal amounts. Electrophoresis: the voltage of the gel concentration part is set to be 80V, and after the band enters the separation gel, the constant voltage is adjusted to be 120V until the bromophenol blue band runs to the bottom end.

(4) Film transfer: preparing a PVDF membrane soaked by methanol, filter paper and black sponge soaked by the membrane transferring solution, separation glue, a membrane transferring clamp and the filter paper, and placing the filter paper in a tray filled with the membrane transferring solution. The rotating film clamp is black, the sponge, the filter paper, the separating gel PVDF film, the other piece of filter paper and the sponge are sequentially placed on the rotating film clamp, and a glass rod is used for carefully removing bubbles in the process. Clamping and placing into a film rotating groove for fixing. The wet transfer method is adopted, the constant current is 0.25A, and the film is transferred for 1-3h according to different molecular weights.

(5) Blocking the immunoblot: the PVDF membrane after membrane conversion is put into a plastic box containing 5% skimmed milk powder, sealed at room temperature for 1 hour, and washed with TBST for 10 minutes. Incubating primary antibody: diluting primary antibody with primary antibody dilution solution of Biyunstian company according to the instruction proportion, putting the transfer printing film into the diluted primary antibody, and incubating overnight in a shaking table at 4 ℃. Hatching a secondary antibody: the following day the membranes were washed with TBST for 10 min X3 times. The transfer film was then transferred to a diluted secondary antibody, incubated at room temperature for 1 hour, and then eluted again with TBST for 10 minutes × 3 times. ECL development: and uniformly delivering the prepared ECL luminous liquid to a strip to be detected, placing the strip for one minute in a dark place, developing by using a chemiluminescence imaging system, and taking a picture.

Example 14: killing effect of MTS detection compound on tumor cells

Taking pancreatic cancer cell strain AspC-1 and lung cancer cell strain A549 colorectal cancer cell strain HCT116 as examples, taking cells in logarithmic phase, centrifuging, discarding supernatant, washing twice with PBS, then counting by resuspension, and evenly inoculating the cells on a 96-well plate at the rate of 500-1500 cells/100 uL per well. Duplicate wells and blanks were set up. The drug was diluted with fresh medium at the desired concentration, 100. Mu.L per well was added to a 96-well plate with cells plated, and the plate was placed in 3Containing CO at 7 ℃ ₂ The incubator of (1) was incubated for 6 days. The reaction was continued for 4 hours by adding 20. Mu.L of MTS solution to the 96-well plate. The absorbance (OD) of the different wells was measured at 490nm wavelength using a microplate reader.

Example 15: test for anti-tumor effect of compound D7 in human lung cancer nude mouse subcutaneous transplantation tumor model

(1) Tumor cells in logarithmic growth phase were collected, washed 2 times with PBS solution, cells were resuspended in serum-free RPMI-1640 medium, the number of desired inoculated cells was calculated, a549 model cells: 2.5X 10 ⁶ /100μL；

(2) Injecting 100 mu L of cell suspension into the right armpit of a nude mouse aged 8 weeks;

(3) When the tumor volume reaches 50-100mm ³ Randomly dividing the mice into 4 groups, namely a solvent control group and 3 groups of drug treatment groups;

(4) Administration: drug-treated groups JQ1 (30 mg/kg), EPZ6438 (30 mg/kg) and D7 (60 mg/kg), solvent control group DMSO (10%, DMSO: castor oil: PBS buffer =1: 8), drug or solvent was intraperitoneally injected at 200 μ L/tube per day;

(5) Two-day tumor growth measurement was started when subcutaneous tumor formation was visible to the naked eye, and the length (L) and width (W) of the tumor body were measured and recorded using a vernier caliper, and the tumor volume calculation formula was V (mm) ³ )＝(L×W ² ) A/2, drawing a tumor growth curve according to the volume of the tumor body;

(6) When the tumor volume of the control group reaches 1000-2000mm ³ The experiment was terminated, the nude mice were sacrificed using dislocation, tumor tissue was left, photographed, recorded and tumor weight was weighed.

To further demonstrate the effect of the compounds of the invention on EZH2 protein levels. In the invention, after 1 mu M of compound D1-D11 or DMSO is used for treating AsPC-1 cells for 48H, the levels of H3K27me3 and c-Myc are measured by using a protein immunoblot (Western blot) experiment, and histone H3 and Actin are used as controls. As shown in figure 1, the results show that compound D7 most significantly reduced H3K27me3 protein levels, and also reduced c-Myc levels.

Further, as shown in figure 2, the effect of varying concentrations of compound D7 of the present invention on EZH2 and BRD4 protein levels. In the invention, after the compound with the specified concentration is treated on the AspC-1 cells for 48 hours, the levels of H3K27me3 and c-Myc are measured by using a protein immunoblotting (Western blot) experiment, and histone H3 and Actin are used as controls. The results show that D7 can inhibit EZH2 and BRD4 enzyme function in a concentration-dependent manner.

Figure 3 shows the effect of compound D7 of the present invention and the existing EZH2 inhibitor EPZ6438, the BRD4 inhibitor JQ1 on the viability of different solid tumor cells. The cell survival rate is measured by using MTS after the compound under the specified concentration is respectively treated on a pancreatic cancer cell strain AsPC-1 and a lung cancer cell strain A549 colorectal cancer cell strain HCT116 days. The results show that compound D7 of the present invention has a stronger killing effect than EPZ6438, JQ 1.

As shown in fig. 4, compound D7 of the present invention has more potent antitumor effect than EPZ6438, JQ1 or both in a nude mouse subcutaneous graft tumor model of a 549.

FIG. 5 shows the antitumor results of different compounds in different tumor cell lines, and it can be seen that compound D7 of the present invention has more potent antitumor effect than EPZ6438, JQ1 or the combination of both.

Table 1 shows the IC of different compounds D1-D11 and EPZ5438, JQ1 in AsPC-1 and H460 cells ₅₀ (μm)。

TABLE 1 IC of different compounds in AsPC-1 and H460 cells ₅₀

From the data in table 1 it can be seen that:

1) When the length of the main chain is 1-14 atoms, the activity of the double-target inhibitor is obviously improved compared with that of EPZ 6438;

2) When the length of the main chain is 3-7 atoms, the activity of the double-target inhibitor is also obviously improved compared with JQ 1;

3) linker length significantly affects the anti-tumor activity of the compound, specifically, the activity of the dual-target inhibitor increases with the increase in the length of the main chain when the length of the main chain is 1 to 7 atoms, the activity of the dual-target inhibitor is significantly improved compared to JQ1 and EPZ6438 when the length of the main chain is 3 to 7 atoms, while the activity is better when the length is 5 to 7 atoms, and the optimal activity is expected when the length is 6 to 7 atoms.

The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.

Claims (10)

1. A dual-target EZH2 and BRD4 inhibitor, characterized by: the double-target inhibitor can inhibit two proteins, namely EZH2 and BRD4 at the same time, and is obtained by coupling a structural fragment for inhibiting the BRD4 protein and a structural fragment for inhibiting the EZH2 protein through a linker, wherein the main chain of the linker contains 1-14 atoms.

2. The dual-target inhibitor of claim 1, wherein: the main chain of the linker contains 5-7 atoms; and/or

The structural fragment for inhibiting the EZH2 protein is selected from EPZ6438, GSK126, PF-06821497 or CPI-1205; and/or

The structural fragment for inhibiting the BRD4 protein is selected from (+) -JQ1, I-BET762, OTX-015, CPI-0610 or TEN-010.

3. The dual-target inhibitor of claim 1, wherein: the main chain of the linker is an alkyl chain, a polyethylene glycol chain or an ether-containing alkyl chain; and/or

At least one end of the backbone is coupled via-NH-to a BRD 4-inhibiting structural fragment or a structural fragment inhibiting the EZH2 protein; and/or

The structural general formula of the double-target inhibitor is shown as formula I:

4. the dual-target inhibitor of any one of claims 1-3, wherein: the linker is selected from one of formulas X1-X15:

/>

5. the dual-target inhibitor of claim 3, wherein: the structural formula is one of formulas D1-D12:

/>

in formulas D1-D12, is selected>

6. A pharmaceutical composition comprises an active ingredient and an auxiliary material, and is characterized in that: the active ingredient comprises the EZH2 and BRD4 double-target inhibitor as defined in any one of claims 1 to 5, or one of the pharmaceutically acceptable salts, crystals and solvates thereof.

7. The pharmaceutical composition of claim 6, wherein: it can be used for treating tumor.

Use of a dual-target inhibitor of EZH2 and BRD4 according to any one of claims 1 to 5 for the preparation of a medicament for the treatment of a tumour.

9. Use according to claim 8, characterized in that: the tumor is a solid tumor or a blood tumor.

10. Use according to claim 9, characterized in that: the solid tumor is selected from lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, renal cancer, uterine cancer, prostate cancer or melanoma; the hematological tumor is selected from leukemia or lymphoma.

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