CDK9 / EZH2 double-target inhibitor as well as pharmaceutical composition and application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to a CDK9 / EZH2 double-target inhibitor as well as a pharmaceutical composition and application thereof, and the CDK9 / EZH2 double-target inhibitor is a compound obtained by coupling a structural fragment for inhibiting a CDK9 protein and a structural fragment for inhibiting an EZH2 protein through a linker. According to the CDK9 and EZH2 double-target inhibitor and the application of the CDK9 and EZH2 double-target inhibitor in tumor resistance, a structural fragment for inhibiting a CDK9 protein and a structural fragment for inhibiting an EZH2 protein are subjected to structural modification and coupled together through a linker, and the length and the connection mode of the linker are adjusted.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a CDK9 / EZH2 dual-target inhibitor and a pharmaceutical composition and application thereof. Background Art

[0002] Cyclin-dependent kinases (CDKs) are members of the serine / threonine kinase family and play an important role in regulating cell proliferation, differentiation, DNA repair and apoptosis. CDKs are generally divided into cell cycle CDKs (CDK1, CDK2, CDK4, CDK6) and transcription CDKs (CDK7-9, CDK11-13, CDK19) according to their specific functions. Among the transcription-related CDKs, CDK9 has attracted special attention from drug developers because it regulates transcription elongation without affecting the cell cycle process. CDK9 can form heterodimers with cyclins (T1, T2 and K), namely positive transcription elongation factor b (P-TEFb). Activated P-TEFb can phosphorylate Serine 2 (Ser2) of the C-terminal domain (CTD) of RNA polymerase II (RNAPII), thereby allowing the elongation phase of transcription to proceed normally. In-depth studies have shown that CDK9 has major signal dysregulation in several tumor cells, including malignant hematological tumors and solid tumors. Mechanistically, inhibition of CDK9 prevents the phosphorylation of RNAPIICTD Ser2 and induces dysregulation of MYC and Mcl-1 protein levels, which has been demonstrated in various malignancies; including adult T-cell leukemia / lymphoma (ATL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), etc.

[0003] Most of the CDK9 inhibitors currently in clinical research are pan-inhibitors, and only a few selective CDK9 inhibitors have entered clinical research (AZD-4573, KB-0742, GFH-009 and VIP-152); and these compounds are basically in clinical phase I / I research or have not made progress, and there are still limitations in research on solid tumors. Studies have found that CDK9 inhibitors can cause abnormal increases in H3K27me3 in DLBCL cell lines, and the combined use of EZH2 inhibitors can inhibit the upregulation of H3K27me3 and achieve a synergistic anti-tumor effect. The CDK9 and EZH2 dual-target inhibitors of the present invention have the function of dual-target inhibition and can be used to treat related tumors. Summary of the invention

[0004] In view of the deficiencies of the above-mentioned prior art, the present invention provides a CDK9 / EZH2 dual-target inhibitor and a pharmaceutical composition and application thereof. The specific technical scheme is as follows:

[0005] The first aspect of the present invention provides a CDK9 / EZH2 dual-target inhibitor, the general structural formula of which is structural formula I:

[0006]

[0007] in:

[0008] The CDK9 pharmacophore represents the ligand that binds to the target protein CDK9;

[0009] Linker represents a covalently linked moiety;

[0010] The EZH2 pharmacophore represents the ligand that binds to the target protein EZH2.

[0011] The CDK9 pharmacophore and the EZH2 pharmacophore moiety can each be independently selected from small molecule compounds, in some embodiments.

[0012] CDK9 pharmacophore part:

[0013] This part represents a part that can bind to CDK9. Generally, the CDK9 pharmacophore is a CDK9 protein inhibitor or a structural fragment having CDK9 protein inhibitory effect. In some embodiments, the structural fragment of the CDK9 pharmacophore is preferably KB-0742, KI-ARV-03, AZD-4573 or SNS-032;

[0014]

[0015] EZH2 pharmacophore part:

[0016] The part refers to a part that can interact with the EZH2 protein. The EZH2 pharmacophore is an EZH2 protein inhibitor or a structural fragment having an EZH2 protein inhibitory effect. In some embodiments, the structural fragment of EZH2 is preferably EPZ6438, GSK126 or C24;

[0017]

[0018] Linker:

[0019] Linker refers to a connecting part that couples the CDK9 pharmacophore and the EZH2 pharmacophore, which can be a chemical bond. Linker can be rigid or flexible. In some preferred embodiments, Linker is flexible.

[0020] In some embodiments, Linker is a linear or branched or cyclic structure comprising 1-30, preferably 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) carbon atoms, each of which may be optionally replaced by one or more (e.g., 2, 3, 4, 5, 6), in particular one heteroatom; wherein the heteroatom is selected from oxygen, sulfur, nitrogen, phosphorus, preferably oxygen, sulfur or nitrogen, more preferably oxygen or nitrogen, in particular oxygen. Each carbon atom may also be optionally replaced by -C(=O)-, -C(=S)-, -S(=O)-, -SO2- and a 3- to 6-membered ring having 0 to 4 heteroatoms, the heteroatoms being selected from oxygen, sulfur, nitrogen, phosphorus.

[0021] In some embodiments, Linker is preferably the following structure:

[0022]

[0023] wherein n is 1, 2, 3, 4, 5, 6, 7 or 8, and m is 1, 2, 3 or 4; and the site of covalent attachment may be any suitable site.

[0024] In some embodiments, the CDK9 pharmacophore is mainly covalently linked to the linker through a nitrogen atom; similarly, the EZH2 pharmacophore can also be covalently linked to the linker through a carbon atom or a heteroatom; the heteroatom is selected from oxygen, sulfur, nitrogen, and phosphorus. Those skilled in the art can select appropriate reaction sites for connection according to the structures of these three parts.

[0025] Furthermore, the structure of the CDK9 / EZH2 dual-target inhibitor is selected from any one of structures I-VII:

[0026]

[0027] wherein n is 1, 2, 3, 5, 6, 7 or 8, and m is 1, 2, 3 or 4;

[0028] The R group is preferably:

[0029]

[0030] In some embodiments, the structure of the CDK9 / EZH2 dual-target inhibitor is:

[0031]

[0032]

[0033]

[0034]

[0035] The second aspect of the present invention provides a pharmaceutical composition comprising one or more of the CDK9 / EZH2 dual-target inhibitors or pharmaceutically acceptable salts thereof according to the first aspect of the present invention and an optional pharmaceutically acceptable carrier.

[0036] Pharmaceutically acceptable carriers refer to conventional drug carriers in the pharmaceutical field, such as: diluents, such as water, etc.; fillers, such as starch, sucrose, etc.; binders, such as cellulose derivatives, alginates, gelatin, polyvinyl pyrrolidone; wetting agents, such as glycerol; disintegrants, such as agar, calcium carbonate and sodium bicarbonate; absorption promoters, such as quaternary ammonium compounds; surfactants, such as cetyl alcohol; adsorption carriers, such as kaolin and bentonite; lubricants, such as talc, calcium stearate, magnesium stearate and polyethylene glycol, etc. In addition, other adjuvants, such as flavoring agents and sweeteners, etc., may be added to the above-mentioned pharmaceutical composition.

[0037] The third aspect of the present invention provides the use of the CDK9 / EZH2 dual-target inhibitor in the preparation of drugs for treating tumors.

[0038] Furthermore, the tumor is a solid tumor or a hematological tumor.

[0039] Furthermore, the solid tumor is selected from lung cancer, colorectal cancer, pancreatic cancer, gastric cancer, bladder cancer, kidney cancer, uterine cancer, prostate cancer or melanoma; the blood tumor is selected from lymphoma, leukemia or multiple myeloma. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The impact of the embodiments of the present invention on the downstream signal pathway;

[0041] Among them, 032 is the CDK9 inhibitor SNS-032; C24 is an EZH2 inhibitor; U-2932 is a wild-type B lymphoma cell line; Karpas422 is a mutant B lymphoma cell line;

[0042] Figure 2 This is a study on the effect of compound D16 of the present invention on inducing cell apoptosis and DNA damage. DETAILED DESCRIPTION

[0043] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0044] In the following examples, the intermediates are numbered the same, i.e., the intermediates with the same number have the same structure. Unless otherwise specified, the raw materials, reagents, methods, etc. used in the examples are all conventional raw materials, reagents, methods in the art.

[0045] Unless otherwise specified, all reaction solvents were purified according to conventional methods. Silica gel (200-300 mesh) for column chromatography was produced by Qingdao Ocean Chemical Branch. GF254 high-efficiency plate was used for thin layer chromatography, which was produced by Yantai Chemical Research Institute. Unless otherwise specified, all solvents were analytically pure reagents, and all reagents used were purchased from Sinopharm Chemical Reagent Co., Ltd. The color was developed using iodine, ultraviolet fluorescence and other methods. The organic solvent was removed under reduced pressure in a rotary evaporator.

[0046] In the following examples, H NMR spectra were recorded using Bruker AMX-400 and Bruker AMX-500 NMR instruments, and the unit of chemical shift δ is ppm. Multiplicities are recorded using the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet J, coupling constant (hertz).

[0047] In the following examples,

[0048] Synthesis of intermediate 6:

[0049]

[0050] Step a

[0051] Weigh compound 1 (1 g, 3.5 mmol), 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (645 mg, 4.3 mmol), HOBT (572 mg, 4.3 mmol) and EDCI (1.2 g, 6.4 mol), add DIPEA 1.9 mL, dissolve with anhydrous dichloromethane, react at room temperature for 3 h, and the reaction is basically completed by TLC monitoring. The organic phase is separated with ethyl acetate (50 mL) and water (25 mL), and the organic phases are combined after three extractions, washed with saturated brine, and then dried over anhydrous sodium sulfate. Column chromatography separation and purification (DCM: MeOH = 20: 1) finally gave 1.3 g of compound 3. Yield: 88%; Properties: white solid. 1 H NMR (400MHz, DMSO-d6) δ11.52(s,1H),8.61(s,1H),8.37(s,1H),8.20(d,J=1.3Hz,1H),7.70(d,J=1.5Hz,1H),5.89(s ,1H),5.09–5.03(m,1H),4.34(d,J=4.9Hz,2H),2.21(s,3H),2.13(s,3H),1.46(d,J=6.6Hz,6H).ESI-MS:m / z=417.307

[0052] Step b

[0053] Weigh 4-Boc-1-(5-bromo-2-pyridyl)piperazine (2g, 5.8mmol), biboronic acid pinacol ester (3g, 8.8mmol), tetrakis(triphenylphosphine)palladium (338mg, 0.3mmol) and potassium acetate (1.7g, 17.5mmol) in a round-bottom flask, add 50mL of 1,4-dioxane to dissolve the raw materials, replace with nitrogen three times and protect with nitrogen; react at 100℃ for 4h, and monitor the reaction by TLC until the raw materials are basically completely converted. Remove the excess dioxane first, then extract with ethyl acetate and water three times, combine the organic layers, wash with saturated NaCl aqueous solution, and dry with anhydrous sodium sulfate. Column chromatography separation and purification (PE:EA=10:1) finally obtain 1.7g of compound 4. Yield: 75%; Properties: white solid. 1 H NMR (400MHz, DMSO-d6) δ8.33(d,J=2.0Hz,1H),7.70(dd,J=8.6,2.0Hz,1H),6.79(d,J=8.6Hz,1H),3 .56(dd,J=6.7,3.9Hz,4H),3.39(d,J=10.6Hz,4H),1.42(s,9H),1.26(s,12H).ESI-MS:m / z=391.512

[0054] Step c

[0055] Compound 3 (1.3 g, 3.1 mmol), compound 4 (1.5 g, 3.7 mmol), Pd(dppf)Cl2 (113 mg, 0.2 mmol) and anhydrous potassium carbonate (1.3 g, 9.3 mmol) were dissolved in 30 mL of 1,4-dioxane, replaced with nitrogen three times, and protected with nitrogen; reacted at 80°C for 3 h, and the reaction was basically completed after TLC monitoring. The excess dioxane was first removed, and then extracted with ethyl acetate and water three times. The organic layers were combined and washed with saturated NaCl aqueous solution and dried over anhydrous sodium sulfate. Column chromatography was separated and purified (PE:EA=5:1), and finally 1.8 g of compound 5 was obtained. Yield: 96%; Properties: milky white solid. 1H NMR(400MHz,DMSO-d6)δ11.52(s,1H),8.67(s,1H),8.59(s,1H),8.35(s,1H),8.08(d, J=10.6Hz,2H),7.84(s,1H),6.98(d,J=8.9Hz,1H),5.89(s,1H),5.76(d,J=1.1Hz,1H), 5.13(p,J=6.5Hz,1H),4.38(d,J=4.9Hz,2H),3.56(d,J=5.4Hz,4H),3.45(s,4H),2.21( s,3H),2.12(s,3H),1.49(d,J=6.5Hz,6H),1.43(d,J=1.1Hz,9H).ESI-MS:m / z=599.736

[0056] Step d

[0057] Dissolve 1.8 g of compound 5 in 5 mL of methanol, then add 2.5 mL of 4 M hydrochloric acid dioxane solution dropwise in an ice bath, warm to room temperature and stir for 1 h. TLC monitoring shows that the reaction is basically complete. Directly spin dry the solvent to obtain 2.1 g of crude product 6. Properties: yellow solid powder. 1 H NMR (400MHz, DMSO-d6) δ8.66(d,J=2.6Hz,1H),8.60(s,1H),8.35(s,1H),8.06(s,2H),7.84(s,1H),6.95(d,J=9.0Hz,1H),5.89( s, 1H), 4.38 (d, J = 4.8Hz, 2H), 3.53 (s, 4H), 2.87 (s, 4H), 2.21 (s, 3H), 2.12 (s, 3H), 1.49 (d, J = 6.6Hz, 6H). ESI-MS: m / z = 499.736.

[0058] Example 1: Synthesis of Compound D1

[0059]

[0060] Step a

[0061] Weigh compound 6 (100 mg, 0.2 mmol) and methyl 4-bromobutyrate (54 mg, 0.3 mmol) in a round-bottom flask, then add K2CO3 (83 mg, 0.6 mmol), react at room temperature for 2 h, and monitor the reaction until it is basically complete. Use DCM (20 mL) and water (10 mL) to separate the organic phase, extract three times, combine the organic layers and wash with saturated NaCl solution, dry over anhydrous sodium sulfate, and separate and purify by column chromatography to obtain 103 mg of compound 7. Yield: 87%; Properties: white foamy solid.1 H NMR(500MHz,Chloroform-d)δ9.86(s,1H),8.43(d,J=1.5Hz,1H),8.32–8.23(m,3H),8.05(d,J=1. 4Hz,1H),7.76(dd,J=7.5,1.5Hz,1H),6.69(d,J=7.5Hz,1H),6.03(p,J=1.0Hz,1H),4.82(hept,J= 6.9Hz,1H),4.19(d,J=8.4Hz,2H),3.71–3.62(m,6H),2.69(t,J=7.1Hz,4H),2.60(t,J=7.1Hz,2H) ,2.38(t,J=7.0Hz,2H),2.30(dd,J=5.0,1.0Hz,6H),1.78(p,J=7.1Hz,2H),1.44(d,J=6.9Hz,5H).

[0062] Step b

[0063] Dissolve compound 7 in 1 mL of methanol, add 2 mL of 1 mol / L NaOH solution dropwise at room temperature, stir at room temperature for half an hour until the raw material is almost completely converted; adjust the pH with 1 mol / L dilute HCl solution until solid precipitates, let stand for a while, and filter to obtain 73 mg of compound 8. Yield: 73%; Properties: milky white solid. 1 H NMR(500MHz,Chloroform-d)δ9.92(s,1H),8.43(d,J=1.5Hz,1H),8.32–8.23(m,3H),8.03(d,J=1.4 Hz,1H),7.76(dd,J=7.5,1.5Hz,1H),6.70(d,J=7.5Hz,1H),6.03(p,J=1.0Hz,1H),4.81(dq,J=13.7, 6.9Hz,1H),4.19(d,J=8.4Hz,2H),3.68(t,J=7.1Hz,4H),2.69(t,J=7.1Hz,4H),2.61(t,J=7.1Hz,2 H), 2.35 (t, J = 7.1Hz, 2H), 2.30 (dd, J = 5.0, 1.0Hz, 6H), 1.79 (p, J = 7.1Hz, 2H), 1.44 (d, J = 6.8Hz, 5H).

[0064] Step c

[0065] Weigh compound 8 (73 mg, 0.12 mmol), KB-0742 (43 mg, 0.15 mmol), HOBT (20 mg, 0.15 mmol) and EDCI (43 mg, 0.23 mmol) in a round-bottom flask, dissolve in anhydrous dichloromethane, and then add 73 μL of DIPEA. React at room temperature for 2 h, and monitor the basic conversion of the reaction raw materials. Separate the organic layer with water and dichloromethane, wash with saturated brine, and dry with anhydrous sodium sulfate. After preparative liquid phase separation and purification, 26 mg of compound D1 was obtained. Yield: 24%; Properties: white solid. 1 H NMR(500MHz,DMSO-d6)δ11.53(s,1H),8.65(d,J=2.6Hz,1H),8.60(t,J=5.0H z,1H),8.35(s,1H),8.05(d,J=6.0Hz,2H),8.00(d,J=2.2Hz,1H),7.90(d,J= 7.3Hz,1H),7.84(s,1H),7.61(d,J=7.6Hz,1H),6.94(d,J=9.0Hz,1H),6.32( d,J=2.2Hz,1H),5.97(s,1H),5.88(s,1H),5.13(m,J=6.7Hz,1H),4.39(d,J=4 .8Hz,2H),4.22(m,J=14.6,7.1Hz,2H),3.60–3.52(m,4H),3.17(s,1H),3.06 –2.97(m,1H),2.56(s,1H),2.43(m,J=9.1,5.4,4.8Hz,1H),2.33(t,J=7.1Hz, 2H),2.21(s,4H),2.15–1.98(m,7H),1.89(m,J=13.5,7.1,6.0Hz,1H),1.77– 1.57(m,8H),1.49(d,J=6.6Hz,7H),1.26–1.21(m,1H),0.76(t,J=7.4Hz,6H).

[0066]

[0067] The synthesis method was based on the synthesis steps of Example 1 to obtain compound D2 (white solid, yield 37%). 1H NMR (500MHz, DMSO-d6) δ11.51(s,1H),8.63(d,J=2.6Hz,1H),8.58(t,J=5.0Hz,1H),8.34(s,1H),8.06–8.00(m,2H),7.99(d,J=2.3Hz,1H),7.8 6–7.80(m,2H),7.60(d,J=7.6Hz,1H),6.91(d,J=8.9Hz,1H),6.30(d,J= 2.3Hz,1H),5.94(s,1H),5.86(s,1H),5.11(m,J=6.6Hz,1H),4.37(d,J= 4.9Hz,2H),4.26–4.14(m,2H),3.53(t,J=4.8Hz,4H),2.46–2.41(m,4H) ,2.30(t,J=7.2Hz,2H),2.20(s,3H),2.16(td,J=7.7,3.8Hz,1H),2.10( s,3H),2.08–1.96(m,4H),1.87(m,J=13.4,7.8,5.9Hz,1H),1.76–1.50( m,7H),1.50–1.40(m,9H),1.21(d,J=4.9Hz,1H),0.75(t,J=7.4Hz,6H).

[0068] Example 3: Synthesis of Compound D3

[0069]

[0070] The synthesis method was based on the synthesis steps of Example 1 to obtain compound D3 (white solid, yield 40%). 1H NMR (500MHz, DMSO-d6) δ11.53(s,1H),8.64(d,J=2.6Hz,1H),8.60(t,J=5.0Hz,1H),8.36(s,1H),8.07–8.01(m,2H),8.00(d,J=2.2Hz,1H),7.88–7. 82(m,2H),7.62(d,J=7.6Hz,1H),6.91(d,J=8.9Hz,1H),6.32(d,J=2.3Hz, 1H),5.95(s,1H),5.87(s,1H),5.12(m,J=6.6Hz,1H),4.39(d,J=4.9Hz,2H ),4.21(m,J=14.4,7.0Hz,2H),3.56–3.50(m,4H),2.44(dd,J=6.7,3.9Hz ,5H),2.29(t,J=7.4Hz,2H),2.21(s,4H),2.12(s,3H),2.04(dt,J=27.4,7 .0Hz,4H),1.88(m,J=13.4,7.6,5.7Hz,1H),1.78–1.57(m,5H),1.54(q,J= 7.4Hz,2H),1.51–1.41(m,10H),1.31–1.21(m,3H),0.76(t,J=7.4Hz,6H).

[0071] Example 4: Synthesis of Compound D4

[0072]

[0073] The synthesis method was based on the synthesis steps of Example 1 to obtain compound D4 (white solid, yield 34%). 1H NMR(500MHz,DMSO-d6)δ11.53(s,1H),8.65(d,J=2.6Hz,1H),8.60(t,J=5.0 Hz,1H),8.36(s,1H),8.04(d,J=8.5Hz,2H),8.00(d,J=2.2Hz,1H),7.84(t,J =3.6Hz,2H),7.62(d,J=7.6Hz,1H),6.92(d,J=8.9Hz,1H),6.32(d,J=2.2Hz, 1H),5.95(s,1H),5.88(s,1H),5.13(m,J=6.6Hz,1H),4.39(d,J=4.8Hz,2H), 4.21(m,J=14.2,7.0Hz,2H),3.53(t,J=4.9Hz,4H),2.46–2.40(m,4H),2.29( t,J=7.2Hz,2H),2.21(s,3H),2.19–2.14(m,1H),2.12(s,3H),2.06(t,J=7.5 Hz,3H),2.03–1.97(m,1H),1.88(dd,J=13.3,7.7,5.7Hz,1H),1.77–1.58(m, 5H),1.54–1.42(m,11H),1.27(dd,J=7.0,3.7Hz,4H),0.76(t,J=7.4Hz,6H).

[0074] Example 5: Synthesis of Compound D5

[0075]

[0076] Step a

[0077] Weigh compound 6 (200 mg, 0.4 mmol) and methyl 2-(2-bromoethoxy)acetate (118 mg, 0.6 mmol) in a round-bottom flask and dissolve in DMF, then add K2CO3 (165 mg, 1.2 mmol) and react at room temperature for 2 h. Monitor the reaction until it is basically complete. Separate the organic phase with DCM (20 mL) and water (10 mL), extract three times, combine the organic layers and wash with saturated NaCl solution, dry over anhydrous sodium sulfate, and separate and purify by column chromatography to obtain 196 mg of compound 9. Yield: 80%; Properties: white foamy solid. 1 H NMR (400 MHz, Chloroform-d) δ11.02(s,1H),8.43(d,J=1.5Hz,1H),8.30(s,1H),8.29–8.23(m,2H),8.02(d,J=1.6Hz,1H),7.7 6(dd,J=7.5,1.5Hz,1H),6.69(d,J=7.5Hz,1H),6.03(p,J=1.0Hz,1H),4.82(m,J=6.9Hz,1H),4.1 9(d,J=8.2Hz,2H),4.04(s,2H),3.77–3.70(m,5H),3.70(d,J=2.7Hz,4H),3.28(t,J=7.1Hz,2H) ,2.66(t,J=7.1Hz,4H),2.30(dd,J=5.1,1.1Hz,6H),1.44(d,J=6.9Hz,5H).ESI-MS: m / z=615.317

[0078] Step b

[0079] Dissolve compound 9 (196 mg, 0.3 mmol) in 1 mL of 1,4-dioxane, add 1 mL of 4M HCl saturated dioxane solution. React at room temperature for 1 h, and monitor that the reaction is basically completed. Directly spin dry the reaction solution to obtain 173 mg of compound 10. Yield: 90%; Properties: light yellow foamy solid. 1 H NMR(400MHz,Chloroform-d)δ11.04(s,1H),8.43(d,J=1.5Hz,1H),8.32–8.23(m,3H),8.05(d,J=1.4 Hz,1H),7.76(dd,J=7.5,1.5Hz,1H),6.69(d,J=7.5Hz,1H),6.03(p,J=1.0Hz,1H),4.82(m,J=6.9Hz,1 H),4.19(d,J=8.4Hz,2H),4.05(s,2H),3.77(t,J=7.1Hz,2H),3.72(t,J=7.1Hz,4H),3.28(t,J=7.0Hz ,2H),2.66(t,J=7.1Hz,4H),2.30(dd,J=5.0,1.0Hz,6H),1.44(d,J=6.9Hz,5H).ESI-MS:m / z=601.301

[0080] Step c

[0081] Compound 10 (50 mg, 0.8 mmol), KB-0742 (34 mg, 0.1 mmol), HOBT (14 mg, 0.1 mmol) and EDCI (29 mg, 0.2 mmol) were placed in a round-bottom flask, dissolved in anhydrous dichloromethane, and then 43 μL of DIPEA was added. The reaction was allowed to proceed for 2 h at room temperature, and the basic conversion of the reaction raw materials was monitored. The organic layer was separated with water and dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After preparative liquid phase separation and purification, 18 mg of compound D5 was obtained. Yield: 25%; Properties: white solid. 1 H NMR (500MHz, DMSO-d6) δ11.52(s,1H),8.64(d,J=2.6Hz,1H),8.58(t,J=5.0Hz,1H),8.35(s,1H),8.04(d,J=9.6Hz,2H),8.00(d,J=2.3Hz ,1H),7.83(s,1H),7.76(d,J=7.8Hz,1H),7.64(d,J=7.6Hz,1H),6.94(d,J=9.0Hz,1H),6.31(d,J=2.3Hz,1H),5.96(s,1H),5.88(s,1H),5 .13(p,J=6.6Hz,1H),4.42–4.29(m,3H),4.23(q,J=7.2Hz,1H),3.89(s,2H),3.63(t,J=5.8Hz,2H),3.56(t,J=5.1Hz,4H),2.58(dt,J=16. 0,5.5Hz,6H),2.21(s,4H),2.12(s,3H),2.10–1.92(m,5H),1.78–1.52(m,7H),1.49(d,J=6.5Hz,6H),1.23(s,6H),0.75(t,J=7.3Hz,6H).

[0082] Example 6: Synthesis of Compound D6

[0083]

[0084] The synthesis method was based on the synthesis steps of Example 5 to obtain compound D6 (white solid, yield 27%). 1H NMR (500MHz, DMSO-d6) δ11.53(s,1H),8.64(d,J=2.5Hz,1H),8.59(t,J=5.0Hz,1H),8.35(s,1H),8.03(d,J=9.4Hz,2H),8.00(d,J=2.2Hz,1H),7. 83(s,1H),7.66(dd,J=17.2,7.7Hz,2H),6.91(d,J=8.9Hz,1H),6.31(d,J =2.2Hz,1H),5.96(s,1H),5.88(s,1H),5.12(m,J=6.6Hz,1H),4.36(dd,J =25.3,6.1Hz,3H),4.23(h,J=7.3Hz,1H),3.89(s,2H),3.60(q,J=6.1,4 .2Hz, 6H), 3.53 (t, J=5.0Hz, 4H), 2.54 (dt, J=9.6, 5.4Hz, 6H), 2.42 (m, J= 9.1,5.4Hz,1H),2.21(s,4H),2.12(s,3H),2.10–1.92(m,4H),1.80–1.52 (m,6H),1.49(d,J=6.5Hz,6H),1.26–1.21(m,1H),0.75(t,J=7.4Hz,6H).

[0085] Example 7: Synthesis of Compound D7

[0086]

[0087] Step a

[0088] Weigh compound 6 (200 mg, 0.4 mmol) and 2-(4-bromobutyl)-1,3-dioxolane (126 mg, 0.6 mmol) in a round-bottom flask and dissolve in DMF, then add triethylamine (117 μL, 1.2 mmol) and react at room temperature for 2 h. Monitor the reaction until it is basically complete. Separate the organic phase with DCM (20 mL) and water (10 mL), extract three times, combine the organic layers and wash with saturated NaCl solution, dry over anhydrous sodium sulfate, and separate and purify by column chromatography to obtain 213 mg of compound 11. Yield: 84%; Properties: white foamy solid. 1HNMR(400MHz,DMSO-d6)δ11.54(s,1H),8.65(s,1H),8.60(t,J=5.0Hz,1H),8.35(s,1H),8.07(s,2H ),7.83(d,J=1.3Hz,1H),6.96(s,1H),5.89(s,1H),5.13(p,J=6.4Hz,1H),4.77(t,J=4.9Hz,1H),4.3 8(d,J=4.9Hz,2H),3.90–3.85(m,2H),3.79–3.73(m,2H),3.54(s,4H),3.09(s,2H),2.45(s,4H),2. 21(s,3H),2.12(s,3H),1.59(s,2H),1.49(d,J=6.6Hz,6H),1.42–1.33(m,2H).ESI-MS:m / z=627.790

[0089] Step b

[0090] Compound 11 (213 mg, 0.4 mmol) was directly dissolved in 1 mL of dilute hydrochloric acid (1 mol / L) and refluxed at 30°C for 1 h, and the acetal was almost completely converted into aldehyde. The excess solvent was removed by rotary evaporation, and the obtained crude product 12 was directly used as the raw material for the next step. 1 H NMR(400MHz,Chloroform-d)δ9.92(s,1H),9.76(t,J=6.2Hz,1H),8.43(d,J=1.5Hz,1H),8.32–8.23(m,3H), 8.03(d,J=1.4Hz,1H),7.76(dd,J=7.5,1.5Hz,1H),6.70(d,J=7.5Hz,1H),6.03(m,J=2.0,1.1Hz,1H),4.82( m,J=6.9Hz,1H),4.19(d,J=8.4Hz,2H),3.69(t,J=7.1Hz,4H),2.69(t,J=7.1Hz,4H),2.49–2.42(m,2H),2.3 0(dd,J=5.0,1.0Hz,6H),2.28–2.21(m,2H),1.62–1.51(m,4H),1.44(d,J=6.8Hz,5H).ESI-MS:m / z=583.737

[0091] Step c

[0092] Compound 12 (50 mg, 0.1 mol) and KB-0742 (30 mg, 0.1 mmol) were dissolved in 1 mL of 1,2-dichloroethane. A catalytic amount of acetic acid was added and the reaction was continued at room temperature for 30 min; then NaHB(OAc)3 (54 mg, 0.2 mmol) was added and the reaction was continued for 1 h. The reaction was monitored by TLC until the raw material was basically converted. The organic phase was separated with DCM (20 mL) and water (10 mL), extracted three times, the organic layers were combined and washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and separated and purified using a preparative chromatography plate to obtain 12 mg of compound D7. Yield: 21%; Properties: white solid. 1 H NMR (500MHz, DMSO-d6) δ8.64(d,J=2.6Hz,1H),8.59(t,J=4.9Hz,1H),8.35(s,1H),8.29(s,1H),8.05(q,J=5.8,4.2Hz,2H),8.01 (d,J=2.3Hz,1H),7.83(s,1H),7.69(d,J=7.6Hz,1H),6.94(d,J=8.9Hz,1H),6.33(d,J=2.2Hz,1H),6.02(s,1H),5.88(s,1H),5.1 3(p,J=6.6Hz,1H),4.38(d,J=4.8Hz,2H),4.32–4.24(m,1H),3.55(t,J=5.1Hz,5H),2.82(t,J=7.5Hz,2H),2.48–2.42(m,5H),2.3 2(t,J=7.2Hz,2H),2.21(s,3H),2.12(s,5H),1.81–1.55(m,8H),1.49(d,J=6.6Hz,8H),1.41–1.32(m,2H),0.77(t,J=7.3Hz,6H).

[0093] Example 8: Synthesis of Compound D8

[0094]

[0095] Step a

[0096] Weigh compound 6 (100 mg, 0.2 mmol) and methyl 3-bromopropionate (50 mg, 0.3 mmol) in a round-bottom flask, add triethylamine (84 μL, 0.6 mmol), react at room temperature for 2 h, and monitor the reaction until it is basically complete. Use DCM (20 mL) and water (10 mL) to separate the organic phase, extract three times, combine the organic layers and wash with saturated NaCl solution, dry over anhydrous sodium sulfate, and separate and purify by column chromatography to obtain 103 mg of compound 13. Yield: 87%; Properties: white foamy solid. 1H NMR (400MHz, DMSO-d6) δ11.52(s,1H),8.70(d,J=2.5Hz,1H),8.59(t,J=5.0Hz,1H),8.35(s ,1H),8.17–8.11(m,1H),8.09(s,1H),7.84(d,J=1.3Hz,1H),7.07(d,J=8.8Hz,1H),.89(s,1 H),5.14(p,J=6.6Hz,1H),4.38(t,J=4.9Hz,2H),3.60(s,3H),3.04(m,7H),2.29(t,J=7.2Hz ,2H),2.21(s,3H),2.11(s,3H),m,1.49(d,J=6.6Hz,6H),1.23(s,1H).ESI-MS:m / z=585.709

[0097] Step b

[0098] Compound 13 (103 mg, 0.2 mmol) was dissolved in 1 mL of methanol, and 879 μL of 1 mol / L NaOH solution was added. The reaction was allowed to react for 1 h at room temperature, and the reaction was monitored to be substantially complete. The pH was adjusted with 1 mol / L dilute HCl solution until solids precipitated. After standing for 10 min, 80 mg of compound 14 was obtained by suction filtration. Yield: 79%; Properties: white solid. 1 H NMR (400MHz, DMSO-d6) δ11.52(s,1H),8.70(d,J=2.5Hz,1H),8.59(t,J=5.0Hz,1H),8.35 (s,1H),8.17–8.11(m,1H),8.09(s,1H),7.84(d,J=1.3Hz,1H),7.07(d,J=8.8Hz,1H),5. 89(s,1H),5.14(p,J=6.6Hz,1H),4.38(t,J=4.9Hz,2H),3.04(s,7H),2.29(t,J=7.2Hz,2 H),2.22(s,3H),2.12(s,3H),m,1.49(d,J=6.6Hz,6H),1.23(s,1H).ESI-MS:m / z=571.291

[0099] Step c

[0100] Compound 14 (80 mg, 0.2 mmol), KI-Arv-03 (44 mg, 0.2 mmol), HOBT (23 mg, 0.2 mmol) and EDCI (49 mg, 0.3 mmol) were placed in a round-bottom flask, dissolved in anhydrous dichloromethane, and then 73 μL of DIPEA was added. The reaction was allowed to proceed for 2 h at room temperature, and the basic conversion of the reaction raw materials was monitored. The organic layer was separated with water and dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After preparative liquid phase separation and purification, 23 mg of compound D8 was obtained. Yield: 20%; Properties: white solid. 1 H NMR(500MHz,DMSO-d6)δ11.54–11.50(m,1H),8.63(d,J=2.5Hz,1H),8.58(t, J=5.0Hz,1H),8.34(s,1H),8.02(dt,J=13.9,5.0Hz,3H),7.98(d,J=2.2Hz,1 H),7.82(s,1H),7.62(d,J=7.7Hz,1H),6.93(d,J=8.9Hz,1H),6.27(d,J=2.2 Hz,1H),5.99(s,1H),5.86(s,1H),5.11(m,J=6.8Hz,1H),4.37(d,J=4.8Hz,2H ),4.20(m,J=28.5,7.0Hz,2H),3.53(t,J=5.0Hz,4H),2.61–2.55(m,4H),2.5 0(s,6H),2.27(t,J=7.2Hz,2H),2.20(s,3H),2.16(m,J=7.7,4.6Hz,1H),2.10 (s,3H),2.03(m,J=19.3,7.2Hz,2H),1.88(m,J=13.3,7.8,5.8Hz,1H),1.77– 1.69(m,2H),1.69–1.63(m,2H),1.47(d,J=6.6Hz,7H),0.89(t,J=7.3Hz,3H).

[0101] Example 9: Synthesis of Compound D9

[0102]

[0103] The synthesis method was based on the synthesis steps of Example 8 to obtain compound D9 (white solid, yield 32%). 1H NMR (500MHz, DMSO-d6) δ11.51(s,1H),8.64(d,J=2.6Hz,1H),8.59(t,J=5.0Hz,1H ),8.35(s,1H),8.17(s,2H),8.04(d,J=8.7Hz,2H),8.00(d,J=2.2Hz,1H),7.86(d, J=7.3Hz,1H),7.83(d,J=1.3Hz,1H),7.62(d,J=7.6Hz,1H),6.93(d,J=8.9Hz,1H) ,6.29(d,J=2.2Hz,1H),6.01(s,1H),5.88(s,1H),5.12(m,J=6.6Hz,1H),4.39(d,J =4.9Hz,2H),4.21(m,J=21.6,7.1Hz,3H),3.60–3.52(m,4H),2.61(dd,J=8.4,6.7 Hz,2H),2.52(d,J=4.3Hz,7H),2.37(t,J=7.0Hz,2H),2.21(s,3H),2.20–2.14(m,1 H),2.12(s,3H),2.09(d,J=7.2Hz,2H),2.07–1.98(m,2H),1.88(m,J=13.3,7.8,5 .7Hz,1H),1.71(m,J=14.9,7.6Hz,3H),1.57–1.42(m,11H),0.92(t,J=7.4Hz,3H).

[0104] Example 10: Synthesis of Compound D10

[0105]

[0106] The synthesis method was based on the synthesis steps of Example 8 to obtain compound D10 (white solid, yield 31%). 1H NMR(500MHz,DMSO-d6)δ8.64(d,J=2.6Hz,1H),8.60(d,J=5.0Hz,1H),8.35(s, 1H),8.18(s,1H),8.07–8.02(m,2H),8.00(d,J=2.2Hz,1H),7.87–7.81(m,2H) ,7.62(d,J=7.6Hz,1H),6.93(d,J=8.9Hz,1H),6.29(d,J=2.2Hz,1H),6.01(s, 1H),5.88(s,1H),5.12(m,J=6.5Hz,1H),4.39(d,J=4.9Hz,2H),4.20(m,J=21.3 ,7.0Hz,3H),3.59–3.51(m,4H),2.61(dd,J=8.4,6.7Hz,2H),2.47(d,J=5.6Hz ,2H),2.33(t,J=7.4Hz,2H),2.21(s,3H),2.19–2.13(m,1H),2.12(s,3H),2.05 (t,J=7.4Hz,3H),2.01(dd,J=9.5,4.3Hz,1H),1.87(s,1H),1.71(q,J=7.3Hz, 3H), 1.48 (t, J=6.9Hz, 11H), 1.28 (h, J=4.9, 4.2Hz, 4H), 0.92 (t, J=7.4Hz, 3H).

[0107] Example 11: Synthesis of Compound D11

[0108]

[0109] The synthesis method was based on the synthesis steps of Example 8 to obtain compound D11 (white solid, yield 42%). 1H NMR (500MHz, DMSO-d6) δ11.61(s,1H),8.72(d,J=2.5Hz,1H),8.67(t,J=5.0Hz,1H),8.4 3(s,1H),8.12(d,J=9.0Hz,2H),8.07(d,J=2.2Hz,1H),7.92(d,J=4.2Hz,2H),7.70(d,J= 7.6Hz,1H),7.00(d,J=8.9Hz,1H),6.36(d,J=2.2Hz,1H),6.08(s,1H),5.95(s,1H),5.20 (m,J=6.6Hz,1H),4.46(d,J=4.8Hz,2H),4.27(m,J=21.7,7.1Hz,2H),3.60(t,J=4.8Hz,4 H),2.69(t,J=7.6Hz,2H),2.58(t,J=2.9Hz,3H),2.50(t,J=5.0Hz,4H),2.35(t,J=7.4Hz ,2H),2.29(s,3H),2.24(m,J=11.8,4.0Hz,1H),2.19(s,3H),2.12(t,J=7.4Hz,3H),2.07 (dd,J=13.5,6.7Hz,1H),1.94(m,J=13.3,6.7Hz,1H),1.79(m,J=7.3Hz,3H),1.62–1.53( m,9H),1.51(d,J=6.9Hz,2H),1.34(s,6H),1.31(d,J=9.2Hz,2H),0.99(t,J=7.3Hz,3H).

[0110] Example 12: Synthesis of Compound D12

[0111]

[0112] Compound 10 (50 mg, 0.8 mmol), KI-ARv-03 (26 mg, 0.1 mmol), HOBT (14 mg, 0.1 mmol) and EDCI (29 mg, 0.2 mmol) were placed in a round-bottom flask, dissolved in anhydrous dichloromethane, and then 43 μL of DIPEA was added. The reaction was allowed to react for 2 h at room temperature, and the basic conversion of the reaction raw materials was monitored. The organic layer was separated with water and dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After preparative liquid phase separation and purification, 18 mg of compound D12 was obtained. Yield: 25%; Properties: white solid. 1H NMR (500MHz, DMSO-d6) δ11.58–11.46(m,1H),8.63(d,J=2.5Hz,1H),8.57(t,J=5.0H z,1H),8.34(s,1H),8.03(d,J=8.6Hz,2H),7.98(t,J=1.9Hz,1H),7.82(s,1H),7.74 (d,J=7.8Hz,1H),7.63(dd,J=7.7,2.5Hz,1H),6.92(dd,J=9.0,2.1Hz,1H),6.27(d, J=2.2Hz,1H),6.00(s,1H),5.86(s,1H),5.10(m,J=6.8Hz,1H),4.37(d,J=4.9Hz,2H) ,4.31(m,J=7.3Hz,1H),4.20(m,J=7.5,6.6Hz,1H),3.88(s,2H),3.61(t,J=5.7Hz,2 H),3.54(t,J=4.9Hz,4H),2.59(t,J=6.3Hz,3H),2.55(q,J=6.6,5.0Hz,5H),2.49(d, J=5.5Hz, 6H), 2.20 (s, 4H), 2.12–2.00 (m, 5H), 1.94 (m, J=13.5, 7.1Hz, 1H), 1.78–1. 68(m,3H),1.67(d,J=7.4Hz,1H),1.57–1.46(m,7H),1.45(s,1H),0.94–0.86(m,3H).

[0113] Example 13: Synthesis of Compound D13

[0114] Step a

[0115] Weigh compound 6 (300 mg, 0.6 mmol) and methyl 5-bromopentanoate (175 mg, 0.9 mmol) in a round-bottom flask, then add anhydrous potassium carbonate (248 mg, 1.8 mmol), react at room temperature for 2 h, and monitor the reaction until it is basically complete. Use DCM (50 mL) and water (25 mL) to separate the organic phase, extract three times, combine the organic layers and wash with saturated NaCl solution, dry over anhydrous sodium sulfate, and separate and purify by column chromatography to obtain 303 mg of compound 15. Yield: 82%; Properties: white foamy solid. 1H NMR(500MHz,Chloroform-d)δ9.86(s,1H),8.43(d,J=1.5Hz,1H),8.30(s,1H),8.29–8.23(m,2H),8.02 (d,J=1.6Hz,1H),7.76(dd,J=7.5,1.5Hz,1H),6.69(d,J=7.5Hz,1H),6.03(dq,J=2.1,1.1Hz,1H),4.82( m,J=6.9Hz,1H),4.19(d,J=8.2Hz,2H),3.69(t,J=7.1Hz,4H),3.64(s,2H),2.70(t,J=7.1Hz,4H),2.52– 2.46(m,2H),2.37–2.31(m,2H),2.28(dd,J=11.0,1.1Hz,6H),1.63–1.50(m,4H),1.44(d,J=6.9Hz,5H).

[0116] Step b

[0117] Dissolve compound 15 (303 mg, 0.6 mmol) in 1 mL of methanol, add 3 mL of 1 mol / L NaOH solution, react at room temperature for 1 h, and monitor the reaction to be basically completed. Adjust the pH with 1 mol / L dilute HCl solution until solid precipitates, let it stand for 10 min, and then filter to obtain 274 mg of compound 16. Properties: white solid.

[0118] Step c

[0119] Compound 16 (80 mg, 0.13 mmol), A1 (42 mg, 0.16 mmol), HOBT (22 mg, 0.2 mmol) and EDCI (46 mg, 0.3 mmol) were placed in a round-bottom flask, dissolved in anhydrous dichloromethane, and then 73 μL of DIPEA was added. The reaction was allowed to proceed for 2 h at room temperature, and the basic conversion of the reaction raw materials was monitored. The organic layer was separated with water and dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After preparative liquid phase separation and purification, 23 mg of compound D13 was obtained. Yield: 20%; Properties: white solid. 1H NMR (400MHz, DMSO-d6) δ11.54(s,1H),10.61(s,1H),8.65–8.57(m,2H),8.36(d,J=1.4Hz,2H),8.26(s,1H),8.09–7.98(m,3H),7.83(d,J =1.2Hz,1H),7.76(d,J=8.0Hz,1H),6.91(d,J=9.0Hz,1H),5.89(s,1H),5.76(s,1H),5.13(m,J=6.6Hz,1H),4.39(d,J=4.8Hz,2H),3.92(s ,2H),3.60(d,J=7.9Hz,1H),3.53(t,J=5.1Hz,4H),2.88(s,2H),2.63(s,1H),2.44(t,J=4.9Hz,4H),2.31(t,J=7.1Hz,2H),2.22(s,3H), 2.12(s,3H),2.07(t,J=7.1Hz,2H),1.90(d,J=11.5Hz,1H),1.78(s,2H),1.57–1.42(m,11H),1.36–1.21(m,11H),1.11(d,J=12.3Hz,1H).

[0120] Example 14: Synthesis of Compound D14

[0121]

[0122] Compound 16 (50 mg, 0.08 mmol), SNS-032 (38 mg, 0.1 mmol), HOBT (28 mg, 0.1 mmol) and EDCI (32 mg, 0.2 mmol) were placed in a round-bottom flask, dissolved in anhydrous dichloromethane, and then 43 μL of DIPEA was added. The reaction was carried out at room temperature for 2 h, and the basic conversion of the reaction raw materials was monitored. The organic layer was separated with water and dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After preparative liquid phase separation and purification, 16 mg of compound D14 was obtained. Yield: 20%; Properties: white solid. 1H NMR (500MHz, DMSO-d6) δ12.31(s,1H),11.53(s,1H),8.64(d,J=2.6Hz,1H),8.59(t,J=4.9Hz,1H),8.35(s,1H),8.08–8.02(m,2H),7.85–7.81(m, 1H),7.38(s,1H),6.94(d,J=8.9Hz,1H),6.70(s,1H),5.88(s,1H),5.12 (p,J=6.6Hz,1H),4.38(d,J=4.8Hz,3H),4.04(s,2H),3.93(d,J=13.6Hz, 1H),3.55(t,J=4.9Hz,4H),3.04(t,J=12.7Hz,1H),2.73(m,J=7.9,3.7H z,1H),2.63–2.54(m,1H),2.46(t,J=5.0Hz,4H),2.34(m,J=9.8,5.6,3.3 Hz,4H),2.21(s,3H),2.12(s,3H),1.81(t,J=14.0Hz,2H),1.58–1.50(m, 1H),1.53(s,2H),1.49(d,J=6.6Hz,8H),1.45–1.36(m,1H),1.16(s,9H).

[0123] Example 15: Synthesis of Compound D15

[0124]

[0125] Compound 12 (50 mg, 0.1 mol) and SNS-032 (39 mg, 0.1 mmol) were dissolved in 1 mL of 1,2-dichloroethane. A catalytic amount of acetic acid was added and the reaction was continued at room temperature for 30 min; then NaHB(OAc)3 (55 mg, 0.3 mmol) was added and the reaction was continued for 1 h. The reaction was monitored by TLC until the raw material was basically converted. The organic phase was separated with DCM (20 mL) and water (10 mL), extracted three times, the organic layers were combined and washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and separated and purified using a preparative chromatography plate to obtain 21 mg of compound D15. Yield: 25%; Properties: white solid. 1H NMR (500MHz, DMSO-d6) δ11.54(s,1H),8.65(d,J=2.5Hz,1H),8.59(d,J=5.1Hz,1H),8.35(s,1H),8.27(s,1H),8.05(d,J=8.3Hz,2H),7.8 3(s,1H),7.38(s,1H),6.95(d,J=8.9Hz,1H),6.71(s,1H),5.88(s,1H),5.13(d,J=6.6Hz,1H),4.38(d,J=4.8Hz,2H),4.04(s,2H),3.55( t,J=5.0Hz,5H),2.90(d,J=10.9Hz,2H),2.46(d,J=9.9Hz,4H),2.30(m,J=15.1,7.3Hz,4H),2.21(s,3H),2.12(s,3H),1.90(t,J=11.3Hz ,2H),1.76(d,J=12.4Hz,2H),1.62(t,J=11.1Hz,2H),1.48(t,J=7.8Hz,10H),1.30(m,J=7.6Hz,2H),1.22(d,J=4.5Hz,1H),1.16(s,9H).

[0126] Example 16: Synthesis of Compound D16

[0127]

[0128] Weigh compound 6 (50 mg, 0.1 mmol), SNS-032 (38 mg, 0.1 mmol), 1,2-dibromoethane (19 mg, 0.1 mmol) and anhydrous potassium carbonate (42 mg, 0.3 mmol) in a round-bottom flask, dissolve the compound in 1 mL of anhydrous DMF, react at room temperature for two hours, and monitor the basic conversion of the raw materials by TLC. The organic phase was separated with DCM (20 mL) and water (10 mL), extracted three times, the organic layers were combined and washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and then separated and purified by preparative liquid phase to obtain 16 mg of compound D16; yield: 17%, properties: white solid. 1H NMR (500MHz, DMSO-d6) δ12.22(s,1H),11.51(s,1H),8.63(d,J=2.6Hz,1H),8.57(t,J=5.0Hz,1H),8.33(s,1H),8.03(q,J =3.8,2.6Hz,2H),7.81(d,J=1.2Hz,1H),7.36(s,1H),6.93(d,J=9.0Hz,1H),6.70(s,1H),5.86(s,1H),5.15–5.07(m,1H) ,4.36(d,J=4.9Hz,2H),4.03(s,2H),3.53(t,J=4.9Hz,4H),2.97(d,J=11.0Hz,2H),2.52(d,J=4.4Hz,4H),2.19(s,3H),2 .10(s,3H),2.03(s,2H),1.74(d,J=3.9Hz,2H),1.61(d,J=11.8Hz,2H),1.47(d,J=6.6Hz,6H),1.21(s,1H),1.15(s,9H).

[0129] Example 17: Synthesis of Compound D17

[0130]

[0131] Weigh compound 16 (50 mg, 0.1 mmol), hu754 (50 mg, 0.1 mmol), HOBT (28 mg, 0.1 mmol) and EDCI (32 mg, 0.2 mmol) in a round-bottom flask, dissolve in anhydrous dichloromethane, and then add 43 μL of DIPEA. React at room temperature for 2 h, and monitor the basic conversion of the reaction raw materials. Separate the organic layer with water and dichloromethane, wash with saturated brine, and dry with anhydrous sodium sulfate. After preparative liquid separation and purification, compound D17 (white solid, 32%) was obtained. 1H NMR(400MHz,DMSO-d6)δ11.55(s,1H),10.60(s,1H),8.67–8.57(m,2H),8.3 5(d,J=6.6Hz,2H),8.26(s,1H),8.09–7.97(m,3H),7.83(d,J=1.2Hz,1H),7. 75(d,J=8.0Hz,1H),6.91(d,J=9.0Hz,1H),5.89(s,1H),5.13(p,J=6.6Hz,1H ),4.39(d,J=4.9Hz,2H),4.12(d,J=5.6Hz,0H),3.93(s,2H),3.60(s,1H),3. 52(t,J=5.0Hz,4H),3.17(d,J=3.9Hz,1H),2.88(s,2H),2.62(s,2H),2.43(t ,J=5.0Hz,4H),2.28(t,J=7.3Hz,2H),2.22(s,3H),2.12(s,3H),2.05(t,J=7 .3Hz,2H),1.90(d,J=12.8Hz,1H),1.78(d,J=6.9Hz,3H),1.50(d,J=6.5Hz,8 H),1.45(d,J=7.3Hz,1H),1.39–1.25(m,5H),1.26(s,6H),1.14–1.04(m,1H)

[0132] Example 18: Synthesis of Compound D18

[0133]

[0134] The synthesis method was similar to Example 17 to obtain compound D18 (white solid, 32%). 1H NMR (400MHz, Methanol-d4) δ8.54(d,J=2.5Hz,1H),8.38(s,1H),8.22(s,1H),8.04–7.96(m,2H),7.92(d,J=1.2Hz,1H),7. 78(d,J=1.3Hz,1H),7.40(s,1H),6.94(d,J=8.9Hz,1H),6.14(s,1H),5.10(p,J=6.7Hz,1H),4.60(s,2H),3.97(s,2H),3.78 (s,1H),3.67(t,J=4.9Hz,5H),3.37(s,2H),3.36(s,1H),3.10(s,2H),2.97(s,2H),2.67(t,J=4.9Hz,4H),2.44(s,3H),2.3 0–2.24(m,3H),2.10(d,J=10.4Hz,2H),2.00(d,J=10.6Hz,2H),1.59(d,J=6.6Hz,5H),1.46(q,J=11.2Hz,3H),1.35(s,6H).

[0135] Example 19: Synthesis of Compound D19

[0136]

[0137] The synthesis method was similar to Example 16, and compound D19 (white solid, yield 24%) was obtained by replacing SNS-032 with hu754. 1 H NMR (400MHz, DMSO-d6) δ9.12(s,1H),8.66(d,J=2.6Hz,1H),8.59(t,J=4.8Hz,1H),8.34(d,J=16.6Hz,2H),8.22( s,1H),8.07(q,J=4.6,3.5Hz,2H),7.97(s,1H),7.84(d,J=1.2Hz,1H),7.68(s,1H),7.50(d,J=7.6Hz,1H),6.97( d,J=8.9Hz,1H),5.89(s,1H),5.14(p,J=6.6Hz,1H),4.39(d,J=4.9Hz,2H),3.94(s,2H),3.57(d,J=5.7Hz,4H),2 .88(s,2H),2.84(s,2H),2.65(s,1H),2.22(s,3H),2.13(s,3H),1.96(s,5H),1.50(d,J=6.5Hz,6H),1.27(s,9H).

[0138] Example 20: Synthesis of Compound D20

[0139]

[0140] Weigh compound 16 (50 mg, 0.1 mmol), hu627 (48 mg, 0.1 mmol), HOBT (28 mg, 0.1 mmol) and EDCI (32 mg, 0.2 mmol) in a round-bottom flask, dissolve in anhydrous dichloromethane, and then add 43 μL of DIPEA. React at room temperature for 2 h, and monitor the basic conversion of the reaction raw materials. Separate the organic layer with water and dichloromethane, wash with saturated brine, and dry with anhydrous sodium sulfate. Prepare liquid phase separation and purification to obtain compound D20. 1 H NMR (400MHz, DMSO-d6) δ11.53(s,1H),9.18(s,1H),8.66(d,J=2.6Hz,1H),8.59(t,J=5.0Hz,1H),8.36(s,1H),8.27(s,1H),8.07(d,J=7 .0Hz,2H),7.84(s,1H),7.69(d,J=7.4Hz,1H),7.53(s,1H),7.41(t,J=7.1Hz,1H),7.22(dd,J=8.4,6.8Hz,1H),7.08(dd,J=11.4,2.5Hz, 1H),6.97(d,J=9.0Hz,1H),6.89(td,J=8.3,2.4Hz,1H),5.89(s,1H),5.14(p,J=6.6Hz,1H),4.39(d,J=4.8Hz,2H),3.77(s,3H),3.59(s ,5H),2.40(s,2H),2.22(s,3H),2.13(s,3H),2.08(t,J=7.0Hz,2H),1.91(s,2H),1.80(s,2H),1.50(d,J=6.6Hz,6H),1.33–1.22(m,7H).

[0141] Example 21: Synthesis of Compound D21

[0142]

[0143] The synthesis method was similar to Example 20 to obtain compound D21 (white solid, 47%). 1H NMR (400MHz, DMSO-d6) δ11.53 (s, 1H), 9.18 (s, 1H), 8.66 (d, J = 2.6Hz, 1H), 8.60(t,J=4.9Hz,1H),8.36(s,1H),8.27(s,1H),8.11–8.03(m,2H),7.84( d,J=1.3Hz,1H),7.60(d,J=8.2Hz,1H),7.54(s,1H),7.41(d,J=7.7Hz,1H) ,7.22(dd,J=8.4,6.8Hz,1H),7.08(dd,J=11.4,2.4Hz,1H),6.97(d,J=8.9 Hz,1H),6.89(td,J=8.4,2.4Hz,1H),5.89(s,1H),5.14(p,J=6.6Hz,1H),4 .39(d,J=4.9Hz,2H),3.77(s,3H),3.61(t,J=4.9Hz,5H),3.45(s,2H),2.9 7(s,2H),2.55(t,J=5.0Hz,4H),2.22(s,3H),2.13(s,3H),1.92(d,J=11.3 Hz,2H),1.80(d,J=12.0Hz,2H),1.50(d,J=6.6Hz,6H),1.44–1.27(m,3H).

[0144] Example 22: Synthesis of Compound D22

[0145]

[0146] The synthesis method was similar to Example 16, and compound D22 (white solid, yield 17%) was obtained by replacing SNS-032 with hu627. 1H NMR(400MHz, Methanol-d4)δ8.18(s,1H),8.02(s,1H),7.57–7.48(m,2H),7.45–7.39(m,2H),7.30(d,J=1.9Hz,1H),7.07–6.99(m,2H), 6.11(s,1H),4.50(s,2H),4.14(t,J=5.2Hz,2H),3.93(d,J=22.2Hz,4H),3.69–3.59(m,1H),3.41–3.33(m,2H),3.16(d,J=7.0Hz,1H),3 .16–3.03(m,4H),2.96(s,2H),2.62(d,J=11.7Hz,1H),2.41(s,3H),2.33(s,3H),2.25(s,3H),2.09(p,J=6.0Hz,5H),1.79–1.71(m,2H) ,1.71–1.56(m,2H),1.37(d,J=12.0Hz,2H),1.34(s,6H),1.30(s,2H),1.29(d,J=15.1Hz,0H),1.20(t,J=7.1Hz,1H),0.99–0.86(m,3H).

[0147] Example 23: Synthesis of Compound D23

[0148]

[0149] Step a

[0150] Weigh compound a (300 mg, 0.6 mmol), compound b (220 mg, 0.8 mmol), Pd(dppf)Cl2 (46 mg, 0.06 mmol) and anhydrous K2CO3 (260 mg, 1.9 mmol) in a two-necked bottle, add 1,4-dioxane 10 mL to dissolve the raw materials, add water 2 mL, replace with nitrogen three times and protect with nitrogen; react at 100 ° C for 4 hours, and monitor the reaction by TLC until the raw materials are basically completely converted. Remove the excess dioxane first, then extract with ethyl acetate and water three times, combine the organic layers, wash with saturated NaCl aqueous solution, and dry with anhydrous sodium sulfate. Column chromatography separation and purification (DCM: MeOH = 20: 1) finally obtain compound 18233 mg, yield: 66%. 1H NMR (400MHz, DMSO-d6) δ11.48(s,1H),8.18(t,J=5.0Hz,1H),7.64–7.51(m,2H),7.36(d,J=1.9Hz,1H),7.1 8(d,J=1.9Hz,1H),7.02–6.94(m,2H),5.86(s,1H),4.65(s,2H),4.29(d,J=4.9Hz,2H),3.94(s,2H),3.83( d,J=11.2Hz,2H),3.57(s,2H),3.25(t,J=11.3Hz,2H),3.13–3.00(m,2H),2.22(d,J=9.8Hz,6H),2.13–2.0 9(m,3H),1.67(d,J=12.5Hz,2H),1.53(td,J=13.0,11.6,6.1Hz,2H),1.08(s,12H),0.83(t,J=6.9Hz,3H).

[0151] Step b

[0152] Compound 18 was dissolved in 2 mL of methanol, and then 3 mL of 1 mol / L NaOH solution was added dropwise at room temperature. After reacting for 1 h at room temperature, the raw material was almost completely converted. The pH was adjusted with 1 M dilute hydrochloric acid solution until solids precipitated. After standing for a while, the filter cake obtained by suction filtration was compound 19. Yield: 86%.

[0153] Step c

[0154] Compound 19 (100 mg, 0.2 mmol), hu754 (88 mg, 0.2 mmol), HOBT (29 mg, 0.2 mmol) and EDCI (63 mg, 0.3 mmol) were placed in a round-bottom flask, dissolved in anhydrous dichloromethane, and then 95 μL of DIPEA was added. The reaction was carried out at room temperature for 2 h, and the basic conversion of the reaction raw materials was monitored. The organic layer was separated with water and dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After preparative liquid phase separation and purification, 87 mg of compound D23 was obtained. Yield: 49%; Properties: white solid. 1H NMR (400MHz, DMSO-d6) δ11.46(s,1H),9.08(s,1H),8.22(s,1H),8.17(t,J=5.0Hz,1H),8.01–7.94(m,2H),7.67(s,1H),7.60–7.54(m, 2H),7.45(d,J=7.5Hz,1H),7.36(d,J=1.9Hz,1H),7.19(d,J=1.8Hz,1H),7.05–6.99(m,2H),5.86(s,1H),4.49(s,2H),4.29(d,J=5.0Hz ,2H),3.94(s,2H),3.87–3.78(m,2H),3.66(d,J=7.7Hz,4H),3.22(s,3H),3.14–2.97(m,4H),2.88(s,2H),2.22(d,J=9.9Hz,6H),2.10 (s,3H),1.92(d,J=11.8Hz,2H),1.80(d,J=12.3Hz,2H),1.66(d,J=12.4Hz,2H),1.58–1.34(m,5H),1.26(s,9H),0.83(t,J=6.9Hz,3H).

[0155] Example 24: Synthesis of Compound D24

[0156]

[0157] The synthesis method was similar to Example 23 to obtain compound D24 (white solid, yield 37%). 1H NMR (400MHz, DMSO-d6) δ11.47(s,1H),9.08(s,1H),8.20(d,J=16.4Hz,2H),7.96(s,1H),7.73(d,J=7.8Hz,1H),7.68(s,1H),7.59–7.51(m,2H ),7.45(d,J=7.6Hz,1H),7.35(d,J=1.9Hz,1H),7.18(d,J=1.8Hz,1H),7.04–6.96(m,2H),5.86(s,1H),4.29(d,J=5.0Hz,2H),4.00(t,J=6.0Hz ,2H),3.94(s,2H),3.83(dd,J=10.9,3.4Hz,2H),3.55(s,1H),3.47(s, 1H),3.30–3.20(m,2H),3.13–2.98(m,2H),2.88(s,2H),2.22(d,J=8.9 Hz,6H),2.12(d,J=4.0Hz,5H),1.91(s,2H),1.80(s,2H),1.71–1.62(m,7H),1.52(qd,J=11.8,4.3Hz,2H),1.27(s,9H),0.83(t,J=6.9Hz,3H).

[0158] Example 25: Synthesis of Compound D25

[0159]

[0160] Step a

[0161] Weigh compound a (300 mg, 0.6 mmol), compound d (247 mg, 0.8 mmol), Pd(dppf)Cl2 (46 mg, 0.06 mmol) and anhydrous K2CO3 (260 mg, 1.9 mmol) in a two-necked bottle, add 1,4-dioxane 10 mL to dissolve the raw materials, add water 2 mL, replace with nitrogen three times and protect with nitrogen; react at 100 ° C for 4 hours, and monitor the reaction by TLC until the raw materials are basically completely converted. Remove the excess dioxane first, then extract with ethyl acetate and water three times, combine the organic layers, wash with saturated NaCl aqueous solution, and dry with anhydrous sodium sulfate. Column chromatography separation and purification (PE: EA = 10: 1) finally obtain 213 mg of compound 17, yield: 57%. 1H NMR(400MHz, DMSO-d6)δ11.46(s,1H),8.18(t,J=5.0Hz,1H),7.60–7.51(m,2H),7.36(d,J=1.8Hz,1H),7.18 (d,J=1.8Hz,1H),7.08–6.99(m,2H),5.86(s,1H),4.36(dd,J=6.2,4.6Hz,2H),4.29(d,J=4.9Hz,2H),3.82(d d,J=6.2,4.6Hz,4H),3.57(s,1H),3.25(d,J=22.3Hz,2H),3.08(q,J=7.0Hz,2H),3.04–2.97(m,1H),2.23(s ,3H),2.20(s,3H),2.10(s,3H),1.66(d,J=12.0Hz,2H),1.53(tt,J=12.5,6.4Hz,2H),0.83(t,J=6.9Hz,3H).

[0162] Step b

[0163] Weigh compound 17 (50 mg, 0.08 mmol), compound hu754 (40 mg, 0.1 mmol) and anhydrous K2CO3 (35 mg, 0.3 mmol) in a round-bottom flask, add 1 mL of DMF to dissolve the raw materials, react at room temperature for 2 h, monitor the reaction by TLC until the raw materials are basically completely converted. Extract three times with water and DCM, combine the organic layers and wash with saturated NaCl aqueous solution, and dry over anhydrous sodium sulfate. After preparative liquid separation and purification, 13 mg of compound D25 was obtained. Yield: 17%, white solid. 1H NMR (400MHz, Methanol-d4) δ8.20(s,1H),7.53–7.49(m,2H),7.41(d,J=2.0Hz,1H),7.28(d,J=1.8Hz,1H),7.19–7.15(m,1H),7.14(s,1 H),7.05–6.99(m,2H),6.90(dd,J=11.1,2.4Hz,1H),6.78(td,J=8.3,2.4Hz,1H),6.10(s,1H),4.49(s,2H),4.14(t,J=5.3Hz,2H),3.91( dq,J=10.1,3.6Hz,2H),3.79(s,3H),3.67–3.59(m,1H),3.39–3.33(m,2H),3.18–3.04(m,5H),2.69–2.60(m,1H),2.39(s,3H),2.31(s, 3H),2.24(s,3H),2.08(p,J=7.8,6.3Hz,4H),1.78–1.71(m,2H),1.63(qd,J=11.7,4.3Hz,2H),1.42–1.25(m,6H),0.89(t,J=7.0Hz,3H).

[0164] Example 26: Synthesis of Compound D26

[0165]

[0166] The synthesis method was similar to Example 25 to obtain compound D26 (white solid, yield 31%). 1H NMR (400MHz, DMSO-d6) δ9.13(s,1H),8.38(s,1H),8.21(s,1H),8.17(t,J=5.0Hz,1H),7.96(s,1H),7.67(s,1H),7.58–7.52(m,2H),7.50(d,J=7. 1Hz,1H),7.34(d,J=1.9Hz,1H),7.17(d,J=1.8Hz,1H),6.99(d,J=2.0Hz,2H),5.86(s,1H),4.29(d,J=4.9Hz,2H),4.00(t,J=6.4Hz,3H),3.93(s, 2H),3.82(d,J=11.3Hz,3H),3.24(t,J=11.5Hz,3H),3.08(q,J=7.1Hz,3 H),3.01(s,1H),2.87(s,2H),2.72(s,2H),2.67(s,1H),2.22(d,J=9.0Hz ,6H),2.10(s,3H),1.97(d,J=16.4Hz,4H),1.73(d,J=7.1Hz,2H),1.66(d ,J=12.0Hz,2H),1.61–1.43(m,6H),1.26(s,10H),0.82(t,J=6.9Hz,3H).

[0167] Example 27: Synthesis of Compound D27

[0168]

[0169] Compound 19 (100 mg, 0.2 mmol), hu627 (85 mg, 0.2 mmol), HOBT (29 mg, 0.2 mmol) and EDCI (63 mg, 0.3 mmol) were placed in a round-bottom flask, dissolved in anhydrous dichloromethane, and then 95 μL of DIPEA was added. The reaction was carried out at room temperature for 2 h, and the basic conversion of the reaction raw materials was monitored. The organic layer was separated with water and dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. Compound D27 (white solid, yield 32%) was obtained after preparative liquid phase separation and purification. 1H NMR (400MHz, DMSO-d6) δ11.47(s,1H),9.18(s,1H),8.27(s,1H),8.18(t,J=5.0Hz, 1H),7.97(d,J=8.0Hz,1H),7.60–7.55(m,2H),7.54(s,1H),7.41(d,J=7.7Hz,1H), 7.37(d,J=1.9Hz,1H),7.28–7.21(m,1H),7.21–7.18(m,1H),7.08(dd,J=11.5,2.5 Hz,1H),7.05–7.00(m,2H),6.90(td,J=8.4,2.4Hz,1H),5.86(s,1H),4.49(s,2H),4 .29(d,J=5.0Hz,2H),3.83(d,J=11.7Hz,2H),3.77(s,3H),3.67(d,J=9.6Hz,1H),3 .44(s,2H),3.26(t,J=11.4Hz,3H),3.14–2.98(m,3H),2.23(d,J=10.1Hz,6H),2.1 1(s,3H),1.93(d,J=11.8Hz,2H),1.80(d,J=12.1Hz,2H),1.67(d,J=12.3Hz,2H),1 .59–1.47(m,2H),1.45–1.34(m,2H),1.27(q,J=11.9Hz,2H),0.83(t,J=6.9Hz,3H).

[0170] Example 28: Synthesis of Compound D28

[0171]

[0172] The synthesis method was similar to Example 27 to obtain compound D28 (white solid, yield 29%). 1H NMR (400MHz, DMSO-d6) δ11.47(s,1H),9.19(s,1H),8.27(s,1H),8.18(t,J=5.0Hz ,1H),7.72(d,J=7.8Hz,1H),7.55(d,J=2.1Hz,1H),7.53(d,J=2.2Hz,2H),7.42(d, J=7.6Hz,1H),7.35(d,J=1.9Hz,1H),7.22(dd,J=8.4,6.7Hz,1H),7.18(d,J=1.8H z,1H),7.08(dd,J=11.5,2.4Hz,1H),7.02–6.97(m,2H),6.89(td,J=8.4,2.4Hz,1H ),5.86(s,1H),4.29(d,J=5.0Hz,2H),4.00(t,J=5.9Hz,2H),3.83(d,J=11.4Hz,2 H),3.77(s,3H),3.57(d,J=14.7Hz,3H),3.30–3.20(m,2H),3.13–2.97(m,3H),2.2 2(d,J=9.2Hz,6H),2.11(s,5H),1.90(s,2H),1.80(s,2H),1.67(dd,J=13.4,8.0H z,7H),1.53(tt,J=12.0,6.0Hz,2H),1.26(d,J=9.8Hz,4H),0.83(t,J=6.9Hz,3H).

[0173] Example 29: Synthesis of Compound D29

[0174]

[0175] Compound 17 (50 mg, 0.08 mmol), compound hu627 (38 mg, 0.1 mmol) and anhydrous K2CO3 (35 mg, 0.3 mmol) were weighed into a round-bottom flask, 1 mL of DMF was added to dissolve the raw materials, and the reaction was monitored by TLC at room temperature for 2 h until the raw materials were almost completely converted. Extracted three times with water and DCM, the organic layers were combined and washed with saturated NaCl aqueous solution, and dried over anhydrous sodium sulfate. Compound D29 (white solid, yield 26%) was obtained after preparative liquid phase separation and purification. 1H NMR(400MHz, Methanol-d4)δ8.16(s,1H),8.00(s,1H),7.53–7.49(m,2H),7.42–7.38(m,2H),7.28(d,J=1.8Hz,1H), 7.03–6.99(m,2H),6.09(s,1H),4.49(s,2H),4.12(t,J=5.2Hz,2H),3.94(s,2H),3.90(dt,J=12.1,3.3Hz,2H),3.67 –3.59(m,1H),3.38–3.33(m,1H),3.17–3.01(m,5H),2.94(s,2H),2.61(d,J=11.7Hz,1H),2.39(s,3H),2.31(s,3H), 2.23(s,3H),2.07(dd,J=8.6,4.7Hz,4H),1.78–1.69(m,2H),1.69–1.55(m,2H),1.32(s,10H),0.88(t,J=6.9Hz,3H).

[0176] Example 30: Synthesis of Compound D30

[0177]

[0178] The synthesis method was similar to Example 29 to obtain compound D30 (white solid, yield 13%). 1H NMR (400MHz, DMSO-d6) δ9.23(s,1H),8.38(s,1H),8.26(s,1H),8.17(t,J=4.9Hz,1H),7.55(s,1H),7.52(d,J=4.8Hz,2H),7.46(d,J=7.6Hz,1H),7. 34(d,J=2.0Hz,1H),7.21(dd,J=8.4,6.7Hz,1H),7.17(d,J=1.9Hz,1H),7. 07(dd,J=11.4,2.4Hz,1H),6.99(d,J=8.6Hz,2H),6.89(td,J=8.5,2.5Hz, 1H),5.86(s,1H),4.28(d,J=4.9Hz,2H),4.00(t,J=6.3Hz,2H),3.82(d,J =11.4Hz,3H),3.76(s,4H),3.25(t,J=11.1Hz,5H),3.13–2.96(m,5H),2.6 5(d,J=28.6Hz,4H),2.21(d,J=8.8Hz,6H),2.10(s,3H),1.93(s,4H),1.78 –1.61(m,5H),1.50(d,J=22.0Hz,7H),1.21(s,4H),0.82(t,J=6.9Hz,3H).

[0179] Biological experimental determination and research

[0180] 1.CDK9 enzyme activity assay

[0181] CDK9 / cycT1, CDK7 / 9tide, ATP and the test compound were added to each well, with a total reaction system of 5 μL, and incubated at 37°C for 120 minutes. Then, according to the instructions of the ADP-Glo ​​Kinase Assay Kit (Promega, V9102), buffer was added in sequence, and luminescence was read on a plate reader (EnVision). IC was calculated using SoftMax Pro 50 value.

[0182] 2. EZH2 enzyme activity assay

[0183] PRC2 complex containing EZH2 Y641F, substrate, SAM and test compound were added to each well, the total reaction system was 10 μL, and the reaction was carried out in the dark at room temperature for 4 hours. Subsequently, 10 μL of Eu-labeled H3K27 Me3 antibody and Streptavidin–XL665 mixture were added to each well, and after incubation at room temperature for 1 hour, the fluorescence values ​​at 620 nm and 665 nm were measured using a multi-label microplate analysis system (PerkinElmer Envision), and the HTRF signal ratio (665 nm / 620 nm) of each well was calculated. The IC of the compound was calculated using SoftMax Pro 5.4.1 software 50 value.

[0184] 3. Cellular IC50 Determination

[0185] In each well of a 96-well plate, 2000 cells were inoculated with 200 μL of fresh culture medium. After incubation with the compound for 7 days, 20 μL of Cell Counting Kit-8 (CCK8) was added to each well. After incubation at 37°C for 2 h, the absorbance of each well was measured at 450 nm using SpectraMAX190 (Molecular Devices), and the IC was calculated using SoftMax Pro. 50 value.

[0186] 4. Western Blot Experiment

[0187] Appropriate amount of human lymphoma cells were plated in 6-well plates. After drug treatment, cells were collected by centrifugation for subsequent experiments. According to the amount of cells, the corresponding amount of 1X loading buffer was added. After lysis, the sample was boiled at 100℃ for 20min. The same volume of sample was taken for SDS-PAGE. After electrophoresis, the protein on the gel was transferred to a nitrocellulose membrane using a GenScript fast transfer instrument. The corresponding bands were cut according to the size of the protein, blocked with TBST containing 5% skim milk powder for 1h, and incubated with primary antibody at 4℃ overnight. The excess primary antibody was eluted with TBST, three times for ten minutes each time, and the secondary antibody was incubated at room temperature for 1h, and then the excess secondary antibody was eluted with TBST, three times for ten minutes each time. Finally, the strips were stained and photographed using a Bio-Rad chromogen.

[0188] 5. Apoptosis Detection

[0189] After an appropriate amount of lymphoma cells were co-incubated with the compound in a six-well plate, they were centrifuged and washed twice with PBS, then stained with an apoptosis detection kit (Vazyme, A211-01). After staining for ten minutes in the dark, they were detected with BD Calibur and data were analyzed with FlowJo_V10.

[0190] Test results

[0191] 1. Study of enzyme inhibition activity and antiproliferative activity in two DLBCL cell lines

[0192] Compared with the positive control compound, these compounds all showed CDK9 / EZH2 dual-target inhibitory activity, among which compounds D15 and D16 not only showed dual-target inhibitory effects at the enzyme level, but also showed excellent anti-proliferative activity in DLBCL cell lines. Therefore, we selected D15 and D16 for downstream signaling pathway inhibition studies.

[0193] According to the structural features of the examples, the positive control compounds used for CDK9 are KI-ARV-03, SNS-032, Hu754 and Hu627; the positive control compounds used for EZH2 are C24 and EPZ6438. The structures are as follows:

[0194]

[0195]

[0196] 2. Effects of representative active compounds on downstream signaling pathways

[0197] The effects of some active compounds on Mcl-1, Myc and H3K27me3 in DLBCL cells were studied by western blot. Figure 1 ). The results showed that when they downregulated Mcl-1 and c-Myc, they could synergistically reduce H3K27me3 caused by CDK9 inhibitors; reflecting the dual functional inhibitory effect of CDK9 / EZH2. In addition, we found that D15 or D16 could significantly reduce the expression of H3K27ac ( Figure 1 AB), high expression of H3K27ac is considered to be an important cause of drug resistance.

[0198] 3. Analysis of the effects of compound D16 on inducing apoptosis and DNA damage in two different DLBCL cell lines

[0199] According to existing literature reports, a single CDK9 inhibitor can induce apoptosis of tumor cells. Figure 2 AB), the dual-target inhibitor D16 showed a more significant ability to induce tumor cell apoptosis compared with the CDK9 inhibitor SNS-032 and the EZH2 inhibitor C24.

[0200] In addition, we found that compound D16 can cause a decrease in the DNA double-strand break repair protein Rad51, and also cause a significant upregulation of the marker protein H2AX for DNA double-strand damage (indicating that DNA damage has accumulated in the cell), and the cleavage of the DNA single-strand repair protein PARP ( Figure 2CD), indicating that tumor cells undergo apoptosis. This biological effect is not seen with the corresponding CDK9 or EZH2 single-target inhibitors at the corresponding dose, suggesting that simultaneous inhibition of dual targets can synergistically cause tumor cell DNA damage and induce apoptosis, with a more potent anti-tumor effect.

[0201] 4. Inhibition of solid tumor lines by compound D16

[0202] The results showed that the CDK9 / EZH2 dual-target inhibitor D16 had better inhibitory activity on solid tumor cell proliferation than the single-target inhibitors SNS-032 or C24.

[0203]

[0204] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A CDK9 / EZH2 dual-target inhibitor, characterized in that: The general structural formula is structural formula Ⅰ: in: The CDK9 pharmacophore represents the ligand that binds to the target protein CDK9; Linker represents a chemical bond or group; The EZH2 pharmacophore represents the ligand that binds to the target protein EZH2.

2. The CDK9 / EZH2 dual-target inhibitor according to claim 1, characterized in that The CDK9 pharmacophore is a CDK9 protein inhibitor or a structural fragment having CDK9 protein inhibitory effect; the EZH2 pharmacophore is an EZH2 protein inhibitor or a structural fragment having EZH2 protein inhibitory effect; the linker is selected from a straight chain, branched chain or cyclic structure containing 1-30 carbon atoms or heteroatoms.

3. The CDK9 / EZH2 dual-target inhibitor according to claim 2, characterized in that: The CDK9 protein inhibitors are KB-0742, KI-ARV-03, AZD-4573 or SNS-032; the EZH2 protein inhibitors are EPZ6438, GSK126 or C24.

4. The CDK9 / EZH2 dual-target inhibitor according to claim 2, characterized in that: Linker has the following structure: wherein n is 1, 2, 3, 4, 5, 6, 7 or 8, and m is 1, 2, 3 or 4.

5. The CDK9 / EZH2 dual-target inhibitor according to claim 2, characterized in that: The structure is shown in Structure I-VII: wherein n is 1, 2, 3, 5, 6, 7 or 8, and m is 1, 2, 3 or 4; Among them, the R group is preferably:

6. The CDK9 / EZH2 dual-target inhibitor according to claim 5, characterized in that The structure is:

7. A pharmaceutical composition, characterized in that Comprising one or more of the CDK9 / EZH2 dual-target inhibitor or pharmaceutically acceptable salts thereof according to any one of claims 1 to 6, and an optional pharmaceutically acceptable carrier.

8. Use of the CDK9 / EZH2 dual-target inhibitor according to any one of claims 1 to 6 in the preparation of a drug for treating tumors.

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

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