A cdk9 / ezh2 dual-target inhibitor, pharmaceutical composition thereof and application thereof

Abstract

The application belongs to the technical field of medicine, and relates to a CDK9 / EZH2 dual-target inhibitor and a pharmaceutical composition and application thereof. The CDK9 / EZH2 dual-target inhibitor is a compound obtained by coupling a structural fragment for inhibiting CDK9 protein and a structural fragment for inhibiting EZH2 protein through a linker. The structural fragment for inhibiting CDK9 protein and the structural fragment for inhibiting EZH2 protein are restructured, coupled together through the linker, and the length and connection mode of the linker are adjusted to obtain the CDK9 and EZH2 dual-target inhibitor and the application thereof in antitumor.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a CDK9 / EZH2 dual-target inhibitor and a pharmaceutical composition and application thereof. BACKGROUND

[0002] Cell cycle-dependent kinases (CDKs) are members of the serine / threonine kinase family, which 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 particular attention from drug developers because it regulates transcriptional elongation without affecting cell cycle progression. CDK9 can form a heterodimer with cyclin (T1, T2 and K), i.e. positive transcription elongation factor b (P-TEFb), and the activated P-TEFb can phosphorylate the 2 serine (Ser2) in the C-terminal domain (CTD) of RNA polymerase II (RNAPII), thereby enabling the elongation phase of transcription to proceed normally. In-depth studies have shown that CDK9 has a major signal disorder in several tumor cells, including malignant hematological tumors and solid tumors. Mechanistically, inhibition of CDK9 can prevent phosphorylation of RNAPII CTD Ser2 and induce dysregulation of MYC and Mcl-1 protein levels, which has been confirmed 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); moreover, these compounds are basically in clinical phase I / I or have no progress, and the research in solid tumors still has limitations. It has been found that CDK9 inhibitors can cause abnormal elevation of H3K27me3 in DLBCL cell lines, and the combined use of EZH2 inhibitors can inhibit the up-regulation of H3K27me3, achieving a synergistic anti-tumor effect. The CDK9 and EZH2 dual-target inhibitor of the present application has the function of dual-target inhibition and can be used for treating related tumors. SUMMARY

[0004] The present application provides a CDK9 / EZH2 dual-target inhibitor and a pharmaceutical composition and application thereof to overcome the deficiencies of the prior art. The specific technical solutions are as follows:

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

[0006]

[0007] Wherein:

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

[0009] The linker represents a covalent connection part;

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

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

[0012] The CDK9 pharmacophore part:

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

[0014]

[0015] The EZH2 pharmacophore part:

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

[0017]

[0018] The linker:

[0019] The linker represents a connection part for coupling the CDK9 pharmacophore and the EZH2 pharmacophore, which can be a chemical bond. The linker can be rigid or flexible. In some preferred embodiments, the linker is flexible.

[0020] In some embodiments, the Linker is a straight chain or branched chain 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 can optionally be 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 can also be optionally replaced by -C(=0)-, -C(=S)-, -S(=0)-, -S02-, 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, the 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; the covalently linked sites can be any suitable sites.

[0024] In some embodiments, the CDK9 pharmacophore moiety is covalently linked to the Linker mainly through a nitrogen atom; similarly, the EZH2 pharmacophore moiety can also be covalently linked to the linker through a carbon atom or a heteroatom, the heteroatom being selected from oxygen, sulfur, nitrogen, phosphorus. One skilled in the art can select a suitable reaction site for connection according to the structure of the three moieties.

[0025] Further, the structure of the CDK9 / EZH2 dual-target inhibitor is selected from any 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 application provides a pharmaceutical composition comprising one or more of the CDK9 / EZH2 dual-targeting inhibitors or pharmaceutically acceptable salts thereof according to the first aspect of the present application and optionally a pharmaceutically acceptable carrier.

[0036] The pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the field of pharmacy, for example: diluents such as water and the like; fillers such as starch, sucrose and the like; binders such as cellulose derivatives, alginate, gelatin, polyvinylpyrrolidone; humectants 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 soap clay; lubricants such as talc, calcium stearate and magnesium stearate and polyethylene glycol and the like. In addition, other adjuvants such as flavoring agents and sweetening agents and the like can also be added to the above pharmaceutical composition.

[0037] The third aspect of the present application provides the use of the CDK9 / EZH2 dual-targeting inhibitor in the preparation of a medicament for treating tumors.

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

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

[0040] Figure 1 Effects of the compounds of the present application on downstream signaling pathways;

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

[0042] Figure 2 Analysis of the effects of the compound D16 of the present application on inducing apoptosis and DNA damage. DETAILED DESCRIPTION

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

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

[0045] All reaction solvents were purified according to the conventional method unless otherwise specified. The silica gel (200-300 mesh) for column chromatography was produced by Qingdao Marine Chemical Factory. The thin layer chromatography used GF254 high efficiency plate, which was produced by Yantai Chemical Institute. All solvents were analytical reagents unless otherwise specified, and the reagents used were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. Color development was performed using iodine, ultraviolet fluorescence, etc. Organic solvents were removed by vacuum evaporation in a rotary evaporator.

[0046] In the following examples, nuclear magnetic resonance hydrogen spectrum was recorded by Bruker AMX-400 and Bruker AMX-500 type nuclear magnetic resonance instrument, and the chemical shift δ was in ppm. The multiplicity was recorded according to 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] 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) were weighed, DIPEA 1.9 mL was added, dissolved in anhydrous dichloromethane, and reacted at room temperature for 3 h. TLC monitoring showed that the reaction was basically complete. The organic phase was separated with ethyl acetate (50 mL) and water (25 mL), and the organic phase was combined after extraction three times, washed with saturated brine, and then dried with anhydrous sodium sulfate. Column chromatography was used for separation and purification (DCM:MeOH = 20:1), and finally 1.3 g of compound 3 was obtained. Yield: 88%; appearance: white solid. 1 H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 8.61 (s, 1H), 8.37 (s, 1H), 8.20 (d, J = 1.3 Hz, 1H), 7.70 (d, J = 1.5 Hz, 1H), 5.89 (s, 1H), 5.09-5.03 (m, 1H), 4.34 (d, J = 4.9 Hz, 2H), 2.21 (s, 3H), 2.13 (s, 3H), 1.46 (d, J = 6.6 Hz, 6H). ESI-MS: m / z = 417.307

[0052] Step b

[0053] 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 for three times and protected with nitrogen; reacted at 80 °C for 3 h, and the reaction was monitored by TLC until the starting material was substantially converted. The excess dioxane was removed first, then extracted with ethyl acetate and water three times, combined the organic layer and washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate. Purified by column chromatography (PE:EA = 5:1), finally 1.8 g of compound 5 was obtained. Yield: 96%; Appearance: milky white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.33 (d, J = 2.0 Hz, 1H), 7.70 (dd, J = 8.6, 2.0 Hz, 1H), 6.79 (d, J = 8.6 Hz, 1H), 3.56 (dd, J = 6.7, 3.9 Hz, 4H), 3.39 (d, J = 10.6 Hz, 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 for three times and protected with nitrogen; reacted at 80 °C for 3 h, and the reaction was monitored by TLC until the starting material was substantially converted. The excess dioxane was removed first, then extracted with ethyl acetate and water three times, combined the organic layer and washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate. Purified by column chromatography (PE:EA = 5:1), finally 1.8 g of compound 5 was obtained. Yield: 96%; Appearance: milky white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 8.67 (s, 1H), 8.59 (s, 1H), 8.35 (s, 1H), 8.08 (d, J = 10.6 Hz, 2H), 7.84 (s, 1H), 6.98 (d, J = 8.9 Hz, 1H), 5.89 (s, 1H), 5.76 (d, J = 1.1 Hz, 1H), 5.13 (p, J = 6.5 Hz, 1H), 4.38 (d, J = 4.9 Hz, 2H), 3.56 (d, J = 5.4 Hz, 4H), 3.45 (s, 4H), 2.21 (s, 3H), 2.12 (s, 3H), 1.49 (d, J = 6.5 Hz, 6H), 1.43 (d, J = 1.1 Hz, 9H). ESI-MS: m / z = 599.736

[0056] Step d

[0057] Compound 5 1.8g was dissolved in 5mL of methanol, then 2.5mL of 4M hydrochloric acid dioxane solution was added dropwise under ice bath conditions, and stirred at room temperature for 1h, TLC monitoring reaction was basically completed. Directly dry the solvent to get 2.1g of crude product 6. Property: yellow solid powder. 1 H NMR (400 MHz, DMSO-d6) δ 8.66 (d, J = 2.6 Hz, 1H), 8.60 (s, 1H), 8.35 (s, 1H), 8.06 (s, 2H), 7.84 (s, 1H), 6.95 (d, J = 9.0 Hz, 1H), 5.89 (s, 1H), 4.38 (d, J = 4.8 Hz, 2H), 3.53 (s, 4H), 2.87 (s, 4H), 2.21 (s, 3H), 2.12 (s, 3H), 1.49 (d, J = 6.6 Hz, 6H). ESI-MS: m / z = 499.736.

[0058] Example 1: Synthesis of compound D1

[0059]

[0060] Step a

[0061] Compound 6 (100mg, 0.2mmol), 4-bromobutyl methyl carbonate (54mg, 0.3mmol) were weighed into a round bottom flask, then K2CO3(83mg, 0.6mmol) was added, and the reaction was carried out at room temperature for 2h, and the reaction was monitored to be basically completed. The organic phase was separated with DCM (20mL) and water (10mL), extracted three times, the organic layer was combined and washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and then column chromatography was used for separation and purification to obtain 103mg of compound 7. Yield: 87%; property: white foam solid.1 H NMR (500 MHz, Chloroform-d) δ 9.86 (s, 1H), 8.43 (d, J = 1.5 Hz, 1H), 8.32 - 8.23 (m, 3H), 8.05 (d, J = 1.4 Hz, 1H), 7.76 (dd, J = 7.5, 1.5 Hz, 1H), 6.69 (d, J = 7.5 Hz, 1H), 6.03 (p, J = 1.0 Hz, 1H), 4.82 (hept, J = 6.9 Hz, 1H), 4.19 (d, J = 8.4 Hz, 2H), 3.71 - 3.62 (m, 6H), 2.69 (t, J = 7.1 Hz, 4H), 2.60 (t, J = 7.1 Hz, 2H), 2.38 (t, J = 7.0 Hz, 2H), 2.30 (dd, J = 5.0, 1.0 Hz, 6H), 1.78 (p, J = 7.1 Hz, 2H), 1.44 (d, J = 6.9 Hz, 5H).

[0062] Step b

[0063] Compound 7 was dissolved in 1 mL of methanol, 2 mL of 1 mol / L NaOH solution was added dropwise at room temperature, and the mixture was stirred at room temperature for half an hour. The raw material was basically completely converted. The pH was adjusted to solid precipitation with 1 mol / L dilute HCl solution, and the filtrate was obtained by standing for a few moments. Yield: 73%; property: white solid. 1 H NMR (500 MHz, Chloroform-d) δ 9.86 (s, 1H), 8.43 (d, J = 1.5 Hz, 1H), 8.32 - 8.23 (m, 3H), 8.05 (d, J = 1.4 Hz, 1H), 7.76 (dd, J = 7.5, 1.5 Hz, 1H), 6.69 (d, J = 7.5 Hz, 1H), 6.03 (p, J = 1.0 Hz, 1H), 4.82 (hept, J = 6.9 Hz, 1H), 4.19 (d, J = 8.4 Hz, 2H), 3.71 - 3.62 (m, 6H), 2.69 (t, J = 7.1 Hz, 4H), 2.60 (t, J = 7.1 Hz, 2H), 2.38 (t, J = 7.0 Hz, 2H), 2.30 (dd, J = 5.0, 1.0 Hz, 6H), 1.78 (p, J = 7.1 Hz, 2H), 1.44 (d, J = 6.9 Hz, 5H).

[0064] Step c

[0065] 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) were weighed into a round bottom flask and dissolved in anhydrous dichloromethane, then 73 μΐ of DIPEA was added. The reaction was monitored at room temperature for 2 h, and the starting material was substantially converted. The organic layer was separated with water and dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After preparative liquid separation and purification, 26 mg of compound D1 was obtained. Yield: 24%; property: white solid. 1 H NMR (500 MHz, DMSO-d6) δ 11.53 (s, 1H), 8.65 (d, J = 2.6 Hz, 1H), 8.60 (t, J = 5.0 Hz, 1H), 8.35 (s, 1H), 8.05 (d, J = 6.0 Hz, 2H), 8.00 (d, J = 2.2 Hz, 1H), 7.90 (d, J = 7.3 Hz, 1H), 7.84 (s, 1H), 7.61 (d, J = 7.6 Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.32 (d, J = 2.2 Hz, 1H), 5.97 (s, 1H), 5.88 (s, 1H), 5.13 (m, J = 6.7 Hz, 1H), 4.39 (d, J = 4.8 Hz, 2H), 4.22 (m, J = 14.6, 7.1 Hz, 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, 4.8 Hz, 1H), 2.33 (t, J = 7.1 Hz, 2H), 2.21 (s, 4H), 2.15 - 1.98 (m, 7H), 1.89 (m, J = 13.5, 7.1, 6.0 Hz, 1H), 1.77 - 1.57 (m, 8H), 1.49 (d, J = 6.6 Hz, 7H), 1.26 - 1.21 (m, 1H), 0.76 (t, J = 7.4 Hz, 6H).

[0066]

[0067] The synthesis method refers to the synthesis steps of Example 1 to obtain compound D2 (white solid, yield 37%). 1H NMR (500 MHz, DMSO-d6) δ 11.51 (s, 1H), 8.63 (d, J = 2.6 Hz, 1H), 8.58 (t, J = 5.0 Hz, 1H), 8.34 (s, 1H), 8.06 - 8.00 (m, 2H), 7.99 (d, J = 2.3 Hz, 1H), 7.86 - 7.80 (m, 2H), 7.60 (d, J = 7.6 Hz, 1H), 6.91 (d, J = 8.9 Hz, 1H), 6.30 (d, J = 2.3 Hz, 1H), 5.94 (s, 1H), 5.86 (s, 1H), 5.11 (m, J = 6.6 Hz, 1H), 4.37 (d, J = 4.9 Hz, 2H), 4.26 - 4.14 (m, 2H), 3.53 (t, J = 4.8 Hz, 4H), 2.46 - 2.41 (m, 4H), 2.30 (t, J = 7.2 Hz, 2H), 2.20 (s, 3H), 2.16 (td, J = 7.7, 3.8 Hz, 1H), 2.10 (s, 3H), 2.08 - 1.96 (m, 4H), 1.87 (m, J = 13.4, 7.8, 5.9 Hz, 1H), 1.76 - 1.50 (m, 7H), 1.50 - 1.40 (m, 9H), 1.21 (d, J = 4.9 Hz, 1H), 0.75 (t, J = 7.4 Hz, 6H).

[0068] Example 3: Synthesis of compound D3

[0069]

[0070] The synthesis method refers to the synthesis steps of Example 1 to obtain compound D3 (white solid, yield 40%). 1H NMR (500 MHz, DMSO-d6) δ 11.53 (s, 1H), 8.64 (d, J = 2.6 Hz, 1H), 8.60 (t, J = 5.0 Hz, 1H), 8.36 (s, 1H), 8.07 - 8.01 (m, 2H), 8.00 (d, J = 2.2 Hz, 1H), 7.88 - 7.82 (m, 2H), 7.62 (d, J = 7.6 Hz, 1H), 6.91 (d, J = 8.9 Hz, 1H), 6.32 (d, J = 2.3 Hz, 1H), 5.95 (s, 1H), 5.87 (s, 1H), 5.12 (m, J = 6.6 Hz, 1H), 4.39 (d, J = 4.9 Hz, 2H), 4.21 (m, J = 14.4, 7.0 Hz, 2H), 3.56 - 3.50 (m, 4H), 2.44 (dd, J = 6.7, 3.9 Hz, 5H), 2.29 (t, J = 7.4 Hz, 2H), 2.21 (s, 4H), 2.12 (s, 3H), 2.04 (dt, J = 27.4, 7.0 Hz, 4H), 1.88 (m, J = 13.4, 7.6, 5.7 Hz, 1H), 1.78 - 1.57 (m, 5H), 1.54 (q, J = 7.4 Hz, 2H), 1.51 - 1.41 (m, 10H), 1.31 - 1.21 (m, 3H), 0.76 (t, J = 7.4 Hz, 6H).

[0071] Example 4: Synthesis of compound D4

[0072]

[0073] The synthesis method refers to the synthesis steps of Example 1 to obtain compound D4 (white solid, yield 34%). 1H NMR (500 MHz, DMSO-d6) δ 11.53 (s, 1H), 8.65 (d, J = 2.6 Hz, 1H), 8.60 (t, J = 5.0 Hz, 1H), 8.36 (s, 1H), 8.04 (d, J = 8.5 Hz, 2H), 8.00 (d, J = 2.2 Hz, 1H), 7.84 (t, J = 3.6 Hz, 2H), 7.62 (d, J = 7.6 Hz, 1H), 6.92 (d, J = 8.9 Hz, 1H), 6.32 (d, J = 2.2 Hz, 1H), 5.95 (s, 1H), 5.88 (s, 1H), 5.13 (m, J = 6.6 Hz, 1H), 4.39 (d, J = 4.8 Hz, 2H), 4.21 (m, J = 14.2, 7.0 Hz, 2H), 3.53 (t, J = 4.9 Hz, 4H), 2.46 - 2.40 (m, 4H), 2.29 (t, J = 7.2 Hz, 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, 5.7 Hz, 1H), 1.77 - 1.58 (m, 5H), 1.54 - 1.42 (m, 11H), 1.27 (dd, J = 7.0, 3.7 Hz, 4H), 0.76 (t, J = 7.4 Hz, 6H).

[0074] Example 5: Synthesis of compound D5

[0075]

[0076] Step a

[0077] Compound 6 (200 mg, 0.4 mmol), 2-(2-bromoethoxy)ethyl acetate (118 mg, 0.6 mmol) were weighed in a round bottom flask and dissolved in DMF, K2CO3 (165 mg, 1.2 mmol) was added and the reaction was allowed to proceed at room temperature for 2 h, the reaction was monitored to be complete. The organic layer was separated with DCM (20 mL) and water (10 mL), extracted three times, the organic layer was combined and washed with saturated NaCl solution, dried over anhydrous sodium sulfate and column chromatography was performed to isolate and purify to obtain 196 mg of compound 9. Yield: 80%; Character: white foamy solid. 1 H NMR (400 MHz, Chloroform-d) δ11.02 (s, 1H), 8.43 (d, J = 1.5 Hz, 1H), 8.30 (s, 1H), 8.29 - 8.23 (m, 2H), 8.02 (d, J = 1.6 Hz, 1H), 7.76 (dd, J = 7.5, 1.5 Hz, 1H), 6.69 (d, J = 7.5 Hz, 1H), 6.03 (p, J = 1.0 Hz, 1H), 4.82 (m, J = 6.9 Hz, 1H), 4.19 (d, J = 8.2 Hz, 2H), 4.04 (s, 2H), 3.77 - 3.70 (m, 5H), 3.70 (d, J = 2.7 Hz, 4H), 3.28 (t, J = 7.1 Hz, 2H), 2.66 (t, J = 7.1 Hz, 4H), 2.30 (dd, J = 5.1, 1.1 Hz, 6H), 1.44 (d, J = 6.9 Hz, 5H). ESI-MS: m / z = 615.317

[0078] Step b

[0079] Compound 9 (196 mg, 0.3 mmol) was dissolved in 1 mL of 1,4-dioxane, and 1 mL of 4 M HCl saturated dioxane solution was added. The reaction was monitored at room temperature for 1 h, and it was found that the reaction was substantially complete. The reaction was directly spin-dried to give 173 mg of compound 10. Yield: 90%; Appearance: light yellow foamy solid. 1 11.02 (s, 1H), 8.43 (d, J = 1.5 Hz, 1H), 8.30 (s, 1H), 8.29 - 8.23 (m, 2H), 8.02 (d, J = 1.6 Hz, 1H), 7.76 (dd, J = 7.5, 1.5 Hz, 1H), 6.69 (d, J = 7.5 Hz, 1H), 6.03 (p, J = 1.0 Hz, 1H), 4.82 (m, J = 6.9 Hz, 1H), 4.19 (d, J = 8.2 Hz, 2H), 4.04 (s, 2H), 3.77 - 3.70 (m, 5H), 3.70 (d, J = 2.7 Hz, 4H), 3.28 (t, J = 7.1 Hz, 2H), 2.66 (t, J = 7.1 Hz, 4H), 2.30 (dd, J = 5.1, 1.1 Hz, 6H), 1.44 (d, J = 6.9 Hz, 5H). ESI-MS: m / z = 615.317

[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 dissolved in a round bottom flask with anhydrous dichloromethane, and then DIPEA 43 μΐ was added. The reaction was monitored at room temperature for 2 h, and the starting material was substantially converted. The organic layer was separated with water and dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After preparative liquid separation and purification, 18 mg of compound D5 was obtained. Yield: 25%; property: white solid. 1 H NMR (500 MHz, DMSO-d6) δ 11.52 (s, 1H), 8.64 (d, J = 2.6 Hz, 1H), 8.58 (t, J = 5.0 Hz, 1H), 8.35 (s, 1H), 8.04 (d, J = 9.6 Hz, 2H), 8.00 (d, J = 2.3 Hz, 1H), 7.83 (s, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.64 (d, J = 7.6 Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.31 (d, J = 2.3 Hz, 1H), 5.96 (s, 1H), 5.88 (s, 1H), 5.13 (p, J = 6.6 Hz, 1H), 4.42 - 4.29 (m, 3H), 4.23 (q, J = 7.2 Hz, 1H), 3.89 (s, 2H), 3.63 (t, J = 5.8 Hz, 2H), 3.56 (t, J = 5.1 Hz, 4H), 2.58 (dt, J = 16.0, 5.5 Hz, 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 = 6.5 Hz, 6H), 1.23 (s, 6H), 0.75 (t, J = 7.3 Hz, 6H).

[0082] Example 6: Synthesis of compound D6

[0083]

[0084] Synthesis method refers to the synthesis steps of Example 5, and compound D6 (white solid, yield 27%) is obtained 1H NMR (500 MHz, DMSO-d6) δ 11.53 (s, 1H), 8.64 (d, J = 2.5 Hz, 1H), 8.59 (t, J = 5.0 Hz, 1H), 8.35 (s, 1H), 8.03 (d, J = 9.4 Hz, 2H), 8.00 (d, J = 2.2 Hz, 1H), 7.83 (s, 1H), 7.66 (dd, J = 17.2, 7.7 Hz, 2H), 6.91 (d, J = 8.9 Hz, 1H), 6.31 (d, J = 2.2 Hz, 1H), 5.96 (s, 1H), 5.88 (s, 1H), 5.12 (m, J = 6.6 Hz, 1H), 4.36 (dd, J = 25.3, 6.1 Hz, 3H), 4.23 (h, J = 7.3 Hz, 1H), 3.89 (s, 2H), 3.60 (q, J = 6.1, 4.2 Hz, 6H), 3.53 (t, J = 5.0 Hz, 4H), 2.54 (dt, J = 9.6, 5.4 Hz, 6H), 2.42 (m, J = 9.1, 5.4 Hz, 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 = 6.5 Hz, 6H), 1.26 - 1.21 (m, 1H), 0.75 (t, J = 7.4 Hz, 6H).

[0085] Example 7: Synthesis of compound D7

[0086]

[0087] Step a

[0088] Compound 6 (200 mg, 0.4 mmol), 2-(4-bromobutyl)-l,3-dioxolane (126 mg, 0.6 mmol) were weighed in a round bottom flask and dissolved in DMF, triethylamine (117 μL, 1.2 mmol) was added and the reaction was allowed to proceed at room temperature for 2 h, monitoring the reaction for completion. 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 column chromatography was used for separation and purification. Yield: 213 mg of compound 11, 84 %; Character: white foamy solid. 1HNMR (400 MHz, DMSO-d6) δ 11.54 (s, 1H), 8.65 (s, 1H), 8.60 (t, J = 5.0 Hz, 1H), 8.35 (s, 1H), 8.07 (s, 2H), 7.83 (d, J = 1.3 Hz, 1H), 6.96 (s, 1H), 5.89 (s, 1H), 5.13 (p, J = 6.4 Hz, 1H), 4.77 (t, J = 4.9 Hz, 1H), 4.38 (d, J = 4.9 Hz, 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 = 6.6 Hz, 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 with 1 mL of dilute hydrochloric acid (1 mol / L) and refluxed at 30 °C for 1 h, and the acetal was basically completely converted into aldehyde. The excess solvent was removed by rotary evaporation, and the obtained crude product 12 could be directly used as the raw material for the next step. 1 H NMR (400 MHz, Chloroform-d) δ 9.92 (s, 1H), 9.76 (t, J = 6.2 Hz, 1H), 8.43 (d, J = 1.5 Hz, 1H), 8.32 - 8.23 (m, 3H), 8.03 (d, J = 1.4 Hz, 1H), 7.76 (dd, J = 7.5, 1.5 Hz, 1H), 6.70 (d, J = 7.5 Hz, 1H), 6.03 (m, J = 2.0, 1.1 Hz, 1H), 4.82 (m, J = 6.9 Hz, 1H), 4.19 (d, J = 8.4 Hz, 2H), 3.69 (t, J = 7.1 Hz, 4H), 2.69 (t, J = 7.1 Hz, 4H), 2.49 - 2.42 (m, 2H), 2.30 (dd, J = 5.0, 1.0 Hz, 6H), 2.28 - 2.21 (m, 2H), 1.62 - 1.51 (m, 4H), 1.44 (d, J = 6.8 Hz, 5H). ESI-MS: m / z = 583.737

[0091] Step c

[0092] Compound 12 (50 mg, 0.1 mmol), 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 allowed to proceed at room temperature for 30 min; after which NaHB(OAc)3 (54 mg, 0.2 mmol) was added and the reaction was allowed to proceed for an additional hour, monitoring the reaction by TLC until the starting material was substantially 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 a saturated NaCl solution, dried over anhydrous sodium sulfate and purified by preparative chromatography plate to obtain 12 mg of compound D7. Yield: 21%; Aspect: white solid. 1 H NMR (500 MHz, DMSO-d6) δ 8.64 (d, J = 2.6 Hz, 1H), 8.59 (t, J = 4.9 Hz, 1H), 8.35 (s, 1H), 8.29 (s, 1H), 8.05 (q, J = 5.8, 4.2 Hz, 2H), 8.01 (d, J = 2.3 Hz, 1H), 7.83 (s, 1H), 7.69 (d, J = 7.6 Hz, 1H), 6.94 (d, J = 8.9 Hz, 1H), 6.33 (d, J = 2.2 Hz, 1H), 6.02 (s, 1H), 5.88 (s, 1H), 5.13 (p, J = 6.6 Hz, 1H), 4.38 (d, J = 4.8 Hz, 2H), 4.32 - 4.24 (m, 1H), 3.55 (t, J = 5.1 Hz, 5H), 2.82 (t, J = 7.5 Hz, 2H), 2.48 - 2.42 (m, 5H), 2.32 (t, J = 7.2 Hz, 2H), 2.21 (s, 3H), 2.12 (s, 5H), 1.81 - 1.55 (m, 8H), 1.49 (d, J = 6.6 Hz, 8H), 1.41 - 1.32 (m, 2H), 0.77 (t, J = 7.3 Hz, 6H).

[0093] Example 8: Synthesis of compound D8

[0094]

[0095] Step a

[0096] Compound 6 (100 mg, 0.2 mmol), 3-bromopropionic acid methyl ester (50 mg, 0.3 mmol) were weighed into a round bottom flask, triethylamine (84 μL, 0.6 mmol) was added and the reaction was allowed to proceed at room temperature for 2 h, monitoring the reaction until it was substantially complete. The organic phase was separated with DCM (20 mL) and water (10 mL), extracted three times, the organic layers were combined and washed with a saturated NaCl solution, dried over anhydrous sodium sulfate and purified by column chromatography to obtain 103 mg of compound 13. Yield: 87%; Aspect: white foamy solid. 1H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 8.70 (d, J = 2.5 Hz, 1H), 8.59 (t, J = 5.0 Hz, 1H), 8.35 (s, 1H), 8.17 - 8.11 (m, 1H), 8.09 (s, 1H), 7.84 (d, J = 1.3 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H), 5.89 (s, 1H), 5.14 (p, J = 6.6 Hz, 1H), 4.38 (t, J = 4.9 Hz, 2H), 3.04 (s, 7H), 2.29 (t, J = 7.2 Hz, 2H), 2.22 (s, 3H), 2.12 (s, 3H), m, 1.49 (d, J = 6.6 Hz, 6H), 1.23 (s, 1H). ESI-MS: m / z = 571.291

[0097] Step b

[0098] Compound 13 (103 mg, 0.2 mmol) was dissolved in 1 mL of methanol, 879 μL of 1 mol / L NaOH solution was added, and the reaction was allowed to proceed at room temperature for 1 h, and the reaction was monitored to be substantially complete. The pH was adjusted with 1 mol / L dilute HCl solution until solid precipitated, and after standing for 10 min, the precipitate was suction filtered to obtain 80 mg of compound 14. Yield: 79%; Appearance: white solid. 1 H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 8.70 (d, J = 2.5 Hz, 1H), 8.59 (t, J = 5.0 Hz, 1H), 8.35 (s, 1H), 8.17 - 8.11 (m, 1H), 8.09 (s, 1H), 7.84 (d, J = 1.3 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H), 5.89 (s, 1H), 5.14 (p, J = 6.6 Hz, 1H), 4.38 (t, J = 4.9 Hz, 2H), 3.04 (s, 7H), 2.29 (t, J = 7.2 Hz, 2H), 2.22 (s, 3H), 2.12 (s, 3H), m, 1.49 (d, J = 6.6 Hz, 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 dissolved in a round bottom flask with anhydrous dichloromethane, and then DIPEA 73 μΐ was added. The reaction was monitored at room temperature for 2 h, and the starting material was substantially converted. The organic layer was separated with water and dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After preparative liquid separation and purification, 23 mg of compound D8 was obtained. Yield: 20%; property: white solid. 1 H NMR (500 MHz, DMSO-d6) δ 11.54 - 11.50 (m, 1H), 8.63 (d, J = 2.5 Hz, 1H), 8.58 (t, J = 5.0 Hz, 1H), 8.34 (s, 1H), 8.02 (dt, J = 13.9, 5.0 Hz, 3H), 7.98 (d, J = 2.2 Hz, 1H), 7.82 (s, 1H), 7.62 (d, J = 7.7 Hz, 1H), 6.93 (d, J = 8.9 Hz, 1H), 6.27 (d, J = 2.2 Hz, 1H), 5.99 (s, 1H), 5.86 (s, 1H), 5.11 (m, J = 6.8 Hz, 1H), 4.37 (d, J = 4.8 Hz, 2H), 4.20 (m, J = 28.5, 7.0 Hz, 2H), 3.53 (t, J = 5.0 Hz, 4H), 2.61 - 2.55 (m, 4H), 2.50 (s, 6H), 2.27 (t, J = 7.2 Hz, 2H), 2.20 (s, 3H), 2.16 (m, J = 7.7, 4.6 Hz, 1H), 2.10 (s, 3H), 2.03 (m, J = 19.3, 7.2 Hz, 2H), 1.88 (m, J = 13.3, 7.8, 5.8 Hz, 1H), 1.77 - 1.69 (m, 2H), 1.69 - 1.63 (m, 2H), 1.47 (d, J = 6.6 Hz, 7H), 0.89 (t, J = 7.3 Hz, 3H).

[0101] Example 9: Synthesis of compound D9

[0102]

[0103] The synthesis method refers to the synthesis steps of Example 8, and compound D9 (white solid, yield 32%) is obtained. 1H NMR (500 MHz, DMSO-d6) δ 11.51 (s, 1H), 8.64 (d, J = 2.6 Hz, 1H), 8.59 (t, J = 5.0 Hz, 1H), 8.35 (s, 1H), 8.17 (s, 2H), 8.04 (d, J = 8.7 Hz, 2H), 8.00 (d, J = 2.2 Hz, 1H), 7.86 (d, J = 7.3 Hz, 1H), 7.83 (d, J = 1.3 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 6.93 (d, J = 8.9 Hz, 1H), 6.29 (d, J = 2.2 Hz, 1H), 6.01 (s, 1H), 5.88 (s, 1H), 5.12 (m, J = 6.6 Hz, 1H), 4.39 (d, J = 4.9 Hz, 2H), 4.21 (m, J = 21.6, 7.1 Hz, 3H), 3.60 - 3.52 (m, 4H), 2.61 (dd, J = 8.4, 6.7 Hz, 2H), 2.52 (d, J = 4.3 Hz, 7H), 2.37 (t, J = 7.0 Hz, 2H), 2.21 (s, 3H), 2.20 - 2.14 (m, 1H), 2.12 (s, 3H), 2.09 (d, J = 7.2 Hz, 2H), 2.07 - 1.98 (m, 2H), 1.88 (m, J = 13.3, 7.8, 5.7 Hz, 1H), 1.71 (m, J = 14.9, 7.6 Hz, 3H), 1.57 - 1.42 (m, 11H), 0.92 (t, J = 7.4 Hz, 3H).

[0104] Example 10: Synthesis of compound D10

[0105]

[0106] The synthesis method refers to the synthesis steps of Example 8 to obtain compound D10 (white solid, yield 31%). 1H NMR (500 MHz, DMSO-d6) δ 8.64 (d, J = 2.6 Hz, 1H), 8.60 (d, J = 5.0 Hz, 1H), 8.35 (s, 1H), 8.18 (s, 1H), 8.07 - 8.02 (m, 2H), 8.00 (d, J = 2.2 Hz, 1H), 7.87 - 7.81 (m, 2H), 7.62 (d, J = 7.6 Hz, 1H), 6.93 (d, J = 8.9 Hz, 1H), 6.29 (d, J = 2.2 Hz, 1H), 6.01 (s, 1H), 5.88 (s, 1H), 5.12 (m, J = 6.5 Hz, 1H), 4.39 (d, J = 4.9 Hz, 2H), 4.20 (m, J = 21.3, 7.0 Hz, 3H), 3.59 - 3.51 (m, 4H), 2.61 (dd, J = 8.4, 6.7 Hz, 2H), 2.47 (d, J = 5.6 Hz, 2H), 2.33 (t, J = 7.4 Hz, 2H), 2.21 (s, 3H), 2.19 - 2.13 (m, 1H), 2.12 (s, 3H), 2.05 (t, J = 7.4 Hz, 3H), 2.01 (dd, J = 9.5, 4.3 Hz, 1H), 1.87 (s, 1H), 1.71 (q, J = 7.3 Hz, 3H), 1.48 (t, J = 6.9 Hz, 11H), 1.28 (h, J = 4.9, 4.2 Hz, 4H), 0.92 (t, J = 7.4 Hz, 3H).

[0107] Example 11: Synthesis of compound D11

[0108]

[0109] The synthesis method refers to the synthesis steps of Example 8 to obtain compound D11 (white solid, yield 42%). 1H NMR (500 MHz, DMSO-d6) δ 11.61 (s, 1H), 8.72 (d, J = 2.5 Hz, 1H), 8.67 (t, J = 5.0 Hz, 1H), 8.43 (s, 1H), 8.12 (d, J = 9.0 Hz, 2H), 8.07 (d, J = 2.2 Hz, 1H), 7.92 (d, J = 4.2 Hz, 2H), 7.70 (d, J = 7.6 Hz, 1H), 7.00 (d, J = 8.9 Hz, 1H), 6.36 (d, J = 2.2 Hz, 1H), 6.08 (s, 1H), 5.95 (s, 1H), 5.20 (m, J = 6.6 Hz, 1H), 4.46 (d, J = 4.8 Hz, 2H), 4.27 (m, J = 21.7, 7.1 Hz, 2H), 3.60 (t, J = 4.8 Hz, 4H), 2.69 (t, J = 7.6 Hz, 2H), 2.58 (t, J = 2.9 Hz, 3H), 2.50 (t, J = 5.0 Hz, 4H), 2.35 (t, J = 7.4 Hz, 2H), 2.29 (s, 3H), 2.24 (m, J = 11.8, 4.0 Hz, 1H), 2.19 (s, 3H), 2.12 (t, J = 7.4 Hz, 3H), 2.07 (dd, J = 13.5, 6.7 Hz, 1H), 1.94 (m, J = 13.3, 6.7 Hz, 1H), 1.79 (m, J = 7.3 Hz, 3H), 1.62 - 1.53 (m, 9H), 1.51 (d, J = 6.9 Hz, 2H), 1.34 (s, 6H), 1.31 (d, J = 9.2 Hz, 2H), 0.99 (t, J = 7.3 Hz, 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 dissolved in a round bottom flask with dry dichloromethane and DIPEA 43 μΐ^was added. The reaction was left to react at room temperature for 2 h, monitoring the reaction until the starting material was almost completely converted. The organic layer was separated with water and dichloromethane, washed with saturated brine and dried over anhydrous sodium sulfate. After purification by preparative liquid chromatography, 18 mg of compound D12 were obtained. Yield: 25%; Aspect: white solid. 1H NMR (500 MHz, DMSO-d6) δ 11.58 - 11.46 (m, 1H), 8.63 (d, J = 2.5 Hz, 1H), 8.57 (t, J = 5.0 Hz, 1H), 8.34 (s, 1H), 8.03 (d, J = 8.6 Hz, 2H), 7.98 (t, J = 1.9 Hz, 1H), 7.82 (s, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.63 (dd, J = 7.7, 2.5 Hz, 1H), 6.92 (dd, J = 9.0, 2.1 Hz, 1H), 6.27 (d, J = 2.2 Hz, 1H), 6.00 (s, 1H), 5.86 (s, 1H), 5.10 (m, J = 6.8 Hz, 1H), 4.37 (d, J = 4.9 Hz, 2H), 4.31 (m, J = 7.3 Hz, 1H), 4.20 (m, J = 7.5, 6.6 Hz, 1H), 3.88 (s, 2H), 3.61 (t, J = 5.7 Hz, 2H), 3.54 (t, J = 4.9 Hz, 4H), 2.59 (t, J = 6.3 Hz, 3H), 2.55 (q, J = 6.6, 5.0 Hz, 5H), 2.49 (d, J = 5.5 Hz, 6H), 2.20 (s, 4H), 2.12 - 2.00 (m, 5H), 1.94 (m, J = 13.5, 7.1 Hz, 1H), 1.78 - 1.68 (m, 3H), 1.67 (d, J = 7.4 Hz, 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]

[0115] Compound 6 (300 mg, 0.6 mmol), 5-bromovaleric acid methyl ester (175 mg, 0.9 mmol) were weighed in a round bottom flask, anhydrous potassium carbonate (248 mg, 1.8 mmol) was added and the reaction was monitored for completion at room temperature for 2 h. The organic layer was separated with DCM (50 mL) and water (25 mL) and extracted three times, the organic layer was combined and washed with saturated NaCl solution, dried over anhydrous sodium sulfate and column chromatography was performed for purification to obtain 303 mg of compound 15. Yield: 82%; Nature: white foamy solid. 1H NMR (500 MHz, Chloroform-d) δ 9.86 (s, 1H), 8.43 (d, J = 1.5 Hz, 1H), 8.30 (s, 1H), 8.29 - 8.23 (m, 2H), 8.02 (d, J = 1.6 Hz, 1H), 7.76 (dd, J = 7.5, 1.5 Hz, 1H), 6.69 (d, J = 7.5 Hz, 1H), 6.03 (dq, J = 2.1, 1.1 Hz, 1H), 4.82 (m, J = 6.9 Hz, 1H), 4.19 (d, J = 8.2 Hz, 2H), 3.69 (t, J = 7.1 Hz, 4H), 3.64 (s, 2H), 2.70 (t, J = 7.1 Hz, 4H), 2.52 - 2.46 (m, 2H), 2.37 - 2.31 (m, 2H), 2.28 (dd, J = 11.0, 1.1 Hz, 6H), 1.63 - 1.50 (m, 4H), 1.44 (d, J = 6.9 Hz, 5H).

[0116] Step b

[0117] Compound 15 (303 mg, 0.6 mmol) was dissolved in 1 mL of methanol, 3 mL of 1 mol / L NaOH solution was added, and the reaction was allowed to proceed at room temperature for 1 h, and the reaction was monitored to be substantially complete. The pH was adjusted with 1 mol / L dilute HCl solution until solid precipitated, and the precipitate was allowed to settle for 10 min, and then suction filtration was performed to obtain 274 mg of compound 16. Property: 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 dissolved in a round-bottom flask with anhydrous dichloromethane, and 73 μL of DIPEA was added. The reaction was allowed to proceed at room temperature for 2 h, and the reaction was monitored to be substantially complete. The organic layer was separated with water and dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After preparative liquid chromatography purification, 23 mg of compound D13 was obtained. Yield: 20%; property: white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.54 (s, 1H), 10.61 (s, 1H), 8.65 - 8.57 (m, 2H), 8.36 (d, J = 1.4 Hz, 2H), 8.26 (s, 1H), 8.09 - 7.98 (m, 3H), 7.83 (d, J = 1.2 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 6.91 (d, J = 9.0 Hz, 1H), 5.89 (s, 1H), 5.76 (s, 1H), 5.13 (m, J = 6.6 Hz, 1H), 4.39 (d, J = 4.8 Hz, 2H), 3.92 (s, 2H), 3.60 (d, J = 7.9 Hz, 1H), 3.53 (t, J = 5.1 Hz, 4H), 2.88 (s, 2H), 2.63 (s, 1H), 2.44 (t, J = 4.9 Hz, 4H), 2.31 (t, J = 7.1 Hz, 2H), 2.22 (s, 3H), 2.12 (s, 3H), 2.07 (t, J = 7.1 Hz, 2H), 1.90 (d, J = 11.5 Hz, 1H), 1.78 (s, 2H), 1.57 - 1.42 (m, 11H), 1.36 - 1.21 (m, 11H), 1.11 (d, J = 12.3 Hz, 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 dissolved in a round bottom flask with dry dichloromethane and DIPEA 43 μΐ^was added. The reaction was monitored for 2 h at room temperature and the starting material was converted substantially. The organic layer was separated with water and dichloromethane, washed with saturated brine and dried over anhydrous sodium sulfate. After preparative liquid separation and purification, 16 mg of compound D14 was obtained. Yield: 20%; Character: white solid. 1H NMR (500 MHz, DMSO-d6) δ 12.31 (s, 1H), 11.53 (s, 1H), 8.64 (d, J = 2.6 Hz, 1H), 8.59 (t, J = 4.9 Hz, 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 = 8.9 Hz, 1H), 6.70 (s, 1H), 5.88 (s, 1H), 5.12 (p, J = 6.6 Hz, 1H), 4.38 (d, J = 4.8 Hz, 3H), 4.04 (s, 2H), 3.93 (d, J = 13.6 Hz, 1H), 3.55 (t, J = 4.9 Hz, 4H), 3.04 (t, J = 12.7 Hz, 1H), 2.73 (m, J = 7.9, 3.7 Hz, 1H), 2.63 - 2.54 (m, 1H), 2.46 (t, J = 5.0 Hz, 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 = 14.0 Hz, 2H), 1.58 - 1.50 (m, 1H), 1.53 (s, 2H), 1.49 (d, J = 6.6 Hz, 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 mmol), 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 allowed to proceed for 30 min at room temperature; after which NaHB(OAc)3(55 mg, 0.3 mmol) was added and the reaction was allowed to proceed for another hour, monitoring the reaction by TLC until the starting material was almost 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 a saturated NaCl solution, dried over anhydrous sodium sulfate and purified by preparative chromatography plate to obtain 21 mg of compound D15. Yield: 25%; Aspect: white solid. 1H NMR (500 MHz, DMSO-d6) δ 11.54 (s, 1H), 8.65 (d, J = 2.5 Hz, 1H), 8.59 (d, J = 5.1 Hz, 1H), 8.35 (s, 1H), 8.27 (s, 1H), 8.05 (d, J = 8.3 Hz, 2H), 7.83 (s, 1H), 7.38 (s, 1H), 6.95 (d, J = 8.9 Hz, 1H), 6.71 (s, 1H), 5.88 (s, 1H), 5.13 (d, J = 6.6 Hz, 1H), 4.38 (d, J = 4.8 Hz, 2H), 4.04 (s, 2H), 3.55 (t, J = 5.0 Hz, 5H), 2.90 (d, J = 10.9 Hz, 2H), 2.46 (d, J = 9.9 Hz, 4H), 2.30 (m, J = 15.1, 7.3 Hz, 4H), 2.21 (s, 3H), 2.12 (s, 3H), 1.90 (t, J = 11.3 Hz, 2H), 1.76 (d, J = 12.4 Hz, 2H), 1.62 (t, J = 11.1 Hz, 2H), 1.48 (t, J = 7.8 Hz, 10H), 1.30 (m, J = 7.6 Hz, 2H), 1.22 (d, J = 4.5 Hz, 1H), 1.16 (s, 9H).

[0126] Example 16: Synthesis of compound D16

[0127]

[0128] 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) were weighed in a round bottom flask and dissolved in 1 mL of dry DMF and allowed to react at room temperature for two hours, monitoring the conversion of the starting material 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 a saturated NaCl solution, dried over anhydrous sodium sulfate and purified by preparative liquid chromatography to obtain 16 mg of compound D16; yield: 17%, aspect: white solid. 1H NMR (500 MHz, DMSO-d6) δ 12.22 (s, 1H), 11.51 (s, 1H), 8.63 (d, J = 2.6 Hz, 1H), 8.57 (t, J = 5.0 Hz, 1H), 8.33 (s, 1H), 8.03 (q, J = 3.8, 2.6 Hz, 2H), 7.81 (d, J = 1.2 Hz, 1H), 7.36 (s, 1H), 6.93 (d, J = 9.0 Hz, 1H), 6.70 (s, 1H), 5.86 (s, 1H), 5.15 - 5.07 (m, 1H), 4.36 (d, J = 4.9 Hz, 2H), 4.03 (s, 2H), 3.53 (t, J = 4.9 Hz, 4H), 2.97 (d, J = 11.0 Hz, 2H), 2.52 (d, J = 4.4 Hz, 4H), 2.19 (s, 3H), 2.10 (s, 3H), 2.03 (s, 2H), 1.74 (d, J = 3.9 Hz, 2H), 1.61 (d, J = 11.8 Hz, 2H), 1.47 (d, J = 6.6 Hz, 6H), 1.21 (s, 1H), 1.15 (s, 9H).

[0129] Example 17: Synthesis of compound D17

[0130]

[0131] 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) were weighed in a round bottom flask and dissolved in dry dichloromethane, then DIPEA 43 μΐ^was added. The reaction was left to react at room temperature for 2 h, monitoring the reaction until the starting material was almost completely converted. The organic layer was separated with water and dichloromethane, washed with saturated brine and dried over anhydrous sodium sulfate. Compound D17 was obtained after preparative liquid chromatography purification (white solid, 32%) 1H NMR (400 MHz, DMSO-d6) δ 11.55 (s, 1H), 10.60 (s, 1H), 8.67 - 8.57 (m, 2H), 8.35 (d, J = 6.6 Hz, 2H), 8.26 (s, 1H), 8.09 - 7.97 (m, 3H), 7.83 (d, J = 1.2 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 6.91 (d, J = 9.0 Hz, 1H), 5.89 (s, 1H), 5.13 (p, J = 6.6 Hz, 1H), 4.39 (d, J = 4.9 Hz, 2H), 4.12 (d, J = 5.6 Hz, 0H), 3.93 (s, 2H), 3.60 (s, 1H), 3.52 (t, J = 5.0 Hz, 4H), 3.17 (d, J = 3.9 Hz, 1H), 2.88 (s, 2H), 2.62 (s, 2H), 2.43 (t, J = 5.0 Hz, 4H), 2.28 (t, J = 7.3 Hz, 2H), 2.22 (s, 3H), 2.12 (s, 3H), 2.05 (t, J = 7.3 Hz, 2H), 1.90 (d, J = 12.8 Hz, 1H), 1.78 (d, J = 6.9 Hz, 3H), 1.50 (d, J = 6.5 Hz, 8H), 1.45 (d, J = 7.3 Hz, 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] Synthesis method according to Example 17 to give compound D18 (white solid, 32%) 1H NMR (400 MHz, Methanol-d4) δ 8.54 (d, J = 2.5 Hz, 1H), 8.38 (s, 1H), 8.22 (s, 1H), 8.04 - 7.96 (m, 2H), 7.92 (d, J = 1.2 Hz, 1H), 7.78 (d, J = 1.3 Hz, 1H), 7.40 (s, 1H), 6.94 (d, J = 8.9 Hz, 1H), 6.14 (s, 1H), 5.10 (p, J = 6.7 Hz, 1H), 4.60 (s, 2H), 3.97 (s, 2H), 3.78 (s, 1H), 3.67 (t, J = 4.9 Hz, 5H), 3.37 (s, 2H), 3.36 (s, 1H), 3.10 (s, 2H), 2.97 (s, 2H), 2.67 (t, J = 4.9 Hz, 4H), 2.44 (s, 3H), 2.30 - 2.24 (m, 3H), 2.10 (d, J = 10.4 Hz, 2H), 2.00 (d, J = 10.6 Hz, 2H), 1.59 (d, J = 6.6 Hz, 5H), 1.46 (q, J = 11.2 Hz, 3H), 1.35 (s, 6H).

[0135] Example 19: Synthesis of compound D19

[0136]

[0137] Synthesis method according to example 16, replacing SNS-032 by hu754 to give compound D19 (white solid, yield 24%) 1 H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.66 (d, J = 2.6 Hz, 1H), 8.59 (t, J = 4.8 Hz, 1H), 8.34 (d, J = 16.6 Hz, 2H), 8.22 (s, 1H), 8.07 (q, J = 4.6, 3.5 Hz, 2H), 7.97 (s, 1H), 7.84 (d, J = 1.2 Hz, 1H), 7.68 (s, 1H), 7.50 (d, J = 7.6 Hz, 1H), 6.97 (d, J = 8.9 Hz, 1H), 5.89 (s, 1H), 5.14 (p, J = 6.6 Hz, 1H), 4.39 (d, J = 4.9 Hz, 2H), 3.94 (s, 2H), 3.57 (d, J = 5.7 Hz, 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 = 6.5 Hz, 6H), 1.27 (s, 9H).

[0138] Example 20: Synthesis of compound D20

[0139]

[0140] 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) were weighed into a round bottom flask and dissolved in dry dichloromethane. DIPEA 43 μL was added. The reaction was allowed to proceed at room temperature for 2 h, monitoring the reaction until the starting material was substantially converted. The organic layer was separated with water and dichloromethane, washed with saturated brine and dried over anhydrous sodium sulfate. Compound D20 was obtained after preparative liquid chromatography purification. 1 H NMR (400 MHz, DMSO-d6) δ 11.53 (s, 1H), 9.18 (s, 1H), 8.66 (d, J = 2.6 Hz, 1H), 8.59 (t, J = 5.0 Hz, 1H), 8.36 (s, 1H), 8.27 (s, 1H), 8.07 (d, J = 7.0 Hz, 2H), 7.84 (s, 1H), 7.69 (d, J = 7.4 Hz, 1H), 7.53 (s, 1H), 7.41 (t, J = 7.1 Hz, 1H), 7.22 (dd, J = 8.4, 6.8 Hz, 1H), 7.08 (dd, J = 11.4, 2.5 Hz, 1H), 6.97 (d, J = 9.0 Hz, 1H), 6.89 (td, J = 8.3, 2.4 Hz, 1H), 5.89 (s, 1H), 5.14 (p, J = 6.6 Hz, 1H), 4.39 (d, J = 4.8 Hz, 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 = 7.0 Hz, 2H), 1.91 (s, 2H), 1.80 (s, 2H), 1.50 (d, J = 6.6 Hz, 6H), 1.33 - 1.22 (m, 7H).

[0141] Example 21: Synthesis of compound D21

[0142]

[0143] Synthesis was performed according to the procedure described in Example 20 to give compound D21 (white solid, 47%) 1H NMR (400 MHz, DMSO-d6) δ 11.53 (s, 1H), 9.18 (s, 1H), 8.66 (d, J = 2.6 Hz, 1H), 8.60 (t, J = 4.9 Hz, 1H), 8.36 (s, 1H), 8.27 (s, 1H), 8.11 - 8.03 (m, 2H), 7.84 (d, J = 1.3 Hz, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.54 (s, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.22 (dd, J = 8.4, 6.8 Hz, 1H), 7.08 (dd, J = 11.4, 2.4 Hz, 1H), 6.97 (d, J = 8.9 Hz, 1H), 6.89 (td, J = 8.4, 2.4 Hz, 1H), 5.89 (s, 1H), 5.14 (p, J = 6.6 Hz, 1H), 4.39 (d, J = 4.9 Hz, 2H), 3.77 (s, 3H), 3.61 (t, J = 4.9 Hz, 5H), 3.45 (s, 2H), 2.97 (s, 2H), 2.55 (t, J = 5.0 Hz, 4H), 2.22 (s, 3H), 2.13 (s, 3H), 1.92 (d, J = 11.3 Hz, 2H), 1.80 (d, J = 12.0 Hz, 2H), 1.50 (d, J = 6.6 Hz, 6H), 1.44 - 1.27 (m, 3H).

[0144] Example 22: Synthesis of compound D22

[0145]

[0146] Synthesis method according to example 16, replacing SNS-032 by hu627 to give compound D22 (white solid, yield 17%) 1H NMR (400 MHz, Methanol-d4) δ 8.18 (s, 1H), 8.02 (s, 1H), 7.57 - 7.48 (m, 2H), 7.45 - 7.39 (m, 2H), 7.30 (d, J = 1.9 Hz, 1H), 7.07 - 6.99 (m, 2H), 6.11 (s, 1H), 4.50 (s, 2H), 4.14 (t, J = 5.2 Hz, 2H), 3.93 (d, J = 22.2 Hz, 4H), 3.69 - 3.59 (m, 1H), 3.41 - 3.33 (m, 2H), 3.16 (d, J = 7.0 Hz, 1H), 3.16 - 3.03 (m, 4H), 2.96 (s, 2H), 2.62 (d, J = 11.7 Hz, 1H), 2.41 (s, 3H), 2.33 (s, 3H), 2.25 (s, 3H), 2.09 (p, J = 6.0 Hz, 5H), 1.79 - 1.71 (m, 2H), 1.71 - 1.56 (m, 2H), 1.37 (d, J = 12.0 Hz, 2H), 1.34 (s, 6H), 1.30 (s, 2H), 1.29 (d, J = 15.1 Hz, OH), 1.20 (t, J = 7.1 Hz, 1H), 0.99 - 0.86 (m, 3H).

[0147] Example 23: Synthesis of compound D23

[0148]

[0149] Step a

[0150] 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) were weighed into a two-necked flask, 1,4-dioxane 10 mL was used to dissolve the raw materials, and 2 mL of water was added, replaced with nitrogen for three times and protected with nitrogen; react at 100 °C for 4 h, monitor the reaction by TLC until the raw material is basically completely converted. First remove the excess dioxane, then extract with ethyl acetate and water three times, wash the combined organic layer with saturated NaCl aqueous solution, and dry over anhydrous sodium sulfate. Purify by column chromatography (DCM:MeOH = 20:1), finally obtain compound 1823 33 mg, yield: 66%. 1H NMR (400 MHz, DMSO-d6) δ 11.48 (s, 1H), 8.18 (t, J = 5.0 Hz, 1H), 7.64 - 7.51 (m, 2H), 7.36 (d, J = 1.9 Hz, 1H), 7.18 (d, J = 1.9 Hz, 1H), 7.02 - 6.94 (m, 2H), 5.86 (s, 1H), 4.65 (s, 2H), 4.29 (d, J = 4.9 Hz, 2H), 3.94 (s, 2H), 3.83 (d, J = 11.2 Hz, 2H), 3.57 (s, 2H), 3.25 (t, J = 11.3 Hz, 2H), 3.13 - 3.00 (m, 2H), 2.22 (d, J = 9.8 Hz, 6H), 2.13 - 2.09 (m, 3H), 1.67 (d, J = 12.5 Hz, 2H), 1.53 (td, J = 13.0, 11.6, 6.1 Hz, 2H), 1.08 (s, 12H), 0.83 (t, J = 6.9 Hz, 3H).

[0151] Step b

[0152] Compound 18 was dissolved with 2 mL of methanol, then 3 mL of 1 mol / L NaOH solution was added dropwise at room temperature, and after 1 h of reaction at room temperature, the raw material was substantially completely converted, and the pH was adjusted with 1 M dilute hydrochloric acid solution until solid precipitated, and after standing for a while, the filter cake was extracted by suction filtration to obtain 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 dissolved in a round-bottom flask with anhydrous dichloromethane, and then 95 μL of DIPEA was added. After 2 h of reaction at room temperature, the raw material was substantially converted. The organic layer was separated with water and dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After preparative liquid separation and purification, 87 mg of compound D23 was obtained. Yield: 49%; property: white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.46 (s, 1H), 9.08 (s, 1H), 8.22 (s, 1H), 8.17 (t, J = 5.0 Hz, 1H), 8.01 - 7.94 (m, 2H), 7.67 (s, 1H), 7.60 - 7.54 (m, 2H), 7.45 (d, J = 7.5 Hz, 1H), 7.36 (d, J = 1.9 Hz, 1H), 7.19 (d, J = 1.8 Hz, 1H), 7.05 - 6.99 (m, 2H), 5.86 (s, 1H), 4.49 (s, 2H), 4.29 (d, J = 5.0 Hz, 2H), 3.94 (s, 2H), 3.87 - 3.78 (m, 2H), 3.66 (d, J = 7.7 Hz, 4H), 3.22 (s, 3H), 3.14 - 2.97 (m, 4H), 2.88 (s, 2H), 2.22 (d, J = 9.9 Hz, 6H), 2.10 (s, 3H), 1.92 (d, J = 11.8 Hz, 2H), 1.80 (d, J = 12.3 Hz, 2H), 1.66 (d, J = 12.4 Hz, 2H), 1.58 - 1.34 (m, 5H), 1.26 (s, 9H), 0.83 (t, J = 6.9 Hz, 3H).

[0155] Example 24: Synthesis of compound D24

[0156]

[0157] Synthesis method refers to Example 23 to obtain compound D24 (white solid, yield 37%) 1H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H), 9.08 (s, 1H), 8.20 (d, J = 16.4 Hz, 2H), 7.96 (s, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.68 (s, 1H), 7.59 - 7.51 (m, 2H), 7.45 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 1.9 Hz, 1H), 7.18 (d, J = 1.8 Hz, 1H), 7.04 - 6.96 (m, 2H), 5.86 (s, 1H), 4.29 (d, J = 5.0 Hz, 2H), 4.00 (t, J = 6.0 Hz, 2H), 3.94 (s, 2H), 3.83 (dd, J = 10.9, 3.4 Hz, 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 = 4.0 Hz, 5H), 1.91 (s, 2H), 1.80 (s, 2H), 1.71 - 1.62 (m, 7H), 1.52 (qd, J = 11.8, 4.3 Hz, 2H), 1.27 (s, 9H), 0.83 (t, J = 6.9 Hz, 3H).

[0158] Example 25: Synthesis of compound D25

[0159]

[0160] Step a

[0161] 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) were weighed into a two-necked flask, 1,4-dioxane 10 mL was used to dissolve the raw materials, and 2 mL of water was added, replaced with nitrogen for three times and protected with nitrogen; react at 100 °C for 4 h, monitor the reaction by TLC until the raw material is basically completely converted. First remove the excess dioxane, then extract with ethyl acetate and water three times, combine the organic layer and wash with saturated NaCl aqueous solution, dry over anhydrous sodium sulfate. Purify by column chromatography (PE:EA = 10:1), finally obtain 213 mg of compound 17, yield: 57%. 1H NMR (400 MHz, DMSO-d6) δ 11.46 (s, 1H), 8.18 (t, J = 5.0 Hz, 1H), 7.60 - 7.51 (m, 2H), 7.36 (d, J = 1.8 Hz, 1H), 7.18 (d, J = 1.8 Hz, 1H), 7.08 - 6.99 (m, 2H), 5.86 (s, 1H), 4.36 (dd, J = 6.2, 4.6 Hz, 2H), 4.29 (d, J = 4.9 Hz, 2H), 3.82 (dd, J = 6.2, 4.6 Hz, 4H), 3.57 (s, 1H), 3.25 (d, J = 22.3 Hz, 2H), 3.08 (q, J = 7.0 Hz, 2H), 3.04 - 2.97 (m, 1H), 2.23 (s, 3H), 2.20 (s, 3H), 2.10 (s, 3H), 1.66 (d, J = 12.0 Hz, 2H), 1.53 (tt, J = 12.5, 6.4 Hz, 2H), 0.83 (t, J = 6.9 Hz, 3H).

[0162] Step b

[0163] Compound 17 (50 mg, 0.08 mmol), compound hu754 (40 mg, 0.1 mmol) and anhydrous K2CO3 (35 mg, 0.3 mmol) were weighed into a round bottom flask, the starting materials were dissolved in 1 mL of DMF, the reaction was monitored by TLC after 2 h at room temperature until the starting material was almost completely converted. Extraction with water and DCM was performed three times, the organic layers were combined and washed with saturated aqueous NaCl, dried over anhydrous sodium sulfate. After preparative liquid phase separation and purification, 13 mg of compound D25 was obtained. Yield: 17%, white solid. 1H NMR (400 MHz, Methanol-d4) δ 8.20 (s, 1H), 7.53 - 7.49 (m, 2H), 7.41 (d, J = 2.0 Hz, 1H), 7.28 (d, J = 1.8 Hz, 1H), 7.19 - 7.15 (m, 1H), 7.14 (s, 1H), 7.05 - 6.99 (m, 2H), 6.90 (dd, J = 11.1, 2.4 Hz, 1H), 6.78 (td, J = 8.3, 2.4 Hz, 1H), 6.10 (s, 1H), 4.49 (s, 2H), 4.14 (t, J = 5.3 Hz, 2H), 3.91 (dq, J = 10.1, 3.6 Hz, 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, 6.3 Hz, 4H), 1.78 - 1.71 (m, 2H), 1.63 (qd, J = 11.7, 4.3 Hz, 2H), 1.42 - 1.25 (m, 6H), 0.89 (t, J = 7.0 Hz, 3H).

[0164] Example 26: Synthesis of compound D26

[0165]

[0166] Synthesis method refers to Example 25 to obtain compound D26 (white solid, yield 31%) 1H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 1H), 8.38 (s, 1H), 8.21 (s, 1H), 8.17 (t, J = 5.0 Hz, 1H), 7.96 (s, 1H), 7.67 (s, 1H), 7.58 - 7.52 (m, 2H), 7.50 (d, J = 7.1 Hz, 1H), 7.34 (d, J = 1.9 Hz, 1H), 7.17 (d, J = 1.8 Hz, 1H), 6.99 (d, J = 2.0 Hz, 2H), 5.86 (s, 1H), 4.29 (d, J = 4.9 Hz, 2H), 4.00 (t, J = 6.4 Hz, 3H), 3.93 (s, 2H), 3.82 (d, J = 11.3 Hz, 3H), 3.24 (t, J = 11.5 Hz, 3H), 3.08 (q, J = 7.1 Hz, 3H), 3.01 (s, 1H), 2.87 (s, 2H), 2.72 (s, 2H), 2.67 (s, 1H), 2.22 (d, J = 9.0 Hz, 6H), 2.10 (s, 3H), 1.97 (d, J = 16.4 Hz, 4H), 1.73 (d, J = 7.1 Hz, 2H), 1.66 (d, J = 12.0 Hz, 2H), 1.61 - 1.43 (m, 6H), 1.26 (s, 10H), 0.82 (t, J = 6.9 Hz, 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 dissolved in a round bottom flask with dry dichloromethane and DIPEA 95 μΐ^was added. The reaction was monitored at room temperature for 2 h and the starting material was converted completely. The organic layer was separated with water and dichloromethane, washed with saturated brine and dried over anhydrous sodium sulfate. Compound D27 was obtained after preparative liquid chromatography purification (white solid, 32% yield) 1H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H), 9.18 (s, 1H), 8.27 (s, 1H), 8.18 (t, J = 5.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.60 - 7.55 (m, 2H), 7.54 (s, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.37 (d, J = 1.9 Hz, 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, 2.4 Hz, 1H), 5.86 (s, 1H), 4.49 (s, 2H), 4.29 (d, J = 5.0 Hz, 2H), 3.83 (d, J = 11.7 Hz, 2H), 3.77 (s, 3H), 3.67 (d, J = 9.6 Hz, 1H), 3.44 (s, 2H), 3.26 (t, J = 11.4 Hz, 3H), 3.14 - 2.98 (m, 3H), 2.23 (d, J = 10.1 Hz, 6H), 2.11 (s, 3H), 1.93 (d, J = 11.8 Hz, 2H), 1.80 (d, J = 12.1 Hz, 2H), 1.67 (d, J = 12.3 Hz, 2H), 1.59 - 1.47 (m, 2H), 1.45 - 1.34 (m, 2H), 1.27 (q, J = 11.9 Hz, 2H), 0.83 (t, J = 6.9 Hz, 3H).

[0170] Example 28: Synthesis of compound D28

[0171]

[0172] Synthetic procedure with reference to example 27 gave compound D28 (white solid, yield 29%) 1H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H), 9.19 (s, 1H), 8.27 (s, 1H), 8.18 (t, J = 5.0 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.55 (d, J = 2.1 Hz, 1H), 7.53 (d, J = 2.2 Hz, 2H), 7.42 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 1.9 Hz, 1H), 7.22 (dd, J = 8.4, 6.7 Hz, 1H), 7.18 (d, J = 1.8 Hz, 1H), 7.08 (dd, J = 11.5, 2.4 Hz, 1H), 7.02 - 6.97 (m, 2H), 6.89 (td, J = 8.4, 2.4 Hz, 1H), 5.86 (s, 1H), 4.29 (d, J = 5.0 Hz, 2H), 4.00 (t, J = 5.9 Hz, 2H), 3.83 (d, J = 11.4 Hz, 2H), 3.77 (s, 3H), 3.57 (d, J = 14.7 Hz, 3H), 3.30 - 3.20 (m, 2H), 3.13 - 2.97 (m, 3H), 2.22 (d, J = 9.2 Hz, 6H), 2.11 (s, 5H), 1.90 (s, 2H), 1.80 (s, 2H), 1.67 (dd, J = 13.4, 8.0 Hz, 7H), 1.53 (tt, J = 12.0, 6.0 Hz, 2H), 1.26 (d, J = 9.8 Hz, 4H), 0.83 (t, J = 6.9 Hz, 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 in a round bottom flask, 1 mL of DMF was used to dissolve the starting materials, the reaction was monitored by TLC after 2 h at room temperature until the starting material was almost completely converted. Extraction with water and DCM three times, the organic layer was combined and washed with saturated aqueous NaCl, dried over anhydrous sodium sulfate. Compound D29 (white solid, yield 26%) was obtained after preparative liquid separation and purification 1H NMR (400 MHz, Methanol-d4) δ 8.16 (s, 1H), 8.00 (s, 1H), 7.53 - 7.49 (m, 2H), 7.42 - 7.38 (m, 2H), 7.28 (d, J = 1.8 Hz, 1H), 7.03 - 6.99 (m, 2H), 6.09 (s, 1H), 4.49 (s, 2H), 4.12 (t, J = 5.2 Hz, 2H), 3.94 (s, 2H), 3.90 (dt, J = 12.1, 3.3 Hz, 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 = 11.7 Hz, 1H), 2.39 (s, 3H), 2.31 (s, 3H), 2.23 (s, 3H), 2.07 (dd, J = 8.6, 4.7 Hz, 4H), 1.78 - 1.69 (m, 2H), 1.69 - 1.55 (m, 2H), 1.32 (s, 10H), 0.88 (t, J = 6.9 Hz, 3H).

[0176] Example 30: Synthesis of compound D30

[0177]

[0178] Synthesis method according to example 29 to give compound D30 (white solid, yield 13%) 1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 8.38 (s, 1H), 8.26 (s, 1H), 8.17 (t, J = 4.9 Hz, 1H), 7.55 (s, 1H), 7.52 (d, J = 4.8 Hz, 2H), 7.46 (d, J = 7.6 Hz, 1H), 7.34 (d, J = 2.0 Hz, 1H), 7.21 (dd, J = 8.4, 6.7 Hz, 1H), 7.17 (d, J = 1.9 Hz, 1H), 7.07 (dd, J = 11.4, 2.4 Hz, 1H), 6.99 (d, J = 8.6 Hz, 2H), 6.89 (td, J = 8.5, 2.5 Hz, 1H), 5.86 (s, 1H), 4.28 (d, J = 4.9 Hz, 2H), 4.00 (t, J = 6.3 Hz, 2H), 3.82 (d, J = 11.4 Hz, 3H), 3.76 (s, 4H), 3.25 (t, J = 11.1 Hz, 5H), 3.13 - 2.96 (m, 5H), 2.65 (d, J = 28.6 Hz, 4H), 2.21 (d, J = 8.8 Hz, 6H), 2.10 (s, 3H), 1.93 (s, 4H), 1.78 - 1.61 (m, 5H), 1.50 (d, J = 22.0 Hz, 7H), 1.21 (s, 4H), 0.82 (t, J = 6.9 Hz, 3H).

[0179] Bioassay determination and research

[0180] 1. CDK9 enzyme activity assay

[0181] Add CDK9 / cycT1, CDK7 / 9tide, ATP and test compound to each well, total reaction volume is 5 μL, incubate at 37 °C for 120 min. Then add buffer according to the instruction of ADP-Glo kinase assay kit (Promega, V9102) and read luminescence on a plate reader (EnVision). Calculate IC 50 values with SoftMax Pro.

[0182] 2. EZH2 enzyme activity assay

[0183] Add EZH2 Y641F-containing PRC2 complex, substrate, SAM and test compound to each well, with a total reaction system of 10 μL, and react for 4 h at room temperature in the dark. Then add 10 μL of Eu-labeled H3K27 Me3 antibody and Streptavidin-XL665 mixture to each well, incubate for 1 h at room temperature, and then measure the fluorescence values at 620 nm and 665 nm using a multi-label microplate analysis system (PerkinElmer Envision), and calculate the HTRF signal ratio (665 nm / 620 nm) of each well. The IC 50 values of the compounds are calculated using SoftMax Pro 5.4.1 software.

[0184] 3. Cell IC50determination

[0185] In each well of a 96-well plate, 2000 cells were inoculated with 200 μL of fresh culture medium. After 7 days of incubation with the compound, 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 50 values were calculated using SoftMax Pro.

[0186] 4. Western Blot experiment

[0187] An appropriate amount of human lymphoma cells was plated in a 6-well plate, and the cells were centrifuged after drug treatment for subsequent experiments. According to the amount of cells, an appropriate amount of 1X loading buffer was added, and the cells were lysed and boiled at 100°C for 20 min. The same volume of sample was taken for SDS-PAGE, and after electrophoresis, the protein on the gel was transferred to a nitrocellulose membrane using a Kinesis rapid transfer apparatus. According to the size of the protein, the corresponding band was cut off, blocked with 5% skim milk in TBST for 1 h, and incubated with the primary antibody at 4°C overnight. The excess primary antibody was washed with TBST for 10 min each time for a total of three times. The secondary antibody was incubated at room temperature for 1 h, and the excess secondary antibody was washed with TBST for 10 min each time for a total of three times. Finally, the band was developed and photographed using a Bio-Rad colorimeter.

[0188] 5. Apoptosis detection

[0189] After co-incubation of an appropriate amount of lymphoma cells with the compound in a six-well plate, the cells were centrifuged and washed twice with PBS, then stained with an apoptosis detection kit (Vazyme, A211-01). After 10 min of staining in the dark, the cells were detected using a BD Calibur and the data were analyzed using FlowJo_V10.

[0190] Detection results

[0191] 1. Enzyme inhibition activity and anti-proliferation activity of two DLBCL cell lines

[0192] These compounds showed CDK9 / EZH2 dual-target inhibition activity compared with positive control compounds, in which compounds D15 and D16 not only showed dual-target inhibition at the enzyme level, but also showed excellent anti-proliferation activity on DLBCL cell lines. Therefore, we selected D15 and D16 for downstream signaling pathway inhibition studies.

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

[0194]

[0195]

[0196] 2. Effect 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 down-regulated Mcl-1 and c-Myc, they could synergistically reduce H3K27me3 caused by CDK9 inhibitors; showing a CDK9 / EZH2 dual-function inhibition effect. In addition, we found that D15 or D16 could significantly reduce the expression of H3K27ac ( Figure 1 A-B), and high expression of H3K27ac was 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 the literature, CDK9 inhibitors alone can induce tumor cell apoptosis. By studying it was found ( Figure 2 A-B), that the dual-target inhibitor D16 showed more obvious ability to induce tumor cell apoptosis compared with CDK9 inhibitor SNS-032 and EZH2 inhibitor C24.

[0200] In addition, we found that compound D16 could cause a decrease in Rad51, a DNA double-strand break repair protein, while also causing a significant up-regulation of H2AX, a marker protein for DNA double-strand damage (indicating accumulation of DNA damage in cells), and cleavage of PARP, a single-strand DNA repair protein ( Figure 2The results showed that the CDK9 / EZH2 dual-target inhibitor D16 had better inhibitory activity on the proliferation of solid tumor cells than the single-target inhibitors SNS-032 or C24.

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

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

[0203]

[0204] The above merely describes preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A CDK9 / EZH2 dual-target inhibitor, characterized in that, Its structure is shown in structure I-III: Where n is 1, 2, 3, 4, 5, 6, 7 or 8, and m is 1, 2, 3 or 4; Wherein, the R group is: 。 2. The CDK9 / EZH2 dual-target inhibitor according to claim 1, characterized in that, The structure is as follows:

3. A pharmaceutical composition, characterized in that, It comprises one or more of the CDK9 / EZH2 dual-target inhibitors or pharmaceutically acceptable salts thereof as described in any one of claims 1-2, and optionally a pharmaceutically acceptable carrier.

4. The use of a CDK9 / EZH2 dual-target inhibitor as described in any one of claims 1-2 in the preparation of a drug for treating tumors.

5. The application according to claim 4, characterized in that, The tumor is either a solid tumor or a hematoma.

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