Indole derivative as well as preparation method and anti-liver cancer application thereof

Abstract

The invention belongs to the technical field of organic compound synthesis and medical application, and particularly relates to an indole derivative as well as a preparation method and anti-liver cancer application thereof. The structure of the compound is shown as a general formula I. The indole derivative with the activity of inhibiting ADAM17 / HDAC, provided by the invention, can inhibit the growth of liver cancer cells at a low dosage and has an obvious anti-metastasis effect, so that the compound plays an important role in preparation of drugs for preventing or treating tumors. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of organic compound synthesis and medical application, and specifically relates to an indole derivative and a preparation method thereof and an application thereof in preventing liver cancer. Background Art

[0002] Globally, liver cancer is listed as one of the "four major cancers in the world", among which hepatocellular carcinoma (HCC) is the most common type of liver cancer. The disease not only has high morbidity and mortality, but also has a high recurrence rate (70%) and is highly invasive. Despite the continuous development of targeted therapy and immunotherapy, their efficacy is still limited, resulting in a five-year survival rate of less than 15% for patients, which has brought a heavy medical burden to patients around the world. At present, a variety of anti-liver cancer drugs approved for clinical use, such as sorafenib and lenvatinib, can delay tumor progression to a certain extent, but their therapeutic effects are often limited, and their ability to control tumor metastasis is significantly insufficient.

[0003] Notch1 is an important receptor in the Notch signaling pathway and plays an important role in the proliferation and apoptosis of HCC. Secondly, it can promote the epithelial-mesenchymal transition (EMT) process, so that liver cancer cells gain stronger invasiveness and migration ability. At the same time, the high expression of Notch1 has been proven to be associated with poor prognosis of liver cancer. Notch1 is regulated by ADAM17 and HDAC. ADAM17 in the integrin-metalloproteinase family (ADAMs) is a key cleavage enzyme in the Notch1 signaling pathway. It directly affects cell differentiation, proliferation and apoptosis by mediating the cleavage and activation of the Notch1 receptor. The ADAM17 inhibitor ZLDI-8 can inhibit the metastasis of hepatocellular carcinoma by reversing the EMT pathway and regulating the Notch signaling pathway. In addition, histone deacetylases (HDACs) also play an important role in the regulation of the Notch signaling pathway. The HDAC inhibitor Valproic acid (VPA) can downregulate the expression of Notch1 and Notch2 at the mRNA level and reduce the expression of Notch1 target gene HES1. The HDAC inhibitor Trichostatin A (TSA) can inhibit EMT induced by the Notch pathway and play a dual role in regulating Notch signaling-mediated smooth muscle cell differentiation. Therefore, it is believed that drugs that dually inhibit ADAM17 and HDAC can achieve the effect of inhibiting liver cancer cell proliferation and inhibiting liver cancer cell metastasis by regulating Notch1 and epigenetic modification. And there are no reports on dual ADAM17 / HDAC inhibitors. Summary of the invention

[0004] The present invention aims to provide an indole derivative and a method for preparing the indole derivative, and also points out the application of the indole derivative in preparing drugs for treating liver cancer.

[0005] Technical solution:

[0006] In a first aspect, the present invention provides an indole derivative or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in general formula I:

[0007]

[0008] Wherein: R1 group is a substituent at position 3 or 4 on the benzene ring; R1 is -OCH3, -N(CH3), -COOCH3, -NHCOCH3, NHCOOC(CH3)3, -F, -CF3, -CN, -SO2CH3 or -Phenyl; X=O, C; n=1,2;

[0009] The R2 group is connected by an amide bond or a hydrazide bond. Wherein R3 is selected from

[0010]

[0011] R4 is selected from

[0012]

[0013] The second aspect of the present invention provides the following compounds or pharmaceutically acceptable salts thereof, selected from:

[0014]

[0015]

[0016] The third aspect of the present invention provides a method for preparing the compound, comprising the following steps:

[0017] (1) Compound 1a-1q is used as a raw material, potassium carbonate and 1-bromo-2-chloroethane are added to an acetonitrile solution of compound 1a-1q, the mixture is heated to 80-100° C. and refluxed for 10-15 hours, cooled to room temperature, the reaction solution is filtered, and the filtrate is concentrated under reduced pressure; after concentration, the crude product is purified by silica gel chromatography to obtain compounds 2a-2q respectively;

[0018] (2) Using compound 1r-s as raw material, triethylamine and p-toluenesulfonyl chloride were added to a dichloromethane solution of 1r-s, and the mixture was reacted at room temperature for 6 to 12 hours. After concentration under reduced pressure, the crude product was purified by silica gel column chromatography to obtain compounds 2r-2s respectively;

[0019] (3) adding potassium carbonate and 2-methylindole-3-carboxaldehyde to a solution of compound 2a-2s in N,N-dimethylformamide, heating the solution to 90-100° C. and reflux for 12-18 hours; diluting the solution with water and extracting the solution with ethyl acetate; drying the mixed organic layer with anhydrous sodium sulfate, filtering the solution and concentrating the solution under reduced pressure; subjecting the crude reaction product to flash column chromatography to obtain compounds 3a-3s, respectively;

[0020] (4) Dissolving compounds 3a-3s and 2-mercapto-4,6-dihydroxypyrimidine in a methanol solution, heating to 70-80° C. for reaction for 2-4 hours; filtering after the reaction, washing the filter cake with methanol to obtain compounds 1-19 respectively;

[0021] (5) Compound 3a and a phenylhydrazide compound were dissolved in anhydrous ethanol, a catalytic amount of acetic acid was added, and the mixture was refluxed at 70 to 80° C. for 2 to 4 hours. After the reaction, the mixture was filtered and the filter cake was washed with ethanol. After vacuum drying, compounds 20 to 25 were obtained respectively.

[0022] (6) Compound 2a and methyl 2-methyl-3-indolecarboxylate were dissolved in N,N-dimethylformamide, refluxed at 95°C for 16 to 20 hours, diluted with water and extracted with ethyl acetate; the mixed organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure; the crude product was dissolved in a methanol solution at 60 to 70°C, slowly cooled and recrystallized to obtain compound 4a; compound 4a was dissolved in a solvent of dioxane: methanol: water = 2:1:1, potassium hydroxide was added, refluxed at 80 to 100°C until the reaction was completed, concentrated under reduced pressure to remove the organic solvent, diluted with water and adjusted to pH 6 to 7, filtered, and the filter cake was dried under vacuum to obtain compound 5a; compound 5a was dissolved in anhydrous dichloromethane, a catalytic amount of N,N-dimethylformamide was added, oxalyl chloride was added dropwise under ice bath and nitrogen protection for 30 minutes, and concentrated under reduced pressure to obtain compound 6a;

[0023] (7) Compound 6a was dissolved in anhydrous pyridine, and an aminobenzoic acid methyl ester compound was added. The reaction was carried out at 80°C to 90°C under nitrogen protection for 5 to 7 hours; hydrochloric acid-water was added to dilute and extracted with ethyl acetate; the mixed organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and purified by slurrying with methanol and petroleum ether: dichloromethane = 3:1 system in sequence, filtered, and the filter cake was vacuum dried, redissolved in methanol: dichloromethane = 1:1 system, and the pH value was adjusted to 11 to 13 with 10M NaOH, and 50% hydroxylamine solution was added dropwise under ice bath conditions. After the reaction was complete, the mixture was concentrated under reduced pressure, diluted with water, and the pH was slowly adjusted to 7 to 8 with 4M HCl, filtered, and the filter cake was vacuum dried to obtain compounds 26-29, respectively.

[0024] Preferably, the phenylhydrazide compound in step (5) is one of 4-phenylsemicarbazide, 4-phenyl-3-thiosemicarbazide, 2-hydroxybenzohydrazide, 2-aminobenzohydrazide, 3-pyridinecarboxyhydrazide and pyrazine-2-carboxyhydrazide.

[0025] Preferably, the methyl aminobenzoate compound in step (7) is one of methyl 4-aminobenzoate, methyl 2-aminobenzoate, methyl 3-aminobenzoate, and methyl 5-aminopyridine-2-carboxylate.

[0026] The fourth aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the compound or a pharmaceutically acceptable salt thereof. Further, the pharmaceutical composition is prepared by adding one or more pharmaceutically acceptable excipients to the compound.

[0027] The fifth aspect of the present invention proposes the use of the compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating liver cancer (including hepatocellular carcinoma, cholangiocarcinoma and mixed liver cancer).

[0028] The sixth aspect of the present invention provides the use of the compound or a pharmaceutically acceptable salt thereof in the preparation of ADAM17 and HDAC inhibitors.

[0029] The seventh aspect of the present invention provides the use of the compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cholangiocarcinoma.

[0030] Beneficial effects:

[0031] Compared with the existing technology, the present invention has the following significant characteristics: the indole derivatives involved have novel structures, high in vitro antiproliferative activity, ADAM17 and HDAC inhibitory activity, and compounds 1-34 have good anti-liver cancer cell proliferation activity, with a liver cancer cell inhibition rate of 0.5 to 15 μM. The preferred compounds were selected for ADAM17 / HDAC targeting confirmation, the effects on liver cancer cell proliferation and apoptosis, the effects on liver cancer cell metastasis, the acute toxicity evaluation on Kunming mice and other related studies, and it was concluded that compound ZSNI-21 inhibits ADAM17 and HDAC at the same time and affects their downstream expression; compound ZSNI-21 can induce apoptosis at a high level; compound ZSNI-21 can reduce the expression of Vimentin and other EMT-related proteins Slug and MMP9, and has the ability to resist liver cancer cell migration; no obvious in vivo toxicity was found at an in vivo dose of 2000 mg / kg.

[0032] In summary, the indole derivatives proposed in the present invention can inhibit the growth of liver cancer cells at a low dose and have obvious anti-metastasis effects. Therefore, this type of compound plays an important role in the preparation of prevention or treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a schematic diagram of the preparation method of compound 1-29;

[0034] Figure 2 is the target affinity of ZSNI-21 for ADAM17 and HDAC; in the figure, A is a schematic diagram of the expression of different targets in liver cancer cells after ZSNI-21 treatment at the thermodynamic level, B is the CETSA analysis of ADAM17 expression in liver cancer cells after ZSNI-21 treatment, C is the CETSA analysis of HDAC1 expression in liver cancer cells after ZSNI-21 treatment, D is the CETSA analysis of HDAC2 expression in liver cancer cells after ZSNI-21 treatment, E is the CETSA analysis of HDAC3 expression in liver cancer cells after ZSNI-21 treatment, and F is the CETSA analysis of HDAC6 expression in liver cancer cells after ZSNI-21 treatment;

[0035] Figure 3 : is the enzyme inhibitory activity of ZSNI-21 against ADAM17 and HDAC; in the figure, A is a schematic diagram of the enzyme activity inhibition of ZSNI-21 against HDAC1; B is a schematic diagram of the enzyme activity inhibition of ZSNI-21 against HDAC2; C is a schematic diagram of the enzyme activity inhibition of ZSNI-21 against HDAC6; D is a schematic diagram of the enzyme activity inhibition of ZSNI-21 against ADAM17;

[0036] Figure 4 Schematic diagram of the effect of ZSNI-21 at different concentrations on the expression of NICD, a downstream pathway of ADAM17, and Acetylated histone H3 and Acetylated histone H4, a downstream pathway of HDAC; Figure A is a schematic diagram of the WB results of the expression of different proteins in liver cancer cells after ZSNI-21 treatment, B is a schematic diagram of the statistical results of NICD expression in liver cancer cells after ZSNI-21 treatment, C is a schematic diagram of the statistical results of Acetylated histone H3 expression in liver cancer cells after ZSNI-21 treatment, and D is a schematic diagram of the statistical results of Acetylated histone H4 expression in liver cancer cells after ZSNI-21 treatment;

[0037] Figure 5 Schematic diagram of ZSNI-21 promoting apoptosis of liver cancer cells in Bel-7402 cell line; Figure A is a colony formation experiment of cells treated with ZSNI-21 at different concentrations; B is a schematic diagram of the statistical results of the colony formation experiment; C is a Hoechst 33342 staining experiment of cells treated with ZSNI-21 at different concentrations, and D is a flow cytometry experiment of cells treated with ZSNI-21 at different concentrations;

[0038] Figure 6 The figure is the in vivo safety evaluation of ZSNI-21; Figure A shows the weight changes of mice within 15 days; Figure B shows the representative H&E staining photos of the main organs of mice in the normal group and the 2000 mg / kg group;

[0039] Figure 7 Figure 1 is a schematic diagram of the anti-metastasis of liver cancer cells by ZSNI-21 in HCC-LM3 cell line; Figure A is a representative image of the wound healing experiment; images were taken at 0 hours and 48 hours after treatment with ZSNI-21 (1.5, 3 and 6 μM), lenvatinib (3 μM) and ZLDI-8 (3 μM), respectively; B is a histogram showing the ratio of wound healing area to total wound area after 48 hours (n=4, *P<0.05, **P<0.01, ***P<0.001); C is immunofluorescence staining of Vimentin in LM3 cell line (scale bar=50 μm); D is Western Blot analysis was performed to detect the expression levels of EMT-related proteins after ZSNI-21 treatment for 48 hours; E is a histogram showing the fluorescence ratio (Vimentin / DAPI) in HCC-LM3 cells (n=4, *P<0.05, **P<0.01, ***P<0.001); FH are the quantitative results of different protein expression levels (n=3, *P<0.05, **P<0.01, ***P<0.001). DETAILED DESCRIPTION

[0040] The present invention is described below by means of specific examples, but the present invention is not limited to these examples.

[0041] The structure of the compound was determined by nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HR-MS). NMR was measured using a Bruker AVANCE-300 / 600 nuclear magnetic resonance spectrometer, the solvent for the measurement was DMSO-d6, and the internal standard was TMS. Column chromatography used 200-300 mesh silica gel (produced by Qingdao Ocean Chemical Plant).

[0042] Example 1: 1-(2-Chloroethoxy)-4-methoxybenzene (Compound 2a)

[0043] like Figure 1 As shown, potassium carbonate (3 equivalents) and 1-bromo-2-chloroethane (3 equivalents) were added to an acetonitrile solution of 4-methoxyphenol (compound 1a) (1 equivalent), which was heated to 90°C and refluxed for 12 hours, then cooled to room temperature (20°C), the salt in the reaction solution was filtered, and the filtrate was concentrated. After concentration, the crude product was purified by silica gel chromatography to obtain the desired product compound 2a. Yield 80%, white crystals. ESI-MS: m / z 187.4 [M+H] + .

[0044] Example 2: 1-(2-Chloroethoxy)-3-methoxybenzene (Compound 2b)

[0045] Compound 1a was replaced by 3-methoxyphenol (Compound 1b), and other conditions were unchanged. The experimental operation of Example 1 was repeated to obtain Compound 2b as white crystals with a yield of 84%. ESI-MS: m / z 187.5 [M+H] + .

[0046] Example 3: 4-(2-chloroethoxy)-1,2-dimethoxybenzene (Compound 2c)

[0047] 3,4-dimethoxyphenol (Compound 1c) was used to replace 1a, and other conditions remained unchanged. The experimental operation of Example 1 was repeated to obtain Compound 2c as white crystals with a yield of 80%. ESI-MS: m / z 217.2 [M+H] + .

[0048] Example 4: 4-(2-chloroethoxy)-N,N-dimethylaniline (Compound 2d)

[0049] 4-Dimethylaminophenol (Compound 1d) was used to replace 1a, and other conditions remained unchanged. The experimental operation of Example 1 was repeated to obtain Compound 2d as white crystals with a yield of 77%. ESI-MS: m / z 200.5 [M+H] + .

[0050] Example 5: 3-(2-Chloroethoxy)-N,N-dimethylaniline (Compound 2e)

[0051] 3-Dimethylaminophenol (Compound 1e) was used to replace 1a, and other conditions remained unchanged. The experimental operation of Example 1 was repeated to obtain Compound 2e as white crystals with a yield of 79%. ESI-MS: m / z 200.4 [M+H] + .

[0052] Example 6: Methyl 4-(2-chloroethoxy)benzoate (Compound 2f)

[0053] The experimental operation of Example 1 was repeated with methyl 4-hydroxybenzoate (Compound 1f) replacing 1a, and other conditions remained unchanged to obtain Compound 2f as white crystals with a yield of 82%. ESI-MS: m / z 215.1 [M+H] + .

[0054] Example 7: Methyl 3-(2-chloroethoxy)benzoate (Compound 2g)

[0055] The experimental operation of Example 1 was repeated with 3-hydroxybenzoic acid methyl ester (Compound 1g) replacing 1a, and other conditions remained unchanged to obtain Compound 2g as white crystals with a yield of 80%. ESI-MS: m / z 215.4 [M+H] + .

[0056] Example 8: N-(4-(2-chloroethoxy)phenyl)acetamide (Compound 2h)

[0057] The experimental procedure of Example 1 was repeated with N-(4-hydroxyphenyl)acetamide (compound 1h) replacing 1a, and other conditions remained unchanged to obtain compound 2h as white crystals with a yield of 79%. ESI-MS: m / z 214.3 [M+H] + .

[0058] Example 9: N-(3-(2-chloroethoxy)phenyl)acetamide (Compound 2i)

[0059] N-(4-hydroxyphenyl)acetamide (compound 1i) was used to replace 1a, and other conditions remained unchanged. The experimental operation of Example 1 was repeated to obtain compound 2i as white crystals with a yield of 79%. ESI-MS: m / z 214.2 [M+H] + .

[0060] Example 10: tert-Butyl (4-(2-chloroethoxy)phenyl)carbamate (Compound 2j)

[0061] The experimental procedure of Example 1 was repeated with 4-(BOC-amino)phenol (Compound 1j) replacing 1a, and other conditions remained unchanged to obtain Compound 2j as white crystals with a yield of 88%. ESI-MS: m / z 271.1 [M+H] + .

[0062] Example 11: 4-(2-Chloroethoxy)benzonitrile (Compound 2k)

[0063] The experimental procedure of Example 1 was repeated with 4-hydroxybenzonitrile (compound 1k) replacing 1a, and other conditions remained unchanged to obtain compound 2k as white crystals with a yield of 82%. ESI-MS: m / z 182.2 [M+H] + .

[0064] Example 12: 3-(2-chloroethoxy)benzonitrile (Compound 21)

[0065] The experimental operation of Example 1 was repeated with 3-hydroxybenzonitrile (Compound 11) replacing 1a, and other conditions remained unchanged to obtain Compound 21 as white crystals with a yield of 80%. ESI-MS: m / z 182.4 [M+H] + .

[0066] Example 13: 1-(2-Chloroethoxy)-4-trifluoromethylbenzene (Compound 2m)

[0067] The experimental procedure of Example 1 was repeated with 4-trifluoromethylphenol (Compound 1m) replacing 1a, and other conditions remained unchanged to obtain Compound 2m as white crystals with a yield of 83%. ESI-MS: m / z 224.1 [M+H] + .

[0068] Example 14: 1-(2-Chloroethoxy)-4-methylsulfonylbenzene (Compound 2n)

[0069] The experimental operation of Example 1 was repeated with 4-methylsulfonylphenol (Compound 1n) replacing 1a, and other conditions remained unchanged to obtain Compound 2n as white crystals with a yield of 89%. ESI-MS: m / z 234.1 [M+H] + .

[0070] Example 15: 4-(2-chloroethoxy)-1,1'-biphenyl (Compound 2o)

[0071] The experimental operation of Example 1 was repeated with 4-hydroxybiphenyl (compound 1o) replacing 1a, and other conditions remained unchanged to obtain compound 2o as white crystals with a yield of 85%. ESI-MS: m / z 233.5 [M+H] + .

[0072] Example 16: 3-(2-Chloroethoxy)-1,1'-biphenyl (Compound 2p)

[0073] The experimental operation of Example 1 was repeated with 3-hydroxybiphenyl (compound 1p) replacing 1a and other conditions unchanged to obtain compound 2p as white crystals with a yield of 86%. ESI-MS: m / z 233.2 [M+H] + .

[0074] Example 17: 2-(2-Chloroethoxy)naphthalene (Compound 2q)

[0075] The experimental procedure of Example 1 was repeated with 2-naphthol (compound 1q) replacing 1a and other conditions unchanged to obtain compound 2q as white crystals with a yield of 83%. ESI-MS: m / z 207.3 [M+H] + .

[0076] Example 18: 4-Fluorophenylethyl 4-methylbenzenesulfonate (Compound 2r)

[0077] like Figure 1As shown, triethylamine (0.5 equivalents) and p-toluenesulfonyl chloride (2 equivalents) were added to a dichloromethane solution of p-fluorophenylethanol (compound 1r) (1 equivalent), and the reaction was carried out in an ice bath (0°C) for 2 hours, and then the solvent was distilled off under reduced pressure. The product was purified by silica gel column to obtain compound 2r. A colorless oil with a yield of 70%. ESI-MS: m / z 295.5 [M+H] + .

[0078] Example 19: 4-methoxyphenethyl p-toluenesulfonate (Compound 2s)

[0079] The experimental procedure of Example 15 was repeated with p-methoxyphenylethanol (compound 1s) replacing 1r and other conditions unchanged to obtain 2s as a light yellow oil with a yield of 56%. ESI-MS: m / z 306.1 [M+H] + .

[0080] Example 20: 1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indole-3-carbaldehyde (Compound 3a)

[0081] like Figure 1 As shown, potassium carbonate (3 equivalents) and 2-methylindole-3-carboxaldehyde (0.8 equivalents) were added to a solution of compound 2a (1 equivalent) in N,N-dimethylformamide, which was heated to 95°C and refluxed for 16 hours. Diluted with water and extracted with ethyl acetate. The mixed organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude reaction product was subjected to flash column chromatography to obtain compound 3a. Yield 65%, light yellow solid. ESI-MS: m / z 310.1 [M+H] + .

[0082] Example 21: 1-(2-(3-methoxyphenoxy)ethyl)-2-methyl-1H-indole-3-carbaldehyde (Compound 3b)

[0083] Compound 2b was used to replace compound 2a, and other conditions were kept unchanged. The experimental operation of Example 20 was repeated to obtain compound 3b as a light yellow solid with a yield of 62%. ESI-MS: m / z 310.1 [M+H] + .

[0084] Example 22: 1-(2-(3,4-dimethoxyphenoxy)ethyl)-2-methyl-1H-indole-3-carbaldehyde (Compound 3c)

[0085] Compound 2c was used to replace compound 2a, and other conditions were unchanged. The experimental operation of Example 20 was repeated to obtain compound 3c as a light yellow solid with a yield of 70%. ESI-MS: m / z 340.5 [M+H] + .

[0086] Example 23: 1-(2-(4-(dimethylamino)phenoxy)ethyl)-2-methyl-1H-indole-3-carbaldehyde (Compound 3d)

[0087] Compound 2d was used to replace compound 2a, and other conditions remained unchanged. The experimental operation of Example 20 was repeated to obtain compound 3d as a light brown solid with a yield of 74%. ESI-MS: m / z 323.2 [M+H] + .

[0088] Example 24: 1-(2-(3-(dimethylamino)phenoxy)ethyl)-2-methyl-1H-indole-3-carbaldehyde (Compound 3e)

[0089] Compound 2e was used to replace compound 2a, and other conditions remained unchanged. The experimental operation of Example 20 was repeated to obtain compound 3e as a light brown solid with a yield of 77%. ESI-MS: m / z 323.3 [M+H] + .

[0090] Example 25: Methyl 4-(2-(3-formyl-2-methyl-1H-indol-1-yl)ethoxy)benzoate (Compound 3f)

[0091] Compound 2f was used to replace compound 2a, and other conditions were kept unchanged. The experimental operation of Example 20 was repeated to obtain compound 3f as a light yellow solid with a yield of 72%. ESI-MS: m / z 338.1 [M+H] + .

[0092] Example 26: Methyl 3-(2-(3-formyl-2-methyl-1H-indol-1-yl)ethoxy)benzoate (Compound 3g)

[0093] Compound 2a was replaced by compound 2g, and other conditions were unchanged. The experimental operation of Example 20 was repeated to obtain compound 3g as a light yellow solid with a yield of 75%. ESI-MS: m / z 338.2 [M+H] + .

[0094] Example 27: N-(4-(2-(3-formyl-2-methyl-1H-indol-1-yl)ethoxy)phenyl)acetamide (Compound 3h)

[0095] Compound 2h was used to replace compound 2a, and other conditions remained unchanged. The experimental operation of Example 20 was repeated to obtain compound 3h as a light yellow solid with a yield of 69%. ESI-MS: m / z 337.2 [M+H] + .

[0096] Example 28: N-(3-(2-(3-formyl-2-methyl-1H-indol-1-yl)ethoxy)phenyl)acetamide (Compound 3i)

[0097] Compound 2i was used to replace compound 2a, and other conditions remained unchanged. The experimental operation of Example 20 was repeated to obtain compound 3i as a light yellow solid with a yield of 65%. ESI-MS: m / z 337.3 [M+H] + .

[0098] Example 29: tert-Butyl (4-(2-(3-carboxaldehyde-2-methyl-1H-indol-1-yl)ethoxy)phenyl)carbamate (3j)

[0099] Compound 2j was used to replace compound 2a, and other conditions were unchanged. The experimental operation of Example 20 was repeated to obtain compound 3j as a light yellow solid with a yield of 61%. ESI-MS: m / z 394.2 [M+H] + .

[0100] Example 30: 4-(2-(3-formyl-2-methyl-1H-indol-1-yl)ethoxy)benzonitrile (Compound 3k)

[0101] Compound 2k was used to replace compound 2a, and other conditions remained unchanged. The experimental operation of Example 20 was repeated to obtain compound 3k as a light yellow solid with a yield of 64%. ESI-MS: m / z 305.1 [M+H] + .

[0102] Example 31: 3-(2-(3-formyl-2-methyl-1H-indol-1-yl)ethoxy)benzonitrile (Compound 31)

[0103] Compound 21 was used to replace compound 2a, and other conditions were kept unchanged. The experimental operation of Example 20 was repeated to obtain compound 31 as a light yellow solid with a yield of 66%. ESI-MS: m / z 305.2 [M+H] + .

[0104] Example 32: 2-Methyl-1-(2-(4-trifluoromethylphenoxy)ethyl)-1H-indole-3-carbaldehyde (Compound 3m)

[0105] Compound 2m was used to replace compound 2a, and other conditions were unchanged. The experimental operation of Example 20 was repeated to obtain compound 3m as a light yellow solid with a yield of 69%. ESI-MS: m / z 347.1 [M+H] + .

[0106] Example 33: 2-Methyl-1-(2-(4-methylsulfonylphenoxy)ethyl)-1H-indole-3-carbaldehyde (Compound 3n)

[0107] Compound 2n was used to replace compound 2a, and other conditions remained unchanged. The experimental operation of Example 20 was repeated to obtain compound 3n as a light yellow solid with a yield of 62%. ESI-MS: m / z 357.1 [M+H] + .

[0108] Example 34: 1-(2-([1,1'-biphenyl]-4-oxy)ethyl)-2-methyl-1H-indole-3-carbaldehyde (Compound 3o)

[0109] Compound 2o was used to replace compound 2a, and other conditions remained unchanged. The experimental operation of Example 20 was repeated to obtain compound 3o as a light yellow solid with a yield of 68%. ESI-MS: m / z 356.2 [M+H] + .

[0110] Example 35: 1-(2-([1,1'-biphenyl]-3-oxy)ethyl)-2-methyl-1H-indole-3-carbaldehyde (Compound 3p)

[0111] Compound 2p was used to replace compound 2a, and other conditions remained unchanged. The experimental operation of Example 20 was repeated to obtain compound 3p as a light yellow solid with a yield of 64%. ESI-MS: m / z 356.3 [M+H] + .

[0112] Example 36: 2-Methyl-1-(2-(naphthalene-2-oxy)ethyl)-1H-indole-3-carbaldehyde (Compound 3q)

[0113] Compound 2q was used to replace compound 2a, and other conditions were unchanged. The experimental operation of Example 20 was repeated to obtain compound 3q as a light yellow solid with a yield of 64%. ESI-MS: m / z 356.3 [M+H] + .

[0114] Example 37: 1-(4-Fluorophenethyl)-2-methyl-1H-indole-3-carbaldehyde (Compound 3r)

[0115] Compound 2r was used to replace compound 2a, and other conditions remained unchanged. The experimental operation of Example 20 was repeated to obtain compound 3r as a light yellow solid with a yield of 82%. ESI-MS: m / z 282.1 [M+H] + .

[0116] Example 38: 1-(4-methoxyphenethyl)-2-methyl-1H-indole-3-carbaldehyde (Compound 3s)

[0117] Compound 2s was used to replace compound 2a, and other conditions remained unchanged. The experimental operation of Example 20 was repeated to obtain compound 3s as a light yellow solid with a yield of 75%. ESI-MS: m / z 293.1 [M+H] + .

[0118] Example 39: 5-((1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)-2-thionedihydropyrimidine-4,6(1H,5H)-dione (Compound 1)

[0119] like Figure 1 As shown, compound 3a (1 equivalent) and 2-mercapto-4,6-dihydroxypyrimidine (1.2 equivalents) were dissolved in a methanol solution, which was heated to 75°C for 3 hours. After the reaction was completed, the solution was filtered while hot, and the filter cake was washed with methanol to obtain compound 1. An orange-yellow solid with a yield of 72%. 1 H NMR (400MHz, DMSO-d6): δ12.16–12.12(m,1H),12.03(d,J=1.9Hz,1H),8.54(s,1H),7.69(d,J=8.1Hz,1H),7.30(dt,J=8. 2,4.2Hz,1H),7.23(d,J=3.9Hz,2H),6.80(s,4H),4.73(t,J=5.0Hz,2H),4.29(t,J=5.1Hz,2H),3.65(s,3H),2.67(s,3H). 13 C NMR (101MHz, DMSO-d6): δ178.24,163.13,159.85,154.32,154.07,152.34,147.23,138.00,126.39,12 4.05,123.57,122.65,115.68(2C),115.07(2C),113.68,111.56,109.85,66.92,55.80,44.17,13.08.

[0120] Example 40: 5-((1-(2-(3-methoxyphenoxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)-2-thionedihydropyrimidine-4,6(1H,5H)-dione (Compound 2)

[0121] Replacing compound 3a with compound 3b and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 2 as an orange-yellow solid with a yield of 75%. 1H NMR (400MHz, DMSO-d6): δ12.15(s,1H),12.04(s,1H),8.55(s,1H),7.70(d,J=8.1Hz,1H),7.31(dt,J=8.3,4.3Hz,1H),7.23(d, J=4.1Hz,2H),7.13(t,J=8.2Hz,1H),6.49–6.40(m,3H),4.75(t,J=5.1Hz,2H),4.34(t,J=5.1Hz,2H),3.68(s,3H),2.68(s,3H). 13 C NMR (101MHz, DMSO-d6): δ178.24,163.12,160.95,159.85,159.55,154.30,147.23,137.97,130.48,126.4 0,124.03,123.58,122.67,113.67,111.57,109.90,107.27,107.06,100.99,66.50,55.57,44.05,13.11.

[0122] Example 41: 5-((1-(2-(3,4-dimethoxyphenoxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)-2-thionedihydropyrimidine-4,6(1H,5H)-dione (Compound 3)

[0123] Replacing compound 3a with compound 3c and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 3 as an orange-yellow solid with a yield of 70%. 1 H NMR (400MHz, DMSO-d6): δ12.17–12.12(m,1H),12.03(s,1H),8.55(s,1H),7 .70(d,J=8.1Hz,1H),7.31(dp,J=8.5,4.3Hz,1H),7.24(d,J=4.0Hz,2H),6.7 9(d,J=8.8Hz,1H),6.46(d,J=2.8Hz,1H),6.38(dd,J=8.7,2.8Hz,1H),4.74 (t,J=5.1Hz,2H),4.30(t,J=5.1Hz,2H),3.65(d,J=8.3Hz,6H),2.68(s,3H). 13C NMR (101MHz, DMSO-d6): δ178.25,163.12,159.85,154.34,152.88,150.13,147.23,143.86,137.95,126.42,1 24.04,123.59,122.69,113.68,113.14,111.55,109.88,104.73,101.15,66.91,56.51,55.95,44.16,13.15.

[0124] Example 42: 5-((1-(2-(4-(dimethylamino)phenoxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)-2-thionedihydropyrimidine-4,6(1H,5H)-dione (Compound 4)

[0125] Replacing compound 3a with compound 3d and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 4 as an orange-yellow solid with a yield of 74%. 1 H NMR (400MHz, DMSO-d6): δ12.14(s,1H),12.03(s,1H),8.54(s,1H),7.69(d,J=8.1Hz,1H),7.29(tt,J=9.5,4.7Hz,1H),7.23(d ,J=4.2Hz,2H),6.78–6.69(m,2H),6.69–6.59(m,2H),4.71(t,J=5.1Hz,2H),4.25(t,J=5.0Hz,2H),2.76(s,6H),2.67(s,3H). 13 C NMR (101MHz, DMSO-d6): δ178.23,163.13,159.85,154.39,150.31,147.22,146.09,138.01,126.39,124. 08,123.57,122.66,115.53(2C),114.64(2C),113.69,111.56,109.78,66.90,44.26,41.53(2C),13.08.

[0126] Example 43: 5-((1-(2-(3-(dimethylamino)phenoxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)-2-thionedihydropyrimidine-4,6(1H,5H)-dione (Compound 5)

[0127] Replacing compound 3a with compound 3e and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 5 as an orange-yellow solid with a yield of 77%. 1H NMR (400MHz, DMSO-d6): δ12.17–12.12(m,1H),12.03(d,J=1.8Hz,1H),8.54(s,1H), 7.70(d,J=8.1Hz,1H),7.31(ddd,J=8.2,5.0,3.5Hz,1H),7.28–7.19(m,2H),7.01(t ,J=8.2Hz,1H),6.28(dd,J=8.3,2.4Hz,1H),6.18(dd,J=7.9,2.3Hz,1H),6.12(t,J= 2.4Hz,1H),4.73(t,J=5.1Hz,2H),4.32(t,J=5.1Hz,2H),2.81(s,6H),2.68(s,3H). 13 C NMR (101MHz, DMSO-d6): δ178.24,163.13,159.84,159.38,154.36,152.18,147.22,137.99,130.07,126.40 ,124.09,123.57,122.67,113.69,111.58,109.82,106.24,102.56,99.19,66.25,44.20,40.57(2C),13.08.

[0128] Example 44: Methyl 4-(2-(3-((4,6-dioxo-2-thionotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methyl-1H-indol-1-yl)ethoxy)benzoate (Compound 6)

[0129] Replacing compound 3a with compound 3f and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 6 as an orange-yellow solid with a yield of 75%. 1 H NMR (400MHz, DMSO-d6): δ12.17(d,J=1.8Hz,1H),12.06(d,J=1.8Hz,1H),8.54(s,1H),7.91–7.80(m,2H),7.72(d,J=8. 0Hz,1H),7.37–7.17(m,3H),7.05–6.94(m,2H),4.80(t,J=5.0Hz,2H),4.45(t,J=5.1Hz,2H),3.78(s,3H),2.69(s,3H). 13C NMR (101MHz, DMSO-d6): δ178.25,166.24,163.10,162.12,159.84,154.15,147.23,137.97,131.69(2C),1 26.37,124.02,123.58,122.75,122.66,114.92(2C),113.66,111.56,110.05,66.87,52.30,43.86,13.08.

[0130] Example 45: Methyl 3-(2-(3-((4,6-dioxo-2-thionotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methyl-1H-indol-1-yl)ethoxy)benzoate (Compound 7)

[0131] Replacing compound 3a with compound 3g and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 7 as an orange-yellow solid with a yield of 78%. 1 H NMR (400MHz, DMSO-d6): δ12.17(d,J=1.8Hz,1H),12.06(d,J=1.8Hz,1H),8.54 (s,1H),7.78–7.69(m,1H),7.52(dt,J=7.8,1.2Hz,1H),7.42–7.35(m,2H),7.3 1(ddd,J=8.2,5.8,2.7Hz,1H),7.24–7.20(m,2H),7.17(ddd,J=8.3,2.7,1.1H z,1H),4.79(t,J=5.0Hz,2H),4.43(t,J=4.9Hz,2H),3.82(s,3H),2.70(s,3H). 13 C NMR (101MHz, DMSO-d6): δ178.25,166.35,163.11,159.85,158.38,154.24,147.24,138.02,131.44,130.47,1 26.35,124.07,123.56,122.65,122.35,120.25,114.59,113.67,111.61,109.94,66.84,52.69,43.96,13.07.

[0132] Example 46: N-(4-(2-(3-((4,6-dioxo-2-thionotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methyl-1H-indol-1-yl)ethoxy)phenyl)acetamide (Compound 8)

[0133] Replacing compound 3a with compound 3h and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 8 as an orange-yellow solid with a yield of 73%. 1 H NMR (400MHz, DMSO-d6): δ12.17(d,J=1.8Hz,1H),12.06(s,1H),9.77(s,1H),8.54(s,1H),7.74–7.66(m,1H),7.48–7.37(m,2H),7.30( ddd,J=8.2,5.0,3.5Hz,1H),7.27–7.17(m,2H),6.87–6.75(m,2H),4.75(t,J=4.8Hz,2H),4.35–4.26(m,2H),2.68(s,3H),1.97(s,3H). 13 C NMR (101MHz, DMSO-d6): δ178.24,168.19,163.12,159.85,154.30,153.99,147.23,137.99,133.46,126.3 9,124.04,123.57,122.65,120.85(2C),114.78(2C),113.67,111.55,109.88,66.58,44.09,24.25,13.09.

[0134] Example 47: N-(3-(2-(3-((4,6-dioxo-2-thionotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methyl-1H-indol-1-yl)ethoxy)phenyl)acetamide (Compound 9)

[0135] Replacing compound 3a with compound 3i and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 9 as an orange-yellow solid with a yield of 69%. 1 H NMR (400MHz, DMSO-d6): δ12.17 (d, J=1.8Hz, 1H), 12.06 (d, J=1.8Hz, 1H), 9.88 (s,1H),8.54(s,1H),7.73(dd,J=8.0,1.1Hz,1H),7.36–7.26(m,1H),7.26–7. 17(m,3H),7.17–7.09(m,1H),7.09–7.03(m,1H),6.56(ddd,J=8.0,2.5,1.3Hz ,1H),4.77(t,J=4.9Hz,2H),4.30(t,J=5.1Hz,2H),2.69(s,3H),1.99(s,3H). 13C NMR (101MHz, DMSO-d6): δ178.25,168.78,163.10,159.84,158.49,154.19,147.21,140.95,138.01,129.95,1 26.35,124.11,123.54,122.63,113.66,112.23,111.51,109.87,109.08,105.72,66.34,44.02,24.43,12.96.

[0136] Example 48: tert-Butyl 4-(2-(3-((4,6-dioxo-2-thionotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methyl-1H-indol-1-yl)ethoxy)phenyl)carbamate (Compound 10)

[0137] Replacing compound 3a with compound 3j and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 10 as an orange-yellow solid with a yield of 70%. 1 H NMR (400MHz, DMSO-d6) δ12.14(s,1H),12.04(s,1H),9.11(s,1H),8.54(s,1H),7.69(d,J=8.1Hz,1H),7.30(qd,J=7.6,6.7,4.2Hz, 3H),7.23(d,J=4.1Hz,2H),6.82–6.74(m,2H),4.74(t,J=5.1Hz,2H),4.29(t,J=5.1Hz,2H),3.17(s,3H),2.67(s,3H),1.44(s,9H). 13 C NMR (101MHz, DMSO-d6) δ178.23,163.12,159.85,154.29,153.46,153.32,147.22,137.98,133.53,126.39,124. 02,123.58,122.66,119.96(2C),114.85(2C),113.67,111.54,109.89,79.16,66.61,44.10,28.62(3C),13.10.

[0138] Example 49: 4-(2-(3-((4,6-dioxo-2-thionotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methyl-1H-indol-1-yl)ethoxy)benzonitrile (Compound 11)

[0139] Replacing compound 3a with compound 3k and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 11 as an orange-yellow solid with a yield of 65%.1 H NMR (400MHz, DMSO-d6): δ12.16(d,J=1.7Hz,1H),12.04(d,J=1.8Hz,1H),8.54(s,1H),7.71(t,J=8.1Hz,3H),7.30(tt, J=8.4,4.0Hz,1H),7.23(d,J=4.1Hz,2H),7.09–7.01(m,2H),4.79(t,J=5.0Hz,2H),4.46(t,J=5.0Hz,2H),2.68(s,3H). 13 C NMR (101MHz, DMSO-d6): δ178.26,163.10,161.71,159.85,154.10,147.23,137.95,134.67(2C),126.3 6,124.01,123.57,122.65,119.46,116.00(2C),113.65,111.56,110.08,103.76,67.05,43.76,13.08.

[0140] Example 50: 3-(2-(3-((4,6-dioxo-2-thionotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methyl-1H-indol-1-yl)ethoxy)benzonitrile (Compound 12)

[0141] Replacing compound 3a with compound 31 and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 12 as an orange-yellow solid with a yield of 70%. 1 H NMR (400MHz, DMSO-d6): δ12.15(d,J=1.8Hz,1H),12.04(d,J=1.8Hz,1H),8.55(s,1H),7.69(d,J=8.1Hz,1H),7.47–7.33 (m,3H),7.31(ddd,J=8.2,5.6,2.9Hz,1H),7.27–7.18(m,3H),4.78(t,J=5.1Hz,2H),4.43(t,J=5.1Hz,2H),2.69(s,3H). 13 C NMR (101MHz, DMSO-d6): δ178.25,163.11,159.86,158.40,154.26,147.24,137.94,131.31,126.35,125.4 0,124.08,123.57,122.65,120.71,118.97,117.81,113.66,112.68,111.55,109.99,67.00,43.82,13.07.

[0142] Example 51: 5-((2-methyl-1-(2-(4-(trifluoromethyl)phenoxy)ethyl)-1H-indol-3-yl)methylene)-2-thionedihydropyrimidine-4,6(1H,5H)-dione (Compound 13)

[0143] Replacing compound 3a with compound 3m and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 13 as an orange-yellow solid with a yield of 71%. 1 H NMR (400MHz, DMSO-d6) δ12.15(d,J=1.8Hz,1H),12.04(d,J=1.8Hz,1H),8.55(s,1H),7.72(d,J=8.1Hz,1H),7.60(d,J=8.6Hz,2H),7.30(d q,J=8.3,4.3Hz,1H),7.23(d,J=4.1Hz,2H),7.07(d,J=8.6Hz,2H),4.80(t,J=5.1Hz,2H),4.45(t,J=5.0Hz,2H),3.17(s,1H),2.69(s,3H). 13 C NMR(101MHz,DMSO-d6)δ178.26,163.10,161.14,159.84,154.13,147.23,137.97,127.45,127.41,1 26.38,126.27,124.01,123.57,122.66,115.42(3C),113.66,111.56,110.06,66.95,43.85,13.09.

[0144] Example 52: 5-((2-methyl-1-(2-(4-(methylsulfonyl)phenoxy)ethyl)-1H-indol-3-yl)methylene)-2-thionedihydropyrimidine-4,6(1H,5H)-dione (Compound 14)

[0145] Replacing compound 3a with compound 3n and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 14 as an orange-yellow solid with a yield of 62%. 1H NMR (400MHz, DMSO-d6) δ12.16(d,J=1.8Hz,1H),12.05(d,J=1.8Hz,1H),8.55(s,1H),7.83–7.75(m,2H),7.71(d,J=8.1Hz,1H),7.31(t t,J=8.4,4.0Hz,1H),7.23(d,J=4.1Hz,2H),7.14–7.06(m,2H),4.80(t,J=5.0Hz,2H),4.48(t,J=5.0Hz,2H),3.11(s,3H),2.69(s,3H).

[0146] 13 C NMR(101MHz,DMSO-d6)δ178.26,163.10,162.07,159.85,154.12,147.24,

[0147] 137.93,133.51,129.66(2C),126.37,124.02,123.58,122.66,115.44(2C),113.66,111.54,110.09,67.16,44.41,43.82,13.11.

[0148] Example 53: 5-((1-(2-([1,1'-biphenyl]-4-oxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)-2-thionedihydropyrimidine-4,6(1H,5H)-dione (Compound 15)

[0149] Replacing compound 3a with compound 3o and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 15 as an orange-yellow solid with a yield of 74%. 1 H NMR (400MHz, DMSO-d6): δ12.15(d,J=1.8Hz,1H),12.04(d,J=1.8Hz,1H),8.56(s,1H),7.73(d,J=8.1Hz,1H),7.59–7.52(m,4H),7.40(t ,J=7.7Hz,2H),7.34–7.26(m,2H),7.24(d,J=4.2Hz,2H),7.01–6.92(m,2H),4.79(t,J=5.1Hz,2H),4.41(t,J=5.1Hz,2H),2.70(s,3H). 13C NMR (101MHz, DMSO-d6): δ178.25,163.13,159.86,157.96,154.29,147.24,140.11,138.01,133.47,129.31(2C),128.24 (2C),127.26,126.64(2C),126.40,124.06,123.58,122.67,115.29(2C),113.69,111.59,109.92,66.62,44.09,13.11.

[0150] Example 54: 5-((1-(2-([1,1'-biphenyl]-3-oxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)-2-thionedihydropyrimidine-4,6(1H,5H)-dione (Compound 16)

[0151] Replacing compound 3a with compound 3p and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 16 as an orange-yellow solid with a yield of 76%. 1 H NMR (400MHz, DMSO-d6): δ12.17–12.12(m,1H),12.04(d,J=1.8Hz,1H),8.55(s,1H) ,7.73(d,J=8.1Hz,1H),7.63–7.54(m,2H),7.43(dd,J=8.4,6.8Hz,2H),7.39–7.27 (m,3H),7.27–7.22(m,2H),7.20(dt,J=7.8,1.2Hz,1H),7.11(t,J=2.1Hz,1H),6.8 8(dd,J=8.1,2.5Hz,1H),4.79(t,J=5.1Hz,2H),4.46(t,J=5.0Hz,2H),2.72(s,3H). 13 C NMR (101MHz, DMSO-d6): δ178.24,163.13,159.86,158.84,154.38,147.24,142.25,140.34,138.02,130.48,129.30(2C),12 8.06,127.27(2C),126.38,124.12,123.58,122.67,119.97,114.17,113.70,112.89,111.62,109.87,66.58,44.14,13.09.

[0152] Example 55: 5-((2-methyl-1-(2-(naphthalene-2-oxy)ethyl)-1H-indol-3-yl)methylene)-2-thionedihydropyrimidine-4,6(1H,5H)-dione (Compound 17)

[0153] Replacing compound 3a with compound 3q and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 17 as an orange-yellow solid with a yield of 65%. 1 H NMR (400MHz, DMSO-d6): δ12.15(d,J=1.9Hz,1H),12.04(d,J=1.8Hz,1H),8.56(s,1H),7.83–7.72(m,4H),7.44(ddd,J=8.2,6.8,1.3Hz ,1H),7.39–7.28(m,3H),7.24(d,J=4.1Hz,2H),7.09(dd,J=8.9,2.5Hz,1H),4.84(t,J=5.1Hz,2H),4.49(t,J=5.1Hz,2H),2.72(s,3H). 13 C NMR (101MHz, DMSO-d6): δ178.25,163.12,159.86,156.20,154.27,147.25,138.01,134.56,129.88,129.08,127.98,12 7.17,126.93,126.41,124.24,124.05,123.60,122.68,118.90,113.69,111.60,109.96,107.25,66.57,44.01,13.13.

[0154] Example 56: 5-((1-(4-fluorophenethyl)-2-methyl-1H-indol-3-yl)methylene)-2-thionedihydropyrimidine-4,6(1H,5H)-dione (Compound 18)

[0155] Replacing compound 3a with compound 3r and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 18 as an orange-yellow solid with a yield of 86%. 1 H NMR (400MHz, DMSO-d6): δ12.15–12.10(m,1H),12.01(d,J=1.8Hz,1H),8.47(s,1H),7.66–7.59(m,1H),7.28(ddd,J=8 .2,5.0,3.5Hz,1H),7.27–7.18(m,4H),7.14–7.03(m,2H),4.52(t,J=7.3Hz,2H),3.06(t,J=7.3Hz,2H),2.40(s,3H).13 CNMR (101MHz, DMSO-d6): δ178.22,163.14,159.80,153.50,147.06,137.73,134.50,134.47,131.42,1 31.34,126.36,124.12,123.62,122.64,115.73,115.52,113.64,111.43,109.66,45.86,34.33,12.47.

[0156] Example 57: 5-((1-(4-methoxyphenethyl)-2-methyl-1H-indol-3-yl)methylene)-2-thionedihydropyrimidine-4,6(1H,5H)-dione (Compound 19)

[0157] Replacing compound 3a with compound 3s and keeping other conditions unchanged, the experimental procedures of Example 39 were repeated to obtain compound 19 as an orange-yellow solid with a yield of 76%. 1 H NMR (400MHz, DMSO-d6) δ12.12(s,1H),12.01(s,1H),8.46(s,1H),7.68–7.62(m,1H),7.30(ddd,J=8.2,4.9,3.5Hz,1H),7.26 –7.19(m,2H),7.14–7.06(m,2H),6.87–6.78(m,2H),4.48(t,J=7.3Hz,2H),3.71(s,3H),3.00(t,J=7.3Hz,2H),2.37(s,3H). 13 C NMR(101MHz,DMSO-d6)δ178.20,163.17,159.81,158.60,153.75,147.04,137.75,130.51(2C),130.1 3,126.37,124.27,123.66,122.66,114.36(2C),113.64,111.44,109.45,55.52,46.20,34.35,12.40.

[0158] Example 58: (E)-2-((1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)-N-phenylhydrazine-1-carboxamide (Compound 20)

[0159] Compound 3a (1 equivalent) and 4-phenylaminourea (1 equivalent) were dissolved in anhydrous ethanol, and a catalytic amount (0.1 equivalent) of acetic acid was added. The mixture was refluxed at 75°C for 3 h. After the reaction, the product was observed to precipitate directly. The filter cake was filtered and washed with ethanol. After vacuum drying, compound 20 was obtained as a white solid with a yield of 87%. 1 H NMR (400MHz, DMSO-d6): δ10.28(s,1H),8.50(s,1H),8.30(s,1H),8.25–8.13(m,1H),7.66–7.59(m,2H),7.57(dt,J=6.6,2.5Hz,1H),7.37–7.2 6(m,2H),7.26–7.13(m,2H),7.07–6.95(m,1H),6.79(d,J=1.3Hz,4H), 4.57(t,J=4.8Hz,2H), 4.21(t,J=5.1Hz,2H), 3.65(s,3H), 2.59(s,3H). 13 C NMR (101MHz, DMSO-d6): δ153.99,153.45,152.52,141.06,139.65,139.13,136.93,129.12(2C),125.10,122.7 1,122.33,121.45,121.30(2C),119.70,115.64(2C),115.07(2C),110.39,107.82,67.37,55.79,42.96,10.84.

[0160] Example 59: (E)-2-((1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)-N-phenylhydrazine-1-thiocarboamide (Compound 21)

[0161] The experimental procedures of Example 58 were repeated with 4-phenyl-3-thiosemicarbazide replacing 4-phenylsemicarbazide and other conditions remaining unchanged to obtain Compound 21 as a light yellow solid with a yield of 86%. 1H NMR (400MHz, DMSO-d6): δ11.45(s,1H),9.55(s,1H),8.58(s,1H),8.24(dd,J=7.7,1.4Hz,1H),7.70–7.61(m,2H),7.61–7.53(m,1H),7.43–7 .31(m,2H),7.27–7.10(m,3H),6.79(t,J=1.1Hz,4H),4.64–4.54(m,2H),4.22(t,J=5.1Hz,2H),3.65(d,J=0.9Hz,3H),2.60(d,J=0.9Hz,3H). 13 C NMR (101MHz, DMSO-d6): δ174.62,154.01,152.50,143.12,141.89,139.77,137.06,128.63(2C),125.36(3C ),124.94,122.57,121.91,121.57,115.66(2C),115.07(2C),110.47,107.54,67.32,55.80,43.11,10.89.

[0162] Example 60: (E)-2-Hydroxy-N'-((1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)benzohydrazide (Compound 22)

[0163] The experimental procedures of Example 58 were repeated with 2-hydroxybenzoylhydrazide replacing 4-phenylaminourea and other conditions remaining unchanged to obtain compound 22 as a white solid with a yield of 89%. 1 H NMR (400MHz, DMSO-d6): δ12.34(s,1H),11.59(s,1H),8.77(s,1H),8.34–8.27(m,1H),7.97–7.90(m,1H),7.57(d,J=8.0Hz,1H),7.48–7.40(m,1H) ,7.20(dtd,J=18.0,7.2,1.3Hz,2H),7.00–6.92(m,2H),6.82–6.76(m,4H ),4.59(t,J=5.2Hz,2H),4.23(t,J=5.2Hz,2H),3.65(s,3H),2.63(s,3H). 13C NMR (101MHz, DMSO-d6): δ164.90,160.32,154.00,152.51,146.18,142.44,137.03,134.09,128.23,125.31,122.5 4,121.89,121.35,119.16,117.89,115.78,115.66(2C),115.07(2C),110.46,108.12,67.35,55.79,43.05,10.82.

[0164] Example 61: (E)-2-amino-N'-((1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)benzohydrazide (Compound 23)

[0165] The experimental procedures of Example 58 were repeated with 2-aminobenzoylhydrazide replacing 4-phenylaminourea and other conditions remaining unchanged to obtain compound 23 as a white solid with a yield of 87%. 1 H NMR (400MHz, DMSO-d6): δ11.23(s,1H),8.69(s,1H),8.30(d,J=7.5Hz,1H),7.61–7.51(m,2H),7.26–7.10(m,4H),6.79(d,J=1.0Hz,4H),6.74( dd,J=8.4,1.2Hz,1H),6.58(ddd,J=8.1,7.1,1.2Hz,1H),6.38(s,2H),4.57(t,J=5.2Hz,2H),4.22(t,J=5.1Hz,2H),3.65(s,3H),2.60(s,3H). 13 C NMR (101MHz, DMSO-d6): δ164.95,154.00,152.52,150.29,144.16,141.39,136.96,132.24,128.46,125.3 3,122.36,121.90,121.07,116.75,115.65(3C),115.06(3C),110.31,108.38,67.38,55.77,42.99,10.73.

[0166] Example 62: (E)-N'-((1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)nicotinic acid hydrazide (Compound 24)

[0167] The experimental procedures of Example 58 were repeated with 3-pyridinecarboxylic acid hydrazide replacing 4-phenylaminourea and other conditions remaining unchanged to obtain compound 24 as a light yellow solid with a yield of 90%.1 H NMR (400MHz, DMSO-d6): δ11.64(s,1H),9.08(dd,J=2.3,0.9Hz,1H),8.75(dd,J=4.8,1.7Hz,1H),8.72(s,1H),8.33–8.21(m,2H),7.61–7. 54(m,2H),7.20(pd,J=7.2,1.4Hz,2H),6.79(d,J=0.9Hz,4H),4.58(q,J=6.5,5.8Hz,2H),4.23(t,J=5.2Hz,2H),3.65(s,3H),2.62(s,3H). 13 C NMR (101MHz, DMSO-d6): δ161.20,154.00,152.52,152.39,148.88,145.55,142.11,137.02,135.67,130.2 0,125.32,124.05,122.51,121.84,121.29,115.66,115.08,110.45,108.09,67.36,55.80,43.05,10.80.

[0168] Example 63: (E)-N'-((1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indol-3-yl)methylene)pyrazine-2-carboxylic acid hydrazide (Compound 25)

[0169] The experimental procedures of Example 58 were repeated with pyrazine-2-carboxylic acid hydrazide replacing 4-phenylaminourea and other conditions remaining unchanged to obtain compound 25 as a light yellow solid with a yield of 84%. 1 H NMR (400MHz, DMSO-d6): δ11.86(s,1H),9.25(d,J=1.5Hz,1H),8.98–8.86(m,2H),8.81–8.73(m,1H),8.33–8.24(m,1H),7.60– 7.51(m,1H),7.24–7.13(m,2H),6.78(d,J=0.7Hz,4H),4.57(t,J=5.3Hz,2H),4.21(t,J=5.2Hz,2H),3.63(s,3H),2.60(s,3H). 13C NMR (101MHz, DMSO-d6): δ158.86,154.00,152.51,147.97,147.01,145.57,144.34,143.70,142.56,137.0 7,125.30,122.52,121.88,121.31,115.67(2C),115.08(2C),110.46,108.25,67.35,55.79,43.06,10.76.

[0170] Example 64: 1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indole-3-carboxylic acid methyl ester (Compound 4a)

[0171] Compound 2a (1 equivalent) and methyl 2-methyl-3-indolecarboxylate (0.8 equivalent) were dissolved in N,N-dimethylformamide, refluxed at 95°C for 16h, diluted with water and extracted with ethyl acetate. The mixed organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was just dissolved in a methanol solution at 65°C, and the temperature was slowly lowered for recrystallization to obtain compound 4a as yellow-green crystals with a yield of 93%. ESI-MS: m / z 340.2 [M+H] + .

[0172] Example 65: 1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indole-3-carboxylic acid (Compound 5a)

[0173] Compound 4a (1 equivalent) was dissolved in a solvent of dioxane: methanol: water = 2: 1: 1, potassium hydroxide (20 equivalents) was added, and the mixture was refluxed at 90°C until the reaction was complete. The organic solvent was removed by concentration under reduced pressure, and then diluted with water and the pH value was adjusted to 6-7 with 4M HCl. The mixture was filtered and the filter cake was dried under vacuum to obtain compound 5a as a white solid with a yield of 90%. ESI-MS: m / z 326.1 [M+H] + .

[0174] Example 66: 1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indole-3-carbonyl chloride (Compound 6a)

[0175] Compound 5a was dissolved in anhydrous dichloromethane, a catalytic amount of N,N-dimethylformamide was added, oxalyl chloride (3 equivalents) was added dropwise under an ice bath and nitrogen protection, and the reaction was continued for 30 minutes, and the mixture was concentrated under reduced pressure to obtain compound 6a. The intermediate was used directly without further purification, with a yield of 88% as a white solid. 1H NMR (400MHz, DMSO-d6): δ7.97–7.91(m,1H),7.71(d,J=8.2Hz,1H),7.27(ddd,J=8.2,7.1,1.2Hz,1H),7.17(ddd,J=8.1,7.2,1 .0Hz,1H),6.86–6.76(m,4H),4.71(t,J=5.1Hz,2H),4.28(t,J=5.1Hz,2H),3.67(s,3H),2.88(s,3H).ESI-MS:m / z345.1[M+H] + .

[0176] Example 67: N-(4-(Hydroxycarbamoyl)phenyl)-1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indole-3-carboxamide (Compound 26)

[0177] Compound 6a (1 equivalent) was dissolved in anhydrous pyridine, methyl 4-aminobenzoate (1.2 equivalent) was added, and the mixture was reacted at 80°C-90°C for 6h under nitrogen protection. Hydrochloric acid-water was added to dilute the mixture and extracted with ethyl acetate. The mixed organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and then purified by slurrying with methanol, petroleum ether: dichloromethane = 3:1 system, filtered, and the filter cake was vacuum dried, redissolved in methanol: dichloromethane = 1:1 system, and the pH value was adjusted to about 12 with 10M NaOH, and 50% hydroxylamine solution (4 equivalents) was added dropwise under ice bath conditions. After the reaction was complete, the mixture was concentrated under reduced pressure, diluted with water, and the pH was slowly adjusted to 7-8 with 4M HCl, filtered, and the filter cake was vacuum dried to obtain compound 26 as a white solid with a yield of 52%. 1 H NMR (400MHz, DMSO-d6): δ9.94(s,1H),7.76(q,J=8.7Hz,4H),7.73–7.69(m,1H),7.61(d,J=8.0Hz,1H),7.18(dt ,J=21.4,7.2Hz,2H),6.83–6.77(m,4H),4.62(t,J=5.2Hz,2H),4.22(t,J=5.1Hz,2H),3.66(s,3H),2.71(s,3H). 13 C NMR (101MHz, DMSO-d6): δ164.63(2C),153.97,152.49,141.60,136.06,127.72(2C),125.91,121.89, 121.05,120.03,119.28(2C),115.58(3C),115.08(3C),110.73,109.53,67.37,55.80,42.87,12.00.

[0178] Example 68: N-(2-(Hydroxycarbamoyl)phenyl)-1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indole-3-carboxamide (Compound 27)

[0179] The experimental procedures of Example 67 were repeated with methyl 2-aminobenzoate replacing methyl 4-aminobenzoate and other conditions remaining unchanged to obtain compound 27 as a white solid with a yield of 48%. 1 H NMR (400MHz, DMSO-d6): δ11.49(s,1H),8.52(d,J=8.3Hz,1H),7.96(d,J=7.6Hz,1H),7.64(dd,J=13.6,7.8Hz,2H),7.47(d,J=8.2Hz ,1H),7.20(p,J=7.8Hz,2H),7.10(q,J=7.6,7.1Hz,1H),6.80(s,4H),4.62(s,2H),4.23(t,J=5.3Hz,2H),3.66(s,3H),2.80(s,3H). 13 C NMR (101MHz, DMSO-d6): δ165.76,163.89,153.99,152.47,142.90,139.48,136.15,131.45,128.04,125.35,122. 52,122.03,121.47(2C),120.60,119.73,115.63(2C),115.08(2C),110.85,108.68,67.25,55.79,42.83,12.20.

[0180] Example 69: N-(2-(Hydroxycarbamoyl)phenyl)-1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indole-3-carboxamide (Compound 28)

[0181] The experimental procedures of Example 67 were repeated with methyl 3-aminobenzoate replacing methyl 4-aminobenzoate and other conditions remaining unchanged to obtain Compound 28 as a white solid with a yield of 49%. 1H NMR (400MHz, DMSO-d6) δ10.91(s,1H),9.86(s,1H),8.16(s,1H),7.84(d,J=8. 0Hz,1H),7.74(d,J=7.7Hz,1H),7.61(d,J=8.0Hz,1H),7.42(d,J=7.7Hz,1H), 7.34(t,J=8.0Hz,1H),7.25–7.11(m,2H),6.81(d,J=1.8Hz,4H),4.62(t,J=5. 3Hz, 2H), 4.23 (t, J = 5.1Hz, 2H), 3.66 (s, 3H), 2.73 (s, 3H), 1.39–1.21 (m, 1H). 13 C NMR(101MHz,DMSO-d6)δ164.23(2C),153.96,152.49,149.36,140.86,140.74,136.00,129.22,126.08,121.72, 120.82,119.92,115.57(2C),115.08(2C),110.60,110.16,109.54,108.24,105.93,67.39,55.79,42.82,11.95.

[0182] Example 70: N-(6-(Hydroxycarbamoyl)pyridin-3-yl)-1-(2-(4-methoxyphenoxy)ethyl)-2-methyl-1H-indole-3-carboxamide (Compound 29)

[0183] Replacing 4-aminobenzoic acid methyl ester with 5-aminopyridine-2-carboxylic acid methyl ester and keeping other conditions unchanged, the experimental procedure of Example 67 was repeated to obtain Compound 29 as a white solid with a yield of 47%. 1 H NMR (400MHz, DMSO-d6) δ11.29(s,1H),10.21(s,1H),8.99(s,1H),8.96–8.91(m,1H),8.35–8.28(m,1H),7.96(d,J=8.5Hz,1H),7.76(d ,J=7.7Hz,1H),7.64(d,J=7.7Hz,1H),7.21(dt,J=12.3,7.2Hz,2H),6.80(s,4H),4.64(s,2H),4.24(s,2H),3.66(s,3H),2.74(s,3H). 13C NMR(101MHz,DMSO-d6)δ164.96,161.84,153.98,152.48,144.49,142.38,140.19,139.15,136.09,127.20,12 5.73,122.62,122.05,121.22,120.13,115.58(2C),115.08(2C),110.85,108.84,67.34,55.79,42.92,12.04.

[0184] Example 71: Evaluation of antiproliferative activity

[0185] The antiproliferative activity of the compounds was evaluated by MTT assay. Cells were seeded in 96-well plates and allowed to adhere overnight. The cells were then exposed to different concentrations of the compounds for 48 h. MTT solution (20 μL, 0.5 mg / mL) was added to each well, and the cells were incubated with MTT at 37 °C for 4 h. After aspirating the supernatant, DMSO (150 μL) was added and the absorbance was read at 490 nm. All assays were repeated three times in parallel. GraphPad prism 8.0 software was used to determine linear regression parameters and calculate IC 50 The values ​​are shown in Table 1 and Table 2.

[0186] Table 1 In vitro antiproliferative activity of Bel-7402 and HCCLM3 in hepatocellular carcinoma

[0187]

[0188]

[0189] Table 2 In vitro antiproliferative activity of RBE in intrahepatic cholangiocarcinoma

[0190]

[0191] As can be seen from Table 1, most of the compounds prepared by the present invention have good anti-proliferation activity against liver cancer cells, and the liver cancer cell inhibition rate of the active compounds is 0.5-15 μM. Especially compounds 1-4, 6-7, 10-13, 17-19, 26-27 and 29, and the aforementioned compounds also exhibit good in vitro anti-proliferation activity against intrahepatic cholangiocarcinoma RBE, as shown in Table 2, indicating that the compounds proposed by the present invention have good anti-proliferation activity against liver cancer cells, especially good anti-proliferation activity against intrahepatic cholangiocarcinoma cells, and can be used in the preparation of anti-liver cancer drugs, especially in the preparation of anti-intrahepatic cholangiocarcinoma drugs.

[0192] Compounds 1-4, 6-7, 10-13, 17-19, 26-27 and 29 are selected as preferred compounds, and one compound is randomly selected from them and recorded as ZSNI-21. The following experiments are continued. Other compounds mentioned in the present invention still have similar technical effects and are not repeated here.

[0193] Example 72: ADAM17 / HDAC targeting confirmation

[0194] CETSA is used to detect the intracellular drug binding efficiency to the target protein. After incubating the cells with the preferred compounds ZSNI-21 and Bel-7402 for 6 hours, the cells were collected with 1 ml of PBS. Centrifuge at 5000 rpm for 3 minutes at 4°C, and resuspend the precipitate in 1 ml of PBS. Then take 80 μL of the suspension and add it to 11 EP tubes respectively. Each EP tube is heated at a specific temperature of 37°C to 77°C for 3 minutes. The cells are then frozen in liquid nitrogen for 3 minutes, followed by 3 freeze-thaw cycles. The supernatant is collected by centrifugation and mixed with NovexTM Tris-Glycine SDS sample buffer (2X) for subsequent immunoblot analysis. As Figure 2 As shown, the preferred compounds prepared by the present invention have good ADAM17 / HDAC targeting activity. To further determine the enzyme activity inhibition activity of the compounds, rhTACE / ADAM17, HDAC1 Fluorogenic Assay Kit, HDAC2 Fluorogenic Assay Kit, and HDAC6 Fluorogenic Assay Kit were used to quantitatively test the target activity. The results are shown in Figure 3 As shown, ZSNI-21 has significant inhibitory activity against ADAM17, HDAC1, HDAC2, and HDAC6, with IC50 of 0.94μM, 1.27μM, 0.845μM, and 0.106μM, respectively.

[0195] WB experiments are used to further verify the effects of the selected compounds on the expression of key proteins downstream of the target, such as Figure 4 As shown, the results showed that the preferred compound ZSNI-21 could significantly inhibit the release of NICD and significantly increase the levels of acetylated histone H3 and acetylated histone H4, among which the inhibition level of NICD was better than that of JG26, and the increase level of acetylated histone was comparable to that of SAHA. This result further verified that the preferred compound simultaneously inhibited ADAM17 and HDAC2 and affected their downstream expression.

[0196] Example 73: Effects of Preferred Compounds on Proliferation and Apoptosis of Hepatoma Cells

[0197] Colony formation assay is a classic experimental method for evaluating cell growth ability and proliferation potential. Bel-7402 cells were cultured at 8×10 2 The cells were inoculated into 6-well plates at a density of cells / well and cultured in a cell culture incubator using complete medium. After the cells adhered overnight, they were treated with gradient concentrations of compound ZSNI-21 and specific concentrations of ZLDI-8 and lenvatinib for 24 hours, and then the medium was replaced with drug-free complete medium. The cells were cultured for two weeks. When clones containing more than 50 cells were counted under a microscope, the medium was discarded and washed with cold PBS, fixed with methanol for 15 minutes, and then washed three times with PBS. At room temperature (20°C), the cells were stained with 1% crystal violet stain for 10 minutes. After washing away the stain, photographs were taken. ImageJ software was used for quantitative analysis to obtain the number of clones. Clone formation rate (%) = number of clones / number of inoculated cells × 100%. The results showed that after BEL-7402 cells were treated with different concentrations of the preferred compounds for 48 hours, the cell proliferation decreased significantly in a concentration-dependent manner ( Figure 5 A and B).

[0198] Hoechst 33342 staining was used to verify whether the preferred compound ZSNI-21 could induce apoptosis in liver cancer cells. Cell morphological changes were observed under an inverted microscope to preliminarily explore the molecular mechanism of apoptosis induction. Bel-7402 cells were plated at 1.5×10 4 The cells were seeded in a 6-well plate, and after the cells adhered overnight, gradient concentrations of the compound were added and incubated for 48 hours. After the incubation, the cells were washed twice with PBS and stained with Hoechst 33342 in the dark. The staining results were imaged and photographed using an inverted fluorescence microscope. Compared with the control group, the cells treated with the compound showed shrinkage of the nucleus and condensation of chromatin, which is a unique phenomenon of cell apoptosis ( Figure 5 C).

[0199] The number of apoptotic cells was analyzed by flow cytometry to evaluate the ability of the preferred compound ZSNI-21 to induce apoptosis of liver cancer cells. Bel-7402 cells were plated at 2×10 5 Seed in a 6-well plate, wait for the cells to adhere overnight, treat with gradient concentrations of compounds for 48 hours, then digest and collect the cells with EDTA-free trypsin, centrifuge at 2000rpm for 5 minutes, and resuspend in pre-cooled 1ml PBS, centrifuge again at 2000rpm for 5 minutes at 4°C, discard PBS, add Binding buffer to resuspend, and then stain according to the Annexin V-FITC / PI double staining cell apoptosis detection kit (KGA108) operating instructions, and use BD FACSCalibur flow cytometer to analyze cell apoptosis. Figure 5As shown in D, compared with the total apoptosis ratio of 7% in the control group, the total apoptosis ratio of the drug-treated group increased from 15.83% to 40.2% in a concentration-dependent manner. The results show that the preferred compound ZSNI-21 can induce apoptosis at a high level.

[0200] Example 74: Acute toxicity evaluation of preferred compounds on Kunming mice

[0201] To evaluate the safety of compound ZSNI-21, C57BL / 6 mice (9-11 weeks old) were divided into a normal group and a 2000 mg / kg administration group (female: male = 1:1). The mice in the administration group were administered once by intragastric perfusion. The abnormal behavior and weight changes of the mice were observed continuously for the first three days after administration, and then observed every two days. The observation period was two weeks in total. No mice in all the administration groups died and no significant weight loss occurred ( Figure 6 A). After the experiment, the main organs of the mice were pathologically sectioned and stained with HE. The results showed that no obvious pathological changes occurred ( Figure 6 B). The above results indicate that compound ZSNI-21 has high in vivo safety.

[0202] Example 75: Effects of Preferred Compounds on Metastasis of Hepatoma Cells

[0203] The highly invasive liver cancer cell HCC-LM3 was used as the research object. The effects of the compounds on the expression of EMT proteins were determined through wound healing experiments, immunofluorescence staining and protein immunoblotting, and then whether the compounds had the ability to resist the migration of liver cancer cells was evaluated.

[0204] 5×10 per well 5 HCC-LM3 cells were seeded into 6-well plates at a density of 10 cells. When the cells attached to the wall and cultured to 90% of each well, the monolayer of cells was scratched with the tip of a 200μL pipette, and the wound area was photographed and recorded at 0 hours. The cells in the control group were grown in a serum-free medium. The drug-treated group was treated with gradient concentrations of the preferred compound ZSNI-21 and specific concentrations of ZLDI-8, lenvatinib and serum-free medium mixed for 48 hours, and the wound area was photographed and recorded at 48 hours. ImagePro Plus software analyzed the images and calculated the migration rate using the formula: migration rate (%) = [1-wound area (48 hours) / area (0 hours)] × 100%. Results of the wound healing assay ( Figure 7 A, B) show significant differences between the drug-treated group and the control group. After 48 hours of treatment with compound ZSNI-21, the migration of HCC-LM3 cells can be effectively inhibited.

[0205] HCC-LM3 cells were plated at 5 × 10 3Inoculate in a 96-well plate, wait overnight for adhesion, and then treat with gradient concentrations of the preferred compound ZSNI-21 for 48 hours. After incubation, wash with PBS, incubate with immunostaining permeabilization solution (Triton X-100) for 15 minutes, wash again, add 10% goat serum blocking solution for 30 minutes, and incubate with anti-Vimentin (Selleck: F0166) at 4°C overnight. After the cells are incubated with the immunofluorescence secondary antibody in the dark for 3 hours, DAPI is added and incubated in the dark for 10 minutes. The results of immunofluorescence were imaged and photographed using an inverted fluorescence microscope. The immunofluorescence results showed that the preferred compound ZSNI-21 can effectively inhibit the expression of Vimentin ( Figure 7 C and E).

[0206] In addition, the results of immunoblotting experiments confirmed that the preferred compounds could reduce the expression of Vimentin and other EMT-related proteins Slug and MMP9 ( Figure 7 D and FG). The above results preliminarily evaluated the ability of the preferred compounds to inhibit the migration of liver cancer cells.

[0207] In summary, the indole derivatives proposed in the present invention as ADAM17 / HDAC inhibitors can be used as ADAM17 / HDAC inhibitors, can inhibit the growth of liver cancer cells at low doses and have obvious anti-metastatic effects, and have the advantages of high in vitro anti-proliferative activity and low toxic reactions. They are suitable for promotion and application in the preparation of tumor treatment drugs, especially in the preparation of lung cancer drugs.

[0208] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An indole derivative or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the compound is shown in general formula I: Wherein: the R1 group is a substituent at position 3 or 4 on the benzene ring; R1 is -OCH3, -N(CH3), -COOCH3, -NHCOCH3, NHCOOC(CH3)3, -F, -CF3, -CN, -SO2CH3 or -Phenyl; X = O, C; n＝1，2； The R2 group is connected by an amide bond or a hydrazide bond. Wherein R3 is selected from R4 is selected from 2. The following compounds or their pharmaceutically acceptable salts are selected from:

3. A method for preparing the compound as claimed in claim 2, characterized in that: The following steps are involved: (1) Compound 1a-1q is used as a raw material, potassium carbonate and 1-bromo-2-chloroethane are added to an acetonitrile solution of compound 1a-1q, the mixture is heated to 80-100° C. and refluxed for 10-15 hours, cooled to room temperature, the reaction solution is filtered, and the filtrate is concentrated under reduced pressure; after concentration, the crude product is purified by silica gel chromatography to obtain compounds 2a-2q respectively; (2) Using compound 1r-s as raw material, triethylamine and p-toluenesulfonyl chloride were added to a dichloromethane solution of 1r-s, and the mixture was reacted at room temperature for 6 to 12 hours. After concentration under reduced pressure, the crude product was purified by silica gel column chromatography to obtain compounds 2r-2s respectively; (3) adding potassium carbonate and 2-methylindole-3-carboxaldehyde to a solution of compound 2a-2s in N,N-dimethylformamide, heating the solution to 90-100° C. and reflux for 12-18 hours; diluting the solution with water and extracting the solution with ethyl acetate; drying the mixed organic layer with anhydrous sodium sulfate, filtering the solution and concentrating the solution under reduced pressure; subjecting the crude reaction product to flash column chromatography to obtain compounds 3a-3s, respectively; (4) Dissolving compounds 3a-3s and 2-mercapto-4,6-dihydroxypyrimidine in a methanol solution, heating to 70-80° C. for reaction for 2-4 hours; filtering after the reaction, washing the filter cake with methanol to obtain compounds 1-19 respectively; (5) Compound 3a and a phenylhydrazide compound were dissolved in anhydrous ethanol, a catalytic amount of acetic acid was added, and the mixture was refluxed at 70 to 80° C. for 2 to 4 hours. After the reaction, the mixture was filtered and the filter cake was washed with ethanol. After vacuum drying, compounds 20 to 25 were obtained respectively. (6) Compound 2a and methyl 2-methyl-3-indolecarboxylate were dissolved in N,N-dimethylformamide, refluxed at 95°C for 16 to 20 hours, diluted with water and extracted with ethyl acetate; the mixed organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure; the crude product was dissolved in a methanol solution at 60 to 70°C, slowly cooled and recrystallized to obtain compound 4a; compound 4a was dissolved in a solvent of dioxane: methanol: water = 2:1:1, potassium hydroxide was added, refluxed at 80 to 100°C until the reaction was completed, concentrated under reduced pressure to remove the organic solvent, diluted with water and adjusted to pH 6 to 7, filtered, and the filter cake was dried under vacuum to obtain compound 5a; compound 5a was dissolved in anhydrous dichloromethane, a catalytic amount of N,N-dimethylformamide was added, oxalyl chloride was added dropwise under ice bath and nitrogen protection for 30 minutes, and concentrated under reduced pressure to obtain compound 6a; (7) Compound 6a was dissolved in anhydrous pyridine, and an aminobenzoic acid methyl ester compound was added. The reaction was carried out at 80°C to 90°C under nitrogen protection for 5 to 7 hours; hydrochloric acid-water was added to dilute and extracted with ethyl acetate; the mixed organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and purified by slurrying with methanol and petroleum ether: dichloromethane = 3:1 system in sequence, filtered, and the filter cake was vacuum dried, redissolved in methanol: dichloromethane = 1:1 system, and the pH value was adjusted to 11 to 13 with 10M NaOH, and 50% hydroxylamine solution was added dropwise under ice bath conditions. After the reaction was complete, the mixture was concentrated under reduced pressure, diluted with water, and the pH was slowly adjusted to 7 to 8 with 4M HCl, filtered, and the filter cake was vacuum dried to obtain compounds 26-29, respectively.

4. The method for preparing the compound according to claim 3, characterized in that: In step (5), the phenylhydrazide compound is one of 4-phenylsemicarbazide, 4-phenyl-3-thiosemicarbazide, 2-hydroxybenzoylhydrazide, 2-aminobenzoylhydrazide, 3-pyridinecarboxylic acid hydrazide and pyrazine-2-carboxylic acid hydrazide.

5. The method for preparing the compound according to claim 3, characterized in that: In step (7), the methyl aminobenzoate compound is one of methyl 4-aminobenzoate, methyl 2-aminobenzoate, methyl 3-aminobenzoate and methyl 5-aminopyridine-2-carboxylate.

6. A pharmaceutical composition, characterized in that Contains pharmaceutically acceptable excipients and the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition is prepared by adding one or more pharmaceutically acceptable excipients to the compound according to any one of claims 1 to 2.

8. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating liver cancer.

9. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of ADAM17 and HDAC inhibitors.

10. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cholangiocarcinoma.

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