DC-Rhoin derivatives and their preparation methods and applications

Abstract

This invention belongs to the field of medicinal chemistry, specifically to DC-Rhoin derivatives. These derivatives, based on DC-Rhoin as a base, are obtained by structurally modifying its C-3 side chain. A total of 22 DC-Rhoin derivatives were synthesized, most of which incorporate nitrogen-containing groups. These nitrogen-containing groups are located in close proximity to reactive residues of the target protein, promoting protonation and subsequent covalent binding, thereby enhancing the covalent binding ability of the inhibitor molecule to the target protein and increasing the inhibitor's activity. Experimental results demonstrate that these DC-Rhoin derivatives exhibit moderate to high anti-tumor proliferation activity and effective anti-migration capabilities.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to a DC-Rhoin derivative and a preparation method and application thereof. Background Art

[0002] Anti-tumor metastasis is currently a hot topic and a difficult issue in anti-tumor drug research. As an important protein that mediates tumor cell migration and invasion, RhoA protein is a highly promising anti-tumor drug target. However, due to the "undruggable" nature of RhoA protein, the development of RhoA protein inhibitors is difficult. Most of the existing RhoA pathway inhibitors are not inhibitors that directly target RhoA protein (Table 1). Rhosin, a small molecule inhibitor that directly targets RhoA protein, was designed and synthesized in 2012 and subsequently used as a positive control for general RhoA inhibitor research. However, Rhosin has a low binding affinity to RhoA protein and can only effectively inhibit RhoA activity in cells at a concentration of more than 30 μM. New, highly active RhoA inhibitors are still to be developed.

[0003] After nearly three years of research, the applicant team discovered a new pocket, called "Clock," on the RhoA protein and successfully developed a small molecule RhoA inhibitor, DC-Rhoin, that covalently binds to this pocket. Studies have shown that DC-Rhoin exhibits activity in inhibiting tumor cell metastasis and proliferation. The discovery of DC-Rhoin, a small molecule covalent inhibitor of RhoA, provides a foundation for further research into RhoA protein inhibitors.

[0004] Table 1 RhoA pathway inhibitors and their target proteins

[0005]

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide DC-Rhoin derivatives and their preparation methods and applications, so as to develop compounds with better activity and drugability.

[0008] To enhance the activity of DC-Rhoin, structural modification is necessary, introducing more structures that can interact with the RhoA protein to improve its non-covalent affinity and extend its residence time (reaction time). Based on the co-crystal structure of DC-Rhoins and RhoA proteins, the non-covalent binding side has a large cavity, which is suitable for structural modification. Therefore, the present invention uses DC-Rhoin as a matrix to synthesize a series of structurally modified DC-Rhoins derivatives on the C-3 side chain. Most of these derivatives have nitrogen-containing groups added. The nitrogen-containing groups are close to the reactive residues of the target protein, which helps promote its protonation and then promote covalent binding, thereby improving the covalent binding ability of the inhibitor molecule with the target protein and enhancing the activity of the inhibitor.

[0009] According to one object of the present invention, a DC-Rhoin derivative is provided, which is a compound represented by formula (I) or a medically acceptable soluble salt of the compound represented by formula (I).

[0010]

[0011] Wherein, when R1 is H, R2 is one of the following groups:

[0012]

[0013] When R1 is 1-methyl-1H-pyrazol-4-yl, R2 is one of the following groups:

[0014]

[0015] It should be noted that the "medically acceptable soluble salts formed by the compounds" in the present invention may be: (1) medically acceptable soluble salts formed by the compounds with inorganic acids and having the pharmacological activity of the corresponding compounds, such as hydrochloric acid, sulfuric acid, nitric acid, etc.; (2) medically acceptable soluble salts formed by the compounds with organic acids and having the pharmacological activity of the corresponding compounds, such as formic acid, methanesulfonic acid, etc.

[0016] According to the second object of the present invention, a method for preparing the above-mentioned DC-Rhoin derivative is provided. When R1 is H, the steps are as follows:

[0017] (1) Benzothiophene-3-carboxylic acid, DMAP, and EDCI were sequentially added to a reaction flask containing DCM, stirred, and then the reactants were added. After reaction at room temperature, H2O, or EtOAc and an equal volume of H2O, or EtOAc and hydrochloric acid solution and H2O were added for extraction; the organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain an intermediate product;

[0018] (2) The intermediate product obtained in step (1) and m-CPBA were added to a reaction flask containing chloroform solution in sequence. After reaction at room temperature, DCM and 5% NaHCO3 solution or EtOAc and 5% NaHCO3 solution or EtOAc and 5% Na2CO3 solution and cold water were added to the reaction flask for extraction, or EtOAc was added dropwise until the reaction solution was clear; the organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain the target product;

[0019] Alternatively, the intermediate product obtained in step (1) and m-CPBA are added to a reaction bottle containing chloroform solution in sequence. After reaction at room temperature, EtOAc is added dropwise with stirring until the reaction solution becomes clear. The mixture is directly added to a 200-300 mesh silica gel sample and separated and purified by silica gel column chromatography to obtain the target product.

[0020] Wherein, the reactant in step (1) is one of tetrahydropyrrolidine, morpholine, 4-methoxypiperidine, 4-phenoxypiperidine, 4-benzoylpiperidine hydrochloride, 3,4-dimethoxyaniline, 3,4-diethoxyaniline, 3,4-methylenedioxyaniline, 6-amino-1,4-benzodioxetine, 3,4-dimethoxyphenol, 4-heptylamine, 3,4-dimethoxyethylamine, 2-(4-methoxyphenoxy)ethylamine, 2-(2-ethoxyphenoxy)ethanol, phenoxyethanol, and 2-phenoxy-1-phenylethanol.

[0021] When R1 is 1-methyl-1H-pyrazol-4-yl, the steps are as follows:

[0022] S1. 5-bromobenzo[b]thiophene-3-carboxylic acid, DMAP, and EDCI were added sequentially to a reaction flask containing DCM, and after stirring, anhydrous ethanol was added. After reaction at room temperature, the solvent was removed by vortexing, EtOAc was added, and the aqueous phase was adjusted to acidic with 0.5N HCl solution. The aqueous phase was supplemented with H2O to 50 mL and extracted; the organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain a first intermediate product;

[0023] S2, the first intermediate product, 1-methylpyrazole-4-boronic acid pinacol ester and Na2CO3 were added to a reaction flask, and under nitrogen protection, a mixture of H2O and DMF was injected, followed by adding Pd(dppf)Cl2 for reaction; the reaction flask was cooled to room temperature, EtOAc and dilute hydrochloric acid solution were poured, and the filtrate was extracted after filtration; the organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure and separated and purified by silica gel column chromatography to obtain a second intermediate product;

[0024] S3, adding the second intermediate product and NaOH to a reaction flask, followed by adding an EtOH / H2O mixture for reaction; cooling the reaction flask to room temperature, pouring EtOAc and dilute hydrochloric acid solution; filtering to obtain a filtrate, performing an extraction operation on the filtrate, treating the organic phase with saturated brine and anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a third intermediate product;

[0025] S4, the third intermediate product, EDCI and DMAP were added to a reaction flask containing DCM in sequence, the reactants were added after stirring, the reaction was carried out at room temperature, the solvent was dried, EtOAc and H2O were added to the reaction flask, the pH was adjusted to weak acidity with dilute hydrochloric acid, and the organic phase was treated with saturated brine and anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a fourth intermediate product;

[0026] S5. The fourth intermediate product and m-CPBA were added to a reaction flask containing chloroform solution in sequence. After the reaction at room temperature, EtOAc and a saturated cold solution of NaHCO3 were added to the reaction flask for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain the target product.

[0027] Wherein, the reactant in step S4 is one of tetrahydropyrrolidine, 4-methoxypiperidine, 4-phenoxypiperidine, 4-benzoylpiperidine hydrochloride, 4-heptylamine, and 3,4-dimethoxyethylamine.

[0028] According to the third object of the present invention, there is provided the use of the above DC-Rhoin derivative in the preparation of RhoA protein inhibitors.

[0029] According to the fourth object of the present invention, there is provided the use of the above-mentioned DC-Rhoin derivatives in the preparation of drugs for treating tumors, in particular in the preparation of drugs for preventing tumor metastasis.

[0030] Figures in the specification

[0031] Figure 1-2 All test compounds inhibited the migration of MDA-MB-231 cells at a concentration of 2.5 μM. Scale bar: 100 μm. Data shown are the mean ± SD of three independent experiments. Compared with the control group: ***P < 0.001, ****P < 0.0001, compared with the DC-Rhoin04 group: ###P < 0.001, ####P < 0.0001.

[0032] Figure 3-4Compound b19 inhibits the migration and invasion of MDA-MB-231 cells in a dose-dependent manner; 24 h, scale bar 1 mm. Compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (a one-way ANOVA). Differences between experimental groups: Migration of a): b19 2 μM vs b19 1 μM, P < 0.05; b19 1 μM vs b19 0.5 μM, P < 0.01; b19 0.5 μM vs b19 0.25 μM, P < 0.01; Invasion of a): b19 4 μM vs b19 0.5 μM, P < 0.01; b19 2 μM vs b19 0.25 μM, P < 0.0001 (a one-way ANOVA).

[0033] Figure 5 The fitted curve of cell migration rate treated with different concentrations of b19 shows that the half-maximal inhibitory concentration of b19 on MDA-MB-231 cell migration is 0.34 μM.

[0034] Figure 6-7 Compound b19 inhibited the migration of MDA-MB-231 cells in a concentration-dependent manner. Compared with the control group, ***P<0.001, ****P<0.0001; compared with b19 4 μM, ###P<0.001, ####P<0.0001 (a one-way ANOVA).

[0035] Figure 8-9 Compound b19 promoted apoptosis in MDA-MB-231 cells at a concentration of 8 μM. Compared with the control group, *P<0.05, **P<0.01, ****P<0.0001 (a one-way ANOVA).

[0036] Figure 10 Fluorescence images of stress fibers in MDA-MB-231 cells stained with phalloidin under a confocal microscope. The three images correspond to the serum-free group (no serum), the serum-treated group (control), and the serum-treated group (b194 μM). Scale bar: 20 μm.

[0037] Figure 11-12Compound b19 dose-dependently downregulated p-MLC levels in MDA-MB-231 cells (ProteinSimple WES automated protein quantification system). The (+) group was starved for 24 hours and then incubated with 10% serum for 15 minutes, while the (-) group remained starved without additional treatment. Data shown are the mean ± SD of three independent experiments. One-way ANOVA: ***P < 0.001, ***P < 0.0001 compared to the control group; #P < 0.05 compared to the rhosin 30μM positive control group and the b19 4μM treatment group.

[0038] Figure 13 Molecular docking experiments between b19 and RhoA based on the crystal structure of RhoA in complex with DC-Rhoin (PDB: 6KX3). a) Close-up view of the interaction between RhoA and DC-Rhoin (PDB: 6KX3). Residues interacting with the ligand are shown as sticks, and hydrogen bonds are represented by black dashed lines. c) Overlay of images in (a) and (b): Residues interacting with the ligand are shown as sticks, DC Rhoin-RhoA hydrogen bonds are represented by orange dashed lines, and b19-RhoA is represented by blue. d) Overlay of images in (a) and (b): RhoA is shown as the surface. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with specific embodiments and accompanying drawings. Unless otherwise specified, the following raw materials are all commercially available.

[0040] 1. Synthesis and Characterization of DC-Rhoin Derivatives

[0041] The synthetic route of DC-Rhoin derivatives is as follows:

[0042]

[0043] Reagents and conditions: (i): EDCI, DMAP, DCM, relevant reactants containing hydroxyl or amino groups, rt; (ii): 1-methylpyrazole boronic acid pinacol ester, Pd(dppf)Cl2, Na2CO3, DMF, H2O, 90°C; (iii): NaOH, H2O, EtOH, 50°C; (iv): m-CPBA, CHCl3, rt.

[0044] In addition, compound b23 is the DC-Rhoin derivative DC-Rhoin04 previously synthesized and published by the applicant team.

[0045] The following is a detailed introduction to the synthesis methods of specific derivatives.

[0046] 1. Synthesis and characterization of intermediates

[0047] (1)Benzo[b]thiophen-3-yl(pyrrolidin-1-yl)methanone / benzo[b]thiophen-3-yl(pyrrolidin-1-yl)methanone(a1)

[0048]

[0049] DMAP (2.81 mmol, 2.0 eq), benzothiophene-3-carboxylic acid (1.40 mmol, 1.0 eq), and EDCI (2.81 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, tetrahydropyrrolidine (2.81 mmol, 2.0 eq) was added. After reacting at room temperature for approximately 4.5 hours, 10 mL of H₂O was added for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 3 / 7, v / v system). The transparent, colorless oil was dissolved and washed with -20°C petroleum ether to obtain 197.3 mg of white solid product a1, with a yield of approximately 60.9%.

[0050] 1 HNMR(400MHz, CDCl3)δ8.01-7.97(m,1H),7.88-7.84(m,1H),7.61(s,1H),7.44-7.39 (m,1H),7.39-7.35(m,1H),3.82-3.59(s,2H),3.59-3.36(s,2H),2.05-1.85(s,4H). 13 CNMR(101MHz, CDCl3)δ164.92,139.76,137.22,133.14,126.92,125.02,124.90,123.89,122.56,49.15,46.17,26.35,24.61.ESI-HRMScalculatedforC 13 H 13 NOS[M+Na] + :254.0610,found254.0626.

[0051] (2)Benzo[b]thiophen-3-yl(morpholino)methanone(a2)

[0052]

[0053] Benzothiophene-3-carboxylic acid (1.12 mmol, 1.0 eq), DMAP (2.24 mmol, 2.0 eq), and EDCI (2.24 mmol, 2.0 eq) were added sequentially to a reaction flask containing 5 mL of DCM. After stirring for 5 minutes, morpholine (2.24 mmol, 2.0 eq) was added. After approximately 8 hours of reaction at room temperature, the solvent was removed by vortexing, and extraction was performed by adding 30 mL of EtOAc and an equal volume of H2O. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 4, v / v system). The transparent, colorless oil was dissolved and washed with -20°C petroleum ether to obtain 135.5 mg of product a2 as a white solid in a yield of approximately 48.9%.

[0054] 1 HNMR (600MHz, CDCl3) δ7.88(d,J=7.9Hz,1H),7.83(d,J=8.0Hz,1H),7.58(s,1H),7.46-7.41(m,1H),7.41-7.36(m,1H),4.08-3.22(m,8H). 13 CNMR(151MHz, CDCl3)δ165.58,139.89,136.86,131.33,127.14,125.21,125.11,122.97,122.82,67.13,47.85,42.80.ESI-HRMScalculated for C 13 H 13 NO2S[M+Na]+:270.0559,found270.0575.

[0055] (3)Benzo[b]thiophen-3-yl(4-methoxypiperidin-1-yl)methanone / benzo[b]thiophen-3-yl(4-methoxypiperidin-1-yl)methanone(a3)

[0056]

[0057] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 4-methoxypiperidine (2.52 mmol, 1.5 eq) was added. After reacting at room temperature for approximately 5.5 hours, the solvent was evaporated, and 50 mL of EtOAc was added. The mixture was then extracted with 20 mL of 0.5 N hydrochloric acid solution and 50 mL of H₂O. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 4, v / v system) to obtain 395.0 mg of product a3 as a colorless, transparent oil in an approximately 85.4% yield.

[0058] 1 HNMR (600MHz, CDCl3) δ7.85(d,J=7.2Hz,1H),7.79(d,J=7.2Hz,1H),7.53(s,1H),7.39(td,J=7.7,1.4Hz,1H),7.36(td,J=7.8,1.5 Hz,1H),4.09(s,1H),3.81-3.58(m,1H),3.58-3.47(m,1H),3.47-3.43(m,1H),3.34(s,3H),3.32-3.06(m,1H),2.10-1.41(m,4H). 13 CNMR(151MHz,CDCl3)δ165.39,139.78,137.00,132.08,126.21,125.02,124.90,12 2.98,122.67,75.28,55.83,44.55,39.33,31.48,30.52.ESI-HRMScalculatedforC 15 H 17 NO2S[M+Na]+:298.0872,found298.0886.

[0059] (4)Benzo[b]thiophen-3-yl(4-phenoxypiperidin-1-yl)methanone / benzo[b]thiophen-3-yl(4-phenoxypiperidin-1-yl)methanone(a4)

[0060]

[0061] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 4-phenoxypiperidine (2.52 mmol, 1.5 eq) was added. After reacting at room temperature for approximately 5.5 hours, the solvent was evaporated, and 50 mL of EtOAc was added. The mixture was then extracted with 20 mL of 0.5 N hydrochloric acid solution and 50 mL of H₂O. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9, v / v system) to obtain 408.6 mg of product a4 as a colorless, transparent oil in an approximately 72.1% yield.

[0062] 1 HNMR (600MHz, CDCl3) δ7.88(d,J=7.9Hz,1H),7.86(d,J=7.8Hz,1H),7.58(s,1H),7.46-7.42(m,1H),7.41-7.37(m,1H),7.31-7.29( m,1H),7.29-7.27(m,1H),6.97(t,J=7.3Hz,1H),6.94(s,1H),6.92(s,1H),4.62-4.54(m,1H),4.24-3.11(m,4H),2.28-1.60(m,4H). 13 CNMR (151MHz, CDCl3) δ165.60,157.12,139.92,137.08,132.07,129.78,126.49,125.17 ,125.05,123.08,122.81,121.42,116.27,71.57,44.23,39.12,31.50,30.68.ESI-HRMS calculatedforC 20 H 19 NO2S[M+Na]+:360.1029,found360.1052.

[0063] (5)Benzo[b]thiophen-3-yl(4-benzoylpiperazin-1-yl)methanone / benzo[b]thiophen-3-yl(4-benzoylpiperazin-1-yl)methanone(a5)

[0064]

[0065] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 4-benzoylpiperidine hydrochloride (2.52 mmol, 1.5 eq) was added and the reaction was allowed to react overnight at room temperature. The solvent was evaporated, and 50 mL of EtOAc was added to the reaction flask. The mixture was extracted with 20 mL of 0.5N hydrochloric acid solution and 50 mL of H₂O. The organic phase was treated with saturated brine and anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate was separated by silica gel column chromatography using a sequential elution system of ethyl acetate / dichloromethane / petroleum ether = 3 / 1 / 6 (v / v / v) → ethyl acetate / dichloromethane = 1 / 1 (v / v). 544.3 mg of product a5, a white solid, was obtained in an approximately 92.5% yield.

[0066] 1 HNMR (600MHz, CDCl3) δ7.87(d,J=7.9Hz,1H),7.81(d,J=7.9Hz,1H),7.59(s,1H),7.51-7.34(m,7H),4.09-3.31(m,8H). 13 CNMR(151MHz,CDCl3)δ170.69,165.70,139.86,136.75,135.11,131.09,130.17,12 8.70,127.38,127.10,125.25,125.13,122.83,47.79,42.55.ESI-HRMScalculated forC 20 H 18 N2O2S[M+Na]+:373.0981,found373.1001.

[0067] (6) N-(3,4-dimethoxyphenyl)benzo[b]thiophene-3-carboxamide / N-(3,4-dimethoxyphenyl)benzo[b]thiophene-3-carboxamide(a6)

[0068]

[0069] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 3,4-dimethoxyaniline (2.52 mmol, 1.5 eq) was added and the reaction was allowed to react overnight at room temperature. The solvent was evaporated, and 50 mL of EtOAc was added to the reaction flask. The mixture was extracted with 20 mL of 0.5 N hydrochloric acid solution (twice) and 50 mL of H₂O. The organic phase was treated with saturated brine and anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate was separated by silica gel column chromatography using two elution systems: ethyl acetate / petroleum ether = 5 / 95 (v / v) → ethyl acetate / dichloromethane / petroleum ether = 3 / 1 / 6 (v / v / v). 325.5 mg of product a6 was obtained as a white solid with a yield of approximately 61.8%.

[0070] 1 HNMR(600MHz, CDCl3)δ8.42(d,J=8.1Hz,1H),7.97(s,1H),7.89(d,J=8.0Hz,1H),7.75(s,1H),7.50-7.47(m,1H), 7.47(s,1H),7.43(t,J=7.6Hz,1H),7.00(dd,J=8.5,2.4Hz,1H),6.85(d,J=8.5Hz,1H),3.91(s,3H),3.88(s,3H). 13 CNMR (151MHz, CDCl3) δ162.16,149.34,146.32,140.42,136.84,132.51,131.48,129.52,125. 49,125.46,124.40,122.77,112.40,111.56,105.42,56.30,56.12.ESI-HRMScalculatedforC 17 H 15 NO3S[M+Na]+:336.0665,found336.0688.

[0071] (7) N-(3,4-diethoxyphenyl)benzo[b]thiophene-3-carboxamide / N-(3,4-diethoxyphenyl)benzo[b]thiophene-3-carboxamide(a7)

[0072]

[0073] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 3,4-diethoxyaniline (2.52 mmol, 1.5 eq) was added and the reaction was allowed to react overnight at room temperature. The solvent was evaporated, and 50 mL of EtOAc was added to the reaction flask. The mixture was extracted with 20 mL of 0.5N hydrochloric acid solution and 50 mL of H₂O. The organic phase was treated with saturated brine and anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate was washed with a 5:95 ethanol:dichloromethane solution by volume. The washed purple-white solid was isolated and purified by silica gel column chromatography (ethyl acetate / dichloromethane = 5 / 95, v / v) to obtain an orange-red crude product. The crude product was then washed with petroleum ether to obtain 253.3 mg of orange-white solid product a7 with a yield of approximately 44.2%.

[0074] 1 HNMR (600MHz, CDCl3) δ8.41 (dt, J=8.1, 1.0Hz, 1H), 7.97 (s, 1H), 7.89 (dt, J= 8.0,1.0Hz,1H),7.77(s,1H),7.47(ddd,J=8.2,7.1,1.2Hz,1H),7.45-7.43( m,1H),7.43-7.40(m,1H),6.99(dd,J=8.6,2.5Hz,1H),6.86(d,J=8.5Hz,1H) ,4.14-4.10(m,2H),4.10-4.06(m,2H),1.47-1.45(m,3H),1.45-1.42(m,3H). 13 CNMR (151MHz, CDCl3) δ162.12,149.20,145.82,140.39,136.85,132.52,131.58,129.51,125.45,12 5.43,124.41,122.74,114.11,112.51,107.03,65.13,64.71,15.01,14.90.ESI-HRMScalculatedfor C 19 H 19 NO3S[M+Na]+:364.0978,found364.0999.

[0075] (8) N-(benzo[d][1,3]dioxol-5-yl)benzo[b]thiophene-3-carboxamide / N-(benzo[d][1,3]dioxol-5-yl)benzo[b]thiophene-3-carboxamide(a8)

[0076]

[0077] DMAP (2.24 mmol, 2.0 eq), benzothiophene-3-carboxylic acid (1.12 mmol, 1.0 eq), and EDCI (2.24 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 3,4-methylenedioxyaniline (1.35 mmol, 1.2 eq) was added. After approximately 3 hours of reaction at room temperature, the solvent was evaporated, and 50 mL of EtOAc was added. The mixture was then extracted with an equal volume of H2O and an equal volume of 5% hydrochloric acid solution. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (acetone / petroleum ether = 1:9, v / v system) to obtain 270.1 mg of the product a8 as an off-white solid in an approximately 81.1% yield.

[0078] 1 HNMR (600MHz, DMSO-d6) δ10.24(s,1H),8.50(s,1H),8.39(d,J=8.0Hz,1H),8.07(d,J=7.7Hz,1H),7. 50-7.46(m,1H),7.46-7.43(m,2H),7.19(dd,J=8.4,2.1Hz,1H),6.92(d,J=8.4Hz,1H),6.02(s,2H). 13 CNMR(151MHz,DMSO-d6)δ161.62,147.01,143.20,139.41,137.11,133.35,131.46,131.14, 125.00,124.94,124.27,122.84,113.14,107.99,102.32,101.01.ESI-HRMScalculatedforC 16 H 11 NO3S[M+Na]+:320.0352,found320.0370.

[0079] (9) N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzo[b]thiophene-3-carboxamide / N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzo[b]thiophene-3-carboxamide(a9)

[0080]

[0081] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 6-amino-1,4-benzodioxetine (2.52 mmol, 1.5 eq) was added and the reaction was allowed to react overnight at room temperature. The solvent was dried, and 50 mL of EtOAc was added to the reaction flask. The mixture was extracted with 20 mL of 0.5 N hydrochloric acid solution and 50 mL of H2O. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane = 5 / 95, v / v system) to obtain 256.7 mg of product a9 as a white solid in a yield of approximately 49.1%.

[0082] 1 HNMR (600MHz, CDCl3) δ8.39(dt,J=8.2,1.0Hz,1H),7.94(s,1H),7.88(dt,J=8.0,1.0Hz,1H),7.69(s,1H),7.46(ddd,J=8.2,7.1,1.2H z,1H),7.42(ddd,J=8.2,7.0,1.3Hz,1H),7.27(d,J=2.5Hz,1H),7.02(dd,J=8.7,2.5Hz,1H),6.85(d,J=8.6Hz,1H),4.28-4.24(m,4H). 13 CNMR (151MHz, CDCl3) δ162.10,143.75,140.89,140.40,136.86,132.48,131.50, 129.51,125.43,124.41,122.72,117.48,114.14,110.37,64.59,64.46.ESI-HRMS calculatedforC 17 H 13 NO3S[M+Na]+:334.0508,found334.0531.

[0083] (10) 3,4-dimethoxyphenylbenzo[b]thiophene-3-carboxylate / 3,4-dimethoxyphenylbenzo[b]thiophene-3-carboxylate (a10)

[0084]

[0085] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 3,4-dimethoxyphenol (2.52 mmol, 1.5 eq) was added and the reaction was allowed to react overnight at room temperature. The solvent was dried by spin drying, and 50 mL of EtOAc was added to the reaction flask. The mixture was then extracted with 20 mL of 0.5 N hydrochloric acid solution and 50 mL of H₂O. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / petroleum ether = 5 / 10 / 85, v / v / v system) to obtain 465.0 mg of product a10 as a white solid in an approximately 88.0% yield.

[0086] 1 HNMR (600MHz, CDCl3) δ8.64(d,J=8.2Hz,1H),8.61(s,1H),7.92(d,J=8.1Hz,1H),7.52(t,J=7.6 Hz,1H),7.45(t,J=7.6Hz,1H),6.94-6.90(m,1H),6.84-6.79(m,2H),3.91(s,3H),3.90(s,3H). 13 CNMR (151MHz, CDCl3) δ161.51,149.67,147.15,144.36,140.14,138.08,136.88,126.44,125. 83,125.42,124.87,122.71,113.29,111.45,106.15,56.38,56.17.ESI-HRMScalculatedforC 17 H 14 O4S[M+Na]+:337.0505,found337.0528.

[0087] (11) N-(heptan-4-yl)benzo[b]thiophene-3-carboxamide / N-(heptan-4-yl)benzo[b]thiophene-3-carboxamide(a11)

[0088]

[0089] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 4-heptylamine (2.52 mmol, 1.5 eq) was added and the reaction was allowed to react overnight at room temperature. The solvent was dried by spin drying, and 30 mL of EtOAc was added to the reaction flask. The mixture was extracted with 20 mL of 0.5 N hydrochloric acid solution and 30 mL of H₂O. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / petroleum ether = 0.5 / 20 / 79.5, v / v / v system) to obtain 378.2 mg of the product a11 as a white solid in an approximately 81.7% yield.

[0090] 1 HNMR (600MHz, CDCl3) δ8.34(d,J=8.1Hz,1H),7.85(d,J=8.0Hz,1H),7.81(s,1H),7.44(t,J=7.2Hz,1H),7.39(t,J= 7.5Hz,1H),5.81(d,J=9.2Hz,1H),4.24-4.16(m,1H),1.61-1.55(m,2H),1.51-1.39(m,6H),0.95(t,J=7.1Hz,6H). 13 CNMR(151MHz, CDCl3)δ163.84,140.41,136.98,132.90,128.54,125.17,124.34,122.67,49.25,37.77,19.40,14.20.ESI-HRMS calculatedforC 16 H 21 NOS[M+Na]+:298.1236,found298.1252.

[0091] (12) N-(3,4-dimethoxyphenethyl)benzo[b]thiophene-3-carboxamide / N-(3,4-dimethoxyphenethyl)benzo[b]thiophene-3-carboxamide(a12)

[0092]

[0093] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 3,4-dimethoxyethylamine (2.52 mmol, 1.5 eq) was added and the reaction was allowed to react overnight at room temperature. The solvent was dried, and 30 mL of EtOAc was added to the reaction flask. The mixture was extracted with 20 mL of 0.5 N hydrochloric acid solution and 30 mL of H2O. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / petroleum ether = 2 / 1 / 7, v / v / v system) to obtain 477.2 mg of white solid product a12 with a yield of approximately 83.2%.

[0094] 1 HNMR (600MHz, CDCl3) δ8.25(d,J=7.8Hz,1H),7.84(d,J=7.5Hz,1H),7.74(s,1H),7.40(ddd,J=8.1,7.1,1.4Hz,1H),7.39-7.35(m,1H),6.81(d,J =8.0Hz,1H),6.79-6.76(m,1H),6.76-6.75(m,1H),6.18(t,J=5.6Hz,1H) ,3.85(s,3H),3.82(s,3H),3.71(q,J=6.6Hz,2H),2.90(t,J=6.9Hz,2H). 13 CNMR(151MHz, CDCl3)δ164.13,149.24,147.89,140.34,136.75,132.29,131.45,129.16,125.19,125 .17,124.22,122.66,120.83,112.10,111.57,56.04,55.95,41.11,35.27.ESI-HRMScalculatedforC 19 H 19 NO3S[M+Na]+:364.0978,found364.1003.

[0095] (13) N-(2-(4-methoxyphenoxy)ethyl)benzo[b]thiophene-3-carboxamide / N-(2-(4-methoxyphenoxy)ethyl)benzo[b]thiophene-3-carboxamide(a13)

[0096]

[0097] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 2-(4-methoxyphenoxy)ethylamine (2.52 mmol, 1.5 eq) was added and the reaction was allowed to react overnight at room temperature. The solvent was dried, and 30 mL of EtOAc was added to the reaction flask. The mixture was extracted with 20 mL of 0.5 N hydrochloric acid solution and 30 mL of H2O. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / petroleum ether = 15 / 20 / 65, v / v / v system) to obtain 533.7 mg of product a13 as a white solid in a yield of approximately 97.0%.

[0098] 1 HNMR (600MHz, CDCl3) δ8.37(d,J=8.1Hz,1H),7.87(s,1H),7.85(d,J=8.0Hz,1H),7.43(t,J=7.5Hz,1H),7.38(t,J=7.5Hz,1 H), 6.85 (d, J = 9.3Hz, 2H), 6.87-6.84 (m, 2H), 6.84-6.81 (m, 1H), 4.11 (t, J = 5.1Hz, 2H), 3.85 (q, J = 5.4Hz, 2H), 3.76 (s, 3H). 13 CNMR(151MHz, CDCl3)δ164.24,154.27,152.67,140.31,136.80,131.95,129.61,125.22, 125.18,124.27,122.64,115.62,114.86,67.54,55.81,39.44.ESI-HRMScalculatedforC 18 H 17 NO3S[M+Na]+:350.0821,found350.0840.

[0099] (14) 2-(2-ethoxyphenoxy)ethylbenzo[b]thiophene-3-carboxylate / 2-(2-ethoxyphenoxy)ethylbenzo[b]thiophene-3-carboxylate (a14)

[0100]

[0101] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 2-(2-ethoxyphenoxy)ethanol (2.52 mmol, 1.5 eq) was added and the reaction was allowed to react overnight at room temperature. The solvent was dried by spin drying, and 30 mL of EtOAc was added to the reaction flask. The mixture was then extracted with 20 mL of 0.5 N hydrochloric acid solution and 30 mL of H₂O. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 2 / 98, v / v system) to obtain 507.1 mg of product a14 as a colorless, transparent oil in an approximately 88.2% yield.

[0102] 1 HNMR (600MHz, CDCl3) δ8.61(d,J=8.2Hz,1H),8.37(s,1H),7.87(d,J=8.0Hz,1H),7.47(t,J=7.5Hz,1H),7.41(t,J=7.6Hz,1H),7.03-6.99(m ,1H),6.98-6.94(m,1H),6.92(s,1H),6.91-6.89(m,1H),4.74-4.70(m ,2H),4.44-4.40(m,2H),4.07(q,J=7.0Hz,2H),1.39(t,J=7.0Hz,3H). 13 CNMR (151MHz, CDCl3) δ162.74,149.71,148.61,140.13,137.20,136.82,127.05,125.58,125.14,124 .94,122.59,122.52,121.22,116.17,114.33,68.06,64.72,63.41,15.02.ESI-HRMScalculatedforC 19 H 18 O4S[M+Na]+:365.0818,found365.0841.

[0103] (15) 2-phenoxyethylbenzo[b]thiophene-3-carboxylate / 2-phenoxyethylbenzo[b]thiophene-3-carboxylate (a15)

[0104]

[0105] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, phenoxyethanol (2.52 mmol, 1.5 eq) was added and the reaction was allowed to react overnight at room temperature. The solvent was evaporated, and 30 mL of EtOAc was added to the reaction flask. The mixture was then extracted with 20 mL of 0.5 N hydrochloric acid solution and 30 mL of H₂O. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / petroleum ether = 1 / 10 / 89, v / v / v) to obtain 270.5 mg of product a15 as a colorless, transparent oil in an approximately 54.0% yield.

[0106] 1 HNMR (600MHz, CDCl3) δ8.57(d,J=8.2Hz,1H),8.37(s,1H),7.84(d,J=8.1Hz,1H),7.45(ddd,J=8.2,7.1,1.1Hz,1H),7.38(ddd, J=8.2,7.0,1.3Hz,1H),7.30-7.26(m,2H),6.98-6.95(m,1H),6.95-6.93(m,2H),4.68(t,J=6.4Hz,2H),4.32(t,J=6.4Hz,2H). 13 CNMR(151MHz,CDCl3)δ162.75,158.69,140.13,137.29,136.80,129.71,126.93,125 .63,125.18,124.86,122.63,121.38,114.85,66.11,63.16.ESI-HRMScalculatedfor C 17 H 14 O3S[M+Na]+:321.0556,found321.0572.

[0107] (16) 2-phenoxy-1-phenylethylbenzo[b]thiophene-3-carboxylate / 2-phenoxy-1-phenylethylbenzo[b]thiophene-3-carboxylate (a16)

[0108]

[0109] Benzothiophene-3-carboxylic acid (1.68 mmol, 1.0 eq), DMAP (6.72 mmol, 4.0 eq), and EDCI (3.36 mmol, 2.0 eq) were added sequentially to a reaction flask containing 10 mL of DCM. After stirring for 5 minutes, 2-phenoxy-1-phenylethanol (2.52 mmol, 1.5 eq) was added and the reaction was allowed to react overnight at room temperature. The solvent was dried, and 30 mL of EtOAc was added to the reaction flask. The mixture was then extracted with 20 mL of 0.5 N hydrochloric acid solution and 30 mL of H₂O. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / petroleum ether = 0.5 / 10 / 89.5, v / v / v) to obtain 530.1 mg of product a16 as a colorless, transparent oil in an approximately 84.3% yield.

[0110] 1 HNMR(600MHz, CDCl3)δ8.65(d,J=8.2Hz,1H),8.45(s,1H),7.88(d,J=8.1Hz,1H), 7.56(d,J=7.1Hz,2H),7.51-7.46(m,1H),7.45-7.42(m,2H),7.42-7.40(m,1H),7 .40-7.36(m,1H),7.33-7.28(m,2H),7.01-6.98(m,1H),6.98-6.94(m,2H),6.45( dd,J=7.8,3.8Hz,1H), 4.50(dd,J=10.5,7.8Hz,1H), 4.36(dd,J=10.5,3.8Hz,1H). 13 CNMR (151MHz, CDCl3) δ161.92,158.67,140.13,137.32,137.23,136.84,129.66,128.86,128.73,127. 09,126.92,125.61,125.18,124.90,122.60,121.41,115.02,74.57,70.69.ESI-HRMScalculatedforC 23 H 18 O3S[M+Na]+:397.0869,found397.0897.

[0111] (17) 5-bromobenzo[b]thiophene-3-carboxylate / ethyl5-bromobenzo[b]thiophene-3-carboxylate(Pre-1)

[0112]

[0113] 5-Bromobenzo[b]thiophene-3-carboxylic acid (7.8 mmol, 1.0 eq), DMAP (31.1 mmol, 4.0 eq), and EDCI (15.6 mmol, 2.0 eq) were added sequentially to a reaction flask containing 25 mL of DCM. After stirring for 5 minutes, anhydrous ethanol (11.7 mmol, 1.5 eq) was added. After reacting at room temperature for approximately 7.75 hours, the solvent was removed by vortexing, and 50 mL of EtOAc was added. The aqueous phase was adjusted to slightly acidic with 0.5 N HCl solution, and the volume was replenished with H2O to 50 mL before extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:99, v / v) to obtain 1.92 g of Pre-1 as a white solid in an approximately 86.3% yield.

[0114] 1 HNMR (600MHz, CDCl3) δ8.77(d,J=1.9Hz,1H),8.39(s,1H),7.72(d,J=8.6Hz,1 H), 7.50 (dd, J = 8.6, 2.0Hz, 1H), 4.42 (q, J = 7.1Hz, 2H), 1.44 (t, J = 7.1Hz, 3H). 13 CNMR(151MHz, CDCl3)δ162.54,138.71,138.42,137.92,128.32,127.69,126.89,123.83,120.04,61.02,14.53.ESI-HRMS calculatedforC 11 H9BrO2S[M+Na]+:306.9399,found306.9408.

[0115] (18) ethyl 5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxylate / ethyl 5-(1-methy l-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxylate (Pre-2)

[0116]

[0117] Pre-1 (6.7 mmol, 1.0 eq), 1-methylpyrazole-4-boronic acid pinacol ester (8.0 mmol, 1.2 eq), and NaCO (20.0 mmol, 3.0 eq) were added to a reaction flask. Under nitrogen, a mixture of 3 mL of H2O and 20 mL of DMF was added, followed by the addition of Pd(dppf)Cl (0.7 mmol, 0.1 eq). The reaction was allowed to proceed at 90°C for approximately 3.3 h. The flask was cooled to room temperature, and 250 mL of EtOAc and an equal volume of dilute hydrochloric acid (30.00 mmol) were added. The filtrate was filtered and extracted. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 5, v / v) to obtain 1.29 g of Pre-2 as a pale orange-white solid in a yield of approximately 67.2%.

[0118] 1 HNMR (600MHz, CDCl3) δ8.72(d,J=1.6Hz,1H),8.38(s,1H),7.87(s,1H),7.83(d,J=8.3Hz,1H),7. 73(s,1H),7.53(dd,J=8.4,1.7Hz,1H),4.43(q,J=7.1Hz,2H),3.97(s,3H),1.45(t,J=7.1Hz,3H). 13 CNMR(151MHz,CDCl3)δ162.93,138.10,137.59,137.20,136.91,130.13,127 .48,127.17,123.34,123.24,122.93,121.16,60.79,39.29,14.55.ESI-HRMS calculatedforC 15 H 14 N2O2S[M+Na]+:309.0668,found309.0684.

[0119] (19) 5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxylicacid / 5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxylicacid(Pre-3)

[0120]

[0121] Pre-2 (3.49 mmol, 1.0 eq) and NaOH (8.73 mmol, 2.5 eq) were added to a reaction flask, followed by an EtOH / H2O mixture, and the reaction was carried out at 50°C for about 40 min. The reaction flask was cooled to room temperature, and 30 mL of EtOAc and an equal volume of dilute hydrochloric acid (9.00 mmol) were poured into it. The residue was filtered to obtain 988.1 mg of a white crude product. The filtrate was subjected to extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 312.0 mg of a white crude product. Finally, 820.9 mg of a light brown crude product of Pre-3 was obtained with a yield of approximately 91.1%.

[0122] 1 HNMR (600MHz, CDCl3) δ8.69(s,1H),8.42(s,1H),7.88(s,1H),7.83(d,J=8.4Hz,1H),7.76(s,1H),7.51(d,J=8.4Hz,1H),3.98(s,3H). 13 CNMR(151MHz, CDCl3)δ164.82,138.38,138.16,137.64,136.60,129.75,127.86,127.15,123.48,123.17,122.97,121.30,39.12.ESI-HRMScalculated forC 13 H 10 N2O2S[M+H]+:259.0536,found259.0552.

[0123] (20) (5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophen-3-yl)(pyrrolidin-1-yl)methanone / (5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophen-3-yl)(pyrrolidin-1-yl)methan one(a17)

[0124]

[0125] Pre-3 (1.16 mmol, 1.0 eq), EDCI (2.32 mmol, 2.0 eq), and DMAP (4.65 mmol, 4.0 eq) were added sequentially to a reaction flask containing 8 mL of DCM. After stirring for 5 minutes, tetrahydropyrrolidine (1.74 mmol, 1.5 eq) was added. The reaction was allowed to proceed at room temperature for approximately 24.5 hours. The solvent was then evaporated, and 30 mL of EtOAc and 30 mL of H₂O were added to the reaction flask. The pH of the aqueous phase was adjusted to a slightly acidic state with dilute hydrochloric acid, followed by extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, and concentrated under reduced pressure to yield 168.6 mg of a17 as a white solid in an approximately 46.7% yield.

[0126] 1 HNMR(600MHz, CDCl3)δ8.10(s,1H),7.84-7.81(m,2H),7.71(s,1H),7.62(s,1H),7.51(d,J=8.4Hz,1 H),3.95(s,3H),3.74(t,J=7.1Hz,2H),3.50(t,J=6.8Hz,2H),2.03-1.97(m,2H),1.93-1.86(m,2H). 13 C NMR (151MHz, CDCl3) δ164.79,138.04,137.73,136.94,132.69,129.70,127.74,127.51,123 .35,123.24,122.86,120.39,49.33,46.25,39.23,26.41,24.62.ESI-HRMScalculatedforC 17 H 17 N3OS[M+Na]+:334.0985,found334.1007.

[0127] (21) (4-methoxypiperidin-1-yl)(5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophen-3-yl)methanone / (4-methoxypiperidin-1-yl)(5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thioph en-3-yl)methanone(a18)

[0128]

[0129] Pre-3 (1.16 mmol, 1.0 eq), EDCI (2.32 mmol, 2.0 eq), and DMAP (4.65 mmol, 4.0 eq) were added sequentially to a reaction flask containing 8 mL of DCM. After stirring for 5 minutes, 4-methoxypiperidine (1.74 mmol, 1.5 eq) was added. The reaction was allowed to proceed at room temperature for approximately 24.5 hours. The solvent was then evaporated, and 30 mL of EtOAc and 30 mL of H₂O were added to the reaction flask. The pH of the aqueous phase was adjusted to a slightly acidic state with dilute hydrochloric acid before extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 4 / 1, v / v) to obtain 216.1 mg of a18 as a colorless, transparent oil in a yield of approximately 52.4%.

[0130] 1 HNMR (600MHz, CDCl3) δ7.87(d,J=1.1Hz,1H),7.83(d,J=8.4Hz,1H),7.80(s,1H),7.67(s,1H),7.53(s,1H),7.50(dd,J=8.3,1.7Hz,1H),4. 25-3.99(m,1H),3.95(s,3H),3.89-3.64(m,1H),3.64-3.52(m,1H),3 .51-3.47(m,1H),3.36(s,3H),2.22-1.84(m,2H),1.84-1.53(m,3H). 13 CNMR(151MHz, CDCl3)δ165.42,137.86,137.83,136.91,131.73,129.84,127.42,126.89,123.48,1 23.08,123.05,119.49,75.30,55.96,44.69,39.39,39.24,31.66,30.51.ESI-HRMScalculatedforC 19 H 21 N3O2S[M+Na]+:378.1247,found378.1262.

[0131] (22) (5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophen-3-yl)(4-phenoxypiperidin-1-yl)methanone / (5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophen-3-yl)(4-phenoxypiperidin-1-yl)methanone(a19)

[0132]

[0133] Pre-3 (1.16 mmol, 1.0 eq), EDCI (2.32 mmol, 2.0 eq), and DMAP (4.65 mmol, 4.0 eq) were added sequentially to a reaction flask containing 8 mL of DCM. After stirring for 5 minutes, 4-phenoxypiperidine (1.74 mmol, 1.5 eq) was added. The reaction was allowed to proceed at room temperature for approximately 24.5 hours. The solvent was then evaporated, and 30 mL of EtOAc and 30 mL of H₂O were added to the reaction flask. The pH of the aqueous phase was adjusted to a slightly acidic state with dilute hydrochloric acid before extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 1, v / v system) to obtain 182.7 mg of a19 as a colorless, transparent oil in an approximately 37.7% yield.

[0134] 1 HNMR (600MHz, CDCl3) δ7.91 (s, 1H), 7.85 (d, J = 8.4Hz, 1H), 7.82 (s, 1H), 7.69 (s, 1H), 7.56 (s, 1H), 7.52 (d, J = 8.3Hz, 1H), 7.31-7.27 (m,2H),6.96(tt,J=7.4,1.0Hz,1H),6.94-6.92(m,1H),6.92-6.90(m,1H),4.64-4.59(m,1H),4.05-3.38(m,7H),2.06-1.80(m,4H). 13 CNMR(151MHz, CDCl3)δ165.50,157.03,137.90,137.85,136.96,131.60,129.90,129.78,127.46,127.03,12 3.54,123.08,121.40,119.52,116.19,71.37,44.26,39.27,38.97,31.55,30.56.ESI-HRMScalculatedforC 24 H 23 N3O2S[M+Na]+:440.1403,found440.1438.

[0135] (23) (4-benzoylpiperazin-1-yl)(5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophen-3-yl)methanone / (4-benzoylpiperazin-1-yl)(5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophen-3-yl)methanone(a20)

[0136]

[0137] Pre-3 (1.16 mmol, 1.0 eq), EDCI (2.32 mmol, 2.0 eq), and DMAP (4.65 mmol, 4.0 eq) were added sequentially to a reaction flask containing 8 mL of DCM. After stirring for 5 minutes, 4-benzoylpiperidine hydrochloride (1.74 mmol, 1.5 eq) was added. The reaction was allowed to proceed at room temperature for approximately 24.5 hours. The solvent was then evaporated, and 30 mL of EtOAc and 30 mL of H₂O were added to the reaction flask. The pH of the aqueous phase was adjusted to a slightly acidic state with dilute hydrochloric acid, followed by extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 4 / 1, v / v) to afford 218.8 mg of product a20 as a white solid in a yield of approximately 43.8%.

[0138] 1 HNMR(600MHz, CDCl3)δ7.88(s,1H),7.84(d,J=8.4Hz,1H),7.80(s,1H),7.67(s,1H),7 .57(s,1H),7.51(d,J=8.4Hz,1H),7.46-7.35(m,5H),3.95(s,3H),3.93-3.38(m,8H). 13 CNMR(151MHz, CDCl3)δ170.75,165.70,137.84,137.58,136.96,135.04,130.71,130.26,130.12,128.76, 127.97,127.37,127.15,123.69,123.15,122.91,119.29,47.90,42.57,39.24.ESI-HRMScalculatedforC 24 H 22 N4O2S[M+Na]+:453.1356,found453.1377.

[0139] (24) N-(heptane-4-yl)-5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxamide / N-(heptan-4-yl)-5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxamide (a21)

[0140]

[0141] Pre-3 (0.77 mmol, 1.0 eq), EDCI (1.55 mmol, 2.0 eq), and DMAP (3.10 mmol, 4.0 eq) were added sequentially to a reaction flask containing 5 mL of DCM. After stirring for 5 minutes, 4-heptylamine (1.16 mmol, 1.5 eq) was added. The reaction was allowed to proceed at room temperature for approximately 24 hours. The solvent was then dried by spin drying, and 30 mL of EtOAc and 30 mL of H₂O were added to the reaction flask. The pH of the aqueous phase was adjusted to a slightly acidic state with dilute hydrochloric acid, followed by extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and washed with petroleum ether to obtain 220.7 mg of a21 as a white solid in an approximately 80.6% yield.

[0142] 1 HNMR (600MHz, CDCl3) δ8.50(s,1H),7.84(s,1H),7.82(d,J=8.4Hz,1H),7.81(s,1H),7.72(s,1H),7.52(d,J=8.4Hz,1H),5.82(d ,J=9.2Hz,1H),4.24-4.16(m,1H),3.94(s,3H),1.63-1.56(m,2H),1.51-1.47(m,2H),1.47-1.40(m,4H),0.95(t,J=7.1Hz,6H). 13 CNMR(151MHz,CDCl3)δ163.80,138.30,137.78,136.88,132.53,129.86,128.92,127.54,12 3.39,123.27,122.91,120.94,49.28,39.20,37.74,19.42,14.23.ESI-HRMScalculatedforC 20 H 25 N3OS[M+Na]+:378.1611,found378.1630.

[0143] (25) N-(3,4-dimethoxyphenethyl)-5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxamide / N-(3,4-dimethoxyphenethyl)-5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxamide (a22)

[0144]

[0145] Pre-3 (0.77 mmol, 1.0 eq), EDCI (1.55 mmol, 2.0 eq), and DMAP (3.10 mmol, 4.0 eq) were added sequentially to a reaction flask containing 5 mL of DCM. After stirring for 5 minutes, 3,4-dimethoxyethylamine (1.16 mmol, 1.5 eq) was added. The reaction was allowed to proceed at room temperature for approximately 24 hours. The solvent was then evaporated, and 30 mL of EtOAc and 30 mL of H₂O were added to the reaction flask. The pH of the aqueous phase was adjusted to a slightly acidic state with dilute hydrochloric acid before extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane / methanol / ethyl acetate = 30 / 1 / 69, v / v / v system) to obtain 198.1 mg of a22 as a white solid in a yield of approximately 60.1%.

[0146] 1 HNMR (600MHz, CDCl3) δ8.44(s,1H),7.80(s,1H),7.78(d,J=8.4Hz,1H),7.72(s,1H),7.64(s,1H),7.49(d,J=8.3Hz,1H),6.80-6.77(m,1 H),6.77-6.75(m,1H),6.75(s,1H),6.38-6.32(m,1H),3.90(s,3H),3.82(s,3H),3.80(s,3H),3.73-3.67(m,2H),2.90(t,J=6.9Hz,2H). 13 CNMR(151MHz, CDCl3)δ164.15,149.08,147.74,138.16,137.56,136.87,131.90,131.38,129.91,129.54,127.41,12 3.36,123.07,122.85,120.79,120.73,111.94,111.39,55.93,55.88,41.08,39.11,35.24.ESI-HRMScalculatedfor C 23 H 23 N3O3S[M+Na]+:444.1352,found444.1375.

[0147] (26) propyl5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxylate (a23)

[0148]

[0149] Pre-3 (0.23 mmol, 1.0 eq), DMAP (0.93 mmol, 4.0 eq), and EDCI (0.46 mmol, 2.0 eq) were added to a reaction flask containing 5 mL of DCM and stirred for 5 minutes before adding n-propanol (0.35 mmol, 1.5 eq). After reacting at room temperature for approximately 4 hours, the solvent was removed by vortexing, and extraction was performed by adding 30 mL of EtOAc and an equal volume of HCl (6.00 mmol). The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 3, v / v system) to obtain 41.1 mg of a23 as a white solid in a yield of approximately 59.5%.

[0150] 1 HNMR (400MHz, CDCl3) δ8.71(dd,J=1.7,0.7Hz,1H),8.37(s,1H),7.85(d,J=0.7Hz,1H),7.81(dd,J=8.4,0.5Hz,1H),7 .70(s,1H),7.51(dd,J=8.4,1.8Hz,1H),4.33(t,J=6.7Hz,2H),3.94(s,3H),1.89-1.79(m,2H),1.07(t,J=7.4Hz,3H). 13 CNMR (101MHz, CDCl3) δ162.98,138.05,137.54,137.10,137.02,130.25,127.32,127.25 ,123.30,123.21,122.87,121.12,66.34,39.19,22.27,10.73.ESI-HRMScalculatedforC 16 H 16 N2O2S[M+Na]+:323.0825,found323.0845.

[0151] 2. Synthesis and characterization of target products

[0152] (1) (1,1-dioxidobenzo[b]thiophen-3-yl)(pyrrolidin-1-yl)methanone / (1,1-dioxidobenzo[b]thiophen-3-yl)(pyrrolidin-1-yl)methanone(b1)

[0153]

[0154] To a reaction flask containing 5 mL of chloroform solution, a1 (0.65 mmol, 1.0 eq) and m-CPBA (1.95 mmol, 3.0 eq) were added sequentially. After approximately 4 h of reaction at room temperature, 50 mL of DCM and an equal volume of 5% NaHCO₃ solution were added to the flask for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 2 / 3, v / v system) to obtain 109.3 mg of product b1 as a white solid in an approximately 63.9% yield.

[0155] 1 HNMR(600MHz, CDCl3)δ7.70(d,J=7.2Hz,1H),7.58-7.55(m,1H),7.55-7.51(m,1H),7.50(d,J=7.2Hz, 1H),6.72(s,1H),3.61(t,J=7.0Hz,2H),3.44(t,J=6.6Hz,2H),1.98-1.92(m,2H),1.92-1.87(m,2H). 13 CNMR(151MHz, CDCl3)δ160.79,139.04,136.76,133.85,131.14,129.66,127.09,124.88,121.54,48.46,46.05,25.99,24.21.ESI-HRMScalculated for C 13 H 13 NO3S[M+Na]+:286.0508,found286.0527.

[0156] (2) (1,1-dioxidobenzo[b]thiophen-3-yl)(morpholino)methanone / (1,1-dioxidobenzo[b]thiophen-3-yl)(morpholino)methanone(b2)

[0157]

[0158] To a reaction flask containing 3 mL of chloroform solution, a2 (0.40 mmol, 1.0 eq) was added, followed by the addition of m-CPBA (1.20 mmol, 3.0 eq) with stirring. After approximately 33 h of reaction at room temperature, most of the solvent was removed by vortexing at low temperature. Extraction was then performed by adding 30 mL of EtOAc and an equal volume of 5% NaHCO₃ solution to the reaction flask. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 2 / 3, v / v system) to afford 90.5 mg of product b2 as a white solid in an approximately 81.0% yield.

[0159] 1 HNMR(600MHz, CDCl3)δ7.78-7.75(m,1H),7.64-7.61(m,1H),7.61-7.58(m,1H),7.42-7.40(m,1H ),6.68(s,1H),3.83-3.80(m,2H),3.80-3.77(m,2H),3.63(t,J=4.7Hz,2H),3.48(t,J=4.Hz,2H). 13 CNMR(151MHz, CDCl3)δ161.50,138.14,136.89,134.09,131.60,129.66,127.14,124.25,121.99,67.02,66.79,47.45,42.37.ESI-HRMScalculated for C 13 H 13 NO4S[M+Na]+:302.0457,found302.0476.

[0160] (3)(1,1-dioxidobenzo[b]thiophen-3-yl)(4-methoxypiperidin-1-yl)methanone / (1,1-dioxidobenzo[b]thiophen-3-yl)(4-methoxypiperidin-1-yl)methanone(b3)

[0161]

[0162] To a reaction flask containing 5 mL of chloroform solution, a3 (0.73 mmol, 1.0 eq) and m-CPBA (1.96 mmol, 2.7 eq) were added under an ice bath and allowed to react overnight at room temperature. After the reaction, 30 mL of EtOAc was added to the reaction solution, followed by extraction with 30 mL of a 5% cold Na2CO3 solution (twice) and 30 mL of cold water. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / petroleum ether = 3 / 1 / 6, v / v / v system) to obtain 188.2 mg of the white solid product b3 in an approximately 83.9% yield.

[0163] 1HNMR(600MHz, CDCl3)δ7.76-7.73(m,1H),7.60-7.58(m,1H),7.58-7.54(m ,1H),7.37-7.34(m,1H),6.65(s,1H),3.94-3.87(m,1H),3.70-3.63(m,1H ),3.63-3.57(m,1H),3.52-3.48(m,1H),3.34(s,3H),3.32-3.27(m,1H),1 .95-1.88(m,1H),1.78-1.75(m,1H),1.75-1.70(m,1H),1.61-1.54(m,1H). 13 CNMR(151MHz,CDCl3)δ161.22,138.91,136.86,133.99,131.43,129.87,126.32,12 4.19,121.83,74.45,56.02,44.02,38.80,31.38,30.09.ESI-HRMScalculatedforC 15 H 17 NO4S[M+Na]+:330.0770,found330.0789.

[0164] (4)(1,1-dioxidobenzo[b]thiophen-3-yl)(4-phenoxypiperidin-1-yl)methanone / (4-benzoylpiperazin-1-yl)(1,1-dioxidobenzo[b]thiophen-3-yl)methanone(b4)

[0165]

[0166] To a reaction flask containing 5 mL of chloroform solution, a4 (0.59 mmol, 1.0 eq) and m-CPBA (1.60 mmol, 2.7 eq) were added under an ice bath and allowed to react overnight at room temperature. After the reaction, 30 mL of EtOAc was added to the reaction solution, followed by extraction with 30 mL of a 5% cold Na2CO3 solution (twice) and 30 mL of cold water. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / petroleum ether = 2 / 1 / 7, v / v / v system) to obtain 180.7 mg of the white solid product b4 in an approximately 82.9% yield.

[0167] 1HNMR(600MHz, CDCl3)δ7.75(d,J=7.0Hz,1H),7.60(td,J=7.5,1.5Hz,1H),7.59-7.5 5(m,1H),7.40(d,J=7.3Hz,1H),7.31-7.27(m,2H),6.97(t,J=7.3Hz,1H),6.91(d,J =7.9Hz,2H),6.69(s,1H),4.62(q,J=4.7,3.9Hz,1H),4.00-3.91(m,1H),3.87-3.81 (m,1H),3.72-3.65(m,1H),3.50-3.41(m,1H),2.04-1.95(m,2H),1.87-1.81(m,2H). 13 CNMR (151MHz, CDCl3) δ161.28,156.79,138.77,136.82,133.99,131.43,129.80,129.77,126.46 ,124.18,121.82,121.55,116.16,70.67,43.60,38.37,31.29,30.00.ESI-HRMScalculatedforC 20 H 19 NO4S[M+Na]+:392.0927,found392.0953.

[0168] (5) (4-benzoylpiperazin-1-yl)(1,1-dioxidobenzo[b]thiophen-3-yl)methanone / (4-benzoylpiperazin-1-yl)(1,1-dioxidobenzo[b]thiophen-3-yl)methanone(b5)

[0169]

[0170] To a reaction flask containing 5 mL of chloroform solution, a5 (0.57 mmol, 1.0 eq) was added, followed by m-CPBA (1.54 mmol, 2.7 eq) under an ice bath, and the reaction was allowed to proceed overnight at room temperature. After the reaction, 30 mL of EtOAc was added to the reaction solution, followed by extraction with 30 mL of a 5% cold Na2CO3 solution (twice) and 30 mL of cold water. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / cyclohexane = 4 / 1 / 5, v / v / v system) to obtain 156.1 mg of the white solid product b5 in a yield of approximately 71.6%.

[0171] 1HNMR (600MHz, CDCl3) δ7.74 (d, J = 6.7Hz, 1H), 7.61-7.56 (m, 2H), 7.46-7.35 (m, 6H), 6.72 (s, 1H), 3.91-3.38 (m, 8H). 13 CNMR (151MHz, CDCl3) δ170.76,161.68,137.97,136.79,134.77,134.08,131.64, 130.43,129.50,128.82,127.33,127.14,124.16,121.99,47.00,42.04.ESI-HRMS calculatedforC 20 H 18 N2O4S[M+Na]+:405.0879,found405.0903.

[0172] (6) N-(3,4-dimethoxyphenyl)benzo[b]thiophene-3-carboxamide1,1-dioxide / N-(3,4-dimethoxyphenyl)benzo[b]thiophene-3-carboxamide1,1-dioxide(b6)

[0173]

[0174] To a reaction flask containing 5 mL of chloroform solution, a6 (0.64 mmol, 1.0 eq) and m-CPBA (1.72 mmol, 2.7 eq) were added under an ice bath and allowed to react overnight at room temperature. After completion of the reaction, 30 mL of EtOAc was added to the reaction solution, followed by extraction with 30 mL of a 5% cold Na2CO3 solution (twice) and 30 mL of cold water. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / cyclohexane = 2 / 1 / 7, v / v / v system) to obtain 56.6 mg of the yellow solid product b6 in a yield of approximately 25.6%.

[0175] 1 HNMR (600MHz, CDCl3) δ8.54(s,1H),7.94(d,J=7.6Hz,1H),7.68(d,J=7.5Hz,1H),7.59(td,J=7.6,1.3Hz,1H),7.54(td,J=7.7 ,1.1Hz,1H),7.39(d,J=2.5Hz,1H),7.12(dd,J=8.6,2.5Hz,1H),7.01(s,1H),6.85(d,J=8.6Hz,1H),3.89(s,3H),3.88(s,3H).13 CNMR(151MHz, CDCl3)δ159.48,149.24,146.85,139.02,136.85,134.22,131.29,130.50,129. 39,128.39,126.12,121.50,112.66,111.48,105.14,56.23,56.11.ESI-HRMScalculatedforC 17 H 15 NO5S[M+Na]+:368.0563,found368.0585.

[0176] (7) N-(3,4-diethoxyphenyl)benzo[b]thiophene-3-carboxamide1,1-dioxide / N-(3,4-diethoxyphenyl)benzo[b]thiophene-3-carboxamide1,1-dioxide(b7)

[0177]

[0178] To a reaction flask containing 5 mL of chloroform solution, a7 (0.59 mmol, 1.0 eq) and m-CPBA (1.59 mmol, 2.7 eq) were added under an ice bath and allowed to react overnight at room temperature. After the reaction, 30 mL of EtOAc was added to the reaction solution, and 30 mL of a 5% cold Na2CO3 solution (twice) and 30 mL of cold water were added for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / cyclohexane = 1 / 1 / 8, v / v / v system) to obtain 66.1 mg of yellow solid product b7 with a yield of approximately 30.0%.

[0179] 1 HNMR(400MHz, CDCl3)δ8.18(s,1H),7.95(d,J=7.0Hz,1H),7.72(dd,J=6.9,1.8Hz,1H),7.64-7.59(m,1H),7.57(td,J=7.7,1.6Hz,1H) ,7.37(d,J=2.5Hz,1H),7.06(dd,J=8.7,2.5Hz,1H),6.98(s,1H),6.88(d,J=8.7Hz,1H),4.11(p,J=7.1Hz,4H),1.46(q,J=7.1Hz,6H). 13CNMR (101MHz, CDCl3) δ159.39,149.15,146.59,139.14,137.03,134.19,131.37,130.36,129.33,128 .34,126.07,121.62,113.85,112.71,106.83,65.06,64.81,14.96,14.88.ESI-HRMScalculatedforC 19 H 19 NO5S[M+Na]+:396.0876,found396.0901.

[0180] (8) N-(benzo[d][1,3]dioxol-5-yl)benzo[b]thiophene-3-carboxamide1,1-dioxide / N-(benzo[d][1,3]dioxol-5-yl)benzo[b]thiophene-3-carboxamide1,1-dioxid e(b8)

[0181]

[0182] To a reaction flask containing 5 mL of chloroform solution, a8 (0.50 mmol, 1.0 eq) and m-CPBA (1.50 mmol, 3.0 eq) were added sequentially. After reacting at room temperature for approximately 2.5 h, EtOAc was added dropwise with stirring until the reaction solution clarified. The sample was directly added to 200-300 mesh silica gel and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9 → 3 / 7, v / v gradient elution). The eluate was concentrated and washed with chloroform to obtain 55.6 mg of the product b8 as a yellow solid in an approximately 33.8% yield.

[0183] 1 HNMR(600MHz,DMSO-d6)δ10.75(s,1H),7.95(d,J=7.2Hz,1H),7.84(d,J=7.6Hz,1H),7.80(s,1H),7.75(td,J=7.6,1.2Hz ,1H),7.69(td,J=7.5,1.1Hz,1H),7.39(d,J=2.1Hz,1H),7.15(dd,J=8.4,2.1Hz,1H),6.94(d,J=8.4Hz,1H),6.03(s,2H). 13CNMR(151MHz,DMSO-d6)δ159.60,147.12,143.91,137.55,136.66,134.23,132.22,131.31, 129.78,129.15,125.26,121.52,113.35,108.13,102.14,101.21.ESI-HRMScalculatedforC 16 H 11 NO5S[M+Na]+:352.0250,found352.0266.

[0184] (9) N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzo[b]thiophene-3-carboxamide 1,1-dioxide / N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzo[b]thiophene-3-carboxamide 1,1-dioxide (b9)

[0185]

[0186] To a reaction flask containing 5 mL of chloroform solution, a9 (0.64 mmol, 1.0 eq) and m-CPBA (1.73 mmol, 2.7 eq) were added under an ice bath and allowed to react overnight at room temperature. After completion of the reaction, 30 mL of EtOAc was added to the reaction solution, followed by extraction with 30 mL of a 5% cold Na2CO3 solution (twice) and 30 mL of cold water. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / cyclohexane = 3 / 2 / 15, v / v / v system) to obtain 70.1 mg of the product b9 as a yellow, fluffy solid in an approximately 31.9% yield.

[0187] 1 HNMR(600MHz,DMSO-d6)δ10.68(s,1H),7.95(d,J=7.2Hz,1H),7.83(d,J=7.5Hz,1H),7.79(s,1H),7.74(td,J=7.6,1.2Hz,1H ),7.69(td,J=7.5,1.1Hz,1H),7.34(d,J=2.5Hz,1H),7.15(dd,J=8.7,2.5Hz,1H),6.86(d,J=8.7Hz,1H),4.27-4.22(m,4H). 13CNMR(151MHz,DMSO-d6)δ159.51,142.96,140.30,137.57,136.67,134.22,131.56,131.29,129 .70,129.18,125.25,121.51,116.91,113.43,109.28,64.18,63.99.ESI-HRMScalculatedforC 17 H 13 NO5S[M+Na]+:366.0407,found366.0435.

[0188] (10) 3,4-dimethoxyphenylbenzo[b]thiophene-3-carboxylate 1,1-dioxide / 3,4-dimethoxyphenylbenzo[b]thiophene-3-carboxylate1,1-dioxide(b10)

[0189]

[0190] To a reaction flask containing 5 mL of chloroform solution, a10 (0.64 mmol, 1.0 eq) and m-CPBA (1.72 mmol, 2.7 eq) were added under an ice bath and allowed to react overnight at room temperature. After completion of the reaction, 30 mL of EtOAc was added to the reaction solution, followed by extraction with 30 mL of a cold 5% Na2CO3 solution (twice) and 30 mL of cold water. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / cyclohexane = 1 / 1 / 8, v / v / v system) to obtain 146.5 mg of product b10 as a white crystalline solid in an approximately 66.1% yield.

[0191] 1 HNMR (600MHz, CDCl3) δ8.26(d,J=7.7Hz,1H),7.77(d,J=7.5Hz,1H),7.64(t,J=7.6Hz,1H),7.59(d,J=7.6Hz,1H), 7.54(s,1H),6.90(d,J=8.6Hz,1H),6.77(dd,J=8.6,2.7Hz,1H),6.75(d,J=2.7Hz,1H),3.90(s,3H),3.89(s,3H). 13CNMR (151MHz, CDCl3) δ160.65,149.77,147.69,143.45,137.48,136.53,134.14,133.48,131. 21,128.44,126.07,121.80,112.70,111.36,105.40,56.33,56.21.ESI-HRMScalculatedforC 17 H 14 O6S[M+Na]+:369.0403,found369.0419.

[0192] (11) N-(heptan-4-yl)benzo[b]thiophene-3-carboxamide1,1-dioxide(b11)

[0193]

[0194] To a reaction flask containing 5 mL of chloroform solution, a11 (0.73 mmol, 1.0 eq) and m-CPBA (1.97 mmol, 2.7 eq) were added under an ice bath and allowed to react overnight at room temperature. After completion of the reaction, 30 mL of EtOAc was added to the reaction solution, followed by extraction with 30 mL of a 5% cold Na2CO3 solution (twice) and 30 mL of cold water. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9, v / v system) to obtain 122.7 mg of the white solid product b11 in a yield of approximately 54.7%.

[0195] 1 HNMR (600MHz, CDCl3) δ7.90(d,J=7.4Hz,1H),7.68(d,J=7.3Hz,1H),7.58(td,J=7.6,1.3Hz,1H),7.54(td,J=7.5,1.2Hz, 1H), 6.86 (s, 1H), 6.42 (d, J = 9.4Hz, 1H), 4.13-4.04 (m, 1H), 1.57-1.50 (m, 2H), 1.47-1.31 (m, 6H), 0.91 (t, J = 7.3Hz, 6H). 13CNMR(151MHz, CDCl3)δ161.31,139.20,137.04,134.09,131.09,129.74,127.77,126.04,121.34,49.80,37.25,19.31,14.05.ESI-HRMScalculatedforC 16 H 21 NO3S[M+Na]+:330.1134,found330.1151.

[0196] (12) N-(3,4-dimethoxyphenethyl)benzo[b]thiophene-3-carboxamide1,1-dioxide / N-(3,4-dimethoxyphenethyl)benzo[b]thiophene-3-carboxamide1,1-dioxide(b12)

[0197]

[0198] To a reaction flask containing 5 mL of chloroform solution, a12 (0.59 mmol, 1.0 eq) and m-CPBA (1.58 mmol, 2.7 eq) were added under an ice bath and allowed to react overnight at room temperature. After the reaction, 30 mL of EtOAc was added to the reaction solution, followed by extraction with 30 mL of a 5% cold Na2CO3 solution (twice) and 30 mL of cold water. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane / cyclohexane = 3 / 1 / 6, v / v / v system) to obtain 105.3 mg of the white oily product b12 in a yield of approximately 47.8%.

[0199] 1 HNMR(600MHz, CDCl3) δ7.80(d,J=7.0Hz,1H),7.71(d,J=6.3Hz,1H),7.60-7.57(m,1H),7.57-7.54(m,1H),6.83(d,J=7.9Hz,1H),6.76 -6.74(m,1H),6.74(s,1H),6.70(s,1H),6.24(d,J=6.1Hz,1H),3.87(s,3H),3.86(s,3H),3.69(q,J=6.6Hz,2H),2.88(t,J=6.9Hz,2H). 13CNMR (151MHz, CDCl3) δ161.65,149.41,148.17,138.87,137.16,134.02,131.30,130.57,129.32,128 .25,125.85,121.62,120.86,111.91,111.61,56.09,56.06,41.13,35.01.ESI-HRMScalculatedforC 19 H 19 NO5S[M+Na]+:396.0876,found396.0897.

[0200] (13) N-(2-(4-methoxyphenoxy)ethyl)benzo[b]thiophene-3-carboxamide1,1-dioxide / N-(2-(4-methoxyphenoxy)ethyl)benzo[b]thiophene-3-carboxamide1,1-dioxide(b13)

[0201]

[0202] To a reaction flask containing 5 mL of chloroform solution, a13 (0.61 mmol, 1.0 eq) and m-CPBA (1.65 mmol, 2.7 eq) were added under an ice bath and allowed to react overnight at room temperature. After completion of the reaction, 30 mL of EtOAc was added to the reaction solution, followed by extraction with 30 mL of a cold 5% Na2CO3 solution (twice) and 30 mL of cold water. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 4, v / v system) to obtain 96.7 mg of white crystalline product b13 in an approximately 44.1% yield.

[0203] 1 HNMR (600MHz, CDCl3) δ7.90 (d, J = 7.5Hz, 1H), 7.67 (d, J = 7.4Hz, 1H), 7.58 (td, J = 7.6, 1.2Hz, 1H), 7.54 (td, J = 7. 5,1.2Hz,1H),6.87-6.83(m,5H),6.81-6.74(m,1H),4.10(t,J=5.0Hz,2H),3.82(q,J=5.3Hz,2H),3.77(s,3H). 13CNMR(151MHz, CDCl3)δ161.87,154.53,152.43,138.56,137.15,134.02,131.26,129.32, 128.75,125.89,121.64,115.68,114.98,66.89,55.88,39.71.ESI-HRMScalculatedforC 18 H 17 NO5S[M+Na]+:382.0720,found382.0739.

[0204] (14) 2-(2-ethoxyphenoxy)ethylbenzo[b]thiophene-3-carboxylate 1,1-dioxide / 2-(2-ethoxyphenoxy)ethylbenzo[b]thiophene-3-carboxylate 1,1-dioxide (b14)

[0205]

[0206] To a reaction flask containing 5 mL of chloroform solution, a14 (0.58 mmol, 1.0 eq) and m-CPBA (1.58 mmol, 2.7 eq) were added under an ice bath and allowed to react overnight at room temperature. After completion of the reaction, 30 mL of EtOAc was added to the reaction solution, followed by extraction with 30 mL of a cold 5% Na2CO3 solution (twice) and 30 mL of cold water. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9 → 15 / 85, v / v gradient elution) to obtain 127.7 mg of product b14 as a colorless, transparent oil in an approximately 58.8% yield.

[0207] 1 HNMR (600MHz, CDCl3) δ8.22(d,J=7.4Hz,1H),7.73(d,J=7.5Hz,1H),7.60(td,J=7.6,1.3Hz,1H),7.56(td,J=7.5,1.2Hz,1H),7.23 (s,1H),7.00-6.95(m,2H),6.93-6.88(m,2H),4.73-4.68(m,2H),4.39-4.36(m,2H),4.06(q,J=7.0Hz,2H),1.40(t,J=7.0Hz,3H). 13CNMR (151MHz, CDCl3) δ161.68,149.75,148.16,137.58,135.90,134.02,133.90,131.01,128.60,126 .18,123.00,121.69,121.14,116.48,114.05,67.60,65.09,64.53,15.01.ESI-HRMScalculatedforC 19 H 18 O6S[M+Na]+:397.0716,found397.0736.

[0208] (15) 2-phenoxyethylbenzo[b]thiophene-3-carboxylate 1,1-dioxide / 2-phenoxyethylbenzo[b]thiophene-3-carboxylate1,1-dioxide(b15)

[0209]

[0210] To a reaction flask containing 5 mL of chloroform solution, a15 (0.67 mmol, 1.0 eq) and m-CPBA (1.81 mmol, 2.7 eq) were added under an ice bath and allowed to react overnight at room temperature. After completion of the reaction, 30 mL of EtOAc was added to the reaction solution, followed by extraction with 30 mL of a cold 5% Na2CO3 solution (twice) and 30 mL of cold water. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9, v / v system) to obtain 167.5 mg of b15 as a white solid in a yield of approximately 75.7%.

[0211] 1 HNMR (600MHz, CDCl3) δ8.22(d,J=7.6Hz,1H),7.74(d,J=7.6Hz,1H),7.61(td,J=7.6,1.3Hz,1H),7.57(td,J=7.7,0.9H z,1H),7.34-7.30(m,2H),7.30(s,1H),7.03-6.99(m,1H),6.94(d,J=7.6Hz,2H),4.73-4.68(m,2H),4.34-4.30(m,2H). 13CNMR (151MHz, CDCl3) δ161.71,158.37,137.57,135.94,134.06,133.84,131.07,129. 82,128.58,126.12,121.75,121.73,114.82,65.49,64.76.ESI-HRMScalculatedforC 17 H 14 O5S[M+Na]+:353.0454,found353.0473.

[0212] (16) 2-phenoxy-1-phenylethylbenzo[b]thiophene-3-carboxylate 1,1-dioxide / 2-phenoxy-1-phenylethylbenzo[b]thiophene-3-carboxylate 1,1-dioxide (b16)

[0213]

[0214] To a reaction flask containing 5 mL of chloroform solution, a16 (0.53 mmol, 1.0 eq) and m-CPBA (1.44 mmol, 2.7 eq) were added under an ice bath and allowed to react overnight at room temperature. After completion of the reaction, 30 mL of EtOAc was added to the reaction solution, followed by extraction with 30 mL of a cold 5% Na2CO3 solution (twice) and 30 mL of cold water. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95, v / v system) to obtain 205.5 mg of b16 as a colorless, transparent oil in an approximately 95.4% yield.

[0215] 1 HNMR (600MHz, CDCl3) δ8.20 (d, J = 7.5Hz, 1H), 7.72 (d, J = 7.3Hz, 1H), 7.58 (td, J = 7. 7,1.2Hz,1H),7.55(t,J=7.4Hz,1H),7.49(d,J=7.9Hz,2H),7.45(t,J=7.3Hz,2H),7 .43-7.39(m,1H),7.32(d,J=8.4Hz,2H),7.30(s,1H),7.01(t,J=7.4Hz,1H),6.94(d ,J=7.8Hz,2H),6.39-6.35(m,1H),4.47-4.42(m,1H),4.31(dd,J=10.9,3.4Hz,1H). 13CNMR(151MHz, CDCl3)δ160.95,158.30,137.46,135.71,135.60,133.98,133.95,131.00,129.77,129. 33,129.08,128.57,126.91,126.07,121.75,121.65,114.91,76.62,70.16.ESI-HRMScalculatedforC 23 H 18 O5S[M+Na]+:429.0767,found429.0794.

[0216] (17)(5-(1-methyl-1H-pyrazol-4-yl)-1,1-dioxidobenzo[b]thiophen-3-yl)(pyrrolidin-1-yl)methanone / (5-(1-methyl-1H-pyrazol-4-yl)-1,1-dioxidobenzo[b]thiophen-3-yl)(pyrrolidin-1-yl)methanone(b17)

[0217]

[0218] To a reaction flask containing 5 mL of chloroform solution, a17 (0.32 mmol, 1.0 eq) and m-CPBA (0.96 mmol, 3.0 eq) were added sequentially. After approximately 25 h of reaction at room temperature, 30 mL of EtOAc and an equal volume of saturated cold NaHCO₃ solution were added to the flask for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (methanol / dichloromethane = 1:99, v / v system) to obtain 69.7 mg of b17 as a white solid in an approximately 63.4% yield.

[0219] 1 HNMR (600MHz, CDCl3) δ7.78(s,1H),7.72(s,1H),7.69(d,J=7.9Hz,1H),7.60(dd,J=7.9,1.5Hz,1H),7.58(d,J=1.4H z,1H),6.73(s,1H),3.93(s,3H),3.67(t,J=6.9Hz,2H),3.51(t,J=6.6Hz,2H),2.02-1.97(m,2H),1.97-1.91(m,2H). 13CNMR(151MHz,CDCl3)δ160.88,138.90,138.58,137.17,133.78,130.76,128.18,127.87,12 7.27,122.22,121.61,121.26,48.76,46.22,39.36,26.13,24.31.ESI-HRMScalculatedfor C 17 H 17 N3O3S[M+Na]+:366.0883,found366.0898.

[0220] (18) (4-methoxypiperidin-1-yl)(5-(1-methyl-1H-pyrazol-4-yl)-1,1-dioxidobenzo[b]thiophen-3-yl)methanone / (4-methoxypiperidin-1-yl)(5-(1-methyl-1H-pyrazol-4-yl)-1,1-dioxidobenzo[b]thiophen-3-yl)methanone(b18)

[0221]

[0222] To a reaction flask containing 5 mL of chloroform solution, a18 (0.42 mmol, 1.0 eq) and m-CPBA (1.26 mmol, 3.0 eq) were added under ice. After approximately 21 h of reaction at room temperature, 30 mL of EtOAc and an equal volume of cold saturated NaHCO3 solution were added to the flask for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (methanol / dichloromethane = 1:99, v / v system) to obtain 120.6 mg of b18 as a white solid in an approximately 74.1% yield.

[0223] 1 HNMR(600MHz, CDCl3)δ7.78(s,1H),7.72(d,J=7.9Hz,1H),7.70(s,1H),7.62(dd,J= 7.9,1.5Hz,1H),7.39(d,J=1.5Hz,1H),6.65(s,1H),3.96(s,3H),3.94-3.89(m,1H) ,3.74-3.69(m,1H),3.67-3.61(m,1H),3.55-3.50(m,1H),3.36(s,3H),3.36-3.32( m,1H),1.97-1.90(m,1H),1.81-1.78(m,1H),1.78-1.75(m,1H),1.65-1.58(m,1H). 13CNMR (151MHz, CDCl3) δ161.29,139.09,138.40,137.20,133.87,130.92,128.15,127.58,126.93,1 22.45,121.20,120.86,74.42,56.10,44.09,39.44,38.88,31.41,30.18.ESI-HRMScalculatedforC 19 H 21 N3O4S[M+Na]+:410.1145,found410.1167.

[0224] (19)(5-(1-methyl-1H-pyrazol-4-yl)-1,1-dioxidobenzo[b]thiophe n-3-yl)(4-phenoxypiperidin-1-yl)methanone / (5-(1-methyl-1H-pyrazol-4-yl)-1,1-dioxidobenzo[b]thiophe n-3-yl)(4-phenoxypiperidin-1-yl)methanone(b19)

[0225]

[0226] To a reaction flask containing 5 mL of chloroform solution, a19 (0.24 mmol, 1.0 eq) and m-CPBA (0.72 mmol, 3.0 eq) were added sequentially. After approximately 25 h of reaction at room temperature, 30 mL of EtOAc and an equal volume of saturated cold NaHCO₃ solution were added to the flask for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (methanol / dichloromethane = 1:99, v / v system) to obtain 96.2 mg of b19 as a colorless, transparent oil in an approximately 89.2% yield.

[0227] 1HNMR(600MHz, CDCl3)δ7.80(s,1H),7.72(d,J=6.3Hz,1H),7.71(s,1H),7.63(dd,J=7.9 ,1.5Hz,1H),7.43(d,J=1.5Hz,1H),7.32-7.27(m,2H),6.97(tt,J=7.4,1.1Hz,1H),6.9 3-6.89(m,2H),6.68(s,1H),4.67-4.61(m,1H),4.05-3.99(m,1H),3.96(s,3H),3.88-3 .80(m,1H),3.76-3.68(m,1H),3.55-3.47(m,1H),2.06-1.98(m,2H),1.90-1.84(m,2H). 13 CNMR(151MHz, CDCl3)δ161.37,156.79,139.10,138.26,137.20,133.88,130.89,129.85,128.16,127.61,127.0 6,122.47,121.64,121.22,120.89,116.16,70.55,43.69,39.44,38.47,31.36,30.08.ESI-HRMScalculatedfor C 24 H 23 N3O4S[M+Na]+:472.1301,found472.1323.

[0228] (20) (4-benzoylpiperazin-1-yl)(5-(1-methyl-1H-pyrazol-4-yl)-1,1-dioxidobenzo[b]thiophen-3-yl)methanone / (4-benzoylpiperazin-1-yl)(5-(1-methyl-1H-pyrazol-4-yl)-1,1-dioxidobenzo[b]thiophen-3-yl)methanone(b20)

[0229]

[0230] To a reaction flask containing 5 mL of chloroform solution, a20 (0.35 mmol, 1.0 eq) and m-CPBA (1.01 mmol, 3.0 eq) were added under an ice bath. After approximately 21 h of reaction at room temperature, 30 mL of EtOAc and an equal volume of saturated cold NaHCO3 solution were added to the flask for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (methanol / dichloromethane = 1:99, v / v system) to obtain 80.1 mg of b20 as a white solid in an approximately 49.5% yield.

[0231] 1 HNMR(600MHz, CDCl3)δ7.79(s,1H),7.73(s,1H),7.74-7.71(m,1H),7.63(dd,J=8.0,1.5H z,1H),7.50-7.33(m,6H),6.70(s,1H),3.96(s,3H),3.93-3.67(m,4H),3.67-3.32(m,4H). 13 CNMR (151MHz, CDCl3) δ170.83,161.77,139.24,137.51,137.23,134.69,133.77,130.55,128.89,128 .16,127.94,127.78,127.20,122.63,121.11,120.81,47.26,42.17,39.47.ESI-HRMScalculatedforC 24 H 22 N4O4S[M+Na]+:485.1254,found485.1292.

[0232] (21) N-(heptane-4-yl)-5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxamide 1,1-dioxide / N-(heptan-4-yl)-5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxamide 1,1-dioxide (b21)

[0233]

[0234] To a reaction flask containing 5 mL of chloroform solution, a21 (0.42 mmol, 1.0 eq) and m-CPBA (1.27 mmol, 3.0 eq) were added under ice. After approximately 25 h of reaction at room temperature, 30 mL of EtOAc and an equal volume of saturated cold NaHCO3 solution were added to the flask for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (methanol / dichloromethane = 0.5 / 99.5 → 1.0 / 49.0, v / v gradient elution) to obtain 153.1 mg of b21 as a yellow solid in an approximately 94.1% yield.

[0235] 1 HNMR (600MHz, CDCl3) δ7.98(d,J=1.4Hz,1H),7.78(s,1H),7.72(s,1H),7.64(d,J=7.9Hz,1H),7.57(dd,J=7.9,1.5Hz,1H),6.90(s,1H) ,6.47(d,J=9.1Hz,1H),4.14-4.05(m,1H),3.93(s,3H),1.59-1.52(m,2H),1.50-1.43(m,2H),1.43-1.33(m,4H),0.93(t,J=7.3Hz,6H). 13 CNMR(151MHz,CDCl3)δ161.36,138.96,138.77,137.15,133.95,130.71,128.53,128.28,12 7.04,122.61,121.96,121.40,49.85,39.36,37.20,19.34,14.09.ESI-HRMScalculatedforC 20 H 25 N3O3S[M+Na]+:410.1509,found410.1537.

[0236] (22) N-(3,4-dimethoxyphenethyl)-5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxamide 1,1-dioxide / N-(3,4-dimethoxyphenethyl)-5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxamide 1,1-dioxide (b22)

[0237]

[0238] To a reaction flask containing 5 mL of chloroform solution, a22 (0.36 mmol, 1.0 eq) and m-CPBA (1.07 mmol, 3.0 eq) were added under ice. After approximately 25 h of reaction at room temperature, 30 mL of EtOAc and an equal volume of saturated cold NaHCO3 solution were added to the reaction flask for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (methanol / dichloromethane = 0.5 / 99.5 → 1.0 / 49.0, v / v gradient elution) to obtain 75.5 mg of b22 as a yellow solid in an approximately 46.2% yield.

[0239] 1 H NMR (600MHz, CDCl3) δ7.90(d,J=1.5Hz,1H),7.81(s,1H),7.72(s,1H),7.66(d,J=7.9Hz,1H),7.60(dd,J=7.9,1.5Hz,1H),6.82-6.80(m,1H ),6.77-6.74(m,2H),6.73(s,1H),6.36(t,J=5.9Hz,1H),3.96(s,3H),3.87(s,3H),3.84(s,3H),3.74-3.68(m,2H),2.89(t,J=6.9Hz,2H). 13 C NMR (151MHz, CDCl3) δ161.74,149.32,148.08,138.91,138.47,137.13,134.06,130.53,130.32,128.89,128 .33,127.22,122.46,122.20,121.38,120.89,111.81,111.49,56.03,56.01,41.11,39.40,35.04.ESI-HRMS calculatedforC 23 H 23 N3O5S[M+Na]+:476.1251,found476.1270.

[0240] (23) propyl5-(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxylate1,1-dioxi de(b23)

[0241]

[0242] To a reaction flask containing 5 mL of chloroform solution, a23 (0.33 mmol, 1.0 eq) was added, followed by m-CPBA (0.90 mmol, 2.7 eq) in an ice bath. After approximately 4.5 h of reaction at room temperature, 50 mL of EtOAc and an equal volume of NaHCO₃ (2.00 mmol) were added to the reaction flask for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 3 / 2, v / v system) to obtain 68.1 mg of b23 as a white solid in a yield of approximately 62.1%.

[0243] 1 HNMR(400MHz, CDCl3)δ8.28(d,J=1.5Hz,1H),7.82-7.79(m,1H),7.72(s,1H),7.68-7.65(m,1H),7.58(dd,J =7.9,1.5Hz,1H),7.30(s,1H),4.32(t,J=6.7Hz,2H),3.95(s,3H),1.85-1.76(m,2H),1.03(t,J=7.4Hz,3H). 13 CNMR (101MHz, CDCl3) δ161.87,138.87,137.18,135.93,134.45,133.74,129.74,128.07 ,126.89,122.62,122.21,121.48,68.02,39.38,21.96,10.52.ESI-HRMScalculatedforC 16 H 16 N2O4S[M+Na]+:355.0723,found355.0739.

[0244] II. Anti-tumor cell proliferation activity of DC-Rhoin derivatives (CCK8 assay)

[0245] 1. Experimental methods

[0246] (1) Seed board

[0247] Trypsinize MDA-MB-231 cells from a T25 culture flask at a cell density of 70%-80%, centrifuge, and count the cells. Dilute the cells to a concentration of 5,000 cells / 100 μL with complete culture medium. Add 100 μL of complete culture medium containing cells to each well of a 96-well plate and incubate in a 37°C CO2 incubator for 24 hours.

[0248] (2) Administration

[0249] Prepare DMSO solutions containing different concentrations of the test compound, and then dilute with complete culture medium to ensure that the DMSO content of the drug-containing complete culture medium in each gradient is 0.1%.

[0250] The original culture medium solution in the 96-well cells was discarded, and complete culture medium was added to the non-drug group. Complete culture medium containing drugs at different concentrations (gradient: 80 / 40 / 20 / 10 / 5 / 2.5 / 1.25 / 0.625 μM) was added to different drug groups, and then cultured in a CO2 incubator at 37°C for 24 h.

[0251] (3) CCK8 detection

[0252] The original culture medium in the 96-well plate was discarded, and 100 μL of complete culture medium containing 10% CCK8 was added to all groups. The cells were incubated at 37°C in the dark for 2-3 h. The absorbance of each well in each group was measured at 450 nm using a microplate reader.

[0253] 2. Experimental results

[0254] The experimental results are shown in Table 1. Most compounds have moderate or high anti-proliferative activity against MDA-MB-231. 50 =4.2μM), b11 (2.4μM), b6 (2.8μM), b19 (2.7μM), b13 (3.0μM), b21 (3.5μM) and b9 (3.7μM) had the highest antiproliferative activity (<5μM). The data on antiproliferative activity showed two most significant structure-activity relationships: First, C-3 carboxamide compounds all showed stronger antiproliferative activity than carboxylate compounds. In b6 and b10, only one of the above factors changed, showing a clear contrast in the intensity of antiproliferative activity. Another significant structure-activity relationship is that the introduction of 1-methyl-1H-pyrazole in C-5 will enhance the antiproliferative activity of C-3 N-heterocyclic ketone derivatives. The IC value of the antiproliferative activity of all C-3 N-heterocyclic ketone derivatives containing 1-methyl-1H-pyrazole in C-5 is 50 The values ​​were lower than those of the corresponding compounds without 1-methyl-1H-pyrazole.

[0255] Table 1 Antiproliferative activity of target products and positive control doxorubicin

[0256] Target product <![CDATA[IC 50 (μM)]]> Target product <![CDATA[IC 50 (μM)]]> Target product <![CDATA[IC 50 (μM)]]> b1 22.8 b9 3.7 b17 14.2 b2 15.5 b10 >80 b18 6.4 b3 11.5 b11 2.4 b19 2.7 b4 4.8 b12 6.5 b20 5.8 b5 24.2 b13 3.0 b21 3.5 b6 2.8 b14 >80 b22 6.5 b7 5.2 b15 >80 Doxorubicin* 4.2 b8 7.0 b16 >40

[0257] 3. Anti-tumor cell migration activity assay of DC-Rhoin derivatives (Transwell)

[0258] 1. Experimental methods:

[0259] (1) Modeling

[0260] The complete medium in a T25 culture flask at a cell density of 70%-80% (MDA-MB-231 cells) was replaced with a serum-free basal medium containing 1% double antibody, and the cells were cultured in a CO2 incubator at 37°C for 24 h.

[0261] (2) Seeding and drug administration

[0262] Prepare a 5 μM DMSO solution containing the test compound and dilute it with serum-free basal medium containing 1% double antibody to ensure that the DMSO content of the drug-containing medium is 0.2%. Add 100 μL of the prepared drug-containing medium to each 1 mL centrifuge tube for use.

[0263] MDA-MB-231 cells in a T25 culture flask were trypsinized, centrifuged, and the cells were counted. The cells were diluted to a concentration of 56,000 cells / 100 μL using serum-free basal medium containing 1% double-antibody.

[0264] Add 100 μL of culture medium containing cells (drug concentration is 2.5 μM, DMSO is 0.1%) to the 1 mL centrifuge tube to be used and gently resuspend. Place 200 μL of resuspended culture medium into the upper chamber of the Transwell. Add 500 μL of complete culture medium containing serum to the lower chamber. Incubate the cells in a 37°C CO2 incubator for 24 hours.

[0265] (3) Cell fixation, staining, and photography

[0266] Aspirate and discard all the culture medium solutions in the upper and lower chambers of the Transwell plate. Wash the upper chamber twice with PBS buffer. Add 1 mL of 4% paraformaldehyde solution to each upper chamber (the overflow solution flows to the lower chamber) and fix the cells for 10 minutes. Wash the upper chamber twice with PBS buffer. Add 1 mL of crystal violet dye solution to each upper chamber (the overflow solution flows to the lower chamber) and stain the cells for 15 minutes. Use tweezers to pick up the upper chamber, pour the crystal violet dye solution in the upper chamber into the waste liquid bottle, place the upper chamber in pure water, and slowly wash away the residual crystal violet dye solution with pure water. Use a pipette to aspirate and discard the remaining pure water, and add 1 mL of pure water to the upper chamber again (the overflow solution flows to the lower chamber). Observe and photograph the Transwell plate under an inverted microscope. The number of cells in the photo was counted using Image-j software.

[0267] 2. Experimental results and discussion

[0268] Due to the close relationship between RhoA targets and cell motility, the effects of the derivatives on the migration activity of MDA-MB-231 cells were further evaluated by Transwell assay to screen derivatives with better RhoA inhibitory properties. 50Compounds b6, b9, b11, b13, b19 and b21 with a concentration of <5 μM were used as test compounds, and DC-Rhoin04 (i.e., b23) was used as a positive control. The anti-MDA-MB-231 cell migration activity of the test compounds was evaluated under the conditions of a dosage concentration of 2.5 μM and an action time of 24 h.

[0269] The experimental results show that ( Figure 1-2 All tested compounds demonstrated effective anti-migration capabilities. Compounds b11, b13, and b19 significantly outperformed the positive control, DC-Rhoin04 (P < 0.0001). Compounds b13 and b19 inhibited the migration of nearly all MDA-MB-231 cells into the lower Transwell chamber within 24 hours.

[0270] Considering the novelty of the structure, compound b19 was selected for further activity study. 4. Inhibitory effect of compound b19 on tumor cell migration and invasion (Transwell)

[0271] 1. Experimental methods

[0272] (1) Modeling

[0273] The complete medium in a T25 culture flask at a cell density of 70%-80% (MDA-MB-231 cells) was replaced with a serum-free basal medium containing 1% double antibody, and the cells were cultured in a CO2 incubator at 37°C for 24 h.

[0274] (2) Matrigel plating (for Transwell invasion assay only, 3.5 h before plating and drug administration)

[0275] Thaw Matrigel overnight at 4°C and place on ice. Dilute the Matrigel (8:1) with pre-chilled serum-free basal medium containing 1% double-stranded antibody. Add 60 μL of this Matrigel dilution to the upper chamber of the Transwell and place in a 37°C CO2 incubator. After incubation for 3 hours, gently aspirate the remaining Matrigel dilution and add 100 μL of serum-free basal medium containing 1% double-stranded antibody to each upper chamber of the Transwell to hydrate the basement membrane for 30 minutes.

[0276] (3) Plate seeding and drug administration

[0277] Prepare DMSO solutions containing different concentrations of the test compound b19 and dilute them with serum-free basal medium containing 1% bispecific antibody, ensuring that the DMSO content of each gradient of drug-containing medium is 0.1%. Add 100 μL of the prepared drug-containing medium to each 1 mL centrifuge tube for use.

[0278] MDA-MB-231 cells in a T25 culture flask were trypsinized, centrifuged, and the cells were counted. The cells were diluted to a concentration of 56,000 cells / 100 μL using serum-free basal medium containing 1% double-antibody.

[0279] Add 100 μL of culture medium containing cells to the 1 mL centrifuge tube to be used and gently resuspend.

[0280] Migration assay: 200 μL of culture medium containing different concentrations of b19 and the same number of cells were placed into the upper chamber of each Transwell (final b19 concentrations: 4, 2, 1, 0.5, and 0.25 μM, DMSO: 0.1%). 500 μL of complete culture medium containing serum was added to the lower chamber. Cells were cultured in a 37°C CO2 incubator for 24 hours.

[0281] Invasion assay: Aspirate the medium used to hydrate the basement membrane in the upper chamber of the Transwell plate. Place 200 μL of medium containing different drug concentrations and the same number of cells into the upper chamber of each Transwell plate (final drug concentrations of 4, 2, 1, 0.5, and 0.25 μM b19, 0.1% DMSO). Add 500 μL of complete medium containing serum to the lower chamber. Incubate the cells at 37°C in a CO2 incubator for 24 hours.

[0282] (4) Cell fixation, staining, and photography

[0283] Aspirate and discard all the culture medium solutions in the upper and lower chambers of the Transwell plate. Wash the upper chamber twice with PBS buffer. Add 1 mL of 4% paraformaldehyde solution to each upper chamber (the overflow solution flows to the lower chamber) and fix the cells for 10 minutes. Wash the upper chamber twice with PBS buffer. Add 1 mL of crystal violet dye solution to each upper chamber (the overflow solution flows to the lower chamber) and stain the cells for 15 minutes. Use tweezers to pick up the upper chamber, pour the crystal violet dye solution in the upper chamber into the waste liquid bottle, place the upper chamber in pure water, and slowly wash away the residual crystal violet dye solution with pure water. Use a pipette to aspirate and discard the remaining pure water, and add 1 mL of pure water to the upper chamber again (the overflow solution flows to the lower chamber). Observe and photograph the Transwell plate under an inverted microscope. The number of cells in the photo was counted using Image-j software.

[0284] 2. Experimental results and discussion

[0285] The experimental results are as follows Figure 3-5As shown. Further Transwell experiments showed that b19 exhibited dose-dependent inhibitory activity on the migration and invasion of MDA-MB-231 cells. 0.25μM b19 still showed significant activity. The fitting curve of cell migration rate at different dosage concentrations showed that the maximum half-inhibitory concentration of b19 on MDA-MB-231 cell migration was 0.34μM. V. Inhibitory effect of compound b19 on tumor cell migration (wound healing assay)

[0286] 1. Experimental methods

[0287] (1) Seed board

[0288] MDA-MB-231 cells in a T25 culture flask at a cell density of 70%-80% were trypsinized and centrifuged for cell counting. The cells were diluted to a concentration of 3 × 10 cells / mL with complete medium. 5 In a 6-well plate, add 2 mL of complete culture medium containing cells to each well and culture in a CO2 incubator at 37°C for 24 h.

[0289] (2) Medication and monitoring

[0290] DMSO solutions containing different concentrations (4 / 2 / 1 μM) of the test compound b19 were prepared and diluted with culture medium containing 1% serum and double antibody to ensure that the DMSO content of the drug-containing complete culture medium in each gradient was 0.1%.

[0291] Using a 200 μL pipette tip, draw two intersecting lines in the center of each well within a 6-well plate. Discard the culture medium in each well. Add culture medium containing 1% serum and dual-antibody to the untreated group, and culture medium containing varying drug concentrations to the different treated groups. Then, photograph the cells at 0 and 24 hours. The cell migration area in the photographs was calculated using Image-J software.

[0292] 2. Experimental results and discussion:

[0293] The experimental results are as follows Figure 6-7 As shown, wound healing assays showed a concentration-dependent anti-migration effect of b19 on MDA-MB-231 cells.

[0294] VI. Pro-apoptotic effect of compound b19 on tumor cells

[0295] 1. Experimental methods

[0296] (1) Seed board

[0297] MDA-MB-231 cells in a T25 culture flask at a cell density of 70%-80% were trypsinized and centrifuged for cell counting. The cells were diluted to a concentration of 3 × 10 cells / mL with complete medium. 5In a 6-well plate, add 2 mL of complete culture medium containing cells to each well and culture in a CO2 incubator at 37°C for 24 h.

[0298] (2) Administration

[0299] DMSO solutions containing different concentrations of the test compound b19 were prepared and then diluted with culture medium containing 1% serum and double antibody to ensure that the DMSO content of the drug-containing complete culture medium in each gradient was 0.1%.

[0300] The culture medium solution in the 6 wells was discarded, and the completed culture medium was added to the non-drug group. The prepared culture medium containing different concentrations (8 / 4 / 2 / 1 μM) of drug b19 was added to different drug groups, and cultured in a CO2 incubator at 37°C for 24 h.

[0301] (3) Sample preparation and testing

[0302] Trypsinize the MDA-MB-231 cells in 6 wells and collect them by centrifugation (300g, 4°C, 5 min). Wash the cells twice with pre-chilled PBS, centrifuging each time (300g, 4°C, 5 min) and discarding the supernatant. Resuspend the cells in 100μL Binding buffer (1×), then add 5μL of Annexin V-FITC and 10μL of PIStaining Solution, mix gently, and incubate at room temperature in the dark for 10-15 min. Resuspend the cells in 400μL Binding buffer (1×). Finally, analyze the samples by flow cytometry.

[0303] 2. Experimental results

[0304] The experimental results are as follows Figure 8-9 As shown, compound b19 has a significant effect of promoting apoptosis of MDA-MB-231 cells at a dosage concentration of 8 μM.

[0305] VII. Inhibitory effect of compound b19 on stress fiber formation

[0306] 1. Experimental methods

[0307] (1) Seed board

[0308] MDA-MB-231 cells in a T25 culture flask at a cell density of 70%-80% were trypsinized and centrifuged for cell counting. The cells were diluted to a concentration of 2 × 10 5 Add 2 mL of complete culture medium containing cells to the confocal culture dish and culture in a CO2 incubator at 37°C for 24 h.

[0309] (2) Administration

[0310] A 4 μM DMSO solution of the test compound b19 was prepared and diluted with serum-free basal medium containing 1% double antibody to ensure that the DMSO content of each gradient of drug-containing medium was 0.1%.

[0311] The culture medium solution in the confocal culture dish was discarded, and 2 mL of 1% double-antibody serum-free basal culture medium was added to the non-drug group (serum-stimulated and non-serum-stimulated groups); 2 mL of culture medium containing 4 μM drug b19 was added to the b19-administered group, and then cultured in a CO2 incubator at 37°C for 24 h.

[0312] (3) Cell treatment

[0313] The cell treatment conditions for different groups are shown in Table 2. 220 μL of serum was added to the serum-stimulated, non-drug group and the b19-treated group (drug concentration 4 μM), and the cells were incubated in a 37°C CO2 incubator for 15 minutes. The non-serum-stimulated, non-drug group remained in the incubator without treatment.

[0314] Table 2 Cell treatment in different groups

[0315]

[0316]

[0317] (4) Phalloidin color development and detection

[0318] Aspirate the culture medium solution in the confocal culture dish and wash the confocal culture dish 3 times with PBS. Add 1 mL of 4% paraformaldehyde and fix the cells on ice for 15 minutes, then wash the confocal culture dish 3 times with PBS. Permeabilize the cells with PBS solution containing 0.5% TritonX-100 for 10 minutes at room temperature, and then wash the confocal culture dish 3 times with PBS. Add 1-5μl SF488-labeled phalloidin stock solution diluted with 200μL PBS to each confocal culture dish for staining, incubate at room temperature for 20 minutes, and then wash the confocal culture dish 3 times with PBS. Add 100μL of anti-fluorescence fading mounting medium containing DAPI to each confocal culture dish, and then observe the cells under a confocal microscope and take pictures.

[0319] 2. Experimental results and discussion

[0320] RhoA regulates actin aggregation, the formation of stress fibers and focal adhesions, the phosphorylation of myosin light chains, and the resulting enhancement of cell contractility. This study evaluated the RhoA inhibitory activity of compound b19 by observing changes in stress fibers (stained with phalloidin) after administration to MDA-MB-231 cells.

[0321] The experimental results are as follows Figure 10 As shown, the three groups of pictures correspond to the serum-free non-drug group (No serum), the non-drug group (Control, serum treatment) and the drug group (b194μM, serum treatment). After serum-starved (Serum-starved) cells are treated with serum, stress fibers are rapidly formed. Compared with the Control group, the stress fibers of the cells in the b194μM drug group were significantly reduced, which is similar to the effect of treatment with Y-27632, an inhibitor of RhoA-related kinase (ROCK, the main effector protein of RhoA) in the literature. The above results show that at a drug concentration of 4μM, compound b19 can inhibit the formation of stress fibers, showing an inhibitory effect on RhoA activity.

[0322] 8. Compound b19 inhibits phosphorylation of RhoA downstream protein MLC

[0323] 1. Experimental methods

[0324] (1) Seed board

[0325] MDA-MB-231 cells in a T25 culture flask at a cell density of 70%-80% were trypsinized and centrifuged for cell counting. The cells were diluted to a concentration of 4 × 10 cells / mL with complete medium. 5 In a 6-well plate, add 2 mL of complete culture medium containing cells to each well and culture in a CO2 incubator at 37°C for 24 h.

[0326] (2) Administration

[0327] Prepare DMSO solutions containing different concentrations of the test compound, and then dilute them with serum-free basal culture medium containing 1% double antibody to ensure that the DMSO content of each gradient of drug-containing culture medium is 0.1%.

[0328] The culture medium solution in the 6 wells was discarded, and 2 mL of 1% double-antibody serum-free basal culture medium was added to the non-drug group (serum-stimulated and non-serum-stimulated groups); 2 mL of culture medium containing different concentrations (4 / 2 / 1 μM) of drug b19 was added to the b19-dosing group; 2 mL of culture medium containing 30 μM Rhosin was added to the positive drug group, and then cultured in a CO2 incubator at 37°C for 24 h.

[0329] (3) Cell treatment

[0330] 220 μL of serum was added to the serum-stimulated non-drug group, b19-administered group, and Rhosin-administered group, and the cells were incubated in a CO2 incubator at 37°C for 15 minutes. The serum-unstimulated non-drug group remained in the incubator without treatment.

[0331] Table 3 Cell treatment in different groups

[0332] Group Name Belong Compound (μM) Serum stimulation 15 minutes Noserum No drug group - - Control No drug group - + b194 μM Drug administration group b19(4) + b192 μM Drug administration group b19(2) + b191 μM Drug administration group b19(1) + Rhosin 30 μM Positive drug group Rhosin(30) +

[0333] (4) Protein extraction

[0334] Aspirate the culture medium from all groups. Wash three times with PBS buffer. After aspirating any remaining PBS, place the 6-well plate on ice and add cell lysis buffer containing protease and phosphatase inhibitors. After lysing the cells on ice for 30 minutes, use a cell scraper and pipette to collect the solution from each well of the 6-well plate. Determine the protein concentration in each well using the BCA assay.

[0335] (5) Sample preparation and testing

[0336] Prepare 5× Master Mix (Loading Buffer) and Ladder (marker, molecular weight standard) according to the instructions for the fully automated Western blotting quantitative analysis system. Dilute the protein sample to 2 μg / μL using 5× Master Mix and Sample Buffer. Denature the prepared sample at 95°C for 5 minutes. After denaturation, remove the sample and cool it on ice for 5 minutes. After cooling, vortex to mix thoroughly, centrifuge briefly, and place on ice until ready to use.

[0337] Using Antibody Diluent II as the solvent, dilute the primary antibody mixtures of MLC + β-actin and p-MLC + β-actin at a ratio of MLC (1:50) + β-actin (1:3000) and p-MLC (1:50) + β-actin (1:3000), respectively. Use a rabbit:mouse (1:1) secondary antibody mixture. Prepare the luminescent solution by mixing Lumino-S and Peroxide in equal proportions and keep on ice until ready to use.

[0338] Following the recommended dosages and procedures, add the markers (molecular weight standards), prepared samples, primary and secondary antibodies, luminescent solution, and wash buffer to the consumables plate. Run the automated Western blotting quantitative analysis system to analyze the samples. Data are processed and analyzed using the accompanying Compass for SW software.

[0339] 2. Experimental results and discussion:

[0340] Myosin II is believed to be involved in the generation of contractile force during cell migration. Myosin II activity is primarily controlled by phosphorylation of its light chain (MLC). This phosphorylation is regulated by two enzymes: MLC kinase and myosin light chain phosphatase (MLCP). Active RhoA (RhoA-GTP) phosphorylates the MYPT1 subunit of MLCP through its primary downstream effector, RhoA-associated kinase (ROCK), leading to the accumulation of phosphorylated myosin light chain (p-MLC) and the consequent increase in cellular contractility and stress fiber formation. Knockdown of RhoA also reduces direct phosphorylation and activation of myosin light chain (MLC), resulting in impaired regulation of the actin cytoskeleton. Therefore, in this study, we evaluated the inhibitory effect of compound b19 on RhoA target activity by measuring p-MLC levels.

[0341] The experimental results are as follows Figure 11-12 Compound b19 dose-dependently downregulated p-MLC levels in MDA-MB-231 cells (P < 0.0001 for b19 1 μM, b19 2 μM, and b19 4 μM), demonstrating an inhibitory effect on RhoA target activity. The b19 concentration required to reduce MLC phosphorylation was in the single digit micromolar range. A 4 μM b19 concentration was more effective than a 30 μM positive control, rhosin.

[0342] IX. Molecular docking experiment of compound b19 and RhoA

[0343] 1. Experimental methods

[0344] (1) Preparation of receptor protein

[0345] The receptor protein selected for this experiment was the crystal structure of RhoA protein containing the covalent inhibitor DC-Rhoin (PDBID: 6KX3), which was published by the applicant team in the paper "Covalent inhibitors allosterically block the activation of Rho family proteins and suppress cancer cell invasion." The RhoA protein was prepared using the "Protein Preparation Wizard" in Schrodinger's Maestro software, including default treatments such as dehydration, hydrogenation, and repair of residue side chains.

[0346] (2) Preparation of ligand

[0347] The structure of compound b19 was drawn and saved as a .SDF file. The small molecule ligand file was imported into Maestro software, and then the small molecule ligand was prepared for docking using "LigPrep" to generate a reasonable 3D conformation.

[0348] (3) Covalent docking

[0349] The coordinates of DC-Rhoin in the RhoA protein crystal structure were selected as the central coordinates of the docking site, and the DC-Rhoin covalent binding site C107A was used as the covalent binding site for the small molecule ligand. The docking box dimensions were similar to those of the covalent inhibitor DC-Rhoin. The covalent binding reaction type was a Michael addition reaction. Finally, the PosePrediction (Thorough) docking mode was used for the covalent docking calculation of compound b19 with the RhoA protein.

[0350] 2. Experimental results

[0351] To investigate the interaction between b19 and RhoA, molecular docking was performed based on the crystal structure of RhoA in complex with DC-Rhoin (PDBID: 6KX3). b19 was covalently bound to the CYS-107 residue in the RhoA pocket via a Michael addition reaction, consistent with its lead compound, DC-Rhoin.

[0352] The docking results showed that b19 could form more and stronger hydrogen bonds with RhoA protein than DC-Rhoin ( Figure 13 b19 binds to the Thr-77 residue of RhoA through the two oxygen atoms of the sulfone group. and PHE-106 residues Form three hydrogen bonds ( Figure 13 .b). Unlike DC-Rhoin, the sulfone group of b19 faces the outside of the protein pocket ( Figure 13 .b).

[0353] When b19 covalently binds to RhoA protein, its benzothiophene ring flips spatially relative to DC-Rhoin ( Figure 13 .d). The 1-methyl-1H-pyrazole of b19 replaces the flexible side chain of DC-Rhoin at the corresponding spatial position and extends to one side of the pocket, entering the groove formed by TRP-58, ASP-59, THR-60 and GLN-63 residues; while the phenoxypyridine of b19 extends to the U-shaped opening formed by GLN-63 and TYR-66 ( Figure 13 .d).

[0354] The above results indicate that compared with the lead compound DC-Rhoin, b19 may produce more and stronger hydrogen bonds near the covalent binding site, thereby giving b19 a higher affinity for the RhoA protein pocket and a more suitable spatial positioning, thereby improving the non-covalent binding ability with the RhoA pocket while promoting covalent binding between the two.

[0355] From the above series of experiments, we can see that the structure-activity relationship shows that the formation of C-3 amide and the introduction of C-5 1-methyl-1H-pyrazole in the DC-Rhoin structure help to enhance the anti-tumor proliferation activity of DC-Rhoin. Among the compounds b1-b22, b19 showed outstanding anti-tumor proliferation activity (IC 50 =2.7 μM), dose-dependent anti-tumor migration (IC 50 At a concentration of 8 μM, b19 significantly promoted the apoptosis of MDA-MB-231 cells.

[0356] In terms of RhoA activity inhibition, b19 can inhibit stress fiber formation. Compared with the 30μM positive control Rhosin, 4μM compound b19 has a stronger inhibitory effect on MLC phosphorylation. Molecular docking studies of the interaction between b19 and RhoA protein revealed that compared to the lead compound DC-Rhoin, b19 may have stronger non-covalent binding and subsequent covalent binding to the RhoA "Clock" pocket, possibly through more hydrogen bonding and higher protein pocket fit.

[0357] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. DC-Rhoin derivative, characterized in that The compound represented by formula (I), or a medically acceptable soluble salt formed by the compound represented by formula (I): Wherein, when R1 is H, R2 is one of the following groups: When R1 is 1-methyl-1H-pyrazol-4-yl, R2 is one of the following groups:

2. The method for preparing the DC-Rhoin derivative according to claim 1, characterized in that: When R1 is H, the steps are as follows: (1) Benzothiophene-3-carboxylic acid, DMAP, and EDCI were sequentially added to a reaction flask containing DCM, stirred, and then the reactants were added. After reaction at room temperature, H2O, or EtOAc and an equal volume of H2O, or EtOAc and hydrochloric acid solution and H2O were added for extraction; the organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain an intermediate product; (2) The intermediate product obtained in step (1) and m-CPBA were added to a reaction flask containing chloroform solution in sequence. After reaction at room temperature, DCM and 5% NaHCO3 solution or EtOAc and 5% NaHCO3 solution or EtOAc and 5% Na2CO3 solution and cold water were added to the reaction flask for extraction, or EtOAc was added dropwise until the reaction solution was clear; the organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain the target product; Alternatively, the intermediate product obtained in step (1) and m-CPBA are added to a reaction bottle containing chloroform solution in sequence. After reaction at room temperature, EtOAc is added dropwise with stirring until the reaction solution becomes clear. The mixture is directly added to a 200-300 mesh silica gel sample and separated and purified by silica gel column chromatography to obtain the target product.

3. The method for preparing the DC-Rhoin derivative according to claim 2, wherein: The reactant in step (1) is one of tetrahydropyrrolidine, morpholine, 4-methoxypiperidine, 4-phenoxypiperidine, 4-benzoylpiperidine hydrochloride, 3,4-dimethoxyaniline, 3,4-diethoxyaniline, 3,4-methylenedioxyaniline, 6-amino-1,4-benzodioxetine, 3,4-dimethoxyphenol, 4-heptylamine, 3,4-dimethoxyethylamine, 2-(4-methoxyphenoxy)ethylamine, 2-(2-ethoxyphenoxy)ethanol, phenoxyethanol, and 2-phenoxy-1-phenylethanol.

4. The method for preparing the DC-Rhoin derivative according to claim 1, characterized in that: When R1 is 1-methyl-1H-pyrazol-4-yl, the steps are as follows: S1. 5-bromobenzo[b]thiophene-3-carboxylic acid, DMAP, and EDCI were added sequentially to a reaction flask containing DCM, and after stirring, anhydrous ethanol was added. After reaction at room temperature, the solvent was removed by vortexing, EtOAc was added, and the aqueous phase was adjusted to slightly acidic with 0.5N HCl solution. The aqueous phase was supplemented with H2O to 50 mL and extracted; the organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain a first intermediate product; S2, the first intermediate product, 1-methylpyrazole-4-boronic acid pinacol ester and Na2CO3 were added to a reaction flask, and under nitrogen protection, a mixture of H2O and DMF was injected, followed by adding Pd(dppf)Cl2 for reaction; the reaction flask was cooled to room temperature, EtOAc and dilute hydrochloric acid solution were poured, and the filtrate was extracted after filtration; the organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure and separated and purified by silica gel column chromatography to obtain a second intermediate product; S3, adding the second intermediate product and NaOH to a reaction flask, followed by adding an EtOH / H2O mixture for reaction; cooling the reaction flask to room temperature, pouring EtOAc and dilute hydrochloric acid solution; filtering to obtain a filtrate, performing an extraction operation on the filtrate, treating the organic phase with saturated brine and anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a third intermediate product; S4, the third intermediate product, EDCI and DMAP were added to a reaction flask containing DCM in sequence, the reactants were added after stirring, the reaction was carried out at room temperature, the solvent was dried, EtOAc and H2O were added to the reaction flask, the pH was adjusted to weak acidity with dilute hydrochloric acid, and the organic phase was treated with saturated brine and anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a fourth intermediate product; S5. The fourth intermediate product and m-CPBA were added to a reaction flask containing chloroform solution in sequence. After the reaction at room temperature, EtOAc and a saturated cold solution of NaHCO3 were added to the reaction flask for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain the target product.

5. The method for preparing the DC-Rhoin derivative according to claim 4, characterized in that: The reactant in step S4 is one of tetrahydropyrrolidine, 4-methoxypiperidine, 4-phenoxypiperidine, 4-benzoylpiperidine hydrochloride, 4-heptylamine, and 3,4-dimethoxyethylamine.

6. Use of the DC-Rhoin derivative according to claim 1 in the preparation of a RhoA protein inhibitor.

7. Use of the DC-Rhoin derivative according to claim 1 in preparing drugs for treating tumors.

8. The use according to claim 7, characterized in that The DC-Rhoin derivative is used in the preparation of anti-tumor metastasis drugs.

Images