Synthesis and in vitro evaluation of benzo[b]thiophene-3-carboxylic acid 1,1-dioxide derivatives as anticancer agents targeting the RhoA/ROCK pathway

Abstract

Rho family GTPases regulate cellular processes and promote tumour growth and metastasis; thus, RhoA is a potential target for tumour metastasis inhibition. However, limited progress has been made in the development of RhoA targeting anticancer drugs. Here, we synthesised benzo[b]thiophene-3-carboxylic acid 1,1-dioxide derivatives based on a covalent inhibitor of RhoA (DC-Rhoin), reported in our previous studies. The observed structure-activity relationship (contributed by carboxamide in C-3 and 1-methyl-1H-pyrazol in C-5) enhanced the anti-proliferative activity of the derivatives. Compound b19 significantly inhibited the proliferation, migration, and invasion of MDA-MB-231 cells and promoted their apoptosis. The suppression of myosin light chain phosphorylation and the formation of stress fibres confirmed the inhibitory activity of b19 via the RhoA/ROCK pathway. b19 exhibited a different binding pattern from DC-Rhoin, as observed in molecular docking analysis. This study provides a reference for the development of anticancer agents targeting the RhoA/ROCK pathway.

Introduction

Rho family GTPases, a subfamily of the Ras superfamily, are crucial drivers of tumour growth and metastasis^1,2. GTPases regulate various biological effects on cellular morphology, movement, and behaviour, including epithelial cell morphogenesis, neurite outgrowth, single or collective cell migration, as well as assembly and contraction of the actin and myosin cable^3–7. A large majority of Rho GTPases act as molecular switches by cycling between an active GTP-bound and an inactive GDP-bound conformation. This cycling between states is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs) (Figure 1)^6–8. RhoA, Rac1, and Cdc42 are the best characterised and most studied Rho family members^1,9. Changes in RhoA expression and an imbalance between active and inactive GTP-bound states promote cancer cell migration and invasion^10–12. RhoA is a potential target for cancer metastasis inhibition. RhoA inhibitors have shown suppression of cancer cell migration and (or) invasion^13–15, and RhoA-related pathways, and their inhibitors, have frequently been used to study the mechanisms by which various target proteins or related pathways affect tumour metastasis^16–20.

Figure 1.

The Rho-GTPase cycle between an active GTP-bound and an inactive GDP-bound conformation. GEFs promote the formation of an active GTP-bound conformation and then its downstream effector proteins subsequently interact with active GTPase^6,7. GAPs enhance the inherent enzyme activity of Rho-GTPase to promote the formation of an inactive GDP-bound conformation^6,7. GDIs are responsible for regulating the spatiotemporal distribution of Rho GTPase between the plasma membrane and cytoplasm^6–8. This figure was drawn for this article by Jinhao Liang, an author of this article.

Due to a potent binding affinity between GTPase and GTP or GDP, the micromolar GTP concentration in cells, and the lack of targetable sites on the surface, the identification of molecules targeting Ras superfamily proteins is difficult^21–23. A crucial breakthrough was the development of K-Ras (a member of the Ras superfamily) inhibitors²⁴, such as Sotorasib and Adagrasib that covalently inhibit K-Ras^G12C by targeting the unprecedented tractable pocket around Cys-12 on K-Ras (Figure 2(a))^25,26. Sotorasib and Adagrasib were approved for the treatment of non-small cell lung cancer harbouring the K-Ras^G12C mutation^27,28.

Figure 2.

The structure of inhibitors of Ras superfamily proteins. (a) The inhibitors of K-Ras^G12C. (b) The inhibitors of RhoA. (c) DC-Rhoin and ACR-895 can covalently bind to Cys-107 residue of RhoA.

Previously, we discovered a new pocket on the RhoA protein and successfully developed a RhoA inhibitor, DC-Rhoin, that can covalently bind with the Cys-107 residue in this pocket¹⁴. DC-Rhoin and its derivative, DC-Rhoin04, inhibit MDA-MB-231 cellular proliferation, migration, and invasion (Figure 2(b))¹⁴. In addition, ACR-895 inhibits RhoA activation via Cys-107 residue interaction (Figure 2(c)), further highlighting Cys-107 as a target residue for RhoA inhibitors²⁹. DC-Rhoin as an inhibitor that binds to RhoA via the Cys-107 residue, and we hypothesised that more active antitumour compounds could be found from DC-Rhoin derivatives.

Therefore, in this study, we aimed to synthesise DC-Rhoin derivatives and determine the anticancer activity of each derivative to study structure-activity relationships and find a compound with potential for further development.

Materials and methods

Chemistry

All reagents and solvents were purchased from chemical companies. Thin layer chromatography (TLC) analysis was used to determine the extent of reactions under UV light (wavelength: 365 nm and 254 nm). TLC/expression-CMS (Advion Interchim Scientific) was used to determine the generation of products and their position in TLC.¹H NMR and ¹³C NMR spectra were evaluated in CDCl₃, MeOD, and DMSO-d₆ on a Bruker AV400 and Bruker AV600 using TMS as the internal reference. High-resolution mass spectra were recorded using an LTQ Orbitrap XL instrument (Thermo Fischer Scientific) with the ESI mode.

Synthesis of ethyl 5-bromobenzo[b]thiophene-3-carboxylate (Pre-1)

A round-bottom flask was charged with 5-bromobenzo[b]thiophene-3-carboxylic acid (7.8 mmol, 1.0 equiv.), DMAP (31.1 mmol, 4.0 equiv.), EDCI (15.6 mmol, 2.0 equiv.), and DCM (25.0 ml). After stirring the reaction mixture for 5 min, anhydrous ethanol was added. The reaction was stirred at room temperature for approximately 7.75 h, and the reaction progress was monitored using TLC-MS. Upon reaction completion, the solvents were evaporated under reduced pressure. Diluted hydrochloric acid (50 ml) was added, and the aqueous phase was extracted using EtOAc. The organic phase was washed with 25 ml of saturated NaCl solution, dried over anhydrous Na₂SO₄, and filtered, and the solvent was concentrated to obtain the crude product. The product (Pre-1) was obtained by column chromatography on silica gel (Eluent: PE/EA = 99:1). White solid; yield: 86%.¹H NMR (600 MHz, CDCl₃) δ 8.77 (d, J = 1.9 Hz, 1H), 8.39 (s, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.50 (dd, J = 8.6, 2.0 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H).¹³C NMR (151 MHz, CDCl₃) δ 162.54, 138.71, 138.42, 137.92, 128.32, 127.69, 126.89, 123.83, 120.04, 61.02, 14.53. ESI-HRMS calculated for C₁₁H₉BrO₂S [M + Na]⁺: 306.9399, found 306.9408.

Synthesis of ethyl 5–(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxylate (Pre-2)

Pre-1 (6.7 mmol, 1.0 equiv.), 1-Methyl-4–(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (8.0 mmol, 1.2 equiv.) and Na₂CO₃ (20.0 mmol, 3.0 equiv.) were added in a round-bottom flask. Under a nitrogen atmosphere, a mixture of H₂O (3 ml) and DMF (20 ml) was added by syringe, followed by the addition of Pd(dppf)Cl₂ (0.7 mmol, 0.1 equiv.). The mixture was stirred at 90 °C for approximately 3.3 h, and the reaction progress was monitored using TLC-MS. Upon reaction completion, the reaction mixture was cooled to room temperature. Subsequently, 250 ml of EtOAc and 250 ml of dilute hydrochloric acid (30.00 mmol) were sequentially poured into the reaction flask. The reaction mixture was filtered, and the filtrate was collected and separated using a separating funnel. Following separation, the organic phase was obtained, washed with 25 ml of saturated NaCl solution, dried over anhydrous Na₂SO₄, filtered, and the solvent was concentrated to obtain the crude product. The product (Pre-2) was obtained by column chromatography on silica gel (Eluent: PE/EA = 5:1). White solid; yield: 67%. ¹H NMR (600 MHz, CDCl₃) δ 8.72 (d, J = 1.6 Hz, 1H), 8.38 (s, 1H), 7.87 (s, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.73 (s, 1H), 7.53 (dd, J = 8.4, 1.7 Hz, 1H), 4.43 (q, J = 7.1 Hz, 2H), 3.97 (s, 3H), 1.45 (t, J = 7.1 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 162.93, 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 calculated for C₁₅H₁₄N₂O₂S [M + Na]⁺: 309.0668, found 309.0684.

Synthesis of 5–(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxylic acid (Pre-3)

Pre-2 (3.49 mmol, 1.0 equiv.) and NaOH (8.73 mmol, 2.5 equiv.) were added to a round-bottom flask, and then a mixture of H₂O (2 ml) and EtOH (10 ml) was added by syringe. The mixture was stirred at 50 °C for approximately 40 min, and the reaction progress was monitored using TLC-MS. Upon reaction completion, the reaction mixture was cooled to room temperature. Following cooling, 30 ml of EtOAc and 30 ml of dilute hydrochloric acid (9.00 mmol) were poured into the flask in sequence. After filtration, the filter residue was collected to afford crude Pre-3. Light brown solid; yield: 91%. ¹H NMR (600 MHz, CDCl₃) δ 8.69 (s, 1H), 8.42 (s, 1H), 7.88 (s, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.76 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 3.98 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 164.82, 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-HRMS calculated for C₁₃H₁₀N₂O₂S [M + H]⁺: 259.0536, found 259.0552.

General synthesis of a1 to a23

A round-bottom flask was charged with benzo[b]thiophene-3-carboxylic acid (or Pre-3, 1.68 mmol, 1.0 equiv.), DMAP (6.72 mmol, 4.0 equiv.), EDCI (3.36 mmol, 2.0 equiv.), and DCM (5 ml). After stirring the reaction mixture for 5 min, a corresponding reactant containing hydroxyl or amino groups (2.02 mmol, 1.2 equiv.) was added. The mixture was stirred at room temperature, and the reaction progress was monitored using TLC-MS. Upon reaction completion, the solvents were evaporated under reduced pressure. Diluted hydrochloric acid was added, and the aqueous phase was extracted using EtOAc. The organic phase was washed with saturated NaCl solution, dried over anhydrous Na₂SO₄, and filtered, and the solvent was concentrated to obtain crude product. The products (a1–a23) were obtained by column chromatography on silica gel.

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

White solid; yield: 61%. ¹H NMR (400 MHz, CDCl₃) δ 8.01–7.97 (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). ¹³C NMR (101 MHz, CDCl₃) δ 164.92, 139.75, 137.22, 133.13, 126.92, 125.02, 124.90, 123.88, 122.56, 49.15, 46.17, 26.35, 24.61. ESI-HRMS calculated for C₁₃H₁₃NOS [M + Na]⁺: 254.0610, found 254.0626.

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

White solid; yield: 49%. ¹H NMR (600 MHz, CDCl₃) δ 7.88 (d, J = 7.9 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.58 (s, 1H), 7.46–7.41 (m, 1H), 7.41–7.36 (m, 1H), 4.08–3.22 (m, 8H). ¹³C NMR (151 MHz, CDCl₃) δ 165.58, 139.89, 136.86, 131.33, 127.14, 125.21, 125.11, 122.97, 122.82, 67.13, 47.85, 42.80. ESI-HRMS calculated for C₁₃H₁₃NO₂S [M + Na]⁺: 270.0559, found 270.0575.

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

Colourless transparent oil; yield: 85%. ¹H NMR (600 MHz, CDCl₃) δ 7.85 (d, J = 7.2 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.53 (s, 1H), 7.39 (td, J = 7.7, 1.4 Hz, 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). ¹³C NMR (151 MHz, CDCl₃) δ 165.39, 139.78, 137.00, 132.08, 126.21, 125.02, 124.90, 122.98, 122.67, 75.28, 55.83, 44.55, 39.33, 31.48, 30.52. ESI-HRMS calculated for C₁₅H₁₇NO₂S [M + Na]⁺: 298.0872, found 298.0886.

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

Colourless transparent oil; yield: 72%. ¹H NMR (600 MHz, CDCl₃) δ 7.88 (d, J = 7.9 Hz, 1H), 7.86 (d, J = 7.8 Hz, 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 = 7.3 Hz, 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). ¹³C NMR (151 MHz, CDCl₃) δ 165.60, 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 calculated for C₂₀H₁₉NO₂S [M + Na]⁺: 360.1029, found 360.1052.

Benzo[b]thiophen-3-yl(4-benzoylpiperazin-1-yl)methanone (a5)

White solid; yield: 93%. ¹H NMR (600 MHz, CDCl₃) δ 7.87 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.59 (s, 1H), 7.51–7.34 (m, 7H), 4.09–3.31 (m, 8H). ¹³C NMR (151 MHz, CDCl₃) δ 170.69, 165.70, 139.86, 136.75, 135.11, 131.09, 130.17, 128.70, 127.38, 127.10, 125.25, 125.13, 122.83, 47.79, 42.55. ESI-HRMS calculated for C₂₀H₁₈N₂O₂S [M + Na]⁺: 373.0981, found 373.1001.

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

White solid; yield: 62%. ¹H NMR (600 MHz, CDCl₃) δ 8.42 (d, J = 8.1 Hz, 1H), 7.97 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.75 (s, 1H), 7.50–7.47 (m, 1H), 7.47 (s, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.00 (dd, J = 8.5, 2.4 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 3.91 (s, 3H), 3.88 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 162.16, 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-HRMS calculated for C₁₇H₁₅NO₃S [M + Na]⁺: 336.0665, found 336.0688.

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

White solid; yield: 44%. 1H NMR (600 MHz, CDCl₃) δ 8.41 (dt, J = 8.1, 1.0 Hz, 1H), 7.97 (s, 1H), 7.89 (dt, J = 8.0, 1.0 Hz, 1H), 7.77 (s, 1H), 7.47 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.45–7.43 (m, 1H), 7.43–7.40 (m, 1H), 6.99 (dd, J = 8.6, 2.5 Hz, 1H), 6.86 (d, J = 8.5 Hz, 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). ¹³C NMR (151 MHz, CDCl₃) δ 162.12, 149.20, 145.82, 140.39, 136.85, 132.52, 131.58, 129.51, 125.45, 125.43, 124.41, 122.74, 114.11, 112.51, 107.03, 65.13, 64.71, 15.01, 14.90. ESI-HRMS calculated for C₁₉H₁₉NO₃S [M + Na]⁺: 364.0978, found 364.0999.

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

Light yellow solid; yield: 81%. ¹H NMR (600 MHz, DMSO-d₆) δ 10.24 (s, 1H), 8.50 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.50–7.46 (m, 1H), 7.46–7.43 (m, 2H), 7.19 (dd, J = 8.4, 2.1 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.02 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 161.62, 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-HRMS calculated for C₁₆H₁₁NO₃S [M + Na]⁺: 320.0352, found 320.0370.

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

White solid; yield: 49%. ¹H NMR (600 MHz, CDCl₃) δ 8.39 (dt, J = 8.2, 1.0 Hz, 1H), 7.94 (s, 1H), 7.88 (dt, J = 8.0, 1.0 Hz, 1H), 7.69 (s, 1H), 7.46 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.42 (ddd, J = 8.2, 7.0, 1.3 Hz, 1H), 7.27 (d, J = 2.5 Hz, 1H), 7.02 (dd, J = 8.7, 2.5 Hz, 1H), 6.85 (d, J = 8.6 Hz, 1H), 4.28–4.24 (m, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 162.10, 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 calculated for C₁₇H₁₃NO₃S [M + Na]⁺: 334.0508, found 334.0531.

N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzo[b]thiophene-3-carboxamide (a10)

White solid; yield: 88%. ¹H NMR (600 MHz, CDCl₃) δ 8.64 (d, J = 8.2 Hz, 1H), 8.61 (s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 6.94–6.90 (m, 1H), 6.84–6.79 (m, 2H), 3.91 (s, 3H), 3.90 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 161.51, 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-HRMS calculated for C₁₇H₁₄O₄S [M + Na]⁺: 337.0505, found 337.0528.

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

White solid; yield: 82%. ¹H NMR (600 MHz, CDCl₃) δ 8.34 (d, J = 8.1 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.81 (s, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 5.81 (d, J = 9.2 Hz, 1H), 4.24–4.16 (m, 1H), 1.61–1.55 (m, 2H), 1.51–1.39 (m, 6H), 0.95 (t, J = 7.1 Hz, 6H). ¹³C NMR (151 MHz, CDCl₃) δ 163.84, 140.41, 136.98, 132.90, 128.54, 125.17, 124.34, 122.67, 49.25, 37.77, 19.40, 14.20. ESI-HRMS calculated for C₁₆H₂₁NOS [M + Na]⁺: 298.1236, found 298.1252.

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

White solid; yield: 83%. ¹H NMR (600 MHz, CDCl₃) δ 8.25 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 7.5 Hz, 1H), 7.74 (s, 1H), 7.40 (ddd, J = 8.1, 7.1, 1.4 Hz, 1H), 7.39–7.35(m, 1H), 6.81 (d, J = 8.0 Hz, 1H), 6.79–6.76 (m, 1H), 6.76–6.75 (m, 1H), 6.18 (t, J = 5.6 Hz, 1H), 3.85 (s, 3H), 3.82 (s, 3H), 3.71 (q, J = 6.6 Hz, 2H), 2.90 (t, J = 6.9 Hz, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 164.13, 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-HRMS calculated for C₁₉H₁₉NO₃S [M + Na]⁺: 364.0978, found 364.1003.

N-(3,4-dimethoxyphenethyl)benzo[b]thiophene-3-carboxamide (a13)

White solid; yield: 97%. ¹H NMR (600 MHz, CDCl₃) δ 8.37 (d, J = 8.1 Hz, 1H), 7.87 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 6.85 (d, J = 9.3 Hz, 2H), 6.87–6.84 (m, 2H), 6.84–6.81 (m, 1H), 4.11 (t, J = 5.1 Hz, 2H), 3.85 (q, J = 5.4 Hz, 2H), 3.76 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 164.24, 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-HRMS calculated for C₁₈H₁₇NO₃S [M + Na]⁺: 350.0821, found 350.0840.

2–(2-ethoxyphenoxy)ethyl benzo[b]thiophene-3-carboxylate (a14)

White solid; yield: 88%. ¹H NMR (600 MHz, CDCl₃) δ 8.61 (d, J = 8.2 Hz, 1H), 8.37 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.47 (t, J = 7.5 Hz, 1H), 7.41 (t, J = 7.6 Hz, 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 = 7.0 Hz, 2H), 1.39 (t, J = 7.0 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 162.74, 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-HRMS calculated for C₁₉H₁₈O₄S [M + Na]⁺: 365.0818, found 365.0841.

2-phenoxyethyl benzo[b]thiophene-3-carboxylate (a15)

Colourless transparent oil; yield: 54%. ¹H NMR (600 MHz, CDCl₃) δ 8.57 (d, J = 8.2 Hz, 1H), 8.37 (s, 1H), 7.84 (d, J = 8.1 Hz, 1H), 7.45 (ddd, J = 8.2, 7.1, 1.1 Hz, 1H), 7.38 (ddd, J = 8.2, 7.0, 1.3 Hz, 1H), 7.30–7.26 (m, 2H), 6.98–6.95 (m, 1H), 6.95–6.93 (m, 2H), 4.68 (t, J = 6.4 Hz, 2H), 4.32 (t, J = 6.4 Hz, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 162.75, 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-HRMS calculated for C₁₇H₁₄O₃S [M + Na]⁺: 321.0556, found 321.0572.

2-phenoxy-1-phenylethyl benzo[b]thiophene-3-carboxylate (a16)

Colourless transparent oil; yield: 84%. ¹H NMR (600 MHz, CDCl₃) δ 8.65 (d, J = 8.2 Hz, 1H), 8.45 (s, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.56 (d, J = 7.1 Hz, 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, 3.8 Hz, 1H), 4.50 (dd, J = 10.5, 7.8 Hz, 1H), 4.36 (dd, J = 10.5, 3.8 Hz, 1H). ¹³C NMR (151 MHz, CDCl₃) δ 161.92, 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-HRMS calculated for C₂₃H₁₈O₃S [M + Na]⁺: 397.0869, found 397.0897.

(5–(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophen-3-yl)(pyrrolidin-1-yl)methanone (a17)

White solid; yield: 47%. ¹H NMR (600 MHz, CDCl₃) δ 8.10 (s, 1H), 7.84–7.81 (m, 2H), 7.71 (s, 1H), 7.62 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 3.95 (s, 3H), 3.74 (t, J = 7.1 Hz, 2H), 3.50 (t, J = 6.8 Hz, 2H), 2.03–1.97 (m, 2H), 1.93–1.86 (m, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 164.79, 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-HRMS calculated for C₁₇H₁₇N₃OS [M + Na]⁺: 334.0985, found 334.1007.

(4-methoxypiperidin-1-yl)(5–(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophen-3-yl)methanone (a18)

Colourless transparent oil; yield: 52%. ¹H NMR (600 MHz, CDCl₃) δ 7.87 (d, J = 1.1 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H), 7.67 (s, 1H), 7.53 (s, 1H), 7.50 (dd, J = 8.3, 1.7 Hz, 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). ¹³C NMR (151 MHz, CDCl₃) δ 165.42, 137.86, 137.83, 136.91, 131.73, 129.84, 127.42, 126.89, 123.48, 123.08, 123.05, 119.49, 75.30, 55.96, 44.69, 39.39, 39.24, 31.66, 30.51. ESI-HRMS calculated for C₁₉H₂₁N₃O₂S [M + Na]⁺: 378.1247, found 378.1262.

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

Colourless transparent oil; yield: 38%. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.82 (s, 1H), 7.69 (s, 1H), 7.56 (s, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.31–7.27 (m, 2H), 6.96 (tt, J = 7.4, 1.0 Hz, 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). ¹³C NMR (151 MHz, CDCl₃) δ 165.50, 157.03, 137.90, 137.85, 136.96, 131.60, 129.90, 129.78, 127.46, 127.03, 123.54, 123.08, 121.40, 119.52, 116.19, 71.37, 44.26, 39.27, 38.97, 31.55, 30.56. ESI-HRMS calculated for C₂₄H₂₃N₃O₂S [M + Na]⁺: 440.1403, found 440.1438.

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

White solid; yield: 44%. ¹H NMR (600 MHz, CDCl₃) δ 7.88 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H), 7.67 (s, 1H), 7.57 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.46–7.35 (m, 5H), 3.95 (s, 3H), 3.93–3.38 (m, 8H). ¹³C NMR (151 MHz, CDCl₃) δ 170.75, 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-HRMS calculated for C₂₄H₂₂N₄O₂S [M + Na]⁺: 453.1356, found 453.1377.

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

White solid; yield: 81%. ¹H NMR (600 MHz, CDCl₃) δ 8.50 (s, 1H), 7.84 (s, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.81 (s, 1H), 7.72 (s, 1H), 7.52 (d, J = 8.4 Hz, 1H), 5.82 (d, J = 9.2 Hz, 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 = 7.1 Hz, 6H). ¹³C NMR (151 MHz, CDCl₃) δ 163.80, 138.30, 137.78, 136.88, 132.53, 129.86, 128.92, 127.54, 123.39, 123.27, 122.91, 120.94, 49.28, 39.20, 37.74, 19.42, 14.23. ESI-HRMS calculated for C₂₀H₂₅N₃OS [M + Na]⁺: 378.1611, found 378.1630.

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

White solid; yield: 60%. ¹H NMR (600 MHz, CDCl₃) δ 8.44 (s, 1H), 7.80 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.72 (s, 1H), 7.64 (s, 1H), 7.49 (d, J = 8.3 Hz, 1H), 6.80–6.77 (m, 1H), 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 = 6.9 Hz, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 164.15, 149.08, 147.74, 138.16, 137.56, 136.87, 131.90, 131.38, 129.91, 129.54, 127.41, 123.36, 123.07, 122.85, 120.79, 120.73, 111.94, 111.39, 55.93, 55.88, 41.08, 39.11, 35.24. ESI-HRMS calculated for C₂₃H₂₃N₃O₃S [M + Na]⁺: 444.1352, found 444.1375.

Propyl 5–(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxylate (a23)

White solid; yield: 60%. ¹H NMR (400 MHz, CDCl₃) δ 8.71 (dd, J = 1.7, 0.7 Hz, 1H), 8.37 (s, 1H), 7.85 (d, J = 0.7 Hz, 1H), 7.81 (dd, J = 8.4, 0.5 Hz, 1H), 7.70 (s, 1H), 7.51 (dd, J = 8.4, 1.8 Hz, 1H), 4.33 (t, J = 6.7 Hz, 2H), 3.94 (s, 3H), 1.89–1.79 (m, 2H), 1.07 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 162.98, 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-HRMS calculated for C₁₆H₁₆N₂O₂S [M + Na]⁺: 323.0825, found 323.0845.

General synthesis of b1 to b23

a1 (or a2-a23, 0.65 mmol, 1.0 equiv.) and m-CPBA (1.95 mmol, 3.0 equiv.) were sequentially added to a round-bottom flask with 5 ml of CHCl₃ at 0 °C. The mixture was stirred at room temperature overnight, and the reaction progress was monitored using TLC-MS. Upon reaction completion, 50 ml of cold saturated sodium bicarbonate solution was added, and the aqueous phase was extracted with EtOAc or DCM. The organic phase was washed with saturated NaCl solution, dried over anhydrous Na₂SO₄, and filtered, and the solvent was concentrated to obtain the crude product. The products (b1–b23) were obtained by column chromatography on silica gel.

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

White solid; yield: 64%. ¹H NMR (600 MHz, CDCl₃) δ 7.70 (d, J = 7.2 Hz, 1H), 7.58–7.55 (m, 1H), 7.55–7.51 (m, 1H), 7.50 (d, J = 7.2 Hz, 1H), 6.72 (s, 1H), 3.61 (t, J = 7.0 Hz, 2H), 3.44 (t, J = 6.6 Hz, 2H), 1.98–1.92 (m, 2H), 1.92–1.87 (m, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 160.79, 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-HRMS calculated for C₁₃H₁₃NO₃S [M + Na]⁺: 286.0508, found 286.0527.

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

White solid; yield: 81%. ¹H NMR (600 MHz, CDCl₃) δ 7.78–7.75 (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 = 4.7 Hz, 2H), 3.48 (t, J = 4.8 Hz, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 161.50, 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-HRMS calculated for C₁₃H₁₃NO₄S [M + Na]⁺: 302.0457, found 302.0476.

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

White solid; yield: 84%. ¹H NMR (600 MHz, CDCl₃) δ 7.76–7.73 (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). ¹³C NMR (151 MHz, CDCl₃) δ 161.22, 138.91, 136.86, 133.99, 131.43, 129.87, 126.32, 124.19, 121.83, 74.45, 56.02, 44.02, 38.80, 31.38, 30.09. ESI-HRMS calculated for C₁₅H₁₇NO₄S [M + Na]⁺: 330.0770, found 330.0789.

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

White solid; yield: 83%. ¹H NMR (600 MHz, CDCl₃) δ 7.75 (d, J = 7.0 Hz, 1H), 7.60 (td, J = 7.5, 1.5 Hz, 1H), 7.59–7.55 (m, 1H), 7.40 (d, J = 7.3 Hz, 1H), 7.31–7.27 (m, 2H), 6.97 (t, J = 7.3 Hz, 1H), 6.91 (d, J = 7.9 Hz, 2H), 6.69 (s, 1H), 4.62 (q, J = 4.7, 3.9 Hz, 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). ¹³C NMR (151 MHz, CDCl₃) δ 161.28, 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-HRMS calculated for C₂₀H₁₉NO₄S [M + Na]⁺: 392.0927, found 392.0953.

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

White solid; yield: 72%. ¹H NMR (600 MHz, CDCl₃) δ 7.74 (d, J = 6.7 Hz, 1H), 7.61–7.56 (m, 2H), 7.46–7.35 (m, 6H), 6.72 (s, 1H), 3.91–3.38 (m, 8H). ¹³C NMR (151 MHz, CDCl₃) δ 170.76, 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 calculated for C₂₀H₁₈N₂O₄S [M + Na]⁺: 405.0879, found 405.0903.

N-(3,4-dimethoxyphenyl)benzo[b]thiophene-3-carboxamide 1,1-dioxide (b6)

Yellow solid; yield: 27%. ¹H NMR (600 MHz, CDCl₃) δ 8.54 (s, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.59 (td, J = 7.6, 1.3 Hz, 1H), 7.54 (td, J = 7.7, 1.1 Hz, 1H), 7.39 (d, J = 2.5 Hz, 1H), 7.12 (dd, J = 8.6, 2.5 Hz, 1H), 7.01 (s, 1H), 6.85 (d, J = 8.6 Hz, 1H), 3.89 (s, 3H), 3.88 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 159.48, 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-HRMS calculated for C₁₇H₁₅NO₅S [M + Na]⁺: 368.0563, found 368.0585.

N-(3,4-diethoxyphenyl)benzo[b]thiophene-3-carboxamide 1,1-dioxide (b7)

Yellow solid; yield: 30%. ¹H NMR (400 MHz, CDCl₃) δ 8.18 (s, 1H), 7.95 (d, J = 7.0 Hz, 1H), 7.72 (dd, J = 6.9, 1.8 Hz, 1H), 7.64–7.59 (m, 1H), 7.57 (td, J = 7.7, 1.6 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 7.06 (dd, J = 8.7, 2.5 Hz, 1H), 6.98 (s, 1H), 6.88 (d, J = 8.7 Hz, 1H), 4.11 (p, J = 7.1 Hz, 4H), 1.46 (q, J = 7.1 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 159.39, 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-HRMS calculated for C₁₉H₁₉NO₅S [M + Na]⁺: 396.0876, found 396.0901.

N-(benzo[d][1,3]dioxol-5-yl)benzo[b]thiophene-3-carboxamide 1,1-dioxide (b8)

Yellow solid; yield: 34%. ¹H NMR (600 MHz, DMSO-d₆) δ 10.75 (s, 1H), 7.95 (d, J = 7.2 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.80 (s, 1H), 7.75 (td, J = 7.6, 1.2 Hz, 1H), 7.69 (td, J = 7.5, 1.1 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 7.15 (dd, J = 8.4, 2.1 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.03 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 159.60, 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-HRMS calculated for C₁₆H₁₁NO₅S [M + Na]⁺: 352.0250, found 352.0266.

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

Yellow solid; yield: 32%. ¹H NMR (600 MHz, DMSO-d₆) δ 10.68 (s, 1H), 7.95 (d, J = 7.2 Hz, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.79 (s, 1H), 7.74 (td, J = 7.6, 1.2 Hz, 1H), 7.69 (td, J = 7.5, 1.1 Hz, 1H), 7.34 (d, J = 2.5 Hz, 1H), 7.15 (dd, J = 8.7, 2.5 Hz, 1H), 6.86 (d, J = 8.7 Hz, 1H), 4.27–4.22 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 159.51, 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-HRMS calculated for C₁₇H₁₃NO₅S [M + Na]⁺: 366.0407, found 366.0435.

3,4-dimethoxyphenyl benzo[b]thiophene-3-carboxylate 1,1-dioxide (b10)

White solid; yield: 66%. ¹H NMR (600 MHz, CDCl₃) δ 8.26 (d, J = 7.7 Hz, 1H), 7.77 (d, J = 7.5 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.54 (s, 1H), 6.90 (d, J = 8.6 Hz, 1H), 6.77 (dd, J = 8.6, 2.7 Hz, 1H), 6.75 (d, J = 2.7 Hz, 1H), 3.90 (s, 3H), 3.89 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 160.65, 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-HRMS calculated for C₁₇H₁₄O₆S [M + Na]⁺: 369.0403, found 369.0419.

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

White solid; yield: 55%. ¹H NMR (600 MHz, CDCl₃) δ 7.90 (d, J = 7.4 Hz, 1H), 7.68 (d, J = 7.3 Hz, 1H), 7.58 (td, J = 7.6, 1.3 Hz, 1H), 7.54 (td, J = 7.5, 1.2 Hz, 1H), 6.86 (s, 1H), 6.42 (d, J = 9.4 Hz, 1H), 4.13–4.04 (m, 1H), 1.57–1.50 (m, 2H), 1.47–1.31 (m, 6H), 0.91 (t, J = 7.3 Hz, 6H). ¹³C NMR (151 MHz, CDCl₃) δ 161.31, 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-HRMS calculated for C₁₆H₂₁NO₃S [M + Na]⁺: 330.1134, found 330.1151.

N-(3,4-dimethoxyphenethyl)benzo[b]thiophene-3-carboxamide 1,1-dioxide (b12)

White oil; yield: 48%. ¹H NMR (600 MHz, CDCl₃) δ 7.80 (d, J = 7.0 Hz, 1H), 7.71 (d, J = 6.3 Hz, 1H), 7.60–7.57 (m, 1H), 7.57–7.54 (m, 1H), 6.83 (d, J = 7.9 Hz, 1H), 6.76–6.74 (m, 1H), 6.74 (s, 1H), 6.70 (s, 1H), 6.24 (d, J = 6.1 Hz, 1H), 3.87 (s, 3H), 3.86 (s, 3H), 3.69 (q, J = 6.6 Hz, 2H), 2.88 (t, J = 6.9 Hz, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 161.65, 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-HRMS calculated for C₁₉H₁₉NO₅S [M + Na]⁺: 396.0876, found 396.0897.

N-(2–(4-methoxyphenoxy)ethyl)benzo[b]thiophene-3-carboxamide 1,1-dioxide (b13)

White solid; yield: 44%. ¹H NMR (600 MHz, CDCl₃) δ 7.90 (d, J = 7.5 Hz, 1H), 7.67 (d, J = 7.4 Hz, 1H), 7.58 (td, J = 7.6, 1.2 Hz, 1H), 7.54 (td, J = 7.5, 1.2 Hz, 1H), 6.87–6.83 (m, 5H), 6.81–6.74 (m, 1H), 4.10 (t, J = 5.0 Hz, 2H), 3.82 (q, J = 5.3 Hz, 2H), 3.77 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 161.87, 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-HRMS calculated for C₁₈H₁₇NO₅S [M + Na]⁺: 382.0720, found 382.0739.

2–(2-ethoxyphenoxy)ethyl benzo[b]thiophene-3-carboxylate 1,1-dioxide (b14)

Colourless transparent oil; yield: 59%. ¹H NMR (600 MHz, CDCl₃) δ 8.22 (d, J = 7.4 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.60 (td, J = 7.6, 1.3 Hz, 1H), 7.56 (td, J = 7.5, 1.2 Hz, 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 = 7.0 Hz, 2H), 1.40 (t, J = 7.0 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 161.68, 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-HRMS calculated for C₁₉H₁₈O₆S [M + Na]⁺: 397.0716, found 397.0736.

2-phenoxyethyl benzo[b]thiophene-3-carboxylate 1,1-dioxide (b15)

White solid; yield: 76%. ¹H NMR (600 MHz, CDCl₃) δ 8.22 (d, J = 7.6 Hz, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.61 (td, J = 7.6, 1.3 Hz, 1H), 7.57 (td, J = 7.7, 0.9 Hz, 1H), 7.34–7.30 (m, 2H), 7.30 (s, 1H), 7.03–6.99 (m, 1H), 6.94 (d, J = 7.6 Hz, 2H), 4.73–4.68 (m, 2H), 4.34–4.30 (m, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 161.71, 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-HRMS calculated for C₁₇H₁₄O₅S [M + Na]⁺: 353.0454, found 353.0473.

2-phenoxy-1-phenylethyl benzo[b]thiophene-3-carboxylate 1,1-dioxide (b16)

Colourless transparent oil; yield: 95%. ¹H NMR (600 MHz, CDCl₃) δ 8.20 (d, J = 7.5 Hz, 1H), 7.72 (d, J = 7.3 Hz, 1H), 7.58 (td, J = 7.7, 1.2 Hz, 1H), 7.55 (t, J = 7.4 Hz, 1H), 7.49 (d, J = 7.9 Hz, 2H), 7.45 (t, J = 7.3 Hz, 2H), 7.43–7.39 (m, 1H), 7.32 (d, J = 8.4 Hz, 2H), 7.30 (s, 1H), 7.01 (t, J = 7.4 Hz, 1H), 6.94 (d, J = 7.8 Hz, 2H), 6.39–6.35 (m, 1H), 4.47–4.42 (m, 1H), 4.31 (dd, J = 10.9, 3.4 Hz, 1H). ¹³C NMR (151 MHz, CDCl₃) δ 160.95, 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-HRMS calculated for C₂₃H₁₈O₅S [M + Na]⁺: 429.0767, found 429.0794.

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

White solid; yield: 63%. ¹H NMR (600 MHz, CDCl₃) δ 7.78 (s, 1H), 7.72 (s, 1H), 7.69 (d, J = 7.9 Hz, 1H), 7.60 (dd, J = 7.9, 1.5 Hz, 1H), 7.58 (d, J = 1.4 Hz, 1H), 6.73 (s, 1H), 3.93 (s, 3H), 3.67 (t, J = 6.9 Hz, 2H), 3.51 (t, J = 6.6 Hz, 2H), 2.02–1.97 (m, 2H), 1.97–1.91 (m, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 160.88, 138.90, 138.58, 137.17, 133.78, 130.76, 128.18, 127.87, 127.27, 122.22, 121.61, 121.26, 48.76, 46.22, 39.36, 26.13, 24.31. ESI-HRMS calculated for C₁₇H₁₇N₃O₃S [M + Na]⁺: 366.0883, found 366.0898.

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

White solid; yield: 74%. ¹H NMR (600 MHz, CDCl₃) δ 7.78 (s, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.70 (s, 1H), 7.62 (dd, J = 7.9, 1.5 Hz, 1H), 7.39 (d, J = 1.5 Hz, 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). ¹³C NMR (151 MHz, CDCl₃) δ 161.29, 139.09, 138.40, 137.20, 133.87, 130.92, 128.15, 127.58, 126.93, 122.45, 121.20, 120.86, 74.42, 56.10, 44.09, 39.44, 38.88, 31.41, 30.18. ESI-HRMS calculated for C₁₉H₂₁N₃O₄S [M + Na]⁺: 410.1145, found 410.1167.

(5–(1-methyl-1H-pyrazol-4-yl)-1,1-dioxidobenzo[b]thiophen-3-yl)(4-phenoxypiperidin-1-yl)methanone (b19)

White solid; yield: 89%. ¹H NMR (600 MHz, CDCl₃) δ 7.80 (s, 1H), 7.72 (d, J = 6.3 Hz, 1H), 7.71 (s, 1H), 7.63 (dd, J = 7.9, 1.5 Hz, 1H), 7.43 (d, J = 1.5 Hz, 1H), 7.32–7.27 (m, 2H), 6.97 (tt, J = 7.4, 1.1 Hz, 1H), 6.93–6.89 (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). ¹³C NMR (151 MHz, CDCl₃) δ 161.37, 156.79, 139.10, 138.26, 137.20, 133.88, 130.89, 129.85, 128.16, 127.61, 127.06, 122.47, 121.64, 121.22, 120.89, 116.16, 70.55, 43.69, 39.44, 38.47, 31.36, 30.08. ESI-HRMS calculated for C₂₄H₂₃N₃O₄S [M + Na]⁺: 472.1301, found 472.1323.

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

White solid; yield: 50%. ¹H NMR (600 MHz, CDCl₃) δ 7.79 (s, 1H), 7.73 (s, 1H), 7.74–7.71 (m, 1H), 7.63 (dd, J = 8.0, 1.5 Hz, 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). ¹³C NMR (151 MHz, CDCl₃) δ 170.83, 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-HRMS calculated for C₂₄H₂₂N₄O₄S [M + Na]⁺: 485.1254, found 485.1292.

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

Yellow solid; yield: 94%. ¹H NMR (600 MHz, CDCl₃) δ 7.98 (d, J = 1.4 Hz, 1H), 7.78 (s, 1H), 7.72 (s, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.57 (dd, J = 7.9, 1.5 Hz, 1H), 6.90 (s, 1H), 6.47 (d, J = 9.1 Hz, 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 = 7.3 Hz, 6H). ¹³C NMR (151 MHz, CDCl₃) δ 161.36, 138.96, 138.77, 137.15, 133.95, 130.71, 128.53, 128.28, 127.04, 122.61, 121.96, 121.40, 49.85, 39.36, 37.20, 19.34, 14.09. ESI-HRMS calculated for C₂₀H₂₅N₃O₃S [M + Na]⁺: 410.1509, found 410.1537.

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

Yellow solid; yield: 46%. ¹H NMR (600 MHz, CDCl₃) δ 7.90 (d, J = 1.5 Hz, 1H), 7.81 (s, 1H), 7.72 (s, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.60 (dd, J = 7.9, 1.5 Hz, 1H), 6.82–6.80 (m, 1H), 6.77–6.74 (m, 2H), 6.73 (s, 1H), 6.36 (t, J = 5.9 Hz, 1H), 3.96 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.74–3.68 (m, 2H), 2.89 (t, J = 6.9 Hz, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 161.74, 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 calculated for C₂₃H₂₃N₃O₅S [M + Na]⁺: 476.1251, found 476.1270.

Propyl 5–(1-methyl-1H-pyrazol-4-yl)benzo[b]thiophene-3-carboxylate 1,1-dioxide (b23, DC-Rhoin04)

White solid; yield: 62%. ¹H NMR (400 MHz, CDCl₃) δ 8.28 (d, J = 1.5 Hz, 1H), 7.82–7.79 (m, 1H), 7.72 (s, 1H), 7.68–7.65 (m, 1H), 7.58 (dd, J = 7.9, 1.5 Hz, 1H), 7.30 (s, 1H), 4.32 (t, J = 6.7 Hz, 2H), 3.95 (s, 3H), 1.85– 1.76 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 161.87, 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-HRMS calculated for C₁₆H₁₆N₂O₄S [M + Na]⁺: 355.0723, found 355.0739.

Cell culture

MDA-MB-231 cells—purchased from HyCyte with cell line authentication by STR profiling, were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gbico) supplemented with 10% foetal bovine serum (FBS, Yeasen) and 1% penicillin– streptomycin (Solarbio) at 37 °C and 5% CO₂.

MCF-7 cells—purchased from HyCyte with cell line authentication by STR profiling, were cultured in MCF-7 cell-specific culture medium at 37 °C and 5% CO₂. This MCF-7 cell-specific culture medium was prepared and purchased from HyCyte, and it included minimum essential medium with 10% FBS, 1% GlutaMAX, 1% non-essential amino acids, 1% sodium pyruvate, 10 µg/mL human recombinant insulin, and 1% penicillin–streptomycin.

A549 cells—purchased from Pricella with cell line authentication by STR profiling, were cultured in Ham’s F-12K (Kaighn’s) Medium (Pricella) supplemented with 10% FBS (Yeasen) and 1% penicillin–streptomycin (Solarbio) at 37 °C and 5% CO₂.

All compounds used in the biological experiments were dissolved in dimethyl sulfoxide (DMSO, Solarbio); the concentration of DMSO in the culture medium was maintained at 0.1%.

Cell proliferation assay

The growth rates of the MDA-MB-231, MCF-7 or A549 cells in the presence of different compounds were determined using a cell counting kit-8 (CCK-8, Beyotime, C0038), according to the manufacturer’s instructions. Briefly, MDA-MB-231 cells (5 × 10³ cells/well) were seeded into 96-well plates (Corning) and cultured for 24 h. Following incubation, the cells were exposed to the DC-Rhoin derivatives or doxorubicin with gradient concentrations for 24 h. The CCK-8 reaction solution (10%) was added to each well, and the cells incubated for 1 to 2 h. After incubation for 1–2 h, the OD value of each well at 450 nm was measured using a Multiskan spectrum (Bio-Tek, Eon). Doxorubicin was used as a positive control.

Migration and Matrigel invasion assays

Migration and Matrigel invasion assays were performed using Transwell plates (Biofil, TCS020024). The Matrigel invasion assays employed invasion chambers coated with Matrigel basement membrane matrix.

MDA-MB-231 cells (in T25 culture flasks) were harvested by trypsinisation and resuspended in a serum-deficient medium containing different concentrations of DC-Rhoin derivatives to a final cell density of 5.0 × 10⁴ µL⁻¹. The resuspended cells (200 µL) were added to the corresponding upper chamber, and 500 µL of DMEM containing 10% FBS was added to the lower chamber. After incubation for 24 h, non-migrated cells on the upper membrane were gently removed by medical cotton, and cells that migrated to the lower membrane were fixed with 4% polyformaldehyde solution, stained with 0.1% crystal violet solution, and visualised under a microscope (Olympus CKX41). The quantity of the cells migrated to the lower membrane was measured using ImageJ 1.50b software (National Institutes of Health).

Wound-healing assay

MDA-MB-231 cells were seeded into 6-well plates (Corning) at a cell density of 3 × 10⁵ ml⁻¹. After being wounding with a 200 µL pipette tip, cells were cultured for 24 h in a serum-deficient medium containing respective-gradient concentrations of b19. Recolonisation of the wounded area was imaged at 0 h and 24 h using a microscope (Olympus CKX41) and measured with ImageJ 1.50b software (National Institutes of Health).

Detection of myosin light chain (MLC) phosphorylation levels

In order to detect the protein levels of MLC and phosphorylated MLC (p-MLC), MDA-MB-231 cells were seeded into 6-well plates (Corning) at a cell density of 2 × 10⁵ ml⁻¹ in serum-deficient medium with or without different concentrations of b19 or Rhosin for 24 h. Cells were stimulated by 10% serum for 15 min before collection. The levels of MLC and p-MLC protein were detected using the anti-MLC (Cell Signalling Technology, 8505s) and anti-p-MLC (Cell Signalling Technology, 3675s) antibodies, and the separation module (Bio-Techne, Catalog # SM-W001) for a WES automated western blot system (Bio-Techne, Catalog # 004–600).

Observation of stress fibres

MDA-MB-231 cells were seeded into a confocal dish (Biosharp, BS-15-GJM) at a density of 2 × 10⁵ ml⁻¹ for 24 h. After removal of the culture medium, cells were further cultured in a serum-deficient medium with or without b19 for an additional 24 h. Next, the cells were stimulated with or without 10% serum for 15 min. The culture medium of the confocal dish was removed. All confocal dishes were washed three times with PBS, and this cleaning operation was performed once before each subsequent addition of different reagents. Cells were fixed with 4% polyformaldehyde solution for 15 min on ice, permeabilised at room temperature with 0.5% Triton X-100 for 10 min, and stained at room temperature with SF488 Phalloidin (Solarbio, CA1640) for 20 min. The fluorescent images were obtained using a laser scanning confocal microscope (Zeiss LSM710).

Cell apoptosis assay

The cell apoptosis assay was conducted by an Annexin V-FITC/PI Apoptosis Detection Kit (Yeason, 40302ES20) using flow cytometry analysis. MDA-MB-231 cells were seeded into 6-well plates (Corning) at a cell density of 3 × 10⁵ ml⁻¹. After being cultured for 24 h in a medium with or without respective gradient concentrations of b19, cells were collected on ice, resuspended with 100 μL of binding buffer, 5 μL of Annexin V-FITC, and 10 μL of PI Staining Solution, and then incubated at room temperature under dark conditions for 15 min. Then, 400 μL of binding buffer was added, and the data were collected using an Agilent NovoCyte Quanteon.

Molecular docking analysis

The crystal structure of RhoA in complex with its inhibitor (DC-Rhoin, PDB code: 6KX3) was selected as the receptor protein for molecular docking analysis. The receptor was prepared using the Protein Preparation Wizard, and b19 was prepared using the LigPrep module of the Schrödinger software package. Consistent with DC-Rhoin, b19 was set to covalently bind with the Cys-107 residue in the RhoA pocket by the Michael addition reaction. A covalent docking of b19 with the RhoA protein was calculated using the Pose Prediction (Through) docking mode of the Schrödinger software package.

Results

Chemical synthesis

5-bromobenzo[b]thiophene-3-carboxylic acid was coupled to EtOH using standard amide coupling (EDCI/DMAP) to obtain Precursor compound 1 (Pre-1). Pre-1 and 1-methyl-4-pyrazole boronic acid pinacol ester were catalysed by Pd(dppf)Cl₂ to form the coupling product, Pre-2. Subsequently, using NaOH as a base, Pre-2 was hydrolysed in a mixed solution of H₂O and EtOH to prepare a precursor containing carboxyl groups, Pre-3. Benzo[b]thiophene-3-carboxylic acid or Pre-3 was coupled to different reactants containing hydroxyl or amino groups to obtain intermediates (a1–a23). These intermediates formed final products (b1–b23) by an oxidation reaction with m-CPBA (Scheme 1). All the target compounds were fully characterised through ¹H NMR, ¹³C NMR, and ESI-HRMS. Except for b23 (DC-Rhoin04), all other final compounds (b1–b22) have been synthesised for the first time and have not previously been reported.

Scheme 1.

Synthesis and Structure of DC-Rhoin Derivatives. Except for b23 (DC-Rhoin04), all other final compounds (b1–b22) have been synthesised for the first time and have not previously been reported. Reagents and conditions: (i): EDCI, DMAP, DCM, corresponding reactants containing hydroxyl or amino groups, rt. (ii): 1-Methyl-4-pyrazole boronic acid pinacol ester, Pd(dppf)Cl₂, Na₂CO₃, DMF, H₂O, 90 °C. (iii): NaOH, H₂O, EtOH, 50 °C; (iv): m-CPBA, CHCl₃, rt.

Biological evaluation

Anti-proliferative activity of compounds

Using a standard CCK-8 assay with doxorubicin as the positive control^30–32, the in vitro effects of DC-Rhoin derivatives, b1–b22, on MDA-MB-231, MCF-7 and A549 cell proliferation were assessed. As shown in Table 1, most compounds exerted a higher anti-proliferative effect on the two types of breast cancer cells (MDA-MB-231 and MCF-7) compared to the lung cancer (A549) cells. Among these compounds, b6, b9, b11, b13, b19, b21, and doxorubicin showed the highest anti-proliferative activity on MDA-MB-231 and MCF-7 cells (IC₅₀ < 5 μM). Only b19 maintained a high anti-proliferative activity on A549 cells (IC₅₀ = 4.8 μM). The anti-proliferative activity on the two types of breast cancer cells revealed two significant structure-activity relationships (Figure 3): (1) All compounds with the carboxamide in C-3 showed stronger anti-proliferative activity than those with the carboxylate ester, and among them, b6 and b10, with only one factor changed in the chemical structure, showed a clear contrast in anti-proliferative activity strength (Table 1). (2) The 1-methyl-1H-pyrazol in C-5 enhanced the anti-proliferative activity of compounds with nitrogen-containing heterocyclic-1-yl methanone (Table 1, b1 vs b17, b3 vs b18, b4 vs b19, and b5 vs b20), and reduced or did not influence the anti-proliferative activity of compounds with relatively flexible side chains (Table 1, b11 vs b21, and b12 vs b22).

Table 1.

The anti-proliferative activity of compounds on tumour cells for 24h..

Compound	IC50 (μM)
	MDA-MB-231	MCF-7	A549
b1	22.8	>40	>40
b2	15.5	12.1	>40
b3	11.5	>40	>80
b4	4.8	10.5	24.5
b5	24.2	7.0	9.0
b6	2.8	2.2	10.0
b7	5.2	2.9	>40
b8	7.0	1.9	9.8
b9	3.7	0.7	7.6
b10	>80	>80	>80
b11	2.4	4.4	13.8
b12	6.5	2.1	11.8
b13	3.0	4.1	11.7
b14	>80	>40	>80
b15	>80	>40	>80
b16	>40	>40	>80
b17	14.2	10.7	>40
b18	6.4	9.8	22.9
b19	2.7	2.0	4.8
b20	5.8	3.4	21.4
b21	3.5	3.8	11.8
b22	6.5	6.8	>40
Doxorubicin	4.2	5.2	5.8

Figure 3.

The structure-activity relationship demonstrated that the carboxamide in C-3 and the 1-methyl-1H-pyrazol in C-5 contributed to antiproliferative activity on two types of breast cancer cells (MDA-MB-231 and MCF-7 cells). The carboxamide in C-3 enhanced anti-proliferative activity on two types of breast cancer cells. The 1-methyl-1H-pyrazol in C-5 showed a further enhancement of this activity when the structure in C-3 is nitrogen-containing heterocyclic-1-yl methanone rather than a relatively flexible side chains.

Effects of compounds on cell migration

Due to the close relationship between RhoA targets and cell motility, we further evaluated the effect of DC-Rhoin derivatives on the migration ability of high metastatic and aggressive MDA-MB-231 cells (Figure 4). The compounds (b6, b9, b11, b13, b19 and b21) that exhibited the most potent anti-proliferative activity were evaluated. DC-Rhoin04, which has been proven to inhibit MDA-MB-231 cell migration¹⁴, was detected using the Transwell assay and was used as a positive control. All tested compounds had an effective anti-migration ability at 2.5 µM. The anti-migration effect of b11, b13, and b19 were significantly greater than that of DC-Rhoin04. b13 and b19 inhibited the migration of almost all the MDA-MB-231 cells to the lower membrane.

Figure 4.

Derivatives of DC-Rhoin suppressed the migration ability of MDA-MB-231 cells at 2.5 µM for 24 h. Scale bars, 100 μm. Data are shown as mean ± SD of three independent experiments and analysed with one-way ANOVA, compared to control: ***P < 0.001, ****P < 0.0001; compared to DC-Rhoin04: ^### P < 0.001, ^#### P < 0.0001.

Inhibition of migration and invasion by b19 for MDA-MB-231 cells

b19 exhibited dose-dependent inhibition activity of MDA-MB-231 cell migration and invasion (Figure 5(a)). Even at low concentrations (0.25 μM), b19 significantly inhibited migration and invasion of MDA-MB-231 cells (Figure 5(a)). Fitting curves of migration rates at different concentrations of b19 showed that the half maximal inhibitory concentration of b19 to inhibit MDA-MB-231 cell migration was 0.34 μM (Figure 5(b)). The wound-healing experiment also showed the anti-migration activity of b19 at 2 μM (Figure 5(c)).

Figure 5.

Compound b19 suppressed the migration and invasion ability of MDA-MB-231 cells in dose-dependent manner. (a) Anti-migration and anti-invasion ability of different concentrations of b19 against MDA-MB-231 cells for 24h. Scale bars, 100 μm. (b) Fitting curves of migration rates at different concentrations of b19; data from (a). (c)Wound-healing assays were used to evaluate the effect of b19 on MDA-MB-231 cell migration. Data are shown as mean ± SD of three independent experiments and analysed with one-way ANOVA, compared to control in (a) or (c): *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001. Migration of (a): b19 2 μM vs b19 1 μM, b19 1 μM vs b19 0.5 μM, b19 0.5 μM vs b19 0.25 μM, P < 0.05. 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. Compared to b19 4 μM in (c): ^### P < 0.001, ^#### P < 0.0001.

b19 inhibited the phosphorylation of MLC

The active state of RhoA (RhoA-GTP) can phosphorylate the MYPT1 subunit of MLC phosphatase (MLCP) through Rho-associated coiled-coil kinase (ROCK), which is a main downstream effector protein of RhoA (Figure 6(a))³³. It causes the accumulation of p-MLC and the subsequent formation of stress fibres (Figure 6(a))^34,35. RhoA knockdown also showed a decrease in MLC¹⁴,³⁵. Therefore, the inhibitory effect of compound b19 on RhoA activity was evaluated by detecting p-MLC levels. Rhosin, an RhoA inhibitor and a common positive control that can significantly downregulate p-MLC levels at 30 μM¹³, was tested simultaneously with b19. b19 downregulated the levels of p-MLC in MDA-MB-231 cells in a dose-dependent manner (Figure 6(b)). In addition, b19 at 4 μM was more potent than Rhosin at 30 μM.

Figure 6.

b19 inhibited the phosphorylation of MLC (R: Rhosin). (a) From the activation of RhoA to the formation of stress fibres. The black line with a single arrow indicates direct activation, the red line indicates inhibition, and the double thin line arrow indicates the final result of the signalling pathway. (b) b19 downregulates the levels of p-MLC in MDA-MB-231 cells in a dose-dependent manner. The group containing (+) was stimulated with 10% serum for 15 min, while the group containing (−) remained in a state of starvation without serum stimulation. Data are shown as mean ± SD of three independent experiments and analysed with one-way ANOVA, compared to control: ****P < 0.0001, **P < 0.01; compared to Rhoin 30 μM: ^#P < 0.05. p-MLC: b19 4 μM vs b19 2 μM, b19 2 μM vs b19 1 μM, P < 0.05.

b19 inhibited the formation of stress fibres

As previously reported, RhoA regulates the formation of stress fibres. After treating serum-starved cells with serum, stress fibres will rapidly form³⁶. RhoA inhibition caused a significant reduction in stress fibres in the cell¹³. The inhibitory effect of b19 on the RhoA/ROCK Pathway was evaluated by observing the changes in stress fibres in MDA-MB-231 cells following treatment. The stress fibres were stained with SF488 Phalloidin and observed under a laser scanning confocal microscope. The stress fibres in the control group (stimulated with serum) increased significantly (Figure 7), while the stress fibres of the 4 μM b19 treatment group were lower than those of the control group. These findings indicate that b19 can inhibit the formation of stress fibres.

Figure 7.

The formation of stress fibres of MDA-MB-231 cells was inhibited by b19 at 4 µM for 24 h. Scale bars, 20 µm. The three fluorescent images of the stress fibres corresponded to No serum (serum-starved cells without serum and b19 treatment), Control (serum-starved cells with serum and without b19 treatment), and 4 μM b19 (serum-starved cells treated with serum and b19 at 4 μM), respectively.

b19 promoted cell apoptosis

In order to investigate the anti-tumour activity of b19 in multiple aspects, we conducted a cell apoptosis assay. b19 promoted apoptosis of MDA-MB-231 cells at 8 μM as shown by the Annexin V-FITC/PI Apoptosis Detection Kit using flow cytometry analysis (Figure 8).

Figure 8.

b19 significantly promoted apoptosis of MDA-MB-231 cells at 8 μM. Data are shown as mean ± SD of three independent experiments and analysed with one-way ANOVA, compared to control: * P < 0.05, ** P < 0.01, **** P < 0.0001.

Molecular docking analysis between b19 and RhoA

To study the interaction between b19 and the RhoA protein, molecular docking analysis was conducted based on the crystal structure¹⁴ of RhoA in complex with its inhibitor, DC-Rhoin. Consistent with DC-Rhoin, b19 was set to covalently bind with the Cys-107 residue in the RhoA pocket via the Michael addition reaction¹⁴.

Molecular docking analysis revealed that b19 forms more and stronger H-bonds than DC-Rhoin (Figure 9(a–c)). Through the two oxygen atoms of the sulphone of b19, three H-bonds formed with the THR-77 (3.0 Å, 3.1 Å) and PHE-106 (3.2 Å) residue near covalent binding sites (Figure 9(b)). Dissimilar to DC-Rhoin, the sulphone of b19 faced the outside of the protein pocket (Figure 9(d)). Due to the 180° flipped benzothiophene compared to DC-Rhoin, the 1-methyl-1H-pyrazole of b19 replaced the flexible side chains of DC-Rhoin in the corresponding spatial location, extending into a groove formed by TRP-58, ASP-59, THR-60 and GLN-63 (Figure 9(b)). Phenoxypyridine of b19 extended towards the U-shaped opening formed by GLN-63 and TYR-66 (Figure 9(b)).

Figure 9.

The docking experiments between b19 and the RhoA protein based on the crystal structure of RhoA in complex with DC-Rhoin¹⁴ (PDB code: 6KX3). (a) A close-up view of the interaction between RhoA and DC-Rhoin¹⁴ (6KX3). The ligand and interacting residues are shown as sticks and hydrogen bonds are indicated by black dotted lines. (b) The docking experiments between b19 and RhoA. (c) The superimposition of a over b. The hydrogen bonds of DC-Rhoin-RhoA are indicated by wheat dotted lines, and the hydrogen bonds of b19-RhoA are shown with blue dotted lines. (d) The superimposition of a over b, while RhoA were shown on the surface-mode.

Discussion

Rho family GTPases regulate cellular movement and are crucial drivers of cancer growth and metastasis⁶. As a family member of Rho, RhoA regulates cell movement through the formation of stress fibres via the RhoA/ROCK pathway and serves as a potential target for the suppression of cancer metastasis^1,13,14.

Recently, K-Ras inhibitors have been successfully developed as anticancer drugs. However, limited progress has been made in drug development targeting Rho-GTPase. Some small molecule inhibitors targeting RhoA or its downstream kinase effectors have been reported. However, their selectivity, efficacy, and toxicity are unsatisfactory.

In this study, we synthesised benzo[b]thiophene-3-carboxylic acid 1,1-dioxide derivatives based on DC-Rhoin, a compound that can covalently bind with the Cys-107 residue in a newly identified RhoA pocket to exert anticancer activity. We found that the DC-Rhoin derivatives exert anti-proliferative activity. In addition, we observed that the carboxamide in C-3 and the 1-methyl-1H-pyrazol in C-5 enhanced the anti-proliferative activity of derivatives. From the perspective of the Michael addition reaction, a possible reason for this structure-activity relationship is that the nitrogen-containing groups in C-3 on the derivatives may induce deprotonation of reactive Cys-107 residues, promote covalent binding, and finally, enhance the activity of the inhibitor³⁷.b19 significantly inhibited migration and invasion of MDA-MB-231 cells in Transwell assays, even at 0.25 μM. The anti-migration activity of b19 (2 μM) was also confirmed in a wound-healing experiment. Suppression of the phosphorylation of the MLC and the formation of stress fibres showed the RhoA/ROCK pathway inhibitory activity of b19. In molecular docking analysis, due to the 180° flipped benzothiophene compared to DC-Rhoin, more and stronger H-bonds near covalent binding sites were generated between the sulphone of b19 and the RhoA residue. The 1-methyl-1H-pyrazole replaced the flexible side chains of DC-Rhoin in the corresponding spatial location and extended into the groove on one side of the pocket, possibly causing higher protein pocket affinity and more suitable spatial positioning for covalent binding. This may be the reason why the 1-methyl-1H-pyrazol in C-5 obviously enhanced the anti-proliferative activity of those derivatives which have a relatively rigid structure in C-3.

Conclusions

To further develop DC-Rhoin, which is covalently bound to an RhoA pocket we previously identified, we synthesised DC-Rhoin derivatives and evaluated their activity on tumour proliferation, migration, invasion and apoptosis. b19 exhibited significant anticancer activity via suppression of the RhoA/ROCK pathway. Therefore, this study serves as a foundation for the development of more DC-Rhoin derivatives based on b19 and the demonstrated structure–activity relationship.

Funding Statement

The research was supported by the National Natural Science Foundation of China (No. 82474216); Natural Science Foundation of Guangdong Province (Nos. 2022A1515010103, 2023B1212060063, and 2023A1515220218); the Science and Technology Program of Guangzhou (Nos. 202002010004 and 202201020488); the Specific Research Fund for TCM Science and Technology of Guangdong Provincial Hospital of Chinese Medicine (No. YN2020QN02), Research Fund for Bajian Talents of Guangdong Provincial Hospital of Chinese Medicine (No. BJ2022KY08), and Special Funds for State Key Laboratory of Dampness Syndrome of Chinese Medicine (Nos. SZ2021ZZ33, and SZ2023ZZ13).

Authors’ contributions

Jinhao Liang designed and synthesised compounds, conducted cell proliferation assay, wound healing assay, migration and Matrigel invasion assays, observation of the stress fibres, molecular docking analysis and wrote the manuscript; Jin Huang conducted detection of the p-MLC and cell apoptosis assay; Jianzhan Yang conducted wound healing assay; Weihong Liang, Yunshan Wu and Haoxiang Li assisted in the synthesis and analysis of compounds; Bo Liu designed and guided the entire project and edited the manuscript. All authors read and contributed to the final manuscript.

Ethics statement

No animal or human studies were involved in this manuscript, therefore ethical approval was not required for this manuscript in accordance with local legislation and institutional requirements.

Disclosure statement

The authors declare that there are no relationships or interests that could have direct or potential influence or impart bias on the work reported in this paper.

Data availability statement

The data supporting this study are available from the corresponding authors upon reasonable request.