Synthesis of pro-apoptotic indapamide derivatives as anticancer agents

Abstract

4-Chloro-3-({[(substitutedamino)carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (1–20) and 4-chloro-3-({[3-(substituted)-4-oxo-1,3-thiazolidine-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide derivatives (21–31) were synthesized from 4-chloro-N-(2-methyl-2,3-dihydroindol-1-yl)-3-sulfamoylbenzamide (indapamide). 4-Chloro-3-({[(4-chlorophenyl) amino) carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide 12 demonstrated the highest proapoptotic activity among all synthesized compounds on melanoma cell lines MDA–MB-435 with 3.7% growth inhibition at the concentration of 10 µM. Compound 12 (SGK 266) was evaluated in vitro using the MTT colorimetric method against melanoma cancer cell line MDA–MB435 growth inhibition for different doses and exhibited anticancer activity with IC₅₀ values of 85–95 µM against melanoma cancer cell line MDA–MB435. In addition, this compound was investigated as inhibitors of four physiologically relevant human carbonic anhydrase isoforms, hCA I, II, IX and XII. The compund inhibited these enzymes with IC₅₀ values ranging between 0.72 and 1.60 µM.

• Apoptosis
• indapamide
• sulfonamide
• sulphonylthiourea
• 4-thiazolidinone

Introduction

In the progress of novel drug discovery, the easiest and most effective way is to work with drug substances with known activity and molecular structure. Basic method is to synthesize the new analogs and homologs of drug substance chosen as precursor with proven biological activity and molecular structure. In this way, it is possible to approach novel drug substances with the same or different biological activity. Moleculer modification offers the researchers the opportunity of improvement in properties of biological efficiency, mechanism of action, administration pathway, toxicity and stability. If it is not possible to obtain drug candidates from precursor drugs, the knowledge will be gained for the synthesis of novel drug substances which could be a key role in research area. Molecular modification methods give the chance to obtain active pharmaceutical ingredients with several pharmacological activities. One of the most significant examples is sulfonamide derivatives which have several biological activities (Figure 1). Sulfonamides represent one of the classical chemotypes associated with potent carbonic anhydrase (CA) inhibition1. CA I and II, CA isoforms, are rather abundant in many tissues and participate in important physiological processes2^,3. Indapamide, 4-chloro-N-(2-methylindolin-1-yl)-3-sulphamoylbenzamide, have been discovered in the 1960s–1970s, when little was known about the various CA isozymes. This drug was a much weaker one (KI of 2520 nM) inhibitor against CA II4.

Figure 1. Schematic representation of sulfonamide derivatives.

It has been declared that sulfonylthiourea and 4-thiazolidinone structures exhibit antitumoral activity5^,6. It has been also indicated that some 4-thiazolidinone derivatives selectively inhibit COX-2 enzyme, an important target for some anticancer studies7. In the present study, indapamide which has indole ring and sulfonamide group was chosen as the starting material and some related 4-thiazolidinone derivatives were synthesized. 4-Chloro-3-({[3-(substituted)-4-oxo-1,3-thiazolidine-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamides (21–31) were synthesized by the addition of ethyl α-bromoacetate and anhydrous sodium acetate in dry ethanol to 4-chloro-3-({[(substitutedamino)carbono-thioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamides (1–20), which were synthesized by the reaction of alkyl/aryl isothiocyanates with indapamide. The structures of the isolated products were determined by the spectral methods and evaluated anticancer activity for the selected compounds.

Experimental methods

Chemistry

General

All chemicals were purchased from Merck (Darmstadt, Germany), Sigma-Aldrich (St. Louis, MO) or Fluka (Buchs, Switzerland). Melting points were determined with a Barnstead melting point apparatus (Barnstead/Electrothermal 9200). Infrared (IR) spectra (KBr disc) were obtained with a Perkin Elmer Spectrum One (Waltham, MA). ¹H NMR and ¹³C NMR spectra in DMSO-d₆ were recorded on a BRUKER AVANCE-DPX (Billerica, MA) spectrometer (400 MHz) and chemical shifts are given in ppm downfield from tetramethylsilane (TMS) as an internal standard using DMSO-d₆ as solvent. Data are reported as follows: chemical shift, multiplicity (br.: broad singlet, d: dublet; m: multiplet, s: singlet and t: triplet), coupling constants (Hz), integration. Elemental analyses were performed on Flash EA 1112 series elemental analyzer (Thermo Finnigan, Italy). Mass spectra were measured on a JMS-700 double-focusing mass spectrometer (JEOL, Akishima, Tokyo, Japan). Follow up of the reactions and checking the purity of the compounds were made by TLC on silica gel protected aluminium sheets (Type 60 F₂₅₄, Merck 1.05550.0001) (Darmstadt, Germany), and the spots were detected by means of UV lamp at λ = 254 nm.

General method for preparation of 4-chloro-3-[({[(substituted)amino]carbonothioyl} amino)sulfonyl]-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamides (1–20)

A solution of indapamide (5 mmol) and anhydrous K₂CO₃ (10 mmol) were refluxed in dry acetone (25 ml) for 30 min. After the addition of appropriate isothiocyanate (5.5 mmol), the mixture was refluxed for 10 h. The completion of reaction was checked by TLC M₁ (dichloromethane:methanol, 10:1, v/v). The solvent was removed under reduced pressure and the residue was triturated in 25 ml of water. The resulting solution was adjusted to pH 2–3 with 2 N HCl, and the precipitate that appeared was collected by filtration, washed with water followed with cold ethanol and dried to give compounds 1–20, respectively.

4-Chloro-3-({[(methylamino)carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl) benzamide (1) (SGK-346)

Light yellow solid, yield: 72%, m.p. 178–182 °C. R_f × 100 value: 20 (M₁). Anal. calc. for C₁₈H₁₉ClN₄O₃S₂ (MW = 438.953 g mol⁻¹): C, 49.25; H, 4.36; N, 12.76. Found: C, 50.22; H, 4.26; N, 12.19%. IR (KBr cm⁻¹): 3435, 3337, 3196 (NH), 3032 (CH arom.), 2962, 2831 (CH aliph.), 1652 (C = O), 1320, 1240 (SO₂), 1160 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.31 (d, 3H), 2.59 (q, 1H), 2.85 (d, 3H), 3.17 (q, 1H), 3.92 (br s, 1H), 6.50 (d, J = 7.8 Hz, 1H), 6.77 (t, 1H), 7.04 (t, 1H), 7.11 (d, 1H), 7.81 (d, J = 8.3 Hz, 1H), 8.16 (d, J = 8.3 Hz, 1H), 8.59 (s, 1H), 9.70 (s, 1H), 10.56 (s, 1H).

4-Chloro-3-({[(ethylamino)carbonothioyl]amino} sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl) benzamide (2) 2 (SGK-259)

Light yellow solid, yield: 74%, m.p. 285 °C. R_f × 100 value: 57.1 (M₁). Anal. calc. for C₁₉H₂₁ClN₄O₃S₂.1/2 H₂O (MW = 452.979 g mol⁻¹): C, 50.38; H, 4.76; N, 12.37. Found: C, 49.59; H, 4.57; N, 11.68%. IR (KBr cm⁻¹): 3435, 3279, 3214 (NH), 3035 (CH arom.), 2958, 2923 (CH aliph.), 1658 (C = O), 1296, 1166 (SO₂), 1150 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.16 (t, 3H), 1.31 (d, 3H), 2.58 (q, 1H), 2.85 (d, 3H), 3.17 (q, 1H), 3.90 (br s, 1H), 6.52 (d, J = 7.6 Hz, 1H), 6.77 (t, 1H), 7.03 (t, 1H), 7.10 (d, 1H), 7.85 (d, J = 8.3 Hz, 1H), 8.14 (d, 1H), 8.50 (s, 1H), 9.70 (s, 1H), 10.60 (s, 1H).

4-Chloro-3-[({[(2-chloroethyl)amino]carbonothioyl}amino) sulfonyl]-N-(2-methyl-2,3-dihydro-1H-indole-1-yl) benzamide (3) (SGK-367)

Light cream-colored solid, yield: 60%, m.p. 282 °C. R_f × 100 value: 59.4 (M₁). Anal. calc. for C₁₉H₂₀Cl₂N₄O₃S₂ (MW = 487.424 g mol⁻¹): C, 46.82; H, 4.14; N, 11.49. Found: C, 46.68; H, 4.21; N, 11.24%. IR (KBr cm⁻¹): 3435, 3197 (NH), 3051 (CH arom.), 2966, 2923 (CH aliph.), 1660 (C = O), 1303, 1169 (SO₂), 1104 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.09 (d, 3H), 2.36 (q, 1H), 2.95 (q, 1H), 3.45 (t, 2H), 3.75 (br s, 1H), 6.30 (d, J = 7.5 Hz, 1H), 6.55 (t, 1H), 6.82 (t, 1H), 6.89 (t, 1H), 7.35 (d, J = 8.7 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H), 8.23 (d, 1H), 10.36 (s, 1H).

4-Chloro-3-({[(propylamino)carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl) benzamide (4) (SGK-260)

Yellow solid, yield: 63%, m.p. 265–267 °C. R_f × 100 value: 60.4 (M₁). Anal. calc. for C₂₀H₂₃ClN₄O₃S₂ (MW = 467.006 g mol⁻¹): C, 51.44; H, 4. 96; N, 12.00. Found: C, 51.96; H, 4.86; N, 12.11%. IR (KBr cm⁻¹): 3437, 3286, 3205 (NH), 3026 (CH arom.), 2959, 2952 (CH aliph.), 1658 (C = O), 1316, 1165 (SO₂), 1153 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 0.90 (t, 3H), 1.32 (d, 3H), 1.60 (m, 2H), 2.60 (q, 1H), 3.18 (q, 1H), 3.95 (br s, 1H), 6.54 (d, J = 7.4 Hz, 1H), 6.77 (t, 1H), 7.05 (t, 1H), 7.11 (d, 1H), 7.87 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H), 8.51 (s, 1H), 9.79 (s, 1H), 10.60 (s, 1H).

4-Chloro-3-({[(isopropylamino)carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (5) (SGK-365)

Light cream-colored solid, yield: 72%. m.p. 302 °C. R_f × 100 value: 45.9 (M₁). Anal. calc. for C₂₀H₂₃ClN₄O₃S₂ (MW = 467.006 g mol⁻¹): C, 51.44; H, 4.96; N, 12.00. Found: C, 50.72; H, 4.58; N, 12.09%. IR (KBr cm⁻¹): 3439, 3332, 3272 (NH), 3047 (CH arom.), 2971, 2929 (CH aliph.), 1660 (C = O), 1308, 1166 (SO₂), 1111 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 0.98 (d, 6H), 1.09 (d, 3H), 2.36 (q, 1H), 2.95 (q, 1H), 3.18 (q, 1H), 3.73 (br s, 1H), 3.92 (t, 1H), 6.31 (t, J = 7.3 Hz, 1H), 6.55 (t, 1H), 6.82 (t, 1H), 6.89 (d, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.93 (d, 1H), 8.29 (s, 1H), 9.45 (s, 1H), 10.37 (s, 1H).

4-Chloro-3-({[(butylamino)carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (6) (SGK-261)

Light yellow solid, yield: 87%, m.p. 240–243 °C. R_f × 100 value: 56.6 (M₁). Anal. calc. for C₂₁H₂₅ClN₄O₃S₂.1/2H₂O (MW = 481.033 g mol⁻¹): C, 51.47; H, 5.35; N, 11.43. Found: C, 51.93; H, 6.02; N, 11.75%. IR (KBr cm⁻¹): 3432, 3265, 3219 (NH), 3039 (CH arom.), 2957, 2928 (CH aliph.), 1664 (C = O), 1354, 1156 (SO₂), 1115 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 0.89 (t, 3H), 1.31 (d, 3H), 1.51 (m, 2H), 2.51 (q, 1H), 3.18 (q, 1H), 3.35 (t, 2H), 3.95 (br s, 1H), 6.52 (d, J = 7.7 Hz, 1H), 6.76 (t, 1H), 7.04 (t, 1H), 7.11 (d, 1H), 7.85 (d, J = 8.3 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.50 (s, 1H), 9.79 (s, 1H), 9.75 (t, 1H), 10.59 (s, 1H).

3-({[(tert-Butylamino)carbonothioyl]amino}sulfonyl)-4-chloro-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (7) (SGK-366)

Light cream-colored solid, yield: 58%. m.p. 298 °C. R_f × 100 value: 54.1 (M₁). Anal. calc. for C₂₁H₂₅ClN₄O₃S₂ (MW = 481.033 g mol⁻¹): C, 52.43; H, 5.24; N, 11.65. Found: C, 53.12; H, 5.17; N, 11.46%. IR (KBr cm⁻¹): 3412, 3294, 3181 (NH), 3001 (CH arom.), 2922, 2899 (CH aliph.), 1654 (C = O), 1304, 1174 (SO₂), 1143 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.09 (d, 3H), 1.20 (s, 9H), 2.37 (q, 1H), 2.95 (q,1H), 3.98 (br s, 1H), 6.31 (d, J = 7.6 Hz, 1H), 6.55 (t, 1H), 6.82 (t, 1H), 6.89 (d, 1H), 7.62 (d, J = 8.2 Hz,1H), 7.93 (d, J = 8.2 Hz, 1H), 8.30 (s, 1H), 9.08 (s, 1H), 10.38 (s, 1H).

4-Chloro-3-({[(cyclohexylamino)carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (8) (SGK-339)

Light yellow solid, yield: 75%, m.p. 195 °C. R_f × 100 value: 56.3 (M₁). Anal. calc. for C₂₃H₂₇ClN₄O₃S₂ (MW = 507.070 g mol⁻¹): C, 54.48; H, 5.37; N, 11.05. Found: C, 55.47; H, 5.67; N, 11.72%. IR (KBr cm⁻¹): 3434, 3255 (NH), 3029 (CH arom.), 2931, 2899 (CH aliph.), 1666 (C = O), 1360, 1113 (SO₂), 1193 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.31 (d, 3H), 1.58 (m, 10H), 2.55 (q, 1H), 3.16 (q,1H), 3.88 (m,1H), 3.97 (br s, 1H), 6.53 (d, J = 7.7 Hz, 1H), 6.77 (t, 1H), 6.98 (d, 1H), 7.08 (d, 1H), 7.85 (d, J = 8.2 Hz, 1H), 8.15 (dd, 1H), 8.50 (d, 1H), 9.69 (d, 1H), 10.55 (s, 1H).

4-Chloro-3-[({[(cyclohexylmethyl)amino]carbonothioyl}amino)sulfonyl]-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (9) (SGK-341)

Light yellow solid, yield: 72%. m.p. 273–275 °C. R_f × 100 value: 59.1 (M₁). Anal. calc. for C₂₄H₂₉ClN₄O₃S₂ (MW = 521.096 g mol⁻¹): C, 55.32; H, 5.61; N, 10.75. Found: C, 55.27; H, 5.32; N, 10.27%. IR (KBr cm⁻¹): 3432, 3260 (NH), 3071 (CH arom.), 2960, 2925 (CH aliph.), 1665 (C = O), 1349, 1114 (SO₂), 1152 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 0.92–1.75 (m, 11H), 1.31 (d, 3H), 2.57 (q, 1H), 3.16 (q, 1H), 3.97 (br s, 1H), 6.51 (d, J = 7.7 Hz, 1H), 6.75 (t, 1H), 7.04 (t, 1H), 7.10 (d, 1H), 7.77 (d, J = 8.3 Hz, 1H), 8.09 (d, J = 8.3 Hz, 1H), 8.45 (s, 1H), 10.55 (s, 1H).

3-{[(Anilinocarbonothioyl)amino]sulfonyl}-4-chloro-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (10) (SGK-262)

Light yellow solid, yield: 67%, m.p. 245 °C. R_f × 100 value: 17.2 (M₁). Anal. calc. for C₂₃H₂₁ClN₄O₃S₂ .2H₂O (MW = 501.022 g mol⁻¹): C, 51.44; H, 4.69; N, 10.43; S, 11,94. Found: C, 51.18; H, 3.83; N, 10.22; S, 11.60%. IR (KBr cm⁻¹): 3414, 3298, 3185 (NH), 3091 (CH arom.), 2961, 2924 (CH aliph.), 1688 (C = O), 1349, 1196 (SO₂), 1115 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.32 (d, 3H), 2.57 (q, 1H), 3.18 (q, 1H), 3.99 (br s, 1H), 6.48 (d, J = 7.7 Hz, 1H), 6.75 (t, 1H), 6.88 (t, 1H), 7.04 (t, 1H), 7.11 (d, 1H), 7.18 (t, 2H), 7. 50 (d, J = 8.2 Hz, 1H), 7.75 (d, 2H), 7.90 (dd, 1H), 8.52 (d, 1H), 9.10 (s, 1H), 10.45 (s, 1H).

4-Chloro-3-[({[(2-chlorophenyl)amino]carbonothioyl}amino) sulfonyl]-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (11) (SGK-340)

Light yellow solid, yield: 63%, m.p. 171–175 °C. R_f × 100 value: 70 (M₁). Anal. calc. for C₂₃H₂₀Cl₂N₄O₃S₂ (MW = 535.467 g mol⁻¹): C, 51.59; H, 3.76; N, 10.46. Found: C, 51.43; H, 3.82; N, 9.22%. IR (KBr cm⁻¹): 3435, 3393, 3250 (NH), 3055 (CH arom.), 2967, 2928 (CH aliph.), 1663 (C = O), 1382, 1201 (SO₂), 1149 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.09 (d, 3H), 2.59 (q, 1H), 3.17 (q, 1H), 3.90 (br s, 1H), 6.31 (d, J = 7.4 Hz, 1H), 6.53 (t, 1H), 6.82 (t, 1H), 6.89 (d, 1H), 7.24–7.44 (m, 4H) 7.73 (d, 1H), 7.97 (d, 1H), 8.03 (s, 1H), 10.37 (s, 1H), 11.33 (s, 1H).

4-Chloro-3-[({[(4-chlorophenyl)amino]carbonothioyl}amino) sulfonyl]-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (12) (SGK-266)

Light yellow solid, yield: 87%, m.p. 261 °C. R_f × 100 value: 56.6 (M₁). Anal. calc. for C₂₃H₂₀Cl₂N₄O₃S₂ (MW = 535.467 g mol⁻¹): C, 51.59; H, 3.76; N, 10.46. Found: C, 52.58; H, 4.45; N, 10.68%. IR (KBr cm⁻¹): 3435, 3245, 3191 (NH), 3052 (CH arom.), 2971, 2928 (CH aliph.), 1666 (C = O), 1363, 1191 (SO₂), 1112 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.31 (d, 3H), 2.59 (q, 1H), 3.17 (q, 1H), 3.99 (m, 1H), 6.53 (d, J = 7.7 Hz, 1H), 6.76 (t, 1H), 7.04 (t, 1H), 7.11 (d, 1H), 7.48 (d, 2H), 7.61 (d, 2H), 7.89 (d, 1H), 8.17 (d, 1H), 8.53 (s, 1H), 9.50 (s, 1H), 10.59 (s, 1H).

4-Chloro-3-[({[(4-fluorophenyl)amino]carbonothioyl}amino) sulfonyl]-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (13) (SGK-265)

Cream-colored solid, yield: 85%, m.p. 205–210 °C. R_f × 100 value: 56 (M₁). Anal. calc. for C₂₃H₂₀ClFN₄O₃S₂.H₂O (MW = 519.013 g mol⁻¹): C, 51.64; H, 4.13; N, 10.43. Found: C, 50.84; H, 3.63; N: 9.57%. IR (KBr cm⁻¹): 3436, 3301, 3256 (NH), 3153 (CH arom.), 3078, 3022 (CH aliph.), 1666 (C = O), 1327, 1236 (SO₂), 1152 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.32 (d, 3H), 2.59 (q, 1H), 3.17 (q, 1H), 3.95 (br s, 1H), 6.55 (d, J = 7,6 Hz, 1H), 6.78 (t, 1H), 7.05 (t, 1H), 7.12 (d, 1H) 7.32 (d, 2H), 7.66 (d, 2H), 7.97 (d, 1H), 8.21 (d,1H), 8.56 (s, 1H), 10.64 (s, 1H), 11.57 (s, 1H).

4-Chloro-3-[({[(2,4-dichlorophenyl)amino]carbonothioyl}amino)sulfonyl]-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (14) (SGK-338)

Cream-colored solid, yield: 61%, m.p. 258–260 °C. R_f × 100 value: 60.2 (M₁). Anal. calc. for C₂₃H₁₉Cl₃N₄O₃S₂ (MW = 569.912 g mol⁻¹): C, 47.72; H, 3.48; N, 9.68. Found: C, 47.02; H, 3.09; N, 8.69%. IR (KBr cm⁻¹): 3435, 3270, 3198 (NH), 3149 (CH arom.), 3015, 2967 (CH aliph.), 1667 (C = O), 1326, 1172 (SO₂), 1125 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.30 (d, 3H), 2.58 (q, 1H), 3.16 (q, 1H), 3.97 (br s, 1H), 6.49 (d, J = 7.7 Hz, 1H), 6.75 (t, 1H), 7.03 (t, 1H), 7.24 (d, 1H), 7.28 (d, 1H), 7.35 (s, 1H), 7.42 (s, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.96 (d, J = 8.2 Hz, 1H), 8.33 (s,1H), 10.45 (s, 1H).

4-Chloro-3-[({[(2-methoxyphenyl)amino]carbonothioyl}amino)sulfonyl]-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (15) (SGK-268)

Light yellow solid, yield: 71%, m.p. 188 °C. R_f × 100 value: 71.4 (M₁). Anal. calc. for C₂₄H₂₃ClN₄O₄S₂ (MW = 531.048 g mol⁻¹): C, 54.28; H, 4.37; N, 10.55. Found: C, 55.20; H, 5.22; N, 10.98%. IR (KBr cm⁻¹): 3409, 3171, 3112 (NH), 3080 (CH arom.), 3000, 2963 (CH aliph.), 1649 (C = O), 1326, 1171 (SO₂), 1151 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.09 (d, 3H), 2.35 (q, 1H), 2.95 (q, 1H), 3.78 (br s, 1H), 6.31 (d, J = 7.2 Hz, 1H), 6.56 (t, 1H), 6.62 (t, 1H), 6.88 (d, 1H) 6.97–7.21 (m, 4H), 7.73 (d, 1H), 7.96 (d,1H), 8.30 (s, 1H), 10.39 (s, 1H), 10.91 (s, 1H).

4-Chloro-3-[({[(4-methoxyphenyl)amino]carbonothioyl}amino)sulfonyl]-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (16) (SGK-269)

Light yellow solid, yield: 78%, m.p. 269 °C. R_f × 100 value: 56.5 (M₁). Anal. calc. for C₂₄H₂₃ClN₄O₄S₂ .2H₂O (MW = 531.048 g mol⁻¹): C, 50.83; H, 4.80; N, 9.88. Found: C, 50.58; H, 4.37; N, 9.61%. IR (KBr cm⁻¹): 3445, 3248, 3200 (NH), 3071 (CH arom.), 2960, 2927 (CH aliph.), 1667 (C = O), 1349, 1187 (SO₂), 1126 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.31 (d, 3H), 2.70 (q, 1H), 3.17 (q, 1H), 3.70 (s, 3H), 3.97 (br s, 1H), 6.48 (d, J = 7.7 Hz, 1H), 6.76 (q, 2H), 7.05 (t, 1H), 7.11 (d, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.60 (d, 2H), 7.89 (d, J = 8.0 Hz, 1H), 8.50 (s, 1H), 8.95 (s, 1H), 10.43 (s, 1H).

4-Chloro-3-[({[(4-cyanophenyl)amino]carbonothioyl}amino) sulfonyl]-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (17) (SGK-263)

Light yellow solid, yield: 38%, m.p. 226 °C. R_f × 100 value: 10.5 (M₁). Anal. calc. for C₂₄H₂₀ClN₅O₃S₂ .2H₂O (MW = 526.032 g mol⁻¹): C, 51.29; H, 4.30; N, 12.46; S, 11.41. Found: C, 50.85; H, 3.46; N, 12.13; S, 11.61%. IR (KBr cm⁻¹): 3346, 3304, 3185 (NH), 3072 (CH arom.), 2962, 2927 (CH aliph.), 2233 (CN), 1693 (C = O), 1421, 1385 (SO₂), 1186 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.32 (d, 3H), 2.59 (q, 1H), 3.17 (q, 1H), 3.83 (br s, 1H), 6.49 (d, J = 7.7 Hz, 1H,), 6.76 (t, 1H), 7.05 (t, 1H), 7.11 (d, 1H), 7.55 (d, 1H), 7.63 (dd, 2H), 7.94 (dd, 1H), 8.00 (dd, 2H), 8.53 (d, 1H), 9.61 (s, 1H), 10.45 (s, 1H). ¹³C NMR (125 MHz) (DMSO-d₆/TMS) δ ppm: 19.07, 36.03, 63.19, 109.11, 119.91, 120.14, 120.18, 124.79, 127.46, 130.16, 132.89, 133.81, 143.69, 152.10, 165.33, 182.93.

3-({[(Benzylamino)carbonothioyl]amino}sulfonyl)-4-chloro-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (18) (SGK-347)

Light yellow solid, yield: 70%, m.p. 244–247 °C. R_f × 100 value: 68.3 (M₁). Anal. calc. for C₂₄H₂₃ClN₄O₃S₂ (MW: 515.049 g mol⁻¹): C, 55.97; H, 4.50; N, 9.68. Found: C, 56.41; H, 4.39; N, 10.57%. IR (KBr cm⁻¹): 3445, 3245 (NH), 3028 (CH arom.), 2965, 2923 (CH aliph.), 1663 (C = O), 1308, 1147 (SO₂), 1112 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.30 (d, 3H), 2.58 (q, 1H), 3.17 (q, 1H), 3.92 (br s, 1H), 4.61 (d, 2H), 6.52 (d, 1H), 6.76 (t, 1H), 7.03(t, 1H), 7.10 (d, 1H), 7.32–7.37 (m, 5H), 7.87(d, J = 8.3 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.50 (s, 1H), 10.23 (t, 1H), 10.59 (s, 1H).

3-({[(Benzoylamino)carbonothioyl]amino}sulfonyl)-4-chloro-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (19) (SGK-348)

Cream-colored solid, yield: 63%, m.p. 246–248 °C. R_f × 100 value: 22.6 (M₁). Anal. calc. for C₂₄H₂₁ClN₄O₄S₂.H₂O (MW = 529.032 g mol⁻¹): C, 52.69; H, 4.24; N, 10.24. Found: C, 52.73, H: 4.29, N: 10.94%. IR (KBr cm⁻¹): 3430, 3263, 3218 (NH), 3035 (CH arom.), 2964, 2846 (CH aliph.), 1662 (C = O), 1349, 1173 (SO₂), 1157 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.32 (d, 3H), 2.59 (q, 1H), 3.18 (q, 1H), 3.98 (br s, 1H), 6.53 (d, J = 7.7 Hz, 1H), 6.77 (t, 1H), 7.05 (t, 2H), 7.11 (d, 1H), 7.51 (t, 2H) 7.63 (t, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.92 (d, 2H), 8.21 (d, J = 8.3 Hz, 1H), 8.68 (d, 1H), 10.65 (s, 1H).

4-Chloro-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)-3-[({[(2-phenylethyl)amino] carbonothioyl}amino)sulfonyl]benzamide (20) (SGK-368)

Light yellow solid, yield: 48%, m.p. 243–244 °C. R_f × 100 value: 59.4 (M₁). Anal. calc. for C₂₅H₂₅ClN₄O₃S₂.H₂O (MW = 529.076 g mol⁻¹): C, 54.88; H, 4.97; N, 10.24. Found: C, 54.67; H, 4.72; N, 10.70%. IR (KBr cm⁻¹): 3436, 3311, 3222 (NH), 3060 (CH arom.), 3019, 2969 (CH aliph.), 1677 (C = O), 1319, 1167 (SO₂), 1150 (C = S). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.09 (d, 3H), 2.37 (q, 1H), 2.67 (t, 2H), 2.95 (q, 1H), 3.38 (t, 2H), 3.75 (br s, 1H), 6.31 (d, J = 7.5 Hz, 1H), 6.55 (t, 1H), 6.84 (t, 1H), 6.89 (d, 1H), 6.99–7.11(m, 5H), 7.61(d, J = 8.1 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 8.28 (s,1H), 10.37 (s, 1H).

General method for preparation of 4-chloro-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)-3-({[3-substituted-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl)benzamide (21–31)

Sulfonylthiourea derivative 1–20 (3.3 mmol) and ethyl bromoacetate (3.6 mmol) were refluxed in absolute ethanol (20 ml) in the presence of anhydrous sodyum acetate (6.6 mmol) for 2 h. The completion of reaction was checked by TLC M₂ (dichloromethane:methanol, 25:1, v/v). The reaction mixture was cooled and poured into ice-cold water (20 ml) and filtered. The precipitate washed with water followed with cold ethanol and dried to give compounds 21–31, respectively.

4-Chloro-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)-3-({[3-methyl-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl) benzamide (21) (SGK-349)

Cream-colored solid, yield: 42%, m.p. 236–241 °C. R_f × 100 value: 48.5 (M₂). Anal. calc. for C₂₀H₁₉ClN₄O₄S₂ (MW = 478.974 g mol⁻¹): C, 50.15; H, 4.00; N, 11.70. Found: C, 51.06; H, 4.28, N: 10.76%. IR (KBr cm⁻¹): 3162 (NH), 3037 (CH arom.), 2966, 2923 (CH aliph.), 1740 (C = O thiazolidinone), 1666 (C = O), 1595 (C = N thia.), 1314, 1195 (SO₂). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.31 (d, 3H), 2.59 (q, 1H), 3.05 (s, 3H), 3.18 (q, 1H), 3.96 (br s, 1H), 4.14 (s, 2H), 6.54 (d, J = 7.6 Hz, 1H), 6.78 (t, 1H), 7.04 (t, 1H), 7.11 (d, 1H), 7.88 (d, 1H), 8.19 (d, 1H), 8.60 (s, 1H), 10.61 (s, 1H).

4-Chloro-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)-3-({[3-ethyl-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl)benzamide (22) (SGK-274)

Yellow solid, yield: 43%, m.p. 216–220 °C. R_f × 100 value: 51.1 (M₂). Anal. calc. for C₂₁H₂₁ClN₄O₄S₂ (MW = 493.001 g mol⁻¹): C, 51.16; H, 4.29; N, 11.36. Found: C, 50.24; H, 4.15; N, 10.62%. IR (KBr cm⁻¹): 3270 (NH), 3049 (CH arom.), 2975, 2933 (CH aliph.), 1741 (C = O thiazolidinone), 1665 (C = O), 1595 (C = N thia.), 1313, 1151 (SO₂). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.04 (t, 3H) 1.31 (d, 3H), 2.59 (q, 1H), 3.17 (q, 1H), 3.63 (q, 2H), 3.95 (br s, 1H), 4.14 (s, 2H), 6.53 (d, 1H), 6.77 (t, 1H), 7.04 (t, 1H), 7.11 (d, 1H), 7.87 (d, J = 8.3 Hz, 1H), 8.18 (d, J = 8.3 Hz, 1H), 8.59 (s, 1H), 10.55 (s, 1H). ¹³C NMR (125 MHz, DMSO-d₆; ppm/δ): 12.48, 19.00, 34.45, 35.97, 38.86, 63.47, 109.32, 120.43, 124.84, 127.49, 127.58, 129.20, 132.76, 132.87, 133.61, 135.07, 138.62, 151.90, 164.23, 172.38, 172.95. HR-MS (EI⁺), m/z (calculated/found): M, 492.0692/M⁺ 492.0680.

4-Chloro-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)-3-({[3-isopropyl-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl)benzamide (23) (SGK-369)

Light orange solid, yield: 56%, m.p. 142–144 °C. R_f × 100 value: 66.4 (M₂). Anal. calc. for C₂₂H₂₃ClN₄O₄S₂ (MW = 507.027 g mol⁻¹): C, 52.11; H, 4.57; N, 11.05. Found: C, 51.65; H, 4.56; N, 10.94%. IR (KBr cm⁻¹): 3264 (NH), 3049 (CH arom.), 2971, 2933 (CH aliph.), 1741 (C = O thiazolidinone), 1673 (C = O), 1607 (C = N thia.), 1315, 1150 (SO₂). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.09 (d, 9H), 2.35 (q, 1H), 2.95 (q, 1H), 3.82 (br s, 1H), 3.89 (s, 2H), 4.37 (m, 1H), 6.32 (d, J = 7.5 Hz, 1H), 6.55 (t, 1H), 6.82 (t, 1H), 6.79 (d, 1H), 7.67 (d, 1H), 7.97 (d,1H), 8.37 (s, 1H), 10.39 (s, 1H).

4-Chloro-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)-3-({[3-butyl-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl)benzamide (24) (SGK-276)

Orange solid, yield: 40%. m.p. 205–210 °C. R_f × 100 value: 70.3 (M₂). Anal. calc. for C₂₃H₂₅ClN₄O₄S₂.H₂O (MW = 521.054 g mol⁻¹): C, 51.25; H, 5.05; N, 10.39. Found: C, 51.56; H, 4.24, N, 10.27%. IR (KBr cm⁻¹): 3266 (NH), 3051 (CH arom.), 2960, 2930 (CH aliph.), 1747 (C = O thiazolidinone), 1668 (C = O), 1594 (C = N thia.), 1317, 1151 (SO₂). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 0.57 (t, 3H), 0.98 (p, 2H), 1.09 (t, 3H), 1.23 (t, 2H), 2.37 (q, 1H), 2.95 (q, 1H), 3.38 (t, 2H), 3.89 (br s, 1H), 3.95 (s, 2H), 6.32 (d, 1H), 6.55 (t, 1H), 6.82 (t, 1H), 6.89 (d, 1H), 7.67 (d, J = 8.2 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 8.37 (s, 1H), 10.39 (s, 1H).

4-Chloro-3-({[3-cyclohexyl-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (25) (SGK-343)

Light yellow solid, yield: 36%. m.p. 125–130 °C. R_f × 100 value: 56.7 (M₂). Anal. calc. for C₂₅H₂₇ClN₄O₄S₂ (MW = 547.091 g mol⁻¹): C, 53.13; H, 5.17; N, 9.91. Found: C, 53.07; H, 4.72; N, 9.19%. IR (KBr cm⁻¹): 3258 (NH), 3050 (CH arom.), 2967, 2931 (CH aliph.), 1739 (C = O thiazolidinone), 1667 (C = O), 1607 (C = N thia.), 1315, 1139 (SO₂). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.30 (d, 3H), 1.19–2.14 (m, 10H), 2.59 (q, 1H), 3.17 (q, 1H), 3.92 (br s, 1H), 4.09 (s, 2H) 4.18 (m, 2H), 6.53 (d, 1H), 6.76 (t, 1H), 7.03 (t, 1H), 7.11 (d, 1H), 7.88 (d, J = 8.3 Hz, 1H), 8.18 (d, J = 8.3 Hz, 1H), 8.57 (s, 1H), 10.59 (s, 1H).

4-Chloro-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)-3-({[4-oxo-3-phenyl-1,3-thiazolidin-2-ylidene]amino}sulfonyl)benzamide (26) (SGK-277)

Cream-colored solid, yield: 65%, m.p. 218–222 °C. R_f × 100 value: 53.3 (M₂). Anal. calc. for C₂₅H₂₁ClN₄O₄S₂.1/2H₂O (MW = 541.043 g mol⁻¹): C, 54.49; H, 4.03; N, 10.19; S, 11.66. Found: C, 54.43; H, 3.06; N, 9.96; S, 11.24%. IR (KBr cm⁻¹): 3238 (NH), 3069 (CH arom.), 2967, 2929 (CH aliph.), 1744 (C = O thiazolidinone), 1664 (C = O), 1547 (C = N thia.), 1306, 1142 (SO₂). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.30 (d, 3H), 3.16 (q, 1H), 3.17 (q, 1H), 3.92 (br s, 1H), 4.27 (s, 2H), 6.52 (d, 1H), 6.76 (t, 1H), 7.04 (t, 1H), 6.96 (d, 1H) 7.27–7.49 (m, 5H), 7.85 (d, J = 8.2 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 8.49 (s,1H), 10.56 (s, 1H).

4-Chloro-3-({[3-(2-chlorophenyl)-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (27) (SGK-344)

Cream-colored solid, yield: 58%, m.p. 103–108 °C. R_f × 100 value: 68.8 (M₂). Anal. calc. for C₂₅H₂₀Cl₂N₄O₄S₂.1/2H₂O (MW: 575.488 g mol⁻¹): C, 51.37; H, 3.62; N, 9.59. Found: C, 50.42; H, 3.25; N, 10.36%. IR (KBr cm⁻¹): 3271 (NH), 3052 (CH arom.), 2971, 2934 (CH aliph.), 1747 (C = O thiazolidinone), 1664 (C = O), 1595 (C = N thia.), 1321, 1153 (SO₂). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.29 (d, 3H), 2.57 (q, 1H), 3.15 (q, 1H), 3.95 (br s, 1H), 4.32 (d, 1H), 4.51 (d, 1H), 6.52 (d, 1H), 6.75 (t, 1H), 7.03 (t, 1H), 7.11 (d, 1H), 7.51 (m, 3H), 7.63 (d, 1H), 7.83 (d, 1H), 8.14 (d, 1H), 8.48 (s, 1H), 10.57 (s, 1H).

4-Chloro-3-({[3-(4-chlorophenyl)-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl) benzamide (28) (SGK-281)

Cream-colored solid, yield: 42%. m.p. 231–235 °C. R_f × 100 value: 58.8 (M₂). Anal. calc. for C₂₅H₂₀Cl₂N₄O₄S₂.1/2H₂O (MW = 575.488 g mol⁻¹): C, 51.37; H, 3.62; N, 9.59. Found: C, 52.28; H, 3.09; N, 9.19%. IR (KBr cm⁻¹): 3239 (NH), 3086 (CH arom.), 2978, 2926 (CH aliph.), 1740 (C = O thiazolidinone), 1662 (C = O), 1597 (C = N thia.), 1319, 1159 (SO₂). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.30 (d, 3H), 2.57 (q, 1H), 3.16 (q, 1H), 3.95 (br s, 1H), 4.74 (s, 2H), 6.53 (d, 1H), 6.76 (t, 1H), 7.03 (t, 1H), 7.11 (d, 1H), 7.62 (d, 2H), 7.69 (d, 2H), 7.80 (d, J = 8.3 Hz, 1H), 8.13 (d, J = 8.3 Hz, 1H), 8.54 (s, 1H), 10.60 (s, 1H).

4-Chloro-3-({[3-(2,4-dichlorophenyl)-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (29) (SGK-342)

Light orange solid, yield: 32%, m.p. 148–150 °C. R_f × 100 value: 75 (M₂). Anal. calc. for C₂₅H₁₉Cl₃N₄O₄S₂ (MW = 603.933 g mol⁻¹): C, 49.23; H, 3.14; N, 9.19. Found: C, 50.12; H, 3.47; N, 8.95%. IR (KBr cm⁻¹): 3263 (NH), 3076 (CH arom.), 2967, 2928 (CH aliph.), 1746 (C = O thiazolidinone), 1678 (C = O), 1607 (C = N thia.), 1328, 1168 (SO₂). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.09 (d, 3H), 2.35 (q, 1H), 2.95 (q, 1H), 3.73 (br s, 1H), 4.30 (d, 1H), 4.69 (d, 1H), 6.32 (d, 1H), 6.55 (t, 1H), 6.82 (t, 1H), 6.89 (d, 1H), 7.53 (q, 2H), 7.63 (d, 1H), 7.84 (s, 1H), 7.93 (d, 1H), 8.34 (s, 1H), 10.39 (s, 1H).

4-Chloro-3-({[3-(4-methoxyphenyl)-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (30) (SGK-284)

Yellow solid, yield: 38%, m.p. 232–236 °C. R_f × 100 value: 55.5 (M₂). Anal. calc. for C₂₆H₂₃ClN₄O₅S₂.1/2H₂O (MW = 571.069 g mol⁻¹): C, 53.83; H, 4.17; N, 9.66. Found: C, 54.58; H, 4.05; N, 9.18%. IR (KBr cm⁻¹): 3219 (NH), 3049 (CH arom.), 2966, 2931 (CH aliph.), 1747 (C = O thiazolidinone), 1668 (C = O), 1608 (C = N thia.), 1304, 1165 (SO₂). ¹H NMR (400 MHz; DMSO-d₆; ppm/δ): 1.08 (d, 3H), 2.36 (q, 1H), 2.94 (q, 1H), 3.54 (s, 3H), 3.72 (br s, 1H), 4.01 (s, 2H), 6.30 (d, 1H), 6.54 (t, 1H), 6.77–6.81 (m, 3H), 6.89 (d, 1H), 6.96 (d, 2H), 7.62 (d, J = 8.2 Hz, 1H), 7.93 (d, J = 8.2 Hz, 1H), 8.27 (s,1H), 10.34 (s, 1H).

4-Chloro-3-({[3-(4-cyanophenyl)-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl) benzamide (31) (SGK-278)

Light cream-colored solid, yield: 63%, m.p. 259–260 °C. R_f × 100 value: 66.4 (M₂). Anal. calc. for C₂₆H₂₀ClN₅O₄S₂.2H₂O (MW = 566.053 g mol⁻¹): C, 51.87; H, 4.02; N, 11.63. Found: C, 51.93; H, 3.14; N, 10.91%. IR (KBr cm⁻¹): 3213 (NH), 3048 (CH arom.), 2990, 2962 (CH aliph.), 1747 (C = O thiazolidinone), 1667 (C = O), 1606 (C = N thia.), 1307, 1143 (SO₂). ¹H NMR (400 MHz; DMSO-d₆; ppm/δδ): 1.29 (d, 3H), 2.58 (q, 1H), 3.17 (q, 1H), 3.95 (br s, 1H), 4.25 (s, 2H), 6.51 (d, 1H), 6.76 (t, 1H), 7.03 (t, 1H), 7.10 (d, 1H), 7.53(d, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.98 (d, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.48 (d, 1H), 10.55 (s, 1H).

Biology

Primary anticancer assay was performed in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda8–11. The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM l-glutamine. For a typical screening experiment, 100 µL of cells were inoculated into 96-well microtiter plates at plating densities ranging from 5000 to 40 000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37 °C, 5% CO₂, 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs. The cytotoxic and/or growth inhibitory effects of the compounds were tested in vitro against the full panel of 60 human tumor cell lines derived from nine neoplastic diseases at 10-fold dilutions. The percentage of growth was evaluated spectrophotometrically versus controls which were not treated with the test agents. Briefly, effect of the compounds on the growth parameters of the different cancer cell lines was evaluated relative to equivalent amounts of DMSO treated controls and expressed as percent growth rate. The compounds were added at 10⁻⁵M concentration for 48 h. Compounds 12, 16, 22, 26, 28 and 30 chosen as prototypes were evaluated against the full panel of 60 human tumor cell lines at a single dose in NCI in vitro primary anticancer assay.

Growth inhibition studies (MDA–MB-435 cell)

Growth inhibition of compound 12 was evaluated in vitro using the MTT colorimetric method against melanoma cancer cell line (MDA–MB-435) at different doses12–15 at Marmara University Faculty of Pharmacy, Department of Pharmaceutical Biotechnology. MTT is cleaved to formazan by the “succinate-tetrazolium reductase” system (EC 1.3.99.1) which belongs to the mitochondrial respiratory chain and is active only in viable cells. These cells were cultured with DMEM (Dulbecco's Modified Eagle Medium; Gibco, Invitrogen) supplemented with 10% FBS (Fetal bovine serum; Gibco, Invitrogen), 1% glutamine and streptomycin–penicillin. Cell viability was determined by trypan blue exclusion assay (10%).

Testing in vitro was done by the method of Woerdenbag et al. with minor modifications15. The MTT metabolic assay was carried out at the seeding density of 1 × 10⁴ cells/well in 96-well flat-bottom cell culture plates with 100 μL of opti-MEM (invitrogen, USA). Following 24-h incubation at 37 °C, 5% CO₂, media was aspirated, compounds were dissolved in DMSO and diluted with medium before addition to the cell cultures at the concentrations of 5 and 10 μg/mL. Cells were incubated for 48 h at 37 °C, 5% CO₂. After the incubation period 10 μL of the MTT labeling reagent [final concentration 0.5 μg/mL (Cell proliferation kit MTT, Roche, Germany)] was added to each well. Samples were incubated for 4–12 h in a humidified atmosphere (e.g. 37 °C, 5.0% CO₂) and 100 μL of the solubilization buffer was added into each well. The plate was allowed to stand overnight in the incubator in a humidified atmosphere (e.g. 37 °C, 5% CO₂) and the formazan precipitates were then solubilized. Absorbance of the formazan product was measured spectrophotometrically at 550 and 690 nm. Statistical analyses were done using unpaired Student’s t-test using Prism 3.0 (GraphPad Software, San Diego, CA).

TUNEL assay

Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining was performed on MDA–MB-435 cell line. Cells were cultured in DMEM supplemented with 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum, 100 units of penicillin/ml and 100 µg of streptomycin/ml at 37 °C in a humidified atmosphere of 5% CO₂ in air. Cells were seeded into 6-well plates at a density of 1.5 × 10⁵ cells/well. Following one-day incubation, medium was replaced and adjacent wells have been inoculated with different concentrations of synthesized compound. Apoptosis was detected after 24 and 48 h, using ApopTag Plus in situ apoptosis detection kit peroxidase (Chemicon) following manufacturer’s protocol with minor modifications. Apoptotic cells were observed brown after the color reaction with DAB (3,3′-diaminobenzidine), while counter staining was managed using methyl green to observe the living cells. Apoptotic cells were detected by standard light microscopy. Live and death cells were counted by two independent observers.

TUNEL assay identifies early stage apoptosis by enzymatically labeling 3′-OH end of DNA strand breaks with modified nucleotides. Since late stage apoptotic and necrotic cells were detached in the adherent cell culture, given counts merely reflects cells in the early apoptotic stage.

Flow-cytometric analysis for apoptotic cell rate by Annexin V-FITC

Test was performed using “Annexin V-FITC apoptosis detection kit” (eBioscience) as described previously16. Briefly, MDA–MB-435 cells (1 × 10⁶) both untreated and treated with indapamide derivatives, were harvested, washed with PBS twice and suspended with binding buffer. The cells were double-stained with Annexin-V-FITC and propidium iodide for 10 min in the dark at room temperature.

CA inhibition studies

Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na₂SO₄ (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO₂ hydration reaction for a period of 10–100 s. The CO₂ concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled–deionized water and dilutions up to 0.01 nM were done thereafter with distilled–deionized water. Inhibitor and enzyme solutions were pre-incubated together for 15 min at room temperature (prior to assay, in order to allow for the formation of the E–I complex). The inhibition constants were obtained by non-linear least-squares methods using PRISM 3, as reported earlier17 and represent the mean from at least three different determinations. CA isoforms were recombinant ones obtained in house as reported earlier17–19.

Results and discussion

Chemistry

In this study, we report the synthesis of indapamide derivatives bearing sulfonylthiourea and 4-thiazolidinone ring systems (Scheme 1). 4-Chloro-3-({[(substitutedamino)carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamides (1–20) were prepared with substituted isothiocyanates and anhydrous K₂CO₃ in dry acetone starting from indapamide. 4-Chloro-3-({[3-(substituted)-4-oxo-1,3-thiazolidine-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamides (21–31) were synthesized by the reaction of ethyl-α-bromoacetate and anhydrous sodium acetate in dry ethanol with the sulfonylthioureas (1–20). Purity of the synthesized compounds was determined by elemental analysis and thin layer chromatography. Structures of these compounds were characterized using FT-IR and ¹H NMR spectral methods. ¹³C NMR, HMBC and HR-MS spectral methods were also used for the compound 22.

Scheme 1. Synthetic route of indapamide derivatives 1–20 and 21–31.

When the IR spectral data of 4-chloro-3-({[(substitutedamino)carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamides (1–20) were evaluated, disappearance of symmetric and asymmetric stretching dual bands at 3313–3320 cm⁻¹ and presence of single band at 3446–3409 cm⁻¹ showed that sulfonylthioureas were formed. C=S stretching bands observed at 1104–1157 cm⁻¹ were another evidence that the reactions took place5^,20–22.

The ¹H NMR spectra of the sulfonylthiourea derivatives (1–20) revealed that CH₃ protons at indoline ring came out as a doublet with the integration three protons at 1.09–1.32 ppm. Non-equivalent H_a and H_b, CH₂ protons of indoline ring, resonated as a doublet of doublet at 2.35–2.59 ppm and another doublet of doublet at 2.95–3.17 ppm, respectively. Likewise, CH protons of the indoline ring were determined as broad singlets at 3.73–3.98 ppm consistent with the values given in the relevant literature23–25. N–H protons of sulfonylthiourea in compounds 1–20 were observed at 9.08–10.6426–28ppm and amide N–H protons were detected at 10.36–11.57 ppm. Sulfonylthiourea N–H peaks appeared with very low intensities or disappeared due to proton exchange with the deuterated solvent29. In the ¹³C NMR spectrum of 4-chloro-3-[({[(4-cyanophenyl)amino]carbonothioyl}amino)sulfonyl]-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide 17 which was selected as a prototype, C = S and cyano groups resonance was observed at 182.93 and 120 ppm, respectively, as an evidence of sulfonylthiourea formation30^,31.

IR spectra of 4-chloro-3-({[3-(substituted)-4-oxo-1,3-thiazolidine-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamides (21–31) exhibited C = O stretching bands of the 4-thiazolidinone at 1739–1747 cm⁻¹ and C = N stretching bands of this rings at 1547–1608 cm⁻¹32^,33. ¹H NMR spectra of compounds 21–31 provided evidence of 4-thiazolidinone structure with –SCH₂ signals observed at 3.88–4.74 ppm as singlets expect compounds 27 and 29 which gave doublet of doublets corresponding to two protons due to chirality32. In order to support ¹H NMR spectral data and provide further evidence for 4-thiazolidinone ring formation, ¹³C NMR, HR-MS and HMBC spectra were recorded for 4-chloro-3-({[-3-ethyl-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide 22 which was chosen as a prototype. The ¹³C NMR spectrum of the compound 22 confirmed the proposed 4-thiazolidinone structure32^,34. The peaks resonated at 172.95, 172.38 and 34.45 ppm for C = N, C = O and S–CH₂, respectively, in this spectrum. HMBC spectrum of compound 22 also verified the structure by long-range ¹H–¹³C couplings as shown in Supplementary Material.

High resolution mass spectrum (HR-MS) of the representative 4-chloro-3-({[3-ethyl-4-oxo-1,3-thiazolidin-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl) benzamide 22 was obtained using electron impact ionization technique. The molecular ion peak observed at m/z 492.0680 Da was within the acceptable limit for molecular weight and the empirical formula of compound 22. The characteristic fragmentations for 4-thiazolidinone were also observed. The main fragmentation product was observed as 1-imino-2-methyl-2,3-dihydro-1H-indolium cation, giving the base peak at m/z 147.0935 Da35^,36.

Biological evaluation

Cancer cell growth inhibitory and apoptotic effect

The effect of selected indapamide derivatives were evaluated at a single concentration of 10⁻⁵M against a panel of 60 cancer cell lines at the NIH-NCI8–11. Compound 12 demonstrated remarkably lowest cell growth promotion among all synthesized compounds on melanoma cell lines MDA–MB-435 (growth promotion −3.70%) and SK-MEL-5 (growth promotion 39.68%), leukemia cell lines K-562 (growth promotion 48.87%), MOLT-4 (growth promotion 35.01) and SR (growth promotion 48.22), CNS cancer cell line SF-295 (growth promotion 31.96%) and ovarian cancer cell line OVCAR-3 (growth promotion 32.45%; Figure 2).

Figure 2. Growth promotion of compound 12 at 10⁻⁵M concentration.

Based on the above results, compound 12 was further investigated for its effect on growth inhibition on melanoma cell line MDA–MB-435 and non-cancer cell line L-929 rat fibroblast cell by MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] assay, at different doses (10, 20, 50, 75, 85 and 90 µM) in dimethylsulfoxide (2.5%) and compared with the control group12–15. Compound 12 exhibited anticancer activity with IC₅₀ value of 85–90 µM against melanoma cell line MDA–MB-435. Growth inhibition data for compound 12 is presented in Figure 3 and for non-cancer cells (L 929 fibroblast) in Figure 4.

Figure 3. Growth inhibition of compound 12 (SGK 266) against melanoma cell line MDA–MB-435.

Figure 4. Growth inhibition of compound 12 (SGK 266) against non-cancer cell line.

It was also investigated whether the effect of compound 12 was mediated via the apoptotic pathway; and if so which apoptotic pathway was responsible for this process. This part of study was performed at the Department of Biophysics, School of Medicine, Marmara University. Adherent cell population was determined by cell count and number of apoptotic cells was detected by TUNEL assay and FACS analysis16. Annexin-V, a well-established technique to determine apoptosis, binds and detects translocation of phosphatidyl serines to the outer membrane, an indication of the beginning of apoptosis37. Flow cytometric analysis of the cell lines was performed using FITC-labeled Annexin-V and propidium iodide, a DNA-binding dye used as an indicator of DNA damage. Our results were introduced in Figure 7, where Q4 and Q2 were regions of early and late stages of apoptosis, respectively, and Q1 involved necrotic cells.

In this study, compound 12 was added to the cell cultures at indicated concentrations (0, 10, 50, 80 and 100 µM) and apoptosis was determined at 24 and 48 h following inoculation. A dose-dependent increase in the number of apoptotic cells can be seen as the concentration rises, which reaches to its maximum at 80 µM for 24-h period. The cell numbers were substantially reduced at 100 µM at the end of 24-h period and at 80 and 100 µM at the end of 48-h period, as serious indicators of cytotoxicity (Figures 5 and 6). Addition of 100 µM substance was completely destructive for the cells which prevented apoptotic analyses due to extremely low cell number and completely distorted morphology (Figure 7).

Figure 5. TUNEL assay of compound 12 (SGK 266) after 24–48 h incubation.

Figure 6. FACS analysis of compound 12 (SGK 266) after 24–48 h incubation.

Figure 7. Representative pictures of MDA–MB-435 cells visualized on a light microscope (400× magnification) at 0 µM (A), 50 µM (B), 80 µM (C) and 100 µM (D) concentrations after 24 h.

In addition, compound 12 have further been investigated for CA inhibition (Table 1). There was no inhibition up to 50 µM inhibitor against hCA I, whereas hCA II, IX and XII was inhibited with IC₅₀ of in the range of 0.72–1.60 µM, being thus a low micromolar inhibitor with a potency similar to clinically used sulfonamide acetazolamide (AAZ) in Supporting information (Figures 8 and 9).

Figure 8. hCA I inhibition of compound 12 (SGK 266) and acetazolamide.

Figure 9. hCA II inhibition of compound 12 (SGK 266) and acetazolamide.

Table 1. Inhibition data of hCA I–XII with compound 12 (SGK 266) and acetazolamide (AAZ) as standard.

	IC50 (µM)
Compounds	hCA I	hCA II	hCA IX	hCA XII
12	>50	1.60	0.72	0.85
AAZ	0.379	0.019	0.047	0.014

Conclusion

The objective of this study was to synthesize and investigate the anticancer activity of new sulfonylthiourea or 4-thiazolidinone derived from indapamide with the hope of discovering new structure leads serving as anticancer agents. Our aim has been verified by the synthesis of two different groups of structure hybrids comprising basically the indole moiety attached to either sulfonylthiourea or 4-thiazolidinone counterparts through various linkages for synergistic purpose. From the preliminary results of cell growth inhibition study, we could conclude that the selected by NIH-NCI compounds 16, 22, 26, 28 and 30 show no significant cell growth inhibition against 60 different cell lines in vitro except compound 12. Compound 12 showed on melanoma cell lines MDA–MB-435, SK-MEL-5, leukemia cell lines K-562 MOLT-4 and SR, CNS cancer cell line SF-295 and ovarian cancer cell line OVCAR-3. Compound 12 (SGK 266) was determined the most marked effect on MDA–MB435 with −3.7% growth promotion at the concentration of 10 µM. Compound 12 evaluated growth inhibition at different doses and found anticancer activity with IC₅₀ value of 85–95 µM against melanoma cancer cell line MDA–MB435. Apoptosis levels of compound 12 were determined for different concentrations at two time points (24 and 48 h) and apoptotic cells were detected by TUNEL assay and FACS analysis. The apoptotic effect of the compound 12 was started at 50 µM following 24-h incubation. At 48-h incubation of compound 12 displayed highly toxic effects on cells as demonstrated by dramatically reduced cells number and clear deformations in shape. The obtained results clearly revealed that compound 12 derived from the indapamide exhibited better growth inhibition and apoptotic effect than their 4-thiazolidinone and other compounds. In addition, this compound was investigated as inhibitors of four physiologically relevant human carbonic anhydrase isoforms, hCA I and II (cytosolic idoforms) as well as hCA IX and XII (transmembrane, tumor-associated isoforms). As seen from data of Table 1, hCA I was not inhibited, whereas the remaining isoforms were inhibited with IC₅₀-s in the range of 0.72–1.60 µM.

Finally, the broad spectrum anticancer activity displayed by compound 12 will be of interest for future derivatization in the hope of finding more active and selective anticancer agents.

Supplementary material available online.

Acknowledgements

The authors are grateful to Dr. Jürgen Gross from the Institute of Organic Chemistry, University of Heidelberg, for his generous help on obtaining HR-EI/FAB mass spectra of the synthesized compounds. We thank the Division of Cancer Research, National Cancer Institute, Bethesda, MD, for the anticancer activity screening. Indapamide was supplied by Sanovel Pharmaceutical Industry Inc.

Declaration of interest

The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of this article. This research was supported by the Scientific and Technical Research Council of Turkey (TUBİTAK), Research Fund Project Number: SBAG-HYD-339 (108S257).

Notes

* This work was partly presented at the 10th International Symposium on Pharmaceutical Sciences, Ankara, Turkey, 26–29 June 2012.