Synthesis and antiproliferative activity of some N′-substituted 2,4-dihydroxybenzothiohydrazides

Abstract

The paper shows that new N'-substituted 2,4-dihydroxybenzocarbothiohydrazides are able to inhibit the in vitro proliferation of human tumor cell lines. The compounds were prepared by the reaction of sulfinylbis[(2,4-dihydroxyphenyl)methanethione] (STB) or its analogs with the hydrazines. The panel of N'-substitution included aryl, pyridinyl and pyrimidinyl rings. The highest antiproliferative activity for N'-(4-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidin-2-yl)-5-ethyl-2,4-dihydroxybenzothiohydrazide (5b) was found. The antiproliferative potency of some compounds was similar to that of cisplatin. Analogs with the Et substituent on benzenediol moiety displayed higher potency than with the unsubstituted one. The influence of N'-substitution on antiproliferative activity of compounds was discussed.

Keywords:

• 2,4-dihydroxybenzothiohydrazide
• antiproliferative activity
• human cancer cell lines
• synthesis

Introduction

Recent drug discovery attempts are highly focused on design and synthesis of small molecules as anticancer agents. Undoubtedly, understanding of the biology of cancer improved in the last decade. One characteristic of cancer cells is their highly proliferative nature. Consequently, inhibition of proliferative pathways is considered to be an effective strategy to fight against cancer and recently much attention has been paid to the discovery and development of new, more potent and selective antiproliferative agents1,2.

Hydrazide moiety or its structural modification is one of the privileged structural fragments in medicinal chemistry having a wide spectrum of activities. This is related to the presence of the –C(=S)–NH– group and the –NH–NH– hydrazine fragment3,4. The therapeutic possibilities of hydrazides were realized after the discovery of isonicotinic acid hydrazide (Figure 1)5. The literature reports that during the past years hydrazide derivatives have been extensively studied for their biological profile and possess a broad bioactivity including antimicrobial6, antimycobacterial7, antitubercular8 antiviral9, antitumor10, anticonvulsant11, anti-inflammatory12 and analgesic13 ones. Tang et al. described a series of benzothiohydrazides as potent thrombocytopenia receptor agonists14. The other derivatives possess inhibitory potency toward monoamine oxidase (MAO) A and B, and acetyl and butyrylcholinesterase15.

Figure 1. Structures of hydrazide-based commercial drugs and natural biologically active agents.

In practice, hydrazides themselves or mixed in more complex drugs have been widely used in medicine and veterinary medicine. The important examples are isoniazid – the antituberculosis agent (Figure 1), iproniazid (Figure 1), acting as a MAO inhibitor16,17 and benserazide (Figure 1), a decarboxylase inhibitor used in combination with levodopa in the treatment of Parkinson’s disease18.

Recently, a small group of naturally occurring hydrazides was enlarged by the discovery of the enehydrazides hydrazidomycins from Streptomyces atratus as congeners of various azoxides. In particular, hydrazidomycin A (Figure 1) proved to be a strong antiproliferative compound against various cancer cell lines with a mean IC₅₀=0.86 μm. It can be considered as a molecule with an amphiphilic character19,20. Another intriguing natural product containing the unusual trisubstituted enehydrazide moiety is geralcin B (Figure 1). It is cytotoxic against MDA231 breast cancer cells with an IC₅₀ of 5 μm21.

Since the anticancer activity of N′-substituted thiohydrazides has not been extensively studied, this investigation deals with the synthesis of 2,4-dihydroxybenzothiohydrazides and evaluation of their antiproliferative activity. The panel of N-modification included pyridinyl and pyrimidinyl substituents which are very important in the drug design. The compounds were modified additionally by lipophilic substituents in a benzenediol moiety. This is a continuation of our studies on design and synthesis of biologically active compounds with the resorcinol moiety22,23. A lot of benzenediol heterocyclic derivatives display a substantial antiproliferative potency, and some of them were studied as potential anticancer agents24–26. These properties prompted our further studies in this area.

Experimental

Chemistry

The IR spectra of the compounds were recorded with a Perkin-Elmer FT-IR 1725X spectrophotometer (Perkin-Elmer Ltd., Beaconsfield, England) (in KBr) or Varian 670 FT-IR spectrometer (ATR). The spectra were made in the range of 600–4000 cm⁻¹. The ¹H NMR and ¹³C NMR spectra were recorded in DMSO-d₆ using a Varian Mercury 400 or a Bruker DRX 500 (Bruker Daltoncs, Inc., Billerica, MA) instrument. Chemical shifts (δ, ppm) were described in relation to tetramethylsilane. The spectra MS (EI, 70 eV) were recorded using the apparatus AMD-604 (Intectra GmbH, Harpstedt, Germany). Elemental analyses (C, H, N) were performed with the use of Perkin-Elmer 2400 instrument (Perkin Elmer, Waltham, MA) and were found to be in good agreement (±0.4%) with the calculated values. The melting point (m.p.) was determined using a Büchi B-540 (Flawil, Switzerland) m.p. apparatus.

The purity of the compounds was examined by HPLC Knauer (Berlin, Germany) with a dual pump, a 20 μl simple injection valve and a UV-visible detector (330 nm). The Hypersil Gold C18 (1.9 μm, 100 × 2.1 mm) column was used as the stationary phase. The mobile phase included different contents of MeOH and acetate buffer (pH 4, 20 nm) as the aqueous phase27. The flow rate was 0.5 ml/min at room temperature. The retention time of an unretained solute (t_o) was determined by the injection of a small amount of acetone dissolved in water. The log k values for 70% or 40% of MeOH (v/v) in the mobile phase are presented. The log k values were calculated as log k = log(t_R − t_o)/t_o, where: t_R, the retention time of a solute; t_o, the retention time of an unretained solute.

General procedure for the synthesis of compounds 1–11

A mixture of 0.01 mol of respective hydrazine and equimolar amounts of STB (1, 2, 3a, 4a, 5a, 6–11) or 5-Cl-STB (3b) or 5-Et-STB (4b, 5b) in 50 ml of MeOH was heated under reflux for 2–3 h stirring. Next, the hot mixture was filtered, and the filtrate was left at room temperature (24 h) or concentrated to small volume. Recrystallization from MeOH/H₂O solution afforded compounds 1–11.

N′-(4-Bromophenyl)-2,4-dihydroxybenzothiohydrazide (1)

Yield: 66%; m.p.: 119–120 °C; log k = 0.197 (70% MeOH). ¹H NMR (100 MHz, DMSO-d₆) δ: 12.30 (s, 1 H, HO-C(2) (exchangeable in D₂O)), 11.54 (s, 1 H, HO-C(4) (exchangeable in D₂O)), 10.35 (s, 1 H, NH (exchangeable in D₂O)), 9.81 (s, 1 H, NH (exchangeable in D₂O)), 7.90 (m, 2 H, H-C(Ar)), 7.69 (d, J = 8.8 Hz, 1 H, H-C(6)), 7.33 (m, 2 H, H-C(Ar)), 6.82 (m, 1 H, H-C(Ar)), 6.28 (m, 1 H, H-C(Ar)) ppm; IR (KBr) V: 3240 (NH, OH), 1593 (C=C), 1488, 1461, 1398, 1287, 1246 (C-OH), 1173, 1118 (C=S), 1071, 1013, 980, 940, 812, 734 cm⁻¹; EI-MS (m/z, %): 339 (M⁺, 14), 338 (14), 324 (8), 306 (23), 292 (9), 226 (12), 208 (6), 186 (13), 171 (88), 153 (100), 137 (68), 124 (7), 108 (24), 92 (41), 80 (13), 77 (15), 65 (51), 63 (26), 52 (17), 39 (18), 36 (4). Anal. Calc. for C₁₃H₁₁BrN₂O₂S (339.21) (%): C, 46.03; H, 3.27; N, 8.26. Found (%): C, 46.07; H, 3.29; N, 8.22.

5-Chloro-N′-(6-chloro-4-(trifluoromethyl)pyridin-2-yl)-2,4-dihydroxybenzothiohydrazide (2)

Yield: 69%; m.p.: 166–167 °C. log k = −0.023 (70% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.96 (s, 1 H), 10.44 (s, 1H), 10.19 (s, 1H), 9.79 (s, 1H), 9.49 (d, J = 4.85 Hz, 1H, pyridine), 7.99 (s, 1H, C-6), 7.29 (d, J = 4.85 Hz, 1H, pyridine) 6.39 (s, 1H, C-3) ppm; ¹³C NMR (125 MHz, DMSO-d₆) δ: 162.0, 162.1, 156.5, 154.5, 149.8, 140.8, 133.5, 123.4, 121.2, 111.5, 111.4, 108.7, 103.8 ppm; EI-MS (m/z, %): 398 (M⁺, 10), 366 (13), 365 (14), 364 (71), 332 (15), 330 (54), 294 (19), 293 (24), 198 (15), 196 (48), 189 (37), 187 (100), 180 (5), 175 (6), 171 (21), 169 (38), 161 (14), 154 (8), 153 (79), 141 (17), 135 (14), 69 (21), 64 (10), 36 (13). Anal. Calc. for C₁₃H₈Cl₂F₃N₃O₂S (398.19) (%): 39.21; H, 2.03; N, 10.55. Found (%): C, 39.30; H, 2.01; N, 10.51.

N′-(4-Cyclopropyl-6-(trifluoromethyl)pyrimidin-2-yl)-2,4-dihydroxybenzothiohydrazide (3a)

Yield: 73%; m.p.: 130–132 °C; log k = 0.291 (40% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.45 (s, 1H, HO-C(2)), 10.91 (s, 1H, HO-C(4)), 10.25 (s, 1H, NH), 10.00 (s, 1H, NH), 8.05 (d, J = 8.7 Hz, 1H, H-C(6)), 7.32 (s, 1H, H-C(pyrimidine)(5)), 6.46–6.38 (m, 2H, H-C(3, 5), 2.21 (m, 1H, H-C(cyclopropan)), 1.13 (m, 4H, CH₂×2) ppm; IR (ATR) V: 3221 (NH, OH), 1595 (C=N, C=C), 1562 (C=C), 1468, 1418, 1338, 1241 (C-F), 1187 (C-OH), 1149 (C=S), 1113, 1020, 936, 842 cm⁻¹; EI-MS (m/z, %): 370 (M⁺, 20), 337 (66), 202 (8), 182 (6), 153 (100), 137 (8), 135 (4), 97 (6), 81 (3), 65 (5), 53 (4), 41 (4), 39 (5). Anal. Calc. for C₁₅H₁₃F₃N₄O₂S (370.35) (%): C, 48.65; H, 3.54; N, 15.13. Found (%): C, 48.75; H, 3.56; N, 15.09.

5-Chloro-N′-(4-cyclopropyl-6-(trifluoromethyl)pyrimidin-2-yl)-2,4-dihydroxybenzothiohydrazide (3b)

Yield: 69%; m.p.: 152–153 °C; log k = −0.324 (70% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.5 (s, 1H), 10.91 (s, 1H), 10.29 (s, 1H), 8.10 (s, 1H, H-C(6)), 7.32 (s, 1H, H-C(pyrimidine)(5)), 6.65 (s, 1H, H-C(3), 2.18 (m, 1H, H-C(cyclopropane)), 1,14 (m, 4H, CH₂×2) ppm; ¹³C NMR (125 MHz, DMSO-d₆): 176.7, 159.6, 156.4, 154.9, 133.1, 121.5, 119.3, 114.8, 111.4, 110.9, 106.3, 103.8, 22.5, 13.5, 11.8; EI-MS (m/z, %): 404 (M⁺, 22), 371 (98), 337 (5), 218 (49), 202 (18), 189 (37), 187 (100), 182 (12), 173 (12), 171 (41), 153 (7), 137 (5), 79 (5), 69 (10). 51 (7), 41 (6). Anal. Calc. for C₁₅H₁₂ClF₃N₄O₂S (404.79) (%): C, 44.51; H, 2.99; N, 13.84. Found (%): C, 46.00; H, 2.98; N, 12.29.

N′-(4-(4-Chlorophenyl)-6-(difluoromethyl)pyrimidin-2-yl)-2,4-dihydroxybenzothiohydrazide (4a)

Yield: 74%; m.p.: 197–198 °C; log k = 0.762 (40% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.65 (s, 1H, HO-C(2)), 11.76 (s, 1H, HO-C(4)), 10.36 (s, 1 H, NH), 10.22 (s, 1H, NH), 8.28 (m, 2H, H-C(2',6'), 8.11 (d, J = 8.7 Hz, 1H, H-C(6)), 7.73 (s, 1H, H-C(pyrimidine)(5)), 7.63 (d, J = 8.7 Hz, 2H, H-C(3′,5′), 6,90 (t, J = 54.3 Hz, 1H, CHF₂), 6.45–6.41 (m, 2 H, H-C(3, 5)) ppm; IR (ATR) V: 3257. 3165 (NH, OH), 1612 (C=N), 1584 (C=C), 1540, 1478, 1454, 1380, 1242 (C-F), 1187 (C-OH), 1113 (C=S), 1092, 1061, 1021, 831, 753 cm⁻¹; EI-MS (m/z, %): 422 (M⁺, 13), 391 (28), 389 (88), 272 (19), 270 (42), 256 (9), 254 (15), 241 (17), 205 (4), 194 (7), 167 (5), 162 (10), 153 (100), 138 (5), 135 (9), 127 (5), 111 (4), 102 (4), 97 (5), 75 (6), 52 (43). Anal. Calc. for C₁₈H₁₃ClF₂N₄O₂S (422.84) (%): C, 51.13; H, 3.10; N, 13.25. Found (%): C, 51.04; H, 3.09; N, 13.20.

N′-(4-(4-Chlorophenyl)-6-(difluoromethyl)pyrimidin-2-yl)-5-ethyl-2,4-dihydroxybenzothiohydrazide (4b)

Yield: 69%; m.p.: 158–159 °C; log k = 0.439 (70% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.31 (s, 1H), 11.85 (s, 1H), 10.33 (s, 1H), 10.23 (s, 1H), 8.23 (m, 2H), 7.98 (s, 1H, pyrimidine), 7.72 (s, 1H), 7.64–7.61 (m, 2H), 6.96 (t, 1H, CHF₂, J = 54.3 Hz), 6.49 (s, 1H, H-C(3)), 2.40 (q, J = 7.5 Hz, 2H, CH₂CH₃), 1.14 (t, J = 7.5 Hz, 3H, CH₃) ppm; EI-MS (m/z, %): 450 (M⁺, 16), 417 (85), 401 (13), 294 (5), 270 (65), 256 (14), 255 (18), 241 (26), 194 (12), 181 (100), 165 (15), 162 (13), 148 (16), 123 (7), 77 (6). Anal. Calc. for C₂₀H₁₇ClF₂N₄O₂S (450.89) (%): C, 53.28; H, 3.80; N, 12.43. Found (%): C, 53.40; H, 3.82; N, 12.39.

N′-(4-(4-Chlorophenyl)-6-(trifluoromethyl)pyrimidin-2-yl)-2,4-dihydroxybenzothiohydrazide (5a)

Yield: 64%; m.p.: 155–156 °C; log k = 0.406 (70% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.53 (s, 1H), 10.55 (s, 1H), 10.26 (s, 1H), 10.20 (s, 1H, NH), 8.38 (d, J = 8.3 Hz, 2H, H-C(benzene)), 8.08 (d, J = 8.7 Hz, 1H, H-C(6)), 7.90 (s,1H, H-C(pyrimidine)), 7.65 (d, J = 8.4 Hz, 2H, H-C(benzene)), 6.44–6.40 (m, 2H, H-C(3,5)) ppm; EI-MS(m/z, %): 440 (M⁺, 22), 409 (14), 407 (67), 290 (8), 288 (17), 272 (12), 155 (8), 153 (100), 135 (6), 97 (7). Anal. Calc. for C₁₈H₁₂ClF₃N₄O₂S (440.83) (%): C, 49.04; H, 2.74; N, 12.71. Found (%): C, 49.14; H, 2.76; N, 12.66.

N′-(4-(4-Chlorophenyl)-6-(trifluoromethyl)pyrimidin-2-yl)-5-ethyl-2,4-dihydroxybenzothiohydrazide (5b)

Yield: 70%; m.p.: 193–194 °C; log k = 0.496 (70% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.47 (s, 1H), 11.53 (s, 1H), 10.53 (s, 1H), 10.18 (s, 1H, NH), 8.32 (d, J = 8.6 Hz, 2H, H-C(benzene)), 7.95 (s, 1H, H-C(6)), 7.91 (s. 1H, H-C(pyrimidine)), 7.64 (m, 2H, H-C(benzene)), 6.49 (s, 1H, H-C(3), 2.42 (q, J = 7.5 Hz, 2H, CH₂CH₃), 1.14 (t, J = 7.5 Hz, 3H, CH₃); EI-MS (m/z, %): 468 (M⁺, 15), 435 (35), 288 (10), 272 (6), 181 (100). Anal. Calc. for C₂₀H₁₆ClF₃N₄O₂S (468.88) (%): C, 51.23; H, 3.44; N, 11.95. Found (%): C, 51.42; H, 3.46; N, 11.89.

N′-(4-(Difluoromethyl)-6-(3,4-dimethylphenyl)pyrimidin-2-yl)-2,4-dihydroxybenzothiohydrazide (6)

Yield: 70%, m.p.: 182–183 °C; log k = 0.839 (40% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.65 (s, 1H, HO-C(2)), 11.76 (s, 1H, HO-C(4)), 10.25 (s, 1H, NH), 10.20 (s, 1H, NH), 8.12 (d, 1H, J = 8.8 Hz, H-C(6)), 8.04 (s, 1H, H-C(pyrimidine)(5)), 7.98 (d, 1H, J = 7.9 Hz, H-C(6′)), 7.67 (s, 1H, H-C(2′)), 7.33 (d, 1H, J = 7.8 Hz, H-C(5′)), 6.87 (t, 1H, J = 54.5 Hz, CHF₂), 6.44 (d, 1H, J = 2.2 Hz, H-C(3)), 6.42 (dd, 1H, J = 8.8 and 2.2 Hz, H-C(5)), 2.33 (s, 3H, CH₃), 2.31 (s, 3H, CH₃) ppm; IR (ATR) V: 3231 (OH, NH), 1641 (C=N), 1587, 1549 (C=C), 1381, 1307, 1237 (C-F), 1167 (C-OH), 1114 (C=S), 1054, 1023, 979, 823 cm⁻¹; EI-MS (m/z, %): 416 (M⁺, 16), 383 (100), 264 (67), 249 (24), 235 (22), 188 (6), 153 (46), 135 (6), 115 (4), 97 (4), 77 (4) cm⁻¹; EI-MS (m/z, %): 416 (M⁺, 16), 383 (100), 264 (67), 249 (24), 235 (22), 188 (6), 153 (46), 135 (6), 115 (4), 97 (4), 77 (4). Anal. Calc. for C₂₀H₁₈ F₂N₄O₂S (416.44) (%): C, 57.68; H, 4.36; N, 13.45. Found (%): C, 57.58; H, 4.38; N, 13.39.

N′-(5-(Difluoromethyl)-4-(4-fluorophenyl)pyrimidin-2-yl)-2,4-dihydroxybenzothiohydrazide (7)

Yield: 74%; m.p.: 175–176 °C; log k = 0.483 (40% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.65 (s, 1H, HO-C(2)), 11.60 (s, 1H, HO-C(4)), 10.33 (s, 1H, NH), 10.21 (s, 1H, NH), 8.34 (m, 2H, H-C(2′,6′), 8.10 (d, J = 8.6 Hz, 1H, H-C(6)), 7.71 (s, 1H, H-C(pyrimidine)(6)), 7.40 (m, 2H, H-C(3′,5′), 6.89 (t, J = 54.36 Hz, 1H, CHF₂), 6.44 (s, 1H, H-C(3)), 6.42 (m, 1H, H-C(5)) ppm; IR (ATR) V: 3224 (NH, OH), 1587 (C=N), 1552, 1468, 1382, 1331, 1237 (C-F), 1192 (C-OH), 1109 (C=S), 1016, 837 cm⁻¹; EI-MS (m/z, %): 406 (M⁺, 16), 373 (100), 254 (38), 239 (17), 238 (17), 225 (11), 178 (6), 153 (83), 146 (11), 122 (5), 97 (5), 95 (5), 51 (3). Anal. Calc. for C₁₈H₁₃ F₃N₄O₂S (406.38) (%): C, 53.20; H, 3.22; N, 13.79. Found (%): C, 54.19; H, 3.23; N, 13.73.

N′-(4-(3,4-Dichlorophenyl)-6-(trifluoromethyl)pyrimidin-2-yl)-2,4-dihydroxybenzothiohydrazide (8)

Yield: 79%; m.p.: 149–151 °C; log k = 0.934 (40% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.55 (s, 1H, HO-C(2)), 10.88 (s, 1H, HO-C(4)), 10.60 (s, 1H, NH), 10.16 (s, 1H, NH), 8.55 (s, 1H, H-C(pyrimidine)(5)), 8,28 (d, J = 8.3 Hz, 1H, H-C(6)), 8.03 (m, 2H, H-C(2′,5′)), 7.85 (dd, 1H, J = 8.3 and 2,8 Hz, H-C(6′)), 6,42 (d, 1H, J = 2 Hz, H-C(3)), 6,38 (dd, 1H, J = 8.1 and 2.2 Hz H-C(5)) ppm; IR (ATR) V: 3250 (NH, OH), 1659 (C=N), 1547 (C=C), 1381, 1240 (C-F), 1164 (C-OH), 1146 (C=S), 1030, 826 cm⁻¹; EI-MS (m/z, %): 475 (M⁺, 3), 441 (33), 322 (14), 306 (6), 293 (6) 196 (4), 153 (100), 135 (8), 97 (6), 69 (4). Anal. Calc. for C₁₈H₁₁Cl₂F₃N₄O₂S (475.27) (%): C, 45.59; H, 2.33; N, 11.79. Found (%): C, 45.49; H, 2.32; N, 11.71.

N′-(4-(Difluoromethyl)-6-(furan-2-yl)pyrimidin-2-yl)-2,4-dihydroxybenzothiohydrazide (9)

Yield: 70%; m.p.: 185–186 °C; log k = −0.163 (70% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.97 (s, 1H), 10.30 (s, 1H), 8.17 (d, J = 8.4 Hz, 1 H, C-6), 8.03 (s, 1 H, pyrimidine), 7.51 (d, J = 3.3 Hz, 1H, furan), 7.38 (s, 1H, furan), 6.89 (t, 1H, CHF₂, J = 54.3 Hz), 6.80 (m, 1 H, furan), 6.50 (d, J =2.2 Hz, 1H, H-C(3)), 6.46 (dd, 1H, J = 8.8 and 2.2, Hz, H-C(5)) ppm. EI-MS (m/z, %): 378 (M⁺, 10), 345 (100), 226 (50), 211 (25), 197 (14), 183 (6), 154 (6), 153 (69), 150 (13), 135 (11), 118 (10), 90 (5), 52 (5). Anal. Calc. for C₁₆H₁₂F₂N₄O₃S (378.35) (%): C, 50.79; H, 3.20; N, 14.81. Found (%): C, 50.99; H, 3.18; N, 14.88.

2,4-Dihydroxy-N′-(4-(1-methyl-1H-pyrazol-4-yl)-6-(trifluoromethyl)pyrimidin-2-yl)benzothiohydrazide (10)

Yield: 70%; m.p.: 211–212 °C; log k = −0.449 (70% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.75 (broad s, 1H), 11.79 (broad s, 1 H), 10.32 (s, 1H), 8.58 (s, 1H, pyrazole), 8.27 (s, 1H, pyrazole), 8.15 (s, 1H, C-6), 7.60 (s, 1H, pyrimidine), 6.49 (s, 1H, H-C(3)), 6.35 (m, 1H, H-C(5)), 3.95 (s, 3H, CH₃) ppm; EI-MS (m/z, %): 410 (M⁺, 21), 378 (17), 377 (85), 259 (4), 258 (28), 243 (11), 202 (5), 153 (100), 108 (5), 42 (6). Anal. Calc. for C₁₆H₁₃F₃N₆O₂S (410.37) (%): C, 46.83; H, 3.19; N, 20.48. Found (%): C, 46.98; H, 3.17; N, 20.59.

2,4-Dihydroxy-N′-(4-(5-methylthiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-yl)benzothiohydrazide (11)

Yield: 77%; m.p.: 202–203 °C; log k = 0.782 (40% MeOH). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.45 (s, 1H, HO-C(2)), 10.37 (s, 1H, NH), 8.09 (d, J = 3.5 Hz, 1H, H-C(thiophene)), 8.07 (d, 1H, J = 9.1 Hz, H-C(6), 7.75 (s, 1H, H-C(pyrimidine)(5)), 6.99 (d, 1H, J = 2.9 Hz, H-C(thiophene), 6.44 (s, 1H, H-C(3)), 6.41 (dd, 1H, J = 8.8 and 2.2 Hz, H-C(5)), 2.53 (s, 3H, CH₃) ppm; IR (ATR) V: 3268 (NH, OH), 1679 (C=N), 1611 (C=C), 1588, 1548 (C=C), 1476, 1399, 1243, 1184 (C-OH), 1142 (C=S), 1112, 993, 839, 806, 774 cm⁻¹; EI-MS (m/z, %): 426 (M⁺, 11), 393 (30), 274 (80), 259 (35), 245 (22), 231 (3), 225 (4), 218 (9), 210 (4), 198 (5), 191 (4), 153 (53), 148 (11), 135 (7), 124 (6), 121 (7), 97 (13), 79 (16), 64 (100), 52 (10), 48 (55), 44 (37) 40 (39), 34 (7). Anal. Calc. for C₁₇H₁₃F₃N₄O₂S₂ (426.44) (%): C, 47.88; H, 3.07; N, 13.14. Found (%): C, 47.98; H, 3.08; N, 13.20.

Biological assays

In vitro antiproliferative assay was performed using the following human cell lines: T47D (breast cancer), SW707 (rectal adenocarcinoma), A549 (non-small cell lung carcinoma), LNCaP cells (prostate cancer), HL-60 (promyelocytic leukemia), HCV29T (bladder cancer) and HT-29 (colon cancer) from the American Type Culture Collection (Rockville, MD) or the Fibiger Institute, Copenhagen, Denmark. Twenty-four hours before the addition of the tested agents, the cells were plated in 96-well plates (Sarstedt Inc, Newton, NC) at a density of 10⁴ cells/well. All cell lines were maintained in the opti-MEM medium supplement with 2 mm glutamine (Gibco, Warsaw, Poland), streptomycin (50 μg/mL), penicillin (50 U/mL) (Polfa, Tarchomin, Poland) and 5% fetal calf serum (Gibco, Grand Island, NY). The cells were incubated at 37 °C in the humid atmosphere saturated with 5% CO₂. The solutions of compounds (l mg/ml) were prepared ex tempore by dissolving the substance in 100 μl of DMSO completed with 900 μl of tissue culture medium. Afterward, the compounds were diluted in the culture medium to reach the final concentrations ranging from 0.1 to 100 μg/ml. The solvent (DMSO) used at the highest concentration in the test did not reveal any cytotoxic activity. Cisplatin was applied as a test referential agent. The cytotoxicity assay was performed after 72 h exposure of the cultured cells at the concentration ranging from 0.1 to 100 μg/ml of the tested agents. The SRB test measuring the cell proliferation inhibition in the in vitro culture was applied28. The cells attached to the plastic were fixed with cold 50% trichloroacetic acid (TCA, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) added on the top of the culture medium in each well. The plates were incubated at 4 °C for 1 h and then washed five times with tap water. The background optical density was measured in the wells filled with the medium, without the cells. The cellular material fixed with TCA was stained with 0.4% sulforhodamine B (Chemie GmbH) dissolved in 1% acetic acid (POCh, Gliwice, Poland) for 30 min. The unbound dye was removed by rinsing (four times) with 1% acetic acid, and the protein-bound dye was extracted with 10 mm unbuffered Tris base (tris(hydroxymethyl)aminomethane, POCh) for determination of optical density (at 540 nm) in a computer-interfaced, 96-well microtiter plate reader Uniskan II (Labsystems, Helsinki, Finland). The compounds were tested in triplicates per experiment. The experiments were repeated at least three times. The IC₅₀ values were calculated using Cheburator 0.9.0 software (www.cheburator.nevozhay.com).

Results and discussion

N′-substituted 2,4-dihydroxybenzocarbothiohydrazides were obtained according to Scheme 1. Compounds (1–11) were prepared by the reaction of sulfinylbis[(2,4-dihydroxyphenyl)methanethione] (STB) or its ethyl (compounds 4b, 5b) or chlorine analogs (compound 3b) with the commercially available hydrazines. STB and its analogs were obtained according to the procedure described previously29. The purity of compounds was monitored by the reversed-phase (RP-18) HPLC chromatography method with methanol–water as the mobile phase.

Scheme 1. Synthesis route of N′-substituted 2,4-dihydroxybenzocarbothiohydrazides (1–11).

All compounds (Figure 2) were characterized by the spectral and mass spectrometry analysis. The ¹H NMR spectra data show bands in the range of 10.6–9.5 ppm characteristic of the protons of –NH–NH– moiety. The region of OH proton signals is similar to that observed for other benzenediol derivatives16. These protons attached to heteroatoms are exchangeable in D₂O. In the spectrum of compounds 3–7 and 8–11 in the range of about 7.7 ppm there is a characteristic singlet which is a signal of the trisubstituted pyrimidine proton. In the case of analog 8, it indicates the slightly larger chemical shift about 8.5 ppm.

Figure 2. Structures of N′-substituted 2,4-dihydroxybenzocarbothiohydrazides (1–11).

Infrared spectroscopy shows the presence of N–H and O–H stretching bands in the range of about 3250 cm⁻¹ characteristics of the hydrazine moiety and the hydroxyl substituents. The absorption bands in the range of 1200–1080 cm⁻¹ may indicate the existence of =C(=S) group.

The EI-MS fragmentation peaks analysis confirmed the structure of the analyzed compounds. In all cases, the molecular ion band M⁺ is registered, however, of weak intensity (10–20%). [C₆H₃(OH)₂C(=S)]⁺ ion (153 m/z) or its analogs: [C₆H₂(OH)₂ClC(=S)]⁺ (187 m/z, compound 3b), [C₆H₂(OH)₂C₂H₅C(–S)]⁺ (181 m/z, compounds 4b, 5b) were observed as the main peaks for most of the compounds. The second principle characteristic fragmentation is related to the desulfhydrylation process of the molecular ion which produces [M-SH]⁺ ions. In some cases, this is the main peak (compounds 7, 9). 135 m/z ion is also a characteristic fragmentary ion corresponding to [C₆H₃(OH)₂CN]⁺. This mass fragmentation pathway is similar to those obtained for 2,4-dihydroxybenzothioamides30.

Compounds were designed and synthesized as potential biologically active agents, so they were evaluated for their antiproliferative potency against human HCV29T (bladder cancer), A549 (non-small lung carcinoma), A498 (renal cancer), LNCaP (prostate cancer), HL-60 (promyelocytic leukemia), MCF-7 (breast cancer) and HT-29 (colon cancer) cells. The cytotoxic activity in vitro was expressed as IC₅₀ (μm), the concentration of the compound that inhibits proliferation rate of the tumor cells by 50% as compared to the control untreated cells. Cisplatin was used as a reference drug. The results of screening are summarized up in Table 1. The data indicate that the considered compounds display good to moderate cytotoxic activity between the 8 and 131 μm IC₅₀ values. Their antiproliferative effect depends explicitly on the type of N′-substitution and the modification of the benzenediol moiety. The most active are pyrimidine derivatives with the additional 4-chlorophenyl substituent on the heterocyclic ring (compounds 4 and 5). The compounds with the additional Et substituent in the benzenediol residue show higher antiproliferative potency compared to the unsubstituted ones (4b, 5b). That effect is similar to that found for other heterocyclic benzenediols prepared by us24,26. LNcaP cells are the most sensitive to the studied compounds and the potency of the most active compounds is similar to that of cisplatin (compounds 4 and 5). HL-60 cells seem to be the most resistant to some studied compounds and most of the derivatives are not active below 100 μg/ml. However, active compounds toward HL-60 cells show a relatively high antiproliferative potency.

Table 1. Antiproliferative activity (IC₅₀*) of N′-substituted 2,4-dihydroxybenzocarbothiohydrazides against the human cancer cell lines.

	IC50/μm
No.	HCV29T	A 498	LNCaP	HL-60	A549	MCF-7	HT-29
2	82.19 ± 0.13	81.96 ± 2.61	76.95 ± 5.20	neg†	79.31 ± 3.80	61.56 ± 30.06	20.63 ± 10.59
3a	76.09 ± 12.07	88.40 ± 10.96	24.95 ± 13.50	neg†	96.50 ± 15.04	58.08 ± 16.93	93.48 ± 2.60
3b	70.18 ± 6.77	80.81 ± 4.22	80.46 ± 14.28	neg†	120.41 ± 34.07	91.48 ± 1.26	81.57 ± 21.69
4a	21.10 ± 6.08	65.18 ± 18.92	16.70 ± 10.86	neg†	59.83 ± 16.84	62.46 ± 17.74	81.12 ± 4.40
4b	18.01 ± 5.77	40.36 ± 12.51	11.69 ± 3.84	8.47 ± 0.33	13.13 ± 0.31	11.87 ± 1.06	12.46 ± 2.26
5a	12.18 ± 5.10	26.99 ± 13.88	7.78 ± 0.23	10.23 ± 3.06	54.89 ± 11.21	55.76 ± 6.63	79.40 ± 7.28
5b	16.63 ± 7.44	40.27 ± 9.73	8.13 ± 1.95	8.06 ± 2.13	17.17 ± 3.24	12.32 ± 3.13	20.15 ± 0.64
6	41.52 ± 4.27	65.43 ± 1.56	32.54 ± 11.09	10.23 ± 1.66	41.69 ± 12.32	32.80 ± 11.45	51.22 ± 20.58
7	57.70 ± 11.34	80.56 ± 12.56	35.80 ± 1.62	neg†	92.18 ± 10.73	69.02 ± 19.42	84.23 ± 6.89
8	ns‡	70.59 ± 4.27	50.54 ± 9.34	neg†	48.24 ± 8.14	49.51 ± 8.14	65.14 ± 2.40
9	82.33 ± 1.32	84.50 ± 1.37	66.39 ± 8.77	neg†	81.04 ± 2.64	65.74 ± 10.57	83.05 ± 0.50
10	130.76 ± 51.20	93.14 ± 16.42	89.65 ± 34.68	neg†	111.53 ± 34.07	81.22 ± 4.87	126.11 ± 22.05
11	68.38 ± 3.17	78.77 ± 4.76	56.33 ± 10.41	neg†	53.77 ± 9.08	55.18 ± 11.23	72.60 ± 2.67
Cisplatin	6.13 ± 2.40	9.83 ± 2.17	14.20 ± 5.63	1.10 ± 0.13	6.77 ± 5.36	11.50 ± 1.60	40.59 ± 11.63

*IC₅₀ (μm) indicates the compound concentration that inhibits the proliferation rate of tumor cells by 50% as compared to the control untreated cells. The values are the mean ± SD of nine independent experiments.

† NEG, negative in the studied concentrations (up to 100 μg/ml).

‡ ns, not studied.

Activity of obtained N′-substituted 2,4-dihydroxybenzocarbothiohydrazides is similar to that for 4-(1,3,4-thiadiazol-2-ilo)benzene-1,3-diols31. 1,3,4-Thiadiazole ring can be considered as a cyclic pseudo analog of carbothiohydrazide moiety. Therefore, one can draw a conclusion that the activity of compounds does not depend on –CSNHNH– atoms arrangement: linear (thiohydrazides) or cyclic (1,3,4-thiadiazoles).

Conclusion

To sum up, the synthesis method of N′-substituted 2,4-dihydroxybenzothiohydrazides was elaborated. The compounds were evaluated for their antiproliferative potency using both solid and hematological human tumor cells. The most active derivatives inhibited the viability of human prostate cancer LNCaP cells by 50% at a concentration of about 8 μm and it was the activity similar to that of cisplatin. The studies confirmed the beneficial influence of the presence of Et substituent in the benzenediol moiety on the antiproliferative activity. The obtained results could be useful for designing and synthesis of new N′-substituted 2,4-dihydroxybenzothiohydrazides of greater antiproliferative potency as potential anticancer agents.

Declaration of interest

The authors declare no conflict of interest.