Study of reactivity of cyanoacetohydrazonoethyl-N-ethyl-N-methyl benzenesulfonamide: preparation of novel anticancer and antimicrobial active heterocyclic benzenesulfonamide derivatives and their molecular docking against dihydrofolate reductase

Abstract

This article describes the synthesis of some novel heterocyclic sulfonamides having biologically active thiophene 3, 4, 5, 6, coumarin 8, benzocoumarin 9, thiazole 7, piperidine 10, pyrrolidine 11, pyrazole 14 and pyridine 12, 13. Starting with 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzenesulfonamide (2), which was prepared from condensation of acetophenone derivative 1 with 2-cyanoacetohydrazide. The structures of the newly synthesized compounds were confirmed by elemental analysis, IR, ¹H NMR, ¹³C NMR, ¹⁹F NMR and MS spectral data. All the newly synthesized heterocyclic sulfonamides were evaluated as in-vitro anti-breast cancer cell line (MCF7) and as in-vitro antimicrobial agents. Compounds 8, 5 and 11 were more active than MTX reference drug and compounds 12, 7, 4, 14, 5 and 8 were highly potent against Klebsiella pneumonia. Molecular operating environment performed virtual screening using molecular docking studies of the synthesized compounds. The results indicated that some prepared compounds are suitable inhibitor against dihydrofolate reductase (DHFR) enzyme (PDBSD:4DFR) with further modification.

Keywords:

• Anticancer
• antimicrobial
• cytotoxicity
• heterocyclic sulfonamides
• molecular docking

Introduction

One of the most important and desirable field in medicinal chemistry, is to find a new specific anticancer agents and to develop new safer, potent and resistance-free antimicrobial drugs. It is evident from the literature survey that benzenesulfonamides exhibited a significant anticancer and antimicrobial activity1–4. Furthermore, benzenesulfonamides derivatives having aromatic heterocyclic moiety possess a wide spectrum of pharmacological activities, such as anticancer5–7, anti-bacterial8–10, anti-inflammatory11 and anti-viral12, hypoglycemic13, diuretic14,15, anti-carbonic anhydrase16 and anti-thyroid17, antimalarial18 and antileprotic19,20 activity. In addition, literature survey exhibited that some of the synthesized benzenesulfonamide derivatives were found to be dihydrofolate reductase (DHFR) inhibitors21–23. On the other hand, the presence of cyanoacetylhydrazono moiety (C=N−N−C(O)−CH₂−CN) in a structure, makes it a versatile and convenient intermediate for the synthesis of a wide variety of heterocyclic compounds. The β-functional nitrile1–4 moiety of the molecule is a favorable unit for addition followed by cyclization or via cycloaddition with numerous reagents providing heterocyclic compounds of different ring sizes with one or several heteroatoms that are interesting as pharmaceuticals agents24–30. Considering this background, some novel heterocyclic sulfonamides as thiophenes 3, 4, 5 and 6, thiazole 7, chromenes 8 and 9, piperidine 10, pyrrolidine 11, tetrahydropyridine 12, pyridine 13, pyrazole 14 and hydrazono 15 derivatives were prepared and tested in terms of anticancer and antimicrobial activity with the purpose of revealing possible leading compounds for development of new anticancer and antimicrobial agents.

Materials and methods

Chemistry

Melting points (°C, uncorrected) were determined in open capillaries on a GallenKemp melting point apparatus (Sanyo Gal-lenKemp, Southborough, UK). Precoated silica gel plates (silica gel 0.25 mm, 60 G F 254; Merck, Germany) were used for thin layer chromatography, dichloromethane/methanol (9.5:0.5 mL) mixture was used as a developing solvent system and the spots were recorded in KBr discs using IR-470 Shimadzu spectrometer (Shi-madzu, Tokyo, Japan). ¹H,¹³C NMR spectra (in DMSO-d₆) were recorded on Bruker Ac-300 ultra shield NMR spectrometer (Bruker, Flawil, Switzerland, δ ppm) at 300 MHz, using TMS as internal standard (¹H and ¹³C) and CFCl₃ (¹⁹F). Electron impact Mass Spectra were recorded on a Shimadzu GC/MS-QP5000 instrument Shimadzu, Tokyo, Japan.

4-(1-(2-(2-Cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzenesulfonamide (2)

To a solution of 1-(4-(N-ethyl-N-methylsulfonyl)phenyl)ethanone (1) (7.23 g, 0.03 mol) in 1,4-dioxane (50 mL), 2-cyanoacetohydrazide (2.97 g, 0.03 mol) was added. The reaction mixture was heated under reflux for 2 h and then left to cool. The solid product formed upon pouring onto ice/water was collected by filtration and recrystallized from ethanol to give 2. Yield, 89%; light brown powder; m.p. 166–168 °C; IR (KBr, cm⁻¹): 3310 (NH), 3095 (CH arom.), 2983, 2943, 2852 (CH aliph.), 2262 (C≡N), 1688 (C=O), 1616 (C=N), 1330, 1160 (SO₂). ¹H NMR (DMSO-d₆): 1.09 (t, 3H, CH₃–CH₂, 3JHH=7.4 Hz), 2.29 (s, 3H, CH₃–C=N), 2.67 (s, 3H, CH₃–N), 3.04 (q, 2H, CH₂–CH₃, 3JHH=7.4 Hz), 4.26 (s, 2H, CH₂–CN), 7.78, 7.98 (dd, 4H, Ar–H, AB system, 3JHH=7.3 Hz), 11.18 (s, 1H, NH, discharged with D₂O). ¹³C NMR (DMSO-d₆): 16.89, 17.73, 24.30, 37.21, 48.43, 114.92, 125.62 (2C), 129.30 (2C), 134.41, 141.68, 155.61, 173.02; MS, m/z (%): 322 (M⁺) (1.4), 122 (100), Anal. Calcd for C₁₄H₁₈N₄O₃S: C, 52.16; H, 5.63; N, 17.38; S, 9.95, found: C, 52.06; H, 5.61; N, 17.32; S, 9.91.

4-(1-(2-(3,5-Diamino-4-cyanothiophene-2-carbonyl)hydrazono)ethyl)-N-ethyl-N-methyl benzenesulfonamide (3)

To a mixture of 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzene sulfonamide (2) (0.5 g, 0.004 mol), malononitrile (0.1 g, 0.004 mol) and 0.5 mL of triethylamine with elemental sulfur (0.05 g 0.004 mol) was refluxed in 30 mL of 1,4-dioxane for 5 h and then left to cool. The solid product formed was collected by filtration and recrystallized from ethanol to give 3. Yield, 77%; black powder; m.p. 230–232 °C; IR (KBr, cm⁻¹): 3406, 3317, 3192 (NH, 2NH₂), 3111 (CH arom.) 2942, 2827 (CH aliph.), 2210 (C≡N), 1687 (C=O), 1624 (C=N), 1398, 1161 (SO₂). ¹H-NMR (DMSO-d₆): 1.22 (t, 3H, CH₃–CH₂, 3JHH=7.4 Hz), 2.46 (s, 3H, CH₃–C=N), 2.6(s, 3H, CH₃–N), 3.23 (q, 2H, CH₂–CH₃, 3JHH=7.4 Hz), 6.5 (s, 4H, 2NH₂, D₂O exchangeable), 7.71–7.92 (2d, 4H, Ar–H, AB system, 3JHH=7.3 Hz), 8.1 (s, 1H, NH, D₂O exchangeable). ¹³C-NMR (DMSO-d₆): 16.8, 17.7, 24.3, 37.5, 76.7, 116.2, 127.9 (2), 128.1 (2), 135.4, 136.1, 139.8, 142.0, 147.8, 149.8, 170.4; MS, m/z (%): 420.10 (M⁺) (0.4), 133 (100), Anal. Calcd. for C₁₇H₂₀N₆O₃S₂: C, 48.56; H, 4.79; N, 19.99; S, 4.25, found: C, 48.59; H, 4.77; N, 19.96; S, 4.27.

Ethyl-2,4-diamino-5-(2-(1-(4-(N-ethyl-N-methylsulfamoyl)phenyl)ethylidene)hydrazine carbonyl)thiophene-3-carboxylate (4)

To a solution of 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzene sulfonamide (2) (0.5 g, 0.004 mol) in 1,4-dioxane (30 mL) containing triethylamine (1 mL) ethyl cyanoacetate (0.24 g, 0.004 mol) together with elemental sulfur (0.05 g, 0.004 mol) were added. The whole reaction mixture was refluxed for 5 h. Then poured onto ice water and the obtained solid was recrystallized from dioxane to give 4. Yield, 65%; pale yellow powder; m.p. 234–236 °C; IR (KBr, cm⁻¹): 3412, 3362, 3194 (NH, 2NH₂), 3107 (CH arom.), 2974, 2937 (CH aliph.), 1730, 1687 (2C=O), 1624 (C=N), 1334, 1161 (SO₂). ¹H-NMR (DMSO-d₆): 1.22 (t, 3H, CH₃–CH₂-N, 3JHH=7.2 Hz), 1.28 (t, 3H, 3JHH = 5.5 Hz, CH₃–CH₂–O), 2.3 (s, 3H, CH₃), 2.4 (s, 3H, N–CH₃), 3.16 (q, 2H, CH₂–CH₃, 3JHH=7.2 Hz), 4.4 (q, 2H, 3JHH = 5.5 Hz, CH₃–CH₂–O), 5.3 (s, 4H, 2NH₂, D₂O exchangeable), 7.8–8.1 (2d, 4H, Ar–H, AB system, 3JHH=7.8 Hz), 9.1 (s, 1H, NH, D₂O exchangeable). ¹³C-NMR (DMSO-d₆): 4.6, 16.8, 17.7, 24.3, 37.8, 59.1, 125.9 (2), 129.0 (2), 132.7, 136.1, 138.7, 141.8, 143.0, 147.8, 148.4, 161.2, 170.0; MS, m/z (%): 466.72 (M⁺) (0.4), 64 (100), Anal. Calcd for C₁₉H₂₅N₅O₅S₂: C, 48.81; H, 5.39; N, 14.98; S, 13.72. Found: C, 48.83; H, 5.37; N, 14.96; S, 13.75.

General method for preparation of 4-(1-(2-(3-amino cycloderivative[b]thiophene-2-carbonyl)hydrazono)ethyl)-N-ethyl-N-methylbenzenesulfonamide (5 and 6)

To a solution of compound 2 (0.5 g, 0.004 mol) in absolute ethanol (30 mL) containing triethylamine (1 mL) either cyclopentanone or cyclohexanone (0.004 mol) together with elemental sulfur (0.05 g, 0.004 mol) were added. The reaction mixture was refluxed for 2 h then poured onto ice/water and the obtained solid was recrystallized from dioxane to give 5 or 6, respectively.

4-(1-(2-(3-Amino-5,6-dihydro-4H-cyclopenta[b]thiophene-2-carbonyl) hydrazono)ethyl)-N-ethyl-N-methylbenzenesulfonamide (5)

Yield, 76%; white powder; m.p. 240–242 °C; IR (KBr, cm¹): 3363, 3194, 3111 (NH, NH₂), 3007 (CH arom), 2974, 2935 (CH aliph.), 1687 (C=O), 1334, 1161 (SO₂). ¹H-NMR (CDCl₃): 1.1 (t, 3H, CH₃–CH₂, 3JHH=7.2 Hz), 2.1–2.2 (m, 2H, 3CH₂ cyclopentane), 2.4 (s, 3H, CH₃–C=N), 2.7 (s, 3H, CH₃–N), 2.9 (q, 2H, CH₂–CH₃, 3JHH=7.2 Hz), 3.1–3.2 (m, 4H, 2CH₂ cyclo), 3.7 (s, 2H, NH₂, D₂O exchangeable), 7.6–7.8 (2d, 4H, Ar–H, AB system, 3JHH=7.7 Hz), 8.3 (s, 1H, NH, D₂O exchangeable). ¹³C-NMR (CDCl₃): 16.6, 17.1, 20.1, 25.9, 28.7, 30.2, 40.4, 125.9 (2), 129.3 (2), 134.2, 136.1, 139.7, 141.6, 143.1, 41.2, 43.3, 170.7. MS, m/z (%): 420 (M⁺) (0.2), 264 (100), Anal. Calcd for chemical formula: C₁₉H₂₄N₄O₃S₂: C, 54.26; H, 5.75; N, 13.32; S, 4.25. Found: C, 54.29; H, 5.72; N, 13.33; S, 4.23.

4-(1-(2-(3-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carbonyl)hydrazono)ethyl)-N-ethyl-N-methylbenzenesulfonamide (6)

Yield, 73%; brown powder; m.p. 232–234 °C; IR (KBr, cm¹): 3363, 3350, 3309 (NH, NH₂), 3086 (CH arom.), 2974, 2935 (CH aliph.), 1685 (C=O), 1624 (C=N), 1334, 1161 (SO₂). ¹H-NMR (DMSO-d₆): 1.1 (t, 3H, CH₃–CH₂, 3JHH=7.2 Hz), 2.1–2.2 (m, 2H, 3CH₂ cyclopentane), 2.4 (s, 3H, CH₃–C=N), 2.7 (s, 3H, CH₃–N), 2.9 (q, 2H, CH₂–CH₃, 3JHH=7.2 Hz), 3.1–3.2 (m, 6H, 3CH₂ cyclo), 3.7 (s, 2H, NH₂, D₂O exchangeable), 7.9–8.1 (2d, 4H, Ar–H, AB system, 3JHH=7.8 Hz), 8.3 (s, 1H, NH, D₂O exchangeable). ¹³C-NMR (DMSO-d₆): 16.7, 17.1, 21.1, 23.4 (2), 24.9, 30.5, 40.4, 127.9 (2), 129.3 (2), 134.2, 136.1, 139.7, 141.6, 143.1, 41.2, 43.2, 170.5. MS, m/z (%): 434 (M⁺) (0.2), 77 (100), Anal. Calcd for C₂₀H₂₆N₄O₃S₂ (434.14): C, 55.28; H, 6.03; N, 12.89; S, 14.76. Found: C, 55.24; H, 6.09; N, 12.86; S, 14.73.

4-(1-(2-(4-Amino-3-phenyl-2-thioxo-2, 3-dihydrothiazole-5-carbonyl)hydrazono)ethyl)-N-ethyl-N-methylbenzenesulfonamide (7)

A mixture of 2 (0.5 g, 0.004 mol) elemental sulfur (0.05 g, 0.004 mol) and phenylisothiocyanate (0.21 g, 0.004 mol) in absolute ethanol (20 mL) was refluxed for 5 h. The reaction mixture was cooled and the obtained solid was recrystallized from acetic acid to give 7; Yield, 68%; yellowish brown powder; m.p. 239–241 °C; IR (KBr, cm¹): 3213, 3196, 3161 (NH, NH₂), 3086 (CH arom.), 2974, 2879 (CH aliph.), 1685 (C=O), 489 (C=N), 1379, 1161 (SO₂), 1201 (C = S). ¹H-NMR (DMSO-d₆): 1.1 (t, 3H, CH₃–CH₂, 3JHH=7.2 Hz), 2.4 (s, 3H, CH₃–C=N), 2.7 (s, 3H, CH₃–N), 2.9 (q, 2H, CH₂–CH₃, 3JHH=7.2 Hz), 5.1 (s, 2H, NH₂, D₂O exchangeable), 7.2–7.5 (m, 5H, Ar–H), 7.8–8.0 (2d, 4H, Ar–H, AB system, 3JHH=7.8 Hz), 11.0 (s, 1H, NH, D₂O exchangeable). ¹³C-NMR (DMSO-d₆): 16.6, 17.2, 37.6, 48.8, 70.8, 127.4 (2), 128.0 (2), 128.4, 129.4 (2), 129.7 (2), 133.9, 140.7, 142.0, 147.2, 49.1, 168.4, 184.2. MS, m/z (%): 489 (M⁺) (0.2), 307 (100), Anal. Calcd for C₂₁H₂₃N₅O₃S₃: C, 51.51; H, 4.73; N, 14.30; S, 19.65. Found: C, 51.53; H, 4.71; N, 14.32; S, 19.66.

General procedure for preparation of coumarin and benzocoumarin derivatives 8 and 9

Equimolecular mixture of 2 (0.5 g, 0.004 mol) and either 2-hydroxybenzaldehyde (0.19 g, 0.004 mol) or 2-hydroxy-1-naphthaldehyde (0.26 g, 0.004 mol) in dioxane (30 mL) containing piperidine (0.5 mL) was refluxed for 3 h. The reaction mixture was poured onto ice/water and the obtained solid was recrystallized from dioxane containing few drops of HCl to give 8 or 9, respectively.

N-ethyl-N-methyl-4-(1-(2-(2-oxo-2H-chromene-3-carbonyl)hydrazono)ethyl)benzene sulfonamide (8)

Yield, 81%; light brown powder; m.p. 235–237 °C; IR (KBr, cm⁻¹): 3308 (NH), 3063 (CH arom.), 2972, 2879 (CH aliph.), 1680, 1672 (2C=O), 1608 (C=N), 1336, 147 (SO₂). ¹H-NMR (CDCl₃): 1.2 (t, 3H, CH₃–CH₂, 3JHH=7.4 Hz), 2.4 (s, 3H, CH₃–C=N), 2.8 (s, 3H, CH₃–N), 3.1 (q, 2H, CH₂–CH₃, 3JHH=7.4 Hz), 7.0–8.0 (m, 8H, Ar–H), 8.7 (s, 1H, CH chromene), 13.5 (s, 1H, NH, D₂O exchangeable). ¹³C-NMR (CDCl₃): 13.1, 4.1, 33.9, 45.1, 14.5, 118.7, 119.8, 124.5, 127.3, 127.5 (2), 129.8, 133.3 (2), 138.1, 142.1, 143.4, 41.8, 43.8, 48.0, 49.2. MS, m/z (%): 427 (M⁺) (1.4), 133 (100), Anal. Calcd for C₂₁H₂₁N₃O₅S: C, 59.00; H, 4.95; N, 9.83; S, 7.50. Found: C, 59.05; H, 4.98; N, 9.80; S, 7.53.

N-ethyl-N-methyl-4-(1-(2-(3-oxo-3H-benzo[f]chromene-2-carbonyl)hydrazono)ethyl)benzene sulfonamide (9)

Yield, 83%; brown powder; m.p. 252–254 °C; IR (KBr, cm¹): 3301 (NH), 3066 (CH arom.), 2974, 2877 (CH aliph.), 1683, 1676 (2C=O), 468 (C=N), 1398, 147 (SO₂). ¹H-NMR (CDCl₃): 1.1 (t, 3H, CH₃–CH₂, 3JHH=7.2 Hz), 2.4 (s, 3H, CH₃–C=N), 2.7 (s, 3H, CH₃–N), 3.1 (q, 2H, CH₂–CH₃, 3JHH=7.2 Hz), 7.3–8.4 (m, 10H, Ar–H), 9.4 (s, 1H, CH chromene), 13.6 (s,1H, NH, D₂O exchangeable). ¹³C-NMR (CDCl₃): 13.1, 4.0, 33.9, 44.9, 112.8, 14.6, 118.7, 122.0, 126.1 (2), 127.4, 127.6, 128.8, 128.9, 129.8 (2), 130.2, 134.6, 138.3, 139.1, 142.1, 41.5, 43.7, 48.1, 49.4. MS, m/z (%): 477 (M⁺) (0.3), 133 (100), Anal. Calcd for C₂₅H₂₃N₃O₅S: C, 62.88; H, 4.85; N, 8.80; S, 6.71. Found: C, 62.85; H, 4.87; N, 8.75; S, 6.73.

4-(1-((3-Cyano-2, 4, 6-trioxopiperidin-1-yl)imino)ethyl)-N-ethyl-N-methylbenzene sulfonamide (10)

To a mixture of 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzene sulfonamide (2) (0.5 g, 0.004 mol), diethyl malonate (0.24 g, 0.004 mol) and 0.5 mL of triethylamine was refluxed in 30 mL of 1,4-dioxane for 5 h and then left to cool. The solid product formed was collected by filtration and recrystallized from ethanol to give 10. Yield, 81%; white powder; m.p. 244–246 °C; IR (KBr, cm⁻¹): 3085 (CH arom.), 2972, 2877 (CH aliph.), 2264 (C≡N), 1685, 1692, 1656 (3C=O), 1624 (C=N), 1334, 1161 (SO₂). ¹H-NMR (DMSO-d₆): 1.22 (t, 3H, CH₃–CH₂, 3JHH=7.3 Hz), 2.5 (s, 3H, CH₃), 2.6 (s, 3H, N–CH₃), 3.23 (q, 2H, CH₂–CH₃, 3JHH=7.3 Hz), 3.6 (s, 2H, CH₂), 4.4 (s, H, CH), 7.6–7.9 (2d, 4H, Ar–H, AB system, 3JHH=7.5 Hz). ¹³C-NMR (DMSO-d₆): 16.8, 17.7, 32.3, 36.2, 40.4, 50.9, 114.9, 125.6 (2), 129.3 (2), 134.4, 141.6, 45.6, 166.0, 170.7, 206.0; MS, m/z (%): 390 (M⁺) (0.1), 307 (100), Anal. Calcd for C₁₇H₁₈N₄O₅S: C, 52.30; H, 4.65; N, 14.35; S, 8.21, found: C, 52.28; H, 4.66; N, 14.38; S, 8.20.

4-(1-((5-Amino-3-cyano-2-oxopyrrolidin-1-yl)imino)ethyl)-N-ethyl-N-ethylbenzene sulfonamide (11)

To a mixture of 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzene sulfonamide (2) (0.5 g, 0.004 mol), 2-chloroacetonitrile (0.12 g, 0.004 mol) and 0.5 mL of triethylamine was refluxed in 30 mL of 1,4-dioxane for 5 h. then left to cool. The solid product formed was collected by filtration and recrystallized from ethanol to give 11. Yield, 77%; light brown powder; m.p. 224–226 °C; IR (KBr, cm⁻¹): 3316, 3207 (NH₂), 3086 (CH arom.), 2942, 2827 (CH aliph.), 2264 (C≡N), 1683 (C=O), 1618 (C=N), 1336, 1161 (SO₂). ¹H-NMR (DMSO-d₆): 1.22 (t, 3H, CH₃–CH₂, 3JHH=7.4 Hz), 2.3 (m, H_a, CH_aH_b), 2.4 (s, 3H, CH₃), 2.5 (m, H_b, CH_aH_b), 2.6 (s, 3H, N–CH₃), 3.2 (m, H, CH), 3.3 (q, 2H, CH₂–CH₃, 3JHH=7.4 Hz), 4.5 (m, H, CH), 7.7–8.1 (2d, 4H, Ar–H, AB system, 3JHH=7.8 Hz). ¹³C-NMR (DMSO-d₆): 14.5, 17.7, 28.1, 35.7, 36.5, 41.4, 64.3, 116.8, 127.4 (2), 129.5 (2), 140.7, 142.1, 170.1, 175.6; MS, m/z (%): 363 (M⁺) (0.1), 264 (100%), Anal. Calcd for C₁₆H₂₁N₅O₃S: C, 52.88; H, 5.82; N, 19.27; S, 8.82, found: C, 52.85; H, 5.80; N, 19.24; S, 8.83.

Ethyl-5-cyano-4-(4-(dimethylamino)phenyl)-1-((1–(4-(N-ethyl-N-methylsulfamoyl)phenyl) ethylidene)amino)-2,6-dioxo-1,2,3,6-tetrahydropyridine-3-carboxylate (12)

In 30 mL of 1,4-dioxane, a mixture of 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzene sulfonamide (2) (0.5 g, 0.004 mol), diethyl-2–(4-(dimethylamino) benzylidene)malonate (0.44 g, 0.004 mol) and 0.5 mL of triethylamine was refluxed for 5 h and then left to cool. The solid product formed was collected by filtration and recrystallized from ethanol to give 12. Yield, 85%; yellow powder; m.p. 243–245 °C; IR (KBr, cm⁻¹): 3109 (CH arom) 2942, 2827 (CH aliph.), 2264 (C≡N), 1685, 1676, 1672 (C=O), 1624 (C=N), 1334, 1161 (SO₂). ¹H-NMR (DMSO-d₆): 1.17 (t, 3H, CH₃–CH₂-N, 3JHH=7.3 Hz), 1.29 (t, 3H, CH₃–CH₂–O, 3JHH=7.1 Hz), 2.32 (s, 3H, CH₃–C=N), 2.42 (s, 3H, CH₃–C=C), 2.68 (s, 3H, CH₃–N), 3.06 (s, 6H, (CH₃)₂N), 3.23 (q, 2H, CH₂–CH₃, 3JHH=7.3 Hz), 3.95 (s, H, O = C–CH–C=O), 4.21 (q, 2H, CH₂–CH₃, 3JHH=7.1 Hz), 6.92–7.20 (2d, 4H, Ar–H, AB system, 3JHH=7.8 Hz), 7.91–8.02 (2d, 4H, Ar–H, AB system, 3JHH=7.9 Hz). ¹³C-NMR (DMSO-d₆): 14.1, 16.8, 17.5, 36.3, 41.1 (2), 41.3, 51.9, 61.4, 95.3, 111.7 (2), 14.8, 127.4 (2), 128.9, 129.5 (2), 129.9 (2), 140.7, 142.1, 40.3, 162.7, 163.4, 165.2, 166.8, 170.7; MS, m/z (%): 565 (M⁺) (0.2), 133 (100), Anal. Calcd for C₂₈H₃₁N₅O₆S: C, 59.45; H, 5.52; N, 12.38; S, 5.67, found: C, 59.47; H, 5.50; N, 12.35; S, 5.69.

4-(1-((6-Amino-3-cyano-4-(4-(dimethylamino)phenyl)-2-oxopyridin-1(2H)-yl)imino)ethyl)-N-ethyl-N-methylbenzenesulfonamide (13)

To a mixture of 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzene sulfonamide (2) (0.5 g, 0.004 mol), 3-(4-(dimethylamino)phenyl)oxirane-2-carbonitrile (0.29 g, 0.004 mol) and 0.5 mL of triethylamine was refluxed in 30 mL of 1,4-dioxane for 6 h and then left to cool. The solid product formed was collected by filtration and recrystallized from ethanol to give 13. Yield, 76%; yellow powder; m.p. 237–239 °C; IR (KBr, cm⁻¹):3124 (CH arom), 2942, 2827 (CH aliph.), 2264 (C≡N), 1687 (C=O), 1624 (C=N), 1336, 1161 (SO₂).¹H-NMR (DMSO-d₆): 1.17 (t, 3H, CH₃–CH₂–N, 3JHH=7.2 Hz), 2.32 (s, 3H, CH₃–C=N), 2.68 (s, 3H, CH₃–N), 3.08 (s, 6H, (CH₃)₂N), 3.23 (q, 2H, CH₂–CH₃, 3JHH=7.2 Hz), 4.51 (s, H, Ar–H), 6.5 (s, 2H, NH₂, D₂O exchangeable), 6.92–7.20 (2d, 4H, Ar–H, AB system, 3JHH=7.5 Hz), 7.91–8.02 (2d, 4H, Ar–H, AB system, 3JHH=8.7 Hz). ¹³C-NMR (DMSO-d₆): 16.9, 17.9, 36.1, 41.3 (2), 41.5, 83.6, 94.5, 111.7 (2), 14.2, 127.4 (2), 128.9, 129.5 (2), 129.6 (2), 140.7, 142.1, 145.3, 40.3, 162.9, 169.8, 170.1; MS, m/z (%):492 (M⁺) (2.8), 208 (100), Anal. Calcd for C₂₅H₂₈N₆O₃S: C, 60.96; H, 5.73; N, 17.06; S, 6.51, found: C, 60.91; H, 5.79; N, 17.01; S, 6.48.

4-(1-(2-(5-Amino-4H-pyrazol-3-yl)hydrazono)ethyl)-N-ethyl-N-methylbenzenesulfonamide (14)

To a mixture of 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzene sulfonamide (2) (0.5 g, 0.004 mol), hydrazine hydrate (0.05 g, 0.004 mol) and 0.5 mL of triethylamine was refluxed in 30 mL of 1,4-dioxane for 6 h and then left to cool. The solid product formed was collected by filtration and recrystallized from ethanol to give 14. Yield, 65%; white powder; m.p. 228–230 °C; IR (KBr, cm⁻¹): 3406, 3365, 3194 (NH, 2NH₂),3109 (CH arom), 2974, 2875 (CH aliph.), 1624 (C=N), 1334, 1161 (SO₂).¹H-NMR (DMSO-d₆): 1.22 (t, 3H, CH₃–CH₂, 3JHH=7.4 Hz), 1.42 (s, 2H, CH₂), 2.32 (s, 3H, CH₃–C=N), 2.66 (s, 3H, CH₃–N), 3.23 (q, 2H, CH₂–CH₃, 3JHH=7.4 Hz), 6.5 (s, 2H, NH₂, D₂O exchangeable), 7.91–8.02 (2d, 4H, Ar–H, AB system, 3JHH=7.3 Hz), 11.22 (s, 1H, NH) discharged with D₂O. ¹³C-NMR (DMSO-d₆): 16.8, 17.7, 32.3, 36.1, 41.4, 127.8 (2), 128.8 (2), 140.4, 142.1, 147.7, 166.1, 166.5; MS, m/z (%): 336 (M⁺) (0.4), 307 (100), Anal. Calcd for C₁₄H₂₀N₆O₂S: C, 49.98; H, 5.99; N, 24.98; S, 9.53, found: C, 49.95; H, 5.97; N, 24.96; S, 9.55.

4-(1-(2-(2-Cyano-3-phenylbut-2-enoyl)hydrazono)ethyl)-N-ethyl-N-methylbenzene sulfonamide (15)

In 30 mL of 1,4-dioxane, a mixture of 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzene sulfonamide (2) (0.5 g, 0.004 mol), acetophenone (0.18 g, 0.004 mol) and 0.5 mL of triethylamine was refluxed for 5 h and then left to cool. The solid product formed was collected by filtration and recrystallized from ethanol to give 15. Yield, 68%; white powder; m.p. 238–240 °C; IR (KBr, cm⁻¹): 3406, 3317, 3207 (NH, 2NH₂), 2942, 2827 (CH aliph.), 2206 (C≡N), 1651 (C=O), 1618 (C=N), 1396, 145 (SO₂).¹H-NMR (DMSO-d₆): 1.17 (t, 3H, CH₃–CH₂, 3JHH=7.3 Hz), 2.32 (s, 3H, CH₃–C=N), 2.42 (s, 3H, CH₃–C=C), 2.66 (s, 3H, CH₃–N), 3.23 (q, 2H, CH₂–CH₃, 3JHH=7.3 Hz), 6.92–7.02 (m, 5H, Ar–H), 7.91–8.02 (2d, 4H, Ar–H, AB system, 3JHH=7.4 Hz), 11.13 (s, 1H, NH) discharged with D₂O. ¹³C-NMR (DMSO-d₆): 14.3, 4.1, 16.7, 36.5, 41.3, 95.3, 14.8, 126.4 (2), 127.5 (2), 127.9, 128.6 (2), 129.5 (2), 139.4, 140.1, 142.3, 147.7, 168.5, 174.1; MS, m/z (%): 424 (M⁺) (0.3), 133 (100), Anal. Calcd for C₂₂H₂₄N₄O₃S: C, 62.24; H, 5.70; N, 13.20; S, 7.55, found: C, 62.25; H, 5.68; N, 13.22; S, 7.53.

In-vitro anticancer screening

In this study, human tumor breast cancer cell line (MCF-7) was used. The cytotoxic activity was measured in-vitro for the newly synthesized compounds using the Sulforhodamine B stain (SRB) assay using the method of Skehan et al.31. Cells were plated in 96-multiwell plate (104 cells/well) for 24 h before treatment with the compound(s) to allow attachment of cell to the wall of the plate. Tested compounds were dissolved in dimethyl sulfoxide.

Different concentrations of the compound under test (0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 μg/mL) were added to the cell monolayer. Triplicate wells were prepared for each individual concentration. Monolayer cells were incubated with the compound(s) for 48 h. at 37 °C and in atmosphere of 5% CO₂. After 48 h, cells were fixed, washed and stained for 30 min with 0.4% (W/V) SRB dissolved in 1% acetic acid. Excess unbound dye was removed by four washes with 1% acetic acid and attached stain was recovered with Tris-EDTA buffer.

Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration is plotted to get the survival curve for breast tumor cell line after the specified time32. The molar concentration required for 50% inhibition of cell viability (IC₅₀) was calculated and compared to the reference drug methotrexate (MTX). The surviving fractions were expressed as means standard error and the results are given in (Tables 1–3).

Table 1. In vitro anticancer screening of the synthesized compounds against human breast cell line (MCF-7).

	Compound concentration (μg/mL)
Cpd No	0.39	0.78	1.56	3.125	6.25	12.5	25	50	IC50*(μg/mL)
2	100 ± 0.0	100 ± 0.0	98.58 ± 0.6	94.43 ± 0.8	87.69 ± 1.5	76.14 ± 4.3	32.19 ± 1.9	14.48 ± 1.1	19.9
3	100 ± 0.0	100 ± 0.0	97.41 ± 0.6	91.38 ± 1.1	85.65 ± 1.5	62.19 ± 4.2	23.61 ± 1.7	10.24 ± 0.4	16.4
4	100 ± 0.0	100 ± 0.0	93.53 ± 0.6	87.82 ± 1.5	70.19 ± 4.6	58.65 ± 3.4	18.27 ± 1.5	7.43 ± 03	4.2
5	100 ± 0.0	100 ± 0.0	100 ± 0.0	98.43 ± 0.6	92.05 ± 0.8	83.17 ± 1.5	35.42 ± 2.7	26.81 ± 1.4	21.2
6	100 ± 0.0	100 ± 0.0	90.31 ± 0.9	79.54 ± 1.5	62.48 ± 3.7	24.96 ± 1.9	11.72 ± 1.0	5.87 ± 0.2	8.33
7	100 ± 0.0	100 ± 0.0	94.91 ± 0.8	89.12 ± 1.2	67.65 ± 4.6	49.18 ± 3.3	28.96 ± 1.7	11.23 ± 0.7	12.2
8	64.4 ± 4.6	56.82 ± 3.9	29.35 ± 2.8	26.84 ± 1.7	14.61 ± 1.1	9.45 ± 1.0	7.38 ± 0.3	3.97 ± 0.2	1.08
9	100 ± 0.0	100 ± 0.0	98.36 ± 0.6	94.65 ± 0.8	86.82 ± 1.1	75.23 ± 1.5	37.38 ± 1.9	10.89 ± 1.0	20.8
10	100 ± 0.0	100 ± 0.0	98.11 ± 0.6	94.76 ± 0.8	88.18 ± 1.2	67.82 ± 4.5	16.27 ± 1.2	7.19 ± 0.5	16.8
11	100 ± 0.0	100 ± 0.0	100 ± 0.0	97.82 ± 0.6	91.54 ± 0.9	78.71 ± 1.5	43.68 ± 3.2	27.92 ± 1.8	22.7
12	100 ± 0.0	100 ± 0.0	94.25 ± 0.7	87.42 ± 1.1	78.36 ± 1.5	61.59 ± 4.6	18.37 ± 1.5	10.94 ± 0.6	4.9
13	100 ± 0.0	100 ± 0.0	98.97 ± 0.6	96.28 ± 0.7	89.31 ± 1.1	77.14 ± 1.5	46.55 ± 3.2	28.72 ± 1.7	23.6
14	100 ± 0.0	100 ± 0.0	98.44 ± 0.6	92.85 ± 0.9	84.93 ± 1.5	68.72 ± 4.6	32.89 ± 1.8	20.64 ± 1.2	19
15	100 ± 0.0	100 ± 0.0	100 ± 0.0	97.89 ± 0.6	91.37 ± 1.1	79.55 ± 1.5	27.18 ± 1.7	14.67 ± 1.1	19.6
MTX	100 ± 0.0	100 ± 0.0	95.32 ± 0.6	89.31 ± 0.8	67.70 ± 0.6	50.12 ± 1.3	29.71 ± 1.5	11.56 ± 0.6	12.3

*Mean of three results obtained from three experiments.

IC₅₀ value: concentration causing 50% inhibition of cell viability.

Table 2. In vitro cytotoxicity screening of the synthesized compounds against normal fibroblasts of baby hamster kidney cell line (BHK).

	Compound concentration (μg/mL)
Cpd No	1.56	3.125	6.25	12.5	25	50	100	200	CC50*(μg/mL)
2	100 ± 0.0	98.13 ± 0.6	94.82 ± 0.8	90.76 ± 1.1	86.38 ± 1.4	71.89 ± 1.5	63.24 ± 3.3	41.06 ± 3.1	160
3	100 ± 0.0	100 ± 0.0	98.71 ± 0.6	94.32 ± 0.9	87.51 ± 1.1	71.24 ± 1.5	52.98 ± 3.8	34.86 ± 1.9	116
4	100 ± 0.0	99.16 ± 0.0	97.24 ± 0.6	91.43 ± 1.2	82.16 ± 1.5	68.74 ± 4.6	50.31 ± 4.1	31.48 ± 2.7	102
5	100 ± 0.0	100 ± 0.0	98.95 ± 0.6	97.83 ± 0.8	91.42 ± 0.9	82.39 ± 1.3	64.95 ± 4.6	48.72 ± 3.1	192
6	98.62 ± 0.6	96.21 ± 0.8	92.87 ± 1.1	81.44 ± 1.4	70.36 ± 4.6	49.21 ± 3.3	37.49 ± 2.7	23.75 ± 1.9	49.1
7	100 ± 0.0	98.74 ± 0.6	95.20 ± 0.9	88.46 ± 1.2	79.21 ± 1.5	65.23 ± 4.6	46.89 ± 3.9	30.46 ± 1.8	91.5
8	96.97 ± 0.7	94.32 ± 0.9	87.21 ± 1.1	78.14 ± 1.4	69.28 ± 4.5	54.13 ± 3.9	39.42 ± 2.8	26.89 ± 1.7	64
9	100 ± 0.0	100 ± 0.0	98.93 ± 0.6	96.12 ± 0.8	87.53 ± 1.1	73.84 ± 1.5	48.62 ± 3.6	31.46 ± 1.9	97.3
10	100 ± 0.0	99.84 ± 0.0	95.86 ± 0.9	90.43 ± 1.3	78.92 ± 1.5	51.38 ± 4.1	39.24 ± 2.7	20.98 ± 1.5	55.7
11	100 ± 0.0	100 ± 0.0	100 ± 0.0	99.27 ± 0.0	94.18 ± 0.9	87.24 ± 1.3	71.43 ± 1.5	53.89 ± 3.9	>200
12	100 ± 0.0	100 ± 0.0	97.13 ± 0.6	90.28 ± 1.1	78.34 ± 1.5	65.26 ± 4.4	44.19 ± 3.2	26.86 ± 1.7	86.2
13	100 ± 0.0	100 ± 0.0	100 ± 0.0	97.28 ± 0.6	91.43 ± 0.9	86.22 ± 1.1	78.19 ± 1.5	60.38 ± 4.6	>200
14	100 ± 0.0	100 ± 0.0	100 ± 0.0	98.29 ± 0.6	90.76 ± 0.1	78.42 ± 1.5	60.87 ± 4.2	41.23 ± 3.1	45
15	100 ± 0.0	99.17 ± 0.0	95.22 ± 0.7	86.91 ± 1.1	74.86 ± 1.5	60.98 ± 4.1	47.51 ± 3.7	36.17 ± 1.9	90.8
MTX	100 ± 0.0	100 ± 0.0	99.10 ± 0.9	93.75 ± 1.2	83.92 ± 1.5	70.14 ± 1.3	52.41 ± 1.5	33.21 ± 1.5	105.2

*Mean of three results obtained from three experiments.

CC₅₀ value: concentration required to inhibit cell proliferation by 50%.

Table 3. CC₅₀, IC₅₀ (in μg/mL and μM) and selectivity index SI of the synthesized compounds against breast carcinoma cell line (MCF-7).

Cpd No	CC50* (μg/mL)	CC50* (μM)	IC50* (μg/mL)	IC50* (μM)	SI
2	160	496.89	19.9	61.8	8.04
3	116	276.19	16.4	39.05	7.07
4	102	218.42	4.2	32.55	6.71
5	192	457.14	21.2	50.48	9.06
6	49.1	113.13	8.33	19.19	5.89
7	91.5	187.12	12.2	24.95	7.5
8	64	149.88	1.08	2.53	59.26
9	97.3	203.98	20.8	43.61	4.68
10	55.7	142.82	16.8	43.08	3.32
11	>200	550.96	22.7	62.53	8.81
12	86.2	42.57	4.9	28.14	5.42
13	>200	406.5	23.6	47.97	8.47
14	45	461.31	19	56.55	8.16
15	90.8	214.4	19.6	46.23	4.63
MTX	105.2	231.72	12.3	27.09	8.55

*Mean of three results obtained from three experiments.

CC₅₀ value: concentration required to inhibit cell proliferation by 50%.

IC₅₀ value: concentration causing 50% inhibition of cell viability.

SI = CC₅₀/IC₅₀ selectivity index.

Cytotoxicity activity

Cell toxicity was monitored by determining the effect of the new sulfonamides on cell morphology and cell viability32. The cytotoxicity assay was monitored on normal fibroblasts of baby hamster kidney (BHK) cell line using 0.1 mL of cell suspension, involving 10 000 cells seeded in each well of a 96-well microtitre plate (Falcon, NJ). Fresh maintenance medium containing different dilutions of the test sample was added after 24 h of seeding. Control cells were incubated without a test sample. The microtitre plates were incubated at 37 °C in a humidified incubator with 5% CO₂ for a period of 48 h. Six wells were used for each concentration of the test sample. After incubation, the culture supernatant was replaced by fresh medium. The cells in each well were then stained and analyzed as described in the antitumor section. The absorbance was detected at 490 nm using a microplate ELISA reader (Sunrise TECAN Inc., Männedorf, Switzerland). The absorbance of untreated cells was considered as 100%. The cell cytotoxic effect (CC₅₀) of each tested compound was calculated using the following formula: percentage of cell cytotoxic effect = [1-(ODt/ODc)] × 100%, where ODt and ODc indicate the absorbance of the test substance and cell control; respectively. The cytotoxic activity against normal fibroblasts of Baby Hamster Kidney (BHK) cells was detected under these experimental conditions with 50% cell cytotoxic concentration (CC₅₀).

Antimicrobial activity

The antibacterial and antifungal activity assays were carried out by using the diffusion plate method33–35. A bottomless cylinder containing a measured quantity (1 mL, mg/mL) of the sample is placed on a plate (9 cm diameter) containing a solid bacterial medium (nutrient agar broth) or fungal medium, which has been heavily seeded with a spore suspension of the test organism. After incubation (24 h for bacteria and 5 days for fungi), the diameter of the clear zone of inhibition surrounding the sample is taken as measure of the inhibitory power of the sample against the particular test organism. The solvent used was DMSO and the concentration of the sample used is 100 μg/mL. The results of antimicrobial activity are summarized in Tables 4 and 5. All the synthesized compounds were evaluated for their antibacterial activity against Gram-positive (Bacillus subtilis, Streptococcus pneumoniae and Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) and fungal strains Aspergillus fumigatus and Candida albicans organisms by diffusion technique33–35.

Table 4. Anti-fungal activity of synthesized compounds*.

	Microorganisms
Cpd No	Aspergillus fumigatus(RCMB 02568)	Candida albicans(RCMB 05036)
2	NA†	NA
3	NA	NA
4	18.3 ± 0.25	NA
5	4.6 ± 0.10	NA
6	NA	NA
7	20.3 ± 0.24	NA
8	17.6 ± 0.58	NA
9	NA	NA
10	17.9 ± 0.36	NA
11	NA	NA
12	20.2 ± 0.20	NA
13	NA	NA
14	17.4 ± 0.16	NA
15	17.6 ± 0.58	NA
Amphotericin B	23.7 ± 0.10	21.9 ± 0.12

*Mean zone of inhibition in mm ± standard deviation (SD); mm: the unit of measurement of inhibition zone (IZ); †NA: No activity. The test was performed using the diffusion agar technique.

Table 5. Anti-bacterial activity of synthesized compounds*.

	Microorganisms
	Gram-positive bacteria			Gram-negative bacteria
Cpd. No	Staphylococcus aureus(RCMB 010027)	Streptococcus pneumoniae(RCMB 010010)	Bacillis subtilis(RCMB 010067)	Pseudomonas aeruginosa(RCMB 010043)	Klebsiella pneumoniae(RCMB 010093)	Escherichia coli(RCMB 010052)
2	NA†	NA	NA	NA	NA	NA
3	NA	NA	NA	NA	NA	NA
4	16.2 ± 0.34	18.3 ± 0.43	20.3 ± 0.53	NA	20.3 ± 0.25	17.8 ± 0.43
5	12.6 ± 0.18	14.8 ± 0.16	16.4 ± 0.12	NA	18.1 ± 0.4	4.2 ± 0.30
6	NA	NA	NA	NA	NA	NA
7	18.7 ± 0.62	19.5 ± 0.42	20.2 ± 0.29	NA	20.9 ± 0.28	19.3 ± 0.13
8	4.4 ± 0.25	16.2 ± 0.4	19.8 ± 0.42	NA	17.4 ± 0.53	4.0 ± 0.43
9	NA	NA	NA	NA	NA	NA
10	4.9 ± 0.44	18.1 ± 0.63	20.0 ± 0.32	NA	NA	13.9 ± 0.46
11	NA	NA	NA	NA	NA	NA
12	19.3 ± 0.16	20.6 ± 0.23	22.8 ± 0.18	NA	21.2 ± 0.29	19.4 ± 0.10
13	NA	NA	NA	NA	NA	NA
14	4.2 ± 0.34	16.1 ± 0.31	18.1 ± 0.25	NA	19.6 ± 0.31	17.5 ± 0.22
15	4.4 ± 0.25	17.7 ± 0.43	19.3 ± 0.53	NA	13.7 ± 0.25	NA
Ampicillin	28.9 ± 0.14	25.3 ± 0.58	26.4 ± 0.34	–	–	–
Gentamicin	–	–	–	26.3 ± 0.4	17.3 ± 0.12	27.3 ± 0.44

*Mean zone of inhibition in mm ± standard deviation (SD); mm: the unit of measurement of inhibition zone (IZ); †NA: No activity. The test was performed using the diffusion agar technique.

Docking and molecular modeling calculations

Materials

All the molecular studies were carried out using Molecular Operating Environment (MOE 2005.06; Chemical Computing Group, Montreal, Canada) as the computational software. All the minimizations were performed with MOE until a RMSD gradient of 0.05 K Cal/mol Å with MMFF94X force field and the partial charges were automatically calculated.

General methodology

The coordinates of the X-ray crystal structure of methotrexate (MTX) bound to dihydrofolate reductase (DHFR) enzyme (PDB ID: 4DFR) were obtained from Protein Data Bank (PDB ID: 1BID). Enzyme structures were checked for missing atoms, bonds and contacts. Hydrogen atoms were added to the enzyme structure. Water molecules and bound ligands were manually deleted. The ligand molecules were constructed using the builder molecule and were energy minimized.

The active site was generated using the MOE-Alpha site finder. Dummy atoms were created from the obtained alpha spheres. Ligands were docked within the dihydrofolate reductase active sites using the MOE-Dock with simulated annealing used as the search protocol and MMFF94X molecular mechanics force field for 8000 interactions. The lowest energy conformation was selected and subjected to an energy minimization using MMFF94X force field. The values of docking results refer to score: for all scoring functions, lower scores indicate more poses that are favorable. The unit for all scoring functions is kcal/mol. E-conf: the energy of the conformer. If there is a refinement stage, this is the energy calculated at the end of the refinement. E-place: score from the placement stage (Placement. A collection of poses is generated from the pool of ligand conformations using one of the placement methods). E-score 1: score from the first rescoring stage. E-score 2: score from the second rescoring stage. E-refine: score from the refinement stage (refinement: energy minimization of the system is carried out using the conventional molecular mechanics setup).

Docking on the active site of dihydrofolate reductase (DHFR)

The recent determination of the three dimensional co-crystal structure of dihydrofolate reductase complexed with the potent inhibitor, methotrexate (MTX) (PDB ID: 4DFR) has led to the development of a model for the topography of the binding site of dihydrofolate reductase.

Results and discussion

Chemistry

The starting material 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methyl benzenesulfonamide (2) was prepared via the condensation reaction between 2-cyanoacetohydrazide and 4-acetyl-N,N-ethylmethylbenzene sulfonamide (1) in refluxing dioxane (Scheme 1)36. The structure of compound 2 was proved on the basis of analytical and spectral data. Thus, IR spectrum of compound 2 showed absorption band at ν = 3310 cm⁻¹, 2262, 1688 cm⁻¹ due to NH, cyano and carbonyl groups, respectively, and 1330, 1160 cm⁻¹ for sulfonyl group. ¹H NMR spectrum revealed the presence of a triplet signal at δ = 1.09 ppm corresponding to methyl of ethyl group (CH₃–CH₂), a singlet signal at 2.29 ppm corresponding to (CH₃–C=N) group, a singlet signal at 2.67 ppm due to (CH₃-N) group, a quarted signal at δ = 3.04 ppm attributed to (CH₃–CH₂–N) group and a singlet signal at δ = 4.26 ppm for (CH₂–CN) group. Moreover, the ¹³C-NMR data exhibited the presence of signals at δ = 17.73 for (CH₃–C=N), δ = 24.30 for (CH₂–C≡N). Further, elucidation for the structure 2 was obtained through studying its chemical reactivity through some chemical reagents.

Scheme 1. Synthesis of starting material 4-(1-(2-(2-cyanoacetyl) hydrazono)ethyl)-N-ethyl-N-methylbenzenesulfonamide (2).

The reaction of 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzenesulfonamide (2) with either malononitrile or ethylcyanoacetate and elemental sulfur in the presence of trimethylamine gave the corresponding thiophene derivatives 3 and 4, respectively. The reaction goes in parallel to the reported Gewald’s thiophene synthesis37, (Scheme 2). Similarly, the reaction of 2 with either cyclopentanone or cyclohexanone and elemental sulfur yielded the corresponding thiophene derivatives 5 and 6, respectively. Formation of 5 and 6 took place according the similar reported reactions of cyclohexanone with methylene reagents and elemental sulfur38. On the other hand, the reaction of 2 with elemental sulfur and phenyl isothiocyanate afforded the thiazole derivative 7. The formation of the latter product took place in parallel to the reported Hanzesch reported reaction39, (Scheme 2). Structures of compound 3, 4, 5, 6 and 7 were based on analytical and spectral data. The chromene derivatives 8 and 9 were obtained in good yield via reaction of 2 with either salicyldehyde or 2-hydroxy naphthalene-1-carbaldehyde (Scheme 2). The reaction goes in analog with the reported literature40,41. ¹HNMR spectrum of 8 and 9 showed the presence of a singlet at δ 9.0 ppm due to the CH of chromene.

Scheme 2. Synthesis of thiophenes 3, 4, 5 and 6, thiazole 7 and chromenes 8 and 9 derivatives.

The reaction of 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzenesulfonamide (2) with diethyl malonate in the presence of trimethylamine gave the corresponding piperidine derivative 10. In the same conditions, the reaction of starting material 2 with 2-chloroacetonitrile and trimethylamine yielded the corresponding pyrrolidine derivative 11. On the other hand, the pyrazole derivative 14 was obtained by reaction of 2 with hydrazine and trimethylamine. The pyridine 13 and tetrahydropyridine 12 derivatives were obtained by action of 3-(4-(dimethylamino)phenyl)oxirane-2-carbonitrile and diethyl-2-(4-(dimethyl amino)benzylidene)malonate respectively. Finally, the compound 2 react with acetophenone in the presence of trimethylamine gave the corresponding hydrazono derivative 15 as shown in Scheme 3.

Scheme 3. Synthesis of piperidine 10, pyrrolidine 11, tetrahydropyridine 12, pyridine 13, pyrazole 14 and hydrazone 15 derivatives.

In vitro anticancer and in vitro cytotoxicity activity

All the newly synthesized compounds were evaluated for their in vitro cytotoxic activity against human breast cancer cell line MCF-7. The relationship between surviving fraction and drug concentration was plotted to obtain survival curve from which the response parameter IC₅₀ value, corresponding to the concentration required for 50% inhibition of cell viability, was calculated and data are presented in Table 1. Methotrexate (MTX) was used as a positive control in this study for comparative purposes (IC₅₀ = 12.3 μg/mL).

The toxicity of the newly synthesized compounds was evaluated against normal fibroblasts of baby hamster kidney cell line (BHK) by determining the CC₅₀ value that correspond to the concentration required to inhibit cell proliferation by 50%. Data are represented in Table 2.

All the results obtained for the newly synthesized compounds are summarized in Table 3 and the selectivity index calculated using the formula SI = CC₅₀/IC₅₀.

From the obtained results in Tables 1–3, we observe that compound 8 having 2-oxo-2H-chromene moiety with SI value 59.26, 3-amino cyclopenta[b]thiophene 5 with SI value 9.06 and 5-amino-3-cyano-2-oxopyrrolidine 11 with SI value 8.81 showed increased activity when compared to MTX with SI value 8.55, while compounds 13, 14, 2, 7 and 3 with SI values 8.47, 8.16, 8.04, 7.50 and 7.07, respectively, were found to be nearly as active as MTX. While the compounds 4, 6, 12, 9, 15 and 10 with SI values 6.71, 5.89, 5.42, 4.68, 4.63 and 3.32, respectively were less active than methotrexate. Furthermore, in compounds with a common structure portion as compounds 5 and 6, we notice that compound 5 (SI value 9.06) having cyclopentane moiety showing high activity as an anticancer agent compared to compound 6 (SI value 5.89) with cyclohexane moiety. However, compound 8 (SI value 59.26) with 2-oxo-2H-chromene moiety is more active than compound 9 (SI value 4.68) with 3-oxo-3H-benzo[f]chromene moiety. Additionally, in comparing compounds 12 (SI value 5.42) and compound 13 (SI value 8.47), we observed that replacing of carbonyl and ethoxycarbonyl groups in compound 12 with amino group and hydrogen atom respectively in compound 13 leads to increase the anticancer activity. It can be concluded from the aforementioned data that, absence of bulky groups in the newly synthesized compounds 5, 8 and 13 leads to increase the anticancer activity, compared to compounds with more bulky groups 6, 9 and 12. It is clear from the present data that the comparison of the SI for the synthesized compounds against human breast cancer cell line (MCF-7) follows the order 8 > 5 > 11 > MTX > 13 > 14 > 2 > 7 > 3 > 4 > 6 > 12 > 9 > 15 > 10. These preliminary results of biological screening of the tested compounds could offer an encouraging framework in this field that may lead to the discovery of potent anticancer agent.

Anti-bacterial and anti-fungal activity

The antimicrobial activity of all the synthesized compounds were examined against different Gram-positive (Bacillus subtilis, Streptococcus pneumoniae and Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) and fungal strains Aspergillus fumigatus and Candida albicans organisms by measuring zone of inhibition. The results of the antimicrobial activity are shown in Tables 4 and 5.

The varied biological activities of the newly synthesized compounds promoted us to synthesize some new derivatives of these ring systems and study their antimicrobial activities. The antifungal activity studies revealed that: compounds 7 and 12 showed moderate effects against Aspergillus fumigatus, however, these compounds are not active against Candida albicans.

On the other hand, compounds 12 display moderate effects against Staphylococcus aureus. Compounds 12 and 7 gave moderate effects against Streptococcus pneumoniae. Compounds 12, 4, 7 and 10 gave moderate effects against Bacillus subtilis. However, these compounds are not active against Pseudomonas aeruginosa. Compound 12 show moderate effect against Escherichia coli. Compounds 12, 7, 4, 14, 5 and 8 afford strong effects against Klebsiella pneumoniae. All the other compounds showed effects against different types of tested microorganisms ranged from negative effects to moderate effects. So we can say that synthesis of new derivatives of these compounds is still an active area of research. The synthesis and study of the antimicrobial activities of new analogous of these compounds will be helpful for medicinal chemist to focus design of novel chemical entities containing tetrahydropyridine, thiazole, thiophene, pyrazole and chromene derivatives as a part of antimicrobial drugs.

Docking and molecular modeling

Thymidylate synthase and dihydrofolate reductase are among the main targets involved in anticancer and antimicrobial activity42,43. Molecular modeling study using MOE44 module was performed in order to rationalize the observed anticancer activity of the newly synthesized compounds. Molecular docking studies further help in understanding the mode of action of the compounds through their various interactions with the active sites of dihydrofolate reductase.

Analysis of ligand–protein interactions in methotrexate-DHFR X-ray complex

The active site revealed that hydrogen bond interactions beside hydrophobic interactions were considered responsible for the observed affinity as it acts as a hydrogen bond donor to the backbone Ile 5 and Ile 94 residues and the side chain Asp 27 residue. It also acts as a hydrogen bond accepter to Arg 52 and Arg 57 residues. This beside many hydrophobic interactions with various amino acid residues: ILe 5, Ala 6, Ala7, Asp 27, Leu 28, Phe 31, Lys 32, Ser 49, Ile 50, Arg 52, Leu 54, Arg 57, Ile 94, Tyr 100 and Thr 113, as shown in (Figure 1).

Figure 1. Ligand–protein interactions in methotrexate-DHFR X-ray complex A) 3D, B) 2D interactions.

Docking simulation study of the synthesized compounds 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15

MOE docking studies of the inhibitors were performed using dihydrofolate reductase co-crystallized with methotrexate (PDB ID: 4DFR) as a template.

Ligand–protein interactions in compound 2-DHFR X-ray complex

The active site revealed the presence of one hydrogen bond interaction as one oxygen atom of SO₂ moiety acted as a hydrogen bond acceptor with the amino acid residues Thr 56 and Ser 59 (2.99 Å and 3.60 Å, respectively) with a strength of 21.6% and 2.5%, respectively. In addition to hydrophobic interactions with the following amino acid residues: Ile 16, Asp 21, Leu 22, Phe 31, Phe 34, Thr 56, Ser 59, Ile 60, Pro 61, Leu 67, Val 115, Gly 117 and Tyr 121, as shown in Figure 2 (Supporting information).

Ligand–protein interactions in compound 3-DHFR X-ray complex

The active site revealed that several molecular interactions were considered responsible for the observed affinity, as one hydrogen atom of 3-amino group acted as a hydrogen bond donor with the amino acid residue Ser 59 (3.31 Å) with a strength of 1.2%. In addition to hydrophobic interactions involving all methyl groups, one oxygen atom of sulfonyl moiety, oxygen atom of carbonyl group, sulfur atom of thiophene, nitrogen atom of 5-amino group and C_2,3,6 of aromatic ring with the following amino acid residues Ile 16, Gly 20, Asp 21, Leu 22, Phe 31, Phe 34, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Leu 67 and Arg 70, as shown in Figure 3 (Supporting information).

Ligand–protein interactions in compound 4-DHFR X-ray complex

The active site illustrated the presence of one hydrogen bond interaction as the hydrogen atom of 2-amino group acted as a hydrogen bond donor with the amino acid residue Phe 31 (2.71 Å) with a strength of 1.6%. Moreover, there is an arene cation interaction between thiophene ring and amino acid residue Arg 70. In addition to hydrophobic interactions involving three methyl groups of ethoxy, ethyledene and N-methyl, C_2,3,6 of aromatic ring, all carbon atoms of thiophene ring, two oxygen atoms of two carbonyl groups and two nitrogen atoms of 2-amino and hydrazine groups with the following amino acid residues Ile 16, Leu 22, Phe 31, Phe 34, Gln 35, Thr 56, Ser 59, Ile 60, Asn 64, Leu 67, Lys 68, Arg 70, Val 14, Tyr 121, as shown in Figure 4 (Supporting information).

Ligand–protein interactions in compound 5-DHFR X-ray complex

The active site showed the presence of several molecular interactions, including two hydrogen bonds. In which both oxygen atoms of SO₂ moiety acted as a hydrogen acceptor via two hydrogen bonds with the amino acid residues Leu 22 and Ser 59 (3.48 Å and 2.87 Å, respectively) with a strength of 1% and 42.2%, respectively. Besides hydrophobic interactions involving methyl and ethyl functions, carbonyl function as well as the other atoms of the compound with the following amino acid residues: Ile 16, Leu 22, Phe 31, Phe 34, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Leu 67, Val 14 and Tyr 121, as shown in Figure 5 (Supporting information).

Ligand–protein interactions in compound 6-DHFR X-ray complex

The active site revealed the presence of hydrogen bond interaction between the hydrogen atom of amino group as it acted as a hydrogen bond donor with the side chain residue; Ser 59 (3.49 Å) with a strength of 0.7%. Moreover, one oxygen atom in SO₂ moiety acted as hydrogen bond acceptor with the side chain residue; Arg 70 (3.26 Å) with a strength of 6.5%. In addition to hydrophobic interactions between one oxygen atom of SO₂ moiety and C_2,3 of phenyl ring with the following amino acid residues: Ile 16, Gly 17, Asp 21, Leu 22, Phe 31, Phe 34, Gln 35, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Leu 67 and Arg 70, as shown in Figure 6 (Supporting information).

Ligand–protein interactions in compound 7-DHFR X-ray complex

The active site revealed that, several molecular interactions were considered responsible for the observed affinity as one oxygen atom of SO₂ moiety acted as a hydrogen acceptor with the amino acid residue Thr 56 (2.82 Å) with a strength of 44%. Besides hydrophobic interactions concerning C_2,3,4 of phenyl ring, methyl groups, oxygen atom of carbonyl function and thiocarbonyl function with the following amino acid residues: Ile 16, Gly 20, Asp 21, Leu 22, Phe 31, Gln 35, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64 and Leu 67, as shown in Figure 7 (Supporting information).

Ligand–protein interactions in compound 8-DHFR X-ray complex

The active site revealed the presence of hydrogen bond interaction between two oxygen atom of SO₂ moiety as they acted as a hydrogen bond acceptor and the amino acid residue Thr 56 (2.66 Å and 3.50 Å; respectively) with a strength of 92.2% and 1.9%, respectively. Moreover, one oxygen atom of SO₂ moiety acted as a hydrogen bond acceptor with the amino acid residue Ser 59 (3.55 Å) with a strength of 2.3%. In addition to hydrophobic interactions involving two methyl groups, oxygen atoms of two carbonyl functions, oxygen atom of quinoline moiety and C_7,8,9 of quinoline moiety with the following amino acid residues: Ile 16, Gly 17, Asp 21, Leu 22, Phe 31, Phe 34, Gln 35, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Leu 67 and Thr 146, as shown in Figure 8 (Supporting information).

Ligand–protein interactions in compound 9-DHFR X-ray complex

The active site revealed the presence of one hydrogen bond interaction as the one oxygen atom of SO₂ moiety acted as a hydrogen bond acceptor with amino acid residue Asn 64 (2.93 Å) with a strength of 12.8%. This beside hydrophobic interaction among the two oxygen atoms of SO₂ moiety, all methyl groups, C_3,4,5,6,7,8 of benzo[f]chromene and oxygen atoms of two carbonyl groups with the following amino acid residues: Ile 7, Ile 16, Asp 21, Leu 22, Phe 31, Phe 34, Gln 35, Ser 59, Ile 60, Asn 64, Leu 67, Lys 68, Arg 70, Val 14 and Tyr 121, as shown in Figure 9 (Supporting information).

Ligand–protein interactions in compound 10-DHFR X-ray complex

The active site illustrated that several molecular interactions were considered responsible for the observed affinity, as one oxygen atom of SO₂ moiety acted as a hydrogen bond acceptor with the amino acid residue Asn 64 (2.66 Å) with a strength of 41.9%. Moreover, oxygen atom of 6-carbonyl function acted as a hydrogen bond acceptor with the amino acid residue Ser 59 (3.66 Å) with a strength of 0.7%. While, oxygen atom of 4-carbonyl function and nitrogen atom of cyano function acted as hydrogen bond acceptors with the amino acid residues Tyr 121 (3.43 Å and 3.28 Å; respectively) with a strength of 1.5% and 0.2%, respectively. In addition to hydrophobic interactions involving other atoms of the compound with the following amino acid residues: Ile 7, Val 8, Ile 16, Leu 22, Phe 31, Phe 34, Thr 56, Ile 60, Pro 61, Asn 64, Leu 67, Val 14 and Tyr 121, as shown in Figure 10 (Supporting information).

Ligand–protein interactions in compound 11-DHFR X-ray complex

The active site revealed the presence of hydrogen bond interaction between one oxygen atom of SO₂ moiety and the side chain residue; Thr 56 (3.25 Å) with a strength of 3.3%. There is also hydrophobic interactions involving two oxygen atoms of SO₂ moiety, methyl functions, amino group, cyano function, C_2,5 of phenyl ring and C_3,4 of pyrrole ring with the following amino acid residues: Ile 7, Val 8, Ile 16, Leu 22, Phe 31, Phe 34, Thr 56, Ile 60, Pro 61, Asn 64, Leu 67, Val 14 and Tyr 121, as shown in Figure 11 (Supporting information).

Ligand–protein interactions in compound 12-DHFR X-ray complex

The active site illustrated the presence of hydrogen bond interaction between two oxygen atom of SO₂ moiety as they acted as a hydrogen bond acceptor and the amino acid residues Gln 35 and Asn 64 (3.75 Å and 3.72 Å; respectively) with a strength of 0.7% and 1.5%, respectively. In addition to hydrophobic interactions involving methyl groups, oxygen atoms of SO₂ moiety and C_2,3 of phenyl ring with the following amino acid residues: Ala 9, Ile 16, Leu 22, Trp 24, Glu 30, Phe 31, Phe 34, Gln 35, Thr 56, Ser 59, Ile 60, Asn 64, Leu 67, Lys 68, Arg 70 and Val 14, as shown in Figure 12 (Supporting information).

Ligand–protein interactions in compound 13-DHFR X-ray complex

The active site revealed the presence of several molecular interactions in which hydrogen atom of amino function acted as a hydrogen bond donor with the amino acid residues Ser 59 (2.80 Å) with a strength of 73.2%. Furthermore, one oxygen atom of SO₂ moiety acted as a hydrogen bond acceptor with the amino acid residue Asn 64 (3.45 Å) with a strength of 3.4%. In addition to hydrophobic interactions involving methyl and ethyl groups, cyano function, two oxygen atoms of SO₂ moiety and C_3,5,6 of phenyl ring with the following amino acid residues: Ala 9, Ile 16, Leu 22, Phe 31, Phe 34, Gln 35, Thr 56, Ser 59, Ile 60 and Asn 64, as shown in Figure 13 (Supporting information).

Ligand–protein interactions in compound 14-DHFR X-ray complex

The active site revealed the presence of arene–arene interaction between the phenyl ring and the amino acid residue Phe 34. Furthermore, one oxygen atom of SO₂ moiety acted as a hydrogen acceptor with the amino acid residue Tyr 121 (2.63 Å) with a strength of 20.1%. In addition to hydrophobic interactions involving other atoms of the compound with many amino acid residues: with the following amino acid residues: Ile 16, Leu 22, Phe 31, Phe 34, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Leu 67, Val 14 and Tyr 121, as shown in Figure 14 (Supporting information).

Ligand–protein interactions in compound 15-DHFR X-ray complex

The active site revealed the presence of several molecular interactions, including two hydrogen bonds. In which both oxygen atoms of SO₂ moiety acted as a hydrogen acceptor via two hydrogen bonds with the amino acid residues Gln 35 and Arg 70 (3.63 Å and 3.07Å, respectively) with a strength of 0.8% and 17.8%, respectively). In addition to hydrophobic interactions involving methyl groups, one oxygen atom of SO₂ moiety and C_2,4,5 of phenyl ring with the following amino acid residues: Ile 16, Gly 20, Asp 21, Leu 22, Phe 31, Gln 35, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64 and Leu 67, as shown in Figure 15 (Supporting information).

Conclusion of the docking and molecular modeling

Docking was performed for the compounds 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 on the dihydrofolate reductase in a trial to predict their mode of action as anticancer drugs. The compounds show several interactions with dihydrofolate reductase enzyme. Particularly noteworthy are the compounds 12, 9, 4, 13, 15 and 7, which suggest that they might exert their action through inhibition of the DHFR enzyme (Table 6). It is worth mentioning that, all the interactions were within a distance range 2.63–3.75 Å, which is considered as a good sign that indicate the absence of clashes between ligands and protein, which indicated that most of these proposed molecules have promising DHFR inhibition selectivity and could be considered as active hits as anticancer agents. It is clear from the present data that the comparison of the docking score energy for tested compounds that the compounds follows the order 12 > MTX > 9 > 4 > 13 > 15 > 7 > 6 > 8 > 5  > 10 > 11 > 3> 2 > 14.

Table 6. Docking score energy of the selective newly synthesized compounds.

Cpd. No	Score	E-conf	E-place	E-score 1	E-score 2	E-refine
2	−20.4574	3.596101	−75.0351	−10.9972	−20.4574	−19.8169
3	−21.4633	41.8881	−109.539	−11.496	−21.4633	−23.3608
4	−24.2934	42.20723	−119.642	−12.193	−24.2934	9.963194
5	−22.1118	42.98828	−81.014	−11.3328	−22.1118	−25.7723
6	−23.0272	30.60611	−108.314	−11.1684	−23.0272	−21.0398
7	−23.4163	73.64976	−98.7849	−12.0301	−23.4163	−4.59358
8	−22.403	52.45129	−112.641	−11.432	−22.403	−17.8918
9	−25.7524	67.21304	−97.5068	−11.616	−25.7524	2.963847
10	−21.8549	−4.1024	−90.8791	−11.5605	−21.8549	−8.5729
11	−21.6592	5.367175	−74.9848	−11.7363	−21.6592	−13.4769
12	−28.6922	73.42744	−103.767	−10.7284	−28.6922	7.603124
13	−24.1631	52.55446	−92.257	−11.5454	−24.1631	−4.2026
14	−20.1099	−5.04918	−87.9892	−10.2547	−20.1099	−13.9655
15	−23.5028	46.12006	−76.1009	−11.4817	−23.5028	−14.7353
MTX	−28.2736	143.6868	−125.016	−12.3235	−28.2736	39.20521

Conclusion

This article proved that compounds having N-ethyl-N-methylbenzene sulfonamide moiety attached to different heterocyclic moieties such as 2-oxo-2H-chromene 8, 3-amino cyclopenta[b]thiophene 5 and 5-amino-3-cyano-2-oxopyrrolidine 11, showed a significant cytotoxic activity against human breast cancer cell line (MCF-7) compared to the reference drug methotrexate (MTX). In addition, compounds 7 and 12 showed moderate activity against Aspergillus fumigatus (fungi) compared to amphotericin B as a reference drug, compounds 12 displayed moderate activity against Staphylococcus aureus (Gram positive). Compounds 12 and 7 gave moderate effects against Streptococcus pneumonia (Gram positive). Compounds 12, 4, 7 and 10 gave moderate effects against Bacillus subtilis (Gram positive) compared to Ampicillin as a reference drug. Compound 12 showed moderate effect against Escherichia coli (Gram negative). Compounds 12, 7, 4, 14, 5 and 8, which are more active than gentamicin as a reference drug against Klebsiella pneumonia as Gram-negative bacteria. The molecular docking of compounds 12, 9, 4, 13, 15 and 7 showed more interaction with DHFR which lead to inhibit this enzyme.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Supplementary material available online