untitled European Journal of Chemistry 6 (4) (2015) 444‐450 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2015 Atlanta Publishing House LLC ‐ All rights reserved ‐ Printed in the USA http://dx.doi.org/10.5155/eurjchem.6.4.444‐450.1316 European Journal of Chemistry Journal webpage: www.eurjchem.com Design and synthesis of new thiophene derivatives together with their antitumor evaluations Mahmoud Ali Abdelaziz Mahmoud Department of Chemistry, Faculty of Science, University of Tabuk, Tabuk, 71491, P.O. Box 741, Kingdom of Saudi Arabia * Corresponding author at: Department of Chemistry, Faculty of Science, University of Tabuk, Tabuk, 71491, P.O. Box 741, Kingdom of Saudi Arabia. Tel.: +966.055.7235368. Fax: +966.04.4262593. E‐mail address: m_mahmoud@ut.edu.sa (M.A.A. Mahmoud). ARTICLE INFORMATION ABSTRACT DOI: 10.5155/eurjchem.6.4.444‐450.1316 Received: 09 September 2015 Accepted: 04 October 2015 Published online: 31 December 2015 Printed: 31 December 2015 A series of new thiophene derivatives (3a,b) have been prepared by the reaction of acetylacetone with either cyanoacetanilide or 2‐cyano‐N‐(p‐tolyl)acetamide and elemental sulfur in the presence of triethylamine as basic catalyst. The two synthesized compounds were used to further synthesize new thiophene derivatives. The structures of all the newly synthesized products have been established on the basis of analytical and spectral data. The antitumor activity of the newly thiophene derivatives was evaluated against six human cancer cell lines, namely gastric cancer (NUGC), colon cancer (DLD1), liver cancer (HA22T and HEPG2), nasopharyngeal carcinoma (HONE1), breast cancer (MCF) and normal fibroblast cells (WI38). KEYWORDS Pyrazole Thiophene Cytotoxicity Breast cancer Antitumor activity Benzo[b]thiophene Cite this: Eur. J. Chem. 2015, 6(4), 444‐450 1. Introduction Thiophenes are versatile reagents for many heterocyclic reactions leading to the formation of important biologically active compounds [1‐6]. In addition, thiophenes derivatives belong to aromatic heterocyclic group consequently; they are important structural fragment in many pharmaceutical and chemical compounds [7,8]. Thiophenes compounds have been found to show nematocidal [9], insecticidal [10], antibacterial [11], antifungal [12], antiviral [13] and antioxidant activity [14]. In fact, many therapeutic drugs contain the thiophene nucleus as the main active moiety through the drug e.g. NSC 652287 (Figure 1) [15]. It was hoped that thiophenes compounds would be as active as the parent compound or that these analogous would, by virtue of its similar chemical structure, combine with the receptor and if not elicit a response of its own, serve effectively as a competitive inhibitor. Thus, when the thiophenes and furans derivatives are prepared, it is conceivable that such an agentmay intensify, mimic or antagonize the physiological activity of the parent substance [16]. 2. Experimental 2.1. Instrumentation Figure 1. Chemical structure of the thiophene NSC 6522872, 5‐bis(5‐ hydroxymethyl‐2‐thienyl)furan. All melting points were determined on an Electrothermal digital melting point apparatus and are uncorrected. IR spectra (KBr discs) were recorded on a FT‐IR plus 460 or Pye Unicam SP‐1000 spectrophotometer. 1H NMR spectra were recorded with Varian Gemini‐200 (200 MHz) and Jeol AS 500 MHz instruments spectra were performed in DMSO‐d6 as solvent using TMS as internal standard and chemical shifts are expressed as δ ppm. MS (EI) spectra were recorded with Hewlett Packard 5988 A GC/MS system and GCMS‐QP 1000 Ex Shimadzu instruments. Analytical data were obtained from the Micro‐Analytical Data Unit at Cairo University and were performed on Vario EL III Elemental analyser. 2.2. Synthesis 2.2.1. General procedure for the synthesis of the thiophene derivatives 3a,b Mahmoud / European Journal of Chemistry 6 (4) (2015) 444‐450 445 Scheme 1 To a solution of acetylacetone (1.0 g, 0.01 mol) in 1,4‐ dioxane (40 mL) containing triethylamine (1.0 mL), either cyanoacetanilide (1.60 g, 0.01 mol) (2a) or 2‐cyano‐N‐(p‐ tolyl)acetamide (2b) (1.74 g, 0.01 mol) and elemental sulfur (0.32 g, 0.01 mol) were added. The reaction mixture, in each case, was heated under reflux for 2 h then left to cool. The solid product formed upon pouring onto ice/water containing few drops of hydrochloric acid was collected by filtration and crystallized from the suitable solvent (Scheme 1). 5‐Acetyl‐2‐amino‐4‐methyl‐N‐phenylthiophene‐3‐carbox‐ amide (3a): Color: Pale yellow crystals from acetic acid. Yield: 74 % (2.03 g). M.p.: 180‐182 °C. IR (KBr, ν, cm‐1): 3473‐3340 (NH2, NH), 3054 (CH, aromatic), 2982 (CH3), 1708, 1687 (2 CO), 1628 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.68 (s, 3H, CH3), 2.97 (s, 3H, CH3), 4.85 (s, 2H, NH2), 7.31‐7.37 (m, 5H, C6H5), 8.38 (s, 1H, NH). Anal. calcd. for C14H14N2O2S: C, 61.29; H, 5.14; N, 10.21. Found: C, 61.03; H, 5.30; N; 10.49%. 5‐Acetyl‐2‐amino‐4‐methyl‐N‐(p‐tolyl) thiophene‐3‐carbox‐ amide (3b): Color: Yellow crystals from acetic acid. Yield: 80 % (2.31 g). M.p.: 220‐222 °C. IR (KBr, ν, cm‐1): 3455‐3332 (NH2, NH), 3058 (CH, aromatic), 2984 (CH3), 1705, 1688 (2 CO), 1625 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.66 (s, 3H, CH3), 2.99 (s, 3H, CH3), 3.11 (s, 3H, CH3), 4.87 (s, 2H, NH2), 7.30‐7.41 (m, 4H, C6H4), 8.36 (s, 1H, NH). Anal. calcd. for C15H16N2O2S: C, 62.48; H, 5.59; N, 9.71. Found: C, 62.31; H, 5.42; N; 9.86%. 2.2.2. Ethyl (5‐acetyl‐4‐methyl‐3‐(phenylcarbamoyl) thiophen‐2‐yl)glycinate (5) To a solution of compound 3a (2.74 g, 0.01 mol) in 1,4‐ dioxane (40 mL) containing potassium hydroxide (0.50 g) ethyl 2‐chloro acetate (1.22 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 2 h then poured onto ice/water containing few drops of hydrochloric acid (till pH = 6) and the formed solid product was collected by filtration. Color: White crystals from ethanol (Scheme 1). Yield: 78 % (2.81 g). M.p.: 195‐197 °C. IR (KBr, ν, cm‐1): 3466‐3323 (2 NH), 3054 (CH, aromatic), 2980 (CH3), 1712‐1686 (3 CO), 1630 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.13 (t, 3H, J = 7.02 Hz, CH3), 2.66 (s, 3H, CH3), 2.83 (s, 3H, CH3), 4.22 (q, 2H, J = 7.02 Hz, CH2), 5.37 (s, 2H, CH2), 7.28‐7.39 (m, 5H, C6H5), 8.31 (s, 1H, NH), 8.40 (s, 1H, NH). Anal. calcd. for C18H20N2O4S: C, 59.98; H, 5.59; N, 7.77. Found: C, 60.17; H, 5.66; N; 7.93%. 2.2.3. General procedure for the synthesis of the 2,3‐dihydro thieno[2,3‐d]pyrimidine derivatives 7a,b To a solution of compound 3a (2.74 g, 0.01 mol) in 1,4‐ dioxane (40 mL) containing triethylamine (0.50 mL) either formaldehyde (0.32 g, 0.01 mol) or benzaldehyde (1.06 g, 0.01 mol) was added. The reaction mixture, in each case, was heated under reflux for 6 h then evaporated under vacuum. 446 Mahmoud / European Journal of Chemistry 6 (4) (2015) 444‐450 The remaining product was triturated with diethyl ether and the formed solid product was collected by filtration (Scheme 1). 6‐Acetyl‐5‐methyl‐3‐phenyl‐2,3‐dihydrothieno[2,3‐d]pyrimi din‐4(1H)‐one (7a): Color: Pale yellow crystals from ethanol. Yield: 66 % (1.89 g). M.p.: 210‐212 °C. IR (KBr, ν, cm‐1): 3458‐ 3312 (2 NH) , 3053 (CH, aromatic), 2982 (CH3) ,1705‐1688 (2 CO), 1628 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.68 (s, 3H, CH3), 2.80 (s, 3H, CH3), 6.41 (s, 2H, CH2), 7.31‐7.38 (m, 5H, C6H5), 8.34 (s, 1H, NH). Anal. calcd. for C15H14N2O2S: C, 62.92; H, 4.93; N, 9.78. Found: C, 62.68; H, 5.04; N, 9.61%. 6‐Acetyl‐5‐methyl‐2,3‐diphenyl‐2,3‐dihydrothieno[2,3‐d]pyri midin‐4(1H)‐one (7b): Color: Orange crystals from ethanol. Yield: 78 % (2.83 g). M.p.: 188‐190 °C. IR (KBr, ν, cm‐1): 3461 (NH), 3056 (CH, aromatic), 2980 (CH3) , 1720‐1687 (2 CO), 1623 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.67 (s, 3H, CH3), 2.82 (s, 3H, CH3), 7.02 (s, 1H, CH), 7.27‐7.35 (m, 10H, 2C6H5), 8.29 (s, 1H, NH). Anal. calcd. for C21H18N2O2S: C, 69.59; H, 5.01; N, 7.73. Found: C, 69.39; H, 4.88; N; 7.47%. 2.2.4. 2‐Amino‐5‐(5‐(cyanomethyl)‐1H‐pyrazol‐3‐yl)‐4‐ methyl‐N‐phenylthiophene‐3‐carboxamide (10) To a solution of compound 3a (2.74 g, 0.01 mol) in 1,4‐ dioxane (40 mL) cyanoacetyl hydrazine (1.00 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 3 h then poured onto ice/water and the formed solid product was collected by filtration (Scheme 1). Color: White crystals from acetic acid. Yield: 69 % (2.32 g). M.p.: > 300 °C. IR (KBr, ν, cm‐ 1): 3479‐3322 (NH2, 2NH), 3052 (CH, aromatic), 2984 (CH3), 2220 (CN), 1688 (CO), 1631 (C=C). 1H NMR (400 MHz, DMSO‐ d6, δ, ppm): 2.68 (s, 3H, CH3), 4.97 (s, 2H, NH2), 5.30 (s, 2H, CH2), 6.89 (s, 1H, pyrazole CH), 7.29‐7.38 (m, 5H, C6H5), 8.27 (s, 1H, NH), 8.35 (s, 1H, NH). Anal. calcd. for C17H15N5OS: C, 60.52; H, 4.48; N, 20.76. Found: C, 60.39; H, 4.77; N; 20.91%. 2.2.5. General procedure for the synthesis benzo[b] thiophene derivatives 13a,b To the dry solid of compound 3a (2.74 g, 0.01 mol) containing ammonium acetate (0.50 g) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added. The whole reaction mixture was heated in an oil bath at 120 °C for 45 min. The formed solid product, upon cooling, was triturated with ethanol and the formed solid was collected by filtration (Scheme 2). 2,5‐Diamino‐6‐cyano‐7‐methyl‐N‐phenylbenzo[b]thiophene‐ 3‐carboxamide (13a): Color: Yellow crystals from acetic acid. Yield: 81 % (2.64 g). M.p.: 112‐115 °C. IR (KBr, ν, cm‐1): 3488‐ 3320 (2NH2, NH), 3056 (CH, aromatic), 2986 (CH3), 2222 (CN), 1689 (CO), 1630 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 3.20 (s, 3H, CH3), 4.77 (s, 2H, NH2), 5.31 (s, 2H, NH2), 7.26‐7.39 (m, 6H, C6H, C6H5), 8.29 (s, 1H, NH). Anal. calcd. for C17H14N4OS: C, 63.34; H, 4.38; N, 17.38. Found: C, 63.39; H, 4.57; N; 17.44%. 2,5‐Diamino‐7‐methyl‐3‐(phenylcarbamoyl)benzo[b]thio phen‐6‐yl propionate (13b): Color: Orange crystals from acetic acid. Yield: 80 % (2.96 g). M.p.: 223‐225 °C. IR (KBr, ν, cm‐1): 3493‐3320 (2NH2, NH), 3057 (CH, aromatic), 2983 (CH3), 2224 (CN), 1689 (CO), 1626 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.16 (t, 3H, J = 7.24 Hz, CH3), 3.19 (s, 3H, CH3), 4.22 (q, 2H, J = 7.24 Hz, CH2), 4.66 (s, 2H, NH2), 5.31 (s, 2H, NH2), 7.31‐ 7.42 (m, 6H, C6H, C6H5), 8.29 (s, 1H, NH). Anal. calcd. for C19H19N3O3S: C, 61.77; H, 5.18; N, 11.37. Found: C, 61.05; H, 5.28; N; 11.60%. 2.2.6. (E)‐2,5‐Diamino‐6‐cyano‐N‐phenyl‐7‐styrylbenzo[b] thiophene‐3‐carboxamide (15) To a solution of compound 13a (3.22 g, 0.01 mol) in 1,4‐ dioxane (40 mL) containing piperidine, benzaldehyde (1.06 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 2 h then poured onto ice/water containing few drops of hydrochloric acid. The formed solid product was collected by filtration (Scheme 2). Color: Orange crystals from ethanol. Yield: 72 % (2.96 g). M.p.: 180‐182 °C. IR (KBr, ν, cm‐1): 3488‐ 3328 (2NH2, NH), 3054 (CH aromatic), 2220 (CN), 1686 (CO), 1628 (C=C). 1H NMR (400MHz, DMSO‐d6, δ, ppm): 4.48 (s, 2H, NH2), 5.33 (s, 2H, NH2), 6.62 (d, 1H, CH=CH), 6.70 (d, 1H, CH=CH), 7.31‐7.48 (m, 11H, C6H, 2C6H5), 8.32 (s, 1H, NH). Anal. calcd. for C24H18N4OS: C, 70.22; H, 4.42; N, 13.65. Found: C, 70.09; H, 4.69; N; 13.81%. 2.2.7. 2,5‐Diamino‐7‐(bromomethyl)‐6‐cyano‐N‐phenyl benzo[b]thiophene‐3‐carboxamide (16) A solution of compound 13a (3.22 g, 0.01 mol) in acetic acid (40 mL) was warmed till 60 °C then bromine solution (0.50 mL, 0.01 mol) in acetic acid (8 mL) was added drop wise with continuous stirring within a period 30 min. The reaction mixture was kept to stir at room temperature for an additional 3 h and the solid product formed upon pouring onto ice/water was collected by filtration (Scheme 2). Color: Pale yellow crystals from acetic acid. Yield: 60 % (2.41 g). M.p.: 140‐142 °C. IR (KBr, ν, cm‐1): 3464‐3348 (2NH2, NH), 3056 (CH aromatic), 2221 (CN), 1689 (CO), 1623 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 4.45 (s, 2H, NH2), 5.36 (s, 2H, NH2), 5.39 (s, 2H, CH2), 7.26‐7.39 (m, 6H, C6H, C6H5), 8.30 (s, 1H, NH). Anal. calcd. for C17H13BrN4OS: C, 50.88; H, 3.27; N, 13.96. Found: C, 50.72; H, 3.61; N; 13.63%. 2.2.8. 2,5‐Diamino‐6‐cyano‐N‐phenyl‐7‐((2‐phenyl hydrazono)methyl)benzo[b]thiophene‐3‐carboxamide (18) To a cold solution (0‐5 °C) of compound 13a (3.22 g, 0.01 mol) in ethanol (50 mL) containing sodium hydroxide benzenediazonium chloride (obtained via the addition of a cold solution of sodium nitrite (0.70 g, 0.01 mol, in water) to a cold solution of aniline (0.94 g, 0.01 mol) in concentrated hydrochloric acid (8 mL, 18 %) with continuous stirring) was added with continuous stirring. The whole reaction mixture was stirred at room temperature for 3 h and the formed solid product was collected by filtration (Scheme 2). Color: Reddish brown crystals from ethanol. Yield: 77 % (3.28 g). M.p.: 150‐ 152 °C. IR (KBr, ν, cm‐1): 3481‐3338 (2NH2, 2NH), 3054 (CH aromatic), 2220 (CN), 1686 (CO), 1630 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 4.46 (s, 2H, NH2), 5.33 (s, 2H, NH2), 6.09 (s, 1H, CH), 7.28‐7.43 (m, 11H, C6H, 2C6H5), 8.32 (s, 1H, NH), 8.43 (s, 1H, NH). Anal. calcd. for C23H18N6OS: C, 64.77; H, 4.25; N, 19.71. Found: C, 64.68; H, 4.49; N; 19.57%. 2.2.9. General procedure for the synthesis of the bithiophene derivatives 19a,b To a solution of compound 13a (2.22 g, 0.01 mol) in 1,4‐ dioxane (40 mL) containing triethylamine (0.50 mL) and elemental sulphur either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added. The whole reaction mixture, in each case, was heated under reflux for 1 h then poured onto ice/water containing few drops of hydrochloric acid. The formed solid product was collected by filtration (Scheme 3). 5,5'‐Diamino‐4'‐cyano‐3‐methyl‐N‐phenyl‐[2,3'‐bithiophene] ‐4‐carboxamide (19a): Color: Yellow crystals from acetic acid. Yield: 83 % (2.94 g). M.p.: 208‐210 °C. IR (KBr, ν, cm‐1): 3473‐ 3321 (2NH2, NH), 3057 (CH aromatic), 2983 (CH3), 2222 (CN), 1689 (CO), 1630 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.84 (s, 3H, CH3), 4.48 (s, 2H, NH2), 5.37 (s, 2H, NH2), 6.89 (s, 1H, thiophene), 7.31‐7.40 (m, 5H, C6H5), 8.30 (s, 1H, NH). Anal. calcd. for C17H14N4OS2: C, 57.61; H, 3.98; N, 15.81. Found: C, 57.83; H, 4.32; N; 15.77%. Mahmoud / European Journal of Chemistry 6 (4) (2015) 444‐450 447 11a, X = CN b, X = COOEt NOAc 120 oC 12a,b 13a, X = CN b, X = COOEt + 14 Piperdine 1,4-Dioxane 15 + Br2 AcOH 16 PhN NCl -+ 17 18 EtOH NaOH 3a + S O H N H2N O X N X S NH O H2N NH2 S O HN X N NH2 S O NH N H2N NH2 O S O HN N NH2 NH2 13a S O NH N H2N NH2 13a + S O NH N H2N NH2 13a S O NH N Br H2N NH2 S O N H N N H N NH2 H2N Scheme 2 Ethyl 5,5'‐diamino‐3‐methyl‐4‐(phenylcarbamoyl)‐[2,3'‐bi thiophene]‐4'‐carboxylate (19b): Color: Yellow crystals from acetic acid. Yield: 74 % (2.96 g). M.p.: 177‐179 °C. IR (KBr, ν, cm‐1): 3460‐3342 (2NH2, NH), 3054 (CH aromatic), 2980, 2877 (CH3,CH2), 1720, 1687 (2CO), 1632 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.14 (t, 3H, J = 7.22 Hz, CH3), 2.89 (s, 3H, CH3), 4.20 (q, 2H, J = 7.22 Hz, CH2), 4.45 (s, 2H, NH2), 5.35 (s, 2H, NH2), 6.92 (s, 1H, thiophene), 7.28‐7.43 (m, 5H, C6H5), 8.32 (s, 1H, NH). Anal. calcd. for C19H19N3O3S2: C, 56.84; H, 4.77; N, 10.47. Found: C, 56.51; H, 4.49; N; 10.37%. 2.2.10. General procedure for the synthesis of the thiophene derivatives (21a‐d) To a solution of either compound 3a (2.74 g, 0.01 mol) or 3b (2.88 g, 0.01 mol) in dimethyl formamide (40 mL) either ethyl cyanoacetate (1.13 g, 0.01 mol) or diethyl malonate (1.60 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 1 h then poured onto ice/water. The solid product, so formed in each case, was collected by filtration (Scheme 3). 5‐Acetyl‐2‐(2‐cyanoacetamido)‐ 4‐methyl‐N‐phenylthio phene‐3‐carboxamide (21a): Color: Yellow crystals from 1,4‐ dioxane. Yield: 79 % (2.70 g). M.p.: 214‐216 °C. IR (KBr, ν, cm‐ 1): 3469‐3360 (2NH), 3056 (CH aromatic), 2982, 2879 (CH3,CH2), 2220 (CN), 1721‐1689 (3CO), 1634 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.86 (s, 3H, CH3), 2.93 (s, 3H, CH3), 5.82 (s, 2H, CH2), 7.25‐7.40 (m, 5H, C6H5), 8.26 (s, 1H, NH), 8.29 (s, 1H, NH). Anal. calcd. for C17H15N3O3S: C, 59.81; H, 4.43; N, 12.31. Found: C, 59.77; H, 4.67; N; 12.09%. 448 Mahmoud / European Journal of Chemistry 6 (4) (2015) 444‐450 Scheme 3 Ethyl 3‐((5‐acetyl‐4‐methyl‐3‐(phenylcarbamoyl)thiophen‐ 2‐yl)amino)‐3‐oxopropanoate (21b): Color: Yellow crystals from 1,4‐dioxane. Yield: 60 % (2.33 g). M.p.: 166‐168 °C. IR (KBr, ν, cm‐1): 3482‐3344 (2NH), 3053 (CH aromatic), 2980, 2883 (CH3,CH2), 1723‐1685 (4CO), 1630 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.15 (t, 3H, J = 7.48 Hz, CH3), 2.83 (s, 3H, CH3), 2.90 (s, 3H, CH3), 4.25 (q, 2H, J = 7.48 Hz, CH2), 5.80 (s, 2H, CH2), 7.29‐7.37 (m, 5H, C6H5), 8.28 (s, 1H, NH), 8.30 (s, 1H, NH). Anal. calcd. for C19H20N2O5S: C, 58.75; H, 5.19; N, 7.21. Found: C, 58.88; H, 4.83; N; 6.93%. 5‐Acetyl‐2‐(2‐cyanoacetamido)‐4‐methyl‐N‐(p‐tolyl) thio phene‐3‐carboxamide (21c): Color: Yellow crystals from 1,4‐ dioxane. Yield: 80 % (2.84 g). M.p.: 170‐172 °C. IR (KBr, ν, cm‐ 1): 3455‐3348 (2NH), 3054 (CH aromatic), 2980, 2883 (CH3,CH2), 2222 (CN), 1723‐1686 (3CO), 1630 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.88 (s, 3H, CH3), 2.90 (s, 3H, CH3), 3.11 (s, 3H, CH3), 5.80 (s, 2H, CH2), 7.29‐7.38 (m, 4H, C6H4), 8.23 (s, 1H, NH), 8.32 (s, 1H, NH). Anal. calcd. for C18H17N3O3S: C, 60.83; H, 4.82; N, 11.82. Found: C, 60.91; H, 4.90; N; 11.69%. Ethyl 3‐((5‐acetyl‐4‐methyl‐3‐(p‐tolylcarbamoyl)thiophen‐ 2‐yl)amino)‐3‐oxopropanoate (21d): Color: Yellow crystals from 1,4‐dioxane. Yield: 66 % (2.66 g). M.p.: 180‐182 °C. IR (KBr, ν, cm‐1): 3489‐3364 (2NH), 3056 (CH aromatic), 2983, 2880 (CH3,CH2), 1720‐1688 (4CO), 1630 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.13 (t, 3H, J = 6.99 Hz, CH3), 2.80 (s, 3H, CH3), 2.92 (s, 3H, CH3), 3.12 (s, 3H, CH3), 4.24 (q, 2H, J = 6.99 Hz, CH2), 5.82 (s, 2H, CH2), 7.27‐7.40 (m, 4H, C6H4), 8.26 (s, 1H, NH), 8.33 (s, 1H, NH). Anal. calcd. for C20H22N2O5S: C, 59.69; H, 5.51; N, 6.96. Found: C, 59.76; H, 5.47; N; 6.73%. 3. Results and discussions 3.1. Chemistry In the present work, we are demonstrating the synthesis of thiophene derivatives together with their uses in heterocyclic synthesis. The cytotoxicity of the newly synthesized products were evaluated against cancer and normal cell lines. Thus, the reaction of acetylactone with either cyanoacetanilide (2a) or 2‐cyano‐N‐(p‐tolyl)acetamide (2b) and elemental sulfur in the presence of triethylamine gave the thiophene derivatives 3a and 3b, respectively. The structures of the latter products were confirmed on the basis of analytical and spectral data. Thus the 1H NMR spectrum of compound 3a showed a two singlets at δ 2.68, 2.97 ppm indicating the presence of two CH3 group, a singlet at δ 4.85 ppm indicating the presence of one NH2 group, a multiplet at δ 7.31‐7.37 ppm corresponding to phenyl group, and one singlet at δ 8.38 for NH group. The reaction of compound 3a with ethyl α‐chloroacetate (4) gave the N‐alkyl product 5. On the other hand, the reaction of compound 3a with either formaldehyde (6a) or benzalde‐ hyde (6b) gave the 2,3‐dihydrothieno [2,3‐d]pyrimidin‐4(1H)‐ one derivatives 7a and 7b, respectively. The analytical and spectral data of compound 7a and 7b were consistence with their respective structures. The 1H NMR spectrum of com‐ pound 7a showed a two singlets at δ 2.68, 2.80 ppm indicating the presence of two CH3 group, a singlet at δ 6.41 ppm indicating the presence of one CH2 group, a multiplet at δ 7.31‐ 7.38 ppm corresponding to phenyl group, and one singlet at δ 8.34 for NH group. Mahmoud / European Journal of Chemistry 6 (4) (2015) 444‐450 449 Table 1. Cytotoxicity of the synthesized compounds against a variety of cancer cell lines a [IC50b (nM)]. Compound Cytotoxocity (IC50 in nM) NUGC DLDI HA22T HEPG2 HONE1 MCF WI38 3a 302 412 129 170 232 222 Na 3b 1255 2238 2065 429 467 180 Na 5 160 206 133 120 189 250 Na 7a 1443 527 2263 2310 230 2188 Na 7b 1320 1142 1754 3175 2048 1370 Na 10 1114 1210 1577 80 1293 126 Na 13a 120 141 159 1092 1042 1190 Na 13b 1113 2020 156 140 110 129 Na 15 30 28 208 233 1008 2170 Na 16 112 131 129 1159 189 1154 Na 18 1128 1078 1105 2120 1068 880 Na 19a 77 42 98 39 1943 590 Na 19b 20 42 120 38 129 78 Na 21a 1148 1260 2230 1287 2280 2266 Na 21b 2055 2072 1079 2693 2228 3332 Na 21c 340 633 48 290 42 1395 Na 21d 269 189 49 260 339 49 Na CHS 828 25 2315 2067 1245 15 18 Na a NUGC, gastric cancer; DLDI, colon cancer; HA22T and HEPG2, liver cancer; HONEI, nasopharyngeal carcinoma; MCF, breast cancer; WI38, normal fibroblast cells. b The sample concentration that produces a 50% reduction in cell growth. The acetyl group present in compound 3a showed interesting activity towards condensation with hydrazide moiety. Thus, the reaction of compound 3a with α‐cyanoacetyl hydrazine (8) in 1,4‐dioxane gave the 3‐thienylpyrazole derivative (10) through the intermediate hydrazide‐hydra‐ zone derivative (9). Moreover, compound 3a was found to be a good candidate towards Knoevenagel condensation reaction through its reaction with active methylene reagents. Interes‐ tingly, compound 3a reacted with either malononitrile (11a) or ethyl cyanoacetate (11b) at 120 °C and the presence of ammonium acetate gave the benzo[b]thiophene derivatives (13a and 13b), respectively. Formation of compound 13a,b took place through the intermediate formation of the condensation products 12a,b. The high yield of compound 13a encouraged us to study some of its chemical reactivity towards some chemical reagents. Interestingly the o‐methyl group present in compound 13a to the cyano group showed acidic nature, this appeared through the reaction of compound 13a with benzaldehyde (14) in the presence of piperidine gave the benzylidene derivative 15. On the other hand, compound 13a reacted with bromine in acetic acid solution gave the bromomethyl derivative 16. Moreover, the reaction of compound 13a with benzenediazonium chloride at 0‐5 °C in the presence of sodium hydroxide gave the phenylhydrazone derivative 18. Structures of compounds 15, 16 and 18 were confirmed on the basis of analytical and spectral data (see experimental section). Next, we moved towards the reactivity of compound 3a towards further thiophene formation through the use of the 2‐ acetyl group. Thus, the reaction of compound 3a with either malononitrile (11a) or ethyl cyanoacetate (11b) and elemen‐ tal sulphur gave the 3‐thienyl thiophene derivatives 19a and 19b, respectively. On the other hand, compounds 3a and 3b reacted with either ethyl cyanoacetate (11b) or diethyl malonate (20) to give the amide derivatives 21a‐d, respect‐ tively. The structures of the latter products were established on the basis of analytical and spectral data (see experimental section). 3.2. Antitumor evaluations 3.2.1. Effect on the growth of human cancer cell lines The heterocyclic compounds, prepared in this study, were evaluated according to standard protocols for their in vitro cytotoxicity against six human cancer cell lines including cells derived from human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), nasopharyngeal carcinoma (HONE1), human breast cancer (MCF) and normal fibroblast cells (WI38). For comparison reasons, CHS 828 was used as standard anticancer drug. All of IC50 values in (nM) are listed in Table 1. 3.2.2. Structure activity relationship From Table 1, the newly synthesized compounds were tested against the six cancer cell lines, the human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), nasopharyngeal carcinoma (HONE1), human breast cancer (MCF) and normal fibroblast cells (WI38). The compounds 3a, 5, 13a, 15, 16, 19a, 19b, 21c and 21d exhibited optimal cytotoxic effect against cancer cell lines with IC50ʼs in the nM range. The reaction of 5‐acetyl‐2‐ amino‐4‐methyl‐N‐phenylthiophene‐3‐carboxamide (3a) with ethyl α‐chloroacetate gave the ethyl 2‐((5‐acetyl‐4‐methyl‐3‐ (phenylcarbamoyl) thiophen‐2‐yl)amino)acetate (5) through which the cytotoxicity decreases against the tested cancer cell lines. Such decrease in cytotoxicity is attributed to CH2COOEt moiety. On the other hand, the reaction of compound 3a with either formaldehyde or benzaldehyde gave the 2,3‐dihydro‐ thieno[2,3‐d]pyrimidin‐4(1H)‐one derivatives 7a and 7b, respectively. The pyrimidine derivatives 7a and 7b showed remarkable cytotoxicity which is more than that of compound 3a against the tested cancer cell lines. Comparing the cytotoxicity of the compound 3a and the 3‐thienylpyrazole derivative 10 exhibited clearly that cyclization of compound 3a to the pyrazole product 10 increases the cytotoxicity of the latter compound. The benzo[b]thiophene derivatives 13a and 13b showed potency against NUGC, HONE1 and MCF cell lines. The benzylidene derivative 15 with CH=CHPh moiety is less potent against the cell lines than compound 13a. On the other hand bromomethyl‐N‐phenylbenzo[b]thiophene 16 with electronegative group the CN and Br showed high potent against NUGC, HONE1 and MCF cell lines. It is obvious that the phenyl hydrazine derivative 18 with the hydrazine moiety showed high cytotoxicity against NUGC, HEPG2, HONE1 and MCF cell lines. It is clear that for the 3‐thienyl thiophene derivatives 19a and 19b, compound 19b with COOEt moiety are less potent than compound 19a with CN moiety. Finally the amide derivatives 21a‐d, compounds 21a with CN moiety and compound 21b with the COOEt moiety are more potent than compound 21c and 21d. Moreover compound 21a showed high cytotoxicity against NUGC, HA22T, HEPG2, HONE1 and MCF cell lines as well as compound 21b showed 450 Mahmoud / European Journal of Chemistry 6 (4) (2015) 444‐450 high cytotoxicity against NUGC, HEPG2, HONE1 and MCF cell lines. 4. Conclusion Many of the synthesized heterocyclic compounds were observed with significant cytotoxicity against most of the cancer cell lines tested (IC50 < 1000 nM). Normal fibroblasts cells (WI38) were affected to a much lesser extent (IC50 > 10,000 nM). Among the tested compounds 3b, 7a, 7b, 18, 21a, and 21b was found to show the highest cytotoxic effect against the 6 cancer cell lines in the range of IC50 33‐442 nM. Broad spectrum antitumor activity was exhibited by compounds 3a, 5, 10, 13a, 13b and 21c. Acknowledgement Mahmoud Ali Abdelaziz Mahmoud would like to thank Prof. Rafat Milad Mohareb, Professor of Organic Chemistry, Faculty of Science, Cairo University, for his helping and completing this work. References [1]. Romagnoli, R.; Baraldi, P. G.; Lopez‐Cara, C.; Cruz‐Lopez, O.; Moorman, A. R.; Massink, A.; IJzerman, A. P.; Vincenzi, F.; Borea, P. A.; Varani, K. Eur. J. Med. Chem. 2015, 101, 185‐204. [2]. Romagnoli, R.; Baraldi, P. G.; Lopez‐Cara, C.; Salvador, M. K.; Preti, D.; Tabrizi, M. A.; Balzarini, J.; Nussbaumer. P.; Bassetto, M.; Brancale, A.; Fu, X. H.; Yang‐Gao, Li. J.; Zhang, S. Z.; Hamel, E.; Bortolozzi, R.; Basso, G.; Viola, G. Bioorg. Med. Chem. 2014, 22(18), 5097‐5109. [3]. Zhang, Q.; Luo, J.; Ye, L.; Wang, H.; Huang, B.; Zhang, J.; Wu, J.; Zhang, S.; Tian, Y. J. Mol. Struct. 2014, 1074, 33‐42. [4]. Mohareb, R. M.; Abbas, N. S.; Ibrahim, R. A. Acta Chim. Slov. 2013, 60, 583‐594. [5]. Lu, X.; Wan, B.; Franzblau, S. G.; You, Q. Eur. J. Med. Chem. 2011, 46(9), 3551‐3563. [6]. Dalvie, D. K.; Kalgutkar, A. S.; Khojasteh‐Bakht, S. C.; Obach, R. S.; O'Donnell, J. P. Chem. Res. Toxicol. 2002, 15, 269‐299. [7]. Kagan, J.; Arora, S. K.; Prakash, I.; Ustunol, A. Heterocycles 1983, 20, 1341‐1345. [8]. Gribble, G. W.; Saulnier, M. G.; Sibi, M. P.; Obaza‐Nutaitis, J. A. J. Org. Chem. 1984, 49, 4518‐4523. [9]. Bakker, J.; Gommers, F. J.; Nieuwenhuis, I.; Wynberg, H. J. Biol. Chem. 1979, 254, 1841‐1844. [10]. Iyengar, S.; Arnason, J. T.; Philogene, B. J.; Murand, P.; Werstink, N. H.; Timmins, G. P. Pestic. Biochem. Physiol. 1987, 29(1), 1‐9. [11]. Matsuura, H.; Saxena, G.; Farmer, S. W.; Hancock, R. E. W.; Towers, G. H. N. Planta Med. 1996, 62, 256‐259. [12]. Chan, G. F. Q.; Towers, G. H. N.; Mitchell, J. C. Phytochem. 1975, 14, 2295‐2296. [13]. Hudson, J. B.; Graham, E. A.; Miki, N.; Towers, G. H. N.; Hudson, L. L.; Rossi, R.; Carpita, A.; Neri, D. Chemosphere 1989, 19, 1329‐1343. [14]. Malmstrom, J.; Jonsson, M.; Cotgreave, I. A.; Hammarström, L.; Sjodin, M.; Engman, L. J. Am. Chem. Soc. 2001, 123, 3434‐3440. [15]. Nobles, W. L.; Blanton, D. W.; Jr, C. J. Pharm. Sci. 1964, 53, 115‐129. [16]. Meotti, F. C.; Silva, D. O.; dos Santos, A. R. S.; Zeni, G.; Rocha, J. B. T.; Nogueira, C. W. Env. Toxicol. Pharm. 2003, 15(1), 37‐44.