A series of 1,3-imidazoles and triazole-3-thiones based thiophene-2-carboxamides as anticancer agents: Synthesis and anticancer activity European Journal of Chemistry 9 (2) (2018) 99-106 European Journal of Chemistry View Journal Online View Article Online A series of 1,3-imidazoles and triazole-3-thiones based thiophene-2- carboxamides as anticancer agents: Synthesis and anticancer activity Reda Ahmed Haggam 1,2,*, Mohamed Gomma Assy 2, Mohamed Hassan Sherif 2 and Mohamed Mohamed Galahom 2 1 Department of Chemistry, Faculty of Science, Islamic University in Madinah, Madinah, 42351, Kingdom of Saudi Arabia rhaggan99@hotmail.com (R.A.H.) 2 Department of Chemistry, Faculty of Science, University of Zagazig, Zagazig, 44519, Egypt m_gomaa59@yahoo.com (M.G.A.), mohasherif@yahoo.com (M.H.S.), mgalahom@yahoo.com (M.M.G.) * Corresponding author at: Department of Chemistry, Faculty of Science, Islamic University in Madinah, Madinah, 42351, Kingdom of Saudi Arabia. Tel: +966.014.537587227 Fax: +966.014.8470354 e-mail: rhaggan99@hotmail.com (R.A. Haggam). 10.5155/eurjchem.9.2.99-106.1701 Received: 18 March 2018 Received in revised form: 09 April 2018 Accepted: 16 April 2018 Published online: 30 June 2018 Printed: 30 June 2018 By addition of semicarbazide or phenylhydrazine hydrochloride to thienoylisothiocyanate (1) resulted in building of thiosemicarbazide derivative (2), triazole derivative (4) and thiophene-2-carboxamide (5), respectively. Basic cyclization of compound 2 led to formation of oxadiazine (3). Synthesis of thiadiazine derivative (6) was achieved via reaction of compound 5 and maleic anhydride in triethyl amine. Heating of compound 5 with ethyl chloroacetate or sodium ethoxide produced thiadiazine derivative (7) and triazolethione (8), respectively. Thiosemicarbazide derivative 11 was synthesized by addition of nicotinic hydrazide to compound 1. Refluxing of compound 11 with lead acetate afforded triazole (13). Moreover, acid and base mediated cyclizations of compound 11 gave thiadiazole (12) and 1,2,4-triazolethione (14) throughout thiophene intermediate, respectively. Addition of ethyl 2-aminothiophene-3-carboxylate to compound 1 formed thiourea (15) which was refluxed with ethoxide giving thiophene-3-carboxylic acid (16). Lastly, nucleophilic addition of amino phenol or ethylene diamine to compound 1 yielded oxazine structure (18) and imidazole derivative (19), respectively. The yields of the synthesized compounds were 61- 95%. The detailed synthesis and spectroscopic data of the new compounds are reported. Thiophene 1,3-Imidazole Isothiocyanates 1,3,4-Thiadiazoles Triazole-3-thiones Anticancer activity Cite this: Eur. J. Chem. 2018, 9(2), 99-106 Journal website: www.eurjchem.com 1. Introduction Aromatic five-membered nitrogen heterocycles have been potential targets of investigations because of their medicinal properties. In the last few decades, the chemistry of 1,2,4- triazole-3-thiones, 1,3,4-thiadiazoles and their fused hetero- cyclic derivatives has received considerable attention owing to their effective biological activities as anticonvulsant [1,2] and antioxidant [3]. Several imidazole derivatives have been published as antiviral [4], anticoagulant [5] and anticancer [6,7] agents. The 1,2,4-triazole rings have been incorporated into a wide variety of therapeutically interesting drug candidates including antiviral (Ribavarin), antimigraine (Rizatriptan) (Figure 1) [8-10], CNS stimulants sedatives and antianxiety [11,12] agents. Compounds connecting to 1,2,4- triazole moiety are famous for powerful antitumorial and anti- HIV agents [13-15]. The synthetic nucleoside ribavirin involving a 1,2,4-triazole nuclus has become an outstanding drug when combined with the pegylated interferon-α, for the treatment of hepatitis C virus infections [16]. We have published some papers containing the synthesis of some new 1,2,4-triazoles attached with several alkyl as well as their activities towards various cyclizing agents [17-20]. As a result, our interest has been recently directed towards the synthesis of some novel azoles and azines of expected antitumor activity [21,22]. 2. Experimental 2.1. Materials All experiments were performed using drying solvents. Thin-layer chromatography (TLC) was performed on a Merck Silica Gel 60F254 with detection by UV light. Products were purified by crystallization. 2.2. Instrumentation We measured the melting points that are uncorrected by an Electro thermal IA 9100 apparatus with open capillary tubes. The IR spectra (KBr disc) were recorded on a Pye Unicam Sp-3-300 or a Shimadzu FTIR 8101 PC infrared spectrophotometer. ABSTRACT RESEARCH ARTICLE KEYWORDS European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2018 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. http://dx.doi.org/10.5155/eurjchem.9.2.99-106.1701 http://dx.doi.org/10.5155/eurjchem.9.2.99-106.1701 https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.9.2.99-106.1701&domain=pdf&date_stamp=2018-06-30 http://www.eurjchem.com/ http://dx.doi.org/10.5155/eurjchem.9.2.99-106.1701 mailto:rhaggan99@hotmail.com mailto:m_gomaa59@yahoo.com mailto:mohasherif@yahoo.com mailto:mgalahom@yahoo.com mailto:rhaggan99@hotmail.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.9.2.99-106.1701&domain=pdf&date_stamp=2018-06-30� 100 Haggam et al. / European Journal of Chemistry 9 (2) (2018) 99-106 N HO OH HO N N O NH2 RizatriptanRibavarin N N N N H N Figure 1. Structure of ribavirin and rizatriptan. The 1H- and 13C-NMR spectra were recorded at a Varian Mercury VX-300 NMR (1H, 300 MHz, 13C, 75.4 MHz) spectrometer using DMSO-d6 as a solvent. All chemical shifts were expressed on the δ (ppm) scale using TMS as an internal standard reference. The coupling constant (Ј) values are given in Hz. Mass spectrometry and analytical data were obtained from the Microanalysis Center at Cairo University, Giza, Egypt. 2.3. Synthesis 2.3.1. Synthesis of N-((thiophene-2-carbonyl)carbamothio- yl)hydrazinecarboxamide hydrochloride (2) A mixture of thiophene-2-carbonylisothiocyanate (1) (3.40 mol) and semicarbazide hydrochloride (3.40 mol) in dioxane: water (1:1, v:v) (50 mL) was stirred at room temperature for 1 h. The reaction mixture was poured into cold water. The formed precipitate was filtered off, washed with cold water, dried and crystallized from ethanol to give compound 2 (Scheme 1). Color: White. Yield: 33%. M.p.: 210-211 °C. FT-IR (KBr, ν, cm-1): 3433, 3317 (NH2), 3156 (NH), 3207, 1671 (C=O), 1667 (C=C), 1267 (C-O). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 7.12-7.86 (m, 3H, thiophene protones), 8.33 (s, 1H, NH), 8.34 (s, 1H, NH), 8.99 (s, 1H, NH), 11.63 (s, 2H, NH2). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 167.1 (1C, NCS), 165.2, 164.3 (2C, 2C=O), 129.8, 128.4, 128.0, 127.5 (Ar-C). MS (EI, m/z (%)): 280 (M+, 100). 128 (4), 110 (100), 83 (30), 69 (35), 57 (60). 2.3.2. Synthesis of 2-hydrazinyl-6-(thiophen-2-yl)-4H-1,3,5- oxadiazine-4-thione (3) A mixture of N-((thiophene-2-carbonyl)carbamothioyl) hydrazinecarboxamide hydrochloride (2) (1.70 mol) and sodium ethoxide (1.70 mol) in ethanol (20 mL) was heated under reflux for 2 h. The reaction mixture was neutralized by 10% HCl. The formed precipitate was filtered off, dried and crystallized from ethanol to give compound 3 (Scheme 1). Color: White. Yield: 89%. M.p.: 279-280 °C. FT-IR (KBr, ν, cm- 1): 3411, 3325 (NH2, NH), 1599 (C=C), 1235 (C-O). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 7.75-7.19 (m, 3H, thiophene protons), 13.61 (s, 2H, D2O exchangeable, NH2), 13.91 (1H, D2O exchangeable, s, NH). 1H NMR (300 MHz, DMSO-d6, δ, H/D- exchange, ppm): 7.75-7.19 (m, 3H, thiophene protons). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 168.1 (1C, NCS), 163.6 (1C, OCN), 155.3 (1C, NCN), 129.5, 128.7, 128.3, 127.2 (Ar-C). MS (EI, m/z (%)): 226 (M+, 15), 183 (100), 124 (35), 110 (40), 70 (25), 69 (30). Anal. calcd. for C7H6N4OS2: C, 37.16; H, 2.67; N, 24.76. Found: C, 37.10; H, 2.53; N, 24.64%. 2.3.3. Synthesis of 1,2-dihydro-1-phenyl-5-(thiophen-2-yl)-1, 2,4-triazole-3-thione (4) A mixture of thiophene-2-carbonylisothiocyanate (1) (3.40 mol) and phenyl hydrazine (3.40 mol) in dioxane (50 mL) was heated under reflux for 2 h. The reaction mixture was poured into water. The formed precipitate was filtered off, washed with water, dried and crystallized from ethanol to give compound 4 (Scheme 1). Color: Yellow. Yield: 63%. M.p.: 175- 176 °C. FT-IR (KBr, ν, cm-1): 3313 (NH), 1635 (C=N), 1615 (C=C). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 7.93-6.72 (m, 8H, ArH’s + thiophene protons), 10.37 (s, 1H, D2O exchangeable, NH). 1H NMR (300 MHz, DMSO-d6, δ, H/D-exchange, ppm): 7.93-6.72 (m, 8H, ArH’s + thiophene protons). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 166.6 (1C, NCS), 156.4 (1C, NCN), 136.5, 129.4, 129.0, 128.6, 128.1, 127.7, 121.1, 119.2 (Ar-C). MS (EI, m/z (%)): 258 (M+, 20), 217 (30), 110 (100), 77 (20). Anal. calcd. for C12H9N3S2: C, 55.57; H, 3.50; N, 16.20. Found: C, 55.49; H, 3.45; N, 15.90%. 2.3.4. Synthesis of N-(1-phenylhydrazinecarbonothioyl)thio- phene-2-carboxamide hydrochloride (5) A mixture of thiophene-2-carbonylisothiocyanate (1) (10.0 mol) and phenyl hydrazine hydrochloride (10.0 mol) in dioxane:water (1:1, v:v) (50 mL) was stirred at room temperature for 1 h. The reaction mixture was poured into cold water. The formed precipitatet was filtered off, washed with cold water, dried and crystallized from ethanol to give compound 5 (Scheme 2). Color: White. Yield: 54%. M.p.: 268- 269 °C. FT-IR (KBr, ν, cm-1): 3406, 3280, (NH2, NH), 1665 (C=O), 1635 (C=C). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 7.98- 7.24 (m, 8H, ArH’s + thiophene protons), 11.62 (s, 2H, NH2), 14.41 (s, 1H, NH). 2.3.5. Synthesis of N-(6-oxo-2-phenyl-5,6-dihydro-1H- furo[2,3-e][1,3,4]thiadiazin-3(2H)-ylidene)thiophene-2- carboxamide (6) A mixture of N-(1-phenylhydrazinecarbonothioyl)thio- phene-2-carboxamide hydrochloride (5) (1.50 mol), maleic anhydride (1.50 mol) and triethyl amine (1.50 mole) in dimethyl formamide (20 mL) was heated under reflux for 6 h. The reaction mixture was neutralized by 10% HCl. The formed precipitate was filtered off, dried and crystallized from ethanol to give compound 6 (Scheme 2 and 3). Color: Brown. Yield: 61%. M.p.: 288-289 °C. FT-IR (KBr, ν, cm-1): 3631, 3217 (NH), 1723 (C=O) (ester), 1667 (C=O) (amide), 1631 (C=N), 1617 (C=C), 1244 (C-O). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 2.95 (s, 2H, CH2CO), 7.98-7.15 (m, 8H, ArH’s + thiophene protons), 12.65 (s, 1H, D2O exchangeable, NH). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 168.3, 165.2 (2C, 2C=O), 156.7 (1C, NCN), 143.1, 146.0, 136.9, 129.7, 129.4, 128.8, 128.2, 127.1, 122.1, 121.2, (Ar-C), 23.8 (1C, CH2). MS (EI, m/z (%)): 357 (M+, 20), 259 (100), 215 (10), 91 (25). Anal. calcd. for C16H11N3O3S2: C, 53.77; H, 3.10; N, 11.76. Found: C, 53.68; H, 2.99; N, 11.70%. 2.3.6. Synthesis of N-(5-hydroxy-3-phenyl-3,6-dihydro-2H- 1,3,4-thiadiazin-2-ylidene)thiophene-2-carboxamide (7) A mixture of N-(1-phenylhydrazinecarbonothioyl)thiophene- 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.2.99-106.1701 Haggam et al. / European Journal of Chemistry 9 (2) (2018) 99-106 101 S 1 4 Dioxane/H2O (1:1) Stirring / r.t. / 1 h 33% S N O N S N H N H 2 3 O H N H N S H N O NH2S O H N H N N H S Ph S O N C S S N NH N Ph S HCl - H2O H2N N H NH3 Cl O H2N N H Ph Dioxane / Reflux 2 h 63% NaOEt/EtOH Reflux 2 h 89% H Scheme 1 2-carboxamide hydrochloride (5) (1.50 mol), ethyl chloro- acetate (1.50 mol) and triethyl amine (1.50 mole) in ethanol (20 mL) was heated under reflux for 6 h. The reaction mixture was neutralized by 10% HCl. The formed precipitate was filtered off, dried and crystallized from ethanol to give compound 7 (Scheme 2). Color: White. Yield: 79%. M.p.: 280- 281 °C. FT-IR (KBr, ν, cm-1): 3631 (OH), 3117 (CH arom.), 1667 (C=O) (amide), 1631 (C=N) 1220 (C-O). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 2.23 (s, 2H, CH2), 8.28-7.15 (m, 8H, ArH’s + thiophene protons), 12.95 (s, 1H, D2O exchangeable, OH). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 165.8 (1C, C=O), 159.1, 156.4 (2C, C=N), 136.1, 129.2, 128.8, 128.4, 127.9, 127.0, 123.1, 121.8 (Ar-C), 34.3 (1C, SCH2). MS (EI, m/z (%)): 317(M+, 100), 271 (20), 200 (30), 110 (30), 91 (10), 77 (50). Anal. calcd. for C14H11N3O2S2: C, 52.98; H, 3.49; N, 13.24. Found: C, 52.93; H, 3.40; N, 13.16%. 2.3.7. Synthesis of 1,2-dihydro-2-phenyl-5-(thiophen-2-yl)- 1,2,4-triazole-3-thione (8) A mixture of N-(1-phenylhydrazinecarbonothioyl)thio- phene-2-carboxamide hydrochloride (5) (1.50 mol) and sodium ethoxide (1.50 mol) in ethanol (20 mL) was heated under reflux for 2 h. The reaction mixture was neutralized by 10% HCl. The formed precipitate was filtered off, dried and crystallized from ethanol to give compound 8 (Scheme 2). Color: Pale white. Yield: 95%. M.p.: 290-291 °C. FT-IR (KBr, ν, cm-1): 3424 (NH), 1635 (C=N), 1597 (C=C). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 8.06-7.18 (m, 8H, ArH’s + thiophene protons), 10.41 (s, 1H, NH). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 167.1 (1C, NCS), 156.1 (1C, NCN), 135.8, 129.7, 129.1, 128.4, 128.3, 127.1, 121.4, 120.2 (Ar-C). MS (EI, m/z (%)): 259 (M+, 100), 200 (10), 149 (5), 108 (10), 91 (25). Anal. calcd. for C12H9N3S2: C, 55.57; H, 3.50; N, 16.20. Found: C, 55.46; H, 3.39; N, 16.10%. 2.3.8. Synthesis of N-(2-isonicotinoylhydrazinecarbonothio- yl)thiophene-2-carboxamide (11) A mixture of thiophene-2-carbonylisothiocyanate (1) (10.0 mol) isonicotinohydrazide (10.0 mol) in dioxane (50 mL) was stirred at room temperature for 1 h. The reaction mixture was poured into water. The formed precipitate was filtered off, washed with water, dried and crystallized from ethanol to give compound 11 (Scheme 4). Color: Yellow. Yield: 58%. M.p.: 212-213 °C. FT-IR (KBr, ν, cm-1): 3419, 3252 (NH), 3018 (CH arom.), 1671, 1666 (2C=O). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 7.98-7.24 (m, 7H, ArH’s + thiophene protons), 11.41 (s, 1H, D2O exchangeable, NH), 11.85 (s, 1H, D2O exchangeable, NH), 12.15 (s, 1H, D2O exchangeable, NH). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 167.1 (1C, NCS), 156.1 (1C, NCN), 135.8, 129.7, 129.1, 128.4, 128.3, 127.1, 121.4, 120.2 (Ar-C). MS (EI, m/z (%)): 306 (M+, 100), 273 (15), 170 (5), 137 (5), 110 (10), 78 (30). 2.3.9. Synthesis of N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2- yl)thiophene-2-carboxamide (12) A mixture of N-(2-isonicotinoylhydrazinecarbonothioyl) thiophene-2-carboxamide (11) (1.20 mol) and sulfuric acid (1.20 mol) in ethanol (20 mL) was heated under reflux for 6 h. The reaction mixture was poured into water. The formed precipitate was filtered off, dried and crystallized from ethanol to give compound 12 (Scheme 4). Color: White. Yield: 95%. M.p.: 320-321 °C. FT-IR (KBr, ν, cm-1): 3386 (NH), 1662 (C=O) (amide), 1637 (C=N), 1628 (C=C). 1H NMR (300 MHz, DMSO- d6, δ, ppm): 8.97-7.30 (m, 7H, ArH’s + thiophene protons), 8.98 (s, 1H, D2O exchangeable, NH). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 162.4 (1C, C=O), 156.4, 152.1 (2C, triazole ring), 149.0, 135.3, 121.4, 137.8, 129.2, 128.4, 127.0 (Ar-C). MS (EI, m/z (%)): 288 (M+, 60), 261 (100), 250 (50), 173 (55), 160 (50), 112 (60). Anal. calcd. for C12H8N4OS2: C, 49.98; H, 2.80; N, 19.43. Found: C, 49.87; H, 2.69; N, 19.38%. 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.2.99-106.1701 102 Haggam et al. / European Journal of Chemistry 9 (2) (2018) 99-106 1 S O N C S Ph N H NH3 Cl Dioxane / H2O (1:1) Stirring / r.t. / 1 h 54% 5 S O H N N NH3 S Ph Cl O O O DMF/TEA Reflux 2 h 61% S O N N NH S Ph O O6 S O N N NH2 S Ph CH2 C O OEt S O N N N S Ph OH 7 Cl OEt OTEA / EtOH Reflux 6 h 79% NaOEt/EtOH Reflux 2 h 95% 8 S HN N N S Ph Scheme 2 9 S O N N NH2 S Ph O O O S O N N NH S Ph O COOH 10 5 S O H N N NH3 S Ph Cl O O O DMF/TEA S O N N NH S Ph O O6 H - H2O Scheme 3 2.3.10. Synthesis of (3-mercapto-5-(pyridin-4-yl)-4H-1,2,4- triazol-4-yl)(thiophen-2-yl)methanone (13) A mixture of N-(2-isonicotinoylhydrazinecarbonothioyl) thiophene-2-carboxamide (11) (1.20 mol) and lead acetate (1.20 mol) in acetic acid (20 mL) was heated under reflux for 6 h. The reaction mixture was poured into water. The formed precipitate was filtered off, dried and crystallized from ethanol to give compound 13 (Scheme 4). Color: Grey. Yield: 83%. M.p.: 327-328 °C. FT-IR (KBr, ν, cm-1): 3108 (CH arom.), 1661 (C=O), 1624 (C=C). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 8.72- 7.27 (m, 7H, ArH’s + thiophene protons), 13.08 (s, 1H, D2O exchangeable, SH). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 168.4 (1C, C=O), 157.6, 152.8 (2C, triazole ring), 149.6, 134.3, 124.2, 129.2, 128.1, 127.6, 120.0 (Ar-C). MS (EI, m/z (%)): 288 (M+, 100), 259 (55), 193 (15), 127 (20), 110 (95), 63 (15). Anal. calcd. for C12H8N4OS2: C, 49.98; H, 2.80; N, 19.43. Found: C, 49.91; H, 2.75; N, 19.35%. 2.3.11. Synthesis of 5-(pyridin-4-yl)-2H-1,2,4-triazole- 3(4H)-thione (14) A mixture of N-(2-isonicotinoylhydrazinecarbonothioyl) thiophene-2-carboxamide (11) (1.20 mol) and potassium hydroxide (1.20 mol) in ethanol (20 mL) was heated under reflux for 6 h. 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.2.99-106.1701 Haggam et al. / European Journal of Chemistry 9 (2) (2018) 99-106 103 1 S O N C S Dioxane / stirring / r.t. 1 h KOH/EtOH Reflux 6 h 95% N H H2N O N S O 12 13 S O H N N N S N N N N N HS Pb(CH3COO)2/AcOH Reflux 6 h S O N N H N N S Hyd. HN N H N N S 14 58% 11 S O H N H N N H S O N H2SO4/EtOH Reflux 6 h 83% -H2O 93% Scheme 4 The reaction mixture was poured into water. The formed precipitate was filtered off, dried and crystallized from ethanol to give compound 14 (Scheme 4). Color: Yellow. Yield: 93%. M.p.: 318-319 °C. FT-IR (KBr, ν, cm-1): 3445 (NH), 3087 (CH arom.), 1648 (C=N). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 8.46-7.15 (m, 4H, ArH’s), 8.97 (s, 1H, D2O exchangeable, NH), 8.98 (s, 1H, D2O exchangeable, NH). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 169.6, 151.5 (2C, triazole ring), 148.1, 134.8, 123.0 (Ar-C). MS (EI, m/z (%)): 178 (M+, 10), 127 (60), 110 (100), 82 (15), 56 (35). Anal. calcd. for C7H6N4S: C, 47.18; H, 3.39; N, 31.44. Found: C, 47.10; H, 3.26; N, 31.33%. 2.3.12. Synthesis of ethyl 2-(3-(thiophene-2-carbonyl)thiou- reido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (15) A mixture of thiophene-2-carbonylisothiocyanate (1) (3.3 mol) and ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene- 3-carboxylate (3.3 mol) in dioxane (50 mL) was heated under reflux for 1 h. The reaction mixture was poured into water. The formed precipitate was filtered off, washed with water, dried and crystallized from acetic acid to give compound 15 (Scheme 5). Color: Yellow. Yield: 70%. M.p.: 236-237 °C. FT-IR (KBr, ν, cm-1): 3412, 3261(2NH), 1735 (C=O) (ester), 1677 (C=O) (amide), 1649 (C=C). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.34 (t, J = 2.3 Hz, 3H, CH3), 1.72 (m, 4H, cyclohexane- 2CH2), 2.63 (t, J = 5.6 Hz, 2H, cyclohexane-CH2), 2.75 (t, J = 5.6 Hz, 2H, cyclohexane-CH2), 4.37 (q, J = 2.3 Hz, 2H, CH2), 7.24 (d, J = 3.6 Hz, 1H, thiophene-Hc), 8.06 (d, J = 4.8 Hz, 1H, thiophene- Ha), 8.38 (t, J = 4.8 Hz, J = 3.6 Hz, 1H, thiophene-Hb), 11.84 (s, 1H, D2O exchangeable, NH), 14.61 (s, 1H, D2O exchangeable, NH). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 167.3 (1C, C=S), 162.4, 161.7 (2C, 2C=O), 150.1, 132.6, 130.8, 129.2, 128.4, 126.2, 125.4, 114.2 (Ar-C), 61.2 (1C, OCH2), 25.8, 23.9, 23.2, 22.5 (4C, 4CH2-cyclohexane), 14.3 (1C, CH3). MS (EI, m/z (%)): 394 (M+, 100), 267 (10), 225 (40), 179 (65), 150 (30), 110 (25). Anal. calcd. for C17H18N2O3S3: C, 51.75; H, 4.60; N, 7.10. Found: C, 51.66; H, 4.52; N, 7.02%. 2.3.13. Synthesis of 2-(3-(thiophene-2-carbonyl)thiourei- do)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid (16) A mixture of ethyl 2-(3-(thiophene-2-carbonyl)thiou- reido)-4, 5, 6, 7-tetrahydrobenzo[b]thiophene-3-carboxylate (15) (1.26 mol) and sodium ethoxide (1.26 mol) in ethanol (20 mL) was heated under reflux for 6 h. The reaction mixture was neutralized by 10% HCl. The formed precipitate was filtered off, dried and crystallized from ethanol to give compound 16 (Scheme 5). Color: Yellow. Yield: 86%. M.p.: 282-283 °C. FT-IR (KBr, ν, cm-1): 3654 (OH), 3424 (NH), 1697 (C=O) (acid), 1676 (C=O) (amide). 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.2.99-106.1701 104 Haggam et al. / European Journal of Chemistry 9 (2) (2018) 99-106 Dioxane Reflux 1 h 86% 70% S NH2 COOEt S N H N H S O S S N H COOH N H S O S S N H N S O O S COOEt HN N H S O S HN H N S O S N H O SN N S NH O S - H2S NH2 NH2 15 16 17 19 SO3H NH2 OH SO3H NH2 O N S S 18 1 S O N C S EtONa Reflux 6 h Dioxane Reflux 2 h 65% Dioxane Reflux 2 h 74% Scheme 5 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.75 (m, 4H, cyclohexane-2CH2), 2.67 (t, J = 5.6 Hz, 2H, cyclohexane-CH2), 2.71 (t, J = 5.6 Hz, 2H, cyclohexane-CH2), 7.23 (d, J = 3.6 Hz, 1H, thiophene-Hc), 8.01 (d, J = 4.8 Hz, 1H, thiophene-Ha), 8.29 (t, J = 4.8 Hz, J = 3.6 Hz, 1H, thiophene-Hb), 11.76 (s, 1H, D2O exchangeable, NH), 13.29 (s, 1H, D2O exchangeable, NH), 14.61 (s, 1H, D2O exchangeable, OH). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 167.4 (1C, C=S), 165.6, 164.7 (2C, 2C=O), 149.8, 132.0, 130.3, 129.4, 128.1, 126.6, 125.0, 114.7 (Ar-C), 25.0, 24.1, 23.4, 22.7 (4C, 4CH2-cyclohexane). MS (EI, m/z (%)): 366 (M+, 100), 287(20), 237(95), 178(45), 150(40), 110(10). Anal. calcd. for C15H14N2O3S3: C, 49.16; H, 3.85; N, 7.64. Found: C, 49.09; H, 3.72; N, 7.54%. 2.3.14. Synthesis of 10-amino-2-(thiophen-2-yl)-4-thioxo- 4H-naphtho[2,3-e][1,3]oxazine-5-sulfonic acid (18) A mixture of thiophene-2-carbonylisothiocyanate (1) (3.34 mol) and 4-amino-3-hydroxynaphthalene-1-sulfonic acid (3.34 mmol) in dioxane (20 mL) was heated under reflux for 2 h. The reaction mixture was poured into water. The formed precipitate was filtered off, washed with water, dried and crystallized from ethanol to give compound 18 (Scheme 5). Color: Brown. Yield: 65%. M.p.: 322-323 °C. FT-IR (KBr, ν, cm- 1): 3652 (OH), 3237 (NH2), 1352(C-O). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 8.80-7.38 (m, 7H, ArH’s + thiophene protons), 9.78-9.74 (br s, 2H, D2O exchangeable, NH2), 11.00 (s, 1H, D2O exchangeable, OH). MS (EI, m/z (%)): 390(M+, 100), 280 (70), 168 (50), 159 (90), 129 (40), 112 (20). Anal. calcd. for C16H10N2O4S3: C, 49.22; H, 2.58; N, 7.17. Found: C, 49.11; H, 2.53; N, 7.14%. 2.3.15. Synthesis of N-(4,5-dihydro-1-(sulfanylene(thiophe- ne-2-carboxamido)methyl)-1H-imidazol-2-yl)thiophene-2- carboxamide (19) A mixture of thiophene-2-carbonylisothiocyanate (1) (3.34 mol) and ethylene diamine (3.34 mol) in dioxane (20 mL) was heated under reflux for 2 h. The reaction mixture was poured into water. The formed precipitate was filtered off, washed with water, dried and crystallized from ethanol to give compound 19 (Scheme 5). Color: White. Yield: 74%. M.p.: 328- 329 °C. FT-IR (KBr, ν, cm-1): 3391, 3290 (NH), 1674, 1665 (2C=O) (amide), 1537 (C=N). 1H NMR (300 MHz, DMSO-d6, δ, ppm): 3.33 (t, J = 2.6 Hz, 2H, CH2), 3.94 (t, J = 2.6 Hz, 2H, CH2), 8.65-7.13 (m, 6H, thiophene protons), 10.80 (s, 1H, D2O exchangeable, NH), 11.40 (s, 1H, D2O exchangeable, NH). 13C NMR (75.4 MHz, DMSO-d6, δ, ppm): 168.41 (1C, C=S), 161.4 (2C, 2C=O), 153.8 (1C, NCN), 130.8, 129.0, 128.3, 119.7 (2Ar- C), 54.4, 52.7 (1C, 2CH2-imidazole). MS (EI, m/z (%)): 366 (M+, 100), 364(M+, 15), 261(10), 185(5), 110(100), 82(20), 57(25). Anal. calcd. for C14H12N4O2S3: C, 46.14; H, 3.32; N, 15.37. Found: C, 46.31; H, 3.20; N, 15.26%. 2.4. Antitumor activity test - tumor cell growth assay Human tumor cell line, MCF-7 (breast adenocarcinoma) MCF-7 was kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). The effects of compounds 6, 7, 8, 12, 13, 15, 16 and 18 on the in vitro growth of human tumor cell line were evaluated according to the method adopted by the National Cancer Institute (NCI, USA) [22-24]. 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.2.99-106.1701 Haggam et al. / European Journal of Chemistry 9 (2) (2018) 99-106 105 Table 1. The inhibitory activities of the synthesized compounds on the growth of MCF-7 cell line. Compound GI50 (μg/L) MCF-7 8 10.29±5.44 12 0.01±0.004 13 0.01±0.008 15 0.01±0.001 16 0.01±0.003 18 2.70±1.24 Doxorubicin 0.04±0.008 Table 2. Evaluation of cytotoxicity of the obtained compounds against MCF-7 cell line. Compound Viability rate (%) IC50 (mg/mL) 0.1 μg/mL 1 μg/mL 10 μg/mL 8 87.26±6.29 82.21±3.98 80.23±5.56 10.49±7.44 12 56.52±4.42 50.89±5.50 55.23±3.90 0.01±0.90 13 54.62±4.53 58.09±6.50 53.34±3.81 0.01±0.50 15 51.32±4.42 55.89±5.50 51.23±3.90 0.01±0.008 16 51.62±4.53 58.09±6.50 51.34±3.81 0.01±0.009 18 93.26±4.19 91.20±3.66 82.09±5.71 2.69±0.04 3. Results and discussion 3.1. Chemistry This research aims at presenting importance of isothio- cyanates as synthetic intermediate for building biologically active heterocycles [25,26]. To begin with addition of semicarbazide hydrochloride to thienoylisothiocyanate (1) [27] in dioxane:water (1:1, v:v) (50 mL) with stirring at room temperature for 1 h afforded thiosemicarbazide derivative 2 as a result of nucleophilic addition to the electrophilic carbon of the isothiocyanate group. An intramolecular basic cyclization of thiophene derivative 2 in sodium ethoxide resulted in oxadiazine nuclus 3 in 89% yield (Scheme 1). Moreover, addition of phenylhydrazine to the heteroallene 1 led to formation of the expected triazole 4 in 63% yield after loss of H2O molecule (Scheme 1). The new structures of the prepared compounds were elucidated on the bases of their spectroscopic data. As an example, the IR spectrum of oxadiazine derivative 3 exhibited characteristic absorption bands at 3411, 3325, 1599 and 1235 cm-1 for NH, NH2, C=N and C=S, respectively. In addition to, absence of carbonyl stretching peak is observed. The 1H NMR of compound 3 showed two singlets with different integration at δ 13.91 and 13.61 ppm for NH and NH2 protons, respect- tively. There is a multiplet in between δ 7.75-7.19 ppm for three protons of the thiophene ring. Disappearance the two peaks at δ 13.91 and 13.61 ppm in D2O spectrum of compound 3 emphasized that the NH and NH2 protons were exchangeable with D2O and this is proof for the correct suggested structure. Also, the mass spectrometry of compound 3 is in agreement with its structure as well as its molecular weight. The 1H NMR spectrum of compound 4 revealed a sharp singlet at δ 10.37 ppm for NH proton which was D2O exchangeable. Addition of phenylhydrazine hydrochloride to thiophene- 2-carbonyl isothiocyanate (1) in dioxane:water (1:1, v:v) produced thiosemicarbazide 5 [23-25]. The synthesis of thiadiazine derivative 6 in 61% yield was achieved via one pot three component reaction of compound 5, maleic anhydride and triethyl amine in DMF (Scheme 2). A mixture of ethyl chloroacetate and thiophene-2-carboxamide hydrochloride 5 was heated for 6 h to yield thiadiazine 7 in 79% yield. Upon heating 5 with sodium ethoxide for 2 h, the triazole 8 was obtained in 95% yield as in Scheme 2. The IR spectrum of compound 8 showed strong bands at 3424, 1597 and 1252 cm-1 for NH, C=N and C=S, respectively. The 1H NMR spectrum of triazole 8 displayed a singlet at δ 14.41 ppm for NH proton which was exchangeable with D2O as well. A multiplet peak is at δ 8.06-7.18 ppm for aromatic protons. The mass spectrometry of compound 8 is in agreement with its suggested structure. The suggested mechanism for establishment of thienoyl- thiadiazine 6 showed that formation of acyclic thienoylimine intermediate 9. Subsequently, the intermediate 9 underwent an intramolecular nucleophilic cyclization to form thiadiazine derivative 10 that was dehydrated giving thienoylthiadiazine structure 6 (Scheme 3). Thiosemicarbazide derivative 11 was synthesized by the addition of nicotinic hydrazide to thiophene-2-carbonyl iso- thiocyanate (1) in dioxane as outlined in (Scheme 4) [23-25]. Depending on reaction condition compound 11 underwent several kinds of heterocyclizations. So, acid mediated cycliza- tion of compound 11 resulted in thiadiazole ring 12 in 95% yield. Heating of compound 11 with lead acetate in acetic acid produced triazole derivative cyclization 13 in 83% yield. On the other hand, base mediated cyclization of compound 11 afforded triazolethione 14 in 93% yield via thiophene inter- mediate (Scheme 4). The thiourea derivative of type 15 was obtained in 70% yield as a result of addition of ethyl 2-amino-4,5,6,7-tetra hydrobenzo[b]thiophene-3-carboxylate in dioxane to thienoyl- isothiocyante 1 (Scheme 5). Upon heating of compound 15 with ethoxide produced thiophene-3-carboxylic acid 16 in 86% yield but the other cyclized product 17 was not formed. Moreover, addition of amino phenol sulfonic acid derivative to heteroallene 1 in dioxane resulted in building the oxazine structure 18 in 65% yield. On the other hand, nucleophilic addition of ethylene diamine in dioxane to thiophene-2- carbonyl isothiocyanate (1) led to formation of imidazole derivative 19 in 74% yield as outlined in Scheme 5. The 1H NMR spectrum of thiophene carboxylic acid 16 revealed three sharp singlets at δ 14.61, 13.29 and 11.76 ppm for OH and 2NH protons which were D2O exchangeable. A triplet peak at δ 8.29 ppm with J = 4.8 and 3.6 Hz for thiophene-Hb. There are two doublets at δ 8.01 and 7.23 ppm for thiophene-Ha and thiophene-Hc. 3.2. Antitumor activity The inhibitory activities of the compounds 8, 12, 13, 15, 16 and 18 were evaluated on the in vitro growth of cancer human cell line breast adenocarcinoma MCF-7. Nearly, all the screened compounds were able to inhibit the growth of the tested human tumor cell line. The results tabulated in Table 1 indicated that Compounds 12, 13, 15 and 16 showed the highest inhibitory effect against MCF-7 cell line compared to the reference doxorubicin. Meanwhile compounds 18 and 8 exhibited high inhibitory effects, which are less than the standard reference in Table 1. 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.2.99-106.1701 106 Haggam et al. / European Journal of Chemistry 9 (2) (2018) 99-106 The cytotoxic and antitumor activities of the compounds 8, 12, 13, 15, 16 and 18 were tested against MCF-7 cells line. The inhibitory activities were detected by using different concentrations as shown in Table 2. Results presented in Table 2 displayed that, compounds 15, 16, 13 and 12 had very strong cytotoxic antitumor activity, while compounds 18 and 8 have strong cytotoxic antitumor activity against MCF-7 cell line. 4. Conclusion In conclusion, we were able to realize the synthesis of a series of new 1,2,4-triazolethione, 1,3,4-thiadiazole, 1,3- imidazole and oxadiazine derivatives bearing thiophene ring system in high yields 61-95%. Compounds were screened for their anticancer activity on cancer human cell lines like breast cancer MCF-7. Most of selected compounds showed remarkable antitumor activity. Compounds 12, 13, 15 and 16 are the most active ones on the cancer cell line due to their reactivity is more than that of the standard doxorubicin on the same cell line. Accordingly, these preliminary results of the newly synthesized thiophene derivatives can serve as a starting point for the development of potent anti-cancer agents. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Author contributions: All authors contributed equally to this work. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. ORCID Reda Ahmed Haggam http://orcid.org/0000-0001-8568-9668 References [1]. Barbuceanu, S. F.; Saramet, G.; Almajan, G. L.; Draghici, C.; Barbuceanu, F.; Bancescu, G. Bioorg. Med. 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Commun. 1990, 55, 1049-1054. Copyright © 2018 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.2.99-106.1701 http://orcid.org/0000-0001-8568-9668 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Materials 2.2. Instrumentation 2.3. Synthesis 2.3.1. Synthesis of N-((thiophene-2-carbonyl)carbamothio- yl)hydrazinecarboxamide hydrochloride (2) 2.3.2. Synthesis of 2-hydrazinyl-6-(thiophen-2-yl)-4H-1,3,5-oxadiazine-4-thione (3) 2.3.3. Synthesis of 1,2-dihydro-1-phenyl-5-(thiophen-2-yl)-1, 2,4-triazole-3-thione (4) 2.3.4. Synthesis of N-(1-phenylhydrazinecarbonothioyl)thio- phene-2-carboxamide hydrochloride (5) 2.3.5. Synthesis of N-(6-oxo-2-phenyl-5,6-dihydro-1H-furo[2,3-e][1,3,4]thiadiazin-3(2H)-ylidene)thiophene-2-carboxamide (6) 2.3.6. Synthesis of N-(5-hydroxy-3-phenyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-ylidene)thiophene-2-carboxamide (7) 2.3.7. Synthesis of 1,2-dihydro-2-phenyl-5-(thiophen-2-yl)-1,2,4-triazole-3-thione (8) 2.3.8. Synthesis of N-(2-isonicotinoylhydrazinecarbonothio-yl)thiophene-2-carboxamide (11) 2.3.9. Synthesis of N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)thiophene-2-carboxamide (12) 2.3.10. Synthesis of (3-mercapto-5-(pyridin-4-yl)-4H-1,2,4-triazol-4-yl)(thiophen-2-yl)methanone (13) 2.3.11. Synthesis of 5-(pyridin-4-yl)-2H-1,2,4-triazole-3(4H)-thione (14) 2.3.12. Synthesis of ethyl 2-(3-(thiophene-2-carbonyl)thiou-reido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (15) 2.3.13. Synthesis of 2-(3-(thiophene-2-carbonyl)thiourei- do)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid (16) 2.3.14. Synthesis of 10-amino-2-(thiophen-2-yl)-4-thioxo-4H-naphtho[2,3-e][1,3]oxazine-5-sulfonic acid (18) 2.3.15. Synthesis of N-(4,5-dihydro-1-(sulfanylene(thiophe-ne-2-carboxamido)methyl)-1H-imidazol-2-yl)thiophene-2-carboxamide (19) 2.4. Antitumor activity test - tumor cell growth assay 3. Results and discussion 3.1. Chemistry 3.2. Antitumor activity 4. Conclusion Disclosure statement ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: