untitled European Journal of Chemistry 5 (4) (2014) 644‐651 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2014 Eurjchem Publishing ‐ Printed in the USA http://dx.doi.org/10.5155/eurjchem.5.4.644‐651.1125 European Journal of Chemistry Journal homepage: www.eurjchem.com Synthesis and antioxidant evaluation of novel sophisticated carboxamides based on 3‐(ethoxycarbonyl)‐4,5,6,7‐tetrahydro‐1‐benzothiophen‐2‐amine Moustafa Ahmed Gouda a,b,* and Serry Atta Atta El Bialy c a Chemistry Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt b Department of Chemistry, Faculty of Science and Arts, Taibah University, Ulla, 41411, Kingdom of Saudi Arabia c Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt *Corresponding author at: Chemistry Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt. Tel.: +2.050.6432235. Fax: +2.050.2246781. E‐mail address: dr_mostafa_chem@yahoo.com (M.A. Gouda). ARTICLE INFORMATION ABSTRACT DOI: 10.5155/eurjchem.5.4.644‐651.1125 Received: 18 July 2014 Received in revised form: 05 August 2014 Accepted: 23 August 2014 Online: 31 December 2014 KEYWORDS Condensation of cyanoacetamide 3 with cycloalkanones and elemental sulfur in the presence of morpholine yielded the bisthiophenes 4 and 5. Also, its (3) condensation with terphthalaldehyde or coupling with p‐phenylenedidiazonium chloride afforded compound 13 and 14, respectively. Furthermore, cyanoacetylation of compound 4 or 5 afforded the cyanoacetamides 6 and 7, respectively. Knoevenagel condensation of compound 7 with aromatic aldehyde afforded the arylidenes 10, 11 and coumarin 12, respectively. Treatment of compound 3 with CS2 in DMF/KOH followed by alkylation reaction with ethyl bromoacetate afforded the triester derivative 16, which gave 3‐aminothiophene 17 upon heating in DMF/TEA. Moreover, refluxing of compound 23 with α‐haloketones afforded 3‐ aminothiophenes 24 and 25. Identity of newly synthesized compounds was established by the spectral data and novel compounds were evaluated as antioxidant agents. Thiocarbamoyl Cyanoacetylation 2‐Aminothiophene Antioxidant evaluation 3,5‐Dimethyl‐1H‐pyrazole 3‐(Ethoxycarbonyl)‐4,5,6,7‐tetrahydro‐1‐ benzothiophen‐2‐amine 1. Introduction Substituted 2‐aminothiophenes are important interme‐ diates in the synthesis of a variety of agrochemicals, dyes and pharmacologically active compounds [1‐3]. The most convergent and well‐established classical approach for the preparation of 2‐aminothiophenes is Gewald’s method [4], which involves multicomponent condensation of a ketone with an activated nitrile and elemental sulfur in the presence of morpholine as a catalyst. 3‐(3,5‐Dimethyl‐1H‐pyrazol‐1‐yl)‐3‐ oxopropanenitrile (2) is a very handy and cheap cyano acetylation reagent, which was first, synthesized and introdu‐ ced in common practice in the late 1950s by Ried and Scheimer [5]. It was successfully applied for the synthesis of various N‐ alkyl and N‐aryl cyanoacetamides [6]. Recently, Gorobets et al. [7], prepare a series from cyanoacetamides via refluxing of 2 with the appropriate amines in toluene. More recently, we used compound 2 for preparation of ethyl 2‐(2‐cyano‐acetylamino)‐ 4,5,6,7‐tetrahydro‐1‐benzothiophene‐3‐carboxylate, 3 [8]. On the other hand, oxidative stress results in oxidative alteration of biological macromolecules such as lipids, proteins and nucleic acids. It is considered to play a pivotal role in the pathogenesis of aging and degenerative diseases [9‐11]. In order to cope with an excess of free radicals produced upon oxidative stress, human bodies have developed sophisticated mechanisms for maintaining redox homeostasis. These protective mechanisms include scavenging or detoxification of reactive oxygen species (ROS), blocking ROS production, sequestration of transition metals, as well as enzymatic and nonenzymatic antioxidant defenses produced in the body, that is, endogenous [12,13] and others supplied with the diet, namely, exogenous ones. Among them, dietary polyphenols have been widely studied for their strong antioxidant capacities and other properties by which cell functions are regulated [14, 15]. Recently, thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives showed interesting biological properties including antioxidant and antitumor activities [16]. In view of the above result we use herein 3 as a building block for the construction of poly‐thiophene ring systems in order to evaluate their antioxidant activity. 2. Experimental 2.1. Instrumentations All melting points are determined on Gallenkamp electric melting point apparatus (uncorrected). Gouda and El‐Bialy / European Journal of Chemistry 5 (4) (2014) 644‐651 645 Scheme 1 Thin layer chroma‐tography (TLC) analysis was carried out on silica gel 60F254 precoated aluminum sheets. The IR spectra were recorded (KBr) on a Mattson 5000 FTIR Spectrophoto‐ meter at the Microanalytical Unit, Faculty of Science, Mansoura University 1H/13C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Bruker 400 NMR spectrometer in the indicated solvents using TMS as an internal reference, at the Georgia State University, Atlanta, Georgia, USA. The mass spectra (EI) were recorded on Kratos MS equipment at the Microanalytical Center, Cairo University, Egypt. Elemental analyses (C, H and N) were carried out at the Microanalytical Center of Cairo University, Egypt. Biological activities were carried out at Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. Ethyl 2‐ amino‐4,5,6,7‐tetrahydro‐1‐benzothiophene‐3‐carboxy late (1) [17], ethyl 2‐[(cyanoacetyl)amino]‐4,5,6,7‐tetrahydro‐1‐benzo thiophene‐3‐carboxylate (3) [8] and (Z)‐ethyl 2‐(2‐cyano‐3‐ mercapto‐3‐(phenylamino)acrylamido)‐4, 5, 6, 7‐tetrahydro benzo[b]thiophene‐3‐carboxylate (23) [8] were reported in the literature 2.2. Synthesis 2.2.1. Reaction of ethyl 2‐[(cyanoacetyl) amino]‐4,5,6,7‐tetra hydro‐1‐benzothiophene‐3‐carboxylate (3) with cyclic ketones and elemental sulfur General procedure: Morpholine (0.33 mL, 3.9 mmol), elemental sulfur (0.141 g, 4.4 mmol) and cyclopentanone (0.34 g, 4 mmol) or cyclohexanone (0.39 g, 4 mmol) were added to a solution of compound 3 (1.17 g, 4 mmol) in ethanol (20 mL). The reaction mixture was refluxed at 60‐80 °C for 6 h and left to stand at room temperature. The separated crystalline product was filtered, dried and recrystallized from ethanol:benzene mixture (3:2, v:v) to give compounds 4 and 5, respectively (Scheme 1). Ethyl 2‐{[(2‐amino‐5,6‐dihydro‐4H‐cyclopenta[b]thien‐3‐yl) carbonyl]amino}‐4,5,6,7‐tetrahydro‐1‐benzothiophene‐3‐carboxy late (4): Color: White powder. Yield: 1.39 g, 89%. M.p.: 241 °C. FT‐IR (KBr, ν, cm‐1): 3415, 3305, 3261 (NH2, NH), 1673, 1616 (C=O). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.30 (t, 3H, J = 6.8 Hz, CH3), 1.71‐1.73 (m, 4H, C5‐2H, C6‐2H, cyclohexane skeleton), 2.37‐2.40 (m, 2H, C5‐2H, cyclopentane skeleton), 2.58‐2.70 (m, 6H, C4‐2H, C7‐2H, cyclohexane skeleton and C4‐ 2H, cyclopentane skeleton), 3.09‐3.18 (m, 2H, C6‐2H, cyclopentane skeleton), 4.28 (q, 2H, J = 6.8 Hz, CH2O), 7.53 (s, 2H, NH2), 11.04 (s, 1H, NH‐CO). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 168.7, 165.3, 161.3, 147.0, 137.6, 129.8, 125.3, 120.3, 110.0, 100.9, 59.9, 29.28, 25.7, 23.4, 22.3, 22.2, 13.8. EI‐MS (m/z (%)): 392 (M++2, 3.13), 390 (M+, 19.9), 344 (3.3), 300 (0.8), 225 (62.9), 178 (100), 166 (73.0), 150 (68.2), 138 (26.7), 122 (54.9), 109 (65.9), 104 (86.8), 90 (32.2), 76 (5.0), 65 (48.2). Anal. calcd. for C19H22N2O3S2: C, 58.44; H, 5.68; N, 7.17. Found: C, 58.40; H, 5.64; N, 7.14%. Ethyl 2‐{[(2‐amino‐4, 5, 6, 7‐tetrahydro‐1‐benzothien‐3‐yl) carbonyl]amino}‐4,5,6,7‐tetrahydro‐1‐benzothiophene‐3‐carboxy late (5): Color: White powder. Yield: 1.45 g, 90%. M.p.: 200 °C. FT‐IR (KBr, ν, cm‐1): 4421, 3397, 3299 (NH, NH2), 1681, 1623 (C=O). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.30 (t, 3H, J = 6.8 Hz, CH3), 1.73‐1.76 (m, 8H, 2C5‐2H, 2C6‐2H , cyclohexane skeleton), 2.50‐2.59 (m, 4H, 2C4‐2H, cyclohexane skeleton), 2.72‐2.78 (m, 4H, 2C7‐2H cyclohexane skeleton), 4.28 (q, 2H, J = 6.8 Hz, CH2O), 7.34 (s, 2H, NH2), 11.10 (s, 1H, NH‐CO). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 165.1, 162.4, 161.6, 147.0, 129.9, 128.3, 125.3, 116.6, 110.1, 105.1, 59.8, 25.9, 25.7, 22.3, 22.2, 13.8. EI‐MS (m/z (%)): 406 (M++2, 2.0), 405 (M++1, 4.47), 404 (M+, 14.0), 325 (3.4), 225 (100), 180 (67.2), 151 (17.4), 91 (15.5), 68 (1.5). Anal. calcd. for C20H24N2O3S2: C, 59.38; H, 5.98; N, 6.92. Found: C, 59.43; H, 6.05; N, 6.97%. 2.2.2. Reaction of 2‐aminothiophene derivatives (4) or (5) with 3‐(3,5‐dimethyl‐1H‐pyrazol‐1‐yl)‐3‐oxopropanenitrile (2) General procedure: A mixture of compound 4 (5.40 g, 14 mmol) or 5 (5.66 g, 14 mmol) and compound 2 [18] (2.28 g, 14 mmol) in dioxane (20 mL), was refluxed for 5 h. The solvent was evaporated under vacuum and the residue was crystallized form ethanol/DMF (5:1, v:v) mixture to give compounds 6 and 7, respectively (Scheme 1). Ethyl 2‐[({2‐[(cyanoacetyl)amino]‐5, 6‐dihydro‐4H‐cyclo penta[b]thien‐3‐yl}carbonyl)amino]‐4,5,6,7‐tetrahydro‐1‐benzo thiophene‐3‐carboxylate (6): Color: White powder. Yield: 4.99 g, 78%. M.p.: 227 °C. FT‐IR (KBr, ν, cm‐1): 3284, 3216 (NH), 2253 (CN), 1670, 1627 (C=O). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.32 (t, 3H, J = 7.2 Hz, CH3), 1.72‐1.74 (m, 4H, C5‐2H, C6‐2H, cyclohexane skeleton), 2.44‐2.47 (m, 2H, C5‐2H, cyclopentane skeleton), 2.63‐2.82 (m, 6H, C4‐2H, C7‐2H, cyclohexane skeleton and C4‐2H, cyclopentane skeleton), 3.09‐3.20 (m, 2H, C6‐2H, cyclopentane skeleton), 4.23 (s, 2H, CH2CN), 4.30 (q, 2H, J = 7.2 Hz, CH2O), 11.40 (s, 1H, NH‐CO), 11.64 (s, 1H, NH‐CO). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 166.2, 160.4, 149.1, 147.3, 137.8, 134.0, 130.4, 126.7, 116.0, 112.2, 111.1, 60.3, 29.0, 28.0, 27.6, 25.7, 23.5, 22.2, 22.1, 13.8. EI‐MS (m/z (%)): 459 (M++2, 0.6), 457 (M+, 1.5), 295 (0.3), 251 (0.6), 233 (12.3), 225 (26.8), 205 (5.4), 178 (37.6), 165 (82.1), 151 (17.1), 104 (13.9), 91 (9.7), 67 (100). Anal. calcd. for C22H23N3O4S2: C, 57.75; H, 5.07; N, 9.18. Found: C, 57.53; H, 5.01; N, 9.22%. 646 Gouda and El‐Bialy / European Journal of Chemistry 5 (4) (2014) 644‐651 Scheme 2 Ethyl 2‐[({2‐[(cyanoacetyl)amino]‐4, 5, 6, 7‐tetrahydrobenzo [b]thien‐3‐yl}carbonyl)amino]‐4, 5, 6, 7‐tetrahydro‐1‐benzothio phene‐3‐carboxylate (7): Color: White powder. Yield: 4.95 g, 75%. M.p.: 237 °C. FT‐IR (KBr, ν, cm‐1): 3293, 3212 (NH), 2260 (CN), 1670, 1623 (C=O). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.31 (t, 3H, J = 6.8 Hz, CH3), 1.74‐1.80 (m, 8H, 2C5‐2H, 2C6‐2H, cyclohexane skeleton), 2.62‐2.80 (m, 8H, 2C4‐2H, 2C7‐2H cyclohexane skeleton), 4.08 (s, 2H, CH2CN), 4.31 (q, 2H, J = 6.8 Hz, CH2O), 11.37 (s, 1H, NH‐CO), 11.46 (s, 1H, NH‐CO). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 165.3, 160.8, 160.3, 145.7, 130.4, 129.2, 128.4, 126.4, 117.3, 115.0, 111.7, 60.2, 25.8, 23.6, 23.5, 22.2, 22.1, 22.0, 13.8. EI‐MS (m/z (%)): 473 (M++2, 0.8), 471 (M+, 7.2), 247 (8.6), 225 (100), 206 (10.3), 179 (52.8), 151 (21.4), 91 (13.3), 68 (50.5). Anal. calcd. for C23H25N3O4S2: C, 58.58; H, 5.34; N, 8.91. Found: C, 58.53; H, 5.30; N, 8.87%. 2.2.3. Reaction of ethyl‐2‐[({2‐[(cyanoacetyl)amino] 4,5,6,7‐ tetrahydrobenzo [b]thien‐3‐yl}carbonyl)amino]‐4,5,6,7‐ tetrahydro‐1‐benzothiophene‐3‐carboxylate (7) with cyclic ketones and elemental sulfur General procedure: Morpholine (0.33 mL), elemental sulphur (0.141 g, 4.4 mmol) and cyclohexanone (0.4 g, 4 mmol) or cyclopentanone (0.34 g, 4 mmol) were added to solution of compound 7 (1.88 g, 4 mmol) in a mixture of ethanol:DMF (4:1, v:v, 25 mL). The reaction mixture was heated under reflux at 60‐80oC for 6 h and left to cool to room temperature. The separated crystalline product was filtered, dried, and recrystallized from DMF:ethanol (2:3, v:v) to give compounds 8 and 9, respectively (Scheme 2). Ethyl‐2‐{[(2‐{[(2‐amino‐5, 6‐dihydro‐4H‐cyclopenta[b]thien‐ 3‐yl)carbonyl]amino}‐5, 6‐dihydro‐4H‐cyclopenta[b]‐thien‐3‐yl) carbonyl]amino}‐4,5,6,7‐tetrahydro‐1‐benzothiophene‐3‐carboxy late (8): Color: White powder. Yield: 1.50 g, 66%. M.p.: 235 °C. FT‐IR (KBr, ν, cm‐1): 3409, 3286, 3261 (NH, NH2), 1668, 1616 (C=O). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.37 (t, 3H, J = 6.8 Hz, CH3), 1.76‐1.85 (m, 8H, 2C5‐2H, 2C6‐2H, cyclohexane skeleton), 2.56‐2.87 (m, 10H, C5‐2H, cyclopentane skeleton, 2C4‐2H, 2C7‐2H, cyclohexane skeleton), 3.05‐3.35 (m, 4H, C4‐2H, C6‐2H, cyclopentane skeleton), 4.33 (q, 2H, J = 6.8 Hz, CH2O), 8.20 (s, 2H, NH2), 11.52 (s, 1H, NH‐CO), 12.44 (s, 1H, NH‐CO). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 170.5, 165.5, 160.9, 147.9, 145.1, 134.6, 134.2, 130.7, 132.0, 122.9, 120.8, 119.1, 118.0, 116.0, 115.2, 112.3, 64.5, 31.4, 31.3, 29.1, 28.2, 27.8, 25.7, 24.1, 24.0, 23.9, 14.4. EI‐MS (m/z (%)): 570 (M++1, 0.12), 569 (M+, 0.13), 430 (3.4), 390 (10.7), 384 (17.7), 370 (14.9), 344 (6.42), 225 (73.5), 205 (26.8), 178 (100), 165 (76.5), 150 (64.3), 138 (25.1), 122 (38.1), 109 (29.2), 103 (27.2), 90 (25.5), 77 (34.1), 64 (27.5). Anal. calcd. for C28H31N3O4S3: C, 59.02; H, 5.48; N, 7.38. Found: C, 58.98; H, 5.45; N, 7.40%. Ethyl‐2‐{[(2‐{[(2‐amino‐4, 5, 6, 7‐tetrahydro‐1‐benzothien‐3‐ yl)carbonyl]amino}‐4,5,6, 7‐tetrahydro‐1‐benzothien‐3‐yl)carbon yl]amino}‐4,5,6,7‐tetrahydro‐1‐benzothiophene‐3‐carboxylate (9): Color: White powder. Yield: 1.61 g, 69%. M.p.: 255 °C. FT‐IR (KBr, ν, cm‐1): 3419, 3282, 3265 (NH, NH2), 1660, 1610 (C=O). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.35 (t, 3H, J = 6.8 Hz, CH3), 1.71‐1.82 (m, 12H, 3C5‐2H, 3C6‐2H, cyclohexane skeleton), 2.77‐2.87 (m, 12H, 3C4‐2H, 3C7‐2H, cyclohexane skeleton ), 4.33 (q, 2H, J = 6.8 Hz, CH2O), 8.20 (s, 2H, NH2), 11.62 (s, 1H, NH‐CO), 11.80 (s, 1H, NH‐CO). EI‐MS (m/z (%)): 584 (M++1, 0.2), 483 (0.2, M+), 465 (0.3), 405 (0.9), 385 (0.4), 357 (0.2), 314 (0.3), 258 (1.7), 224 (8.4), 179 (100), 150 (67.0), 123 (61.2), 128 (2.1), 117 (39.1), 91 (29.6), 76 (41.2), 50 (38.1). Anal. calcd. for C29H33N3O4S3: C, 59.66; H, 5.70; N, 7.20. Found: C, 59.61; H, 5.72; N, 7.18%. 2.2.4. Reaction of ethyl‐2‐[({2‐[(cyanoacetyl)amino]‐4,5,6,7‐ tetrahydrobenzo[b]thien‐3‐yl}carbonyl)amino]‐4,5,6,7‐tetra hydro‐1‐benzothiophene‐3‐carboxylate (7) with aromatic aldehydes General procedure: A mixture of compound 7 (1.17 g, 2.5 mmol), 4‐(dimethylamino) benzaldehyde (0.37 g, 2.5 mmol), 4‐ (piperidin‐1‐yl)benzaldehyde (0.47g, 2.5 mmol), or 2‐hydroxy benzaldehyde (0.31 g, 2.5 mmol) in ethanol (15 mL) containing piperidine (0.2 mL) was stirred at 80 °C for 4 h. The separated crystals was filtered, dried and washed with hot DMF to give compounds 10‐12, respectively (Scheme 2). Ethyl 2‐({[2‐({(2E)‐2‐cyano‐3‐[4‐(dimethylamino)phenyl] prop‐2‐enoyl}amino)‐4, 5, 6, 7‐tetrahydro‐1‐benzothien‐3‐yl] carbonyl}amino)‐4,5,6,7‐tetrahydro‐1‐benzothiophene‐3‐carboxy late (10): Color: Red needle crystals. Yield: 1.43 g, 95%. M.p.: > 320 °C. FT‐IR (KBr, ν, cm‐1): 3293, 3183 (2NH), 2196 (CN), 1688, 1614 (C=O). EI‐MS (m/z (%)): 605 (M++3, 29.4), 604 (M++2, 35.3), 255 (100), 180 (70.6), 179 (76.5), 156 (52.9), 107 (29.4), 98 (35.3), 77 (70.6). Anal. calcd. for C32H34N4O4S2: C, 63.76; H, 5.69; N, 9.29. Found: C, 63.69; H, 5.66; N, 9.31%. Gouda and El‐Bialy / European Journal of Chemistry 5 (4) (2014) 644‐651 647 Scheme 3 Ethyl‐2‐({[2‐({(2E)‐2‐cyano‐3‐[4‐(piperidin‐1‐yl)phenyl] prop‐2‐enoyl}amino)‐4,5,6, 7‐tetrahydro‐1‐benzothien‐3‐yl] carbonyl}amino)‐4,5,6, 7‐tetrahydro‐1‐benzothiophene‐3‐ carboxylate (11): Color: Red crystals. Yield: 1.52 g, 95%. M.p.: 306 °C. FT‐IR (KBr, ν, cm‐1): 3446 (br, NH), 2191(CN), 1671, 1631 (C=O), 1606 (C=N). EI‐MS (m/z (%)): 597 (M+‐OEt, 18.5), 417 (48.1), 355 (33.3), 239 (29.6), 174 (100), 128 (18.5), 90 (14.8), 66 (37.0), 51 (18.5). Anal. calcd. for C35H38N4O4S2: C, 65.39; H, 5.96; N, 8.72. Found: C, 65.41; H, 5.93; N, 8.70. Ethyl 2‐{[(2‐{[(2‐imino‐2H‐chromen‐3‐yl)carbonyl]amino}‐4, 5, 6, 7‐tetrahydro‐1‐benzothien‐3‐yl)carbonyl]amino}‐4, 5, 6, 7‐ tetrahydro‐1‐benzothiophene‐3‐carboxylate (12): Color: Yellow powder. Yield: 1.28 g, 89%. M.p.: 260 °C. FT‐IR (KBr, ν, cm‐1): 3413, 3340, 3255 (NH), 1670, 1623 (C=O). EI‐MS (m/z (%)): 568 (M+ ‐7, 0.21), 404 [M+‐3‐(carbonyliminocoumarine), 0.32], 384 (M+, 0.4), 358 (0.5), 224 (12.9), 179 (100), 164 (11.6), 151 (42.8), 134 (12.3), 123 (20.4), 117 (36.6), 97 (23.3), 86 (41.6), 74 (31.4), 50 (49.8). Anal. calcd. for C30H29N3O5S2: C, 62.59; H, 5.08; N, 7.30. Found: C, 62.53; H, 5.03; N, 7.34%. 2.2.5. Synthesis of ethyl 2‐((E)‐2‐cyano‐3‐(4‐((E)‐2‐cyano‐3‐ (3‐(ethoxycarbonyl)‐4,5,6, ‐tetrahydrobenzo‐[b]thiophen‐2‐ ylamino)‐3‐oxoprop‐1‐enyl)phenyl)acrylamido)‐4,5,6,7‐ tetrahydrobenzo‐[b]thiophene‐3‐carboxylate (13) A mixture of compound 3 (0.73 g, 2.5 mmol) and terphthal aldehyde (0.17 g 1.25 mmol) in ethanol (15 mL) containing piperidine (0.2 mL) was stirred at 80 °C for 4 h. The separated crystals was filtered, dried and crystallized from DMF:ethanol (4:1, v:v) mixture to give compound 13 (Scheme 3). Color: Yellowish green powder. Yield: 0.56 g, 77%. M.p.: 239 °C. FT‐IR (KBr, ν, cm‐1): 3184, (NH), 2195 (CN), 1729, 1677, 1621 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 1.34 (m, 6H, 2CH3), 1.75‐1.82 (m, 8H, 2C5‐2H, 2C6‐2H, cyclohexane skeleton ), 2.82‐3.12 (m, 8H, 2C4‐2H, 2C7‐2H, cyclohexane skeleton), 4.42 (m, 4H, 2CH2O), 7.70‐8.0 (m, 6H, Ar‐H, 2CH=), 11.45 (s, 1H, NH‐CO), 11.75 (s, 1H, NH‐CO). EI‐MS (m/z (%)): 679 (M+‐3, 0.1), 543 (0.2), 499 (0.2), 453 (0.2), 404 (0.3), 384 (1.2), 359 (7.8), 336 (19.3), 290 (8.9), 246 (21.5), 224 (27.4), 205 (33.4), 178 (70.0), 150 (91.3), 126 (43.9), 122 (72.9), 111 (82.8), 90(60.0), 83(72.1), 68 (100). Anal. calcd. for C36H34N4O6S2: C, 63.32; H, 5.02; N, 8.21. Found: C, 63.41; H, 5.06; N, 8.23%. 2.2.6. Synthesis of ethyl‐(E)‐2‐cyano‐2‐(2‐(4‐((E)‐2‐cyano‐1‐ (3‐(ethoxycarbonyl)‐4,5,6,7‐tetrahydrobenzo[b]thiophen‐2‐ ylacetamido)hydrazono)phenylhydrazono)‐N‐(4,5,6,7‐tetra hydrobenzo [b]thiophen‐2‐yl)acetamido‐3‐ carboxylate (14) To a well stirred cooled solution of p‐phenylenediamine (0.27 g, 2.5 mmol) in conc. HCl (3 mL), a solution of NaNO2 (0.4 g, 5.8 mmol in 5 mL H2O) was added dropwise. The above cooled diazonium salt solution was added slowly to a well stirred solution of 2 (1.46 g, 5 mmol) in pyridine (20 mL). The reaction mixture was stirred for 2 h. The crude product was filtered off, dried and recrystallize from DMF:EtOH (5:2, v:v) mixture to give compound 14 (Scheme 3). Color: Scarlet red crystals. Yield: 1.57 g, 88%. M.p.: 305 °C. FT‐IR (KBr, ν, cm‐1): 3218, 3166 (NH), 2220, (CN), 1666, 1625 (C=O), 1488 (N=N). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.38‐1.41 (m, 6H, 2CH3), 1.76‐1.82 (m, 8H, 2C5‐2H, 2C6‐2H, cyclohexane skeleton ), 2.70‐ 2.93 (m, 8H, 2C4‐2H, 2C7‐2H, cyclohexane skeleton), 4.41‐4.43 (m, 4H, 2CH2O), 7.93‐8.44 (m, 4H, Ar‐H), 10.45 (s, 1H, NH‐CO), 10.65 (s, 1H, NH‐CO), 12.45 (br, 1H, NH=N), 12.54 (br, 1H, NH=N). EI‐MS (m/z (%)): 723 (M++1/2H2O, 0.1), 513 (M+, 0.2), 467 (0.6), 436 (0.8), 396 (5.5), 292 (10.1), 246 (28.7), 225 (27.5), 206 (91.4), 178 (100), 150 (65.0), 121 (44.3), 115 (38.0), 104 (31.9), 90 (65.5), 67 (65.3), 64 (52.9). Anal. calcd. for C34H34N8O6S2: C, 57.13; H, 4.79; N, 15.68. Found: C, 57.18; H, 4.73; N, 15.62%. 2.2.7. Synthesis of ethyl 2‐(2‐cyano‐3,3‐bis(2‐ethoxy‐2‐oxo ethylthio)acrylamido)‐4,5,6,7‐tetrahydrobenzo[b]thiophene ‐3‐carboxylate (16) To a cold suspension of KOH (1.16 g, 20 mmole) in dry dimethylformamide (25 mL) were added the nitrile derivative 3 (1.45 g, 5 mmole) subsequently carbon disulphide (1.14 g, 15 mmol) slowly dropwise under stirring over a period of 15 min. while the temperature of the mixture was maintained at 5‐10 °C. The mixture was stirred at room temperature for 12 h. Then cooled again at 0 °C, ethyl bromoacetate (1.67 g, 10 mmol) was added dropwise over a period of 10 min and left to stand at room temperature for 24 h. The mixture was poured onto ice cold‐water. The resulting precipitate was filtered off, dried and crystallized from ethanol:DMF (1:5, v:v) mixture to give compound 16 (Scheme 4). Color: Yellow sheet crystals. Yield: 2.19 g, 81%. M.p.: 178 °C. FT‐IR (KBr, ν, cm‐1): 3140 (NH), 2170 (CN), 1729, 1677, 1621 (C=O). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.14 (t, 3H, J = 6.8 Hz, CH3), 1.25 (t, 3H, J = 6.8 Hz, CH3), 1.28 (t, 3H, J = 6.8 Hz, CH3), 1.75‐1.78 (m, 4H, C5‐2H, C6‐2H, cyclohexane skeleton), 2.65‐2.79 (m, 4H, C4‐2H, C7‐2H, cyclohexane skeleton ), 3.17 (br, 4H, 2CH2), 4.07 (q, 2H, J = 6.8 Hz, CH2O), 4.18 (q, 2H, J = 6.8 Hz, CH2O), 4.31 (q, 2H, J = 6.8 Hz, CH2O), 12.66 (s, 1H, NH‐CO). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 169.4, 166.5, 166.4, 163.9, 145.9, 143.1, 132.7, 127.9, 115.2, 61.4, 61.0, 60.2, 37.1, 36.7, 25.6, 23.6, 22.2, 22.1, 13.8, and 13.6. EI‐MS (m/z (%)): 540 (M+, 0.8), 529 (3.9), 424 (35.3), 385 (2.8), 366 (5.2), 338 (2.0), 224 (20.6), 205 (39.5), 177 (76.4), 150 (69.0), 134 (63.9), 119 (52.1), 108 (81.9), 101 (69.9), 83(76.3), 76 (100), 63 (87.3). Anal. calcd. for C23H29N2O7S3: C, 51.09; H, 5.22; N, 5.18. Found: 51.15; H, 5.18; N, 5.19%. 2.2.8. Synthesis of ethyl 2‐(4‐amino‐2‐(2‐ethoxy‐2‐oxoethyl thio)‐5‐(ethoxycarbonyl) thiophene‐3‐carboxamido)‐4,5,6,7‐ tetrahydrobenzo[b]thiophene‐3‐carboxylate (17) A mixture of compound 16 (0.54 g, 1.0 mmol) and TEA (0.2 mL) in DMF (15 mL) was stirred at 140 °C. 648 Gouda and El‐Bialy / European Journal of Chemistry 5 (4) (2014) 644‐651 Scheme 4 The mixture was cooled, poured into cooled water, the separated crystals was filtered, dried and crystallize from DMF:EtOH (1:2, v:v) mixture to give compound 17 (Scheme 4). Color: Brown powder. Yield: 0.36 g, 66%. M.p.: 192 °C. FT‐IR (KBr, ν, cm‐1): 3411, 3280, 3261 (NH, NH2), 1714, 1677, 1614 (C=O). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.21‐1.28 (m, 9H, 3CH3), 1.74‐1.77 (m, 4H, C5‐2H, C6‐2H, cyclohexane skeleton), 2.65‐2.79 (m, 4H, C4‐2H, C7‐2H, cyclohexane skeleton), 3.16 (s, 2H, CH2), 4.26‐4.31 (m, 6H, J = 6.8 Hz, 3CH2O), 5.08 (br, 2H, NH2), 12.03 (s, 1H, NHCO). EI‐MS (m/z (%)): 541 (M++1, 0.2), 525 (3.5), 464 (1.1), 422 (1.9), 390 (1.7), 377 (35.2), 346 (2.6), 238 (5.3), 225 (30.2), 206 (19.3), 177 (35.2), 165 (10.1), 150 (46.5), 120 (89.1), 108 (95.3), 93 (38.7), 90 (45.1), 77 (100), 68 (41.7), 57 (86.6). Anal. calcd. for C23H28N2O7S3: C, 51.09; H, 5.22; N, 5.18. Found: 51.05; H, 5.26; N, 5.15%. 2.2.9. Synthesis of ethyl 2‐(2‐cyano‐2‐(4‐methyl‐3‐phenyl thiazol‐2(3H)‐ylidene)acetamido)‐4,5,6,7‐tetrahydrobenzo‐ [b]thiophene‐3‐carboxylate (19) and ethyl 2‐(2‐cyano‐2‐(4‐ oxo‐3‐phenylthiazolidin‐2‐ylidene)acetamido)‐4,5,6,7‐tetra hydrobenzo[b]thiophene‐3‐carboxylate (20) To a cold suspension of finely divided KOH (0.11 g, 2 mmol) in dry dimethylformamide (10 mL), the cyanoacetamide derivative 3 (0.58 g, 2 mmol) followed by phenyl isothio cyanate (0.27 g, 2 mmol) was added. The mixture was stirred at room temperature for 12 h, and then cooled again to 5‐0 °C, treated with the chloroacetone (0.18 g, 2 mmol) or ethyl bromoacetate (0.24 g, 2 mmol) and left to stand at room temperature for 24 h, the mixture was poured into ice cold water. The resulting precipitate was filtered off, dried and crystallized from DMF: ethanol (5:2, v:v) mixture to afford compounds 19 and 20, respectively (Scheme 5). Ethyl 2‐{[(2Z)‐2‐cyano‐2‐(4‐methyl‐3‐phenyl‐1, 3‐thiazol‐ 2(3H)‐ylidene)acetyl]amino}‐4, 5, 6, 7‐tetrahydro‐1‐benzothio phene‐3‐carboxylate (19): Color: Pale yellow powder. Yield 0.59 g, 63%. M.p.: 206 °C. (Lit. [8], 205 °C). Ethyl 2‐(2‐cyano‐2‐(4‐oxo‐3‐phenylthiazolidin‐2‐ylidene)acet amido)‐4, 5, 6, 7‐tetrahydrobenzo[b]thiophene‐3‐carboxylate (20): Color: Brown powder. Yield: 0.51 g, 55%. M.p.: > 320 °C. (Lit. [8] > 320 °C). 2.2.10. Reaction of ethyl 2‐{[(2E)‐3‐anilino‐2‐cyano‐3‐ mercaptoprop‐2‐enoyl]amino}‐4,5,6,7‐tetrahydro‐1‐benzo thiophene‐3‐carboxylate (23) with α‐halogen carbonyl compounds Chloroacetone (0.18 g, 2 mmol) or ethyl bromoacetate was added in solution containing ethanol (10 mL), triethylamine (0.2 mL) and compound 23 (0.86 g, 2 mmole). The reaction mixture was refluxed for 3 h. The obtained product after addition of water was filtered, dried and crystallized from ethanol:benzene (1: 3, v:v) to give compounds 24 and 25, respectively (Scheme 5). Ethyl 2‐{[(5‐acetyl‐4‐amino‐2‐anilino‐3‐thienyl) carbonyl] amino}‐4, 5, 6, 7‐tetrahydro‐1‐benzothiophene‐3‐carboxylate (24): Color: Brown crystals. Yield 0.67 g, 69%. M.p.: 230 °C. FT‐ IR: 3234 (NH, NH2, br), 1671, 1610 (C=O). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 1.21 (t, 3H, J = 7.2 Hz, CH3), 1.74‐1.90 (m, 4H, C5‐2H, C6‐2H), 2.10 (s, 3H, CH3), 2.63‐2.77 (m, 4H, C4‐2H, C7‐ 2H), 4.42 (q, 2H, J = 7.2 Hz, CH2O), 7.1‐7.5 (m, 8H, NH2, NH, Ar‐ H), 11.82 (s, 1H, NH‐CO). EI‐MS (m/z (%)): 484 (M++1, 8.9), 451 (6.9), 437 (7.6), 259 (15.0), 242 (22.0), 225 (41.0), 216 (47.9), 178 (100), 150 (66.1), 143 (37.7), 121 (41.5), 116 (37.0), 90 (36.7), 76 (64.9), 50 (25.8). Anal. calcd. For C24H25N3O4S2: C, 59.61; H, 5.21; N, 8.69. Found: C, 59.56; H, 5.15; N, 8.75%. Ethyl 2‐(3‐amino‐2‐(ethoxycarbonyl)‐5‐(phenylamino)thio phene‐4‐carboxamido)‐4, 5, 6, 7‐tetrahydrobenzo[b]thiophene‐3‐ carboxylate (25): Color: Brown crystals. Yield 0.81 g, 79%. M.p.: 283 oC. FT‐: 3366, 3200 (NH, NH2), 1664, 1621 (C=O). EI‐MS (m/z (%)): 515 (M++2, 1.9), 514 (M++1, 21.8), 468 (18.7), 422 (3.5), 394 (2.8), 288 (4.4), 243 (71.7), 225 (81.1), 215 (77.8), 178 (100), 170 (36.4), 150 (66.9), 142 (60.8), 122 (49.4), 115 (33.8), 90 (31.9), 77 (67.4). Anal. calcd. for C25H27N3O5S2 :C, 58.46; H, 5.30; N, 8.18. Found: C, 58.40; H, 5.36; N, 8.13%. 2.3. Antioxidant screening 2.3.1. Superoxide anion radical scavenging assay The assay was performed according to Nishmiki et al. [19], with minor modifications. Test solution (0.1 mL, 1 mg/mL of sample solution (DMSO)) in 0.1 M phosphate buffer pH = 7.4, 62.5 µL of 468 µM NADH solution, 62.5 µL of 156 µM nitroblue tetrazolium (NBT) solution and 62.5 µL of 60 µM phenazine Gouda and El‐Bialy / European Journal of Chemistry 5 (4) (2014) 644‐651 649 Table 1. Assay for superoxide anion radical scavenging and Bleomycin‐dependent DNA damage (DNA) Compound no Antioxidant activity (Superoxide anion radical scavenging, %) Absorbance of samples Ascorbic Acid 66.1 0.091 1 58.6 0.078 3 48.6 0.127 4 31.2 0.080 5 35.7 0.079 6 45.7 0.113 7 61.4 0.136 8 55.8 0.125 9 41.4 0.118 10 38.6 0.157 11 10.3 0.151 12 55.7 0.102 13 6.1 0.123 14 35.6 0.145 16 58.9 0.169 17 37.1 0.134 23 57.1 0.098 24 20.0 0.100 25 18.6 0.126 * All compounds were dissolved in DMSO:MeOH (1:1, v:v) and tested at the final concentration of 0.1 mL of 1 mg/mL. Scheme 5 methophosphate (PMS) solution were added to a microwell plate and incubated at room temperature for 5 min. and the abundance at 560 nm was measured against blank samples. The capacity of scavenging super oxide radicals was calculated using the following formula: Scavenging activity = [A (control) ‐ A (test)/ A (control)] × 100 (1) The (NBT+NADH+PMS) solution without sample solution was used as control. 2.3.2. Bleomycin‐dependent DNA damage The assay was performed according to Aeschlach et al. [20], with minor modifications. The reaction mixture (0.5 mL) contained DNA (0.5 mg/mL), bleomycin sulfate (0.05 mg/mL), MgCl2 (5 mM), FeCl3 (50 mM) and samples to be tested (0.1 mL of 1 mg/mL). L‐Ascorbic acid was used as a positive control. The mixture was incubated at 37 °C for 1 h. The reaction was terminated by addition of 0.05 mL EDTA (0.1 M). The color was developed by adding thiobarbituric acid (TBA) (0.5 mL) (1%, w:v) and HCl (0.5 mL) (25%, v:v) followed by heating at 80 °C for 10 min. After centrifugation, the extent of DNA damage was measured by the increase in absorbance at 532 nm (Table 1). 3. Results and discussion 3.1. Chemistry Schemes 1‐4 describe the syntheses of the target molecules. The starting ethyl 2‐[(cyanoacetyl)amino]‐4,5,6,7‐tetrahydro‐ 1‐benzothiophene‐3‐carboxylate 3 [8] was prepared according 650 Gouda and El‐Bialy / European Journal of Chemistry 5 (4) (2014) 644‐651 Figure 1. Structure activity relationship of the more potent antioxidant compounds. to the previously reported method via refluxing of ethyl 2‐ amino‐4,5,6,7‐tetrahydro‐1‐benzothiophene‐3‐carboxylate (1) [17] with 3‐(3,5‐dimethyl‐1H‐pyrazol‐1‐yl)‐3‐oxopropane nitrile (2) [18]. Multicomponent condensation of cyclopentanone or cyclo hexanone with compound 3 and elemental sulfur containing morpholine as a catalyst afforded the bithiophene derivatives 4 and 5, respectively. Compounds 4 and 5 form hydrogen bonds between carbonyl of esters and NH groups of amide and between amino groups and amidic groups. Because the IR spectra revealed low stretching vibrations of ester and amidic carbonyl groups and the 1H NMR spectra show the presence of NH singlet signal at 11.04 and 11.10 ppm, respectively, similar behaviour was reported [21]. Moreover, cyanoacetylation of compound 4 and 5 under the same previous conditions afforded the cyanoacetamide derivatives 6 and 7. Treatment of compound 7 with cyclopentanone or cyclohexanone and elemental sulfur in ethanol containing morpholine afforded the corresponding trithiophene derivatives 8 and 9, respectively. Compound 8 and 9 formed three hydrogen bonds (Scheme 1). Knoevenagel condensation of compound 7 with 4‐N,N‐ dimethylaminobezaldehyde, 4‐piperdin‐1‐ylbenzaldehyde or salicyaldehyde in ethanol containing a catalytic amount of piperidine afforded the corresponding (E) arylidenes 10, 11 and coumarin derivative 12, respectively [22] (Scheme 2). Furthermore, condensation of compound 3 with terphthaldehyde in ethanol containing a catalytic amount of piperidine achieved the (E) bis‐arylidene derivative 13 [23]. Coupling of compound 3 with benzene‐1,4‐bis(diazonium) dichloride afforded the (E) hydrazo derivative 14 [21] (Bioisostere of compound 13) (Scheme 3). On the other hand, stirring of compound 3 with carbon disulphide in DMF containing potassium hydroxide followed by in situ addition of ethyl bromoacetate afforded the triester derivative 16 via the intermediate 15, which afforded the dithiophene 17 upon heating in DMF containing a catalytic amount of triethylamine (Scheme 4). Attempting for preparation of compounds 21 and 22, which were bioisostere of compound 16 via reaction of compound 3 with phenyl isothiocyanate in dry DMF in the presence of potassium hydroxide followed by addition of reaction with chloroacetone or ethyl bromoacetate was failed. Although both compound 21 and 22 were formed in situ, subsequently cyclized to the 2‐{[(2Z)‐2‐cyano‐2‐(4‐methyl‐3‐ phenyl‐1,3‐thiazol‐2(3H)‐ylidene)acetyl]amino}‐4,5,6,7‐tetra hydro‐1‐benzothiophene‐3‐carboxylate (19) and ethyl 2‐{[(2Z) ‐2‐cyano‐2‐(4‐oxo‐3‐phenyl‐1,3‐thiazolidin‐2‐ylidene)acetyl] amino}‐4,5,6,7‐tetrahydro‐1‐benzothiophene‐3‐carboxylate (20), respectively [8]. Acidification of the intermediate potassium salt 15 afforded compound 23 [8], refluxing of compound 23 with chloroacetone or ethyl bromoacetate afforded the dithiophene derivatives 24 and 25 (Bioisoster of compound 17), respectively (Scheme 5). The structure of new synthesized compounds was established on the basis of their elemental analyses and spectral data (IR, 1H, 13C NMR and mass spectral data, c.f. Experimental section). 3.2. Biological Assays 3.2.1. Superoxide anion radical scavenging assay The antioxidant activity of the new synthesized compounds was evaluated following the protocols published by Nishimiki et al. [19]. The biological data showed clearly that compounds 1, 7, 8, 12, 16 and 23 have good activities, while compounds 3‐6, 9, 10, 14 and 17 exhibited moderate activities. On the other hand, the anther compounds exhibited weak activities (Table 1). Thus, it would appear that introducing of cyanoacetamide, thiocarbamoyl, thiophene and coumarin moieties enhances the antioxidant properties of 2‐aminothiophene derivatives. All compounds were dissolved in DMSO:MeOH (1:1, v:v) and tested at the final concentration of 0.1 mL of 1 mg/mL. The extent of DNA damage is expressed by increase of absorbance at 520 nm. The synthesized compounds were test for Bleomycin‐dependent DNA damage (Table 1) and showed that compounds 1, 4, 5, 12, 23 and 24 have an ability to protect DNA from the induced damage by Bleomycin. By comparing the results obtained of antioxidant of the compounds reported in this paper to their structures, the following structure activity relationship (SAR's) were postulated: (i) 2‐Aminothiophene derivatives 1, 4 and 5 are more potent than ascorbic acid which may be attributed to the replacement of furan moiety with the thiophene moiety and presence of amino group. (ii) Compounds 3, 6 and 7 (N‐substituted‐2‐amino thiophene) is less potent than compounds 1, 4 and 5, Gouda and El‐Bialy / European Journal of Chemistry 5 (4) (2014) 644‐651 651 respectively which may be due to decrease of basicity of amino group. (iii) Sulfanyl derivative 23 is more potent than 3 which may be attributable to presence of thiocarbamoyl moiety or may be due to oxidation of S‐H to S‐S and presence of further conjugation. (iv) Compounds 12 and 24 exhibited high antioxidant activity which may be due presence of imino coumarin and 3‐aminothiophene moiety (Figure 1). 4. 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