untitled European Journal of Chemistry 8 (3) (2017) 240‐247 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2017 Atlanta Publishing House LLC ‐ All rights reserved ‐ Printed in the USA http://dx.doi.org/10.5155/eurjchem.8.3.240-247.1599 European Journal of Chemistry Journal webpage: www.eurjchem.com Heteroaromatization with 4‐phenyldiazenyl‐1‐naphthol. Part II: Synthesis of some new benzochromens, benzochromenopyrimidines, benzochromenotriazolopyrimidines, benzochromenopyrimidotriazepine and antimicrobial activities Ibrahim Ali Radini 1, Hany Mostafa Hamed 1,2, Mohammed Abdo Yahya Kharir 1 and Ashraf Hassan Fekry Abd Elwahab 1,2,* 1 Department of Chemistry, Faculty of Science, Jazan University, 2097, Jazan, Kingdom of Saudi Arabia 2 Chemistry Department, Faculty of Science, Al‐Azhar University, 11884, Nasr City, Cairo, Egypt * Corresponding author at: Department of Chemistry, Faculty of Science, Jazan University, 2097, Jazan, Kingdom of Saudi Arabia. Tel.: +966.054.0963753. Fax: +966.017.3230028. E‐mail address: ash_abdelwahab@yahoo.com (A.H.F.A. Elwahab). ARTICLE INFORMATION ABSTRACT DOI: 10.5155/eurjchem.8.3.240-247.1599 Received: 12 June 2017 Received in revised form: 12 July 2017 Accepted: 12 July 2017 Published online: 30 September 2017 Printed: 30 September 2017   Synthesis of several new of benzochromenes (4‐7), benzochromenopyrimidines (8 and 9), 14‐(4‐chlorophenyl)‐12‐(phenyldiazenyl)‐14H‐benzo[7,8]chromeno[3,2‐e][1, 2, 4]triazolo[1, 5‐c]pyrimidines, 4‐amino‐16‐(4‐chlorophenyl)‐14‐(phenyldiazenyl)‐16H‐benzo[7', 8']chro‐ meno[2',3':4,5]pyrimido[1,6‐b][1,2,4]triazepine‐3‐carbonitrile (10a‐e, 13) and 9‐(benz‐ ylideneamino)‐7‐(4‐chlorophenyl)‐5‐(phenyldiazenyl)‐7, 9‐dihydro‐8H‐benzo[7,8]chromeno [2,3‐d]pyrimidin‐8‐imine (12), form starting from 2‐amino‐4‐(p‐chlorophenyl)‐6‐phenyl‐ diazenyl‐4H‐benzochromene‐3‐carbonitrile (3). The structure of these new compounds was confirmed using IR, 1H NMR and 13C NMR as well as MS spectroscopy. The structure activity relationship studies of the target compounds in agreement with the in vitro essays and confirmed higher potent antimicrobial activity against some of the tested microorganisms. The structure‐activity relationship study revealed that the antimicrobial activity of benzochromenopyrimido triazepine nucleus was more beneficial than benzochromeno‐ triazolopyrimidine nucleus for antimicrobial activity. KEYWORDS Benzochromene Antimicrobial activities Benzochromenopyrimidine 4‐Phenenyldiazenyl‐1‐naphthol Benzochromenotriazolopyrimidine Benzochromenopyrimidotriaziepine Cite this: Eur. J. Chem. 2017, 8(3), 240‐247 1. Introduction Nowadays, synthetic azo compounds are widely used in different application fields, such as medicines, cosmetics, food, paints, plastics, shipbuilding, automobile industry, cable manufacture [1‐12]. Moreover azo compounds are known for their antineoplastics [13], antidiabetics [14], antiseptics [15], antibacterial [16] and antitumor [17]. Benzochromene repre‐ sent a class of heterocyclic compounds endowed with potent antimicrobial agents [18‐23], antileishmanial [24,25], anti‐ cancer [26,27], antiproliferative [28], antioxidant [29,30], hypertensive [31], antitumor [32‐35] effects and activities, as well as for the treatment of Alzheimer’s disease [36], schizoph‐ renia disorders [37], and fused chromene ring systems also displayed blood platelet antiaggregating [38], antihistaminic [39], analgesic [40‐42], hypolipidemic [43], DNA breaking and mutagenicity activities [44]. The promising results of previous studies [18,23,45] prompted us to further extend our research towards the synthesis of annulation of heterocyclic systems of potential biological application. In continuation of our previous work we are reporting here the synthesis of some more analogues of benzochromene moiety as a base unit and antimicrobial activities. The structure activity relationships (SAR) are discussed in this work to correlate between the substituent effects and the activities that aid in drug design. 2. Experimental 2.1. Instrumentation Melting points were determined with a Stuart Scientific Co., Ltd. apparatus. IR spectra were determined as KBr pellets on a Jasco FT/IR 460 plus spectrophotometer. 1H NMR and 13C NMR spectra were recorded using a Bruker AV 400 MHz spectrometer. Mass spectra were measured on a Shimadzu GC/MS‐QP5050A spectrometer. Radini et al. / European Journal of Chemistry 8 (3) (2017) 240‐247 241 OH N N O N N Cl CN NH2 CN CN H Cl EtOH / Pip. + O N N Cl CN N OCH2CH3 CH3 O N N Cl CN N OCH2CH3 45 1 2 3 4 5 67 8 9 10 a b c d fg h e Reflux 1 2 Ac2O CH3C(OC2H5)3 Ac2O CH(OC2H5)3 3 Scheme 1 All the reagents and solvents were purchased from Sigma‐ Aldrich or S.D. fine chemicals limited and used without further purification. Thin‐layer chromatography (TLC) was performed using Merck silica gel 60F254 pre‐coated plates (0.25 mm) and silica gel (particle size 60‐120 mesh) used for column chromatography. Elemental analyses were carried out at the Regional Centre for Mycology and Biotechnology (RCMP, Al‐ Azhar University, Cairo, Egypt) and the results were within ±0.3% of calculated values. 2.2. Synthesis 2.2.1. Synthesis of 2‐amino‐4‐(4‐chlorophenyl)‐6‐(phenyl diazenyl)‐4H‐benzo[h]chromene‐3‐carbonitrile (3) A solution of 4‐phenyldiazenyl‐1‐naphthol (1) (2.48 g, 10 mmol) in EtOH (20 mL) was treated with 2‐(4‐chloro benzylidene)malononitrile (2) (1.88 g, 10 mmol) and piperidine (0.5 mL) under reflux 2 hr. The reaction mixture was heated until complete precipitation. The solid product which formed was collected by filtration and recrystallized from dioxane. This compound was prepared according to the literature [45] (Scheme 1). Color: Orange solid. Yield: 90 %. M.p.: 245‐246 °C. FT‐IR (KBr, ν, cm–1): 3417, 3327, 3207 (NH2), 3001, 2960, 2812 (CH‐str.), 2196 (CN), 1666 (C=C), 1510 (N=N). 1H NMR (400 MHz, CDCl3, δ, ppm): 8.93‐7.24 (m, 14H, Ar‐H), 4.95 (s, 1H, H‐4), 4.84 (brs, 2H, NH2, exchangeable by D2O). 13C NMR (100 MHz, CDCl3, δ, ppm): 158.59 (C‐2), 152.94 (C‐6), 144.70, 142.63, 133.44, 131.41, 133.29, 129.45, 129.20, 129.14, 127.82, 127.43, 123.75, 123.71, 123.28, 123.17, 120.90, 112.25 (Ar‐C), 116.98 (CN), 61.33 (C‐3), 40.74 (C‐4). MS (EI, m/z (%)): 438 (M++2, 1.0), 436 (M+, 20.4), 77 (100). Anal. calcd. for C26H17ClN4O: C, 71.48; H, 3.92; N, 12.82. Found: C, 71.01; H, 3.40; N, 12.39%. 2.2.2 Synthesis of 4H‐benzo[h]chromene derivatives 4 and 5 General procedure: A mixture of compound 3 (4.36 g, 10 mmol), triethyl orthoformate or triethyl orthoacetate (10 mmol) and Ac2O (30 mL) was refluxed for 3 hr. The solvent was removed under reduced pressure and the resulting solid was crystallized from benzene to give compound 4 and 5, respectively (Scheme 1). Ethyl (Z)‐N‐(4‐(4‐chlorophenyl)‐3‐cyano‐6‐((E)‐phenyl diazenyl)‐4H‐benzo[h]chromen‐2‐yl)formimidate (4): Color: Yellow solid. Yield: 83%. M.p.: 238‐240 °C. FT‐IR (KBr, ν, cm–1): 2977, 2936, 2874 (CH‐str.), 2201 (CN), 1668 (C=C), 1491 (N=N). 1H NMR (400 MHz, CDCl3, δ, ppm): 8.94 (s, 1H, N=CH), 8.92‐7.20 (m, 14H, Ar‐H), 5.01 (s, 1H, H‐4), 4.54 (q, J = 7.2 Hz, 2H, CH2), 1.44 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3, δ, ppm): 159.44 (C‐2), 158.63 (N=C), 145.73 (C‐6), 144.60, 142.66, 137.54, 133.80, 133.64, 132.73, 132.60, 131.55, 131.33, 129.52, 129.30, 127.80, 127.48, 123.69, 120.28, 120.90, 120.85 (Ar‐C), 116.97 (CN), 81.30 (C‐3), 64.51 (CH2), 40.95 (C‐4), 18.45 (CH3). MS (EI, m/z (%)): 494 (M++2, 9.12.), 492 (M+, 23.70), 77 (100). Anal. calcd. for C29H21ClN4O2: C, 70.66; H, 4.29; N, 11.37. Found: C, 70.34; H, 4.02; N, 11.08%. Ethyl (Z)‐N‐(4‐(4‐chlorophenyl)‐3‐cyano‐6‐((E)‐phenyl diazenyl)‐4H‐benzo[h]chromen‐2‐yl)acetimidate (5): Color: Yellow solid. Yield: 80%. M.p.: 241‐243 °C. FT‐IR (KBr, ν, cm–1): 2979, 2940, 2887 (CH‐str.), 2199 (CN), 1667 (C=C), 1495 (N=N). 1H NMR (400 MHz, CDCl3, δ, ppm): 8.93‐7.25 (m, 14H, Ar‐H), 5.05 (s, 1H, H‐4), 4.37 (q, J = 7.2 Hz, 2H, CH2), 2.19 (s, 3H, CH3), 1.43 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3, δ, ppm): 168.46 (N=C), 159.99 (C‐2), 152.97, 146.39, 144.77, 142.25, 133.63, 131.51, 131.27, 129.60, 129.30, 127.87, 127.41, 124.08, 123.68, 121.30 (Ar‐C), 118.09 (C‐3), 116.07 (CN), 64.05 (CH2), 42.64 (C‐4), 18.40 (CH3), 13.94 (CH3). MS (EI, m/z (%)): 506 (M+, 7.65), 44 (100). Anal. calcd. for C30H23ClN4O2: C, 71.07; H, 4.57; N, 11.0. Found: C, 70.90; H, 4.36; N, 10.67 %. 242 Radini et al. / European Journal of Chemistry 8 (3) (2017) 240‐247 Scheme 2 2.2.3. Synthesis of N'‐(4‐(4‐chlorophenyl)‐3‐cyano‐6‐((E)‐ phenyldiazenyl)‐4H‐benzo[h] chromen‐2‐yl)formimidamide (6) A stream of NH3 gas was passed through compound 4 (4.92 g, 10 mmol) in methanol (20 mL) at room temperature for 1 h. Then the mixture was left overnight. The solid product was collected and crystalized from benzene (Scheme 2). Colour: Pale yellow solid. Yield: 77%. M.p.: 266‐268 °C. FT‐IR (KBr, ν, cm–1): 3342, 3328, (NH2) 2980, 2962, 2894 (CH‐str.), 2203 (CN), 1664 (C=C), 1501 (N=N). 1H NMR (400 MHz, CDCl3, δ, ppm): 8.88 (s, 1H, N=CH), 8.87‐7.35 (m, 16H, Ar‐H + NH2), 5.22 (s, 1H, H‐4). 13C NMR (100 MHz, CDCl3, δ, ppm): 160.21 (C‐2), 155.47 (N=CH), 146.43 (C‐6), 152.87, 144.43 143.48, 132.42, 132.06, 131.20, 130.40, 129.97, 129.38, 128.89, 128.09, 124.16, 123.53, 123.29, 121.85, 120.19 (Ar‐C), 117.66 (CN), 112.65 (C‐4a), 72.81 (C‐3), 41.85 (C‐4). MS (EI, m/z (%)): 465 (M++ 2, 1.09), 463 (M+, 3.84), 77 (100). Anal. calcd. for C27H18ClN5O: C, 69.90; H, 3.91; N, 15.10. Found: C, 69.64; H, 3.72; N, 14.86%. 2.2.4. Synthesis of N‐benzylidene‐N'‐(4‐(4‐chlorophenyl)‐3‐ cyano‐6‐((E)‐phenyldiazenyl)‐4H‐benzo[h]chromen‐2‐yl) formimidamide (7) A mixture of compound 6 (4.63 g, 10 mmol), benzaldehyde (0.01 mol), dioxane (20 mL) and piperidine (0.5 mL) was refluxed for 2 h. The solid product was collected by filtration and crystallized from dioxane (Scheme 2). Colour: Yellow solid. Yield: 70%. M.p.: 300‐302 °C. FT‐IR (KBr, ν, cm–1): 2989, 2970, 2885 (CH‐str.), 2220 (CN), 1667 (C=C), 1546 (N=N). 1H NMR (400 MHz, CDCl3, δ, ppm): 9.01 (s, 1H, N=CH), 8.77 (s, 1H, N=CH), 8.70‐7.63 (m, 19H, Ar‐H + NH2), 5.81 (s, 1H, H‐4). 13C NMR (100 MHz, CDCl3, δ, ppm): 166.54 (C‐2), 159.20 (N=CH), 148.01 (C‐6), 153.32, 145.21 144.02, 133.10, 132.92, 132.53, 131.65, 130.72, 130.51, 129.80, 129.52, 128.75, 128.34, 127.66, 127.43, 124.20, 123.84, 123.50, 121.92, 120.38 (Ar‐C), 117.01 (CN), 112.89 (C‐4a), 72.45 (C‐3), 41.53 (C‐4). MS (EI, m/z (%)): 552 (M++2, 1.45), 551 (M+, 3.76), 77 (100). Anal. calcd. for C34H22ClN5O: C, 73.98; H, 4.02; N, 12.69. Found: C, 73.70; H, 3.84; N, 12.53 %. 2.2.5. Synthesis of 7‐(4‐chlorophenyl)‐5‐(phenyldiazenyl)‐ 7,9‐dihydro‐8H‐benzo[7,8]chromeno [2,3‐d]pyrimidine‐8‐ thione (8) Gaseous hydrogen sulfide was bubbled through compound 4 (4.92 g, 10 mmol) in presence of triethylamine (0.5 mL) in methanol for 2 hours. The solid formed was collected to give compound 8 and recrystallized from benzene (Scheme 2). Color: Pale brown solid. Yield: 76%. M.p.: 280‐282 °C. FT‐IR (KBr, ν, cm–1): 3220 (NH), 3010, 3000, 2987 (CH‐str.), 1668 (C=C), 1560 (N=N), 1310 (C=S). 1H NMR (400 MHz, CDCl3, δ, ppm): 8.95 (s, 1H, N=CH), 8.74‐7.62 (m, 15H, Ar‐H + NH), 5.73 (s, 1H, H‐4). 13C NMR (100 MHz, CDCl3, δ, ppm): 183.62 (C‐8), 161.04 (C‐11a), 156.63 (C‐10), 153.63, 144.41, 143.50, 132.61, 132.33, 131.28, 130.49, 129.90, 129.40, 128.91, 128.23, 124.38, 123.75, 123.38, 122.15, 121.23 (Ar‐C), 111.90 (C‐7a), 42.05 (C‐7). Radini et al. / European Journal of Chemistry 8 (3) (2017) 240‐247 243 O N N Cl NN NH N O N N Cl NN N N 9 R CH a) CH(OC2H5)3 or HCOOH b) CH3C(OC2H5)3 or CH3COCl c) PhCOCl d) NCCH2COOC2H5 e) ClCH2COCl O N N Cl NN N H N S CS2 / alc. KOH 10a; R = H 10b; R = CH3 10c; R = Ph 10d; R = CH2CN 10e; R = CH2Cl 11 12 O N N Cl NN N N O N N Cl NN N N -H2 10c PhCHO / et han ol Pip er di ne O N N Cl NN CN NC OC2H5 H N N NH2 CN O N N Cl NN N NH2 C CN N 13 H H Dioxane/ pip. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 56 7 8 9 10 11 12 13 14 16 1 2 345 a b c de f a b c e d fg123 4 5 6 Scheme 3 MS (EI, m/z (%)): 481 (M+ + 1, 1.41), 480 (M+, 9.33), 69 (100). Anal. calcd. for C27H17ClN4OS: C, 67.43; H, 3.56; N, 11.65. Found: C, 67.11; H, 3.25; N, 11.37 %. 2.2.6. Synthesis of 7‐(4‐chlorophenyl)‐8‐imino‐5‐(phenyl diazenyl)‐7H‐benzo[7,8]chromeno[2,3‐d]pyrimidin‐9(8H)‐ amine (9) A solution of compound 4 (4.92 g, 10 mmol), hydrazine hydrate (5 mL, 99%), in ethanol (50 mL) was stirred at room temperature for h. The yellow solid product formed was washed with cold ethanol, dried and recrystallized from dioxane (Scheme 2). Color: Yellow solid. Yield: 85%. M.p.: 225‐ 227 °C. FT‐IR (KBr, ν, cm–1): 3320, 3280 (NH2), 3210 (NH), 2954, 2921, 2881 (CH‐str.), 1647 (C=N), 1544 (N=N). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 8.83 (s, 1H, H‐10), 8.82‐7.31 (m, 14H, Ar‐H), 6.81 (br, 1H, NH), 5.74 (s, 2H, NH2, exchangeable by D2O), 5.45 (s, 1H, H‐7). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 155.87 (C‐11a), 152.89 (C‐8), 151.43 (C‐10), 146.78, 144.06, 143.62, 131.96, 131.03, 130.44, 129.93, 129.08, 128.77, 128.54, 128.06, 124.11, 123.49, 123.28, 121.66 (Ar‐C), 40.63 (C‐7), 112.84 (C‐7a). MS (EI, m/z (%)): 480 (M++ 2, 12.28), 478 (M+, 26.86), 77 (100). Anal. calcd. for C27H19ClN6O: C, 67.71; H, 4.00; N, 17.55. Found: C, 67.45; H, 3.84; N, 17.24 %. 2.2.7. Synthesis of triazolo derivatives 10a‐e General procedure: A mixture of compound 9 (4.78 g, 10 mmol), triethyl orthoformate or formic acid, (0.01 mol), acetyl chloride or triethyl orthoacetate (0.01 mol), benzoyl chloride (10 mmol) and chloro acettylchloirde (10 mmol) in dry benzene (20 mL) was refluxed for 3 h to give compound 10a‐c and 10e, while a mixture of compound 9 (0.01 mol), ethyl cyanoacetate (10 mmol) in absolute ethanol (20 mL) was refluxed for 5 h to give compound 10d, respectively, the solvent was extracted and the resulting product was recrystal‐ lized from 1,4‐dioxane (Scheme 3). 14‐(4‐Chlorophenyl)‐12‐(phenyldiazenyl)‐14H‐benzo[7, 8] chromeno[3,2‐e][1,2,4]triazolo [1,5‐c]pyrimidine (10a): Color: Pale yellow solid. Yield: 83%. M.p.: 320‐322 °C. FT‐IR (KBr, ν, cm–1): 3001, 2961, 2888 (CH‐str.), 1650 (C=N), 1547 (N=N). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 9.06 (s, 1H, H‐2), 8.92‐6.88 244 Radini et al. / European Journal of Chemistry 8 (3) (2017) 240‐247 (m, 15H, Ar‐H + H‐5), 5.80 (s, 1H, H‐14). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 160.11 (C‐6a) 152.74 (C‐12), 151.34 (C‐2), 150.62 (C‐14b), 144.91 (C‐8b), 142.76, 142.27, 141.10, 132.51, 132.10, 131.01, 130.33, 129.70, 129.10, 128.32, 128.02, 123.96, 123.40, 122.66, 121.43 (Ar‐C), 117.73 (C‐14a), 40.51 (C‐14). MS (EI, m/z (%)): 490 (M++2, 1.90), 488 (M+, 3.64), 127 (100). Anal. calcd. for C28H17ClN6O: C, 68.78; H, 3.50; N, 17.19. Found: C, 68.60; H, 3.32; N, 17.01%. 14‐(4‐Chlorophenyl)‐2‐methyl‐12‐(phenyldiazenyl)‐14H‐ benzo[7, 8]chromeno[3, 2‐e][1, 2, 4]triazolo[1, 5‐c]pyrimidine (10b): Color: Pale yellow solid. Yield: 80%. M.p.: > 360 °C. FT‐ IR (KBr, ν, cm–1): 3010, 2975, 2897 (CH‐str.), 1643 (C=N), 1549 (N=N). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 9.62 (s, 1H, H‐5) 8.90‐7.34 (m, 14H, Ar‐H), 6.09 (s, 1H, H‐14), 2.46 (s, 3H, CH3). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 161.44 (C‐6a), 153.93 (C‐12), 153.32 (C‐2), 152.82 (C‐14b), 146.70 (C‐8b), 121.55, 123.29, 123.52, 124.07, 128.29, 129.18, 129.94, 130.77, 131.11, 132.09, 143.29, 141.16, 132.41, 128.74 (Ar‐C), 118.70 (C‐14a), 112.91 (C‐13a), 104.09 (C‐13), 40.30 (C‐14), 14.50 (CH3). MS (EI, m/z (%)): 504 (M+ + 2, 24.46), 502 (M+, 65.05), 77 (100). Anal. calcd. for C29H19ClN6O: C, 69.25; H, 3.81; N, 16.71. Found: C, 69.04; H, 3.63; N, 16.54%. 14‐(4‐Chlorophenyl)‐2‐phenyl‐12‐(phenyldiazenyl)‐14H‐ benzo[7, 8]chromeno[3, 2‐e][1, 2, 4]triazolo[1, 5‐c]pyrimidine (10c): Color: Yellow solid. Yield: 76%. M.p.: 275‐277 °C. FT‐IR (KBr, ν, cm–1): 3012, 2976, 2899 (CH‐str.), 1641 (C=N), 1548 (N=N). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 8.87 (s, 1H, H‐5), 8.78‐6.82 (m, 19H, Ar‐H), 6.01 (s, 1H, H‐14). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 161.44 (C‐6a), 153.93 (C‐12), 153.32 (C‐2), 152.82 (C‐14b), 146.70 (C‐8b), 143.90, 143.29, 141.16, 132.41, 132.09, 131.11, 130.77, 129.93, 129.45, 128.97, 128.77, 123.93, 123.65, 123.45, 121.33 (Ar‐C), 118.70 (C‐14a), 112.91 (C‐13a), 104.09 (C‐13), 37.95 (C‐14). MS (EI, m/z (%)): 565 (M++1, 2.49), 564 (M+, 6.50), 77 (100). Anal. calcd. for C34H21ClN6O: C, 72.27; H, 3.75; N, 14.87. Found: C, 72.05; H, 3.52; N, 14.62 %. 2‐(14‐(4‐Chlorophenyl)‐12‐(phenyldiazenyl)‐14H‐benzo [7,8]chromeno[3, 2‐e][1, 2, 4]triazolo[1,5‐c]pyrimidin‐2‐yl)aceto nitrile (10d): Color: Yellow solid. Yield: 74%. M.p.: 268‐270 °C. FT‐IR (KBr, ν, cm–1): 2973, 2962, 2901 (CH‐str.), 2240 (CN), 1636 (C=N), 1545 (N=N). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 9.76 (s, 1H, H‐5), 8.90‐7.30 (m, 14H, Ar‐H), 6.09 (s, 1H, H‐14), 4.46 (s, 2H, CH2). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 161.44 (C‐6a), 153.93 (C‐12), 153.32 (C‐2), 152.82 (C‐14b), 146.70 (C‐8b), 143.90, 143.29, 141.16, 132.41, 132.09, 131.11, 130.77, 129.94, 129.18, 128.74, 128.29, 124.07, 123.52, 123.29, 121.55 (Ar‐C), 117.73 (C‐14a), 116.92 (CN), 112.81 (C‐ 13a), 101.69 (C‐13), 40.61 (C‐14), 18.43 (CH2). MS (EI, m/z (%)): 529 (M+ + 2, 34.39), 527 (M+, 81.52), 77 (100). Anal. calcd. for C30H18ClN7O: C, 68.25; H, 3.44; N, 18.57. Found: C, 68.05; H, 3.16; N, 18.31 %. 2‐(Chloromethyl)‐14‐(4‐chlorophenyl)‐12‐(phenyldiazenyl)‐ 14H‐benzo[7,8]chromeno[3,2‐e][1,2,4]triazolo[1,5‐c]pyrimidine (10e): Color: Yellow solid. Yield: 70%. M.p.: > 360 °C. FT‐IR (KBr, ν, cm–1): 2961, 2934, 2890 (CH‐str.), 1654 (C=N), 1542 (N=N). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 9.84 (s, 1H, H‐5), 8.94‐7.32 (m, 14H, Ar‐H), 6.10 (s, 1H, H‐14), 4.51 (s, 2H, CH2). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 161.48 (C‐6a), 153.96 (C‐12), 153.36 (C‐2), 153.04 (C‐14b), 147.71 (C‐8b), 144.74, 143.45, 141.43, 132.40, 132.11, 131.15, 130.58, 129.63, 129.33, 128.66, 128.40, 124.35, 123.81, 123.63, 121.73 (Ar‐C), 117.70 (C‐14a), 112.85 (C‐13a), 101.72 (C‐13), 40.73 (C‐14), 18.60 (CH2). MS (EI, m/z (%)): 537 (M+ + 1, 0.83), 536 (M+, 1.44), 77 (100). Anal. calcd. for C29H18Cl2N6O: C, 64.82; H, 3.38; N, 15.64. Found: C, 64.65; H, 3.08; N, 15.32 %. 2.2.8. Synthesis of 14‐(4‐chlorophenyl)‐12‐(phenyl diazenyl)‐14H‐benzo[7,8]chromeno[3,2‐e][1,2,4]triazolo [1,5‐c]pyrimidine‐2(3H)‐thione (11) A mixture of compound 9 (4.78 g, 10 mmol) with carbon disulfide (0.01 mol) and potassium hydroxide (10 mmol) in ethanol (15 mL) was heated under reflux for 5 h. After removal of ethanol, water was added and the resulting alkaline solution was acidified with acetic acid and the precipitate formed collected by filtration, washed with water and dried and crystallized from 1,4‐dioxane (Scheme 3). Color: Yellow solid. Yield: 69%. M.p.: 290‐292 °C. FT‐IR (KBr, ν, cm–1): 3300 (NH), 3020, 2951, 2850 (CH‐str.), 1646 (C=N), 1533 (N=N), 1043 (C=S). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 10.21 (s, 1H, NH), 8.73‐6.69 (m, 14H, Ar‐H), 8.95 (s, 1H, H‐5), 5.74 (s, 1H, H‐14). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 170.03 (C=S), 162.32 (C‐6a), 157.21 (C‐14b), 156.92 (C‐2), 155.83 (C‐12), 148.64 (C‐ 8b), 145.30, 144.65, 142.86, 133.60, 132.76, 131.95, 130.86, 129.72, 129.52, 128.53, 128.27, 124.31, 123.96, 123.54, 121.95 (Ar‐C), 118.54 (C‐14a), 113.05 (C‐13a), 108.11 (C‐13), 40.10 (C‐14). MS (EI, m/z (%)): 522 (M+ + 2, 3.34), 520 (M+, 6.05), 63 (100). Anal. calcd. for C28H17ClN6OS: C, 64.55; H, 3.29; N, 16.13. Found: C, 64.31; H, 3.12; N, 15.89 %. 2.2.9. Synthesis of 9‐(benzylideneamino)‐7‐(4‐chloro phenyl)‐5‐((E)‐phenyldiazenyl)‐7,9‐dihydro‐8H‐benzo[7,8] chromeno[2,3‐d]pyrimidin‐8‐imine (12) A mixture of compound 9 (4.78 g, 10 mmol), benzaldehyde (10 mmol), piperidine (0.05 mL) and dioxane (30 mL) was refluxed for 6 h. The solvent was extracted and the resulting product was recrystallized from 1,4‐dioxane (Scheme 3). Color: Yellow solid. Yield: 72%. M.p.: 242‐244 °C. FT‐IR (KBr, ν, cm–1): 3211 (NH), 3068, 3010, 2964 (CH‐str.), 1637(C=N), 1528 (N=N). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.07 (br, 1H, NH), 8.89 (s, 1H, N=CH), 8.50 (s, 1H, H‐10), 8.35‐6.91 (m, 19H, Ar‐H), 6.05 (s, 1H, H‐7). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 163.83 (C‐8), 160.98 (C‐11a), 158.42 (C‐10), 156.56 (N=CH), 151.65 (C‐11a), 144.88, 144.51, 137.55, 134.64, 132.64, 130.23, 129.62, 128.91, 127.54, 126.74, 126.33, 124.54, 124.01, 121.74, 119.82, 117.57, 102.79 (Ar‐C), 97.60 (C‐7a), 39.36 (C‐7). MS (EI, m/z (%)): 568 (M+ + 2, 005), 566 (M+, 1.34), 356 (100). Anal. calcd. for C34H23ClN6O: C, 72.02; H, 4.09; N, 14.82. Found: C, 71.88; H, 3.92; N, 14.67 %. 2.2.10. Synthesis of 4‐amino‐16‐(4‐chlorophenyl)‐14‐ (phenyldiazenyl)‐16H‐benzo[7',8'] chromeno[2',3':4,5] pyrimido[1,6‐b][1,2,4]triazepine‐3‐carbonitrile (13) A mixture of compound 9 (4.78 g, 10 mmol), 2‐ (ethoxymethylene)malononitrile (0.01 mol), piperidine (0.05 mL) and ethanol (30 mL) was refluxed for 5 h. The solvent was extracted and the resulting product was recrystallized from N,N‐dimethylformamide (DMF) (Scheme 3). Color: Pale yellow solid. Yield: 68%. M.p.: 290‐292 °C. FT‐IR (KBr, ν, cm–1): 3340, 3332 (NH2), 3001, 2986, 2923 (CH‐str.), 2217 (CN), 1670 (C=N), 1507 (N=N). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 5.72 (s, 1H, H‐16), 8.89 (s, 1H, N=CH), 8.50 (s, 1H, H‐7), 8.76‐6.83 (m, 16H, Ar‐H + NH2). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 162.21 (C‐8a), 160.11 (C‐6a), 154.72 (C‐2), 151.20 (C‐4), 149.70 (C‐7), 147.71, 145.53, 144.34, 133.21, 133.01, 132.81, 132.62, 130.70, 130.18, 129.62, 129.15, 124.16, 123.90, 123.48, 123.26, 121.59, 121.43 (Ar‐C), 116.83 (CN), 105.62 (C‐ 3), 92.31 (C‐16a), 39.81 (C‐16). MS (EI, m/z (%)): 555 (M+ + 1, 0.92), 554 (M+, 1.25), 77 (100). Anal. calcd. for C31H19ClN8O: C, 67.09; H, 3.45; N, 20.19. Found: C, 68.85; H, 3.17; N, 19.89 %. 2.3. Antimicrobial Assay All the newly synthesized compounds 3‐13 were screened for their in vitro antimicrobial activity at 25 µg/mL to determine the zone of inhibition against four Gram‐positive bacteria: Staphylococcus aureus (RCMB 000106) and Bacillis subtilis (RCMB 000108) and two Gram‐negative pathogenic bacteria: Pseudomonas aeruginosa (RCMB 000102) and Radini et al. / European Journal of Chemistry 8 (3) (2017) 240‐247 245 Table 1. Antimicrobial activity of the new compounds. Compound a Minimum inhibitory concentration (MIC) (μg/mL) b Bacterial strains Fungal strains Gram‐positive bacteria Gram‐negative bacteria S. aureus B. subtilis P. aeruginosa E. coli A. fumigatus C. albicans 3 13.2±0.3 15.1±0.2 16.6±01 NA NA NA 4 16.1±0.1 18.2±0.1 NA NA NA NA 5 13.2±0.3 15.1±0.2 NA NA NA NA 6 12.9±0.1 18.1±0.1 NA NA NA NA 7 15.1±0.1 12.1±0.3 NA NA NA NA 8 17.1±0.1 14.9±0.1 18.2±0.1 NA NA NA 9 18.1±0.1 15.5±0.2 17.6±02 18.8±0.1 16.7±0.1 13.7±0.2 10a 23.2±0.1 20.5±0.2 19.6±01 24.2±0.1 23.7±0.2 21.7±0.2 10b 20.4±0.2 21.3±0.1 22.1±0.2 24.1±0.1 20.9±0.2 23.1±0.1 10c 22.6±0.1 20.9±0.2 20.3±01 24.2±0.3 23.9±0.1 24.6±0.2 10d 28.1±0.3 27.2±0.1 25.3±02 26.1±0.4 25.2±0.1 23.2±0.2 10e 26.1±0.1 23.8±0.2 25.6±01 23.6±0.3 29.1±0.6 20.3±0.1 11 16.9±0.3 18.5±0.1 14.9±01 16.1±0.2 19.2±0.4 14.8±01 12 20.5±0.3 19.8±0.1 21.7±0.2 28.7±0.1 27.6±0.2 23.1±0.3 13 25.7±0.1 28.5±0.3 27.3±01 27.2±0.3 23.6±0.3 22.8±0.2 Ampicillin 27.4±0.1 29.4±0.7 26.3±0.3 28.5±0.1 ‐ ‐ Mycostatine ‐ ‐ ‐ ‐ 29.5±0.1 27.1±0.1 a c = 1 mg/mL of new compounds in DMF b NA = Not active, Diameter of the hole = 6 mm, Data are expressed in the form of mean±SD. Escherichia coli (RCMB 000103) using standard antibiotics (Ampicillin) as reference drugs, and two fungi: Aspergillus fumigatus (RCMB 002003) and Candida albicans (RCMB 005002) using standard antibiotics (Mycostatine) as reference drugs. The activities of these compounds were tested by agar diffusion method using Mueller‐Hinton agar medium for bacteria and Sabouraud’s agar medium for fungi [46,47]. The tested compounds were dissolved in N,N‐dimethylformamide to give a solution of 1 mg/mL. The inhibition zones (diameter of the hole) were measured in millimeters (6 mm) at the end of an incubation period of 48 h at 28 °C; N,N‐dimethyl‐ formamide showed no inhibition zone. The inhibitory effects of the synthetic compounds against these organisms are given in Figure 1 and Table 1. 0 5 10 15 20 25 30 35 In h ib it io n z o n e d ia m et er Compounds S. aureus B. subtilis P. aeruginosa E. coli A. fumigatus C. albicans Figure 1. Antimicrobial activity of the tested compounds compared to ampicillin and mycostatine. 3. Results and discussion Condensation of 4‐phenyldiazenyl‐1‐naphthol (1) with 2‐ (p‐chlorobenzylidene)malonoitrile (2) in ethanolic piperidine afforded the corresponding 2‐amino‐4‐(p‐chlorophenyl))‐6‐ phenyldiazenyl‐4H‐naphtho[1,2‐b]pyran‐3‐carbonitrile (3) [45], while treatment of compound 3 with triethyl orthofor‐ mate or triethyl orthoacetate in acetic anhydride for 5 h afforded ethyl N‐(4‐(4‐chlorophenyl)‐3‐cyano‐6‐(phenyl diazenyl)‐4H‐benzo[h]chromen‐2‐yl)formimidate (4) and ethyl N‐(4‐(4‐chlorophenyl)‐3‐cyano‐6‐(phenyldiazenyl)‐4H‐ benzo[h]chromen‐2‐yl)acetimidate (5), respectively (Scheme 1). The structure of compound 4 and 5 were in accord with its spectroscopic data. The IR spectrum of compounds showed the principal absorption band at 2201 and 2199 cm–1, indicating the presence of a cyano group (CN) and disappear of amino group (NH2) in the molecule. Thus, the 1H NMR spectrum of compound 4 and 5 showed a singlet at δ 5.01 and δ 5.05 ppm for the 4H‐pyran, a triplet at δ 1.44‐1.43 ppm and a quartet at δ 4.37‐4.54 ppm indicating ethoxy group, a singlet at δ 8.94 ppm to the N=CH group and a singlet at δ 2.19 ppm to the CH3 group. Reaction of compound 4 with ammonia (NH3) gas in methanol at room temperature for 1 h yielded the open chain product N'‐(4‐(4‐chlorophenyl)‐3‐cyano‐6‐(phenyldiazenyl)‐ 4H‐benzo[h]chromen‐2‐yl)formimidamide (6), follow by benz‐ aldehyde under reflux afforded Schiff base product 7, while treatment of compound 4 with hydrogen sulfide in ethanol/ triethyl amine at room temperature for 2 h afforded 7‐(4‐ chlorophenyl)‐5‐(phenyldiazenyl)‐7,9‐dihydro‐8H‐benzo[7,8] chromeno[2,3‐d]pyrimidine‐8‐thione 8. Reaction of compound 4 with hydrazine hydrate in ethanol at room temperature for 2 h afforded 7‐(4‐chlorophenyl)‐8‐imino‐5‐(phenyldiazenyl)‐ 7H‐benzo[7,8]chromeno[2,3‐d]pyrimidin‐9(8H)‐amine (9) (Scheme 2). The structure of compounds 6‐9 were established by spectral data. The IR spectrum of compound 6 showed absorp‐ tions at ν 3342, 3328 (NH2), 2203 cm‐1 (CN), while compound 7 showed absorption at 3220 (NH), 1310 cm‐1 (C=S) and compound 9 showed absorptions at 3320, 3280 (NH2), 3210 cm‐1 (NH). The 1H NMR of compound 6 showed chemical shifts at δ 8.88 (s, 1H, N=CH), 5.22 (s, 1H, H‐4), while compound 9 showed chemical shifts at δ 8.83 (s, 1H, H‐10), 6.81 (br, 1H, NH), 5.74 (s, 2H, NH2) and 5.45 (s, 1H, H‐7). While the 13C NMR of compound 6 showed δ 41.85 (C‐4), 72.81 (C‐3), 117.66 (CN), 155.47 (N=CH) and 160.21 (C‐2). 13C NMR of compound 8 showed δ 42.05 (C‐7), 111.90 (C‐7a), 156.63 (C‐10), 161.04 (C‐ 11a) and 183.62 (C‐8). 13C NMR of compound 9 showed δ 40.63 (C‐7), 151.43 (C‐10) and 152.89 (C‐8). The mass spectra of compounds 6‐9 displayed [M+] ion peaks m/z 463 (M+, 3.84), 551 (M+, 3.76), 480 (M+, 9.33) and 478 (M+, 26.86), respectively. Condensation of compound 9 with carboxylic acid deri‐ vatives such as formic acid or triethyl orthoformate, acetyl chloride or triethyl orthoacetate, benzoyl chloride, ethyl cyanoacetate, and chloroacetyl chloride, afforded benzo‐ chromenotriazolo pyrimidines 10a‐e, respectively. Reaction of compound 9 with benzaldehyde in ethanol/piperdine gave the open chain product 9‐(benzylideneamino)‐7‐(4‐chlorophenyl) ‐5‐((E)‐phenyldiazenyl)‐7,9‐dihydro‐8H‐benzo[7,8]chromeno [2,3‐d]pyrimidin‐8‐imine, 12. Compound 10c was also 246 Radini et al. / European Journal of Chemistry 8 (3) (2017) 240‐247 prepared by cyclization of compounds 12 in dioxane/ piperidine solution under reflux as confirmed by the M.p., mixed M.p., and their identical IR, NMR and MS spectra. Treatment of compound 9 with carbon disulfide in alcoholic potassium hydroxide solution gave the 14‐(4‐chlorophenyl)‐ 12‐(phenyldiazenyl)‐14H‐benzo[7, 8]chromeno[3, 2‐e][1, 2, 4] triazolo[1,5‐c]pyrimidine‐2(3H)‐thione, while reaction of compound 9 with 2‐(ethoxymethylene)malononitrile in dioxane under reflux afforded 4‐amino‐16‐(4‐chlorophenyl)‐ 14‐(phenyldiazenyl)‐16H‐benzo[7', 8']chromeno[2', 3':4, 5] pyrimido [1,6‐b][1,2,4]triazepine‐3‐carbonitrile (Scheme 3). The structure of compounds 10‐13 were established by spectral data. The IR spectrum of compound 11 showed absorptions at 3300 (NH), 1043 cm−1 (C=S), compound 12 showed absorptions at 3211 cm‐1 (NH) and compound 13 showed absorptions at 3340, 3332 (NH2), 2217 cm−1 (CN). The 1H NMR of compound 10b showed chemical shifts at δ 9.62 (s, 1H, H‐5), 6.09 (s, 1H, H‐14), 2.46 ppm (s, 3H, CH3), compound 10d showed chemical shifts δ 9.76 (s, 1H, H‐5), 6.09 (s, 1H, H‐ 14), 4.46 ppm (s, 2H, CH2), compound 11 showed chemical shifts δ 10.21 (s, 1H, NH), 8.95 (s, 1H, H‐5), 5.74 ppm (s, 1H, H‐ 14) and compound 13 showed chemical shifts δ 5.72 (s, 1H, H‐ 16), 8.89 (N=CH), 8.50 (s, 1H, H‐7), 8.76‐6.83 ppm (m, 16H, Ar‐ H + NH2), while the 13C NMR of compound 10a showed δ 40.51 (C‐14), 151.34 ppm (C‐2), compound 10b showed δ 14.50 (CH3), 40.30 (C‐14), 153.32 ppm (C‐2), compound 10d showed δ 18.43 (CH2), 40.61 (C‐14), 116.92 (CN), 153.32 ppm (C‐2) and compound 13 showed δ at 39.81 (C‐16), 116.83 (CN), 149.70 (C‐7), 151.20 (C‐4), 154.72 ppm (C‐2). The mass spectra of compounds 10‐13 displayed [M+] ion peaks m/z 488 (M+, 3.64), 502 (M+, 65.05), 564 (M+, 6.50), 527 (M+, 81.52), 536 (M+, 1.44), 520 (M+, 6.05), 566 (M+, 1.34) and 554 (M+, 1.25), respectively. The structure activity relationship studies of compounds 3‐13 revealed that compounds 10d, e, 12 and 13 with inhibitory effects of 28.1±0.3, 29.1±0.6, 28.7±0.1 and 28.5±0.3 µg/mL good activities than the against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Aspergillus fumigatus, respectively, compared to the standard antibiotics, ampicillin and mycostatine as reference drugs. Other compounds showed almost equipotent activities or were inactive, implying that the benzochromenotriazolopyrimidine and benzochromenepyrimidine nucleus was more active than the benzochromene and 2‐methyleneaminobenzochromene. Compounds 10a‐e, 12, and 13 were found to be the with inhibitory effects ranging 27.4±0.1 to 29.4±0.7 µg/mL more activities as compared to the standard antibiotics ampicillin and mycostatine, while compounds 9 and 11 with inhibitory effect of 13.7±0.2 to 19.2±0.4 µg/mL were exhibited moderate activities as compared to the standard antibiotics ampicillin and mycostatine and compounds 3 and 8 showed moderate activity against Pseudomonas aeruginosa with inhibitory effect ranging 16.6±0.1 and 18.2±0.1 µg/mL as compared to the standard antibiotics ampicillin, while compounds 3, 4, 5, 6 , 7 and 8 moderate to weak activity against Staphylococcus aureus, Bacillis subtilis and Pseudomonas aeruginosa with inhibitory effect ranging 13.2±0.3 and 18.1±0.1 µg/mL as compared to the standard antibiotics ampicillin. Compounds 4, 5, 6 and 7 inactive against Pseudomonas aeruginosa, while compounds 3, 4, 5, 6, 7 and 8 inactive against Escherichia coli, and Aspergillus fumigatus, respectively, compared to the standard antibiotics, ampicillin and mycostatine as reference drugs. 4. Conclusions In conclusion, a series of novel methyleneaminobenzo‐ chemene, benzochromenotriazolopyrimidine were synthe‐ sized successfully in good yield, starting from 4H‐benzo‐ chromene derivative. All the new compounds were fully spectroscopically characterized. The title compounds were synthesized as new compounds with antimicrobial activity in vitro. Compounds 4, 5, 6 and 7 inactive against Pseudomonas aeruginosa, while compounds 3, 4, 5, 6, 7 and 8 inactive against Escherichia coli, and Aspergillus fumigatus, respect‐ tively, compared to the standard antibiotics, ampicillin and mycostatine as reference drugs, while compounds 10a‐e, 12, 13 showed high to good activities compared to the standard antibiotics, The structure‐activity relationship study revealed that the antimicrobial activity of benzochromenopyrimido‐ triazepine nucleus was more beneficial than benzochromeno‐ triazolopyrimidine nucleus for antimicrobial activity. Acknowledgements The authors profoundly thank the Regional Center for Mycology & Biotechnology (RCMP), Al‐Azhar University, Cairo, Egypt, for providing the facilities to determine the anti‐ bacterial and antifungal activities. The authors thank the Deanship of Scientific Research of Jazan University to support the research. References [1]. 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