untitled European Journal of Chemistry 8 (1) (2017) 76‐81 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.8.1.76-81.1542 European Journal of Chemistry Journal webpage: www.eurjchem.com Synthesis and characterization of new imidazolidineiminothione and bis‐imidazolidineiminothione derivatives as potential antimicrobial agents Yousry Ahmed Ammar 1, Samir Youssef Abbas 2, Marwa Ahmed Mohamed Shehab El‐Sharief 3, Mohamed Abd El‐Rashid Salem 1,4,* and Ahmed Ragab Mohamed 1 1 Chemistry Department, Faculty of Science, Al‐Azhar University, Nasr City, Cairo, 11284, Egypt 2 Organometallic and Organometalloid Chemistry Department, National Research Centre, Cairo, 12622, Egypt 3 Applied Organic, Chemistry Department, National Research Centre, Cairo, 11284, Egypt 4 Chemistry Department, Faculty of Science and Arts, King Khalid University, Mohail Assir, 900, Kingdom of Saudi Arabia * Corresponding author at: Chemistry Department, Faculty of Science, Al‐Azhar University, Nasr City, Cairo, 11284, Egypt. Tel.: +966.536.563810. Fax: +966.536.563810. E‐mail address: m_eltayyeb@hotmail.com (M.A. Salem). ARTICLE INFORMATION ABSTRACT DOI: 10.5155/eurjchem.8.1.76-81.1542 Received: 03 January 2017 Received in revised form: 26 January 2017 Accepted: 29 January 2017 Published online: 31 March 2017 Printed: 31 March 2017   A series of new imidazolidineiminothiones (2‐4) and bis‐imidazolidineiminothiones (5) were synthesized through the cycloaddition reaction of N‐arylcyanothioformamides (1) with some electrophilic reagents. Structure of imidazolidineiminothione derivatives were established based on spectroscopic IR, 1H NMR, 13C NMR, MS and elemental analyses data. These compounds were screened for their antibacterial and antifungal activities. Among the synthesized compounds, imidazolidineiminothione derivative 3a showed about 25% less potent effect than Ampicillin against S. epidermidis and B. subtilis (MIC, 0.49 μg/mL) and about 50 % less potent effect than Amphotericin B against A. clavatus and G. Candidum. Bis‐ imidazolidineiminothione derivative 5a was equipotent to the Gentamycin in inhibiting the growth of N. gonorrhoeae (MIC, 0.49 μg/mL), and displayed 50% less active than Amphotericin B against A. clavatus. KEYWORDS Cycloaddition Antifungal activity Antibacterial activity Imidazolidine iminothiones N‐Arylcyanothioformanilides Bis‐Imidazolidine iminothiones Cite this: Eur. J. Chem. 2017, 8(1), 76‐81 1. Introduction Infectious diseases caused by bacteria and fungi remain a major worldwide health problem due to rapid development of resistance to the existing antibacterial and antifungal drugs. So, the medical community faces a serious problem against infections caused by the pathogenic microbes and needs effective therapies and search for novel antimicrobial agents. The quest for new and effective antimicrobial agents, resistant to the mechanisms of defense of these bacteria, is of para‐ mount importance [1‐3]. Imidazoles and their fused derivatives are key components of great many bioactive compounds of both natural and synthetic origin [4] such as histidine, purines and biotin. Anti‐ microbial activity was stated among derivatives possessing aromatic substituents at the imidazole nitrogen e.g. N‐acyl and 5‐arylidene derivatives of hydantoin and 4‐thiohydantoin [5,6]. Moreover, previous reports demonstrated that synthetic imidazoles act either as inhibitors of α‐adrenoceptor mediated events in platelets [7] or inducers of platelets activation [8]. Imidazolidineiminothiones and the various heterocycles derived from them were shown to exhibit an interesting and a wide range of pharmacological effects including antitumor, antiviral, antimicrobial and antifungal strains [9‐15]. N‐Substi‐ tuted cyanothioformamides were used as a key intermediate for a number of interesting heterocyclic ring closures such as imidazole [16], oxazole [17], and thiazole [18,19]. In parti‐ cular, their reactions with isocyanates [16] and isothiocya‐ nates [17] give 1,3‐disubstituted imidazolidine systems. In view of the above‐mentioned findings, and as a continuation of our effort to identify new candidates [9‐15], which are crucial in designing new, potent, selective, and less toxic antimicrobial agents, its report herein the synthesis and antimicrobial evaluation of some novel imidazolidineimino thione. The target compounds were rationalized so as to comprise some pharmacophores that are believed to be responsible for the biological activity of some relevant chemotherapeutic such as the carbonyl, thiocarbonyl, imino and cyano groups functionalities. The substitution pattern of the imidazolidineiminothione ring was carefully selected in order to confer different electronic environment to the molecules. Ammar et al. / European Journal of Chemistry 8 (1) (2017) 76‐81 77 2a, Ar = 3,4-Cl2-C6H3 2b, Ar = 4-CH3-C6H4 2c, Ar = 4-OCH3-C6H4 N H CN S 1a EtOH, Et3N (cat), Reflux, 3 hr NC N N Ar CN F N NH S NH NC N N Ar F N N S NH NC N N Ar F N NH S N NC N N Ar F NCS i - KCN ii - HCl F Scheme 1 2. Experimental All the melting points are uncorrected. IR (KBr) spectra were measured on Shimadzu 440 spectrometer. 1H NMR and 13C NMR spectra were obtained in DMSO‐d6 on a Varian Gemini 300 MHz spectrometer using TMS as internal standard. The chemical shifts are reported as δ ppm units at the Micro‐ analytical Center in National Research Center, Egypt. Mass spectra were obtained on GCMS/QP 1000 Ex mass spectro‐ meter at 70 eV at National Research Center, Egypt. Elemental analyses were carried out at the Department of Chemistry, Faculty of Science, Cairo University, Egypt. Microbiology screening was carried out in the Regional Center for Microbiology and Biotechnology (RCMB), Antimicrobial unit test organisms, Al‐Azhr University, Cairo, Egypt. 2.1. Chemistry 2.1.1. Synthesis of 2‐(aryldiazenyl)‐2‐(1‐(4‐fluorophenyl)‐4‐ imino‐5‐thioxoimidazolidin‐2‐ylidene)acetonitriles (2a‐c) A mixture of cyanothioformamide (1a) (0.01 mol), arylhydrazonomalononitriles (0.01 mol) and of triethylamine (3 drops) in absolute ethanol was heated under reflux for 4 h. left to cool. The solid product was collected and recrystallized from dioxane to give compounds 2a‐c (Scheme 1). 2‐((3,4‐Dichlorophenyl)diazenyl)‐2‐(1‐(4‐fluorophenyl)‐4‐ imino‐5‐thioxoimid‐azolidin‐2‐ylidene)acetonitrile (2a): Color: Gray. Yield: 73 %. M.p.: 258‐260 °C. IR (KBr, ν, cm‐1): 3410 (NH), 2201 (C≡N), 1671 (C=N), 1574 (N=N), 1186 (C=S). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 6.43‐7.64 (m, 7H, Ar‐H), 8.87 (br, 1H, NH; exchangeable with D2O), 9.81 (br, 1H, NH, exchangeable with D2O). MS (EI, m/z (%)): 418 (M+; 24.7), 419 (M+1; 28.2), 245 (12.6), 174 (18.4), 159 (19.2), 145 (100), 109 (56.8), 95 (67.5), 75 (36.4). Anal. calcd. for C17H9Cl2FN6S: C, 48.70; H, 2.16; N, 20.05. Found: C, 48.48; H, 2.02; N, 20.18%. 2‐(1‐(4‐Fluorophenyl)‐4‐imino‐5‐thioxoimidazolidin‐2‐ylide ne)‐2‐(p‐tolyldiazenyl)acetonitrile (2b): Color: Gray. Yield: 72 %. M.p.: 190‐191 °C. IR (KBr, ν, cm‐1): 3401 (NH), 2213 (C≡N), 1671 (C=N), 1584 (N=N), 1182 (C=S). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 2.26 (s, 3H, CH3), 6.73‐7.52 (m, 8H, Ar‐H), 8.63 (br, 1H, NH; exchangeable with D2O), 9.56 (br, 1H, NH, exchangeable with D2O). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 21.4 (CH3), 105.1, 113.3, 116.7, 116.9, 122.5 (2C), 129.9 (2C), 130.7, 130.8, 135.4, 141.5, 150.8, 160.5, 161.9, 164.6, 188.2 (C=S). MS (EI, m/z (%)): 364 (M+; 54.2 %), 365 (M+1; 19.2), 264 (22.4), 119 (21.2), 9 (100), 65 (65.8). Anal. calcd. for C18H13FN6S: C, 59.33; H, 3.60; N, 23.06. Found: C, 59.46; H, 3.81; N, 23.42%. 2‐(1‐(4‐Fluorophenyl)‐4‐imino‐5‐thioxoimidazolidin‐2‐ylide ne)‐2‐(4‐methoxy‐phenyldiazenyl)acetonitrile (2c): Color: White. Yield: 71%. M.p.: 260‐262 °C. IR (KBr, ν, cm‐1): 3401 (NH), 2214 (CN), 1666 (C=N), 1585 (N=N), 1137 (C=S). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 3.86 (s, 3H, OCH3), 6.81‐ 7.53 (m, 8H, Ar‐H), 8.52 (br, 1H, NH, exchangeable with D2O), 9.45 (br, 1H, NH, exchangeable with D2O). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 56.1 (OCH3), 105.2, 114.6, 116.7, 116.9 (2C), 122.5 (2C), 124.5 (2C), 130.3 (2C), 130.7, 147.2, 160.4, 161.9, 163.6, 187.8 (C=S). MS (EI, m/z (%)): 380 (M+; 77.2 %), 380 (M+1; 22.2), 235 (22.4), 107 (100), 93 (44.8) 77 (15.2). Anal. calcd. for C18H13FN6OS: C, 56.83; H, 3.44; N, 22.09. Found: C, 57.11; H, 3.28; N, 21.97 %. 2.1.2. Synthesis of 1‐butyl‐5‐imino‐3‐(aryl)‐4‐thioxo imidazolidin‐2‐ones (3a‐c) To a solution of compound 1a,b and d (0.01 mol) in ether (20 mL), n‐butylisocyanate (0.01 mol) and triethylamine (0.5 mL) were added; the reaction mixture was stirred at room temperature for 2 hr. The obtained products were crystallized from absolute ethanol to form compounds 3a‐c (Scheme 2). 1‐Butyl‐3‐(4‐fluorophenyl)‐5‐imino‐4‐thioxoimidazolidin‐2‐ one (3a): Color: White. Yield: 83 %. M.p.: 80‐82 °C. IR (KBr, ν, cm‐1): 3250 (NH), 2960 (CH‐aliph), 1766 (C=O), 1660 (C=N). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 0.91 (t, 3H, J = 11.5 Hz, CH3), 1.33 (h, 2H, J = 12.0 Hz, CH2), 1.67 (p, 2H, J = 12.0 Hz, CH2), 3.68 (t, 2H, J = 11.5 Hz, CH2), 7.35‐7.42 (m, 2H, Ar‐H), 7.54‐7.58 (m, 2H, Ar‐H), 9.47 (br, 1H, NH, exchangeable with D2O). Anal. calcd. for C13H14FN3OS: C, 55.90; H, 5.05; N, 15.04. Found: C, 56.14; H, 4.89; N, 15.11%. 1‐Butyl‐5‐imino‐4‐thioxo‐3‐p‐tolylimidazolidin‐2‐one (3b): Color: Yellow. Yield: 79 %. M.p.: 95‐97 °C. IR (KBr, ν, cm‐1): 3250 (NH), 2951 (CH‐aliph), 1765 (C=O), 1660 (C=N). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 0.95 (t, 3H, J = 11.5 Hz, CH3), 1.32 (h, 2H, J = 11.5 Hz, CH2), 1.67 (p, 2H, J = 12.0 Hz, CH2), 2.36 (s, 3H, CH3), 3.67 (t, 2H, J = 12.0 Hz, CH2), 7.32 (s, 4H, Ar‐H), 9.44 (br, 1H, NH; exchangeable with D2O). 78 Ammar et al. / European Journal of Chemistry 8 (1) (2017) 76‐81 Scheme 2 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 13.5 (CH3), 19.3 (CH2), 20.7 (CH3), 20.8 (CH2), 29.0 (CH2), 127.2, 127.3, 129.4, 129.6, 130.6, 138.9, 153.8 (C=N), 154.6 (C=O), 183.1 (C=S). Anal. calcd. for C14H17N3OS: C, 61.06; H, 6.22; N, 15.26. Found: C, 61.32; H, 6.42; N, 15.52%. 1‐Butyl‐5‐imino‐3‐(4‐methoxyphenyl)‐4‐thioxoimidazolidin‐ 2‐one (3c): Color: Gray. Yield: 80 %. M.p.: 110‐112 °C. IR (KBr, ν, cm‐1): 3242 (NH), 2951 (CH‐aliph), 1763 (C=O), 1656 (C=N). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 0.91 (t, 3H, J = 11.5 Hz, CH3), 1.3 (h, 2H, J = 11.5 Hz, CH2), 1.69 (p, 2H, J = 11.5 Hz, CH2), 3.69 (t, 2H, J = 12.0 Hz, CH2), 3.81 (s, 3H, OCH3), 7.05 (d, 2H, J = 8.5 Hz, Ar‐H), 7.36 (d, 2H, J = 8.5 Hz, Ar‐H), 9.42 (br, 1H, NH; exchangeable with D2O). Anal. calcd. for C14H17N3O2S: C, 57.71; H, 5.88; N, 14.42. Found: C, 57.62; H, 5.74; N, 14.23%. 2.1.3. Synthesis of 1‐(aryl)‐4‐imino‐3‐(3‐isocyanato‐4‐ methylphenyl)‐5‐thioxoimid‐azolidin‐2‐ones (4a‐d) A mixture of cyanothioformamides 1b‐e (0.01 mol) in ether (20 mL), toluenediisocyanate (0.01 mol) and triethylamine (0.5 mL) were added, the reaction mixture was stirred at room temperature for 0.5 hr. The obtained products were crystallized from proper solvent to afford 5‐imino‐4‐ thioxo‐2‐imidazolidinones 4a‐d (Scheme 2). 4‐Imino‐3‐(3‐isocyanato‐4‐methylphenyl)‐5‐thioxo‐1‐p‐tolyl imidazolidin‐2‐one (4a): Color: Gray. Yield: 77 %. M.p.: 217‐ 219 °C. IR (KBr, ν, cm‐1): 3238 (NH), 2971 (CH‐aliph), 2272 (NCO), 1775 (C=O), 1667 (C=N). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 1.82 (s, 3H, CH3), 2.33 (s, 3H, CH3), 7.05‐7.53 (m, 7H, Ar‐H), 9.52 (s, 1H, NH, exchangeable with D2O). MS (EI, m/z (%)): 350 (M+, 14.2), 351 (M+1, 3.5), 149 (35.9), 148 (100), 122 (13.2), 107 (12.7), 92 (10.8), 77 (27.4). Anal. calcd. for C18H14N4O2S: C, 61.70; H, 4.03; N, 15.99. Found: C, 61.42; H, 4.12; N, 15.77%. 4‐Imino‐3‐(3‐isocyanato‐4‐methylphenyl)‐1‐(3‐methoxy phenyl)‐5‐thioxoimidazolidin‐2‐one (4b): Color: Beige. Yield: 81 %. M.p.: 233‐235 °C. IR (KBr, ν, cm‐1): 3343 (NH), 2968 (CH‐ aliph), 2270 (NCO), 1779 (C=O), 1665 (C=N). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 2.35 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 7.07‐7.97 (m, 7H, Ar‐H), 9.60 (s, 1H, NH, D2O exchanagable). Anal. calcd. for C18H14N4O3S: C, 59.01; H, 3.85; N, 15.29. Found: C, 59.21; H, 3.77; N, 15.42%. 4‐Imino‐3‐(3‐isocyanato‐4‐methylphenyl)‐1‐(4‐methoxy phenyl)‐5‐thioxoimidazolidin‐2‐one (4c): Color: Gray. Yield: 78 %. M.p.: 248‐250 °C. IR (KBr, ν, cm‐1): 3352 (NH), 2924 (CH‐ aliph), 2267 (NCO), 1778 (C=O), 1666 (C=N). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 2.34 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 7.08‐7.52 (m, 7H, Ar‐H), 9.70 (s, 1H, NH, exchangeable with D2O). Anal. calcd. for C18H14N4O3S: C, 59.01; H, 3.85; N, 15.29. Found: C, 58.85; H, 3.72; N, 15.33%. 1‐(3‐Ethoxyphenyl)‐4‐imino‐3‐(3‐isocyanato‐4‐methylphen yl)‐5‐thioxoimidazolidin‐2‐one (4d): Color: Beige. Yield: 79 %. M.p.: 216‐218 °C. IR (KBr, ν, cm‐1): 3346 (NH), 2978 (CH‐ aliph), 2271 (NCO), 1779 (C=O), 1665 (C=N). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 1.34 (t, 3H, J = 6.9 Hz, CH3), 2.35 (s, 3H, CH3), 4.05 (q, 2H, J = 6.9 Hz, CH2), 7.07‐7.97 (m, 7H, Ar‐H), 9.60 (s, 1H, NH, exchangeable with D2O). Anal. calcd. for C19H16N4O3S: C, 59.99; H, 4.24; N, 14.73. Found: C, 60.15; H, 4.13; N, 14.59%. 2.1.4. Synthesis of 3,3'‐(4‐methyl‐1,3‐phenylene)bis(1‐ (aryl)‐5‐imino‐4‐thioxoimid‐azolidin‐2‐ones (5a‐d) A mixture of cyanothioformamides 1b‐e (0.02 mol) in ether (20 mL), toluenediisocyanate (0.01 mol) and triethy‐ lamine (0.5 mL) were added, the reaction mixture was stirred at room temperature for 1 hr. The obtained products were crystallized from ethanol to afford bis‐imidazolidineimino thiones 5a‐d (Scheme 2). 3,3'‐(4‐Methyl‐1,3‐phenylene)bis(4‐imino‐5‐thioxo‐1‐p‐tolyl imidazolidin‐2‐one) (5a): Color: Beige. Yield: 83 %. M.p.: 165‐ 167 °C. IR (KBr, ν, cm‐1): 3304 (br, 2NH), 2922 (CH‐aliph), 1779 (br, 2C=O), 1665 (br, 2C=NH), 1219 (br, 2C=S). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 2.28 (s, 6H, 2CH3), 2.39 (s, 3H, CH3), 7.08‐8.47 (m, 11H, Ar‐H), 9.58 (s, 1H, NH, exchangeable with D2O), 9.59 (s, 1H, NH, D2O exchangeable). Anal. calcd. for C27H22N6O2S2: C, 61.58; H, 4.21; N, 15.96. Found: C, 61.44; H, 4.15; N, 15.77%. 3,3'‐(4‐Methyl‐1,3‐phenylene)bis(4‐imino‐1‐(3‐methoxy phenyl)‐5‐thioxoimidazolidin‐2‐one) (5b): Color: Gray. Yield: 85 %. M.p.: 139‐141°C. IR (KBr, ν, cm‐1): 3340 (NH), 2923 (CH‐ Ammar et al. / European Journal of Chemistry 8 (1) (2017) 76‐81 79 aliph), 1779 (br, C=O), 1664 (br, C=NH), 1223 (br, C=S). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 2.36 (s, 3H, CH3), 3.80 (s, 6H, 2OCH3), 7.09‐7.49 (m, 11H, Ar‐H), 9.61 (s, 1H, NH, exchangeable with D2O), 9.71 (s, 1H, NH, exchangeable with D2O). Anal. calcd. for C27H22N6O4S2: C, 58.05; H, 3.97; N, 15.04. Found: C, 58.14; H, 3.88; N, 15.17%. 3,3'‐(4‐Methyl‐1,3‐phenylene)bis(4‐imino‐1‐(4‐methoxyphe nyl)‐5‐thioxoimidazolidin‐2‐one) (5c): Color: Gray. Yield: 84 %. M.p.: 248‐250 °C. IR (KBr, ν, cm‐1): 3232 (br, 2NH), 2927 (CH‐ aliph), 1778 (br, 2C=O), 1666 (br, 2C=NH), 1167 (br, 2C=S). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 2.34 (s, 3H, CH3), 3.83 (s, 6H, 2OCH3), 7.11‐7.78 (m, 11H, Ar‐H), 9.66 (s, 1H, NH, exchangeable with D2O), 9.70 (s, 1H, NH, exchangeable with D2O). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 17.9 (CH3), 55.9 (2OCH3), 114.8 (3C), 126.4, 128.0, 129.4 (3C), 129.5 (3C), 131.3, 131.5, 132.1, 137.2, 154.1 (2C), 154.3, 154.5, 160.1 (3C), 183.5 (C=S), 183.7 (C=S). Anal. calcd. for C27H22N6O4S2: C, 58.05; H, 3.97; N, 15.04. Found: C, 57.89; H, 4.04; N, 14.89%. 3,3'‐(4‐Methyl‐1,3‐phenylene)bis(1‐(3‐ethoxyphenyl)‐4‐imi no‐5‐thioxoimidazolidin‐2‐one) (5d): Color: Gray. Yield: 80 %. M.p.: 148‐150 °C. IR (KBr, ν, cm‐1): 3343 (br, 2NH), 2977 (CH‐ aliph), 1779 (br, 2C=O), 1665 (br, 2C=NH). 1H NMR (300 MHz, DMSO‐d6, δ, ppm): 1.36 (t, 6H, J = 6.9 Hz, CH3), 2.27 (s, 3H, CH3), 4.04‐4.06 (q, 4H, J = 7.1 Hz, CH2), 7.07‐7.79 (m, 11H, Ar‐ H), 9.70 (s, 1H, NH; exchangeable with D2O), 9.74 (s, 1H, NH; exchangeable with D2O). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 15.0 (2CH3), 17.9 (CH3), 65.4 (2CH2), 114.5 (3C), 115.9, 120.2 (3C), 126.4, 128.0, 130.4 (3C), 131.5, 132.1, 134.9, 154.0, 154.1 (2C), 154.3, 159.3 (3C), 183.3 (2C=S). Anal. calcd. for C29H26N6O4S2: C, 59.37; H, 4.47; N, 14.32. Found: C, 59.23; H, 4.31; N, 14.19%. 2.2. Antimicrobial activity Chemical compounds were individually tested against a panel of Gram positive and Gram negative bacterial pathogens and fungi. Antimicrobial tests were carried out by the agar well diffusion method using 100 μL of suspension containing 1×108 CFU/mL of pathological tested bacteria and 1×106 CFU/mL of fungi spread on nutrient agar and Sabouraud dextrose agar media, respectively. After the media had cooled and solidified, wells (10 mm in diameter) were made in the solidified agar and loaded with 100 μL of tested compound solution prepared by dissolving 5 mg of the chemical compound in one mL of dimethyl sulfoxide (DMSO). The inoculated plates were then incubated for 24 h at 37 °C for bacteria, and 72 h at 28 °C for fungi. Negative controls were prepared using DMSO employed for dissolving the tested compound. Ampicillin, gentamycin and amphotericin B (1 mg/mL) were used as standard for antibacterial and antifungal activity, respectively. After incubation time, antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms and compared with that of the standard. Antimicrobial activities were expressed as inhibition diameter zones in millimeters (mm). The experiment was carried out in triplicate and the average zone of inhibition was calculated. 2.2.1. Minimal inhibitory concentration (MIC) measurement The microdilution susceptibility test in Müller‐Hinton Broth (Oxoid) and Sabouraud Liquid Medium (Oxoid) was used for the determination of antibacterial and antifungal activity, respectively. Stock solutions of the tested compounds, ampicillin, gentamycin, amphotericin B and sulfisoxazole were prepared in DMF at concentrations 1000 µg/mL. Each stock solution was diluted with standard method broth (Difco) to prepare serial twofold dilutions in the range of 500‐0.007 µg/mL. 10 mL of the broth containing about 106 CFU/mL of test bacteria or 104 CFU/mL of the test fungus was added to each well of 96‐well microtiter plate. The sealed microplates were incubated at 37 °C for 24 h for antibacterial activity and at 25 °C for 48 h for antifungal activity in a humid chamber. At the end of the incubation period, the minimal inhibitory concentrations (MIC) values were recorded as the lowest concentrations of the substance that had no visible turbidity. Control experiments with DMF and uninoculated media were run parallel to the test compounds under the same conditions. 3. Results and discussion 3.1. Chemistry N‐arylcyanothioformamide derivative 1a‐d were prepared by treating arylisothiocyanate with potassium cyanide according to literature procedures [10,14]. Reaction of N‐(4‐ florophenyl)cyanothioformamide derivative (1a) with aryl hydrazonomalononitrile derivatives in ethanol in the presence of triethylamine as a catalyst under reflux condition afforded imidazolidineiminothione derivatives (2a‐c) containing arylazo moiety in good yields (Scheme 1). The structure of the synthesized compounds was corroborated on the basis of spectral and analytical results. Infrared spectra of these compounds revealed absorption bands corresponding to NH, C≡N, C=N and C=S functional groups. The 1H NMR spectra of compounds 2a‐c exhibited two singlet signals around δ 8.60 (C=NH) and 9.60 ppm (NH imidazole ring) along with aromatic protons. 13C NMR spectra of compounds 2b,c displayed signals at δ 187.8‐188.2 ppm due to C=S. The reaction mechanism was assumed to proceed through a nucleophilic attack of the nitrogen atom of the cyanothioformamide moiety (1a) to the cyano group in arylhydrazonomalononitrile to form inter‐ mediate that followed by intramolecular cyclization to yield the cyclized products 2 as illustrated in Scheme 1. Treating an ethereal solution of various N‐arylcyano thioformamides (1a,b,d) with an equimolar amount of butyl isocyanate, followed by addition of a few drops of triethylamine as catalyst afforded imidazolidineiminothione derivatives (3a‐c) (Scheme 2). The structure of compound 3 has been assigned as a reaction product on the basis of analytical and spectral data. The IR spectra of compounds 3a‐c displayed absorption bands around 3250 cm‐1 due to NH and 1765 cm‐1 due to C=O function. The 1H NMR spectra of compounds 3a‐c exhibited triplet, sextet, quintet and triplet signals around δ 0.91, 1.33, 1.67 and 3.68 ppm, specific for butyl protons, respectively. Also, 1H NMR spectra showed another specific broad signal around δ 9.42 ppm for NH proton. 13C NMR spectrum of compound 3b displayed four signals at δ 13.5, 19.3, 20.8 and 29.0 ppm for butyl carbons, three signals at δ 153.8, 154.6 and 183.1 ppm for C=N, C=O and C=S, respectively. 5‐Imino‐5‐thioxo‐2‐imidazolidinone derivatives (3a‐c) were furnished as the sole products, indicating that the ring closing reaction proceeds via a single path, which involves attack via the nitrogen atom. The behavior of the cyanothioformanilides towards 2,4‐diisocyana totoluene was discussed. Thus, equimolar reaction between cyanothioformanilides (1b‐e) and 2,4‐diisocyanato‐toluene in ether and in the presence of triethylamine caused cyclo‐ addition reaction to furnish 5‐thioxoimidazo‐lidin‐2‐ones (4a‐ d). On the other hand, bis‐imidazolidineiminothiones (5a‐d) were obtained via the reaction of cyanothioformanilide (1) with 2,4‐diisocyanato‐toluene (2:1 molar ratio) under the same reaction condition (Scheme 2). The structures of compounds 4 and 5 have been assigned as a reaction product on the basis of analytical and spectral data. IR spectra of compound 4 showed signal around 2270 cm‐1 due to the presence of isocyanate group while IR spectra of compound 5 showed the complete disappearance of this signal. Moreover IR spectra of compounds 4 and 5 showed the presence of three signals around 3340, 1779 and 1665 cm‐1 due to the presence of three groups NH, C=O and C=N, respectively. 80 Ammar et al. / European Journal of Chemistry 8 (1) (2017) 76‐81 Table 1. Antimicrobial activity of the synthesized compounds against the pathological organisms expressed as inhibition diameter zones in millimeters (mm) based on well diffusion assay. Compound Gram positive bacteria Gram negative bacteria Fungi S. aureus S. epidermidis B. subtilis N. gonorrhoeae E. coli K. pneumoniae A. fumigatus A. clavatus G. candidum 2a 17.8±0.48 14.9±0.23 17.2±0.48 16.9±0.41 17.5±0.43 14.6±0.53 17.6±0.35 12.7±0.43 11.7±0.43 2b 0 0 0 0 0 0 0 0 0 2c 0 0 0 0 0 0 0 0 0 3a 28.2±0.54 25.4±0.47 28.6±0.51 19.8±0.63 20.1±0.48 24.5±0.58 19.0±0.12 17.3±0.26 19.8±0.26 3b 11.5±0.43 10.3±0.23 16.5±.042 12.3±0.43 10.6±0.26 9.5±0.34 23.3±0.54 22.6±0.57 26.9±0.38 4a 20.9±0.44 19.7±0.59 21.8±0.47 17.3±0.32 18.8±0.54 16.47±0.54 16.6±0.34 15.6±0.43 13.9±0.32 4b 15.9±0.45 14.8±0.24 17.2±0.64 13.2±0.36 15.3±0.35 17.4±0.38 15.7±0.36 14.6±0.43 12.8±0.38 4c 10.2±0.42 9.3±0.50 12.2±0.63 0 0 0 9.6±0.23 9.8±0.46 11.5±0.27 5a 22.7±0.65 20.8±0.45 22.7±0.56 21.4±0.41 22.5± 0.36 21.4±0.36 18.7±0.28 20.93±0.38 24.8±0.54 5b 0 0 12.23±0.2 0 0 0 0 10.9±0.34 9.8±0.27 5c 19.9±0.64 20.8±0.32 21.7±0.37 20.9±0.45 20.9±0.36 22.1±0.53 19.7±0.28 21.8±0.27 22.7±0.39 Ampicillin 28.9±0.14 25.4±0.18 29.8±0.35 ‐ ‐ ‐ ‐ ‐ ‐ Gentamycin ‐ ‐ ‐ 22.3±0.58 23.4±0.3 26.3±0.15 ‐ ‐ ‐ Amphotericin B ‐ ‐ ‐ ‐ ‐ ‐ 23.7±0.10 21.9±0.12 25.4±0.16 The 1H NMR spectra of compounds 4 and 5 displayed characteristic signals due aliphatic protons and broad signals assignable the NH. The 13C NMR spectra of compounds 4 and 5 displayed characteristic signals for the carbon of CH3, OCH3, C=NH, C=O and C=S groups. 3.2. Antimicrobial activity The search for newer antibacterial and antifungal agents is still in continuation; due to the rapid development of the resistance among bacteria and fungi. Imidazolidineimino thione derivatives may comprise a new class of antimicrobial agents with diminished resistance. Therefore, the aim of the present investigation is to synthesize different series of imidazolidineiminothione derivatives which bearing various substituent at C2, N1 and N3. Accordingly, these compounds were synthesized and tested for their expected effect(s) against selected Gram‐positive, Gram‐negative bacteria and fungi species. 3.2.1. Antibacterial and antifungal activities The synthesized compounds were tested in vitro for antibacterial and antifungal activities against the following strains: three Gram‐positive bacteria, Staphylococcus aureus (RCMB 010027), Staphylococcus epidermidis (RCMB 010024) and Bacillus subtilis (RCMB 010063); three Gram‐negative bacteria, Neisseria gonorrhoeae (RCMB 010079), Escherichia coli (RCMB 010052) and Klebsiella pneumoniae (RCMB 010093) and three Fungi, Aspergillus fumigatus (RCMB 02564), Aspergillus clavatus (RCMB 02593) and Geotrichum candidum (RCMB 05096), and the results were summarized in Table 1. The synthesized compounds were tested for anti‐ microbial activity by the agar diffusion method [20] using a 1 cm microplate well diameter and a 100 μL of each concentration. Antimicrobial tests were carried out using 100 μL of tested compound solution prepared by dissolving 5 mg of the chemical compound in 1 mL of dimethyl sulfoxide (DMSO). Ampicillin, gentamycin and amphotericin B (1 mg/mL) were used as standard references for Gram positive bacteria, Gram negative bacteria and antifungal activity, respectively. Certain aspects of the structure activity relation‐ ships of the prepared compounds were clearly highlighted. The results of the antimicrobial screening demonstrated the following assumptions about the structural activity relationship (SAR). Incorporating arylazo moiety as in structure 2 had a detrimental effect on antimicrobial activity. Structure 2 has at C2 side chain ending with arylazo moiety (Aryl: 2a; 3,4‐dichlorophenyl, 2b; 4‐methylphenyl, 2c; 4‐ methoxyphenyl). The presence of 3,4‐dichlorophenyl moiety (2a) resulted in the highest antimicrobial activity among all the compounds investigated in this structure. The presence of 3,4‐dichlorophenyl moiety exhibited moderate activity against most of the tested organisms. 4‐Methylphenyl (2b) and 4‐ methoxyphenyl (2c) showed no activity against all tested organisms. Imidazolidineiminothione derivatives (3a‐c) contain 4‐fluorophenyl (3a), 4‐methylphenyl (3b), 4‐methoxy phenyl (3c) moieties at N3 and butyl moiety at N1: Regarding the effect of the aryl substitution at N3, the type of the substitutions on the benzene ring of aryl moiety is important. It was noticed that the presence of 4‐fluorophenyl in compound 3a showed the maximum activities against most the test bacterial strains which showed near the reference drug. On the other hand, compound 3a showed moderate to good activity against the tested fungi strains. Unlike compound 3a, 4‐methylphenyl (3b) and 4‐methoxyphenyl (3c) moieties showed moderate activities against most the test bacterial strains and showed high activity against the tested fungi near the reference drug. Imidazolidineiminothione derivatives (4a‐ d) contains 4‐methylphenyl (4a), 3‐methoxylphenyl (4b), 4‐ methoxyphenyl (4c), 3‐ethoxylphenyl (4d) moieties at N1 and 3‐isocyanato‐4‐methylphenyl moiety at N3. Regarding the effect of the aryl substitution at N1, it was noticed that the presence of methylphenyl moiety (4a) resulted in the highest antimicrobial activity among all the compounds investigated in this structure. The presence of methylphenyl moiety exhibited moderate to good activity against most of the tested organisms while 4‐methoxyphenyl (4c) showed weak to moderate activity against all tested organisms. Bis‐imidazolidineimino thione derivatives (5a‐d) contains 4‐methyl‐phenyl (5a), 3‐ methoxyphenyl (5b), 4‐methoxyphenyl (5c), 3‐ethoxylphenyl (5b), moieties at N1 and methylphenylene linker at N3. Regarding the effect of the aryl substitution at N1, It was noticed that the presence of para substitution at phenyl moiety resulted in the highest antimicrobial activity among all the compounds investigated in this structure. Bis‐compounds 5a,c with two imidazolidineiminothione moiety showed activity in some cases equipotent to the reference drug; especially against fungi. 3.2.1.1. MIC of the most active compounds Minimum inhibitory concentration (MIC) of the most active synthesized compounds 3a, 5a and 5c was evaluated in vitro using the related reference [21]. The results of minimum inhibitory concentration were depicted in Table 2. Regarding the effect of each substituent at C2 and those at N1 and N3 against bacterial and fungal strains, results of antimicrobial activity in this study revealed that: Compound 3a showed about 25% less potent effect than Ampicillin against S. epidermidis and B. subtilis (MIC, 0.49 μg/mL) and showed about 50% less potent effect than Amphotericin B against A. clavatus and G. Candidum. Ammar et al. / European Journal of Chemistry 8 (1) (2017) 76‐81 81 Table 2. Minimum inhibitory concentration (µg/mL) of the more potent synthesized compounds against the pathological organisms. Compound Gram positive bacteria Gram negative bacteria Fungi S. aureus S. epidermidis B. subtilis N. gonorrhoeae E. coli K. pneumoniae A. fumigatus A. clavatus G. candidum 2a 0.98 0.49 0.49 15.63 7.81 15.63 1.95 0.98 0.24 5a 0.49 1.95 1.95 0.49 3.9 15.63 0.98 0.98 1.95 5c 1.95 1.95 0,98 1.95 0.49 1.95 0.98 7.81 1.95 Ampicillin 0.03 0.12 0.15 ‐ ‐ ‐ ‐ ‐ ‐ Gentamycin ‐ ‐ ‐ 0.49 0.24 0.12 ‐ ‐ ‐ Amphotericin B ‐ ‐ ‐ ‐ ‐ ‐ 0.12 0.49 0.12 Compound 5a was equipotent to the Gentamycin in inhibiting the growth of N. gonorrhoeae (MIC, 0.49 μg/mL), and displayed 50% less activity compared to Amphotericin B against A. clavatus. Compound 5c showed about 50% less potent effect than Gentamycin against E. coli (MIC, 0.49 µg/mL). 4. Conclusion The objective of the present study was to synthesize and investigate the antimicrobial activities of some new imidazo‐ lidineiminothione which bearing various substituent at N1 and others at N3 beside various substituent at C2 with the hope of discovering new structure leads serving as potent antimicro‐ bial agents. Our aim has been verified by the synthesis of imidazolidineiminothione, imidazolidineimino thione contain‐ ing arylazo moiety and bis‐imidazolidine‐iminothione deriva‐ tives. It is worth mentioning that, imidazole dineiminothione derivative 3a contain 4‐fluorophenyl at N3 and butyl moiety at N1 showed the maximum activities against most the test bacterial strains which showed near the reference drug. Bis‐ imidazolidineiminothione 5a was equipotent to the Gentamycin in inhibiting the growth of N. gonorrhoeae (MIC, 0.49 μg/mL), and displayed 50% less activity compared to Amphotericin B against A. clavatus. 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