untitled European Journal of Chemistry 8 (1) (2017) 96‐100 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.8.1.96-100.1543 European Journal of Chemistry Journal webpage: www.eurjchem.com One‐pot synthesis and antimicrobial activity of new 4,6‐disubstituted‐3,4‐dihydropyrimidine‐2(1H)‐thiones Mahmoud Al‐Refai 1,*, Mohammad Ibrahim 1, Abdullah Al‐Fawwaz 2 and Armin Geyer 3 1 Department of Chemistry, Faculty of Science, Al Al‐Bayt University, Al‐Mafraq, 25113, Jordan 2 Department of Biological Sciences, Faculty of Science, Al Al‐Bayt University, Al‐Mafraq, 25113, Jordan 3 Faculty of Chemistry, Philipps University Marburg, Hans‐Meerwein‐Straße 4, Marburg, 35032, Germany * Corresponding author at: Department of Chemistry, Faculty of Science, Al Al‐Bayt University, Al‐Mafraq, 25113, Jordan. Tel.: +962.2.6297000/2141. Fax: +962.2.6297021. E‐mail address: mahmoud_alrefai@aabu.edu.jo (M. Al‐Refai). ARTICLE INFORMATION ABSTRACT DOI: 10.5155/eurjchem.8.1.96-100.1543 Received: 12 January 2017 Received in revised form: 05 February 2017 Accepted: 08 February 2017 Published online: 31 March 2017 Printed: 31 March 2017   A series of 3,4‐dihydropyrimidine‐2(1H)‐thiones (3a‐i) were synthesized in moderate yields via a one‐pot reaction of 3‐acetyl‐2,5‐diclorothiophene (1), aryl aldehydes (2a‐i) and thiourea in methanolic solution of potassium hydroxide under reflux conditions. All newly synthesized compounds were characterized by extensive NMR analysis, including 1D NMR experiments (1H and 13C) and 2D NMR experiments (COSY, HMBC and HSQC), as well as ESI‐ MS and HRESI‐MS data. The antimicrobial activity of all new compounds (3a‐f) was tested against bacteria and fungi. Thione derivative (3c) only showed activity against Staphylococcus aureus, Bacillus subtilis and Aspergillus niger. KEYWORDS Thione Thiourea Pyrimidine Thiophene Pyrimidine‐2‐thione Antimicrobial activity Cite this: Eur. J. Chem. 2017, 8(1), 96‐100 1. Introduction Heterocyclic compounds containing thiones have shown a wide range of pharmacological activities, such as antimicrobial [1‐4], antidepressant [5], antitubercular [6], antihistamines [7], anti‐HIV [8], and others [9‐12]. Pyrimidine rings, on the other hand, are important substructures in natural products like nucleic acids and vitamin B1 [13,14]. Different pyrimi‐ dines have shown a pharmaceutical importance because of their biological activity such as antifungal [15], antiviral [16‐ 18], anti‐inflammatory [19], antileishmanial [20] and anti‐ cancer [20,21]. In the view of these facts, we report the synthesis, characterization, and antimicrobial activity of new diaryl‐ substituted pyrimidine‐2‐thiones via a one‐pot reaction (Scheme 1). 2. Experimental 2.1. Materials Thiourea was purchased from Aldrich. 3‐Acetyl‐2,5‐ dichlorothiophene (1) have been prepared according to literature procedure [22,23]. Solvents were dried and distilled according to standard methods. 2.2. Instrumentation 1H and 13C NMR spectra were recorded at 300 K on Bruker spectrometers (300‐600 MHz). Chemical shifts δ are given in parts per million (ppm) and were determined from the center of the respective coupling pattern (s: singlet, d: doublet, dd: doublet of doublet, t: triplet). The solvent signals were used as internal standard (DMSO‐d6: δ (1H) 2.50 ppm, δ (13C) 39.52 ppm). ESI‐HMRS measurements were performed on a LTQ‐FT mass spectrometer (Thermo Fisher Scientific). Thin layer chromatography (TLC) monitoring of the reaction was carried out using analytical TLC plates coated with silica gel (60F254, Merck). Preparative column chroma‐ tography was carried out at room temperature with compressed air using flash silica gel (particle size 40‐60 mm, Merck), with the mobile phase being CHCl3:pentane mixture. 2.3. Synthesis 2.3.1. General procedure for the synthesis of thiones (3a‐i) Al‐Refai et al. / European Journal of Chemistry 8 (1) (2017) 96‐100 97 Scheme 1 3‐Acetyl‐2,5‐dichlorothiophene (1) was added to the aromatic aldehydes (2a‐i) in methanolic solution of potassium hydroxide (0.01 mol, in 50 mL). The mixture was stirred for 2 hours at room temperature, and then thiourea (0.03 mol) was added and refluxed for about 10 hours. The reaction mixture was cooled, poured into ice water (150 mL), and then neutralized with hydrochloric acid. The obtained solid was filtered off, air‐dried, and purified using column chroma‐ tography (Scheme 1). 4‐(2‐Bromophenyl)‐6‐(2,5‐dichlorothiophen‐3‐yl)‐3,4‐dihyd ropyrimidine‐2(1H)‐thione (3a): Color: Pale yellow. Yield: 39%. 1H NMR (600 MHz, DMSO‐d6, δ, ppm): 5.33 (d, J = 4.7 Hz, 1H, H‐ 5), 5.41 (dd, J = 4.7, 2.7 Hz, 1H, H‐4), 7.13 (s, 1H, H‐4'), 7.26 (dt, J = 8.4, 1.6 Hz, 1H, H‐4"), 7.40 (dd, J = 9.3, 1.6 Hz, 1H, H‐6"), 7.49 (t, J = 7.7 Hz, 1H, H‐5"), 7.61 (d, J = 8.4 Hz, 1H, H‐3"), 9.01 (bs, 1H, NH‐3), 9.95 (bs, 1H, NH‐1). 13C NMR (150 MHz, DMSO‐ d6, δ, ppm): 175.5 (CS‐2), 142.1 (Cq‐1"), 132.8 (CH‐3"), 131.4 (Cq‐6), 129.5 (CH‐4"), 128.5 (CH‐5"), 128.3 (CH‐6"), 127.4 (CH‐4'), 127.3 (Cq‐3'), 124.9 (Cq‐5'), 123.7 (Cq‐2'), 120.0 (Cq‐ 2"), 102.4 (CH‐5), 54.6 (CH‐4). MS (+ESI, m/z (%)): 443 ([M+Na+2]+, 30), 863 ([2M+Na]+, 13), 1283 ([3M+Na]+, 26). HRMS (+ESI, m/z): 440.8657 [M+Na]+, 442.8629 [M+Na+2]+, 444.8605 [M+Na+4]+, 446.8578 [M+Na+6]+, (calcd. for C14H9BrCl2N2S2Na, 440.8660). 4‐(3‐Bromophenyl)‐6‐(2,5‐dichlorothiophen‐3‐yl)‐3,4‐dihyd ropyrimidine‐2(1H)‐thione (3b): Color: Pale yellow. Yield: 53%. 1H NMR (600 MHz, DMSO‐d6, δ, ppm): 5.15 (dd, J = 4.9, 3.1 Hz, 1H, H‐4), 5.32 (d, J = 4.9 Hz, 1H, H‐5), 7.22 (s, 1H, H‐4'), 7.38 (m, 2H, H‐5",6"), 7.53 (m, 2H, H‐2",4"), 9.13 (bs, 1H, NH‐3), 9.93 (bs, 1H, NH‐1). 13C NMR (150 MHz, DMSO‐d6, δ, ppm): 175.2 (CS‐2), 146.6 (Cq‐1"), 132.3 (Cq‐6), 131.5 (CH‐5"), 131.0 (CH‐2"), 129.8 (CH‐4"), 128.3 (CH‐4'), 127.9 (Cq‐3'), 126.1 (CH‐6"), 125.4 (Cq‐5'), 124.4 (Cq‐2'), 122.4 (Cq‐3''), 104.5 (CH‐ 5), 54.4 (CH‐4). MS (‐ESI, m/z (%)): 419 ([M‐H]‐, 100), 421 ([M‐H+2]‐, 49), 423 ([M‐H+4]‐, 9). HRMS (‐ESI, m/z): 418.8663 [M‐H]‐, 420.8632 [M‐H+2]‐, 422.8598 [M‐H+4]‐, (calcd. for C14H8BrCl2N2S2, 418.8840). 6‐(2,5‐Dichlorothiophen‐3‐yl)‐4‐(4‐fluorophenyl)‐3,4‐dihyd ropyrimidine‐2(1H)‐thione (3c): Color: Pale yellow. Yield: 61%. 1H NMR (600 MHz, DMSO‐d6, δ, ppm): 5.14 (dd, J = 4.8, 3.0 Hz, 1H, H‐4), 5.29 (d, J = 4.8 Hz, 1H, H‐5), 7.21 (s, 1H, H‐4'), 7.25 (t, J = 9.3 Hz, 2H, H‐3",5"), 7.40 (dd, J = 8.9, 5.8 Hz, 2H, H‐2",6"), 9.10 (bs, 1H, NH‐3), 9.88 (bs, 1H, NH‐1). 13C NMR (150 MHz, DMSO‐d6, δ, ppm): 175.0 (CS‐2), 162.9, 161.3 (d, JCF = 251.20 Hz, Cq‐4"), 140.3 (Cq‐1"), 132.3 (Cq‐6), 129.2, 129.1 (d, JCF = 8.61 Hz, CH‐2",6"), 128.3 (CH‐4’), 127.7 (Cq‐3'), 125.3 (Cq‐5'), 124.3 (Cq‐2'), 116.0, 115.8 (d, JCF = 21.65 Hz, CH‐3",5"), 104.9 (CH‐5), 54.3 (CH‐4). HRMS (+ESI, m/z): 380.9461 [M+Na]+, 382.9433 [M+Na+2]+, 384.2077 [M+Na+4]+, (calcd. for C14H9Cl2FN2S2Na, 380.9460). 4‐(3‐Bromo‐4‐methoxyphenyl)‐6‐(2, 5‐dichlorothiophen‐3‐ yl)‐3,4‐dihydropyrimidine‐2(1H)‐thione (3d): Color: Pale yellow. Yield: 64%. 1H NMR (500 MHz, DMSO‐d6, δ, ppm): 3.86 (s, 3H, OCH3‐4"), 5.09 (dd, J = 4.7, 2.6 Hz, 1H, H‐4), 5.29 (d, J = 4.5 Hz, 1H, H‐5), 7.17 (d, J = 8.6 Hz, 1H, H‐5"), 7.22 (s,1H, H‐4'), 7.35 (dd, J = 8.5, 2.2 Hz, 1H, H‐6"), 7.56 (d, J = 2.2 Hz, 1H, H‐2"), 9.08 (bs, 1H, NH‐3), 9.48 (bs, 1H, NH‐1). 13C NMR (125 MHz, DMSO‐d6, δ, ppm): 175.0 (CS‐2), 155.5 (Cq‐4"), 137.7 (Cq‐1"), 132.4 (Cq‐6), 131.7 (CH‐2"), 127.8 (CH‐4'), 127.8 (CH‐6"), 127.6 (Cq‐3'), 125.4 (Cq‐5'), 124.3 (Cq‐2'), 113.5 (CH‐5"), 110.9 (Cq‐3"), 104.7 (CH‐5), 56.8 (4"‐OCH3), 53.9 (CH‐4). MS (+ESI, m/z (%)): 451 ([M+H]+, 100), 453 ([M+H+2]+, 49), 455 ([M+H+4]+, 9). HRMS (+ESI, m/z): 450.8922 [M+H]+ , 452.8889 [M+H+2]+, 454.8854 [M+H+4]+, (calcd. for C15H12BrCl2N2OS2, 450.8924). 6‐(2,5‐Dichlorothiophen‐3‐yl)‐4‐(2,4‐dimethoxyphenyl)‐3,4‐ dihydropyrimidine‐2(1H)‐thione (3e): Color: Pale yellow. Yield: 58%. 1H NMR (500 MHz, DMSO‐d6, δ, ppm): 3.78 (s, 3H, OCH3‐ 4"), 3.81 (s, 3H, OCH3‐2"), 5.22 (d, J = 4.8 Hz, 1H, H‐5), 5.27 (dd, J = 4.8, 2.4 Hz, 1H, H‐4), 6.59 (d, J = 3.1 Hz, 1H, H‐3"), 6.60 (d, J = 9.0, 1H, H‐5"), 7.13 (d, J = 9.0 Hz, 1H, H‐6"), 7.17 (s, 1H, H‐4'), 8.73 (bs, 1H, NH‐3), 9.75 (bs, 1H, NH‐1). 13C NMR (125 MHz, DMSO‐d6, δ, ppm): 175.7 (CS‐2), 160.6 (Cq‐4"), 157.0 (Cq‐2"), 132.4 (Cq‐6), 128.2 (CH‐4'), 128.1 (CH‐6"), 127.3 (Cq‐3'), 125.3 (Cq‐5'), 124.2 (Cq‐1"), 123.91 (Cq‐2'), 105.4 (CH‐5"), 104.7 (CH‐5), 99.0 (CH‐3"), 56.1 (OCH3‐2"), 55.8 (OCH3‐4") 49.8 (CH‐4). HRMS (‐ESI, m/z): 398.9802 [M‐H]‐, 400.9771 [M‐ H+2]‐, 402.8047 [M‐H+4]‐, (calcd. for C16H13Cl2N2O2S2, 398.9801). 6‐(2,5‐Dichlorothiophen‐3‐yl)‐4‐(2,5‐dimethoxyphenyl)‐3,4‐ dihydropyrimidine‐2(1H)‐thione (3f): Color: Pale yellow. Yield: 62%. 1H NMR (500 MHz, DMSO‐d6, δ, ppm): 3.71 (s, 3H, OCH3‐ 5"), 3.77 (s, 3H, OCH3‐2"), 5.29 (d, J = 2.2 Hz, 2H, H‐4,5), 6.79 (d, 1H, J = 3.1 Hz, H‐6"), 6.86 (dd, J = 8.8, 3.1Hz, 1H, H‐4"), 6.96 98 Al‐Refai et al. / European Journal of Chemistry 8 (1) (2017) 96‐100 Table 1. Antimicrobial activity of thione derivatives and the standard antibiotics (3a‐f) *. Chemical compounds Bacterial strains Fungal strains Escherichia coli Staphylococcus aureus Bacillus subtilis Aspergillus niger Penicillium sp 3a -- -- -- -- -- 3b -- -- -- -- -- 3c - + ++ - - 3d -- - - -- -- 3e -- -- - -- -- 3f -- -- -- -- -- Cephalaxin.H2O (12.5 mg/mL) -- ++ +++ NT NT Amphotericin B (20 µg/mL) NT NT NT -- - * 0‐10 mm (‐‐), 11‐15 mm (‐), 16‐20 mm (+), 21‐25 mm (++), over 25 mm (+++), NT: Not Tested. (d, J = 9.0 Hz, 1H, H‐3"), 7.16 (s, 1H, H‐4’), 8.82 (bs, 1H, NH‐3), 9.83 (bs, 1H, NH‐1). 13C NMR (150 MHz, DMSO‐d6, δ, ppm): 176.1 (CS‐2), 153.9 (Cq‐5"), 149.9 (Cq‐2"), 132.9 (Cq‐1"), 132.3 (Cq‐6), 128.1 (CH‐4'), 127.5 (Cq‐3'), 125.3 (Cq‐5'), 124.0 (Cq‐ 2'), 114.0 (CH‐6"), 112.7 (CH‐4"), 112.6 (CH‐3"), 104.4 (CH‐5), 56.5 (OCH3‐2"), 55.9 (OCH3‐5"), 50.3 (CH‐4). HRMS (‐ESI, m/z): 398.9803 [M‐H]‐, 400.9773 [M‐H+2]‐, 402.9743 [M‐H+4]‐, (calcd. for C16H13Cl2N2O2S2, 398.9801). 6‐(2,5‐Dichlorothiophen‐3‐yl)‐4‐(4‐isopropylphenyl)‐3,4‐di hydropyrimidine‐2(1H)‐thione (3g): Color: Pale yellow. Yield: 54%. 1H NMR (600 MHz, DMSO‐d6, δ, ppm): 1.19 (d, J = 6.6 Hz, 6H, CH(CH3)2‐4"), 2.88 (sp, J = 6.9 Hz, 1H, CH(CH3)2‐4"), 5.07 (dd, J = 4.8, 2.7 Hz, 1H, H‐4), 5.27 (d, J = 4.8 Hz, 1H, H‐5), 7.18 (s, 1H, H‐4'), 7.26 (d, J = 8.3 Hz, 2H, H‐2",6"), 7.28 (d, J = 8.3 Hz, 2H, H‐3",5"), 8.98 (bs, 1H, NH‐3), 9.74 (bs, 1H, NH‐1). 13C NMR (150 MHz, DMSO‐d6, δ, ppm): 174.4 (CS‐2), 147.9 (CH‐4"), 141.0 (Cq‐1"), 131.8 (Cq‐6), 127.6 (CH‐4'), 127.0 (Cq‐3'), 126.6 (CH‐2",6"), 126.4 (CH‐3",5"), 124.8 (Cq‐5'), 123.6 (Cq‐2'), 104.5 (CH‐5), 54.4 (CH‐4), 33.1 (CH(CH3)2‐4"), 23.8 (CH(CH3)2‐ 4"). MS (+ESI, m/z (%)): 405 ([M+Na]+, 48), 407 ([M+Na+2]+, 37). 409 ([M+Na+4]+,18). MS (‐ESI, m/z (%)): 381 ([M‐H]‐, 100), 383 ([M‐H+2]‐, 71), 385 ([M‐H+4]‐, 7). HRMS (‐ESI, m/z): 381.0059 [M‐H]‐, 383.0021 [M‐H+2]‐, 384.9999 [M‐H+4]‐ (calcd. for C17H15Cl2N2S2, 381.0059). HRMS (+ESI, m/z): 405.0008 [M+Na]+, 406.9975 [M+Na+2]+, 408.9970 [M+Na+4]+, (calcd. for C17H16Cl2N2S2Na, 405.0024). 6‐(2,5‐Dichlorothiophen‐3‐yl)‐4‐(naphthalen‐1‐yl)‐3,4‐dihyd ropyrimidine‐2(1H)‐thione (3h): Color: Pale yellow. Yield: 62%. 1H NMR (500 MHz, DMSO‐d6, δ, ppm): 5.42 (d, J = 4.8 Hz, 1H, H‐ 5), 5.96 (dd, J = 4.6, 2.8 Hz, 1H, H‐4), 7.17 (s, 1H, H‐4'), 7.52 (d, J = 7.3 Hz, 1H, H‐10"), 7.59 (m, 3H, H‐2",5",6"), 7.92 (d, J = 8.3 Hz, 1H, H‐9"), 7.99 (dd, J = 7.7, 1.5 Hz, 1H, H‐7"), 8.25 (d, J = 8.3 Hz, 1H, 4"), 9.10 (bs, 1H, NH‐3), 9.94 (bs, 1H, NH‐1). 13C NMR (125 MHz, DMSO‐d6, δ, ppm): 175.8 (CS‐2), 139.5 (Cq‐1"), 134.0 (Cq‐8"), 132.3 (Cq‐6), 129.7 (Cq‐3"), 129.2 (CH‐7"), 128.6 (CH‐9"), 128.1 (CH‐4'), 127.4 (Cq‐3'), 127.0 (CH‐5"), 126.4 (CH‐5"), 126.3 (CH‐2"), 125.4 (Cq‐5'), 124.8 (CH‐10"), 124.1 (Cq‐2'), 123.7 (CH‐4"), 105.2 (CH‐5), 52.5 (CH‐4). HRMS (+ESI, m/z): 390.9890 [M+H]+, 392.9861 [M+H+2]+, 394.9833 [M+H+4]+, (calcd. for C18H13Cl2N2S2, 390.9892). 4‐(Anthracen‐9‐yl)‐6‐(2,5‐dichlorothiophen‐3‐yl)‐3,4‐dihyd ropyrimidine‐2(1H)‐thione (3i): Color: Pale yellow. Yield: 41%. 1H NMR (600 MHz, DMSO‐d6, δ, ppm): 5.20 (d, J = 2.9 Hz, 1H, H‐ 5), 6.96 (d, J = 2.9 Hz, 1H, H‐4), 7.25 (s, 1H, H‐4'), 7.57 (m, 4H, H‐4",5",11",12"), 8.60 (d, J = 8.8 Hz, 2H, H‐3",13"), 8.16 (d, J = 8.3 Hz, 2H, H‐6",10"), 8.68 (s, 1H, 8"), 9.09 (bs, 1H, NH‐3), 10.01 (bs, 1H, NH‐1). 13C NMR (120 MHz, DMSO‐d6, δ, ppm): 175.2 (CS‐2), 133.0 (Cq‐1"), 132.5 (Cq‐6), 131.5 (Cq‐7",9"), 130.2 (Cq‐2",14"), 129.7 (CH‐6", 10"), 129.1 (CH‐8"), 128.2 (CH‐4'), 127.1 (Cq‐3'), 126.7 (CH‐4",12"), 125.5 (CH‐5",11"), 125.4 (Cq‐5'), 124.7 (CH‐3",13"), 124.3 (Cq‐2'), 105.6 (CH‐5), 51.1 (CH‐4). MS (‐ESI, m/z (%)): 439 ([M‐H]‐, 39), 441 ([M‐ H+2]‐, 13). HRMS (‐ESI, m/z): 438.9903 [M‐H]‐, 440.9873 [M‐ H+2]‐, (calcd. for C22H13Cl2N2S2, 438.9903). 2.4. Biological activity The in vitro antimicrobial activity of pyrimidine‐2‐thione derivatives (3a‐f) was determined by the wells diffusion method [24‐26] (Table 1). For these assays, cultures of the following microorganisms were used: two Gram‐positive (Staphylococcus aureus (ATCC 6538) and Bacillus subtilis (ATCC 6633)), one Gram‐negative (Escherichia coli (ATCC 25922)) bacteria, and fungal strains (Aspergillus niger and Penicillium sp. which were isolated from local environments). Bacterial cultures were maintained on nutrient agar medium, while fungal cultures were maintained on potato dextrose agar (PDA). Suspensions of the tested micro‐ organisms were spread on the solid nutrient agar medium and PDA plates. 25±2 mg of each thione derivatives (3a‐f) were dissolved in 2 mL of dimethyl sulfoxide, then 100 μL of each chemical compound (12.5 mg/mL) were added to agar wells. A positive control containing microbial culture without chemical compound and a negative control containing only the medium were performed as well. At the end of the incubation time (24 h at 37 °C for bacteria and 25 °C for fungi), positive anti‐ bacterial and antifungal activities were established by the presence of a measurable inhibition zone and recorded in its width (mm) which includes the well diameter. Each test was performed in three replicates. Cephalaxin.H2O at concentration 12.5 µg/mL was used as standard against bacteria while Amphotericin B at concentration 20 µg/mL was used as standard antifungal agents. 3. Results and discussion 3.1. Preparation and characterization of 3,4‐dihydro pyrimidine‐2(1H)‐thiones The one‐pot synthesis of 4,6‐disubstituted‐3,4‐dihydro‐ pyrimidine‐2(1H)‐thiones (3a‐i) was carried out by the reac‐ tion of 3‐acetyl‐2,5‐dichlorothiophene (1) with aromatic aldehydes (2a‐i) in methanolic solution of potassium hyd‐ roxide at room temperature. Thiourea was then added under reflux to give the 3,4‐dihydropyrimidine‐2(1H)‐thiones (3a‐i) in moderate yields (Scheme 1). The newly synthesized pyrimidine 2‐thiones (3a‐i) were characterized by ESIMS, HRMS, 1D and 2D NMR techniques. The complete data are present in the experimental section. The 1H NMR spectra showed for each compound two exchangeable proton signals in the range of δ 9.48‐10.01 ppm, and δ 8.73‐9.13 ppm corresponding to NH‐1 and NH‐3, res‐ pecttively. The two pyrimidine protons H‐4 and H‐5 were resonated in the range of δ 5.07‐5.43 ppm except in case of the anthracene substituent, where the H‐4 was appeared at  6.96 ppm. Furthermore, the thiophene proton H‐4' was revealed in the range of δ 7.13‐7.25 ppm. In the 13C NMR spectra of compounds (3a‐i), the C=S carbons were resonated downfield between δ 174.4‐176.1 ppm. The two signals in the range of δ 102.4‐105.6 ppm and δ 49.8‐54.6 ppm were assigned to C‐5 and C‐4 of the pyrimidine nucleus, respectively. The complete assignments of 1H and 13C NMR resonances were confirmed by H‐H COSY, HSQC, and HMBC experiments. As an example, Figure 1 shows the HSQC spectrum of compound 3f. The ESI‐MS spectra revealed the right molecular ion peaks characteristics of isotopic chlorine clusters confirming the presence of two chlorine atoms. Al‐Refai et al. / European Journal of Chemistry 8 (1) (2017) 96‐100 99 (a) (b) Figure 1. (a) HMBC and (b) HSQC, H,H COSY (▬), HMBC (→) correlations (DMSO‐d6, 600 MHz) of 6‐(2,5‐dichlorothiophen‐3‐yl)‐4‐(2,5‐dimethoxyphenyl)‐ 3,4‐dihydropyrimidine‐2(1H)‐thione (3f). (a) (b) Figure 2. Zone of Inhibition obtained by the wells diffusion method for compound 3c, against (a) Staphylococcus aureus and (b) Bacillus subtilis. The molecular formulas were determined by measuring the HRESI‐MS spectra, which came in good agreement with the calculated values. 3.2. Biological Screening In the present study, antimicrobial activity of new thione compounds (3a‐f) against one Gram‐negative bacteria (Escherichia coli), two Gram‐positive bacteria (Staphylococcus aureus, Bacillus subtilis); and two fungal strains isolated from local environments (Aspergillus niger, and Penicillium sp.) were tested using diffusion well technique [24‐26]. The antimicrobial activity of all tested thiones (3a‐f) and standards antimicrobial agents (Cephalaxin.H2O and Amphotericin B) were given in Table 1. The results showed that there was a difference in antimicrobial activity between different thione compounds. All thiones did not showed any activity against all tested species, except thione derivative (3c), which showed a good activity against Gram positive bacteria (Staphylococcus aureus and Bacillus subtilis), and weak activity against Gram negative bacteria (Escherichia coli) and the two fungal strains (Aspergillus niger and Penicillium sp), Figure 2. 4. Conclusions In the present work, new pyrimidine‐2‐thione derivatives have been successfully synthesized and characterized using different spectroscopic techniques. Most of the newly synthesized compounds tested against bacteria and fungi. Except thione derivative (3c), all of the tested compounds showed no activity against selected microorganisms. Acknowledgements We are grateful to Al Al‐Bayt University (Mafraq, Jordan) and the DFG (Germany) for financial support. References [1]. Eweiss, N. F.; Bahajaj, A. A.; Elsherbini, E. A. J. Heterocycl. Chem. 1986, 23, 1451‐1458. 100 Al‐Refai et al. / European Journal of Chemistry 8 (1) (2017) 96‐100 [2]. Hussein, M. A.; El‐Shorbagi, A. N.; Khallil, A. R. Arch. Pharm. 2001, 334, 305‐308. [3]. Aboul‐Fadl, T.; Hussein, M. A.; El‐Shorbagi, A. N.; Khallil, A. R. Arch. Pharm. 2002, 335, 438‐442. [4]. El‐Emam, A. A.; Al‐Deeb, O. A.; Al‐Omar, M.; Lehmann, J. Bioorg. Med. Chem. 2004, 12, 5107‐5113. [5]. Kane, J. M.; Dudley, M. W.; Sorensen, S. M.; Miller, F. P. J. Med. Chem. 1988, 31, 1253‐1258. [6]. Rajesh, S. M.; Kumar, R. S.; Libertsen, L. A.; Perumal, S.; Yogeeswari, P.; Sriram, D. Bioorg. Med. Chem. Lett. 2011, 21, 3012‐3016. [7]. Cale, A. D.; Gero, T. W.; Walker, K. R.; Lo, Y. S.; Welstead, W. J.; Jaques, L. W.; Johnson, A. F.; Leonard, C. A.; Nolan, J. C.; Johnson, DN. J. Med. Chem. 1989, 32, 2178‐2199. [8]. Rao, A.; Carbone, A.; Chimirri, A.; De Clercq, E.; Monforte, A. M.; Monforte, P.; Pannecouque, C.; Zappala, M. Il Farm. 2002, 57, 747‐ 751. [9]. Krauze, A. A.; Vitolinya, R. O.; Zarin’sh, G. V.; Pelche, Y. E.; Kalme, Z. A.; Kimenis, A. A. Pharm. Chem. J. 1985, 19, 313‐318. [10]. Yarim, M.; Sarac, S.; Ertan, M.; Kilic, F. S.; Erol, K. Arzneimittelforschung 2002, 52, 27‐33. [11]. Wujec, M.; Pitucha, M.; Dobosz, M.; Kosikowska, U.; Malm, A. Acta Pharm. 2004, 54, 251‐260. [12]. Sosnicki, J. G.; Lukasz, S.; Kurzawski, M.; Peruzynska, M.; Maciejewska, G.; Drozdzik, M. Org. Biomol. Chem. 2014, 12, 3427‐ 3440. [13]. Price, D.; May, E. L.; Pickel, F. D. J. Am. Chem. Soc. 1940, 62, 2818‐ 2820. [14]. Obermeyer, H. G.; Chen. , L. J. Biol. Chem. 1945, 159, 117‐122. [15]. Chen, Q.; Zhu, X. L.; Jiang, L. L.; Liu, Z. M.; Yang, G. F. Eur. J. Med. Chem. 2008, 43, 595‐603. [16]. Nugent, R. A.; Schlachter, S. T.; Murphy, M. J.; Cleek, G. J.; Poel, T. J.; Wishka, D. G.; Graber, D. R.; Yagi, Y.; Keiser, B. Y.; Olmsted, R. A.; Kopta, L. A.; Swaney, S. M.; Poppe, S. M.; Morris, J.; Tarpley, W. G.; Thomas, R. C J. Med. Chem. 1998, 41, 3793‐3803. [17]. Hockova, D.; Holy, A.; Masojidkova, M.; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J. Bioorg. Med. Chem. 2004, 12, 3197‐3202. [18]. Brunelle, M. N.; Lucifora, J.; Neyts, J.; Villet, S.; Holy, A.; Trepo, C.; Zoulim, F. Antimicrob. Agents Chemother. 2007, 51, 2240‐2243. [19]. Costa, E. V.; Pinheiro, M. L. B.; Xavier, C. M.; Silva, J. R.; Amaral, A. C. F.; Souza, A. D.; Barison, A.; Campos, F. R.; Ferreira, A. G.; Machado, G. M.; Leon, L. L. J. Nat. Prod. 2006, 69, 292‐294. [20]. Xie, F.; Zhao, H.; Zhao, L.; Lou, L.; Hu, Y. Bioorg. Med. Chem. Lett. 2009, 19, 275‐278. [21]. Ghorab, M. M.; Ragab, F. A.; Alqasoumi, S. I.; Alafeefy, A. M.; Aboulmagd, S. A. Eur. J. Med. Chem. 2010, 45, 171‐178. [22]. Bachjman, G. B.; Heise, L. V. J. Am. Chem. Soc. 1948, 70, 2368‐2387. [23]. Al‐Refai, M.; Ibrahim, M. M.; Geyer, A.; Marsch, M.; Ali, B. F. J. Chem. Crystallogr. 2016, 46, 331‐340. [24]. Alsohaili, S. A.; Al‐Fawwaz A. T. Eur. Sci. J. 2014, 10, 156‐165. [25]. Gowri, M.; Ananthalakshmi, S.; Therese Punitha, J. Int. J. of Pharm. Life Sci. 2013, 4, 2780‐2784. [26]. Chenni, M.; El Abed, D.; Rakotomanomana, N.; Fernandez, X.; Chemat, F. Molecules 2016, 21, 113, 1‐16.