HETEROCYCLES

Paper | Regular issue | Vol. 85, No. 5, 2012, pp. 1141-1154
Received, 26th February, 2012, Accepted, 30th March, 2012, Published online, 6th April, 2012.
DOI: 10.3987/COM-12-12456

■ Synthesis, Characterization and Evaluation of Antimicrobial Activity of Some Novel 1,2,4-Triazoles and 1,3,4-Thiadiazoles Bearing Imidazole Nuclues

Mohamed Reda Aouad,* Nadjet Rezki, Mohamed Kasmi, Linda Aouad, and Merieme A. Rezki

Department of Chemistry, Faculty of Science, Taibah University, Madinah Munawwarah, Saudi Arabia

Abstract

A series of N1-[(4,5-di and 1,4,5-triphenylimidazol-2-yl)thioacetyl]-N4-alkyl/aryl-thiosemicarbazides 3-7 were synthesized from (4,5-di and 3,4,5-triphenylimidazol-2-yl)thioacetic acid hydrazide 1, 2. The treatment of compounds 3-7 with NaOH gave 5-[(4,5-di and 1,4,5-triphenylimidazol-2-yl)-3-thiomethyl]-4-alkyl/aryl-2H-1,2,4-triazoles-3-thione 8-12, while the acidic treatment of compounds 3-7 afforded 5-[(4,5-di- and 1,4,5-triphenylimidazol-2-yl)-3-thiomethyl]-2-alkyl/arylamino-1,3,4-thiadiazoles 13-17. Moreover, potassium hydrazinecarbothionates 18, 19 were obtained from the reaction of acyl hydrazides 1, 2 with carbon disulfides in basic media and converted into 4-amino-1,2,4-triazole-3-thiones 20, 21 and 1,3,4-thiadiazole-2-thiols 22, 23 by the treatment with hydrazine hydrate and sulfuric acid, respectively. All newly synthesized compounds were screened for their antimicrobial activity.

INTRODUCTION
1,2,4-Triazole and its derivatives belong to a class of exceptionally active compounds possessing a wide spectrum of biological properties, including antibacterial,1 antimicrobial,2,3 antihypertensive,4 analgesic,5 antiviral,6 antioxidant,7 anti-inflammatory,8 antitumor,9,10 anti-HIV,11 pesticidal,12 insecticidal,13 herbicidal14 and fungicidal activity.15 Moreover, it was reported that compounds having triazole moieties, such as vorozole, letrozole and anastrazole appeared to be very effective aromatase inhibitors, which in turn prevented breast cancer.16-18
Furthermore, 1,3,4-thiadiazole nucleus takes part in the structure of several biologically active compounds, including antibacterial,19-22 antifungal,21,22 antitubercular,23-25 analgesic,26 antiinflammatory,21,22,26 antidepressant,27 leishmanicidal28 activities.
Heterocycles containing an imidazole moiety constitute a class of compounds possessing a wide spectrum of biological activities such as anti-bacterial,29 antiasthmatic30 and antiulcerative31 properties.
The binding of two or three heterocyclic rings having different sites or mode of action has commanded the world-wide attention of many research groups because of their high potential to exhibit antimicrobial activity.32-34
In view of these facts, the aim of the present study is to obtain 1,2,4-triazole and 1,3,4-thiadiazole derivatives carrying imidazole moiety as antimicrobial agents.

RESULTS AND DISCUSSION
Chemistry
The synthesis of the 1,2,4-triazole and 1,3,4-thiadiazole derivatives is illustrated and outlined in Scheme 1.

The key intermediates 1 and 2 were prepared from ethyl(4,5-di- and 1,4,5-triphenylimidazol-2-yl)-thioacetate following the literature method.35 Treatment of the acid hydrazides 1 and 2 with various alkyl/aryl isothiocyanates in ethanol gave corresponding N1-[(4,5-di- and 1,4,5-triphenyl)thioacetyl]-N4-alkyl/aryl-thiosemicarbazides 3-7 in good yield.
The structure of the compounds investigated 3-7 was confirmed by their IR spectra which displayed absorption peaks in the range of 3240-3365 cm-1 for NH, 1696-1703 cm-1 for C=O and 1296-1319 cm-1 corresponding to C=S stretching vibrations. Their 1H NMR spectra showed a multiplet at δH 7.13-7.91 ppm for aromatic protons. The CONH and CSNH protons were observed as singlets at 8.05-8.56 and 10.08-10.36 ppm, respectively confirming the formation of acid thiosemicarbazides (controlled with D2O). The carbon signals of these groups were recorded between 163 and 184.16 ppm.
Thiosemicarbazides undergo different cyclization reactions to give five member heterocycles. The product of cyclization depends on the reagent used. This cyclization leads to the formation of 1,3,4-oxadiazole ring through the oxidative cyclization of thiosemicarbazides using iodine and potassium iodide in ethanolic sodium hydroxide. 36-37 On the other hand, the 1,2,4-triazole and 1,3,4-thiadiazole derivatives were obtained by the treatment of thiosemicarbazides with sodium hydroxide and sulfuric acid, respectively.32,36-38
Therefore, the thiosemicarbazides 3-7 on heating with 10% aqueous NaOH underwent smooth cyclization through dehydration to afford 5-[(4,5-di- and 1,4,5-triphenylimidazol-2-yl)-3-thiomethyl]-4-alkyl/aryl-2H-1,2,4-triazoles-3-thione 8-12.
These compounds displayed 1H and 13C NMR spectra and elemental analyses consistent with the assigned structures. The IR spectra of the triazoles 8-12 exhibited N-H bands in the region 3307-3341 cm-1. The absorption bands at 1610-1630 cm-1 are due to the presence of C=N stretching of the triazole ring system. Absence of the C=O absorptions in 8-12 provided definitive proof for the formation of new products.
The exhibited chemical shifts obtained from 1H NMR spectra were all supported the proposed structures of 8-12. The 1H NMR of 10-12 chosen as prototypes showed single NH triazole resonances in the 14.40-14.47 ppm regions (controlled with D2O). The SCH2 protons resonated at 4.62-4.79 ppm. Additional signals belonging to phenyl ring were observed in the aromatic region in the 1H and 13C NMR spectra of these compounds. Moreover, C=S group resonated at 181.80-183.64 ppm in the 13C NMR spectra of compounds 8-12 confirming the presence of these compounds in their thione forms.
5-[(4,5-Di- and 1,4,5-triphenylimidazol-2-yl)-3-thiomethyl]-2-alkyl/arylamino-1,3,4-thiadiazoles 13-17 were obtained by cyclization of 3-7 by treating with cold concentrated sulfuric acid.
The IR spectra of the synthesized thiadiazoles 13-17 showed absorption peaks in the region 1613-1645 cm-1 due to C=N stretching vibrations. In the 1H NMR spectra of 13-17, the singlets of CONH and CSNH of thiosemicarbazides had disappeared, and the NH proton at 2-position of 1,3,4-thiadiazoles ring appeared as singlet at 10.21-10.35 ppm (controlled with D2O).
On the other hand, in ethanol solution of potassium hydroxide, carbohydrazides 1, 2 interacted with carbon disulfide to potassium hydrazine carbodithionates 18 and 19, which were used as precursors for the synthesis of 4-amino-1,2,4-triazole-3-thiones 20, 21 and 1,3,4-thiadiazole-2-thiols 22, 23 (scheme 2).

The refluxing of 18, 19 and hydrazine hydrate led to the preparation of 4-amino-5-[(4,5-di and 1,4,5-triphenylimidazol-2-yl)-3-thiomethyl]-2H-1,2,4-triazole-3-thiones 20, 21 in good yields. NMR spectral characteristic of the amino-1,2,4-triazoles 20, 21, revealed in their 1H NMR spectra the two characteristic singlets of the NH2 and NH (triazole) in the regions 7.90-8.03 ppm and 14.15-14.20 ppm, respectively (controlled with D2O). In the NMR spectra of compounds 20 and 21, the exocyclic C=S signals were observed at 182.30 and 183.76 ppm, respectively.
In sulfuric acid medium, the precursors 18, 19 formed 5-[(4,5-di and 1,4,5-triphenylimidazol-2-yl)-3-thiomethyl]-1,3,4-thiadiazole-2-thiols 22, 23. In the 13C NMR spectra of compounds 22, 23, the absence of the signals >180 ppm attributed to the thiocarbonyl carbons (C=S), confirmed the formation of the thiadiazoles in their thiol forms.
In addition, the chemical shifts of SH thiadiazole protons were detected as singlet at 13.92-13.98 ppm (controlled with D2O), which confirmed the thiol form. The remaining protons and carbons were observed at the expected regions (see experimental part).

Antimicrobial activity
The antibacterial and antifungal activity, assay (Table 1) indicated that the acid hydrazides 1 and 2 show slight activity only against E. coli and P. aeruginosa. 1-[(4,5-Diphenylimidazol-2-yl)thioacyl]-4-methylthiosemicarbazide (3) displayed moderate to good activities against all tested microorganisms except C. albicans and C. tropicalis, whereas its 2-fluorophenyl analogue 4 displayed good activity against all bacterial and yeast strains. On the other hand, 1-[(1,4,5-triphenylimidazol-2-yl)thioacyl]-4-methylthiosemicarbazide (5) showed moderate antimicrobial activities towards E. coli and P. aeruginosa, while 1-[(1,4,5-triphenylimidazol-2-yl)thioacyl]-4-(2-fluorophenyl)thiosemicarbazide (6) showed good activity against all bacterial and yeast strains. 1-[(1,4,5-Triphenylimidazol-2-yl)thioacyl]-4-phenylthiosemicarbazide (7) indicated excellent activity against E. coli, P. aeruginosa, C. albicans and C. tropicalis. The antimicrobial activity of 4-alkyl/aryl-5-[(4,5-di- and 1,4,5-triphenylimidazole-2-yl)-thiomethyl]-2H-1,2,4-triazole-3-thiones 8-12 divulged that compound 9 showed good antimicrobial activity against tested bacterial and good antifungal activity against C. albicans.
Compounds 10 and 11 possessing three phenyl groups on imidazole ring exhibited excellent antibacterial activity against S. aureus and B. subtilis and good activity against E. coli and P. aeruginosa. The compound 12 having phenyl group on N-4 triazole ring showed excellent antifungal activity than antibacterial action. On other hand, the antimicrobial activity of the thiadiazoles 13-16 revealed that all the tested compounds possessed moderate to good inhibition, compounds 13 and 14 showed comparatively moderate activity against E. coli and P. aeruginosa and good inhibition towards C. albicans and C. tropicalis. Similarly, compounds 15 and 16 also showed significant activity against all tested microbial strains. In contradiction, compound 17 possessing phenyl substitution on thiadiazole ring displayed no activity.
The conversion of hydrazide structure in compound 1 to 1,2,4-triazole ring of 4-amino-5-[(4,5-diphenylimidazol-2-yl)thiomethyl]-2H-1,2,4-triazole-3-thione (20) caused excellent antimicrobial activities against all bacterial strains and C. albicans. 5-[(4,5-Diphenylimidazol-2-yl)thiomethyl]-1,3,4-thiadiazole-2-thiol (21), which is the thiadiazole derivative of compound 1, demonstrated slight activity only towards E. coli. The conversion of compound 2 to compound 22 caused important antimicrobial activity. On the contrary to the activity of compound 22, compound 23, which was obtained from the reaction of compound 2 with H2SO4, indicated no activities towards all microbial strains.
Based on the activity data of all the synthesized compounds, it is concluded that imidazole bearing 1,2,4-triazole moiety exhibits better antibacterial activity than 1,3,4-thiadiazole moiety. Moreover, the presence of amino group at N-4 of triazole ring seems to have marginal effect on biological activity. In fact, upon N-amination the antibacterial activity marginally increases.
Interestingly, it was found that thiadiazole derivatives 13-16 are more active against fungal pathogens as compared to bacterial pathogens.
The antimicrobial activity of all the synthesized compounds could be attributed to the presence of triazole ring. This ring is incorporated into a wide variety of drugs used in medical therapy.

CONCLUSION
The research study reports the successful synthesis and antimicrobial activity of new 4-alkyl/aryl-2H-1,2,4-triazole-3-thiones, 4-amino-2H-1,2,4-triazole-3-thiones, 2-aminoalkyl/aryl-1,3,4-thiadiazoles and 1,3,4-thiadiazoles-3-thiols, carrying biologically active imidazole ring. Their antimicrobial activity study revealed that some of the compounds tested showed moderate to excellent antibacterial and antifungal activities against pathogenic strains.

EXPERIMENTAL
Chemistry
Melting points were determined on a Melt-temp apparatus and are uncorrected. 1H and 13C NMR spectra were recorded on a Jeol 500 MHz spectrometer. The IR spectra were measured as potassium bromide pellets using a Perkin-Elmer 1430 series FTIR spectrometer. The microanalyses for C, H and N were performed on Perkin-Elmer elemental analyzer.
General method for the synthesis of compounds 3-7. A mixture of corresponding compound 1 or 2 (10 mmol) and the appropriate isothiocyanate derivatives (10 mmol) was refluxed in EtOH for 6 h. The solution was cooled and a white solid appeared. The obtained precipitate was filtered and recrystallized from ethanol to afford the desired product.
1-[(4,5-Diphenylimidazol-2-yl)thioacetyl]-4-methylthiosemicarbazide (3). Yield 88%, mp 158 °C; IR: 3355, 3270 (NH), 1700 (C=O), 1315 cm-1 (C=S); 1H NMR (500 MHz, DMSO-d6) δH 2.85 (3H, s, CH3), 4.70 (2H, s, SCH2), 7.18-7.63 (10H, m, arH), 8.05 (1H, s, NH), 8.55 (1H, s, NH), 10.10 (1H, s, NH), 12.54 (1H, s, NH imidazole); 13C NMR (125 MHz, DMSO-d6) δc 34.40 (CH3), 40.97 (CH2), (127.10, 127.94, 128.81, 129.21, 129.98, 131.66, 135.75, 137.89) arC, 163.00 (C=O), 181.70 (C=S). Anal. Calcd (%) for C19H19N5OS2: C, 57.41; H, 4.82; N, 17.62. Found: C, 57.30; H, 4.98; N, 17.52.
1-[(4,5-Diphenylimidazol-2-yl)thioacetyl]-4-(2-fluorophenyl)thiosemicarbazide (4). Yield 80%, mp 200-201 °C; IR: 3343, 3265 (NH), 1701 (C=O), 1296 cm-1 (C=S); 1H NMR (500 MHz, DMSO-d6) δH 4.61 (2H, s, SCH2), 7.23-7.76 (14H, m, arH), 8.10 (1H, s, NH), 8.56 (1H, s, NH), 10.08 (1H, s, NH), 12.43 (1H, s, NH imidazole); 13C NMR (500 MHz, DMSO-d6) δC 41.75 (SCH2), (125.33, 127.56, 128.07, 128.78, 129.15, 129.87, 130.45, 132.65, 131.80, 135.89, 138.24, 141.34) arC, 167.89 (C=O), 182.49 (C=S). Anal. Calcd (%) for C24H20FN5OS2: C, 60.36; H, 4.22; N, 14.66. Found: C, 60.12; H, 4.45; N, 14.46.
1-[(1,4,5-Triphenylimidazol-2-yl)thioacetyl]-4-methylthiosemicarbazide (5). Yield 84%, mp 167 °C; IR: 3345 and 3258 (NH), 1698 (C=O), 1319 cm-1 (C=S). 1H NMR (500 MHz, DMSO-d6) δH 2.92 (3H, s, CH3), 4.74 (2H, s, SCH2), 7.15-7.78 (15H, m, arH), 8.20 (1H, s, NH), 8.36 (1H, s, NH), 10.24 (1H, s, NH); 13C NMR (125 MHz, DMSO-d6) δC 35.61 (CH3), 40.46 (CH2), (126.87, 127.33, 127.27, 128.09, 128.74, 129.45, 129.81, 131.80, 136.09, 138.24) arC, 166.35 (C=O), 183.05 (C=S). Anal. Calcd (%) for C25H23N5OS2: C, 63.40; H, 4.89; N, 14.79. Found: C, 63.28; H, 4.80; N, 14.52.
1-[(1,4,5-Triphenylimidazol-2-yl)thioacetyl]-4-(2-fluorophenyl)thiosemicarbazide (6). Yield 80%, mp 216-217 °C; IR: 3346 and 3279 (NH), 1703 (C=O), 1310 cm-1 (C=S); 1H NMR (500 MHz, DMSO-d6) δH 4.61 (2H, s, SCH2), 7.13-7.91 (19H, m, arH), 8.15 (1H, s, NH), 8.27 (1H, s, NH), 10.19 (1H, s, NH); 13C NMR (125 MHz, DMSO-d6) δC 41.94 (SCH2), (124.77, 126.45, 127.80, 127.98, 128.22, 128.51, 128.86, 129.05, 129.74, 132.11, 136.41, 138.40, 141.60) arC, 163.78 (C=O), 182.32 (C=S). Anal. Calcd (%) for C30H24FN5OS2: C, 65.08; H, 4.37; N, 12.65. Found: C, 64.89; H, 4.13; N, 12.51.
1-[(1,4,5-Triphenylimidazol-2-yl)thioacetyl]-4-phenylthiosemicarbazide (7). Yield 78%, mp 145 °C. IR: 3365 and 3240 (NH), 1696 (C=O), 1300 cm-1 (C=S); 1H NMR (500 MHz, DMSO-d6) δH 4.80 (2H, s, SCH2), 7.20-7.75 (20H, m, arH), 8.10 (1H, s, NH), 8.25 (1H, s, NH), 10.34 (1H, s, NH),13C NMR (125 MHz, DMSO-d6) δC 40.07 (SCH2), (127.88, 127.97, 128.14, 128.46, 129.80, 129.89, 130.24, 130.68, 131.80, 132,90, 136.65, 138.24) arC, 163.70 (C=O), 184.16 (C=S). Anal. Calcd (%) for C30H25N5OS2: C, 67.26; H, 4.70; N, 13.07. Found: C, 67.09; H, 4.55; N, 13.19.

General procedure for the synthesis of compounds 8-12. A solution of the corresponding thiosemicarbazide 3-7 (10 mmol) in 2 N NaOH was refluxed for 8 h. The resulting solution was cooled to room temperature and acidified with 37% HCl. The precipitate formed was filtered, washed with water and recrystallized from EtOH to afford the desired compounds.
4-Methyl-5-[(4,5-diphenylimidazol-2-yl)thiomethyl]-2H-1,2,4-triazole-3-thione (8). Yield 82%, mp 185-186 °C; IR: 3330 (NH), 1630 (C=N), 1306 cm-1 (C=S); 1H NMR (500 MHz, DMSO-d6) δH 3.42 (3H, s, CH3), 4.72 (2H, s, SCH2), 7.20-7.60 (10H, m, arH), 12.54 (1H, s, NH imidazole), 14.28 (1H, s, NH triazole); 13C NMR (125 MHz, DMSO-d6) δC 33.88 (CH3), 38.43 (CH2), (126.68, 127.80, 128.20, 129.56, 129.75, 130.98, 134.78, 137.20) arC, 155.36 (triazole C=N), 182.45 (C=S). Anal. Calcd (%) for C19H17N5S2: C, 60.13; H, 4.52; N, 18.45. Found: C, 60.29; H, 4.42; N, 18.61.
4-(2-Fluorophenyl)-5-[(4,5-diphenylimidazol-2-yl)thiomethyl]-2H-1,2,4-triazole-3-thione (9). Yield 77%, mp 230-231 °C; IR: 3341 (NH), 1624 (C=N), 1300 cm-1 (C=S); 1H NMR (500 MHz, DMSO-d6) δH 4.71 (2H, s, SCH2), 7.18-7.73 (14H, m, arH), 12.45 (1H, s, NH imidazole), 14.37 (1H, s, NH triazole); 13C NMR (125 MHz, DMSO-d6) δC 40.76 (SCH2), (125.33, 127.42, 127.80, 128.16, 128.51, 129.40, 130.70, 131.93, 134.38, 139.06, 141.56) arC, 154.52 (triazole C=N), 183.61 (C=S). Anal. Calcd (%) for C24H18FN5S2: C, 62.72; H, 3.95; N, 15.24. Found: C, 62.95; H, 4.10; N, 15.07.
4-Methyl-5-[(1,4,5-triphenylimidazol-2-yl)thiomethyl]-2H-1,2,4-triazole-3-thione (10). Yield 80%, mp 209-211 °C; IR: 3307 (NH), 1615 (C=N), 1297 cm-1 (C=S); 1H NMR (500 MHz, DMSO-d6) δH 3.50 (3H, s, CH3), 4.79 (2H, s, SCH2), 7.24-7.82 (15H, m, arH), 14.45 (1H, s, NH triazole); 13C NMR (125 MHz, DMSO-d6) δC 33.54 (CH3), 39.22 (CH2), (126.60, 127.09, 127.41, 127.69, 128.40, 129.16, 130.00, 131.56, 135.78, 137.68) arC, 154.78 (triazole C=N), 181.80 (C=S). Anal. Calcd (%) for C25H21N5S2: C, 65.91; H, 4.65; N, 15.37. Found: C, 66.08; H, 4.51; N, 15.28.
4-(2-Fluorophenyl)-5-[(1,4,5-triphenylimidazol-2-yl)thiomethyl]-2H-1,2,4-triazole-3-thione (11). Yield 79%, mp 243-244 °C; IR: 3339 (NH), 1627 (C=N), 1313 cm-1 (C=S); 1H NMR (500 MHz, DMSO-d6) δH 4.62 (2H, s, SCH2), 7.10-7.77 (19H, m, arH), 14.40 (1H, s, NH triazole); 13C NMR (125 MHz, DMSO-d6) δC 39.46 (SCH2), (123.87, 126.45, 127.24, 128.34, 128.56, 129.24, 129.45, 130.50, 131.79, 131.78, 136.05, 138.24, 140.68) arC, 152.95 (triazole C=N), 183.00 (C=S). Anal. Calcd (%) for C30H22FN5S2: C, 67.27; H, 4.14; N, 13.07. Found: C, 67.43; H, 4.30; N, 12.90.
4-Phenyl-5-[(1,4,5-triphenylimidazol-2-yl)thiomethyl]-2H-1,2,4-triazole-3-thione (12). Yield 75%, mp 167-168 °C. IR: 3320 (NH), 1610 (C=N), 1308 cm-1 (C=S); 1H NMR (500 MHz, DMSO-d6) δH 4.74 (2H, s, SCH2), 7.19-7.90 (20H, m, arH), 14.47 (1H, s, NH triazole); 13C NMR (125 MHz, DMSO-d6) δC 38.73 (SCH2), (127.29, 127.70, 128.34, 128.98, 129.50, 130.05, 130.38, 130.72, 131.44, 132,67, 136.16, 139.78) arC, 152.84 ( triazole C=N), 182.90 (C=S). Anal. Calcd (%) for C30H23N5S2: C, 69.60; H, 4.48; N, 13.53. Found: C, 69.74; H, 4.66; N, 13.59.

General method for the synthesis of compounds 13-17. A mixture of the corresponding thiosemicarbazide 3-7 (10 mmol) in cold concentrated sulfuric acid (30 mL) was stirred for 30 min. Then, the mixture was allowed reach to cool to room temperature. After stirring for an additional 3 h, the resulting solution was poured into ice-cold water and made alkaline to pH 8 with ammonia. The precipitated product was filtered and recrystallized from EtOH to afford the desired product.
5-[(4,5-Diphenylimidazol-2-yl)thiomethyl]-2-(N-methylamino)-1,3,4-thiadiazole (13). Yield 73%, mp 249-250 °C; IR: 3230 (NH), 1645 and 1624 cm-1 (C=N); 1H NMR (500 MHz, DMSO-d6) δH 3.34 (3H, s, CH3), 4.72 (2H, s, SCH2), 7.20-7.67 (10H, m, arH), 10.22 (1H, s, NH), 12.40 (1H, s, NH imidazole); 13C NMR (125 MHz, DMSO-d6) δC 32.65 (CH2), 35.12 (CH3), (125.90, 127.45, 127.77, 129.09, 129.57, 131.60, 134.89, 136.73) arC, 154.45 and 157.05 (thiadiazole C=N). Anal. Calcd (%) for C19H17N5S2: C, 60.13; H, 4.52; N, 18.45. Found: C, 60.30; H, 4.39; N, 18.36.
5-[(4,5-Diphenylimidazol-2-yl)thiomethyl]-2-[N-(2-fluorophenyl)]amino-1,3,4-thiadiazole (14). Yield 72%, mp 269-270 °C; IR: 3246 (NH), 1629 and 1613 cm-1 (C=N); 1H NMR (500 MHz, DMSO-d6) δH 4.66 (2H, s, SCH2), 7.14-7.82 (14H, m, arH), 10.33 (1H, s, NH), 12.42 (1H, s, NH imidazole); 13C NMR (125 MHz, DMSO-d6) δC 39.05 (SCH2), (123.88, 126.73, 127.23, 127.80, 128.34, 128.70, 129.57, 131.88, 134.50, 137.19, 140.80) arC, 153.57 and 157.30 (thiadiazole C=N). Anal. Calcd (%) for C24H18FN5S2: C, 62.72; H, 3.95; N, 15.24. Found: C, 62.86; H, 3.76; N, 15.41.
5-[(1,4,5-Triphenylimidazol-2-yl)thiomethyl]-2-(N-methylamino)-1,3,4-thiadiazole (15). Yield 71%, mp 260-261 °C; IR: 3245 (NH), 1639 and 1616 cm-1 (C=N); 1H NMR (500 MHz, DMSO-d6) δH 3.18 (3H, s, CH3), 4.76 (2H, s, SCH2), 7.21-7.70 (15H, m, arH), 10.26 (1H, s, NH); 13C NMR (125 MHz, DMSO-d6) δC 32.97 (CH2), 36.00 (CH3), (126.21, 126.78, 127.64, 127.80, 128.33, 128.96, 130.15, 130.67, 135.90, 137.46) arC, 153.94 and 159.14 (thiadiazole C=N). Anal. Calcd (%) for C25H21N5S2: C, 65.91; H, 4.65; N, 15.37. Found: C, 65.78; H, 4.81; N, 15.50.
5-[(1,4,5-Triphenylimidazol-2-yl)thiomethyl]-2-[N-(2-fluorophenyl)]amino-1,3,4-thiadiazole (16). Yield 74%, mp 279-280 °C; IR: 3278 (NH), 1638 and 1613 cm-1 (C=N); 1H NMR (500 MHz, DMSO-d6) δH 4.63 (2H, s, SCH2), 7.13-7.80 (19H, m, arH), 10.21 (1H, s, NH); 13C NMR (125 MHz, DMSO-d6) δC 40.33 (SCH2), (123.87, 126.23, 127.67, 127.89, 128.75, 128.90, 129.34, 130.41, 131.51, 135.70, 137.66, 140.22) arC, 153.11 and 157.50 (thiadiazole C=N). Anal. Calcd (%) for C30H22FN5S2: C, 67.27; H, 4.14; N, 13.07. Found: C, 67.01; H, 3.97; N, 13.32.
5-[(1,4,5-Triphenylimidazol-2-yl)thiomethyl]-2-(N-phenylamino)-1,3,4-thiadiazole (17). Yield 67%, mp 223-224 °C; IR: 3217 (NH), 1640 and 1617 cm-1 (C=N); 1H NMR (500 MHz, DMSO-d6) δH 4.83 (2H, s, SCH2), 7.10-7.78 (20H, m, arH), 10.35 (1H, s, NH); 13C NMR (125 MHz, DMSO-d6) δC 33.82 (SCH2), (126.55, 127.86, 128.35, 129.14, 130.21, 130.42, 131.50, 131.67, 132,51, 135.83, 138.46) arC, 154.67 and 157.08 (thiadiazole C=N). Anal. Calcd (%) for C30H23N5S2: C, 69.60; H, 4.48; N, 13.53. Found: C, 69.47; H, 4.60; N, 13.40.

General method for the synthesis of compounds 18 and 19. Carbon disulfide (15 mmol) was added dropwise to a solution of 1 or 2 (10 mmol) in absolute EtOH (30 mL) containing potassium hydroxide (15 mmol) at 0 ºC. The reaction was stirred at room temperature for 16 h, and then cooled and diluted with Et2O. The precipitate was filtered, washed with Et2O and dried. The potassium dithiocarbazinates 18 and 19 were obtained in nearly quantitative yield and used without further purification as it were moisture sensitive.
General method for the synthesis of compounds 20 and 21. Hydrazine hydrate (95%, 20 mmol) was added to a suspension of the potassium salt 18 or 19 (10 mmol) in water (10 mL) and the mixture was refluxed with stirring for 4 h. After cooling, it was diluted with water then acidified with aqueous hydrochloric acid. The precipitate was filtered, washed with water and recrystallized from EtOH to give yellow needles.
4-Amino-5-[(4,5-diphenylimidazol-2-yl)thiomethyl]-2H-1,2,4-triazole-3-thione (20). Yield 77%, mp 190 °C; IR: 3210-3320 (NH, NH2), 1632 (C=N), 1308 cm-1 (C=S); 1H NMR (500 MHz, DMSO-d6) δH 4.59 (2H, s, SCH2), 7.26-7.56 (10H, m, arH), 7.90 (2H, s, NH2), 12.31 (1H, s, NH imidazole), 14.15 (1H, s, NH triazole); 13C NMR (125 MHz, DMSO-d6) δC 36.16 (SCH2), (125.70, 126.38, 127.28, 128.44, 129.35, 130.92, 134.71, 136.08) arC, 153.08 (triazole C=N), 182.30 (C=S). Anal. Calcd (%) for C18H16N6S2: C, 56.82; H, 4.24; N, 22.09. Found: C, 57.02; H, 4.31; N, 21.93.
4-Amino-5-[(1,4,5-triphenylimidazol-2-yl)thiomethyl]-2H-1,2,4-triazole-3-thione (21). Yield 76%, mp 229 °C; IR: 3224-3330 (NH, NH2), 1620 (C=N), 1296 cm-1 (C=S); 1H NMR (500 MHz, DMSO-d6) δH 4.66 (2H, s, SCH2), 7.12-7.60 (15H, m, arH), 8.03 (2H, s, NH2), 14.20 (1H, s, NH triazole); 13C NMR (125 MHz, DMSO-d6) δC 37.20 (SCH2), (126.63, 126.86, 127.08, 127.67, 128.11, 128.84, 129.20, 130.48, 134.63, 136.52) arC, 152.14 ( triazole C=N), 183.76 (C=S). Anal. Calcd (%) for C24H20N6S2: C, 63.13; H, 4.42; N, 18.41. Found: C, 63.29; H, 4.60; N, 18.59.
General method for the synthesis of compounds 22 and 23. A mixture of the corresponding dithiocarbazate 18 or 19 (10 mmol) in cold concentrated sulfuric acid (30 mL) was stirred for 30 min. Then, the mixture was allowed reach to cool to room temperature. After stirring for an additional 5 h, the resulting solution was poured into ice-cold water and made alkaline to pH 8 with ammonia. The precipitated product was filtered and recrystallized from EtOH to afford the desired product.
5-[(4,5-Diphenylimidazol-2-yl)thiomethyl]-1,3,4-thiadiazole-2-thiol (22). Yield 68%, mp 260-261 °C; IR: 2615 (SH), 1635, 1617 cm-1 (C=N); 1H NMR (500 MHz, DMSO-d6) δH 4.65 (2H, s, SCH2), 7.18-7.60 (10H, m, arH), 12.40 (1H, s, NH imidazole), 13.92 (1H, s, SH); 13C NMR (500 MHz, DMSO-d6) δC 34.50 (CH2), (126.24, 126.66, 127.50, 127.92, 128.71, 129.87, 134.56, 137.34) arC, 153.12 and 157.94 (thiadiazole C=N). Anal. Calcd (%) for C18H14N4S3: C, 56.52; H, 3.69; N, 14.65. Found: C, 56.38; H, 3.86; N, 14.52.
5-[(1,4,5-Triphenylimidazol-2-yl)thiomethyl]-1,3,4-thiadiazole-2-thiol (23). Yield 66%, mp 239-240 °C; IR: 2600 (SH), 1640, 1621 cm-1 (C=N); 1H NMR (500 MHz, DMSO-d6) δH 4.72 (2H, s, SCH2), 7.20-7.73 (15H, m, arH), 13.98 (1H, s, SH); 13C NMR (125 MHz, DMSO-d6) δC 35.38 (CH2), (126.00, 126.51, 127.13, 127.63, 129.81, 130.69, 135.38, 137.93) arC, 154.26 and 159.40 (thiadiazole C=N). Anal. Calcd (%) for C24H18N4S3: C, 62.85; H, 3.96; N, 12.22. Found: C, 63.03; H, 3.89; N, 12.37.
Antimicrobial activity
All bacterial and yeast strains were obtained from the microbiology Laboratory-Faculty of medicine, University of Djillali Liabes (Sidi-Bel-Abbes-Algeria) and were as follows: Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Candida tropicalis and Candida albicans. All the newly synthesized 1,2,4-triazoles and 1,3,4-thiadiazoles were screened for their antimicrobial and antifungal activity. Thus, they were dissolved in dimethylformamide (DMF) to prepare chemical stock solution of 10 mg/1 mL.
Agar-well diffusion method was the simple screening method used for this study.39-40 Thus, each microorganism was suspended in Mueller Hinton (MH) broth and diluted approximately to 106 colony forming unit (cfu)/mL. They were “flood-inoculated” onto the surface of MH agar and sabouraud Dextrose Agar and then dried. For Candida tropicalis and Candida albicans, SDA were used. Five millimeter diameter wells were cut from the agar using a sterile cork-borer, and 50 µL of the chemical substances was delivered into the wells. The plates were incubated for 16-18 h at 37 ºC. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organism. Ampicillin (10 µg) and Fluconazole (100 µg) were standard drugs. Dimethylformamide was used as solvent controls. The antimicrobial activity results are summarized in Table 1.

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