Synthesis and antibacterial activity of some new 1,2,4-triazole derivatives bearing carbohydrazide moiety European Journal of Chemistry 11 (2) (2020) 113-119 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2020 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. http://dx.doi.org/10.5155/eurjchem.11.2.113-119.1968 European Journal of Chemistry View Journal Online View Article Online Synthesis and antibacterial activity of some new 1,2,4-triazole derivatives bearing carbohydrazide moiety Haitham Husein Al-Sa’doni 1, Fatima-Azzahra Delmani 2, Abdullah Mohammed Al Balushi 1, Ala’a Hamed Al-Ahmad 1, Sondos Omar Alsawakhneh 1 and Yaseen Ahmad Al-Soud 1,* 1 Department of Chemistry, Faculty of Science, Al Al-Bayt University, Al-Mafraq, Jordan haitham_alsadoni@yahoo.com (H.H.A.), abd_sohar@yahoo.com (A.M.A.), alaa_hamed59@yahoo.com (A.H.A.), chom_s_omr@icloud.com (S.O.A.), alsoud@aabu.edu.jo (Y.A.A.) 2 Department of Biology, Faculty of Science, Jerash University, 26150 Jerash, Jordan fa.delmani@jpu.edu.jo (F.A.D) * Corresponding author at: Department of Chemistry, Faculty of Science, Al Al-Bayt University, Al-Mafraq, Jordan. e-mail: alsoud@aabu.edu.jo (Y.A. Al-Soud). 10.5155/eurjchem.11.2.113-119.1968 Received: 12 February 2020 Received in revised form: 29 February 2020 Accepted: 24 March 2020 Published online: 30 June 2020 Printed: 30 June 2020 In this study, a series of 1,2,4-triazol-3-carbohydrazide derivatives and compound of 1,2,4- triazole-3-(4H)-thion have been synthesized. Structures and purity of the new compounds were confirmed by the use of their chromatographic and spectral data besides microanalysis. Four different bacterial stains for the study of the biological activity of compounds 6g, 7c, 7g and 7i; two Gram-positive strains, and two Gram-negative strains have been used. Compound 6g was found to be the most active of the four tested compounds against Pseudomonas aeruginosa, Bacillus cereus and Staphylococcus aureus, with an inhibition zone diameter of 16, 9, and 10 mm, respectively. Calculating the minimal inhibitory concentration value (MIC) for the positive drugs who formed an inhibition zone in the agar well diffusion method, we found that both compounds 6g and 7i were the most active of the four tested compounds against Pseudomonas aeruginosa and Bacillus cereus with an MIC value of 0.5 µg/mL for both bacteria. These results suggest that these two compounds could be considered as potential antibacterial agents against a range of bacteria. Synthesis 1,2,4-Triazoles Carbohydrazides Antibacterial activity Broth dilution method 1,2,4-Triazole-3-(4H)-thion Cite this: Eur. J. Chem. 2020, 11(2), 113-119 Journal website: www.eurjchem.com 1. Introduction Antibiotic resistance is becoming a serious threat to public health and there is an urgent need for new and improved antimicrobial agents [1-5]. The occurrence of 1,2,4-triazole system in numerous biologically active molecules has been recognized to possess activities, such as analgesic [6], antibacterial [7-9], antifungal [10,11], anti-inflammatory [12], antitumor [13,14], antitrypanosomal [15], antiproliferative [16] and antibiotics properties [17]. Literature survey reveals that important chemothera- peutics, such as ribavirin (antiviral) [18,19], fluconazole (antifungal) [20,21], alprazolam (tranquilizer) [22], trapidil (hypotensive) [19], vorozole and letrozole (nonsteroidal competitive aromatase inhibitor) [19,21,23,24] (Figure 1), that consist of substituted 1,2,4-triazole ring. In previous work, we report the synthesis of a variety of 1,5-dialkyl-1H-1,2,4-triazoles bearing different precursors such as N-alkylphthalimides [25], D-mannopentitol-1-yl-1,2,4- triazoles [26], benzotriazoles [27], 3'-triazolo-thymidines [28], 1,4-disubstituted piperazines [29]. Additionally, we have synthesized a series of 1,2,4-triazoles derivatives as CYP enzyme inhibitors [30]. On the basis of these pharmacological activity of 1,2,4- triazole derivatives, as well as the antimicrobial activity of some carbohydrazide compounds prompted us to synthesize a series of new 1,5-disubstituted 1,2,4-triazole derivatives bearing carbohydrazide moiety as antibacterial agents, via the cycloaddition of ethyl cyanoacetate with different reactive cumulenes. 2. Experimental 2.1. Materials and methods Chemical names follow IUPAC nomenclature. Starting materials were purchased from Aldrich, Acros, Lancaster, Merck, or Fluka and were used without purification. Reaction progress was monitored by thin layer chromatography (TLC) on Alugram SIL G UV254 (Macherey-Nagel). ABSTRACT RESEARCH ARTICLE KEYWORDS http://dx.doi.org/10.5155/eurjchem.11.2.113-119.1968 https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.11.2.113-119.1968&domain=pdf&date_stamp=2020-06-30 http://www.eurjchem.com/ http://dx.doi.org/10.5155/eurjchem.11.2.113-119.1968 mailto:haitham_alsadoni@yahoo.com mailto:abd_sohar@yahoo.com mailto:alaa_hamed59@yahoo.com mailto:chom_s_omr@icloud.com mailto:alsoud@aabu.edu.jo mailto:fa.delmani@jpu.edu.jo mailto:alsoud@aabu.edu.jo http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.11.2.113-119.1968&domain=pdf&date_stamp=2020-06-30� 114 Al-Sa’doni et al. / European Journal of Chemistry 11 (2) (2020) 113-119 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.113-119.1968 N N N CONH2 OHOH HOH2C Ribavirin N N N Cl N N N H3C Vorozole N N N CH3 N Ph Cl Alprazolam N N N CNNC Letrozole N N N N H3C N CH3 CH3 Trapidil Fluconazole F F N N N OH N N N Figure 1. Drugs bering 1,2,4-triazole ring used in medicine. Melting points were measured on a Mettler FP1 melting point apparatus and are uncorrected. All new compounds were analyzed for C, H, and N using a 2400 CHN Elemental Analyzer by Perkin Elmer. The observed results agreed with the calculated percentages to within ±0.4%. 1H and 13C-NMR spectra were recorded on a Bruker DRX-300 instrument. Chemical shifts are given in parts per million (ppm), and tetramethylsilane (TMS) was used as internal standard for spectra obtained in CDCl3. Mass spectra (ESI) were measured on a TSQ Quantum (Thermo Electron Corporation) instrument with a RP18 100-3 column (Macherey Nagel). 2.2. Synthesis 2.2.1 General procedure for synthesis of compounds 6 and 7 Compound 4 (0.092 g, 0.5 mmol) or compound 5 (0.099 g, 0.5 mmol), reacted with substituted sulfonyl chlorides (0.6 mmol) in chloroform (20 mL) in the presence of pyridine (5 drops) and stirred for 48 h at room temperature. Then residue was evaporated to dryness and the residue was partitioned between CHCl3 (40 mL) and water (3×40 mL). The organic phase was evaporated to dryness (Na2SO4). The product was then filtered and washed with diethyl ether followed by recrystallization from CH2Cl2 / ether to afford compounds 6 or 7 (Scheme 1 and 2). Benzene sulfonic acid N'[2-(1-ethyl-5-methyl-1H-[1,2,4]tria zole-3-yl)-acetyl]-hydrazide (6a): Color: Dark brown crystals. Yield: 63 %. M.p.: 175-177 °C. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.26 (t, 3H, CH2CH3), 2.33 (s, 3H, -CH3C=N), 3.29 (s, 2H, CH2C=O), 4.02 (q, 2H, CH2CH3), 7.49 (t, 2H, Ar-H), 7.59-7.64 (t, 1H, Ar-H), 7.79 (d, 2H, Ar-H), 9.92 (bs, 1H, NH), 10.23 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6, δ, ppm): 11.5 (C5'), 15.1 (C1''), 33.6 (C3'), 43.0 (C1'), 125.9, 128.0, 129.2, 133.3 (Ar), 151.2 (C-3), 155.8 (C-5), 167.1 (C=O). MS (ESI, m/z): 324 (M + H)+. Anal. calcd. for C13H17N5O3S: C, 48.29; H, 5.30; N, 21.66. Found: C, 48.55; H, 5.44; N, 21.89%. 4-Chlorobenzene sulfonic acid N'[2-(1-ethyl-5-methyl-1H-[1, 2,4]triazole-3-yl)-acetyl]-hydrazide (6b): Color: Milky crystals. Yield: 68 %. M.p.: 160-162 °C. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.28 (t, 3H, CH2CH3), 2.34 (s, 3H, CH3C=N), 2.54 (s, 2H, CH2C=O), 4.03 (q, 2H, CH2CH3), 7.56 (d, 2H, Ar-H), 7.78 (d, 2H, Ar-H), 10.00 (bs, 1H, NH), 10.26 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6, δ, ppm): 11.6 (C5'), 15.2 (C1''), 33.8 (C3'), 42.8 (C1'), 129.3, 130.0, 138.2, 138.4 (Ar), 152.0 (C-3), 156.3 (C-5), 167.4 (C=O). MS (ESI, m/z): 358, 360 (M + H)+. Anal. calcd. for C13H16ClN5O3S: C, 43.64; H, 4.51; N, 19.57. Found: C, 43.85; H, 4.72; N, 19.80(%). 3-Trifluoromethyl-benzene sulfonic acid N'[2-(1-ethyl-5- methyl-1H-[1,2,4]triazole-3-yl)-acetyl]-hydrazide (6c): Color: White crystals. Yield: 72 %. M.p.: 206-208 °C. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.27 (t, 3H, CH2CH3), 2.32 (s, 3H, CH3C=N), 3.28 (s, 2H, CH2C=O), 4.01 (q, 2H, CH2CH3), 7.76 (t, 1H, Ar-H), 8.01-8.10 (m, 3H, Ar-H), 10.25 (bs, 1H, NH), 10.35 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6, δ, ppm): 11.6 (C5'), 15.2 (C1''), 33.8 (C3'), 42.8 (C1'), 124.4 , 124.5, 130.0, 130.1, 130.8, 132.2, 140.9 (Ar), 152.1 (C-3), 156.3 (C-5), 167.5 (C=O). MS (ESI, m/z): 392 (M + H)+. Anal. calcd. for C14H16F3N5O3S: C, 42.96; H, 4.12; N, 17.89. Found: C, 42.77; H, 4.21; N, 18.09(%). 2,5-Dichlorobenzene sulfonic acid N'[2-(1-ethyl-5-methyl- 1H-[1,2,4]triazole-3-yl)-acetyl]-hydrazide (6g) [31]. 2-Thiophene sulfonic acid N'[2-(1-ethyl-5-methyl-1H-[1,2,4] triazole-3-yl)-acetyl]-hydrazide (6i): Color: Brown crystals. Yield: 73 %. M.p.: 211-213 °C. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.28 (t, 3H, CH2CH3), 2.33 (s, 3H, CH3C=N), 2.54 (s, 2H, CH2C=O), 4.02 (q, 2H, CH2CH3), 7.09-7.95 (m, 3H, Thiophene- H), 9.99 (bs, 1H, NH), 10.24 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6, δ, ppm): 11.6 (C5'), 15.2 (C1''), 33.8 (C3'), 42.8 (C1'), 128.0, 133.8, 134.3, 139.3 (Thiophene-C), 152.0 (C-3), 156.3 (C-5); 167.4 (C=O). MS (ESI, m/z): 330 (M + H)+. Anal. calcd. for C11H15N5O3S2: C, 40.11; H, 4.59; N, 21.26; Found: C, 40.30; H, 4.74; N, 21.51(%). 7-Methoxy-4-methyl-2-oxo-2H-chromene-6-sulfonic acid N' [2-(1-ethyl-5-methyl-1H-[1, 2, 4]triazole-3-yl)-acetyl]-hydrazide (6k): Color: White crystals. Yield: 63 %. M.p.: 222-224 °C. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.21 (t, 3H, CH2CH3), 2.26 (s, 3H, CH3C=N), 2.42 (s, 3H, CH3-Coumarin), 3.27 (s, 2H, CH2C=O), 3.92 (s, 3H, OCH3), 4.01 (q, 2H, CH2CH3), 6.34 (s, 1H, Ar-H), 7.22 (s, 1H, Ar-H), 8.00 (s, 1H, Ar-H), 9.65 (bs, 1H, NH), 10.16 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6, δ, ppm): 11.5 (C5'), 15.6 (C1''), 18.5 (CH3-Coumarin), 33.6 (C3'), 42.8 (C1'), 57.8 (O-CH3), 101.6, 112.3, 112.5, 124.2, 127.7, 151.9, 153.5, 156.3 (Ar), 158.2 (C-3), 159.9 (C-5), 160.7, 167.6 (C=O). MS (ESI, m/z): 436 (M + H)+. Anal. calcd. for C18H21N5O6S: C, 49.65; H, 4.86; N, 16.08. Found: C, 49.87; H, 4.99; N, 16.29 (%). 4-Chlorobenzene sulfonic acid N'[2-(1,5-diethyl-1H-[1,2,4] triazole-3-yl)-acetyl]-hydrazide (7b): Color: White crystals. Yield: 76 %. M.p.: 110-112 °C. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.18 (t, 3H, C-CH2CH3), 1.26 (t, 3H, N-CH2CH3), 2.70 (q, 2H, C-CH2CH3), 3.34 (s, 2H, CH2C=O), 4.03 (q, 2H, N-CH2CH3), 7.55 (d, 2H, Ar-H), 7.78 (d, 2H, Ar-H), 10.07 (bs, 1H, NH), 10.31 (bs, 1H, NH). Al-Sa’doni et al. / European Journal of Chemistry 11 (2) (2020) 113-119 115 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.113-119.1968 Scheme 1 13C NMR (75 MHz, DMSO-d6, δ, ppm): 12.2 (C5'''), 15.4 (C1''), 18.7 (C5''), 33.9 (C3'), 42.6 (C1'), 129.3, 130.0, 138.2, 138.4 (Ar), 156.4 (C-3), 156.5 (C-5), 167.4 (C=O). MS (ESI, m/z): 372, 374 (M + H)+. Anal. calcd. for C14H18ClN5O3S: C, 45.22; H, 4.88; N, 18.83. Found: C, 45.41; H, 4.97; N, 19.11(%). 3-Trifluoromethyl-benzene sulfonic acid N'[2-(1,5-diethyl- 1H-[1,2,4]triazole-3-yl)-acetyl] -hydrazide (7c) [31]. 4-Nitrobenzene sulfonic acid N'[2-(1,5-diethyl-1H-[1,2,4] triazole-3-yl)-acetyl]-hydrazide (7d): Color: White crystals. Yield: 81 %. M.p: 145-147 °C. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.17 (t, 3H, C-CH2CH3), 1.26 (t, 3H, N-CH2CH3), 2.69 (q, 2H, C-CH2CH3), 3.33 (s, 2H, CH2C=O), 4.02 (q, 2H, N-CH2CH3), 8.04 (d, 2H, Ar-H), 8.30 (d, 2H, Ar-H), 10.41 (bs, 1H, NH), 10.45 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6, δ, ppm): 12.2 (C5'''), 15.4 (C1''), 18.7 (C5''), 33.9 (C3'), 42.6 (C1'), 124.4, 129.7, 145.4, 149.9 (Ar), 150.2 (C-3), 156.3 (C-5), 167.6 (C=O). MS (ESI, m/z): 383 (M + H)+. Anal. calcd. for C14H18N6O5S: C, 43.97; H, 4.74; N, 21.98. Found: C, 44.26; H, 4.94; N, 22.22(%). 2,5-Dichlorobenzene sulfonic acid N'[2-(1,5-diethyl-1H-[1,2, 4] triazole-3-yl)-acetyl]-hydrazide (7g) [31]. 2-Thiophene sulfonic acid N'[2-(1,5-diethyl-1H-[1,2,4]tria zole-3-yl)-acetyl]-hydrazide (7i) [31]. 4,7-Dimethyl-2-oxo-2H-chromene-6-sulfonic acid N'[2-(1,5- diethyl-1H-[1,2,4]triazole-3-yl)-acetyl]-hydrazide (7j): Color: Yellow crystals. Yield: 71 %. M.p.: 225-227 °C. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.09 (t, 3H, C-CH2CH3), 1.21 (t, 3H, N- CH2CH3), 2.44 (s, 3H, CH3-Coumarin), 2.61 (q, 2H, C-CH2CH3), 3.33 (s, 2H, CH2C=O), 2.73 (s, 3H, CH3-Coumarin), 3.94 (q, 2H, N-CH2CH3), 6.48 (s, 1H, Ar-H), 7.41 (s, 1H, Ar-H), 8.12 (s, 1H, Ar-H), 10.12 (bs, 1H, NH), 10.26 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6, δ, ppm): 12.2 (C5'''), 15.4 (C1''), 18.3 (CH3- Coumarin), 18.6 (C5''), 20.7 (CH3-Coumarin), 33.6 (C3'), 42.5 (C1'), 114.9, 117.4, 120.3, 127.6, 133.7, 143.4, 153.1, 156.3 (Ar), 155.6 (C-3), 156.4 (C-5), 159.7, 167.8 (C=O). MS (ESI, m/z): 434 (M + H)+. Anal. calcd. for C19H23N5O5S: C, 52.65; H, 5.35; N, 16.16. Found: C, 52.89; H, 5.56; N, 16.37(%). 2.2.2. General procedure for synthesis of compounds 8 Compound 5 (0.099g, 0.5 mmol), reacted with substituted carbonyl chlorides (0.6 mmol) in chloroform (20 mL) in the presence of pyridine (5 drops) and stirred for 48 h at room temperature. 116 Al-Sa’doni et al. / European Journal of Chemistry 11 (2) (2020) 113-119 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.113-119.1968 Cl Cl O2N NO2 CF3 S NO2 Me O Me O OH MeO O Me O OMe Cl N N O S G O N H N N H CH2CH3H3CCH2 O N N O S G O N H N N H CH2CH3H3C O N N N CH2CH3H3CCH2 N NH N S G N N O C G N H N N H CH2CH3H3CCH2 O 1 2 3 4 5 5 ' 1 ' 1 '' 3 ' 5 ''5 ''' 1 2 1 ' 1 '' 3 3 ' 4 5 ih j k G = a b c d e f g 6 7 8 9 Scheme 2 Then residue was evaporated to dryness and the residue was partitioned between CHCl3 (40 mL) and water (3×40 mL). The organic phase was evaporated to dryness (Na2SO4). The product was then filtered and washed with diethyl ether followed by recrystallization from CH2Cl2/ether to afford compounds 8 (Scheme 1 and 2). 3-Methoxy-benzoic acid N'[2-(1,5-diethyl-1H-[1,2,4]triazole- 3-yl)-acetyl]-hydrazide (8f): Color: Brown crystals. Yield: 80 %. M.p.: 118-120 °C. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.21 (t, 3H, C-CH2CH3); 1.31 (t, 3H, N-CH2CH3); 2.71 (q, 2H, C-CH2CH3), 3.33 (s, 2H, CH2C=O); 3.54 (s, 3H, OCH3), 4.02 (q, 2H, N- CH2CH3), 7.12-7.17(m, 1H, Ar-H), 7.43 (bs, 3H, Ar-H), 10.13 (bs, 1H, NH), 10.42 (bs, 1H, NH). 13C NMR (300 MHz, DMSO-d6, δ, ppm): 12.3 (C5'''), 15.5 (C1''), 18.7 (C5''), 34.3 (C3'), 42.6 (C1'), 55.7 (OCH3), 112.8, 118.2, 119.4, 120.2, 130.1, 134.3 (Ar), 156.7 (C-3), 159.6 (C-5), 166.5, 167.8 (C=O). MS (ESI, m/z): 332 (M + H)+. Anal. calcd. for C16H21N5O3: C, 57.99; H, 6.39; N, 21.13. Found: C, 58.25; H, 6.50; N, 21.38(%). 3,5-Dinitro-benzoic acid N'[2-(1, 5-diethyl-1H-[1, 2, 4]tria zole-3-yl)-acetyl]-hydrazide (8h): Color: Brown crystals. Yield: 66 %. M.p.: 137-139 °C. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.19 (t, 3H, C-CH2CH3), 1.29 (t, 3H, N-CH2CH3), 2.69 (q, 2H, C- CH2CH3), 3.33 (s, 2H, CH2C=O), 4.05 (q, 2H, N-CH2CH3), 7.36- 9.01 (m, 3H, Ar-H), 8.09 (bs, 2H, NH). 13C NMR (75 MHz, DMSO-d6, δ, ppm): 12.3 (C5'''), 15.5 (C1''), 18.7 (C5''), 34.3 (C3'), 42.6 (C1'), 122.4, 124.4, 128.2, 128.4, 135.0, 136.7 (Ar), 148.8 (C-3), 150.0 (C-5), 164.4, 167.7 (C=O). MS (ESI, m/z): 392 (M + H)+. Anal. calcd. for C15H17N7O6: C, 46.04; H, 4.38; N, 25.05. Found: C, 46.26; H, 4.51; N, 25.27(%). 2.2.3. Synthesis of 5-((1,5-diethyl-1H-1,2,4-triazol-3-yl) methyl)-4-(4-hydroxyphenyl)-2,4-dihydro-3H-1,2,4- triazole-3-thione (9e) Compound 5 (0.099 g, 0.5 mmol), reacted with 4- isothiocyanato-phenol (0.091 g, 0.6 mmol) in toluene (20 mL) in the presence of pyridine (5 drops) and stirred for 48 h at room temperature. Then residue was evaporated to dryness and the residue was partitioned between CHCl3 (40 mL) and water (3×40 mL). The organic phase was evaporated to dryness (Na2SO4). The product was then filtered and washed with diethyl ether followed by recrystallization from CH2Cl2 / ether to afford compounds (9e): Color: Brown crystals. Yield: 70 %. M.p.: 140-142 °C. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 1.01 (t, 3H, C-CH2CH3), 1.22 (t, 3H, N-CH2CH3), 2.54 (q, 2H, C- CH2CH3), 3.52 (s, 2H, CH2C=O), 3.98 (q, 2H, N-CH2CH3), 5.52 (bs, 1H, OH), 6.73 (d, 2H, Ar-H), 7.07 (d, 2H, Ar-H), 10.29 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6, δ, ppm): 12.3 (C5'''), 15.4 (C1''), 18.7 (C5''), 34.6 (C3'), 42.8 (C1'), 115.2, 128.3, 130.7, 155.8 (Ar), 156.9, 157.1, 168.0, 181.7 (C=S). MS (ESI, m/z): 331 (M + H)+. Anal. calcd. for C15H18N6OS: C, 54.53; H, 5.49; N, 25.44. Found: C, 54.74; H, 5.68; N, 25.66(%). 2.3. Evaluation of antibacterial activity A preliminary test for the antibacterial activity of compounds 6g, 7c, 7g and 7i was tested by two methods, the agar well diffusion method [32] and the broth dilution method [33] using different bacterial strains. 2.3.1. Test bacteria A number of bacterial isolates were used in this study, two Gram-positive strains, Bacillus cereus (ATCC11778) and Staphylococcus aureus (ATCC25923); and two gram-negative strains, Pseudomonas aeruginosa (ATCC27853) and Shigella sp (ATCC23354). 2.3.2. Preparation of the test compounds and controls Four compounds were selected for this biological study, named, 6g, 7c, 7g and 7i. 10 µg of each compound were dissolved in 1 mL dimethyl sulfoxide (DMSO) in small sterile plastic cups and stored in dark until use. DMSO was used as a negative control, whereas penicillin (10 µg/mL) was used as a positive control. 2.3.3. Agar well diffusion method Mueller-Hinton agar medium was used as a growth medium. Media were dispensed in Petri dishes to produce Mueller-Hinton agar plates; plates were then inoculated with the test bacterium. The inoculum originated from a fresh bacterial culture with an O.D. of about 0.5 for all cultures. 6 mm-holes were punched in the solidified Mueller-Hinton agar plates using a cork borer; wells were then filled with 50 μL of each compound. Penicillin was used as a positive control, while DMSO was used as a negative control. Al-Sa’doni et al. / European Journal of Chemistry 11 (2) (2020) 113-119 117 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.113-119.1968 Table 1. Inhibition zone diameters in millimeters (mm) around the wells of the active compounds against different bacteria. Compound P. aeruginosa S. aureus B. cereus Shigella sp. 6g 16 9 10 - 7c - - 10 - 7g - - 8 - 7i 15 - 10 - Penicillin (Positive control) - 27 10 35 DMSO (Negative control) - - - - The plates were then incubated upright at 37 °C for 24 hours. Growth-free inhibition zones were then measured to the nearest millimeter. Each test was repeated twice. 2.3.4. Broth dilution method The broth dilution method was used to determine the minimum inhibitory concentration (MIC) for each active compound [34]. Different volumes of nutrient broth, fresh culture, and the test chemical were mixed in the wells of a sterile microwell plate to yield the following final concentrations: 0.5, 1.0 and 2.0 µg/mL. DMSO and penicillin replaced the test chemical in the negative and the positive control experiments, respectively. MIC was expressed as the lowest dilution causing an inhibition of growth judged by lack of turbidity. A well containing the broth only (blank) was used for comparison. The plates were then incubated at 37 °C for 24 hours. 3. Results and discussion 3.1. Synthesis The biological importance of 1,2,4-triazole prompted us to synthesize new 1,5-1H-1,2,4-triazole derivatives containing a carbohydrazide backbone, which would be favorable in achieving some specificity of pharmacological action in view of the development of effective clinical antibacterial drugs. The triazolehydrazides 4 and 5 which prepared by the reaction of the reactive intermediates 2 prepared in situ from the dichloride 1, were reacted via the cycloaddition reaction with ethyl cyanoacetate 3 to give, after spontaneous rearran- gement [35,36], the triazolehydrazides 4 and 5 [27]. These compounds were used as starting materials for the synthesis of the carbohydrazide derivatives 6 and 7, by reaction with the appropriate substituted sulfonyl chlorides in 63-81% yield. Triazole derivative 8 synthesized by reaction with appropriate substituted carbonyl chlorides in CHCl3 for 18 h in 66-80% yield (Scheme 1). Next, the hydrazides 5 were used in synthesis of different 1,2,4-triazole-3-thiones. Thus, treatment of compound 5 with p-hydroxyphenyl isothiocyanate in toluene in the presence of pyridine as base afforded 1,2,4- triazole-3-thione 9e (70%). The 1H NMR and 13C NMR spectra of all prepared compounds are in total agreement with the suggested structures. DEPT experiments were employed to differentiate secondary and quaternary carbons from primary and tertiary carbons. Additional support of the proposed structures comes from mass spectral data; mass spectra of the prepared compounds showed the correct molecular ions (M+·), as suggested by their molecular formulas. The 1H NMR spectra of compounds 6a-c, 6i and 6k showed similar signal patterns. Triazole-CH3 appeared as singlets at in the region δ 2.33-2.44 ppm while the protons CH2CH3 appeared at higher field in the region δ 2.21-2.28 ppm. The quartets in the region around δ 4.1 ppm were attributed to the CH2CH3, whereas the singlets in the region δ 2.54-3.34 ppm were assigned to CH2C=O. The 1H NMR spectra of compounds 6a-c, 6i and 6k were characterized by the presence of the O=CNH which appeared as broad singlets in the region δ 9.65- 10.25 ppm, whereas the broad singlets in the region δ 10.16- 10.35 ppm were assigned to NHSO2. The singlets at δ 3.33 ppm for the compounds 8f and 8h were assigned to CH2C=O. The 13C NMR spectra of compounds 6a-c, 6i and 6k contained the resonance signals C-3 and C-5 of the triazole ring at higher field between δ 151.2-158.2 and 155.8-159.9 ppm, respect- tively, whereas the signals resonated at δ 150.2-156.4 and 156.3-156.5 ppm were attributed to C-3 and C-5 of the derivatives of compound 7, respectively. 3.2. Antibacterial activity Four compounds 6g, 7c, 7g and 7i were selected for testing their antibacterial activity using two methods: the agar well diffusion method and the broth dilution method. Table 1 shows the results obtained from the agar well diffusion method; we found that compound 6g was active against three strains out of four, Pseudomonas aeruginosa (ATCC27853), Staphylococcus aureus (ATCC25923) and Bacillus cereus (ATCC 11778) with an inhibition zone diameter of 16, 9, and 10 mm, respectively; Shigella sp. (ATCC23354) was resistant to this chemical, no inhibition zone was formed. Compound 7c was active against B. cereus (ATCC 11778) only with an inhibition zone diameter of 10 mm. Penicillin was used as a positive control with an inhibition zone diameter of 27, 10, and 35 mm respectively against S. aureus, B. cereus (ATCC 11778), and Shigella sp; and DMSO was used as negative control for the tested bacteria. Compound 7g was active against B. cereus (ATCC 11778) only with an inhibition zone diameter of 8 mm, but not against P. aeruginosa, S. aureus, and Shigella sp. Compound 7i was active against P. aeruginosa and B. cereus with an inhibition zone diameter of 15 and 10 mm, respectively; S. aureus and Shigella sp. were resistant to this compound (Table 1). As shown in Table 1, compounds 6g and 7i were active against the Gram-negative bacterium P. aeruginosa (16 and 15 mm, respectively); the bacterium was resistant to penicillin, it did not show any inhibitory zone against it. P. aeruginosa is one of the widely distributed pathogens in nature and considered one of the agents responsible for community-acquired infections [32], it’s considered an opportunistic pathogen known to have the ability to rapidly develop resistance to multiple classes of antibiotics [37]. Epidemiological studies showed that infections caused by drug-resistant P. aeruginosa are related with serious complications including death [38,39]. Both compounds showed similar activity against B. cereus showing an inhibition zone of 10 mm, the same inhibition zone was found with the positive control penicillin. B. cereus, a Gram positive, spore forming bacterium responsible for food poisoning due the production of toxins [40]; it’s one of the causative agents of foodborne outbreaks worldwide [41-43]. Unfortunately, none of the tested compounds showed any activity against Shigella sp. one of the leading causes of diarrheal diseases worldwide and one of the most resistant pathogens against many antibiotics [44,45]. Regarding the bacterial strain S. aureus, only compound 6g was active against it with an inhibition zone diameter of 9 mm. Different minimal inhibitory concentrations (MIC) values were registered for each compound that showed an inhibition zone in the agar well diffusion test. 118 Al-Sa’doni et al. / European Journal of Chemistry 11 (2) (2020) 113-119 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.113-119.1968 Table 2. Minimal inhibitory concentration values (µg/ml) of the tested chemicals against their sensitive strains. Compound P. aeruginosa S. aureus B. cereus 6g 0.5 2 0.5 7c NT NT 1 7g NT NT 2 7i 0.5 NT 0.5 Penicillin (positive control) NT 0.5 0.5 *NT: Not tested, because these compounds did not show any bacterial activity against the targeted bacterium using the agar well diffusion method. The MIC values are shown in Table 2; compounds 6g and 7i were the most active ones of all the tested compounds against P. aeruginosa and B. cereus with an MIC value of 0.5 µg/mL against both bacteria; compound 6g had lower activity against S. aureus with an MIC value of 2 µg/mL whereas compound 7i was not tested because it did not show any antibacterial activity against this bacterium in the agar well diffusion test. Compound 7c showed an intermediate antibacterial activity with an MIC value of 1 µg/mL against B. cereus. The MIC value of 7g against B. cereus was also high (2 µg/mL) compared with the values found with the other compounds. Compound 7c and 7g were not tested against S. aureus nor tested against P. aeruginosa because they did not show any antibacterial activity against these two bacteria using the agar well diffusion method (Table 2). When comparing compound 6g and 7i (the most active ones of the 4 tested compounds against this panel of bacterial stains) with penicillin, we found that both drugs were having similar MIC values as those found with penicillin (0.5 µg/mL) against B. cereus, penicillin was not active against P. aeruginosa but active against S. aureus (MIC value 0.5 µg/mL). These results suggest that both compounds 6g and 7i could be selected as potential antibacterial agents with an activity similar or stronger than penicillin against some bacterial stains. 4. Conclusion We report the synthesis and in vitro antibacterial evaluation of new 1,2,4-triazole derivatives. The four tested compounds, 6g and 7i were the most active ones against P. aeruginosa (one of the most resistant bacteria against many drugs) and B. cereus ATCC11778 (MIC 0.5 µg/mL), followed by compound 7c against B. cereus ATCC11778 (MIC 1 µg/mL). These results suggest that compound 6g and 7i could be good candidates as antibacterial agents; further studies could be done on other bacterial strains as well as the study of the interactions caused by these compounds with the bacterial DNA. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Author contributions: All authors contributed equally to this work. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. ORCID Fatima-Azzahra Delmani http://orcid.org/0000-0002-0030-7139 Abdullah Mohammed Al Balushi http://orcid.org/0000-0002-9655-3784 Ala’a Hamed Al-Ahmad http://orcid.org/0000-0002-1411-8937 Sondos Omar Alsawakhneh http://orcid.org/0000-0003-2477-3027 Yaseen Ahmad Al-Soud http://orcid.org/0000-0003-3357-3801 References [1]. Jubie, S.; Prabitha, P.; Rajesh, K. R.; Kalirajan, R.; Gayathri, R.; Sankar, S.; Elango, K. Med. Chem. Res. 2012, 21, 1403-1410. [2]. Prakash, O.; Aneja, D. K.; Hussain, K.; Lohan, P.; Ranjan, P.; Arora, S.; Sharma, C.; Aneja, K. R. Eur. J. Med. Chem. 2011, 46, 5065-5073. [3]. Laxminarayan, R.; Duse, A.; Wattal, C.; Zaidi, A. K.; Wertheim, H. F.; Sumpradit, N.; Vlieghe, E.; Hara, G. L.; Gould, I. M.; Goossens, H.; Greko, C.; So, A. D.; Bigdeli, M.; Tomson, G.; Woodhouse, W.; Ombaka, E.; Peralta, A. Q.; Qamar, F. N.; Mir, F.; Kariuki, S.; Bhutta, Z. A.; Coates, A.; Bergstrom, R.; Wright, G. D.; Brown, E. D.; Cars, O. Lancet Infect. Dis. 2013, 13, 1057-1098. [4]. Laxminarayan, R.; Heymann, D. L. Bio. Med. J. 2012, 344, e1567. [5]. Siwek, A.; Staczek, P.; Stefanska, J. Eur. J. Med. Chem. 2011, 46, 5717- 5726. [6]. Turan-Zitouni, G.; Kaplanciki, Z. A.; Erol, K.; Kilic, F. S. Il Farmaco 1999, 54, 218-223. [7]. Mari, S. K.; Bantwal, S. H.; Nalilu, S. K. Eur. J. Med. Chem. 2008, 43, 309-314. [8]. Ulusoy, N.; Gursoy, A.; Otuk, G. Il Farmaco 2001, 56, 947-952. [9]. Guzeldemirci, N. U.; Kucukbasmac, O. Eur. J. Med. Chem. 2010, 45, 63- 68. [10]. Jin, R.; Liu, J.; Zhang, G.; Li, J.; Zhang, S.; Guo, H. Chem. Biodiver. 2018, 15(9), e1800263. [11]. Wei, Q. L.; Zhang, S. S.; Gao, J.; Li, W. H; Xu, L. Z.; Yu, Z. G. Bioorg. Med. Chem. 2006, 14, 7146-7153. [12]. Shehy, M. F.; Abu-Hashem, A.; El-Telbani, E. M. Eur. J. Med. Chem. 2010, 45, 1906-1911. [13]. Al-Soud, Y. A.; Al-Dweri, M. N.; Al-Masoudi, N. A. Il Farmaco 2004, 59, 775-783. [14]. Boraei, A. T. A.; Gomaa, M. S.; Ashry, S. H.; Duerkop, A. Eur. J. Med. Chem. 2017, 125, 360-371. [15]. Papadopoulou, M. V.; Bloomer, W. D.; Rosenzweig, H. S.; Chatelain, E.; Kaiser, M.; Wilkinson, S. R.; MaKenzie, C.; Ioset, J. R. J. Med. Chem. 2012, 55, 5554-5565. [16]. Li, Y. H.; Zhang, B.; Yang, H. K.; Li, Q.; Diao, P. C.; You, W. W.; Zhao, P. L. Eur. J. Med. Chem. 2017, 125, 1098-1106. [17]. Xia, Z. P.; Wang, X. D.; Wang, P. F.; Zhou, Y.; Zhang, J. W.; Zhang, L.; Zhou, J.; Zhou, S. S.; Ouyang, H.; Lin, X. Y.; Mustapa, M.; Reyinbaike, A.; Zhu, H. L. Eur. J. Med. Chem. 2014, 80, 92-100. [18]. Kucukguzel, S. G.; Cıkla-Suzgun, P. Eur. J. Med. Chem. 2015, 97, 830- 870. [19]. Sztanke, K.; Tuzimski, T.; Rzymowska, J.; Pasternak, K.; Kandefer- Szerszen, M. Eur. J. Med. Chem. 2008, 43, 404-419. [20]. Orek, C.; Koparir, P.; Koparıi, M. Spectrochim. Acta A 2012, 97, 923- 934. [21]. Zhao, P. L.; Ma, W. F.; Duan, A. N.; Zou, M.; Yan, Y. C.; You, W. W.; Wu, S. G. Eur. J. Med. Chem. 2012, 54, 813-822. [22]. Hou, Y. P.; Sun, J.; Pang, Z. H.; Lv, P. C.; Li, D. D.; Yan, L.; Zhang, H. J.; Zheng, J.; Zhao, E. X.; Zhu, H. L. Eur. J. Med. Chem. 2011, 19, 5948- 5954. [23]. Puthiyapurayil, P.; Poojary, B.; Chikkanna, C.; Buridipad, S. K. Eur. J. Med. Chem. 2012, 57, 407-416. [24]. Takahashi, K.; Yamagishi, G.; Hiramatsu, T.; Hosoya, A.; Onoe, K.; Doi, H.; Nagata, H.; Wada, Y.; Onoe, H.; Watanabe, Y.; Hosoya, T. Bioorg. Med. Chem. 2011, 19, 1464-1470. [25]. Al-Soud, Y. A.; Al-Masoudi, N. A. Pharmazie 2001, 56, 372-375. [26]. Al-Masoudi, N. A.; Al-Soud, Y. A.; Lagoja, I. Carbohydr. Res. 1999, 318, 67-74. [27]. Al-Masoudi, N. A.; Al-Soud, Y. A.; El-Ferawnah, A. R. S. Bioorg. Med. Chem. 2003, 11, 1701-1708. [28]. Al-Soud, Y. A.; Al-Masoudi, N. A. Heteroat. Chem. 2003, 14, 298-303. [29]. Al-Soud, Y. A.; Al-Masoudi, N. A. Farmaco 2004, 59, 41-46. [30]. Al-Soud, Y. A.; Heydel, M.; Hartmann, R. W. Tetrahedron Lett. 2011, 52, 6372-6375. [31]. Jacob, H. J.; Irshaid, F. I.; Al-Soud, Y. A.; Al-Balushi, A. M.; Al-Arqan, H. R. Adv. Stud. Biol. 2013, 5, 303-318. [32]. Rojas, J. J.; Ochoa, V.; Ocampo, S.; Munoz, J. BMC Complement Altern. Med. 2006, 6(1), 1-6. [33]. Cappuccino, J.; Sherman N., A Laboratory Manual, 7th Edition, http://orcid.org/0000-0002-0030-7139 http://orcid.org/0000-0002-9655-3784 http://orcid.org/0000-0002-1411-8937 http://orcid.org/0000-0003-2477-3027 http://orcid.org/0000-0003-3357-3801 Al-Sa’doni et al. / European Journal of Chemistry 11 (2) (2020) 113-119 119 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.113-119.1968 Benjamin Cummings Publishing Company, USA, 2008. [34]. CLSI, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, Approved Standard, 9th edition, CLSI document M07-A9. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA, 2012. [35]. Wang, Q.; Jochims, J. C.; Kohlbrandt, S.; Dahlenburg, L.; Al-Talib, M.; Hamed, A.; Ismail, A. E. Synthesis 1992, 1992(7), 710-718. [36]. Wang, Q.; Al-Talib, M.; Jochims, J. C. Chem. Ber. 1994, 127, 541-547. [37]. Lister, P. D.; Wolter, D. J.; and Hanson, N. D. Clin. Microbiol. Rev. 2009, 22, 582-610. [38]. Aloush, V.; Navon-Venezia, S.; Seigman-Igra, Y.; Cabili, S.; Carmeli, Y. Antimicrob. Agents Chemother. 2006, 50, 43-48. [39]. Gasink, L. B.; Fishman, N. O.; Weiner, M. G.; Nachamkin, I.; Bilker, W. B.; Lautenbach, E. Am. J. Med. 2006, 119, 19-25. [40]. Organji, S. R.; Abulreesh, H. H.; Elbanna, K.; Osman, G. E. H.; Khider, M. Asian Pac. J. Trop. Biomed. 2015, 5, 515-520. [41]. Stenfors Arnesen, L. P.; Fagerlund, A.; Granum, P. E. FEMS Microbiol. Rev. 2008, 32, 579-606. [42]. Bottone, E. J. Clin. Microbiol. Rev. 2010, 23, 382-398. [43]. Bennett, S. D.; Walsh, K. A.; Gould, H. A. Clin. Infect. Disea. 2013, 57, 425-433. [44]. Peirano, G.; Souza, F. S.; Rodrigues, D. P. Mem. Inst. Oswaldo Cruz. 2006, 101, 245-250. [45]. Rosewell, A.; Ropa, B.; Posanai, E.; Dutta, S. R.; Mola, G.; Zwi, A.; MacIntyre, C. R. Emerg. Infect. Dis. 2010, 16, 1797-1799. Copyright © 2020 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Materials and methods 2.2. Synthesis 2.2.1 General procedure for synthesis of compounds 6 and 7 2.2.2. General procedure for synthesis of compounds 8 2.3. Evaluation of antibacterial activity 2.3.1. Test bacteria 2.3.2. Preparation of the test compounds and controls 2.3.3. Agar well diffusion method 2.3.4. Broth dilution method 3. Results and discussion 3.1. Synthesis 3.2. Antibacterial activity 4. Conclusion Disclosure statement ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: