The newly synthesized compounds were screened for their antimicrobial activities; some of them were found to be active towards the test microorganisms as the results demonstrated that the synthesized compounds exhibited a broad spectrum of activity with minimum inhibitory concentration (MIC) values of 31.3–500 µg/mL against gram-positive and gram-negative bacteria, Candia albicans and Saccharomyces cerevisiae.
Turkish Journal of Chemistry http://journals.tubitak.gov.tr/chem/ Research Article Turk J Chem (2013) 37: 812 823 ă ITAK c TUB ⃝ doi:10.3906/kim-1212-66 Synthesis and biological activities of methylenebis-4H -1,2,4-triazole derivatives ¨ Yıldız UYGUN, Hacer BAYRAK,∗ Havva OZKAN Department of Chemistry, Faculty of Sciences, Karadeniz Technical University, Trabzon, Turkey Received: 27.12.2012 • Accepted: 10.05.2013 • Published Online: 16.09.2013 • Printed: 21.10.2013 Abstract: 5,5 ′ -Methylenebis(4–phenyl-4 H -1,2,4-triazole-3-thiol) (2) was synthesized starting from hydrazinecarbothioamide compound (1) Treatment of compound with ethyl bromoacetate produced diethyl 5,5 ′ -{methylenebis[(4phenyl-4 H -1,2,4-triazole-5,3-diyl)thio]}diacetate (3), which was converted to the corresponding diacetohydrazide derivative (4) by treatment with hydrazine hydrate The reaction of compound with several aldehydes produced the corresponding arylidene hydrazides, 5a–d Syntheses of Mannich bases 6a–c were carried out by the treatment of compound with several amines in the presence of formaldehyde (4{[5-({5-[(4-Amino-2-chlorophenyl)thio]-4-phenyl-4 H -1,2,4triazol-3-yl}methyl)-4-phenyl-4 H -1,2,4-triazol-3-yl]thio}-3-chlorophenyl)amine (8) was prepared by reduction of nitro groups of 3,3 ′ -methylenebis{5-[(2-chloro-4-nitrophenyl)thio]-4-phenyl-4 H -1,2,4-triazole} (7) that were obtained from the condensation of with 3,4-dichloronitrobenzene The newly synthesized compounds were screened for their antimicrobial activities; some of them were found to be active towards the test microorganisms as the results demonstrated that the synthesized compounds exhibited a broad spectrum of activity with minimum inhibitory concentration (MIC) values of 31.3–500 µ g/mL against gram-positive and gram-negative bacteria, Candia albicans and Saccharomyces cerevisiae All compounds displayed lower activity in this series against the microorganisms with MIC values of 31.3–500 µ g/mL than did the compared control drugs of ampicillin, streptomycin, and fluconazole Key words: 1,2,4-Triazole, morpholine, furan, Schiff base, Mannich base, antimicrobial activity Introduction 1,2,4-Triazole moiety has been incorporated into a number of therapeutically important agents 1,2 Itraconazole, fluconazole, voriconazole, 3,4 triazolam, alprazolam, etizolam and furacilin, ribavirin, hexaconazole, triadimefon, 10 myclobutanil, 11 rizatriptan, 12 and fluotrimazole 13 are known drugs containing 1,2,4-triazole rings It is well known that one of the main aims of therapeutic treatment is the control of serious infections, along with the prevention and treatment of some infectious complications due to other therapeutic procedures such as cancer chemotherapy and surgery In some cases such as tuberculosis, cancer, or AIDS, and in cases of organ transplantation, fungal infections became an important complication and a major cause of morbidity and mortality in immune-compromised individuals 14 However, in recent years, much attention has been focused on the treatment of infections caused by multidrug-resistant bacteria and fungi resulting from the widespread use and misuse of the known antimicrobial agents 15 This serious global health problem requires efforts toward the design and synthesis of new antimicrobial agents that are effective against pathogenic microorganisms resistant to currently available drugs ∗ Correspondence: 812 h.bayrak@ktu.edu.tr UYGUN et al./Turk J Chem In our recent studies, we obtained some bitriazolyl compounds containing another heterocyclic group responsible for biological activity such as morpholine, thiomorpholine, or a furan nucleus 16 Moreover, some 4-arylidenamino-1,2,4-triazoles including morpholine or a piperazine nucleus synthesized in our laboratory were found to possess antimicrobial activity 17 In organic syntheses, the compounds incorporating amino and mercapto groups together have been accepted as useful intermediates for further reactions In this connection, the synthesis of some triazolothiadiazoles or triazolothiadiazines having antimicrobial activity has been performed starting from 4-amino-5-mercapto1,2,4-triazoles in our laboratories The amino and mercapto groups, which can also react with electrophiles, are considered as ready-made nucleophilic centers for the synthesis of condensed heterocyclic rings Some 1,2,4-triazole derivatives containing N -methylpiperazine moiety, which were obtained by Mannich reactions, have been reported as antimicrobial agents More recently, some Mannich bases possessing antimicrobial activity have been synthesized in our laboratory between 4-arylidenamino-1,2,4-triazoles and methyl piperazine or morpholine 18 In the present study, as an extension of our previous studies on the synthesis of nitrogenated heterocycles with potential chemotherapeutic activities, synthesis and antimicrobial activity screening studies of some new 1,2,4-triazole derivatives have been performed Results and discussion The synthetic route of the compounds is shown in Figures and Synthesis of 5,5 ′ -methylenebis(4-phenyl4H -1,2,4-triazole-3-thiol) (2) was performed by cyclocondensation of 2,2 ′ -(1,3-dioxopropane-1,3-diyl)bis(N phenylhydrazinecarbothioamide) (1) in basic media In the IR and NMR spectra of compound 2, the absence of any signals pointing toward a carbonyl group supported the ring cyclization In addition, in the H NMR spectrum, the presence of the signal pointing toward –SH groups that was observed at 13.89 ppm as a D O exchangeable singlet integrating protons pointed toward a cyclic form for this compound The reaction of with ethyl bromoacetate in the presence of sodium ethoxide produced diethyl 5,5 ′ -{methylenebis[(4-phenyl-4H 1,2,4-triazole-5,3-diyl)thio]}diacetate (3) In the H and 13 C NMR spectra of compound 3, additional signals originating from ester moiety were recorded at the related chemical shift values, while the signal due to the –SH function disappeared Compound was converted to a hydrazide derivative with hydrazine hydrate in good yield The IR and H NMR spectra of the hydrazide compound (4) exhibited signals belonging to the -NHNH function Several aldehydes were treated with compound to give the corresponding Schiff bases (5a–d) in good yields In the H NMR spectra of compounds 5a–d, additional signals originating from arylidene moiety were observed at the aromatic region between 8.04 and 8.34 ppm The signal originating from the –NHNH group of parent compound disappeared in the H NMR and IR spectra of compounds 5a–d It was reported that arylidene hydrazides may exist as E/Z geometrical isomers about the -N = CH double bond and as cis/trans amide conformers 18−20 A literature survey 19 revealed that dimethyl-d6 sulfoxide solution causes the emergence of the geometrical E isomer in higher percentages, whereas less polar solvents support the Z isomer by an intramolecular hydrogen bond In the present study, the NMR spectral data were taken in dimethyl-d6 sulfoxide solution and no signals pointing to the Z isomer were present However, due to the cis/trans conformers of the E geometrical isomer of compounds 5a–d, certain signals were recorded as sets in the NMR spectra The signals observed as sets at 7.96–8.36 ppm and 10.08–12.05 ppm were attributed to the -N=CH bond and –NH functions of cis- and trans-conformers, respectively The upfield lines of -N= CH and 2NH protons were attributed to the cis-conformer of the amide structure and downfield 813 UYGUN et al./Turk J Chem O O H2NNH S i NHNH2 NH O O S HNNH NHNH NH ii N N N N O N N N N N N O S N S N iii EtO HS OEt SH iv N N N N O O S N N S NHNH2 H2NNH Comp Ar OCH3 5a 5b v HO Cl N N N N O O S N N 5c S Cl NHN CHAr ArCH NNH 5d Br 5a-d Figure Synthetic pathway for the preparation of compounds 1–5 BrCH COOEt; iv : H NNH ; v : ArCHO 814 i : PhNCS; ii: NaOH; iii: Na (k) , EtOH, UYGUN et al./Turk J Chem O O N N NH N N N N N N S NH S 6a NH i O HS N N N N N N N N N N N N S NH O S SH ii 6b iii O O N N iv S Cl O2N S N N N N N N N N N N N N S Cl 6c S NO2 v Cl H2N S N N N N N N Cl S NH2 Figure Reaction and conditions for the synthesis of compounds 6a–c, 7, and i : 2-morpholinoethanamine, HCHO; ii: furan-2-ylmethanamine, HCHO; iii: morpholine, HCHO; iv : Na(k), EtOH, 3,4-dichloronitrobenzene; v : H NNH , Pd-C 815 UYGUN et al./Turk J Chem lines of the protons of the same group to the trans-conformer 21 Furthermore, the -OH group of compound 5b was seen as different singlets due to the existence of cis/trans-amide conformers The treatment of with several primary and secondary amines in the presence of formaldehyde afforded the corresponding Mannich bases (6a–c) With this conversion, additional signals derived from amine moiety and methylene linkage were observed at the related chemical shift values in the H- and 13 C NMR spectra of compounds 6a–c, while no signal pointing toward the –SH function is present Treatment of compound with 3,4-difluoronitrobenzene resulted in the formation of 3,3 ′ -methylenebis{5-[(2-chloro-4-nitrophenyl)thio]-4-phenyl-4H -1,2,4-triazole} (7), and in the NMR spectra there are additional signals of aromatic protons instead of the –SH protons The nitro groups of this compound were then reduced to amine groups using hydrazine hydrate as the hydrogen source in the presence of Pd/C catalyst In the H NMR spectrum of compound 8, the presence of a D O exchangeable signal at 5.78 ppm integrating protons confirmed the reduction The absorption bands due to amino groups were observed at 3337 and 3210 cm −1 in the IR spectrum of All of the newly synthesized compounds gave elemental analysis results consistent with the proposed structures In addition, molecular masses of the newly synthesized compounds were confirmed by the appearance of [M] + , [M + 1] + , [M + 2] + , or [M + Na] + ion peaks at corresponding m/z values in the mass spectra of these compounds All of the newly synthesized compounds were tested for their antimicrobial activities, and only the positive results are presented in the Table According to the results obtained, compound displayed better activity against Staphylococcus aureus and Enterococcus faecalis, which are gram-positive cocci, and Bacillus cereus, which is a gram-positive spore bacillus, than against enteric bacteria Escherichia coli and Yersinia pseudotuberculosis and gram-negative bacillus Pseudomonas aeruginosa This compound (1) displayed complete inactivity towards Mycobacterium smegmatis, an atypical tuberculosis factor, and Candida albicans and Saccharomyces cerevisiae, which are yeast-like fungi According to the obtained results, it can be concluded that the conversion of carbothioamide moiety into the 1,2,4-triazole nucleus in compound and the further substitution reactions leading to the formation of ester (3) and hydrazide (4) derivatives resulted in the inactivity against the test microorganisms Schiff base derivatives (5a–c) of compound exhibited antibacterial activity against the test microorganisms with minimum inhibitory concentration (MIC) values varying between 62.5 and 250 µ g/mL Moreover, marginal antifungal activities were observed for compounds 5a–c against Candida albicans and Saccharomyces cerevisiae Among the Mannich bases (6a–c), compound 6c was found to have the most potent antibacterial activity against the test microorganisms, except for Mycobacterium smegmatis, with MIC values between 62.5 and 125 µg/mL Furthermore, compound 6c displayed slight antifungal activity against Saccharomyces cerevisiae with a MIC value of 250 µ g/mL Motivated by these findings and in continuation of our ongoing efforts, we would like to report here the synthesis and investigation of biological activities of new bitriazolyl derivatives incorporating various heterocyclic rings responsible for biological activity in a single structure Experimental All of the chemicals were purchased from Fluka Chemie AG (Buchs, Switzerland) and used without further purification Melting points of the synthesized compounds were determined in open capillaries on a Bă uchi B-540 melting point apparatus and are uncorrected Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 aluminum sheets The mobile phase was acetone and diethyl ether (1:2), and detection was done using UV light IR spectra were recorded as potassium bromide pellets using a PerkinElmer 816 UYGUN et al./Turk J Chem Table Screening for antimicrobial activity of the compounds 1, 5a–d, and 6a–c Comp no 5a 5b 5c 5d 6a 6b 6c Amp Strep Flu Ec 62.5 62.5 62.5 62.5 250 250 250 62.5 Minimal inhibition concentration values (µg/mL) Yp Pa Sa Ef Bc Ms Ca 62.5 62.5 31.3 31.3 31.3 125 62.5 125 31.3 62.5 62.5 500 125 62.5 125 31.3 62.5 62.5 500 125 62.5 125 31.3 62.5 62.5 250 250 125 125 250 125 500 250 500 500 500 250 250 500 500 500 250 62.5 62.5 125 62.5 62.5 32 > 128 2