Synthesis of new derivatives of hydrazinecarbothioamides and 1,2,4-triazoles, and an evaluation of their antimicrobial activities J. Serb. Chem. Soc. 80 (12) 1461–1470 (2015) UDC 547.298.4’264–304.3+547.78’367+ JSCS–4811 542.913:615.28–188 Original scientific paper 1461 Synthesis of new derivatives of hydrazinecarbothioamides and 1,2,4-triazoles, and an evaluation of their antimicrobial activities LAURA I. SOCEA1*, GABRIEL SARAMET2, CONSTANTIN DRAGHICI3, BOGDAN SOCEA4, VLAD D. CONSTANTIN4 and MANUELA A. RADU-POPESCU5 1Organic Chemistry Department, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, 6 Traian Vuia Street, 020956, Bucharest, Romania, 2Pharma- ceutical Technology Department, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, 6 Traian Vuia, 020956 Bucharest, Romania, 3“C. D. Nenitzescu” Institute of Organic Chemistry, Romanian Academy, 202B Splaiul Independentei, 060023 Bucharest, Romania, 4Faculty of General Medicine, “Carol Davila” University of Medicine and Pharmacy, St. Pantelimon Emergency Hospital, 340–342, Soseaua Pantelimon Street, 021623, Bucharest, Romania and 5General and Pharmaceutical Microbiology Department, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, 6 Traian Vuia Street, 020956, Bucharest, Romania (Received 27 February, revised 15 May, accepted 18 May 2015) Abstract: A new series of hydrazinecarbothioamides 6–9 bearing a 5H-diben- zo[a,d][7]annulene moiety were synthesized. Cyclization of 6–9 in NaOH sol- ution produced the corresponding 4H-1,2,4-triazole-3-thiols 10–13, which proved to be axial isomers. The thioethers 14–17 were prepared by alkylation of 10–13 with methyl iodide. All new compounds were characterized by ele- mental analysis, and IR, UV, 1H-NMR and 13C-NMR spectroscopy. An eval- uation for antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Salmonella enterica subsp. ent- erica serovar Typhimurium, Shigella flexneri and Candida albicans was performed. Keywords: acylhydrazinecarbothioamide; 1,2,4-triazole-3-thiol; dibenzo[a,d]- [7]annulene; antimicrobial activity. INTRODUCTION Bacterial infection remains a serious threat to human lives because of their capacity to develop resistance to existing antibiotics, which is an increasing pub- lic health problem. For this reason, obtaining new types of antibacterial agents is a very important task. * Corresponding author. E-mail: laurasocea@gmail.com doi: 10.2298/JSC150227039S _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 1462 SOCEA et al. The tricyclic framework of 5H-dibenzo[a,d][7]annulene constitutes an int- egral part of the structure of molecules that are known to be effective for the treatment of depressive disorders.1,2 Analogs of 5H-dibenzo[a,d][7]annulene, such as protriptyline and demexiptiline, are well known tricyclic antidepressants, which are used in the treatment of migraines, tension headaches, anxiety, psy- chosis, aggression and violent behavior. The anti-allergic drug cyproheptadine (Cyp) is known to have inhibitory activity for L-type calcium channels in addi- tion to histamine and serotonin receptors.3 Recently, studies that were trying to detect other possible pharmacological actions of already known tricyclic antidepressants have received increasing inter- est.4–13 Dibenzo[a,d][7]annulene moieties are incorporated in biologically active compounds that exhibit muscarinic receptor antagonist properties and are useful in the treatment of Parkinson’s disease, tardive dyskinesia and motion sickness.4 Dibenzo[a,d][7]annulene derivatives exhibit antidiabetic,5 antiparasitic,6 metalloprotease inhibitory7 and antimicrobial activity.8–13 Munoz-Bellido et al. realized an extensive study that demonstrated the intense antibacterial activities presented by some antidepressant from the group of serotonin recapture inhi- bitors, such as clomipramine and sertraline.11 Similarly some psychiatric agents, such as protriptyline or cyclobenzaprine, are associated with some chemotherapy agents (sulfathiazole) that enhance the antibacterial activity of the latter and red- uce the MIC up to 50 %.11 1,2,4-Triazole derivatives are known to show biological properties including antimicrobial,14–19 anticancer,20 anti-inflammatory,21,22 anticonvulsant,23 anti- viral,24,25 antitubercular,25 hypolipidemic,26 antioxidant activities27,28 and others. Several compounds containing 1,2,4-triazole rings are used in therapy. For example, fluconazole, terconazole and itraconazole are used as antimicrobial drugs, while vorozole, letrozole and anastrozole are non-steroidal drugs used for the treatment of cancer.29 Other examples are ribavirin (antiviral agent), riza- triptan (antimigraine agent) and alprazolam (anxiolytic agent),30 beside others. Furthermore, a number of substituted hydrazinecarbothioamide were found to exhibit antifungal,12,31–36 tuberculostatic,36 cytostatic,37 anticonvulsant,38 antiviral39 and antioxidant activities.28,40 Considering the above data, in continuation of ongoing research on biologic- ally active compounds, the synthesis of new hydrazinecarbothioamides and 1,2,4- -triazoles bearing the 5H-dibenzo[a,d][7]annulene moiety and their antimicrobial activities were considered.41,42 EXPERIMENTAL Chemistry All reactants and solvents of the highest purity were obtained commercially and used without further purification. Melting points were determined on a Boetius apparatus and are _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ NEW HYDRAZINECARBOTHIOAMIDES AND 1,2,4-TRIAZOLES 1463 uncorrected. The UV–Vis spectra were recorded on a SPECORD 40 Analytik Jena spectro- meter, in methanol (2.5×10-5 M) in the wavelength range 200–600 nm. The IR spectra were recorded in KBr pellets using a Vertex 70 Bruker spectrometer. Elemental analyses were performed on an ECS-40-10-Costeh micro-dosimeter (and the values were within ±0.4 % of the theoretical ones). The NMR spectra were recorded on a Varian Gemini 300 BB instrument operating at 300 MHz for 1H- and 75 MHz for 13C-NMR, using DMSO-d6 as solvent for the hydrazinecarbothioamides and CDCl3 for the 1,2,4-triazole compounds. Chemical shifts (δ in ppm) were assigned according to the internal standard signal of tetramethylsilane in DMSO-d6 (δ = 0 ppm). Coupling constants, J, are expressed in Hz. Analytical and spectral data of the synthesized compounds are given in the Supple- mentary material to this paper. General procedure for the preparation of N-substituted 2-(5H-dibenzo[a,d][7]annulen-5-yla- cetyl)-hydrazinecarbothioamides (6–9) A mixture of 2-(5H-dibenzo[a,d][7]annulen-5-yl)acetohydrazide (1, 4 mmol) and the required isothiocyanate 2–5 (4 mmol) in absolute ethanol (30–50 mL) was refluxed for 6–12 h. On cooling the reaction mixture to room temperature, a precipitate appeared. This was filtered off and recrystallized from ethanol to obtain the desired compound. General procedure for the preparation of 4-substituted 5-(5H-dibenzo[a,d][7]annulen-5-yl- methyl)-4H-1,2,4-triazole-3-thiols (10–13) A solution of the required hydrazinecarbothioamide (6–9, 1 mmol) in 8 mL of 8 % NaOH solution was refluxed for 3–9 h and then filtered. After cooling, the filtrate was neutralized with acetic acid. The obtained white precipitate was filtered and recrystallized from CHCl3:petroleum ether (1:2, V:V, boiling range: 60–80 °C). General procedure for the preparation of 4-substituted 3-(5H-dibenzo[a,d][7]annulen-5- ylmethyl)-5-(methylsulfanyl)-4H-1,2,4-triazoles (14–17) To a solution of sodium ethoxide (1mmol of sodium in 10 mL of absolute ethanol) was added the required triazole 10–13 (1 mmol). The reaction mixture was stirred at room tem- perature until a solution was obtained. To this solution, methyl iodide (1 mmol) was added and stirring continued for 10 h. The reaction mixture was poured into ice water and the precipitate was filtered off, washed with water and recrystallized from ethanol. Antimicrobial activity The antibacterial and antifungal activities of the compounds were investigated by the broth microdilution method, in 96 flat-bottomed wells microplates (Nunc, Denmark). Dimethyl sulfoxide was used as the solvent for the preparation of stock solutions of the compounds, to obtain a concentration of 2048 μg mL-1. The antimicrobial actions of the newly-synthesized compounds were tested against 6 reference bacterial strains, i.e., Staphylo- coccus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, Salmonella enterica subsp. enterica serovar Typhimu- rium ATCC 14028, Shigella flexneri ATCC 12022, and one reference yeast strain, i.e., Can- dida albicans ATCC 90028. Gentamicin was used as a positive control for S. aureus, P. aeruginosa and E. coli, and fluconazole for C. albicans. Bacterial susceptibility testing was performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) M100-S16 and European Committee on Antimicrobial Susceptibility Testing (EUCAST).43,44 All 96 wells of the microplate were filled in with 100 μL of Müller–Hinton broth (cation- -adjusted) when testing compounds against bacteria and Sabouraud broth when testing against _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 1464 SOCEA et al. the yeast. Series of two-fold dilutions of the newly-obtained compounds were performed in Műller–Hinton or Sabouraud broth. In the case of the reference bacterial strains, the inoculum was prepared by suspending 5 distinct colonies from a 24 h culture obtained on Columbia Blood Agar (BioMérieux, France), in a tube with Brain Heart Infusion broth (BHI broth). After vortex mixing and adjusting the density to the turbidity of the 0.5 McFarland standard, the bacterial suspension was diluted 1:100 in BHI broth, in order to obtain the working inoculum. Afterwards, each well of the microdilution plates containing 100 μL of Müller– –Hinton broth with compound was inoculated within 15 min with 100 μL of the bacterial inoculum, including the growth control wells, but not the sterility control wells that were filled with 200 μL of compound-free Müller–Hinton broth. In the case of the reference yeast strains, the inoculum was prepared by suspending 5 distinct colonies from a 24 h culture obtained on Sabouraud dextrose agar, in a tube with 5 mL of sterile distilled water. After vortex mixing and adjusting the density to the turbidity of the 0.5 McFarland standard, the fungal suspension was diluted in sterile distilled water in order to obtain a working inoculum. Each well of the microdilution plates containing 100 μL of Sabouraud broth with compound was inoculated with 100 μl yeast inoculum within 15 min, including the growth control wells, but not the sterility control wells, which were filled only with 200 μL compound-free Sabouraud broth. After performing the inoculum controls from the growth control wells, the microplates were incubated at 37 °C for 24 h.48-50 The lowest concentration of each compound able to inhibit visible microbial growth was considered the minimum inhibitory concentration (MIC) value. RESULTS AND DISCUSSION Chemistry The reaction sequences employed for the syntheses of the title compounds are shown in Scheme 1. In the present work, N-aryl-2-(5H-dibenzo[a,d][7]- annulen-5-ylacetyl)hydrazinecarbothioamides 6–9 were synthesized by nucleo- philic addition of 2-(5H-dibenzo[a,d][7]annulen-5-yl)acetohydrazide (1) to the aryl isothiocyanates 2–5, in absolute ethanol under reflux. 2-(5H-Dibenzo[a,d]- [7]annulen-5-yl)acetohydrazide (1) was prepared starting from dibenzosuber- enone, according to a literature method.42,43,46 Synthesis of the new 4-alkyl/aryl-5-(5H-dibenzo[a,d][7]annulen-5-yl- methyl)-4H-1,2,4-triazole-3-thiols 10–13 (that exist in equilibrium with their thione tautomer) consisted in intramolecular cyclization of acylhydrazine- carbothioamides 6–9 in 8 % sodium hydroxide solution under reflux.41,46,47 The treatment of 1,2,4-triazoles 10–13 with methyl iodide in basic media yielded the 4-substituted 3-(5H-dibenzo[a,d][7]annulen-5-ylmethyl)-5- (methylsulfanyl)-4H-1,2,4-triazoles 14–17 and not the N-methylated derivatives. Analytical and spectral data of the newly synthesized compounds All the synthesized compounds were analyzed by IR, UV–Vis, 1H-NMR and 13C-NMR spectroscopy. The analytical and spectral data of the new compounds are given in the Supplementary material to this paper. _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ NEW HYDRAZINECARBOTHIOAMIDES AND 1,2,4-TRIAZOLES 1465 Infrared spectra of new hydrazinecarbothioamides 6–9 showed a new band at 1249–1258 cm–1 due to the stretching vibration of the C=S groups. This fact confirmed the addition of the 2-(5H-dibenzo[a,d][7]annulen-5-yl)acetohydra- zides to different isothiocyanates. The C=O and N–H stretching bands were pre- sent at 1673–1699 and 3141–3359 cm–1, respectively. The hydrazinecarbothioamides 6–9 were present as two conformational iso- mers, 5′-axial and 5′-equatorial in about 3:1 ratio, interconvertible by inversion of the middle ring, which was confirmed by their 1H-NMR spectra.42 In 6–9-axial isomers, the H-5′(eq) is deshielded, manifested as a triplet at 4.62–4.65 ppm (J in range 7.0–7.3 Hz), whereas the CH2-12′ protons are shielded by the double bond, showing a doublet at 2.57–2.60 ppm (Scheme S-1 of the Supplementary material to this paper). Double bonds shield H-5′ axial, while aromatic rings deshield H-5′ equatorial, because of the current cycle. The H-5′(eq) appeared at δ = 4.62–4.65 ppm (triplet) and H-5′(ax) appeared at δ 3.74–3.76 ppm (triplet).42 The signals of NH protons appeared as singlets between 9.53–10.12 ppm and the double bond protons H-10′ and H-11′ appeared as a singlet at 7.02–7.03 ppm. The 13C-NMR spectrum of compounds 6–9 showed a narrow δ domain (123–140 ppm) with C-10′ and C-11′ easily recognizable at δ 130–131 ppm, cor- responding to the dibenzo[a,d][7]annulene moiety. C=S carbon atom could be responsible for the appearance of a signal at δ ≈ 181 ppm. Cyclization of 6–9 to 10–13 was proved by the IR spectra that showed the disappearance of the νC=O band. It appears that in KBr pellets, the 1,2,4-triazole- -3-thiols 10–13 exist in their thionic tautomeric form.41 Scheme 1. Synthetic route to the title compounds. _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 1466 SOCEA et al. The presence of a single conformational isomer (the axial one) of the tri- azoles 10–13 was confirmed by their 1H-NMR spectra. Cyclization of 6–9 to 10– –13 and the subsequent reactions produced the loss of the minor equatorial isomer, probably due to an increased solubility in acidic water. H-5′(eq) appears at δ 4.35–4.45 ppm (triplet, J = 7.9 Hz) and the CH2-12′ protons manifest as doublets at 2.67–2.93 ppm in 10–13. The NH signals of 6–9 totally disappeared, and were replaced by singlets at δ 11.44–11.90 ppm, attributable to the SH pro- ton. Thus, in solution, the above equilibrium was shifted towards the thiolic form. Conversion of hydrazinecarbothioamides 6–9 to the triazoles 10–13 was also confirmed by the 13C-NMR spectra. A new quaternary carbon signal (for C-3) appeared at δ 166.57–167.97 ppm (Scheme S-2 of the Supplementary material) simultaneously with the disappearance of the C=S signal of 6–9 (δ = 181 ppm). Furthermore, a new signal for C-5 of 10–13 appeared at δ ≈ 155.5 ppm, instead of the C=O signal from 6–9 at δ 169–170 ppm. A new band in 2929–2983 cm−1 region, due to presence of methyl group (νCH3) in the IR spectra confirmed the structures of compounds 14–17, obtained by alkylation of the triazoles 10–13 with methyl iodide. Proof of S-alkylation that led to the formation of compounds 14–17 was given by the disappearance of the C=S stretching band in the IR spectra. The presence of new signals at 14.8 ppm corresponding to CH3 group in the 13C-NMR spectra of compounds 14–17 was the most significant proof of alkyl- ation of triazoles 10–13 with methyl iodide. Heterocyclic carbons C-3 and C-5 from these methylated compounds resonated at 154.82–155.11 ppm (more shielded than the C-3 heterocyclic carbon from the 1,2,4-triazoles 10–13) and δ 151.28– –151.54 ppm, respectively. 1H-NMR spectra of the 3-(methylsulfonyl)-1,2,4-triazoles indicated the presence of a single conformational isomer, the axial one, except for triazole 15, which exists as two isomers, 5′-axial and 5′-equatorial in about 1:1 ratio. Antimicrobial activity The antimicrobial activities of all products were investigated in vitro against S. aureus, P. aeruginosa, E. coli, B. subtilis, S. enterica subsp. enterica serovar Typhimurium, S. flexneri and C. albicans by the dilution method. The MIC values were determined using the dilution method with dimethyl sulfoxide as solvent. Dimethyl sulfoxide showed no antimicrobial activity against the tested strains. The MIC values (μg mL–1) for the new compounds against the strains are presented in Table I. The investigation of the antimicrobial activity of the compounds was per- formed in duplicate. As control, S. aureus, E. coli and P. aeruginosa were tested against gentamicin, and C. albicans against fluconazole by the broth micro- _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ NEW HYDRAZINECARBOTHIOAMIDES AND 1,2,4-TRIAZOLES 1467 dilution method.44,45,49–51 The MIC value of gentamicin was 2 μg mL–1 for all tested strains and the MIC value of fluconazole was 2 μg mL–1 for the reference strain. TABLE I. In vitro antimicrobial activity of compounds 6–17 as MIC values (μg mL-1) Compd. Bacterial strains Yeast S. aureus P. aeruginosa E. coli B. subtilis S. enterica subsp. enterica serovar Typhimurium S. flexneri C. albicans 6 256 16 512 >1024 256 1024 >1024 7 512 256 >1024 >1024 256 256 >1024 8 >1024 1024 512 >1024 1024 512 >1024 9 512 256 512 >1024 512 512 >1024 10 512 >1024 64 128 64 512 >1024 11 256 128 512 128 >1024 1024 >1024 12 256 512 512 128 >1024 512 >1024 13 512 64 >1024 >1024 512 512 >1024 14 256 128 512 128 >1024 512 128 15 256 128 512 128 512 512 128 16 256 256 512 256 128 512 128 17 512 512 512 256 256 512 256 Gentamicin 2 2 2 – – – – Fluconazole – – – – – – 2 The antimicrobial screening data revealed that all the newly synthesized compounds exhibited weaker antimicrobial activities compared to those of the control drugs. For the reference bacterial strains, the MIC values of the compounds ranged between: 16–1024 μg mL–1 for the hydrazinecarbothoamides 6–9, 64–1024 μg mL–1 for the 1,2,4-triazole-3-thiols 10–13 and 128–512 μg mL–1 for the methyl- sulfamyl-1,2,4-triazoles 14–17. Hydrazinecarbothioamide 6 with a chlorine atom presented the strongest action against P. aeruginosa (MIC 16 μg mL–1). 1,2,4-Triazole-3-thiol 13 with a 4-(2-phenylethyl) fragment presented the strongest action against P. aeruginosa (MIC 64 μg mL–1). Comparing the results of this study, it was observed: a) compounds con- taining 4-chlorophenyl group had moderate antibacterial activity, hydrazinecar- bothioamides against S. aureus, S. enterica subsp. enterica serovar Typhimurium and P. aeruginosa, 1,2,4-triazole-3-thiol against E. coli and S. enterica subsp. enterica serovar Typhimurium, and methylsulfanyl-1,2,4-triazole against P. aeruginosa; b) hydrazinecarbothioamides containing 4-chlorophenyl or 4-bro- mophenyl had better action compared to derivatives containing 4-iodophenyl or 4-(2-phenylethyl); c) the presence of methylsulfanyl-1,2,4-triazole ring in the structures 14–17 were favorable for the activity against the bacterial strains; d) _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 1468 SOCEA et al. hydrazinecarbothioamides 6–9 and 1,2,4-triazole-3-thiols 10–13 were almost inactive against C. albicans but methylsulfanyl-1,2,4- triazole showed moderate activity against fungus strain. CONCLUSIONS In this paper, the synthesis and characterization of four new acyl hydra- zinecarbothioamides, four 4H-1,2,4-triazole-3-thiol derivatives and four methyl- sulfanyl-1,2,4-triazoles containing the 5H-dibenzo[a,d]annulene moiety were presented. The structures of new compounds were confirmed by spectral data (IR, UV, 1H-NMR and 13C-NMR) and elemental analysis. All the compounds were investigated for their antimicrobial activity against S. aureus, P. aerugi- nosa, E. coli, B. subtilis, S. enterica subsp. enterica serovar Typhimurium, S. flexneri and C. albicans. The antibacterial screening data are given for all the tested compounds. The data indicated weak antibacterial activity, except for compound 6 (which pre- sented good action against P. aeruginosa), 10 (which presented moderate action on S. enterica subsp. enterica serovar Typhimurium and E. coli) and 13 (which presented a moderate action on P. aeruginosa). Based on the MIC values pre- sented by the tested compounds, it could be concluded that, in general, the deri- vatives containing a chlorine or bromine atom had better antibacterial activity against the tested strains. SUPPLEMENTARY MATERIAL Physical, analytical and spectral data for the synthesized compounds are available elec- tronically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgement. This work was supported by the University of Medicine and Pharm- acy “Carol Davila” Bucharest, Project No. 28331/04.11.2013. И З В О Д СИНТЕЗА НОВИХ ДЕРИВАТА ХИДРАЗИНКАРБОТИОАМИДА И 1,2,4-ТРИАЗОЛА И ЊИХОВА АНТИМИКРОБНА АКТИВНОСТ LAURA I. SOCEA1, GABRIEL SARAMET2, CONSTANTIN DRAGHICI3, BOGDAN SOCEA4, VLAD D. CONSTANTIN4 и MANUELA A. RADU-POPESCU5 1 Organic Chemistry Department, Faculty of Pharmacy, „Carol Davila“ University of Medicine and Pharmacy, 6 Traian Vuia Street, 020956, Bucharest, Romania, 2 Pharmaceutical Technology Department, Faculty of Pharmacy, „Carol Davila“ University of Medicine and Pharmacy, 6 Traian Vuia, 020956 Bucharest, Romania, 3 “C.D. Nenitzescu” Institute of Organic Chemistry, Romanian Academy, 202B Splaiul Independentei, 060023 Bucharest, Romania, 4 Faculty of General Medicine, „Carol Davila“ University of Medicine and Pharmacy, St. Pantelimon Emergency Hospital, 340–342, Soseaua Pantelimon Street, 021623, Bucharest, Romania и 5 General and Pharmaceutical Microbiology Department, Faculty of Pharmacy, „Carol Davila“ University of Medicine and Pharmacy, 6 Traian Vuia Street, 020956, Bucharest, Romania Синтетисана је серија деривата хидразинкарботиоамида 6–9 који садрже 5H-дибен- зо[a,d][7]ануленски део. Циклизацијa деривата 6–9 у раствору NaOH даје одговарајуће аксијалне изомере 4H-1,2,4-триазол-3-тиола 10–13. Тиоетри 14–17 су добијени алкило- вањем деривата 10–13 јодметаном. Сва нова једињења окарактерисана су елементалном _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ NEW HYDRAZINECARBOTHIOAMIDES AND 1,2,4-TRIAZOLES 1469 анализом, IR, UV, 1H-NMR и 13C-NMR спектроскопијом. Извршено је испитивање анти- микробне активности према Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028, Shigella flexneri ATCC 12022 и Candida albicans ATCC 90028. (Примљено 27. фебруара, ревидирано 15. маја, прихваћено 18. маја 2015) REFERENCES 1. P. E. Holtzheimer III, C. B. Nemeroff, NeuroRx. 3 (2006) 42 2. P. K. Gillman, Br. J. Pharmacol. 151 (2007) 737 3. K. G. Wu, T. H. Li, T. Y. Wang, C. L. Hsu, C. J. Chen, Int. J. Immunopathol. Pharmacol. 25 (2012) 231 4. M. Hibert, L. Van Hijfte, M. Richards, P. Mose (Merrell Pharmaceuticals, Inc.) US5508280 A, 1996 5. J. Inoue, Y. S. Cui, L. Rodriguez, Z. Chen, P. F. Kador, Eur. J. Med. Chem. 34 (1999) 399 6. A. T. Evans, S. L. Croft, Biochem. 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