Synthesis and antimicrobial evaluation of some novel 1,2,4-triazole and 1,3,4-thiadiazole derivatives

Abstract This study presents the synthesis and spectral analysis of new derivatives of 1,2,4-triazole-3-thione and 1,3,4-thiadiazole. New compounds were prepared by cyclization reaction of acyl thiosemicarbazide derivatives in the presence of alkaline and acidic media. All synthesized compounds were screened for their in vitro antibacterial activity by using the agar dilution technique. Six of the compounds had potential activity against Gram-positive bacteria (minimal inhibitory concentration [MIC] = 15.63–500 μg/mL). Some compounds showed good activity especially against Bacillus subtilis ATCC 6633 (MIC = 15.63–250 μg/mL), Staphylococcus aureus ATCC 25923 (MIC = 31.25–250 μg/mL), and Micrococcus luteus ATCC 10240 (MIC = 125–250 μg/mL). Graphical abstract


Introduction
For the last few decades, there has been a tremendous growth of research in the synthesis of nitrogen and sulfur containing heterocyclic derivatives because of their utility in various applications, such as pharmaceuticals, propellants, explosives, and pyrotechnics.
In continuation of our research program on the synthesis of 1,2,4-triazole and 1,3,4-thiadiazole compounds exhibiting biologic activity, it was thought to be interesting to synthesize new antimicrobial agents, especially when the development of resistance of pathogenic bacteria toward available antibiotics is rapidly becoming a major worldwide problem. The designing of new compounds to deal with resistant bacteria has become one of the most important areas of antibacterial research today. In addition, primary and opportunistic microbial infections continue to increase rapidly because of the increased number of immunocompromised patients.
Keeping in mind the above facts, we designed and synthesized series of some new 1,2,4-triazole-3-thione and 1,3,4-thiadiazole derivatives and evaluated their in vitro antibacterial activity.

Chemistry
The substituted 1,2,4-triazole and 1,3,4-thiadiazole derivatives are generally obtained by the cyclization reaction of thiosemicarbazide derivatives, which is dependent not only on the pH of the medium, but also on the nature of substituents in thiosemicarbazide derivatives Pachuta-Stec, 1995, 1996). The presence of alkaline media usually promotes the reaction of cyclization to obtain 1,2, 4-triazole systems, whereas in acidic media, 1,3,4-thiadiazole derivatives were obtained. 4,5-Diphenyl-4H-1,2,4-triazole-3-thione 1 was a starting material for the synthesis of new compounds, which consist of two 1,2,4-triazole systems or 1,2,4-triazole and 1,3, 4-thiadiazole systems connected with the S-methylene group. Compound 1 was obtained by the cyclization reaction of 1,4-diphenyl thiosemicarbazide in alkaline media. In the next step, compound 1, which can exist in two tautomeric forms, was submitted to the reaction with ethyl bromoacetate in the presence of sodium ethanolate. The reaction let us obtain ethyl 2- [(4,5-diphenyl-4H-1,2,4triazol-3-yl)sulfanyl] acetate (2). The direction of this reaction to form a thio derivative of compound 1 was revealed and confirmed by X-ray crystallography . The mechanism of this reaction as a nucleophilic substitution on the sulfur atom had been studied and investigated earlier (Wujec and Paneth, 2007).
Subsequently, compound 2 was converted to hydrazide 3 in reaction with 100 % hydrazine hydrate. Then, reactions of hydrazide 3 with various isothiocyanates were performed in two ways.
All new thiosemicarbazide derivatives 4a-l were obtained by heating reactants in an oil bath; temperatures were selected experimentally (t = 50-110°C). Thiosemicarbazide derivatives 4a, c, d were products of the reaction of hydrazide 3 with appropriate isothiocyanates in the presence of diethyl ether carried in room temperature.
A new group of compounds, which consist of two 1,2, 4-triazole-3-thione derivatives 5a-i, were acquired in cyclization reaction with 2 % aqueous solution of sodium hydroxide of new acyl thiosemicarbazide derivatives 4a-i.
The mechanism of cyclization of thiosemicarbazide was investigated earlier (Siwek and Paneth, 2007). It was proved that the direction of cyclization is dependent on the nature of substituents and acidic or alkaline media (Siwek et al., 2010).
The structure of all obtained compounds was confirmed by elementary analysis, IR and 1 H NMR spectra. Some of the compounds were also submitted to 13 C NMR and MS spectra analyses. The crystal structure of the representative compound 2 was determined by the single-crystal X-ray analysis. The reactions were performed according to Schemes 1 and 2.
In the IR spectra of the thiosemicarbazide derivatives 4a-l, the following characteristic absorption bands were observed: about 1,700 cm -1 corresponding to the C=O group and in the range of 1,300 cm -1 corresponding to the C=S group. Compounds which consist of two 1,2,4-triazole systems 5a-i, 9, 10 had absorption bands: about 1,300 cm -1 (C=S group), about 1,500 cm -1 (C-N group), in the range of 1,600 cm -1 (C=N group), and about 3,100-3,200 cm -1 (NH group). Then, in the IR spectra of the new derivatives of 1,3,4-thiadiazole 6a-i, the following characteristic absorption bands were observed: in the range of 1,500 cm -1 corresponding to the C-N group and in the range of 1,600 cm -1 corresponding to the C=N group and about 3,200 cm -1 for the NH group. Compounds 7a-i, 11 had a characteristic absorption band at about 1,700 cm -1 for the C=O group. 1 H NMR spectra of the thiosemicarbazide derivatives 4a-l show three proton signals typical for the NH group in the d 8.32-12.87 ppm range, whereas for the new compounds consisting of two 1,2,4-triazole system 5a-i, 9, 10, one proton signal of the NH group was observed in the d 13.62-14.13 ppm range. The 1,3,4-thiadiazole derivatives 6a-i had one typical proton signal of the NH group in the d 9.35-10.47 ppm range. Derivatives of N,N-disubstituted acetamide 7a-i had one proton signal of the CH 3 group in the d 2.06-2.16 ppm range. Compound 11 had one proton signal for the OH group (d 13.68 ppm) and for the pyrrolidine substituent. Similarly, 4,5-disubstituted-2-(pyrrolidin-1-ylmethyl)-1,2,4-triazole-3-thione 12 had one typical proton signal for the NH group (d 14.68 ppm) and for the pyrrolidine substituent.
Compound 2 crystallizes in the monoclinic space group P2 1 / n with one molecule in the asymmetric unit of the crystal. The diffraction study confirmed that the molecule contained the 1,2,4-triazole ring, substituted at C3, N4, and C5 atoms by thioacetate moiety and two phenyl rings, respectively (Fig. 1). The chain of atoms from S1 to ethyl C4 is almost planar (rmsd = 0.006 Å ); a higher twist (4.56°) is observed around the C4-O1 bond in the solid state. The best plane of the atoms of thioacetate unit intersects that of the 1,2,4-triazole ring at the angle of 81.4(1)°. The carbonyl C2=O2 group in 2 is cis oriented with respect to the thioether S1 atom. What is more, it seems to be preferred in thioacetate derivatives in the solid state (CSD, V.5.33, Allen, 2002). The geometric parameters of the ester group are within normal ranges (International Tables for  Crystallography, 1995). Likewise, the S1-C3 and S1-C1 distances, being of 1.738(2) and 1.789(3) Å , are in agreement with the single thioether C-S bonds. The most characteristic feature of the crystal of 2 is the presence of centrosymmetric molecular dimers. The ''head-to-head'' oriented molecules within the dimer form short S1ÁÁÁO2 i [3.268(3) Å ; (i) 1x, -y, -z] contacts which might be attractive in their nature (Ramasubbu and Parthasarathy, 1989).

Microbiology
On the basis of the preliminary results obtained by the agar dilution method, it was shown that some of the newly Scheme 1 Synthesis of new derivatives of thiosemicabrazide, 1,2,4-triazole-3-thione and 1,3,4-thiadiazole synthesized compounds had the potential activity against reference strains of Gram-positive bacteria. None of the compounds had inhibitory effect on the Gram-negative bacteria growth.
According to The somewhat lower activity against reference strains of Gram-positive bacteria was shown by compound 5c (MIC values from 250 to 1,000 lg/mL). According to our results, MICs of cefuroxime, which has been extensively used to treat bacterial infections, were 0.24-1.95 lg/mL for Staphylococcus species and 0.49-62.5 lg/mL for the other Gram-positive bacteria.
With our research, it has been established that the introduction of the benzoyl group in thiosemicarbazide and the benzyl group in 1,3,4-thiadiazole derivative yielded active compounds endowed with a wide spectrum of antimicrobial activities.
The compounds 4l and 6h with potential activity against the reference strains of Gram-positive bacteria may be regarded as precursor compounds for searching for new derivatives showing antimicrobial activity against pathogenic (e.g. S. aureus) or opportunistic (e.g. S. epidermidis, M. luteus, B. subtilis, or B. cereus) bacteria.

Chemistry
Melting points were determined in Fisher-Johns blocks (Pittsburgh, US) and presented without any corrections. The IR spectra (m, cm -1 ) were recorded in KBr tablets using a Specord IR-75 spectrophotometer (Germany). The NMR spectra were recorded on a Bruker Avance 300 apparatus (Bruker BioSpin GmbH, Rheinstetten/Karlsruhe, Germany) in dimethyl sulfoxide (DMSO)-d 6 with TMS as the internal standard, and chemical shifts are given in ppm (d-scale). The MS spectra were recorded on a Thermo- Finnigan Trace DSQ GC MS apparatus (Waltham, Massachusetts, US). Chemicals were purchased from Merck Co., or Lancaster and used without further purification. The purity of the obtained compounds was checked by TLC on aluminum oxide 60 F 254 plates (Merck Co., Whitehouse Station, New Jersey, US), in a CHCl 3 /C 2 H 5 OH (10:1, v/v) solvent system with UV visualization (k = 254 nm).
Elemental analysis of the obtained compounds was performed for C, H, N, S. The maximum percentage differences between calculated and found values for each element were within the error and amounted to ±0.4 %.
Crystal data for 2 C 18 H 17 N 3 O 2 S, colorless prism, 0.45 9 0.29 9 0.14 mm 3 , monoclinic, P2 1 /n, a = 11.692 (1)  Single-crystal diffraction data were measured at room temperature on an Oxford Diffraction Xcalibur diffractometer with the graphite-monochromated Mo Ka radiation (k = 0.71073). The programs CrysAlis CCD and CrysAlis Red (Oxford Diffraction, Xcalibur CCD System, 2006) were used for data collection, cell refinement, and data reduction. The intensity data were corrected for Lorentz and polarization effects. The structure was solved by direct methods using SHELXS-97 and refined by the full-matrix least-squares on F 2 using the SHELXL-97 (Sheldrick, 2008). All non-hydrogen atoms were refined with anisotropic displacement parameters. All H-atoms were positioned geometrically and allowed to ride on their parent atoms with U iso (H) = 1.2 U eq (C).   (2) Method A 0.23 g (10 mmol) of sodium was added to 5 mL of anhydrous ethanol. The solution was placed in a three-necked flask equipped with reflux condenser and closed with a tube of CaCl 2 and mercury stirred. The content was mixed till the sodium dissolved completely and then 2.53 g (10 mmol) of 4,5-diphenyl-4H-1,2,4-triazole-3-thione (1) was added. Then, 1.22 mL ethyl bromoacetate was added drop by drop. The content of the flask was mixed for 4 h and left at room temperature for 12 h. Then, 10 mL of anhydrous ethanol was added and heated for 1 h. The mixture was filtered of inorganic compounds. After cooling, the precipitate was filtered and crystallized from ethanol.

Method B
2.53 g (10 mmol) of 4,5-diphenyl-4H-1,2,4-triazole-3thione (1) was dissolved in 10 mL of N,N-dimethylformamide. Then, 1 g of potassium carbonate and 1.22 mL of ethyl bromoacetate were added to the solution. The content of the flask was refluxed for 2 h. The mixture was filtered of inorganic compounds. Then, the distilled water was added and the precipitated compound was filtered, dried, and crystallized from ethanol.

Derivatives of thiosemicarbazide (4a-l)
General method (for compounds 4a-l) A mixture of 3.25 g (10 mmol) of hydrazide (3) and 10 mmol appropriate isothiocyanate was heated in an oil bath at 50-110°C for 8-20 h. The product was washed with diethyl ether to remove unreacted isothiocyanate. Then it was filtered, dried, and crystallized from ethanol 4a-c, d, g-l, butanol 4e, or methanol 4f.

Method B (for compounds 6a, d)
20 mL of 10 % ethanolic solution of hydrochloric acid was added to thiosemicarbazide 4a, d and the reaction mixture was heated under reflux for 1 h. Subsequently, the solution was left at room temperature for 24 h. The precipitate formed was separated by filtration, dried, and crystallized from ethanol.

5-Aminoallyl
A mixture of 10 mmol of appropriate 2,5-disubstituted-1, 3,4-thiadiazole 6a-i in 5 mL of acetic anhydride was heated under reflux for 2 h. Distilled water was added to the reaction mixture and it was allowed to cool. The resulting precipitate was filtered and washed with distilled water. The residue was purified by recrystallization from ethanol.

Materials and methods
All synthesized compounds were preliminarily tested for their in vitro antibacterial activity against Gram-positive and -negative reference bacterial strains and next by the broth microdilution method against the selected bacterial strains.
Panel reference strains of aerobic bacteria from the American Type Culture Collection, including six Grampositive bacteria, S. aureus ATCC 25923, S. aureus ATCC 6538, S. epidermidis ATCC 12228, B. subtilis ATCC 6633, B. cereus ATCC 10876, M. luteus ATCC 10240, and four Gram-negative bacteria, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453, Pseudomonas aeruginosa ATCC 9027, were used. Microbial suspensions with an optical density of 0.5 McFarland standard 150 9 10 6 CFU/mL (CFUs-colony forming units) were prepared in sterile 0.85 % NaCl. All stock solutions of the tested compounds were prepared in DMSO. The medium with DMSO at the final concentration and without the tested compounds served as the controlno microbial growth inhibition was observed.
Preliminary antimicrobial potency in vitro of the tested compounds was screened using the agar dilution method on the basis of the bacterial growth inhibition on the Mueller-Hinton agar containing the compounds at a concentration of 1,000 lg/mL. The plates were poured on the day of testing. 10 lL of each bacterial suspension was put onto the prepared solid media. The plates were incubated at 37°C for 18 h (Bourgeois et al., 2007).
The antibacterial activity in vitro of the potentially active compounds was determined by the broth microdilution method on the basis of MIC, usually defined as the lowest concentration of the compound at which there was no visible growth of microorganisms (White et al., 2002). Determination of the MIC value was achieved by the broth microdilution method according to a CLSI (Clinical and Laboratory Standards Institute) recommendation with some modifications (2008). The 96-well microplates were used; 198 lL of Mueller-Hinton broth with a series of twofold dilutions of the tested compound in the range of the final concentrations from 0.24 to 1,000 lg/mL was inoculated with 2 lL of microbial suspension (total volume per each well-200 lL). After incubation (at 35°C for 18 h), spectrophotometric measurements of optical density (OD 600 ) of the bacterial cultures with the tested compounds were performed in order to determine MIC. OD 600 of bacterial cultures in the medium without the tested compounds was used as a control. The blank control wells with twofold dilution of each of the tested compounds added to the Mueller-Hinton broth without bacterial suspension were incubated under the same conditions. Cefuroxime, belonging to the second generation of cephalosporins, was used as a control antimicrobial agent.