Synthesis and Antimicrobial Activity of Some New 1,3,4-thiadiazole and 1,2,4-triazole Compounds Having a D,l-methionine Moiety

New 1,3,4-thiadiazole, 5a-e, and 1,2,4-triazolecompounds 6a-c, containing a D,L-methionine moiety were synthesized by intramolecular cyclization of 1,4-disubstituted thiosemicarbazides 4a-e in acid and alkaline media, respectively. The potential antimicrobial effects of the synthesized compounds were investigated using the Staphylococcus aureus ATCC 25923, Bacillus antracis ATCC 8705, Bacillus cereus ATCC 10987, Sarcina lutea ATCC 9341 and Escherichia coli ATCC 25922 strains. The newly synthesized compounds exhibited promising activities against Bacillus antracis and Bacillus cereus.

Taking these observations into account in the present study, some new 1,3,4-thiadiazoles and 1,2,4-triazoles having a D,L-methionine moiety have been synthesized and their structures were characterized by 1 H-NMR, IR spectroscopy and elemental analysis.The potential antimicrobial activity and degree of toxicity of the synthesized compounds were also investigated.

Results and Discussion
The synthesis of new 1,3,4-thiadiazole and 1,2,4-triazole compounds was performed in several steps.In the first step, 2-(3-nitrophenyl)-4-(2-methylthioethyl)-∆ 2 -5-oxazolinone (2) was obtained by cyclization of N-(3-nitrobenzoyl)-D,L-methionine (1) in the presence of acetic anhydride.This intermediate, through reaction with 98% hydrazine hydrate solution in a dioxane medium, gave the hydrazide of N-(3-nitrobenzoyl)-D,L-methionine (3), which, in the next step was reacted with different isothiocyanates whereupon new 1,4-disubstituted thiosemicarbazides 4a-e were obtained (Scheme 1).Scheme 1. Synthesis of the intermediates 4a-e.In the last step new 1,3,4-thiadiazole, 5a-e, and 1,2,4-triazole compounds 6a-c were obtained by intramolecular cyclization of the thiosemicarbazides 4a-e in acid and alkaline media, respectively (Scheme 2).The structures of all the synthesized compounds were confirmed by IR, 1 H-NMR spectral measurements and elemental analysis.The IR spectra of compounds 4a-e showed intense absorption bands within the 3181-3367 cm -1 range that were attributed to NH and NH 2 function vibrations.The absorption band of the -C=O function appears in the 1641-1644 cm -1 region and the bands which appear at 1171-1173 cm -1 were attributed to the −C=S function.In the 1 H-NMR spectra, the proton signals due to the NH group were recorded between 9.07-10.80ppm and the aromatic protons signals appear at 7.19-8.74ppm.
In the IR spectra of compounds 5a-e the absorption band of the -NH-CO function appears at 2916-3302 cm -1 and the absorption band of the C=N group was identified at 1431-1433 cm -1 .In the 1 H-NMR spectra the proton signal due to the -NH-CO function was observed as a singlet between 9.06-9.07ppm.
The IR spectra of compounds 6a-c showed an intense absorption band between 2589-2592 cm -1 that was attributed to the −SH function and in the 1 H-NMR spectra, the proton signal due to the SH group appeared as a singlet at 12.60-12.80ppm.
Moreover, the elemental analysis results were all in a good agreement with the structures proposed for compounds 2, 3, 4a-e, 5a-e and 6a-c.

Biological activity
The potential antimicrobial activity of compounds 4a-e, 5a-e and 6a-c towards five standard bacterial strains was investigated.From the date presented in Table 1 it may be seen that thiosemicarbazides 4a-e exhibit relatively good activity against Staphylococcus aureus and Escherichia coli, but are only slightly active against Bacillus antracis, Bacillus cereus and Sarcina lutea strains.Upon cyclization of thiosemicarbazides to the corresponding thiadiazoles 5a-e and triazoles 6a-c the activity towards Staphylococcus aureus and Escherichia coli decreases, while the activity against Bacillus antracis and Bacillus cereus increases.Concerning the activity towards the Sarcina lutea strain, it was noted that the activity of the triazoles is comparable to that of the thiosemicarbazideas, but the thiadiazoles are inactive.The most active compounds against Bacillus antracis and Bacillus cereus are 5c (a thiadiazole compound) and 6c (a triazole compound), both with a 4-methylphenyl moiety on the heterocyclic ring.This could be explained by electropositive effect of methyl group attached to the phenyl moiety because of the known favorable influence of electron donating groups on the potency of the heterocyclic nuclei.Further investigations are in progress.The synthesized compounds were also investigated for their toxicity (Table 2) and it was observed that all tested compounds had a low toxicity.

General
All melting points were determined on a Melt-Temp R apparatus equipped with a digital thermometer and are uncorrected.The combustion analysis was performed on an Elemental Exeter Analytical CE 440 Apparatus.The IR spectra were measured as potassium bromide pellets on a Digilab Scimitar Series spectrophotometer; the wave numbers are given in cm -1 .The 1 H-NMR spectra were recorded in DMSO-d 6 solutions on Brucker ARX-300 spectrometer ( 1 H: 300 MHz) at ambient temperature.Chemical shifts were recorded as δ values in parts per millions (ppm) and were indirectly referenced to tetramethylsilane via the residual solvent signal (2.49 for 1 H).All chemical reagents were obtained from the Aldrich Chemical Company.
N-(3-nitrobenzoyl)-D,L-methionylhydrazide (3, 0.78 g, 0.0025 mol) was dissolved in methanol (10 mL) and a solution of the appropriate isothiocyanate (0.0025 mol) in methanol (10 mL) was added.The reaction mixture was heated at 70-80 ºC for two hours.After cooling the solvent was evaporated under reduced pressure and the solid was dried under vacuum at room temperature.The crude product was purified by crystallization from ethanol.

General procedure for the synthesis of 1,3,4-thiadiazole compounds 5a-e.
To corresponding thiosemicarbazide 4a-e (0.006 mol) concentrated H 2 SO 4 (1 mL) was added under stirring.The reaction mixture was stirred at room temperature for one hour and then added dropwise to cold water.The obtained solid was dried under vacuum at 45 ºC.The crude product was purified by crystallization from ethanol.General procedure for the synthesis of 1,2,4-triazole compounds 6a-c.

Antimicrobial activity assessment
The test microorganisms used to evaluate the potential antimicrobial activity of the new synthesized compounds were: Staphylococcus aureus ATCC 25923, Bacillus antracis ATCC 8705, Bacillus cereus ATCC 10987, Sarcina lutea ATCC 9341 and Escherichia coli ATCC 25922.All the new compounds were weighed and dissolved in dimethylsulphoxide (DMSO) to prepare an extract stock solution of 100 mg/mL.The antimicrobial effects of the substances were quantitatively tested in the respective broth media by using double dilution and the Minimal Inhibitory Concentration (MIC) values (µg/mL) were determined [17].The antibacterial assays were performed in Mueller-Hinton broth (MH) at pH 7.3.The MIC was defined as the lowest concentration that showed no growth.Dimethylsulfoxide (DMSO) with dilution of 1:10 was used as solvent control.

Toxicity study
The acute toxicity was estimated by intraperitoneal administration of the compounds as a suspension in Tween 80 to groups of fourteen mice, each weighting 20-25 g, according to the classical laboratory methodology [18].The animals were observed and the death rate ascertained after 7 days.

Table 2 .
The DL50 values of the tested compounds.

. DL 50 mg/Kg body Comp. DL 50 mg/Kg body
The solid compound obtained was dried under vacuum at 40-45 ºC.