Synthesis and Antimicrobial Activity of Bis [ 4-methoxy-3-( 6-aryl-7 H-[ 1 , 2 , 4 ] triazolo [ 3 , 4-b ] [ 1 , 3 , 4 ] thiadiazin-3-yl ) phenyl ] methanes and Bis [ ( triazolo [ 3 , 4-b ] thiadiazepin-3-yl ) phenyl ] methanes

A series of novel bis[4-methoxy-3-(6-aryl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-3-yl)phenyl]methanes and bis[(triazolo[3,4-b]thiadiazepin-3-yl)phenyl]methanes (5a–e and 6a–e) has been synthesized and characterized by IR, H and C NMR, MS and elemental analysis. All the newly synthesized compounds were screened for their antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Klobsinella aerogenes and Chromobacterium violaceum and antifungal activity against Candida albicans, Aspergillus fumigatus, Trichophyton rubrum and Trichophyton mentagrophytes. Compounds 5b, 5d, 5e, 6b, 6c and 6e exhibited potent activity against the tested bacteria and fungi, and emerged as potential molecules for further development.

Owing to the immense importance and varied bioactivities exhibited by triazolo-thiadiazines and thiadiazepines and in continuation of our work on biologically active heterocycles, [43][44][45][46][47][48][49][50][51] we were stimulated to integrate thiadiazines moieties in a triazole framework, since these systems possess well documented antimicrobial activity.In this connection, some bis-heterocyclic compounds such as bis-triazolo thiadiazines and triazolo-thiadiazepines have been synthesized and evaluated for their antibacterial and antifungal activity.For the synthesis of target com-Srinivas: Synthesis and Antimicrobial Activity of ... pounds, 4-amino-1, 2, 4-triazol-3-thione is used as an intermediate because the amino and mercapto groups are appropriate nucleophile centers for the synthesis of fused heterocyclic compounds.

Results and Discussion
5,5'-Methylenebis(2-hydroxybenzoic acid) (2), required for the synthesis of the title compounds, was prepared according to the procedure described in the literature. 52Compound 2 on reaction with methyl iodide, in the presence of aq.KOH at 80 °C, furnished 5-(3-formyl-4methoxybenzyl)-2-methoxybenzoic acid (3).The condensation of 3 with thiocarbohydrazide at melt temperature for 3 h afforded bis[4-methoxy-3-[4-amino-5-sulfanyl-4H-1,2,4-triazol-3-yl]phenyl]methane (4) as a yellow solid (Scheme 1).IR spectrum of 4 showed two absorption bands in the region of 3335-3235 and 2596 cm -1 assigned to NH 2 and SH groups, two absorption bands at1554 and 1512 cm -1 attributable to C=N vibrations, providing a strong evidence for the formation of a triazole ring. 1 H NMR spectrum of 4 showed two signals at δ 2.17 and 5.47 ppm corresponding to -SH and -NH 2 protons, respectively.The aromatic protons appeared in the region δ 6.62-9.93ppm in accord with the structure. 13C NMR spectrum of 4 showed signals at δ 156.6 and 134.4 ppm corresponding to the 3-C and 5-C of the triazole moiety, respectively.The other signals observed were at the expected chemical shifts with appropriate integrals.In addition, elemental analysis is also consistent with the structure proposed for 4.

Antibacterial Evaluation
All the newly synthesized compounds 5a-e and 6a-e were screened for their antibacterial activity against Gram-positive bacteria viz.Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC 96), and Gram-negative bacteria viz.Klobsinella aerogenes (MTCC 39) and Chromobacterium violaceum (MTCC2656) by disc diffusion method. 53For the antibacterial assay, standard inoculums (1-2 × 107 c.f.u/mL 0.5 McFarland standards) were introduced onto the surface of sterile agar plates, and a sterile glass spreader was used for even distribution of the inoculums.The discs measuring 6.26 mm in diameter were prepared from Whatman no. 1 filter paper and sterilized by dry heat at 140 °C for 1 h.The sterile discs previously soaked in a known concentration of the test compounds were placed in the nutrient agar medium.The plates were inverted and incubated for 24 h at 37 °C.The inhibition zones were measured and compared with the standard drug streptomycin.The zone of inhibition data are presented in Table 1.The antibacterial screening revealed that all the tested compounds 5a-e showed moderate to good inhibition towards all the tested strains.Compounds 5b, 5d, 5e, 6b, 6c and 6e exhibited potent inhibitory activity compared to the standard drug at the tested concentrations.

Antifungal Evaluation
Compounds 5a-e and 6a-e were also evaluated for in vitro antifungal activity against four fungi viz.Candida albicans (ATCC 10231), Aspergillus fumigates (HIC 6094), Trichophyton rubrum (IFO 9185) and Trichophyton mentagrophytes (IFO 40996) by agar diffusion method. 52For the antifungal assay Sabourands agar media was prepared by dissolving peptone (1 g), D-glucose (4 g) and agar (2 g) in distilled water (100 mL) and adjusting the pH to 5.7.Normal saline was used to make a suspension of spore of fungal strain for lawning.A loopful of particular fungal strain was transferred to 3 mL saline to get a suspension of corresponding species.20 mL of agar media was poured into each petri-dish, excess of suspension was decanted and the plates were dried by placing in an incubator at 37 °C for 1 h.Using agar punch, wells were made and each well was labeled.A control was also prepared in triplicate and maintained at 37 °C for 3-4 days.The C. albicans was grown for 48 h at 28 °C in YPD broth (1% yeast extract, 2% peptone and 2% dextrose), harvested by centrifugation and then washed twice with sterile distilled water.A. fumigatus, T. rubrum and T. mentagrophytes were plated in potato dextrose agar (PDA) (Difco) and incubated at ted at 28 °C for two weeks.Spo-Srinivas: Synthesis and Antimicrobial Activity of ... res were washed three times with sterile distilled water and re-suspended in distilled water to obtain an initial inoculums size of 10 5 spores/mL.The zones of inhibition were determined and compared with the standard drug amphotericin B (Table 2).Results of antifungal activity showed that most of the new compounds, i.e. 5b, 5d, 5c, 6b, 6c and 6e were active with moderate to good activity.

Experimental
Commercial grade reagents were used as supplied.Solvents except analytical reagent grade were dried and purified according to literature when necessary.Reaction progress and purity of the compounds were checked by thin-layer chromatography (TLC) on pre-coated silica gel F254 plates from Merck and were visualized either by exposure to UV light or dipping in 1% aqueous KMnO 4 solution.Silica gel chromatographic columns (60-120 mesh) were used for separations.All melting points are uncorrected and measured using Fisher-Johns apparatus.IR spectra were recorded as KBr disks on a Perkin-Elmer FT IR spectrometer.The 1 H NMR and 13 C NMR spectra were recorded on a Varian gemini spectrometer (300 MHz for 1 H and 75 MHz for 13 C).Chemical shifts are reported as δ ppm against TMS as internal reference and coupling constants (J) are reported in Hz units.Mass spectra were recorded on a VG micro mass 7070H spectrometer.Elemental analyses (C, H, N) determined by a Perkin-elmer 240 CHN elemental analyzer were within ±0.4% of theoretical.

Preparation of bis[ [4-methoxy-3-[ [4-amino-5-sulfanyl-4H-1,2,4-triazole-3-yl] ]phenyl] ]methane (4):
A mixture of compound 3 (0.01 mol) and thiocarbohydrazide (0.02 mol) was heated until the contents melted.The reaction was maintained at this temperature for 3 h.The fused mass thus obtained was treated with sodium bicarbonate solution to dissolve the unreacted compound 3.It was then washed with water and collected by filtration.The product was recrystallized from a mixture of dioxane and ethanol to afford the compound  [1,2,4] ]triazolo[ [3,4-b] ][ [1,3,4] ]thiadiazol)phenyl] ] methanes 5a-e: A mixture of compound 4 (0.01 mol) and the corresponding phenacylbromide (0.02 mol) in absolute ethanol (20 mL), was refluxed for 6 h.The reaction mixture was concentrated and cooled to room temperature, and the remaining solvent was removed under reduced pressure, then diethyl ether (25 mL) was added and the reaction mixture was left at 0 °C for overnight.The precipitated solid was filtered off; the crude product thus obtained was purified by column chromatography on silica gel with hexane-ethyl acetate as eluent to afford the pure compounds 5a-e.a Streptomycin (50 μg / disc) was used as positive reference and compounds 5a-e and 6a-e (50 μg / disc) were screened.a Amphotericin (100 μg / disc) was used as positive reference and compounds 5a-e and 6a-e (100 μg / disc) were screened.

Table 1 .
Antibacterial activity of 5a

Table 2 .
Antifungal activity of 5a-e and 6a-e