Microwave assisted synthesis and antimicrobial screening of fused triazoles

In the present study, a series of N -{4-[(4-amino-5-sulfanyl-4 H -1,2,4-triazol-3-yl)methyl]-1,3-thiazol-2-yl}-2-substituted amide derivatives ( 1a-d ) were synthesized in good yields and characterized. The compounds were evaluated for their preliminary in vitro antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhosa and then were screened for antitubercular activity against Mycobacterium tuberculosis H37 Rv strain by the MABA assay method. The antibacterial data of the tested compounds indicated that compounds 6b , 6c , 8a , 8b and 8c showed better activity against bacteria compared to reference drugs. The in vitro antitubercular activity reports of compounds tested against M. tuberculosis strain H37 Rv showed better activity by 8a , 8b and 8c .


Introduction
Tuberculosis remains the major cause of death over the world and the emergence of multi-drug resistant tuberculosis has made the condition most alarming.Up to 4% of all tuberculosis cases worldwide are resistant to more than one antitubercular drug because of incomplete or partial therapy. 1 Therefore, there is an urgent demand for a new class of antitubercular agent with a different mode of action and it has led medicinal chemists to explore a wide variety of chemical structures.While reports [2][3][4] are available stating the emergence of thiazoles and/or triazoles as potent antitubercular agents, appreciation of these findings, coupled with our observation that, today, the trend in antimycobacterial drug design [5][6][7] is to join together two or three molecules having different sites or mechanisms of action, initiated our construction of compounds containing both the thiazole and triazole ring systems in the same matrix to serve as a new scaffold towards the development of novel antimycobacterial agents.
Microwave assisted reactions 8 using dry media 9 have attracted much interest because of the simplicity in operation, greater selectivity and rapid synthesis of a variety of heterocyclic compounds. 10Thus, it was thought worthwhile to synthesize the title compounds using a Green Route, the MORE (Microwave Organic Reaction Enhancement) method.

Scheme 1
Compounds 1a-d were synthesized according to the literature. 11Compounds 1a-d, adsorbed on acidic alumina 12 , were treated with acetic anhydride at 0 °C to yield 2a-d.The transformed

Antitubercular activity
The above-synthesized products were screened against M. tuberculosis using the Microplate Alamar Blue Assay (MABA) 13 on a High Throughput Screening (HTS) machine at 25 µg/mL and lower concentrations using M. tuberculosis H37Ra as a surrogate for the virulent H37Rv strain.The results are shown in Table 2.The results of MABA have been found comparable to the standard BACTEC 460 system based assay.The standard antitubercular drugs Rifamycin, Isoniazid, p-aminosalicylic acid, Ethambutol and Ethionamide (MIC range 0.3-3 µg/mL) were taken as positive controls.We have also performed a cytotoxicity analysis of the abovesynthesized compounds, using neutral red uptake by using the Vero-C-1008 cell line at various concentrations (6.25 µg/mL to 50 µg/mL), none of them were found toxic.Hence the activities of the above-synthesized compounds were not due to cytotoxicity of the compounds.During the preliminary screening, four compounds 1a-d were tested (Table 2) at 25 µg/mL concentration for their antimycobacterial activity.One of the compounds, viz.1b exhibited 96% inhibition at this concentration while other compounds exhibited less than 90% inhibition at the same concentration.
Thus, we have considered 1b as a lead molecule and subsequent structural modifications were carried out.As a first step towards lead optimization, the amino group was protected to the corresponding acetamide 2a-d, however, all of these modifications resulted in a substantial decrease in activity.The next structural modification made was a dimeric product of 2a-d but this change also resulted in a substantial loss of biological activity.
Compounds 4b and 4c showed 97% inhibition at 25 µg/mL (Table 2), which was obtained by S-alkylation with acetonitrile.Thus, looking at the activity, it was decided to modify the structure at the SH group.In order to optimize the sulfhydryl component, four compounds 5a-d were synthesized and investigated, which revealed loss of activity.A further modification of compounds 5a-d produced compounds 6a-d.The results of the antimycobacterial activity are quite interesting because all of these compounds have shown inhibition above 90% at 25 µg/mL (Table 2).
On the other hand, in secondary screening at 12.5 µg/mL concentration these compounds were found to have decreased antimycobacterial activity.Compounds 6a-d were selected for further studies as they had a protected and free amino group, which opened an area for further modification at this point.Compounds 7a-l were obtained by treatment with acid chlorides, which ultimately showed decreased antimycobacterial activity.Furthermore, compounds 6a-d were investigated after conversion to Schiff bases with benzaldehyde, all of the compounds 8a-d showed more than 95% inhibition at 25 µg/mL concentration and, more interestingly, compounds 8a, 8b and 8c showed more than 94% inhibition at 12.5 µg/mL concentration.Although we have not been able to substantially enhance the activity of these compounds in the present study, the data presented here are encouraging and deserve further investigation.

Antimicrobial activity
The compounds listed in Table 1 were screened for antimicrobial activity against different microorganisms under the following conditions.Method: Well diffusion method, 14 Medium: The nutrient agar medium, Solvent: Chloroform.Concentrations: 50µM and 100 µM.Condition: 24 hours at 24-28 °C, Standard: The antibiotic Gentamycin The nutrient agar medium, 20 mL was poured into the sterile petri dishes.To the solidified plates, wells were made using a sterile cork borer 10 mm in diameter.The 24 hour subcultured bacteria was inoculated in the petri-plates, with a sterile cotton swab dipped in the nutrient broth medium.After inoculating, the compounds were dissolved separately with the chloroform solvent and poured into the wells with varying concentrations ranging from 50 and 100 µM using a micropipette.The plates were left over 24 hours at 24-28 °C.The antibiotic Gentamycin was used as a standard for comparative study.The percentage of inhibition was calculated by the formula % Inhibition = Diameter of the inhibition zone x 100 From these data, it has been found that all the compounds tested showed a broad spectrum of inhibitory properties.From the antibacterial screening it was observed that all the compounds exhibited activity against all the organisms employed.Looking at the structure activity relationship, marked inhibition in bacteria was observed with compounds 6b, 6c, 8a, 8b and 8c, whereas 1d, 3a, 4b, 4c, and 4d have shown moderate activity and others showed the least activity.

Experimental Section
General Procedures.The melting points were recorded on an Electrothermal apparatus and are uncorrected. 1H NMR spectra were recorded on a Bruker Avance 300 MHz instrument using CDCl 3 as solvent (chemical shifts in δ' ppm) using TMS as internal standard; mass spectra on a Finning LCQ mass spectrometer.Microwave irradiation was carried out in a Raga Scientific Microwave System, Model RG31L at 2450 MHz.Elemental analyses were performed on a Heracus CHN-Rapid Analyser.The purity of the compounds was checked on silica gel coated Al plates (Merck).

Table 2 .
Percent inhibition at 25µg/mL concentration