The synthesis of compounds was achieved as described previously and purity was confirmed by melting point, 1H and 13C NMR spectroscopy .
All synthesized compounds were screened for their minimum inhibitory concentration (MIC, µg⁄mL) against selected four Gram-positive, viz. Staphylococcus aureus (NCIM–2079), Bacillus subtilis (NCIM–2063), Bacillus cereus (NCIM–2156), Staphylococcus Epidermidis and four Gram-negative bacterial strains viz. Pseudomonas aeruginosa (NCIM–2036), Escherichia coli (NCIM–2065), Proteus Mirabilis and Proteus vulgaris by the broth dilution method as recommended by the National Committee for Clinical Laboratory Standards with minor modifications . Cefixime was used as standard antibacterial agent. Solutions of the test compounds and reference drug were prepared in dimethyl sulfoxide (DMSO) at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.56 µg ⁄ mL. Nine tubes were prepared in duplicate with the second set being used as MIC reference controls (16–24 h visual). After sample preparation, the controls were placed in a 37 °C incubator and read for macroscopic growth (clear or turbid) the next day. Into each tube, 0.8 mL of nutrient broth was pipetted (tubes 2–8); tube 1 (negative control) received 1.0 mL of nutrient broth and tube 9 (positive control) received 0.9 mL of nutrient. Tube 1, the negative control, did not contain bacteria or antibiotic. The positive control, tube 9, received 0.9 mL of nutrient broth because it contained bacteria but not antibiotic. The test compounds were dissolved in DMSO (100 µg⁄mL); 0.1 mL of increasing concentration of the prepared test compounds is serially diluted from tube 2 to tube 7 from highest (100 µg mL–1) to lowest (1.56 µg mL–1) concentration (tubes 2–8 containing 100, 50, 25, 12.5, 6.25, 3.125 and 1.56 µg mL–1). After this process, each tube was inoculated with 0.1 mL of the bacterial suspension whose concentration corresponded to 0.5 McFarland scale (9×108 cells⁄mL), and each bacterium was incubated at 37 °C for 24 h at 150 rpm. The final volume in each tube was 1.0 mL. The incubation chamber was kept humid. At the end of the incubation period, MIC values were recorded as the lowest concentration of the substance that gave no visible turbidity, that is, no growth of inoculated bacteria.
Time Kill Assay
The S. aureus in logarithmic growth phase were diluted to ∼106 CFU/mL and exposed to concentrations equivalent to ½ × MIC, 1 × MIC, 2 × MIC, 4 × MIC and 8 × MIC (in triplicate) of compound 7l and 7m in tryptic soy broth. Aliquots (100 μL) were collected from each treatment after 0, 2, 4, 6 and 24 h of incubation at 37 °C and subsequently serially diluted in PBS. The bacteria were then transferred to tryptic soy agar plates and incubated
at 37 °C for 18−20 h before viable CFU/mL was determined .
DNA Gyrase Inhibition Study
The DNA gyrase A (S. aureus) assay of compound 7m was carried out as previously reported . Briefly, the S. aureus was cultured in medium B (2 g yeast extract, 10 g polypeptone, 1.2 g (NH4)2SO4, 8 g Na2HPO, 2g KH2PO4, 0.2 g MgSO4, 4 g glucose in 1L distilled water). DNA gyrase purification, Supercoiling, and decatenation were executed as reported by F. Blanche . The IC50 (μg/mL) was determined from the dose response curve.
In vivo anti-bacterial activity
The antibacterial activity of compound 7m was performed using female albino Wistar rats of 40–45 days old, 180–200g b.w distributed in 5 groups (n = 6/each) . Briefly, S. aureus was grown in the Müeller-Hinton broth (MH) after reaching the log phase of growth; the suspension was centrifuged at1000xg for 15min. The supernatant was discarded, and the bacterial pellet was re-suspended in phosphate buffer saline (PBS) to achieve a concentration of 4x106 CFU/mL. All rats were inoculated intravenously (iv.) with 0.2mL of S. aureus. After 1h of inoculation, rats were treated with the vehicle (sterile PBS), Compound (three gradient dose) (p.o). A sham group was injected with sterile PBS in place of the bacterial suspension. Positive control group rats were treated with 60mg/kg of amoxicillin (p.o). To perform the quantitative evaluation of the bacteria in the blood of all groups, the blood samples were taken by retroorbital puncture of rats after 1 h of treatment and the blood samples plated on blood agar plates. The plates were incubated at 37 °C in ambient air overnight. The colonies of S. aureus were counted by colony counter. All experiments were done aseptically, while samples and contaminated materials were sterilized before being disposed.
Metabolic Liability Prediction
The cytochrome P450-mediated sites of metabolism were determined using the web server of RS Predictor by uploading the SDF file format of the compound 7m .
The web platform (http://www-metaprint2d.ch.cam.ac.uk/) by uploading the SMILES string of compound 7m was used to determine the SOM. The fingerprint matching was set to Loose, with the following values of parameters: number of exact levels: 2; similarity threshold: 1.0; first weight: 1.0; second weight: 1.0; third weight: 1.0; fourth weight: 0.75; fifth weight: 0.5; and sixth weight: 0.25.
The synthesized compounds, clubbed thiazole–1,3,5-triazine derivatives were screened for their antifungal activity minimum inhibitory concentration (MIC) against C. albicans NCIM–3102, C. glabrata NCIM–3266, C. neoformans NCIM–3542, and A. niger NCIM–620 using modified broth microdilution method recommended by CLSI, using fungicidal amphotericin B as standard controls  The Candida spp. and C. neoformans strains were subcultured on Sabouraud dextrose agar at 35 ± 1 °C for 24 h and 48 h respectively, while A. niger strains were subcultured 35 ± 1 °C for 4 days. The Candida spp. and C. neoformans suspensions were diluted with modified broth, RPMI 1640 medium at pH 4.5 in comparison to 0.5 McFarland standard to afford final target inocula of 5.0 × 103 and 5.0 × 105 for Candida spp. and C. neoformans respectively and A. niger inoculum was made to 4.0 × 104 colony forming units (CFU)/mL at pH 7.3 with the same RPMI 1640 medium in 5% Alamar blue. The fungal inocula were added to the samples to achieve a final volume of 200 μL. Eight serial dilutions, starting with 20.0000 μg/mL of the compounds (dissolved in dimethyl sulfoxide, DMSO) were made using 20% DMSO in normal saline to afford least concentration of 0.1563 μg/mL ( = 0.16 μg/mL) and transferred in duplicate to 96-well flat-bottom microplates. All organisms were examined at 630 nm prior to and after incubation (Candida spp. at 37 ± 1 °C, 18 to 24 h; while for C. neoformans and A. niger at 35 ± 1 °C, 72 h). The lowest test concentration that allowed no detectable growth (or no more than 20% growth for fluconazole, for A. niger, no color change from blue to pink) was defined as the minimum inhibitory concentration (MIC).
Statistical analysis and ethical approval
The results were expressed in terms of mean±standard deviation (SD). One-way analysis of variance (ANOVA) was used, followed by the Student–Newman–Keuls test when statistical difference was detected among the groups. Values of p>0.05 were considered significant. The GraphPad Prism©version5.01 for Windows (GraphPad Software, USA) was used for statistical analysis. The animal experiment was performed as per the Guide for the Care and Use of Laboratory Animals of and was permitted by the Institutional Animal Ethics Committee of SHUATS, Allahabad.