All synthesized compounds were screened for their minimum inhibitory concentration (MIC, µg mL–1) against a panel of three Gram-positive, viz. Staphylococcus aureus (NCIM–2079), Bacillus subtilis (NCIM–2063), Bacillus cereus (NCIM–2156) and three Gram-negative bacterial strains viz. Pseudomonas aeruginosa (NCIM–2036), Escherichia coli (NCIM–2065), and Proteus vulgaris for determination of their antibacterial efficacy using cefixime as a standard drug and results were presented in Table 1. It was depicted from the results that entire set of molecules showed mild to moderate activity and even in some cases showed potent activity against tested microorganism. It is noteworthy to mention that the presence of substituent have marked effect on the activity profile as seen in the case of compound 7b which showed excellent activity than its non-substituted counterpart, compound 7a. Further, isomeric replacements of substituent have not significantly altered the activity profile except in the case E. coli where compound 7c exhibit equipotent activity to the reference drug. In the case of compounds 7 (d-g) having electron withdrawing group on the R along with –OH and –OCH3 with different positions showed pronounced inhibition of Gram negative microorganism in comparison to Gram positive microorganism. To our surprise, drastic upsurge in activity against Gram positive microorganisms was reported by compound 7 (h-m) having electron withdrawing groups on both the positions. Among the selected compounds, 7l was found to exhibit considerable activity against entire panel of organisms. 7l showed considerable activity against S. aureus, B. subtilis and B. cereus. It showed moderate activity against P. aeruginosa, E. coli and P. vulgaris. Additionally, the introduction o-chloro in the place of m-fluoro on the phenyl of 1,3,5-traizine (compound 7m) renders the molecule significantly more active. The antibacterial activity was increased in the case of B. subtilis, while equipotent activity was achieved against S. aureus, and B. cereus as compared to standard. In the case of P. aeruginosa,E. coliand P. vulgaris, compound 7m showed moderate activity. The introduction non-halogenated electron withdrawing group showed drastic decline in activity against entire tested microorganisms, compound 7k. In the case of compounds having electron donating groups viz.,, 7n and 7o has showed remarkable inhibition of Gram-positive microorganisms.
On the basis of antibacterial results, structure-activity relationship studies suggested that nature of substituent has strong influence on the activity of the compounds, Figure 1. It has been noted that presence of electron withdrawing groups render target analogues more active against Gram positive microorganisms than Gram negative and activity was more confined to the presence of halogen atoms e.g. 7j, 7l and 7n. On the other hand, the antibacterial profile has been significantly improved by the presence of electron donating substituent against Gram negative microorganism viz. 7b, 7c, 7n and 7o. This could be explained on the basis of the perturbations brought by these atoms which are very useful to modulate the steric effect on phenyl ring of drug and which in turn alter the ease of penetration of molecule in bacterial cell wall resulting better activity profile.
The molecules were also tested for the determination of antibiofilm activity against S. aureus and B. subtilis. As presented in Table 1, it has been found that, majority of the compounds showed mild to no activity. Among the tested analogues, compound 7m showed more potent activity as compared to cefixime, followed by 7l. The rest of the active analogues displayed moderate activity, while no activity in the case of compound 7a, 7c, 7i, 7k and 7o.
Time kill assay
This assay is widely used for the determination of antimicrobial efficacy of the agent in relation to time against the challenge microorganism. Thus, two most potent molecules (7l and 7m) were further subjected to time kill assay against S. aureus and the results have been presented in Figure 2. It has been found that both compound (7l and 7m) showed bacteriostatic action over the duration of time, with most prominent activity in the case of 7m.
DNA Gyrase Inhibition Assay
In reference to the excellent antibacterial activity of target analogues, in the next part, we are interested in determination of the mechanism of action of most active analogue 7l and 7m against the DNA gyrase enzyme. The gyrase enzyme is responsible for the introduction of negative supercoils into DNA. It was believed to take part in the initiation and elongation stages during replication and transcription of the bacterial organisms. Thus, its inhibition by a novel class of inhibitor offers distinctive advantage in terms of growing bacterial resistance. The inhibitory profile of compound 7l and 7m was determined against the DNA gyrase isolated from S. aureus. The results as presents in Figure 3, confirmed that, both the analogues showed significant inhibition of DNA gyrase of S. aureus. Particularly, compound 7m was proved as an efficient inhibitor of gyrase enzyme than 7m.
Docking analysis with DNA Gyrase enzyme
To further exemplify the difference in the mode of interaction with the target receptor, both the compounds were allowed to dock with DNA gyrase protein model using LigandFit module of Discovery Studio 2.5 (Accelrys, Inc., San Diego, CA, USA). On close inspection of the docking interaction as shown in Figure 4, it might be suggested that due to excellent intermolecular H-bonds with Asn54, Asp57, Glu58, Arg144, compound 7l showed excellent interaction with DNA gyrase. Moreover, compound 7m showed prolific interaction with vital key residues, such as, Glu58, Val130, Ile175, Ile186 via numerous H-bond with no pi-cation interaction shown in Table 2. Thus, it has been confirmed that, our designed molecules exhibit significant inhibition of DNA gyrase vital for bacterial organisms. It also found in agreement with above inhibition study.
In vivo antibacterial activity against S. aureus infection model
The in vivo antibacterial activity of compound 7m was presented in Figure 5. The results suggested that, compound 7m showed significant inhibition of S. aureus infection in test animal. Compound 7m showed dose-dependent reduction of viability of bacteria with maximum activity in 25mg/kg treated group. The compound 7m does not found as efficient as ciprofloxacin as standard.
Metabolic Liability of 7m
The drugs used clinically are subjected to metabolic process which causes degradation of the molecule in various fragments to increase the polarity for enhanced excretion. The metabolic processes are also frequently associated with the production of metabolite that is toxic in nature. Therefore, in early drug discovery process, the prediction of metabolites of candidate molecule is very vital to prevent iteration in the subsequent procedures and to minimize the possible liabilities and risks coupled with biotransformation. Concerning the excellent antibacterial activity of compound 7m, it is worthwhile to define its metabolic liability. In past decade, the computational (in-silico) techniques have significantly contributed for the prediction of metabolites of xenobiotics. These techniques are found more advantageous than the wet experiments and virtually provide prior knowledge about the molecule of interest. In the present study, we have utilized two diverse program, such as, Regioselectivity-Predictor (RS-Predictor) and MetaPrint2D React for the prediction of metabolic liability of compound 7m. The FDA approved drugs are majorly metabolized by the Cytochrome P450s (CYPs) in site-specific manner to generate metabolites. Therefore, the RS-Predictor was utilized to identify experimentally observed site of metabolism (SOM) within the top two rank positions for substrate sets of each CYP isozyme (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4). The RS-Predictor calibrates an isozyme-specific regioselectivity QSAR (using topological and quantum chemical descriptors) from a set of known isozyme substrates and metabolites by treating each substrate as an individual competition between candidate SOMs. The results of prediction have been given in Figure 6. It has been found that, in majority of CYP isoenzymes, the fluoro containing phenyl group is highly labile SOM, except in the case of CYP2A6 and CYP2D6. On the other hand, many other fragments of the molecule were identified as moderately and fairly labile.
The metabolic process is quite multifaceted that, no single software can able to offer all the information and hence amalgamation of approaches could afford more pragmatic and precise view of biotransformation. Thus, the compound 7m was further analysed with MetaPrint2D-React. It is a metabolic product predictor developed by Unilever Cambridge, Centre for Molecular Science Informatics, University of Cambridge, UK. It utilizes historic metabolic data for SOM, as enumerated by the circular fingerprints which assumes most favourable site to undergo Phase I metabolism. It further finds the similarity between the known SOM and sites that not to be metabolized. The Symyx® Metabolite database was used for the generation of data for the MetaPrint2D, where, for each transformation, the differences between the structure of the reactant and product are identified: groups added or eliminated, bonds broken or made and bonds whose order has changed. To make results simpler, only Phase I reaction (defined as the addition of a single oxygen atom; covering hydroxylation, oxidation and epoxidation) and eliminations (e.g., dealkylation, ester and amide hydrolysis) are taken in account. For an addition, the neighbouring atom next to the added oxygen is marked as a reaction centre. In the case of elimination, a bond gets broken, and both atoms connected by the bond are considered to be reaction centres. Thus, the results of the study as shown in Figure 7, suggested that phenyl of 1,3,5-triazine are considered as a major SOM for the compound 7m in humans. On output, each atom in a 7m was colour coded by its normalized occurrence ratio (NOR) computed and provided in Table 3. A high NOR indicate a more frequently reported site of metabolism in the metabolite database. The normalised occurrence ratio does not indicate how likely a molecule is to be metabolised, but rather the relative likelihood of metabolism occurring at a particular site in the molecule, assuming it is metabolised.
As depicted in Table 4, target compounds exhibits mild to moderate activity against the tested fungal microorganism’s viz., Candida albicans, Candida glabrata, Cryptococcus neoformans and Aspergillus niger. It has been observed that, none of the compounds exhibit significant activity against A. niger and C. neoformans except compound 7d. Entire set of compounds displayed significant inhibition of Candida Spp where the pattern of inhibition is much more prominent against C. albicans than C. glabrata. Compound 7d displayed significant activity against the entire tested organism and more specifically, it found equipotent to standard drug against C. neoformans. On close inspection of Table 4, it has been observed that there is no specific correlation exists between substituent and the inhibitory activity. However, to improve this activity profile and get insight about structure–activity relationships, further studies are currently underway in our laboratory and reported subsequently.