People have been ailing regularly nowadays and conventional antibiotics have become less potent due to drug resistance. The molecular architecture of the antibiotics issued to patients has only marginally changed since their discovery, thus this has caused microorganisms to adapt or survive the repeated administration of the same drugs over time, causing medication to be less effective, and people becoming sicker because the lack of new, different and potent antibiotic structures has enabled bacteria to mutate. This presented the pressing need to develop pyrazole compounds because they suit the category of being structurally different and have been reported to exhibit excellent antibacterial activities. To obtain pyrazole derivatives, Microwave-Assisted Organic Synthesis (MAOS) was utilized to synthesize ten novel compounds of (4Z)-4-arylidene-4,5-dihydro-3-methyl-5-oxopyrazole-1-carbothioamide derivatives (4a-4j) by a one-pot multicomponent reaction (MCR) methodology that is ecofriendly and adhering to green chemistry principles, occurring by catalyst-free in a waterethanol solvent system. The methodology to access novel pyrazoles (4a-4j) in this study is superior to conventional pyrazole synthesis because it requires harsh reaction conditions and expensive catalysts, which deviates from green chemistry. All synthesized compounds (4a-4j) were fully characterized and confirmed by 1H NMR, 13C NMR, 2D NMR, FTIR and TOF-MS. Antimicrobial study was performed against two Gram-negative and two Gram-positive strains viz. Escherichia Coli (E.Coli), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus Aureus (S. Aureus), and Streptococcus pneumoniae (S. pneumoniae), respectively, and amoxicillin was used as the reference drug. It was found that only 4c and 4j exhibited activity against E. coli and their Minimum Inhibitory Concentration ( MIC) values were 1.38 and 2.50 mg/mL, respectively. In contrast, Amoxicillin displayed a lower MIC value of 0.0306 mg/mL, thereby suggesting that amoxicillin is more effective than the synthesized pyrazoles against E. coli. Pyrazoles 4a, 4d, and 4g showed no antibacterial activity against P. aeruginosa. Compounds 4b, 4c, 4e, 4f, 4h, 4i, and 4j displayed varying MIC values ranging from 0.212 to 0.625 mg/mL against S. aureus, 4i being 0.212 mg/mL the lowest MIC value. Amoxicillin displayed a lower MIC value than all the pyrazoles against P. aeruginosa. Against S. pneumoniae, 4j demonstrated an excellent antibacterial potential with a lower MIC value of 0.0156 mg/mL compared to that of amoxicillin (0.0306 mg/mL). The other pyrazoles displayed marginally higher MIC values than amoxicillin; 4g showed no activity for S. aureus. However, 4i displayed the lowest MIC value, suggesting its potential as an antibacterial agent. It was percieved that the unsubstituted phenyl ring in 4i contributed to its enhanced potency against test bacteria. Molecular docking studies were performed to predict the binding sites and affinities of the pyrazoles, revealing that they primarily target Penicillin Binding Proteins (PBPs) in the test bacteria. Docking scores for E. coli ranged from -6.7 to -7.9 kcal/mol and compound 4f exclusively exhibited the best docking score of -8.1 kcal/mol against E. coli better than amoxicillin’s docked score of -7.0 kcal/mol. Docking scores for P. aeruginosa ranged from - 6.8 to -7.7 kcal/mol. Compounds 4g and 4j displayed the highest negative docking scores of - 7.7 kcal/mol, outperforming amoxicillin thereby suggesting their potential as inhibitors, however 4b and 4f were comparable. Furthermore, in silico simulation, Molecular Dynamics (MD) studies were conducted for 4g and 4j. Docking scores for S. pneumoniae range from - 7.2 to -8.1 kcal/mol. Compound 4j displayed the highest negative docking score. Agreement between docking and in vitro results reinforces 4j as a potential inhibitor. The pyrazoles exhibited docking scores between -5.5 and -6.6 kcal/mol for S. aureus with 4j showing potential as an inhibitor. While lower than other PBPs, some pyrazoles are comparable to amoxicillin. Furthermor, the pyrazoles exhibited LogP (Lipophilicity) values oscillating between 1 and 2.5, indicating better bioavailability. Amoxicillin, partitioned close to an aqueous phase, showed by a negative LogP value of -0.29. Amoxicillin’s LD50 (lethal dose for killing 50% of the bacteria) is 15 g/kg, suggesting non-toxicity orally. Among pyrazoles, only compound 4c shares this safety profile as amoxicillin, qualifying 4c to be an orally administered antibacterial agent. Other pyrazoles fall into toxicity class 4, necessitating alternative administration routes, such as IV (Intravenous) or IM (Intramuscular). All studied compounds showed no affinity for BBB (Blood-Brain Barrier) permeability or P-glycoprotein binding, indicating they can traverse the system without hindrance from nonspecific enzymes or tissues. Compound 4d does not inhibit any CYP (Cytochrome P450) isoenzymes, similar to amoxicillin. Compound 4e selectively inhibits CYP 3A4, relevant for drug metabolism.
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