NEW MULTI-TARGET DIRECTED p-NITROPHENYL HYDRAZONES; CONCEPTION, DESIGN, SYNTHESIS, IN-VIVO ANTI-INFLAMMATORY EVALUATION, AND IN-SILICO STUDIES AGAINST COX-2, 5-LOX, AND H+/K+ ATPASE

Abstract

NSAIDs and coxibs are associated with serious gastroinstestinal and cardiovascular side effects on their long term usage. Five hydrazones having chloro, methoxy, and nitro group substitutions were designed following a patent and extensive literature review. The designed compounds were evaluated in-silico for their pharmacokinetic and toxicity profile. Synthesis of the hydrazones was achieved through mechanochemistry (solvent-free) and conventional methods. Structures of the synthesized hydrazones were determined using spectroscopic methods namely; FTIR and NMR analyses. The synthesized hydrazones were evaluated for their anti-inflammatory activity in-vivo using Carageenan induced-edema in mice. Molecular docking study was conducted using crystal structures of COX-2, 5-LOX, and H +/K + ATPase.

All the designed compounds demonstrated plausible pharmacokinetics and apparent safety profile. These compounds exhibited statistically significant (p ≤ 0.005) anti-inflammatory activities at all doses in carrageenan-induced paw edema in the mice model. Careful analysis of their anti-inflammatory activity indicated possibly time-dependent edema inhibition at each dose. Moreso, compound TMB345 exhibited superior activity at 10mg/kg to reference drugs used, and also dose-dependent anti-inflammatory activity. However, all the synthesized hydrazones indicated a slow onset of action (4.58% - 23.66%) compared to those of reference drugs 35.50% and 33.21% for piroxicam and celecoxib respectively, this onset of action was improved as the dose increased. All the synthesized compounds notwithstanding indicated superior and longer duration of action (52.05% - 87.30%) compared to celecoxib (49.18%) and piroxicam (60.25%) used as reference drugs. Interestingly, all the p- nitrophenylhydrazones exhibited potent inhibition of biological targets used for the docking simulation. Although, celecoxib as a selective COX-2 inhibitor demonstrated marked lower binding energy compared to any of the compounds. This suggests that it is more selective for COX-2 compared to the designed compounds. Compound TMB345 had infinitesimally higher binding energy compared to omeprazole. Compound TMB345 had superior interactions in-silico with COX-2, 5-LOX, and H+/K+ ATPase compared to their approved marketed inhibitors celecoxib, zileuton, and omeprazole respectively. Also, compounds TMB345 formed two hydrogen bond interactions with key amino acids; SER-516 which is one of the two most important interactions for COX-2 related anti-inflammatory activity.

Also, the designed compounds exhibited high inhibitory potency against the proton pump (PP) demonstrating good interactions with key amino acids; CYS-813 and CYS-822 responsible for proton pump (PP) inhibitory activity. Therefore, compound TMB345 has been identified as a lead compound.