Bi‐heterocyclic benzamides as alkaline phosphatase inhibitors: Mechanistic comprehensions through kinetics and computational approaches

Abstract

Novel bi‐heterocyclic benzamides were synthesized by sequentially converting 4‐(1H‐indol‐3‐yl)butanoic acid (1) into ethyl 4‐(1H‐indol‐3‐yl)butanoate (2), 4‐(1H‐indol‐3‐yl)butanohydrazide (3), and a nucleophilic 5‐[3‐(1H‐indol‐3‐yl)propyl]‐1,3,4‐oxadiazole‐2‐thiol (4). In a parallel series of reactions, various electrophiles were synthesized by reacting substituted anilines (5a–k) with 4‐(chloromethyl)benzoylchloride (6) to afford 4‐(chloromethyl)‐N‐(substituted‐phenyl)benzamides (7a–k). Finally, the nucleophilic substitution reaction of 4 was carried out with newly synthesized electrophiles, 7a–k, to acquire the targeted bi‐heterocyclic benzamides, 8a–k. The structural confirmation of all the synthesized compounds was done by IR, 1H NMR, 13C NMR, EI‐MS, and CHN analysis data. The inhibitory effects of these bi‐heterocyclic benzamides (8a–k) were evaluated against alkaline phosphatase, and all these molecules were identified as potent inhibitors relative to the standard used. The kinetics mechanism was ascribed by evaluating the Lineweaver–Burk plots, which revealed that compound 8b inhibited alkaline phosphatase non‐competitively to form an enzyme–inhibitor complex. The inhibition constant Ki calculated from Dixon plots for this compound was 1.15 μM. The computational study was in full agreement with the experimental records and these ligands exhibited good binding energy values. These molecules also exhibited mild cytotoxicity toward red blood cell membranes when analyzed through hemolysis. So, these molecules might be deliberated as nontoxic medicinal scaffolds to render normal calcification of bones and teeth.

Synthesis, kinetic studies and in-silico investigations of novel quinolinyl-iminothiazolines as alkaline phosphatase inhibitors

“…In addition, Kinetic mechanism analysis was conducted to determine the mechanism of inhibition. The most potent molecule was selected on the basis of IC 50 in order investigate the competitive or non-competitive enzyme inhibition by following our previously reported method 57 . The detailed procedure for alkaline phosphate inhibition assay and kinetic mechanism analysis is given in supplementary file .…”

Potent Alkaline Phosphatase Inhibitors, Pyrazolo-Oxothiazolidines: Synthesis, Biological Evaluation, Molecular Docking, and Kinetic Studies

“…Primarily based on the IC 50 findings, we selected the most effective inhibitor 7g for CIAP to determine the mechanism of enzyme inhibition using our published method [ 70 ]. The inhibitor ( 7g ), and substrate p -NPP concentrations were used at 0.00, 0.023, 0.045 and 0.091 µM, 10, 5, 2.5, 1.25, 0.625 and 0.3125 mM, respectively.…”

Current status ofN-,O-,S-heterocycles as potential alkaline phosphatase inhibitors: a medicinal chemistry overview

“…Dixon plots were used to compute the compound's inhibitory constant K i , which came out at 1.15 mM. 156 Ashraf et al (2019) reported the synthesis and molecular docking of N-(5-(alkylthio)-1,3,4-oxadiazol-2-yl)methylbenzamide analogs as potential inhibitors of the AP. The aim was to evaluate the effect of alkylthio substitution on the inhibitory activity and binding affinity of these compounds towards AP.…”