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

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, 1 H NMR, 13 C 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.