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.

Label-free electrochemical impedimetric immunosensor for sensitive detection of IgM rheumatoid factor in human serum.

The authors report a label-free and direct detection of rheumatoid factor- Immunoglobulin M (IgM-RF) based on an impedimetric-interdigitated wave type microelectrode array (IDWµE). IDWµE was functionalized with a self-assembled monolayer (SAM) of thioctic acid for antigen immobilization. The SAM functionalized IDWµE were characterized by atomic force microscopy, Energy Dispersive X-Ray Spectroscopy, and X-ray photoelectron spectroscopy. The covalent immobilization of IgG-Fc onto the SAM modified electrode arrays was characterized morphologically via AFM and electrochemically via cyclic voltammetry and electrochemical impedance spectroscopy. Impedimetric measurements in the presence of redox probe (Potassium ferrocyanide and potassium ferricyanide) showed a significant change in both the impedance spectrum and charge transfer resistance upon IgM-RF binding. Impedance measurements were target specific and linear with an increase in IgM-RF concentrations between 1 and 200 IU mL-1 in redox probe and human serum, respectively. The sensor showed detection limits of 0.6 IU mL-1 in the presence of redox probe and 0.22 IU mL-1 in the human serum. The biosensor exhibited good reproducibility (relative standard deviation (RSD), 4.96%) and repeatability (RSD, 2.31%) with an acceptable selectivity towards IgM-RF detection. The sensor also showed a good stability for 3 weeks at 4 °C in 1X PBS. Therefore, the sensitive and stable immunosensor exhibiting IDWµE features can be integrated with a miniaturized potentiostat to develop a sensing system that detects IgM-RF for point-of-care applications.