Design, Synthesis, In Silico, and Biological Evaluation of Novel Chalcone–Benzamide Conjugates as Alkaline Phosphatase Inhibitors

Abstract Alkaline phosphatase (ALP) is a zinc-dependent metalloenzyme that catalyzes the hydrolysis of phosphate monoesters and is essential to several important physiological processes, such as bone mineralization and signal transduction. The clinical correlation of dysregulated ALP activity with a wide range of disease types, including bone disorders, hepatobiliary diseases, and certain malignancies, highlights the therapeutic importance of developing and identifying potent calf intestinal alkaline phosphatase (CIALP) inhibitors. Eleven new chalcone–benzamide derivatives (3a–3k), containing structurally different substituents, were synthesized and extensively investigated by spectroscopic methods such as IR, 1H NMR, 13C NMR, and low-resolution EI-MS analysis. All synthesized compounds were screened for DPPH radical-scavenging activity, with ascorbic acid as the standard (IC50 = 18.68 μM), and CIALP inhibitory activity, with KH2PO4 as the standard (IC50 = 2.80 ± 0.065 μM). In the DPPH assay, compound 3k showed the highest antioxidant activity (IC50 = 21.21 μM), followed by 3h (29.94 μM), 3j (39.92 μM), and 3c (53.22 μM), closest to the reference standard. In the enzyme inhibition assay, compounds 3d (IC50 = 3.93 ± 0.314 μM), 3h (IC50 = 4.49 ± 1.312 μM), and 3c (IC50 = 5.69 ± 1.124 μM) demonstrated the most potent enzyme inhibitory activities. Docking of the synthesized compounds was performed against the crystal structure of placental alkaline phosphatase (PLAP, PDB ID: 1EW2), used as a structural homologue in the absence of a deposited CIALP crystal structure, which showed that all synthesized compounds had good binding energies ranging from −7.9 to −8.3 kcal/mol; this narrow range is within the resolution limits of AutoDock scoring and did not correlate with experimental enzyme-inhibitory potency across the series. Prioritized lead candidates were found to have good oral bioavailability (expected to be above 90% absorption), acceptable physicochemical properties (compliant with Lipinski’s rule of five), and manageable toxicity risk. Together, the integrated in vitro biological and in silico computational data suggested that compounds 3h, 3c, and 3j are promising dual-function leads with both antioxidant and enzyme-inhibitory activity, while compound 3d stands out as a potent, structurally distinct enzyme inhibitor─making the benzamide scaffold a promising structural framework for the rational optimization of multitarget therapeutics.