Design and Synthesis of 4-Arylazo Pyrazole Carboxamides as Dual AChE/BChE Inhibitors: Kinetic and In Silico Evaluation

TL;DR

Scientists designed and tested new compounds that strongly inhibit two enzymes linked to neurological diseases, with some performing better than a standard drug.

Contribution

A new series of 4-arylazo pyrazole carboxamides was synthesized and shown to inhibit AChE and BChE more effectively than tacrine.

Findings

Compound 5e inhibited AChE with a KI of 20.86 nM, outperforming tacrine.

Most compounds showed better BChE inhibition than the reference compound.

Molecular dynamics simulations confirmed stable binding of the top compounds in enzyme active sites.

Abstract

Background/Objectives: Pyrazole carboxamides are widely used as adaptable medicinal-chemistry scaffolds and have been explored as cholinesterase (ChE) inhibitor chemotypes. In this work, we prepared a new series of 4-arylazo-3,5-diamino-N-tosyl-1H-pyrazole-1-carboxamides 5(a–m) and evaluated their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), supported by structure-based computational analyses. Methods: Thirteen derivatives 5(a–m) were synthesized, fully characterized with analytical techniques (FT-IR, H NMR, and C NMR), and tested in vitro against AChE and BChE, with tacrine (THA) used as the reference inhibitor. Docking calculations were used to examine plausible binding modes. The top-ranked complexes (7XN1–5e and 4BDS–5i) were further examined by 100 ns explicit-solvent molecular dynamics (MD) simulations in Cresset Flare, followed by RMSD/RMSF analysis and contact-persistence profiling. Predicted ADME/Tox. properties were also assessed to identify potential developability issues. Results: The series showed strong ChE inhibition, and several compounds were more potent than THA. Compound 5e (4-nitro) was the most active AChE inhibitor (KI = 20.86 ± 1.61 nM) compared with THA (KI = 164.40 ± 20.84 nM). For BChE, the KI values ranged from 31.21 to 87.07 nM and exceeded the reference compound’s activity. MD trajectories supported stable binding in both systems (10–100 ns mean backbone RMSD: 2.21 ± 0.17 Å for 7XN1–5e; 1.89 ± 0.11 Å for 4BDS–5i). Most fluctuations were confined to flexible regions, while key contacts remained in place, consistent with the docking models. ADME/Tox. predictions suggested moderate lipophilicity but generally low aqueous solubility; all compounds were predicted as non-BBB permeant, and selected liabilities were flagged (e.g., carcinogenicity for 5e/5g/5h/5i; nephrotoxicity for 5f/5g). Conclusions: The 4-arylazo-3,5-diamino-N-tosyl-1H-pyrazole-1-carboxamide scaffold delivers low-nanomolar ChE inhibition, with docking and MD supporting stable binding modes. Future optimization should prioritize solubility improvement and mitigation of predicted toxicities and metabolic liabilities, especially given the predicted lack of BBB permeability for CNS-directed applications.

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