The design and synthesis of cysteine protease inhibitors containing isoxazole bearing warheads against Leishmania donovani.

Cysteine proteases are critical drug targets for Trypanosomatids, crucial for the host cell invasion, survival and establishing infection. The current therapeutic regimen for leishmaniasis is inadequate, necessitating the search for safer anti-leishmanial agents. A fourteen-member library of new isoxazole derivatives was synthesised using a 1, 3-dipolar cycloaddition approach and then evaluated for antileishmanial activity. Ligands PB, PM and PE were found most effective against intramacrophage amastigotes of Leishmania donovani with EC50 values of 2.42μM, 2.93 μM and 5.19μM, respectively, compared to Amphotericin-B (IC50 2.15 μM). They also inhibited the promastigote stage of the parasite with an EC50 of 9.44 µM, 11.99µM and 14.81 µM, respectively. Mechanistic in silico studies against cysteine protease, followed by in vitro corroboration using Bz-Arg-AMC hydrochloride fluorogenic peptide substrate ascertained the promising potential of PM (IC50 10.34 μM) as a cysteine protease inhibitor. Compounds fared well in the toxicity screen against THP-1 human monocytic cells. Compounds PB, PM and PE emerged as potent hits, and PM with high activity and low toxicity can be further developed into a potent lead molecule (s) to fight Leishmania donovani. The design of isoxazole-based small molecule libraries presents a promising strategy for the development of safer and potent antileishmanial agents targeting cysteine proteases.