The Effect of Deuteration and Homologation of the Lactam Ring of Nirmatrelvir on Its Biochemical Properties and Oxidative Metabolism

Abstract

This study explores the relationship between structural alterations of nirmatrelvir, such as homologation and deuteration, and metabolic stability of newly synthesized derivatives. We developed a reliable synthetic protocol toward dideutero-nirmatrelvir and its homologated analogues with high isotopic incorporation. Deuteration of the primary metabolic site of nirmatrelvir provides a 3-fold improvement of its human microsomal stability but is accompanied by an increased metabolism rate at secondary sites. Homologation of the lactam ring allows the capping group modification to decrease and delocalize the molecule’s lipophilicity, reducing the metabolic rate at secondary sites. The effect of deuteration was less pronounced for the 6-membered lactam than for its 5-membered analogue in human microsomes, but the trend is reversed in the case of mouse microsomes. X-ray data revealed that the homologation of the lactam ring favors the orientation of the drug’s nitrile warhead for interaction with the catalytic sulfur of the SARS-CoV-2 Mpro, improving its binding. Comparable potency against SARS-CoV-2 Mpro from several variants of concern and selectivity over human cysteine proteases cathepsin B, L, and S was observed for the novel deuterated/homologated derivative and nirmatrelvir. Synthesized compounds displayed a large interspecies variability in hamster, rat, and human hepatocyte stability assays. Overall, we aimed to apply a rational approach in changing the physicochemical properties of the drug to refine its biochemical and biological parameters.

A Structural Comparison of Oral SARS-CoV-2 Drug Candidate Ibuzatrelvir Complexed with the Main Protease (Mpro) of SARS-CoV-2 and MERS-CoV

“…However, the second-generation oral drug candidate ibuzatrelvir has overcome this problem. As reported in our previous paper, two soft spots related to metabolic stability were identified in nirmatrelvir: the CH 3 group at P2 and next to the nitrogen at P1. The P2 group is more susceptible to metabolism.…”

“…Ibuzatrelvir, similar to nirmatrelvir, contains an electrophilic nitrile warhead for covalent interaction with the thiol of M pro catalytic cysteine. There are several crystal structures of nitrile warheads in complex with the SARS-CoV-2 M pro from us and other groups. ,− The SARS-CoV-2 M pro has also been crystallized in complex with many different covalent inhibitors containing a variety of warhead functional groups. , For example, a ketoamide compound was shown bound in the active site from the group of Hilgenfeld, and we reported a crystal structure of the SARS-CoV-2 M pro with GC373, an aldehyde . Our interest in the SARS-CoV-2 main protease and its inhibitors − prompted us to examine how ibuzatrelvir inhibits SARS-CoV-2 M pro in vitro and explore its interaction with the M pro of another coronavirus, MERS-CoV.…”