# Identification and Exploration of a Series of SARS-Cov‑2 MPro Cyano-Based Inhibitors Revealing Ortho-Substitution Effects within the P3 Biphenyl Group

## TL;DR

This paper explores the development of a new SARS-CoV-2 protease inhibitor by modifying a compound's structure to improve its effectiveness and selectivity.

## Contribution

The study introduces a novel approach using ortho-substituted biphenyl groups to enhance inhibitor potency and binding.

## Key findings

- Compound 6a showed submicromolar potency against SARS-CoV-2 MPro.
- Ortho-substitution in the P3 benzamide improved biochemical and cellular activity.
- Compound 22e, with a proline modification, achieved high potency and selectivity.

## Abstract

Starting from a simple scaffold hopping exercise based
on our previous
exploration of cysteine protease inhibitors against legumain, compound 6a was identified as a starting point for the development
of a SARS-CoV-2 main protease (MPro) inhibitor. Compound 6a displayed submicromolar biochemical potency in the ultrasensitive
assay developed by Drag and co-workers. Through an iterative structure–activity
relationship campaign, we discovered an unexpected improvement in
both biochemical and cellular potency through the incorporation of
an ortho substituent within the P3 benzamide. X-ray crystallography
revealed that incorporation of the ortho substituent caused a subtle
but important binding enhancement of the P1 glutamate group within
the MPro S1 pocket. While incorporation of the ortho substituent
improved the potency, the off-target selectivity against a panel of
cysteine proteases and cell activity remained suboptimal. Further
scanning of the P2 core revealed that incorporation of the 3.1.0 proline
could address these issues and afford compound 22e, a
highly potent and cellularly active MPro inhibitor.