N-acylbenzimidazoles as selective Acylators of the catalytic cystein of the coronavirus 3CL protease

The 3CL protease (3CLpro, Mpro) plays a key role in the replication of the SARS-CoV-2 and was validated as therapeutic target by the development and approval of specific antiviral drugs (nirmatrelvir, ensitrelvir), inhibitors of this protease. Moreover, its high conservation within the coronavirus family renders it an attractive therapeutic target for the development of anti-coronavirus compounds with broad spectrum activity to control COVID-19 and future coronavirus diseases. Here we report on the design, synthesis and structure-activity relationships of a new series of small covalent reversible inhibitors of the SARS-CoV-2 3CLpro. As elucidated thanks to the X-Ray structure of some inhibitors with the 3CLpro, the mode of inhibition involves acylation of the thiol of the catalytic cysteine. The synthesis of 60 analogs led to the identification of compound 56 that inhibits the SARS-CoV-2 3CLpro with high potency (IC50 = 70 nM) and displays antiviral activity in cells (EC50 = 3.1 μM). Notably, compound 56 inhibits the 3CLpro of three other human coronaviruses and exhibit a good selectivity against two human cysteine proteases. These results demonstrate the potential of this electrophilic N-acylbenzimidazole series as a basis for further optimization. [Display omitted] •Novel non-peptidomimetic covalent reversible SARS-CoV-2 3CLpro inhibitors were discovered by HTS and optimized.•X-Ray structure revealed a covalent binding to the catalytic Cys145 thanks to the electrophilic N-acylbenzimidazole warhead.•56 inhibits four 3CL proteases of α-CoV and β-CoV including SARS-CoV-2 and is selective against 2 human cysteine proteases.•56 displayed antiviral activity on 2 human coronaviruses in cells.