<i> <b>N</b> </i> -Alkyl Sulfamates as a New Class of nsP2 Cysteine Protease Inhibitors with Broad-Spectrum Antialphaviral Activity

The emergence of mosquito-borne alphaviruses that cause chronic arthritis or encephalitis underscores the urgent need for broad-spectrum antiviral therapeutics. The viral nsP2 cysteine protease, which is essential for alphavirus replication, is a promising antiviral target. Vinyl sulfone covalent inhibitors potently inhibit nsP2 protease but suffer from glutathione reactivity and species-dependent systemic clearance catalyzed by glutathione <i>S</i>-transferase. To address these liabilities, we explored alternative electrophilic warheads and identified acetamide inhibitors bearing <i>N</i>-alkyl sulfamate warheads with improved biochemical and antiviral profiles. 2-((5-(2-Ethoxyphenyl)-1<i>H</i>-pyrazol-3-yl)amino)-2-oxoethyl methylsulfamate emerged as a lead compound with potency against New and Old World alphaviruses, low GSH reactivity, and proteome-wide selectivity. Despite its promising antialphaviral activity, 2-((5-(2-ethoxyphenyl)-1<i>H</i>-pyrazol-3-yl)amino)-2-oxoethyl methylsulfamate exhibited rapid clearance due to hepatic glucuronidation. Structure-activity studies revealed modifications that improve metabolic stability while retaining antiviral activity. These findings introduce sulfamate acetamides as a new class of covalent nsP2 protease inhibitors and direct-acting pan-alphavirus drugs.