Grayson D. Pipher

The ongoing emergence and reemergence of RNA viruses from multiple viral families highlights the urgent need for broad‐spectrum antiviral therapeutics. To address this issue, a series of 4′‐azido fleximer nucleoside analogs were synthesized and evaluated for broad‐spectrum antiviral activity against pathogens of epidemic/pandemic concern. One compound, KAD‐039, featuring a 2′, 3′‐diisobutyrate‐5′‐ProTide prodrug, exhibited potent antiviral activity across flaviviruses, filoviruses, and coronaviruses, with EC ₅₀ values ranging from 1 to 10 µM and minimal cytotoxicity (CC ₅₀ > 50 µM). Pharmacokinetic evaluation of KAD‐039 demonstrated high plasma stability in dog, monkey, and human plasma, with half‐lives ranging from 19.9 to 28 h but exhibited poor stability in human liver microsomes ( t _1/2 = 6.1 min). To help elucidate the mechanism of action of KAD‐039 against these viral families, computational molecular docking studies were performed using the expected triphosphate active form within the cap/GTP‐binding pockets of several viral methyltransferases and showed that KAD‐039TP docked into all of the viral MTases with similar or higher predicted affinity compared to the natural substrate. In addition, KAD‐039TP displayed reduced predicted affinity for the human N7 methyltransferase, suggesting low host toxicity. These results highlight 4′‐azido fleximer analogs as promising broad‐spectrum antivirals, potentially functioning through viral methyltransferase inhibition, and support further investigation.