Development of Lipopeptides as Orthoflavivirin Inhibitors with Low Micromolar Broad-Spectrum Antiorthoflaviviral Activity

Orthoflaviviral infections increasingly impact the global population; no specific therapeutic treatments are available. The orthoflaviviral protease NS2B-NS3 is a promising target for antiviral drug development. Here, we present the design, synthesis, structure-activity relationship (SAR), and in vivo PK study of a novel lipopeptide scaffold emerging from exploration of the previously investigated polycationic geminoids <b>4-6</b>. The <i>N</i>-palmitoyl moiety is essential for protease inhibition; optimization of the peptide sequence led to lipopeptides <b>73</b> and <b>79</b>, which selectively inhibited dengue virus (DENV2) NS2B-NS3 and exhibited low micromolar antiviral potency in DENV2-, West Nile virus (WNV)-, and Zika virus (ZIKV)-infected cells without significant cytotoxicity. Compound <b>73</b> (Palmitoyl-Lys-Ala-d-Ala-Lys-NH₂) demonstrated a favorable in vivo pharmacokinetic profile in BALB/c mice following intravenous (IV), intraperitoneal (IP), and subcutaneous (SC) administration, showing stability and good tolerability. Herein, we detail the SAR of the lipopeptide scaffold and suggest its potential for in vivo therapeutic application administered 20 mg/kg subcutaneously <i>b.i.d.</i>.