Discovery of 4-Quinazolinone-Containing Phenylalanine Derivatives as Potent, Resistant-Tolerant HIV Capsid Inhibitors

The HIV-1 capsid (CA) is a validated antiviral target that plays critical roles in both the early and late stages of the viral life cycle. Using structure-based strategy, we designed and synthesized a series of phenylalanine derivatives containing a 4-quinazolinone scaffold as novel HIV-1 CA inhibitors. Among them, IC-2i exhibited potent antiviral activity in MT-4 cells against HIV-1 NL4-3 (EC₅₀ = 0.65 ± 0.27 nM) and effectively protected cells from HIV-1 IIIB infection. SPR revealed that IC-2i interacts strongly with CA hexamers (K _D = 2.7 ± 0.5 nM) with an extended residence time and competes with host factors CPSF6 and NUP153, disrupting CA assembly and disassembly. IC-2i retained activity against lenacapavir (LEN)-resistant strains, such as N74D (11-fold shift vs. 20-fold for LEN). Crystallographic analysis revealed that IC-2i binds at the CA NTD-CTD interface and forms hydrogen bonds with Thr107 and Ser41 (NTD-NTD interface). Pharmacological evaluation demonstrated favorable properties, including good plasma stability, low toxicity (SI > 1571), and suitable pharmacokinetics with a prolonged half-life following subcutaneous administration (T _1/2 = 19.9 h). Overall, this study identifies 4-quinazolinone-based phenylalanine derivatives as promising HIV-1 CA inhibitors and highlights IC-2i as a potential long-acting therapeutic candidate for HIV-1 treatment.