Further exploring the tolerant region II: Identification of 2,4,5-trisubstituted pyrimidines as HIV-1 reverse transcriptase allosteric inhibitors with desirable antiviral activities and reduced cytotoxicity.

Here, we report a series of 2,4,5-trisubstituted pyrimidines as potent HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) by further exploring the potential tolerant region II within NNIBP. Most compounds were identified with potent inhibitory activity against HIV-1 wild-type (WT) strain with lower cytotoxicity. Among them, 14l exhibited the most outstanding antiviral activity (EC50 = 6.50-52.9 nM) against WT and a panel of mutant HIV-1 strains with much-reduced cytotoxicity (CC50 = 228 μM) and higher selectivity index (SI = 31434) than that of the lead 36a (CC50 = 45.6 μM; SI = 20550). Furthermore, the detailed molecular modeling studies revealed that the 3-thienyl substituent of 14l engages in multiple stable contacts with conserved residues within tolerant region II, providing a structural basis for the observed potent antiviral efficacy and enhanced anti-resistance profile. These findings collectively position 14l as a promising candidate for subsequent development as a novel lead compound.