Exploring the role of conformational restriction in 5-alkyl-6-benzyl-2-{[(methylthio)methyl]thio}pyrimidin-4(3H)-ones. Effects in HIV-1 infected cells and reverse transcriptase enzymes

Conformational restriction is a powerful strategy previously used to optimize several HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI) series, including 3,4-dihydro-2-alkoxy-6-benzyl-4-oxopyrimidines (DABOs). Herein, we report the design, synthesis, and biological evaluation of a focused series of conformationally restricted 5-alkyl-2-{[(methylthio)methyl]thio}-6-(1-phenylethyl)-DABO (MTM-S-DABOs) compounds (16d-f, 17d-f, 18d-f, and 19d-f) comparing them with the corresponding unconstrained 6-phenylmethyl analogues (16a-c, 17a-c, 18a-c, and 19a-c). The introduction of a methyl group at the C6 α-benzylic position of the pyrimidinone core of MTM-S-DABOs markedly enhanced the antiviral potency and reverse transcriptase inhibition when combined with less bulky C5 substituents, whereas it generally reduced activity if combined with C5 isopropyl substitution. Among the investigated analogues, the 2,6-difluorophenyl (16e) and the 2-chloro-6-fluorophenyl (17d) derivatives emerged as the most promising racemic compounds, showing subnanomolar activity against wt HIV-1, low nanomolar potency against the mutant K103 N and the multidrug-resistant clinical isolate IRLL98, and (sub)micromolar activity against Y181C, Y188L, and K103N + Y181C strains. Enzymatic assays confirmed HIV-1 reverse transcriptase (RT) as the primary target and supported the cellular SAR trends. Enantioseparation of 16e and 17d combined with CD studies and the evaluation of the antiviral activity both in cells and with recombinant RTs revealed a pronounced enantioselectivity, with the R enantiomers displaying superior antiviral profiles compared with the corresponding S enantiomers and racemates. Notably, (R)-16e and (R)-17d combined subnanomolar activity against wt HIV-1 and IRLL98 with high selectivity and potent inhibition of resistant RT variants. These findings identify conformationally restricted MTM-S-DABOs, particularly their R enantiomers, as highly potent second-generation NNRTI leads for further optimization.