Dihydro-alkoxy-benzyl-oxopyrimidines (DABOs) are a family of potent NNRTIs developed in the past decade. Attempts to improve their potency and selectivity led to thio-DABOs (S-DABOs), DATNOs, and difluoro-thio-DABOs (F 2-S-DABOs). More recently, we reported the synthesis and molecular modeling studies of a novel conformationally constrained subtype of the S-DABO series characterized by the presence of substituents on the methylene linkage connecting the pyrimidine ring to the aryl moiety (Mai, A., et al. J. Med. Chem. 2001, 44, 2544-2554). Now we report the computer-aided design, synthesis, and biological evaluation of four new DABO prototypes (5-alkyl-2-cyclopentylamino-6-[1-(2,6-difluorophenyl)alkyl]-3, 4-dihydropyrimidin-4(3H)-ones, F2-NH-DABOs) in which the sulfur atom of the related F2-S-DABOs is replaced by an amino group. For these studies, we used as a reference model the cocrystallized MKC-442/RT complex. Docking studies with Autodock of the newly designed F2-NH-DABOs on the ligand-derived RT confirmed the findings previously described for the F 2-S-DABOs. The F2-NH-DABO binding mode resembles that reported for F2-S-DABOs, with the difference that the NH moiety at the C-2 position represents a new anchor site for ligand/enzyme complexation. The predicted inhibition constant (Ki) values by the internal scoring function of Autodock, and the predicted IC50 values by the application of a VALIDATE II/HIV-RT model strongly suggested the synthesis of the designed amino-DABOs. F2-NH-DABOs were shown to be highly active in both anti-RT and anti-HIV biological assays with IC50 and EC 50 comparable with that of the reference compound MKC-442. Interestingly, 2-cyclopentylamino-6-[1-(2,6-difluorophenyl)ethyl]-3,4-dihydro-5-methyl pyrimidin-4(3H)-one (9d) was active against the Y181C HIV-1 mutant strain at submicromolar concentration, with a resistance value similar to that of efavirenz, the last FDA-approved NNRTI for AIDS therapy, and 2-fold lower than that of its 2-cyclopentylthio counterpart 8d. The introduction in 9d of a new anchor point (pyrimidine C-2 NH group), with the formation of a new hydrogen bond with Lys101, could compensate for the lack of positive hydrophobic ligand/NNBP interactions due to the Tyr181 to Cys181 mutation.