Structure-based design, synthesis, and biological evaluation of conformationally restricted novel 2-alkylthio-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4(3H)-ones as non-nucleoside inhibitors of HIV-1 reverse transcriptase

5-Alkyl-2-(alkylthio)-6-(2,6-difluorobenzyl)-3,4-dihydropyrimidin-4(3H)-one s (S-DABOs, 2) have been recently described as a new class of human immunod eficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inh ibitors (NNRTIs) active at nanomolar concentrations (Mai, A. et al. J. Med. Chem. 1999, 42, 619-627). In pursuing our lead optimization efforts, we de signed novel conformationally restricted S-DABOs, 3, featuring a methyl at the benzylic carbon (Y = Me) and at the pyrimidine 5-position (R = Me). Con formational analyses and docking simulations suggested that the presence of both methyls would significantly reduce conformational flexibility without compromising, in the R enantiomers, the capability of fitting into the RT non-nucleoside binding pocket. To develop structure-activity relationships, we prepared several congeners of type 3 belonging to the thymine (R = Me) and uracil (R = H) series, featuring various 2-alkylthio side chains (X = M e, i-Pr, n-Bu, i-Bu, s-Bu, c-pentyl, and c-hexyl) and aryl moieties differe nt from the 2,6-difluorophenyl (Ar = phenyl, 2,6-dichlorophenyl, 1-naphthyl ). Moreover, alpha -ethyl derivatives (Y = Et) were included in the synthet ic project in addition to alpha -methyl derivatives (Y = Me). All of the ne w compounds were evaluated for their cytotoxicity and anti-HIV-1 activity i n MT-4 cells, and some of them were assayed against highly purified recombi nant wild-type HIV-1 RT using homopolymeric template primers. The results w ere expressed as CC50 (cytotoxicity), EC50 (anti-HIV-1 activity), SI (selec tivity, given by the CC50/EC50 ratio), and IC50 (RT inhibitory activity) va lues. In the 2,6-difluorobenzylthymine (R = Me) series, methylation of the benzylic carbon improved anti-HIV-1 and RT inhibitory activities together w ith selectivity. Compound 3w (Ar = 2,6-F-2-Ph, R = Y = Me, X = c-pentyl) tu rned out the most potent and selective among the S-DABOs reported to date ( CC50 > 200 muM, EC50 = 6 nM, IC50 = 5 nM, and SI > 33 333). Assays performe d on the pure enantiomer (+)-3w, much more active than (-)-3w, yielded the following results: CC50 > 200 muM, EC50 = 2 nM, IC50 = 8 nM, and SI > 100 0 00, under conditions wherein MKC-442 was less active and selective (CC50 > 200 muM, EC50 = 30 nM, IC50 = 40 nM, SI > 6666). The 2,6-difluorophenylethy lthymines (R = Me) were generally endowed with higher potency compared with the uracil counterparts (R = H). In the 2,6-difluorophenyl series the best and the least performant 2-alkylthio side chains were the 2-c-pentylthio a nd the 2-methylthio, respectively. When the methyl at the benzylic carbon w as replaced by an ethyl, activity was retained or decreased slightly, thus suggesting that the dimensions of the cavity within the RT hosting this sub stituent would not be compatible with groups larger than ethyl. Aryl moieti es different from the 2,6-difluorophenyl (phenyl, 1-naphthyl, 2,6-dichlorop henyl) were generally detrimental to activity, consistent with a favorable electronic effect exerted by the 2,6-fluorines on a putative charge-transfe r interaction between the aromatic moieties of the inhibitor and Tyr188.