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

Abstract

5-Alkyl-2-(alkylthio)-6-(2,6-difluorobenzyl)-3,4-dihydropyrimidin-4(3H)-ones (S-DABOs, 2) have been recently described as a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) active at nanomolar concentrations (Mai, A. et al. J. Med. Chem. 1999, 42, 619-627). In pursuing our lead optimization efforts, we designed novel conformationally restricted S-DABOs, 3, featuring a methyl at the benzylic carbon (Y = Me) and at the pyrimidine 5-position (R = Me). Conformational 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 = Me, i-Pr, n-Bu, i-Bu, s-Bu, c-pentyl, and c-hexyl) and aryl moieties different from the 2,6-difluorophenyl (Ar = phenyl, 2,6-dichlorophenyl, 1-naphthyl). Moreover, alpha-ethyl derivatives (Y = Et) were included in the synthetic project in addition to alpha-methyl derivatives (Y = Me). All of the new compounds were evaluated for their cytotoxicity and anti-HIV-1 activity in MT-4 cells, and some of them were assayed against highly purified recombinant wild-type HIV-1 RT using homopolymeric template primers. The results were expressed as CC(50) (cytotoxicity), EC(50) (anti-HIV-1 activity), SI (selectivity, given by the CC(50)/EC(50) ratio), and IC(50) (RT inhibitory activity) values. In the 2,6-difluorobenzylthymine (R = Me) series, methylation of the benzylic carbon improved anti-HIV-1 and RT inhibitory activities together with selectivity. Compound 3w (Ar = 2,6-F(2)-Ph, R = Y = Me, X = c-pentyl) turned out the most potent and selective among the S-DABOs reported to date (CC(50) > 200 microM, EC(50) = 6 nM, IC(50) = 5 nM, and SI > 33 333). Assays performed on the pure enantiomer (+)-3w, much more active than (-)-3w, yielded the following results: CC(50) > 200 microM, EC(50) = 2 nM, IC(50) = 8 nM, and SI > 100 000, under conditions wherein MKC-442 was less active and selective (CC(50) > 200 microM, EC(50) = 30 nM, IC(50) = 40 nM, SI > 6666). The 2,6-difluorophenylethylthymines (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 and the 2-methylthio, respectively. When the methyl at the benzylic carbon was replaced by an ethyl, activity was retained or decreased slightly, thus suggesting that the dimensions of the cavity within the RT hosting this substituent would not be compatible with groups larger than ethyl. Aryl moieties different from the 2,6-difluorophenyl (phenyl, 1-naphthyl, 2,6-dichlorophenyl) were generally detrimental to activity, consistent with a favorable electronic effect exerted by th...

Chiral HPLC separation and absolute configuration of novel S‐DABO derivatives

“…10 One compound related to the above derivatives and having two stereogenic centers was resolved into four stereoisomers by HPLC on Chiralpak IA chiral stationary phase (CSP) using ethyl acetate-based eluents. 11 The assignment of absolute configuration by X-ray diffraction analysis was not possible because of unfavorable physical properties of isolated stereoisomers and difficulty to obtain crystalline derivatives.…”

Substrate‐Induced Stable Enzyme–Inhibitor Complex Formation Allows Tight Binding of Novel 2‐Aminopyrimidin‐4(3H)‐ones to Drug‐Resistant HIV‐1 Reverse Transcriptase Mutants

“…Compounds MC1838 and MC1839, which share a chiral center at the benzylic position, were prepared and tested as racemates. In previous DABO studies, [9][10][11]15] the R enantiomer of related derivatives was predicted as the most active; studies are in progress to confirm this hypothesis for this series as well.…”

“…[2] Since then, a great number of oxopyrimidines have been synthesized and tested as anti-HIV-1 agents to obtain more potent and selective compounds. Structure-activity relationship (SAR) profiles of DABOs together with molecular modeling investigations of their putative binding modes have shown that the presence of a C2-alkoxy (DABOs), [3][4][5] -alkylthio (S-DABOs), [6][7][8][9] or -alkylamino (NH-DABOs) [10,11] side chain is a structural determinant for the antiviral activity of these derivatives. Moreover, 2,6-difluoro substitution on the C6-phenylmethyl moiety and the introduction of a methyl group at the pyrimidine C5-position and on the methylene bridge that connects the pyrimidine and phenyl rings (benzylic position) furnished new conformationally restricted compounds with nanomolar activity against wild-type HIV-1 and sub-micromolar activity against the Y181C mutant strain.…”

“…Moreover, 2,6-difluoro substitution on the C6-phenylmethyl moiety and the introduction of a methyl group at the pyrimidine C5-position and on the methylene bridge that connects the pyrimidine and phenyl rings (benzylic position) furnished new conformationally restricted compounds with nanomolar activity against wild-type HIV-1 and sub-micromolar activity against the Y181C mutant strain. [8][9][10][11] Cross-docking experiments performed by us on 68 different NNRTI-RT complexes suggested that a double N,N substitution on the 2-amino-DABO template can confer an implicit increased capacity for the molecules to re-adopt different experimental non-nucleoside binding site (NNBS) conformations by preventing the C2-NH group from establishing a further hydrogen bond in the NNBS. This information potentially leads to more flexible RT inhibitors with a broader range of activity against NNRTI-resistant mutants.…”

Computational modeling studies on anti-HIV-1 non-nucleoside reverse transcriptase inhibition by dihydroalkoxybenzyloxopyrimidines analogues: an electrotopological atomistic approach

“…Sudbeck et al have performed derivatization of DABO by modelling studies of potent 1-[(2-hydroxy ethoxy) methyl]-6-(phenylthio) thymine (HEPT) and by the substitution of benzyl moiety with phenyl thio moiety at the C-6 position of the pyrimidine ring [29]. A structure-activity relationship (SAR) profile of DABOs together with molecular modeling investigation on their putative binding mode have shown that the presence of a C-2-alkoxy (DABOs), C-2-alkylthio (S-DABOs), or C-2-alkylamino (NH-DABOs) side chain is the structural determinant for the antiviral activity of these derivatives, with the length and size of the C-2 side chain having only modulator effects on potency [30][31][32][33][34]. The 2,6-difluoro substitution at the C-6-benzyl moiety of S-DABOs and NH-DABOs produced favorable π-stacking interactions with the Tyr188 side chain into the non-nucleoside binding site (NNBS), leading to compounds (F2-S-DABOs and F2-NH-DABOs) active in the nanomolar range.…”