Several 6-phenylselenenyl-substituted acyclouridine derivatives were prepared for evaluation as antiviral agents. Lithiation of the tert-butyldimethylsilyl-protected acyclonucleosides 4a-f with lithium diisopropylamide at -78 degrees C, followed by reaction with diphenyl diselenide as an electrophile, and subsequent removal of the protecting group with tetra n-butylammonium fluoride gave 1-[(2-hydroxyethoxy)methyl]-6-(phenylselenenyl)uracils 6a-f in 50-70% overall yield. The potency and spectrum of activity of compounds 6a-f against HIV-1 in vitro was similar to HEPT (1), a related 6-phenylthio acyclonucleoside. However, whereas HEPT inhibited HIV-1 reverse transcriptase, the selenium-containing derivatives were ineffective, suggesting a different mechanism of action. Of significance was the finding that the 6-phenylselenenyl acyclonucleosides inhibited also HIV-2 in primary human lymphocytes.
Lithiation of 5-bromo-2,4-bis(benzyloxy)pyrimidine (3) with n-BuLi at -80 degrees C followed by the addition of diphenyl diselenide or diphenyl disulfide as an electrophile furnished the corresponding 5-(phenylhetera)-2,4-bis(benzyloxy)pyrimidine, which on exposure to trimethylsilyl iodide in CH2-Cl2 at room temperature yielded the 5-(phenylhetera)uracils in 70-75% yield. Similarly, the 6-(phenylhetera)uracils were prepared from 6-bromo-2,4-bis(benzyloxy)pyrimidine (10). 1-[(2-Hydroxyethoxy)methyl]-5-(phenylselenenyl)uracil (PSAU, 18) and 1-(ethoxymethyl)-5-(phenylselenenyl)uracil (17) were synthesized by the electrophilic addition of benzeneselenenyl chloride to the acyclic uracils under basic conditions. These compounds were evaluated for their ability to inhibit dihydrouracil dehydrogenase (DHUDase, E.C. 1.3.1.2), orotate phosphoribosyltransferase (OPRTase, E.C. 2.4.2.10), uridine phosphorylase (UrdPase, E.C. 2.4.2.3), and thymidine phosphorylase (dThdPase, E.C. 2.4.2.4). 5-(Phenylselenenyl)uracil (PSU, 6) and 5-(phenylthio)uracil (PTU, 7) inhibited DHUDase with apparent K(i) values of 4.8 and 5.4 microM, respectively. The corresponding 6-analogues, compounds 13 and 14, demonstrated inhibitory activity against OPRTase. PTU as well as PSU and its riboside, 2'-deoxyriboside, and acyclonucleosides were inhibitors of UrdPase, with PSAU (18) being the most potent with an apparent K(i) value of 3.8 microM. None of the compounds evaluated had any effect on dThdPase. Interestingly, most of the compounds showed modest selective anti-human-immunodeficiency-virus activity in acutely infected primary human lymphocytes.
Acyclic 6-phenylselenenyl- and 6-phenylthiopyrimidine derivatives are potent and specific inhibitors of human immunodeficiency virus type 1 (HIV-1). The development of in vitro resistance to two derivatives, 5-ethyl-1-(ethoxymethyl)-(6-phenylthio)-uracil (E-EPU), was evaluated by serial passage of HIV-1 in increasing concentrations of inhibitor. HIV-1 variants exhibiting > 500-fold resistance to E-EPSeU and E-EPU were isolated after sequential passage in 1, 5, and 10 microM inhibitor. The resistant variants exhibited coresistance to related acyclic 6-substituted pyrimidines and the HIV-1-specific inhibitors (+)-(5S)-4,5,6,7-tetrahydro-5- pyrimidines and the HIV-1-specific inhibitors (+)-(5S)-4,5,6,7-tetrahydro-5- methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jk]benzodiazepin-2(1H)- thione (TIBO R82150) and nevirapine, but remained susceptible to 3'-azido-3'-deoxythymidine, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and phosphonoformic acid. DNA sequence analysis of reverse transcriptase (RT) derived from E-EPSeU-resistant virus identified a Tyr (TAT)-to-Cys (TGT) mutation at either codon 188 (Cys-188; 9 of 15 clones) or codon 181 (Cys-181; 5 of 15 clones). The same amino acid changes were found in RT from E-EPU-resistant virus, but the Cys-181 mutation was more common (9 of 10 clones) than the Cys-188 mutation (1 of 10 clones). Site-specific mutagenesis and production of mutant recombinant viruses demonstrated that both the Cys-181 and Cys-188 mutations cause resistance to E-EPSeU and E-EPU. Of the two mutations, the Cys-188 substitution produced greater E-EPSeU and E-EPU resistance. The predominance of the Cys-188 mutation in E-EPSeU-resistant variants has not been noted for other classes of HIV-1 specific RT inhibitors. HIV-1 resistance is likely to limit the therapeutic efficacy of acyclic 6-substituted pyrimidines if they are used as monotherapy.
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