Myristoylated derivatives of 2',3'-didehydro-2',3'-dideoxythymidine (stavudine) bi-functional prodrugs with potent anti-HIV-1 activity and low cytotoxicity.

BACKGROUND: To improve in vitro antiviral activity and selectivity of stavudine (d4T), a range of its bi-functional prodrugs, 5'-O-myristoylated derivatives, have been synthesized. METHODS: Stavudine 5'-O-myristoylated esters were synthesized using modified Parang's procedure. The cytotoxicity and anti-HIV activity was evaluated in the established MT-4 cell line. The level of p24 protein in culture medium was assayed, and EC50 and EC90 values were determined. RESULTS: Excellent anti-HIV activity was obtained for stavudine derivatives 2',3'-didehydro-2',3'-dideoxy-5'-O-(11-thioethylundecanoyl) thymidine, 2',3'-didehydro-2',3'-dideoxy-5'-O-(12-thioethyldodecanoyl) thymidine and 5'-O-(12-azidododecanoyl)-2',3'-didehydro-2',3'-dideoxythymidine with C10 and C11 alkyl chains bearing thioethyl- and azido- substituents. These prodrugs were more potent than the parent stavudine, as is clear from their EC50 values: 2',3'-didehydro-2',3'-dideoxy-5'-O-(11-thioethylundecanoyl) thymidine (R=CO(CH2)10SC2H5, EC50 0.06 µM), 2',3'-didehydro-2',3'-dideoxy-5'-O-(12-thioethyldodecanoyl) thymidine (R=CO(CH2)11SC2H5, EC50 0.09 µM) and 5'-O-(12-azidododecanoyl)-2',3'-didehydro-2',3'-dideoxythymidine (R=CO(CH2)11N3, EC50 0.06 µM), while 50% cytotoxic concentration was >16.65 µM, >7.5 µM and >18.53 µM, respectively. CONCLUSIONS: Overall data demonstrate that compounds 2',3'-didehydro-2',3'-dideoxy-5'-O-(11-thioethylundecanoyl) thymidine, 2',3'-didehydro-2',3'-dideoxy-5'-O-(12-thioethyldodecanoyl) thymidine and 5'-O-(12-azidododecanoyl)-2',3'-didehydro-2',3'-dideoxythymidine are very potent and selective anti-HIV agents and could be useful in treatment of HIV infections of the central nervous system.

Thiated derivatives of 2',3'-dideoxy-3'-fluorothymidine: synthesis, in vitro anti-HIV-1 activity and interaction with recombinant drug resistant HIV-1 reverse transcriptase forms.

Various thiated analogues of thymine 2',3'-dideoxy-3'-fluoronucleoside (FLT) and their 5'-monophosphates and 5'-triphosphates were prepared with the use of modified multistep procedures. The thiated analogues of FLT and FLTMP were evaluated against the wild type and drug- and multidrug-resistant strains of HIV-1, using the replicative phenotyping format of the deCIPhR assay, and showed potent inhibition of drug-resistant HIV-1 strains at low cytotoxicity. Additionally, inhibition of recombinant drug resistant forms of reverse transcriptase from single and multiple HIV-1 mutants by the synthesized 5'-triphosphates was investigated. The strongest inhibition was observed for K103N and Δ67 mutants and the most potent anti-HIV-1 activity against drug resistant strains and the lowest cytotoxicity was exerted by S4FLTMP and FLTMP which may be regarded as potential anti-HIV/AIDS agents.

Partial selective inhibition of HIV-1 reverse transcriptase and human DNA polymerases γ and ß by thiated 3'-fluorothymidine analogue 5'-triphosphates.

3'-Deoxy-3'-fluorothymidine (FLT, alovudine(®)) belongs to the most potent agents inhibiting HIV-1 replication. Its 5'-triphosphate (FLTTP) is a potent inhibitor of HIV-1 reverse transcriptase (HIV RT). Unfortunately, FLT exerts substantial hematologic toxicity both in vitro and in vivo. It was suggested that this toxicity may be related to inhibition of human DNA polymerases, especially mitochondrial DNA polymerase γ, by nucleoside analogue 5'-triphosphates leading to termination of DNA synthesis and mitochondrial dysfunction. To decrease the toxicity of FLT, its thiated analogues, 4-SFLT and 2-SFLT, were previously synthesized and shown to be potent inhibitors of HIV-1 with low in vitro cytotoxicity. To explain this phenomenon in the present study the synthesis of 5'-triphosphates of thiated FLT analogues was undertaken and their interaction with recombinant HIV-1 RT and human DNA polymerases γ (pol γ) and ß (pol ß) was investigated. It was shown that 3'-deoxy-3'-fluoro-4-thiothymidine 5'-triphosphate (4-SFLTTP) and 3'-deoxy-3'-fluoro-2-thiothymidine 5'-triphosphate (2-SFLTTP) were, similarly to FLTTP, potent competitive inhibitors of HIV-1 RT, with K(i)(app) values of 0.091 and 0.022 µM respectively. It is of interest that 2-SFLTTP, a compound in an unusual syn conformation around the glycosidic bond was an uncompetitive inhibitor of human mitochondrial DNA pol γ with K(i)(app) of 0.174 µM, while 4-SFLTTP in anti conformation inhibited this enzyme similarly to FLTTP, i.e., non-competitively, with K(i)(app) of 0.055 µM. Both 4-SFLTTP and 2-SFLTTP were competitive inhibitors of human DNA pol ß, with K(i)(app) values of 16.84 and 4.04 µM, respectively. The results point to partially selective inhibition of HIV RT by thiated 3'-fluorothymidine 5'-triphosphate analogues. Of special interest is that 2-SFLTTP, showing syn conformation, is a less potent inhibitor of human mitochondrial pol γ than 4-SFLTTP and FLTTP, both in the anti conformation, and has a higher inhibitory activity against HIV-1 RT than 4-SFLTTP. Moreover, the parent nucleoside 2-SFLT possessing the syn conformation shows a more potent anti-HIV-1 activity and a better selectivity index than its 4-thio isomer in the anti conformation (Matthes et al., 1989; Poopeiko et al., 1995), 2-SFLT is a potent and selective anti-HIV-1 agent with the selectivity index 4-fold higher than that of FLT. Findings regarding the mechanisms of antiviral and cytotoxic activities of FLT and its thioanalogues are discussed.

Synthesis and anti-HIV properties of novel 6-phenylselenenyl-5-propyluracils.

Novel 6-phenylselenenyl-5-propyluracils were synthesized from 5-propyluracil with the use of regioselective synthesis to give 1-[(2-hydroxyethoxy)-methyl]-6-phenylselenenyl-5-propyluracil (6), 1-ethoxymethyl-6-phenylselenenyl-5-propyluracil (9) and 1-benzyloxymethyl-6-phenylselenenyl-5-propyluracil (10). Interaction of these compounds with recombinant HIV-1 reverse transcriptase (RT) was evaluated using a non-isotopic colorimetric method. Compounds 9 and 10 exerted potent HIV RT inhibition (IC(50) 0.06 and 0.05 microM respectively) while compound 6 showed moderate inhibition (IC(50) = 3.5 microM). Potent anti-HIV-1 activity in MT-2 cells inoculated by a syncythia-inducing HIV-1 (cat #3 strain) laboratory isolate was exerted by compounds 9 and 10 (EC(50) 0.62 microM and 0.025 microM, respectively), while compound 6 showed only moderate activity (IC(50) = 4.1 microM). In addition, compound 10 showed very good in vitro therapeutic index (TI > 2046), indicating that it is a potential anti-HIV/AIDS drug.