It has been proposed that the preference of 3‘-azido-3‘-deoxythymidine (AZT) for the extreme 3 E (south) conformation, as observed in its X-ray structure, is responsible for its potent anti-HIV activity. However, it has also been suggested that the antipodal north conformation may be required for the strong interaction of AZT 5‘-triphosphate with its target enzyme, HIV reverse transcriptase (RT). To resolve this issue, we have constructed two conformationally rigid carbocyclic analogues of AZT which are locked permanently into opposite 2 E (north) and 3 E (south) conformations in order to test the ability of the corresponding 5‘-triphosphates to inhibit RT. The two isomeric carbocyclic analogues of AZT, (N)-methano-carba-AZT (1) and (S)-methano-carba-AZT (2), were constructed on a bicyclo[3.1.0]hexane template that exhibits a rigid pseudoboat conformation, capable of mimicking the furanose pucker in the classical north and south conformations that are characteristic of standard nucleosides. The unique conformational properties of 1 and 2 observed by both X-ray and solution NMR studies showed the existence of the same invariant conformations in solution and in the solid state. In addition, differences observed in the outcome of the Mitsunobu inversion of a secondary hydroxyl function attempted with both bicyclo[3.1.0]hexane nucleoside analogues could be explained by the rigid pseudoboat nature of this system. In one case, the bicyclic system facilitated formation of an anhydronucleoside intermediate, whereas in the other it completely prevented its formation. The chemically synthesized 5‘-triphosphates of 1 and 2 were evaluated directly as RT inhibitors using both a recombinant enzyme and enzyme obtained and purified directly from wild-type viruses. The results showed that inhibition of RT occurred only with the conformationally locked 2 E (N)-methano-carba-AZT 5‘-triphosphate. This inhibition was equipotent to and kinetically indistinguishable from that produced by AZT 5‘-triphosphate. The antipodal 3 E (S)-methano-carba-AZT 5‘-triphosphate, on the other hand, did not inhibit RT.

2',3'-Dideoxy purine nucleosides have anti-HIV activity in vitro and the inosine analogue is being clinically evaluated. The instability of these compounds toward acidic conditions complicates oral administration. The effect of the addition of a fluorine atom to the 2'-position was investigated by preparing the fluorine-containing 2'-erythro and 2'-threo isomers of ddA and the threo isomer of ddI. All fluorine-containing compounds were indefinitely stable to acidic conditions which completely decomposed ddI (1) and ddA (2) in minutes. While the fluorine-containing erythro isomer, 5, was inactive, the threo isomers, 2'-F-dd-ara-A (3) and 2'-F-dd-ara-I (4), were just as potent and active in protecting CD4+ ATH8 cells from the cytopathogenic effects of HIV-1 as the parent drugs. Exposure to pH 1 at 37 degrees C prior to testing destroyed the activity of ddA and ddI but left the anti-HIV properties of 3 and 4 unchanged. The fluorinated analogues also protected cells exposed to HIV-2 and inhibited gag gene product expression but not as effectively as the parent compounds. The fluorine-containing analogues appear to be somewhat more toxic in vitro to the antigen- and mitogen-driven proliferation of immunocompetent cells than their corresponding parent compounds.

A major obstacle to the success of gene therapy strategies with ME180 tumors. Treatment of tumors with Ad.TK RC that directly target cancer cells is the poor vector distriwithout GCV resulted in a similar antitumor effect, conbution within solid tumors. To address this problem, we firming that the replicating vector has an oncolytic effect. developed an E1b 55 kDa attenuated, replicationWhen GCV was initiated 3 days after Ad.TK RC injection, competent adenovirus (Ad.TK RC ) which expresses the hersurvival of mice with each tumor type was greatly propes simplex-1 thymidine kinase (HSVtk) gene to sensitize longed, with 60% of animals with ME180 tumors surviving tumors to ganciclovir (GCV). Efficacy of this combined for over 160 days. These results confirm that both the strategy was tested in nude mice with subcutaneous oncolysis caused by a replicating virus and suicide/prodrug human A375 melanoma and ME180 cervical carcinomas.gene therapy with HSVtk/GCV have potent antitumor Intratumoral injection of a replication-defective adenoviral effects. When combined, these two approaches are compvector expressing HSVtk (Ad.TK) followed by GCV treatlementary resulting in a significantly improved treatment ment resulted in doubling of the survival time of mice bearoutcome. ing A375 tumors and 20% long-term survival of mice