BACKGROUND: To improve the existing combination therapies of infection with the human immunodeficiency virus (HIV) and to cope with virus strains that are resistant to multiple drugs, we initiated a search for effective inhibitors of HIV integrase, the enzyme responsible for inserting the viral cDNA into the host cell chromosome.RESULTS: We have now identified a series of 5H-pyrano[2,3-d:-6,5-d']dipyrimidines that block the replication of various strains of HIV-1 and HIV-2. The most potent congener, 5-(4-nitrophenyl)-2,8-dithiol-4,6-dihydroxy-5H-pyrano[2,3-d:-6,5-d']dipyrimidine (V-165), inhibited the replication of HIV-1(III(B)) in MT-4 cells at a 50% effective concentration (EC(50)) of 8.9 microM, which is 14-fold below its cytotoxic concentration. V-165 was equally active against virus strains that were resistant toward inhibitors of viral entry or reverse transcriptase. In combination regimens in cell culture, V-165 acted subsynergistically with zidovudine or nelfinavir and synergistically with nevirapine. V-165 inhibited both reverse transcriptase and integrase activities in enzymatic assays at micromolar concentrations, but only a close correlation was found between the anti-HIV activity observed in cell culture and the inhibitory activity in the integrase strand transfer assays. Time-of-addition experiments indicated that V-165 interfered with the viral replication cycle at a time point coinciding with integration. Quantitative Alu-PCR corroborated that the anti-HIV activity of V-165 is based upon the inhibition of proviral DNA integration.CONCLUSIONS: Based on their mode of action, which is different from that of clinically approved anti-HIV drugs, PDPs are good candidates for further development into new drugs and to be included in future combination regimens.