Publications

Starting from ethyl (1S,4R)-4-hydroxy-2-methylcyclopent-2-ene-1-carboxylate, as a common enantiopure building block readily obtained by enzymatic kinetic resolution of the corresponding racemic derivative, 3′-methylcarbovir, 3′-methylabacavir, and three modified analogues were synthesized via the linear construction of the purine heterocycles. These derivatives were evaluated as potential agents against HIV-1, HIV-2, and other important viral pathogens. None of the new compounds had significant antiviral activity at a concentration of 100 µg˙mL-¹, which was the highest concentration tested.

Two prominent members of the ATP-binding cassette superfamily of transmembrane proteins, multidrug resistance 1 (MDR1) P-glycoprotein and multidrug resistance protein 1 (MRP1), can mediate the cellular extrusion of xenobiotics and (anticancer) drugs from normal and tumor cells. The MRP subfamily consists of at least six members, and here we report the functional characterization of human MRP5. We found resistance against the thiopurine anticancer drugs, 6-mercaptopurine (6-MP) and thioguanine, and the anti-HIV drug 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in MRP5-transfected cells. This resistance is due to an increased extrusion of PMEA and 6-thioinosine monophosphate from the cells that overproduce MRP5. In polarized Madin-Darby canine kidney II (MDCKII) cells transfected with an MRP5 cDNA construct, MRP5 is routed to the basolateral membrane and these cells transport S-(2,4-dinitrophenyl)glutathione and glutathione preferentially toward the basal compartment. Inhibitors of organic anion transport inhibit transport mediated by MRP5. We speculate that MRP5 might play a role in some cases of unexplained resistance to thiopurines in acute lymphoblastic leukemia and/or to antiretroviral nucleoside analogs in HIV-infected patients.

A varicella-zoster virus (VZV) strain resistant to 5-iodo-2'-deoxyuridine (IdUrd) and 5-bromo-2'-deoxyuridine (BrdUrd) but sensitive to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVdUrd) was isolated. The 2'-deoxythymidine (dThd) kinase of this mutant (Ito) strain was characterized; it was much less efficient in phosphorylating dThd, 2'-deoxycytidine, and BrdUrd than were the dThd kinases from wild-type (CaQu, Kobayashi) VZV strains. The Ito dThd kinase had a markedly decreased affinity for dThd, 2'-deoxycytidine, and BrdUrd but only a slightly decreased affinity for IVdUrd than had the wild-type VZV dThd kinase. BrdUrd was incorporated to a much lesser extent in VZV (Ito strain)-infected cells than wild-type VZV-infected cells, but IVdUrd was incorporated in Ito VZV-infected cells as efficiently as in wild-type VZV-infected cells. While resistant to IdUrd and BrdUrd, the Ito strain was susceptible to inhibitors of de novo thymidylate biosynthesis such as aminopterin.

2'-Deoxyuridines with a five-membered heterocyclic substituent in the 5-position were synthesized by palladium-catalyzed coupling reactions of 5-iodo-2'-deoxyuridine with the activated heteroaromatics. Further modification of the compound with the 5-thien-2-yl substituent gave 5-(5-bromothien-2-yl)-2'-deoxyuridine and 5-(5-chlorothienyl-2-yl)-2'-deoxyuridine. Both compounds show potent and selective activity against herpes simplex virus type 1 and varicella-zoster virus.

By using an assay system based on a human hepatoblastoma cell line (HB611) that continuously synthesizes hepatitis B virus DNA, the following compounds were found to inhibit hepatitis B virus DNA synthesis at concentrations that were significantly lower than their minimum cytotoxic concentrations: 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, 9-(phosphonylmethoxyethyl)adenine, 2',3'-dideoxy-2',3'-didehydrocytidine, and 2',3'-dideoxycytidine. The most potent compound was PMEDAP (50% effective concentration, 0.02 micrograms/ml). The selective index, or ratio of the 50% cytotoxic concentration to 50% effective concentration, of PMEDAP was greater than 750.

The study of strategies for the treatment of human cytomegalovirus (HCMV) infection starts with the discovery of compounds that are potent and selective inhibitors of HCMV replication. Selectivity means that the window between the concentrations that block viral replication on the one hand and those that affect host cell functions on the other is sufficiently wide. Because HCMV induces an obvious cytopathic effect (CPE) in human fibroblast cultures, potential antiviral activity can easily be scored microscopically. For this type of assay, a relatively low input of virus should be used, so that the CPE does not appear within the first 2-3 d after infection. If too high a multiplicity of infection (MOI) is used, the input virus will result in a substantial amount of CPE shortly (i.e., within 24 h) after infection. As the CPE induced by this input virus will not be blocked by compounds that inhibibit new progeny virus formation, the use of a high MOI will mask a potential inhibitory effect of a given compound on HCMV replication. Scoring of CPE can be done either by counting the HCMV-induced plaques (although this is a rather labor-intensive method for large-scale screening purposes) or simply by scoring the CPE on a scale of 1-5 (or 10). Each assay should always be validated by using a reference compound with proven anti-HCMV activity (e.g., ganciclovir, cidofovir, foscarnet).

Abstract Conjugate addition of an anionic nucleophile (nucleobase) to an active triple bond (α, β unsaturated carboxylate or phosphonate) was used for preparing α-ethenyl carboxylate or phosphonate derivatives of purines and pyrimidines.