2′,3′‐Dideoxyadenosine (ddA), 2′,3′‐didehydro‐2′,3′‐dideoxyadenosine (d4A) and their lipophilic 5′‐monophosphate triester (aryloxyphosphoramidate) prodrugs were evaluated for their anti‐retrovirus and anti‐hepatitis B virus activity in various cell culture models. The aryloxyphosphoramidate derivatives of ddA (Cf 1093) and d4A (Cf 1001) showed markedly superior (100–1000‐fold) efficacies than the parent drugs against human immunodeficiency virus type 1 (HIV‐1), HIV‐2, simian immunodeficiency virus (SIV), Moloney murine sarcoma virus (MSV) and human hepatitis B virus (HBV) replication regardless of the cell type in which the virus replication was studied (i.e., human T‐lymphocyte CEM, MT‐4, Molt/4 and C8166 cells, peripheral blood lymphocytes (PBL), monocyte/macrophages (M/M), murine embryo fibroblasts and human hepatocyte cells). Also the selectivity index (ratio of cytotoxic concentration/antivirally effective concentration) of both aryloxyphosphoramidate prodrugs was markedly increased. In particular the d4A prodrug Cf 1001 showed a selectivity index of 300–3000 as compared with 2–3 for the parental d4A in established laboratory cell lines. Also Cf 1001 had a selectivity index of 400–650 in HIV‐1‐infected PBL and M/M, respectively. Both Cf 1001 and Cf 1093 were equally efficient as 3TC (lamivudine) in inhibiting HBV replication in hepatocytes, and rank among the most potent HIV and HBV inhibitors reported so far in cell culture.

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A novel membrane-soluble prodrug of the 5-monophosphate derivative of 3TC containing a phenyl group and the methyl ester of L-alanine linked to the phosphorus through a phosphoramidate bond with the primary amino moiety (designated Cf 1109) was prepared. The 3TC prodrug proved less potent an inhibitor of HIV-1 and HIV-2 replication in CEM cell cultures than 3TC, but lost only 20-fold antiviral potency in 2-deoxycytidine kinase-deficient CEM/dCK 0 cells compared with a more than 2,000-fold decrease of activity of 3TC. In contrast, 3TC and Cf 1109 proved equally highly effective in inhibiting HBV release in supernatants of HBV-transfected Hep G2 2.2.15 cell cultures (50% effective concentration Ç 0.02 mM). Both compounds easily selected for highly resistant HIV-1 strains at a comparable speed of breakthrough. The mutant viruses contained an 184-Ile and/or 184-Val amino acid change in their reverse transcriptase. Our data are suggestive for a relatively poor delivery of 3TC-MP in the intact CEM cells but a remarkably high delivery of 3TC and/or 3TC-MP in the intact Hep G2 2.2.15 cells. ᭧ 1996 Academic Press, Inc.The most recently approved 2,3-dideoxynucleoside (ddN) derivative for treatment of HIV infections is 2,3-dideoxy-3-thiacytidine (3TC, lamivudine) (Fig. 1). This drug is also subject of clinical trials in human hepatitis B virus (HBV)-infected patients. In contrast with the other approved (b-D) ddN derivatives has 3TC the b-L(0) isomeric conformation. 3TC needs to be converted to its 5-triphosphate derivative by cellular enzymes to be inhibitory to the reverse transcriptase (RT) of HIV, and is endowed with low if any cytotoxicity (1-5). However, the first activation (phosphorylation) step is catalysed by cytosolic 2-deoxycytidine kinase (dCK) which has a relatively poor affinity for 3TC (K i /K m Ç 50) resulting in a moderate efficiency of conversion of 3TC to its 5-monophosphate derivative (6). In addition, a potential drawback of 3TC is the rapid emergence of drug-resistant HIV-1 strains (7-11). Indeed, mutant virus strains that emerged in the presence of 3TC were shown to contain an amino acid change from methionine (Met) to valine (Val) or isoleucine (Ile) at position 184 of the RT. Such virus is highly resistant to the inhibitory effects of 3TC in cell culture (7-11), and is reported to show low-level cross-resistance to 2,3-dideoxycytidine (ddC) (Fig. 1) and 2,3-dideoxyinosine (ddI).In an attempt to circumvent the first intracellular phosphorylation step of 3TC, we synthesized the arylphosphoramidate derivative of 3TC containing a methylester of L-alanine linked to the 5-phosphate of 3TC-MP through its primary amino group (Fig. 1). The corresponding 2,3-didehydro-2,3-dideoxythymidine (d4T, stavudine) analogue (So324) was previously shown to be able to deliver directly the 5-monophosphate derivative of d4T into intact cells and to efficiently bypass the first (thymidine kinase-directed) phosphorylation of d4T (12,13). The antiviral potency and resistance profile of Cf 1109 is described compared to 3TC.

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The anti-human immunodeficiency virus (HIV) activity of aryloxyphosphoramidate protides of a number of anti-HIV nucleoside analogues was assessed in resting primary monocyte-macrophages (M/M). While 2,3-dideoxythymidine (d4T), 2,3-dideoxyadenosine (ddA), and 2,3-dideoxy-2,3-didehydroadenosine (d4A) protides showed an anti-HIV activity that was 25-to 625-fold greater than the parent nucleotides d4T, ddA, and d4A, respectively, other aryloxyphosphoramidate protides showed similar or even lower anti-HIV activities than their parent compounds. This variable anti-HIV effect is most likely related to the different dynamics of intracellular nucleoside monophosphate release from the protides. Our results indicate the potential advantage of therapeutic use of this approach for some nucleotide analogues to affect HIV replication in M/M, one of the major reservoirs of HIV in vivo.In all body compartments, resting monocyte-macrophages (M/M) can be infected by human immunodeficiency virus (HIV) (1, 12-14, 27, 34, 37). Because of their resistance to the cytopathic effect of the virus, M/M are considered the most relevant reservoir of HIV in the body (10,18,26,31) and a crucial target for a successful therapeutic approach (2,3,35).M/M are characterized by some cellular peculiarities that affect not only virus replication (6, 28; S. Aquaro, P. Bagnarelli, M. Clementi, T. Guenci, R. Calio', and C. F. Perno, Program Abstr. 6th Conf. Retrovir. Opportunistic Infect., abstr. 607, 1999.) but also the activity of antiviral drugs (31, 33). Indeed, their resting status, and thus their limited DNA synthesis, does not require, for physiological functions, high intracellular levels of 2Ј-deoxynucleotide pools. As a consequence, and despite the limited phosphorylation of anti-HIV nucleoside analogues typically found in M/M, the ratio of the triphosphate forms of nucleoside reverse transcriptase inhibitors (NRTIs) to their natural triphosphorylated 2Ј-deoxynucleotide counterparts is higher than that found in lymphocytes (4). Thus, not surprisingly, the currently approved NRTIs for clinical use, i.e., azidothymidine (AZT), 2Ј,3Ј-dideoxyinosine, 2Ј,3Ј-dideoxycytidine (ddC), (Ϫ)(L)-3Ј-thia-2Ј,3Ј-dideoxycytidine (3TC), 2Ј,3Ј-didehydro-2Ј,3Ј-dideoxythymidine (d4T), show anti-HIV efficacies in M/M greater than those found in lymphocytes (4).Despite this increased anti-HIV efficacy in M/M, the in vivo antiviral activity of most NRTIs in sequestered compartments is still suboptimal for a number of reasons, including the limited penetration of most NRTIs in sanctuaries (11, 16) and the high expression of p170 glycoprotein in M/M (17), that may affect the overall intracellular drug concentrations in M/M. Therefore, attempts should be made to increase the intracellular concentration of the triphosphate forms of the NRTIs in M/M.To conveniently bypass the first phosphorylation step of NRTIs, masked nucleoside 5Ј-monophosphate (MP) prodrugs have been synthesized. These compounds were designed to act as monophosphorylated membrane-soluble prodrugs of the ...