Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine.

The phosphoramidate triester prodrugs of anti-human HIV 2', 3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-monophosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2', 3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metabolism of triester prodrugs of d4T and AZT using pure enzymes or different biological media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-containing intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme preparations. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alkaline phosphatase, and phosphodiesterase.     

Substrate specificity of human deoxycytidine kinase toward antiviral 2',3'-dideoxynucleoside analogs.

Deoxycytidine (dCyd) kinase has been purified to homogeneity from human leukemic spleen, and the capacity of the enzyme to phosphorylate 2',3'-dideoxynucleoside (ddN) analogs that are clinically effective inhibitors of human immunodeficiency virus (HIV) replication was evaluated. Cytosine-containing ddN analogs, such as 2',3'-dideoxycytidine, 2',3'-dideoxy-2',3'-dehydrocytidine, and cytallene, were efficiently phosphorylated by dCyd kinase, while no phosphorylation of purine-containing ddN analogs was detected. dCyd kinase was completely inactive toward 2',3'-dideoxyadenosine (ddAdo), 2',3'-dideoxyinosine, 2',3'-dideoxyguanosine, and adenallene, although it was capable of phosphorylating both 2'-deoxyadenosine (dAdo) and 2'-deoxyguanosine (dGuo). The abilities of wild type and mutant human T lymphoblastoid CEM cells to accumulate ddAdo in situ and in vitro were also ascertained. Comparison of the abilities of intact wild type CEM cells and derivatives deficient in nucleoside transport, dCyd kinase, and/or adenosine (Ado) kinase to accumulate [3H]ddAdo-derived radioactivity revealed no significant differences among the wild type and mutant strains. However, ddAdo phosphorylating activity was decreased in extracts from Ado kinase-deficient cells but not in lysates prepared from cells genetically deficient in dCyd kinase activity. In comparative growth rate experiments, wild type, nucleoside transport-deficient, and dCyd kinase-deficient CEM cells were equally sensitive to ddAdo toxicity, while, interestingly, a deficiency in Ado kinase correlated with a 5-fold decreased growth sensitivity to the purine ddN. Insertion of an adenine phosphoribosyltransferase deficiency into the CEM cell lines did not influence ddAdo toxicity or incorporation rate. These results imply that Ado kinase may be an important factor in ddAdo phosphorylation by CEM cells. Furthermore, these studies demonstrate that cytosine- and purine-containing ddNs are transported and activated by independent pathways and, therefore, have important implications for anti-HIV therapy in that pyrimidine and purine ddNs might be used in combination for the treatment of acquired immunodeficiency syndrome.     

Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5

The human multidrug resistance proteins MRP4 and MRP5 are organic anion transporters that have the unusual ability to transport cyclic nucleotides and some nucleoside monophosphate analogs. Base and nucleoside analogs used in the chemotherapy of cancer and viral infections are potential substrates. To assess the possible contribution of MRP4 and MRP5 to resistance against these drugs, we have investigated the transport mediated by MRP4 and MRP5. In cytotoxicity assays, MRP4 conferred resistance to the antiviral agent 9-(2-phosphonomethoxyethyl)adenine (PMEA) and high-performance liquid chromatography analysis showed that, like MRP5, MRP4 transported PMEA in an unmodified form. MRP4 also mediated substantial resistance against other acyclic nucleoside phosphonates, whereas MRP5 did not. Apart from low-level MRP4-mediated cladribine resistance, the cytotoxicity of clinically used anticancer nucleosides was not influenced by overexpression of MRP4 or MRP5. In contrast, MRP5 mediated efflux of the pyrimidine-based antiviral 2',3'-dideoxynucleoside 2',3'-didehydro-2',3'-dideoxythymidine 5'-monophosphate (d4TMP) and its phosphoramidate derivative alaninyl-d4TMP from cells loaded with the 2',3'-didehydro-2',3'-dideoxythymidine prodrugs cyclosaligenyl-d4TMP and aryloxyphosphoramidate d4TMP (So324), respectively. Moreover, only inside-out membrane vesicles derived from MRP5-overexpressing cells accumulated alaninyl-d4TMP. Cellular efflux and vesicular uptake studies were carried out to further compare transport mediated by MRP4 and MRP5 and showed that dipyridamole, dilazep, nitrobenzyl mercaptopurine riboside, sildenafil, trequinsin and MK571 inhibited MRP4 more than MRP5, whereas cyclic nucleotides and monophosphorylated nucleoside analogs were equally poor inhibitors of both pumps. These results strongly suggest that the affinity of MRP4 and MRP5 for nucleotide-based substrates is low.     

Potent nonclassical nucleoside antiviral drugs based on the N,N-diarylformamidine concept

New formamidine-3TC (3TC = 2',3'-dideoxy-3'-thiacytidine) analogues have been synthesized through various methods, and their antiviral activities (HIV, HBV) have been evaluated in vitro. Anti-HIV-1 in acutely infected MT-4 cells and peripheral blood monocellular cells (PBMCs) showed that compounds substituted by N,N-diarylformamidine side chains at the 4-N nucleic base position (compounds 3 and 8-11) had at least equivalent anti-HIV activity as 3TC (EC50 = 0.5 and 11.6 microM, respectively). Moreover, the newly synthesized compounds demonstrated higher anti-HBV activity (EC50 ranging from 0.01 to 0.05 microM) compared to the parent nucleoside 3TC (EC50 = 0.2 microM). It should be underlined that these new promising derivatives inhibited HIV in cells of a macrophage lineage, which are known to be cellular reservoir for HIV. These results were particularly of interest, since the antiviral activities appeared not to be mediated through the formamidine bond hydrolysis and consequently the release of free 3TC. These new analogue series were found to be highly stable to hydrolysis even after prolonged incubation in different biological media (t(1/2) ranged from 48 to 120 h). This enzymatic stability, coupled to the fact that no delay in the antiviral response was observed compared to the free 3TC antiviral response, suggest that this new N,N-diarylformamidine nucleoside series should not be considered as classical prodrugs.     

Improved uptake and retention of lipophilic prodrug to improve treatment of HIV

Dideoxynucleosides currently in use for anti-HIV therapy have been found to be inefficient in passing through the blood-brain barrier to enter and maintain therapeutic drug levels in brain, a very significant reservoir of HIV. The low bioavailability of these drugs combined with the bone marrow toxicity of AZT (3'-azido, 3'-deoxythymidine, Zidovudine), resulting in anemia and leukopenia, pancreatitis with ddI (2',3'-dideoxyinosine, Didanosine) and painful peripheral neuropathy in case of ddC (2',3-dideoxycytosine, Zalcitabine) are the limiting factors in their use. In addition, the emergence of strains of HIV resistant to AZT, the most commonly used drug, further restricts its use. Thus the control of AIDS and its complications, needs special therapeutic approaches to combat the disease. In order to overcome these limitations, AZT and ddI have been synthesized as ester-linked ceramide- and phosphatidylcholine-linked prodrugs possessing therapeutic attributes lacking in the parent compounds. There is greater uptake and longer retention of these prodrugs in NIH/3T3 cells in vitro. Pretreatment with our prodrugs blocked infection of these cells by Moloney murine leukemia virus (M-MuLV) for an extended period, which the parent drugs failed to do. When human CD4+ HeLa cells were continuously exposed to the AZT prodrug, subsequent infection of these cells by HIV was blocked. Similar results were obtained with NIH/3T3 cells exposed to M-MuLV. AE(6)C, a prodrug of AZT linked to ceramide via a cleavable ester bond and a six carbon linker, was less toxic to both mouse and human bone marrow progenitor cells than free AZT. Most significantly, the prodrugs concentration was greater and the retention longer, in well known sanctuaries for HIV, such as the brain, testes and thymus.     

cycloSal-Pronucleotides of 2'-fluoro-ara- and 2'-fluoro-ribo-2',3'- dideoxyadenosine as a strategy to bypass a metabolic blockade

Novel, lipophilic cycloSal triesters 4a-c and 5a-c were synthesized, respectively, from the ara- and ribo-configurated 2'-fluorinated-2', 3'-dideoxyadenosines 2 and 3. The cycloSal phosphotriesters were used as tools to study the effects of the two different sugar pucker conformations induced by two opposite configurations of the fluorine substituent at C2' of the dideoxyribose moiety. F-ara-ddA (2) is known to be an active anti-HIV agent, whereas the ribo-analogue 3 is inactive. Hydrolysis studies with the triester precursors 4a-c and 5a-c showed selective formation of the monophosphates of 2 and 3. The lipophilicity of the triester prodrugs was considerably increased by the cycloSal mask with respect to ddA (1), F-ara-ddA (2), and F-ribo-ddA (3). Phosphotriesters 4 and 5 proved to be completely resistant to ADA and AMPDA deamination. In parallel experiments, ribo-nucleoside 3 showed a 50-fold faster deamination rate relative to the ara-analogue 2. Against HIV in CEM cells, the phosphotriesters 4 proved to be 10-fold more potent than the parent nucleoside 2. Furthermore, the prodrugs 4 were active against MSV-induced transformation of C3H/3T3 fibroblasts, while 2 was inactive. More interestingly, the ribo-configurated phosphotriesters 5, prepared from the inactive F-ribo-ddA (3), showed a level of anti-HIV activity that was even higher than that of F-ara-ddA (2). Our findings clearly prove that the application of the cycloSal-pronucleotide concept to F-ribo-ddA (3) overcomes a metabolic blockade in the formation of the corresponding monophosphate.     

Synthesis and anti-HIV activity of different sugar-modified pyrimidine and purine nucleosides

A series of base-modified pyrimidine 3'-azido-2',3'-dideoxynucleosides and 3'-substituted purine and pyrimidine 2',3'-dideoxynucleosides have been synthesized and evaluated for their inhibitory activity against human immunodeficiency virus (HIV) replication in MT-4 cells. The following pyrimidine derivatives emerged as the most potent and/or selective inhibitors of HIV-induced cytopathogenicity (in order of decreasing selectivity: 3'-azido-3'-deoxythymidine (AZT), 3'-azido-2',3'-dideoxyuridine (AzddUrd), 3'-azido-2',3'-dideoxy-5-methylcytidine (AzddMeCyd), 3'-fluoro-ddUrd (FddUrd), 3'-fluoro-ddThd (FddThd), the N4-hydroxylated derivative of AzddMeCyd and the N4-methylated derivative of AzddMeCyd. Among the purine 2',3'-dideoxynucleosides, 3'-azido-2',3'-dideoxyguanosine (AzddGuo), 3'-fluoro-ddGuo (FddGuo), and 3'-fluoro-2,6-diaminopurine 2',3'-dideoxynucleoside (FddDAPR) were the most selective inhibitors of HIV replication.     

Antiretroviral activity and cytotoxicity of novel zidovudine (AZT) derivatives and the relation to their chemical structure

Zidovudine (AZT) was the first nucleoside analogue licensed for the treatment of HIV infection. Efforts have continuously been made to improve the therapeutic characteristics of this drug, most of them focussed on prodrugs design. Here we describe the anti-HIV-1 activity and cytotoxicity of six novel AZT derivatives namely 3'-azido-3'-deoxy-5'-O-oxalyl-N-valinethymidine, 3'-azido-3'-deoxy-5'-O-oxalyl-N-leucinethymidine, 3'-azido-3'-deoxy-5'-O-oxalyl-N-isoleucinethymidine, 3'-azido-3'-deoxy-5'-O-oxalyl-N-phenylalaninethymidine, 3'-azido-3'-deoxy-5'-O-oxalylthymidine acid, 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine and 5-chloro-6-hydroxy-5,6-dihydro-3'-azido-3'-deoxythymidine which were perfectly characterized. AZT-Val, AZT-Leu, AZT-iLeu, AZT-Phen, AZT-Ac and AZT-Iso have shown a similar or higher selectivity index than that of AZT itself, in one or both of the different cell cultures used (PBMC and MT2). However, AZT-ClOH showed no anti-HIV activity. These results suggest that using amino acids in the design of AZT derivatives improves AZT activity.     

New antiviral nucleoside prodrugs await application

In this review, we intend to highlight outstanding concepts of antiviral nucleoside prodrugs which have been developed in recent years, so as to improve the efficacy of a given antiviral drug or to overcome some drug deficiencies. Examples of antiviral carrier-linked nucleoside prodrugs or nucleoside bioprecursors are described, and their active mechanisms discussed. The described nucleoside prodrugs are classified in two structural classes: prodrugs bearing molecular modifications on the sugar moiety and prodrugs bearing molecular modifications on the nucleic base. Despite the important research work accomplished through out the world during the last few years in developing improved antiviral drugs for the treatment of HIV (human immunodeficiency virus), HBV (hepatitis B virus), HCV (hepatitis C virus), HSV (herpes simplex virus), HCMV (human cytomegalovirus), etc infections, only few nucleoside antiviral prodrugs are marketed, while promising prodrugs deriving from original concepts were developed. The most relevant concepts are discussed: (1) - pronucleotide approach allows the design of prodrugs, which by-pass the first kinase phosphorylation step; (2) - drug design based on Bodor's concept for brain delivery improved drugs and (3) - 5'-O-carbonate nucleosides and deaminase approaches, which allow active drug regeneration. Nonetheless, none of these innovative models have reached the market.     

Anti-retrovirus activity of 3'-fluoro- and 3'-azido-substituted pyrimidine 2',3'-dideoxynucleoside analogues

The 3'-fluoro-and 3'-azido-substituted derivatives of 2',3'-dideoxythymidine (ddThd), 2',3'-dideoxyuridine (ddUrd), 2',3'-dideoxy-5-ethyluridine (ddEtUrd) and 2',3'-dideoxycytidine (ddCyd) have been synthesized and evaluated for their anti-retrovirus activity [against human immunodeficiency virus (HIV) and murine Moloney sarcoma virus (MSV)]. Based on their 50% effective doses the most potent inhibitors of HIV replication in human MT4 lymphocytes were: FddThd (0.001 microM), AzddThd (0.004 microM), FddUrd (0.04 microM) and AzddUrd (0.36 microM). Their selectivity indexes were 197, 5000, 500 and 677, respectively. In contrast, none of the 3'-substituted ddEtUrd derivatives had a marked antiviral effect. The 2',3'-dideoxynucleoside analogues showed poor, if any, substrate affinity for (bacterial) dThd phosphorylase. AzddThd and FddThd inhibited human dThd kinase to a much greater extent (Ki/Km: 0.66 and 3.4, respectively) than did AzddUrd or FddUrd (Ki/Km: 71 and 171, respectively). The Ki/Km values of FddCyd and AzddCyd for human dCyd kinase were about 60. Although phosphorylation is a prerequisite for the anti-retrovirus activity of the 2',3'-dideoxynucleoside derivatives, there is no close correlation between the anti-retrovirus potency of the 3'-fluoro- and 3'-azido-substituted ddUrd, ddThd, ddEtUrd and ddCyd derivatives and their affinity for dThd kinase or dCyd kinase.     

3'-substituted 2',3'-dideoxynucleoside analogues as potential anti-HIV (HTLV-III/LAV) agents

A series of 2',3'-unsaturated and 3'-substituted 2',3'-dideoxynucleoside analogues of purines and pyrimidines have been synthesized and evaluated for their inhibitory activity against human immunodeficiency virus (HIV). The 2',3'-unsaturated analogues of 2',3'-dideoxycytidine (ddeCyd) and 2',3'-dideoxythymidine (ddeThd), 3'-azido-2',3'-dideoxythymidine (AzddThd), 3'-fluoro-2',3'-dideoxythymidine, 2',3'-dideoxycytidine (ddCyd), and 2',3'-dideoxyadenosine (ddAdo) emerged as the most potent inhibitors of HIV-induced cytopathogenicity in the human T lymphocyte cell lines ATH8 and MT4. In ATH8 cells ddCyd, ddeCyd, and ddAdo had the highest therapeutic index whereas in MT4 cells AzddThd, ddThd, ddCyd, and ddAdo were the most selective. Derivatives from ddThd in which the substituent group was linked to the 3'-carbon atom via a thio, sulfonyl, or oxygen bridge were far less inhibitory to HIV than was AzddThd.     

Escherichia coli mediated biosynthesis and in vitro anti-HIV activity of lipophilic 6-halo-2',3'-dideoxypurine nucleosides

A series of 6-substituted 2',3'-dideoxypurine ribofuranosides (ddP) was enzymatically synthesized with live E. coli in an effort to enhance the lipophilicity of this class of anti-human immunodeficiency virus (HIV) compounds and thereby facilitate drug delivery into the central nervous system. All 6-halo-substituted ddPs were substantially more lipophilic, as defined by their octanol-water partition coefficient (P), than their nonhalogenated congeners 2',3'-dideoxyinosine (ddI) or 2',3'-dideoxyguanosine (ddG). For this class of compounds, log P's ranged from +0.5 to -1.2 in the following order: 6-iodo, 2-amino-6-iodo greater than 6-bromo, 2-amino-6-bromo greater than 6-chloro, 2-amino-6-chloro greater than 6-fluoro, 2-amino-6-fluoro much greater than ddG greater than ddI. These compounds were evaluated in vitro for ability to suppress the infectivity, replication, and cytopathic effect of HIV. 2-Amino-6-fluoro-, 2-amino-6-chloro-, and 6-fluoro-ddP exhibited a potent activity against HIV comparable to that of ddI or ddG and completely blocked the infectivity of HIV without affecting the growth of target cells. The comparative order of in vitro anti-HIV activity was 2-amino-6-fluoro, 2-amino-6-chloro, 6-fluoro greater than 2-amino-6-bromo greater than 2-amino-6-iodo, 6-chloro greater than 6-bromo greater than 6-iodo. These compounds also exhibited potent in vitro activity against HIV-2 and 3'-azido-3'-deoxythymidine-resistant HIV-1 variants. All 2-amino-6-halo-ddPs and 6-halo-ddPs were substrates for adenosine deaminase (ADA) and were converted to ddG or ddI, respectively. In the presence of the potent ADA inhibitor 2'-deoxycoformycin, 6-halo-substituted ddPs failed to exert an in vitro antiretroviral effect. These dideoxypurine nucleoside analogues represent a new class of lipophilic prodrugs of ddG and ddI that possess the potential for more effective therapy of HIV-induced neurologic disorders.