Conjugates of phosphorylated zalcitabine and lamivudine with SiO2 nanoparticles: Synthesis by CuAAC click chemistry and preliminary assessment of anti-HIV and antiproliferative activity.

Conjugates of phosphorylated dideoxynucleoside antiviral drugs dideoxycytidine (zalcitabine) and lamivudine with SiO2 nanoparticles were obtained via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry between a nucleoside triphosphate containing an alkynyl group at the γ-phosphate or azidothymidine triphosphate and SiO2 nanoparticles containing alkyl azide or alkynyl groups, respectively. 4-(Prop-2-yn-1-yloxy)butylamino group has been attached to the γ-phosphate group of dideoxycytidine (zalcitabine) and lamivudine 5'-triphosphates via the phosphoramidate linkage. New compounds were shown to be potent killers of human colon carcinoma cells. Anti-HIV activity of the conjugates was demonstrated as well. The conjugates of phosphorylated lamivudine and dideoxycytidine (zalcitabine) showed higher potency than the parent nucleosides. The conjugate of phosphorylated azidothymidine was less active against HIV-1 than the parent nucleoside probably because of the replacement of its 3'-azido group by 1,2,3-triazole ring. These results show an opportunity for using SiO2 nanoparticles as a transport for delivering phosphorylated nucleosides to cells in order to increase their efficiency as antiviral and anticancer drugs.

Structural basis for processivity and antiviral drug toxicity in human mitochondrial DNA replicase.

The human DNA polymerase gamma (Pol γ) is responsible for DNA replication in mitochondria. Pol γ is particularly susceptible to inhibition by dideoxynucleoside-based inhibitors designed to fight viral infection. Here, we report crystal structures of the replicating Pol γ-DNA complex bound to either substrate or zalcitabine, an inhibitor used for HIV reverse transcriptase. The structures reveal that zalcitabine binds to the Pol γ active site almost identically to the substrate dCTP, providing a structural basis for Pol γ-mediated drug toxicity. When compared to the apo form, Pol γ undergoes intra- and inter-subunit conformational changes upon formation of the ternary complex with primer/template DNA and substrate. We also find that the accessory subunit Pol γB, which lacks intrinsic enzymatic activity and does not contact the primer/template DNA directly, serves as an allosteric regulator of holoenzyme activities. The structures presented here suggest a mechanism for processivity of the holoenzyme and provide a model for understanding the deleterious effects of Pol γ mutations in human disease. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity.

Vibrational and thermal analyses of multicomponent crystal forms of the anti-HIV drugs lamivudine and zalcitabine.

The vibrational and thermal characterizations of four multicomponent molecular crystals of lamivudine, namely, lamivudine hydrochloride anhydrate (1), lamivudine hydrochloride monohydrate (2), lamivudine duplex I (3), with a 8:2:2:1:4 lamivudine:maleic acid:HCl:(CH3)2CHOH:H2O stoichiometry, being all three more soluble in water than the commercial solid form of lamivudine, and lamivudine maleate (4), have been performed here by infrared (IR) and Raman spectroscopy, differential scanning calorimetry (DSC), and thermogravimetry (TG). Furthermore, the vibrational spectra of zalcitabine hydrochloride (5), isostructural to 1 but with a methylene moiety in the 3'-position of the five-membered ring instead of sulfur in lamivudine, have also been measured in order to point out the role of this molecular substitution and conformation in the vibrational modes of the salts. In fact, scattering bands at the high frequency range relative to CH stretching modes are not superimposable in the Raman spectra of 1 and 5, even though these crystal forms are assembled with the same molecular conformation and intermolecular packing. At the same time, the structural similarity between 1 and 5 can be reflected in their IR spectra, as in the carbonyl and iminium stretching bands shifted to lower frequencies as consequence of their hydrogen bonding engagement. Furthermore, a scattering band at 3057 cm(-1) is observed only in the Raman spectra of crystal forms present with their 5'-CH2OH moiety in-gauche conformation, namely, 2-4. It is absent in the Raman spectra of 1 and 5 whose 5'-CH2OH moiety adopts (+)gauche conformation. In-gauche conformation, the 5'-OH oxygen is pointed toward one of the two aromatic CH hydrogens. Consequently, there is formation of an intramolecular hydrogen bond between them, shifting the aromatic CH stretching band to a lower frequency. The DFT calculations have also revealed in-phase and out-of-phase couplings of the two aromatic CH stretchings in the Raman spectra of 1, which is without intramolecular hydrogen bond due to (+)gauche conformation of 5'-CH2OH moiety. Both coupled vibrational modes are observed in the corresponding experimental spectrum as a single peak because of their similar frequencies. On contrary, aromatic CH stretching modes are not coupled in 2 due to the intramolecular hydrogen bond, resulting in resolution of the Raman bands. Thermal events in DSC and TG curves of 1 and 2 are also in agreement with crystal stoichiometry as observed from single-crystal X-ray diffraction analysis.

Interfacial behavior of PEGylated lipids and their effect on the stability of squalenoyl-drug nanoassemblies.

Squalenoyl-gemcitabine (Sq-Gem) and squalenoyl-dideoxycytidine (Sq-ddC) are amphiphilic prodrugs that self-assemble in water to form nanoassemblies (NAs) with well-defined structures and size. However, like other drug nanocarriers, these nanoassemblies are rapidly cleared from the blood stream by the reticulo-endothelial system. By adding squalenoyl-PEG (Sq-PEG) or cholesterol-PEG (Chol-PEG) to the squalenoyl prodrugs, composite nanoassemblies (CNAs) were formed, with different sizes and structures. The effect of the PEG-lipids on the formation and stability of these nanoassemblies was assessed by transmission electron microscopy, quasi-elastic light scattering and surface tension measurements in various conditions. The results revealed different stabilities with time for Sq-ddC and Sq-Gem nanoassemblies in aqueous medium, the latter being much less stable than the former. They also demonstrated that the presence of Sq-PEG or Chol-PEG in composite Sq-ddC nanoassemblies contributed to their rapid destabilization. The analysis of the adsorption kinetics of Sq-PEG into a prodrug monolayer below and above its critical aggregation concentration allowed getting a better insight into prodrug-lipopolymer molecular interactions, and their consequences on the formation of composite prodrug nanoassemblies.

Sensitivity of the polymerase of vesicular stomatitis virus to 2' substitutions in the template and nucleotide triphosphate during initiation and elongation.

The RNA synthesis machinery of non-segmented negative-sense RNA viruses comprises a ribonucleoprotein complex of the genomic RNA coated by a nucleocapsid protein (N) and associated with polymerase. Work with vesicular stomatitis virus (VSV), a prototype, supports a model of RNA synthesis whereby N is displaced from the template to allow the catalytic subunit of the polymerase, the large protein (L) to gain access to the RNA. Consistent with that model, purified L can copy synthetic RNA that contains requisite promoter sequences. Full processivity of L requires its phosphoprotein cofactor and the template-associated N. Here we demonstrate the importance of the 2' position of the RNA template and the substrate nucleotide triphosphates during initiation and elongation by L. The VSV polymerase can initiate on both DNA and RNA and can incorporate dNTPs. During elongation, the polymerase is sensitive to 2' modifications, although dNTPs can be incorporated, and mixed DNA-RNA templates can function. Modifications to the 2' position of the NTP, including 2',3'-ddCTP, arabinose-CTP, and 2'-O-methyl-CTP, inhibit polymerase, whereas 2'-amino-CTP is incorporated. The inhibitory effects of the NTPs were more pronounced on authentic N-RNA with the exception of dGTP, which is incorporated. This work underscores the sensitivity of the VSV polymerase to nucleotide modifications during initiation and elongation and highlights the importance of the 2'-hydroxyl of both template and substrate NTP. Moreover, this study demonstrates a critical role of the template-associated N protein in the architecture of the RNA-dependent RNA polymerase domain of L.

Anti-HIV efficacy and biodistribution of nucleoside reverse transcriptase inhibitors delivered as squalenoylated prodrug nanoassemblies.

Due to their hydrophilic nature, most nucleoside reverse transcriptase inhibitors (NRTIs) display a variable bioavailability after oral administration and a poor control over their biodistribution, thus hampering their access to HIV sanctuaries. The limited cellular uptake and activation in the triphosphate form of NRTIs further restrict their efficacy and favour the emergence of viral resistance. We have shown that the conjugation of squalene (sq) to the nucleoside analogues dideoxycytidine (ddC) and didanosine (ddI) leads to amphiphilic prodrugs (ddC-sq and ddI-sq) that spontaneously self-organize in water as stable nanoassemblies of 100-300 nm. These nanoassemblies can also be formulated with polyethylene glycol coupled to either cholesterol (Chol-PEG) or squalene (sq-PEG). When incubated with peripheral blood mononuclear cells (PBMCs) in vitro infected with HIV, the NRTI-sq prodrugs enhanced the antiviral efficacy of the parent NRTIs, with a 2- to 3-fold decrease of the 50% effective doses and a nearly 2-fold increase of the selectivity index. This was also the case with HIV-1 strains resistant to ddC and/or ddI. The enhanced antiviral activity of ddI-sq was correlated with an up to 5-fold increase in the intracellular concentration of the corresponding pharmacologically active metabolite ddA-TP. The ddI-sq prodrug was further investigated in vivo by the oral route, the preferred route of administration of NRTIs. Pharmacokinetics studies performed on rats showed that the prodrug maintained low amounts of free ddI in the plasma. Administration of (3)H-ddI-sq led to radioactivity levels higher in the plasma and relevant organs in HIV infection as compared to administration of free (3)H-ddI. Taken together, these results show the potential of the squalenoylated prodrugs of NRTIs to enhance their absorption and improve their biodistribution, but also to enhance their intracellular delivery and antiviral efficacy towards HIV-infected cells.

Complete conformational space of the potential HIV-1 reverse transcriptase inhibitors d4U and d4C. A quantum chemical study.

A comprehensive quantum-chemical conformational analysis of two nucleoside analogues, 2',3'-didehydro-2',3'-dideoxyuridine (d4U) and 2',3'-didehydro-2',3'-dideoxycytidine (d4C), is reported. The electronic structure calculations were performed at the MP2/6-311++G(d,p)//B3LYP/6-31++G(d,p) level of theory. It was found that d4U and d4C adopt 20 conformers and 19 conformers, respectively, which correspond to local minima on the respective potential energy landscapes. QTAIM and NBO analyses show that the d4U and d4C conformers are stabilised by a complicated network of specific intramolecular interactions, which includes conventional (OHO) and non-conventional (CHO, CHHC) H-bonds as well as closed-shell van der Waals (CO) contacts. A satisfactory linear correlation was found between Grunenberg's compliance constants for closed-shell intramolecular interactions and their energy. It is shown that there are no conformational obstacles for incorporation of d4U and d4C into the double helical A and B forms of DNA. The less pronounced biological activity of d4U as compared to 2',3'-didehydro-2',3'-dideoxythymidine (d4T) is most likely due to the presence of the bulky methyl group at the 5-position of d4T, which can be recognised by target enzymes.

Strong antiviral activity of the new l-hydroxycytidine derivative, l-Hyd4FC, in HBV-infected human chimeric uPA/SCID mice.

BACKGROUND: Suppression of viral replication with nucleoside/nucleotide inhibitors has been shown to greatly improve the outcome of chronic HBV infection. ß-l-nucleoside analogues, especially ß-l-deoxycytidine derivatives represent one of the most efficient groups of antiretroviral compounds. We recently described that hydroxylation of the amino group of these ß-l-deoxycytidine derivatives preserved their strong HBV inhibitory activity in vitro, but strongly reduced their cytotoxicity. From this new group of compounds we selected ß-l-2',3'-didehydro-2',3'-dideoxy-N(4)-hydroxy-5-fluorocytidine (l-Hyd4FC) for a first in vivo investigation. The aim of this study was to determine the antiviral activity of l-Hyd4FC in HBV-infected human liver chimeric urokinase plasminogen activator (uPA)/SCID mice. METHODS: Stably infected animals (median 6×10(7) HBV DNA/ml) were injected daily with either l-Hyd4FC (50 mg/kg) or saline as controls. Mice treated with lamivudine served to compare the in vivo antiviral potency of l-Hyd4FC. Virological changes were determined by quantitative PCR. RESULTS: Treatment with l-Hyd4FC for 4 weeks induced a 2-log reduction of viraemia, while a median 1.5-log decline was achieved with lamivudine. Intrahepatically, l-Hyd4FC induced a median eightfold decline of viral activity (relaxed circular DNA/covalently closed circular DNA), and threefold reduction of pregenomic RNA/GAPDH levels. No significant decline of subgenomic HBV transcripts, as well as of circulating hepatitis B e antigen and hepatitis B surface antigen was detected. Maintenance of human serum albumin concentrations throughout the study, negative TUNEL staining and occurrence of viral rebound after drug withdrawal indicated that l-Hyd4FC was not toxic in human hepatocytes. CONCLUSIONS: Administration of l-Hyd4FC in uPA/SCID mice harbouring HBV-infected human hepatocytes demonstrated the high antiviral potency of this drug in vivo. Such characteristics make l-Hyd4FC a good candidate for further investigations a as potential HBV therapeutic agent.

Cytosine deoxyribonucleoside anti-HIV analogues: a small chemical substitution allows relevant activities.

The search for new nucleoside analogue compounds targeting the virally encoded reverse transcriptase was developed by modifying the nucleoside structure to create inhibitor compounds. In this review, the structure-activity relationship of antiviral compounds synthesised from the naturally existing cytosine deoxyribonucleoside (dC) was evaluated. The line of research starting from dC led to the synthesis of 2',3'-dideoxycytidine (ddC; zalcitabine), 2',3'-dideoxy-3'-thiacytidine (3TC; lamivudine) and 2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC; emtricitabine) and looks very interesting because each product comes from a single small change in the chemical structure of the former compound, resulting in a progressive improvement in terms of activity, pharmacokinetics, tolerability and emergence of resistance.

Squalenoylation: a generic platform for nanoparticular drug delivery.

Squalene is a triterpene widely distributed in nature that is an intermediate in the cholesterol biosynthesis pathway. The remarkable dynamic folded conformation of squalene has been used to chemically conjugate this lipid with various therapeutic molecules to construct nanoassemblies of 100-300 nm. In this review, we discuss the new concept of "squalenoylation" through application to anticancer (i.e. gemcitabine, paclitaxel, cisplatin etc.…) or antiviral (ddI, ddC) compounds. In a lego-type approach, it is also possible to construct multifunctional nanoparticles endowed with additional imaging functionalities (i.e. "Nanotheragnostics"). This new nanotechnology platform is expected to have important applications in pharmacology.

Hepatitis C virus NS5B polymerase exhibits distinct nucleotide requirements for initiation and elongation.

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3'-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (K(app)(m) approximately 100-400 mum) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 10(3)- to 10(4)-fold, revealing a much reduced nucleotide requirement for elongation (K(app)(m) approximately 0.03-0.09 microm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 microm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3'-end of the HCV(-)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.

Development of a sensitive and specific liquid chromatography/mass spectrometry method for the determination of tenofovir in human plasma.

A sensitive and specific method for the quantitation of tenofovir (TFV) in human plasma by liquid chromatography/electrospray ionization mass spectrometry was developed and validated. Plasma samples were prepared by solid-phase extraction performed on Waters Oasis cation-exchange cartridges (30 mg). Chromatographic separation was performed isocratically on a reversed-phase Waters Atlantis dC18 column (2.0x100 mm, 3 microm). The mobile phase consisted of a hydroxylamine/acetic acid buffer (pH 6.75) and methanol (93:7, v/v). The acquisition was performed in selected ion monitoring mode for the protonated molecular ions [M+H]+ of m/z 288.2 for TFV and 212.2 for the internal standard, zalcitibine. The method was fully validated to determine its specificity, recovery, linearity and sensitivity, accuracy and precision. The analytical range was set at 1-750 ng/mL using a 200 microL plasma sample, with a mean coefficient of determination (r2) of 0.9969. The mean accuracies for the calibration standards ranged from -5.0 to 4.3%, while the precisions were within 1.2 and 6.4%. Intra-assay and inter-assay mean accuracies for three quality control concentrations (2, 60, and 600 ng/mL) ranged from -6.1 to 10.7%, while the precisions were within 1.3 and 9.1%. TFV was shown to be stable under normal storage and assay conditions; no degradation was seen when stored at -20 degrees C or -80 degrees C for up to 6 months, and after 16 h at room temperature in the injection matrix. The present method provides an accurate, precise, and sensitive tool for TFV quantitation and was successfully applied to an external proficiency-testing program and pharmacokinetic analysis.

The triphosphate of beta-D-4'-C-ethynyl-2',3'-dideoxycytidine is the preferred enantiomer substrate for HIV reverse transcriptase.

The enantioselective synthesis of the beta-d (1) enantiomer of 4'-C-ethynyl-2',3'-dideoxycytidine confirms an earlier stereochemical assignment that was strictly based on the ability of HIV reverse transcriptase and its M184V mutant to discriminate between the d- and l-configuration of nucleoside 5'-triphosphates.

Pharmacokinetics of the antiviral agent beta-L-3'-fluoro-2',3'-didehydro-2',3'-dideoxycytidine in rhesus monkeys.

beta-L-3'-Fluoro-2',3'-didehydro-2',3'-dideoxycytidine (L-3'-Fd4C) is a potent and selective antiretroviral nucleoside with activity against lamivudine-resistant human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV) in vitro. The pharmacokinetics of L-3'-Fd4C were characterized in three rhesus monkeys given single intravenous and oral doses. A two-compartment open model was fitted to the plasma and urine data. Plasma concentrations declined in a biexponential fashion with an average beta half-life of 12.45 h and central and steady-state volumes of distribution of 0.43 and 1.90 liters/kg, respectively. The average systemic and renal clearance values were 0.23 and 0.08 liters/kg, respectively, and the apparent mean terminal half-life of the oral dose was 12.5 h. The serum concentrations exceeded the 90% effective concentration value for lamivudine-resistant and wild-type HIV-1 after oral administrations. A large variation was observed in the oral bioavailability, which ranged from 15 to 31%. To determine whether the bioavailability may be improved by using a basic buffer solution, the oral dose was repeated to the same animals in a sodium bicarbonate solution. The bioavailability of L-3'-Fd4C administered with sodium bicarbonate was not significantly different from the bioavailability when the oral dose was administered in the absence of buffer (P = 0.49), suggesting that further development of this compound may warrant other approaches, such as development of a prodrug to improve its oral absorption.

A 4'-C-ethynyl-2',3'-dideoxynucleoside analogue highlights the role of the 3'-OH in anti-HIV active 4'-C-ethynyl-2'-deoxy nucleosides.

4'-C-ethynyl-2'-deoxynucleosides belong to a novel class of nucleoside analogues endowed with potent activity against a wide spectrum of HIV viruses, including a variety of resistant clones. Although favorable selectivity indices were reported for several of these analogues, some concern still exists regarding the 3'-OH group and its role in cellular toxicity. To address this problem, we removed the 3'-OH group from 4'-C-ethynyl-2'-deoxycytidine (1a). This compound was chosen because of its combined high potency and low selectivity index. The removal of the 3'-OH was not straightforward; it required a different synthetic approach from the one used to synthesize the parent compound. Starting with glycidyl-4-methoxyphenyl ether, the target 4'-C-ethynyl-2',3'-dideoxycytidine analogue (rac-1h) was obtained after 13 steps. In a cellular assay, rac-1h was completely inactive (0.001-10 microM) against HIV(LAI), demonstrating the critical importance of the 3'-OH for antiviral activity. To determine whether the role of the 3'-OH was essential for the phosphorylation of the compound by cellular kinases or for inhibition of DNA polymerization, we synthesized and tested the 5'-triphosphate (rac-1h-TP) for its ability to inhibit HIV reverse transcriptase (RT). rac-1h-TP was slightly more potent than AZT-5'-triphosphate against wild-type HIV RT, suggesting that the role of the 3'-OH is crucial only for the activation of the drug by cellular kinases. The lipase-catalyzed resolution of rac-1h into ent-1h (beta-D-dideoxyribo) and ent-14 (beta-L-dideoxyribo) and the synthesis of the corresponding 5'-triphosphates established the stereochemical assignment based on HIV RT's preference for the beta-D-enantiomer, which was confirmed by assaying against the M184V variant, an RT mutant with a marked preference for incorporating nucleosides in the D-configuration.

Investigating the effects of stereochemistry on incorporation and removal of 5-fluorocytidine analogs by mitochondrial DNA polymerase gamma: comparison of D- and L-D4FC-TP.

Enantiomers of beta-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D/L-D4FC) are nucleoside analog reverse transcriptase inhibitors (NRTIs) currently under investigation as antiviral agents. One of the major problems of NRTIs is toxicity to mitochondria. It has been shown that mitochondrial toxicity of NRTIs can correlate with incorporation and removal of these compounds by mitochondrial DNA polymerase (Pol gamma). Mechanistic studies have shown that, if activated, NRTIs are incorporated more efficiently by HIV-1 reverse transcriptase (RT) and less efficiently by Pol gamma, the corresponding nucleosides are considered to be more selective. In the present study, in order to predict potential DNA Pol gamma-related mitochondrial toxicity of D- and L-D4FC, the incorporation and removal of the monophosphate form of these compounds by Pol gamma were studied using transient kinetic methods. Our cell-free results showed that Pol gamma incorporated the natural D-isomer significantly more efficiently than the unnatural L-isomer. However, the removal rates of these enantiomers from the chain-terminated primers were almost identical. While these results suggest that D-D4FC may present more mitochondrial toxicity than L-D4FC in cell-free assays, we have previously shown that HIV-1 RT prefers D-D4FC-TP as a substrate over the L-isomer, particularly in the case of mutant forms of RT associated with nucleoside drug resistance such as M184V. Since the effectiveness of NRTIs is a balance between efficiency of incorporation by wild-type and drug-resistant forms of HIV-1 RT and mitochondrial toxicity, our kinetic results suggest that both enantiomers may show promise as potential therapeutics.

Synthesis of phosphorothioamidites derived from 3'-thio-3'-deoxythymidine and 3'-thio-2',3'-dideoxycytidine and the automated synthesis of oligodeoxynucleotides containing a 3'-S-phosphorothiolate linkage.

The synthesis of N4-benzoyl-5'-O-dimethoxytrityl-2',3'-dideoxy-3'-thiocytidine and its phosphorothioamidite is described for the first time, together with a shortened procedure for the preparation of 5'-O-dimethoxytrityl-3'-deoxy-3'-thiothymidine and its corresponding phosphorothioamidite. The first fully automated coupling procedure for the incorporation of a phosphorothioamidite into a synthetic oligodeoxynucleotide has been developed, which conveniently uses routine activators and reagents. Coupling yields using this protocol were in the range of 85-90% and good yields of singularly modified oligonucleotides were obtained. Coupling yields were also equally good when performed on either a 0.2 or 1 micro mol reaction column, thus facilitating large scale syntheses required for mechanistic studies.

Synthesis and biological evaluation of some 5-nitro- and 5-amino derivatives of 2'-deoxycytidine, 2',3'-dideoxyuridine, and 2',3'-dideoxycytidine.

In this article, we describe the synthesis of 5-nitro-1-(2-deoxy-alpha-D-erythro-pentofuranosyl)cytosine (4alpha), 5-nitro-1-(2-deoxy-beta-D-erythro-pentofuranosyl)cytosine (4beta), 5-amino-1-(2-deoxy-alpha-D-erythro-pentofuranosyl)cytosine (5alpha), 5-nitro-1-(2-deoxy-beta-D-erythro-pentofuranosyl)cytosine (5beta), 5-nitro-1-(2,3-dideoxy-beta-D-ribofuranosyl)uracil (6beta), 5-amino-1-(2,3-dideoxy-alpha,beta-D-ribofuranosyl)uracil (7), 5-nitro-1-(2,3-dideoxy-alpha,beta-D-ribofuranosyl)cytosine (8) and 5-amino-1-(2,3-dideoxy-beta-D-ribofuranosyl)cytosine (9beta). The prepared compounds were tested for their activity against HIV and HBV viruses, but they did not show significant activity.