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A series of novel phosphorylated derivatives of didanosine were designed and docking studies were performed with a fusion protein of the Newcastle disease virus (NDV), to develop antiviral compounds against NDV. Based on the docking scores and binding affinities, three derivatives were selected. These compounds were synthesized and characterized by IR, (1) H, (13) C, (31) P, and CHN analysis and mass spectra. They were assessed for their in vitro antiviral activity in DF-1 cells; DDI-10 showed better antiviral activity as evidenced by significant reduction in plaque formation and cytopathic effects. DDI-10 was further evaluated in NDV-infected chicken; the survival rates and antioxidant enzyme levels in brain, liver, and lung tissues were estimated. Superoxide dismutase and catalase were significantly raised, and lipid peroxidation and HA titer levels were decreased upon treatment with 1.5 mg/kg body weight of DDI-10 than with 3 mg/kg body weight of DDI. Further histopathological alterations in NDV-infected tissues were restored in chicken treated with DDI-10. Thus, based on the results from in silico, in vitro, and in vivo assays, the novel phosphorylated DDI-10 might be considered as potent antiviral compound for NDV infection in chicken., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Owing to the promising antiviral activity of amino acid ester-substituted phosphorylated nucleosides in the present study, a series of phosphorylated derivatives of emtricitabine and didanosine substituted with bioactive amino acid esters at P-atom were synthesized. Initially, molecular docking studies were screened to predict their molecular interactions with hemagglutinin-neuraminidase protein of Newcastle disease virus and E2 protein of human papillomavirus. The title compounds were screened for their antiviral ability against Newcastle disease virus (NDV) by their in ovo study in embryonated chicken eggs. Compounds 5g and 9c exposed well mode of interactions with HN protein and also exhibited potential growth of NDV inhibition. The remaining compounds exhibited better growth of NDV inhibition than their parent molecules, i.e., emtricitabine (FTC) and didanosine (ddI). In addition, the in vitro anticancer activity of all the title compounds were screenedagainst HeLa cell lines at 10 and 100 μg/mL concentrations. The compounds 5g and 9c showed an effective anticancer activity than that of the remaining title compounds with IC50 values of 40 and 60 μg/mL, respectively. The present in silico and in ovo antiviral and in vitro anticancer results of the title compounds are suggesting that the amino acid ester-substituted phosphorylated FTC and ddI derivatives, especially 5g and 9c, can be used as NDV inhibitors and anticancer agents for the control and management of viral diseases with cancerous condition.

The aim of this study was to explore the potential of novel oleic acid (OA) derivatives as buccal permeation enhancers for the delivery of didanosine (ddI). The OA derivatives, i.e. ester derivative (OA1E), the dicarboxylic acid derivative (OA1A) and the bicephalous dianionic surfactant (OA1ANa) were synthesized and their effects were compared to the parent OA. OA, OA1E, OA1A and OA1ANa at 1% w/w all showed potential for enhancing the buccal permeability of ddI with enhancement ratio (ER) of 1.29, 1.33, 1.01 and 1.72, respectively. OA1ANa at 1% w/w demonstrated the highest flux (80.30 ± 10.37 µg cm(-2 )h), permeability coefficient (4.01 ± 0.57 × 10(-3) cm h(-1)) and ER (1.72). The highest flux for ddI (144.00 ± 53.54 µg cm(-2 )h) was reported with OA1ANa 2% w/w, which displayed an ER of 3.09 more than that with ddI alone. At equivalent concentrations, OA1ANa (ER = 3.09) had a significantly higher permeation-enhancing effect than its parent OA (ER = 1.54). Histomorphological studies confirmed that OA1ANa at all concentrations (0.5, 2.0 and 6.0% w/w) had no adverse effects on the mucosae. Morphological changes such as vacuoles formation and increased intercellular spaces were attributed to the buccal permeation-enhancing effect of OA1ANa. This study demonstrated the potential of novel OA derivatives as buccal permeation enhancers. OA1ANa at 2% w/w was also identified as the optimal novel OA derivative to widen the pool of fatty acid derivatives as chemical permeation enhancers for buccal drug delivery.

New nanomedicines could improve drug accumulation in HIV sanctuaries and ameliorate their antiretroviral efficiency. In this view, we propose herein a combined strategy based on a biomimetic prodrug of ddI and its formulation in well-characterized lipid nanoobjects. The glycerolipidic prodrug of ddI (ProddINP) has been synthesized and its bulk structure was characterized. An appropriate formulation of this prodrug has been designed using a rational approach combining different physicochemical techniques. The high incorporation ratio of the prodrug into dipalmitoylphosphatidylcholine (DPPC) bilayers was determined by DSC. Then two liposome preparation methods were compared, with respect to size, incorporation yield and molecular/supramolecular organization of vesicles. The best liposomal formulation of ProddINP has been checked to keep intact the anti-HIV activity of ddI. This formulation was finally compared to ddI after oral route in rat. The animal experiments evidenced the increase of ddI blood half life (3-fold) and its enhanced accumulation as prodrug form at 24h in numerous organs and especially intestine after administration of ProddINP in comparison with free drug. Finally, the tested liposomal formulation of ProddINP seems to be a promising approach to eradicate HIV infection from intestinal sanctuaries where the virus can concentrate., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Five peptidomimetic prodrugs of didanosine (DDI) were synthesized and designed to improve bioavailability of DDI following oral administration via targeting intestinal oligopeptide transporter (PepT1) and enhancing chemical stability. The permeability of prodrugs was screened in Caco-2 cells grown on permeable supports. 5'-O-L-valyl ester prodrug of DDI (compound 4a) demonstrated the highest membrane permeability and was selected as the optimal target prodrug for further studies. The uptake of glycylsarcosine (Gly-Sar, a typical substrate of PepT1) by Caco-2 cells could be inhibited by compound 4a in a concentration-dependent manner. The Caco-2 cells were treated with 0.2 nM leptin for enhanced PepT1 expression. The uptake of compound 4a was markedly increased in the leptin-treated Caco-2 cells compared with the control Caco-2 cells, both of which were obviously inhibited by 20 mM Gly-Sar. The K(m) and V(max) values of kinetic study of compound 4a transported by PepT1 in Caco-2 cells were 0.91 mM and 11.94 nmol/mg of protein/10 min, respectively. The chemical stability studies were performed in simulated gastric fluid (SGF), phosphate buffers under various pH conditions, rat tissue homogenates and plasma at 37 °C. The concentrations of DDI could not be detected in the two minutes in SGF. But compound 4a could significantly increase DDI acidic stability, and its t(½) was extended to as long as 36 min in SGF. Compound 4a was stable in pH 6.0 phosphate buffer but could be quickly transformed into DDI in plasma and tissue homogenates. The oral absolute bioavailability of DDI was 47.2% and 7.9% after compound 4a and DDI were orally administered to rats at a dose of 15 mg/kg, respectively. The coadministration with antiacid agent could also suggest that compound 4a was more stable under harsh acidic conditions compared with DDI. Compound 4a bioavailability in rats was reduced to 33.9% when orally co-administered with Gly-Sar (100 mg/kg). The In Vivo bioactivation mechanism of compound 4a was investigated by comparing the levels of DDI and compound 4a in the jugular and portal veins in rats. The plasma concentration of intact compound 4a was very low in portal veins and could hardly be detected in the jugular vein. In conclusion, compound 4a could significantly improve the oral bioavailability of DDI in rats through PepT1-mediated absorption and enhanced acidic stability, followed by rapid and mostly intracellular bioactivation, the majority in the intestinal cells but the minority in the liver. Additionally, the prodrug strategy targeted to intestinal PepT1 could offer a promising strategy to improve oral bioavailability of poorly absorbed didanosine.

The success of nucleoside reverse transcriptase inhibitors (NRTIs) in treating HIV-1 infection and reducing mother-to-child transmission of the virus during pregnancy is accompanied by evidence that NRTIs cause long-term health risks for cancer and mitochondrial disease. Thus, agents that mitigate toxicities of the current combination drug therapies are needed. Previous work had shown that the NRTI-drug pair zidovudine (AZT)-didanosine (ddI) was highly cytotoxic and mutagenic; thus, we conducted preliminary studies to investigate the ability of the active moiety of amifostine, WR1065, to protect against the deleterious effects of this NRTI-drug pair. In TK6 cells exposed to 100 muM AZT-ddI (equimolar) for 3 days with or without 150 muM WR1065, WR1065 enhanced long-term cell survival and significantly reduced AZT-ddI-induced mutations. Follow-up studies were conducted to determine if coexposure to AZT and WR1065 abrogated the antiretroviral efficacy of AZT. In human T-cell blasts infected with HIV-1 in culture, inhibition of p24 protein production was observed in cells treated with 10 muM AZT in the absence or presence of 5-1,000 muM WR1065. Surprisingly, WR1065 alone exhibited dose-related inhibition of HIV-1 p24 protein production. WR1065 also had antiviral efficacy against three species of adenovirus and influenza A and B. Intracellular levels of unbound WR1065 were measured following in vitro/in vivo drug exposure. These pilot study results indicate that WR1065, at low intracellular levels, has cytoprotective and antimutagenic activities against the most mutagenic pair of NRTIs and has broad spectrum antiviral effects. These findings suggest that the activities have a possible common mode of action that merits further investigation., (Copyright 2009 Wiley-Liss, Inc.)

Self-assembled drug delivery systems (SADDS) are defined as the self-assemblies of amphiphilic prodrugs, integrating prodrugs, molecular self-assembly and nanotechnology for drug targeting and controlled release. Cholesteryl-succinyl didanosine (CSD) and cholesteryl-adipoyl didanosine (CAD) nanoparticulate systems in water were previously prepared and optimized. In this paper, the in vitro and in vivo behavior of them was investigated. Precipitation occurred when they were mixed with acid solutions due to rapid production of hypoxanthine and subsequent disruption of supramolecular structures. They showed pH-dependent degradation and kept relatively stable in the neutral pH range. CSD is more stable than CAD due to the shorter spacer and poloxamer protection. CSD showed different degradation rates in various plasma with the descending order of rat, mouse, rabbit, dog and human. The half-life (t(1/2)) of CSD is 9 days in rat plasma, and 5.9 days in rat liver homogenates. CAD has a faster degradation than CSD though the t(1/2) in rat liver homogenates is long to 23 h. CSD nanoparticulates showed no significant anti-HIV effect in MT4 cell model because of very slow degradation. CSD nanoparticulates showed the distribution t(1/2) of 7.6 min after bolus intravenous (i.v.) administration to rats, and the site-specific distribution in liver, lung and spleen with the high t(1/2) of 10 days in liver. The factors affecting achievement of successful SADDS are discussed.

Self-assembled nanostructures were obtained through injecting cholesteryl-succinyl didanosine (CSD) solutions in tetrahydrofuran (THF) into water. The incorporation of THF leads to CSD flexible bilayers. Spherical vesicles of CSD initially formed based on hydrophobic interaction and transformed to short nanotubes with THF leaving. A large proportion of THF led to formation of long flexible nanoribbons, also transforming to short nanotubes after removing THF because of rigid bilayers recovering. Cholesteryl-adipoyl didanosine (CAD) only formed spherical vesicles due to its long and flexible tails. A triblock copolymer, poloxamer 188 (P188) could insert into CSD monolayers at the air/water interface. P188 stabilized CSD nanoparticulate systems by incorporation and adsorption. The optimal formulation of nanoparticulate systems was identified and the appropriate ratio of CSD/P188 and CSD concentration in injected solutions were the keys. A highly concentrated system had a narrow size distribution, allowing heat sterilization at 100 degrees C, and resisting aggregation under high gravity accelerations in spite of the low zeta potential of -18.5 mV. A gel-liquid crystalline phase transition at 47 degrees C occurred. The system may become promising self-assembled drug delivery systems (SADDS) for anti-HIV therapy.

A series of prodrugs of didanosine were synthesized in an effort to enhance the anti-HIV activity. The 5'-OH function of didanosine was esterified with different aryl piperazine acetic acid derivatives and evaluated for anti-HIV-1 activity in MT-4 cell line using the MTT assay method. Among the synthesized compounds, (tetrahydro-5-(1,6-dihydro-6-oxopurin-9-yl)furan-2-yl)methyl 2-(4-(4-chlorophenyl)piperazin-1-yl)acetate (4b) was found to be the most potent compound with EC50 of 0.64 microM and was not toxic to the MT-4 cells up to 1000 microM with a selectivity index of > 1562. Compound 4b was found to be seven times more potent than the parent drug didanosine (EC50 of 4.8 microM) in vitro. In vitro hydrolysis of the various esters in human plasma indicated that these agents were relatively stable toward plasma esterases with t1/2 ranging from 20-60 min.

Purpose: Previous attempts to ascertain the role of uptake and efflux transporters in the oral absorption of anti-HIV dideoxypurine nucleosides have been inconclusive. A novel in situ intestinal perfusion technique with complete mesenteric arterial/venous cannulation was used to examine the asymmetry of ileal dideoxynucleoside permeability under near in vivo conditions., Methods: Intestinal perfusions were performed in the rat ileum, with cannulation of the artery and vein immediately entering and leaving the segment. Urea and mannitol were used as passive permeability markers, and the directional transport of 2',3'-dideoxyinosine (ddI), 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), and 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA) were examined., Results: Urea and mannitol exhibited symmetric permeability (PLtoB = PBtoL), whereas F-ddI and ddI showed significantly higher permeability in blood-to-lumen transport (PBtoL > PLtoB). PBtoL for F-ddA exceeded PLtoB, but the difference did not reach significance at p < 0.05. PBtoL for ddI was demonstrated to be saturable with increasing ddI concentrations, but PLtoB was independent of ddI concentration. PBtoL for ddI was shown to be dependent on sodium concentration and inhibited by probenecid., Conclusions: Symmetric transport was demonstrated for urea and mannitol as expected for these passive permeability markers. F-ddI and ddI were shown to be preferentially transported from blood to lumen. The basolateral to luminal transport of ddI is saturable, inhibited by probenecid, and sodium ion dependent. These results are consistent with carrier-mediated uptake on the basolateral membrane.

A series of new homo and heterodimers of ddI has been synthesized. A glutarate diester spacer was used to covalently couple ddI onto ddI, AZT or d4T.

Enhanced central nervous system (CNS) delivery of certain poorly penetrating 2',3'-dideoxynucleosides has been achieved by designing prodrugs that are substrates for enzymes, such as adenosine deaminase (ADA), that are present at high activities in brain tissue. In this study, the potential role of adenosine deaminase localized within the endothelial cells of the blood-brain barrier (BBB) in providing enhanced intracellular and CNS delivery of an ADA-activated prodrug is assessed in vitro using cell culture models of the BBB. The kinetics of uptake and bioconversion of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA), a model ADA-activated prodrug of 2'-beta-fluoro-2',3'-dideoxyinosine, were determined in primary cultured bovine brain microvascular endothelial cells. Model-based simulations of CNS availability derived from in vitro estimates of parameters for simple passive diffusion and ADA-catalyzed deamination suggest that ADA that is localized within the BBB plays an important role in the conversion of F-ddA to 2'-beta-fluoro-2',3'-dideoxyinosine during its passage across the BBB. Consistent with in vivo observations, these simulations demonstrate that elevated levels of certain enzymes, such as ADA, in the brain microvascular endothelial cells of the BBB may be exploited in the design of brain-targeted prodrugs or drug-carrier conjugates, which brain tissue selectively converts.

There is still a need for direct determination (i.e. without dephosphorylation) of nucleoside reverse transcriptase inhibitor (NRTI) triphosphorylated nucleotides in the peripheral-blood mononuclear cells (PBMCs) of HIV-positive patients. The objective of this paper was first to improve our previously described direct liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for stavudine triphosphate (d4T-TP). Preparation of PBMCs was modified to reduce degradation of d4T-TP during cell preparation and to simplify this step for routine use in clinical units. The performance of several HPLC columns was compared in order to improve the stability of peak shape over time. The SMT C(18) column was replaced by a Supelcogel ODP-50, thereby reducing two-fold the concentration of the first standard. Various internal standards were compared to optimize peak shape and remove an interfering peak in LC. 2-Chloroadenosine 5prime prime or minute-triphosphate was chosen as the most appropriate internal standard. Substitution of the narrowbore column by a microbore column (150 x 0.32 mm) is also presented and discussed. Secondly, this improved method was successfully applied to the simultaneous determination of d4T-TP, dideoxyadenosine triphosphate (ddA-TP) and lamivudine triphosphate (3TC-TP) in PBMCs, which is useful in view of the common use of NRTI combinations. The method was subsequently applied to clinical samples from HIV-positive patients receiving antiretroviral therapy containing d4T, ddl and/or 3TC. This method can be used simply and routinely on approximately 200 samples per week, using commercially available instruments and with a simple cell lysis as sample treatment., (Copyright 2002 John Wiley & Sons, Ltd.)

A sensitive precolumn derivatization method has been developed to measure the 5'-triphosphate of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA, lodenosine), a new anti-HIV drug, in human lymphocytes by HPLC using fluorescence detection. Reaction of chloroacetaldehyde with F-ddA triphosphate in extracts from human lymphocytes produces a highly fluorescent etheno adduct. This derivative is then separated and quantitated by reverse-phase paired-ion chromatography. Degradation of natural nucleic acid ribosides, such as ATP, using periodate oxidation simplifies the chromatogram and minimizes interference with detection of the target analyte. This method, modeled using cultured MOLT-4 T-lymphocytes, achieves a linear detector response for peak area measurements over the range 2.5 to 22.5 pmol (50-450 nM using 50 microl sample). Analyte recovery is greater than 90%, and the method achieves a limit of detection and limit of quantitation of 1.4 and 2.5 pmol per HPLC injection (50 microl sample containing cellular extract from 2.5 x 10(6) cells), respectively. Application of this method to measure F-ddATP in peripheral blood mononuclear cells from HIV-infected patients treated with F-ddA at 3.2 mg/kg twice daily for 22 days shows F-ddATP levels which range from 1.5 to 3.5 pmol/10(6) cells., (Copyright 2001 Academic Press.)

The syntheses of the 2'-deoxy and 2',3'-dideoxynucleosides of 2,8-diaza-3-deazainosine and the 2',3'-dideoxynucleoside of 2-aza-3-deazainosine were achieved and the pathways leading to these novel nucleosides are described. The preparation of the 2',3'-dideoxynucleoside (1) of 2-aza-3-deazainosine involved deoxygenation of the 2'-deoxy-3'-imidazolide intermediate with n-Bu3SnH and AlBN. The latter nucleoside was synthesized from the known 2'-deoxy derivative of 2-aza-3-deazainosine. The three-step synthesis of 1 from the 2'-deoxy analogue was accomplished in 40% overall yield. Rather than synthesize the corresponding 2',3'-dideoxynucleoside (2) of 2,8-diaza-3-deazainosine in the same manner, i.e. deoxygenation of the 2'-deoxynucleoside, a more cost-effective route was chosen. This pathway involved reductive cleavage of the 5'-protected, 2',3'-thiocarbonate derivative to furnish a mixture of the 2'- and 3'-deoxy isomers. This mixture was not separated, but was deoxygenated by the aforementioned imidazolide method. Using this methodology, 2 was prepared in 23% overall yield from 2,8-diaza-3-deazainosine. Nucleosides 1 and 2 were evaluated for antiretroviral activity and were found to be inactive.

A single and multiple dose pharmacokinetic (PK) study was conducted in rats following oral administration of 2'-fluoro-2',3'-dideoxyadenosine (FddA) and 2'-fluoro-2',3'-dideoxyinosine (FddI) at three dose levels. Six rats/gender were assigned to one of the three FddA or FddI dose levels: 40, 250, and 1000 mg/kg/day. Three rats/gender were assigned to the PK study on day 1, while the remaining 3 rats/gender were assigned to the PK study on day 14. The rats received the appropriate doses of either FddA or FddI orally by gavage once a day for 14 days. Serial blood samples up to 24 h and cumulative urine samples (0-24 h) were collected on both days 1 and 14. Plasma and urine samples were analyzed for the concentrations of intact FddA and/or FddI using a validated assay. The data were subjected to non-compartmental PK analyses. Over the dose range of 40-1000 mg/kg. both FddA and FddI exhibited dose dependent pharmacokinetics in rats. Following FddA administration, there was a rapid and extensive in vivo conversion of FddA to FddI; FddI was the major circulating moiety as reflected by Cmax and AUC values (generally 2-3-fold greater than those of FddA at each dose level) as well as the amount excreted (%UR) in the urine. In contrast, following FddI administration, Cmax, AUC, and %UR values were 2-5-fold lower as compared to the FddI generated from FddA administration at each dose level, which also suggested that FddI was not absorbed as extensively as FddA. Based on the findings of this study, FddA is an excellent prodrug of FddI.

This study investigates the mechanisms of absorption and the role of intestinally localized purine salvage pathway enzymes on the ileal availabilities of 2',3'-dideoxyinosine (ddI), a substrate for purine nucleoside phosphorylase (PNP); 2'-fluoro-2',3'-dideoxyinosine (F-ddI), a non-PNP substrate; and 6-chloro-2',3'-dideoxypurine (6-Cl-ddP), an adenosine deaminase (ADA) activated prodrug of ddI. The potential for increasing the intestinal availability of 6-Cl-ddP through the use of ADA inhibitors, namely, 2'-deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), is also explored. Drug permeability coefficients across the intestinal epithelium were determined in in situ perfusions in the mesenteric vein cannulated rat ileum based on both drug appearance in blood (Pblood) and disappearance from the lumen (Plumen) and their paracellular and transcellular components were estimated by comparison to the permeabilities of two paracellular markers, mannitol and urea. Values of Pblood for ddI were determined to be (1.1 +/- 0.3) x 10(-6) cm/s, in close agreement with the value of (1.0 +/- 0.3) x 10(-6) cm/s obtained for F-ddI, a PNP resistant analogue of ddI having virtually the same molecular size and lipophilicity as ddI. This indicates that PNP may not play an important role in the low intestinal absorption of ddI. The Pblood for 6-Cl-ddP, (19 +/- 2) x 10(-6) cm/s, was 4.5-fold lower than Plumen, (84 +/- 12) x 10(-6) cm/s, which means that 77 +/- 6% of 6-Cl-ddP was metabolized during its intestinal transport, thus qualitatively accounting for the low oral bioavailability (7%) of 6-Cl-ddP observed in vivo in rats. Extensive intracellular metabolism of 6-Cl-ddP by ADA was confirmed by the high concentrations of ddI found both in the intestinal lumen and blood during 6-Cl-ddP perfusions and by a rate of ddI appearance in blood which was approximately 10-fold higher than ddI controls. Co-perfusion of the potent, hydrophilic ADA inhibitor DCF (Ki = 0. 001-0.05 nM) with 6-Cl-ddP led to only partial inhibition of intestinal ADA, while complete inhibition was obtained using the less potent but more lipophilic inhibitor EHNA (Ki = 1-20 nM). Hence, EHNA may be used to improve intestinal absorption of 6-Cl-ddP in vivo.

AZT-P-ddI is an antiviral heterodimer composed of one molecule of 3'-azido-3'-deoxythymidine (AZT) and one molecule of 2',3'-dideoxyinosine (ddI) linked through their 5' positions by a phosphate bond. The metabolic fate of the dimer was studied with isolated rat, monkey, and human hepatocytes and was compared with that of its component monomers AZT and ddI. Upon incubation of double-labeled [14C]AZT-P-[3H]ddI in freshly isolated rat hepatocytes in suspension at a final concentration of 10 microM, the dimer was taken up intact by cells and then rapidly cleaved to AZT, AZT monophosphate, ddI, and ddI monophosphate. AZT and ddI so formed were then subject to their respective catabolisms. High-performance liquid chromatography analyses of the extracellular medium and cell extracts revealed the presence of unchanged dimer, AZT, 3'-azido-3'-deoxy-5'-beta-D-glucopyranosylthymidine (GAZT), 3'-amino-3'-deoxythymidine (AMT), ddI, and a previously unrecognized derivative of the dideoxyribose moiety of ddI, designated ddI-M. Trace extracellular but substantial intracellular levels of the glucuronide derivative of AMT (3'-amino-3'-deoxy-5'-beta-D-glucopyranosylthymidine [GAMT]) were also detected. Moreover, the extent of the formation of AMT, GAZT, and ddI-M from the dimer was markedly lower than that with AZT and ddI alone by the hepatocytes. With hepatocytes in primary culture obtained from rat, monkey, and human, large interspecies variations in the metabolism of AZT-P-ddI were observed. While GAZT and ddI-M, metabolites of AZT and ddI, respectively, as well as AZT 5'-monophosphate (MP) and ddI-MP were detected in the extracellular media of all species, AMT and GAMT were produced only by rat and monkey hepatocytes. No such metabolites were formed by human hepatocytes. The metabolic fate of the dimer by human hepatocytes was consistent with in vivo data recently obtained from human immunodeficiency virus-infected patients.

Purpose: This study examines the central nervous system (CNS) delivery of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA) and 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), acid stable analogues of dideoxyadenosine (ddA) and dideoxyinosine (ddI) having reduced susceptibility to purine salvage pathway enzymes important in the metabolism of ddA and ddI, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), respectively. Their CNS delivery compared to that for ddI provides insight into the role of brain tissue ADA and PNP in these processes., Methods: Brain and cerebrospinal fluid (CSF) concentration-time profiles were obtained for F-ddI during and after intravenous infusions of F-ddI, and for both F-ddA and F-ddI after F-ddA infusions in normal rats or rats pre-treated with the ADA inhibitor 2'-deoxycoformycin (DCF). Rate constants for CNS entry, efflux and metabolism were estimated by computer fits using plasma concentration-time profiles as the driving force functions., Results: The CNS delivery of F-ddI did not differ significantly from that for ddI. F-ddA, which is more lipophilic than F-ddI, provided higher brain (approximately 8x) and CSF (approximately 11x) concentrations of total dideoxynucleoside (F-ddA and F-ddI) compared to F-ddI. Deamination by brain tissue ADA to form F-ddI reduced CNS levels of intact F-ddA but provided higher brain parenchyma (5x) and CSF/plasma (3x) ratios of F-ddI relative to F-ddI controls. Thus, F-ddA functions in part as a CNS-activated prodrug of F-ddI. DCF pre-treatment inhibited brain tissue ADA, abolishing the prodrug effect, and enhancing F-ddA concentrations in both brain parenchyma (5x) and CSF (6x)., Conclusions: PNP metabolism does not appear to play a role in the low CNS delivery of ddI. On the other hand, deamination of F-ddA by brain tissue ADA is an important process, such that F-ddA functions in part as a CNS-activated prodrug of F-ddI. Enhanced CNS uptake of intact F-ddA can be achieved with ADA inhibition.

In the search for prodrugs of 2',3'-dideoxyinosine (ddI) with potential for improving the delivery of the nucleoside analogue to the lymphatic system, we synthesized dipalmitoylphosphatidyl-2',3'-dideoxyinosine (DPP-ddI) and its pharmacokinetics were investigated in mice. The disposition of ddI in plasma and lymph nodes was examined following intravenous and oral administration of parent nucleoside (100 mg/kg) and DPP-ddI (400 mg/kg, equivalent to 100 mg/kg of ddI). Concentrations of ddI were determined by HPLC. Pharmacokinetic parameters were estimated by area/moment analysis. Intravenous administration of DPP-ddI resulted in a pattern of lower peak concentrations of ddI and more sustained exposure of parent nucleoside in plasma and lymph nodes compared to administration of the parent nucleoside. Both ddI and DPP-ddI yielded similar AUC values in lymph nodes. Oral administration of the prodrug resulted in lower concentrations and AUC values of ddI in plasma and lymph nodes when compared to administration of the parent nucleoside. The bioavailability of ddI following ddI and DPP-ddI administration was 15 and 8%, respectively. The results of the present study demonstrate that DPP-ddI administered intravenously shows potential for targeting and sustaining level of ddI in the nodular lymphatic tissues.

3'-Azido-3'-deoxythymidilyl-(5',5')-2',3'-dideoxy-5'-inosinic acid (AZT-P-ddI, IVX-E-59, Scriptene) is a heterodimer composed of one molecule of 3'-azido-3'-deoxythymidine (zidovudine or AZT) and one molecule of 2',3'-dideoxyinosine (didanosine or ddI) linked through their 5' positions by a phosphate bond. AZT-P-ddI exhibits enhanced antiviral activity and selectivity in vitro compared with AZT and ddI alone. The pharmacokinetics of AZT-P-ddI were studied in 12 patients with human immunodeficiency virus (HIV) who had CD4+ cell counts higher than 200 cells/mm3. Isotopic preparations of (14C)-AZT-P-(3H)-ddI were administered intravenously (50 mg and 100 mg) to eight patients; 1 month later these patients were crossed over to oral administration (100 mg and 200 mg). A second group of patients (n = 4) received only a 450-mg oral dose of AZT-P-ddI. Plasma levels of unchanged AZT-P-ddI after intravenous infusion declined rapidly and were undetectable 0.75 hours after the end of infusion, whereas the parent compound was not detected after oral administration, indicative of a very rapid metabolism. The parent entity was enzymatically cleaved in vivo yielding the two constituent drugs AZT and ddI, which were subsequently subjected to their respective pharmacokinetic and metabolic processes. The beta-glucuronide derivative of AZT (GAZT) represented the major metabolite of AZT, but there were no detectable levels of the toxic metabolite 3'-amino-3'-deoxythymidine (AMT). A major and previously unrecognized in vivo metabolite of ddI, referred as ddI-M, was detected in plasma and urine. Analysis by high-field proton nuclear magnetic resonance and mass spectrometry led to the identification of ddI-M as being R(-)-dihydro-5-(hydroxymethyl)-2(3H)-furanone. The formation of AZT and ddI metabolites was increased after oral administration of AZT-P-ddI compared with the intravenous infusion, with an area under the concentration-time curve (AUC) ratio of metabolite to AZT and metabolite to ddI being 7.7 and 5.7 (oral) and 3.8 and 1.1 (intravenous), respectively. The newly identified ddI-M exhibited sustained plasma levels for extended time periods with an apparent elimination half-life (t1/2) of approximately 10 hours after oral administration of AZT-P-ddI. Recovery of radioactivity associated with 3H and 14C in urine was essentially complete within 48 hours after oral and intravenous administration of AZT-P-ddI. The oral bioavailability of AZT (64.7-67.3%) and ddI (33.6-42.9%) and the other pharmacokinetic parameters were consistent with previous data reported with each nucleoside analog alone or in combination therapy.

In an effort to improve the pharmacokinetic properties and tissue distribution of 2'-F-ara-ddI, two lipophilic prodrugs, 6-azido-2'-3'-dideoxy-2'-fluoro-beta-D- arabinofuranosylpurine (FAAddP, 4) and N6-methyl-2'-3'-dideoxy-2'-fluoro-beta-D-arabinofuranosyladenine (FMAddA, 5), were synthesized and their biotransformation was investigated in vitro and in vivo, in mice. Compounds 4 and 5 were synthesized via the intermediate 2. For the in vitro studies, FAAddP and FMAddA were incubated in mouse serum, liver homogenate, and brain homogenate. FAAddP was metabolized in liver homogenate by the reduction of the azido to the amino moiety followed by deamination, yielding 2'-F-ara-ddI. The conversion of FAAddP to 2'-F-ara-ddA was mediated by microsomal P-450 NADPH reductase system, as shown by the liver microsomal assay. FAAddP was also converted to 2'-F-ara-ddI at a slower rate in the brain than in the liver. FMAddA, however, was stable in brain homogenate and was slowly metabolized in the liver homogenate. Metabolic conversion of FMAddA in vitro was stimulated by the addition of adenosine deaminase. In the in vivo metabolism study, FAAddP underwent reduction to 2'-F-ara-ddA followed by deamination to 2'-F-ara-ddI. FMAddA did not result in increased brain delivery of 2'-F-ara-ddI in vivo, probably due to the slow conversion as observed in the in vitro studies. However, there was an increase in the half-life of 2'-F-ara-ddI produced from FMAddA. This report is the first example in the design of prodrugs using the azido group for adenine- and hypoxanthine-containing nucleosides. This interesting and novel approach can be extended to other antiviral and anticancer nucleosides.

Enzymes of the purine salvage pathway play an important role in altering the in vivo pharmacokinetics of 2',3'-dideoxypurine nucleosides. This study examines the pharmacokinetics of enzyme-resistant 2'-beta-fluoro analogues of 2',3'-dideoxyinosine (ddI) and 2',3'-dideoxyadenosine (ddA). 2'-beta-Fluoro-2',3'-dideoxyinosine (F-ddI) is an acid-stable analogue of ddI that is highly resistant to purine nucleoside phosphorylase, the principal enzyme in ddI metabolism. 2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA), an acid-stable and purine nucleoside phosphorylase-resistant analogue of ddA, is converted in vivo to F-ddI by adenosine deaminase (ADA) but is a much poorer substrate for this enzyme than is ddA. Both F-ddA and F-ddI have been shown to have activity against human immunodeficiency virus in vitro, and F-ddA has been selected by the National Cancer Institute for clinical trials as a new human immunodeficiency virus reverse transcriptase inhibitor. The pharmacokinetics of F-ddI and ddI were compared at equivalent doses in chronically catheterized rats. Because ddI and F-ddI are isosteres having nearly identical lipophilicity, this comparison is likely to reflect primarily metabolic differences. The clearance of F-ddI was substantially reduced, in comparison with that of ddI (27.3 ml/min/kg vs. 90.9 ml/min/kg), resulting in higher systemic concentrations at steady state and prolonged retention of F-ddI after termination of infusions, consistent with a significant metabolic component in the clearance of ddI. Concentrations of F-ddA and F-ddI during and after infusions of F-ddA were determined in both untreated and 2'-deoxycoformycin-pretreated rats. In untreated rats, F-ddA was rapidly eliminated from plasma, with a total clearance of 68.5 ml/kg/min. Metabolic clearance of F-ddA to F-ddI accounted for 58% of this value (bioconversion t1/2 = 9.8 +/- 1.9 min). Pretreatment with 2'-deoxycoformycin, an ADA inhibitor, reduced the clearance of F-ddA to 23.8 ml/min/kg, leading to 2.9 +/- 0.4-fold higher steady-state plasma concentrations of F-ddA, in agreement with a 2.5-fold enhancement predicted by a compartmental model assuming complete ADA inhibition.

A high-performance liquid chromatographic assay was developed and validated for a simulataneous determination of 2'-fluoro-2' 3'-dideoxyadenosine (FddA) and its metabolite, 2'-fluoro-2',3'-dideoxyinosine (Fddl) in dog plasma and urine. In vitro, FddA and Fddl exhibit activity against human immunodeficiency virus (HIV). A solid phase extraction was applied to extract FddA, Fddl, and the internal standard (IS; 3',5'-anhydrothymidine) from the biomatrices. The processed samples were chromatographed using a C8 column coupled with a mobile phase consisting of monobasic phosphate, dibasic phosphate, ethylene glycol monomethyl ether, and water. Detection was performed at 257 nm. The nominal retention times were 9, 14, and 26 min for Fddl, IS, and FddA, respectively. The lower limits of quantitation were 0.1 and 2.0 micrograms/mL in plasma and urine, respectively, for both analytes. The accuracy of the assay deviated < or = 10% from the nominal concentrations, and the precision was < or = 14% coefficient of variation. In either matrix, both analytes were stable for at least three freeze-thaw cycles and in the injection media for at least 54 h. The extraction recoveries of the analytes were greater than 80%. The application of this assay was demonstrated in a preliminary pharmacokinetic study of FddA and Fddl in dogs. Two male dogs per dose level received a 100, 250, or 500 mg/kg oral dose of FddA once daily for 14 days. The early appearance of Fddl in plasma (0.25 h; the first sampling time) and greater plasma levels of Fddl than FddA (> 50-fold of Cmax), suggested that the conversion of FddA to Fddl was rapid and extensive. Renal excretion appeared to be the major route of elimination of Fddl.

Nucleotide heterodimers were synthesized and examined for their inhibitory effects on the replication of human immunodeficiency virus type 1 (HIV-1), including HIV-1 reverse transcriptase (RT) inhibitor-resistant mutants. 3'-Azido-3'-deoxythymidilyl-(5')-phospho-(5')-6-[(3', 5'-dimethylphenyl)thio]-5-ethyl-1-[(2-hydroxyethoxy)methyl]uracil (AZT-P-E-HEPU-dM) and 3'-azido-3'-deoxythymidilyl-(5')-phospho-(5')-2', 3'-dideoxyinosine (AZT-P-ddI) proved to be highly potent and selective inhibitors of HIV-1 (IIIB strain) in MT-4 cells. The mechanism of inhibition by these heterodimers may be attributed to their degradation and the formation of each constituent. AZT-P-E-HEPU-dM was also markedly inhibitory to an AZT-resistant mutant (HIV-1-IIIB/AZT) and an E-HEPU-dM-resistant mutant (HIV-1-IIIB-R). However, AZT-P-ddI was found to have a less inhibitory effect on HIV-1-IIIB/AZT than on HIV-1-IIIB. The heterodimers of (5',5') AZT and ribavirin (AZT-P-Ribavirin) and (5',5') ddI and ribavirin (ddI-P-Ribavirin) were also synthesized: AZT-P-Ribavirin inhibited HIV-1 replication, but ddI-P-Ribarvirin did not.

A series of 6-substituted amino analogs of 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl) purines (F-ddN) has been synthesized and characterized with the objective of finding compounds which might be superior to existing drugs for the treatment of HIV in the central nervous system. These compounds are intended to be more lipophilic than the currently approved anti-HIV drugs for better blood-brain barrier penetration. Subsequent adenosine deaminase (ADA)-catalyzed hydrolysis of these prodrugs in the brain is expected to produce the anti-HIV agent, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)hypoxanthine (F-ddI). The new compounds, synthesized from the corresponding 6-chloro analog, include F-ddN which contain methylamino, ethylamino, dimethylamino, hydroxylamino, methoxyamino, benzyloxyamino, hydrazino, and nitro substituents in the 6-position. The 6-nitro analog was isolated as an unexpected product during the preparation of the 6-chloro derivative. Among the analogs with anti-HIV activity, the ethylamino and dimethylamino compounds are ca. 100 times more lipophilic than ddI or F-ddI. As expected, 2'-fluoro substitution protects the compounds from acid-catalyzed glycosylic cleavage. Only the hydroxylamino and nitro analogs underwent any nonenzymatic hydrolysis at pH 1.0 or 7.4. This reaction, however, results in hydrolysis of the group in the 6-position rather than glycosylic bond cleavage. ADA catalyzes the hydrolysis of the 6-substituents at rates which vary from slightly slower (NO2, 1.7x) to much slower (NHEt, 5000x) than F-ddA. The 6-dimethylamino analog is the only compound which possesses anti-HIV activity (ED50 18 microM) without ADA hydrolysis. With the exception of the two inactive alkoxyamino compounds, the other prodrugs exhibited cellular protection in the HIV-1/PHA-PBM system with IC50 potencies of 7-40 microM.

Mononucleoside phosphotriester derivatives of dideoxynucleosides, following intracellular enzymatic activation, are likely to liberate their parent 5'-nucleoside monophosphate. Prodrugs of 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-didehydro-2',3'- dideoxythymidine (d4T), 2',3'-dideoxyinosine (ddl) and 2',3'-dideoxyadenosine (ddA) were evaluated for their anti-HIV1 activities in monocyte-derived macrophages, cells which are known to have low levels of nucleoside kinases. Prodrugs were found to be much more active, or just as active, as the corresponding dideoxynucleoside. Furthermore, their selectivity index was enhanced by a factor of 2 to 200.

A series of 6-halo-(F-, Cl-, Br-, I-) and 6-alkoxy-(OMe-, OEt-) 9-(2,3-dideoxy-2-fluoro-beta-D-threopentofuranosyl) purines (F-ddN) have been synthesized and characterized with the objective of finding compounds which might be superior to existing drugs for the treatment of HIV in the central nervous system. These compounds, which contain lipophilic 6-substituents, were chosen as acid-stable prodrugs for the anti-HIV-active F-ddN, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl) hypoxanthine (F-ddI), because of their potential to increase blood-brain-barrier penetration relative to F-ddI. All the new compounds were more lipophilic than the currently approved anti-AIDS drugs. Partition coefficient increases of 30- and 110-fold were achieved, relative to didanosine (ddI), for the 6-chloro- and 6-ethoxy analogues. 2'-Fluoro substitution abolished the pH 1, acid-catalyzed cleavage of the nucleoside glycosylic bond. However, pH 1, acid-catalyzed hydrolysis of the 6-fluoro substituent to produce F-ddI was observed to occur at a rate (t1/2 0.54 h) which was ca. 40-170 times faster than that of the other prodrugs. The utility of the F-ddNs as prodrugs for F-ddI depends upon their ability to act as substrates for adenosine deaminase. The relative rates of adenosine deaminase-catalyzed prodrug hydrolysis to F-ddI varied by a factor of > 25,000 with the 6-fluoro- and 6-ethoxy analogues reacting the fastest and slowest, respectively. All of the prodrugs possessed anti-HIV activity in the phytohemagglutinin-stimulated peripheral blood mononuclear cell test system and a qualitative correlation exists between prodrug anti-HIV activity and adenosine deaminase hydrolysis rates.

With increasing awareness of the mitochondrial toxicity associated with certain 2',3'-dideoxynucleosides used in anti-human immunodeficiency virus therapy, procedures for quantitative analyses of drug effects on mitochondrial DNA (mtDNA) have assumed enhanced importance. For this reason we have developed a method to measure the copy numbers of mtDNA in cultured MOLT-4 cells. First a hybrid competitive DNA template was synthesized by conventional polymerase chain reaction (PCR), using two custom-synthesized 40-mer composite primers incorporating mitochondrial displacement loop sequences linked by a non-mitochondrial cDNA template (a 76-base pair sequence from the tat/rev region of human immunodeficiency virus cDNA). For the competitive assay, increasing known copy numbers of the hybrid competitive template were added as an internal control to samples containing total cellular DNA. With this approach, two competitive PCR products were generated, 1) a mitochondrial displacement loop-derived fragment (182 base pairs) and 2) a competitive DNA template-derived fragment (156 base pairs). Absolute quantitation was achieved by radiometric comparison of the relative amounts of the two products. To test the versatility of this method, varying amounts of competitive template (6.6 x 10(4) to 6.6 x 10(9) copies) were used with a fixed quantity of total cellular DNA taken from cells cultured for 9 days in the presence or absence of selected pyrimidine and purine dideoxynucleosides. The results showed that the copy number of cellular mtDNA is 823 +/- 71 copies/cell in MOLT-4 cells. Little selective depletion of mtDNA, compared with total cellular DNA, was seen with the purine dideoxynucleosides examined; however, when the cells were exposed to the pyrimidine dideoxynucleoside 2',3'-dideoxycytidine (50 nM) for 9 days, mtDNA content was specifically depleted, although total cellular DNA decreased by only 10%. Thus, in addition to the presently used methods of assessing mitochondrial impairment, i.e., Southern blot analysis and electron microscopy, the competitive PCR method provides a third and convenient assay, with particular applicability to determination of mtDNA in very small numbers of cells.

In order to enhance the brain delivery of 2'-F-ara-ddI,2'-F-ara-ddP 6 was synthesized and its in vitro and in vivo bioconversion reaction studied. For the study, a new efficient synthetic method for 2'-F-ara-ddP 6 was developed from 5-benzoyl-1,2-O-isopropylidene-3-deoxyribose 1. For in vitro study 2'-F-ara-ddP was incubated in pH 2, mouse liver homogenate, and mouse serum at 37 degrees C. No degradation was observed in pH 2 and serum, while in liver homogenate 2'-F-ara-ddP was almost completely converted to 2'-F-ara-ddI within 20 min (t1/2 = 3.54 min). In order to determine the role of xanthine oxidase in the conversion of 2'-F-ara-ddP to 2'-F-ara-ddI, in vitro studies were conducted in phosphate buffer (pH 7.4) in the presence or absence of allopurinol, in which the half-lives of 2'-F-ara-ddP were 7.4 and 3.4 h, respectively, indicating the conversions were catalyzed by the xanthine oxidase. A similar experiment with aldehyde oxidase isolated from the human liver did not affect the biotransformation. The biotransformation was also detected in the brain homogenate, although the rate of conversion was low and incomplete. In order to assess the bioconversion in vivo, pharmacokinetic studies of 2'-F-ara-ddP and 2'-F-ara-ddI were conducted in mice. The maximum serum concentrations of 2'-F-ara-ddI administered itself and as 2'-F-ara-ddP reached 48.1 +/- 10.00 and 89.3 +/- 26.0 microM and were observed in 1 and 0.25 h, respectively. The data indicate that 2'-F-ara-ddI is absorbed at a slower rate than that of 2'-F-raa-ddP. The bioavailability of the prodrug after oral administration was 60.7%. The concentration of 2'-F-ara-ddI following oral administration of 2'-ara-ddI was close to the detection limits while 2'-F-ara-ddI was detected at significantly higher concentrations in the brain after oral administration of 2'-F-ara-ddP. From this study, we have administered the enhanced brain delivery of anti-HIV nucleoside utilizing an in vivo biotransformation system.

The purine dideoxynucleosides 2'-beta-fluoro-2',3'-dideoxyadenosine (2'-beta-F-ddAdo), 2'-beta-fluoro-2',3'-dideoxyinosine, and 2'-beta-fluoro-2',3'-dideoxyguanosine (2'-beta-F-ddGuo) are active inhibitors of the replication of the human immunodeficiency virus (HIV) in the ATH8 assay system, with 2'-beta-F-ddAdo and 2'-beta-fluoro-2',3'- dideoxyinosine showing activity and potency equivalent to those of their respective parent compounds, 2',3'-dideoxyadenosine (ddAdo) and 2',3'-dideoxyinosine. Because inhibitors of IMP dehydrogenase such as ribavirin and tiazofurin stimulate the 5'-phosphorylation and consequently the anti-HIV activity of the three nonfluorinated parent compounds (ddAdo, 2',3'-dideoxyinosine, and 2',3'-dideoxyguanosine), we have undertaken a study in MOLT-4 cells to determine whether a similar stimulatory effect is observed with their 2'-beta-fluorinated analogs. The 5'-phosphorylation of all the fluoro compounds was found to be greatly enhanced by low levels (10 microM) of either ribavirin or tiazofurin, with the greatest increase being seen with 2'-beta-F-ddAdo, where stimulation of the formation of the 5'-mono-, di-, and triphosphorylated nucleotides was approximately 20-fold, 6-fold, and 5-fold, respectively. These increases were approximately 3-fold greater than the increases seen with the nonfluorinated parent compound ddAdo. In the case of 2'-beta-F-ddGuo, the greatest stimulation (8-fold) was seen in the formation of the 5'-diphosphate. In parallel with the increased phosphorylation of 2'-beta-F-ddAdo and 2'-beta-F-ddGuo, the anti-HIV potency of these two compounds at the 5 microM level was approximately doubled in the presence of ribavirin (5 microM).

The acute cardiotoxic potential of single dosages of FddA (2'-fluoro-2',3'-dideoxyadenosine) and FddI (2'-fluoro-2',3'-dideoxyinosine) was investigated in 6- to 9-week-old rats. Both nucleoside analogs were administered orally at 1000 and 2000 mg/kg and intravenously at 500 or 1000 mg/kg. For comparative purposes, additional groups of rats received 2'-deoxyadenosine or the 2-fluororibose moiety common to both the FddA and FddI molecules. The effects of two adenosine receptor antagonists, caffeine and theophylline, on the cardiotoxicity induced by FddA were also investigated. Deaths occurred within a few hours to a few days in FddA-treated rats given 2000 mg/kg orally or 500 mg/kg intravenously and in FddI-treated rats given 1000 mg/kg intravenously. Microscopic examination of the hearts revealed myocardial degeneration and necrosis for all rats that died and myocardial fibrosis for many survivors. No deaths or cardiac lesions were observed after administration of 2'-deoxyadenosine or the 2-fluororibose moiety. FddA was more cardiotoxic than FddI in rats at equivalent dosages administered either orally or intravenously. Based on the anatomic findings, all deaths were attributed to cardiac lesions. The administration of high, oral dosages of caffeine and theophylline accentuated the acute cardiotoxicity of FddA in rats.

Five ester prodrugs of 2'3'-dideoxyinosine (DDI) were synthesized for the purpose of improving oral bioavailability. The prodrugs, acetate (C2-DDI), octanoate (C8-DDI), stearate (C18-DDI), benzoate (Bz-DDI), and hemisuccinate (Suc-DDI) were proved to quantitatively regenerate their parent drug by enzymatic hydrolysis. Though the chemical stability of the prodrugs under acidic conditions was not improved, their solubility in water was significantly decreased by esterification, except for Suc-DDI. Bioavailability was evaluated by oral administration to rats. Two hydrophobic prodrugs (C8-DDI and Bz-DDI) showed higher absolute bioavailability (23.5% and 31.0%, respectively) than did DDI (15.2%), though that of C2-DDI (11.5%) and Suc-DDI (4.5%) was poor.

The x-ray crystal structures of two new anti-HIV compounds, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine (2'-F-dd-araA) and 9-(2,3-dideoxy-2-fluoro-beta-D-threo- pentofuranosyl)hypoxanthine (2'-F-dd-aral), have been determined at two temperatures. Both crystals are in the space group P2(1)2(1)2(1), and their structures were solved by direct methods. Least-squares refinement produced final R-factors of 0.027 for the 2'-F-dd-araA structure and of 0.044 for the 2'-F-dd-aral structure, respectively. The latter structure contains a two-fold disordered conformation of the sugar moiety. All three conformers (one for 2'-F-dd-araA and two for 2'-F-dd-aral) adopt an anti chi CN glycosyl torsion angle. The sugar in the 2'-F-dd-araA structure has a C2'-endo pucker conformation, whereas the sugar in the 2'-F-dd-aral structure has a mixture of C2'-endo and C3'-endo pucker conformations. When the sugar adopts the C2'-endo conformation, the torsion angle about the C4'-C5' bond is in a transgauche+ conformation. In contrast, when the sugar adopts the C3'-endo conformation, the torsion angle about the C4'-C5' bond is in a gauche(+)-gauche- conformation. The C2'-F bond distance is 1.406(3) A, similar to that found in other aliphatic C-F bonds. The results suggest that the 2'-fluoro-2',3'-dideoxyarabinosyl nucleosides do not have a strong preference for either C2'-endo or C3'-endo sugar pucker.

Combinations of an amidoalkylphosphocholine, 8, and AZT have been found to cause an apparent synergistic action in suppressing infectious HIV-1 replication. In addition, amidoalkyl, oxyalkyl, and thioalkyl ether lipids have been chemically linked to anti-HIV-1 nucleosides (AZT and DDI) through phosphate and phosphonate linkages. These conjugates have shown promising in vitro anti-HIV-1 activity. Also, the conjugates have a 5-10-fold reduction in cell cytotoxicity compared to AZT alone. The most active compound, an amidoalkyl ether lipid-AZT conjugates, 4A, was found to have a differential selectivity of 1793 in a syncytial plaque assay. In comparison, AZT alone has a value of 1281.