Synthesis of New Homo and Heterodimers of 2′,3′-Dideoxyinosine (ddi) Using Ester Linkages

Abstract

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.     

High-performance liquid chromatographic analysis of 2',3'-dideoxyinosine in biological samples

A high-performance liquid chromatographic analysis for the anti-AIDS drug 2',3'-dideoxyinosine (ddI) in rat plasma and urine, with a limit of detection of 0.2 μg/ml and requiring a sample size of 100 μl is described. Diluted plasma or urine samples were extracted using a C₁₈ solid-phase extraction column. Retention of ddI on more polar solid-phase extraction columns was insufficient for sample clean-up. This method is useful for pharmacokinetic studies of ddI in small rodents.     

Strategy for Industrial Scale Production of Dideoxyinosine: Enzymatic Deamination of Dideoxyadenosine by Adenosine Deaminase

Abstract

A procedure has been devised for the production of dideoxyinosine (ddI) in high yield and purity. The method employs adenosine deaminase to deaminate 2′,3′-dideoxyadenosine (ddA) to ddI. On the basis of systematic evaluation of various reaction conditions, a substrate concentration of 0.5 M ddA and an enzyme concentration of 5.6 units per ml were selected, and all buffers were eliminated. Under these conditions ddA was quantitatively converted to ddI within 4 hrs. Addition of 25% DMSO to the reaction facilitated the isolation of ddI as a result of the differential solubilities of the product and substrate, but did not affect the enzymatic reaction. An industrial scale reaction has been designed on the basis of the data presented.     

Synthesis of 2',3'-dideoxyinosine via radical deoxygenation

A synthetic method for 2',3'-dideoxyinosine (ddI) from inosine was established via radical deoxygenation of N1,5'-O-diprotected-2',3'-bis-S-methyl dithiocarbonate of inosine derivatives. The radical deoxygenation proceeded smoothly to give the desired dideoxy compounds in good yields using 1-ethylpiperidinium hypophosphite and triethylborane. Benzyl or p-methoxybenzyl protection of inosine at the N1, 5'-O-positions were effective for the ddI synthesis.     

Prenatal developmental toxicity evaluation of 2',3'-dideoxyinosine (ddI) and 2',3'-didehydro-3'-deoxythymidine (d4T) co-administered to Swiss Albino (CD-1) mice

BACKGROUND: In pregnant women, antiretroviral drugs improve maternal health and reduce vertical transmission of human immunodeficiency virus to the infant. However, few nonclinical studies have examined the potential for adverse drug interactions. METHODS: On gestational days (GD) 6-16, mice were dosed with vehicle, ddI (360, 1440, or 2,880 mg/kg/day, p.o.), d4T (60, 240, or 480), or ddI/d4T combinations (360/60, 1,440/240, or 2,880/480). Daily doses were divided into two equal parts that were administered >or=6-hr apart. Body weight, clinical signs, and feed consumption were monitored. Pregnancies (22-24/group) were confirmed at necropsy. Maternal liver and gravid uterine weights (GUW), uterine implants (resorption, live or dead fetus), fetal body weight, gender, and morphologic anomalies (external, visceral, skeletal) were recorded. RESULTS: Maternal body weight, clinical signs, and GUW were unaffected. Maternal weight change corrected for GUW was greater than controls at 60 and 480 d4T. Relative feed consumption during treatment was increased relative to controls at 1,440 and 2,880 ddI and 2,880/480 ddI/d4T. Relative maternal liver weight was elevated above controls at 240 and 480 d4T and 2,880/480 ddI/d4T, and above the constituent dose of ddI at 1,440/240 and 2,880/480 ddI/d4T. Liver weight was not affected by ddI and there was no significant drug interaction. Prenatal mortality and morphologic anomalies were not increased. Fetal body weight showed only a decreasing trend for ddI/d4T, no effect for ddI or d4T, and no statistically significant drug interaction. CONCLUSIONS: In pregnant mice, ddI/d4T combinations were not associated with well-defined developmental toxicity or adverse drug interactions.     

Neurotoxicity caused by didanosine on cultured dorsal root ganglion neurons

The purpose of the present study was to investigate whether didanosine (ddI) directly causes morphological and ultrastructural abnormalities of dorsal root ganglion (DRG) neurons in vitro. Dissociated DRG cells and organotypic DRG explants from embryonic 15-day-old Wistar rats were cultured for 3 days and then exposed to ddI (1 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) for another 3 days and 6 days, respectively. Neurons cultured continuously in medium served as normal controls. The diameter of the neuronal cell body and neurite length were measured in dissociated DRG cell cultures. Neuronal ultrastructural changes were observed in both culture models. ddI induced dose-dependent decreases in neurite number, length of the longest neurite in each neuron, and total neurite length per neuron in dissociated DRG cell cultures with 3 days treatment. There were no morphological changes seen in organotypic DRG cultures even with longer exposure time (6 days). But ddI induced ultrastructural changes in both culture models. Ultrastructural abnormalities included loss of cristae in mitochondria, clustering of microtubules and neurofilaments, accumulation of glycogen-like granules, and emergence of large dense particles between neurites or microtubules. Lysosome-like large particles emerged inconstantly in neurites. ddI induced a neurite retraction or neurite loss in a dose-dependent manner in dissociated DRG neurons, suggesting that ddI may partially contribute to developing peripheral neuropathy. Cytoskeletal rearrangement and ultrastructural abnormalities caused by ddI in both culture models may have a key role in neurite degeneration.     

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes.

In the present study, one has determined the relative role of plasma membrane equilibrative (Na+-independent) ENT nucleoside transport proteins (particularly ENT2) in the uptake of antiviral nucleoside analogues for comparison with the previously reported drug transport properties of concentrative (Na+-dependent) CNT nucleoside transport proteins. The human and rat nucleoside transport proteins hENT1, rENT1, hENT2 and rENT2 were produced in Xenopus oocytes and investigated for their ability to transport three 3'-deoxy-nucleoside analogues, ddC (2'3'-dideoxycytidine), AZT (3'-azido-3'-deoxythymidine) and ddI (2'3'-dideoxyinosine), used in human immunodeficiency virus (HIV) therapy. The results show, for the first time, that the ENT2 transporter isoform represents a mechanism for cellular uptake of these clinically important nucleoside drugs. Recombinant h/rENT2 transported ddC, ddI and AZT, whilst h/rENT1 transported only ddC and ddI. Relative to uridine, h/rENT2 mediated substantially larger fluxes of ddC and ddI than h/rENT1. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 transport-positive for AZT and enhanced the uptake of both ddC and ddI, identifying this region as a major site of 3'-deoxy-nucleoside drug interaction.     

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative,nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes

In the present study, one has determined the relative role of plasma membrane equilibrative (Na⁺-independent) ENT nucleoside transport proteins (particularly ENT2) in the uptake of antiviral nucleoside analogues for comparison with the previously reported drug transport properties of concentrative (Na⁺-dependent) CNT nucleoside transport proteins. The human and rat nucleoside transport proteins hENT1, rENT1, hENT2 and rENT2 were produced in Xenopus oocytes and investigated for their ability to transport three 3'-deoxy-nucleoside analogues, ddC (2' 3'-dideoxycytidine), AZT (3'-azido-3'-deoxythymidine)and ddI (2' 3'-dideoxyinosine), used in human immunodeficiency virus (HIV) therapy. The results show, for the first time, that the ENT2 transporter isoform represents a mechanism for cellular uptake of these clinically important nucleoside drugs. Recombinant h/rENT2 transported ddC, ddI and AZT, whilst h/rENT1 transported only ddC and ddI. Relative to uridine, h/rENT2 mediated substantially larger fluxes of ddC and ddI than h/rENT1. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 transport-positive for AZT and enhanced the uptake of both ddC and ddI, identifying this region as a major site of 3'-deoxy-nucleoside drug interaction.     

Nucleoside Phosphorylases from Clostridium Perfringens in the Synthesis of 2′,3′-Dideoxyinosine

Four Clostridium perfringens phosphorylases were subcloned, overexpressed and analyzed for their substrate specificity. DeoD(1) and PunA could use a variety of purine substrates, including an antiviral drug 2′,3′-dideoxyinosine (ddI). In one-pot synthesis using Clostridium phosphorylases, 2′,3′-dideoxyuridine and hypoxanthine were converted to ddI at yield of about 30%.     

Design of the New Molecular Transport Systems for the Nucleosides-Pharmacophores Carrying.

Abstract

The structure design of a specific and nonspecific transport systems for nucleosides was proposed. A number of model compounds were synthesized.

# AZT-2,3′-dideoxy-3′-azidothymidine. d4T-3′-deoxy-2′,3′-didehydrothymidine, ddC -2,3′-dideoxcytidine. ddI -2′.3′-dideoxyinosine. DMAP-4-N,N-dimetylaminopyridyne, DCC-dicyclohexylcarbodiimide.     

Structural analysis of 2',3'-dideoxyinosine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine and 2',3'-dideoxycytidine by 500-MHz 1H-NMR spectroscopy and ab-initio molecular orbital calculations.

Solution structure of anti-AIDS drug, 2',3'-dideoxyinosine (ddI) has been assessed by NMR spectroscopy and pseudorotational analysis in conjunction with its analogues: 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyguanosine (ddG) and 2',3'-dideoxycytidine (ddC). The absence of 3'-hydroxyl groups in these compounds has prompted us to establish the relationship between proton-proton and corresponding endocyclic torsion angles in the 2',3'-dideoxyribofuranose moiety on the basis of five available crystal structures of 2',3'-dideoxynucleosides. A subsequent pseudorotational analysis on ddI (1), ddA (2), ddG (3) and ddC (4) shows that the twist C2'exo-C3'-endo forms of sugar are overwhelmingly preferred (75-80%) over the C2'-endo envelope forms. The phase angles (P) for North and South conformers with the corresponding puckering amplitude (psi m) for ddI (1), ddA (2) and ddG (3) are as follows: PN = 0.1 degrees, PS = 161 degrees and psi m = 34.1 degrees for ddI (1); PN = 1.4 degrees, PS = 160 degrees and psi m = 34.2 degrees for ddA (2) and PN = 2.4 degrees, PS = 163 degrees and psi m = 33.6 degrees for ddG (3). The predominant North conformer of ddC (4) is intermediate between twist C2'-exo-C3'-endo and C3'-endo envelope (P = 10.9 degrees) with a psi m of 34.7 degrees. Note that these preponderant North-sugar structures (approx. 75-80%) found in the solution studies of ddI (1), ddA (2), dG (3) and ddC (4) are not reflected in the X-ray crystal structures of 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures denosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures are found to be in the South-type geometry (ddA crystalizes in C3'-exo envelope form, while ddC adopts the form intermediate between the C3'-exo envelope and C3'-endo-C4'-exo twist form). This means that X-ray structures of ddA (2) and ddC (4) only represent the minor conformer of the overall pseudorotamer population in solution. An assumption that the structure of the pentofuranose sugar (i.e. P and psi m) participating in conformational equilibrium described by the two-state model remains unchanged at different temperatures has been experimentally validated by assessing five unknown pseudorotational parameters with eight unique observables (3J1'2', 3J1'2", 3J2'3', 3J2'3", 3J2"3', 3J2"3", 3J3'4' and 3J3"4') for 2',3'-dideoxynucleosides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis of 2',3'-dideoxy-2',3'-didehydro nucleosides via a serendipitous route

This paper describes a "green" synthesis of 2',3'-unsaturated 2',3'-dideoxynucleosides via an electrochemical reaction. Using this approach d4T, d4U, ddA and ddI can be synthesized in high yields.     

Increased hematopoietic toxicity following administration of interferon-å with combination dideoxynucleoside therapy (zidovudine plus DDI) administered in normal mice

Because of the urgency to develop drugs which will effectively combat HIV infection, many combination therapies which have proved effective against HIV in vitro have undergone, or are undergoing clinical trial. Unfortunately many of drugs are being used without rigorous and exhaustive preclinical evaluation to assess their potential to develope hematopoeitic toxicity. We report here the results of two in vivo studies performed to analyze the effect of combined zidovudine (AZT) plus didanosine (ddl) therapy, either with or without interferon-å (IFN-å), on murine hematopoiesis. Normal C57BL/6 female mice were administered AZT (1.0 mg/ml) plus dose-escalation ddI (0.1, 1.0 and 2.5 mg/ml) placed in their drinking water. Control mice received IFN-å (100 units/ml) alone. Mice were serially bled and sacrified over a six-week period for assessment of hematopoietic toxicity measured by peripheral blood indices and assays of hematopoietic progenitors, i.e., erythroid (BFU-E), myeloid (CFU-GM), and megakaryocyte (CFU-Meg) cultured from bone marrow and spleen. AZT plus dose-escalation ddI decreased the hematocrit and white blood cell count when administered to normal mice compared to untreated controls during the six-week examination period. Marrow derived BFU-E, CFU-GM, and CFU-Meg were all reduced, however an increase was observed from the spleen for all three progenitor cell types. Use of IFN-å, in addition to combination AZT plus ddI further decreased the hematocrit, white blood cell and platelets. Marrow derived CFU-GM and CFU-Meg were increased slightly and only marginally for BFU-E with a similar response observed from the spleen. These results demonstrate that combination AZT plus ddI when used in vivo may produce synergistic hematopoietic toxicity, and that the addition of IFN-å to this treatment regimen increases this toxicity. These data indicate caution when this therapeutic approach is suggested for patients infected with HIV. If used, these patients wil require careful monitoring for blood cel toxicity.     

SYNTHESIS OF 2′,3′-DIDEOXY-2′,3′-DIDEHYDRO NUCLEOSIDES VIA A SERENDIPITOUS ROUTE

This paper describes a “green” synthesis of 2′,3′-unsaturated 2′,3′-dideoxynucl-eosides via an electrochemical reaction. Using this approach d4T, d4U, ddA and ddI can be synthesized in high yields.     

Homo and heterodimers of ddI, d4T and AZT: influence of (5'-5') thiolcabonate-carbamate linkage on anti-HIV activity

New homo and heterodimers of ddI, d4T and AZT with (5-5) thiolcarbonate-carbamate linkages have been prepared with the aim of testing them against wild type and NNRTI resistant HIV mutants. The prepared dimers showed a low activity in comparison to the parent drug.     

Liquid chromatographic-tandem mass spectrometric assay for the simultaneous determination of didanosine and stavudine in human plasma, bronchoalveolar lavage fluid, alveolar cells, peripheral blood mononuclear cells, seminal plasma, cerebrospinal fluid and tonsil tissue

We have developed a sensitive, high-pressure liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method for the simultaneous determination of didanosine (ddI) and stavudine (d4T) in human plasma, bronchoalveolar lavage fluid (BALF), alveolar cells (AC), peripheral blood mononuclear cells (PBMC), seminal plasma, cerebrospinal fluid (CSF), and tonsil tissue. Plasma, AC, PBMC and CSF were run with an isocratic HPLC method, while BALF supernatant, semen, and tonsil tissue utilized a gradient elution. Samples were prepared by solid phase extraction. Detection was by electrospray positive ionization with multiple reaction monitoring mode. The lower limits of quantitation for both ddI and d4T were 2.0 ng/ml in plasma; 0.5 ng/ml in CSF; 0.4 ng/ml in AC, PBMC, and BALF; 1.0 ng/ml in seminal plasma; and 0.01 ng/mg in tonsil tissue.