DNA adductomics aided rapid screening of genotoxic impurities using nucleosides and 3D bioprinted human liver organoids.

Current genotoxicity assessment methods are mainly employed to verify the genotoxic safety of drugs, but do not allow for rapid screening of specific genotoxic impurities (GTIs). In this study, a new approach for the recognition of GTIs has been proposed. It is to expose the complex samples to an in vitro nucleoside incubation model, and then draw complete DNA adduct profiles to infer the structures of potential genotoxic impurities (PGIs). Subsequently, the genotoxicity is confirmed in human by 3D bioprinted human liver organoids. To verify the feasibility of the approach, lansoprazole chloride compound (Lanchlor), a PGI during the synthesis of lansoprazole, was selected as the model drug. After confirming genotoxicity by Comet assay, it was exposed to different models to map and compare the DNA adduct profiles by LC-MS/MS. The results showed Lanchlor could generate diverse DNA adducts, revealing firstly its genotoxicity at molecular mechanism of action. Furthermore, the largest variety and content of DNA adducts were observed in the nucleoside incubation model, while the human liver organoids exhibited similar results with rats. The results showed that the combination of DNA adductomics and 3D bioprinted organoids were useful for the rapid screening of GTIs.

Genotoxicity assessment of potentially mutagenic nucleoside analogues using ToxTracker®.

Nucleoside analogues have long been designed and tested in cancer treatment and against viral infections. However, several early compounds were shown to have mutagenic properties as a consequence of their mode-of-action. This limited their use, and several have been discontinued for lengthy treatments or altogether. Nonetheless, nucleoside analogues remain an attractive modality for virally driven diseases, of which many still are without proper treatment options. To quantitatively assess the genotoxic mode-of-action of a panel of nucleoside analogues, we applied the ToxTracker® reporter assay. Many of the early nucleoside analogues showed a genotoxic response. The more recently developed nucleoside analogues, Remdesivir and Molnupiravir that are currently being repurposed for Covid-19 treatment, had a different profile in ToxTracker and did not induce the genotoxicity reporters. Our analyses support the metabolite GS-441524 over the parent analogue Remdesivir. In contrast, Molnupiravir was devoid of clear cellular toxicity while its active metabolite (EIDD-1931) was cytotoxic and induced several biomarkers. Nucleoside analogues continue to be attractive treatment options upon viral infections. ToxTracker readily distinguished between the genotoxic analogues and those with different profiles and provides a basis for clustering and potency ranking, offering a comprehensive tool to assess the toxicity of nucleoside analogues.

Phenoxazine nucleoside derivatives with a multiple activity against RNA and DNA viruses.

Emerging and re-emerging viruses periodically cause outbreaks and epidemics all over the world, eventually leading to global events such as the current pandemic of the novel SARS-CoV-2 coronavirus infection COVID-19. Therefore, an urgent need for novel antivirals is crystal clear. Here we present the synthesis and evaluation of an antiviral activity of phenoxazine-based nucleoside analogs divided into three groups: (1) 8-alkoxy-substituted, (2) acyclic, and (3) carbocyclic. The antiviral activity was assessed against a structurally and phylogenetically diverse panel of RNA and DNA viruses from 25 species. Four compounds (11a-c, 12c) inhibited 4 DNA/RNA viruses with EC50 ≤ 20 µM. Toxicity of the compounds for the cell lines used for virus cultivation was negligible in most cases. In addition, previously reported and newly synthesized phenoxazine derivatives were evaluated against SARS-CoV-2, and some of them showed promising inhibition of reproduction with EC50 values in low micromolar range, although accompanied by commensurate cytotoxicity.

Repurposing Nucleoside Analogs for Human Coronaviruses.

Coronavirus disease 2019 (COVID-19) is a serious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or CoV-2). Some reports claimed certain nucleoside analogs to be active against CoV-2 and thus needed confirmation. Here, we evaluated a panel of compounds and identified novel nucleoside analogs with antiviral activity against CoV-2 and HCoV-OC43 while ruling out others. Of significance, sofosbuvir demonstrated no antiviral effect against CoV-2, and its triphosphate did not inhibit CoV-2 RNA polymerase.

Anti-norovirus activity of C7-modified 4-amino-pyrrolo[2,1-f][1,2,4]triazine C-nucleosides.

Synthetic nucleoside analogues characterized by a C-C anomeric linkage form a family of promising therapeutics against infectious and malignant diseases. Herein, C-nucleosides comprising structural variations at the sugar and nucleobase moieties were examined for their ability to inhibit both murine and human norovirus RNA-dependent RNA polymerase (RdRp). We have found that the combination of 4-amino-pyrrolo[2,1-f][1,2,4]triazine and its 7-halogenated congeners with either a d-ribose or 2'-C-methyl-d-ribose unit resulted in analogues with good antiviral activity against murine norovirus (MNV), albeit coupled with a significant cytotoxicity. Among this series, 4-aza-7,9-dideazaadenosine notably retained a strong antiviral effect in a human norovirus (HuNoV) replicon assay with an EC50 = 0.015 µM. This study demonstrates that C-nucleosides can be used as viable starting scaffolds for further optimization towards the development of nucleoside-based inhibitors of norovirus replication.

A Humanized Yeast Phenomic Model of Deoxycytidine Kinase to Predict Genetic Buffering of Nucleoside Analog Cytotoxicity.

Knowledge about synthetic lethality can be applied to enhance the efficacy of anticancer therapies in individual patients harboring genetic alterations in their cancer that specifically render it vulnerable. We investigated the potential for high-resolution phenomic analysis in yeast to predict such genetic vulnerabilities by systematic, comprehensive, and quantitative assessment of drug-gene interaction for gemcitabine and cytarabine, substrates of deoxycytidine kinase that have similar molecular structures yet distinct antitumor efficacy. Human deoxycytidine kinase (dCK) was conditionally expressed in the Saccharomycescerevisiae genomic library of knockout and knockdown (YKO/KD) strains, to globally and quantitatively characterize differential drug-gene interaction for gemcitabine and cytarabine. Pathway enrichment analysis revealed that autophagy, histone modification, chromatin remodeling, and apoptosis-related processes influence gemcitabine specifically, while drug-gene interaction specific to cytarabine was less enriched in gene ontology. Processes having influence over both drugs were DNA repair and integrity checkpoints and vesicle transport and fusion. Non-gene ontology (GO)-enriched genes were also informative. Yeast phenomic and cancer cell line pharmacogenomics data were integrated to identify yeast-human homologs with correlated differential gene expression and drug efficacy, thus providing a unique resource to predict whether differential gene expression observed in cancer genetic profiles are causal in tumor-specific responses to cytotoxic agents.

A case of entecavir-induced Fanconi syndrome.

Acquired Fanconi syndrome has been associated with the long-term ingestion of several nucleoside analogs used to treat chronic hepatitis B virus infection. However, the nucleoside analog entecavir has not been found to cause nephrotoxicity. We report a case of entecavir-induced Fanconi syndrome. Our patient was a 73-year-old man admitted to our hospital because of renal dysfunction. He also presented with hyperaminoaciduria, renal diabetes, phosphaturia, hypophosphatemia, hypokalemia, hypouricemia, and hyperchloremic metabolic acidosis, supporting a diagnosis of Fanconi syndrome. In this case, the cause of Fanconi syndrome was most likely entecavir, which had been administered as needed depending on his renal function for 5 years. After drug discontinuation and replacement with tenofovir alafenamide fumarate therapy once a week, the patient's kidney function recovered and electrolyte anomalies partially improved. We highlight the fact that entecavir may induce severe renal dysfunction, which can cause the development of Fanconi syndrome; therefore, close monitoring of proximal tubular function is recommended during entecavir therapy.

Metabolism of 4-pyridone-3-carboxamide-1ß-d-ribonucleoside (4PYR) in primary murine brain microvascular endothelial cells (mBMECs).

4-pyridone-3-carboxamide-1ß-D-ribonucleoside (4PYR) is a derivative of nicotinamide found physiologically in human body fluids that can be metabolized to mono-, di- or triphosphate derivatives (4PYMP, 4PYDP and 4PYTP respectively) and an analogue of NAD - the 1-ß-D-ribonucleoside-4-pyridone-3-carboxamide adenine dinucleotide (4PYRAD) in human cells. The European Uremic Toxin Work Group (EUTox) has classified 4PYR as a uremic toxin that adversely affects endothelium. This study aimed to investigate the metabolism of 4PYR in murine brain microvascular endothelial cells (mBMECs). Incubation of mBMECs with 4PYR was carried out for 0, 24, 48 or 72 h. After incubation, a medium was removed and cellular concentrations of ATP, ADP, NAD, 4PYMP and 4PYRAD were analyzed using reversed-phase HPLC. 4PYR was metabolized by mBMECs to 4PYMP and 4PYRAD that reached concentrations of 2 ± 0.7 and 0.6 ± 0.2 nmol/mg protein (mean ± SEM), respectively, after 72 h incubation. However, unlike with endothelial cells studied so far this process has no effect on energy balance in the cell as indicated by maintained ATP/ADP ratio and adenine and nicotinamide intracellular pools. Further studies are required to explain whether the difference in 4PYR metabolism is related to differences between species or organs.

Addressing the selectivity and toxicity of antiviral nucleosides.

Nucleoside and nucleotide analogs have played significant roles in antiviral therapies and are valued for their impressive potency and high barrier to resistance. They have been approved for treatment of herpes simplex virus-1, HIV, HBV, HCV, and influenza, and new drugs are being developed for the treatment of RSV, Ebola, coronavirus MERS, and other emerging viruses. However, this class of compounds has also experienced a high attrition rate in clinical trials due to toxicity. In this review, we discuss the utility of different biochemical and cell-based assays and provide recommendations for assessing toxicity liability before entering animal toxicity studies.

Presente y futuro de darunavir para el tratamiento de la infección por virus de la inmunodeficiencia humana / Present and future of darunavir for the treatment of human immunodeficiency virus infection

Darunavir es un fármaco antirretroviral que, desde su comercialización en 2007, ha sido esencial en el tratamiento de los pacientes infectados por VIH. Los primeros ensayos clínicos posicionaron a darunavir 600 mg 2 veces al día potenciado con ritonavir como un fármaco básico, en combinación con otros, en cualquier pauta de rescate avanzado. Posteriormente, los ensayos clínicos en pacientes naïve situaron a darunavir como uno de los fármacos preferentes en las combinaciones de inicio de tratamiento con darunavir, posición en la que sigue estando en 2016, a pesar de la llegada de los inhibidores de la integrasa, en la mayoría de guías de práctica clínica. En estos años ha demostrado también su eficacia en la simplificación a monoterapia bajo ciertas condiciones y su utilidad en diferentes escenarios clínicos como uno de los componentes de diferentes biterapias. Dada la necesidad de adaptar el tratamiento antirretroviral a la realidad de cada paciente, hay algunos escenarios (inmunodepresión grave, no disponer de genotipo basal en el momento del inicio del tratamiento, dudas respecto a adherencia, embarazo, toxicidad por nucleósidos y, por supuesto, rescate) en los que darunavir, por su potencia, barrera genética y experiencia de uso, puede aportar un valor añadido. La reciente comercialización de darunavir potenciado con cobicistat en comprimido único y los ensayos clínicos en marcha (simplificación a darunavir + lamivudina, tratamiento en pacientes naïve con alafenamida/emtricitabina/darunavir/cobicistat, etc.) se antojan avances considerables que mejorarán la tolerancia y la comodidad de uso del fármaco y sus escenarios potenciales (AU)

Darunavir is an antiretroviral agent that, since it was marketed in 2007, has been essential in the treatment of HIV-infected patients. The first clinical trials demonstrated that twice-daily, ritonavir-boosted darunavir 600 mg was an essential drug, in combination with others, in any advanced rescue strategy. Subsequently, clinical trials in treatment-naïve patients revealed darunavir to be a preferred drug in initial combination regimens, which continues to be the case in 2016, despite the arrival of integrase inhibitors, in most clinical practice guidelines. In the last few years, the efficacy of darunavir has also been demonstrated in simplification to monotherapy under certain conditions, as well as its utility in distinct clinical scenarios, such as its use as one of the components of dual therapy. Given the need to adapt antiretroviral therapy to the reality of each patient, there are some scenarios (severe immunodepression, the lack of an available baseline genotype at treatment initiation, doubts about adherence, pregnancy, nucleoside toxicity and, obviously, rescue therapy) in which darunavir, because of its potency, genetic barrier and the wide experience of its use, can provide added value. The recent commercialisation of cobicistat-boosted darunavir in single tablet form and ongoing clinical trials (simplification to darunavir + lamivudina, its use in treatment-naïve patients with alafenamide/emtricitabine/darunavir/cobicistat, etc.) represent considerable advances that will improve the tolerability and ease of use of the drug and increase its potential scenarios (AU)

Synthesis of a Novel Class of 1,3-oxathiolane Nucleoside Derivatives of T- 705 and Evaluation of Their Anti-influenza A Virus and Anti-HIV Activity.

BACKGROUND: T-705 (Favipiravir) is a broad spectrum antiviral agent approved for stockpiling in Japan and currently in Phase 3 testing in the United States. Against influenza, it acts as a prodrug, converted intracellularly to selectively inhibit viral RNA-dependent RNA polymerase or similar enzymes. This is regarded as a novel antiviral mechanism of action, reducing crossresistance to other existing anti-influenza drugs. OBJECTIVE: To develop new analogs, a class of 1,3-oxathiolane nucleoside derivatives of T-705 was designed and synthesized in this work. RESULTS: Anti-influenza activity and Anti-HIV activity of these compounds were evaluated. Compound 1a displayed activity against A H1N1 with an IC50 of 40.4 µmol/L. Compound 1b showed weak activity against HIV with a viral suppression rate of 70-80% at 30 µmol/L. CONCLUSION: A class of 1,3-oxathiolane nucleoside derivatives of T-705 was designed and synthesized, and one of them was identified as a novel scaffold against viral infection.

Risk of cancer in children exposed to antiretroviral nucleoside analogues in utero: The french experience.

All nucleoside analogues for treating HIV infection, due to their capacity to integrate into and alter human DNA, are experimentally genotoxic to some extent. The long-term oncogenic risk after in utero exposure remains to be determined. Cancer incidence in uninfected children exposed to nucleos(t)ide reverse transcriptase inhibitors (NRTIs) was evaluated, by cross-checking against the National Cancer Registry, in the French perinatal study of children born to HIV+ mothers. Twenty-one cancers were identified in 15,163 children (median age: 9.9 years [interquartile range (IQR): 5.8-14.2]) exposed to at least one NRTI in utero between 1990 and 2014. Five of these children were exposed to zidovudine monotherapy, and 15 to various combinations, seven of which included didanosine. Overall, the total number of cases was not significantly different from that expected for the general population (SIR = 0.8[0.47-1.24]), but the number of cases after didanosine exposure was twice that expected (SIR = 2.5 [1.01-5.19]). Didanosine accounted for only 10% of prescriptions but was associated with one-third of cancers. In multivariate analysis, didanosine exposure was significantly associated with higher risk (HR = 3.0 [0.9-9.8]). This risk was specifically linked to first-trimester exposure (HR = 5.5 [2.1-14.4]). Three cases of pineoblastoma, a very rare cancer, were observed, whereas 0.03 were expected. Two were associated with didanosine exposure. Despite reassuring data overall, there is strong evidence to suggest that didanosine displays transplacental oncogenicity. These findings cannot be extrapolated to other NRTIs, but they highlight the need for comprehensive evaluations of the transplacental genotoxicity of this antiretroviral class. Environ. Mol. Mutagen., 60:404-409, 2019. © 2017 Wiley Periodicals, Inc.

Synthesis, fungicidal evaluation and 3D-QSAR studies of novel 1,3,4-thiadiazole xylofuranose derivatives.

1,3,4-Thiadiazole and sugar-derived molecules have proven to be promising agrochemicals with growth promoting, insecticidal and fungicidal activities. In the research field of agricultural fungicide, applying union of active group we synthesized a new set of 1,3,4-thiadiazole xylofuranose derivatives and all of the compounds were characterized by 1H NMR and HRMS. In precise toxicity measurement, some of compounds exhibited more potent fungicidal activities than the most widely used commercial fungicide Chlorothalonil, promoting further research and development. Based on our experimental data, 3D-QSAR (three-dimensional quantitative structure-activity relationship) was established and investigated using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques, helping to better understand the structural requirements of lead compounds with high fungicidal activity and environmental compatibility.

Structure-activity relationship analysis of mitochondrial toxicity caused by antiviral ribonucleoside analogs.

Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3'-deoxy or 2'-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (Ki) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2' or 4' mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity.

DRAM Is Involved in Regulating Nucleoside Analog-Induced Neuronal Autophagy in a p53-Independent Manner.

The widespread use of combined anti-retroviral therapy (cART) has not decreased the prevalence of HIV-1-associated neurocognitive disorder (HAND), a type of neurodegenerative disease, even though cART effectively inhibits virus colonization in the central nervous system. Therefore, anti-retroviral agents cannot be fully excluded from the pathogenesis of HAND. Our previous study reported that long-term nucleoside analogue (NA) exposure induced mitochondrial toxicity in the cortical neurons of HAND patients and mice, but the exact mechanism of NA-associated neurotoxicity has remained unclear. Alteration of autophagy can result in protein aggregation and the accumulation of dysfunctional organelles, which are hallmarks of some neurodegenerative diseases. In this study, we first found increased autophagy in cortical autopsy specimens of AIDS patients. We then found that a low dose of NAs could stimulate autophagy in primary cultured neurons, while a high dose of NAs could induce only neuronal apoptosis. The level of NA-induced Bcl-2 and Bax expressions determined whether neuronal autophagy or apoptosis occurred. Furthermore, the level of NA-induced neuronal apoptosis correlated with the dysfunction of cellular DNA polymerase gamma. Damage-regulated autophagy modulator (DRAM) overexpression was also involved in NA-induced neuronal autophagy. p53 played a role in the regulation of NA-induced neuronal apoptosis, but its role in NA-associated neuronal autophagy was uncertain. Our results suggest that DRAM is involved in the regulation of NA-induced neuronal autophagy in a p53-independent manner. Further research is needed to investigate the underlying mechanism.

Síntesis y actividad citotóxica de conjugados de la uridina con triterpenos en células de cáncer de mama / Synthesis and cytotoxic activity of conjugates of the uridine with triterpenes on breast cancer cells

Objetivos: Sintetizar conjugados del acetónido de la uridina con triterpenos (colesterol y 3β-5α,8α- endoperoxido-colest-6-en-3-ol) y ácido succínico como puente. Métodos: Se preparó el acetónido de la uridina en acetona mediante catálisis ácida. Se prepararon los succinatos de los esteroles con anhídrido succínico y catalizador nucleofílico 4-N,N-dimetilamino-piridina (DMAP). Los conjugados 1 y 2 se sintetizaron mediante la esterificación de Steglich, con agente de acoplamiento N,N’-diciclohexilcarbodiimida (DCC)y DMAP. Los compuestos se caracterizaron por espectroscopia de RMN (1H RMN y 13C RMN) y espectrometría de masas. Los derivados se evaluaron sobre líneas celulares de ovario de hámster chino (CHO-K1) y de cáncer de mamá (MCF-7). Resultados: Se obtuvieron derivados conjugados del acetónido de la uridina con dos triterpenos con rendimientos superiores al 80%. Los conjugados de uridina con triterpenos no presentaron inhibición significativa de la viabilidad celular sobre las líneas celulares MCF-7 y CHO-K1, tampoco se evidenció una relación dosis-respuesta para los compuestos evaluados. Conclusiones: El método de esterificación con agentes de acoplamiento permitió obtener conjugados de la uridina con triterpenos empleando el ácido succínico como puente. Sin embargo los derivados de uridina obtenidos no presentaron actividad citotóxica significativa (p < 0,05) sobre las líneas celulares evaluadas

Aims: Synthesize of uridine acetonide conjugates with triterpenoids (cholesterol and 3β-5α,8α-endoperoxide- cholest-6-en-3-ol) and succinic acid as linking. Methods: The acetonide derivative of uridine was prepared with acid catalysis in acetone. Sterols succinates were prepared with succinic anhydride and nucleophilic catalyst 4-N,N-dimethylamino-pyridine (DMAP). The conjugates were synthesized by Steglich method with N,N’-dicyclohexylcarbodiimide (DCC) Coupling agent and DMAP. The compounds were characterized by NMR spectroscopy (1H NMR, 13C NMR), and mass spectrometry. The derivatives were assessed in Chinese Hamster Ovary (CHO) and breast cancer (MCF-7) cell lines. Results: The conjugates of uridine acetonide with two triterpenes were obtained with yields higher than 80%. The conjugates prepared don’t showed significant inhibition of cell viability on MCF-7 and CHO cell lines, furthermore these substances did not show a relationship dose-response. Conclusions: The esterification method with coupling agents allowed obtained uridine conjugates with triterpenoids. However the uridine derivatives don’t showed significant cytotoxic activity (p < 0,05) against cell lines evaluated

The pivotal role of uridine-cytidine kinases in pyrimidine metabolism and activation of cytotoxic nucleoside analogues in neuroblastoma.

Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine as well as the pharmacological activation of several cytotoxic pyrimidine ribonucleoside analogues. In this study, we investigated the functional role of two isoforms of UCK in neuroblastoma cell lines. Analysis of mRNA coding for UCK1 and UCK2 showed that UCK2 is the most abundantly expressed UCK in a panel of neuroblastoma cell lines. Transient and stable overexpression of UCK2 in neuroblastoma cells increased the metabolism of uridine and cytidine as well as the cytotoxicity of 3-deazauridine. Knockdown of endogenous UCK2 as well as overexpression of UCK1 resulted in decreased metabolism of uridine and cytidine and protected the neuroblastoma cells from 3-deazauridine-induced toxicity. Subcellular localization studies showed that UCK1-GFP and UCK2-GFP were localized in the cell nucleus and cytosol, respectively. However, co-expression of UCK1 with UCK2 resulted in a nuclear localization of UCK2 instead of its normal cytosolic localization, thereby impairing its normal function. The physical association of UCK1 and UCK2 was further demonstrated through pull-down analysis using his-tagged UCK. The discovery that UCK2 is highly expressed in neuroblastoma opens the possibility for selectively targeting neuroblastoma cells using UCK2-dependent pyrimidine analogues, while sparing normal tissues.

Rational Development of Nucleoside Diphosphate Prodrugs: DiPPro-Compounds.

The bio-reversible protection of nucleoside diphosphates is summarized. The design, hydrolytic characteristics, and the antiviral activity of these prodrugs of NDPs are described. In contrast to earlier attempts, the DiPPro-approach [ß-(bis(acyloxybenzyl) nucleoside diphosphates)] leads to the successful delivery of the desired nucleoside diphosphates. The stability towards hydrolysis is dependent on the specific acyl moieties in the bis(acyloxybenzyl) unit as well as on the particular nucleoside analogue. Hydrolysis studies in aqueous PBS buffer (pH 7.3), 20 % human plasma in PBS, RPMI-1640 culture medium, and CEM cell extracts were carried out. Contrary to a high chemical and plasma stability, the compounds showed a very low half-life in CEM cell extracts, and efficiently released the nucleoside analogues diphosphates, e.g. of AZT, d4T and BVDU. Two additional types of cycloSal- NDP prodrugs were studied but neither proved to be useful as nucleoside diphosphate prodrugs. In summary, the results led to the development of a new series of non-symmetric nucleoside diphosphate prodrugs that selectively delivered the nucleoside diphosphate in cell extracts.

Effect of 4-pyridone-3-carboxamide ribonucleoside (4PYR)-potential cardiovascular toxin in perfused rat heart.

We recently described a new nicotinamide derivative: 4-pyridone-3-carboxamide ribonucleoside (4PYR) and its conversion to intracellular metabolites (4PYR monophosphate: 4PYMP and 4PYR adenylate diphosphate: 4PYRAD). The aim of this study was to clarify the metabolism and physiological effects of brief exposure to 4PYR in perfused rat heart. Rat hearts were perfused in Langendorff mode. After 15 min equilibration, 100 µM 4PYR (or solvent in controls) was infused into coronary circulation for 5 min. Coronary flow was recorded with electromagnetic flow meter and left ventricular mechanical function was assessed with intraventricular baloon by constructing pressure-volume relations. After perfusion hearts were freeze-clamped and analyzed using HPLC for phosphocreatine, creatine, ATP with metabolites as well as 4PYR metabolites. 4PYR infused into the coronary circulation was rapidly converted in the heart into 4PYMP and 4PYRAD with concentrations reaching 85.6±46.9 and 43.9±6.4 nmol/g dry weight, respectively, while control concentrations were below 20 nmol/g. 4PYR had no effect on baseline coronary flow (11.9±2.3 ml/min versus 11.0±2.7 ml/min in control) or stimulated by shear stress (23.2±4.5 ml/min versus 23.1±5.2 ml/min in control). Both systolic and diastolic left ventricular mechanical function were not affected by 4PYR. No difference was noted for heart rate. Myocardial concentrations of ATP or phosphocreatine were also not affected by 4PYR. We conclude that 4PYR has no immediate effect on coronary endothelium or cardiomyocyte functions such as coronary flow, rhythm, diastolic properties, or contractility despite rapid incorporation into intracellular metabolites. This study also indicates the lack of effect on purinergic receptors.

Synthesis of novel 2'-fluoro-3'-hydroxymethyl-5'-deoxythreosyl phosphonic acid nucleoside analogues as antiviral agents.

A series of purine 5'-deoxyphosphonate analogues were designed and synthesized to mimic naturally occurring purine monophosphate from 1,3-dihydroxyacetone as starting material. The discovery of threosyl phosphonate nucleoside (PMDTA, EC50 = 2.53 µM) as a potent anti-HIV agent has led to the synthesis and biological evaluation of 2',3'-modified 5'-deoxyversions of the threosyl phosphonate nucleosides. The synthesized 2'-fluoro-3'-hydroxymethyl 5'-deoxythreosyl phosphonic acid nucleoside analogues 14, 18, 23, and 27 were tested for anti-HIV activity as well as cytotoxicity. The adenine analogue 18 exhibits weak in vitro anti-HIV-1 activity (EC50 = 19.2 µM).