RESUMEN
This review depicts in vitro and in vivo results obtained with nucleotide prodrugs (pronucleotides) bearing S-acyl-2-thioethyl (SATE) groups as esterase-labile phosphate protections. New developments are illustrated by the design of mononucleoside mixed phosphoester derivatives leading to the selective intracellular delivery of the corresponding 5'-mononucleotide through two different enzyme-mediated activation steps.
Asunto(s)
Antivirales/farmacología , Nucleótidos/farmacología , Profármacos/farmacología , Animales , Antivirales/síntesis química , Antivirales/química , División Celular/efectos de los fármacos , VIH-1/efectos de los fármacos , Virus de la Hepatitis B/efectos de los fármacos , Humanos , Cinética , Nucleótidos/síntesis química , Nucleótidos/química , Organofosfatos/química , Organofosfatos/metabolismo , Organofosfatos/farmacocinética , Profármacos/síntesis química , Profármacos/química , Relación Estructura-Actividad , Factores de TiempoRESUMEN
The synthesis of new 3'-deoxy-3'-[4-(pyrimidin-1-yl)methyl-1,2,3-triazol-1-yl]-thymidine 6a-f, from 3'-azido-3'-deoxy-5'-O-monomethoxytrityl-thymidine is described. The key step is the 1,3-dipolar cycloaddition between the azido group of the protected AZT 3 and N-1-propargylpyrimidine derivatives 2a-f. All new derivatives 6a-f were evaluated for their inhibitory effects against the replication of HIV-1 (IIIB), HIV-2 (ROD). No marked activity was found.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Tiazoles/química , Timidina/análogos & derivados , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Línea Celular , Ciclización , VIH-1/efectos de los fármacos , Humanos , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/metabolismo , Timidina/químicaRESUMEN
The synthesis and the study of two phosphorothiolate derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and glucosyl residues associated to the phosphorus atom by a 2-oxyethyl link, are reported. These derivatives could be considered as prototypes of a new series of nucleotide prodrugs (pronucleotides).
Asunto(s)
Antivirales/síntesis química , Nucleósidos/síntesis química , Profármacos/síntesis química , Zidovudina/síntesis química , Antivirales/química , Estructura Molecular , Nucleósidos/farmacología , Profármacos/química , Zidovudina/análogos & derivados , Zidovudina/químicaRESUMEN
The synthesis, anti-HIV activity and stability studies of a H-phosphonamidate derivative of 3'-azido-2',3'-dideoxythymidine (AZT) incorporating a N,N-diisopropylamino residue as first model of alkylamino group are reported. The results demonstrate that such phosphorylated structure exerts its biological effects via chemical hydrolysis into the corresponding H-phosphonate, precursor of the parent nucleoside.
Asunto(s)
Fármacos Anti-VIH/síntesis química , VIH/efectos de los fármacos , Nucleósidos/síntesis química , Nucleótidos/síntesis química , Profármacos/síntesis química , Zidovudina/análogos & derivados , Zidovudina/síntesis química , Línea Celular , Diseño de Fármacos , Estabilidad de Medicamentos , Humanos , Hidrólisis , Estructura Molecular , Organofosfonatos , Linfocitos TRESUMEN
N-Acetyl oligonucleotides and their prodrugs were synthesized on photolabile solid support. Tm studies showed a decrease of hydridization for N-acetyl A and G and an increase for N-acetyl C. In cells extract, acetyl groups were hydrolysed.
Asunto(s)
Oligonucleótidos/síntesis química , Profármacos/síntesis química , Acetilación , Secuencia de Bases , Hidrólisis , Indicadores y Reactivos , Conformación de Ácido Nucleico , Nucleósidos/química , Oligonucleótidos/química , Profármacos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
MALDI-TOF mass spectrometry has been used to characterize solid-supported oligonucleotides containing natural and non-natural and non-nucleoside moieties and a variety of internucleosidic linkages including phosphate and phosphite triesters and H-phosphonate diesters. This technique was used to follow the reactions involved in oligonucleotide synthesis; this enabled direct control of the elongation and optimization of the coupling process.
Asunto(s)
Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/síntesis química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Bases , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
beta-L-Thymidine (L-dT) and beta-L-2'-deoxycytidine (L-dC) are potent and highly specific inhibitors of hepatitis B virus (HBV) replication both in vivo and in vitro (50% effective concentrations, 0.19 to 0.24 microM in 2.2.15 cells). The intracellular metabolisms of L-dT and L-dC were investigated in HepG2 cells and primary cultured human hepatocytes. L-dT and L-dC were extensively phosphorylated in both cell types, with the 5'-triphosphate derivative being the predominant metabolite. In HepG2 cells, the 5'-triphosphate levels were 27.7 +/- 12.1 and 72.4 +/- 1.8 pmol/10(6) cells for L-dT and L-dC, respectively. In primary human hepatocytes, the 5'-triphosphate levels were 16.5 +/- 9.8 and 90.1 +/- 36.4 pmol/10(6) cells for L-dT and L-dC, respectively. Furthermore, a choline derivative of L-dCDP was detected at concentrations of 15.8 +/- 1.8 and 25.6 +/- 0.1 pmol/10(6) cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to L-dC, the 5'-monophosphate and 5'-triphosphate derivatives of beta-L-2'-deoxyuridine (L-dUMP and L-dUTP, respectively) were also observed, reaching intracellular concentrations of 6.7 +/- 0.4 and 18.2 +/- 1.0 pmol/10(6) cells, respectively. In human hepatocytes, L-dUMP and L-dUTP were detected at concentrations of 5.7 +/- 2.4 and 43.5 +/- 26.8 pmol/10(6) cells, respectively. It is likely that deamination of L-dCMP by deoxycytidylate deaminase leads to the formation of L-dUMP, as the parent compound, L-dC, was not a substrate for deoxycytidine deaminase. The intracellular half-lives of L-dTTP, L-dCTP, and L-dUTP were at least 15 h, with intracellular concentrations of each metabolite remaining above their respective 50% inhibitory concentrations for the woodchuck hepatitis virus DNA polymerase for as long as 24 h after removal of the drug from cell cultures. Exposure of HepG2 cells to L-dT in combination with L-dC led to concentrations of the activated metabolites similar to those achieved with either agent alone. These results suggest that the potent anti-HBV activities of L-dT and L-dC are associated with their extensive phosphorylation.
Asunto(s)
Antivirales/farmacología , Carcinoma Hepatocelular/metabolismo , Desoxicitidina/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B/tratamiento farmacológico , Hepatocitos/efectos de los fármacos , Neoplasias Hepáticas/metabolismo , Timidina/farmacología , Cromatografía Líquida de Alta Presión , Desoxicitidina/metabolismo , Semivida , Hepatitis B/virología , Humanos , Fosforilación , Timidina/metabolismo , Células Tumorales CultivadasRESUMEN
The biological evaluation of mononucleotide prodrugs (pronucleotides) of various nucleoside reverse transcriptase inhibitors (NRTIs) such as zidovudine (AZT), zalcitabine (ddC) and lamivudine (3TC) was reported in human T-lymphoid MOLT-4/8 cells which were grown continuously for more than 1 year in a medium containing cytarabine (Ara-C). In this cell line, expression of deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) was decreased in comparison to parental cells (3.8 and 2.9-fold, respectively). The lower mRNA level of TK1 correlated significantly with lower enzyme activity, whereas no dCK activity was detectable. In Ara-C-resistant cells, anti-HIV-1 effects of ddC, 3TC and AZT were more than 100-fold lower compared with parental cells. In contrast, the corresponding mononucleoside phosphotriesters bearing S-acyl-2-thioethyl (SATE) groups as biolabile phosphate protection retained anti-HIV-1 activity due to their ability to bypass the first monophosphorylation step catalyzed by dCK or TK1. The results demonstrate that in vitro selection of T-lymphoid cells in the presence of Ara-C results in cross-resistance to deoxycytidine (ddC, 3TC) and thymidine (AZT) analogs and that these cellular resistance mechanisms can be bypassed by the use of bis(SATE) pronucleotides.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Profármacos/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , División Celular , Línea Celular , Citarabina/farmacología , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina Quinasa/metabolismo , Resistencia a Medicamentos , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Pruebas de Sensibilidad Microbiana , Linfocitos T/virología , Timidina/análogos & derivados , Timidina/farmacología , Timidina Quinasa/metabolismoRESUMEN
The chemical synthesis of some 4-substituted 1-[1-(2-hydroxyethoxy)-methyl-1,2.3-triazol-(4 and 5)-ylmethyl]-1-H-pyrazolo[3,4-d]pyrimidines 12a,b, 13a,b and 14-23 as acyclic nucleosides is described. Treatment of (2-acetoxyethoxy)methylbromide with sodium azide afforded (2-acetoxyethoxy)methylazide 9. The heterocycles 6a,b were alkylated, separately, with propargyl bromide to obtain. regioselectively, 4-(methyl and benzyl)thio-1-(prop-2-ynyl)-1H-pyrazolo[3,4-d]pyrimidines 7a,b. These N-alkylated products were condensed with compound 9 via a 1,3-dipolar cycloaddition reaction to obtain, after separation and deprotection, 1,4- and 1,5-regioisomers 12a,b and 13a,b. The deprotected acyclic nucleosides 12a and 13a served as precursors for the preparation of 4-amino (14 and 15), 4-methylamino (16 and 17). 4-benzylamino (18 and 19), 4-methoxy (20 and 21) and 4-hydroxy (22 and 23) analogues. Compounds 7a,b and all deprotected acyclic nucleosides were evaluated for their inhibitory effects against the replication of HIV-(IIIB) and HIV-2(ROD) in MT-4 cells and for their anti-tumor activity. No marked activity was found. However, initial evaluation of 6a,b, 7a,b. 12a,b, 13a,b and 14-23 showed that compound 7b has marked activity against M. tuberculosis.
Asunto(s)
Nucleósidos/química , Nucleósidos/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Antivirales/síntesis química , Antivirales/farmacología , Línea Celular , VIH/metabolismo , Humanos , Modelos Químicos , Nucleósidos/síntesis química , Pirimidinas/síntesis química , TemperaturaRESUMEN
Three simple, related nucleosides, beta-L-2'-deoxycytidine (LdC), beta-Lthymidine (LdT), and beta-L-2'-deoxyadenosine (LdA), have been discovered to be potent, specific and selective inhibitors of the replication hepatitis B virus (HBV), as well as the closely related duck and woodchuck hepatitis viruses (WHV). Structure-activity relationship analysis indicates that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific anti-hepadnavirus activity. The simple nucleosides had no effect on the replication of 15 other RNA and DNA viruses, and did not inhibit human DNA polymerases (alpha, beta and gamma) or compromise mitochondrial function. The nucleosides are efficiently converted intracellularly into active triphosphate metabolites that have a long half-life. Once-daily oral administration of these compounds in the woodchuck efficacy model of chronic HBV infection reduced viral load by as much as 10(8) genome equivalents/ml serum and there was no drug-related toxicity. In addition, a decline in WHV surface antigen (WHsAg) paralleled the decrease in viral load. This class of nucleosides displays an excellent overall safety profile. The first compound, LdT, has already entered clinical trials and LdC, currently being developed as a prodrug, is expected to enter the clinic in the near future. These compounds have the potential for use in combination therapy with the goal of achieving superior viral suppression and diminishing the onset of resistance.
Asunto(s)
Antivirales/uso terapéutico , Hepatitis B/tratamiento farmacológico , Nucleósidos/uso terapéutico , Animales , Antivirales/farmacocinética , Modelos Animales de Enfermedad , Humanos , Pruebas de Sensibilidad Microbiana , Nucleósidos/farmacocinéticaRESUMEN
Oxidative stress and glutathione deficiency seem to play a major role in the pathogenesis of HIV infection, as suggested by the increased survival of HIV-infected patients treated with N-acetylcysteine, a prodrug of glutathione. However, beneficial effects of GSH-replenishing drugs are restricted in vivo by the high concentrations needed to obtain biological effects and their low bioavailability. In this study, we evaluated the antiretroviral and antioxidant activities of new more lipophilic GSH-replenishing molecules, in macrophages infected in vitro with HIV-1. In these experimental conditions, a prodrug of N-acetylcystéine and beta-mercaptoethylamine, I-152 demonstrated a potent anti-HIV activity, increased intracellular GSH level, and decreased TNF-alpha production. Altogether, these results suggest that I-152 could be beneficial as adjuvant therapy of antiretrovirals in HIV-infected patients, especially in those with damages to the central nervous system or with mitochondrial damages associated with highly active antiretroviral therapy.
Asunto(s)
Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Fármacos Anti-VIH/farmacología , Antioxidantes/farmacología , Cisteamina/análogos & derivados , Cisteamina/farmacología , Glutatión/fisiología , VIH-1/efectos de los fármacos , Macrófagos/virología , Profármacos/farmacología , Acetilcisteína/toxicidad , Butionina Sulfoximina/farmacología , Células Cultivadas , Cisteamina/toxicidad , Evaluación Preclínica de Medicamentos , VIH-1/fisiología , Humanos , Macrófagos/metabolismo , Estrés Oxidativo , Factor de Necrosis Tumoral alfa/biosíntesis , Replicación Viral/efectos de los fármacosRESUMEN
A short TCCT Me-SATE prooligonucleotide was successfully synthesized using 2-(tert-butyldiphenyloxymethyl) benzoyl protecting group, after its removal by means of trimethylsilyl chloride and water.
Asunto(s)
Oligonucleótidos/síntesis química , Siloxanos/química , Cromatografía Líquida de Alta Presión , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Compuestos de Trimetilsililo/químicaRESUMEN
Synthesis of Z and E ethenyl acyclonucleosides (6a-e and 7a-e) via Michael addition of nucleobases with the diethyl acetylenedicarboxylate is described. The structures of compounds have been confirmed by spectral data. New compounds were found to be inactive against DNA and RNA viruses.
Asunto(s)
Virus ADN/efectos de los fármacos , Hidrocarburos Acíclicos/síntesis química , Hidrocarburos Acíclicos/farmacología , Nucleósidos/síntesis química , Nucleósidos/farmacología , Virus ARN/efectos de los fármacos , Antineoplásicos/química , Antineoplásicos/farmacología , Antivirales/química , Antivirales/farmacología , División Celular/efectos de los fármacos , Línea Celular , Tamaño de la Célula/efectos de los fármacos , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Hidrocarburos Acíclicos/química , Isomerismo , Espectroscopía de Resonancia Magnética , Nucleósidos/química , Espectrofotometría Ultravioleta , Relación Estructura-ActividadRESUMEN
Fluorescein labeled Me-SATE T20 models were synthesized. The high uptake of these prooligos in HeLa cells was confirmed by fluorescence microscopy, flow cytometry and by spectrofluorometry.
Asunto(s)
Oligonucleótidos/farmacocinética , Timidina/análogos & derivados , Fluoresceínas/análisis , Fluoresceínas/química , Células HeLa , Humanos , Microscopía Fluorescente , Oligonucleótidos/análisis , Nucleótidos de Pirimidina/análisis , Nucleótidos de Pirimidina/farmacocinética , Timidina/análisis , Timidina/farmacocinéticaRESUMEN
The fate of a dodecathymidine prodrug in cell extract was monitored by MALDI-TOF MS. This technique allows a facile identification and a relative quantification of metabolites produced. We showed that the relative peak intensities were similar to the relative metabolite proportions that permitted the determination of their half-lives. The oligonucleotide prodrug was fully metabolized to yield the T12 phosphorothioate likely through a carboxyesterase mediated mechanism.
Asunto(s)
Oligonucleótidos/farmacocinética , Profármacos/farmacocinética , Nucleótidos de Pirimidina/farmacocinética , Timidina/análogos & derivados , Biotransformación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Synthesis, biological activities and decomposition kinetics of novel phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-tButyl-2-thioethyl (tBuSATE) group and L-tyrosinyl residues are reported. All the derivatives appeared to be potent inhibitors of HIV-1 replication in various cell culture experiments. The proposed decomposition process of these mixed phosphotriesters may involve successively an esterase and then a phosphodiesterase activation.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Profármacos/síntesis química , Profármacos/farmacología , Zidovudina/análogos & derivados , Fármacos Anti-VIH/química , Células Cultivadas , Diseño de Fármacos , Estabilidad de Medicamentos , Ésteres/síntesis química , Ésteres/química , Ésteres/farmacología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Profármacos/química , Replicación Viral/efectos de los fármacos , Zidovudina/síntesis química , Zidovudina/farmacologíaRESUMEN
A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3'-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.
Asunto(s)
Antivirales/farmacología , Desoxirribonucleósidos/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Animales , Antivirales/química , Desoxiadenosinas/química , Desoxiadenosinas/farmacología , Desoxicitidina/química , Desoxicitidina/farmacología , Desoxirribonucleósidos/química , Virus de la Hepatitis B de la Marmota/efectos de los fármacos , Virus de la Hepatitis B de la Marmota/fisiología , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/tratamiento farmacológico , Humanos , Relación Estructura-Actividad , Especificidad por Sustrato , Timidina/química , Timidina/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
The synthesis and the study of new mononucleoside phosphoramidate diesters bearing S-acyl-2-thioethyl (SATE) groups and an alkylamino residue are reported. The studied compounds appear to be able to deliver the corresponding 5'-mononucleotide inside the cells, and could be considered as prototypes for a new kind of mononucleotide prodrugs (pronucleotides).
Asunto(s)
Amidas/síntesis química , Fármacos Anti-VIH/síntesis química , Nucleótidos/síntesis química , Ácidos Fosfóricos/síntesis química , Profármacos/síntesis química , Amidas/farmacocinética , Amidas/farmacología , Fármacos Anti-VIH/farmacocinética , Fármacos Anti-VIH/farmacología , Humanos , Nucleótidos/farmacocinética , Nucleótidos/farmacología , Ácidos Fosfóricos/farmacocinética , Ácidos Fosfóricos/farmacología , Profármacos/farmacocinética , Profármacos/farmacologíaRESUMEN
Oligonucleotide models bearing 6, 12 or 18 histamine residues were synthesized on solid support and labeled with fluorescein. Only the oligo with 6 histamine residues showed a high uptake in HeLa cells with a nuclear localization. Experiment a 4 degrees C or with bafilomicyn A1 suggest that uptake proceeded by an endocytosis mechanism followed by a destabilization of the membrane. Once in the cytoplasm the oligo reached rapidly the nucleus.
Asunto(s)
Imidazoles/farmacocinética , Macrólidos , Oligonucleótidos/farmacocinética , Antibacterianos/farmacología , Núcleo Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Fluoresceína/química , Células HeLa , Histamina/análogos & derivados , Histamina/farmacocinética , Humanos , Imidazoles/química , Microscopía Fluorescente , Oligonucleótidos/química , Organofosfonatos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Timidina/análogos & derivados , Timidina/farmacocinéticaRESUMEN
Improvements of an "on-line cleaning" HPLC method for analysis of biological samples are presented: (i) the use of cleaning precolumns filled with hydrophobic stationary phases instead of the hydrophilic ones previously used to eliminate the biological matrix: (ii) the combination in the mobile phase of anionic and cationic pairing reagents in order to retain on the precolumn all the metabolites, whatever their hydrophilicity and ionicity are. Such modifications allowed to study the biotransformation of prodrugs of 5-Fluorouracil, designed to act as antitumoral pronucleotides.