RESUMEN
To develop an understanding of the structure-activity relationships for the inhibition of orthopoxviruses by nucleoside analogues, a variety of novel chemical entities were synthesized. These included a series of pyrimidine 5-hypermodified acyclic nucleoside analogues based upon recently discovered new leads, and some previously unknown "double-headed" or "abbreviated" nucleosides. None of the synthetic products possessed significant activity against two representative orthopoxviruses; namely, vaccinia virus and cowpox virus. They were also devoid of significant activity against a battery of other DNA and RNA viruses. So far as the results with the orthopoxviruses and herpes viruses, the results may point to the necessity for nucleoside analogues 5'-phosphorylation for antiviral efficacy.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Nucleósidos/síntesis química , Nucleósidos/farmacología , Orthopoxvirus/efectos de los fármacos , Virus ARN/efectos de los fármacos , Antivirales/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Nucleósidos/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The antiviral activity of a new series of thymidine analogs was determined against vaccinia virus (VV), cowpox virus (CV), herpes simplex virus, and varicella-zoster virus. Several compounds were identified that had good activity against each of the viruses, including a set of novel 5-substituted deoxyuridine analogs. To investigate the possibility that these drugs might be phosphorylated preferentially by the viral thymidine kinase (TK) homologs, the antiviral activities of these compounds were also assessed using TK-deficient strains of some of these viruses. Some of these compounds were shown to be much less effective in the absence of a functional TK gene in CV, which was unexpected given the high degree of amino acid identity between this enzyme and its cellular homolog. This unanticipated result suggested that the CV TK was important in the mechanism of action of these compounds and also that it might phosphorylate a wider variety of substrates than other type II enzymes. To confirm these data, we expressed the VV TK and human TK1 in bacteria and isolated the purified enzymes. Enzymatic assays demonstrated that the viral TK could efficiently phosphorylate many of these compounds, whereas most of the compounds were very poor substrates for the cellular kinase, TK1. Thus, the specific phosphorylation of these compounds by the viral kinase may be sufficient to explain the TK dependence. This unexpected result suggests that selective phosphorylation by the viral kinase may be a promising new approach in the discovery of highly selective inhibitors of orthopoxvirus replication.
Asunto(s)
Antivirales/metabolismo , Antivirales/farmacología , Timidina Quinasa/metabolismo , Virus Vaccinia/enzimología , Secuencia de Aminoácidos , Herpesviridae/efectos de los fármacos , Humanos , Cinética , Datos de Secuencia Molecular , Orthopoxvirus/efectos de los fármacos , Fosforilación , Timidina Quinasa/químicaRESUMEN
5-Formyl-2'-deoxyuridine-3',5'-diacetate was converted to a small library of 5-substituted pyrimidine nucleoside N-acylamino acid amides by means of a Ugi multicomponent reaction. The reaction allowed introduction of various substituents at the acyl moiety, at the amino acid alpha-amide group, and at the amino acid carboxyl function. Evaluation of these novel 5-substituted nucleosides against vaccinia virus and cowpox virus provided one compound with discernable activity against cowpox virus but five- to eightfold less active than the Cidofovir standard. More promising activity was seen for the inhibition of Leishmania donovani promastigotes. Several synthetic products showed antileishmanial activity in the 10(-5)M range. When compared to earlier studies demonstrating anti-orthopoxviral and antileishmanial activity of 5-substituted pyrimidine nucleosides, these results imply that the 5-(N-acylamino acid amide)-derivatized pyrimidine nucleosides may possess more steric bulk, greater hydrophobicity, and more flexibility than is compatible with these particular biological activities.
Asunto(s)
Antiprotozoarios/síntesis química , Antiprotozoarios/farmacología , Antivirales/síntesis química , Antivirales/farmacología , Desoxiuridina/análogos & derivados , Leishmania/efectos de los fármacos , Nucleósidos/síntesis química , Nucleósidos/farmacología , Animales , Virus de la Viruela Vacuna/efectos de los fármacos , Desoxiuridina/síntesis química , Desoxiuridina/farmacología , Indicadores y Reactivos , Espectrometría de Masas , Relación Estructura-Actividad , Virus Vaccinia/efectos de los fármacosRESUMEN
2',5'-Oligoadenylate tetramer (2-5A) has been chemically conjugated to short HIV-1 Tat peptides to provide 2-5A-tat chimeras. Two different convergent synthetic approaches have been employed to provide such 2-5A-tat bioconjugates. One involved generation of a bioconjugate through reaction of a cysteine terminated Tat peptide with a alpha-chloroacetyl derivative of 2-5A. The second synthetic strategy was based upon a cycloaddition reaction of an azide derivative of 2-5A with a Tat peptide bearing an alkyne function. Either bioconjugate of 2-5A-tat was able to activate human RNase L. The union of 2-5A and Tat peptide provided an RNase L-active chimeric nucleopeptide with the ability to be taken up by cells by virtue of the Tat peptide and to activate RNase L in intact cells. This strategy provides a valuable vehicle for the entry of the charged 2-5A molecule into cells and may provide a means for targeted destruction of HIV RNA in vivo.
Asunto(s)
Nucleótidos de Adenina/química , Fármacos Anti-VIH/síntesis química , Sistemas de Liberación de Medicamentos , Productos del Gen tat/química , Productos del Gen tat/farmacocinética , Oligorribonucleótidos/química , Proteínas Recombinantes de Fusión/farmacocinética , Nucleótidos de Adenina/farmacocinética , Nucleótidos de Adenina/farmacología , Alquinos , Fármacos Anti-VIH/farmacocinética , Fármacos Anti-VIH/farmacología , Azidas , Permeabilidad de la Membrana Celular/efectos de los fármacos , Endorribonucleasas/efectos de los fármacos , Productos del Gen tat/farmacología , Humanos , Oligorribonucleótidos/farmacocinética , Oligorribonucleótidos/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacocinética , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
The following structurally diverse 5-substituted-2'-deoxyuridine nucleosides displayed potent in vitro antileishmanial activity: 5-formyl, 5-(2,2,-dicyanovinyl)-, 5-(2-cyano-2-ethoxycarbonylvinyl), 5-(2-cyano-2-methoxycarbonylvinyl)-, 5-(2-amino-3-cyano-5-oxo-5,6,7,8-tetrahydro-4H-chromen-4-yl)- and related congeners, and the 5-(3-methyl-5-oxo-1-phenyl-4,5-dihydro-4H-pyrazol-4-ylidene) group.
Asunto(s)
Antiprotozoarios/síntesis química , Leishmania donovani/efectos de los fármacos , Nucleósidos de Pirimidina/síntesis química , Nucleósidos de Pirimidina/farmacología , Animales , Antiprotozoarios/farmacología , Concentración 50 Inhibidora , Relación Estructura-ActividadRESUMEN
Two privileged drug scaffolds have been hybridized to create the novel heteromorphic nucleoside 5-(2-amino-3-cyano-5-oxo-5,6,7,8-tetrahydro-4H-chromen-4-yl)-1-(2-deoxypentofuranosyl)pyrimidine-2,4(1H,3H)-dione (2). Compound 2 inhibited the replication of two orthopoxviruses, vaccinia virus (VV) (EC(50) = 4.6 +/- 2.0 microM), and cowpox virus (CV) (EC(50) = 2.0 +/- 0.3 microM). Compound 2 exhibited reduced activity against a thymidine kinase (TK) negative strain of CV, implying a requirement for 5'-monophosphorylation for antiorthopoxvirus activity. Compound 2 was efficiently phosphorylated by VV TK, establishing that VV TK is more promiscuous than previously believed.
Asunto(s)
Antivirales/síntesis química , Benzopiranos/síntesis química , Orthopoxvirus/efectos de los fármacos , Nucleósidos de Pirimidina/síntesis química , Antivirales/química , Antivirales/farmacología , Benzopiranos/química , Benzopiranos/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Virus de la Viruela Vacuna/efectos de los fármacos , Virus de la Viruela Vacuna/enzimología , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Humanos , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/farmacología , Piel/citología , Relación Estructura-Actividad , Timidina Quinasa/genética , Virus Vaccinia/efectos de los fármacosRESUMEN
The nucleoside 5-formyl-2'-deoxyuridine has been used as a starting point for the generation of novel 5-substituted pyrimidine nucleosides that are shown to possess significant antiviral activity against two representative orthopoxviruses, namely vaccinia virus and cowpox virus.
Asunto(s)
Guerra Biológica/prevención & control , Virus de la Viruela Vacuna/efectos de los fármacos , Nucleósidos de Pirimidina , Viruela/prevención & control , Virus Vaccinia/efectos de los fármacos , Animales , Humanos , Pruebas de Sensibilidad Microbiana , Nucleósidos de Pirimidina/síntesis química , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/farmacologíaRESUMEN
To provide potential new leads for the treatment of orthopoxvirus infections, the 5-position of the pyrimidine nucleosides have been modified with a gem diether moiety to yield the following new nucleosides: 5-(dimethoxymethyl)-2'-deoxyuridine (2b), 5-(diethoxymethyl)-2'-deoxyuridine (3b), 5-formyl-2'-deoxyuridine ethylene acetal (4b), and 5-formyl-2'-deoxyuridine propylene acetal (5b). These were evaluated in human foreskin fibroblast cells challenged with the vaccinia virus or cowpox virus. Of the four gem diether nucleosides, only the dimethyl gem diether congener showed significant antiviral activity against both viruses. This antiviral activity did not appear to be related to the decomposition to the 5-formyl-2'-deoxyuridine, which was itself devoid of anti-orthopoxvirus activity in these assays. Moreover, at the pH of the in vitro assays, 2b was very stable with a decomposition (to aldehyde) half-life of >15 d. The anti-orthopoxvirus activity of pyrimidine may be favored by the introduction of hydrophilic moieties to the 5-position side chain.
Asunto(s)
Antivirales/síntesis química , Desoxiuridina/análogos & derivados , Desoxiuridina/síntesis química , Orthopoxvirus/efectos de los fármacos , Animales , Antivirales/química , Antivirales/farmacología , Virus de la Viruela Vacuna/efectos de los fármacos , Desoxiuridina/química , Desoxiuridina/farmacología , Estabilidad de Medicamentos , Esterasas/química , Éteres/síntesis química , Éteres/química , Éteres/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Humanos , Técnicas In Vitro , Hígado/enzimología , Piel/citología , Porcinos , Virus Vaccinia/efectos de los fármacos , Agua/químicaRESUMEN
Synthetic hybridization of two privileged drug scaffolds, pyrazolone on the one hand and pyrimidine nucleoside on the other, resulted in the generation of two novel 5-substituted pyrimidine nucleosides with potent in vitro antiviral activity against two representative orthopoxviruses, vaccinia virus and cowpox virus.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Nucleósidos/química , Nucleósidos/farmacología , Orthopoxvirus/efectos de los fármacos , Orthopoxvirus/fisiología , Pirazolonas/química , Pirazolonas/farmacología , Pirimidinas/química , Antivirales/química , Estructura Molecular , Nucleósidos/síntesis química , Pirazolonas/síntesis química , Relación Estructura-Actividad , Replicación ViralRESUMEN
A series of synthetic oligonucleotide phosphorothioate 15-mers were generated against specific sequences in the West Nile virus RNA genome. These antisense oligonucleotides targeted (1) conserved features of the West Nile virus RNA genome that may be expected to lead to inhibition of virus replication since such features play essential roles in the virus lifecycle; (2) G-quartet oligonucleotides with potential facilitated uptake properties and that also targeted conserved sequences among a range of West Nile virus strains. Several formulations with significant in vitro antiviral activity were found. Among the active oligonucleotides were examples that targeted both C-rich RNA sequences of the West Nile RNA genome as well as recognized conserved sequences key to West Nile virus replication. Since the antiviral activity of the latter oligonucleotides diminished upon 2'-O-methyl substitution, it is likely that their activity involves RNase H-catalyzed RNA degradation. One G-rich oligonucleotide that did not target a West Nile virus RNA sequence also was found. These results suggest the potential of antisense strategies for the control of West Nile virus replication if the attendant problem of oligonucleotide delivery can be adequately addressed.
Asunto(s)
Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Replicación Viral/efectos de los fármacos , Replicación Viral/genética , Virus del Nilo Occidental/fisiología , Animales , Chlorocebus aethiops , Células Vero , Virus del Nilo Occidental/efectos de los fármacos , Virus del Nilo Occidental/genéticaRESUMEN
Efficient regioselective synthesis of nucleoside conjugates was achieved by cycloaddition reaction of azides and alkynes using sodium ascorbate/ CuSO4 system as a catalyst. These 16 novel thymidine analogues were obtained in excellent yields (75-100%), employing mild reaction conditions with a broad scope of structural modification. For the compounds tested, no specific antiviral effects could be witnessed against a broad range of viruses.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Antivirales/síntesis química , Timidina/análogos & derivados , Timidina/farmacología , Triazoles/síntesis química , Triazoles/farmacología , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Diseño de Fármacos , Modelos Moleculares , Relación Estructura-Actividad , Triazoles/uso terapéuticoRESUMEN
A convergent synthetic approach was used to conjugate 2',5'-oligoadenylate (2-5A, p5'A2' [p5'A2'](n)()p5'A) to phosphorodiamidate morpholino oligomers (morphants). To provide requisite quantities of 2-5A starting material, commercially and readily available synthons for solid-phase synthesis were adapted for larger scale solution synthesis. Thus, the tetranucleotide 5'-phosphoryladenylyl(2'-->5')adenylyl(2'-->5')adenylyl(2'-->5')adenosine (p5'A2'p5'A2'](2)p5'A2', tetramer 2-5A, 9) was synthesized starting with 2',3'-O-dibenzoyl-N(6),N(6)-dibenzoyl adenosine prepared from commercially available 5'-O-(4-monomethoxytrityl) adenosine. Coupling with N(6)-benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(tert-butyldimethylsilyl) adenosine-2'-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite, followed by oxidization and deprotection, generated 5'-deprotected dimer 2-5A. Similar procedures lengthened the chain to form protected tetramer 2-5 A. The title product 9 p5'A(2'p5'A)(3) (tetramer 2-5A) was obtained through phosphorylation of the terminal 5'-hydroxy of the protected tetramer and removal of remaining protecting groups using concentrated ammonium hydroxide-ethanol (3:1, v/v) at 55 degrees C and tetrabutylammonium fluoride (TBAF) in THF at room temperature, respectively. The 2-5A-phosphorodiamidate morpholino antisense chimera 11 (2-5A-morphant) was synthesized by covalently linking an aminolinker-functionalized phosphorodiamidate morpholino oligomer with periodate oxidized 2-5A tetramer (p5'A2'[p5'A2'](2)p5'A). The resulting Schiff base was reduced with cyanoborohydride thereby transforming the ribose of the 2'-terminal nucleotide of 2-5A N-substituted morpholine. RNase L assays demonstrated that this novel 2-5A-antisense chimera had significant biological activity, thereby providing another potential tool for RNA ablation.
Asunto(s)
ADN sin Sentido/síntesis química , Ribonucleasa H/metabolismo , Antineoplásicos/síntesis química , Antivirales/síntesis química , Catálisis , ADN sin Sentido/análogos & derivados , ADN sin Sentido/farmacología , Endorribonucleasas/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Morfolinas , Morfolinos , ARN Viral/efectos de los fármacos , ARN Viral/metabolismoRESUMEN
Sequence-specific 3-deazaadenosine (c(3)A)-substituted analogues of trimeric 2',5'-oligoadenylate, p5'A2'p5'A2'p5'A, were synthesized and evaluated for their ability to activate human RNase L (EC 3.1.2.6) aiming at the elucidation of the nitrogen-3 role in this biochemical process. Substitution of either 5'-terminal or 2'-terminal adenosine with c(3)A afforded the respective analogues p5'(c(3)A)2'p5'A2'p5'A and p5'A2'p5'A2'p5'(c(3)A) that were as effective as the natural tetramer itself as activators of RNase L (EC(50)=1nM). In contrast, p5'A2'p5'(c(3)A)2'p5'A showed diminished RNase L activation ability (EC(50)=10nM). The extensive conformational analysis of the c(3)A-substituted core trimers versus the parent natural core trimer by the (1)H and (13)C NMR, and CD spectroscopy displayed close stereochemical similarity between the natural core trimer and (c(3)A)2'p5'A2'p5'A and A2'p5'A2'p5'(c(3)A) analogues, thereby strong evidences for the syn base orientation about the glycosyl bond of the c(3)A residue of the latter were found. On the contrary, an analogue A2'p5'(c(3)A)2'p5'A displayed rather essential deviations from the spatial arrangement of the parent natural core trimer.
Asunto(s)
Adenina/química , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Nitrógeno/química , Tubercidina/química , Tubercidina/farmacología , Animales , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Activación Enzimática/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Estructura Molecular , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Fosfatos/química , Estereoisomerismo , Tubercidina/síntesis química , Tubercidina/metabolismoRESUMEN
2-5A trimer [5'-monophosphoryladenylyl(2'-5')adenylyl(2'-5')adenosine] activates RNase L. While the 5'-terminal and 2'-terminal adenosine N(6)-amino groups play a key role in binding to and activation of RNase L, the exocyclic amino function of the second adenylate (from the 5'-terminus) plays a relatively minor role in 2-5A's biological activity. To probe the available space proximal to the amino function of the central adenylate of 2-5A trimer during binding to RNase L, a variety of substituents were placed at that position. To accomplish this, the convertible building block 5'-O-dimethoxytrityl-3'-O-(tert-butyldimethylsilyl)-6-(2,4-dinitrophenyl)thioinosine 2'-(2-cyanoethylN,N-diisopropylphosphoramidite) was prepared as a synthon to introduce 6-(2,4-dinitrophenyl)thioinosine into the middle position of the 2-5A trimer during automated synthesis. Post-synthetic treatment with aqueous amines transformed the (2,4-dinitrophenyl)thioinosine into N(6)-substituted adenosines. Assays of these modified trimers for their ability to bind and activate RNase L showed that activation activity could be retained, albeit with some sacrifice compared to unmodified p5'A2'p5'A2'p5'A. Thus, the spatial domain about this N(6)-amino function could be available for modifications to enhance the biological potency of 2-5A analogues and to ligate 2-5A to targeting vehicles such as antisense molecules.
Asunto(s)
Endorribonucleasas/metabolismo , Tioinosina/análogos & derivados , Tioinosina/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Humanos , Insectos , Tioinosina/síntesis químicaRESUMEN
Respiratory syncytial virus (RSV) is a leading cause of respiratory disease in infants, young children, immunocompromised patients, and the institutionalized elderly. Previous work had shown that RNase L, an antiviral enzyme of the interferon system, could be recruited to cleave RSV genomic RNA by attaching tetrameric 2prime prime or minute-5prime prime or minute-linked oligoadenylates (2-5A) to an oligonucleotide complementary to repetitive gene-start sequences within the RSV genome (2-5A antisense). A 2prime prime or minute-O-methyl RNA-modified analog of the lead 2-5A anti-RSV chimera is shown here to have enhanced antiviral activity in cell culture studies while also cleaving RSV genomic RNA in an RNase L- and sequence-specific manner. When administered intranasally to RSV-infected African green monkeys, this chimera reduced nasal RSV replication by up to four log(10) units in a dose- and time-dependent manner.
Asunto(s)
Nucleótidos de Adenina , Antivirales/uso terapéutico , Oligonucleótidos Antisentido/uso terapéutico , Oligorribonucleótidos , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitiales Respiratorios/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Administración Intranasal , Animales , Antivirales/administración & dosificación , Antivirales/farmacología , Línea Celular , Chlorocebus aethiops , Endorribonucleasas/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacología , ARN Viral/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/genéticaRESUMEN
Respiratory syncytial virus (RSV) continues as an emerging infectious disease not only among infants and children, but also for the immune-suppressed, hospitalized and the elderly. To date, ribavirin (Virazole) remains the only therapeutic agent approved for the treatment of RSV. The prophylactic administration of palivizumab is problematic and costly. The quest for an efficacious RSV antiviral has produced a greater understanding of the viral fusion process, a new hypothesis for the mechanism of action of ribavirin, and a promising antisense strategy combining the 2'-5' oligoadenylate antisense (2-5A-antisense) approach and RSV genomics.