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1.
J Med Chem ; 63(18): 10380-10395, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32816483

RESUMO

Chronic hepatitis C (CHC) is a major liver disease caused by the hepatitis C virus. The current standard of care for CHC can achieve cure rates above 95%; however, the drugs in current use are administered for a period of 8-16 weeks. A combination of safe and effective drugs with a shorter treatment period is highly desirable. We report synthesis and biological evaluation of a series of 2',3'- and 2',4'-substituted guanosine nucleotide analogues. Their triphosphates exhibited potent inhibition of the HCV NS5B polymerase with IC50 as low as 0.13 µM. In the HCV replicon assay, the phosphoramidate prodrugs of these analogues demonstrated excellent activity with EC50 values as low as 5 nM. A lead compound AL-611 showed high levels of the nucleoside 5'-triphosphate in vitro in primary human hepatocytes and in vivo in dog liver following oral administration.

2.
J Med Chem ; 62(9): 4555-4570, 2019 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-30951311

RESUMO

We report the synthesis and biological evaluation of a series of 4'-fluoro-2'- C-substituted uridines. Triphosphates of the uridine analogues exhibited a potent inhibition of hepatitis C virus (HCV) NS5B polymerase with IC50 values as low as 27 nM. In an HCV subgenomic replicon assay, the phosphoramidate prodrugs of these uridine analogues demonstrated a very potent activity with EC50 values as low as 20 nM. A lead compound AL-335 (53) demonstrated high levels of the nucleoside triphosphate in vitro in primary human hepatocytes and Huh-7 cells as well as in dog liver following a single oral dose. Compound 53 was selected for the clinical development where it showed promising results in phase 1 and 2 trials.


Assuntos
Alanina/análogos & derivados , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Nucleotídeos de Uracila/farmacologia , Uridina/análogos & derivados , Alanina/síntese química , Alanina/farmacologia , Animais , Antivirais/síntese química , Linhagem Celular Tumoral , Cães , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Humanos , Inibidores da Síntese de Ácido Nucleico/síntese química , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosforamidas , Pró-Fármacos/síntese química , Replicon/efeitos dos fármacos , Nucleotídeos de Uracila/síntese química , Nucleotídeos de Uracila/metabolismo , Uridina/síntese química , Uridina/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores
3.
PLoS One ; 11(5): e0154097, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27163448

RESUMO

ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.


Assuntos
Antivirais/farmacologia , Benzazepinas/farmacologia , Citidina Trifosfato/análogos & derivados , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Farmacorresistência Viral/genética , Niacinamida/análogos & derivados , Mutação Puntual , Proteínas Virais/antagonistas & inibidores , Linhagem Celular Tumoral , Citidina Trifosfato/farmacologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Combinação de Medicamentos , Sinergismo Farmacológico , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Expressão Gênica , Humanos , Niacinamida/farmacologia , RNA Viral/antagonistas & inibidores , RNA Viral/biossíntese , RNA Viral/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
4.
J Med Chem ; 59(10): 4611-24, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27120583

RESUMO

Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action. Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed, prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells, and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are also presented.


Assuntos
Antivirais/farmacologia , Nucleosídeos/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Piridazinas/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Feminino , Humanos , Células Madin Darby de Rim Canino/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/química , Piridazinas/síntese química , Piridazinas/química , Piridinas/síntese química , Piridinas/química , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade
5.
PLoS Pathog ; 11(6): e1004995, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26098424

RESUMO

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.


Assuntos
Antivirais/farmacologia , Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Chlorocebus aethiops , Humanos , RNA Viral/genética , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais/genética
6.
J Med Chem ; 58(4): 1862-78, 2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25667954

RESUMO

Respiratory syncytial virus (RSV) is a leading pathogen of childhood and is associated with significant morbidity and mortality. To date, ribavirin is the only approved small molecule drug, which has limited use. The only other RSV drug is palivizumab, a monoclonal antibody, which is used for RSV prophylaxis. Clearly, there is an urgent need for small molecule RSV drugs. This article reports the design, synthesis, anti-RSV activity, metabolism, and pharmacokinetics of a series of 4'-substituted cytidine nucleosides. Among tested compounds 4'-chloromethyl-2'-deoxy-2'-fluorocytidine (2c) exhibited the most promising activity in the RSV replicon assay with an EC50 of 0.15 µM. The 5'-triphosphate of 2c (2c-TP) inhibited RSV polymerase with an IC50 of 0.02 µM without appreciable inhibition of human DNA and RNA polymerases at 100 µM. ALS-8176 (71), the 3',5'-di-O-isobutyryl prodrug of 2c, demonstrated good oral bioavailability and a high level of 2c-TP in vivo. Compound 71 is a first-in-class nucleoside RSV polymerase inhibitor that demonstrated excellent anti-RSV efficacy and safety in a phase 2 clinical RSV challenge study.


Assuntos
Antivirais/farmacologia , Desoxicitidina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Pró-Fármacos/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírus Sinciciais Respiratórios/enzimologia , Animais , Antivirais/administração & dosagem , Antivirais/química , Cricetinae , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxicitidina/síntese química , Desoxicitidina/química , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Descoberta de Drogas , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Haplorrinos , Humanos , Masculino , Conformação Molecular , Poli(ADP-Ribose) Polimerases/metabolismo , Pró-Fármacos/administração & dosagem , Pró-Fármacos/química , Ratos , Ratos Sprague-Dawley , Infecções por Vírus Respiratório Sincicial/virologia , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
7.
Nucleic Acids Symp Ser (Oxf) ; (52): 643-4, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18776544

RESUMO

In our search for improved therapeutic agents against HCV we synthesized 7-deaza-7-ethynyl-2'-C-methyladenosine (1) and its 2'-deoxy-2'-fluoro analogue 2. The corresponding nucleoside triphosphates were efficient chain terminators of the HCV NS5b polymerase with IC(50)'s of 0.75 microM and 0.4 microM respectively. However, only the ribo-nucleoside 1 exhibited activity in a Huh7 cell based replicon assay with an EC(50) of 0.09 microM. In order to overcome the lack of activity of the fluoro analogue 2 we synthesised several phosphoroamidate prodrugs.


Assuntos
Antivirais/síntese química , Hepacivirus/efeitos dos fármacos , Tubercidina/análogos & derivados , Antivirais/química , Antivirais/farmacologia , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Tubercidina/síntese química , Tubercidina/química , Tubercidina/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores
8.
Artigo em Inglês | MEDLINE | ID: mdl-15892257

RESUMO

3'-Azido-3',5-dideoxythymidine 5'-phosphonate and 3',5'-dideoxy-5'-difluoromethylenethymidine 5'-phosphonate were prepared by multistep syntheses. The nucleoside 5'-phosphonates were converted to their triphosphates and triphosphate mimics (P3Ms) containing beta,gamma-difluoromethylene, beta,gamma-dichloromethylene, or beta,gamma-imodo by condensation with pyrophosphate or pyrophosphate mimics, respectively. Inhibition of HIV-1 reverse transcriptase by the nucleoside P3Ms is briefly discussed.


Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , Mimetismo Molecular , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Timidina/análogos & derivados , Nucleotídeos de Timina/síntese química , Zidovudina/análogos & derivados , Didesoxinucleotídeos , Estrutura Molecular , Inibidores da Transcriptase Reversa/química , Relação Estrutura-Atividade , Timidina/síntese química , Timidina/química , Timidina/farmacologia , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia , Zidovudina/síntese química , Zidovudina/química , Zidovudina/farmacologia
9.
J Med Chem ; 48(7): 2695-700, 2005 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-15801860

RESUMO

The triphosphates of antiviral 2',3'-dideoxynucleosides (ddNs) are the active chemical species that inhibit viral DNA synthesis. The inhibition involves incorporation of ddNMP into DNA and subsequent chain termination. A conceivable strategy for antiviral drugs is to employ nucleoside 5'-triphosphate mimics that can entirely bypass cellular phosphorylation. AZT 5'-alpha-R(P)-borano-beta,gamma-(difluoromethylene)triphosphate (5'-alphaB-betagammaCF(2)TP) has been identified as a potent inhibitor of HIV-1 reverse transcriptase (HIV-1 RT). This work was aimed at confirming that 5'-alphaB-betagammaCF(2)TP is a useful generic triphosphate moiety and can render antiviral ddNs with potent inhibitory effects on HIV-1 RT. Thus, 10 ddNs were converted to their 5'-alphaB-betagammaCF(2)TPs via a sequence (one-pot) of reactions: formation of an activated phosphite, formation of a cyclic triphosphate, boronation, and hydrolysis. Other synthetic routes were also explored. All ddN 5'-alphaB-betagammaCF(2)TPs tested exhibited essentially the same level of inhibition of HIV-1 RT as the corresponding ddNTPs. A conclusion can be made that 5'-alphaB-betagammaCF(2)TP is a generic and promising triphosphate mimic (P3M) concerning HIV-1 RT inhibition and serum stability. It is anticipated that use of 5'-alphaB-betagammaCF(2)TP as P3M moiety will lead to the discovery of a new class of anti-HIV agents.


Assuntos
Fármacos Anti-HIV/síntese química , Compostos de Boro/síntese química , Desoxirribonucleotídeos/síntese química , Transcriptase Reversa do HIV/metabolismo , Inibidores da Transcriptase Reversa/síntese química , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Compostos de Boro/química , Compostos de Boro/metabolismo , Bovinos , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Técnicas In Vitro , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/metabolismo , Estereoisomerismo
10.
J Med Chem ; 48(4): 1199-210, 2005 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-15715486

RESUMO

Several triphosphates of modified nucleosides (1-6) were identified as inhibitors (IC(50) = 0.08-3.8 microM) of hepatitis C virus RNA-dependent RNA polymerase (RdRp). Although the initial SAR developed by determining the ability of the triphosphates to inhibit the in vitro activity of the HCV RdRp identified several potent inhibitors, none of the corresponding nucleosides exhibited significant inhibitory potency in a cell-based replicon assay. To improve upon the activity, bis(tBu-S-acyl-2-thioethyl) nucleoside 5'-monophosphate esters (7-12) were synthesized, and these derivatives exhibited improved potency compared to the corresponding nucleosides in the cell-based assay. Analysis of the intracellular metabolism demonstrated that the S-acyl-2-thioethyl (SATE) prodrug is metabolized to the 5'-triphosphate 40- to 155-fold more efficiently compared to the corresponding nucleoside. The prodrug approach involving bis(tBuSATE)cytidine 5'-monophosphate ester significantly reduced the deamination of cytidine derivatives by cellular deaminases. Additionally, chromosomal aberration studies with the SATE prodrug in cells showed no statistically relevant increase in aberrations compared to the concurrent controls.


Assuntos
Citidina Monofosfato/análogos & derivados , Citidina Monofosfato/síntese química , Citidina/análogos & derivados , Citidina/química , Hepacivirus/efeitos dos fármacos , Organofosfatos/síntese química , Pró-Fármacos/síntese química , Animais , Células CHO , Linhagem Celular Tumoral , Aberrações Cromossômicas/induzido quimicamente , Cricetinae , Cricetulus , Citidina Monofosfato/química , Citidina Monofosfato/farmacologia , Hepacivirus/genética , Humanos , Organofosfatos/química , Organofosfatos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , RNA Viral/antagonistas & inibidores , Trítio , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacos
11.
Nucleosides Nucleotides Nucleic Acids ; 24(10-12): 1651-64, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16438041

RESUMO

Nucleoside reverse transcriptase inhibitors (NRTIs) are prodrugs which require three intracellular phosphorylation steps to yield their corresponding, biologically active, nucleoside triphosphate. In order to circumvent this often inefficient phosphorylation cascade, a plausible approach is to provide the active species directly in the form of a stabilized nucleoside triphosphate mimic. We have previously shown that such a mimic, namely 5'-alpha-Rp-borano-beta,gamma-(difluoromethylene)triphosphate (5'-alphaBCF2TP) is a generic triphosphate mimic that is biologically stable and can render antiviral ddNs with potent inhibitory activity against HIV-1 RT. Herein we report the synthesis and activity against HIV-1 RT of several ddN 5'-alpha-modified-beta,gamma(difluoromethylene)triphosphate mimics with either a non-bridging calphaP-thio (5'-alphaSCF2TP) or alpha-P-seleno (5'-alpha SeCF2TP) modification. One compound, namely, AZT-5'-alpha-P-seleno-beta,gamma-(difluoromethylene)triphosphate (diastereomer I), was identified as a potent inhibitor of HIV-1 RT (Ki = 64 nM) and represents the first report of HIV-1 RT inhibition data for a nucleotide bearing an alpha-P-seleno modification. These triphosphate mimics may be useful in the investigation of enzyme mechanism and may have interesting properties with respect to drug resistance and polymerase selectivity.


Assuntos
Fármacos Anti-HIV/síntese química , Didesoxinucleosídeos/síntese química , Transcriptase Reversa do HIV/antagonistas & inibidores , Compostos Organosselênicos/síntese química , Inibidores da Transcriptase Reversa/síntese química , Sulfetos/síntese química , Fármacos Anti-HIV/química , Didesoxinucleosídeos/química , Transcriptase Reversa do HIV/química , Compostos Organosselênicos/química , Inibidores da Transcriptase Reversa/química , Sulfetos/química
12.
J Med Chem ; 47(27): 6902-13, 2004 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-15615539

RESUMO

In search of active nucleoside 5'-triphosphate mimics, we have synthesized a series of AZT triphosphate mimics (AZT P3Ms) and evaluated their inhibitory effects on HIV-1 reverse transcriptase as well as their stability in fetal calf serum and in CEM cell extracts. Reaction of AZT with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, followed by treatment of the phosphite intermediate 2 with pyrophosphate analogues, yielded the cyclic triphosphate intermediates 4b-4f, which were subjected to boronation and subsequent hydrolysis to give AZT 5'-alpha-borano-beta,gamma-bridge-modified triphosphates 6b-6f in moderate to good yields. Reaction of the cyclic intermediate 4d with iodine, followed by treatment with a series of nucleophiles, afforded the AZT 5'-beta,gamma-difluoromethylene-gamma-substituted triphosphates (7b-7i). Several different types of AZT P3Ms containing alpha-P-thio (or dithio) and beta,gamma-difluoromethylene (13,14), alpha,beta-difluoromethylene and gamma-P-methyl(or phenyl) (15,16), and alpha-borano-beta,gamma-difluoromethylene and gamma-O-methyl/phenyl (11,12) were also synthesized. The effectiveness of the compounds as inhibitors of HIV-1 reverse transcriptase was determined using a fluorometric assay and a poly(A) homopolymer as a template. A number of AZT P3Ms exhibited very potent inhibition of HIV-1 reverse transcriptase. Modifications at the beta,gamma-bridge of triphosphate rendered the AZT P3Ms 6b-6f with varied activities (K(i) from 9.5 to >>500 nM) while modification at the alpha,beta-bridge of triphosphate led to weak AZT P3M inhibitors. The results imply that the AZT P3Ms were substrate inhibitors, as is AZT triphosphate. The most active compound, AZT 5'-alpha-R(p)()-borano-beta,gamma-(difluoromethylene)triphosphate (AZT 5'-alphaB-betagammaCF(2)TP) (6d-I), is as potent as AZT triphosphate with a K(i)() value of 9.5 nM and at least 20-fold more stable than AZT triphosphate in the serum and cell extracts. Therefore, for the first time, a highly active and stable nucleoside triphosphate mimic has been identified, which is potentially useful as a new type of antiviral drug. The promising triphosphate mimic, 5'-alpha-borano-beta,gamma-(difluoromethylene)triphosphate, is expected to be valuable to the discovery of nucleotide mimic antiviral drugs.


Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , Inibidores da Transcriptase Reversa/síntese química , Zidovudina/análogos & derivados , Estabilidade de Medicamentos , Humanos , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Relação Estrutura-Atividade
13.
J Am Chem Soc ; 126(46): 15006-7, 2004 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-15547979

RESUMO

Chemical modification of nucleic acids at the 2'-position of ribose has generated antisense oligonucleotides (AONs) with a range of desirable properties. Electron-withdrawing substituents such as 2'-O-[2-(methoxy)ethyl] (MOE) confer enhanced RNA affinity relative to that of DNA by conformationally preorganizing an AON for pairing with the RNA target and by improving backbone hydration. 2'-Substitution of the ribose has also been shown to increase nuclease resistance and cellular uptake via changes in lipophilicity. Interestingly, incorporation of either 2'-O-[2-(methylamino)-2-oxoethyl]- (NMA) or 2'-O-(N-methylcarbamate)-modified (NMC) residues into AONs has divergent effects on RNA affinity. Incorporation of 2'-O-NMA-T considerably improves RNA affinity while incorporation of 2'-O-NMC-T drastically reduces RNA affinity. Crystal structures at high resolution of A-form DNA duplexes containing either 2'-O-NMA-T or 2'-O-NMC-T shed light on the structural origins of the surprisingly large difference in stability given the relatively minor difference in chemistry between NMA and NMC. NMA substituents adopt an extended conformation and use either their carbonyl oxygen or amino nitrogen to trap water molecules between phosphate group and sugar. The conformational properties of NMA and the observed hydration patterns are reminiscent of those found in the structures of 2'-O-MOE-modified RNA. Conversely, the carbonyl oxygen of NMC and O2 of T are in close contact, providing evidence that an unfavorable electrostatic interaction and the absence of a stable water structure are the main reasons for the loss in thermodynamic stability as a result of incorporation of 2'-O-NMC-modified residues.


Assuntos
RNA/química , Timidina/análogos & derivados , Materiais Biomiméticos/química , Cristalografia por Raios X , DNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , Timidina/química
14.
J Med Chem ; 47(21): 5284-97, 2004 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-15456273

RESUMO

Hepatitis C virus infection constitutes a significant health problem in need of more effective therapies. We have recently identified 2'-C-methyladenosine and 2'-C-methylguanosine as potent nucleoside inhibitors of HCV RNA replication in vitro. However, both of these compounds suffered from significant limitations. 2'-C-Methyladenosine was found to be susceptible to enzymatic conversions by adenosine deaminase and purine nucleoside phosphorylase, and it displayed limited oral bioavailability in the rat. 2'-C-Methylguanosine, on the other hand, was neither efficiently taken up in cells nor phosphorylated well. As part of an attempt to address these limitations, we now report upon the synthesis and evaluation of a series of heterobase-modified 2'-C-methyl ribonucleosides. The structure-activity relationship within this series of nucleosides reveals 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine as potent and noncytotoxic inhibitors of HCV RNA replication. Both 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine display improved enzymatic stability profiles as compared to that of 2'-C-methyladenosine. Consistent with these observations, the most potent compound, 4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine ribonucleoside, is orally bioavailable in the rat. Together, the potency of the 2'-C-methyl-4-amino-pyrrolo[2,3-d]pyrimidine ribonucleosides and their improved pharmacokinetic properties relative to that of 2'-C-methyladenosine suggests that this class of compounds may have clinical utility.


Assuntos
Antivirais/síntese química , Hepacivirus/genética , RNA Viral/antagonistas & inibidores , Ribonucleosídeos/síntese química , Adenosina Desaminase/química , Administração Oral , Animais , Antivirais/química , Antivirais/farmacocinética , Disponibilidade Biológica , Linhagem Celular , Estabilidade de Medicamentos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Fosforilação , Purina-Núcleosídeo Fosforilase/química , RNA Viral/biossíntese , Ratos , Ribonucleosídeos/química , Ribonucleosídeos/farmacocinética , Relação Estrutura-Atividade
15.
Antimicrob Agents Chemother ; 48(10): 3944-53, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15388457

RESUMO

Improved treatments for chronic hepatitis C virus (HCV) infection are needed due to the suboptimal response rates and deleterious side effects associated with current treatment options. The triphosphates of 2'-C-methyl-adenosine and 2'-C-methyl-guanosine were previously shown to be potent inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of viral RNA in cells. Here we demonstrate that the inclusion of a 7-deaza modification in a series of purine nucleoside triphosphates results in an increase in inhibitory potency against the HCV RdRp and improved pharmacokinetic properties. Notably, incorporation of the 7-deaza modification into 2'-C-methyl-adenosine results in an inhibitor with a 20-fold-increased potency as the 5'-triphosphate in HCV RdRp assays while maintaining the inhibitory potency of the nucleoside in the bicistronic HCV replicon and with reduced cellular toxicity. In contrast, while 7-deaza-2'-C-methyl-GTP also displays enhanced inhibitory potency in enzyme assays, due to poor cellular penetration and/or metabolism, the nucleoside does not inhibit replication of a bicistronic HCV replicon in cell culture. 7-Deaza-2'-C-methyl-adenosine displays promising in vivo pharmacokinetics in three animal species, as well as an acute oral lethal dose in excess of 2,000 mg/kg of body weight in mice. Taken together, these data demonstrate that 7-deaza-2'-C-methyl-adenosine is an attractive candidate for further investigation as a potential treatment for HCV infection.


Assuntos
Antivirais , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/metabolismo , Tubercidina/farmacologia , Tubercidina/farmacocinética , Animais , Técnicas de Cultura , Farmacorresistência Viral , Feminino , Genótipo , Hepacivirus/enzimologia , Hepatite C/enzimologia , Humanos , Células Jurkat , Dose Letal Mediana , Camundongos , Polinucleotídeo Adenililtransferase/metabolismo , RNA/biossíntese , RNA Polimerase II/metabolismo , Timidina/farmacologia , Replicação Viral/efeitos dos fármacos
16.
J Med Chem ; 47(9): 2283-95, 2004 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-15084127

RESUMO

As part of a continued effort to identify inhibitors of hepatitis C viral (HCV) replication, we report here the synthesis and evaluation of a series of nucleoside analogues and their corresponding triphosphates. Nucleosides were evaluated for their ability to inhibit HCV RNA replication in a cell-based, subgenomic replicon system, while nucleoside triphosphates were evaluated for their ability to inhibit in vitro RNA synthesis mediated by the HCV RNA-dependent RNA polymerase, NS5B. 2'-C-Methyladenosine and 2'-C-methylguanosine were identified as potent inhibitors of HCV RNA replication, and the corresponding triphosphates were found to be potent inhibitors of HCV NS5B-mediated RNA synthesis. The data generated in the cell-based assay demonstrated a fairly stringent structure-activity relationship around the active nucleosides. Increase in steric bulk beyond methyl on C2, change in the stereo- or regiochemistry of the methyl substituent, or change of identity of the heterobase beyond that of the endogenous adenine or guanine was found to lead to loss of inhibitory activity. The results highlight the importance of the ribo configuration 2'- and 3'-hydroxy pharmacophores for inhibition of HCV RNA replication in the cell-based assay and demonstrate that inclusion of the 2'-C-methylribonucleoside pharmacophore leads to increased resistance to adenosine deaminase and purine nucleoside phosphorylase mediated metabolism.


Assuntos
Hepacivirus/química , Nucleosídeos de Purina/síntese química , Ribonucleosídeos/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Adenosina Desaminase/química , Ligação de Hidrogênio , Metilação , Conformação Molecular , Nucleosídeos de Purina/química , Purina-Núcleosídeo Fosforilase/química , Purinas/química , Ribonucleosídeos/química , Ribose/química , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química
17.
Org Lett ; 5(12): 2017-20, 2003 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-12790517

RESUMO

[structure: see text] Oligonucleotides with a novel, 2'-O-[2-[2-(N,N-dimethylamino)ethoxy]ethyl] (2'-O-DMAEOE) modification have been synthesized. This modification, a cationic analogue of the 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, exhibits high binding affinity to target RNA (but not to DNA) and exceptional resistance to nuclease degradation. Analysis of the crystal structure of a self-complementary oligonucleotide containing a single 2'-O-DMAEOE modification explains the importance of charge factors and gauche effects on the observed antisense properties. 2'-O-DMAEOE modified oligonucleotides are ideal candidates for antisense drugs.


Assuntos
Etano/química , Éteres/química , Oligonucleotídeos Antissenso/química , Animais , Sequência de Bases , Estabilidade de Medicamentos , Etano/análogos & derivados , Modelos Moleculares , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Hibridização de Ácido Nucleico , Oligonucleotídeos Antissenso/metabolismo , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Diester Fosfórico Hidrolases/metabolismo , RNA Complementar/metabolismo , Venenos de Serpentes/enzimologia , Espectrometria de Massas por Ionização por Electrospray , Eletricidade Estática , Estereoisomerismo , Relação Estrutura-Atividade
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