Marija Prhavc

A 7-Deaza-Adenosine Analog Is a Potent and Selective Inhibitor of Hepatitis C Virus Replication with Excellent Pharmacokinetic Properties

ABSTRACT 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.

2′-O-Carbamate-containing oligonucleotides: synthesis and properties

Abstract In order to evaluate the effect of a new 2′-carbohydrate modification on the hybridization properties of oligonucleotides, uridine 2′- O -carbamates were synthesized and incorporated into DNA strands. The key intermediate in the synthesis, a mixed succinimide carbonate 2 , was treated with various amines to give 2′- O -carbamates 3 . Thermal melting studies of modified oligonucleotides revealed that the presence of the 2′- O -carbamate modification significantly destabilized DNA/RNA duplexes. A molecular-modeling study indicated that unfavorable steric interactions between the hydrogen of the NH group from the carbamate substituent and the anomeric hydrogen of the sugar residue on the same strand of the duplex may be the contributing factor causing destabilization.

Synthesis and Anti-Influenza Activity of Pyridine, Pyridazine, and Pyrimidine C-Nucleosides as Favipiravir (T-705) Analogues

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.

2′,3′-Dideoxynucleoside 5′-β,γ-(Difluoromethylene) Triphosphates With α-P-Thio or α-P-Seleno Modifications: Synthesis and Their Inhibition of HIV-1 Reverse Transcriptase

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′-α-R p -borano-β,γ-(difluoromethylene) triphosphate (5′-αBCF 2 TP) is a generic triphosphate mimic that is biologically stable and can render antiviral ddNs with potent inhibitory activity against HIV-1 RT.[1] [2] Herein we report the synthesis and activity against HIV-1 RT of several ddN 5′-α-modified-β,γ-(difluoromethylene) triphosphate mimics with either a non-bridging α-P-thio (5′-αSCF 2 TP) or α-P-seleno (5′-αSeCF 2 TP) modification. One compound, namely, AZT-5′-α-P-seleno-β,γ-(difluoromethylene) triphosphate (diastereomer I), was identified as a potent inhibitor of HIV-1 RT (K i = 64 nM) and represents the first report of HIV-1 RT inhibition data for a nucleotide bearing an α-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.

Preparation and Utility of 5-β-D-Ribofuranosyl-1H-tetrazole as a Key Synthon for C-Nucleoside Synthesis

Abstract A synthesis of 5-β-D-ribofuranosyl-1H-tetrazole (2) and 5-β-D-ribo-furanosyl-1,3,4-oxadiazole-2(3H)-one (9) derivatives is described. Ring transformations of 2 have been investigated in an effort to establish the stability of this synthon for further use in dipolar cycloaddition reactions.

Carbohydrate Modifications in Antisense Oligonucleotide Therapy: New Kids on the Block

Abstract Chemical modifications to improve the efficacy of an antisense oligonucleotide are designed to increase the binding affinity to target RNA, to enhance the nuclease resistance, and to improve cellular delivery. Among the different sites available for chemical modification in a nucleoside building block, the 2′-position of the carbohydrate moiety1 has proven to be the most valuable for various reasons: (1) 2′-modification can confer an RNA-like 3′-endo conformation to the antisense oligonucleotide. Such a preorganization for an RNA like conformation2,3,4,5 greatly improves the binding affinity to the target RNA; (2) 2′-modification provides nuclease resistance to oligonucleotides; (3) 2′-modification provides chemical stability against potential depurination conditions pharmacology evaluations and correlation with pharmacokinetic changes are emerging from these novel chemical modifications. Analytical chemistry of modified oligonucleotides before and after biological administration of antisense oli...

Synthesis of 5′-Triphosphate Mimics (P3Ms) of 3′-Azido-3′,5′-Dideoxythymidine and 3′,5′-Dideoxy-5′-Difluoromethylenethymidine as HIV-1 Reverse Transcriptase Inhibitors

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 β,γ-difluoromethylene, β,γ-dichloromethylene, or β,γ-imodo by condensation with pyrophosphate or pyrophosphate mimics, respectively. Inhibition of HIV-1 reverse transcriptase by the nucleoside P3Ms is briefly discussed.

Novel regioselective N-alkylations of 5-substituted-2H-tetrazoles

Regioselective alkylation of 5-substituted-2H-tetrazoles1 to 2-alkyl derivatives 3 was achievedvia decar☐ylative alkylation with alkyl cyanoformates. Lesser selectivity was observed with chloroformates.

The Preparation and Utility of 5-β-D-Ribofuranosyl-(2H)-Tetrazole as a Key Synthon for C-Nucleoside Chemistry

Abstract A synthesis of 5-β-D-ribofuranosyl-2H-tetrazole (1) 5-β-D.-ribofuranosyl-oxadiazoles, diethyl-δ2 -pyrazoline-4,5-dicarboxylate and diethylpyrazole-4,5-dicarboxylate derivatives is described. Ring transformations of1, has been investigated in an effort to establish the stability of this synthon for further use in dipolar cycloaddition reactions.

SYNTHESIS OF PYRAZOLO[4,3-C]PYRIDINE C-RIBONUCLEOSIDES VIA AN EFFECTIVE TETRAZOLE TO PYRAZOLE TRANSFORMATION

Abstract Methyl 2-[4-methoxycarbonyl-5-(β-D-ribofuranosyl)-1H-pyrazolyl-3]-acetate (7a) obtained from ribofuranosyltetrazole 3 by conjugative addition to dimethyl 1,3-allenedicarboxylate or dimethyl 3-chloro-2-pentenedioate after electrocyclization of 2-propenyltetrazole 5 was used as a suitable intermediate to provide pyrazolo [4,3-c]pyridine C-ribonucleosides 1 and 2 related to 7-substituted 3-deazaxanthosine and -guanosine analogs.