Zhi Hong

Viramidine, a Prodrug of Ribavirin, Shows Better Liver-Targeting Properties and Safety Profiles Than Ribavirin in Animals

Ribavirin, part of the current first line combination therapy for the treatment of chronic hepatitis C, may cause haemolytic anaemia and poses a significant challenge to the clinical management of the disease. Viramidine, a prodrug of ribavirin, is currently under development. In-vitro partition demonstrated that viramidine had less association with RBCs than ribavirin in rat, monkey and man, and thus has less liability for haemolytic anaemia than ribavirin. In a whole body autoradiography study in rats following oral dosing (30 mg/kg) of [14C]ribavirin or [14C]viramidine to monkeys, viramidine produced 32% higher radioactivity in the liver than ribavirin, indicating a better liver-targeting properties. In portal vein-cannulat-ed cynomolgus monkeys following single oral dosing (30 mg/kg) of [3H]viramidine or [3H]rib-avirin, viramidine retained 3X higher radioactivity in the liver than ribavirin. Viramidine dosing also produced a higher viramidine to ribavirin ratio in portal plasma than in systemic plasma, indicating that the liver was the main site for the viramidine conversion to ribavirin and subsequent trapping of the drug. After multiple oral dosing (10 mg/kg) of [14C]ribavirin or [14C]viramidine to monkey, viramidine yielded three times the drug level in the liver but only half in RBCs compared to rib-avirin. Viramidine and ribavirin had comparable toxicity profiles in a 28-day toxicity study in rats. In contrast, viramidine had much better safety profiles than ribavirin in a 28-day toxicity study in monkeys. In conclusion, viramidine has better liver-targeting properties and safety profiles than ribavirin in animals.

Dinucleotide Analogues as Novel Inhibitors of RNA-Dependent RNA Polymerase of Hepatitis C Virus

ABSTRACT Replication of hepatitis C virus (HCV) RNA is catalyzed by the virally encoded RNA-dependent RNA polymerase NS5B. It is believed that the viral polymerase utilizes a de novo or primer-independent mechanism for initiation of RNA synthesis. Our previous work has shown that dinucleotides were efficient initiation molecules for NS5B in vitro (W. Zhong, E. Ferrari, C. A. Lesburg, D. Maag, S. K. Ghosh, C. E. Cameron, J. Y. Lau, and Z. Hong, J. Virol. 74:9134-9143, 2000). In this study, we further demonstrated that dinucleotide analogues could serve as inhibitors of de novo initiation of RNA synthesis directed by HCV NS5B. Both mononucleotide- and dinucleotide-initiated RNA syntheses were affected by dinucleotide analogues. The presence of the 5′-phosphate group in the dinucleotide compounds was required for efficient inhibition of de novo initiation. Optimal inhibitory activity also appeared to be dependent on the base-pairing potential between the compounds and the template terminal bases. Because the initiation process is a rate-limiting step in viral RNA replication, inhibitors that interfere with the initiation process will have advantages in suppressing virus replication. The use of dinucleotide analogues as inhibitor molecules to target viral replication initiation represents a novel approach to antiviral interference.

Synthesis of nucleoside libraries on solid support. I. N2, N6-disubstituted diaminopurine nucleosides

Starting with 2‐iodo‐6‐chloro‐9‐(β‐D‐ribofuranosyl)purine, a library of more than 1,300 N2,N6‐polysubstituted diaminopurine nucleosides was created. The starting material was condensed with a polystyrene monomethoxytrityl resin and a pool of primary and secondary amines was used to displace the 6‐chloro atom with high regio‐selectivity. The 2‐iodo was subsequently displaced by various primary amines. Nucleosides were cleaved from the resin with hexafluoroisopropanol solutions. A majority of compounds reached a purity of more than 80% without the need for any type of purification. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.

Synthesis of Nucleoside Libraries on Solid Support. II. 2,6,8‐Trisubstituted Purine Nucleosides Using 8‐Bromoguanosine as Precursor

A series of 2,6,8‐trisubstituted purine nucleoside libraries was prepared by parallel solid‐phase synthesis using 8‐bromoguanosine as a common synthetic precursor. Polystyrene‐methoxytrityl chloride resin was linked to the N2 or O5′ position of the guanosine analogues. 8‐Bromoguanosine was derivatized at the C8 position via carbon‐carbon bond formation. Nucleophilic aromatic substitution at C2 and/or C6 positions with various amines produced two series of purine nucleoside libraries with very diverse substitution. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.

Conversion of viramidine to ribavirin in vivo by adenosine deaminase and its inhibition by 2'-deoxycoformycin.

Previously we reported that viramidine is a prodrug of ribavirin and that adenosine deami-nase catalyses viramidine deamination to ribavirin in vivo. This in vivo study explores this prodrug conversion in rats and inhibition by a potent adenosine deaminase inhibitor, 2′-deoxyco-formycin. We found that conversion of viramidine to ribavirin was viramidine dose-dependent in rat plasma. A single intravenous dose of 0.25 mg/kg 2′-deoxycoformycin suppressed orally administered viramidine conversion to ribavirin in plasma by 50%. The inhibition was 2′-deoxycoformycin dose-dependent and a single dose of 2 mg/kg decreased the ribavirin/viramidine area under the concentration—time curve between 0 h and 6 h ratio by 2.5-fold. These findings provide strong evidence that adenosine deaminase plays a major role in converting viramidine to ribavirin in vivo.

Solid-phase parallel synthesis of 4-β-D-ribofuranosylpyrazolo[4,3-d]pyrimidine nucleosides

The synthesis of pyrazolo[4,3-d]pyrimidine nucleoside library using solid-phase parallel synthesis methodology is described. Glycosylation of the trimethylsilyl (TMS) derivative of 1- and 2-(methyl)-1H and 2H-pyrazolo[4,3-d]pyrimidine-5,7-(4H,6H)-dione (5) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of TMS triflate provided two novel protected nucleosides 6 and 7. The structures of 6 and 7 were assigned by 1H and 2D NMR experiments. Nucleosides 6 and 7 were then transformed to the key intermediates 12 and 15 respectively. Reaction of 12 and 15 with MMTCl resin in the presence of 2,6-lutidine afforded the necessary scaffolds B and C. Different amines (96) were introduced selectively by nucleophilic substitution on scaffolds B and C using solid-phase parallel semi-automated synthesizer. Cleavage of the products from the solid support with 30% HFIP in a parallel fashion yielded nucleoside libraries simultaneously, and they were analyzed and characterized by high-throughput LC-MS.