David Bernard Smith

The Novel Nucleoside Analog R1479 (4′-Azidocytidine) Is a Potent Inhibitor of NS5B-dependent RNA Synthesis and Hepatitis C Virus Replication in Cell Culture

Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4′-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC50 = 1.28 μm) with similar potency compared with 2′-C-methylcytidine (IC50 = 1.13 μm). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mm. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5′-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a Ki of 40 nm. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3′-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3′-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2′-C-MeATP and other 2′-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.

Biochemical Study of the Comparative Inhibition of Hepatitis C Virus RNA Polymerase by VX-222 and Filibuvir

ABSTRACT Filibuvir and VX-222 are nonnucleoside inhibitors (NNIs) that bind to the thumb II allosteric pocket of the hepatitis C virus (HCV) RNA-dependent RNA polymerase. Both compounds have shown significant promise in clinical trials and, therefore, it is relevant to better understand their mechanisms of inhibition. In our study, filibuvir and VX-222 inhibited the 1b/Con1 HCV subgenomic replicon, with 50% effective concentrations (EC50s) of 70 nM and 5 nM, respectively. Using several RNA templates in biochemical assays, we found that both compounds preferentially inhibited primer-dependent RNA synthesis but had either no or only modest effects on de novo-initiated RNA synthesis. Filibuvir and VX-222 bind to the HCV polymerase with dissociation constants of 29 and 17 nM, respectively. Three potential resistance mutations in the thumb II pocket were analyzed for effects on inhibition by the two compounds. The M423T substitution in the RNA polymerase was at least 100-fold more resistant to filibuvir in the subgenomic replicon and in the enzymatic assays. This resistance was the result of a 250-fold loss in the binding affinity (Kd) of the mutated enzyme to filibuvir. In contrast, the inhibitory activity of VX-222 was only modestly affected by the M423T substitution but more significantly affected by an I482L substitution.

Physicochemical properties of the nucleoside prodrug R1626 leading to high oral bioavailability.

The nucleoside analog R1479 is a potent and highly selective inhibitor of NS5b-directed hepatitis C virus (HCV) RNA polymerase in vitro. Because of its limited permeability, lipophilic prodrugs of R1479 were screened. Selection of the prodrug involved optimization of solubility, permeability, and stability parameters. R1626 has dissociation constant, intrinsic solubility, log partition coefficient (n-octanol water), and Caco-2 permeability of 3.62, 0.19 mg/mL, 2.45, and 14.95 × 10−6 cm/s, respectively. The hydrolysis of the prodrug is significantly faster in the Caco-2 experiments than in hydrolytic experiments, suggesting that the hydrolysis is catalyzed by enzymes in the cellular membrane. Using GastroPlus™, the physical properties of R1626 successfully predict the dose dependence of the pharmacokinetics in humans previously studied. The program predicts that if the particle size of R1626 is less than 25 μm, it will be well absorbed. Prodrugs with a solubility of greater than 100 μg/mL and permeability in the Caco-2 assay greater than 3 × 10−6 cm/s are expected to achieve a high fraction absorbed.

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 2: Synthesis and structure―activity relationships of benzothiazine-substituted quinolinediones

A new series of benzothiazine-substituted quinolinediones were evaluated as inhibitors of HCV polymerase NS5B. SAR studies on this series revealed a methyl sulfonamide group as a high affinity feature. Analogues with this group showed submicromolar potencies in the HCV cell based replicon assay. Pharmacokinetic and toxicology studies were also performed on a selected compound (34) to evaluate in vivo properties of this new class of inhibitors of HCV NS5B polymerase.

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 4: structure-based design, synthesis, and biological evaluation of benzo[d]isothiazole-1,1-dioxides

A series of benzo[d]isothiazole-1,1-dioxides were designed and evaluated as inhibitors of HCV polymerase NS5B. Structure-based design led to the incorporation of a high affinity methyl sulfonamide group. Structure-activity relationship (SAR) studies of this series revealed analogues with submicromolar potencies in the HCV replicon assay and moderate pharmacokinetic properties. SAR studies combined with structure based drug design focused on the sulfonamide region led to a novel and potent cyclic analogue.

The Florisil® catalyzed [1,3]-sigmatropic shift of allyl phenyl ethers — An entryway into novel mycophenolic acid analogues

Abstract Florisil ® was found to be effective in promoting the [1,3]-sigmatropic shift of mycophenolic acid related allyl phenyl ethers. Several novel mycophenolic acid analogues were thus prepared. Through a crossover experiment using two deuterated analogues of the model system, the reaction was shown to be intramolecular.

Effects of Spray Volume and Droplet Size on Herbicide Deposition and Common Cocklebur (Xanthium strumarium) Control1

Abstract: A field study was conducted in 1992 and 1993 to identify the spray volume and droplet size combinations to optimize control of common cocklebur (Xanthium strumarium) from acifluorfen by maximizing target deposition. In many instances, acifluorfen controlled common cocklebur better using either small (250 µm) or large (450 µm) spray droplets when applied at the lower carrier volumes of either 56 or 112 L/ha. When sprays were applied at 169 L/ha, there was little difference in control between droplet sizes. Deposition of acifluorfen was determined in 1993. Stepwise regression indicated that acifluorfen deposition amount is less important than environmental conditions for common cocklebur control. Relative humidity was the most significant variable for determining common cocklebur control with acifluorfen. Nomenclature: Acifluorfen, 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid; common cocklebur, Xanthium strumarium L. #3 XANST. Additional index words: Acifluorfen, deposition, efficacy, XANST. Abbreviations: VMD, volume median diameter; WAT, weeks after treatment.

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 3: synthesis and optimization studies of benzothiazine-substituted tetramic acids.

Benzothiazine-substituted tetramic acids were discovered as highly potent non-nucleoside inhibitors of HCV NS5B polymerase. X-ray crystallography studies confirmed the binding mode of these inhibitors with HCV NS5B polymerase. Rational optimization of time dependent inactivation of CYP 3A4 and clearance was accomplished by incorporation of electron-withdrawing groups to the benzothiazine core.

Utility of 4,6-dichloro-2-(methylthio)-5-nitropyrimidine. An efficient solid-phase synthesis of olomoucine

4,6-Dichloro-2-(methylthio)-5-nitropyrimidine has been utilized as a building block for an efficient nine-step synthesis of olomoucine. The methodology reported herein is applicable to the regiocontrolled solution and solid-phase synthesis of libraries of highly substituted purines, as well as other related scaffolds.

On the utility of α-heteroatom substituted orthoesters in the Johnson Claisen rearrangement

Abstract α-Heteroatom substituted orthoesters were prepared and found to undergo the Johnson Claisen rearrangement with a variety of allylic alcohols giving γ,δ-unsaturated α-heteroatom substituted esters in fair to excellent yields. The diastereoselectivity of the process was examined.