Wonsuk Chang

Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977

A phosphoramidate prodrug of 2′-deoxy-2′-α-fluoro-β-C-methyluridine-5′-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixture of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot analysis showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the production of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5′-monophosphate form, PSI-7411. siRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, respectively.

Effect of artemisinins and other endoperoxides on nitric oxide-related signaling pathway in RAW 264.7 mouse macrophage cells

Artemisinin is the active principle of the Chinese herb Artemisia annua L. In addition to its anti-malarial activity, artemisinin and its derivatives have been shown to exert profound anti-cancer activity. The endoperoxide moiety in the chemical structure of artemisinin is thought to be responsible for the bioactivity. Here, we analyzed the cytotoxicity and the ability of artemisinin, five of its derivatives, and two other endoperoxides to inhibit generation of nitric oxide (NO). In the RAW 264.7 mouse macrophage cell line, the well-established model cell line to analyze NO generation, artesunate revealed the highest ability to inhibit NO production among all compounds tested. In cytotoxicity assays (XTT assay), the IC(50) value of RAW 264.7 cells for artesunate was determined to be 3.1+/-0.7 microM. In order to associate the cytotoxic effects with specific alteration in gene expression related to NO metabolism and signaling, whole genome mRNA microarray analyses were conducted. RAW 264.7 cells were treated with artesunate using DMSO as vehicle control followed by microarray analysis. A total of 36 genes related to NO metabolism and signaling were found to be differentially expressed upon exposure to artesunate. Apart from NO-related genes, the expression of genes associated with other functional groups was also analyzed. Out of 24 functional groups, differential expression was most prominent in genes involved in cell-to-cell signaling and interactions. Further refinement of this analysis showed that the pathways for cAMP-mediated signaling and Wnt/beta-catenin signaling were most closely related to changes in mRNA expression. In conclusion, NO generation and signaling play a role in exhibiting cytotoxic activity of artesunate. In addition, other signaling pathways also contribute to the inhibitory effect of artesunate towards RAW 264.7 cells pointing to a multi-factorial mode of action of artesunate.

Malaria-Infected Mice Are Cured by Oral Administration of New Artemisinin Derivatives

In four or five chemical steps from the 1,2,4-trioxane artemisinin, a new series of 23 trioxane dimers has been prepared. Eleven of these new trioxane dimers cure malaria-infected mice via oral dosing at 3 x 30 mg/kg. The clinically used trioxane drug sodium artesunate prolonged mouse average survival to 7.2 days with this oral dose regimen. In comparison, animals receiving no drug die typically on day 6-7 postinfection. At only 3 x 10 mg/kg oral dosing, seven dimers prolong the lifetime of malaria-infected mice to days 14-17, more than double the chemotherapeutic effect of sodium artesunate. Ten new trioxane dimers at only a single oral dose of 30 mg/kg prolong mouse average survival to days 8.7-13.7, and this effect is comparable to that of the fully synthetic trioxolane drug development candidate OZ277, which is in phase II clinical trials.

Discovery of a Novel Class of Potent HCV NS4B Inhibitors: SAR Studies on Piperazinone Derivatives

HTS screening identified compound 2a (piperazinone derivative) as a low micromolar HCV genotype 1 (GT-1) inhibitor. Resistance mapping studies suggested that this piperazinone chemotype targets the HCV nonstructural protein NS4B. Extensive SAR studies were performed around 2a and the amide function and the C-3/C-6 cis stereochemistry of the piperazinone core were essential for HCV activity. A 10-fold increase in GT-1 potency was observed when the chiral phenylcyclopropyl amide side chain of 2a was replaced with p-fluorophenylisoxazole-carbonyl moiety (67). Replacing the C-6 nonpolar hydrophobic moiety of 67 with a phenyl moiety (95) did not diminish the GT-1 potency. A heterocyclic thiophene moiety (103) and an isoxazole moiety (108) were incorporated as isosteric replacements for the C-6 phenyl moiety (95), resulting in significant improvement in GT-1b and 1a potency. However, the piperazonone class of compounds lacks GT-2 activity and, consequently, were not pursued further into development.

Malaria-infected mice live until at least day 30 after a new monomeric trioxane combined with mefloquine are administered together in a single low oral dose.

In only five simple steps and 48% overall yield from the natural trioxane artemisinin, the thermally and hydrolytically stable trioxane fluoroanilide 4b has been prepared. Upon one oral dose of only 6.8 mg/kg of monomeric trioxane 4b combined with 20 mg/kg of mefloquine hydrochloride, all of the malaria-infected mice lived until at least day 30 post infection. Of the five mice in this surviving group, four (80%) were completely cured (no parasites in their blood) and one mouse had 4% blood parasitemia. Importantly, the efficacy of this ACT chemotherapy using monomeric trioxane 4b plus mefloquine hydrochloride is considerably better than the efficacy under the same conditions using the popular trioxane drug artemether plus mefloquine hydrochloride.