Synthesis of novel diflunisal hydrazide hydrazones as anti-hepatitis C virus agents and hepatocellular carcinoma inhibitors

Hepatitis C virus (HCV) infection is a main cause of chronic liver disease, leading to liver cirrhosis and hepatocellular carcinoma (HCC). The objective of our research was to develop effective agents against viral replication. We have previously identified the hydrazide hydrazone scaffold as a promising hepatitis C virus (HCV) and hepatocelluler inhibitor. Herein we describe the design a number of 2',4'-difluoro-4-hydroxy-N'-(arylmethylidene) biphenyl-3-carbohydrazide (3a-t) as anti-HCV and anticancer agents. Results from evaluation of anti-HCV activity indicated that most of the synthesized hydrazone derivatives inhibited viral replication in the Huh7/Rep-Feo1b and Huh 7.5-FGR-JCI-Rluc2A reporter systems. Antiproliferative activities of increasing concentrations of 2',4'-difluoro-4-hydroxy-N'-(2-pyridyl methylidene)biphenyl-3-carbohydrazide 3b and diflunisal (2.5-40 mu M) were assessed in liver cancer cell lines (Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU-475) with sulforhodamine B assay for 72 h. Compound 3b with 2-pyridinyl group in the hydrazone part exhibited promising cytotoxic activity against all cell lines with IC50 values of 10, 1034 16.21 4.74, 9.29 and 8.33 mu M for Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU-475 cells, respectively, and produced dramatic cell cycle arrest at SubG1/G0 phase as an indicator of apoptotic cell death induction. (C) 2015 Elsevier Masson SAS. All rights reserved

Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors

The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B–RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC50 values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors

Persistent Growth of a Human Plasma-Derived Hepatitis C Virus Genotype 1b Isolate in Cell Culture

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNAI (VA RNAI), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-α/β-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2′-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics

Anti-TAR Polyamide Nucleotide Analog Conjugated with a Membrane-Permeating Peptide Inhibits Human Immunodeficiency Virus Type 1 Production

The emergence of drug-resistant variants has posed a significant setback against effective antiviral treatment for human immunodeficiency virus (HIV) infections. The choice of a nonmutable region of the viral genome such as the conserved transactivation response element (TAR element) in the 5′ long terminal repeat (LTR) may potentially be an effective target for drug development. We have earlier demonstrated that a polyamide nucleotide analog (PNA) targeted to the TAR hairpin element, when transfected into cells, can effectively inhibit Tat-mediated transactivation of HIV type 1 (HIV-1) LTR (T. Mayhood et al., Biochemistry 39:11532-11539, 2000). Here we show that this anti-TAR PNA (PNA(TAR)), upon conjugation with a membrane-permeating peptide vector (transportan) retained its affinity for TAR in vitro similar to the unconjugated analog. The conjugate was efficiently internalized into the cells when added to the culture medium. Examination of the functional efficacy of the PNA(TAR)-transportan conjugate in cell culture using luciferase reporter gene constructs resulted in a significant inhibition of Tat-mediated transactivation of HIV-1 LTR. Furthermore, PNA(TAR)-transportan conjugate substantially inhibited HIV-1 production in chronically HIV-1-infected H9 cells. The mechanism of this inhibition appeared to be regulated at the level of transcription. These results demonstrate the efficacy of PNA(TAR)-transportan as a potential anti-HIV agent

Synthesis and Characterization of Celecoxib Derivatives as Possible Anti-Inflammatory, Analgesic, Antioxidant, Anticancer and Anti-HCV Agents

A series of novel N-(3-substituted aryl/alkyl-4-oxo-1,3-thiazolidin-2-ylidene)-4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamides 2a–e were synthesized by the addition of ethyl a-bromoacetate and anhydrous sodium acetate in dry ethanol to N-(substituted aryl/alkylcarbamothioyl)-4-[5-(4-methylphenyl)-3-(trifluoro-methyl)-1H-pyrazol-1-yl]benzene sulfonamides 1a–e, which were synthesized by the reaction of alkyl/aryl isothiocyanates with celecoxib. The structures of the isolated products were determined by spectral methods and their anti-inflammatory, analgesic, antioxidant, anticancer and anti-HCV NS5B RNA-dependent RNA polymerase (RdRp) activities evaluated. The compounds were also tested for gastric toxicity and selected compound 1a was screened for its anticancer activity against 60 human tumor cell lines. These investigations revealed that compound 1a exhibited anti-inflammatory and analgesic activities and further did not cause tissue damage in liver, kidney, colon and brain compared to untreated controls or celecoxib. Compounds 1c and 1d displayed modest inhibition of HCV NS5B RdRp activity. In conclusion, N-(ethylcarbamothioyl)-4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (1a) may have the potential to be developed into a therapeutic agent

Microwave assisted synthesis of some novel Flurbiprofen hydrazide-hydrazones as anti-HCV NS5B and anticancer agents

The synthesis of a new series of flurbiprofen hydrazide-hydrazones using microwave assisted reactions is described. Substituted aldehydes were condensed with flurbiprofen hydrazide by microwave irradiation to corresponding hydrazones. Synthesis of N'-[(4-bromothiophen-2-yl)methylidene]-2-(2-fluorobiphenyl-4-yl) propanehydrazide (3o) employing microwave assisted process resulted in higher yields, in faster time and with less chemical waste compared to traditional techniques. (2-fluorobiphenyl-4-yl)-N'-(phenylmethylidene)propanehydrazide (3p) and N'-[(2-chloro-6-fluorophenyl) methylidene]-2-(2-fluorobiphenyl-4-yl)propanehydrazide (3s) inhibited the growth of a leukemia cancer cell line HL-60 (TB) by 66.37% and an ovarian cancer cell line OVCAR-4 by 77.34% (single dose, 10 mu M), respectively at the National Cancer Institute (NCI), but had no significant effect on a panel of sixty human tumor cell lines. Flurbiprofen hydrazide- hydrazones were weak inhibitors of hepatitis C virus NS5B polymerase activity with N'-[(5-ethylfuran-2-yl)methylidene]-2-(2-fluorobiphenyl-4-yl)propanehydrazide (3m) being the most active of this series. Binding mode investigations of compound 3m suggested that allosteric pocket (AP).B may be the potential binding site for flurbiprofen hydrazones and these results will also assist in further derivatization of 3m using the green chemistry approach and improve the potency of S-flurbiprofen hydrazide- hydrazones