Inhibition of hepatitis B virus gene expression and replication by helioxanthin and its derivative.

Chronic hepatitis B virus (HBV) infection continues to be an important worldwide cause of morbidity and mortality. All the currently approved therapeutic drugs have their limitations: interferon-alpha (IFN-alpha) has limited efficacy and a high incidence of adverse effects; nucleoside analogues are very efficient HBV DNA inhibitors, but resistance occurs eventually. Therefore, it is important to develop new non-nucleoside/nucleotide agents with different modes of action that can be used for antiviral combination therapy. Here, we report on a novel class of compounds, helioxanthin and its derivative 5-4-2, which had potent anti-HBV activities in HepG2.2.15 cells, with the EC50s of 1 and 0.08 microM, respectively. The lamivudine-resistant HBV, L526M/M550V double mutant strain, was also sensitive to helioxanthin and 5-4-2. This class of compounds not only inhibited HBV DNA, but also decreased HBV mRNA and HBV protein expression. The EC50 of HBV DNA inhibition was consistent with the EC50 of HBV 3.5 Kb transcript inhibition, which was 1 and 0.09 microM for helioxanthin and 5-4-2 respectively. Western blot analysis of cell lysate from HepG2.2.15 cells showed that the core protein expression decreased in a dose-dependent manner after drug treatment. In conclusion, helioxanthin and 5-4-2 are potentially unique new anti-HBV agents, which possess a different mechanism of action from existing therapeutic drugs. Both compounds inhibited HBV RNA and protein expression in addition to inhibiting HBV DNA.

Synthesis and antiviral activity of helioxanthin analogues.

A series of natural product analogues based on helioxanthin (2), with particular attention to modification of the lactone ring and methylenedioxy group, were synthesized and evaluated for their antiviral activities. Among them, lactam derivative 18 and helioxanthin cyclic hydrazide 28 exhibited significant in vitro antiviral activity against hepatitis B virus (EC(50) = 0.08 and 0.03 microM, respectively). Compound 18 showed the most potent antiviral activity against hepatitis C virus (55% inhibition at 1.0 microM). Compound 12, an acid-hydrolyzed product of helioxanthin cyclic imide derivative 9, was found to exhibit broad-spectrum antiviral activity against hepatitis B virus (EC(50) = 0.8 microM), herpes simplex virus type 1 (EC(50) = 0.15 microM) and type 2 (EC(50) < 0.1 microM), Epstein-Barr virus (EC(50) = 9.0 microM), and cytomegalovirus (EC(50) = 0.45 microM). Helioxanthin lactam derivative 18 also showed marked inhibition of herpes simplex virus type 1 (EC(50) = 0.29 microM) and type 2 (EC(50) = 0.16 microM). The cyclic hydrazide derivative of helioxanthin 28 and its brominated product 42 exhibited moderately potent activities against human immunodeficiency virus (EC(50) = 2.7 and 2.5 microM, respectively). Collectively, these molecules represent a novel set of antiviral compounds with unique structural features.

Increased anti-P-glycoprotein activity of baicalein by alkylation on the A ring.

The aqueous extract of Scutellariae baicalensis Georgi has inhibitory activity against P-gp 170, a multiple drug resistant gene product. Baicalein, one of the major flavones, was found to be responsible for this activity. The hydroxyl groups of the A ring of baicalein were systematically alkylated in order to assess the effect of such modifications on the activity against P-gp 170. The impact of the baicalein modifications on activity against the growth of a human nasopharyngeal cancer cell line KB and its P-gp 170 overexpressing cell line KB/MDR were also examined. The results indicate that alkylation of R5 of baicalein does not have a major impact on the interaction with P-gp 170, whereas alkylation of R6 or R7 alone or both, could enhance the interaction of baicalein with P-gp 170 as well as the amount of intracellular accumulation of vinblastine, a surrogate marker for the activity of P-gp 170 pump of KB/MDR cells. In this case, the optimal linear alkyl functionality is a propyl side chain. These modifications could also alter the activity of compounds inhibiting cell growth. Among the different compounds synthesized, the most potent molecule against P-gp 170 is 5-methoxy-6,7-dipropyloxyflavone (23). Its inhibitory activity against P-gp 170 is approximately 40 times better, based on EC50 (concentration of the compound enhancing 50% of the intracellular vinblastine accumulation in the KB/MDR cells) and 3 times higher, based on Amax (the intracellular vinblastine accumulation of the KB/MDR cells caused by the compound) as compared to baicalein. Compound 23 is also a more selective inhibitor than baicalein against P-gp 170, because its cytotoxicity is less than that observed for baicalein. The growth inhibitory IC50 of compound 23 against KB and KB/MDR cells are about the same, suggesting that compound 23 is unlikely to be a substrate of P-gp 170 pump. Acetylation of R6, R7 or both could also decrease EC50 and increase Amax. Acetylated compounds are more toxic than baicalein, and their potency against cell growth is compromised by the presence of P-gp 170, suggesting that these compounds are substrates of P-gp 170. Benzylation of R6 or R7 but not both also enhanced anti-P-gp170 activity and potency against cell growth; however, the presence of P-gp 170 in cells did not have an impact on their sensitivity to these molecules, suggesting that the benzylated compounds are inhibitors but not substrates of P-gp 170, and perhaps have a different mechanism of action. In conclusion, the substitutions of R6 and R7 hydroxyl groups by alkoxy groups, acetoxy groups, or benzyloxy groups could yield compounds with different modes of action against P-gp 170 with different mechanisms of action against cell growth.

Lignans of Rosa multiflora roots.

Five known lignans, (+)-pinoresinol (1), (+)-8-hydroxypinoresinol (2), (-)-dehydrodiconiferyl alcohol (3), (+)-trans-dehydrodiconiferyl alcohol (4), and (-)-olivil (5), were isolated from the roots of Rosa multiflora for the first time. Their structures were determined using spectroscopic data.

New pregnane glycosides from Cynanchum ascyrifolium.

Two new pregnane glycosides, cynascyrosides D and E, were isolated from the roots of Cynanchum ascyrifolium. The structures of these compounds were determined on the basis of spectroscopic and chemical evidence as cynajapogenin A 3-O-alpha-L-cymaropyranosyl-(1-->4)-beta-D-digitoxopyranosyl-(1-->4)-beta-L-cymaropyranoside and cynajapogenin A 3-O-beta-D-glucopyranosyl-(1-->4)-alpha-L-diginopyranosyl-(1-->4)-beta-L-cymaropyranosyl-(1-->4)-beta-D-digitoxopyranoside.