Yu-Huan Li

Alkaloids from the Root of Isatis indigotica

Seventeen new alkaloids (1-17) and 14 known analogues have been isolated from an aqueous extract of the root of Isatis indigotica. The structures and absolute configurations of these compounds were determined by extensive spectroscopic data analysis, including 2D NMR, single-crystal X-ray crystallography using anomalous scattering of Cu Kα radiation, and electronic circular dichroism spectra calculations based on the quantum-mechanical time-dependent density functional theory. Compounds 1, 2, and 3 are the first examples of natural products with unique linkages between a molecule of 2-(4-methoxy-1H-indol-3-yl)acetonitrile and 2-(1H-indol-3-yl)acetonitrile, 2-(4-methoxy-1H-indol-3-yl)acetonitrile, and 4-hydroxyphenylethane, respectively. Compounds (-)-4 and (+)-4 represent the first natural products with the pyrrolo[2,3-b]indolo[5,5a,6-b,a]quinazoline skeleton. Some structural assignments for the new alkaloids suggest that the assignments made for certain previously reported alkaloids require revision. Compounds 1-3 and arvelexin (18) show antiviral activity against the influenza virus A/Hanfang/359/95 (H3N2), with IC(50) values of 3.70-12.35 μM, and 17 inhibits Coxsackie virus B3 replication with an IC(50) of 6.87 μM.

Design and synthesis of oxymatrine analogues overcoming drug resistance in hepatitis B virus through targeting host heat stress cognate 70.

Heat-stress cognate 70 (Hsc70) is a host protein required for hepatitis B virus (HBV) replication, and oxymatrine (1) suppresses Hsc70 expression. Taking Hsc70 as a target against HBV, 22 analogues of 1 defined with substituents at position 1, 13, or 14 were synthesized and evaluated for their activity on Hsc70 mRNA expression. The SAR revealed that (i) the oxygen atom at the 1-position was not essential, (ii) increasing electron density on the ring D reduced the activity, and (iii) introducing a proper substituent at the 13- and/or 14-position(s), especially electron-withdrawing groups, might enhance the activity. Among the analogues, 6b possessing 13-ethoxyl afforded an increased activity in respect to 1. Importantly, it was active for either wild-type or lamivudine-resistant HBV, as its target is host Hsc70 but not viral enzymes. LD(50) of 6b in mice was over 750 mg/kg in oral route. We consider compound 6b promising for further investigation.

Oxymatrine inhibits hepatitis B infection with an advantage of overcoming drug-resistance

Oxymatrine (OMTR) is an anti-hepatitis drug used in China. Its mechanism of action is unknown. Recently, we found that OMTR inhibits hepatitis B virus (HBV) via down-regulating the expression of heat-stress cognate 70 (Hsc70), a host protein required for HBV DNA replication. Goal of this study was to assess the effect of OMTR on clinical HBV drug-resistance. OMTR monotherapy (oral, 12 months) reduced blood HBV DNA by 96% and HBeAg by 70% in the chronic hepatitis B (CHB) patients resistant to lamivudine (n = 17), equal to its efficacy in the naïve CHB cohort (n = 20). Liver biopsy study showed that OMTR treatment caused a decrease of Hcs70 mRNA in liver cells, parallel with a reduction of intracellular HBV DNA. Combination of lamivudine with OMTR (n = 15) (oral, 12 months) showed an enhanced anti-HBV effect as compared to lamivudine monotherapy (n = 25). The incidence of drug resistance against lamivudine in the combination group was significantly lower than that in the lamivudine group (1/15 vs 7/25; p<0.01). The results were further confirmed in vitro. Treatment of HBV(+) HepH2215 cells with sub-optimal dose of OMTR for 8 months suppressed HBV replication without inducing drug resistance, whereas lamivudine monotherapy caused drug-resistant mutation in 3 months. Combination of OMTR with lamivudine prevented HBV from developing drug resistance.

Enantiomers of an indole alkaloid containing unusual dihydrothiopyran and 1,2,4-thiadiazole rings from the root of Isatis indigotica.

A pair of enantiomers (1a and 1b) of an indole alkaloid containing dihydrothiopyran and 1,2,4-thiadiazole rings was isolated from an aqueous extract of the root of Isatis indigotica. The structures and absolute configurations of the enantiomers were determined by extensive spectroscopic analysis, especially 2D NMR, modified Moshers method, and electronic CD (ECD). The proposed biosynthetic pathway and preliminary investigations of the biological activity of compounds 1a and 1b against influenza virus A/Hanfang/359/95 (H3N2) and HSV-1 are also discussed.

Small molecular compounds that inhibit hepatitis C virus replication through destabilizing heat shock cognate 70 messenger RNA

Host heat shock cognate 70 (Hsc70) protein is packaged into hepatitis C viral (HCV) particles as a structural component of the virus in the assembly process. It helps HCV RNA release into the cytoplasm in the next infection cycle. The goal of this study is to investigate whether chemically down‐regulating host Hsc70 expression could be a novel strategy to interrupt HCV replication. Compounds were screened with an Hsc70 messenger RNA (mRNA) assay. IMB‐DM122 was found to be an effective and safe inhibitor for Hsc70 mRNA/protein expression in human hepatocytes. IMB‐DM122 inhibited HCV replication through destabilization of Hsc70 mRNA, and the half‐life of host Hsc70 mRNA was reduced by 78% after the compound treatment. The Hsc70 mRNA 3′ untranslated region sequence is the element responsible for the effect of IMB‐DM122 on Hsc70 mRNA. The compound appears to be highly efficient in inhibiting Hsc70‐related HCV replication. Treatment of the HCV‐infected hepatocytes with IMB‐DM122 reduced the virion encapsidation of Hsc70, and therefore disrupted HCV replication and the infection cycle. IMB‐DM122 showed considerable good safety in vitro as well as in vivo with no indication of harmful effect on liver and kidney functions. Conclusion: Hsc70 might be a new drug target and mechanism to inhibit HCV proliferation. (HEPATOLOGY 2010;)

Glucosylated caffeoylquinic acid derivatives from the flower buds of Lonicera japonica.

Three new glucosylated caffeoylquinic acid isomers (1–3), along with six known compounds, have been isolated from an aqueous extract of the flower buds of Lonicera japonica. Structures of the new compounds were determined by spectroscopic and chemical methods as (−)-4-O-(4-O-β-d-glucopyranosylcaffeoyl)quinic acid (1), (−)-3-O-(4-O-β-d-glucopyranosylcaffeoyl)quinic acid (2), and (−)-5-O-(4-O-β-d-glucopyranosylcaffeoyl)quinic acid (3), respectively. In the preliminary in vitro assays, two known compounds methyl caffeate and 2ʹ-O-methyladenosine showed inhibitory activity against Coxsackie virus B3 with IC50 values of 3.70 μmol/L and 6.41 μmol/L and SI values of 7.8 and 12.1, respectively.

1,2,3-Triazole-containing derivatives of rupestonic acid: Click-chemical synthesis and antiviral activities against influenza viruses

Two series of rupestonic acid derivatives, (1-substituted-1H-1,2,3-triazol-4-yl)methyl 2-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate and N-(1-substituted-1H-1,2,3-triazol-4-yl)methyl 2-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide were easily and efficiently synthesized via click chemistry. These compounds were tested for their in vitro activities against various strains of influenza A virus (H1N1, oseltamivir resistant H1N1, H3N2) and influenza B virus. The results showed that nine compounds were active against the H1N1 strain of influenza A virus and among them the best one 14a, was as active as the reference drugs, Oseltamivir and Ribavirin. Some of them were also active on the Oseltamivir resistant H1N1 strain. In regards to influenza B virus, twenty-one compounds over thirty were active and seven of them 7b, 8b, 9b, 10a, 11b, 12b, 13b showed better activity than Ribavirin. The structure-activity relationship of these compounds is discussed on the basis of each type of the viruses studied. Furthermore, four best representative compounds 7b, 10a, 12b and 14a were evaluated in a plaque assay experiment using MDCK cells and RBV as control compound and the results showed that 7b, 10a and 12b were better than RBV in inhibiting plaque formation, in good accordance with their anti-influenza B activities.

Antiviral glycosidic bisindole alkaloids from the roots of Isatis indigotica

Seven new glycosidic bisindole alkaloids, isatindigobisindolosides A–G (1–7), were isolated from an aqueous extract of the Isatis indigotica roots. Their structures including absolute configurations were determined by spectroscopic and chemical methods, together with calculations of electronic circular dichroism (ECD) spectra based on the quantum-mechanical time-dependent density functional theory. In the NMR spectra of 1–3, it is found that integration of H-2 and intensity of C-2 are affected not only by a substitution group at C-2 but also by solvents. Influences of the glucopyranosyloxy on the calculated ECD spectra of the glycosidic bisindole alkaloids are discussed. Compounds 2, 5, and 6 showed antiviral activity against both the influenza virus A/Hanfang/359/95 (H3N2) and Coxsackie virus B3 with IC50 values of 8.4–100.0 μM.

Host apolipoprotein b messenger RNA‐editing enzyme catalytic polypeptide‐like 3G is an innate defensive factor and drug target against hepatitis C virus

Host cellular factor apolipoprotein B messenger RNA (mRNA)‐editing enzyme catalytic polypeptide‐like 3G (hA3G) is a cytidine deaminase that inhibits a group of viruses including human immunodeficiency virus‐1 (HIV‐1). In the continuation of our research on hA3G, we found that hA3G stabilizing compounds significantly inhibited hepatitis C virus (HCV) replication. Therefore, this study investigated the role of hA3G in HCV replication. Introduction of external hA3G into HCV‐infected Huh7.5 human hepatocytes inhibited HCV replication; knockdown of endogenous hA3G enhanced HCV replication. Exogenous HIV‐1 virion infectivity factor (Vif) decreased intracellular hA3G and therefore enhanced HCV proliferation, suggesting that the presence of Vif might be an explanation for the HIV‐1/HCV coinfection often observed in HIV‐1(+) individuals. Treatment of the HCV‐infected Huh7.5 cells with RN‐5 or IMB‐26, two known hA3G stabilizing compounds, increased intracellular hA3G and accordingly inhibited HCV replication. The compounds inhibit HCV through increasing the level of hA3G incorporated into HCV particles, but not through inhibiting HCV enzymes. However, G/A hypermutation in the HCV genome were not detected, suggesting a new antiviral mechanism of hA3G in HCV, different from that in HIV‐1. Stabilization of hA3G by RN‐5 was safe in vivo. Conclusion: hA3G appears to be a cellular restrict factor against HCV and could be a potential target for drug discovery. (HEPATOLOGY 2011;)

Indole alkaloid glucosides from the roots of Isatis indigotica.

Abstract Five new indole alkaloid glucosides named isatindigotindolosides A-E (1–5), along with three known analogs (6–8), were isolated from an aqueous extract of the Isatis indigotica roots. Their structures including the absolute configurations were determined based on comprehensive spectroscopic data analysis, combined with chemical methods and electronic circular dichroism spectra calculations. In the preliminary assays, compounds 1, 6 and 7 showed antiviral activity against influenza virus A/Hanfang/359/95 (H3N2) with IC50 values of 14.6–33.3 μM. Compound 1 also exhibited inhibitory effect against nitric oxide (NO) production in microglial cell BV2 with an inhibition ratio of 93.0% at 10 μM.