Anti-HBV and cytotoxic activities of pyranocoumarin derivatives

https://doi.org/10.1016/j.bmc.2008.12.007Get rights and content

Abstract

Four natural pyranocoumarins clausenidin (1), nordentatin (2), clausarin (3), and xanthoxyletin (4) were isolated from the medicinal plant Clausena excavata. Recently, we found that 1 and 2 suppressed hepatitis B virus surface antigen in HepA2 cells, and in addition, 13 showed cytotoxic activity against four human cancer cell lines (A549, MCF7, KB, and KB-VIN). To explore the SAR of 14, 17 pyranocoumarin analogues (521) were designed and synthesized. Among these analogues, 5 and 10 were the most potent against hepatitis B virus with EC50 values of 1.14 and 1.34 μM, respectively. The most interesting result in the cytotoxicity assay was the significant activity of 1, 5, and 6 against the multi-drug resistant cell line, KB-VIN, without activity against the KB cell line. These data suggest that these three compounds could be useful hits for developing MDR-inverse drugs.

Introduction

Coumarins are a widely distributed and important class of natural compounds with broad pharmacological activities. They occur commonly in plants belonging to the families Rutaceae, Simaroubaceae, Meliaceae, and Burseraceae. We previously identified pyranocoumarins clausenidin 1, nordentatin 2, clausarin 3, and xanthoxyletin 4 from the roots of Clausena excavata (Rutaceae),1 which are used as a traditional remedy to treat viral hepatitis, cough, asthma, fever, headache, dermatological conditions, and gastrointestinal diseases.2, 3, 4 During our continuing screening for antiviral and cytotoxic activities of traditional drugs and herbal materials, we found that 1 and 2 exhibited strong suppressive activity on hepatitis B virus surface antigen (HBsAg) production in human hepatoma cells. Furthermore, 13 showed potent cytotoxicity against A549, MCF7, KB, and KB-VIN human cancer cell lines. Therefore, we synthesized additional analogues of the isolated pyranocoumarins to evaluate structure–activity relationships and develop more selective and potent anti-hepatitis B virus (anti-HBV) or antitumor agents.

Hepatitis B virus (HBV) infection frequently results in both acute and chronic hepatitis and remains a major health problem worldwide. The number of chronic HBV-infected people is estimated to be more than 400 million worldwide with an annual death toll of 1.2 million per year.5 HBV is also associated with a high risk (25–40%) of developing cirrhosis and hepatocellular carcinoma leading to significant mortality.6, 7, 8 Immunization against HBV has helped to prevent new infections,9 but millions of chronically infected patients will eventually succumb to the infection sequence unless treated with currently available therapies. Currently, the most promising anti-HBV agents are interferon-α (IFN-α) and nucleoside analogues. The efficacy of IFN-α is limited and often associated with severe adverse effects.10 Nucleoside analogues such as lamivudine (3TC), adefovir dipivoxil (bis-POMPMEA) and penciclovir (PCV) inhibit reverse transcriptase activity of the viral polymerase and have demonstrated clinical utility.11 However, some chronic HBV-infected and liver transplant patients often experience a recurrence of HBV after a period of antiviral treatment with 3TC or PCV. This recurrence is due to the emergence of viral resistance.12, 13 Genotypic resistance emerges in 14–32% of patients within the first 12 months of 3TC therapy, increasing to 40% within two years of treatment and 57% by the third year.14 Adefovir is usually well-tolerated and significantly reduces serum HBV DNA levels.5 However, the primary limitations of adefovir therapy are dose-related side effects such as nephrotoxicity, lactic acidosis, and severe hepatomegaly with steatosis, and cessation of therapy may result in serious hepatitis. Therefore, novel classes of non-nucleoside anti-HBV agents with different modes of action are urgently needed.

In our studies, certain natural pyranocoumarins not only strongly suppressed HBsAg production in human hepatoma HepA2 cells, but also showed profound cytotoxic activity against four human cancer cell lines. In view of the novel structural template, we decided to synthesize additional analogues of the isolated pyranocoumarins to determine structure–activity relationships (SAR) and possibly develop more selective and potent anti-HBV or antitumor agents. Herein, we report our semi-synthesis of several new pyranocoumarin derivatives in excellent yields by using hydrogenation, epoxidation, and methylation techniques. Preliminary SAR studies on anti-HBV and cytotoxic activities are also discussed in this paper (Fig. 1).

Section snippets

Chemistry

We have seen limited reports of chemical modification on pyranocoumarins 14;15, 16, 17, 18 therefore, we probed several structural changes, including hydrogenation, epoxidation, and methylation, at positions C-5, C-6, C-7, C-2′, C-3′, C-2′′, and C-3′′. Accordingly, semi-synthetic analogues 521 were prepared from 1–4 as illustrated in Scheme 1, Scheme 2.

Compounds 1, 2, and 3 were reacted separately with methyl iodide in basic medium to yield 7, 11, and 16, respectively. Catalytic hydrogenation

Results and discussion

In our screening program, HepA2 cells were plated into 24-well plates and allowed to attach overnight. The medium was subsequently changed to serum-free DMEM, and various concentrations of semi-synthetic pyranocoumarin analogues were added. The production of HBsAg in the medium was determined after 48 h incubation. The 50% efficacy concentrations (EC50, in μM) for these analogues are listed in Table 1.

Among the tested pyranocoumarin analogues, 1, 5, 10, 11, and 12 had anti-HBV EC50 values of

General experimental procedures

Melting points were determined on a Yanaco MP-S3 melting point apparatus and are uncorrected. IR spectra were determined in KBr discs on a Shimadzu FT-IR Prestige 21. 1H and 13C NMR spectra were recorded on a Bruker Avance-300 NMR spectrometer, using tetramethylsilane (TMS) as internal standard; all chemical shifts were reported in ppm (δ). All MS and HRMS spectra (EI) were obtained on a VG-70-250S mass spectrometer. Elemental microanalysis were determined by Elementar Vario EL III and gave

Acknowledgments

The authors are grateful for financial support from the National Science Council, Taiwan, and Republic of China (NSC 94-2113-M-006-008) awarded to T. S. Wu and also thankful to the National Research Institute of Chinese Medicine, Taiwan, Republic of China, for partial financial support of this research. Thanks are also due in part to support from NIH grant CA-17625 from the National Cancer Institute awarded to K. H. Lee.

References and notes (21)

  • L. Fu et al.

    Biochem. Pharmacol.

    (1998)
  • N.W.Y. Leung et al.

    Hepatology

    (2001)
  • T. Tomimatsu et al.

    Tetrahedron

    (1972)
  • R.D.H. Murray et al.

    Tetrahedron

    (1984)
  • S.C. Huang et al.

    Phytochemistry

    (1997)
  • Sasaki, S. Khoyo Taiwan Minkan Yakyo Shokubutsu Shi, Khobunkan, Taipei, 1924, p...
  • K. Boonsong et al.

    Phytother. Res.

    (2005)
  • S. Arunrat et al.

    Planta Med.

    (2003)
  • A.H. Malik et al.

    Ann. Int. Med.

    (2000)
  • M.H. Chang et al.

    New Eng. J. Med.

    (1997)
There are more references available in the full text version of this article.

Cited by (97)

  • A quick and convenient <sup>1</sup>H quantitative NMR method for determination of bioactive pyranocoumarins from Clausena excavata

    2021, Phytochemistry Letters
    Citation Excerpt :

    The main biologically active constituents found in the rhizomes and roots of C. excavata extracts are clausenidin, dentatin, and nordentatin (Fig. 1). These pyranocoumarins possess diverse bioactivities, including antibacterial (Sunthitikawinsakul et al., 2003; Wu and Furukawa, 1982), anticancer (Chakthong et al., 2016; Sharif et al., 2011; Sripisut et al., 2012; Su et al., 2009), and anti-HIV (Kongkathip et al., 2005) responses. This plant has promising use for the treatment of HIV infection.

View all citing articles on Scopus
View full text