Elsevier

Bioorganic & Medicinal Chemistry

Volume 12, Issue 21, 1 November 2004, Pages 5545-5551
Bioorganic & Medicinal Chemistry

Brasilicardins B–D, new tricyclic terpernoids from actinomycete Nocardia brasiliensis

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Abstract

Three new tricyclic terpenoids, brasilicardins B–D (2–4), were isolated together with brasilicardin A (1), a potent immunosuppressive compound, from the cultured broth of a pathogenic actinomycete Nocardia brasiliensis IFM0406, and the structures and stereochemistry were determined by spectroscopic data and a single crystal X-ray diffraction analysis. The immunosuppressive and cytotoxic activities of 2–4 were examined in the comparison with 1.

Introduction

During our search for bioactive substances from pathogenic actinomycetes of the genus Nocardia,1 we have previously isolated brasilicardin A (1), a novel tricyclic terpenoid consisting of an anti/syn/anti-perhydrophenanthrene skeleton with two sugars and an amino acid side-chain, from Nocardia brasiliensis IFM-0406.2 This unique terpenoid moiety was shown to be biosynthesized from glucose via the nonmevalonate pathway.3 Brasilicardin A (1) exhibits potent immunosuppressive activity in mouse mixed lymphocyte reaction (MLR) assay and cytotoxic activity against adriamycin-resistant murine lymphoma cells. Its immunosuppressive potency is compatible to those of cyclosporin A or ascomycin.4 Further investigation on the extract of this strain resulted in the isolation of three new congeners, brasilicardins B–D (2–4), and the structures and stereochemistry were determined by spectroscopic data and a single crystal X-ray diffraction analysis. In this paper we describe the isolation and structure elucidation of 2–4 and their immunosuppressive activities.

Section snippets

Results and discussion

The supernatant of the fermentation broth (80 L) was subjected to a Diaion HP-20 column (50% MeOH aq → MeOH), in which fractions eluted with MeOH were separated on a silica gel and C18 columns, and centrifugal partition chromatography followed by C18 HPLC (MeOH/H2O and MeOH/H2O/CF3CO2H) to afford brasilicardins B (2, 6.6 mg), C (3, 55 mg), and D (4, 23 mg) together with a known related compound, brasilicardin A (1, 428 mg).

Brasilicardin B {2, [α]D23 +17 (c 1.0, MeOH)} was obtained as a colorless

General experimental procedure

The 3.35 ppm resonance of residual CH3OH and 49.8 ppm of CD3OD were used as internal references for 1H and 13C NMR spectra, respectively. FAB mass spectra were obtained using nitrobenzylamine as a matrix.

Cultivation

The voucher specimen of Nocardia brasiliensis (strain IFM 0406) was deposited at the Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University (deposit No. FERM BP-5498).4 This actinomycete was grown in the broth [glycerol (2.0%), polypepton (1.0%), and meat extract (0.5%) in H2O, pH 

Acknowledgements

We thank Ms. S. Oka and Miss. M. Kiuchi, Center for Instrumental Analysis, Hokkaido University, for ESIMS and FABMS measurements. This work was partly supported by a Grant-in-Aid from the Takeda Science Foundation and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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