Colossolactones, New Triterpenoid Metabolites from a Vietnamese Mushroom Ganoderma colossum § Peter Kleinwa ¨ chter, † Ngo Anh, ‡ Trinh Tam Kiet, ‡ Brigitte Schlegel, † Hans-Martin Dahse, † Albert Ha ¨ rtl, † and Udo Gra ¨ fe* ,† Hans-Kno ¨ ll-Institute for Natural Products Research, Beutenbergstrasse 11, D-07745 Jena, Germany, and Mycological Research Center, Hanoi State University, 334 Nguyen Trai Street, Hanoi, Vietnam Received September 8, 2000 Seven new triterpenoid metabolites (colossolactones; 1-7) were isolated from a fruiting body of Ganoderma colossum, and their structures were determined by MS and NMR methods. The fungal family Ganodermataceae is represented by more than 200 species, which mostly occur in subtropical and tropical regions. 1 Some members of the Ganoder- mataceae, such as Ganoderma lucidum and Ganoderma applanatum, are used in Asian folk medicine for treatment of some diseases. 2 Numerous terpenoid compounds have been reported as components of these medically important species. 3-5 However, little information is available on metabolites of other representatives of these genera. In this paper we report the structure of seven new triterpenoid metabolites, colossolactones A-G(1-7), isolated from Ganoderma colossum Donk (Ganodermataceae) (Chart 1). A fruiting body of Ganoderma colossum (ca. 200 g wet weight) was collected on a trunk of Delonix regia (Fabaceae) in Hue city, Thua Thien-Hue province, Vietnam. It was characterized taxonomically as a representative of the Ganoderma family and species G. colossum (synonymous: Polyporus colossus, Dendrophagus colossus) due to the reticulated cell wall structure and other morphological features. 4 For the isolation of metabolites 1-7 the lyophilized fruiting body of G. colossum was extracted with 1 L of 1:1 CHCl 3 /MeOH and subsequently with 1 L of ethyl acetate. Compounds 1-7 were isolated from the residue of the evaporated extract by several subsequent chromato- graphic steps. The molecular formulas were determined by HREIMS showing [M] + and respective diagnostic frag- ments. The IR spectra attested to the presence of carbonyl groups due to absorbances in the range 1696-1717 cm -1 . UV absorbances (λ max 326-328 nm) of compounds 4-7 suggested the occurrence of a triene-lactone chromophore. The structures of the new sterol-type metabolites 1-7 were determined conclusively by 1D and 2D 1 H and 13 C NMR spectroscopy. The proton broad-band decoupled 13 C NMR and DEPT spectra suggested the number and binding type of the skeleton carbons and the substitution pattern. A prominent feature was the occurrence of lactone car- bonyls in 2-7 and conjugated double bonds in 4-7. The 1 H- 1 H COSY and TOCSY spectra were particularly helpful for the assignment of overlapping proton signals of the individual rings. The sequence of carbon and hydrogen atoms was settled by heteronuclear 2D NMR experiments (HSQC, HMBC). The observable C-H long-range couplings (HMBC) of the methyl groups at the quaternary ring carbons were attributable to a sterol-type ring system. Assignment of the relative stereochemistry of 1-7 was supported by the observable NOE correlations of the methyl protons with neighboring protons of the rings and other methyl substituents in the NOESY and ROESY spectra. Measurements of optical rotation confirmed the chiral nature of 1-7. Assignments of NMR signals are given in the Experimental Section. Structures such as 3-7 containing a seven-membered lactone as the triterpenoid ring A and a δ-lactone side chain at C-17 have not been reported previously for fungal metabolites. However, representatives of this structural type such as schisanlactones, kadsulactone A, kadsudilac- tone, and lancilactones were isolated from the stems and roots of plants such as Schisandra sp., Kadsura heteroclita, K. coccinea, and K. lancilimba, used as folk medicines for the treatment of rheumatism, stomachache, and entero- gastritis. 6-9 Colossolactones (1-7) displayed no antimicrobial activity against a spectrum of bacteria and fungi but moderate cytotoxicity against L-929, K-562, and HeLa cells with IC 50 values ranging from 15 to 35 μg/mL. Moreover, they inhibited 3R-hydroxysteroid dehydrogenase (3R-HSD) in concentrations comparable to indomethacin as standard drug, suggesting antiinflammatory properties for 1-7. 10 Experimental Section General Experimental Procedures. HREIMS were taken with a AMD 402 double-focusing mass spectrometer (AMD Intectra, Harpstedt, Germany). ESIMS was measured with a Quattro triple quadrupole instrument (VG Biotech, Altrin- chem, England) and HRESIMS with MAT 95 XL (Finnigan, Bremen, Germany). IR spectra were recorded on a Shimadzu IR-470 spectrophotometer. 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE DRX 500 spectrometer using TMS as internal standard. Optical rotations were measured with a Propol instrument (Dr. Kernchen Optical Works, Seelze, Germany). Melting points are uncorrected. Organism. The fruiting body of Ganoderma colossum was characterized by the following morphological features: basid- iocarps annual, up to 35 cm diameter, 5-8 cm thickness; context structure spongy, white when fresh, chamoid when dried; basidiospore ovoid, yellow, bitunicate; space well struc- ture has reticulation clearly; basidiospore size 9-17 × 14-20 μm. A specimen was deposited in the fungal culture collection of the Mycological Center, University Hanoi, Vietnam. Extraction and Isolation. The fruiting body (200 g wet weight) was extracted with 1 L of CHCl 3/MeOH and, subse- § Dedicated to Prof. Gu ¨ nter Adam on the occasion of his 65th birthday. * To whom correspondence should be addressed. Tel: (+49) (3641) 656700. Fax: (+49) (3641) 656705. E-mail: UGRAEFE@pmail.hki-jena.de. † Hans-Kno ¨ll-Institute. ‡ Mycological Center. 236 J. Nat. Prod. 2001, 64, 236-239 10.1021/np000437k CCC: $20.00 © 2001 American Chemical Society and American Society of Pharmacognosy Published on Web 01/19/2001