Calbistrin E and Two Other New Metabolites from an Australian Isolate of Penicillium striatisporum Michael Stewart, † Robert J. Capon,* ,† Ernest Lacey, ‡ Shaun Tennant, ‡ and Jennifer H. Gill ‡ Centre for Molecular Biodiversity, Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland 4072, Australia, and Microbial Screening Technologies Pty. Ltd., Building A 28-54 Percival Road, Smithfield, New South Wales 2164, Australia Received November 29, 2004 An Australian isolate of Penicillium striatisporum collected near Shalvey, New South Wales, exhibited selective antifungal activity against Candida albicans versus Saccharomyces cerevisiae. Bioassay-directed fractionation yielded members of the rare class of fungal metabolites known as the calbistrins. These included a new example of this structure class, calbistrin E (1), as well as the known polyenes calbistrin C(2) and deformylcalbistrin A (3). Also recovered from P. striatisporum were new triene and butenolide acids, striatisporin A (4) and striatisporolide A (5), together with the known fungal metabolites versiol (6) and (+)-hexylitaconic acid (7). Structures for all metabolites were determined by detailed spectroscopic analysis. During a search for novel bioactive fungal metabolites we examined an unusual Penicillium species (MST-F9530) isolated from a construction site in the western suburbs of Sydney, Australia. Identified as P. striatisporum, this strain had not previously been isolated in Australia, and furthermore, published accounts of its chemistry were limited to a single 1994 Japanese patent, 1 which reported a pair of neoplasm inhibitors for which molecular struc- tures were not assigned. It is noteworthy that during the taxonomic upheavals that have characterized the genus Penicillium, P. striatisporum was at one time regarded as a synonym for P. restrictum. 2 Although now designated as a distinct species, 3 P. restrictum has been reported to produce two unrelated classes of antifungal polyenes, the calbistrins 4 and the restricticins, 5-7 as well as azaphilone- type antibiotics with angiotensin II inhibitory activity. 8,9 Whereas Penicillium species can be difficult to identify without extensive growth and media studies, 10 P. striatisporum can be readily differentiated by the unusual striated ornamentation on the spore surface. 11 In our hands P. striatisporum displayed selective antifungal activity against Candida albicans compared with Saccharomyces cerevisiae, a characteristic that proved to be media depend- ent and correlated with levels of secondary metabolite production. The occurrence of a rare Penicillium species with promising bioactivity and metabolite profiles prompted a more detailed chemical investigation. HPLC analysis of the fermentation extract of MST-F9530 indicated the presence of an extensive family of metabolites containing a polyene moiety. The occurrence of a distinctive chromophore, together with the selective antifungal activity against C. albicans, was suggestive of the calbistrins, a rare class of antifungal metabolites first reported in 1993 from P. restrictum. 4,12 Limited to only four known examples, the calbistrins exist as pairs of E/Z isomers about Δ 8′,9′ , with calbistrins A (8) and B (9) being tricyclic acetals, and calbistrins C (2) and D (10) being bicyclic reduced ana- logues. The calbistrins are known to be unstable to light, base, and aprotic solvents, properties that add to the challenge of isolation and characterization. For example, calbistrin A (8) is known to undergo a base-mediated retro- aldol degradation to yield deformylcalbistrin A (3). 13 The complete absolute stereochemistry of calbistrin A (8) (and by inference 2, 3, 9, and 10) was resolved in 1997 by total synthesis. 14 The calbistrins reportedly display potential as cholesterol-lowering agents, 15 as antifungals, 16,17 and as promoters of nerve growth factor production. 18 This current report represents the first reoccurrence of calbistrins in the primary literature since their isolation in 1993 and syn- thesis in 1997 and extends the scope of the calbistrin molecular motif to include a new example, calbistrin E (1), which is a tricyclic dehydro analogue of calbistrin A (8). It also describes the novel polyene diacid striatisporin A (4), which can be viewed as a truncated homologue of the calbistrin side chain, and the new butenolide striatisporolide A(5). A solvent extract obtained from the solid phase culture of Penicillium striatisporum (MST-F9530), sourced from a soil sample collected near Shalvey, New South Wales, Australia, displayed potent antifungal activity against Candida albicans (assay titer 128) but not Saccharomyces cerevisiae (assay titer 0). Bioassay-directed fractionation of this extract employing C 18 SPE and HPLC yielded the known compound calbistrin C (2) (LD 99 50 µg/mL), whereas careful analysis of LC/MS (DAD and ESI) and NMR data on associated fractions provided evidence supportive of the presence of all other known calbistrins (8, 9, and 10). Further fractionation efforts yielded pure samples of de- formylcalbistrin A (3) as well as a new but unstable calbistrin analogue, calbistrin E (1). More polar fractions yielded two calbistrin-like components in the form of the new diacid striatisporin A (4) and the known polyketide versiol (6) 19,20 (LD 99 50 µg/mL), as well as the new buteno- lide striatisporolide A (5) and the known plant growth regulator (+)-hexylitaconic acid (7). 21 Structures were assigned to the known compounds 2, 3, 6, and 7 based on spectroscopic comparisons to literature data. Structure arguments for the new compounds 1, 4, and 5 are detailed below. It is worthwhile noting that prior to being isolated as versiol from Aspergillus versicolor, 19,20 6 was isolated 22,23 and patented 24 as the antifungal agent LL-N313 from Spororamia affinis. High-resolution ESI(+)MS analysis of 1 revealed a pseudomolecular ion (M + Na) supporting a molecular * To whom correspondence should be addressed. Tel: +61 7 3346 2979. Fax: +61 7 3346 2101. E-mail: r.capon@imb.uq.edu.au. † The Institute for Molecular Bioscience. ‡ Microbial Screening Technologies Pty. Ltd. 581 J. Nat. Prod. 2005, 68, 581-584 10.1021/np049614y CCC: $30.25 © 2005 American Chemical Society and American Society of Pharmacognosy Published on Web 03/18/2005