Antifungal 3-Butylisocoumarins from Asteraceae-Anthemideae D. Engelmeier, † F. Hadacek,* ,† O. Hofer,* ,‡ G. Lutz-Kutschera, ‡ M. Nagl, ‡ G. Wurz, ‡ and H. Greger † Comparative and Ecological Phytochemistry Department, Institute of Botany, Rennweg 14, A-1030 Vienna, and Institute of Organic Chemistry, Wa ¨ hringerstrasse 38, A-1090 Vienna, University of Vienna, Austria Received March 26, 2003 Seven new naturally occurring 3-butylisocoumarins were isolated and identified from lipophilic extracts of aerial as well as underground organs: corfin (17) and 3′-hydroxycorfin (18) from the roots of Chamaemelum mixtum and (-)-(R)-2′-methoxydihydroartemidin (5), (+)-(S,R)-epoxyartemidin (6a), dracumerin (12), (+)-(R)-(E)-3′-hydroxyartemidin (13), and capillarin isovalerate (20) from various organs of Artemisia dracunculus (tarragon). Furthermore, six known derivatives, artemidiol (7), (E/Z)-artemidin (11), capillarin (19), artemidinol (21), 8-hydroxyartemidin (22), and 8-hydroxycapillarin (23), were obtained. The antifungal activities of all naturally occurring derivatives were determined in a germ-tube inhibition test against a susceptible strain of rice blast fungus Pyricularia grisea. The 3-butyl side-chain is a prerequisite for high activity. Eleven structurally related synthetic derivatives were additionally tested to explore the influence of structural characteristics on activity. Benlate, blasticidin S, kresoxim-methyl, griseofulvin, and the carrot phytoalexin 6-methoxymellein all served as positive controls. The most recent review 1 of isocoumarins listed more than 160 natural products that occur in a wide range of organ- isms, including bacteria, fungi, and plants. Moreover, these compounds were also detected in secretions of termites and ants. 6-Methoxymellein was one of the first derivatives to be isolated from a plant source. This compound gained some attention because it was identified to cause the bitter taste that spoils the quality of stored carrots. 2 In vitro experiments suggested that the formation of 6-meth- oxymellein was actually induced by fungal infection, and subsequently, a number of studies explored its function as a phytoalexin. 3 Polyketide synthases are involved in the biosynthesis of most naturally occurring isocoumarins, e.g., in 6-meth- oxymellein, 4 or in the mycotoxin ochratoxin A. 5 However, isocoumarins isolated from Artemisia capillaris Thunb., A. dracunculus L. (tarragon), and several other genera of the Asteraceae tribe Anthemideae were found to descend from fatty acid precursors on the basis of feeding experiments. 6 Thus, we suggest that these isocoumarins deserve a separate status within isocoumarins as 3-butylisocou- marins. A preliminary TLC bioautography, using Cladosporium herbarum (Pers.: Fries) Link, indicated that extracts of various sources of A. dracunculus as well the only source of Chamaemelum mixtum (L.) All. contained different antifungal 3-butylisocoumarins. These findings and the fact that 6-methoxymellein was already known to form a potent antifungal natural product prompted us to explore the antifungal activities of naturally occurring 3-butylisocou- marins. Previous phytochemical analyses of various sources of the polymorphic A. dracunculus and other closely related species had already led to the identification of different 3-butylisocoumarins. 7,8 Thus, we refocused on various sources of A. dracunculus grown from seed that were obtained from different botanical gardens. HPLC-UV fingerprinting of lipophilic crude extracts intended to detect as much structural variety as possible. As a result, five derivatives (5, 6a, 12, 13, and 20) turned out to represent hitherto unpublished structures. In addition, two further new isocoumarins, 17 and 18, were isolated and identified from the roots of C. mixtum collected in Corfu Island, Greece. Aside from additional hydroxyl groups on carbon atom 5 or 8 on the aromatic ring system (21-23), structural diversity mainly resulted from various transformations of the butyl side-chain. To obtain more information concern- ing the structure-activity relationships of this type of isocoumarins, 11 additional derivatives were synthesized. 9 In bioautography on TLC plates, diffusion often impairs the determination of inhibitory concentrations. 10 Conse- quently, germ-tube inhibition tests were carried out in broth microdilutions to obtain a comparability of results. A susceptible strain of the rice blast fungus Pyricularia grisea (Cooke) Sacc. [teleomorph Magnaporthe grisea (Her- bert) Barr], 11 which has already served as a test organism in our laboratory to detect other antifungal natural prod- ucts, 12 was used to evaluate the antifungal activity of the available 3-butylisocoumarins. Results and Discussion 3-Butylisocoumarins are characterized by typical UV spectra. Modifications of the chromophore system by sub- stituents affect the intensity and position of maxima, in particular between 270 and 320 nm. Additional hydroxy- lation of the aromatic ring causes a bathochromic shift of the maximum in the wavelength range from 340 to 360 nm. 13 These spectroscopic characteristics permit the direct detection of isocoumarins in HPLC-UV analyses. A hexaploid 7 strain of A. dracunculus from the Botanical Garden in Novosibirsk, Russia (AR-789), accumulated a larger amount of compound 12. The UV spectrum could be clearly differentiated from that of the co-occurring isomeric mixture of E/Z-artemidin (11) by a bathochromic shift from 280-305 nm to 300-315 nm. This suggested an extension of the chromophore. 13 Both 11 and 12 occurred as a pair of E/Z-isomers that were largely inseparable by HPLC: the isomers of artemidin (11) were at least partially resolved, and those of 12 eluted as a single peak. Only GC supplied a baseline resolution of the corresponding 2′-(E)- and 2′-(Z)-isomers of 11 and 12. In comparison with * To whom correspondence should be addressed. Tel: +43 1 4277 54072. Fax: +43 1 4277 9541. E-mail: franz.hadacek@univie.ac.at (F.H.). Tel: +43 1 4277 52107. Fax: +43 1 4277 9521. E-mail: otmar.hofer@univie.ac.at (O.H.). † Institute of Botany. ‡ Institute of Organic Chemistry. 19 J. Nat. Prod. 2004, 67, 19-25 10.1021/np0301339 CCC: $27.50 © 2004 American Chemical Society and American Society of Pharmacognosy Published on Web 12/12/2003