~ Pergamon PII: S0031-9422(97)00897-2 Phytochemistry, Vol. 48, No. 7, pp. 1139 1143, 1998 i' 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0031 9422/98 $19.00+0.00 SPHAEROPSIDONE AND EPISPHAEROPSIDONE, PHYTOTOXIC DIMEDONE METHYL ETHERS PRODUCED BY SPHAEROPSIS SAPINEA F. SP. CUPRESSI GROWN IN LIQUID CULTURE ANTONIO EVIDENTE,* LORENZO SPARAPANO,fOLGA FIERRO,GIOVANNIBRUNO,f FEDERICO GIORDANO + and ANDREAMOTTA§ Dipartimento di ScienzeChimico-Agrarie, Universitfidi Napoli Federico 11, Via Universitfi 100, 80055 Portici, Italy; t Dipartimento di Patologia Vegetale, Universitfidi Bari, Via Amendola 165/A, 70126 Bari, Italy; +Dipartimento di Chimica, Universitfidi Napoli Federico II, Via Mezzocannone 4, 80134 Napoli, Italy; § Istituto per la Chimica di Molecole di lnteresse Biologicodel CNR¶], Via Toiano 6, 80072 Arco Felice~Italy (Received in revisedjbrm 16 September 1997) Key Word Index--Cupressus sempervirens; cypress; Sphaeropsis sapinea f.s. cupressi; cypress canker disease; phytotoxins; tetraketides; dimedone methyl ethers; sphaeropsidone; epi- sphaeropsidone. Abstract--Two phytotoxic dimedone methyl ethers, named sphaeropsidone and episphaeropsidone, were isolated from Sphaeropsis sapinea f. sp. cupressi, a phytopathogenic fungus causing a canker disease of Italian cypress (Cupressus sempervirens L.). The same fungus produced the sphaeropsidins A, B and C, which are three phytotoxins recently chemically characterized as pimarane diterpenes. Sphaeropsidone and epi- sphaeropsidone were characterized, using essentially spectroscopic methods, as two new phytotoxic dis- ubstituted 7-oxabicyclo[4.1.0]hept-3-en-2-ones,which are epimers at C-5. Assayed on severed twigs of cypress, sphaeropsidone caused browning and necrosis on Cupressus macrocarpa, no symptoms on C. sempervirens and chlorosis on C. arizonica. Episphaeropsidone caused necrosis on C. macrocarpa, browning and necrosis on C. sempert,irens and necrosis on C. arizonica. On the non-host plant tomato, both phytotoxins caused wilting. In a microbial assay, both compounds showed an inhibitory effect on the growth of five fungal species tested. The growth of Verticillium dahliae was enhanced by both dimedone methyl ethers. © 1998 Elsevier Science Ltd. All rights reserved INTRODUCTION Sphaeropsis sapinea f.sp. cupressi is a fungal pathogen which causes stem and branch canker of Italian cypress (Cupressus sempervirens L.) in areas limited to the Mediterranean coast. Such a canker differs from Seiridium canker, which is a common disease of cypress in the Mediterranean area [1]. Recently, the main phytotoxin and two toxic struc- turally related metabolites were isolated from culture extracts of S. sapinea f. sp. cupressi. All of them were chemically characterized as pimarane diterpenes, and were named sphaeropsidins A, B and C, respectively. The latter was identified as a new tricyclic acid pim- arane diterpene [1, 2]. Moreover, it was demonstrated for the first time that Diplodia mutila, another ubiqui- tous fungus which induces canker formation, pro- duced sphaeoropsidins A and C [2]. * Author to whom correspondence should be addressed. ¶ Part of the Istituto Nazionale di Chimica dei Sistemi Biologici. The organic extracts of the culture filtrates of S. sapinea f. sp. cupressi contained lower concentrations of at least three other phytotoxic substances. Two of them appeared to be strictly related compounds which were more polar than sphaeropsidins, and from which they were structurally different. This paper describes the isolation, and chemical and biological characterization of new toxic metabolites, which proved to be epimeric dimedone methyl ethers. They were called sphaeropsidone and epi- sphaeropsidone (1 and 2, respectively). The structures and the relative configuration were confirmed by X- ray analysis while the absolute stereochemistry was deduced by circular dichroism and NMR. RESULTS AND DISCUSSION The crude oily residue (1.20 g) obtained by extrac- tion of the culture filtrates (3 1) of S. sapinea f. sp. cupressi with EtOAc was fractionated using a com- bination of CC and prep TLC to yield sphaeropsidins A, B and C and two more polar metabolites (1 and 1139