ent-Beyerane diterpenoids from the heartwood of Excoecaria parvifolia Mary H. Grace a,1 , Juan A. Faraldos b,1 , Mary Ann Lila a , Robert M. Coates b, * a Department of Natural Resources and Environmental Sciences, University of Illinois at Urbana–Champaign, Urbana, 1201 S Dorner Drive, Urbana, IL 61801, USA b Department of Chemistry, University of Illinois at Urbana–Champaign, 600 South Mathews Avenue, 470 RAL, Box 40, Urbana, IL 61801, USA Received 17 August 2006; received in revised form 6 October 2006 Available online 29 December 2006 Abstract Chromatographic fractionations of the toluene extract of the heartwood of Excoecaria parvifolia collected in Australia resulted in the isolation of 12 beyerane diterpenes (1–12), and the triterpene, lupeol. Four of the isolated diterpenoids (5–7 and 12) have unusual struc- tures: ent-3-oxa-beyer-15-en-2-one, (5); ent-15,16-epoxy-2-hydroxy-19-norbeyer-1,4-dien-3-one (6); methyl ent-2,4-seco-15,16-epoxy-4- oxo-3,19-dinorbeyer-15-en-2-oate (7); and ent-2,17-dihydroxy-19-norbeyer-1,4,15-trien-3-one (12). The structures were established by spectroscopic analyses, NMR data comparisons with similar diterpenes, and chemical correlations. All the diterpenes are assumed to have the same absolute configuration as the co-occurring (+)-stachenol (4). Diosphenol 2 and nor-lactone 5 exhibited significant potency in bioassays for cytotoxic activity against leukemia cells (L1210). Plausible biosynthetic pathways are proposed to explain the origin of the diterpene metabolites. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Excoecaria parvifolia; Euphorbiaceae; Heartwood; Diterpenoids; ent-Beyeranes; Diosphenols; L1210; Cytotoxicity 1. Introduction The Excoecaria genus in the family Euphorbiaceae is comprised of about 40 species, which are distributed throughout tropical Africa, Asia, and northwestern Austra- lia (Wills, 1973; Wiriyachitra et al., 1985). Many plants in this genus are recognized as skin irritants and tumor pro- moters (Erickson et al., 1995). The most widely reported species is the mangrove, Excoecaria agallocha L. the latex and leaves of which may cause blindness, and have long been used as fish poison and poison arrow heads (Ohigashi et al., 1974). The bark and wood of Excoecaria plants have been applied in Thai medicine as a remedy against flatulence (Karalai et al., 1994). In Sri Lanka, the smoke of the burn- ing wood is used in treatment of leprosy, while the root pounded with ginger is an embrocation for swelling hands and feet (Jayaweera, 1980). E. parvifolia J. Muell., an ende- mic species of the wet–dry savannah of northern Australia also known as the Gutta percha tree, has not yet been chem- ically investigated (Maguire and Saenger, 2000). In this paper, we describe the isolation and structure determination of four novel diterpenoids (5–7 and 12, Fig. 1), as well as the partial synthesis of the first three. In addition, the wide-spread triterpene alcohol lupeol (Burns et al., 2000) and the following known ent-diterpe- noids were isolated by chromatographic means: stachenone (1)(Baarschers et al., 1962), nor-dienone 2, nor-keto ester 3, stachenol (4) (Chalmers et al., 1977; Munkombwe et al., 1997), dihydroxy dienone 8, keto acid 9, rhizophorin C(10), and the 6 epimer of 8 (11). The beyerene diosphe- nols 2, 8, and 11 were previously isolated from the heart- wood of the South African species Spirostachys africana (syn. E. africana)(Munkombwe et al., 1997). The nor-keto acid 9 and its methyl ester (3), prepared from 9 and dia- zomethane, were subsequently reported in a later investiga- tion of Spirostachys africana (Munkombwe et al., 1998). Keto ester 3 is herein isolated as a natural product for 0031-9422/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2006.11.010 * Corresponding author. Tel.: +1 217 333 4280; fax: +1 217 244 8024. E-mail address: rmcoates@uiuc.edu (R.M. Coates). 1 These co-authors made approximately equal contributions to the work reported. www.elsevier.com/locate/phytochem Phytochemistry 68 (2007) 546–553 PHYTOCHEMISTRY