Sequestered Chemistry of the Arminacean Nudibranch Leminda millecra in Algoa Bay, South Africa Kerry L. McPhail, † Michael T. Davies-Coleman,* ,† and John Starmer ‡ Department of Chemistry, Rhodes University, Grahamstown, South Africa, and P.O. Box 3035 Koror, Palau 96940 Received February 23, 2001 Extracts of the endemic nudibranch Leminda millecra collected in Algoa Bay, South Africa, yielded four known metabolites, millecrones A (1) and B (2), isofuranodiene (5), and (+)-8-hydroxycalamenene (9), and nine new compounds, algoafuran (7), cubebenone (8), and a series of seven triprenylquinones and hydroquinones (21-27). A subsequent GC-MS survey of extracts of 21 of L. millecra’s possible octocoral prey species in Algoa Bay identified the soft coral Alcyonium fauri as the source of 1 and the gorgonian Leptogorgia palma as the species producing 2 and 8. The endemic South African nudibranch Leminda mille- cra Griffiths, 1985 (family Lemindidae, suborder Armina- cea) is a translucent pink nudibranch with a blue-edged mantle that is expanded into well-developed parapodia. This species, lacking external gills or cerata and possessing a distinct internal morphology, is the single representative of a relatively new family of Arminacean nudibranchs. 1 L. millecra is reported to occur from the Cape Peninsula to Kwazulu Natal, 1 and we have observed this species to be particularly abundant at depths of 20-40 m, feeding on octocorals (mostly gorgonians), in Algoa Bay, near the coastal city of Port Elizabeth. In their earlier chemical study of L. millecra collected from the Transkei coast (500 km northeast of Algoa Bay), Pika and Faulkner 2 provided the first conclusive evidence of an octocoral diet for this species. Spicules characteristic of the soft corals Alcyonium foliatum, A. valdiviae, and Capnella thyrsoidea were found in the nudibranch’s gut, while the skeletal structures of the sequestered metabolites, millecrones A (1) and B (2) and the millecrols A (3) and B (4), were suggestive of their octocoral origin. 2 Unfortunately, Pika and Faulkner’s in- vestigation was hampered by a paucity of nudibranch material, and they noted the presence of three minor metabolites in their L. millecra extracts in insufficient amounts for structure elucidation. 2 Therefore, given the abundance of L. millecra in Algoa Bay and our ongoing interest in identifying bioactive metabolites sequestered by Southern African nudibranchs and sea hares, 3 we present here the results of our investigation of the sequestered chemistry of L. millecra in Algoa Bay. Results and Discussion A total of 32 specimens of L. millecra were collected using scuba from several reefs in Algoa Bay in October 1998 and again in February 1999. The two collections of L. millecra were independently steeped in acetone and the acetone extracts concentrated and partitioned between EtOAc and water. The 1 H NMR spectra of the two EtOAc fractions were very similar, and they were consequently combined (1.78 g) and chromatographed over Si gel using a hexane/ EtOAc solvent gradient. Of the seven initial chromatogra- phy fractions thus obtained, the three major fractions [1 (322 mg), 3 (440 mg), and 4 (373 mg)] were adjudged, from the plethora of methyl, oxymethine, and olefinic proton resonances in their 1 H NMR spectra, to be worthy of further investigation. Additional chromatography over Si gel followed by normal-phase HPLC of the nonpolar fraction 1 yielded two known compounds: millecrone A (93 mg, 2.9 mg/animal), spectroscopically identical ( 1 H, 13 C, IR, and [R] D ) to 1 isolated previously from the Transkei specimens of L. millecra, 2 and isofuranodiene (5, 11.0 mg, 0.3 mg/animal). 4 The bicyclic structure of 5 was indicated from the six degrees of unsaturation implied by the molecular formula C 15 H 20 O, established from HRFABMS data, and the pres- ence of eight olefinic resonances in the 13 C NMR spectrum of this compound. Four of the latter 13 C resonances [δ 149.7 (s), 136.0 (d), 121.9 (s), and 118.9 (s)], together with an aromatic proton singlet (δ 7.06) in the 1 H NMR spectrum of 5, confirmed the presence of a furan moiety and led us to the structure of isofuranodiene. 4 Although our 1 H NMR data were almost identical to those reported for isofura- nodiene and furanodiene (6), 5 two vinyl methyl resonances (δ 16.2 and 16.5) in the 13 C NMR spectrum of 5 were consistent with an E configuration for both ring olefins as reported for isofuranodiene. 4 Interestingly, Bowden et al. 4 have proposed that the E,Z configuration of furanodiene, initially suggested three decades ago without recourse to 13 C NMR data, 5 is incorrect and that 5 and 6 are the same compound from a comparison of the vinyl methyl proton NMR chemical shifts of 5 with those published for 6 (Δδ ( 0.02 ppm). Bicyclic, fused furanodecane metabolites appear to be confined to octocorals and three, double-bond posi- tional isomers of 5 have been isolated from Xenia soft corals, 4 the Nephtheidae soft coral Lemnalia africana, 6 and one Pseudopterogorgia species. 7 Millecrone B (2, 19 mg, 0.6 mg/animal) was obtained as one of the major compounds in initial chromatography fraction 3 after exhaustive normal-phase HPLC of this fraction. The spectroscopic and physical data of millecrone B were identical with those of 2 isolated during the earlier investigation of L. millecra. 2 In addition to 2, fraction 3 yielded two new sesquiterpenes, algoafuran (7, 1.5 mg, 0.05 mg/animal) and cubebenone (8, 129 mg, 4 mg/animal), and the known 8-hydroxycalamenene (9, 16 mg, 0.5 mg/animal). HRFABMS data revealed the molecular formula of C 17 H 22 O 2 (274.15671, Δ mmu +0.2) for algoafuran (7), which, in conjunction with standard acetate NMR signals (δ C 171.1 and δ Η 2.07), suggested that 7 was a sesquiter- pene monoacetate. The presence of the single acetate * To whom correspondence should be addressed. Tel: +27 46 603 8264. Fax: +27 46 6225109. E-mail: M.Davies-Coleman@ru.ac.za. † Department of Chemistry, Rhodes University. ‡ P.O. Box 3035 Koror, Palau. 1183 J. Nat. Prod. 2001, 64, 1183-1190 10.1021/np010085x CCC: $20.00 © 2001 American Chemical Society and American Society of Pharmacognosy Published on Web 09/05/2001