Cadiolides A and B, New Metabolites from an Ascidian of the Genus Botryllus Cameron J. Smith, † Robert L. Hettich, ‡ Jamaluddin Jompa, § Akbar Tahir, § Michelle V. Buchanan, ‡ and Chris M. Ireland* ,† Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112-9453, Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6365, and Faculty of Marine, and Fisheries Sciences University of Hasanuddin, Ujung Pandang, Indonesia Received January 30, 1998 Marine ascidians are prolific producers of metabolites derived from amino acids. 1 Metabolites produced from phenylalanine or tyrosine are especially plentiful and include the tunichromes, 2 lamellarins, 3 the lukianols, 4 rigidin, 5 the polycitrons, 6 the ningalins 7 and the botryl- lamides. 8 The rubrolides, 9 isolated from Ritterella rubra, are examples of non-nitrogenous members of this group. We now wish to report the isolation of cadiolides A (1) and B (2) from an Indonesian Botryllus sp. The cadi- olides do not contain nitrogen and are related in structure to the rubrolides, but possess a novel carbon skeleton. Specimens of Botryllus sp. were collected by hand using scuba (-3 to -15 m) near Barrang Caddi in Indonesia and kept frozen until workup. Thawed samples of Botryllus sp. were homogenized and extracted with either EtOH or MeOH to give a crude extract. This extract was partitioned between hexane, CHCl 3 , and aqueous MeOH. The aqueous MeOH soluble material was further frac- tionated by size-exclusion chromatography using Sepha- dex LH-20 (MeOH), resulting in the separation of a number of highly colored bands including yellow, orange, red, and black. Samples were inspected by TLC and 1 H NMR spectroscopy and purified further by gradient silica flash chromatography (0-10% MeOH in CHCl 3 ) to give rubrolide A (3) and cadiolides A (1) and B (2) as amorphous solids. Cadiolide A (1) was obtained as an orange amorphous solid that yielded an abundant ion cluster in both the negative ion ES-MS and MALDI-MS spectra centered at m/z 715 (M - H) - . The positive ion MALDI-MS spectrum also showed an abundant ion cluster, this time centered at m/z 717 (M + H) + . This isotopic packet corresponded to the presence of four bromine atoms (Figure 1, the top portion is the measured mass spectrum, and the bottom portion is the calculated mass spectrum for the proposed ion C 24 H 13 Br 4 O 6 + ). Note the excellent agreement in the relative abundances of the isotopic peaks. The most abundant ion was measured to be m/z 716.7398, which corresponds within 0.4 ppm to the most abundant calculated ion for C 24 H 13 79 Br 2 81 Br 2 O 6 + at m/z 716.7401. This is consistent with the molecular formula C 24 H 12 79 Br 2 81 Br 2 O 6 possessing 17 degrees of unsaturation. The 13 C NMR spectrum contained only 18 resonances, implying that there was some element(s) of symmetry present in a highly unsaturated aromatic structure. Only six proton resonances, all with chemical shifts greater that δ 6, were observed in the 1 H NMR spectrum, and integration showed that four of the signals accounted for two protons each (δ 6.88, 7.55, 7.76, and 7.89), one for a single proton (δ 6.33) and one exchangeable signal accounted for three protons (δ 10.41). The UV spectrum showed absorptions at 254 and 406 nm, demonstrating the aromaticity and extended conjugation of 1. The IR spectrum showed absorptions at 1721 and 1683 cm -1 . This, coupled with carbon resonances at δ 152.4, 157.4, 159.8, 165.5 and 185.0, indicated the presence of a number of aromatic hydroxyl groups and conjugated carbonyl groups. The broad three-proton resonance at δ 10.26 in the 1 H NMR spectrum was assigned to three exchangeable phenolic protons. An HMQC experiment established the proton and carbon one-bond connectivities in the struc- ture. In particular, correlations were observed between carbons resonating at δ 116.2, 132.9, 133.5, and 133.7 and protons resonating at δ 6.88, 7.55, 7.76, and 7.89, respectively. This coupled with the observation of three- bond proton-carbon connectivities in an HMBC experi- ment between carbon resonances at δ 116.2, 132.9, 133.5, and 133.7 and proton resonances at δ 6.88, 7.55, 7.76, and 7.89, respectively, suggested the presence of a series * To whom correspondence should be addressed. Phone: (801) 581 8305. FAX: (801) 585 6208. E-mail: cireland@deans.pharm.utah.edu. † University of Utah. ‡ Oak Ridge National Laboratory. § Faculty of Marine and Fisheries Sciences, University of Hasanud- din. (1) Davidson, B. S. Chem. Rev. 1993, 93, 1771. (2) Bruening, R. C.; Olitz, E. M.; Furukawa, K.; Nakanishi, K.; Kustin, K. J. Am. Chem. Soc. 1985, 107, 5298. (3) (a) Andersen, R. J.; Faulkner, D. J.; Cun-heng, H.; Van Duyne, G. D.; Clardy, J. J. Am. Chem. Soc. 1985, 107, 5492. (b) Lindquist, N.; Fenical, W.; Van Duyne, G. D.; Clardy, J. J. Org. Chem. 1988, 53, 4570. (4) Yoshida, W. Y.; Lee, K. K.; Carroll, A. R.; Scheuer, P. J. Helv. Chim. Acta 1992, 75, 1721. (5) Kobayashi, J.; Cheng, J.-F.; Kikuchi, Y.; Ishibashi, M.; Yama- mura, S.; Ohizumi, Y.; Ohta, T.; Nozoe, S. Tetrahedron Lett. 1990, 31, 4617. (6) Rudi, A.; Goldberg, I.; Stein, Z.; Frolow, F.; Benayahu, Y.; Schleyer, M.; Kashman, Y. J. Org. Chem. 1994, 59, 999. (7) Kang, H.; Fenical, W. J. Org. Chem. 1997, 62, 3254. (8) McDonald, L. A.; Swersey, J. C.; Ireland, C. M.; Carroll, A. R.; Coll, J. C.; Bowden, B. F.; Fairchild, C. R.; Cornell, L. Tetrahedron 1995, 51, 5237. (9) Miao, S.; Anderson, R. J. J. Org. Chem. 1991, 56, 6275.