Axinellamines A-D, Novel Imidazo-Azolo-Imidazole Alkaloids from the Australian Marine Sponge Axinella sp. Sylvia Urban, † Priscila de Almeida Leone, † Anthony R. Carroll, † Gregory A. Fechner, † Jill Smith, † John N. A. Hooper, ‡ and Ronald J. Quinn* ,† Queensland Pharmaceutical Research Institute, Griffith University, Brisbane, Queensland 4111, Australia, and Queensland Museum, South Brisbane, Queensland 4101, Australia Received June 1, 1998 Four imidazo-azolo-imidazole alkaloids, axinellamines A-D, have been isolated from an Australian marine sponge, Axinella sp. (order: Halichondrida: family: Axinellidae). These compounds contain a unique perhydrocyclopenta-imidazo-azolo-imidazole carbon skeleton. Three of these compounds had bactericidal activity against Helicobacter pylori at 1000 µM. Introduction Bromopyrrole-containing alkaloids such as hymenidin and oroidin have been isolated from sponges of the genera Axinella and Agelas. 1-3 More complex bromopyrroles which also contain guanidine moieties such as 2,3- dibromostyloguanidine and dimers such as dibromoag- eliferin acetate have been isolated from Stylotella au- rantium 4 and Agelas conifera, 5,6 respectively. In this report the isolation, characterization, and identification of four imidazo-azolo-imidazole bromopyrrole-contain- ing metabolites, axinellamines A-D (1-4) from an Australian marine sponge, Axinella sp., is described. Axinellamines B-D(2-4) had bactericidal activity against Helicobacter pylori, a gram negative bacterium associated with pepticular and gastric cancer, at 1000 µM. Results and Discussion Bioassay guided fractionation of the crude methanol extract of Axinella sp. revealed that fractions inhibiting the growth of H. pylori consistently had ion peaks at m/z 846 and 860 in the electrospray mass spectrum (ESIMS). The application of LC/positive ESIMS, using reverse- phase C18 HPLC, allowed chromatographic conditions to be established. A larger scale purification of the crude methanol extract was successfully achieved by gel per- meation chromatography (LH-20 Sephadex, using metha- nol as the eluant) followed by reverse-phase HPLC (using 1% trifluoroacetic acid (TFA) with acetonitrile/H 2 O gra- dients), resulting in the isolation of axinellamines A-D (1-4). The structures of axinellamines A-D(1-4) were deduced from 1D and 2D NMR data (Tables 1, 2, and 4) and MS analysis. The positive electrospray mass spectra of 1 and 2 displayed clusters of ion peaks [(M + H) - 2CF 3 COOH] + at m/z 842/844/846/848/850/852, while the negative electrospray mass spectra displayed clusters of molecular ion peaks [M - H] - at m/z 1069/1071/1073/ 1075/1077/1079, which was consistent with a ClBr 4 isotope pattern. The positive electrospray mass spectra of 3 and 4 displayed clusters of ion peaks [(M + H) - 2CF 3 COOH] + at m/z 856/858/860/862/864/866, while the negative electrospray mass spectra displayed clusters of molecular ion peaks [M - H] - at m/z 1083/1085/1087/ 1089/1091/1093, which were also consistent with a ClBr 4 isotope pattern with the incorporation of an additional 14 mass units compared with 1 and 2. Axinellamines A (1) and B (2) were thus isomeric, as were axinellamines C(3) and D (4), and were deduced to have the molecular formulas C 22 H 23 Br 4 ClN 10 O 4 ‚2CF 3 COOH for 1 and 2 and * To whom correspondence should be addressed. Tel.: +61 7 3849 1366. Fax: +61 7 3849 1292. E-mail: R.Quinn@qpri.gu.edu.au. † Griffith University. ‡ Queensland Museum. (1) Kobayashi, J.; Ohizumi, Y.; Nakamura, H.; Hirata, Y. Experentia 1986, 42, 1176-1177. (2) Kobayashi, J.; Ohizumi, Y.; Nakamura, H.; Hirata, Y.; Waka- matsu, K.; Miyazawa, T. Experentia 1986, 42, 1064-1065. (3) Walker, R. P.; Faulkner, D. J.; Van Engen, D.; Clardy, J. J. Am. Chem. Soc. 1981, 103, 6772-6773. (4) Keifer, P. A.; Schwartz, R. E.; Koker, M. E. S.; Hughes, R. G., Jr.; Rittschof, D.; Rinehart, K. L. J. Org. Chem. 1991, 56, 2965-2975. (5) Kato, T.; Shizuri, Y.; Izumida, H.; Yokoyama, A.; Endo, M. Tetrahedron Lett. 1995, 36, 2133-2136. (6) Williams, D. H.; Faulkner, D. J. Tetrahedron 1996, 52, 5381- 5390. 731 J. Org. Chem. 1999, 64, 731-735 10.1021/jo981034g CCC: $18.00 © 1999 American Chemical Society Published on Web 01/14/1999