Antineoplastic Agents. 380. Isolation and X-ray Crystal Structure Determination of Isoaaptamine from the Republic of Singapore Hymeniacidon sp. and Conversion to the Phosphate Prodrug Hystatin 1 1 George R. Pettit,* ,† Holger Hoffmann, † James McNulty, † Kerianne C. Higgs, † Alison Murphy, † David J. Molloy, † Delbert L. Herald, † Michael D. Williams, † Robin K. Pettit, † Dennis L. Doubek, † John N. A. Hooper, ‡ Leslie Albright, § Jean M. Schmidt, † Jean-Charles Chapuis, † and Larry P. Tackett † Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-2404, Sessile Marine Invertebrates, Queensland Museum, South Brisbane, Queensland 4101, Australia, and Department of Biology, Western Oregon State University, Monmouth, Oregon 97361 Received October 2, 2002 By use of bioassay (murine P388 lymphocytic leukemia cell line) guided isolation procedures, extracts of the Republic of Singapore marine sponge Hymeniacidon sp. were found to contain demethyl- oxyaaptamine (1) and aaptamine (3) as prominent cancer cell growth inhibitory constituents accompanied by the trace, albeit more active, component isoaaptamine (4). The isolation, X-ray structure elucidation, and antineoplastic and antimicrobial activities of isoaaptamine (4) have been summarized. Because of instability, isoaaptamine (4) was converted to a stable sodium phosphate prodrug designated hystatin 1 (7). Marine Porifera continue to be a rapidly expanding source of new drug candidates 2,3 with unprecedented structures 4-6 and very important biological activities. 7,8 From the onset 6 of our investigations directed at exploring marine organisms as sources of new anticancer drug candidates, we have continued to evaluate marine sponge constituents. 6-8 In 1992, during our marine invertebrate survey in the Republic of Singapore, we located a previously unknown species of sponge belonging to the genus Hyme- niacidon (order Halichondrida) that yielded methanol- dichloromethane extracts active against the murine P388 lymphocytic leukemia cell line. A 500 kg (wet wt) re- collection of the organism was employed to isolate the cancer cell line inhibitory components utilizing the P388 cell line to guide the separations. The sponge methanol-water and methanol-dichlo- romethane extracts (see Experimental Section) were par- titioned between water and dichloromethane. The dichlo- romethane phase was partitioned between hexane and methanol-water (9:1) to give polar fraction A. The initial aqueous phase was next extracted with ethyl acetate followed by 1-butanol to yield alcohol-soluble fraction B (1.08 kg). A series of Sephadex LH-20 gel permeation and partition chromatographic steps were used to separate fraction A and led to the previously known demethy- loxyaaptamine (1) 9 and the dimethyl ketal 2, likely an artifact of the isolation process. 9 Demethyloxyaaptamine (1) was isolated in gram quantities and exhibited a P388 ED 50 of 0.31 µg/mL, while the much less abundant dimethyl ketal 2 was inactive (P388 ED 50 59 µg/mL). Fraction B was separated, and aaptamine (3) 10a was also isolated in gram quantities along with the minor (10 -5 % yields) constituent isoaaptamine (4), 11 each as the hydro- chloride salt. The cancer cell growth inhibitory activities of compounds 1, 3, and 4 are summarized in Table 1. The structures of compounds 1, 2, and 3 were readily deduced from spectral properties and literature compari- sons. 9,10a However, the structure of compound 4 proved to be troublesome. Analysis of the 1 H and 13 C NMR spectra indicated that it was nearly identical to isoaaptamine (4), which was first reported by Fedoreev. 11a Subsequently, it was again isolated by Kashman 11b and Shen. 11c In our experiments, all of the proton and carbon signals were shifted downfield slightly owing to its isolation as the * To whom correspondence should be addressed. Tel: 480-965-3351. Fax: 480-965-8558. † CRI. We dedicate this contribution to the memory of Dr. Harry B. Wood (July 30, 1919-August 7, 2002), an outstanding pioneer of anticancer drug development. ‡ Queensland Museum. § Western Oregon State University. 506 J. Nat. Prod. 2004, 67, 506-509 10.1021/np0204592 CCC: $27.50 © 2004 American Chemical Society and American Society of Pharmacognosy Published on Web 02/28/2004