Exiguamide, a New Spirocyclic Sesquiterpene from the Marine Sponge Geodia exigua that Inhibits Cell Fate Specification During Sea Urchin Embryogenesis Mylene M. Uy, a Shinji Ohta, b, * Mihoko Yanai, b Emi Ohta, b Toshifumi Hirata a and Susumu Ikegami c, * a Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan b Instrument Center for Chemical Analysis, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan c Department of Applied Biochemistry, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan Received 24 June 2002; accepted 9 August 2002 Abstract—A new nitrogen-containing bicyclic spirosesquiterpene designated exiguamide which inhibited cell fate specification dur- ing sea urchin embryogenesis has been isolated from the marine sponge Geodia exigua. Its structure was determined by interpreta- tion of spectral data and X-ray crystallographic analysis. # 2002 Elsevier Science Ltd. All rights reserved. During the early development of sea urchins, the cell division synchrony in all blastomeres lasts only until the third cleavage. In the fourth cleavage, the four cells of the animal tier split meridionally into eight blastomeres, each having the same volume. The vegetal tier, however, undergoes an unequal equatorial cleavage to produce four large cells, the macromeres, and four smaller micromeres at the vegetal pole. Micromeres give rise to spiculogenetic primary mesenchyme cells at later developmental stages. The sea urchin embryo provides an excellent model system to study the molecular mechanisms of cell fate specifica- tion. This report describes the purification and the structure elucidation of a new nitrogen-containing bicyclic spiro- sesquiterpene designated exiguamide (1) which is capable of inhibiting the formation of micromeres without affecting cell division thereby producing spicule-deficient larvae. The marine sponge Geodia exigua Thiele (order Astro- phorida, family Geodiidae; 160 g, wet weight) collected off Oshima, Kagoshima Prefecture, Japan in July 2001, was cut into small pieces and steeped in MeOH. The concentrated MeOH extracts were partitioned between water and hexane. The bioactive hexane-soluble fraction (1.1 g) was subjected to ODS column chromatography using 0–100% MeOH in H 2 O as eluent. The bioactive fraction was purified by silica gel column chromato- graphy using 0–100% ethyl acetate in hexane as eluent, followed by crystallization from aqueous MeOH to furnish 1 (8.0 mg; 0.005% wet weight) as colorless crys- tals, mp 139–140  C, [a] D 25 +31.7  (c 0.08, CHCl 3 ). The (+)-FABMS and ()-FABMS data of 1 exhibited pseudomolecular ion peaks at m/z 250 and m/z 248 corresponding to [M+H] + and [MH]  , respectively. The molecular formula of 1 was established to be C 16 H 27 NO on the basis of high-resolution FABMS data (m/z 250.2174 [M+H] + , Á +0.3 mmu). The IR spec- trum displayed absorption bands at 3323 (NH) and 1657 cm 1 (amide and C¼C). Inspection of the 1 H and 13 C NMR spectra (Table 1) together with DEPT and HMQC spectral data revealed the presence of three ali- phatic methyls, an olefinic methyl, four aliphatic methy- lenes, four aliphatic methines, an olefinic methine, a formyl and two quaternary carbons. 1 H– 1 H COSY corre- lations between the NH proton and both H-6 and H-16 provided evidence of the occurrence of the formylamino 0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(02)00662-5 Bioorganic & Medicinal Chemistry Letters 12 (2002) 3037–3039 *Corresponding author. Tel.: +81-824-24-7487; fax: +81-824-24- 7486; e-mail: ohta@sci.hiroshima-u.ac.jp