New Sesquiterpene Carbonimidic Dichlorides and Related Compounds from the Sponge Stylotella aurantium Musri Musman, Junichi Tanaka, and Tatsuo Higa* Department of Chemistry, Biology, and Marine Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan Received July 5, 2000 Five new sesquiterpenes (1-5) having a carbonimidic dichloride or an aldehyde function have been isolated, together with seven known related compounds (6-12), from the sponge Stylotella aurantium. The structures of the new compounds were elucidated from spectral data. The absolute stereochemistry of the previously reported reticulidin A (10) was determined. Four of the new compounds showed cytotoxicity with a range of IC 50 values of 0.1-1 µg/mL against several tumor cell lines. We recently reported the isolation and structure elucida- tion of two new sesquiterpene carbonimidic dichlorides (8, 10) from the nudibranch Reticulidia fungia. 1 Inasmuch as related compounds have earlier been described only from a few species of sponges, 2-6 it was evident that the nudibranch constituents originated from a sponge. How- ever, we were unable to locate a plausible species in the vicinity of the nudibranch collection site on Irabu Island, Okinawa. More recently we examined cytotoxic constitu- ents of a sponge collected from a coral reef of Iriomote Island, located 150 km west of Irabu, and later identified as Stylotella aurantium, the same species that yielded carbonimidic dichlorides in Australia. 6 Our sample also gave sesquiterpene carbonimidic dichlorides, including five new congeners (1-5), which were responsible for the cytotoxicity of the lipophilic extract of the sponge. As described earlier, 1 our interest in these compounds is two- fold: their biological activity, such as cytotoxicity, 1 and biogenesis of the functionality. 6 In this note we describe the isolation and structures of the new members having this functionality (1, 4, 5) and two new related compounds (2, 3). A sample (90 g, wet wt) of S. aurantium Kelly-Borges and Bergquist (family Axinellidae) was extracted with acetone, and the concentrated extract was partitioned between ethyl acetate and water. The EtOAc extract (0.65 g) was separated on Si gel followed by preparative TLC and/or HPLC to afford sesquiterpenes 1-12 in yields ranging from 1.5 to 12.0 mg. Compound 1,[R] D +4.5° (c 0.20, CHCl 3 ), was isolated as a colorless oil. The molecular formula C 16 H 23 NCl 4 was determined by observing a molecular ion at m/z 369 in ESIMS and by HREIMS at m/z 334.0870 ([M - Cl] + ). The presence of a carbonimidic dichloride functional group was inferred from a carbon signal at δ 127.0 s and also by IR absorption at 1655 cm -1 , as reported earlier. 2-6 The 1 H and 13 C NMR spectra of 1 indicated the presence of two exomethylenes [δ 4.66 s, 4.95 s, 5.07 s, 5.19 s; δ 109.2 t (C-14), 114.4 t (C-15), 145.6 s (C-6), 146.3 s (C-9)], two chlorine-bearing methines [δ 3.90 dd, 4.60 t; δ 62.2 d (C-10), 70.9 d (C-3)], a methylene bearing a nitrogen [δ 3.84 d; δ 59.3 t (C-11)], and two methyls [δ 0.84 s, 1.16 s; δ 15.9 q (C-12), 27.2 q (C-13)]. These data, together with the unsaturation requirement, suggested 1 to be monocyclic. Connectivity was made by interpreting 2D NMR (COSY, HMQC, HMBC) spectra. The presence of a six-membered ring was shown by COSY (H-1R/H-2R, H-2R/H-3, H-1R/ H-5, H-1R/H-14a, H-1/H-14b, and H-5/H-14ab) and HMBC data (H-1R/C-2,-3,-5,-6,-14, H-2R/C-1,-3,-4,-6, H-3/C-4,- 12,-13, H-5/C-3,-4,-6,-8,-14, and H 3 -12,-13/C-3,-4,-5). Con- nectivity between the ring and the terminal carbonimidic dichloride was also made by COSY (H-5/H-7ab,-8ab; H-7ab/ H-8ab, H-8ab/H-15ab, H-10/H-11,-15b) and HMBC (H-10/ C-15, H 2 -11/C-16) cross-peaks. Relative stereochemistry in the ring was elucidated as shown by observing positive NOEs (H-3/H-5, H-3/H 3 -12, H-7/H 3 -13). The stereochem- istry at C-10 remains to be solved. Compound 2,[R] D +16° (c 0.13, CHCl 3 ), was obtained as a colorless glass. EIMS of 2 showed a molecular ion at m/z 270. High-resolution measurement of this peak gave a molecular formula C 15 H 23 ClO 2 . The formula indicated four degrees of unsaturation. The NMR spectra displayed sig- nals for three methyl singlets [δ 1.11 (H 3 -12), 1.11 (H 3 -13), 1.14 (H 3 -11); δ 17.8 q, 23.9 t, 30.4 q], R,-unsaturated aldehyde [δ 10.02 d; δ 127.5 d (C-14), 163.5 s (C-8), 190.1 d (C-15)], and two methines bearing a chlorine and/or a hydroxyl [δ 3.93 d, 4.18 q; δ 71.9 d (C-2), 76.3 d (C-3)]. The * To whom correspondence should be addressed. Tel.: 81-98-895-8558. Fax: 81-98-895-8538. E-mail thiga@sci.u-ryukyu.ac.jp. 111 J. Nat. Prod. 2001, 64, 111-113 10.1021/np0003342 CCC: $20.00 © 2001 American Chemical Society and American Society of Pharmacognosy Published on Web 12/08/2000