Negombins A-I, New Chlorinated Polyfunctional Diterpenoids from the Marine Sponge Negombata Species Amira Rudi, ² Yehuda Benayahu, ‡ and Yoel Kashman* ,² School of Chemistry, Raymond and BeVerly Sackler Faculty of Exact Sciences and Department of Zoology, Tel AViV UniVersity, Ramat AViV Tel AViV 69978, Israel kashman@post.tau.ac.il Received March 21, 2007 ABSTRACT Nine novel compounds designated negombins A-I (1-9) were isolated, together with latrunculin, from the Tanzanian sponge Negombata sp. The nine are sacculatane type diterpenes, previously only known from liverworts. The structures of the compounds were elucidated by interpretation of MS and 1D and 2D NMR spectra. A possible biogenesis initiated by the naturally rare chloronium ion is suggested, possibly hinting to a guest microorganism as the source of the compounds. Compound 4 is toxic to brine shrimp larvae. In the framework of searching for bioactive compounds from marine invertebrates 1,2 and our long-standing interest in the metabolites of the sponge Latrunculia magnifica 3 (presently Negombata sp.), 4 we investigated three specimens of this sponge collected at Pemba Island, Tanzania 5 The ethyl acetate extract of the freeze-dried sponge (5 g dry weight) was separated by sequential chromatographies on Sephadex LH-20 (eluting with hexane/CHCl3/MeOH 2:1:1) and silica gel (eluting with hexane/ethyl acetate) to afford negombins A-I(1-9) in quantities of 2-12 mg each. The EIMS of 1 6 exhibited a molecular ion [M] + at m/z 352 for which a formula of C 20 H 29 O 3 Cl, with six degrees of unsaturation, was determined by HRMS. The IR (1725, 1708, 1678 cm -1 ) together with the 1 H NMR spectra (δ H 9.56s, 9.70d) suggested the presence of two aldehyde groups. The 1 H NMR and 13 C NMR experiments (Table 1) revealed in addition to the two CHO groups (δc 193.0d, 203.8d), the presence of two trisubstituted double bonds (δc 138.2s, 152.8d, most likely conjugated to a CO, and 123.7d, 132.1s; δ H 7.05q and 5.07t) a hydroxymethine (δc 72.0d, δ H 3.68q) and, in agreement with the MS peak-cluster, a chloromethine group (δc 61.0d, δ H 4.55dd). The above functionalities account for four of the six degrees of unsaturation of 1, suggesting a bicyclic structure for negombin A. The COSY spectrum revealed the presence of three spin systems (a-c) as shown in Figure 1. HMBC correlation, (Table 1 and Figure 1) established the complete planar structure of 1. Key starting points for interpretation of the CH correlations were the ones * To whom correspondence should be addressed. Phone: 972-3-6408419. Fax: 972-3-6409293. ² School of Chemistry. ‡ Department of Zoology. (1) Chill, L.; Rudi, A.; Benayahu, Y.; Schleyer, M.; Kashman, Y. Org. Lett. 2004, 6, 755-758. (2) Sorek, H.; Rudi, A.; Gueta, S.; Reyes, F.; Martin, J. M.; Aknin, M.; Gaydou, E.; Vacelet, J.; Kashman, Y. Tetrahedron 2006, 62, 8838-8843. (3) Spector, I.; Shochet, N.; Kashman, Y.; Groweiss, A. Science 1983, 219 (4584) 493-495. (4) Kelly-Borges, M.; Vacelet, J. Memoris Queensland Museum 1995, 38, 477-503. (5) The red orange sponge is growing on steep reef walls exposed to strong water currents and was collected at a depth of 22-25 m. Voucher specimens (ZMTAU PO 25464-25466) are deposited at the Zoological Museum, Tel Aviv University, Israel. A new collection and comprehensive work is required for identification of the sponge which is close to Sigmosceptrella another genus, besides the Negombata, of the Podospongi- idae family. (6) Negombin A (1), an oil. [R] 20 D -10.8 (c 0.9 CHCl3) (for 1 H and 13 C NMR see Table 1). IR (CHCl3) νmax 3054, 2986, 2036, 1708, 1678, 1272 cm -1 . EIMS m/z 352 [M] + (20), 334 [M - H2O] + (35), 309 (20), 281 (20), 221 (43), 157 (50), 69 (100). HREIMS m/z [M - H2O] + 334.1687 (calcd for C20H27O2Cl, 334.1693). ORGANIC LETTERS 2007 Vol. 9, No. 12 2337-2340 10.1021/ol070633j CCC: $37.00 © 2007 American Chemical Society Published on Web 05/16/2007