Ciliatamides A-C, Bioactive Lipopeptides from the Deep-Sea Sponge Aaptos ciliata # Yoichi Nakao,* ,†,‡ Shizuka Kawatsu, † Chikane Okamoto, † Masaaki Okamoto, † Yoshitsugu Matsumoto, † Shigeki Matsunaga, † Rob. W. M. van Soest, § and Nobuhiro Fusetani* ,⊥ Graduate School of Agricultural and Life Sciences, The UniVersity of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan, Institute for Systematics and Ecology, UniVersity of Amsterdam, 1090 GT Amsterdam, The Netherlands, and Graduate School of Fisheries Sciences, Hokkaido UniVersity, 3-1-1, Minato-cho, Hakodate, 041-8611, Japan ReceiVed January 15, 2008 Three lipopeptides, ciliatamides A-C(1–3), were isolated from the deep-sea sponge Aaptos ciliata, and their structures were elucidated on the basis of spectroscopic and chemical methods. Ciliatamides A (1) and B (2) were found to be antileishmanial, while 2 also exhibited marginal cytotoxicity to HeLa cells. Leishmaniasis is a disease caused by an obligate intracellular parasite of the genus Leishmania and includes several types of disease ranging from self-healing ulcers to fatal visceral leishma- niasis. 1 Pentavalent antimonials are the most common drugs for the treatment of leishmaniasis, 2 but have cardiotoxic effects. Therefore, alternative treatments are urgently required. In the course of a drug discovery project from Japanese marine invertebrates, we have reported the isolation of renieramycin A from the sponge Neopetrosia sp. as a potential antileishmanial agent. 3 Subsequently, we found significant antileishmanial activity in the organic extract of the deep-sea sponge Aaptos ciliata. Fractionation of the extract yielded three new lipopeptides, named ciliatamides A-C(1-3). Herein, we report the isolation, structure elucidation, and biological activity of these compounds. The CHCl 3 -soluble portion of the sponge extract was fractionated by a modified Kupchan procedure 4 to yield an active CHCl 3 layer. This layer was further separated by ODS flash chromatography, silica gel column chromatography, and repetitive reversed-phase HPLC to yield ciliatamides A (1) (1.7 × 10 -4 % yield based on wet weight) and B (2) (4.4 × 10 -4 %), as yellowish oils. Another collection of the same sponge was similarly processed to afford ciliatamide C (3) (1.8 × 10 -3 %). Ciliatamide A (1) gave a molecular formula of C 26 H 39 N 3 O 3 as established by HRFABMS. The 1 H NMR spectrum revealed the presence of two amide NH groups (δ 7.74 and 7.66), a phenyl group [δ 7.22 (2H), 7.21 (2H), and 7.17], and a terminal vinyl group [δ 5.79 and 4.88 (2H)], which was supported by 13 C NMR signals for three amide carbonyl carbons (δ 177.1, 177.0, and 171.0), six sp 2 carbons [δ 138.5, 130.0 (2C), 129.5 (2C), and 127.5], and two olefinic carbons (δ 140.0 and 114.6). Analysis of the HSQC spectrum identified a terminal vinyl group, an N-methyl, 12 sp 3 methylenes, 2 sp 3 methines, and a monosubstituted phenyl group. Interpretation of the COSY spectrum led to connectivities H-2-H- 4, H-7-H-9, H-12-H-13, and H-22-H-26, suggesting the presence of a fatty acid as well as phenylalanine (Phe) and lysine (Lys) residues. Interpretation of HMBC data could assign the two amino acid moieties as N-methylphenylalanine (MePhe) and Lys (Figure 1). The connection of the acyl group and MePhe through the N-methyl amide was established by HMBC correlations (Me-20/C-1 and C-12), while CO (MePhe) and NH-22 (Lys) were connected by a HMBC cross-peak (NH-22/C-11). The identification of deca-9-enoic acid was straightforward from HMBC cross-peaks (H-8/C-10 and C-6, H-7/C-6 and C-5, H-6/C-8, C-7, C-5, and C-4, H-5/C-7, C-6, C-4, and C-3, H-4/C-6 and C-5, and H-2/C-1). Judging from the degrees of unsaturation, 1 must contain a ring, which was realized by HMBC correlations between NH-26/C-22 and H-26/C-21, thereby permitting the construction of an ǫ-caprolactam moiety. Comparison of the 1 H NMR data of 1 with those of acetyl Lys and R-acetoamide-ǫ-caprolactam prepared from Lys and R-amino-ǫ- caprolactam, respectively, substantiated this assignment (Figure 2). Marfey’s analysis 5 of the acid hydrolysates of 1 disclosed the L-configuration of both Lys and MePhe. Thus, the structure of ciliatamide A (1) was assigned as shown. # Dedicated to Dr. G. Robert Pettit of Arizona State University for his pioneering work on bioactive natural products. * Authors to whom correspondence should be addressed. (Y.N.) Tel/ Fax: +81-3-5286-2568. E-mail: ayocha@waseda.jp. (N.F.) Tel/Fax: +81- 138-40-8884. E-mail: anobu@fish.hokudai.ac.jp. † The University of Tokyo. ‡ Present address: School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan. § University of Amsterdam. ⊥ Hokkaido University. Figure 1. Key COSY and HMBC correlations for ciliatamide A (1). J. Nat. Prod. 2008, 71, 469–472 469 10.1021/np8000317 CCC: $40.75  2008 American Chemical Society and American Society of Pharmacognosy Published on Web 02/08/2008