Arch Pharm Res Vol 32, No 12, 1695-1698, 2009 DOI 10.1007/s12272-009-2205-y A New C29-sterol with a Cyclopropane Ring at C-25 and 26 from the Vietnamese Marine Sponge Ianthella sp. Nguyen Huu Tung1,2, Chau Van Minh1, Phan Van Kiem1, Hoang Thanh Huong1, Tran Thu Ha1, Nguyen Tien Dat1, Nguyen Xuan Nhiem1,2, Nguyen Xuan Cuong1, Jae-Hee Hyun3, Hee-Kyoung Kang3, and Young Ho Kim2 1 Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Nghiado, Caugiay, Hanoi, Vietnam, 2College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea, and 3School of Medicine, Institute of Medical Sciences, Jeju National University, Jeju 690-756, Korea (Received July 23, 2009/Revised September 25, 2009/Accepted September 27, 2009) One new C29 sterol with a cyclopropane ring at C-25 and C-26, aragusteroketal B (1), and aragusterol B (2) were isolated from the Vietnamese marine sponge Ianthella sp. Their structures were elucidated by extensive spectroscopic analyses. Both 1 and 2 showed moderate cytotoxic activity against MCF-7, SK-Hep-1, and HeLa cell lines with IC50 in the range of 12.8-27.8 µM. Key words: Sponge, Ianthella sp., Sterol, Aragusteroketal B, Cytotoxic activity INTRODUCTION MATERIALS AND METHODS There are numerous novel sterols, particularly in terms of unique side chain structures such as those with high degree of alkylation and unusual function groups from marine sponges (Faulkner, 1993). In which, sterols with a cyclopropane ring at C-25 and C26 are relatively rare and first reported in 1978 from sponges, the Petrosia ficiformis (Sica and Zollo, 1978) and Halichondria sp. (Ravi et al., 1978). Some of these analogs have been reported to show biological activities including anti-inflammation and cytotoxicity (Iguchi et al., 1994; Kobayashi et al., 1996; Mandeau et al., 2005). Our previous study on the Vietnamese marine sponge Ianthella sp. resulted in the isolation of three sterols showing anticancer properties (Tung et al., 2009). In the continuation of this work, we isolated an additional new sterol of this skeleton, named aragusteroketal B (1), together with a known one, aragusterol B (2). This paper deals with their structure elucidation and cytotoxic activity as well. General procedures The following instruments were used to obtain physical data: optical rotations, JASCO DIP-360 digital polarimeter; IR spectra, Perkin-Elmer 577 spectrometer; NMR spectra, Bruker DRX 500 NMR spectrometers; FABMS and HRFABMS, JEOL JMS-AX505 HR-5890 series spectrometer. For column chromatography (CC), silica gel (70-230 and 230-400 mesh, Merck) and YMC RP-18 resins (3050 µm, Fuji Silysia Chemical Ltd.) were used. For thin layer chromatography (TLC), Kieselgel 60 F254 (Merck 1.05715) or RP-18 F254s (Merck) plates were used; and spots were visualized by spraying with 10% aqueous H2SO4 solution follow by heating. Correspondence to: Young Ho Kim, College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea Tel: 82-42-821-5933, Fax: 82-42-823-6566 E-mail: yhk@cnu.ac.kr Animal materials The marine sponge Ianthella sp. (2.4 kg, wet weight) were collected at Namyet island, Khanh Hoa province in March, 2007, and were kept frozen (-20oC) until used; it was identified by Dr. Do Cong Thung, Institute of Marine Resources and Environment, Vietnam Academy of Science and Technology (VAST). The specimen vouchers of the Ianthella sp. (No 20070306) were deposited at Institute of Natural Products Chemistry and Institute of Marine Resources and Environment, VAST. 1695 1696 N. H. Tung et al. Extraction and isolation The frozen sample Ianthella sp. was ground and exhaustively extracted by the ultrasound-assisted manner with methanol (5 L × 3, each one day) at room temperature, and combined MeOH extract was concentrated in vacuo to give a black gum residue (70 g). The residue was suspended in H2O (0.5 L), and partitioned with EtOAc (0.5 L × 3). Next, the EtOAcsoluble portion (9.3 g) was subjected to a silica gel Table I. 1H- and a 13 column eluted with a gradient of MeOH in CHCl3 (30:1-1:1, v/v) to give four fractions (1a-d). The fraction 1d was repeatedly chromatogaphed using a silica gel column with an eluent of hexane-EtOAc (5:1) to give three subfractions (2a-c). Thereafter, fraction 2b was further chromatographed using a reversed-phase C-18 YMC column with an eluent of MeOH-acetone-H2O (5:1:3) to afford 2 (6.5 mg). Finally, repeated column chromatography of the fraction 2c (1.8 g) using silica C NMR Data for 1 and 2 1 C no. δCa,b 1 35.18 2 28.60 3 100.60 4 35.61 5 6 42.60 28.62 7 31.59 8 9 10 11 34.07 52.95 35.94 29.31 12 13 14 15 78.21 48.87 55.07 23.68 16 25.29 17 18 19 20 21 22 64.40 9.81 11.76 75.89 28.93 34.15 23 31.94 24 25 26 27 39.26 27.31 13.12 11.78 28 29 OCH3 OCH3 19.40 20.45 47.69 47.73 2 δHa,c (J in Hz) 1.58, 1.32, 1.90, 1.45, 1.63, 1.30, 1.31, 1.29, 1.18, 1.68, 0.88, 1.32, 0.83, m m m m m m m m m m m m m 1.73, m 1.27, m 3.36, dd (11.0, 4.5) 0.96, 1.68, 1.20, 1.71, 1.49, 1.65, 0.84, 0.81, m m m m m t (6.5) s s 1.16, 1.55, 1.32, 1.58, 1.30, 0.66, 0.16, 0.50, 0.16, 0.11, 1.03, 0.93, 3.14, 3.19, s m overlapped m m m m m m m d (6.0) d (6.5) s s Measured in CDCl3, b at 125 MHz, c at 500 MHz δCa,b 38.65 38.35 211.1 6 44.81 46.78 29.10 31.31 33.98 52.72 35.84 29.40 77.91 48.90 54.85 23.62 25.24 64.35 9.81 11.63 75.95 28.98 34.07 31.94 39.24 27.29 13.13 11.75 19.40 20.47 δHa,c(J in Hz) 2.04, 1.36, 2.42, 2.33, m m dt (6.5, 3.5) m 2.20, ddd (14.5, 6.0, 2.5) 2.10, t (14.5) 1.53, m 1.30 - 1.40, m 1.72, 0.88, 1.38, 0.83, m overlapped m m 1.76, m 1.36, m 3.37, dd (11.0, 4.5) 1.02, 1.70, 1.22, 1.72, 1.50, 1.65, 0.86, 1.01, m m m m m t (6.5) s s 1.16, 1.55, 1.32, 1.62, 1.32, 0.68, 0.18, 0.50, 0.18, 0.12, 1.03, 0.94, s m overlapped br t (10.5) m m m m m m d (6.0) d (6.5) A New C29-sterol from the Marine Sponge Ianthella sp. 1697 gel (n-hexane-EtOAc, 3:2) and reversed-phase C-18 YMC (MeOH-H2O-acetone, 5:1:2) resulted in 1 (10.2 mg). Aragusteroketal B (1) White amorphous powder; [α]D20 +13o (c 0.50, CHCl3); IR (KBr) νmax 3440, 2942, 1092 cm-1; positive FABMS m/z 491 [M+H]+; positive HRFABMS m/z 491.4094 [M+H]+ (Calcd for C31H55O4 491.4100); 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz): see Table I. Aragusterol B (2) White powder; m.p. 194-195oC; [α]D20 +4o (c 0.50, CHCl3); positive FABMS m/z 445 [M+H]+; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz): see Table I. MTT assay Cell growth inhibition by different samples was analyzed using colorimetric MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide] assay based on the reported protocol (Mosmann, 1983). RESULTS AND DISCUSSION The sponge sample was extracted with MeOH, then the combined MeOH extract was partitioned to EtOAc and H2O-soluble portions. The EtOAc-soluble portion was chromatographed using silica gel and C-18 YMC resin columns to afford 1 and 2 (Fig. 1). The IR spectrum of 1 displayed absorptions at 3440 cm-1 (OH group) and 1092 cm-1 (ether linkage). The molecular formula of 1, C31H54O4, was established based on HRFABMS (found at m/z 491.4094 [M+H]+, calcd for C31H55O4 491.4100) and FABMS (observed peak at m/z 491 [M+H]+). The 1H and 13C NMR data of 1 and 2 are summarized in Table I. The assignments were confirmed by HMQC, HMBC, COSY, and NOESY spectra. The NMR data suggested 1 and 2 belonging to C29 steroids with C10 side chain containing a cyclopropane ring at C-25 and C-26. The presence of the cyclopropane ring was defined due to upfield resonance signals, typically, in their 1H NMR (H-25, H-26, and H-27) and 13C NMR (C-26 and C-27) (Kobayashi et al., 1996; Mandeau et al., 2005; Tung et al., 2009). In addition, five methyl groups were observed in the 1H NMR spectra of 1 and 2 including three singlets (H-18, H-19, and H-21) and two doublets (H-28 and H-29) which were integrated relatively for 3H, respectively. The 13C NMR spectrum of 2 disclosed one carbonyl carbon (δC 211.16, C-3) and two hydroxy-bearing carbons Fig. 1. Chemical structures of 1 and 2 (δC 77.91, C-12 and δC 75.95, C-20). The other NMR data of 2 were well in agreement with aragusterol B (Iguchi et al., 1994). To the best of our knowledge, aragusterol B (2) was isolated for the first time from Ianthella species. The 1H- and 13C NMR spectra of 1 were in difference from those of aragusterol B (2) by the apperance of two methoxy groups (δC 47.69 and 47.73; δH 3.14 and 3.19 (each 3H, s)), together with a downfield quaternary carbon signal (δC 100.60) instead of carbonyl carbon at δC 211.16 (C-3) in compound 2, which indicated the existence of a dimethylketal group at C-3 like aragusteroketals A and C isolated from the sponge Xestospongia sp. (Kobayashi et al., 1996). Additionally, in the HMBC spectrum of 1, the cross correlations of two methoxy protons (δH 3.14 and 3.19) with C-3 at δC 100.60 further confirmed the dimethylketal structure, respectively (Fig. 2). Furthermore, as shown in Fig. 2, the double quantum filtered correlation spectroscopy (DQF COSY) expreriment on 1 indicated the presence of partial structures written in bold lines. Relative stereochemistry of the side chain of 1 and 2 was established from careful comparison of their 1H and 13C spectra with literature and NOESY experiments (Iguchi et al., 1994; Kobayashi et al., 1996; Tung et al., 2009; Umeyama et al., 2000). Consequently, the 17â-orientation of the side chain was disclosed by NOESY cross-peaks of H-12/H-21 and H-14/H-17. The 1698 N. H. Tung et al. ACKNOWLEDGEMENTS This work was supported by the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0093815) and the Vietnam KC10.20/06-10 Project. The authors are grateful to Dr. Do Cong Thung, VAST, for his kind identification of the sponge, and Korean Basic Science Institute (KBSI) for NMR and MS measurements. REFERENCES Fig. 2. COSY and significant HMBC correlations for 1 Fig. 3. Key NOESY correlations of the side chain of 1 and 2 Table II. Cytotoxicity data of compounds 1 and 2 Compound 1 2 Mitoxantroneb Cell line a MCF-7 SK-Hep-1 HeLa 16.9 12.8 7.1 25.3 18.5 8.7 24.6 27.8 8.2 a Results are expressed as IC50 values (µM), and values < 50 µM are considered to be active. b Positive control geometry of the cyclopropane ring was deduced to be E by the NOESY cross-peak of H-25/H-28 (Fig. 3). Their cytotoxic activities were tested against a panel of three human tumor cell lines, including breast (MCF-7), liver (SK-Hep-1), and cervical (HeLa) using the MTT assay. As the results (Table II), compounds 1 and 2 showed moderate cytotoxic activity against all three cell lines with IC50 values of 16.9, 25.3, and 24.6 µM for 1; 12.8, 18.5, and 27.8 µM for 2, respectively. Faulkner, D. J., Marine natural reports. Nat. Prod. Rep., 10, 497-539 (1993). Iguchi, K., Shimura, H., Taira, S., Yokoo, C., Matsumoto, K., and Yamada, Y., Aragusterol B and D, New 26,27Cyclosterols from the Okinawan Marine Sponge of the Genus Xestospongia. J. Org. Chem., 59, 7499-7502 (1994). Kobayashi, M., Chen, Y. J., Higuchi, K., Aoki, S., and Kitagawa, I., Marine natural products. XXXVII. Aragusteroketals A and C, two novel cytotoxic steroids from a marine sponge of Xestospongia sp. Chem. Pharm. Bull., 44, 1840-1842 (1996). Mandeau, A., Debitus, C., Aries, M. 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