New -Caryophyllene-Derived Terpenoids from the Formosan Soft Coral Sinularia gibberosa

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1 Ó 006 The Chemical Society of Japan Bull. Chem. Soc. Jpn. Vol. 7, No. 0, 7 (006) 7 New -Caryophyllene-erived Terpenoids from the Formosan Soft Coral Sinularia gibberosa Shin-Pin Chen, Chih-ua Chao, o-cheng uang, Yang-Chang Wu, Chung-Kuang Lu, ; Chang-Feng ai, 6 and Jyh-orng Sheu epartment of Marine Biotechnology and esources, National Sun Yat-sen University, Kaohsiung 0, Taiwan epartment of Chemical and Materials Engineering, Cheng Shiu University, Kaohsiung, Taiwan Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 07, Taiwan National Museum of Marine Biology and Aquarium, Checheng, Pingtung, Taiwan Institute of Marine Biotechnology, National ong wa University, Checheng, Pingtung, Taiwan 6 Institute of ceanography, National Taiwan University, Taipei 06, Taiwan eceived February, 006; sheu@mail.nsysu.edu.tw Seven new -caryophyllene-derived terpenoids ( 7) were isolated from EtAc extracts of the Formosan soft coral Sinularia gibberosa. The structures of compounds 7 were elucidated on the basis of extensive spectroscopic analyses and by comparison with the spectral data of related metabolites. Cytotoxicity evaluation of the above metabolites towards a limited panel of cancer cell lines also will be described. Soft corals belonging to the genus Sinularia (Alcyoniidae) have been found to be a rich source of structurally unique and biologically active diterpenoids and norditerpenoids. uring the course of our investigation on bioactive natural products from marine invertebrates, a series of metabolites including steroids, sesquiterpenoids, diterpenoids, and norditerpenoids have been discovered. ur present study on the chemical constituents of a soft coral Sinularia gibberosa, which was collected off the coast of northeastern Taiwan, has led to the isolation of seven new -caryophyllene-derived terpenoids ( 7) (Chart ). We describe herein the isolation, structure elucidation, and biological activity of these compounds. Compound was isolated as a colorless oil, ½Š þ: (c 0.6, CCl ). Its -ESI-MS had a pseudomolecular ion peak at m=z 7.6 ½M þ NaŠ þ (calcd for C 6 Na, 7.6), corresponding to the molecular formula of C 6. Thus, the compound was determined to have five degrees of unsaturation. The characteristic NM signals [.6 (, s, Me); C 7. (C-),. (Me)] and an I absorption at 7 cm indicated the presence of a methyl ester functional group. Furthermore, two methyls ( C 6., C and 7.0, C ), one trisubstituted epoxide ( C., C and 6., C), and one exocyclic double bond ( C.0, C and 0.6, C) were assigned from the C NM and EPT spectra of compound. The above functionalities revealed that compound is a tricyclic compound, including the presence of an epoxide. Using NM spectroscopic analysis (CSY, MQC, and MBC), the caryophyllene-based skeleton of compound was elucidated (Fig. ). The relative stereochemistry of compound (Fig. ) was established from NE correlations observed in a NESY experiment. In the NESY spectrum of compound, - interacted 7 : = 0: = C 0 MeC 6 0 : = Ac Chart. 0 7 Ac : = 6: = Ac 7: = : = : = 6 0 Published on the web ctober 0, 006; doi:0.6/bcsj.7.7

2 Bull. Chem. Soc. Jpn. Vol. 7, No. 0 (006) New Terpenoids from Sinularia gibberosa MeC and Ac Ac 6 7 Fig.. Selective CSY and MBC correlations of compounds 7. with - but not with -, -, and -, suggesting that - is situated on the opposite face of the compound from -, -, and -. It was found that the relative stereochemistry of compound is the same as that of a known metabolite., Also, we have previously isolated a related norcaryophyllene, compound, from a soft coral of the same genus Sinularia. 6 Thus, a (S,S,S,,S )-configuration for compound was suggested. Gibberosin A () was obtained as a colorless oil, ½Š þ: (c.0, CCl ). The molecular formula of compound,c, was established by -ESI-MS, and it was determined to have six degrees of unsaturation. The C NM spectrum of compound had signals for carbons (Table ), including those of two ketones ( C. and 07.6), one trisubstituted epoxide ( C., C and 6.7, C), and one exocyclic double bond ( C., C and 0., C). From a comparison of and C NM spectral data of compound (Tables and ) with those of compound and nanolobatin C (0), 6 it was found that compound contains the same ring structure as that of compounds and 0, and the acetoxy-bearing methine at C- in compound 0 was replaced by a ketone in compound, which was also confirmed by the MBC and CSY correlations as shown in Fig.. Thus, the planar structure of compound was fully established. From the NE correlations observed in the NESY spectrum of (Fig. ), - interacted with -, but not with -, -7, and -. Also, - did not interact with -. This suggests that - has an -orientation and -, -7, and - have -orientation. Therefore, the relative configurations on C-, C-, C-, C-, and C- in compound were found to be the same as those of compound. : = CMe : = : = Ac : = 6: = 7: = Ac 0 7 Fig.. Key NESY correlations of compounds 7. Gibberosin B () was obtained as a colorless oil, ½Š þ: (c 0., CCl ). According to the -ESI-MS (m=z 7.0, ½M þ NaŠ þ ) and C NM data, its molecular formula is C 0 0 Na. Therefore, compound was determined to have six degrees of unsaturation. The and C NM spectral data were very similar to those of a known compound 6 (Tables and ) except that a carbon signal at 7. (C-) in compound was shifted to. in compound. Furthermore, the NM spectrum of compound showed an additional signal at 7. (br s) which was considered to be a hydroperoxy proton. In other words, the hydroxy group in compound was replaced by a hydroperoxy group in compound. The double bond between C- and C- was determined to have a trans geometry based on the coupling constant (J ¼ : z) between - ( 6., d) and - ( 6., d). The relative configurations on C-, C-, C-, C-, and C- in compound were found to be the same as those of compounds and, on the basis of the NE correlations observed in a NESY experiment as shown in Fig.. Gibberosin C () was isolated as a colorless oil, ½Š þ: (c 0., CCl ). Its -ESI-MS established the same molecular formula, C 0 0, as that of compound. The and C NM data of compound (Tables and, respectively) are very similar to those of compound. Careful inspection

3 S.-P. Chen et al. Bull. Chem. Soc. Jpn. Vol. 7, No. 0 (006) Table. C NM Spectral ata of Compounds 7 C# aþ aþ aþ bþ cþ 6 aþ 7 bþ 7. (C) dþ. (C). (C). (C). (C). (C). (C) 7.6 (C ). (C ). (C ).6 (C ). (C ).0 (C ). (C ).6 (C ). (C ). (C ). (C ).6 (C ). (C ).7 (C ). (C). (C).6 (C) 60.7 (C). (C). (C).7 (C) 6. (C) 6.7 (C) 6.6 (C) 6.0 (C) 6.7 (C) 6.6 (C) 6. (C) (C ) 0. (C ) 0. (C ) 6. (C ) 0. (C ) 0. (C ) 0. (C ) 7.7 (C ). (C ). (C ). (C ).6 (C ). C ).7 (C ) 0.6 (C) 0. (C) 0.6 (C).6 (C).6 (C) 0. (C) 0. (C) 7. (C) 7. (C) 7. (C).6 (C). (C) 7. (C) 7. (C) 0. (C ).0 (C ). (C ) 7.6 (C ). (C ). (C ). (C ).0 (C). (C) 7.7 (C) 6. (C) 7. (C). (C).7 (C) 6. (C ). (C) 0. (C) 0. (C). (C).6 (C). (C) 7. (C) 0.6 (C ).0 (C).0 (C). (C ) 6. (C ). (C ).0 (C ) 6. (C ). (C). (C). (C ) 6. (C). (C) 7.0 (C ) 07.6 (C). (C). (C). (C). (C).6 (C) 6 0. (C ). (C ). (C ). (C ). (C ) 6. (C ) 7 6. (C ). (C ). (C ). (C ).7 (C ). (C ). (C ) 6. (C ) 6. (C ) 7. (C ) 7.0 (C ) 7.0 (C ) 7. (C ). (C ). (C ). (C ).0 (C ). (C ) 0 7. (C ). (C ) 6. (C ) 6. (C ) 7. (C ) Ac 70.7 (C) 7. (C). (C ). (C ) Me. (C ) a) Spectra recorded at 00 Mz in CCl at C. b) Spectra recorded at Mz in CCl at C. c) Spectra recorded at 7 Mz in CCl at C. d) Attached protons were deduced by EPT spectra. The values are in ppm downfield from TMS. of the CSY and MBC spectral data of compound showed that compound had a planar structure similar to that of compound (Fig. ). The relative stereochemistry of compound was elucidated by analysis of the vicinal coupling constants and NE correlations observed in a NESY experiment. The double bond between C- and C- was also determined to have a trans geometry based on the coupling constant (J ¼ : z) between - ( 6., d) and - ( 6., d). In the NESY spectrum of compound, - interacted with -0 but not with -, -, and -, suggesting that - and -0 have an -orientation and -, -, and - have a -orientation. Therefore, compound was determined to be a C-, C- diastereomer of compound. Gibberosin () has the molecular formula C based on its -ESI-MS and NM spectral data (Tables and, respectively). Comparison of the C NM spectral data of compound with those of compound (Table ) suggested that compound is another caryophyllene-derived diterpeneoid. From careful inspection on the and C NM spectral data of compound, C- was assigned to have a saturated prenyl substituent. Also, two methyl groups (. (6, s) and C.) were attached to C-, and an acetoxy group ( C. and 70.7; I absorption at 7 cm ) was found to be attached to C- ( C.; ( -6 and -7)., 6, s). Based on the data, the molecular framework of compound was determined. In addition, the stereochemistry of compound was established based on key NE correlations (Fig. ). Gibberosin E (6) was isolated as a colorless oil, ½Š þ:0 (c 0., CCl ). The molecular formula of compound 6 was determined to be C 0 0 based on -ESI-MS, and it has six degrees of unsaturation. The and C NM data of compound 6 revealed that this compound has the same ring skeleton as those of compounds. An I absorption at 670 cm corresponded to a trisubsituted double bond. In the NM spectrum, no signal was observed for an acetoxy group; however, an additional olefinic proton (., J ¼ 7:, 7.,. z), as compared to that of compound was observed. Based on these findings, together with the correlations observed in the CSY and MBC spectra (Fig. ), compound 6 was determined to have a planar structure. The relative stereochemistry of caryophyllene-based moiety for compound 6 was found to be the same as that of compound, determined from the NE correlations observed in a NESY spectrum (Fig. ). Gibberosin F (7) was obtained as a colorless oil, ½Š þ6:7 (c 0.6, CCl ). The molecular formula of compound 7 was determined to be C based on -ESI-MS. The and C NM data are very similar to those of compound 6. By comparison of the NM spectral data of compound 6 with those of compound 7, it was found that a vinyl methyl attached to C- in compound 6 was replaced by an acetoxy-bearing methylene in compound 7. The MBC cross peaks from -7 to C-, and -6 to C- and the acetoxy carbonyl carbon confirmed the above observation. The double bound between C- and C- was determined to have a cis geometry based on the NE interaction between - and -7. The caryophyllene moiety in compounds,, and 7 now are known to have the same relative stereochemistry at the asymmetric carbons, C-, C-, C-, C-, and C- as that in compound. Thus, the relative configurations of these five compounds were assigned to be S,S,S,,S. ow-

4 0 Bull. Chem. Soc. Jpn. Vol. 7, No. 0 (006) New Terpenoids from Sinularia gibberosa Table. NM Spectral ata of Compounds 7 # aþ aþ aþ bþ cþ 6 aþ 7 bþ.6 dd. dd.6 dd. dd. m. dd. m (.6,.6) dþ (0.0, 0.0) (0.0, 0.0) (0.0, 0.0) (.6,.6). m. m. m.7 m.7 m.0 m. m. m. m. m.7 m.7 m. m.7m.0 m.0 ddd.0 m.0 m.0 m.07 ddd.07 ddd (.,.,.6) (.,.,.) (.0,.0,.0).0 m.0 ddd. m.7 m.0 m.0 ddd.0 m (.,.,.6) (.,.,.6). dd. dd. dd.00 dd. dd. dd. dd (0.,.) (0.,.6) (0.,.6) (.,.) (0.,.6) (0.,.0) (0.0,.) 6.7 m. m. m.7 m. m.7 m. m.7 m. m.0 m. m.0 m. m 7. m. m.0 m. m.0 m. m.0 m. m.7 m.6 m. m. m.6 m. m.6 q.6 m.7 q. q.6 q.6 q.70 m (0.0) (.0) (.0) (.6) (0.0) 0. m. m.7 m.7 m.7 m. m. m.76 m.77 m.7 m.0 m.77 m.77 m. m. s.66 m 6. d 6. d. m.06 dd. dd (.) (.) (.0, 7.) (.0, 6.). dd. dd (.0, 7.) (.0, 6.). s.7 m 6. d 6. d.0 m. dd m eþ. dd (.) (.) (7., 7.,.) (6., 6.).0 s. s 6. s. s. s. s.6 s. s 7. s. s.0 s. s.7 s. s.0 s. s. s. s. s. s.0 s.0 s. s.0 s. s.0 s.0 s.00 s.0 s.00 s.00 s.00 s 0. s. s. s.0 s.0 s Ac.7 s.06 s Me.6 s 7. s 7. s a) Spectra recorded at 00 Mz in CCl at C. b) Spectra recorded at 00 Mz in CCl at C. c) Spectra recorded at 00 Mz in CCl at C. The values are in ppm downfield from TMS. d) J value (in z) in parentheses. e) m is equal to septet. ever, compound was assumed to possess a (S,,,-,S ) configuration as - and -0 were found to be positioned on the face. Cytotoxicity of metabolites 7 toward a limited panel of cancer cell lines was evaluated. Compound had weak cytotoxicity towards ep G (uman hepatocellular carcinoma), A (human lung carcinoma), and MA-MB- (human breast carcinoma) cell lines with IC 0 s of.7,., and. mgml, respectively. Also, metabolite 6 had weak cytotoxicity (IC 0 6. mgml ) towards MA-MB-. The other metabolites were inactive toward the growth of the above three cancer cell lines. Experimental General Experimental Procedures. ptical rotations were measured on a Jasco IP-000 digital polarimeter. I spectra were recorded on a Jasco FT-00 infrared spectrophotometer. NM spectra were recorded on a Bruker Avance PX00 FT- NM at 00 Mz for and 7 Mz for C or on a Varian Unity INVA 00 FT-NM at 00 Mz for and Mz for C, in CCl using TMS as an internal standard. Low-resolution mass data and -MS data were recorded by ESI FT-MS on a Bruker APEX II mass spectrometer. Silica gel (Merck, 0 00 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-, 0. mm) were used for analytical TLC. igh-performance liquid chromatography (PLC) was performed on a itachi L-700 apparatus equipped with a Bischoff refractive index detector, or a itachi L-700 UV detector and with the Merck ibar Si-60 column (0 mm, 7 mm). Animal Material. The soft coral Sinularia gibberosa was collected by hand using scuba equipment off the coast of northeastern Taiwan, in May, 00, at a depth of 0 m, and was stored in a freezer until extraction. A voucher specimen was deposited in the epartment of Marine Biotechnology and esources, National Sun Yat-sen University (specimen No ).

5 S.-P. Chen et al. Bull. Chem. Soc. Jpn. Vol. 7, No. 0 (006) Extraction and Isolation. The soft coral S. gibberosa (. kg fresh wt) was collected and freeze dried. The freeze-dried material was minced and extracted exhaustively with EtAc. The EtAc extract (. g) was fractionated by using open column chromatography on silica gel using hexane and hexane EtAc mixtures of increasing polarity to yield fractions. Fraction 6, eluted with hexane/etac (:), was subjected to Sephadex L-0 column ( 0 cm) using Acetone, which was followed by normal phase PLC eluted with hexane/acetone (: to 7:) to afford compounds (0.7 mg) and 6 (. mg). Fraction, eluted with hexane/etac (:), was further purified by silica gel column, using hexane/etac (:) as eluent to afford compound (0. mg). A combined mixture of fractions 7 was purified by normal phase PLC by using hexane/etac (:) and then hexane/acetone (:) as eluents to afford compound (. mg). Fraction 0, eluted with hexane/etac (:), was further purified by normal phase PLC using hexane/acetone (: to :) to give compounds 7 (.0 mg), (0. mg), and (. mg). Methyl-(S,S,S,,S )-,-epoxycaryophyllen-- oate (): Colorless oil; ½Š þ: (c 0.6, CCl ); I (KBr) max 7, 6 cm ; NM and C NM data, see Tables and ; ESI-MS m=z 7 (½M þ NaŠ þ ); -ESI-MS m=z 7.6 ½M þ NaŠ þ (calcd for C 6 Na, 7.6). Gibberosin A (): Colorless oil; ½Š þ: (c.0, CCl ); I (KBr) max 76, 70, 6 cm ; NM and C NM data, see Tables and ; ESI-MS m=z 7 (½M þ NaŠ þ ); -ESI- MS m=z 7. ½M þ NaŠ þ (calcd for C Na, 7.6). Gibberosin B (): Colorless oil; ½Š þ: (c 0., CCl ); I (KBr) max 6, 76, 6, 6 cm ; NM and C NM data, see Tables and ; ESI-MS m=z 7 (½M þ NaŠ þ ); -ESI-MS m=z 7.0 ½M þ NaŠ þ (calcd for C Na, 7.0). Gibberosin C (): Colorless oil; ½Š þ: (c 0., CCl ); I (KBr) max 6, 76, 6, 6 cm ; NM and C NM data, see Tables and ; ESI-MS m=z 7 (½M þ NaŠ þ ); -ESI-MS m=z 7.0 ½M þ NaŠ þ (calcd for C Na, 7.0). Gibberosin (): Colorless oil; ½Š þ6: (c 0., CCl ); I (KBr) max 7, 70, 6 cm ; NM and C NM data, see Tables and ; ESI-MS m=z (½M þ NaŠ þ ); -ESI- MS m=z. ½M þ NaŠ þ (calcd for C Na,.). Gibberosin E (6): Colorless oil; ½Š þ:0 (c 0., CCl ); I (KBr) max 76, 670, 6 cm ; NM and C NM data, see Tables and ; ESI-MS m=z (½M þ NaŠ þ ); -ESI- MS m=z.6 ½M þ NaŠ þ (calcd for C 0 0 Na,.). Gibberosin F (7): Colorless oil; ½Š þ6:7 (c 0.6, CCl ); I (KBr) max 7, 76, 670, 6 cm ; NM and C NM data, see Tables and ; ESI-MS m=z (½M þ NaŠ þ ); -ESI-MS m=z.7 ½M þ NaŠ þ (calcd for C - Na,.). Cytotoxicity Testing. Compounds were assayed for cytotoxicity against ep G, A, and MA-MB- cancer cells using the MTT [-(,-dimethylthiazol--yl)-,-diphenyltetrazolium bromide] method. Freshly trypsinized cell suspensions were seeded into a 6-well microtiter plate at densities of cells per well and then the test compounds were added from MS-diluted stock solutions. After days in culture, attached cells were incubated with MTT (0. mg ml, h) and subsequently dissolved in MS. The absorbency at 0 nm was then measured using a microplate reader. The IC 0 is the concentration of agent that reduced cell growth by 0% under the experimental conditions. Financial support of this work provided by National Science Council of Taiwan (NSC--B-0-00) awarded to J.-. Sheu was greatly appreciated. eferences J. W. Blunt, B.. Copp, M.. G. Munro, P. T. Northcote, M.. Prinsep, Nat. Prod. ep. 00,,, and references therein. J.-. Sheu, A. F. Ahmed,.-T. Shiue, C.-F. ai, Y.-. Kuo, J. Nat. Prod. 00, 6, 0, and references therein. S.-L. Wu, P.-J. Sung, J.-. Su, J.-. Sheu, J. Nat. Prod. 00, 66,. J.-. Sheu, L.-F. uang, S.-P. Chen, Y.-L. Yang, P.-J. Sung, G.-. Wang, J.-. Su, C.-. Chao, W.-P. u, J.-J. Wang, J. Nat. Prod. 00, 66, 7. J.-. Sheu, G.-. Wang, C.-Y. uh, K. Soong, J. Nat. Prod. 00, 66, A. F. Ahmed, J.-. Su,.-T. Shiue, X.-J. Pan, C.-F. ai, Y.-. Kuo, J.-. Sheu, J. Nat. Prod. 00, 67,. 7 A. F. Ahmed, J.-. Su, Y.-. Kuo, J.-. Sheu, J. Nat. Prod. 00, 67, 07. C.-. Chao, L.-F. uang, Y.-L. Yang, J.-. Su, G.-. Wang, M. Y. Chiang, Y.-C. Wu, C.-F. ai, J.-. Sheu, J. Nat. Prod. 00, 6, 0. A. F. Ahmed, M.-. Wu, G.-. Wang, Y.-C. Wu, J.-. Sheu, J. Nat. Prod. 00, 6, 0. 0 J.-. Su,.-C. uang, C.-. Chao, L.-Y. Yan, Y.-C. Wu, C.-C. Wu, J.-. Sheu, Bull. Chem. Soc. Jpn. 00, 7, 77. Y.-J. Tseng, A. F. Ahmed, C.-F. ai, M. Y. Chiang, J.-. Sheu, rg. Lett. 00, 7,. J. Jakupovic, E. Ellmauerer, F. Bohlmann,. M. King,. obinson, Phytochemistry 6,, 7. W.-. Abraham, L. Ernst, B. Stumpf, Phytochemistry 0,,. a) J.-. Sheu, S.-Y. uang, C.-Y. uh, J. Nat. Prod. 6,,. b) J.-. Sheu, S.-Y. uang, G.-. Wang, C.-Y. uh, J. Nat. Prod. 7, 60, 0. M. C. Alley,. A. Scudiero, A. Monks, M. L. ursey, M. J. Czerwinski,. L. Fine, B. J. Abbott, J. G. Mayo,.. Shoemaker, M.. Boyd, Cancer es.,,.