Letter pubs.acs.org/OrgLett
Tortuosenes A and B, New Diterpenoid Metabolites from the Formosan Soft Coral Sarcophyton tortuosum Kuan-Hua Lin,† Yen-Ju Tseng,† Bo-Wei Chen,† Tsong-Long Hwang,‡ Hsing-Yin Chen,§ Chang-Feng Dai,⊥ and Jyh-Horng Sheu*,†,∥,# †
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan § Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan ⊥ Institute of Oceanography, National Taiwan University, Taipei 106, Taiwan ∥ Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan # Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 402, Taiwan ‡
S Supporting Information *
ABSTRACT: Tortuosenes A and B (1 and 2), possessing new diterpenoid molecular structures, were isolated from the Formosan soft coral Sarcophyton tortuosum along with a known cembrane, emblide (3). The structures of 1 and 2 were established by extensive spectroscopic analysis, and the absolute configuration of 1 was determined by TDDFT ECD calculations. Tortuosene A was found to display significant inhibitory effects on the generation of the superoxide anion in Nformyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced neutrophils.
S
oft corals of the genus Sarcophyton have been shown to be a rich source of the cembranoidal diterpene.1 Many cembranes discovered recently from Formosan soft corals of this genus were also found to exhibit attractive cytotoxic and anti-inflammatory activity.2−7 The soft coral Sarcophyton tortuosum (Tixier−Durivault, 1946) has been recognized as rich sources of the biscembranoids.8−11 Our current chemical investigation on the Taiwanese S. tortuosum has led to the isolation of two new diterpenoids, tortuosenes A and B (1 and 2), and the known compound, emblide (3)12 (Figure 1). The structures of the new natural products were determined by extensive spectroscopic analysis. The absolute configuration of tortuosene A (1) was determined by the application of the TDDFT ECD approach. The cytotoxicity and anti-inflammatory activities of 1−3 were also evaluated. The soft coral S. tortuosum (1.3 kg, wet weight) was collected by hand using SCUBA off the coast of Lanyu Island of Taiwan, in August 2008, and stored in a freezer before extraction. The freeze-dried organism was minced and extracted exhaustively with EtOAc. The EtOAc-soluble fraction (10.2 g) was chromatographed on silica gel to yield 25 fractions. Fraction 17, eluted with n-hexane−EtOAc (3:1), was purified again by using silica gel (n-hexane−EtOAc, 3:1), and then by normalphase HPLC, eluting with n-hexane−EtOAc (5:1) to afford 3 © 2014 American Chemical Society
Figure 1. Structures of 1−3.
(30.1 mg). Fractions 18 and 19, eluting with n-hexane−EtOAc (2:1 and 1:1, respectively), were combined and purified by Received: December 23, 2013 Published: February 13, 2014 1314
dx.doi.org/10.1021/ol403723b | Org. Lett. 2014, 16, 1314−1317
Organic Letters
Letter
normal-phase HPLC using n-hexane−acetone (3:1) as eluent to afford a mixture that was further separated by normal-phase HPLC using n-hexane−EtOAc (3:1) to afford 1 (32.5 mg) and 2 (2.5 mg). Tortuosene A (1), [α]25D +59 (c 0.83, CHCl3), was obtained as a white powder and had the molecular formula C23H32O7 as determined by HRESIMS (m/z calcd 443.2046; found 443.2043, [M + Na]+), requiring eight degrees of unsaturation. The infrared spectrum of 1 showed absorptions of hydroxyl (νmax 3499 cm−1) and carbonyl (νmax 1725 cm−1) groups. The 13 C NMR spectroscopic data of 1 (Table 1) revealed the
Figure 2. Key COSY and HMBC correlations of 1.
Table 1. 1H and 13C NMR Chemical Shifts for Compound 1
was possible to establish four proton sequences from H-2 to H3; H2-5 to H-7; H2-9 to H2-10; H2-13 and H2-14; and H3-16 to H3-17 through H-15. The HMBC experiment used to connect the above substructures was found to show the following key correlations: H-2 to C-1, C-4, C-12, C-14 and C-20; H-3 to C1, C-5 and C-18; H-7 to CO of 7-OAc; one proton of H2-10 (δ 2.58) to C-12; H2-13 to C-11; both H3-16 and H3-17 to C-1; and H3-19 to C-7, C-8, and C-9. In the NOESY spectrum of 1 (Figure 3), correlations were observed between H-2 and one proton of H2-5 (δ 2.97), which
1 1
C/H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 7-OAc 18-COOMe
Ha
3.24, d, 8.0d 6.63, d, 8.0 2.64, 2.97, 1.89, 2.02, 4.56,
m td, 13.2, 5.2 m m d, 10.4
1.60, 1.92, 2.58, 2.65,
m m m m
2.30, 2.43, 1.62, 1.96, 1.70, 0.82, 0.96,
dd, 18.0, 6.8 m m m m d, 6.8 d, 6.8
1.42, s 1.97, s 3.80, s
13 b
C
74.6, Cc 49.5, CH 138.1, CH 133.1, C 24.4, CH2 27.1, CH2 75.5, CH 84.8, C 33.0, CH2 37.9, CH2 199.7, C 118.6, C 20.5, CH2 25.5, CH2 33.2, CH 15.6, CH3 15.8, CH3 166.9, C 20.2, CH3 161.6, C 21.0, CH3 169.5, C 52.0, CH3
Figure 3. Key NOESY correlations of 1.
further correlated with one of proton of H2-6 (δ 2.02) and H319 (δ 1.42, s); H3-19 with both of the above-mentioned H-5 and H-6 but not with H-7 (δ 4.56); and H-7 with another H-6 (δ 1.89) but not with H3-19; therefore, assuming the βorientation of H-2, it was suggested that H3-19 is β-oriented and H-7 is α-oriented. The relative configuration of C-1 could not be determined as the NOE correlations between H-2 and both H-15 and H3-16 might be observed in two C-1 diastereomers. We further measured the NOESY spectrum of 1 in pyridine-d5 and observed the NOE correlation of 1-OH (δ 5.70, s) and H-2 (δ 3.61, d, J = 7.6 Hz) and all the correlations found in CDCl3 (see Supporting Information). Thus, the βorientation of hydroxyl group at C-1 was determined. Tortuosene B (2), [α]25D +12 (c 0.71, CHCl3), was isolated as white power. The molecular formula C21H28O6 of 2 was established by HRESIMS (m/z calcd 399.1783; found 399.1785, [M + Na]+). Furthermore, it was shown that the 1 H and 13C NMR data of 2 (Table 2) were similar to those of 1 except that the resonances of the acetoxy group in 1 were absent in the NMR spectra of 2. In addition, the acetoxy groupcontaining methine carbon at δ 75.5 in 1 was shifted downfield to δ 207.2 in 2. Thus, tortuosene B (2) is a derivative of 1 with 7-keto functional group.
a
Spectrum recorded at 400 MHz in CDCl3. b100 MHz in CDCl3. c Deduced from DEPT. dJ values (Hz) in parentheses.
presence of 23 carbon signals, which were assigned with the assistance of DEPT spectrum into five methyls (including an oxygenated carbon resonating at δ 52.0 and one acetate methyl resonating at δ 21.0), six sp3 methylenes, three sp3 methines (including one oxygenated carbon resonating at δ 75.5), two sp3 oxygenated quaternary carbons (δ 84.8 and 74.6), one sp2 methine, and six sp2 quaternary carbons that included three carbonyl groups (δ 199.7, 169.5 and 166.9) and three olefinic quaternary carbons (δ 161.6, 133.1, and 118.6). From the 1H NMR spectrum of 1, the resonances of one olefinic proton (δ 6.63, d, J = 8.0 Hz) and five methyls (δ 3.80, s; 1.97, s; 1.42, s; 0.96, d, J = 6.8 Hz; 0.82, d, J = 6.8 Hz) were also observed. The above results suggest a terpenoid origin for 1. The COSY and HMBC (Figure 2) correlations were further used to establish the molecular skeleton of 1. From the COSY spectrum of 1, it 1315
dx.doi.org/10.1021/ol403723b | Org. Lett. 2014, 16, 1314−1317
Organic Letters
Letter
Table 2. 1H and 13C NMR Chemical Shifts for Compound 2
Scheme 1. Proposed Biosynthetic Pathway for 1 and 2
2 1
C/H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 18-COOMe
Ha
3.34, d, 8.8d 6.42, d, 8.8 2.82, 3.38, 2.70, 3.25,
1.63, 2.43, 2.58, 2.63,
2.27, 2.47, 1.54, 1.94, 1.62, 0.82, 0.95,
m m m m
m m m m
dd, 18.0, 6.8 m m dd, 13.6, 6.8 m d, 6.8 d, 6.8
1.58, s 3.79, s
13 b
C
74.7, Cc 49.3, CH 137.7, CH 133.4, C 25.0, CH2 37.7, CH2 207.2, C 88.1, C 26.9, CH2 37.2, CH2 200.7, C 119.2, C 20.7, CH2 25.6, CH2
Figure 5. Molecular skeleton of tortuosane.
33.3, CH 15.6, CH3 15.8, CH3 166.4, C 24.4, CH3 160.1, C 52.2, CH3
20 and epoxidized at C-7/C-8 double bond to form an aldehydocembrane 5, which is structurally related to the known compound 3. 5 was further cyclized from C-2 to C-20 via the acid-catalyzed nucleophilic attack of C-1/C-2 double bond to the carbonyl group. The 2,3,4,5-tetrahydrooxepine might be formed by the following acid-catalyzed ring-opening of the epoxide by nucleophilic attack of 20-OH at C-8. C-12/C-20 double bond was arisen from the migration of C-11/C-12 double bond (Scheme 1). To explore the biological properties of isolated compounds, the cytotoxicity of 1−3 was evaluated against a limited panel of cancer cell lines, including human erythroleukemia (K562), leukemia (CCRF-CEM), breast carcinoma (T47D) and lymphoid T (MOLT 4) carcinoma cells, using the Alamar Blue assay.14,15 The results showed that 1−3 were not cytotoxic toward these four cancer cell lines. The in vitro anti-inflammatory effects of the compounds 1−3 were evaluated by measuring their ability in suppressing superoxide anion generation and elastase release in human neutrophils, using the previously described procedures.16,17 At a concentration of 10 μM, compound 1, but not 2 and 3, exhibited significant inhibition (56.0 ± 3.1%) toward N-formylmethionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)induced superoxide anion (O2•−) generation. At the same concentration, 2 and 3 weakly inhibited the elastase release (13.7 ± 3.5 and 29.2 ± 6.1%, respectively), while 1 was found to be inactive. The IC50 of 1 against the superoxide anion generation was further measured to be 7.3 ± 0.8 μM.
a
Spectrum recorded at 400 MHz in CDCl3. b100 MHz in CDCl3. c Deduced from DEPT. dJ values (Hz) in parentheses.
The absolute configuration of tortuosene A (1) was determined on the basis of the TDDFT ECD method.13 The CD spectrum of 1 in methanol displays a negative Cotton effect at 232 (Δε = −14.9) and a positive Cotton effect at 278 (Δε = +26.1). The calculated ECD spectrum of 1 was found to match well with the experimental ECD (see Figure 4), allowing the absolute configuration of tortuosene A (1) to be assigned as (1R,2R,7S,8R). Structurally, tortuosenes A and B (1 and 2) belong to a new C-2/C-20-cyclized cembranoid skeleton, for which we suggest the name tortuosane (Scheme 1, Figure 5). A plausible biosynthetic pathway for 1 and 2 from the cembranoid-type compound was proposed as shown in Scheme 1. The postulated cembranoidal precursor 4 could be oxidized at C-
■
ASSOCIATED CONTENT
S Supporting Information *
1
H and 13C NMR, COSY, HSQC, HMBC, and NOESY spectra of 1 and 2 in CDCl3 and 1H, 13C NMR, HSQC, and NOESY spectra of 1 in pyridine-d5, and experimental details including general procedures, bioactivity testing and DFT calculations. This material is available free of charge via the Internet at http://pubs.acs.org.
Figure 4. Experimental CD spectrum of 1 compared with the ECD spectrum calculated for the (1R,2R,7S,8R) enantiomer of 1. 1316
dx.doi.org/10.1021/ol403723b | Org. Lett. 2014, 16, 1314−1317
Organic Letters
■
Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel:+886-7-5252000. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS Financial support of this work was provided by the National Science Council (NSC-100-2320-B-110-001-M2) and Ministry of Education (01C030205) of Taiwan awarded to J.-H.S.
■
REFERENCES
(1) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat. Prod. Rep. 2013, 30, 237−323. (2) Wang, S.-K.; Hsieh, M.-K.; Duh, C.-Y. Mar. Drugs 2012, 10, 1433−1444. (3) Cheng, S.-Y.; Wang, S.-K.; Chiou, S.-F.; Hsu, C.-H.; Dai, C.-F.; Chiang, M. Y.; Duh, C.-Y. J. Nat. Prod. 2010, 73, 197−203. (4) Cheng, Y.-B.; Shen, Y.-C.; Kuo, Y.-H.; Khalil, A. T. J. Nat. Prod. 2008, 71, 1141−1145. (5) Lin, W.-Y.; Lu, Y.; Su, J.-H.; Wen, Z.-H.; Dai, C.-F.; Kuo, Y.-H.; Sheu, J.-H. Mar. Drugs 2011, 9, 994−1006. (6) Lin, W.-Y.; Su, J.-H.; Lu, Y.; Wen, Z.-H.; Dai, C.-F.; Kuo, Y.-H.; Sheu, J.-H. Bioorg. Med. Chem. 2010, 18, 1936−1941. (7) Lin, W.-Y.; Lu, Y.; Chen, B.-W.; Huang, C.-Y.; Su, J.-H.; Wen, Z.H.; Dai, C.-F.; Kuo, Y.-H.; Sheu, J.-H. Mar. Drugs 2012, 10, 617−626. (8) Su, J.-Y.; Long, K.-H.; Pang, T.-S. J. Am. Chem. Soc. 1986, 108, 177−178. (9) Leone, P. A.; Bowden, B. F.; Carroll, A. R.; Coll, J. C.; Meehan, G. V. J. Nat. Prod. 1993, 56, 521−526. (10) Zeng, L.-M.; Lan, W. J.; Su, J.-Y.; Zhang, G.-W.; Feng, X.-L.; Liang, Y.-J.; Yang, X.-P. J. Nat. Prod. 2004, 67, 1915−1918. (11) Jia, R.; Guo, Y.-W.; Chen, P.; Yang, Y.-M.; Mollo, E.; Gavagnin, M.; Cimino, G. J. Nat. Prod. 2007, 70, 1158−1166. (12) Toth, J. A.; Burreson, B. J.; Scheuer, P. J.; Finer-Moore, J.; Clardy, J. Tetrahedron 1980, 36, 1307−1309. (13) Diedrich, C.; Grimme, S. J. Phys. Chem. A 2003, 107, 2524− 2539. (14) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2000, 267, 5421−5426. (15) Nakayama, G. R.; Caton, M. C.; Nova, M. P.; Parandoosh, Z. J. Immunol. Methods 1997, 204, 205−208. (16) Hwang, T.-L.; Su, Y.-C.; Chang, H.-L.; Leu, Y.-L.; Chung, P.-J.; Kuo, L.-M.; Chang, Y.-J. J. Lipid. Res. 2009, 50, 1395−1408. (17) Hwang, T.-L.; Li, G.-L.; Lan, Y.-H.; Chia, Y.-C.; Hsieh, P.-W.; Wu, Y.-H.; Wu, Y.-C. Free Radical Biol. Med. 2009, 46, 520−528.
1317
dx.doi.org/10.1021/ol403723b | Org. Lett. 2014, 16, 1314−1317
