Novel cyclic sesquiterpene peroxides from the Formosan
soft coral Sinularia sp.
Chih-Hua Chao,
aChi-Hua Hsieh,
aShin-Pin Chen,
aChung-Kuang Lu,
b,cChang-Feng Dai,
dYang-Chang Wu
eand Jyh-Horng Sheu
a,*a
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan, ROC
b
National Museum of Marine Biology and Aquarium, Checheng, Pingtung 944, Taiwan, ROC
cInstitute of Marine Biotechnology, National Dong Hwa University, Checheng, Pingtung 944, Taiwan, ROC d
Institute of Oceanography, National Taiwan University, Taipei 106, Taiwan, ROC
eGraduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC
Received 30 November 2005; revised 16 January 2006; accepted 23 January 2006
Abstract—Four novel cyclic peroxide-containing sesquiterpenes (1–4), with a c-alkylidene-a-methyl-a,b-unsaturated c-lactone moiety, have been isolated from a Formosan soft coral of the genus Sinularia. Their structures were elucidated mainly by extensive 1D and 2D NMR experiments.
2006 Elsevier Ltd. All rights reserved.
Soft coral of the genus Sinularia has been found to be a rich source of bioactive secondary metabolites.1Cyclic
peroxides are of great interest because they often exhibit a wide spectrum of biological activities including anti-parasitic and cytotoxic activities against cancer cells.2–4
During the course of our investigation on the bioactive chemical constituents from marine invertebrates,5–15
four novel sesquiterpenoids, sinularioperoxides A–D (1–4) possessing a cyclic peroxide and a c-alkylidene-a-methyl-a,b-unsaturated c-lactone moieties, have been isolated from a soft coral Sinularia sp., collected off the northeastern Taiwan coast in May 2004, at a depth of 10 m. We describe herein the isolation and structure elucidation of these compounds.
The organism (1.0 kg fresh wt) was collected and freeze dried. The freeze-dried material was minced and extracted exhaustively with EtOH. The organic extract was concentrated to an aqueous suspension and parti-tioned between EtOAc and water. The EtOAc extract (9.8 g) was fractionated by open column chromatogra-phy on silica gel using n-hexane and n-hexane–EtOAc
mixtures of increasing polarity. A fraction eluted with n-hexane/EtOAc (1:4) was subjected to Sephadex LH-20 column (2· 90 cm) using acetone and followed by normal phase HPLC (n-hexane/acetone, 8:1) to afford compounds 1 (5.0 mg), 2 (1.0 mg), 3 (1.1 mg), and 4 (3.0 mg).
Sinularioperoxide (1),½a25D 2 (c 1.64, CHCl3), was
iso-lated as a colorless oil. Its HRESIMS exhibited a pseudomolecular ion peak at m/z 303.1209 [M+Na]+, corresponding to the molecular formula C15H20O5
(calcd 303.1208). Thus, 1 possesses six degrees of unsatu-ration. The IR spectrum of 1 was found to exhibit absorptions of hydroxy (3366 cm1), carbon–carbon double bond (1668 cm1), and lactone carbonyl groups (1757 cm1). The characteristic NMR signals [dH 7.04
(1H, s, H-3), 5.66 (1H, s, H-5), and 2.01 (3H, s, H3-13); dC 170.7 (C-1), 129.1 (C-2), 138.7 (C-3), 146.2
(C-4), 117.5 (C-5), and 10.5 (C-13)] and UV absorption at kmax273 nm indicated the presence of
c-alkylidene-a-methyl-a,b-unsaturated c-lactone moiety.16,17 Signals
resonating at d 135.4 (s) and 127.5 (d) in 13C NMR spectrum of 1 suggested the presence of a trisubstituted double bond. The above functionalities account for five of the six degrees of unsaturation in the molecule of 1, suggesting that there should be an additional ring in the molecule of sinularioperoxide A. The gross structure of metabolite 1 was further established by the 2D NMR
0040-4039/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetlet.2006.01.107
Keywords: Sinularioperoxide; Sinularia; Sespuiterpene; Cyclic peroxide; Soft coral.
* Corresponding author. Tel.: +886 7 5252000x5030; fax: +886 7 5255020; e-mail:sheu@mail.nsysu.edu.tw
studies, particularly in 1H–1H COSY, HMQC, and HMBC experiments. The correlations of1H–1H COSY revealed two spin systems, as depicted inFigure 1. Its HMBC spectrum showed many informative correla-tions, such as H-5 to C-3, C-4, and C-6, H3-13 to
C-1, C-2, and C-3, H3-14 to C-5, C-6, and C-7; and
H3-15 to C-9, C-10, and C-11 (Fig. 1). Moreover,
acet-ylation of 1 yielded 1a. It was found that the NMR signal of H2-12 in 1 at d 4.21 was downfield shifted to
d4.61 in 1a (Tables 1 and 2), confirming that the
hydr-oxy group of 1 should be attached at C-12. From the above 2D NMR data, the molecular formula obtained from the HRESIMS, and the reductive cleavage of 1a by zinc powder and acetic acid in EtOAc to afford diol 1b18,19 (99% yield) (Fig. 2 and Table 2) confirmed the
cyclic peroxide linkage between C-6 and C-9. Combina-tion of the above observaCombina-tions led to the establishment of the planar structure of 1.
The relative stereochemistry of 1 was determined by the NOE correlations observed in a NOESY experiment and also by the analysis of the coupling constant of H-9. It was found that this proton (d 4.44) displayed a large coupling constant (J = 11.0 Hz), revealing its axial orientation. In the NOESY spectrum of 1, H-9 was found to show NOE interactions with both H-8a and H-7a, and H3-14 exhibited NOE correlation with H-7a
and H-7b. Thus, H3-14 must be equatorially oriented,
and positioned on the a face (Fig. 3). The E geometry was assigned for the 10,11-double bond on the basis of the observation of an NOE correlation between H3-15
and H2-12. The Z geometry of both 2,3- and 4,5-double
bonds was established by the observation of the NOE correlations of H3-13/H-3 and H-3/H-5. Therefore, the
relative structure of 1 was established unambiguously. Compound 2, ½a25D 40 (c 0.80, CHCl3), revealing IR
absorptions at 3422, 1755, and 1668 cm1, and UV absorption (MeOH) at kmax (log e) 272 nm (4.19), was
isolated as a colorless oil. Its HRESIMS exhibited a pseudomolecular ion peak at m/z 303.1206 [M+Na]+ and established the same molecular formula as that of 1. Thus, 2 is an isomer of 1. It was found that except for H-9, the1H NMR spectral data of 2 are very similar to those of 1. Inspection of the 13C NMR data of com-pounds 1 and 2 also showed obvious differences between the carbon shifts at C-9 and C-15. The carbon resonance
O O HO O O 13 1 4 9 12 14 15 11 6 5 1 O O O O 2 HO O O HO O O 3 O O HO 4 O O O O HO O O 1 4 9 12 14 15 11 6 5 13 HMBC 1H−1H COSY
Figure 1. Selective1H–1H COSY and HMBC correlations of 1.
Table 1.1H and13C NMR spectral data of compounds 1–4
C # 1 2 3 4 1 Ha 13Cb 1Ha 13Cb 1Ha 13Cb 1Ha 13Cb 1 170.7 (s)d 170.7 (s) 170.6 (s) 170.2 (s) 2 129.1 (s) 129.2 (s) 129.6 (s) 131.6 (s) 3 7.04 s 138.7 (d) 7.06 s 138.6 (d) 7.00 s 138.7 (d) 7.56 s 136.8 (d) 4 146.2 (s) 146.2 (s) 146.8 (s) 148.8 (s) 5 5.66 s 117.5 (d) 5.66 s 117.3 (d) 5.27 s 116.4 (d) 5.60 s 115.8 (d) 6 81.0 (s) 81.1 (s) 80.3 (s) 79.5 (s) 7 a1.80 m 34.0 (t) 1.80 m 33.7 (t) 2.12 m, 2H 31.6 (t) 1.92 m, 2H 34.0 (t) b2.58 ddd (13.0, 3.5, 3.5)c 2.59 ddd (12.0, 3.5, 3.5) 8 a1.70 m 25.4 (t) 1.58 m 25.4 (t) 1.83 m 23.1 (t) 1.81 m 23.7 (t) b1.75 m 1.83 m 1.98 m 2.02 m 9 4.44 d (11.0) 84.8 (d) 4.87 dd (10.3, 2.5) 80.2 (d) 4.41 dd (8.0, 3.5) 83.8 (d) 4.44 br d (9.5) 84.5 (d) 10 135.4 (s) 136.0 (s) 136.0 (s) 135.6 (s) 11 5.63 t (6.5) 127.5 (d) 5.62 t (6.5) 129.7 (d) 5.70 t (6.5) 126.7 (d) 5.71 t (6.5) 127.3 (d) 12 4.21 d (6.5), 2H 59.1 (t) 4.16 dd (12.8, 6.8) 58.5 (t) 4.25 d (6.5), 2H 59.3 (t) 4.26 d (6.5), 2H 59.2 (t) 4.21 dd (12.8, 7.3) 13 2.01 s 10.5 (q) 2.02 s 10.5 (q) 2.01 s 10.5 (q) 2.01 s 10.8 (q) 14 1.41 s 25.2 (q) 1.41 s 25.4 (q) 1.57 s 22.6 (q) 1.56 s 24.0 (q) 15 1.68 s 14.0 (q) 1.69 s 19.5 (q) 1.75 s 13.8 (q) 1.75 s 13.7 (q) aSpectra recorded at 500 MHz in CDCl 3at 25C. b Spectra recorded at 125 MHz in CDCl3at 25C. c
J values (in Hz) in parentheses.
d
Multiplicity deduced by DEPT and indicated by usual symbols.
of C-9 at d 84.8 in 1 was found to be upfield shifted to d 80.2 in 2, while that of C-15 at d 14.0 in 1 was shifted to lower field (d 19.5) in 2 (Table 1). The above differences may be due to the E geometry of 10,11-double bond in 1 has been converted to Z geometry in 2. Those findings were further confirmed by an NOE correlation between H3-15 and H-11 (Fig. 3). This phenomenon can be
explained by the significant c-effect20,21 arising from
the steric compression between 11-CH2OH and C-15
in 1 and 11-CH2OH and C-9 in 2. After detailed
exami-nation of 1D and 2D NMR, the structure of 2 was established and named as sinularioperoxide B.
Sinularioperoxide C (3), ½a25D 2 (c 1.68, CHCl3), was
isolated as a colorless oil. The molecular formula 3 was determined by HRESIMS and was found to be identical to that of 1. The IR (mmax 3366, 1755, and
1672 cm1) and UV (kmax 272 nm) spectral data of 3
are almost the same as those of 1. Thus, 3 is the geomet-ric isomer of both 1 and 2. By comparison of the NMR data of 3 with those of 1, the obvious differences were observed for chemical shifts of C-14 (d 22.6 in 3 and 25.2 in 1), H-5 (d 5.27 in 3 and 5.66 in 1), and H-7 (d
2.12, 2H, in 3 and 1.80 and 2.58 in 1) (Table 1). These findings suggested that the equatorial methyl H3-14 in
1 might be converted to the axial orientation in 3. The above findings were further confirmed by the detailed inspection of the NOESY spectrum, which showed NOE correlations of H-9/H-8a and H-8b/H3-14
(Fig. 3). Thus, the molecular structure of 3, including the relative configuration, was fully determined. HRESIMS of sinularioperoxide D (4), appeared as a colorless oil with an ½a25D value of2 (c 1.68, CHCl3),
exhibited a pseudomolecular ion peak at m/z 303.1206 [M+Na]+ and established the same molecular formula as those of 1–3. The IR and UV spectra of 4 showed absorptions at 3418, 1766, and 1666 cm1 and 273 nm, respectively. By comparison of the 1H and 13C NMR spectra of 3 and 4, these two compounds have very sim-ilar spectral data except for the deviation between pro-ton signals of H-5 in 3 (d 5.27) and 4 (d 5.60) (Table 1). The NOESY spectra of 3 and 4 were also similar except for the lack of an NOE correlation between H-3 and H-5 in 4 (Fig. 3). Therefore, 4 should contain an E double bond between C-4 and C-5 and the structure of this metabolite was determined unambiguously. Preliminary biological activity screening revealed that these four compounds are not active against the growth of a limited panel of cancer cell lines, including A549 (human lung carcinoma), HepG2 (human hepatocellular carcinoma), MCF7 and MAD-MB-231 (both human breast carcinoma) cells. The results of further biological activity screening will be reported elsewhere in the future.
Table 2.1H and13C NMR chemical shifts of derivatives 1a and 1b
C # la 1b 1Ha 13Cb 1Hc 13Cd 1 170.7 sf 170.3 s 2 129.1 s 129.0 s 3 7.05 s 138.7 d 6.99 s 138.6 d 4 146.2 s 146.3 s 5 5.66 s 117.4 d 5.25 s 120.0 d 6 81.0 s 72.8 s 7 1.80 m 34.0 t 1.80 m 39.1 t 2.60 br d (10.7)e 1.90 m 8 1.70 m 25.5 t 1.67 m 29.7 t 1.75 m 1.30 m 9 4.45 br d (7.5) 84.5 d 4.05 t (6.2) 77.3 d 10 137.7 s 143.0 s 11 5.54 t (6.2) 122.3 d 5.59 t (6.5) 119.6 d 12 4.61 d (6.8) 60.7 t 4.62 d (6.8) 60.9 t 13 2.02 s 10.4 q 2.00 s 10.5 q 14 1.41 s 25.2 q 1.48 s 29.0 q 15 1.71 s 14.0 q 1.68 s 12.3 q OAc 2.06 s 20.9 q 2.06 s 21.0 q 170.9 s 171.0 s a Spectra recorded at 300 MHz in CDCl3at 25C. b Spectra recorded at 75 MHz in CDCl3at 25C. c Spectra recorded at 400 MHz in CDCl3at 25C. d Spectra recorded at 100 MHz in CDCl3at 25C. eJ values (in Hz) in parentheses.
fMultiplicity deduced by DEPT and indicated by usual symbols.
O O AcO O O 1a HO HO AcO O O S * R * R* 1b Zn, HOAc/EtOAc S*
Figure 2. Conversion of 1a to diol 1b.
H H H O O HO O O H H 13 1 4 9 12 14 15 11 6 5 1 H H H O O O O H H 2 HO H O O HO H O O H 3 H O O HO H H 4 O O
Figure 3. Selective NOESY correlations of 1–4.
It has to be noted here that cyclic peroxides 1–4 are the brand-new type of cyclic peroxy terpenoids with a c-alkylidene-a-methyl-a,b-unsaturated c-lactone moiety. To the best of our knowledge, this type of terpenoids was discovered for the first time.
Acknowledgments
Financial support of this work provided by Ministry of Education and National Science Council of Taiwan awarded to J.-H.S. is greatly appreciated.
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