HETEROCYCLES

Note | Regular issue | Vol. 78, No. 6, 2009, pp. 1557-1562
Received, 19th December, 2008, Accepted, 11th March, 2009, Published online, 12th March, 2009.
DOI: 10.3987/COM-08-11634

■ Scyphiphorins C and D, Two New Iridoid Glycosides from the Chinese Mangrove Scyphiphora hydrophyllacea

Shu-Hong Tao, Shu-Hua Qi, Si Zhang,* Zhi-Hui Xiao, and Qing-Xin Li

Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China

Abstract

Two new iridoid glycosides with a lignan substituent, named scyphiphorins C (1) and D (2), were isolated from the stem bark of a Chinese mangrove Scyphiphora hydrophyllacea Gaertn. f., together with five known iridoid glycosides, 10-O-acetylgeniposidic acid (3), 7-deoxy-8-epi-loganic acid (4), mussaenoside (5), 7-deoxygardoside (6), and 10-deoxygeniposidic acid (7). The structures of compounds 1 and 2 were elucidated on the basis of spectroscopic data, especially 2D NMR techniques.

Rubiaceae family of mangrove plants has only one species, Scyphiphora hydrophyllacea Gaertn. f. that mainly distributed along the seashore of India, Malaysia, Australia and Hainan Island of China. Pharmacological studies revealed that the ethanol extract of the twigs of S. hydrophyllacea showed inhibitory effect towards human hepatoma SMMC-7721 cell line with IC50 value of 15.1 μg/ml.1 And previous chemical investigations on this plant uncovered over twenty compounds, including iridoids, triterpenoids, steroids, flavonoids, coumarins.1-5 Further investigation on the stem bark of the same plant resulted in the discovery of two new iridoid glycosides with a lignan substituent, scyphiphorins C (1) and D (2), together with five known iridoid glycosides, 10-O-acetylgeniposidic acid6 (3), 7-deoxy-8-epi-loganic acid7 (4), mussaenoside7 (5), 7-deoxygardoside8 (6), and 10-deoxygeniposidic acid9 (7) (Figure 1). To date, iridoid glycosides with such kind of lignan substituent haven’t been reported. The structure arguments for the two new compounds 1 and 2 are detailed below.

The ethanolic extract of the stem bark of S. hydrophyllacea was subjected to sequential extraction with petroleum ether, EtOAc, and n-BuOH . The resulting n-BuOH extract was chromatographed on silica gel and followed by preparative reverse-phase C18 HPLC to yield compounds 1-7.
Compound 1 was obtained as white amorphous powder, and had a molecular formula of C36H40O17 established by HR-ESI-MS ([M+Na]+, m/z 767.2134 ). Consequently, 1 had 17 degrees of unsaturation. The IR (KBr) absorption bands at 3420, 1715, 1709, 1605, and 1495 cm-1 indicated the existence of hydroxyl, carbonyl group, and aromatic ring. The 1H and 13C NMR data (Table 1) indicated that 11 units of the 17 unsaturations came from nine carbon-carbon double bonds, and two carbonyl groups. Therefore, the other six units of unsaturations came from six rings. In the 1H NMR spectrum of 1, the presence of a trans carbon-carbon double bond [δH 7.63, 6.37 (each 1H, d, J = 15.9 Hz)], another six olefinic protons [δH 7.54, 7.11, 7.03, 5.90 (each 1H, s), and 6.72 (2H, s)], two methoxy groups [δH 3.83 (6H, s)] were recognized. DEPT experiments revealed that 1 had two tertiary methyls (two methoxy), four methylenes, eighteen methines (eight olefinic) and twelve quaternary carbons (including two carbonyls). Among these signals, six carbons（δC 100.6 d, 78.4 d, 78.0 d, 74.9 d, 71.6 d, 62.9 t）belonging to a glucopyranosyl moiety. And the signal at δH 4.75 (1H, d, J = 7.8 Hz) suggested the relative configuration of the glucopyranosyl moiety was β-configuration.

Moreover, Ten signals at δC 153.2 d, 139.9 s, 131.4 d, 113.0 s, 98.4 d, 63.7 t, 47.5 d, 40.0 t, 36.7 d, were similar to those of 7-deoxy-8-epi-loganic acid7 (4), mussaenoside7 (5), 7-deoxygardoside8 (6), 10-deoxygeniposidic acid9 (7), and especially 10-O-acetylgeniposidic acid6 (3), which indicated that 1 should have the same geniposidic acid nucleus with a special substituent at C-10.
In addition, the NMR data showed the signals of two tetrasubstituted phenyl rings [δH 7.11, 7.03 (each 1H, s), and 6.72 (2H, s)], one trans carbon-carbon double bond [δH 7.63, 6.37 (each 1H, d, J = 15.9 Hz)], two methines [δH 5.62 (1H, d, J = 6.3 Hz), 3.55 (1H, m); δC (89.7 d, 55.3 t)] and one methylene [δH 3.88 (2H, m); δC 64.8 d] that attributed to an aliphatic –O-CH-CH-CH2-O– link. These NMR data were similar to those of cassyformin10 and cimicifugic acid,11 which suggested that 1 had a dihydrobenzofuran type neolignan moiety. In the HMBC spectrum (Figure 2), the correlations between H-7΄ and C-9΄, C-1΄, H-8΄ and C-9΄, C-2΄or C-6΄, H-7˝ and C-1˝, C-2˝or C-6˝, C-8˝, C-9˝, H-8˝ and C-5΄, C-6΄, C-9˝, C-7˝, C-1˝ confirmed the determination of the neolignan moiety. Furthermore, the HMBC correlations between H-10 and C-8, C-7, C-9′ showed that the neolignan moiety was located at C-10 of the iridoid aglycone. In particular, the NOESY interactions between H-7˝ and H-9˝, H-8˝ and H-2˝/6˝ indicated trans configuration of the dihydrofuran ring. Based on the above results, the structure of 1, named scyphiphorin C, was elucidated as shown in Figure 1.

Compound 2 was isolated as a white amorphous powder. Its molecular formula was established as C35H38O16 by HR-ESI-MS ([M+Na]+, m/z 737.2028 ). The 1H and 13C NMR spectral data of 2 (Table 1) were very similar to those of 1, except for the presence of a 1,3,5-trisubstituted phenyl ring [δH 7.30, 7.21, 7.20 (each 1H, s), δC 148.9 s, 148.3 s, 133.1 s, 119.9 d, 116.6 d, 110.9 d] instead of a 1,3,4,5-tetrasubstituted phenyl ring in compound 1. The NMR data of the 1,3,5-trisubstituted phenyl ring and the HMBC correlations between methoxy proton and C-5˝，H-7˝ and C-1˝, C-2˝, C-6˝ suggested that there was a 3-OH-5-OCH3-trisubstituted phenyl ring attached to C-7˝. More confirming evidences about the structural elucidation of 2 were shown in its HMBC spectrum (Figure 2). The NOESY interactions between H-7˝ and H-9˝, H-8˝ and H-2˝/6˝ also indicated the 7˝/8˝-trans configuration. Thus the structure of 2, named scyphiphorin D, was characterized as shown in Figure 1.

EXPERIMENTAL
General NMR spectra were recorded in methanol-d4 or pyridine-d5 using a Bruker AV-500 spectrometer (500MHz for 1H NMR and 125MHz for 13C NMR) with tetramethylsilane as the internal standard. UV spectra were obtained on a Varian cary 100 conc UV-Visible spectrophotometer and IR spectra were mesured on a Bruker VECTOR22 infrared spectrophotometer. HR-ESI-MS spectral were recorded on a Bruker Bio TOF Q MS spectrometer in positive ion mode. Optical rotation: POLAPTRONIC HNQW5 high resolution polarimeter. Preparative HPLC was carried out on ODS colums (250 × 10 mm i.d., Phenomenex luna) with a Waters 996 photodiode array detector. For Column Chromatography (CC), silica gel (200-300 mesh) (Qingdao Mar. Chem. Ind. Co. Ltd.), macroporous resin (D101)(Nankai University Chemical Plant), Sephadex LH-20 (Pharmacia), and RP-18 silica gel (40-60 mesh) (Merck) were used.
Plant Material The stem bark of Scyphiphora hydrophyllacea Gaertn. f. was collected in April 2005 from Wenchang, Hainan province, P. R. China. The identification of the plant was performed by Professor Si Zhang, the South China Sea Institute of Oceanology, Chinese Academy of Sciences. A voucher specimen (No. GKLMMM017) is deposited in the South China Sea Institute of Oceanology, Chinese Academy of Sciences.
Extraction and isolation The dried stem bark (10 kg) of S. hydrophyllacea was extracted with 95% ethanol at rt for four times (6 days per time). The extract was concentrated under reduced pressure. The viscous residue (560 g) was suspended in water and extracted successively with petroleum ether, EtOAc, and n-BuOH. The n-BuOH extract (150 g) was chromatographed on silica gel column and eluted using CHCl3/MeOH system (100:0―1:1) to yield Fractions 1-9. Fr.1 was refined in CHCl3/MeOH (1:1) to yield 3 (70 mg). Fr. 4 was separated by CC (RP-18 Si gel, H2O/MeOH 50:50) to yield 4 (7 mg). Fr. 7 was purified with preparative HPLC (H2O/MeOH 55:45) to yield 1 (8 mg) and 2 (10 mg). Fr. 8 was subjected to CC (Si gel, CHCl3/MeOH 5:1―1:1) to give 5 (30 mg), 6 (180 mg). Fr. 9 was purified by CC (RP-18 Si gel, H2O/MeOH 50:50) to afford 7 (25 mg).
Scyphiphorin C (1): White amorphous powder. +28.0 (c 8.0, MeOH); UV λmax (MeOH) nm (log ε): 230 (4.29), 329 (4.10). IR (KBr) cm-1: 3420, 2960, 1715, 1709, 1605, 1495, 1509, 1268, 1073. 1H and 13C NMR (see Table 1). HR-ESI-MS m/z 767.2134 ([M+Na]+, C36H40NaO17+, Calcd. 767.2158).
Scyphiphorin D (2): White amorphous powder. +27.0 (c 10.0, C5H5N). UV λmax (MeOH) nm (log ε): 228 (4.20), 331 ( 3.96). IR (KBr) cm-1: 3425, 2950, 1716, 1710, 1603, 1504, 1274, 1065. 1H and 13C NMR (see Table 1). HR-ESI-MS m/z 737.2028 ([M+Na]+, C35H38NaO16+, Calcd 737.2052).

ACKNOWLEDGEMENTS
Support for this work from China National Key Program for Base Research（2005CCA04800）is gratefully acknowledged.

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