Chinese Journal of Chemistry, 2006, 24, 393—395 Full Paper

* E-mail: jiangb@mail.sioc.ac.cn or huangh2003@mail.sioc.ac.cn; Tel.: 0086-21-54925201; Fax: 0086-21-64166128 Received November 21, 2005; revised December 19, 2005; accepted January 25, 2006.

© 2006 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Ring-B Linked Bidihydroflavonoids from Thuidium kanedae Sak

ZHAO, Xina,b(赵鑫) JIANG, Hai-Xiab(姜海霞) HUANG, Hao*,b(黄浩) ZHU, Rui-Lianga(朱瑞良) JIANG, Biao*,b(姜标)

a School of Life Science, East China Normal University, Shanghai 200062, China b Department of Modern Organic Synthesis, Shanghai Institute of Organic Chemistry,

Chinese Academy of Sciences, Shanghai 200032, China

A new bidihydroflavonoid, thuidinin 1, together with 5 known compounds including one known biflavonoid 3',3'''-binaringenin (2) and four other known natural products pentadecane (3), methyl β-orcinolarboxylate (4), β-sitosterol (5) and daucostenol (6), were isolated from leaves of Thuidium kanedae Sak. Their structures were es-tablished on the basis of 1D and 2D spectroscopic analysis. The absolute stereochemistry of compounds 1 and 2 was determined by CD spectra.

Keywords Thuidium kanedae Sak, bidihydroflavonoid, spectroscopic analysis, CD spectrum

Introduction

Thuidium kanedae Sak is a moss of the genus Thuidium in the Thuidiaceae family. In 1979, triterpe-noids were isolated from the plant.1 Chalcones and their antibacterial activity were further reported later in 1991.2 No further chemical studies on this plant have been published since then. This paper reports the isola-tion and identification of a new biflavonoid, thuidinin 1, together with 5 known compounds 2—6. Their struc-tures were elucidated as 4',4''',5",7,7"-pentahydroxy- 5-methoxy-3',3'''-biflavanone (1), 3',3'''-binaringenin (2),3 pentadecane (3),4 methyl β-orcinolarboxylate (4),5 β-sitosterol (5)6 and daucostenol (6)6 based on extensive spectroscopic data analyses and comparisons with those in literatures. To the best of our knowledge, compound 1 is a new compound. The 2R absolute configuration of compounds 1 and 2 was assigned for the first time (Fig-ure 1). In addition, 3—6 were isolated for the first time from T. kanedae Sak.

Experimental

General procedures

Melting points were determined on an X4 apparatus and uncorrected. 1H, 13C NMR, COSY, HMQC, HMBC and NOESY spectra were recorded on a Bruker DRX- 400 MHz NMR spectrometer with TMS as internal standard. Optical rotations were measured on a Perkin Elmer 243B polarimeter. Mass spectral measurements were performed on a Fining 4021 or Fining MAT 8403 gas chromatography/mass spectrometer at 70 eV and mass data were tabulated as m/z. CD spectra measure-ments were performed using a JASCO-710 CD spec-tropolarimeter. Silica gel of 200—300 mesh, silica

Figure 1 Bidihydroflavonoids from Thuidium kanedae Sak.

gel H and silica gel GF254 were purchased from Marine Chemical Factory, Qindao, China. Sephadex LH-20 was purchased from Sanling Co., Japan.

Biological material

T. kanedae Sak was collected in Zhejiang Province of China, in June 2004 and identified by Professor Youfang Wang of East China of Normal University. A voucher specimen (No. 20040326) was deposited in the college of Life of East China of Normal University, Shanghai, China.

Cytotoxic activity

Cell viability was determined by measuring MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, Sigma) dye absorbance of living cells. Briefly, 1 d after cells (5000 cells per well) were seeded in 96-well microtiter plates, the cells were treated with 10 µmmol•L－1 compounds 1 and 2. DMSO alone was used as a control. Cells were incubated at 37 ℃ in a hu-midified atmosphere containing 5% CO2. 3 d after the treatment, 15 µL MTT solution (1 g•L－1) was added to

394 Chin. J. Chem., 2006, Vol. 24, No. 3 ZHAO et al.

© 2006 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

each well and the plates were incubated for an addi-tional 4 h at 37 ℃. To achieve solubilization of the formazan crystal formed in viable cells, 50 µL MTT lysis solution (10% isobutanol, 20% SDS and 0.02 mmol•L－1 hydrochloric acid) was added to each well before absorbance at 550/650 nm was measured. Each experiment was performed in triplicate and repeated at least twice.

Extraction and isolation

The whole plant of T. kanedae Sak was air-dried for 2 weeks and ground mechanically to give 2 kg powder. The powder was extracted with 95% EtOH three times at room temperature, and the solvent was evaporated in vacuo. The residue was partitioned in H2O and ex-tracted with petroleum ether and ethyl acetate three times, respectively. The petroleum ether and EtOAc extracts were concentrated in vacuo to afford 48 g and 15 g of residue, respectively. The EtOAc portion was subjected to column chromatography (CC) on silica gel, using CHCl3/ Me2CO (from 10∶0 to 5∶5, V∶V) as eluent. By combining the fractions with TLC (GF254) monitor-ing, 7 fractions were obtained. Fraction 3 (～288 mg) was rechromatographed on a silica gel column eluting with petroleum ether/EtOAc (19∶1 to 3∶1, V∶V) to produce compound 3 (15 mg). Fraction 5 was applied to silica gel column and developed with CHCl3/Me2CO (from 9∶1 to 1∶1, V∶V) to yield 1 (53 mg), 2 (46 mg) and 6 (10 mg). LH-20 column chromatography was em-ployed to further purify compound 1 (47 mg) and 2 (38 mg). The petroleum portion was applied to CC on silica gel, eluted with petroleum ether/EtOAc (from 19∶1 to 1∶1, V∶V) to yield 4 (18 mg) and 5 (122 mg).

Thuidinin 1: Light yellow amorphous powder. m.p.

241 — 243 ℃ ; 20D[ ]α 12.34 (c 1.0, CH3OH); CD

(CH3OH, 20 ℃) ∆OD292＝＋25.661(mdeg), ∆OD319＝

－9.549 (mdeg); 1H and 13C NMR spectral are liated in Table 1.

3',3'''-Binaringenin (2): Light yellow amorphous powders. m.p. 241 — 243

℃ ; 20D[ ]α 10.17 (c 1.0,

CH3OH); CD (CH3OH, 20 ℃ ) ∆OD277＝＋30.045 (mdeg), ∆OD319＝－12.350 (mdeg); 1H and 13C NMR spectral data are also listed in Table 1. 3',3'''-Binaringenin is a known compound, that was identified by its 1H and 13C NMR spectra. The HMBC data of 2 are reported for the first time (Table 1).

Pentadecane (3): Colorless liquid. IR (KBr) νmax: 2924, 2851, 1689, 1464, 1029, 807 cm－1; EIMS (70 eV) m/z: 213 ([M＋H]＋), 197, 179, 155, 141, 127, 111, 99, 85, 71, 57.

Methyl β-orcinolarboxylate (4): Colorless crystals. m.p. 140—142 ℃; 1H NMR (CDCl3) δ: 12.03 (s, 1H, OH), 6.21 (s, 1H), 5.17 (br s, 1H, OH), 3.92 (s, 3H, OMe), 2.46 (s, 3H, CH3), 2.10 (s, 3H, CH3).

β-Sitosterol (5): Colorless needles. m.p. 138—140 ℃; it gave positive result in Libermann-Burchard test; EIMS (70 eV) m/z: 414 (M＋).6

Daucostenol (6): White amorphous powder. m.p. 268—270 ℃, its IR, 1H and 13C NMR spectral data were consistent with literature values.6

Results and discussion

The AcOEt-soluble fraction and petroleum-soluble portion of the whole plant of T. kanadae Sak were separated by repeated column chromatography and re-crystallization to give a new biflavanone, thuidinin 1, and 5 known compounds 2—6.

Table 1 1H, 13C, and HMBC NMR spectra data of compounds 1 and 2 (C5D5N, 400 MHz)

1 2 No.

δH δC HMBC δH δC HMBC

2/2'' 5.60 (br d, J＝13.0 Hz) 80.0/80.0 H-2', 6', 3/H-2''', 6''', 3'' 5.50 (dd, J＝12.0, 3.0 Hz) 80.2 H-2'/2'', 6', 3/H-2''',6''', 3''

3 ax/3'' ax 3.00 (dd, J＝17.0, 13.0 Hz) 46.5/43.7 3.46 (dd, J＝17.0, 12.0 Hz) 43.8

3 eq/3'' eq 3.40 (1 dd, J＝7.0, 3.0 Hz) 3.00 (dd, J＝17.0, 3.0 Hz)

4/4'' 189.2/196.9 H-3, 2/H-3'', 2'' 196.8 H-2/2'', H-3/3'', H-8/8''

5/5'' 163.7/165.4 H-6, OMe/H-6'' 165.5 H-6/H-6''

6/6'' 6.50 (br s)/6.48 (br s) 94.9/96.5 H-8/H-8'' 6.50 (br s) 96.6 H-8/H-8''

7/7'' 166.6/169.0 H-8/H-8'' 169.4 H-8/H-8''

8/8'' 6.60 (br s)/6.51 (br s) 97.3/97.6 H-6/H-6'' 6.53 (br s) 97.7 H-6/H-6''

9/9'' 165.9/164.4 164.5

10/10'' 105.0/103.1 H-8/H-8'' 103.1 H-8/H-8''

CH3O 3.96 (s) 56.2

1'/1''' 130.3/130.8 H-2, 2' 129.7 H-2, 2'

2'/2''' 7.90 (br s) 131.4/131.3 7.90 (br s) 131.2

3'/3''' 128.0/128.0 128.5

4'/4''' 157.5/157.3 H-2', 5', 6'/H-2''', 5''', 6''' 158.7 H-2', 5', 6'/H-2''', 5''', 6'''

5'/5''' 7.48 (d, J＝8.0 Hz) 117.8/117.7 H-2', 6'/H-2''', 6''' 7.48 (d, J＝8.0 Hz) 118.4 H-2', 6'/H-2''', H-6'''

6'/6''' 7.70 (d, J＝8.0 Hz) 127.8/127.8 H-2', 5'/H-2''', 5''' 7.70 (d, J＝8.0 Hz) 128.0 H-2', H-5'/H-2''', H-5'''

Bidihydroflavonoids Chin. J. Chem., 2006 Vol. 24 No. 3 395

© 2006 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The known compounds were determined as 3',3'''- binaringenin (2),3 pentadecane (3),4 methyl β-orcino- larboxylate (4),5 β-sitosterol (5)6 and daucostenol (6)6, respectively, from their MS and 1H NMR spectra, which were identical to the reported data.

Thuidinin 1 was obtained as light yellow amorphous powder with the molecular formula of C31H24O10 as de-termined by the ESIMS and NMR spectra. The 1H NMR spectrum of compound 1 displayed two sets of charac-teristic ABX type signals accounting for six protons at δ 5.60 (br d, J＝13.0 Hz, 2H, H-2, 2''), 3.40 (dd, J＝17.0, 13.0 Hz, 2H, H-3, 3'', trans) and 3.00 (dd, J＝17.0, 3.0 Hz, 2H, H-3, 3'', cis), and each set corresponded to a dihydropyrone moiety in flavanone structure, indicating a biflavanone skeleton for compound 1. Other features in the 1H NMR spectrum were a single methoxyl (3.96, s), two sets of three ABX aromatic proton signals at δ 7.90 (br s, 2H, H-2', 2'''), 7.70 (d, J＝8.0 Hz, 2H, H-6', 6''') and 7.48 (d, J＝8.0 Hz, 2H, H-5', 5'''), typically as-signable to 2' (2'''), 5' (5''') and 6' (6''') protons in B-rings of the biflavanone skeleton. The four singlet signals at δ 6.48 (br s, 1H), 6.50 (br s, 1H), 6.51 (br s, 1H) and 6.60 (br s, 1H) were attributed to the H-6 (H-6'') and H-8 (H-8'') of the two A-rings, which generally resonates at higher field. These data suggest that 1 is a biflavanone interflavonyl merged from two similar flavanones, and the only difference between two flavanone units is that the hydroxyl group in one monomer was replaced by methoxyl group in the other. Furthermore, it could be concluded that both flavanone units possessed 5,7-dioxygenated substituents in ring A and 1,3,4- trisubstituted pattern in the ring B, which was further corroborated by the 13C NMR data.

The 13C NMR of compound 1 gave 31 signals, in-cluding eight oxygenated aromatic carbons (δ 164.0, 166.6, 165.9, 157.5, 157.3, 164.4, 169.0 and 165.4), which confirmed 6 oxygenated substituents in rings A and B of the biflavanone compound 1. Of these oxygen-ated substituents, one methoxyl group was readily ap-parent from the appearance of carbon signal at δ 56.2 (s) in the 13C NMR spectrum as well as its 1H NMR signal at 3.96 (s). Thus, five hydroxyl groups in 1 could be inferred from the molecular formula of C31H24O10. Careful analysis of the 13C NMR of compound 1 clearly verified the biflavanone 1 to be composed of two similar flavanone units as most carbon signals appeared in pairs with very similar chemical shifts except the single methoxyl carbon at δ 56.2 (s) and two carbonyl signals with quite different chemical shifts at δ 196.9 and 189.2 assignable to C-4 and C-4'', respectively, indicative of different substitutes at C-5 and C-5'' in the two A rings.

The methoxyl group at C-5 and the interflavanonyl linkage at 3', 3''' were concluded with evidences from 2D NMR spectra. The location of the methoxyl group at C-5 was assigned based on the observed NOESY corre-lation from δH 3.96 (OMe-5) to δH 6.50 (H-6). The HMBC correlations from the oxymethine signal C-2

(C-2') at δ 80.0 (d) to the singlet proton signal H-2' (2''') at δ 7.90 (br s, 2H) and H-6' (6''') at δ 7.70 (d, J＝8.0 Hz, 2H) established that C-3' (C-3''') was substituted, further HMBC correlations for H-2' (2''')/C-3' (C-3''') and H-2' (2''')/C-4' (C-4''') substantiated the interflavanonyl link-age at 3', 3'''. Evidence for the connection was also ob-tained from NOESY cross-peaks between H-2 and H-2' as well as between H-2 and H-6' which unambiguously proved a 3', 3''' linkage between the subunits of the pro-posed dimmer. The CD spectrum of compound 1 dis-played positive and negative cotton effects at 292 and 319 nm, respectively. This observation indicated 2R absolute stereochemistry for the biflavanone 1,7 a rarely encountered absolute configuration among natural fla-vonoids.8-10 A combination of 2D NMR techniques, in-clusive of 1H-1H COSY, HMQC, HMBC and NOESY experiments, enabled the assignments of 1H NMR and 13C NMR data for compound 1 (Table 1 and Figure 2). Consequently, the structure of compound 1 was deter-mined as 4',4''',5'',7,7''-pentahydroxy-5-methoxy-3',3''- biflavanone named thuidinin. To our knowledge, it has not been reported before.

Compounds 1 and 2 were evaluated for their cyto-toxic activity against A549 human cancer cell line, no cytotoxic activity was observed for compound 1 and 2 under assayed condition.

Figure 2 The key HMBC (from C to H with single arrow curves) and selected NOE correlations (double arrow curves) of 1.

References

1 Asakawa, Y.; Tokunaga, N.; Toyota, M. J. Hattori Bot. Lab. 1979, 45, 395.

2 Zheng, W.-F. CHENIA 1996, 1, 64. 3 Seeger, T.; Geiger, H.; Zinsmeister, H. D.; Rozdzinski, W.

Phytochemistry 1993, 34, 295. 4 Cong, P.-Z.; Su, K.-M. Dictionary of Analytical Chemistry,

Vol. 9, Chemical Industry Press, Beijing, 2000, (in Chi-nese).

5 Ding, Z.-H.; Ding, J.-K.; Lou, J.-F. Acta Bot. Yunnanica 1990, 12, 99 (in Chinese).

6 Li, Y.-L.; Wang, H.-L.; Suo, Y.-R. Acta Bot. Boreali- Occidentalia Sin. 2004, 24, 1292.

7 Gaffield, W. Tetrahedron 1970, 26, 4093. 8 Seeger, T.; Geiger, H.; Zinsmeister, H. D. Z. Naturforsch. C

1992, 47, 667. 9 Sievers, H.; Burkhardt, G.; Becker, H.; Zinsmeister, H. D.

Phytochemistry 1992, 31, 3233. 10 Su, B.-N.; Park, E. J.; Vigo, J. S.; Graham, J. G.; Cabieses,

F.; Fong, H. H. S.; Pezzuto, J. M.; Kinghorn, A. D. Phyto-chemistry 2003, 63, 335.

(E0511215 ZHAO, C. H.)