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Three new phenolic compounds from Dalbergia odorifera ab

a

a

a

Hao Wang , Wen-Hua Dong , Wen-Jian Zuo , Hui Wang , Hui-Min b

a

a

Zhong , Wen-Li Mei & Hao-Fu Dai a

Key Laboratory of Biology and Genetic Resources of Tropical Crops, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Ministry of Agriculture, Haikou571101, China b

College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao266042, China Published online: 06 Dec 2014.

To cite this article: Hao Wang, Wen-Hua Dong, Wen-Jian Zuo, Hui Wang, Hui-Min Zhong, Wen-Li Mei & Hao-Fu Dai (2014): Three new phenolic compounds from Dalbergia odorifera, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.968559 To link to this article: http://dx.doi.org/10.1080/10286020.2014.968559

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Journal of Asian Natural Products Research, 2014 http://dx.doi.org/10.1080/10286020.2014.968559

Three new phenolic compounds from Dalbergia odorifera Hao Wangab, Wen-Hua Donga, Wen-Jian Zuoa, Hui Wanga, Hui-Min Zhongb, Wen-Li Meia* and Hao-Fu Daia*

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a Key Laboratory of Biology and Genetic Resources of Tropical Crops, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Ministry of Agriculture, Haikou 571101, China; bCollege of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China

(Received 26 February 2014; final version received 18 September 2014) Three new phenolic compounds (1 – 3) were isolated from the heartwood of Dalbergia odorifera T. Chen. (Leguminosae). Their structures were established based on spectroscopic methods including 1D and 2D NMR (HSQC, COSY, HMBC and ROESY). Compound 2 exhibited cytotoxicity against BEL-7402 tumor cell lines. Keywords: leguminosae; Dalbergia odorifera; phenolic compounds; cytotoxic activity

1.

Introduction

The heartwood of Dalbergia odorifera T. Chen. (Leguminosae) has been used in traditional Chinese medicine to treat blood disorder, ischemia, swelling, necrosis and rheumatic pain, as a famous South China Medicine [1]. Phytochemical studies showed that D. odorifera mainly contained flavonoids and volatile oil, which showed a variety of biological activities, such as anti-inflammatory, antioxidant, antimicrobial, and antiplatelet activities [2 – 14]. And the types of flavonoids isolated from D. odorifera include pterocarpans, neoflavones, isoflavans, isoflavones, flavanones, isoflavanones, and so on. In our present investigation of the bioactive principles of this plant, three new phenolic compounds (1 – 3) (Figure 1) were isolated from the heartwood of D. odorifera. All the compounds were evaluated for their cytotoxicity against chronic myelogenous leukemia cell line (K562), human gastric carcinoma cell line (SGC-7901), and human hepatocellular carcinoma cell line (BEL-7402). This paper describes the

isolation, structural elucidation, and bioactivities of these compounds from the heartwood of D. odorifera. 2.

Results and discussion

Compound 1 was obtained as a yellow amorphous solid. The molecular formula was determined to be C24H22O6 from the molecular ion peak [M 2 H]2 at m/z 405.1340 in the negative HR-ESI-MS. 1H NMR data of 1 revealed the presence of a monosubstituted benzene ring (dH 7.61, 2H, br d, J ¼ 7.1 Hz; dH 7.35, 2H, br t, J ¼ 7.1 Hz; dH 7.27, 1H, br t, J ¼ 7.1 Hz), four singlet aromatic protons (dH 6.99, 6.54, 6.47 and 5.77), three methoxyl groups (dH 3.86, 3.78 and 3.75), and two sets of methylene protons (dH 3.04– 3.10, 2.69 – 2.77, 2.71 – 2.80 and 2.48 – 2.55). The 13C NMR spectrum of 1 showed signals for 16 aromatic and olefinic carbons along with 2 carbonyls (dC 186.6 and 182.7), 3 methoxyls (dC 56.5, 56.4 and 56.1), 1 quaternary carbon (dC 79.8) connected to an oxygen atom, and 2 methylenes (dC 29.4 and 22.5) as edited by

*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis

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H. Wang et al.

5'

2' H3CO 7 H3CO 6

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HO 7

8

10 11

4'

3'

8a O 1' 2

1"

6' 6"

HO 5

O

5" 3 4a 4 5 O 2" 4" OCH3 3" 1 8

6 5

4

8

7

13 3

3a

7'

C H3CO 6

12

A

9

7a

1'

O 2 6'

3' 5'

H3CO

1 8a O 4a 4

3 O

O

8'

9'

2 O

1'

2' 3'

3

HO 13'

6'

11'

4' OCH3

10'

B

5' 4'

12'

O 2'

14'

16' 15'

OCH3

2

Figure 1. Chemical structures of compounds 1 – 3.

the DEPT and HSQC experiments. The 1 H – 1H COSY cross-peaks between protons at dH 3.04– 3.10 (1H, m) and dH 2.48 –2.55 (1H, m) confirmed one coupling sequence among two sets of methylene protons of C-3 (dC 29.4) and C-4 (dC 22.5). Two aromatic protons at 7.61 ppm of monosubstituted benzene ring exhibited HMBC cross-peaks with one quaternary carbon C-2 (dC 79.8). Furthermore, the HMBC correlations from H-3 (dH 3.04– 3.10, 1H, m; 2.69– 2.77, 1H, m) to C-2 and C-10 (dC 141.1), and from H-4 (dH 2.71– 2.80, 1H, m; 2.48 –2.55, 1H, m) to C-4a (dC 111.9), C-5 (dC 112.2) and C-8a (dC 146.5) revealed a skeleton of flavan. The HMBC also showed correlations from H-

H3CO H3CO

O

O

O

OCH3

H-H COSY correlations key HMBC correlations key ROESY correlations

Figure 2. Key H– H COSY, HMBC, and ROESY correlations of 1.

600 (dH 6.99, 1H, s) to C-100 (dC 149.0), C-200 (dC 186.6), C-400 (dC 158.2) and C-500 (dC 182.7), and from H-300 (dH 5.77, 1H, s) to the same four carbons. These HMBC correlations around the carbonyl carbons suggested a benzoquinone system. Except for the monosubstituted benzene ring, the rest chemical shifts of 1 were similar to those of calussequinone, an isoflavan with a benzoquinone attached at C-3, which was also isolated from the heartwood of D. odorifera T. Chen. (Leguminosae) [15]. The connectivity between C-2 (dC 79.8) and C-100 (dC 149.0) was deduced by the HMBC correlations from H-3 (dH 3.04 – 3.10, 1H, m; 2.69 –2.77, 1H, m) to C-100 (dC 149.0) and from H-600 (dH 6.99, 1H, s) to C-2 (dC 79.8). One methoxy group (dC 56.1) present on this quinone ring was revealed by its HMBC correlation from protons at 3.75 ppm to the corresponding carbon C-400 (dC 158.2) and from H-600 (dH 6.99, 1H, s) to C-2 (dC 79.8) and C-400 (dC 158.2). In the ROESY spectrum, the ROE correlations between the methoxy protons with H-300 (dH 5.77, 1H, s) also supported the position. The locations of other two methoxy groups were supported by the HMBC correlations from two methoxy protons (dH 3.78, 3.86) to their linked

Journal of Asian Natural Products Research H3CO

O

O

3 O

O

O

+ H3CO

m/ z 77

m/ z 329

O

OCH3

OCH3

H3CO

O

O

m/ z 137

m/ z 242

O

O H3CO OCH3

O

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H3CO H3CO

O

O O

m/ z 406

OH

O

+

H3CO

H3CO

m/ z 138

H3CO

O

OCH3

H3CO

CO

O

O O

OCH3

m/ z 406

O m/ z 240

m/ z 166

O OCH3

OCH3 C2H4

m/ z 253

m/ z 225

CO

H3CO

OH

O

O H3CO

O

m/ z 241

OCH3

m/ z 167

O

m/ z 225

OCH3

O

OCH3

m/ z 225

Figure 3. Proposed fragmentation pathway of compound 1.

carbons (dC 149.2 and 143.7), respectively, and from H-5 (dH 6.47, 1H, s) to C-4 (dC 22.5) and C-7 (dC 143.7), and also supported by the ROE correlations between the methoxy protons (dH 3.78) with H-5 (dH 6.47, 1H, s) and the methoxy protons (dH 3.86) with H-8 (dH 6.54, 1H, s). These 2D NMR (HSQC, COSY, HMBC, and ROESY) connectivities (Figure 2) finally established the structure of 1 as 6,7-dimethoxy-2-(4-methoxy-benzoquinonyl)-flavan, and an MS fragmentation pathway of 1 is proposed in Figure 3. Compound 2 was obtained as a yellow amorphous solid. The molecular formula was determined to be C33H30O7 from the molecular ion peak [M 2 H]2 at m/z 537.1926 in the negative HR-ESI-MS. The 1 H NMR spectrum revealed signals corresponding to nine aromatic protons resonating in the range from dH 7.5 to 6.5,

and two singlet methine protons (dH 7.11 and 6.90) in the aromatic region, four olefinic proton signals (dH 6.21, 6.21, 5.76 and 5.72) in the middle range, three methoxyl groups at 3.90, 3.80 and 3.09 ppm, as well as two sets of methylene protons (dH 3.97 and 3.84; 2.69 and 2.63). The 13C NMR spectrum showed signals for 24 aromatic and/or olefinic carbons along with 1 carbonyl (dC 186.5), 3 methoxyls (dC 56.6, 56.3 and 53.0), 1 quaternary carbon (dC 77.7) connected to an oxygen atom, and 2 methylenes (dC 42.1 and 26.5) as edited by the DEPT and HSQC experiments. The sequence of C80 – C-90 –C-100 was assigned on the basis of 1H – 1H COSY cross-peaks between the proton at dH 5.76 (1H, ddd, J ¼ 15.7, 8.2, 6.4 Hz) with protons at dH 2.63 (1H, dd, J ¼ 13.1, 6.4 Hz), 2.69 (1H, dd, J ¼ 13.1, 8.2 Hz) and 6.21 (1H, d, J ¼ 15.7 Hz).

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H. Wang et al.

HO O O

H3CO

H3CO

OCH3

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HO

H–H COSY correlations key HMBC correlations key ROESY correlations

Figure 4. Key H– H COSY, HMBC, and ROESY correlations of 2.

Further structure elucidation of 2 was performed using an HMBC experiment. One monosubstituted benzene ring (A), one disubstituted benzene ring (B), and one tetrasubstituted benzene ring (C) were confirmed by HMBC correlations (Figures 1 and 4). The B ring was considered to be connected to C-100 (dC 134.1) to form a phenylpropenyl moiety, as HMBC long-range correlations were observed from H-100 (dH 6.21, 1H, d, J ¼ 15.7 Hz) to C-120 (dC 112.8) and C-160 (dC 119.2), and from H-90 (dH 5.76, 1H, ddd, J ¼ 15.7, 8.2, 6.4 Hz) to C-110 (dC 138.7). The HMBC correlations from H-60 (dH 6.21, 1H, br s) to C-20 (dC 186.5), C-40 (dC 173.2), C-50 (dC 77.7) and C-70 (dC 26.5), and from H-30 (dH 5.72, 1H, s) to C10 (dC 138.3), C-20 (dC 186.5), C-50 (dC 77.7) suggested a quinone system, which was quite likely a six-membered ring. The correlations from H-90 (dH 5.76, 1H, ddd, J ¼ 15.7, 8.2, 6.4 Hz) on the phenylpropenyl moiety to C-50 (dC 77.7) on the quinone ring, and from H-80 (dH 2.69, 1H, dd, J ¼ 13.1, 8.2 Hz; 2.63, 1H, dd, J ¼ 13.1, 6.4 Hz) to C-40 (dC 173.2) and C-60 (dC 142.8) revealed that the linkage of

the phenylpropenyl chain was at the quaternary carbon ( dC 77.7) on the quinone ring. The HMBC correlations from H-4 (dH 7.11, 1H, s), H-9 (dH 7.45, 1H, br d, J ¼ 7.4 Hz), and H-13 (dH 7.45, 1H, br d, J ¼ 7.4 Hz) to C-3 (dC 119.1) proved that rings A and C were connected with C-3 (dC 119.1). Furthermore, H-70 (dH 3.97, 1H, dd, J ¼ 17.4, 1.4 Hz; 3.84, 1H, dd, J ¼ 17.4, 1.4 Hz) showed cross-peaks with C-3 (dC 119.1), C-2 (dC 149.9), C-10 (dC 138.3), C-20 (dC 186.5), and C-60 (dC 142.8) which suggest that the quinone ring was connected with rings A and C through the sequence of C-3 – C-2 – C-70 – C-10 . Two methoxyl groups present in the quinone ring were revealed by HMBC correlations from protons at 3.09 ppm to the corresponding carbons C-50 (dC 77.7), and from protons at 3.80 ppm to C-40 (dC 173.2). The ROE correlations between the methoxy protons at 3.09 ppm with H-60 (dH 6.21, 1H, br s) and between the methoxy protons at 3.80 ppm with H-30 (dH 5.72, 1H, s) in the ROESY spectrum also supported the methoxyl group positions. The remaining methoxyl group was attached at C-6 by HMBC correlation from protons at 3.90 ppm to C-6 (dC 145.2), and HMBC correlations from H-4 (dH 7.11, 1H, s) to C-3 (dC 119.1) and C-6 (dC 145.2). The ROE correlations between the methoxy protons at 3.90 ppm with H-7 (dH 6.92, 1H, s) in the ROESY spectrum also supported the position. The HMBC correlations from hydroxyl proton at 5.56 ppm to C-4 (dC 104.0), C-5 (dC 142.9), and C-6 (dC 145.2), and from hydroxyl proton at 5.19 ppm to C-130 (dC 156.0), C-120 (dC 112.8), and C-140 (dC 114.8) revealed that C-5 (dC 142.9) and C130 (dC 156.0) were substituted by the hydroxyl group, respectively. Then according to the molecular formula, only one oxygen atom was left, so that the two remaining oxygenated aromatic and/or olefinic carbons at 148.6 and 149.9 ppm were expected to form an ether linkage, which was most likely between C-7a (dC

Journal of Asian Natural Products Research

5 O OCH3

HO

HO O

H3CO

C O

H3CO m/ z 181

O

O

H3CO

O

m/ z 240

H3CO

H3CO

HO

OCH3

OCH3

m/ z 181

m/ z 482

OCH3

m/ z 181

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O HO HO

O H3CO

O

OCH3

O H3CO

H3CO OCH3

HO

m/ z 420

O

H3CO m/ z 167

H3CO

O OCH3

m/ z 538

OCH3

m/ z 167 OCH3 HO

+

m/ z 133

O O

HO H3CO

m/ z 405

O

H3CO

OCH3

H3CO

OCH3

m/ z 167

Figure 5. Proposed fragmentation pathway of compound 2.

148.6) and C-2 (dC 149.9) to form a furan ring. These 2D NMR (HSQC, COSY, HMBC and ROESY) connectivities (Figure 4) finally established the structure of 2 as 2-[4,5-dimethoxy-5-(3-m-hydroxyphenyl-trans-allyl)cyclohexa-3,6-dien2-on-1-ylmethyl]-5-hydroxy-6-methoxy3-phenylbenzofuran. The 1H and 13C NMR spectroscopic data were similar to those of 2-[4,5-dimethoxy-5-(3-phenyltrans-allyl)cyclohexa-3,6-dien-2-on-1ylmethyl]-5-hydroxy-6-methoxy-3-phenylbenzofuran isolated from Dalbergia cochinchinensis [16]. The difference was the presence of one more oxygenated aromatic carbon, instead of an aromatic carbon bearing a proton, which resulted in the appearance of a disubstituted benzene ring in 2 instead of a monosubstituted benzene ring. Thus, the structure of compound 2 was confirmed, and named

phenylbenzofuran I. An MS fragmentation pathway of 2 is proposed in Figure 5. Compound 3 was obtained as a colorless amorphous solid with the molecular formula C16H12O6 as established by HREIMS molecular peak at m/z 300.0639 [M]þ. The 1H NMR data of compound 3 showed one pattern of ABX proton signals at dH 7.79 (1H, d, J ¼ 8.7 Hz), 6.70 (1H, dd, J ¼ 8.7, 2.2 Hz), and 6.49 (1H, d, J ¼ 2.2 Hz), and another pattern at dH 6.78 (1H, d, J ¼ 8.6 Hz), 6.57 (1H, d, J ¼ 2.5 Hz), and 6.41 (1H, dd, J ¼ 8.6, 2.5 Hz); one methylene group (dH 4.73, 2H, s); and one methoxyl group (dH 3.75). The 13C NMR spectrum of 3 showed one carbonyl carbon (dC 181.4), five oxygenated aromatic carbons, one quaternary aromatic carbon, six aromatic carbons bearing a proton, one quaternary carbon (dC 107.0), one methylene carbon (dC

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H. Wang et al. HO

O O O

O

OCH3 key HMBC correlations key ROESY correlations

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Figure 6. Key correlations of 3.

HMBC

and

ROESY

71.1), and one methoxyl group (dC 56.2) as edited by the DEPT and HSQC experiments. The HMBC correlations from H-5 (dH 7.79, 1H, d, J ¼ 8.7 Hz) to C-7 (dC 166.6) and C-8a (dC 164.2); and from H-6 (dH 6.70, 1H, dd, J ¼ 8.7, 2.2 Hz) to C-8 (dC 103.7) and C-4a (dC 113.1) established a benzene ring, together with the HMBC correlations from H-2 (dH 4.73, 2H, s) to C-3 (dC 107.0), C-4 (dC 181.4), and C-8a (dC 164.2) which revealed a skeleton of chromanone (Figure 6). A hydroxyl group was located at C-7 by HMBC correlation peaks from the H-5 (dH 7.79, 1H, d, J ¼ 8.7 Hz) to C-4 (dC 181.4) and C-7 (dC 166.6). The only methoxy group was

HO

attached to another trisubstituted benzene ring by the HMBC correlation from methoxy protons at dH 3.75 to C-40 (dC 156.6), and by the ROE correlations between the same methoxy protons with H-30 (dH 6.57, 1H, d, J ¼ 2.5 Hz) and H-50 (dH 6.41, 1H, dd, J ¼ 8.6, 2.5 Hz). Then according to the molecular formula, only two oxygen atoms were left, which were most likely to be connected to the quaternary carbon (dC 107.0), and two remaining oxygenated aromatic carbons (dC 141.9, 148.7) to form a five-membered heterocyclic ring. Thus, the molecular skeleton was deduced as spiro[benzo[d ][1,3]dioxole-20 ,3-chroman]4-one, as no HMBC long-range correlations were observed from H-2 (dH 4.73, 2H, s) to any carbons in the benzene ring with three oxygenated aromatic carbons. A similar compound isolated from the same sample before was 40 -methoxy-20 ,3,7-trihydroxyisoflavanone [7]. The main difference was the appearance of the five-membered heterocyclic ring in 3, which changed greatly the chemical shifts of C-3 and C-10 . Comprehensive analysis of the 1H and 13C NMR data and 2D NMR (HSQC, COSY, HMBC and ROESY) spectra established the structure of compound 3, which was

O

O O

C

O

OCH3

m/ z 191

O

m/ z 136

O

OCH3

O

m/ z 164

m/ z 95

OCH3

CO HO

O

HO O

O O

CO

O

m/ z 272

O

HO

O

O OCH3

m/ z 300

OCH3

HO

HO

HO

O

m/ z 123

OCH3

OH

HO

OH

CO

CO O

m/ z 149

O

m/ z 177 O

m/ z 137 O

Figure 7. Proposed fragmentation pathway of compound 3.

m/ z 109

Journal of Asian Natural Products Research Table 1. Cytotoxicity of compounds 1 – 3 against three human cancer cell lines. IC50 (mM) Compound

K562

SGC-7901

BEL-7402

2 1, 3 Paclitaxela

. 40 . 40 5.97

. 40 . 40 1.87

33.5 .40 7.38

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a

Positive control.

proposed to be 7-hydroxy-50 -methoxyspiro [benzo[d ][1,3]dioxole-2 0 ,3-chroman]-4one as shown in Figure 6. And an MS fragmentation pathway of 3 is proposed in Figure 7. We tried to obtain single crystals of 1– 3 but failed, so that their absolute configurations could not be determined by X-ray diffraction. In this study, all of the compounds were evaluated for the antitumor activities against K562, SGC7901, and BEL-7402 cell lines, using the MTT method, with paclitaxel as the positive control (IC50 ¼ 5.97 mM against K562, IC50 ¼ 1.87 mM against SGC-7901, and IC50 ¼ 7.38 mM against BEL-7402). The results showed that compound 2 exhibited cytotoxicity against BEL-7402 tumor cell lines with IC50 value of 33.5 mM (Table 1). Other compounds tested were inactive against the three tumor cell lines (IC50 value . 40 mM). 3. 3.1

Experimental General experimental procedures

Optical rotations were recorded using a Rudolph Autopol III polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). CD spectra were acquired on a Chirascan spectrometer (Applied Photophysics Ltd, Leatherhead, UK). Melting points were determined on a Beijing Taike X-5 stage apparatus (Beijing Taike Instrument Company, Beijing, China) and are uncorrected. UV spectra were recorded on a Shimadzu UV-2550 spectrometer (Beckman, Brea, CA, USA). IR absorp-

7

tions were obtained on a Nicolet 380 FT-IR instrument using KBr pellets (Thermo, Pittsburgh, PA, USA). 1H, 13C, and 2D NMR spectra were recorded on Bruker Avance 500 NMR spectrometers (Bruker, Bremen, Germany), using TMS as an internal standard. HRMS were measured with an API QSTAR Pulsar mass spectrometer (Bruker) or Waters Autospec Premier (Waters Corporation, Milford, MA, USA). Column chromatography (CC) was performed with silica gel (60 – 80, 200 – 300 mesh, silica gel H, 10 – 40 mm, Marine Chemical Industry Factory, Qingdao, China) and Sephadex LH-20 (Merck, Darmstadt, Germany). TLC was carried out on silica gel G precoated plates (Marine Chemical Industry Factory), and spots were detected by spraying with 5% H2SO4 in EtOH followed by heating. Paclitaxel (with the purity above 99.5%) was produced by Jiangsu Yew (Yew Pharmaceutical Co., Ltd, Wuxi, China). 3.2

Plant material

The dried heartwood of D. odorifera was purchased from the Haikou Free Market of Agricultural Products, Hainan Province, China, in October 2010. The specimen was identified by Dr Xi-Long Zheng of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, where a voucher specimen (No. 20101009) has been deposited. 3.3 Extraction and isolation The dried and crushed heartwood of D. odorifera (8.4 kg) was extracted three times with 95% (v/v) ethanol (50 l) at room temperature for three weeks totally. The solution was evaporated under reduced pressure to obtain an extract (0.9 kg), which was suspended in distilled water and then partitioned with petroleum ether, ethyl acetate, and n-butanol, respectively. The EtOAc-soluble residue (477 g) was sub-

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H. Wang et al.

mitted to CC over silica gel (silica H, 3.1 kg, 15 cm £ 50 cm) and eluted with CHCl3 – MeOH (v/v, 1:0, 100:1, 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1, 0:1, each 8.0 l) of increasing polarity resulting in 18 fractions (Fr.1–Fr.18). Fr.3 (6.2 g) was subjected to silica gel CC (200–300 mesh, 2.5 cm £ 48 cm, 60 g) with a gradient elution of petroleum ether–Me2CO (1:0; 100:1; 50:1; 25:1; 15:1; 10:1; 3:1; 0:1, each 1.5 l), resulting in 27 fractions (Fr.3.1–Fr.3.27). Fr.3.15 was separated by Sephadex LH-20 column (1.4 cm £ 42 cm) using CHCl3 – MeOH (1:1), followed by silica gel eluted with petroleum ether–CHCl3 (2:8) to obtain 1 (3 mg). Fr.6 (51.3 g) was subjected to silica gel CC (silica H, 5.0 cm £ 23 cm, 200 g) with a gradient elution of petroleum ether– Me2CO (50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, 0:1, each 2.0 l), resulting in 30 fractions (Fr.3.1–Fr.3.30). Fr.6.24 was separated by Sephadex LH-20 (1.4 cm £ 42 cm, 23 g) using CHCl3 – MeOH (1:1), followed by silica gel eluted Table 2.

with CHCl3 to give 2 (20.7 mg). Fr.8 (60.2 g) was subjected to silica gel CC (silica H, 5.0 cm £ 24 cm, 200 g) with a gradient elution of petroleum ether–Me2CO (100:1, 50:1, 25:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 0:1, each 1.8 l), resulting in 24 fractions (Fr.8.1–Fr.8.24). Fr.8.11 was separated by Sephadex LH-20 column (1.4 cm £ 42 cm) using CHCl3 –MeOH (1:1), followed by silica gel eluted with CHCl3 – MeOH (100:1) to obtain 3 (3.5 mg).

3.3.1 6,7-Dimethoxy-2-(4-methoxybenzoquinonyl)-flavan (1) C24H22O6, yellow amorphous solid; mp 145–1488C; ½a30 D 2 5:5 (c 0.1, MeOH); UV (MeOH): lmax (log 1) 265 (4.48), 290 (4.25), 300 (4.18) nm; CD (MeOH): D1220 nm 2 0.29, D1235 nm 2 0.45, D1249 nm 2 0.59; IR (KBr): y max 2927, 1640, 1600, 1506, 1448, 1200, 1121, 1019, 830, 696 cm21; 1H and 13C NMR spectral data: see Table 2; EIMS: m/z 406 [Mþ] (96),

1

H (500 MHz) and 13C (125 MHz) NMR spectral data of compound 1 (d, ppm and J, Hz).

Position

dC

2 3 4 4a 5 6 7 8 8a 10 20 30 40 50 60 100 200 300 400 500 600 6-OCH3 7-OCH3 400 -OCH3

79.8 (s) 29.4 (t) 22.5 (t) 111.9 (s) 112.2 (d) 149.2 (s) 143.7 (s) 101.2 (d) 146.5 (s) 141.1 (s) 126.8 (d) 128.5 (d) 128.2 (d) 128.5 (d) 126.8 (d) 149.0 (s) 186.6 (s) 109.1 (d) 158.2 (s) 182.7 (s) 131.5 (d) 56.5 (q) 56.4 (q) 56.1 (q)

dH 3.04 –3.10 (1H, m); 2.69– 2.77 (1H, m) 2.71 –2.80 (1H, m); 2.48– 2.55 (1H, m) 6.47 (1H, s) 6.54 (1H, s) 7.61 (1H, 7.35 (1H, 7.27 (1H, 7.35 (1H, 7.61 (1H,

br d, 7.1) br t, 7.1) br t, 7.1) br t, 7.1) br d, 7.1)

5.77 (1H, s) 6.99 (1H, 3.78 (3H, 3.86 (3H, 3.75 (3H,

s) s) s) s)

Journal of Asian Natural Products Research 329 (12), 253 (23), 242 (38), 241 (48), 240 (100), 225 (22), 167 (92), 166 (36), 138 (16), 137 (3), 77 (9); HR-ESI-MS: m/z 405.1340 [M 2 H]2 (calcd for C24H21O6, 405.1344).

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3.3.2

Phenylbenzofuran I (2)

C33H30O7, yellow amorphous solid; mp 110 –1138C; ½a30 D þ 24:0 (c 0.1, MeOH); UV (MeOH): lmax (log 1) 209 (4.46), 296 (3.92) nm; CD (MeOH): D1220 nm þ 4.15, D1212 nm 2 1.45, D1231 nm þ 1.35; IR (KBr): y max 3447, 2928, 2279, 1614, Table 3. Position

1449, 1371, 1219, 1156 cm21; 1H and 13 C NMR spectral data: see Table 3; EIMS: m/z 538 [Mþ] (3), 482 (19), 420 (21), 405 (48), 302 (20), 240 (66), 181 (45), 167 (71), 133 (100), 77 (20); HRESI-MS: m/z 537.1926 [M 2 H]2 (calcd for C33H29O7, 537.1919). 3.3.3 7-Hydroxy-5 0 -methoxyspiro[benzo [d][1,3]dioxole-2 0 ,3-chroman]-4-one (3) C16H12O6, yellow amorphous solid; mp 121 –1248C; ½a30 D 2 200 (c 0.05, MeOH);

1

H (500 MHz) and 13C (125 MHz) NMR spectral data of compound 2 (d, ppm and J, Hz).

dC

2 3 3a 4 5 6 7 7a 8 9 10 11 12 13 10 20 30 40 50 60 70

149.9 (s) 119.1 (s) 121.3 (s) 104.0 (d) 142.9 (s) 145.2 (s) 94.7 (d) 148.6 (s) 132.4 (s) 128.7 (d) 129.0 (d) 127.4 (d) 129.0 (d) 128.7 (d) 138.3 (s) 186.5 (s) 104.9 (d) 173.2 (s) 77.7 (s) 142.8 (d) 26.5 (t)

80

42.1 (t)

90 100 110 120 130 140 150 160 6-OCH3 40 -OCH3 50 -OCH3 5-OH 130 -OH

9

122.9 (d) 134.1 (d) 138.7 (s) 112.8 (d) 156.0 (s) 114.8 (d) 129.8 (d) 119.2 (d) 56.6 (q) 56.3 (q) 53.0 (q)

dH

7.11 (1H, s) 6.92 (1H, s) 7.45 (1H, br d, 7.4) 7.37 (1H, br t, 7.4) 7.30 (1H, br t, 7.4) 7.37 (1H, br t, 7.4) 7.45 (1H, br d, 7.4) 5.72 (1H, s) 6.21 (1H, br s) 3.97 (1H, dd, 17.4, 1.4) 3.84 (1H, dd, 17.4, 1.4) 2.69 (1H, dd, 13.1, 8.2) 2.63 (1H, dd, 13.1, 6.4) 5.76 (1H, ddd, 15.7, 8.2, 6.4) 6.21 (1H, d, 15.7) 6.51 (1H, t, 2.1) 6.70 (1H, d, 7.7) 7.08 (1H, t, 7.7) 6.70 (1H, dd, 7.7, 2.1) 3.90 (3H, s) 3.80 (3H, s) 3.09 (3H, s) 5.56 (1H, s) 5.19 (1H, s)

10

H. Wang et al.

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Table 4. 1H (500 MHz) and 13C (125 MHz) NMR spectral data of compound 3 (d, ppm and J, Hz). Position

dC

2 3 4 4a 5 6 7 8 8a 10 20 30 40 50 60 40 -OCH3

71.1 (t) 107.0 (s) 181.4 (s) 113.1 (s) 130.7 (d) 112.9 (d) 166.6 (s) 103.7 (d) 164.2 (s) 141.9 (s) 148.7 (s) 98.1 (d) 156.6 (s) 106.1 (d) 108.9 (d) 56.2 (q)

Support Science and Technology Subject (2013BAI11B04), and Major Technology Project of Hainan Province (ZDZX2013008-4).

dH 4.73 (2H, s)

7.79 (1H, d, 8.7) 6.70 (1H, dd, 8.7, 2.2) 6.49 (1H, d, 2.2)

6.57 (1H, d, 2.5) 6.41 (1H, dd, 8.6, 2.5) 6.78 (1H, d, 8.6) 3.75 (3H, s)

UV (MeOH): lmax (log 1) 205 (4.47), 234 (4.12), 291 (4.10) nm; CD (MeOH): D1206 nm þ 3.18, D1 209 nm þ 3.22, D1232 nm þ 1.44; IR (KBr): y max 3431, 2924, 2859, 1612, 1493, 1457, 1376, 1248, 1199, 1152, 1106, 1033 cm21; 1H and 13C NMR spectral data: see Table 4; EIMS: m/z 300 [Mþ] (55), 272 (15), 191 (17), 177 (12), 167 (35), 164 (97), 149 (100), 137 (24), 136 (10), 123 (39), 111 (40), 109 (42), 97 (58), 95 (27); HREIMS: m/z 300.0639 [M]þ (calcd for C16H12O6, 300.0634). 3.4 Bioassay of cytotoxic activity Human chronic myelogenous leukemia cell line (K562), human gastric carcinoma cell line (SGC-7901), and human hepatocellular carcinoma cell line (BEL-7402) were obtained from the Cell Bank of Type Culture Collection of Shanghai Institute of Cell Biology, the Chinese Academy of Sciences. The specific experimental procedures were same as those described previously [17]. Acknowledgments This research was financially supported by Special Fund for Agro-scientific Research in the Public Interest (201303117), National

References [1] The State Pharmacopoeia Commission of P.R. China, Pharmacopoeia of the People’s Republic of China (Chemical Industry Press, Beijing, 2000), Vol. 1. [2] Q. Zhao, J.X. Guo, and Y.Y. Zhang, J. Chin. Pharm. Sci. 9, 1 (2000). [3] S.C. Chan, Y.S. Chang, J.P. Wang, S. C. Chen, and S.C. Kuo, Planta Med. 64, 153 (1998). [4] S.H. Lee, J.Y. Kim, G.S. Seo, Y.C. Kim, and D.H. Sohn, Inflamm. Res. 58, 257 (2009). [5] Z.J. Cheng, S.C. Kuo, S.C. Chan, F.N. Ko, and C.M. Teng, Biochem. Biophys. Acta 1392, 291 (1998). [6] X. Yu, W. Wang, and M. Yang, Food Chem. 104, 715 (2007). [7] X.B. Zhao, W.L. Mei, M.F. Gong, W.J. Zuo, H.X. Bai, and H.F. Dai, Molecules 16, 9775 (2011). [8] Y. Tao and Y. Wang, Fitoterapia 81, 393 (2010). [9] H. Lu, Y.F. Cao, C.M. Hu, X.Y. Sun, H. Li, Y. Liu, X.G. Fu, and H.Z. Sun, Fitoterapia 84, 208 (2013). [10] D.S. Lee and G.S. Jeong, Eur. J. Pharm. 728, 1 (2014). [11] D.S. Lee, B. Li, N.K. Im, Y.C. Kim, and G.S. Jeong, Int. Immunopharmacol. 16, 114 (2013). [12] D.S. Lee, B. Li, S. Keo, K.S. Kim, G.S. Jeong, H. Oh, and Y.C. Kim, Int. Immunopharmacol. 17, 828 (2013). [13] C. Lee, J.W. Lee, Q.H. Jim, D.S. Jang, S.J. Lee, D.H. Lee, J.T. Hong, Y. Kim, M.K. Lee, and B.Y. Hwng, Bioorg. Med. Chem. Lett. 23, 4263 (2013). [14] D.S. Lee, K.S. Kim, W. Ko, B. Li, S. Keo, G.S. Jeong, H. Oh, and Y.C. Kim, Phytother. Res. (2014). [15] C.W. Choi, Y.H. Choi, M. Cha, Y.S. Kim, G.H. Yon, Y. Kim, S.U. Choi, Y.H. Kim, and S.Y. Ryu, J. Korean Soc. Appl. Biol. Chem. 52, 375 (2009). [16] O. Shirota, V. Pathak, S. Sekita, M. Satake, Y. Nagashima, Y. Hirayama, Y. Hakamata, and T. Hayashi, J. Nat. Prod. 66, 1128 (2003). [17] Y.X. Li, W.L. Mei, W.J. Zuo, Y.X. Zhao, W.H. Dong, and H.F. Dai, Phytochem. Lett. 5, 41 (2012).

Three new phenolic compounds from Dalbergia odorifera.

Three new phenolic compounds (1-3) were isolated from the heartwood of Dalbergia odorifera T. Chen. (Leguminosae). Their structures were established b...
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