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Cytotoxic flavanes from Uraria clarkei a

a

a

a

Zhi-Yong Jiang , Xi-Shan Bai , Hui Liang , Chao Wang , Wen-Juan a

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Li , Jun-Ming Guo & Xiang-Zhong Huang

a

a

Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education, School of Chemistry and Biotechnology, Yunnan University of Nationalities, Kunming, 650500, China Published online: 05 Aug 2013.

To cite this article: Zhi-Yong Jiang, Xi-Shan Bai, Hui Liang, Chao Wang, Wen-Juan Li, Jun-Ming Guo & Xiang-Zhong Huang (2013) Cytotoxic flavanes from Uraria clarkei, Journal of Asian Natural Products Research, 15:9, 979-984, DOI: 10.1080/10286020.2013.822368 To link to this article: http://dx.doi.org/10.1080/10286020.2013.822368

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Journal of Asian Natural Products Research, 2013 Vol. 15, No. 9, 979–984, http://dx.doi.org/10.1080/10286020.2013.822368

Cytotoxic flavanes from Uraria clarkei Zhi-Yong Jiang, Xi-Shan Bai, Hui Liang, Chao Wang, Wen-Juan Li, Jun-Ming Guo and Xiang-Zhong Huang* Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education, School of Chemistry and Biotechnology, Yunnan University of Nationalities, Kunming 650500, China

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(Received 17 December 2012; final version received 2 July 2013) Two new flavanes, (2R)-40 -hydroxy-20 ,5,7-trimethoxyflavane (1) and (2R,4R)-20 ,40 dihydroxy-5,7-dimethoxyflavan-4-ol (2), were isolated from Uraria clarkei, together with two known compounds 5,7-dimethoxy-40 -hydroxyflavan (3) and 5,7,40 trimethoxyflavan (4). The structures of the new flavanes were characterized by analyses of the MS, IR, UV, CD, 1D, and 2D NMR data. Cytotoxicity test suggested that compounds 1 – 4 possessed slight activity against K-562 and Hela cell lines, with the IC50 values ranging from 26.6 to 56.3 mM. Keywords: Uraria clarkei; flavane; cytotoxicity; (2R)-40 -hydroxy-20 ,5,7-trimethoxyflavan; (2R,4R)-20 ,40 -dihydroxy-5,7-dimethoxyflavan-4-ol

1. Introduction Uraria clarkei, mainly distributed in southwest of China, India, and Vietnam [1], has long been used as the folk medicine for the treatment of postpartum hypogalactia, hemoptysis, and venomous snake bite in China [2]. Previous research suggested that the main chemical constituents in the Uraria genus were flavonoids [3–7]. There was no phytochemical investigation on this plant up to now. In our ongoing efforts to discover antitumor metabolites from natural sources, the ethanol extracts of the aerial part of U. clarkei were found to contain cytotoxic active constituents. With the aim of searching for the active ingredients, the aerial part of U. clarkei was phytochemically investigated to afford two new flavanes, besides two known compounds 5,7-dimethoxy-40 hydroxyflavan (3) and 5,7,40 -trimethoxyflavan (4). By extensive analyses of the MS, UV, IR, CD, and NMR data, the two new flavanes was characterized as (2R)-40 hydroxy-20 ,5,7-trimethoxyflavan (1) and

(2R,4R)-20 ,40 -dihydroxy-5,7-dimethoxyflavan-4-ol (2) (Figure 1). Cytotoxicity bioassay suggested that compounds 1 – 4 possessed slight activity against K-562 and Hela cell lines. This paper reports the isolation and structural elucidation of two new compounds and the cytotoxicity of isolates. 2.

Results and discussion

Compound 1 was obtained as pale yellow powder and had the molecular formula C18H20O5, determined by the positive HRESI-MS at m/z 317.1349 [M þ H]þ. Its IR spectrum displayed the presence of hydroxyl (3389 cm21) and aromatic ring (1606, 1501, 1456 cm21) functions. The 1H NMR spectrum (Table 1) of compound 1 exhibited one ortho-coupled aromatic proton at d 7.29 (1H, d, J ¼ 8.0 Hz, H-60 ); three meta-coupled aromatic protons at d 6.40 (1H, d, J ¼ 2.4 Hz, H-30 ), 6.15 (1H, d, J ¼ 2.4 Hz, H-8), and 6.10 (1H, d, J ¼ 2.4 Hz, H-6); and one ortho-meta-coupled aromatic

*Corresponding author. Email: [email protected] q 2013 Taylor & Francis

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Z.-Y. Jiang et al. MeO MeO 7

2' MeO 7

9 O 2 5 OMe

1'

4'

OH

4'

OH

4 5 OMe OH

4

2

MeO

O

O

OMe

OH

OMe

OMe

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2'

2 1'

1

MeO

HO

1 9 O

4

3

Figure 1. The structures of compounds 1 – 4.

proton at d 6.42 (1H, dd, J ¼ 8.0, 2.4 Hz, H-50 ), besides three methoxyl groups [d 3.76 (6H, s), 3.80 (3H, s)] and one oxygenated methine [d 5.33 (1H, dd, J ¼ 10.4, 2.4 Hz)]. The 13C NMR spectrum (Table 1) showed 18 carbon signals, of which five quaternary Table 1. 1H NMR (400 MHz) and CDCl3 (d in ppm, J in Hz).

13

carbons occupied by an oxygen function were presented (d 159.1, 158.6, 157.4, 156.7, 156.4), together with three methoxyls at d 55.5, 55.4, and 55.4 and one oxygenated tertiary carbon at d 72.2. Analyses of the NMR data suggested that compound 1 had a

C NMR (100 MHz) spectral data of compounds 1 and 2 in

1 Position 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 5-OMe 7-OMe 20 -OMe

2

dH

dC

dH

dC

5.33 (1H, dd, 10.4, 2.4) 2.18 (1H, m) 1.98 (1H, m) 2.60 – 2.76 (2H, m) – 6.10 (1H, d, 2.4) – 6.15 (1H, d, 2.4) – – – – 6.40 (1H, d, 2.4) – 6.42 (1H, dd, 8.0, 2.4) 7.29 (1H, d, 8.0) 3.80 (3H, s) 3.76 (3H, s) 3.76 (3H, s)

72.2 28.2

5.28 (1H, dd, 1.6, 5.4) 2.18 (1H, m) 2.29 (1H, m) 5.67 (1H, dd, 4.4, 2.8) – 6.04 (1H, d, 2.4) – 5.99 (1H, d, 2.4) – – – – 6.36 (1H, d, 2.4) – 6.40 (1H, dd, 8.4,2.4) 7.22 (1H, d, 8.4) 3.87 (3H, s) 3.73 (3H, s) –

67.4 26.7

19.5 158.6 91.3 159.1 93.5 156.7 103.8 122.1 157.4 98.8 156.4 107.1 127.6 55.5 55.4 55.4

62.1 159.1 91.6 161.9 92.9 154.8 103.3 114.1 154.7 103.4 157.4 108.2 131.8 55.9 55.3 –

Journal of Asian Natural Products Research HO

MeO O

MeO

O

MeO

981

OH

OH

OMe

OMe 1

OH 2

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Figure 2. Key HMBC (H ! C) and COSY (H—H) correlations of compounds 1 and 2.

flavane skeleton and shared a structure similar to that of erycibenun E [8]. The planar structure of compound 1 differed from that of erycibenun E [8] mainly in the replacement mode at the ring B. As shown in the HMBC spectrum (Figure 2), the correlation between the methoxy at d 3.76 (3H, s) and C-20 at d 157.4 established the location of the methoxyl, and the crosspeaks between H-2 at d 5.33 (1H, dd, J ¼ 10.4 and 2.4 Hz) and C-20 at d 157.4, C-60 at d 127.6 and C-10 at d 122.1 suggested that the methoxyl group should be linked at the C-20 . The substitution of 40 -hydroxy at ring B in compound 1 was determined by analyses of the coupling constant of H-30 , H-50 , and H-60 in the 1H NMR spectrum (Table 1), as well as the HMBC correlations (Figure 2). To characterize the absolute configuration of C-2, the CD curve was recorded, and the positive Cotton effect at 278.5 nm manifested that C-2 should be in R configuration [9]. The 2R configuration could also be supported by the positive optical rotation value of compound 1

(a negative value of [a ]D for erycibenun E (2S) according to the literature [8]). Finally, the structure of compound 2 was elucidated as (2R)-40 -hydroxy-20 ,5,7-trimethoxyflavane. The other HMBC (Figure 2) and RPOESY (Figure 3) correlations further verified the above deduce. Compound 2 was obtained as a pale yellow powder. Its HR-ESI-MS gave the quasi-molecular ion peak at m/z 341.1005 [M þ Na]þ, in agreement with the molecular formula C17H18O6. The IR spectrum showed the absorptions for hydroxyl (3370 cm21) and aromatic ring (1603, 1506, 1451 cm21) functions. Similarly, the 1 H NMR spectrum (Table 1) also displayed the proton signals due to one ortho-coupled aromatic proton at d 7.22 (1H, d, J ¼ 8.4 Hz, H-60 ), three meta-coupled aromatic protons at d 6.36 (1H, d, J ¼ 2.4 Hz, H-30 ), 6.04 (1H, d, J ¼ 2.4 Hz, H-6), and 5.99 (1H, d, J ¼ 2.4 Hz, H-8), and one ortho-metacoupled proton at d 6.40 (1H, dd, J ¼ 8.4, 2.4 Hz, H-50 ), suggesting the similar replacement in the aromatic rings as compound 1,

MeO MeO

O

HO OH

MeO

O

H OMe H 1

Figure 3. Selected ROESY (H

OMe OH 2

H) correlations of compounds 1 and 2.

OH

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together with two methoxyl groups and two oxygenated methines. The 13C NMR (DEPT) spectrum (Table 1) exhibited 17 carbon resonances involving two aromatic rings, two methoxyls, two methines linked with an oxygen, and one methylene. Comparing 1D and 2D NMR data with those of compound 1 demonstrated that both compounds, possessing the flavane skeleton, had a 5,7-dimethoxy substitution in ring A. The main difference between the two new compounds was that there was a hydroxyl located at the C-4 of compound 2, and the existence of two hydroxyls in ring B. The restriction of the two hydroxyl groups to the C-20 and C-40 of compound 2 was concluded by the HMBC correlations (Figure 2) between H-2 [d 5.28 (1H, dd, J ¼ 1.6 and 5.4 Hz)] and C-10 (d 114.1), C-20 (d 154.7) and C-60 (d 131.8), and H-60 [d 7.22 (1H, d, J ¼ 8.4 Hz)] and C-2 (d 67.4), as well as by analyses of the coupling constants of H-30 , 50 , and 60 appearing in the 1H NMR spectrum (Table 1). In order to determine the relative configuration, a ROESY experiment (Figure 3) was conducted in which the correlation between H-2 and H-4 was observed, implying that H-2 and H-4 were in the same side. The positive Cotton effect at 242 and 280 nm allowed the assignment of the 2R,4R-configuration as the previous report [9]. Accordingly, compound 2 was elaborated as (2R,4R)-20 ,40 -dihydroxy-5,7dimethoxyflavan-4-ol (2). By comparing the spectral data with those in the literature, two known compounds were identified as 5,7-dimethoxy40 -hydroxyflavan (3) [10] and 5,7,40 trimethoxyflavan (4) [11], which were also isolated from this plant for the first time. All the isolates were assayed for their cytotoxic activities against K-562 and Hela cell lines. As summarized in Table 2, compounds 1 –4 exhibited slight antitumor activity in vitro, suppressing K-562 cell lines with IC50 values of 30.7, 54.6, 45.8, and 56.3 mM, and Hela cell lines with the IC50 values of 26.6, 43.6, 40.6, and 49.3 mM, respectively.

Table 2. 1 – 4.

Cytotoxic activities of compounds IC50 (mM)

Compounds

K-562

Hela

1 2 3 4 cis-Platinum

30.7 54.6 45.8 56.3 9.7

26.6 43.6 40.6 49.3 12.5

In conclusion, compounds 3 and 4, previously isolated from Erycibe expansa in Thailand, had been found to possess inhibitory effects on NO production [8]. Our work showed that flavanes 1 – 4 had cytotoxic activity. The flavonoids were the main constituents in U. clarkei according to our preceding research. This is the first report about the phytochemical investigation on this plant and it would be helpful for better exploiting the folk medicine. 3. 3.1

Experimental General experimental procedures

UV spectra were obtained on a Shimadzu UV-210A (Shimadzu, Tokyo, Japan) spectrophotometer. CD curves were recorded by a Jasco J720 (Jasco, Inc., Tokyo, Japan) spectrometer. IR spectra were measured by Bio-Rad FTS-135 (Bio-Rad, Berkeley, CA, USA) spectrometer. 1D and 2D NMR spectra were recorded by Bruker AM-400 NMR (Bruker Corporation, Zu¨rich, Switzerland), with tetramethylsilane as internal standard. ESI-MS and HR-ESI-MS were acquired on a LC –MS (Finnigan LCQ Deca XP MAX; Finnigan Corp., San Jose, CA, USA) and a Q-TOF micro mass spectrometer (Waters, Milford, MA, USA), respectively. Column chromatography (CC) was carried out on silica gel (SiO2; 200– 300 mesh; Qingdao Meigao Chemical Company, Qingdao, China) and Sephadex LH-20 (40 –70 mm; Pharmacia Fine Chemical Co. Ltd, Uppsala, Sweden).

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3.2 Plant material The aerial parts of U. clarkei were collected in Simao, Yunnan Province, China, in August 2009, and identified by Dr Jingmei Lu from Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (No. 2009-08-03) has been deposited in the Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education, Yunnan University of Nationalities. 3.3 Extraction and isolation The dried and powdered aerial part of U. clarkei (8 kg) was extracted with 95% EtOH (70 liters) under reflux for three times. The extract was concentrated in vacuum, and then partitioned between water, petroleum ether, EtOAc, and n-BuOH, respectively, to provide a petroleum ether fraction (100 g), EtOAc fraction (80 g), and n-BuOH fraction (200 g). The EtOAc fraction was fractionated by silica gel CC with gradient elution [petroleum ether:EtOAc; 20:1, 10:1, 5:1, 2:1, 1:1, 0:1 (v/v)] to present six fractions (Frs 1–6). Fr. 3 (7 g) was subjected to silica gel CC (150 g, 2.5 cm £ 50 cm) and eluted with CHCl3:MeOH (98:2, 95:5, 90:10) to afford three fractions (Frs 3a–3c). Fr. 3b (1.0 g) was subjected to silica gel CC (100 g, CHCl3:Me2CO, 70:30), followed by Sephadex LH-20 CC (CHCl3:MeOH; 1:1) to obtain compounds 1 (80 mg), 3 (205 mg), and 4 (116 mg). Fr. 3c (500 mg) was isolated on silica gel CC (40 g, CHCl3:Me2CO, 80:20), and further purified by HPLC (MeOH:H2O; 75:25, v/v) to yield compound 2 (150 mg). 3.3.1 (2R)-4 0 -Hydroxy-2 0 ,5,7trimethoxyflavane (1) Pale yellow powder. ½a21:8 D þ 23:7 (c ¼ 0.23, MeOH). UV (MeOH) lmax (log 1): 256 (3.48), 277 (3.61) nm. CD (c ¼ 0.1, MeOH) D1210 nm þ 3.39, D1278 nm þ 0.23; IR (KBr): nmax 3389, 2943, 1606, 1501, 1456,

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1285, 1198, 1110, 1049 cm21. For 1H and 13 C NMR spectral data, see Table 1. ESI-MS (positive): m/z 317 [M þ H]þ. HR-ESI-MS (positive): m/z 317.1349 [M þ H]þ (calcd for C18H21O5, 317.1353). 3.3.2 (2R,4R)-2 0 ,4 0 -Dihydroxy-5,7dimethoxyflavan-4-ol (2) Pale yellow powder. ½a20:5 D 2 15:1 (c ¼ 0.14, MeOH). UV (MeOH) lmax (log 1): 258 (3.56), 278 (3.73) nm. CD (c ¼ 0.109, MeOH) D1242 nm þ 1.26, D1280 nm þ 0.25; IR (KBr): nmax 3370, 2951, 1603, 1506, 1451, 1270, 1107, 1010 cm21. For 1H and 13C NMR spectral data, see Table 1. ESI-MS (positive): m/z 319 [M þ H]þ. HR-ESI-MS (positive): m/z 341.1005 [M þ Na]þ (calcd for C17H18O6Na, 341.1001). 3.4 Cytotoxic assay Cytotoxic activities were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method [12] using K-562 and Hela cell lines. Briefly, the cell suspensions (200 ml) at a density of 5 £ 104 cells ml21 were plated in 96 well microtiter plates and incubated for 24 h at 37.88C in a humidified incubator at 5% CO2. The test compound solution (2 ml in DMSO) at different concentrations was added to each well and further incubated for 72 h under the same conditions. Then, 20 ml of the MTT solution was added to each well and incubated for 4 h. The old medium (150 ml) containing MTT was then gently replaced by DMSO and pipetted to dissolve any formazan crystals formed. Absorbance was then determined on a Spectra Max Plus plate reader at 540 nm. Dose – response curves were generated and the IC50 values were defined as the concentration of compound required to inhibit cell proliferation by 50%. cisPlatinum, an approved agent for the treatment of many tumors, was used as the positive control.

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Acknowledgments

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This work was financially supported by the National Natural Science Foundation of China (NSFC Nos 21162041, 21262047), the Science Foundation of the Education Department of Yunnan Province (2012J071), and the Independent Innovation Project for postgraduates (Nos 11HXYJS03, 2011HXDZB04) from the Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education, Yunnan University of Nationalities. We are grateful to Dr Chun-Suo Yao from the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, for the registration of CD curve.

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[3] C. Wang, H. Liang, J. Guo, X.Z. Huang, X.F. Liu, and J. Wang, China J. Chin. Mater. Med. 36, 2676 (2011). [4] M.M. Rahman, S. Gibbons, and A.I. Gray, Phytochemistry 68, 1692 (2007). [5] C. Wang, H. Liang, J.M. Guo, X.Z. Huang, X.F. Liu, and J. Wang, Chin. J. Ethnomed. Ethnopharm. 2, 28 (2011). [6] Y. Chen, X.L. Si, S. Wei, and X.J. Xu, Chin. Tradit. Patent Med. 31, 266 (2009). [7] Y.Y. Wang, X.Q. Zhang, L.M. Gong, H.L. Ruan, H.F. Pi, and Y.H. Zhang, Chin. Pharm. J. 44, 1217 (2009). [8] T. Morikawa, F.M. Xu, H. Matsuda, and M. Yoshikawa, Chem. Pharm. Bull. 54, 1530 (2006). [9] D. Slade, D. Ferreira, and J.P.J. Marais, Phytochemistry 66, 2177 (2005). [10] A. Okamoto, T. Ozawa, H. Imagawa, and Y. Arai, Agric. Biol. Chem. 50, 1655 (1986). [11] A.N. Flavan, A.J. Birch, and M. Salahuddin, Tetrahedron Lett. 32, 2211 (1964). [12] T. Mosmann, J. Immunol. Methods 65, 55 (1983).

Cytotoxic flavanes from Uraria clarkei.

Two new flavanes, (2R)-4'-hydroxy-2',5,7-trimethoxyflavane (1) and (2R,4R)-2',4'-dihydroxy-5,7-dimethoxyflavan-4-ol (2), were isolated from Uraria cla...
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