Three new cytotoxic sesquiterpenoids from Solanum lyratum

Phytochemistry Letters 6 (2013) 453–456

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Three new cytotoxic sesquiterpenoids from Solanum lyratum Fang Yao a, Qin-Lan Song b, Lei Zhang a, Gui-Sheng Li a, Sheng-Jun Dai a,* a b

School of Pharmaceutical Science, Yantai University, Yantai 264005, People’s Republic of China Shandong University of Traditional Chinese Medicine Affiliated Hospital, Jinan 250011, People’s Republic of China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 5 March 2013 Received in revised form 14 May 2013 Accepted 17 May 2013 Available online 4 June 2013

In our continuing effort to discover more new cytotoxic sesquiterpenoid from Solanum lyratum collected from different districts of China, three new sesquiterpenoids, attributable to eudesmane-type (1, named solajiangxin D) and vetispirane-type (2–3, named solajiangxins E and 2-hydroxysolajiangxin E), respectively, were isolated. Their structures were elucidated on the basis of integrated spectroscopic techniques, mainly HR-FABMS, 1D and 2D NMR (1H–1H COSY, HMQC, HMBC and ROESY). In vitro, compounds 1–3 were found to show significant cytotoxicities against three human cancer lines (P-388, HONE-1, and HT-29), and gave ED50 values in the range 2.1–3.7 mg/ml. ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Solanum lyratum Solanaceae Sesquiterpenoids Solajiangxins D–E 2-Hydroxysolajiangxin E Cytotoxic activity

1. Introduction The genus Solanum, which is one of the largest genera of the Solanaceae, is widely distributed in tropical and temperate zones and is a rich source of active secondary metabolites. Many species belonging to this genus always draw the attention of numerous chemical researchers due to their biologically active constituents, including steroids, steroidal alkaloids and their glycosides (Zhou et al., 2006; Lu et al., 2011). Solanum lyratum, commonly known as ‘‘Bai-Ying’’ in traditional Chinese medicine and ‘‘Back-Mo-Deung’’ in traditional Korean medicine, respectively, is a perennial herb, and has been used as anti-anaphylactic (Kang et al., 1998), antiinflammatory (Zhang et al., 2012), anti-tumor (Hsu et al., 2008), immunomodulatory (Yang et al., 2010), and anti-oxidant (Kuo et al., 2009) agents. In previous phytochemical studies on S. lyratum collected from the Linyi district, Shandong Province, China, six new sesquiterpenoids were separated and most of them showed significant cytotoxic activities (Dai et al., 2009; Ren et al., 2009; Yue et al., 2012). As part of our ongoing search for more biologically active sesquiterpenoids, we investigated the whole plant of S. lyratum collected in Zhangshu district, Jiangxi Province, China. The EtOH extract of this species was successively partitioned with CHCl3 and EtOAc. The CHCl3 fraction was concentrated in vacuo and

* Corresponding author. Tel.: +86 535 6706025; fax: +86 535 6706036. E-mail address: [email protected] (S.-J. Dai).

sequentially subjected to column chromatography over silica gel and Sephadex LH-20 to give three new sesquiterpenoids. By means of extensive spectroscopic analyses, their structures were identified as eudesmane-type (1, named solajiangxin D) and vetispiranetype (2–3, named solajiangxins E and 2-hydroxysolajiangxin E), respectively. In addition, three new compounds were screened for cytotoxicity against P-388 (mouse lymphocytic leukemia), HONE1 (human nasopharyngeal) and HT-29 (human colon adenocarcinoma) cells. Herein we report on the isolation, structural elucidation and cytotoxicity of the three new sesquiterpenoids. 2. Results and discussion Compound 1 was isolated and purified as white powder. The molecular formula was established as C15H24O4 by HR-FAB mass spectroscopy, which displayed a quasi-molecular ion peak at m/z 269.1758 [M+H]+. The IR spectrum showed absorption bands at 3359 (br), 1705 and 1633 cm1, which were assignable to hydroxyl, carbonyl and double bond groups. The 1H NMR spectrum of 1 showed signals corresponding to one tertiary methyl group (dH 0.53, 3H, s, H3-14), one secondary methyl group (dH 1.02, 3H, d, J = 7.0 Hz, H3-13), two olefinic protons (dH 4.40, 1H, s, Ha-15; 4.74, 1H, s, Hb-15), two oxygenated methine protons (dH 3.03, 1H, d, J = 9.0 Hz, H-1; 3.30, 1H, m, H-2), two hydroxyl groups (dH 5.79, 1H, br s, C1-OH; 4.84, 1H, br s, C2-OH), and one carboxyl moiety (dH 11.39, 1H, s, C11-OH). The 13C NMR displayed 15 carbon resonances and the distortionless enhancement by polarization transfer (DEPT) spectrum was consistent with the presence of two

1874-3900/$ – see front matter ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2013.05.012

F. Yao et al. / Phytochemistry Letters 6 (2013) 453–456

454

OH 14 1

HO

9

O

5

3

11 7

H

14

7

12

3

O

5

OH

11

9

1 15

13

15

12

1

H

O

3'

14

7

13

O

O 1'

2'

3 5

12 11

9

2

13

1 15

OH

O

3'

O 1'

2'

3

Fig. 1. The structures of new sesquiterpenoids isolated from Solanum lyratum.

OH HO

O H H

O OH

H

H 1

O

O

2

Fig. 2. Key HMBC correlations for compounds 1–2.

methines bearing hydroxyl groups (dC 84.5, C-1; 70.3, C-2), a carbonyl carbon (dC 177.1, C-12), and two olefinic carbons (dC 147.8, C-4; 107.3, C-15), as well as two methyls, four methylenes (sp3 hybridized), three methines (sp3 hybridized), and one quaternary carbon (sp3 hybridized). These signal patterns indicated 1 could be a sesquiterpenoid with an eudesmane-type skeleton (Guilhon and Mu¨ller, 1998). In the heteronuclear multiple bond connectivity (HMBC) spectrum of 1 (Fig. 2), the long-range correlations from H3-14 to C-1, from C1-OH to C-1 and C-10, and from C2-OH to C-2 and C-3 confirmed that two hydroxyl groups were connected to C-1 and C-2, respectively, while correlations from H3-13 to C-12 and from H-11 to C-12 demonstrated that the carboxyl moiety was connected to C-11. Based on the above data and comprehensive 2D NMR experiments (1H–1H COSY, HMQC, HMBC), the structure of 1 was unambiguously established as shown in Fig. 1. The relative configuration of 1 was elucidated by 1H–1H coupling constant and the ROESY spectrum (Fig. 3). The large coupling value of H-1 (J = 9.0 Hz) with H-2 indicated H-1 and H-2 must be in the axial position. In the ROESY experiment, the cross peaks were observed from H3-14 to H-2 and C1-OH, as well as from H-5 to H-1 and H-7. Thus, H3-14 and H-2 were on the same molecular plane (b-configuration), while H-1, H-5, and H-7 were on the opposite side of the molecular plane (a-configuration). Compound 2 was isolated as a colorless viscous oil, and the molecular formula was determined to be C18H28O3 by HR-FABMS, which displayed a quasi-molecular ion peak at m/z 293.2112 [M+H]+. The IR spectrum showed absorption band at 1660 cm1, indicating the presence of one a,b-unsaturated ketone group (Coxon et al., 1974). The 1H NMR spectrum revealed the presence

H HO

Ha Hb

CH3

HO

COOH H

H

1

H

CH3

O

Hb H Ha

H

2

Fig. 3. Selected NOE correlations for compounds 1–2.

O

O

of the following fragments: five methyl groups at dH 1.00 (3H, d, J = 7.0 Hz, H-15), 1.31 (3H, s, H-13), 1.40 (3H, s, H-20 ), 1.41 (3H, s, H30 ), and 1.95 (3H, s, H-14); a tri-substituted double bond unit at dH 5.75 (1H, s, H-7); and an oxygenated methylene group at dH 3.73 (1H, d, J = 8.4 Hz, Ha-12), 3.86 (1H, d, J = 8.4 Hz, Hb-12). Detailed examination of the 1H–1H COSY spectrum revealed two spin systems. The first spin system included the signals of a methine (dH 2.15, 1H, m, H-2) and three methylenes (dH 1.98, 1H, dd, J = 7.8, 13.4 Hz, Ha-1; 1.62, 1H, dd, J = 11.6, 13.4 Hz, Hb-1; 1.83, 1H, m, Ha3; 1.68, 1H, m, Hb-3; 1.67, 1H, m, Ha-4; 1.89, 1H, m, Hb-4). The second spin system was traced from a methine (dH 2.10, 1H, m, H10), a methylene (dH 2.65, 1H, dd, J = 4.6, 16.8 Hz, Ha-9; 2.20, 1H, dd, J = 4.3, 16.8 Hz, Hb-9) and a methyl (dH 1.00, 3H, d, J = 7.0 Hz, H15). The 13C NMR (Table 1) and DEPT spectra showed 18 carbon resonances including five methyls, five methylenes (one oxygenated, four sp3 hybridized), three methines (two sp3 hybridized, one olefinic), and five quaternary carbons (two oxygenated, one olefinic, one carbonyl and one sp3 hybridized). Careful analysis of the above 18 carbon signals indicated that 15 were due to a vetispirane-type sesquiterpenoid skeleton (Anderson et al., 1977), and 3 to an isopropylidene group. In the HMBC experiment (Fig. 2), the long-range correlations from H3-14 at dH 1.95 to C-6 at dC 166.3 and C-7 at dC 125.6, as well as from H-7 at dH 5.75 to C-6 at dC 166.3, C-8 at dC 199.0 and C-14 at dC 20.9 clearly positioned the double bond across C-6/C-7 and carbonyl group at C-8. Based on the above data and comprehensive 2D NMR experiments (1H–1H COSY, HMQC, HMBC), the structure of 2 was established as shown in Fig. 1. The relative configuration of 2 could be deduced from analyses of the 1H–1H coupling constants and NOESY spectrum (Fig. 3). The coupling constants between H-10 and Ha-9, as well as H-10 and Hb-9 were observed to be 4.6 Hz, and 4.3 Hz, respectively, this suggested the cyclohexane ring is adopting half-chair conformation with H3-15 predominantly in a pseudoaxial position. The NOE correlations of H-1a/H3-15, H-1a/H-2, H-2/H12a, H-12a/H3-20 , H-1b/H-10 and H-1b/H3-13 indicated that H-1a, H-2, H-12a, H3-15 and H3-20 were co-facial and a-orientation, while H-10 and H3-13 were on the opposite side of the molecular plane and thus b-orientation. Compound 3 was isolated and purified as a colorless viscous oil, which was shown to have a molecular formula of C18H28O4 by HRFABMS that displayed a quasi-molecular ion peak at m/z 309.2061 [M+H]+. The 1H and 13C NMR spectra of 3 (Table 1) together with the DEPT experiment suggested that the structure of 3 (Fig. 1) closely resembled that of 2, the only marked difference was that an oxygenated quaternary carbon at dC 84.0 (C-2) was observed, instead of a methine at dC 47.5 (C-2) as seen in 2. With the aid of the NOESY data, it was readily confirmed that 3 had the same relative configuration at C-2, C-5, C-10, and C-11 as 2. Preliminary cytotoxicity screening revealed that three isolated new sesquiterpenoids (1–3) exhibited significant cytotoxicities against P-388 (mouse lymphocytic leukemia), HONE-1 (human nasopharyngeal) and HT-29 (human colon adenocarcinoma) cells as shown in Table 2.

F. Yao et al. / Phytochemistry Letters 6 (2013) 453–456

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Table 1 NMR spectroscopic data for compounds 1–3.a,b No.

1 (DMSO-d6)

2 (CDCl3)

3 (DMSO-d6)

dH

dC

dH

dC

dH

dC

1

3.03 (d, 9.0)

84.5 CH

1.98 (dd, 7.8, 13.4, Ha-1) 1.62 (dd, 11.6, 13.4, Hb-1)

37.4 CH2

1.99 (d, 14.2, Ha-1) 1.75 (d, 14.2, Hb-1)

44.6 CH2

2 3

3.30 (m) 1.93 (br t, 12.7, Ha-3) 2.41 (dd, 5.4, 12.7, Hb-3)

70.3 CH 43.1 CH2

2.15 (m) 1.83 (m, Ha-3) 1.68 (m, Hb-3)

47.5 CH 28.3 CH2

147.8 C

1.67 (m, Ha-4) 1.89 (m, Hb-4)

33.9 CH2

4

2.02 (m, Ha-3) 1.62 (m, Hb-3) 1.85 (m, Ha-4) 1.84 (m, Hb-4)

84.0 C 35.3 CH2 33.8 CH2

5

1.73 (br d, 11.6)

46.9 CH

49.9 C

49.7 C

6

1.11 1.41 2.18 1.09 1.48

(m, Ha-6) (m, Hb-6) (m) (m, Ha-8) (m, Hb-8)

27.7 CH2

166.3 C

166.5C

44.6 CH 23.5 CH2

5.75 (s)

125.6 CH 199.0 C

5.59 (s)

124.5 CH 198.3 C

1.87 (m, Ha-9) 1.08 (m, Hb-9)

36.5 CH2

2.65 (dd, 4.6, 16.8, Ha-9) 2.20 (dd, 4.3, 16.8, Hb-9) 2.10 (m)

42.8 CH2

2.66 (dd, 5.1, 16.5, Ha-9) 2.03 (dd, 4.3, 16.5, Hb-9) 2.30 (m)

43.2 CH2

73.0 CH2

71.9 CH2

24.8 CH3 20.9 CH3

3.62 4.05 1.28 1.88

0.90 (d, 7.0, 3H)

7 8

9 10 11

39.0 C 39.8 CH

1.49 (m)

12 13 14

1.02 (d, 7.0, 3H) 0.53 (s, 3H)

13.9 CH3 11.1 CH3

3.73 3.86 1.31 1.95

15

4.40 (s, H-15a) 4.74 (s, H-15b)

107.3 CH2

1.00 (d, 7.0, 3H)

15.8 CH3

1.40 (s, 3H) 1.41 (s, 3H)

109.2 C 26.7 CH3 27.0 CH3

10 20 30 C1-OH C2-OH COOH a b

177.1 C

(d, 8.4, Ha-12) (d, 8.4, Hb-12) (s, 3H) (s, 3H)

38.4 CH 82.1 C

5.79 (br s) 4.84 (br s) 11.39 (s)

(d, 8.8, Ha-12) (d, 8.8, Hb-12) (s, 3H) (s, 3H)

1.27 (s, 3H) 1.32 (s, 3H) 4.53 (s)

39.8 CH 84.5 C

22.9 CH3 20.4 CH3 16.1 CH3 108.7 C 27.0 CH3 27.2 CH3

Chemical shift values were in ppm and J values (in Hz) were presented in parentheses. The assignments were based on HMQC, HMBC, and 1H–1H COSY experiments.

Table 2 Cytotoxicitya of compounds 1–3 against cultured P-388, HONE-1 and HT29 cancer cell lines. Compounds

Cell lines ED50 (mg/ml) P-388

HONE-1

HT-29

Etoposideb Cisplatinb 1 2 3

2.1 1.9 3.1 2.7 2.6

1.6 1.7 3.7 2.1 2.8

1.9 1.8 3.6 2.4 2.5

a For significant cytotoxic activity of pure compounds, an ED50 of 4.0 mg/ml is required. b Positive control substance.

3. Experimental 3.1. General experimental procedures Optical rotations were measured on a Perkin-Elmer 241 polarimeter. UV spectra were obtained on a Shimadzu UV-160 spectrophotometer. IR spectra were recorded on a Perkin-Elmer 683 infrared spectrometer with KBr disks. FABMS and HR-FABMS were recorded on an Autospec-Ultima ETOF MS spectrometer. NMR spectra were recorded on a Varian Unity BRUKER 400 at 400 MHz (1H) and 100 MHz (13C), with TMS as the internal standard. Silica gel (200–300 mesh) for column chromatography (CC) and silica gel GF254 for preparative TLC were obtained from Qingdao Marine Chemical Factory, Qingdao, People,s Republic of China. Precoated plates of silica gel GF254 were used for TLC, and detected under UV light.

3.2. Plant material S. lyratum Thunb. was collected in Zhangshu district, Jiangxi Province, People’s Republic of China, in September 2010, and identified by professor Gui-Sheng Li, School of Pharmaceutical Science, Yantai University. The whole plants of S. lyratum were harvested and air-dried at room temperature in the dark. A voucher specimen (YP10082) has been deposited at the herbarium of the School of Pharmaceutical Science, Yantai University. 3.3. Extraction and isolation The air-dried whole plant (50.0 kg) of S. lyratum was finely cut and extracted three times (1 h 3) with refluxing EtOH. Evaporation of the solvent under reduced pressure provided the ethanolic extract. The extract was dissolved and suspended in H2O (3.0 l) and partitioned with CHCl3 (5  8 l) and EtOAc (5  8 l). The CHCl3 fraction (191.7 g) was initially subjected to column (10 cm  120 cm) chromatography on silica gel (200– 300 mesh, 3.0 kg), eluted with cyclohexane–acetone [v/v, 95:5 (5.0 l), 90:10 (5.0 l), 85:15 (10.0 l), 80:20 (10.0 l), 75:25 (10.0 l), 70:30 (10.0 l), 60:40 (5.0 l) and 50:50 (5.0 l)] to give eight fractions. Fraction 4 (8.4 g) was separated by CC over silica gel [eluted by petroleum ether–acetone (v/v, 100:0–70:30)] and preparative TLC [cyclohexane–acetone (v/v, 7:3)], and subsequently purified on Sephadex LH-20 [100 g, eluting with CHCl3– CH3OH (v/v, 10:40)] to give compounds 2 (59 mg) and 3 (67 mg). Fraction 5 (7.3 g) was separated by CC over silica gel [eluted by petroleum ether–acetone (v/v, 95:5–65:35)], giving 1 (107 mg).

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3.4. Solajiangxin D (1) White powder, ½a29 D +16.4 (c 0.63, MeOH). IR (KBr) nmax: 3359 (br), 1705, 1633, 1466, 1385, 1227, and 1028 cm1. FABMS m/z: 269.3 [M+H]+. HR-FABMS m/z: 269.1758 [M+H]+ (Calcd. for C15H24O4, 269.1753). For 1H and 13C NMR spectroscopic data, see Table 1.

are grateful to Ms. Wen-Yan Wang, Li Shen (School of Pharmaceutical Science, Yantai University) for the measurements of FABMS, HR-FABMS, UV, IR and NMR spectra, respectively. The authors also gratefully acknowledge Mr. Liang Ye (School of Pharmaceutical Science, Yantai University) for the bioactivity screenings.

References 3.5. Solajiangxin E (2) Colorless viscous oil, ½a29 D 106.3 (c 0.77, CHCl3). UV (MeOH) lmax: 243 nm. IR (KBr) nmax: 1660, 1617, 1464, 1385, 1025 and 893 cm1. FABMS m/z: 293.4 [M+H]+. HR-FABMS m/z: 293.2112 [M+H]+ (Calcd. for C18H28O3, 293.2117). For 1H and 13C NMR spectroscopic data, see Table 1. 3.6. 2-Hydroxysolajiangxin E (3) Colorless viscous oil, ½a29 D 94.1 (c 0.79, CHCl3). UV (MeOH) lmax: 243 nm. IR (KBr) nmax: 3339, 1660, 1615, 1462, 1385, 1023 and 895 cm1. FABMS m/z: 309.3 [M+H]+. HR-FABMS m/z: 309.2061 [M+H]+ (Calcd. for C18H28O4, 309.2066). For 1H and 13 C NMR spectroscopic data, see Table 1. 3.7. Cytotoxic bioassays Cytotoxicity was determined against P-388 (mouse lymphocytic leukemia), HONE-1 (human nasopharyngeal) and HT-29 (human colon adenocarcinoma) tumor cells (provided by the Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College) using the MTT assay method and anti-cancer drugs, etoposide and cisplatin, as positive controls. The experimental details of this assay were carried out according to a previously described procedure (Hou et al., 1995; Ren et al., 2009). Acknowledgements This study was financially supported by the Natural Science Foundation of Shandong Province (No. ZR2009CZ004). The authors

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