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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
New sesquiterpenoid derivatives from Solanum lyratum and their cytotoxicities a
a
a
a
Gui-Sheng Li , Fang Yao , Lei Zhang , Xi-Dian Yue & Sheng-Jun a
Dai a
School of Pharmaceutical Science, Yantai University, Yantai, 264005, China Published online: 29 Oct 2013.
To cite this article: Gui-Sheng Li, Fang Yao, Lei Zhang, Xi-Dian Yue & Sheng-Jun Dai (2014) New sesquiterpenoid derivatives from Solanum lyratum and their cytotoxicities, Journal of Asian Natural Products Research, 16:2, 129-134, DOI: 10.1080/10286020.2013.839664 To link to this article: http://dx.doi.org/10.1080/10286020.2013.839664
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 2, 129–134, http://dx.doi.org/10.1080/10286020.2013.839664
New sesquiterpenoid derivatives from Solanum lyratum and their cytotoxicities Gui-Sheng Li, Fang Yao, Lei Zhang, Xi-Dian Yue and Sheng-Jun Dai* School of Pharmaceutical Science, Yantai University, Yantai 264005, China
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(Received 21 April 2013; final version received 25 August 2013) Three new sesquiterpenoid isopropylidene derivatives, named solajiangxins H and I (1 and 2) and 7-hydroxylsolajiangxin I (3), were isolated from the whole plant of Solanum lyratum. Their structures were elucidated on the basis of integrated spectroscopic techniques, mainly HR-FAB-MS, 1D NMR, and 2D NMR (1H– 1H COSY, HMQC, HMBC, and NOESY). In vitro, compounds 1– 3 were found to show significant cytotoxicity against three cancer cells (P-388, HONE-1, and HT-29), and gave IC50 values in the range of 3.2– 7.7 mM. Keywords: Solanum lyratum; sesquiterpenoid isopropylidene derivatives; solajiangxins H and I; 7-hydroxylsolajiangxin I; 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 has 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 [1,2]. Solanum lyratum, commonly known as ‘Bai-Ying’ in traditional Chinese medicine and ‘Back-Mo-Deung’ in traditional Korea medicine, is a perennial herb and has been used as anti-anaphylactic, anti-inflammatory, antitumor, immunomodulatory, and antioxidant agents [3–7]. In the 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 [8–10]. As part of our ongoing search for more biologically active sesquiterpenoids, we investigated the aerial parts of S. lyratum collected in Zhangshu district, Jiangxi
Province, China. Three new sesquiterpenoid isopropylidene derivatives, attributable to eudesmane-type (1, named solajiangxin H) and vetispirane-type (2 and 3, named solajiangxin I and 7-hydroxylsolajiangxin I), were obtained (Figure 1). In addition, three new compounds were screened for cytotoxicity against P-388, HONE-1, and HT-29 cells. Herein, we report on the isolation, structural elucidation, and the evaluation of cytotoxic effects of three new sesquiterpenoid derivatives.
2. Results and discussion Compound 1 was obtained as a colorless viscous oil. The molecular formula was established as C18H28O4 by HR-FAB mass spectrum, which displayed a quasi-molecular ion peak at m/z 309.2061 [M þ H]þ. The IR spectrum showed absorption bands at 3337 and 1645 cm21, which were assignable to hydroxyl and conjugated carbonyl groups. The 1H NMR spectrum of 1 showed signals corresponding to five tertiary methyl groups at dH
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
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G.-S. Li et al. 14
OH 14
7
9
1
2' 7
O
5 9
1
O 13
15
13
3'
11
5
3
O
1'
O
3
12
2 R=H
O
2'
1'
3 R = OH
1
12
11
RO
15
3'
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Figure 1. The structures of compounds 1 – 3 isolated from S. lyratum.
1.32 (3H, s, H-13), 1.17 (3H, s, H-14), 1.78 (3H, s, H-15), 1.41 (3H, s, H-20 ), and 1.44 (3H, s, H-30 ); an oxygenated methylene at dH 3.75 (d, J ¼ 8.6 Hz, Ha-12) and 3.92 (d, J ¼ 8.6 Hz, Hb-12); and an oxygenated methine at dH 3.82 (dd, J ¼ 12.7, 5.4 Hz, H-1). Detailed examination of the 1H– 1H COSY experiment revealed two spin systems. The first spin system included the signals of three methylenes (dH 1.95, br t, J ¼ 14.8 Hz, Ha-6; 2.75, br d, J ¼ 14.8 Hz, Hb-6; 1.84, m, Ha-8; 1.50, m, Hb-8; 1.32, m, Ha-9; 2.18, m, Hb-9) and a methine (dH 1.59, m, H-7). The second spin system was traced from a methylene (dH 2.56, dd, J ¼ 16.4, 12.7 Hz, Ha-2; 2.66, dd, J ¼ 16.4, 5.4 Hz, Hb-2) and a methine (dH 3.82, dd, J ¼ 12.7, 5.4 Hz, H-1). The 13C NMR spectrum displayed 18 carbon resonances, and the DEPT spectrum was consistent with the presence of five methyls, five methylenes (one oxygenated), two methines (one oxygenated), and six quaternary carbons (two olefinic and one carbonyl). Careful analyses of above signal patterns indicated the pre-
sence of a eudesmane-type sesquiterpenoid skeleton and an isopropylidene group [11]. In the HMBC experiment (Figure 2), the correlations of H3-14 with C-1 and C-5, of H-1 with C-2, C-3, and C-5, and of H3-15 with C-3, C-4, and C-5 clearly positioned the double bond across C-4/C-5, the carbonyl group at C-3, and the hydroxyl moiety at C-1, respectively. Based on above data and comprehensive 2D NMR experiments (1H– 1H COSY, HMQC, and HMBC), the structure of 1 was established as shown in Figure 1. The relative configuration of 1 was determined from analyses of the 1H – 1H coupling constants and the NOESY spectrum (Figure 3). The coupling constants of H-1/Ha-2 and H-1/ Hb-2 were observed to be 12.7 and 5.4 Hz, respectively, suggesting that the cyclohexane ring is adopting a half-chair conformation with H-1 in an axial position and C1 –OH in an equatorial position [12]. Furthermore, the nuclear overhauser effect (NOE) correlations of Ha-9/Ha-1, Ha-9/H7, H3-14/Ha-6, H3-14/Ha-8, Ha-8/H3-13, and Ha-6/H3-13 indicated that H-1 and H-7
OH
O O H
O
O
O O
1
Figure 2. Key HMBC correlations for compounds 1 and 2.
2
Journal of Asian Natural Products Research
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H
O H
OH
Hb O
H H H3C
CH3
H
H
H3C H
1
O
H
H H
H H H
H O
O
CH3
CH3
H
O CH3
CH3 CH3
Ha
2
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Figure 3. Selected NOE correlations for compounds 1 and 2.
were in a-orientation, while H3-14 was in b-orientation (Figure 2). 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 293.2113 [M þ H]þ. The IR spectrum gave absorption band at 1658 cm21, indicating the presence of conjugated carbonyl group. The 1H NMR spectrum revealed the presence of the following fragments: five methyl groups at dH 1.01 (3H, d, J ¼ 7.1 Hz, H-15), 1.33 (3H, s, H-13), 1.39 (3H, s, H-20 ), 1.41 (3H, s, H-30 ), and 1.96 (3H, s, H-14); a trisubstituted double bond unit at dH 5.76 (s, H-7); and an oxygenated methylene group at dH 3.73 (d, J ¼ 8.7 Hz, Ha-12) and 3.80 (d, J ¼ 8.7 Hz, Hb-12). The 1H– 1H COSY spectrum displayed two spin systems. The first spin system included the signals of a methine (dH 1.99, m, H-2) and three methylenes (dH 2.16, dd, J ¼ 13.2, 7.8 Hz, Ha-1; 1.72, dd, J ¼ 13.2, 12.0 Hz, Hb-1; 1.63, m, Ha-3; 1.85, m, Hb-3; 1.67, m, Ha-4; 1.89, m, Hb-4). The second spin system was due to a methine (dH 2.10, m, H-10), a methylene (dH 2.64, dd, J ¼ 16.8, 4.8 Hz, Ha-9; 2.22, dd, J ¼ 16.8, 4.3 Hz, Hb-9), and a methyl (dH 1.01, 3H, d, J ¼ 7.1 Hz, H3-15). The 13C NMR and DEPT spectra showed 18 carbon resonances, including 5 methyls, 5 methylenes (1 oxygenated), 3 methines, and 5 quaternary carbons. Detailed examination of above 18 carbon signal patterns revealed the presence of a vetispirane-type sesquiterpenoid skeleton and an isopropylidene group [13]. The
locations of the double bond and carbonyl group were established from the HMBC spectrum. The long-range correlations of H3-14 with C-6 and C-7, as well as of H-7 with C-6, C-8, and C-14 definitively positioned the double bond across C-6/C-7 and the carbonyl group at C-8. Based on above data and comprehensive 2D NMR experiments (1H– 1H COSY, HMQC, and HMBC), the structure of 2 was determined as shown in Figure 1. The relative configuration of the stereogenic centers of 2 was elucidated by 1H – 1H coupling constants, NOE results as well as comparison of its physical and spectral data with those of the similar compound. The coupling constants of H-10/Ha-9 and H10/Hb-9 were observed to be 4.8 and 4.3 Hz, respectively, proving that the cyclohexane ring is adopting a half-chair conformation with H-10 predominantly in a pseudoequatorial position and H3-15 in a pseudoaxial position [14]. In the NOE experiment (Figure 3), the NOE correlations of Ha-1/H3-15, Ha-1/H-2, Ha-1/H313, H-2/H3-13, and Hb-1/H-10 were observed. Furthermore, its optical rotation value (½a29 D 2 76:3. c ¼ 0.76, in CHCl3) and NMR data of chiral carbons (Table 1) were in agreement with those of the previously reported sesquiterpenoid, which was isolated from Solanum tuberosum [14]. According to the above analyses, it was indicated that H3-13 and H3-15 were in a-orientation while H-10 was in b-orientation. Compound 3 was isolated as a colorless viscous oil, which was shown to have
b
a
22.4 CH3 16.2 CH3 11.0 CH3 26.8 CH3 27.1 CH3
3.75 (d, 8.6, Ha-12) 3.92 (d, 8.6, Hb-12) 1.32 (s, 3H) 1.17 (s, 3H) 1.78 (s, 3H)
1.41 (s, 3H) 1.44 (s, 3H)
41.4 C 82.8 C 72.4 CH2
37.5 CH2
1.39 (s, 3H) 1.41 (s, 3H)
3.73 (d, 8.7, Ha-12) 3.80 (d, 8.7, Hb-12) 1.33 (s, 3H) 1.96 (s, 3H) 1.01 (d, 7.1, 3H)
2.64 (dd, 16.8, 4.8, Ha-9) 2.22 (dd, 16.8, 4.3, Hb-9) 2.10 (m)
5.76 (s)
1.63 (m, Ha-3) 1.85 (m, Hb-3) 1.67 (m, Ha-4) 1.89 (m, Hb-4)
2.16 (dd, 13.2, 7.8, Ha-1) 1.72 (dd, 13.2, 12.0, Hb-1) 1.99 (m)
dH
2 (CDCl3)
Chemical shift values were in ppm and J values (in Hz) were presented within parentheses. The assignments were based on HMQC, HMBC, and 1H – 1H COSY experiments.
13 14 15 10 20 30 – OH
10 11 12
9
46.1 CH 22.2 CH2
161.1 C 29.3 CH2
5 6
7 8
129.7 C
4
1.95 (br t, 14.8, Ha-6) 2.75 (br d, 14.8, Hb-6) 1.59 (m) 1.84 (m, Ha-8) 1.50 (m, Hb-8) 1.32 (m, Ha-9) 2.18 (m, Hb-9)
197.1 C
42.3 CH2
74.4 CH
dC
3
2.56 (dd, 16.4, 12.7, Ha-1) 2.66 (dd, 16.4, 5.4, Hb-1)
2
dH
1 (CDCl3)
H and 13CNMR spectroscopic data for compounds 1 – 3a,b.
3.82 (dd, 12.7, 5.4)
1
1
No.
Table 1.
24.1 CH3 21.2 CH3 16.2 CH3 109.2 C 26.9 CH3 27.3 CH3
39.1 CH 83.0 C 73.7 CH2
42.8 CH2
125.7 CH 198.8 C
50.4 C 166.3 C
33.9 CH2
29.4 CH2
47.2 CH
36.6 CH2
dC
dH
3 (DMSO-d6)
1.27 (s, 3H) 1.32 (s, 3H) 4.60 (s)
3.61 (d, 8.8, Ha-12) 3.99 (d, 8.8, Hb-12) 1.27 (s, 3H) 1.87 (s, 3H) 0.90 (d, 7.0, 3H)
2.66 (dd, 16.5, 5.1, Ha-9) 2.03 (dd, 16.5, 4.3, Hb-9) 2.30 (m)
5.59 (s)
1.65 (m, Hb-3) 1.85 (m, Ha-3) 1.89 (m, Ha-4) 1.84 (m, Hb-4)
1.96 (d, 14.3, Ha-1) 1.92 (d, 14.3, Hb-1)
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22.7 CH3 20.4 CH3 16.3 CH3 109.2 C 26.8 CH3 27.1 CH3
40.5 CH 84.9 C 71.9 CH2
43.2 CH2
124.4 CH 198.3 C
50.2 C 166.8 C
33.7 CH2
36.4 CH2
84.1 C
43.6 CH2
dC
132 G.-S. Li et al.
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Journal of Asian Natural Products Research a molecular formula of C18H28O4 by HRFAB-MS that displayed a quasi-molecular ion peak at m/z 309.2060 [M þ H]þ. The 1 H and 13C NMR spectra of 3 (Table 1), together with the DEPT experiment, suggested that the structure of 3 (Figure 1) closely resembled to that of 2, the only marked difference was that an oxygenated quaternary carbon at dC 84.1 (C-2) was observed, instead of a methine at dC 47.2 (C-2) as seen in 2. With the aid of the NOESY data and optical rotation value, 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 new sesquiterpenoid derivatives (1 – 3) exhibited significant cytotoxicities against P-388, HONE-1, and HT-29 cells as shown in Table 2.
3.
Experimental
3.1 General experimental procedures Optical rotations were measured on a Perkin-Elmer 241 polarimeter (PerkinElmer Corporation, Waltham, MA, USA). UV spectra were obtained on a Shimadzu UV-160 spectrophotometer (Shimadzu Corporation, Tokyo, Japan). IR spectra were recorded on a Perkin-Elmer 683 infrared spectrometer with KBr disks Table 2. Cytotoxicitya of compounds 1 – 3 against cultured P-388, HONE-1, and HT29 cancer cell lines. Growth inhibition constant (IC50)a [mM] Compounds Etoposide Cisplatinb 1 2 3
b
P-388
HONE-1
HT-29
2.3 ^ 0.8 2.2 ^ 0.3 3.2 ^ 0.2 3.3 ^ 0.8 3.6 ^ 0.7
1.9 ^ 0.3 2.2 ^ 0.4 4.8 ^ 0.6 5.5 ^ 0.4 5.2 ^ 0.7
2.3 ^ 0.5 2.0 ^ 0.3 5.4 ^ 0.4 7.7 ^ 0.6 7.3 ^ 0.5
IC50 represents means ^ standard deviation of three independent replicates. The IC50 greater than 10 mM was considered to indicate no cytotoxicity. b Positive control substance. a
133
(PerkinElmer Corporation, Waltham, USA). FAB-MS and HR-FAB-MS were recorded on an Autospec-Ultima ETOF MS spectrometer (Bruker BioSpin, Ettlingen, Germany). NMR spectra were recorded on a Varian Unity BRUKER 400 at 400 MHz (1H) and 100 MHz (13C), with tetramethylsilane as the internal standard (Bruker BioSpin). Silica gel (200–300 mesh) for column chromatography (CC) and silica gel GF254 for preparative thin layer chromatography (TLC) were obtained from Qingdao Marine Chemical Factory, Qingdao, 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, 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 shadow. A voucher specimen (No. 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. The solvent was concentrated in vacuo to yield a crude extract, which was then dissolved and suspended in H2O (3.0 liters) and partitioned with CHCl3 and EtOAc. The CHCl3 fraction (191.7 g) was initially subjected to CC (10 cm £ 120 cm) on silica gel (200 –300 mesh, 3.0 kg), eluted with cyclohexane:acetone (v/v, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 60:40, and 50:50) to give eight fractions. Fraction 3 (5.6 g) was separated by CC over silica gel [eluting in a gradient with cyclohexane:ethyl acetate (v/v, 100:0 – 70:30)],
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giving 2 (47 mg) and a mixture (189 mg). The mixture was further separated by preparative TLC [chloroform:ethyl acetate (v/v, 90:10)], and subsequently purified on Sephadex LH-20 [100 g, eluting with CHCl3:CH3OH (v/v, 10:40)] to give 1 (102 mg) and 3 (61 mg).
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3.3.1
Solajiangxin H (1)
Colorless viscous oil, ½a29 D 2 54:1 (c ¼ 0.81, CHCl3). UV (MeOH) lmax: 249 nm. IR (KBr) nmax: 3337, 1645, 1467, 1379, 1086, 1025, and 860 cm21. For 1H and 13C NMR data, see Table 1. FAB-MS m/z: 309.5 [M þ H]þ. HR-FAB-MS m/z: 309.2061 [M þ H]þ (calcd for C18H29O4, 309.2066). 3.3.2
Solajiangxin I (2)
Colorless viscous oil, ½a29 D 2 76:3 (c ¼ 0.76, CHCl3). UV (MeOH) lmax: 243 nm. IR (KBr) nmax: 1658, 1617, 1465, 1385, 1025, and 893 cm21. For 1H and 13C NMR data, see Table 1. FAB-MS m/z: 293.3 [M þ H]þ. HR-FAB-MS m/z: 293.2113 [M þ H]þ (calcd for C18H29O3, 293.2117). 3.3.3
7-Hydroxylsolajiangxin I (3)
Colorless viscous oil, ½a69 D 2 69:1 (c ¼ 0.78, CHCl3). UV (MeOH) lmax: 243 nm. IR (KBr) nmax: 3340, 1663, 1615, 1464, 1385, 1026, and 891 cm21. For 1H and 13C NMR data, see Table 1. FAB-MS m/z: 309.3 [M þ H]þ. HR-FAB-MS m/z: 309.2060 [M þ H]þ (calcd for C18H29O4, 309.2066). 3.4
Antitumoral cytotoxic bioassays
Cytotoxicity was determined against P-388 (mouse lymphocytic leukemia), HONE-1 (human nasopharyngeal), and HT-29 (human colon adenocarcinoma) cells using the methyl thiazolyl tetrazolium assay method. The experimental details of this assay were carried out according to a previously described procedure [9,15].
Acknowledgments This study was financially supported by the Natural Science Foundation of Shandong Province (No. ZR2009CZ004). The authors are grateful to Ms Wen-Yan Wang and Li Shen (School of Pharmaceutical Science, Yantai University) for the measurements of FAB-MS, HR-FAB-MS, UV, IR, and NMR spectra, respectively. The authors also gratefully acknowledge Mr Hong-Bo Wang (School of Pharmaceutical Science, Yantai University) for the bioactivity screenings.
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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
New sesquiterpenoid derivatives from Solanum lyratum and their cytotoxicities a
a
a
a
Gui-Sheng Li , Fang Yao , Lei Zhang , Xi-Dian Yue & Sheng-Jun a
Dai a
School of Pharmaceutical Science, Yantai University, Yantai, 264005, China Published online: 29 Oct 2013.
To cite this article: Gui-Sheng Li, Fang Yao, Lei Zhang, Xi-Dian Yue & Sheng-Jun Dai (2014) New sesquiterpenoid derivatives from Solanum lyratum and their cytotoxicities, Journal of Asian Natural Products Research, 16:2, 129-134, DOI: 10.1080/10286020.2013.839664 To link to this article: http://dx.doi.org/10.1080/10286020.2013.839664
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 2, 129–134, http://dx.doi.org/10.1080/10286020.2013.839664
New sesquiterpenoid derivatives from Solanum lyratum and their cytotoxicities Gui-Sheng Li, Fang Yao, Lei Zhang, Xi-Dian Yue and Sheng-Jun Dai* School of Pharmaceutical Science, Yantai University, Yantai 264005, China
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(Received 21 April 2013; final version received 25 August 2013) Three new sesquiterpenoid isopropylidene derivatives, named solajiangxins H and I (1 and 2) and 7-hydroxylsolajiangxin I (3), were isolated from the whole plant of Solanum lyratum. Their structures were elucidated on the basis of integrated spectroscopic techniques, mainly HR-FAB-MS, 1D NMR, and 2D NMR (1H– 1H COSY, HMQC, HMBC, and NOESY). In vitro, compounds 1– 3 were found to show significant cytotoxicity against three cancer cells (P-388, HONE-1, and HT-29), and gave IC50 values in the range of 3.2– 7.7 mM. Keywords: Solanum lyratum; sesquiterpenoid isopropylidene derivatives; solajiangxins H and I; 7-hydroxylsolajiangxin I; 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 has 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 [1,2]. Solanum lyratum, commonly known as ‘Bai-Ying’ in traditional Chinese medicine and ‘Back-Mo-Deung’ in traditional Korea medicine, is a perennial herb and has been used as anti-anaphylactic, anti-inflammatory, antitumor, immunomodulatory, and antioxidant agents [3–7]. In the 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 [8–10]. As part of our ongoing search for more biologically active sesquiterpenoids, we investigated the aerial parts of S. lyratum collected in Zhangshu district, Jiangxi
Province, China. Three new sesquiterpenoid isopropylidene derivatives, attributable to eudesmane-type (1, named solajiangxin H) and vetispirane-type (2 and 3, named solajiangxin I and 7-hydroxylsolajiangxin I), were obtained (Figure 1). In addition, three new compounds were screened for cytotoxicity against P-388, HONE-1, and HT-29 cells. Herein, we report on the isolation, structural elucidation, and the evaluation of cytotoxic effects of three new sesquiterpenoid derivatives.
2. Results and discussion Compound 1 was obtained as a colorless viscous oil. The molecular formula was established as C18H28O4 by HR-FAB mass spectrum, which displayed a quasi-molecular ion peak at m/z 309.2061 [M þ H]þ. The IR spectrum showed absorption bands at 3337 and 1645 cm21, which were assignable to hydroxyl and conjugated carbonyl groups. The 1H NMR spectrum of 1 showed signals corresponding to five tertiary methyl groups at dH
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
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OH 14
7
9
1
2' 7
O
5 9
1
O 13
15
13
3'
11
5
3
O
1'
O
3
12
2 R=H
O
2'
1'
3 R = OH
1
12
11
RO
15
3'
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Figure 1. The structures of compounds 1 – 3 isolated from S. lyratum.
1.32 (3H, s, H-13), 1.17 (3H, s, H-14), 1.78 (3H, s, H-15), 1.41 (3H, s, H-20 ), and 1.44 (3H, s, H-30 ); an oxygenated methylene at dH 3.75 (d, J ¼ 8.6 Hz, Ha-12) and 3.92 (d, J ¼ 8.6 Hz, Hb-12); and an oxygenated methine at dH 3.82 (dd, J ¼ 12.7, 5.4 Hz, H-1). Detailed examination of the 1H– 1H COSY experiment revealed two spin systems. The first spin system included the signals of three methylenes (dH 1.95, br t, J ¼ 14.8 Hz, Ha-6; 2.75, br d, J ¼ 14.8 Hz, Hb-6; 1.84, m, Ha-8; 1.50, m, Hb-8; 1.32, m, Ha-9; 2.18, m, Hb-9) and a methine (dH 1.59, m, H-7). The second spin system was traced from a methylene (dH 2.56, dd, J ¼ 16.4, 12.7 Hz, Ha-2; 2.66, dd, J ¼ 16.4, 5.4 Hz, Hb-2) and a methine (dH 3.82, dd, J ¼ 12.7, 5.4 Hz, H-1). The 13C NMR spectrum displayed 18 carbon resonances, and the DEPT spectrum was consistent with the presence of five methyls, five methylenes (one oxygenated), two methines (one oxygenated), and six quaternary carbons (two olefinic and one carbonyl). Careful analyses of above signal patterns indicated the pre-
sence of a eudesmane-type sesquiterpenoid skeleton and an isopropylidene group [11]. In the HMBC experiment (Figure 2), the correlations of H3-14 with C-1 and C-5, of H-1 with C-2, C-3, and C-5, and of H3-15 with C-3, C-4, and C-5 clearly positioned the double bond across C-4/C-5, the carbonyl group at C-3, and the hydroxyl moiety at C-1, respectively. Based on above data and comprehensive 2D NMR experiments (1H– 1H COSY, HMQC, and HMBC), the structure of 1 was established as shown in Figure 1. The relative configuration of 1 was determined from analyses of the 1H – 1H coupling constants and the NOESY spectrum (Figure 3). The coupling constants of H-1/Ha-2 and H-1/ Hb-2 were observed to be 12.7 and 5.4 Hz, respectively, suggesting that the cyclohexane ring is adopting a half-chair conformation with H-1 in an axial position and C1 –OH in an equatorial position [12]. Furthermore, the nuclear overhauser effect (NOE) correlations of Ha-9/Ha-1, Ha-9/H7, H3-14/Ha-6, H3-14/Ha-8, Ha-8/H3-13, and Ha-6/H3-13 indicated that H-1 and H-7
OH
O O H
O
O
O O
1
Figure 2. Key HMBC correlations for compounds 1 and 2.
2
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H
O H
OH
Hb O
H H H3C
CH3
H
H
H3C H
1
O
H
H H
H H H
H O
O
CH3
CH3
H
O CH3
CH3 CH3
Ha
2
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Figure 3. Selected NOE correlations for compounds 1 and 2.
were in a-orientation, while H3-14 was in b-orientation (Figure 2). 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 293.2113 [M þ H]þ. The IR spectrum gave absorption band at 1658 cm21, indicating the presence of conjugated carbonyl group. The 1H NMR spectrum revealed the presence of the following fragments: five methyl groups at dH 1.01 (3H, d, J ¼ 7.1 Hz, H-15), 1.33 (3H, s, H-13), 1.39 (3H, s, H-20 ), 1.41 (3H, s, H-30 ), and 1.96 (3H, s, H-14); a trisubstituted double bond unit at dH 5.76 (s, H-7); and an oxygenated methylene group at dH 3.73 (d, J ¼ 8.7 Hz, Ha-12) and 3.80 (d, J ¼ 8.7 Hz, Hb-12). The 1H– 1H COSY spectrum displayed two spin systems. The first spin system included the signals of a methine (dH 1.99, m, H-2) and three methylenes (dH 2.16, dd, J ¼ 13.2, 7.8 Hz, Ha-1; 1.72, dd, J ¼ 13.2, 12.0 Hz, Hb-1; 1.63, m, Ha-3; 1.85, m, Hb-3; 1.67, m, Ha-4; 1.89, m, Hb-4). The second spin system was due to a methine (dH 2.10, m, H-10), a methylene (dH 2.64, dd, J ¼ 16.8, 4.8 Hz, Ha-9; 2.22, dd, J ¼ 16.8, 4.3 Hz, Hb-9), and a methyl (dH 1.01, 3H, d, J ¼ 7.1 Hz, H3-15). The 13C NMR and DEPT spectra showed 18 carbon resonances, including 5 methyls, 5 methylenes (1 oxygenated), 3 methines, and 5 quaternary carbons. Detailed examination of above 18 carbon signal patterns revealed the presence of a vetispirane-type sesquiterpenoid skeleton and an isopropylidene group [13]. The
locations of the double bond and carbonyl group were established from the HMBC spectrum. The long-range correlations of H3-14 with C-6 and C-7, as well as of H-7 with C-6, C-8, and C-14 definitively positioned the double bond across C-6/C-7 and the carbonyl group at C-8. Based on above data and comprehensive 2D NMR experiments (1H– 1H COSY, HMQC, and HMBC), the structure of 2 was determined as shown in Figure 1. The relative configuration of the stereogenic centers of 2 was elucidated by 1H – 1H coupling constants, NOE results as well as comparison of its physical and spectral data with those of the similar compound. The coupling constants of H-10/Ha-9 and H10/Hb-9 were observed to be 4.8 and 4.3 Hz, respectively, proving that the cyclohexane ring is adopting a half-chair conformation with H-10 predominantly in a pseudoequatorial position and H3-15 in a pseudoaxial position [14]. In the NOE experiment (Figure 3), the NOE correlations of Ha-1/H3-15, Ha-1/H-2, Ha-1/H313, H-2/H3-13, and Hb-1/H-10 were observed. Furthermore, its optical rotation value (½a29 D 2 76:3. c ¼ 0.76, in CHCl3) and NMR data of chiral carbons (Table 1) were in agreement with those of the previously reported sesquiterpenoid, which was isolated from Solanum tuberosum [14]. According to the above analyses, it was indicated that H3-13 and H3-15 were in a-orientation while H-10 was in b-orientation. Compound 3 was isolated as a colorless viscous oil, which was shown to have
b
a
22.4 CH3 16.2 CH3 11.0 CH3 26.8 CH3 27.1 CH3
3.75 (d, 8.6, Ha-12) 3.92 (d, 8.6, Hb-12) 1.32 (s, 3H) 1.17 (s, 3H) 1.78 (s, 3H)
1.41 (s, 3H) 1.44 (s, 3H)
41.4 C 82.8 C 72.4 CH2
37.5 CH2
1.39 (s, 3H) 1.41 (s, 3H)
3.73 (d, 8.7, Ha-12) 3.80 (d, 8.7, Hb-12) 1.33 (s, 3H) 1.96 (s, 3H) 1.01 (d, 7.1, 3H)
2.64 (dd, 16.8, 4.8, Ha-9) 2.22 (dd, 16.8, 4.3, Hb-9) 2.10 (m)
5.76 (s)
1.63 (m, Ha-3) 1.85 (m, Hb-3) 1.67 (m, Ha-4) 1.89 (m, Hb-4)
2.16 (dd, 13.2, 7.8, Ha-1) 1.72 (dd, 13.2, 12.0, Hb-1) 1.99 (m)
dH
2 (CDCl3)
Chemical shift values were in ppm and J values (in Hz) were presented within parentheses. The assignments were based on HMQC, HMBC, and 1H – 1H COSY experiments.
13 14 15 10 20 30 – OH
10 11 12
9
46.1 CH 22.2 CH2
161.1 C 29.3 CH2
5 6
7 8
129.7 C
4
1.95 (br t, 14.8, Ha-6) 2.75 (br d, 14.8, Hb-6) 1.59 (m) 1.84 (m, Ha-8) 1.50 (m, Hb-8) 1.32 (m, Ha-9) 2.18 (m, Hb-9)
197.1 C
42.3 CH2
74.4 CH
dC
3
2.56 (dd, 16.4, 12.7, Ha-1) 2.66 (dd, 16.4, 5.4, Hb-1)
2
dH
1 (CDCl3)
H and 13CNMR spectroscopic data for compounds 1 – 3a,b.
3.82 (dd, 12.7, 5.4)
1
1
No.
Table 1.
24.1 CH3 21.2 CH3 16.2 CH3 109.2 C 26.9 CH3 27.3 CH3
39.1 CH 83.0 C 73.7 CH2
42.8 CH2
125.7 CH 198.8 C
50.4 C 166.3 C
33.9 CH2
29.4 CH2
47.2 CH
36.6 CH2
dC
dH
3 (DMSO-d6)
1.27 (s, 3H) 1.32 (s, 3H) 4.60 (s)
3.61 (d, 8.8, Ha-12) 3.99 (d, 8.8, Hb-12) 1.27 (s, 3H) 1.87 (s, 3H) 0.90 (d, 7.0, 3H)
2.66 (dd, 16.5, 5.1, Ha-9) 2.03 (dd, 16.5, 4.3, Hb-9) 2.30 (m)
5.59 (s)
1.65 (m, Hb-3) 1.85 (m, Ha-3) 1.89 (m, Ha-4) 1.84 (m, Hb-4)
1.96 (d, 14.3, Ha-1) 1.92 (d, 14.3, Hb-1)
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22.7 CH3 20.4 CH3 16.3 CH3 109.2 C 26.8 CH3 27.1 CH3
40.5 CH 84.9 C 71.9 CH2
43.2 CH2
124.4 CH 198.3 C
50.2 C 166.8 C
33.7 CH2
36.4 CH2
84.1 C
43.6 CH2
dC
132 G.-S. Li et al.
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Journal of Asian Natural Products Research a molecular formula of C18H28O4 by HRFAB-MS that displayed a quasi-molecular ion peak at m/z 309.2060 [M þ H]þ. The 1 H and 13C NMR spectra of 3 (Table 1), together with the DEPT experiment, suggested that the structure of 3 (Figure 1) closely resembled to that of 2, the only marked difference was that an oxygenated quaternary carbon at dC 84.1 (C-2) was observed, instead of a methine at dC 47.2 (C-2) as seen in 2. With the aid of the NOESY data and optical rotation value, 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 new sesquiterpenoid derivatives (1 – 3) exhibited significant cytotoxicities against P-388, HONE-1, and HT-29 cells as shown in Table 2.
3.
Experimental
3.1 General experimental procedures Optical rotations were measured on a Perkin-Elmer 241 polarimeter (PerkinElmer Corporation, Waltham, MA, USA). UV spectra were obtained on a Shimadzu UV-160 spectrophotometer (Shimadzu Corporation, Tokyo, Japan). IR spectra were recorded on a Perkin-Elmer 683 infrared spectrometer with KBr disks Table 2. Cytotoxicitya of compounds 1 – 3 against cultured P-388, HONE-1, and HT29 cancer cell lines. Growth inhibition constant (IC50)a [mM] Compounds Etoposide Cisplatinb 1 2 3
b
P-388
HONE-1
HT-29
2.3 ^ 0.8 2.2 ^ 0.3 3.2 ^ 0.2 3.3 ^ 0.8 3.6 ^ 0.7
1.9 ^ 0.3 2.2 ^ 0.4 4.8 ^ 0.6 5.5 ^ 0.4 5.2 ^ 0.7
2.3 ^ 0.5 2.0 ^ 0.3 5.4 ^ 0.4 7.7 ^ 0.6 7.3 ^ 0.5
IC50 represents means ^ standard deviation of three independent replicates. The IC50 greater than 10 mM was considered to indicate no cytotoxicity. b Positive control substance. a
133
(PerkinElmer Corporation, Waltham, USA). FAB-MS and HR-FAB-MS were recorded on an Autospec-Ultima ETOF MS spectrometer (Bruker BioSpin, Ettlingen, Germany). NMR spectra were recorded on a Varian Unity BRUKER 400 at 400 MHz (1H) and 100 MHz (13C), with tetramethylsilane as the internal standard (Bruker BioSpin). Silica gel (200–300 mesh) for column chromatography (CC) and silica gel GF254 for preparative thin layer chromatography (TLC) were obtained from Qingdao Marine Chemical Factory, Qingdao, 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, 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 shadow. A voucher specimen (No. 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. The solvent was concentrated in vacuo to yield a crude extract, which was then dissolved and suspended in H2O (3.0 liters) and partitioned with CHCl3 and EtOAc. The CHCl3 fraction (191.7 g) was initially subjected to CC (10 cm £ 120 cm) on silica gel (200 –300 mesh, 3.0 kg), eluted with cyclohexane:acetone (v/v, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 60:40, and 50:50) to give eight fractions. Fraction 3 (5.6 g) was separated by CC over silica gel [eluting in a gradient with cyclohexane:ethyl acetate (v/v, 100:0 – 70:30)],
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giving 2 (47 mg) and a mixture (189 mg). The mixture was further separated by preparative TLC [chloroform:ethyl acetate (v/v, 90:10)], and subsequently purified on Sephadex LH-20 [100 g, eluting with CHCl3:CH3OH (v/v, 10:40)] to give 1 (102 mg) and 3 (61 mg).
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3.3.1
Solajiangxin H (1)
Colorless viscous oil, ½a29 D 2 54:1 (c ¼ 0.81, CHCl3). UV (MeOH) lmax: 249 nm. IR (KBr) nmax: 3337, 1645, 1467, 1379, 1086, 1025, and 860 cm21. For 1H and 13C NMR data, see Table 1. FAB-MS m/z: 309.5 [M þ H]þ. HR-FAB-MS m/z: 309.2061 [M þ H]þ (calcd for C18H29O4, 309.2066). 3.3.2
Solajiangxin I (2)
Colorless viscous oil, ½a29 D 2 76:3 (c ¼ 0.76, CHCl3). UV (MeOH) lmax: 243 nm. IR (KBr) nmax: 1658, 1617, 1465, 1385, 1025, and 893 cm21. For 1H and 13C NMR data, see Table 1. FAB-MS m/z: 293.3 [M þ H]þ. HR-FAB-MS m/z: 293.2113 [M þ H]þ (calcd for C18H29O3, 293.2117). 3.3.3
7-Hydroxylsolajiangxin I (3)
Colorless viscous oil, ½a69 D 2 69:1 (c ¼ 0.78, CHCl3). UV (MeOH) lmax: 243 nm. IR (KBr) nmax: 3340, 1663, 1615, 1464, 1385, 1026, and 891 cm21. For 1H and 13C NMR data, see Table 1. FAB-MS m/z: 309.3 [M þ H]þ. HR-FAB-MS m/z: 309.2060 [M þ H]þ (calcd for C18H29O4, 309.2066). 3.4
Antitumoral cytotoxic bioassays
Cytotoxicity was determined against P-388 (mouse lymphocytic leukemia), HONE-1 (human nasopharyngeal), and HT-29 (human colon adenocarcinoma) cells using the methyl thiazolyl tetrazolium assay method. The experimental details of this assay were carried out according to a previously described procedure [9,15].
Acknowledgments This study was financially supported by the Natural Science Foundation of Shandong Province (No. ZR2009CZ004). The authors are grateful to Ms Wen-Yan Wang and Li Shen (School of Pharmaceutical Science, Yantai University) for the measurements of FAB-MS, HR-FAB-MS, UV, IR, and NMR spectra, respectively. The authors also gratefully acknowledge Mr Hong-Bo Wang (School of Pharmaceutical Science, Yantai University) for the bioactivity screenings.
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