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Callistephus A, a novel sesquiterpene from the Callistephus chinensis flower
G Model
PHYTOL 818 1–4 Phytochemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol 1 2 3 4 5 6 7 8 9
Callistephus A, a novel sesquiterpene from the Callistephus chinensis flower , Jia-qing Cao a,b, Zhen-ting Liu c, Lei Yuan a,b, Gang Chen a,b, Jing-xin Liu c, , Yu-qing Zhao a,b,*
Q1 Xiao-shu Zhang a,b
Fan-zhi Qu
a,b
a
School of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China c Chifeng MinYi Natural Anthocyanins Company Limited, Inner Mongolia Autonomous Region, 024000, People’s Republic of China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 September 2014 Received in revised form 27 October 2014 Accepted 29 October 2014 Available online xxx
Two new compounds callistephus A (1) and callistephus B (2) together with 4,6-dihydroxyphenyl-1butanone-2-b-D-O-glucopyranoside (3), were isolated and purified from the Callistephus chinensis flower. Their structures were identified by the interpretation of spectroscopic data. X-ray crystallographic and analysis of quantum chemical ECD calculation were applied to determine the absolute configuration. Of them, callistephus A represents a rare sesquiterpene with new carbon skeleton and also its possible biogenesis was proposed. ß 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Keywords: Callistephus chinensis Sesquiterpene X-ray diffraction Electronic circular dichroism (ECD)
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
1. Introduction The genus Callistephus chinensis belongs to the Asteraceae family. Previous pharmacological studies have shown that Asteraceae extracts display various biological activities including anti-diabetes, antioxidant properties and inhibitory effects against bacteria and viruses. Moreover, the extracts are also known to prevent indigestion, pneumonia, hepatitis, and tumors (Matsuda et al., 2002; Cheng et al., 2005; Zhu et al., 2005; Gorzalczany et al., 2013). However, limited information on phytochemical or pharmacological studies of C. chinensis flower (CCF) was available. As a continue research about CCF (Zhang et al., 2013), in this present work, study on structural determination of CCF extract resulted in the isolation of one rare sesquiterpene along with one new and one known compounds. Herein, we present the isolation and structural elucidation of these new compounds. Their structures were elucidated as callistephus A (1) callistephus B (2) and 4,6-dihydroxyphenyl-1-butanone-2-b-D-O-glucopyranoside (3) (Li et al., 2008). To the best of our knowledge, all of these Q3 compounds were isolated from the CCF for the first time (Fig. 1).
* Corresponding author at: School of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China. Q2 Tel.: +86 24 23986521; fax: +86 24 23986521. E-mail addresses: [email protected], [email protected] (Y.-q. Zhao).
2. Results and discussion
30
Compound 1 was purified as white crystals. The molecular formula compound 1 was determined as C20H28O5 by HR-ESI-MS (m/z 371.1830 [M+Na]+, calcd 371.1829), with seven degrees of unsaturation. The structure of 1 was elucidated by interpretation of its NMR spectra, including 1H, 13C, HSQC, and HMBC. The 1H NMR spectrum (Table 1) showed signals of five methyl groups (dH 1.29, 1.33, 1.35, 1.88, and 1.99), several multiplets (dH 1.47–2.08), two olefinic protons (dH 6.12 and 5.51), and an oxygenated proton (dH 5.07). Twenty carbon resonances were observed in its 13C NMR data (Table 1). Using the DEPT technique, five methyls, four methylenes, four methines (including an oxygenated and two olefinic carbons), and seven quaternary carbons (including two lactone carbonyls) were found in 1. 1H–1H COSY correlations of H2-1/H-2/H-11 and HMBC correlations from H2-1 to C-3 and C-11 and from H-4 to C-11 and C-15 constructed a six-member ring (A). 1 H–1H COSY correlations of H2-7/H2-8/H2-9 and HMBC correlations from H-11 to C-9 and C-10, from H3-12 to C-9, C-10, and C-11, and from H3-13 to C-5, C-6, C-7, and C-14 allowed the establishment of a seven-member ring (B). An olefinic ester group (dC 16.0, 20.6, 127.4, 138.9, and 168.3) was assigned at C-2 and deduced from HMBC correlations from H-2 to C-16 and from H-18 to C-16. An ester bond was found in the B fragment (Fig. 2). The connection of rings A and B was established by HMBC correlations
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
http://dx.doi.org/10.1016/j.phytol.2014.10.030 1874-3900/ß 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Please cite this article in press as: , X.- Zhang., et al., Callistephus A, a novel sesquiterpene from the Callistephus chinensis flower. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.10.030
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PHYTOL 818 1–4 X.- Zhang et al. / Phytochemistry Letters xxx (2014) xxx–xxx
2
Fig. 1. Structures of 1–3.
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from H2-1 to C-10, from H-4 to C-7, and from H-11 to C-1, C-3, C-9, and C-10, which indicate that C-5 and C-11 were shared by rings A and B. Finally, the planar structure of 1 was established with a rare 6/7 skeleton. The relative configuration of 1 was established by analyzing its NOESY correlations. Significant NOE correlations among H-2/H-4, H-4/H-11, and H-2/H3-15 suggested that the atoms were co-facial (Fig. 3). X-ray diffraction analysis (Fig. 4) finally confirmed the structure proposed above Thus, the structure of 1 was confirmed, and the compound was named callistephus A. From a biogenetic point of view, compound 1 is structurally related to himachalane, a small group of 6/7 skeleton sesquiterpenoids found mainly in Asteraceae. The biogenesis of himachalane framework starts with farnesyl diphosphate, which after several steps leads to himachalane cation, a key intermediary in the biosynthesis of several compounds (Radulovic and Denic, 2013). Compound 1 seems to derive from himachalane cation by a range of rearrangement and oxidation reactions (Scheme 1). Compound 2 presented as a white amorphous powder possessing a molecular formula of C12H12O5, as determined by the positive HR-ESI-MS (m/z 259.0576 [M+Na]+, calcd 259.0577) with seven degrees of unsaturation. In the IR spectrum (nmax, cm1), 2 revealed bands characteristic of the hydroxyl group (3415 and 3400) and aromatic (1597, 1502, and 1463) absorption. Structural identification of compound 2 and gymnastone by 1H and 13C NMR (Table 1) spectroscopy yielded similar spectra but with some changes in the side chains (Dat et al., 2004; Ma et al., 2010a,b). The 13C NMR spectrum of 2 showed only two
Table 1 NMR spectroscopic data for compounds 1 and 2 (d in ppm, J in Hz). No
1 (CDCl3)
dH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.58, m 2.08, dt, 12.0, 4.1 5.07, dd, 12.3, 3.8 551, d, 2.9
1.47, 1.86, 1.59, 1.70, 1.78, 1.82,
2 (DMSO-d6)
dC
m m m m m m
2.66, dt, 12.0, 3.8 1.33, s 1.35, s 1.29, s
6.12, qd, 7.2, 1.4 1.88, m 1.99, dd, 7.2, 1.4
dH
dC
29.2
165.6
78.3 72.9 127.4 138.9 46.1 40.1
103.2 106.9 157.1 116.2 113.0 147.3
6.72, s 7.04, s
8.12, s
21.1
135.9
33.4
204.5
82.0 46.4 27.7 21.9 175.3 24.3 168.3 127.6 139.8 20.6 16.0
2.68, 4.65, 11.0, 3.65,
s dd, 5.6 m
Fig. 2. Key 1H–1H COSY and HMBC correlations of 1 and 2.
oxygen-attached carbon signals (dC 68.4 and 64.2), and its 1H spectrum showed one hydroxyl methylene peak [dH 4.65 (1H, dd, J = 11.0, 5.6)]. The structure of 1 was also confirmed by HMBC experiments (Fig. 2), which showed the correlations from H-3 to C8, from H-4 to C-9, from H-7 to C-9 and C-10, from H-12 to C-2. HSQC and HMBC experiments allowed deduction of the planar structure of 2. On the basis of this deduction, the predicted ECD curve of 2 was calculated by the quantum chemical method at the [B3LYP/6-31G (d)] level using the GAUSSIAN 09 program, and the predicted ECD curve of 12R-2 was similar to the experimental one (Fig. 5). These findings demonstrated the absolute configuration of 2 as 12R. Thus, the structure of 2 was confirmed, and the compound was named callistephus B.
82 83 84 85 86 87 88 89 90 91 92 93 94
3. Experimental
95
3.1. General experimental procedures
96
Optical rotations were measured on a Perkin-Elmer polarimeter. IR Spectra: Bruker IFS-55 spectrophotometer; KBr pellets; cm1. HR-TOF-MS: BIC micro TOF-Q mass spectrometer; in m/z (rel %). The circular dichroism (CD) spectra were measured on a Bio-Logic Science (MOS-450, France). The X-ray crystallographic data were collected on a Bruker Smart CCD diffractometer using graphite-monochromated Mo Ka radiation. NMR spectra were recorded on Bruker AV-600 spectrometer with TMS as internal standard, J in Hz. Column chromatography (cc): silica gel (SiO2:200– 300 mesh, Qingdao Marine Chemical Group, Co); Sephadex LH-20 (pharmacia, Co). Prep HPLC (Beijing CXTH3000 system): P3000 pump, UV3000 spectrophotometric detector at 210 nm, YMC C18 reversed-phase column (5 mm, 10 250 nm; flow rate3.0 mL/min).
97 98 99 100 101 102 103 104 105 106 107 108 109
3.2. Plant material
110
The flowers of Asteraceae for CCF were collected from Inner Mongolia Autonomous Region, Republic of China, in 2012 and
111 112
27.4 68.4 64.2
Fig. 3. NOESY correlations of 1.
Please cite this article in press as: , X.- Zhang., et al., Callistephus A, a novel sesquiterpene from the Callistephus chinensis flower. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.10.030
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PHYTOL 818 1–4 X.- Zhang et al. / Phytochemistry Letters xxx (2014) xxx–xxx
3
Fig. 4. X-ray crystal structure of 1.
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authenticated by Prof. Jincai Lu of Shenyang Pharmaceutical University. A voucher specimen (No. 20120124) was deposited in the Herbarium of Shenyang Pharmaceutical University (Shenyang, People’s Replublic of China). The extract of CCF was provided from Tianjin Jianfeng Natural Products Research and Development Co and extracted with ethanol (50%) three times at 50 8C.
119
3.3. Extraction and isolation
120 121 122 123 124 125 126 127 128 129 130 131 132
The extract (100 g) was suspended in water (2000 mL) and partitioned with ethyl acetate and n-butanol successively, to yield the ethyl acetate and n-butanol extracts. The ethyl acetate extract (70 g) was subjected to silica gel column chromatography (300– 400 mesh, 1.5 kg), using a step-wise system of CH2Cl2/MeOH (100:0–5:1, v/v) gradient elution, to afford nine fractions (Fr 1–Fr Q4 10) on the basis of TLC composition. Fr 3 (1.2 g) was subjected to Sephadex LH-20 column chromatography (CC), using CH2Cl2/ MeOH (1:1, v/v) elution to give five fractions L1–L5. Fraction L2 gave compound 1 (27 mg) by recrystallization. Fraction L5 (0.4 g) was subjected to silica gel CC, with petroleum ether–acetone (40:1, 20:1, 10:1, v/v) gradient elution, to yield compound 2. Fr 5 (3.5 g) was subjected to silica gel CC, with CH2Cl2/MeOH (50:1–5:1, v/v)
gradient elution, to yield five fractions S1–S5. Fraction S4 (0.5 g) was subjected to repeated Sephadex LH-20 (CH2Cl2/MeOH, 1:1, v/v) CC, and further purified using preparative HPLC (YMC, ODS S-5, 55% MeOH), affording compounds 3 (8 mg).
133 134 135 136
3.3.1. Callistephus A (1) Colorless crystal (CH2Cl2); ½a25 D 23:9 (c 0.125, CH2Cl2); IR (KBr) vmax 3425, 2944, 1730, 1690, 1643 cm1; HR-ESI-MS: (m/z 371.1830 [M+Na]+, calcd 371.1829); 1H NMR (600 MHz, CH2Cl2) and 13C NMR (125 MHz, CH2Cl2) spectroscopic data, see Table 1.
137 138 139 140 141
3.3.2. Callistephus B (2) White amorphous powder (MeOH); ½a25 D þ 19:2 (c 0.125, MeOH); IR (KBr) vmax 3433, 1740, 1587, 1419 cm1; HR-ESI-MS (m/z 259.0576 [M+Na]+, calcd 259.0577); 1H NMR (600 MHz, DMSO) and 13C NMR (125 MHz, DMSO) spectroscopic data, see Table 1.
142 143 144 145 146 147
3.4. X-ray crystallographic analysis of 1
148
The crystal of 1 belongs to an orthorhombic system, space group P212121 with a = 10.5339 (37) A˚, b = 13.2349 (46) A˚, c = 13.4690
149 150
Scheme 1. Possible biosynthesis of compound 1.
Please cite this article in press as: , X.- Zhang., et al., Callistephus A, a novel sesquiterpene from the Callistephus chinensis flower. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.10.030
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PHYTOL 818 1–4 X.- Zhang et al. / Phytochemistry Letters xxx (2014) xxx–xxx
4
Fig. 5. Comparison of the experimental and calculated ECD spectra of 2 (Boltzmann averaged).
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(47) A˚, a = b = g = 90.008, V = 1877.7818 (0) A˚3, Z = 8, Dc = 1232 g/ cm3, T = 293.15 K, l (Mo Ka) = 0.71074 A˚, the final R1 = 0.0782, wR2 = 0.1612 (w = 1/sjFj2), and S = 1.014 observed reflections with I > 2s(I). The structures were solved by direct methods and refined by full matrix least-squares on F2 using the SHELXTL-97 software package.
157 Q5 Uncited reference 158
Dai et al. (2008).
Acknowledgments
159
The research was supported by E&T modern center for Natural Q6 Products of Liaoning Province of China (No. 2008402021) and Construction of R&D institute of state original new drug at Benxi of Liaoning Province (2009ZX09301-012-105B). We are glad to acknowledge the Analytical Center of Shenyang Pharmaceutical University for NMR measurements.
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References
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Cheng, W., Li, J., You, T., Hu, C., 2005. Anti-inflammatory and immunomodulatory activities of the extracts from the inflorescence of Chrysanthemum indicum Linne´. J. Ethnopharmacol. 101, 334–337. Dai, Y., He, X.J., Zhou, G.X., Kurihara, H., Ye, W.C., Yao, X.S., 2008. Acylphloroglucinol glycosides from the fruits of Pyracantha fortuneana. J. Asian Nat. Prod. Res. 10, 111–117. Dat, N.T., Kiem, P.V., Cai, X.F., Shen, Q.H., Bae, K., Kim, Y.H., 2004. Gymnastone, a new Benzofuran derivative from Gymnaster koraiensis. Arch. Pharm. Res. 27, 1106–1108. Gorzalczany, S., Moscatelli, V., Ferraro, G., 2013. Artemisia copa aqueous extract as vasorelaxant and hypotensive agent. J. Ethnopharmacol. 13, 217–221. Li, R.T., Weng, Z.Y., Pu, J.X., Sun, H.D., 2008. Chemical constituents from Schisandra sphenanthera. Chin. Chem. Lett. 19, 696–698. Ma, Z.Z., Deng, G., Lee, D.Y.W., 2010a. Novel neoclerodane diterpene derivatives from the smoke of salvinorin A. Tetrahedron Lett. 51, 5207–5209. Ma, Z.Z., Deng, G., Dai, R.H., Xu, W., Lee, D.Y.W., 2010b. Thermal degradation products derived from the smoke of Salvia divinorum leaves. Tetrahedron Lett. 51, 5480–5482. Matsuda, H., Morikawa, T., Toguchida, I., Harima, S., Yoshikawa, M., 2002. Medicinal Flowers VI. Absolute stereostructures of two new flavanone glycosides and a phenylbutanoid glycoside from the flowers of Chrysanthemum indicum L.: their inhibitory activities for rat lens aldose reductase. Chem. Pharm. Bull. 50, 972–975. Radulovic, N.S., Denic, M.S., 2013. Essential oils from the roots of echinops bannaticus Rochelex Schrad and Echinops sphaerocephalus L. (Asteraceae): chemotaxonomic and biosynthetic Aspects. Chem. Biodiv. 10, 658–676. Zhang, X.S., Liu, Z.T., Bi, X.L., Liu, J.X., Li, W., Zhao, Y.Q., 2013. Flavonoids and its derivatives from Callistephus chinensis flowers and their inhibitory activities against a-glucosidase. EXCLI J. 12, 956–966. Zhu, S.Y., Yang, Y., Yu, H.D., Zou, G.L., 2005. Chemical composition and antimicrobial activity of the essential oils of Chrysanthemum indicum. J. Ethnopharmacol. 96, 151–158.
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Please cite this article in press as: , X.- Zhang., et al., Callistephus A, a novel sesquiterpene from the Callistephus chinensis flower. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.10.030
PHYTOL 818 1–4 Phytochemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol 1 2 3 4 5 6 7 8 9
Callistephus A, a novel sesquiterpene from the Callistephus chinensis flower , Jia-qing Cao a,b, Zhen-ting Liu c, Lei Yuan a,b, Gang Chen a,b, Jing-xin Liu c, , Yu-qing Zhao a,b,*
Q1 Xiao-shu Zhang a,b
Fan-zhi Qu
a,b
a
School of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China c Chifeng MinYi Natural Anthocyanins Company Limited, Inner Mongolia Autonomous Region, 024000, People’s Republic of China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 September 2014 Received in revised form 27 October 2014 Accepted 29 October 2014 Available online xxx
Two new compounds callistephus A (1) and callistephus B (2) together with 4,6-dihydroxyphenyl-1butanone-2-b-D-O-glucopyranoside (3), were isolated and purified from the Callistephus chinensis flower. Their structures were identified by the interpretation of spectroscopic data. X-ray crystallographic and analysis of quantum chemical ECD calculation were applied to determine the absolute configuration. Of them, callistephus A represents a rare sesquiterpene with new carbon skeleton and also its possible biogenesis was proposed. ß 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Keywords: Callistephus chinensis Sesquiterpene X-ray diffraction Electronic circular dichroism (ECD)
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
1. Introduction The genus Callistephus chinensis belongs to the Asteraceae family. Previous pharmacological studies have shown that Asteraceae extracts display various biological activities including anti-diabetes, antioxidant properties and inhibitory effects against bacteria and viruses. Moreover, the extracts are also known to prevent indigestion, pneumonia, hepatitis, and tumors (Matsuda et al., 2002; Cheng et al., 2005; Zhu et al., 2005; Gorzalczany et al., 2013). However, limited information on phytochemical or pharmacological studies of C. chinensis flower (CCF) was available. As a continue research about CCF (Zhang et al., 2013), in this present work, study on structural determination of CCF extract resulted in the isolation of one rare sesquiterpene along with one new and one known compounds. Herein, we present the isolation and structural elucidation of these new compounds. Their structures were elucidated as callistephus A (1) callistephus B (2) and 4,6-dihydroxyphenyl-1-butanone-2-b-D-O-glucopyranoside (3) (Li et al., 2008). To the best of our knowledge, all of these Q3 compounds were isolated from the CCF for the first time (Fig. 1).
* Corresponding author at: School of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China. Q2 Tel.: +86 24 23986521; fax: +86 24 23986521. E-mail addresses: [email protected], [email protected] (Y.-q. Zhao).
2. Results and discussion
30
Compound 1 was purified as white crystals. The molecular formula compound 1 was determined as C20H28O5 by HR-ESI-MS (m/z 371.1830 [M+Na]+, calcd 371.1829), with seven degrees of unsaturation. The structure of 1 was elucidated by interpretation of its NMR spectra, including 1H, 13C, HSQC, and HMBC. The 1H NMR spectrum (Table 1) showed signals of five methyl groups (dH 1.29, 1.33, 1.35, 1.88, and 1.99), several multiplets (dH 1.47–2.08), two olefinic protons (dH 6.12 and 5.51), and an oxygenated proton (dH 5.07). Twenty carbon resonances were observed in its 13C NMR data (Table 1). Using the DEPT technique, five methyls, four methylenes, four methines (including an oxygenated and two olefinic carbons), and seven quaternary carbons (including two lactone carbonyls) were found in 1. 1H–1H COSY correlations of H2-1/H-2/H-11 and HMBC correlations from H2-1 to C-3 and C-11 and from H-4 to C-11 and C-15 constructed a six-member ring (A). 1 H–1H COSY correlations of H2-7/H2-8/H2-9 and HMBC correlations from H-11 to C-9 and C-10, from H3-12 to C-9, C-10, and C-11, and from H3-13 to C-5, C-6, C-7, and C-14 allowed the establishment of a seven-member ring (B). An olefinic ester group (dC 16.0, 20.6, 127.4, 138.9, and 168.3) was assigned at C-2 and deduced from HMBC correlations from H-2 to C-16 and from H-18 to C-16. An ester bond was found in the B fragment (Fig. 2). The connection of rings A and B was established by HMBC correlations
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
http://dx.doi.org/10.1016/j.phytol.2014.10.030 1874-3900/ß 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Please cite this article in press as: , X.- Zhang., et al., Callistephus A, a novel sesquiterpene from the Callistephus chinensis flower. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.10.030
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PHYTOL 818 1–4 X.- Zhang et al. / Phytochemistry Letters xxx (2014) xxx–xxx
2
Fig. 1. Structures of 1–3.
54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81
from H2-1 to C-10, from H-4 to C-7, and from H-11 to C-1, C-3, C-9, and C-10, which indicate that C-5 and C-11 were shared by rings A and B. Finally, the planar structure of 1 was established with a rare 6/7 skeleton. The relative configuration of 1 was established by analyzing its NOESY correlations. Significant NOE correlations among H-2/H-4, H-4/H-11, and H-2/H3-15 suggested that the atoms were co-facial (Fig. 3). X-ray diffraction analysis (Fig. 4) finally confirmed the structure proposed above Thus, the structure of 1 was confirmed, and the compound was named callistephus A. From a biogenetic point of view, compound 1 is structurally related to himachalane, a small group of 6/7 skeleton sesquiterpenoids found mainly in Asteraceae. The biogenesis of himachalane framework starts with farnesyl diphosphate, which after several steps leads to himachalane cation, a key intermediary in the biosynthesis of several compounds (Radulovic and Denic, 2013). Compound 1 seems to derive from himachalane cation by a range of rearrangement and oxidation reactions (Scheme 1). Compound 2 presented as a white amorphous powder possessing a molecular formula of C12H12O5, as determined by the positive HR-ESI-MS (m/z 259.0576 [M+Na]+, calcd 259.0577) with seven degrees of unsaturation. In the IR spectrum (nmax, cm1), 2 revealed bands characteristic of the hydroxyl group (3415 and 3400) and aromatic (1597, 1502, and 1463) absorption. Structural identification of compound 2 and gymnastone by 1H and 13C NMR (Table 1) spectroscopy yielded similar spectra but with some changes in the side chains (Dat et al., 2004; Ma et al., 2010a,b). The 13C NMR spectrum of 2 showed only two
Table 1 NMR spectroscopic data for compounds 1 and 2 (d in ppm, J in Hz). No
1 (CDCl3)
dH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.58, m 2.08, dt, 12.0, 4.1 5.07, dd, 12.3, 3.8 551, d, 2.9
1.47, 1.86, 1.59, 1.70, 1.78, 1.82,
2 (DMSO-d6)
dC
m m m m m m
2.66, dt, 12.0, 3.8 1.33, s 1.35, s 1.29, s
6.12, qd, 7.2, 1.4 1.88, m 1.99, dd, 7.2, 1.4
dH
dC
29.2
165.6
78.3 72.9 127.4 138.9 46.1 40.1
103.2 106.9 157.1 116.2 113.0 147.3
6.72, s 7.04, s
8.12, s
21.1
135.9
33.4
204.5
82.0 46.4 27.7 21.9 175.3 24.3 168.3 127.6 139.8 20.6 16.0
2.68, 4.65, 11.0, 3.65,
s dd, 5.6 m
Fig. 2. Key 1H–1H COSY and HMBC correlations of 1 and 2.
oxygen-attached carbon signals (dC 68.4 and 64.2), and its 1H spectrum showed one hydroxyl methylene peak [dH 4.65 (1H, dd, J = 11.0, 5.6)]. The structure of 1 was also confirmed by HMBC experiments (Fig. 2), which showed the correlations from H-3 to C8, from H-4 to C-9, from H-7 to C-9 and C-10, from H-12 to C-2. HSQC and HMBC experiments allowed deduction of the planar structure of 2. On the basis of this deduction, the predicted ECD curve of 2 was calculated by the quantum chemical method at the [B3LYP/6-31G (d)] level using the GAUSSIAN 09 program, and the predicted ECD curve of 12R-2 was similar to the experimental one (Fig. 5). These findings demonstrated the absolute configuration of 2 as 12R. Thus, the structure of 2 was confirmed, and the compound was named callistephus B.
82 83 84 85 86 87 88 89 90 91 92 93 94
3. Experimental
95
3.1. General experimental procedures
96
Optical rotations were measured on a Perkin-Elmer polarimeter. IR Spectra: Bruker IFS-55 spectrophotometer; KBr pellets; cm1. HR-TOF-MS: BIC micro TOF-Q mass spectrometer; in m/z (rel %). The circular dichroism (CD) spectra were measured on a Bio-Logic Science (MOS-450, France). The X-ray crystallographic data were collected on a Bruker Smart CCD diffractometer using graphite-monochromated Mo Ka radiation. NMR spectra were recorded on Bruker AV-600 spectrometer with TMS as internal standard, J in Hz. Column chromatography (cc): silica gel (SiO2:200– 300 mesh, Qingdao Marine Chemical Group, Co); Sephadex LH-20 (pharmacia, Co). Prep HPLC (Beijing CXTH3000 system): P3000 pump, UV3000 spectrophotometric detector at 210 nm, YMC C18 reversed-phase column (5 mm, 10 250 nm; flow rate3.0 mL/min).
97 98 99 100 101 102 103 104 105 106 107 108 109
3.2. Plant material
110
The flowers of Asteraceae for CCF were collected from Inner Mongolia Autonomous Region, Republic of China, in 2012 and
111 112
27.4 68.4 64.2
Fig. 3. NOESY correlations of 1.
Please cite this article in press as: , X.- Zhang., et al., Callistephus A, a novel sesquiterpene from the Callistephus chinensis flower. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.10.030
G Model
PHYTOL 818 1–4 X.- Zhang et al. / Phytochemistry Letters xxx (2014) xxx–xxx
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Fig. 4. X-ray crystal structure of 1.
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authenticated by Prof. Jincai Lu of Shenyang Pharmaceutical University. A voucher specimen (No. 20120124) was deposited in the Herbarium of Shenyang Pharmaceutical University (Shenyang, People’s Replublic of China). The extract of CCF was provided from Tianjin Jianfeng Natural Products Research and Development Co and extracted with ethanol (50%) three times at 50 8C.
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3.3. Extraction and isolation
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The extract (100 g) was suspended in water (2000 mL) and partitioned with ethyl acetate and n-butanol successively, to yield the ethyl acetate and n-butanol extracts. The ethyl acetate extract (70 g) was subjected to silica gel column chromatography (300– 400 mesh, 1.5 kg), using a step-wise system of CH2Cl2/MeOH (100:0–5:1, v/v) gradient elution, to afford nine fractions (Fr 1–Fr Q4 10) on the basis of TLC composition. Fr 3 (1.2 g) was subjected to Sephadex LH-20 column chromatography (CC), using CH2Cl2/ MeOH (1:1, v/v) elution to give five fractions L1–L5. Fraction L2 gave compound 1 (27 mg) by recrystallization. Fraction L5 (0.4 g) was subjected to silica gel CC, with petroleum ether–acetone (40:1, 20:1, 10:1, v/v) gradient elution, to yield compound 2. Fr 5 (3.5 g) was subjected to silica gel CC, with CH2Cl2/MeOH (50:1–5:1, v/v)
gradient elution, to yield five fractions S1–S5. Fraction S4 (0.5 g) was subjected to repeated Sephadex LH-20 (CH2Cl2/MeOH, 1:1, v/v) CC, and further purified using preparative HPLC (YMC, ODS S-5, 55% MeOH), affording compounds 3 (8 mg).
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3.3.1. Callistephus A (1) Colorless crystal (CH2Cl2); ½a25 D 23:9 (c 0.125, CH2Cl2); IR (KBr) vmax 3425, 2944, 1730, 1690, 1643 cm1; HR-ESI-MS: (m/z 371.1830 [M+Na]+, calcd 371.1829); 1H NMR (600 MHz, CH2Cl2) and 13C NMR (125 MHz, CH2Cl2) spectroscopic data, see Table 1.
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3.3.2. Callistephus B (2) White amorphous powder (MeOH); ½a25 D þ 19:2 (c 0.125, MeOH); IR (KBr) vmax 3433, 1740, 1587, 1419 cm1; HR-ESI-MS (m/z 259.0576 [M+Na]+, calcd 259.0577); 1H NMR (600 MHz, DMSO) and 13C NMR (125 MHz, DMSO) spectroscopic data, see Table 1.
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3.4. X-ray crystallographic analysis of 1
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The crystal of 1 belongs to an orthorhombic system, space group P212121 with a = 10.5339 (37) A˚, b = 13.2349 (46) A˚, c = 13.4690
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Scheme 1. Possible biosynthesis of compound 1.
Please cite this article in press as: , X.- Zhang., et al., Callistephus A, a novel sesquiterpene from the Callistephus chinensis flower. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.10.030
G Model
PHYTOL 818 1–4 X.- Zhang et al. / Phytochemistry Letters xxx (2014) xxx–xxx
4
Fig. 5. Comparison of the experimental and calculated ECD spectra of 2 (Boltzmann averaged).
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(47) A˚, a = b = g = 90.008, V = 1877.7818 (0) A˚3, Z = 8, Dc = 1232 g/ cm3, T = 293.15 K, l (Mo Ka) = 0.71074 A˚, the final R1 = 0.0782, wR2 = 0.1612 (w = 1/sjFj2), and S = 1.014 observed reflections with I > 2s(I). The structures were solved by direct methods and refined by full matrix least-squares on F2 using the SHELXTL-97 software package.
157 Q5 Uncited reference 158
Dai et al. (2008).
Acknowledgments
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The research was supported by E&T modern center for Natural Q6 Products of Liaoning Province of China (No. 2008402021) and Construction of R&D institute of state original new drug at Benxi of Liaoning Province (2009ZX09301-012-105B). We are glad to acknowledge the Analytical Center of Shenyang Pharmaceutical University for NMR measurements.
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Please cite this article in press as: , X.- Zhang., et al., Callistephus A, a novel sesquiterpene from the Callistephus chinensis flower. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.10.030