Letter - spectral assignment Received: 8 July 2014
Revised: 19 September 2014
Accepted: 26 September 2014
Published online in Wiley Online Library: 16 October 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4168
Two new sesquiterpenoids from Artemisia annua Hai-bo Li,a,b,c,d† Yang Yu,b† Zhen-zhong Wang,a,d Jun Yang,b Wei Xiaoa,c,d** and Xin-sheng Yaob*
Introduction Artemisia annua L., a fragrant annual herb belonging to the family Asteraceae (formerly Compositae), is widely distributed in the temperate zones of Europe, Asia and North America. The leaves of A. annua, popularly known as ‘qinghao’, have been traditionally used throughout the ages as a treatment for malaria, inflammation and intermittent fever.[1,2] In 1972, the artemisinin [qinghaosu (QHS)], an endoperoxide sesquiterpene lactone, was first discovered as one of the main antimalarial constituents from A. annua, which opened a new research era in the malarial chemotherapy. Since then, both QHS and ‘qinhao’ soon became well known worldwide. The phytochemical investigations on the other components led to the discovery and isolation of sesquiterpenoids, flavonoids, coumarins, triterpenoids and phenolics.[3–8] Among them, the dominated sesquiterpenoids, mainly including amorphane and cadinane sesquiterpenoids, possessed unusual highly oxygenated nature for the formation of hydroperoxides and endoperoxides. Guaiane-type sesquiterpenoids are known to possess antitumor activities and cytotoxic effects on human cancer cell lines.[9–12] Our current investigation for the unusual sesquiterpenoids from the entitled plant resulted in the isolation of one new guainane and one new cadinane type sesquiterpenoids (1 and 2) (Fig. 1). Herein, we report their isolation and structural elucidation by the extensive analysis of 1D and 2D NMR, HR-ESI-MS, calculated ECD and crystal X-ray diffraction.
The1H–1H COSY spectrum of 1 revealed the precence of a proton spin system (H-8/H-9/H-10/14-CH3) in bold as shown in Fig. 2. In the HMBC spectrum, the correlations between 14-CH3 and C-1/C-9/ C-10, between H-6 and C-1/C-5/C-7, as well as between H-8 and C-6/C-7 indicated the presence of a cycloheptane unit. The HMBC correlations between 15-CH3 and C-3/C-4/C-5 and between H-2 and C-1/C-3/C-5 allowed the establishment of a substitued α, β-unsaturated cyclopentanone unit. Furthermore, the presence and the location of a hydroxyisopropyl were established by the HMBC correlations between 12, 13-CH3 and C-7/C-11. According to the molecular formula evidence, three hydroxy groups were connected at C-7, C-8 and C-11, respectively. Notably, the structure possessed a hydroperoxy at C-1, which was interpreted by low-field chemical shift (δC 94.0). Based on these data, the gross structure of 1 was determined to be 7, 8, 11-trihydroxy-1-hydroperoxy-4guaien-3-one. The relative configuration of 1 was clarified from the NOESY experiment. The NOESY cross peaks were observed between H-10 (δH 1.86)/H-8 (δH 4.06) and H-8 (δH 4.06)/H-9α (δ 1.21) suggesting that H-10 and H-8 were all on the same face (α) of the molecule. The hydroxyisopropyl was β-orientation, which was found in most sesquiterpenoids with a known stereochemistry.[14,15] The β-configuration of hydroperoxy at C-1 was deduced by the 1H NMR data of 14-CH3 (δ 1.06) because the value of the chemical shift of 14-CH3 is δH 1.07 for β-configuration and δH 0.77 for α-orientation.[9] The absolute configuration at C-1 of 1 was determined by the CD spectrum. The CD spectrum of 1 showed a positive Cotton effect at
Results and discussion
244
Compound 1 was isolated as a yellow amorphous solid. The HR-ESIQ/TOF-MS gave [M + H]+ ion at m/z 301.1646 (calcd. for 301.1651), corresponding to the molecular formula C15H24O6, with four degrees of unsaturation. The1H NMR spectrum of 1 displayed singlets of three methyl groups linked to quaternary carbons at δH 1.36 (3H, s, H-12), 1.38 (3H, s, H-13) and 1.76 (3H, s, H-15), as well as a doublet of one methyl linked to tertiary carbon at δH 1.06 (3H, d, J = 7.1 Hz, H-14). The13C NMR and DEPT-135 spectra revealed the precence of 15 carbon signals, including a carbonyl (δC 209.7), a tetrasubstituted double bond (δC 144.6 and 166.3), three oxygenated quaternary carbons (δ 94.0, 79.1 and 79.0), three methylenes (δC 40.7, 37.5 and 29.8), two methines (δC 77.4 and 34.1), as well as four methyl groups (δC 26.4, 25.4, 18.5 and 8.9). The comparison of these data with those of 1β-hydroxytorilolone[13] permited the full assignment of the resonances for 1 as shown in Table 1.
Magn. Reson. Chem. 2015, 53, 244–247
* Correspondence to: Xin-sheng Yao, Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China. E-mail: tyaoxs@jnu. edu.cn ** Wei Xiao, Jiangsu Kanion Pharmaceutical Co. Ltd. and State Key Lab of New-Tech for Chinese Medicine Pharmaceutical Process, Lianyungang 222001, China. E-mail: [email protected] †
The first two authors contributed equally to this work.
a Jiangsu Kanion Pharmaceutical Co. Ltd., Lianyungang 222001, China b Institute of Traditional Chinese Medicine and Natural Products College of Pharmacy, Jinan University, Guangzhou 510632, China c Nanjing University of Chinese Medicine, Nanjing 210000, China d State Key Lab of New-Tech for Chinese Medicine Pharmaceutical Process, Lianyungang 222001, China
Copyright © 2014 John Wiley & Sons, Ltd.
1D and 2D NMR spectra, crystal X-ray diffraction and electronic circular dichroism (ECD)
1
λmax 249 nm and a negative Cotton effect at λmax 312 nm, which is opposite to (1R,7R,8R,10R)-7, 8,11-trihydroxyguai-4-en3-one 8-O-β-D-glucopyranoside,[16] suggesting a S configuration at C-1 for 1. To confirm that, the electronic circular dichroism (ECD) computation was performed at the B3LYP/6-31g(d) level.[17,18] As a result, the calculated ECD showed high agreement with the experimental spectrum as shown in Fig. 3. From the previous evidence, the absolute configuration of 1 was elucidated to be 1S,7R,8R,10S. Compound 2 was obtained as a white needle crystal. The HRESI-Q/TOF-MS gave [M + Na]+ ion at m/z 305.1365 (calcd. for 305.1365), corresponding to the molecular formula C15H22O5, with five degrees of unsaturation. The 13C NMR and DEPT-135 spectra
1a δC
1
1.35, m 2.61, ddd (16.8, 12.0, 4.4) 2.42, ddd (16.8, 12.0, 4.4)
Experimental
2b
δH (J in Hz)
94.0
δC
δH (J in Hz)
45.9
1.38, m 1.81, m
2.38, d (17.4, Hα) 2
40.7
33.6 2.28, d (17.4, Hβ)
3
209.7
4 5
144.6 166.3
6
29.8
7
79.0
8
77.4
38.7
211.9 178.4
General procedure
2.22, d (13.2, Hα) 52.5 2.55, dd, (11.6, 11.6) 2.76, d (13.2, Hβ) 44.6 4.06, dd (10.8, 2.6)
9
37.5
10 11 12
1.66, ddd (14.4, 10.8, 10.8, Hα) 34.1 1.86, m 79.1 26.4 1.36, s
13
25.4
1.38, s
14 15
18.5 8.9
1.06, d (7.1) 1.76, s
2.74, td (11.6, 2.8) 1.85, m (Hα)
UPLC-ESI-Q/TOF-MS spectra were acquired using a Waters Snapt G2 mass spectrometer. Optical rotations were determined on a JASCO P-1020 digital polarimeter in CH3OH. IR spectra were measured on a JASCO FT/IR-480 plus spectrometer. UV spectra were recorded in
24.7 1.55, m (Hβ) 1.74, m (Hβ)
1.21, brdd (14.4, 2.6, Hβ)
a
) correlations of 1 and 2.
of 2 displayed 15 carbon signals, including three carbonyls (δC 211.9, 178.4 and 169.7), one terminal double bond (δC 144.7 and 125.8), four methines (δC 52.5, 45.9, 44.6 and 34.4), four methylenes (δC 38.7, 36.3, 33.6 and 24.7), as well as two methyl groups (δC 29.9 and 20.0), suggesting a skeleton of sequiterpene. In 1H–1H COSY spectrum, two proton spin systems, H-1/H-6/H-7/H-8/H-9/H-10/ H-14 and H-1/H-2/H-3, permitted us to establish the partial structure of a methyl substitued six-member ring. A carbon skeleton of sesquiterpene was determined by key HMBC correlations between H-13 and C-7/C-11/C-12, between H-6 and C-5, as well as between H-15 and C-3/C-4 (Fig. 2). By comparison with the molecular formula C15H22O5, two hydroxyl groups should be linked to C-12 and C-5, respectively. Thus, the gross structure of 2 was elucidated as shown in Fig. 1. The relative configuration of 2 was determined through inspection of the NOESY spectrum. The NOE correlations between H-1 and H-7 and between H-6 and H-10 suggested α-orientation of H-1 and H-7 and β-orientation of the H-6 and H-10. Moreover, a single-crystal X-ray diffraction experiment was conducted with CuKα, and the Flack parameter of 0.1 (2) allowed an unambiguous assignment of the absolute configuration of all the chiral centers as 1S,6R,7R,10R[19] (Fig. 4). Crystallographic data for structure 2 has been deposited at the Cambridge Crystallographic Data Centre (CCDC 1012340). Therefore, the structure of 2 was elucidated as (1S,6R,7R,10R)- 6-carboxy-10-methyl-α-methylene-1-(1-oxobutyl)cyclohexaneacetic acid.
Table 1. NMR spectroscopic data for compounds 1 and 2 (in CD3OD, δ in ppm and J in Hz) Pos.
1
Figure 2. Key HMBC (H → C) and H– H COSY(
Figure 1. Chemical structures of compounds 1 and 2.
36.3
1
1.16, brdd (10.3, 2.8, Hα) 34.4 1.34, m 144.7 169.7 6.19, brs (Hα) 125.8 5.68, brs (Hβ) 20.0 0.93, d (6.4) 29.9 2.12, s
13
Magn. Reson. Chem. 2015, 53, 244–247
Figure 3. The experimental and calculated ECD of 1.
Copyright © 2014 John Wiley & Sons, Ltd.
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245
600 MHz for H and 150 MHz for C NMR. 1 13 400 MHz for H and 100 MHz for C NMR.
b
H.-b. Li et al. MHz, SW = 4006.4 Hz, AQ = 0.128 s, RD = 2.0 s, NS = 1; for NOESY, SF = 400.13 MHz, SW = 6410.3 Hz, AQ = 0.0799 s, RD = 2.0 s, NS = 16.
Plant material The dried leaves of A. annua were purchased from Ji’an Medical Material Market, Jiangxi, China, in April 2009 and identified by Professor Zhou Wu (Jiangsu Kanion Pharmaceutical Co. Ltd.). A voucher specimen 2009AA206 was deposited in Jiangsu Kanion Pharmaceutical Co. Ltd., Lianyungang, China.
Extraction and isolation Figure 4. X-ray crystal structure of compound 2.
CH3OH using a JASCO V-550 UV/Vis spectrometer. CD spectra were recorded on a JASCO J-810 CD spectrometer. A single-crystal X-ray diffraction was measured on Agilent SuperNova. Diaion HP-20 (Mitsubishi-Chemical, Japan), silica gel (200–300 mesh; Qingdao Marine Chemical Ltd., China), ODS (60–80 μm; Merck), Toyopearl HW-40 (TOSOH Co., Japan) and Sephadex LH-20 (50 μm; Amersham Pharmacia Biotech, Sweden) were used for column chromatography (CC). MPLC separations were carried out on a BÜCHI C-610 series. Analytical HPLC was performed on a Shimadzu LC-6AB series pump equipped with a UV detector and a Cosmosil 5C18-MS-II column (5 μm; i.d. 250 × 4.6 mm, Nacalai Tesque Inc., Japan). HPLC separations were performed using a Shimadzu LC-6AD series pump equipped with a UV detector and a Cosmosil 5C18-MS-II column (5 μm; i.d. 250 × 20 mm, Nacalai tesque Inc., Japan).
Nuclear magnetic resonance spectra
246
Nuclear magnetic resonance spectra were recorded on a Bruker Avance-600/-400 spectrometer equipped with a BBO-5 mm probe in 0.5 ml CD3OD at 300 K. Solvent signals were used as internal standard (CD3OD: δH = 3.30, δC = 49.0 ppm). The pulse conditions for 600 MHz spectrometer were as follows: For 1H, spectrometer frequency (SF) = 600.13 MHz, spectral width (SW) = 12 376.2 Hz, pulse 90° width (P1) = 12.0 μs, Fourier transform size (SI) = 65 536, acquisition time (AQ) = 2.65 s, line broadening (LB) = 0.1 Hz, relaxation delay (RD) = 1.0 s, number of scans (NS) = 16; for 13C, SF = 150.90 MHz, SW = 35 971.2 Hz, pluse 90° width (P1) = 8.1 μs, SI = 32 768, AQ = 0.91 s, LB = 1 Hz, RD = 2.0 s, NS = 3652; for HSQC, SF = 600.13 MHz for 1H and 150.90 MHz for 13 C, SW 1H = 2948.1 Hz, SW 13C = 24 998.9 Hz, AQ = 0.017 s, RD = 1.4 s, NS = 8; for HMBC, SF = 600.13 MHz for 1H and 150.90 MHz for 13C, SW 1H = 2948.1 Hz, SW 13C = 33 518.1 Hz, AQ = 0.69 s, RD = 1.0 s, NS = 32; for 1H–1H COSY, SF = 600.13 MHz, SW = 2948.1 Hz, AQ = 0.35 s, RD = 1.8 s, NS = 8; for NOESY, SF = 600.13 MHz, SW = 6127.5 Hz, AQ = 0.17 s, RD = 2.0 s, NS = 64. The pulse conditions for 400 MHz spectrometer were as follows: For 1H, SF = 400.13 MHz, SW = 6410.3 Hz, P1 = 14.0 μs, SI = 65 536, AQ = 2.56 s, LB = 0.3 Hz, RD = 2.0 s, NS = 1; for 13C, SF = 100.61 MHz, SW = 24 154.6 Hz, P1 = 7.5 μs, SI = 32 768, AQ = 0.68 s, LB = 1 Hz, RD = 2.0 s, NS = 200; for HSQC, SF = 400.13 MHz for 1H and 100.61 MHz for 13C, SW 1H = 4006.4 Hz, SW 13C = 24 154.6 Hz, AQ = 0.128 s, RD = 2.5 s, NS = 1; for HMBC, SF = 400.13 MHz for 1H and 100.61 MHz for 13C, SW 1H = 4006.4 Hz, SW 13C = 24 154.6 Hz, AQ = 0.128 s, RD = 2.0 s, NS = 8; for 1H–1H COSY, SF = 400.13
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The dried leaves of A. annua (3.5 kg) was pulverized and refluxed with 70% (v/v) EtOH (8 l, 3 × 2 h). After evaporation of EtOH in vacuo, the aqueous residue was subjected to CC over HP-20 macroporous adsorptive resins, eluted with water and 30 and 95% ethanol in successive to yield three fractions (1–3). Fraction 3 (60 g, 95% EtOH eluate) was separated over a silica gel column (SiO 2, 200–300 mesh, Φ 9.8 × 62 cm) eluted with a CHCl3-MeOH in gradient to yield nine crude fractions (3A–3I). Subfraction 3A (6.5 g, CHCl 3-MeOH, 95 : 5, eluant) was separated by CC over Sephadex LH-20 (100 g, Φ 2 × 100 cm) eluted with CHCl 3-CH3 OH (1 : 1) in gradient. The subfraction 3A-2 (4.5 g, CHCl3–MeOH, 1 : 1, eluant) was then separated by CC over ODS (50 μm, Φ 5 × 38 cm) eluted with MeOH-H 2O in gradient. The subfraction 3A-2-c (MeOH-H2O, 7 : 3, eluant) was further purified by preparative HPLC (CH 3 CN-H 2O, 1 : 4) to afford compound 1 (5.5 mg, tR 28.4 min). Subfraction 3B (4.8 g, CHCl3 -MeOH, 90:10, eluant) was submitted to repeated ODS, Toyopearl HW-40 columns eluted with MeOH/H2O, followed by preparative HPLC (MeOH : H2O, 5.5 : 4.5) to yield compound 2 (35.7 mg, tR 37.4 min). Physical and spectroscopic data of new compounds (1S,7R,8R,10S)-7,8,11-trihydroxy-1-hydroperoxy-4-guaien-3-one (1)
Yellow amorphous solid. [α]25 D -29.3 (c = 2, MeOH). UV (MeOH) λmax nm (log ε): 235.7 (4.33); IR (KBr) νmax cm 1: 3304, 2976, 1677, 1630, 1455, 1384, 1077; CD (c = 0.16, MeOH): 249.2 nm (Δε 0.99), 312.4 nm (Δε 1.95); HR-ESI-Q/TOF-MS: m/z 301.1646 ([M + H]+, C15H25O+6 , calcd. for 301.1651); 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), refer to Table 1. (1S,6R,7R,10R)-6-carboxy-10-methyl-α-methylene-1-(1-oxobutyl)-cyclohexanea cetic acid (2)
White needle crystal. [α]25 D -16.5 (c = 2, MeOH). UV (MeOH) λmax nm (log ε): 204 (2.89); IR (KBr) νmax cm 1: 3500–2500, 1723, 1695; CD (c = 0.14, MeOH): 206.8 nm (Δε 3.73), 227.3 nm (Δε 1.58), 251.4 nm (Δε 1.24), 286.7 nm (Δε 1.18); HR-ESI-Q/TOF-MS: m/z 305.1365 ([M + Na]+, C15H22O5Na+, calcd. for 305.1365). 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz), refer to Table 1. Quantum chemical ECD calculation of 1
The conformational analysis for compound 1 was carried out by the MMFF94s force field. The lowest energy conformer was sbmitted to the ECD calculation in methanol solution at [B3LYP/6-31g(d)] level with the Boltzmann model, and the predicted ECD curve of compound 1 was similar to the experimental one.
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2015, 53, 244–247
1D and 2D NMR spectra, crystal X-ray diffraction and electronic circular dichroism (ECD) X-ray crystallographic analysis of compound 2
Upon crystallization from MeOH using the vapor diffusion method, needles of 2 were obtained. Crystal Data for C15H22O5 (M = 282.33): monoclinic, space group P21, a = 5.37 760(13) Å, b = 28.5292(7) Å, c = 10.4393(3) Å, β = 99.637(2)°, V = 1578.98 (7) Å3, Z = 2, T = 150.01(10) K, μ(Cu Kα) = 0.730 mm 1, Dcalc = 1.186 g/mm3, 14 952 measured reflections, 5235 independent reflections [Rint = 0.0552]. The final R1 was 0.0467, and wR2 was 0.1247 [I > 2σ(I)]. The goodness of fit on F2 was 1.027. Flack parameter = 0.1 (2). Crystallographic data for structure 2 has been deposited at the Cambridge Crystallographic Data Centre (CCDC 1012340). Acknowledgements This work was supported by the Natural Science Foundation of Jiangsu Province (BK20140441) and the National Basic Research Projects (973 program) of the Ministry of Science and Technology of China (2010CB735604) and the Programme of Introducing Talents of Discipline to Universities (B13038). We thank Yun-wen Chen for helping to calculate ECD.
References
[3] R. S. Bhakuni, D. C. Jain, R. P. Sharma, S. Kumar. Curr. Sci. 2001, 80, 35. [4] G. D. Brown. Phytochemistry 1993, 32, 391. [5] G. D. Brown. Phytochemistry 1994, 36, 637. [6] S. L. Yang, M. F. Roberts, J. D. Phillipson. Phytochemistry 1989, 28, 1509. [7] G. Q. Zheng. Planta Med. 1994, 60, 54. [8] J. A. Marco, J. F. Sanz, J. F. Bea. Pharmazie 1990, 45, 382. [9] H. W. Park, S. U. Choi, N. I. Baek, S. H. Kim, J. S. Eun, J. H. Yang, D. K. Kim. Arch. Pharm. Res. 2006, 29, 131. [10] E. Rodriguez, G. H. N. Towers, J. C. Mitchell. Phytochemistry 1976, 15, 1573. [11] M. G. Lee, K. T. Lee, S. G. Chi. Biol. Pharm. Bull. 2001, 24, 303. [12] W. Xiao, X. Li, N. Li, M. Bolati, X. J. Wang, X. G. Jia, Y. Q. Zhao. Fitoterapia 2011, 82, 983. [13] T. Yi, L. Zhang, H. W. Fu, S. L. Yang, J. K. Tian. Helv. Chim. Acta 2009, 92, 2769. [14] J. H. Ryu, Y. S. Jeong. Arch. Pharm. Res. 2001, 24, 532. [15] C. Labbe, F. Faini, J. Coll, P. Carbonell. Phytochemistry 1998, 49, 793. [16] K. Machida, K. Oyama, M. Ishii, R. Kakuda, Y. Yaoita, M. Kikuchi. Chem. Pharm. Bull. 2000, 48, 746. [17] J. Ren, J. X. Jiang, L. B. Li, T. G. Liao, R. R. Tian, X. L. Chen, S. P. Jiang, C. U. Pittman Jr., H. J. Zhu. Eur. J. Org. Chem. 2009, 23, 3987. [18] P. J. Stephens, J. J. Pan, K. J. Krohn. J. Org. Chem. 2007, 72, 7641. [19] H. D. Flack. Acta Crystallogr., Sect. A 1983, 39, 876.
Supporting information
[1] A. R. Bilia, P. Melillo de Malgalhaes, M. C. Bergonzi, F. F. Vincieri. Phytomedicine 2006, 13, 487. [2] T. T. Hien, N. J. White. Lancet 1993, 341, 603.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
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Magn. Reson. Chem. 2015, 53, 244–247
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc
Revised: 19 September 2014
Accepted: 26 September 2014
Published online in Wiley Online Library: 16 October 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4168
Two new sesquiterpenoids from Artemisia annua Hai-bo Li,a,b,c,d† Yang Yu,b† Zhen-zhong Wang,a,d Jun Yang,b Wei Xiaoa,c,d** and Xin-sheng Yaob*
Introduction Artemisia annua L., a fragrant annual herb belonging to the family Asteraceae (formerly Compositae), is widely distributed in the temperate zones of Europe, Asia and North America. The leaves of A. annua, popularly known as ‘qinghao’, have been traditionally used throughout the ages as a treatment for malaria, inflammation and intermittent fever.[1,2] In 1972, the artemisinin [qinghaosu (QHS)], an endoperoxide sesquiterpene lactone, was first discovered as one of the main antimalarial constituents from A. annua, which opened a new research era in the malarial chemotherapy. Since then, both QHS and ‘qinhao’ soon became well known worldwide. The phytochemical investigations on the other components led to the discovery and isolation of sesquiterpenoids, flavonoids, coumarins, triterpenoids and phenolics.[3–8] Among them, the dominated sesquiterpenoids, mainly including amorphane and cadinane sesquiterpenoids, possessed unusual highly oxygenated nature for the formation of hydroperoxides and endoperoxides. Guaiane-type sesquiterpenoids are known to possess antitumor activities and cytotoxic effects on human cancer cell lines.[9–12] Our current investigation for the unusual sesquiterpenoids from the entitled plant resulted in the isolation of one new guainane and one new cadinane type sesquiterpenoids (1 and 2) (Fig. 1). Herein, we report their isolation and structural elucidation by the extensive analysis of 1D and 2D NMR, HR-ESI-MS, calculated ECD and crystal X-ray diffraction.
The1H–1H COSY spectrum of 1 revealed the precence of a proton spin system (H-8/H-9/H-10/14-CH3) in bold as shown in Fig. 2. In the HMBC spectrum, the correlations between 14-CH3 and C-1/C-9/ C-10, between H-6 and C-1/C-5/C-7, as well as between H-8 and C-6/C-7 indicated the presence of a cycloheptane unit. The HMBC correlations between 15-CH3 and C-3/C-4/C-5 and between H-2 and C-1/C-3/C-5 allowed the establishment of a substitued α, β-unsaturated cyclopentanone unit. Furthermore, the presence and the location of a hydroxyisopropyl were established by the HMBC correlations between 12, 13-CH3 and C-7/C-11. According to the molecular formula evidence, three hydroxy groups were connected at C-7, C-8 and C-11, respectively. Notably, the structure possessed a hydroperoxy at C-1, which was interpreted by low-field chemical shift (δC 94.0). Based on these data, the gross structure of 1 was determined to be 7, 8, 11-trihydroxy-1-hydroperoxy-4guaien-3-one. The relative configuration of 1 was clarified from the NOESY experiment. The NOESY cross peaks were observed between H-10 (δH 1.86)/H-8 (δH 4.06) and H-8 (δH 4.06)/H-9α (δ 1.21) suggesting that H-10 and H-8 were all on the same face (α) of the molecule. The hydroxyisopropyl was β-orientation, which was found in most sesquiterpenoids with a known stereochemistry.[14,15] The β-configuration of hydroperoxy at C-1 was deduced by the 1H NMR data of 14-CH3 (δ 1.06) because the value of the chemical shift of 14-CH3 is δH 1.07 for β-configuration and δH 0.77 for α-orientation.[9] The absolute configuration at C-1 of 1 was determined by the CD spectrum. The CD spectrum of 1 showed a positive Cotton effect at
Results and discussion
244
Compound 1 was isolated as a yellow amorphous solid. The HR-ESIQ/TOF-MS gave [M + H]+ ion at m/z 301.1646 (calcd. for 301.1651), corresponding to the molecular formula C15H24O6, with four degrees of unsaturation. The1H NMR spectrum of 1 displayed singlets of three methyl groups linked to quaternary carbons at δH 1.36 (3H, s, H-12), 1.38 (3H, s, H-13) and 1.76 (3H, s, H-15), as well as a doublet of one methyl linked to tertiary carbon at δH 1.06 (3H, d, J = 7.1 Hz, H-14). The13C NMR and DEPT-135 spectra revealed the precence of 15 carbon signals, including a carbonyl (δC 209.7), a tetrasubstituted double bond (δC 144.6 and 166.3), three oxygenated quaternary carbons (δ 94.0, 79.1 and 79.0), three methylenes (δC 40.7, 37.5 and 29.8), two methines (δC 77.4 and 34.1), as well as four methyl groups (δC 26.4, 25.4, 18.5 and 8.9). The comparison of these data with those of 1β-hydroxytorilolone[13] permited the full assignment of the resonances for 1 as shown in Table 1.
Magn. Reson. Chem. 2015, 53, 244–247
* Correspondence to: Xin-sheng Yao, Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China. E-mail: tyaoxs@jnu. edu.cn ** Wei Xiao, Jiangsu Kanion Pharmaceutical Co. Ltd. and State Key Lab of New-Tech for Chinese Medicine Pharmaceutical Process, Lianyungang 222001, China. E-mail: [email protected] †
The first two authors contributed equally to this work.
a Jiangsu Kanion Pharmaceutical Co. Ltd., Lianyungang 222001, China b Institute of Traditional Chinese Medicine and Natural Products College of Pharmacy, Jinan University, Guangzhou 510632, China c Nanjing University of Chinese Medicine, Nanjing 210000, China d State Key Lab of New-Tech for Chinese Medicine Pharmaceutical Process, Lianyungang 222001, China
Copyright © 2014 John Wiley & Sons, Ltd.
1D and 2D NMR spectra, crystal X-ray diffraction and electronic circular dichroism (ECD)
1
λmax 249 nm and a negative Cotton effect at λmax 312 nm, which is opposite to (1R,7R,8R,10R)-7, 8,11-trihydroxyguai-4-en3-one 8-O-β-D-glucopyranoside,[16] suggesting a S configuration at C-1 for 1. To confirm that, the electronic circular dichroism (ECD) computation was performed at the B3LYP/6-31g(d) level.[17,18] As a result, the calculated ECD showed high agreement with the experimental spectrum as shown in Fig. 3. From the previous evidence, the absolute configuration of 1 was elucidated to be 1S,7R,8R,10S. Compound 2 was obtained as a white needle crystal. The HRESI-Q/TOF-MS gave [M + Na]+ ion at m/z 305.1365 (calcd. for 305.1365), corresponding to the molecular formula C15H22O5, with five degrees of unsaturation. The 13C NMR and DEPT-135 spectra
1a δC
1
1.35, m 2.61, ddd (16.8, 12.0, 4.4) 2.42, ddd (16.8, 12.0, 4.4)
Experimental
2b
δH (J in Hz)
94.0
δC
δH (J in Hz)
45.9
1.38, m 1.81, m
2.38, d (17.4, Hα) 2
40.7
33.6 2.28, d (17.4, Hβ)
3
209.7
4 5
144.6 166.3
6
29.8
7
79.0
8
77.4
38.7
211.9 178.4
General procedure
2.22, d (13.2, Hα) 52.5 2.55, dd, (11.6, 11.6) 2.76, d (13.2, Hβ) 44.6 4.06, dd (10.8, 2.6)
9
37.5
10 11 12
1.66, ddd (14.4, 10.8, 10.8, Hα) 34.1 1.86, m 79.1 26.4 1.36, s
13
25.4
1.38, s
14 15
18.5 8.9
1.06, d (7.1) 1.76, s
2.74, td (11.6, 2.8) 1.85, m (Hα)
UPLC-ESI-Q/TOF-MS spectra were acquired using a Waters Snapt G2 mass spectrometer. Optical rotations were determined on a JASCO P-1020 digital polarimeter in CH3OH. IR spectra were measured on a JASCO FT/IR-480 plus spectrometer. UV spectra were recorded in
24.7 1.55, m (Hβ) 1.74, m (Hβ)
1.21, brdd (14.4, 2.6, Hβ)
a
) correlations of 1 and 2.
of 2 displayed 15 carbon signals, including three carbonyls (δC 211.9, 178.4 and 169.7), one terminal double bond (δC 144.7 and 125.8), four methines (δC 52.5, 45.9, 44.6 and 34.4), four methylenes (δC 38.7, 36.3, 33.6 and 24.7), as well as two methyl groups (δC 29.9 and 20.0), suggesting a skeleton of sequiterpene. In 1H–1H COSY spectrum, two proton spin systems, H-1/H-6/H-7/H-8/H-9/H-10/ H-14 and H-1/H-2/H-3, permitted us to establish the partial structure of a methyl substitued six-member ring. A carbon skeleton of sesquiterpene was determined by key HMBC correlations between H-13 and C-7/C-11/C-12, between H-6 and C-5, as well as between H-15 and C-3/C-4 (Fig. 2). By comparison with the molecular formula C15H22O5, two hydroxyl groups should be linked to C-12 and C-5, respectively. Thus, the gross structure of 2 was elucidated as shown in Fig. 1. The relative configuration of 2 was determined through inspection of the NOESY spectrum. The NOE correlations between H-1 and H-7 and between H-6 and H-10 suggested α-orientation of H-1 and H-7 and β-orientation of the H-6 and H-10. Moreover, a single-crystal X-ray diffraction experiment was conducted with CuKα, and the Flack parameter of 0.1 (2) allowed an unambiguous assignment of the absolute configuration of all the chiral centers as 1S,6R,7R,10R[19] (Fig. 4). Crystallographic data for structure 2 has been deposited at the Cambridge Crystallographic Data Centre (CCDC 1012340). Therefore, the structure of 2 was elucidated as (1S,6R,7R,10R)- 6-carboxy-10-methyl-α-methylene-1-(1-oxobutyl)cyclohexaneacetic acid.
Table 1. NMR spectroscopic data for compounds 1 and 2 (in CD3OD, δ in ppm and J in Hz) Pos.
1
Figure 2. Key HMBC (H → C) and H– H COSY(
Figure 1. Chemical structures of compounds 1 and 2.
36.3
1
1.16, brdd (10.3, 2.8, Hα) 34.4 1.34, m 144.7 169.7 6.19, brs (Hα) 125.8 5.68, brs (Hβ) 20.0 0.93, d (6.4) 29.9 2.12, s
13
Magn. Reson. Chem. 2015, 53, 244–247
Figure 3. The experimental and calculated ECD of 1.
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600 MHz for H and 150 MHz for C NMR. 1 13 400 MHz for H and 100 MHz for C NMR.
b
H.-b. Li et al. MHz, SW = 4006.4 Hz, AQ = 0.128 s, RD = 2.0 s, NS = 1; for NOESY, SF = 400.13 MHz, SW = 6410.3 Hz, AQ = 0.0799 s, RD = 2.0 s, NS = 16.
Plant material The dried leaves of A. annua were purchased from Ji’an Medical Material Market, Jiangxi, China, in April 2009 and identified by Professor Zhou Wu (Jiangsu Kanion Pharmaceutical Co. Ltd.). A voucher specimen 2009AA206 was deposited in Jiangsu Kanion Pharmaceutical Co. Ltd., Lianyungang, China.
Extraction and isolation Figure 4. X-ray crystal structure of compound 2.
CH3OH using a JASCO V-550 UV/Vis spectrometer. CD spectra were recorded on a JASCO J-810 CD spectrometer. A single-crystal X-ray diffraction was measured on Agilent SuperNova. Diaion HP-20 (Mitsubishi-Chemical, Japan), silica gel (200–300 mesh; Qingdao Marine Chemical Ltd., China), ODS (60–80 μm; Merck), Toyopearl HW-40 (TOSOH Co., Japan) and Sephadex LH-20 (50 μm; Amersham Pharmacia Biotech, Sweden) were used for column chromatography (CC). MPLC separations were carried out on a BÜCHI C-610 series. Analytical HPLC was performed on a Shimadzu LC-6AB series pump equipped with a UV detector and a Cosmosil 5C18-MS-II column (5 μm; i.d. 250 × 4.6 mm, Nacalai Tesque Inc., Japan). HPLC separations were performed using a Shimadzu LC-6AD series pump equipped with a UV detector and a Cosmosil 5C18-MS-II column (5 μm; i.d. 250 × 20 mm, Nacalai tesque Inc., Japan).
Nuclear magnetic resonance spectra
246
Nuclear magnetic resonance spectra were recorded on a Bruker Avance-600/-400 spectrometer equipped with a BBO-5 mm probe in 0.5 ml CD3OD at 300 K. Solvent signals were used as internal standard (CD3OD: δH = 3.30, δC = 49.0 ppm). The pulse conditions for 600 MHz spectrometer were as follows: For 1H, spectrometer frequency (SF) = 600.13 MHz, spectral width (SW) = 12 376.2 Hz, pulse 90° width (P1) = 12.0 μs, Fourier transform size (SI) = 65 536, acquisition time (AQ) = 2.65 s, line broadening (LB) = 0.1 Hz, relaxation delay (RD) = 1.0 s, number of scans (NS) = 16; for 13C, SF = 150.90 MHz, SW = 35 971.2 Hz, pluse 90° width (P1) = 8.1 μs, SI = 32 768, AQ = 0.91 s, LB = 1 Hz, RD = 2.0 s, NS = 3652; for HSQC, SF = 600.13 MHz for 1H and 150.90 MHz for 13 C, SW 1H = 2948.1 Hz, SW 13C = 24 998.9 Hz, AQ = 0.017 s, RD = 1.4 s, NS = 8; for HMBC, SF = 600.13 MHz for 1H and 150.90 MHz for 13C, SW 1H = 2948.1 Hz, SW 13C = 33 518.1 Hz, AQ = 0.69 s, RD = 1.0 s, NS = 32; for 1H–1H COSY, SF = 600.13 MHz, SW = 2948.1 Hz, AQ = 0.35 s, RD = 1.8 s, NS = 8; for NOESY, SF = 600.13 MHz, SW = 6127.5 Hz, AQ = 0.17 s, RD = 2.0 s, NS = 64. The pulse conditions for 400 MHz spectrometer were as follows: For 1H, SF = 400.13 MHz, SW = 6410.3 Hz, P1 = 14.0 μs, SI = 65 536, AQ = 2.56 s, LB = 0.3 Hz, RD = 2.0 s, NS = 1; for 13C, SF = 100.61 MHz, SW = 24 154.6 Hz, P1 = 7.5 μs, SI = 32 768, AQ = 0.68 s, LB = 1 Hz, RD = 2.0 s, NS = 200; for HSQC, SF = 400.13 MHz for 1H and 100.61 MHz for 13C, SW 1H = 4006.4 Hz, SW 13C = 24 154.6 Hz, AQ = 0.128 s, RD = 2.5 s, NS = 1; for HMBC, SF = 400.13 MHz for 1H and 100.61 MHz for 13C, SW 1H = 4006.4 Hz, SW 13C = 24 154.6 Hz, AQ = 0.128 s, RD = 2.0 s, NS = 8; for 1H–1H COSY, SF = 400.13
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The dried leaves of A. annua (3.5 kg) was pulverized and refluxed with 70% (v/v) EtOH (8 l, 3 × 2 h). After evaporation of EtOH in vacuo, the aqueous residue was subjected to CC over HP-20 macroporous adsorptive resins, eluted with water and 30 and 95% ethanol in successive to yield three fractions (1–3). Fraction 3 (60 g, 95% EtOH eluate) was separated over a silica gel column (SiO 2, 200–300 mesh, Φ 9.8 × 62 cm) eluted with a CHCl3-MeOH in gradient to yield nine crude fractions (3A–3I). Subfraction 3A (6.5 g, CHCl 3-MeOH, 95 : 5, eluant) was separated by CC over Sephadex LH-20 (100 g, Φ 2 × 100 cm) eluted with CHCl 3-CH3 OH (1 : 1) in gradient. The subfraction 3A-2 (4.5 g, CHCl3–MeOH, 1 : 1, eluant) was then separated by CC over ODS (50 μm, Φ 5 × 38 cm) eluted with MeOH-H 2O in gradient. The subfraction 3A-2-c (MeOH-H2O, 7 : 3, eluant) was further purified by preparative HPLC (CH 3 CN-H 2O, 1 : 4) to afford compound 1 (5.5 mg, tR 28.4 min). Subfraction 3B (4.8 g, CHCl3 -MeOH, 90:10, eluant) was submitted to repeated ODS, Toyopearl HW-40 columns eluted with MeOH/H2O, followed by preparative HPLC (MeOH : H2O, 5.5 : 4.5) to yield compound 2 (35.7 mg, tR 37.4 min). Physical and spectroscopic data of new compounds (1S,7R,8R,10S)-7,8,11-trihydroxy-1-hydroperoxy-4-guaien-3-one (1)
Yellow amorphous solid. [α]25 D -29.3 (c = 2, MeOH). UV (MeOH) λmax nm (log ε): 235.7 (4.33); IR (KBr) νmax cm 1: 3304, 2976, 1677, 1630, 1455, 1384, 1077; CD (c = 0.16, MeOH): 249.2 nm (Δε 0.99), 312.4 nm (Δε 1.95); HR-ESI-Q/TOF-MS: m/z 301.1646 ([M + H]+, C15H25O+6 , calcd. for 301.1651); 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), refer to Table 1. (1S,6R,7R,10R)-6-carboxy-10-methyl-α-methylene-1-(1-oxobutyl)-cyclohexanea cetic acid (2)
White needle crystal. [α]25 D -16.5 (c = 2, MeOH). UV (MeOH) λmax nm (log ε): 204 (2.89); IR (KBr) νmax cm 1: 3500–2500, 1723, 1695; CD (c = 0.14, MeOH): 206.8 nm (Δε 3.73), 227.3 nm (Δε 1.58), 251.4 nm (Δε 1.24), 286.7 nm (Δε 1.18); HR-ESI-Q/TOF-MS: m/z 305.1365 ([M + Na]+, C15H22O5Na+, calcd. for 305.1365). 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz), refer to Table 1. Quantum chemical ECD calculation of 1
The conformational analysis for compound 1 was carried out by the MMFF94s force field. The lowest energy conformer was sbmitted to the ECD calculation in methanol solution at [B3LYP/6-31g(d)] level with the Boltzmann model, and the predicted ECD curve of compound 1 was similar to the experimental one.
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2015, 53, 244–247
1D and 2D NMR spectra, crystal X-ray diffraction and electronic circular dichroism (ECD) X-ray crystallographic analysis of compound 2
Upon crystallization from MeOH using the vapor diffusion method, needles of 2 were obtained. Crystal Data for C15H22O5 (M = 282.33): monoclinic, space group P21, a = 5.37 760(13) Å, b = 28.5292(7) Å, c = 10.4393(3) Å, β = 99.637(2)°, V = 1578.98 (7) Å3, Z = 2, T = 150.01(10) K, μ(Cu Kα) = 0.730 mm 1, Dcalc = 1.186 g/mm3, 14 952 measured reflections, 5235 independent reflections [Rint = 0.0552]. The final R1 was 0.0467, and wR2 was 0.1247 [I > 2σ(I)]. The goodness of fit on F2 was 1.027. Flack parameter = 0.1 (2). Crystallographic data for structure 2 has been deposited at the Cambridge Crystallographic Data Centre (CCDC 1012340). Acknowledgements This work was supported by the Natural Science Foundation of Jiangsu Province (BK20140441) and the National Basic Research Projects (973 program) of the Ministry of Science and Technology of China (2010CB735604) and the Programme of Introducing Talents of Discipline to Universities (B13038). We thank Yun-wen Chen for helping to calculate ECD.
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Additional supporting information may be found in the online version of this article at the publisher’s web site.
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