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Three new labdane diterpenes from Loxocalyx urticifolius
Phytochemistry Letters 19 (2017) 55–59
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Three new labdane diterpenes from Loxocalyx urticifolius Min Zhaoa , Da-Le Guoa , Lv-Yi Yuanb , Yu-Cheng Guc , Lei Chena , Li-Sheng Dinga,* , Yan Zhoua,* a CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, PR China b Southwest University for Nationalities, Chengdu 610041, PR China c Syngenta Jealott’s Hill International Research Centre, Berkshire RG42 6EY, UK
A R T I C L E I N F O
Article history: Received 9 September 2016 Received in revised form 9 November 2016 Accepted 21 November 2016 Available online xxx Keywords: Loxocalyx urticifolius Lamiaceae Labdane diterpene Cytotoxic activity
A B S T R A C T
Three new labdane diterpenes, namely loxocalyxin D (1), loxocalyxin E (2) and 13-epiloxocalyxin E (3), were isolated from the whole plants of Loxocalyx urticifolius Hemsl. Their structures were elucidated by extensive spectral analysis and chemical methods. The absolute configuration of loxocalyxin D (1) was confirmed by X-ray crystallographic analysis. The cytotoxic activities of the isolated labdane diterpenes were evaluated against the four human cancer cell lines A549, HepG2, HL-60 and MCF-7. 13epiloxocalyxin E (3) exhibited selective cytotoxicity against A549 and MCF-7 with the IC50 values of 22.4 and 47.3 mM, respectively. © 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Loxocalyx (Lamiaceae), a small endemic genus with only two species, is mainly distributed in the south of China (Zhang et al., 1994). The whole plants of Loxocalyx urticifolius Hemsl. have long been used as a traditional Chinese medicine to treat rheumatism and dysentery (Zhang et al., 2001). Our previous investigations on the constituents of L. urticifolius demonstrated that terpenoids are the main components (He et al., 2012). However, there is still no report on any biological activity of the terpenoids of this plant. Our recent phytochemical studies based on bioassay-guided fractionation led to the isolation of three new labdane diterpenes, namely loxocalyxin D (1), loxocalyxin E (2) and 13-epiloxocalyxin E (3) from the EtOAc extract of L. urticifolius. Their structures (Fig. 1) were unequivocally determined by extensive spectroscopic analysis and chemical methods. The absolute configuration of loxocalyxin D (1) was confirmed by X-ray crystallographic analysis. This paper describes the isolation, structure elucidation and cytotoxicity evaluation of the isolated diterpenes.
Compound 1 was obtained as colorless crystals. Its molecular formula C21H28O5 was established by the HRESIMS spectrum (m/z 361.2011 [M + H]+, calcd. for C21H29O5, 361.2015). The 1H and 13C NMR spectrum of 1 showed five methyl singlets at dH 0.87, 1.32, 1.90, 2.11, and 3.61 (3H, s), and their corresponding carbons at dC 15.0, 17.0, 23.5, 14.1, and 52.3; two olefinic groups at dH 5.82 (s) and 5.87 (s), and their corresponding carbons at dC 117.2 and 128.1; two ester carboxy groups at dC 172.3 and 178.6; and an aldehydic carboxy group at dC 197.1, which were characteristic for the labdane diterpene (He et al., 2012). A b-methyl-a,b-unsaturated-ketone group in ring B was confirmed by the HMBC correlations between H-17 and C-6/C-7/C-8/C-9, and between H-7 and C-5/C-9. A b-methyl-a,b-unsaturated-g -lactone ring was identified by the presence of characteristic resonances at dC 85.8 (C-12), 168.3 (C13), 117.2 (C-14), 172.3 (C-15), and 14.1 (C-16), which was supported by the HMBC correlations between H-12 and C-15, and between H16 and C-13/C-14/C-15 (Fig. 2). In addition, the 1H-1H COSY correlations of H-12/H-11, and H-11/H-9 suggested that C-12 of the b-methyl-a,b-unsaturated-g -lactone ring was connected to C-11, and which was connected to C-9 of ring B (Fig. 2). This was further confirmed by the HMBC correlations. The relative stereochemistry of 1 was assigned on the basis of NOE correlations (Fig. 3). An X-ray diffraction experiment using the anomalous scattering of Cu Ka radiation was conducted to determine the absolute configuration of 1 (Fig. 4). The results confirmed the absolute configuration of 1
* Corresponding authors. E-mail addresses: [email protected] (L.-S. Ding), [email protected] (Y. Zhou).
http://dx.doi.org/10.1016/j.phytol.2016.11.010 1874-3900/© 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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M. Zhao et al. / Phytochemistry Letters 19 (2017) 55–59
Fig. 1. Structures of compounds 1–3.
Fig. 2. Key HMBC and 1H-1H COSY correlations for compounds 1–3.
as 4S, 5S, 9R, 10S, 12R. Based on the above evidence, the structure of 1 was identified as (4S,5S,9R,10S,12R)-6-oxolabd-7-ene-12(15)olide-19-oate, and named loxocalyxin D. Compound 2 was obtained as a white amorphous powder. Its molecular formula was determined to be C21H30O6 from the [M + H]+ peak at m/z 379.2123 in the HRESIMS spectrum (calcd. for C21H31O6, 379.2121). Its spectroscopic features suggested 2 to be a labdane diterpene. After comparison of the NMR spectra of 2 and loxocalyxin A (He et al., 2012), the only difference was the disapperance of a methoxy group in 2, and the C-15 of 2 was found to be shifted downfield by 4.8 ppm. A methyl ester product 2a was produced from 2 using Steglich esterification with CH3OH. This ester product 2a was identified as loxocalyxin A by HRESIMS and NMR analysis, which was consistent with literature data (He et al., 2012). On the basis of the evidence, the structure of 2 was identified as (4S,5S,9R,10S,13S)-6,12-dioxolabd-7-ene-19-oate-15oic acid, and named loxocalyxin E. Compound 3 was obtained as a white amorphous powder. It was an isomer of 2 with the molecular formula C21H30O6 according to the HRESIMS spectrum (m/z 379.2124 [M + H]+, calcd. for
C21H31O6, 379.2121). The NMR spectroscopic data of 3 were almost identical with those of 2 except only a small difference of the chemical value of C-13 (Table 1), which suggested it was a C-13 epimer of 2. Steglich esterification of 3 with CH3OH could produce its methyl ester product 3a. Compound 3a was proved to be 13epiloxocalyxin A by comparing the NMR data of 3a with those of 13-epiloxocalyxin A (He et al., 2012). Thus, the structure of 3 was identified as (4S,5S,9R,10S,13R)-6,12-dioxolabd-7-ene-19-oate-15oic acid, and named 13-epiloxocalyxin E. In order to explore the potential medicinal function of L. urticifolius for antineoplastic activity, all of the isolated compounds were evaluated for their cytotoxic activities against the four human cancer cell lines A549, HepG2, HL-60 and MCF-7. The cytotoxic activities of the isolated compounds 1–3 were determined using the MTT colorimetric assay, and the results were shown in Supplementary data (Table 1.). Compound 3 exhibited selective cytotoxicity against A549 and MCF-7 with the IC50 values of 22.4 and 47.3 mM, respectively. All of the isolated compounds 1–3 were inactive (IC50 > 50 mM) to the normal human hepatocyte cell line (L02).
M. Zhao et al. / Phytochemistry Letters 19 (2017) 55–59
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3. Experimental 3.1. General experimental procedures Melting points were determined by using a BOT-X6 apparatus. Optical rotations were recorded on a Perkin Elmer Model 341 Polarimeter. IR measurements were obtained on a PerkinElmer one FT-IR spectrometer (KBr). The 1D and 2D NMR spectra were recorded on a Bruker Ascend 400 spectrometer using TMS as an internal standard. The ESIMS and HRESIMS were performed on a Waters XevoTM TQ mass spectrometer and a Bruker MicrOTOF QII mass spectrometer, respectively. Semi-preparative HPLC was performed on a LC 3000 liquid chromatography (ChuangXingTongHeng Science And Technology Co., Beijing) equipped with a Kromasil RP-C18 column (10 250 mm, 5 mm) and a LC 3000 UV detector (detection wavelength: 205 nm). Column chromatography (CC) was performed with silica gel (75 mm, Qingdao Marine Chemical Factory, China), ODS (50 mm, YMC, Japan), and Sephadex LH-20 (50 mm, Amersham Pharmacia Biotech, UK). Fractions were monitored by TLC detection which was achieved by spraying the silica gel plates (Qingdao Marine Chemical Factory, China) with 20% H2SO4-EtOH solution followed by heating. All of the solvents used were of analytical grade. 3.2. Plant material Whole plants of L. urticifolius were collected from Jinfoshan in Chongqing City, P. R. China, in Auguest 2015 and identified by Mr. Si-Rong Yi (Chongqing Institute of Medicinal Plant Cultivation, Chongqing). A voucher specimen (No. CIBT89) was deposited at Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, P. R. China. 3.3. Extraction and isolation Fig. 3. Key NOESY correlations for compounds 1–3.
Air-dried and powdered plant material of L. urticifolius (1.25 kg) was extracted three times (each for 3 days) with MeOH (6 L 3) at room temperature and concentrated under vacuum to yield a crude extract (248 g). The crude extract was suspended in H2O and then partitioned successively with petroleum ether and EtOAc. The EtOAc extract (18 g) was subjected to silica gel CC (75 mm, 5 40 cm, 200 g) and eluted with a gradient of petroleum etheracetone (30:1; 20:1; 10:1; 8:2:; 7:3:; 3:2; 1:1) to obtain five fractions A–E. Fractions A–E were evaluated for their cytotoxic activities against the human cancer cell line A549, and fraction C and D showed better cytotoxic activities against A549 with the IC50 values of 23.2 and 17.8 mM, respectively (Table 2 of Supplementary data). Fraction C (2.2 g) was submitted to Sephadex LH-20 CC (50 mm, 2 150 cm, 250 g), with MeOH-H2O (75:25, v/v) to yield compound 1 (165 mg). Fraction D (5.6 g) was subjected to CC (ODS, 50 mm, 2 30 cm, 200 g), and eluted with 30%, 50%, 70%, 90% and 100% MeOH-H2O to give five subfractions (a-e). Fraction D-c (125 mg) was purified by semi-preparative HPLC using MeOH-H2O (65:35, v/v; 4 mL/min) to yield compounds 2 (13 mg), and 3 (10 mg). 3.3.1. Loxocalyxin D (1) Colorless crystals (CHCl3); C21H28O5; mp 173–175 C; ½a20 D + 4 (c 0.1, CHCl3); IR (KBr) ymax 2946, 1760, 1722, 1664, 1439, 1249, 1145 cm1; HRESIMS: m/z 361.2011 [M + H]+ (calcd. 361.2015); for 1 H NMR and 13C NMR spectroscopic data, see Table 1. 3.3.2. Loxocalyxin E (2)
Fig. 4. X-ray crystal structure of compound 1.
White amorphous powder; C21H30O6; ½a20 D 139 (c 0.1, CHCl3); IR (KBr) ymax 3442, 2928, 1716, 1669, 1647, 1436, 1379, 1253, 1146,
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M. Zhao et al. / Phytochemistry Letters 19 (2017) 55–59
Table 1 13 C (100 MHz in CDCl3) and 1H (400 MHz in CDCl3) NMR spectroscopic data of compounds 1–3. 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 19-OCH3 a
2
3
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
37.7 17.5 37.1 43.0 60.2 197.1 128.1 160.2 52.9 42.4 29.2 85.8 168.3 117.2 172.3 14.1 23.5 17.0 178.6 15.0 52.3
1.43 m, 1.76 m 1.62a 1.52 m, 1.62a – 3.11 s – 5.87 s – 2.81 br s-like – 1.66 m, 1.96 ddd (15.6, 3.4, 3.1) 4.80 br d (10.7) – 5.82 s – 2.11 s 1.90 s 1.32 s – 0.87 s 3.61 s
37.3 17.5 37.4 43.1 59.8 197.7 127.5 160.5 49.2 41.3 38.3 211.2 42.3 37.3 177.4 17.5 22.3 17.1 179.0 15.8 52.4
1.27 m, 1.70 m 1.60a 1.51 m, 1.61a – 3.18 s – 5.88 s – 3.26 dd (8.2, 1.8) – 2.70 dd (19.0, 1.8), 2.80 dd (19.0, 8.2) – 3.08 m 2.39 dd (17.0, 4.5), 2.90 dd (17.0, 9.6) – 1.21 d (6.7) 1.75 s 1.36 s – 0.89 s 3.64 s
37.3 17.8 37.3 43.1 59.8 197.8 127.7 160.0 48.7 41.3 39.2 211.4 41.8 37.3 177.4 17.3 22.2 17.2 179.0 15.8 52.4
1.28 m, 1.67 m 1.60a 1.52 m, 1.62a – 3.19 s – 5.88 s – 3.28 dd (8.6, 1.6) – 2.71 dd (19.1, 1.6), 2.83 dd (19.1, 8.6) – 3.07 m 2.42 dd (17.0, 4.5), 2.93 dd (17.0, 9.7) – 1.19 d (6.7) 1.75 s 1.36 s – 0.89 s 3.64 s
Overlapped with other signals.
1058 cm1; HRESIMS: m/z 379.2123 [M + H]+ (calcd. 379.2121); for 1 H NMR and 13C NMR spectroscopic data, see Table 1.
HPLC to give the ester product. The product 2a was identified to be loxocalyxin A, and the product 3a was identified to be 13epiloxocalyxin A by NMR analysis and HRESIMS.
3.3.3. 13-Epiloxocalyxin E (3) White amorphous powder; C21H30O6; ½a20 D 102 (c 0.1, CHCl3); IR (KBr) ymax 3443, 2929, 1716, 1669, 1436, 1379, 1254, 1189, 1146, 1057 cm1; HRESIMS: m/z 379.2124 [M + H]+ (calcd. 379.2121); for 1 H NMR and 13C NMR spectroscopic data, see Table 1. 3.4. X-ray crysttallographic analysis of compound 1 The single-crystal X-ray diffraction data for 1 was collected on a Bruker APEX DUO CCD diffractometer with Cu-Ka radiation (l = 1.54178 Å) at 100(2) K. The structure was solved by direct method using SHELXS-97 (Sheldrick, 2008) and refined with fullmatrix leastsquares calculations on F2 using SHELXS-97 (Sheldrick, 2008). Crystal data for compound 1, C21H28O5, M = 360.43, a = 11.8287(2) Å, b = 11.8293(2) Å, c = 13.5073(2) Å, a = 90 , b = 90 , g = 90 , V = 1890.01(5) Å3, T = 100(2) K, space group P212121, Z = 4, m(CuKa) = 0.725 mm1, 10,371 reflections measured, 3157 independent reflections (Rint = 0.0311). The final R1 values were 0.0311 (I > 2s (I)). The final wR(F2) values were 0.0803 (I > 2s (I)). The final R1 values were 0.0312 (all data). The final wR(F2) values were 0.0803 (all data). The goodness of fit on F2 was 1.069. Flack parameter = 0.01(4). The crystallographic data for structure 1 in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC1502845. These data can be obtained free of charge from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; and e mail: deposit@ccdc cam ac uk. 3.5. Steglich esterification of compounds 2 and 3 Each compound (8 mg) was dissolved in anhydrous CH2Cl2 (2 mL). 1 mg DMAP, 1 mL CH3OH, and 20 mg DCC was added to the reaction mixture at 0 C and was stirred at 0 C for 6 h. After precipitated urea was filtered off, the filtrate was evaporated under vacuum. The residue was dissolved in CH2Cl2. The CH2Cl2 solution was washed twice with 0.5 N HCl and with saturated NaHCO3 solution, and then dried over MgSO4. (Neises and Steglich, 1978) The solvent was concentrated and purified by semi-preparative
3.6. Cytotoxicity assay The cytotoxic activities of compounds 1–3 against A549 (human alveolar basal epithelial cancer cell line), HepG2 (human liver hepatoma cancer cell line), HL-60 (human promyelocytic leukemia cancer cell line) and MCF-7 (human breast cancer cell line) were determined by the MTT assay (Ni et al., 2009; Zhao et al., 2016). The cancer cell lines were cultured in RPMI-1640 medium containing 100 U/mL penicillin, 100 mg/ mL streptomycin, and 10% fetal bovine serum at 37 C in a 5% CO2 atmosphere. These cells were seeded into each well of 96-well plates at a density of 1.5 105 cells/mL and incubated for 24 h. The tested compounds (0.5, 2, 10, and 50 mM) were added into triplicate wells, and were incubated for another 72 h at 37 C. 0.05 mg (10 mL of a 5 mg/mL solution) MTT was added into each well, and the plate was incubated for another 4 h. DMSO (100 mL) was then added to dissolve the formazan crystals. The absorbance was recorded immediately on a microplate reader (Amersham Pharmacia Biotech., USA) at a wavelength of 570 nm. The cytotoxic activities are expressed as IC50, which calculated as the concentrations of the tested compounds resulting in 50% reduction of absorption compared to the untreated controls. Dose-response curves were plotted for the samples, and the IC50 values were determined as the mean of three independent experiments. Acknowledgments This work was financially supported grants from the National Natural Sciences Foundation of China (21402157, 21572219 and 21572221) and a Syngenta Postgraduate Fellowship awarded to Min Zhao. Appendix A. Supplementary data Full spectroscopic data (NMR, IR, ESIMS, and HRESIMS) and cytotoxic activities of compounds (1–3) can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2016.11.010.
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References He, D.H., Xu, X.M., Zhou, Y., Deng, W.L., Xia, B., Ding, L.S., 2012. Terpenoids from Loxocalyx urticifolius. Helv. Chim. Acta 95, 1136–1143. Neises, B., Steglich, W., 1978. Simple method for the esterification of carboxylic acids. Angew. Chem. Int. Ed. Engl. 17, 522–524. Ni, G., Zhang, Q.J., Zheng, Z.F., Chen, R.Y., Yu, D.Q., 2009. 2-Arylbenzofuran derivatives from Morus cathayana. J. Nat. Prod. 72, 966–968. Sheldrick, G.M., 2008. A short history of SHELX. Acta Crystallogr. A 64, 112–122.
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Zhang, H.Y., Zhang, Z.Y., 1994. The Handbook of Traditional Chinese Medicine Sources in China. Science Press, Beijing pp. 1085. Zhang, X.R., Wang, M.A., Zhang, Y., Wang, M.K., Ding, L.S., 2001. Chemical constituents of Loxocalyx urticifolius Hemsl. China J. Chin. Mater. Med. 26, 692– 694. Zhao, M., Da-Wa, Z.M., Guo, D.L., Fang, D.M., Chen, X.Z., Xu, H.X., Gu, Y.C., Xia, B., Chen, L., Ding, L.S., Zhou, Y., 2016. Cytotoxic triterpenoid saponins from Clematis tangutica. Phytochemistry 130, 228–237.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Three new labdane diterpenes from Loxocalyx urticifolius Min Zhaoa , Da-Le Guoa , Lv-Yi Yuanb , Yu-Cheng Guc , Lei Chena , Li-Sheng Dinga,* , Yan Zhoua,* a CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, PR China b Southwest University for Nationalities, Chengdu 610041, PR China c Syngenta Jealott’s Hill International Research Centre, Berkshire RG42 6EY, UK
A R T I C L E I N F O
Article history: Received 9 September 2016 Received in revised form 9 November 2016 Accepted 21 November 2016 Available online xxx Keywords: Loxocalyx urticifolius Lamiaceae Labdane diterpene Cytotoxic activity
A B S T R A C T
Three new labdane diterpenes, namely loxocalyxin D (1), loxocalyxin E (2) and 13-epiloxocalyxin E (3), were isolated from the whole plants of Loxocalyx urticifolius Hemsl. Their structures were elucidated by extensive spectral analysis and chemical methods. The absolute configuration of loxocalyxin D (1) was confirmed by X-ray crystallographic analysis. The cytotoxic activities of the isolated labdane diterpenes were evaluated against the four human cancer cell lines A549, HepG2, HL-60 and MCF-7. 13epiloxocalyxin E (3) exhibited selective cytotoxicity against A549 and MCF-7 with the IC50 values of 22.4 and 47.3 mM, respectively. © 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Loxocalyx (Lamiaceae), a small endemic genus with only two species, is mainly distributed in the south of China (Zhang et al., 1994). The whole plants of Loxocalyx urticifolius Hemsl. have long been used as a traditional Chinese medicine to treat rheumatism and dysentery (Zhang et al., 2001). Our previous investigations on the constituents of L. urticifolius demonstrated that terpenoids are the main components (He et al., 2012). However, there is still no report on any biological activity of the terpenoids of this plant. Our recent phytochemical studies based on bioassay-guided fractionation led to the isolation of three new labdane diterpenes, namely loxocalyxin D (1), loxocalyxin E (2) and 13-epiloxocalyxin E (3) from the EtOAc extract of L. urticifolius. Their structures (Fig. 1) were unequivocally determined by extensive spectroscopic analysis and chemical methods. The absolute configuration of loxocalyxin D (1) was confirmed by X-ray crystallographic analysis. This paper describes the isolation, structure elucidation and cytotoxicity evaluation of the isolated diterpenes.
Compound 1 was obtained as colorless crystals. Its molecular formula C21H28O5 was established by the HRESIMS spectrum (m/z 361.2011 [M + H]+, calcd. for C21H29O5, 361.2015). The 1H and 13C NMR spectrum of 1 showed five methyl singlets at dH 0.87, 1.32, 1.90, 2.11, and 3.61 (3H, s), and their corresponding carbons at dC 15.0, 17.0, 23.5, 14.1, and 52.3; two olefinic groups at dH 5.82 (s) and 5.87 (s), and their corresponding carbons at dC 117.2 and 128.1; two ester carboxy groups at dC 172.3 and 178.6; and an aldehydic carboxy group at dC 197.1, which were characteristic for the labdane diterpene (He et al., 2012). A b-methyl-a,b-unsaturated-ketone group in ring B was confirmed by the HMBC correlations between H-17 and C-6/C-7/C-8/C-9, and between H-7 and C-5/C-9. A b-methyl-a,b-unsaturated-g -lactone ring was identified by the presence of characteristic resonances at dC 85.8 (C-12), 168.3 (C13), 117.2 (C-14), 172.3 (C-15), and 14.1 (C-16), which was supported by the HMBC correlations between H-12 and C-15, and between H16 and C-13/C-14/C-15 (Fig. 2). In addition, the 1H-1H COSY correlations of H-12/H-11, and H-11/H-9 suggested that C-12 of the b-methyl-a,b-unsaturated-g -lactone ring was connected to C-11, and which was connected to C-9 of ring B (Fig. 2). This was further confirmed by the HMBC correlations. The relative stereochemistry of 1 was assigned on the basis of NOE correlations (Fig. 3). An X-ray diffraction experiment using the anomalous scattering of Cu Ka radiation was conducted to determine the absolute configuration of 1 (Fig. 4). The results confirmed the absolute configuration of 1
* Corresponding authors. E-mail addresses: [email protected] (L.-S. Ding), [email protected] (Y. Zhou).
http://dx.doi.org/10.1016/j.phytol.2016.11.010 1874-3900/© 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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M. Zhao et al. / Phytochemistry Letters 19 (2017) 55–59
Fig. 1. Structures of compounds 1–3.
Fig. 2. Key HMBC and 1H-1H COSY correlations for compounds 1–3.
as 4S, 5S, 9R, 10S, 12R. Based on the above evidence, the structure of 1 was identified as (4S,5S,9R,10S,12R)-6-oxolabd-7-ene-12(15)olide-19-oate, and named loxocalyxin D. Compound 2 was obtained as a white amorphous powder. Its molecular formula was determined to be C21H30O6 from the [M + H]+ peak at m/z 379.2123 in the HRESIMS spectrum (calcd. for C21H31O6, 379.2121). Its spectroscopic features suggested 2 to be a labdane diterpene. After comparison of the NMR spectra of 2 and loxocalyxin A (He et al., 2012), the only difference was the disapperance of a methoxy group in 2, and the C-15 of 2 was found to be shifted downfield by 4.8 ppm. A methyl ester product 2a was produced from 2 using Steglich esterification with CH3OH. This ester product 2a was identified as loxocalyxin A by HRESIMS and NMR analysis, which was consistent with literature data (He et al., 2012). On the basis of the evidence, the structure of 2 was identified as (4S,5S,9R,10S,13S)-6,12-dioxolabd-7-ene-19-oate-15oic acid, and named loxocalyxin E. Compound 3 was obtained as a white amorphous powder. It was an isomer of 2 with the molecular formula C21H30O6 according to the HRESIMS spectrum (m/z 379.2124 [M + H]+, calcd. for
C21H31O6, 379.2121). The NMR spectroscopic data of 3 were almost identical with those of 2 except only a small difference of the chemical value of C-13 (Table 1), which suggested it was a C-13 epimer of 2. Steglich esterification of 3 with CH3OH could produce its methyl ester product 3a. Compound 3a was proved to be 13epiloxocalyxin A by comparing the NMR data of 3a with those of 13-epiloxocalyxin A (He et al., 2012). Thus, the structure of 3 was identified as (4S,5S,9R,10S,13R)-6,12-dioxolabd-7-ene-19-oate-15oic acid, and named 13-epiloxocalyxin E. In order to explore the potential medicinal function of L. urticifolius for antineoplastic activity, all of the isolated compounds were evaluated for their cytotoxic activities against the four human cancer cell lines A549, HepG2, HL-60 and MCF-7. The cytotoxic activities of the isolated compounds 1–3 were determined using the MTT colorimetric assay, and the results were shown in Supplementary data (Table 1.). Compound 3 exhibited selective cytotoxicity against A549 and MCF-7 with the IC50 values of 22.4 and 47.3 mM, respectively. All of the isolated compounds 1–3 were inactive (IC50 > 50 mM) to the normal human hepatocyte cell line (L02).
M. Zhao et al. / Phytochemistry Letters 19 (2017) 55–59
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3. Experimental 3.1. General experimental procedures Melting points were determined by using a BOT-X6 apparatus. Optical rotations were recorded on a Perkin Elmer Model 341 Polarimeter. IR measurements were obtained on a PerkinElmer one FT-IR spectrometer (KBr). The 1D and 2D NMR spectra were recorded on a Bruker Ascend 400 spectrometer using TMS as an internal standard. The ESIMS and HRESIMS were performed on a Waters XevoTM TQ mass spectrometer and a Bruker MicrOTOF QII mass spectrometer, respectively. Semi-preparative HPLC was performed on a LC 3000 liquid chromatography (ChuangXingTongHeng Science And Technology Co., Beijing) equipped with a Kromasil RP-C18 column (10 250 mm, 5 mm) and a LC 3000 UV detector (detection wavelength: 205 nm). Column chromatography (CC) was performed with silica gel (75 mm, Qingdao Marine Chemical Factory, China), ODS (50 mm, YMC, Japan), and Sephadex LH-20 (50 mm, Amersham Pharmacia Biotech, UK). Fractions were monitored by TLC detection which was achieved by spraying the silica gel plates (Qingdao Marine Chemical Factory, China) with 20% H2SO4-EtOH solution followed by heating. All of the solvents used were of analytical grade. 3.2. Plant material Whole plants of L. urticifolius were collected from Jinfoshan in Chongqing City, P. R. China, in Auguest 2015 and identified by Mr. Si-Rong Yi (Chongqing Institute of Medicinal Plant Cultivation, Chongqing). A voucher specimen (No. CIBT89) was deposited at Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, P. R. China. 3.3. Extraction and isolation Fig. 3. Key NOESY correlations for compounds 1–3.
Air-dried and powdered plant material of L. urticifolius (1.25 kg) was extracted three times (each for 3 days) with MeOH (6 L 3) at room temperature and concentrated under vacuum to yield a crude extract (248 g). The crude extract was suspended in H2O and then partitioned successively with petroleum ether and EtOAc. The EtOAc extract (18 g) was subjected to silica gel CC (75 mm, 5 40 cm, 200 g) and eluted with a gradient of petroleum etheracetone (30:1; 20:1; 10:1; 8:2:; 7:3:; 3:2; 1:1) to obtain five fractions A–E. Fractions A–E were evaluated for their cytotoxic activities against the human cancer cell line A549, and fraction C and D showed better cytotoxic activities against A549 with the IC50 values of 23.2 and 17.8 mM, respectively (Table 2 of Supplementary data). Fraction C (2.2 g) was submitted to Sephadex LH-20 CC (50 mm, 2 150 cm, 250 g), with MeOH-H2O (75:25, v/v) to yield compound 1 (165 mg). Fraction D (5.6 g) was subjected to CC (ODS, 50 mm, 2 30 cm, 200 g), and eluted with 30%, 50%, 70%, 90% and 100% MeOH-H2O to give five subfractions (a-e). Fraction D-c (125 mg) was purified by semi-preparative HPLC using MeOH-H2O (65:35, v/v; 4 mL/min) to yield compounds 2 (13 mg), and 3 (10 mg). 3.3.1. Loxocalyxin D (1) Colorless crystals (CHCl3); C21H28O5; mp 173–175 C; ½a20 D + 4 (c 0.1, CHCl3); IR (KBr) ymax 2946, 1760, 1722, 1664, 1439, 1249, 1145 cm1; HRESIMS: m/z 361.2011 [M + H]+ (calcd. 361.2015); for 1 H NMR and 13C NMR spectroscopic data, see Table 1. 3.3.2. Loxocalyxin E (2)
Fig. 4. X-ray crystal structure of compound 1.
White amorphous powder; C21H30O6; ½a20 D 139 (c 0.1, CHCl3); IR (KBr) ymax 3442, 2928, 1716, 1669, 1647, 1436, 1379, 1253, 1146,
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Table 1 13 C (100 MHz in CDCl3) and 1H (400 MHz in CDCl3) NMR spectroscopic data of compounds 1–3. 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 19-OCH3 a
2
3
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
37.7 17.5 37.1 43.0 60.2 197.1 128.1 160.2 52.9 42.4 29.2 85.8 168.3 117.2 172.3 14.1 23.5 17.0 178.6 15.0 52.3
1.43 m, 1.76 m 1.62a 1.52 m, 1.62a – 3.11 s – 5.87 s – 2.81 br s-like – 1.66 m, 1.96 ddd (15.6, 3.4, 3.1) 4.80 br d (10.7) – 5.82 s – 2.11 s 1.90 s 1.32 s – 0.87 s 3.61 s
37.3 17.5 37.4 43.1 59.8 197.7 127.5 160.5 49.2 41.3 38.3 211.2 42.3 37.3 177.4 17.5 22.3 17.1 179.0 15.8 52.4
1.27 m, 1.70 m 1.60a 1.51 m, 1.61a – 3.18 s – 5.88 s – 3.26 dd (8.2, 1.8) – 2.70 dd (19.0, 1.8), 2.80 dd (19.0, 8.2) – 3.08 m 2.39 dd (17.0, 4.5), 2.90 dd (17.0, 9.6) – 1.21 d (6.7) 1.75 s 1.36 s – 0.89 s 3.64 s
37.3 17.8 37.3 43.1 59.8 197.8 127.7 160.0 48.7 41.3 39.2 211.4 41.8 37.3 177.4 17.3 22.2 17.2 179.0 15.8 52.4
1.28 m, 1.67 m 1.60a 1.52 m, 1.62a – 3.19 s – 5.88 s – 3.28 dd (8.6, 1.6) – 2.71 dd (19.1, 1.6), 2.83 dd (19.1, 8.6) – 3.07 m 2.42 dd (17.0, 4.5), 2.93 dd (17.0, 9.7) – 1.19 d (6.7) 1.75 s 1.36 s – 0.89 s 3.64 s
Overlapped with other signals.
1058 cm1; HRESIMS: m/z 379.2123 [M + H]+ (calcd. 379.2121); for 1 H NMR and 13C NMR spectroscopic data, see Table 1.
HPLC to give the ester product. The product 2a was identified to be loxocalyxin A, and the product 3a was identified to be 13epiloxocalyxin A by NMR analysis and HRESIMS.
3.3.3. 13-Epiloxocalyxin E (3) White amorphous powder; C21H30O6; ½a20 D 102 (c 0.1, CHCl3); IR (KBr) ymax 3443, 2929, 1716, 1669, 1436, 1379, 1254, 1189, 1146, 1057 cm1; HRESIMS: m/z 379.2124 [M + H]+ (calcd. 379.2121); for 1 H NMR and 13C NMR spectroscopic data, see Table 1. 3.4. X-ray crysttallographic analysis of compound 1 The single-crystal X-ray diffraction data for 1 was collected on a Bruker APEX DUO CCD diffractometer with Cu-Ka radiation (l = 1.54178 Å) at 100(2) K. The structure was solved by direct method using SHELXS-97 (Sheldrick, 2008) and refined with fullmatrix leastsquares calculations on F2 using SHELXS-97 (Sheldrick, 2008). Crystal data for compound 1, C21H28O5, M = 360.43, a = 11.8287(2) Å, b = 11.8293(2) Å, c = 13.5073(2) Å, a = 90 , b = 90 , g = 90 , V = 1890.01(5) Å3, T = 100(2) K, space group P212121, Z = 4, m(CuKa) = 0.725 mm1, 10,371 reflections measured, 3157 independent reflections (Rint = 0.0311). The final R1 values were 0.0311 (I > 2s (I)). The final wR(F2) values were 0.0803 (I > 2s (I)). The final R1 values were 0.0312 (all data). The final wR(F2) values were 0.0803 (all data). The goodness of fit on F2 was 1.069. Flack parameter = 0.01(4). The crystallographic data for structure 1 in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC1502845. These data can be obtained free of charge from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; and e mail: deposit@ccdc cam ac uk. 3.5. Steglich esterification of compounds 2 and 3 Each compound (8 mg) was dissolved in anhydrous CH2Cl2 (2 mL). 1 mg DMAP, 1 mL CH3OH, and 20 mg DCC was added to the reaction mixture at 0 C and was stirred at 0 C for 6 h. After precipitated urea was filtered off, the filtrate was evaporated under vacuum. The residue was dissolved in CH2Cl2. The CH2Cl2 solution was washed twice with 0.5 N HCl and with saturated NaHCO3 solution, and then dried over MgSO4. (Neises and Steglich, 1978) The solvent was concentrated and purified by semi-preparative
3.6. Cytotoxicity assay The cytotoxic activities of compounds 1–3 against A549 (human alveolar basal epithelial cancer cell line), HepG2 (human liver hepatoma cancer cell line), HL-60 (human promyelocytic leukemia cancer cell line) and MCF-7 (human breast cancer cell line) were determined by the MTT assay (Ni et al., 2009; Zhao et al., 2016). The cancer cell lines were cultured in RPMI-1640 medium containing 100 U/mL penicillin, 100 mg/ mL streptomycin, and 10% fetal bovine serum at 37 C in a 5% CO2 atmosphere. These cells were seeded into each well of 96-well plates at a density of 1.5 105 cells/mL and incubated for 24 h. The tested compounds (0.5, 2, 10, and 50 mM) were added into triplicate wells, and were incubated for another 72 h at 37 C. 0.05 mg (10 mL of a 5 mg/mL solution) MTT was added into each well, and the plate was incubated for another 4 h. DMSO (100 mL) was then added to dissolve the formazan crystals. The absorbance was recorded immediately on a microplate reader (Amersham Pharmacia Biotech., USA) at a wavelength of 570 nm. The cytotoxic activities are expressed as IC50, which calculated as the concentrations of the tested compounds resulting in 50% reduction of absorption compared to the untreated controls. Dose-response curves were plotted for the samples, and the IC50 values were determined as the mean of three independent experiments. Acknowledgments This work was financially supported grants from the National Natural Sciences Foundation of China (21402157, 21572219 and 21572221) and a Syngenta Postgraduate Fellowship awarded to Min Zhao. Appendix A. Supplementary data Full spectroscopic data (NMR, IR, ESIMS, and HRESIMS) and cytotoxic activities of compounds (1–3) can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2016.11.010.
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References He, D.H., Xu, X.M., Zhou, Y., Deng, W.L., Xia, B., Ding, L.S., 2012. Terpenoids from Loxocalyx urticifolius. Helv. Chim. Acta 95, 1136–1143. Neises, B., Steglich, W., 1978. Simple method for the esterification of carboxylic acids. Angew. Chem. Int. Ed. Engl. 17, 522–524. Ni, G., Zhang, Q.J., Zheng, Z.F., Chen, R.Y., Yu, D.Q., 2009. 2-Arylbenzofuran derivatives from Morus cathayana. J. Nat. Prod. 72, 966–968. Sheldrick, G.M., 2008. A short history of SHELX. Acta Crystallogr. A 64, 112–122.
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Zhang, H.Y., Zhang, Z.Y., 1994. The Handbook of Traditional Chinese Medicine Sources in China. Science Press, Beijing pp. 1085. Zhang, X.R., Wang, M.A., Zhang, Y., Wang, M.K., Ding, L.S., 2001. Chemical constituents of Loxocalyx urticifolius Hemsl. China J. Chin. Mater. Med. 26, 692– 694. Zhao, M., Da-Wa, Z.M., Guo, D.L., Fang, D.M., Chen, X.Z., Xu, H.X., Gu, Y.C., Xia, B., Chen, L., Ding, L.S., Zhou, Y., 2016. Cytotoxic triterpenoid saponins from Clematis tangutica. Phytochemistry 130, 228–237.