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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Three new ursane-type triterpenoids from the aerial parts of Isodon excisoides a
a
a
a
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Ke Jiao , Huo-Yun Li , Peng Zhang , Hui-Fang Pi , Han-Li Ruan & a
Ji-Zhou Wu a
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Tongji School of Pharmaceutical Sciences, Huazhong University of Science and Technology, Wuhan, 430030, China Published online: 24 Oct 2013.
To cite this article: Ke Jiao, Huo-Yun Li, Peng Zhang, Hui-Fang Pi, Han-Li Ruan & Ji-Zhou Wu (2013) Three new ursane-type triterpenoids from the aerial parts of Isodon excisoides, Journal of Asian Natural Products Research, 15:9, 962-968, DOI: 10.1080/10286020.2013.837458 To link to this article: http://dx.doi.org/10.1080/10286020.2013.837458
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Journal of Asian Natural Products Research, 2013 Vol. 15, No. 9, 962–968, http://dx.doi.org/10.1080/10286020.2013.837458
Three new ursane-type triterpenoids from the aerial parts of Isodon excisoides Ke Jiao, Huo-Yun Li, Peng Zhang*, Hui-Fang Pi, Han-Li Ruan and Ji-Zhou Wu Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Tongji School of Pharmaceutical Sciences, Huazhong University of Science and Technology, Wuhan 430030, China
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(Received 14 June 2013; final version received 20 August 2013) Three new ursane-type triterpenoids, 2a,3b-dihydroxy-11a,12a-epoxy-urs-28,13bolide (1), 2a,3b,24-trihydroxy-11a,12a-epoxy-urs-28,13b-olide (2), and 2a,3a,24trihydroxy-11,20(30)-dien-urs-28,13b-olide (6), together with six known ursane-type triterpenoids (3 – 5, 7– 9), were isolated from the EtOAc extract of the aerial parts of Isodon excisoides. Their structures were elucidated on the basis of 1D NMR and 2D NMR analyses as well as HRMS experiments. Keywords: Isodon excisoides; ursane; triterpenoid
1.
Introduction
The genus Isodon (formally Rabdosia) comprising about 150 species of undershrubs, subundershrubs, or perennial herbs is mainly distributed in tropical and subtropical Asia [1]. It is a cosmopolitan and important genus of the Labiatae family, and has attracted considerable attention as a fertile source of ent-kaurane diterpenoids that have been verified to be the main bioactive constituents [2]. Besides the entkauranoids, triterpenoids were found to be another type of rich constituents in the Isodon plants, but previous investigations rarely led to the isolation of new triterpenoids [3 – 5]. Isodon excisoides (Sun ex C.H. Hu) C. Y. Wu et H.W. Li is a perennial herb mainly distributed in Hubei, Sichuan, and Yunnan at the altitudes of 700 – 3000 m [6]. Previous research on the chemical constituents on Isodon excisoides had led to the isolation of five ent-kaurane diterpenoids [7 – 9]. However, in our recent phytochemical investigation of this plant grown in a different ecological environment, three new ursane-type triterpenoids, 2a,3b-
dihydroxy-11a,12a-epoxy-urs-28,13bolide (1), 2a,3b,24-trihydroxy-11a,12aepoxy-urs-28,13b-olide (2), and 2a,3a,24trihydroxy-11,20(30)-dien-urs-28,13bolide (6), together with six known ursanetype triterpenoids isodonadenanthin (3) [4], 2a,3a,24-trihydroxy-11a,12a-epoxy-urs20(30)-en-28,13b-olide (4) [10], 2a,3a, 24-trihydroxy-11-en-urs-28,13b-olide (5) [11], 2a,3a,24-trihydroxy-12(13)-en-urs28-oic acid (7) [12,13], 2a,3a-dihydroxy12(13)-en-urs-28-oic acid (8) [14], and 2a,3b,24-trihydroxy-12(13)-en-urs-28-oic acid (9) [15] (Figure 1), were obtained. The structures of the known compounds were identified by the comparison of their NMR spectral data with those reported in the literature. Herein, we report the isolation and structure elucidation of those new compounds. 2.
Compound 1 was isolated as a white amorphous powder. Its molecular formula was determined to be C30H46O5 based on its HR-ESI-MS data at m/z 509.3230 [M þ Na]þ. The IR spectrum of 1 showed
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
Results and discussion
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Figure 1. The structures of compounds 1 – 9.
absorption bands for hydroxyl group at 3355 cm21 and for g-lactone at 1764 cm21. 1H NMR spectrum (Table 1) exhibited five tertiary methyl signals at dH 1.05 (3H, s, H-25), 1.08 (3H, s, H-24), 1.14 (6H, s, H-26, 27), and 1.24 (3H, s, H-23); two secondary methyl signals at dH 0.88 (3H, d, J ¼ 6.3 Hz, H-30) and 1.31 (3H, d, J ¼ 5.8 Hz, H-29); and four oxygenated methine signals at dH 3.09 (1H, d, J ¼ 3.8 Hz, H-12), 3.29 (1H, dd, J ¼ 3.8, 2.0 Hz, H-11), 3.39 (1H, d, J ¼ 9.3 Hz, H3), and 4.18 (1H, ddd, J ¼ 12.4, 9.3, 4.3 Hz, H-2). The 13C NMR (Table 1) and distortionless enhancement by polarization transfer spectra revealed 30 carbon signals, including seven methyl carbons at dC 16.4 (C-27), 17.1 (C-24), 17.5 (C-29), 18.5 (C-25), 19.4 (C-30), 20.5 (C-26), and 28.8 (C-23); four oxygenated methine carbons at dC 54.8 (C-11), 56.4 (C-12), 68.1 (C-2), and 83.7 (C-3); as well as one carbonyl
group at dC 178.7 (C-28). The remaining carbon signals were observed and assigned to seven methylene, five methine, and six quaternary carbons. The quaternary carbon signals at dC 88.9 (C-13) and 178.7 (C-28), along with the IR absorption at 1764 cm21, indicated that 1 possessed a glactone ring between C-13 and C-28. These NMR spectral data indicated that compound 1 was an ursane-type triterpenoid [16]. Comparing the 13C NMR data (Table 1) of 1 with those of 3b-hydroxy11a,12a-epoxy-urs-28,13b-olide [17] showed that 1 had the same skeleton. The difference was that the C-2 (dC 26.7) in 3b-hydroxy-11a,12a-epoxy-urs-28,13bolide shifted upfield to dC 68.1 corresponding to an additional proton signal at dH 4.18 (1H, ddd, J ¼ 12.4, 9.3, 4.3 Hz, H-2) in compound 1, which suggested that the additional hydroxy was at C-2 in compound 1. The 1H – 1H COSY correlation of
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Table 1.
1
H and 13C NMR spectral data for compounds 1 (C5D5N), 2 (CD3OD), and 6 (C5D5N).
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1
2
No.
dH, J (Hz)
dC
1a 1b 2
2.57 dd (12.4, 4.3) 1.53 m 4.18 ddd (12.4, 9.3, 4.3) 3.39 d (9.3)
47.4
3 4 5 6a 6b 7a 7b 8 9 10 11 12 13 14 15a 15b 16a 16b 17 18 19 20 21a 21b 22a 22b 23 24a 24b 25 26 27 28 29 30a 30b
0.96 m 1.52 m 1.52 m 1.23 m 1.04 m 1.77 m 3.29 dd (3.8, 2.0) 3.09 d (3.8) 1.69 m 0.98 m 2.10 td (13.2, 5.7) 1.29 m 1.81 m 1.77 m 0.83 m 1.40 m 1.30 m 1.85 m 1.57 m 1.24 s 1.08 s 1.05 s 1.14 s 1.14 s 1.31 d (5.8) 0.88 d (6.3)
68.1 83.7 39.8 54.9 18.0 31.5 41.5 51.9 37.7 54.8 56.4 88.9 41.7 27.0 23.0 45.2 60.3 37.4 40.3 30.6 31.8 28.8 17.1 18.5 20.5 16.4 178.7 17.5 19.4
dH, J (Hz) 2.26 m 1.12 m 3.87 ddd (9.6, 9.6, 4.6) 3.10 d (9.6) 0.96 m 1.67 m 1.60 m 1.35 m 1.20 m
3.25 dd (3.6, 2.0) 2.97 d (3.6) 1.63 m 1.15 m 2.27 m 1.31 m
46.1 67.7 84.3 43.0 54.8 17.8
41.3 51.5 37.1 54.5 55.9 89.5 41.1 26.4 22.4
1.85 m 1.81 m 0.97 m 1.59 m 1.38 m 1.75 m 1.51 m 1.23 s 4.01 d (11.2) 3.46 d (11.2) 1.13 s 1.06 s 1.16 s
H-1/H-2/H-3 and the HMBC (Figure 2) correlations of H-2 with C-1 and C-3 further confirmed this elucidation. In the NOESY spectrum, H-3 correlated with Ha-1, H-5, and H-23 (Figure 3). Thus, the relative stereochemistry of the 3-OH group was elucidated as b-orientation. The NOE correlations of H-2 with H-24 and H-25, H-11 with H-25, and H-12 with
dC
31.2
1.64 m
1.22 d (5.0) 1.01 d (5.2)
6
45.2 60.2 37.2 40.0 30.1 31.1 22.0 64.3 17.6 19.4 15.4 180.1 16.4 18.4
dH, J (Hz) 2.20 m 1.85 m 4.52 ddd (10.9, 3.0, 2.0) 4.63 d (2.0) 1.81 m 1.83 m 1.83 m 1.39 m 1.12 m 2.24 m 5.56 dd (10.3, 2.9) 6.13 d (10.3) 1.60 m 1.09 m 2.25 m 1.35 m 1.62 m 2.64 q (6.2) 2.18 m 1.87 m 1.92 m 1.57 m 1.67 s 4.08 d (10.7) 3.86 d (10.7) 1.01 s 1.16 s 1.03 s 1.04 d (6.4) 4.78 s 4.66 s
dC 42.7 65.7 74.0 44.9 48.4 18.0 31.6 42.0 53.3 37.5 128.7 133.5 88.7 42.0 25.4 22.7 44.9 60.9 36.2 151.5 31.6 33.0 23.6 64.4 19.3 19.0 15.5 178.5 16.2 107.4
H-18 implied that both the 2-OH group and the 11-12-epoxy group had aorientations. Therefore, the structure of compound 1 was assigned as 2a,3bdihydroxy-11a,12a-epoxy-urs-28,13bolide (Figure 1). Compound 2 was also obtained as a white amorphous powder. HR-ESI-MS gave one quasi-molecular ion peak at m/z
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Figure 2.
Selected 1H – 1H COSY (
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) and HMBC ( ! ) correlations of compounds 1, 2, and 6.
Figure 3. Key NOESY ( $ ) correlations of compounds 1, 2, and 6.
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525.3194 [M þ Na]þ corresponding to the molecular formula C30H46O6. The 1H and 13 C NMR spectral data of compound 2 were similar to those of 1 (Table 1), except for an additional hydroxyl. In the HMBC spectrum of 2, the proton signals at dH 3.46 and 4.01 (H2-24) showed longrange correlations with C-3, C-4, C-5, and C-23 (Figure 2), suggesting that the hydroxy group was at C-24, which was further confirmed by the NOE correlations of the proton signal at dH 4.01 (H-24) with H-2 and H-25 (Figure 3). Thus, the structure of 2 was assigned as 2a,3b,24-trihydroxy-11a,12a-epoxyurs-28,13b-olide (Figure 1). Compound 6 was obtained as a white amorphous powder. Its HR-ESI-MS revealed a quasi-molecular ion at m/z 485.3268 [M þ H]þ, indicating a molecular formula of C30H44O5. The IR spectrum showed absorption bands for hydroxyl group at 3421 cm21 and for g-lactone at 1742 cm21. The 13C NMR spectral data of 6 (Table 1) were similar to those of compound 5 [11], except for an additional olefinic bond (dH 4.66, 4.78; dC 107.4, 151.5). In the HMBC spectrum of 6, the correlations of H2-30 (dH 4.66, 4.78) with C-19 (dC 36.2), C-20 (dC 151.5), and C-21 (dC 31.6); H3-29 (dH 1.04) with C-20 (dC 151.5); and H-22 (dH 1.92) with C-20 (dC 151.5; Figure 2) indicated that the additional olefinic bond was between C-20 and C-30. In the NOESY spectrum, the a-orientations of 2-OH and 3OH were elucidated by the correlations of H-2 with H-24 and H-25, and H-3 with Hb-1 and H-24 (Figure 3). Therefore, the structure of compound 6 was assigned as 2a,3a,24-trihydroxy-11,20(30)-dien-urs28,13b-olide (Figure 1). 3. 3.1
Experimental General experimental procedures
Melting points were measured using an XT4-100X micro-melting point apparatus (Tech Instrument Corporation, Beijing, China) and are uncorrected. Optical
rotations were obtained on a PerkinElmer 341 polarimeter (Perkin-Elmer, Foster City, CA, USA). IR spectra were detected by a Vertex 70 spectrometer (Bruker Corporation, Karlsruhe, Germany) with KBr disks. The NMR spectra were run on a Bruker UltrashieldTM 400 plus spectrometer (1H NMR, 400 MHz; 13C NMR, 100 MHz; Bruker BioSpin, Fa¨llanden, Switzerland), both with tetramethylsilane as an internal standard. HR-ESI-MS data were recorded on Thermo Scientific LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, CA, USA). Semi-preparative HPLC was carried out on an Agilent 1100 Series apparatus (Agilent Technologies, Santa Clara, CA, USA), equipped with an Alltech evaporative light scattering detector (Alltech, Dearfield, IL, USA). The semi-preparative HPLC column of YMCPack ODS-A SH-343-5 (YMC, Komatsu City, Japan) was used. Column chromatography was carried out on silica gel (200 –300 mesh; Qingdao Haiyang Chemical Co., Ltd, Qingdao, China) and MCI gel CHP20P (75 – 150 mm; Mitsubishi Chemical Corporation, Japan). Fractions were monitored by thin layer chromatography (GF254; Qingdao Haiyang Chemical Co., Ltd, Qingdao, China), and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in ethanol. 3.2 Plant material The aerial parts of Isodon excisoides were collected in Enshi, Hubei Province, China, in October 2009, and identified by Prof. J.Q. Li (Wuhan Botanical Garden, Chinese Academy of Sciences). A voucher specimen (No. ICC-20091013) was deposited at the Faculty of Pharmaceutical Sciences, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China. 3.3 Extraction and isolation The air-dried and coarsely powdered aerial parts of Isodon excisoides (5 kg) were
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Journal of Asian Natural Products Research macerated in 95% ethanol for 3 days at room temperature for three times. All extracts were pooled together and concentrated by rotary evaporator under reduced pressure at 558C to yield a crude extract (450 g), which was suspended in H2O and then successively partitioned with petroleum ether, EtOAc, and n-BuOH. The EtOAc extract (130 g) was chromatographed on a silica gel column, eluted with a gradient solvent system (CHCl3: Me2CO, 100:0, 20:1, 5:1, 2:1, 1:1, 0:100) to afford six fractions (Fr. 1 –6). Fr. 2 (10.22 g) was decolorized on an MCI gel column (MeOH:H2O, 90:10) and then chromatographed on a silica gel column (CHCl3:MeOH, 60:1 – 30:1) to afford four subfractions (Fr. 2.1 –2.4). Compounds 1 (1.7 mg), 3 (7.5 mg), and 8 (3.6 mg) were isolated from Fr. 2.4 (1.14 g) by silica gel column chromatography (petroleum ether: Me2CO, 30:1 –5:1). Fr. 4 (16.53 g) was decolorized on an MCI gel CHP20P column (MeOH:H2O, 90:10) and then chromatographed on a silica gel column (CHCl3:MeOH, 40:1 – 10:1) to afford four subfractions (Fr. 4.1– 4.4). Fr. 4.3 (0.96 g) was chromatographed on a silica gel column (CHCl3:Me2CO, 10:1 – 2:1) to afford four subfractions (Fr. 4.3.1– 4.3.4). Fr. 4.3.2 (95 mg) was further purified by semi-preparative HPLC (MeOH:H 2O, 75:25, 2.0 ml/min) to obtain compounds 2 (2.1 mg) and 9 (8.2 mg). Fr. 5 (14.76 g) was chromatographed on a silica gel column (CHCl3:MeOH, 30:1 – 2:1) to afford five fractions (Fr. 5.1 –5.5). Fr. 5.1 (2.36 g) was chromatographed on a silica gel column (CHCl3:Me2CO, 5:1 –1:1) to afford five subfractions (Fr. 5.1.1– 5.1.5). Compound 4 (3.6 mg) was isolated from Fr. 5.1.1 (0.26 g) by silica gel column chromatography (CHCl3:MeOH, 50:1). Fr. 5.3 (1.88 g) was chromatographed on a silica gel column (CHCl3:Me 2CO, 5:1 – 1:1) to afford four subfractions (Fr. 5.3.1 –5.3.4). Fr. 5.3.2 (0.29 g) was chromatographed on a silica gel column (CHCl3:MeOH, 50:1 –30:1) to afford two
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subfractions (Fr. 5.3.2.1 – 5.3.2.2). Fr. 5.3.2.1 (62 mg) was further purified by semi-preparative HPLC (MeOH:H 2O, 70:30, 2.0 ml/min) to obtain compounds 5 (3.3 mg), 6 (1.0 mg), and 7 (2.5 mg). 3.3.1 2a,3b-Dihydroxy-11a,12a-epoxyurs-28,13b-olide (1) White amorphous powder; m.p. 301 – 3028C; ½a25 D þ 23:74 (c 0.30, MeOH); IR (KBr) nmax (cm21): 3355, 2940, 2855, 1765, 1031; 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS (positive-ion mode): m/z 509.3230 [M þ Na]þ (calcd for C30H46O5Na, 509.3243). 3.3.2 2a,3b,24-Trihydroxy-11a,12aepoxy-urs-28,13b-olide (2) White amorphous powder; m.p. 316 – 3188C; ½a25 D þ 9:00 (c 0.20, MeOH); IR (KBr) nmax (cm21): 3421, 2925, 2858, 1769, 1685, 1056; 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS (positive-ion mode): m/z 525.3194 [M þ Na] þ (calcd for C30 H46O 6Na, 525.3192). 3.3.3 2a,3a,24-Trihydroxy-11,20(30)dien-urs-28,13b-olide (6) White amorphous powder; m.p. 294 – 2968C; ½a25 D þ 1:22 (c 1.48, MeOH); IR (KBr) nmax (cm21): 3421, 2930, 2865, 1742, 1640, 1028; 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS (positive-ion mode): m/z 485.3268 [M þ H]þ (calcd for C30H45O5, 485.3267). Acknowledgments This work was financially supported by the opening fund of Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Huazhong University of Science and Technology. We are grateful to pharmacist C.R. Wang (Enshi Food and Drug Administration, Hubei Province, China) for medicinal material collection.
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References [1] H.D. Sun, S.X. Huang, and Q.B. Han, Nat. Prod. Rep. 23, 673 (2006). [2] H.D. Sun, Y.L. Xu, and B. Jiang, Diterpenoids from Isodon Species (Science Press, Beijing, 2001), pp. 93 –105. [3] H.D. Sun, Y.L. Xu, and B. Jiang, Diterpenoids from Isodon Species (Science Press, Beijing, 2001), p. 1. [4] B. Jiang, Q.B. Han, W. Xiang, Z.W. Lin, and H.D. Sun, Acta Botanica Yunnanica 24, 663 (2002). [5] F. Wang, X.M. Li, and J.K. Liu, Chem. Pharm. Bull. 57, 525 (2009). [6] Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinice Edita, Flora Reipublicae Popularis Sinicae (Science Press, Beijing, 1977), Vol. 66, p. 514. [7] L. Ding, H. Wang, G.A. Liu, and D.J. Yang, J. Chem. Res. 2004, 697 (2004). [8] J.X. Zhang, Y.X. Wang, Z.A. He, F.L. Yan, and H.D. Sun, Chin. Chem. Lett. 20, 201 (2009).
[9] Y.X. Wang, L.L. Zhu, Z.A. He, and J.X. Zhang, Chin. Chem. Lett. 21, 610 (2010). [10] W. Zhao, J.X. Pu, X. Du, Y.L. Wu, Y. Zhao, F. He, H.B. Zhang, Y.B. Xue, W.L. Xiao, G.Q. Chen, and H.D. Sun, Arch. Pharm. Res. 34, 2007 (2011). [11] Y. Sashida, K. Ogawa, T. Yamanouchi, H. Tanaka, Y. Shoyama, and I. Nishioka, Phytochemistry 35, 377 (1994). [12] J. Sakakibara and T. Kaiya, Phytochemistry 22, 2547 (1983). [13] H. Kojima, H. Tominaga, S. Sato, and H. Ogura, Phytochemistry 26, 1107 (1987). [14] H. Kojima and H. Ogura, Phytochemistry 25, 729 (1986). [15] Y.H. Xiao, A.L. Zhang, and G.L. Zhang, Nat. Prod. Res. Dev. 19, 978 (2007). [16] S.B. Mahato and A.P. Kundu, Phytochemistry 37, 1517 (1994). [17] A.V. Tkachev, A.Y. Denisov, Y.V. Gatilov, I.Y. Bagryanskaya, S.A. Shevtsov, and T.V. Rybalova, Tetrahedron 50, 11459 (1994).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Three new ursane-type triterpenoids from the aerial parts of Isodon excisoides a
a
a
a
a
Ke Jiao , Huo-Yun Li , Peng Zhang , Hui-Fang Pi , Han-Li Ruan & a
Ji-Zhou Wu a
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Tongji School of Pharmaceutical Sciences, Huazhong University of Science and Technology, Wuhan, 430030, China Published online: 24 Oct 2013.
To cite this article: Ke Jiao, Huo-Yun Li, Peng Zhang, Hui-Fang Pi, Han-Li Ruan & Ji-Zhou Wu (2013) Three new ursane-type triterpenoids from the aerial parts of Isodon excisoides, Journal of Asian Natural Products Research, 15:9, 962-968, DOI: 10.1080/10286020.2013.837458 To link to this article: http://dx.doi.org/10.1080/10286020.2013.837458
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2013 Vol. 15, No. 9, 962–968, http://dx.doi.org/10.1080/10286020.2013.837458
Three new ursane-type triterpenoids from the aerial parts of Isodon excisoides Ke Jiao, Huo-Yun Li, Peng Zhang*, Hui-Fang Pi, Han-Li Ruan and Ji-Zhou Wu Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Tongji School of Pharmaceutical Sciences, Huazhong University of Science and Technology, Wuhan 430030, China
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(Received 14 June 2013; final version received 20 August 2013) Three new ursane-type triterpenoids, 2a,3b-dihydroxy-11a,12a-epoxy-urs-28,13bolide (1), 2a,3b,24-trihydroxy-11a,12a-epoxy-urs-28,13b-olide (2), and 2a,3a,24trihydroxy-11,20(30)-dien-urs-28,13b-olide (6), together with six known ursane-type triterpenoids (3 – 5, 7– 9), were isolated from the EtOAc extract of the aerial parts of Isodon excisoides. Their structures were elucidated on the basis of 1D NMR and 2D NMR analyses as well as HRMS experiments. Keywords: Isodon excisoides; ursane; triterpenoid
1.
Introduction
The genus Isodon (formally Rabdosia) comprising about 150 species of undershrubs, subundershrubs, or perennial herbs is mainly distributed in tropical and subtropical Asia [1]. It is a cosmopolitan and important genus of the Labiatae family, and has attracted considerable attention as a fertile source of ent-kaurane diterpenoids that have been verified to be the main bioactive constituents [2]. Besides the entkauranoids, triterpenoids were found to be another type of rich constituents in the Isodon plants, but previous investigations rarely led to the isolation of new triterpenoids [3 – 5]. Isodon excisoides (Sun ex C.H. Hu) C. Y. Wu et H.W. Li is a perennial herb mainly distributed in Hubei, Sichuan, and Yunnan at the altitudes of 700 – 3000 m [6]. Previous research on the chemical constituents on Isodon excisoides had led to the isolation of five ent-kaurane diterpenoids [7 – 9]. However, in our recent phytochemical investigation of this plant grown in a different ecological environment, three new ursane-type triterpenoids, 2a,3b-
dihydroxy-11a,12a-epoxy-urs-28,13bolide (1), 2a,3b,24-trihydroxy-11a,12aepoxy-urs-28,13b-olide (2), and 2a,3a,24trihydroxy-11,20(30)-dien-urs-28,13bolide (6), together with six known ursanetype triterpenoids isodonadenanthin (3) [4], 2a,3a,24-trihydroxy-11a,12a-epoxy-urs20(30)-en-28,13b-olide (4) [10], 2a,3a, 24-trihydroxy-11-en-urs-28,13b-olide (5) [11], 2a,3a,24-trihydroxy-12(13)-en-urs28-oic acid (7) [12,13], 2a,3a-dihydroxy12(13)-en-urs-28-oic acid (8) [14], and 2a,3b,24-trihydroxy-12(13)-en-urs-28-oic acid (9) [15] (Figure 1), were obtained. The structures of the known compounds were identified by the comparison of their NMR spectral data with those reported in the literature. Herein, we report the isolation and structure elucidation of those new compounds. 2.
Compound 1 was isolated as a white amorphous powder. Its molecular formula was determined to be C30H46O5 based on its HR-ESI-MS data at m/z 509.3230 [M þ Na]þ. The IR spectrum of 1 showed
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
Results and discussion
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Figure 1. The structures of compounds 1 – 9.
absorption bands for hydroxyl group at 3355 cm21 and for g-lactone at 1764 cm21. 1H NMR spectrum (Table 1) exhibited five tertiary methyl signals at dH 1.05 (3H, s, H-25), 1.08 (3H, s, H-24), 1.14 (6H, s, H-26, 27), and 1.24 (3H, s, H-23); two secondary methyl signals at dH 0.88 (3H, d, J ¼ 6.3 Hz, H-30) and 1.31 (3H, d, J ¼ 5.8 Hz, H-29); and four oxygenated methine signals at dH 3.09 (1H, d, J ¼ 3.8 Hz, H-12), 3.29 (1H, dd, J ¼ 3.8, 2.0 Hz, H-11), 3.39 (1H, d, J ¼ 9.3 Hz, H3), and 4.18 (1H, ddd, J ¼ 12.4, 9.3, 4.3 Hz, H-2). The 13C NMR (Table 1) and distortionless enhancement by polarization transfer spectra revealed 30 carbon signals, including seven methyl carbons at dC 16.4 (C-27), 17.1 (C-24), 17.5 (C-29), 18.5 (C-25), 19.4 (C-30), 20.5 (C-26), and 28.8 (C-23); four oxygenated methine carbons at dC 54.8 (C-11), 56.4 (C-12), 68.1 (C-2), and 83.7 (C-3); as well as one carbonyl
group at dC 178.7 (C-28). The remaining carbon signals were observed and assigned to seven methylene, five methine, and six quaternary carbons. The quaternary carbon signals at dC 88.9 (C-13) and 178.7 (C-28), along with the IR absorption at 1764 cm21, indicated that 1 possessed a glactone ring between C-13 and C-28. These NMR spectral data indicated that compound 1 was an ursane-type triterpenoid [16]. Comparing the 13C NMR data (Table 1) of 1 with those of 3b-hydroxy11a,12a-epoxy-urs-28,13b-olide [17] showed that 1 had the same skeleton. The difference was that the C-2 (dC 26.7) in 3b-hydroxy-11a,12a-epoxy-urs-28,13bolide shifted upfield to dC 68.1 corresponding to an additional proton signal at dH 4.18 (1H, ddd, J ¼ 12.4, 9.3, 4.3 Hz, H-2) in compound 1, which suggested that the additional hydroxy was at C-2 in compound 1. The 1H – 1H COSY correlation of
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Table 1.
1
H and 13C NMR spectral data for compounds 1 (C5D5N), 2 (CD3OD), and 6 (C5D5N).
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1
2
No.
dH, J (Hz)
dC
1a 1b 2
2.57 dd (12.4, 4.3) 1.53 m 4.18 ddd (12.4, 9.3, 4.3) 3.39 d (9.3)
47.4
3 4 5 6a 6b 7a 7b 8 9 10 11 12 13 14 15a 15b 16a 16b 17 18 19 20 21a 21b 22a 22b 23 24a 24b 25 26 27 28 29 30a 30b
0.96 m 1.52 m 1.52 m 1.23 m 1.04 m 1.77 m 3.29 dd (3.8, 2.0) 3.09 d (3.8) 1.69 m 0.98 m 2.10 td (13.2, 5.7) 1.29 m 1.81 m 1.77 m 0.83 m 1.40 m 1.30 m 1.85 m 1.57 m 1.24 s 1.08 s 1.05 s 1.14 s 1.14 s 1.31 d (5.8) 0.88 d (6.3)
68.1 83.7 39.8 54.9 18.0 31.5 41.5 51.9 37.7 54.8 56.4 88.9 41.7 27.0 23.0 45.2 60.3 37.4 40.3 30.6 31.8 28.8 17.1 18.5 20.5 16.4 178.7 17.5 19.4
dH, J (Hz) 2.26 m 1.12 m 3.87 ddd (9.6, 9.6, 4.6) 3.10 d (9.6) 0.96 m 1.67 m 1.60 m 1.35 m 1.20 m
3.25 dd (3.6, 2.0) 2.97 d (3.6) 1.63 m 1.15 m 2.27 m 1.31 m
46.1 67.7 84.3 43.0 54.8 17.8
41.3 51.5 37.1 54.5 55.9 89.5 41.1 26.4 22.4
1.85 m 1.81 m 0.97 m 1.59 m 1.38 m 1.75 m 1.51 m 1.23 s 4.01 d (11.2) 3.46 d (11.2) 1.13 s 1.06 s 1.16 s
H-1/H-2/H-3 and the HMBC (Figure 2) correlations of H-2 with C-1 and C-3 further confirmed this elucidation. In the NOESY spectrum, H-3 correlated with Ha-1, H-5, and H-23 (Figure 3). Thus, the relative stereochemistry of the 3-OH group was elucidated as b-orientation. The NOE correlations of H-2 with H-24 and H-25, H-11 with H-25, and H-12 with
dC
31.2
1.64 m
1.22 d (5.0) 1.01 d (5.2)
6
45.2 60.2 37.2 40.0 30.1 31.1 22.0 64.3 17.6 19.4 15.4 180.1 16.4 18.4
dH, J (Hz) 2.20 m 1.85 m 4.52 ddd (10.9, 3.0, 2.0) 4.63 d (2.0) 1.81 m 1.83 m 1.83 m 1.39 m 1.12 m 2.24 m 5.56 dd (10.3, 2.9) 6.13 d (10.3) 1.60 m 1.09 m 2.25 m 1.35 m 1.62 m 2.64 q (6.2) 2.18 m 1.87 m 1.92 m 1.57 m 1.67 s 4.08 d (10.7) 3.86 d (10.7) 1.01 s 1.16 s 1.03 s 1.04 d (6.4) 4.78 s 4.66 s
dC 42.7 65.7 74.0 44.9 48.4 18.0 31.6 42.0 53.3 37.5 128.7 133.5 88.7 42.0 25.4 22.7 44.9 60.9 36.2 151.5 31.6 33.0 23.6 64.4 19.3 19.0 15.5 178.5 16.2 107.4
H-18 implied that both the 2-OH group and the 11-12-epoxy group had aorientations. Therefore, the structure of compound 1 was assigned as 2a,3bdihydroxy-11a,12a-epoxy-urs-28,13bolide (Figure 1). Compound 2 was also obtained as a white amorphous powder. HR-ESI-MS gave one quasi-molecular ion peak at m/z
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Figure 2.
Selected 1H – 1H COSY (
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) and HMBC ( ! ) correlations of compounds 1, 2, and 6.
Figure 3. Key NOESY ( $ ) correlations of compounds 1, 2, and 6.
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525.3194 [M þ Na]þ corresponding to the molecular formula C30H46O6. The 1H and 13 C NMR spectral data of compound 2 were similar to those of 1 (Table 1), except for an additional hydroxyl. In the HMBC spectrum of 2, the proton signals at dH 3.46 and 4.01 (H2-24) showed longrange correlations with C-3, C-4, C-5, and C-23 (Figure 2), suggesting that the hydroxy group was at C-24, which was further confirmed by the NOE correlations of the proton signal at dH 4.01 (H-24) with H-2 and H-25 (Figure 3). Thus, the structure of 2 was assigned as 2a,3b,24-trihydroxy-11a,12a-epoxyurs-28,13b-olide (Figure 1). Compound 6 was obtained as a white amorphous powder. Its HR-ESI-MS revealed a quasi-molecular ion at m/z 485.3268 [M þ H]þ, indicating a molecular formula of C30H44O5. The IR spectrum showed absorption bands for hydroxyl group at 3421 cm21 and for g-lactone at 1742 cm21. The 13C NMR spectral data of 6 (Table 1) were similar to those of compound 5 [11], except for an additional olefinic bond (dH 4.66, 4.78; dC 107.4, 151.5). In the HMBC spectrum of 6, the correlations of H2-30 (dH 4.66, 4.78) with C-19 (dC 36.2), C-20 (dC 151.5), and C-21 (dC 31.6); H3-29 (dH 1.04) with C-20 (dC 151.5); and H-22 (dH 1.92) with C-20 (dC 151.5; Figure 2) indicated that the additional olefinic bond was between C-20 and C-30. In the NOESY spectrum, the a-orientations of 2-OH and 3OH were elucidated by the correlations of H-2 with H-24 and H-25, and H-3 with Hb-1 and H-24 (Figure 3). Therefore, the structure of compound 6 was assigned as 2a,3a,24-trihydroxy-11,20(30)-dien-urs28,13b-olide (Figure 1). 3. 3.1
Experimental General experimental procedures
Melting points were measured using an XT4-100X micro-melting point apparatus (Tech Instrument Corporation, Beijing, China) and are uncorrected. Optical
rotations were obtained on a PerkinElmer 341 polarimeter (Perkin-Elmer, Foster City, CA, USA). IR spectra were detected by a Vertex 70 spectrometer (Bruker Corporation, Karlsruhe, Germany) with KBr disks. The NMR spectra were run on a Bruker UltrashieldTM 400 plus spectrometer (1H NMR, 400 MHz; 13C NMR, 100 MHz; Bruker BioSpin, Fa¨llanden, Switzerland), both with tetramethylsilane as an internal standard. HR-ESI-MS data were recorded on Thermo Scientific LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, CA, USA). Semi-preparative HPLC was carried out on an Agilent 1100 Series apparatus (Agilent Technologies, Santa Clara, CA, USA), equipped with an Alltech evaporative light scattering detector (Alltech, Dearfield, IL, USA). The semi-preparative HPLC column of YMCPack ODS-A SH-343-5 (YMC, Komatsu City, Japan) was used. Column chromatography was carried out on silica gel (200 –300 mesh; Qingdao Haiyang Chemical Co., Ltd, Qingdao, China) and MCI gel CHP20P (75 – 150 mm; Mitsubishi Chemical Corporation, Japan). Fractions were monitored by thin layer chromatography (GF254; Qingdao Haiyang Chemical Co., Ltd, Qingdao, China), and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in ethanol. 3.2 Plant material The aerial parts of Isodon excisoides were collected in Enshi, Hubei Province, China, in October 2009, and identified by Prof. J.Q. Li (Wuhan Botanical Garden, Chinese Academy of Sciences). A voucher specimen (No. ICC-20091013) was deposited at the Faculty of Pharmaceutical Sciences, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China. 3.3 Extraction and isolation The air-dried and coarsely powdered aerial parts of Isodon excisoides (5 kg) were
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Journal of Asian Natural Products Research macerated in 95% ethanol for 3 days at room temperature for three times. All extracts were pooled together and concentrated by rotary evaporator under reduced pressure at 558C to yield a crude extract (450 g), which was suspended in H2O and then successively partitioned with petroleum ether, EtOAc, and n-BuOH. The EtOAc extract (130 g) was chromatographed on a silica gel column, eluted with a gradient solvent system (CHCl3: Me2CO, 100:0, 20:1, 5:1, 2:1, 1:1, 0:100) to afford six fractions (Fr. 1 –6). Fr. 2 (10.22 g) was decolorized on an MCI gel column (MeOH:H2O, 90:10) and then chromatographed on a silica gel column (CHCl3:MeOH, 60:1 – 30:1) to afford four subfractions (Fr. 2.1 –2.4). Compounds 1 (1.7 mg), 3 (7.5 mg), and 8 (3.6 mg) were isolated from Fr. 2.4 (1.14 g) by silica gel column chromatography (petroleum ether: Me2CO, 30:1 –5:1). Fr. 4 (16.53 g) was decolorized on an MCI gel CHP20P column (MeOH:H2O, 90:10) and then chromatographed on a silica gel column (CHCl3:MeOH, 40:1 – 10:1) to afford four subfractions (Fr. 4.1– 4.4). Fr. 4.3 (0.96 g) was chromatographed on a silica gel column (CHCl3:Me2CO, 10:1 – 2:1) to afford four subfractions (Fr. 4.3.1– 4.3.4). Fr. 4.3.2 (95 mg) was further purified by semi-preparative HPLC (MeOH:H 2O, 75:25, 2.0 ml/min) to obtain compounds 2 (2.1 mg) and 9 (8.2 mg). Fr. 5 (14.76 g) was chromatographed on a silica gel column (CHCl3:MeOH, 30:1 – 2:1) to afford five fractions (Fr. 5.1 –5.5). Fr. 5.1 (2.36 g) was chromatographed on a silica gel column (CHCl3:Me2CO, 5:1 –1:1) to afford five subfractions (Fr. 5.1.1– 5.1.5). Compound 4 (3.6 mg) was isolated from Fr. 5.1.1 (0.26 g) by silica gel column chromatography (CHCl3:MeOH, 50:1). Fr. 5.3 (1.88 g) was chromatographed on a silica gel column (CHCl3:Me 2CO, 5:1 – 1:1) to afford four subfractions (Fr. 5.3.1 –5.3.4). Fr. 5.3.2 (0.29 g) was chromatographed on a silica gel column (CHCl3:MeOH, 50:1 –30:1) to afford two
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subfractions (Fr. 5.3.2.1 – 5.3.2.2). Fr. 5.3.2.1 (62 mg) was further purified by semi-preparative HPLC (MeOH:H 2O, 70:30, 2.0 ml/min) to obtain compounds 5 (3.3 mg), 6 (1.0 mg), and 7 (2.5 mg). 3.3.1 2a,3b-Dihydroxy-11a,12a-epoxyurs-28,13b-olide (1) White amorphous powder; m.p. 301 – 3028C; ½a25 D þ 23:74 (c 0.30, MeOH); IR (KBr) nmax (cm21): 3355, 2940, 2855, 1765, 1031; 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS (positive-ion mode): m/z 509.3230 [M þ Na]þ (calcd for C30H46O5Na, 509.3243). 3.3.2 2a,3b,24-Trihydroxy-11a,12aepoxy-urs-28,13b-olide (2) White amorphous powder; m.p. 316 – 3188C; ½a25 D þ 9:00 (c 0.20, MeOH); IR (KBr) nmax (cm21): 3421, 2925, 2858, 1769, 1685, 1056; 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS (positive-ion mode): m/z 525.3194 [M þ Na] þ (calcd for C30 H46O 6Na, 525.3192). 3.3.3 2a,3a,24-Trihydroxy-11,20(30)dien-urs-28,13b-olide (6) White amorphous powder; m.p. 294 – 2968C; ½a25 D þ 1:22 (c 1.48, MeOH); IR (KBr) nmax (cm21): 3421, 2930, 2865, 1742, 1640, 1028; 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS (positive-ion mode): m/z 485.3268 [M þ H]þ (calcd for C30H45O5, 485.3267). Acknowledgments This work was financially supported by the opening fund of Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Huazhong University of Science and Technology. We are grateful to pharmacist C.R. Wang (Enshi Food and Drug Administration, Hubei Province, China) for medicinal material collection.
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