Chemical shift assignments of two oleanane triterpenes from Euonymus hederaceus

Chemical shift assignments of two oleanane triterpenes from Euonymus hederaceus first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:panyuanjiang@zju.edu.cn †E-mail:panyuanjiang@zju.edu.cn panyuanjiang@zju.edu.cn Department of Chemistry, Zhejiang University, Hangzhou 310027, China Department of Chemistry, Zhejiang University, Hangzhou 310027, China 1 H-NMR and 13 C-NMR assignments of 12-oleanene-3,11-dione (compound 1) were completely described for the first time through conventional 1D NMR and 2D shift-correlated NMR experiments using 1 H- 1 HCOSY, HMQC, HMBC techniques. Based on its NMR data, the assignments of 28-hydroxyolean-12-ene-3,11-dione (compound 2) were partially revised. Euonymus hederaceus 12-oleanene-3,11-dione 28-hydroxyolean-12-ene-3,11-dione 1D-NMR 2D-NMR Assignment INTRODUCTION 1 Euonymus hederaceus champ.ex Benth (Celastraceae) has been used as a traditional medical plant (Chang et al., 1996 1996 ). Two compounds, 12-oleanene-3,11-dione and 28-hydroxyolean-12-ene-3,11-dione (Fig. 1 1 ) were isolated by its phytochemical investigation. The complete 1 H and 13 C chemical shift assignments of 12-oleanene-3,11-dione are described here for the first time with 1D and 2D NMR, correcting some data in Yuan et al.( 1994 1994 ). The 13 C assignments of 28-hydroxyolean-12-ene-3,11-dione were partially revised by Shirota et al.( 1996 1996 ). Compound 1: R=CH 3 ; Compound 2: R=CH 2 OH MATERIALS AND METHODS 1 The barks and stems of Euonymus hederaceus champ.ex Benth, collected in October 2003 in Suicang County, Zhejiang Province, were identified by Dr. WU Bin. After crushing them, the obtained powder was extracted thrice by methanol, once a week to give an extract (500 g) after removing methanol. The extract was partitioned between petroleum ether and water. The petroleum ether soluble fraction (33 g) was applied to a silica gel column using petroleum ether and ethyl acetate as a gradient eluent system. Compound 1 and impure compound 2 were obtained when the eluent volume ratios of petroleum ether to ethyl acetate were 10:1 and 6:1, respectively. Compound 2 was further separated and purified by preparative HPLC. 1 H and 13 C NMR spectra were measured on Bruker AVANCE DMX 500 NMR, using CDCl 3 as solvent and TMS as internal reference. RESULT AND DISCUSSION 1 Compound 1 was isolated as white powder of m.p. 241~245 °C, and gave responded positively to the Lieberman-Buchard test for triterpenes. The molecular formula was deduced as C 30 H 46 O 2 by ESI-MS: 437.2 (C 30 H 46 O 2 − , [M-H] − ). The 1 H-NMR data (Table 1 1 ) showed singlet signals for eight angular methyl groups. The 13 C-NMR and DEPT spectra showed 30 signals for eight methyl, nine methylene, four methine and nine quaternary carbons including a keto carbonyl and an α, β-unsaturated keto carbonyl. HMQC experiments revealed 1 H- 13 C direct correlation between the protons of eight methyl groups and corresponding carbons, and in the observe, olean-type triterpene skeleton was deduced (Mahato and Kundu, 1994 1994 ). The resonance assignments for eight methyl were at δ H 1.07, 1.10, 1.27, 1.18, 1.37, 0.85, 0.91, 0.89 to H-23, 24, 25, 26, 27, 28, 29, 30 respectively. The protons of two methyl groups ( δ H 1.10, δ C 26.5 and δ H 1.07, δ C 21.6), and two methylene groups ( δ H 2.95, δ C 40.0 and δ H 2.60, δ C 34.5) had 1 H- 13 C long-range correlation with the carbon at δ C 217.4, establishing the carbonyl to be C-3. The hydrogen signal appearing at δ H 5.62 (s), corresponding to the only present olefinic hydrogen atom correlates to the carbon atom at δ C 128.3 in the HMQC spectrum. The olefinic C-13 atom was quaternary and appeared at 171.3, and a keto C-atom appearing at 199.7 suggested that compound 1 had an olean-12-ene-11-ketone skeleton (Shirota et al., 1996 1996 ). These assumptions were confirmed by the HMQC and HMBC spectra, for the H-12 olefinic proton ( δ H 5.62) indicated 1 H- 13 C long range correlations with the methine carbons C-9 ( δ C 61.2), C-14 ( δ C 43.7); the H-9 proton ( δ H 2.44) with C-11 ( δ C 199.7); the H-18 proton ( δ H 2.14) with C-13 ( δ C 29.02) in the HMBC spectrum (Table 1 1 ). Therefore, compound 1 was considered to be 12-oleanene-3,11-dione. It was isolated in 1970 (Govindachari et al., 1970 1970 ). The 13 C-NMR assignments for this compound have not reported until 1994 (Yuan et al., 1994 1994 ). In this study, the 1 H- and 13 C-NMR assignments for compound 1 were carried out on the basis of DEPT, HMQC, 1 H- 1 H COSY (homonuclear correlation spectroscopy) and HMBC experiments. Our result revises some assignments in Yuan et al.( 1994 1994 ). The assignments are different from those in Shirota et al.( 1996 1996 ) The assignments are different from those in Yuan et al.( 1994 1994 ) Multiplicities by DEPT experiments in parentheses s: quaternary; d: CH; t: CH 2 ; q: methyl C atom; m: Multiplicities by 1 H-NMR experiment; br: broad form Multiplicities by 1 H-NMR experiment 2 Atom number 3 Compound 1 Compound 2 δ C δ H HMBC (carbon) δ C C-1 240.0t b b 2.95m, 1.41m C-9, C-25 040.0t C-2 234.5t e e 2.60m, 2.35m / 234.2t C-3 217.4s e e / C-1, C-2, C-23, C-24 217.4s C-4 248.0s e e / C-2, C-23 048.0s C-5 255.6d e e 1.28 C-7, C-25, C-24 055.7d C-6 219.0t e e 1.54, 1.03 / 19.1t C-7 232.3t e e 1.64, 1.45 C-6, C-26 32.3t C-8 245.5s e e / C-6, C-27 45.5s C-9 261.2d e e 2.44 1H s C-12, C-25, C-26 61.3d C-10 236.9s e e / C-6, C-9, C-25 36.9s C-11 199.7s e e / C-9 199.6s C-12 128.2d e e 5.62 1H s C-18 128.4d C-13 171.3s e e / C-18 170.2s C-14 243.7s e e / C-9, C-26, C-12 43.8s C-15 226.7t e e 1.18, 1.81br C-27 26.2t C-16 226.6t e e 0.98, 2.09 C-22, C-28 21.6t a a C-17 232.6s e e / C-15, C-21 37.0s C-18 247.9d e e 2.14m C-12, C-28 43.0d C-19 245.5t e e 1.64, 1.10 C-30, C-29, C-21 45.3t C-20 231.3s e e / C-30 31.3s C-21 234.7t e 1.37, 1.18 C-22, C-30 34.1t C-22 236.7t e e 1.45, 1.31 C-28 30.9t a a C-23 21.6q e e 1.07 3H s C-24 21.7q C-24 26.7q e e 1.10 3H s C-23 26.5q C-25 215.9q e e 1.27 3H s C-9 15.9q C-26 218.9q e e 1.18 3H s C-9 18.7q C-27 223.6q e e 1.37 3H s C-15 23.6q C-28 229.0q e e 0.85 3H s C-16, C-22 69.9q C-29 23.3q e e 0.91 3H s C-19, C-30 23.8q C-30 33.8q e e 0.89 3H s C-21, C-29 33.3q Compound 2 has the molecular formula C 30 H 46 O 3 and similar chemical shifts with the compound 1, and was different from the compound 1 in that one tertiary methyl group signal at δ C 29.02 disappeared and one methylene connecting to hydroxyl appeared at δ C 69.9. We determined the structure of compound 2 to be 28-hydroxyolean-12-ene-3,11-dione by comparing the NMR data to data in Shirota et al.( 1996 1996 ). The γ-effect was supposed to result in C-16, C-18, C-22 in the upfield shifts of 0~9×10 −6 (Mahato and Kundu, 1994 1994 ) when the hydroxyl group replaced the 28-methyl group in the carbon assignments of compound 1, so that, the assignments of this compound in Shirota et al.( 1996 1996 ) were partially revised (Table 1 1 ). Researching the 13 C-NMR rule of the triterpenoids is helpful for deducing structures of unknown analogs; the complete assignments of compound 1 and assignment corrections for compound 2 to some extent offer scientific material for future research. References ZF Chang , GP Lu , J Wei , FX Song , FW Liu . The performance settlement of of traditional officinal Euonymus plants (xu wan) . Chinese Journal of Information on Traditional Chinese Medicine , 1996 . 3 ( 3 ): 24 - 26 . (in Chinese) . 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Studies on triterpenoid constituents isolated from the roots of sabia schumamniana 10.1631/jzus.2005.B0719 Project (Nos. 20375036 and 20472073) supported by the National Natural Science Foundation of China 2004-11-24 2005-04-23 2005-07-29 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 6B