Chemical shift assignments of two oleanane triterpenes from Euonymus hederaceus

Abstract

¹H-NMR and ¹³C-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 ¹H-¹HCOSY, HMQC, HMBC techniques. Based on its NMR data, the assignments of 28-hydroxyolean-12-ene-3,11-dione (compound 2) were partially revised.

INTRODUCTION

Euonymus hederaceus champ.ex Benth (Celastraceae) has been used as a traditional medical plant (Chang et al., 1996). Two compounds, 12-oleanene-3,11-dione and 28-hydroxyolean-12-ene-3,11-dione (Fig.1) were isolated by its phytochemical investigation. The complete ¹H and ¹³C 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). The ¹³C assignments of 28-hydroxyolean-12-ene-3,11-dione were partially revised by Shirota et al.(1996).

Fig. 1.

The structure of compounds 1 and 2

Compound 1: R=CH₃; Compound 2: R=CH₂OH

MATERIALS AND METHODS

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.

¹H and ¹³C NMR spectra were measured on Bruker AVANCE DMX 500 NMR, using CDCl₃ as solvent and TMS as internal reference.

RESULT AND DISCUSSION

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 C₃₀H₄₆O₂ by ESI-MS: 437.2 (C₃₀H₄₆O₂ ⁻, [M-H]⁻). The ¹H-NMR data (Table 1) showed singlet signals for eight angular methyl groups. The ¹³C-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 ¹H-¹³C 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). 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 ¹H-¹³C 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). These assumptions were confirmed by the HMQC and HMBC spectra, for the H-12 olefinic proton (δ _H 5.62) indicated ¹H-¹³C 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). Therefore, compound 1 was considered to be 12-oleanene-3,11-dione. It was isolated in 1970 (Govindachari et al., 1970). The ¹³C-NMR assignments for this compound have not reported until 1994 (Yuan et al., 1994). In this study, the ¹H- and ¹³C-NMR assignments for compound 1 were carried out on the basis of DEPT, HMQC, ¹H-¹H COSY (homonuclear correlation spectroscopy) and HMBC experiments. Our result revises some assignments in Yuan et al.(1994).

Table 1.

¹H-NMR, ¹³C-NMR, HMQC, HMBC for compound 1 and compound 2

Atom number	Compound 1			Compound 2
	δC	δH	HMBC (carbon)	δC
C-1	240.0tb	2.95m, 1.41m	C-9, C-25	040.0t
C-2	234.5te	2.60m, 2.35m	/	234.2t
C-3	217.4se	/	C-1, C-2, C-23, C-24	217.4s
C-4	248.0se	/	C-2, C-23	048.0s
C-5	255.6de	1.28	C-7, C-25, C-24	055.7d
C-6	219.0te	1.54, 1.03	/	19.1t
C-7	232.3te	1.64, 1.45	C-6, C-26	32.3t
C-8	245.5se	/	C-6, C-27	45.5s
C-9	261.2de	2.44 1H s	C-12, C-25, C-26	61.3d
C-10	236.9se	/	C-6, C-9, C-25	36.9s
C-11	199.7se	/	C-9	199.6s
C-12	128.2de	5.62 1H s	C-18	128.4d
C-13	171.3se	/	C-18	170.2s
C-14	243.7se	/	C-9, C-26, C-12	43.8s
C-15	226.7te	1.18, 1.81br	C-27	26.2t
C-16	226.6te	0.98, 2.09	C-22, C-28	21.6ta
C-17	232.6se	/	C-15, C-21	37.0s
C-18	247.9de	2.14m	C-12, C-28	43.0d
C-19	245.5te	1.64, 1.10	C-30, C-29, C-21	45.3t
C-20	231.3se	/	C-30	31.3s
C-21	234.7te	1.37, 1.18	C-22, C-30	34.1t
C-22	236.7te	1.45, 1.31	C-28	30.9ta
C-23	21.6qe	1.07 3H s	C-24	21.7q
C-24	26.7qe	1.10 3H s	C-23	26.5q
C-25	215.9qe	1.27 3H s	C-9	15.9q
C-26	218.9qe	1.18 3H s	C-9	18.7q
C-27	223.6qe	1.37 3H s	C-15	23.6q
C-28	229.0qe	0.85 3H s	C-16, C-22	69.9q
C-29	23.3qe	0.91 3H s	C-19, C-30	23.8q
C-30	33.8qe	0.89 3H s	C-21, C-29	33.3q

^aThe assignments are different from those in Shirota et al.(1996)

^eThe assignments are different from those in Yuan et al.(1994)

^bMultiplicities by DEPT experiments in parentheses

s: quaternary; d: CH; t: CH₂; q: methyl C atom; m: Multiplicities by ¹H-NMR experiment; br: broad form Multiplicities by ¹H-NMR experiment

Compound 2 has the molecular formula C₃₀H₄₆O₃ 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). 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) 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) were partially revised (Table 1).

Researching the ¹³C-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.