New 18(4→3)-Abeo-Abietanoids from Tripterygium wilfordii

Abstract

Three 18(4→3)-abeo-abietanoids, a new natural product and two new compounds, named tripordolides A–C (1–3), were isolated from the leaves of Tripterygium wilfordii. Their structures were elucidated on the basis of their spectroscopic analysis, and the absolute configuration of compounds was confirmed by CD and X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation. Compounds 1 and 3 showed moderate inhibitory activities against NO production in lipopolysaccharide-induced (LPS) RAW 264.7 macrophages in vitro.

1. Introduction

Tripterygium wilfordii, also called “leigongteng”, is a member of the Celastracrae family [1], and was commonly used in Traditional Chinese Medicines to cure IgA nephropathy cancer, systemic lupus erythematosus, ankylosing spondylitis, psoriasis, and idiopathic diseases [2,3,4]. Earlier chemical studies on T. wilfordii have led to the isolation of alkaloids [5,6,7], diterpenes [8], triterpenes [9], sesquiterpenes [10,11,12], and lignans [13], which showed a wide range of biological activity, including anti-fertility, anti-inflammatory, immunosuppressive, anti-HIV, and anti-tumor [14]. In order to explore structurally and biologically interesting natural products from T. wilfordii, three new 18(4→3)-abeo-abietanoids (Figure 1), containing a new natural product and two new compounds, named tripordolides A–C (1–3), were isolated. Herein, we describe the isolation, structural elucidation, and activities of these compounds.

2. Results and Discussion

Tripordolide A (1) was obtained as a colorless needle, and gave a positive reaction in the kedde reagent test on the TLC, which suggested the structure of compound 1 contained the lactone ring. Its molecular formula of C₂₀H₂₆O₇ was determined on the basis of HRESIMS at m/z 379.17532 [M + H]⁺ (calculated for C₂₀H₂₇O₇: 379.17513), indicating eight degrees of unsaturation. The ¹H NMR spectrum data of 1 (

Table 1. ¹H and ¹³C NMR data of compounds 1–3.

NO.	1		2		3
	δHa (mult., J/Hz)	δCb	δHc (mult., J/Hz)	δCd	δHc (mult., J/Hz)	δCd
1	1.59, m; 1.89, m	27.5	7.22, d (8.0)	124.6	1.34, m; 2.18, m	29.8
2	2.03, m; 2.15, m	17.1	7.70, d (8.0)	123.1	2.12, m; 2.25, m	17.4
3		122.8		124.4		122.8
4		164.8		146.5		164.0
5	2.49, m	35.1		128.7	2.54, m	40.8
6	1.64, m; 1.53, m	24.6	2.50, m; 2.50, m	25.0	1.68, m; 2.06, m	30.2
7	3.96, d (2.8)	74.3	3.62, t (1.9)	58.3	4.22, m	70.6
8		75.9		60.4		131.7
9		76.0		60.7		137.8
10		40.2		138.5		35.6
11	6.12, d (overlap)	134.0	3.68, d (5.0)	66.7	4.45, br d (8.0)	62.0
12	6.10, d (overlap)	131.1	4.10, m	69.7	3.34, d (overlap)	55.9
13		82.7		74.9		61.9
14	3.99, d (6.0)	77.6	3.58, dd (7.8, 5.2)	74.8	4.47, d (4.4)	67.5
15		70.5		73.6		69.6
16	1.12, s	24.3	1.23, s	25.2	1.27, s	27.4
17	1.12, s	27.4	1.41, s	26.8	1.26, s	26.1
18		173.5		170.4		173.5
19	4.77, m	70.4	5.42, m	68.9	4.82, m	70.3
20	1.00, s	13.1			1.05, s	21.5
7-OH					5.34, d (5.4)
8-OH	4.86, s
9-OH	4.03, s
11-OH					5.02, d (8.0)
14-OH	4.90, d(6.0 )				4.86, d (4.4)
15-OH	4.15, s				4.17, s

^a In DMSO-d6 (500 MHz); ^b In DMSO-d6 (125 MHz). ^c In DMSO-d6 (600 MHz); ^d In DMSO-d6 (150 MHz).

) exhibited signals of three methyl groups [δ_H 1.12 (6H, s, H₃-16 and H₃-17)], two aromatic methines [δ_H 6.10 (1H, d, overlap) and 6.12 (1H, d, overlap)], an oxygenated methylene [δ_H 4.77(2H, m)], and two oxygenated methines [δ_H 3.96(1H, d, J = 2.8 Hz, H-7; 3.99 (1H, d, J = 6.0 Hz)]. The ¹³C NMR and DEPT spectra demonstrated 20 carbons resonances, which contained a carbonyl, seven oxygenated carbons (four quaternary carbons, a methylene, and two methines), two aromatic quaternary carbons, and two aromatic methenes. According to this information, the skeleton of 1 was recognized to be an 18(4→3)-abeo-abietane [15], which contained five rings and four hydroxy groups to be satisfied with its indices of hydrogen deficiency. Its planar structure and ¹H, ¹³C NMR assignments could be confirmed with an analysis of the two-dimensional NMR (Figure 2). A ring of α, β-unsaturated-γ-lactone was confirmed based on the HMBC correlation from H₂-19 [δ_H 4.77 (2H, m)] to C-18(δ_C 173.5) and C-3(δ_C 122.8). Structure of rings A and B could be determined by the ¹H, ¹H COSY correlations of H-1/H-2 and H-5/H-6/H-7, and the HMBC correlation of H₃-20 [δ_H 1.00 (3H, s)]/C-1 (δ_C 27.5), C-5 (δ_C 35.1), C-9 (δ_C 76.0), and C-10 (δ_C 40.2). The HMBC Correlations from H-12 to C-9, C-13 (δ_C 82.7), C-14 (δ_C 77.6), and C-15 (δ_C 70.5); from H-14 [δ_H 3.99 (1H, d, J = 6.0 Hz)] to C-8 (δ_C 75.9) and C-13 corroborated the ring C. The four hydroxy groups were assigned to C-8, C-9, C-14, and C-15, indicated by the HMBC correlations of 8-OH [δ_H 4.86 (1H, s)]/C-7 (δ_C 74.3) and C-8, 9-OH [δ_H 4.03 (1H, s)]/C-10, C-11 (δ_C 134.0), and C-12 (δ_C 131.1), 15-OH [δ_H 4.15 (1H, s)]/C-13, C-15, C-16 (δ_C 24.3), and C-17 (δ_C 27.4); the ¹H, ¹H COSY correlation from H-14 to 14-OH [δ_H 4.90 (1H, d, J = 6.0 Hz)]. Finally, the fifth ring was an oxide bridge between C-7 and C-13 (furan ring) to meet with the HRESIMS data.

NOESY analysis was used to deduce the relative configuration of 1 (Figure 3). Orientations were confirmed by the NOE correlations of H₃-20/8-OH, 9-OH and H-6β; of H-6β /H-7/H-14. Finally, the proposed structure of 1 was confirmed by an X-ray crystallography analysis using anomalous scattering of Cu Kα radiation (Figure 4). Accordingly, the absolute configuration of 5S, 7R, 8S, 9R, 10S, 13S, and 14S was established based on the value of the Flack absolute structure parameter—0.02(11).

Tripordolide B (2) was obtained as a colorless oil and showed a [M + Na]⁺ ion peak at m/z 399.1052 (calculated for 399.1050) in the HRESIMS, which corresponded to the molecular formula C₁₉H₂₀O₈. Some obvious signals, a ring of α, β-unsaturated-γ-lactone [δ_H 5.42 (2H, m, H-17); δ_C 170.4 (C-18), 68.9 (C-19), 124.4 (C-3), and 146.5 (C-4)] and an oxygen isopropyl group [δ_H 1.23, 1.41 (each 3H, s, H₃-16 and H₃-17); δ_C 25.2(C-16), 26.8 (C-17), and 73.6 (C-15)], indicated 2 was also an 18(4→3)-abeo-abietane derivative (Table 1) and similar to a less 20-CH₃ and aromatized nor-abietane, tripterlide F [16]., This was proven by the HMBC correlations of H-1 [δ_H 7.22 (1H, d, J = 8.0 Hz)]/C-4, C-5 (δ_C 128.7) and C-9 (δ_C 60.7); of H-2 [δ_H 7.70 (1H, d, J = 8.0 Hz)]/C-4, C-10 (δ_C 138.5) and C-18 (Figure 2). Some major differences between the two compounds in the chemical shift values of C-12 and C-13 were observed, which could be attributed to the breakage of a trisubstituted epoxide between C-12 (δ_C 69.7) and C-13 (δ_C 74.9). Furthermore, compared to the tripterlide F, the oxygen isopropyl group of 2 also lead to the lower field of C-15 (δ_C 73.6), C-16 (δ_C 25.2), and C-17 (δ_C 26.8). The relative configuration was established by the NOESY correlations of H-12/H-1 and of H-7/H-14/H-12 (Figure 5). Thus, 2 was elucidated as shown, named tripordolide B.

Tripordolide C (3) was obtained as a colorless oil and had the molecular formula C₂₀H₂₆O₇, deduced from the [M + Na]⁺ ion peak at m/z 401.1574 (calculated 401.1571). Its ¹H and ¹³C NMR characteristics (Table 1) could be concluded; 3 was also an 18(4→3)-abeo-abietane derivative. Its planar structure has been confirmed by an analysis of a two-dimensional NMR (Figure 2). HMBC correlation of 15-OH [δ_H 4.17 (1H, s)]/C-15 (δ_C 69.6), C-16 (δ_C 27.4), and C-17 (δ_C 26.1); ¹H, ¹H COSY correlations of 7-OH [δ_H 5.34 (1H, d, J = 5.4 Hz)]/H-7 [δ_H 4.22 (1H, m)], 11-OH [δ_H 5.02 (1H, d, J = 8.0 Hz)]/H-11[δ_H 4.45 (1H, br d, J = 8.0 Hz)], and 14-OH [δ_H 4.86 (1H, d, J = 4.4 Hz)]/H-14 [δ_H 4.47 (1H, d, J = 4.4 Hz)] suggested that the four hydroxy groups were located at C-7, C-11, C-14, and C-15. HMBC correlations from H₃-20 [δ_H 1.05 (1H, s)] to C-5 (δ_C 40.8), C-10 (δ_C 35.6), and C-11 (δ_C 62.0), and from H-14 [δ_H 4.47 (1H, d, J = 4.4 Hz)] to C-8 (δ_C 131.7), C-9 (δ_C 137.8), C-12 (δ_C 55.9), and C-13 (δ_C 61.9) indicated a double bond between C-8 and C-9. Moreover, a trisubstituted epoxide between C-12 and C-13 was formed to meet with its indices of hydrogen deficiency, which also led the chemical shift of C-12 (δ_C 55.9) and C-13 (δ_C 61.9) to upfield. The relative configuration was established by the NOESY correlations of H-12/H-11/H-20 and of H-6β/H-7/H-14-OH (Figure 5). Therefore, 3 was elucidated as shown, named tripordolide C.

Three 18(4→3)-abeo-abietanes were evaluated for their inhibitory activities against NO production in lipopolysaccharide-induced (LPS) RAW 264.7 macrophages in vitro. Compounds 1 and 3 inhibited NO production in mouse peritoneal macrophage 48.4 ± 5.7% and 53.5 ± 4.8% at a concentration of 10 μM, while the positive control dexamethasone gave an inhibitory ratio of 63.1 ± 4.6% at an identical concentration.

3. Experimental Section

3.1. General

A Bruker APEX DUO diffractometer with Cu Kα radiation ((Bruker-Biospin, Billerica, MA, USA) was used to collect the X-ray data of compound 1. A JASCO P-2000 polarimeter (JASCO, Easton, MD, USA), JASCO V650 (Thermo Scientific, Waltham, MA, USA), spectrophotometer, and XT-5B micro melting point apparatus (Aihuishi Ltd. Shanghai, China) were used to collect optical rotations, UV spectra, and melting points, respectively. VNS-600 and Bruker-500 (Bruker-Biospin, Billerica, MA, USA) spectrometers were used to collected NMR spectra. The Agilent 1100 series LC/MSD ion trap mass spectrometer (Agilent, Waldbronn, Germany) was used to collect HRESIMS spectra. Preparative HPLC was performed on a Shimadzu LC-6AD instrument with a SPD-20A detector (Shimadzu Corp., Tokyo, Japan), using a YMC-Pack ODS-A column (2 × 25 cm, 5 μm, YMC, Tokyo, Japan). Column chromatography was performed with silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), polyamide (60–100 mesh, Changfeng Chemical Inc., Jiangsu, China), and ODS (50 μm, YMC, Tokyo, Japan).

3.2. Plant Material

The leaves of T. wilfordii were collected in Taining, Fujian, China, in September 2009 and identified by Professor Lin Ma from the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. A voucher specimen (No. 20090034) is deposited at the herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences, and Peking Union Medical College, China.

3.3. Extraction and Isolation

The air-dried and powdered leaves of Tripterygium wilfordii (100 kg) were extracted two times with 80% EtOH (800 L) at 80 °C for 2 h. After filtration and evaporation ethanol under reduced pressure at 50 °C, the aqueous residue was diluted with H₂O and then partitioned three times with ethyl acetate (30 L). The remaining water extract was subjected to passage over polyamide by elution with water and 30%, 60%, and 95% EtOH-H₂O. Then the water elution was subjected to passage over D101 macroporous resin by elution successively with H₂O, 30%, 60%, and 95% EtOH-H₂O (Fractions B₁-B₄). Fraction B₃ (338.5 g) was subjected to passage over a bergmeal column eluting successively with ethyl acetate, ethanol, and methanol (Fractions C₁–C₃). Fraction C₂ (151.3 g) was chromatographed on a silica gel column eluting successively with a solvent gradient system (CHCl₃−MeOH, 15:1−0:1) to afford 7 fractions (D₁−D₇). Fraction D₄ (3.36 g) was passed over an PRP-512A macroporous resin column with MeOH-H₂O (10%, 30%, 60% and 100%) to give four fractions (E₁–E₄), and the fraction E₃ (0.45 g) was purified by preparative HPLC (MeOH-H₂O, 35:65, v/v, detected at 210 nm, 8 mL/min) to give 1 (5.3mg), 2 (1.3 mg), and 3 (1.0 mg).

3.4. Spectral Data

Single crystals of C₂₀H₂₆O₇ were recrystallized from MeOH mounted in inert oil and transferred to the cold gas stream of the diffractometer.

Crystal data of tripordolide A (1): C₂₀H₂₆O₇, M = 378.41, monoclinic, a = 7.7259(3) Å, b = 7.3200(3) Å, c = 15.4365(5) Å, β = 98.081(3)°, U = 864.33(5) Å3, T = 105(2), space group P21 (no. 4), Z = 2, μ(Cu Kα) = 0.912, 6799 reflections measured, 3061 unique (R_int = 0.0282) which were used in all calculations. The final wR(F₂) was 0.0901 (all data). Flack parameter was −0.02(11).

Crystallographic data for the structure of tripordolide A (1) have been deposited in the Cambridge Crystallographic Data Centre (deposition numbers: CCDC 1844652). Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk.

Characterization Data of Tripordolides A–C (1–3)

Tripordolide A (1): colorless needle; [α]${}_{\mathrm{D}}^{25}$—32.5 (c 0.1 MeOH); UV (MeOH) λ_max (log ε) 220 (3.53) nm; ¹H NMR (DMSO-d₆, 500 MHz) and ¹³C NMR (DMSO-d₆, 125 MHz), see Table 1; HRESIMS m/z 379.17532 [M + H]⁺ (calculated for C₂₀H₂₇O₇, 379.17513). (Supplementary Materials).

Tripordolide B (2): colorless oil (MeOH); [α]${}_{\mathrm{D}}^{25}$—35.1 (c 0.1, MeOH); IR (microscope) ν_max 3394, 2921, 1757, 1646, 1419, 1323, 1050, and 1027 cm⁻¹; ¹H NMR (DMSO-d₆, 600 MHz) and ¹³C NMR (DMSO-d₆, 150 MHz), see Table 1; HRESIMS m/z 399.1052 [M + Na]⁺ (calculated for C₁₉H₂₀NaO₇, 399.1050). (Supplementary Materials).

Tripordolide C (3): colorless oil (MeOH); [α]${}_{\mathrm{D}}^{25}$—77.3 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 220 (4.02) nm; ¹H NMR (DMSO-d₆, 600 MHz) and ¹³C NMR (DMSO-d₆, 150 MHz), see Table 1; HRESIMS m/z 401.1574 [M + Na]⁺ (calculated for C₂₀H₂₆NaO₇, 401.1571). (Supplementary Materials).

3.5. Biological Activities

The inhibitory effects of three compounds on NO production in LPS-induced RAW264.7 macrophage were evaluated. Dexamethasone was used a positive control. Concrete operation method was according to described in the literature [17].

4. Conclusions

In this research, no more than 30 18(4→3)-abeo-abietanes were isolated from the plant. Phytochemical studies on T. wilfordii have led to three abeo-abietanoids, a new natural product, and two new compounds, named tripordolides A–C (1–3). Tripordolide A (1) was reported in a patent as a synthetic product, but none of the spectra data was used as reference [18]. Its structure was elucidated on the basis of their spectroscopic analysis, and its absolute configuration was confirmed by an X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation. Notably, tripordolide A (1) was the first example of 18(4→3)-abeo-abietanes, which possessed 7/13 oxygen bridges in the natural products. Compounds 1 and 3 showed moderate inhibitory activities against NO production in LPS-induced RAW 264.7 macrophage in vitro.