A New Insecticidal Sesquiterpene Ester from Celastrus Angulatus

Abstract

A new sesquiterpene polyol ester with a β-dihydroagarofuran skeleton, NW37 (1), and three known compounds NW13 (2), NW16 (3) and NW35 (4) were isolated by bioassay-guided fractionation from the highly polar MeOH extracts of the root bark of Celastrus angulatus. Their chemical structures were elucidated mainly by analyses of MS and NMR spectral data. The insecticidal activity of compound 1 against 4^th instar Mythimna separata larvae with a KD₅₀ value of 252.3 μg·g^-1 was demonstrated.

1. Introduction

Various β-dihydroagarofuran sesquiterpene polyol esters and pyridine alkaloids, some of which exhibit insect antifeedant, insecticidal, antitumor, reversing multidrug resistance, anti-HIV, and immunosuppressive activities, have been obtained from the plants of the Celastraceae family [1,2,3,4,5,6,7,8,9,10]. Celastrus angulatus, a plant of the this family, is widely distributed in China and used for the treatment of rheumatism in traditional Chinese medicine and as an insecticide [11,12]. In our previous studies, some antifeedant, narcotic, and insecticidal ingredients were isolated from the toluene extracts of the root bark of C. angulatus. To obtain a sufficient number of compounds for QSAR research on their insecticidal activity against Mythimna separata, the chemical constituents from the root bark of C. angulatus were re-investigated guided by activity-guided fractionation. These studies have led to the isolation of a novel sesquiterpene polyol ester NW37 (1). In this paper, the isolation, structure elucidation and insecticidal activity of compound 1 were presented.

2. Results and Discussion

Four sesquiterpene polyol esters 1-4 were isolated from the MeOH extracts of the root bark of C. angulatus by macroporous resin column chromatography and RP-HPLC, and their structures were elucidated on the basis of UV, HR-ESI-MS and NMR spectroscopic evidence. Compound 1, a white powder, analyzed for C₃₈H₅₂O₁₄ by HR-ESI-MS (m/z 750.3695 [M+NH₄] ⁺, calculated 750.3700), and NMR spectra data (Table 1). Its IR spectrum revealed characteristic ester absorptions at 1,741 cm^-1, and a free hydroxyl absorption at 3,510 cm^-1. The UV spectrum contained an aromatic moiety (232 and 275 nm). The NMR spectra suggested the presence of three acetate esters, δ C 169.85 (CO), 169.60 (CO), 169.47 (CO), 21.59 (CH₃), 21.28 (CH₃), 20.53 (CH₃), δ H 2.10 (3H, s), 2.07 (3H, s), 1.46 (3H, s), one benzoate ester, δ C 164.64 (CO), 133.96 (CH), 130.34 (2×CH), 128.60 (2×CH), 128.52 (C), δ H 8.00 (2H, d, J=7.0 Hz), 7.59 (1H, t, J=7.0 Hz), 7.45 (2H, t, J=7.0 Hz) and two α-methylbutanoate esters, δ C 176.68 (CO), 175.42 (CO), 41.28 (CH), 41.22 (CH), 26.65 (CH₂), 26.58 (CH₂),16.68 (CH₃), 16.49 (CH₃), 11.82 (CH₃), 11.67 (CH₃), δ H 2.59 (1H, m), 2.50 (1H, m), 1.80 (2H, m), 1.55 (2H, m), 1.25 (3H, d, J=2.0 Hz), 1.23 (3H, d, J=2.0 Hz), 0.96 (6H, m). The ¹H-NMR of 1 showed the presence of three methyl groups at δ 1.49 (3H, s, H-13), 1.65 (3H, s, H-14), 1.62 (3H, s, H-15). Based on the published literature [13,14], the ¹H-¹H COSY spectrum signals at δ 5.62 (1H, d, J=3.5 Hz, H-1), 5.56 (1H, dd, J=3.5 Hz, 3.0, H-2), 6.25 (1H, s, H-6), 5.32 (1H, d, J=3.0 Hz, H-8)and 5.68 (1H, s, H-9) can be assigned to five protons attached to carbon atoms bearing secondary ester groups, while signals at δ 4.87 (1H, d, J=10.0 Hz, H-12a) and δ 4.83 (1H, d , J=10.0Hz, H-12b) can be assigned to the two protons attached to carbon atoms bearing primary ester groups.

The ¹³C-NMR (DEPT) spectrum of the parent skeleton of 1 showed three methyls at δ 24.72, 25.83 and 29.76, one methylene at δ 42.27, one methylene attached to an oxygen function at δ 65.72, one methine at δ 53.35, five methines attached to an oxygen function at δ 71.09, 68.26, 75.58, 76.32 and 72.31, one quaternary carbon at δ 54.20, and three quaternary carbons attached to an oxygen function at δ 70.06, 83.69 and 91.64, whose chemical shifts were very similar to those of reported β-dihydro-agarofurans. It was thus determined that compound 1 was a β-dihydroagarofuran sesquiterpene substituted with three acetate, one benzoate and two α-methylbutanoate esters. The ester group distributions were determined from the HMBC spectrum, which showed cross-peaks between H-9 and the carbonyl at δ 164.64 of the benzoate ester, H-12, H-8 and the carbonyl at δ 176.68, 175.42 of the two α-methylbutanoate esters, H-1, H-2, H-6 and the carbonyls at δ 169.85, 169.60, 169.47 of three acetate esters, respectively. In the molecular skeleton of β-dihydroagarofuran sesquiterpenes, H-1 and H-6 have axial stereochemistry. From the results of the NOESY spectrum of 1, the correlation between H-6 and H-9 indicated the presence of H-9_eq and the correlation between H-14 and H-8 indicated the presence of H-8_eq (Figure 2).Therefore, compound 1 was identified as 1β,2β,6α-triacetoxy-8β,12-di-(α-methyl)butanoyl-9α-benzoyloxy-4α-hydroxy-β-dihydroagarofuran.

Figure 1. The structures of compounds 1-4.

Figure 1. The structures of compounds 1-4.

NW13(2), NW16(3) and NW35(4) were known compounds, and there were characterized as 1β,2β,6α,8α,12-pentaacetoxy-9α-benzoyloxy-4α-hydroxy-β-dihydroagarofuran (2) [13], 1β,2β,6α,8β-tetraacetoxy-9β-benzoyloxy-12-isobutanoyloxy-4α-hydroxy-β-dihydroagarofuran (3)[14] and 1β,2β,8α,12-tetraacetoxy-9β-benzoyloxy-β-dihydroagarofuran (Angulatueoid B, 4) [15] on the basis of UV, IR, ¹H- and ¹³C-NMR spectroscopic evidence.

Table 1. The NMR data of compound 1. (CDCl_3, ¹H-NMR at 500 MHz, ¹³C-NMR at 125MHz, respectively)

No.	Δ C (DEPT)	δH (J, Hz)	HMBC
1	71.09 CH	5.62 (1H, d, J=3.5 Hz)	C-2,C-10, C=O of Ac
2	68.26 CH	5.56 (1H, dd, J=3.5 Hz, J=3.0 Hz)	C-10, C=O of Ac
3	42.27 CH2	2.24 (1H, m), 2.00 (1H, m)	C-1,C-2,C-4,C-5,C-13
4	70.06 C
5	91.64 C
6	75.58 CH	6.25 (1H,s)	C-5,C-7,C-8,C-10,C-11, C=O of Ac
7	53.35 CH	2.37 (1H, d, J=3.0 Hz)	C-5,C-6,C-8,C-9
8	76.32 CH	5.32 (1H, d, J=3.0 Hz)	C=O of MeBut
9	72.31 CH	5.68 (1H, s)	C-5,C-7,C-8,C-10,C-12, C=O of Bz
10	54.20 C
11	83.69 C
12	65.72 CH2	4.87 (1H,d, J=10.0 Hz) 4.83 (1H,d, J=10.0 Hz)	C-1,C-5,C-9,C-10, C=O of MeBut
13	24.72 CH3	1.49 (3H, s)	C-4, C-5
14	25.83 CH3	1.65 (3H, s)	C-7, C-11
15	29.76 CH3	1.62 (3H, s)	C-7, C-11
Ac	169.85 (CO), 21.59 (CH3)	2.10 (3H, s)
Ac	169.60 (CO), 21.28 (CH3)	2.07 (3H, s)
Ac	169.47 (CO), 20.53 (CH3)	1.46 (3H, s)
MeBut	176.68 (CO) 41.28 (CH), 26.65 (CH2), 11.82 (CH3), 16.68 (CH3)	2.59 (1H, m), 1.80 (2H, m), 1.25 (3H, d, J=2.0 Hz), 0.96 (3H, m)
MeBut	175.42 (CO) 41.22 (CH), 26.58 (CH2), 11.67 (CH3), 16.49 (CH3)	2.50 (1H, m), 1.55 (2H, m), 1.23 (3H, d, J=2.0 Hz), 0.96 (3H, m)
Bz	164.64 (CO), 133.96 (CH), 130.34 (2×CH), 128.60 (2×CH), 128.52 (C)	8.00 (2H, d, J=7.0 Hz), 7.59 (1H, t, J=7.0 Hz), 7.45 (2H, t, J=7.0 Hz)

Table 1. The NMR data of compound 1. (CDCl_3, ¹H-NMR at 500 MHz, ¹³C-NMR at 125MHz, respectively)

No.	Δ C (DEPT)	δH (J, Hz)	HMBC
1	71.09 CH	5.62 (1H, d, J=3.5 Hz)	C-2,C-10, C=O of Ac
2	68.26 CH	5.56 (1H, dd, J=3.5 Hz, J=3.0 Hz)	C-10, C=O of Ac
3	42.27 CH2	2.24 (1H, m), 2.00 (1H, m)	C-1,C-2,C-4,C-5,C-13
4	70.06 C
5	91.64 C
6	75.58 CH	6.25 (1H,s)	C-5,C-7,C-8,C-10,C-11, C=O of Ac
7	53.35 CH	2.37 (1H, d, J=3.0 Hz)	C-5,C-6,C-8,C-9
8	76.32 CH	5.32 (1H, d, J=3.0 Hz)	C=O of MeBut
9	72.31 CH	5.68 (1H, s)	C-5,C-7,C-8,C-10,C-12, C=O of Bz
10	54.20 C
11	83.69 C
12	65.72 CH2	4.87 (1H,d, J=10.0 Hz) 4.83 (1H,d, J=10.0 Hz)	C-1,C-5,C-9,C-10, C=O of MeBut
13	24.72 CH3	1.49 (3H, s)	C-4, C-5
14	25.83 CH3	1.65 (3H, s)	C-7, C-11
15	29.76 CH3	1.62 (3H, s)	C-7, C-11
Ac	169.85 (CO), 21.59 (CH3)	2.10 (3H, s)
Ac	169.60 (CO), 21.28 (CH3)	2.07 (3H, s)
Ac	169.47 (CO), 20.53 (CH3)	1.46 (3H, s)
MeBut	176.68 (CO) 41.28 (CH), 26.65 (CH2), 11.82 (CH3), 16.68 (CH3)	2.59 (1H, m), 1.80 (2H, m), 1.25 (3H, d, J=2.0 Hz), 0.96 (3H, m)
MeBut	175.42 (CO) 41.22 (CH), 26.58 (CH2), 11.67 (CH3), 16.49 (CH3)	2.50 (1H, m), 1.55 (2H, m), 1.23 (3H, d, J=2.0 Hz), 0.96 (3H, m)
Bz	164.64 (CO), 133.96 (CH), 130.34 (2×CH), 128.60 (2×CH), 128.52 (C)	8.00 (2H, d, J=7.0 Hz), 7.59 (1H, t, J=7.0 Hz), 7.45 (2H, t, J=7.0 Hz)

The insecticidal activities of compounds 1-4 against 4^th instar larvae of Mythimna separata were tested by the leaf disc method (for thr methodology see [13,14,16,17]). The result showed that the KD₅₀ value for compound 1 was 252.3 μg· g^-1. The symptoms displayed by the Mythimna separata indicated that these compounds have stronger insecticidal but not narcotic or antifeedant activities. On comparison of the KD₅₀ data of compounds 1-4 presented in Table 2 and other compounds isolated in our laboratory, such as celangulatin C (KD₅₀=280.4 μg· g^-1), celangulatin F (KD₅₀=201.5 μg· g^-1) and angulatin A (KD₅₀=300.9 μg· g^-1) (for structures see Figure 3 ) [18], it was very interesting to note that compound 4 exhibited weaker activities than compound 1-3 and other compounds. For the structure of these compounds, it is obvious that the stereochemistry and the type of the ester groups at C-1 and C-2 in these compounds are similar, and the differences between them are the substitution groups at C-8, C-9 and C-12. In addition, the protons of C-4 and C-6 of compound 4 were not substituted by hydroxyl or ester groups, which indicated that the C-4 and C-6 substituents have a positive effect on the insecticidal activity. Moreover, these results suggested that the substitutes and stereochemistry of C-8, C-9, and C-12 play important roles in these compounds [13,18,19,20].

Table 2. The KD₅₀ data of 1~4 and other compounds.

Compounds	KD50 (μg· g-1)
1	252.3
2	290.1
3	360.2
4	884.3
Celangulatin C	280.4
Celangulatin F	201.5
Angulatin A	300.9

Table 2. The KD₅₀ data of 1~4 and other compounds.

Compounds	KD50 (μg· g-1)
1	252.3
2	290.1
3	360.2
4	884.3
Celangulatin C	280.4
Celangulatin F	201.5
Angulatin A	300.9

Figure 3. The structures of celangulatin C, celangulatin F and angulatin A.

Figure 3. The structures of celangulatin C, celangulatin F and angulatin A.

3. Experimental

3.1. General

Melting points were measured on a Yanagimoto apparatus and are uncorrected. Optical rotations were measured on a Perkin-Elmer 341 polarimeter (USA). IR spectra were determined on an IR-450 instrument (KBr plate). ¹H-NMR, ^1-C-NMR, DEPT, COSY, HMQC, HMBC, and NOESY spectra were recorded on Bruker Avance 500 MHz NMR Spectrometer with CDCl₃ as solvent and TMS as internal standard. HR-ESI-MS was obtained on a Bruker Apex II mass spectrometer. Finnigan LCQ Advantage MAX LC/MS, equiped with Surveyor DAD detector and Hypersil ODS₂ C₁₈ column (4.6×250 mm, 5 μm, Dalian Elite Analytical Instruments Co., Ltd., P.R. China), was used to analyse the samples. Compounds were purified with a Waters 600E HPLC apparatus equipped with a Hypersil ODS₂ C₁₈ preparative column (20 × 250 mm, 10 μm, Dalian Elite Analytical Instruments Co., Ltd., P.R. China) , MeOH-H₂O (55: 45) as eluent, UV detector set at 230 nm.

3.2. Plant material

The root bark of C. angulatus was collected in Qinling mountain, Taibai County, Shaanxi Province, People’s Republic of China, in October 2007, authenticated by Dr. Hua Yi of the College of Life Sciences, Northwest Agricultural & Forestry University, and dried in the shade (at room temperature). Voucher specimens (samples no. NWAU2007-A18) were deposited at the College of Plant Protection, Northwest Agricultural & Forestry University.

3.3. Extraction and isolation

The dried and pulverized root bark (2.0 kg) of C. angulatus was extracted four times with MeOH (6.0L) under reflux. The extracted material (120 g) was adsorbed in a D101 macroporous resin (Hebei Cangzhou Chemical Co., Ltd., P.R. China) column (5.0×150 cm) and eluted with MeOH-H₂O (5:5, 6:4, 7:3), and 100 fractions of ca. 500 mL each were collected. After removal of the solvents under reduced pressure, fractions were analysed by LC/DAD/MS, and similar ones were combined. The insecticidal activity of every fraction was assayed. Then the fractions which containing unknown sesquiterpene polyol esters were selected for further purification by RP-HPLC column, affording four compounds: NW37 (1, 75 mg), NW13 (2, 78 mg), NW16 (3, 92mg) and NW35 (4, 35mg).

Compound 1: C₃₈H₅₂O₁₄, white powder, -12.0° (CH₃COCH₃, c 1.20); IR v: 3510,2926, 1741, 1632, 1380, 1232, 1060,891, 712 cm^-1; UV: 232, 275 nm; ESI-MS (MS/MS): m/z (%) 755 [M+Na] ⁺ (17), 695 [M+Na-AcOH] ⁺ (80), 653 [M+Na-MeBuOH] ⁺ (100), 633[M+Na-BzOH]⁺ (21), 593 [M+Na-AcOH-MeBuOH] ⁺(12). ¹H- and ¹³C-NMR (CDC_l3) see Table 1. Major NOESY correlations Figure 2.

Figure 2. Major NOESY correlations in 1.

Figure 2. Major NOESY correlations in 1.

3.4. Insecticidal activity

Toxic leaf discs of known area were treated with known amounts of the test samples dissolved in acetone (acetone and celangulin V were used as negative and positive control). The 4^th instar larvae of M. separata were fed with the discs for 12 h (repeated 10 times for each sample). After 24 h, the numbers of knocked-down larvae (symptoms: the larvae were narcotized and could not move; the bodies were immobilized and very soft; and the response disappeared completely) were recorded, and the toxicity was ascertained by estimating the median knock-down dose (KD₅₀ value) of the test sample [14].