Antifeedant Triterpenoids from the Seeds and Bark of Lansium domesticum cv Kokossan (Meliaceae)

Two tetranortriterpenoids, kokosanolide A (1) and C (2) were isolated from the seeds and three onoceranoid-type triterpenoids: kokosanolide B (3), 8,14-secogammacera-7,14-diene-3,21-dione (4) and a 1.5:0.5 mixture of 8,14-secogammacera-7,14(27)-diene-3,21-dione (5) and compound 4 were isolated from the bark of kokossan (Lansium domesticum). Complete 1H- and 13C-NMR data of the triterpenoids 1-5 are reported. The triterpenoids’ structures were elucidated primarily by means of high field 1D- and 2D-NMR, IR and HRMS spectral data. Triterpenoids 1-5 exhibited moderate to strong antifeedant activity against the fourth instar larvae of Epilachna vigintioctopunctata.

During the course of our continuing search for novel antifeedant compounds from tropical Meliaceae plants, the methanol extract of L. domesticum showed strong antifeedant activity against the fourth instar larvae of Epilachna vigintioctopunctata. Herein, we report the 1 H and 13 C NMR data and structural elucidation for these compounds 1-5 isolated from seed and bark extracts of the plant. The structures of compounds 1, 3 and 5 were established previously by X-ray diffraction [7][8][9].

Results and Discussion
Liquid-liquid partitioning of the MeOH extract of the seed of L. domesticum cv Kokossan into nhexane, EtOAc and aqueous MeOH fractions gave the n-hexane fraction (4 g) as the most active one, with 100% antifeedant activity at 1% concentration. Purification of the n-hexane fraction using silica gel 60 open column chromatography led to the isolation of compounds 1 and 2. In addition, the MeOH extract of the bark of L. domesticum was partitioned between n-hexane and ethyl acetate to give the ethyl acetate fraction. A crude ethyl acetate fraction was subjected to vacuum column chromatography on silica gel 60 and further purified by silica gel column chromatography to yield compounds 3-5.
Correlations between H-6/H-5/H-20 indicated that methyl group and tetrahydrofuran ring should be αorientation. Thus, the gross structure of tetranortriterpenoid 1 was elucidated as a hexacyclic ring system. The structure and relative stereochemistry were further elucidated by using single-crystal Xray diffraction analysis [7]. An ORTEP drawing of 1 is shown in Figure 3. Consequently, the structure of tetranortriterpenoid 1 was established to be a tetranortriterpenoid and was named kokosanolide A. Kokosanolide C (2) was obtained as colorless needle-like crystals from n-hexane-EtOAc and decomposed during the measurement of its melting point. The UV spectrum showed an absorption maximum at 275 nm (ε 4,500), indicating the presence of an α-β-unsaturated ketone. The IR spectrum showed bands which were ascribable to hydroxyl (ν max 3,563 cm −1 ), a ester carbonyl (ν max 1,758 cm −1 ) and unsaturated ketone (ν max 1,704 cm −1 ). The 1 H-and 13 C-NMR (Table 1) spectra of 2 were quite similar to those of 1, except for the absence of the ketone signal at δ 208.5 and appearance of a geminal proton signal at [δ H 2.18 (1H, m), 1.71 (1H, m); δ C 20.9]. In the HMBC spectrum of 2, long range correlations were observed between the signals at δ 1.72 and 2.18 and the carbon signals at δ 76.1 (C-2), 47.8 (C-4) and 55.9 (C-5), suggesting that compound 2 was a 3-deoxo derivative of compound 1 and it was thus named kokosanolide C.
Kokosanolide B (3) was obtained as cubic crystals, m.p. 148-150 °C, from n-hexane-EtOAc. The molecular formula of 3 was determined to be C 30 H 48 O 3 by LC-ESI-MS data (m/z 456.6892, [M+H] + ), and combined with the 1 H-and 13 C-NMR spectral data (Table 2), thus required seven degrees of unsaturation. IR absorption bands at 3,749, 1,705, 1,384 and 1,261 cm −1 suggested the presence of hydroxyl, carbonyl, and gem-dimethyl functionalities, respectively. Analysis of 1 H-and 13 C-NMR data, DEPT and the HMQC spectra of 3 revealed the presence of thirty signals: three sp 2 and five sp 3 quaternary carbons, one sp 2 and four sp 3 methines, nine sp 3 methylenes, and eight methyl groups. Among them, one sp 2 methine (δ C 121.7; δ H (1H, 5.41, m) was ascribed to the isolated double bond, while two carbonyl carbons (δ 216.9 and 217.1 ppm) and one sp 3 carbon (δ 74.0) were assigned to those bearing an oxygen atom (C-3, C-21 and C-14, respectively), suggesting that compound 3 was an onoceranoid-type triterpenoid [5]. The positions of the ketones, hydroxyl and isolated double bond were further determined by the COSY and HMBC experiments (Figure 4), H-7 (δ 5.41) showed correlations to C-6 (δ 28.9), C-9 (δ 55.5) and C-8 (δ 135.3), suggesting that an isolated double bond is located at C-7 and C-8. The methylene signals at C-2 (δ 2.23) revealed correlations to carbonyl signal at δ 216.9, whereas H-5 (δ 1.57) was correlated to C-4 (δ 47.6) and C-3 (δ 216.9), indicating that one of the carbonyl moeity and gem-dimethyl are placed at C-3 and C-4, respectively. Furthermore, the methylene signals at C-20 (δ 2.26) showed correlations to the carbonyl signal at δ 217.1, whereas the methine signal at C-17 (δ 1.42) showed correlations to C-22 (δ 47.6 ) and C-21 (δ 217.1), thus suggesting the other carbonyl group and gem-dimethyls were located at C-21 and C-22, respectively. An oxygenated tertiary carbon signal was revealed to be C-14 by the correlation between methine signal at C-13 (δ C 1.12 ) to oxygenated carbon at δ C 74.0 ppm and correlation between methylene signals at C-15 (δ C 1.46 ) to oxygenated carbon at δ C 74.0 ppm. Thus, the structure of onoceranoidtype triterpenoid 3 was determined as 8,14-secogammacera-14-hydroxy-7-ene-3,21-dione and was named kokosanolide B. The structure and relative stereochemistry were also elucidated by using single-crystal X-ray diffraction analysis [8]. An ORTEP drawing of 3 is shown in Figure 5.   (Table 2), requires eight degrees of unsaturation. Compound 4 showed no absorption maxima in the UV spectrum indicating the absence of a conjugated double bond. The IR spectrum showed bands which were ascribed to a ketone (ν max 1,708 cm −1 ), isolated double bond (ν max 1,662 cm −1 ) and gem-dimethyl (ν max 1,430 and 1,360 cm −1 ). The 13 C-NMR spectrum of 4 showed 15 signals, similar to those of kokosanolide B, suggesting that 4 has a symmetrical structure. The essential differences between the NMR spectra of 4 and kokosanolide B consisted of the absence of a hydroxyl group and appearance of a double bond [δ 5.43 (1H, m), δ H 122.1 and 135.3] and fifteen carbon signals, suggesting that 4 was a dehydroxy derivative of kokosanolide B. In order to determine the connectivity of the partial structure due to a newly double bond, HMBC experiments were carried out. The signal of olefinic proton H-15 (δ 5.43) was correlated to C-14 (δ 135.3), C-13 (δ 55.6) and C-16 (δ 30.1), indicating that a new double bond was located at C-14 and C-15, suggesting that 4 has two similar unit structure. Consequently, compound 4 was identified as a 8,14-secogammacera-7,14-diene-3,21-dione [5].

General
Melting points were measured on an Electrothermal melting point apparatus and are uncorrected. Optical rotations were recorded on a Perkin-Elmer 341 polarimeter. The UV spectra were obtained on a UV Ultraspec 3000 Pro spectrophotometer. The IR spectra were recorded on a Perkin-Elmer 1760X FT-IR in KBr. The mass spectra were recorded with a Mariner Biospectrometry-Finnigan instrument. 1 H-and 13 C-NMR spectra were obtained with a JEOL JNM A-500 spectrometer using TMS as internal standard. All ORTEP diagrams were obtained from previous reports. Chromatographic separations were carried out on silica gel 60 (Merck). TLC plates were precoated with silica GF 254 (Merck, 0.25 mm) and detection was achieved by spraying with 10% H 2 SO 4 in ethanol, followed by heating.

Plant material
The bark and seed of L. domesticum cv Kokossan were collected in Cililin District, Bandung, West Java Province, Indonesia in March 2006. The plant was identified by the staff of the Laboratory of Plant Taxonomy, Department of Biology, Padjadjaran University, Indonesia. A voucher specimen (No. 10184) was deposited at the herbarium of the Padjadjaran University.

Extraction and isolation
Dried seeds of L. domesticum cv kokossan (2 kg) were extracted exhaustively with methanol 12 L at room temperature for 3 days. The resulting methanol extract (84 g) was partitioned between nhexane (2.5 L) and 10% aqueous methanol (2.5 L) to give an n-hexane soluble fraction (4 g) after removal of the solvent. The n-hexane extract was subjected to column chromatography on silica gel 60 using a n-hexane and dichloromethane (8:2). The fraction eluted with n-hexane-dichloromethane (6:4) was further separated by column chromatography on silica gel (n-hexane-ethyl acetate 7:3) to give 1 (150 mg) and 2 (26 mg).