Rings D-seco and B,D-seco tetranortriterpenoids from root bark of Entandrophragma angolense

Rings D-seco and B,D-seco tetranortriterpenoids from root bark of Entandrophragma angolense

Phytochemistry 72 (2011) 1854–1858 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Rin...

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Phytochemistry 72 (2011) 1854–1858

Contents lists available at ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Rings D-seco and B,D-seco tetranortriterpenoids from root bark of Entandrophragma angolense Tienabe K. Nsiama a,1, Hiroaki Okamura a, Toshiyuki Hamada a, Yoshiki Morimoto b, Matsumi Doe b, Tetsuo Iwagawa a, Munehiro Nakatani a,⇑ a b

Department of Chemistry and Bioscience, Faculty of Science, Kagoshima University, 1-21-35 Korimoto, Kagoshima 890-0065, Japan Analytical Division, Graduate School of Science, Osaka City University, 3-3-7 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan

a r t i c l e

i n f o

Article history: Received 16 October 2010 Received in revised form 27 March 2011 Available online 11 July 2011 Keywords: Entandrophragma angolense Meliaceae Root bark Tetranortriterpenoids Limonoids Gedunin Methyl angolensate

a b s t r a c t Investigation of the root bark extract of Entandrophragma angolense led to identification of two gedunintype limonoids 5-hydroxy-7-deacetoxy-7-oxogedunin (1) and 5,6-dehydro-7-deacetoxy-7-oxogedunin (2), and three methyl angolensate derivatives, 6-deacetoxydomesticulide D (3), 6-deacetoxydomesticulide D 21-methylether (4), and entangosin (5), together with known compounds, methyl angolensate (6), 6-acetoxymethyl angolensate (7) and secomahoganin (8). Their structures were established by extensive NMR experiments in conjunction with mass spectrometry. Entangosin is a rare example of a limonoid derivative having a fully O-substituted furan moiety. Ó 2011 Published by Elsevier Ltd.

1. Introduction In an investigation on plants with antifeedant activity, the extract of the root bark of Entandrophragma angolense, collected in DR Congo, showed potent antifeedant activity against Spodoptera insects (Nakatani et al., 2004; Nsiama et al., 2008a,b; Saad et al., 2003). This plant belongs to the Meliaceae family, and plants of this family are known to produce bitter principles (limonoids) which have been reported for their wide range of activities including anti-HIV (Sunthitikawinsakul et al., 2003), antimalarial (Bickii et al., 2000; Omar et al., 2003; Saewan et al., 2006), cytotoxicity against cell lines (Miller et al., 2004; Uddin et al., 2007; Zhou et al., 2005) in addition to insect antifeedants (Carpinella et al., 2002; Nakatani et al., 2002; Nihei et al., 2006). E. angolense is distributed in tropical Africa where it is widely used in ethnomedical treatment of various gastrointestinal disorders including peptic ulcers in human and as an antimalarial (Njar et al., 1995). The occurrence and discovery of gedunin from this plant contributed greatly in the development of the chemistry of limonoids (Taylor, 1984). Previously, we reported the isolation and the antifeedant property of mexicanolide-type limonoids from this plant (Nsiama et al., ⇑ Corresponding author. E-mail address: [email protected] (M. Nakatani). Present address: Department of Oncology, London Regional Cancer Program, London Health Science Centre, 790 Commissioners Road East, London, Ontario, Canada N6A 4L6. 1

0031-9422/$ - see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.phytochem.2011.05.014

2008a). Herein, we report the isolation and the structural determination of five new compounds 1–5 and three known compounds from the root bark extract of this plant. 2. Results and discussion The ethyl acetate soluble portion of the methanol extract of E. angolense was subjected to solvent partition between methanol– water (2:1) and CHCl3. The CHCl3 layer was purified by silica gel chromatography followed by C18 reversed phase HPLC to give the five new compounds, 5-hydroxy-7-deacetoxy-7-oxogedunin (1),

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5,6-dehydro-7-deacetoxy-7-oxogedunin (2), 6-deacetoxydomesticulide D (3), 6-deacetoxydomesticulide D 21-methylether (4) and entangosin (5), along with the known compounds, methyl angolensate (6) (Akisanya et al., 1960), methyl 6-acetoxyangolensate (7) (Connolly et al., 1967) and secomahoganin (8) (Kadota et al., 1989). Compound 1 was obtained as white amorphous solid. Its molecular formula C26H30O7, deduced from the HRFAB-MS spectrum (pseudomolecular ion at m/z 455.2092, [M+H]+) and 13C NMR and DEPT spectra, corresponds to 12 degrees of unsaturation. The IR spectrum displayed bands at 3420 (hydroxyl), 1720–1736 (carbonyl or/and a,b-unsaturated), 1649 (carbon–carbon double) and 875 cm1 (furan ring). The analysis of the 1H and 13C NMR spectroscopic data fully explained that six elements of unsaturation were present as double bonds (three carbon–carbon including furan ring, two ketones and one ester); therefore, the molecule is pentacyclic. The NMR spectroscopic data of 1 (Table 1) showed signals of five tertiary methyl groups, three methylenes, eight methines (five olefinics) and nine non-hydrogenated carbons, and the presence of one b-furanyl moiety (d 6.35, 7.39 and 7.42; each one proton). All protons directly bonded to carbon atoms were assigned by 1 H–13C shift-correlated measurement (HMQC). The data from decouplings and subsequent 2D NMR studies using the COSY, HMBC and NOESY, suggested that compound 1 was gedunin-type limonoid (Khalid et al., 1989; Mitsui et al., 2006). The oxygenated quaternary carbon at d 62.9, assigned as C-5, displayed a cross-peak with an hydroxyl proton at (d 1.67) suggesting location of this group at this position. In addition, the methylene protons at d 2.33 (d, J = 13.2 Hz) and 3.41 (d, J = 13.2 Hz) were assigned as H2-6, due to the HMBC correlation observed with the carbon signal at d 209.2 (C-7) and the oxymethine carbon at C-5. The relative stereochemistry of 1 was established by NOE experiments. The NOESY spectrum showed close similarities with

O H H

H

H

H

H

O

H H

O

O H O

O H

Fig. 1. Selected HMBC correlations for 2.

reported NOE data of gedunin-type compounds (Khalid et al., 1989; Mitsui et al., 2006). The NOE cross-peaks observed between: H-17 and H-12b, H-11b and Me-30, H-17 and Me-30, and Me-19 and Me-30 indicated b-orientation of these protons. The a-orientation of the H-9 was deduced by a NOE cross-peak shared with the Me-18. The b-orientation of the 14,15-epoxide moiety was deduced by the weak NOE correlation observed between H-15 and Me-18, and H-15 and Me-30, as observed also in gedunin. These results are consistent with the stereochemistry shown for 1, and the compound was assigned as 5-hydroxy-7-deacetoxy-7-oxogedunin. Compound 2 was isolated as a white amorphous solid. Its HRFAB-MS spectrum displayed a pseudo-molecular ion at m/z 437.1971, [M+H]+. Combined with 13C NMR spectroscopic data, the molecular formula of this compound was established as C26H28O6 (13 degrees of unsaturation). The following characteristic absorptions observed in IR spectrum 1736–1750, 1650 and 873 cm1 corresponded to the presence of carbonyl, carbon - carbon double, and furan ring, respectively.

Table 1 NMR spectroscopic data (400 MHz, CDCl3) of compounds 1 and 2. Position

1 2 3 4 5 6a b 7 8 9 10 11a b 12a b 13 14 15 16 17 18 19 20 21 22 23 28 29 30 OH-5

5-Hydroxy-7-deacetoxy-7-oxogedunin (1)

5,6-Dehydro-7-deacetoxy-7-oxogedunin (2)

dC

dH (J in Hz)

dC

dH (J in Hz)

160.9 132.9 211.6 53.3 62.9 123.5

7.13, d (6.2) 6.29, d (6.2)

152.2 125.7 200.8 49.4 166.2 123.5

6.89, d (10.0) 6.00, d (10.0)

209.2 52.1 46.7 48.2 16.4 29.4 38.0 66.6 55.3 167.2 77.9 19.8 26.4 120.3 141.1 109.8 143.1 28.5 21.0 17.8

2.33, d (13.2) 3.41, d (13.2)

2.16, dd (11.8,3.8) 1.88, 1.94, 1.47, 1.80,

m m ddd (9.4,7.0,2.5) ddd (18.1,9.4,4.4)

4.29, s 5.54, s 1.15, s 1.06, s 7.42, 6.35, 7.39, 1.01, 1.40, 1.52, 1.67,

s br s br s s s s s

198.6 48.5 44.5 41.4 18.9 32.8 37.4 64.9 52.2 166.7 77.7 20.6 22.6 120.1 141.0 109.8 143.1 25.5 28.2 17.2

6.03, s

2.40, br d 1.97, 1.81, 1.45, 1.92,

dd (13.5,8.0) m o dd (13.9,7.3)

3.95, s 5.44, s 1.03, s 1.50, s 7.42, 6.38, 7.39, 1.40, 1.45, 1.08,

s br s t (1.4) s s s

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Table 2 NMR spectroscopic data (400 MHz, CDCl3) of compounds 3–5. Position

6-Deacetoxydomesticulide D (3) dC

1 2a b 3 4 5 6a b 7 8 9 10 11a b 12a b 13 14 15a b 16 17 18 19 20 21 22 23 28 29 30a b OMe-7 OMe-21 OMe-23 a

77.4 39.3 212.7 48.1 43.0 32.6 174.0 144.8 49.6 43.9 23.8 29.3 41.9 80.0 33.5 169.3 79.9 14.2 21.7 163.7 98,1 121.7 169.3 25.8 21.4 112.3 52.3

dH (J in Hz) 3.53, dd (6.3,3.5) 2.49, dd (14.5,3.5) 2.91, dd (14.5,6.3)

2.84, br d (10.1) 2.63, dd (16.5,10.1) 2.24a

2.24 1.66, 2.24, 1.31, 2.06,

a

m o m m

2.88, d (18.1) 2.60, d (18.1) 5.62, s 0.93, s 0.96, s 6.14, br s 6.24, br s 1.00, 1.19, 5.20, 4.92, 3.72,

s s br s br s s

6-Deacetoxydomesticulide D 21-methylether (4) dC 77.3 39.6 213.0 47.8 42.8 32.9 173.8 145.6 49.4 44.0 23.4 28.8 41.8 79.5 33.6 169.5 77.6 13.3 21.4 134.9 102.3 147.6 168.6 26.7 21.1 117.9 51.9 57.4

dH (J in Hz) 3.48, t (5.8) 2.60, dd (14.3,5.2) 2.81, dd (14.3,6.5)

2.92, d (10.3) 2.57, dd (16.0,7.7) 2.25, dd (16.0,5.3)

2.18, d (4.9) 2.25, 1.60, 1.07, 2.25,

o m o o

2.89, d (18.2) 2.58, d (18.2) 5.71, s 0.90, s 0.87, s 5.77, s 7.18, s 1.09, 1.18, 5.17, 4.99, 3.71, 3.59,

s s s s s s

Entangosin (5) dC 76.4 39.9 213.0 47.9 42.7 32.8 173.6 145.3 49.2 44.3 23.5 27.6 41.6 80.3 33.3 169.9 83.8 14.4 21.4 82.1 109.9 76.1 112.6 27.4 21.1 112.2 52.0 56.5 55.5

dH (J in Hz) 3.39, t (6.4) 2.60, dd (13.8,5.8) 2.74, dd (13.8,7.0)

2.86, br d 2.53, dd (15.9,9.9) 2.28, br d (15.9)

2.11, br d (5.1) 1.63, 2.17, 2.01, 1.95,

m br dd (15.8,5.3) br dd (13.1,5.3) ddd (19.7, 13.1, 5.3)

2.80, d (18.0) 2.50, d (18.0) 5.19, s 1.09, s 0.79, s 4.92, s 4.20, d (3.2) 4.99, d (3.2) 1.10, s 1.17, s 5.14,s 4.85, s 3.71, s 3.51, s 3.45, s

Signals were overlapped.

The NMR spectroscopic data of compound 2 showed very close similarities with those of 1, implying that both compounds possess the same basic skeleton. Unlike compound 1, 2 displayed no signal due to the hydroxyl group. Also, the presence of a tri-substituted double bond at d 166.2 (C-5) and 123.5 (C-6) was recognized. In addition to an HMBC correlation observed between the olefinic proton with C-5, this proton showed also HMBC correlations with the carbon signals at d 198.6 (C-7) (Fig. 1). From these findings, 2 was assigned as 5,6-dehydro-7-deacetyl-7-oxogedunin. Similarly to 1, its relative stereochemistry was determined by NOE considerations as shown. Compound 3 was obtained as an amorphous powder. Its HRFAB-MS spectrum showed molecular ion at m/z: 503.2286 [M+H]+ in accordance with the molecular formula C27H34O9, indicating 11 degrees of unsaturation. The IR spectrum showed absorptions due to hydroxyl (3300 cm1) and several carbonyls (1736–1753 cm1). Analysis of the 13C NMR spectroscopic data (Table 2) indicated that 3 contains one ketone, three ester carbonyls and four olefinic carbons taking into account six degrees of unsaturation. Hence, the five remaining were attributed to four saturated rings. The 1H, 13C NMR and HMQC spectra also indicated the presence of four quaternary methyls, six methylenes, six methines and eight quaternary carbons. The NMR spectroscopic data of this compound were closely similar to those of domesticulide D (Saewan et al., 2006) including the characteristic 21-hydroxy-21,23-butenolide with an hemiacetal carbon at d 98.1 (C-21) and an esteric carbonyl carbon at d 169.3 (C-23). However, the absence of signal due to an acetyl group

was noted, and it was deduced that 3 is the 6-deacetoxydomesticulide D. The relative stereochemistry of 3 was clarified by both the coupling constants and the NOE experiments. The NOE cross-peaks observed between: H2-6 and Me-19, H-5 and Me-28, explained the alpha-orientation of H-5. The H-9 proton exhibited NOE correlations with Me-18, H-11a, and H-15a. Thus, the a-orientation of these protons could be explained. NOE cross-peaks were also observed between H-17 and H-12b, H-11b, and H-15b indicated the b-orientation of these protons. The NOE cross-peak between H-1 and Me-29 indicated the b-orientation of H-1. Compound 4 showed no molecular ion peak in the mass spectrum but a fragment ion peak was observed at m/z 485.2203 [M+CH3OH], due to the labile nature of the methoxy acetal group. The molecular formula of C28H36O9 was deduced from the mass spectrum in conjugation with the 13C NMR and DEPT spectra. The NMR spectra of 4 showed close similarities to those of 3 except for observation of additional NMR signals (d 57.4 and d 3.59) due to a methoxy group. This suggested that 4 possess the same basic skeleton as 3. The location of the methoxy group was established by the HMBC correlation of methoxy methyl protons to the hemiacetal carbon signal at d 102.3 (C-21). Thus, 4 has been identified as seco-tetranortritepenoid bearing a c-methoxybutenolide at C-17. It has been assigned as 6-deacetoxydomesticulide D 21-methylether. Similarly to 3, the relative stereochemistry of 4 was determined by NOE experiments. Thus, structure 4 has been determined as shown.

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NOE connectivity between OMe-21 and H-23 leads to the conclusion they are on the other face of tetrahydrofuran ring. Thus, structure 5 is proposed as shown. The antifeedant activity of the isolated compounds (1–5) against the third-instar larvae of Spodoptera littoralis (Boisd.) was briefly evaluated. The gedunin derivatives were weakly active at 1000 ppm, corresponding to a concentration of ca. 20 lg/leafcm2, while the domesticulides showed moderate activities at the same concentration. 3. Conclusion The investigation of the extract of the root bark of E. angolense led to identification of eight limonoids derivatives including five new structures. These new compounds have the particularity of being A-seco limonoid of gedunin type (1 and 2) and B,D-seco compound of andirobin type (3–5). Among the known compounds, this is the first report on the isolation of 6-acetoxymethyl angolensate and secomahoganin from this plant. Entagosin is a rare tetranortriterpenoid derivative possessing a 3,4-dihydroxy-2,5-dimethoxytetrahydrofuran ring, a fully O-substituted furan moiety, as side-chain. 4. Experimental 4.1. General

Fig. 2. Significant NOE correlations observed in 5; structure has been refined using MM2 force field.

Entangosin (5) was isolated as an amorphous solid. In the HRMS, as in 4, the molecular ion was not observed but fragment ion due to the loss of methanol was detected at m/z 535.2524. Thus the molecular formula was deduced as C29H42O11 from these mass spectroscopic data in conjugation with analyses of the 13C NMR and DEPT spectra. The IR spectrum showed absorption bands for carbonyl groups (1762–1720 cm1) and double bonds (1652 cm1). The 1H and 13C NMR spectroscopic data of 5 showed close similarities to those of 3 and 4 besides resonances of the C-17 sidechain. The 13C NMR spectrum of 5 indicated two hemiacetal carbon signals at d 109.9 and 112.6, two oxygenated carbons at 82.1 and 76.1 (instead of tri-substitued double as observed in 3 and 4), while the 1H NMR spectrum suggested two methoxy methyl groups at d 3.51 and 3.45. The proton at d 4.99 (H-23), coupled with the proton at d 4.20 (H-22) in the COSY, showed HMBC cross-peaks with another hemiacetal carbon signal at d 109.9 (C-21), the quaternary oxygenated carbon at d 82.1 (C-20) and the methoxy carbon at d 55.5 (d 3.51), permitting also the location of the methoxy group at C-23. The remaining methoxy group was placed at C-21 by its coupling with the hemiacetal carbon at d 109.9 (C21) bearing the proton observed as a single at d 4.92 (H-21). The HMBC cross-peak between the methoxy methyl protons and C-20 also supported the above mentioned location. Thus, it was concluded that the C-17 side-chain is a 20,22-dihydroxy-21,23-dimethoxytetrahydrofuran. Such side-chains have been found in salvinicins A and B, two natural products from a plant in the Lamiaceae (Harding et al., 2005). The relative stereochemistry of 5 was determined by NOE experiments (Fig. 2) as for 3 and 4. In the NOE spectrum, H-17 correlated with H-21, H-22 and OMe-23. These correlations could suggest that these protons and the methoxy are oriented in the same face of the tetrahydrofuran ring. The

Optical rotations were measured, at room temperature, in CHCl3 on a JASCO DIP-370S. IR (Film) and UV (MeOH) spectra were recorded on JASCO FT/IR 5300 and Shimadzu UV-210A spectrophotometers, respectively. The 1H and 13C spectra were acquired at room temperature at 600 and 400 MHz on a JEOL FX-600 spectrometer, whereas HREIMS were obtained using a JEOL JMS XD303 instrument. Thin layer chromatography (TLC) was performed using E. Merck Silica gel 60 F254 precoated plates (0.25 mm), with column chromatography (CC) carried out using silica gel 60 (70– 230 mesh). Preparative high pressure liquid chromatography was performed on Waters lBondapak C18 column by using H2O:MeOH (30:70 ? 35:65, v/v) and H2O:MeCN (40:60 ? 45:65, v/v) as solvent systems. 4.2. Plant material Root bark of E. angolense was collected in July 2002 at Parc Botanique de Kisantu in the DR Congo. The plant material was identified by junior lecturer Paul Malumba Kamba of Faculty of Agriculture/University of Kinshasa. A voucher specimen (Devred 496) was deposited in the herbarium of Institut National pour l’Etude et la Recherche Agronomique of Kinshasa University. 4.3. Extraction and isolation of compounds 1–5 Air-dried root bark of E. angolense (1.3 kg) was extracted with MeOH (3  4 L) at room temperature for four weeks to give a crude extract (112.6 g) after solvent removal under vacuum. Upon addition of EtOAc (350 ml), two phases were obtained, and the insoluble part was removed by filtration. The filtrate was concentrated in vacuo to provide a complex mixture (38 g) which was suspended in MeOH:H2O (2:1) and extracted with CHCl3 (3  300 ml). The CHCl3 extract (4.4 g) was fractioned by silica gel CC with CH2Cl2:MeOH (1:99 ? 5:99, v/v) solvent system. The limonoid fraction (1.2 g), eluted with MeOH:CH2Cl2 (1:99, v/v), was further fractioned by middle pressure chromatography using n-hexane– acetone as solvent system, and the elution with 3:97, 6:94 and 10:90 v/v of the solvent system gave three main limonoid fractions. The first (351 mg) and the second fractions (82 mg) were purified

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through HPLC with H2O:MeOH (25:75 ? 30:70, v/v) followed by H2O:CH3CN (40:60 ? 45:55, v/v) gradient as eluent. The first fraction gave methyl angolensate 6 (74.6 mg), compound 3 (4.7 mg) and 6-acetoxymethyl angolensate 7 (2.1 mg), while the second fraction gave compound 4 (3.5 mg) and compound 5 (12.6 mg). The third and last limonoid fraction (112.1 mg) led to secomahoganin 8 (3.2 mg), compound 2 (5.1 mg) and compound 1 (2 mg) after purification through HPLC using successively H2O:MeOH (30:70 ? 35:65, v/v) followed by H2O:CH3CN (40:60 ? 45:55, v/ v) as eluent. 4.3.1. 5-Hydroxy-7-deacetoxy-7-oxogedunin (1) Amorphous solid; ½a25 D ¼ þ52:2 (c 0.18, CHCl3); UV (MeOH) kmax (log e) 215 (4.16) nm; IR (film) cmax 3643, 2935, 1795, 1640, 1550, 835 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRFAB-MS m/z 455.2092 ([M+H]+, calcd for C26H31O7, 455.2070). 4.3.2. 5,6-Dehydro-7-deacetoxy-7-oxogedunin (2) Amorphous solid; ½a25 D ¼ þ42:1 (c 0.13, CHCl3); UV (MeOH) kmax (log e) 239 (3.62) nm; IR (film) cmax 2929, 1780, 1650, 1538, 837 cm1; For 1H and 13C NMR spectroscopic data, see Table 1; HRFAB-MS m/z 437.1971 ([M+H]+, calcd for C26H29O6, 437.1964). 4.3.3. 6-Deacetoxydomesticulide D (3) Amorphous solid; ½a25 D ¼ 38 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 215 (4.12) nm; IR (film) cmax 3436, 1724, 1692, 933 cm1; for 1H and 13C NMR spectroscopic data, see Table 2; HRFAB-MS m/z 503.2286 ([M+H]+, calcd for C27H35O9, 503.2281). 4.3.4. 6-Deacetoxydomesticulide D 21-methylether (4) Amorphous solid; ½a25 D ¼ 42 (c 0.28, CHCl3); UV (MeOH) kmax (log e) 216 (4.49) nm; IR (film) cmax 2972, 1741, 1458, 935 cm1; for 1H and 13C NMR spectroscopic data, see Table 2; HRFAB-MS m/z 485.2203, ([MCH3OH]+, calcd for C27H33O8, 485.2175). 4.3.5. Entangosin (5) Amorphous solid; ½a25 D ¼ 27 (c 0.15, CHCl3); UV (MeOH) kmax (log e) 203 (3.90) nm; IR (film) cmax 3447, 2947, 1734, 1386, 812 cm1; for 1H and 13C NMR spectroscopic data, see Table 2; HRFAB-MS m/z 535.2524 ([MCH3OH]+), calcd for C28H39O10, 535.2543). 4.4. Antifeedant test The antifeedant activity of the isolated compounds was tested by the conventional leaf disk method using Chinese cabbage (Brassica campestris L. var chinensis) against the third-instar larvae of S. littoralis (Boisduval) (Wada and Munakata, 1968). Acknowledgment We would like to thank Mr. Crispin Mulaji and Mr. Paul Malumba, University of Kinshasa, for collection and identification of Meliaceae plants.

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