Phytochemistry 54 (2000) 107110

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New isoavones from Ceiba pentandra

F.N. Ngounou a, A.L. Meli a, D. Lontsi a, B.L. Sondengam a, Atta-Ur-Rahman b, M. Iqbal Choudhary b,*, Shahid Malik b, Farzana Akhtar b
b a Department of Organic Chemistry, Faculty of Science, University of Yaounde 1, P.O. Box 812, Yaounde, Cameroon International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan

Received 6 October 1999; received in revised form 28 December 1999

Abstract Two new isoavones, pentandrin (1) and pentandrin glucoside (2), were isolated from the stem barks of Ceiba pentandra along with b-sitosterol and its 3-O-b-D-glucopyranoside, which was isolated for the rst time from this plant. The structures of these compounds were elucidated with the help of spectroscopic techniques, while the structure of 1 was unambiguously conrmed by single-crystal X-ray diraction studies. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Isoavones; Ceiba pentandra; Pentandrin; Pentandrin glucoside; X-ray diraction

1. Introduction

2. Results and discussion Compound 1, C18H16O7, was isolated from the ethyl acetate extract of the stem bark of C. pentandra, as yellow solid crystals. The UV spectrum of 1 showed absorptions at 325, 262, 236, 203 nm indicating the presence of substituted aromatic rings and a, b unsaturated ketone in the molecule (Agrawal, 1989). The IR spectrum showed absorptions at 3404 (OH), 1658 (CO) and 1461 (C1C) cm1. The 1 H-NMR spectra [CDCI3, 400 MHz] of 1 (Table 1) showed signals characteristic of the isoavone moieties. Two 1H doublets at d 6.39 and 6.37 J6, 8 2X3 Hz) represented the aromatic H-8 and H-6 meta to each other. Two 1H doublets at d 6.72 and 6.68 J2 H , 6 H 1X8 Hz) were assigned to meta coupled aromatic H-6 ' and H-2 '. A 1H singlet at d 7.86 was assigned to H-2. Another 1H singlet appeared at d 12.8 was due to the hydrogen bonded C-5 hydroxyl proton. Three 3H singlets at d 3.91, 3.89 and 3.86 indicated the presence of three methoxyl groups. The 13 C-NMR spectra [CDCI3, 100 MHz, BB, DEPT, (Table 1)] of 1 showed resonances for all 18 carbons with three methoxy, ve methine and 10 quaternary carbons (Murthy and Rao, 1986). The oxygen-

Ceiba pentandra L. Gaertn. is a tree upto 50 m high which occurs in areas ranging from tropical America to Asia through Africa (Aubreville and Leroy, 1975). The plant is well reputed in the African traditional medicine for the treatment of many illnesses, such as, headache, dizziness, constipation, mental troubles and fever. It is also used as diuretic (Busson et al., 1965; Bouquet and Debray, 1974; Irvine, 1961). Previous work on C. pentandra described the isolation of a number of sesquiterpenoids (Rao et al., 1993). In our systematic search for active metabolites from Cameroonian medicinal plants, we have investigated the stem bark extract of this plant and isolated two new isoavones: pentandrin (1) and pentadrin 5'-O-b-D-glucoside (2), the structures of which were elucidated with the help of single crystal X-ray diraction technique as well as NMR spectroscopy.
* Corresponding author. Fax: +92-21-496-3373. E-mail address: [email protected] (M.I. Choudhary).

0031-9422/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 1 - 9 4 2 2 ( 0 0 ) 0 0 0 3 5 - 2

108 Table 1 1 H - and

F.N. Ngounou et al. / Phytochemistry 54 (2000) 107110

13

C-NMR for Compounds 1 (CDCl3) and 2 (DMSO) 1

1

Carbon number

2
13

Hd J (Hz)

Cd

Hd J (Hz)

13

Cd

2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 10 20 30 40 50 60 OCH3 OCH3 OCH3

7.86 (s ) 6.37, (d, 6.39, (d, 6.68, (d, 6.72, (d, 3.86 (s ) 3.89 (s ) 3.91 (s )

2.3) 2.3)

1.8)

1.8)

153.2 123.7 180.6 162.7 98.3 165.6 92.5 157.8 106.2 126.6 108.6 152.3 135.8 149.4 105.7 55.8 56.1 61.0

8.43 (s ) 6.95, (d, 6.98, (d, 6.60, (d, 6.37, (d, 4.91, (d, 3.74 (s ) 3.79 (s ) 3.84 (s )

2.3) 2.3)

1.8)

1.8) 7.5)

155.1 122.3 180.0 161.7 98.3 165.3 92.4 157.4 105.3 125.8 110.1 152.7 138.5 150.6 107.7 101.0 77.1 76.7 73.4 69.9 60.8 56.0 56.1 60.3

bearing carbons resonated at d 165.6 (C-7), 162.7 (C5), 157.8 (C-9), 153.2 (C-2), 152.3 (C-3 '), 149.4 (C-5 ') and 135.8 (C-4 '). The carbonyl carbon and the three methoxy carbons appeared at d 180.6, 61.0, 56.1 and 55.8, respectively. Direct one-bond 1 Ha13 C connectivities of 1 were established by HMQC (hetronuclear multiple quantum coherence) spectrum which showed that H-2 d 7.86) was connected with C-2 d 153.2). Similarly, H-6 d 6.37) and H-8 d 6.39) showed connectivity with C-6 d 98.3) and C-8 d 92.5), respectively. H-6 ' d 6.72) and H-2 ' d 6.68) showed HMQC connectivity with d 105.7 (C-6 ') and 108.6 (C-2 '), respectively. The long-range hetronuclear interactions observed in HMBC spectrum

(heteronuclear multiple bond connectivity) for 1 are shown in Fig. 1. The HREI MS of 1 showed the M+ at m/z 344.0845 corresponding to the molecular formula C18H16O7 (calcd. 344.0847) with 118 of unsaturation in the molecule, eight of which were accounted for by the two benzene rings, one by the pyrane ring, one by the carbonyl group and one by the double bond in the pyrane ring. The fragment at m/z 167.0294 [C8H6O4+ H]+ resulted from the retro Diels Alder cleavage indicated

Fig. 1. Important HMBC interactions in Compound 1.

Fig. 2. A computer generated ORTEP drawing of 1. The hydrogens shown are at arbitrary positions and rened isotropically.

F.N. Ngounou et al. / Phytochemistry 54 (2000) 107110

109

the presence of one hydroxyl and one methoxy substituent in ring A (Chibber and Sharma, 1979). On the basis of above spectral data, the compound 1 was deduced as 5,5 '-dihydroxy- 7,3 ',4 '-trimethoxy isoavone. Subsequent single-crystal X-ray diraction analysis (Fig. 2) conrmed structure 1 for this compound. The molecule showed planarity throughout the structure. The average CC bond distance in the aro matic rings is 1.386 (2) A. Compound (2), C24H26O12, was isolated as a yellowish amorphous solid. The UV spectrum of 2 showed absorptions at 325, 262, 237 and 204 nm (Agrawal, 1989). The IR spectrum showed absorptions at 3406 (OH), 1657 (CO) and 1463 (C1C) cm1. The 1 H-NMR [DMSO, 300 MHz, (Table 1)] spectrum of compound 2 was distinctly similar to 1, with additional signals of a sugar moiety. The anomeric signal appeared as a doublet at d 4.91 J1 HH , 2 HH 7X5 Hz), while the other methine protons of the sugar moiety appeared between d 3.195.35. The 13 C-NMR spectrum of 2 also showed similarity with that of 1 except for the addition of six carbon signals of a sugar moiety which appeared at d 101.0, 77.1, 76.7, 73.4, 69.9 and 60.8, assigned to C-10, C-20, C-30, C-40, C-50 and C-60, respectively. The substitution of the sugar moiety at C-5 ' was inferred by the important HMBC interactions between anomeric H-10 and C-5 '. The presence of a hydrogen-bonded OH signal at d 12.85 also indicated that there was only C5 ' position available for the attachment of sugar moiety. The HMBC interactions are shown in Fig. 3. The HREI MS of compound 2 showed the M+ at m/z 506.1472 corresponding to the molecular formula C24H26O12 (calcd. 506.1424) with 128 of unsaturation. The M+ was further conrmed by FAB MS [M + 1]+ at m/z 507.1472. The EI MS showed the highest peak at m/z 344.0847 [M+ 162] indicating the presence of a sugar moiety in the molecule, which was further conrmed by 1 H- and 13 C-NMR spectra. The rest of the mass fragmentation of 2 was similar to compound 1.

On the basis of above spectral data, compound 2 was deduced to be the glycoside of 1. 2.1. Acid hydrolysis of 2 Compound 2 (5 mg) was dissolved in MeOHdistilled H2O (1:1). Then, 5% HCl (5 ml) solution was added in it and the solution was reuxed for 7 h at 608C. After cooling, MeOH was evaporated in vacuo. The reaction mixture was extracted thrice with chloroform to remove the aglycone part of the molecule, which was identied as 1 on the basis of Co-TLC, EI MS and 1 H-NMR. The residue obtained after removal of the acid was compared with standard sugars on silica gel plates (E. Merck Art. No. 5554) using n(7:20:12:7:6). BuOHEtOAc-iso-PrOHHOAcH2O The TLC was developed through multiple run. Spots were visualized with aniline phthalate reagent and the sugar was identied to be D-glucose. 3. Experimental Vacuum liquid chromatography (n-hexane:ethyl acetate and ethyl acetate:methanol) was performed using silica gel (type 60, Merck). Thin layer chromatography was carried out using Merck precoated silica gel sheets (0.25 mm, 60 F254). Column chromatography was performed using silica gel (230400 mesh). Ceric sulphate spray reagent and UV light (366 nm) were used for the detection of the compounds. UV spectra were recorded on a Hitachi UV 3200 spectrophotometer. IR spectra were recorded on a JASCO 302-A spectrophotometers. Melting points were measured on a Gallenkamp melting point apparatus and were uncorrected. FAB MS measurements were carried out on a JEOL-HX 110 mass spectrometer. EI MS were recorded on a Varian MAT 311A mass spectrometer. The 1 H-NMR spectra were recorded on Bruker AM 400 and 300 MHz NMR spectrometers, while 13 C-NMR spectra were recorded at 100 and 75 MHz on the same instruments. Single crystal X-ray data were collected on Bruker P4 diractometer (previously Nicolet). 3.1. Crystal data and X-ray crystal structure determination of pentandrin (1) Crystals of 1 suitable for X-ray analysis were obtained by recrystallization from methanol. 3.2. Crystal data Light yellow prismatic crystals, dimensions 0.30 0.25 0.30 mm3, C18H16O7; M+ = 344.0847 amu; Monoclinic, b 97X630 (2)8, a 7X8110 (10), b

Fig. 3. Important HMBC interactions in Compound 2.

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F.N. Ngounou et al. / Phytochemistry 54 (2000) 107110

7X9510 (10), c 25X177 (2) A, V 1549X8 (3) A3; space 3 group P21, Z 4, Dx 1X476 Mg m , m 0X970 mm1, F000 720; temperature = 293 (2) K. 3.3. Data collection Bruker P4 diractometer (previously Nicolet), y-2y scan type, graphite-monochromated CuKa radiations; 3621 reections were measured 3X5 ` y ` 1358 of which 2877 were unique. Three standard reections were measured after every 97 reections, which showed no signicant crystal decay. Data was corrected for Lorentz and polarization eects. 3.4. Structure analysis and renement The crystal structure was solved by direct methods and rened by full-matrix least squares on F 2 values [SHELXTL vr. 5.03]. Non-hydrogen atoms were rened with anisotropic temperature factors. Hydrogen atoms were included at calculated positions and rened in the riding mode. Final values of the residuals R and wR2 [for 2877 reections with I b 2sI were 0.0469 and 0.1345, respectively. The highest and lowest peaks in nal dierence Fourier map were 0.274 and 0.255 eA3. Table of atomic coordinates, bond lengths, bond angles and list of structure factors have been deposited at the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 IEZ, UK. 3.5. Plant material The stem barks of Ceiba pentandra L. Gaertn. were collected at Mvolye Hill, Yaounde zone, Central Province of Cameroon, in May, 1998. A voucher specimen (# HNC 43623) was deposited at the National Herbarium (Yaounde, Cameroon). 4. Extraction and isolation The stem barks of the plant was cut into small pieces, air-dried and pulverized. The resulting powder (10.3 kg) was macerated thrice at room temperature with MeOH for three days. The MeOH extracts were combined and concentrated to dryness under reduced pressure to obtain a solid (750 g), which was successively fractionated with n-hexane, dichloromethane and ethyl acetate. The ethyl acetate fraction showed moderate antifungal activity against Trichophyton schoenleinii, T. simii and Microsporum canis fungi. The

ethyl acetate extract (50 g) was puried by vacuum liquid chromatography on silica gel (250 g). Elution with n-hexaneethyl acetate (85:15) aorded pentandrin (1) (15.1 mg, 1.46 104% yield) as yellow crystals in MeOH. Elution with n-hexaneethyl acetate (1:4) furnished (2) (13.3 mg, 1.30 104% yield). During this investigation, b-sitosterol and its 3-O-b-Dglucopyranoside were also isolated. Pentandrin (1), C18H16O7; yellow solid crystal; m.p. 1591608; UV (MeOH) lmax : 325, 262, 236, 203 nm; IR nKBr cm1: 3404 (OH), 1658 (CO), 1461 (C1C) max cm1. HREI MS: m/z: 344.0845 (calcd. 344.0847) [C18H16O7], 167.0294 [C8H7O4, calcd. 167.0340]; 1 HNMR (400 MHz, CDCl3): d Table 1, 13 C-NMR (100 MHz, CDCl3): d Table 1. Pentandrin glucoside (2), C24H26O12; yellow amorphous; m.p. 1801828; UV (MeOH) lmax : 325, 262, 237 204 nm; IR nKBr cm1: 3406 (OH), 1657 (CO), 1463 max (C1C) cm1. HREI MS: m/z: 506.1472 (calcd. 506.1424) [C24H26O12], 344.0845 (calcd. 344.0847) [C18H16O7], 167.0294 [C8H7O4, calcd. 167.0340]; 1 HNMR (300 MHz, DMSO): d Table 1, 13 C-NMR (75 MHz, DMSO): d Table 1.

Acknowledgements The travel of F.N.N. was supported by the Third World Academy of Sciences (Italy). We are also thankful to Dr. Paulette Bisseck for providing the plant material. One of us (Shahid Malik) acknowledge the nancial support of COMSATS, Islamabad.

References
Agrawal, P.K., 1989. Carbon-13 NMR of avonoids. Elsevier Science Publishers, Amsterdam, p. 5. Aubreville, A., Leroy, J.J., 1975. Flore du Cameroun 19, Museum National d' Histoire Naturelle, Laboratoire de Phanerogamie, Paris, 11. Bouquet, A., Debray, M., 1989. Plantes Medicinales de la Cote d'Ivoire. ORSTOM, Paris, p. 52. Busson, F., Jagger, P., Lunuen, P., Pinta, M., 1965. Plantes Alimentaires de l'Ouest Africain, Etude Botanique et Chimique. ORSTOM, Paris, p. 308. Chibber, S.S., Sharma, R.P., 1979. Phytochemistry 18, 1583. Irvine, F.R., 1961. Woody plants of Ghana. Oxford University Press, London, p. 190. Murthy, M.S.R., Rao, E.V., 1986. Magnetic Resonance in Chemistry 24, 225. Rao, K.V., Sreeramulu, K., Gunasekar, D., 1993. J. Nat. Prod. 56, 2041.