Phytochemistry 64 (2003) 575–581 www.elsevier.com/locate/phytochem Diterpenoids from Neoboutonia glabrescens (Euphorbiaceae) Alembert T. Tchindaa,1, Apollinaire Tsopmoa, Mathieu Tenea, Pierre Kamnainga, David Ngnokama, Pierre Tanea, Johnson F. Ayafora,,, Joseph D. Connollyb,*, Louis J. Farrugiab a Department of Chemistry, University of Dschang, Box 67, Dschang, Cameroon b Chemistry Department, The University of Glasgow G12 8QQ, Scotland, UK Received 29 January 2003; received in revised form 27 February 2003 Dedicated to the memory of Professor Jeffrey B. Harborne Abstract Glabrescin, a daphnane diterpenoid, neoboutonin, a degraded diterpenoid with a novel skeleton, and neoglabrescins A and B, two rhamnofolane derivatives, have been isolated from the stem bark of Neoboutonia glabrescens Prain (Euphorbiaceae), together with the known tigliane derivative, baliospermin, and the known daphnane, montanin. Other constituents include squalene, 3-acetylaleuritolic acid, oleanolic acid and sitosterol, and the phenolic compounds 9-methoxy-1,7-dimethylphenanthrene and 2,3,8tri-O-methylellagic acid. The structures were assigned on the basis of spectral studies and comparison with published literature data. The structures of neoglabrescins A and B were derived for their acetylated derivatives and, in the case of neoglabrescin A, confirmed by X-ray crystallographic analysis. # 2003 Elsevier Ltd. All rights reserved. Keywords: Neoboutonia glabrescens; Euphorbiaceae; Glabrescin; Daphnanes; Neoboutonin; Rhamnofolanes; Neoglabrescins A and B 1. Introduction The genus Neoboutonia (Euphorbiaceae) is widely distributed in tropical West Africa and represented by the species N. diaguissensis Beille, N. manii Benth, N. glabrescens Prain and N. melleri Prain var vellutina Prain. These species, with the exception of N. diaguissensis, grow in the anglophone part of Cameroon (Hutchison, 1958). The chemistry of this genus has not been extensively studied. However, tigliane derivatives and triterpenoids have been reported (Zhao et al., 1998) from the leaves of N. melleri. N. glabrescens Prain is a soft wooded tree of about 1.7 m height, which grows in open spaces in forests (Hutchison, 1958). It has skin irritant properties and is used in Cameroon ethnomedi- * Corresponding author. Tel.: +44-141-330-5499; fax: +141-3304888. E-mail address: joec@chem.gla.ac.uk (J.D. Connolly). 1 Present address: IMPM, Centre for the Study of Medicinal Plants and Traditional Medicine, Yaoundé, Cameroon. , Deceased. 0031-9422/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0031-9422(03)00158-4 cine against worms, abdominal and stomach pains, and malaria (Thomas et al., 1989). 2. Results and discussion In the course of our on-going research on Cameroonian medicinal plants used traditionally to treat human parasitic diseases (Tchuendem et al., 1999; Ayafor et al., 1994) we have studied the CH2Cl2–MeOH (1:1) extract of the stem bark of Neoboutonia glabrescens Prain. In addition to the known daphnane montanin (2), the known tigliane baliospermin (Ogura et al., 1978), 9-methoxy-1,7-dimethylphenanthrene (Long et al., 1997), 2,3,8-tri-O-methylmethylellagic acid (Nawwar et al., 1994; Yazaki and Hillis, 1976), 3-acetylaleuritolic acid (Woo and Hildebert, 1977; McLean et al., 1987), oleanolic acid, squalene, and sitosterol, two compounds glabrescin (1) and neoboutonin (3) were isolated. Acetylation of a polar fraction from the CH2Cl2–MeOH (1:1) extract of the stem bark with a mixture of pyridineacetic anhydride afforded the acetates of two new rhamnofolane derivatives, neoglabrescins A (4) and B 576 A.T. Tchinda et al. / Phytochemistry 64 (2003) 575–581 (5). The structure of neoglabrescin A was confirmed by X-ray crystallographic analysis.
Glabrescin was obtained as a yellow oil, [a]22 D +82.0 (c 0.35, CHCl3). The molecular formula was deduced as C48H79O9 from analysis of the 13C NMR and DEPT data and the EI mass spectrum (m/z 798, [M]+). The IR spectrum showed characteristic bands at 3465 (hydroxyl), 1763 (ester) and 1697 cm1 (a,b-unsaturated cyclopentenone). The proton and carbon signals (Table 1), which were very close to those of the daphnane derivative montanin (2) (Ogura et al., 1978), were assigned unambiguously to the daphnane diterpenoid framework using a combination of 1H–1H COSY, HMQC and HMBC experiments. It was apparent that glabrescin contained an ortho-ester function (
119.8) and an ester attached to C-20. In the HMBC spectrum the ester carbonyl (
173.8) showed correlations to the characteristic AB proton pattern of 2H-20 [
3.85 (d, J=11.9 Hz, H-20A) and 4.78 (d, J=11.9 Hz, H-20B)]. Of particular note in the 1H NMR spectrum were the resonances of two primary methyl groups (6H,
0.90, t, Table 1 NMR spectral data of glabrescin (1) and montanin (2) in CDCl3 1 13 C NMR spectral data of 2 Carbon
C (ppm)
H HMBC correlations
(multiplicity, J) (H to C) (ppm) 1 2 3 4 5 6 7 8 9 10 11 12 161.5 136.9 210.1 72.6 70.3 59.6 64.5 36.9 79.1 48.4 35.1 36.8 7.61 13 14 15 16 17 18 19 20 84.4 82.1 146.7 111.6 19.4 20.7 10.3 66.2 10 20 120 100 200 –(CH2)n– 1600 119.8 35.2 14.5 173.8 34.5 23.1–32.3 14.5 a 4.28, s 3.34, s 2.92 (d, 2.5) 3.80a 2.49, m 1.67/2.22 (dd, 14.3, 8.7) 3, 4, 9, 19 161.7 137.0 210.3 72.6 3, 4, 6, 7, 10, 20 72.3 60.8 5, 6, 8, 9, 14, 20 64.7 6, 7, 9, 10, 11, 13, 14 37.0 79.1 2, 3, 5, 11 48.5 8, 9, 10, 13, 18 35.2 9, 14, 15, 18 36.8 4.36 (d, 2.5) 7, 9, 9, 12, 13, 15 4.91/ 5.04, s 1.80, s 1.18 (d, 7.1) 1.82, s 3.85/4.78 (d, 11.9) 13, 15, 17 13, 15, 16 9, 11, 12 1, 2, 3 5, 6, 1 1.96/1.96a 0.90a 84.5 82.1 146.6 111.5 19.4 20.7 10.3 65.6 119.7 35.2 14.5 2.34 (t, 7.4) 23.1–32.3 0.90a Coupling constants not determined due to overlapping. J=7.0 Hz, Me-120 and Me-1600 ), one doublet methyl (
1.18, d, J=7.1 Hz, Me-18), two vinyl methyls [
1.80 (Me-17) and 1.82 (Me-19)], two methylene protons [
4.91 (H-16A) and 5.04 (H-16B)] and a deshielded olefinic proton [
7.61 (H-1)]. In addition to the methylene proton signals observed for 2H-12, integration identified 48 other methylene protons overlapping at
1.27–1.29 and 1.61–1.64 which could be assigned to the side chains. The presence of the ester carbonyl, the orthoester carbon and two primary methyl groups suggested the existence of two fatty chains. The mass fragments observed in the EI mass spectrum at m/z 183 and 239 could be assigned to lauroyl (CH3(CH2)10CO+) and palmitoyl (CH3(CH2)14CO+) ion fragments respectively. Daphnane derivatives with an ortho-ester involving a palmitoyl moiety have not yet been reported. In contrast, 20-palmitoyloxy daphnane diterpenoids are commonly found in the Euphorbiaceae (Kupchan et al., 1976; Adolph et al., 1984; Jolad et al., 1983). Since montanin (2) also occurs in this extract, it is reasonable to assume that glabrescin is montanin 20-palmitate (1). Neoboutonin 3 was obtained as pale yellow crystals from MeOH–CH2Cl2. The EIMS showed a molecular ion peak at m/z 286 while 1H- and 13C-NMR spectra (Table 2) indicated the presence of sixteen nonexchangeable protons, two exchangeable protons and seventeen carbon atoms. This was consistent with the molecular formula C17H18O4, whose nine double bonds equivalents could be accommodated by a ketone, a naphthalene ring system and an additional ring. The UV spectrum showed absorption maxima at lmax 237 and 334 nm, corresponding to a conjugated aromatic system. The 1H NMR spectrum of 3 revealed the presence of three methyl singlets at
1.12, 1.45 and 2.24 Table 2 1 H and 13C NMR spectral data of neoboutonin (3) Carbon
C 1 3 4 5 6 7 8 9 10 11 12 13 14 15 18 19 OMe 205.3 64.6 44.7 167.6 97.9 146.6 119.6 132.6 120.9 107.2 159.2 128.2 125.5 17.5 26.3 26.9 57.0
H HMBC connectivities (H to C) 4.06 (s) 1, 4, 18, 19 6.73 (s) 4, 5, 7, 8, 10 8.12 (s) 8, 10, 12, 13 7.87 2.24 1.45 1.12 4.00 7, 9, 12, 15 12, 13, 14 3, 4, 5, 19 3, 4, 5, 18 7 (s) (s) (s) (s) (s) 577 A.T. Tchinda et al. / Phytochemistry 64 (2003) 575–581 biaceae) (Kokpol et al., 1990) and the phenanthrene derivatives from Domohinea perrieri (Euphorbiaceae) (Long et al., 1997) are also all degraded diterpenoids. Acetylation of a polar fraction of the crude extract followed by chromatography afforded the acetates of two rhamnofolane derivatives neoglabrescins A (4) and B (5). Neoglabrescin A tetraacetate (4a) had a molecular formula C25H32O11 as deduced from its EIMS, which showed a molecular ion peak at m/z 508. Its spectroscopic properties (Table 3) clearly indicated its trisnorditerpenoid nature. Its 13C and DEPT spectra revealed seventeen skeletal carbons consisting of two methyl groups, three methylenes, seven methines and five quaternary carbon atoms, including a carbonyl group, a vinyl carbon and three oxygenated carbons. The compound was a tetraacetate as shown by the presence of four methyl singlets at
2.01, 2.08, 2.08 and 2.19 correlating to four ester carbonyls in the HMBC spectrum. The placement of three of these acetoxy groups was achieved using the HMBC correlations observed and a methoxyl group at
4.00. The substituents on the carbon skeleton were positioned using the correlations observed in the HMBC spectrum. Especially important were those between H-3 and C-1, C-10, C-18, and C-19, H-6 and C-5, C-7, C-8, and C-10, H-11 and C-10 and C-12 and H-14 and C-7 and C-12. The proposed structure 3 was further supported by the correlations observed in the NOE difference spectra. Irradiation of the methyl at
2.24 (Me-15) resulted in an increase of the intensity of H-14. The H-3 (
4.06) proton showed NOEs with both Me-18 (
1.45) and Me-19 (
1.12), that with the former being greater. The absolute configuration of the sole chiral center was not determined. It seems likely that neoboutonin (3), which has a novel carbon skeleton, is closely related to 1,7-dimethyl-9-methoxyphenanthrene (Long et al., 1997), another constituent of the extract, and that they both are isoprenoid in origin. The numbering system used for neoboutonin reflects its putative biogenetic origin. It is reasonable to assume that trigonostemone from Trigostemon reidioides (Euphor- Table 3 1 H and 13C NMR spectral data of compound 4a (CDCl3) and 5a (CD3OD) 4a 5a Carbon
C
H Mult (J in Hz)
C
H Mult (J in Hz) HMBC (H!C) 1 2 3 4 5 6 7 8 9 10 11 12a 12b 13 14a 14b 15 16 17 18 19 20a 20b CH3CO– 124.2 141.5 77.3 94.3 78.4 88.7 76.9 45.8 70.7 55.8 39.5 45.8 5.50 – 5.85 – 6.34 – 4.17 2.36 – 2.95 2.18 2.29 2.45 – 3.06 2.33 d (1.6) 128.8 145.5 81.6 81.4 83.3 84.2 79.2 61.4 73.8 51.8 42.8 49.6 5.75 – 5.59 – 5.03 – 4.08 2.97 – 2.97 2.39 2.06 2.23 – 3.20 s 4.19 s 1, 2,–OAc s 7, 10,–OAc CH3CO– 208.8 39.5 – – – 14.3 13.6 62.7 170.8 170.7 170.5 170.0 21.6 21.2 21.1 21.1 1.00 1.67 4.95 4.09 – – – – 2.01 2.08 2.19 2.08 d (1.4) s d (3.8) overlapping bs overlapping overlapping dd (15.4, 12.2) t (15.3) dd (15.5, 1.5) 209.3 58.8 84.2 30.5 26.0 17.9 13.6 64.9 173.5 172.8 172.2 – 1.24 1.18 0.92 1.50 4.65 4.31 – – – 21.8 21.5 21.2 1.91 1.96 2.09 d (6.6) t (1.3) d (12.5) d (12.5) s s s s Assignments are based on HMBC, HMQC and 1H–1H COSY experiments. d (10.7) dd (10.8, 10.7) bs m overlapping dd (16.3, 11.8) overlapping 15, 17 15, 16 9, 11, 12 1, 2 s s d (9.2) s d (11.8) d (11.8) –OAc s s s –OAc –OAc –OAc 578 A.T. Tchinda et al. / Phytochemistry 64 (2003) 575–581 between H-3 (
5.85, d, J=1.4 Hz), H-5 (
6.34, s), H20a (
4.95, d, J=12.5 Hz), H-20b (
4.09, d, J=12.5 Hz) and three carbonyl esters. The IR spectrum showed a strong band at 3628 cm1 corresponding to a hydroxyl group whose proton correlated in the HMBC spectrum with the carbon atom at
70.7 (C-9). These observations enabled us to attach the fourth acetoxy group at C-6 (
88.7). The vinyl proton (
5.50, d, J=1.6 Hz, H-1) and methyl (
1.67, t, J=1.3 Hz, Me-19) form part of the methylcyclopentene ring commonly found in diterpenoids from the Euphorbiaceae. The HMBC correlations observed between H-1 and C-2 (
141.5) as well as between Me-19 and C-1 (
124.2) further confirmed the presence of the double bond. The 1H–1H COSY spectrum delineated the partial connectivities H7/H-8/2H-14 and Me-18/H-11/2H-12. The chemical shifts of H-12a (
2.29, m), H-12b (
2.45, dd, J=15.4, 12.2 Hz), H-14a (
3.06, t, J=15.3 Hz) and H-14b (
2.33, dd, J=1.5, 15.5 Hz) showed that they were adjacent to a carbonyl function (
208.8, C-13). This was confirmed by the cross-peaks observed in the HMBC spectrum between H-14 and C-13. The secondary methyl group Me-18, a common feature of the cyclohexane ring of these derivatives appeared at
H 1.00 (d, J=6.6 Hz). An uncommon feature was the presence of a C-4/C-7 ether linkage. HMBC correlations were observed between H-1, H-7 and C-4 (
94.3), enabling us to suggest the presence of this bridge. The relative stereochemistry of 1 was determined by NOE experiments. Irradiation of H-3 enhanced the intensities of H5, H-10 and Me-19. NOEs were also observed between H-8, H-11 and H-12a, H-7, H-14b and H-20b as well as between H-5, H-3, H-10 and H-20a. The ether linkage was thus deduced to be b-oriented. Although rhamnofolane diterpenoids have been isolated from the Euphorbiaceae family (Stuart and Barrett, 1969; Jakupovic et al., 1988) this is the first time they have been found in the genus Neoboutonia. The structure of 4a was confirmed by a single-crystal X-ray analysis (Fig. 1). Thus neoglabrescin A has the structure and stereochemistry shown in (4) and appears to have been derived by attack of a 4b-OH on a 6a,7a-epoxide precursor. The IR spectrum of the unacetylated mixture containing neoglabrescin A showed only ketonic carbonyl absorption, indicating that compound 4 had no esters present. Neoglabrescin A is a new trisnor-rhamnofolane derivative with an unusual 4,7-ether linkage. The loss of three carbons is readily explained by a retroaldol reaction of the typical C-14 hydroxyisopropyl group of rhamnofolane derivatives. The FABMS of neoglabrescin B triacetate (5a) displayed pseudomolecular ion peaks [M+Na]+ and [M+H]+ at m/z 547 and 525, respectively, consistent with the molecular formula C26H36O11. Three acetoxy groups were identified in the 1H NMR spectrum as methyl singlets at
1.91, 1.96 and 2.09 showing HMBC correlations with ester carbonyls at
172.2, 172.8 and 173.5. The twenty remaining carbon atoms, consisting of four methyl groups, two methylenes, eight methines and six quaternary carbons, were assigned to a rhamnofolane diterpenoid framework. Similarities were observed between the NMR spectral data of compound 5a (Table 3) and those of 4a, with the additional presence of an isopropyl group including two methyl singlets at
1.18 (Me-17) and 1.24 (Me-16) directly attached to a downfield oxygen-bearing carbon at
84.1 (C-15). The 1H NMR and DEPT spectra showed that C-14 was a methine, bearing the isopropyloxy group. Moreover, the chemical shift of C-4 (
94.3) shifted to 81.4 ppm in 5a, indicating the lack of esterification at this position. The large coupling constant (J=10.7 Hz) between H-7 and H-8 showed that the two protons were trans. NOE interactions were observed between Me-16, H-14 and H-7, Me-17, H-20b and H-8 as well as between H-7, H-5 and H-14. These observations led to the conclusion that the isopropyloxy group was attached to C-7 through an ether linkage. The downfield chemical shift of C-15 (
84.2) was consistent with this conclusion. The ether ring was deduced to be b-oriented. HMBC correlations between the protons at
5.59 (s, H-3), 5.03 (s, H-5), 4.31 (d, J=11.8, H-20b) and 4.65 (d, J=11.8 Hz, H-20a) and the ester carbonyls showed that the acetates were attached at C-3, C-5 and C-20. The remaining carbons and protons were assigned by analysis of further 1H, 13C NMR, 1H–1H COSY, HMQC and HMBC data and by comparison with the NMR spectral data of 4a. Thus neoglabrescin B (5) is a new rhamnofolane derivative which appears to have been derived by attack of a 15OH on a 6a,7a-epoxide precursor. Fig. 1. ORTEP diagram of neoglabrescin A tetraacetate (4a). A.T. Tchinda et al. / Phytochemistry 64 (2003) 575–581 3. Experimental 3.1. General experimental procedures Optical rotations were measured on an AA Series Automatic Polaar 2000 polarimeter. Melting points were determined by means of a Reitchert apparatus and are uncorrected. Mass spectra (70 eV) were recorded with a Jeol JMS 700 apparatus. The UV spectra were obtained with a Shimadzu 3101 PC instrument and the IR spectra determined with a Jasco FT-IR 410 apparatus. 1H (400.6 MHz) and 13C (100.13 MHz) Nmr spectra were recorded in CDCl3 (with its signals at
7.25 and 77.0 ppm as standard reference) or in CD3OD (with its signals at
3.21 and 49.4 ppm as standard reference) with a Brüker DPX 400 apparatus. NMR data acquisition and processing were performed with the aid of the XWIN NMR software package. NOE experiments were carried out using a Brüker AM 360 instrument. For MPLC, the chromatotron ser. no. 36B connected to a FMI pump QD (flow rate 10 ml/mn) was used with plates (2 mm) prepared with silica gel 60 PF254 contain- 579 ing CaSO4. CC was run on Merck silica gel 60 and Sephadex LH-20, while TLC was carried out on silica gel 60 GF254 pre-coated plates with detection accomplished by spraying with 50% H2SO4 followed by heating at 100
C. 3.2. Plant material The stem bark of Neoboutonia glabrescens, Prain was collected at Mundemba (South-West, Cameroon) in July 1997. Mr. Paul Mezili, a retired botanist of the Cameroon National Herbarium, authenticated the plant material. Voucher specimens (BUD 0407) have been deposited at the Herbarium of the Botany Department of the University of Dschang. 3.3. Extraction and isolation The dried and ground stem bark (2 kg) of N. glabrescens was extracted with a mixture of MeOH–CH2Cl2 (1:1) (4 l) to yield a crude organic extract (120 g) on drying. This extract was dissolved in MeOH–H2O (1:4) 580 A.T. Tchinda et al. / Phytochemistry 64 (2003) 575–581 and extracted sequentially with CH2Cl2, EtOAc and n-BuOH. The combined CH2Cl2 and EtOAc extracts (68 g) were subjected to CC on Si gel, eluting with hexane-EtOAc followed by EtOAc–MeOH of increasing polarities to afford four main fractions A–D. Fraction A (1.5 g, eluted with EtOAc–hexane 1:4) gave squalene (10 mg) and sitosterol (14 mg). Fraction B (3 g, eluted with EtOAc–hexane 2:3) was passed over a Si gel column with CH2Cl2 as eluent to yield a sub-fraction which was further purified by gel permeation through Sephadex LH-20 [MeOH–CH2Cl2 (1:4)] to give glabrescin (1) (53 mg) as an orange oil and 3-b acetylaleuritolic acid (8 mg). Fraction C (3.5 g) eluted from the column with EtOAc–hexane (3:2) was separated on a Si gel column using mixtures of EtOAc–hexane of increasing polarity followed by repeated gel permeation chromatography through Sephadex LH-20 [CH2Cl2–hexane (1:4)] to give montanin (2) (700 mg), oleanolic acid (11 mg), baliospermin (50 mg), and a mixture which was purified by prep tlc [Me2CO-CH2Cl2 (3:17)] to afford neoboutonin (3) (13 mg). Finally, fraction D (4 g, eluted with MeOH–EtOAc 1:9) was purified over a column with Me2CO–CH2Cl2 (1:9) to give 2,3,8-tri-O-methylellagic acid (11 mg) and 9-methoxy-1,7-dimethylphenanthrene (14 mg) as a white powder. The crude extract was passed through a silica gel column, eluting with hexane–EtOAc and EtOAc–MeOH mixtures of increasing polarity. The polar fraction (450 mg) obtained with EtOAc–MeOH (95:5) was treated with pyridine-acetic anhydride (1:1; 50 ml) and left overnight at rt. Concentration under reduced pressure yielded an acetylated mixture which was purified by MPLC using the chromatotron with CH2Cl2–MeOH of increasing polarity as eluent. The fraction obtained with 2% CH2Cl2–MeOH furnished neoglabrescin A tetraacetate (4a) (32 mg) while the 3% CH2Cl2–MeOH afforded neoglabrescin B triacetate (5a) (6 mg). 3.3.1. Glabrescin (1) Orange oil; [a]24 D +82 (c 0.35, CHCl3); IR (CHCl3) nmax 3465, 1736, 1697, 1458, 1378, 1159, 1115 cm1; 1H and 13C NMR data see Table 1; EIMS (70 eV) m/z (rel. int.) [M]+ 798 (6), 770 (11), 643 (1), 615 (2), 599 (5), 571 (10), 548 (2), 543 (9), 542 (6), 527 (17), 499 (13), 342 (32), 325 (70), 283 (47), 183 (35), 161 (33), 57 (100); anal. C 72.15%, H 9.82%, calc. for C48H79O9, C 72.14%, H 9.84%. 3.3.2. Neoboutonin (3) Pale yellow crystals (hexane–EtOAc); mp 277–278
C; [a]20 D 41 (c 0.2, MeOH); UV MeOH lmax (log e) 334 (3.3), 237 (2.8); IR (CHCl3) nmax 3430, 1628, 1105, 470 cm1; 1H and 13C NMR data see Table 2. EIMS (70 eV) m/z (rel. int.) [M]+ 286 (80), 271 (100), 256 (50), 255 (65), 241 (50), 227 (12), 211 (12), 149 (20), 57 (20); anal. C 71.33%, H 6.33%, calc. for C17H18O4, C 71.31%, H 6.34%. 3.3.3. Neoglabrescin A tetraacetate (4a) Colorless crystals from acetone/petroleum ether; mp
247–248
; [a]20 D 64.9 (c 0.7 CHCl3); IR nmax (KBr): 3628, 2977, 1745, 1515, 1422, 1363,1227, 1046 cm1; 1H (400.6 MHz) and 13C (100.13 MHz) NMR see Table 3; EIMS m/z 508 [M+ C25H32O11] (0.5), 466 (1), 448 (25), 406 (3), 388 (12), 328 (60), 286 (80), 268 (100), 263 (52), 221 (75), 163 (30), 150 (33), 108 (62). 3.3.4. Neoglabrescin B triacetate (5a) Colorless crystals from acetone/petroleum ether; mp 215–216
; [a]20 D +8.9 (c 0.09 MeOH); IR nmax (KBr) 3439, 2969, 1743, 1689, 1631, 1371, 1260, 1060 cm1; 1H and 13C NMR see Table 3; FABMS m/z 547[ M+Na]+ (67), 525 [M+H]+ (28), 524 [M+,C26H36O11, absent], 507 (3), 465 (2), 445 (3), 405 (2), 387 (3), 307 (9), 289 (13), 273 (6), 195 (6), 176 (9), 154 (100). 4. Experimental details of crystal structure determination Details of data collection procedures and structure refinements are given in Table 4. A single crystal of suitable size was attached to a glass fibre using silicone grease, and mounted on a goniometer head in a general position. The crystal was cooled over a period of 0.5 h in the cold stream of the Oxford instruments Cryostream. Data were collected on a Enraf-Nonius KappaCCD diffractometer, running under Nonius collect software, and using graphite monochromated X-radiation (l=0.71073 Å) precise unit cell dimensions were determined by post-refinement of the setting angles of a significant portion of the data using Scalepack (Otwinowski and Minor, 1997). The frame images were integrated using Denzo (SMN) and resultant raw intensity files processed using a locally modified version of DENZOX. No absorption corrections were deemed necessary. Data were sorted and merged using SORTAV (Blessing, 1997). The structures were solved by direct methods using SIR-97. All non-H atoms were allowed under anisotropic thermal motion. C–H hydrogen atoms were included at calculated positions, with C–H=0.96 Å, and were refined with a riding model and with Uiso set to 1.2 times of the attached C-atom. The O-H hydrogen atoms were found from difference maps and refined with a riding model. Refinement with SHELXL97-2 (Sheldrick, 1997) using full-matrix leastsquares on F2 and all the unique data and with the weighting scheme w=[s(Fo)2+(AP)2+BP]1 where P=[F2o/3+2F2c /3] and A=0.0405, B=0.1822 converged to the residuals shown in Table 4. The absolute configuration could not be determined experimentally from refinement of the Flack absolute parameter, and the known absolute configurations were assigned. Calculations using Platon indicated that there were no voids in A.T. Tchinda et al. / Phytochemistry 64 (2003) 575–581 Table 4 Crystallographic data of 4a Compound formula C25H32O11 Compound color Colorless Mr 508.51 Space group P212121 crystal system Orthorhombic a/Å 8.9990 (3) b/A 9.9599 (3) c/Å 28.5395 (11) 2557.97 (15) V/Å3 Z 4 Dcalc/gcm3 1.32 F(000) 1080 m(MoKa)mm1 0.104 Temperature/K 100 Crystal size/mm 0.20.20.02  angle/deg 2.17–27.08 No. of data collected 9938 No. of unique data 5345 hkl range 11!11;-12!12;-36!36 0.0514 Rint No. of data in refinement 5345 No. of refined parameters 332 Final R[I>2s(I)](all data) 0.0594 (0.1226) 0.0975 (0.116) R2w[I>2s(I)](all data) Goodness of fit S 1.036 Flack absolute structure parameter 0.9 (12) Largest remaining feature in election 0.239–0.246 density map/eA3 Max shift/esd in last cycle 0.001 R ¼ ðjF0 j  jFC jÞ=ðFo ÞwR2 n 
2 o1=2 ¼  w Fo2  F2c   Rint ¼ Fo2  Fc2 ðmeanÞ=Fo2 (summation is carried out only where more than one symmetry equivalent is averaged). the lattice capable of containing any solvent molecules. Thermal ellipsoids were obtained using the program ORTEP-3 for Windows (Farrugia, 1997). All calculations were carried out using the WinGX package of crystallographic programs (Farrugia, 1999). Crystallographic data for structure 4a have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC173033. Copies of the data can be obtained free of charge on application to CCDC, e-mail: deposit@ccdc.cam.ac.uk. Tables of observed and calculated structure factors are also available from L. J. Farrugia on request. Acknowledgements The authors are grateful to IPICS (International Programme in the Chemical Sciences) for financial support including a travel grant to the University of Glasgow (A.T.T.). This paper is also dedicated to the memory of Professor Johnson Foyere Ayafor who died in a car accident on November 10, 2000. 581 References Adolph, W., Hecker, E., 1984. On the active principles of the spurge family. X. Skin irritants, cocarcinogens, and cryptic cocarcinogens from the latex of the Manchineel tree. J. Nat. Prod. 47, 482–496. Ayafor, J.F., Tchuendem, M.H.K., Nyasse, B., Tillequin, F., Anke, H., 1994. Novel bioactive diterpenoids from Aframomum aulacocarpus. J. Nat. Prod. 57, 917–923. Blessing, R.H., 1997. Outlier treatment in data merging. J. Appl. Cryst. 30, 421–426. Farrugia, L.J., 1997. ORTEP-3 for Windows- a version of ORTEP-III with a Graphical User Interface (GUI). J. Appl. Cryst. 30, 565. Farrugia, L.J., 1999. WinGX suite for small-molecule single-crystal crystallography. J. Appl. Cryst. 32, 837–838. Hutchison, J., 1958. Flora of West Tropical Africa. In: Keay, R.W.J. (Ed.), 2nd edition, part 2. Crown Agents for Overseas Governments and Administrations, London, p. 404. Jakupovic, J., Grenz, M., Schemeda-Hirschmann, G., 1988. Rhamnofolane derivatives from Jatropha grossidentata. Phytochemistry 27, 2997–2998. Jolad, S.D., Hoffmann, T.J.J., Timmermann, B.N., Schram, K.H., Cole, J.R., Bates, R.B., Klenck, R.E., Tempesta, M.S., 1983. Daphnane diterpenoids from Wikstroemia monticola. Wikstrotoxins A-D, Huratoxin and Excoecariatoxin. J. Nat. Prod. 46, 675–680. Kokpol, U., Thebpatiphat, S., Boonyaratavej, S., Chedchuskulcai, V., Ni, C.-Z, Clardy, J., Chaichantipyuth, C., Chittawong, V., Miles, D.W., 1990. Structure of trogonostemone, a new phenanthrenone derivative from the Thai plant Trigonostemon reidioides. J. Nat. Prod. 53, 1148–1151. Kupchan, S.M., Shizuri, Y., Summer, W.C., Haynes, H.R., Leighton, A.P., Sickles, B.R., 1976. Isolation and structural elucidation of new potent and antileukemic diterpenoid esters from Gnidia species. J. Org. Chem. 41, 3851–3852. Long, L., Lee, S.K., Chai, H.-B., Rasoanaivo, P., Gao, Q., Navarro, H., Wall, M.E., Wani, M.C., Farnsworth, N.R., Cordell, G.A., Pezzuto, J.M., Kinghorn, A.D., 1997. Novel bioactive phenanthrene derivatives from Domohinea perrieri. Tetrahedron 53, 15663–15670. McLean, S., Perpick-Dumont, M., Reynolds, W.F., Jacobs, H., Lachmensing, S.S., 1987. Unambiguous structure and nuclear magnetic resonance characterization of two triterpenoids of Maptounea guianensis by two-dimensional nuclear magnetic resonance spectroscopy. Can. J. Chem. 65, 2519–2525. Nawwar, M.A.M., Hussein, S.A.M., Merfort, I., 1994. NMR spectral analysis of polyphenols from Punica granatum. Phytochemistry 36, 793–798. Ogura, M., Koike, K., Cordell, G.A., 1978. Potential anticancer agents VIII. Constituents of Baliospermum montanum. Planta Med. 33, 128–143. Otwinowski, Z., Minor, W., 1997. In: Carter, C.W., Sweete, R.M. (Eds), Macromolecular crystallography. Part A. Academic Press, London, pp 307–326. Sheldrick, G.M., 1997. SHELXL-9797-102 Release 97-2. Program for Crystal Structure Refinement. University of Göttingen, Germany. Stuart, K.L., Barrett, M.A., 1969. A phorbol derivative from Croton rhamnifolius. Tetrahedron Lett. 10, 2399–2400. Tchuendem, M.H.K., Mbah, J.A., Tsopmo, A., Ayafor, J.F., Sterner, O., Okunji, C., Iwu, M.M., Schuster, B.M., 1999. Anti-plasmodial sesquiterpenoids from the African Reneilmia cincinnata. Phytochemistry 52, 1095–1099. Thomas, D.W., Thomas, J.M., Bromley, W.A., 1989. Korup Ethnobotany Survey. WWF, Surrey, UK. Woo, S.W., Hildebert, W., 1977. 3-Acetylaleuritolic acid from the seeds of Phytolacca americana. Phytochemistry 16, 1845–1846. Yazaki, Y., Hillis, W.E., 1976. Polyphenols of Eucalyptus globulus, E. regnans and E. deglupta. Phytochemistry 15, 1180–1182. Zhao, W., Wolfender, J.-L., Mavi, S., Hostettmann, K., 1998. Diterpenes and sterols from Neoboutonia melleri. Phytochemistry 48, 1173–1175.