Diterpenes from the leaves of Croton zambesicus

Phytochemistry 65 (2004) 1165–1171 www.elsevier.com/locate/phytochem Diterpenes from the leaves of Croton zambesicus Sebastien Blocka, Chiara Baccellia, Bernard Tinantb, Luc Van Meerveltc, Raoul Rozenbergd, Jean-Louis Habib Jiwand, Gabriel Llabrèse, Marie-Claire De Pauw-Gilletf, Joelle Quetin-Leclercqa,* a Laboratoire de Pharmacognosie, Unité CHAM, Université Catholique de Louvain, UCL 72.30-CHAM, Av. E. Mounier, 72, 1200 Bruxelles, Belgium b Unité CSTR, Département de Chimie, UCL, Place Pasteur 1, 1348 Louvain-la-Neuve, Belgium c Biomolecular Architecture, Chemistry Department, K.U. Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium d Laboratoire de Spectrométrie de Masse, UCL, Place Pasteur 1, 1348 Louvain-la-Neuve, Belgium e Département de Physique Expérimentale, Université de Liège, Allée du 6 Août, 17, 4000 Liège, Belgium f CRCE, Histologie-Cytologie Université de Liège, Rue de Pitteurs, 20, 4020 Liège, Belgium Received 9 December 2003; accepted 17 February 2004 Abstract Two new trachylobane- and one isopimarane-type diterpenoids: ent-18-hydroxy-trachyloban-3-one; ent-trachyloban-3-one; isopimara-7,15-dien-3b-ol, were isolated from the leaves of Croton zambesicus, together with trans-phytol, b-sitosterol, a-amyrin and stigmasterol. The structures were determined by extensive NMR techniques and X-ray analysis. The cytotoxicity of these compounds has been evaluated on cancer and non-cancer cell-lines. # 2004 Elsevier Ltd. All rights reserved. Keywords: Croton zambesicus; Euphorbiaceae; Diterpene; Trachylobane; Pimarane; Cytotoxicity 1. Introduction Croton zambezicus Muell. Arg. (Euphorbiaceae) (Syn. C. amabilis Muell. Arg., C. gratissimus Burch.) is a shrub or small tree reaching 10 m in height. It’s a Guineo-Congolese species widespread in Tropical Africa (Adjanohoun et al., 1989). The leaf decoction is used in Benin as anti-hypertensive, anti-microbial (urinary infections) and to treat fever associated with malaria (Adjanohoun et al., 1989; Watt and Breyer-Brandwikj, 1962). The genus Croton is well known for its diterpenoid content and a lot of different types of diterpenes (phorbol esters, clerodane, labdane, kaurane, trachylobane, pimarane, etc.) have been isolated from this genus. There is very little literature concerning the phytochemical study of Croton zambesicus although if this plant is widely used in African traditional medicine. Labdane, clerodane and trachylobane diterpenes have been * Corresponding author. Tel.: +32-2-7647254; fax: +32-27647253. E-mail address: leclercq@cham.ucl.ac.be (J. Quetin-Leclercq). 0031-9422/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2004.02.023 identified in the stem bark of Croton zambesicus (Ngadjui et al., 2002). Recently we have identified a new cytotoxic trachylobane diterpene from the leaves of C. zambesicus (Block et al., 2002). In order to continue our investigations on the composition of the cytotoxic dichloromethane extract of the leaves we have isolated and characterised two new trachylobane and one isopimarane diterpenes together with trans-phytol, a-amyrin and sterols. 2. Results and discussion HSCCC separation of the dichloromethane extract from the leaves of C. zambesicus gave 21 fractions. These fractions were further purified by MPLC. From these fractionations, we isolated five diterpenes: ent-trachyloban-3b-ol (Block et al., 2002), ent-18-hydroxy-trachyloban-3-one (1), isopimara-7,15-dien-3b-ol (2), enttrachyloban-3-one (3) and trans-phytol (4) together with a-amyrin and sterols: b-sitosterol and stigmasterol. All these compounds were isolated for the first time from the leaves of C. zambesicus. 1166 S. Block et al. / Phytochemistry 65 (2004) 1165–1171 Fig. 1. View and atom labelling of one molecule from the asymmetric unit of 1 (Spek, 1998). Compound 1 was isolated as a white crystal, whose molecular formula, C20H30O2, was established by HREIMS. Infrared absorptions at 3520 and 1702 cm
1 provided evidence of respectively hydroxyl and carbonyl groups. The presence of a cyclopropane ring was deduced from the 1H NMR spectrum that exhibits two signals at
H 0.62 and 0.87 ppm (H-12 and H-13 respectively) and by the 13C NMR spectrum that shows signals at
C 20.4 (C-12), 24.2 (C-13), 22.5 (C-16) ppm. From these observations and comparison with NMR data from closely related structures (Block et al., 2002; Midiwo et al., 1997; Hasan et al., 1982; Arnone et al., 1979; Leong et al., 1997) we could conclude that compound 1 belongs to the trachylobane series of diterpene. The presence of the carbonyl group was confirmed by the signal at
C 219.1 ppm in the 13C spectrum. The primary alcohol was revealed by the two doublets at
H 3.63 and 3.37 ppm in the 1H spectrum and by the signal at
C 66.8 ppm in the 13C spectrum (Fig. 1). Long range 1H-13C correlations (HMBC) between the three protons at
H 0.99 (Me-19) and the 13C NMR signal at
C 66.8 and X-ray analysis supported the C-18 position for the hydroxyl group. The position of the ketone at C-3 was also deduced from HMBC correlations between the protons at
H 2.22 (H-2a), 2.62 (H2b), 0.99 (Me-19), 3.63 (H-18a), 3.37 (H-18b) and the ketonic carbon at
C 219.1. The full 1H and 13C NMR assignments were established with HMQC correlations. X-ray crystallographic analysis was conducted to confirm the structure of 1. All the naturally-occurring trachylobane diterpenes isolated so far belong to the enantio series and comparison with other ent-trachylobanes confirms the ent-configuration of 1 (Block et al., 2002; Midiwo et al., 1997; Hasan et al., 1982; Arnone et al., 1979; Leong et al., 1997). 1 is then identified as ent-18-hydroxy-trachyloban-3-one. Compound 2 was isolated as a white solid with a molecular composition of C20H32O as inferred from HR-EIMS. The IR spectrum showed absorption bands for a hydroxyl group (3314 cm
1) and for a mono-substituted double bond (3099, 1639, 909 cm
1). The combined analysis of the 13C NMR and DEPT spectra revealed the presence of 20 carbon signals assigned to four methyls, seven methylenes, five methine among which one tertiary alcohol and two olefinic carbons and four quaternary carbons. The occurrence in the 1H spectrum of three dd at
H 5.80 (J=10.8 and 17.6 Hz, H-15), at
H 4.93 (J=1.6 and 17.6 Hz, H-16B) and at
H 4.87 (J=1.6 and 10.8 Hz, H-16A) associated with the presence of a broad doublet at
H 5.37 (J=3.5 Hz, H-7) and four singlets corresponding to methyl groups suggested a pimarane type skeleton (Lago et al., 2000). The position of the alcohol on the skeleton of the pimarane was determined as C-3 by the HMBC correlation between the proton at
H 3.26 (H-3) and the 13C NMR signals
C 27.4 (C-2) and 37.8 (C-4) and by the 1H–1H 1167 S. Block et al. / Phytochemistry 65 (2004) 1165–1171 COSY correlation between the protons
H 3.26 (H-3) and 1.62 (H-2). The equatorial position of the hydroxyl group at C-3 was deduced by the observation of the coupling constants of the dd at 3.26 (J=4.7 and 10.9 Hz, H-3a). The position of the double bound between C-7 and C-8 was defined by the 1H-1H COSY correlation between the protons
H 5.37 (H-7) and
H 1.97 (H-6). The full 1H and 13C NMR assignments were established with HMQC correlations. The stereochemistry at C-13 was established by comparison of the 13 C NMR chemical shifts of C-15, C-16 and C-17 with those of isopimarane diterpenoids, showing an equatorial position for the Me-17 and an axial position for the vinyl group (Beier, 1978; Wenkert and Buckwalter, 1972; Rasoamiaranjanahary et al., 2003; Polonsky et al., 1970; Anjaneyulu et al., 2003; Lago et al., 2000). Compound 2 was finally identified as isopimara-7,15-dien3b-ol. This compound has already been synthesised from virescenol A (Polonsky et al., 1970; Ceccherelli et Table 1 13 C and 1H NMR spectroscopic data for diterpenes 1 and 3 in CDCl3. 13 C NMR at 125 MHz for 1 and 100 MHz for 3. 1H NMR at 500 MHz for 1 and 400 MHz for 3. Chemical shifts are given in ppm; multiplicities and coupling constant J (in parentheses) in Hz Position 1
C 1a 1b 2a 2b 3 4 5a 6a 6b 7a 7b 8 9a 10 11a 11b 12a 38 35 219.1 52.5 52.3 20.6 37.8 40.4 49.2 37.5 19.7 20.4 13a 24.2 14a 14b 33.5 15a 15b 16 17 18a 50.2 22.5 20.5 66.8 18b 19 20 16.8 14.4 3
H 1.46 m 1.46 m 2.22 m 2.62 m – – 1.21 m 1.43 m 1.43 m 1.25 m 1.83 m – 1.59 m – 1.94 m 1.72 m 0.62 d (7.8) 0.87 dd (3.1, 7.8) 1.24 m 2.09 d (11.9) 1.44 d 1.27 d – 1.14 s 3.63 d (11.4) 3.37 d (11.4) 0.99 s 1.18 s
C 38 34.1 217.5 47.6 55.5 21.1 38.3 40.4 52.4 37.7 19.6 20.5 24.2 33.2 2.07 d (12.0) 50.2 22.5 20.4 26 21.5 14.1
H 1.44 m 1.72 m 2.30 m 2.55 m – – 1.24 m 1.41 m 1.41 m 1.24 m 1.80 m – 1.22 m – 1.93 m 1.74 m 0.61 d (7.6) 0.85 dd (3.2, 7.6) 1.23 m 1.44 1.26 – 1.13 1.05 d d s s 1.01 s 1.10 s al., 1985) and isolated from the leaves of Guarea macrophylla (Lago et al., 2000) but this is the first pimaranetype diterpene isolated from C. zambesicus. Moreover, comparison of NMR data of 2 with those reported by Lago (Lago et al., 2000) shows very good agreement excepted for the chemical shifts of C-2 (27.4) and C-6 (23.1) that are inverted. Our assignments were confirmed by the HMBC correlation between the proton at
H 3.26 (H-3) and the 13C NMR signal
C 27.4 (C-2) and by the 1H–1H COSY correlations between the protons
H 3.26 (H-3) and 1.62 (H-2) and between the protons
H 5.37 (H-7) and
H 1.97 (H-6) and by comparison with closely related structure (Ansell et al., 1993; Meragelman et al., 2003; Aiyar et al., 1969, 1971). Complete 13C and 1H NMR assignments of 2 are presented in Table 2. Compound 3 was isolated as a colourless oil. Its molecular formula was determined as C20H30O by HREIMS analysis. Compound 3 contained a carbonyl group as inferred from the IR absorption band at 1706 cm
1. The trachylobane skeleton of this compound was, as compound 1, identified by the presence of the cyclopropane ring signals in the 1H (
H 0.61 and 0.85 ppm respectively for H-12 and H-13) and 13C (
C 20.5, 24.2 and 22.5 ppm respectively for C-12, C-13 and C-16) Table 2 NMR assignments of compound 2 in CDCl3 (13C at 100 MHz and 1H at 400 MHz). Chemical shifts are given in ppm; multiplicities and coupling constant J (in parentheses) in Hz Position 2
C 1a 1b 2a 2b 3a 4 5a 6a 6b 7 8 9a 10 11a 11b 12a 12b 13 14a 14b 15 16A 38.6 150.3 109.2 16B 17 18 19 20 21.4 28.3 15.6 14.9 27.4 79.3 37.8 50 23.1 121.4 135.4 51.9 37.3 20.1 36.1 35.3 45.9
H 1.25 1.84 1.62 1.62 3.26 – 1.16 1.97 1.97 5.37 – 1.63 – 1.57 1.39 1.36 1.53 – 1.95 1.95 5.80 4.87 4.93 m m m m dd (4.7, 10.9) 0.86 1.00 0.90 0.87 s s s s dd (4.9, 11.9) m m bd (3.5) m m m m m m m dd (10.8, 17.6) dd (1.6, 10.8) dd (1.6, 17.6) 1168 S. Block et al. / Phytochemistry 65 (2004) 1165–1171 NMR spectra. The presence of the carbonyl was deduced from the signal at
C 217.5 ppm on the 13C spectrum and the position on C-3 was deduced from HMBC spectra showing correlation between the protons at
H 2.55 (H-2b) and 1.05 (H-18) and the carbon at
C 217.5. The stereochemistry of 3 was based on biosynthetic considerations (all natural trachylobanes isolated up to now belong to the enantio series) and on comparison of spectral data from 1 and closely related compounds (Kapingu et al., 2000; Ngouela et al., 1998; Arnone et al., 1979; Leong et al., 1997). 3 is then identified as ent-trachyloban-3-one. In order to complete the study on the cytotoxic activity of the dichloromethane extract of C. zambesicus, the isolated diterpenes, aamyrin and sterols were tested in vitro against cancer (HeLa, HL-60) and non-cancer (WI-38) cell lines. The results are presented in Table 3. The biological activities of trachylobane diterpenes are poorly known but recently we have shown that ent-trachyloban-3b-ol possesses cytotoxic activities on HeLa cells (IC50 on HeLa cells=7.3 mg/ml). The cytotoxicities of compounds 1 and 3 are a little bit lower but no clear specificity between cell lines could be observed even if 3 is 2.5 more active on HeLa cells (cancer cell line) than on WI-38 (non-cancer cell line). Different biological properties have been described for various pimarane derivatives, including antimicrobial and spasmolytic (Vlietinck, 1987), antihypertensive (Ohashi et al., 2000), antituberculosis (Ulubelen et al., 1997), antifungal (Rasoamiaranjanahary et al., 2003) and antiinflammatory described as international patent (Suh et al., 1999). Studies have also demonstrated that pimarane derivatives inhibited the tumor-promoting effect of TPA (12-O-tetradecanoylphorbol 13-acetate) and were slightly cytotoxic (Minami et al., 2002; Chang et al., 2000) suggesting an interesting cancer chemopreventive potential. The results obtained on the cytotoxicity of compound 2 confirm the weak cytotoxic activity of pimarane diterpenes. In comparison to the other diterpenes, trans-phytol (4) shows a similar range of activity than trachylobanes. The cytotoxic activity of Table 3 Cytotoxicity data for compounds 1–4a 3. Experimental 3.1. General High Speed Counter-Current Chromatography was performed on a HSCCC Kromaton III, SEAB. An Omnifit glass column (OM 6427 15750 mm) packed with Lichroprep Si 60 (15–25 mM, Merck) was used for MPLC. Analytical TLC was performed on precoated silica gel 60 F254 plates (Merck) and detection was achieved by spraying with sulfuric anisaldehyde, followed by heating 5 min at 105  C. The IR spectra were recorded on a Perkin Elmer FTIR 286. The optical rotation values were obtained on a Perkin-Elmer 241 spectropolarimeter in CH2Cl2 solution. UV spectra were measured on a Uvikon 933 (Kontron) spectrophotometer. NMR spectra of compounds 2 and 3 were recorded on a Bruker Avance DRX-400 spectrometer in CDCl3 at 400 MHz (1H) and 100 MHz (13C), at 25  C. NMR spectra of compound 1 were recorded on a Bruker Avance 500 at 500 MHz (1H) and 125 MHz (13C); d in ppm rel. to Me4Si (internal standard). HR-EIMS was recorded at 70 eV in an AutoSpec 6 F mass spectrometer and EIMS at 70 eV on a Finnigan TSQ7000 triple quadrupole; m/z (rel. intensity in%). 3.2. Plant material The aerial parts of C. zambesicus were collected in the surroundings of Cotonou (Benin) in December 2000 and identified by botanist Prof. V. Adjakidje (Université d’Abomey-Calavi-Benin). A voucher specimen has been deposited at the herbarium of the Belgian national botanical garden at Meise (BR S.P. 848.108). 3.3. Extraction and isolation Cell linesb Compound HeLa HL-60 WI-38 1 2 3 4 Camptothecin 12.2 2.1 25.3 3.3 9.6 1.6 13.8 1.3 0.5 mM 12.71.2 28.94.0 12.41.9 16.42.0 0.1 mM 18.32.7 32.63.6 23.83.2 13.81.7 0.6 mM a phytol is due to an induction of apoptosis (Komiya et al., 1999). Finally, in agreement with literature data (Chaturvedula et al., 2002; Awad et al., 2000; Moghadasian, 2000), b-sitosterol, a-amyrin and stigmasterol were not cytotoxic at the tested concentrations (IC50 > 30 mg/ml on every cell lines). Results are expressed as mean of IC50 values (mg/ml)SEM of three independent experiments. b HeLa, human cervix carcinoma; HL-60, human promyelocytic leukemia; WI-38, non-cancer human lung fibroblast. Air-dried and powdered leaves (580 g) were percolated at room temperature with dichloromethane to give 34 g of extract. Part of this extract (5 g) was fractionated by HSCCC using the two phases solvent system heptane–acetonitrile–dichloromethane (10:7:3) (descending mode, mobile phase: lower phase, flow rate: 2 ml/min, fraction collection: 4 min/tube, rotation: 500 rpm, volume of column: 1000 ml). 21 fractions (F1– F21) were obtained. F6 (315 mg) was separated by MPLC on silicagel 60 (15–25 mM) eluted with Tol– CH3CN (93:7) giving 6 fractions (F61–F66). Fraction S. Block et al. / Phytochemistry 65 (2004) 1165–1171 F65 (59.4 mg) was finally purified by MPLC on silicagel 60 (15–25 mM) eluted with Tol–EtOAc–CH3CN (91:8:1) to give compound 1 (20 mg). Fraction F9 (350.6 mg) was separated by MPLC on silicagel 60 (15–25 mM) eluted with Tol–EtOAc (98:2) giving 8 fractions (F91– F98). Fraction F94 (49.5 mg) was purified by MPLC on silicagel 60 (15–25 mM) with Tol–EtOAc (96:4) as mobile phase to afford compound 2 (14 mg). Fraction F12 contained ent-trachyloban-3b-ol, previously identified in the plant (Block et al., 2002). Fraction F14 (256 mg) was applied to MPLC on silicagel 60 (15–25 mM) eluted with Tol–EtOAc (93:8). 7 fractions (F141–F147) were obtained. Fraction F142 was purified by MPLC on silicagel 60 (15–25 mM), using Tol–EtOAc (90:10) as mobile phase to give trans-phytol (4) (8 mg). Fraction F144 was purified by MPLC on silicagel 60 (15–25 mM), Tol–EtOAc (92:8) was used as mobile phase and 25 mg of compound 3 were obtained. F17 gave a-amyrin and b-sitosterol. F18 gave stigmasterol. 3.4. ent-18-Hydroxy-trachyloban-3-one (1)  White crystals [CH2Cl2]. ½22 D :
77 (CH2Cl2, c 0.1);
1 UV lmax nm (log ): 218 (2.23); IR NaCl max cm : 3520 (OH), 2988, 2928, 2859, 1702 (C¼O), 1460, 1444, 1417, 1380, 1256, 1209, 1164, 1094, 1082, 1047, 1011, 975, 844, 757; 1H and 13C are given in Tables 1; EI-MS 70 eV m/z (rel. int.): 302 [M]+ (30), 284 [M
H2O]+ (45), 272 (48), 269 (26), 257 (16), 246 (7), 215 (6), 201(5), 187 (4), 185 (2), 159 (1), 145 (1), 107 (3), 105 (17), 93 (22), 91 (42), 81 (36), 79 (83), 55 (100). HR-EIMS m/z: 302.2249 [M]+ (calc. for C20H30O2 302.2246). 3.5. Isopimara-7,15-dien-3-ol (2)  Amorphous powder. ½22 D : +15 (CH2Cl2, c 0.1); UV NaCl lmax nm (log ): 226 (2.61); IR max cm
1: 3314 (OH), 2953, 2926, 2968, 1669, 1463, 1378, 1366, 1002; 1H and 13 C are given in Table 2; EI-MS 70 eV m/z (rel. int.): 288 [M]+ (5), 273 [M–CH3]+ (8), 270 [M
H2O]+ (4), 255 (26), 245 (9), 227 (7), 213 (9), 200 (17), 185 (19), 171 (19), 145 (44), 134 (50), 132 (66), 131 (99), 129 (100), 119 (87), 105 (69), 91 (24). HR-EIMS m/z: 288.2448 [M]+ (calc. for C20H32O 288.2453). 3.6. ent-Trachyloban-3-one (3)  Colorless oil. ½22 D :
37 (CH2Cl2, c 0.1); UV lmax nm
1 (log ): 228 (2.78); IR NaCl max cm : 2969, 2933, 2860, 1706 (C¼O), 1458, 1384, 1367, 1261, 1202, 1112, 1082, 1010, 844; 1H and 13C are given in Table 1; EI-MS 70 eV m/z (rel. int.): 286 [M]+ (77), 271 [M
CH3]+ (17), 253 (1), 230 (23), 215 (12), 200 (11), 173 (5), 159 (8), 145 (12), 131 (8), 119 (14), 107 (11), 105 (100), 93 (16), 91 (15), 81 (8), 79 (10), 55 (9). HR-EIMS m/z: 286.2293 [M]+ (calc. for C20H30O 286.2296). 1169 3.7. X-Ray structure analysis of compound 1 Colourless crystals were obtained by slow evaporation from a dichloromethane solution. C20H30O2, Mr=302.44, monoclinic, space group P 21, a=7.311(1), b=42.210(1), c=10.903(1) Å, =91.10(1) , V=3363.8(1) Å3, Z=8, Dx=1.20 g cm
3, =0.577 mm
1, F(000)=1328, T=120 K. A total of 29,725 reflections were collected using a Bruker SMART 6000 CCD detector and CuKa radiation (l=1.54178 Å). 6532 independent refection (Rint=0.052). The structure was solved by direct methods with SHELXS-97 (Sheldrick, 1997) and refined by least-squares using F2 values and anisotropic thermal parameters for non-hydrogen atoms with SHELXL-97 (Sheldrick, 1997). The H atoms of the hydroxyl groups were localized from difference Fourier maps; all the other H atoms were calculated and included in the refinement with a common isotropic temperature factor. Final R values are: R=0.041 for 6370 observed reflections, R (all data)=0.042, wR2=0.109, S=1.02, Flack parameter=0.08(14). The data have been deposit with the Cambridge Crystallographic Data Centre (Nr CCDC 222992). The four independent molecules are similar except that the conformation of ring I (C1–C2–C3–C4–C5– C10) is clearly a less flattened chair in molecule 3 (labelled C301–C310) than in the three other ones (in molecule 3, the endocyclic torsion angles are :
52, 47,
43, 48,
54 and 54 while the mean values for molecules 1 2 and 4 are
50, 34,
29, 40,
54 and 58 ). Selected average bond lengths are (Å): C(3)– O(22)=1.220(3), C(20)–O(21)=1.425(3). The four OH groups are hydrogen-bonded to a C¼O of another molecule making infinite one-dimensional chains. 3.8. Cytotoxicity assay HeLa (human cervix carcinoma cells) and WI-38 (human lung fibroblast) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL) supplemented with 10% fetal calf serum (Gibco BRL) and antibiotics (100 IU penicillin/ml, 100 mg streptomycin/ml). HL-60 (human promyelocytic leukemia) cells were routinely grown in suspension in RPMI 1640 medium (Gibco BRL) containing 0.33% l-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, antibiotics (100 IU penicillin/ml, 100 mg streptomycin/ml) and supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL). Cells were incubated at 37  C in a humidified atmosphere containing 5% CO2. Stock solutions of compounds were prepared at 10 mg/ml in DMSO and stored at 4  C. The cytotoxicity of the compounds on HeLa and WI-38 cells was evaluated using the tetrazolium salt MTT (Sigma) colorimetric method based on the cleavage of the reagent by dehydrogenases in viable 1170 S. Block et al. / Phytochemistry 65 (2004) 1165–1171 cells. Briefly, 5000 HeLa or WI-38 cells per well were seeded in 100 ml of DMEM in 96-well microculture plates for 24 h. After 24 h adaptation, 100 ml of medium containing various drug concentrations were added to each well, while control cells received fresh medium containing analogous DMSO concentrations. Each concentration was tested in at least 8 wells. After 72 h incubation, the medium was replaced by 100 ml DMEM (without serum) medium containing 10 ml of MTT solution (3 mg/ml in PBS). After 45 min in the incubator, the medium was removed and 100 ml of DMSO were added to each well. The plates were shaked and optical densities were recorded at two wavelengths (570 nm and 620 nm), against a background control as blank (100 ml of pure DMSO). The cytotoxicity on HL-60 cells was evaluated using another tetrazolium salt, WST-1 (Boehringer). Briefly, 50000 HL-60 cells in 100 ml of RPMI 1640 medium were seeded in each well of a 96-well plate. 100 ml of fresh medium containing various drug concentrations were added to each well while control cells received fresh medium with analogous concentrations of DMSO. Each concentration was tested in at least 8 wells. After 72 h treatment, each well was supplemented with 10 ml of WST-1 and then incubated for 45 min. Afterwards the plates were shaken and the optical density was measured at 450 and 620 nm against a background control as blank on a microplate reader. For the 3 cell lines, the relative optical density was expressed as percent of the control cells considered as 100%. In each case, camptothecin (Sigma) was used as positive control. IC50 determination was achieved via regression analysis of the results of at least 5 different concentrations of each drug. Results are mean  SEM of 3 independent experiments. Acknowledgements The authors wish to thank M.C. Fayt for its skillfull technical assistance. We also thank Professor J. Hanuise for the optical rotation measurement and Prof. R. Flammang for HR-EIMS analysis. This work was supported by a grant and founds from the ‘‘fonds spécial de recherche’’ of the Catholic University of Louvain and from the ‘‘Région Wallonne’’ (project : CORD/LEVE 383/conv.114713 075665 for De Pauw-Gillet). References Adjanohoun, E.J., Adjakidje, V., de Souza, S., 1989. Contribution aux Études Ethnobotaniques et Floristiques en République Populaire du Bénin, Vol. 1. Agence de Coopération Culturelle et Technique, Paris, pp. 245. Aiyar, V.N., Rao, P.S., Sachdev, G.P., Seshadri, T.R., 1969. Isolation and constitution of deoxyoblongifoliol from Croton oblongifolius. Indian J. Chem. 7, 838–839. Aiyar, V.N., Seshadri, T.R., 1971. Chemical components of Croton oblongifolius IV. Constitution of oblongifoliol and deoxyoblongifoliol. Indian J. Chem. 9, 1055–1059. Anjaneyulu, A.S.R., Rao, V.L., Sreedhar, K., 2003. Agallochins J-L, new isopimarane diterpenoids from Excoecaria agallocha L. Nat. Prod. Res. 17, 27–32. Ansell, S.M., Pegel, K.H., Taylor, D.A.H., 1993. Diterpenes from the timber of 20 Erythroxylum species. Phytochemistry 32, 953–959. Arnone, A., Mondelli, R., St Pyrek, J., 1979. 13C NMR spectroscopy of natural substances, IV—13C NMR studies of trachylobane diterpenes: complete carbon assignment. Org. Magn. Res. 12, 429– 431. Awad, A.B., Downie, A.C., Fink, C.S., 2000. Inhibition of growth and stimulation of apoptosis by b-sitosterol treatment of MDA-MB-231 human breast cancer cells in culture. Int. J. Mol. Med. 5, 541–545. Beier, R., 1978. Stereochemical assignment at the C-13 carbon of pimaradienes by carbon-13 NMR A reassessment. Org. Magn. Res. 11, 586. Block, S., Stévigny, C., De Pauw-Gillet, M.-C., de Hoffmann, E., Llabrès, G., Adjakidjé, V., Quetin-Leclercq, J., 2002. ent-Trachyloban-3b-ol, a new cytotoxic diterpene from Croton zambesicus. Planta Medica 68, 647–649. Ceccherelli, P., Curini, M., Marcotullio, M.C., 1985. 3b,1-Oxidoisopimara-7,15-diene as intermediate in the conversion of virescenol B into isopimara-7,15-dien-19-ol. J. Chem. Perkin Trans. I 2173–2175. Chang, L.C., Song, L.L., Park, E.J., Luyengi, L., Lee, K.J., Farnsworth, N.R., Pezzuto, J.M., Kinghorn, A.D., 2000. Bioactive constituents of Thuja occidentalis. J. Nat. Prod. 63, 1235–1238. Chaturvedula, V.S.P., Schilling, J.K., Miller, J.S., Andriantsiferana, R., Rasamison, V.E., Vincent, E., Kingston, D.G.I., 2002. Two new triterpene esters from the twigs of Brachylaena ramiflora from the Madagascar rainforest. J. Nat. Prod. 65, 1222–1224. Hasan, C.H., Healey, T.M., Waterman, P.G., 1982. 7b-Acetoxytrachyloban-18oic acid from the stem bark of Xylopia quintasii. Phytochemistry 21, 177–179. Kapingu, M.C., Guillaume, D., Mbwambo, Z.H., Moshi, M.J., Uliso, F.C., Mahunnah, R.L.A., 2000. Diterpenoids from the roots of Croton macrostachys. Phytochemistry 54, 767–770. Komiya, T., Kyohkon, M., Ohwaki, S., Eto, J., Katsuzaki, H., Imai, K., Kataoka, T., Yoshioka, K., Ishii, Y., Hibasami, H., 1999. Phytol induces programmed cell death in human lymphoid leukemia Molt 4B cells. Int. J. Mol. Med. 4, 377–380. Lago, J.H.G., Brochini, C.B., Roque, N.F., 2000. Terpenes from the leaves of Guarea macrophylla (Meliaceae). Phytochemistry 55, 727– 731. Leong, Y.W., Harrison, L.J., 1997. ent-Trachylobane diterpenoids from liverwort Mastigophora diclados. Phytochemistry 45, 1457– 1459. Meragelman, T.L., Silva, G.L., Mongelli, E., Gil, R.R., 2003. ent-Pimarane type diterpenes from Gnaphalium gaudichaudianum. Phytochemistry 62, 567–572. Midiwo, J.O., Owuor, F.A.O., Juma, B.F., Waterman, P.G., 1997. Diterpenes from the leaf exudate of Psiadia punctulata. Phytochemistry 45, 117–120. Minami, T., Wada, S., Tokuda, H., Tanabe, G., Muraoka, O., Tanaka, R., 2002. Potential antitumor-promoting diterpenes from the cones of Pinus Luchuensis. J. Nat. Prod. 65, 1921–1923. Moghadasian, M.H., 2000. Pharmacological properties of plant sterols: in vivo and in vitro observations. Life Sci. 67, 605–615. Ngadjui, B.T., Abegaz, B.M., Keumedjio, F., Folefoc, G.N., Kapche, G.W.F., 2002. Diterpenoids from the stem bark of Croton zambesicus. Phytochemistry 60, 345–349. Ngouela, S., Nyasse, B., Tsamo, E., Brochier, M., Morin, C., 1998. A trachylobane diterpenoid from Xylopia aethiopica. J. Nat. Prod. 61, 264–266. Ohashi, K., Bohgaki, T., Matsubara, T., Shibuya, H., 2000. Indonesian medicinal plants XXIII. Chemical structures of two new S. Block et al. / Phytochemistry 65 (2004) 1165–1171 migrated pimarane-type diterpenes, neoorthosiphols A and B, and suppressive effects on rat thoracic aorta of chemical constituents isolated from the leaves of Ortosiphon aristatus (Lamiaceae). Chem. Pharm. Bull. 48, 433–435. Polonsky, J., Baskevitch, Z., Bellavita, N.C., Ceccherelli, P., 1970. Structures des virescenol A et B, metabolites d’Oospora virescens (Link) Wallr. Bull. Soc. Chim. de France 5, 1912–1918. Rasoamiaranjanahary, L., Guilet, D., Martson, A., Randimbivololona, F., Hostettmann, K., 2003. Antifungal isopimaranes from Hypoestes serpens. Phytochemistry 64, 543–548. Sheldrick, G.M., 1997. SHELXS-97 and SHELXL-97. Program for the Solution and Refinement of Crystal Structures. University of Göttingen, Germany. Spek, A., 1998. PLATON, Molecular Geometry Program. University of Utrecht, The Netherlands. 1171 Suh, Y.G., Choi, Y.H., Lee, H.K., Kim, Y.H., Park, H.S., 1999. Diterpene derivatives and anti-inflammatory analgesic agents comprising the same. PCT Int. Appl. WO 9937600 A1, p. 53. Ulubelen, A., Topcu, G., Johansson, C.D., 1997. Norditerpenoids and diterpenoids from Salvia multicaulis with antituberculous activity. J. Nat. Prod. 60, 1275–1280. Vlietinck, A.J. 1987. Biologically active natural compounds. In: Hostettmann, K., Lea, P.J. (Eds.), Proceedings of the PSE, vol. 27. Oxford University Press, Oxford, UK. Watt, J.M., Breyer-Brandwikj, M.G., 1962. The Medicinal and Poisonous Plants of Southern and Eastern Africa, second ed. E. and S. Livingstone Ltd., London, UK. Wenkert, E., Buckwalter, B.L., 1972. Carbon-13 nuclear magnetic resonance spectroscopy of naturally occuring substances X. Pimaradienes. J. Am. Chem. Soc. 94, 4367–4368.