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Three clerodane diterpenoids from Croton eluteria Bennett
Phytochemistry 57 (2001) 1209–1212 www.elsevier.com/locate/phytochem
Three clerodane diterpenoids from Croton eluteria Bennett Claire Vigor*, Nicolas Fabre, Isabelle Fouraste´, Claude Moulis Laboratoire Pharmacophores Redox, Phytochimie et Radiobiologie, Faculte´ des Sciences Pharmaceutiques, 35 chemin des Maraıˆchers, F-31062 Toulouse, France Received 13 November 2000; received in revised form 29 March 2001
Abstract Three furanoid clerodanes have been isolated from the stem bark of Croton eluteria Bennett. Their structures have been established by spectroscopic methods. The compounds were named cascarillin B (7a-acetoxy-3,4,15,16-diepoxy-12-oxo-cleroda13(16),14-dien-20-al), cascarillin C (7a-acetoxy-15,16,12,20-diepoxy-20-hydroxy-cleroda-3,4,13(16),14-triene) and cascarillin D (7aacetoxy-3,4,15,16-diepoxy-cleroda-13(16),14-dien-20-al). # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Croton eluteria Bennett; Euphorbiaceae; Furanoid diterpene; Clerodane
1. Introduction Cascarilla bark, obtained from Croton eluteria Bennett (Euphorbiaceae) is a shrub or small tree indigenous to the Bahama Islands. It was reported to be balsamic, digestive, hypotensive, narcotic, stomachic and tonic (Greenish, 1924). It has been used as a traditional medicine for many applications such as for dysentery, dyspepsia, malaria and fever (Duke, 1984). From the phytochemical point of view, some terpene derivatives were previously reported and specially some ent-clerodane furano-diterpenoids: cascallin, cascarillone and cascarillin A (Halsall et al., 1965; Hegnauer, 1966; Claude-Lafontaine et al., 1976; Hagedorn and Brown, 1991). In this paper, we describe the isolation and structural elucidation of three new related compounds from the stem bark of C. eluteria Bennett.
2. Results and discussion The molecular formula C22H28O6 of compound 1 was determined by ESI mass spectrometry mesured in the positive ion mode (m/z 389.2 [M+H]+, m/z 411.2 [M+Na]+). Its 13C NMR decoupled spectrum (Table 1) * Corresponding author. Tel.: +33-5-6225-6844; fax: +33-5-61554330. E-mail address: [email protected] (C. Vigor).
showed 22 signals in agreement with the molecular formula. The characteristic NMR signals for a furan ring were present in the 1H and 13C NMR spectra (H 6.64 br s, 7.40 br s, 8.03 br s; C 108.6, 144.6, 147.5 and 128.9 Cq), as were those of an aldehyde group (H 10.18 s; C 206.0), an ester carbonyl group (C 170.3) and a keto group (C 193.6). These functional groups account for five of the six oxygens, and the remaining oxygen was involved in an epoxide unit as indicated by a singlet signal at H 2.85 and C 62.0. In addition, four methyl groups were present and were identified from their chemical shifts and coupling patterns as a doublet for C-17 (H 1.01 d, J=7.3 Hz; C 13.7), two singlets (C-18: H 1.12 s; C 19.9 and C-19: H 1.06 s; C 19.2) and an acetyl group (H 2.04 s; C 21.6). Considering the quaternary carbons (C 36.3 and 56.0), the C–H group (C 48.5) and their heteronuclear correlations displayed in the HMBC spectrum, these signals could be assigned, respectively, to carbons C-5, C-9 and C-10 of the decalin moiety. The position of the aldehyde group was deduced from the HMBC spectrum which exhibited cross peaks between the aldehyde carbon and H-8, H-11 and the aldehyde proton with C-9, C-11 (Fig. 1). Its chemical shift agrees with an aldehyde group that we assigned to C-9. The location of the epoxide group (C-3–C-4) was deduced from the HMBC spectrum correlations between C-3 (C 62.0) and H-1a, H-2a, H-18 and C-4 (C 65.1) with H-18, H19, H-6a, H-6b, H-2a; Fig. 1. In the same way, the acetoxy group was located on position C-7 (correlations
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between acetate protons (H 2.04 s) and C-7), and the keto group in position C-12 (correlations between the quaternary carbon signal at C 193.6 and H-11a, H-11b and H-14; Fig. 1).
The 13C NMR signal of the shielded Me-19 (C 19.2) suggested a trans A/B ring junction for decalin as previously discussed by Manabe and Nishino (1986). The a orientation for the C-3–C-4 epoxy group was proposed on the basis of the 1H NMR signals of the shielded Me19 protons (H 1.06 s) (Manabe and Nishino, 1986). Finally, an a orientation for the acetoxy group was proposed on the basis of the J value J 8b 7b=4 Hz which is in accordance with axial-equatorial coupling and corresponds to an approximate dihedral angle H-7b–H-
8b of 70 . This was supported by the coupling constant values of H-6 (see Table 1) and the values reported in the literature (Bedir et al., 1999; Tori et al., 1999). Thus, these data allowed us to establish 1, as 7a-acetoxy-3,4,15,16-diepoxy-12-oxo-cleroda-13(16),14-dien-20al, which we named cascarillin B. The positive ESI mass spectrum of compound 2 showed peaks at m/z 397.2, m/z 413.2 and m/z 337.0 corresponding to [M+Na]+, [M+K]+, and [M– OCOCH3+Na]+ respectively, suggesting a C22H30O5 formula. The acetoxy group was still present as supported by the ESI mass spectral data, the carbon at dC 172.4, and three protons at H 2.06 ppm. The HMBC spectrum did not allow us to locate the acetoxy group. But, the 1H–1H COSY spectrum showed a cross peak between H-17 (H 1.13, d, J=7.3 Hz) and H-8 (H 1.66, dd, J=7.3, 3 Hz). The latter proton (J=3 Hz) correlates with a deshielded proton at H 5.03 ppm (m) whose chemical shift is in agreement with a CH-7a-acetoxy as already described for 1. Both epoxide and aldehyde groups were absent from 2 and replaced by a double bound and a lactol function, respectively. This was shown by the characteristic 1H and 13C NMR signals at H 5.22 ppm (H-3), C 122.3 (C-3) and C 144.6 (C-4) for the first one, and at H 5.85 ppm (H-20), C 101.6 (C-20) and H 5.08 ppm (H-12), C 73.2 (C-12) for the second one. The spectrum unambiguously revealed the C-3–C-4 unsaturation (cross peaks between H-18/C-4 and H-18/ C-3). This was confirmed by the 1H–1H COSY correlations (H-18/H3). In the same way, the C-20 carbon (C 101.6), involved in the lactol function, was correlated in the HMBC spectrum with H-8, H-11b and H-12. The correlations of C-9 with H-10, H-17, H-11a, H-11b and H-20, confirmed this result. The methine (C-12) at C 73.2 was correlated with H-11b and H-20, and this proton displayed a cross peak with C-16 and C-20. Thus, the structure of 2 was established as 7a-acetoxy15,16,12,20-diepoxy-20-hydroxy-cleroda-3,4,13(16),14triene, which we named cascarillin C. The molecular formula of compound 3 was determined as C22H30O5 based on ESI mass spectrum [M+Na]+ (m/z 397.3) and [M+K]+ (m/z 413.3). Comparison of the spectral data of this compound with that of 1 indicated that compound 3 differed from 1 through the lack of a keto group in position C-12. These results led us to propose the structure 7a-acetoxy-3,4,15,16-diepoxy-cleroda13(16),14-trien-20-al for compound 3, which we named cascarillin D.
3. Experimental 3.1. Instrumentation 1
H NMR (400 MHz) and J mod. 13C NMR (100 MHz) were obtained with CD3OD or CDCl3 as solvent,
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C. Vigor et al. / Phytochemistry 57 (2001) 1209–1212 Table 1 1 H and 13C NMR data for compounds 1, 2 in CD3OD and 3 in CDCl3 ( in ppm, multiplicities, J in Hz) H
C
Position
1
1a 1b 2a 2b 3 4 5 6a 6b 7 8 9 10 11a 11b 12
1.37 m 1.17 m 1.95 m 1.42 m 2.85 s – – 2.09 dd (14.6–3.7) 1.76 dd (14.6–3.7) 5.25 dd (3.7-3) 2.63 dd (7.3–4) – 2.04 s 3.37 d (17.8) 2.76 d (17.8) –
2.00 m 1.85 m 5.22 br s – – 2.17 dd (14.6–2.6) 1.47 dd (14.6–3.4) 5.03 ddd (3.4–3–2.8) 1.66 dd (7.3–3) – 1.62 br s 2.64 dd (13.3–7.2) 1.92 dd (13.3–8.9) 5.08 dd (8.9–7.2)
– 6.64 br s 7.40 br s 8.03 br s 1.01 d (7.3) 1.12 s 1.06 s 10.18 s 2.04 s –
– 6.63 br s 7.40 br s 7.45 br s 1.13 d (7.3) 1.59 m 1.27 s 5.85 s 2.06 s –
13 14 15 16 17 18 19 20 OCOCH3 OCOCH3
2
3 2.04 m
1.61 m 1.38 m 2.13 m 1.76 m 3.00 br s – – 2.13 m 1.84 dd (14.5–3.7) 5.29 dd (3.6-3) 2.14 m – 3.31 dd (4.9–3.3) 1.99 m 1.65 m a: 2.35 dd (12.5–4.9) b: 2.28 dd (12.5–4.9) – 6.32 br s 7.4 br s 7.32 br s 1.15 d (7. 3) 1.19 s 1.07 s 10.14 d (0.7) 2.07 s –
1
2
3
16.6
23.5
17.1
27.6
29.6
29.0
62.0 65.1 36.3 39.9
122.3 144.6 39.7 42.1
63.5 66.7 37.6 41.1
74.2 37.3 56.0 48.5 38.5
77.3 47.3 55.5 52.1 45.3
75.9 38.5 54.9 49.9 32.2
193.6
73.2
18.4
128.9 108.6 144.6 147.5 13.7 19.9 19.2 206.0 21.6 170.3
131.7 111 144.3 140.9 15.4 18.5 22.7 101.6 21.4 172.4
126 111.8 144.3 140.1 13.5 20.1 19.5 208.1 21.3 172.1
ion mode) mass spectra were recorded on an Autospec 6F mass spectrometer manufactured by Micromass. 3.2. Plant material Croton eluteria Bennett, collected in Ecuador, was purchased from Richard et Frappa, Aubagne, France. Voucher specimens were identified by Professor Isabelle Fouraste´ and deposited at the herbarium of the Pharmacognosy Laboratory, Faculty of Pharmacy (Toulouse). 3.3. Extraction and isolation
Fig. 1. Significant HMBC couplings of 1.
on a Bru¨cker ARX-400 spectrometer. Chemical shifts are given in (ppm) using the solvent peaks, H 4.87, C 48 ppm and H 7.23, C 77 ppm, respectively, as standards ; ESI–MS: 3.5 kV (MeOH–CHCl3) (Perkin Elmer API Sciex 365 Mass spectrometer); HR–FAB (positive
Air-dried powdered bark (1 kg) of C. eluteria Bennett was extracted with Me2CO at room temperature (24 h). The extract (70 g) was taken up in CHCl3 and chromatographed on silica gel columns eluted with various proportions of CHCl3 and MeOH. The fraction containing the main furanoid diterpenes (revealed on TLC by Ehrlich reagent) was evaporated to dryness and separated on 3 successive silica gel (70–200 mm) columns eluted with a hexane–EtOAc gradient. Further purification of the crude diterpene by reversephase chromatography (Mega Band Elut Varian C18 cartridge: MeOH–H2O 6:4), followed by medium
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pressure (10 bars) silica gel chromatography (silica 60A˚, CC chromagel SDS, 6–35 mm) eluted with toluene–EtOAc 95:5 and then another reverse-phase chromatography (C18 cartridge MeOH–H2O 1:1) gave pure compound 1. Concerning compounds 2 and 3, they were obtained by successive medium pressure (10 bars) silica gel chromatography (silica 60 A˚, CC chromagel SDS, 6–35 mm) eluted with a toluene–EtOAc gradient, followed by reverse-phase chromatography (C 18 cartridge: MeOH– H2O 13:7; 14:6) and preparative HPLC (Varian Dynamax 100 A˚, MeOH–H2O 7:3, 10 ml min 1, 204 nm; MeOH–H2O 15:5, 10 ml min 1, 204 nm). 3.3.1. Cascarillin B (1) C22H28O6; yellow resin (126 mg); mp 68–70 C; [a]20 D 29.1 (CHCl3; c 1.65); UV lmax CHCl3 nm (log ": 275 (3.1); ESI–MS, m/z: 389.2 [M+H]+; 411.2 [M+Na]+; HR–FABMS (positive mode) m/z: 329.174 [M– C2H4O2+H]+; C20H24O4+H requires, 329.175; 1H NMR, 13C NMR: Table 1. 3.3.2. Cascarillin C (2) C22H30O5; clear yellowish resin (11 mg); mp 60–62 C; 47.6 (CHCl3; c 0.227); UV lmax CHCl3 nm (log ": [a]20 D 244 (2.9); ESI–MS, m/z: 397.2 [M+Na]+; 413.2 [M+K]+; 337.0 [M–OCOCH3+Na]+; HR–FABMS (positive mode) m/z: 357.207 [M–H2O+H]+; C22H28O4+H requires, 357.207; 1H NMR, 13C NMR: Table 1.
3.3.3. Cascarillin D (3) C22H30O5; clear yellowish resin (6 mg); mp 64–66 C; 23.6 (CHCl3; c 0.356); ESI–MS, m/z: 397.3 [a]20 D [M+Na]+; 413.3 [M+K]+; HR-FABMS (positive mode) m/z: 375.216 [M+H]+; C22H30O5+H requires, 275.217; 1H NMR, 13C NMR: Table 1.
References Bedir, E., Tasdemir, D., C¸alis, I., Zerbe, O., Sticher, O., 1999. Neoclerodane diterpenoids from Teucrium polium. Phytochemistry 51, 921–925. Claude-Lafontaine, A., Ruillard, M., Cassan, J., Azzaro, M., 1976. New terpene derivatives, constituents of the essential oil of Cascarilla. Bull. Soc. Chim. Fr., 88–90. Duke, J.A., 1984. CRC Handbook of Medicinal Herbs. CRC Press, Boca Raton. Greenish, H.G., 1924. A Text Book of Materia Medica, 4th Edition. J&A Churchill, London. Hagedorn, M.L., Brown, S., 1991. The constituents of Cascarilla oil (Croton eluteria Bennett). Flavour and Fragrance Journal 6, 193–204. Halsall, T.G., Oxford, A.W., Rigby, W., 1965. The structure of cascarillin A, an epoxy-furanoid diterpene. Chem. Commun. 11, 218– 219. Hegnauer, R., 1996. Chemotaxonomie der Pflanzen, Vol 4. Bick La¨user, Basel and Stuttgart. Manabe, S., Nishino, C., 1986. Stereochemistry of cis-clerodane diterpenes. Tetrahedron 42, 3461–3470. Tori, M., Katto, A., Masakazu, S., 1999. Nine new clerodane diterpenoids from rhizomes of Solidago altissima. Phytochemistry 52, 487–493.
Three clerodane diterpenoids from Croton eluteria Bennett Claire Vigor*, Nicolas Fabre, Isabelle Fouraste´, Claude Moulis Laboratoire Pharmacophores Redox, Phytochimie et Radiobiologie, Faculte´ des Sciences Pharmaceutiques, 35 chemin des Maraıˆchers, F-31062 Toulouse, France Received 13 November 2000; received in revised form 29 March 2001
Abstract Three furanoid clerodanes have been isolated from the stem bark of Croton eluteria Bennett. Their structures have been established by spectroscopic methods. The compounds were named cascarillin B (7a-acetoxy-3,4,15,16-diepoxy-12-oxo-cleroda13(16),14-dien-20-al), cascarillin C (7a-acetoxy-15,16,12,20-diepoxy-20-hydroxy-cleroda-3,4,13(16),14-triene) and cascarillin D (7aacetoxy-3,4,15,16-diepoxy-cleroda-13(16),14-dien-20-al). # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Croton eluteria Bennett; Euphorbiaceae; Furanoid diterpene; Clerodane
1. Introduction Cascarilla bark, obtained from Croton eluteria Bennett (Euphorbiaceae) is a shrub or small tree indigenous to the Bahama Islands. It was reported to be balsamic, digestive, hypotensive, narcotic, stomachic and tonic (Greenish, 1924). It has been used as a traditional medicine for many applications such as for dysentery, dyspepsia, malaria and fever (Duke, 1984). From the phytochemical point of view, some terpene derivatives were previously reported and specially some ent-clerodane furano-diterpenoids: cascallin, cascarillone and cascarillin A (Halsall et al., 1965; Hegnauer, 1966; Claude-Lafontaine et al., 1976; Hagedorn and Brown, 1991). In this paper, we describe the isolation and structural elucidation of three new related compounds from the stem bark of C. eluteria Bennett.
2. Results and discussion The molecular formula C22H28O6 of compound 1 was determined by ESI mass spectrometry mesured in the positive ion mode (m/z 389.2 [M+H]+, m/z 411.2 [M+Na]+). Its 13C NMR decoupled spectrum (Table 1) * Corresponding author. Tel.: +33-5-6225-6844; fax: +33-5-61554330. E-mail address: [email protected] (C. Vigor).
showed 22 signals in agreement with the molecular formula. The characteristic NMR signals for a furan ring were present in the 1H and 13C NMR spectra (H 6.64 br s, 7.40 br s, 8.03 br s; C 108.6, 144.6, 147.5 and 128.9 Cq), as were those of an aldehyde group (H 10.18 s; C 206.0), an ester carbonyl group (C 170.3) and a keto group (C 193.6). These functional groups account for five of the six oxygens, and the remaining oxygen was involved in an epoxide unit as indicated by a singlet signal at H 2.85 and C 62.0. In addition, four methyl groups were present and were identified from their chemical shifts and coupling patterns as a doublet for C-17 (H 1.01 d, J=7.3 Hz; C 13.7), two singlets (C-18: H 1.12 s; C 19.9 and C-19: H 1.06 s; C 19.2) and an acetyl group (H 2.04 s; C 21.6). Considering the quaternary carbons (C 36.3 and 56.0), the C–H group (C 48.5) and their heteronuclear correlations displayed in the HMBC spectrum, these signals could be assigned, respectively, to carbons C-5, C-9 and C-10 of the decalin moiety. The position of the aldehyde group was deduced from the HMBC spectrum which exhibited cross peaks between the aldehyde carbon and H-8, H-11 and the aldehyde proton with C-9, C-11 (Fig. 1). Its chemical shift agrees with an aldehyde group that we assigned to C-9. The location of the epoxide group (C-3–C-4) was deduced from the HMBC spectrum correlations between C-3 (C 62.0) and H-1a, H-2a, H-18 and C-4 (C 65.1) with H-18, H19, H-6a, H-6b, H-2a; Fig. 1. In the same way, the acetoxy group was located on position C-7 (correlations
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between acetate protons (H 2.04 s) and C-7), and the keto group in position C-12 (correlations between the quaternary carbon signal at C 193.6 and H-11a, H-11b and H-14; Fig. 1).
The 13C NMR signal of the shielded Me-19 (C 19.2) suggested a trans A/B ring junction for decalin as previously discussed by Manabe and Nishino (1986). The a orientation for the C-3–C-4 epoxy group was proposed on the basis of the 1H NMR signals of the shielded Me19 protons (H 1.06 s) (Manabe and Nishino, 1986). Finally, an a orientation for the acetoxy group was proposed on the basis of the J value J 8b 7b=4 Hz which is in accordance with axial-equatorial coupling and corresponds to an approximate dihedral angle H-7b–H-
8b of 70 . This was supported by the coupling constant values of H-6 (see Table 1) and the values reported in the literature (Bedir et al., 1999; Tori et al., 1999). Thus, these data allowed us to establish 1, as 7a-acetoxy-3,4,15,16-diepoxy-12-oxo-cleroda-13(16),14-dien-20al, which we named cascarillin B. The positive ESI mass spectrum of compound 2 showed peaks at m/z 397.2, m/z 413.2 and m/z 337.0 corresponding to [M+Na]+, [M+K]+, and [M– OCOCH3+Na]+ respectively, suggesting a C22H30O5 formula. The acetoxy group was still present as supported by the ESI mass spectral data, the carbon at dC 172.4, and three protons at H 2.06 ppm. The HMBC spectrum did not allow us to locate the acetoxy group. But, the 1H–1H COSY spectrum showed a cross peak between H-17 (H 1.13, d, J=7.3 Hz) and H-8 (H 1.66, dd, J=7.3, 3 Hz). The latter proton (J=3 Hz) correlates with a deshielded proton at H 5.03 ppm (m) whose chemical shift is in agreement with a CH-7a-acetoxy as already described for 1. Both epoxide and aldehyde groups were absent from 2 and replaced by a double bound and a lactol function, respectively. This was shown by the characteristic 1H and 13C NMR signals at H 5.22 ppm (H-3), C 122.3 (C-3) and C 144.6 (C-4) for the first one, and at H 5.85 ppm (H-20), C 101.6 (C-20) and H 5.08 ppm (H-12), C 73.2 (C-12) for the second one. The spectrum unambiguously revealed the C-3–C-4 unsaturation (cross peaks between H-18/C-4 and H-18/ C-3). This was confirmed by the 1H–1H COSY correlations (H-18/H3). In the same way, the C-20 carbon (C 101.6), involved in the lactol function, was correlated in the HMBC spectrum with H-8, H-11b and H-12. The correlations of C-9 with H-10, H-17, H-11a, H-11b and H-20, confirmed this result. The methine (C-12) at C 73.2 was correlated with H-11b and H-20, and this proton displayed a cross peak with C-16 and C-20. Thus, the structure of 2 was established as 7a-acetoxy15,16,12,20-diepoxy-20-hydroxy-cleroda-3,4,13(16),14triene, which we named cascarillin C. The molecular formula of compound 3 was determined as C22H30O5 based on ESI mass spectrum [M+Na]+ (m/z 397.3) and [M+K]+ (m/z 413.3). Comparison of the spectral data of this compound with that of 1 indicated that compound 3 differed from 1 through the lack of a keto group in position C-12. These results led us to propose the structure 7a-acetoxy-3,4,15,16-diepoxy-cleroda13(16),14-trien-20-al for compound 3, which we named cascarillin D.
3. Experimental 3.1. Instrumentation 1
H NMR (400 MHz) and J mod. 13C NMR (100 MHz) were obtained with CD3OD or CDCl3 as solvent,
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C. Vigor et al. / Phytochemistry 57 (2001) 1209–1212 Table 1 1 H and 13C NMR data for compounds 1, 2 in CD3OD and 3 in CDCl3 ( in ppm, multiplicities, J in Hz) H
C
Position
1
1a 1b 2a 2b 3 4 5 6a 6b 7 8 9 10 11a 11b 12
1.37 m 1.17 m 1.95 m 1.42 m 2.85 s – – 2.09 dd (14.6–3.7) 1.76 dd (14.6–3.7) 5.25 dd (3.7-3) 2.63 dd (7.3–4) – 2.04 s 3.37 d (17.8) 2.76 d (17.8) –
2.00 m 1.85 m 5.22 br s – – 2.17 dd (14.6–2.6) 1.47 dd (14.6–3.4) 5.03 ddd (3.4–3–2.8) 1.66 dd (7.3–3) – 1.62 br s 2.64 dd (13.3–7.2) 1.92 dd (13.3–8.9) 5.08 dd (8.9–7.2)
– 6.64 br s 7.40 br s 8.03 br s 1.01 d (7.3) 1.12 s 1.06 s 10.18 s 2.04 s –
– 6.63 br s 7.40 br s 7.45 br s 1.13 d (7.3) 1.59 m 1.27 s 5.85 s 2.06 s –
13 14 15 16 17 18 19 20 OCOCH3 OCOCH3
2
3 2.04 m
1.61 m 1.38 m 2.13 m 1.76 m 3.00 br s – – 2.13 m 1.84 dd (14.5–3.7) 5.29 dd (3.6-3) 2.14 m – 3.31 dd (4.9–3.3) 1.99 m 1.65 m a: 2.35 dd (12.5–4.9) b: 2.28 dd (12.5–4.9) – 6.32 br s 7.4 br s 7.32 br s 1.15 d (7. 3) 1.19 s 1.07 s 10.14 d (0.7) 2.07 s –
1
2
3
16.6
23.5
17.1
27.6
29.6
29.0
62.0 65.1 36.3 39.9
122.3 144.6 39.7 42.1
63.5 66.7 37.6 41.1
74.2 37.3 56.0 48.5 38.5
77.3 47.3 55.5 52.1 45.3
75.9 38.5 54.9 49.9 32.2
193.6
73.2
18.4
128.9 108.6 144.6 147.5 13.7 19.9 19.2 206.0 21.6 170.3
131.7 111 144.3 140.9 15.4 18.5 22.7 101.6 21.4 172.4
126 111.8 144.3 140.1 13.5 20.1 19.5 208.1 21.3 172.1
ion mode) mass spectra were recorded on an Autospec 6F mass spectrometer manufactured by Micromass. 3.2. Plant material Croton eluteria Bennett, collected in Ecuador, was purchased from Richard et Frappa, Aubagne, France. Voucher specimens were identified by Professor Isabelle Fouraste´ and deposited at the herbarium of the Pharmacognosy Laboratory, Faculty of Pharmacy (Toulouse). 3.3. Extraction and isolation
Fig. 1. Significant HMBC couplings of 1.
on a Bru¨cker ARX-400 spectrometer. Chemical shifts are given in (ppm) using the solvent peaks, H 4.87, C 48 ppm and H 7.23, C 77 ppm, respectively, as standards ; ESI–MS: 3.5 kV (MeOH–CHCl3) (Perkin Elmer API Sciex 365 Mass spectrometer); HR–FAB (positive
Air-dried powdered bark (1 kg) of C. eluteria Bennett was extracted with Me2CO at room temperature (24 h). The extract (70 g) was taken up in CHCl3 and chromatographed on silica gel columns eluted with various proportions of CHCl3 and MeOH. The fraction containing the main furanoid diterpenes (revealed on TLC by Ehrlich reagent) was evaporated to dryness and separated on 3 successive silica gel (70–200 mm) columns eluted with a hexane–EtOAc gradient. Further purification of the crude diterpene by reversephase chromatography (Mega Band Elut Varian C18 cartridge: MeOH–H2O 6:4), followed by medium
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pressure (10 bars) silica gel chromatography (silica 60A˚, CC chromagel SDS, 6–35 mm) eluted with toluene–EtOAc 95:5 and then another reverse-phase chromatography (C18 cartridge MeOH–H2O 1:1) gave pure compound 1. Concerning compounds 2 and 3, they were obtained by successive medium pressure (10 bars) silica gel chromatography (silica 60 A˚, CC chromagel SDS, 6–35 mm) eluted with a toluene–EtOAc gradient, followed by reverse-phase chromatography (C 18 cartridge: MeOH– H2O 13:7; 14:6) and preparative HPLC (Varian Dynamax 100 A˚, MeOH–H2O 7:3, 10 ml min 1, 204 nm; MeOH–H2O 15:5, 10 ml min 1, 204 nm). 3.3.1. Cascarillin B (1) C22H28O6; yellow resin (126 mg); mp 68–70 C; [a]20 D 29.1 (CHCl3; c 1.65); UV lmax CHCl3 nm (log ": 275 (3.1); ESI–MS, m/z: 389.2 [M+H]+; 411.2 [M+Na]+; HR–FABMS (positive mode) m/z: 329.174 [M– C2H4O2+H]+; C20H24O4+H requires, 329.175; 1H NMR, 13C NMR: Table 1. 3.3.2. Cascarillin C (2) C22H30O5; clear yellowish resin (11 mg); mp 60–62 C; 47.6 (CHCl3; c 0.227); UV lmax CHCl3 nm (log ": [a]20 D 244 (2.9); ESI–MS, m/z: 397.2 [M+Na]+; 413.2 [M+K]+; 337.0 [M–OCOCH3+Na]+; HR–FABMS (positive mode) m/z: 357.207 [M–H2O+H]+; C22H28O4+H requires, 357.207; 1H NMR, 13C NMR: Table 1.
3.3.3. Cascarillin D (3) C22H30O5; clear yellowish resin (6 mg); mp 64–66 C; 23.6 (CHCl3; c 0.356); ESI–MS, m/z: 397.3 [a]20 D [M+Na]+; 413.3 [M+K]+; HR-FABMS (positive mode) m/z: 375.216 [M+H]+; C22H30O5+H requires, 275.217; 1H NMR, 13C NMR: Table 1.
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