Phytochemistry 64 (2003) 631–635 www.elsevier.com/locate/phytochem Secondary metabolites from Cedrelopsis grevei (Ptaeroxylaceae) Dulcie A. Mulhollanda,*, Dashnie Naidooa, Milijaona Randrianarivelojosiaa,b, Peter K. Cheplogoia, Philip H. Coombesa a Natural Products Research Group, School of Pure and Applied Chemistry, University of Natal, Durban, 4041, South Africa b Malaria Research Group, BP 1274- Antananarivo (101)- Institut Pasteur de Madagascar, Madagascar Received 16 January 2003; received in revised form 5 May 2003 Dedicated to the memory of Professor Jeffrey B. Harborne Abstract From the hexane extract of the stem bark of Cedrelopsis grevei (Ptaeroxylaceae) was isolated the triterpenoid derivative, cedashnine, and the quassinoid, cedphiline, along with cedmiline, scoparone, b-amyrin and sitosteryl glucoside. # 2003 Elsevier Ltd. All rights reserved. Keywords: Ptaeroxylaceae; Cedrelopsis grevei; Triterpenoid derivative; Quassinoid; Cedmiline; Cedashnine; Cedpetine; Scoparone; b-Amyrin; Sitosteryl glucoside 1. Introduction The secondary metabolites isolated from the Madagascan species Cedrelopsis grevei (Ptaeroxylaceae) vary greatly from specimen to specimen investigated. We have recently reported the isolation of two limonoid derivatives, cedmiline and cedmilinol from the bark of a specimen collected at Ankarafantsika in the wetter north of Madagascar (Mulholland et al., 1999a). The bark and wood of specimens collected in the drier south have yielded a range of coumarins and chromones including cedrelopsin, greveichromenol, greveiglycol, heteropeucenin, peucenin, alloptaeroxylin, ptaeroxylinol, ptaeroglycol, ptaeroxylin, (Mulholland et al., 1999b; Dean et al.,1967; Dean and Robinson, 1971; Dean and Taylor, 1966; Eshiett and Taylor, 1968; McCabe et al., 1967; Schulte et al., 1973), cedrecoumarins A and B, (Mulholland et al., 2002) whereas the fruit contains prenylated chalcones and flavanones (Koorbanally et al., 2003). A further specimen (08-99/ MJ.MDul,TAN) was collected during the flowering period from Ankarafantsika in an attempt to isolate more of the limonoid derivatives. However, cedmiline (1), a related hexanortriterpenoid, cedashnine (2), a * Corresponding author. Tel.: +27 31 260 1108; fax: +27 31 260 3091. E-mail address: mulholld@nu.ac.za (D.A. Mulholland). 0031-9422/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0031-9422(03)00342-X quassinoid, cedphiline (3), the known coumarin, scoparone (4), and the common phytosterols, b-amyrin and sitosteryl glucoside were isolated. 2. Results and discussion The hexane extract of the stem bark of Cedrelopsis grevei yielded the limonoid derivative, cedmiline (1), isolated previously from this source (Mulholland et al., 1999a) and the related compound, cedashnine (2). Cedashnine 2 differs from cedmiline 3 only in the structure of the side chain. In cedmiline 3, the side chain occurs as a furan ring, but in cedashnine 2, a 21hydroxy-23,21-butenolide ring is present. Cedashnine 2 is a hexanortriterpenoid with a molecular formula of C24H28O8. The IR spectrum showed bands at 3381 cm
1 (OH stretch), 1710 and 1756 cm
1 (C¼O stretch). The presence of a ring A a,b-unsaturated lactone was indicated by a pair of doublets at 6.37 (H-1) and 5.90 (H-2, J=12.8 Hz) and resonances at 153.9, 118.4 and 167.5 ascribable to C-1, C-2 and C-3 respectively. The C-4 oxygenated quaternary carbon resonance occurred at 84.8. Ring B was expanded incorporating C-30 as previously reported for cedmiline 3 and cedmilinol (Mulholland et al., 1999a). This was indicated by the chemical shifts of C-7 (209.0) and the diastereotopic H-30 protons (3.57 and 2.46 ABq, J=12.3 Hz). 632 D.A. Mulholland et al. / Phytochemistry 64 (2003) 631–635 Resonances ascribed to H-5 and H-6a and b each occurred as double doublets at 3.09 (J=4.0, 7.8), 2,78 (J=4.0, 17.0) and 2.46 (J=7.8, 17.0) respectively. The COSY spectrum showed coupling between the H-9 resonance (1.99) and the two H-11 resonances which were, in turn, seen to be coupled to the two H-12 resonances. The H-17 resonance occurred as a singlet at 5.25. NOESY correlations between H-17 and H-9, H12a and H-18 indicated that H-17 was in the a-orientation as was shown to be the case with cedmiline 3. The ketone carbonyl resonance at 212.3 showed HMBC correlations with H-17, 3H-18 and H-30b and thus was placed at C-14. The chemical shift of 82.7 for C-8, as in cedmiline 3, confirmed a C-17, C-8 ether linkage. Thus the basic tetracyclic structure of cedashnine 2 was confirmed to be the same as in cedmiline 3. However, no resonances ascribable to a furan ring were present. Subtracting the number of atoms and double bond equivalents required for the tetracyclic structure, left C4H3O3 and three double bond equivalents for the sidechain which could be assigned to a 21-hydroxy23,21-butenolide ring. Double bond carbon resonances were seen at 120.2 (C-20) and 154.0 (C-22), a lactone carbonyl carbon at 167.1 (C-23) and a hemiacetal carbon resonance at 98.0 (C-21). The HSQC spectrum enabled the assignment of singlets at 6.25 and 6.10 to H-21 and H-22 respectively. The fact that the COSY spectrum showed no correlation between the two proton singlets and the HMBC spectrum showed correlations between both C-22 and C-21 and H-17 confirmed the above assignments. It was surprising that only one epimer was present for this compound. Usually in compounds of this type (Cheplogoi and Mulholland, 2003) some resonances are paired in the 1H and 13C NMR spectra because of the hemiacetal existing as a pair of C-21 epimers. A model was constructed to try to explain this observation. It was seen that hydrogen bonding can occur between the hydroxyl group proton at C-21 and the oxygen of the 8,17-ether if the hydroxyl group is a-orientated. Supporting evidence for this was a correlation observed in the NOESY spectrum between H-21 (which would have to be b) and the 3H-18 resonance. This was shown to be possible from the model. Thus an S configuration would occur at C-21. Thus structure (2) is proposed for cedashnine and is supported by HMBC and NOESY correlations as given in Table 1. HRMS of cedphiline (3), showed a molecular ion at m/z 502.25593 indicating a formula of C28H38O8. A peak at m/z 442 indicated the loss of an acetic acid molecule indicating the presence of an acetate group. The IR spectrum showed a hydroxyl stretch band (3500 cm
1) and carbonyl absorptions at 1745 and 1719 cm
1. The 13C NMR spectrum showed the presence of 28 carbon atoms including two for an acetate group and one for a methoxyl group and thus indicated the presence of a C25 quassinoid of type D (Polonsky, 1985). A conspicuous pair of doublets at 2.59 and 2.64 (J=13.7 Hz) were assigned to two H-17 protons. These protons showed HMBC correlations to C-20 ( 164.2, C), C-21 ( 74.5, CH2) and C-22 ( 120.1, CH) of a butenolide ring. Resonances at 4.73 (2H) and 5.92 (s, 1H) were assigned to 2H-21 and H-22. The resonance at 173.1 showed HMBC correlations with 2H-21 and H-22 and 633 D.A. Mulholland et al. / Phytochemistry 64 (2003) 631–635 was assigned to C-23. The methyl group three proton singlets in the 1H NMR spectrum were assigned to 3H19 (H 1.00), 3H-18 (H 1.32) and 3H-30 (H 1.34). The following HMBC correlations were observed: the 3H-19 resonance to C-1 (C 81.0) and C-9 (C 53.8), the 3H-30 resonance to C-7 (C 78.8) and C-9 (C 53.8) and the 3H-18 resonance to C-17 (C 42.5). The methyl group proton doublet at H 0.93 (J=6.4 Hz) showed HMBC correlations to C-3 (C 37.5), C-4 (C 29.1) and C-5 (C 44.2) and was assigned to 3H-28. The C-28 resonance appeared at C 20.0. The carbonyl carbon resonance at C 164.9 was assigned to C-16 of the ring D lactone. The C-16 resonance showed HMBC correlations to the H-15 resonance at H 5.83. The H-7 and H-9 resonances appeared at H 4.14 and 1.89 respectively in the 1H NMR spectrum. The H-9 resonance (H 1.89) showed HMBC correlations with C-1 (C 81.0), C-5 (C 44.2), C-7 (C 78.8), C-8 (C 39.1), C-10 (C 42.1), C-11 (C 70.7), C-14 (C 172.4), C-19 (C 11.8), and C-30 (C 23.0). The H-5 and H-1 resonances appeared at H 1.24 and 3.08 respectively. The H-1 resonance (H 3.08) was superimposed with the H-2 resonance at H 3.08 and this created problems in assigning the stereochemistry of H-1 and H-2. The resonances at C 81.0, 80.6 and C 56.6 were assigned to C-1, C-2 and the methoxyl group carbon respectively by use of the HSQC spectrum. The methoxyl group proton resonance showed HMBC correlation to C-2 (C 80.6), which confirmed that the methoxyl group was attached at C-2. On biosynthetic grounds, 3H-19 and 3H-30 are b-orientated and H-5, H9, 3H-18 and 3H-28 are a-orientated. The H-1/H-2 superimposed resonance showed NOESY correlations with 3H-19, H-4, H-5, H-9, the methoxyl group proton resonance and a very weak correlation with 3H28. In order to distinguish between NOESY correlations with H-2 and H-3, the compound was acetylated. Although the NOESY spectrum of the acetylated product was weak due to the small amount of material available for acetylation, it was clear that the H-1 resonance had shifted to become superimposed with the H21 correlation at H 4.73 and showed NOESY correlations with H-5 and H-9 confirming that H-1 was a and hence the hydroxyl group at C-1, b. Upon acetylation of 3, the H-2 resonance moved under the methoxyl group proton resonance at 3.33, but a strong NOESY correlation could be seen with the 3H19 and H-4 resonances confirming b-stereochemistry for H-2. The H-6 resonance at H 1.82 showed NOESY correlations with H-4 and 3H-19, which indicated a borientation for this H-6 proton. The C-6 resonance appeared at C 25.4 and the remaining H-6a resonance was shown to occur at H 2.05 by use of the Table 1 NMR spectral data for cedashnine (2) C 13C / ppm (CD3OD) 1H / ppm (CD3OD) HMBC (C!H) COSY NOESY 1 2 3 4 5 6 154.0 118.4 167.5 84.8 51.9 46.2 (CH) (CH) (C) (C) (CH) (CH2) 19 2 1 2, 11a, 19 1 6a, 6b 5, 6b 5, 6a 6a, 9, 28, 30a 5, 6b, 28 6a, 19, 29 7 8 9 10 11 209.0 82.7 64.4 48.5 21.3 (C) (C) (CH) (C) (CH2) 6.37 (d, J=12.9 Hz) 5.90 (d, J=12.9 Hz) – – 3.09 (dd, J=4.0, 7.8 Hz) a) 2.78 (dd, J=4.0, 17.0 Hz) b) 2.46 (dd, J=7.8, 17.0 Hz) – – 1.99 (m) – a) 1.88 (m) b) 2.06 (m) a) 2.06 (m) b) 1.60 (m) – – 5.25 (s) 0.97 (s) 1.22 (s) – 6.25 (s) 6.10 (s) – 1.43 (s) 1.53 (s) a) 3.57 (d, J=12.3 Hz) b) 2.46 (d, J=12.3 Hz) 11a 5, 11a, 17 9, 11b, 12a, 12b 11a, 12b 11a, 12b 11a, 11b, 12a 1, 9, 12a 12b, 19 11a, 17, 18 11b, 18 12 13 14 17 18 19 20 21 22 23 28 29 30 41.4 (CH2) 51.1 212.3 79.6 13.6 18.6 120.2 98.0 154.0 167.1 31.1 19.8 44.6 (C) (C) (CH) (CH3) (CH3) (C) (CH) (CH) (C) (CH3) (CH3) (CH2) 2, 28, 29 1, 19, 28, 29 30b 6b, 30a, 30b 30a, 30b 19, 30b 2, 19 12a 17, 18 17, 18 17, 18, 30b 18 9, 12a, 18, 22 12a, 12b, 17, 21, 22 1, 6b, 11b, 29, 30b 1 17 17 18, 30b 17, 18 29 28 5, 6a, 29 6b, 19, 28 5, 30b 21, 30a 30b 30a 634 D.A. Mulholland et al. / Phytochemistry 64 (2003) 631–635 HSQC spectrum. Both the H-6a and H-6b resonances were seen to be coupled in the COSY spectrum to H-5 and H-7. The two H-3 proton resonances appeared at H 0.90 and 2.08 in the 1H NMR spectrum The acetate group proton resonance appeared at H 1.94 in the 1H NMR spectrum of 3. The carbonyl carbon resonance of the acetate group appeared at C 170.8 and showed HMBC correlations with H-11 at H 5.64, which indicated that the acetate group was attached at C-11 (C 70.7). The H-11 resonance showed a NOESY correlation to 3H-19, which suggested a b-orientation for this H-11 proton, leaving the acetate group with an aorientation. The H-11 resonance also showed HMBC correlations with C-9 (C 53.8), C-12 (C 41.5) and C-13 (C 39.6). The two H-12 resonances appeared as a doublet (J=16.3 Hz) at H 2.00 and a double doublet (J=16.3, 5.3 Hz) at H 2.42. It was not possible to distinguish between the H-12a and H-12b resonances as both H-12 resonances showed a NOESY correlation with H-11 and the resonance at H 2.42 showed a NOESY correlation with the superimposed 3H-18/3H- 30 resonance. All other NOESY correlations agreed with a model of cedphiline (3). The known compounds scoparone (4), and b-amyrin were also isolated from the hexane extract and sitosteryl glucoside was isolated from the ethyl acetate extract. These compounds were identified using NMR spectroscopy and structures confirmed by comparison against literature values (Matida et al., 1996; Ahmad, 1994; Duddeck and Kaiser, 1982) This is the first report of the isolation of a quassinoid from outside the Simaroubaceae family. It is interesting that both limonoids and quassinoids have only been found to occur together only in the Cedrelopsis genus of the Ptaeroxylaceae family and the Harrisonia genus of the Simaroubaceae. The similarity between the highly rearranged limonoids from Cedrelopsis and Harrisonia also supports a close link between the Ptaeroxylaceae and the Harrisonia genus of the Simaroubaceae. We have previously noted close similarities between compounds isolated from the Cneoraceae and Ptaeroxylaceae families (Mulholland and Mahomed, 2000) Table 2 NMR spectral data for cedphiline (3) 13C / ppm (CDCl3) C 1 2 3 81.0 (CH) 80.6 (CH) 37.5 (CH2) 4 5 6 29.1 (CH) 44.2 (CH) 25.4 (CH2) 7 8 9 10 11 12 78.8 39.1 53.8 42.1 70.7 41.5 (CH) (C) (CH) (C) (CH) (CH2) 13 14 15 16 17 39.6 172.4 116.4 164.9 42.5 (C) (C) (CH) (C) (CH2) 18 19 20 21 22 23 28 30 Oac Me OMe OAc C=O 31.2 11.8 164.2 74.5 120.1 173.1 20.0 23.0 21.8 56.6 170.8 (CH3) (CH3) (C) (CH2) (CH) (C) (CH3) (CH3) (CH3) (CH3) (C) a 1H / ppm (CDCl3) a 3.08 3.08a a) 0.90 (m) b) 2.08 (m) 1.38 (m) 1.24 (m) a) 2.08 (m) b) 1.82 (m) 4.14 (m) – 1.89 (d, J=5.6 Hz) – 5.64 (m) a) 2.00 (d, J=16.3 Hz)b b) 2.42 (dd, J=16.3, 5.3 Hz)b – – 5.83 (s) – a) 2.59 (d, J=13.7 Hz) b) 2.64 (d, J=13.7 Hz) 1.32 (s) 1.00 (s) – 4.73 (2H) (d, J=1.1 Hz) 5.92 (s) – 0.93 (d, J=6.4 Hz) 1.34 (s) 1.94 (s) 3.33 (s) – HMBC (C!H) COSY NOESY 9, 19 1, 3a, OMe 28 2 1, 3b, 3a 2, 3b, 4 2, 3a 3a 6a, 6b 5, 6a, 7 5, 6b, 7 6b, 6a 5, 9, OMe 4, 19, OMe 3b 2, 3a, 28, OMe 2, 3b, 19, 28 1, 9, 28 6b, 7, 28 4, 6a, 7, 19, 30 6a, 6b, 30 28 9, 19, 28 9, 30 9, 15, 30 1, 7, 11, 12b, 19, 30 9, 19 9, 12b 11, 17a, 17b, 18 11 9, 12b, 12a 11, 12b 11, 12a 1, 5 12a, 12b, 19 11, 12b 11, 12a, 18/30 11, 12b, 12a, 17a, 17b, 18 9, 12b, 15, 17b, 18, 30 17a, 18, 21 15 12a, 18 12b, 12a, 17a, 17b 1, 9 17a, 17b, 21, 22 17a, 17b, 22 17a, 17b, 21 21, 22 7, 9 17b 17a 15, 17b, 18, 21, 22 17a, 18, 21, 22 12a, 15, 17a, 17b, 21, 22 2, 4, 6b, 11, 30 15, 17a, 17b, 18/30 17a, 17b, 18/30 4, 5, 6a 6b, 7, 19 1, 2, 3b 11, AcO Me Resonances superimposed, NOESY correlations could be distinguished by examining the NOESY spectrum of an acetylated product. The H-12a and H-12b resonances may be interchanged. Both showed NOESY correlations with H-11 and one showed a correlation with the superimposed 3H-18, 3H-30 resonance. b D.A. Mulholland et al. / Phytochemistry 64 (2003) 631–635 3. Experimental Stem bark of Cedrelopsis grevei Baill. (Ptaeroxylaceae) was collected from Ankarafantsika in the north-west forest of Madagascar and identified by Dr. M. Randrianarivelojosia and a voucher specimen retained (0899/MJ.MDul,TAN). The dried, milled stem bark (1.7 kg) was extracted successively using a Soxhlet apparatus with hexane, ethyl acetate and methanol for 48 h with each solvent. The hexane extract (143 g) yielded, after cc over silica gel (Merck 9385) using 5 g of extract, b-amyrin (55 mg) and scoparone (4) (14 mg). A sample of 8 g of the ethyl acetate extract (39 g) was separated as above and yielded cedmiline (1), (50 mg), cedphiline (3), (45 mg), cedashnine (2), (25 mg) and sitosteryl b-dglucopyranoside (21 mg). The 1H NMR spectrum of the methanol extract indicated the presence of sugars only, so was not investigated further. IR spectra were recorded with a Nicolet Impact 400 D spectrometer on sodium chloride plates and calibrated against an air background. HRMS were obtained using a Kratos High Resolution MS 9/50 spectrometer at the Cape Technikon. UV spectra were recorded with a Varian DMS 300 UV–visible spectrophotometer using dichloromethane as solvent. 1H and 13C NMR spectra were recorded on a Varian Unity Inova 400 MHz NMR spectrometer. Optical rotations were measured at room temperature using either a Perkin Elmer 241 Polarimeter or an Optical Activity AA-5 Polarimeter together with a series A2 stainless steel (4200 mm) unjacketed flow tube. Cedashnine (2): white crystalline (25 mg); m.p. 220– 221  C; HRMS: 444.17759 (C24H28O8 req. 444.17842), 426.16951(M+
H2O), 316.16656, 277.07193, 258.12538, 175.10432, 121.06497, 108.05742 (100%); IR: max(NaCl): 3381, 2861, 1756, 1710 cm
1; [a]D+15.38 (c 0.26, CH3OH). For NMR spectral data, see Table 1. Cedphiline (3): white crystalline (45 mg); m.p.160– 161  C; HRMS: 502.25593 (C28H38O8 req. 502.25667), 85.06565 (100%), 119.08579 (19.48%), 345.20736 (18.59%), 410.2087 (27.90%), 442.23434 (M+– CH3COOH) (21.08%), 459.23942 (M+–CH3C¼O) (7.61%); IR: max(NaCl): 3500, 2917, 2855, 1745, 1719 cm
1; [a]D
43.97 (c 0.348, CH2Cl2). For NMR spectral data, see Table 2. Acknowledgements This research was funded by the University of Natal Research Fund and the Foundation for Research and 635 Development. We gratefully acknowledge the Wellcome Trust Equipment grant number 052451 for the provision of the 400 MHz NMR spectrometer. We are grateful to Mr. Dilip Jagjivan and Dr. P. Boshoff for running NMR and mass spectra and Dr. Harison Rabarison and Mr. Romain for their assistance in obtaining and identifying plant material. References Ahmad, V.U., 1994. Handbook of Natural Products Data, volume 2, Pentacyclic Triterpenoids. Elsevier Science B.V., London. pp. 21-22. Cheplogoi, P.K., Mulholland, D.A., 2003. Tetranortriterpenoid derivatives from Turraea parvifolia (Meliaceae). Phytochemistry 62, 1173–1178. Dean, F.M., Parton, B., Somvichien, N., Taylor, D.A.H., 1967. Chromones, containing an oxepin ring, from Ptaeroxylon obliquum. Tetrahedron Letters 36, 3459–3464. Dean, F.M., Robinson, M.L., 1971. Heartwood chromones of Cedrelopsis grevei. Phytochemistry 10, 3221–3227. Dean, F.M., Taylor, D.A.H., 1966. Extractives from East African timbers. II. Ptaeroxylon obliquum. J. Chem. Soc. (C) 114–116. Duddeck, H., Kaiser, M., 1982. 13NMR Spectroscopy of Coumarin Derivatives. Organic Magnetic Resonance 20, 55–73. Eshiett, I.T., Taylor, D.A.H., 1968. The isolation and structure elucidation of some derivatives of dimethylallylcoumarin, chromone, quinoline, and phenol from Fagara species and from Cedrelopsis grevei. J. Chem. Soc. (C) 481–484. Koorbanally, N.A., Randrianarivelojosia, M., Mulholland, D.A., Quarles van Ufford, L., van den Berg, A.J.J., 2003. Extractives from the seed of Cedrelopsis grevei. Phytochemistry 62, 1225–1226. Matida, A.K., Rossi, M.H., Blumenthal, E.E.A., Schuquel, I.T.A., Malheiros, A., Vidotti, G.J., 1996. 3-b-O-b-d-Glucopyranosylsitosterol in species of Labiatae, Verbenaceae and Apocynaceae. Ann. Assoc. Bras. Quim. 45, 147–151. McCabe, P.H., McCrindle, R., Murray, R.D.H., 1967. Constituents of sneezewood, Ptaeroxylon obliquum. I. Chromones. J. Chem. Soc. (C) 145–151. Mulholland, D.A., Mahomed, H., Randrianarivelojosia, M., Lavaud, C., Massiot, G., Nuzillard, J., 1999a. Limonoid derivatives from Cedrelopsis grevei. Tetrahedron 55, 11547–11552. Mulholland, D.A., Kotsos, M., Mahomed, H.A., Randrianarivelojosia, M., 1999b. The chemistry of the Ptaeroxylaceae. Nig. J. Nat. Prod. Med. 3, 13–16. Mulholland, D.A., Kotsos, M., Mahomed, H.A., Koorbanally, N.A., Randrianarivelojosia, M., van Ufford, L.Q., van den Berg, A.A.J., 2002. Coumarins from Cedrelopsis grevei (Ptaeroxylaceae). Phytochemistry 61, 919–922. Mulholland, D.A., Mahomed, H., 2000. Isolation of Cneorubin X, an unusual diterpenoid from Ptaeroxylon obliquum (Ptaeroxylaceae). Biochemical Systematics and Ecology 28, 713–716. Polonsky, J., 1985. Quassinoid Bitter Principles II. Progress in the Chemistry of Organic Natural Products 47, 221–264. Schulte, K.E., Ruecker, G., Klewe, U., 1973. Components of medicinal plants. XXVIII. Some constituents of the bark of Cedrelopsis grevei. Arch. Pharm. (Weinheim, Ger.) 306, 857–865.