Phytochemistry 61 (2002) 919–922 www.elsevier.com/locate/phytochem Coumarins from Cedrelopsis grevei (Ptaeroxylaceae) Dulcie A. Mulhollanda,*, Maria Kotsosa, Hamdani A. Mahomeda, Neil A. Koorbanallya, Milijaona Randrianarivelojosiaa,b, Linda Quarles van Uffordc, Albert J.J. van den Bergc a Natural Products Research Group, School of Pure and Applied Chemistry, University of Natal, Durban, 4041, South Africa b Laboratory of Pharmacology, EES Sciences, University of Antananarivo, BP 906, Antananarivo, 101, Madagascar c Department of Medicinal Chemistry, Faculty of Pharmacy, University of Utrecht, PO Box 80082, 3508 TB Utrecht, The Netherlands Received in revised form 26 August 2002 Abstract The stem bark of Cedrelopsis grevei Baill. has yielded the first reported examples of 5-prenylated coumarins, cedrecoumarin A and B as well as the known coumarins, cedrelopsin, scoparone, O-methylcedrelopsin and norbraylin, and the known chromones ptaeroglycol and ptaeroxylinol. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cedrelopsis grevei; Ptaeroxylon obliquum; Ptaeroxylaceae; b-Amyrin; Scoparone; O-methylcedrelopsin; Norbraylin; Cedrecoumarin A; Cedrecoumarin B; Ptaeroglycol; Ptaeroxylinol; a and b estrogen receptor agonist; Superoxide anion scavenger 1. Introduction The monotypic genus Ptaeroxylon has given difficulty to botanists, who have placed this genus in the Sapindaceae, Rutaceae and most popularly, in the Meliaceae. In their generic monograph on the Meliaceae, Styles and Penington (1975) state that the genus Cedrelopsis is closely related to Ptaeroxylon and very similar to it in morphology and in the structure of the secondary xylem. The pollen of Cedrelopsis and Ptaeroxylon have been found to be very similar, unlike that of any known Meliaceous pollen grain but similar to that of some Rutaceae. They concluded that Cedrelopsis and Ptaeroxylon do not belong to the Meliaceae and that there is insufficient evidence to place them in either the Sapindaceae or Rutaceae. Thus, these genera are now considered to form a separate family, the Ptaeroxylaceae, which has been shown to be chemically distinct from both the Rutaceae and Meliaceae and to contain a range of chromones and some unusual coumarins (Dean et al., 1967a, b; Dean and Taylor, 1966; Eshiett and Taylor, 1968; Dean and Robinson, 1971). * Corresponding author. Tel.: +27-31-260-1108; fax: +27-31-2603091. E-mail address: mulholld@nu.ac.za (D.A. Mulholland). Cedrelopsis grevei Baill. is one of seven species of the genus Cedrelopsis which is confined to Madagascar. This species is commonly referred to as ‘‘Katrafay’’ by the local people, and it is believed to relieve muscular fatigue when the bark is soaked in bath water. Two specimens have been investigated, one specimen from the wetter north-western part of Madagascar which resulted in the isolation of the pentacyclic triterpenoid, b-amyrin, as well as two novel limonoidderived compounds, a pentanortriterpenoid, cedmilinol 1, and a hexanortriterpenoid, cedmiline 2 (Mulholland et al., 1999; Fig. 1). The isolation of these limonoid derivatives led to the investigation of the second specimen collected in the drier south of Madagascar. Compounds which have been isolated previously from Cedrelopsis grevei include the 6,7-oxygenated chromones ptaeroxylin (Eshiett and Taylor, 1968), alloptaeroxylin, alloptaeroxylin methyl ether, peucinin, heteropeucinin, greveiglycol and ptaeroglycol (Dean and Robinson, 1971) as well as the coumarin cedrelopsin (Eshiett and Taylor, 1968). Ptaeroxylin, alloptaeroxylin (Dean and Taylor, 1966) and ptaeroglycol (Dean et al., 1967b) have also been isolated from the South African Ptaeroxylon obliquum (Ptaeroxylaceae). 0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(02)00440-5 920 D.A. Mulholland et al. / Phytochemistry 61 (2002) 919–922 Fig. 1. Cedmilinol (1), cedmiline (2), cedrecoumarin A (3) and cedrecoumarin B (4) (arrows indicate NOE correlations). 2. Results and discussion In the present study, the stem bark of C. grevei Baill. obtained from the dry southern part of Madagascar was investigated. The species furnished b-amyrin, the 6,7-dioxygenated coumarins cedrelopsin, scoparone, O-methylcedrelopsin, norbraylin and two novel coumarins, cedrecoumarin A 3 and cedrecoumarin B 4 (Fig. 1), as well as the known chromones ptaeroglycol and ptaeroxylinol. The cedrecoumarins are of particular interest as they are the first examples of 5-prenylated coumarins to be reported. Cedrecoumarin A 3 was found to be an angular pyranocoumarin and the structural isomer of norbraylin. The high resolution mass spectrum of this compound exhibited a molecular ion at m/z 244.0731, indicating the molecular formula C14H12O4. The nine double bond equivalents present suggested the presence of a threering coumarin. A fragment peak at m/z 229 ([M-15]+) is typical of pyranocoumarins and results from the loss of one of the geminal methyl groups, to form the stable benzopyrylium ion (Murray et al., 1982). The IR spectrum showed peaks at 3370 (O–H stretching), and 1710 cm
1 (C¼O stretching), the latter frequency being typical for the a-pyrone carbonyl stretching (Murray et al., 1982). The 1H NMR spectrum of 3 showed two one proton doublets at dH 8.08 and 6.19 which were assigned to H-4 and H-3 of the coumarin nucleus based on HMBC correlations with C-2. Resonances at dH 1.43 (23H), 5.90 (1H) and 6.79 (1H) were assigned to 3H-40 and 3H-50 , H-20 and H-10 of the pyran ring. The carbon atom, C-10 of the pyran ring was bonded to C-5 based on results of HMBC and NOESY experiments. Firstly, a NOESY correlation was seen between H-10 and H-4 and this could only occur if C-10 was attached to C-5. Secondly, the HMBC spectrum showed correlations between H-10 and C-4a, C-5 and C-6, confirming attachment of the isoprenyl group at C-5. The one proton singlet at dH 6.63 in the 1H NMR spectrum of 3 was allocated to H-8 as a correlation was observed between this proton and C-4a and C-6 in the HMBC spectrum. No correlations with H-8 were seen in the NOESY spectrum, supporting this assignment. The remaining hydroxy group was placed at C-7. The remaining quaternary carbons were assigned using the HMBC spectrum (Table 1.). The high resolution mass spectrum of cedrecoumarin B 4 showed a molecular formula C16H18O4 (m/z 274.1191). The IR spectrum showed C–H aromatic stretching at 3010 cm
1, a carbonyl stretching band at 1730 cm
1, and a C¼C stretching band at 1605 cm
1. This carbonyl stretch frequency is typical of the a-pyrone carbonyl group stretch of a coumarin (Murray et al., 1982). The 1H NMR spectrum of this compound indicated it was a prenylated coumarin with two methoxy groups. Two one-proton doublets at dH 8.00 (J=9.5 Hz) and 6.31 (J=9.5 Hz) were allocated to H-4 and H-3 respectively as in cedrecoumarin A 3 and the corresponding 13C NMR resonances occurred at dC 143.3 and 113.1. The isoprenyl group was again placed at C-5 based on NOE experiments. Irradiation of H-4 gave a positive NOE for the two-proton doublet at dH 3.65 which was assigned to 2H-10 . Irradiation of the 2H-10 (dH 3.65) resonance showed a NOE enhancement of H4, H-20 , the methoxy group proton resonance at dH 3.81 and the vinyl methyl group proton resonance at dH 1.87. Thus a methoxy group was placed at C-6 and the methyl proton resonance was assigned to 3H-50 . Irradiation of the H-20 (dH 5.10) resonance gave a NOE enhancement of the 2H-10 resonance and the methyl proton resonance at dH 1.73 which could be assigned to 3H-40 . The resonances at dC 25.4, 123.6, 25.9 and 18.2 in the 13C NMR spectrum were assigned to C-10 , C-20 , C-40 and C-50 , respectively. Irradiation of the resonance at dH 6.94 attributed to H-8 showed NOE enhancement of only the methoxy group proton resonance at dH 3.98. The fact that enhancement of only one methoxy group proton resonance occurred on irradiation of the signal at dH 6.94 confirms the assignment of this resonance to H-8. Remaining 13C NMR assignments are given in Table 1. Cedrecoumarin B decomposed on standing. Cedrecoumarin A was found to be active at 10 mg/ml in assays for agonistic activity on ER (estrogen receptor) a and ER b using human embryonal kidney cells transfected with either ER a or ER b, and both with the luciferase reporter gene (Kuiper et al., 1998). The activity of cedrecoumarin A on ER b was more pronounced (60% of the maximum stimulation by 10
11 M 17-bestradiol) in comparison with ER a (40%). However, 921 D.A. Mulholland et al. / Phytochemistry 61 (2002) 919–922 Table 1 1 H and 13C NMR data of cedrecoumarin A 3 and cedrecoumarin B 4 3, 1Ha Pos. 2 3 4 4a 5 6 7 8 8a 6-OCH3 7-OCH3 10 20 30 40 50 a b c d 6.19, d (9.7) 8.08, d (9.7) 6.63, s 6.79, d (10.0) 5.90, d (10.0) 1.43, s 1.43, s 3, 13Cb 3, HMBC correlations 162.50 111.40 140.28 107.31 117.64 137.84 149.99 102.63 150.43 – – 116.72 133.49 76.49 26.25 26.25 H-3,4 3, NOESY interactions 4, 1Hc H-4 H-3,10 6.31, d (9.5) 8.00, d (9.5) H-10 3,4,8 H-4,10 ,20 H-10 ,20 ,8 H-8 H-4 H-4,8 6.94, s H-4,20 H-10 ,3H-40 ,3H-50 H-4,20 H-10 , 3H-40 , 3H-50 3.81, s 3.98, s 3.65, d (6.5) 5.10, m H-10 ,20 H-10 ,20 H-10 ,20 H-10 ,20 1.73, s 1.87, s 4, 13Cd 163.5 113.1 143.3 112.2 133.9 154.0 158.1 99.7 145.3 61.4 56.7 25.4 123.6 133.7 25.9 18.2 4, NOE interactions H-4 H-3, 2H-10 H-8 7-OCH3 2H-10 H-4,20 ,3H-50 , 6-OCH3 2H-10 , 3H-40 H-20 2H-10 300 MHz, CD3OD. 100 MHz, CD3OD. 300MHz, CD3OD, Sample decomposed—no HMBC/NOESY spectra obtained. 75MHz, CD3OD. activity was low compared to the standard genistein which is active in the same ER a and b assays at 30 ng/ ml (80 and 160%, respectively). Cedrecoumarin A was found to inhibit luminol-enhanced chemiluminescence of reactive oxygen metabolites generated by human polymorphonuclear leukocytes activated with opsonized zymosan (IC50 3.2 mg/ml) and to scavenge superoxide anions in a cell free system (IC50 3.0 mg/ml) suggesting anti-inflammatory activity for this compound (Smit et al., 2000; Van den Worm, 2001). 3. Experimental 3.1. General IR spectra were recorded with a Nicolet Impact 400 D spectrometer on sodium chloride plates and calibrated against an air background. EIMS were obtained using a Finnigan 1020 spectrophotometer operating at 70 eV. HRMS were obtained using a Kratos High Resolution MS 9/50 spectrometer. 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 Gemini 300 MHz spectrometer in CD3OD. HMBC and NOESY spectra for 3 were recorded on a Varian Unity Inova 400 MHz spectrometer. 3.2. Extraction and isolation The stem bark of C. grevei was collected in Madagascar by Dr. M. Randrianarivelojosia and a voucher specimen (No.: 002-Mj/M.Dul) was retained at the University of Antananarivo in Madagascar. A sample of the dried powdered stem-bark (1051.7 g) was extracted with hexane using a Soxhlet apparatus, to yield 35.8 g of extract. A 7.5 g sample of the hexane extract was examined and after repeated column chromatography over silica gel (Merck 9385), using dichoromethane/ethyl acetate in varying proportions, yielded b-amyrin (105 mg) (Razdan et al., 1987), the coumarins cedrelopsin (5 mg) (Eshiett and Taylor, 1968), scoparone (13 mg) (Razdan et al., 1987), O-methylcedrelopsin (10 mg) (Kokwaro et al.,1983), norbraylin (3 mg) (Deshmukh et al., 1976) and two oxepin ring chromones, ptaeroglycol (26 mg) (Dean et al., 1967b; Dean and Robinson, 1971) and ptaeroxylinol (31 mg) (Dean et al., 1967b) which were identified by comparison of their physical and spectroscopic data against literature values as referenced above. The novel coumarins cedrecoumarin A 3 and cedrecoumarin B 4 were also isolated. 3.2.1. Cedrecoumarin A 3 9-Hydroxy-7,7-dimethyl-2H,7H-benzo[1,2-b:3,4-b]dipyran-2-one (13 mg), yellow gum, HRMS m/z 244.0731 (C14H12O4 requires 244.0735) EIMS m/z (rel. int.) 244 (26), 229 (100), 201 (10), 100 (1); IR vmax (cm
1): 3370, 1710; UV lmax nm (log "): 316 (2.6), 275 (1.8), 232 (4.1); 1 H and 13C NMR data are given in Table 1. 3.2.2. Cedrecoumarin B 4 6,7-Dimethoxy-5-(3-methyl-2-butenyl)-2H-1-benzopyran-2-one, 6,7-dimethoxy-5-prenylcoumarin (12 mg), yellow gum, HRMS m/z 274.1191 (C16H18O4 requires 274.1205) EIMS m/z (rel. int.) 274 (90), 244 (8), 233 922 D.A. Mulholland et al. / Phytochemistry 61 (2002) 919–922 (17), 205 (12), 167 (145), 149 (100); IR vmax (cm
1) : 3010, 1730, 1605; UV lmax nm (log "): 335 (1.9), 296 (1.9), 259, 231 (4.1); 1H and 13C NMR data are given in Table 1. Acknowledgements This research was funded by the University of Natal Research Fund and the Foundation for Research and Development. We gratefully acknowledge the Wellcome Trust Equipment grant number 053547 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 Mr. A. Rakotozafy and Dr. J. Ranaivoravo for their assistance in obtaining plant material. References Dean, F.M., Parton, B., Somvichien, N., Taylor, D.A.H., 1967a. The coumarins of Ptaeroxylon obliquum. Tetrahedron Lett. 36, 2147. Dean, F.M., Parton, B., Somvichien, N., Taylor, D.A.H., 1967b. Chromones containing an oxepin ring from Ptaeroxylon obliquum. Tetrahedron Lett. 36, 3459. Dean, F.M., Robinson, M.L., 1971. The heartwood chromones of Cedrelopsis grevei. Phytochemistry 10, 3221. Dean, F.M., Taylor, D.A.H., 1966. Extractives from East African Timbers. Part II. Ptaeroxylon obliquum. J. Chem. Soc. C, 114. Deshmukh, M.N., Deshpande, V.H., Rama Rao, A.V., 1976. Two new coumarins from Toddalia aculeate. Phytochemistry 15, 1419. Eshiett, I.T., Taylor, D.A.H., 1968. The isolation and structural elucidation of some derivatives of dimethylallyl-coumarin, chromone, quinoline and phenol from Fagara species and from Cedrelopsis grevei. J. Chem. Soc. C, 481. Kokwaro, J.O., Messana, I., Galeffi, G., Pattamia, M., Marini Bettolo, G.B., 1983. Coumarins from Xanthoxylum usambarense. Planta Medica 47, 251. Kuiper, G.G.J.M., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., Van der Saag, P.T., Van der Burg, B., Gustafsson, J.A., 1998. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor b. Endocrinology 193, 4252–4263. Mulholland, D., Mahomed, H.A., Kotsos, M., Randrianarivelojosia, M., Lavaud, C., Massiot, G., Nuzillard, J.M., 1999. Limonoid derivatives from Cedrelopsis grevei. Tetrahedron 55, 11547. Murray, R.D.H., Mendez, J., Brown, S.A., 1982. The Natural Coumarins—Occurrence, Chemistry and Biochemistry. John Wiley and Sons Limited, Chichester. Razdan, T.K., Qadri, B., Harkar, S., Waight, E.S., 1987. Chromones and coumarins from Skimmia laureola. Phytochemistry 26, 2063. Smit, H.F., Kroes, B.H., Van den Berg, A.J.J., Van der Wal, D., Van den Worm, E., Beukelman, C.J., Van Dijk, H., Labadie, R.P., 2000. Immunomodulatory and anti-inflammatory activity of Picrorhiza scrophulariiflora. J. Ethnopharmacol. 73, 101–109. Styles, B.T., Pennington, T.D., 1975. A generic monograph of the Meliaceae. Blumea 22, 476. Van den Worm, E., 2001. Investigations on Apocynin, a Potent NADPH Oxidase Inhibitor. PhD thesis, University of Utrecht.