0031-9422(94)00605-9 Pergamon Phytochemistry, Vol. 38, No. 1, pp. 217-219, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/95 $9.50 + 0.00 FAGAROPSINE, A DEGRADED LIMONOID GLUCOSIDE FROM FAGAROPSIS GLABRA JOEL BOUSTIE,* MARIE-JosI~ RESPAUD,CLAUDE MOULIS, CATHERINELAVAUD,~JACQUELINEGLEYE and ISABELLE FOURASTt~ Laboratoire de Pharmacognosie, Facult6 de Pharmacie, Universit6 de Toulouse III, 35 Chemin des Maraichers-31062 Toulouse, Cedex, France; *Laboratoire de Pharmacognosie et de Mycologie, Facult6 de Pharmacie, Universit6 de Rennes I, 2 Av. du Pr. L6on Bernard-35043 Rennes, Cedex, France; tLaboratoire de Pharmacognosie, Facult6 de Pharmacie, Universit6 de Reims, 51 Rue Cognacq-Jay-51096 Reims, Cedex, France (Received in revisedform 1 July 1994) Key Word lndex--Faoaropsis olabra; Rutaceae; limonoid; degraded limonoid glycoside; fagaropsine. Al~tract--Phytochemical studies of the alcoholic-soluble portion of Fagaropsis glabra have resulted in the isolation of fagaropsine, a degraded limonoid glycoside. Its absolute structure was elucidated as 1-O-fl-D-giucopyranosyl-4ct-(3'furanyl)-7fl-hydroxy-4a~t,8~-dimethyl-4,4a,5,6,7,8-hexahydro-3-benzopyran-2-one on the basis of spectral data. INTRODUCTION 5' Previous investigations of Fagaropsis species related the presence in this genus of benzophenanthridine alkaloids [1, 2] and limonoids of the limonidic tetranortriterpenoid [1, 33 and degraded limonoid class 1,3-5]. The various biological activities of these limonoids are of agricultural and medicinal interest 1-63. In the field of antifeedants research, structure-activity relationships 1-7] led chemists to focus on the C/D ring of these compounds I-8,93 which are naturally encountered as degraded limonoids. In addition to previous degraded limonoids found in F. glabra [3, 53, we report here the isolation and identification of the first glucosylated degraded limonoid glucoside (I) called fagaropsine, which differs in the D-ring glycosylation pattern from other reported fl-D-limonoid glycosides [ 103. RESULTSAND DISCUSSION As the electronic impact mass spectrum of 1 did not afford any positive result, the molecular weight was determined by FAB mass spectrometry. The positive ion FAB mass spectrum showed the pseudomolecular ion peak [M + H 3 + at m/z 441 corresponding to C21H2sOlo and the base peak at ra/z 279 indicated the presence of a hexose [(M + H)-(180 + H20)3 +. The IR spectrum of 1 exhibited a carbonyl absorption at 1712 cm- t suggesting a pyrano-type aglycone with a conjugated 6-1actonic ring. These results were corroborated by an extensive analysis of the NMR data (Tables 1 and 2). The comparison with signals observed for degraded iimonoids we previously isolated [3, 5], in addition to signals corresponding to a sugar moiety, indicated a glycosylated compound of the pyroangolenside type [8, 11]. The coupling con217 4 ' / ~ X , Xol, -9 n o ~ . 2" r 3./on ~. . \OH stant J = 6.5 Hz of the anomeric proton resonating at 64.78 and the 13C and 1H NMR signals were consistent with a fl-D-glucose substitution. The molecular mass indicated a further oxygen substituent. Homodecoupling experiments and a 2D COSY-45 homonuclear spectrum clarified most of the ambiguous 1H coupling systems and confirmed the presence of a H-7 carbinylic signal located at 63.86 and partially overlapping with H-6A of the glucose. The broad singlet shape of H-7 suggested its equatorial position which was confirmed by ROEs observed between H-7, and the two H-6 and Me-8. In addition, the absence of ROEs between H-7 and the sugar protons suggested a C-10-glucosidic substitution. Conclusive evidence was given by the HMBC spectrum where a 3J u_ c cross-peak appeared between C- 1 (6139.3) of the aglycone and H-1 (64.78) of the sugar. Moreover, the 218 J. BOUSTIE et al. Table 1. 1HNMR (300 MHz) data of fagaropsine in CDaOD H Table 2. 13C NMR (75 MHz) data of fagaropsine in CD3OD Gated Dec. 6 J (Hz) C Aglycone 4 5ax 5©q 6ax 6cq 7 8 Me-4a Me-8 2' 4' 5' fl-D-glucose l" 2" 3" 4" 5" 6"A 6"B 5.26 s 1.85 td (12.5, 6.0) 1.13" 1.95 tt (12.5, 2.5) 1.68 br dd (12.5, 6.0) 3.84 br s* 3.53 qt (7.6, 1.5) 1.16s 1.20 d (7.6) 7.59 dd (1.7, 0.8) 6.50 dd (2.0, 0.8) 7.53 dd (2.0, 1.7) 4.78 d (6.5) 3.41" 3.39* 3.38* 3.34* 3.84* dd (12.0, 2.1) 3.68 dd (12.0, 5.0) *Overlapped signals. assignment of the IH and x3c N M R resonances of I was supported by the HMBC spectrum and the sequence of the sugar protons which appeared as unresolved signals at 63.30-3.45 in the 1D 1HNMR, can be determined as H-2", 3", 4", 5" (Table 1). The high field position of the angular methyl group signal in the t H N M R spectrum indicated its position cis to the furan ring ( "-- + 0.4 ppm in trans-isomers) [8] and supported the normal limonoid stereochemistry. The comparison with NMR spectra of previously identified degraded limonoids [3-5, 11] and particularly dictamdiol, an aglycone isomer [3, 12], suggested that ring C had a chair conformation. A W-like coupling (1.5 Hz) was present between H-6eq (61.68) and H-8 (63.53) which inferred the axial (~t)-position of the 8-methyl group. Thus, the structure of fagaropsine (1) was deduced to be 1-O-~-D-glucopyranosyl-(4R,4aR,7S,8S)-4-(3'-furanyl)-7hydroxy-4a,8-dimethyl-4,4a,5,6,7,8-hexahydro-2H-3-benzopyran-2-one as the nomenclatural system adopted refers to its limonoid structure. This compound is the first natural degraded limonoid isolated in a glucosylated form. It suggests the presence in this species of another biosynthetic route in limonoid metabolic glycosylation in contrast to the numerous 17O-/~-glucopyranosyl limonoids found in Citrus [ 10, 13] or Tetradium [14] species. Aglycone 1 2 4 4a 5 6 7 8 8a 9 10 2' 3' 4' 5' fl-D-glucose 1" 2" 3" 4" 5" 6" ~ 139.3 s 165.2 s 82.6 d 39.6 s 28.9 t 23.5 t 71.2 d 37.0 d 155.0 s 19.7 q 19.5 q 142.9 dd 121.2 br s lll.2dd 144.5 ddd 105.2 d 75.4 d 77.8 d 71.1 d 78.5 d 62.5 t dc- H (Hz) (149.5) (129.5) (130.0) (145.3) (133.3) (129.5) (129.5) (199.0,10.5) (174.0,14.0) (204.2,10.0, 9.0) (134.4) (145.7) (141.9) (145.3) (141.3) (142.2) CDaOD at 300.13 and 75.45 MHz, respectively, using TMS as int. standard. FAB-MS were recorded in glycerol and glycerol + KI matrix. Plant material. Fagaropsis glabra Capuron trunk bark was collected in the Sambava country (NE of Malagasy Republic) and authenticated at source by the ORSTOM centre of Tananarive where a voucher specimen is deposited with the reference number : 59. Extraction and isolation. Dried powdered trunk bark (900 g) of F. glabra was defatted with petrol and then successively extracted with CH2C12 (12 1) and EtOH 90 ° (15 I). The ethanolic extract was chromatographed over Amberlite XAD-4. The extract eluted with M e O H H 2 0 (3 : 1) contained compounds reacting with Ehrlich reagent and was flash-chromatographed over silica gel. Crude fagaropsine was eluted with E t O A c - M e O H (9:1) and purified over a Bond Elut Cla eluted with M e O H - H 2 0 (3:2) to afford 12 mg of 1. Fagaropsine 1. Mp 165-170°; UV 2mM~ °n nm (log e): 204 (3.8), 238sh; FT-IR Vma Karx c m - :3430 (hydroxy), 1712, 1650 (conjugated 6-1actone), 1504, 1070, 1026, 928, 876, 764, 728 (3-substituted furan); FAB-MS m/z (rel. int.): 441 ([M + H]+; C21H2sOlo) (100), 279 [(M + H) - (glucose + H20-] + (90); IH and 13CNMR: Tables 1 and 2. EXPERIMENTAL General. Mps" uncorr. Analyt. TLC was on silica gel GF254 and furanyl compounds were visualized with Ehrlich reagent spray followed by immersing in HCI vapour. XH and 13CNMR spectra were obtained in Acknowledgements--We sincerely thank Dr P. Uriac (Rennes) for his structural advice. We also thank Dr Montsarrat B. (CNRS Toulouse) for recording the mass spectra. Liminoid glucoside from Fagaropsis glabra REFERENCES 1. Waterman, P. G. and Khalid, S. A. (1981) Biochem. Syst. Ecol. 9, 45. 2. Blaise, A. J., Marion, C. and Winternitz, F. (1986) J. Nat. Prod. 49, 724. 3. Boustie, J., Gleye, J., Blaise, A. and Fourast6, I. (1992) Planta Med. 58, 228. 4. Blaise, A. J. and Winternitz, F. (1985) Phytochemistry 24, 2379. 5. Boustie, J., Moulis, C., Gleye, J., Fourast6, I., Servin, P. and Bon, M. (1990) Phytochemistry 29, 1699. 6. Champagne, D. E., Koul, O., Isman, M. B., Scudder, G. G. E. and Towers, G. H. N. (1992) Phytochemistry 32, 377. 7. Bentley, M. D., Rajab, M. S., Mendel, M. J. and Alford, A. R. (1990) J. Agric. 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