J. Nat. Prod. 2000, 63, 473-476
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Flavonol-Cinnamate Cycloadducts and Diamide Derivatives from Aglaia laxiflora Yuan-Jian Xu,† Xiao-Hua Wu,‡ B. K. H. Tan,‡ Yee-Hing Lai,† J. J. Vittal,† Z. Imiyabir,§ L. Madani,§ K. S. Khozirah,⊥ and S. H. Goh*,† Department of Chemistry, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Department of Pharmacology, National University of Singapore, Singapore, Forest Research Center, Sepilok, Sabah, Malaysia, and Forest Research Institute of Malaysia, Kepong, Kuala Lumpur, Malaysia Received September 15, 1999
Leaf extracts of the Malaysian plant Aglaia laxiflora provided two cytotoxic compounds, a new rocaglaol rhamnoside (1), a known rocaglaol (2), new (but inactive) flavonol-cinnamaminopyrrolidine adducts (36), and their probable biosynthetic precursors (7 and trimethoxyflavonol). All structures were elucidated primarily by 2D NMR spectroscopy. The structure and stereochemistry of aglaxiflorin A (3) were confirmed by single-crystal X-ray crystallography. Aglaia species of the family Meliaceae are of ethnomedicinal value1,2 and have insecticidal3 and cytotoxic4 properties. Study of the Malaysian plant A. laxiflora has led to the isolation of a cytotoxic cyclopentatetrahydrobenzofuranrhamnoside (1), a derivative of rocaglaol (2), and several new bicyclic diamides (3-6), which appear to be cycloadducts of likely biosynthetic precursors such as (+)-odorinol (7) and trimethoxylflavonol. Results and Discussion Compound 1 exhibited a peak at m/z 651[M - H](C35H39O12, FABMS negative ion mode). The IR spectrum showed a strong band at 1744 cm-1, corresponding to an ester, and strong bands at 1148 and 1124 cm-1, typical of sugars, indicating that 1 was a glycoside. The 1H NMR spectrum showed four methoxyl groups, one phenyl group, one disubstituted phenyl group, and one oxygenated benzene group with two meta protons. Other peaks of the 1H NMR spectrum reflected the presence of a modified rhamnose unit. The 13C NMR data were similar to a known rocaglamide glycoside,6 except that 1 contained an additional acetyl group on the glycoside moiety. The acetyl group was determined to be at 2′ of the rhamnose, inasmuch as the 2′ proton was significantly shielded (δ 5.52). This conclusion was confirmed by a HMBC crosspeak H-2′/CdO. The 3′-methoxyl group was determined by a NOE-difference experiment; irradiation of the threeproton signal at δ 3.45 caused enhancements of the oneproton signals at δ 5.52 (H-2′) and 3.70 (H-3′). The modified rhamnose was placed at the 11-position because of the observed HMBC cross-peak H-1′/C-11 and the H-1′/H-11 NOE correlation. The glycosidic bond is R, because H-1′ has a small coupling constant (1.6 Hz) with H-2′. The absolute configuration of rocaglaol (2) is known from enantioselective synthesis,7 and the rhamnoside residue has been found to have no significant influence on the rotation of this class of compounds.4 It is therefore likely the absolute sterochemistry of 1 is as depicted, based on similarity of the sign and magnitude of the rotation of 1 to the parent compound 2 and its derivatives.4 Thus, 1 * To whom correspondence should be addressed. Tel.: (65) 8743511. Fax: (65) 7791691. E-mail:
[email protected]. † Department of Chemistry, National University of Singapore. ‡ Department of Pharmacology, National University of Singapore. § Forest Research Center, Sepilok. ⊥ Forest Research Institute of Malaysia.
10.1021/np990454d CCC: $19.00
was assigned as 11-R-(2′-acetoxy-3′-methoxyrhamnosyl)rocaglaol, the second rocaglaol glycoside isolated from nature.6
© 2000 American Chemical Society and American Society of Pharmacognosy Published on Web 03/22/2000
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Journal of Natural Products, 2000, Vol. 63, No. 4
Figure 1. X-ray structure of aglaxiflorin A (3).
Aglaxiflorin A (3) was isolated as colorless needles, [R]25D +8.1°, and its molecular formula was determined as C38H44N2O10 by HRFABMS. The IR spectrum showed absorption bands at 3467 (OH), 3400 (NH), 1742 (an unconjugated ester CdO), 1647, and 1615 cm-1 (amide carbonyl). Being amides, rotamers were detectable in both the 1H and 13C NMR as two sets of signals of unequal intensity when CDCl3 was used as solvent, but in methanold4 only one major set of peaks was prominent. The 1H NMR spectrum disclosed three methoxyl groups. In addition, three aromatic rings related to those observed for rocaglaol2-4 (2) were deduced to be one phenyl group, one parasubstituted phenyl, and one aromatic ring with meta positions unsubstituted. Analysis of the 1H and 13C NMR spectra of 3 indicated a skeleton similar to those found in aglains.5 However, the low-field signal (δC 76.0) indicated that 3 had a new 2-hydroxy-2-methylbutyryl group. The HMBC and NOESY spectra confirmed the structure of 3 as depicted. Notable HMBC correlations were H-10/C-1, -9, -11, -19, -20, -24 and H-12/C-1, -2, -13, CH3CO. Relative stereochemical assignments were from NOE difference and NOESY correlations, e.g. H-10/H-12, -20, -24; H-11/H-20, -24, -27; and H-12/H-10, -14, -18. A single-crystal X-ray diffraction experiment established the definitive structure and relative stereochemistry of aglaxiflorin A (3) (Figure 1). The trans H-10/H-11 stereochemistry and the relative proximity of H-11 and H-27 were also revealed. Assuming that 3 is derived from the isolated (+)-odorinol 7, which has known absolute stereochemistry, the absolute configuration for 3 was assigned as depicted. The 27S configuration has also been postulated for some aglains from A. argentea.5 Aglaxiflorin B (4), [R]25D -105.5°, an isomer of aglaxiflorin A (3), gave M+ at m/z 688.2979 (HREIMS), which is consistent with C38H44N2O10 (688.2996). The IR spectrum showed an ester band at 1732 cm-1 and two amide bands at 1651 and 1617 cm-1. The NMR and HMBC data were similar to those of 3, indicating that its structure differed in the configurations of C-10 and C-11. NOESY correlations H-12/H-11 and H-11/H-12, -20, -24 support the relative stereochemical assignments shown for 4. Aglaxiflorin C (5), [R]25D +34.9°, another isomer of 3 and 4, had molecular formula C38H44N2O10 (HREIMS). The IR spectrum of 5 exhibited absorption bands at 3507 (OH), 3405 (NH), 1749 (an unconjugated ester carbonyl), 1648, and 1620 cm-1 (amide carbonyls). The 1D NMR and HMBC data showed significant differences, compared to those of 3 and 4. With NOESY correlations for H-11/H-10, -20, -24,CH3CO but not for H-12/H-10, -11, the stereochemical
Xu et al.
assignments for C-12, H-10β, H-11R were made for 5 as shown. The chemical shifts for H-10 and H-11 and the corresponding carbon resonances indicated that the pyrrolidamide and phenyl groups are attached at carbons 10 and 11, respectively. This was confirmed from the HMBC correlations H-11/C-1, -2, -19, -20 and NOESY correlations H-12/OH-1,H-14. Protons in the shielding cones of the aromatic rings were significantly shifted upfield, e.g. OCH3-3 and H-27 at δ 3.06 and δ 5.38, respectively. Accurate mass measurement confirmed the molecular formula of 6 as C36H42N2O9 corresponding to a deacetylated 3. The IR spectrum showed OH and NH absorption bands at 3482 and 3399 cm-1, respectively, and amide carbonyl bands at 1621 and 1610 cm-1; the ester band found in 3 was absent. 13C NMR data showed some similarities to those of 3, but marked differences were found for C-11, C-12, H-11, and H-12. The structure of 6 was determined from the NOESY correlations H-12/H-14 and HMBC correlations H-11/C-1, -2, -19, -20. Diamides of aminopyrrolidine with cinnamic and 2methylbutanoic (or derivatives) acids (7-9) have previously been isolated from Aglaia species.1,3 Odorinol, in particular, has been isolated in two enantiomeric form from the same species.1,3 (+)-Odorinol (7), presently isolated from A. laxiflora, had a rotation similar in magnitude but opposite to that of (-)-odorinol for which the absolute stereochemistry is known. Thus, the enantiomeric structure is likely to be as shown, and this is corroborated by the X-ray structure as for 3 (Figure 1). It is likely that the cycloadducts (3-6) from Aglaia are cycloaddition products of cinnamamidopyrrolidine derivatives with trimethoxyflavonol. An acid-catalyzed cycloaddition of flavonol to a cinnamyl derivative could lead to aglaxiflorin intermediates, which could subsequently undergo a Wagner-Meerwein rearrangement to rocaglaoltype compounds. Rocaglaol (2) and its rhamnoside derivative (1) were determined to be the main cytotoxic compounds, while the diamide 7 had previously been found to have antileukemic activity.4 Aglaxiflorins A-D (3-6) were, however, inactive to P-388 and MOLT4 cell lines. Experimental Section General Experimental Procedures. Optical rotations at 25° were taken on a Perkin-Elmer 241 polarimeter. EIMS was run on a Micromass VG 7035 mass spectrometer at 70 ev. NMR spectra were recorded by Bruker ACF 300 [300 MHz (1H) and 75 MHz (13C)] and AMX [500/400 MHz (1H) and 125/ 100 MHz (13C)] instruments using CDCl3, with TMS as an internal standard unless otherwise stated. IR spectra were recorded on a Bio-Rad FT-IR spectrometer, and UV spectra were recorded on a Hewlett-Packard 8452A diode array spectrometer. X-ray data collection was carried out on a Siemens CCD SMART system. Liquid chromatography was performed on Si gel (Kieselgel 60, particle size 0.040-0.063 mm) and Sephadex LH-20. TLC was run on Si gel precoated glass plates (Merck Si gel 60 F254). Plant Material. The leaves of A. laxiflora (Meliaceae) were collected from Mt. Tawai, Kinabatangan, Sabah, Malaysia, in 1996, and identified by L. Madani. A voucher specimen (SAN135189) was deposited at the herbarium of the Forest Research Centre, Sepilok, Sandakan, Sabah, Malaysia. Cytotoxicity. Cytotoxic activity was determined as described previously:8 rocaglaol (2) (IC50 ) 0.023, 0.034,
