Chemical Constituents from Bombacopsis glabra ( Pasq . ) A . Robyns : Complete 1 H and 13 C NMR Assignments and X Ray Structure of 5-Hydroxy-3 , 6 , 7 , 8 , 4 '-pentamethoxyflavone

Do extrato hexânico da casca do caule de Bombacopsis glabra (Bombacaceae) foram isolados a flavona 5-hidroxi-3,6,7,8,4’-pentametoxiflavona (1) e os triterpenos lupenona, 9,19-ciclolanost23-eno-3β,25-diol (2), (24R)-9,19-ciclolanost-25-eno-3β,24-diol (3) e (24S)-9,19-ciclolanost-25eno-3β,24-diol (4). As estruturas foram determinadas por espectroscopia de RMN de C e de H (1D e 2D) e EM, e ainda, por comparação com dados da literatura para os triterpenos. A confirmação inequívoca da estrutura da flavona 1 foi realizada por um estudo de difração de raios X. As cinco substâncias foram isoladas pela primeira vez de espécies de Bombacaceae.


Introduction
The Bombacaceae family comprises 28 genera and 200 species. 1 Despite the economic significance of Ceiba and Ochroma species as sources of kapok and balsa wood, respectively, little is known about the chemistry of plants from this family. 2,3As part of our program of study of the Brazilian flora, we have carried out the first investigation of the chemical constituents of the bark from Bombacopsis glabra (Pasq.) A. Robyns.

Results and Discussion
Although compound 1 has been previously synthesised 8 and isolated from Stachys aegyptiaca 9 but a full 1 H NMR assignment have not been made.In the present study, all 1 H and 13 C shifts were unambiguously and directly assigned by HMQC, HMBC and nOe difference experiments.
The 2 J HC and 3 J HC couplings by HMBC are shown in Figure 1.Also, 1 H-1 H nOe enhancements were seen from H2',6' to 13-CH 3 (1.3 %) and 15-CH 3 (1.0 %) and from 12-CH 3 to the almost overlapping resonances of 11-CH 3 and/ or 13-CH 3 (2.3 % in total).These enhancements defined the structure unambiguously, with only trivial reliance on shift data.The complete assignments are listed in Table 1.
The crystal structure of 1 (Figure 2) confirmed the substitution pattern deduced from NMR.The benzopyran ring (C) is essentially coplanar with ring A. However, the methoxyphenyl ring (B) is twisted 23º from the plane of ring C. A similar result has been found for other flavones. 10he methoxy group lies in the plane of the phenyl ring.This is in agreement with Bolt and Bauch 11 where the methyl groups of -methoxynaphthalene derivatives lie in the plane of the aromatic ring, syn to Cα.However, the methoxy groups at carbons C3, C6, C7 and C8 are not coplanar with ring A, their torsion angles being 110.8(4)º, 66.7(6)º, -62.1(5)º and -74.0(5)º respectively, presumably because of steric hindrance.In contrast, one of the methyl groups in the slightly less hindered 8-hydroxy-1,2,3-trimethoxybenzo[a]anthracene-7,12-dione, lies in the plane of the aromatic ring. 12though pre-irradiation at δ 8.17An intramolecular hydrogen bond exists between O3 and O2 [O3 … O2 2.572(4) Å; H3 … O2 1.61(9) Å; O3-H3 1.02(9) Å; O3-H3 … O2 156 (8)].This is consistent with the absence of nOe enhancement at 11-CH 3 upon irradiation of H3 (Figure 2).The 1 H NMR spectrum of compound 2 showed, besides other signals, resonances for cyclopropane methylene (δ 0.54 and 0.31, 1H, d, each), seven methyls and two olefinic hydrogens (δ 5.59, m) suggesting a cycloartenol type triterpene.The detailed analysis of the 1 H and 13 C NMR spectra led us to propose the structure of 9,19-cyclolanost-23-ene-3β,25-diol.The 23Z isomer has been previously isolated 13 and also the 3-acetate of this compound, having a E double bond. 14As the signals of H-23 and H-24 appeared as a multiplet centered at δ 5.59, a direct assignment of the double bond geometry was difficult, but the data were similar to those reported by Greca et al 13 for the Z isomer.Using two-dimensional NMR techniques (COSY, HMQC and HMBC), it was possible to assign unequivocal the 1 H and 13 C NMR spectra.Some of the 13 C NMR assignments made by us differ slightly from those given by Greca et al., 13 but were in full agreement with the data reported by Kamisato et al. 15 Although compound 2 was identified many years ago, only now a complete unambiguous assignment of the 1 H NMR spectrum has been achieved.
Compound 3 was isolated as a white solid and its 1 H NMR and 13 C NMR spectra were similar to those of compound 2. The EIMS showed a peak at m/z 442, corresponding to the molecular formula C 30 H 50 O 2 , suggesting that 3 was an isomer of 2. The major differences were relative to the signals corresponding to the side chain.In the 1 H NMR spectrum, resonances at δ 4.95 (m) and another at δ 4.82 (m) were observed, consistent with the presence of a =CH 2 group, and this was confirmed by the signals at δ 147.36 (C-25) and 111.31 (C-26) in the 13 C NMR spectrum.Furthermore, the location of the double bond between C-25 and C-26 was proposed, due to the absence of Me-26 signal at δ 1.7, when compared with corresponding signal in the 1 H NMR spectrum of the compound 2, and also by the coupling observed in the 1 H 1 H-COSY spectrum between olefine hydrogen (H-26 and H-26') and Me-27.
The chemical shift at high frequency observed for H-24 (δ 4.0, m), when compared with H-3, suggested that the hydroxyl group should be placed next to the double bond (at C-24).This position was further confirmed by couplings observed in the 1 H 1 H-COSY spectrum for the side chain hydrogens: 21-CH 3 and H-20; H-20 and H-22/H-22'; H-22/H-22' and H-23/H-23'; and finally, between H-23/H-23' and H-24.
Compounds 4 and 3 were obtained as a mixture.The duplicity of most of the signals of the side chain, observed in the 13 C NMR spectrum of the mixture, and the coincidence of the other signals, suggested that they are epimers at C-24.By comparison of the 13 C chemical shifts of compounds 3 and 4 with literature data, 14 they were identified as (24R)-9,19-cyclolanost-25-ene-3β,24-diol and (24S)-9,19-cyclolanost-25-ene-3β,24-diol, respectively.The complete assignments of the 13 C NMR spectra of compounds 2, 3 and 4 are shown in Table 2.

General experimental procedures
Melting points were determined on a MQAPF-301 apparatus and are uncorrected.The 1 H and 13 C NMR spectra were obtained on a Bruker DPX 400 (at 400 MHz and 100.6 MHz, respectively) and DPX 300 (at 300 MHz and 75 MHz, respectively) instruments.HMQC and HMBC experiments were optimized for 1 J CH 145 Hz and 2 and 3 J CH 7.7 Hz, respectively.Mass spectra were obtained on a V.G.Analytical ZAB-IF spectrometer, at 70 eV.Infrared spectra were registered on a Perkin Elmer FTIR PARAGON 1000 spectrophotometer.X-ray crystallographic data were collected on a Siemens SMART CCD area-detector diffractometer.UV data were obtained on a Varian CARY 50 spectrophotometer.Column chromatography and TLC plates were prepared using silica gel Merck.

Crystal structure determination of compound 1
Crystals of 1 suitable for single crystal x-ray diffraction studies were obtained as orange plates by recrystallization from methanol.Data were collected using a Siemens SMART CCD area-detector diffractometer.A multi-scan absorption correction was applied using SADABS. 16The structure was solved by direct methods and refined by fullmatrix least-squares on F 2 for all dara using SHELXL 97. 17 Hydrogen atoms were added at calculated positions and refined using a riding model.Anisotropic displacement displacement parameters were used for all non-H atoms.

Table 2 .
13C NMR assignment for compounds 2, 3 and 4, and 1 H assignment for compound 2, assigned via COSY, HMBC, HMQC and selected nOe-difference spectra a,b,c,d The assignments with same letter can be changed.# (a) = axial e (e) = equatorial