New Flavonoids and Coumarins from Platymiscium floribundum Vogel

Dois novos flavonoides, 3,4,10-tri-hidróxi-9-metoxi-pterocarpano and 2’,4’-di-hidróxi4’-metoxi-β-hidroxichalcanonol, foram isolados a partir de Platymiscium floribundum, em adição a homopterocapina, 2’,4’,4-tri-hidroxichalcona, 7,3’,5’-tri-hidroxiflavanona, 7,3’-di-hidróxi8,5’-di-metoxiisoflavanona, 8-hidróxi-5,6,7-tri-metoxicumarina, 6-hidróxi-7,8-di-metoxicumarina, 6,7,8-tri-metoxicumarina, 6,7-di-metoxicumarina, 8-hidróxi-6,7-di-metoxicumarina, 3β-acetóxi28-hidróxi-olean-12-eno, 1,2,3-tri-metoxi-5-alilbenzeno, 3,4-di-metoxicinamaldeído, e β-sitosterol e estigmasterol como mistura. A caracterização estrutural de todos os compostos foi estabelecida com base em métodos espectroscópicos, particularmente ressonância magnética nuclear (RMN) 1D e 2D e comparação com dados da literatura.


Introduction
Platymiscium floribundum Vog. is a medium sized tree which belongs to the Fabaceae-Papilionoideae family.In the Brazilian Northeast, this plant is popularly known as "sacambu" and "jacaranda-do-litoral" and has been used by the local population in folk medicine as an antiinflammatory agent. 1 In addition, the wood has showed high commercial value in carpentry. 2revious chemical investigation of P. floribundum led to the isolation of isoflavonoids and pterocarpans from the heartwood. 1Since then, several activities such as cytotoxic, 1 antimitotic 3 and apoptosis in HL-60 human leukemia cells 4 of the flavonoids have been evaluated.

Results and Discussion
Compound 1 was obtained as a white solid with mp 108.2-110.0o C and [a] D 20 + 272 o (CH 3 OH, c 0.02).The IR (infrared) spectrum showed absorption bands at 3379 and 1636 cm -1 , characteristic of hydroxyl groups and aromatic skeleton, respectively.Its molecular formula was established by the negative mode HRESIMS (high-resolution electronspray ionization mass spectra) peak at m/z 301.0713 [M -H] -(calcd.for C 16 H 14 O 6 -H, 301.0712).
All these data were supported by the 13 C CPD (composite pulse decoupling) spectrum along with the 13 C DEPT-135 (distortionless enhancement by polarization transfer) that revealed 16 carbon atoms corresponding to one methyl, one methylene, 6 methines and 8 non-hydrogenated carbons.However, the confirmation of the above suggestion for 1 was supported by the HSQC (heteronuclear single-quantum correlation spectroscopic) and HMBC (heteronuclear multiple-bond correlation spectroscopic) experiments, which allowed the unequivocal assignments of its 13 C and 1 H NMR data.The assignments of the relative positions of the methoxyl and the hydroxyl groups at C-9 and C-10, respectively, were defined in the HMBC spectrum that showed cross-peaks of the methyl protons at d 3.79 (−OCH 3 ) and the hydrogens at d 6.68 (H-7) and 6.43 (H-8), with the carbon at d 150.53 (C-9), in addition to the correlation of the signal at d 6.43 (H-8) with the carbon at d 132.49(C-10).Moreover, the correlation of hydrogen at d 6.88 (H-1) with the carbons at d 145.91 (C-4a), 80.70 (C-11a) and 147.19 (C-3), besides the correlations between the hydrogen at d 6.53 (H-2) with the carbons at d 147.19 (C-3), 113.91 (C-1a) definitively established that the other two hydroxyl groups were located at the C-3 and C-4 in the tetrassubstituted aromatic A-ring.Compound 1 is closely related to metilocarpan D, previously isolated from Metilotus alba 5 except for the presence of one hydroxyl group at C-3 instead of a methoxyl group.
It is well known from the literature that, according to biogenetical regulations, the hydrogens (6a and 11a) at the B/C rings junction of all natural pterocarpans are always cis, either a,a or β,β, thus leading to only two enantiomeric forms.It is also known, through CD (circular dichroism) and/or ORD (optical rotatory dispersion) analyses, that (-) optical rotation can be associated with a,a positioning (6aR, 11aR), while the (+) optical rotation can be associated with the β,β positioning (6aS, 11aS) of both series. 5From the (+) optical rotation of compound 1, it could be assumed an (6aS, 11aS) absolute configuration for it.As expected, the CD spectrum of 1 should a similar profile of that from (+)-pterocarpin, and almost a mirror image of (-)-maackiain, what is in agreement with the suggested (6aS, 11aS) absolute stereochemistry for 1. 5 Thus, it was possible to characterize 1 as the (6aS,11aS)-3,4,10-trihydroxy-9-methoxypterocarpan.
Compound 2, a white solid with mp 185. in the HRESIMS spectrum.The absorption bands at 3418 and 1632 cm -1 in the IR spectrum were consistent with hydroxyl and carbonyl groups, respectively.
The 1 H NMR spectrum showed hydrogens relative to one methoxyl group at d 3.79 (s, −OCH 3 ), a methylene at d 2.92 (dd, J 14.2, 6.3 Hz, H-ab) and 3.14 (dd, J 14.2, 3.8 Hz, H-aa), and a oxymethine hydrogen at d 5.22 (dd, J 6.3, 3.8 Hz, H-β).The presence of a AA'BB' system in the aromatic region at d 7.06 (d, J 8.5 Hz, H-2/6) and 6.82 (d, J 8.5 Hz, H-3/5) suggested the existence of a para-disubstituted benzene ring that was evidenced by the double intensity of the proton for two methines.The other aromatic ring was characterized as an ABX spin system, corresponding to a 1',2',4' substitution, by the signals at d 7.57 (d, J 8.4 Hz, H-6'), 6.46 (d, J 8.4 Hz, H-5') and 6.43 (bs, H-3').The 13 C NMR spectrum showed 16 absorptions including one carbonyl, one methoxyl group, one methylene (d 42.1), one oxymethine (d 72.7) and twelve aromatic carbons (d 103.9-165.3).On the basis of HMBC and HMQC spectral analysis, the structure of compound 2 was further determined, and all proton and carbon signals were fully assigned.In the HMBC spectrum, the hydrogens at d 7.57 (H-6'), 3.14 (Ha-a) and 2.92 (Ha-b) correlated with the carbonyl carbon at d 202.9 (C-b'), the hydrogens at 7.57 (H-6'), and 6.43 (H-3') with the carbon at d 165.6 (C-2'), and the hydrogens at d 6.46 (H 3'/5') and 7.57 (H-6') with the carbon at d 163.5 (C-4').These findings indicated that the hydroxyl groups were located at C-2' and C-4' on the A ring in the chalcanonol skeleton.On the other hand, the long-range correlation observed between the aromatic hydrogens at d 6.82 (H-3/5), 7.02 (H-2/6) and the methoxyl at d 3.79 with the carbon at d 158.6 (C-4) allowed the attachment of the methoxyl group to the C-4 carbon of the para-substituted aromatic ring.Thus, the structure of compound 2 was established as the new 2',4'-dihydroxy-4-methoxy-β-hydroxychalcanonol.

Experimental
General procedures IR spectra were recorded on an ABB BOMEN FT-IR 1000 spectrometer, using KBr pellets.The NMR spectra were performed on a Bruker Avance DRX 500 spectrometer, equipped with an inverse detection probehead and z-gradient accessory working at 499.9 ( 1 H) and at 124.97 MHz ( 13 C), respectively.All pulse sequences are standard in the Bruker XWIN-NMR software, and all experiments were conducted at the room temperature.The samples, dissolved either in CDCl 3 , CD 3 OD or C 5 D 5 N (0.6 ml), were transferred to 5 mm tubes.High resolution mass spectra were recorded on an UltrOTOF-Q (quadrupole orthogonal time-of-flight) mass spectrometer (LC-IP-TOF model 225-07100-34, Shimadzu) by negative ionization mode of the ESI source.HPLC (high performance liquid chromatographic) analyses were performed on a Shimadzu chromatograph (Japan) equipped with a ternary pump (Shimadzu LC-20AT) and PDA (photodiode array) detector (Shimadzu SPD-M20A), using Phenomenex columns (analytical: 250 × 4.6 mm, 5 μm; semi-preparative: 250 × 10 mm, 10 μm).HPLC grade n-hexane, CHCl 3 and EtOAc were purchased from Tedia Co (Brazil, São Paulo) and the HPLC grade water was obtained by a Milli-Q purification system (Millipore, Bedford, USA).Column chromatography was performed either over silica gel 60 (VETEC, 70-230 and 40-63 mesh) or Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala, Sweden).TLC (thin layer chromatograph) was performed on precoated silica gel aluminum sheets (Merck) and the compounds were visualized by UV detection and by spraying with vanillin/perchloric acid/EtOH solution, followed by 5 min heating.

Plant material
An entire P. floribundum specimen was collected in Acarape County, Ceará State, Brazil.A voucher specimen (No. 31052) was identified by Dr. Edson Paula Nunes and deposited at the Herbário Prisco Bezerra (EAC), Departamento de Biologia, Universidade Federal do Ceará, Fortaleza-CE, Brazil.