Amburosides C-H and 6-O-Protocatechuoyl Coumarin from Amburana cearensis

Amburana cearensis A. C. Smith (syn.: Torresea cearensis Fr. All.Fabaceae), known as “cumaru” or “imburana-decheiro”, is a native tree from northeastern Brazil, where its trunk bark and seeds are popularly used for the treatment of respiratory affections such as asthma and bronchitis. The hydroalcohol extract from trunk bark and some of its constituents demonstrated bronchodilator, antiinflammatory and analgesic activities in pre-clinical trials. We have previously reported the isolation of coumarin, phenol acids, flavonoids, and the phenol glucoside amburoside A from the trunk bark of A. cearensis. A cytotoxic activity assay revealed that both the flavonoids isokaempferide and kaempferol inhibit the sea egg development as well as tumor cell lines. Amburoside A showed protective effects against CCl 4 -induced hepatotoxicity in rats, and presented a significant neuroprotective effect on mesencephalic cells against 6-hydroxydopamine-induced neurotoxicity. In addition, isokaempferide and amburoside A also showed effects on rodent inflammatory processes and myeloperoxidase activity in human neutrophils. Continuing with the earlier phytochemical and pharmacological studies on A. cearensis, it is reported now the isolation and structural elucidation of six new amburosides C-H (1-6) from the seeds, besides a new 6-coumaryl protocatechuate (7) from the trunk bark. In addition, known compounds were characterized as coumarin, 6-hydroxycoumarin, isokaempferide, formononetin, amburoside A, (E)-o-coumaric acid and vanillic acid. The structure elucidation was performed by spectroscopic techniques such as IR, HRMS, 1D and 2D NMR, and comparison to spectral data from literature.


Introduction
Amburana cearensis A. C. Smith (syn.: Torresea cearensis Fr. All.-Fabaceae), known as "cumaru" or "imburana-decheiro", is a native tree from northeastern Brazil, where its trunk bark and seeds are popularly used for the treatment of respiratory affections such as asthma and bronchitis. 1 The hydroalcohol extract from trunk bark and some of its constituents demonstrated bronchodilator, antiinflammatory and analgesic activities in pre-clinical trials. 2 We have previously reported the isolation of coumarin, phenol acids, flavonoids, and the phenol glucoside amburoside A from the trunk bark of A. cearensis. 3 A cytotoxic activity assay revealed that both the flavonoids isokaempferide and kaempferol inhibit the sea egg development as well as tumor cell lines. 4 Amburoside A showed protective effects against CCl 4 -induced hepatotoxicity in rats, 5 and presented a significant neuroprotective effect on mesencephalic cells against 6-hydroxydopamine-induced neurotoxicity. 6 In addition, isokaempferide and amburoside A also showed effects on rodent inflammatory processes and myeloperoxidase activity in human neutrophils. 7 Continuing with the earlier phytochemical and pharmacological studies on A. cearensis, it is reported now the isolation and structural elucidation of six new amburosides C-H (1-6) from the seeds, besides a new 6-coumaryl protocatechuate (7) from the trunk bark. In addition, known compounds were characterized as coumarin, 6-hydroxycoumarin, isokaempferide, formononetin, amburoside A, (E)-o-coumaric acid and vanillic acid. The structure elucidation was performed by spectroscopic techniques such as IR, HRMS, 1D and 2D NMR, and comparison to spectral data from literature.

Results and Discussion
Compound 1, a brown solid, showed a molecular formula of C 22 H 24 O 11 as established from its HRESIMS quasi-molecular ion at m/z 487.1302 [M + Na] + . The 1 H NMR spectrum showed singlet signals relative to one methyl group at d 2.02 (s) and to an oxymethylene at d 5.23 (s). An axial anomeric proton at d 4.91 (d, J 7.4 Hz, H-1´´), and signals in the region of d 4.91-3.39, suggested the presence of a sugar unit, whose b-D-anomeric configuration was judged based on the large value of its coupling constant (J 7.4 Hz). The presence of an A 2 B 2 system in the aromatic region at d 7.37 (d, J 8.6, 2H) and 7.08 (d, J 8.6, 2H), suggested the existence of a para-disubstituted benzene ring. A second aromatic ring was characterized as an ABX spin system, corresponding to a 1,2,4 substitution by the signals at d 7.43 (d, J 2.1), 7.41 (dd, J 8.4; 2.1) and 6.79 (d, J 8.4). The 13 C NMR spectral data of 1 confirmed the b-glucopyranosyl unit by the signals at d 102.3 (C-1´´), 75.0 (C-2´´), 78.0 (C-3´´), 72.0 (C-4´´), 75.6 (C-5´´) and 64.8 (C-6´´), 8 together with 14 further signals ascribable for the aglycone, that were assigned two carbonyls, a methyl group, an oxymethylene and twelve aromatic carbons, including the symmetric para-substituted benzene ring.
On the basis of HMBC and HSQC spectral analysis, the structure of compound 1 was further determined and all proton and carbon signals were fully assigned. In the HMBC spectrum, the anomeric hydrogen at d 4.91 (H-1´´) did correlate with the oxygenated aromatic carbon at d 159.0 (C-4), which in turn, also correlated with the hydrogens at d 7.08 (H-3/5) and 7.37 (H-2/6). These findings indicated that the b-D-glucosyl moiety was located at C-4 of the parasubstituted aromatic ring. The acetyl group was attached to C-6´´of the glucose unit, as evidenced by the HMBC crosspeaks of oxymethylene protons at d 4.39 and 4.24 (2H-6´´) and the methyl protons at d 2.02 (CH 3 -2´´´) with the carbonyl at d 172.9 (C-1´´´). On the other hand, the long-range correlation observed between the oxymethylene singlet at  Compound 4 was isolated as a dark brown solid, and showed [M + Na] + peak at m/z 597.1256 in the HRESIMS spectrum, corresponding to the molecular formula C 27 H 26 O 14 . Its 1 H NMR data resemble those of compound 2, except for the absence of an AMX system and the appearance of a signal at d 7.10 (s, H-2´´´/6´´´) integrating for two hydrogens suggesting the presence of a galloyl group in 4. This was supported by the 13   On the basis of coumarin biosynthesis, it was initially proposed the protocatechuate-group linked to coumarin at C-7 position. 9,10 However, the spectroscopic evidences led us to reject this proposition and to suggest the ester bridge through the oxygenated C-6. The placement of substituent at C-6 was supported by the dipolar coupling observed on 2D-NOESY spectrum between the hydrogens at d 8.02 (d, J 9.6 Hz, H-4) and 7.21 (d, J 2.9 Hz, H-5). 3 J HMBC correlations of the hydrogens at d 7.25 (H-7) and the carbons at d 114.6 (C-5) and 149.0 (C-9) reinforced the structural proposition. Moreover, 7-hydroxy coumarin and its derivatives show a 13 C NMR chemical shift at about d 102-104 assigned to C-8. 11 No signal was observed in this range on the 13 C NMR spectrum of 7, the most shielded hydrogenated carbon of 7 appears at d 114.6. Thus, compound 7 was named as the new protocatechuoyl derivative of 6-hydroxy-coumarin.
In addition, the precursor of compound 7, 6-hydroxycoumarin was isolated from seeds of A. cearensis and characterized by physical and spectroscopic methods. Comparative analysis of 6-hydroxycoumarin (mp 221.7-222.9 °C, t R = 11.7 min) with an authentic sample of umbelliferone and with literature data (mp 230-233 °C, t R = 13.1 min) allowed to distinguish them. 12,13 According to biogenetic rules, monoxygenatedcoumarins are preferentially substituted at C-7 position yielding umbelliferone and its derivatives, due to the common precursor of this chemical class, p-coumaric acid, which is biosynthentized by the shikimate pathway from either tyrosine-deamination or p-hydroxylation of cinnamic acid. 9,10 Nevertheless, despite a large occurrence of simple coumarins oxygenated at the C-7 position, 6-hydroxycoumarin was also found in some species like Bidens parviflora (Asteraceae), Paeonia suffruticosa (Paeoniaceae) and Hydrangea chinensis (Hydrangeaceae). [13][14][15] Among the isolated compounds, isokampferide and coumarin have been isolated from the trunk bark, however formononetin and (E)-o-coumaric acid are being reported for first time for the genus Amburana. Vanillic acid and amburoside A already described for the trunk bark, are now being identified from seeds. 3,17 Experimental General experimental procedures NMR experiments were performed on a Bruker DRX-500 spectrometer, operating at 500 MHz for 1 H, and at 125 MHz for 13 C. NMR samples were dissolved in deuterated solvents (Cambridge Isotope Laboratories, Andover, USA) and referenced to their residual undeuterated portions for protons and to the central peak of the deuterated carbons. IR spectra were recorded on a Perkin-Elmer FT-IR 1000 spectrometer (Waltham, USA), using KBr pellets. High resolution mass spectra were recorded on an UltrOTOF-Q mass spectrometer Bruker Daltonics, Billerica, USA and LC-IT-TOF model 225-07100-34-SHIMADZU, by positive or negative ionization modes of the ESI source. Melting points (uncorrected) were determined from a Mettler Toledo FP82HT apparatus (Columbus, USA), adopting a heating rate of 2 °C min -1 . Column chromatography was performed either over silica gel 60 (VETEC, 70-230 mesh) or Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala, Sweden). TLC was performed on precoated silica gel aluminum sheets (Merck) and monitored by UV detection and exposure to iodine vapour. Reverse phase chromatography was performed on C18 Sep-Pak® Waters cartridge. HPLC analysis were performed on a Waters 1525 chromatograph (Milford, USA) equipped with a binary pump and photodiode-array detector (Waters-2996 PDA), using Waters X-Terra RP-18 columns (analytical: 250 × 4.6 mm, 5 μm; semi-preparative: 250 ×10 mm, 10 μm) at 35 ºC in a thermostatic oven. HPLC grade methanol was purchased from Tedia Co (Fairfield, USA) and the HPLC grade water (18 mW) was obtained by a Milli-Q purification system (Millipore, Bedford, USA).

Plant material
The trunk barks of Amburana cearensis A. C. Smith were collected at the Quixeramobim County (Ceará State, Northeast of Brazil) in September 2002 while the seeds were purchased from a herbal store in Fortaleza City in June 2001. Voucher specimens (# 837 and 847) were deposited at the Herbário Prisco Bezerra (EAC) and identified by Dr. Afrânio G. Fernandes, from the Department of Biology, Federal University of Ceará.

Extraction and isolation
Around 1.5 kg of powdered seeds of A. cearensis were previously macerated in hexane (1.8 L) for 24 h, yielding a yellowish oil (312 g) containing a precipitate that after being separated, was characterized as coumarin (53.5 g, mp 68 °C). 10 Then, 1.1 kg of the remaining residue were extracted in small aliquots (100 g) with ethanol on a Sohxlet apparatus, affording a dark brown extract (144.0 g, 9.6% m ext /m seed ), after pooling together all ethanol solutions and vacuum distillation of the solvent. Powdered dried trunk barks (4.2 kg) of A. cearensis were subjected to the same extraction procedure yielding also a brown ethanol extract (228.0 g, 5.4% m ext /m trunk ).
The ethanol extract of the trunk barks (228.0 g) was suspended in H 2 O-MeOH (2:1) and subjected to partition with EtOAc (3 ×150 mL). The EtOAc fraction (64.5 g) was purified by chromatography on silica gel 60 by elution with   ), and a mixture of amburoside G (3, 5.7 mg) and amburoside H (6, 4.0 mg), which was posteriorly reinjected and purified. The known compounds were identified by 1 H and 13 C NMR, in addition to comparison with literature data. Furthermore, 6-hydroxycoumarin (t R = 11.7 min) and an authentic sample of umbelliferone (t R = 13.1 min) were compared by HPLC (analytical Waters X-Terra RP-18 column), using the same elution gradient, but changing the running time (20 min) and flow rate (1.0 mL min -1 ).

Supplementary Information
Supplementary data (IR, HRMS, 1 H and 13 C NMR spectra of compounds 1-7) are available free of charge at http://jbcs.sbq.org.br, as PDF file.