Two Unusual Epicatechin and Epiafzelechin Derivatives from Mascagnia pubiflora , a Plant Toxic to Cattle

Dois novos e incomuns derivados de epicatequina e epiafzelequina contendo unidades feniloctanóides glicosiladas e denominados mascagninas A e B foram isolados, juntamente com quercetina-3-O-α-L-ramnopiranosil-(1→6)-β-D-glucopiranosídeo, das partes aéreas de Mascagnia pubiflora (Malpighiaceae), uma planta tóxica para o gado. Suas estruturas foram determinadas pela combinação de espectroscopia de ressonância magnética nuclear (RMN) uni e bidimensionais e dados de espectroscopia de massas (MS).


Introduction
In Brazil, poisoning by plants is one of the main causes of death to adult cattle and the number of plants which are considered to be toxic to ruminants is constantly increasing. 1,2Therefore, considerable losses due to cattle intoxication by ingestion of seeds, leaves or roots of toxic plants have become a significant problem in cattle producing areas.In the state of Mato Grosso do Sul, Brazil's greatest producer of pasture-herd cattle, M. pubiflora (Mascagnia pubiflora ) (A. Juss.)Griseb., a liana popularly known as "cipó prata" and "corona" that belongs to the Malpighiaceae family, is the main causative agent of sudden bovine death as a consequence of heart attack, due to a peracute poisoning. 2,3The cardiotoxic activity of M. pubiflora was studied by Saad et al. 4 in 1970, and attempts to isolate and characterize the active principles pointed to the possibility of the presence of a biologically active glycoside. 4Considering the economic losses due to the toxic activity of M. pubiflora on grazing animals and the absence of a more detailed phytochemical study, we decided to investigate the chemical composition of the aerial parts of a specimen of M. pubiflora growing in the "Pantanal" border of Mato Grosso do Sul.Herein, we report on the isolation of two novel and unusual epicatechin and epiafzelechin derivatives bearing glucosylated phenyloctanoid units, named mascagnins A and B, together with quercetin-3-Oα-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside.
Compound 1 was obtained as an amorphous powder and its molecular formula was suggested to be C 55 H 70 O 24 on the basis of the quasi-molecular ion peak at m/z 1113.3706[M-H] -in the high resolution electron spray ionization mass spectrum (HRESIMS) and on NMR data.The molecular formula pointed to twenty-one degrees of unsaturation for the compound.Its IR spectrum showed absorption bands for hydroxyl (3410 cm -1 ) and aromatic (1617, 1510 cm -1 ) functionalities.The 1 H and 13 C NMR spectra of 1 (Table 1) exhibited signals for fifteen aromatic quaternary carbons (nine oxygenated) and nine aromatic methine carbons, thus indicating the presence of four aromatic rings in its structure.In addition, signals attributable to carbons of aliphatic moieties were assigned to two methyls, nine methylenes, six methines (four oxymethines), and two oxygenated quaternary carbons.Additionally, twelve carbon signals corresponding to two sugar residues were also observed and were assigned to two D-glucopyranosyl units. 5,6The anomeric centers were each determined to have a β-configuration based on the coupling constant values of the α-oriented anomeric proton signals, which were observed as a two-proton doublet at d H 4.64 (J 7.3 Hz). 5,6his, in turn, showed cross-peak correlations in the heteronuclear single quantum coherence (HSQC) spectrum with the signals at d 104.5 and 104.8, accounting for the two anomeric carbons, whose chemical shifts around 100 ppm indicated the O-glucosidic nature of 1. 5,6 Three of the four aromatic rings were found to be 1,2,4-trisubstituted, as ascertained by the connectivities observed in 1 H-1 H correlation spectroscopy (COSY) spectrum, in which three spin systems could be assigned by the signals at d 6.57 (dd, J 8.3 and 1.9 Hz), 7.00 (d, J 8.3 Hz) and 6.62 (d, J 1.9 Hz) [I]; d 6.51 (dd, J 8.3 and 1.9 Hz), 6.99 (d, J 8.3 Hz) and 6.60 (d, J 1.9 Hz) [II] and d 6.75 (dd, J 8.2 and 1.6 Hz), 6.78 (d, J 8.2 Hz) and 6.96 (d, J 1.6 Hz) [III].In addition, the respective HSQC and heteronuclear multiple bond correlations (HMBC) along with the corresponding aromatic carbon resonances indicated the presence of one alkyl and two ortho-oxygenated substituents in each of the aromatic rings.The absence of other proton resonances in the aromatic region of the 1 H NMR spectrum as well as the presence of six quaternary carbon signals ascribable to aromatic carbons in the 13 C NMR spectrum defined the fourth aromatic ring in the structure of 1 as a hexasubstituted one.The chemical shifts assigned to the carbons of this ring at d 99.5, 105.3, 105.6, 150.9, 151.7 and 154.0 matched to an 1,3,5-trioxygenated aromatic ring bearing alkyl residues at the 2, 4 and 6 positions.
Another spin system detected in the 1 H-1 H COSY spectrum consisted of two broad singlets at d H 4.19 (1H) and 4.68 (1H), one double doublet at d 2.95 (1H, J 16.7 and 4.0 Hz) and one broad doublet at d 2.82 (1H, J 16.7 Hz).In the HSQC spectrum, cross-peaks were observed between the last two signals and the carbon signal at d 29.5 as well as between the two broad singlets at d 4.19 and 4.68 and the carbon signals at d 67.1 and 79.9, respectively.This information, along with the presence of the aforementioned hexasubstituted aromatic ring and the spin system III, which in turn showed correlations in the HSQC spectrum with the methine carbons at d 119.3, 116.4 and 115.1, respectively, were in accordance with the presence of an epicatechin residue in the structure of 1 bearing alkyl side chains at carbons C-6 and C-8 of ring A. 7,8 The remaining carbon resonances in the aliphatic region of the 13 C NMR and distortionless enhancement by polarization transfer (DEPT) spectra suggested two C 8 carbon-containing chains, each composed of four sp 3 methylenes in the region of d 31.6 to 39.7, two sp 3 methines, one being oxymethinic (d 70.0/70.8,d 24.9), one tertiary methyl group (d 28.5) and one sp 3 quaternary carbon indicative of the presence of a hemiacetal functionality (d 98.8/ 98.9).An interesting feature of the 13 C NMR spectrum of 1 was the appearance of these sets of signals, which showed very similar chemical shifts and the same multiplicities in the DEPT spectrum, the same being observed for the signals ascribed to the carbons of the two remaining 1,2,4-trisubstituted aromatic rings as well as the signals of their corresponding hydrogens (the abovementioned spin systems I and II).This led to the assumption that 1 had identical groups, which formed two disjoint spin-systems, linked to ring A of the epicatechin unit.On the basis of correlations discernible from 1 H-1 H COSY, HSQC and HMBC experiments, they were characterized as being two glucosylated phenyloctanoid units, as shown in Figure 1.A cross-peak between the anomeric proton of the two glucosyl moieties at d 4.64 and the oxygenated carbon of the two trisubstituted aromatic rings at d 145.9 (attributed to C-12a) and d 144.7 (attributed to C-12b) in the HMBC spectrum revealed the linkage between these two groups.The HMBC connectivities between H-14a (d 6.57) and C-12a and between H-14b (d 6.51) and C-12b provided further support for these assignments.Likewise, the correlations between the carbon signals at d 139.4 (C-9a) and 139.6 (C-9b) with the proton signals ascribed to, respectively, H-8a (d 2.46) and H-8b (d 2.30 and 2.57) established the linkage between the aromatic rings and the benzylic methylene group of the two aliphatic eight-carbon chains.The signals of these methylene protons, in turn, were found to have long-range correlations with the signal of the corresponding vicinal carbons (C-7a and C-7b, d 39.7).The remaining degrees of unsaturation suggested by the molecular formula of 1 indicated that the eight-carbon moieties must have one ring system each.This information, together with the above-mentioned structural fragments, and the evidence of substitution at C-6 and C-8 positions of the epicatechin unit, were strongly indicative that the carbons of these alkyl side chains formed, along with C-6/C-8 and the oxygen atoms at C-5/C-7 positions of the epicatechin core, a six-membered hemiacetal bearing a methyl group bonded to the hemiacetal carbon.Accordingly, the attachment of C-1a/C-1b to C-6/C-8 of the epicatechin skeleton was deduced unambiguously from the correlations between H-1a/H-1b ( in the HMBC spectrum were in accordance with these assignments.The stereochemistry at C-6a and C-6b could not be elucidated while the stereochemical relationships of H-1a/H-1b with the methyl groups C-4a/C-4b were determined to be syn, as revealed by analysis of the nuclear Overhauser enhancement spectroscopy (NOESY) spectrum.Further evidence of structure 1 was given by additional two-and three-bond correlations discernible in the HMBC spectrum (Table 1).
In summary, all the spectral data provided the basis to determine the structure 1 for mascagnin A, an unusual epicatechin derivative, which has not been previously reported.Compound 2, to which was assigned the trivial name mascagnin B, displayed a quasi-molecular ion peak at m/z 1097.3313[M-H] -in the HRESIMS spectrum, which was sixteen mass units lower than that of 1 and in agreement with the molecular formula C 55 H 70 O 23 .The 1 H and 13 C NMR spectra of 2 are closely comparable to those of 1 (Table 2).However, the signals of ring B of the epicatechin moiety were not present, suggesting that 2 differed from 1 only in the nature of the flavan-3-ol nucleus.Accordingly, the presence of a typical pair of AA'BB' doublets at d H 7.32 (2H, J 8.2 Hz, H-2' and H-6') and d H 6.80 (2H, J 8.2 Hz, H-3' and H-5') was clearly indicative of a para-hydroxy substituted ring B in the structure of 2. This could also be deduced from the carbon resonances at d C 131.8 (C-1'), 128.9 (C-2',C-6'), 115.9 (C-3', C-5') and 157.9 (C-4') and from the connectivities observed between the proton and carbon signals of ring B in the HSQC and HMBC spectra (Table 2).These data can be accounted for in 2 if the epicatechin nucleus of 1 is replaced by a flavan-3-ol moiety of the epiafzelechintype.The spectral data reported in the literature for epiafzelechin supported this assumption. 7,9The remaining 1 H and 13 C NMR assignments of mascagnin B were based on the information afforded by DEPT, HSQC and HMBC experiments and also by comparison with the data obtained for 1 (Table 2).Thus, the structure of 2, a novel epiafzelechin derivative, was established as that depicted in Figure 1.
The biogenetic origin of the phenyloctanoid residues found in the unique epicatechin and epiafzelechin derivatives 1 and 2 can be postulated as arising from chain extension of a C 6 -C 3 unit with three malonyl-Co A units, followed by hydrolysis and decarboxylation of the terminal thioester function of the polyketide moiety.
The 1 H and 13 C NMR spectral data of compound 3 (Figure 1) were found to be in good agreement with those of a di-O-glycosylflavonol of the rutinoside-type which was identified as the known quercetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (rutin). 5,10n spite of the wide occurrence of epicatechin and epiafzelechin as plant constituents, the isolation of flavan-3-ol derivatives bearing glucosylated phenyloctanoid units from M. pubiflora in the present work is noteworthy, since only two records are found in the literature on the chemical constituents of members of the genus Mascagnia: the aforementioned preliminary chemical study of M. pubiflora 4 and the reported isolation of two naphtho-γpyrone glycosides from M. rigida. 11

Plant material
The aerial parts of M. pubiflora were collected in Aquidauana, Mato Grosso do Sul, Brazil, in August 2006.The plant material was identified by Dr. Arnildo Pott from the Empresa Brasileira de Pesquisa Agropecuária, EMBRAPA-CPAO, Campo Grande, MS, Brazil, where a voucher specimen (No. 5218) is deposited.