Flavonoids from Lonchocarpus muehlbergianus

The light petroleum extract from the roots of Lonchocarpus muehlbergianus Hassl contained nine flavonoids, including six new ones. These are 2,4-cis-2,4,5,8-tetramethoxy-(2 ́ ́,3 ́ ́:6,7)-furanoflavan; 2,4-cis-4hydroxy-2,5,8-trimethoxy-(2 ́ ́,3 ́ ́:6,7)-furanoflavan; 2,4-cis-2-prenyloxy-4,5,8-trimethoxy-(2 ́ ́,3 ́ ́:6,7)-furanoflavan; 2,4-cis-2-prenyloxy-4-hydroxy-5,8-dimethoxy-(2 ́ ́,3 ́ ́:6,7)-furanoflavan; 2’,5’,6’-trimethoxy9-(1,1-dimethylallyoxy)-[2 ́ ́,3 ́ ́:3 ́,4 ́]-furanochalcone; 5,6-dimethoxy-(2 ́ ́,3 ́ ́:7,8)-furanoflavone, identified by analysis of their spectral data (UV, IR, 1H and 13C NMR, 2D-NMR, NOE and MS). The natural occurrence of 2,4-dioxygenated flavan derivatives is being reported for the first time. Quantitative analysis of the petrol extract, by using reversed-phase HPLC, showed that the most abundant flavonoid in the extract is 2,4-cis-2,4,5,8tetramethoxy-(2 ́ ́,3 ́ ́:6,7)-furanoflavan.


INTRODUCTION
In continuation of our studies on the flavonoids of the Lonchocarpus species (Leguminosae), occurring in Brazil, we have examined L. muehlbergianus Hassl, which was allocated in Lonchocarpus subgenus Punctati together with L. subglaucescens (Magalhães et al. 1996).
Phytochemical data, obtained with several Lonchocarpus species previously investigated, allowed the characterization of many secondary metabolites mainly consisting of flavonoid structural types.Up to the present nothing is found in the literature about the natural occurrence of 2,4-dioxygenated flavans, while in Lonchocarpus, the occurrence of 4-EIMS: direct probe insert at 70 eV.HREIMS: measurements were made on a VGAuto Spec-Fisions Instrument.HPLC: UV detector, reversed-phase column Varian C18, MCH, 10 µm (300 mm × 4.0 mm) and isocratic elution with CH 3 CN:H 2 O (70:30) as the mobile phase, at a flow rate of 0.8 ml/min.CC and TLC: silica gel 35-70 mesh, flash chromatography: silica gel 230-400 mesh.

Plant Material
Roots of L. muehlbergianus were collected in the Ecological Park -Unicamp, Campinas (SP) in February 1992.Voucher specimens have been deposited at the herbarium (A.M.G.A. Tozzi 95-30) of Campinas State University (UNICAMP), Campinas-SP, Brazil.

RESULTS AND DISCUSSION
The petrol extract from the roots of L. muehlbergianus was submitted to adsorption chromatographic separation analysis (column, chromatotron, TLC and preparative TLC) furnishing nine flavonoids (1-9, Fig. 1).
Flavans 1-4, showed similar 1 H NMR spectra (Table I) as all of them have resonances for the hydrogens of a furan ring, two aromatic methoxyl groups and an unsubstituted B ring, where two of its hydrogens are deshielded.By comparison with flavan 7 1 H NMR data (Magalhães et al. 1996), the lack of H-2 signal and a much simpler multiplet in the region expected for C-3 hydrogens, suggest that flavans 1-4 have an OR group (R = methoxyl or prenyl) at C-2 causing the paramagnetic shift of H-2´and H-6´hydrogens.The 13 C NMR spectrum (Table II) and DEPT (90 • and 135 • ) allowed the assignment of all carbons; a peak around δ 100 (C • ) in place of a peak around δ 77 (CH) was taken as evidence of an OR group at C-2.In the NOE difference spectra irradiation of the methoxyl group hydrogens at C-4 enhanced the signal of one aromatic methoxyl group while irradiation of H-3´´hydrogens caused enhancement of H-2´´and of the same aromatic methoxyl group signal, indicating a linear fusion of furan with A ring (Fig. 2).
Flavan 1 was isolated as a yellow oil.Its HREIMS showed a molecular ion [M] + of m/z 370.1333, corresponding to C 21 H 22 O 6 (required M + 370.1416).The base peak at m/z 236 [C 12 H 12 O 5 ] + corresponds to the fragment bearing A ring from Cring retro Diels-Alder (RDA) cleavage, while those at m/z 337 (93%) and m/z 307 (76%) can be represented by I and II fragments (Fig. 3).The UV spectrum indicated that the A and B rings are unconjugated.The 1 H NMR spectrum (Table I) also showed the signals of two aliphatic methoxyl groups which were located at C-2 and C-4, in accordance with the double doublets assigned to the H-4 and 2H-3 hydrogens.The respective chemical shifts were confirmed by COSY (Table III) and NOE data (Fig. 4).
Flavan 2 was also isolated as a yellow oil and showed a similar UV spectrum.Its HREIMS showed a molecular ion [M] + of m/z 356.1208, corresponding to C 20 H 20 O 6 (required M + 356.1260), which is fourteen mass units lower than flavan 1, suggesting that one methoxyl was replaced by one hydroxyl.The base peak at m/z 222 [C 11 H 10 O 5 ] + , originating from RDA cleavage of the C-ring, indicates that the hydroxyl group is part of the fragment including A-ring.The 1 H NMR spectrum (Table I) showed all resonances of the A and B rings that were seen in flavan 1, except for a signal corresponding to an aliphatic methoxyl group and the presence of a much more complex multiplet for H-4 as well as a doublet integrating for one hydrogen which was attributed to a hydroxyl group at C-4.The chemical shifts of C-ring hydrogens and carbons were confirmed by COSY (Table III) and HETCOR (Table III).
The 1 H NMR (Table I) and 13 C NMR (Table II) spectra of flavans 3 and 4 show the resonances for an O-prenyl group.In the case of flavan 3 there is also a resonance for an aliphatic methoxyl group while in flavan 4 there are the multiplets for a hydroxyl group at C-4 as in flavan 2. The MS spectrum of flavan 3 showed [M] + at m/z 424 while in that of flavan 4 [M + ] at m/z 410 is again fourteen mass units lower, confirming the replacement of a methoxyl group by a hydroxyl group.Both spectra showed a peak at m/z 324, which can be respectively rationalized by the simultaneous loss of methyl-1,1dimethyl-1-vinyl ether [CH 3 OC(CH 3 ) 2 CH = CH 2 ] and 1,1-dimethyl-1-vinyl alcohol [HOC(CH 3 ) 2 CH= CH 2 ] (Fig. 5).The molecular structure was also sup- H-3 ax , H-3 eq H-3 ax , H-3 eq C-4 orted by COSY (Table IV) and NOE differential experiments (Fig. 2).
The 1 H NMR spectrum of flavonoid 5 (Table V) is very similar to that of dibenzoylmethane 8 (Magalhães et al. 1997) because both have resonances for the hydrogens of an unsubstituted aromatic ring, three aromatic methoxyl groups and one dimethylallyl group.The singlet of a methinic hydrogen on 5, however, is deshielded suggesting a corresponding enol ether.Through a NOE experiment it was observed that irradiation of the H-8 enhanced (21%) H-2 and H-6 signals, while irradiation of H-2 and H-6 has not caused an enhancement of dimethylallyl resonances, suggesting that flavonoid 5 is the Z regioisomer of the corresponding 9-OR enol ether of 8.In the 13 C NMR spectrum two signals of low intensity at δ 191.6 and δ 169.1 were attributed to C-7 and C-9, respectively while a signal at δ 107,4 was attributed to C-8.Many weak signals in the 13 C-NMR spectrum may be due to the presence of several tautomeric forms.In the UV spectrum, an additional band at λ max 341 nm was attributed to the cinamoyl system, now present in the enol ether.The main peaks in the MS spectrum can be explained by the simultaneous loss of methoxyl and 1,1-dimethyl-1-vinyl radicals to give a flavone type fragment m/z 322 (83%), followed by the loss of a methyl radical to give the fragment m/z 307 (100%) (Fig. 6).
The The 1 H NMR (Table V) and 13 C NMR (Table II) spectral data are compatible with a flavone analogous of flavan 7.
Flavan 7 was isolated in much lower amounts from L. subglaucescens (Magalhães et al. 1996), when its structure was established by 1 H NMR, 13 C NMR and NOE data.We now report additional spectral data (UV, IR and MS) including the use of 2D-NMR techniques such as COSY, HETCOR and COLOC (Table VI) which support its molecular constitution.The UV spectrum is in accordance with the lack of conjugation between the aromatic rings while the MS spectrum displayed a molecular ion at m/z 340 [M] + , together with peaks at m/z 236 [C 12 H 12 O 5 ] + and 104 [C 8 H 8 ] + corresponding to fragments derived from C ring RDA cleavage.Flavan 7 was submitted to a hydrolysis reaction.After reaction work up two major products were obtained: 7a and 7b while some of the starting material was recovered.
The structure of 7a was deduced through the comparison of its 1 H NMR spectrum with that of flavan 7 (Table V).The summation of the coupling constant values obtained for H-2, H-3 and H-4 signals [J 2,3ax + J 2,3eq = 14.1 Hz and J 4,3ax (3.0) + J 4,3eq (2.0) = 5.0 Hz], now are nearly those attributed to trans relative configuration.The H-4 signal also appeared as a triplet with a coupling constant of J =3.0 Hz (Clark-Lewis 1968, Bolger et al. 1966).So in flavan 7, the ''triplet'' corresponding to H-4 signal is in fact, a superimposed double doublet.Finally, the downfield chemical shift value of H-2 is strong evidence that it is being deshielded by the 4-OMe group which is on the same side of the C-ring in 7a.These findings suggest that the relative configuration of 4,5,7-trimethoxy-8-prenyl flavan, previously iso-  et al. 1988) is also 2:4-trans, since in its 1 H NMR spectrum the signal at δ 5.29 (1H, dd, J = 12 and 4 Hz) is very close to that of H-2 in 7a (Table V).
The 1 H NMR spectrum of 7b (Table V) closely resembled that of 7a, except for the lack of a singlet corresponding to the methoxyl group at C-4 and the presence of an absorption corresponding to hydroxyl groups (δ 2.63).Significant differences caused by the presence of the hydroxyl group, however, were observed in the 13 C NMR spectrum (Table II); the chemical shifts that were assigned to C-3 and C-4 appeared deshielded (+3.5 ppm) and shielded (−8.3 ppm), respectively.The NOE spectrum exhibited the expected interactions (Fig. 7).The IR spectrum showed the presence of a hydroxyl group (ν = 3364 cm -1 ).The spectral data obtained for compound 9 agree with those previously reported (Nascimento et al. 1976, Nascimento andMors 1981).Additionally a NOE experiment was carried out in order to confirm the angular closure of the dimethylchromene ring in the A-ring and the location of the methoxyl group on C-6. for a scholarship awarded to Ivani da Silva Blanco, to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support and to Dr. Carol Collins for writing revision.Palavras-chave: Lonchocarpus muehlbergianus, Leguminosae, flavonóides, flavanas.

Fig. 3 -
Fig. 3 -Fragments corresponding to the most abundant peaks in the mass spectrum of 1.

Fig. 5 -Fig. 6 -
Fig. 5 -Rationalization of a pathway leading to a common fragment in the mass spectra of 3 and 4.

TABLE IV COSY ( 1 H-1 H) data of flavans 3 and 4.
The MS spectrum displayed the molecular ion peak at m/z 326 [M] + , other significant peaks at m/z 308 [M -H 2 O] + and those originating from RDA cleavage of the C-ring at m/z 222 [C 11 H 10 O 5 ] + and m/z 104 [C 8 H 8 ] + .