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Boletín de la Sociedad Chilena de Química

Print version ISSN 0366-1644

Bol. Soc. Chil. Quím. vol.47 no.4 Concepción Dec. 2002

http://dx.doi.org/10.4067/S0366-16442002000400006 

A NEW LIGNAN FROM
THE PATAGONIAN VALERIANA CARNOSA Sm.

Pedro Cuadra and Víctor Fajardo

Departamento de Ciencias y Recursos Naturales, Facultad de Ciencias,
Universidad de Magallanes, Casilla 113-D, Punta Arenas, Chile

(Received: January 3, 2002 - Accepted: July 3, 2002)

ABSTRACT

In addition to the known caffeoyl methyl ester, two pinoresinol type lignans were isolated from the whole plant of Valeriana carnosa Sm. One of these metabolites is

1-hydroxypinoresinol (1), which has previously been isolated from the genera Olea and Fraxinus. The other one (2) was identified as a novel compound and was characterised by using 1H (including NOE experiments), and 13C-NMR spectroscopic techniques and mass spectrometry.

KEYWORDS:

RESUMEN

Además del conocido éster metílico del ácido cafeico, se aislaron dos lignanos del tipo del pinoresinol utilizando plantas enteras de Valeriana carnosa Sm. Uno de estos metabolitos es el 1-hidroxipinoresinol (1), el cual fue previamente aislado desde plantas de los géneros Olea y Fraxinus; y el otro (2) se identificó como un compuesto nuevo y fue caracterizado mediante técnicas espectroscópicas de RMN-1H (incluyendo experimentos de ENO) y RMN-13C y espectrometría de masas.

PALABRAS CLAVES:

INTRODUCTION

Valeriana carnosa is an annual shrub which grows on the islands spread along the Patagonian canals of Chile (66º 25’- 73º 40’ W; 48º 36’-56º 30’ S), where rain falls for over 300 days of the year, and this plant has been used for years by ancient local natives and nowadays because of the particular medicinal properties attributed to it (antioxidant, antihypertensive, vasodilative, anti-stress, among others). Frequently, lignan type compounds have been involved in these biological activities1-4.

Several phytochemical studies carried out on Valeriana and on other taxonomically related plant genera such as Fraxinus5, Eucomia6 and Olea7,8 have reported the presence of pinoresinol derivatives, valepotriates and alkaloids as major components in the plant extracts. In contrast, this study did not reveal the presence of either valepotriates or alkaloids. Instead, two compounds with a lignan skeleton were isolated and their chemical structures were determined by using NMR (1H and 13C) spectroscopy including NOE experiments. In this paper, a novel di-hydroxylated pinoresinol-type lignan is described.

RESULTS AND DISCUSSION

Compound 1 showed the characteristic reaction (black spots) for pinoresinol-type lignans on TLC plates when sprayed with H2SO4 and heated. The HR EIMS spectrum showed the molecular ion peak at m/z 374.1896 which fits the molecular formula C20H22O7, as expected for a lignan. The fragmentation pattern observed in the EIMS spectrum (the base peak at m/z 151 and the fragments at m/z 280, 272, 152, 137 and 123), and its UV spectrum, which showed an alkaline bathochromic shift of absorption maxima, both suggest a pinoresinol-type lignan containing at least one phenolic group. The stereochemistry of 1 was elucidated by using NOEDS. In this case, the strong interactions observed between H-6b and H-8b, and H-4a and H-5a , and the absence of interactions between H-2b and H-4a , firmly support an equatorial/equatorial substitution pattern with both H-2band H-6bin a cis relative configuration. In this way, 1 was identified as (+)-1-hydroxy-2,6-di(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[ 3.3.0] octane (see figure 1), which has previously been isolated from Olea and Fraxinus species by Tsukamoto et al.5,7,8,10. All spectroscopic data are in complete agreement with those reported in the literature.


Fig. 1. The exo-exo conformation of 1 and 2 diastereomers
Compound 2 showed the same reaction as 1 on TLC plates. Its HR EIMS spectrum showed the molecular ion peak at m/z 390.389 which fits with the molecular formula C20H22O8 -as for a lignan- and the base peak at m/z 151. Other peaks at m/z 374.296 and 278 were identified as indicated by the literature for the fragmentation of these compounds9,11. The strong peaks at m/z 152, 137 and 123 were attributed to fragments containing the 4-hydroxy-3-methoxyphenyl groups of pinoresinol9,12. The bathochromic shift observed in the absorption maxima after addition of NaOH also suggests the presence of a pinoresinol-type lignan with one or more phenolic groups13. In the 1H-NMR spectrum a group of three-two proton signals was observed at d =6.96 (dd; J= 1.7, 8.0 Hz), 6.90 (d; J= 1.7 Hz) and 6.85 ppm (d; J= 8.0 Hz), which were attributed to 6 protons distributed in two 1,2,4-trisubstituted benzene rings, and a singlet at d =3.83 ppm due to two methoxyl groups presumably located at positions C-3’ and C-3’’ (see table 1). This evidence shows the presence of two identically 3,4-disubstituted phenyl groups with one methoxyl group on each, as in caffeic acid derivatives, also identified in this work. The NOE analysis confirmed the location of the methoxyl groups at C-3’ and C-3’’ on the aromatic rings. Other signals of the 1H-NMR spectrum, the singlets at d = 4.93 (2H) and 4.06 (4H) ppm, were attributed to two benzylic protons of a dioxabicyclooctane ring structure, as in pinoresinol-type lignans, H-2/H-6, and to four alicyclic protons (H-4a ,b /H-8a ,b ) of the same ring, respectively. These assignments are well correlated with those reported in the literature for benzylic protons for the exo-exo (diequatorial) diasteromers of 2,6-diaryl-3,7-dioxabyciclo[ 3.3.0] octanes9. In this way, compound 2 was partially identified as 2,6-di(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[ 3.3.0] octane.

Table I. 1H-NMR data for 1-hydroxypinoresinol (1) and 1,5-dihydroxypinoresinol (2) and its tetracetate derivative (3). d/ppm, (J = Hz), CDCl3, TMS.

Continuing the structural analysis, the 13C-NMR-GASPE spectrum of 2 was correlated with those of known lignans (see table 2). As reported by Pelter et al.,9 and Chiba et al., 197713, the chemical shifts of C-1’ and C-1" are sensitive to changes in the substituents of the aromatic rings and to their stereochemistry. Likewise, the chemical shifts of C-1, C-5, C-2,6 and C-4,8 are sensitive to changes in the substituents on the bicyclic ring system and to their stereochemistry but not to changes in the aryl groups. Based on the appreciable differences of chemical shifts between these carbon atoms, the signals for 10 carbon atoms seen in the GASPE spectrum of 2, were assigned. Six of them are in identical di-oxygenated aromatic rings at d =109.81 (C-2’/C-2"), 114.43 (C-6’/C-6"), 119.87 (C-5’/C-5"), 127.51 (C-1’/C-1"), 145.60 (C-3’/C-3") and 146.67 (C-4’/C-4"). The peak at d =87.48 ppm was assigned to two oxygen bearing carbons (C-1/C-5). The substituents on these carbons and the other two located on positions C-4’’ and C-4’ of the aromatic rings, must be four hydroxyl groups because of the absence of signals for additional methoxyl groups. The upfield shifts observed in C-1’ and C-1" are due to the g substituent effect of the axial alcoholic hydroxyl groups attached to C-1 and to C-5. Another two signals at d =86.88 and 76.33 were attributed to the four alicyclic carbon atoms linked to an oxygen atom in the dioxabicyclo-octane ring (C-2/C-6 and C-4/C-8, respectively). The remaining peak at d =55.80 ppm is due to the methoxyl groups at C-3’ and C-3". Thus, it was clearly demonstrated that 2 contains a 1,5-dihydroxy-2,6-diaryl-3,7-dioxabicyclo[ 3.3.0] octane ring arranged in a completely symmetric distribution with no differences rising from each half of the molecule.

Table II. Comparison among the 13C-NMR chemical shifys of 1,5-dihydroxypinoresinol (2), its tetraacetates 1-hydroxypinoresinol (1 and 1*) (d/ppm, CDCl3. TMS)

Because of the symmetric distribution of substituents in 2, as evidenced in both the 13C-NMR and 1H-NMR spectra, the relative position of the aryl groups at C-2 and the hydroxyl groups located at positions C-1 and C-5, is restricted only to two stereoisomers: either axial/axial or equatorial/equatorial. In a recent study, Lutz et al.,14 stated that the oxolane ring annelation of pinoresinol-type lignans is always cis and both rings are in an envelope conformation, with the oxygen atom pointing away from the plane of the other four carbon atoms. In order to assign the stereochemistry on carbons C-1/5 and C-2/6, NOE 1H-NMR spectroscopy was used.

Irradiation of H-6b of 2 gave a clear NOE on the resonance of H-8b and vice-versa. This evidence shows that H-6b and H-8b are in cis relationship because there are usually no effects found between trans-1,3-oriented protons (see figures 1 and 2). The strong effects between H-2b (d =4.64 ppm) and H-8b (d =3.92 ppm) and between H-2b /H-6’ and H-6b /H-6"(d =6.96) also indicate that both H-2b and H-6b are located on the b -side of the dioxabicyclooctane ring leaving the 4-hydroxy-3-methoxyphenyl group on the a -side. This places H-6b and H-2b in a relative cis configuration. These effects are in agreement with those observed in tetrahydrofurofuran derivatives with a so-called "equatorial/equatorial" substitution pattern14. All other NOE data (see table 2) are compatible with a chair/boat conformation with both phenyl groups in pseudo-axial positions.

Chemical derivatization of the hydroxyl groups (per-acetylation) was another piece of evidence to corroborate this structure. Thus, the acetylation shift experiments provided more evidence for the stereochemistry of 2. Upon acetylation, a tetraacetate was obtained. Its 1H-NMR spectra showed two signals for phenolic (d =2.30 and 2.32 ppm) and another two alcoholic acetoxy groups (d =1.56 and 1.80 ppm). The NMR downfield shift observed for the cis protons to a 1-axial acetoxy group in (+)-1-acetoxy-2-(2-methoxy-4,5-methylenedioxy-phenoxy)-6-(2-methoxy-4,5-methylenedioxyphenyl)-3,7-dioxabicyclo[ 3.3. 0] octane15, was also seen for H-4a (0.87 ppm) and H-8a (1.07 ppm) after acetylation of 2 (see table 1). These results are in complete agreement with the NOEDS spectra of the tetraacetate, which showed the same interactions observed in 2. In this way, the stereochemistry and the relative positions of the aryl substituents on both rings, was determined using NOE difference spectroscopy. These results are summarised in figure 2. On the basis of all this evidence, 2 was identified as the novel lignan (+)-1,5-dihydroxy-2(S),6(S)-di(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[ 3.3.0] octane.


Fig. 2. A 3D - structure of 2, obtained by computer imaging (Chem Draw 3D Mcintosh) showing the major interactions observed after running NOE experiments.

EXPERIMENTAL

Valeriana carnosa was collected during the summer of 1997, in Sierra Baguales, Ultima Esperanza province, Chile (50º 44’ S, 72º 22’ W; 600 m a.s.l.). Six kg of dried whole plants (mainly roots) were extracted in cold MeOH (20 l x 2) for 48 h. The MeOH extract (500 g) was pre-fractionated by flash column chromatography with different solvent mixtures: 100% petroleum ether (40-60º); 100% CHCl3 and CHCl3-MeOH (increasing polarity). The more polar fractions (50 ml each) were further partitioned through a combination of CC and TLCP techniques, using silica gel (Kieselgel 60; 0.063-0.200 mm; 70-230 mesh ASTM) as adsorbent. In this way, compound 1 (yield: 0,3% dry wt.) was obtained as pure crystals (mp 201º) from fractions 32-40 (50 ml each) and was further purified by TLCP (kieselgel 60; 2.0 mm; 20 x 20 cm) eluted with CHCl3:MeOH=17:3 (v/v) mixture. Likewise, compound 2 was collected from more methanolic fractions and was purified by TLCP with CHCl3:MeOH=95:5 (v/v) and Et2O:AcOEt=5:3 (v/v), as solvent mixtures. Per-acetylation of 2 (yield: 0,1% dry wt.) was carried out with acetic anhydride in pyridine and stirring for 24 h at room temperature. From the fractions eluted with CHCl3/MeOH mixtures a third compound was obtained. It came out continually in several fractions and was crystallized in cold MeOH (0º C, 24 h) as white needles (mp 55-56 º). Its structure was completely elucidated by using the same spectroscopic techniques as for 1 and 2. In this way, this compound was identified as trans-3,4-dihydroxyphenyl propenoate methyl ester (yield: 1% dry wt.)

The chemical structures of these compounds were determined by using a combination of 1H-NMR, 13C-NMR-GASPE, NOEDS, UV and mass spectroscopy. The 1H and 13C NMR spectra were recorded using Bruker spectrometers (WM-360; AM-500) at 360 (13C at 90 MHz) and 500 MHz (13C at 125 MHz). GASPE and NOE experiments were performed with the same spectrometers. All spectra were recorded for CDCl3 solutions. EIMS spectra were recorded in a Kratos MS 9/50 spectrometer at 90 eV. The UV spectra were recorded with a Shimadzu UV-160A spectrophotometer, using methanolic solutions of the compounds. All the NMR and EIMS spectra were obtained at the Chemistry Department of Pennsylvania State University.

ACKNOWLEDGEMENTS

We thank the Dirección de Investigación of the Universidad de Magallanes (PR-F4-01RN-98) for financial support and the PSU facilities for recording all the spectra.

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