Phytochemical constituents from the seeds of Calycotome villosa subsp. intermedia

1 Introduction

Calycotome villosa (Poiret) Link (Leguminosae), a shrub that grows mostly in cool places, is very common in the Mediterranean area (Gibbs, 1968; Tutin, 1972). However, C. villosa (Poiret) Link subsp. intermedia (C. Presl) Quezel and Santa, a 50–150 cm spiny shrub with yellow flowers during the spring season, grow especially in the north of Africa and Spain (Greuter et al., 1989). Calycotome villosa subsp. intermedia has intricate, angular, pubescent branches, which are green when young and become greyish when mature. It shows sharp terminations and alternate leaves; the lower leaves are elongate, oval and trifoliate, and covered by sericeous down. The flowers are yellow and grouped, and have a bell-shaped calyx, thickly downy legume with a protuberant upper rib, and round dark olivaceous seeds. This shrub grows mostly in cool places, on asperities from 500 to 900 m above sea level (Camarda and Valsecchi, 1983). Calycotome villosa subsp. intermedia is not used in the Moroccan folk medicine. Medicinal uses have been reported only for C. villosa (Poiret) Link as antitumoral agent (Hartwell, 1982) and for the treatment of furuncle, cutaneous abscess and chilblain in the Sicilian folk medicine (Lentini et al., 1993).

As a part of a systematic research study on the constituents of Moroccan natural plants, we have reported, in previous phytochemical studies of Calycotome villosa subsp. intermedia, the isolation and characterization of flavone glucosides from the leaves and flowers (El Antri et al., 2004a) and tetrahydroisoquinoline alkaloids and isoquinoline-N-oxide alkaloid from the seeds (El Antri et al., 2004b,4c). Flavonoid compounds exhibit varied biological effects and have been identified as antitumor agents, antioxidants, and free radical scavengers (Manthy and Buslig, 1998; Bors et al., 1990). Substituted-tetrahydroisoquinoline alkaloids, in which C-1 is a quaternary stereogenic center, have been reported to display very interesting biological and pharmacological properties (He et al., 2000; Herbert, 1985). In a continuing search on the seeds of this species, the methanolic extract was found to contain a complex mixture of compounds. The present study describes the isolation and the structural elucidation of a new dihydroisoquinoline-N-oxide alkaloid together with a paraben derivative.

2 Materials and methods

3 Results and discussion

The seeds of Calycotome villosa subsp. intermedia were extracted with hexane and then with hot methanol. The methanolic extract was purified in the usual way for the isolation of alkaloids (Dalton, 1979). Fractionation and silica gel column chromatography yielded the new compound 1 and the known compound 2 (Scheme 1) besides the previously isolated alkaloids (El Antri et al., 2004b,4c). Compound 1 was the less polar of all alkaloids found in the methanolic extract of the seeds of Calycotome villosa subsp. intermedia.

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Scheme 1

A = phenyl ring and B = sofa six-membered ring.

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Compound 1 was isolated in a crystalline form and completely characterized. The molecular formula of 1 was determined to be C₁₂H₁₅NO₄ on the basis of ESI-MS (m/z = 238 [M+H]⁺) and elemental analysis. In the UV spectrum of 1, the maximum bands are 203, 240 and 331 nm. Its IR spectrum showed absorption bands at 3459-3349, 2970, 1610, 1520, 1465, 1274, 1142, 1029 and 850 cm⁻¹. The ¹H NMR spectrum of 1 showed two aromatic protons at δ 6.83 (s, H-8) and 6.66 (s, H-5), two signals at δ 4.01 (t, 2H, J = 7.5 Hz) and 3.01 (t, 2H, J = 7.5 Hz) typical of H-4 and H-3 protons of a dihydroisoquinoline nucleus, and two singlets at δ 3.83 and 3.82 corresponding to the methoxyl groups at C-6 and C-7, which, in addition to the ¹³C NMR spectrum and a prominent peak at m/z = 238 in the ESI mass spectrum, confirmed the dihydroisoquinoline skeleton in the molecule. The remaining ¹H and ¹³C NMR data of 1 (singlet at δ 4.73 (2H), three CH₂, two CH and five quaternary carbons) as well as the presence of an ion at m/z = 190 [C₁₂H₁₅NO₄ + H − H₂O − NO]⁺ in the ESI-MS, evidenced a dihydroisoquinoline N-oxide in the molecule. Thus, the structure of 1 was determined as a 1-hydroxymethyl-6,7-dimethoxy-3,4-dihydroisoquinoline-N-oxide, which is a new natural product.

Compound 2 was isolated as colorless needles, with molecular formula C₈H₈O₃ deduced from the molecular ion peak at m/z 152 in the EI-MS and supported by ¹H NMR and ¹³C NMR data. The mass spectrum of compound 2 showed molecular ion peak at m/z 152 with ions corresponding to loss of OCH₃ (M − 31; m/z = 121) followed by loss of the carbonyl group (M − 31 − 28; m/z = 93). The ¹H NMR spectrum analysis revealed the presence of a pair of doublets at δ 7.96 (d, J = 8.2 Hz, H-3 and H-5) and 6.88 (d, J = 8.2 Hz, H-2 and H-6), each integrating to two protons, suggesting the presence of two aromatic hydrogen in ortho position. Each singlet of hydroxyl and methoxyl signals was observed at δ 5.60 and 3.92, respectively. Its ¹³C NMR spectrum showed six resonances (two quaternary carbons, two CH groups, one carboxyl and one methoxyl) and the signals of carboxyl C⚌O (δ 167.0) and methoxyl (δ 159.8). Accordingly, compound 2 was assigned as a paraben derivative named methyl-4-hydroxybenzoate (Scheme 1). The physical and spectroscopic data found are in concordance with literature values (Cheng et al., 2001; Perveen et al., 2009). Although there are many types of paraben derivatives by organic synthesis (Crombie et al., 1979; Hirota et al., 1981), methyl-4-hydroxybenzoate 2 was isolated for the first time from Calycotome villosa subsp. intermedia.

The crystal structure of 1 has been determined on single crystal. Crystallographic data are given in Table 1. Selected geometrical parameters, bond lengths and angles are listed in Table 2 according to the numbering scheme displayed in Fig. 1. The unit cell contains two molecules C₁₂H₁₅NO₄. The main part of the molecule of alkaloid 1 is the dihydroisoquinoline molecular fragment, which consists of the phenyl ring (A) and the sofa six-membered ring (B) (see Scheme 1). The deformation of the molecule can be expressed in terms of the distorsion angles between its different parts. According to the values of the corresponding distorsion angles: C3 C4 C5 C10–175.5(2), C7 C6 C5 C4–176.7(2), C10 C5 C6 C1 176.3(2), C2 C1 C6 C7 177.1(2), C1 C2 C3 O31–178.4(2), we state that the bonds C5–C10 and C6–C7 are in the plane of the ring. Same could be concluded about the orientation of the methoxy groups. The O31–C32 methoxy group lies in the plane of the ring (C4 C3 O31 C32 is −4.2°) while the methoxy group O21 C22 is out of the plane (C1 C2 O21 C22 is 16.7°). The hydroxyl group O72 has a gauche arrangement with respect to the H72–O72–C71–C7 torsion angle (65.0°).

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Figure 1

Perspective view of the molecule of 1. Thermal ellipsoids drawn at 50% probability levels.

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A one restriction to the freedom of the molecule’s motion is imposed by intramolecular H-bonds, between H72 and O81 (2.711(2) Å). More such interactions are used to extend the molecular network in the three dimensions. Fig. 2 depicts a projection on the [0 0 1]. One can remark the formation of the “dimers” and their propagation in the ab-plane using H–O interactions.

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Figure 2

Projection of the crystal structure of alkaloid 1 onto [0 0 1].

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4 Conclusion

The isolation and spectroscopic characterization of a new alkaloid together with a paraben derivative from the seeds of Calycotome villosa subsp. intermedia was described. The structure of the new alkaloid 1 was solved by X-ray analysis. Paraben derivative 2 was isolated for the first time from Calycotome villosa subsp. intermedia.