Isolation and X-ray Crystal Structure of Tetrahydroisoquinoline Alkaloids from Calycotome Villosa Subsp. Intermedia

Two tetrahydroisoquinoline alkaloids were extracted from the alkaloid fraction of a methanol extract of the seeds of Calycotome Villosa Subsp. intermedia. Their structures were established as


Introduction
The isoquinoline alkaloids form one of the largest groups of compounds found in a variety of plant families [1][2][3][4].With more than 50 different compounds found in nature, 1,2,3,4-tetrahydroisoquinoline (THIQ) and especially its derivatives with alkoxy substituents on the aromatic rings constitute the largest group within the simple isoquinoline alkaloids.These natural alkaloids are generally optically active compounds possessing important clinical applications such as analgesics, antihypertensives, smooth or skeletal muscle relaxants, antispasmodics, antitussives, antimalarials, narcotics and antipyretics [5].It is worth noting that 1-substituted-THIQs, in which C-1 is a quaternary stereogenic centre, have been reported to display very interesting biological and pharmacological properties [3,6,7].For instance, 1-methyl-and 1-phenyltetrahydroisoquinoline are involved in the treatment of Parkinson's and other nervous system diseases [8][9][10].Previous phytochemical studies resulted in the isolation of two flavone glucosides from the flowers and leaves of Calycotome villosa Subsp.intermedia [11].In continuation of our chemical investigations on this plant, we describe in this paper the isolation and unambiguous structure elucidation of two alkaloids, with the chemical formulae C 12 H 17 NO 3 and C 12 H 18 NO 3 Cl and here labelled 1 and 2, respectively.

NH
Confirmation of the structure was obtained from X-ray diffraction of single crystals of 1 grown by slow evaporation from methanol.As expected, 1 has a tetrahydrosubstituted isoquinoline structure [20][21][22][23].The molecular structure and numbering scheme are shown in Figure 1.All bond lengths and angles are reasonable within experimental error (Table 1).The C5-C10 aromatic ring is planar (r.m.s.deviation = 0.0094 Å).The methoxy groups at C7 and C8 atoms are slightly rotated around the C7-O71 and C8-O81 bonds (Figure 1), the dihedral angles between the plane of the aromatic ring and the planes defined by atoms C7, O71, C72 and C8, O81, C82 being respectively 12.7° and 7.5°.The C4 atom lies almost in the plane of the aromatic ring, whereas atom C1 is slightly displaced from it (the deviation of atoms C4 and C1 from the ring plan are 0.003 and 0.045 Å, respectively).The C1-C10 and C4-C5 bonds are in the plane of the ring (angles with the normal to the plane are 91.0 and 90.6°, respectively), whereas the two remaining bonds, C8-O81 and C7-O71, are slightly out of the plane (angles with the normal to the plane are 91.4 and 88.6°, respectively).As expected, the heterocyclic ring of tetrahydroisoquinoline adopts a half chair conformation.
Alkaloid However, the low basicity of 2, its low solubility in apolar organic solvents and its high solubility in water led us to think that alkaloid 2 was a salt.In an attempt to confirm this, an X-ray crystallographic study was performed.Suitable crystals for X-ray studies were obtained from methanol by slow evaporation.A view of 2 is shown in Figure 2, while selected molecular dimensions are reported in Table 2. Bond lengths and angles values fall in the expected range [23].The independent unit of the crystal consist of the protonated main molecule and Cl -anion.As previously found for alkaloid 1, the main skeleton of 2 is formed by a tetrahydroisoquinoline moiety.The chloride ion Cl -interacts with the tetrahydroisoquinoline by means of weak H-bonds, with the strongest one [(N8-H8A…Cl1)] at 3.091 Å.The crystallographic data obtained for the alkaloid 2 confirmed the previous spectroscopic attributions and the structure was assigned as (S)-7-hydroxymethyl-2-3-dimethoxy-7,8,9,10tetrahydroisoquinoline chloride.

Conclusions
Two alkaloids were isolated from the seeds of Calycotome Villosa Subsp.intermedia.Their structures were solved by X-ray analysis.Isolation and characterization of other alkaloids from this plant are currently under investigation.

General
Melting points were measured in open capillary tubes in a Büchi 530 apparatus and are uncorrected.UV-visible spectra were obtained on a Varian Cary 3E spectrophotometer, and IR spectra were recorded on a Pye Unicam Perkin-Elmer spectrophotometer. 1 H-and 13 C-NMR spectra were recorded in CDCl 3 and DMSO-d 6 on a Bruker (Wiessembourg, France) AM 300 spectrometer (300 and 75 MHz, for 1 H-and 13 C-NMR, respectively) and chemical shifts are given as δ values with TMS as an internal standard.[α] 20  D values were measured using an ADP 220 polarimeter (Bellingham + Stanley LDT).ESI-MS data were obtained on a Quattro II tandem quadripole mass spectrometer (Micromass, Manchester, UK) fitted with an electrospray ionisation.Silica gel GF 254 was used for TLC.Spots on chromatograms were detected under UV light (254 nm) and by Dragendorff's reagent.Column chromatography (CC) was carried out on silica gel 60 (70-230 mesh).

Plant material
Seeds of Calycotome Villosa Subsp.intermedia were collected from the aerial part of the plant in June 2001 and again in June 2002 from Zrireg valley, plateau of Tazzeka, area of Taza, Morocco.

Extraction and isolation
The powdered seeds (100 g) were first extracted with hexane for 24 h and then with methanol for 48 h using a Soxhlet apparatus.The methanolic solution was evaporated to dryness and the resulting crude extract was dissolved in a 5% hydrochloric acid solution and extracted first with hexane and then with CH 2 Cl 2 .The aqueous solution was made basic to pH 10 with concentrated ammonia and extracted three times each with 200 mL of CH 2 Cl 2 .The collected organic phases were dried over anhydrous sodium sulfate and evaporated under reduced pressure to give 1.75 g of the crude alkaloid extract.This was extracted with acetone and the residual product was dissolved in methanol (25 mL) and kept at room temperature overnight to give a crystalline powder.The precipitate obtained was filtered off and washed three times with CH 2 Cl 2 to yield 0.15 g of a purified alkaloid 2. The combined organic extract (CH 2 Cl 2 + CH 3 COCH 3 ) was concentrated and chromatographed on silica gel.The fraction eluted with 10% methanol in CH 2 Cl 2 was concentrated to dryness to give pure compound 1 as colorless crystals (0.5 g).These products were recrystallized from methanol to afford crystals suitable for X-ray analysis.

Figure 1 .
Figure 1.ORTEP view of 1 showing the atomic labeling scheme, with H atoms removed for clarity.Thermal ellipsoids drawn at 50% probability levels.

Figure 2 .
Figure 2. ORTEP view of (2) showing the atomic labelling scheme, with H atoms omitted for clarity.Thermal ellipsoids drawn at 50% probability levels.

Table 1 .
Selected bond lengths and angles of 1.

Table 2 .
Selected bond lengths and angles of 2.

Table 3 .
Crystal and experimental data of 1 and 2