Limonoids from Spiranthera odoratissima St. Hil

Abstracts

Eleven substances have been isolated from the roots of Spiranthera odoratissima, two new limonoids, the known limonoid limonin, three furoquinoline alkaloids, dictamnine, gamma-fagarine and skimmianine, three beta-indoloquinazoline alkaloids, rutaecarpine, evodiamine and 1-hydroxyrutaecarpine, the coumarin aurapten and beta-sitosterol. Structure elucidation has been carried out by IR as well as 1D and 2D NMR; the new structures were also confirmed by X-ray crystallographic analyses.

Spiranthera odoratissima; Rutaceae; limonoids; alkaloids; X-ray diffraction

Onze substâncias foram isoladas das raízes de Spiranthera odoratissima: dois novos limonóides, o limonóide já conhecido limonina, três alcalóides furoquinolínicos (dictamina, gama-fagarina e esquimianina), três alcalóides beta-indoloquinazolínicos (rutaecarpina, evodiamina e 1-hidroxirutaecarpina), a cumarina aurapteno e beta-sitosterol. A elucidação estrutural dessas substâncias foi realizada através de técnicas espectrais como IV e RMN em uma e duas dimensões; as estruturas novas foram confirmadas por difração de raios-X.

ARTICLE

Limonoids from Spiranthera odoratissima St. Hil

Tereza A. N. Ribeiro^I; Eliane A. da Silva Ndiaye^I; Eudes da S. Velozo^II; Paulo C. Vieira^{*, III}; Javier Ellena^IV; Paulo T. de Sousa Júnior^I

^IDepartamento de Química, Universidade Federal de Mato Grosso, Av. Fernando Correa s/n, 78060-900 Cuiabá - MT, Brazil

^IIFaculdade de Farmácia, Universidade Federal da Bahia, Rua Barão de Geremoaba s/n, 40170-290 Salvador - BA, Brazil

^IIIDepartamento de Química, Universidade Federal de São Carlos, CP 676, 13565-905 São Carlos - SP, Brazil

^IVInstituto de Física, Universidade de São Paulo, CP 369, 13560-970 São Carlos - SP, Brazil

ABSTRACT

Eleven substances have been isolated from the roots of Spiranthera odoratissima, two new limonoids, the known limonoid limonin, three furoquinoline alkaloids, dictamnine, g-fagarine and skimmianine, three b-indoloquinazoline alkaloids, rutaecarpine, evodiamine and 1-hydroxyrutaecarpine, the coumarin aurapten and b-sitosterol. Structure elucidation has been carried out by IR as well as 1D and 2D NMR; the new structures were also confirmed by X-ray crystallographic analyses.

Keywords:Spiranthera odoratissima, Rutaceae, limonoids, alkaloids, X-ray diffraction

RESUMO

Onze substâncias foram isoladas das raízes de Spiranthera odoratissima: dois novos limonóides, o limonóide já conhecido limonina, três alcalóides furoquinolínicos (dictamina, g-fagarina e esquimianina), três alcalóides b-indoloquinazolínicos (rutaecarpina, evodiamina e 1-hidroxirutaecarpina), a cumarina aurapteno e b-sitosterol. A elucidação estrutural dessas substâncias foi realizada através de técnicas espectrais como IV e RMN em uma e duas dimensões; as estruturas novas foram confirmadas por difração de raios-X.

Introduction

Spiranthera odoratissma St. Hil., is a shrub found in the central Brazilian savannah, as well as in Bolivia.¹ In Mato Grosso state it is known by the vernacular name of "manacá", being used in folk medicine to treat syphilis, rheumatism, kidney infections, urinary retention, abdominal pains, gout, acne and boil.² This plant has already been investigated from chemistry viewpoint. From a specimen collected in Bahia were isolated furoquinoline alkaloids; coumarins and terpenes.³ The Rutaceae family is characterized by the abundance of anthranilic acid derived alkaloids coumarins, limonoids and flavonoids mainly.⁴

We have been interested in the chemistry of Rutaceae,^5-8 and as part of ongoing work on this family, in this study we describe the isolation and the identification of two new limonoids, as well as the known limonin. Also described are the isolation and identification of the furoquinoline alkaloids g-fagarine dictamnine, skimmianine, the b-indoloquinazoline alkaloids rutaecarpine, evodiamine and 1-hydroxy-rutaecarpine, the coumarin aurapten and b-sitosterol.

Experimental

General experimental procedures

Melting points were measured in a Mettler FP-80 apparatus and are uncorrected. Specific rotations were determined in a Perkin-Elmer 341 polarimeter. The IR spectra were obtained in a Bomem FT-IR MB100 equipment with the samples in KBr pellets. ¹H and ¹³C NMR spectra were measured in Bruker AC-200 (200 MHz), ARX-400 (400 MHz) and Varian Mercury-300 (300 MHz) apparatus. The chemical shifts (d) are expressed in ppm and the coupling constants (J) in Hertz; TMS was used as internal standard, as well as the residual hydrogen from the solvents (CDCl₃ and DMSO-d₆). Radial preparative chromatography (RPC) was carried out with a Chromatotron apparatus and were performed with Merck Kiesegel 60 PF254. Column chromatography (CC) was performed with silica gel 60 (Merck, 63-230 µm), and flash column chromatography with silica (Merck, 43-63 µm). Analytical thin layer chromatography (TLC) was carried out with Merck Kiesegel 60 F254 (0.25 mm) plates.

Plant material

The roots from S. odoratissima were collected at Cuiabá-Barão de Melgaço road (Km 1) in December 1999. A voucher specimen was deposited at Central Herbarium of Universidade Federal de Mato Grosso (registration # 24246).

Extraction and isolation of compounds

The roots of S. odoratissima (3.3 kg) were macerated at room temperature with dichloromethane (3 x 8L), with occasional stirring, during 7 days. The dichloromethane extract-DCE (126 g; 3.82%) was obtained after filtration and solvent removal in vacuo. Subsequent extraction was performed with methanol (3 x 8L) using the same procedure above, affording the methanol extract-ME (120 g; 3.64%).

An aliquot of DCE (20.0 g) was submitted to conventional acid-base extraction (HCl 1%; 4 x 100 mL) and the alkaloid fraction (190 mg) was chromatographed by RPC, in a 4 mm radial plate, with hexane-CHCl₃ (8:2; 100 mL); CHCl₃ (250 mL) and CHCl₃-MeOH (95:5; 100 mL), affording 6 fractions. The furoquinoline alkaloids g-fagarine (15 mg), dictamnine (8 mg) and skimmianine (30 mg) were isolated after washing fractions 2, 3 and 4 with Et₂O.

Another DCE fraction (42.5 g) was filtered in a silica column (400 g) with petrol ether (0.5 L), CH₂Cl₂ (2 L), CHCl₃ (1 L), CHCl₃-MeOH (99:1; 2 L), CHCl₃-MeOH (9:1; 1 L), MeOH (1 L) and MeOH-0.1%HOAc (0.5 L), affording fractions A to G, respectively.

Column chromatography (CC) was carried out on fraction B (23.0 g) employing the gradient solvent system: hexane-CH₂Cl₂ (1:1; 450 mL), CH₂Cl₂ (200 mL), CH₂Cl₂-MeOH (99:1; 200 mL), (95:5; 200 mL), (9:1; 600 mL) and MeOH (200 mL), affording 8 fractions after TLC analysis. Fraction 2 [from hexane-CH₂Cl₂ (1:1)] was chromatographed by RPC (hexane- CH₂Cl₂; 9:1; 7:3; 1:1), CH₂Cl₂ and CHCl₃-MeOH (99:1), affording the coumarin aurapten (50 mg), after washing the crude hexane-CH₂Cl₂ (7:3) fraction with EtOH. Fraction 4 [from hexane-CH₂Cl₂ (99:1)] was submitted to medium pressure chromatography using the gradient solvent system: hexane-CH₂Cl₂ (2:8; 300 mL), (1:9; 70 mL), CH₂Cl₂ (150 mL), CH₂Cl₂-MeOH (9:1; 250 mL). Aurapten (109 mg) was obtained after washing with Et₂O the combined hexane-CH₂Cl₂ (2:8) fractions; b-sitosterol was obtained after washing the combined hexane-CH₂Cl₂ (1:9) fractions with EtOH; 1 (30 mg) was obtained after washing the combined CH₂Cl₂-MeOH (95:5 and 9:1) fractions with EtOH.

Fraction D (10 g) was submitted to medium pressure chromatography using the solvent system: CH₂Cl₂ (300 mL), CH₂Cl₂-CH₃CN (98:2; 200 mL), (95:5; 300 mL), (7:3; 200 mL); (1:1; 300 mL) and MeOH (200 mL). The combined CH₂Cl₂-CH₃CN (98:2 and 95:5) fractions (2.5 g) were re-submitted to medium pressure chromatography employing the gradient hexane-CHCl₃ (1:9; 400 mL), CHCl₃ (200 mL), CHCl₃-MeOH (95:5; 300 mL), (9:1; 300 mL), (1:1; 200 mL) and MeOH (200 mL). Limonin (3) (130 mg) precipitated after EtOH addition to the combined hexane-CHCl₃ (1:1) and CHCl₃ fractions. The supernatant liquid was chromatographed by CC in hexane-CHCl₃ (2:8; 270 mL), CHCl₃ (50 mL), CHCl₃-CH₃CN (8:2; 200 mL), (6:4; 150 mL), (3:7; 100 mL), CH₃CN (50 mL), CH₃CN-EtOH (7:3; 100 mL) and (1:1; 150 mL). Skimmianine (30 mg) was obtained from the combined hexane-CHCl₃ (2:8) and CHCl₃ fractions after solvent removal and washing the solid with EtOH. The combined CHCl₃-MeOH (95:5) fractions were chromatographed by CC using CH₂Cl₂ (50 mL), CH₂Cl₂-EtOAc (8:2; 300 mL), (7:3; 300 mL), (3:7; 200 mL), EtOAc (200 mL), EtOAc-MeOH (8:2; 200 mL) and MeOH (100 mL). The combined CH₂Cl₂-EtOAc (8:2) and (7:3) fractions were submitted to RPC with CHCl₃, CHCl₃-MeOH (95:5), (9:1) and EtOAc-MeOH (9:1). Limonin (3) (60 mg) was obtained after solvent removal from fraction CHCl₃-MeOH (9:1).

ME (30 g) was suspended in MeOH-H₂O (7:3; 100 mL) and extracted with CH₂Cl₂ (3 x 100 mL), EtOAc (3 x 100 mL) and n-BuOH (3 x 100 mL). The CH₂Cl₂ fraction (2.7 g) was submitted to CC in CH₂Cl₂ (1.7 L), CH₂Cl₂-MeOH (99:1; 2.2 L), (98:2; 0.8 L), (95:5; 1.5 L); (9:1; 0.7 L), (7:3; 0.6 L), (1:1; 0.8 L) and MeOH (0.9 L). The combined CH₂Cl₂-MeOH (99:1) fractions (550 mg) were submitted to CC in CH₂Cl₂ (300 mL), CH₂Cl₂-MeOH (99:1; 150 mL), (97:3; 50 mL), (95:5; 350 mL), (9:1; 150 mL), (1:1; 50 mL) and MeOH (50 mL). A precipitate (80 mg) was obtained by adding EtOH to the combined CH₂Cl₂ fractions. Flash CC was performed with this precipitate, employing CH₂Cl₂ (150 mL), CH₂Cl₂-MeOH (99:1; 100 mL), (95:5; 100 mL), (1:1; 50 mL) and MeOH (50 mL). Rutaecarpine (4) (7 mg) was isolated after solvent removal from the combined CH₂Cl₂ fractions; evodiamine (5) (5 mg) was isolated after solvent removal from the combined CH₂Cl₂-MeOH (99:1) fractions. The combined CH₂Cl₂-MeOH (95:5 and 1:1) fractions were submitted to flash CC in CH₂Cl₂ (40 mL) and CH₂Cl₂-MeOH (99:1; 30 mL). 1-hydroxyrutaecarpine (6) (4 mg) and 2 (7 mg) were isolated from the second CH₂Cl₂ fraction and the third CH₂Cl₂-MeOH (99:1), respectively.

Compound (1)

[a]²⁵ (CHCl₃, c. 0.1): -26 ºC. IR (KBr) n_max/ cm^-1: 3405, 1767, 1741, 1714 . ¹H and ¹³C NMR (CDCl₃): Tables 1 and 2.

Compound (2)

[a]²⁵ (CHCl₃, c. 0.05): -20 ºC. IR (KBr) n_max/ cm^-1: 1766, 1742, 1707. ¹H and ¹³C NMR (CDCl₃): Tables 1 and 2.

Single crystal X-ray analysis

Low temperature X-ray diffraction data collections were performed at 120(2) K, on an Enraf-Nonius Kappa-CCD diffractometer equipped with an Oxford Cryosystem liquid N₂ device, using graphite-monochromated MoK a radiation (0.71073 Å). Data were collected up to 50° in 2q, with a redundancy of 4 in the phi scans and omega scans with kappa offsets modes. The final unit cell parameters were based on all reflections. Data collections were made using the COLLECT program;⁹ integration and scaling of the reflections were performed with the HKL Denzo-Scalepack system of programs.¹⁰ No absorption corrections were applied.

The structures were solved by direct methods with SHELXS 86¹¹ and SHELXS-97.¹² The models were refined by full-matrix least squares on F² with SHELXL-97.¹³ All the hydrogen atoms were stereochemically positioned and refined with the riding model.¹² Hydrogen atoms of the CH and CH₂ groups were set isotropic with a thermal parameter 20% greater than the equivalent isotropic displacement parameter of the atom to which each one was bonded. This percentage was set to 50% for the hydrogen atoms of the CH₃ groups. Data collections and experimental details for the complexes are summarized in Table 3. The programs SHELXL-97,¹³ and ORTEP-3¹⁴ were used within WinGX¹⁵ to prepare materials for publication. Atomic coordinates, bond lengths and angles, and thermal parameters have been deposited at the Cambridge Crystallographic Data Centre (see below).

Results and Discussion

Limonoid (1) has been isolated from the dichloromethane extract from the roots of S. odoratissima as a white amorphous solid (mp 174-177 ºC), presenting [a]_D²⁵ (CHCl₃): – 26º. The IR spectrum has shown absorptions at 3405 (OH), 1767 (lactone), 1741 (a,b-unsaturated ester) and 1714 (ketone).

¹H NMR spectrum presented signals related to the b-substituted furan ring, where H-21 and H-23 appeared as multiplets at d 7.41 and at d 7.36, respectively. The b-furan hydrogen H-22 appeared as a double doublet at d 6.37 (J= 1.7, 0.8 Hz). The hydrogen H-17, from the d-epoxylactone ring, was observed at d 5.54 (s, 1H).

The ¹H and ¹³C NMR spectra from (1) has shown similarities with the correlated spectra of (3), also isolated in this study, as shown in Tables 1 and 2. The absence of two doublets, associated to the geminal hydrogens at C-19, very common in structures related to (3), displaying a A,D-seco ring,¹⁶ when linked with the low intensity, quaternary carbon signal observed at d 182.5, from the DEPT experiment, was a good indication for the presence of a carbonyl group at C-19 in 1. HMBC spectrum has shown long distance coupling (J³) between H-1, H-9 and C-19, confirming that C-19 in 1 is oxidized.

The a,b-unsaturated ester moiety, detected by the IR spectrum, was further confirmed by ¹H NMR (d 3.71, s, 3H; -CO₂CH₃), as well as by the signals at d 165.5 (-CO₂CH₃) and d 52.3 (-CO₂CH₃) in ¹³C NMR. The olefinic hydrogens H-1 and H-2 were detected as two doublets at d 6.65 and d 6.12 respectively; the cis configuration of the double bond could be determined by the coupling constant value (J_1,2 = 12.4 Hz). HSQC spectrum showed correlation between these two protons and the carbons at d 151.6 (C-1) and d 123.2 (C-2), respectively.

The DEPT ¹³C NMR spectrum presented two signals related to methylenic carbons at d 38.1 (C-6) and d 41.2 (C-12). HMBC experiment has shown H-6 correlation with C-7 (d 207.3; J²) and C-4 (d 87.6; J³). The methylenic hydrogen H-6 resonated at d 3.24 (dd, J= 18.4, 10.4 Hz, H-6_ax) and d 2.74 (dd, J= 18.4, 2.5 Hz, H-6_eq); COSY spectrum has shown coupling of both hydrogen with H-5 at d 3.09 (dd, J= 10.4, 2.5 Hz).

Reasonably similar values have been observed when comparing the ¹³C NMR data for rings B and D in compounds (1) and (3) (Table 2). DEPT experiment, however, presented three signals related to methylenic carbons in (3) and only two signals related to methylenic carbons in (1). The hydroxyl group, shown to be present in (1) by the IR spectrum, nevertheless, should be located at C-11 or C-12 in ring C. Furthermore, the carbinolic hydrogen appeared as a broad doublet at d 4.51 (J= 6.7 Hz, 1H), which has been associated to d 66.4 signal (HSQC), indicating that H-11 couples only with one H-12_eq. No coupling was observed between H-11 and H-9 and H-12_ax. The HMBC spectrum showed a long distance coupling (J³) with C-8 (d 48.5), confirming that the hydroxylated carbon is C-11. The a stereochemistry of the OH group was determined by X-ray crystallography (Figure 2). The H-12 methylenic hydrogens were observed at d 1.83 (d, J= 15 Hz, H-12_ax) and d 1.61 (m, H-12_eq). HMBC showed long range coupling (J³) between H-12_ax with C-9 (d 43.9), C-14 (d 65.1), C-17 (d 77.8) and C-18 (d 19.2).

Four singlets (3H) relative to the methyl groups, were observed at d 1.02 (H-18), d 1.16 (H-30), d 1.44 (H-29) and d 1.49 (H-28). The correct attribution of each methyl group position was carried out by using HMBC and HSQC experiments. The long distance correlation of d 1.02 (Me-18) with C-12 (d 41.2), C-13 (d 36.5), C-14 (d 65.1), C-17 (d 77.8), and d 1.16 (C-30) with C-7 (d 207.3), C-8 (d 48.5), C-9 (d 43.9) and C-14 (d 65.1), could be easily observed in HMBC spectrum.

The structure presented for compound (1) was definitely confirmed by single crystal X-ray diffraction (Figure 2).

The second new limonoid (2), was obtained from the methanol extract of the roots of S. odoratissima, as a white solid presenting a high mp (> 300 ºC) and [a]_D²⁵ -20 (CHCl₃). The IR spectrum presented absorptions at 1766 (lactone), 1742 (a,b-unsaturated ester) and 1707 (ketone).

Compound (2) ¹H NMR spectrum presented high similarity with the one from compound (1), indicating a limonoid with a b-substituted furan ring, as shown by the two a-furan hydrogen at d 7.39 (m) and the b-furan hydrogen at d 6.34 (m). An additional signal was observed at d 2.10 (s, 3H), being attributed to the presence of an extra methyl group in (2).

¹³C NMR spectrum presented 29 signals, showing that compound (2) should have two more carbons than the analogous (1); a quaternary carbon at d 170.5 and a methyl group at d 21.2 were observed by the DEPT experiment indicating the presence of an acetyl group in this compound.

These ¹H and ¹³C NMR data, in association with the absence of a hydroxyl absorption in IR spectrum led to the conclusion that the hydroxyl group was esterified in (2). Single crystal X-ray diffraction has confirmed the proposed structure for (2) (Figure 3).

The known compounds were identified through comparison of their spectral data with the ones in the literature, limonin (3),¹⁷ dictamnine, skimmianine;¹⁸ the b-indoloquinazoline alkaloids rutaecarpine (4), evodiamine (5)¹⁹ and 1-hydroxyrutaecarpine (6),²⁰ and the coumarin aurapten.²¹ The isolated alkaloids and limonoids have been described as typical metabolites from the Rutaceae.²²

Supplementary Information

Crystallographic data (excluding structure factors) for the structures in this paper has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no CCDC 238939 and 238940. Copies of the data can be obtained, free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: [phone]; or e-mail: [e-mail]).

Acknowledgements

The authors are grateful to Dr. Antônio Gilberto Ferreira from Laboratório de Ressonância Magnética Nuclear (Universidade Federal de São Carlos) for obtaining NMR spectroscopic data. Financial assistance from FAPEMAT, CAPES, CNPq, FAPESP is acknowledged.

Received: September 23, 2004

Published on the web: August 30, 2005

FAPESP helped in meeting the publication costs of this article

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Publication Dates

• Publication in this collection
  20 Jan 2006
• Date of issue
  Nov 2005

History

• Received
  23 Sept 2004