Limonoids from Spiranthera odoratissima St . Hil

Onze substâncias foram isoladas das raízes de Spiranthera odoratissima: dois novos limonóides, o limonóide já conhecido limonina, três alcalóides furoquinolínicos (dictamina, γ-fagarina e esquimianina), três alcalóides β-indoloquinazolínicos (rutaecarpina, evodiamina e 1-hidroxirutaecarpina), a cumarina aurapteno e β-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. 3The Rutaceae family is characterized by the abundance of anthranilic acid derived alkaloids coumarins, limonoids and flavonoids mainly. 4e have been interested in the chemistry of Rutaceae, [5][6][7][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 γ-fagarine dictamnine, skimmianine, the β-indoloquinazoline alkaloids rutaecarpine, evodiamine and 1-hydroxyrutaecarpine, the coumarin aurapten and β-sitosterol.

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. 1 H and 13 C NMR spectra were measured in Bruker AC-200 (200 MHz), ARX-400 (400 MHz) and Varian Mercury-300 (300 MHz) apparatus.The chemical shifts (δ) 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 (CDCl 3 and DMSO-d 6 ).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%).

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 N 2 device, using graphite-monochromated MoK α radiation (0.71073 Å).Data were collected up to 50° in 2θ, 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. 10No absorption corrections were applied.
The structures were solved by direct methods with SHELXS 86 11 and SHELXS-97. 12The models were refined by full-matrix least squares on F 2 with SHELXL-97. 13All the hydrogen atoms were stereochemically positioned and refined with the riding model. 12Hydrogen atoms of the CH and CH 2 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 CH 3 groups.Data collections and experimental details for the complexes are summarized in Table 3.The programs SHELXL-97, 13 and ORTEP-3 14 were used within WinGX 15 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).
The 1 H and 13 C 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 δ 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 (J 3 ) between H-1, H-9 and C-19, confirming that C-19 in 1 is oxidized.
The structure presented for compound (1) was definitely confirmed by single crystal X-ray diffraction (Figure 2).
Compound (2) 1 H NMR spectrum presented high similarity with the one from compound (1), indicating a limonoid with a β-substituted furan ring, as shown by the two α-furan hydrogen at δ 7.39 (m) and the β-furan hydrogen at d 6.34 (m).An additional signal was observed at δ 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 δ 170.5 and a methyl group at δ 21.2 were observed by the DEPT experiment indicating the presence of an acetyl group in this compound.
These 1 H and 13 C 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).

Supplementary Information
Crystallographic data (excluding structure factors) for the structures in this paper has been deposited with the

Table 3 .
Crystal data and structure refinement.