A Novel 3,9-(1,2,3-Trioxocine)-Type Steroid of Rauia nodosa (Rutaceae)

A new natural product, a 3,9-(1,2,3-trioxocine)-type steroid, named rauianodoxy (6), was isolated from Rauia nodosa, together with five steroids: sistostenone (1), stigmastenone (2), sitosterol (3), stigmasterol (4) and ergosterol peroxide (5), one coumarin, O-geranylosthenol (7), and three alkaloids, N-methylflindersine (8), zantobungeanine (9) and veprissine (10). Compounds 5–8 were isolated for the first time in the genus Rauia. These compounds were characterized on the basis of their spectral data, mainly one and two-dimensional NMR, and mass spectra, also involving comparison with the literature data. Theoretical studies at the DFT level reveal structural parameters for the 1,2,3-trioxole bridge compatible with known structures containing a similar group.


Introduction
The Rutaceae family is represented by 155 genera and 1600 species distributed in the tropical and temperate regions of the World.This family is commonly found in Tropical America, South Africa, Asia and Australia [1].The Rutaceae family is characterized by an abundance of anthranilic acid derived alkaloids, coumarins, limonoids and flavonoids, with different types of biological activities [2].The biological importance of compounds from the Rutaceae family can be highlighted by the Pilocarpus genus that synthesizes large concentrations of the alkaloid pilocarpine used to treat glaucoma [3].
The Rauia genus, taxonomically found in the Rutaceae family, Rutoideae subfamily, Galipeae tribe (formerly Cusparieae) and Galipeinae subtribe (before Cuspariinae) [4] has been little studied.This genus includes five species: R. spicata, R. subtruncata, R. nodosa, R. prancei and R. resinosa.The last three species can be found in the North, Northeast and Southeast Brazil.The phytochemical study of R. resinosa revealed the presence of alkaloids and coumarins [5].
In the present paper, we describe the isolation and characterization of a novel 3,9-(1,2,3-trioxocine)type steroid named rauianodoxy (6), together with nine known compounds: five steroids, a mixture of sistostenone (1) and stigmastenone (2), a mixture of sitosterol (3) and stigmasterol (4) and ergosterol peroxide (5), one coumarin, O-geranylosthenol (7), and three alkaloids, a mixture of N-methylflindersine (8) and zantobungeanine (9), and veprissine (10).All these natural products were isolated for the first time in this species and the 3,9-(1,2,3-trioxocine)-type steroid named rauianodoxy ( 6) is mentioned for the first time on record.The compounds 5-8 were isolated for the first time in the Rauia genus.The structures were established on the basis of spectral data, mainly 1 H and 13 C (1D and 2D) NMR spectra, mass spectrometry and by comparison with literature data.Complementary results describing structural parameters of the novel steroid were obtained from calculations at the DFT level.
Table 1. 1 H-(500 MHz) and 13 C-(125 MHz) NMR of rauianodoxy (6), including results obtained by heteronuclear 2D shift-correlated HSQC and HMBC, in CDCl3 as solvent and TMS used as internal reference.Chemical shifts (δ, ppm) and coupling constants (J, Hz, in parenthesis).Scheme 1. Proposed fragmentation mechanisms to justify peaks (positive modes, only peaks classified as principal) obtained in a methanol/water solution of compound 6.

HSQC
The relative stereochemistry of rauianodoxy ( 6) was determined from the coupling constants of relevant hydrogen atoms and from the observed 1 H-1 H-NOESY, which showed cross-peaks assigned to dipolar interaction (spatial proximity, as shown in Figure 2).Thus, the observed hydrogen H-8 spatial interactions with both methyl groups 3H-19 and 3H-18 indicated that these hydrogen atoms are in    The results of the extensive application of 1D and 2D NMR spectral techniques were also used to confirm the structure and to establish the complete 1 H and 13 C chemical shift assignments of 6 (Table 1).
The HRESIMS obtained for the compound 6, essential to postulate the presence of one additional oxygen atom and to define the molecular formulae C28H44O3 by [ A few natural ozonides (structures containing the 1,2,3-trioxocine group) as precursors in ozonolysis of olefins, have been discovered in higher plant species [16][17][18].However, plant ozonides could be formed from cuticle olefins with ozone from the air, or alternatively inside cells, as ozone may enter the mesophyll of the plants [19,20] causing necrosis of the leaves.
Compound 6 was mainly characterized by one and two-dimensional NMR and mass spectra, but theoretical studies at the DFT level have also been used.A model was performed to access structural details of the suggested structure and to verify the stability of the 1,2,3-trioxocine bridge connecting two atoms, C3 and C9, spatially not very close to each other in compound 6.The modeling results revealed that the proposed bridge is in fact stable, with bonding distances comparable to those of known compounds (models) containing similar bridges.In the optimized LSDA/pBP86/DN* compound 6 structure, the 1,2,3-trioxocine bridge is part of two adjacent eight-membered rings which adopt two different conformations: a distorted chair-like conformation and a distorted boat-like conformation (Figure 3).The C(3)-O(1) distance was 1.470 Å and C(9)-O(3) distance was 1.520 Å. Structures containing the proposed 1,2,3-trioxocine bridge are scarce, so it is difficult to obtain structural data of known compounds to compare with the proposed structure; the majority of 1,2,3-trioxocine bridge-containing species reported in the literature came from reactions between O3 and small alkenes and cycloalkenes, which are quite unstable.Therefore, in order to verify the adequacy of the modeled structure, we modeled by the same procedure the structures of the known products of the reactions of cyclopentene and cyclohexene with O3 [21], both containing trioxole groups.The C-O distances in the compound that results from the reaction between O3 and cyclopentene (compound 11 in Figure 1), after geometry optimization, were 1.439 and 1.440 Å.In the case of the cyclohexene derivative (compound 12 in Figure 1), these distances were 1.425 and 1.442 Å.The longer bond distances obtained for compound 6 could be attributed to the long distance between the atoms to which O( 1 3) angle is 112.57°, a value compatible with a sp 3 character for the oxygen atoms.Thus, the structure of the new 3,9-(1,2,3-trioxocine)-type steroid isolated from Rauia nodosa was here established for the first time as a novel natural product and named rauianodoxy (6).

General Experimental Procedures
ESI-MS (high resolution) mass spectra were obtained by using a ESI-TOF-MS Shimadzu mass spectrometer (SHIMADZU, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, Brazil), using the positive ion mode of analysis.Chromatographic purifications were carried out by using silica gel 60 (0.063-0.200 mm). 1 H-and 13 C-NMR spectra were measured on a Brüker Ultrashield 500 Plus spectrometer (BRÜKER, Universidade Federal Rural do Rio de Janeiro, Seropédica, Brazil), operating at 500 ( 1 H) and 125 ( 13 C) MHz.CDCl3 was used as solvent with TMS as internal reference.Chemical shifts are given in the δ scale (ppm) and coupling constants (J) in Hz.One dimensional (1D) 1 H-and 13 C-NMR spectra were acquired under standard conditions by using a direct detection 5 mm 1 H/ 13 C dual probe.Standard pulse sequences were used for two dimensional spectra by using a multinuclear inverse detection 5 mm probe with field gradient.
A theoretical study was implemented to verify the viability of the molecular structure containing the 1,2,3-trioxocine bridge.The Spartan'10 (Wavefunction, Inc., Universidade Federal Rural do Rio de Janeiro, Seropédica, Brazil) program was used in all steps of the theoretical study.The structure was initially submitted to a Monte Carlo conformational search with the MMFF94 molecular mechanics method [22].This procedure generated a number of conformers and the geometry of the most stable conformer was then fully reoptimized in two steps: first with the semiempirical molecular orbital PM3 method [23], and finally with the pBP86/DN* DFT method.The pBP86, composed of the Becke 1988 exchange functional [24] and the Perdew 86 correlation functional [25], provides a perturbative implementation of the non-local Becke-Perdew model.The DN* basis set is derived from numerical atomic solutions, incorporating polarization functions on heavy (i.e., non-hydrogen) atoms, which adds some flexibility within the basis set.

Plant Material
Stems of Rauia nodosa (Rutaceae) were collected in May 2011 at Vale Cia Reserve, Linhares, Espírito Santo, Brazil.The voucher specimen of R. nodosa was deposited at Vale Cia herbarium, under the code CRVD-3301.

Extraction and Isolation
Stems of Rauia nodosa (Rutaceae), immediately after collection, were dried at room temperature until a constant weight (about one week) and ground in hammer mills.The dried and powdered material (4.8 kg) was extracted with hexane.The obtained solutions (48 L) were distilled under reduced pressure in a rotary evaporator furnishing 4.47 g of crude hexane extract.
The hexane extract was chromatographed over silica gel column with a gradient of hexane/ethyl acetate to afford twenty one fractions.Fraction 9 (835.6 mg) was rechromatographed over a silica gel column with a gradient of hexane/ethyl acetate furnishing six fractions.Fraction 9.2 (62.8 mg) was rechromatographed over a silica gel column with a gradient of hexane/ethyl acetate affording the compounds 1+2 (5.1 mg).Fraction 9.5 (260.9 mg) was rechromatographed over a silica gel column with a gradient of hexane/ethyl acetate furnishing seven fractions and 3+4 (11.5 mg) and the fraction 9.5.6 (55.6 mg) was rechromatographed over a silica gel column with a gradient of hexane/ethyl acetate yielding compound 7 (29.8mg).
/z 428 + m/z 428+ Na+ β-orientations (axial-axial); the relative configuration S assigned to the CH-20 carbon atom was based on the special interaction between 3H-18 and H-20, as shown in Figure2.
) and O(3) atoms are attached, C(3) and C(9), which are part of the rigid steroid ring system, and also to 1,3-diaxial steric interactions between the O(1) and O(3) atoms with H atoms. Accordingly, in a simplified model of compound 6, containing only rings A and B (compound 13 in Figure 1), modeled by the same procedure, the C(3)-O(1) and C(9)-O(3) distances were also longer, 1.464 Å was 1.475 Å, respectively.The 1,2,3-trioxocine bridge is not symmetrical in the steroid derivative, with the O(1)-O(2) distance is equal to 1.507 Å and the O(2)-O(3) distance is equal to 1.471 Å.The corresponding bond distances were almost identical in the model compound 11, 1.474 and 1.470 Å, but were clearly different in the case of compounds 12 and 13, 1.579 and 1.423 Å, 1.538 and 1.444 Å, respectively.In compound 6, the O(1)-O(2)-O(