Diterpene and other Constituents from Stemodia maritima ( Scrophulariaceae )

Um novo diterpeno, (5S*,8S*,9R*,10S*)-11β,12β-epoxi-9α-hidróxi-19(4→3)abeo-abieta3,13-dieno-19,18-olideo, e as substâncias conhecidas estemodina, D-manitol, ácido betulínico, uma mistura de 3β-O-β-D-glicopiranosil-β-sitosterol e 3β-O-β-D-glicopiranosilestigmasterol, e 5,7,4’-triidróxi-3,8,3’-trimetoxiflavona, foram isolados das folhas e talos de Stemodia maritima. A elucidação estrutural de todas as substâncias baseou-se na interpretação de dados espectrais, principalmente RMN (1D e 2D) e espectrometria de massa (EM), envolvendo comparação com valores descritos na literatura.


Introduction
Stemodia Benth. is one of Scrophulariaceae genus and occurs in tropical and subtropical regions of the world. 1 Although Stemodia comprises about 40 species, the chemical investigation of this genus is restricted to five species 4 from which flavonoids, 2,3 labdane diterpenes 4,5 and diterpenes derivatives with a rare tetracyclic skeletal, named stemodane, were isolated.This later class of diterpenes seems to be chemomarkers of Stemodia. 6. maritima Linn. is a very common shrub that widely grows in Northeast Region of Brazil, near the sea coast, where it is known as "melosa".It has been used to treat stomachache, dropsy and swelling by local population, although toxic symptoms was reported in cattle. 7][10] The chemical composition and larvicidal activity of its essential oil were recently reported. 11n the course of the phytochemical investigation of S. maritima from the Northeast Region of Brazil, herein we report the non-volatile composition of this species.A new diterpene, (5S*,8S*,9R*,10S*)-11β,12β-epoxy-9α-hydroxy-19(4→3)abeo-abieta-3,13-diene-19,18-olide (1), together with the known compounds stemodin (2) (Figure 1), D-mannitol, betulinic acid, a mixture of 3β-O-β-D-glucopyranosyl-β-sitosterol and 3β-O-β-Dglucopyranosylstigmasterol, and 5,7,4'-trihydroxy-3,8,3'trimethoxyflavone were isolated from the leaves and stems of this plant.Structural elucidation of all compounds was based on the interpretation of spectral data, meanly NMR (1D and 2D) and MS, and comparison with literature data.
The 1 H NMR spectrum (  The aforementioned data were coherent with a non aromatic abietane-type diterpene that displays an epoxy ring, a tertiary hydroxyl group, an α,β-unsaturatedγ-lactone system and two double bonds, having some similarities with the diterpene triptolide. 12he location of these functions in the abietane skeleton was deduced through additional HMBC analysis (Table 1   is generally present in abietane-type diterpenoids. 13Finally, the butenolide ring involving the carbons C-3, C-4, C-18 and C-19 was located by the correlations of the methylene hydrogens at d H 4.72 and 4.68 (2H-19) with C-4 (d C 162.0, 2 J), C-3 (d C 125.3, 3 J) and C-18 (d C 173.9, 3 J).
Compound 2 was obtained as colorless crystal and its molecular formula C 20 H 34 O 2 was deduced by EIMS ([M] + , m/z 306) and 1 H and 13 C NMR analysis.Its IR spectrum showed hydroxyl absorption at ν max 3311 cm -1 .All spectral data were in accordance with the structure of the stemodin (2), a stemodane-type diterpene previously isolated from Stemodia species. 6,8ompound 2 was submitted to acetylation with pyridine and acetic anhydride (see Experimental section), yielding 2a 8 (Figure 1).The 1D and 2D NMR spectral data of 2 and of its acetyl derivative (2a) were also used to complete 1 H and 13 C chemical shifts described in Table 2. Dipolar interactions observed from 1 H-1 H-NOESY analysis of 2 are summarized in Figure 3.
The other isolated compounds were identified on the basis of their spectral analysis and comparison with the literature data.

General experimental procedures
Melting points were obtained from a Mettler FP82HT apparatus and are uncorrected.IR spectra were recorded using a Perkin Elmer 1000 FT-IR spectrophotometer.Optical rotations were measured on a Perkin Elmer 341 polarimeter.High resolution electrospray ionization mass spectra (ESI-MS/MS), in positive mode, was performed on a QTOF Micromass spectrometer (QqTOF, Micromass-UK). 1 H and 13 C NMR spectra were recorded on a Bruker Avance DRX-500 (500 MHz for 1 H and 125 MHz for 13 C); chemical shifts are given in ppm relative to residual CHCl 3 (7.27 and 77.23 ppm).Silica Gel 60 (Merck, 230-400 mesh) was used for analytical TLC.Silica gel 60 (Merck, 60 F 254 , 0.2 mm) was used for column chromatography.All compounds were visualized on TLC by spraying with vanillin/perchloric acid/EtOH followed by heating.

Extraction and isolation
The fresh stems (200.0 g) of S. maritima were exhaustively extracted with ethanol, at room temperature, to obtain a crude material, composed by a precipitate, which was recrystalized from methanol to give D-mannitol 14 (80.0 mg, 0.04%).
The structures of known compounds were established by 1D 1 H and 13 C ({ 1 H} and DEPT) and 2D 1 H-1 H-COSY, HSQC and HMBC NMR spectral data (Table 2) and by comparison of their spectroscopy data with those reported in the literature. 6
Magnética Nuclear (CENAUREMN), Universidade Federal do Ceará (UFC), Fortaleza-CE, for the support to perform of the NMR (1D and 2D) spectra and to the Laboratório Thomson de Espectrometria de Massas, IQ-Unicamp, Campinas-SP, Brazil, for the high resolution mass spectra.

Figure 2 .
Figure 2. Fragments postulated to justify some of principal peaks observed in EIMS of 1.
was collected during the flowering stage in September 2006 along the Flexeiras Beach, Ceara Cost, Northeast of Brazil.The plant was identified by Dr. F. S. Cavalcanti and Prof. E. P. Nunes from the Herbário Prisco Bezerra (EAC), Universidade Federal do Ceará, Fortaleza, Brazil, where a voucher specimen (# 38483) is deposited.

Table
1) revealed the presence of an isopropyl group (d H 1.03, d, J 6.8 Hz, 3H-16; d H 1.05, d, J 6.8 Hz, 3H-17; d H 2.62, sep, J 6.8 Hz, H-15), a methyl group at d H 1.01 (3H, s, 3H-20) attached to quaternary carbon, two oxygenated methine hydrogens at d H 3.66 (dd, J 2.5 and 1.9 Hz, H-11) and d H 4.40 (brs, H-12), compatible with the presence of an epoxy ring, two deshielded hydrogen from a oxygenated methylene group at d H 4.72 (brdd, J 17.2 and 1.6 Hz, H-19α) and d H 4.68 (brdd, J 17.2 and 1.6 Hz, H-19β), and an olefinic hydrogen at d H 5.24 (brd, J 5.0 Hz, H-14).Analysis of BB and DEPT 135° 13 C NMR spectra (Table 1) revealed 20 lines, in accordance with the molecular formula C 20 H 26 O 4 .From these data it is possible to deduce the presence of the six non-protonated carbons: one carbonyl group (d C 173.9), three sp 2 carbons, one oxygenated sp 3 carbon and one non-oxygenated sp 3 carbon.Additionally, it was observed six methine carbons, including two sp 3 oxygenated at d C 66.6 and 59.7 and one sp 2 at d C 121.8; five methylene carbons, one of them oxygenated at d C 70.4, and three methyl carbons.