Diterpene and other constituents from Stemodia maritima (Scrophulariaceae)

SHORT REPORT

Diterpene and other constituents from Stemodia maritima (Scrophulariaceae)

Francisco E. A. Rodrigues^I; Jefferson Q. Lima^{I, II}; Maria da Conceição F. de Oliveira^I; Jackson N. Vasconcelos^I; Gilvandete M. P. Santiago^{I, III}; Jair Mafezoli^I; Raimundo Braz-Filho^IV; Angela M. C. ArriagaI, ^*

^ICurso de Pós-Graduação em Química, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, CP 6036, 60451-970 Fortaleza-CE, Brazil

^IICurso de Engenharia Ambiental, Instituto Federal do Ceará, Campus Juazeiro do Norte, 63040-000 Juazeiro do Norte-CE, Brazil

^IIIDepartamento de Farmácia, Universidade Federal do Ceará, 60430-370 Fortaleza-CE, Brazil

^IVCentro de Ciências e Tecnologias, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28013-603 Campos dos Goytacazes-RJ, Brazil

ABSTRACT

A new diterpene, (5S*,8S*,9R*,10S*)-11β,12β-epoxy-9α-hydroxy-19(4→3) abeo-abieta-3,13-diene-19,18-olide, together with the known compounds stemodin, D-mannitol, betulinic acid, a mixture of 3β-O-β-D-glucopyranosyl-β-sitosterol and 3β-O-β-D-glucopyranosylstigmasterol and 5,7,4'-trihydroxy-3,8,3'-trimethoxyflavone were isolated from the leaves and stems of Stemodia maritima. Structural elucidation of all compounds was based on interpretation of spectral data, mainly NMR (1D and 2D) and MS, including comparison with values described in the literature.

Keywords:Stemodia maritima, Scrophulariaceae, diterpenes, steroids, flavonoids

RESUMO

Um novo diterpeno, (5S*,8S*,9R*,10S*)-11β,12β-epoxi-9α-hidróxi-19(4→3) abeo-abieta-3,13-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 species⁴ 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

S. 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 Stemodane diterpenes, including glycosides derivatives, possessing antiviral and cytotoxic properties were isolated from this species.^6,8-10 The chemical composition and larvicidal activity of its essential oil were recently reported.¹¹

On 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-D-glucopyranosylstigmasterol, 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.

Results and Discussion

The molecular formula of compound 1 was established through HR-ESI-MS, which showed the quasi-molecular ion peak at m/z 331.1799 ([M+1]⁺, corresponding to the molecular formula C₂₀H₂₆O₄ and indicating eight degrees of unsaturation. EIMS from 1 showed the molecular ion peak at m/z 330 (C₂₀H₂₆O₄, 5%) and additional peaks at m/z 315 (C₁₉H₂₃O₄, 7%) and m/z 287 [C₁₇H₁₉O₄, 100%], attributed to fragments 1a and 1b, respectively (Figure 2). The presence of a hydroxyl absorption (ν_max 3433 cm^-1) and an α,β-unsatured-γ-lactone (ν_max 1729 cm^-1) was inferred from its IR spectrum.

The ¹H NMR spectrum (Table 1) revealed the presence of an isopropyl group (δ_H 1.03, d, J 6.8 Hz, 3H-16; δ_H 1.05, d, J 6.8 Hz, 3H-17; δ_H 2.62, sep, J 6.8 Hz, H-15), a methyl group at δ_H 1.01 (3H, s, 3H-20) attached to quaternary carbon, two oxygenated methine hydrogens at δ_H 3.66 (dd, J 2.5 and 1.9 Hz, H-11) and δ_H 4.40 (brs, H-12), compatible with the presence of an epoxy ring, two deshielded hydrogen from a oxygenated methylene group at δ_H 4.72 (brdd, J 17.2 and 1.6 Hz, H-19α) and δ_H 4.68 (brdd, J 17.2 and 1.6 Hz, H-19β), and an olefinic hydrogen at δ_H 5.24 (brd, J 5.0 Hz, H-14).

Analysis of BB and DEPT 135° ¹³C NMR spectra (Table 1) revealed 20 lines, in accordance with the molecular formula C₂₀H₂₆O₄. From these data it is possible to deduce the presence of the six non-protonated carbons: one carbonyl group (δ_C 173.9), three sp² carbons, one oxygenated sp³ carbon and one non-oxygenated sp³ carbon. Additionally, it was observed six methine carbons, including two sp³ oxygenated at δ_C 66.6 and 59.7 and one sp² at δ_C 121.8; five methylene carbons, one of them oxygenated at δ_C 70.4, and three methyl carbons.

The aforementioned data were coherent with a non aromatic abietane-type diterpene that displays an epoxy ring, a tertiary hydroxyl group, an α,β-unsaturated-γ-lactone system and two double bonds, having some similarities with the diterpene triptolide.¹²

The location of these functions in the abietane skeleton was deduced through additional HMBC analysis (Table 1), which revealed the following long-range correlations: the epoxy hydrogens at δ_H 3.66 (H-11) with C-13 (δ_C 140.1, ³J) and at δ_H 4.4 (H-12) with C-13 (δ_C 140.1, ²J) and C-14 (δ_C 121.8, ³J); the isopropyl hydrogen at δ_H 2.62 (H-15,) with C-13 (δ_C 140.1, ²J) and C-12 (δ_C 66.6, ³J); the olefin hydrogen at δ_H 5.24 with C-12 (δ_C 66.6, ³J), C-13 (δ_C 140.1, ²J) and C-15 (δ_C 28.6, ³J). The position of the hydroxyl group at C-9 was established based in the correlations of this oxymethine carbon (δ_C 67.9) with the hydrogen of the methyl group (3H-20, δ_H 1.01, ³J), which is generally present in abietane-type diterpenoids.¹³ Finally, 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 δ_H 4.72 and 4.68 (2H-19) with C-4 (δ_C 162.0, ²J), C-3 (δ_C 125.3, ³J) and C-18 (δ_C 173.9, ³J).

The relative configuration of 1 (Figure 3) was assigned by the analysis of the ¹H-¹H-NOESY spectrum. The β-orientation of the epoxy function (11,12β-epoxide) was determined by the dipolar interactions of the hydrogen at δ_H 3.66 (H-11) with 2H-1 (δ_H 1.77 and 1.36). In addition, the methyl signal at δ_H 1.01 (3H-20) exhibited cross-peaks with the hydrogens at δ_H 2.86 (H-8), δ_H 2.20 (H-2β) and δ_H 1.62 (H-6β). The hydrogen at δ_H 2.51 (H-5) showed dipolar interaction with the hydrogens at δ_H 1.77 (H-1α), 1.67 (H-6α) and 1.07 (H-7α). Based on these correlations, the hydroxyl group at C-9 was established at α position (Figure 3). Therefore, all these data allowed to establish the structure of 1 as (5S*,8S*,9R*,10S*)-11β,12β-epoxy-9α-hydroxy-19(4→3) abeo-abieta-3,13-diene-19,18-olide.

Compound 2 was obtained as colorless crystal and its molecular formula C₂₀H₃₄O₂ was deduced by EIMS ([M]⁺, m/z 306) and ¹H and ¹³C 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.^6,8

Compound 2 was submitted to acetylation with pyridine and acetic anhydride (see Experimental section), yielding 2a⁸ (Figure 1). The 1D and 2D NMR spectral data of 2 and of its acetyl derivative (2a) were also used to complete ¹H and ¹³C chemical shifts described in Table 2. Dipolar interactions observed from ¹H-¹H-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.

Experimental

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). ¹H and ¹³C NMR spectra were recorded on a Bruker Avance DRX-500 (500 MHz for ¹H and 125 MHz for ¹³C); chemical shifts are given in ppm relative to residual CHCl₃ (7.27 and 77.23 ppm). Silica Gel 60 (Merck, 230-400 mesh) was used for analytical TLC. Silica gel 60 (Merck, 60 F₂₅₄, 0.2 mm) was used for column chromatography. All compounds were visualized on TLC by spraying with vanillin/perchloric acid/EtOH followed by heating.

Plant material

S. maritima 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.

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-mannitol¹⁴ (80.0 mg, 0.04%).

The aqueous extract obtained after the essential oil extraction (hydrodistillation) of the fresh stems of S. maritima was submitted to liquid-liquid partition with hexane/MeOH (3:7). The hexane fraction (340.0 g) was submitted to column chromatography on silica gel column, using a gradient solvent system of hexane and CH₂Cl_2. Chromatography of the subfraction hexane (380.0 mg) using hexane/EtOAc mixtures with increasing polarity yielded betulinic acid¹⁵ (8.5 mg, 0.0025%). Successive flash chromatography of CH₂Cl₂ subfraction (2.0 g) using 0-100% CH₂Cl₂/EtOAc provided a mixture of 3β-O-β-D-glucopyranosyl-β-sitosterol and 3β-O-β-D-glucopyranosylstigmasterol¹⁶ (8.2 mg, 0.0024%).

After extraction of the essential oils from the leaves of S. maritime by hydrodistillation, the aqueous extract was subjected to liquid-liquid partition with ethyl acetate. The organic fraction (4.0 g) was chromatographed over silica gel with CHCl₃, EtOAc and MeOH to afford three subfractions F1-F3. Successive flash column chromatography of F1 (1.2 g), previously eluted from CHCl₃, yielded 2 (45.3 mg, 1.13%) after elution with CHCl₃/hexane 7:3. From these same column, fraction CHCl₃/hexane 9:1 (180.0 mg) was also obtained and rechromatographed over silica gel using the same eluent system to afforded 5,7,4'-trihydroxy-3,8,3'-trimethoxyflavone¹⁷ (6.5 mg, 0.0019%) and 1 (15.6 mg, 0.39%).

(5S*,8S*,9R*,10S*)-11β,12β-epoxy-9α -hydroxy-19(4→3)abeo-abieta-3,13-diene-19,18-olide (1)

Crystalline Solid; mp 264.6-266.5 °C; IR (film, KBr) ν_max/ cm^-1: 3433, 2962, 2866, 1729, 1663, 1453, 1344, 1036; HREIMS, m/z 331.1799, required m/z 331.1909; [α]²⁵_D = -12.9^° (c 1.0, CHCl₃).

Stemodin ( 2 )

Crystalline Solid; mp 189.9-192.4 °C; IR (film, KBr) ν_max/ cm^-1: 3311, 2954, 1463,1367, 1217,1032; EIMS, m/z 306 (M^+.), 291, 288, 273, 232, 217, 161, 94.

The structures of known compounds were established by 1D ¹H and ¹³C ({¹H} and DEPT) and 2D ¹H-¹H-COSY, HSQC and HMBC NMR spectral data (Table 2) and by comparison of their spectroscopy data with those reported in the literature.⁶

Acetylation of 2

To a solution of compound 2 (24.0 mg) in pyridine (0.5 mL) were added Ac₂O (1.0 mL) and catalytic amount of DMAP. The mixture was stirred for 5 h at room temperature. Subsequent workup afforded a residue that was chromatographed using hexane/CHCl₃ (1:1), hexane/CHCl₃ (1:3) as eluent to yield compound 2a⁸ (12.0 mg, 50.0%) as a colorless solid.

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br, as PDF file.

Acknowledgments

The authors acknowledge the financial support provided by Brazilian Agencies CNPq, CAPES, FUNCAP and PRONEX, and are grateful to Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ-RJ-Brazil) for the fellowship to visitant research and grants and to the Centro Nordestino de Aplicação e Uso da Ressonância 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.

Received: June 24, 2009

Web Release Date: March 25, 2010

Supplementary Information

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Publication Dates

• Publication in this collection
  14 Oct 2011
• Date of issue
  2010

History

• Received
  24 June 2009