New triterpene isolated from Eschweilera longipes (Lecythidaceae)

Abstracts

The phytochemical studies of Eschweilera longipes Miers (Lecythidaceae) have led to the identification of a new triterpene 3beta, 24-dihydroxyfriedelane, the known 1beta, 2beta, 3beta, 19beta-tetrahydroxyurs-12-en-28-oic acid (1beta-hydroxyeucaphic acid) besides the saponin sitosterol 3betaO-betaD-glucopyranoside. The structures were established from the IR, NMR and mass spectra data including 2D NMR experiments of natural substances and of the acetyl derivative of the new triterpene.

Eschweilera longipes; Lecythidaceae; triterpenoids

O estudo fitoquímico de Eschweilera longipes Miers (Lecythidaceae) conduziu a identificação de um novo triterpeno 3beta, 24-diidroxifriedelano, do ácido 1beta, 2beta, 3beta, 19beta-tetraidroxiursa-12-en-28-óico conhecido como ácido 1beta-hidroxieucáfico além da saponina 3betaO-betaD-glucopiranosilsitosterol. As estruturas foram estabelecidas com análise de dados espectrais de IV, massas e RMN incluindo experimentos 2D das substâncias naturais e do derivado acetilado do triterpeno novo.

Eschweilera longipes; Lecythidaceae; triterpenoides

New triterpene isolated from Eschweilera longipes (Lecythidaceae)

PATRÍCIA M. DA COSTA; MÁRIO G. DE CARVALHO

Departamento de Química, ICE, Universidade Federal Rural do Rio de Janeiro

23851-970 Seropédica, RJ, Brazil

^{Correspondence}

ABSTRACT

The phytochemical studies of Eschweilera longipes Miers (Lecythidaceae) have led to the identification of a new triterpene 3b, 24-dihydroxyfriedelane, the known 1b, 2b, 3b, 19b-tetrahydroxyurs-12-en-28-oic acid (1b-hydroxyeucaphic acid) besides the saponin sitosterol 3bO-bD-glucopyranoside. The structures were established from the IR, NMR and mass spectra data including 2D NMR experiments of natural substances and of the acetyl derivative of the new triterpene.

Key words:Eschweilera longipes, Lecythidaceae, triterpenoids.

RESUMO

O estudo fitoquímico de Eschweilera longipes Miers (Lecythidaceae) conduziu a identificação de um novo triterpeno 3b, 24-diidroxifriedelano, do ácido 1b, 2b, 3b, 19b-tetraidroxiursa-12-en-28-óico conhecido como ácido 1b-hidroxieucáfico além da saponina 3bO-bD-glucopiranosilsitosterol. As estruturas foram estabelecidas com análise de dados espectrais de IV, massas e RMN incluindo experimentos 2D das substâncias naturais e do derivado acetilado do triterpeno novo.

Palavras-chave: Eschweilera longipes, Lecythidaceae, triterpenoides.

INTRODUCTION

Lecythidaceae is a pantropical family (about 25 genera and 400 species) with the greatest concentration of genera in tropical South America (Brito 1986).

Species of this family have been reported as showing pharmacological activities and the chemical study of some species as Petersianthus macrocarpus, Barringtonia acutangula and Cereya arborea, allowed the identification of pentacyclic triterpenes, saponins, elagic acid and indolo[2,1-b]quinazolinic alkaloids (Pant and Rastogi 1979, Das and Mahato 1983, Pal et al. 1991, Massiot et al. 1992 and Bergman 1989).

Eschweilera longipes Miers is a tree that occurs in the north and north-east of Brazil and has been used in the wood industry and in construction. Only triterpenes have been found in the Eschweilera genera. Two previous papers report the isolation of ten known triterpenes along with sitosterol, stigmasterol, a-tocopherol and tocotrienol from E. longipes (Carvalho et al. 1998) and three pentacyclic triterpenoids which were isolated from the bark and leaves of E. rabeliana (Carvalho et al. 1995).

MATERIALS AND METHODS

GENERAL EXPERIMENTAL PROCEDURE

Mp's are uncorrected. NMR spectra were measured in Pyridine-d₆, MeOD₄ or CDCl₃ solutions and recorded on a Bruker (200 and 500 MHz for ¹H and 50.3 and 100 MHz for ¹³C, respectively) and on a GEOL (400 MHz for ¹H and 100 MHz for ¹³C) spectrometer using TMS as internal standard. High resolution mass spectra were obtained using a VG Auto Spec-300 spectrometer; FT-IR spectra were recorded in KBr disks on a Perkin-Elmer 1600 spectrometer. Chromatography was performed using Aldrich silica gel with suitable granulation for column and preparative TLC. The visualization of spots was done by UV (254 and 366 nm) and exposure to iodine vapor.

PLANT MATERIAL

The wood and leaves were collected in the Amapá State. A voucher specimen (nº 00358) is deposited in the Amapaense Herbarium HAMAB of the Museu Angelo Moreira da Costa Lima-IEPA, Macapá, Amapá, Brazil.

EXTRACTION AND ISOLATION

The dried leaves (0.7Kg) were extracted exhaustively by CH₂Cl₂ maceration at room temperature. The solvent was removed under vacuum to yield a residue (10.38g). This residue was chromatographed on silica gel column starting with CH₂Cl₂ and successive mixtures of CH₂Cl₂-EtOAc, EtOAc-MeOH and finally, MeOH as eluent to afford 70 fractions of 50 mL each. The 23-40 fractions, eluted with EtOAc-MeOH (9:1), gave a colourless solid (1, 70mg, mp 340ºC) which is insoluble in CDCl₃. The derivative 1a was prepared dissolving 1 in a mixture of pyridine and Ac₂O (1:1) and the solution was allowed to stand for 24h at room temperature. The usual work-up gave a residue which was dried under vacuum and crystallized from AcOEt to yield the diacetate (1a, 65mg, mp 318ºC).

The dried wood (1.0 Kg) was extracted exhaustively by MeOH maceration at room temperature. The solvent was removed under vacuum to yield a residue (47.7g). This residue was dissolved in MeOH:H₂O (8:2) and extracted with dichloromethane. The fraction CH₂Cl₂ was chromatographed on silica gel column using CH₂Cl₂ and successive mixtures of CH₂Cl₂-EtOAc, EtOAc-MeOH and finally, MeOH as eluent to afford 220 fractions of 50mL each. The 9-17 fractions, eluted with CH₂Cl₂, gave a colourless solid (2, 40mg, mp 285ºC) soluble in MeOH. The 70-82 fractions, eluted with EtOAc-MeOH (9:1), gave colourless solid (3, 40mg, mp 290ºC). The derivative 3a was prepared dissolving 3 in a mixture of pyridine and Ac₂O (1:1) and working up as usual.

RESULTS AND DISCUSSION

The chromatographic fractionation of the dichloromethane extract from the leaves of Eschweilera longipes afforded two triterpene, 1 and 2, besides the saponin sitosterol 3bO-bD-glucopyranoside (3).

The IR spectrum of 1 showed absorption bands attributed to hydroxyl (3450 cm^-1), (1100 and 1050 cm^-1) and very strong at 2950 and 2960 cm^-1 suggesting a terpenoid with primary and secondary alcohol. The difficulty to dissolve it in CDCl₃ led to prepare the acetyl derivative treating it with pyridine and Ac₂O (1:1).

The ¹H NMR spectrum of 1a displayed singlet signals for seven tertiary methyl groups of a pentacyclic triterpene and two signals at 1.96 (s, 3H) and 2.00 (s, 3H) of acetyl groups. The signals at 4.40 and 4.60 (d, J = 13 Hz) are typical of two methylene hydrogens. The H-3 was represented by the signal at 4.94 (br d, J = 2.4 Hz). The comparative analysis of HBBD and DEPT ¹³C NMR spectra was used to recognize the signals corresponding to six quaternary carbons, two monoxigenated (^dCH74.5 and ^dCH265.1) besides signals of seven methyl, eleven methylene, four methyne groups and two acetyl groups (d170.1, 170.0, 21.2 and 21.1). Those data allowed to propose the molecular formula C₃₀H₅₀O₂ (O=C-CH₃)₂ that was confirmed by HRMS with M⁺ 528.41740 Da [calcd for C₃₀H₃₅(O₂CCH₃)₂ 528.41808]. Thus, these spectral data, the 2D experiments (¹H-¹H-COSY, ¹H-¹³C-COSY-ⁿJ_CH, n=1,2,3) and comparison with ¹³C NMR spectroscopic values described in the literature for acetyl friedelinol (Carvalho et al. 1995, Mahato and Kundu 1994 and Ahmad and Atta-ur-Rahman 1994) show the absence of signal at 15.7 (CH₃-24) in the fridelinol. This observation and the difference of the C-5 and C-6 chemical shift of 1 and those of acetyl friedelinol led to locate one acetyl group at C-24. The prominent peaks in the HRMS at m/z 455 (1b, 21,5%, M- CH₂OCOCH₃), 395 [1c, 8,3%, M- (CH₂OCOCH₃ + HOCOCH₃)], 344 (1d, 33,9%, C₂₅H₄₄), 274 (1e, 17,3%, C₂₀H₃₄) 255 (1f, 15%) and 205 (1g, 23.8%, C₁₅H₂₅), Figure 1, also suggested the presence of two acetyl groups in the C-3 and C-24 carbons. The NOE observed between H-24/H-25, H-24/H-23, H-24/H-1 in the NOESY spectra of 1a (3b, 24-diacetylfriedelane) was used to confirm the structure of the new triterpene (1) as 3b, 24-dihydroxyfriedelane, Figure 1. The complete ¹H and ¹³C NMR (1D and 2D) assignments of 1 and 1a are described in Table I.

Fig. 1
- Structures for compounds isolated from E. longipes, acethyl derivatives and forprominent peaks in the HRMS.

Compound 2 was characterized as 1b-hydroxyeucaphic acid by analysis of IR, NMR ¹H and ¹³C (HBBD and DEPT) and 2D experiments (¹H-¹H-COSY, ¹H-¹³C-COSY, ⁿJ_CH, n=1,2,3) and EI-MS spectra including comparison of the ^dH chemical shifts in pyridine registered in the literature (Guang et al. 1989). The NOE signal between H-1/H-5, H-1/H-9, H-2/H-24, H-2/H-25, H-3/H-24, H-3/H-23, H-11/H-25, H-18/H-29, H-18/H-12 and H-12/H-29 observed in the NOESY spectrum were used to confirm the structure of 2 as 1b, 2b, 3b, 19b-tetrahydroxyurs-12-en-28-oic acid. The better resolution of the ABC system (H-2, H-1 and H-3) and the absence of ¹³C NMR data of 2 in the literature led us to make the complete assignment of ^dH and ^dC in methanol. The EIMS spectra data were used to confirm the structure. ^dH (MeOD₄, 200 MHz): 3.39 (d, 8.4Hz, H-1), 3.63 (dd, 8.4 and 3.2 Hz, H-2), 3.45 (d, 3.2 Hz, H-3), 1.3 (m, H-5), 2.1 (m, H-9), 5.20 (brs, H-12), 2.47 (s, H-18), 0.96 (s, H-23), 0.87 (s, H-24), 1.00 (s, H-25), 0.78 (s, H-26), 1.34 (s, H-27), 1.18 (s, H-29), 0.92 (d, 6.6Hz, H-30); ^dC (MeOD₄, 50.3 MHz): 79.9 (C-1), 70.4 (C-2), 79.3 (C-3), 39.1 (C-4), 48.2 (C-5), 18.0 (C-6), 32.8 (C-7), 41.2 (C-8), 48.0 (C-9), 37.4 (C-10), 25.2 (C-11), 129.3 (C-12), 137.3 (C-13), 43.2 (C-14), 29.4 (C-15), 28.3 (C-16), 48.2 (C-17), 53.3 (C-18), 72.2 (C-19), 41.2 (C-20), 26.9 (C-21), 37.6 (C-22), 27.7 (C-23), 21.0 (C-24), 11.6 (C-25), 16.4 (C-26), 23.5 (C-27), 180.0 (COOH), 25.3 (C-29), 15.2 (C-30); EIMS, m/z (%): 504(10%), 264(35%), 246(15%), 201(45%), 173(20%) and 146(100%).

The spectrometric analysis of IR, ¹H and ¹³C (PND and DEPT) NMR of 3 including comparison with literature data (Chaurasia and Wichtl 1987) were used to identify the saponin 3 as sitosterol 3bO-bD-glucopyranoside, Figure 1.

ACKNOWLEDGMENTS

The authors are grateful to CNPq, CAPES and FAPERJ for research fellowships and financial support. We thank to CENAUREMN-FINEP (Centro Nordestino de Aplicação e Uso de Ressonância Magnética Nuclear), UFCE, Fortaleza, Ceará, Brazil for ¹H (500 MHz) and ¹³C (125 MHz) NMR spectra. We thank to UENF (Universidade Estadual do Norte Fluminense) for ¹H (400 MHz), ¹³C (100 MHz), NOESY and MS spectra and to UNICAMP for HR-MS spectra.

Manuscript received on May 22, 2002;

accepted for publication on November 17, 2002;

presented by FERNANDO GALEMBECK

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Publication Dates

• Publication in this collection
  17 Apr 2003
• Date of issue
  Mar 2003

History

• Accepted
  17 Nov 2002
• Received
  22 May 2002