A New ent-Clerodane Diterpene from Hymenaea courbaril var. altissima

Short Report

A New ent-Clerodane Diterpene from Hymenaea courbaril var. altissima

Raquel T. Nogueira^a, Rosana A. Giacomini^a, George J. Shepherd^b and Paulo M. Imamura^*a

^a Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970, Campinas - SP, Brazil

^b Instituto de Biologia, Universidade Estadual de Campinas, CP 6109, 13083-970, Campinas - SP, Brazil

Da casca do tronco de Hymenaea courbaril var. altissima (Ducke) foi isolado um novo ent-clerodano na forma de éster metílico, após esterificação com diazometano, o (-)-(5R,8S,9S,10R)-clerod-3-en-15-oato de metila. Foram isolados também dois ent-labdanos conhecidos: o (-)-copalato de metila e (-)-eperuato de metila. Do extrato AcOEt do epicarpo foi isolado, após esterificação com diazometano, o (-)-zanzibarato de metila. As estruturas dos compostos foram elucidadas com base na análise de dados espectroscópicos.

A new ent-clerodane and two known ent-labdanes were isolated from the barks of Hymenaea courbaril var. altissima (Ducke). The new diterpene was identified, after esterification with diazomethane, as methyl (-)-(5R,8S,9S,10R)-clerod-3-en-15-oate and known diterpenes were identified as methyl (-)-copalate and methyl (-)-eperuate. From the EtOAc extract of seed pods a known methyl (-)-zanzibarate was isolated. The structures of isolated compounds were elucidated based on spectroscopic studies.

Keywords: Hymenaea courbaril var. altissima, ent-clerodane, ent-labdane

Introduction

Hymenaea is a genus widely distributed from Central to South America and is known popularly as "jatobá", "jataí" or "jutaí".¹ The wood is used for woodwork and the resin extract is locally used as a remedy for many purposes.² The chemical composition of the resin obtained from some species of Hymenaea is already known in the literature³ and recently we reported the isolation of two new ent-clerodane derivatives from the seed pods resin of H. courbaril var. stilbocarpa.⁴ We now describe the isolation of two known diterpenes (1, 2) and a novel ent-clerodane (3) from the ethyl acetate extract of H. courbaril var. altissima (Ducke) barks, which grows mainly in a subtropical forest on the Atlantic Coast,⁵ and the isolation of a known diterpene (9) from the ethyl acetate extract of its seed pods. Another five compounds (4-8) were also identified by GC/MS analyses.

Results and Discussion

The ethyl acetate extract of H. courbaril var. altissima barks was esterified with diazomethane and submitted to a combination of column chromatography (silica gel) and preparative TLC on SiO₂/AgNO₃ (5%, w/w), to afford a new diterpene, methyl (-)-(5R,8S,9S,10R)-clerod-3-en-15-oate (3), along with the known methyl (-)-copalate (1)⁶ and methyl (-)-eperuate (2).⁷ Through GC/MS analysis of the methylated extract, besides compounds 1-3, five known diterpenes were also identified by their retention indices and by co-injection with authentic samples as: methyl isoozate (4),⁴ methyl ozate (5),⁴ methyl kovalenate (6),⁴ methyl (5S^*,8S^*,9S^*,10R ^*)-clero-3,13-dien-15-oate (7)⁴ and methyl (5R^*,8S^*,9S^*,10R ^*)-clero-3,13-dien-15-oate (8).⁴

The ethyl acetate extract of seed pods of H. courbaril var. altissima was esterified with diazomethane and submitted to a combination of column chromatography (silica gel), preparative TLC on SiO₂/AgNO₃ (5%, w/w) and RP-HPLC to give a known methyl (-)-zanzibarate (9).⁸ Through GC/MS analysis of the methylated extract, three known diterpenes: methyl isoozate (4), methyl ozate (5) and methyl kovalenate (6) were also identified by retention indices and by co-injection with authentic samples.⁴

Methyl (-)-(5R,8S,9S,10R)-clerod-3-en-15-oate (3) was obtained as colorless oil, [a]_D²⁰¾ 54.0 (c 0.6, CHCl₃) and HRMS data (m/z 320.27156) suggested the molecular formula C₂₁H₃₆O₂. The ¹H NMR spectrum of 3 showed two signals at d 0.89 (s, 3H) and 1.05 (s, 3H), corresponding to the methyl groups attached to a quaternary carbon and two signals at d 0.90 (d, J 7.0 Hz, 3H) and 0.96 (d, J 6.6 Hz, 3H), corresponding to the methyl groups attached to a methine carbon. Signal for an additional methyl group, attached to a sp² carbon at d 1.59 (brd, J 1.5 Hz, 3H) coupled to a vinylic hydrogen at d 5.20 (brs, 1H), was observed.

The full ¹H and ¹³C NMR signals assignment was based on homonuclear and heteronuclear 2D experiments (COSY-90, HSQC) and by comparison with the ¹³C NMR spectral data of the previously isolated compound 8,⁴ which showed a good agreement for the chemical shifts of carbons belonging to the decalin moiety.

The ¹H NMR spectrum also revealed signals for methylene hydrogens (d 2.13, dd, J 9.0 and 4.4 Hz; 2.35 dd, J 9.0 and 4.4 Hz) adjacent to a carbomethoxyl group and cross peaks in the COSY spectrum showing their coupling to a methinic hydrogen at d 1.88 (m). This hydrogen was, in turn, connected to the methyl group at d 0.96 and methylene hydrogens at d 1.33 (m). The ¹³C NMR data connectivity analyses and comparison of ¹³C NMR data of the side chain of 3 with those of 2 were in good agreement. The chemical shift of carbon C-19, that appeared at d 20.6, confirms the trans relationship for A/B ring.⁴ The assignments for all signals were fully confirmed by HSQC and HMBC experiments.

Experimental

General

¹H- and ¹³C-NMR spectra were recorded in CDCl₃ solution at 300 MHz and 75 MHz, respectively, with a Bruker AC 300/P spectrometer (internal standard TMS). IR spectra of neat samples were obtained on a Perkin-Elmer 1600 series FTIR. Mass spectra of purified compounds were obtained at 70 eV on an Hewlett-Packard 5990/5970 system equipped with a J&W Scientific DB-5 fused silica capillary column (30 m x 0.25 mm x 0.25 mm). High-resolution mass spectra (HRMS) were performed with a VG 7070E spectrometer by electron impact with beam energy of 70 eV. Optical rotations were measured with a Carl Zeiss photoelectric polarimeter.

Isolation

The seed pods and barks of Hymenaea courbaril var. altissima (Ducke) were collected in March 1994 at Fazenda Santa Elisa (IAC- Campinas), São Paulo State, Brazil. A voucher specimen was deposited in the Herbarium of IB-UEC (# no. GS 01/03). Dry barks (360 g) were powdered and extracted with EtOAc (3L) for 5 days at room temperature. After filtration and evaporation of solvent at reduced pressure a crude extract (34 g) was obtained as an oily resin. A sample of resin (789 mg) was esterified with diazomethane and submitted successively for purification using silica gel column chromatography (CHCl₃: MeOH 1% to 5%, v/v), prep TLC on SiO₂/AgNO₃ (5%, w/w) eluting with a mixture of different solvents and polarity (petroleum ether/EtOAc/CH₂Cl₂) to yield (-)-methyl copalate (1) {11 mg, [a]_D²⁰¾19.0 CHCl₃, c 0.7; lit.,⁶ [a]_D²⁰¾12.9}, methyl eperuate (2) {15 mg, [a]_D²⁰¾24.0 CHCl₃, c 1.1; lit.⁷, [a]_D²⁰¾26.0} and methyl (-)-(5R,8R,9R,10S) clerod-3-en-15-oate (3) {12 mg, [a]_D²⁰¾54.0 CHCl₃, c 0.6}. GC/MS analysis of the barks methylated extract indicated: methyl isoozate (4) (3.7%), methyl ozate (5) (3.9%), methyl kovalenate (6) (3.2%), methyl (5S^*,8S^*,9S^*,10R ^*)-cleroda-3,13-dien-15-oate (7) (2.3%) and methyl (5R^*,8S^*,9S^*,10R ^*)-cleroda-3,13-dien-15-oate (8) (0.8%).

Dry seed pods (2.5 kg) were powdered and extracted with n-hexane (6L) for 5 days at room temperature. After filtration and evaporation of solvent at reduced pressure a crude extract (110 g) was obtained as an oily resin. A sample of resin (5 g) was submitted to acid and base extraction to furnish acidic fraction (3.3 g). Treatment with excess diazomethane gave esterified extract (3.4 g) which was submitted to successive purification by silica gel column chromatography (petroleum ether : EtOAc, 95:5, v/v) to yield (-)-methyl zanzibarate (6) as colorless crystals {150 mg, mp 132-134 ^oC, [a]_D²⁰¾37.5 CHCl₃, c 2.0; lit.,⁸ [a]_D¾37.0, CHCl₃, c 1.0}. GC/MS analysis of the esterified extract indicated the presence of methyl isoozate (4) (5.7%), methyl ozate (5) (46.5%) and methyl kovalenate (6) (10.5%).

Methyl (-)-(5R,8S,9S,10R)-clerod-3-en-15-oate (3)

Viscous oil, [a]_D²⁰¾54.0 (CHCl₃, c 0.6); HREIMS m/z: 320.27156 [M]⁺ (calcd 320.27153 for C₂₁H₃₄O₂); IR n_max / cm^-1: 1741, 1458, 1436, 1381, 1162, 1096, 1012 (film); ¹H NMR see Table 1; ¹³C NMR see Table 1.

Acknowledgements

This work was performed with financial support of FAPESP. RTN thanks CAPES and FAEP/UNICAMP for fellowships and RAG acknowledges FAPESP. We also thank Dr. L.H.B. Baptistella and Dr. C.H. Collins for helpful discussion and suggestions.

References

Received: August 31, 2001

Published on the web: April 15, 2002

FAPESP helped in meeting the publication costs of this article.

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Publication Dates

• Publication in this collection
  01 July 2002
• Date of issue
  June 2002

History

• Accepted
  15 Apr 2002
• Received
  31 Aug 2001