Macrocarpane, a new sesquiterpene skeleton from the leaves of Porcelia macrocarpa

Abstracts

The volatile oil and the hexane extract from the leaves of Porcelia macrocarpa (Warm.) R.E. Fries, Annonaceae, were submitted to chromatographic separations. Nine sesquiterpenes (alpha-cubebene, alpha-copaene, germacrene-D, bicyclogermacrene, gamma-cadinene, delta-cadinene, spathulenol, globulol and cis-cubenol) and one diterpene (phytol) were identified in the volatile oil (83.1 % in weight). Two sesquiterpenes (spathulenol and macrocarp-11(15)-en-8-ol), the latter with a novel skeleton, one diterpene (phytol) and two steroids (sistosta-5,25-dien-3beta-ol and sitosta-5,22,25-trien-3beta-ol) were isolated from the hexane extract. These compounds were characterized by ¹H and 13C NMR spectroscopy, including bidimensional analysis and mass spectrometry.

macrocarpane; spathulenol; steroids; sesquiterpenes; Porcelia macrocarpa

O óleo volátil e o extrato hexânico das folhas de Porcelia macrocarpa (Warm.) R.E. Fries, Annonaceae, foram submetidos a fracionamentos cromatográficos. Nove sesquiterpenos (alfa-cubebeno, alfa-copaeno, germacreno-D, biciclogermacreno, gama-cadineno, delta-cadineno, espatulenol, globulol e cis-cubenol) além de um diterpeno (fitol) foram identificados no óleo volátil, correspondendo a 83.1% (em massa) do total de constituintes. Dois sesquiterpenos (espatulenol e macrocarp-11(15)-en-8-ol), o último apresentando um novo esqueleto estrutural, um diterpeno (fitol), além de dois esteróides (sitosta-5,25-dien-3beta-ol e sitosta-5,22,25-trien-3beta-ol), foram isolados do extrato hexânico. As substâncias foram caracterizadas através de experimentos de RMN de ¹H e 13C, incluindo análise bidimensional, além de espectrometria de massas.

ARTICLE

Macrocarpane, a new sesquiterpene skeleton from the leaves of Porcelia macrocarpa

Mariana H. Chaves^I; João Henrique G. Lago^II; Nídia F. Roque^{II, III}

^IInstituto de Química, Universidade Federal do Piauí, Campus Ininga, 64049-550 Teresina, PI, Brazil

IIInstituto de Química, Universidade de São Paulo, CP 26077, 05513-970 São Paulo, SP, Brazil

IIIInstituto de Química, Universidade Federal da Bahia, 40170-290 Salvador, BA, Brazil

^{Address to correspondence}

RESUMO

O óleo volátil e o extrato hexânico das folhas de Porcelia macrocarpa (Warm.) R.E. Fries, Annonaceae, foram submetidos a fracionamentos cromatográficos. Nove sesquiterpenos (a-cubebeno, a-copaeno, germacreno-D, biciclogermacreno, g-cadineno, d-cadineno, espatulenol, globulol e cis-cubenol) além de um diterpeno (fitol) foram identificados no óleo volátil, correspondendo a 83.1% (em massa) do total de constituintes. Dois sesquiterpenos (espatulenol e macrocarp-11(15)-en-8-ol), o último apresentando um novo esqueleto estrutural, um diterpeno (fitol), além de dois esteróides (sitosta-5,25-dien-3b-ol e sitosta-5,22,25-trien-3b-ol), foram isolados do extrato hexânico. As substâncias foram caracterizadas através de experimentos de RMN de ¹H e ¹³C, incluindo análise bidimensional, além de espectrometria de massas.

ABSTRACT

The volatile oil and the hexane extract from the leaves of Porcelia macrocarpa (Warm.) R.E. Fries, Annonaceae, were submitted to chromatographic separations. Nine sesquiterpenes (a-cubebene, a-copaene, germacrene-D, bicyclogermacrene, g-cadinene, d-cadinene, spathulenol, globulol and cis-cubenol) and one diterpene (phytol) were identified in the volatile oil (83.1 % in weight). Two sesquiterpenes (spathulenol and macrocarp-11(15)-en-8-ol), the latter with a novel skeleton, one diterpene (phytol) and two steroids (sistosta-5,25-dien-3b-ol and sitosta-5,22,25-trien-3b-ol) were isolated from the hexane extract. These compounds were characterized by ¹H and ¹³C NMR spectroscopy, including bidimensional analysis and mass spectrometry.

Keywords: macrocarpane, spathulenol, steroids, sesquiterpenes, Porcelia macrocarpa

Introduction

The chemical constitution of Porcelia macrocarpa was described in four previous papers. In the first one, acetogenins from seeds were reported.¹ Two other papers report the presence of amides, lignanamides and alkaloids from the branches.^2,3 The polar constituents, such as aminoacids, trimethylamonium salts and glycosilated flavonoids were also described.⁴ From our previous studies with P. macrocarpa we describe herein the identification of nine sesquiterpenes and one diterpene in the volatile oil and two sesquiterpenes, one diterpene and two steroids in the hexane extract, both from the leaves. The ¹H and ¹³C NMR data of 10, including bidimensional analysis, showed a novel sesquiterpene skeleton, which was denominated macrocarpane.

Results and Discussion

The hexane extract of the leaves of P. macrocarpa was submitted to chromatographic separations to give two sesquiterpenes: spathulenol (7) and macrocarp-11(15)-en-8-ol (10), one diterpene: phytol (11) and two steroids: sistosta-5,25-dien-3b-ol (12) and sitosta-5,22,25-trien-3b-ol (13). The sesquiterpene 7, the diterpene 11 and the steroids 12 and 13 were identified by analysis of their ¹H and ¹³C NMR data and comparison with data described in the literature.^5-8

The EIMS spectrum of 10 showed the molecular ion peak at m/z 220 Da. The ¹³C NMR spectra (BBD and DEPT 135°) displayed fifteen signals refering to three methyls, four methylenes, six methines, as well as two quaternary carbon atoms, suggesting the occurrence of an oxygenated sesquiterpene with molecular formula C₁₅H₂₄O. The signals at d 146.0 (C) and d 105.7 (CH₂) were indicative of an exocyclic methylenic group, and the single signal at the carbinolic region [d 77.4 (CH)] confirms the presence of one hydroxyl group in the molecule of 10.

The ¹H NMR spectrum showed three methyl groups at d 0.79 (s), d 0.93 (s) and d 0.93 (d, J 3.0 Hz), the latter being linked to a methinic carbon atom. This spectrum also showed a signal at d 0.51 (m), suggesting the presence of a cyclopropane ring in the structure of 10. The observation of a double-multiplete at d 48.6, in the gated ¹³C NMR spectrum with J 163 Hz, characteristic of three member ring,⁹ confirms the cyclopropane ring in the molecule. The HMQC spectrum showed long-range correlations between carbons and hydrogens (Table 1). These results associated with those showed in ¹H-¹H COSY spectrum indicated the connectivities in the molecule, and suggested a decahydroazulene derivative with a cyclopropane ring. The mutual long range coupling between the signals at d 1.48 (H-1) and d 4.82 (H-15), d 2.28 (H-10) and d 1.29 (H-5), the latter being a W coupling, corroborates the suggested structure and indicated the b orientation to cyclopropane ring. This spectrum also showed the coupling between the hydrogens at C-9 and those at C-10 and C-8; thus, the two methyl groups should be at C-7. The presence of these groups interrupted the ¹H-¹H connectivities in the seven member ring. Therefore, the cyclopropane group could only be at the five-carbon ring. The mutual coupling observed among the signals at d 0.51 (H-4) and d 1.29 (H-5), and d 1.18 (H-3) located the cyclopropane ring between C-3/C-5. This situation was corroborated by the correlations between H-3/H-2 and H-3/H-6, observed in the ¹H-¹H COSY spectrum. HMBC long range correlations confirm the proposed structure. The small coupling constants of H-1 (W_½ = 8 Hz) indicated a cis ring junction between the five and seven member rings. The chemical shift of methyl group at C-4 (d 21.8) suggested the presence of this group at a position, free of d interactions. This sesquiterpenic skeleton has not been described in the literature yet, and was denominated macrocarpane. Therefore, the structure of 10 was defined as macrocarp-11(15)-en-8-ol.

The biosynthetic pathway proposed to 10 should involve a himachalane derivative which was not detected in this study, but has been isolated from Ferula latipinna (Umbelliferae),¹⁰ which could be formed by cyclization of bicyclogermacrene (Figure 1). In the volatile oil, the absence of 10 and the reduced amount of 7 (3.0%) could be explained by the predominance of germacrene-D (37.8%) and bicyclogermacrene (27.5%). In the hexane extract, these sesquiterpenes were not detected, indicating that 7 and 10 should be formed from 4,¹¹ corroborating the proposed biosynthetic pathway.

The volatile oil from the leaves of P. macrocarpa was submitted to chromatographic separationand the fractions obtained were analysed by GC.¹² The sesquiterpenes 3, 4, 7, 8 and 9 and the diterpene 11 were characterized as the main compounds in mixtures by analysis of their ¹H and ¹³C NMR spectra. The comparison with the literature values confirmed the proposed structures^5,13-15. Additionally, the crude volatile oil was submitted to analysis by GC/MS. The sesquiterpenes 1, 2, 5 and 6 were identified. This analysis confirms the identification of the sesquiterpenes 3, 4 and, 7, and the diterpene 11, which were characterized by NMR. Using this methodology, which is a combination of three spectrometric techniques (NMR, GC, GC/MS), nine sesquiterpenes, and one diterpene (83.1 % in weight) were identified in the volatile oil (Table 2).

Experimental

Plant material

All the leaves of P. macrocarpa (Warm.) R.E. Fries were collected from the same specimen, in March 1992 (hexane extract), and in December 2000 (volatile oil) at the Jardim Botânico of São Paulo State. A voucher specimen has been deposited in the herbarium of the Instituto Botânico, São Paulo, Brazil under reference SP76791.

Extraction and isolation of the compounds

Dried powdered leaves (750 g) of P. macrocarpa were extracted with hexane three times at room temperature. The concentrated hexane extract was partitioned between MeOH/H₂O (9:1, v/v) and hexane. The hexanic part (10 g) was submitted to precipitation with MeOH from which were obtained the waxy material (9 g), and the MeOH soluble material (1 g). The MeOH/H₂O phase (6 g) and the MeOH soluble material (1 g) were pooled together, chromatographed on silica gel column and eluted with increasing amounts of EtOAc in hexane, from which were obtained four groups (I-IV), after TLC on silica gel analyses. The group I (351 mg) was submitted to flash chromatography on silica gel and eluted with hexane-EtOAc (6:4, v/v) yielding 7 (77 mg). Group II (720 mg) was methylated with diazomethane, submitted to flash chromatography on silica gel and eluted with hexane-EtOAc (97:3, v/v) giving two mixtures A (359 mg), and B (30 mg). The ¹H NMR spectrum of A showed the occurrence of fatty acid esters. Fraction B was applied to a Sephadex LH-20 column and eluted with hexane-CH₂Cl₂ (1:4, v/v) to yield 11 (10 mg). Group III (483 mg) was submitted to flash chromatography on silica gel and eluted with hexane-[CH₂Cl₂-MeOH (98:2, v/v)] (6:4, v/v) to give two mixtures C (30 mg) and D (200 mg). Mixture C was comprised of two steroids (12 and 13). Mixture D was applied to a Sephadex LH-20 column and eluted with hexane-CH₂Cl₂ (1:4, v/v) to give a mixture of one sesquiterpene and steroidal material. The purification by prep. TLC on silica gel with 15% of AgNO₃ [hexane-EtOAc (6:4, v/v)] yielded 10 (15 mg).

The fresh plant material (2250 g) was hydrodistilled using a Clevenger type apparatus to give the crude volatile oil (735 mg; yield 0.03%). Part of this material (500 mg) was submitted to flash chromatography on silica gel and eluted with CH₂Cl₂ and CH₂Cl₂-MeOH (95:5 and 9:1) affording mixtures containing 1 + 3 + 4 (fr 4, 137 mg), 3 + 4 (fr 6, 25 mg), 11 (fr 34, 16 mg), 7 (fr 38, 12 mg), 8 + 9 (fr 39, 52 mg). The remaining part of the crude volatile oil was subjected to analysis by GC/MS to identify the sesquiterpenes 1, 2, 5 and 6.

General

Silica gel 60 (Merck) was used for chromatography: 63-200 mm for CC, 40-63 mm for flash chromatography, PF₂₅₄ (5-45 mm) for preparative TLC. IR spectra were obtained as KBr pellets in a Perkin-Elmer Infrared Spectrometer model 1750. Sephadex LH-20 (Sigma) was used for molecular exclusion chromatography. NMR spectra were recorded at 300 and 500 MHz for ¹H and 75 and 125 MHz for ¹³C on Brüker DPX-300 and DRX-500 spectrometers using CDCl₃ as solvent and internal standard. The GC analysis was performed in a Hewlett-Packard 5890 series II (using helium as carrier gas) equipped with a FID detector and a capillary column HP-5, crosslinked 5% phenyl in 95% methyl silicone (30 m x 0.32 mm, film thickness 0.25 mm) with a automatic injector (HP 7673) and an electronic integrator (HP 3396A). The temperature programming was from isothermal 100 °C for 2 min, 100°-240 °C at 5°C.min^-1, then isothermal at 240 °C for 5 min. The (FID) injector and detector temperatures were 180 °C and 260 °C, respectively. The GC/MS analyses were carried out in an EIMS 70 eV Hewlett-Packard HP-5973 coupled with a Hewlett-Packard HP-6890 with a DB-50 column (30 m x 0.25 mm, film thickness 0.25 mm) using the same temperature programming conditions describe above. The identification of the compounds was done by comparing retention indices (determined relatively to the retention times of a series of n-alkanes) and mass spectra to those of authentic samples.

Macrocarp-11(15)-en-8-ol (10): colourless oil. IR (KBr) n_max cm^-1: 3402 (OH), 3108 (cyclopropane ring), 2945 (C-H), 1637 (>C=CH₂), 919, 888. EIMS (70 eV) m/z (rel. int.) 220 [M]⁺(58), 202 [M-H₂O]⁺(7), 187 [C₁₄H₁₉]⁺(17), 177 [C₁₂H₁₇O]⁺(22), 159 [C₁₂H₁₅]⁺(100), 145(24), 135(16), 133(27), 131(32), 121(16), 119(32), 117(30), 109(28), 107(23), 105(48), 95(14), 93(44), 91(55), 81(23), 79(32), 77(31), 69(13), 67(32), 65(14), 57(12), 55(36), 53(22). ¹H and ¹³C NMR: see table 1.

Acknowledgements

The authors are grateful to Dra. Maria Claudia Marx Young from Instituto de Botânica/SMS for the plant material provision. This work was supported by CAPES, CNPq and FAPESP.

Received: November 1, 2001

Published on the web: October 29, 2002

FAPESP helped in meeting the publication costs of this article.

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Publication Dates

• Publication in this collection
  07 Apr 2003
• Date of issue
  Jan 2003

History

• Received
  01 Nov 2001