Macrocarpane , a New Sesquiterpene Skeleton from the Leaves of 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 (α-cubebeno, α-copaeno, germacreno-D, biciclogermacreno, γ-cadineno, δ-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-3β-ol e sitosta-5,22,25-trien-3β-ol), foram isolados do extrato hexânico. As substâncias foram caracterizadas através de experimentos de RMN de H e C, incluindo análise bidimensional, além de espectrometria de massas.


Introduction
The chemical constitution of Porcelia macrocarpa was described in four previous papers.In the first one, acetogenins from seeds were reported. 1Two other papers report the presence of amides, lignanamides and alkaloids from the branches. 2,3The polar constituents, such as aminoacids, trimethylamonium salts and glycosilated flavonoids were also described. 4From 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 1 H and 13 C NMR data of 10, including bidimensional analysis, showed a novel sesquiterpene skeleton, which was denominated macrocarpane.
The 1 H NMR spectrum showed three methyl groups at δ 0.79 (s), δ 0.93 (s) and δ 0.93 (d, J 3.0 Hz), the latter being linked to a methinic carbon atom.This spectrum also showed a signal at δ 0.51 (m), suggesting the presence of a cyclopropane ring in the structure of 10.The observation of a double-multiplete at δ 48.6, in the gated 13 C 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 1 H-1 H 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 δ 1.48 (H-1) and δ 4.82 (H-15), δ 2.28 (H-10) and δ 1.29 (H-5), the latter being a W coupling, corroborates the suggested structure and indicated the β 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 1 H-1 H 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 δ 0.51 (H-4) and δ 1.29 (H-5), and δ 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 1 H-1 H 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 (δ 21.8) suggested the presence of this group at a position, free of δ 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 separation and 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 1 H and 13 C NMR spectra.The comparison with the literature values confirmed the proposed structures 5,[13][14][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).

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/H 2 O (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/H 2 O 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 1 H 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-CH 2 Cl 2 (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-[CH 2 Cl 2 -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-CH 2 Cl 2 (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 AgNO 3 [hexane-EtOAc (6:4, v/v)] yielded 10 (15 mg).
The fresh plant material (2250 g) was hydrodistilled

General
Silica gel 60 (Merck) was used for chromatography: 63-200 µm for CC, 40-63 µm for flash chromatography, PF 254 (5-45 µm) 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 1 H and 75 and 125 MHz for 13 C on Brüker DPX-300 and DRX-500 spectrometers using CDCl 3 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 µm) 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 µm) 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.15)-en-8-ol (10)  (13), 67(32), 65 (14), 57 (12), 55(36), 53(22). 1 H and 13 C NMR: see table 1.

Table 2 .
Chemical constituition of the crude volatile oil from leaves of P. macrocarpa Hypothetic biosynthetic pathway of sesquiterpenes 7 and 10 from 3 and 4.