Resin Diterpenes from Austrocedrus chilensis

Seventeen diterpenes belonging to the labdane, abietane and isopimarane skeleton classes were isolated from the resin of the Chilean gymnosperm Austrocedrus chilensis and identified by spectroscopic and spectrometric methods. The diterpene 12-oxo-labda-8(17),13E-dien-19 oic acid is reported for the first time as a natural product and 14 diterpenes are reported for the first time for the species.


Introduction
The gymnosperm tree Austrocedrus chilensis (D. Don) Florin et Boutelje (Syn.: Austrocedrus chilensis (D.Don) Pic. Ser. et Bizz) (Cupressaceae) is known as "ciprés de cordillera" and grows in sandy soils in the Eastern Andean slopes up to 2,000 m over sea level. The plant leaves has been used as a sudorific and the powdered fruits to treat diarrhea [1]. Little information exists on other medicinal uses of the plant, but the wood is highly appreciated. The monoterpene carvacrol and β-thujaplicin (4-isopropyl tropolone = hinokitiol) and the flavonoid taxifolin were isolated from the wood of A. chilensis [2]. An ethanol extract from the aerial parts of the tree, including leaves, twigs and stems, afforded the lignan desoxypodophyllotoxin and the diterpenes 8,20-dihydroxy-8(11),13-abietadien-12one and pisiferol [3][4][5]. Flavonoids were identified from a 70% ethanol extract of leaves and branchlets OPEN ACCESS of A. chilensis [6], while sugiol and another unidentified diterpene were isolated from a tree bark methanol extract [7].
The investigation of gymnosperm resin composition has been carried out using chromatographic means for the isolation and further identification of the constituents by spectroscopic and spectrometric methods [9][10][11]. New techniques used to characterize the resin constituents include gas chromatography coupled to mass spectrometry (GC-MS) [12][13][14][15], and proton magnetic resonance spectroscopy [16].
Following our studies on native South American plants, we have now examined the composition of resin exudates from a mature population of Austrocedrus chilensis trees to establish suitable conditions for metabolic profiling.

Results and Discussion
Seventeen diterpenes with isopimarane, labdane and abietane skeletons were isolated from the resin of Austrocedrus chilensis, and identified by spectroscopic and spectrometric methods. Fourteen of these diterpenes are reported for the species for the first time. The compounds were identified by spectroscopic means and by comparison of their spectral data with literature values. The 13 C-NMR data of compounds 4, 6, 12-14a is shown in Table 1. The structures of the compounds identified in the resin are shown in Figure 1. From the compounds identified in the present work, only sandaracopimaric acid (compound 5), ferruginol (compound 15), and 7-oxoferruginol (sugiol, compound 17) were previously reported [8] in a resin sample of this species collected in southern Chile. On the other hand, the previously reported diterpenes 2,3-dehydroferruginol and 6,7-dehydroferruginol [8] were not identified in our samples. The differences can be explained due to different plant populations and collection time.   [17]. In a new article, additional labdanes, abietanes and pimaranes were identified, including junicedric acid, 13-epi-cupressic acid methyl ester, copalol, 13-oxo-14,15-dinorlabd-8(17)-en-19 oic acid methyl ester, trans-communic acid, cis-communic acid, 19-acetoxyferruginol, sugiol methyl ether, 6α-hydroxydemethyl-cryptojaponol, 5,6-dehydrosugiol methyl ether, cupresol, nejukol, isopimarinol and isopimaric acid [18].
The compound 14 was isolated as the corresponding methyl ester and is described as a new natural product. The structure of compound 14 follows from the HR-MS indicating a molecular formula C 20 H 30 O 3 and C 21 H 32 O 3 after methylation (compound 14a), accounting for six degrees of unsaturation (i.e., three double bonds, two rings and one carbonyl function). The IR spectrum of the methyl ester shows an ester function and α,β-unsaturated ketone at 1,724 and 1,672 cm −1 , respectively. In the 1 H-NMR spectrum, an olefinic side chain proton at δ 6.77 (H-14) coupled with two allyl methyl groups at δ 1.84 (H-15) and 1.74 ppm (H-16), indicates an α,β-unsaturated ketone system in the side chain of the diterpene. The E-configuration of the side chain double bond follows from the chemical shift of the olefinic proton and the C-15 methyl group and is in agreement with the data reported for related diterpenes isolated from the liverwort Scapania undulata [19].
While the composition of resin samples from a female and a male tree showed the same main compounds, the relative ratio of the diterpenes was different. This fact can be related to several factors, including gender, individual, seasonal, or response to pathogens, among others. However, a much larger number of samples should be analyzed to disclose the significance of the present findings. The establishment of a chromatographic method for the fast and reliable identification of the resin constituents opens new possibilities for the comparative study of populations of this tree as well as the response of A. chilensis to environmental stress and microorganisms [43][44][45][46], including the pathogenic fungus Phytophthora austrocedrae.

General
Optical rotations were obtained for solutions in CHCl 3 (concentrations expressed in g/100 mL) on a Jasco DIP 370 polarimeter (Jasco Analytical Instruments, Easton, MD, USA). IR spectra were recorded on a Nicolet Nexus FT-IR instrument (Thermo Electron Corporation, Waltham, MA, USA). All NMR experiments were performed on a Bruker Avance 400 NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) equipped with a 5 mm inverse detection z-gradient probe. The 1 H and 13 C spectra (at 400 and 100 MHz, respectively) were measured at room temperature (22-23 °C) using CDCl 3 as solvent. Chemical shifts are given on the  scale and were referenced to residual CHCl 3 at 7.25 ppm for 1 H spectra and to the solvent at 77.00 ppm for 13 C spectra. One-dimensional 1 H and 13 C-NMR spectra were acquired under standard conditions. The pulse programs of the COSY, gHSQC, gHMBC experiments were taken from the Bruker software library. Homonuclear twodimensional spectra (COSY) and inverse proton-detected heteronuclear two-dimensional spectra (gHSQC) were acquired in the phase-sensitive mode and gHMBC spectra were acquired in the absolute value mode. The data for the gHSQC spectra were collected in a 1024 × 256 matrix with a spectral width of 4  Compounds were characterized by electron-ionization (EI) mass spectra. Retention time (Rt, min) and MS fragmentation patterns of the known compounds were compared with literature.

Plant Material
The resin of Austrocedrus chilensis was collected from a mature tree population growing at Las Trancas, VIII Region, Chile (36°54′03′′S, 31°32′47′′W). Voucher herbarium specimens have been deposited at the Herbario de la Universidad de Talca and were identified by Patricio Peñailillo. The voucher specimens correspond to the individuals from those the resin was obtained.
The fraction pool 15-19 (1.92 g) was chromatographed on a RP-8 silica gel column with MeOH/H 2 O 9:1 as the mobile phase in three batches. Some 90 fractions of 5 mL each were collected and pooled after TLC to afford a mixture of E-communic acid (main compound) and Z-communic acid (compounds 12 and 13) in a 3:1 ratio (350 mg) and sandaracopimaric acid (90 mg, 5). A representative sample of the fractions 1-32 (136 mg) was treated with diazomethane. After preparative TLC (silica gel, PE/EtOAc/acetone 9:2:1) 51 mg of torulosic acid methyl ester (10a) and 33 mg of a mixture of torulosic acid methyl ester (10a) and the alcohol 7 were obtained. After methylation, preparative TLC of fraction pool 20-23 (toluene/DCM/Et 2 O 4.5:4.5:1) yielded 12.3 mg of compound 14a and 13 mg of a mixture of the compounds 6 and 7 in a 4:1 ratio.

Conclusions
The composition of A. chilensis resin was established by spectroscopic and spectrometric means. A GC-MS method was developed for the fast identification of the diterpene constituents in the resin, setting the conditions for new studies on the physiological response of the tree under different environmental stimuli.