New Pinane Derivatives Found in Essential Oils of Calocedrus decurrens

The main objective of this study was to determine the chemical composition of essential oils (EOs) obtained from leaf, old branches, and young branches of a coniferous species Calocedrus decurrens acclimated to Corsica. The analytical investigation was conducted by GC(RI), GC-MS, pc-GC, and NMR. C. decurrens leaf, old branches, and young branches EOs contained α-pinene (11.2; 56.6; 22.3%), myrcene (13.4; 8.4; 9.7%), Δ-3-carene (31.3; 5.2; 11.1%), limonene (6.4; 5.1; 5.5%), terpinolene (6.9; 1.5; 3.2%), and pin-2-en-8-ol (4.2; 4.5; 10.4%) as major components, respectively. Special attention was paid to purifying and identifying four unusual pinane derivatives: pin-2-en-8-ol, pin-2-en-8-yl Acetate, pin-2-en-8-al, and methyl pin-2-en-8-oate. The last two are reported for the first time.


Introduction
Over the last century, the French National Forest Office ("Office National des Forêts") has introduced several coniferous species in Corsica. These introductions were made for the purposes of testing the growing abilities of these species out of their natural ecosystems. In the course of our studies, several essential oils from these introduced species such as Pinus halepensis [1,2], Larix decidua, Pseudotsuga menziesii, Pinus ponderosa, Sequoiadendron giganteum, and Cryptomeria japonica [3] were investigated. However, another species formerly introduced in Corsica, Calocedrus decurrens, was still not studied.
C. decurrens, commonly known as incense cedar, is an aromatic conifer belonging to the Cupressaceae family, native to Oregon and California. Its natural area extends to 31 • at 46 • parallel of north latitude and it is found between 900 and 2500 m altitude. It is widely used in reforestation in the USA because of its rusticity and its adaptation in difficult sites (limestone, shallow, dry land). The bark of Calocedrus is thick, reaching 20 cm, and is an excellent protection against forest fires which are very frequent in its native area. Furthermore, C. decurrens wood is considered as precious wood, owing to its highly aromatic wood, and is often used for the manufacture of pencils. Its wood is also highly valued for its decorative appearance and durability in carpentry, interior cladding, and furniture [4,5].
In France, this species was introduced around 1850. Since, it has been widespread in many parks and gardens, particularly in the Mediterranean area, for example, Gréoux-les-Bains and Châteauvert [5]. By contrast, in Corsica, the C. decurrens species was introduced in 1989 in the forest of Ospedale.
In this study, we focus on the analytical investigation of essential oils obtained from leaf, old branches, and young branches of C. decurrens acclimated to Corsica. We also give a comparison of their chemical compositions with EOs obtained from trees grown in their native areas. Special attention was paid to the identification of unusual pinane derivatives, such as pin-2-en-8-ol (A) and pin-2-en-8-yl Acetate (B), and two additional compounds, pin-2-en-8-al (C) and methyl pin-2-en-8-oate (D), never reported so far in the literature.

Identification of Pin-2-en-8-ol (A)
The first unknown constituent named A was identified neither by computer matching against commercial and lab-constructed SM libraries, nor by NMR using our in-house 13 C-NMR databases. As determined by GC(RI) and GC-MS, the compounds A, accounted for 4.2%, 4.5% and 10.3% in the leaf, old branches, and young branches essential oils of C. decurrens. RI values measured for this compound, 1189/1800 (RI a /RI p ), suggested a monoterpenic alcohol.
The young branches essential oil containing 10.3% of A was subjected to column chromatography (CC) on silica gel using a gradient of solvents (pentane/Et 2 O). Fraction F3 (eluted at 100% Et 2 O), contained 51.8% of the unknown compound. Compound A was finally isolated from F3 by means of preparative capillary-gas chromatography (pc-GC), with 95% purity (GC).
The 13 C-NMR spectrum of the isolated compound displayed 10 signals. Taken altogether, MS and NMR data, especially those provided by the DEPT spectra (2 C, 3 CH, 3 CH 2 and 2 CH 3 ) and by 1 H and 13 C-NMR (occurrence of C=CH and methylene alcohol function at 69.00 ppm), suggested the formula C 10 H 16 O.
By taking into account the degree of unsaturation and the multiplicity of carbon signals, compound A corresponded to a bicyclic unsaturated monoterpene alcohol. Moreover, from characteristic chemical shifts and signals multiplicity of H-7a/7b (Table 2), we deduced the occurrence of bicyclic unsaturated monoterpene alcohol having a pinane skeleton.
The CH 2 at 69.00 ppm indicated a primary alcohol, potentially located on carbons 8, 9 or 10. The possibility of a hydroxy group on C-10 was immediately discarded because the 13 C-NMR values of adjacent carbons 2 and 10 remained close to those of α-pinene [10] ( Table 2). The lack of HMBC correlations between hydrogens at 3.50-3.54 ppm (H-8b/8a) and C-2 confirmed the previous observation.
The 13 C-NMR data of pin-2-en-9-ol are available in the literature [11], and did not match with that of compound A (Table 2). Compound A was thus identified as pin-2-en-8-ol (A). In the case of α-pinene, methyl-8 and methyl-9 strongly differ in their respective 13 C-NMR chemical shifts, due to the presence of a γ-steric effect between C-9 and C-2 [10]. The structure was further corroborated by examination of the NOESY spectrum, (i) showing no correlation between hydrogens at 3.50-3.54 ppm (H-8b/8a) and the olefinic proton H-3; and (ii) by the presence of a clear correlation between methyl-9 (0.95 ppm) and H-3. The total assignment of compound A was finally achieved by the detailed examination of 2D-NMR data ( Table 2). We noticed that the compound A was partially described over thirty years ago by De Pascual Teresa et al. [12], with a partial description of 1 H-NMR chemical shifts. In the course of this investigation, we reported for the first time the 13 C-NMR values, along with an assignment of 1 H and 13 C chemical shifts.

Identification of Pin-2-en-8-yl Acetate (B)
Compound B is the second unknown component of SM libraries and 13 C-NMR databases, which was present at 0.6, 0.6, and 1.4% in leaf, old branches, and young branches essential oils, respectively. Retention indices for B (RIa/RIp = 1310/1683) suggested an ester. The presence of common odd-electron ions (OE •+ : 84, 92, 134 m/z) in both mass spectra of A and B indicated a possible structural relationship between the two compounds. Moreover, the presence of OE •+ at m/z = 134 potentially indicated a [M •+ − AcOH] loss from the molecular ion observed at m/z = 194 ( Figure 2). Compound B was finally isolated from a fraction of the young branches essential oil. Fraction F2 (eluted with 95/5 pentane/Et 2 O) contained 8.0% of B, which was subsequently isolated by pc-GC with 76% purity (GC).
The 13 C-NMR spectrum of B displayed twelve signals. Multiple DEPT experiments allowed for differentiating of three quaternary carbons, three CH, three CH 2 , and three CH 3 . Moreover, the quaternary carbon at 170.48 ppm and the methylene at 70.58 ppm confirmed the presence of an ester. The presence of a singlet at 1.72 ppm in the 1 H spectrum correlated to a methyl signal at 20.53 ppm in the HSQC spectrum suggested an acetate group. This was confirmed by the correlation between H-12 and C-11 in the HMBC spectrum. Altogether, MS and NMR data permitted to envisage a pinane ester having the formula C 12 H 18 O 2 . By comparison of its 13 C-NMR data with that of pin-2-en-8-ol (A), we noted that only C-8 and C-6 were impacted by the presence of the acetate. C-8 moved upfield by 1.58 ppm, and C6 moved downfield by 1.76 ppm. Such variations of chemical shifts are commonly encountered when alcohols are compared to their corresponding acetates [13].
As final proof, the examination of the NOESY spectrum allowed us to notice correlations between hydrogen at 0.96 ppm (H-9) with H-3, and the absence of a correlation between hydrogens at 4.26-4.32 ppm (H-8b/8a) and H-3, thus confirming the orientation of the acetate in exo-position to the main cycle. All these data permitted the identification of compound B as pin-2-en-8-yl Acetate ( Table 3). As with compound A, B was partially described by De Pascual Teresa et al. [12], with some 1 H-NMR chemical shifts and MS data. Herein, we detail a full set NMR data, among which includes: (i) a complete 1 H-NMR spectrum; (ii) 13 C-NMR values (described for the first time); and (iii) full 2D-NMR data. Interestingly, Adams et al. reported in 2006 the chemical composition of a leaf essential oil from C. decurrens [8]. In their study, the authors mentioned an unidentified component at RI = 1330 (DB-5). Although their RI value is shifted 20 points compared to our value, owing to the difference of stationary phase, the MS fragmentation previously reported is very similar to that we observed for B.

Identification of Pin-2-en-8-al (C)
A third compound (C), having RI values at 1102/1460 (RIa/RIp), could not be identified by GC-MS and 13 C-NMR analysis, even by using all of the computerized commercial MS libraries at our disposal, as well as our laboratory-built 13 C-NMR data library-it was unknown from our MS and 13 C-NMR databases. This component was present in leaf, old branches, and young branches EO of C. decurrens in 1.6%, 0.8%, and 2.0%, respectively. The examination of the MS spectrum showed a M •+ at m/z = 150, suggesting the possible presence of an aldehyde function. However, despite our many tests, any attempt of purification by pc-GC failed, since the isolated product was never pure, prompting us to adopt another strategy.
Thus, the pyridinium chlorochromate (PCC) oxidation of pin-2-en-8-ol (A) available in F3 at 51.8% yielded an aldehyde (45.6% in oxidized F3 named F3'), perfectly corresponding with both the RI values and MS data observed for compound C. The compound was further purified by means of column chromatography (CC) on silica gel using the following gradient of solvents pentane/Et2O 95/5 to yielded F3'-1 (11.6 mg) containing 80% of pin-2-en-8-al (C). However, we noticed a rapid oxidation of this aldehyde between GC (directly recorded after CC) and NMR/GC-MS analysis (recording after 2 h). The compound C seemed to oxidize under atmospheric conditions-indeed, GC-MS and NMR analysis indicated the formation of corresponding acid: pin-2-en-8-oic acid (E) (Figure 2 and Table 4).
The 13 C-NMR spectrum displayed ten signals with strong intensity corresponding at the compound C. Subsequent DEPT experiments allowed us to determine the multiplicity of the different carbon signals, and suggested from NMR values, once more, a component having the pinane skeleton (C 10 H 14 O). The methine (CH) observed at 204.19 ppm confirmed the presence of the aldehyde. Additionally, the 13 C chemical shifts of the two olefinic carbons were similar to those of pin-2-en-8-ol (A), and a comparison with the chemical shifts reported for pin-2-en-9-al [11] confirmed we had isolated its epimer. Finally, after examination of all 2D-NMR data, compound C was unambiguously identified as pin-2-en-8 al (C) ( Table 4).
Herein again, pin-2-en-8-al (C) might correspond to an unknown compound reported by Adams et al. in 2006 [8]. Both the RI value (1120) and MS data published by the authors are in fair concordance with our results, thus suggesting it is probably the same molecule. A fourth compound (D) respectively present in 3.0%, 1.8% and 3.6% in leaf, old branches, and young branches oils remained unknown from our MS and 13 C-NMR database. Its RI values, 1207/1543 (RIa/RIp), suggested an oxygenated monoterpene. Fraction F2 (eluted at 95/5 pentane/Et 2 O) obtained from the young branches EO contained 17.8% of compound D. This compound was isolated from F2 by means of pc-GC, giving D in 91% purity (GC).
The MS spectrum indicated an M •+ at m/z = 180. The 13 C-NMR spectrum exhibited eleven signals having the following multiplicity: three quaternary carbons, three CH, two CH 2 , and three CH 3 . The two chemical shifts at 51.47 ppm (CH 3 -O) and 178.28 ppm indicated the presence of a methyl ester. Here again, the degree of unsaturation calculated from the formula C 11 H 16 O 2 suggested a pinane derivative. Interestingly, by comparison of chemical shifts of the compound D with those of pin-2-en-8-ol (A), we noticed that only C-6, C-8, and C-11 were impacted by the presence of the ester. The comparison with 13 C-NMR values of pin-2-en-8-oic acid (E) was even more revealing-indeed, all chemical shifts are very similar excepted: (i) C-8, which was moved downfield by 6.63 ppm (indicating an ester function) and (ii) C-11, which was not present in (E) and being characteristic of methoxy group. As described above for others compounds, the analysis of the NOESY spectrum indicated that the ester function is positioned on C-8 (Table 5). According to the HMBC spectrum, the signal at 51.47 ppm, characteristic of a methoxy group, was correlated with C-8, thus confirming the presence of a methyl ester.
The essential oils obtained from the different parts of Calocedrus decurrens from Corsica are qualitatively close in terms of chemical composition, but they present significant differences related to their content of each major monoterpenes. Taken altogether, these EOs are qualitatively close to that described by Adams et al. and Von Rudloff [7,8]. Even if it was difficult to make any further comparison since the old and young branches were studied for the first time, we can nevertheless note the total absence of phenolic compounds in our samples.
From an ecological point of view, the C. decurrens species seems to be adaptable to the hard conditions in Corsica, and could be a good alternative for reforestation in some areas, due to its strong resistance against the forest fire.

Plant Material, Isolation of Essential Oils
The experimental Ospedale forest (southeast of the Corsica) located at 960 m altitude (GPS coordinates: 41 • 39,578 N, 009 • 11,413 E) was created by the French National Forest Office ("Office National des Forêts") in 1989 [14]. Thus, these trees are about thirty years old.
We harvested leaf, old branches (parts of branches previously growing), and young branches (parts of branches actually growing), that we will sometimes refer to as, respectively: L; OB, and YB.
A voucher specimen was deposited at the Conservatoire Botanique National de Corse, (Corte, France), under accession numbers GG2776.

Preparation of pin-2-en-8-al (C)
In a 50 mL round bottom flask equipped with a reflux condenser, 225.6 mg (1.05 mmol) of pyridinium chlorochromate (PCC) and 31.1 mg (0.23 mmol) of sodium acetate were suspended in 2 mL of anhydrous CH 2 Cl 2 . Fraction F3 (76.2 mg) containing 51.8% of A (39.5 mg, 0.26 mmol of A) was diluted in 3 mL of CH 2 Cl 2 and added to the PCC solution under stirring in one portion. After 7 h, 20 mL of dry Et 2 O was added and the supernatant decanted from the black gum. The insoluble residue was washed thoroughly 3 times with 10 mL portions of anhydrous Et 2 O whereupon it became a black granular solid. The combined organic layers were passed through a short pad of Florisil, and the solvent was evaporated to yield fraction F3' (42.1 mg) containing 45.6% of compound C. The compound was further purified by means of column chromatography on silica gel (60-200 µm, 60 Å, 30 g) using the following gradient of solvents: pentane/Et 2 O 95/5 to yielded F3'-1 (11.6 mg) containing 80% of pin-2-en-8-al (C).