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Auto-oxidation of Ent-beyer-15-en-19-al isolated from the essential oil of the heartwood of Erythroxylum monogynum Roxb.: formation of 15,16-epoxy-ent-beyeran-19-oic acid and other products

Abstract

Chemical investigation of the essential oil obtained from the heartwood of Erythroxylum monogynum Roxb. yielded three beyerene type diterpenoids ent-beyer-15-ene (1), ent-beyer-15-en-19-ol (erythroxylol A) (2) and ent-beyer-15-en-19-al (3). Ent-beyer-15-en-19-al (3) was found to be unstable at room temperature, giving rise to hitherto unknown 15,16-epoxy-ent-beyeran-19-oic acid (4). This conversion involves the auto-oxidation of a C-4 axial aldehyde group of an ent-beyer-15-ene diterpenoid with the concurrent epoxidation of the C-15 double bond. This is the first report of the auto-oxidation of an aldehyde group to a carboxylic acid group with the concurrent epoxidation of a double bond in the same compound. Further investigation of this observation under controlled conditions resulted in the isolation and identification of ent-beyer-15-en-19-oic acid (5), two new epoxy hydroperoxides, 15,16-epoxy-19-nor-ent-beyeran-4α-hydroperoxide (6a), 15,16-epoxy-18-nor-ent-beyeran-4β-hydroperoxide (6b), and two new hydroperoxides, ent-beyer-19-nor-15-en-4α-hydroperoxide (7), ent-beyer-18-nor-15-en-4β-hydroperoxide (8) and ent-beyer-18-nor-15-en-4β-ol (9). Identification of these compounds was carried out by the extensive usage of spectroscopic data including 1D and 2D NMR. The acid 5 and the alcohol 9 have been reported previously as natural products from Elaeoselinum asclepium and Erythroxylum monogynum. The mechanistic basis of this auto-oxidation reaction is discussed.

Introduction

Erythroxylum monogynum Roxb. (Erythroxylaceae), is a small evergreen tree indigenous to Sri Lanka and India. The oil obtained by the distillation of its heartwood has been used traditionally as a wood preservative [1] and in the local perfumery industry. Many diterpenoids belonging to the kaurane and beyerane groups have been reported from this plant [2]. In continuing our search for industrially useful essential oils in Sri Lanka, we initiated an investigation of the heartwood of E. monogynum. The essential oil obtained by the initial hydrodistillation of the heartwood contained a mixture of monoterpenoids along with three high molecular weight compounds, one of which was identified as ent-beyer-15-ene (1) by Gas chromatography–mass spectrometry (GC–MS) and confirmed by 1H and 13C NMR data. With the view to identifying the remaining two high molecular weight compounds, steam distillation of the heartwood of E. monogynum has been carried out initially at 40 psi for 24 h (Stage 1) exhaustively to remove the monoterpene fraction and then at 70 psi for additional 12 h (Stage 2) to obtain mainly the high molecular weight fraction. The essential oil obtained in the Stage 2 showed the presence of three compounds on GC–MS analysis, one of which was identified as ent-beyer-15-ene (1). The remaining two compounds were identified as erythroxylol A (2) and an unsaturated diterpene aldehyde, ent-beyer-15-en-19-al (3) by the analysis of their 1H and 13C NMR data. This is the first report of 3 from this plant as well as the complete assignment of its 1H and 13C NMR data supported by 2D NMR spectroscopic data. Compound 3 has been reported previously as an unstable oil from the timber of Erythroxylum zambesicum. Its structure had been established by its conversion to the corresponding alcohol (erythroxylol A, 2), and the observation of an aldehyde group, three tertiary methyl groups and a cis double bond in its 1H NMR spectrum [3]. Prior to the report by Ansell [3] it had been reported as a semisynthetic product obtained from the oxidation of erythroxylol A (2) and to be a low melting solid (m. p. 63–65 °C), stable at 0 °C under nitrogen. The structure had been established by reducing it to ent-beyer-15-ene via its ethylene thioacetal [4]. Compound 3 has also been reported from Viguiera grammatoglossa [5] and Myriocephalus stuartii [6].

Compound 3 was found to be unstable at room temperature converting into more polar compounds. Of these the major compound was isolated and identified as 15,16-epoxy-ent-beyeran-19-oic acid (4). According to published data, this is the first report of the auto-oxidation of an aldehyde group to a carboxylic acid group with the concurrent epoxidation of a double bond in the same compound. Prompted by this observation we set-up an experiment to study this auto-oxidation process. In this experiment when 3 was exposed to air as a solution in cyclohexane at room temperature (28–30 °C) it underwent auto-oxidation to give two major products and five minor products. Of the two major products, the more polar one was found to be 4 while the other major compound was identified as ent-beyer-15-en-19-oic acid (5). Further investigation of the reaction mixture enabled us to isolate and identify the five minor compounds produced during this auto-oxidation process as, 15,16-epoxy-19-nor-ent-beyeran-4α-hydroperoxide (6a), 15,16-epoxy-18-nor-ent-beyeran-4β-hydroperoxide (6b), ent-beyer-19-nor-15-en-4α-hydroperoxide (7), ent-beyer-18-nor-15-en-4β-hydroperoxide (8), and ent-beyer-18-nor-15-en-4β-ol (9). Herein we report the occurrence of 3 in E. monogynum along with its complete assignment of 1H and 13C NMR data and the structure elucidation of compounds 4, 5, 6a, 6b, 7–9 (Fig. 1) utilizing their spectroscopic data. The plausible pathway of the formation of these compounds during the auto-oxidation of 3 is also discussed.

Fig. 1
figure1

Structures of ent-beyer-15-ene (1), ent-beyer-15-en-19-ol (erythroxylol A) (2), ent-beyer-15-en-19-al (3) and auto-oxidation products of 3

Results and discussion

Heartwood of E. monogynum was subjected to steam distillation in two stages. The oil obtained in stage 1 was found to contain a mixture of monoterpenoids along with three high molecular weight compounds by GC–MS analysis (see Additional file 2). The oil obtained in stage 2 was shown to consist of mainly the above three major compounds by GC–MS analysis. The oil obtained in stage 2 was subjected to silica gel column chromatography to isolate these three compounds.

Compound 3, a viscous liquid, was identified as ent-beyer-15-en-19-al from the following spectroscopic data. It showed the molecular ion at m/z 286.2 [M]+. Its 1H and 13C NMR spectra (Tables 1 and 2) together with distortionless enhancement by polarization transfer (DEPT)135 and heteronuclear single quantum coherence (HSQC) data showed the presence of three methyls attached to quaternary carbons (δH 0.99 s, 1.00 s, 0.60 s and δC 24.4, 24.8, 14.6), eight methylenes, five methines, two of which are olefinic [δH 5.68 d (J = 5.6 Hz), 5.46 d (J = 5.6 Hz); δC 134.5, 136.8] and one which is aldehydic [δH 9.75 d (J = 1.4 Hz); δC 205.9] and four quaternary carbons accounting for C20H30O. Because the aldehyde carbonyl and the olefinic double bond accounted for two degrees of unsaturation, it was evident that 3 possesses a tetracyclic framework. NMR data together with connectivity of quaternary methyl groups, non-protonated carbons, methylenes and methines which were established by the analysis of heteronuclear multiple bond correlations (HMBC) (Fig. 2) showed that it had an ent-beyerene skeleton. Beyerane diterpenoids are common to Erythroxylum species [3, 4]. The tertiary methyl group at δH 1.00 (s) assigned to CH3-18 showed HMBC correlations with C-3 (δC 34.3), C-4 (δC 48.3), C-5 (δC 56.8) and the aldehyde carbonyl carbon (δC 205.9), placing the methyl and aldehyde groups at C-4. The tertiary methyl group at δH 0.99 showed HMBC correlations with the olefinic carbon at δC 136.8 (C-16), methylene carbons at δC 32.9 (C-12) and 61.0 (C-14) and quaternary carbon at δC 43.7 (C-13), placing it at C-13. The remaining tertiary methyl group at δH 0.60 showed HMBC correlations with the methine carbons at δC 56.8 (C-5), and 51.7 (C-9) and with methylene carbon at δC 38.7 (C-1) placing it at C-10. The olefinic proton at δH 5.46 assigned to H-16 showed HMBC correlations with the quaternary carbon at δC 43.7 (C-13) while the remaining olefinic proton at δH 5.68 showed HMBC correlation with the quaternary carbon at δC 48.9 (C-8) placing it at C-15, while both the olefinic protons showed HMBC correlations with the methylene carbon at δC 61.0 (C-14). Compound 3 underwent reduction with NaBH4 to give ent-beyer-15-en-19-ol (erythroxylol A) (2) confirming its structure as ent-beyer-15-en-19-al.

Table 1 1H NMR (400 MHz) Spectroscopic Data (δ) of Compounds 3, 4, 6a, 6b, 7, and 8 in CDCl3
Table 2 13C NMR (100 MHz) Spectroscopic Data (δ) of Compounds 3, 4, 6a, 6b, 7, and 8 in CDCl3
Fig. 2
figure2

Selected HMBCs of 3

The remaining two compounds isolated from the essential oil were identified as ent-beyerene (1) by its GC–MS data [7] and comparison with reported 13C NMR data [8] (Additional file 2: Table S1) and ent-beyer-15-en-19-ol (erythroxylol A) (2) by the analysis of its 1H and 13C NMR data and comparison with reported 13C NMR data [8] (Additional file 2: Table S1). These two compounds have been reported previously from the timber of E. monogynum [4].

Thin layer chromatographic (TLC) analysis of a solution of compound 3 in cyclohexane showed that the compound was unstable when exposed to air and decomposed to a number of compounds, all of which were more polar than the parent compound. It was observed that the decomposition of 3 was prevented by the addition of butylated hydroxy toluene (BHT) to the solution supporting the view that the decomposition was an auto-oxidation occurring through a free radical mechanism. Compound 3 was found to be stable when stored at 0 °C under nitrogen in the absence of a solvent.

Periodic TLC analysis of a solution of 3 in cyclohexane indicated a changing pattern of spots characteristic of a radical reaction, which stabilized after 14 days to give a pattern of six spots, two of which were present in larger amounts than the others. Compound 4, the more polar one of the two major products was obtained as a viscous liquid, analyzed for C20H30O3 by a combination of high-resolution electro-spray ionization mass spectrometry (HRESIMS) and 13C NMR spectroscopy. The 1H and 13C NMR spectra (Tables 1 and 2) together with HSQC data of 4 indicated that its structure was very similar to that of 3. In comparing the 13C NMR spectrum of 4 with that of 3, the oxidation of the aldehyde group to a carboxylic acid group is clearly indicated by the appearance of a signal at δC 183.9 and the loss of the signal at δC 205.9. This structural change is also reflected in the 1H NMR spectrum, where the signal due to aldehyde proton at δH 9.75 in 3 is absent in 4. Epoxidation of the double bond is indicated by the loss of two olefinic CH groups [δH 5.68 d, (J = 5.6 Hz), 5.46 d, (J = 5.6, Hz); δC 134.5, 136.8] and the appearance of two new oxygenated methines [δH 3.43 d, (J = 3.0 Hz), 3.04 d, (J = 3.0 Hz); δC 55.9, 60.1). The tertiary methyl group at δH 1.25 assigned to 18-H3 [9] showed HMBC correlations with C-3 (δC 37.7), C-4 (δC 43.7), C-5 (δC 56.9) and the carboxyl carbonyl carbon (δC 183.9), placing the methyl and carboxylic acid groups at C-4. The tertiary methyl group at δH 1.02 showed HMBC correlations with the oxygenated methine carbon at δC 60.1 (C-16) and methylene carbon at δC 46.6 (C-14) placing it at C-13. The remaining tertiary methyl group at δH 0.84 showed HMBC correlations with the methine carbons at δC 56.9 (C-5), and 55.7 (C-9) placing it at C-10. The oxygenated methine proton at δH 3.04 (d, J = 3.0 Hz) assigned to H-16 showed HMBC correlation with the quaternary carbon at δC 38.9 (C-13) and the remaining oxygenated methine proton at δH 3.43 (d, J = 3.0 Hz) showed HMBC correlation with the quaternary carbon at δC 44.3 (C-8) placing it at C-15, while both the oxygenated methine protons showed HMBC correlations with the methylene carbon at δC 46.6 (C-14). Connectivity of remaining carbons was established by the HMBC correlations as shown in Fig. 3a. Irradiation of 15-H [δH 3.43 d (J = 3.0 Hz)] in the Selective nuclear Overhauser effect spectroscopy (NOESY) Gradient experiment exhibited enhancement of 20-H3 (δH 0.84 s) (Fig. 3b) (Additional file 2: Figure S15) suggesting the α- orientation of the 15-H, confirming the formation of epoxide from the β-face (exo epoxide). The formation of the epoxide brings about clear differentiation of the two H atoms on 14-C with one H moving up-field to δH 0.53 as a doublet with a coupling constant of 11.0 Hz, typical for a geminal coupling. Thus, structure of 4 was determined to be 15,16-epoxy-ent-beyeran-19-oic acid.

Fig. 3
figure3

a Selected HMBCs and b NOEs observed in Selective NOESY gradient experiments of 4

Compound 5, the less polar major product was obtained as a colorless viscous liquid. The 1H and 13C NMR spectroscopic data together with HSQC and DEPT135 of 5 (Additional file 2: Table S2 and Figures S16–S21) showed very close resemblance of its structure to those of 3 and 4. The presence of a carboxylic acid C = O was indicated by the signal at δC 183.8 in its 13C NMR spectrum while the presence of the two olefinic protons as in the case of 3 were clearly evident by the presence of the signals at δH 5.73 d (J = 5.7 Hz); δC 134.8 (CH-15) and δH 5.45 d (J = 5.7 Hz); δC 136.5 (CH-16). Thus 5 was identified as ent-beyer-15-en-19-oic acid by the comparison of its 13C NMR data with reported data for ent-beyer-15-en-19-oic acid (Additional file 2: Table S2), which has been isolated from Elaeoselinum asclepium [10]. As the reported 13C NMR data of this compound was not supported by 2D NMR (HSQC and HMBC) spectroscopic data and its 1H NMR data was not available, we assigned the 1H and 13C NMR data of 5 with the help of HSQC and HMBC correlations (Additional file 2: Table S2).

The most polar minor auto-oxidation product (6) obtained as a colorless viscous liquid was determined to be a mixture of two isomers 6a and 6b epimeric at C-4 in 1:2 ratio from the following evidence. GC–MS analysis showed two peaks in the GC, both of which showed the same [M]+m/z 306.4; 1H NMR spectrum of 6 showed two doublets at δH 3.40 (J = 3.0 Hz) and δH 3.47 (J = 3.0 Hz) in 2:1 ratio and 6 tertiary methyl groups and 13C NMR spectrum showed 37 carbon signals. 1H and 13C NMR spectroscopic data (Tables 1 and 2) together with HSQC and DEPT135 revealed that it contained eight quaternary carbons of which two are oxygenated (δC 84.7 and 84.0), eight methines of which four are oxygenated [δH 3.40 d (J = 3.0 Hz), 3.03 d (J = 3.0 Hz), 3.47 d (J = 3.0 Hz), 3.02 d (J = 3.0 Hz); δC 55.9, 60.16, 56.0, 60.20], sixteen methylenes and six tertiary methyls (δH 0.92 s, 1.02 s, 1.13 s, 1.03 s, 1.01 s, 1.30 s; δC 15.6, 21.5, 18.3, 16.0, 21.5, 24.7) of which two overlapped at δC 21.5 in the 13C NMR spectrum. These data suggested that these two isomers lack a carbon atom from each of these two isomers and presence of 15(16) epoxide as in the case of compound 4. 13C NMR spectrum of 6 did not show either CHO or CO2H carbon signals but showed two signals at δC 84.7 and 84.0 for oxygenated quaternary carbons indicating that the 18 (or 19) C has been lost from the beyerane skeleton and a hydroperoxide (-OOH) group has been attached to 4-C, suggesting the possibility of these two being C-4 epimers of each other. Analysis of HMBC correlations (Fig. 4) permitted the unambiguous assignment of NMR signals of each of the epimers (Tables 1 and 2).

Fig. 4
figure4

Selected HMBCs of 6a and 6b

Differentiation of epimers 6a and 6b and the relative configuration at C-4 in 6b was achieved by a series of Selective NOESY Gradient experiments (Fig. 5). Irradiation of the signal at δH 0.92 (20-H3) in a Selective NOESY Gradient experiment caused enhancement of the methyl signal at δH 1.13 (19-H3) and the oxygenated methine proton at δH 3.40 (15-H) suggesting that these two methyl groups and the oxygenated methine proton belong to the epimer 6b and both methyl groups are on the same side of the molecule confirming α-orientation of the C-4 methyl group. Further it is evident that the H-15 is also α-orientated confirming the formation of epoxide from the β-face (exo epoxide) as in the case of 4. Irradiation of the methyl signal at δH 1.13 (19-H3) caused enhancement of signal at δH 0.92 (20-H3) confirming the above (Additional file 2: Figures S29 and S30). When the signal at δH 1.30 [18-H3 (Me at C-4)] of 6a was irradiated in a Selective NOESY Gradient experiment no enhancement of signals was observed confirming the β-orientation of this methyl group (Additional file 2: Figure S31).

Fig. 5
figure5

3D structures of 6a and 6b showing NOE observed in Selective NOESY gradient experiments of 6b

Methyl protons at δH 1.13 (19-H3) in compound 6b showed HMBC correlations with the quaternary carbon at δC 84.7 (C-4), methylene carbon at δC 35.3 (C-3) and methine carbon at δC 50.5 (C-5) placing this methyl at C-4. Further the methylene proton signals at δH 1.72 and 1.40 (2-H2) and the methine proton signal at δH 1.54 (H-5) showed HMBC correlations with the quaternary carbon at δC 84.7 (C-4). The methyl protons at δH 0.92 assigned to CH3-20 showed HMBC correlations to methylene carbon at δC 38.3 (C-1), quaternary carbon at δC 38.4 (C-10), and methine carbons at 50.5 (C-5) and 56.3 (C-9). The protons of remaining methyl group at δH 1.02 (17-H3) showed HMBC correlations to quaternary carbon at δC 39.0 (C-13), methine carbon at δC 60.16 (C-16), methylene carbons at δC 35.5 (C-12) and 46.7 (C-14). Methine protons of the epoxide ring at δH 3.03 (H-16) and δH 3.40 (H-15) showed HMBC correlations to quaternary carbons δC 39.0 (C-13) and δC 44.2 (C-8) respectively while both of them showed correlations to δC 46.7 (C-14). The remaining key HMBC correlations useful for the establishment of connectivity in the molecule are shown in Fig. 4.

It appears that the remaining three methyl signals in the 1H NMR spectrum of 4 responsible for tertiary methyl groups belong to the epimer 6a. The methyl protons at δH 1.30 (18-H3) in compound 6a showed HMBC correlations with the quaternary carbon at δC 84.0 (C-4), methylene carbon at δC 34.9 (C-3) and methine carbon at δC 55.9 (C-5) placing this methyl at C-4. Methyl protons at δH 1.03 (20-H3) showed HMBC correlations with the methylene carbon at δC 39.3 (C-1) and methine carbons at δC 55.9 (C-5) and δC 56.1 (C-9). The remaining methyl group at δH 1.01 (17-H3) showed HMBC correlations to quaternary carbon at δC 39.1 (C-13), methine carbon at δC 60.20 (C-16), methylene carbons at δC 35.4 (C-12) and 46.9 (C-14). Methine protons of the epoxide ring at δH 3.47 (15-H) and δH 3.02 (16-H) showed HMBC correlations to quaternary carbons δC 44.1 (C-8) and 39.1 (C-13) respectively while both of them showed correlations to δC 46.9 (C-14). The remaining key HMBC correlations useful for the establishment of connectivity in the molecule are shown in Fig. 4. Although these two epimers were inseparable under normal phase chromatographic techniques, it was possible to separate them by reverse phase analytical TLC and the two compounds were subjected to HRESIMS to determine their molecular formulae. Compound 6a analyzed for C19H30O3, m/z 307.22594 [M + H]+ (calcd. for C19H31O3, 307.22746), m/z 305.21215 [M – H] (calcd. for C19H29O3, 305.21180) and 6b analyzed for C19H30O3, m/z 307.22617 [M + H]+ (calcd. for C19H31O3, 307.22746), m/z 305.21217 [M—H] (calcd. for C19H29O3, 305.21180). These data confirmed the structures of 6a and 6b as15,16-epoxy-19-nor-ent-beyeran-4α-hydroperoxide and 15,16-epoxy-18-nor-ent-beyeran-4β-hydroperoxide respectively. The 1H and 13C NMR spectra obtained for these two samples were found to be consistent with the assignments made for 6a and 6b based on the above analysis of the spectra of their mixture 6 (Additional file 2: Figures S32–S37).

Compound 7 obtained as a colorless viscous liquid, analyzed for C19H30O2 by a combination of HRESIMS and 13C NMR spectroscopy. 1H and 13C NMR spectroscopic data (Tables 1 and 2) together with HSQC and DEPT135 of compound 7 showed the presence four quaternary carbons of which one is oxygenated (δC 84.2), four methines of which two are olefinic [δH 5.71 d (J = 5.7 Hz), 5.45 d (J = 5.7 Hz); δC135.2, 136.4], eight methylenes and three tertiary methyls (δH 0.99 s, 1.28 s, and 0.85 s; δC 24.9, 24.8, and 15.5). Comparison of this data with the corresponding data for 6a/6b suggested that 7 could be a C-4 hydroperoxide similar to 6a or 6b but with a C-15 olefinic double bond. Compound 8 obtained as a colorless viscous liquid, analyzed for C19H30O2 by a combination of HRESIMS and 13C NMR spectroscopy. 1H and 13C NMR spectroscopic data (Tables 1 and 2) together with HSQC and DEPT135 of compound 8 also showed the presence four quaternary carbons of which one is oxygenated (δC 85.0), four methines of which two are olefinic [δH 5.67 d (J = 5.7 Hz), 5.46 d (J = 5.7 Hz); δC 135.0, 136.6], eight methylenes and three tertiary methyls (δH 0.99 s, 1.12 s, and 0.74 s; δC 24.9, 18.4, and 14.9) suggesting that this could be the C-4 epimer of 7. Unambiguous assignment of 1H and 13C NMR signals of each of the compounds 7 and 8 was enabled by the analysis of HMBC correlations of respective compounds (Fig. 6).

Fig. 6
figure6

Selected HMBC correlations of 7 and 8

Since 7 and 8 are C-4 epimers of each other it was necessary to establish the stereochemistry at C-4 in these two compounds. Comparison of the 1H and 13C NMR chemical shifts of C-4 methyl groups (18-H3 or 19-H3) and 20-H3 of these two compounds with those of 6a and 6b (Tables 1 and 2) indicated that the hydroperoxide group in 7 is α-oriented and in 8 it is β-oriented. This was further confirmed by series of Selective NOESY Gradient experiments. Irradiation of the signal at δH 0.74 (20-H3) of 8 in a Selective NOESY Gradient experiment caused enhancement of the methyl signal at δH 1.12 (19-H3) suggesting that these two methyls are in the same side of the molecule indicating the α-orientation of the C-4 methyl group. Irradiation of the methyl signal at δH 1.12 (19-H3) caused enhancement of signal at δH 0.74 (20-H3) further confirming the above suggestion (Additional file 2: Figures S50 and S51). Irradiation of the signal at δH 0.85 (20-H3) or δH 1.28 (18-H3) of 7 did not cause enhancements of either signals (Additional file 2: Figures S43 and S44) suggesting that these two methyl groups are not in the same face and hence suggested the β-orientation of methyl group at C-4 (18-H3). Thus, the compounds 7 and 8 were identified as ent-beyer-19-nor-15-en-4α-hydroperoxide and ent-beyer-18-nor-15-en-4β-hydroperoxide respectively.

Compound 9 was obtained as a white, amorphous solid. Comparison of 1H and 13C NMR spectroscopic data (Additional file 2: Table S3) together with HSQC and DEPT135 of compound 9 with those of 7/8 suggested that this is a C-4 alcohol with C-15 olefinic double bond. Although both ent-beyer-18(19)-nor-15-en-α- and β-ols were known, their spectroscopic assignments were not supported by 2D NMR (HSQC and HMBC) spectroscopic data. Hence, we assigned the 1H and 13C NMR data of this compound with the help of HSQC and HMBC data. The HMBC correlations useful for the assignment of 1H and 13C NMR signals confirming the structure of compound 9 are shown in (Additional file 2: Table S3). Stereochemistry at C-4 has been established by carrying out Selective NOESY Gradient experiments. Irradiation of the signal at δH 0.71 (20-H3) of 9 in a Selective NOESY Gradient experiment caused enhancement of the methyl signal at δH 1.14 (19-H3) indicating that these two methyl groups are in the same side of the molecule confirming α- orientation of the C-4 methyl group. Irradiation of the methyl signal at δH 1.14 (19-H3) caused enhancement of signal at δH 0.71 (20-H3) further confirming the above α- orientation of the C-4 methyl group (Additional file 2: Figures S57 and S58). Thus, the compound 9 was identified as ent-beyer-18-nor-15-en-4β-ol, which has been isolated previously as a natural product from E. monogynum [11].

The susceptibility of 4-axial aldehyde groups in the diterpenes towards auto-oxidation giving rise to carboxylic acids and hydroperoxides via radical mechanisms has been previously reported [10, 12, 13]. Although 3 was known to be an unstable compound, there have been no previous reports on the products obtained from the auto-oxidation of 3. The formation of the epoxy compounds 4, 6a and 6b during the auto-oxidation of 3 can be rationalized by considering the steps involved in the auto-oxidation of aldehydes to carboxylic acids. Auto-oxidation of an aldehyde to the corresponding carboxylic acid is a facile reaction and takes place via a free radical mechanism [14, 15]. Acyl peroxy radicals and per-acids are generated as intermediates during the reaction (Scheme 1). Both these species are capable of epoxidizing an olefinic double bond. Thus, both catalyzed and uncatalyzed processes for the epoxidation of an olefin coupled to the oxidation of an aldehyde to the corresponding carboxylic acid by molecular oxygen have been reported [16,17,18,19,20].

Scheme 1
scheme1

Auto-oxidation of an aldehyde group with concurrent epoxidation of a carbon–carbon double bond

We envisage the epoxidation taking place via an intermolecular reaction between a C-19 peroxy group and the C-15,16 olefin group giving rise to the exo-epoxide because the approach of the two species to form an endo-epoxide would be sterically hindered. Steric hindrance of the α-face of the molecule exerted by the axial 20-methyl group would also allow epoxidation to compete with the usual bimolecular termination reaction (Scheme 1) between the peroxy acid and the aldehyde to give two molecules of carboxylic acids. This interpretation is supported by the observation that the C-20,29 double bond of betulonaldehyde does not undergo epoxidation during the auto-oxidation of its unhindered C-28 formyl group [21] which can be approached without hindrance from the β-face of the molecule. It is interesting to note that betulonaldehyde on auto-oxidation gave in addition to betulonic acid, two epimeric C-17 hydroperoxides which would correspond to 7 and 8 in the current study. In a related process, 14-hydroxypimara-8,15-dien-19-oic acid has been isolated from the auto-oxidation of pimara-8(14),15-dien-19-al (which also has an axial aldehyde group) [12]. It has been proposed that the 14-hydroxy compound arises from the ring opening hydrolysis followed by dehydration of the corresponding 8(14)-epoxy carboxylic acid on the basis of chromatographic evidence, although such an epoxy carboxylic acid has not been isolated from the reaction mixture from the auto-oxidation reaction.

The formation of the epimeric epoxy hydroperoxides 6a and 6b, the epimeric hydroperoxides 7 and 8, and the alcohol 9 can be explained (Scheme 2) as arising from the reactions of the tertiary cycloalkyl radical at C-4 which can be formed by the decarbonylation of the acyl radical and decarboxylation of the acyloxy radical that are formed during the auto-oxidation process [12, 15].

Scheme 2
scheme2

Possible pathways of formation of the epimeric epoxy hydroperoxides 6a and 6b, epimeric hydroperoxides 7 and 8, and the alcohol 9

The ease of decarbonylation of tertiary acyl radicals is well known. The decarbonylation and decarboxylation reactions are further aided by the loss of steric strain when the sp3 carbon (C-4) changes to a planar sp2 carbon removing the 1,3-diaxial interaction of the radical on C-19 with the 20 α- methyl group. The 20 α- methyl group also directs the approach of molecular oxygen and the peracid group to preferentially approach the planar C-4 radical from the β- face by sterically hindering the α- face approach. This results in the excess of 6b over 6a, as observed in the 1H NMR spectrum of 6, (the mixture of 6a and 6b) isolated by column chromatography of the auto-oxidation reaction mixture. Further, of the two possible epimeric alcohols, only the β- alcohol 9 could be detected. However, we note that the preference for the β- face approach of molecular oxygen is not reflected in the relative isolated yields of the hydroperoxides 7 and 8, probably due to experimental losses of yield. Although 9 has been reported as a natural product isolated from the timber of E. monogynum [11], our results support the suggestion by Caputo [13] and Tanaka [12] that it was an artifact arising from the auto-oxidation of 3.

The auto-oxidation of 3 is inhibited when it is found as a component of the essential oil of E. monogynum by the presence of other components such as 4, which can act as radical chain breakers. However, we note that no trace of 3 could be detected by GC analysis in a 1 year old sample of the essential oil which had been stored under ambient conditions.

Conclusions

The auto-oxidation of the aldehyde group of ent-beyer-15-en-19-al to a carboxylic acid group is a facile process and takes place both with and without the concurrent epoxidation of the 15,16-double bond. Our results suggest that 4-hydroxy-19-nor-ent-beyer-15-ene which has been reported previously as a natural product from E. monogynum may be an artefact arising from the auto-oxidation reaction. Steric hindrance exerted by the axial 20-methyl group plays a determining role in the product distribution of the auto-oxidation reaction. The usage of the essential oil from the heartwood of E. monogynum in perfumery will be limited by the instability in the presence of oxygen of ent-beyer-15-en-19-al, which is a major component of the oil.

Experimental

General experimental procedures

Optical rotations were measured in CHCl3 with a BioBase Automatic polarimeter BK-P2. IR spectra were recorded as Attenuated total reflections spectra on a Perkin Elmer Spectrum 2. 1D and 2D NMR spectra were recorded in CDCl3 with a Bruker Ascend 400 spectrometer at 400 Mz for 1H NMR and 100 MHz for 13C NMR using residual CHCl3 as the internal reference. High resolution MS were measured on a Q-Exactive (ThermoScientific) equipment, with H-ESI source. Thin layer chromatography was carried out on Merck analytical normal phase (G60, F254, 0.2 mm) and reverse phase (Silica gel 60 RP-18, 0.25 mm) plates. Preparative layer chromatography was carried out on Analtech normal phase plates (G60, 0.5 mm). All solvents used for chromatography were of AR grade from Sigma-Aldrich. Compounds were visualized by spraying with anisaldehyde-sulfuric acid reagent made by mixing anisaldehyde (0.5 ml) with glacial acetic acid (10 ml), followed by 85 ml of methanol and concentrated sulfuric acid (5 ml). Dry column chromatography was carried out using Analtech silica gel (35–75 micron, 150 A). Column fractions were analyzed by TLC and similar fractions were combined and evaporated under reduced pressure. GC–MS analysis was carried out using an Agilent 7890 A GC system equipped with 5975C inert XL MSD Triple–Axis Detector, and HP-5 MS fused silica capillary with a (5% Phenyl)-methylpolysiloxane stationary phase (30 m × 0.25 mm Id, x 0.25 µm film thickness) capillary column. Helium (99.999%) was used as carrier gas. Mass spectra were acquired in the EI mode at 70 eV within the range of 40.5 to 500 mass units.

Plant material

The heartwood of E. monogynum was collected from a tree found at a home garden in Weerawila, Sri Lanka (6˚14ʹ 56.9ʹʹ N, 81˚13ʹ 46.6ʹʹ E). The species was identified by Prof. D. S. A. Wijesundara, National Institute of Fundamental Studies, Hantane, Kandy, Sri Lanka (former Director of the Royal Botanic Gardens, Peradeniya and the National Herbarium of Sri Lanka). A voucher specimen (Voucher no.43-002-010) was deposited in the herbarium at the R & D division at LINK Natural Products, Sri Lanka.

Extraction of essential oil

The dried heartwood (46 kg) of E. monogynum was cut into 8 mm pieces and subjected to steam distillation in two stages. A steam pressure of 40 psi was used, and the flow rate of the condensate was 500–600 mL min−1 during the first stage (24 h) to obtain 76.8 g of a pale yellow, light oil. Distillation was continued for an additional 12 h (stage 2) at a steam pressure of 70 psi and a condensate flow rate of 800–900 mL min−1 to obtain 40.8 g of a thick oil. Both fractions were dried over anhydrous sodium sulfate and stored at − 4 °C in a refrigerator. GC–MS analysis of the oil obtained in stage 2, indicated the presence of three major compounds, of which two were identified as ent-beyer-15-ene (1) and erythroxylol A (2). GC–MS analysis of the oil obtained in stage 1 showed that it also contained these three compounds, in lower concentrations along with lower boiling monoterpenoids.

Ent-beyer-15-ene Diterpenoids from the essential oil of E. monogynum

The oil obtained in stage 2 of the above steam distillation (1.08 g) was subjected to dry column chromatography using gradient elution with hexane-dichloromethane and 70 fractions (10 mL) were collected. These were analyzed by TLC and fractions containing same compound were combined. Fraction 2 on evaporation under reduced pressure gave ent-beyer-15-ene (1, 230 mg, 21.00%) as a colorless, odiferous viscous liquid. Fractions 3–15 gave ent-beyer-15-en-19-al (3, 118 mg, 1.09%) as a colorless odiferous viscous liquid and fractions 38–60 gave erythroxylol A (2, 180 mg, 1.66%) as an off-white amorphous powder.

Ent-beyer-15-ene (1)

A colorless viscous liquid; \({[\upalpha ]_{{\text{D}}}}^{{25}}\) +26.5° (c 0.061, CHCl3); IR (FT-ATR) νmax 2920, 2844, 1386, 1364,750 cm−1; GC–MS (tR 36.7 min) m/z 272.3, M+. Identity of Ent-beyer-15-ene was established by comparison with reported GC–MS and 13C NMR data [7, 8] (Additional file 2: Table S3).

Erythroxylol A (2)

An off white amorphous solid; \({[\upalpha ]_{{\text{D}}}}^{{25}}\) +24.24° (c 0.0033, CHCl3); IR (FT-ATR) νmax 3380, 2922, 2865, 2845, 1727, 1448, 1379, 1364, 1025. 973, 749; 1H and 13C NMR spectroscopic data (Additional file 2: Table S3); GC–MS (tR 40.24 min) m/z 288.3, M+. Identity of erythroxylol A was established by comparison with reported 13C NMR data [8].

Ent-beyer-15-en-19-al (3)

A colorless viscous liquid;\({[\upalpha ]_{{\text{D}}}}^{{25}}\) +27.55° (c 0.075, CHCl3); IR (FT-ATR) νmax 2931, 2866, 2846, 1716, 1450, 751 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; GC–MS (tR 39.3 min) m/z 286.3, M+.

Auto-oxidation of ent-beyer-15-en-19-al (3)

Ent-beyer-15-en-19-al) (3, 118 mg) was dissolved in cyclohexane 50 mL and kept at room temperature (29 ± 2 °C) and monitored daily by TLC (mobile phase, CH2Cl2; visualizing agent, anisaldehyde-sulfuric acid reagent) for 2 weeks. During the initial period a variable pattern of spots was observed with some spots being transient while others were more long-lasting. A stable TLC pattern of spots was obtained towards the end of the 2-weeks period. It was observed that 3 has been converted into at least six different compounds. The total reaction mixture was chromatographed over a column of dry silica gel (15 g) sequentially eluting with n-hexane (200 mL), n-hexane:CH2Cl2 (95:5) (200 mL), n-hexane: CH2Cl2 (90: 10) (200 mL), n-hexane:CH2Cl2 (80:20) (500 mL), n-hexane:CH2Cl2 (75:25) (600 mL), n-hexane:CH2Cl2 (70:30) (200 mL), n-hexane:CH2Cl2 (60:40) (200 mL), n-hexane:CH2Cl2 (50:50) (200 mL), n-hexane:CH2Cl2 (40:60) (200 mL), CH2Cl2 (300 mL), CH2Cl2: EtOAc (90:10) (300 mL), and CH2Cl2: EtOAc (80:20) (200 mL). A total of 453 fractions (F1–F453) were collected (F1–F132, 10 mL each and F133–F 453, 5 mL each). The fractions were analyzed by TLC and similar fractions were combined and the solvents were evaporated under reduced pressure. F420–F452 gave 4 (25.5 mg, 19.5%), F163 – F212 gave 3 (32.9 mg 26.5%) and F85–F96 gave 7 (4.5 mg, 3.7%). F358–F389 gave 6 (9.5 mg, 7.5%) which was found to be a mixture of epimers, 6a and 6b by the analysis of NMR spectroscopic data. This mixture of epimers (8.0 mg) was separated by reverse phase TLC using MeOH: H2O (9:1) as the eluent (double development, path length 20 cm) to obtain 6a (2.0 mg) and 6b (3.0 mg). Fractions F105– F132 gave a colorless oily mass (7.0 mg) which gave 8 (3.2 mg, 2.2%) and 9 (1.7 mg 1.5%) on separation by preparative TLC using CH2Cl2 as the eluent (double development, path length 20 cm).

15,16-epoxy-ent-beyeran-19-oic acid (4)

A colorless viscous liquid; [α]25D–24° (c 0.00525, CHCl3); IR (FT-ATR) νmax 2946, 2849, 1693, 1454, 1257, 847 cm-1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z, 319.22638 [M + H]+ (calculated for C20H31O3, 319.22746).

Ent-beyer-15-en-19-oic acid (5)

A colorless viscous liquid; [α]25D +10.9° (c 0.0044, CHCl3); IR (FT-ATR) νmax 2945, 2846, 1693, 1451, 1255, 753 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; GC–MS (tR 20.77 min), m/z 302.2, M+.

15,16-epoxy-19-nor-ent-beyeran-4α-hydroperoxide (6a)

A colorless viscous liquid; [α]25D +72.7° (c 0.00165, CHCl3); IR (FT-ATR) νmax 3357. 2925, 2868, 2860, 1725, 1455, 1382, 992, 81,846,820,752, 497 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2. HRESIMS m/z 307.22594 [M + H]+ (calcd. for C19H31O3, 307.22746), m/z 305.21215 [M–H] (calcd. for C19H29O3, 305.21180).

15,16-epoxy-19-nor-ent-beyeran-4β-hydroperoxide (6b)

Colorless viscous liquid; [α]25D + 66.7 (c 0.0009, CHCl3); IR (FT-ATR) νmax 3316, 2945, 2926, 2850, 1729, 1455, 1370, 996, 882, 847, 814, 747, 498 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2. HRESIMS, m/z 307.22617 [M + H]+ (calcd. for C19H31O3, 307.22746), m/z 305.21217 [M–H] (calcd. for C19H29O3, 305.21180).

Ent-beyer-19-nor-15-en-4α-hydroperoixde (7)

A colorless viscous liquid; [α]25D +21.82° (c 0.00275, CHCl3); IR (FT-ATR) νmax 3330, 2922, 2846, 1451, 1365, 1187, 749 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2. HRESIMS, m/z 289. 21728 [M–H]¯ (calculated for C19H29O2, 289.21689).

Ent-beyer-18-nor-15-en-4β-hydroperoixde (8)

A colorless viscous; [α]25D +50.53° (c 0.00095, CHCl3); IR (FT-ATR) νmax 3393, 2924, 2848, 1452, 1383, 1187, 751 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2. HRESIMS, m/z 289. 21732 [M–H]¯ (calculated for C19H29O2, 289.21689).

Ent-beyer-18-nor-15-en-4β-ol (9)

A colorless viscous liquid; [α]25D +53.33° (c 0.00075, CHCl3); IR (FT-ATR) νmax 3376, 2923, 2850, 1740, 1454, 1383, 1364, 1187, 751 cm−1; 1H and 13C NMR spectroscopic data, (Additional file 2: Table S2). HRESIMS, m/z 257. 22601 [M + H–H2O]+ (calculated for C19H30O, 257.22707).

Reduction of Ent-beyer-15-en-19-al (3) to Erythroxylol A (2)

Ent-beyer-15-en-19-al (3, 58.5 mg, 0.20 mmol) was dissolved in 25 mL of dry MeOH (dried over molecular sieve-4A) and added excess sodium borohydride. The reaction mixture was kept overnight at room temperature. The reaction mixture was poured into 50 ml of distilled water and acidified with 2 M hydrochloric acid. The acidified reaction mixture was partitioned with CH2Cl2 (100 ml x 3) and the organic phase was evaporated under reduce pressure to obtain 49 mg of a crude product as white solid which was purified by column chromatography on dry silica eluting with hexane (200 mL) hexane: CH2Cl2 (95:5) (300 mL) to obtain erythroxylol A (2, 30 mg, 51.2%) whose identity was confirmed by comparison with the sample of erythroxylol A isolated by us (TLC, GC-MS).

Availability of data and materials

The data supporting the conclusions of this article is included within the article and its additional files.

Abbreviations

BHT:

Butylated hydroxy toluene

DEPT:

Distortionless enhancement by polarization transfer

GC–MS:

Gas chromatography-mass spectrometry

HMBC:

Heteronuclear multiple bond correlation

HRESIMS:

High resolution electro-spray ionization mass spectrometry

HSQC:

Heteronuclear single quantum coherence

NOESY:

Nuclear overhauser effect spectroscopy

TLC:

Thin layer chromatography

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Acknowledgements

We thank Prof. Cecília Maria Alves de Oliveira, Regional Center for Technological Development and Innovation (CRTI), Federal University of Goiás, Brazil, for high resolution mass spectroscopic data. We also thank Dr. Nuwan Deleepa De Silva, Sri Lanka Institute of Nanotechnology (SLINTEC) and Prof. Nilwala Kottegoda, University of Sri Jayewardenepura, Sri Lanka, for NMR spectroscopic data. Mr. Duminda Jayewardena and Mr. D. Nishantha H. K Samarajeewa, Link Natural Products Pvt. Ltd are acknowledged for their skillful technical assistance.

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TMSGT carried out the experimentation, participated in analysis of spectroscopic data and contributed to the preparation of the manuscript. KTDDeS and CP contributed to the analysis of data and GMKBG elucidated the structures and contributed to the preparation of the manuscript. AMA conceptualized the study design and contributed to the spectroscopic data analysis and the preparation of the manuscript. DSAW identified the plant material and participated in the preparation of the manuscript. All authors read and approved the final manuscript.

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Correspondence to G. M. Kamal Bandara Gunaherath.

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Tennakoon, T.M.S.G., Gunaherath, G.M.K.B., De Silva, K.T.D. et al. Auto-oxidation of Ent-beyer-15-en-19-al isolated from the essential oil of the heartwood of Erythroxylum monogynum Roxb.: formation of 15,16-epoxy-ent-beyeran-19-oic acid and other products. BMC Chemistry 14, 18 (2020). https://doi.org/10.1186/s13065-020-00671-9

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Keywords

  • Erythroxylaceae
  • Erythroxylum monogynum
  • Essential oil
  • Auto-oxidation
  • Diterpenoids
  • Epoxy bayeranes
  • Hydroperoxides
  • Axial aldehyde group
  • 1D and 2D NMR