Diterpenes from Erythroxylum barbatum

Das raízes de Erythroxylum barbatum foram isolados dois novos diterpenos entrosan-1ona-5α,15ξ,16-triol (1) e ent-11α-acetoxi-devadarano-15ξ,16-diol (2), além do ent-devadarano15ξ,16-diol (erythroxidiol X, 3) e do procesterol. Investigação do lenho do caule forneceu o ent-devadarano-11α,15ξ,16-triol (erythroxitriol Q, 4), o ent-devadarano-7β,15ξ,16-triol (hidroxidevadarool, 5), o β-sitosterol e seu glicosídeo. A caracterização estrutural, inclusive a estereoquímica relativa dos compostos, foi estabelecida com base em métodos espectroscópicos, particularmente RMN uni e bidimensional, e comparação com dados da literatura.


Introduction
The genus Erythroxylum comprises 250 species, which are widely distributed throughout the tropics, with large areas of diversity in South America, Africa and Madagascar.6][7] However, with exception of the cocaineproducing species E. coca and E. novogranatense, 8 few attention has been focused on the phytochemical analysis of this genus and the chemical constituents of many species used in traditional medicine still remain unknown.

Results and Discussion
Compound 1 was isolated as a colorless resin.The IR spectrum showed characteristic absorption of hydroxy group at 3421 cm -1 and keto group at 1708 cm -1 .
The 1 H NMR spectrum displayed signals for four angular methyl groups [δ H 1.18 (6H) and 1.00 and 0.58].In addition, were observed three carbinolic hydrogens at δ H 3.34 (dd, J 9.4, 2.9 Hz, H-15); δ H 3.76 (dd, J 10.7, 2.9 Hz, H-16a) and 3.54 (dd, J 10.7, 9.4 Hz, H-16b) in a typical ABC pattern suggesting the presence of a 1,2-glycol system.These observations were further confirmed by the 1 H-1 H COSY experiment through the cross-peaks for the geminal coupling of the methylene hydrogens at δ H 3.76 and 3.54 (2H-16), and for both of them with the hydrogen at δ H 3.34 (H-15).Further, a spin subsystem constituted by two adjacent methylene hydrogens at δ H 2.39 (m, H-2) and 1.48 (m, H-3), which were placed between a quaternary carbon and the keto group was characterized.
The 13 C NMR spectrum exhibited resonances for four methyl groups, eight methylenes, three methines, and five non-hydrogenated carbons, one of which was associated to a carbonyl group (δ C 212.6).
Pimarane, isopimarane and rosane skeletons have four angular methyl groups and can be differentiated by the relative stereochemistry at C-3 and the position of the methyl at C-20, respectively.Rosanes arise from migration of the C-10 methyl group of pimaranes to the C-9 and occur in both enantiomeric series.The placement of a methyl group at C-9 in a rosane skeleton for compound 1 was readily established by detailed examination of the HMBC spectrum, through correlations of the hydrogen at δ H 2.05 (H-10) with the methyl group at δ C 11.9 (C-20) and the carbon at δ C 24.2 (C-6).Furthermore, were observed correlations for the methylene hydrogens at δ H 1.48 (H-3) with the carbons at δ C 29.5 (C-19) and 29.3 (C-18), and of the methylene group at δ H 1.40 and 1.09 (H-6) with the carbons at δ C 62.8 (C-10) and 40.2 (C-8), respectively.In the same way, correlations of the signal relative to methyl groups at δ H 1.18 (H-18, H-19) and of the hydrogen at δ H 1.70 (H-7) with the carbon at δ C 71.2 (C-5), indicated that the location of the tertiary alcohol at C-5 could be assumed.The position of the carbonyl group at C-1 was deduced by the correlation of its 13 C NMR chemical shift at δ C 212.6 with the hydrogens at δ H 2.39 (H-2) and 1.48 (H-3).The absolute configuration of 1 was not ascertained particularly because its levorotatory property, however, it is reasonable to assume that it possess an absolute stereochemistry belonging to the enantio-series, considering the entrosane skeleton of diterpenoids isolated previously from Erythroxylum species 5,7 that led to the "Absolute Stereochemistry Biogenetic Rule" suggested by Sukh Dev. 14 Thus, the structure of 1 could be identified as the new ent-rosan-1-one-5α,15ξ,16-triol.
Compound 2, a white amorphous solid, showed a molecular ion at m/z 364, compatible with a molecular formula C 22 H 36 O 4 .The IR spectrum revealed absorption of hydroxyl group at 3456 cm -1 , carbonyl ester at 1724 cm -1 and C-O stretching at 1249 cm -1 .
Comparative analysis of BB and DEPT- 13 C NMR (Table 1) spectra revealed 22 signals.From these data one can easily deduce the presence of three oxygen bearing saturated carbons at δ C 62.5 (CH 2 ), 78.2 (CH) and 80.6 (CH), and 18 other sp 3 not functionalized carbon signals (4 methyls, 8 methylenes, 2 methines and 4 quaternaries), in addition of a characteristic carbonyl of an ester function at δ C 162.5.Carbonyl carbons of acetate derivatives of alcohols usually appear around 170 ppm, thus the relative shielding of the acetate carbonyl (∆δ ~ 8.0 ppm) may indicates stereochemical implications related to positioning either as α-equatorial or β-axial as observed for other devadarane diterpenoids. 12The unambiguous assignment of all carbon and hydrogens was possible for the HMQC spectrum analysis (Table 1).
Besides the other NMR data already discussed, further evidences of a devadarane skeleton merged from the observed long-range correlations in the HMBC spectrum.In this experiment were observed correlation signals between hydrogens of the cyclopropane moiety at δ H 0.14 and 0.53 (2H-19) with carbons at δ C 22.6 (C-18), 33.9 (C-3) and 50.3 (C-10).Like compound 1, correlations of the oxymethine hydrogen at δ H 3.32 (H-15) with the carbons at δ C 19.7 (C-17), 35.2 (C-14) and 36.6 (C-12) confirmed the position of the ethyl 1,2-glycol system at C-13 and provided additional proof for the suggested structure.The acetyl group at C-11 was confirmed due to the correlation of the oxymethine hydrogen at δ H 4.82 (H-11) with the carbons at δ C 7.7 (C-20) and 50.3 (C-10) and 162.5 (C-22).The shielding effect observed for the 13 C NMR signal of the C-20 methyl group at δ C 7.7, when compared with related devadarane diterpenoids (~δ C 12.0), 10 is attributed to a γ-gauche shielding effect due the presence of the equatorial oxy group at C-11.On the basis of biogenetic arguments, along with the knowledge of occurrence of ent-devadarane diterpenoids only in Erythroxylum spp., we can suggest that compound 2 also belongs to the enantio series.From the foregoing evidence, compound 2 was identified as the new natural α-acetoxydevadarane-15ξ,16-diol, although it was cited previously in the literature as the chemical derivative of compound 4, 12 this paper comprises the first report of 2 as a natural product.
The NOESY experiment was an important tool to reach the final relative stereochemistry of compound 5.In particular, the orientation of the H-7 was determined to be axial, as revealed by the diagnostic NOE crosspeak for the hydrogen H-7 at δ H 3.42 with the CH 3 -20 at δ H 0.81.This was in agreement with the relative stereochemistry already reported for hydroxydevadarool, isolated previously from Erythroxylum monogynum. 13p to now the occurrence of devadarane diterpenoids in higher plants is restricted to species of the Erythroxylum genus.Their presence is remarkable and the potential as chemical markers was already discussed. 13Physical and spectroscopic data comparison of compounds 3, 4, and 5 with those from literature made possible their identification as ent-devadaran-15ξ,16-diol (erythroxydiol X), 10 ent-devadaran-11α,15ξ,16-triol (erythroxytriol Q) 12 and ent-devadaran-7β,15ξ,16-triol (hydroxydevadarool), 13 respectively.To the best of our acknowledge this is the first report of compounds 4 and 5 13 C NMR spectroscopic data.

General procedures
Melting points were obtained on a Mettler FP82HT apparatus and are uncorrected.IR spectra were recorded

Extraction and isolation
Roots (1.7 kg) and trunk heartwood (3.3 kg) of E. barbatum were separately pulverized and extracted with hexane at room temperature.Both plant residues from the hexane extraction were extracted with ethanol.All the solvents were removed under reduced pressure to give the correspondent extracts.
The EtOH extract (30.0 g) from roots was dissolved in a mixture of MeOH:H 2 O 1:1 and submitted to partition successively with hexane, CHCl 3 , EtOAc and MeOH.The CHCl 3 fraction (7.0 g) was submitted to silica gel flash chromatography using CHCl 3 and EtOAc as binary mixtures with increasing polarity, to give several fractions which were combined according to TLC analysis.Fraction 23 (1.2 g) was further purified over Sephadex LH-20 by elution with MeOH to afford compound 1 (13.0 mg).Coarse chromatography on a silica gel column of the hexane extract (3.2 g) by elution with hexane, CH 2 Cl 2 , EtOAc and MeOH yielded the correspondent fractions.The EtOAc fraction (0.8 g) was further purified over Sephadex LH-20 by elution with MeOH to furnished 15 fractions.Successive flash chromatography of fraction 5-11 furnished the pure compounds 2 (16.2 mg), 3 (4.0mg) and procesterol (9.7 mg).