New Diterpenoids from Leaves of Guarea macrophylla ( Meliaceae )

A fase em hexano do extrato etanólico das folhas de Guarea macrophylla (Meliaceae) foi submetida a fracionamento cromatográfico. Esses procedimentos permitiram o isolamento de um novo diterpenóide: 7α-hidroperóxido-isopimara-8(14),15-dieno-2α,3β-diol, um novo norditerpenóide: 19-nor-isopimara-7,15,4(18)-trien-3-ona além de sete diterpenóides de estruturas conhecidas. As estruturas dos compostos isolados foram definidas através de análise dos dados espectrométricos e comparação com aqueles descritos na literatura.


Introduction
As part of ours studies of Guarea species we have investigated the chemical composition of the hexane phase of the ethanol extract from leaves of G. macrophylla.This species grows in Brazil from Rio Grande do Sul to Rio de Janeiro and Minas Gerais States extending to Mato Grosso and Brasilia, and is hold also in the Amazon region. 1 An earlier investigation of the dichloromethane extract of the leaves yielded one monoterpene, four sesquiterpenes, five diterpenes and one triterpene, 2 indicating, for the first time, the co-occurrence of diterpenes and triterpenes in members of Meliaceae.In another study, nine related cycloartane triterpenoids were also detected in the leaves. 3In the volatile oils from the leaves and stem bark were detected several sesquiterpene and diterpene derivatives as well as fatty acids. 4,5In the present investigation we report the isolation and structural determination of two new minor diterpenoid derivatives: 7α-hydroperoxy-isopimara-8( 14),15-diene-2α,3β-diol (1) and 19-nor-isopimara-7,15,4(18)-trien-3-one (2), in addition to seven known diterpenoids (3-9).All these compounds, except 1, 2, 7 and 8, have been described previously in this plant.Structures were elucidated by analysis of their spectrometric data and comparison with data described in the literature.

Results and Discussion
The crude EtOH extract from the leaves of G. macrophylla was partitioned between hexane and aqueous ethanol.The hexane phase was submitted to chromatographic separation on silica gel and Sephadex LH-20 to yield one new diterpenoid: 7α-hydroperoxyisopimara-8( 14),15-diene-2α,3β-diol (1), and one new J. Braz.Chem.Soc.
Compound 1 was obtained as a white amorphous powder whose partial molecular formula C 20 H 32 O 3 was deduced by analysis of the 13 C NMR spectra (BBD and DEPT 135°) and LREIMS.The 1 H NMR spectrum showed three dd at δ H 4.94 (J 11.1 and 1.2 Hz, 1H), 4.97 (J 17.9 and 1.2 Hz, 1H) and 5.79 (J 17.9 and 11.1 Hz, 1H) and a broad singlet at 5.70 (1H).These signals associated with four methyl signals at δ H 1.08, 1.04, 0.87, 0.86, suggested the presence of an isopimarane diterpene skeleton. 6The 13 9 ) as well as two oxygenated methines at δ C 68.6 (CH) and 83.6 (CH), which were attributed, respectively, to C-2 and C-3. 10 These assignments were confirmed by analysis of the 1 H-and DQ-COSY NMR spectra, which showed the correlated signals of H-2 and H-3 at δ H 3.66 (ddd, J 11.7, 9.6 and 4.2 Hz, 1H) and 3.05 (d, J 9.6 Hz, 1H), respectively, and the comparison with 13 C NMR data reported to model compound M3. 11The configuration of the hydroxyl groups were confirmed such as 2α and 3β based on the value of the coupling constants, which were indicative of two transdiaxial couplings [H-2β/H-3α (J 9.6 Hz) and H-2β/H-1α (J 11. 7 Hz)] and one axial-equatorial coupling [H-2β/H-1β (J 4.2 Hz)], in agreement with H-2β and H-3α coupling constant values reported for the model M5. 10 The 1 H NMR spectrum showed also one dd at δ H 4.34 (J 3.9 and 2.1 Hz, 1H) and one s at δ H 7.41 (1H, exchangeable with D 2 O), suggesting the presence of a hydroperoxy group at C-7, which could be confirmed by comparing the 1 H and 13 C NMR data of 1 with those reported to the diterpene methyl-7β-hydroperoxypimara-8( 14),15-dien-19-oate (M4). 12In the light of the above observations, the molecular formula of 1 was defined as C 20 H 32 O 4 .However, the LREIMS spectrum did not show the molecular ion peak at m/z 336 Da but a fragment at m/z 320 Da instead, which is indicative of the lost of one oxygen atom, similar to the fragmentation observed to other hydroperoxyde derivatives. 13Therefore, the structure of 1 was elucidated as 7α-hydroperoxyisopimara-8( 14),15-diene-2α,3β-diol.
Compounds 3 to 9 were identified by analysis of their LREIMS, 13 C and 1 H NMR spectra and comparison with data reported in the literature. 14,15This is the first report of compounds 7 and 8 from the genus Guarea.
Several sesquiterpenes, diterpenes and triterpenes (cycloartane derivatives only) have been identified in G. macrophylla, while limonoids (meliacins), which are produced by the oxidative degradation of the side chain from tirucalane/euphane triterpenes, had so far not been detected.However, a biogenetic pathway proposed to the formation of 1 and 2 showed that the oxidative/degradative tendency, characteristic of Meliaceae, should be observed in G. macrophylla.