Two New Cneorubin Related Diterpenes from the Leaves of Guarea guidonia ( Meliaceae )

Dois novos diterpenos do tipo das cneorubinas, além de dois conhecidos, foram isolados das folhas da Guarea guidonia (Meliaceae). Um dos novos diterpenos possui o sistema trans biciclo [5.1.0] octano, encontrado m uito raramente entre os produtos n aturais. As estruturas foram determinadas com base na análise de dados espectr ais. A análise dos espectros de RMN dos diterpenos conhecidos permitiu uma re-atribuição de dados da literatura.


Introduction
Guarea guidonia (L) Sleumer is a brazilian Meliaceae species occurring all over the country.The leaves of G. guidonia have already been chemically investigated and different compositions have been found for specimens collected in different country regions [1][2][3] .We are now investigating the leaves of a specimen collected in Campo Grande-MS, western of Brazil.
This paper describes the isolation and identification of two known (1 and 2) and two novel (3 and 4) diterpenes besides sitosterol, stigmasterol, β-selinene and a mixture of unidentified hydrocarbon sesquiterpenes obtained from the hexane fraction of the methanolic extract of G. guidonia leaves.Despite the report of the known compounds from Cneorum tricoccom, Cneoracea, 19 years ago 4 , it is particularly surprising that they have not been identified from any other natural source.Furthermore, no structurally related natural products have been isolated from plant sources.There is only a report about the isolation of emmottene, a related diterpene from Briareum polyanthes, a Bermudian gorgonian 5 .

Results and discussion
The 13 C NMR data of diterpenes 1 and 2 isolated from Guarea guidonia are identical to those reported for cneorubin Y and X, respectively 4 .Comparison of the 13 C and 1 H NMR data of 2 with those reported for the aromadendrane sesquiterpene, spathulenol, 6 showed that the diterpene and the sesquiterpene have identical ring system, including the stereochemistry (Tables 1 and 2).However, the five and seven membered rings of cneorubin X (2) are not cis-fused as previously proposed 7 but trans-fused.The analysis of the 2D NMR ( 1 H-1 H COSY and 13 C-1 H COSY) spectra allowed the reassignment of the NMR data for 1 and 2 .The former has the same ring system as the sesquiterpene bicyclogermacrene 8 .3) confirmed that the relative stereochemistry of the molecule is the same as in spathulenol.The diterpenes 3 and 4 have not been described yet in the literature.Their molecular formula (C 20 H 32 O 2 ) have been deduced by the EI/MS and 13 C NMR data.Although the1 H NMR spectra of these terpenes reveal significative difference in the region between δ 0.5 and 2.0 (Table 2), the analysis of other spectral data ( 13 C NMR, 13 C-1 H COSY and HMBC) showed that both have an identical planar structure and differ from that of 2 only due to the presence of an additional hydroxyl group Table 3. Correlation observed in the NOESY spectra for 3 and 4 (400 MHz, δ, CDCl 3 ).

4
The 13 C NMR chemical shift of the exocyclic methylene (δ 106.5) 8 as well as the H-6 and H-7 chemical shifts (ca δ 0.7 ) 8 observed in the 1 H NMR of 4 suggested that the five and seven membered rings are also trans fused in this diterpene.The NOESY spectrum of 4 (Table 3) showed almost the same spatial correlation as in 3.The most important of them is the correlation between H-15 and H-1 which is larger in 4 than in 3, indicating that both compounds have H-1 and the methyl group at C-4 on the same side of the molecule.Considering that the diterpene 4 showed a higher Rf value than 3 on silica gel TLC plate, one can conclude that the hydroxyl groups of 3 have stronger interaction with the silanol groups of the gel.A possible intramolecular hydrogen bonding, between these OH groups in 4, as consequence of a epimerization at C-16, in relation to 3, would weaken the interaction of the diterpene with the silica gel.This situation would modify the conformation of the ring system, and also justify the difference in the NMR spectra.Nevertheless the results obtained with molecular modeling experiments showed that an intramolecular hydrogen bonding between the hydroxyl groups in 4 increase the energy of the compound.The side chain in 4 is also at C-12, as shown by the 13 C NMR data, so the difference between 3 and 4 must be in the cyclopropyl junction.The overlaping of the signals of H-6 and H-7 in the 1 H NMR spectrum of 4 made that analysis difficult.The observation of a spatial correlation between H-5 and H-13 in 4 and not in 3, as well as between H-15 and H-16 in 3 but not in 4 agrees with the proposed modification.No spatial correlation is observed between H-5 and H-6 or H-7 but a small interaction is observed between H-1 and H-6 or H-7 in 4. As in 3 we could see a correlation between H-1 and H-6, but not between H-1 and H-7, we can suggest that the correlation in 4 is also between H-1 and H-6.By these observations we proposed that 3 and 4 are epimers at C-7, so the last one has a trans bicyclo[5.1.0]system ring as in emmottene 5 .Inspection of molecular model showed that in 4 the methyl group at C-4 is closer to C-1 than in 3, justifying a strong nOe between H-1 and H-15 observed in the NOESY spectrum.This observation also agrees with the shielding of the cyclopentane ring carbons in the 13 C NMR spectrum of the diterpene 4, in relation to those of 3. The structures of 4 and of emmottene 5 suggest that a revision in the stereochemistry of aromadendrane like natural products is necessary.We named the skeleton of diterpenes 2-4 as cneorubinane.
It is interesting to mention that species of Cneoraceae family also produce meliacins, tetranortriterpenes that characterise the chemical constitution of Meliaceae species.

General Procedures
The IR spectra were registered as KBr pellets in a Perkin Elmer FT 1750 spectrometer.The low resolution mass spectra were obtained in a INCOS 50 Finnigan-Mat instrument operating at 70 eV.The 1 H and 13 C NMR spectra, using CDCl 3 as solvent and TMS as internal reference, were run in a Bruker AC 200 and a Varian U-400.For the column separations Merck silica gel, 63-200 mm and 40-63 mm was used.For preparative TLC Merck silica gel 60 GF 254 was employed.

Plant material
Leaves of Guarea guidonia (L.) Sleumer were collected in February 1994, in Campo Grande, Mato Grosso do Sul, Brazil.A voucher was deposited under the number 1870 in the Herbarium of the Universidade Federal do Mato Grosso do Sul.

Extraction and isolation of the compounds
Dried leaves of G. guidonia (1100 g ) were extracted at room temperature with methanol.After distillation of the solvent, 72 g of the extract were obtained.This material was submitted to a partition between hexane and MeOH/ H 2 O (95:5).The hexane phase (47 g) was dissolved in hot MeOH and kept at 4 o C during 24 h.After filtration, the soluble part was concentrated affording 25 g of a solid residue.This residue was submitted to chromatography on a silica gel column eluted with gradient mixtures of hexane and ethyl acetate.The first fractions were submitted to further purification on silica gel column, eluted with gradient mixtures of hexane and CH 2 Cl 2, and to preparative TLC using silica gel impregnated with AgNO 3 .β-Selinene (40 mg), an unidentified sesquiterpene mixture (60 mg) and 1 (60 mg) and 2 (20 mg) were isolated.The intermediary fractions afforded a mixture of sitosterol and stigmasterol (50 mg).The more polar fractions were chromatographed over silica gel column eluted with step gradient mixtures of hexane and isopropanol yielding 4 (25 mg) and 3 (20 mg).