Terpenoid Constituents from Leaves of Guarea kunthiana

Das folhas de Guarea kunthiana foram obtidos um novo diterpeno com esqueleto do tipo caurano (ent-caur-16-eno-2-ona), além de oito diterpenos (ent-caur-16-eno, ent-3αe 3β-hidroxicaur16-eno, kolavelool, kolavenol, kolavenal, ent-13-epi-óxido de manoíla e (-)-neftenol), quatro sesquiterpenos (alismol, alismóxido, espatulenol e 4β,10α-aromadendranodiol), poliprenol-12 e αe δ-tocoferóis. Kolavenal está sendo relatado pela primeira vez como produto natural, assim como a ocorrência de diterpenos do tipo cembrano e ent-caurano na família Meliaceae.


Results and Discussion
A crude ethanol extract of the leaves of Guarea kunthiana was partitioned between methanol-H 2 O (9:1) and hexane.The hydromethanolic solution was further partitioned with ethyl acetate.Compounds 1-10 and 14-16 were isolated from the hexane soluble fraction while compounds 11-13 and further amounts of 5, 6 and 10 were obtained from the ethyl acetate soluble fraction, after a combination of column chromatography on silica gel, gel filtration and preparative TLC separations.The known compounds were identified by comparison of their physical and spectroscopic (IR, 1 H and 13 C NMR, MS) data with those of authentic samples and/or reported in the literature.
7][8] Although widely distributed in a number of plants, no records related to the isolation of kaurene-type diterpenes have hitherto been reported in the Meliaceae.The molecular formula of 3, obtained as an amorphous solid, was established as C 20 H 30 O from HREIMS data ([M + ] m/z 286.2296).Its IR spectrum was consistent with the presence of a carbonyl (1714 cm -1 ) and an exomethylene (881 cm -1 ) group, while the 1 H NMR spectrum (Table 1), displaying the characteristic pair of broad singlets due to the exocyclic methylene group, was very similar to that of 1 (ent-kaur-16-ene).The only significant difference referred to the presence of two double doublets at δ 2.50 (H-1 eq , J 12.6 and 1.9 Hz) and δ 2.15 (H-3 eq , J 13.6 and 1.9 Hz) and two broad doublets at δ 1.90 (H-1 ax , J 12.6 Hz) and δ 2.24 (H-3 ax , J 13.6 Hz).The 13 C NMR spectrum (Table 1) showed, in addition to the signals at δ 155.1 (C) and 103.4 (CH 2 ) attributed to the exocyclic double bond at C-16 in a kaurene skeleton, a ketone carbon signal at δ 212.0.Accordingly, the molecular ion observed in the HREIMS at m/z 286.2296 was 14 mass units higher than that of 1.Comparison of 13 C NMR data of 1 and 3 indicated that they had identical constituting rings B, C and D, therefore indicating that the carbonyl was located on the A ring of compound 3.The deshielding of the A-ring methylene carbons in 3 when compared to those of 1 (∆δ 14-15) suggested that the carbonyl group was located at the C-2 position.This proposal was in agreement with the chemical shifts and multiplicities of H-1 and H-3 as aforementioned.
Accordingly, the 1 H-1 H COSY spectrum of 3 showed the geminal coupling between H-1 ax and H-1 eq as well as between H-3 ax and H-3 eq .Likewise, this spectrum depicted the long-range couplings between H-1 eq and H-3 eq , H-1 ax and H-20 and H-3 ax and H-19.Unambiguous assignment of H-1 and H-3 was also achieved by the long-range correlations observed in the HMBC spectrum between H-1 and C-9, C-10, C-20 and between H-3 and C-4, C-18 and C-19 (Table 1).On the other hand, the cross-peak correlations in the HMQC spectrum of H-1 and H-3 with the carbon signals at δ 56.1 and δ 56.3, respectively, as well as the long range connectivities shown by the HMBC spectrum between the signal ascribed to H-20 (δ 1.00) and δ 56.1 and between H-18 (δ 1.03) and δ 56.3 confirmed the assignments of C-1 and C-3, respectively.The significant HMBC correlations noticed between the signals of H-1 and H-3 and that of the carbonyl carbon further supported the location of this group at C-2.The remaining quaternary carbons in the structure of 3 were assigned according to the 1 H- 13 C long range correlation data provided in Table 1.On the basis of its negative [α] D value, it is suggested that 3 belongs to the ent series, similarly to other analogous kaurenoid diterpenes. 8,9Thus 3 was characterized as ent-kaur-16-en-2-one.
Compound 4 which showed a molecular ion at m/z 288 in EIMS and a negative specific rotation value, was assumed to be a C-3 epimeric derivative of 2 on the basis of the close resemblance of their 1 H and 13 C NMR spectra, except for the chemical shift and coupling constant of H-3 and in some resonance values of A-ring carbons.In the 1 H NMR spectrum of a broad singlet at δ 3.39, instead of a double doublet at δ 3.18 (J 10.2, 5.8 Hz) ascribed to H-3 in 2, determined the equatorial nature of H-3 and the resulting axial orientation of the C-3 hydroxyl group.As expected, an upfield shift of the C-3 resonance (δ 76.1) was observed in 4 when compared to that of 2 (δ 79.1).Likewise, the axial orientation of the hydroxyl accounted for the γ-effect on C-1 and C-5 which were shielded by 5.4 and 6.2 ppm, respectively.Therefore, compound 4 was determined to be ent-3α-hydroxy-kaur-16-ene, which is described for the first time in the Meliaceae, yet previously reported from Annona senegalensis (Annonaceae) 10 and as a reduction product of ent-kaur-16-en-3-one. 7 Identity was further confirmed by comparison with literature data ( 1 H and EIMS). 7The 13 C NMR spectral data of 4, not previously reported in literature, are now presented in Table 1.The unambiguous assignment of the carbon resonances of 4, mainly those of C-18, C-19 and C-20 methyls, was based on information provided by HMQC, HMBC and NOESY experiments.
Diterpenes 5 and 6 were characterized as ent-clerodane derivatives with a ∆ 3 double bond, in addition to a hydroxyl group and a double bond in the C-9 side chain.The optical rotation values and spectral characteristics ( 1 H and 13 C NMR, MS and IR) of 5 and 6 agreed with those of kolavelool 8,11 and kolavenol, 8,12 respectively.The first was already obtained from Guarea trichilioides 13 while kolavenol was hitherto unreported in the Meliaceae.
The 1 H and 13 C NMR spectra of 6 and 7 were almost identical, except for the presence of an aldehyde (δ H 9.95 d, 7.6 Hz; δ C 191.3) in 7 in place of the hydroxymethylene group at C-15.The carbon resonances at δ 127.2 and 165.5 together with a carbonyl absorption band at 1675 cm -1 in the IR spectrum also confirmed this assumption.Therefore compound 7 was characterized as ent-(E)-cleroda-3,13-dien-15-al whose spectroscopic properties (IR and 1 H NMR) and optical rotation value were very similar to those described for kolavenal, a compound obtained by MnO 2 oxidation of 6. 3 Thus compound 7 is reported for the first time as a genuine natural product.Its 13 C NMR data, not reported before now and presented in Table 1, readily support the assignment of the double bond configuration in the side chain as (E), on the basis of the chemical shifts of C-12 (δ 34.4) and C-16 methyl group (δ 17.8).In similar 13-en-15-al ent-clerodane derivatives, e. g., 3R*,4R*-dihydroxyclerod-13E-en-15-al and the corresponding (Z) isomer, the presence of a carbonyl at C-15 accounts for the γ-effect observed at C-12 methylene carbon in the (Z) isomer (δ 26.9) with respect to the corresponding resonance in the (E) isomer (δ 34.5).On the other hand, the same effect is observed at the C-16 methyl group in the (E) isomer (δ 17.1) when compared to the corresponding resonance of the (Z) isomer (δ 24.7). 14he 1 H and 13 C NMR spectral data of compound 8, in addition to the cross-peak correlations and the long-range connectivities observed in the HMQC and HMBC spectra, respectively, defined 8 as a carbomonocyclic diterpene of the cembrane class, characterized as a cembra-3,7,11triene-15-ol.These results, together with the negative optical rotation value of 8, agreed with those reported earlier for (-)-nephthenol, (1R,3E,7E,11E)-cembra-3,7,11triene-15-ol. 4,8The great majority of cembranoids of plant origin occurs in tobacco (Nicotiana tabacum, Solanaceae) and in Coniferae species, although they are widely distributed in marine organisms and insects. 5,8However, (-)-nephthenol was only described in marine invertebrates (e.g.soft corals). 4,5Thus, this is, to the best of our knowledge, the first report of the isolation of a cembrane-type diterpene from Meliaceae, as well as of the occurrence of (-)-nephthenol in a plant taxon.
The structure of 9 was shown to be of a labdane-type diterpenoid from its 1 H and 13 C NMR spectral data, which showed striking resemblance with those of (+)-13-epimanoyl oxide, 15 a compound which differs from 9 by having a specific rotation of opposite sign. 15The latter diterpene was then characterized as ent-13-epi-manoyl oxide, which was previously isolated from members of other plant families, e.g.Cistaceae and Flacourtiaceae 16 but, until now, not yet reported in the Meliaceae.
The sesquiterpenes 10 -12 obtained in the present work were found to be identical in their spectral data with spathulenol (data compared with those of an authentic sample), alismol 17,18 and alismoxide, 17 respectively.Direct comparison of the spectral data of 13 with those of 4β,10αaromadendranediol proved their identity. 19,20This sesquiterpene was first isolated as the dextrorotatory isomer from the soft coral Sinularia mayi 19 and later obtained in the (-) form in only few plant species, e. g.Brasilia sickii. 20n the basis of the negative sign of the specific rotation of 13, its structure was therefore established as (-)-4β,10αaromadendranediol.Apart from 10, these sesquiterpenes are described for the first time in the Meliaceae.
The polyprenol 14 was identified as ficaprenol-12 (also known as polyprenol-12) by comparison of its 1 H and 13 C NMR spectral data with literature. 21This compound, which has been obtained from leaves of several angiosperms, was also isolated from Guarea jamaicensis. 22lthough members of the Meliaceae, including those belonging to the genus Guarea that occur in Brazil, are well-known for the occurrence of sesqui-, di-, tri-and tetranortriterpenoids, 1,13,23 the isolation for the first time of cembranoid and kaurenoid diterpenes in this family is noteworthy for its chemosystematic relevance.Also worthy of mention is the fact that, unlike the specimen of G. kunthiana found in Ecuador whose aerial parts yielded one limonoid as the only natural compound, no limonoids were detected in the leaves of the specimen collected in Brazil and investigated in the present work.

Experimental
General experimental procedures IR spectra were recorded as KBr pellets on a Bomem-Hartmann & Braun FT IR spectrometer. 1 H and 13 C NMR spectra were measured on a Bruker DPX-300 spectrometer at 300 MHz ( 1 H) and 75 MHz ( 13 C), using CDCl 3 as a solvent and internal reference.Standard pulse sequences were used for homo -and heteronuclear correlation experiments.EIMS data were obtained at 70 eV on a Shimadzu QP-5000 (Departamento de Química, UFMS, MS, Brazil) and on a HP 5973 MSD spectrometer (Instituto de Química, UFBA, BA, Brazil).HREIMS data were obtained at 70 eV on a VG Autospec spectrometer (Instituto de Química, UNICAMP, SP, Brazil).Optical rotations were determined on a Perkin-Elmer 341 polarimeter (Na filter, λ = 589 nm).

Plant material
The leaves of Guarea kunthiana A. Juss.were collected in Campo Grande, MS, Brazil, in September 2000.The plant material was identified by one of the authors (U.R.) and Dr. Germano Guarin Neto (Universidade Federal de Mato Grosso, MT, Brazil).Voucher specimens (11536) are deposited at the CGMS Herbarium (UFMS), Campo Grande, MS, Brazil.

3, 4 and 7 in CDCl 3
a Chemical shifts in δ from TMS; b Coupling constants (J in Hz) are given in parentheses.