Phytochemistry of Trattinnickia burserifolia , T . rhoifolia , and Dacryodes hopkinsii : Chemosystematic Implications

O estudo de Trattinnickia burserifolia levou ao isolamento dos triterpenos conhecidos ursanos α-amirenona, α-amirina, 3-epi-α-amirina, 3α,16β-diidroxiurs-12-eno; oleananos β-amirenona, βamirina, 3-epi-β-amirina, 3α,16β-diidroxiolean-12-eno; tirucalanos ácidos 3α-hidroxitirucal-8,24dien-21-óico, 3α-hidroxitirucal-7,24-dien-21-óico, e 3-oxotirucal-8,24-dien-21-óico; damaranos dammarenediol-II e 3α,20(S)-diidroxidamar-24-eno. Além desses foram ainda isolados o monoterpeno novo 2(S*)-fenilacetoxi-4(R*)-p-menta-1(7),5-dieno, e os triterpenos novos 3βfenilacetoxiurs-12-eno, 3β-fenilacetoxiolean-12-eno e 3β,16β,11α-triidroxiurs-12-eno. Os triterpenos de T. burserifolia, T. rhoifolia e Dacryodes foram analisados em mistura. Os espectros de RMN C mostraram que os principais triterpenos eram α-amirina e β-amirina em T. burserifolia; α-amirina, βamirina, 3-epi-α-amirina, 3-epi-β-amirina, lupenona, ácidos 3α-hidroxitirucal-8,24-dien-21-óico e 3α-hidroxitirucal-7,24-dien-21-óico em T. rhoifolia; α-amirina, β-amirina, lupeol, tirucalol, sitosterol e estigmasterol em D. hopkinsii. A quimiossistemática da tribo Protieae é discutida.


Introduction
The Burseraceae has usually been considered to contain 21 genera and nearly 600 species.Engler (1931) classified these genera into three tribes. 1The Protieae consist of four genera exhibiting many morphological characters regarded as primitives.Three Protieae genera occur in tropical America and one in Asia.The following group the Boswellieae contain eight genera centred in Africa and Asia.In contrast, the Canarieae, represented by nine genera, appear more advanced in their morphology.This tribe is predominantly Paleotropical, therefore, two genera occur in South America.Later Lam (1932)  recognised these tribes but replaced the name Boswellieae by Bursereae. 2repidospermum Hook. is a member of the Protieae and consists of five species distributed in the tropical South America.Swart in 1942 on morphological grounds described the genus Hemicrepidospermum to accommodate C. rhoifolium. 3However, other aspects of Lima et al.J. Braz.Chem.Soc.
their morphology have led Daly (1989) to consider Hemicrepidospermum a section of Crepidospermum; the two sections have three and two species, respectively. 4The following tropical S. American genera of the Protieae, Tetragastris and Protium, have long been considered closely related, in fact, many specimens of each genus have been mistakenly referred to the other. 4Garuga is the only representative of Asian Protieae and its morphology is easily recognisable. 4rattinnickia was also a member of the Protieae, however, morphological and anatomical evidence have led Daly (1989) to transfer it into the Canarieae and to propose a taxonomic position close to Dacryodes. 4ithin tribe Protieae phytochemical data were not available for Crepidospermam, Tetragastris, Trattinnickia and Dacryodes.As part of our chemosystematic interest in the Brazilian Burseraceae, we recently reported the phytochemical investigation of Crepidospermam rhoifolium Benth.and Tetragastris altissima (Aublet) Swart. 5Thus, we have now examined the resin, stem bark and branches of Trattinnickia burserifolia Engl., T. rhoifolia var.willdenowii Engl.and Dacryodes hopkinsii Daly. 4
The two new triterpenes 2 and 3 showed a single spot on TLC in various solvent systems and attempts to separate this mixture into its constituents were not successful.They also showed the spectral characteristics of a phenylacetoxyl substituent.The 1 H and 13 C NMR spectra of this mixture in addition to signals described above for phenylacetoxyl, revealed resonances for C-1 and H-1 to C-30 and H-30 in close agreement with those for α-amyrin (5) and β-amyrin (7), respectively 7,10 (Table 2).The downfield shift of the signals for C-3 (δ 81.4) and H-3 (δ 4.48) in the 1 H and 13 C NMR spectra, when compared with 5 and 7, determined the position of the phenylacetoxyl at C-3 in both the compounds of the mixture.The phenylacetoxyl present at C-3β was evident by resonance at δ 4.48 with a large coupling constant (J 11.0 and 5.1 Hz).The structure of the new natural products were thus established as 3βphenylacetoxyurs-12-ene (2) and 3β-phenylacetoxyolean-12-ene (3).
The new triterpene 4 was identified on the basis of the following data.The 1 H NMR spectrum indicated the presence of three signals characteristics of protons attached to a carbon adjacent to an oxygen atom (δ 3.23, dd, J 10.4 and 5.8 Hz; 4.23, dd, J 11.1 and 5.2 Hz; 4.27, dd, J 8.7 and 3.2 Hz), one olefinic proton (δ 5.24, d, J 3.2 Hz), and eight methyl groups, six of them on quaternary carbons and two of them on a methine group, suggesting a urs-12-ene skeleton.From the HMBC experiments (Table 3) the observed correlations between the two methyl protons at δ 0.77 and 0.97 and the 13  , allowing the assignment of these to C-11, C-13 and C-12, respectively.Moreover, the existence of correlations between H-12 and the 13 C signals at δ 59.9 (CH) and 44.3 (quaternary carbon) led to their assignments as C-18 and C-14, respectively.A fourth methyl proton at δ 1.20 (δ C 24.3) was attributed to H 3 -27 by its correlations with the C-13 (δ 141.3) and C-14 (δ 44.3) signals.The H 3 -27 signal also showed a cross-peak with the signal at δ 43.6, confirming a methyl group at C-8.In the same way, the unsubstituted C-15 emerged from the correlation between the H 3 -27 signal and the 13 C signal at δ 36.0 ( 3 J; CH 2 ), which showed one-bond correlation with the 1 H signal at δ 1.36 (m).This signal was coupled to the 1 H signal at δ 4.23 (dd, J 11.1 and 5.2 Hz), requiring the presence of a hydroxyl function at C-16.The coupling constants indicated that the hydroxyl group was attached β (equatorial) to C-16 and was coupled only to H 2 -15, indicating C-17 fully substituted.This was supported by the relationship of the H-16 (δ 4.23) signal to the 13 C signal at δ 21.9, which showed one-bond correlation with the methyl proton at δ 0.73, and long-range correlation with the 13 C signals for C-18, C-16 and at δ 38.5 (quaternary carbon) and 35.2 (CH 2 ).The signals at δ H 0.73, δ C 21.9, 38.5 and 35.2 were then assigned to H 3 -28, C-28, C-17 and C-22, respectively.A sixth methyl proton at δ 1.05 (δ 18.0) was attributed to H 3 -26 by its correlation with the C-9 and C-14.H 3 -26 signal also showed cross peaks with the 13 C signal at δ 43.6 (quaternary carbon) and 33.7 (CH 2 ), which were attributed to C-8 and C-7, respectively.A seventh methyl proton at δ H 0.82 (d, J 6.3 Hz; δ C 17.8) was attributed to H 3 -29 by its correlation with the C-18 signal.H 3 -29 signal also showed cross peaks with the 13 C signal at δ 39.1 (CH) and (or) 39.5 (CH), suggesting a methine for C-20, indicating a methyl group to be located at C-20 and confirming a urs-12-ene skeleton.Thus, the eighth methyl proton at δ H 0.91 (d, J 5.9 Hz; δ C 21.5) was attributed to H 3 -30.The signal for C-21 was established as δ 30.4 (CH 2 ; δ H 1.42 m, by HSQC) by the existence of a correlation between the H 3 -30 signal and this 13 C signal.
The stereochemistry suggested for 4 was based on the biosynthesis of urs-12-enes.However, for C-3, C-11 and C-16 the stereochemistry were assigned by coupling constants and NOESY experiments.A model shows that, in compound 4, ring A is nearer to a chair conformation, in which H-3 and H-5 are on the α-side of the molecule.This was supported by NOESY experiments (Table 3), which showed correlation of the signal of H-3α (δ 3.23; OH-3β) with the signal of H-5α (δ 0.72 m, by HSQC).Moreover, the existence of a correlation from H-3 to H 3 -23 (δ 0.97) confirmed that Me-23 is in the α-configuration.In addition, the signal of H-11 (δ 4.27) showed cross-peaks with the signals of H 3 -25 (δ 1.06) and H 3 -26 (δ 1.05), suggesting a spatial proximity of H-11 to Me-25 and Me-26, which requires 11-OH to be in the α-configuration.The  .Based in the above evidence the structure of this compound was thus established as 3β,16β,11α-trihydroxyurs-12-ene (4).The structural assignment was also supported by comparison of the 13 C NMR spectrum (Table 4) with those of 3β,16βdihydroxyurs-12-ene (11) 7 and 3α,11α-dihydroxyurs-12ene (12). 10In order to confirm of the assignments for 6a and 8a discussed below, 4 was acetylated.This reaction involved dehydration of C-11 alcohol and acetylation of the C-3 and C-16 hydroxyl groups to give 3β,16βdiacetoxyurs-9(11),12-diene (4a).The 1 H NMR spectrum of 4a revealed the downfield shift of the signals for H-3 (δ 4.51, dd, J 11.4 and 4.9 Hz) and H-16 (δ 5.46, dd, J 11.4 and 5.5 Hz).From the HMBC experiments (Table 3) the observed correlations between the two methyl protons at δ 0.88 and 0.90 and the 13 C signals at δ 79.5, 50.0 ( 3 J; CH), 36.8 ( 2 J; quaternary carbon), 27.1 ( 3 J, CH 3 ) and 15.7 ( 3 J, CH 3 ) led to their assignments as C-3, C-5, C-4, C-23 and C-24, respectively.The oxymethine proton at δ 4.51 showed long-range correlation with the 13 C signal at δ 170.0 and with the C-24 (δ 15.7) and C-23 (δ 27.1) signals, confirming this proton signal to H-3 and allowing the assignment of the signal at δ 170.0 to C-3 acetoxyl group.Moreover, the existence of correlations between H 3 -28 (δ 0.91) and the 13 C signals at δ 69.9 (CH), 57.8 (CH), 36.3 (quaternary carbon) and 34.4 (CH 2 ) led to their assignments as C-16, C-18, C-17 and C-22, respectively.Thus, the  13 C signal at δ 122.2 led to their assignments as H-18 and C-12.Thus, the second olefinic carbon at δ 114.6 was attributed to C-11.
The ursa-9(11),12-diene system was also supported by the 13 C NMR spectrum which agreed closely with published data for 3β-acetoxyurs-9(11),12-diene (13). 10In the HMBC experiments several other long-range correlations were observed, which also confirmed the attribution of all the 13 C signals of the molecule (Table 3 and 4).

Experimental
General NMR on a Bruker DRX 400, with TMS as internal standard; ESI-MSMS: low resolution on a triple quadrupole Micromass Quattro LC instrument, equipped with a "Z-spray" ion source; GC-MS: Shimadzu GC-17A gas chromatograph fitted with a fused silica DB-5 (30 m x 0.25 mm ID, 0.25 µm film thickness) capillary column with helium as the carrier gas at a flow rate of 1.6 mL min -1 .The temperature was programmed initially at 60 °C for 2 min, then increased with a rate of 3 °C min -1 to 240 °C.The injection was split and its temperature was 225 °C.The interface temperature was 250 °C.The chromatograph was coupled to a Shimadzu QP5000 mass selective detector at 70 eV; IR (BOMEN -Ft/IR).[α] D : Perkin Elmer 241 instrument; IR (KBr, BOMEN -Ft/IR); R-HPLC: Recycling High-Performance Liquid Chromatography on a model Shimadzu LC-6AD; the column used was a Shim-pack Prep-Sil (H), 250 mm X 20 mm, 5 mm particle size, 100 Å pore diameter; eluant: CHCl 3 ; flow rate: 8.0 mL min -1 and 5.0 mL min -1 ; detection (Shimadzu SPD-6AV): UV λ 254 nm.
The MeOH fraction from the concentrated CHCl 3soluble fraction of resin was subjected to column chromatography over silica gel eluting with a hexane-EtOAc-MeOH gradient to afford 15 fractions.These fractions were combined in 7 groups on the basis of analytical TLC.The 7 groups were monitored by 1 H NMR (200 MHz) and were examined only those which showed features of taxonomic interest.Group 4 (fractions G4F5-8) was three times flash rechromatographed on silica gel eluting with CH 2 Cl 2 -EtOAc 7:3, then CH 2 Cl 2 -EtOAc 7:3 and finally CH 2 Cl 2 -MeOH 95:5, affording 4 (2.6 mg).Compound 4 was allowed to react overnight with an excess of Ac 2 O in pyridine.Work-up as usual yielded 3β,16βdiacetoxyurs-9(11),12-diene (4a).

Table 4 .
13C NMR spectrum data for compounds 4