A novel D:A-friedooleanane triterpenoid and other constituents of the stem bark of Dichapetalum barteri Engl.

A novel pentacyclic triterpenoid, 2 β -hydroxy-3-oxo-D:A-friedoolenanan-29-oic acid has been isolated from the methanol extract of the stem bark of Dichapetalum barteri, and characterised by spectroscopic methods. Also reported for the first time from the plant are two lupane-type triterpenoids betulinic acid and betulonic acid, the friedelane triterpenoids syn :canophyllal, syn :canophyllol, friedelan-3-one and friedelan-3 α -ol (epifriedelinol), the more common triterpenoids β -sitosterol and stigmasterol, as well as seven long chain esters of ferulic acid.


Introduction
Dichapetalum barteri belongs to the small family Dichapetalaceae (syn.Chailletiaceae), which is distributed throughout the world's tropical and subtropical regions. 1 Some members of the family are known to be highly toxic, particularly to livestock, due to the presence of fluorinated carboxylic acids. 2 The fluorinated acids have been extensively studied.However, very little is known of the non-fluorinated constituents of this plant genus.N-methyl alanine and N-methyl serine have been reported from Dichapetalum cymosun, 3 while friedelin has been found in D. gelonoides. 4 As part of our systematic investigation of the family Dichapetalaceae occurring in Ghana, we recently reported a new class of triterpenoids never reported from any other plant family.Eight of these have so far been reported, which we named Dichapetalins A to H. 5,6 Biogenetically, their basic structures are characterised by the addition of a C 6 -C 2 -unit to a cyclodammarane skeleton.These compounds exhibit strong and selective in-vitro cytotoxicity.
In the present paper, we report the results of studies on the relatively rare species, D. barteri, which is said to have strong rodenticidal activity 7 and is also toxic to goats. 8ractionation of the methanol extract of the stem bark of the plant yielded a novel pentacyclic triterpenoid identified as 2β-hydroxy-3-oxo-D:A-friedooleanan-29-oic acid 1, which is isomeric with 2α-hydroxy populnonic acid (2α -hydroxy-octandronic acid) 2b and 2α-hydroxy-3-oxo-D:A-friedooleanan-30-oic acid 3a.
Also obtained from the petrol and acetone extracts were several known triterpenoids including the two lupane-type triterpenoids betulinc acid and betulonic acid, as well as seven long-chain esters of E-ferulic acid with the straight chain C 23 to C 29 alkan-1-ols.

Results and Discussion
Column chromatography of the methanol extract of the stem bark of the plant gave five main fractions.Further purification of fraction 2 gave compound 1 as off-white needle-like crystals, mp 199-201 o C, brown with anisaldehyde reagent.MS gave a molecular ion peak at 472, C 30 H 48 O 4 . 1 H-NMR showed signals for six angular methyl singlets at δ 0.71, 0.86, 0.99, 1.05, 1.07 and 1.31ppm as well as a methyl doublet (J = 7.8Hz) at δ 0.96 ppm, which was assigned to methyl protons at position 23.A signal at δ 2.28 ppm (1H, q, J = 7.8Hz), was assigned to a proton at C-4 vicinal to the C-3 carbonyl carbon.The proton geminal to the OH group at C-2 appears as a multiplet at δ 4.12, compared to the C-2 proton in the α-isomer 2b which occurs at 3.96 and that of compound 3a (δ 4.00).The remaining 23 protons were assigned as methylene and methine protons occurring between δ 2.52 and 1.15 ppm.A broad signal at δ 3.7, removed by H-D exchange, confirmed the presence of an OH group.The assignment of the 1 H-NMR signals is summarised in Table 1.
Comparison of the melting point, 1 H and 13 C-NMR spectra (Tables 1 and 2) of compound 1 with those of populnonic acid (3-oxo-D:A friedooleanan-29-oic acid) 2a, 2α-hydroxy populnonic acid methyl ester (syn.2α-hydroxy-3-oxo-D:A-friedooleanan-29-oicacid methyl ester) 2c, 3-oxo-D:A-friedooleanan-30-oic acid methyl ester 3b and the acetate of 2α-hydroxy-3-oxo D:A-friedooleanan-30-oic acid methyl ester 3c suggests that compound 1 is different from all these related compounds with which it has the same basic skeleton, and confirms the presence and orientation of the carboxyl and the hydroxyl groups at C-20 and C-2 respectively, and the carbonyl group at C-3. 13 C-NMR of the compound showed a singlet at δ 212.The HMBC measurements (Fig. 1) showed that compound 1 exhibited two-bond couplings between H-4 and the C-3 carbonyl carbon, between H-4 and C-5, as well as between H-1 and C-10.There is also a three-bond coupling between H-1 and C-3 and H-1 and C-5, confirming the structure of ring A. The protons of the angular methyl H-30 couple with the carboxyl carbon, locating the carboxyl carbon to be attached to C-20.The proton at C-2 is coupled to C-1 only, unlike 2b and its derivatives 2c and 2d in which this proton is coupled to C-3, C-4and C-10.
The mass spectrum gave the molecular ion at m/z 472, with other significant peaks at 426 (M+ -HCOOH), 411 (426-CH 3 ), 290 and 289, representing a fraction resulting from cleavage of ring D followed by loss of H, 207, 189, 177, 175.The base peak appeared at m/z 155, due to a fraction resulting from loss of CH 3 and H 2 O from M + followed by cleavage of ring D.
In addition to compound 1 a number of other known triterpenoids including the cytotoxic agent betulinic acid, betulonic acid, canophyllal, canophyllol, friedelin and a mixture of βsitosterol and stigmasterol were also isolated for the first time from the plant.
HPLC separation of a white crystalline solid obtained from repeated column chromatography of one of the fractions of the acetone extract gave seven homologous long chain esters of E-ferulic acid (4a-4g), with molecular ions M + at m/z 516, 530, 544, 558, 572, 586 and 600.The most abundant of these was E-ferulic acid hexacosyl ester (4d), the others (4a, 4b, 4c, 4e, 4f, and 4g) being the tricosyl, tetracosyl, pentacosyl, heptacosyl, octacosyl and nonacosyl esters respectively.These compounds were characterised by spectroscopic methods (UV, including shift behaviour on addition of NaOH, 1 H NMR, 13 C NMR, MS) and comparison of the resulting data with those reported in the literature. 11,12All these compounds are reported for the first time in Dichapetalum barteri.

Table 1 .
33e remaining 27 signals in the13C-NMR are sp3-hybridised signals occurring between 75 and 6.5 ppm.DEPT measurements indicated the presence of 8 quaternary, 5 tertiary (CH), 10 secondary (CH 2 ) and 7 primary (CH 3 ) carbons.The assignment of the 13 C-signals using DEPT measurements are as summarised in Table1.Comparison of the 13 C-signals of 1 with those of 2a, 2b, 3b and 3c is as summarised in table3. 1 H and 13 C-NMR data for compound 1 4 ppm, assigned to a C-3 ketone, and a doublet at δ 75.2 ppm due to carbon 2. A signal at δ 183.3 ppm was attributed to a carboxyl carbon at C-29. *Interchangeable values **Signals not well resolved