Biotransformation of betulinic and betulonic acids by fungi

Article abstract of DOI:10.1016/j.phytochem.2006.12.007

Betulinic acid (1), a triterpenoid found in many plant species, has attracted attention due to its important pharmacological properties, such as anti-cancer and anti-HIV activities. The closely related, betulonic acid (2) also has similar properties. In order to obtain derivatives potentially useful for detailed pharmacological studies, both compounds were submitted to incubations with selected microorganisms. In this work, both were individually metabolized by the fungi Arthrobotrys, Chaetophoma and Dematium, isolated from the bark of Platanus orientalis as well as with Colletotrichum, obtained from corn leaves; such fungal transformations are quite rare in the scientific literature. Biotransformations with Arthrobotrys converted betulonic acid (2) into 3-oxo-7β-hydroxylup-20(29)-en-28-oic acid (3), 3-oxo-7β,15α-dihydroxylup-20(29)-en-28-oic acid (4) and 3-oxo-7β,30-dihydroxylup-20(29)-en-28-oic acid (5); Colletotrichum converted betulinic acid (1) into 3-oxo-15α-hydroxylup-20(29)-en-28-oic (6) acid whereas betulonic acid (2) was converted into the same product and 3-oxo-7β,15α-dihydroxylup-20(29)-en-28-oic acid (4); Chaetophoma converted betulonic acid (2) into 3-oxo-25-hydroxylup-20(29)-en-28-oic acid (7) and both Chaetophoma and Dematium converted betulinic acid (1) into betulonic acid (2). Those fungi, therefore, are useful for mild, selective oxidations of lupane substrates at positions C-3, C-7, C-15, C-25 and C-30.

Full text of DOI:10.1016/j.phytochem.2006.12.007

PHYTOCHEMISTRY
Phytochemistry 68 (2007) 834–839
www.elsevier.com/locate/phytochem
Biotransformation of betulinic and betulonic acids by fungi
a
b
a
a,
*
Denise Z.L. Bastos , Ida C. Pimentel , Daniel A. de Jesus , Br a´ s H. de Oliveira
a
Universidade Federal do Paran a´ , Departamento de Qu ı´ mica, CP19081, 81531-970 Curitiba-PR, Brazil
b
Universidade Federal do Paran a´ , Departamento de Patologia B a´ sica, Curitiba-PR, Brazil
Received 9 August 2006; received in revised form 5 December 2006
Available online 26 January 2007
Abstract
Betulinic acid (1), a triterpenoid found in many plant species, has attracted attention due to its important pharmacological properties,
such as anti-cancer and anti-HIV activities. The closely related, betulonic acid (2) also has similar properties. In order to obtain deriv-
atives potentially useful for detailed pharmacological studies, both compounds were submitted to incubations with selected microorgan-
isms. In this work, both were individually metabolized by the fungi Arthrobotrys, Chaetophoma and Dematium, isolated from the bark of
Platanus orientalis as well as with Colletotrichum, obtained from corn leaves; such fungal transformations are quite rare in the scientific
literature. Biotransformations with Arthrobotrys converted betulonic acid (2) into 3-oxo-7b-hydroxylup-20(29)-en-28-oic acid (3), 3-oxo-
7b,15a-dihydroxylup-20(29)-en-28-oic acid (4) and 3-oxo-7b,30-dihydroxylup-20(29)-en-28-oic acid (5); Colletotrichum converted betu-
linic acid (1) into 3-oxo-15a-hydroxylup-20(29)-en-28-oic (6) acid whereas betulonic acid (2) was converted into the same product
and 3-oxo-7b,15a-dihydroxylup-20(29)-en-28-oic acid (4); Chaetophoma converted betulonic acid (2) into 3-oxo-25-hydroxylup-20(29)-
en-28-oic acid (7) and both Chaetophoma and Dematium converted betulinic acid (1) into betulonic acid (2). Those fungi, therefore,
are useful for mild, selective oxidations of lupane substrates at positions C-3, C-7, C-15, C-25 and C-30.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Keywords: Betulinic acid; Betulonic acid; Biotransformation; Triterpene, Arthrobotrys; Colletotricum; Chaetophoma; Dematium
1
. Introduction
malaria properties, whereas betulonic acid (2) has anti-
inflammatory, anti-melanoma and anti-viral activity prop-
erties (Yogeeswari and Sriram, 2005). Due to such proper-
ties, the preparation of derivatives of these compounds for
structure–activity relationship studies, has attracted recent
attention, with target positions being mainly the hydroxyl
moiety at C-3 and the carboxyl group (Yogeeswari and Sri-
ram, 2005; Cichewicz and Kouzi, 2004). The chemical pro-
cedures used, however, were limited to activated positions
in the molecule.
In order to target positions more difficult to be function-
alized by chemical methods, microbial procedures have
also been used. On one occasion (Chatterjee et al., 1999),
betulinic acid (1) was glycosylated by a species of Cunning-
hamella, and in another report (Chatterjee et al., 2000) oxi-
dation at C-3 and hydroxylation at C-1, C-7, C-11 and C-
Betulinic acid (1) and betulonic acid (2) are naturally
occurring triterpenes found in many plants. For example,
Betulinic acid (1) has been found in Ziziphus spp. (Su
et al., 2002), Syzygium spp. (Chang et al., 1999), Diospyros
spp. (Mallavadhani et al., 2004), Paeonia spp. (Lin et al.,
1
998), and Doliocarpus spp. (de Oliveira et al., 2002). How-
ever, one of the most widely reported sources of it is the
birch tree (Betula spp.), where it can be found in high con-
centration (Yogeeswari and Sriram, 2005; Cichewicz and
Kouzi, 2004). Both (1) and (2) are valuable compounds
because they exhibit important biological properties. Betu-
linic acid (1) has been reported to have anti-melanoma,
anti-neuroblastoma, anti-leukemia, anti-HIV, and anti-
15 by Bacillus megaterium was described. In yet another
*
study, the use of Bacillus megaterium, Cunninghamella ele-
gans and Mucor mucedo resulted in metabolites oxidized at
Corresponding author. Tel.: +55 41 33613395; fax: +55 41 33613186.
E-mail address: bho@ufpr.br (B.H. de Oliveira).
0
031-9422/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.12.007

D.Z.L. Bastos et al. / Phytochemistry 68 (2007) 834–839
835
C-1, C-3, C-6, C-7, C-11 and C-15 (Kouzi et al., 2000),
Table 1
1
3
C NMR spectroscopic data for 3-oxo-7b,15a-dihydroxylup-20(29)-en-
whereas the methyl ester of betulinic acid (1) was obtained
by incubation with Nocardia (Zhang et al., 2005). Betulonic
acid (2) has also been subjected to biotransformation. It
was transformed by Chetomium longisrotre to produce
metabolites hydroxylated at C-7 and C-15, together with
a derivative whose ring A was opened at C-3 (Akihisa
et al., 2002).
Microbiological transformation of organic compounds
can be useful in two ways during drug development. The
derivatives may have improved properties when compared
to the lead compound, or they may be identical to the
metabolites of those compounds when they are adminis-
tered to mammals. In this case, microbiological transfor-
mation can be a viable alternative to chemical synthesis
for preparation of drug metabolites (Azerad, 1999). There
are few reports on biotransformation by fungi of triter-
penes in general, and of lupanes in particular. The objective
of this work, therefore, was to obtain new derivatives of
betulinic (1) and betulonic (2) acids, potentially useful for
pharmacological studies, using the fungi Arthrobotrys,
Chaetophoma, Dematium, and Colletotrichum.
2
2
8-oic acid (5), 3-oxo-15a-hydroxylup-20(29)-en-28-oic acid (6) and 3-oxo-
5-hydroxylup-20(29)-en-28-oic acid (7) (CDCl ; 100 MHz)
3
C
5
6
7
1
39.5
34.1
39.9
34.2
29.9
34.2
2
3
4
5
6
7
8
9
0
11
12
3
4
5
6
218.1
46.7
52.1
31.0
72.4
47.9
48.7
37.0
21.1
25.1
37.2
48.6
68.9
41.5
54.6
49.7
46.5
149.9
29.7
36.8
26.4
21.1
15.5
10.7
8.3
218.1
47.3
54.6
19.8
37.0
42.3
50.4
37.2
21.5
25.5
38.0
47.4
70.9
42.3
55.2
48.9
46.5
149.8
31.0
36.9
26.6
21.0
16.0
16.3
8.2
218.1
47.0
51.4
20.8
33.7
40.5
42.1
41.4
22.7
25.8
38.4
42.9
29.4
32.5
56.4
49.3
47.0
150.9
30.7
37.3
27.3
20.4
60.4
16.2
14.6
179.0
109.5
19.4
1
1
1
1
1
17
18
1
2
2
2
2
9
0
1
2
3
2
. Results and discussion
24
2
2
2
2
5
6
7
8
29
30
Betulinic acid (1) was isolated from the bark of Platanus
orientalis, and betulonic acid (2) was prepared from it by
oxidation with Jones’ reagent. The spectroscopic data for
both compounds were consistent with their structures and
compatible with published data (Peng et al., 1998; Gonz-
alez et al., 1983). Both compounds were then submitted
to biotransformation by fungi isolated from plants.
The use of epiphytic and endophytic microorganisms
may be a useful strategy for the biotransformation of nat-
ural compounds found in the same plant source. The fungi
Arthrobotrys, Chaetophoma and Dematium were isolated
from Platanus orientalis, the same source of betulinic acid
180.2
110.0
19.5
180.5
110.1
19.5
(a methylene group at 33.7 ppm) was absent but another
methine signal appeared instead at 72.8 ppm. The hydrox-
ylation at C-7 was further confirmed by the paramagnetic
shifts of 11.3 and 7.2 ppm of the neighboring C-6 and C-
8 respectively (from to 19.7 and 40.7 to 31.0 and 47.9).
The stereochemistry at C-7 was deduced from the multi-
1
(
1) used in this work, whereas Colletotrichum was from
plicity of corresponding C–H signal in the H NMR spec-
corn leaves (Bastos et al., 2004). Colletotrichum has been
successfully used in the biotransformation of steroids giv-
ing more highly oxidized metabolites (Wilson et al., 1999).
Biotransformation of betulonic acid (2) with Arthrobot-
rys led to isolation of three metabolites. The spectroscopic
data of the first two were compatible with those of the
known 3-oxo-7b-hydroxylup-20(29)-en-28-oic acid (3) and
trum (3.8 ppm). It was a double doublet (J = 6.4 and
15.6 Hz) compatible with an axial (a) position for that pro-
ton. The product, therefore, is 3-oxo-7b,30-dihydroxylup-
20(29)-en-28-oic acid (5).
The fungus Colletotrichum was used for biotransforma-
tion of betulinic acid (1) to produce a more polar com-
pound as determined by TLC. Its molecular formula was
ꢀ
3
-oxo-7b,15a-dihydroxylup-20(29)-en-28-oic acid (4),
respectively (Akihisa et al., 2002). The third metabolite
5) had a molecular formula established as C H O from
established as C H O from its HRMS ([MꢀH] , m/z
3
0
46
4
469.3380). Its IR spectrum showed the expected carboxyl
carbonyl absorption at 1694 cm and also a new carbonyl
absorption at 1706 cm , indicating the formation of a
ꢀ
1
(
3
0
46
5
ꢀ
1
ꢀ1
its HRMS ([MꢀH] , m/z 485.3343). The H NMR spec-
trum, when compared to the first metabolite, showed a
new peak at 3.8 ppm integrating for one proton, indicating
possible hydroxylation at a methylene carbon. This was
ketone functionality, which was absent in the parent com-
1
pound. The H NMR spectrum of the metabolite was sim-
ilar to that of the parent compound showing characteristic
signals corresponding to six methyl groups. However, it
lacked a resonance for H-3 indicating oxidation of the
corresponding secondary alcohol to a ketone. Moreover,
a new signal appeared at 4.05 ppm integrating for one pro-
1
3
confirmed by analysis of the C NMR spectrum (Table
), which showed nine methylenes (DEPT), instead of 10
1
for the parent compound, as well as an extra methine sig-
nal. The resonance corresponding to C-7 in the substrate

836
D.Z.L. Bastos et al. / Phytochemistry 68 (2007) 834–839
ton, indicating possible hydroxylation at a methylene car-
The biotransformation of betulinic acid (1) by Chaeto-
phoma and by Dematium resulted in the formation of betu-
lonic acid (2). The same transformation was previously
reported by the use of Bacillus megaterium (Kouzi et al.,
2000). It is interesting to note the similarity of this 3-OH
oxidation reaction to that of cholestanol compounds into
cholestanones, catalyzed by microbial cholesterol oxidases
(Motteran et al., 2001). Similar enzymes might also be pres-
ent in plants because betulinic (1) and betulonic (2) acids
have been found together in plants (Chiang et al., 2005).
1
3
bon. Those differences were also noted in the C NMR
spectrum (Table 1), where the resonance corresponding
to C-3 of betulinic acid (1) (a methylene at 78.2 ppm) was
absent and a new methine signal appeared at 70.9 ppm.
The resonances of C-14 and C-16 were also subject to para-
magnetic shifts from 42.6 and 32.4 to 47.2 and 42.3, respec-
tively, indicating hydroxylation had occurred at C-15. An
HMQC experiment established a connection between C-
1
5 and H-15 (d : 70.9 and d : 4.05). Further evidence
C H
was provided by the signal of the methyl group (C-27).
The c effect provided by the hydroxyl at C-15 produced a
shift from 14.8 ppm in betulinic acid (1) to 8.2 ppm in the
metabolite. The stereochemistry at C-15 was inferred from
3. Concluding remarks
1
the multiplicity of the corresponding resonance in the H
The results obtained with Arthrobotrys, Chaetophoma
and Dematium showed that the use of microorganisms iso-
lated from the same source as the substrate increase the
likelihood of obtaining derivatives using this approach.
The oxidation reactions with Choletotrichum, however,
were similar to those obtained by biotransformation of ste-
roids by the same fungus genus (Wilson et al., 1999) thus
showing that its oxidases and monooxygenases have broad
substrate specificity towards their polynuclear terpenes.
The use of the fungi described in this work is, therefore,
a viable procedure for obtaining derivatives of betulinic
(1) and betulonic (2) acids. The derivatives obtained result
from oxidation reactions, which are of normal occurrence
during mammalian metabolism. Those compounds could
potentially be useful in the study of the metabolism of bet-
ulinic (1) and betulonic (2) acids in mammals.
NMR spectrum (4.05 ppm). It was a double doublet
(
J = 4.8 Hz and 11.2 Hz) indicating an axial position for
H-15, which otherwise would be a broad singlet. The equa-
torial (a) orientation for the hydroxyl is, therefore, the
most likely, and the product is 3-oxo-15a-hydroxylup-
2
0(29)-en-28-oic acid (6).
The biotransformation of betulonic acid (2) by Colletotri-
chum resulted in formation of two more polar compounds as
determined by TLC. The first was identical to that of 6 pro-
duced from biotransformation of betulinic acid (1) by the
same fungus. The second metabolite was more polar than
the first, as determined by TLC. The spectroscopic data
were, however, identical to that of 5 obtained from the bio-
transformation of betulonic acid (2) by Arthrobotrys as
described above. It has also been described previously as a
result of biotransformation of betulonic acid (2) by Cheto-
mium longirostre (Akihisa et al., 2002).
The biotransformation of betulonic acid (2) by Chaeto-
phoma resulted in formation of a more polar compound as
determined by TLC. Its molecular formula was established
4. Experimental
4.1. General experimental details
ꢀ
as C H O from its HRMS ([MꢀH] , m/z 469.3380). Its
3
0
46
4
1
H NMR spectrum, when compared to that of the substrate,
Mps were determined in a Quimis apparatus, model
Q340523. Optical rotations were measured using a Belling-
ham Stanley apparatus, model D, whereas IR spectra were
recorded in KBr discs using a Bio-Rad spectrometer model
FTS3500GX. NMR spectra were acquired with Bruker
spectrometer models Avance DRX400 and Avance DPX
200 using TMS as internal standard; 1D and 2D experi-
ments (DEPT, 1H-1H COSY, HMQC, HMBC, NOESY)
were carried out for structure determinations. Mass spectra
showed five methyl signals, instead of six. The signal of C-25
was missing and a new methylenic group appeared at
1
3
4
.10 ppm. Hydroxylation at C-25 was confirmed by
C
NMR spectroscopic analysis (Table 1), which showed a res-
onance at 60.4 ppm, and shifts on the absorptions of the
neighboring C-1, C-5 and C-9 (from 39.7, 55.0 and 49.9 to
2
9.9, 51.4 and 42.1, respectively). The product, therefore,
is 3-oxo-25-hydroxylup-20(29)-en-28-oic acid (7).
2
9
2
0
3
3
0
R H C
2
1
2
3
4
1
9
(2) R = R = R = R = H
2
2
1
2
3
4
4
(
3) R = OH, R =R = R =H
R
2
5
1
18
1
12613
CO H
CO H
1
2
3
4
2
2
1
4
16
(4) R =R =OH, R =R =H
2
8
15
1
1
3
2
4
0
(5) R =R =OH, R =R =H
3
5
7
27
3
7
2
1
3
4
2
R
1
(6) R =R =R =H, R =OH
HO
O
R
1
2
3
4
(
7) R =R =R =H, R =OH
2
4
23
1

D.Z.L. Bastos et al. / Phytochemistry 68 (2007) 834–839
837
were recorded in an API 3000 System from Applied Biosys-
4.5. General biotransformation procedure
tems/MDS Sciex, whereas high resolution mass spectra
employed a Micromass Q-Tof from Waters. Solvents were
distilled before use. TLC was performed on 0.2 mm thick
silica prepared plates from Merck (art. # 5554). Spot visu-
alizations were made by spraying with H SO /EtOH (1:1,
v/v) followed by heating and inspection under UV light.
Vacuum liquid CC was performed with silica Si-60 from
Merck (art. # 7744), using appropriate mixtures of hex-
ane/EtOAc. Planar centrifugal chromatography was per-
formed with a Chromatotron (Harrison Research, Palo
Alto, CA, USA) model 7924T. CC was performed on silica
from Merck (art. # 7736) whereas preparative HPLC was
carried out using a Rainin system with a pump (model
SD-200), pump head 50 (50 ml/min part # R007101065),
UV detector (UV-1) and Dynamax C18 column (21.4 ·
Stock cultures of fungi were stored on potato dextrose
agar slants, at 4 ꢁC. Seed cultures of each fungus were
obtained by transferring fungi from stock cultures to a
ꢀ
1
liquid medium containing (g l ) glucose (10), corn steep
liquor (8) and yeast extract (2). The experiments were
conducted in conical flasks (1 l), containing the same
liquid medium (200 ml) inoculated with the fungi. The
flasks were shaken at 30 ꢁC and 150 rpm for 48 h. The
substrates, previously dissolved in a minimum volume
(ꢁ2 ml) of acetone (betulonic acid, 2) or DMSO (betuli-
nic acid, 1), were evenly distributed among the flasks,
except one, kept as a control, and the reaction was
allowed to proceed for 7 more days. The mycelium was
then removed by filtration, and the biomass was extracted
with EtOAc (100 ml), for 30 min, in an ultrasonic bath
and filtered again. The broth was extracted with the same
solvent (3 · 100 ml) and all extracts were combined and
2
4
2
2
50 mm, 8 lm, art # R00083221C). Flow rate used was
0 ml/min and eluate monitored at 210 nm.
dried (anhydr. Na SO ). After filtration, the solvents were
4
.2. Isolation of betulinic acid (1)
2
4
evaporated under reduced pressure, and the crude extract
was fractionated by chromatography. Controls were car-
ried out in order to verify the action of the medium
The bark of Platanus orientalis was collected in Curitiba
(
Brasil), with a voucher specimen deposited at the herbar-
ium of UFPR (#47812). The dried pulverized bark
100.0 g) was continuously extracted with CH Cl in a Soxh-
(without fungus) on the substrates and the presence of
similar metabolites on the fungi cultures (without
substrates).
(
2
2
let apparatus for 3 h. After solvent evaporation, a portion of
the extract (4.0 g) was submitted to vacuum liquid CC on
silica (80.0 g), with elution using hexane/EtOAc mixtures.
A solid was obtained (0.69 g), which was characterized as
betulinic acid (1) by spectroscopic methods and comparison
with literature data (Peng et al., 1998).
4.6. Biotransformation of betulonic acid (2) by Arthrobotrys
The crude extract obtained from the biotransformation
of 2 (1.280 g) was fractionated by CC on silica. Elution
with hexane–EtOAc (2:1 v/v) led to the isolation of metab-
olites 3 (21 mg, 1.64% yield), 4 (8 mg, 0.62% yield) and 5
4
.3. Preparation of betulonic acid (2)
(17 mg, 1.33% yield).
Betulinic acid (1) (1.0 g, 2.26 mmol) was dissolved in
acetone (50 ml) and the mixture was cooled in an ice bath.
Jones’ reagent was then added slowly (10 ml), with stirring,
and the reaction was allowed to proceed for 1.5 h. Then
4.6.1. 3-Oxo-7b-hydroxylup-20(29)-en-28-oic acid (3)
2
5
M.p. 165–168 ꢁC; ½aꢂ þ 17 (MeOH; c0.6); HRMS m/z:
D
ꢀ
469.3380 [MꢀH] (calcd for C H O : 470.3396). ESI-MS
3
0
46
4
ꢀ
MeOH (25 ml) was added, followed by H O (40 ml). The
ꢀ45 eV, m/z (rel. int.) 469.4 [MꢀH]
(100), 451
2
ꢀ
ꢀ
solvents were evaporated under reduced pressure, and the
aqueous residue was extracted with EtOAc (2 · 40 ml).
The organic phase was dried, filtered and the solvent evap-
orated. The solid residue was then subjected to silica CC
affording a solid, which was characterized as betulonic acid
[MꢀHꢀH O] (2.0), 424 [MꢀHꢀCOOH] (8.0), 407
2
+
ꢀ
[MꢀHꢀOHꢀCOOH] (2.0), 411 [MꢀHꢀOHꢀC H O]
3
5
ꢀ
1
1
(5.0); IR
m
/cm
:
3438, 2956, 1694.
H NMR
max
(400 MHz, CDCl ) d: 0.92 (3H, s, H-25), 1.02 (3H, s, H-
3
26), 1.00 (3H, s, H-24), 1.07 (3H, s, H-27), 1.28 (3H, s,
(
2) by spectroscopic methods and comparison with litera-
H-23), 1.69 (3H, s, H-30), 3.84 (1H, dd, J = 5.2 Hz and
1
3
ture data (Gonzalez et al., 1983).
15.2 Hz, H-7), 4.61 (1H, s, H-29), 4.77 (1H, s, H-29).
C
NMR (100 MHz, CDDl ): d 39.3 (t, C-1), 34.0 (t, C-2),
3
4
.4. Fungi
218.0 (s, C-3), 46.6 (s, C-4), 52.4 (d, C-5), 32.5 (t, C-6),
74.4 (d, C-7), 48.9 (s, C-8), 49.8 (d, C-9), 36.9 (s, C-10),
Isolation and identification of fungi are described else-
21.4 (t, C-11), 25.5 (t, C-12), 37.2 (d, C-13), 48.6 (s, C-
14), 68.9 (d, C-15), 41.5 (t, C-16), 54.6 (s, C-17), 49.7 (d,
C-18), 46.5 (d, C-19), 149.9 (d, C-20), 29.7 (t, C-21), 36.8
(t, C-22), 26.4 (q, C-23), 21.1 (q, C-24), 15.5 (q, C-25),
10.7 (q, C-26), 8.3 (q, C-27), 180.2 (s, C-28), 110.0 (t, C-
29), 19.5 (q, C-30).
where (Bastos et al., 2004). They were deposited at the cul-
ture collection of the Department of Basic Pathology of
Universidade Federal do Paran a´ : Arthrobotrys (DPB134)
Colletotrichum (DPB136), Chaetophoma (DPB125) and
Dematium (DPB157).

838
D.Z.L. Bastos et al. / Phytochemistry 68 (2007) 834–839
4
(
.6.2. 3-Oxo-7b,15a-dihydroxylup-20(29)-en-28-oic acid
4)
4.8. Biotransformation of betulonic acid (2) by
Colletotrichum
25
M.p. 280–282 ꢁC; ½aꢂ þ 20 (MeOH; c0.3); HRMS m/z:
D
ꢀ
4
85.3323 [MꢀH] (calcd for C H O :486.3345). ESI-MS
The crude EtOAc extract obtained from the biotransfor-
mation of 2 (640 mg) was fractionated by planar centrifu-
gal chromatography, and then by CC on silica gel, using
mixtures of hexane–EtOAc (2:1 v/v) as eluent. The first
metabolite isolated (6, 19 mg; 2.97% yield) was identical
to that obtained from the biotransformation of betulinic
acid (1) by Colletotrichum, and the second one (5, 11 mg;
1.72% yield) was the same as that obtained for biotransfor-
mation of betulonic acid (2) by Arthrobotrys.
3
0
46
5
ꢀ
ꢀ
45 eV, m/z (rel. int.) 485.4 [MꢀH] (100), 467 [Mꢀ
ꢀ
ꢀ
HꢀH O]
(55.9), 440 [MꢀHꢀCOOH]
(26.1), 423
2
ꢀ
ꢀ
[
(
MꢀHꢀOHꢀCOOH] (3.7), 411 [MꢀHꢀOHꢀC H O]
3
5
ꢀ
1
1
3.8); IR m /cm : 3467, 2945, 1706, 1688; H NMR
max
(
400 MHz, CDCl ) d: 0.93 (3H, s, H-25), 1.03 (3H, s, H-
3
2
6), 1.04 (3H, s, H-24), 1.02 (3H, s, H-27), 1.10 (3H, s,
H-23), 1.69 (3H, s, H-30), 3.80 (1H, dd, J = 6.4 Hz and
1
7
5.6 Hz, H-7), 3.90 (1H, dd, J = 6.8 Hz and 15.6 Hz, H-
1
3
), 4.63 (1H, s, H-29), 4.72 (1H, s, H-29). C NMR
(
(
100 MHz, CDDl ): d 39.5 (t, C-1), 34.1 (t, C-2), 218.0
s, C-3), 46.9 (s, C-4), 52.1 (d, C-5), 31.0 (t, C-6), 72.8 (d,
3
4.9. Biotransformation of betulonic acid (2) by
Chaetophoma
C-7), 47.9 (s, C-8), 48.7 (d, C-9), 37.0 (s, C-10), 21.1 (t,
C-11), 25.1 (t, C-12), 37.2 (d, C-13), 48.6 (s, C-14), 68.9
(
4
2
The crude extract obtained from the biotransformation
of 2 (640 mg) was fractionated by vacuum liquid chroma-
tography on silica. Elution with hexane and hexane/EtOAc
produced a solid (27 mg; 4.22% yield) which was character-
d, C-15), 41.5 (t, C-16), 54.6 (s, C-17), 49.7 (d, C-18),
6.5 (d, C-19), 149.9 (d, C-20), 29.7 (t, C-21), 36.8 (t, C-
2), 26.4 (q, C-23), 21.1 (q, C-24), 15.5 (q, C-25), 10.7 (q,
C-26), 8.3 (q, C-27), 180.2 (s, C-28), 110.0 (t, C-29), 19.5
q, C-30).
ized as 3-oxo-25-hydroxylup-20(29)-en-28-oic acid (7).
(
25
M.p. 215–218 ꢁC; ½aꢂ ꢀ 12:7 (MeOH; c0.3); HRMS m/z:
D
ꢀ
4
69.3380 [MꢀH] (calcd for C H O : 470.3396). ESI-
3
0
46
4
4
.6.3. -Oxo-7b,30-dihydroxylup-20(29)-en-28-oic acid (5)
ꢀ
MS ꢀ45 eV, m/z (rel. int.) 469.3 [MꢀH] (100), 451
25
M.p.: 168–171 ꢁC; ½aꢂ þ 14:2 (MeOH; c0.3); HRMS m/
ꢀ
ꢀ
D
[MꢀHꢀH O] (2.2), 424 [MꢀHꢀCOOH] (9.0), 407
2
ꢀ
z: 485.3343 [MꢀH] (calcd for C H O : 486.3345). ESI-
+
3
0
46
5
[
1
MꢀHꢀOHꢀCOOH] (5.0); IR m 3403, 2951, 1700,
max
ꢀ
MS ꢀ45 eV, m/z (rel. int.) 485.4 [MꢀH] (100), 440
ꢀ1
1
688 cm ; H NMR (CDCl , 400 MHz) d 1.00 (3H, s,H-
3
ꢀ
ꢀ
[
(
2
MꢀHꢀCOOH]
(23.8), 423 [MꢀHꢀOHꢀCOOH]
ꢀ ꢀ1
24), 1.01 (3H, s, H-27), 1.23 (3H, s, H-26), 1.28 (3H, s,
52.4), 411 [MꢀHꢀC H O] (38.0); IR m /cm : 3487,
3
5
max
H-23), 1.70 (3H, s, H-30), 3.12 (2H, m, H-25), 3.20 (2H,
1
945, 1694; H NMR (400 MHz, CDCl ) d: 0.92 (3H, s,
3
ddd, J = 4.4, 17, 26 Hz, H-12), 4.61 (1H, s, H-29), 4.76
H-25), 0.99 (3H, s, H-24), 1.02 (3H, s, H-26), 1.08 (3H, s,
H-27), 1.27 (3H, s, H-23), 3.85 (1H, dd, J = 4.8 Hz and
13
(
1H, s, H-29). For C NMR spectrum, see Table 1.
1
4
5.6 Hz, H-7), 4.13 (2H, m, H-30), 4.92 (1H, s, H-29),
.98 (1H, s, H-29); C NMR spectrum, see Table 1.
1
3
4.10. Biotransformation of betulinic acid (1) by
Chaetophoma
4
.7. Biotransformation of betulinic acid (1) by
Colletotrichum
The crude extract from the biotransformation 1 (618
mg) was fractionated by vacuum liquid chromatography
on silica. Elution with hexane and hexane/EtOAc produced
a solid (5.8 mg; 0.94% yield) which was characterized as
betulonic acid (2) by comparison with spectroscopic data
identical to that prepared previously.
The extract obtained from the biotransformation of 1
(
640 mg) was fractionated by planar centrifugal chroma-
tography on a silica rotor (2 mm thick). Elution with hex-
ane–EtOAc (8:2 v/v) produced a white solid (23 mg;),
which was refractionated by prep. HPLC on a C18 column.
Elution with CH CN-H O (4:1) provided a white solid
4.11. Biotransformation of betulinic acid (1) by Dematium
3
2
(
1
2
15 mg; 2.34% yield) which was characterized as 3-oxo-
The extract from the biotransformation of 1 (640 mg)
5a-hydroxylup-20(29)-en-28-oic acid (6). M.p. 274–
25
was fractionated by vacuum liquid chromatography on
silica. Elution with hexane and hexane/dichloromethane
produced a solid (4 mg; 0.62% yield), which was character-
ized as betulonic acid (2) identical to that prepared
previously.
76 ꢁC; ½aꢂ ꢀ 11:5 (MeOH; c0.3); HRMS m/z: 469.3380
D
ꢀ
[
MꢀH] (calcd for C H O : 470.3396). ESI-MS ꢀ45
3
0
46
4
ꢀ
ꢀ
eV, m/z (rel. int.) 469.3 [MꢀH] (100), 451 [MꢀHꢀH O]
2
ꢀ
(
1.2), 424 [MꢀHꢀCOOH] (10.0), 407 [MꢀHꢀOHꢀ
+
ꢀ1
1
COOH] (3.0); IR m /cm : 2945, 1706, 1694;
H
max
NMR (400 MHz, CDCl ) d: 0.94 (3H, s, H-25), 1.01 (3H,
3
s, H-26), 1.01 (3H, s, H-24), 1.08 (3H, s, H-27), 1.26 (3H,
s, H-23), 1.70 (3H, s, H-30), 4.05 (1H, dd, J = 4.8 Hz and
Acknowledgements
1
1.2 Hz, H-15), 4.64 (1H, s, H-29), 4.74 (1H, s, H-29); for
C NMR spectrum, see Table 1.
1
3
The financial support of FINEP and CAPES (Brazil) are
greatly acknowledged.

D.Z.L. Bastos et al. / Phytochemistry 68 (2007) 834–839
839
References
Lin, H.C., Ding, H.Y., Wu, Y.C., 1998. Two novel compounds from
Paeonia suffruticosa. J. Nat. Prod. 61, 343–346.
Mallavadhani, U.V., Satyanarayana, K.V., Mahapatra, A., 2004. Quan-
titative evaluation of anticancer marker levels of an Ayurvedic
preparation, ‘‘Virala’’. Pharm. Biol. 42, 338–341.
Akihisa, T., Takamine, Y., Yoshizumi, K., Tokuda, H., Kimura, Y., Ukiya,
M., Nakahara, T., Yokochi, T., Ichiishi, E., Nishino, H., 2002. Microbial
transformations of two lupane-type triterpenes and anti-tumor-promot-
ing effects of the transformation products. J. Nat. Prod. 65, 278–282.
Azerad, R., 1999. Microbial models for drug metabolism. In: Faber, K.
Motteran, L., Pilone, M.S., Molla, G., Ghisla, S., Pollegioni, L., 2001.
Cholesterol oxidase from Brevibacterium sterolicum – the relationship
between covalent flavinylation and redox properties. J. Biol. Chem.
(Ed.),. Advances in Biochemical Engineering/Biotechnology, vol. 63.
Springer, Berlin/Heidelberg, pp. 169–218.
Bastos, D.Z.L., Pimentel, I.C., Dykstra, C., Gabardo, J., Oliveira, B.H.,
2
76, 18024–18030.
de Oliveira, B.H., Santos, C.A.M., Espindola, A.P.D.M., 2002. Determi-
nation of the triterpenoid, betulinic acid, in Doliocarpus schottianus by
HPLC. Phytochem. Anal. 13, 95–98.
2
004. Fungos associados a` casca do caule de Platanus orientalis L.
Estudos de Biologia 26, 37–41.
Peng, C., Bodenhausen, G., Qiu, S.X., Fong, H.H.S., Farnsworth,
N.R., Yuan, S.G., Zheng, C.Z., 1998. Computer-assisted structure
elucidation: application of cisoc-sec to the resonance assignment and
structure generation of betulinic acid. Magn. Reson. Chem. 36, 267–
Chang, C.W., Wu, T.S., Hsieh, Y.S., Kuo, S.C., Chao, P.D.L., 1999.
Terpenoids of Syzygium formosanum. J. Nat. Prod. 62, 327–328.
Chatterjee, P., Pezzuto, J.M., Kouzi, S.A., 1999. Glucosidation of
betulinic acid by Cunninghamella species. J. Nat. Prod. 62, 761–763.
Chatterjee, P., Kouzi, S.A., Pezzuto, J.M., Hamann, M.T., 2000. Biotrans-
formation of the antimelanoma agent betulinic acid by Bacillus
megaterium ATCC 13368. Appl. Environ. Microbiol. 66, 3850–3855.
Chiang, Y.M., Chang, J.Y., Kuo, C.C., Chang, C.Y., Kuo, Y.H., 2005.
Cytotoxic triterpenes from the aerial roots of Ficus microcarpa.
Phytochemistry 66, 495–501.
2
78.
Su, B.N., Cuendet, M., Farnsworth, N.R., Fong, H.H.S., Pezzuto, J.M.,
Kinghorn, A.D., 2002. Activity-guided fractionation of the seeds of
Ziziphus jujuba using a cyclooxygenase-2 inhibitory assay. Planta Med.
6
8, 1125–1128.
Yogeeswari, P., Sriram, D., 2005. Betulinic acid and its derivatives:
a review on their biological properties. Curr. Med. Chem. 12,
Cichewicz, R.H., Kouzi, S.A., 2004. Chemistry, biological activity and
chemotherapeutic potential of betulinic acid for the prevention and
treatment of cancer and HIV infection. Med. Res. Rev. 24, 90–114.
Gonzalez, A.G., Amaro, J., Fraga, B.M., Luis, J.G., 1983. 3-Oxo-6b-
hydroxyolean-18-en-28-oic acid from Orthopterygium huancuy. Phyto-
chemistry 22, 1828.
6
57–666.
Wilson, M.R., Gallimore, W.A., Reese, P.B., 1999. Steroid transforma-
tions with Fusarium oxysporum var. cubense and Colletotrichum musae.
Steroids 64, 834–843.
Zhang, J., Cheng, Z.-H., Yu, B.-Y., Cordell, G.A., Qiuc, S.X., 2005.
Novel biotransformation of pentacyclic triterpenoid acids by Nocardia
sp. NRRL 5646. Tetrahedron Lett. 46, 2337–2340.
Kouzi, S.A., Chatterjee, P., Pezzuto, J.M., Hamann, M.T., 2000.
Microbial transformations of the antimelanoma agent betulinic acid.
J. Nat. Prod. 63, 1653–1657.