Microbial hydroxylation of bufalin by Cunninghamella blakesleana and Mucor spinosus

Article abstract of DOI:10.1021/np0500023

The microbial transformation of a cytotoxic bufadienolide, bufalin (1), was carried out using two strains of filamentous fungi. Cunninghamella blakesleana catalyzed the specific 12α-hydroxylation of bufalin and produced 12α-hydroxybufalin (2) and 7β,12α-dihydroxybufalin (3) as the major metabolites, together with 7β-hydroxybufalin (4) and 12β-hydroxybufalin (5) in low yields. Two minor products were isolated from the culture broth of Mucor spinosus and were identified as 7β,15α- dihydroxybufalin (6) and 5β,7β-dihydroxybufalin (7), respectively. Metabolites 2, 3, 6, and 7 are new compounds, and their structures were fully characterized by NMR and MS spectroscopy.

Full text of DOI:10.1021/np0500023

626
J. Nat. Prod. 2005, 68, 626-628
Microbial Hydroxylation of Bufalin by Cunninghamella blakesleana and Mucor
spinosus
Min Ye,† Jian Han,† Guangzhong Tu,‡ Dongge An,‡ and Dean Guo*,†
The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University,
Xueyuan Road 38, Beijing 100083, People’s Republic of China, and Beijing Institute of Microchemistry, Beijing 100091,
People’s Republic of China
Received January 1, 2005
The microbial transformation of a cytotoxic bufadienolide, bufalin (1), was carried out using two strains
of filamentous fungi. Cunninghamella blakesleana catalyzed the specific 12R-hydroxylation of bufalin
and produced 12R-hydroxybufalin (2) and 7â,12R-dihydroxybufalin (3) as the major metabolites, together
with 7â-hydroxybufalin (4) and 12â-hydroxybufalin (5) in low yields. Two minor products were isolated
from the culture broth of Mucor spinosus and were identified as 7â,15R-dihydroxybufalin (6) and 5â,7â-
dihydroxybufalin (7), respectively. Metabolites 2, 3, 6, and 7 are new compounds, and their structures
were fully characterized by NMR and MS spectroscopy.
Bufalin, cinobufagin, and resibufogenin are the major
bufadienolides isolated from the traditional Chinese drug
ChanSu.1,2 They are steroids with a characteristic R-pyrone
ring at C-17 and possess strong antitumor activity. Previ-
ous reports showed that they exhibit significant inhibitory
effects against human myeloid leukemia cells, human
hepatoma cells, and prostate cancer cells with IC50 values
of 10-9-10-10 mol/L. These activities are mediated by the
induction of cell apoptosis and cell differentiation.3-6 For
several years we have focused on the biotransformation of
these bufadienolides to obtain new analogues with im-
proved cytotoxicity and increased water solubility, which
are of essential importance for the development as new
drugs. More than 40 biotransformed products have been
obtained, so far, including 18 new compounds.7-10 The
major biotransformation reactions involved hydroxylation
at the 1â-, 5â-, 7â-, 12â-, and 16R-positions. In this paper,
we report the specific 12R-hydroxylation of bufalin (1) by
Cunninghamella blakesleana AS 3.970. Two new minor
products from the culture broth of Mucor spinosus AS
3.3450 are also described.
A screen of 20 fungal strains for the biotransformation
of bufalin had been carried out as described in our previous
paper.10 Two new significant peaks were observed in the
HPLC chromatogram of the biotransformed products from
C. blakesleana AS 3.970. This biotransformation was scaled
up in the present study, and the two target products were
obtained by silica gel column chromatography and pre-
parative liquid chromatography. Their structures were
identified as 12R-hydroxybufalin (2) and 7â,12R-dihydroxy-
bufalin (3). Two minor products were also purified and
characterized as the known 7â-hydroxybufalin (4) and 12â-
hydroxybufalin (5),10 respectively. The biotransformation
of bufalin by M. spinosus AS 3.3450 to afford 12 metabolites
was reported previously, mainly involving the hydroxyla-
tion at C-7â, C-12â, and C-16R.10 In this study, two new
minor products were obtained by preparative liquid chro-
matography and identified as 7â,15R-dihydroxybufalin (6)
and 5â,7â-dihydroxybufalin (7). The structures (Figure 1)
were characterized by mass spectrometry and extensive
Figure 1. Biotransformation of bufalin (1) by Cunninghamella
blakesleana and Mucor spinosus.
NMR techniques, including 1H NMR, 13C NMR, DEPT,
HMQC, HMBC, and NOESY.
The atmospheric pressure chemical ionization mass
spectrum (APCI-MS) of 2 gave a [M + H]+ ion at m/z 403,
suggesting the molecular formula C24H34O5. When com-
pared to bufalin, an additional oxygen-bearing tertiary
carbon appeared at δ 74.0. According to the HMBC
spectrum, it showed long-range couplings with 18-CH3 (δ
0.52) and H-17 (δ 3.13), indicating the introduction of a
hydroxyl group at C-12. In contrast to 12â-hydroxylated
bufadienolides, where C-18 appeared at remarkably higher
fields (∼δ 10.5),10 the C-18 of 2 resonated at δ 18.0,
* To whom correspondence should be addressed. Tel: 86-10-82801516.
Fax: 86-10-82802700. E-mail: gda@bjmu.edu.cn.
† Peking University.
‡ Beijing Institute of Microchemistry.
10.1021/np0500023 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/07/2005

Notes
Journal of Natural Products, 2005, Vol. 68, No. 4 627
Table 1. Cytotoxic Activities of the Biotransformed Products
against Human Cancer Cell Lines (n ) 3)a
indicating R-configuration of the C-12 hydroxyl group.
There was little effect on the chemical shift of C-18 due to
their γ-trans positions. In accordance, C-9 (δ 27.9) and C-17
(δ 44.1) shifted upfield by 6.9 and 6.0 ppm when compared
to bufalin, respectively, since they are in the γ-gauche
positions of 12R-OH. On the basis of the above evidence,
compound 2 was identified as 12R-hydroxybufalin.
IC50 (µmol/L)
compound
Bel-7402b
BGC-823c
HeLad
1
2
3
5
7
7.0 × 10-3
4.1 × 10-1
2.9
4.5 × 10-2
4.2 × 10-1
5.3
2.8 × 10-2
6.0 × 10-2
4.7 × 10-1
7.6 × 10-3
0.022
The APCI mass spectrum of 3 gave a [M + H]+ ion at
m/z 419, suggesting the molecular formula C24H34O6. The
13C NMR spectrum showed two additional oxygenated
methine signals at δ 68.9 and 73.7, suggesting that 3 was
a dihydroxylated product of bufalin. Similar to 2, the signal
at δ 73.7 showed HMBC correlations with 18-CH3 (δ 0.54)
and H-17 (δ 3.15), and C-18 appeared at δ 18.1. Thus
metabolite 3 was also hydroxylated at C-12R. The other
signal (δ 68.9) corresponded to an unusually broad peak
at δ 3.80 in the HMQC spectrum, and C-6 (δ 36.5) and C-8
(δ 46.3) were shifted downfield by 10.0 and 5.1 ppm when
compared to bufalin, respectively. This evidence supported
the hydroxylation at C-7. The broad signal of H-7 (δ 3.80)
resulted from its axial-axial couplings with H-6â and H-8,
indicating that 7-OH should be in the â-configuration.
Accordingly, the signal of 14-OH (δ 5.34) appeared at a
rather low field, as a result of the intramolecular hydrogen
bonding of 7â-OH with 14-OH. Therefore, compound 3 was
identified as 7â,12R-dihydroxybufalin.
Compounds 6 and 7 were obtained as minor products
from M. spinosus. The APCI mass spectrum of 6 gave a
[M + H]+ ion at m/z 419, suggesting the molecular formula
C24H34O6. The 13C NMR spectrum showed two additional
oxygen-bearing signals at δ 68.5 and 79.0, suggesting that
6 was a dihydroxylated product of bufalin. Similar to 3,
C-8 of 6 shifted downfield to δ 46.5, and 14â-OH to δ 5.45,
suggesting that a 7â-OH had been introduced. In addition,
C-16 (δ 40.6) shifted downfield by 12.2 ppm when compared
to bufalin, indicating an additional hydroxylation at C-15.
The NOE enhancement of H-15 (δ 4.21) with 14â-OH (δ
5.45) supported the R-configuration of 15-OH. Thus, com-
pound 6 was identified as 7â,15R-dihydroxybufalin.
The APCI mass spectrum of 7 gave a [M + H]+ ion at
m/z 419, suggesting the molecular formula C24H34O6. An
oxygenated quaternary carbon appeared at δ 73.2, which
showed HMBC correlation with 19-CH3 (δ 0.82), suggesting
hydroxylation at C-5. In accordance, C-10 shifted downfield
to δ 39.6, while C-1 shifted upfield to δ 24.8 due to a
γ-gauche effect. The other new signal appeared at δ 69.9.
Its corresponding proton signal (δ 3.63) appeared as a very
broad peak, and 14â-OH shifted downfield to δ 5.42. This
evidence indicated the presence of a 7â-hydroxyl group.
Therefore, compound 7 was identified as 5â,7â-dihydroxy-
bufalin.
The two major products 2 and 3 from C. blakesleana were
both hydroxylated at C-12R, and they constituted more
than 90% of the total metabolites. This reaction could thus
be considered as a specific one. Indeed, 7â-hydroxybufalin
(4) was completely converted into 7â,12R-dihydroxybufalin
(3) as the only product, within 2 days when it was added
to the cultures of C. blakesleana. Although a number of
products hydroxylated at the 1â-, 5â-, 7â-, 11â-, 12â-, and
16R-positions had been reported before, this is the first
report of 12R-hydroxylation of bufadienolides. Interestingly,
we had discovered the specific 12â-hydroxylation of bufa-
dienolides by Alternaria alternata AS 3.4578.9 These two
reactions allowed the directed and stereoselective synthesis
of 12-OH bufadienolides.
2.6 × 10-2
3.1 × 10-2
n.d.e
n.d.
a The data for compounds 1 and 5 were adopted from the
literature.10 b Human hepatoma cells. c Human gastric cancer
cells. d Human cervical carcinoma cells. e n.d., not determined.
by the MTT method.12 The results are given in Table 1.
12R-Hydroxybufalin (2) showed significant inhibitory ef-
fects against all three tested cancer cell lines. The IC50
values ranged from 4.2 × 10-1 to 6.0 × 10-2 µmol/L.
However, its effects were apparently weaker than 12â-
hydroxybufalin (5), which exhibited almost comparable
activities to bufalin.10 Compound 3 was further hydroxy-
lated at C-7â, and its activities were about 10-fold weaker
than 2. 5â,7â-Dihydroxybufalin (7) was significantly inhibi-
tory to HeLa cells, and the IC50 value was 0.022 µmol/L.
Most dihydroxylated bufadienolides we obtained previously
showed weak cytotoxicities, and compound 7 appeared to
be an exception. Its activities against Bel-7402 and BGC-
823 cells, together with those of compound 6, however, were
not determined due to the limited amounts of pure com-
pounds. Their potential as drug candidates still needs
further evaluation.
Experimental Section
General Experimental Procedures. Melting points were
determined with an XT4A apparatus and are uncorrected.
Optical rotations were measured with a Perkin-Elmer 243B
polarimeter. UV spectra were recorded with a TU-1901 UV-
vis spectrophotometer. IR spectra were determined in KBr
with an Avatar 360 FT-IR spectrophotometer. 1H and 13C NMR
spectra were obtained on a Bruker DRX-500 spectrometer (500
MHz for 1H NMR and 125 MHz for 13C NMR) in DMSO-d6 at
ambient temperature with tetramethylsilane (TMS) as the
internal standard. The chemical shifts (δ values) are given in
parts per million (ppm) relative to TMS at 0 ppm. The coupling
constants (J values) are reported in hertz (Hz). APCI mass
spectra were measured on a Finnigan LCQ Advantage mass
spectrometer in the positive ion mode. High-resolution mass
spectra (HR-MS) were obtained on a Bruker APEX II Fourier
transform ion cyclotron resonance (FT-ICR) mass spectrom-
eter. A SpectraSERIES HPLC apparatus (Thermo Quest) with
a 100 µL loop was used for preparative liquid chromatography.
Samples were separated on a YMC ODS-A column (5 µm, φ
10 × 250 mm). The flow rate was 2.0 mL/min, and the
detection wavelength was 296 nm. Bufalin (1) was isolated
from the Chinese drug ChanSu and unambiguously identified
by NMR and MS techniques.11 The purity was determined to
be >99.5% by HPLC analysis. Silica gel (200-300 mesh) for
column chromatography was purchased from Qingdao Marine
Chemical Corporation, Qingdao, China. All chemical solvents
used for product isolation were of analytical grade.
Microorganisms and Culture Media. Cunninghamella
blakesleana AS 3.970 and Mucor spinosus AS 3.3450 were
purchased from China General Microbiological Culture Col-
lection Center, Beijing, China, and were maintained on potato
agar slants at 4 °C. Fermentations were carried out in a potato
medium consisting of 20 g of potato extract, 20 g of glucose,
and 1000 mL of distilled H2O. The media were sterilized in
an autoclave at 121 °C and 1.06 kg/cm2 for 30 min.
Biotransformation of Bufalin (1) by C. blakesleana AS
3.970. Mycelia of C. blakesleana from agar slants were
aseptically transferred to 250 mL Erlenmeyer flasks contain-
In vitro cytotoxic activities of the biotransformed prod-
ucts were determined with three human cancer cell lines

628 Journal of Natural Products, 2005, Vol. 68, No. 4
Notes
ing 80 mL of liquid potato medium. The fungus was incubated
at 25 °C on a rotary shaker (180 rpm) in the dark for 24 h to
make a stock inoculum. Then 5 mL of the inoculum was added
to each of the 10 1-L flasks containing 350 mL of potato
medium. After 36 h incubation, a total amount of 200 mg of
bufalin dissolved in 10 mL of EtOH was distributed equally
among the 10 flasks. The incubation was allowed to continue
for four additional days on the shaker. The cultures were then
pooled and filtered through Whatman no. 1 filter paper. The
filtrate was extracted with 3 L of EtOAc three times. The
organic extract was concentrated and evaporated to dryness
in a rotary evaporator under reduced pressure at 60 °C to yield
0.43 g of a brownish solid. The extract was subjected to silica
gel column chromatography (55 g, 200-300 mesh, φ 2 × 30
cm) and eluted in 100 mL fractions with petroleum ether (60-
90 °C) and acetone (4:1 to 1:1, v/v), gradually increasing the
proportion of acetone. Fraction 2 was purified to yield 4 (3.2
mg). Fractions 3-7 were subjected to preparative liquid
chromatography and eluted with MeOH-H2O (45:55, v/v) to
give 2 (52.7 mg), 3 (34.6 mg), and 5 (2.9 mg). Their purities
were above 95%, as determined by HPLC/UV determination.
12r-Hydroxybufalin (2): white powder; C24H34O5; mp
204.0, 298.0 nm; 1H NMR (DMSO-d6, 500 MHz) δ 7.69 (1H, d,
J ) 10.0 Hz, H-22), 7.52 (1H, s, H-21), 6.27 (1H, d, J ) 10.0
Hz, H-23), 5.51 (1H, d, J ) 4.0 Hz, 7-OH), 5.45 (1H, s, 14-
OH), 4.86 (1H, d, J ) 3.0 Hz, 15-OH), 4.21 (2H, br s, H-7,
H-15), 4.18 (1H, br s, 3-OH), 3.82 (1H, br s, H-3), 2.68 (1H, t,
J ) 8.5 Hz, H-17), 0.83 (3H, s, H-19), 0.58 (3H, s, H-18); 13C
NMR (DMSO-d6, 125 MHz) δ 161.3 (s, C-24), 148.9 (d, C-21),
147.2 (d, C-22), 122.0 (s, C-20), 114.4 (d, C-23), 84.1 (s, C-14),
79.0 (d, C-15), 68.5 (d, C-7), 64.4 (d, C-3), 48.9 (d, C-17), 46.7
(s, C-13), 46.5 (d, C-8), 40.6 (t, C-16), 39.0 (t, C-12), 37.1 (t,
C-6), 36.7 (d, C-5), 34.7 (s, C-10), 34.3 (t, C-4), 32.7 (d, C-9),
29.3 (t, C-1), 27.5 (t, C-2), 22.5 (q, C-19), 20.8 (t, C-11), 18.2
(q, C-18); APCI-MS m/z 419 [M + H]+; HR-FT-ICRMS m/z
419.2432 (calcd for C24H35O6, 419.2428).
5â,7â-Dihydroxylbufalin (7): white powder; C24H34O6; mp
162-165 °C; [R]25 -14.1° (c 0.19, MeOH); UV λmax(MeOH)
D
1
205.0, 301.0 nm; H NMR (DMSO-d6, 500 MHz) δ 7.93 (1H,
dd, J ) 10.0 and 2.0 Hz, H-22), 7.51 (1H, d, J ) 2.0 Hz, H-21),
6.26 (1H, d, J ) 10.0 Hz, H-23), 5.67 (1H, br s, 7-OH), 5.42
(1H, s, 14-OH), 5.20 (1H, br s, 3-OH), 4.84 (1H, br s, 5-OH),
3.95 (1H, br s, H-3), 3.63 (1H, br s, H-7), 0.82 (3H, s, H-19),
0.60 (3H, s, H-18); 13C NMR (DMSO-d6, 125 MHz) δ 161.3 (s,
C-24), 149.2 (d, C-21), 147.5 (d, C-22), 122.5 (s, C-20), 114.3
(d, C-23), 84.4 (s, C-14), 73.2 (s, C-5), 69.9 (d, C-7), 66.3 (d,
C-3), 49.9 (d, C-17), 47.3 (s, C-13), 45.3 (d, C-8), 44.6 (t, C-6),
40.2 (t, C-12), 39.6 (s, C-10), 37.9 (t, C-4), 36.3 (d, C-9), 33.2 (t,
C-15), 28.4 (t, C-16), 27.0 (t, C-2), 24.8 (t, C-1), 21.4 (t, C-11),
16.8 (q, C-19), 16.7 (q, C-18); APCI-MS m/z 419 [M + H]+; HR-
FT-ICRMS m/z 419.2425 (calcd for C24H35O6, 419.2428).
150-153 °C; [R]25 +11.7° (c 1.27, MeOH); UV λmax (MeOH)
D
208.0, 300.0 nm; IR νmax (KBr) 3452, 2937, 2870, 1709, 1628,
1536, 1450, 1376, 1242, 1134, 1032, 947 cm-1 1H NMR
;
(DMSO-d6, 500 MHz) δ 7.94 (1H, dd, J ) 10.0 and 2.0 Hz,
H-22), 7.43 (1H, d, J ) 2.0 Hz, H-21), 6.28 (1H, d, J ) 10.0
Hz, H-23), 4.61 (1H, d, J ) 3.5 Hz, 12-OH), 4.16 (1H, d, J )
2.5 Hz, 3-OH), 4.03 (1H, s, 14-OH), 3.88 (1H, br s, H-3), 3.40
(1H, br s, H-12), 3.13 (1H, dd, J ) 9.5 and 6.0 Hz, H-17), 0.83
(3H, s, H-19), 0.52 (3H, s, H-18); 13C NMR (DMSO-d6, 125
MHz) δ 161.4 (s, C-24), 148.8 (d, C-21), 147.9 (d, C-22), 123.5
(s, C-20), 114.2 (d, C-23), 83.4 (s, C-14), 74.0 (d, C-12), 64.6 (d,
C-3), 51.5 (s, C-13), 44.1 (d, C-17), 41.4 (d, C-8), 35.7 (d, C-5),
34.5 (s, C-10), 33.5 (t, C-15), 33.1 (t, C-4), 30.4 (t, C-16), 29.5
(t, C-1), 29.0 (t, C-11), 27.9 (d, C-9), 27.6 (t, C-2), 26.4 (t, C-6),
23.7 (q, C-19), 20.9 (t, C-7), 18.0 (q, C-18); APCI-MS m/z 403
[M + H]+; HR-FT-ICRMS m/z 403.2474 (calcd for C24H35O5,
403.2479).
Bioassay. Human hepatoma Bel-7402 cells, human gastric
cancer BGC-823 cells, and human cervical carcinoma HeLa
cells were maintained in RPMI 1640 medium (GIBCO/BRL,
Rockville, MD) supplemented with 10% (v/v) fetal bovine
serum and cultured in 96-well microtiter plates for the assay.
Appropriate dilutions (10-3-102 µmol/L) of the test compounds
were added to the cultures. After incubation at 37 °C, 5% CO2
for 72 h, the survival rates of the cancer cells were evaluated
by the MTT method.12 The activity was shown as IC50 value,
which is the concentration (µmol/L) of test compound to give
50% inhibition of cell growth. Results are expressed as the
mean value of triplicate determinations.
7â,12r-Dihydroxybufalin (3): white powder; C24H34O6; mp
165-167 °C; [R]25 +13.1° (c 1.14, MeOH); UV λmax (MeOH)
D
206.0, 298.0 nm; IR νmax (KBr) 3415, 2935, 1706, 1627, 1537,
1448, 1245, 1139, 1031, 953 cm-1 1H NMR (DMSO-d6, 500
;
Acknowledgment. We thank the National Outstanding
Youth Foundation by NSF of China (Grant No. 39925040) and
Trans-Century Training Program Foundation for the Talents
by the Ministry of Education for financial support.
MHz) δ 7.95 (1H, dd, J ) 10.0 and 2.0 Hz, H-22), 7.43 (1H, d,
J ) 2.0 Hz, H-21), 6.27 (1H, d, J ) 10.0 Hz, H-23), 5.47 (1H,
d, J ) 4.5 Hz, 7-OH), 5.34 (1H, s, 14-OH), 4.70 (1H, d, J ) 4.0
Hz, 12-OH), 4.19 (1H, d, J ) 3.0 Hz, 3-OH), 3.85 (1H, s, H-3),
3.80 (1H, br s, H-7), 3.41 (1H, s, H-12), 3.15 (1H, br s, H-17),
0.86 (3H, s, H-19), 0.54 (3H, s, H-18); 13C NMR (DMSO-d6,
125 MHz) δ 161.4 (s, C-24), 148.7 (d, C-21), 148.0 (d, C-22),
123.3 (s, C-20), 114.3 (d, C-23), 84.4 (s, C-14), 73.7 (d, C-12),
68.9 (d, C-7), 64.2 (d, C-3), 51.0 (s, C-13), 46.3 (d, C-8), 44.0 (d,
C-17), 36.6 (d, C-5), 36.5 (t, C-6), 34.8 (t, C-15), 34.2 (t, C-4),
34.2 (s, C-10), 30.3 (t, C-16), 29.1 (t, C-1, C-11), 27.4 (t, C-2),
27.2 (d, C-9), 23.6 (q, C-19), 18.1 (q, C-18); APCI-MS m/z 419
[M + H]+; HR-FT-ICRMS m/z 419.2422 (calcd for C24H35O6,
419.2428).
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Biotransformation of Bufalin (1) by M. spinosus AS
3.3450. The biotransformation procedure was the same as
described in our previous paper.10 A total amount of 800 mg
of bufalin was used. The biotransformation products were
separated on a silica gel column chromatography (135 g, 200-
300 mesh, φ 3 × 45 cm) and eluted in 100 mL fractions with
petroleum ether (60-90 °C) and acetone (4:1 to 1:1, v/v).
Fractions 38-55 [petroleum ether-acetone (2:1)] were sub-
jected to preparative liquid chromatography and eluted with
MeOH-H2O (50:50, v/v) to afford 6 (2.1 mg) and 7 (2.4 mg).
7â,15r-Dihydroxybufalin (6): white powder; C24H34O6; mp
(7) Ye, M.; Dai, J.; Guo, H.; Cui, Y.; Guo, D. Tetrahedron Lett. 2002, 43,
8535-8538.
(8) Ye, M.; Ning, L.; Zhan, J.; Guo, H.; Guo, D. J. Mol. Catal. B: Enzymol.
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(9) Ye, M.; Qu, G.; Guo, H.; Guo, D. Appl. Environ. Microbiol. 2004, 70,
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251-253 °C; [R]25 -7.4° (c 0.14, MeOH); UV λmax(MeOH)
NP0500023
D