Microbial transformation of three bufadienolides by Nocardia sp. and some insight for the cytotoxic structure-activity relationship (SAR)

Article abstract of DOI:10.1016/j.bmcl.2007.09.065

Resibufogenin, cinobufagin, and bufalin are cytotoxic steroids isolated from the Chinese drug Chan'su. Biotransformation of these three bufadienolides by Nocardia sp. NRRL 5646 was investigated. Notably, resibufogenin was converted to 3-acetyl 15β-hydroxyl bufotalin, via an unprecedented 14β,15β-epoxy ring cleavage and a regio-selective acetoxylation. This product showed significantly increased cytotoxic activity. The regio-selective acetylation of the 3-OH was also involved in the other reactions. The structures of metabolites were established by ESI-LC/MS and 2D NMR techniques. The in vitro cytotoxic activities against human cancer cell lines of the substrates and the transformed products were determined by the MTT method and their structure-activity relationship (SAR) was discussed. This investigation provided a useful approach to prepare new bufadienolides and the SAR research.

Full text of DOI:10.1016/j.bmcl.2007.09.065

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Bioorganic & Medicinal Chemistry Letters 17 (2007) 6062–6065
Microbial transformation of three bufadienolides by Nocardia
sp. and some insight for the cytotoxic structure–activity
relationship (SAR)
Jian Zhang,a,b Yang Sun,b Ji-Hua Liu,a Bo-Yang Yua,* and Qiang Xub
aDepartment of Complex Prescription of TCM, China Pharmaceutical University, Nanjing 210038, People’s Republic of China
bState Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University,
Nanjing 210093, People’s Republic of China
Received 5 July 2007; revised 12 September 2007; accepted 19 September 2007
Available online 22 September 2007
Abstract—Resibufogenin, cinobufagin, and bufalin are cytotoxic steroids isolated from the Chinese drug Chan’su. Biotransforma-
tion of these three bufadienolides by Nocardia sp. NRRL 5646 was investigated. Notably, resibufogenin was converted to 3-acetyl
15b-hydroxyl bufotalin, via an unprecedented 14b,15b-epoxy ring cleavage and a regio-selective acetoxylation. This product showed
significantly increased cytotoxic activity. The regio-selective acetylation of the 3-OH was also involved in the other reactions. The
structures of metabolites were established by ESI-LC/MS and 2D NMR techniques. The in vitro cytotoxic activities against human
cancer cell lines of the substrates and the transformed products were determined by the MTT method and their structure–activity
relationship (SAR) was discussed. This investigation provided a useful approach to prepare new bufadienolides and the SAR
research.
Ó 2007 Elsevier Ltd. All rights reserved.
Chan’su, also called toad venom or toad poison, which
is made of the skin secretions of giant toads, including
Bufo gargarizans and B. melanostictus, is a popular Chi-
nese Traditional Medicine (TCM). It has been used
extensively in clinics, either alone or in combination
with other herbal ingredients in the forms of brand com-
plex formula such as Liu-Shen-Wan and She-Xian-Bao-
Xin-Wan. Bufadienolides, the major active constituents
of Chan’su, are C-24 steroids, the characteristic struc-
tural feature of which is a doubly unsaturated six mem-
bered lactone ring (a-pyrone) on position 17b.
Furthermore, these compounds are characterized by
the trans-junction of rings B and C and usually the
cis-junction of rings C and D.1,2 Resibufogenin, cino-
bufagin, and bufalin are the three major components
of Chan’su. And their contents in the crude drug could
be as high as 5–10% of the dry weight. They have been
reported to exhibit significant inhibitory activities
against human myeloid leukemia cells (K562, U937,
ML1, and HL60), human hepatoma cells (SMMC7221),
and prostate cancer cells (LNCaP, DU105, and PC3).
The activities are mediated by induction of cell apopto-
sis and cell differentiation, and the regulations of a vari-
ety of genes and proteins are involved in the process.3–5
Biotransformation is an alternative tool in the structural
modification of complex natural products due to its
great capabilities to catalyze novel reactions and its re-
gio- and stereo-selectivity.6,7 Microorganisms, especially
actinomycetes, are well known as efficient and selective
catalysts. In previous papers we reported the biotrans-
formation of some complex natural products and ob-
tained a series of new products.8–11 Therefore, it is
envisioned that biotransformation of bufadienolides
may provide some analogues which could be utilized
for screening for new activities or better activity/side ef-
fects profile. By studying the cytotoxicities of the ob-
tained products, we can explore the structure and
activity relationship (SAR) of such compounds (Fig. 1).
Nocardia sp. NRRL 5646, one of the commonly used
microbes, for biotransformation because of its broad-
spectrum of enzymatic capability such as carboxylic acid
and aldehyde reduction, phenol methylation, and skele-
Keywords: Biotransformation; Nocardia sp. NRRL 5646; Bufadieno-
lide; Resibufogenin; Cinobufagin; Bufalin; SAR.
*
Corresponding author. Tel.: +86 25 83313080; fax: +86 25
85391042; e-mail: boyangyu59@163.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.065

J. Zhang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6062–6065
Table 1. 13C NMR spectral data of compounds 1–3a
6063
O
C
1
2
3
C
1
2
3
O
1
29.5
27.9
66.7
33.3
36.0
25.8
21.1
39.3
33.6
35.5
20.8
39.4
45.3
30.6
26.1
70.6
30.8
37.3
25.2
21.0
39.2
33.9
35.5
21.3
39.5
45.3
30.5
26.0
70.6
30.7
37.2
25.2
21.1
39.3
33.6
35.4
21.1
39.5
47.7
14
15
16
17
18
19
20
21
22
23
24
74.7
60.0
32.4
47.8
16.9
23.8
74.5
60.0
32.5
47.7
17.0
23.8
85.1
2
3
74.2
75.1
47.7
16.8
23.7
4
5
6
7
122.3
149.6
147.0
115.2
162.0
122.5
150.7
147.3
115.2
161.7
170.3
21.3
121.5
150.6
148.7
115.2
160.3
170.3
21.3
8
R4
9
10
11
12
13
R2
R3
C@O
CH3
R1
Compound 1 R1=OH, R2= R3=O, R4=H
Compound 2 R1=OAc, R2= R3=O, R4=H
a Recorded in pyridine-d5.
carbon by the DEPT experiments, and the disappear-
ance of one signal at d 32.5 (C-16) of one secondary car-
bon showed that the structure of 3 was very similar to
that of cinobufagin, which contains one acetoxyl group
at C-16. When compared that to resibufogenin, the C-14
and C-15 epoxy ring signals shifted downfield by
10.4 ppm and 14.2 ppm, this suggested the cleavage of
the epoxy ring. By the literature study of the NMR data
of the bufalin, cinobufagin, bufotalin, and their deri-
vates,14–18 we confirmed that compound 3 was a hydrox-
ylated bufotalin derivate. In accordance, the HMBC
spectrum showed the long-range correlation between
H-15 (d 3.57) and C-14 (d 85.1). The stereochemistry
of 15-OH was established as b-configuration by compar-
ing the carbon signals with those in 15b-hydroxyl bufa-
lin (the chemical shift of the C-15 with the
a-configuration hydroxy group is 78.2 ppm).19 In the
NOESY spectrum, a correlation between H-15 and
H-7(d 0.94) suggested that H-15 was a-oriented and
the hydroxyl group was b, thereby compound 3 was fur-
ther identified as 3-acetyl 15b-hydroxyl bufotalin
(Figs. 2 and 3).
Compound 3 R1=OAc R2= R3=OH, R4=OAc
Compound 4 R1=OH, R2= R3=O, R4=OAc
Compound 5 R1=OAc, R2= R3=O, R4=OAc
Compound 6 R1=OH, R2=OH, R3=R4=H
Compound 7 R1=OAc, R2=OH, R3=R4=H
Figure 1. Structures of resibufogenin (1), cinobufagin (4), and bufalin
(6) and related biotransformation products.
ton re-arrangement of quinovic acid glycosides via
methyl migration,8 was found to convert the substrates
efficiently to less polar metabolites. In this report, we de-
scribe the unique biotransformation results of resibufog-
enin, cinobufagin, and bufalin upon microbe Nocardia
sp. and the cytotoxic activity of the related compounds.
The substrates were incubated at a preparative-scale
with microbe Nocardia sp. according to the standard
two-stage fermentation protocol. The work-up and iso-
lation of metabolites were carried out similarly as de-
scribed previously.12
Structurally, it is particularly noteworthy that com-
pound 3, a bufotalin derivative, does not have the
14b,15b-epoxy group in the skeleton. As we all know,
the presence of a 14b hydroxy group or a 14b,15b-epoxy
A 100 mg sample of 1 (C24H32O4, Mr = 384) was used
for the preparative-scale incubation for 120 h. The incu-
bation cultures were filtered and the broth was extracted
with EtOAc. The pooled EtOAc solution was dried over
anhydrous Na2SO4, and the solvent was removed in a
vacuum. The resulting residue was subjected to silica
gel chromatographic separation which afforded two
metabolites, M-1 (2, 12% yield based on the starting
material 1) and M-2 (3, 29%).
O
O
M-1 (2) was obtained as white powder. Extensive mass
and NMR spectroscopic studies revealed it to be 3-acet-
yl resibufogenin on the basis of its NMR data (Table 1)
with literature values.13
OAc
The ESI-MS spectrum of M-2 (3) showed the [M-H]À
ion peak at 501.2, suggesting the molecular formula
C28H38O8. Comparing the NMR spectrum with that of
compound 2, there was one additional acetoxyl group
signals and the signal at d 75.1 was assigned as a tertiary
OH
OH
AcO
Figure 2. Key HMBC correlations for 3-acetyl 15b-hydroxyl bufo-
talin (3).

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J. Zhang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6062–6065
Table 2. Cytotoxic activities of the biotransformation products against
human cancer cell lines (n = 3)
H
O
O
a
CH3
Compound
IC50 (lmol/L)
CH3
H
HepG2
HeLa
BGC-823
H
H
Taxol
0.82 0.06
>50
>50
0.02 0.01
>50
>50
1.15 0.14
7.89 0.21
19.80 1.37
3.37 0.25
8.50 0.12
4.11 0.55
0.99 0.36
0.70 0.06
OH
H
H
1
2
3
4
5
6
7
3.10 0.44
5.20 0.17
6.44 0.41
0.64 0.09
0.74 0.06
0.45 0.06
0.76 0.06
1.09 0.11
0.73 0.07
1.14 0.32
OAc
H
OH
HO
H
H
H
a Data were expressed as means SD of three experiments and each
includes triplicate sets.
Figure 3. Selected NOESY correlations for 3-acetyl 15b-hydroxyl
bufotalin (3).
group is assumed to be an integral part of the natural
genins. The large variety of known genin is chiefly due
to the number and position of the substituents.2 This
is the first successful direct conversion of the resibufog-
enin to a bufotalin derivative, a reaction which should
be very difficult to conduct through organic synthesis.
From the point view of SAR research, the 16-acetoxyl
group and 14b-hydroxyl group are essentially important
for the activities of bufadienolides, thus the disappear-
ance of 14b, 15b-epoxy ring would increase the cytotox-
icity. So it is envisioned that compound 3 would be more
active than corresponding resibufogenin analogues. The
cytotoxic activities of the biotransformed products were
evaluated by the MTT method. Compound 3 showed
strong inhibitory activities against human hepatoma
HepG2 cells, human gastric cancer BGC-823 cells, and
human cervical carcinoma HeLa cells with IC50 values
of 2.8, 0.50, and 3.1 lmol/L, respectively. Its cytotoxic-
ity increased as high as bufalin which is one of the most
active bufadienolides isolated from Chan’su. The results
were in agreement with the structure–activity relation-
ships previously proposed by Ye and Kamano very
well.19–21 During the past years, there were many reports
on the biotransformation of bufadienolides, the metab-
olites were mostly hydroxylated products,18,19,22–27 the
epoxide hydrolase catalyzed epoxy ring cleavage was
first observed. It was very helpful to study the biosynthe-
sis pathways of the bufadienolides with or without
epoxy ring. The product with enhanced cytotoxicity also
encouraged us to explore the structure diversity and the
SAR of bufadienolides.
which has two hydroxyl groups on C-3 and C-14 was
also studied to explore the regio-selectivity of the acety-
lation. Only one metabolite 3-acetyl bufalin (7) was
found. It showed great regio-selectivity of the biotrans-
formation of bufadienolides by Nocardia sp. The acety-
lated products of compounds 5 and 7 showed little
weaker but still potent cytotoxicities than the corre-
sponding substrates. These results provided guidance
for the future directed synthesis of bufadienolides of
pharmaceutical interest (Table 2).
Nocardia sp. NRRL 5646 contains an astonishing array
of interesting and useful enzymes, many of which are
similar to those found in mammals. A soluble form of
nitric oxide synthase(NOSNOC), carboxylic acid reduc-
tase, aryl aldehyde oxidoreductase, aldehyde reductase,
guanylate cyclase, and ornithine transcarbamoylase,
have been characterized in this organism.27–30 And the
unique catalytic capability of Nocardia sp. to the bufadi-
enolides deserves further exploration to enhance the
chemical diversity of bufadienolides. Such studies could
provide new platforms for combinatorial synthesis31–33
and the development of new bufadienolide antitumor
drugs.
Acknowledgments
This work was supported by grants from National Nat-
ural Science Foundation of China (No. 20602040) and
grants from the State Ministry of Education (No.
02118). We also thank the China Postdoctoral Science
Foundation (No. 20060400916). Nocardia sp. was ob-
tained from Prof. J.P.N. Rosazza, University of Iowa.
To investigate the capability and selectivity of the en-
zyme system in Nocardia sp. to hydrolyze the 14b,15b-
epoxy ring in the bufadienolides, cinobufagin (4) was
employed for parallel biotransformation under the same
conditions. It was found that compound 4 can only be
metabolized to 3-acetyl cinobufagin (5). It was interest-
ing to note that the minor structure difference between
compounds 1 and 4 is an acetoxy group at position of
C-16, but the biotransformation result showed great dif-
ference. The explanation for this observation could be
either the blockade of the access of the substrate to
the active domain of the hydrolase enzyme due to the
16-acetoxyl group, or more likely, the hydrolysis,
hydroxylation, and acetylation are consecutive processes
with the assumption that the catalytic active domains
are proximate. The biotransformation of bufalin (6)
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.09.065.
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