Bufogargarizins A and B: Two novel 19-norbufadienolides with unprecedented skeletons from the venom of Bufo bufo gargarizans

Article abstract of DOI:10.1002/chem.201000847

Figure Presented Toad venom: Bufogargarizins A and B with unprecedented skeletons were discovered from the venom (middle) of Bufo bufo gargarizans (left). The absolute configurations were elucidated by spectroscopic data, X-ray crystal diffraction, modified Mosher's method and computational analysis.

Full text of DOI:10.1002/chem.201000847

COMMUNICATION
DOI: 10.1002/chem.201000847
Bufogargarizins A and B: Two Novel 19-Norbufadienolides with
Unprecedented Skeletons from the Venom of Bufo bufo gargarizans
Hai-Yan Tian,[a, c] Lei Wang,[a, b] Xiao-Qi Zhang,[a, b] Ying Wang,[a, b] Dong-Mei Zhang,[a, b]
Ren-Wang Jiang,[a, b] Zhong Liu,[a] Jun-Shan Liu,[a] Yao-Lan Li,[a, b] and Wen-Cai Ye*[a, b, c]
Toad venom, secreted from the postauricular and skin
glands of Bufo bufo gargarizans Cantor or Bufo melanostic-
tus Schneider, is useful as a chemical weapon against the
natural enemies of toads. The dried toad venom has also
been widely used as a traditional Chinese medicine in the
treatment of superficial infection, odontalgia and skin
cancer, although this secretion is known for its toxicity.[1]
Bufadienolides bearing a d-lactone ring at the C-17 in the
steroidal skeleton had been proved to be active ingredients
of toad venom, since these compounds showed significant
cardiotonic, anesthetic, and antitumor activities.[2–3] To date,
more than forty bufadienolides have been isolated from the
venom of toads.[4–6] Bufalin, a main active component from
toad venom, had been reported to induce apoptosis in vari-
ous human cancer cell lines such as human leukemia HL60
and U937 cells, and the action mechanism was involved to
influence the expression of apoptosis-related genes as bcl-2,
c-myc, and Tiam 1.[7–8] Furthermore, bufadienolides were
known to be specific inhibitors of Na+/K+ ATPase, which
was reported as a potential target for anticancer drugs refer-
ring to the recent discovery of its signaling pathways.[9–10]
The above pharmacological properties implied the promis-
ing therapeutic use of those compounds in, for example,
cancer treatment. In our research for structurally unique
and biologically interesting bufadienolides, two novel 19-
norbufadienolides, bufogargarizins
A
(1) and
B
(2)
(Scheme 1), with two unprecedented carbon skeletons, to-
Scheme 1. Chemical structures of 1 and 2 (the carbon atoms were num-
bered from rings A to E; No. 19 wasn’t marked since the Me-19 was lost
in 1 and 2).
gether with three presumably biosynthetic related inter-
mediates 3,[11] 4, and 6 were isolated from the venom of
Bufo bufo gargarizans. Herein, we describe the isolation
and structure elucidation with the absolute configurations of
1 and 2. In addition, a plausible biogenetic pathway of bufo-
gargarizins A (1) and B (2) was also proposed.
[a] H.-Y. Tian, Dr. L. Wang, Dr. X.-Q. Zhang, Dr. Y. Wang,
Dr. D.-M. Zhang, Prof. Dr. R.-W. Jiang, Dr. Z. Liu, J.-S. Liu,
Prof. Dr. Y.-L. Li, Prof. Dr. W.-C. Ye
Institute of Traditional Chinese Medicine and Natural Products
Jinan University, Guangzhou 510632 (P. R. China)
Fax : (+86)20-8522-1559
The roughly powdered toad venom was extracted with
95% ethanol under ultrasonic irradiation. The EtOH extract
was filtered and concentrated under reduced pressure to
afford a residue, which was then dissolved in 20% ethanol
and partitioned with CH2Cl2. The extract, which showed sig-
nificant cytotoxic activity, was then purified by chromatogra-
phy on silica gel, reversed-phase C18 silica gel, and prepara-
tive HPLC columns to yield compounds 1–4, and 6.
Compound 1 was obtained as a colorless block. The quasi-
molecular ion at m/z 459.2023 [M+H]+ in its HR-ESI-MS
indicated that the molecular formula of 1 was C25H30O8. The
UV absorption maximum at 295 nm and IR band at
E-mail: chywc@yahoo.com.cn
[b] Dr. L. Wang, Dr. X.-Q. Zhang, Dr. Y. Wang, Dr. D.-M. Zhang,
Prof. Dr. R.-W. Jiang, Prof. Dr. Y.-L. Li, Prof. Dr. W.-C. Ye
Guangdong Province Key Laboratory of Pharmacodynamic
Constituents of TCM and New Drugs Research, Jinan University
Guangzhou 510632 (P.R. China)
[c] H.-Y. Tian, Prof. Dr. W.-C. Ye
Department of Phytochemistry, China Pharmaceutical University
Nanjing 210009 (P. R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000847.
Chem. Eur. J. 2010, 16, 10989 – 10993
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
10989

1725 cmÀ1 implied the presence of d-lactone ring. The analy-
sis of NMR spectra revealed that 1 possessed twenty-five
carbons, including a methyl (dH 0.84, s), an acetyl group [dH
1.85 (1H, s); dC 171.6, 20.3], and a d-lactone ring [dH 6.23
(d, J=8.0 Hz, 1H), 7.36 (d, J=1.0 Hz, 1H), 8.01 (dd, J=8.0,
tween H-4b (dH 2.85, dd, J=10.0, 10.0 Hz, 1H) and C-6 (dC
64.5), and between H-2b (dH 2.10, m, 1H) and C-10 (dC
81.4), as well as between H2-7 (dH 1.58, m, 2H) and C-10 in-
dicated that rings A and B were connected via C-6 and C-10
bond. The detailed interpretation of HMBC correlations al-
lowed the establishment of the planar structure of 1, which
was a novel 19-norbufadienolide with 7/5/6/5 carbon rings.
In order to determine the relative configuration of 1, the
ROESY spectrum was extensively analyzed (for details see
Supporting Information). However, the uncertainty of the
conformation of the seven-membered ring made it difficult
to determine the orientation of hydroxyl group at C-10 by
NOE correlations. Fortunately, crystals suitable for single-
crystal X-ray diffraction were grown from methanol solu-
tion. The relative configuration of 1 was unequivocally de-
duced by the result of X-ray diffraction analysis
(Figure 1).[12] Meanwhile, some useful conformational infor-
1
1.0 Hz, 1H); dC 114.1, 118.2, 150.8, 153.6, and 163.9]. The H
and 13C NMR signals assigned to fragment A (Scheme 2)
were very similar to that of cinobufotalin (6)[6a] (Scheme 3),
1
Scheme 2. Key H–1H COSY and HMBC correlations of 1 and 2.
Figure 1. X-ray crystal structure of 1.
mation could be obtained from the crystal structure: the
plane of d-lactone ring was nearly perpendicular to the ring
D, making H-21 (dH 7.36, d, J=1.0 Hz) and H-17 (dH 2.98,
d, J=8.6 Hz) into the same orientation, which well account-
ed for the NOE correlation between H-21 and H-17; the
seven-membered ring A and six-membered ring C were
both in chair conformation; the hydroxyl group at C-10 was
b-oriented, and 1 remained the A/B cis, B/C trans, as well as
C/D cis ring junctions.
Subsequently, the modified Mosherꢁs method was applied
to determine the absolute configuration of 1.[13] Comparison
1
of the H NMR chemical shifts between (S)- and (R)-MTPA
esters of 1 led to the assignment of S configuration of C-3
(Scheme 4). Therefore, the structure of 1 was fully estab-
lished and deduced to possess the 3S, 6S, 8R, 9S, 10S, 13R,
14S, 15R, 16R, and 17R configurations.
Compound 2 was isolated as an amorphous powder and
was shown to have the same molecular formula as 1 by its
HR-ESI-MS. Comparison of the NMR data of 2 (Table 1)
with those of 1 indicated that 2 also had fragment A in its
Scheme 3. Plausible biogenetic pathway for 1 and 2.
1
structure (Scheme 2). The H,1H COSY spectrum revealed
indicating that 1 had the same substructure as 6, with an
epoxy at C-14 and C-15, as well as an acetyl group at C-16.
The 1H,1H COSY spectrum revealed the presence of the
spin systems in bold as shown in Scheme 2. The HMBC cor-
relations between H-3 (dH 3.62, m) and C-5 (dC 209.9), be-
the presence of the partial units in bold as shown in
Scheme 2. The HMBC correlations between H-1 (dH 2.04,
2H) and C-9 (dC 212.7), between H-3b (dH 1.41, dd, J=12.0,
10.0 Hz, 1H) and C-5 (dC 39.7), as well as between H-6 [dH
1.58 (1H, H-6a); 1.67 (1H, H-6b)] and C-4 (dC 79.9) re-
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Bufogargarizins A and B
COMMUNICATION
H-5b (dH 1.00, dd, J=14.1, 11.2 Hz), between H-5b and H-
3b (dH 1.17, dd, J=12.0, 10.0 Hz), as well as between H-3b
and 2-OH (dH 4.58, d, J=5.2 Hz), indicating that those pro-
tons were b-oriented. In contrast, the correlations between
H-10 (dH 2.97, dd, J=8.7, 8.7 Hz) and H-8 (dH 2.69, ddd, J=
11.9, 11.3, 3.9 Hz), between H-10 and 4-OH (dH 4.74, s), be-
tween 4-OH and H-2 (dH 4.20, m), between H-17 (dH 2.88,
d, J=9.3 Hz) and H-16 (dH 5.48, dd, J=9.3, 1.2 Hz), as well
as between H-16 and H-15 (dH 3.74, d, J=1.2 Hz) revealed
these protons were a-oriented. These findings suggested 2
also possessed A/B cis, B/C trans, and C/D cis ring junctions.
Nevertheless, in contrast to the b-oriented OH and H at the
junction positions of rings A and B in 1, the corresponding
OH and H were found to be a-oriented in 2. To confirm the
most reasonable conformation of 2, a combination of com-
putational analysis based on SYBYL software and interpre-
tation of NMR data was applied.[14] Coincidentally, the
seven-membered ring B in 2 also had the chair conforma-
tion as 1 when it was in a state of low energy (for details,
see Experimental Section). Thus, the relative configuration
of 2 was established and shown in Figure 2. As 1, the result
of the modified Mosherꢁs method (Scheme 4) suggested that
the absolute configuration of C-2 was S. Therefore, the
structure of 2 was elucidated and assigned to have 2S, 4R,
7R, 8S, 10R, 13R, 14S, 15R, 16R, and 17R configurations.
Scheme 4. Dd values (dSÀdR) for the MTPA esters of 1 and 2.
vealed the presence of 5/7 consecutive carbocycles via C-4
and C-10 bond (Scheme 2). Furthermore, the HMBC corre-
lations between H-11a (dH 1.97, 1H) and C-9 (dC 212.7),
and between H-7 (dH 1.74, 1H) and C-15 (dC 61.0) implied
that rings B and C were linked through C-7 and C-8 bond.
Consequently, 2 was inferred to be an unprecedented 19-
norbufadienolide with a 5/7/6/5 carbocycle system.
The ROESY spectrum (recorded in [D6]DMSO) revealed
correlations (Figure 2) between Me-18 (dH 0.74, s, 3H) and
H-7 (dH 1.64, ddd, J=11.3, 11.3, 3.5 Hz), between H-7 and
Table 1. 1H and 13C NMR data of compounds 1–2 (CD3OD, J in Hz).[a,b]
1
dH
2
dH
2[c]
dH
1
dH
2
dH
2[c]
dH
No.
No.
dC
dC
dC
dC
dC
dC
1a
b
1.05
1.95 (m)
34.0
2.04
2.04
35.6
1.84
1.84
34.6
11a
b
1.51
1.51
21.4
1.97
1.48
24.3
1.80
22.8
1.41 (dd,
14.1, 10.0)
1.53
1.70 (ddd,
13.5, 3.0, 3.0)
–
2a
b
1.85 (m)
2.10 (m)
32.3
70.8
54.9
4.38 (m)
–
71.7
51.9
79.9
4.20 (m)
–
69.4
51.0
77.3
12a
b
1.53
1.87
41.7
1.59
1.79
39.4
37.5
3a
b
3.62 (m)
–
1.94
1.80
13
14
–
–
47.0
73.5
–
–
46.5
72.3
44.7
70.9
1.41 (dd,
12.0, 10.0)
–
1.17 (dd,
12.0, 10.0)
–
–
4a
b
2.68 (dd,
10.0, 4.6)
2.85 (dd,
10.0, 10.0)
–
15
16
17
18
3.80 (d,
1.3)
5.46 (dd, 76.7
8.6, 1.3)
2.98 (d,
8.6)
0.84 (s)
60.8
3.76 (brs) 61.0
3.74 (d,
1.2)
5.48 (dd,
9.3, 1.2)
2.88 (d,
9.3)
59.6
74.4
48.8
16.9
–
–
5.49 (d,
8.6)
2.94 (d,
8.6)
76.4
51.2
17.5
118.1
153.7 7.47 (d,
1.0)
150.7 7.82 (dd,
8.0, 1.0)
5a
b
209.9 1.95
1.20 (dd,
39.7
1.82
38.3
50.9
17.8
118.2
–
1.00 (dd,
14.1, 11.2)
1.50
0.86 (s)
0.74 (s)
14.1, 11.2)
1.58
1.67
6a
b
–
64.5
26.8
22.4
39.9
20.8
37.7
20
21
–
–
–
115.8
152.3
3.17 (dd,
8.4, 8.4)
1.58
1.50
7.36 (d,
1.0)
8.01 (dd, 150.8 8.00 (dd,
8.0, 1.0)
6.23 (d,
8.0)
153.6 7.38 (d,
1.0)
7a
b
–
–
22
148.3
112.9
160.7
8.0, 1.0)
114.1 6.24 (d,
8.0)
1.58
–
1.74
1.64 (ddd,
11.3, 11.3, 3.5)
2.69 (ddd,
11.9, 11.3, 3.7)
–
23
114.1 6.24 (d,
8.0)
164.0
8a
37.8
2.73 (ddd,
11.9, 11.3, 3.7)
–
–
3.10 (dd,
8.2, 8.2)
58.1
55.6
24
–
163.9
–
–
b
2.34 (m)
1.09
–
COCH3
–
171.6
20.3
–
171.6
20.3
–
169.3
20.2
9
10
58.0
81.4
212.7
62.8
–
210.7 COCH3 1.85 (s)
61.3
1.86 (s)
–
1.82 (s)
4.58 (d,
5.2)
2.97 (dd,
8.2, 8.2)
2-OH
–
4-OH
–
–
4.74 (s)
[a] Assignments were established by COSY, HSQC and HMBC spectra. [b] Overlapped signals were reported without designating multiplicity. [c] 1H and
13C NMR data were recorded in [D6]DMSO.
Chem. Eur. J. 2010, 16, 10989 – 10993
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10991

W.-C. Ye et al.
Experimental Section
Extraction and isolation: The dried venom (1.5 kg) was ground into
rough powder and then extracted with 95% ethanol under ultrasonic ir-
radiation. The EtOH extract was filtered and concentrated under re-
duced pressure to afford a residue (900 g), which was then dissolved in
20% ethanol and partitioned with CH2Cl2. The CH2Cl2 solution was com-
bined and concentrated to afford a residue (321 g). The residue was puri-
fied by chromatography on silica gel (200–300 mesh), eluted with cyclo-
hexane/acetone 5:1, 3:1, and 1:1 to yield 15 fractions (fractions 1–15).
Compound 6 (1.5 g) was obtained by recrystallization of fraction 7. Frac-
tion 10 was separated using silica gel eluted with chloroform/ethanol
100:2, 95:5, and 90:10 to afford 11 subfractions (fractions 10a–10k). Frac-
tion 10c was further separated by preparative HPLC eluted with acetoni-
Figure 2. Key ROESY correlations of 2.
trile/water/TFA 23:77:0.05 to yield
1 (8.0 mg), 2 (5.5 mg), and 3
(36.0 mg). 4 (80.0 mg) was obtained from fraction 10g using the same
chromatography method.
Compounds 1 and 2 had unusual 7/5/6/5 and 5/7/6/5 ring
systems, respectively, instead of the 6/6/6/5 skeleton present-
ed in common bufadienolides, which interested us to pre-
sume the biogenetic pathway among these compounds. Ci-
nobufotalin (6), 19-hydroxylcinobufotalin (5) and 19-oxoci-
nobufotalin (4) were three known ingredients of toad
venom.[15] The aldehyde group at C-10 in 4 should be
formed by oxidation of the corresponding hydroxymethyl
group in 5, which was an oxide of 6, as the formation of resi-
bufagin and hellebrigenin.[5,6a,16] Then 4 might be changed
into 3 through a Baeyer–Villiger reaction.[17] After undergo-
ing an oxidation procedure of 3, an presumably intermediate
with diketone unit was formed.[18] Finally, 1 and 2 should be
yielded through two intramolecular aldol condensation pro-
cedures of the intermediate (Scheme 3).[19]
Computational methods: The structure of 2 was calculated by using mo-
lecular modeling software package SYBYL 7.0 (Tripos, St Louis, MO,
USA). All hydrogen atoms were added and overlaid with key correla-
tions observed in the ROESY spectrum. The energy was minimized for
1000 steps using the Tripos force field and Powell method and the termi-
nation setting was 0.001 kcalmolÀ1 ꢂAÀ1. Then a grid search was carried
out with an interval of 58 for each rotatable bond to obtain the lowest
energy conformation.
Bufogargarizin A (1): Colorless block crystals; m.p. 164–1658C; [a]D24
=
À24.58 (c=0.2, in CH3OH); 1H and 13C NMR data, see Table 1. IR
(KBr): nmax =3418, 1725, 1635, 1536, 1379, 1244, 1128, 1050, 954, 883, 839,
792, 754, 662 cmÀ1; UV (CH3OH): lmax (loge)=203 (3.1), 295 nm (2.7);
HR-ESI-MS: m/z: calcd for C25H31O8: 459.2013; found: 459.2020
[M+H]+.
Bufogargarizin
CH3OH); 1H and 13C NMR data, see Table 1; IR (KBr): nmax =3441,
1706, 1633, 1537, 1456, 1374, 1245, 1058, 966, 889, 841, 809, 782, 666 cmÀ1
B
(2): amorphous powder; [a]2D6 =+4.28 (c=0.1, in
;
Of all the tested bufadienolides, 6 and bufalin (as a posi-
tive control) showed potent antiproliferative effects on
HeLa and HepG 2 cell lines, with IC50 values ranging from
0.1 to 3 mm. Compound 4 also exhibited cytotoxic activities
with IC50 values of 4.01Æ0.51 mm on HeLa cells and 7.84Æ
0.13 mm on HepG 2 cells, indicating that the presence of
10b-aldehyde group made no significant difference in cyto-
toxicity on the two cell lines. Compound 3, a 19-nor deriva-
tive of 6, showed weak antiproliferative effect on HeLa cells
with IC50 value of 35.56Æ4.19 mm, and no apparent effect on
HepG 2 cells, suggesting 10b-hydroxyl substitution de-
creased the activities on these cancer cell lines. However,
the antiproliferative effects of 1 and 2 were notably drop-
ped, implying that the changes of rings A and B could great-
ly decrease the cytotoxic activity of bufadienolides on the
two cancer cells (for details, see Supporting Information).
Bufogargarizins A (1) and B (2) were the first examples
of bufadienolides with unusual alterations of rings A and B.
The isolation and structure elucidation including absolute
configurations of these compounds has added to a diverse
and complex array of bufadienolide family. The plausible
biogenetic pathway for 1 and 2 was reasonable and interest-
ing. Bioassay result further confirmed that the essential ster-
oidal skeleton is necessary for cytotoxic activities of these
bufadienolides. Further chemistry and biological studies for
such interesting compounds and other bufadienolides from
the venom are currently ongoing.
UV (CH3OH): lmax (loge)=203 (2.9), 295 nm (2.7); HR-ESI-MS: m/z:
calcd for C25H31O8: 459.2013; found: 459.2017 [M+H]+.
Acknowledgements
This work was supported by the National Natural Science Foundation of
China for Outstanding Young Scientists (No. 30625039), Program for
Changjiang Scholars (to W.C.Y.), National Natural Science Foundation of
China (No. 90913020), Natural Science Foundation of Guangdong Prov-
ince (No. 9451063201002969), New Century Excellent Talents Scheme
(NCET-08-0612), and China Postdoctoral Science Foundation (No.
20090460786).
Keywords: biogenetic
pathway
·
bufadienolide
·
cytotoxicity · natural products · structure elucidation
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COMMUNICATION
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1.344 MgmÀ3, m(CuKa)=0.859 mmÀ1, F
ACHTUNGTRENNUNG ACHTUNGTRENN(UGN 000)=1016. Data collection
was performed on a SMART CCD by using graphite monochromat-
ed radiation (l=1.54184 ꢃ) under low temperature (nitrogen gas);
7123 unique reflections were collected to qmax =62.538, in which
5485 reflections were observed [F2 >4s(F2)]. The structures were
solved by direct methods (SHELXTL version 5.1) and refined by
full-matrix least-squares on F2. In the structure refinements, non-hy-
drogen atoms were refined anisotropically. Hydrogen atoms bonded
to carbons were placed on the geometrically ideal positions by the
“ride on” method. Hydrogen atoms bonded to oxygen were located
by the difference Fourier method and were included in the calcula-
tion of structure factors with isotropic temperature factors. The final
Received: April 5, 2010
Published online: August 16, 2010
Chem. Eur. J. 2010, 16, 10989 – 10993
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10993