New cytotoxic bufadienolides from the biotransformation of resibufogenin by Mucor polymorphosporus

Article abstract of DOI:10.1016/j.tet.2005.07.012

Resibufogenin is a cytotoxic steroid isolated from the Chinese drug ChanSu. The biotransformation of resibufogenin by Mucor polymorphosporus afforded 22 products, and 15 of them were new. The transformation reactions involved hydroxylations at C-1β, C-5, C-7α, C-7β, C-12α, C-12β and C-16α, as well as epimerization or dehydrogenation of 3-OH. Hydroxylations at C-12α, C-12β and C-16α were the major reactions, each giving products in >5% yields, whereas the other products were obtained in fairly low yields. Some of the products showed decreased but still potent cytotoxicities. This investigation provided a useful approach to prepare new bufadienolides and most of them were difficult to obtain by chemical means.

Full text of DOI:10.1016/j.tet.2005.07.012

Tetrahedron 61 (2005) 8947–8955
New cytotoxic bufadienolides from the biotransformation
of resibufogenin by Mucor polymorphosporus
Min Ye,a Jian Han,a Dongge An,b Guangzhong Tub and Dean Guoa,
*
aThe State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University,
Xueyuan Road 38, Beijing 100083, China
bBeijing Institute of Microchemistry, Beijing 100091, China
Received 31 January 2005; revised 1 July 2005; accepted 5 July 2005
Available online 26 July 2005
Abstract—Resibufogenin is a cytotoxic steroid isolated from the Chinese drug ChanSu. The biotransformation of resibufogenin by Mucor
polymorphosporus afforded 22 products, and 15 of them were new. The transformation reactions involved hydroxylations at C-1b, C-5, C-7a,
C-7b, C-12a, C-12b and C-16a, as well as epimerization or dehydrogenation of 3-OH. Hydroxylations at C-12a, C-12b and C-16a were the
major reactions, each giving products in O5% yields, whereas the other products were obtained in fairly low yields. Some of the products
showed decreased but still potent cytotoxicities. This investigation provided a useful approach to prepare new bufadienolides and most of
them were difficult to obtain by chemical means.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
number of new peaks were observed by HPLC in the
incubation mixture, whereas no corresponding peak was
found in control tests. The peaks showed UV absorption
maxima at 294–298 nm, which is characteristic for the
a-pyrone ring of bufadienolides. Thus, they should be the
biotransformed products of resibufogenin. In the preparative
biotransformation, a total amount of 800 mg of resibufo-
genin was fed to the microbial cultures. After 6 days
incubation, the products were recovered by extracting with
ethyl acetate. The obtained extract was subjected to ODS
column chromatography and preparative HPLC to afford 22
pure compounds, which were structurally characterized by
Biotransformation is an important tool in the structural
modification of organic compounds, especially natural
products, due to its significant regio- and stereo-selec-
tivities.1,2 Filamentous fungi have frequently been used to
catalyze selective hydroxylation reactions, which are
usually difficult to achieve by chemical means.3 In the
past several years, we have studied the biotransformation of
several natural bufadienolides, including bufalin, cinobufa-
gin and resibufogenin, and obtained more than 30 products,
22 of them being new compounds.4–8 These substrates are
the major effective constituents of the Chinese drug
ChanSu,9 and possess significant anticancer activities.10–13
Our prospective aim is to discover new bufadienolide
derivatives with more potent bioactivities and improved
physico-chemical properties as drug candidates. Herein we
report the biotransformation of resibufogenin by the
filamentous fungus Mucor polymorphosporus.
MS and extensive NMR techniques, including 1H NMR, 13
C
NMR, DEPT, HMQC, HMBC, 1H–1H COSY and NOESY.
These products were 7b-hydroxyl resibufogenin (2),14
3-epi-7b-hydroxyl resibufogenin (3), marinobufagin
(5-hydroxyl resibufogenin, 4),15 5,7b-dihydroxyl resibufo-
genin (5), 7a-hydroxyl resibufogenin (6), 12b-hydroxyl
resibufogenin (7),15 3-epi-12b-hydroxyl resibufogenin (8),
5,12b-dihydroxyl resibufogenin (9), 12a-hydroxyl resi-
bufogenin (10),14 3-epi-12a-hydroxyl resibufogenin (11),
5,12a-dihydroxyl resibufogenin (12), 1b,12a-dihydroxyl
resibufogenin (13), 3-oxo-12a-hydroxyl resibufogenin (14),
12-oxo-resibufogenin (15), 16a-hydroxyl resibufogenin
(16),16 3-epi-16a-hydroxyl resibufogenin (17), 3-oxo-16a-
hydroxyl resibufogenin (18),17 7b,16a-dihydroxyl resi-
bufogenin (19), 12b,16a-dihydroxyl resibufogenin (20),
1b,16a-dihydroxyl resibufogenin (21), 12a,16a-dihydroxyl
resibufogenin (22), and 3-oxo-D4-resibufogenin (23).18
2. Results and discussion
In the screening test, resibufogenin (1) was completely
metabolized in the cultures of M. polymorphosporus. A
Keywords: Biotransformation; Bufadienolide; Cytotoxicity; Mucor poly-
morphosporus; Resibufogenin.
Corresponding author. Tel.: C86 10 8280 1516; fax: C86 10 8280 2700;
e-mail: gda@bjmu.edu.cn
*
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.07.012

8948
M. Ye et al. / Tetrahedron 61 (2005) 8947–8955
Figure 1. Chemical structures of compounds 1–23.
Table 1. 13C NMR spectral data for compounds 1–12 (125 MHz, DMSO-d6)
C
1
2
3
4
5
6
7
8
9
10
11
12
1
2
29.4t
27.5t
29.0t
27.4t
30.2t
34.3t
24.8t
27.3t
24.6t
27.1t
29.4t
27.6t
29.4t
27.4t
30.3t
34.7t
24.9t
27.2t
29.3t
27.5t
30.3t
34.8t
24.7t
27.3t
3
4
64.5d
33.0t
64.2d
34.0t
69.6d
37.0t
66.4d
36.5t
66.2d
37.7t
64.4d
34.4t
64.5d
32.9t
69.7d
36.0t
66.4d
36.5t
64.5d
33.1t
69.8d
36.1t
66.4d
36.7t
5
6
7
8
35.5d
25.6t
20.2t
33.1d
38.5d
35.0s
20.6t
36.2d
34.1t
41.5d
34.5t
74.1s
34.0t
22.4t
32.2d
41.6d
40.4s
21.1t
73.2s
42.2t
35.4d
36.5t
35.6d
25.5t
20.4t
32.4d
33.8d
34.7s
29.4t
72.9d
50.7s
73.5s
59.2d
31.6t
41.7d
11.4q
23.6q
122.1s
150.5d
147.5d
114.2d
161.0s
41.1d
26.0t
20.6t
32.6d
34.6d
34.2s
29.2t
72.8d
50.7s
73.5s
59.3d
31.6t
41.7d
11.4q
23.0q
122.1s
150.5d
147.5d
114.2d
161.0s
73.4s
34.0t
22.4t
31.5d
36.9d
40.0s
29.9t
72.8d
50.5s
73.5s
59.3d
31.6t
41.6d
11.3q
16.7q
122.1s
150.5d
147.4d
114.2d
161.0s
35.6d
25.7t
20.2t
33.0d
30.8d
34.6s
28.4t
74.0d
48.9s
72.5s
61.2d
34.8t
42.7d
17.3q
23.4q
122.8s
150.1d
147.7d
114.3d
161.1s
41.2d
26.2t
20.5t
33.2d
31.6d
34.1s
28.3t
74.0d
48.8s
72.5s
61.2d
34.8t
42.7d
17.3q
22.8q
122.7s
150.1d
147.7d
114.3d
161.1s
73.5s
34.1t
22.5t
32.1d
34.2d
40.0s
29.0t
74.2d
48.7s
72.6s
61.3d
34.8t
42.7d
17.2q
16.5q
122.7s
150.1d
147.7d
114.3d
161.1s
66.2d
40.3d
37.3d
34.6s
21.0t
66.3d
40.4d
38.0d
34.1s
20.7t
67.1d
39.8d
40.0d
40.4s
21.3t
63.6d
36.5d
31.8d
35.9s
20.2t
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
38.2t
38.2t
38.2t
38.3t
38.1t
37.4t
44.6s
74.0s
59.3d
31.6t
46.1d
16.5q
23.7q
122.0s
150.5d
147.4d
114.1d
161.0s
45.1s
76.2s
61.1d
31.2t
45.8d
16.3q
23.6q
121.5s
150.8d
147.2d
114.2d
161.0s
45.1s
76.2s
61.1d
31.2t
45.7d
16.3q
23.1q
121.5s
150.8d
147.2d
114.2d
161.0s
44.4s
73.4s
59.4d
31.5t
46.0d
16.4q
16.7q
121.9s
150.5d
147.3d
114.1d
161.0s
44.9s
76.1s
61.1d
31.1t
45.6d
16.3q
16.7q
121.5s
150.8d
147.2d
114.2d
161.0s
45.2s
72.2s
63.3d
31.7t
45.3d
16.3q
23.3q
122.2s
150.4d
147.4d
114.1d
161.0s

M. Ye et al. / Tetrahedron 61 (2005) 8947–8955
Table 2. 13C NMR spectral data for compounds 13–23 (125 MHz, DMSO-d6)
8949
C
13
14
15
16
17
18
19
20
21
22
23
1
2
71.8d
32.0t
35.9t
36.7t
29.1t
27.3t
29.4t
27.6t
30.4t
34.8t
35.9t
36.7t
29.0t
27.5t
29.4t
27.5t
71.8d
32.1t
29.3t
27.5t
34.9t
33.5t
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
66.7d
33.0t
30.1d
25.2t
19.9t
33.2d
32.6d
40.0s
28.3t
73.9d
48.7s
72.4s
61.2d
34.8t
42.7d
17.3q
18.4q
122.7s
150.1d
147.7d
114.3d
161.1s
211.6s
41.6t
43.0d
25.5t
19.7t
32.8d
31.6d
34.3s
28.4t
74.0d
48.9s
72.3s
61.2d
34.8t
42.7d
17.2q
21.8q
122.7s
150.1d
147.7d
114.3d
161.1s
64.4d
32.8t
35.7d
25.2t
20.4t
32.5d
34.4d
35.0s
37.3t
210.5s
59.8s
73.4s
59.6d
32.1t
37.6d
17.5q
23.0q
120.8s
151.1d
114.5d
147.3d
160.9s
64.4d
33.0t
35.4d
25.6t
20.2t
33.1d
38.5d
35.0s
20.6t
69.7d
36.0t
41.0d
26.0t
20.4t
33.3d
39.2d
34.5s
20.4t
211.5s
41.6t
42.9d
25.4t
19.6t
32.9d
38.5d
34.8s
20.7t
64.1d
34.0t
36.1d
34.2t
66.2d
40.4d
37.5d
34.5s
21.1t
64.4d
33.0t
35.6d
25.5t
20.4t
32.4d
33.8d
34.7s
29.4t
73.8d
50.0s
74.4s
61.2d
75.6d
54.5d
11.7q
23.6q
120.4s
150.3d
147.3d
114.3d
160.9s
66.6d
32.9t
29.9d
25.0t
19.9t
33.3d
40.2d
39.5s
20.5t
64.4d
33.1t
35.6d
25.6t
20.1t
32.8d
30.9d
34.6s
28.1t
73.1d
48.0s
72.7s
61.9d
75.3d
53.8d
17.5q
23.5q
120.2s
150.5d
147.1d
114.4d
160.8s
197.9s
123.2d
170.1s
31.3t
26.3t
33.0d
51.9d
38.3s
20.6t
39.0t
39.0t
39.0t
39.7t
38.9t
37.8t
43.9s
74.9s
61.4d
75.6d
58.4d
17.0q
23.7q
120.2s
150.4d
147.1d
114.3d
160.9s
43.9s
74.9s
61.4d
75.6d
58.3d
17.0q
23.1q
120.2s
150.4d
147.1d
114.3d
160.9s
43.9s
74.8s
61.4d
75.6d
58.5d
17.0q
22.1q
120.2s
150.5d
147.1d
114.3d
160.9s
44.6s
77.0s
62.3d
75.2d
58.0d
16.9q
23.7q
119.8s
150.7d
147.0d
114.4d
160.8s
43.8s
74.8s
61.3d
75.5d
58.3d
17.0q
18.7q
120.1s
150.5d
147.1d
114.3d
160.9s
44.4s
73.4s
59.4d
31.4t
45.9d
16.4q
17.1q
121.8s
150.6d
147.3d
114.2d
161.0s
Fifteen of them (3, 5, 6, 8, 9, 11–15, 17, 19–22) were new
compounds. The chemical structures were illustrated in
Figure 1. Compounds 2, 7, 10, 16 and 23 had been obtained
previously by chemical or enzymatic means, and 18 had
been reported as a metabolite of cinobufagin in rat liver
microsomes. However, their NMR data were determined
and assigned here for the first time.
The products were fully characterized by comparing their
NMR spectra with the substrate or related compounds. The
general NMR rules of bufadienolides we previously
reported were extensively utilized in this procedure.7 The
NMR data are given in Tables 1–5.
All the bufadienolides gave significant [MCH]C ions in the
1
Table 3. H NMR spectral data for compounds 2–9 (500 MHz, DMSO-d6, J in Hz)
H
1
2
3
4
5
6
7
8
9
1.42m
1.35m
1.42m
1.32m
3.83br s
1.82m
1.28m
1.77m
1.60m
1.25m
3.54br s
1.50m
1.18m
1.68m
0.97m
3.31br s
1.43m
1.41m
1.38m
1.58m
1.37m
3.57br s
1.68m
1.25m
1.52m
1.46m
3.99br s
1.70m
1.25m
1.50m
1.42m
3.95br s
1.42m
1.38m
1.44m
1.31m
3.78br s
1.76m
1.25m
1.65m
2.42m
1.29m
3.78br s
1.43m
1.35m
1.45m
1.35m
3.87br s
1.78m
1.16m
1.72m
1.72m
1.08m
1.27m
0.94m
1.52m
1.22m
1.69m
0.97m
3.38br s
1.57m
1.33m
1.32m
1.71m
1.16m
1.29m
0.96m
1.81m
1.63m
1.57m
1.17m
3.28br s
1.70m
1.24m
1.54m
1.45m
3.99br s
2.07m
1.28m
2
3
4
2.08br d (11.5) 1.92m
1.29m
1.40m
5
6
1.52m
1.14m
1.42m
0.92m
1.86m
1.54m
1.48m
1.24m
1.60m
1.41m
3.58s
2.33dd
(15.0,10.5)
1.80m
2.52d (10.5)
0.66 (3H,s)
0.85 (3H,s)
7.52d (2.5)
7.75dd
(10.0,2.5)
6.25d (10.0)
5.21d (3.5)
4.80s
1.42m
1.38m
3.40br s
1.52m
1.16m
1.38m
0.90m
1.80m
1.55m
1.60m
1.25m
3.30m
7
8
9
11
1.82m
1.68m
1.46m
1.18m
1.63m
1.44m
4.08s
2.44dd
(15.0,10.5)
1.90d (15.0)
2.55d (10.0)
0.71 (3H,s)
0.90 (3H,s)
7.54d (2.0)
7.70dd
(10.0,2.0)
6.26d (10.0)
4.22d (2.5)
1.82t (10.0)
1.72m
1.45m
1.20m
1.64m
1.42m
4.11s
1.80t (10.0)
1.45m
1.51m
1.22m
1.62m
1.45m
4.06s
2.41dd
(15.0,10.0)
1.90m
2.56d (10.0)
0.70 (3H,s)
0.86 (3H,s)
7.54d (2.5)
7.70dd
(9.5,2.5)
6.26d (9.5)
5.25br s
4.91s
1.94br d (11.5) 1.80m
2.05m
1.50m
1.28m
1.57m
1.40m
4.17s
2.35dd
(15.0,11.5)
1.77m
2.45d (11.5)
0.65 (3H,s)
0.88 (3H,s)
7.52d (2.5)
7.77dd
1.65m
1.58m
1.18m
3.28m
12
15
16
3.55s
2.30dd
(15.0,10.5)
1.82m
3.02d (10.5)
0.56 (3H,s)
0.88 (3H,s)
7.43s
7.67d
(10.0)
3.58s
2.32dd
(15.0,10.5)
1.84m
3.03d (10.5)
0.56 (3H,s)
0.87 (3H,s)
7.44d (2.0)
7.67dd
(10.0,2.0)
6.25d (10.0)
4.50d (4.0)
3.55s
2.29dd
(15.0,11.0)
1.82m
3.02d (10.5)
0.56 (3H,s)
0.84 (3H,s)
7.43s
7.66d
(10.0)
2.46m
1.92d (15.5)
2.56d (10.0)
0.71 (3H,s)
0.88 (3H,s)
7.54d (2.5)
7.70dd
(9.5,2.5)
6.26d (9.5)
4.53d (4.0)
17
18
19
21
22
(9.5,2.5)
6.25d (9.5)
4.03d (3.0)
23
6.25d (10.0)
4.21br s
6.25d (10.0)
5.22d (3.5)
4.82s
3-OH
5-OH
7-OH
12-OH
3.64s
3.67s
3.62s
4.26d (2.5)
4.77d (4.5)
4.79d (5.0)
4.80d (5.0)

8950
M. Ye et al. / Tetrahedron 61 (2005) 8947–8955
Table 4. 1H NMR spectral data for compounds 10–15 and 23 (500 MHz, DMSO-d6, J in Hz)
H
1
10
11
12
13
14
15
23
1.39m
1.32m
1.50m
1.31m
3.88br s
1.85m
1.17m
1.68m
1.75m
1.06m
1.36m
1.02m
1.80m
2.18dt (2.0,11.0)
1.55m
1.42m
1.47m
1.28m
1.66m
0.94m
3.37br s
1.62m
1.33m
1.31m
1.72m
1.17m
1.34m
1.03m
1.85brt (12.0)
2.26brt (11.0)
1.55m
1.70m
1.22m
1.55m
1.36m
4.01br s
2.10m
1.32m
3.58br s
1.94m
1.35m
2.42m
1.98m
1.44m
1.27m
1.42m
1.34m
3.86br s
1.78m
1.16m
1.75m
1.82m
1.11m
1.40m
1.07m
2.30dt (3.5,13.5)
1.92dt (3.5,13.5)
2.52t (14.0)
2.19dd (14.0,3.5)
2.02m
1.62m
2.38m
2.16m
2
1.72m
1.68m
4.01br s
1.88m
1.29m
1.94m
1.70m
1.16m
1.42m
1.35m
1.90m
2.04m
1.45m
1.42m
3.54br s
3
4
2.70t (14.0)
1.85br d (15.0)
1.74m
1.76m
1.18m
1.38m
1.12m
1.96m
2.36m
5.63s
5
6
1.55m
1.12m
1.42m
0.95m
1.84m
2.08m
1.58m
1.40m
3.54m
2.35m
2.23m
1.74m
0.93m
2.06m
1.24m
1.57m
1.39m
7
8
9
11
1.62m
1.56m
3.60br s
1.40m
3.57br s
12
3.57br s
1.67dt (3.0,11.5)
1.42m
3.54s
15
16
3.57s
2.32dd
3.58s
2.34brt (14.5)
3.57s
2.32m
3.57s
2.32m
3.63s
2.32m
3.64s
2.08dd
2.30dd
(14.0,11.0)
1.68m
(15.0,10.0)
1.84m
(15.0,10.0)
1.82d (15.0)
2.55d (9.5)
0.73 (3H,s)
1.16 (3H,s)
7.54d (2.5)
7.75dd (9.5,2.5)
6.26d (9.5)
1.70m
1.71m
1.68m
1.71m
17
18
19
21
22
23
1-OH
3-OH
5-OH
12-OH
3.08d (10.0)
0.61 (3H,s)
0.88 (3H,s)
7.47d (2.0)
7.74dd (10.0,2.0)
6.25d (10.0)
3.07d (10.0)
0.61 (3H,s)
0.86 (3H,s)
7.48s
7.74d (10.0)
6.25d (10.0)
3.08d (10.0)
0.61 (3H,s)
0.84 (3H,s)
7.48s
7.74d (10.0)
6.25d (10.0)
3.07d (10.5)
0.61 (3H,s)
0.97 (3H,s)
7.47s
7.54d (9.5)
6.25d (9.5)
4.82d (7.5)
5.17d (3.5)
3.11br d (10.5)
0.64 (3H,s)
0.96 (3H,s)
7.50s
7.75d (9.5)
6.26d (9.5)
3.80d (10.0)
0.90 (3H,s)
0.95 (3H,s)
7.54d (2.5)
7.65dd (10.0,2.5)
6.27d (10.0)
4.18d (2.5)
4.72d (4.5)
4.49d (4.0)
4.79d (3.5)
5.19d (3.5)
4.79s
4.75d (4.0)
4.23d (3.0)
4.72d(3.0)
4.74d (4.5)
Table 5. 1H NMR spectral data for compounds 16–22 (500 MHz, DMSO-d6, J in Hz)
H
1
16
17
18
19
20
21
22
1.42m
1.42m
1.44m
1.35m
3.89br s
1.80m
1.18m
1.70m
1.75m
1.08m
1.32m
1.02m
1.91m
1.68m
1.46m
1.15m
1.76m
1.65m
3.44s
1.70m
1.52m
1.72m
0.96m
3.39br s
1.65m
1.32m
1.80m
1.74m
1.21m
1.30m
1.02m
1.88m
1.68m
1.46m
1.15m
1.74m
1.62m
3.46s
1.98m
1.34m
2.34m
2.01m
1.44m
1.38m
1.42m
1.36m
3.86br s
1.65m
1.32m
1.78m
1.60m
1.28m
3.55br s
1.44m
1.35m
1.43m
1.32m
3.89br s
1.75m
1.18m
1.72m
1.72m
1.08m
1.30m
0.92m
1.82m
1.65m
1.58m
1.18m
3.62br s
3.62br d (7.5)
1.43m
1.35m
1.52m
1.34m
3.90br s
1.80m
1.18m
1.69m
1.76m
1.07m
1.32m
1.02m
1.91m
2.17m
1.65m
1.47m
3.65br s
2
1.72m
1.66m
4.01br s
1.88m
1.32m
1.93m
1.70m
1.18m
1.38m
1.04m
1.96m
1.54m
1.30m
1.21m
1.72m
1.63m
3.45s
3
4
2.71m
1.84m
1.72m
1.78m
1.21m
1.38m
1.12m
1.96m
1.80m
1.54m
1.31m
1.84m
1.65m
3.52s
5
6
7
8
9
11
1.88t (10.0)
1.72m
1.42m
1.20m
1.79m
1.68m
3.92s
4.08br s
12
15
16
3.42s
4.03d (4.5)
3.41s
3.87d (11.0)
4.00d (3.5)
4.01br s
4.01br s
3.99d (4.5)
17
2.43s
2.44s
2.46
2.49s
2.92s
2.42s
2.89s
(overlapped)
0.67 (3H,s)
0.98 (3H,s)
7.56s
18
19
21
22
0.63 (3H,s)
0.90 (3H,s)
7.54d (2.0)
7.60dd
0.63 (3H,s)
0.88 (3H,s)
7.55d (2.0)
7.60dd
0.69 (3H,s)
0.92 (3H,s)
7.57s
0.53 (3H,s)
0.89 (3H,s)
7.44s
0.63 (3H,s)
0.99 (3H,s)
7.54d (2.5)
7.60dd
0.64 (3H,s)
0.90 (3H,s)
7.56d (2.5)
7.61dd
7.61d (10.0)
7.59d (10.0)
7.51d (10.0)
(10.0,2.0)
6.25d (10.0)
(10.0,2.0)
6.25d (10.0)
(10.0, 2.5)
6.25d (10.0)
4.82d (7.5)
5.18d (4.5)
(10.0,2.5)
6.26d (10.0)
23
6.26d (10.0)
6.27d (10.0)
6.26d (10.0)
4.24d (3.0)
1-OH
3-OH
7-OH
12-OH
16-OH
4.22d (2.5)
5.47d (4.0)
4.54d (4.5)
5.53d (3.0)
4.25d (2.5)
3.53s
4.21d (3.0)
4.75d (5.0)
5.46d (4.5)
5.94d (2.5)
5.21d (11.0)
5.46d (4.5)
5.62br s
5.45d (4.5)

M. Ye et al. / Tetrahedron 61 (2005) 8947–8955
8951
atmospheric pressure chemical ionization (APCI) mass
spectra. The mono- or dihydroxylation of resibufogenin
could be deduced by the [MCH]C ions at m/z 401 or m/z
417, respectively. And a 2-unit decrease of [MCH]C
indicated the presence of a carbonyl group. The exact
position and stereo-configuration of the hydroxyl or
carbonyl groups, however, were determined by NMR
spectroscopy. Any substitution led to stable and character-
compound
resibufogenin.
5
was identified as 5,7b-dihydroxyl
The 13C NMR spectrum of 6 showed an extra oxygenated
methine signal at d 63.6. Its HMBC correlation with H-8 (d
1.94) indicated that C-7 was hydroxylated. When compared
to 2, the signal of H-7 (d 3.78) appeared as a much narrower
peak, suggesting the a-configuration of 7-OH. In accord-
ance, C-9 (d 31.8) resonated at a much higher field than that
of 2 (d C-937.3), due to the g-gauche effect of 7a-OH. The
NOE enhancement of 7-OH (d 4.26) with H-4a (d 1.25)
was also observed. Therefore, compound 6 was identified as
7a-hydroxyl resibufogenin.
1
istic changes in the H and 13C NMR spectra, as we had
summarized.7 Upon the introduction of 1b-OH, C-19 was
shifted upfield to d 17–18 due to g-gauche effect, and C-10
was shifted downfield by 4–5 ppm. The 1b- and 3b-
hydroxyl groups were both resonated at much lower fields
(d 4.8–5.2) because of their intra-molecular hydrogen
bonding. The W-type long-range coupling between H-1a
and H-3a could be observed in the 1H–1H COSY spectrum.
When 3-OH was epimerized into a-configuration, H-3b
Compounds 7 and 8 were both hydroxylated at 12b-position
since their C-18 signals shifted upfield to d 11.4, and C-13
downfield to d 50.7. In addition, the NOESY spectra showed
NOE enhancements of H-12 (d 3.28) with H-16a (d 2.32)
and H-17 (d 3.03). However, their A-ring carbon signals
were very different. The H-3 signal (d 3.38) of 8 appeared as
a very broad peak, suggesting that its 3-OH should be in
a-configuration. Thus, they were identified as 12b-hydroxyl
resibufogenin (7) and 3-epi-12b-hydroxyl resibufogenin (8),
respectively. The presence of 12b-OH in compound 9 was
established in a similar manner, and was confirmed by the
HMBC correlation of H-18 (d 0.56) with C-12 (d 72.8). In
addition, a new quaternary carbon appeared at d 73.4, and
C-10 was shifted downfield to d 40.0, while C-19 upfield to
d 16.7. All evidences supported the presence of 5-OH.
The HMBC correlation of H-19 (d 0.84) with C-5 was
also observed. Therefore, compound 9 was identified as
5,12b-dihydroxyl resibufogenin.
appeared as a very broad peak (W ⁄ Z20–25 Hz) due to
1
2
3
its Jaa couplings with H-2a and H-4a. Similar to 1b-OH,
the introduction of 5-OH led to the upfield shift of C-19 to d
16–17, and the downfield shift of 3-OH. When a 7b-OH was
present, H-7a appeared as a very broad peak due to its 3Jaa
couplings with H-6b and H-8. When 7-OH was in the
a-configuration, however, the H-7b signal was much
narrower, and C-9 was shifted upfield to d 31–33 due to
the g-gauche effect of 7a-OH. Both 12a-OH and 12b-OH
resulted in 3–4 ppm upfield shift of C-17, 4–6 ppm
downfield shift of C-13, and 0.5 ppm downfield shift of
H-17. The epimers could be differentiated by the chemical
shift of C-18, which was shifted upfield to d 10–12 at the
presence of a 12b-OH due to g-gauche effect, while was
hardly affected by 12a-OH. The 16a-OH could be
characterized by the 12–14 ppm downfield shift of C-17.
In addition, H-17 appeared as a sharp singlet since the
dihedral angle of H-17 and H-16b was approximately 908.
This feature was different from 16b-OH bufadienolides,
where H-17 appeared as a doublet (JZ8.5–9.5 Hz). The
above rules were applied to the structural elucidation of
biotransformation products.
Compounds 10–14 were identified as 12a-hydroxylated
derivatives of resibufogenin, since their C-13 signals were
shifted downfield to d 48.7–48.9, C-17 shifted upfield to d
42.7, and C-18 appeared at d 17.2–17.3. The chemical shift
of C-18 could hardly be affected by 12a-OH since they were
in g-trans positions. In the HMBC spectrum of compound
11, H-18 (d 0.61) had a long-range coupling with C-12
(d 74.0). The signal of C-9 was shifted upfield by 6.9 ppm
when compared to resibufogenin, due to the g-gauche effect
of 12a-OH. The NOE enhancement of 12a-OH (d 4.79)
with H-17 (d 3.07) was also observed in the NOESY
spectrum (Fig. 2). The 3a-OH of 11 was established by the
broad peak of H-3 (d 3.37). Therefore, compound 11 was
identified as 3-epi-12a-hydroxyl resibufogenin. A carbonyl
signal appeared at d 211.6 in the 13C NMR spectrum of 14,
and C-2 (d 36.7) and C-4 (d 41.6) were significantly shifted
downfield, suggesting the presence of C-3 carbonyl group.
The HMBC spectrum showed long-range couplings of C-3
with H-2 and H-4. Thus, compound 14 was identified as
3-oxo-12a-hydroxyl resibufogenin. It could be considered
as an intermediate in the epimerization from 12a-hydroxyl
resibufogenin (10) to 11. For compound 12, a quaternary
carbon appeared at d 73.5, C-10 shifted downfield to d 40.0,
and C-19 upfield to d 16.5, suggesting the hydroxylation at
C-5. Both 3-OH (d 5.19) and 5-OH (d 4.79) shifted
remarkably downfield due to intra-molecular hydrogen
bonding effect. Thus, the structure of 12 should be 5,12a-
dihydroxyl resibufogenin. The NMR spectra of 13 was very
similar to that of 12, with C-10 shifted downfield to d 40.0,
C-19 upfield to d 18.4, and 3-OH downfield to d 5.17.
The 13C NMR spectrum of 2 showed an additional
oxygenated methine signal at d 66.2, and C-8 appeared at
a lower field at d 40.3, suggesting the hydroxylation at C-7.
According to the HMQC spectrum, the signal of H-7
(d 3.54) appeared as a very broad peak, indicating the
b-configuration of 7-OH. Thus, compound 2 was identified
as 7b-hydroxyl resibufogenin. The presence of 7b-OH in
compound 3 was established by comparing its NMR data
with 2. In addition, the HMBC spectrum showed a long-
range coupling of H-8 (d 1.82) with C-7 (d 66.3). However,
the A-ring carbon signals were very different from those of
2, but similar to 3-epi-7b-hydroxyl bufalin,7 indicating that
the 3-OH should be in the a-configuration. Thus, compound
3 was identified as 3-epi-7b-hydroxyl resibufogenin. A
comparison of the NMR data of 5 with those of 2 and
5-hydroxyl resibufogenin (4) suggested that compound 5
was hydroxylated both at C-5 and C-7b. The quaternary
carbon signal at d 73.2 should thus be assigned to C-5. In
accordance, C-19 was shifted upfield to d 16.7, and 3-OH
was shifted downfield to d 5.25 because of its hydrogen
bonding with 5-OH. The signal of H-8 (d 1.80) appeared as a
triplet (JZ10.0 Hz) due to its 3Jaa couplings with H-7a and
H-9, and confirmed the b-configuration of 7-OH. Therefore,

8952
M. Ye et al. / Tetrahedron 61 (2005) 8947–8955
Figure 2. Key HMBC (left) and NOESY (right) correlations of compound 11.
However, the additional oxygenated signal at d 71.8 was
assigned as a methine carbon by the DEPT experiment, and
suggested the hydroxylation at C-1b. Compound 13 was
thus identified as 1b,12a-dihydroxyl resibufogenin. In the
13C NMR spectrum of 15, a carbonyl signal appeared at d
210.5. It showed HMBC correlations with H-18 (d 0.90) and
H-17 (d 3.80), which allowed its assignment to C-12. In
accordance, C-11 (d 37.3) and C-13 (d 59.8) were shifted
downfield remarkably by 16.7 and 15.2 ppm, respectively,
when compared to resibufogenin. Thus, compound 15 was
identified as 12-oxo-resibufogenin. It could be considered as
the transformation intermediate from 7 to 10.
each showed NOE enhancements with H-5 (d 1.93). In
addition, the signal of C-17 was shifted downfield to d 58.3,
suggesting the presence of 16-OH. The NOESY spectrum
showed NOE enhancements of H-16 (d 3.99) with H-22
(d 7.60), and 16-OH (d 5.45) with H-17 (d 2.42), and
established the a-configuration of 16-OH. In accordance,
the signal of H-17 appeared as a sharp singlet. Thus,
compound 21 was identified as 1b,16a-dihydroxyl resi-
bufogenin. The C-13 signal of 22 was shifted downfield
to d 48.0 and C-18 appeared at d 17.5, suggesting the
hydroxylation at C-12a. In addition, downfielded C-17 (d
53.8) and a sharp singlet for H-17 (d 2.89) suggested the
presence of 16a-OH. They were confirmed by the HMBC
correlations of H-18 (d 0.64) with C-12 (d 73.1), and H-16
(d 3.87) with C-20 (d 120.2), respectively. Thus, compound
22 was identified as 12a,16a-dihydroxyl resibufogenin.
Compounds 16–22 were characterized as 16a-hydroxylated
derivatives of resibufogenin. In the 13C NMR spectrum of
compound 17, a new oxygenated methine signal appeared at
d 75.6, and C-17 shifted downfield to d 58.3, suggesting the
presence of 16-OH. It was confirmed by the HMBC
correlations of H-16 (d 4.01) with C-20 (d 120.2) and
C-13 (d 43.9). The signal of H-17 appeared as a singlet,
consistent with the a-configuration of 16-OH. In the
NOESY spectrum, the NOE enhancements of H-16b
(d 4.01) with H-21 (d 7.55) and H-22 (d 7.60) were
observed. The 3a-OH of 17 was established by the broad
peak of H-3 (d 3.39). Thus, compound 17 was identified as
3-epi-16a-hydroxyl resibufogenin. In the 13C NMR spec-
trum of 18, a carbonyl signal appeared at d 211.5, and C-2
(d 36.7) and C-4 (d 41.6) resonated at relatively lower fields.
Its structure was established as 3-oxo-16a-hydroxyl
resibufogenin. Compounds 19–22 were dihydroxylated
derivatives of resibufogenin, giving [MCH]C ions at m/z
417. Their structures were identified by comparing with
structurally related compounds and confirmed by 2D NMR
spectra. The additional carbon signal of 19 at d 66.2 showed
long-range correlation with H-8 (d 1.88), and its correspond-
ing proton signal at d 3.55 appeared as a very broad peak,
suggesting the hydroxylation at C-7b. Thus, compound 19
was identified as 7b,16a-dihydroxyl resibufogenin. The
signal of C-18 in compound 20 was shifted upfield to d 11.7,
and C-13 shifted downfield to d 50.0, suggesting the
hydroxylation at C-12b, and allowed the identification of
compound 20 as 12b,16a-dihydroxyl resibufogenin. In the
13C NMR spectrum of 21, an additional methine signal
appeared at d 71.8, the signal of C-10 was shifted downfield
to d 39.5 and C-19 upfield to d 18.7, suggesting that C-1 was
hydroxylated. The 1H–1H COSY spectrum showed a
W-type long-range coupling of H-1 (d 3.62) with H-3
(d 4.01), suggesting the b-configuration of 1-OH. Both
1-OH (d 4.82) and 3-OH (d 5.18) were resonated at much
lower fields due to intra-molecular hydrogen bonding, and
Most of the biotransformed products of resibufogenin by M.
polymorphosporus were mono- or dihydroxylated deriva-
tives at various positions, including C-1b, C-5, C-7a, C-7b,
C-12a, C-12b and C-16a. The epimerization and dehy-
drogenation at C-3 were also observed. Hydroxylations at
C-12a, C-12b and C-16a were found to be the major
reactions, each giving products in O5% isolated yields,
while the other products were only obtained in fairly low
yields. Previously, we had reported the biotransformation of
bufalin by M. spinosus, where the major reactions involved
hydroxylations at C-7b, C-12b and C-16a, and 7b-
hydroxylated products constituted more than 50% of the
total metabolites.7 Since the structures of resibufogenin and
bufalin only differed in the 14b,15b-epoxy ring, this ring
appeared to inhibit 7b-hydroxylation of bufadienolides due
to its close spatial position with C-7. This assumption could
be evidenced by our latest results from the substrate
specificity investigation of bufadienolide 12b-hydroxyl-
ation by Alternaria alternata.19
For natural bufadienolides, hydroxyl substitutions usually
occur at C-5, C-11a or C-12b, and bufadienolides with a
hydroxyl group at C-1b, C-7a, C-7b or C-16a obtained in
this study are rarely seen.20 Our result obviously indicated
the great potential of biotransformation in the synthesis of
natural product-derived new compounds.
Mucor species are of the most frequently used microbial
catalysts, and have been reported to catalyze the hydroxyl-
ation of a variety of natural products, including steroids,
diterpenes and sesquiterpenes, leading to arrays of
hydroxylated products in most of the reactions.21–27 The
potent hydroxylation capabilities of Mucor species should

M. Ye et al. / Tetrahedron 61 (2005) 8947–8955
8953
Table 6. Cytotoxic activities of the biotransformed products against human cancer cell lines (nZ3)
Compound
IC50 (mmol/L)
Bel-7402
BGC-823
HeLa
Taxol
1
2
3
4
6
7
8
9
10
11
12
13
14
15
16
17
19
20
21
22
23
3.4!10K1
1.3!10K1
2.7
28.7
4.3
1.0
1.1!10K1
2.2
14.0
10.8
3.7
1.5!10K2
1.0!10K2
5.0!10K1
3.9
1.4
2.1
4.9
3.5
3.9
8.0!10K1
37.0
7.2
45.9
16.8
14.4
35.7
97.3
41.8
17.3
5.4
O100
38.1
O100
25.2
18.5
14.4
5.8
33.4
17.8
8.2
3.4
19.1
78.4
5.8
4.2
4.6
44.6
34.7
55.4
6.7
2.9
4.5
57.0
53.8
30.5
30.5
19.0
O100
O100
O100
10.0
20.3
4.3
5.6
3.6
be due to their cytochrome P450 enzyme systems.1,2
Obviously, there could be a family of monohydroxylases
responsible for the hydroxylation reactions. Their properties
warrant further investigation so that the biotransformation
process could be monitored more directly and efficiently.
The chemical shifts (d values) were given in parts per
million (ppm) relative to TMS at 0 ppm. The coupling
constants (J values) were reported in hertz (Hz). Mass
spectra were measured on a Finnigan LCQ Advantage mass
spectrometer equipped with an APCI source. High-
resolution mass spectra (HR-MS) were obtained on a
Bruker APEX II Fourier transform ion cyclotron resonance
(FT-ICR) mass spectrometer.
The cytotoxic activities of the biotransformed products were
evaluated by the MTT method (Table 6). The results were in
agreement with the structure-activity relationships pre-
viously proposed by Ye et al. and Kamano et al.7,28,29
Resibufogenin showed strong inhibitory activities against
human hepatoma Bel-7402 cells, human gastric cancer
BGC-823 cells and human cervical carcinoma HeLa cells,
with IC50 values of 0.13, 0.11, and 0.01 mmol/L, respec-
tively. All the products showed less potent activities. The
7b- or 12b-hydroxylations reduced the activities by less
than 10 folds, and the resultant products still showed
significant cytotoxic effects. However, the epimerization or
dehydrogenation of 3-OH, and 12a-hydroxylation remark-
ably reduced the activities. All the 16a-hydroxylated
products showed very weak cytotoxic activities, with IC50
values O10 mmol/L. These results provided guidance for
the future directed synthesis of bufadienolides of pharma-
ceutical interest.
3.2. Chemicals
Resibufogenin (1) was isolated from the Chinese drug
ChanSu, and unambiguously identified by NMR and MS
techniques. The purity was determined to be O99.5% by
HPLC analysis. Chromatorex ODS (100–200 mesh) for
column chromatography was purchased from Fuji Silysia
Chemical Ltd., Japan. All chemical solvents used for
products isolation were of analytical grade or higher.
3.3. Microorganism and culture media
The fungal strain of Mucor polymorphosporus AS 3.3443
was purchased from China General Microbiological Culture
Collection Center (Beijing, China) and maintained on
potato agar slants at 4 8C. Fermentations of fungi 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 8C and 1.06 kg/
cm2 for 30 min.
3. Experimental
3.1. General
Melting points were determined with an XT4A apparatus
(uncorrected). Optical rotations were measured with a
Perkin-Elmer 243B polarimeter. UV spectra were detected
with a TU-1901 UV–vis spectrophotometer. IR spectra were
recorded in KBr with an Avatar 360 FT-IR spectro-
3.4. Preparative HPLC conditions
For the isolation of biotransformation products, a Spectra-
SERIES HPLC apparatus (Thermo Quest) with a 100 mL
loop was used. Samples were separated on a YMC ODS-A
column (5 mm, :10!250 mm). The flow rate was 2.0 mL/
min, and the detection wavelength was 296 nm.
1
photometer. H and 13C NMR spectra were recorded on a
Bruker DRX-500 spectrometer (500 MHz for 1H NMR and
125 MHz for 13C NMR) in DMSO-d6 at ambient tempera-
ture with tetramethylsilane (TMS) as the internal standard.

8954
M. Ye et al. / Tetrahedron 61 (2005) 8947–8955
3.5. Biotransformation procedure
3379, 2934, 2868, 1705, 1632, 1538, 1454, 1130, 1033 cmK1
;
APCI-MS (m/z): 401 [MCH]C; HR-FT-ICRMS m/z calcd
Mycelia of M. polymorphosporus from agar slants were
aseptically transferred to 250 mL Erlenmeyer flasks
containing 80 mL of liquid potato medium. The fungus
was incubated at 25 8C on a rotary shaker (180 rpm) in the
dark for 24 h to make a stock inoculum. An amount of 8 mL
of the inoculum was then added to each of the 40 1-L flasks
containing 350 mL of potato medium. After 24 h incu-
bation, a total amount of 800 mg of resibufogenin dissolved
in 40 mL of ethanol was distributed equally among the 40
flasks. The incubation was allowed to continue for
additional 6 days on the shaker. The cultures were then
pooled and filtered in vacuo. The filtrate was extracted with
10 L of ethyl acetate for three times. The organic extract
was evaporated to dryness in a rotary evaporator under
reduced pressure at 60 8C to yield 2.7 g of a brownish solid.
for C24H33O5 [MCH]C, 401.2328, found 401.2322; 13C
and H NMR data, see Tables 1 and 3.
1
3.6.5. 5,12b-Dihydroxyl resibufogenin (9). White powder;
C24H32O6; mp 149–152 8C; [a]2D5 C7.9 (c 0.4, MeOH);
UVlmax(MeOH): 205.0, 297.0 nm; IRnmax(KBr): 3408,
2938, 1712, 1631, 1537, 1452, 1040 cmK1; APCI-MS
(m/z): 417 [MCH]C; HR-FT-ICRMS m/z calcd for
C24H33O6 [MCH]C, 417.2276, found 417.2268; 13C and
1H NMR data, see Tables 1 and 3.
3.6.6. 3-epi-12a-Hydroxyl resibufogenin (11). White
powder; C24H32O5; mp 139–140 8C; [a]2D5 C3.7 (c 0.4,
MeOH); UVlmax(MeOH): 204.0, 300.0 nm; IRnmax(KBr):
3427, 2936, 2867, 1712, 1631, 1537, 1453, 1127, 1046 cmK1
;
APCI-MS (m/z): 401 [MCH]C; HR-FT-ICRMS m/z calcd
3.6. Isolation and purification of biotransformation
products
for C24H33O5 [MCH]C, 401.2328, found 401.2323; 13C
and H NMR data, see Tables 1 and 4.
1
The sample was separated on an ODS open column (:3 cm
by 30 cm) and eluted with MeOH–H2O (10:90–80:20, v/v)
to afford five fractions (I–V). Fraction I was subjected to
preparative HPLC and eluted with MeOH–H2O (45:55, v/v)
to yield 9 (7.5 mg). Fraction II was separated by preparative
HPLC and eluted with MeOH–H2O (47.5:52.5, v/v) to give
12 (28.9 mg), 19 (8.4 mg), and 20 (25.8 mg). Fraction III
was subjected to preparative HPLC and eluted with MeOH–
H2O (50:50, v/v) to yield 5 (1.6 mg), 7 (48.0 mg), and 13
(14.0 mg). Fraction IV was separated by preparative HPLC
and eluted with MeOH–H2O (58:42, v/v) to yield 2
(10.7 mg), 3 (3.4 mg), 4 (2.1 mg), 6 (2.0 mg), 8 (43.7 mg),
10 (39.7 mg), 11 (5.9 mg), 14 (3.0 mg), 15 (2.8 mg), 16
(58.6 mg), 21 (3.2 mg), 22 (3.8 mg), and 23 (2.8 mg).
Fraction V was subjected to preparative HPLC and eluted
with MeOH–H2O (75:25, v/v) to yield 17 (7.1 mg) and 18
(1.0 mg). Purities of the above products were O95%,
determined by HPLC-UV means.
3.6.7. 5,12a-Dihydroxyl resibufogenin (12). White pow-
der; C24H32O6; mp 146–148 8C; [a]2D5 C23.4 (c 1.2,
MeOH); UVlmax(MeOH): 204.0, 300.0 nm; IRnmax(KBr):
3420, 2936, 1712, 1632, 1538, 1452, 1377, 1223, 1128,
1040 cmK1; APCI-MS (m/z): 417 [MCH]C; HR-FT-
ICRMS m/z calcd for C24H33O6 [MCH]C, 417.2276,
found 417.2268; 13C and 1H NMR data, see Tables 1 and 4.
3.6.8. 1b,12a-Dihydroxyl resibufogenin (13). White
powder; C24H32O6; mp 164–166 8C; [a]2D5 C5.2 (c 0.6,
MeOH); UVlmax(MeOH): 205.0, 299.0 nm; IRnmax(KBr):
3410, 2935, 1714, 1633, 1537, 1451, 1377, 1228, 1134,
1051 cmK1; APCI-MS (m/z): 417 [MCH]C; HR-FT-
ICRMS m/z calcd for C24H33O6 [MCH]C, 417.2276,
found 417.2267; 13C and 1H NMR data, see Tables 2 and 4.
3.6.9. 3-Oxo-12a-hydroxyl resibufogenin (14). White
powder; C24H30O5; mp 131–133 8C; [a]2D5 C6.2 (c 0.2,
MeOH); UVlmax(MeOH): 206.0, 298.0 nm; IRnmax(KBr):
3444, 2937, 1707, 1635, 1538, 1454, 1378, 1119, 1051 cmK1
;
3.6.1. 3-epi-7b-Hydroxyl resibufogenin (3). White pow-
der; C24H32O5; mp 249–251 8C; [a]2D5 C5.2 (c 0.2, MeOH);
UVlmax(MeOH): 204.0, 298.0 nm; APCI-MS (m/z): 401
[MCH]C; HR-FT-ICRMS m/z calcd for C24H33O5 [MC
APCI-MS (m/z): 399 [MCH]C; HR-FT-ICRMS m/z calcd
for C24H31O5 [MCH]C, 399.2171, found 399.2164; 13C
and H NMR data, see Tables 2 and 4.
1
1
H]C, 401.2328, found 401.2319; 13C and H NMR data,
see Tables 1 and 3.
3.6.10. 12-Oxo-resibufogenin (15). White powder;
C24H30O5; mp 209–210 8C; [a]2D5 C35.1 (c 0.2, MeOH);
UVlmax(MeOH): 204.0, 298.0 nm; IRnmax(KBr): 3492,
2930, 1713, 1539, 1453, 1373, 1126, 1038 cmK1; APCI-
MS (m/z): 399 [MCH]C; HR-FT-ICRMS m/z calcd for
C24H31O5 [MCH]C, 399.2171, found 399.2163; 13C and
1H NMR data, see Tables 2 and 4.
3.6.2. 5,7b-Dihydroxyl resibufogenin (5). White powder;
C24H32O6; mp 224–227 8C; [a]2D5 C32.1 (c 0.1, MeOH);
UVlmax(MeOH): 206.0, 299.0 nm; APCI-MS (m/z): 417
[MCH]C; 13C and H NMR data, see Tables 1 and 3.
1
3.6.3. 7a-Hydroxyl resibufogenin (6). White powder;
C24H32O5; mp 138–140 8C; [a]2D5 C11.7 (c 0.1, MeOH);
UVlmax(MeOH): 204.0, 299.0 nm; IRnmax(KBr): 3437,
2928, 1713, 1634, 1538, 1453, 1125, 1037 cmK1; APCI-
MS (m/z): 401 [MCH]C; HR-FT-ICRMS m/z calcd for
C24H33O5 [MCH]C, 401.2328, found 401.2322; 13C and
1H NMR data, see Tables 1 and 3.
3.6.11. 3-epi-16a-Hydroxyl resibufogenin (17). White
powder; C24H32O5; mp 140–142 8C; [a]2D5 K24.1 (c 0.4,
MeOH); UVlmax(MeOH): 205.0, 299.0 nm; IRnmax(KBr):
3417, 2934, 2867, 1712, 1631, 1536, 1454, 1374, 1241,
1128, 1040 cmK1; APCI-MS (m/z): 401 [MCH]C; HR-FT-
ICRMS m/z calcd for C24H33O5 [MCH]C, 401.2328, found
1
401.2321; 13C and H NMR data, see Tables 2 and 5.
3.6.4. 3-epi-12b-Hydroxyl resibufogenin (8). White
powder; C24H32O5; mp 233–235 8C; [a]2D5 C12.1 (c 1.0,
MeOH); UVlmax(MeOH): 206.0, 299.0 nm; IRnmax(KBr):
3.6.12. 7b,16a-Dihydroxyl resibufogenin (19). White
powder; C24H32O6; mp 127–130 8C; [a]2D5 K33.4 (c 0.5,

M. Ye et al. / Tetrahedron 61 (2005) 8947–8955
3. Mahato, S. B.; Garai, S. Steroids 1997, 62, 332–345.
8955
MeOH); UVlmax(MeOH): 206.0, 298.0 nm; IRnmax(KBr):
3492, 3284, 2932, 1708, 1630, 1539, 1453, 1261, 1141,
1036 cmK1; APCI-MS (m/z): 417 [MCH]C; HR-FT-
ICRMS m/z calcd for C24H33O6 [MCH]C, 417.2276,
found 417.2265; 13C and 1H NMR data, see Tables 2 and 5.
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3.6.13. 12b,16a-Dihydroxyl resibufogenin (20). White
powder; C24H32O6; mp 159–160 8C; [a]2D5 K3.2 (c 1.1,
MeOH); UVlmax(MeOH): 203.0, 299.0 nm; IRnmax(KBr):
3419, 2936, 2879, 1711, 1632, 1537, 1453, 1237, 1134,
1035 cmK1; APCI-MS (m/z): 417 [MCH]C; HR-FT-
ICRMS m/z calcd for C24H33O6 [MCH]C, 417.2276,
found 417.2267; 13C and 1H NMR data, see Tables 2 and 5.
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3.6.14. 1b,16a-Dihydroxyl resibufogenin (21). White
powder; C24H32O6; mp 188–190 8C; [a]2D5 K20.7 (c 0.2,
MeOH); UVlmax(MeOH): 205.0, 296.0 nm; APCI-MS
(m/z): 417 [MCH]C; HR-FT-ICRMS m/z calcd for
C24H33O6 [MCH]C, 417.2276, found 417.2269; 13C and
1H NMR data, see Tables 2 and 5.
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3.6.15. 12a,16a-Dihydroxyl resibufogenin (22). White
powder; C24H32O6; mp 136–140 8C; [a]2D5 K42.1 (c 0.2,
MeOH); UVlmax(MeOH): 205.0, 298.0 nm; IRnmax(KBr):
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3400, 2934, 1714, 1631, 1536, 1452, 1237, 1132, 1034 cmK1
;
APCI-MS (m/z): 417 [MCH]C; HR-FT-ICRMS m/z calcd
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for C24H33O6 [MCH]C, 417.2276, found 417.2269; 13C
and H NMR data, see Tables 2 and 5.
1
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3.7. Bioassay
18. Stache, U.; Radscheit, K.; Haede, W.; Fritsch, W. Liebigs
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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,
Maryland, USA) supplemented with 10% (v/v) fetal bovine
serum and cultured in 96-well microtiter plates for the assay.
Appropriate dilutions (10K3–102 mmol/L) of the test
compounds were added to the cultures. After incubation at
37 8C, 5% CO2 for 72 h, the survival rates of the cancer cells
were evaluated by the MTT method.30 The activity was
shown as the IC50 value, which is the concentration (mmol/
L) of test compound to give 50% inhibition of cell growth.
Results were expressed as the mean value of triplicate
determinations. Taxole was selected as the positive control.
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Acknowledgements
26. Collins, D. O.; Ruddock, P. L. D.; Chiverton de Grasse, J.;
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479–488.
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.
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References and notes
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