Bufospirostenin A and Bufogargarizin C, Steroids with Rearranged Skeletons from the Toad Bufo bufo gargarizans

Article abstract of DOI:10.1021/acs.jnatprod.6b01018

Bufospirostenin A (1) and bufogargarizin C (2), two novel steroids with rearranged A/B rings, were isolated from the toad Bufo bufo gargarizans. Compound 1 represents the first spirostanol found in animals. Compound 2 is an unusual bufadienolide with a cycloheptatriene B ring. Their structures were elucidated by spectroscopic analysis, single crystal X-ray diffraction analysis, and computational calculations.

Full text of DOI:10.1021/acs.jnatprod.6b01018

Note
pubs.acs.org/jnp
Bufospirostenin A and Bufogargarizin C, Steroids with Rearranged
Skeletons from the Toad Bufo bufo gargarizans
Hai-Yan Tian,† Li-Jun Ruan, Tong Yu, Qing-Fei Zheng, Nan-Hao Chen, Rui-Bo Wu,
,⊥
†,⊥
†,⊥
‡
§
§
†
,†
,†
,†
Xiao-Qi Zhang, Lei Wang,* Ren-Wang Jiang,* and Wen-Cai Ye*
†Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's
Republic of China
‡
State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of
Sciences, Shanghai 200032, People's Republic of China
§
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People's Republic of China
*
S Supporting Information
ABSTRACT: Bufospirostenin A (1) and bufogargarizin C (2), two
novel steroids with rearranged A/B rings, were isolated from the
toad Bufo bufo gargarizans. Compound 1 represents the first
spirostanol found in animals. Compound 2 is an unusual
bufadienolide with a cycloheptatriene B ring. Their structures
were elucidated by spectroscopic analysis, single crystal X-ray
diffraction analysis, and computational calculations.
oads are amphibians, and there are 34 genera and 410
1
,2
T
species all over the world. Toads have long been3
recorded as a valuable source of traditional Chinese medicine.
The medicinal parts of toads include toad skin, toad venom,
3
toad liver, and toad bile. The venom of toad, locally called
ChanSu, is used to treat toothaches, heart failure, and other
4
conditions. HuaChanSu injections, prepared from the skin of
the toad Bufo bufo gargarizans or B. melanostictus, have been
shown to be a valuable anticancer drug for the treatment of
5
many kinds of cancers in China. Chemical components found
were each extracted with 95% EtOH, respectively. Then the
in toads include bufadienolides, indole alkaloids, C steroids,
23
concentrated extract of the bile was suspended in H O and
2
6
−11
peptides, and bile acids.
Among them, bufadienolides were
reported to be the main active ingredients, which inhibit Na/K
ATPase to regulate several important cellular processes and
play potential therapeutic roles in various diseases. In our
previous studies, some new bufadienolides in the toad venom
partitioned with cyclohexane, EtOAc, and n-BuOH, and that of
the venom was dissolved in 20% EtOH and partitioned with
CH Cl . Compound 1 was isolated from the EtOAc fraction of
2
2
12
the bile, and 2 from the CH Cl part of the venom extract.
2 2
Compound 1 was obtained as a colorless block. A protonated
1
3,14
+
were found.
As a continuing investigation of the chemical
molecule at m/z 429.2999 [M + H] in its HRESIMS spectrum
constituents of Bufo species, bufospirostenin A (1), a
spirostanol with an unprecedented 5/7/6/5/5/6 ring system,
and bufogargarizin C (2), a bufadienolide with an unusual
cycloheptatriene B ring, were isolated from the bile and venom
of the toad Bufo bufo gargarizans. Herein, we report the
isolation, structure elucidation, and Na/K ATPase (NKA)
inhibitory activities of 1 and 2.
indicated that the molecular formula of 1 was C27
H
40
O
4
, with
eight degrees of unsaturation. The H NMR spectrum showed
the presence of three olefinic protons [δ 4.74 and 4.77 (1H
1
H
2
each, br s, H -27); 5.65 (1H, m, H-6)], three methyl signals [δH
0
7
.83 (3H, s, H -18), 1.09 (3H, s, H -19), and 0.95 (3H, d, J =
3
3
.0 Hz, H -21)], and four oxygenated methine protons [δ 3.93
3
H
(
1H, m, H-3); 4.43 (1H, dd, J = 13.6, 7.7 Hz, H-16); 3.82 and
The gallbladders and venom of toads were collected from
Baoying Toad Breeding Base, Baoying country, Jiangsu
province of China. The concentrated bile and dried venom
Received: November 7, 2016
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b01018
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
Table 1. H and 13C NMR Data for 1 and 2 (δ in ppm)
1
a
b
c
b
c
1
2
1
2
No.
1
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
No.
δ ,
δH (J in Hz)
δC, type
δH (J in Hz)
C
53.4, CH
38.3, CH
2.81, t (9.5)
2.16, ov
73.4, CH
29.2, CH
5.23, d (5.9)
1.71, m
15α
β
33.8, CH
2
2.14, m
34.3, CH
2
2
1.60, ov
2
α
2
2
2
2
1.31, ov
2.33, m
β
1.75, ov
2.18, ov
16α
β
81.9, CH
4.43, dd (3.6,7.7)
29.2, CH
1.75, m
3
71.9, CH
44.3, CH
3.93, m
73.7, CH
36.5, CH
4.73, m
2.03, m
4
β
5
6
α
2.48, dd (14.3,6.5)
2.17, ov
2.44, d (16.5)
3.31, dd (16.5,5.5)
17
18
19α
β
64.2, CH
16.9, CH
15.9, CH
1.77, ov
0.83, s
1.09, s
49.4, CH
23.4, CH
35.2, CH
2.60, dd (9.5,7.5)
0.78, s
3
3
3
2
144.9, C
128.5, C
1.94, d (15.9)
2.18, ov
123.1, CH
33.7, CH
5.65, m
1.79, m
2.10, m
1.48, ov
1.34, m
127.2, CH
127.8, CH
6.89, d (7.8)
6.97, d (7.8)
7
β
8
9
a
2
20
21
22
23α
b
43.0, CH
14.9, CH
110.5, C
34.0, CH
1.94, m
120.4, C
3
2
2
0.95, d (7.0)
148.3, CH
145.1, CH
115.8, CH
7.30, br s
39.0, CH
61.2, CH
75.0, C
137.0, C
129.7, C
138.6, C
22.0, CH
7.34, dd (9.6,2.6)
6.34, d (9.6)
1.74, ov
1.70, ov
2.25, m
2.56, m
1
1
0
1α
24.1, CH
2
2
1.48, ov
2.00, m
1.73, ov
1.17, m
2
2
2.49, dt (16.8,5.5)
2.73, m
24α
β
29.5, CH
161.9, C
β
12α
41.0, CH
33.7, CH
1.67, m
25
26α
β
145.2, C
65.8, CH
β
1.60, ov
2
2
3.82, d (11.9)
4.27, d (11.9)
4.74, br s
1
1
3
41.8, C
42.1, C
4
57.1, CH
1.27, m
50.0, CH
2.77, m
27α
β
109.0, CH
4.77, br s
a
b
1
13
c
1
ov: overlapped proton resonances. Measured in CD OD, 300 MHz for H NMR, 75 MHz for C NMR. Measured in CDCl , 400 MHz for H
3
3
NMR, 100 MHz for 13C NMR.
4
.27 (1H each, d, J = 11.9 Hz, H -26)]. The 13C NMR and
established a 5/7 ring system. The detailed interpretation of the
HMBC correlations (Figure 1a) allowed the establishment of
the planar structure of 1.
2
DEPT spectra revealed the existence of 27 carbon signals.
Based on the extensive analysis of COSY, HSQC, HMBC, and
ROESY spectra, the H and C NMR signals of 1 were
1
13
In order to determine the relative configurations of 1, the
assigned as shown in Table 1. Comparison of the NMR data of
ROESY spectrum was extensively analyzed (Supporting
15,16
1
with those of neoruscogenin
suggested that 1 had the
Information, Figure S2). NOE correlations between H
-19
3
same substructure as neoruscogein in rings C, D, E, and F.
Further structural information on 1 came from the inspection
of the COSY spectrum, which demonstrated the presence of
three isolated spin systems as shown in Figure 1a. The HMBC
correlations between H -4 and C-6, between H -19 and C-1,
and H-2β/H-4β/H-8, and between H -18 and H-8/H-20
3
suggested these protons were in the same orientation.
Furthermore, NOE correlations between H-1 and H-3α/H-
7
1
α/H-9, between H-14 and H-16/H-7α, as well as between H-
7 and H-16 indicated those protons had the same orientation.
2
3
Crystals suitable for single-crystal X-ray diffraction analysis were
obtained from a MeOH solution of 1. Therefore, the relative
configuration of 1 was fully established by X-ray crystallo-
graphic analysis (Figure 3). A relatively small Flack parameter
between H-2β and C-10, and between H-7β and C-5 indicated
rings A and B were connected via a C-1 to C-5 bond, which
0
.0 (3) from the final refinement enabled the determination of
the absolute configurations of 1 to be 5Z, 1R, 3S, 8S, 9S, 10R,
1
3S, 14S, 16S, 17R, 20S, and 22R, which was also consistent
1
7,18
with biosynthetic considerations.
Thus, the complete
structure of compound 1 was established, which was named
as bufospirostenin A.
Compound 2 was isolated as a white powder. A protonated
molecule at m/z 363.1959 [M + H]+ indicated that the
molecular formula of 2 was C H O , with 12 degrees of
24
26
3
unsaturation. The UV absorption maximum at 300 nm suggests
1
the presence of a 2H-pyran-2-one moiety. In the H NMR
spectrum, characteristic signals for a 2H- pyran-2-one moiety
[
δH 6.34 (1H, d, J = 9.6 Hz) and 7.30 (1H, br s), 7.34 (1H, dd,
J = 9.6, 2.6 Hz)], one pair of mutually coupled olefinic protons
δH 6.89 (1H, d, J = 7.8 Hz) and 6.97 (1H, d, J = 7.8 Hz)], two
[
oxygenated protons [δ 4.73 (1H, m), 5.23 (1H, d, J = 5.9
H
Hz)], and one angular methyl [δ 0.78 (3H, s)] were observed.
H
The analysis of the 13C NMR and DEPT spectra permitted
differentiation of 24 carbon resonances into one methyl, seven
methylenes, nine methines, and seven nonprotonated carbons.
This evidence suggested that 2 was a bufadienolide derivative.
Figure 1. COSY and key HMBC correlations of 1 (a) and 2 (b).
B
DOI: 10.1021/acs.jnatprod.6b01018
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Journal of Natural Products
Note
Via combined analysis of the COSY, HSQC, HMBC, and
1
13
ROESY spectra of 2, the H and C NMR signals were
unambiguously assigned (Table 1).
In the COSY spectrum, five spin systems could be observed
as shown in bold face in Figure 2b. In the HMBC spectrum, the
Figure 3. (a) Key NOE correlations of 2; (b) conformational analysis
of rings A/B in 2: an optimized stereoisomer shows the dihedral angles
between H-1 and H-2α/H-2β and the spatial distance between H-1
1
9
and H-11α/H-11β.
Figure 2. X-ray crystal structure of 1.
correlations between H-17 and C-21/C-22 indicated that the
2
H-pyran-2-one moiety was connected to C-17 as for typical
bufadienolides. The HMBC correlations between H -18 and C-
3
1
2/C-13/C-14/C-17 revealed the angular methyl was attached
at C-13. This evidence suggested the presence of the rings C/
D/E. Furthermore, HMBC correlations between H-1 and C-5/
C-10, between H-6 and C-4/C-8/C-10, between H-7 and C-5,
as well as between H-19α (δ 1.94) and C-1 supported the
H
establishment of rearranged A/B rings, containing a rare
cycloheptatriene ring B. Moreover, the connection of the C/B
rings was accomplished by HMBC correlations between H-14
and C-7/C-8/C-9 and between H -11/H -12 and C-9. In
2
2
addition, taking account of the molecular formula of 2, as well
Figure 4. Experimental ECD spectrum of 2 in MeOH and the
calculated ECD spectra of two optimized stereoisomers.
as the deshielded chemical shifts of H-3 (δ 4.73) and C-3 (δC
H
7
3
3.7) compared with those of a common bufadienolide (δH
.89 and δ 67.1, Supporting Information), the structure of 2
C
should possess one more degree of unsaturation corresponding
to a four membered cyclic ether constructed by an oxygen
bridge between C-1 and C-3. The above evidence allowed the
establishment of the planar structure of 2.
The relative configuration of 2 could be determined by a
ROESY experiment (Figure 3a). The NOE correlations
between H -18 and H-14/H-21 indicated that H-14 was in
the same β-orientation as the C-18 angular methyl group and
the C-17 lactone ring. According to a comparison with our
previously reported bufadienolides
matched well with the experimental spectrum, which was
strong evidence that it was the most reasonable absolute
configuration. It is quite interesting that the signal of H-1
21
displayed as a doublet with a coupling constant of 5.9 Hz in the
1
H NMR spectrum, though the vicinal C-2 bears two protons.
Through a conformation analysis, it was found that the dihedral
angle between H-1 and H-2α was 99.8°, corresponding to a
coupling constant close to zero, while that between H-1 and H-
2β was 31.6°, in line with a coupling constant of 5.9 Hz based
3
8
,14
22
and on biosynthetic
on the Karplus equation (Figure 3). Moreover, the analysis of
considerations, the absolute configurations of C-13, C-14, and
C-17 were deduced to be 13S, 14S, and 17S. However, the
configuration of the oxetane group in ring A was still unclear
due to an uncertain conformation of rings A/B. In order to
establish the full absolute configuration of 2, a quantum
chemical calculation was carried out. Aiming to get one
reasonable configuration, two optimized stereoisomers, (1R,
the spatial distances between the protons illustrated well the
NOE correlations observed in its ROESY spectrum (Figure
3b). The spatial distances between H-1 and H-11α/H-11β were
4.2 and 3.9 Å due to the arch-shape of the molecule, supporting
the NOE correlations between those protons. Thus, the
structure of 2, named bufogargarizin C, was fully established.
Many traditional Chinese medicines containing bufadieno-
lides or other steroid-like compounds have been reported to
have a cardioactive effect and promote the blood circulation via
inhibition of NKA. Thus, compounds 1 and 2 together with
bufalin (the main bufadienolide in Bufo bufo gargarizans) were
evaluated for their inhibitory activities against NKA. At 12.5
and 25 μM, compound 1 showed 21% and 43% inhibition of
NKA, respectively. At the same concentrations, bufalin showed
complete inhibition of NKA, while compound 2 was inactive,
3R, 13S, 14S, 17S)-2 and (1S, 3S, 13S, 14S, 17S)-2, were built
based on the X-ray single crystal structure of 8,9-didehydro-14-
deoxybufalin (Supporting Information), which was a structur-
ally modified bufadienolide derivative with the same C, D, and
23
2
0
E rings as 2. Furthermore, their ECD spectra were calculated
at the level of PBE0/6-311++G (2d, 2p) with the PCM solvent
continuum model and MeOH as the solvent, and the
stereoisomer with the 1R, 3R configurations (Figure 4)
C
DOI: 10.1021/acs.jnatprod.6b01018
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Journal of Natural Products
Note
which suggested that a bufadienolide with a rearranged A/B
ring structure is not favorable for NKA inhibition.
Bufospirostenin A (1). Colorless needle crystals; mp 187−189 °C;
27
[
α]D + 54 (c 0.1, MeOH); UV (MeOH): λ (log ε) 208 (3.57) nm;
max
−1
1
13
IR (KBr): 3414, 2956, 2935, 1617, 1204 cm ; H and C NMR data,
Tables 1 and S1; HRESIMS: m/z 429.29985 [M + H] (calcd for
In the present work we report two novel steroids from toad
bile and venom, respectively. It is very intriguing for toads to
synthesize such interesting A/B ring rearranged steroids.
Compound 1 has an unusual 5/7/6/5/5/6 ring system,
which is the first example of a spirostanol with alteration of
rings A and B. Compound 2 is a steroid presumably
biosynthesized from a normal bufadienolide, through a series
of steps to form an unprecedented 6/7/6/5/5 ring system
containing a cycloheptatriene in its backbone. The discovery of
these compounds enriches the structural patterns of the steroid
family. The biosynthetic pathways and the endogenous
biological functions of these compounds in toads need further
investigations.
+
C H O , 429.29994).
Bufogargarizin C (2). White powder; [α]D + 20 (c 0.1, MeOH);
2
7
41
4
25
UV (MeOH): λmax (log ε) 300 (3.80) nm; ECD (0.02 mg/mL,
MeOH), λmax (Δε) 342 (−2.63), 263 (10.89), and 231 (−12.40) nm;
−
1
1
13
IR (KBr): 2927, 2858, 1715, 1666 cm ; H and C NMR
spectroscopic data, Tables 1, S2 and S3; ESI-MS: m/z 363 [M +
+
+
+
H] , 385 [M + Na] and 747 [2M+Na] ; HRESIMS: m/z 363.1959
+
[
M + H] (calcd for C H O , 363.1955).
24 27 3
Biological Assay. Na/K-ATPase from pig kidney microsomal
membranes was prepared by treatment with SDS and purified by
2
3
differential centrifugation. The specific activities of the Na/K-
ATPase of various kidney preparations were in the range 900 to 1200
μmol Pi/mg/h, which was more than 95% of the total ATPase activity.
The inhibitory activities of different concentrations of compounds 1, 2,
and bufalin (purity over 98% by HPLC analysis) on 0.5 μg of Na/K-
ATPase were determined by measuring the release of inorganic
phosphate from ATP over a 15 min period at 37 °C in an assay
EXPERIMENTAL SECTION
■
General Experimental Procedures. The melting point was
obtained with an X-5 melting point apparatus without correction.
Optical rotations were measured in MeOH on a Jasco P-1020
polarimeter at room temperature. UV spectra were recorded in MeOH
on a Jasco V-550 UV/vis spectrophotometer. ECD spectra were
obtained on a Jasco J-810 spectropolarimeter. NMR spectra were
acquired with Bruker AV-300, AV-400, and AV-600 spectrometers
using TMS as an internal standard. HRESIMS spectra were measured
on an Agilent 6210 ESI/TOF mass spectrometer. The X-ray
diffraction study was carried out on an Agilent Gemini S Ultra with
Cu Kα radiation. Column chromatographic methods were carried out
on commercial silica gel (100−400 mesh, Qingdao Marine Chemical
medium containing 100 mM NaCl, 20 mM KCl, 1 mM MgCl , 1 mM
2
2+
EGTA, 20 mM Tris-HCl, and 2 mM ATP-Mg mixture, pH 7.4. The
absorbance at 620 nm was measured with a microplate reader
(
SpectraMax 340PC384, Molecular Devices) after treatment using the
24
Biomol Green Reagent.
ASSOCIATED CONTENT
■
*
S
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b01018.
Plant). TLC analyses were carried out using precoated silica gel GF2
54
plates (Qingdao Marine Chemical Plant). Analytical high-performance
liquid chromatography (HPLC) was carried out on an Agilent 1200
system chromatograph equipped with a Quat Pump and DAD
detector. Semipreparative HPLC (semi-RP-HPLC) was performed on
a WUFENG LC-100 system equipped with an UV detector using a
COSMOSIL Packed 5C -MS-II column (particle size 5 μm, 250 × 10
Detailed NMR spectra; HR-ESI-MS, ESI-MS, UV
spectra; and X-ray crystal data of 1 and 8,9-didehydro-
14-deoxybufalin (PDF)
18
X-ray crystallography data (cif) for 1 (CCDC 1434619)
mm). All solvents used in column and HPLC were of analytical grade
Shanghai Chemical Plant) and chromatographic grade (Fisher
(CIF)
(
X-ray crystallography data (cif) for 8,9-didehydro-14-
Scientific), respectively.
Animal Materials. The gallbladders and venom of toads were
collected from Baoying Toad Breeding Base in Jiangsu province of
China, and authenticated as Bufo bufo gargarizans Cantor by Guang-
Xiong Zhou (Jinan University, Guangzhou, P. R. China). They were
sacrificed according to a procedure approved by the Institute Animal
and Use Committee of Jinan University (No. 20130729001) and in
accordance with the National Institutes of Health’s Guide for the Care
and Use of Laboratory Animals (seventh edition).
deoxybufalin (CCDC 1471863) (CIF)
AUTHOR INFORMATION
■
*
Corresponding Authors
Tel: +86-20-85221559. Fax: +86-20-85221559. E-mail:
chyewc@gmail.com (W. C. Ye).
Extraction and Isolation. The collected gallbladders (240 g) and
venom (1.5 kg) were extracted thoroughly with 95% EtOH under
ultrasonic condition (40 min × 3, 40 °C). The combined EtOH
extracts were filtered and concentrated under reduced pressure to
afford 24.0 g of extract for the bile and 900.0 g for the venom. The
*Tel: +86-20-85221559. Fax: +86-20-85221559. E-mail:
trwjiang@jnu.edu.cn (R. W. Jiang).
*
Tel: +86-20-85221559. Fax: +86-20-85221559. E-mail:
cpuwanglei@126.com (L. Wang).
ORCID
concentrated residue of bile was then suspended in H O and
2
partitioned with cyclohexane, EtOAc, and n-BuOH, and those of the
Wen-Cai Ye: 0000-0002-2810-1001
Author Contributions
H.-Y.T., L.-J.R., and T.Y. contributed equally to this work.
venom were dissolved in 20% EtOH and partitioned with CH Cl2
2
three times. The EtOAc fraction (1.6 g) of the bile extract was
subjected to silica gel column chromatography (200−300 mesh) with
a gradient elution of CH Cl /MeOH (from 50:1 to 2:1) to yield 15
⊥
2
2
Notes
subfractions (TBE-1 to 15). Compound 1 (1.9 mg, t 24.6 min) was
R
The authors declare no competing financial interest.
separated from fraction TBE-2 by semipreparative HPLC eluting with
8
2% MeOH/H O. The CH Cl portion of the venom extract was then
2 2 2
chromatographed on silica gel (10 times, 200−300 mesh), eluting with
cyclohexane-−cetone (5:1, 3:1, 1:1) to yield 15 fractions (Fr. 1 to 15).
Fr. 8 was separated by reversed-phase C18 silica gel, eluting with
ACKNOWLEDGMENTS
■
This research work was financially supported by National
Natural Science Foundation of China (81102518, 81473336,
and 81573315) and by the Pearl River Science and Technology
Program for Hai-Yan Tian (201506010020).
MeOH/H O gradients (20:80−90:10) to give six subfractions (Fr.
2
8
.1−Fr. 8.6). Fr. 8.6 was purified by preparative HPLC using MeOH/
2
H O (72:28) as eluent to yield 2 (2.0 mg).
D
DOI: 10.1021/acs.jnatprod.6b01018
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Journal of Natural Products
Note
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DOI: 10.1021/acs.jnatprod.6b01018
J. Nat. Prod. XXXX, XXX, XXX−XXX