Article pubs.acs.org/jnp
C23 Steroids from the Venom of Bufo bufo gargarizans Hai-Yan Tian,†,⊥ Shi-Lin Luo,‡,⊥ Jun-Shan Liu,†,§ Lei Wang,† Ying Wang,†,§ Dong-Mei Zhang,† Xiao-Qi Zhang,† 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 ‡ Department of Phytochemistry, China Pharmaceutical University, Nanjing 210009, People’s Republic of China § JNU-HKUST Joint Laboratory for Neuroscience & Innovative Drug Research, Jinan University, Guangzhou 510632, People’s Republic of China S Supporting Information *
ABSTRACT: Five new C23 steroids (1−5) together with five known bufadienolides (6−10) were isolated from the venom of Bufo bufo gargarizans (ChanSu in Chinese). The structures of the new steroids were elucidated by extensive spectroscopic methods in combination with X-ray diffraction analysis. Among these C23 steroids, only compound 3 showed cytotoxicities against HepG2 and A549 cancer cells, with respective IC50 values of 26.8 ± 8.3 and 45.6 ± 2.5 μM. In contrast, the bufadienolides (7−10) displayed potent inhibitory activities against these cancer cells, with respective IC50 values in the ranges 0.5−5.5 and 0.6−6.5 μM, but relatively less cytotoxicity on normal mouse spleen cells. In addition, the Na+/K+-ATPase inhibitory activities of 2, 5, and 7 revealed that the lactone moiety of a bufadienolide was important for the inhibitory activity.
T
steroids were determined. We report herein the structural elucidation and biological activities.
he venom of the Asiatic toad Bufo bufo gargarizans (ChanSu in Chinese) was used as a traditional Chinese medicine to treat superficial infections, odontalgia, and heart failure.1 In the current Chinese Pharmacopoeia, ChanSu has been reported to contain diverse chemical constituents, including cardiac steroids, indole alkaloids, and organic acids.2 In a previous investigation of toad venom, we isolated and characterized two new C23 steroids possessing a unique side chain at C-17. These substances were cleavage products of known bufadienolides, which were the main active ingredients of toad venom. These compounds are also known for their severe cardiotoxicity associated with specific inhibition of Na+/ K+ ATPase.5−7 As part of an ongoing effort aimed at elucidating the chemical diversity of this Bufo species, we isolated five new C23 steroids (1−5) together with five known bufadienolides, resibufogenin (6),8 bufalin (7),9,10 cinobufagin (8),10 bufotalin (9),11 and telocinobufagin (10).10 Structural studies, utilizing a combination of NMR, HRESIMS, and X-ray diffraction methods, demonstrate that 1 is a new C23 steroid with a unique acetal cage-like structural feature, compound 2 is the first example of a naturally occurring C23 steroid bearing a C-17 methyl butyrate side chain, compound 3 possesses an α,βunsaturated formyl unit at C-17, and steroids 4 and 5 share the same skeleton possessing a dihydropyran ring that bridges C-14 and C-21 in both structures. In addition, the cytotoxicities against HepG2 and A549 cancer cells and mouse spleen cells and the Na+/K+-ATPase inhibitory activities of the these © 2013 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION The molecular formula of 1 was established as C23H34O4 by analysis of its HRESIMS spectrum, exhibiting a quasi-molecular ion at m/z 375.2531 [M + H]+. The IR spectrum exhibited a characteristic absorption for a hydroxy group (3449 cm−1). The 1 H NMR spectrum contained two methyl singlets [δH 0.88 (3H, s); 1.11 (3H, s)], a methyl doublet [δH 1.19 (3H, d, J = 5.6 Hz)], and resonances for four oxymethine protons [δH 3.10 (1H, q, J = 5.6 Hz); 3.89 (1H, br s); 4.50 (1H, m); 4.57 (1H, d, J = 1.3 Hz)]. Twenty-three carbon resonances, including three methyl, eight methylene, eight methine, and four quanternary carbons were observed in the 13C NMR spectrum. Comparison of the NMR data for 1 with those of resibufogenin (6, Figure S1 in Supporting Information)8 indicated that the 1H and 13C NMR signals for the protons and carbons in the A, B, and C rings had similar chemical shifts, except for the absence of a 2Hpyran-2-one moiety in 1 compared to 6. Further information about the structure of 1 came from inspection of its 1H−1H COSY spectrum, which demonstrates the presence of three spin systems represented in bold face in Figure 1. The correlations between H-21 (δH 4.57) and C-14 (δC 91.1)/C-15 (δC 78.0) in the HMBC spectrum indicated Received: March 5, 2013 Published: September 19, 2013 1842
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Figure 2. Key NOESY correlations of 1 and 4.
bufadienolides. Moreover, the distinct NOE correlation between H-21 (δH 4.55) and H-22 (δH 3.10) suggested that these two protons were in the same β-orientation. In spite of the above NOESY correlation data, we were unable to assign the configuration of C-20 and the orientation of the oxirane ring. However, crystals suitable for X-ray diffraction analysis (Figure 3) were obtained from an MeOH
Figure 3. ORTEP plot of single-crystal X-ray data of 1.
solution of 1. The results showed that compound 1 was composed of five six-membered and two five-membered rings and an oxirane ring. Rings D, E, F, and G were cross-linked through an acetal moiety that formed a cage-like structure. It was noteworthy that the C-20−C-22 bond length was short (1.468 Å), in accord with the existence of an oxirane ring.12 A final refinement resulted in a suitably low Flack parameter 0.01(18) that permitted assignment of the absolute configuration of 1 as 1S, 5R, 8R, 9S, 10S, 13R, 14S, 15R, 17S, 20R, 21R, and 22S.13 Thus, 1 was determined as (3β,5β,14β,15β,20R,21R,22S)-14,21:15,21:20,22-triepoxy-24norcholan-3-ol. The molecular formula of 2 was determined as C24H34O4 on the basis of the presence of a quasi-molecular ion at m/z 409.2348 [M + Na]+ in its HRESIMS spectrum. Characteristic IR absorption bands indicated the existence of hydroxy (3532 cm−1) and carbonyl (1722, 1698 cm−1) groups. The 1H NMR spectrum of 2 contained resonances corresponding to two angular methyl groups (δH 1.17 and 1.08), a methoxy group (δH 3.67, s), and two olefinic protons [δH 5.99 (t, J = 2.4 Hz), 7.29 (d, J = 2.4 Hz)]. Analysis of the 13C NMR and DEPT spectra showed that 2 contained 24 carbons, including one methoxy and two methyl groups, nine methylene, six methine, and four quaternary carbons, and two carbonyl groups. The
Figure 1. Key 1H−1H COSY and HMBC correlations of 1 and 2.
that ether bridges were present between C-21 and C-14 as well as C-21 and C-15 and that these bridges comprised a cage-like structure with an acetal functionality at C-21 (δC 104.1). Moreover, HMBC correlations between H-23 (δH 1.19) and C20 (δC 62.2), as well as H-22 (δH 3.10) and C-21, suggested the presence of a C-21→C-20→C-22→C-23 fragment in 1. Taking into account the atomic composition of this compound, an oxirane ring encompassing C-20 and C-22 should also be present. Taken together, compound 1 was determined as a new C23 steroid with an acetal cage-like moiety, as shown in Figure 1. The relative configuration of 1 was determined by analysis of NOESY data, which showed correlations between H3-19 (δH 0.88) and H-5 (δH 1.68), H3-19 and H-8 (δH 1.70), H3-18 (δH 1.11) and H-8, and the 3-OH proton (δH 4.18) and H-5. These observations suggested that all of these protons have the same β-orientation. Furthermore, NOESY correlations between H-4α (δH 1.84) and H-9 (δH 1.39), H-9 and H-7α (δH 1.15), and H7α and H-15 (δH 4.48) indicated that these protons had an αorientation (Figure 2). Therefore, 1 possessed the same A/B cis, B/C trans, and C/D cis ring system found in the known 1843
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to the 2H-pyran-2-one moiety in 9 with the signals of a terminal double bond [δH 4.64 (1H, br d, J = 11.6 Hz), 4.75 (1H, br d, J = 17.4 Hz), 6.28 (1H, dd, J = 17.4, 11.6 Hz); δC 136.5, 106.6] and a trisubstituted double bond [δH 6.34 (1H, s); δC 117.8, 144.3] in 4. The HMBC spectrum of 4 displayed correlations between H-21 (δH 6.34) and C-17 (δC 46.9), H-22 (δH 6.28) and C-21 (δC 144.3), H-21 and C-22 (δC 136.5), and H2-23 (δH 4.64 and 4.75) and C-20 (δC 117.8), indicating the presence of a conjugated diene moiety in the side chain. The characteristic HMBC correlation between H-21 (δH 6.34) and C-14 (δC 90.2) suggested that a dihydropyran ring was formed between C-14 and C-21. The relative configuration of 4 was determined by analysis of NOESY correlations (Figure 2). Accordingly, the structure of 4 was determined as (3β,5β,14β,16β,20Z)-16-acetoxy-14,21-epoxy-24-norcholan20,22-dien-3-ol. The quasi-molecular ion at m/z 359.2581 [M + H]+ in the HRESIMS spectrum of 5 suggested a molecular formula of C23H34O3. UV and IR spectroscopic data indicated that 5 contained a conjugated diene group. Comparison of the NMR data of 5 with those of 4 revealed that both substances possessed the same C-17 side chain. The main difference in their NMR spectra was the absence of an acetoxy group at C-16 and the presence of a hydroxy group at C-5 in 5, which was subsequently confirmed by analysis of the 1H−1H COSY, HSQC, and HMBC spectra. The relative configuration of 5 was assigned by analysis of the NOESY spectrum. Accordingly, the structure of 5 was determined as (3β,5β,14β)-14,21-epoxy-24norcholan-20,22-diene-3,5-diol. A biological investigation was carried out to determine if these C23 steroids inhibit Na+/K+-ATPase from porcine cerebral cortex. At concentrations as high as 50 μM, compounds 2 and 5 did not inhibit the enzyme (other C23 steroids were not tested due to the limited materials). In contrast, bufalin (7), a typical bufadienolide, was a potent inhibitor of Na+/K+-ATPase, with an IC50 value of 0.011 ± 0.001 μM (Figure 5), which was comparable with the positive
NMR data showed that 2 had the same steroid skeleton as that in the known bufadienolides. The HMBC correlations between Me-18 (δH 1.17) and C-12 (δC 36.6)/C-14 (δC 153.8)/C-17 (δC 174.8), H-15 (δH 5.99) and C-8 (δC 36.3)/C-13 (δC 53.9), and H-16 (δH 7.29) and C-13 (53.9)/C-14 (δC 153.8) (Figure 1) suggested that a conjugated diene group was present in ring D. The HMBC spectrum also displayed cross-peaks between H2-22 (δH 3.02) and C-20/C-24 (δC 173.7), H2-23 (δH 2.64) and C-20/C-24, and the methoxy group (δH 3.67) and C-24, indicating the existence of a linear side chain (Figure 1). Futhermore, the HMBC correlation between H-16 and C-20 (δC 192.4) revealed that the side chain was linked to C-17. The relative configuration of 2 was confirmed by X-ray diffraction analysis (Figure 4), and the complete structure was
Figure 4. ORTEP plot of single-crystal X-ray data of 2.
determined to be (3β,5β)-methyl 3-hydroxy-20-oxo-21-norcholan-14,16-dien-24-oate. Compound 2 is the first naturally occurring 21-nor steroid containing a methyl butyrate side chain. It is noteworthy that 2 could be detected in the crude CH2Cl2 extract by LC-MS analysis (Figure S2, Supporting Information), confirming that 2 was not an artifact. HRESIMS analysis of 3 showed a quasi-molecular ion at m/z 417.2639 [M + H] + (calcd for C25H37O5: 417.2636), corresponding to a molecular formula of C25H36O5. Absorption bands at 3542 and 1719 cm−1 in the IR spectrum of 3 demonstrated the presence of hydroxy and carbonyl groups. A comparison of 1H and 13C NMR data of 3 with those of cinobufagin (8, Figure S1 in the Supporting Information) revealed that they possessed a similar tetracylic A−D ring system. In addition, HMBC correlations observed between H21 (δH 10.10) and C-22 (δC 151.0), and Me-23 (δH 2.17) and C-20 (δC 135.1), indicated the existence of a C4 side chain in 3. Moreover, an HMBC correlation between H-16 (δH 5.37) and C-20 showed that the C4 unit was connected to C-17. Finally, the NOESY correlation between H-21 (δH 10.10) and Me-23 (δH 2.17) indicated a Z-double bond. Accordingly, the structure of 3 was identified as (3β,5β,14β,16β,20Z)-16-acetoxy-14,15epoxy-3-hydroxy-24-norcholan-20(22)-en-21-al. The molecular formula of 4 was determined as C25H36O4 through analysis of its HRESIMS spectrum, which contained a quasi-molecular ion at m/z 423.2522 [M + Na]+ (calcd for C25H36O4Na: 423.2509). The existence of a maximum absorption at 250 nm in the UV spectrum and an absorption band at 1663 cm−1 in the IR spectrum suggested that 4 contained a conjugated diene moiety. Comparison of the 1H and 13C NMR data of 4 with those of bufotalin (9, Figure S1 in Supporting Information) revealed they contained similar A−D ring systems. The main difference between the 1D NMR spectra of 4 and 9 was the replacement of signals corresponding
Figure 5. Inhibitory effects of compounds 2 and 7 (ouabain as positive control) on Na+/K+-ATPase. GraphPad Prism software (version 5) was used to fit sigmoid curves to estimate the slopes and IC50 for the dose/inhibition relationships. The inhibitor concentrations are displayed on a logarithmic scale. The data are expressed as the mean of a minimum of three independent experiments.
control (ouabain). The observations indicated that removal of the lactone ring in bufadienolides greatly decreased the interactions between steroids with Na+/K+-ATPase. Moreover, the cytotoxicities of steroids 1−10 against HepG2 and A549 cancer cell lines were evaluated. Compounds 1, 2, 4, and 5 were found to be nearly inactive against these cancer cells, while 3 1844
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Table 1. 1H and 13C NMR Data for 1−5 (methanol-d4, J in Hz)a 1b position 1α 1β 2α 2β 3α 4α 4β 5 6α 6β 7α 7β 8 9 10 11α 11β 12α 12β 13 14 15α 15β 16α 16β 17 18 19 20 21 22 23
δH (J in Hz) 1.23, 1.40 1.33, 1.55 3.89, 1.84 1.16, 1.68 1.77, 1.10 1.15 1.41 1.70 1.39
m
30.0
m
28.0
br s
65.1 33.4
m m
1.15 1.53 1.51 1.31
4.50, m 2.32, dt (4.0, 13.5) 1.84 1.73 1.11, s 0.88, s 4.57, d (1.3) 3.10, q (5.6) 1.19, d (5.6)
24 3-OH OCH3 COCH3 COCH3
2 δC
36.1 26.0 20.9 36.3 36.3 35.2 20.6 33.1 45.7 91.1 78.0 34.5 46.6 18.5 24.2 62.2 104.1 58.8 13.9
δH (J in Hz) 1.49 1.49 1.47 1.47 4.07, 1.93, 1.37 1.80, 1.30, 2.04, 1.71, 1.66, 2.38, 1.37
3 δH (J in Hz)
δC
30.3
1.52 1.52 1.53 1.53 4.15, br s 1.34, m 1.89, m 1.77, m 1.2, br d (7.7) 1.88 0.94, m 1.48 1.40 1.56
29.5
27.9 m m
66.9 33.3
br d (12.1) m m m m m
36.4 26.1
1.47 1.38 0.82, m 2.44, m
4
δC
23.6 36.3 44.5 35.9 20.8 36.6
1.36, 1.33, 1.42, 1.89,
m m m m
27.9 66.8 33.2 36.0 27.5 20.7 33.0 39.3 35.5 21.0 39.6
5.99, t (2.4)
53.9 153.8 117.9
3.59, s
44.4 72.5 59.9
7.29, d (2.4)
141.3
5.37, d (9.4)
75.3
1.17, s 1.08, s
174.8 18.9 23.8 192.4
3.49, d (9.4) 0.77, s 0.97, s
3.02, td (6.9, 3.1) 2.64, m
33.2 28.3
10.09, s 7.10, m 2.17, d (7.7)
44.5 16.4 23.7 135.1 191.3 151.0 13.3
1.98, s
170.0 20.8
5
δH (J in Hz) 1.30, 1.51, 1.65, 1.50 4.06, 1.98 1.32 1.77 1.21, 1.91 1.20 1.74 1.72 1.73
δC
m m m
30.9
m
67.7 34.2
28.5
37.4 27.3
m
21.6 39.3 36.2 36.4 21.6
1.51 1.32 1.57 1.32
33.7 41.3 90.2 39.6
2.70, m 1.87, m 5.48, m
79.6
2.70, s 1.01, s 0.99, s 6.34, s 6.28, dd (17.4, 10.8) 4.64, br d (10.8) 4.75, br d (17.4)
46.9 16.2 24.2 117.8 144.3 136.5 106.6
δH (J in Hz) 1.40, 1.82, 1.58, 1.69 4.13, 2.20 1.47,
δC
m m m
26.3
m
69.2 38.0
28.6
m 76.4 35.8
1.33, m 1.69 1.14 1.86 1.71 1.61
24.1 38.6 40.2 41.6 22.2
1.53 1.38 1.43 1.57
2.09, 1.90, 1.78, 2.07 2.70, 1.00, 0.97,
33.1 42.1 91.3 33.4
m m m
32.0
s s s
6.16, s 6.17, dd (17.2, 10.8) 4.65, br d (10.8) 4.87, br d (17.2)
46.9 16.0 17.5 123.2 143.4 135.3 105.8
173.7 4.18, d (3.1) 3.67, s
51.7 173.5 21.3
1.91 s
a Assignments were made by analysis of COSY, HSQC, and HMBC spectra. Overlapping signals are reported without designating multiplicity. b1H and 13C NMR data were recorded in DMSO-d6.
Table 2. Cytotoxic Activities of 1−10 on HepG2 and A549 Cancer Cells IC50 (μM)a compound doxorubicin 1 2 3 4 5 a
HepG2 0.3 ± >100 >100 26.8 ± 64.8 ± 68.7 ±
0.1
8.3 19.9 0.0
IC50 (μM)a A549
compound
0.1 ± 0.0 >100 >100 45.6 ± 2.5 >100 >100
6 7 8 9 10
HepG2 43.3 0.6 5.5 0.5 2.4
± ± ± ± ±
5.0 0.0 0.1 0.1 0.1
A549 >100 0.6 ± 6.5 ± 0.9 ± 3.0 ±
0.1 0.5 0.2 0.5
Data are presented as the mean value ± SEM of three experiments performed in four replicates
with an IC50 value of 0.5 ± 0.1 μM. The bioassay results from this effort support the earlier findings that the mechanism of cytotoxicity of the bufadienolides was related to their Na+/K+ATPase inhibition properties.15 In addition, compounds 1−10 were tested against mouse spleen cells to evaluate their toxicities. Compounds 1−4 and 6 had no cytotoxicity against
was found to display weak activity, with respective IC50 values of 26.8 ± 8.3 and 45.6 ± 2.5 μM. In contrast, 6, 8, 9, and 10 exhibited stronger cytotoxic activities against the two cancer cells (Table 2).14 Among them, compound 6 was the most potent against the A549 cell line, with an IC50 value of 0.6 ± 0.1 μM, while 9 was the most active steroid against HepG2 cells, 1845
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cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 375.2531 [M + H]+ (calcd for C23H35O4, 375.2530). (3β,5β)-Methyl (3-hydroxy-20-oxo-21-norcholan-14,16-dien-24oate) (2): colorless needles; mp 101−102 °C; [α]26 D +207 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (2.96), 309 (3.04) nm; IR (KBr) νmax 3532, 1722, 1698, 1642, 1523, 1439, 1375, 1262, 1221, 1022, 1097, 1017, 800 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 409.2348 [M + Na]+ (calcd for C24H34O4Na, 409.2349). (3β,5β,14β,16β,20Z)-16-Acetoxy-14,15-epoxy-3-hydroxy-24norcholan-20(22)-en-21-al (3): amorphous powder; [α]26 D +250 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 238 (2.62) nm; IR (KBr) νmax 3541, 2937, 1719, 1688, 1671, 1383, 1252, 1036 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 417.2639 [M + H]+ (calcd for C25H37O5, 417.2636). (3β,5β,14β,16β,20Z)-16-Acetoxy-14,21-epoxy-24-norcholan20,22-dien-3-ol (4): amorphous powder; [α]24 D −63 (0.1, MeOH); UV (MeOH) λmax (log ε) 203 (2.74), 250 (2.95) nm; IR (KBr) νmax 3418, 2938, 2350, 1730, 1663, 1251 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 423.2522 [M + Na]+ (calcd for C25H36O4Na, 423.2509). (3β,5β,14β)-14,21-Epoxy-24-norcholan-20,22-diene-3,5-diol (5): amorphous powder; [α]26 D −40 (0.1, MeOH); UV (MeOH) λmax (log ε) 203 (2.73), 253 (3.10) nm; IR (KBr) νmax 3341, 2945, 1628, 1382, 1165, 860 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 359.2581 [M + H]+ (calcd for C23H34O3, 359.2581). X-ray Analysis. The structures were solved by using direct methods (SHELXTL version 5.1) and refined by using full-matrix least-squares treatment on F2. In the structure refinements, nonhydrogen atoms were refined anisotropically. Hydrogen atoms bonded to carbons were placed at geometrically ideal positions by using the “ride on” method. Hydrogen atoms bonded to oxygen were located by employing the difference Fourier method and were included in the calculation of structure factors with isotropic temperature factors. (3β,5β,14β,15β,20R,21R,22S)-14,21:15,21:20,22-Triepoxy-24norcholan-3-ol (1): colorless plates from MeOH, C23H34O4, orthorhombic, P212121, a = 7.5100(1) Å, b = 9.9176(1) Å, c = 26.4060(3) Å, β = 97.542(2)°, V = 1966.75(4) Å3, Z = 4, dx = 1.265 Mg/m3, μ(Cu Kα) = 0.672 mm−1, F(000) = 816. Data collection was performed on a SMART CCD using graphite-monochromated radiation (λ = 1.54184 Å) under room temperature (nitrogen gas); 3139 unique reflections were collected to θmax = 62.66°, in which 2987 reflections were observed [F2 > 4σ(F2)]. The final R = 0.0333, Rw = 0.0349, and S = 1.046. (3β,5β)-Methyl (3-hydroxy-20-oxo-21-norcholan-14,16-dien-24oate) (2): colorless needles from MeOH, C24H34O4, triclinic, P1, a = 7.2695(5) Å, b = 10.9062(9) Å, c = 14.3200(12) Å, β = 79.719(2)°, V = 1101.53(15) Å3, Z = 1, dx = 1.220 Mg/m3, μ(Mo Kα) = 0.084 mm−1, F(000) = 440. Data collection was performed on a SMART CCD using graphite-monochromated radiation (λ = 0.71093 Å) under room temperature (nitrogen gas); 6364 unique reflections were collected to θmax = 26.00°, in which 5337 reflections were observed [F2 > 4σ(F2)]. The final R = 0.0346, Rw = 0.1076, and S = 1.129. Crystallographic data of 1 and 2: CCDC 848897 and 848898 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Bioassays. Inhibition of Na+/K+-ATPase Enzymatic Activity. The Na+/K+-ATPase inhibitory activities were determined by using a known colorimetric method.16,17 Na+/K+-ATPase from porcine cerebral cortex was obtained from Sigma-Aldrich. The inhibitory activities of different concentrations of compounds on 0.03−0.06 U/ ml of Na+/K+-ATPase were determined by measuring the release of inorganic phosphate from ATP over a 15 min period at 37 °C in a medium containing Na+ 125 mM, K+ 2.8 mM, Mg2+ 4.5 mM, EDTA 0.5 mM, and ATP 5 mM, buffered to pH 7.80 using Tris (24 mM), as reflected by the absorbance at 655 nm after colorization using the Taussky−Shorr reagent. GraphPad Prism software (version 5) was used to fit sigmoid curves and estimate the slopes and IC50 values.
this normal cell line (IC50 > 100 μM), while 5 showed a weak effect on mouse spleen cells, with an IC50 value of 79.4 ± 3.8 μM. Compounds 7−10 showed relatively low cytotoxicities (nearly 100% survival rate at a concentration of 5 μM) on the mouse spleen cells, in contrast to their effects on the above cancer cell lines. Further investigations are being carried out to explore relationships between the chemical structures and bioactivities of bufadienolides and related steroids.
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EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were determined using an X-5 melting point apparatus and are reported without correction. Optical rotations were measured using MeOH solutions on a Jasco P-1020 polarimeter at room temperature. UV spectra were obtained using MeOH solutions on a Jasco V-550 UV/vis spectrophotometer. IR spectra were recorded on a Jasco FT/IR-480 Plus Fourier transform infrared spectrometer using KBr pellets. NMR spectra were obtained on a Bruker AV-400 or AV-600 spectrometer. ESIMS spectra were obtained on a Finnigan LCQ Advantage Max ion trap mass spectrometer. HRESIMS was measured on an Agilent 6210 ESI/TOF mass spectrometer. Precoated silica gel GF254 plates (Qingdao Marine Chemical Plant, Qingdao, P. R. China) were used for TLC analysis. Column chromatography was carried out on silica gel (200−400 mesh, Qingdao Marine Chemical Plant, Qingdao, P. R. China), reversed-phase C18 silica gel (Merk, Darmstadt, Germany), and Sephadex LH-20 (Pharmacia Biotec AB, Uppsala, Sweden). All solvents used in column chromatography and HPLC were of analytical grade (Shanghai Chemical Plant, Shanghai, P. R. China) and chromatographic grade (Fisher Scientific, NJ, USA), respectively. Animal Material. The venom of Bufo bufo gargarizans was collected from Baoying Toad Breeding Base in Jiangsu Province of China in August 2008 and authenticated by Professor Guang-Xiong Zhou (Jinan University). A specimen (No. 2008082001) was deposited in the Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, P. R. China. Collection. The toad was first washed using clean water. Then its body and head were fixed with one hand. The toad was squeezed to provoke it to secrete more venom. An aluminum clamp was subsequently used to press the parotid gland moderately and swiftly. A white liquid ran off, which was collected using a watch glass. The collected venom was filtered through nylon sieves with screen mesh 80 and subsequently was spread on a glass plate (30 cm × 15 cm × 3 mm). The venom was finally dried in the shade to form a dark brown plate. About 2500 toads were used to afford about 1.5 kg of dried venom. The protocol was approved by the Animal Care and Use Committee of the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, China. Extraction and Isolation. The dried and roughly powdered venom (1.5 kg) was extracted by using 95% EtOH (12 L) under ultrasonic conditions (40 min, 40 °C). The crude extract was concentrated under reduced pressure to provide a residue (900 g), which was subsequently partitioned between CH2Cl2 and H2O. The concentrated CH2Cl2 layer (321 g) was subjected to silica gel (200− 300 mesh) using a mixture of cyclohexane−acetone (5:1, 3:1, and 1:1) as eluents to give 15 fractions (Fr. 1 to Fr. 15). Fr. 3 was subjected to reversed-phase C18 silica gel eluted by a gradient of MeOH−H2O (30:70 to 90:10) to afford a mixture that was further purified by preparative HPLC using MeOH−H2O (70:30) as the eluent to yield 1 (0.7 mg), 3 (3.2 mg), 4 (4.2 mg), 5 (6.3 mg), 6 (77.2 mg), and 10 (478.0 mg). Fr. 5 was chromatographed on silica gel eluted by a gradient of cyclohexane−acetone (5:1, 4:1, 3:1, 2:1, and 1:1) to yield 7 (2.4 g) and 8 (2.1 g). Fr. 6 was purified by preparative HPLC (MeOH−H2O, 65:35) to yield 9 (94.6 mg). Similarly, Fr. 7 was purified by preparative HPLC (MeOH−H2O, 78:22) to give 2 (5.0 mg). (3β,5β,14β,15β,20R,21R,22S)-14,21:15,21:20,22-Triepoxy-24norcholan-3-ol (1): colorless plates from MeOH; mp 132−133 °C; [α]25 D +86 (c 0.1, CHCl3); IR (KBr) νmax 3449, 2937, 1263, 1090, 1035 1846
dx.doi.org/10.1021/np400174f | J. Nat. Prod. 2013, 76, 1842−1847
Journal of Natural Products
Article
Splenocyte Processing. KM mice were sacrificed, and the spleens were removed and homogenized in PBS by a homogenizer. The suspension was centrifuged, and the pellet was lysed in red blood cell lysis buffer (Tris-base 2.6 g/L and NH4Cl 7.47 g/L, pH = 7.2) for 5 min and filtered by a 300-mesh sieve.18 The mouse spleen lymphocytes were used to test the cytotoxicity of bufadienolides. The Laboratory Animal Care and Use Committee of Jinan University approved all the experimental procedures. Cytotoxicity Assay. HepG2 and A549 cancer cell lines were purchased from the American Type Culture Collection (ATCC). The cytotoxicities of 1−10 were measured by using the previously described MTT assay,19 with doxorubicin as a positive control.
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(10) Li, W. X.; Sun, H.; Li, Q.; Zhang, X. Q.; Ye, W. C.; Yao, X. S.; Li, W. X.; Sun, H.; Li, Q.; Zhang, X. Q.; Ye, W. C.; Yao, X. S. Zhongcaoyao 2007, 38, 183−185. (11) Cao, X. T.; Wang, D.; Wang, N.; Dai, Y. H.; Cui, Z. Shengyang Yaoke Daxue Xuebao 2009, 26, 778−281. (12) Jiang, R. W.; Ma, S. C.; But, P. P. H.; Mak, T. C. W. J. Chem. Soc., Perkin Trans. 1 2001, 22, 2920−2923. (13) Niu, C. S.; Ho, D. M.; Zask, A.; Ayral-Kaloustian, S. Bioorg. Med. Chem. Lett. 2010, 20, 1535−1538. (14) Zhang, D. M.; Liu, J. S.; Tang, M. K.; Yiu, A.; Cao, H. H.; Jiang, L.; Chan, J. Y. W.; Tian, H. Y.; Fung, K. P.; Ye, W. C. Eur. J. Pharmacol. 2012, 692, 19−28. (15) Ioannis, P.; Eleftherios, P. D. Nat. Rev. Drug Discovery 2008, 7, 926−935. (16) Taussky, H. H.; Shorr, E. J. Biol. Chem. 1953, 202, 675−685. (17) Motomi, M.; Tsutomu, M.; Toshifumi, A.; Shino, M.; Masanori, Y.; Jennifer, F. M.; Vincent, P. B. J. Nat. Prod. 1998, 61, 1476−1481. (18) Allen, R. G.; Lafuse, W. P.; Galley, J. D.; Ali, M. M.; Ahmer, B. M. M.; Bailey, M. T. Brain Behav. Immun. 2012, 26, 371−382. (19) Zhang, D. M.; Wang, Y.; Tang, M. K.; Chan, Y. W.; Lam, H. M.; Ye, W. C. Biochem. Biophys. Res. Commun. 2007, 362, 759−765.
ASSOCIATED CONTENT
S Supporting Information *
The structures of the known compounds 6−10, HRESIMS, IR, UV, 1D and 2D NMR spectra of 1−5, crystal data of 1 and 2, and detection of compound 2 by LC-MS are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86 20 8522 0936. Fax: +86 20 8522 1559. E-mail:
[email protected] (W.-C. Ye);
[email protected] (R.-W. Jiang). Author Contributions ⊥
H.-Y. Tian and S.-L. Luo contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported financially by the Program for Changjiang Scholars and Innovative Research Team in University (IRT0965), the Joint Fund of NSFC-Guangdong Province (No. U0932004), National Natural Science Foundation of China (Nos. 90913020 and 21311095), and China Postdoctoral Science Foundation (No. 20110490915). The authors thank Prof. Xue-Long Sun for advice on the manuscript.
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REFERENCES
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dx.doi.org/10.1021/np400174f | J. Nat. Prod. 2013, 76, 1842−1847
