b JNU-HKUST Joint Laboratory for Neuroscience & Innovative Drug Research, Ji'nan University, Guangzhou 510632, China;
c School of Traditional Chinese Medicine, Southern Medical University, Southern Medical University, Guangzhou 510515, China
Toad venom is a product of the skin secretion of Bufo bufo gargarizans Cantor or Bufo melanostrictus Schneider,which is widely used as a traditional Chinese medicine for the treatment of superficial infection,odontalgia,and heart failure . The main active constituents of the toad venom are bufadienolides,which possess A/B cis,B/C trans and C/D cis ring junctures with a δ-lactone ring at the C-17 position in the steroidal skeleton [2, 3]. The bufadienolides showed significant cardiotonic,anesthetic,and anti-cancer activities [4, 5, 6]. These compounds had been reported to show anti-proliferative activities against some cancer cells,such as HepG2,HL60,and U937 [7, 8]. In our previous research,we had reported a series of new bufadienolides,including two new ones with unprecedented skeletons . Some of them showed potent cytotoxic activities . Recently,it was proved that they may act as a novel class of activators of ClC-3 chloride channel and induce apoptosis of cancer cells . As a continued project for search of biologically active bufadienolides from the toad venom,three new C-3 dehydrated bufadienolides (Fig. 1) were isolated. Though dehydrated bufadienolides have been identified from plant sources before,and more than one hundred bufadienolides were identified from the toad venom of different species ,to our best knowledge,it was the first time to isolate the C-3 dehydrated bufadienolides from the animal sources. In addition,compounds 2 and 3 showed potent cytotoxicity with IC50 values less than 10 mmol/L against A549 and HepG2 cancer cell lines.2. Experimental
The dried and roughly powdered venom (1.5 kg) was extracted by 95% ethanol under ultrasonic condition (40 min,40 ℃). The extract was concentrated under reduced pressure to provide a residue (900 g),which was subsequently partitioned between methylene dichloride (CH2Cl2) and water. The CH2Cl2 solution was evaporated to give a residue (321 g),which was then subjected to silica gel (200-300 mesh),eluted with cyclohexane-acetone (5:1, 3:1 and 1:1) to give 15 fractions (Fr.1-15). Fr. 8 was separated by reverse-phase C18 silica gel,eluted with methanol-water gradients (20:80-90:10) to give six subfractions (Fr. 8.1-Fr. 8.6). Fr.8.5 was purified by preparative HPLC using methanol-water (70:30) as eluent to yield 1 (7.0 mg),and Fr.8.6 was purified by preparative HPLC using methanol-water (72:28) as eluent to yield 2 (3.0 mg) and 3 (2.0 mg),respectively.3. Results and discussion
Compound 1 was obtained as an amorphous powder. [a]D 26-45 (c 0.10,CH3OH). The quasi-molecular ion [M+Na]+ at m/z 421.1987 (calcd. for C24H30NaO5: 421.1985) in its HR-ESI-MS spectrum indicated that the molecular formula of 1 was C24H30NaO5. The 1H NMR spectrum of 1 exhibited signals of two angular methyls at δH 0.51 (Me-18),1.24 (Me-19),a δ-lactone ring [δHH 7.45 (d,J = 2.5 Hz, H-21),7.91 (dd,J = 9.7,2.5 Hz,H-22),6.26 (d,J = 9.7 Hz,H-23)] and two coupled olefinic methine protons δH 5.77 (br δ,J = 10.2 Hz,H- 3) and 5.43 (br d,J = 10.2 Hz,H-4). Analysis of 13C NMR and DEPT- 135 spectra revealed that 1 possessed twenty-four carbon signals, including two sp2 methine carbons at δC 128.5 and 132.5,one oxygenated methine at δC 80.7,a ketone at δC 211.4,an oxygenated quaternary carbon at δC 84.3. All the 1H NMR and 13C NMR signals of 1 were assigned by an extensive analysis of 1H-1H COSY,HSQC, and HMBC data (Table 1) and comparison of theNMRdata of 1 with those of bufarenogin  showed they were very similar,except for the appearance of a double bond and the absence of the 3a-H signal in 1. The 1H-1H COSY spectrum (Fig. 2) revealed the correlations between H-3 to H-2a (δH 2.19)/H-2β (δH 1.88),H-3 to H-4,and H-4 to H-5,which displayed the presence of a spin system from C-2 to C-5. The HMBC correlations between H-4 and C-6 (δC 28.0)/C-10 (δC 34.6) were observed in the HMBC spectrum (Fig. 2). These data indicated that the double bond should be located between C-3 and C-4. Thus,1 was elucidated as 5β,12β-12,14- dihydroxy-11-oxobufa-3,20,22-trienolide (Fig. 1).
Compound 2 was obtained as an amorphous powder. The molecular formula C24H30NaO5 was deduced by its quasi-molecular ion [M+Na]+ at m/z 421.1986 (calcd. 421.1985) in the HR-ESI-MS spectra. The NMR data (Table 1) of 2 were very similar to 1,but the chemical shifts of the protons and carbons for the double bond were different,indicating the position of double bond in 2 was different from 1. The correlations between H-1α (δH 2.63) and H-2 (δH 5.50),H-2 and H-3 (δH 5.64),H-3 and H-4β (δH 1.90) in the 1H-1H COSY spectrum,together with correlations between CH2-1 and C-3,H-4 and C-2,and H-2 and C-10 (δC 34.2) in the HMBC spectrum indicated that the position of the double bond in 2 was between C-2 and C-3. Thus,the structure of 2 was established as (5b,12b)-12,14-dihydroxy-11-oxobufa-2,20,22-trienolide (2) (Fig. 1).
Compound 3 was obtained as an amorphous powder. The molecular formula C24H30NaO5 was established by its HR-ESI-MS data (m/z 421.1987 [M+Na]+,calcd. for C24H30NaO5: 421.1985). The 1H NMR and 13C NMR data (Table 1) revealed that 3 was an isomer of 1 and 2. The NMR data for carbons and protons assigned to the double bond had similar chemical shifts to 1,indicating the position of the double bond in 3 was also between C-3 and C-4. The above result was confirmed by the correlations between H-1β(δH 1.35) and H-2β (δH 2.39),H-2α (δH 2.01) and H-3 (δH 5.77),H-3 and H-4 (δH 5.34),and H-4 and H-5 (δH 2.11) in the 1H-1H COSY spectrum,as well as the HMBC correlations between H-1α(δH 1.95) and C-3 (δC 129.2),H-6α (δH 1.89) and C-4 (δC 132.4). The major difference between 1 and 3 was found in the chemical shifts of H- 12 (δH 4.2) and C-12 (δC 84.1) (Table 1),which were more downfield than those of 1,indicating that the configuration of 12- OH in 3 might be α-oriented. Comparison of NMR data of 3 with those of the known pseudobufarenogin  confirmed that they have the same structural features of B,C,and D rings with 11- carbonyl and 12α-OH functionalities. Thus,the structure of 3 was established as (5β,12α)-12,14-dihydroxy-11-oxobufa-2,20,22-trienolide (3) (Fig. 1).
Compounds 1-3 are three 3-dehydrated bufadienolides. In our previous study,we synthesized dehydrated bufalin derivatives with hydrochloric acid. The results showed that dehydration commonly took place at 14-OH,and C-3 dehydrated bufadienolides cannot be obtained in this condition . Furthermore, hydroxyl group often dehydrates in acid or heating conditions; however,no acid or heating were used in our experimental procedures. To further confirm if compounds 1-3 were natural products,LC-MS analysis of the CH2Cl2 extract of the toad venom was performed,which showed that compounds 1-3 could be detected in the LC-MS chromatogram (see Supporting Information), which confirmed that compounds 1-3 were not artifacts.
The cytotoxic activities of 1-3 were evaluated on A549 and HepG2 cancer cells. Compound 1 with a double bond between C-3 and C-4 and a beta-hydroxyl group at C-12 showed a moderate inhibitory effect against these two cancer cells with IC50 values of 14.77 ± 1.20 and 22.51 ± 3.54 mmol/L,respectively; while compound 2 with a double bond between C-2 and C-3 showed significant inhibitory activities against these two cancer cells with IC50 values of 4.95 ± 0.08 and 8.9 ± 2.40 mmol/L,respectively,indicating that the position of the double bond is important for activities. Compound 3 possessing a double bond between C-3 and C-4 and an alpha-oriented hydroxyl group at C-12 showed similar potency as 2 but stronger than 1 on the two cancer cells with IC50 values of 3.63 ± 0.52 and 4.95 ± 0.56 mmol/L,respectively,indicating that the configuration of 12-OH might also be an important factor for the cytotoxic activities.
Our present work on venom of Bufo bufo gargarizans yielded three new C-3 dehydrated bufadienolides. To the best of our knowledge,it was the first time to isolate the C-3 dehydrated bufadienolides from the animal sources. Both compounds 2 and 3 showed potent cytotoxicities with IC50 values less than 10 mmol/L on A549 and HepG2 cancer cells.Acknowledgments
This work was supported by National Natural Science Foundation of China (No. 81102518),and Postdoctoral Granted Financial Support (No. 20110490915) and the Guangdong High Level Talent Scheme (R.W.J.).Appendix A. Supplementary data
Supplementary material related to this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2014.02.006.
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