Synthesis and biological evaluation of Combretastatin A-4 derivatives containing a 3’-O-substituted carbonic ether moiety as potential antitumor agents
© Ma et al.; licensee Chemistry Central Ltd. 2013
Received: 21 August 2013
Accepted: 25 November 2013
Published: 5 December 2013
Combretastatin A-4 (CA-4), which is an excellent antineoplastic agent, was isolated from Combretum caffrum. To date, structural modification studies of CA-4 have focused predominantly on the construction of new therapeutic agents for drug discovery. As a part of our ongoing work towards the modification of natural products, we have focused on the 3’-O-substituent groups in the B-ring of CA-4 under the hypothesis that these novel derivatives will possess good bioactivities and behave as effective antiproliferative pro-drugs.
A series of novel CA-4 derivatives, which contained a 3’-O-substituted carbonic ether moiety, were synthesized and evaluated for their antitumor activities against four tumor cell lines, including MDA-MB-231, MCF-7, K562 and A549 cells. These derivatives exhibited clear antitumor activities, and CA-4E, in particular, showed the highest bioactivity of all of the derivatives tested against all four tumor cell lines, with IC50 values in the range of 1 to 180 nM. Based on its high bioactivity, CA-4E was subsequently selected to investigate the antitumor mechanism of these synthetic compounds. The cell cycle results demonstrated that CA-4E induced time- and dose-dependent G2/M arrest in a similar manner to CA-4, although its effect was more powerful than that of CA-4, and the apoptosis data showed that CA-4E induced cellular apoptosis in a dose-dependent manner.
The newly synthesized CA-4 derivatives exhibited good antitumor activities in vitro, with CA-4E, in particular, showing the highest bioactivity of all of the compounds tested. Furthermore, CA-4E induced time- and dose-dependent G2/M arrest and cellular apoptosis in a dose-dependent manner. Taken together, these results suggest that CA-4E should be subjected to further investigation as a potential anticancer drug candidate.
Results and discussion
CA-4 is recognized as an important parent compound, in terms of its biological effects, and hundreds of structural modified CA-4 derivatives have been synthesized and reported [15, 16], where the modifications have traditionally been affected using either a Wittig reaction or a Perkin condensation reaction. Using the Wittig reaction, the CA-4 product is generally formed as a mixture with the corresponding trans isomer, which can be difficult to remove, and this can lead to complications in any follow-up work . In contrast, the Perkin condensation proceeds stereoselectively to afford CA-4 as the major product . The use of the Perkin condensation reaction therefore provides better access to the CA-4 derivatives and, in contrast to the Wittig reaction, does not require extensive purification procedures.
Biological activity evaluation
WST-1-based cell cytotoxicity assay
In vitro cytotoxicities against four different tumor cell lines
As shown in Table 1, all of the synthesized compounds exhibited good in vitro cytotoxicity against all four of the tumor cell lines tested, with IC50 values of less than 800 nM in all cases, except for compound 14. Compound 7 (CA-4E) showed the greatest cytotoxicity of all of the compounds tested against all four cell lines with IC50 values in the range of 1 to 180 nM. It is noteworthy that CA-4E showed lower cytotoxicities than those of CA-4. Compound 6 was only weakly cytotoxic compared to CA-4, except against the MCF-7 cells. Compounds 8a, 8b, 9a, 9b, 10, 11 and 12 displayed similar cytotoxicities to the parent compound CA-4 against the MDA-MB-231, MCF-7 and A549 cells. However, compounds 13 and 14, especially 14, showed much lower cytotoxicities than CA-4, and these lower cytotoxicities were attributed to the size of the 3’-O-substituted carbonic ether moiety, in that the long side chain in 14 most likely prevented the binding of the active moiety to the colchicine site of tubulin. These results therefore suggested that the modification of CA-4 with bulky substituents at the C-3’ position would lead to a reduction in the activity of the inhibitors.
Cell cycle analysis
Effect on apoptosis
FCC was performed with 200–300 mesh silica gel (10-40 μm, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China). The reactions were monitored by thin-layer chromatography using silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.), and the plates were visualization by UV light. Melting points were measured on a Gallenkamp melting point apparatus (Beijing, China). NMR spectra were recorded on a Bruker Avance 300 MHz NMR spectrometer (Bruker, Billerica, Massachusetts, USA) at 300 (1H) and 75 (13C) MHz using TMS as an internal reference standard. HRMS were conducted on an LTQ-Qrbitrap XL (Thermo-Fisher, Cambridge, Massachusetts, USA). The experimental procedures for preparation of CA-4 and the target compounds 6-14, as well as copies of 1H-NMR and 13C-NMR, can be found in Additional file 2.
Biological activity evaluation
Cell culture and antitumor activity
Four human tumor cell lines were tested in the current study, including MDA-MB-231 breast cancer, MCF-7 breast cancer, K562 leukemia, A549 lung cancer cells, which were obtained from the Immune Pharmacological Research Institute at Shandong University. All of the test compounds were dissolved in DMSO at 10 μM, and subsequently diluted to the appropriate concentration prior to the addition to the cells. All four human tumor cell lines were cultured in an RPMI 1640 medium (GIBCO) supplemented with 10% bovine fetal calf serum. The cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2 in an incubator. The cancer cells were seeded in 96-well plates and treated with different concentrations of the synthesized compounds after 6 h of incubation. Five replicate wells were used for each concentration, and the concentration of DMSO used in each case never exceeded 0.1%, so that it would not affect the growth of the cells. The treated cells were incubated for 72 h and 10 μL of the cell proliferation reagent WST-1 was then added to each well. The wells were then incubated at 37°C under 5% CO2 in a humidified incubator for 2 h. The absorbance was measured in a microplate reader at 450/630 nm. The IC50 values were then calculated according to the percentage of growth in the presence of the test compounds.
Cell cycle analysis
For the cell cycle analysis experiments, MCF-7 cells (3 × 105) were seeded in 6-well plates and treated with different concentrations of CA-4 and CA-4E (i.e., 0, 1.5, and 3 nM). The cells were then incubated for 24 and 32 h before being washed twice with ice-cold PBS, harvested, fixed with ice-cold PBS in 75% ethanol and stored at 4°C overnight. The cells were then incubated with RNase A (0.1 mg/mL) at 37°C for 45 min, and then stained with propidium iodide (0.1 mg/mL) for 30 min on ice in the absence of light. The DNA contents of 10,000 events were measured by flow cytometery and the cell cycle profiles were analyzed on the basis of the DNA content histograms .
Cell apoptosis with FITC-Annexin V/PI double staining
Trypsin without EDTA was used to digest and collect the control group and the cells treated with 1.5 and 3 nM of CA-4 and CA-4E. Flow cytometry was performed according to the manufacturer’s procedure provided with the apoptosis detection kit. The MCF-7 cells were washed twice with PBS and centrifuged at 650 × g for 5 min. A binding buffer suspension (500 μL) was added to the cells followed by 5 μL of the FITC-Annexin V mix, and the resulting mixture was held at 4°C for 20 min. Two and a half microliters of the PI mix was then added to the mixture, and the resulting cell suspension was held at 4°C in the absence of light for 15 min. Flow cytometry was performed using a BD FACS Caliber instrument (BD Biosciences, San Jose, USA).
In summary, we have successfully synthesized a novel series of CA-4 analogues bearing a 3’-O-substituted carbonic ether moiety and evaluated their antitumor activities against four tumor cell lines using a WST-1-based cytotoxicity assay. The results revealed that all of the synthesized compounds exhibited high levels of antitumor activity, with most of the compounds exhibiting similar levels of bioactivity to CA-4. Compound CA-4E, in particular, showed much higher levels of bioactivity than CA-4, with IC50 values in the range of 1 to 180 nM. For this reason, CA-4E was selected as the best representative of the synthesized compounds to investigate the antitumor mechanism of these analogues by assessing the effect of CA-4E on the cell cycle and apoptosis. The cell cycle results demonstrated that CA-4E induced time- and dose-dependent G2/M arrest in the same way as CA-4, although the effect of CA-4E was more pronounced than that of CA-4. Furthermore, the apoptosis data showed that CA-4E induced cellular apoptosis in a dose-dependent manner. Taken together, these results suggest that the anticancer activity of CA-4E is worthy of further study, with CA-4E representing a potential new anticancer drug candidate.
This work was supported by the National Natural Science Foundation of China (Grant no. 81274031). We kindly thank Prof. Jian Zhang (Immune Pharmacological Institute, School of Pharmaceutical Sciences, Shandong University) for her assistance with the biological activity research.
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