Synthesis and biological evaluation of 1,2,3-triazole linked aminocombretastatin conjugates as mitochondrial mediated apoptosis inducers
Introduction
Tubulin, a globular protein, has emerged as one of the valuable molecular targets in anticancer drug discovery. It exists as dimeric (α and β) subunits in the microtubules, which plays a pivotal role in many cellular process such as maintenance of skeletal integrity of cell, cell signaling and segregation of chromosomes during mitosis.1, 2 Several chemical agents are known to target the microtubule dynamics by binding to different domains of the tubulin protein and prevent the polymerization or depolymerization of the microtubules resulting in mitotic spindle arrest.3 In nature, the bark of the South African tree Combretum caffrum contains cis stilbene derivatives named as combrestatins, which are well known to effectively bind at the colchicine site of the tubulin and inhibit the polymerization resulting in the arrest of cell proliferation.4 Combretastatin A-4 (CA-4, 1) is the lead compound of this class with high cytotoxic potency against murine lymphocytic leukemia, human ovarian and colon cancer cell lines. However it suffers from limitation of poor aqueous solubility and to overcome this, prodrug CA-4P (2) and other synthetic analogues have been developed, which are undergoing clinical trials.5, 6, 7, 8, 9 The structure activity relationship studies of CA-4 suggested the importance of trimethoxy substitution on the ring A and the crucial role played by cis-configuration of the olefic bond to impart activity to this pharmacophoric structure has been well established. The ring B has been reported to be tolerant to structural modification to enhance the pharmacological profile of the molecule. Particularly, the aminocombretastatins such as AVE-8063 (3), AVE-8062 (4) with an amino and amino-serine groups replacing the hydroxyl group at the C-3 position of ring B showed potent antitubulin activity and cytotoxicity.
In recent years 1,2,3-triazoles have emerged as attractive and desirable scaffolds in the development of potential drug molecules in medicinal chemistry. Molecules with triazole nucleus are known to display broad spectrum of biological properties such as antifungal, anticancer,10, 11, 12, 13 anti-allergic, antibacterial, anti-HIV, anticonvulsant, anti-inflammatory and antitubercular activities. Interestingly, triazole hybrids of some biologically important natural products14, 15, 16, 17, 18 have been developed and recently N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide (5) have been evaluated as antimicrotubule agents against selective cancer cell lines.19
We have been involved in the design and synthesis of new heterocyclic based compounds as potential anticancer agents that could target the tubulin polymerization process.20 In the present work, we have explored the structural modification of the ring B of CA-4 by incorporating the triazole moiety on different positions via an amide linkage. These conjugates are evaluated for their cytotoxic activity and studied for the mechanistic aspects which include the inhibition of tubulin polymerization and apoptosis induction.
Section snippets
Chemistry
The synthesis of these triazole linked combretastatin conjugates (6a–e, 7a–e, and 8a–e) was accomplished as illustrated in Scheme 1, Scheme 2. The synthesis of conjugates with amino group at C-3 of ring B (6a–e and 7a–e) started with a double bond forming Wittig reaction between the nitro benzaldehydes (10, 11) and 3,4,5-trimethoxybenzyl triphenylphosphonium bromide (9) in presence of sodium hydride in anhydrous dichloromethane to provide a mixture of nitro Z-stilbenes (12,13) and E-stilbenes (
Cytotoxicity
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay24 was performed to evaluate the cytotoxic potential of these compounds against selected human cancer cell lines which include HeLa (cervical) HepG2 (liver) A549 (lung), ACHN (renal) and MCF-7 (breast). The results are summarized as IC50 values in Table 1 and are compared with CA-4. The assay revealed that these compounds showed significant cytotoxic activity with IC50 values ranging from 0.044–9.5 μM against different
Molecular modeling studies
A molecular modeling study was performed to explain the binding mode of conjugates 6d and 7c within tubulin protein domains to correlate the polymerization inhibitory activities of these compounds (Fig. 10). Coordinates of protein structure of tubulin–colchicine was obtained from the Protein Data Bank (PDB ID 3E22). Docking was accomplished into the colchicine binding sites of tubulin using AutoDock 4.2 software and the important interactions are discussed. Results showed that the triazole ring
Conclusion
In conclusion, a new series of 1,2,3-triazole linked aminocombretastatin conjugates (6a–e to 8a–e) have been synthesized and evaluated for their anticancer potential against selected human cancer cell lines. All the synthesized compounds showed potent anticancer activity against the tested cancer cell lines and two of them (6d and 7c) showed IC50 values 0.053 and 0.044 μM, respectively, against A549 cancer cell line. Flow cytometric analysis of these compounds showed the arrest of the cell cycle
Experimental protocol
All chemicals and reagents were obtained from Aldrich (Sigma–Aldrich, St. Louis, MO, USA), Lancaster (AlfaAesar, Johnson Matthey Company, Ward Hill, MA, USA) or Spectrochem Pvt. Ltd (Mumbai, India) and were used without further purification. Reactions were monitored by TLC, performed on silica gel glass plates containing 60 G F-254, and visualization was achieved by UV light or iodine indicator. Column chromatography was performed with Merck 60–120 mesh silica gel. 1H spectra were recorded on
Evaluation of in vitro anti-cancer activity
The cytotoxic activity of the compounds was determined using MTT assay. 1 × 104 cells/well were seeded in 200 μL DMEM, supplemented with 10% FBS in each well of 96-well microculture plates and incubated for 24 h at 37° C in a CO2 incubator. Compounds, diluted to the desired concentrations in culture medium, were added to the wells with respective vehicle control. After 48 h of incubation, 10 μL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg/mL) was added to each well and the
Molecular modeling studies
All the compounds under study and reference compounds 3D structures were built and optimised using Gaussian 03 W. These optimised 3D structures were utilised for Docking. All the compounds were Docked using AutoDockTools46 software package. The co crystallised structure of colchicine site tubulin downloaded from the PDB data bank (http://www.rcsb.org/pdb/index.html; PDB code: 3E22).47
Acknowledgments
B.S acknowledge CSIR-UGC, New Delhi and for the award of Senior Research Fellowship. We also acknowledge CSIR for financial support under the 12th Five Year plan project ‘Affordable Cancer Therapeutics (ACT)’ (CSC0301).
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