Design, Synthesis And Biological Evaluation Of 1-Methylindeno[1,2-C]Pyrazoles As Novel Tubulin Inhibitors

Cancer (malignant tumor), one of the main causes of patient death, seriously threats the life and health of human beings. The biggest difference between cancer and normal cells is that the proliferation of cancer cells is abnormally frequent and often uncontrolled, which makes tumour cell growth heavilydependent on the dynamic instability of tubulin/microtubule involving in polymerization and depolymerization. In addition, the process of the rapid cancer cell proliferation and tumor metastasis relies heavily on the supply of nutrients surrounding blood vessels, which lead to angiogenesis actively within tumors. However, endothelial cells that participate in the angiogenesis of tumors are not mature, which greatly depend on the microtubule cytoskeleton to maintain their shape. Because of their vital role in mitosis and growth of tumors, microtubules have been attractive targets for several disease conditions and especially for cancer.Tubulin inhibitors constitute one of the most effective classes of chemotherapeutics and are used extensively for the treatment of a wide variety of human cancers advanced disease. At present, there are four well known microtubule binding sites:the taxane binding site, the laulimalide binding site, the vinca domain and the colchicine binding site. the taxane site and the Laulimalide site inhibitors are microtubule stabilizers, which stabilize microtubules and disturb microtubules depolymerization, while the later two groups are tubulin-polymerization inhibitors, which inhibit microtubule polymerization.Novel small molecule tubulin inhibitors (such as CA4P, OXi4503) interacting with the colchicine site can be used as antimitotic agents and vascular disrupting agents, which demonstrated broad antitumor activity, significant antitumor activity versus multi-drug-resistant tumor phenotypes and activity for inhibiting the spread of tumor. For the above reasons, tubulin inhibitors targeting colchicine site have pulling in a considerable attention as anticancer agents.Studies based on colchicine binding site, including the pharmacophore model of inhibitors, colchicine site inhibitor binding domains and structural characteristics of inhibitors, not only provide the basis for understanding the interaction between proteins and inhibitors but alao lay a theoretical foundation.for more rational drug design based on the target structure.Based on the crystal structure of colchicine binding site and following the characteristics of the binding site and the structure model of inhibitors, we design a series of 1-methylindeno[1,2-c]pyrazoles as novel tubulin inhibitors. In combination with computer aided drug design and virtual screening, a total of 16 compounds were designed and synthesized, and their structures were confirmed by’H-NMR,13C-NMR, ESI-MS, Elemental analysis. Then, they were evaluated for the inhibitory activities against tumor cells in vitro by the MTT assay. All compounds showed potent anticancer activity against K562, PC3, HeLa, HepG2 and MCF-7 cancer cell lines. Most of them showed similar or improved activity compared to positive control ABT-751. Especially, compounds LL-01 and LL-04 exhibited a remarkable improvement of antiproliferative activity (nM ranges) relative to ABT-751. Bioassays indicated that compounds LL-01 and LL-04 not only inhibit tubulin polymerization like colchine but also arrest the cell cycle at the G2/M phase.Compounds LL-01 and LL-04 can form multiple hydrogen bondings with colchicine binding site, which may contribute to obviously improved antitumor cell proliferation activity compared to ABT-751. For example, the acetamide NH2 group at the 7-position in LL-01 acts as hydrogen bond donor and interacts with Ser178α, Thr179α of a-tubulin, which are located in the interface of α,β-tubulin, while ABT-751 does not have any interaction in this part. In addition, the amino group of m-ethoxyaniline as hydrogen bond donor interacts with Thr240β, and the ethoxy oxygen atom as hydrogen bond acceptor can combine with Tyr202β and Asn 167β, respectively.Through the above research, we have found highly potent tubulin inhibitors, which are conducting futher antitumor pharmacology studies. The study also provide the basis for further structural modification and structure-activity relationship study.