Design, Synthesis, And Antitumor Activity Of A Series Of Novel Compounds As Potent Antitubulin Agents

Maligant tumour is one of the diseases that seriously threaten peoples’health, being the second most in the dead patient of the world, which seriously threats the life human beings. And more, as environmental pollution and oversized life pressure are actually serious, more and more young man suffer from cancer illness, and it is urgent take efficacious measures to prevent, detect and treat tunlor. The proliferation of cancer cells is abnormally frequent and often uncontrolled. Chromosome mobilization depends on the dynamic instability of tubulin/microtubule involving in polymerization and depolymerization in mitotic chromosomes metaphase. Microtubules, important components of cytoskeleton, are found in virtually all eukaryotic cells, which can form centriole, flagllum and nerviduct with other proteins. Microtubules are involved in many cellular processes,including maintenance of cell shape, cell invasion an migration, growth and development of tumour cells, intracellular transport and cell signaling. As microtubules play the important role in the process of tumour cell proliferation, they have been the attractive target and efficacious drug targets for the development of novel anti-cancer chemotherapeutics. MTAs (Microtubul e targeting agents) are known to interact with tubulin through at least four bindin g sites:the laulimalide, taxane, vincaalkaloid, and colchicine. Similar to paclitaxel, Laulimalide can promote the tubulin-microtubule assembly, but binds to a different siteon the microtubules. Given simple structure and proper physiochemieal features of tubulin-colchicine binding site and success of the taxanes and vinca alkaloids, which have established tubulin as a valid target in cancer therapy, more and more researches that focused on developing colchicine-like compounds for cancer treatment have been reported. Our research focuses on the identification and modification of kinds of bioactive small molecules from references and means of Computer Aided Drug Design using indole as a basic skeleton. The present work report the synthesis, active screening and structure-activity relationships of five series of 109 indole derivatives on the basis of introducing pharmacophore skeleton, such as piperazine and CA-4 scaffold, all these synthesized compounds were firstly reported and characterized by elemental analysis,1H NMR, and MS spectra. The results show that some of compounds present potent inhibiting tubulin polymerization and anticancer activity in vitro and in vivo as concluded following part:(1) A series of 20 novel tubulin inhibitors containing indole skeletons has been synthesized and tested their biological activities. Compound A27 excellent the most potent antiproliferative activity (IC5O= 0.241±0.08μM against A549 cell lines) and tubulin inhibitory activity (IC50=10.23±0.9μM). Further apoptosis experiment showed that compound Al could inhibit inhibitory activity in A549 cells and induce apoptosis. Computer simulation results showed that compound A27 can bind well to tubulin-colchicine site that provide a strong theoretical support for tubulin inhibitor studies.(2) CA-4 strongly inhibits the polymerization of tubulin by binding to the colchicine-binding site. The structure of CA-4, can be divide into three pharmacophore components, a linking bridge, A-ring containing 3,4,5-trimethoxy, and B-ring.Based on the structure and activity of CA-4, three series of 83 novel Combretastatin A-4 (CA-4) Analogues were designed and synthesized, and tested their antiproliferative activities on six cancer cell lines (A549, MCF-7, Hela, HepG2, HT-29, and SSC-4). Compound D33 exhibited the most potent inhibitory activity with an IC50 value of 0.08±0.01μM for A549, compared with positive control CA-4(IC5o=0.13±0.02μM). Compound D33 can induce A549 apotosis by mitochondrial dysfunction and intracellular ROS accumulation pathway with the change of expression of apoptosis-related proteins. The primary action of compound D33 was to inhibit microtubule assembly from intracellular tubulin pool. In the cell free system, compound D33 can inllibite tulin polymerjzation. in the live cell system, compound D33 caused wild microtubule de-polymerization and whole cytoskeleton network disruption. Using computational molecular docking as well as tubulin competition binding assay we identified colchicine pocket on tubulin as the compound D33’s selective binding site. Further, compound D33 can induce G2/M-phase arrest with the down-regulated expression of Cyclin B1 and Cdc2 and the up-regulation of p21. In vivo study, compound D33 effectively inhibited A549 xenograft tumor growth without causing significant loss of body weight.(3) Structural modifications at the link-bridge based Combretastatin A-4 (CA-4) and compound B9-D33 resulted in highly potent compounds. A total of 6 novel 1-indolyl acetate-5-nitroimidazole derivatives were designed and synthesized. Among them, compound E4 was identified as a potent antiproliferative agent against a panel of human cancer cell lines with nM IC50 values ranging from 15 to 2200 nM. In contrast, its cytotoxic effects on three normal human cells(293T, Macrophage, L02) were minimal. Compound E4 can induce G2/M cell cycle arrest. Cellular studies have revealed that the induction of apoptosis by compound E4 was associated with a collapse of mitochondrial membrane potential, accumulation of reactive oxygen species, alterations in the expression of some cell cycle-related proteins (Cyclin B1, Cdc25c, Cdc2, P21) and some apoptosis-related proteins (Bax, PARP, Bcl-2, Caspase3). The docking mode showed the binding posture of colchicine and compound E4 are similar in the colchicine-binding pocket of tubulin, as confirmed by colchicine-tubulin competitive binding assay, tubulin polymerization inhibitory activity, extracellular protein expression determination assay and confocal immunofluorescence microscopy. Animal studies show that compound E4 monotherapy yielded 61.9% inhibition of tumor growth for Hela in nude mice at 15 mg/kg, which suggest that compound E4 is a promising new antimitotic agent with clinical potential.