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Novel imidazo[4,5-b]pyridine derived tubulin polymerization inhibitors

Background Imidazo-pyridine derivatives possess great therapeutic potential and significance in the drug discovery due to the wide range of their biological features. Out of all biological activities displayed by imidazo-pyridine derivatives, one of the most important is antitumor activity while they can easily interact with essential biomolecules due to the structural similarity with purine.[1] Considerable interest in the development of novel chemotherapeutics is focused on novel heteroaromatic compounds that achieve antitumor activity by inhibition of tubulin polymerization. [2] Aims We have designed novel imidazo[4, 5-b]pyridine derived acrylonitriles, described their synthesis and structural characterization. Antiproliferative activity was tested in vitro against nine human cancer cells in order to study the influence of the substituents placed at the phenyl ring and at the N-atom of the imidazo[4, 5-b]pyridine nuclei. Both experimental and computational methods confirmed the same biological target. Combination of docking and molecular dynamic simulation further explained interaction with biological target. Methods Main precursors were prepared by the uncatalyzed microwave assisted amination starting from 2-chloro-3-nitro-pyridine followed by the reduction of nitro group to amino. 2-cyanomethlyimidazo[4, 5-b]pyridines were obtained in the cyclocondensation reaction with ethyl cyanoacetate, while targeted acrylonitriles were prepared in condensation reaction with chosen aromatic aldehydes. The structures of newly prepared compounds were confirmed by means of 1H and 13C NMR spectroscopy as well as MS spectrometry. Antiproliferative activity in vitro was evaluated on human cancer cell lines from different cancer types and most potent derivatives were tested on normal cells as well as on multidrug resistant strains. Results The successful amination was confirmed by the appearance of the signals related to the protons of methyl and isobutyl group in the aliphatic part of NMR spectra as well as the signals related to the protons of NH group. The reduction of nitro group gave another signal related to the protons of amino group in 1H NMR spectra. Due to the appearance of the signal related to methylene group in the aliphatic part of spectra as well as the disappearance of signal related to amino group, the formation of 2-cyanomethylimidazo[4, 5-b]pyridine nuclei has been confirmed. Finally, in the both spectra of targeted imidazo[4, 5-b]pyridine derived acrylonitriles, we might observe the formation of one novel singlet which corresponds to the proton of acrylonitrile group. Targeted compounds were obtained in low to moderate yields. Some of the prepared acrylonitriles were obtained as a mixture of E- and Z-isomers and could not be efficiently separated by the column chromatography. Prepared derivatives were tested for their antiproliferative activity in vitro on nine human cancer cell lines. Obtained results revealed that the most active compounds have an isobutyl chain at the N-atom of the imidazo[4, 5-b]pyridine nuclei bearing either N, N-dimethylamino or N, N-diethylamino group at the phenyl ring Most of the tested compounds lacked activity, while compounds bearing both isobutyl group and p-dialkylamino moiety showed antiproliferative activity in submicromolar range of inhibitory concentration (IC50 0.2 to 0.6 μM) and were chosen for further biological evaluation. Additional biological results suggest that imidazo[4, 5-b]pyridine derived acrylonitriles described herein are promising chemotypes with the potential to inhibit the growth of a divers panel of cancer cell types, maintaining their activity against multidrug-resistant cancer cells. Even at high concentrations, the selected acrylonitriles had little or no effect on the viability of normal cells, resulting in a promising selectivity window. The most potent derivatives dose dependently inhibits the polymerization of tubulin, a well-established target for anticancer agents, while they did not bind to the colchicine binding site. Finally, a scratch-wound assay showed that lead compound was able to inhibit cancer cell migration at subtoxic concentrations. A combination of docking and molecular dynamic simulations demonstrated that observed antitumor activities are predominantly relying on the E-isomers of studied ligands, as they are both thermodynamically more stable and show increased binding affinities over their Z-analogues. In addition, it revealed that smaller ligands bind to the allosteric site close to the taxane site, which is buried deeper within the β-subunit, where they exhibit stronger affinities, yet are only marginally getting in the way of tubulin polymerization. In contrast, the most potent ligands, contain an additional p-dialkylamino moiety, which directs their binding towards extended colchicine site βIII on the surface among interacting tubuline subunits, where they interfere with the tubulin polymerization and reveal submicromolar activities. Conclusion Within this research we have selected lead molecules as potent tubulin polymerization inhibitors. Obtained results revealed that the most active compounds have an isobutyl chain at the N-atom of the imidazo[4, 5-b]pyridine nuclei bearing either N, N-dimethylamino or N, N-diethylamino group at the phenyl ring as E-isomers. Most potent compounds showed activity on multi drug resistant strains while not affecting viability of normal cells.

imidazo[4, 5-b]pyridine, acrylonitrile, antiproliferative activity

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