Design And Biological Evaluation Of N-methyl Indole Dihydropyrazole Derivatives And Near-infrared Ferrous Probe

Cancer has now become the leading cause of human death in countries around the world in the 21st century.Among many cancers,colorectal cancer ranks in the top five in terms of morbidity and mortality.At present,the main treatment methods for colon cancer mainly include surgical treatment,radiotherapy,and chemotherapy,which have great toxic and side effects.Clinically,there is an urgent need for highly effective low-toxic small molecule drugs for inhibiting the proliferation and migration of colorectal cancer cells.APC protein is a tumor suppressor protein.The mechanism of APC-Asef interaction leading to the proliferation and migration of colorectal cancer is that the pathological APC signal is activated,blocking the negative regulation of Asef,leading to human colorectal cancer proliferation migrate.Knocking out APC or Asef or through small molecule inhibitors can effectively inhibit this interaction.In this project,we chose to block the APC protein in the APC-Asef interaction as a target,draw on the similar binding mechanism of drugs,and use computer-aided simulation of molecular docking to design a new class of N-methylindole dihydropyrazole derivatives as an inhibitor of APC-Asef interaction.A total of 20 target compounds Q1-Q20 were synthesized,and the structures were confirmed and characterized by hydrogen spectroscopy,carbon spectroscopy,mass spectrometry,and elemental analysis.And evaluate the biological activity of the target compound.The anti-proliferation experiment results of MTT method show that most of the compounds can significantly inhibit the proliferation of tumor cells,and the drugs have better inhibitory effect on human colorectal cancer cells HCT116,SW480 and HT29.Compound Q19 had the best inhibitory activity,and the GI₅₀ of HCT116,SW480,and HT29 were 1.37±0.53?M,10.9±0.87?M,1.22±0.33?M,respectively.It is better than the positive control drug Regorafenib.Moreover,compound Q19 has low cytotoxicity and can significantly inhibit the APC-Asef interaction.The IC₅₀ for the inhibition of APC-Asef interaction in vitro can reach 1.02±0.10?M.Apoptosis and membrane potential experiments indicate that compound Q19 can cause concentration-dependent depolarization of HCT116 cell mitochondria and induce apoptosis of HCT116 cells in a dose-dependent manner.The existence of strong interaction forces such as hydrogen bonds and?bonds in the analysis of molecular docking model results also confirmed that Q19 can interact with APC protein.Therefore,N-methylindole dihydropyrazole derivatives can be used as potential APC-Asef interaction inhibitors,and provide important clues for the future screening of more effective APC-Asef interaction inhibitors.The ability of iron to circulate between different oxidation states and regulate various physiological processes such as oxygen transport,enzyme reactions,DNA synthesis,etc.is necessary for organisms to survive in an oxygen-rich environment.However,everything from cancer to various diseases is related to the imbalance of iron homeostasis.Therefore,it is very necessary to develop a ferrous ion detector that can be applied inside the body.This subject is based on the N-redox mechanism,using the naphthalene ring as the basic skeleton,combined with morpholine and a six-membered heterocyclic ring containing malononitrile,and designed and synthesized a"turn-on"type based on molecular activity Near-infrared fluorescent probe YTP.Through fluorescence spectroscopy,the optical properties of YTP were tested,and it was found that the addition of ferrous iron significantly changed the optical properties of the probe.After the reflection of YTP and ferrous iron,the Stokes shift was greater than 200,which has the potential for application in living imaging.In vitro activity tests show that the response time of YTP and ferrous iron is extremely short,and within 20 s,YTP has completely reacted with ferrous iron.The concentration test shows that the fluorescence intensity of YTP is linearly correlated with the concentration of ferrous iron in the concentration of 0-45?M.The calculated detection limit of YTP is 0.86?M.Through testing selectivity of YTP,anti-interference and p H stability,it was found that this probe's response to ferrous iron is specific and not affected by other metal ions,anions,amino acids and other substrates.The effect of p H on the probe is also very small,meetingthe requirements of in vivo complex environment testing.The fluorescence confocal microscope imaging experiment proved that the near-infrared probe YTP has the potential to detect ferrous iron in living cells.Compared with the reported ferrous iron detection probes,YTP has the advantages of high response sensitivity,rapid reaction time,and high stability.Because it is a near-infrared probe,it can penetrate tissue deeply and cause less damage to biological samples.It can provide a real-time and efficient detection tool for ferrous concentration detection in the environment and the body.