Design,Synthesis,and Biological Evaluation Of Diarylpyrazole Derivatives As Novel Antitumor Agents Targeting Microtubules

Objective:Combretum-A4（CA-4）isolated from Combretum caffrum,a natural chain alkane,has attracted a lot of attention from researchers since it was considered as an anti-mitogenic drug.The aim of this paper is to design and synthesize novel CA-4 class of antitumor drugs targeting microtubules,which will provide the best possible help to address the medical community’s therapeutic strategies against tumors.Methods:Twenty target compounds were designed based on the principles of computer-aided drug design and bioelectronic equipartition;a series of target compounds were synthesized using Suzuki coupling（Suzuki）and structurally characterized using mass spectrometry（MS）,nuclear magnetic resonance hydrogen spectroscopy（¹H-NMR）and nuclear magnetic resonance carbon spectroscopy(¹³C-NMR);two human cancer cell lines（human gastric cancer SGC-7901 cell line and human cervical cancer He La cell line）were selected by MTT method to test anti-proliferative activity of the synthesized 20 target compounds in vitro,and the most active compound was screened out for 20s;the effect of compound 20s on tubulin polymerization was determined by tubulin inhibition assay;cell cycle arrest and apoptosis of He La cells induced by compound 20s were detected by flow cytometry;damage degree of tubulin skeleton on compound 20s was observed by confocal microscopy;molecular docking was conducted to investigate its effect on tubulin polymerization at molecular level.Results:（1）The results of MTT experiments,in which CA-4 was used as a control,showed that compound 20s exhibited moderate micromolar activity.Compound 20s showed the strongest activity against He La cells with an IC₅₀ value of 1.9±0.11μM.（2）In the microtubulin aggregation inhibition assay,compared with the control group,it can be seen that compound 20s had significantly inhibited the aggregation of microtubulin.（3）In the cell cycle,He La cells were treated with three indicated concentrations of compound20s at 2-fold IC₅₀,5-fold IC₅₀ and 10-fold IC₅₀,and it was observed that the percentage of cells in the G₂/M phase of the He La cell cycle increased from 18.17%to 50.63%,compared with only 5.48%in the control group;meanwhile,in the apoptosis assay,He La cells were also treated with the three indicated concentrations of compound 20s at 1-fold IC₅₀,2-fold IC₅₀ and3-fold IC₅₀,and the total number of cells with early apoptosis（Q3）and late apoptosis（Q2）was 15.13%,22.73%and 32.7%,compared with only 7.1%in the control group.（4）He La cells treated with Compound 20s exhibited changes in the shape of the nucleus under confocal microscopy,and the microtubule network became disordered,disorganized and contracted,and even multinucleated.（5）Molecular docking showed that the oxygen atom of 3,4,5-trimethoxy of compound 20s contributed to the hydrogen bonding with residueβASP251 and the nitrogen atom of indole formed a hydrogen bond with residueβASN349.Conclusion:This paper designed 20 of the compounds by using pyrazole five-membered rings to replace the unstable double bonds in the CA-4 molecule.These compounds were synthesized by Suziki reaction in two steps,and the structures of 20 target compounds were verified by MS,¹H-NMR and ¹³C-NMR;MTT experiments confirmed that compound 20s had the strongest biological activity and the most potent effect on He La cells;microtubulin polymerization inhibition assay showed that compound 20s could inhibit microtubule protein aggregation,and we speculated that the target of compound 20s might be microtubule protein;cell cycle and apoptosis assay also further verified that compound 20s induced apoptosis by blocking He La cells in G₂/M phase;immunofluorescence assay confirmed that compound 20s disrupted microtubules and inhibited microtubule assembly;molecular docking also confirmed that compound 20s might be binding to the colchicine binding site on microtubulin and thus influencing microtubulin polymerization.These preliminary results recommend that compound 20s is likely to be a microtubule destabilizer that deserves further investigation.