Design,Synthesis And Anti-Colon Cancer Activity Evaluation Of Highly Potent Small Molecule Inhibitors Of APC-Asef Interaction

Cancer has become the leading killer of human health in the 21st century.This is especially true in developing countries,which not only have similar incidence rates as developed countries,but also tend to have higher mortality rates.As the largest developing country in the world,cancer is a difficult problem that Chinese medicine has to solve.In recent years,the morbidity and mortality of colon cancer in China has increased rapidly,and it has become the third largest malignant tumor.Moreover,with the acceleration of China's urbanization process and aging population distribution,the harm degree of colon cancer will further increase in the future.Therefore,the research and development of highly effective and low-toxicity targeted colon cancer specific drugs has become a hot spot of contemporary pharmaceutical research.APC(Adenomatous polyposis coli)protein,also known as Adenomatous polyp protein,plays a major role in tumor inhibition in the body.Clinical studies found that corresponds to the APC protein in patients with colon cancer genes often frameshift mutations?cur,causing the APC protein expression is not complete,only expressed as n-terminal ARM structure domain part of APC protein amino acids of the truncated,nor its physiological function,with the body of guanosine monophosphate impact factor Asef(APC-stimulated guanine nucleotide exchange factor)is selected,causing a series of physiological function lesion,promote the growth of colon cancer in the body and metastasis.Therefore,if the interaction between the pathological changes of apc-asef protein is blocked,the growth and metastasis of colon cancer in human body will be curbed,which is of great importance for the treatment of colon cancer.Blocking the interaction between truncated APC protein and Asef protein has become an important idea in the treatment of colon cancer.To block this study of interaction of APC-Asef as the research target,based on the group under the condition of existing active structural framework,using the computer aided drug design technology of APC and Asef type lesions truncated conjugate protein crystal analysis calculation,at the center of the lesion type truncation APC and Asef operation area as the center and the design can be truncated with pathological changes in the area type drugs of small molecules,effectively combine the APC with Asef competitive with pathological changes of APC protein,and make the original has been combined with pathological changes of truncation type APC and Asef swim to leave,The interaction between APC protein and Asef protein was blocked from the perspective of spatial hindrance and separation.A total of four series of novel apc-asef protein interaction small molecule inhibitors were designed and synthesized,of which 83 compounds were synthesized for the first time.The chemical structure was characterized by 1H NMR,MS and other common analytical methods.The skeleton configurations of four compounds were analyzed by X-ray single crystal diffraction technology.And screening of biological activity in vivo and in vitro experiments,respectively from the molecular level,cellular level,animals living level three aspects to evaluate the various series of compounds against colon cancer activity,some of these compounds as APC-Asef protein interaction between small molecule inhibitors showed a better inhibitory activity of colon cancer,the paper is as follows:(1)Two series of Apc-Asef protein interaction small molecule inhibitors with dihydropyrazole triazole as the basic framework were designed by CADD and synthesized.A total of 58 target compound was firstly synthesized,and two single crystal A13 and B15 furtherly determined the skeleton of the series compounds configurations.The inhibitory activity of APC-Asef protein interaction,in vitro anti-tumor cell proliferation activity,apoptosis induction and cell adhesion were studied.The results showed that the representative compound A13 of dihydropyrazole triazole derivatives containing naphthalene moieties Al-A32 had the optimal APC-Asef protein interaction inhibitory activity and HCT116 antiproliferation activity,with IC50 values of 0.83±0.03?M and 0.97±0.03,?M,respectively.B15,as the representative compound of dihydropyrazole triazole derivativeS containing benzooxy-heterocyclic skeleton B1-B26,has the optimal APC-Asef protein interaction inhibitory activity and HCT116 antiproliferation activity,with IC50 values of 0.74±0.01 ?M and 0.75±0.02?M,respectively.A13 and B15 were better than positive control drug Regorafenib(IC50,HCT116=1.44±0.13 ?M).A13 and B15 could effectively induce apoptosis of HCT116 cells and reduce their metastasis.Besides,the experimental results of subcutaneous transplantation tumor model of colon cancer in nude mice further demonstrated that compound B15,as an apc-asef protein interaction inhibitor,had a better effect on the treatment of colon cancer.(2)On the basis of the skeleton analysis of dihydropyrazole triazole derivatives,25 dihydropyrazole thiomorpholine derivatives containing naphthalene moieties C1-C25 were designed and synthesized by computer aided drug analysis.The inhibitory activity of compounds C1-C25 on the APC-Asef interaction was evaluated at the molecular level by Fluorescence Polarization and Native-PAGE experiments.The inhibitory activity of the compound C8 was optimal,with the IC50 of 0.13±0.01 ?M.There is a competitive inhibition relationship with Asef for the binding of the diseased APC protein.The anti-colon cancer activity of this series of compounds was evaluated at the cellular.level by in vitro Cell Anti-proliferative Activity,Cytotoxicity,Flow Cytometry Detection,Laser Confocal,Cell Adhesion and Transwell Invasion Assay.The results showed that the series of compounds C1-C25 had a tendency to inhibit colon cancer HCT116.The representative compound C8 behaved the best inhibitory activity with an IC50 value of 0.39±0.01?M,which was superior to the positive control drug Regorafenib(IC50=1.15±0.03?M).Compound C8 could effectively induce apoptosis,metastasis and invasion of HCT116 cells.In addition,with the construction of HCT116 cells subcutaneously transplanted ectopic tumor model and orthotopic transplantation tumor model in nude mice and study of HE staining,the representative compound C8 could potently inhibited the growth of colon cancer in vivo and have good biological tolerance,from the macroscopic and microscopic perspectives.From the above,the representative compound C8 was a promising anti-colon cancer drug lead compound targeting APC-Asef interaction inhibitor.