Antitumor Activity And Mechanism Of Novel Quinazoline Derivatives And Their Application In Cell Imaging

Malignant tumor is one of the major diseases that seriously threaten human health.Its treatment and cure have been the life mission for generations of scientists and clinicians.Many quinazoline derivatives as targeted antitumor drugs,including gefitinib,erlotinib and lapatinib,have been used in clinical,and showed effectively at the beginning of tumor treatment.However,the problems such as drug resistance and liver toxicity usually were induced after taking the drugs for a period of time in patients.Therefore,it is urgent to develop new antitumor drugs with high potency,low toxicity and low resistance.In this study,we learn the prevalence and the treatment of tumor,epidermal growth factor receptor?EGFR?and its related signal transduction pathways,as well as mitochondria,and their regulation in tumor development;investigate the current status and mechanism of quinazoline antitumor drugs in marketed and clinical trials;summarize the research progress on the structural modification and structure-activity relationship of quinazoline antitumor compounds,and its application in biological detection.On the basis of the above,we observed the antitumor activities,probable mechanism,structure-activity relationship and application in cell imaging of the compounds in our laboratory.The main research contents and results are as follows.1.The antiproliferative activities of 158 compounds?A-G class?on five human tumor cell lines in our laboratory were detected using methyl thiazolyl tetrazolium?MTT?assay.The results showed that there were significant differences in the inhibitory activities of the compounds of calss A-G.Among them,the inhibitory activities of 6-?5-substituted furan-2-yl?quinazolines?Class A?,4-?4-?E?-?propen-1-yl?phenyl-amino?quinazolines?Class B?,6,7-dimorpholinoalkoxy quinazolines?Class C?,6-fluoroquinazolines?Class D?and isatin-quinazoline hybrids?Class F?were higher,while the inhibitory activities of 1-deoxynojirimycin?1-DNJ?-quinazoline hybrids?Class E?and other novel heterocyclic compounds?Class G?were lower.In particular,4-?3-chloro-4-?3-fluorobenzyloxy?phenylamino?-6-?5-hydroxymethylfuran-2-yl?quinaz-oline?T5?of class A,4-?4-?E?-?propen-1-yl?phenylamino?-6,7-bis-?3-morpholino-propoxy?quinazoline?T25?of calss B,4-?3-chloro-4-?3-fluorobenzyloxy?phenyl-amino?-6,7-bis?3-morpholinopropoxy?quinazoline?T31?of class C and 4-(3-chloro-4-??3-fluorobenzyloxy?phenylamino?-6-fluoro-7-?piperazin-1-yl?quinazoline?T44?of class D could significantly inhibit the proliferation of tumor cells,and the inhibitory effect presented a better dose-dependent manner.The IC₅0 values of T5 against SW480,A431,A549 and NCI-H1975 were 5.58,3.64,7.35 and 3.01 ?M,respectively.The IC₅0 values of T25 against SW480,A431,A549 and NCI-H1975 were 6.08,1.35,8.83 and 5.53 ?M,respectively.The IC₅0 values of T31 against SW480,A431,A549,HCC827 and NCI-H1975 were 3.27,1.31,1.65,0.37 and 4.87?M,respectively.The IC₅0 values of T44 against SW480,A431,A549,HCC827 and NCI-H1975 were 1.75,0.98,1.55,0.71 and 2.30 ?M,respectively.The analysis of structure-activity relationship showed that 6,7-dimorpholino substitution among compounds of class B,3-chloro-4-?3-fluorobenzyloxy?substitution at 4-position of quinazoline among compounds of class C and piperazinyl or 1-?diethylaminoethyl?piperazinyl substitution at 7-position of quinazoline among compounds of class D could strengthen the antiproliferative activities of compounds;when the isatin structure units were assigned in 5-position of furan and diethylaminoethyl were used to modify N1 of isatin unit among compounds of calss F,the antiproliferative activities could be effectively enhanced;compared with other heterocyclic?pyrrole-triazine,arylamino-a-glucofuranose,matrine?derivatives tested in this study,compounds containing quinazoline core showed higher antiproliferative activities.2.The inhibitory effects of compounds with high antiproliferative activities against wild type EGFR?EGFRwt?were tested using enzyme-linked immuno sorbent assay?ELISA?,the results showed that some of the tested compounds have certain inhibitory effects against EGFRwt.Especially compound T5,T25 and T31 could significantly inhibit the activity of EGFRwt,and the IC₅0 values were 5.06,20.72 and 6.99 nM,respectively.The binding mode between compound T5,T25,T31 and EGFR were analyzed using molecular docking through Sybyl software,and the results indicated that two hydrogen bonds were formed between T5,T25 and EGFRwt,respectively;four hydrogen bonds were formed between T31 and EGFRwt,and two of them were located in Asp-Phe-Gly?DFG?motif of EGFR.After A549 cells were treated with T5,T25 and T31,respectively,the effects of these compounds on EGFR and its downstream signaling pathways were analyzed by western blot?WB?.The results showed that compound T5,T25 and T31 could block the EGF/EGFR signaling pathway,and inhibit its downstream signaling pathways?PI3K/AKT and RAS-RAF-MEK-ERK?.The inhibitory effects of T31 against mutant EGFR activities was detected by commercial methods,and the results showed that T31 could significantly inhibit EGFRT790M activity with IC₅0 value of 9.28 nM.Cell apoptosis and cell cycle analysis indicated that T31 could induce apoptosis of A549 cells and arrest cell cycle in G0/G1 phase.In summary,compound T5 and T25 might be potent EGFRwt inhibitors,and compound T31 might be a potent EGFRwt and EGFRT790M inhibitor.3.Based on the antiproliferative activities of compounds against A549 cells,the CoMFA and CoMSIA model were established using Sybyl software,and the three-dimensional quantitative structure activity relationship?3D-QSAR?of EGFRwt-targeted compounds?Class A,B and C?were analyzed.The results indicated that the activities of compounds could be enhanced by appropriate modification at 4-,6-or 7-positions of quinazoline,including increasing the bulk of substituent of phenylamino group and electropositivity of phenylamino of 4-position,increasing the H-bond donor and hydrophobic group of 4-position,electron-donating groups and increasing the bulk of substituent at 6-and 7-position,reducing the H-bond donor of substituent at 6-position,increasing hydrophobic group at 6-position,increasing H-bond receptors and hydrophilic groups at 7-position.4.The inhibitory activities of compound among class E against EGFRwt were tested using ELISA,and the results showed that among them,4-?3-chloro-4-fluorophenylamino?-6-methoxy-7-?3-??2R,3R,4R,5S?-2-hydroxymethyl-3,4,5-trihydroxy-l-piperidyl?propoxy?quinazoline?T73?could effectively suppresse the activity of EGFRwt,and the IC₅0 value was 1.79 nM.Since the activity of a-glucosidase could be inhibited by T73 with IC₅0 value of 0.39 ?M in our previous study.The above results indicated that the bioavailability of the compound could be improved through the structural modification of T73,then dual targeting inhibitors targeting EGFRwt and a-glucosidase could be expected to be developed,which will give a new hope for the treatment of diabetic patients with tumor.5.The inhibitory effects of compound T44 from class D on EGFR and EGFR signaling pathway were investigated using ELISA and WB,respectively,and the results showed there were no significant inhibitory effects of T44 on EGFR.The kinase spectrum were screened using human protein tyrosine kinase?PTK?phosphorylation antibody array assay,and the effects of T44 against seventy-one PTK were evaluated.The results indicated the targeting protein of T44 was not EGFR,but ALK,ACK1,BTK,ABL1or BMX,which need to be further studied.Cell apoptosis,cell cycle,and cell clone forming assay were demonstrated that T44 could induce the apoptosis of A549 cells,arrest the cell cycle in G0/G1 phase,and significantly inhibit the formation of A549 cell clones.The study on the optical properties of T44 showed T44 had good fluorescence performance.It turned out that T44 could be rapidly absorbed by cells,emitted blue fluorescence in cells,and located in the mitochondria through analyzing its intracellular imaging and subcellular localization using microscopic imaging.The effect of T44 on mitochondrial function was studied by using flow cytometry combined the corresponding labeling reagents and WB,the results showed that T44 could reduce the mitochondrial membrane potential and increase the level of reactive oxygen in mitochondria.In addition,T44 could reduce the expression level of caspase 9 and caspase 3,increase the expression level of cleaved caspase 9 and cleaved caspase 3.The results of zebrafish model transplanted A549 tumor cells showed that T44 could inhibit the proliferation and migration of A549 cells in zebrafish.In conclusion,T44 is a potential antitumor candidate compound which played its antitumor role and induced apoptosis mainly through regulating the function of mitochondria.6.The study on the optical properties of 4-(3-chloro-4-??3-fluoro-benzyloxy?phenylamino?-6-fluoro-7-?4-?2-methoxyethyl?piperazin-1-yl?quinazoline?T52?from class D showed that T52 had good fluorescence performance.The cytotoxicities of T52 were low,there was no obvious effect on cell proliferation resulting from MTT assay after A549,Hela,LO2,HepG2,MCF-7 and HEK293A cells were treated with T52.Besides,the results of microscopic imaging showed that T52 was capable of fluorescence imaging in cells and location in mitochondria.In short,T52 had certain application potential in cell imaging.Moreover,the crystal structure of compound T52 was analyzed by X-ray diffraction,which would contribute to further research on T52.