Design, Synthesis And Bioactive Evaluation Of Quinazoline Compounds As Antitumor Agents

Quinazoline drugs were initially used and developed in antimalarial or antibacterial field, and its various derivatives can be extracted from plants directly.In the past decade, protein kinases (PK) have become a class of important targets for cancer drug development. Due to its good physical and chemical properties, quinazoline skeleton as a superior template of kinase inhibitors has been widely used in antitumor drug design. Currently most of the marketed or clinical kinase inhibitors contain the quinazoline moiety, particularly EGFR inhibitors (Erlotinib, Lapatinib, Afatinib, Figure1). These three inhibitors perform different binding modes at the ATP pocket. Erlotinib can bind to the active conformation of EGFR, which is referred to as type I inhibitor; Lapatinib can bind to the unactive conformation of EGFR, which is referred to as type II inhibitor; Afatinib can form one covalent bond with the CYS residue in the binding site, which is referred to as type III inhibitor. The development of three inhibitors could almost reflect all the aspects of kinase inhibitors. Thus, the modification based on quinazoline skeleton has been one of the hot spots in the field of medicinal chemistry. This thesis could be divided into the two parts:the first part mainly focused on the study of quinazoline scaffold, and described the procedure about design and synthesis of96novel quinazoline derivatives (2-20～2-4、3a～3j、6a～6j、3-11～3-41、3-5a～3-5j�'�3-6a～3-6j), and finally completed the preliminary evaluation of these compounds as anticancer agent. The results showed most of the compounds performed well in the anticancer activity assay; The second part focoused on the design of dual EGFR/c-Met kinase inhibitors. Followed by structure-based drug design principles,116novel quinazoline derivatives have been designed, and subsequently14target compounds were obtained by means of combination of molecular docking and3D-QSAR model. The work of synthesis about these14compounds will be completed in our group. The detail above was summarized below.Part Ⅰ(1) Several EGFR kinase inhibitors under the clinical development had the skeleton of acrylamide at the6-position of quinazoline rings (Figure2). This moiety could form one covalent bond with the cysteine residue at the ATP active site of EGFR kinase motif. Thereby this led to the higher affinity towards the target protein. Based on the acrylamide structure,25kinds of novel quinazoline derivatives containing cinnamic amide moiety (2-20-2-44) were designed, synthesized and evaluated as EGFR inhibitors in this part. The starting compound2-amino-4-nitrobenzonitrile reacted with N,N-Dimethylformamide dimethyl acetal, and then the cyclization reaction of the intermediate with the substituted aniline could generate the4-anilinoquinazoline scaffold. Based on this skeleton, the nucleophilic reaction with the corresponding cinnamoyl chloride could produce the target compounds. All the compounds were validated by’HNMR and ESI-MS. The melting point and yield of each compound was measured. All the compounds were screened by the MTT method, and subsequently several potent compounds obtained in the cancer cell experiment were evaluated by the EGFR inhibition assay. Most of compounds exhbited potential antiproliferative activity against murine melanoma cells (B16-F10) and non-small cell lung cancer cells (A549) antiproliferative activity. The SAR (Structure-activity relationship) displayed that compounds with the meta-substituent of the cinnamamide ring performed better than compounds with the ortho-and para-position of that, irrespective of the type of substituent. Kinase inhibitory assay showed compound2-43(Figure2) performed best among these compounds as well as the A431cells inhibition assay, of which the IC50values could reach up to0.12μM and0.19μM, respectively, comparable to the positive molecule Erlotinib (0.03μM and0.27μM, respectively). To further investigate the mode of action of compound2-43at the binding site of EGFR target protein, we performed molecular docking experiments. The results showed that the compound2-43can be well fitted into the ATP binding pocket of EGFR protein kinase; Compared to the original ligand Erlotinib, the two molecules nearly possessd the same action mode.(2) most of protein kinase inhibitors investigated under the clinical research were reversible ATP competitive inhibitors. Generally, the modifications around quinazoline scaffold were clustered at the4-position or the6,7-position of quinazoline rings. Based on this, four groups of quinazoline derivatives (A, B, C and D) were designed, and synthesized as reversible kinase inhibitors in this part, in which there were71new compounds in all. All the compounds were validated by1HNMR and ESI-MS. The melting point and yield of each compound was measured. Compounds in A group (3a～3j and6a～6j) primarily targeted VEGFR2protein kinase while compounds in the remaining three groups (B:3-10～3-22; C:3-5a～3-5j and3-6a～3-6j; D:3-23～3-41) mainly targeting EGFR protein kinase. Through the preliminary screening of the activity, Most of compounds displayed potential kinase inhibitory activity. Finally we obtained four kinds of lead compounds (Figure2):3j,3-19,3-5b and3-35. Compound3j had a strong inhibitory effect against VEGFR2, with an IC50value of2.72nM, and kinase selectivity assays revealed that the compound has a high specificity towards VEGFR2. The remaining three compounds containing different structural framework exhibited to different extent EGFR inhibitory activity. It is noteworthy that the compound3-35can simultaneously inhibit the kinase activity of EGFR and HER2, with the IC50values of0.12μM and0.096μM, respectively.Part ⅡOverexpression of c-Met kinase is closely related with EGFR resistance, and combination therapy of EGFR inhibitor with c-Met inhibitor could obivously improved the progression free survival (PFS) of cancer patients. Based on this survey, we gained insight into the binding site similarity between EGFR and c-Met; Decomposed the important EGFR and c-Met inhibitors collected from the protein data bank (PDB) into the2/3ring fragments; Compared their similarity between fragments for the crucial templates in the development of dual EGFR/c-Met inhibitors; Designed116novel dual EGFR/c-Met inhibitors by the fragment-based drug design method; Finally obtained the14target molecules screened by molecular docking and3D-QSAR. It’s worth noting that in the molecular docking compounds2R2-A-12and2R2-A-6performed better than compound4-1(Figure3) reported by the Vichem company. The work of synthesis will be completed in our group.