Design,Synthesis,Biological Evalution And Salt Screening Of EGFR Inhibitors

Cancer has become the first leading cause of death among people before the age of70 in most regions or countries in the world.Traditional cytotoxic drugs inhibit normal cells while killing tumor cells.With the rapid development of molecular biology and cell biology,inhibitors based on cell signal transduction pathway have gradually become a hot research area in the development of anti-tumor drugs.Epidermal growth factor receptor(EGFR)is a tyrosine protein kinase that includes an extracellular binding site,a helical transmembrane domain,and an intracellular tyrosine kinase domain.When the receptor binds to a ligand and forms a dimer with another receptor,it can activate the signaling pathway in the cell,making the normal cell growth,cell apoptosis,and cell proliferation process uncontrollable and causing tumors.EGFR is overexpressed in many human malignancies,such as lung,breast,esophageal,pancreatic,and head and neck cancers.Till now,a number of drugs targeting EGFR have been approved for the treatment of tumors,including small molecule compounds such as Gefitinib,Erlotinib,etc.,and macromolecular monoclonal antibodies such as Cetuximab,Nimotuzumab,etc.The successful launch of these drugs further proved the reliability of EGFR as a target in anticancer drug development.According to the structure-activity relationship of EGFR small molecule kinase inhibitors,in this thesis,a quinazoline ring was used as the core structure of the inhibitors.Anilines with different substituents and different heterocyclic structures on the side chain were screened.34 compounds were designed and synthesized.Structures of all the compounds were confirmed by H-NMR,C-NMR,F-NMR and high-resolution mass spectrometry.The inhibitory activity of EGFR kinase was tested for these 34 target compounds,in which 15 compounds had comparable or stronger inhibitory activity compared with Gefitinib.According to the structure classification,11 compounds were selected for pharmacokinetic evaluation experiments in rats.Among them,compounds II-P23 and II-P31 were well absorbed in rats,and their bioavailability reached 76.8% and 101.8%,respectively,which are equivalent or better compared with Gefitinib.Compound II-P23 and II-P31 which have better inhibitory activity on EGFR kinase and show higher pharmacokinetic absorption and bioavailability in rats,were selected to carry out in vivo xenograft tumor inhibition experiments in nude mice,including CDX models inoculated with tumor cells and PDX model with tissue derived from patient’s tumor.The results showed that compound II-P31 could significantly inhibit the growth of transplanted tumors in nude mice in models of esophageal cancer,pancreatic cancer,NSCLC,head and neck cancer,and epidermoid cancer.With II-P31 as a candidate compound for further development,15 acids were screened for salt-forming reactions with free base II-P31,in which 8 salts were obtained as single crystals.For the salt with a single crystal form,the salt ratio,melting point test,and the pharmacokinetic study in beagle dogs were carried out.The results indicated that the exposure of mesylate II-P31 increased by 75% compared to the free base,which greatly improved the bioavailability of the drug.With mesylate II-P31 as a candidate salt form,experiments including hygroscopicity,influencing factors and accelerated experiments were carried out.The stability of mesylate II-P31 was investigated,and results showed that the mesylate II-P31 was stable under high temperature,high humidity,light and other conditions.Therefore,mesylate II-P31 was finally chosen.The synthesis process of mesylate II-P31 was developed.A commercially available V-1 was used as the starting material,and the chlorinated intermediate V-2 was generated through the chlorination reaction.The chlorinated intermediate V-2 can be produced in an industrial scale with optimized process.Compound V-2 undergoes nucleophilic substitution with 3-chloro-4-fluoroaniline to generate intermediate V-4,which then undergoes hydrolysis reaction under alkaline conditions to generate intermediate V-5.Compound V-5 reacts with 1-bromo-3-chloropropane,and intermediate V-6 is formed by nucleophilic substitution reaction.Compound V-6 reacts with heterocyclic compound V-7·HCl,generating free base Larotinib,which was then salified with methanesulfonic acid.The final API mesylate Larotinib is obtained through recrystallization process as an off-white powder.The whole route has mild reaction conditions and simple processing.The whole process has been scaled up to a scale of100 kilograms,with a total yield of 51.7% and an average yield of 89.9% per step.All impurities in intermediates with a level of more than 0.10% was identified.The level of all individual impurities in the final API were less than 0.10%,and the purity of the final API was as high as 99.9%.All intermediates and target compounds have been characterized by H-NMR,C-NMR and high-resolution mass spectrometry.Among them,mesylate Larotinib has been characterized by single-crystal X-ray diffraction,and the structure and configuration was confirmed,consistent with the proposed structure.