Design,Synthesis And Anti-tumor Activity Of Quinazoline Compounds And Analogues Targeting Epidermal Growth Factor Receptor

Malignant tumor seriously threatens human health.Recent studies have found that abnormal activation of protein tyrosine kinases(PTK)is closely related to tumor occurrence.Protein tyrosine kinase is one of the largest protein super-families.This type of kinase functions as enzyme that catalyzes the transfer of the ?-phosphate group from adenosine triphosphate to tyrosine of substrates.Epidermal growth factor receptor(EGFR)is the typical receptor tyrosine kinase(RTK)and has been intensively studied,which contains the EGFR1(EGFR,HER1,c-erbB1),HER2(EGFR2,c-erbB2),HER3(EGFR3,c-erbB3),HER4(EGFR4,c-erbB4).EGFR gene is located on 7p11-13,whose gene is responsible for the transcription of transmembrane glycoprotein of 1186 amino acid residues.EGFR is divided into three regions,including the extracellular domain the transmembrane region and intracellular region.EGFR extracellular domain contains two ligand binding regions with high affinity to epidermal growth factor(EGF),and two rich cysteine regions.There are 12 glycosylation sites connected with N-glycosidic bond in the extracellular domain,which can influence the binding of EGFR with ligands and dimerization of EGFR kinase.The transmembrane region is an alpha helical structure with a single strand consisting of 23 amino acid residues,which fixes the receptor on the cell membrane.The intracellular region consists of 542 amino acid residues,which is the EGFR kinase domain.The intracellular region can be divided into 3 sub-regions such as the membrane-proximal region,tyrosine kinase region and a C-terminal region.The membrane-proximal region is an important region mediating intracellular dimerization of EGFR.The tyrosine kinase region can be divided into the hinge domain,solvent domain and hydrophobic domain according to the different ATP binding sites.The hinge domain refers to binding site of adenosine in ATP.The solvent domain refers to the binding site with ATP sugar base and phosphate.The hydrophobic domain is catalytic site regulating tyrosine kinase.The hydrophobic domain starts with a DFG sequence(Asp-Phe-Gly),and ends with a APE(Ala-Pro-Glu)sequence,whose middle part contains a ring-shaped structure with three tyrosine,which forms the dynamic loop.C-terminal region consists of 229 amino acids,containing at least 5 tyrosine phosphorylation sites.When the EGFR ligands such as EGF combines with EGFR,EGFR undergoes dimerization,then ATP binds to EGFR kinase binding sites,kinase conformation flips,which activiates the kinase,resulting in the phosphorylation of C-terminal tyrosine residues.Upon phosphorylation,tyrosine residues become binding site of signal transducers and adaptor proteins,which activates various signaling molecules and signaling downstream pathways,inducing cell proliferation,differentiation,migration,adhesion and anti apoptosis.Some studies have showed that EGFR kinase is over-expressed in a variety of tumors,such as breast cancer,lung cancer,liver cancer and gastric cancer.Once the ligand of EGFR binds to EGFR,monomer EGFR forms homodimer or heterodimmer.This would trigger signal transduction of EGFR,promote the ATP to bind with kinase in the cell membrane,and activate EGFR kinase.This would lead to phosphorylation of tyrosine residues in C-terminal region,and phosphorylation of tyrosine becomes binding sites of signal transducers.Some studies have showed that signal transducers include Grb-2,Nck,Crk,Shc,Src,Chk,PI3K and phospholipase C,etc.There are three main signal pathways related to EGFR kinase activation,such as PI3K/Akt pathway,Ras/Raf/MEK/ERK pathway and STAT3 pathway.In an early study,structure of EGFR has not been clearly known,the researchers try to find EGFR kinase inhibitors from natural products,and then modify these natural moleculars in order to obtain small-molecule compounds with higher biological activity,lower toxicity,higher selectivity and bioavailability.With high throughput screening technology,the researchers found out some quinazoline heterocycle compounds,such as compound PD0153035 in the small molecular compound library,which shows obviously inhibitory effect on the EGFR kinase.The discovery renders the researchers to find more quinazoline heterocyclic compounds as EGFR inhibitors.In the next few years,a series of quinazoline derivatives emerged,all of which have good inhibitory effect on EGFR kinase.Through the analysis of the structure of EGFR kinase and the crystal complex structure of quinazoline compounds binding to EGFR,researchers found that the nitrogen atom in quinazoline ring forms the hydrogen bonds with the hinge region of EGFR kinase,which is in the same way of ATP adenosine cyclic mode.This combination is weaker than the binding force with intermolecular covalent bond,which is called a reversible inhibitor.In the process of clinical treatment of cancer,researchers demonstrated that this class of compound has no effect on tumor cell with the KRAS gene mutations,no obvious effect on tumor cell without mutations,and no effect on the tumor cell whose genes mutate near to the ATP binding site of EGFR.A related explanation is as follows:the mutation of KRAS gene in tumor causes that the growth of the cancer cell is not dependent on the activation of EGFR signaling pathway.However,the gene mutation in ATP binding sites,which is near the EGFR kinase,such as L858R mutation,enhances the binding of reversible inhibitor to EGFR binding sites of ATP.This showed the limitations of this class of compounds in clinical anti-tumor.Clinical study found that the mutation of the threonine at position 790th threonine(T)mutated into methionine(M)in EGFR kinase domain causes the occurrence of acquired drug resistance of tumor cells.Researchers explained at molecular level that the higher steric hindrance of side chain of M than T prevents the combination of EGFR small molecule drugs with EGFR kinase domain.In drug design,therefore,the researchers use pyrimidine ring to replace quinazoline ring,reducing the steric hindrance of M caused by side chain effect to the drug binding.Then olefin and kinase sulfhydryl groups in the molecule of solvent region cysteine were introduced to form covalent bonds,which is difficult to dissociate.Hence researchers designed a series of compounds,such as CO-1686,AZD9291,Afatinib.Afatinib has been in the market,treated for EGFR kinase mutant(L858R/T790M)of patients with non-small cell lung cancer.This is due to the introduction of an EGFR kinase solvent region cysteine sulfhydryl groups in the compound,forming a covalent bond,which is difficult to dissociate.This class of compounds showed more strong binding force with ATP binding site than the reversible inhibitor,which is combined with EGFR kinase ATP binding site by hydrogen bonds.Therefore,this type of compounds is known as irreversible inhibitor.This class of compounds are bioisosteres of quinazoline ring by the analysis by bioisosterism principles,such asphthalazine,pyrrolo[2,3-d]pyrimidine,pyrazolo[3,4-d]pyrimidine derivatives,which has a pyrimidine fused five-member heterocyclic ring or six-member heterocyclic nucleus,and they have good inhibition of EGFR kinase activity.On the basis of the understanding,analysis of EFGR kinase structures,structural characteristics of inhibitors and the mechanisms of resistance,this paper aims to design a series of inhibitors of EGFR kinase in four aspects,and Quinazoline compounds and analogues was used as the core of the molecular backbone.(1)increasing the hydrogen bond acceptor in the quinazoline(2)increasing the hydrogen bond donor on the quinazoline(3)introduction different-size hydrophobic groups for EGFR kinase activation hydrophobic loop in the quinazoline(4)Introducing substituent groups with different sizes and polarities in quinazolines,aimed at the hydrophobic region and the solvent region of EGFR kinase.Then we design and synthesize a series of compounds,investigate the inhibitory effect on EGFR kinase by fluorescence resonance energy transferring method,and perform molecular dynamics simulation on the the complex of compound with good activity docked into the binding site of EGFR to verify the rationality of the design of compounds.This will provide a theoretical basis for further design and synthesis of small molecular inhibitors with high efficiency.Research objectives,methods and results are as follows1.Design,molecular docking and CLogD prediction of small molecular compoundsObjectiveA new series of substituted quinazolines were designed and synthesized based on the binding mode of EGFR with ATP and the mechanism of resistance to clinical drugs.MethodsQuinazoline was used as the template to explore more novel epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs).Then we consulted the mode of other receptor tyrosine kinase small molecule inhibitors,such as Crizotinib(c-Met inhibitor),and designed the novel series of substituted quinazoline compounds and analogues,the structure formula was as follows.To improve the binding affinity of compounds to kinase,substituent was introduced on 2,4,6,and/or 8 position of quinazoline.The Suflex-Dock program was used to determine whether the quinazoline compounds and analogues have the same binding site as lead compounds.MarvinSketch 6.2.0 was used for prediction of CLogD values of designed compounds.ResultsWe have designed 18 small molecular compounds,which have been proved that there are no similar compounds reported via the search of SciFinder.The molecular docking using Suflex-Dock program showed 5.47-7.98 scores and that the compounds can enter the kinase ATP binding region.Specifically,1-nitrogen atom(N)in the quinazoline ring could form hydrogen bond with the kinase hinge region.R3 substituent could be close to the solvent region of kinase.R2 substituent can also extend into the hydrophobic region of kinase.Through prediction of CLogD values in the MarvinSketch 6.2.0,designed small molecule compounds showed good CLogD value less than 5.19.2.Synthesis and wave spectrum characterization of small molecule compoundsObjectiveWe aimed to synthesize novel quinazoline compounds and analogues.MethodsSubstituted quinazolines were obtained through cyclization,chlorination,and coupling reaction by using 2-amino-5-bromo benzoic acid and amides as start materials.2-amino quinazolines were obtained through cyclization,deamidination,condensation,and coupling reaction by using 2-amino-5-bromo benzoic acid and dicyandiamide as start materials.Substituted pyrido[2,3-d]pyrimidines were obtained through bromination,cyclization,chlorination,and coupling reaction by using 2-aminonicotinic acid as start material.Results18 Quinazoline compounds and analogues which have not been reported in the literature were obtained in moderate yields.And the structures of compounds and intermediates were confirmed by 1H NMR,13C NMR and ESI-MS.3.The activity evaluation of compoundsObjectiveWe aimed to determine the inhibition activity on EGFR kinases and cancer cells of 18 target compounds.MethodsWe used the Z'-LYTE fluorescence resonance energy transferring technology in Life company to test the inhibition activity on kinase ErbB1,EGFR(L858R),EGFR(L858R,M790T)and HER2.Anti-tumor activity of compounds was test on cancer cell by MTT method using breast cancer cell line SK-BR-3,MDA-MB-231,MCF-7,BT-474 and lung cancer cell line A549.Gefitinib was used as positive drug.ResultsThe compound I-4 inhibited the activity of ErbB1with IC50 of 44.1 nM,and had no effects on EGFR(ErbB1)T790M,HER2 and HER4.The compound I-4 had inhibitory effect on SK-BR-3 breast cancer cell line with an IC50 of 64nM.4.Molecular dynamics simulationObjectiveWe aimed to clarify the mechanism of compound I-4 binding to EGFR kinase.MethodMolecular dynamics simulation and free energy calculation were used to clarify the mechanism of compound I-4 binding to EGFR kinase.ResultsFree energy analysis revealed that van der Waals interactions and the nonpolar solvation energy provided the substantial driving force for the binding process.The favorable Coulomb interactions within the protein-inhibitor complex are counteracted by the unfavorable electrostatics of desolvation upon binding.Furthermore,per-residue free energy decomposition analysis revealed that the most favorable contribution came from Leu718,Val726,Lys745,Leu788,Thr790,Leu792,Met793,Cys797,and Leu844.Particularly,the interaction energies of Met793 and Leu844 residue were-2.26899Kcal/mol and-2.32687 Kcal/mol,respectively,which was very important to the binding of receptor-ligand.Conclusion:In this paper,three types of quinazoline compounds and analogues were designed and synthesized.The compound 1-4 inhibited the activity of ErbB1,with IC50 of 44.1 nM,and had no effects on EGFR(ErbB1)T790M,HER2 and HER4.The compound 1-4 had inhibitory effect on the breast cancer cell line SK-BR-3 with an IC50 of 64nM.Free energy analysis revealed that van der Waals interactions and the nonpolar solvation energy provided the substantial driving force for the binding process of ?-4 and EGFR.Furthermore,per-residue free energy decomposition analysis revealed that the most favorable contribution came from Met793 and Leu844 residue.