Design,Synthesis And Preliminary Anti-cancer Activity Evaluation Of Novel Nitrogen-Containing Aromatic Heterocyclic Derivatives As AKT Inhibitors

The phosphatidylinositol-3 kinase(PI3K)-AKT-mTOR pathway is one of the most commonly dysregulated pathways in all of cancer,with somatic mutations and copy number alteration.This pathway plays an important role in cell signaling,cell proliferation,survival,dif-ferentiation and apoptosis.The mutation and abnormal activation of the pathway molecule are closely related to the occurrence and development of tumors and drug resistance of tumor.AKT is a key node in the PI3K/AKT/mTOR signaling pathway and abnormal activation or overexpression of AKT has been found in many tumors such as prostate cancer,lymphoma,leukemia,myeloma,breast cancer,colon cancer,pancreatic cancer,melanoma.Activated AKT regulates cell signaling through phosphorylation of a series of downstream substrates(GSK3?,Bad,Caspase 9,FOXO,ASK1)that promote cell growth,proliferation,differentiation,survival,and inhibition of apoptosis.In recent years,AKT has become one of the hot targets in anticancer research.To date,four kinds of Akt inhibitors with different mechanisms of action have been reported,including ATP-competitive inhibitors,phosphatidylinositol analog inhibitors,allosteric inhibitors and pseudosubstrate inhibitors.Among them,the most widely studied are ATP competitive inhibitors and allosteric inhibitors.At present,a number of ATP competitive AKT inhibitors(AZD5363,GDC0068,AT13148,GSK2141795,GSK2110183)and allosteric inhibitors(ARQ092,BAY1125976,MK2206)have been advanced into clinical trial study.ATP competitive AKT inhibitors mainly combine the AKT protein with the three mainly important parts,including the Hinge domain in the AKT catalytic domain(mainly containing Glu228 and Ala230 amino acid residues),hydrophobic pocket P-loop domain of AKT(mainly by Lys158,Gly162,Lys179,Leul81 and other amino acid composition),AKT Acid hole(mainly containing Glu234 and Glu278 amino acid residues).In this dissertation,we selected the AKT inhibitor E19 which was reproted previously by our group as the lead compound.Based on the binding model of AKT inhibitors with AKT kinase and lead compound E19,five series of novel nitrogen-containing aromatic heterocyclic derivatives(AD,AE,AF,AG,AH)were designed and synthesized and evaluated as AKT Inhibitors via using the principles of medicinal chemistry,computer-aided drug design and biochemistry methods.Finally,we discovered novel high selective AKT inhibitors with robust anti-activity.Firstly,based on lead compound E19,three novel series of pyrrolopyrimidine derivatives(AD,AE,AF)were designed,synthesized and evaluated as AKT1 inhibitors via replacing piperazine of compound E19 with more flexible aminopiperidine,introducing hydroxyl or amino side chain at the a-position of the carbonyl group and different halogens at the 5-position on the pyrrolopyrimidine core.The majority of compounds exhibited weak inhibitory activity against AKT1 kinase.Among all the compounds,compounds AE10,AF21,AF22,AF28,AF31 showed the most AKT1 inhibitory activity with the IC50 values of 0.60 ± 0.12 ?M?0.29 ± 0.11?M?0.69 ± 0.15 ?M?0.23 ± 0.10 ?M?0.36 ± 0.13 ?M,respectively,which were less than that of positive control GSK690693(IC50=21.3 ± 2.1 nM).The structure and activity relationship of the three series of pyrrolopyrimidine derivatives demonstrated that the more flexible aminopiperidine chain had a detrimental effect on AKT1 inhibitory activity and majority of the hydroxyl or amino side chain made little contribution on AKT1 inhibitory activity.Whereas the isopropylamine methylene and piperidine ring side chain could form hydrogen bonds with the Glu278 residues in acid hole of AKT1,which could enhance the inhibitory activity against AKT1.The introduction of chlorine or bromine substitution at the C-5 position of the pyrrolopyrimidine ring favors the activity of the compound.In addition,compounds AE10,AF21,AF22,AF28,AF31 exhibited comparable anti-proliferative activity against PC-3 cell line with positive control GSK690693.Especially,the growth inhibitory activity of AE10 exhibited about 5 times higher than that of GSK690693 against PC-3 cell line.These compounds exhibited desirable anti-proliferative effect against MCL cell lines with IC50 values at a low micromolar level,which is relatively better than that of positive control GSK690693 and Ibrutinib.Hence,compounds AE10,AF21,AF22,AF28,AF31 might serve as lead compound for further exploitation and optimization of AKT1 inhibitors.The novel series of compounds(AG)were designed,synthesized and evaluated as AKT1 inhibitors by modifying and optimizing compounds AE10,AF21,AF22 and AF31 using fragment splicing and scaffold hopping strategies.Most of these compounds showed moderate AKT1 inhibitory activity and anti-proliferative activity against tumor cells.Among them,compounds AG16 and AG18 exhibited the most AKT1 inhibitory activity with IC50 values of 7.1 ± 1.2 nM and 8.8 ± 1.3 nM,respectively,which were about 3 times higher than that of the positive control GSK690693(IC50=21.3±2.1 nM).Compound AG18 has exhibited robust inhibition activity against most of AGC kinase family kinases,at the concentration of 1 ?M.Compound AG18 is a pan-AKT inhibitor and because of the high homology of the AGC family kinases,compound AG18 lacked selectivity for RSK1,PKA,p70S6K kinases.The growth inhibitory activity of compounds AG16 and AG18 against PC-3 cell line was about 5 times higher than that of GSK690693(IC50=14.1 ± 1.6 ?M)with IC50 values of 2.9 ± 0.7 ?M and 3.0 ± 0.6 ?M,respectively,and they also exhibited robust anti-proliferative activity against multiple MCL cell lines with IC50 values at the low micromolar level(about 1?M),which was better than that of GSK690693 and Ibrutinib.Further analysis of the structure-activity relationship of the AG series of compounds showed different purine analogue rings had a great effect on AKT1 inhibitory activity.The compounds(AG9-AG18)containing pyrazolopyrimidine ring,purine ring and quinazoline ring had stronger inhibitory activity against AKT1 than those of compounds containing aminopyrimidine ring,5-methylpyrrolopyrimidine ring and thienopyrimidine ring(AG1-AG8).Wherein the compounds containing pyrazolopyrimidine ring exhibited the most AKT1 inhibitory activity.The different substituent groups at C-3 position of pyrazolopyrimidine had significant differences on activity of compounds.The compounds with chlorine or bromine at C-3 position of pyrazolopyrimidine exhibited much more potency than those of compounds with hydrogen at C-3 position(AG15-AG18 vs AG13-AG14).The different R1 group on the benzene ring also had a great effect on the AKT1 inhibitory activity.Compounds with bromine-substituted(R1)showed much stronger AKT1 inhibitory activity than those of compounds with 2,4-dichloro-substituted(R1).Preliminary research indicated that compounds AG16 and AG18 inhibited the phosphorylation of GSK3? in PC-3 cells in a dose-dependent manner and induced cell apoptosis time-dependently at low micromolar level.Moreover,compound AG18 could also shorten G2 phase,prolong S phase of PC-3 cell line,block S cycle.In human liver microsomal metabolism stability experiments,the half-life of compounds AG16 and AG18 are T1/2=15.8 min and T1/2=1 8.5 min,respectively,indicating that compounds AG16 and AG18 metabolized rapidly in human liver microsomes.The next work will focus on improving the selectivity of compounds AG16 and AG18 over AKT kinase and the stability of compounds AG16 and AG18 in liver microsomes.On the basis of the previously identified compound AF28,the novel series of compounds(AH)were designed,synthesized and evaluated as AKT1 inhibitors via fragment splicing strategy.Most of the compounds showed strong AKT1 inhibitory activity and anti-proliferative activity against PC-3 cell line.Among them,compounds AH8 and AH10 exhibited comparable inhibitory activity against AKT1 with GSK690693(IC50=21.3±2.1 nM)with IC50 values of 24.3±1.6 nM and 26.9±1.7 nM,respectively.Further analysis of the structure-activity relationship of the AH series of compounds showed that bromine or chlorine substituted pyrazolopyrimidine ring was more favorable for AKT1 inhibitory activity than purine ring and quinazoline ring and different substituents(R1)on the benzene ring had a great effect on AKT1 activity.Compounds with fluorine substitution(R1)exhibited much weaker AKT1 inhibitory activity than those of compounds with bromine or chlorine substitution(R1),probably due to the electronegativity of F was stronger than Cl or Br.Preliminary mechanism research indicated compound AH8 not only inhibited the phosphorylation of GSK3? in a dose-dependent manner but also inhibited the phosphorylation level of AKT,indicating that compound AH8 not only targeted AKT and probably also targeted on the upstream of AKT targets such as PI3K.Compound AH8 also induced apoptosis in a dose-dependent manner with apoptotic rate of 27.83%at a concentration of 10?M,which was higher than that of the positive control GSK690693(14.34%).In addition,compound AH8 showed robust anti-proliferative activity against PC-3 and multiple lymphoma cell lines at low mircomolar level(IC50=1?4?M).In human liver microsomal metabolism stability experiments,the half-life of compound AH8 is more than 145 min,indicating that compound AH8 possesses high metabolic stability in human liver microsomes.Therefore,AH8 might serve as candidate drug for further development.