Design,Synthesis And Biological Evaluation Of Novel Cdk4/6 Inhibitors

Cancer is one of the major diseases affecting human health and has become an important public health problem in the world.Cancer almost has the highest mortality rate in both developed and developing countries.According to statistics from WHO,there were 18.1 million new cancer cases in the world in 2018,and the number of cancer deaths in 9.6 million.Among them,the cancer death rate in developing countries is higher than that in developed countries,accounting for 57.0%of the world patients,and the death toll is as high as 65.0%of the world.In China,the incidence and mortality of cancer has increased year after year,making it one of the major public health problems that seriously threaten the health of the Chinese population.According to the latest statistics from 2018 maligants tumor deaths accounted for 23.9%of all deaths.The annual medical expenses caused by malignant tumors exceeds 220 billion RMB.This paper reviews the Cdk signaling pathway and introduces the research progress of Cdk inhibitors.On the basis of summarizing the structure-activity relationship of Cdk inhibitors,combined with computer-aided drug design technology,a novel highly selective Cdk4/6 inhibitor was designed based on pyrazole quinazoline structure.The different sites of the mother nucleus were modified and improved,and a total of 75 compounds in 4 series were synthesized,which were not reported in the literature.Specifically,it includes pyrazoloquinazoline,phenyl substituted pyrazoloquinazoline and pyrazoloquinazolinecarboxamide.All compounds were confirmed by MS and NMR,and the purity of the compounds were confirmed by high performance liquid chromatography.The N,N-dimethylformamide dialkyl acetal was used as the alkyl source to achieve different nitrogen alkylation reactions of N-H compounds.The reaction has the advantages of cheap raw materials,easy operation,mild reaction conditions,broad substrate scope and no metal participation.By studying the effects of solvents,temperature,reaction time,and the amount of N,N-dimethylformamide dialkyl acetal on the reaction,the optimal reaction conditions were obtained.This paper explored the synthetic route of Palbociclib.The optimized process reduces the reaction step and increases the yield,providing a new reference for the industrial production of Palbociclib.The 75 synthesed compounds was tested for Cdk2,4,6 inhibitory activity,under 0.5?mol/L concentration.The arylamine-substituted moiety in the A-type compounds showed a higher inhibition rate;the amide compounds in the B-type compounds show a high inhibition rate,and the substitution of a small group on the nitrogen atom of the amide is better than the substitution with a large group;in the C-type compounds,the compounds that substituted at the p-position of benzene ring has a higher inhibition rate;and the compounds containing the heterocyclic ring structure in the D-type compounds exhibit greater activity and selectivity than that containing benzene ring structuure.Furthermore,the compound C-19,C-21,D-8,D-10,D-13 keep the equvalent activity with positive compound Palbociclib(Cdk4 IC₅₀=0.010?mol/L,Cdk6 IC₅₀=0.060?mol/L).Compound D-10(Cdk4 IC₅₀=0.010?mol/L,Cdk6 IC₅₀=0.060?mol/L)has the best activity and selectivity,the selectivity to Cdk4,6 is 70 times and 27 times that of Cdk2,respectively.Based on the results of the inhibitory activity of the synthesized compounds on Cdk2,4,6 we have concluded the structure-activity relationship between pyrazolo[4,3-h]quinazoline and Cdk4/6:1)The pyrimidine ring moiety in the pyrazolo[4,3-h]quinazoline forms two critical hydrogen bonds with Val101 in the hinge region.2)The 2-position of the mother nucleu is substituted with a2-aminopyridine group,which greatly improves the selectivity.3)The 4-position substitued with a methyl group has a better activity on Cdk2 no substitution is better for Cdk4/6.4)The compound that substitued at the 3-position of the mother nucleu with amide is optimal,and the attachment of a larger substituent to the amide nitrogen atom reduces the activity.The proliferation inhibition experiments of four Cdk4/6-dependent sensitive cell lines MCF-7,HCT116,PC-9 and NCI-H1975 cells were carried out with 17 compounds.The experimental results show that most of the compounds have strong inhibitory effects on the proliferation of these four tumor cells,and the best inhibitory effect on MCF-7.IC₅₀ values of multiple compounds for MCF-7is less than 200 nM.Among them,the compounds C-1,D-8,D-9,D-13(MCF-7 IC₅₀=0.51?mol/L,0.21?mol/L,0.19?mol/L,0.13?mol/L)were comparable to the activity of drug Palbociclib(MCF-7 IC₅₀=0.93?mol/L);the activities of D-8(MCF-7 IC₅₀=0.13?mol/L)and D-10(MCF-7IC₅₀=0.19?mol/L)were 7.2 times and 4.9 times more potent then that of Palbociclib,respectively.After pharmacological evaluation,some compounds were evaluated for their drug properties.First,two physical and chemical properties of the compound drug properties were determined and calculated.Compounds C-19,C-21,D-8,D-9,D-10,C-19 and D-11 keep the equvilant ClogP value with Palbociclib.The ClogP value of most compounds is less than 5,indicating that the tested compound has a better lipid-water distribution.After pharmacological evaluation and physical and chemical properties determination,the best compoumd D-10 was selected for pharmacokinetic study in rats.The results showed that the oral half-life of D-10 was as long as 1.65 h,and the maximum plasma concentration(C_max)was 30?g/mL.The concentration of D-10 in plasma reached the highest at 20 min,and the blood concentration became stable after 150 min,which had good pharmacokinetic properties.