Discovery And SARs Of 5-chloro-N~4-phenyl-N~2-(pyridin-2-yl) Pyrimidine-2,4-diamine Derivatives As Oral Available And Dual CDK 6 And 9 Inhibitors With Potent Anti-tumor Activity

The cyclin-dependent kinases?CDKs?are promising therapeutic targets for cancer therapy.Palbociclib,Ribociclib and Abemaciclib,as selective CDK4/6 inhibitor,have been approved for treatment of breast cancer.However,frequent adverse reactions and drug resistance significantly limited the clinical prognoses.Studies have revealed that the resistance to CDK4/6 inhibitors was primarily attributed to the overexpression of CDK9.Therefore,development of CDK6 and 9 dual inhibitors is an effective strategy to overcome CDK4/6 inhibitors resistance in clinic.However,CDK6 and 9 dual inhibitors have not been precedented.Herein,we describe our efforts toward the discovery of a series of CDK6 and 9 dual inhibitors.In this study,we designed and synthesized a number of small-molecule compounds based on the binding pocket of the target proteins.Systematically study the structure-activity relationship of these inhibitors,verify the targeting of these compounds and clarify the mechanism of action.Comprehensively explore the experience and prospects of multi-target inhibitors in the development of anti-tumor drugs.In the beginning,we found a compound?hit 8?with dual inhibitory activities of CDK6 and CDK9 through pre-screening in our compound library.Its kinase inhibition IC₅₀ for CDK6 and 9 is 6.65 and 3.43 ?M,respectively,and it has a certain ability to inhibit tumor cell proliferation(IC₅₀ = 38.6 ? 56.2 ?M).Starting with hit 8,we analysis the interaction between the amino acid residues in the target protein and novel compounds and optimized the compounds with computer-aided drug design.The direction of optimization is guided by the kinase inhibitory activity,and the optimization effect of the compound is evaluated by the antitumor activity.CDK6/9 kinase inhibitory activity test and antitumor activity screening were performed simultaneously.With step-by-step optimization,dual CDK6/9 inhibitors were developed to meet our expectations.In order to balance the selectivity,safety,and effectiveness of the target compound during development,we added the CDK2 kinase activity test to improve selectivity and added the L02 cell proliferation test to evaluate the compound's toxicity to normal cells.Through step-by-step optimization and structure-activity relationship analysis,a 5-chloro-pyrimidine-2,4-diamine derivative,compound 66,was obtained that met our expectation.It had similar inhibitory efficacy with both target and showed potent antitumor activity.Its inhibitory effect on CDK6 and CDK9 kinases reached 40 n M,and its in vitro antitumor activity was increased to 4.01 ? 8.08 ?M,while it has little effect on other CDK family proteins.To clarify the interaction of compound 66 with the target protein,we performed targeted verification experiments.First,we constructed recombinant plasmids of the target proteins and successfully expressed and purified the active CDK6 and CDK9 recombinant proteins through the Hek293 T cells.Compound 66 was then incubated with the corresponding active recombinant protein for a period of time,and the direct interaction between the small molecule compound and the target protein was characterized by cellular thermal shift assay?CETSA?and microscale thermophoresis?MST?analysis.These studies show that compound 66 has a strong affinity for CDK6 and CDK9 and can improve their thermal stability,which proves that small molecule 66 has a direct interaction with the target proteins.In addition,we have conducted further studies on the mechanism of action of compound 66,including cell physiology,cell cycle,apoptosis,and reactive oxygen species testing,and clarified its role in regulating downstream signaling pathways.These studies show that compound 66 can significantly inhibit the protein functions of CDK6 and CDK9,and affect key cellular physiological processes such as DNA damage repair,cell cycle and apoptosis by regulating downstream signaling pathways,which ultimately kill tumor cells.In order to further study the physicochemical and druggability properties of the compound 66,we tested the aqueous solubility,lipid-water partition coefficient,hepatocyte metabolism and CYP inhibition affect.The results show that compound 66 is poor in aqueous solubility,but its hydrochloride has good solubility.In addition,the lipid-water partition coefficient?Log P?of compound 66 is 1.23.These properties indicate that compound 66 is suitable for oral administration and conforms to the Lipinski's Rule of Five.Hepatocyte metabolism and CYP inhibition determination have shown that compound 66 has acceptalbe metabolic stability and no obvious CYP inducing effect.It is worth noting that compound 66 is metabolized faster in mice than in humans.In order to test the in vivo activity of compound 66,we further studied the safety,pharmacokinetic characteristics,and in vivo efficiacy of the compound through animal models.The half-life?t1/2?,mean residence time?MRT?,and bioavailability were calculated by measuring the residual rate of compound 66 in SD rats.The LD50 of compound 66 on ICR mice was tested to evaluate its toxicity.The in vivo activity test was evaluated by a nude mouse tumor-bearing model of MDA-MB-231 breast cancer cells.Pharmacokinetic tests show that the oral half-life of Compound 66 in SD rats is 3.24 hours,and the average residence time is 5.08 hours,which is a medium level,but its bioavailability is poor,only 28.9%.The toxicity test showed the antiproliferative IC₅₀ on L02 cells is greater than 100 ?M,and the LD50 on ICR mice is greater than 500 mg/kg.These indicated the toxicity of compound 66 in vitro and in vivo is not obvious.Moreover,the tumor inhibition rate?compared with the control group?in the tumor-bearing experiment reached 79.4%.These results show that compound 66 not only inhibits the growth of a variety of tumor cells in vitro,but also has excellent antitumor activity in vivo.At the same time,it has good druggable properties and can be administered by oral administration.These results indicate that compound 66 is a very promising anti-tumor compound.In summary,starting from hit 8,a series of 5-chloro-pyrimidine-2,4-diamine derivatives were designed,synthesized and evaluated for inhibitory potency on both kinase activity and cell proliferation.With step-by-step optimization,compound 66 was discovered as the most active dual CDK6/9 inhibitor with balancing potency against two targets and selectivity over CDK2 compared with current clinical therapeutics Palbociclib and Abemaciclib.Importantly,compound 66 significantly inhibited tumor growth in xenograft mouse model with no obvious toxicity,indicating the promising therapeutic potential for cancer treatment.The above results are of great importance in the development of dual CDK6/9 inhibitors for cancer therapy.