Design,Synthesis And Biological Evaluation Of Indolin-2-one And Quinazoline-based P21 Activated Kinase 4(PAK4) Inhibitors

p21-activated kinases(PAKs)are members of the STE20 family of serine/threonine kinases that lie downstream of Rac1 and Cdc42.The PAK family is comprised of six members and is subdivided into two groups group I(PAK1-3)and group Ⅱ(PAK4-6)based on sequence and structural homology.PAK4 is the most well characterized group Ⅱ PAK family members.PAK4 plays a very important role in cytoskeleton reconstruction,proliferation,survival,migration and invasion.PAK4 are amplified,overexpressed,or hyperactivated in several tumor subtypes.Given the important functions of PAK4 were closely association with occurrence,development of tumor.It has become a new target in cancer therapy.In the first part:Discovery of indolin-2-one derivatives as potent PAK4 inhibitors:design,synthesis and biological evaluation.The first round of structure modification:Guided by superposition of the crystal conformation of PF-3758309 at the ATP-binding site of PAK4 with the predicted binding modes of Sunitinib with the PAK4 and utilizing a pharmacophore hybridization approach,a novel series of substituted indolin-2-one derivatives were designed,synthesized and evaluated for their in vitro biological activities against PAK4.Compound 6g inhibited the activity of PAK4 with an IC50 value of 27 nM,its IC50 values against A549 cell line and MCF-7 cell line,in which PAK4 has been found to be overexpressed,were 0.83 μM和 2.11 μM respectively.Apoptosis analysis in A549 cells suggested that compound 6g delayed cell cycle progression by arresting cells in the G2/M phase of the cell cycle,retarding cell growth.Further investigation demonstrated that compound 6g strongly inhibited migration and invasion of A549 cells.Western blot analysis indicated that compound 6g potently inhibited the PAK4/LIMK1/cofilin signalling pathways.The second round of structure modification:We further embarked upon a structure guided modification of 3 position of the indolin-2-one core.Based upon the docking model of compound 6g bound to PAK4,it was reasonable to assume that the exocyclic alkene of the indolin-2-one core provided the appropriate vector to access the hydrophobic region,delimited by Leu447 and Val379.We envisioned that the introduction of phenyl to the discussed above region to form new derivatives might strengthen hydrophobic interactions with PAK4 and improve potency.The SAR of substitution around the phenyl was subsequently explored.The representative compound 8a exhibited excellent enzyme inhibition(PAK4 IC50=25 nM)and cellular potency(A549 IC50=0.58 μM,HCT116 IC50=0.095 μM).An X-ray structure of compound 8a bound to PAK4 was obtained.Crystallographic analysis confirmed predictions from molecular modelling and helped refine SAR results.In addition,Compound 8a displayed focused multi-targeted kinase inhibition.Further profiling of compound 8a revealed it showed poor metabolic stability in human and rat liver microsomes.The third round of structure modification:To address the poor metabolic stability of compound 8a,we substituted terminal primary alcohol of(S)-phenylglycinol with N,N-dimethylamine.Unfortunately,whether PAK4 inhibitory activity or metabolic stability in rat liver microsomes,no satisfactory results were obtained.Our further investigations were still being in progress.In the second part:Quinazoline-based PAK4 inhibitors:Design,synthesis ans optimation.1)Design,synthesis and evaluation of carbamate prodrugs of CZh-226.CZh-226 is a highly selective PAK4 inhibitors.However,the oral bioavailability of CZh226 was low(1.12%).We have explored a series of carbamate prodrugs of CZh-226,which the NH of piperazine was masked to form more lipophilic species in an attempt to improve oral absorption.The potency of each prodrug in inhibiting PAK4 was determined.The chemical stability of prodrugs was examined in PBS(pH 7.4),SGF(pH 1.2),SIF(pH 6.8).The metabolic stability was studied in rat liver S9 and plasma.The prodrugs that showed a promising conversion rate to CZh-226 in rat plasma were submitted to oral administration studies in SD rats and compared to CZh-226.Prodrugs were orally administered at the equimolar doses of 10 mg kg-1 of CZh-226,and the values of AUC0-24h of CZh-226 were measured.2)Design,synthesis and biological evaluation of 4-aminoquinazoline-2-carboxamide derivatives as potent and selective PAK4 inhibitors.Guided by X-ray crystallography of CZh-226 and PAK4,a series of novel 4aminoquinazoline-2-carboxamide PAK4 inhibitors were designed and synthesized to overcome CZh-226’s low oral bioavailability based on the principles of bioisosterism and fragment substitution.We explored chemical modifications of 2,4,6-positions quinazoline core.Structure-activity relationships of these CZh-226 derivatives were discussed based on PAK4 inhibitory activities.Specifically,those that maintained the low nanomolar binding affinity for PAK4 equal to CZh-226 were further evaluated toward PAK1 inhibitory activities and pharmacokinetics properties in rat.Taken together,these results established the foundation of PAK4-oriented in vivo antitumor pharmacodynamic evaluation and provided novel insights into the development of potent,selective and orally available small-molecule PAK4 inhibitors as anti-cancer agents.