Discovery Of Novel Small Molecules With Favorable Anti-prostate Cancer Activity And Druggability

Prostate cancer(PCa)is a malignancy seriously threatening male health,and no cure has yet been established for advanced metastatic PCa.PCa is hormone dependent,and specifically,the survival and proliferation of PCa cells is dependent on androgen receptor(AR)signaling,making blockade of AR signaling a central therapeutic strategy.Androgen deprivation therapy(ADT),blocking the endogenous synthesis of androgen by surgical or chemical means,is the standard treatment and significantly prolongs the survival of PCa patients.However,most of the patients relapse and progress into a stage termed castration-resistant PCa(CRPC).The pathology of CPRC is driven by three main forces:1)restoration of AR signaling;2)AR bypass signaling;3)AR independence.The mechanism of AR signaling recovery involes AR amplification,AR mutation,as well as adrenal androgen and intraprostatic androgen synthesis.CRPC developed by restored AR signaling,although resistant to ADT,remains AR signaling dependent.AR bypass signaling refers to replacement of AR signaling function by activated glucocorticoid receptor signaling,and complete AR independence refers to aberrant activation of PI3K-AKT signaling,MAPK signaling,WNT signaling,or DNA repair defects.Diverse targeted agents have entered clinical evaluation as CRPC therapeutics,including the 2nd generation of AR antagonists enzalutamide and apalutamide targeting AR signaling,CYP17 inhibitor abiraterone blocking adrenal androgen synthesis,AKT inhibitor ipatasertib targeting PI3K-AKT signaling,as well as PARP inhibitor targeting DNA repair defects.Here our work started from the molecules showing anti-PCa activity in the previous work of our laboratory,further evaluation of anti-PCa activity and druggability was performed for these molecules,and a total of eight series of novel molecules with diverse scaffolds was designed and synthesized based on principle of medicinal chemistry and means of computer aided drug design,of which anti-PCa acvitity and druggability was evaluated,leading to the identification of molecules with promising cellular anti-PCa acitivity and in vitro ADME property.A series of diphenyl substituted thiohydantoin derivatives was developed based on enzalutamide and apalutamide via an analogue-based drug design strategy.Several compounds in this series,including 5i,5j,5k,5s and 5t,exhibited high antiproliferative activity in PCa LNCaP cells and high inhibitory activity against prostate specific antigen(PSA).Further evaluation conducted in this thesis revealed that all five compounds had potent AR antagonistic activity,of which compounds 5j,5k and 5s showed significantly higher acitivity compared with enzalutamide.An evaluation of metabolic stability in human liver microsomes(HLMs)showed that compounds 5j,5k and 5s with terminal methyl substitution could be metabolized by HLMs especially for 5i with a half life of 4.7 min.Meanwhile,compounds 5s and 5t with diverse amide groups showed high stability(T1/2>2h).Further evaluation demeonstrated 5t with high stability in human plasma and high plasma protein binding property,as well as high permeability in a PAMPA assay.Based on this,exploration of in vivo anti-PCa pharmacological and pharmacokinetic behavior for 5t could be warranted.Isatin(indole-2,3-dione)derivatives were reported to suppress proliferation and induce apoptosis in tumor cells,and consistently,a series of 1-(3,4-dichlorobenzyl)-2,3-dione-indoline derivatives developed by our laboratory showed high antiproliferative and apoptotic activity in mantle cell lymphoma cell lines.In this thesis,a series of novel isatin derivatives containing a thiohydantoin moiety was developed via a scaffold merging stratety.Compounds D1(IC50=0.58 ?M),D2(IC50=0.35 ?M)and D5(IC50=0.96 ?M)were screened out with highly selective antiproliferative activity against LNCaP cells,along with D7 showing high antiproliferative activity in both LNCaP(IC50=1.33 ?M)and PC-3(IC50=0.70 ?M)cells.Unfortunately,compound D1 failed to show apoptotic induction in LNCaP cells.In further evaluation of druggability for compounds D1 and D5,rapid metabolization was observed for D1 in both human and mouse liver microsomes(T1/2<10 min),while D5 showed a relatively higher stability(T1/2? 30 min).In human plasma,D1 could also be metabolized with a high plasma protein binding level,while D5 showed an almost complete combination with plasma protein.In PAMPA assay,D1 and D5 showed high and poor permeability,respectively.1-Methyl-1H-pyrazole derivatives 10d,10e and 10gshowed high antiproliferative activity and moderate PSA inhibitory activity in LNCaP cells.Luciferase reporter gene assay revealed 10d and 10e as potent AR antagonists comparable with enzalutamide,however,10e could be metabolized by HLMs with a half life of 32.7 min.Aiming for an elevation of metabolic stability,a series of 5-methyl-1H-pyrazole derivatives was developed via a fragment splicing strategy.Several compounds in this series showed superior antiproliferative activity in LNCaP cells compared with enzalutamide and bicalutamide,and among them,compounds A13 and A14 showed higher AR antagonistic activity than bicalutamide.Significantly improved stability was achieved for both A13 and A14(T1/2>2 h)compared with 10e in HLMs,meeting our design expectation.Compound A13 also showed high stability and high protein binding in human plasma,as well as a high permeability in PAMPA assay5-Methyl-1H-pyrazole derivative A13 demonstrated ideal in vitro ADME property but moderate antiproliferative activity in PCa cells,further structural modification of A13 was conducted,leading to three series of pyrazole based compounds.Evaluation of antiproliferative activity in LNCaP cells screened out three compounds 12F(IC50=1.55 ?M),7C(IC50=1.55 ?M)and 16C(IC50=2.92 ?M)with significantly improved antiproliferative acitivity compared with bicalutamide(IC50=12.02 ?M)and enzalutamide(IC50=16.96 ?M).Compounds 12F and 16C showed high stability in HLMs,especially for 16C with on metabolism detected in both human and mouse liver microsomes,while 7C was moderately stable.All three compounds showed high stability and protein binding in human plasma,as well as high permeability in PAMPA assayAnother structural optimizing strategy on A13 focused on modification of the pyrazole core,and three series of novel compounds based on a pyrrolopyrimidine,pyrazolopyrimide or purine core were developed.Preliminary screening was conducted via an assessment of antiproliferative activity in PCa cell lines LNCaP,PC-3 and 22Rvl,leading to the identification of compounds 8K(IC50=0.57 ?M),22H(IC50=1.15 ?M)and 17N(IC50=0.56 ?M)with selectively antiproliferaretive activity in LNCaP cells,compound 18N(IC50=1.96 ?M)with highly selective activity in PC-3 cells,as well as compound 6N showing high toxicity in both LNCaP(IC50=0.82 ?M)and PC-3(IC50=0.54 ?M)cells.Further evaluation of in vitro ADME property showed that compounds 8k,22H,6N and 18N was highly stable in HLMs(T1/2=3-7 h)while 17N could be metabolized by both human and mouse liver microsomes(T1/2 ?15 min).All five compounds showed high stability and high protein binding in human plasma.In PAMPA assay,compounds 8K and 17N showed high permeability while 22H,6N and 18N were pooly permeable.Pyrazolopyrimidine derived compounds AG16,AG18 and 10h involving a piperazine moiety were identified with potent AKT inhibitory activity and antiproliferative activity in PC-3 cells in our previous work.Further evaluation of in vitro ADME property was conducted in this thesis.Compounds AG16 and AG18 with an isopropyl amino side chain could be metabolized by HLMs(T1/2?15 min),while 10h with a piperidin-4-yl side chain showed high stability(T1/2>2 h).10h was stable in human plasma and showed moderate combination with plasma protein,however,it showed a poor permeability in the PAMPA assay.With the purpose of discovering and developing novel small molecules with favorable anti-PCa activity and druggability,works in this dissertation were conducted in two main directions.First,we performed further evaluation of anti-PCa activity and pharmacokinetic property for compounds(such as 5t,10e and 10h)identified in our laboratory's previous work,which provided a basis for future in vivo evaluation.Secondly,based on thiohydantoin derivative 5t and pyrazole derivative 10e,iterative structural optimization was conducted and resulted in the design and synthesis of a total of 133 compounds in eight series.Systemic evaluation of in vitro anti-PCa activity was performed and screened out the compounds with high cellular anti-PCa activity,of which in vitro ADME property was evaluated.Promising compounds(A13,12F,16C,8K,6N and 18N et al)were identified with high cellular anti-PCa activity and favorable in vitro ADME property,as well as unsatisfactory compounds with defects like poor metabolic stability(D1)or low permeability(22H)for which further optimization was required.These data laid foundation for a further exploration of anti-PCa functional mechanism,in vivo pharmacological and pharmacokinetic property,and finally,the development of potential anti-PCa drug candidates.