Design,Synthesis And Antitumor Activity Of 1-ethyl Pyrazole Derivatives Targeting Androgen Receptor

Currently,malignancy has become the leading cause of death in China.The mortality of cancer has become NO.1 in the last 20 years and with an upward trend during the last few years.Prostate cancer is the most common cancer in males and is the second leading cause of cancer-related death.It can be predicted that prostate cancer will become the most common cancer for males in China,which will seriously influencing human health.Androgen receptor(AR)is usually over expressed in prostate cancer and has been found to play an important role to control prostate proliferation and progress.AR is a key target in the treatment of prostate cancer.Androgen receptor belongs to steroid receptor and contains 919 amino acid residues.It can be divided into three major domains,namely the N-terminal transcription domain(NTD,coded by exon 1),the DNA binding domain(DBD,coded by exon 2 and 3),and the ligand-binding domain(LBD,coded by exon 4-8).A small hinge region crosses DBD and LBD.A nuclear localization signal(NLS)spans the region between DBD and the hinge region.The AR LBD has the similar three dimensional structures with other agonist-bound steroid receptors such as estrogen receptor.The LBD of AR consists of 11α-Helices(H1,H3～H12)and two β-turns,arranging in three layers.The H3,H5,H10 and H11 form the main section of LBD.Testosterone and dihydrotestosterone(DHT)are endogenous ligands of AR.Testosterone is usually converted to DHT by 5a-reductase.Although both testosterone and DHT could bind to the AR,DHT has a higher affinity profile.AR is located in the cytoplasm without a ligand presence and binds to heat shock proteins(HSPs).Once the LBD of AR bond DHT,AR is dissociated from HSPs with a conformational change generating a hydrophobic groove of activation function-2 on the surface and thanslocates to the nucleus as a homodimer.Ligand-activated AR binds to the specific androgen response elements in the DNA and interacts with coactivators through AF 2.The interaction of coactivators with AF 2 triggers the recruitment of RNA polymerase II and other transcriptional factors to form transcription machinery for target genes(TM),which are involved in regulation of cell proliferation and survival of prostate cells.Based on the critical roles of AR in PCa,hormone therapy i.e.androgen derivation by using AR antagonists or surgical castration has been developed as a main treatment of PCa,namely androgen ablation therapy.Hormone therapy is initially effective but ultimately PCa progresses to castration-resistant prostate cancer(CRPC).The mechanism of CRPC has not been completely described,but most researchers considered that it is related to AR signaling pathway.CRPCs usually have more active AR due to overexpression of AR.AR antagonist can be divided into two types according to the feature of structures,namely the steroidal AR antagonists and nonsteroidal AR antagonists.The disadvantages of poor selectivity,low oral bioavailablility,cross-reactivity with other steroid receptors limited the clinical use of steroid AR antagonists,while nonsteroidal antagonists can overcome these problems to a degree.For example,flutamide nilutamide and bicalutamide,have been approved for the treatment of prostate cancer.But long term use of these drugs can cause drug resistance for patients which limit the use of these compounds.The second generation AR antagonists such as MDV3100(approved by FDA in 2012)were used to treat CRPC expressing excess AR.Another AR antagonist ARN509 on clinical Phase Ⅱ trials also shows inhibition in AR-overexpressing prostate cancer cells.In conclusion,discovering drugs targeting AR has been a hot point in the last several years.Discovering new skeletons of AR antagonists is significant for the treatment of CRPC.We designed pharmacophore based on virtual screen,446073 compounds were screened from ZINK database.Through docking with W741L and wild type AR,41 compounds were selected.We choose 9 compounds and detect the inhibition for LNCaP cell lines and PC-3 cell lines.Among these compounds,compound 7 possesses inhibitory activity against LNCaP cells and PC-3 cells.Comparing bicalutamide,compound 7 MDV3100,the common features are summarized.New skeletons bearing pyrazole were designed.Docking analysis was conducted for these compounds.The result displayed most compounds can form hydrogen bonds with key amino acids in AR LBD.In addition,compounds can enter into the "V" pocket of AR.The experiment of inhibitory activity for PC-3 cell lines and LNCaP cell lines were conduct and the antagonist activity was also conducted.Through the above experiment,we selected some compounds possess AR antagonist activity and inhibitory activity for prostate cancer cell lines.The scheme started with substituted acetophenone.Through nuclear addition and elimination cyclization,ethylation,hydrolysis reaction,we get series of acids.Finaly,the acid reaction with substituted phenylamide and the target compounds were obtained.We get 18 compounds.The structures of these compounds were identified through MS,HRMS and 1H-NMR.Cell inhibitory activity announced that some compounds possessed inhibitory activity against AR over-expressed LNCaP cell lines.And compound 5f and compound 5g possess antagonist activity against AR,39.54%and 33.47%respectively.The structure-activity relationship of these compounds indicated that the hydrogen bond acceptors are indispensible.For the left aromatic ring,fluorine substituted is better than methyl and trifluoromethyl.For the right aromatic ring,the meta-positon substituent is better than the para-position substituent and compounds bearing trifluromethyl and bromine on the para-position are the most potent compounds.