Design,Synthesis And Anti-prostate Cancer Activity Of Thiohydantoin And Pyrazole Derivatives As Novel Androgen Receptor Antagonists

Research BackgroundProstate cancer (PCa) is the most common noncutaneous malignancy threating male. Although the incidence and mortality of PCa in China are much lower than that in western countries. They have been increasing rapidly in recent years, with the improving of people’s living standard and medical diagnositic method.Early stages of local PCa can be successfully treated with surgery and radiation therapy. Since Huggins and Hodges recognized that PCa depends on androgen and androgen receptor (AR) signaling pathway for growth, androgen deprivation therapy (ADT), including surgical or medical castration and administration of AR antagonists to reach maximal androgen blockade, has been considered as standard therapy for men with nonlocal or metastatic disease. ADT commonly leads to an initial response with suppression of prostate specific antigen (PSA) levels in 80-90% of patients. However, the fundamental problem is that virtually all patients show disease progression at a median of 18-24 months after the beginning of ADT, despite maintenance of castrate testosterone serum level less than 20ng/dl. This stage of PCa is referred to as castration-resistant PCa (CRPC). There is no available therapy for CRPC patients now. Multiple mechanisms have been proposed to explain CRPC and much evidence exists proved that the progression of CRPC is associated with the reactivation of AR. The recent notion that AR remains an important target even in castration-resistant seting, has led to an increasing interest in developing novel AR antagonists as therapeutic option for CRPC.Based on the crystal structure of AR ligand binding pocket (LBD) and the interaction pattern of AR antagonists with AR LBD, we employed multiple drug design and discovery stratergies of medicinal chemistry, chemicobiology and computer chemistry to design and synthesis novel AR antagonists as candidate drug for the treatment of CRPC.MethodWe designed and synthesized a series of thiohydantoin derivatives by tiny modification of the second generation AR antagonist Enzalutamide. The thiohydantoin derivatives were prepared using 2-substituted-4-nitrobenziotic acid as starting materials by condensation reation, reduction reaction, strecker reaction and thiohydantoin cyclization reation. We determined their ability of inhibiting PSA expression and AR transcriptional activity induced by DHT and their anti-proliferation activity towards prostate cancer cell lines LNCaP and PC-3 by real time PCR, luciferase reporter gene assay and MTT assay.In order to discover AR antagonists with novel scaffolds, we designed a virtual screening method based on pharmacophore screening and molecule docking screening. The 3D-pharmacophore model was generated on the basis of the structures of five known AR antagonists by the GALAHAD module of computer aided drug design software SYBLE XI.1. The UNITY module was used to generate 3D search query to perform screening on commercial compounds database. Then the screened molecules were docked to wildtype AR (3B5R) and mutant AR (1Z95) respectively using the Surflex dock module. The screened molecules were evaluated for their ability of binding to AR, inhibiting PSA expression and cell growth of LNCaP and PC-3 by fluorescence polarization assay, real time PCR and MTT assay respectively.We optimized the structure of hit compound T3 obtained from virtual screening to improve the biological activity. The linker of T3 was replaced to carbonyl or carboxamide group, different substituents on two aryl ring were also investigated to conclude structure activity relationship (SAR). This series compounds were syhthesized using substituted acetophenone as starting material by Claisen condensation reaction, parazole cyclization reation, N-alkylation reaction, hydrolisis reaction, friedel-crafts reaction and condensation reaction. The inhibitory effects on DHT induced PSA expression and the anti-proliferation activity towards LNCaP and PC-3 of the synthesized compounds were determined by real time PCR and MTT assay respectively.In this thesis,40 target compounds were synthesized and the stuctures of them were identified by 1H-nuclear magnetic resonance (’H-NMR),13C-nuclear magnetic resonance (13C-NMR) and high resolution mass spectra (HRMS). The purity of the target compounds were also determined by high performance liquid chromatography (HPLC) and the melting point by RY-1 microscopic melting point apparatus. Most of the target compounds are novel and not reported in previous literature.Results and DisccusionIn this work, a series of thiohydantoin derivatives were synthesized and evaluated for their abilities of inhibiting prostate cancer cell proliferation, AR transcriptional acitivity and AR target gene PSA expression in prostate cancer cells. Most of these compounds, like Enzalutamide, showed selective cell growth inhibition towards AR positive LNCaP cells and inhibited AR target gene PSA expression. Among these compounds, compound 7t was the most active to inhibit LNCaP cell growth (IC50= 1.78 μM), approximately 15-folds more potent than Enzalutamide (IC50= 23.58μM). Compounds 7i and 7j not only inhibit LNCaP cell growth and AR transcriptional activty, but also inhibit AR negative PC-3 cell growth, suggesting that both compounds function through both AR-dependent and/or AR-independent pathways. Compounds 7i and 7j may have advantage over Enzalutamide for CRPC treatment and worth of further study.In order to discover AR antagonists with novel scaffolds, we designed a virtual screening method based on pharmacophore screening and molecule docking screening. The parmacophore model generated in the virtual screening procedure contains six features including three hydrophobic centers and three hydrogen bond acceptors and partially match the pharmacophore features proposed in previous literature.9 Small molecules with different scaffolds were obtained througn the virtual screening procedure. The inhibitory effects on AR transcriptional activity, prostate cancer cell proliferation and the binding affinity to AR of the hit molecules were evaluated. All these molecules demonstrated ability of androgen displacement and inhibiting DHT induced PSA expression in LNCaP cells, indicating that the virtual screening method is reliable. Among these hit molecules, compound T3 with a structure of diary 1 pyrazole, exhibited moderate inhibitory activity to AR transcriptional ability, similar binding affinity to AR and comparable anti-proliferative activity to that of Enzalutamide and it could be considered as a lead compound for further optimization and development of novel AR antagonists.Through the structure optimization of lead T3, we obtained pyrazole-5-keto derivatives and pyrazole-5-carboxamide derivatives. Pyrazole-5-keto derivatives were prepared by friedel-craft reaction, this reaction restrict the diversity of substituents on benzene ring and we only obtained two pyrazole-5-keto derivatives. We investigated the effects of different substituents on benzene ring on PSA inhibitory activity, concluding the SAR of pyrazole-5-carboxamide derivatives as follows:1) compounds with CF3 or F at the Ri position are more avctive than compounds with C1、Br or CH3 at the same position; 2) there is no obvious difference between methyl and ethyl sustituent at the R2 position; 3) compounds with the singal substituents at R4 position are more potent than compounds with same singal substituents at R3 or R5 position, compounds with the singal substituents of Br, Cl, CF3, CN or nitro group at R4 position is better than that with functional group of ethynyl, F or H; 4) the introduction of halogen at R5 position can improve the activity, if the substituent at R4 position is CF3,.The most promising compounds in this series were H25 and H43, comparing with hit compound T3,1) H25 and H43 showed improved effects on inhibiting PSA expression, the inhibitory rates of H25 and H43 at the concentration of 10μM are 100.00% and 87.90% respectively, however the inhibitory rates of T3 and Enzalutamide are 60.71% and 96.79% respectively; 2) H25 and H43 were 3-4 fold more potent than lead T3 in the ability of inhibiting LNCaP growth, the GI50 of H25, H43 and hit T3 were7.73, 3.54 and 16.52μM respectively; 3) H25 and H43 show strong inhibiting effects on the cell growth of PC-3. All the results above indicated that these compounds may inhibit cell growth through both AR-dependent and/or AR-independent pathway, the detail machnism worth of further study.ConclusionBased on the crystal structure of AR ligand binding pocket (LBD) and the interaction pattern of AR antagonists with AR LBD, we designed and synthesized a series of thiohydantoin derivatives of Enzalutamide, meanwhile we discovered hit compound T3 with potent activities by pharmacophore-and docking-based virtual screening and subsequent structure optimization of T3 generated pyrazole-5-keto derivatives and pyrazole-5-carboxamide derivatives. The SAR of pyrazole-5-carboxamide derivatives were also investigated. By preliminary in vitro biological evaluation, we found several compounds worthy of further study. What mentioned above provide basis for the further design of novel AR antagonist.