By WT bicalutamide directly by mutating the corresponding residues. Each Ser, Thr, Tyr and OH was allowed to rotate, to optimize a hydrogen bond with the ligand are kept other parts of the protein rigid. The compounds were cozy GOLD classification. The analysis of NMR and mass spectra of the chemical structure of the compound 3 of compound 3 was verified by 1H NMR and mass spectrometric analyzes. The compound 3 Wei It solid. 1H-NMR: d 8.48, 8.08, 8.02, 7.90, 7.63 7.68, 7.58 7.62, 7.54, 7.47 7.55, 7.39, 6, 36, 6.14 6.16, 6.08, 5.00 5.06, 4.82 4.84. TOF-MS-MZ 580.13. The cell lines LNCaP Lines and laptops were purchased from American Type Culture Collection, 3rd The HEK293 cell line was kindly provided by Dr. Lin RT CEP-18770 available. LNCaP and PC 3 growth inhibition were androgen starvation for 2 days in phenol red-free RPMI 1640 containing 10% FBS and activated carbon were removed at a density of 7103 cells per well seeded in 6 t 96-well plates. After overnight incubation, the cells with fresh phenol red-free RPMI 1640 designated by 10% FBS and 0.1 nM DHT CS were complements erg Exposed to DMSO vehicle and test compounds in concentrations 72 h Living cells were evaluated by MTT assay. Experiments were performed in quadruplicate and repeated twice. Luciferase assays for AR transactivation assays were performed as previously described. Briefly, PC 3 cells in 24-well plates were seeded t transiently transfected with MMTV-luciferase reporter plasmid and TSF or transmission ARexpressing Renilla luciferase using zero LipofectamineTM 2000 reagent according to the manufacturer’s protocol. The pCMV and pCMV T877A AR AR H874Y plasmids were kind donations from Dr. S. Srivastava The plasmids pCMV and pCMV W741C AR weights were obtained from Dr. Liang and Dr. Nian Song OSMU Ogawa provided. Five hours after transfection, the medium against phenol red-free RPMI 1640 with carbon was erg Complements VER Deducted changed 10% FBS. After a further 16 h
the transfected cells were DMSO vehicle, 0.1 nM DHT is exposed, and designated compounds in concentrations in the presence and absence of 0.1 nM DHT for 24 h. Luciferase activity was t detected by the two dispensing system according to the manufacturer luciferase s protocol. The ratio Ratio divided by firefly luciferase luminescence inducible by the luminescence of the Renilla luciferase control was normalized to that of 0.1 nM DHT. The experiments were performed in triplicate and repeated at least twice. Luciferase assays for the activation of NF JB transfections for luciferase assay were performed in HEK293 cells. Subconfluent HEK293 cells on 24-well plates seeded t transiently treated with 50 ng of reporter pRLTK, cotransfected 100 ng of NF jB Luc reporter and 100 ng of plasmids or RelA IKKb, as indicated by the method of the Copr Zipitation of phosphate calcium. The TOTAL GE of DNA were kept constant by supplementation with empty vector. 24 h after transfection, reporter gene activity Th bydual luciferase reporter assay measured in order according to the manufacturer’s instructions. RT-PCR of LNCaP cells were was in phenol red-free RPMI 1640 ergs complements with 10% FBS for 48 h CS. Starved cells of androgens were with DMSO vehicle control, 0.1 nM DHT and bicalutamide and the compound 3-1 treated IM and 2.5 in the presence of 0.1 nM DHT for 24 h.