obtaiMurine macrophages as ls RAW264.7 cells were obtained from the American Type Culture Collection. Macrophages ASA404 derived from bone marrow were isolated from the bone marrow of the femur C57 BL6 M Nozzles branch prepared as described above. Antique Body for COX-2 and iNOS were obtained from Cayman Chemical, w During the fight against GPX 1 and GAPDH were Fitzgerald Industries and Abcam are. Purified ovine COX-1 and COX-2 recombinant human were obtained from Cayman Chemical, and were used without further purification. Validated TaqMan probes for the analysis of real-time PCR of COX-2 and TNF expression were purchased from Applied Biosystems. 2.2. Synthesis of celecoxib celecoxib was prepared using the method described above. Melting points were recorded on a Fisher melting point Johnson. Unless otherwise stated, 1H NMR spectra were recorded with a Bruker 500 MHz Ger t. Chemical shifts are reported in ppm relative to tetramethylsilane at 0 ppm au S reported. All coupling constants are given in Hertz. Signals as s, d, t, m and cited dt. Low resolution and high Elektronensto MS scans were performed on a 4000 Q Trap hybrid triple quadruple linear ion trap instrument installation proteomics Penn State Cancer Institute at Penn State Hershey College of Medicine, Hershey, Pennsylvania. HighRes Send MS were Instrumentation Center Chemistry, State University of New York at Buffalo, New York performed. TLC was performed on aluminum supports, precoated sheets of silica gel. Celecoxib 1H NMR: 2.33, 7.20, 7.21 7.23, 7.51, 7.55, 7.88. Methyl 2,4 dioxo butanoate 4 has been reported in the literature.
All materials and reagents were purchased from Sigma Aldrich Chemical Co. and used without further purification. 2.2.1. Preparation of 1 5 p tolyl 1H-pyrazol 3 carboxylic Methylester A L Solution of diketone 1 and hydrazine in 100 ml of methanol was stirred at room temperature for 15 min heated, stirred for 3 hours, then allowed to stand overnight at room temperature. Addition of dilute HCl formed from a solid white S color, filtered, washed with water and dried. The residue was recrystallized with EtOAc hexane to give 2 as a pure isomer. Mp 118 120, 1 H-NMR: 2.40, 4.00, 4.89, 7.05, 7.13, 7.19, 7.52, 7.92, MS: 371.0, 340.2 , 232.1. 2.2.2. Preparation of 4-benzenesulfonamide, under nitrogen to a cooled L Solution of two cyclical high pyrazole in dry THF, LiAlH 4 was added in small Droxinostat portions for 20 min. Subsequently End the ice bath is removed and the resulting suspension was stirred overnight at room temperature. The mixture was in zersto Enem ice containing 1N Na2SO4 L Solution for 30 minutes was followed by extraction with EtOAc left. The EtOAc extracts were dried over anhydrous MgSO 4, filtered and concentrated to give by 3. MP 96 98, 1 H NMR: 2.33, 4.52, 5.24, 6.59, 7.15, 7.22, 7.41, 7.44, 7.81, MS: 344.1, 326, a. 2.2.3. Preparation of 4-benzenesulfonamide A mixture of alcohol 3, triethylamine, p toluenesulfonyl, and anhydrous lithium chloride in 100 ml of dry THF was added under reflux for heated for 16 h. The reaction mixture was diluted with EtOAc and washed with 1 N HCl, saturated Ttigter NaHCO 3 and water, dried over MgSO4, filtered and evaporated to give the crude