Our data showed that DZ1 can inhibit EBV LMP1 induced promoter activity of cycl

Melting points were determined on a Kofler hot stage apparatus and are uncorrected. 1H NMR spectra were recorded using a Bruker 500 MHz spectrometer, and chemical shifts were expressed as with tetramethylsilane as an internal standard. The IR spectra were obtained on a Shimadzu 470 spectropho tometer. The mass spectra were run on a Finnigan Amuvatinib 850879-09-3 TSQ 70 spectrometer at 70 eV. Elemental analyses were carried out on a CHN O rapid elemental analyzer for C, H, and N, and the results were within 0. 4% of the theoret ical values. General procedure for the peparation of 2 amino 4 aryl 4H naphthopyran 3 carbonitrile derivatives 4a n In general, diammonium hydrogen phosphate was added to a mixture of or B naphtol, substituted aryl aldehyde, and malonitrile in ethanol and water.<br><br> The reaction mixture buy AT-406 was stirred at room temperature for 4 h. After cooling, the precipitated solid was filtered, washed with cold ethanol, and crystallized from the same solvent. The synthesized compounds were characterized by IR, 1H NMR, mass spectrpscopy and elemental analysis. Physicochemical and spectral proper ties of compounds 4a n were consistent with the previ ously reported data. c Src kinase activity assay The effect of synthesized compounds on the activity of c Src kinase was determined by HTScan Src Kinase Assay Kit, catalogue number 7776 from Cell Signaling Technology, according to manufacturers proto col. Streptavidin coated plates were purchased from Pierce. In brief, the kinase reaction was started with the incubation of the 12. 5 uL of the reaction cocktail with 12.<br><br> 5 uL of prediluted compounds for 5 min at room temperature. ATP substrate cocktail was added to the mixture. The biotinylated substrate contains the residues surrounding tyrosine AG-490 133550-30-8 160 of signal transduction protein and has a sequence of EGIYDVP. The reaction mixture was incubated for 30 min at room temperature. The kinase reaction was stopped with the addition of 50 uL of 50 mM EDTA. The reaction solution was transferred into 96 well streptavidin plates, diluted with 75 uL double distilled water, and incubated at room temperature for 60 min. At the end of the incuba tion, the wells were washed three times with 200 uL of 0. 05% Tween 20 in PBS buffer. After that to each well was added 100 uL of phosphotyrosine antibody and the wells were incubated for another 60 min. After washing three times with 0.<br><br> 05% Tween 20 in PBS T, the wells were incubated with 100 uL secondary anti mouse IgG antibody, which was HRP conjugated for next 30 min at room temperature. The wells were washed five times with 0. 05% Tween 20 in PBS and then were incubated with 100 uL of 3,3,5,5 tetramethylbenzidine dihydrochloride sub strate for 5 min. The reaction was stopped by adding 100 uL well of stop solution to each well and mixed well and read the absorbance at 450 nm using a microplate reader. IC50 values of the compounds were calculated using ORIGIN 6. 0 software. IC50 is the concentration of the compound that inhibited enzyme activity by 50%. All the experiments were carried out in triplicate. Cell culture and cell proliferation assay Cell culture Breast carcinoma BT 20 cell lines were obtained from American Type Culture Collection.