Ls the first fluorescently labeled optical imaging agent validated for COX-2-targeted in vivo imaging of inflammation and cancer.EXPERIMENTAL PROCEDURES Chemistry. Standard approaches had been utilized for the synthesis of fluorescent derivatives of NSAIDs and COXIBs. A wide wide variety of carboxylic acid-containing core compounds (e.g., indomethacin, an iodoindomethacin, an indolyl carboxamide analog of indomethacin (reverse indomethacin), flurbiprofen, ketoprofen, or even a carboxylic acid derivative of celecoxib) were tethered through a series of alkyl, aryl piperazinyl, or polyethylene glycol linkers to a diverse array of bulky organic fluorophore moieties. The fluorophores incorporated dansyl, dabsyl, coumarin, fluorescein, rhodamine, alexa-fluor, nile blue, cy5, cy7, near IR, and IR dyes, as well as some lanthanide chelators (Figure 1). Synthetic procedures, as well as analytical and spectroscopic characterization of all compounds, are described in Supporting Info. Fluorometry. Steady state fluorescence excitation and emission spectra were determined for each and every compound having a Spex 1681 Fluorolog spectrofluorometer, equipped with a 450 W xenon arc lamp. The excitation and emission monochromator slit widths had been 1-2 mm. The solvent utilized was pH 7 buffer. Inhibition Assay Employing Purified COX-1 and COX-2. Inhibition of purified ovine COX-1 or mouse COX-2 by test compounds was assayed by a previously described system,27 which quantifies the conversion of [1-14C]arachidonic acid todx.doi.org/10.1021/bc300693w | Bioconjugate Chem. 2013, 24, 712-Bioconjugate Chemistry [1-14C]prostaglandin solutions. Reaction mixtures of 200 L consisted of hematin-reconstituted protein in one hundred mM TrisHCl, pH 8.0, 500 M phenol, and [1-14C]arachidonic acid (50 M, 55-57 mCi/mmol, Perkin-Elmer). For the timedependent inhibition assay, hematin-reconstituted COX-1 (44 nM) or COX-2 (66 nM) was preincubated at 25 for 17 min and 37 for three min with varying inhibitor concentrations in dimethylsulfoxide followed by the addition of [1-14C]arachidonic acid (50 M) for 30 s at 37 . Reactions were terminated by solvent extraction in diethyl ether/ methanol/1 M citrate buffer, pH four.1867923-49-6 In stock 0 (30:4:1).867065-85-8 Chemical name The phases were separated by centrifugation at 2000g for 2 min, and also the organic phase was spotted on a thin-layer chromatography plate (EMD Kieselgel 60, VWR).PMID:33387184 The plate was created in ethyl acetate/methylene chloride/glacial AcOH (75:25:1) at four . Radiolabeled merchandise had been quantified using a radioactivity scanner (Bioscan, Inc., Washington, DC. The percentage of total merchandise observed at various inhibitor concentrations was divided by the percentage of solutions observed for protein samples preincubated for the same time with dimethyl sulfoxide. Cell Culture and In Vitro Intact Cell Metabolism Assay. Inhibition of COX-2 in intact cells by test compounds was assayed by a previously described technique.27 Briefly, RAW264.7, ATCC TIB-71 murine macrophage-like cells (passage number 8-15, mycoplasma adverse by a polymerase chain reaction detection system) were grown in Dulbecco’s Modified Eagle Medium (DMEM) + ten heat-inactivated fetal bovine serum to 40 confluence (6-well plates, Sarstedt). The cells had been activated for 7 h in two mL serum-free DMEM with 200 ng/mL bacterial lipopolysaccharide (Calbiochem) and 10 U/mL interferon gamma (Calbiochem) to induce COX-2 expression. Human 1483 head and neck squamous cell carcinoma (HNSCC) cells (passage 8-18, mycoplasma adverse by a polymerase chain reac.