Development of Fluorescence Imaging Probes for Labeling COX-1 in Live Ovarian Cancer Cells

Abstract

Recent experimental evidence demonstrated an aberrant overexpression of cyclooxygenase-1 (COX-1) in various cancers, which has stimulated the development of COX-1-selective inhibitors as promising anticancer drugs and cancer imaging agents. Herein we describe the synthesis and validation of 3-(furan-2-yl)-N-aryl 5-amino-pyrazoles as a novel class of COX-1 inhibitors, including molecular docking studies. Among all tested compounds, 4-(5-azido-3-(furan-2-yl)-1H-pyrazol-1-yl)benzoic 17 displayed a favorable COX-1 inhibition and selectivity profile (COX-1 IC₅₀ = 0.1 μM, SI >1000 over COX-2). Compound 17 was selected as a lead structure for developing the novel COX-1-selective fluorescent probe 22. Fluorescent probe 22 was prepared via click chemistry by installing a nitro-benzoxadiazole motif as a fluorophore into the 3-(furan-2-yl)-N-aryl 5-amino-pyrazole scaffold. Fluorescence probe 22 was tested in ovarian cancer cell line OVCAR-3, confirming its usefulness for targeting and visualizing COX-1 in living cells with confocal microscopy.

Figure 1

Selective COX-1 inhibitors containing a central five-membered heterocycle.

Figure 2

Synthesis of compounds 12–18.

Figure 3

Synthesis of fluorescent compound 22.

Figure 4

Confocal microscopy experiments with fluorescence imaging probe 22 in OVCAR-3 cells. Cells were treated with (a) PBS (control) and (b–d) 10 μM of 22. (b) COX-1 staining due to the cellular uptake of 22 (nuclear staining not shown). (c) Merged image showing nuclei (blue) and COX-1 staining (green) as a result of the cellular uptake of conjugate 22 and cell membrane labeling (red). (d) OVCAR-3 cells were pretreated with 50 μM of SC560 before the treatment with 22. All images are on the same scale as that indicated in panel a.

Figure 5

Molecular docking of compounds 17 (left) and 22 (right) in the COX-1 isozyme binding site (PDB ID: 1EQG).