Carboranyl Derivatives of Rofecoxib with Cytostatic Activity against Human Melanoma and Colon Cancer Cells

Owing to the involvement of cyclooxygenase-2 (COX-2) in carcinogenesis, COX-2-selective inhibitors are increasingly studied for their potential cytotoxic properties. Moreover, the incorporation of carboranes in structures of established anti-inflammatory drugs can improve the potency and metabolic stability of the inhibitors. Herein, we report the synthesis of carborane-containing derivatives of rofecoxib that display remarkable cytotoxic or cytostatic activity in the micromolar range with excellent selectivity for melanoma and colon cancer cell lines over normal cells. Furthermore, it was shown that the carborane-modified derivatives of rofecoxib showed different modes of action that were dependent on the cell type.

*All hydrogen atoms were calculated on idealized positions. The compound shows an extreme tendency for dendritic growth and after several attempts, only a very small pure single crystal could be isolated from the cluster.    with an inter-assay coefficient of variation of 3.6%.

Cell viability tests.
The number of viable cells in culture was estimated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and CV (crystal violet) tests. Cells were incubated in the presence of compounds 4a-c, 5 and 6 for 48 h, then the viability was measured as described. 4 The results were expressed as a percentage of a value obtained for the untreated control cells that was arbitrarily set to be 100%. A non-linear regression analysis of obtained results was done by using GraphPad Prism software to calculate IC50 values.
A375 cells were treated with 5 for 48 h, trypsinized, and stained with AnnexinV-FITC (Ann) or propidium iodide (PI), or apostat as proposed by the manufacturer. Cells were analyzed by CyFlow® Space Partec using the PartecFloMax® software.

Cell staining with CFSE.
To quantify cell division rate, cells were incubated for 10 min at 37 °C in the presence of CFSE (1 µM) and upon removal of dye, cells were exposed to an IC50 dose of 5 for 48 h and subsequently quantified using flow cytometry.

Wound healing test.
Cell motility and proliferation was analyzed using wound healing assay. A375 and 518A2 cells were cultivated in 6-well plates until 80% of confluence in monolayers was reached. The scratch was made using a sterile pipette tip which was drawn across the center of the wells. The scratched cell layers were washed twice with fresh PBS to remove loose or damaged cells. Cells were than cultivated in the presence of an IC50 dose of 5 for 48 h, washed and finally fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. The wounds were digitally photographed using a Nikon inverted microscope TS2 with 5 MPIX camera.

Measurement of intracellular nitric oxide
The intracellular dye 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF, 5 µM) was used for quantification of NO. After treatment for 48 h, the cells were incubated in RPMI, without phenol red, for 1 h at 37 °C. Subsequently the dye (DAF) was removed and the cells where additionally incubated for 15 min in serum/phenol red free conditions, with an aim to complete de-esterification of intracellular diacetates. Finally, cells were resuspended in PBS and analyzed as described.

Statistics
Cells were treated with a range of concentrations in triplicate; IC50 concentrations were calculated from at least three independent experiments. Significance of the differences between various treatments was calculated by the analysis of variance (ANOVA), followed by the Student-Newman-Keuls test. A p value less than 0.05 was considered significant.

Molecular docking studies
Ligand structures were constructed with Avogadro 1.1.1. 6 Ligand geometries were optimized with the xtb code 7 using the GFN2 parameter set. The atomic partial charges for each ligand were derived at the HF/6-31+G* level according to the RESP 8-11 procedure using Gaussian 09 12 and antechamber. The COX-2 crystal structure PDB ID: 4Z0L 17 was downloaded from www.rcsb.org. 13 All ligands and non-standard residues except for the heme groups, and all water molecules were removed with the UCSF Chimera package. 14    2.0 3.0 1.5 3.5 / *The selectivity index was calculated as a ratio between the IC50 of the compounds cytotoxicity determined for macrophages and the IC50 of the compounds cytotoxicity determined for the given cell line (IC50 macrophages / IC50 cancer). No value was calculated for 6, as it showed no effect on the viability of the tested cell lines up to 50 µM