4-[(8-Ethynyl, 8-vinyl or 8-ethynyl-methyl)-6-chromanoyl]-benzoic and 2-[4-[(8-ethynyl, 8-vinyl or 8-ethynyl-methyl)-6-chromanoyl]-phenyl]-acetic acid, their esters and salts having cytochrome P450RAI inhibitory activity

#1. Field of the Invention

#The present invention is directed to novel compounds which inhibit the enzyme cytochrome P450RAI. More particularly, the present invention is directed to 4-[(8-ethynyl, 8-vinyl or 8-ethynyl-methyl)-6-chromanoyl]-benzoic and 2-[4-[(8-ethynyl, 8-vinyl or 8-ethynyl-methyl)-6-chromanoyl]-phenyl]-acetic acid, their esters and salts which inhibit either the enzyme cytochrome P450RAI1 or the enzyme cytochrome P450RAI2, or both enzymes.

#2. Background Art

#FIG. 1 is a schematic representation of the P450RAI cell based assay utilized to evaluate the ability of the compounds of the invention to inhibit the Cytochrome P450RAI enzyme.

#P450RAI-1 and P450RAI-2 Cell-Based Inhibitor Assay:

#FIG. 1 shows a schematic diagram of the P450RAI-1 and P450RAI-2 cell based assay. P450RAI-1 stably transfected HeLa cells, or P450RAI-2 stably transfected HeLa cells, as applicable, are maintained in 100 millimolar tissue culture dishes in Modified Eagle's Medium (MEM) containing 10% Fetal Bovine Serum (FBS) and 100 μg/ml hygromycin. Exponentially growing cells are harvested by incubating in trypsin. Cells are then washed with 1× Phosphate Buffered Saline (PBS) and plated in a 48-well plate at 5×105 cells in 0.2 ml MEM medium containing 10% FBS and 0.05 μCi [3H]-RA in the presence or absence of increasing concentrations of the test compounds. The compounds are diluted in 100% DMSO and then added in triplicate wells at either 10, 1 or 0.1 μM final concentration. As a positive control for RA metabolism inhibition, cells are also incubated with ketoconazole at 100, 10 and 1 μM. Cell are incubated for 3 hours at 37° C. The retinoids are then extracted using the procedure of Bligh et al. (1959) Canadian Journal of Biochemistry 37, 911-917, modified by using methylenechloride instead of chloroform. The publication Bligh et al. (1959) Canadian Journal of Biochemistry 37, 911-917 is specifically incorporated herein by reference. The water soluble radioactivity is quantified using a β-scintillation counter. IC50 values represent the concentration of inhibitor required to inhibit all-trans-RA metabolism by 50 percent and are derived manually from log-transformed data. The IC50 values obtained in this assay for several preferred compounds of the invention with both the RAI-1 and RAI-2 enzymes are disclosed in Table 1 below.

#Assays of Retinoid-Like or Retinoid Antagonist and Inverse Agonist-Like Biological Activity