Abstract
N‐(Dicyclohexyl)acetylpiperidine‐4‐benzylidene‐4‐carboxylic acid (1) is an excellent in vitro inhibitor of 5α‐reductase (5αR). Compound 1 showed, however, much lower inhibition activity of 5αR in vivo than in vitro, which might be caused by poor membrane permeability. The methyl ester of 1 (1a) was therefore tested as a model prodrug to see if it has better permeability properties than the corresponding acid 1. It was also monitored that this methyl ester was cleaved into the active compound 1 within the DU145 cells. Quantitative matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) and high‐performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) methods were established with reliable linearity factors (0.996 for MALDI‐TOFMS and 0.998 for HPLC/MS/MS) and reproducibility (relative standard deviation = 6.5% for MALDI‐TOFMS and 2.8% for HPLC/MS/MS). The samples for MS analysis were effectively prepared from the cell homogenates using solid‐phase extraction, with a high recovery of 90% on average. The intracellular amount of 1a (1.7 nmol) was much higher than that of 1 (0.032 nmol) in DU145 cells after 6 h of incubation. After incubation with the ester (1a), the cleaved acid (1) was detected within the cells. The concentration of acid 1 (0.045 nmol) in this experiment was higher than the acid content (0.032 nmol) after direct incubation with 1. Surprisingly, high amounts of the cleaved compound 1 were found outside the cells after 6 h of incubation with 1a. Copyright © 2004 John Wiley & Sons, Ltd.