Quantitative analysis of 5 alpha-reductase inhibitors in DU145 cells using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry and high-performance liquid chromatography/tandem mass spectrometry.
N-(Dicyclohexyl)acetylpiperidine-4-benzylidene-4-carboxylic acid (1) is an excellent in vitro inhibitor of 5 alpha-reductase (5 alpha R). Compound 1 showed, however, much lower inhibition activity of 5 alpha R 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.
Pharmaceutical and Medicinal Chemistry, Saarland University, Postfach 151150, D-66041 Saarbruecken, Germany.
Min-Jung
MJ
Kang
Sonal
S
Mathur
Rolf W
RW
Hartmann
D058891:5-alpha Reductase Inhibitors; D000230:Adenocarcinoma / Q000737:chemistry* / Q000201:enzymology; D001565:Benzoates / Q000032:analysis*; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000032:analysis*; D006801:Humans; D008297:Male; D010880:Piperidines / Q000032:analysis*; D011355:Prodrugs / Q000032:analysis*; D011471:Prostatic Neoplasms / Q000737:chemistry* / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D058891:5-alpha Reductase Inhibitors; D001565:Benzoates; D004791:Enzyme Inhibitors; C488696:N-(dicyclohexyl)acetylpiperidine-4-benzylidene-4-carboxylic acid; D010880:Piperidines; D011355:Prodrugs