title: Evaluation of cell permeation of a potent 5alpha-reductase inhibitor using MALDI-TOF MS.
abstract: N-(Dicyclohexyl)acetyl-piperidine-4-benzylidene-4-carboxylic acid (1), although a very potent in vitro 5alpha-steroid reductase (5alphaR) type 2 inhibitor, showed only marginal in vivo activity in rats. Since this could be due to hindered cellular uptake of the carboxylic acid, acid (1) and its corresponding methyl ester (1a) were compared with respect to their permeation properties. In the parallel artificial membrane permeation assay (PAMPA), 1a showed a higher %flux of 55 versus 6 for 1. Considering the high potency of 1 and better permeation of 1a, the use of 1a as a prodrug for 1 was explored using the human prostate carcinoma cell line DU145. Esterase activity, a prerequisite for this prodrug concept was detected employing 4-nitrophenyl acetate (4-NPA) as a substrate. After incubation of DU145 cells with 1 and 1a, respectively, permeated 1a and its hydrolysis to 1 were unequivocally observed by MALDI-TOF MS analyses, whereas 1 could not be detected inside the cells above the detection limit. Regarding biological activity, 1a showed a stronger inhibition of 5alphaR in intact DU145 cells than 1 (IC50 values, 4 microM and > 10 microM for 1a and 1, respectively). These results suggest that the in vivo activity of 1 might be increased by the use of its methyl ester prodrug 1a.
authors.affiliation: Pharmaceutical and Medicinal Chemistry, Saarland University, D-66041 Saarbruecken, Germany.
authors.forename: Sonal
authors.initials: S
authors.lastname: Mathur
authors.forename: Franck
authors.initials: F
authors.lastname: Picard
authors.forename: Ulrich
authors.initials: U
authors.lastname: Dossou
authors.forename: Catherine
authors.initials: C
authors.lastname: Barassin
authors.forename: Stefanie B
authors.initials: SB
authors.lastname: Seidel
authors.forename: Min-Jung
authors.initials: MJ
authors.lastname: Kang
authors.forename: Rolf W
authors.initials: RW
authors.lastname: Hartmann
mesh_terms: D001565:Benzoates / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects* / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D042944:Cholestenone 5 alpha-Reductase / Q000037:antagonists & inhibitors*; D004354:Drug Screening Assays, Antitumor; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004950:Esterases / Q000378:metabolism; D004952:Esters / Q000737:chemistry / Q000494:pharmacology*; D018120:Finasteride / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D008297:Male; D008567:Membranes, Artificial; D008968:Molecular Conformation; D010880:Piperidines / Q000737:chemistry / Q000494:pharmacology*; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D001565:Benzoates; D004791:Enzyme Inhibitors; D004952:Esters; D008567:Membranes, Artificial; C488696:N-(dicyclohexyl)acetylpiperidine-4-benzylidene-4-carboxylic acid; D010880:Piperidines; D018120:Finasteride; D042944:Cholestenone 5 alpha-Reductase; D004950:Esterases