A Real-Time Fluorescence Assay for MeasuringN-Dealkylation

Abstract

A real-time fluorescence assay system using a series of 9-N-(alkylamino)acridine derivatives (methyl, ethyl, n-propyl, n-butyl, n-pentyl, and benzyl) that are N-dealkylated to 9-aminoacridine (9AA) is described. The product, 9AA, is approximately 27-fold more fluorescent than the substrates using excitation and emission wavelengths of 405 and 455 nm, respectively. Tests using expressed CYP1A1, 1A2, 3A4, 3A5, 1B1, 2C9, 2C19, and 2D6 indicated that N-dealkylase activity is specific for CYP1A1 and CYP2D6. CYP2D6 N-dealkylated methyl, ethyl, n-propyl, and n-butyl substrates, whereas CYP1A1 N-dealkylated these plus the n-pentyl derivative. Activities using 5 microM 9-N-(alkylamino)acridine substrates ranged from 0.1 to 0.9 pmol 9AA/min/pmol P450. Kinetic constants for CYP1A1 N-dealkylation of the 9-N-(methylamino)acridine (MAA) and 9-N-(ethylamino)acridine (EAA) were K(m) 1.09 +/- 0.68 and 0.35 +/- 0.21 microM and the V(max) 61.9 +/- 48.5 and 113.8 +/- 8.4 pmol 9AA/min/pmol CYP1A1, respectively. Kinetic constants for CYP2D6 N-dealkylation of MAA and EAA were K(m) 7.9 +/- 5.4 and 3.2 +/- 1.6 microM, and V(max) 501 +/- 35.4 and 702.7 +/- 257 pmol 9AA/min/pmol CYP2D6, respectively. The experimental binding energies (DeltaG(bind)) were calculated for MAA with CYP1A1 and CYP2D6 to be -8.266 and -7.074 kcal/mol, respectively. The DeltaG(bind) values for EAA with CYP1A1 and CYP2D6 were -8.950 and -7.618 kcal/mol, respectively. The substrates were suitable for monitoring N-dealkylase activity in microsomal preparations (human, rat, and monkey hepatic preparations) and human hepatocellular carcinoma cell suspensions. Assays were conducted by monitoring reactions either in 96-well microtiter plates using a fluorescence plate reader or in cuvettes using a spectrofluorimeter.

Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes

“…CYP2D6 produced fluorescent 7-hydroxyl metabolites only by O-demethylation of the 7-methoxy derivatives (compounds 9 and 10). Known profluorescent CYP2D6 substrates include 3- [2-(N,N-diethyl-N-methylammonium)-ethyl]-7-methoxy-4methylcoumarin (AMMC), MFC, MAMC and some 9-N-aminoacridine derivatives, all of which are also catalyzed by several other CYP forms (Chauret et al, 2001;Rendic, 2002;Mayer et al, 2007). However, AMMC exhibits some degree of CYP selectivity, as CYP1A1, 1A2, 1B1 and 2B6 oxidize it only weakly (Chauret et al, 2001).…”

A general fluorescence off/on strategy for fluorogenic probes: Steric repulsion-induced twisted intramolecular charge transfer (sr-TICT)

“…Since CYP3A4 activity is one of the most important markers for differential maturation of hepatocytes and intestinal epithelial cells (34), fluorogenic probes for CYP3A4 activity would also be useful for noninvasively monitoring maturation. Some fluorogenic probes for CYP3A4 activity have been reported, but these have various limitations, such as low selectivity, low sensitivity, and short absorbance/fluorescence wavelengths (26,28,(35)(36)(37). In addition, although the existing fluorogenic probes for CYPs activity usually use the o-dealkylation or hydroxylation activity as a fluorescence off/on switch (26,28), our design strategy uses the N-dealkylation activity and therefore may show different reactivity and selectivity from the existing probes.…”

Pd-catalyzed one-pot approach for installation of 9-aminoacridines via Buchwald-Hartwig amination and cycloaromatization

“…9-Aminoacridines have been constructed using multi-step reactions, the final stage of the synthesis being nucleophilic substitution of chlorine or another nucleofuge in position 9 of the acridine molecule (SNAr reaction). [28][29][30] Bauer synthesized the 9-aminoacridine through Chichibabin reaction of acridine with sodium amide in dimethylaniline at 150 o C. 31 However, in an attempt to reproduce this experiment, the authors 32 obtained a different result which led the isolation of three compounds -the starting material acridine (14%), 9aminoacridine (31%), and 9,9′-diacridanyl. Pang and co-workers reported efficient tandem reaction to construct N-aryl acridines with easily accessible o-cyanoanilines and diaryliodonium salts.…”