Stereoselective metabolism of fenoxaprop‐ethyl and its chiral metabolite fenoxaprop in rabbits

The stereoselective metabolism of the enantiomers of fenoxaprop‐ethyl (FE) and its primary chiral metabolite fenoxaprop (FA) in rabbits in vivo and in vitro was studied based on a validated chiral high‐performance liquid chromatography method. The information of in vivo metabolism was obtained by intravenous administration of racemic FE, racemic FA, and optically pure (−)‐(S)‐FE and (+)‐(R)‐FE separately. The results showed that FE degraded very fast to the metabolite FA, which was then metabolized in a stereoselective way in vivo: (−)‐(S)‐FA degraded faster in plasma, heart, lung, liver, kidney, and bile than its antipode. Moreover, a conversion of (−)‐(S)‐FA to (+)‐(R)‐FA in plasma was found after injection of optically pure (−)‐(S)‐ and (+)‐(R)‐FE separately. Either enantiomers were not detected in brain, spleen, muscle, and fat. Plasma concentration–time curves were best described by an open three‐compartment model, and the toxicokinetic parameters of the two enantiomers were significantly different. Different metabolism behaviors were observed in the degradations of FE and FA in the plasma and liver microsomes in vitro, which were helpful for understanding the stereoselective mechanism. This work suggested the stereoselective behaviors of chiral pollutants, and their chiral metabolites in environment should be taken into account for an accurate risk assessment. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Stereoselective metabolism of propranolol glucuronidation by human UDP‐glucuronosyltransferases 2B7 and 1A9

Stereoselective metabolism of propranolol side‐chain glucuronidation was studied for two recombinant human uridine diphosphate glucuronosyltransferases (UGTs), UGT1A9 and UGT2B7. The S‐ and R‐propranolol side‐chain glucuronides produced in the incubation mixtures were assayed simultaneously by RP‐HPLC with fluorescent detector. The excitation and emission wavelengths were set at 310 nm and 339 nm, respectively. UGT1A9 prefers catalyzing S‐enantiomer to R‐enantiomer and the intrinsic clearance (CL_int) ratios of S‐enantiomer to R‐enantiomer are 3.8 times and 6.5times for racemic propranolol and individual enantiomers, respectively. UGT2B7, however, catalyzes slightly less S‐enantiomer than R‐enantiomer and the CL_int ratio of S‐enantiomer to R‐enantiomer is 0.8 times. The high concentration of racemic propranolol (>0.57 mmol/l) and individual enantiomers (>0.69 mmol/l) exhibited substrate inhibition of glucuronidation for UGT2B7, but only the S‐enantiomer (>0.44 mmol/l) in racemic propranolol exhibited substrate inhibition for UGT1A9. The substrate inhibition constants (K_si) were all similar (P > 0.05). Drug–drug interactions were also found between S‐ and R‐enantiomer glucuronidation metabolisms by UGT1A9 and UGT2B7. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Integration of metabolomics and in vitro metabolism assays for investigating the stereoselective transformation of triadimefon in rainbow trout

Triadimefon is a systemic agricultural fungicide of the triazole class whose major metabolite, triadimenol, also a commercial fungicide, provides the majority of the actual fungicidal activity, i.e., inhibition of steroid demethylation. Both chemicals are chiral: triadimefon has one chiral center with two enantiomers while its enzymatic reduction to triadimenol produces a second chiral center and two diastereomers with two enantiomers each. All six stereoisomers of the two fungicides were separated from each other using a chiral BGB‐172 column on a GC‐MS system so as to follow stereospecificity in metabolism by rainbow trout hepatic microsomes. In these microsomes the S‐(+) enantiomer of triadimefon was transformed to triadimenol 27% faster than the R‐(?) enantiomer, forming the four triadimenol stereoisomers at rates different from each other. The most fungi‐toxic stereoisomer (1S,2R) was produced at the slowest rate; it was detectable after 8 h, but below the level of method quantitation. The triadimenol stereoisomer ratio pattern produced by the trout microsomes was very different from that of the commercial triadimenol standard, in which the most rat‐toxic pair of enantiomers (known as “Diastereomer A”) is about 85% of the total stereoisomer composition. The trout microsomes produced only about 4% of “Diastereomer A”. Complementary metabolomic studies with NMR showed that exposure of the separate triadimefon enantiomers and the racemate to rainbow trout for 48 h resulted in different metabolic profiles in the trout liver extracts, i.e., different endogenous metabolite patterns that indicated differences in effects of the two enantiomers. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Enantioselective synthesis and teratogenicity of propylisopropyl acetamide,a CNS‐active chiral amide analogue of valproic acid

Propylisopropyl acetamide (PID), an amide analogue of the major antiepileptic drug valproic acid (VPA), possesses favorable anticonvulsant and CNS properties. PID contains one chiral carbon atom and therefore exists in two enantiomeric forms. The purpose of this work was to synthesize the two PID enantiomers and evaluate their enantiospecific teratogenicity. Enantioselective synthesis of PID enantiomers was achieved by coupling valeroyl chloride with optically pure (4S)‐ and (4R)‐benzyl‐2‐oxazolidinone chiral auxiliaries. The two oxazolidinone enolates were alkylated with isopropyl triflate, hydrolyzed, and amidated to yield (2R)‐ and (2S)‐PID. These two PID enantiomers were obtained with excellent enantiomeric purity, exceeding 99.4%. Unlike VPA, both (2R)‐ and (2S)‐PID failed to exert teratogenic effects in NMRI mice following a single 3 mmol/kg subcutaneous injection. From this study we can conclude that individual PID enantiomers do not demonstrate stereoselective teratogenicity in NMRI mice. Due to its better anticonvulsant activity than VPA and lack of teratogenicity, PID (in a stereospecific or racemic form) has the potential to become a new antiepileptic and CNS drug. Chirality 11:645–650, 1999. © 1999 Wiley‐Liss, Inc.     

Influence of Gasoline Inhalation on the Enantioselective Pharmacokinetics of Fluoxetine in Rats

Fluoxetine is used clinically as a racemic mixture of (+)‐(S) and (–)‐(R) enantiomers for the treatment of depression. CYP2D6 catalyzes the metabolism of both fluoxetine enantiomers. We aimed to evaluate whether exposure to gasoline results in CYP2D inhibition. Male Wistar rats exposed to filtered air (n = 36; control group) or to 600 ppm of gasoline (n = 36) in a nose‐only inhalation exposure chamber for 6 weeks (6 h/day, 5 days/week) received a single oral 10‐mg/kg dose of racemic fluoxetine. Fluoxetine enantiomers in plasma samples were analyzed by a validated analytical method using LC‐MS/MS. The separation of fluoxetine enantiomers was performed in a Chirobiotic V column using as the mobile phase a mixture of ethanol:ammonium acetate 15 mM. Higher plasma concentrations of the (+)‐(S)‐fluoxetine enantiomer were found in the control group (enantiomeric ratio AUC_{(+)‐(S)/(–)‐(R)} = 1.68). In animals exposed to gasoline, we observed an increase in AUC_0‐∞ for both enantiomers, with a sharper increase seen for the (–)‐(R)‐fluoxetine enantiomer (enantiomeric ratio AUC_{(+)‐(S)/(–)‐(R)} = 1.07), resulting in a loss of enantioselectivity. Exposure to gasoline was found to result in the loss of enantioselectivity of fluoxetine, with the predominant reduction occurring in the clearance of the (–)‐(R)‐fluoxetine enantiomer (55% vs. 30%). Chirality 25:206–210, 2013. © 2013 Wiley Periodicals, Inc.     

Enantioseparation of mandelic acid on vancomycin column: Experimental and docking study

So far, no detailed view has been expressed regarding the interactions between vancomycin and racemic compounds including mandelic acid. In the current study, a chiral stationary phase was prepared by using 3-aminopropyltriethoxysilane and succinic anhydride to graft carboxylated silica microspheres and subsequently by activating the carboxylic acid group for vancomycin immobilization. Characterization by elemental analysis, Fourier transform infrared spectroscopy, solid-state nuclear magnetic resonance, and thermogravimetric analysis demonstrated effective functionalization of the silica surface. R and S enantiomers of mandelic acid were separated by the synthetic vancomycin column. Finally, the interaction between vancomycin and R/S mandelic acid enantiomers was simulated by Auto-dock Vina. The binding energies of interactions between R and S enantiomers and vancomycin chiral stationary phase were different. In the most probable interaction, the difference in mandelic acid binding energy was approximately 0.2 kcal/mol. In addition, circular dichroism spectra of vancomycin interacting with R and S enantiomers showed different patterns. Therefore, R and S mandelic acid enantiomers may occupy various binding pockets and interact with different vancomycin functions. These observations emphasized the different retention of R and S mandelic acid enantiomers in vancomycin chiral column.     

Enantioseparation of Mandelic Acid Enantiomers With Magnetic Nano‐Sorbent Modified by a Chiral Selector

In this study, R(+)‐α‐methylbenzylamine‐modified magnetic chiral sorbent was synthesized and assessed as a new enantioselective solid phase sorbent for separation of mandelic acid enantiomers from aqueous solutions. The chemical structures and magnetic properties of the new sorbent were characterized by vibrating sample magnetometry, transmission electron microscopy, Fourier transform infrared spectroscopy, and dynamic light scattering. The effects of different variables such as the initial concentration of racemic mandelic acid, dosage of sorbent, and contact time upon sorption characteristics of mandelic acid enantiomers on magnetic chiral sorbent were investigated. The sorption of mandelic acid enantiomers followed a pseudo‐second‐order reaction and equilibrium experiments were well fitted to a Langmuir isotherm model. The maximum adsorption capacity of racemic mandelic acid on to the magnetic chiral sorbent was found to be 405 mg g^?1. The magnetic chiral sorbent has a greater affinity for (S)‐(+)‐mandelic acid compared to (R)‐(?)‐mandelic acid. The optimum resolution was achieved with 10 mL 30 mM of racemic mandelic acid and 110 mg of magnetic chiral sorbent. The best percent enantiomeric excess values (up to 64%) were obtained by use of a chiralpak AD‐H column. Chirality 27:835–842, 2015. © 2015 Wiley Periodicals, Inc.     

Human tyrosinase is able to oxidize both enantiomers of rhododendrol

Racemic RS‐4‐(4‐hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was used as a topical skin‐whitening agent until it was recently reported to induce leukoderma. We then showed that oxidation of RD with mushroom tyrosinase rapidly produces RD‐quinone, which is quickly converted to RD‐cyclic quinone and RD‐hydroxy‐p‐quinone. In this study, we examined whether either or both of the enantiomers of RD can be oxidized by human tyrosinase. Using a chiral HPLC column, racemic RD was resolved optically to R(?)‐RD and S(+)‐RD enantiomers. In the presence of a catalytic amount of l ‐dopa, human tyrosinase, which can oxidize l ‐tyrosine but not d ‐tyrosine, was found to oxidize both R(?)‐ and S(+)‐RD to give RD‐catechol and its oxidation products. S(+)‐RD was more effectively oxidized than l ‐tyrosine, while R(?)‐RD was less effective. These results support the notion that the melanocyte toxicity of RD depends on its tyrosinase‐catalyzed conversion to toxic quinones and the concomitant production of reactive oxygen species.     

Synthesis of enantiopure planar chiral bis‐(para)‐pseudo‐meta‐type [2.2]paracyclophanes

A new type of planar chiral (R_p)‐ and (S_p)‐4,7,12,15‐tetrasubstituted [2.2]paracyclophanes was prepared from racemic 4,7,12,15‐tetrabromo[2.2]paracyclophane as the starting substrate. Regioselective lithiation and transformations afforded racemic bis‐(para)‐pseudo‐meta‐type [2.2]paracyclophane (4,15‐dibromo‐7,12‐dihydroxy[2.2]paracyclophane). Its optical resolution was performed by the diastereomer method using a chiral camphanoyl group as the chiral auxiliary. The diastereoisomers were readily isolated by simple silica gel column chromatography, and the successive hydrolysis afforded (R_p)‐ and (S_p)‐bis‐(para)‐pseudo‐meta‐type [2.2]paracyclophanes ((R_p)‐ and (S_p)‐4,15‐dibromo‐7,12‐dihydroxy[2.2]paracyclophanes). They can be used as pseudo‐meta‐substituted chiral building blocks.     

A New Biosensor for Chiral Recognition Using Goat and Rabbit Serum Albumin Self‐Assembled Quartz Crystal Microbalance

Quartz crystal microbalance (QCM) biosensor was used for the chiral recognition of five pairs of enantiomers by using goat serum albumin (GSA) and rabbit serum albumin (RbSA) as chiral selectors. Serum albumin (SA) was immobilized on the QCM through the self‐assembled monolayer technique, and the surface concentration of GSA and RbSA were 8.8 × 10^?12 mol cm^?2 and 1.2 × 10^?11 mol cm^?2, respectively. The QCM biosensors showed excellent sensitivity and selectivity. Meanwhile, the chiral recognition of SA sensors was quite species dependent. There were differences between GSA and RbSA sensors in the ability and the preference of chiral recognition. To R,S‐1,2,3,4‐tetrahydro‐1‐naphthylamine (R,S‐1‐TNA), R,S‐1‐(4‐methoxyphenyl)ethylamine (R,S‐4‐MPEA), and R,S‐1‐(3‐methoxyphenyl)ethylamine (R,S‐3‐MPEA), the preference of the stereoselective SA‐drug binding of the two kinds of SA sensors were consistent. However, to R,S‐2‐octanol (R, S‐2‐OT) and R,S‐methyl lactate (R,S‐MEL), the two kinds of SA sensors had opposite chiral recognition preference. Moreover, the interactions of SA and the five pairs of enantiomers have been further investigated through ultraviolet (UV) and fluorescent (FL) spectra. The UV/FL results were in accordance with the consequence of QCM. Chirality 24:804–809, 2012. © 2012 Wiley Periodicals, Inc.     

Separation of ephedrine and pseudoephedrine enantiomers using a polysaccharide‐based chiral column: A normal phase liquid chromatography–high‐resolution mass spectrometry approach

Chiral considerations are found to be very much relevant in various aspects of forensic toxicology and pharmacology. In forensics, it has become increasingly important to identify the chirality of doping agents to avoid legal arguments and challenges to the analytical findings. The scope of this study was to develop an liquid chromatography–mass spectrometry (LCMS) method for the enantiomeric separation of typical illicit drugs such as ephedrines (ie, 1S,2R(+)‐ephedrine and 1R,2S(?)‐ephedrine) and pseudoephedrine (ie, R,R(?)‐pseudoephedrine and S,S(+)‐pseudoephedrine) by using normal phase chiral liquid chromatography–high‐resolution mass spectrometry technique. Results show that the Lux i‐amylose‐1 stationary phase has very broad and balancing‐enantio‐recognition properties towards ephedrine analogues, and this immobilized chiral stationary phase may offer a powerful tool for enantio‐separation of different types of pharmaceuticals in the normal phase mode. The type of mobile phase and organic modifier used appear to have dramatic influences on separation quality. Since the developed method was able to detect and separate the enantiomers at very low levels (in pico grams), this method opens easy access for the unambiguous identification of these illicit drugs and can be used for the routine screening of the biological samples in the antidoping laboratories.     

Enantioselective Degradation of the Chiral Fungicides Metalaxyl and Furalaxyl by Brevibacillus brevis

For almost four decades, the chiral fungicides metalaxyl and furalaxyl have been in use in plant protection on a global scale. Both substances are distributed as racemic mixtures, yet the desirable interference in nucleic acid synthesis of harmful fungi only occurs by the (‐)‐R‐enantiomer. As enantioselective degradation in Scheyern (Germany) and Yaoundé (Cameroon) soils has been documented, the influence of 50 isolated microorganisms on the R/S ratio was investigated. A high‐pressure liquid chromatography method with a chiral column to separate enantiomers of metalaxyl and furalaxyl, and subsequent detection by tandem mass spectrometry, was employed. Only one of these microorganisms, a strain of Brevibacillus brevis, showed an enantioselective degradation pattern in liquid culture; the respective (‐)‐R‐enantiomers were preferably degraded. Moreover, (‐)‐R‐furalaxyl was degraded faster in cultures supplemented simultaneously with both fungicides of the same concentration. Chirality 25:336–340, 2013. © 2013 Wiley‐Liss Inc. Chirality 00:000‐000:, 2013. © 2013 Wiley Periodicals, Inc.     

Separation and toxicity of salithion enantiomers

Enantioseletive toxicities of chiral pesticides have become an environmental concern recently. In this study, we evaluated the enantiomeric separation of salithion on a suite of commercial chiral columns and assessed the toxicity of enantiomers toward butyrylcholinesterase and Daphnia magna. Satisfactory separations of salithion enantiomers could be achieved on all tested columns, that is, Chiralcel OD, Chiralcel OJ, and Chiralpak AD column. However, the Chiralpak AD column offered the best separation and was chosen to prepare micro‐scale of pure salithion enantiomers for subsequent bioassays. The first and second enantiomers eluted on the Chiralpak AD column were further confirmed to be (?)‐S‐salithion and (+)‐R‐salithion, respectively. The half inhibition concentrations to butyrylcholinesterase of racemate, (+)‐R‐salithion, and (?)‐S‐salithion were 33.09, 2.92, and 15.60 mg/l, respectively, showing (+)‐R‐enantiomer being about 5.0 times more potent than its (?)‐S‐form. However, the median lethal concentrations (96 h) of racemate, (+)‐R‐salithion, and (?)‐S‐salithion toward D. magna were 3.54, 1.10, and 0.36 μg/l, respectively, suggesting that (?)‐S‐salithion was about 3.0 times more toxic than (+)‐R‐form. Racemic salithion was less toxic than either of the enantiomers in both bioassays, suggesting that antagonistic interactions might occur between the enantiomers during the toxication action. This work reveals that the toxicity of salithion toward butyrylcholinesterase and D. magna is enantioselective, and this factor should be taken into consideration in the environmental risk assessment of salithion. Chirality 2009. © 2009 Wiley‐Liss, Inc.     

Introduction of axial chirality in a planar aromatic ligand results in chiral recognition with DNA

Dimerization of a hydroxycarbazole produces an axially chiral biaryl, BICOL ( 2 ). One enantiomer (R)‐ 2 , is capable of enantioselective binding to different polymorphs of DNA. The biaryl (R)‐ 2 was shown by fluorescence and circular dichroism to induce a shift of Z‐DNA to B‐DNA. The opposite enantiomer (S)‐ 2 shows no specific binding. The significant difference in behaviour between the two enantiomers (S)‐ 2 and (R)‐ 2 is in line with molecular modelling studies which show two very different binding geometries between the enantiomers with each polymorph of DNA. Copyright © 2010 John Wiley & Sons, Ltd.     

The stereoselective sulfate conjugation of 4'-methoxyfenoterol stereoisomers by sulfotransferase enzymes

The presystemic sulfate conjugation of the stereoisomers of 4′‐methoxyfenoterol, (R,R′)‐MF, (S,S′)‐MF, (R,S′)‐MF, and (S,R′)‐MF, was investigated using commercially available human intestinal S9 fractions, a mixture of sulfotransferase (SULT) enzymes. The results indicate that the sulfation was stereospecific and that an S‐configuration at the β‐OH carbon of the MF molecule enhanced the maximal formation rates with (S,R′)‐MF (S,S′)‐MF (R,S′)‐MF ≈ (R,R′)‐MF, and competition studies demonstrated that (S,R′)‐MF is an effective inhibitor of (R,R′)‐MF sulfation (IC₅₀ = 60 μM). In addition, the results from a cDNA‐expressed human SULT isoform screen indicated that SULT1A1, SULT1A3, and SULT1E1 can mediate the sulfation of all four MF stereoisomers. Previously published molecular models of SULT1A3 and SULT1A1 were used in docking simulations of the MF stereoisomers using Molegro Virtual Docker. The models of the MF‐SULT1A3 and MF‐SULT1A1 complexes indicate that each of the two chiral centers of MF molecule plays a role in the observed relative stabilities. The observed stereoselectivity is the result of multiple hydrogen bonding interactions and induced conformational changes within the substrate–enzyme complex. In conclusion, the results suggest that a formulation developed from a mixture of (R,R′)‐MF and (S,R′)‐MF may increase the oral bioavailability of (R,R′)‐MF. Chirality 24:796–803, 2012. © 2012 Wiley Periodicals, Inc. ¹      

Resolution of RS-abscisic acid and the separation of abscisic acid metabolites from plant tissue by high-performance liquid chromatography

Attempts to resolve the enantiomers of racemic abscisic acid (ABA) by high-performance liquid chromatograpy on a chiral stationary-phase column were unsuccessful. However, reduction of RS-methyl ABA (RS-Me-ABA) with sodium borohydride generates a new chiral centre and one of the two isomeric products, the RS-Me-1′,4′-cis-diol of ABA, was separated into its enantiomers by high-performance liquid chromatography on an optically active Pirkle column.High-performance liquid chromatography on a μBondapak C₁₈ column separated the metabolites and conjugates of [2-¹⁴C]ABA fed to tomato shoots. The resolution method was used to measure the relative proportions of R and S enantiomers in the free acid liberated from conjugates of ABA.     

An oxorhenium complex bearing a chiral cyclohexane‐1‐olato‐2‐thiolato ligand: Synthesis,stereochemistry, and theoretical study of parity violation vibrational frequency shifts

In our effort towards measuring the parity violation energy difference between two enantiomers, a simple chiral oxorhenium complex 5 bearing enantiopure 2‐mercaptocyclohexan‐1‐ol has been prepared as a potential candidate species. Vibrational circular dichroism revealed a chiral environment surrounding the rhenium atom, even though the rhenium is not a stereogenic center itself, and enabled to assign the (1S,2S)‐(?) and (1R,2R)‐(+) absolute configuration for 5 . For both compound 5 and complex 4 , previously studied by us and bearing a propane‐2‐olato‐3‐thiolato ligand, relativistic calculations predict parity violating vibrational frequency differences of a few hundreds of millihertz, above the expected sensitivity attainable by a molecular beam Ramsey interferometer that we are constructing.     

Lack of Enantioselectivity in the SULT1A3‐catalyzed Sulfoconjugation of Normetanephrine Enantiomers: An In Vitro and Computational Study

(1R)‐Normetanephrine is the natural stereoisomeric substrate for sulfotransferase 1A3 (SULT1A3)‐catalyzed sulfonation. Nothing appears known on the enantioselectivity of the reaction despite its potential significance in the metabolism of adrenergic amines and in clinical biochemistry. We confronted the kinetic parameters of the sulfoconjugation of synthetic (1R)‐normetanephrine and (1S)‐normetanephrine by recombinant human SULT1A3 to a docking model of each normetanephrine enantiomer with SULT1A3 and the 3′‐phosphoadenosine‐5′‐phosphosulfate cofactor on the basis of molecular modeling and molecular dynamics simulations of the stability of the complexes. The K_M, V_max, and k_cat values for the sulfonation of (1R)‐normetanephrine, (1S)‐normetanephrine, and racemic normetanephrine were similar. In silico models were consistent with these findings as they showed that the binding modes of the two enantiomers were almost identical. In conclusion, SULT1A3 is not substrate‐enantioselective toward normetanephrine, an unexpected finding explainable by a mutual adaptability between the ligands and SULT1A3 through an “induced‐fit model” in the catalytic pocket. Chirality, 25:28‐34, 2012.© 2012 Wiley Periodicals, Inc.     

Enhanced Resolution of a Secondary Alcohol by Hydrolysis of a Bichiral Ester Catalyzed by Lipase from Candida cylindracea

The effect of a chiral centre in the acyl group on the resolution of esters prepared from a racemic alcohol was investigated. R-2-chloropropionic acid afforded a higher enantiomeric ratio than S-2-chioropropionic acid in the hydrolysis of the corresponding esters of racemic 1-phenylethanol catalyzed by Candida cylindracea lipase. Even when a mixture of esters prepared from racemic acid and racemic alcohol was used for resolution of the alcohol, a noteworthy high enantioselectivity was observed. The hydrolysis of a bichiral ester offers an amplification in the resolution of enantiomers of alcohols by the combination of a chemical diastereoselectivity and an enzymatic enantio- and diastereoselectivity.     

Steady‐State Plasma Concentration of Donepezil Enantiomers and Its Stereoselective Metabolism and Transport In Vitro

The aim of the present study was to elucidate the differences in the plasma concentration of two enantiomers of donepezil in Chinese patients with Alzheimer's disease (AD) and investigate in vitro stereoselective metabolism and transport. Donepezil enantiomers were separated and determined by LC‐MS/MS using D5‐donepezil as an internal standard on a Sepax Chiralomix SB‐5 column. In vitro stereoselective metabolism and transport of donepezil were investigated in human liver microsomes and MDCKII‐MDR1 cell monolayer. Pre‐dose (Css‐min) plasma concentrations were determined in 52 patients. The mean plasma level of (R)‐donepezil was 14.94 ng/ml and that of (S)‐donepezil was 23.37 ng/ml. One patient's plasma concentration of (R)‐donepezil was higher than (S)‐donepezil and the ratio is 1.51. The mean plasma levels of (S)‐donepezil were found to be higher than those of (R)‐donepezil in 51 patients and the ratio of plasma (R)‐ to (S)‐donepezil varies from 0.34 to 0.85. In the in vitro microsomal system, (R)‐donepezil degraded faster than (S)‐donepezil. V_max of (R)‐donepezil was significantly higher than (S)‐donepezil. The P‐gp inhibition experiment shown that the P_app of the two enantiomers was higher than 200 and the efflux ratios were 1.11 and 0.99. The results of the P‐gp inhibition identification experiment showed IC50 values of 35.5 and 20.4 μM, respectively, for the two enantiomers. The results indicate that donepezil exhibits stereoselective hepatic metabolism that may explain the differences in the steady‐state plasma concentrations observed. Neither (R)‐ nor (S)‐donepezil was a P‐gp substance and the two enantiomers are highly permeable through the blood–brain barrier. Chirality 25:498–505, 2013. © 2013 Wiley Periodicals, Inc.