Synthesis, biological evaluation and 3D-QSAR of 1,3,5-trisubstituted-4,5-dihydro-(1H)-pyrazole derivatives as potent and highly selective monoamine oxidase A inhibitors

The present report provides a extended study of the chemistry, the inhibitory activity against monoamino oxidases (MAO), and molecular modeling including the 3D-QSAR hypothesis of 1,3,5-trisubstituted-4,5-dihydro-(1H)-pyrazole derivatives. Four series of about eighty novel pyrazoline derivatives were prepared and investigated for their ability to inhibit the activity of the A and B isoforms of MAO selectively. Most of the new synthesized compounds proved more reversible, potent, and selective inhibitors of MAO-A than of MAO-B, and could be taken into account to develop the search further in this field, knowing that reversible and selective MAO-A inhibitors are used as antidepressant and antianxiety drug. The 30 most active compounds show inhibitory activity on MAO-A in the 8.6 x 10(-8) - 9.0 x 10(-9)M range. Moreover, it should be pointed out that for most of them a high IC(50) > or = 10(-9)M value is associated with a high A-selectivity (Selectivity Index MAO-B/MAO-A in the 10,000-16,250 range). Furthermore, due to the presence of a chiral centre at the C5 position of the pyrazole moiety, we performed the semi-preparative chromatographic enantioseparation of the most potent, selective, and chiral compounds. The separated enantiomers were then submitted to in vitro biological evaluation, and from the results of these experiments it has been possible to point out a difference in inhibiting the two isoforms selectively between the racemic mixture and the single enantiomers. The molecular modeling work was carried out combining the Glide docking approach with CoMFA with the aim to rationalize the structure-activity relationships of each pyrazoline inhibitor toward MAO-A and MAO-B isoforms and to derive a suitable selectivity model.     

Synthesis, molecular modeling studies, and selective inhibitory activity against monoamine oxidase of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-(1H)- pyrazole derivatives

A novel series of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-(1H)-pyrazole derivatives have been synthesized and investigated for the ability to inhibit selectively the activity of the A and B isoforms of monoamine oxidase (MAO). All the synthesized compounds show high activity against both the MAO-A and the MAO-B isoforms with Ki values between 27 and 4 nM and between 50 and 1.5 nM, respectively, except for a few derivatives whose inhibitory activity against MAO-B was in the micromolar range. Knowing that stereochemistry may be an important modulator of biological activity, we performed the semipreparative chromatographic enantioseparation of the most potent, selective, and chiral compounds. The separated enantiomers were then submitted to in vitro biological evaluation. The selectivity of the (-)-(S)-1 enantiomer against MAO-B increases twice and a half, while the selectivity of the (-)-(S)-4 enantiomer against MAO-A triples. Both the MAO-A and MAO-B isoforms respectively of the 1O5W and 1GOS models deposited in the Protein Data Bank were considered in the computational study. The docking study was carried out using several computational approaches with the aim of proposing possible binding modes of the MAO enantioselective compounds 1 and 4.     

Synthesis and structure-activity relationships of 1-substituted 4-(1,2-diphenylethyl)piperazine derivatives having narcotic agonist and antagonist activity

Racemates and enantiomers of 1-substituted 4-[2-(3-hydroxyphenyl)-1-phenylethyl]piperazine derivatives (3-18) were synthesized, and their analgesic and other pharmacological activities and structure-activity relationships were investigated. The S-(+) enantiomers of 2a, 5, 7, 9, 10, and 15-18 had a stronger analgesic activity than their R-(-) enantiomers; analgesic activity of the strongest one [(S)-(+)-10] was 105 times as potent as that of morphine. The S-(+) enantiomers of these compounds had the opposite configuration to that of morphine with respect to its (C-9) asymmetric center but the same configuration to that of the tyrosine residue of Met5-enkephalin. The R-(-) enantiomers of 16 and 18 showed narcotic antagonist activity, but the S-(+) enantiomers did not. (R)-(-)-18 had analgesic and narcotic antagonist activities comparable to pentazocine but showed no significant physical dependence liability. From these results, it is suggested that these compounds show an uncommon enantioselectivity in comparison with morphine and its surrogates, and belong to a new series of compounds having a potent analgesic activity.     

Irreversible Inhibition of Rat Liver Mitochondrial MAO A and MAO B by Enantiomers of Deprenyl and α-Methylpargyline

Using rat liver mitochondrial monoamine oxidase (MAO) A and MAO B, the possible influence of stereochemical factors upon the irreversible inhibition by propargylamine derivatives has been studied using the enantiomers of deprenyl and of α-methylpargyline. Whether studying the inhibition of MAO A or MAO B, little difference was found among enantiomeric pairs in the first-order rate constant (k₂) for formation of the enzyme inhibitor adduct. Similarly, and with the exception of (S)-d -(+)-deprenyl (k₂ = 0 or an extremely low value at MAO A), the computed value of k₂ for the individual enantiomers showed little variation between MAO A and MAO B. These results suggest that inhibitor selectivity towards a particular form of the enzyme is determined predominantly at the competitive phase of the inhibition.     

Synthesis and selective inhibitory activity of 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole derivatives against monoamine oxidase

A novel series of 1-acetyl-3-(4-hydroxy- and 2,4-dihydroxyphenyl)-5-phenyl-4,5-dihydro-(1H)-pyrazole derivatives 1-12 have been synthesized and investigated for the ability to selectively inhibit the activity of the A and B isoforms of monoamine oxidase (MAO). The new synthesized compounds 1-12 proved to be more reversible, potent, and selective inhibitors of MAO-A than of MAO-B. Knowing that stereochemistry may be an important modulator of biological activity, we performed the semipreparative chromatographic enantioseparation of the most potent, selective, and chiral compounds, 6 and 11. The separated enantiomers were then submitted to in vitro biological evaluation while increasing their inhibitory activity and A selectivity. The (-)-6 enantiomer shows K(i(MAO-A)) = 2 nM and SI = 165 000, (+)-6 shows K(i(MAO-A)) = 6 nM and SI = 166 666, (-)-11 shows K(i(MAO-A)) = 4 nM and SI = 80 000, and (+)-11 shows K(i(MAO-A)) = 7 nM and SI = 38 571.     

Adenosine deaminase inhibitors. Synthesis and biological evaluation of C1' and nor-C1' derivatives of (+)-erythro-9-(2(S)-hydroxy-3(R)-nonyl)adenine.

The synthesis of various chiral derivatives of (+)-erythro-9-(2-hydroxy-3-nonyl)adenine, (+)-EHNA, from (2S,3R)-3-amino-1,2-O-isopropylidene-1,2-nonanediol by condensation with 5-amino-4,6-dichloropyrimidine is described. The compounds synthesized were C1'- and nor-C1'-(+)-EHNA derivatives. When tested with calf spleen ADA, C1'-OH- and nor-C1'-(+)-EHNA had comparable inhibitory activity that was 1 order of magnitude lower than that of (+)-EHNA. Potency was reduced further in nor-C1' derivatives.     

(+)-4-dimethylamino-2, α-dimethylphenethylamine (FLA 336(+)), a selective inhibitor of the a form of monoamine oxidase in the rat brain

(+)-4-Dimethylamino-2, α-dimethylphenethylamine (FLA 336(+)) and its N-demethylated secondary amino derivative FLA 788(+) were examined for their monoamine oxidase (MAO) inhibitory effects in the rat brain. They were found to be reversible and very selective inhibitors of the A form of monoamine oxidase in vitro and in vivo after oral administration. FLA 788(+) was 2–6 times more active than FLA 336(+) in vitro depending on the assay technique employed but the two compounds had similar potency after oral administration. Both compounds inhibited competitively the deamination of 5-hydroxytryptamine by hypothalamic mitochondria. Although the irreversible inhibitor clorgyline was 60 times more potent than FLA 336(+) in vitro, it was equipotent with FLA 336(+) and FLA 788(+) in the rat brain after oral administration. There was a high correlation between the log plasma concentration of FLA 788(+) and the MAO inhibition in hypothalamic slices. The plasma concentration of the metabolite FLA 788(+) exceeded that of FLA 336(+) after oral administration of the latter compound. Thus, the MAO inhibition produced by FLA 336(+) in vivo, appears in part to be due to the metabolite FLA 788(+).     

Stereochemical aspects of parachloroamphetamine metabolism: Rabbit liver microsomal metabolism of RS-, R(-)-, and S(+)-para-chloroamphetamine

With the aid of a chiral derivatizing reagent and a sensitive and specific nitrogen-phosphorus gas Chromatographic assay, metabolism of para-chloroamphetamine (PCA) enantiomers has been studied following incubation of racemic (RS)-, R(?) and S(+)-PCA with rabbit liver microsomal preparations. Significant metabolism of racemic PCA and the individual enantiomers was observed following incubation in rabbit liver microsomal preparations. Metabolism required viable microsomes, NADPH and molecular oxygen, and the rate of metabolism increased following pretreatment with phenobarbital. Incubation of racemic PCA resulted in more rapid metabolism of the S(+) enantiomer than of the R(?) enantiomer. When the enantiomers were incubated individually, each enantiomer was metabolized more rapidly than when incubated as part of a racemic mixture, and the R(?) enantiomer was metabolized more rapidly than the S(+) enantiomer.     

Stereoselective inhibition of prolactin secretion by (-)-HW-165, a novel 3-PPP congener; further support for similarities between central DA autoreceptors and pituitary lactotroph DA receptors

The novel atypical dopamine (DA) receptor agonist HW-165 (trans-7-hydroxy-1,2,3,4,4a,5,6,10b-octahydrobenzo(f)quinoline), a 'rigid' 3-PPP congener, and its enantiomers were investigated with regard to their actions on prolactin (PRL) release in rats. Racemic HW-165 dose dependently inhibited the elevation of PRL secretion induced by monoamine synthesis disruption (NSD 1015) combined either with abolished DA nerve impulse flow (GBL) or with monoamine depletion (reserpine). Racemic HW-165 was roughly equipotent to 3-PPP but 5-10-fold less potent than apomorphine. The PRL inhibitory action of racemic HW-165 was stereoselectively prevented by the DA antagonist (+)-butaclamol. Since the chiral aspect of lactotroph DA receptor activation by 'atypical' DA receptor agonists has not been covered in previous investigations the effects of the HW-165 enantiomers were now studied. Our findings suggest that the PRL inhibitory properties of racemic HW-165 reside in its (-) enantiomer, (+)-HW-165 being devoid of activity. Taken together, the results reinforce the concept that the pituitary lactotroph DA receptors mediating the inhibition of PRL release are similar, notably also from a stereochemical point of view, to central DA receptor sites with presumed high agonist sensitivity, such as DA autoreceptors.     

Enantioselective uptake of BOF-4272, a xanthine oxidase inhibitor with a chiral sulfoxide, by isolated rat hepatocytes.

The transport mechanisms of the enantiomers of BOF-4272, a new drug for the treatment of hyperuricemia, were studied using freshly prepared rat hepatocytes. BOF-4272 consists of S(-) and R(+) enantiomers due to a chiral center in the sulfoxide moiety. The uptake of these BOF-4272 enantiomers by hepatocytes was found to be temperature and dose dependent. The temperature-dependent uptake of the S(-) and R(+) enantiomers showed saturation kinetics. The Km values for the S(-) and R(+) enantiomers were 59.3 and 25.7 microM, respectively, which was a significant difference (p < 0.05). However, the maximal uptake rate was comparable for both enantiomers. Metabolic inhibitors such as antimycin, oligomycin, rotenone, carbonylcyanide m-chlorophenyl hydrazone, and carbonyl cyanide-p-(trifluromethoxy)-phenylhydrazone significantly inhibited uptake of the R(+) enantiomer, but had little effect on uptake of the S(-) enantiomer. Ouabain (an inhibitor of Na+/K(+)-ATPase) and p-nitrobenzylthioinosine (NBMPR, a nucleoside transporter inhibitor) showed no significant effects on the uptake of either enantiomer. Organic anions such as taurocholate and cholate reduced the uptake of both enantiomers. These results suggest that the hepatic uptake of both BOF-4272 enantiomers is not due to simple diffusion but also involves carrier-mediated uptake. We suggest that the carrier-mediated uptake of BOF-4272 enantiomers includes both NBMPR-insensitive facilitated diffusion and an active transport system in liver plasma membrane, and that the enantioselective uptake of BOF-4272 is due to differences in affinity for the active transporter.     

Synthesis and Properties of cis- and trans-4-Hydroxypraziquantel

The title compound is the main metabolite of the anthelmintic agent praziquantel. The synthesis or cis- and trans-4-OH derivatives (as well as their enantiomers) of praziquantel is described. Thus, racemic, (+)- and (-)-pyrazinoisoquinolin-4-one ( 1 ) was reacted either with 4-oxocyclohexane carboxylic acid chloride or with 4-oxocyclohexane carboxylic acid in the presence of DCC to give the racemic, (+)- and (-)-ketone 2 . Racemic, (+)- and (-)-ketone 2 , respectively, was reduced with K-Selectride yielding the racemic, (+)- and (-)-cis-4-OH derivatives 3a . or with NaBH₄ producing racemic, (+)-and (-)-trans-4-OH derivatives 3b . The physicochemical properties (MS, NMR, HPLC) of both isomers and their enantiomers are reported.     

Synthesis of fenthion sulfoxide and fenoxon sulfoxide enantiomers: effect of sulfur chirality on acetylcholinesterase activity

Earlier reports have demonstrated that recombinant flavin-containing monooxygenase 1 (FMO1) catalyzes the oxidation of the organophosphate pesticide fenthion to (+)-fenthion sulfoxide in a stereoselective fashion. In order to elucidate the absolute configuration of the sulfoxide metabolite produced, we established an efficient synthesis of both enantiomers of fenthion sulfoxide, which were transformed into chiral fenoxon sulfoxides using a two-step protocol. The use of chiral oxidants, namely, N-(phenylsulfonyl)(3,3-dichlorocamphoryl) oxaziridines, afforded enantioenriched fenthion sulfoxides with high ee (>82%) from the parent sulfide. Single recrystallizations afforded chiral fenthion sulfoxides with >99% ee, measured by chiral HPLC analysis. The absolute configuration of the (+)-sulfoxide generated from fenthion metabolism by FMO1 was determined to be (R)-(+)-fenthion sulfoxide, confirmed by X-ray crystallographic analysis of the (S)-(-)-antipode. Inhibition of human recombinant (hrAChE) and electric eel (eeAChE) acetylcholinesterase were assayed with fenthion, fenoxon, and the racemates and enantiomers of fenthion sulfoxide and fenoxon sulfoxide. Results revealed stereoselective inhibition with (R)-(+)-fenoxon sulfoxide when compared with that of (S)-(-)-fenoxon sulfoxide (IC50 of 6.9 and 6.5 microM vs 230 and 111 microM in hrAChE and eeAChE, respectively). Fenthion sulfoxide (R or S enantiomers) did not present anti-AChE properties. Although the stereoselective sulfoxidation of fenthion to (R)-(+)-fenthion sulfoxide by FMO represents a detoxification pathway, the results of this study support the notion that subsequent oxidative desulfuration of (R)-(+)-fenthion sulfoxide (in vivo) may represent a critical bioactivation pathway, resulting in the production of (R)-(+)-fenoxon sulfoxide, a potent AChE inhibitor.     

Enantioseparation of erythro-mefloquine and its analogues in capillary electrophoresis

The enantioseparations of the chiral antimalaria drug (R,S)-erythro-alpha-(2-piperidyl)-2,8-bis(trifluoromethyl)4-quinolinemethanol (erythro-mefloquine, erythro-MQ) and its analogues were studied by capillary electrophoresis (CE) using cyclodextrins (CDs) as chiral selectors. The emphasis was put on the enantiomer affinity pattern of MQ towards different CDs as well as on simultaneous enantioseparations of erythro-MQ and its structural analogues. All three native CDs resolved the enantiomers of erythro-MQ and the enantiomer affinity pattern was the same, i.e. (+)-erythro-MQ was the more tightly bond enantiomer. However, the affinity pattern of erythro-MQ enantiomers was opposite in the case of heptakis-(2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD), heptakis-(2,3-di-O-methyl-6-sulfo)-beta-CD (HDMS-beta-CD), heptakis-(3-O-methyl-2,6-di-O-sulfo)-beta-CD (HMdiSu-beta-CD) and randomly sulfated beta-CD (SU-beta-CD). Randomly hydroxyalkylated and acetylated derivatives of CDs appeared to be suitable chiral selectors for simultaneous enantioseparation of erythro-MQ and its analogues.     

Liquid chromatographic chiral stationary phases in pharmaceutical analysis: determination of trace amounts of the (-)-enantiomer in (+)-amphetamine

A rapid and accurate method was developed for the determination of the enantiomeric composition of amphetamine preparations. Amide derivatives of the amphetamine enantiomers are first formed by using achiral 2-naphthoyl chloride. The resulting enantiomeric amides are then chromatographed on a commercially available chiral stationary phase. The capacity factors (k') of (-)- and (+)-2-naphthoylamphetamine are 20 and 22, respectively, and the separation factor (alpha) for the two enantiomers is 1.08. The method allows detection of as little as 0.5% of the (-)-enantiomer in (+)-amphetamine and is applicable to both bulk drug and single-tablet analyses.     

Pharmacological and toxicological testing of the enantiomers of two chiral fomocaine alkylmorpholine derivatives in comparison to their in vitro interactions on drug metabolism in rats

Between the stereoisomers of amide-type local anaesthetics differences have been noticed with respect to pharmacokinetics and side effects, but not regarding local anaesthetic capacity. Therefore, only S-(-)-ropivacaine has been introduced into clinical practice and with bupivacaine both the racemate and the S-(-)-enantiomer (levobupivacaine) are available by now. Based on this background, the aim of the present study was to evaluate if there are also dissimilarities to be found both in the toxicity and in the effectiveness of the enantiomers of two newly synthesized chiral fomocaine alkylmorpholine derivatives, OW3 and OW13, with an additional C2-chain in 2- or an additional C3-chain in 3-position at the morpholine ring, respectively. For this purpose, in vitro the interaction capacity with cytochrome P450 (CYP)-mediated monooxygenase and oxidase functions was investigated using rat liver 9000 g supernatants or microsomes. In vivo LD50, paresis of the N. ischiadicus and surface and conduction anaesthesia (cornea, N. ischiadicus) were tested in rats. The enantiomers of both OW3 and OW13 caused a concentration dependent inhibition of all CYP-mediated model reactions investigated. With all model reactions the (-)-enantiomer of OW3 was less effective than the (+)-form, whereas the opposite was the case with OW13. Also toxicity was lower with the (-)-enantiomer of OW3 and with the (+)-form of OW13 than with the respective counterparts. With both derivatives no clear-cut dissimilarities were noticed in the local anaesthetic capacity of the enantiomers. None of the four compounds caused paresis. Thus, similar to amide-type local anaesthetics, also with the enantiomers of chiral fomocaine alkylmorpholine derivatives differences in pharmacokinetic properties and toxicity could be demonstrated.     

Permanently charged chiral 1,4-dihydropyridines: molecular probes of L-type calcium channels. Synthesis and pharmacological characterization of methyl(omega-trimethylalkylammonium) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate iodide, calcium channel antagonists

We report the synthesis of the single enantiomers of permanently charged dihydropyridine derivatives (DHPs with alkyl linker lengths of two and eight carbon atoms) and their activities on cardiac and neuronal L-type calcium channels. Permanently charged chiral 1,4-dihydropyridines and methyl (omega)-trimethylalkylammonium) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate iodides were synthesized in high optical purities from (R)-(-) and (S)-(+)-1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-3-+ ++pyridinecarboxylic acid, obtained by resolution of racemic 1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-3-pyridi necarboxylic acid. Competition binding experiments with radioligand [3H]-(+)-PN200-110 and the block of whole cell barium currents through L-type calcium channels in GH4C1 cells show that the compounds with the eight-carbon alkyl linker optimally block the L-type Ca2+ channels, and that the S-enantiomer is more potent than the R-enantiomer.     

Resolution, absolute configuration, and cholinergic enantioselectivity of (-)- and (+)-c-2-methyl-r-5-[(dimethylamino)methyl]-1,3-oxathiolane t-3-oxide methiodide and related sulfones

As a continuation of previous studies on chiral cholinergic agonists carrying a 1,3-oxathiolane nucleus, the enantiomers of c-2-methyl-r-5-[(dimethylamino)methyl]-1,3-oxathiolane t-3-oxide methiodide ((+)- and (-)-1) and of cis-2-methyl-5-[(dimethylamino)methyl]-1,3-oxathiolane 3,3-dioxide ((+)- and (-)-2) were obtained and their absolute configurations established by synthesis. The cholinergic potency of the four isomers was evaluated in vitro on guinea pig ileum and frog rectus abdominis models, and the results show that (-)-1, which has the same absolute configuration as L-(+)-muscarine, is a selective and potent muscarinic agent. The (+)-1 enantiomer is some hundred times less potent than (-)-1 on the muscarinic guinea pig ileum while, on the same tissue, the corresponding sulfone derivatives ((+)- and (-)-2) show no enantioselectivity.     

Recent advances in the study of (–)clausenamide: chemistry,biological activities and mechanism of action

Clausenamide (clau) is one of seven novel compounds isolated from Clausena lansium (Lour) skeels. Clau is unusual in that it contains 4 chiral centers yielding 8 pairs of enantiomers. After identification of the configuration of these enantiomers, the synthesis of 16 enantiomers, including optically active clau and (+) and (–)clau was carried out. During this study, many stereochemical and synthetic difficulties were solved and the Baldwin principle was updated. Production scale is now sufficient to meet the needs of clinical practice. In a pharmacological study numerous models and indicators showed that (–)clau is the active enantiomer, while (+)clau is inactive and elicits greater toxicity than (–)clau. The principal pharmacological effects of (–)clau are to increase cognition, demonstrated in ten models of memory impairment, as well as to inhibit β-amyloid (Aβ) toxicity, blocking neurofibrillary tangle formation by inhibiting the phosphorylation of tau protein. This anti-dementia effect is characterized by increased synaptic plasticity both in efficacy and in structure and provides new support for the theory that synaptic loss is the main cause of dementia. (–)Clau is considered to be a promising drug candidate for treatment of Alzheimer׳s disease and other neurodegenerative disorders.KEY WORDS: (–)Clausenamide, Enantiomers, Cognition, Alzheimer׳s disease pathology, Tau, High phosphorylation, Synaptic plasticity     

Enantiospecificity of glutamate carboxypeptidase II inhibition

Two representative glutamate carboxypeptidase II (GCP II) inhibitors, 2-(hydroxypentafluorophenylmethyl-phosphinoylmethyl)pentanedioic acid 2 and 2-(3-mercaptopropyl)pentanedioic acid 3, were synthesized in high optical purities (>97%ee). The two enantiomers of 2 were prepared from previously reported chiral intermediates, (R)- and (S)-2-(hydroxyphosphinoylmethyl)pentanedioic acid benzyl esters 8. The synthesis of (R)- and (S)-3 involves the hydrolysis of (R)- and (S)-3-(2-oxo-tetrahydro-thiopyran-3-yl)propionic acids, (R)- and (S)-11, the corresponding optically pure thiolactones delivered by chiral chromatographic separation of the racemic 11. GCP II inhibitory assay revealed that (S)-2 is 40-fold more potent than (R)-2. In contrast, both enantiomers of 3 inhibited GCP II with nearly equal potency. The efficacy observed in subsequent animal studies with these enantiomers correlated well with the inhibitory potency in a GCP II assay.     

Enantioselective quantification of carvedilol in human plasma by HPLC in heavily medicated heart failure patients

A simple, specific, sensitive, inexpensive and rapid HPLC method for enantioselective quantification of carvedilol in human plasma was developed in this study. S(−)- and R(+)-carvedilol and R(+)-propranolol as the internal standard were extracted from human plasma by liquid–liquid extraction using methyl tert-butyl ether. Enantioseparation was performed on a reverse-phase C18 Phenomenex Luna 5micron 150 mm × 2 mm column after chiral derivatization with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate. The mobile phase was a mixture of water and acetonitrile. The peaks were detected using a fluorescence detector, where the excitation and emission wavelengths were set at 242 and 344 nm, respectively. The limits of quantification for the S(−)- and R(+)-carvedilol enantiomers were both 0.5 ng/ml. Combined intra- and inter-day variations for both enantiomers were less than 8.3%. The combined accuracy for both enantiomers ranged from 91.7 to 104.7%. This method was used to assay the carvedilol enantiomers in human plasma samples obtained from heavily medicated heart failure patients within the framework of a clinical trial.