Pharmacological evaluation of both enantiomers of (R,S)-BM-591 as thromboxane A2 receptor antagonists and thromboxane synthase inhibitors

The aim of this work is to evaluate the anti-thromboxane activity of two pure enantiomers of (R,S)-BM-591, a nitrobenzene sulfonylurea chemically related to torasemide, a loop diuretic. The drug affinity for thromboxane A2 receptor (TP) of human washed platelets has been determined. In these experiments, (R)-BM-591 (IC50 = 2.4+/-0.1 nM) exhibited a significant higher affinity than (S)-BM-591 (IC50 = 4.2+/-0.15 nM) for human washed platelets TP receptors. Both enantiomers were stronger ligands than SQ-29548 (IC50 = 21.0+/-1.0 nM) and sulotroban (IC50 = 930+/-42 nM), two reference TXA2 receptor antagonists. Pharmacological characterisations of (S)-BM-591 and (R)-BM-591 were compared in several models. Thus, (R)-BM-591 strongly prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) and U-46619 (1 microM) while (S)-BM-591 showed a lower activity. On isolated tissues pre-contracted by U-46619, a stable TXA2 agonist, (S)-BM-591 was more potent in relaxing guinea-pig trachea (EC50 = 0.272+/-0.054 microM) and rat aorta (EC50 = 0.190+/-0.002 microM) than (R)-BM-591 (EC50 of 9.60+/-0.63 microM and 0.390+/-0.052 microM, respectively). Moreover, at 1 microM, (R)-BM-591 totally inhibited TXA2 synthase activity, expressed as TXB2 production from human platelets, while at the same concentration, (S)-BM-591 poorly reduced the TXB2 synthesis (22%). Finally, in rats, both enantiomers lost the diuretic activity of torasemide. In conclusion, (R)-BM-591 exhibits a higher affinity and antagonism on human platelet TP receptors than (S)-BM-591 as well as a better thromboxane synthase inhibitory potency. In contrast, (S)-BM-591 is more active than the (R)-enantiomer in relaxing smooth muscle contraction of rat aorta and trachea guinea pig. Consequently, (R)-BM-591 represents the best candidate for further development in the field of thrombosis disorders.     

Characterization of novel imidazole derivative, JM-8686, a potent inhibitor of allene oxide synthase

The inhibitory properties of a first synthetic jasmonic acid biosynthesis inhibitor, JM-8686, were investigated. Steady-state kinetic analysis indicates that the compound is a competitive inhibitor of allene oxide synthase (AOS) with a K(i) value of approximate 0.62+/-0.15 microM. Dialysis experiment indicates that AOS inactivation by JM-8686 is reversible. The optical difference spectroscopy analysis of JM-8686 and AOS interaction indicates that JM-8686 induced type II binding spectra with a K(d) value of approximate 1.6+/-0.2 microM, suggesting that JM-8686 binds to the prosthetic heme iron of AOS. Comparison of the inhibitory potency of the compound against HPL (CYP74B) from tomato revealed that JM-8686 was a highly selective inhibitor for AOS.     

Facile entry to (-)-(R)- and (+)-(S)-mexiletine

The title compounds, 1a and 1b, have been synthesized in a three-step sequence starting from (-)-(S) and (+)-(R)-propylene oxide, respectively, in acceptable overall yields. The enantiomeric excess values for 1a and 1b were 96% and 93% respectively, as assessed by HPLC analysis on a chiral stationary phase of the corresponding N-acetyl derivatives. The synthetic route herein presented may represent a facile entry to highly enriched mexiletine enantiomers, alternative to those previously reported in the literature.     

Synthesis and antipicornavirus activity of (R)- and (S)-1-[5-(4'-chlorobiphenyl-4-yloxy)-3-methylpentyl]-3-pyridin-4-yl-imidazolidin-2-one

The new pyridyl imidazolidinone derivative, 1-[5-(4'-chlorobiphenyl-4-yloxy)-3-methylpentyl]-3-pyridin-4-yl-imidazolidin-2-one (+/-)-1a, was synthesized and found to have an excellent antiviral activity against EV71 (IC50 = 0.009 microM). Therefore, both the enantiomers, (S)-(+)-1a and (R)-(-)-1a, have been prepared starting from readily available monomethyl (R)-3-methylglutarate (7) as a useful chiral building block and their antiviral activity was evaluated in a plaque reduction assay. Interestingly, we observed that the enantiomer (S)-(+)-1a was 10-fold more active against enterovirus71 (EV71) (IC50 = 0.003 microM) than the corresponding enantiomer (R)-(-)-1a (IC50 = 0.033 microM). Similar results were found against all five strains (1743, 2086, 2231, 4643, and BrCr) of EV71 tested. This demonstrated that the absolute configuration of the chiral carbon atom at the 3-position of the alkyl linker considerably influenced the anti-EV71 activity of these pyridyl imidazolidinones.     

Design and synthesis of novel imidazole derivatives as potent inhibitors of allene oxide synthase(CYP74)

Allene oxide synthase (AOS) is a key enzyme in the oxylipin pathway in plants leading to jasmonic acid and other jasmonates (JAs), important signal mediators of defense signal networks in plants. AOS uses hydroperoxylinolenic acid as an oxygen donor as well as the substrate, thus the biochemical conversion of 13(S)-hydroperoxylinolenic acid to allene oxide can proceed in the absence of oxygen and NADPH. We have designed the synthesized of a series of novel imidazole derivatives and tested them in a bioassay as AOS inhibitors using a purified recombinant AOS enzyme isolated from Arabidopsis and expressed in E. coli. Among the derivatives prepared, heptyl 8-[1-(2,4-dichlorophenyl)-2-imidazolylethoxy]octanoate (k) was found to be the most potent inhibitor, with an IC₅₀ of 10±5 nM, which is 250,000-fold and 1,000,000-fold more potent than the known AOS inhibitors, acetylsalicyclic acid (2.5 mM) and ketoconazole (10 mM), respectively.     

Resolution, absolute stereochemistry, and enantiopharmacology of the GluR1-4 and GluR5 antagonist 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid

The phosphono amino acid, (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propio nic acid (ATPO), is a structural hybrid between the NMDA antagonist (RS)-2-amino-7-phosphonoheptanoic acid (AP7) and the AMPA and GluR5 agonist, (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA). ATPO has been resolved into (S)-ATPO and (R)-ATPO using chiral HPLC, and the absolute stereochemistry of the two enantiomers was established by an X-ray crystallographic analysis of (R)-ATPO. (S)-ATPO and (R)-ATPO were characterized pharmacologically using rat brain membrane binding and electrophysiologically using the cortical wedge preparation as well as homo- or heteromeric GluR1-4, GluR5-6, and KA2 receptors expressed in Xenopus oocytes. (R)-ATPO was essentially inactive as an agonist or antagonist in all test systems. (S)-ATPO was an inhibitor of the binding of [(3)H]AMPA (IC(50) = 16 +/- 1 microM) and of [(3)H]-6-cyano-7-nitroquinoxaline-2,3-dione ([(3)H]CNQX) (IC(50) = 1.8 +/- 0.2 microM), but was inactive in the [(3)H]kainic acid and the [(3)H]-(RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([(3)H]CPP) binding assays. (S)-ATPO did not show detectable agonist effects at any of the receptors under study, but antagonized AMPA-induced depolarization in the cortical wedge preparation (IC(50) = 15 +/- 1 microM). (S)-ATPO also blocked kainic acid agonist effects at GluR1 (K(i) = 2.0 microM), GluR1+2 (K(i) = 3.6 microM), GluR3 (K(i) = 3.6 microM), GluR4 (K(i) = 6.7 microM), and GluR5 (K(i) = 23 microM), but was inactive at GluR6 and GluR6+KA2. Thus, although ATPO is a structural analog of AP7 neither (S)-ATPO nor (R)-ATPO are recognized by NMDA receptor sites.     

2-Amino-3-(3-hydroxy-1,2,5-thiadiazol-4-yl)propionic acid: resolution, absolute stereochemistry and enantiopharmacology at glutamate receptors

In order to identify new subtype-selective (S)-glutamate (Glu) receptor ligands we have synthesized (RS)-2-amino-3-(3-hydroxy-1,2,5-thiadiazol-4-yl)propionic acid [(RS)-TDPA]. Resolution of (RS)-TDPA by chiral chromatography was performed using a Crownpac CR(+) column affording (R)- and (S)-TDPA of high enantiomeric purity (enantiomeric excess=99.9%). An X-ray crystallographic analysis revealed that the early eluting enantiomer has R-configuration. Both enantiomers showed high affinity as well as high agonist activity at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, determined using a [(3)H]AMPA binding assay and an electrophysiological model, respectively. The affinities and agonist activities obtained for (R)-TDPA (IC(50)=0.265 microM and EC(50)=6.6 microM, respectively) and (S)-TDPA (IC(50)=0.065 microM and EC(50)=20 microM, respectively) revealed a remarkably low AMPA receptor stereoselectivity, (S)-TDPA showing the highest affinity and (R)-TDPA the most potent agonist activity. In addition, (S)-TDPA was shown to interact with synaptosomal Glu uptake sites displacing [(3)H](R)-aspartic acid (IC(50 ) approximately 390 microM). An enantiospecific and subtype-selective agonist activity was observed for (S)-TDPA at group I metabotropic Glu (mGlu) receptors (EC(50)=13 microM at mGlu(5) and EC(50)=95 microM at mGlu(1)).     

Separation and quantitation of (R)- and (S)-atenolol in human plasma and urine using an alpha 1-AGP column

A method for the determination of (R)- and (S)-atenolol in human plasma and urine is described. The enantiomers of atenolol are extracted into dichloromethane containing 3% heptafluorobutanol followed by acetylation with acetic anhydride at 60 degrees C for 2 h. The acetylated enantiomers were separated on a chiral alpha 1-AGP column. Quantitation was performed using fluorescence detection. A phosphate buffer pH 7.1 (0.01 M phosphate) containing 0.25% (v/v) acetonitrile was used as mobile phase. The described procedure allows the detection of less than 6 ng of each enantiomer in 1 ml plasma. The relative standard deviation is 4.4% at 30 ng/ml of each enantiomer in plasma. The plasma concentration of (R)- and (S)-atenolol did not differ significantly in two subjects who received a single tablet of racemic atenolol. The R/S ratio of atenolol in urine was approximately 1.     

Endothelium-dependent relaxation of rat aorta to a histamine H(3) agonist is reduced by inhibitors of nitric oxide synthase, guanylate cyclase and Na,K-ATPase

The possible involvement of different effector systems (nitric oxide synthase, guanylate cyclase, beta-adrenergic and muscarinic cholinergic receptors, cyclooxygenase and lipoxygenase, and Na(+),K(+)-ATPase) was evaluated in a histamine H(3) receptor agonist-induced ((R)alpha-methylhistamine, (R)alpha-MeHA) endothelium-dependent rat aorta relaxation assay. (R)alpha-MeHA (0.1 nM - 0.01 mM) relaxed endothelium-dependent rat aorta, with a pD(2) value of 8.22 +/- 0.06, compared with a pD(2) value of 7.98 +/- 0.02 caused by histamine (50% and 70% relaxation, respectively). The effect of (R)alpha-MeHA (0.1 nM - 0.01 mM) was competitively antagonized by thioperamide (1, 10 and 30 nM) (pA(2) = 9.21 +/- 0.40; slope = 1.03 +/- 0.35) but it was unaffected by pyrilamine (100 nM), cimetidine (1 muM), atropine (10 muM), propranolol (1 muM), indomethacin (10 muM) or nordthydroguaiaretic acid (0.1 mM). Inhibitors of nitric oxide synthase, L-N(G)-monomethylarginine (L-NMMA, 10 muM) and N(G)-nitro-L-arginine methylester (L-NOARG, 10 muM) inhibited the relaxation effect of (R)alpha-MeHA, by approximately 52% and 70%, respectively). This inhibitory effect of L-NMMA was partially reversed by L-arginine (10 muM). Methylene blue (10 muM) and ouabain (10 muM) inhibited relaxation (R)alpha-MeHA-induced by approximately 50% and 90%, respectively. The products of cyclooxygenase and lipoxygenase are not involved in (R)alpha-MeHA-induced endothelium-dependent rat aorta relaxation nor are the muscarinic cholinergic and beta-adrenergic receptors. The results also suggest the involvement of NO synthase, guanylate cyclase and Na(+),K(+)-ATPase in (R)alpha-MeHA-induced endothelium-dependent rat aorta relaxation.     

Differential effects of (R)-, (R, S)- and (S)-8-hydroxy-2-(di-n-propylamino)tetralin on hippocampal serotonin release and induction of hypothermia in awake rats

The effects of (R)- and (S)-optical isomers of 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and of the racemate (R,S)-8-OH-DPAT on serotonin (5-HT) release in the ventral hippocampus of awake rats and on induction of the whole-body hypothermia were studied. Extracellular 5-HT levels were determined by a newly developed high-sensitive HPLC method based on derivatization with benzylamine and fluorescence detection. The basal levels of 5-HT in 20 min microdialysates from rats perfused with Ringer solution or with Ringer solution containing 1 microM citalopram were 6.3 +/- 1.3 fmol/20 microl and 36.1 +/- 4.2 fmol/20 microl (n=20), respectively. The reduction of hippocampal 5-HT levels induced by subcutaneous (s.c.) administration of (R,S)-8-OH-DPAT (0.3 mg/kg) was significantly attenuated by the presence of 5-HT reuptake inhibitor citalopram in Ringer solution only at its peak value at 40 min (maximal reduction to 60% compared to 46% of control values in Ringer-perfused rats), whereas the overall effects were comparable at both experimental conditions. Injection of (R)-8-OH-DPAT (0.3 mg/kg s.c.) caused further reduction of 5-HT levels, to 49% and 41%, respectively, whereas (S)-8-OH-DPAT (0.3 mg/kg s.c.) caused maximal reduction of 5-HT levels only to 74% of controls in both perfusion groups. Similar pattern and time-courses were observed in rats with hypothermia induced by injection of 8-OH-DPAT enantiomers, where (R,S), (R)-forms were about two-times more potent than the (S)-isomer. It is concluded that the acute systemic dose of (R)-, (S)- and (R,S)-8-OH-DPAT enantiomers exerted enantiomer-specific effects on 5-HT(1A) receptor-mediated function both at the presynaptic and postsynaptic sites as revealed by monitoring hippocampal 5-HT levels and body temperature.     

Synthesis and analysis of the enantiomers of calmidazolium,and a 1H NMR demonstration of a chiral interaction with calmodulin

Calmidazolium {R24571, 1-[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy]ethyl]-1H-imidazolium chloride} is a potent calmodulin inhibitor. This paper describes the synthesis and properties of the enantiomers of calmidazolium from the enantiomers of miconazole {1(N)-(2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl))-ethyl imidazole}, prepared from the racemate by chiral preparative scale high performance liquid chromatography. Overlap between ligand and protein resonances in the aromatic region of the ¹H NMR spectrum of the calmidazolium-calmodulin complexes has been obviated by preparation of the protein with all of its nine phenylalanine rings deuterated (Phe-d₅ calmodulin). This has been accomplished by the overexpression of calmodulin derived from Trypanosoma brucei rhodiesiense in E. coli in a medium supplemented with ring-deuterated phenylalanine. The kinetics of binding of each enantiomer are slow on the ¹H NMR time scale as judged by the behaviour of the H2 resonance of Histidine-107, which is clearly visible under the sample conditions used. The aromatic spectral regions of the protein-bound (+) and (−) enantiomers contrast strikingly, reflecting differences in bound environment and/or conformation. Chirality 8:545–500, 1996. © 1997 Wiley-Liss, Inc.     

Asymmetric chloronicotinyl insecticide, 1-[1-(6-chloro-3-pyridyl)ethyl]-2-nitroiminoimidazolidine: preparation,resolution and biological activities toward insects and their nerve preparations

The asymmetric chloronicotinyl insecticide, 1-[1-(6-chloro-3-pyridyl)ethyl]-2-nitroiminoimidazolidine, was prepared, and the absolute configurations of the enantiomers were determined by an X-ray analysis. The insecticidal activity against the housefly measured with metabolic inhibitors showed the (S) enantiomer to be slightly more active than the (R) isomer. Electrophysiological measurements on the American cockroach central nerve cord showed the compounds to elicite the impulses and subsequently blocked them. The neuroblocking potency of the (S) isomer was 5.9 microM, while that of the (R) isomer was as high as 73 microM. The molar concentrations required for 50% inhibition of the specific binding of [3H]imidacloprid to the housefly head membrane preparation were respectively 0.19 microM and 0.95 microM for the (S) and (R) isomers. This enatioselectivity ratio was smaller than 35 for nicotine isomers but greater than 2 for epibatidine isomers.     

Syntheses of quinolone hydrochloride enantiomers from synthons (R)- and (S)-2-methylpiperazine

A series of R and S enantiomers of 7-(3-methylpiperazin-1-yl) quinolone derivatives were synthesized from (R)- and (S)-tert-butyl 2-methylpiperazine-1-carboxylate and tested for their antibacterial activities on 14 kinds of bacteria. Although no distinct difference in in vitro antibacterial activities was observed, 2-64-fold difference between R and S enantiomers was observed in approximately 52% of cases.     

Synthesis and biological evaluation of 1-(2,4,5-trisubstituted phenyl)-3-(5-cyanopyrazin-2-yl)ureas as potent Chk1 kinase inhibitors

Based on the X-ray crystallography of our lead compound 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-cyanopyrazin-2-yl)urea in the checkpoint kinase 1 (Chk1) enzyme, we modified R4, and to a lesser extent, R2, and R5 of the phenyl ring, and made a variety of N-aryl-N'-pyrazinylurea Chk1 inhibitors. Enzymatic activity less than 20 nM was observed in 15 of 41 compounds. Compound 8i provided the best overall results in the cellular assays as it abrogated doxorubicin-induced cell cycle arrest (IC50=1.7 microM) and enhanced doxorubicin cytotoxicity (IC50=0.44 microM) while displaying no single agent activity.     

Development and validation of a chiral liquid chromatographic method, based on Chiralpak to quantify enantiomers of (+/-)-DRF 2725 in rat plasma: lack of inversion of ragaglitazar (S(-)-DRF 2725) to its antipode in plasma

A selective, accurate and reproducible high-performance liquid chromatographic (HPLC) method for the separation of individual enantiomers of DRF 2725 [R(+)-DRF 2725 and S(-)-DRF 2725 or ragaglitazar] was obtained on a chiral HPLC column (Chiralpak). During method optimization, the separation of enantiomers of DRF 2725 was investigated to determine whether mobile phase composition, flow-rate and column temperature could be varied to yield the base line separation of the enantiomers. Following liquid-liquid extraction, separation of enantiomers of DRF 2725 and internal standard (I.S., desmethyl diazepam) was achieved using an amylose based chiral column (Chiralpak AD) with the mobile phase, n-hexane-propanol-ethanol-trifluoro acetic acid (TFA) in the ratio of 89.5:4:6:0.5 (v/v). Baseline separation of DRF 2725 enantiomers and I.S., free from endogenous interferences, was achieved in less than 25 min. The eluate was monitored using an UV detector set at 240 nm. Ratio of peak area of each enantiomer to I.S. was used for quantification of plasma samples. Nominal retention times of R(+)-DRF 2725, S(-)-DRF 2725 and I.S. were 15.8, 17.7 and 22.4 min, respectively. The standard curves for DRF 2725 enantiomers were linear (R(2) > 0.999) in the concentration range 0.3-50 microg/ml for each enantiomer. Absolute recovery, when compared to neat standards, was 70-85% for DRF 2725 enantiomers and 96% for I.S. from rat plasma. The lower limit of quantification (LLOQ) for each enantiomers of DRF 2725 was 0.3 microg/ml. The inter-day precisions were in the range of 1.71-4.60% and 3.77-5.91% for R(+)-DRF 2725, S(-)-DRF 2725, respectively. The intra-day precisions were in the range of 1.06-11.5% and 0.58-12.7% for R(+)-DRF 2725, S(-)-DRF 2725, respectively. Accuracy in the measurement of quality control (QC) samples was in the range 83.4-113% and 83.3-113% for R(+)-DRF 2725, S(-)-DRF 2725, respectively. Both enantiomers and I.S. were stable in the battery of stability studies viz., bench-top (up to 6 h), auto-sampler (up to 12 h) and freeze/thaw cycles (n = 3). Stability of DRF 2725 enantiomers was established for 15 days at -20 degrees C. The application of the assay to a pharmacokinetic study of ragaglitazar [S(-)-DRF 2725] in rats is described. It was unequivocally demonstrated that ragaglitazar does not undergo chiral inversion to its antipode in vivo in rat plasma.     

Enantioseparation, absolute configuration determination, and anticonvulsant activity of (+/-)-1-(4-aminophenyl)-7,8-methylenedioxy-1,2,3,5-tetrahydro-4H-2,3-benzodiazepin-4-one

The resolution of 1-(4-aminophenyl)-7,8-methylenedioxy-1,2,3,5-tetrahydro-4H-benzodiazepin-4-one (+/-)-(R,S)-2 was accomplished by chiral HPLC. The absolute configuration of (+)-2, determined by X-ray crystallographic analysis, was R. The in vivo anticonvulsant activity of the enantiomers (+)-(R)-2 and (-)-(S)-2 is reported. It has been also demonstrated that compound (+/-)-(R,S)-2 in vivo undergoes oxidative metabolism to derivative 1.     

Synthesis, resolution, stereochemistry, and molecular modeling of (R)- and (S)-2-acetyl-1-(4'-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline AMPAR antagonists

Recently we identified (R,S)-2-acetyl-1-(4'-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (6) as a potent non-competitive AMPA receptor antagonist able to prevent epileptic seizures. We report here the optimized synthesis of compound 6, its resolution by chiral preparative HPLC, and the absolute configuration of (R)-enantiomer established by X-ray diffractometry. The biological tests of the single enantiomers revealed that higher anticonvulsant and antagonistic effects reside in (R)-enantiomer as also suggested by molecular modeling studies.     

Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers.

Stereoselective drug-channel interactions may help to elucidate the molecular basis of voltage-gated potassium channel block by local anesthetic drugs. We studied the effects of the enantiomers of bupivacaine on a cloned human cardiac potassium channel (hKv1.5). This channel was stably expressed in a mouse Ltk- cell line and studied using the whole-cell configuration of the patch-clamp technique. Both enantiomers modified the time course of this delayed rectifier current. Exposure to 20 microM of either S(-)-bupivacaine or R(+)-bupivacaine did not modify the activation time constant of the current, but reduced the peak outward current and induced a subsequent exponential decline of current with time constants of 18.7 +/- 1.1 and 10.0 +/- 0.9 ms, respectively. Steady-state levels of block (assessed with 250-ms depolarizing pulses to +60 mV) averaged 30.8 +/- 2.5% (n = 6) and 79.5 +/- 3.2% (n = 6) (p < 0.001), for S(-)- and R(+)-bupivacaine, respectively. The concentration dependence of hKv1.5 inhibition revealed apparent KD values of 27.3 +/- 2.8 and 4.1 +/- 0.7 microM for S(-)-bupivacaine and R(+)-bupivacaine, respectively, with Hill coefficients close to unity, suggesting that binding of one enantiomer molecule per channel was sufficient to block potassium permeation. Analysis of the rate constants of association (k) and dissociation (l) yielded similar values for l (24.9 s-1 vs. 23.6 s-1 for S(-)- and R(+)-bupivacaine, respectively) but different association rate constants (1.0 x 10(6) vs. 4.7 x 10(6) M-1 s-1 for S(-)- and R(+)-bupivacaine, respectively). Block induced by either enantiomer displayed a shallow voltage dependence in the voltage range positive to 0 mV, i.e., where the channel is fully open, consistent with an equivalent electrical distance delta of 0.16 +/- 0.01. This suggested that at the binding site, both enantiomers of bupivacaine experienced 16% of the applied transmembrane electrical field, referenced to the inner surface. Both bupivacaine enantiomers reduced the tail current amplitude recorded on return to -40 mV and slowed their time course relative to control, resulting in a "crossover" phenomenon.(ABSTRACT TRUNCATED AT 400 WORDS)     

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.