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.     

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 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.     

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)).     

Resolution, configurational assignment, and enantiopharmacology at glutamate receptors of 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) and demethyl-ACPA

We have previously described (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) as a potent agonist at the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of (S)-glutamic acid (Glu) receptors. We now report the chromatographic resolution of ACPA and (RS)-2-amino-3-(3-carboxy-4-isoxazolyl)propionic acid (demethyl-ACPA) using a Sumichiral OA-5000 column. The configuration of the enantiomers of both compounds have been assigned based on X-ray crystallographic analyses, supported by circular dichroism spectra and elution orders on chiral HPLC columns. Furthermore, the enantiopharmacology of ACPA and demethyl-ACPA was investigated using radioligand binding and cortical wedge electrophysiological assay systems and cloned metabotropic Glu receptors. (S)-ACPA showed high affinity in AMPA binding (IC(50) = 0.025 microM), low affinity in kainic acid binding (IC(50) = 3.6 microM), and potent AMPA receptor agonist activity on cortical neurons (EC(50) = 0.25 microM), whereas (R)-ACPA was essentially inactive. Like (S)-ACPA, (S)-demethyl-ACPA displayed high AMPA receptor affinity (IC(50) = 0.039 microM), but was found to be a relatively weak AMPA receptor agonist (EC(50) = 12 microM). The stereoselectivity observed for demethyl-ACPA was high when based on AMPA receptor affinity (eudismic ratio = 250), but low when based on electrophysiological activity (eudismic ratio = 10). (R)-Demethyl-ACPA also possessed a weak NMDA receptor antagonist activity (IC(50) = 220 microM). Among the enantiomers tested, only (S)-demethyl-ACPA showed activity at metabotropic receptors, being a weak antagonist at the mGlu(2) receptor subtype (K(B) = 148 microM).     

Stereochemistry of the Enzymatic Reduction of 2-(4-Methoxybenzyl)-1-Cyclohexanone by Solanum Aviculare Cells in vitro

During the investigation of chemical properties of the dicyclic system of insect juvenile hormone analogues, biotransformation of 2-(4-methoxybenzyl)-1-cyclohexanone (1) by plant cell cultures was studied. Among other components, the cis-(1S, 2S)- and cis-(1R, 2R)-2-(4-methoxybenzyl)-1-cyclohexanol enantiomers 2a and 2b were found in the reaction mixture. Higher stereoselectivity of the biotransformation was observed for trans-(1S, 2R)-enantiomer 3a formation, which occurred in at least 60% of calculated enantiomeric excess.     

Asymmetric synthesis and stereochemical structure-activity relationship of (R)- and (S)-8-[1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethoxy] octanoic acid heptyl ester, a potent inhibitor of allene oxide synthase

The preparation of both enantiomers of 8-[1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethoxy] octanoic acid heptyl ester (JM-8686), a potent inhibitor of allene oxide synthase, has been achieved using 2,4-dichlorophenacyl bromide as a starting material. The key step was the asymmetric reduction of 1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethanone with chiral BINAL-H. The products were purified by chiral high-performance liquid chromatography (HPLC) to afford pure (R)-JM-8686 and (S)-JM-8686. The inhibitory activities and binding affinities of these enantiomers toward allene oxide synthase were determined. We found that the inhibition potency of (R)-JM-8686 is approximately 200 times greater than that of (S)-JM-8686, with IC(50) values of approximately 5+/-0.2 nM and 950+/-18 nM, respectively. The dissociation constants of (R)-JM-8686 and (S)-JM-8686 with respect to the recombinant allene oxide synthase were approximately 1.4+/-0.3 microM and 4.8+/-0.6 microM, respectively.     

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.     

(R)- and (S)-5-hydroxy-2-(dipropylamino)tetralin (5-OH DPAT): assessment of optical purities and dopaminergic activities

Racemic 5-hydroxy-2-(dipropylamino)tetralin (5-OH DPAT), a potent and selective dopamine (DA) D2-receptor agonist, was resolved into the enantiomers by a new method. The enantiomers of 5-OH DPAT were determined by chiral ion-pair chromatography using N-benzyloxycarbonylglycyl-L-proline as the counter ion. The enantiomeric purity of (R)-5-OH DPAT was found to be greater than 99.7%. The ability of the enantiomers to change the rat brain DOPA levels was evaluated in vivo. The results indicate that (R)-5-OH DPAT is a weakly potent DA D2-receptor antagonist.     

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.     

Intermolecular chiral assemblies in R(-) and S(+) 2-butanol detected by microcalorimetry measurements

Supramolecular chiral assemblies of R(-) and S(+) 2-butanol, in their neat form or when dissolved in their nonchiral isomer isobutanol, were evaluated by isothermal titration calorimetry (ITC) ensuing mixing. Dilution of 0.5 M solution of R(-) 2-butanol in isobutanol into the latter liberated heat of several calories per mole, which was approximately double than that obtained in parallel dilutions of S(+) 2-butanol in isobutanol. The ITC dilution profiles indicated an estimate of about 100 isobutanol solvent molecules surrounding each of the 2-butanol enantiomers, presumably arranged in chiral configurations, with different adopted order between the isomers. Mixings of neat R and S 2-butanol were followed by endothermic ITC profiles, indicating that, in racemic 2-butanol, both the supramolecular order and the intermolecular binding energies are lower than in each of the neat chiral isomers. The diversion from symmetrical ITC patterns in these mixings indicated again a subtle difference in molecular organization between the neat enantiomers. It should be noted that the presence of impurities, α-pinene and teterhydrofuran, at a level totaling 0.5%, did not influence the ITC heat flow profiles. The findings of this study demonstrate for the first time that chiral solutes in organic solvents are expected to acquire asymmetric solvent envelopes that may be different between the enantiomers, thus broadening this phenomenon beyond the previously demonstrated cases in aqueous solutions.     

Enantioselective sulfation of β2-receptor agonists by the human intestine and the recombinant M-form phenolsulfotransferase

The β₂-receptor agonist class of drugs is metabolized in humans almost exclusively by sulfate conjugation. The objective of this investigation was to determine the influence of chemical structure on the stereoselectivity of the sulfoconjugation of these chiral drugs. The pure enantiomers of six β₂-agonists, including those clinically most widely used, were all effectively sulfated both by the cytosol of the human intestine and the recombinant human M-form phenolsulfotransferase (PST). Whereas the apparent K_m values (K_m,app) for the sulfation of the individual drug enantiomers by the intestinal cytosol varied widely, ranging from 4.8 μM for (S)-isoproterenol to 889 μM for (S)-albuterol, these K_m,app values were highly correlated with those obtained with M-PST (correlation coefficient 0.994). In contrast, the M-PST V_max,app values were similar for all drug enantiomers, ranging from 276 to 914 pmol min⁻¹ mg⁻¹ protein, implying that substrate binding to M-PST by far is the main determinant of the sulfation activity. For isoproterenol, the K_m,app for M-PST was 6.1 times higher for the active (R)- than for the inactive (S)-enantiomer. For other β₂-agonists, the stereoselectivity decreased towards unity as the K_m,app increased. However, for albuterol, containing a hydroxymethyl substituent at the aromatic ring, the stereoselectivity was dramatically reversed, with 10 times higher K_m,app for the inactive (S)- than for the active (R)-enantiomer. Chirality 10:800–803, 1998. © 1998 Wiley-Liss, Inc.     

Biological significance of the enantiomeric purity of drugs

The knowledge that enantiomers of chiral compounds may differ widely in biological activity, qualitatively as well as quantitatively, is not new. Nevertheless most of the pharmacological data available to date on chiral drugs are obtained from experiments with racemates which assume that the biological activity generally resides in one of the enantiomers. With the advancements made in stereospecific synthesis and stereoselective analysis of drugs pharmacologists are now offered new possibilities to explore the steric aspects of drug action. This survey will discuss pharmacological data obtained with enantiomer pairs of phenylethylamine derivatives which interact with adrenergic mechanisms. The degree of resolution is seldom specified in published work on stereoselectivity of drugs. In a recent study from our laboratory the enantiomers of the β₂-adrenoceptor agonist formoterol and their diastereomers have been evaluated. We found that the (R;R)-enantiomer was by far the most potent. However, the relative potencies obtained for the (R;S)-, (S;R), and (S;S)- isomers were critically dependent on the degree of enantiomeric purity. It is concluded that the certainty of potency ratios observed for chiral drugs is limited by the enantiomeric purity and by unspecific effects of the least active enantiomer at very high concentrations. © 1993 Wiley-Liss, Inc.     

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.     

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.     

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.     

Diphenylethanediamine (DPEDA) derivatives as chiral selectors: IV. A comparison of 3,5-dinitrobenzoylated (S,S)- and (S,R)-DPEDA-derived chiral stationary phases with Pirkle's standard (R)-phenylglycine-derived phase in normal phase HPLC

Undecanoyl bound 3,5-dinitrobenzoyl-(S,R)-1,2-diphenylethane-1,2-diamine [(1S,2R)-DNB-DPEDA] as chiral selector (SO) has been synthesized and used as a chiral stationary phase (CSP II) for normal-phase enantioselective HPLC. It is compared with the already published diastereomeric (1S,2S)-DNB-DPEDA-derived CSP I and with the “standard” Pirkle DNB-(R)-phenylglycine-derived CSP III. Chromatographic data for about 100 racemic analytes reveal that CSP II is able to separate especially well enantiomers of derivatized aromatic carboxylic acids and analytes having a benzyl substituent bound at the chiral center. However, CSP I was found to be superior to CSP II and III in its general applicability and its ability to resolve enantiomers of heterocyclic drugs. © 1994 Wiley-Liss, Inc.     

Assignment of absolute configuration and optical purity determination of (R)- and (S)-econazole nitrate by enantioselective HPLC: Method development and application

A method is described for the synthesis and optical purity determination of (?)-(R)- and (+)-(S)-econazole via the optically pure intermediates, (R)- and (S)-imidazolylethanol, which are available by chromatographic resolution or by fractional crystallization of diastereomeric O,O′-disubstituted (R*;R*)- or (S*;S*)-tartaric acid monoesters of the parent imidazolylethanol racemate. Furthermore, this method allows the chromatographic assignment of the absolute configuration of the chiral center of the imidazolylethanol enantiomers and consequently of econazole enantiomers. In addition, a direct liquid chromatographic enantioseparation method for the determination of the optical purity of (R)- and (S)-econazole and other chiral imidazoles on a protein type CSP (OVM) is described and applied to confirm chromatographically the absolute configuration evaluations. © 1994 Wiley-Liss, Inc.     

Biomimetic conversion of (3S)-(-)-neodictyoprolenol to optically pure (1S,2R)-(-)-dictyopterene B,marine algal sex pheromone

Both enantiomers of (3S)-(-)- and (3R)-(+)-Neodictyoprolenol [(3S,5Z,8Z)-(-)-1,5,8-undecatrien-3-ol] were successfully converted to the algal sex pheromone, (1S,2R)-(-)-dictyopterene B and (1R,2S)-(+)-dictyopterene B in high enantiomeric purities (e. e. > 99%), respectively, by the biomimetic reaction involving phosphorylation and elimination under a mild condition.