Kinetic Study on the Base-Catalyzed Imine-Enamine Tautomerism of a Chiral Biologically Active Isoxazoline Derivative by HPLC on Amylose Tris(3,5-dimethylphenylcarbamate) Chiral Stationary Phase.

Isoxazoline is a nitrogen- and oxygen-containing five-membered heterocyclic scaffold with diverse biological profiles such as antimicrobial, fungicidal, anticancer, antiviral, analgesic and anti-inflammatory activity. Accordingly, the use of this peculiar structural framework in drug discovery is a successful strategy for the development of new drug candidates. Here, a chiral saccharin/isoxazoline hybrid was considered to investigate the tendency of the imine moiety of the heterocyclic ring to tautomerize to the enamine form in the presence of a basic catalyst. The pseudo-first-order rate constants for the base-catalyzed tautomerization process were measured in different solvents and at different temperatures by off-column kinetic experiments based on the amylose (3,5-dimethylphenylcarbamate)-type chiral stationary phase. The kinetic results obtained in this study may be a useful aid in the perspective of designing experimental conditions to control the stereointegrity of these types of pharmacologically active compounds and drive their synthesis toward the preferred, imine or enamine, tautomer.

Improving the performance of nadolol stereoisomers' preparative separation using Chiralpak IA by SMB chromatography.

The pseudobinary preparative separation of nadolol stereoisomers is performed by simulated moving bed chromatography (SMB). Using the Chiralpak IA adsorbent, a new 25:75:0.1 (v/v/v) methanol-acetonitrile-diethylamine solvent composition was selected to perform the experimental SMB separation and compare it with the previous results obtained using pure methanol. Using a 2 g L-1 total feed concentration of an equimolar mixture of the four stereoisomers of nadolol, the more retained component was fully recovered (100% purity and 100% recovery), with a system productivity of 0.77 g L-1  hour-1 and a solvent consumption of 9.62 L g-1 . Comparing these results with the ones previously reported using 100:0.1 methanol-diethylamine solvent composition, this work shows that the 25:75:0.1 methanol-acetonitrile-diethylamine is a better alternative for the preparative separation of nadolol stereoisomers by SMB chromatography. These results are confirmed by simulation of the SMB operation for higher feed concentrations, by comparing the performances of the two solvent compositions using the data obtained experimentally through the measurement of the adsorption equilibrium isotherms and the kinetic data obtained for both solvents. The new experimental and simulation results stress out that the performance of the preparative separation can be improved by a careful selection of the solvent composition.

Validation of an enantioselective LC-MS/MS method to quantify enantiomers of (±)-OTX015 in mice plasma: Lack of in vivo inversion of (-)-OTX015 to its antipode.

A highly sensitive, specific and enantioselective assay has been validated for the quantitation of OTX015 enantiomers [(+)-OTX015 and (-)-OTX015] in mice plasma on LC-MS/MS-electrospray ionization as per regulatory guidelines. Protein precipitation was used to extract (±)-OTX015 enantiomers and internal standard (IS) from mice plasma. The active [(-)-OTX015] and inactive [(+)-OTX015] enantiomers were resolved on a Chiralpak-IA column using an isocratic mobile phase (0.2% ammonia/acetonitrile 20 : 80, v/v) at a flow rate of 1.2 mL/min. The total run time was 6.0 min. (+)-OTX015, (-)-OTX015 and IS eluted at 3.34, 4.08 and 4.77 min, respectively. The MS/MS ion transitions monitored were m/z 492 → 383 for OTX015 and m/z 457 → 401 for IS. The standard curves for OTX015 enantiomers were linear (r2 > 0.998) in the concentration range 1.03-1030 ng/mL. The inter- and intraday precisions were in the range 2.20-13.3 and 8.03-12.1% and 3.80-14.4 and 8.97-13.6% for (+)-OTX015 and (-)-OTX015, respectively. Both the enantiomers were found to be stable in a battery of stability studies. This novel method has been applied to the study of stereoselective oral pharmacokinetics of (-)-OTX015 and unequivocally demonstrated that (-)-OTX015 does not undergo chiral inversion to its antipode in vivo in mice.

Studies on Pidotimod Enantiomers With Chiralpak-IA: Crystal Structure, Thermodynamic Parameters and Molecular Docking.

Pidotimod, a synthetic dipeptide, has two chiral centers with biological and immunological activity. Its enantiomers were characterized by x-ray crystallographic analysis. A chiral stationary phase (CSP) Chiralpak-IA based on amylose derivatized with tris-(3, 5-dimethylphenyl carbamate) was used to separate pidotimod enantiomers. The mobile phase was prepared in a ratio of 35:65:0.2 of methyl-tert-butyl-ether and acetonitrile trifluoroaceticacid. In addition, thermodynamics and molecular docking methods were used to explain the enantioseparation mechanism by Chiralpak-IA. Thermodynamic studies were carried out from 10 to 45 °C. In general, both retention and enantioselectivity decreased as the temperature increased. Thermodynamic parameters indicate that the interaction force between the pidotimod enantiomer (4S, 2'R) and IA CSP is stronger and their complex model is more stable. According to GOLD molecular docking simulation, Van der Waals force is the leading cause of pidotimod enantiomers separation by IA CSP.

Stereolability of chiral ruthenium catalysts with frozen NHC ligand conformations investigated by dynamic-HPLC.

The stereolability of chiral Hoveyda-Grubbs II type ruthenium complexes bearing N-heterocyclic carbene (NHC) ligands with Syn-phenyl groups on the backbone and Syn- or Anti-oriented o-tolyl N-substituents was studied by resorting to dynamic high-performance liquid chromatography (D-HPLC). A complete chromatographic picture of the involved stereoisomers (four for Anti- and two for Syn-complexes) was achieved at very low temperatures (-53°C and -40°C respectively), at which the NHC-Ru bond rotations were frozen out. Inspection of the chromatographic profiles recorded at higher temperatures revealed the presence of plateau zones between the couples of either Syn or Anti stereoisomers, attesting to the active interconversion between the eluted species. Such dynamic chromatograms were successfully simulated through procedures based on both theoretical plate and classical stochastic models. The good superimposition achieved between experimental and simulated chromatographic profiles allowed determination of the related isomerization energy barriers (ΔGisom (#) ), all derived by rotation around the NHC-Ru bond. The obtained diastereomerization barriers between the Anti isomers were found in very good agreement with those previously measured by experimental nuclear magnetic resonance (NMR) and assessed through Density Functional Theory (DFT) calculations. With the same approach, for the first time we also determined the enantiomerization barrier of the Syn isomer. Focused changes to the structure of complex Syn, studied by a molecular modeling approach, were found suitable to strongly reduce the stereolability arising from rotation around the NHC-Ru bond.

Dried blood spot analysis of (+) and (-) darunavir enantiomers on immobilized amylose tris-(3, 5-dimethylphenylcarbamate) LC and its application to pharmacokinetics.

Dried blood spot analysis is an innovative novel blood sampling technique gaining interest in drug discovery and development processes owing to its inherent advantages over the conventional whole blood, plasma or serum sample collection. The present manuscript describes the development and validation of a highly sensitive and precise method of evaluation of pharmacokinetics of (+) and (-) darunavir enantiomers on rat dried blood spots. The enantiomers on rat dried blood spots were extracted into methanol and separated by LC on a Chiralpak IA column using hexane and ethanol containing 0.1% DEA (75:25, v/v) as a mobile phase at 20°C; both the enantiomers were detected at 266 nm using a photodiode array detector. The method was validated in terms of selectivity, linearity, accuracy, precision and stability as per the US Food and Drug and Administration guidelines. The hematocrit effect on extraction recovery was evaluated and the mean recoveries of (-) and (+) enantiomers of darunavir from dried blood spots were found to be 85.76 and 88.91% respectively. The intra- and inter-day precision and accuracy were 3.1-8.4 and 0.8-4.8% respectively. The developed method was successfully applied to a pharmacokinetic study of (+) and (-) enantiomers of darunavir on rat dried blood spots.

Enantiomeric separation of indoxacarb on an amylose-based chiral stationary phase and its application in study of indoxacarb degradation in water.

Direct semipreparative enantioseparation of indoxacarb was performed on a semipreparative Chiralpak IA column using normal-phase high-performance liquid chromatography (HPLC) with n-hexane-isopropanol-ethyl acetate (70:20:10) mixture as mobile phase. Degradation of indoxacarb (2.33S + 1R) and its two enantiopure isoforms in three aqueous buffer solutions and four water samples collected from natural water sources was then elucidated by HPLC analysis on Chiralpak IA column. Degradation of all three indoxacarbs complied with first-order kinetics and demonstrated linearity with regression coefficients R(2) > n0.88. Indoxacarb (2.33S + 1R) underwent enantioselective degradation in river water, rain water, and buffer solution of pH 7.0. Enantiopure S-(+)-indoxacarb and R-(-)-indoxacarb were both found to be configurationally stable in water.

Simultaneous enantio- and diastereo-selective high-performance liquid chromatography separation of paroxetine on an immobilized amylose-based chiral stationary phase under green reversed-phase conditions.

A simple and green high-performance liquid chromatography method for the separation of paroxetine from its enantiomeric and diastereomeric impurities has been developed. The simultaneous chromatographic resolution was carried out on the amylose-based Chiralpak IA-3 chiral stationary phase using the mixture ethanol-water-diethylamine 80:20:0.1 (v/v/v) as a mobile phase. The effects of substitution of ethanol with methanol or acetonitrile and changes in column temperature on selectivity have been carefully investigated. The optimized single-run HPLC protocol allows the baseline separation of the enantiomers of paroxetine without suffering from interference from five other chiral and achiral impurities reported in the monograph of the European Pharmacopoeia.

Preliminary kinetic evaluation of an immobilized polysaccharide sub-2µm column using a low dispersion supercritical fluid chromatograph.

The performance of a 3×50mm, 1.6µm dp column with an immobilized polysaccharide stationary phase (ChiralPak IA-U) was evaluated for efficiency, and pressure drop, with respect to flow rate and modifier concentration using supercritical fluid chromatography (SFC). This appears to be the first such report using such a column in SFC. A unique low dispersion (ultra-high performance) SFC was used for the evaluation. The minimum reduced plate height of 2.78, indicates that the maximum efficiency was similar to or better than coated polysaccharide columns. Selectivity was different from ChiralPak AD, with the same chiral selector, as reported by many others. At high flows and high methanol concentrations, pump pressures sometimes approached 600bar. With 5% methanol, pressure vs. flow rate was non-linear suggesting turbulent flow in the connector tubing. The optimum flow rate (Fopt) at 40% methanol was ≈0.8mL/min, where the column efficiency was highest. At 5% methanol, Fopt increased to ≈1.6mL/min, but efficiency degraded noticeably. The differences in Fopt suggests that the solute diffusion coefficients are a strong function of modifier concentration. Several sub-1min separations, including a 7.5s separation, are presented.

Analytical and semipreparative high performance liquid chromatography enantioseparation of bicalutamide and its chiral impurities on an immobilized polysaccharide-based chiral stationary phase.

Direct HPLC separation of enantiomers of Bicalutamide (BCT), a non-steroidal antiandrogen used for the treatment of prostate cancer, was performed by using the immobilized amylose-based Chiralpak IA chiral stationary phase (CSP). Enantioselective conditions were achieved using standard normal phase mixtures n-hexane-alcohol (ethanol or 2-propanol) and a "non-standard" mobile phase containing ethyl acetate (EA). The chromatographic behaviour of the IA CSP under these elution modes was evaluated and compared at different temperatures. The eluent mixture n-hexane-EA-ethanol 100-30-5 (v/v/v) and the column temperature of 40°C were identified as the best operational conditions to carry out semipreparative enantioseparations on a 1-cm I.D. IA column. Using this protocol, about 960mg of (R)-BCT, which is the enantiomer with the almost entire anti-androgenic activity of BCT, per day could be isolated. The analytical and semipreparative HPLC resolution of chiral impurities of BCT, and their empiric absolute configuration assignment by circular dichroism correlation method are also presented.

Pharmaceutical-enantiomers resolution using immobilized polysaccharide-based chiral stationary phases in supercritical fluid chromatography.

Since their introduction on the market the applicability of immobilized polysaccharide-based chiral stationary phases in high-performance liquid chromatography has been thoroughly investigated. These immobilized phases have the benefit to be applicable with a wide range of modifiers, potentially extending the application range of the polysaccharide-based stationary phases. Because an increasing number of stationary phases are being introduced in the field of chiral chromatography it is important to evaluate their enantioselectivity in different techniques in order to get an idea about their applicability. In this study, three immobilized chiral polysaccharide-based stationary phases (Chiralpak IA, IB, and IC) are evaluated in supercritical fluid chromatography (SFC) with a test set of pharmaceutical racemates. This is done in a three-fold manner: their performance is evaluated (1) using traditional modifiers, (2) using mixtures of atypical modifiers, and (3) the results were compared to those on coated stationary phases with an equivalent chiral selector. To get a visual overview of the enantioselective patterns of the different chromatographic systems (mobile and stationary phase combinations), a Principal Component Analysis is performed, which allows determining the (dis)similarity between individual systems. To assess the complementarity cumulative success rates are determined. The immobilized chiral stationary phases prove to yield high cumulative success rates.