Paul Richert

Applications of simulated moving-bed chromatography to the separation of the enantiomers of chiral drugs

Although most preparative chiral separations have been performed in the conventional batch-mode process, interest in simulated moving-bed (SMB) chromatography is growing, because it permits large amounts of mobile phase to be saved and productivity increased, thus reducing production costs. Based on the examples of three different drugs, the usefulness of this technique for the separation of enantiomers on a preparative scale has been demonstrated. Compared to the batch elution chromatography, a reduction of the mobile phase consumption of respectively 81% and 84% has been achieved for the separation of the enantiomers of the antiasthmatic agent formoterol and of the antitussive agent guaifenesin. Furthermore, a higher throughput could be reached under SMB conditions. The influence of feed rate and extract on the separation has also been investigated. The results show that these two factors considerably affect purity and productivity, and constitute important parameters for finely adjusting the chromatographic conditions depending on the requirements. For all racemates, both enantiomers could be obtained with a purity ranging between 99 and 99.9%.

Development and validation of chiral high-performance liquid chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester.

A stereospecific HPLC method for the quantitation of CGP 49309 in samples of its corresponding enantiomer valsartan has been developed and validated. The enantiomeric separation was achieved on a 5 micron silica-bonded, alpha 1-acid glycoprotein column (Chiral AGP) with a phosphate buffer, pH 7, containing 2% (v/v) 2-propanol as a mobile phase. The linearity was established in the range 0.1-4% (r > 0.999). The limit of quantitation was 0.1% and the limit of detection was 0.04%. The accuracy of the method was found to be 96.7% (average). For the precision (repeatability), a relative standard deviation value of 2.4% was found. Similarly, a stereoselective HPLC method was also developed and validated for the quantitation of the enantiomer of the starting material used for the synthesis of valsartan, namely (R)-valinebenzyl ester tosylate. Baseline resolution of the enantiomers of valinebenzyl ester tosylate could be achieved on the chiral crown ether column Crownpak CR (Daicel) at 50 degrees C using water-methanol-trifluoroacetic acid (850:150:1, v/v) as a mobile phase. The linearity was established in the range 0.5-5% (r > 0.999). The accuracy of the method was found to be 100.5% (average). For the precision (repeatability), a relative standard deviation value of 3.4% was found. Both methods were found to be suitable for the analysis of the respective analytes.

Simulated moving bed chromatographic resolution of a chiral antitussive.

The behavior of a laboratory simulated moving bed (SMB) unit for continuous chromatographic separation of enantiomers has been considered. This was applied to the resolution of a chiral antitussive agent, guaifenesin, on Chiralcel OD, during an experimental campaign involving nineteen runs. The application of recently developed criteria for the design and optimization of SMB units allows us to understand and rationalize the experimental results, as well as to indicate how to optimize the separation performances. A three-step procedure to determine the adsorption isotherms needed to apply these criteria is proposed; it is reliable and may be applied also where pure components are not available.

Conformational Refinement of Hydroxamate-Based Histone Deacetylase Inhibitors and Exploration of 3-Piperidin-3-ylindole Analogues of Dacinostat (LAQ824)

Inspired by natural product HDAC inhibitors, we prepared a series of conformationally restrained HDAC inhibitors based on the hydroxamic acid dacinostat (LAQ824, 7). Several scaffolds with improved biochemical and cellular potency, as well as attenuated hERG inhibition, were identified, suggesting that the introduction of molecular rigidity is a viable strategy to enhance HDAC binding and mitigate hERG liability. Further SAR studies around a 3-piperidin-3-ylindole moiety resulted in the discovery of compound 30, for which a unique conformation was speculated to contribute to overcoming increased lipophilicity and attenuating hERG binding. Separation of racemate 30 afforded 32, the R enantiomer, which demonstrated improved potency in both enzyme and cellular assays compared to dacinostat.

A Novel Class of Oral Direct Renin Inhibitors: Highly Potent 3,5-Disubstituted Piperidines Bearing a Tricyclic P3–P1 Pharmacophore

A small library of fragments comprising putative recognition motifs for the catalytic dyad of aspartic proteases was generated by in silico similarity searches within the corporate compound deck based on rh-renin active site docking and scoring filters. Subsequent screening by NMR identified the low-affinity hits 3 and 4 as competitive active site binders, which could be shown by X-ray crystallography to bind to the hydrophobic S3-S1 pocket of rh-renin. As part of a parallel multiple hit-finding approach, the 3,5-disubstituted piperidine (rac)-5 was discovered by HTS using a enzymatic assay. X-ray crystallography demonstrated the eutomer (3S,5R)-5 to be a peptidomimetic inhibitor binding to a nonsubstrate topography of the rh-renin prime site. The design of the potent and selective (3S,5R)-12 bearing a P3(sp)-tethered tricyclic P3-P1 pharmacophore derived from 3 is described. (3S,5R)-12 showed oral bioavailability in rats and demonstrated blood pressure lowering activity in the double-transgenic rat model.

(+)-4-Phosphonophenylglycine (PPG) a new group III selective metabotropic glutamate receptor agonist

A new synthesis of (R,S)-PPG (4-phosphonophenylglycine) and the separation of the protected enantiomers leading after deprotection to (+)- and (-)-PPG are described. Pharmacological characterization at the group III metabotropic glutamate receptors hmGluR4a and hmGluR7b revealed (+)-PPG as the active enantiomer.

Effect of aromatase inhibitors on estrogen 2-hydroxylase in rat liver

The effect of aromatase inhibitors, 4-hydroxyandrostenedione, CGS 16949A and aminoglutethimide on the inhibition of estrogen 2-hydroxylase activity in rat liver microsomes in vitro and on its induction in vivo has been examined. Estrogen 2-hydroxylase was found to have over twice the affinity for estradiol compared to estrone. Using high pressure liquid chromatography and employing estradiol as a substrate, the IC50 values were 2.2, 98, 110 and 908 microM for the reference compound ketoconazole and the aromatase inhibitors, 4-hydroxyandrostenedione, CGS 16949A and aminoglutethimide, respectively. Similar IC50 values were obtained using estrone as a substrate and by a tritiated water method employing estradiol as a substrate. The Km value for estrogen 2-hydroxylase with estradiol as a substrate using a tritiated water method was 4.3 microM with a Vmax of 11.89 nmol/h/mg. Ketoconazole, CGS 16949A and aminoglutethimide exhibited non-competitive inhibition whereas 4-hydroxyandrostenedione appeared to be a competitive inhibitor of estrogen 2-hydroxylase. The Ki values were 2.6, 72, 114 and 958 microM for ketoconazole, 4-hydroxyandrostenedione, CGS 16949A and aminoglutethimide, respectively. All three aromatase inhibitors were weak inhibitors of estrogen 2-hydroxylase as compared to the reference drug, ketoconazole. Following treatment of rats with aminoglutethimide (40 mg/kg/day; i.p.; for 3 days), estrogen 2-hydroxylase activity was increased by 28 and 30% using estradiol and estrone as substrates, respectively. Following treatment of rats with CGS 16949A (2 mg/kg/day; p.o.; for 3 days), the corresponding increase in estrogen 2-hydroxylase activity was 48 and 44%. The results of this study indicate that the aromatase inhibitors, aminoglutethimide and CGS 16949A are only weak inhibitors of estrogen 2-hydroxylase activity in vitro and show no evidence of inhibition in vivo. On the contrary, there was some evidence to suggest that both aminoglutethimide and CGS 16949A induce estrogen metabolism following repeated administration. Therefore, aminoglutethimide and CGS 16949A may lower estrogen levels not only by primarily inhibiting their synthesis but also by inducing the metabolism of estrogens.

Hilfsmittel für die präparative hochleistungs-flüssigkeitschromatographie

Abstract New aids for preparative high-performance liquid chromatography A new preparative separation system is described. It consists of a column, a splitting system which allows the controlled withdrawal of parts of the eluate for subsequent detection and a peak detector connected to a fraction collector.