Fluorinated piperidine acetic acids as gamma-secretase modulators.

We report herein a novel series of difluoropiperidine acetic acids as modulators of gamma-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective beta-difluorination with Selectfluor. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Abeta42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250mg/kg per day, AUC(0-24)=2100microMh) did not exhibit Notch-related effects.

Impact of an error in the column hold-up time for correct adsorption isotherm determination in chromatography II. Can a wrong column porosity lead to a correct prediction of overloaded elution profiles?

The adsorption isotherm was determined for phenol in methanol/water on a C-8 stationary phase using frontal analysis in staircase mode, assuming different total column porosities, from 1 to 87%. Each set of adsorption isotherm data, with a certain column porosity, was fitted to various adsorption models and the generated parameters were used to calculate overloaded elution band profiles that were compared with experiments. It was found that the bi-Langmuir model had an optimum fit for a porosity that corresponds well with the value found experimentally. The adsorption energy distribution (AED) calculations and error analysis confirmed a bimodal energy distribution. It was also found that band profiles can be accurately predicted with a quite arbitrary chosen porosity, under prerequisite that a wrong but flexible adsorption model is chosen instead of the correct one. The latter result is very useful for quick optimizations of preparative separations where the exact value of the column porosity is not available.

Impact of an error in the column hold-up time for correct adsorption isotherm determination in chromatography I. Even a small error can lead to a misunderstanding of the retention mechanism.

The impact of a realistic error in the column hold-up time on the determination of the adsorption isotherm model was systematically investigated. Frontal analysis and the inverse method were used for the accurate determination of the adsorption isotherm. The true retention times of the breakthrough curves were used with a known hold-up time as reference. The adsorption isotherms were calculated using the same procedure that is used for real experimental adsorption isotherms, where the true hold-up time is unknown. The raw data were analyzed with calculations of adsorption energy distributions (AEDs), Scatchard plots, fitting to different rival adsorption models and finally their ability to predict true profiles. The results show that for a true Langmuir and bi-Langmuir model with an underestimated hold-up time the error may lead to a more heterogeneous model and for overestimated cases false adsorption processes like multi-layer adsorption or solute-solute interaction are assumed. The Scatchard plots for data obtained using a Langmuir adsorption isotherm are nonlinear and the AEDs show clear deviations from Langmuir behavior already at small deviations from the true hold-up time at a moderate surface coverage. The inverse method confirms the result that was obtained from the frontal analysis procedure.

Multiparallel microfluidic high-performance liquid chromatography for high-throughput normal-phase chiral analysis.

The suitability of the Eksigent Express 800 microfluidic eight-channel HPLC instrument for multiparallel normal-phase chiral analysis in support of high-throughput pharmaceutical process research was investigated. Analysis of test mixtures containing the two enantiomers of benzoin and the closely related (R,S)-dihydrobenzoin, was carried out in a 96-well microplate, affording rapid (<2 h) and accurate assessment of enantiopurity. In a second example, use of the instrument to support high-throughput catalyst screening of the asymmetric hydrogenation of a prochiral unsaturated ester is presented, in which method development (gradient screening of four columns and two eluents, followed by optimization to afford a fast analytical method) and analysis of a 96-well microplate was carried out within a single working day. This represents a considerable improvement over conventional analysis techniques that usually take several days to complete.

Investigation of the stability of Chiralpak AD chiral stationary phase under various solvent conditions and development of a method to identify stationary phase-derived polymer contamination.

The stability of Chiralpak AD chiral stationary phase under various solvent conditions was investigated. An analytical method for the detection of the presence of solubilized Chiralpak AD coating was developed using CD spectroscopy (CD signal at 245 nm). In addition, NMR analysis of the solubilized polymer revealed a characteristic signal for the 3,5-dimethylphenyl carbamate methyl protons at around 2.5 ppm. Both of these methods may be helpful in detecting contamination by the Chiralpak AD polymer or in the study of CSP solvent compatibility.

Fast methods of enantiopurity determination for the Soai reaction: towards a general enantioenrichment detector?

The Soai reaction system possesses a remarkable combination of characteristics (enantioselective autocatalysis, strong positive nonlinearity leading to asymmetric amplification, ability to be triggered by wide variety of exogenous enantioenriched materials) that make it a potential starting point for a generalized detector for enantioenrichment. In this study we apply standard approaches used in pharmaceutical process research to the problem of developing a rapid method for analysis of the enantiopurity of the Soai reaction product. Several methods for rapid analysis (<1 min per sample) are described, including an approach using chiral supercritical fluid chromatography (SFC) and an approach using achiral chromatography with circular dichroism (CD) detection. Some thoughts on incorporation into a device for generalized enantioenrichment detection are presented.

Multiparallel chiral method development screening using an 8-channel microfluidic HPLC system.

The Eksigent Express 800 8-channel microfluidic HPLC system was investigated for carrying out multiparallel screening and development of fast normal phase chiral separations. In contrast to the familiar automated sequential chiral method development approaches that often afford a next day result, the multiparallel approach offers the exciting possibility of near "real time" method development, often affording an optimized method in less than 1 h. In this study, four column types (300 microm i.d.) with two different mobile phases are screened using a universal standard gradient approach. Interestingly, parallel method optimization following initial screening was shown to sometimes lead to surprising and unanticipated outcomes, emphasizing the value of the multiparallel screening approach. A variety of standard test racemates were analyzed, with optimized separation methods for most in the 1- to 2-min range. These results compare favorably with results obtained on a single channel conventional HPLC system using 4.6-mm i.d. columns. In addition, isocratic methods developed on the microbore columns are readily translated to the larger column format.

Development of a gradient elution preparative high performance liquid chromatography method for the recovery of the antibiotic ertapenem from crystallization process streams.

A gradient elution preparative chromatography method was developed for the recovery of the antibiotic ertapenem from crystallization mother-liquor streams. The preparative HPLC method that was developed on the lab-scale employs an analytical size column of conventional dimensions (25 cm x 0.46 cm) packed with Kromasil C8 stationary phase. Gradient elution was used with aqueous acetic acid and acetonitrile as mobile phases. A target of processing approximately 30 mg of ertapenem per half an hour at a flow rate of 1.5 mL/min with high yield and adequate rejection of all major impurities was achieved. This corresponds to a productivity of approximately 0.6 kg ertapenem as free acid per kilogram of stationary phase per day (kkd). The scalability of the method was demonstrated by using a 5 cm i.d. column configuration to generate 10 g of purified ertapenem. This work complements a previous study improving on the productivity and throughput of the method by employing gradient elution and the use of crystallization to remove some key impurities that are chromatographically difficult to resolve [A. Vailaya, P. Sajonz, O. Sudah, V. Capodanno, R. Helmy, F.D. Antia, J. Chromatogr. A 1079 (2005) 80].

Challenges in the analytical method development and validation for an unstable active pharmaceutical ingredient.

A sensitive high-performance liquid chromatography (HPLC) impurity profile method for the antibiotic ertapenem is developed and subsequently validated. The method utilizes an Inertsil phenyl column at ambient temperature, gradient elution with aqueous sodium phosphate buffer at pH 8, and acetonitrile as the mobile phase. The linearity, method precision, method ruggedness, limit of quantitation, and limit of detection of the impurity profile HPLC method are found to be satisfactory. The method is determined to be specific, as judged by resolving ertapenem from in-process impurities in crude samples and degradation products that arise from solid state thermal and light stress, acid, base, and oxidative stressed solutions. In addition, evidence is obtained by photodiode array detection studies that no degradate or impurity having a different UV spectrum coeluted with the major component in stressed or unstressed samples. The challenges during the development and validation of the method are discussed. The difficulties of analyzing an unstable active pharmaceutical ingredient (API) are addressed. Several major impurities/degradates of the API have very different UV response factors from the API. These impurities/degradates are synthesized or prepared by controlled degradation and the relative response factors are determined.

Exploiting pH mismatch in preparative high-performance liquid chromatographic recovery of ertapenem from mother liquor streams.

Preparative chromatography was successfully employed to recover ertapenem from mother liquor streams. The recovery process involved concentration of mother liquor stream by evaporation, purification by reversed-phase preparative high-performance liquid chromatography (HPLC), and removal of chromatographic solvents in the recovered fractions by evaporation. HPLC feed was prepared by stripping off the organic solvents from the mother liquor using a wiped-film evaporator. Purification was first carried out on a 25 cm x 0.46 cm analytical column packed with 10-microm Kromasil C8 particles and then scaled up to a 25 cm x 5 cm preparative column. Gram-level recovery of ertapenem with high purity was achieved by exploiting a novel approach based on pH mismatch between the feed and the eluent. Purified ertapenem streams from preparative HPLC runs were combined, evaporated and recycled into the crystallizer for ertapenem isolation.

Influence of the column hold-up time measurement accuracy on the prediction of chromatographic band profiles.

The influence of the column hold-up time measurement accuracy on the determination of equilibrium isotherms by classical frontal analysis and the prediction of overloaded elution band profiles were investigated. The ideal model of chromatography in combination with a Langmuir isotherm was used. Breakthrough curves and overloaded elution profiles were computer generated with a known hold-up time value (true hold-up time). Then these data were evaluated the same way as it is done with experimental chromatographic data where the true hold-up time is unknown, i.e. to determine the equilibrium isotherm by the frontal analysis procedure, to fit the isotherm data to the Langmuir model and then to predict chromatographic band profiles using, e.g. the ideal model of chromatography. A comparison of overloaded elution profiles obtained with different deviations of the hold-up time from its true value shows that the effect of its measurement error is significant in preparative liquid chromatography because the isotherm is usually strongly nonlinear in this case.