Maximizing kinetic performance in supercritical fluid chromatography using state-of-the-art instruments.

Recently, there has been a renewed interest in supercritical fluid chromatography (SFC), due to the introduction of state-of-the-art instruments and dedicated columns packed with small particles. However, the achievable kinetic performance and practical possibilities of such modern SFC instruments and columns has not been described in details until now. The goal of the present contribution was to provide some information about the optimal column dimensions (i.e. length, diameter and particle size) suitable for such state-of the-art systems, with respect to extra-column band broadening and system upper pressure limit. In addition, the reliability of the kinetic plot methodology, successfully applied in RPLC, was also evaluated under SFC conditions. Taking into account the system variance, measured at ∼85µL(2), on modern SFC instruments, a column of 3mm I.D. was ideally suited for the current technology, as the loss in efficiency remained reasonable (i.e. less than 10% decrease for k>6). Conversely, these systems struggle with 2.1mm I.D. columns (55% loss in N for k=5). Regarding particle size, columns packed with 5µm particles provided unexpectedly high minimum reduced plate height values (hmin=3.0-3.4), while the 3.5 and 1.7µm packing provided lower reduced plate heights hmin=2.2-2.4 and hmin=2.7-3.2, respectively. Considering the system upper pressure limit, it appears that columns packed with 1.7µm particles give the lowest analysis time for efficiencies up to 40,000-60,000 plates, if the mobile phase composition is in the range of 2-19% MeOH. The 3.5µm particles were attractive for higher efficiencies, particularly when the modifier percentage was above 20%, while 5µm was never kinetically relevant with modern SFC instruments, due to an obvious limitation in terms of upper flow rate value. The present work also confirms that the kinetic plot methodology could be successfully applied to SFC, without the need for isopycnic measurements, as the difference in plate count between predicted and experimental values obtained by coupling several columns in series (up to 400mm) was on average equal to 3-6% and with a maximum of 13%.

The evaluation of 25 chiral stationary phases and the utilization of sub-2.0µm coated polysaccharide chiral stationary phases via supercritical fluid chromatography.

A rapid screening method to identify the best conditions for chiral separations is described. We analyzed a representative set of 80 racemic compounds against 25 different chiral stationary phases with three different mobile phases to identify the combination of columns and mobile phases that will separate the most compounds on the initial screen. While the OD separated the largest number of compounds, we found the best combination of six columns to be the AD, AS, AY, CC4, ID and Whelk-O1. The second team included the CCC, Cellulose-1, Cellulose-3 or OJ, IA, IE and IF. All 80 compounds were separated with a resolution range of 0.65-15.36. Screening the covalently bonded phases provided separation for 79 of the 80 compounds. We also found ethanol (0.1% NH4OH) separated more compounds than methanol (0.1% NH4OH) or isopropanol (0.1% NH4OH). As part of this study, we also compared the effectiveness of stationary phases that have the same chiral selector. Finally, we demonstrated the effectiveness of using a fast, 1.5-min screening method that utilizes a 1.7µm coated polysaccharide chiral stationary phase.

Identification of C-2 hydroxyethyl imidazopyrrolopyridines as potent JAK1 inhibitors with favorable physicochemical properties and high selectivity over JAK2.

Herein we report on the structure-based discovery of a C-2 hydroxyethyl moiety which provided consistently high levels of selectivity for JAK1 over JAK2 to the imidazopyrrolopyridine series of JAK1 inhibitors. X-ray structures of a C-2 hydroxyethyl analogue in complex with both JAK1 and JAK2 revealed differential ligand/protein interactions between the two isoforms and offered an explanation for the observed selectivity. Analysis of historical data from related molecules was used to develop a set of physicochemical compound design parameters to impart desirable properties such as acceptable membrane permeability, potent whole blood activity, and a high degree of metabolic stability. This work culminated in the identification of a highly JAK1 selective compound (31) exhibiting favorable oral bioavailability across a range of preclinical species and robust efficacy in a rat CIA model.

Step-economic synthesis of (+)-crocacin C: a concise crotylboronation/[3,3]-sigmatropic rearrangement approach.

The step-economic total synthesis of (+)-crocacin C has been achieved in 20% yield from commercially available starting materials. This approach requires the isolation of only 8 intermediates and can provide a reliable supply of (+)-crocacin C for the development of new antifungal and crop protection agents.

Discovery and optimization of C-2 methyl imidazopyrrolopyridines as potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2.

Herein we report the discovery of the C-2 methyl substituted imidazopyrrolopyridine series and its optimization to provide potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2. The C-2 methyl substituted inhibitor 4 exhibited not only improved JAK1 potency relative to unsubstituted compound 3 but also notable JAK1 vs JAK2 selectivity (20-fold and >33-fold in biochemical and cell-based assays, respectively). Features of the X-ray structures of 4 in complex with both JAK1 and JAK2 are delineated. Efforts to improve the in vitro and in vivo ADME properties of 4 while maintaining JAK1 selectivity are described, culminating in the discovery of a highly optimized and balanced inhibitor (20). Details of the biological characterization of 20 are disclosed including JAK1 vs JAK2 selectivity levels, preclinical in vivo PK profiles, performance in an in vivo JAK1-mediated PK/PD model, and attributes of an X-ray structure in complex with JAK1.

The use of ammonium hydroxide as an additive in supercritical fluid chromatography for achiral and chiral separations and purifications of small, basic medicinal molecules.

This study describes using 0.1% of a 28-30% ammonium hydroxide solution as an additive to alcohol modifiers in SFC to improve chromatographic peak shapes for basic molecules. Ammonium hydroxide's high volatility leaves no residual additive in the purified sample unlike classical additives in preparative chromatography such as diethylamine and triethylamine. We demonstrate that the silica support is stable despite having ammonium hydroxide in the modifier by running a durability study for over 350 h (105 L of solvent, 105,000 column volumes) on an analytical Chiralcel OJ column and a second study for 30 h (7.2 L, 14,400 column volumes) on an analytical Lux Cellulose-1 column. The peak shape of small, basic molecules is greatly improved with the use of ammonium hydroxide and this improvement is very similar to those having 0.1% diethylamine as a mobile phase additive. Electrospray ionization is also enhanced with the presence of ammonium hydroxide compared with that of diethylamine. We have found that the age of the 28-30% bottle of ammonium hydroxide solution can have significant effects on the chromatography and we describe how this can be overcome. Finally, we analyzed 23 racemic and basic compounds on six different chiral stationary phases and found there to be very little chiral selectivity difference between ammonium hydroxide and diethylamine, triethylamine, ethanolamine and isopropylamine.

A high throughput approach to purifying chiral molecules using 3µm analytical chiral stationary phases via supercritical fluid chromatography.

In this study we describe a fast 2.5 min gradient chiral screening method that utilizes 3 µm particles CSPs. An empirical approach to scale-up from the 2.5 min gradient method to an isocratic preparative method is described. We also evaluate the use of 5 µm preparative columns that are 150 mm in length versus the industry standard of 250 mm. Finally, we evaluate eleven different CSPs against 46 compounds, 23 commercially available and 23 internal compounds from a variety of projects. All 46 compounds were separated using the 2.5 min gradient method. Assuming an R(s) of 1.0 or greater, the Chiralpak AD column from Chiral Technologies proved most useful, followed by the Cellulose-1 from Phenomenex. The Cellulose-4, a novel stationary phase from Phenomenex, provided the third most separations of the eleven columns tested. For the 46 compounds tested, the Chiralcel OJ column from Chiral Technologies outperformed Phenomenex's version, the Lux Cellulose-3.