Advancing stereoisomeric separation of an atropisomeric Bruton's tyrosine kinase inhibitor by using sub-2 µm immobilized polysaccharide-based chiral columns in supercritical fluid chromatography

Highlights

• Successful separation of four atropisomers of Bruton's tyrosine kinase inhibitor BMS-986142 achieved on achiral and chiral columns in HPLC and on chiral column packed with sub-2 μm in SFC.

• Excellent diastereomeric selectivity towards BMS-986142 atropisomers afforded by cellulose-based chiral stationary phases in general.

• Better efficiency, productivity and sensitivity demonstrated by the separation on sub-2 μm IB-U column in 3.0 mm I.D. when compared to the same type columns in 4.6 mm I.D. in SFC.

• Inadequacy of modern SFC instrument for column packed with 1.6 μm particles in 3.0 mm I.D. × 100 mm dimension revealed by kinetic performance analysis with compound BMS-986142.

Abstract

BMS-986142 is a Bruton's tyrosine kinase inhibitor under development to treat several disease types. The compound contains three chiral elements: one chiral center and two chiral axes, resulting in three potential atropisomeric impurities in its drug substance and drug products. Separation of BMS-986142 atropisomers has been successfully achieved on an achiral polar-embedded C18 column in reversed-phase liquid chromatography (RPLC) and on polysaccharide-based chiral columns in RPLC and supercritical fluid chromatography (SFC). Compared to the RPLC chiral separation, the SFC atropisomeric separation on a sub-2 μm immobilized cellulose-based column is much more efficient and environmentally friendly. The analysis time in SFC was reduced by 8-fold compared to that in RPLC, and the method sensitivity in SFC on the sub-2 μm chiral column in 3.0 mm I.D. was 2 to 4-fold better than that on 3 μm chiral columns in 4.6 mm I.D.. Furthermore, our study suggests that the contribution to band broadening from the extra column volume (ECV) of modern commercial SFC instrument was not negligible for a 3.0 mm I.D. × 100 mm column packed with 1.6 μm particles. This result reaffirms that there is a great need for further improvement of SFC instrument design in order to realize the full theoretical efficiency of both sub-2 μm achiral and chiral columns.

Introduction

The pharmaceutical industry was completely changed after the thalidomide disaster came to light over 60 years ago [1], and chirality became a central topic of developing chiral drugs [2]. The majority of chiral pharmaceutical compounds have a point-centered chirality with fully substituted carbon as a stereogenic center. Another less common source of chirality is atropisomerism, where stereochemistry originates from restricted rotation along a bond axis due to steric hindrance or electronic constraints. A fascinating characteristic of an atropisomer is that the chiral molecule undergoes slow configuration interconversion, and this racemization process has a half-life of >1000s depending on the rotational energy barrier and other factors, such as temperature, physical form, chemical environment, etc. [3], [4], [5]. As a lesson learned from the thalidomide disaster, the stereolability of a chiral drug is always avoided in drug molecule design, because the resulting stereoisomer and the drug may differ in biological activities leading to dissimilar pharmacological, toxicological and pharmacokinetic profiles. Consequently, atropisomerism had remained as an uncharted field in pharmaceutical research for many years. In recent years, as researchers have gained more knowledge and better control of atropisomerism, the pharmaceutical industry has started to accept drug-like atropisomers in the search for better medicines and to address unmet medical needs [6], [7], [8], [9], [10].

BMS-986142 (Fig. 1) is a drug candidate under development as a Bruton's tyrosine kinase (BTK) inhibitor to treat several disease types [8], [9], [10]. The most noticeable structural feature of this compound is the complexity of chirality: besides a chiral center at the carbazole ring, it contains two bonds of hindered rotation that lead to two diastereomeric axes. Molecular modeling revealed that the energy barrier was estimated to be 28 kcal/mol and 26 kcal/mol for the carbazole-aryl CC bond and the aryl-quinazolinedione CN bond rotation, respectively [10]. These energy levels lead to one major and two minor atropisomer forms observed upon rotation (Fig. 1). However, the rotational barriers of BMS-986142 are high enough to give the compound excellent conformational stability at ambient temperature, which allows the compound to be isolated, processed, formulated and used for clinical studies.

During the drug discovery and pharmaceutical development of single atropisomeric drugs, a series of special challenges arising from the intrinsic changeability of atropisomers have been encountered [8], [9], [10], [11], [12], [13]. From an analytical perspective, it can be a difficult task to reliably characterize drug substances and drug products with atropisomerism. Due to sample instability, it is desired to have analytical methods that enable fast analysis to ensure accurate determination of the impurity profile of atropisomeric drugs. Chiral chromatographic techniques based on packed columns, such as liquid chromatography (LC) and SFC, are two effective tools employed in pharmaceutical laboratories to analyze stereoisomeric impurities of atropisomeric drugs [11], [12], [13], [14], [15], [16]. In recent years, many exciting advances in LC and SFC instrumentation, as well as in column technologies, particularly introduction of sub-2 μm fully porous particles (FPPs) and sub-3 μm superficially porous particles (SPPs), have successfully pushed the time boundaries of chiral separations into seconds or even sub-second regimes [17], [18], [19], [20]. SFC, which employs low viscosity and high diffusivity liquid carbon dioxide (CO₂) with or without a small percentage of polar organic modifier as mobile phases, is an appealing technology to us in the analysis of atropisomeric drugs due to its orthogonality to RPLC, analysis speed, and greenness in conjunction with chiral columns packed with sub-2 μm particles.

Analytical methods that are capable of separating BMS-986142 and its three potential atropisomers are needed for several purposes, such as in-process atropisomeric impurity control; drug substance and drug product GMP release; evaluation of the racemization risk around both chiral axes in BMS-986142; understanding all the parameters that affect the interconversion of all four atropisomers; the kinetics of the interconversion process; etc. Due to the diversity in the study requirements, we decided to take several approaches to address the atropisomeric separation problem. For quality control purposes, since atropisomers of BMS-986142 are also diastereomers, we dedicated our method development efforts to achiral columns to develop a method that separated all organic impurities, including the atropisomers. For chemical development purposes, we needed a chiral method that could resolve as many stereoisomers as possible, since theoretically there are four more stereoisomers besides atropisomers for BMS-986142. In the meantime, we needed a fast method that would allow us to evaluate the risks and kinetics of interconversion of the atropisomers. In this paper, we focus our interest on developing a fast atropisomeric separation with recently available commercial polysaccharide-based chiral columns packed with sub-2 μm particles for SFC. We also compare the atropisomeric separation of BMS-986142 on sub-2 μm chiral columns in SFC with those on conventional 3 μm and 5 μm chiral columns in the aspects of atropisomeric separation, analysis speed, sensitivity, and kinetic performance.