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
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.
Section snippets
Materials and chemicals
HPLC grade methanol (MeOH), isopropyl alcohol (IPA), acetonitrile (MeCN), sodium phosphate monobasic anhydrous, phosphoric acid and racemic trans-stilbene oxide (TSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was processed with a Millipore Milli-Q™ water system from Thermo Fisher Scientific (Waltham, MA, USA). Food grade carbon dioxide (CO2) was supplied from Airgas (Piscataway, NJ, USA). BMS-986142 active pharmaceutical ingredient (API), formulated tablets, and
LC separation of BMS-986142 atropisomers
BMS-986142 possesses three chiral elements that theoretically results in eight stereoisomers. Since the chirality of the carbazole chiral center was controlled upstream in the synthesis (the enantiomeric impurity of chiral starting material was typically less than 0.5%), only four possible atropisomeric diastereomers caused by rotation about two chiral axes were observed in the final API (Fig. 1). As all four atropisomers are diastereomers to each other, it is possible to resolve them by either
Conclusions
Separation of BMS-986142 atropisomers has been successfully achieved on an achiral polar-embedded C18 column in RPLC and on polysaccharide-based chiral columns in RPLC and SFC. Compared to the RP chiral separation, the separation on immobilized IB-type columns in SFC are more efficient. The implementation of IB-U column with a sub-2 μm particle size enabled us to further enhance the atropsiomeric separation and develop a more environmental-friendly analytical method. Not only was the chiral
CRediT authorship contribution statement
Brian Lingfeng He: Conceptualization, Methodology, Writing - original draft. Nicole G. Kleinsorge: Data curation. Ling Zhang: Methodology, Writing - review & editing. Brent Kleintop: Writing - review & editing.
Declaration of Competing Interest
The authors declare no interest of conflict for the whole content of this manuscript.
Acknowledgments
The authors would like to greatly thank Dr. Jonathan Shackman and Dr. Qinggang Wang for constructive discussion and reviewing of the manuscript. The authors also want to thank Judy Lin for discussion in HPLC method development, and Dr. Thomas Razler and the whole BMS-986142 team at BMS, New Brunswick, for providing the materials of BMS-986142 API, three related atropisomers and drug product.
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