As vixotrigine (1) entered
a later clinical phase
for trigeminal neuralgia (ZakrzewskaJ. M.
Zakrzewska, J. M.
Lancet Neurol.201716291300), the development
of a sustainable late-stage process was required to meet the supply
needs for formulation optimization, phase 3 clinical trials, and registration
stability batches (this is the expected commercial formulation). In
this article, we describe how the process was streamlined from the
early supply route (GiblinG.
Giblin, G.
10.1021/acs.oprd.0c00382Org. Process
Res. Dev.2020) and a comprehensive control strategy was established.
Process improvements included improving safety and scalability for
a temperature-sensitive Grignard reaction, simplifying unit operations,
removal of heterogenous conditions, and route redesign to afford a
high yielding, one-pot sequential alkylation and amidation. Improvement
in the salt formation step, combined with wet milling, resulted in
improved particle properties with enhanced flow properties of the
final active pharmaceutical ingredient. The process mass intensity
was improved 65% while maintaining drug substance purity at more than
99.8%. This new process has been scaled up to generate metric ton
quantities of drug substance.
A two-step pharmaceutical manufacturing process was developed for the large-scale preparation of 6-chloro-9-((4methoxy-3,5-dimethylpyridin-2-yl)methyl)-9H-purin-2-amine methanesulfonic acid salt (4) from commercially available starting materials. In the first step, the benzylpurine free base (3) was prepared by benzylation of 6-chloro-9H-purin-2-amine (1) with 2-(chloromethyl)-4-methoxy-3,5-dimethylpyridine hydrochloride (2). The benzylpurine free base was then directly converted into the methanesulfonic acid salt. It was necessary to charge the pyridine hydrochloride 2 in portions into the mixture of K 2 CO 3 (−325 mesh) and the chloropurine compound 1 in dimethylacetamide (DMA). The major regioisomeric impurity (6), formed by N 7 benzylation, and inorganic salts were removed by filtration. Treatment of the DMA filtrate with MsOH afforded the target salt with negligible degradation. In the second step, recrystallization of the crude salt from DMSO−EtOAc with seeding gave crystalline API in high yield and purity despite the hydrolytic instability of the product in solution.
B cell receptor (BCR) mediated activation of nuclear factor κB (NF-κB) is key to humoral immunity. CARMA1 (CARD11) is essential for BCR mediated NF-κB activation by interacting with Bcl10 and MALT1. Besides these two main players, few interaction partners of the CARMA1 complex are known. Here we identified new interaction partners of CARMA1. We generated two rabbit antisera against mouse CARMA1 to immunopurify endogenous CARMA1 from lysates of mouse B cells. Nik-binding protein (NIBP), Ras-GAP SH3 binding protein 2 (G3BP1) and Trk-fused gene (Tfg) were identified by peptide mass fingerprinting in immunopurified CARMA1 complexes. The interaction of Tfg and CARMA1 was confirmed by co-immunoprecipitation and Blue native polyacrylamide gel electrophoresis using the anti CARMA1 and newly generated anti Tfg antibodies. This analysis revealed that CARMA1 formed complexes of 600-1000 kDa. Additionally, Tfg was found in complexes of 500-600 kDa which increased in size to ∼740 kDa upon overexpresssion.
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