CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) is the prototypical human constitutive androstane receptor (hCAR) activator, serving as a lead compound for novel ligand design. This compound has two stereoisomers, and the activity of each is currently unknown. In this work, we aimed to characterize the stereochemistry and behavior in solution of CITCO in order to better understand this pharmacological tool. Using spectroscopic and crystallographic methods, we discovered that CITCO is synthesized stereoselectively as the E isomer regardless of the reaction conditions. The Z isomer forms in solution and is isolable. NMR kinetic data shows that CITCO experiences time- and concentration-dependent stereoisomerization in solution, both from E to Z and from Z to E. The equilibrium position is concentration-dependent. When assaying conditions mimicking those used for biological hCAR activation, rapid isomerization ensues, leading to a mixture of isomers in less than 24 hours. As hCAR activation reporter assays usually take 24 hours to measure activity, our finding suggests that all CITCO activity reported so far comes from a mixture of stereoisomers regardless of the original sample composition. This explains the fact that both pure isomers display seemingly identical activities. Molecular modeling simulations suggest each isomer has a unique binding pose for hCAR. Further studies are needed to unambiguously determine the biological activity and binding mode of each isomer in order to undertake rational hCAR activator design.

Stereoisomerization of the human CAR activator CITCO complicates its use as a reference ligand

ACS Fall 2020 Virtual Meeting

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