# Synthesis and evaluation of enantiomeric quinoline-2-carboxamides: positron emission tomography imaging agents for the translocator protein

## TL;DR

Researchers synthesized and tested two versions of a PET imaging agent for tracking inflammation, finding both work well despite a key genetic variation.

## Contribution

The study provides a detailed characterization of LW223 enantiomers as polymorphism-insensitive TSPO PET agents.

## Key findings

- The R-enantiomer of LW223 has 7.5-fold higher affinity for TSPO than the S-enantiomer.
- Both enantiomers remain insensitive to the rs6971 polymorphism in human brain tissue.
- Molecular docking studies revealed enantiomer-specific interactions with TSPO.

## Abstract

The translocator protein (TSPO) is a key biomarker for inflammation. Positron emission tomography (PET) imaging of TSPO has emerged as a valuable tool for investigating multiple disorders throughout the body. We previously reported the development of [18F]LW223, a quinoline-2-carboxamide bearing an R-configured side chain, which binds TSPO independently of the rs6971 human polymorphism and enables quantification of macrophage-driven inflammation in the course of myocardial infarction. In the present study, we provide a comprehensive molecular and biological characterisation of LW223 and its S-enantiomer, further supporting their potential as PET imaging agents for human inflammatory processes. Two synthetic routes were developed: one enabling direct multigram-scale synthesis of LW223, and another allowing late-stage (radio)fluorination. The latter was applied for the synthesis of the S-enantiomer. Binding assays using homogenised human brain tissue revealed that the S-enantiomer exhibits 7.5-fold lower affinity (Ki = 4.5 ± 0.7 nM) than the R-enantiomer, yet remains insensitive to rs6971 polymorphism. Molecular docking studies with the X-ray structure of wild-type TSPO from Bacillus cereus provided insights into enantiomer-specific binding interactions. Collectively, these findings advance our understanding of LW223 as a TSPO-targeted PET ligand for human inflammatory disease.

Synthesis and biological testing of the R- and S-enantiomers of LW223, a third generation TSPO PET imaging agent using human brain tissue showed that despite a 7.5-fold difference in affinity, both are insensitive to rs6971 polymorphism.