Development of 18F-Labelled Quinazolinone Derivatives for PET Imaging of Myocardial GHSR (Ghrelin Receptor)

Introduction: This work presents the design and synthesis of various fluorinated structural analogues of quinazolinone small molecules, their binding affinity evaluation, and their development and application as [¹⁸F]radiotracers for imaging of the ghrelin receptor, also known as the growth hormone secretagogue receptor (GHSR). Previously, we reported a series of quinazolinone ligands for the GHSR with sub-nanomolar receptor affinity.¹ We now disclose to discovery of a fluorine-containing derivative well suited for radiolabeling and describe preliminary evaluation in a canine model of myocardial infarction.

Methods: Quinazolinone analogues were modeled in the GHSR binding site using molecular docking analysis. Multi-step synthetic routes were conducted for non-radioactive derivatives and precursors. Compounds were purified by silica flash chromatography and characterized by NMR spectroscopy and HRMS. The affinity for GHSR was determined using a radioligand binding assay using [¹²⁵I-His⁹]ghrelin and varying concentrations of the test compound. Radiolabelling was conducted using azeotropically dried ¹⁸F, potassium carbonate and Kryptofix 2.2.2. followed by reaction with the tosylate precursor at 100 °C. The radiolabelled compound was purified by semi-preparative HPLC. The sensitivity and in vivo and ex vivo specificity of the lead candidate, [¹⁸F]LCE470, was evaluated in a canine model of surgically-induced myocardial infarction using PET-MRI, which allowed for anatomic localization of tracer uptake within different regions of the left ventricle.

Results: Molecular docking predicted space within the quinazolinone-bound GHSR binding pocket for a small fluorine-containing substituent that would not sterically hinder binding of the radioligand. Six derivatives were synthesized and GHSR affinity was determined, with IC₅₀ values ranging from 353 nM to 0.33 nM. The lead candidate, LCE470, has an IC₅₀ of 0.33 nM and cLog D_7.4 of 2.53. This is the highest known receptor binding affinity for a radiolabelled GHSR ligand. Radiolabelling of the tosylate precursor proceeded as a one-step radiofluorination method to produce [¹⁸F]LCE470 in 6-18% radiochemical yield, >99% purity and molar activities ranging from 17 to 220 GBq/µmol (n=11). PET/MRI imaging using [¹⁸F]LCE470 was conducted in a canine preclinical model of myocardial infarction. In vivo blocking studies in healthy hounds and ex vivo blocking studies in myocardial tissue showed specificity of [¹⁸F]LCE470, and sensitivity was demonstrated by positive correlation (p<0.007) between in vivo tracer uptake and GHSR abundance.

Conclusions: Through molecular docking, synthesis of quinazolinone analogues and receptor affinity evaluation, [¹⁸F]LCE470 was discovered as a sub-nanomolar PET imaging agent targeting the cardiac GHSR (ghrelin receptor).