Efficient radiosynthesis and preliminary autoradiography evaluation of a potent 11C-labeled LRRK2 inhibitor (2019)

1102 Objectives: Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene is one of the most common causes for Parkinson’s disease (PD), which are related with ~10% of familial cases and 2% of sporadic cases [1]. Current PD therapy targeting LRRK2 mainly focuses on the development of potent and selective LRRK2 inhibitors. Recently, 3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)(morpholino)methanone (1) was identified as a promising LRRK2 inhibitor (IC50 = 9 nM) [2].[1] The goal of this project was to carry out an efficient radiosynthesis of [11C]1 and perform preliminary evaluation via in vitro autoradiography studies.
 Methods: LRRK2 inhibitor 1 and its precursor (3-hydroxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)(morpholino)methanone (desmethyl-1) were synthesized in a parallel manner. In specific, the cross-coupling reaction between benzoic acid 2 and morpholine readily proceeded to give amide 3, which was subsequently reduced to the key amine intermediate 4. Another intermediate 6 was obtained through nucleophilic substitution of 2,4-dichloro-5-(trifluoromethyl)pyrimidine 5 with methylamine. Finally, the cross-coupling reaction between 4 and 6 readily occurred to deliver the desired LRRK2 inhibitor 1. Boron tribromide mediated demethylation of 1 readily proceeded to forge the corresponding precursor desmethyl-1. Compound 1 was radiolabeled with [11C]CH3I by heating a solution of desmethyl-1 (1.5 mg) and NaOH (1 M, 4 μL) in DMF (0.3 mL) at 80 oC for 5 min. The lipophilicity of [11C]1 was measured using liquid-liquid partition between n-octanol and PBS (‘shake flask method’). The binding specificity was confirmed via in vitro autoradiography studies in rat, monkey and human brain sections.
 Results: LRRK2 inhibitor 1 was synthesized in 27% yield over 3 steps and the precursor desmethyl-1 was obtained in 18% overall yield over 4 steps. The LRRK2 inhibitor [11C]1 was isolated in 19% non-decay corrected radiochemical yield (relative to [11C]CH3I) with high radiochemical purity (>99%) and molar activity (>37 GBq/µmol). No signs of radiolysis was observed for [11C]1 up to 90 min after formulation in saline containing 10% ethanol. The logD value of [11C]1 was determined to be 2.93 ± 0.02 (n = 3), indicating a high possibility of brain penetration. In vitro autoradiography studies confirmed the binding specificity of [11C]1 in rat, monkey and human brain. In rat brain sections, heterogeneous distribution of [11C]1 was observed with high bound radioactivity in hippocampus and cerebral cortex, and low bound radioactivity in cerebellum, which was consistent with LRRK2 expression in rat brain. Pretreatment with PF-06447475 (10 μM), a known LRRK2 inhibitor, remarkably reduced the bound activity in hippocampus and cerebral cortex, which was also observed in the autoradiography studies of monkey and human brain sections.
Conclusion: We have successfully radiolabeled a LRRK2 inhibitor 1 in high radiochemical yield and high molar activity. Reasonable lipophilicity and in vitro binding specificity was demonstrated, which may pave the way for future development and evaluation of novel LRRK2 PET tracers.
Acknowledgements: We thank Drs. Thomas Brady, Neil Vasdev and Elijahu Livni (Nuclear Medicine, MGH) for their helpful discussion.
Reference: [1] (a) C. Paisan-Ruiz et al., Neuron 2004, 44, 595-600; (b) A. Zimprich et al., Neuron 2004, 44, 601-607; [2] A. A. Estradaet al., J. Med. Chem. 2012, 55, 9416-9433.