Radiosynthesis and Preclinical Evaluation of [¹¹C]PF-06447475 for LRRK2 imaging

Objectives: Parkinson’s disease (PD) is a debilitating neurodegenerative disorder with unclear underlying mechanism and targets for therapy. Extensive studies have demonstrated Leucine-rich repeat kinase 2 (LRRK2) as a novel and promising target for PD therapy, since LRRK2 mutations are found to be related with ~10% of familial PD patients and 2% of sporadic cases. Recently, PF-06447475 (3-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)benzonitrile) was identified as a LRRK2 inhibitor with excellent activity and selectivity (IC₅₀ = 3 nM) [1]. The objective of this project was to perform isotopic labeling of PF-06447475 with carbon-11, and preliminary in vitro autoradiography in rodents.

Methods: The LRRK2 inhibitor PF-06447475 and the corresponding bromide precursor 1 were synthesized according to literature procedure [1]. Briefly, the commercially available 4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine 2 was initially protected with the SEM group under basic conditions to give compound 3. Pd-catalyzed cross-coupling reaction between 3 and (3-cyanophenyl)boronic acid followed by TFA-promoted deprotection of the SEM group proceeded smoothly to forge chloropyrimidine 4. Finally, nucleophilic substitution of 4 with morpholine readily occurred to deliver the desired LRRK2 inhibitor PF-06447475. The bromide precursor 1 was synthesized in a similar manner to that of PF-06447475 except that nucleophilic substitution with morpholine occurred prior to the Pd-catalyzed cross-coupling reaction with (3-cyanophenyl)boronic acid. Radiosynthesis of [¹¹C]PF06447475 was carried out through cuprous iodide promoted cross-coupling reaction between bromide precursor and [¹¹C]NH₄CN in dimethylformamide (DMF) at 180 ^oC for 5 min. The binding specificity was validated through in vitro autoradiography (baseline and blocking) on rat brain tissues.

Results: [¹¹C]PF06447475 was isolated in an average 8.5% radiochemical yield (ca. 3.3 GBq; non-decay corrected from starting [¹¹C]CO₂ at end of synthesis) with high radiochemical purity (>99%) and molar activity (greater than 90 GBq/μmol) within 35 min. No sign of radiolysis was detected up to 90 min after formulation in saline containing 0.8% ascorbic acid, 0.7% Tween 80 and 2.7% ethanol. Further in vitro autoradiography studies confirmed the binding specificity of [¹¹C]PF06447475 to LRRK2. Heterogeneous distribution was observed and several regions exhibited high bound radioactivity including striatum, cerebral cortex and hippocampus, while low bound radioactivity was detected in pons and cerebellum, which was consistent with the distribution of LRRK2 in rat brains. Pretreatment studies with PF-06447475 (10 μM) led to higher than 85% reduction of the bound activity in striatum, cerebral cortex and hippocampus, as well as abolishment of heterogeneity in different brain regions.

Conclusions: We have successfully radiolabeled a potent LRRK2 inhibitor PF06447475 with [¹¹C]NH₄CN in high radiochemical yield and high molar activity. In vitro autoradiography studies demonstrated the binding specificity of [¹¹C]PF06447475. Further characterization including PET studies to evaluate its brain-penetration ability and in vivo binding specificity, ex vivo whole body distribution and radiometabolic analysis is underway and will be reported on due course. References: [1] J. Med. Chem. 2015, 58, 419-432.