Lewis Acid-Facilitated Radiofluorination of MN3PU: A LRRK2 Radiotracer

Abstract

Background: Temperature-sensitive radiopharmaceutical precursors require lower reaction temperatures (<100 °C) during nucleophilic radiofluorination in order to avoid compound thermolysis, often resulting in sub-optimal radiochemical yields (RCYs). To facilitate nucleophilic aromatic substitution (SNAr) of nucleofuges commonly used in radiofluorination (e.g., nitro group), we explored the use of Lewis acids as nucleophilic activators to accelerate [18F]fluoride incorporation at lower temperatures, and thereby increasing RCYs for thermolabile activated precursors. Lewis acid-assisted radiofluorination was exemplified on the temperature-sensitive compound 1-(4-(4-morpholino-7-neopentyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)phenyl)-3-(6-nitropyridin-3-yl)urea (MN3PU, compound 3) targeting leucine-rich repeat kinase 2 (LRRK2), an important target in the study of Parkinson’s disease and various cancers. Methods: To a vessel containing dried K[18F]F-K222 complex, a solution of precursor MN3PU ((3), 1 mg; 1.8 μmol) and Lewis acid (6 μL of 0.2 μmol: chromium II chloride (A), ferric nitrite (B) or titanocene dichloride (C)) in 500 μL of N,N-dimethylformamide (DMF) (with 10% t-BuOH for B) were added. Reactions were stirred for 25 min at 90 °C. In parallel, reactions were conducted without the addition of Lewis acids for baseline comparison. After purification via preconditioned Sep-Pak C18 plus cartridges, aliquots were analyzed by analytical radio-HPLC. Results: Non-decay corrected radiochemical yields (ndc RCYs) for [18F]FMN3PU (7) were improved from 1.7 ± 0.7% (no addition of Lewis acids) to 41 ± 1% using Cr(II) and 37 ± 0.7% using Ti(II)-based Lewis acids, with radiochemical purities of ≥96% and molar activities (Am) of up to 3.23 ± 1.7 Ci/μmol (120 ± 1.7 GBq/μmol). Conclusion: RCYs of [18F]FMN3PU (7) improved from ~5% using conventional nucleophilic radiofluorination, up to 41 ± 1% using Lewis-acid supported SNAr.

Imaging Leucine-Rich Repeat Kinase 2 In Vivo with 18F-Labeled Positron Emission Tomography Ligand

“…Positron emission tomography (PET) has emerged as an ideal noninvasive imaging technique to probe in vivo distribution, expression, and activity of a biological target. − Benefiting from its high sensitivity and ability for quantification, a suitable LRRK2-targeted PET probe enables in vivo LRRK2 mapping and allows us to measure target occupancy for providing guidance of dose selection and/or safety margins in the brain, which thereafter would substantially facilitate the clinical translation of novel LRRK2 inhibitors. − Meanwhile, an LRRK2-targeted PET ligand would provide a molecular imaging tool for studying physiopathological changes in neurodegenerative diseases including PD and strengthen our understanding of disease progression. To guide the discovery of novel PET neuroligands, we have previously disclosed a series of design and selection criteria, including multiparameter optimization (MPO) for CNS drugs and PET probes by incorporating physicochemical and in vitro absorption, distribution, metabolism, and excretion (ADME) properties based on our drug candidate database. − Specifically, CNS PET ligand candidates should manifest the following attributes: (1) excellent binding affinity (usually in the subnanomolar or nanomolar range) and selectivity (>30–100 fold); (2) high BBB penetration with high passive membrane permeability (RRCK Papp A-B > 5 × 10 –6 cm/s), low interaction with P-glycoprotein (P-gp) transporter (MDR1 BA/AB ≤ 2.5), and favorable physicochemical parameters, including MPO score (CNS PET MPO > 3); and (3) low risk of nonspecific binding with an unbound fraction in the brain (Fu_b) and plasma (Fu_p) greater than 0.05.…”

Evaluation of [18F]PF-06455943 as a Potential LRRK2 PET Imaging Agent in the Brain of Nonhuman Primates

“…In this study, we evaluated a promising LRRK2 PET ligand [ 18 Animal Care and Use Committee at the Massachusetts General Hospital (protocol #: 2003N000338), which abides by the Institute of Laboratory Animal Resources guidelines. All animals were deprived of food for 12 h prior to the study.…”

“…However, whole-body biodistribution studies showed limited brain uptake in rodents, indicating potential limitation for further translational development . Building upon previous effort by our group and others, we have successfully developed [ 18 F]­PF-06455943 as a novel potential PET ligand for LRRK2 imaging . Biochemistry assays and autoradiography studies show that [ 18 F]­PF-06455943 has good binding affinity (IC 50 = 3.6 nM) and selectivity toward LRRK2, good brain permeability, and a predicted low risk of nonspecific binding with favorable unbound fraction in the brain and plasma …”

“…al and Schaffer et. al , independently developed LRRK2 PET ligands [ 18 F]­1 & [ 18 F]­2 and [ 18 F]­FMN3PA & [ 18 F]­FMN3PU, respectively, but comprehensive pharmacological and pharmacokinetic information was elusive for all of these radioligands (Supporting Information; Figure S1).…”

Recent Advances in Synthetic Methodologies to Form C-18F Bonds

“…However, 12 is temperature-sensitive and thermally decomposes in polar, aprotic solvents (e.g., DMF and DMSO), thus causing undesirable byproducts under the general conditions of radiofluorination. Schaffer et al ( Malik et al, 2020 ) demonstrated that by using chromium (II) chloride and titanocene dichloride as Lewis acids, it was possible to achieve non-decay corrected radiochemical yields (ndc RCYs) for [ 18 F]MN3PU up to 41 ± 1% and 37 ± 0.7%, respectively ( Figure 3 , Reaction 3).…”