Site-Specific and Residualizing Linker for 18F Labeling with Enhanced Renal Clearance: Application to an Anti-HER2 Single-Domain Antibody Fragment.

1624 Objectives: Positive allosteric modulators of muscarinic acetylcholine receptor subtype 4 (M4) have been identified as promising activators for the treatment of neurological disorders and neurodegenerative diseases, including schizophrenia, Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB). We have identified 1-(6,7-dimethyl-4-(methylamino) -1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-yl)-2-(trans-2-(6-fluoropyridin-3-yl)cyclopropyl)ethan-1-one (7) as a lead molecule for M4-specific PET ligand attributed to its high potency (IC50 = 0.8 nM),[1] feasibility for 18F-labeling on 2-substituted pyridine scaffold and favorable in vitro brain permeability prediction (MPO score of 5.83 and logBB of -0.16). The goal of the project was to synthesize the standard and precursor molecules and perform radiolabeling of compound 7 with fluorine-18 as well as preliminary evaluation of [18F]7 in rodents. Methods: The leading compound 7 was synthesized as summarized in Scheme 1A.The cyclization of compound 1 with Et2Zn and CH2I2 led to intermediate 2 in 80% yield. Through Suzuki coupling reaction of compound 2 and 5-bromo-2-fluoropyridine 3, compound 4 was obtained in 25% yield. By deprotection and oxidation with RuCl3 and NaIO4, compound 4 wasconverted to carboxylic acid 5 in 53% yield. Through condensation reactions between acid 5 and amine 6, the desired product 7 was obtained in 86% yield. The synthesis of the labeling precursor for compound 7 was showed in Scheme 1B.Through Suzuki coupling reaction of compound 2 and 5-bromo-2-nitropyridine 8, compound 9 was obtained in 73% yield. By deprotection and oxidation with RuCl3 and NaIO4, compound 9 wasconverted to carboxylic acid 10 in 95% yield. After condensation reaction of acid 10 and amine 6, the precursor 11 was obtained in 63% yield. 18F-labeled radiotracer [18F]7 was obtained by reacting with nitro precursor 11 with [18F]ﬂuoride using potassium carbonate as eluting base (Scheme 1C). Then the binding specificity in vitro of [18F]7 was evaluated by autoradiography studies on Sprague-Dawley rat brain. Subsequently, dynamic PET acquisitions (0-60 min) were conducted with [18F]7 in Sprague-Dawley rats. Results: The synthesis of candidate compound 7 was obtained from commercially available compound 1 in four steps with overall yield of 9%. The radiochemical yield of [18F]7 was 2% (non-decay corrected) with greater than 99% radiochemical purity, and the molar activity was higher than 37 GBq/μmol (1.0 Ci/μmol). The results of autoradiography studies are shown in Figure 2. At the baseline, the distribution of radioactivity was mainly concentrated in the striatum, cerebral cortex, thalamus and hippocampus, which was consistent with the expression pattern of M4 in the brain. In the blocking study, pretreatment with unlabeled 7 resulted in visible reduction of radioactivity in striatum and cerebral cortex which are reported as the M4 rich brain regions. PET images of [18F]7 aredisplayed inFigure 1B. [18F]7 showed limited brain uptake (ca. 0.55 SUV peak uptake) with marginal difference in the cerebral cortex, cerebellum, striatum, hippocampus and thalamus. Conclusions: The 18F-labeling ligand [18F]7 (also named [18F]M4R-1911)was obtained in a reasonable radiochemical yield, high radiochemical purity and high molar activity. While in vitro autoradiography studies showed that [18F]7 hadlow-to-moderate in vitro binding specificity, PET studies confirmed that [18F]7 was not a suitable tracer for in vivo imaging of M4 due to limited brain permeability. Further in-depth medicinal chemistry studies are necessary to design new M4 ligands with improved brain permeability and high specific binding for PET imaging studies. References: [1] WO2018234953.