Design, Synthesis, and Evaluation of (2-Aminocyclopropyl)phenyl Derivatives as Novel Positron Emission Tomography Imaging Agents for Lysine-Specific Demethylase 1 in the Brain

Abstract

Dysregulation of histone H3 lysine 4 (H3K4) methylation is implicated in the pathogenesis of neurodevelopmental disorders. Lysine-specific demethylase 1 (LSD1) determines the methylation status of H3K4 through flavin adenine dinucleotide (FAD)-mediated histone demethylation. Therefore, LSD1 inhibition in the brain can be a novel therapeutic option for treating these disorders. Positron emission tomography (PET) imaging of LSD1 allows for investigating LSD1 expression levels under normal and disease conditions and validating target engagement of therapeutic LSD1 inhibitors. This study designed and synthesized (2aminocyclopropyl)phenyl derivatives with irreversible binding to LSD1 as PET imaging agents for LSD1 in the brain. We optimized lipophilicity of the lead compound to minimize the risk of nonspecific binding and identified 1e with high selectivity over monoamine oxidase A and B, which are a family of FAD-dependent enzymes homologous to LSD1. PET imaging in a monkey showed a high uptake of [ 18 F]1e to regions enriched with LSD1, indicating its specific binding to LSD1.

Design, Synthesis, and Evaluation of [18F]T-914 as a Novel Positron-Emission Tomography Tracer for Lysine-Specific Demethylase 1

“…The highest uptake of [ 18 F] 1g was found in the cerebellum, followed by the anterior cingulate, frontal lobe, caudate nucleus, and pons. These observations of heterogeneity across brain regions were consistent with known LSD1 expression levels Figure D focuses on the cerebellum (highest uptake of [ 18 F] 1g ) and pons (lowest uptake of [ 18 F] 1g ) at both baseline and following unlabeled 1g (3.0 mg/kg) pretreatment.…”

“…These observations of heterogeneity across brain regions were consistent with known LSD1 expression levels. 12 Figure 4D focuses on the cerebellum (highest uptake of [ 18 F]1g) and pons (lowest uptake of [ 18 F]1g) at both baseline and following unlabeled 1g (3.0 mg/kg) pretreatment. The reduction in the uptake of [ 18 F]1g in the cerebellum following 1g pretreatment indicates displaceable specific binding of [ 18 F]1g.…”

“…Homogeneous timeresolved fluorescence detection system (Cisbio) was used to estimate in vitro LSD1 demethylation activity as described in the previous report. 12 In Vitro MAOA/B Enzyme Assay. MAOA/B enzyme activity was measured with MAO-Glo Assay (Promega) as described in the previous report.…”

“…MAOA/B enzyme activity was measured with MAO-Glo Assay (Promega) as described in the previous report. 12 Analysis of FAD Adducts with Compounds in LSD1 Protein by High-Resolution Mass Spectrometry. Human recombinant LSD1 protein (125 μM) was treated with 1 mM 1g at room temperature for 1 h in 50 mM Tris-HCl (pH 8.0) to form 1g-FAD adduct in LSD1.…”

“…Based on these considerations, the 4-(2-aminocyclopropyl)­benzamide derivative, 1a , which contained a 2-fluoropyridine, was chosen as the lead compound for developing a 18 F-labeled LSD1 PET tracer (Figure ). 1a inhibited the enzyme activity of human recombinant LSD1 with 11 nM of concentration for 50% inhibition (IC 50 ). The efflux ratio of 1a for multidrug resistance 1 (MDR1) was 1.3, suggesting that 1a had good brain permeability.…”