Preclinical evaluation of ¹¹C-JNJ55511118 and ¹¹C-JNJ56022486 for imaging subtype-selective AMPA receptor

Objectives: Modulating transmembrane AMPA (a-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid) receptor regulatory proteins (TARPs) has emerged as a novel and alternative strategy towards pharmacotherapies for neuronal hyperactivity related CNS disorders such as epilepsy, PD, multiple sclerosis and schizophrenia. The development of TARP γ-8 subtype selective AMPA receptor antagonists, such as JNJ55511118 (1), JNJ56022486 (2) and LY3130481, is particularly promising for therapy and diagnosis of pathologic disorders within forebrain.[1][2] The goal of the project was to carry out ¹¹C-isotopologue labeling of two novel TARP γ-8 dependent AMPA receptor antagonists JNJ55511118 and JNJ56022486, and perform preliminary PET studies and in vitro autoradiography in rodents. Methods: The synthesis of labeling precursor 5 was achieved through PdCl₂(dtbpf)-catalyzed cross-coupling of aryl boric acid 3 with 4-bromobenzene-1,2-diamine 4. The precursor 8 was assembled via a sequence of PdCl₂(dppf)-catalyzed coupling of aryl iodide 6 with 4-amino-3-nitrophenyl borate 7, followed by reduction Fe-mediated reduction in aqueous HCl solution. Subsequent treatment of diamine 5 and 8 with 1,1’-carbonyldiimidazole readily generated the corresponding standard 1 and 2, respectively. Radiosynthesis of [¹¹C]1 and [¹¹C]2 was performed using two ¹¹C-carbonylation labeling strategies from [¹¹C]COCl₂ and [¹¹C]CO₂, respectively. Dynamic PET scans (60 min) were conducted in Sprague-Dawley rats. In vitro autoradiography (baseline and blocking) were also performed on frozen rat brain tissue to validate the binding specificity. Results: The standard compounds 1 and 2 were achieved in 50% yield over two steps and 61% yield over three steps, respectively, and their corresponding precursor 5 and 8 were obtained in 73% and 68% yields, respectively. Both ¹¹C-carbonylayion strategies readily delivered the isotopologue of [¹¹C]1 and [¹¹C]2 in more than 10% isolated radiochemical yields (ca. 2.22 GBq; non-decay corrected from starting [¹¹C]CO₂) with high radiochemical purity (>99%) and high specific activity (greater than 40 GBq/μmol) within 45 min (end of synthesis). The compound [¹¹C]1 synthesized either from [¹¹C]COCl₂ or [¹¹C]CO₂ showed no signs of radiolysis up to 90 min after formulation (10% ethanol in saline). In preliminary PET studies, while AMPA antagonist [¹¹C]2 showed limited brain uptake (0.3 SUV), [¹¹C]1 is brain permeable with peak uptake of 1 SUV. We further utilize in vitro autoradiography to confirm binding specificity of [¹¹C]2, which showed heterogeneous distribution and reasonable bound radioactivity in the region of hippocampus, in accordance with the brain distribution of TARP γ-8 AMPA receptor. Pretreatment studies with JNJ56022486 (3 mg/kg) showed moderate binding with statistic significance compared to baseline study.

Conclusions: We have successfully radiolabeled two potent TARP γ-8 dependent AMPA receptor antagonists in high radiochemical yields. In vivo PET study demonstrated the brain-penetration of [¹¹C]1 and in vitro autoradiography confirmed the specific binding of [¹¹C]2. Further characterization including blocking studies with structurally-diverse AMPA antagonists, ex vivo whole body distribution and radiometabolite analysis will be performed in order to evaluate and develop potent TARP γ-8 dependent AMPA receptor PET tracers. References: [1] J Pharmacol Exp Ther. 2016, 357, 394-414; [2] Nat Med. 2016, 22, 1496-1501.