Synthesis and preclinical evaluation of [11C]uPSEM792 for PSAM4-GlyR based chemogenetics

Abstract

Chemogenetic tools are designed to control neuronal signaling. These tools have the potential to contribute to the understanding of neuropsychiatric disorders and to the development of new treatments. One such chemogenetic technology comprises modified Pharmacologically Selective Actuator Modules (PSAMs) paired with Pharmacologically Selective Effector Molecules (PSEMs). PSAMs are receptors with ligand-binding domains that have been modified to interact only with a specific small-molecule agonist, designated a PSEM. PSAM⁴ is a triple mutant PSAM derived from the α7 nicotinic receptor (α7^{L131G,Q139L,Y217F}). Although having no constitutive activity as a ligand-gated ion channel, PSAM⁴ has been coupled to the serotonin 5-HT₃ receptor (5-HT₃R) and to the glycine receptor (GlyR). Treatment with the partner PSEM to activate PSAM⁴-5-HT₃ or PSAM⁴-GlyR, causes neuronal activation or silencing, respectively. A suitably designed radioligand may enable selective visualization of the expression and location of PSAMs with positron emission tomography (PET). Here, we evaluated uPSEM792, an ultrapotent PSEM for PSAM⁴-GlyR, as a possible lead for PET radioligand development. We labeled uPSEM792 with the positron-emitter, carbon-11 (t_1/2 = 20.4 min), in high radiochemical yield by treating a protected precursor with [¹¹C]iodomethane followed by base deprotection. PET experiments with [¹¹C]uPSEM792 in rodents and in a monkey transduced with PSAM⁴-GlyR showed low peak radioactivity uptake in brain. This low uptake was probably due to high polarity of the radioligand, as evidenced by physicochemical measurements, and to the vulnerability of the radioligand to efflux transport at the blood-brain barrier. These findings can inform the design of a more effective PSAM⁴ based PET radioligand, based on the uPSEM792 chemotype.

Figure 1

Structures of varenicline, uPSEM792, other uPSEMs, and [¹⁸F]ASEM.

Scheme 1

Synthesis of compound 2, as a precursor for the ¹¹C-labeling of uPSEM792, and reference compounds uPSEM792 and uPSEM793. Note that the products from 1 are racemic.

Figure 2

¹³C-NMR spectra (for aliphatic carbon region) verifying radiolabeling position in [¹¹C]uPSEM792. (A) ¹³C-NMR spectrum of reference uPSEM792. (B) ¹³C-NMR spectrum of reference uPSEM793. (C) ¹³C-NMR spectrum of the carrier in [¹¹C/¹³C]uPSEM792 isolated through HPLC from the reaction deploying [¹¹C]iodomethane plus a known amount of [¹³C]iodomethane (conditions: K₂CO₃–K 2.2.2 at 80 °C in DMF). (D) ¹³C-NMR spectra of the carrier in [¹¹C/¹³C]uPSEM792 and [¹¹C/¹³C]uPSEM793 isolated through HPLC from the reaction deploying [¹¹C]iodomethane plus a known amount of [¹³C]iodomethane (conditions: K₂CO₃–K 2.2.2 at 60 °C in MeOH).

Scheme 2

Radiosynthesis of [¹¹C]uPSEM792.

Figure 3

[¹¹C]uPSEM792 has low uptake in wild-type rat brain. (A) Whole brain time-activity curve after intravenous injection of [¹¹C]uPSEM792 into rats (mean ± range; n = 2). (B) Transaxial PET image of radioactivity concentration (SUV; summed from 0 to 120 min) of entire rat head including brain. Note the brain region, which is pointed out with the arrow, lies within the circle, and has conspicuously low radioactivity uptake.

Figure 4

Intravenously administered [¹¹C]uPSEM792 gives more polar radiometabolites in rat brain and plasma. Reversed phase HPLC radiochromatographic profiles are shown for rat brain (A) and plasma (B) at 100 min after intravenous administration of [¹¹C]uPSEM792.

Figure 5

[¹¹C]uPSEM792 gives higher average whole brain radioactivity uptake in dual efflux transporter knockout mice than in wild-type mice after i.v. injection of [¹¹C]uPSEM792.

Figure 6

[¹¹C]uPSEM792 has low uptake in non-transduced healthy and PSAM⁴-GlyR monkey brain. (A) Time-activity curves for whole brain after i.v. injection of [¹¹C]uPSEM792 into non-transduced healthy and PSAM⁴-GlyR monkey, and for target (PSAM⁴-GlyR injected site, Left-amygdala), and mirror (opposite contralateral site, Right amygdala) (B) MRI (left) and PET images (summed from 0 to 120 min) for PSAM⁴-GlyR monkey whole brain (middle) and non-transduced healthy monkey whole brain (right).

Figure 7

(A) Reversed phase HPLC analysis of non-transduced healthy monkey plasma sampled at 120 min after [¹¹C]uPSEM792 injection showed that most of the radioactivity eluted as radiometabolites before unchanged radioligand. (B) By 78 min radiometabolites represented half of the radioactivity in plasma in non-transduced healthy monkey. (C) In PSAM⁴-GlyR monkey injected with [¹¹C]uPSEM792, radiometabolites accounted for 50% of radioactivity in plasma at 30 min.