ABP688, a novel selective and high affinity ligand for the labeling of mGlu5 receptors: Identification, in vitro pharmacology, pharmacokinetic and biodistribution studies

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Abstract

[11C]ABP688 (2) has recently been demonstrated to be a useful PET tracer for in vivo imaging of the metabotropic glutamate receptors type 5 (mGluR5) in rodents. We describe here the identification and preclinical profiling of ABP688 and its tritiated version [3H]ABP688, and show that its high affinity (Kd = 2 nM), selectivity, and pharmacokinetic properties fulfill all requirements for development as a PET tracer for clinical imaging of the mGlu5 receptor.

Introduction

Based on amino-acid sequence, pharmacology, and second messenger coupling, metabotropic glutamate receptors (mGluRs) have been clustered into three groups (I–III).1 Group I receptors (mGluR1 and mGluR5) are coupled to the phospholipase C intracellular pathway, Group II (mGluR2 and mGluR3) and Group III (mGluR4, 6, 7, and 8) are negatively linked to adenylyl cyclase.

In contrast to the glutamate-gated ion channels (NMDA, AMPA, and kainate receptors), which mediate the glutamatergic neurotransmission in the CNS, mGlu receptors belong to the ‘family 3’ G protein-coupled receptors and have been shown to play a modulatory role in the glutamatergic synaptic transmission, either by regulating ion channel activity or by neurotransmitter release. The recent discovery of small molecules that selectively interact with receptors of Group I (mGlu1 and mGlu5) and II (mGlu2 and mGlu3) has facilitated significant advances in the understanding of the roles of these receptors in brain physiology and pathophysiology.2

The identification of MPEP (1, 2-methyl-6-(phenylethynyl)-pyridine, Fig. 1), a selective, non-competitive, and brain-penetrable mGlu5 receptor antagonist,3 allowed the exploration of the therapeutic potential of mGlu5 receptor antagonism. Results from a variety of behavioral studies revealed that—with the exception of the benzodiazepines—mGlu5 receptor antagonists exhibit the widest and most robust anxiolytic activity in pre-clinical models seen to date.4, 5, 6 Upcoming clinical studies, involving the use of specific mGlu5 receptor antagonists, should soon indicate whether the preclinical anxiolytic-like effects translate into anxiolytic activity in man.

As comprehensive as it might be, the preclinical profile of a novel mGlu5 antagonist, as for other CNS drugs, does not allow an accurate prediction of the adequate drug dosing in humans. Positron emission tomography (PET) with receptor specific ligands is an imaging method which has been proven useful for studying these parameters in humans.7, 8

To date, a number of mGlu5 receptor antagonists have been successfully used to label mGlu5 receptors in vitro.9, 10 However, the development of these ligands into useful PET tracers has so far not met with success11, 12 and only very recently a series of 11C and 18F-derivatives of MTEP and MTEB (2-methyl-4-phenylethynyl-thiazole), allowing PET imaging in rhesus monkey, has been described.13

In parallel to our discovery program aiming at the identification of mGlu5 receptor antagonists, we intended to identify a suitable PET ligand to support the clinical development of a pharmaceutical agent. Our target criteria for a potential PET ligand were: (1) a log P below 4; (2) an easy radiolabeling in the last synthesis step; (3) a high affinity (<10 nM) for mGluR5 and selectivity (>100-fold) over other receptors; (4) a rapid brain uptake and elimination; and (5) the absence of brain-penetrating radiolabeled metabolites. In order to allow for displacement studies and for determination of receptor occupancy by therapeutic drug candidates, any potential tracer would have to be competitively and fully displaced by drug candidates binding to the allosteric binding pocket at the mGlu5 receptor.

Initially, a series of PET candidates was synthesized based on the prototypic antagonist MPEP. A 11C or 18F label was introduced in positions C-3 of the phenyl ring or C-6 of the pyridine ring, which were shown to tolerate modifications without compromising the affinity for the allosteric binding site.14, 15 Four of these initial derivatives were tested in vivo in rat to determine their brain uptake and distribution but failed to meet the target profile, by exhibiting a combination of a relatively high lipophilicity, high non-specific binding, insufficient affinity or limited metabolic stability.

We subsequently revisited the library of compounds which had been synthesized during the medicinal chemistry program for mGluR5 antagonists and we focused on a series of derivatives with a cyclohexenone oxime instead of the aromatic ring contained in MPEP. The introduction of an oxime significantly decreased lipophilicity and improved water solubility, as compared to the precursor series. The most promising candidate identified was ABP688 (2), a high affinity and selective mGluR5 antagonist with a calculated log P of 2.4 (Fig. 1).

Since this compound fulfilled all of our criteria in terms of physicochemical properties, we decided to further explore its potential as a PET imaging ligand for the mGlu5 receptor. Here, we report on the preclinical characterization of ABP688 and its tritiated species [3H]ABP688. This characterization allowed the selection of this ligand for development as a PET tracer.16

Section snippets

Synthesis of ABP688 (2) and its labeling precursor 7

The synthesis of ABP688 (2) and of the precursor of [3H]ABP688, oxime 7, started from commercially available 2-bromo-6-methyl-pyridine. A Sonogashira17 cross coupling with 2-methyl-3-butin-2-ol yielded the tertiary alcohol (3), which was easily and in good yields converted into the alkynyl-pyridine (4), by base-catalyzed acetone elimination (Scheme 1). A second Sonogashira18 coupling with 3-bromo-cyclohex-2-enone (5)19 led directly to ketone 6, which was then readily transformed into the

Potency and selectivity in functional assays

ABP688 was originally shown to be a potent antagonist of quisqualate-induced phosphoinositol (PI) accumulation in L(tk-) cells expressing recombinant human mGluR5 (hmGluR5), with an IC50 value of 2.4 nM (95% CI: 0.5–12 nM) (Fig. 2A). In the same preparation, ABP688 completely inhibited glutamate-induced calcium release with an IC50 value of 2.3 nM (95% CI: 2.1–2.5) nM (data not shown) and had no effect, up to 10 μM, on ATP-induced PI accumulation via stimulation of endogenously expressed purinergic

Discussion

Initial efforts to identify a mGluR5-selective PET tracer based on the structural framework of the selective antagonist MPEP failed to produce a valid PET tracer. It is only very recently that a first series of 11C and 18F-derivatives of MTEP and MTEB (2-methyl-4-phenylethynyl-thiazole) has been described, allowing in vivo PET imaging in rhesus monkey.

The aim of our program was to identify a molecule with an improved tracer profile compared to the MPEP series, particularly a higher affinity,

Chemicals

Reagents and solvents were purchased and used without further purification. Column chromatography was performed with Merck Silica Gel 60 (0.040–0.063 mm). Thin-layer chromatography to monitor reactions and to identify product-containing fractions was carried out using glass plates pre-coated with silica gel (60 F254) and was developed in a UV-chamber (254 and/or 365 nm).

Analytics

High-resolution mass spectra were acquired on a 9.4T APEX III Fourier Transform mass spectrometer (Bruker Daltonik GmbH, Bremen,

Acknowledgments

The authors thank H. P. Müller, W. Inderbitzin, R. Felber (Chemistry); D. Fehlmann, E. Shuepbach (autoradiography); P. Guntz (animal PK); R. Masero, R. Lehmann (Radiochemistry) C. Stierlin, N. Reymann (in vitro pharmacology), J. Hamon, Y. Gezahegen, T. Luethi, L. Sampson, S. Staub, and S. Vidal (cross-selectivity panel)for excellent technical support, E. Francotte for stability testing and HPLC analysis, Analytics Department (G. Bovermann, L. Oberer) for assignment of isomers, K. Kaupmann, J.

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