Radiosynthesis and evaluation of [11C]YM-202074 as a PET ligand for imaging the metabotropic glutamate receptor type 1

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Abstract

Introduction

Developing positron emission tomography (PET) ligands for imaging metabotropic glutamate receptor type 1 (mGluR1) is important for studying its role in the central nervous system. N-cyclohexyl-6-{[N-(2-methoxyethyl)-N-methylamino]methyl}-N-methylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-202074) exhibited high binding affinity for mGluR1 (Ki=4.8 nM), and selectivity over other mGluRs in vitro. The purpose of this study was to label YM-202074 with carbon-11 and to evaluate in vitro and in vivo characteristics of [11C]YM-202074 as a PET ligand for mGluR1 in rodents.

Methods

[11C]YM-202074 was synthesized by N-[11C]methylation of its desmethyl precursor with [11C]methyl iodide. The in vitro and in vivo brain regional distributions were determined in rats using autoradiography and PET, respectively.

Results

[11C]YM-202074 (262–630 MBq, n=5) was obtained with radiochemical purity of >98% and specific activity of 27–52 GBq/μmol at the end of synthesis, starting from [11C]CO2 of 19.3–21.5 GBq. In vitro autoradiographic results showed that the high specific binding of [11C]YM-202074 for mGluR1 was presented in the cerebellum, thalamus and hippocampus, which are known as mGluR1-rich regions. In ex vivo autoradiography and PET studies, the radioligand was specifically distributed in the cerebellum, although the uptake was low. Furthermore, the regional distribution was fairly uniform in the whole brain by pretreatment with JNJ16259685 (a mGluR1 antagonist). However, radiometabolite(s) was detected in the brain.

Conclusions

From these results, especially considering the low brain uptake and the influx of radiometabolite(s) into brain, [11C]YM-202074 may not be a useful PET ligand for in vivo imaging of mGluR1 in the brain.

Introduction

Metabotropic glutamate receptors (mGluRs) are a family of G-protein-coupled receptors activated by the neurotransmitter glutamate [1], [2]. They have been classified into three major groups with eight subtypes based on their sequence homology, signal transduction mechanism, and pharmacology. Group 1 mGluRs (mGluR1 and mGluR5) mobilize intracellular calcium by stimulating phospholipase C and up- or down-regulate neuronal excitability. Group 2 (mGluR2 and mGluR3) and Group 3 (mGluR4, mGluR6, mGluR7 and mGluR8) inhibited adenylate cyclase and hence reduce synaptic transmission. mGluR1 is mainly a postsynaptic receptor and is thought to be involved in disorders such as cerebellar ataxia, Parkinson's disease, anxiety, mood disorders, stroke, epilepsy and pain [3].

7-Hydroxyiminocyclopropan[b]chromen-1α-carboxylic acid ethyl ester (CPCCOEt) was identified as a noncompetitive ligand for mGluR1 (Fig. 1) [4], [5]. However, CPCCOEt has low binding affinity for mGluR1. Later, several new noncompetitive mGluR1 antagonists (BAY36-7620 [6], R214127 [7], JNJ16259685 [8], 4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2,3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1(2H)-carboxamide (FTIDC) [9], 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-298198) [10], and N-cyclohexyl-6-{[N-(2-methoxyethyl)-N-methylamino]methyl}-N-methylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-202074) [11]) with higher potency than CPCCOEt have been developed (Fig. 1).

Recently, many positron emission tomography (PET) ligands for selective mGluR5 imaging have been reported, such as [11C]MPEP [12], [11C]ABP688 [13], [18F]FE-DABP688 [14], [18F]F-PEB [15], and [18F]SP203 [16]. In contrast, to our knowledge, only two mGluR1-selective PET ligands have been reported: [11C]JNJ-16567083 [17] and [18F]MK-1312 [18]. High uptake of [11C]JNJ-16567083 was observed in the rat cerebellum, but no PET image was shown [17]. Additionally, there have been no further reports using [11C]JNJ-16567083. As for [18F]MK-1312, PET studies in monkeys, and in vitro binding assay using monkey and human brain homogenates have been performed. However, the abstract did not provide a PET image and basic characteristics (binding affinity, metabolism and so on) of [18F]MK-1312 [18].

YM-298198 and YM-202074 are thiazolo[3,2-a]benzimidazole derivatives, whose chemical structures are different from [11C]JNJ-16567083 and [18F]MK-1312 (Fig. 1). YM-202074 (Ki=4.8 nM) exhibited higher binding affinity for mGluR1 than YM-298198 (Ki=19 nM) [10], [11]. Moreover, YM-202074 showed the 50% inhibitory concentration (IC50) value of 8.6 nM for mGluR1 and no inhibition up to 1 μM for mGluR5 (68% inhibition at 10 μM) [11].

In this study, we chose YM-202074 as a new potent and selective mGluR1 PET ligand candidate. The purposes of this study were as follows: (1) to synthesize N-cyclohexyl-6-{[N-(2-methoxyethyl)-N-[11C]methylamino]methyl}-N-methylthiazolo[3,2-a]benzimidazole-2-carboxamide ([11C]YM-202074, Fig. 2); and (2) to evaluate its in vitro and in vivo characteristics as a PET ligand for mGluR1 in rodents.

Section snippets

Chemicals

All chemicals and solvents were of analytic or high-performance liquid chromatography (HPLC) grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Wako Pure Industries (Osaka, Japan). Proton nuclear magnetic resonance (1H-NMR) spectra were recorded on a JNM-GX-270 spectrometer (JEOL, Tokyo, Japan). High-resolution fast atom bombardment mass spectra (HRFABMS) were obtained on a NMS-SX 102-SX spectrometer (JEOL). Column chromatography was carried out on Kieselgel gel 60 F254

Results

YM-202074 and desmethyl YM-202074 were synthesized according to the reaction sequences shown in Fig. 2. The reaction of 1 with thionyl chloride, following by coupling with N-(2-methoxyethyl)methylamine, produced YM-202074 as an authentic sample at a total yield of 77%. Desmethyl YM-202074 was prepared from 1 at a yield of 65% via a similar route to synthesize YM-202074. The lipophilicity (LogD and cLogD) of YM-202074 is 2.7 and 2.5, respectively.

[11C]YM-202074 was synthesized by N-[11

Discussion

Only two mGluR1-selective PET ligands have been reported, [11C]JNJ-16567083 [17] and [18F]MK-1312 [18]. It is important to develop mGluR1 PET ligands with different chemical structures to investigate the function and involvement of mGluR1 in various brain disorders (cerebellar ataxia, Parkinson's disease, anxiety, mood disorders, stroke, epilepsy, pain), and to measure the receptor occupancy of mGluR1 for preclinical studies (e.g., dose-finding studies). In this study, we synthesized [11

Conclusion

In this study, we synthesized [11C]YM-202074, and evaluated its in vitro and in vivo binding characteristics and metabolism. This radioligand is highly potent and specific for mGluR1 in vitro, but its brain uptake was low. Furthermore, its radiometabolite(s) entering the brain confounded ex vivo autoradiography and PET studies. From these results, [11C]YM-202074 may not be a useful PET ligand for in vivo imaging of mGluR1 in the brain. We are continuing to develop useful mGluR1-selective PET

Acknowledgments

The authors thank Mr. Hisashi Suzuki (National Institute of Radiological Sciences, Chiba, Japan), and Mrs. Yuichiro Yoshida, Masanao Ogawa, and Kenji Furutsuka (SHI Accelerator Service Co., Ltd., Tokyo, Japan) for their technical support with radiosynthesis and analysis, and Drs. Chie Itoh, Toramatsu and Maki Okada (National Institute of Radiological Sciences, Chiba, Japan) for acquiring the PET and MRI images. We are also grateful to the staff of the National Institute of Radiological Sciences

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