Synthesis, radiolabelling and in vitro and in vivo evaluation of a novel fluorinated ABP688 derivative for the PET imaging of metabotropic glutamate receptor subtype 5.

(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-(2-(3-(18)F-fluoropropoxy)ethyl) oxime ([(18)F]-PSS223) was evaluated in vitro and in vivo to establish its potential as a PET tracer for imaging metabotropic glutamate receptor subtype 5 (mGluR5). [(18)F]-PSS223 was obtained in 20% decay corrected radiochemical yield whereas the non-radioactive PSS223 was accomplished in 70% chemical yield in a S(N)2 reaction of common intermediate mesylate 8 with potassium fluoride. The in vitro binding affinity of [(18)F]-PSS223 was measured directly in a Scatchard assay to give K(d) = 3.34 ± 2.05 nM. [(18)F]-PSS223 was stable in PBS and rat plasma but was significantly metabolized by rat liver microsomal enzymes, but to a lesser extent by human liver microsomes. Within 60 min, 90% and 20% of [(18)F]-PSS223 was metabolized by rat and human microsome enzymes, respectively. In vitro autoradiography on horizontal rat brain slices showed heterogeneous distribution of [(18)F]-PSS223 with the highest accumulation in brain regions where mGluR5 is highly expressed (hippocampus, striatum and cortex). Autoradiography in vitro under blockade conditions with ABP688 confirmed the high specificity of [(18)F]-PSS223 for mGluR5. Under the same blocking conditions but using the mGluR1 antagonist, JNJ16259685, no blockade was observed demonstrating the selectivity of [(18)F]-PSS223 for mGluR5 over mGluR1. Despite favourable in vitro properties of [(18)F]-PSS223, a clear-cut visualization of mGluR5-rich brain regions in vivo in rats was not possible mainly due to a fast clearance from the brain and low metabolic stability of [(18)F]-PSS223.

Efficient (18)F-labeling of large 37-amino-acid pHLIP peptide analogues and their biological evaluation.

Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pH(e) < 7). Labeling of peptides with [(18)F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known "click" methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC "click chemistry" for the simple and efficient (18)F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and an L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[(18)F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥98%. The subsequent Cu(I)-catalyzed "click" reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [(18)F]-D-WT-pHLIP and [(18)F]-L-K-pHLIP were obtained with total radiochemical yields of 5-20% after HPLC purification. The total reaction time was 85 min including formulation. In vitro stability tests revealed high stability of the [(18)F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65% and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [(18)F]-D-WT-pHLIP and the negative control [(18)F]-L-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be useful for the (18)F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [(18)F]-pHLIP analogues as potential PET tracers.

In vitro and in vivo evaluation of [18F]-FDEGPECO as a PET tracer for imaging the metabotropic glutamate receptor subtype 5 (mGluR5).

(E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-2-(2-(18)F-fluoroethoxy)ethyl oxime, ([(18)F]-FDEGPECO), a novel high affinity radioligand for the metabotropic glutamate receptor subtype 5 (mGluR5) was assessed for its potential as a PET imaging agent. In vitro autoradiography on rat brain slices resulted in a heterogeneous and displaceable binding to mGluR5-rich brain regions. [(18)F]-FDEGPECO showed high stability in rat plasma and brain homogenate as well as in human plasma and microsomes. Good blood-brain barrier passage was predicted from an in vitro transport assay with P-glycoprotein-transfected hMDR1-MDCK cells. In vivo PET imaging on rats revealed specific uptake of radioactivity in the mGluR5-rich brain regions such as hippocampus, striatum and cortex while the cerebellum, a region with low mGluR5-expression, showed negligible uptake. Blockade experiments by co-injection of [(18)F]-FDEGPECO and M-MPEP (6mg/kg), an antagonist for mGluR5, reduced the level of radioactivity in mGluR5-regions to that of the cerebellum, pointing to an effective blockade of specifically bound [(18)F]-FDEGPECO. Postmortem biodistribution studies at 15min p.i. confirmed the distribution pattern observed in PET. HPLC analysis of rat brain extracts indicated that 98.5% and 91% of the total radioactivity were parent compound at 5min and 17min p.i., respectively. Taken together, the high affinity and the high in vivo specificity of [(18)F]-FDEGPECO for mGluR5 in the rat brain as well as the lack of in vivo defluorination make this new [(18)F]-labeled ABP688 derivative a suitable ligand for the preclinical PET imaging of mGluR5. These favorable characteristics warrant further evaluation in humans.

Syntheses and pharmacological characterization of novel thiazole derivatives as potential mGluR5 PET ligands.

Four novel thiazole containing ABP688 derivatives were synthesized and evaluated for their binding affinity towards the metabotropic glutamate receptor subtype 5 (mGluR5). (E)-3-((2-(Fluoromethyl)thiazol-4-yl)ethynyl)cyclohex-2-enone O-methyl oxime (FTECMO), the ligand with the highest binding affinity (K(i)=5.5+/-1.1 nM), was labeled with fluorine-18. [(18)F]-FTECMO displayed optimal lipophilicity (log D(pH7.4)=1.6+/-0.2) and high stability in rat and human plasma as well as sufficient stability in rat liver microsomes. In vitro autoradiography with [(18)F]-FTECMO revealed a heterogeneous and displaceable binding in mGluR5-rich brain regions. PET imaging with [(18)F]-FTECMO in Wistar rats, however, showed low brain uptake. Uptake of radioactivity into the skull was observed suggesting in vivo defluorination. Thus, although [(18)F]-FTECMO is an excellent ligand for the detection of mGluR5 in vitro, its in vivo characteristics are not optimal for the imaging of mGluR5 in rats in vivo.

Structure-activity relationships of fluorinated (E)-3-((6-methylpyridin-2-yl)ethynyl)cyclohex-2-enone-O-methyloxime (ABP688) derivatives and the discovery of a high affinity analogue as a potential candidate for imaging metabotropic glutamate recepors subtype 5 (mGluR5) with positron emission tomography (PET).

The metabotropic glutamate receptor subtype 5 (mGluR5) is recognized to be involved in numerous brain disorders. In an effort to obtain a fluorine-18 labeled analogue of the mGluR5 PET tracer [(11)C]ABP688, 13 novel ligands based on the core structure of ABP688 were synthesized. Molecules in which the methyl group at the oxime functionality of ABP688 was replaced by fluorobenzonitriles, fluoropyridines, and fluorinated oxygen containing alkyl side chains were investigated. Substituents at the oxime functionality are well tolerated and resulted in five candidates with K(i) values below 10 nM. The most promising candidate, (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone-O-2-(2-fluoroethoxy)ethyloxime (38, K(i) = 3.8 nM), was radiolabeled with fluorine-18. Scatchard analysis of [(18)F]38 which modeled best for two sites pointed to high binding affinity (K(D1) = 0.61 +/- 0.19 nM and K(D2) = 13.73 +/- 4.69 nM) too. These data strongly suggest the further evaluation of [(18)F]38 as a candidate for imaging the mGluR5.