Synthesis and Characterization of Fluorine-18 labeled N-(4-Chloro-3-((fluoromethyl-d₂)thio)phenyl)picolinamide for Imaging of mGluR4 in Brain

Abstract

We have synthesized and characterized [¹⁸F]-N-(4-chloro-3-((fluoromethyl-d₂)thio)phenyl)-picolinamide ([¹⁸F]15) as a potential ligand for the PET imaging of mGluR4 in the brain. Radioligand [¹⁸F]15 displays CNS drug-like properties, including mGluR4 affinity, potent mGluR4 PAM activity and selectivity against other mGluRs, as well as sufficient metabolic stability. Radiosynthesis was carried out in two steps. The radiochemical yield of [¹⁸F]15 was 11.6 ± 2.9% (n = 7, decay corrected) with a purity of 99% and a molar activity of 84.1 ± 11.8 GBq/μmol. Ex vivo biodistribution studies showed reversible binding of [¹⁸F]15 in all investigated tissues including the brain, liver, heart, lungs, and kidneys. PET imaging studies in male Sprague-Dawley rats showed that [¹⁸F]15 accumulates in the brain regions known to express mGluR4. Pretreatment with the unlabeled mGluR4 PAM compounds 13 (methylthio analog) and 15 showed significant dose-dependent blocking effects. These results suggest that [¹⁸F]15 is a promising radioligand for PET imaging mGluR4 in the brain.

Graphical Abstract

INTRODUCTION

Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting more than six million people worldwide.^1–2 PD results from the progressive loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc), causing dysfunction of the basal ganglia (BG) motor circuit.^3–4 The current pharmacotherapy aims to replace missing dopamine by using the dopamine precursor levodopa (L-DOPA). This treatment provides symptomatic relief and is successful in the early PD medication period,⁵ however, as the disease progresses L-DOPA becomes less effective and produces debilitating side effects such as L-dopa-induced dyskinesia (LID).^6–7 Hence the development of a disease modifying therapy for PD remains a challenge for researchers.

Over the last decade, metabotropic glutamate receptor 4 (mGluR4) has gained interest due to the potential benefits in treating numerous neuronal diseases such as PD, LID and other disorders.^{6, 8–9} The mGluR4 receptor is expressed at multiple synapses in desired regions of the BG motor circuit, mainly localized presynaptically in the striatum, hippocampus, thalamus, and cerebellum.^10–12 As a group III mGluR, mGluR4 interacts with the G_ai/o subunit of G-protein that negatively couples with adenylate cyclase to inhibit cAMP dependent signal pathways.^{1, 13} Its activation reduces presynaptic neurotransmitter release, subsequently, decreasing output of the indirect pathway. This is a promising strategy to normalize the BG circuitry in PD and ultimately reduce or eliminate PD symptoms.^{3, 5, 8, 10} Consequently, this approach has opened a new avenue for developing nondopaminergic treatments for PD and for identifying novel disease modifying therapeutics.

As a family C GPCR, the activation of mGluR4 can be accomplished or enhanced by two different mechanisms: orthosteric agonists or positive allosteric modulators (PAMs). Although most orthosteric ligands lack clear subtype-selectivity and/or blood–brain barrier (BBB) penetration, few examples of selective and brain penetrant orthosteric agonists such as LSP4-2022 have been reported.^14–15 Since allosteric modulators may offer some benefits such as subtype-selectivity, retained physiology of the receptor and the saturation effects, substantial effort has been focused on development of mGluR4 PAMs as new therapeutics for neurological diseases such as PD and LID.⁶ Hundreds of mGluR4 PAMs have been reported and/or patented since 2009.^16–17 Figure 1 shows some representative mGluR4 PAMs, of which compounds 1-10 have demonstrated antiparkinsonian activity in animal models of PD.^18–28 Compound 11 is a potent mGluR4 PAM that has been used as the reference compound for mGluR4 PAM activity.²⁸ Prexton Therapeutics (Prexton) announced in 2017 the launch of phase II clinical trial of its investigational drug candidate, PXT002331 (10, Foliglurax), in PD.⁶

Figure 1.

Representative mGluR4 PAMs.

An efficient PET radioligand for mGluR4 could be an important tool for understanding the role of mGluR4 in healthy and disease conditions, and also for the development of new drugs targeting this receptor.^{9, 29} PET offers picomolar sensitivity and is a fully translational technique; therefore, extensive research efforts have been directed towards the development of PET ligands suitable for in vivo probing of mGluR4 (Figure 2).^30–33 In the past, we have studied the pharmacokinetics of [¹¹C]4, ([¹¹C]ML-128), [¹⁸F]12 ([¹⁸F]KALB001), [¹¹C]13 ([¹¹C]mG4P012 or [¹¹C]KALB012) and [¹¹C]14 by using PET imaging in rats. PET studies with [¹⁸F]12 and [¹¹C]13 were conducted also in non-human primates.³¹ These PET radiotracers have been designed and/or developed based on mGluR4 PAMs.

Figure 2.

PET tracers for mGluR4. We have previously synthetized and reported the tracers [¹¹C]4³¹, [¹⁸F]12³², [¹¹C]13³³ and [¹¹C]14³³.

Among these PET ligands, the in vitro data revealed that compound 13 has sufficient mGluR4 binding affinity and selectivity over other mGluRs, as well as a suitable metabolic stability.³² PET studies in rats showed that [¹¹C]13 accumulated rapidly into the brain and had higher uptake, slower washout and 25% better contrast than [¹¹C]4, indicating improved imaging characteristics as a PET radiotracer for mGluR4.³² The improved pharmacological properties and the enhanced imaging characteristics indicated that [¹¹C]13 is a useful PET radiotracer for mGluR4 in biological research and drug development.

Recently [¹¹C]13 (renamed [¹¹C]PXT012253 by Prexton Therapeutics) has been used as a biomarker during the preclinical development of a potential therapeutic drug, PXT0002331 (10, an mGluR4 PAM), for PD and LID.^34–35 The radiotracer [¹¹C]13 was reported to display binding in mGluR4-expressing regions in the brain of cynomolgus monkeys. Competition of [¹¹C]13 with 10 showed high specific binding in the total distribution volume, which is useful for target occupancy or longitudinal studies. This data further supports [¹¹C]13 as a promising PET radioligand for mGluR4 in the monkey brain and for further development in human subjects.

Development of a fluorine-18 labeled PET tracer for imaging mGluR4 is important since fluorine-18 is often the radionuclide of choice for both its physical and nuclear characteristics. Its half-life is sufficient to carry out relatively extensive imaging protocols when compared to what is possible with carbon-11. This facilitates kinetic studies and high-quality metabolic and plasma analysis, while the low positron energy associated with fluorine-18 decay leads to high imaging resolution. We reported a fluorine-18 labeled PET tracer for mGluR4 ([¹⁸F]12, [¹⁸F]KALB001) in 2014, which showed good affinity to mGluR4 and sufficient brain uptake³¹; however, this phthalimide derivative was not stable under neutral condition and required formulation in an acidic buffer (pH 4.0). Further development of mGluR4 PET tracers indicated that [¹¹C]13 had more favorable characteristics for imaging³² and it initiated designing a new fluorine-18 labeled PET tracer, [¹⁸F]mG4P027 ([¹⁸F]15).

There are three possible positions to introduce fluorine-18: the 6-pyridyl, 4-chloro and 3-methylthio moieties (Chart 1). SAR results³⁶ indicate that 4-fluoro substitution of the chloro-atom gives reduced binding affinity which follows the order: Cl < H < F < I < Br. On the other hand, incorporation of a fluorine atom at the 6-pyrindyl position also slightly lowers the affinity and affects its metabolic stability. Next step was to evaluate the position of 3-phenyl, which is sensitive to substitutions. Our previous results showed that the 3-fluoromethoxy compound had an improved affinity compared to that of the 3-methoxy compound. Since the fluoromethylthio group may not be metabolically stable, we applied a 3-dideuteriumfluoromethylthio moiety to replace the 3-methylthio group as shown in Chart 1. Deuterium isotope effects have been used to reduce in vivo metabolic rates. The incorporation of dideuterium into [¹⁸F]15 could reduce the rate of defluorination initiated by cleavage of the C–H bond without altering the binding affinity to mGluR4.

Chart 1.

The design of [¹⁸F]15 from the structure of [¹¹C]13.

Herein, we report our results on the chemical synthesis, in vitro pharmacochemical properties, radiolabeling, ex vivo biodistribution and PET imaging in rats of this new fluorine-18 labeled PET ligand for mGluR4.

RESULTS AND DISCUSSION

Chemistry.

The novel unlabeled mGluR4 ligand 15 was prepared to study its in vitro pharmacochemical properties (Scheme 1). The reaction of methylene-d₂ bis(tosylate) 16 with cesium fluoride was carried out in t-amyl alcohol at 80°C to give fluoromethyl-d₂ 4-methylbenzenesulfonate 17 in 48% yield. It was found that the choice of solvent used in this reaction was very important. If the reaction was carried out in acetonitrile, the yield was 5%. The thiophenol precursor 18 had been used for the radiolabeling of [¹¹C]13, which was prepared from 1-chloro-nitrobenzene via multi-steps.³² In order to support the translational study of [¹¹C]13, a new synthetic route was developed for synthesis of 18 with optimized reaction conditions, resulting in an improved yield and a more efficient workup procedure.³⁷ The synthesis of compound 15 was achieved by treating 17 with the thiophenol precursor 18 in 92% yield.

Scheme 1.

Synthesis of compound 15.

In vitro pharmacochemical properties.

To evaluate compound 15, the pharmacochemical properties including affinity to mGluR4, mGluR4 PAM activity, selectivity to other mGluRs, lipophilicity, plasma protein binding, metabolic and solution stabilities were determined. In this study compound 15 was compared to the previously reported mGluR4 PET ligands: compounds 4, 13 and others.

The tritium-labeled radioligand [³H]4 (N-(4-chloro-3-(methoxy-t₃)phenyl)picolinamide) was prepared for competitive binding assay (supporting information).^{31, 38} Compounds 4, 8, 13 and 15 were characterized with the competitive binding assay using mGluR4 transfected CHO cells by increasing the concentration of test compound from 0.01 nM to 10 μM in the presence of 2 nM of [³H]4, in which the binding affinities to mGluR4 were described as IC₅₀ values (Table 1).

Table 1.

Affinity to mGluR4.

Compound	4	8	13	15
IC50±SE (nM)	5.1±1.1	3.2±0.6	3.5±0.7	3.5±0.5
log(IC50±SE)	−8.29±0.09	−8.49±0.07	−8.46±0.08	−8.46±0.06

As displayed in Table 1, compound 15 has a similar IC₅₀ value (3.5±0.5 nM) as that of 8 (3.2±0.6 nM) and 13 (3.5±0.7 nM) and has slightly improved affinity compared to 4 (5.1±1.1 nM). The results also confirmed that compound 15 binds to the same allosteric site of mGluR4 as that of the other three compounds.

The mGluR4 PAM activity was determined using Promega’s split luciferase based GloSensor cAMP biosensor assay.^39–40 The mGluR4 PAM activity of 15 was evaluated in presence of EC₂₀ concentration of agonist (1 μM L-SOP) by measuring changes in intracellular cAMP concentration, the relevant second messenger mechanism. Compound 11, one of the most potent mGluR4 PAMs, was used as the reference compound for the assay. As shown in Figure 3, the EC₅₀ values of 11 and 15 were 55 nM and 324 nM, respectively, suggesting that 15 is a moderately potent mGluR4 PAM. In the mGluR4 Gi fold shift assay, 15 shifts the agonist (L-SOP) response curves to the left 7.5-fold at 3.0 μM in 98.3% Glu Max efficacy.

Figure 3.

Quantification of the allosteric effects of mGluR4 PAM 15 by using a cAMP assay. (Left) mGluR4 Gi PAM activity assay, in which mGluR4 PAM (11 or 15) potentiates the effect induced by an EC₂₀ glutamate concentration (EC₅₀: 324 nM for 15; 55 nM for 11); (Right) mGluR4 Gi fold shift assay, in which 15 shifts the agonist (L-SOP) response curves to the left 7.5-fold at 3.0 μM in 98.3% Glu Max efficacy. Data represents the average of at least three independent determinations performed in triplicate.

The selectivity of 15 was also analyzed among the various mGluR subtypes, in which the Gq coupled receptors (mGluR1 and mGluR5) were tested using Ca²⁺ mobilization assay and the Gi/o coupled receptors (mGluR2, mGluR3, mGluR4, mGluR6 and mGluR8) using the cAMP assay. Results demonstrated that 15 has selectivity against other mGlu receptors (Table S1) and that 15 has mGluR4 agonist activity (EC₅₀ = 2.75 μM), hence, 15 is an mGluR4 ago-PAM.

Lipophilicity has a major effect on BBB penetration, ADMET properties and pharmacological activity. The values of lipophilicity (cLogP) and tPSA for 15 were calculated by using ChemDraw as 2.95 and 41.46, respectively. The LogD_7.4 of 15 was 3.82 as measured with a scaled-down shake flask method using [¹⁸F]15 (Table 2) and is in the range normally considered favorable for a PET ligand.⁴²

Table 2.

Pharmacochemical properties.

Compound	MW	tPSA	HBD	cLogP	Log D7.4	PPB
15	298.76	41.46	1	2.95	3.82	93.1%

As the PET tracer is delivered into the bloodstream, it can bind to albumin, a1-acid glycoprotein and lipoproteins. Plasma protein binding (PPB) reduces the free drug in the bloodstream and inhibits BBB penetration.⁴² The PPB value was obtained with an ultrafiltration assay by using [¹⁸F]15. As Table 2 shows, the PPB of 15 was 93.1%, which gives a suitable free tracer concentration available to cross the BBB.

After iv injection PET tracers encounter plasma decomposition by hydrolytic enzymes in the blood and are carried into the liver where they face diverse hepatic metabolic reactions such as phase I oxidations by CYP and flavin monooxygenases (FMO). Unstable compounds often have high clearance (Cl_int) and short half-life (t_1/2) resulting in poor in vivo pharmacokinetics (PK) and unsatisfactory pharmacological performance. The in vitro plasma and liver microsomal stability of 4, 13 and 15 were studied by incubating the compounds in rat serum and rat liver microsomes as well as NADPH cofactor, respectively, using previously published methods.^43–44 Diltiazem and propranolol were used as co-assay QC controls for plasma and microsomal stability assays, respectively, to ensure that the assays were operating properly, and that the activity of the plasma and microsomes were consistent with established criteria. As Table 3 shows, 4, 13 and 15 are more stable than diltiazem in rat plasma. The results also show that 15 exhibits excellent microsomal stability and is more stable than 4 and 13, in which the suitable hepatic clearance was predicted. The solution stability of 15 was evaluated at pH 5.0, 7.4 and 9.4, respectively.⁴⁵ The results indicate that 15 is relatively stable in pH ranging from 5.0 to 9.4 (Table 3).

Table 3.

Metabolic and solution stabilities.

Compound	The plasma stability	The microsome stability		The solution stability
	The intact ± SEM at 120 (min%)	t1/2 (min)	Clint (μL/min /mg protein)	The intact ± SEM at 120 min (%)
				pH = 5.0	pH = 7.4	pH = 9.4
4	98.2 ± 6.3	14.4	96.2	92.7 ± 3.1	97.3 ± 1.1	89.1 ± 2.1
13	91.5 ± 2.5	25.3	54.8	85.9 ± 0.8	87.9 ± 3.4	89.3 ± 1.3
15	84.5 ± 10.2	57.3	24.2	87.1 ± 0.9	88.3 ± 1.9	84.3 ± 1.8
Diltiazem	49.0 ± 10.6	--	--	94.7 ± 1.4	99.0 ± 5.7	69.8 ± 2.6
Propranolol	--	3.6	390	--	--	--

The in vitro pharmacological studies reveal that compound 15 has many CNS drug-like properties, including appropriate mGluR4 affinity, potent mGluR4 PAM activity and selectivity against other mGluRs, and suitable lipophilicity as well as PPB, adequate metabolic stability and solution stability. Based on these results, compound 15 was selected for radiolabeling and in vivo evaluation as a potential mGluR4 PET radioligand.

Radiochemistry.

As shown in Scheme 2 the radiolabeling of [¹⁸F]15 was carried out in two steps: 1) generation of [¹⁸F]fluoromethyl-d₂ 4-methylbenzenesulfonate [¹⁸F]17 by [¹⁸F]fluorination of methylene-d₂ bis(4-methylbenzenesulfonate) 16; 2) the radiolabeling of the thiophenol precursor 18 with [¹⁸F]17.

Scheme 2.

Radiosynthesis of [¹⁸F]15.

Compound [¹⁸F]17 was prepared from the reaction of 16 in acetonitrile and t-butanol with non-carrier-added (nca) [¹⁸F]fluoride in the presence of tetrabutylammonium bicarbonate at 85 °C for 10 min. The radioactive product was a mixture of [¹⁸F]17 and the side product [¹⁸F]19, which was purified by semi-preparative HPLC, eluting with a solution of water and acetonitrile (50:50) at a flow rate of 4 mL/min to give [¹⁸F]17 in 20% yield. The obtained [¹⁸F]17 reacted with the thiophenol precursor 18 in DMSO at 150 ˚C for 10 min, resulting the desired product [¹⁸F]15 in over 95% conversion. The radiolabeled product was purified by semi-preparative HPLC, eluting with 0.1% formic acid solution of water and acetonitrile (40:60) at a flow rate of 4 mL/min to produce [¹⁸F]15. The final product was obtained after eluting 0.5 mL of ethanol followed by 4.5 mL of sterile saline through the C₁₈ Sep-Pak column resulting the final formulation of [¹⁸F]15 in saline containing 10% ethanol with 11.6% ± 2.9% radiochemical yield (RCY, n = 7, decay corrected) and over 99% radiochemical purity. The total synthesis of [¹⁸F]15 took 2.5–3.5 h with the molar activity 84.1 ± 11.8 GBq/μmol (n = 7).

Ex vivo Biodistribution Studies.

Ex vivo biodistribution studies were carried out in normal male Sprague Dawley rats to quantify accumulation of the tracer in different organs, as well as determine metabolic pathways. These studies showed reversible binding of [¹⁸F]15 in all investigated tissues with maximum uptake between 10 and 20 min except in the kidneys, where excretion of urine can affect tissue activity samples. Liver had the highest accumulation of 1.33±0.12 % ID/g at 20 min followed by kidneys 0.93±0.57 % ID/g at 30 min, lungs 0.58±0.17 % ID/g at 20 min, heart 0.50±0.08 % ID/g at 20 min and brain 0.41±0.18 % ID/g at 10 min. (Figure 4).

Figure 4.

Biodistribution of [¹⁸F]15.

PET imaging.

For in vivo characterization with PET imaging, [¹⁸F]15 was synthesized nine times and 30 studies were conducted in male Sprague−Dawley rats comprising 15 baseline studies and 9 blocking studies by using 13 as a blocking agent with doses of 1, 2 or 3 mg/kg and 6 self-blocking studies by using “cold” 15 as blocking agent with the same doses as 13. These studies demonstrate that [¹⁸F]15 crosses BBB and accumulates in the brain areas known to express mGluR4 (Figure 5).

Figure 5.

Eight consecutive coronal slices and midbrain axial and sagittal slices fused with the anatomical borderlines of different brain areas show the distribution of [¹⁸F]15 in the rat brain (Sprague Dawley). The images show the average accumulation of [¹⁸F]15 between 1 and 15 min after administration of the radioactivity. Note that the maximum color code refers to the maximum pixel value in each image. The borderlines of different brain areas and their names are color coded to match the corresponding TACS in Figure 6.

Time-activity curves (TACs) show fast reversible binding in the striatum, thalamus, cerebellum and cortex (Figure 6). The maximum average uptake at the time interval of 1–10 min after administering of the ligand was in the thalamus, 1.77±0.26 % ID/cc. The binding of [¹⁸F]15 in the rat brain was blocked in the whole brain (22–25%) by using 13 and even dose dependently in specific brain areas like striatum, 18, 20 and 28% using doses of 1, 2 or 3 mg/kg. Self-blocking with the same doses of 15 had similar blocking effect with the dose of 2 mg/kg but was significantly lower with 1 mg/kg and higher with 3 mg/kg compared to blocking with 13 (Figure 7). This PET tracer will be further developed in preclinical research with an aim for translational studies.

Figure 6.

Time-activity curves show fast accumulation and washout in all investigated brain areas. The highest accumulation was observed in the thalamus. The baseline data is averaged from five normal male rats (Sprague Dawley) with weight between 205–225 g. (left) The follow up period of 60 min shows that after 20 min the washout from striatum is low while the cerebellar activity stays nearly constant. (right) Accumulation in the different brain parts can be seen separately during the first 10 min. This image shows the highest accumulation in the thalamus achieved about 2 min after administration of the ligand.

Figure 7.

Blocking was calculated as a percent change from the baseline values. Blocking studies show that both 13 and 15 inhibit [¹⁸F]15 binding dose dependently. The blocking effect using 13 with the doses of 1, 2 or 3 mg/kg was from 8 to 32 percent in different brain areas while using 15 it was from 1 to 61 percent from the baseline values. However, the blocking effect with the dose of 2 mg/kg 15 was at the same level as with 13.

SUMMARY

In this study, we have designed, synthesized and characterized a new fluorine-18 labeled PET ligand for imaging mGluR4 in the brain. This radioligand displays CNS drug-like properties, including good mGluR4 affinity, potent mGluR4 PAM activity and selectivity against other mGluRs, suitable lipophilicity as well as PPB, adequate metabolic and solution stability. The radiolabeling of [¹⁸F]15 was carried out in two steps. The PET imaging studies in male Sprague−Dawley rats demonstrate that [¹⁸F]15 crosses BBB and accumulates the brain areas known to express mGluR4. Pretreatment with the unlabeled compounds 13 and 15 show the significant dose-dependent blocking effects. The results suggest that [¹⁸F]15 is a promising candidate for PET imaging of mGluR4 in the brain and for translational studies in PD and/or other disorders.

EXPERIMENTAL SECTION

Animal procedures.

The animal studies were approved and done under strict supervision of Subcommittee on Research Animals of the Massachusetts General Hospital and Harvard Medical School and performed in accordance with the Guide of NIH for the Care and Use of Laboratory Animals.

Materials and Methods.

All reagents and starting materials were obtained from the commercial sources including Sigma-Aldrich (St. Louis, MO), Thermo Fisher Scientific, Oakwood Products, Inc., Matrix Scientific and used as received. The reactions were monitored by TLC using a UV lamp monitored at 254 nm. If necessary, the reactions were also checked by LC−MS using the Agilent 1200 series HPLC system coupled with a multiwavelength UV detector and a model 6310 ion trap mass spectrometer (Santa Clara, CA) equipped with a Luna C₁₈ column (Phenomenex, 100 × 2 mm, 5 μ, 100 Å). The RP-HPLC was carried out by using a 7-min gradient method (LC-Method 1): eluent A: 0.1% formic acid/ H₂O; eluent B: 0.1% formic acid/CH₃CN; gradient: 5% B to 95% B from 0 to 3 min, 95% B from 3 to 4.5 min, 95% to 5% B from 4.5 to 5 min, 5% B from 5 to 7 min; flow rate at 0.7 mL/min. The silica gel used in flash column chromatography was from Aldrich (Cat. 60737, pore size 60 Å, 230–400 mesh). The products were identified by LC−MS as well as ¹H NMR, ¹³C NMR and ¹⁹F NMR using a Varian 500 MHz spectrometer. All NMR samples were dissolved in chloroform-d (CDCl₃) containing tetramethylsilane as a reference standard. Chemical shifts were expressed as ppm and calculated downfield or upfield from the NMR signal of reference standard. J was expressed as Hz, and its splitting patterns were reported as s, d, t, q, or m. HRMS was acquired using a DART-SVP ion source (IonSense, Saugus, MA) attached to a JEOL AccuTOF 4G LC-plus mass spectrometer (JEOLUSA,Peabody, MA) in positive-ion mode from Prof. Peter Caravan’s Laboratory. Unless otherwise specified, the purity of all new compounds was over 95% determined by HPLC.

Chemistry.

Fluoromethyl-d₂ 4-methylbenzenesulfonate (17).

To the solution of methylene-d₂ bis(tosylate) (16, 9.01g, 25.2 mmol) in tert-amyl alchol (150.0 mL) was added CsF (3.83 g, 25.2 mmol). The mixture was stirred at 80 ˚C for 2.0 h. A large amount of white solid precipitated out. After filtration, the filtrate was condensed under vacuum. The resulting residue was chromatographed on silica gel by eluting with ethyl acetate and hexane (1:30 to 1:15) to afford 17 as a colorless oil (2.49 g, 48%) and 3.02 g of starting material 16 was recovered. ¹H NMR (500 MHz, CDCl₃) δ 7.83 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 2.46 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 145.6, 133.8, 129.9 (2C), 127.9 (2C), 97.6 (dp, J = 26.5 Hz, J = 229.3 Hz), 21.7. ¹⁹F NMR (470 MHz, CDCl₃) δ −154.5 (p, J = 9.4 Hz). LC-MS (method 1): t R = 3.67 min, (ESI) m/z calcd. for C₈H₂₂D₂FNO₃S 224.0; found 224.0 [M + NH₄]⁺.

N-(4-Chloro-3-((fluoromethyl-d₂)thio)phenyl)picolinamide (15).

To the solution of N-(4-chloro-3-mercaptophenyl)picolinamide hydrogen chloride salt (18, 150.0 mg, 0.5 mmol) and 17 (154.5 mg, 0.75 mmol) in acetonitrile (7.0 mL) were added K₂CO₃ (690 mg, 5.0 mmol) and KI (83.0 mg, 0.5 mmol). The resulting mixture was heated to reflux for 2 h. The solvent was removed under vacuum and the residue was dissolved in DCM (20 mL) and water (20 mL). The water phase was further washed with DCM twice (20 mL). The organic layer was combined, dried over anhydrous Na₂SO₄ and concentrated under vacuum. The residue was chromatographed on silica gel by eluting with EtOAc and hexane (1:3) to afford the title product as a white powder (137.2 mg, 92%). ¹H NMR (500 MHz, CDCl₃) δ 10.08 (s, 1H), 8.62 (ddd, J = 4.7, 1.6, 0.9 Hz, 1H), 8.29 (dt, J = 7.8, 1.0 Hz, 1H), 7.92 (td, J = 7.7, 1.7 Hz, 1H), 7.86 (d, J = 1.7 Hz, 1H), 7.51 (ddd, J = 7.6, 4.7, 1.2 Hz, 1H), 7.44 (dd, J = 8.7, 2.3 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H). ¹⁹F NMR (470 MHz, CDCl₃) δ - 150.9 (dt, J = 16.2, 8.2 Hz). ¹³C NMR (126 MHz, CDCl₃) δ 162.1, 149.3, 148.0, 137.8, 137.3, 134.3, 130.2, 128.62 (d, J = 2.4 Hz), 126.7, 122.5, 120.6, 119.5, 85.8 (dp, J = 24.0, 217.2 Hz). LC-MS (method A1): t R = 4.05 min, (ESI) m/z calcd. for C₁₃H₉D₂ClFN₂OS 299.0; found 298.9 [M + H]⁺. HRMS (m/z) calcd. for C₁₃H₉D₂ClFN₂OS 299.0390; found 299.0388 [M + H]⁺.

Radiochemistry Procedure.

[¹⁸F]Fluoride was generated by a GE PETtrace 16.5 MeV cyclotron (GE Healthcare, Waukesha, WI, USA) using ¹⁸O-enriched water (Isoflex Isotope, San Francisco, CA) with proton bombardment. Fluorine-18 labeling of [¹⁸F]15 was accomplished in two steps. First, [¹⁸F]fluoride in ¹⁸O-enriched water was passed through a QMA Sep-Pak Cartridge (Waters, Milford, MA) to trap [¹⁸F]fluoride ions, which was washed off by 0.5 mL of the aqueous solution of tetrabutylammonium hydrogen carbonate (75 mM, from ABX advanced biochemical compounds). Acetonitrile (1.0 mL) was added to the solution and the solvents were evaporated at 115 °C in a stream of nitrogen. In order to remove water completely, 1.0 mL of acetonitrile was added and evaporated in a stream of nitrogen three more times. To the residue containing [¹⁸F]fluoride was added 16 (12.0 mg) in 0.7 mL of acetonitrile and t-BuOH (4:1). The resulting solution was heated to 85 °C for 10 min and then cooled to room temperature followed by addition of 1.5 mL of water. The mixture was then purified by a semi-preparative HPLC (Waters 4000 system equipped with an Xbridge BEH C₁₈ OBD column: 130 Å, 5 μ, 10 × 250 mm) by eluting with a solution of water and acetonitrile (50:50) at a flow rate of 4 mL/min to give the fractions containing [¹⁸F]17. The combined fraction was diluted with water to 40 mL and loaded on a C₁₈ Sep-Pak column. The column was further dried through a stream of nitrogen for 20–30 min. [¹⁸F]17 was washed off the C₁₈ Sep-Pak column by 0.7 mL of dry DMSO to a reaction vessel containing 2.0 mg of 18 and cesium carbonate (3.0 – 5.0 mg). The resulting mixture was heated to 150 ˚C for 10 min and then cooled to rt, followed by addition of the HPLC eluents (2.5 mL, 0.1% formic acid solution of water and acetonitrile 40:60). The mixture was then purified using the semi-preparative HPLC (Waters 4000 system equipped with an Xbridge BEH C₁₈ OBD column: 130 Å, 5 μ, 250 × 10 mm) by eluting with a 0.1% formic acid solution of water and acetonitrile (40:60) at a flow rate of 4 mL/min. The fraction containing [¹⁸F]15 was diluted with water to 40 mL and loaded on a C₁₈ Sep-Pak column to give the final formulation of [¹⁸F]15 in saline containing 10% ethanol with 11.6% ± 2.9% radiochemical yield (RCY, n = 7, decay corrected). The purity of [¹⁸F]15 was over 99% that was analyzed by an analytical HPLC (Waters 2487 series equipped with a UV detector and a BIOSCAN radioactivity detector and an ACE 5 C₁₈-AR column: 250 × 10 mm, 5 μ). Identity of [¹⁸F]15 was confirmed by co-injection of the cold compound 15 in HPLC analysis. The total synthesis of [¹⁸F]15 took 2.5–3.5 h with the molar activity 84.1 ± 11.8 GBq/μmol (n =7).

Determination of Log D.

An aliquot (10 μL, 74 kBq) of [¹⁸F]15 was added to a test tube containing 2.5 mL of octanol and 2.5 mL of phosphate buffer solution (pH 7.4). The test tube was mixed by vortex for 2 min and then centrifuged for 2 min to fully separate the aqueous and organic phase. The samples taken from the octanol layer (0.1 mL) and the aqueous layer (1.0 mL) were saved for radioactivity measurement. An additional aliquot of the octanol layer (2.0 mL) was carefully transferred to a new test tube containing 0.5 mL of octanol and 2.5 mL of phosphate buffer solution (pH 7.4). The previous procedure (vortex mixing, centrifugation, sampling, and transfer to the next test tube) was repeated until six sets of aliquot samples had been prepared. The radioactivity of each sample was measured using PerkinElmer Wizard2 2480 gamma-counter. The log D of each set of samples was calculated as the following: Log D_7.4 = Log (radioactivity of octanol layer × 10 / radioactivity of PBS layer)

Plasma Protein Binding Assay.

An aliquot of radiolabeled compound [¹⁸F]15 in saline (10 μL, 74 kBq) was added to a sample of human plasma (0.5 mL). The mixture was gently mixed by repeated inversion and incubated for 10 min at room temperature. Following incubation, a small sample (10 μL) was removed to determine the total radioactivity in the plasma sample (A_T; A_T = A_bound + A_unbound). The upper part of the Centrifree tube was discarded, and an aliquot (10 μL) from the bottom part of the tube was removed to determine the amount of unbound radioactivity (A_unbound) that passed through the membrane (molecular weight cutoff 30 kD). The radioactivity of each sample was measured using PerkinElmer Wizard2 2480 gamma-counter. Plasma protein binding was derived by the following equation: %unbound= A_unbound x 100/A_T.

Ex vivo Studies of Biodistribution.

For biodistribution studies the normal male Sprague Dawley rats were anesthetized with isoflurane/nitrous oxide (1–1.5% isoflurane) to install catheter into the tail vain for administration of radioactivity ([¹⁸F]15, 22±3 MBq, iv, mass 0.072±0.09 μg). Total of 15 rats in deep anesthesia (isoflurane 4% and cervical dislocation) were sacrificed in a group of three at five different time points (5, 10, 20, 30, and 60 min) after injection of [¹⁸F]15. Major organs including lung, heart, liver, spleen, kidney, muscle, midbrain, cortex, cerebellum and blood were harvested to determine radioactivity. The tissue samples were weighted, and the radioactivity was measured with the standard samples of [¹⁸F]15 using Wizard2 2480, Perkin Elmer, CA. Radioactivity of the tissue samples was determined as percent of the injected activity per gram of the tissue.

PET Imaging of [¹⁸F]15 in Rats.

Altogether 24 normal male Sprague Dawley rats were used for the imaging studies comprising 15 baseline studies and 15 blocking studies. Rats were anaesthetized with isoflurane/nitrous oxide (1.0–1.5% isoflurane, with oxygen flow of 1–1.5 L/min), and catheter was installed into tail vein for the administration of [¹⁸F]15. Dynamic volumetric PET data was acquired with a PET-CT scanner for 60 min (Triumph-II, Tri-Foil Imaging, Northridge, CA). The vital signals such as heart rate and respiration rate were monitored during scanning period. PET data acquisition was started immediately after administration of radioactivity with the dose range of 18–34 MBq (mass 0.059±0.111 μg) depending on the size of the rat followed by CT imaging to obtain anatomical information and data for attenuation correction of PET data. PET data was processed by using maximum-likelihood expectation-maximization (MLEM) algorithm with 30 iterations to dynamic volumetric images, and corrected for uniformity, scatter, and attenuation. The CT data was processed by the modified Feldkamp algorithm using matrix volumes of 512×512×512 and pixel size of 170 μm. Co-registration of CT and PET images and analysis of PET images were carried out using PMOD3.2 software (PMOD Technology, Zurich, Switzerland).

For blocking studies, the same scanning protocols were used as for the baseline studies. For mGluR4 blocking experiments, 1, 2 or 3 mg/kg of 13 or 15 dissolved in a saline solution with 10% DMSO, 5% Tween-20 and 85% PBS was injected 1 min before iv administration of [¹⁸F]15.

ABBREVIATIONS USED

Cl_int

the intrinsic clearance

G_i

adenylate cyclase inhibitory G-protein

L-SOP

L-Serine-O-phosphate

TAC

time-activity curve

%ID/g

percentage of injected dose per gram of wet tissue