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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Bioorg Med Chem. Author manuscript; available in PMC 2010 July 1.
Published in final edited form as:
PMCID: PMC2719852
NIHMSID: NIHMS125015

Synthesis and Biological Evaluation of Novel Carbon-11 Labeled Pyridyl Ethers: Candidate Ligands for In Vivo Imaging of α4β2 Nicotinic Acetylcholine Receptors (α4β2-nAChRs) in the brain with Positron Emission Tomography

Abstract

The most abundant subtype of cerebral nicotinic acetylcholine receptors (nAChR), α4β2, plays a critical role in various brain functions and pathological states. Imaging agents suitable for visualization and quantification of α4β2 nAChRs by positron emission tomography (PET) would present unique opportunities to define the function and pharmacology of the nAChRs in the living human brain. In this study, we report the synthesis, nAChR binding affinity, and pharmacological properties of several novel 3-pyridyl ether compounds. Most of these derivatives displayed a high affinity to the nAChR and a high subtype selectivity for α4β2-nAChR. Three of these novel nAChR ligands were radiolabeled with the positron-emitting isotope 11C and evaluated in animal studies as potential PET radiotracers for imaging of cerebral nAChRs with improved brain kinetics.

Introduction

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that are formed by combinations of various α and β subunits. The subunit combinations define different receptor subtypes with distinct biophysical, physiological and pharmaceutical properties as well as different locations throughout the nervous systems.14 Many studies have shown that nAChRs could play important roles in brain functions and are also involved in various brain pathologies such as Alzheimer’s disease, Parkinson’s disease, schizophrenia, anxiety, and nicotine addiction.58

The in vivo imaging of nAChRs with positron emission tomography (PET) promises to improve our understanding of various CNS disorders. During the last decade there has been considerable interest in the development of suitable PET radioligands for imaging nAChRs, in particular, the most abundant α4β2-nAChR subtype. The current development of nAChR PET radioligands has mostly focused on the analogs of (S)-3-(1-methylpyrrolidin-2-yl)pyridine (nicotine, 1) and 2-(6-Chloro-3-pyridinyl)-7-azabicyclo[2.2.1]heptane (epibatidine, 2) and, more recently, on a series of compounds that were developed by Abbott Laboratories, including (S)-3-(azetidin-2-ylmethoxy)pyridine (A-85380, 3) and (S)-3-((1-methylpyrrolidin-2-yl)methoxy)pyridine (A-84543, 4).9,10

The interested reader will be able to find more details on the development of these nAChR PET radioligands in several recent reviews.1118

Presently, only two radiotracers, 2-[18 F]fluoro-A-85380 (5), and 6-[18F]fluoro-A-85380 (6) (Fig 2), are available for studying α4β2 nAChRs in human brain using PET.1925 The slow brain kinetics of these radiotracers hamper quantification of the nAChR since it takes 4–7 hours of PET scanning for the tracer radioactivity to reach a spatial-temporal steady state.26 The most recent review about the nAChR PET radioligands27 describes several analogs of 5 with improved brain kinetics, but the combined imaging properties of these radiolabeled A-85380 analogs in animals were not sufficiently promising for human studies.

Figure 2
Radiolabeled 3-pyridyl ether compounds as PET radiotracers for imaging nAChR.

Previously, we developed [11C]CH3-PVC (7),2830 (Fig 2) a radiolabeled analog of A-84543 (4) with faster kinetics in the Rhesus monkey brain than those of 5 and 6. The accelerated brain kinetics of 7 are thought to be associated with its higher lipophilicity and, consequently, better blood-brain barrier (BBB) permeability. However, the binding potential of 7 in Rhesus monkey brain was not sufficiently high for use in human subjects. We suggested that compounds that are structurally similar to 7, and with similar lipophilicity but with a higher binding affinity, would display better imaging properties. We now report the synthesis, in vitro characterization, radiosynthesis, in vivo regional brain distribution in rodents and PET imaging in nonhuman primates of several novel analogs of 7 with improved imaging properties.

Results and Discussion

Chemistry

Scheme 1 and Scheme 2 outline the synthesis of 16 and 25. All novel compounds were characterized by 1H NMR, MS, and elemental analysis prior to biological evaluation. The key intermediates 14, 23a and 23b were obtained by Mitsunobu reaction. The final N-methyl derivative 16 was prepared by deprotection of 14 with TFA followed by N-methylation with 37% HCHO/NaBH3CN. Compounds 25a and 25b were obtained by heating of 23a and 23b with 37% HCHO/HCOOH.

Scheme 1
Reagents: (a) NaOH, 4-bromomethyl-2-fluoropyridine, MeOH; (b) HNO3/H2SO4; (c) POCl3, quinoline; (d) Fe, H2O, AcOH; (e) HBF4, NaNO2/H2O; then Ac2O; (f) 1 M KOH, MeOH; (g) DEAD, PPh3, THF. (h) CF3COOH/CH2Cl2. (i) aqueous HCHO/NaBH3CN.
Scheme 2
Reagents: (a) NaOH, 2-chloro-5-(chloromethyl)pyridine, MeOH; (b) HNO3/H2SO4; (c) POCl3, quinoline; (d) Fe, H2O, AcOH; (e) HBF4, NaNO2/H2O, Ac2O; (f) 1 M KOH, MeOH; (g) DEAD, PPh3, THF; (h) CF3COOH/CH2Cl2. (i) aqueous HCHO, HCOOH.

Lipophilicity

The partition coefficient between octanol and phosphate buffer at pH 7.4 (logD7.4) of compounds 16, 25 and the previously described 26 ((S)-2-chloro-2'-fluoro-5-((1-methylpyrrolidin-2-yl)methoxy)-3,4'- bipyridine)31 were determined by a conventional shake-flask method (Table 1). These lipophilicity values correspond to an optimal range (1.5–3) for the most known PET radiotracers.32

Table 1
Comparison of lipophilicity and binding affinities of (−)-nicotine, (±)epibatidine, 2-F-A-85380, 7 and new 3-pyridyl ether compounds 15, 16, 24a, 25a, 25b and 26

Structure – binding affinity relationship

Our previous studies demonstrated that [11C]Me-PVC (7)30 (Figure 2), a radiolabeled analog of A-84543 (4), exhibited substantially more rapid brain kinetics than those of 2-[18F]fluoro-A-85380, the only available radioligand for imaging of nAChR in the human brain, which required 6–8 h of PET scanning due to its slow brain kinetics. Despite the optimized brain kinetics [11C]Me-PVC (7) was not a good candidate for human studies because of its relatively low binding potential31. We hypothesized that an analog of 7 with higher binding affinity to α4β2 nAChRs might exhibit a higher binding potential while its brain kinetics remains sufficiently rapid. One of the important molecular properties of Me-PVC (7) is high conformational flexibility.29 We hypothesized that replacement of the vinyl linker between the two pyridine rings with the more flexible-CH2-O- linker could potentially enhance the binding affinity. This hypothesis was a driving force for the synthesis of the diether series 15, 16 (Scheme 1) and 24 and 25 (Scheme 2), the analogs of 7 with improved conformational flexibility.

Binding affinities of all new nAChR ligands were determined in competition binding experiments using stably expressed transfected cell lines (α2β2-, α2β4-, α3β2-, α3β4-, α4β2- and α4β4-nAChR subtypes)33 and the radioligand [3H]epibatidine (Table 1). Subtype selectivity is a critical issue for the effectiveness and safety of nAChR ligands. α4β2 subtype and α3β4 subtype represent the main ganglionic nAChR population. The α3β4 subtype is found in many sympathetic ganglia, while the α4β2 subtype is the predominant nAChR in the forebrain; therefore, the affinity ratios of ligands at these subtypes can help to predict the likelihood of autonomic nervous system side effects of ligands aimed at the predominant receptor subtype in the forebrain. Thus we compared the affinities of these ligands at the α3β4 subtype with their affinities at the α4β2 subtype. The binding affinities of 16 and 25a are very high and comparable with the binding affinity of epibatidine (Table 1). The previously synthesized ligand 26 (Fig 7)31 that does not have a linker between two pyridine rings showed similar binding affinity but its α4β2/α3β4 selectivity was greater than the selectivity of 16 and 25a. Comparison of the α4β2-nAChR binding affinity of more rigid 26 with more flexible 16 and 25a suggests that molecular flexibility within this series is not crucial parameter. The R-enantiomer 25b showed substantially lower binding affinity at the α2β2 and α4β2 subtypes than the S-enantiomer 25a.

Figure 7
Time-radioactivity curves in thalamus and cerebellum of microPET study with [11C]26 in a male Wistar rat (weight 300 g). Baseline study with [11C]26 (0.6 mCi, specific radioactivity = 8200 mCi/µmol)

Radiochemistry

Compounds 16, 25a and 26 were selected for further radiolabeling and animal studies because they exhibited the highest binding affinity at α4β2-nAChR within the series. Although compound 24a displayed even higher binding affinity at α4β2-nAChRs than compounds 16, 25a and 26, it was not further studied as radioligand owe to the lack of methyl group making 11C-labelling difficult.

The straightforward preparation of [11C]16 and [11C]25a was accomplished via N-methylation of the nor-methyl precursors 15 and 24a with the [11C]CH3I in N,N-dimethylformamide as shown in Scheme 3. The final products [11C]16 and [11C]25a were prepared in high radiochemical yield ([11C]16 − 18.4±8.8% (n=4), [11C]25a - 29±7.2% (n=3)). The radiochemical punty was greater than 98% and specific radioactivity were: 8022±5592 (n=4) mCi/µmol for [11C]16 and 3646±1233 mCi/µmol (n=3) for [11C]25a (nondecay corrected from the end of 11CH3I synthesis).

Scheme 3
Radiosynthesis of [11C]16 and [11C]25a

In vivo studies

The novel radioligands [11C]16, [11C]25a and the previously synthesized [11C]26 (Fig. 7)31 were studied in rodents and baboon as potential probes for PET imaging of central nAChR.

Rodent Studies

In the mouse study with [11C]25a, brain radioactivity displayed regional distribution that was consistent with the known distribution of α4β2-nAChRs.35 Brain uptake was highest in the thalamus and lowest in the cerebellum (Fig. 3). The maximal thalamus/cerebellum ratio peaked at 45 min post-injection to a value of 3.8.

Figure 3
Regional brain time-uptake curves of [11C]25a in mice, mean ± SD (0.07 mCi per animal, S.A. = 4900 mCi/µmol).

The promising preliminary brain distribution data in mice suggested a necessity for more comprehensive microPET imaging studies. MicroPET experiments with [11C]25a demonstrated excellent imaging properties of the radioligand in the rat brain (Fig 4, Fig 5) with very high uptake of radioactivity in the nAChR-rich thalamus and cortical regions and low accumulation of radioactivity in the nAChR-poor cerebellum. Compounds [11C]16 and [11C]26 demonstrated a similar distribution pattern in the rat brain (Fig. 6, Fig. 7).

Figure 4
Small animal PET study with [11C]25a. Summed (20–90 min) image of [11C]25a (0.4 mCi, specific radioactivity = 4500 mCi/µmol) in a male Wistar rat (weight 153 g).
Figure 5
Time-radioactivity curves in thalamus and cerebellum of PET studies with [11C]25a in a male Wistar rat (weight 150 g). Left panel: Time-uptake curves of [11C]25a in the rat brain regions. Right panel: blockade study of [11C]25a (0.4 mCi, specific radioactivity ...
Figure 6
Time-radioactivity curves in thalamus and cerebellum of microPET studies with [11C]16 in a male Wistar rat (weight 360 g). Left panel: Baseline study with [11C]16 (0.4 mCi, specific radioactivity = 5500 mCi/µmol) Right panel: blockade study of ...

Blocking studies with [11C]25a and [11C]16 were performed by injection of cytisine, a selective α4β2-nAChR agonist. The study showed a dramatic reduction of the regional radioactivity uptake in the rat thalamus, whereas little displacement of radioactivity from the rat cerebellum was observed (Fig. 5, Fig. 6) suggesting that in vivo binding of [11C]25a and [11C]16 is specifically mediated by nAChR.

Baboon PET studies

Based on the promising results from the rodent experiments with [11C]16, [11C]25a and [11C]26, PET studies in baboons were performed. All three compounds exhibited rapid accumulation of radioactivity in the baboon brain with rapid washout from the cerebellum and slow washout from the thalamus during first 90 min after injection (Fig 8Fig 10). The thalamus-to-cerebellum radioactivity ratios increased and approached a plateau in 90–120 min post injection (Fig. 8Fig. 10) suggesting that [11C]16, [11C]25a and [11C]26 manifest faster brain kinetics than has been found for 5. PET modeling demonstrated comparable thalamic binding potential values (BPTh = 0.7–0.8) of all three radioligands ([11C]16, [11C]25a and [11C]26) and lower than that of 5 (Fig. 11).

Figure 8
Time profiles of radioactivity concentrations in the baboon thalamus and cerebellum (left axis), and thalamus-to-cerebellum ratios (right axis) of [11C]25a
Figure 10
Thalamus/Cerebellum ratio of accumulated radioactivity (%SUV = %Standardized Uptake Value) versus time in the baseline PET experiments with [11C](−)-26 (JHU85270) in baboon.
Figure 11
Histogram of binding potential estimates in the thalamus of 5 (Rhesus monkey)20 and [11C]16, [11C]25a and [11C]26 (baboon)

The blood metabolite analysis of the radioligands [11C]16, [11C]25a and [11C]26 revealed substantial amounts of unidentified radiolabeled metabolites with greater retention time on the reverse-phase HPLC chromatogram (Fig. 12) that are likely to be lipophilic and, perhaps, penetrate BBB. Non-specific binding of the lipophilic radiometabolites could be a reason of the relatively high non-specific binding and modest BP values of [11C]16, [11C]25a and [11C]26.

Figure 12
HPLC analysis of baboon plasma at 1 h post administration of [11C]16. Radioligands [11C]25a and [11C]26 exibit similar metabolic HPLC profile.

Conclusion

Two new α4β2-nAChR ligands, [11C]16 and [11C]25a have been synthesizedand radiolabeled. These two radioligands and our recently developed radioligand [11C]2631 , have been evaluated in rats and nonhuman primates in this study. Competition binding assay with cell lines stably expressing various nAChR subtypes demonstrated that they have high affinities for β2-containing nAChR subtypes, which are predominate in the CNS. Regional brain distribution studies in rodents showed high specific accumulation of [11C]16, [11C]25a and [11C]26 in the nAChR-rich regions. PET imaging studies in baboons demonstrated that [11C]16, [11C]25a and [11C]26 label central nAChRs and the baboon brain kinetics of all three radioligands are sufficiently rapid for quantification of 120 min images. Despite the high binding affinities of [11C]16, [11C]25a and [11C]26 their thalamic binding potential values in baboons are lower than that of compound 5, perhaps, due to formation of radiolabeled lipophilic metabolites.

Experimental Section

Chemicals were obtained from Aldrich (Milwaukee, WI) and used as received. Column chromatography was carried out using E. Merck silica gel 60F (230–400 mesh). Analytical thin-layer chromatography (TLC) was performed on aluminum sheets coated with silica gel 60 F254 (0.25 mm thickness, E. Merck, Darmstadt, Germany). Melting points were determined with a Fisher-Johns apparatus and are not corrected. 1H NMR spectra were recorded with a Varian-400 NMR spectrometer at nominal resonance frequencies of 400 MHz in CDCl3 or DMSO-d6 (referenced to internal Me4Si at δH 0 ppm). The chemical shifts (δ) were expressed in parts per million (ppm). First order J values were given in Hertz. High resolution mass spectrometry was performed at the University of Notre Dame Mass Spectrometry facility. Elemental analyses were determined by Galbraith Laboratories, Inc. (Knoxville, TN). The HPLC system consisted of two Waters model 590EF pumps, two Rheodyne model 7126 injectors, an in-line Waters model 441 UV detector (254 nm) and a single sodium iodide crystal flow radioactivity detector. All HPLC chromatograms were recorded with Varian Galaxie software (version 1.8). The analytical and semi-preparative chromatography were performed using Phenomenex Luna C-18 10 µm columns (analytical 4.6 × 250 mm and semi-preparative 10 × 250 mm). A dose calibrator (Capintec 15R) was used for all radioactivity measurements. [11C]Methyl iodide was prepared using a General Electric Methyl Iodide Microlab from [11C]carbon dioxide produced by a General Electric PETtrace biomedical cyclotron.

3-((2-fluoropyridin-4-yl)methoxy)pyridin-2-ol (8)

2,3-dihydroxypyridine (0.556 g, 5 mmol) was added portion-wise to a solution of NaOH (0.22 g, 5 mmol) in methanol (5 mL) followed by addition of a solution of 4-(bromomethyl)-2-fluoropyridine (0.95 g, 5 mmol) in MeOH (2 mL) dropwise during 10 min. The mixture was stirred at room temperature for 36 h. After evaporation of methanol, the residue was diluted with water (20 mL) and extracted with CHCl3 (6 × 30 mL). The combined organic extracts were dried over MgSO4, filtered and concentrated in vacuo. Recrystallization in ethyl acetate produced a white solid (0.89 g, 81%); mp 216–217 °C; 1H NMR (400 MHz, DMSO-d6/TMS) δ 11.72 (s, 1H), 8.26 (d, J=5.2 Hz, 1H), 7.39 (m, 1H), 7.20 (s, 1H), 7.00 (dd, J=1.6 Hz, 6.8 Hz, 1H), 6.92 (dd, J=1.6 Hz, 8.0 Hz, 1H), 6.10 (t, J=6.4 Hz, 13.6 Hz, 1H), 5.17 (s, 2H); HRMS calculated for C11H10FN2O2: [M+H] m/z=221.0726, found: 221.0721.

3-((2-fluoropyridin-4-yl)methoxy)-5-nitropyridin-2-ol (9)

The compound 8 (440 mg, 2 mmol) was carefully dissolved in chilled H2SO4 (96%, 1.5 mL) and a mixture of HNO3 (70%) /H2SO4 (96%) (0.2 mL of each) was added dropwise at 0–5 °C. The reaction mixture was stirred at 0–5 °C for 20 min and poured into 20 g crashed ice. The yellow precipitate was filtered off, washed with cold water (5 × 5 mL) and dried (382 mg, 72%); mp 229–230 °C; 1H NMR (400 MHz, DMSO-d6/TMS) δ 12.82 (s, 1H), 8.40 (d, J=2.8 Hz, 1H), 8.29 (d, J=5.2 Hz, 1H), 7.54 (d, J=2.8 Hz, 1H), 7.42 (d, J=5.2 Hz, 1H), 7.22 (s, 1H), 5.31 (s, 2H); HRMS calculated for C11H9FN3O4: [M+H] m/z=266.0577, found: 266.0579.

2-Chloro-3-(2-fluoro-4-(pyridinyl)methoxy)-5-nitropyridine (10)

Phosphoryl chloride (0.45 mL, 4.86 mmol) was added dropwise to a magnetically stirred mixture of 9 (318 mg, 1.2 mmol) and quinoline (0.15 mL, 1.2 mmol). The mixture was blanketed with argon and heated at 120 °C for 2 h. Upon the complete consumption of the precursor, as indicated by TLC, the mixture was cooled in the ice bath and diluted with 20 mL of H2O. The resulting brown precipitate was filtered and recrystallized from ethanol to yield sand-colored crystals (289 mg, 85%); mp 121–122 °C; 1H NMR (400 MHz, CDCl3/TMS) δ 8.93 (d, J=2.4 Hz, 1H), 8.32 (d, J=4.8 Hz, 1H), 8.01 (d, J=2.4 Hz, 1H), 7.32 (d, J=4.0 Hz, 1H), 7.12 (d, J=0.8 Hz, 1H), 5.33 (s, 2H); HRMS calculated for C11H8FN3O3: [M+H] m/z=284.0238, found: 284.0240.

6-Chloro-5-((2-fluoropyridin-4-yl)methoxy)pyridin-3-amine (11)

The precursor 10 (490 mg, 1.72 mmol) was added to a mixture of water (5.1 mL) and glacial acetic acid (6.8 mL). Iron powder (407 mg, 7.3 mmol) was then added to the reaction flask, and the mixture was heated to 60 °C for 1 h. TLC analysis of the reaction mixture indicated a complete consumption of the precursor. Water (15 mL) was added to the reaction flask, the mixture was neutralized with potassium hydroxide pellets to pH = 8 and extracted with ethyl acetate (3 × 40 mL). The ethyl acetate layer was evaporated to yield yellow solid (358 mg, 82%); mp 145–147 °C; 1H NMR (400 MHz, CDCl3/TMS) δ 8.26 (d, J=5.2 Hz, 1H), 7.56 (d, J=2.4 Hz, 1H), 7.29 (m, 1H), 7.08 (s, 1H), 6.56 (d, J=2.0 Hz, 1H), 5.15 (s, 2H). 3.79 (br s, 2H); HRMS calculated for C11H10ClFN3O: [M+H] m/z=254.0496, found: 254.0498.

6-Chloro-5-((2-fluoropyridin-4-yl)methoxy)pyridin-3-yl acetate (12)

Compound 11 (305 mg, 1.2 mmol) was added to HBF4 (2.4 mL, 48%) at 0 °C. A solution of sodium nitrite (126 mg, 1.8 mmol) in water (0.9 mL) was added dropwise over a period of 20 min and the mixture was stirred for 1 h at 0 °C. The resulting solid was filtered, washed with cold diethyl ether (3 × 5 mL), and air-dried for 15 min. The solid was then dissolved in acetic anhydride (5 mL) and heated for 1 h to 85 °C. The solvent was evaporated, and the resulting residue was dissolved in diethyl ether (35 mL). The diethyl ether solution was washed with H2O (2 × 20 mL), dried over MgSO4, and evaporated under reduced pressure to give a residue which was purified by silica gel chromatography (Hexanes/ Ethyl acetate 1:1). The product was obtained as white solid (230 mg, 65%); mp 102–103 °C; 1H NMR (400 MHz, CDCl3/TMS) δ 8.28 (d, J=4.8 Hz, 1H), 7.93 (d, J=2.4 Hz, 1H), 7.28 (m, 1H), 7.12 (d, J=2.4 Hz, 1H), 7.08 (s, 1H), 5.18 (s, 2H), 2.34 (s, 3H); HRMS calculated for C13H11ClFN2O3: [M+H] m/z=297.0442, found: 297.0431.

2-Chloro-3-(2-fluoro-4-(pyridinyl)methoxy)-5-hydoxypyridine (13)

6-Chloro-5-((2-fluoropyridin-4-yl)methoxy)pyridin-3-yl acetate 12 (0.59 g, 1.99 mmol) was added to a mixture of KOH (4 mL, 1 M) and MeOH (4 mL) at 5 °C. After it was stirred for 1 h in an ice bath, TLC showed the starting material completely consumed. The solution was made acidic (pH 5), with glacial acetic acid and the resulting solid was filtered and dried. Compound 13 was obtained as a white powder (0.43 g, 85%); mp 187–188 °C; 1H NMR (400 MHz, DMSO-d6/TMS) δ 10.34 (s, 1H), 8.29 (d, J=5.2 Hz, 1H), 8.29 (d, J=2.8 Hz, 1H), 7.58 (d, J=2.4 Hz, 1H), 7.41 (d, J=5.2 Hz, 1H), 7.21 (s, 1H), 7.02 (d, J=2.4 Hz, 1H), 5.35 (s, 2H); HRMS calculated for C11H9Cl2N2O2: [M+H] m/z=255.0336, found: 255.0349.

2-Chloro-5-((1-(tert-butoxycarbonyl)-2-(S)-pyrrolidinyl)methoxy)-3-(2-fluoro-4-(pyridinyl)methoxy)pyridine (14)

Diethyl azodicarboxylate (0.365 mL, 2.03 mmol) and triphenylphosphine (535 mg, 2.03 mmol) were mixed in THF (10 mL) at 0 °C, under argon for 30 min. N-Boc-L-prolinol (409 mg, 2.03 mmol) and compound 13 (398 mg, 1.56 mmol) were then added to the reaction flask, and the mixture was stirred at room temperature for 36 h. The solvent was evaporated under reduced pressure at 55 °C, and the crude oil was purified via flash chromatography (Hexane/Ethyl acetate 2:1 to 1:1). Compound 14 was obtained as colorless oil (478 mg, 70%); 1H NMR (400 MHz, CDCl3/TMS) δ 8.26 (m, 1H), 7.75 (d, J=2.4 Hz, 1H), 7.42 (s, 1H), 7.29 (m, 1H), 7.08 (m, 1H), 5.29 (s, 1H), 5.17 (s, 1H), 4.25 (m, 1H), 4.06–4.12 (m, 1H), 3.90 (m, 1H), 3.29–3.49 (m, 2H), 1.91–2.06 (m, 4H), 1.48 (s, 9H); HRMS calculated for C21H26ClFN3O4: [M+H] m/z=438.1596, found: 438.1587.

2-Chloro-3-(2-fluoro-4-(pyridinyl)methoxy)-5-(2-(S)-pyrrolidinyl)methoxy)-pyridine (15)

TFA (2 mL) was added to a solution of the precursor 14 (370 mg, 0.84 mmol) in CH2Cl2 (3 mL) at 0 °C. The mixture was stirred at 0–5 °C for 2 h until the completion of the reaction, as monitored by TLC, the solvent was evaporated via rotary evaporation at 50 °C. Compound 15 was obtained via flash chromatography on a short silica gel column (CHCl3/MeOH 6:1 to 3:1) as colorless oil (TFA salt, 820 mg, 76%). 1H NMR (400 MHz, CD3OD) δ 8.23 (d, J=5.6 Hz, 1H), 7.77 (d, J=2.4 Hz, 1H), 7.43 (d, J=5.2 Hz, 1H), 7.28 (d, J=2.0 Hz, 1H), 7.21 (s, 1H), 5.36 (s, 2H), 4.34 (m, 1H), 4.17 (m, 1H), 3.90 (m, 1H), 3.26–3.31 (m, 2H), 1.82–2.24 (m, 4H); HRMS calculated for C16H18ClFN3O2: [M+H] m/z=338.1071, found: 338.1058. Anal. calculated for C16H17ClFN3O2·8TFA·H2O, C, 30.29; H, 2.15; N, 3.31 Found, C, 30.16; H, 2.19; N, 3.61.

2-Chloro-3-(2-fluoro-4-(pyridinyl)methoxy)-5-((1-methyl-2-(S)-pyrrolidinyl)methoxy)pyridine (16)

A mixture of the secondary amine 15 (170 mg, 0.5 mmol) and 37% formaldehyde (0.20 mL) in acetonitrile (2 mL) was stirred for 20 min at room temperature, then sodium cyanoborohydride (50 mg, 0.8 mmol) was added in small portions. After stirring for 15 min at room temperature, acetic acid was carefully added until the solution tested neutral on wet pH paper. Stirring was continued for 2 h, HOAc being added occasionally to maintain the pH near neutrality. The solvent was evaporated at reduced pressure, and 15 mL of 5% K2CO3 was added to the residue. The resulting mixture was extracted with three 20 mL portions of CHCl3. The combined CHCl3 extracts were washed with water 10 mL, dried with anhydrous Na2SO4 and evaporated in vacuo to give a residue. After purification by column chromatography on silica gel with CHCl3/MeOH (15:1), the product was obtained as a white solid (146 mg, 83%); 1H NMR (400 MHz, CDCl3/TMS) δ 8.26 (d, J=5.2 Hz, 1H), 7.75 (d, J=2.8 Hz, 1H), 7.28 (m, 1H), 7.08 (s, 1H), 6.84 (d, J=2.8 Hz, 1H), 5.16 (s, 2H), 3.92–4.02 (m, 2H), 3.13 (m, 1H), 2.65 (m, 1H), 2.47 (s, 3H), 2.33 (m, 1H), 1.70–2.06 (m, 4H). Anal. Calcd. for C17H19ClFN3O2·0.25·H2O, C, 57.29; H, 5.52; N, 11.80; Found: C, 57.53; H, 5.67; N, 11.82.

3-((6-chloropyridin-3-yl)methoxy)pyridin-2-ol (17)

2,3-dihydroxypyridine (5.56 g, 50 mmol) was added portion-wise to a solution of NaOH (2 g, 50 mmol) in methanol (30 ml), followed by addition of a solution of 2-chloro-5-(chloromethyl)pyridine (8.1 g, 50 mmol) in MeOH (10 mL) dropwise during 30 min. The mixture was stirred at room temperature for 96 h, and then heated at 40 °C for 13 h. After evaporation of methanol, the residue was diluted with water and extracted with CHCl3 (6 × 100 mL). The combined organic extracts were dried over MgSO4, filtered and concentrated in vacuo. Recrystallization in ethyl acetate afforded 17 (9.2 g, 78%) as white solid; mp 201–202 °C; 1H NMR (400 MHz, CDCl3/TMS) δ 13.00 (s, 1H), 8.46 (d, J=2.4 Hz, 1H), 7.81 (d, J=2.4 Hz, 1H), 7.36 (d, J=8.4 Hz, 1H), 7.08 (dd, J=1.6 Hz, 6.4 Hz, 1H), 6.82 (dd, J=1.6 Hz, 6.4 Hz, 1H), 6.20 (t, J=7.0 Hz and 14.0 Hz), 5.14 (s, 2H). HRMS calculated for C11H10ClN2O2: [M+H] m/z=237.0431, found: 237.0452.

3-((6-chloropyridin-3-yl)methoxy)-5-nitropyridin-2-ol (18)

Compound 17 (4.8 g, 20.3 mmol) was added to magnetically stirred H2SO4 (96%, 15 mL) chilled with an ice-bath. Then, a cold mixture of HNO3/H2SO4 (2 mL+2 mL) was added dropwise during 20 min. The resulting mixture was stirred at 0–5 °C for 25 min and poured into 150 mL of icy water. The yellow precipitate was filtered off, washed with cold water (5 × 60 mL) and dried (4.57 g, 80%); mp 211–212 °C; 1H NMR (400 MHz, DMSO-d6/TMS) δ 12.77 (s, 1H), 8.51 (s, 1H), 8.38 (m, 1H), 7.95 (dd, J=2.4 Hz, 8.4 Hz, 1H), 7.60 (m, 2H), 5.21 (s, 2H); HRMS calculated for C11H9ClN3O4: [M+H] m/z=282.0282, found: 282.0276.

2-Chloro-3-(2-chloro-5-(pyridinyl)methoxy)-5-nitropyridine (19)

Compound 18 (5.49 g, 19.5 mmol) was added to quinoline (2.35 mL, 19.6 mmol). The reaction flask was cooled to 5 °C, and phosphoryl chloride (7.3 mL, 78 mmol) was added dropwise. The mixture was blanketed with argon and heated at 120 °C for 2 h. Upon complete consumption of the precursor, as indicated by TLC, the mixture was cooled to room temperature and 100 mL of H2O was added. The mixture was then cooled to 0 °C and the resulting brown solid was filtered. Re-crystallization from ethanol gave sand-colored crystals, (5.22 g, 89%); mp 151–152 °C; 1H NMR (400 MHz, CDCl3/TMS) δ 8.92 (d, J=2.4 Hz, 1H), 8.55 (d, J=2.0 Hz, 1H), 8.04 (d, J=2.4 Hz, 1H), 7.84 (dd, J=2.4 Hz, 8.0 Hz, 1H), 7.45 (d, J=8.4 Hz, 1H), 5.26 (s, 2H); HRMS calculated for C11H8Cl2N3O3: [M+H] m/z=299.9943, found: 299.9938.

5-Amino-2-chloro-3-(2-chloro-5-(pyridinyl)methoxy)pyridine (20)

Compound 19 (5.19 g, 17.3 mmol) was added to a solution of H2O (51 mL) and glacial acetic acid (68 mL). Iron powder (4.06 g, 72.9 mmol) was then added to the reaction flask, and the mixture was heated to 60 °C. After 1 h, TLC monitoring of the reaction indicated complete consumption of the precursor. H2O (100 mL) was added to the reaction flask, and the mixture was made basic, pH > 8, with potassium hydroxide pellets. The mixture was then extracted with ethyl acetate (3 × 50 mL) and evaporated to a yellow solid (3.97 g, 85%); mp 152–153 °C; 1H NMR (400 MHz, CDCl3/TMS) δ 8.46 (d, J=1.6 Hz, 1H), 7.82 (dd, J=2.2 Hz, 8.2 Hz, 1H), 7.53 (d, J=2.4 Hz, 1H), 7.39 (d, J=8.0 Hz, 1H), 6.59 (d, J=2.0 Hz, 1H), 5.09 (s, 2H), 3.76 (br s, 2H); HRMS calculated for C11H10Cl2N3O: [M+H m/z=270.0210, found: 270.0183.

5-Acetyloxy-2-chloro-3-(2-chloro-5-(pyridinyl)methoxy)pyridine (21)

Amine 20 (3.1 g, 11.5 mmol) was added to HBF4 (24 mL, 48%) and cooled to 0 °C. A solution of sodium nitrite (1.21 g, 17.5 mmol) and H2O (9 mL) was added dropwise over a period of 1 h. After the addition of sodium nitrite was complete, the mixture was stirred for 1 h at 0 °C. The resulting solid was filtered, washed with cold diethyl ether (3 × 20 mL), and air-dried for 15 min. The solid was then dissolved in acetic anhydride (47 mL) and heated for 1 h to 70–85 °C. The solvent was evaporated, and the resulting residue was dissolved in diethyl ether (60 mL). The diethyl ether solution was washed with H2O (2 × 20 mL), dried over MgSO4, and evaporated under reduced pressure to give a residue which was purified by silica gel chromatography (Hexanes/ Ethyl acetate 1:1). Compound 21 was obtained as white solid (2.34 g, 72%); mp 134–135 °C; 1H NMR (400 MHz, CDCl3/TMS) δ 8.48 (d, J=2.0 Hz, 1H), 7.92 (d, J=2.0 Hz, 1H), 7.81 (dd, J=2.4 Hz, 8.4 Hz, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.16 (d, J=2.0 Hz, 1H), 5.13 (s, 2H), 2.34 (s, 3H); HRMS calculated for C13H11Cl2N2O3: [M+H] m/z=313.0147, found: 313.0161.

2-Chloro-3-(2-chloro-5-(pyridinyl)methoxy)-5-hydroxypyridine (22)

Compound 21 (1.22 g, 3.9 mmol) was added to a mixture of 1M KOH solution (6.3 mL) and MeOH (10 mL) at 5 C. After it was stirred for 2 h in an ice bath, TLC showed the starting material completely consumed. The solution was made acidic to pH 5, with glacial acetic acid (~1.5 mL) and the resulting solid was filtered and dried. The product was obtained as a white powder (0.87 g, 82%); mp 228–229 °C; 1H NMR (400 MHz, DMSO-d6/TMS) δ 10.32 (s, 1H), 8.53 (d, J=2.0 Hz, 1H), 7.95 (dd, J=2.4 Hz, 8.0 Hz, 1H), 7.60 (d, J=8.4 Hz, 1H), 7.57 (d, J=2.4 Hz, 1H), 7.09 (d, J=2.8 Hz, 1H), 5.26 (s, 2H); HRMS calculated for C11H9Cl2N2O2: [M+H] m/z=271.0041, found: 271.0024.

2-Chloro-5-((1-(tert -butoxycarbonyl)-2-(S)-pyrrolidinyl)methoxy)-3-(2-chloro-5-(pyridinyl)methoxy)pyridine (23a)

Diethyl azodicarboxylate (0.105 mL, 0.6 mmol) and triphenylphosphine (158 mg, 0.6 mmol) were mixed in anhydrous THF (2 mL) at 0 °C, under argon for 30 min. N-Boc-L-prolinol (121 mg, 0.6 mmol) and precursor 22 (121 mg, 0.45 mmol) were then added to the reaction flask, and the mixture was stirred at room temperature for 24 h. The solvent was evaporated under reduced pressure at 55 °C, and the crude oil was purified via flash chromatography (Hexane/Ethyl acetate 2:1 to 1:1). Compound 23a was obtained as colorless oil (112 mg, 55%); 1H NMR (400 MHz, CDCl3/TMS) δ 8.51 (s, 1H), 7.86 (d, J=8.0 Hz, 1H), 7.74 (d, J=2.4 Hz, 1H), 7.47 (s, 1H), 7.39 (m, 1H), 5.25 (s, 2H), 4.24 (m, 1H), 4.09 (m, 1H), 3.88 (m, 1H), 3.39 (m, 2H), 1.88–2.00 (m, 4H), 1.48 (s, 9H); HRMS calculated for C21H26Cl2N3O4: [M+H] m/z=454.1300, found: 454.1299.

2-Chloro-5-(1-(tert-butoxycarbonyl)-2-(R)-pyrrolidinyl)methoxy)-3-(2-chloro-5-(pyridinyl)methoxy)pyridine (23b)

Diethyl azodicarboxylate (0.21 mL, 1.2 mmol) and triphenylphosphine (316 mg, 1.2 mmol) were mixed in anhydrous THF (5 mL) at 0 °C, under argon for 30 min. N-Boc-D-prolinol (242 mg, 1.2 mmol) and precursor 22 (296 mg, 1.1 mmol) were then added to the reaction flask, and the mixture was stirred at room temperature for 36 h. The solvent was evaporated under reduced pressure at 55 °C, and the crude oil was purified via flash chromatography (Hexane/Ethyl acetate 2:1 to 1:1). Compound 23b was obtained as colorless oil (340 mg, 68%); 1H NMR (400 MHz, CDCl3/TMS) δ 8.51 (s, 1H), 7.86 (d, J=8.0 Hz, 1H), 7.74 (d, J=2.4 Hz, 1H), 7.47 (s, 1H), 7.39 (d, J=8.4 Hz, 1H), 5.25 (s, 2H), 4.24 (m, 1H), 4.09 (m, 1H), 3.88 (m, 1H), 3.29–3.39 (m, 2H), 1.88–2.00 (m, 4H), 1.48 (s, 9H).

5-(((S)-pyrrolidin-2-yl)methoxy)-3-((6-chloropyridin-3-yl)methoxy)-2-chloropyridine (24a)

TFA (5 mL) was added to a solution of 23a (820 mg, 1.8 mmol) in CH2Cl2 (6 mL) at 0 °C. The mixture was stirred at 0–5 °C for 2 h until the completion of the reaction, as monitored by TLC, the solvent was evaporated via rotary evaporation at 50 °C. Compound 24a was obtained via flash chromatography on a short silica gel column (CHCl3/MeOH 10:1) as colorless oil (510 mg, 80%). 1H NMR (400 MHz, CDCl3/TMS) δ 8.48 (d, J=2.8 Hz, 1H), 7.82 (dd, J=2.4 Hz, 8.0 Hz, 1H), 7.73 (d, J=2.4 Hz, 1H), 7.41 (d, J=8.4 Hz, 1H), 6.88 (d, J=2.8 Hz, 1H), 5.12 (s, 2H), 3.93–3.97 (m, 1H), 3.85–3.89 (m, 1H), 3.53–3.57 (m, 1H), 2.98–3.03 (m, 2H), 1.93–1.98 (m, 1H), 1.79–1.87 (m, 3H), 1.53–1.58 (m, 1H). HRMS calculated for C16H18Cl2N3O2: [M+H] m/z=354.0776, found: 354.0777. Anal. Calcd. for C16H17Cl2N3O2 TFA0·5H2O, C, 45.28; H, 4.01; N, 8.81; Found, C, 45.66; H, 4.16; N, 8.70.

5-(((R)-pyrrolidin-2-yl)methoxy)-3-((6-chloropyridin-3-yl)methoxy)-2-chloropyridine (24b)

The compound 24b was prepared via 23b using the procedure that is described for preparation of 24a. The product 24b was obtained as colorless oil. Yield: 82%. 1H NMR (400 MHz, CDCl3/TMS) δ 8.48 (d, J=2.8 Hz, 1H), 7.81 (dd, J=2.4 Hz, 8.4 Hz, 1H), 7.74 (d, J=2.4 Hz, 1H), 7.40 (d, J=8.0 Hz, 1H), 6.89 (d, J=2.8 Hz, 1H), 5.12 (s, 2H), 3.95–3.98 (m, 1H), 3.87–3.91 (m, 1H), 3.53–3.58 (m, 1H), 2.98–3.04 (m, 2H), 2.09 (br s, 1H), 1.94–2.02 (m, 1H), 1.78–1.89 (m, 2H), 1.52–1.61 (m, 1H). Anal. Calcd. for C16H17Cl2N3O2·TFA·H2O, C, 44.53; H, 4.16; N, 8.66; Found: C, 44.36; H, 4.00; N, 8.58.

2-Chloro-3-(2-chloro-5-(pyridinyl)methoxy)-5-((1-methyl)-2-(S)-pyrrolidinyl)methoxy)pyridine (25a)

Compound 24a (145 mg, 0.32 mmol) was dissolved in a mixture of formic acid (0.35 mL) and formalin (0.7 mL), refluxed for 8 h, and cooled to room temperature. The reaction mixture was poured into 5% K2CO3 solution in water (20 mL). The aqueous mixture was extracted with CHCl3 (4 × 15 mL), the combined extracts were washed with water (20 mL), dried over Na2SO4, and the solvent was removed. The residue was chromatographed on silica gel (CHCl3/MeOH 15:1) to give the product as a white solid (76 mg, 65%); 1H NMR (400 MHz, CDCl3/TMS) δ 8.48 (d, J=2.4 Hz, 1H), 7.82 (dd, J=2.4Hz, 8.8 Hz, 1H), 7.73 (d, J=2.4 Hz, 1H), 7.40 (d, J=8.4 Hz, 1H), 6.89 (d, J=2.8 Hz, 1H), 5.11 (s, 2H), 3.93–4.02 (m, 2H), 3.13 (m, 1H), 2.65 (m, 1H), 2.47 (s, 3H), 2.30 (m, 1H), 2.02 (m, 1H), 1.74–1.83 (m, 3H); HRMS calculated for C17H20Cl2N3O2: [M+H] m/z=368.0933, found: 368.0929; Anal. Calcd. for: C17H19Cl2N3O2, C, 55.45; H, 5.20; N, 11.41; Found, C, 55.30; H, 5.36; N, 11.43.

2-Chloro-3-(2-chloro-5-(pyridinyl)methoxy)-5-((1-methyl)-2-(R)-pyrrolidinyl)methoxy)pyridine (25b)

This compound was prepared similar to compound 25a. The product was obtained as white solid. Yield: 72%. 1H NMR (400 MHz, CDCl3/TMS) δ 8.48 (d, J=2.0 Hz, 1H), 7.82 (dd, J=2.4Hz, 8.4 Hz, 1H), 7.73 (d, J=2.4 Hz, 1H), 7.40 (d, J=8.4 Hz, 1H), 6.89 (d, J=2.4 Hz, 1H), 5.11 (s, 2H), 3.93–4.02 (m, 2H), 3.13 (m, 1H), 2.65 (m, 1H), 2.47 (s, 3H), 2.30 (m, 1H), 2.02 (m, 1H), 1.72–1.86 (m, 3H); HRMS calculated for C17H20Cl2N3O2: [M+H] m/z=368.0933, found: 368.0926; Anal. Calcd. for C17H19Cl2N3O2·0.25·H2O, C, 54.90; H, 5.29; N, 11.31. Found. C, 54.69; H, 5.32; N, 11.23.

Radiochemistry

2-Chloro-3-(2-fluoro-4-(pyridinyl)methoxy)-5-((1-[11C]methyl-2-(S)-pyrrolidinyl)methoxy)pyridine ( [11C]16)

Precursor 15 (1–2 mg) was dissolved in 200 µL of anhydrous DMF, capped in a small V-vial and cooled to −40 °C. [11C]Methyl iodide was swept by argon flow into the solution. After the radioactivity reached a plateau, the vial was assayed in the dose calibrator and then heated at 80 °C for 5 min. Water (200 µL) was added and the solution was injected onto the semi-preparative HPLC column (Phenomenex Luna C-18 10 µm column, semi-preparative 10 × 250 mm, 30:70 v/v CH3CN/0.1 M ammonium formate, 12 mL/min). The retention time of 15 was 3.8 min. The product [11C]16 peak, having retention time of 5.2 min, was collected into a flask containing 50 mL water. The mixture was transferred through a Waters C-8 Sep-Pak Plus. The product was eluted with 1 mL ethanol into a vial and diluted with 9 mL of 0.9% saline. The final product [11C]16 was then analyzed by analytical HPLC (Phenomenex Luna C-18 10 µm columns, analytical 4.6 × 250 mm, 30:70 v/v CH3CN/0.1 M ammonium formate, 3 mL/min, tR=2.5 min) to determine the radiochemical purity (>98%) and the specific radioactivity at the time of synthesis. The total synthesis time was 35 min from EOB with an average radiochemical yield of 18.4±8.8% and specific radioactivity of 8022±5592 (n=4) mCi/µmol (nondecay corrected from the end of 11CH3I synthesis).

2-Chloro-3-(2-chloro-5-(pyridinyl)methoxy)-5-((1-[11C]methyl)-2-(S)-pyrrolidinyl)methoxy)pyridine ( [11C]25a)

Precursor 24a (1.5 mg) was dissolved in 200 µL of anhydrous DMF, capped in a small V-vial and cooled to −40 °C. [11C]Methyl iodide was swept by argon flow into the vial. After the radioactivity reached a plateau, the vial was assayed in the dose calibrator and then heated at 80 °C for 5 min. Water (200 µL) was added and the solution was injected onto the semi-preparative HPLC column (Phenomenex Luna C-18 10 µm column, semi-preparative 10 × 250 mm, 32:68 v/v CH3CN/0.1 M ammonium formate, 8 mL/min). The retention time of normethyl precursor 24a was 4.7 min. The product peak, having retention time of 7.7 min, was collected into a flask containing 50 mL water. The water solution was transferred through a Waters C-8 Sep-Pak Plus. The product was eluted with 1 mL ethanol into a vial and diluted with 9 mL of 0.9% saline. The final product [11C]25a was then analyzed by analytical HPLC (Phenomenex Luna C-18 10 µm columns, analytical 4.6 × 250 mm, 40:60 v/v CH3CN/0.1 M ammonium formate, 2 mL/min, tR=3.5 min) to determine the radiochemical purity (>98%) and the specific radioactivity at the time of synthesis. The total synthesis time was 40 min from EOB with an average radiochemical yield of 29±7.2% and specific radioactivity of 3646±1233 mCi/µmol (nondecay corrected from the end of 11CH3I synthesis).

Animal Studies

All animal studies were approved by the Animal Care and Use Committee of the Johns Hopkins University.

Mouse studies

Baseline Study. Male, CD-1 mice weighing 25–30 g from Charles River Laboratories, (Wilmington, MA) were used for biodistribution studies. The animals were sacrificed by cervical dislocation at 5, 15, 30 and 60 min after intravenous injection of radiotracer in 200 µL saline vehicle into a lateral tail vein (3 animals per time-point). The brains were rapidly removed and dissected on ice. The brain regions of interest were blotted, weighed and their radioactivity content was determined in an automated γ-counter with a counting error below 3%. Aliquots of the injectate were prepared as standards and their radioactivity content was counted along with the tissue samples. The percent of injected dose per gram of tissue (%ID/g tissue) was calculated. To assess binding specificity, blocking studies were performed in the same manner as described above except that 0.2 mL of a solution containing the blocking dose in saline was administered 5 min prior to subcutaneous (s.c.) injection of the radioligand.

Micro PET studies with rats

A male Wistar rat (Charles River) was anesthetized by intraperitoneal administration of a combination of ketamine (72 mg/kg), xylazine (6 mg/kg), and acepromazine (6 mg/kg) and positioned on the bed of the GE eXplore Vista small-animal PET scanner (GE Medical Systems, Waukesha, WI) and kept anesthetized with isoflurane (0.5%–1%; approximately 1 L/min). The radiotracer (11.1 MBq or 0.3 mCi in 0.2 mL of saline) or blocking agent cytisine were injected via the tail vein. After the radioligand injection, the PET images were acquired using a 28 dynamic frame protocol (90 min total; 3 × 20s, 3 × 40s, 5 × 60s, 6 × 120s, 8 × 300s, 3 × 600s). PET images were reconstructed using a 2D OSEM algorithm after subtracting the scatter component from the sonogram images. Mean images were created and they were used for drawing regions of interest (ROIs) on the thalamus and the cerebellum. For each region the ROI was drawn on two adjacent slices, then the ROIs were applied to the dynamic images to generate time-activity curves.

Baboon PET experiments

Male baboons (Papio anubis) (20–26 kg) were studied in baseline control experiments. The animals were fasted for 12 hours prior to the PET study. Anesthesia was given initially with intramuscular injection of 20 ml (9 mg/kg) Saffan® (Schering-Plough, Middlesex, U.K.). The baboon was intubated and anesthesia was maintained with a constant infusion of Saffan® diluted with isotonic saline (1:4) at an average flow rate that corresponded to 7.5 mg/kg/h. Circulatory volume was maintained by infusion of isotonic saline. An arterial catheter was inserted for blood sampling. Physiological vital signs including heart rate, ECG, blood pressure and oxygen saturation were continuously monitored throughout the study. The animal was positioned in a high resolution research tomograph (ECAT HRRT) brain PET scanner (CPS Innovations, Inc., Knoxville, TN). A custom thermoplastic mask was molded to the shape of the baboon head and attached to the PET scanner bed to reproducibly position the animal and restrict motion during the PET scan.

A 6 min transmission scan with a 1 mCi Germanium-68 source was initially performed for attenuation correction. Then dynamic PET data were acquired in 3D list mode during a 120 min period following a bolus injection of the radioligand ([11C]16 (21 mCi, specific radioactivity = 2900 mCi/µmol) or [11C]24a (17.5 mCi, specific radioactivity = 2800 mCi/µmol) or ([11C]26 (31 mCi, specific radioactivity = 11600 mCi/µmol) in a list mode. Arterial blood was sampled rapidly initially and with prolonging intervals throughout the scan.

Volumes of interest (VOIs) were defined on SPGR (spoiled gradient) MRI acquired in the Signa 1.5 Tesla scanner (GE Medical Systems, Milwaukee, WI) for thalamus and cerebellum. VOIs were transferred to PET space using MRI-to-PET coregistration parameters 37 and applied on PET frames to generate time-radioactivity curves (TACs) of the regions. Fractions of un-metabolized radio-tracers in plasma for arterial blood samples that underwent HPLC analysis were fitting with a sum of two exponentials to obtain metabolite-corrected plasma TACs. A two-tissue compartmental model with four parameters (i.e., K1 and k2 for blood-to brain and brain-to-blood clearance rate constants across the blood-brain-barrier, and k3 and k4 for association and dissociation rate constants to and from receptors) was used to describe the kinetics of radiotracers. Three parameters (K1, k3, and k4) were estimated in thalamus by setting the K1-k2 ratio to the estimate obtained in cerebellum.38 A fixed fraction (3.5%) of total plasma TAC was subtracted from tissue TAC at each PET frame to remove the radioactivity in vasculature in tissue. The outcome variable, binding potential was given as the k3-k4 ratio39,40 The possibility that lipophilic metabolites might enter the brain was ignored in tracer kinetics modeling.

Metabolite Analysis in Baboon Plasma

Metabolites of [11C]16, [11C]25a and 11C]26 in baboon were studied using a general method previously developed for PET radiotracers41 . Specifically, arterial blood samples were withdrawn at 5, 15, 30, 60, and 90 min intervals up to 90 min postinjection and plasma was analyzed for the presence of the parent radioligands and their radiolabeled metabolites. Briefly, 3 mL of plasma in 8 M urea were passed through a capture column (19 × 4.6 mm Strata-X, Phenomenex, Torrance, CA) at 2 mL/min, followed by 1% acetonitrile in water to wash plasma proteins from the column. The effluent from the capture column, containing only highly polar components, flows through a dual BGO detector (Bioscan, Washington, DC). The solvent was then switched to a mixture of 35% acetonitrile/65% 0.1M aqueous ammonium bicarbonate (2 mL/min) for elution of the radiolabeled components bound to the capture column onto the analytical column (Gemini C18, 4.6 × 250 mm, Phenomenex, Torrance, CA).

Figure 1
Resentative nAChR ligands – main lead compounds for development of nAChR PET radioligands.
Figure 9
Thalamus and cerebellum time-uptake curves and thalamus/cerebellum ratio of accumulated radioactivity versus time in the baseline PET experiments with [11C]16 in baboon

Acknowledgments

The authors thank Paige Finley for her help with the animal experiments, Robert C. Smoot for radiochemistry assistance, and James Fox, David J. Clough and Karen Edmonds for PET scanner operation. We are grateful to Judy W. Buchanan for editorial help. This research was supported by the Department of Radiology of Johns Hopkins University School of Medicine and U.S. Public Health Service grants from the National Institutes of Health (DA020777 and MH079017). The National Institute of Mental Health Psychoactive Drug Screening Program (Grant N01 MH32004) supported some of the in vitro studies.

Footnotes

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