(Trk) Inhibitor for PET Imaging

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Brief Article Cite This: J. Med. Chem. 2018, 61, 1737−1743

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Identification of [18F]TRACK, a Fluorine-18-Labeled Tropomyosin Receptor Kinase (Trk) Inhibitor for PET Imaging Vadim Bernard-Gauthier,*,†,×,◇ Andrew V. Mossine,‡,◇ Anne Mahringer,§ Arturo Aliaga,∥ Justin J. Bailey,† Xia Shao,‡ Jenelle Stauff,‡ Janna Arteaga,‡ Phillip Sherman,‡ Marilyn Grand’Maison,⊥ Pierre-Luc Rochon,# Björn Wan̈ gler,∇ Carmen Wan̈ gler,○ Peter Bartenstein,◆ Alexey Kostikov,# David R. Kaplan,¶ Gert Fricker,§ Pedro Rosa-Neto,∥ Peter J. H. Scott,‡,∞,◇ and Ralf Schirrmacher*,†,◇ †

Department of Oncology, Division of Oncological Imaging, University of Alberta, Edmonton, AB T6G 2R3, Canada Department of Radiology, Division of Nuclear Medicine, The University of Michigan Medical School, Ann Arbor, Michigan 48109, United States § Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg 69120, Germany ∥ Translational Neuroimaging Laboratory, McGill Centre for Studies in Aging, Douglas Mental Health University Institute, Montreal, QC H4H 1R3, Canada ⊥ Biospective Inc., Montreal, QC H4P 2R2, Canada # McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montreal, QC H3A 2B4, Canada ∇ Molecular Imaging and Radiochemistry and ○Biomedical Chemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, Mannheim 68167, Germany ◆ Department of Nuclear Medicine, Ludwig-Maximilians-University of Munich, Munich 81377, Germany ¶ Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada ∞ The Interdepartmental Program in Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States ‡

S Supporting Information *

ABSTRACT: Changes in expression and dysfunctional signaling of TrkA/B/C receptors and oncogenic Trk fusion proteins are found in neurological diseases and cancers. Here, we describe the development of a first 18F-labeled optimized lead suitable for in vivo imaging of Trk, [18F]TRACK, which is radiosynthesized with ease from a nonactivated aryl precursor concurrently combining largely reduced P-gp liability and improved brain kinetics compared to previous leads while displaying high on-target affinity and human kinome selectivity.

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hematological cancers.12,13 Clinical pan-Trk inhibitors have shown striking efficacy en route to approval in phase I/II trials in genomically defined patients, indicating that highly diverse malignancies driven by similarly diverse NTRK fusions may be treated with common targeted inhibitors.12,14−16 Trk inhibitors in clinical trials are now being explored in CNS primary tumors and metastases.17,18 Given those advances, there is a need for comparative clinical studies of the spatiotemporal alterations in TrkA/B/C densities between healthy individuals and patients with neurodegenerative diseases or cancer, and the determination of target engagement of Trk antineoplastic kinase inhibitor drugs19,20 in the context of neurooncological malignancies. An impediment to performing these studies, however, is the lack of suitable nondestructive analytic tools applicable for imaging of patients

INTRODUCTION Tropomyosin receptor kinases A, B, and C (TrkA, TrkB, and TrkC) constitute a family of single-pass transmembrane receptor tyrosine kinases with highly homologous intracellular kinase domains encoded by the NTRK1, NTRK2, and NTRK3 genes, respectively.1,2 TrkA/B/C are primarily found in various neuronal subsets where their activation mediates neuronal survival and differentiation in the developing and mature central (CNS) and peripheral (PNS) nervous systems.3,4 Considerable evidence has emerged over the past decades that link perturbed Trk signaling or expression to a plethora of neurological disorders and neurodegenerative diseases including Alzheimer’s (AD), Parkinson’s (PD), and Huntington’s (HD) diseases.5−11 Abnormal overexpression and activation of Trk in non-neural tissue are also found in numerous cancers.12 Perhaps most significantly, NTRK1/2/3 chromosomal rearrangements leading to oncogenic Trk proteins bearing intact Trk kinase domains have been identified in multiple solid and © 2017 American Chemical Society

Received: November 1, 2017 Published: December 19, 2017 1737

DOI: 10.1021/acs.jmedchem.7b01607 J. Med. Chem. 2018, 61, 1737−1743

Journal of Medicinal Chemistry

Brief Article

radiolabeling of a series of 6-(2-(3-fluorophenyl)pyrrolidin-1yl)imidazo[1,2-b]pyridazine Trk inhibitors which led to the identification of the first Trk radiotracer clinical lead, [11C]-(R)8 ([11C]-(R)-IPMICF16, Figure 1B).27 In the non-human primate (NHP) and human brains in vivo, highest uptake following [11C]-(R)-8 injection was found in regions associated with pronounced levels of TrkB/C, the two most abundant Trk paralogs present in the CNS. Radiotracer [11C]-(R)-8 was also used as an in vitro tool to quantify TrkB/C levels in healthy versus AD brains using autoradiography.27 While enabling the first steps toward the clinical study of Trk in vivo using PET, this radiotracer is labeled with carbon-11 (11C t1/2 = 20.3 min) which requires on site production and exhibits overall slow brain kinetics and P-gp efflux liability. Here, we sought to identify a kinome-selective 18F-labeled Trk inhibitor lead (18F t1/2 = 109.8 min) accessible in a single radiosynthetic step and compatible with large scale automated production to facilitate distribution and use in upcoming studies while mitigating P-gp liability of our initial 11C-lead and improving brain kinetics in NHPs.

in vivo. In recent years, the repurposing of Trk-binding compounds, primarily diverse kinase inhibitors, as positron emission tomography (PET) neuroimaging probes (1−8, Figure 1A) has been explored.21−26 We recently reported the

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RESULTS AND DISCUSSION Owing to the unique potency/selectivity properties attained with the inhibitor series which led to the initial validation of [11C]-(R)-8,24,27 we aimed to identify a poor or non-P-gp substrate within the 6-(2-(3-fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazine core scaffold series. We performed a screen of our racemic 3-fluorophenyl)pyrrolidine-based Trk inhibitor library using a P-gp calcein-AM cellular assay to reprioritize those analogs based on efflux profile. This approach led to the identification of 9, the only derivative displaying markedly reduced P-gp interaction compared to the structurally analogous lead 8 (Figure 2D,E; Supporting Information Figure 1). Whereas 8 displayed an EC50 of 1.3 μM for P-gp, the

Figure 1. (A) Chemical structures of Trk-targeted PET radiotracers and (B) optimization rationale of the [11C]-(R)-8 lead.

Figure 2. Development and in vitro assessment of [18/19F]-(R)-9. (A) View of the predicted type I binding mode of (R)-9 (space fill model) with TrkA (gray ribbons, PDB code 4PMT). (B) Surface rendering of (R)-9 (sticks) bound to the ATP binding site of TrkA (gray surface). (C) Comprehensive kinase selectivity profile of (R)-9 against 369 kinases. Kinases are ordered alphabetically and data represented as radar chart with 100.0%, 50.0%, and 0% activity relative to control (200 nM, n = 2) ([γ-33P]ATP-based enzymatic assay performed by Reaction Biology). (D, E) Calcein-AM cellular assays. Calcein-AM assays were conducted in human P-gp overexpressing Madin−Darby canine kidney (MDCKII) cells. In all cases, intracellular fluorescence in the absence of test compounds was set as 100%. Valspodar was used as positive control for P-gp. Shown is a comparison of the results obtained from Calcein-AM assay for P-gp with 8 (EC50 = 1.3 μM for P-gp) and 9 (EC50 ≫ 50 μM for P-gp). (F) Venn diagram showing overlapping kinase targets between (R)-9, 2, and 4 with or without CNS expression (cutoffs based on refs 20 and 21.). (G, H) Regional quantification for the in vitro human brain tissue autoradiography experiments with [18F]-(R)-9 in human prefrontal cortex (G) and cerebellum (H) (n = 4−6). Data are expressed as the mean ± SD: (∗∗) P ≤ 0.01, (∗∗∗) P ≤ 0.001, (∗∗∗∗) P ≤ 0.0001. 1738

DOI: 10.1021/acs.jmedchem.7b01607 J. Med. Chem. 2018, 61, 1737−1743

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ROS1 (IC50 = 46.0 nM; 148- to 307-fold TrkB/C selectivity), and ACK1 (IC50 = 53.7 nM; 173- to 358- fold TrkB/C selectivity). In addition, (R)-9 displayed low potencies toward TXK and PIM3; both inhibited at an approximate IC50 of 200 nM. Taken together, the in vitro assessments confirmed the exquisite affinity and kinome selectivity of our new fluorinated inhibitor lead. We envisioned that the 18F-labeled isotopologue of (R)-9 could be attained using the recently described copper-mediated nucleophilic radiofluorination methods starting from an inactivated aryl pinacolboronic ester or boronic acid precursor.29,30 To this end, the necessary labeling precursor (R)-10 was obtained via amide coupling between intermediate (R)-12 and the commercially available 4-aminophenylboronic acid pinacol ester (Scheme 1). The proof-of-principle manual radiosynthesis of [18F]-(R)-9 using [18F]KF/K222 aliquots (∼50 MBq, 1.4 mCi) dried following conventional conditions and base content with [Cu(OTf)2(pyr)4] in DMF (20 min, 110 °C)29 resulted in radiochemical conversions (RCCs) of 30% as determined by radio-TLC (Radiochemistry section in Supporting Information). For the production of [18F]-(R)-9 on a preparative scale, we used weakly basic 18F− elution conditions with KOTf/K2CO3 (73:1 molar ratio).30−32 Under such conditions, and using either [Cu(OTf)2(pyr)4] or Cu(OTf)2 (with added pyridine) as metal sources, [18F]-(R)-9 was obtained in 1.9 ± 0.2% isolated radiochemical yields (RCYs, n = 5). Those conditions were suitable for full automation and large scale production (up to 55.5 GBq−1.5 Ci of starting nocarrier-added (n.c.a.) 18F−) (see Radiochemistry section in Supporting Information). An up to 4-fold improvement in RCYs could be achieved using DMA/n-BuOH (2:1) as reaction medium combined with an 18F− source eluted beforehand with Et4NHCO3 in MeOH (single evaporation, no azeotropic drying, 8% n.d.c. RCY).33 Under these conditions, the analysis of the crude reaction revealed 25−30% of radiochemical incorporation of 18F−. Importantly, under all aforementioned conditions, initially measured molar activities (Am) were at least 1 order of magnitude below expected values (∼15 GBq/μmol) which led to the investigation of the content of the UV peak corresponding to [18F]-(R)-9/(R)-9. Using HPLC column screening,34 it was found that the protodeboronated sideproduct (R)-13, whose formation could not be avoided under radiofluorination conditions, coeluted alongside [18F]-(R)-9 when C18 HPLC columns were used for separation and quality control (QC) leading to poor effective Am. Proper removal of (R)-13 was crucial in light of the excellent pan-Trk potency of this derivative (IC50 for TrkA/B/C of 0.522, 99% radiochemical purity (Scheme 1) and >100 GBq/μmol Am suitable for in vitro and in vivo experiments (see Radiochemistry section in Supporting Information). With the tracer in hand, we conducted in vitro autoradiography using human brain tissue and in vivo NHP PET imaging studies. To identify specific TrkB/C binding of [18F](R)-9 in human brains, we used the selective pan-Trk inhibitor 4 (GW441756, Figure 1A) and a fluorinated GW2580 derivative 2, one of the most selective kinase inhibitors for

structural modifications found in 9 led to a drastic erosion of the P-gp liability with an EC50 well beyond 50 μM (highest tested concentration) under the same conditions (efflux reductions were also observed in BODIPY-prazosin (BCRP) assays, Supporting Information Figure 2). Importantly, we noted that the installation of a simplified 4-F phenyl fragment in place of the 3-F-4-OMe phenyl as the solvent-exposed amide moiety (Figure 2A,B) on the racemic derivatives was associated with a small decrease in TrkB/C potencies in the order of 7.3and 4.3 fold, respectively.24 As modest reductions in Trk potencies were sought to ensure a new lead with faster brain kinetics in vivo and considering that the fluoroaryl amide moiety offers a potential position for labeling, 9 was selected for further development. The synthesis of the required R-enantiomer28 (R)-9 was straightforward using the enantiopure carboxylic acid (R)-1225 and 4-fluoroaniline (Scheme 1). The potencies (IC50) of (R)-9 Scheme 1. Synthesis of (R)-9, Radiosynthesis of [18F]-(R)-9, and HPLC Quality Controla

a Reagents and conditions: (a) 4-fluoroaniline or aniline, DIPEA, HBTU, DMF, rt, 63−70%; (b) 4-aminophenylboronic acid pinacol ester, DIPEA, HBTU, DMF, rt, 62%; (c) [18F]Et4NF ([18F]TEAF), [Cu(OTf)2(pyr)4], DMA/n-BuOH, 20 min, 110 °C, then PFP HPLC.

in [γ-33P]ATP-based enzymatic assays were 4.21, 0.15, and 0.31 nM for human TrkA, TrkB and TrkC, respectively, consistent with data previously obtained with the corresponding racemic counterpart (Supporting Information Figure 3). We next determined inhibitory constants (Ki) toward all Trk paralogs (Supporting Information Figure 4). While the Ki values for TrkA that is expressed at low levels in the CNS were comparable between (R)-8 and (R)-9 (2.80 ± 0.16 nM versus 2.65 ± 0.71 nM), the measured Ki values for TrkB and TrkC that are more highly expressed in the CNS were both favorably reduced by ∼6-fold moving from (R)-8 to (R)-9 (Ki(TrkB) = 0.32 ± 0.10 nM; Ki(TrkC) = 0.14 ± 0.05 nM). To ascertain the effect of the structural changes on selectivity, large scale kinase profiling was performed (Figure 2C). The mapping of secondary inhibitions of (R)-9 in a 369 kinase screen was found to be similar overall to (R)-8 (Table 1 in Supporting Information). This assay used a 200 nM single concentration cutoff to estimate human kinome targets for which our lead displayed ∼1000-fold selectivity (based on TrkB/C). Specific potency data were obtained for the only three off-target kinases inhibited over their IC50 at the cutoff concentration, specifically PIM1 (IC50 = 25.5 nM; 82- to 170-fold TrkB/C selectivity), 1739

DOI: 10.1021/acs.jmedchem.7b01607 J. Med. Chem. 2018, 61, 1737−1743

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TrkB/C.22,23,35−37 Figure 2F shows the overlapping kinase targets between (R)-9, 2, and 4 with or without CNS expression. Co-incubation of [18F]-(R)-9 with 2 (type I inhibitor) or 4 (type II inhibitor) led to significant reduction in the human prefrontal cortex and cerebellum (Δ = −24 to 36%; (∗∗) P ≤ 0.01 to (∗∗∗∗) P ≤ 0.0001, Figure 2G,H). In NHP baseline PET imaging experiments, radiotracer [18F]-(R)9 rapidly crossed the blood-brain barrier (BBB) and displayed heterogeneous brain uptake, consistent with previous data with [11C]-(R)-8 (Figure 3A, Figure 4 top panel) and the known

Reduction in Am led to reductions of [18F]-(R)-9 uptake in TrkB/C-rich regions (Figure 3, Figure 4), while white matter remained unchanged. Hence, the ratios of tracer uptake in thalamus, cerebellum, and cortex relative to subcortical white matter (SUVR, summed 10−30 min) varied significantly between conditions and were 3.0−3.6 at 245 GBq/μmol, 2.4−3.0 at 173 GBq/μmol, and 1.9−2.4 at 15 GBq/μmol.

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CONCLUSION In summary, we have described an 18F-labeled Trk radiotracer that, based on our assessment so far, is suitable for use in PET imaging studies in vivo. During the development of [18F]-(R)-9, we methodically addressed crucial limitations of our previous lead 11C-lead regarding efflux, potency/brain kinetics, and isotopic label. Further imaging studies with [18F]-(R)-9 (referred as [18F]TRACK, previously referred as [18F]-(R)IPMICF1734) including the first human evaluation and detailed quantification will be reported in due course.

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EXPERIMENTAL SECTION

General Methods. All moisture sensitive reactions were carried out in oven-dried flasks under nitrogen atmosphere with dry solvents. Reagents and solvents were purchased at the highest commercial quality from Fisher, Sigma-Aldrich, Alfa-Aesar, or Synthonix and were used without further purification unless specified otherwise. Compounds used for blocking in autoradiography studies, 2 and 4 (GW441756), were synthesized as previously reported22,23 or purchased from Aldrich. 1H NMR and 13C NMR spectra were recorded on an Agilent/Varian DD2MR two channel 400 MHz spectrometer, an Agilent/Varian VNMRS two-channel 500 MHz spectrometer, or an Agilent/Varian Inova four-channel 500 MHz spectrometer in CDCl3 or DMSO-d6, and peak positions are given in parts per million using TMS as internal standard. Peaks are reported as s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, m = multiplet, b = broad; coupling constant(s) in Hz; integration. High resolution mass spectra (HRMS) analysis was obtained from the Regional Center for Mass Spectrometry of The Chemistry Department of the Université de Montréal (LC-MSD-TOF Agilent). Compounds tested for biological evaluation were >95% pure (HPLC). HPLC method A: Phenomenex LUNA C18 column (100 Å, 250 mm × 10 mm, 10 μm). Elution at 4.0 mL min−1 with a mixture of MeCN (A) and 20 mM NaH2PO4 (B) isocratic at 60% A and 40% B. HPLC method B: Phenomenex LC analytic LUNA C18 column (100 Å, 250 mm × 4.6 mm, 5 μm). Elution at 0.7 mL min−1 with a mixture of MeCN (A) and H2O (B) isocratic at 75% A and 25% B. The synthesis of compound (R)-12 was performed as previously reported.27 The synthesis of compound (R)-9 (HPLC method A (λ = 254 nm), tR = 15.0 min, purity 97%) was performed as previously described for the corresponding racemic compound using enantiopure (R)-12.24,27 General information relating to radiochemistry is provided in Supporting Information. Chemistry and Radiochemistry. Chemistry. (R)-6-(2-(3Fluorophenyl)pyrrolidin-1-yl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)imidazo[1,2-b]pyridazine-3-carboxamide ((R)10). DIPEA (43 mL, 0.25 mmol) was added to a solution of (R)-6(2-(3-fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazine-3-carboxylic acid (33 mg, 0.10 mmol) in DMF (3 mL). HBTU (38 mg, 0.10 mmol) was then added in one portion, and the reaction mixture was stirred at 23 °C for 5 min. A solution of 4-aminophenylboronic acid pinacol ester (22 mg. 0.10 mmol) in DMF (1 mL) was added dropwise, and the reaction mixture was stirred at 23 °C for 16 h. The reaction mixture was diluted with ethyl acetate (50 mL), washed with water (25 mL) and brine (25 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (1 → 5% MeOH in CH2Cl2) to afford 40 mg of the title compound (62%). Physical State: white amorphous solid. Rf = 0.13 (5:95 MeOH/

Figure 3. Rhesus monkey regional brain time−activity curves after intraveneous injection of [18F]-(R)-9 at (A) Am = 245 GBq/μmol, (B) Am = 173 GBq/μmol, and (C) Am = 15 GBq/μmol.

Figure 4. Representative in vivo PET imaging of [18F]-(R)-9 in the rhesus monkey brain at high (top panel) and low effective (lower panel) Am: CB, cerebellum; CC, corpus callosum; Ctx, cortex; TH, thalamus (10−30 min postinjection summed images).

distribution of TrkB/C. Regional uptake was most pronounced within TrkB/C-rich gray matter regions (SUVpeak of 0.9, 0.8, and 0.6 in the cerebellum, thalamus, and cortex, respectivtly, 3− 5 min postinjection) and signifcantly lower in subcortical white matter (∼0.2 SUV0−90min). Despite the similar extent of brain uptake compared to our 11C-lead, [18F]-(R)-9 was, as intended, characterized by faster brain kinetics with the rapid reach of SUVpeak in the early phase followed by apparent reversible kinetic and progressive washout over the ensuing 90 min scan (gray matter ΔSUVpeak‑90 min = 25−30%). In comparison, more potent [11C]-(R)-8 displayed steady increase in brain uptake over 60 min postinjection.27 Comparison from high to low effective Am versions of [18F](R)-9 revealed the presence of specific binding (Figure 3). 1740

DOI: 10.1021/acs.jmedchem.7b01607 J. Med. Chem. 2018, 61, 1737−1743

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CH2Cl2, UV light). HRMS (ESI+): m/z calcd for C29H31[10B]FN5O3 (M + H)+, 527.2613; found 527.2615. 1H NMR (400 MHz, CDCl3) δ = 10.70 (br s, 1H), 8.31 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 9.8 Hz, 1H), 7.61 (br d, J = 7.4 Hz, 2H), 7.32 (dt, J = 5.9, 8.0 Hz, 1H), 7.03−6.94 (m, 2H), 6.91 (br d, J = 9.5 Hz, 1H), 6.53 (br d, J = 9.8 Hz, 1H), 5.09 (br d, J = 8.1 Hz, 1H), 4.00 (br d, J = 6.2 Hz, 1H), 3.86− 3.76 (m, 1H), 2.57 (br s, 1H), 2.20−2.08 (m, 3H), 1.36 (s, 12H). 13C NMR (126 MHz, CDCl3) δ = 163.3 (d, J = 247.7 Hz, 1C), 157.1, 152.1, 144.8 (br d, J = 5.9 Hz, 1C), 140.7, 138.2, 138.1, 135.9, 130.8 (br d, J = 8.0 Hz, 1C), 127.2, 122.5, 121.2 (d, J = 2.6 Hz, 1C), 119.2, 114.7 (d, J = 21.1 Hz, 1C), 112.6 (d, J = 22.2 Hz, 1C), 111.0, 83.7, 62.1, 48.7, 35.9, 24.9, 22.7. (R)-6-(2-(3-Fluorophenyl)pyrrolidin-1-yl)-N-phenylimidazo[1,2-b]pyridazine-3-carboxamide ((R)-13). To a solution of the (R)-6-(2-(3fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazine-3-carboxylic acid (9.0 mg, 0.028 mmol), analine hydrochloride (7.0 mg, 0.054 mmol), and DIPEA (29 μL, 0.166 mmol) in 1 mL of DMF was added HBTU (15.8 mg, 0.042 mmol). After 24 h at 23 °C, the reaction was diluted with CH2Cl2 (30 mL) and washed with H2O (2 × 25 mL), brine (25 mL), and dried over anhydrous sodium sulfate. The organic solution was dried to an orange solid and purified by flash column chromatography (1 → 2% MeOH in CH2Cl2) to afford 7.1 mg of the title compound (63%). Physical state: white amorphous solid. Rf = 0.39 (5:95 MeOH/CH2Cl2, UV light). HRMS (ESI+): m/z calcd for C24H20FN5O (M + H)+, 401.1652; found 401.1646. HPLC method B (λ = 254 nm); tR = 11.4 min; purity, >99%. 1H NMR (400 MHz, CDCl3) δ = 10.60 (br s, 1H), 8.32 (s, 1H), 7.72 (d, J = 9.8 Hz, 1H), 7.58 (br d, J = 5.4 Hz, 1H), 7.37 (t, J = 7.9 Hz, 2H), 7.31 (dt, J = 5.8, J = 7.9 Hz, 1H), 7.15 (tt, J = 1.1, 7.4 Hz, 1H), 7.04−6.96 (m, 2H), 6.93 (td, J = 2.1, 9.6 Hz, 1H), 6.56 (d, J = 10.1 Hz, 1H), 5.11 (d, J = 7.9 Hz, 1H), 4.07−3.97 (m, 1H), 3.87−3.77 (m, 1H), 2.65−2.51 (m, 1H), 2.25−2.10 (m, 3H). 13C NMR (126 MHz, CDCl3) δ = 163.3 (d, J = 247.7 Hz), 157.2, 152.1, 144.8 (d, J = 6.2 Hz), 138.2, 138.0, 138.0, 130.7 (d, J = 8.0 Hz), 129.1, 127.2, 124.3, 122.6, 121.1 (d, J = 2.8 Hz), 120.4, 114.7 (d, J = 21.1 Hz), 112.6 (d, J = 22.2 Hz), 110.9, 62.1, 48.7, 35.9, 22.8. Radiochemistry. The complete descriptions of the radiochemical syntheses for in vitro studies at the McConnell Brain Imaging Center (McGill University) and in vivo studies at the University of Michigan PET Center (University of Michigan) are provided in Supporting Information. Briefly (McGill site), no-carrier-added (n.c.a) aqueous 18 − F was produced by a 18O(p,n)18F nuclear reaction on an enriched [18O]water target of the cyclotron (IBA Cyclon 18/9 MeV cyclotron, McGill site). The drying of 18F−, radiolabeling, and purification were carried out using semiautomated radiosynthesis module Scintomics GRP (Germany) equipped with a preparative HPLC (Knauer) with radioactivity and UV detector and a homemade manifold setup. [18F]F−/H2O (220−500 mCi) was passed through an unconditioned Sep-Pak Light 46 mg QMA cartridge (Waters) as an aqueous solution in 18O-enriched water from the male side. The cartridge was then flushed with methanol (3 mL), and 18F− was then eluted with 450 μL of a tetraethylammonium bicarbonate solution in methanol (1 mg/ mL) followed by methanol (500 μL) from the female side into a conical Wheaton vial (5 mL). The solvent was removed at 100 °C in vacuum, first under a stream of argon (100 mL/min), then in closed system, and the vacuum was quenched with air. The reaction vial was then charged with precursor 10 (5.2 mg, 10 μmol) and [Cu(OTf)2(pyr)4] (6.8 mg, 10 μmol) in a mixture of dimethylacetamide (500 μL) and 1-butanol (250 μL), and the mixture was allowed to react for 20 min at 110 °C. The crude mixture was then cooled, diluted with HPLC eluent (1.5 mL, 50% MeCN, 50% 50 mM NH4HCO3), and injected onto HPLC through a nylon filter to remove residual copper. Collected from HPLC [18F]-(R)-9 was diluted with 20 mL of H2O and passed through a preconditioned (5 mL of EtOH followed by 10 mL of water) Sep-Pak C18 Light cartridge and then eluted with 5 mL of EtOH and used directly in the autoradiography experiments. Quality control of the isolated [18F]-(R)-9 was performed using HPLC method B. The radiochemically pure [18F]-(R)-9 was obtained in 8% RCY (EOS, nondecay corrected isolated yield from 18F−/H2O;

nonoptimized), >99% radiochemical purity, and effective Am of 77 GBq/μmol.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01607. Supplementary tables and figures, detailed methods for biological evaluation, docking analysis, radiochemistry, in vitro autoradiography, non-human primate PET imaging, and NMR spectra (PDF) Molecular formula strings and some data (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*V.B.-G.: phone, 617-726-6869; e-mail, vbernard-gauthier@ mgh.harvard.edu. *R.S.: phone, 780-248-1829; e-mail, [email protected]. ORCID

Ralf Schirrmacher: 0000-0002-7098-3036 Present Address ×

V.B.-G.: Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital and Department of Radiology, Harvard Medical School, 55 Fruit St., White 427, Boston, Massachusetts, 02114, United States.

Author Contributions ◇

V.B.-G and A.V.M. contributed equally to the work, and P. J. H. S. and R.S. contributed equally to the work. All authors performed and/or conceptually contributed to experiments. V.B.-G and R.S. wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Gassan Massarweh, Robert Hopewell, and Dean Jolly from the Montreal Neurological Institute for radionuclide production. This work was financially supported by Canada Foundation for Innovation (CFI) Project 203639 to R.S., Cancer Research Society and C7 Council to R.S., Natural Science and Engineering Research Council of Canada (NSERC) to R.S., and Weston Brain Institute to R.S. and U.S. Department of Energy/National Institute of Biomedical Imaging and Bioengineering (Grant DE-SC0012484) to P.J.H.S.

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ABBREVIATIONS USED AM, acetoxymethyl; BCRP, breast cancer resistance protein; BODIPY, boron dipyrromethene; Ci, curie; CNS, central nervous system; HBTU, 3-[bis(dimethylamino)methyliumyl]3H-benzotriazol 1-oxide hexafluorophosphate; DIPEA, N,Ndiisopropylethylamine; DMA, dimethylacetamide; EC50, maximal effective concentration; GBq, gigabecquerel; IC50, maximal inhibitory concentration; Ki, inhibitory constant; NHP, nonhuman primate; NTRK, neurotrophic receptor kinase (gene); PET, positron emission tomography; P-gp, P-glycoprotein; SD, standard deviation; SUV, standardized uptake value; TKI, tyrosine kinase inhibitor; TLC, thin layer chromatography; Trk, tropomyosin receptor kinase 1741

DOI: 10.1021/acs.jmedchem.7b01607 J. Med. Chem. 2018, 61, 1737−1743

Journal of Medicinal Chemistry

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Brief Article

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DOI: 10.1021/acs.jmedchem.7b01607 J. Med. Chem. 2018, 61, 1737−1743