(Trk) Inhibitor for PET I - ACS Publications - American Chemical Society

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Brief Article

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 S. Sherman, Marilyn Grand'Maison, Pierre Luc Rochon, Björn Wängler, Carmen Wängler, Peter Bartenstein, Alexey Kostikov, David R. Kaplan, Gert Fricker, Pedro Rosa-Neto, Peter J. H. Scott, and Ralf Schirrmacher J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 7

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 Wängler#, Carmen WänglerΨ, Peter Bartensteinς, Alexey Kostikov∇, David R. KaplanΦ, Gert Fricker§, Pedro Rosa-Neto∥, Peter J. H. Scott‡,•,⊗, 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, MI, 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, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, Mannheim 68167, Germany. Ψ Biomedical Chemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, Mannheim 68167, Germany. ς Department of Nuclear Medicine, Ludwig-Maximilians-University of Munich, Munich, 81377, Germany. Φ 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, MI 48109, United States. ‡

KEYWORDS. Tropomyosin receptor kinase, Trk, positron emission tomography, PET, neuroimaging, fluorine-18, copper-mediated radiofluorination, Trk inhibitor.

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 non-activated aryl precursor concurrently combining largely reduced P-gp liability and improved brain kinetics compared to previous leads, while displaying both high on-target affinity and human kinome selectivity.

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 both the developing and mature central (CNS) and peripheral (PNS) nervous systems.3,4 Considerable evidence has emerged over the last 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 over-expression and activation of Trk in non-neural tissue is 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 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 anti-neoplastic kinase inhibitor drugs19,20 in the context of neurooncological malignancies. An impediment to performing these studies, however, is the lack of suitable non-destructive analytic tools applicable for imaging of patients 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 radiolabeling of a series of 6-(2-(3fluorophenyl)pyrrolidin-1-yl)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 both the nonhuman primate (NHP) and human brains in vivo, highest uptake following [11C]-(R)-8 injection was found in regions associated with pronounced


Page 2 of 7

Page 3 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry 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 towards 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 Pgp liability of our initial 11C-lead and improving brain kinetics in NHPs.

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 7.3- and 4.3 fold respectively.24 As modest reductions in Trk potencies were sought in order to ensure a new lead with faster brain kinetics in vivo, and considering that the fluoroaryl amide moiety offers a potential position for labeling, compound 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 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 (Supplementary Figure 3). We next determined inhibitory constants (Ki) towards all Trk paralogs (Supplementary 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 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 vitro biology section 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; 82-170-fold TrkB/C selectivity), ROS1 (IC50 = 46.0 nM; 148-307-fold TrkB/C selectivity) and ACK1 (IC50 = 53.7 nM; 173-358- fold TrkB/C selectivity). In addition, compound (R)-9 displayed low potencies towards TXK and PIM3 – both inhibited at an approximate IC50 value of 200 nM. Taken together, the in vitro assessments confirmed the exquisite affinity and kinome selectivity of our new fluorinated inhibitor lead.

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

Scheme 1. Synthesis of (R)-9, radiosynthesis of [18F]-(R)-9 and HPLC quality controla

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-1yl)imidazo[1,2-b]pyridazine core scaffold series. We performed a screen of our racemic 3-fluorophenyl)pyrrolidinebased Trk inhibitor library using a P-gp Calcein-AM cellular assay in order to re-prioritize those analogs based on efflux profile. This approach led to the identification of compound 9, the only derivative displaying markedly reduced P-gp interaction compared to the structurally analogous lead 8 (Figure 2D,E; Supplementary Figure 1). Whereas 8 displayed an EC50 value of 1.3 µM for P-gp, the 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, Supplementary Figure 2). Importantly, we noted that the

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

2



media 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 one 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, co-eluted 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 the light of the excellent pan-Trk potency of this derivative (IC50 for TrkA/B/C of 0.522, < 0.050 and < 0.050 nM respectively, Scheme 1, Supplementary Figure 5). The optimal preparative methods for the purification of [18F]-(R)-9 exploited a pentafluorophenyl (PFP) HPLC column which enabled complete separation of the tracer from (R)-13 (> 2 min peak separation, Scheme 1). The combination of the DMA/nBuOH reaction conditions and the PFP separation finally afforded [18F]-(R)-9 in practical isolated n.d.c. RCYs, > 99% radiochemical purity (Scheme 1) and > 100 GBq/µmol Am suitable for in vitro and in vivo experiments (see Radiochemistry section in Supporting Information).

([18F]TEAF), [Cu(OTf)2(pyr)4], DMA/nBuOH, 20 min, 110°C then PFP HPLC.

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 (ca. ≈ 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/nBuOH (2:1) as reaction

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 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. 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 Ref. 20 and Ref. 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 expressed as mean ± SD. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

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 both 2 (type I inhibitor) or 4 (type II inhibitor) led to significant reduction in the human prefrontal cortex and cerebellum (∆ = -24-36%;

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

3


Page 4 of 7

Page 5 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry (lower panel) Am. CB, cerebellum; CC, corpus callosum; Ctx, cortex; TH, thalamus (10-30 min post-injection summed images).

**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 distribution of TrkB/C. Regional uptake was most pronounced within TrkB/C-rich gray matter regions (SUVpeak 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 SUV0-90 min). 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 ∆SUV peak-90 min = 25-30 %). In comparison, more potent [11C]-(R)-8 displayed steady increase in brain uptake over 60 min postinjection.27

CONCLUSION In summary, we have described an 18F-labeled Trk radiotracer which, 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.

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, AlfaAesar 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 respectively. 1H NMR and 13C NMR spectra were recorded on an Agilent/Varian DD2 MR 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 d6-DMSO 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 × 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 × 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 reported27. The synthesis of compound (R)-9 (HPLC method A (λ = 254 nm), Rt = 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 SI Appendix. Chemistry and Radiochemistry. Chemistry. (R)-6-(2-(3Fluorophenyl)pyrrolidin-1-yl)-N-(4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)phenyl)imidazo[1,2-b]pyridazine-3carboxamide ((R)-10). DIPEA (43 mL, 0.25 mmol) was added to a solution of (R)-6-(2-(3-fluorophenyl) pyrrolidin-1yl)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

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.

Comparison from high to low effective Am versions of [18F](R)-9 revealed the presence of specific binding (Figure 3). 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.

Figure 4. Representative in vivo PET imaging of [18F]-(R)-9 in the rhesus monkey brain at high (top panel) and low effective

4



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/CH2Cl2, UV light). HRMS (ESI+): m/z calc. 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-(3Fluorophenyl)pyrrolidin-1-yl)-N-phenylimidazo[1,2b]pyridazine-3-carboxamide ((R)-13). To a solution of the (R)6-(2-(3-fluorophenyl) pyrrolidin-1-yl)imidazo [1,2b]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 DMF was added HBTU (15.8 mg, 0.042 mmol). After 24 hours at 23°C, the reaction was diluted with CH2Cl2 (30 mL) and washed with H2O (2 X 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 calc. for C24H20FN5O (M + H)+: 401.1652, found 401.1646. HPLC method B (λ = 254 nm); Rt = 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 SI Appendix. Briefly (McGill Site), no-carrieradded (n.c.a) aqueous 18F- 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 semi-automated radiosynthesis module Scintomics GRP (Germany) equipped with a preparative HPLC (Knauer) with radioactivity and UV detector and a home-made 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 18 F 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 H2O and passed through a preconditioned (5 mL EtOH followed by 10 mL water) Sep-Pak C18 Light cartridge and then eluted with 5 mL 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, non-decay corrected isolated yield from 18F-/H2O; non-optimized), > 99% radiochemical purity and effective Am of 77 GBq/µmol.

ASSOCIATED CONTENT Supporting Information Available: Supplementary tables and figures, detailed method for biological evaluation, docking analysis, radiochemistry, in vitro autoradiography, non-human primate PET imaging, NMR spectra and molecular formula strings.

AUTHOR INFORMATION Corresponding Authors *For V.B.-G.: phone, 617-726-6869; E-mail, vbernardgauthier@ mgh.harvard.edu. *For R.S.: phone, 780-248-1829; E-mail, [email protected].

ORCID Vadim Bernard-Gauthier: 0000-0002-1588-7981 Ralf Schirrmacher: 0000-0002-7098-3036 Peter J. H. Scott: 0000-0002-6505-0450 Author Contributions All authors performed and/or conceptually contributed to experiments. V.B.-G and R. S. wrote the manuscript. All authors have given approval to the final version of the manuscript. ∞ V.B.-G and A.V.M. contributed equally to the work. ⊗P. J. H. S. and R. S. contributed equally to the work. Funding This work was financially supported by Canada Foundation for Innovation (CFI) project no. 203639 to R.S, Cancer Research Society and C7 Council to RS, Natural Science and Engineering Research Council of Canada (NSERC) to RS and Weston Brain Institute to RS and US Department of Energy / National Institute of Biomedical Imaging and Bioengineering (DE-SC0012484) to PJHS. Notes The authors declare no competing financial interest. Present Address

5


Page 6 of 7

Page 7 of 7

Journal of Medicinal Chemistry ♦

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

10. Baydyuk, M.; Nguyen, M. T.; Xu, B. Chronic deprivation of TrkB signaling leads to selective lateonset nigrostriatal dopaminergic degeneration. Exp Neurol 2011, 228, 118-125. 11. Gines, S.; Bosch, M.; Marco, S.; Gavalda, N.; DiazHernandez, M.; Lucas, J. J.; Canals, J. M.; Alberch, J. Reduced expression of the TrkB receptor in Huntington's disease mouse models and in human brain. Eur. J. Neurosci. 2006, 23, 649-658. 12. Vaishnavi, A.; Le, A. T.; Doebele, R. C. TRKing down an old oncogene in a new era of targeted therapy. Cancer Discov. 2015, 5, 25-34. 13. Martin-Zanca, D.; Hughes, S. H.; Barbacid, M. A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences. Nature 1986, 319, 743-748. 14. Doebele, R. C.; Davis, L. E.; Vaishnavi, A.; Le, A. T.; Estrada-Bernal, A.; Keysar, S.; Jimeno, A.; VarellaGarcia, M.; Aisner, D. L.; Li, Y. L.; Stephens, J.; Morosini, D.; Tuch, B. B.; Fernandes, M.; Nanda, N.; Low, J. A. An oncogenic NTRK fusion in a patient with soft-tissue sarcoma with response to the tropomyosin-related kinase inhibitor LOXO-101. Cancer Discovery 2015, 5, 1049-1057. 15. Farago, A. F.; Le, L. P.; Zheng, Z.; Muzikansky, A.; Drilon, A.; Patel, M.; Bauer, T. M.; Liu, S. V.; Ou, S. H.; Jackman, D.; Costa, D. B.; Multani, P. S.; Li, G. G.; Hornby, Z.; Chow-Maneval, E.; Luo, D.; Lim, J. E.; Iafrate, A. J.; Shaw, A. T. Durable clinical response to Entrectinib in NTRK1-rearranged non-small cell lung cancer. J. Thorac. Oncol. 2015, 10, 1670-1674. 16. Dolgin, E. Loxo TRK inhibitor data wows oncologists. Nat Biotechnol. 2017, 35, 694-695. 17. Ardini, E.; Menichincheri, M.; Banfi, P.; Bosotti, R.; De Ponti, C.; Pulci, R.; Ballinari, D.; Ciomei, M.; Texido, G.; Degrassi, A.; Avanzi, N.; Amboldi, N.; Saccardo, M. B.; Casero, D.; Orsini, P.; Bandiera, T.; Mologni, L.; Anderson, D.; Wei, G.; Harris, J.; Vernier, JM.; Li, G.; Felder, E.; Donati, D.; Isacchi, A.; Pesenti, E.; Magnaghi, P.; Galvani, A. Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications. Mol Cancer Ther. 2016, 15, 628-639. 18. Cook, PJ.; Thomas, R.; Kannan, R.; de Leon, E. S.; Drilon, A.; Rosenblum, M. K.; Scaltriti, M.; Benezra, R.; Ventura, A. Somatic chromosomal engineering identifies BCAN-NTRK1 as a potent glioma driver and therapeutic target. Nat Commun. 2017, 8:15987. 19. Bailey, J. J. Schirrmacher, R.; Farrell, K.; BernardGauthier, V. Tropomyosin receptor kinase inhibitors: an updated patent review 2010-2016 Part I. Expert Opin. Ther. Patents 2017, 27, 733-751. 20. Bailey, J. J.; Schirrmacher, R.; Farrell, K.; BernardGauthier, V. Tropomyosin receptor kinase inhibitors: an updated patent review 2010-2016 Part II. Expert Opin. Ther. Patents 2017, 27, 831-849. 21. Bernard-Gauthier, V.; Boudjemeline, M.; Rosa-Neto, P.; Thiel, A.; Schirrmacher, R. Towards tropomyosinrelated kinase B (TrkB) receptor ligands for brain imaging with PET: radiosynthesis and evaluation of 2-(4[18F]fluorophenyl)-7,8-dihydroxy-4H-chromen-4-one and 2-(4-([N-methyl-(11)C]-dimethylamino)phenyl)-

(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.

ACKNOWLEDGMENT We are grateful to Gassan Massarweh, Robert Hopewell and Dean Jolly from the Montreal Neurological Institute for radionuclide production.

ABBREVIATIONS 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, non-human primate, NTRK, neurotrophic receptor kinase (gene); PET, positron emission tomography; P-gp, Pglycoprotein, SD, standard deviation; SUV, standardized uptake value, TKI, tyrosine kinase inhibitor; TLC, thin layer chromatography; Trk, tropomyosin receptor kinase.

REFERENCES 1. 2.

3.

4.

5.

6.

7.

8.

9.

Deinhardt K, C. M. Trk receptors. Handb Exp Pharmacol. 2014, 220, 103-119. Chao, M. V. Neurotrophins and their receptors: a convergence point for many signalling pathways. Nat Rev Neurosci 2003, 4, 299-309. Martin-Zanca, D.; Barbacid, M.; Parada, L. F. Expression of the Trk protooncogene is restricted to the sensory cranial and spinal ganglia of neural crest origin in mouse development. Gene Dev 1990, 4, 683-694. Klein, R.; Parada, L. F.; Coulier, F.; Barbacid, M. Trkb, a novel tyrosine protein-kinase receptor expressed during mouse neural development. Embo Journal 1989, 8, 3701-3709. Allen, S. J.; Wilcock, G. K.; Dawbarn, D. Profound and selective loss of catalytic TrkB immunoreactivity in Alzheimer's disease. Biochem Bioph Res Co 1999, 264, 648-651. Ginsberg, S. D.; Che, S. L.; Wuu, J.; Counts, S. E.; Mufson, E. J. Down regulation of trk but not p75(NTR) gene expression in single cholinergic basal forebrain neurons mark the progression of Alzheimer's disease. J. Neurochem. 2006, 97, 475-487. Savaskan, E.; Muller-Spahn, F.; Olivieri, G.; Bruttel, S.; Otten, U.; Rosenberg, C.; Hulette, C.; Hock, C. Alterations in trk A, trk B and trk C receptor immunoreactivities in parietal cortex and cerebellum in Alzheimer's disease. Eur Neurol 2000, 44, 172-180. Baydyuk, M.; Nguyen, M. T.; Xu, B. Chronic deprivation of TrkB signaling leads to selective lateonset nigrostriatal dopaminergic degeneration. Exp Neurol 2011, 228, 118-125. Haque, N. S.; Hlavin, M. L.; Fawcett, J. W.; Dunnett, S. B. The neurotrophin NT4/5, but not NT3, enhances the efficacy of nigral grafts in a rat model of Parkinson's disease. Brain Res 1996, 712, 45-52.

6



22.

23.

24.

25.

26.

27.

28.

7,8-dihydroxy-4H-chromen-4-one. Bioorg Med Chem. 2013, 21, 7816-7829. Bernard-Gauthier, V.; Schirrmacher, R. 5-(4-((4[18F]fluorobenzyl)oxy)-3-methoxybenzyl)pyrimidine2,4-diamine: A selective dual inhibitor for potential PET imaging of Trk/CSF-1R. Bioorg Med Chem Lett 2014, 24, 4784-4790. Bernard-Gauthier, V.; Aliaga, A.; Aliaga, A.; Boudjemeline, M.; Hopewell, R.; Kostikov, A.; RosaNeto, P.; Thiel, A.; Schirrmacher, R. Syntheses and evaluation of carbon-11 and fluorine-18-radiolabeled pan-tropomyosin receptor kinase (Trk) inhibitors: exploration of the 4-aza-2-oxindole scaffold as Trk PET imaging agents. ACS Chem. Neurosci. 2015, 6, 260-276. Bernard-Gauthier, V.; Bailey, J. J.; Aliaga, A.; Kostikov, A.; Rosa-Neto, P.; Wuest, M.; Brodeur, G. M.; Bedell, B. J.; Wuest, F.; Schirrmacher, R. Development of subnanomolar radiofluorinated (2pyrrolidin-1-yl)imidazo[1,2-b]pyridazine pan-Trk inhibitors as candidate PET imaging probes. MedChemComm 2015, 6, 2184-2193. Bernard-Gauthier, V.; Mahringer, A.; Vesnaver, M.; Fricker, G.; Schirrmacher, R. Design and synthesis of a fluorinated quinazoline-based type-II Trk inhibitor as a scaffold for PET radiotracer development. Bioorg. Med. Chem. Lett. 2017, 27, 2771-2775. Taylor, N. J.; Emer E.; Preshlock, S.; Schedler, M.; Tredwell, M.; Verhoog, S.; Mercier, J.; Genicot, C.; Gouverneur, V. Derisking the Cu-mediated 18Ffluorination of heterocyclic positron emission tomography radioligands. J Am Chem Soc. 2017, 139, 8267-8276. Bernard-Gauthier, V.; Bailey, J. J.; Mossine, A. V.; Lindner, S.; Vomacka, L.; Aliaga, A.; Shao, X.; Quesada, C. A.; Sherman, P.; Mahringer, A.; Kostikov, A.; Grand’Maison, M.; Rosa-Neto, P.; Soucy, J.-P.; Thiel, A.; Kaplan, D. R.; Fricker, G.; Wängler, B.; Bartenstein, P.; Schirrmacher, R.; Scott, P. J. H. A Kinome-wide selective radiolabeled TrkB/C inhibitor for in vitro and in vivo neuroimaging: synthesis, preclinical evaluation and first-in-human. J. Med. Chem. 2017, 60, 6897-6910. Choi, H. S.; Rucker, P. V.; Wang, Z. C.; Fan, Y.; Albaugh, P.; Chopiuk, G.; Gessier, F.; Sun, F. X.; Adrian, F.; Liu, G. X.; Hood, T.; Li, N. X.; Jia, Y.; Che, J. W.; McCormack, S.; Li, A.; Li, J.; Steffy, A.; Culazzo, A.; Tompkins, C.; Phung, V.; Kreusch, A.; Lu, M.; Hu, B.; Chaudhary, A.; Prashad, M.; Tuntland, T.; Liu, B.; Harris, J.; Seidel, H. M.; Loren, J.; Molteni, V. (R)-2Phenylpyrrolidine substituted imidazopyridazines: A

29.

30.

31.

32.

33.

34.

35.

36.

37.

new class of potent and selective pan-TRK inhibitors. ACS Med. Chem. Lett. 2015, 6, 562-556. Tredwell, M.; Preshlock, S. M.; Taylor, N.J.; Gruber, S.; Huiban, M.; Passchier, J.; Mercier, J.; Genicot, C.; Gouverneur, V. A general copper-mediated nucleophilic 18F fluorination of arenes. Ang. Chem. Int. Ed. 2014, 53, 7751-7755. Mossine, A. V.; Brooks, A. F.; Makaravage, K. J.; Miller, J. M.; Ichiishi, N.; Sanford, M. S.; Scott, P. J. H. Synthesis of [18F]arenes via the copper-mediated [18F]fluorination of boronic acids. Org. Lett. 2015, 17, 5780-5783. Mossine, A. V.; Brooks, A. F.; Ichiishi, N.; Makaravage, K. J.; Sanford, M. S.; Scott, P. J. H. Development of customized [18F]fluoride elution techniques for the enhancement of copper-mediated late-stage radiofluorination. Sci. Rep. 2017, 7, 233. Zlatopolskiy, B. D.; Zischler, J.; Krapf, P.; Zarrad, F.; Urusova, E. A.; Kordys, E.; Endepols, H.; Neumaier, B. Copper-mediated aromatic radiofluorination revisited: Efficient production of PET tracers on a preparative scale. Chem. Eur. J. 2015, 21, 5972-5979. Zischler, J.; Kolks, N.; Modemann, D.; Neumaier, B. Zlatopolskiy, B. D. Alcohol-enhanced Cu-mediated radiofluorination. Chem. Eur. J. 2017, 23, 3251-3256. Mossine, A. V.; Brooks, A. F.; Bernard-Gauthier, V.; Bailey, J. J.; Ichiishi, N.; Schirrmacher, R. Sanford, M. S.; Scott, P. J. H. Automated synthesis of PET radiotracer by copper-mediated 18F-fluorination of organoborons: Importance of the order of addition and competing protodeborylation. J. Label. Compd. Radiopharm. 2017, doi: 10.1002/jlcr.3583. Wood, E. R.; Kuyper, L.; Petrov, K. G.; Hunter, R. N. 3rd; Harris, P. A.; Lackey, K. Discovery and in vitro evaluation of potent TrkA kinase inhibitors: oxindole and aza-oxindoles. Bioorg. Med. Chem. Lett. 2004, 14, 953-957. Davis, M. I.; Hunt, J. P.; Herrgard, S.; Ciceri, P.; Wodicka, L. P.; Pallares, G.; Hocker, M.; Treiber, D. K.; Zarrinkar, P. P. Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol. 2011, 29, 10461051. Elkins, J. M.; Fedele, V.; Szklarz, M.; Abdul Azeez, K. R.; Salah, E.; Mikolajczyk, J.; Romanov, S.; Sepetov, N.; Huang, X.-P.; Roth, B. L.; Haj Zen, A. A.; Fourches, D.; Muratov, E.; Tropsha, A.; Morris, J.;Teicher, B. A.; Kunkel, M.; Polley, E.; Lackey, K. E.; Atkinson, F. L.; Overington, J. P.; Bamborough, P.; Müller, S.; Price, D. J.; Willson, T. M.; Drewry, D. H.; Knapp, S.; Zuercher, W. J. Comprehensive characterization of the published kinase inhibitor set. Nat Biotechnol. 2016, 34, 95-103.

Insert Table of Contents artwork here

7


Page 8 of 7

(Trk) Inhibitor for PET I - ACS Publications - American Chemical Society

Recommend Documents