Identification of Three Novel Radiotracers for Imaging Aggregated Tau

Article pubs.acs.org/jmc

Identification of Three Novel Radiotracers for Imaging Aggregated Tau in Alzheimer’s Disease with Positron Emission Tomography Luca C. Gobbi,*,† Henner Knust,† Matthias Körner,† Michael Honer,† Christian Czech,† Sara Belli,† Dieter Muri,† Martin R. Edelmann,† Thomas Hartung,† Isabella Erbsmehl,† Sandra Grall-Ulsemer,† Andreas Koblet,† Marianne Rueher,† Sandra Steiner,† Hayden T. Ravert,‡ William B. Mathews,‡ Daniel P. Holt,‡ Hiroto Kuwabara,‡ Heather Valentine,‡ Robert F. Dannals,‡ Dean F. Wong,‡,§,∥ and Edilio Borroni† †

Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland Department of Radiology, §Department of Psychiatry, and ∥Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States

‡

S Supporting Information *

ABSTRACT: Aggregates of tau and beta amyloid (Aβ) plaques constitute the histopathological hallmarks of Alzheimer’s disease and are prominent targets for novel therapeutics as well as for biomarkers for diagnostic in vivo imaging. In recent years much attention has been devoted to the discovery and development of new PET tracers to image tau aggregates in the living human brain. Access to a selective PET tracer to image and quantify tau aggregates represents a unique tool to support the development of any novel therapeutic agent targeting pathological forms of tau. The objective of the study described herein was to identify such a novel radiotracer. As a result of this work, we discovered three novel PET tracers (2-(4-[11C]methoxyphenyl)imidazo[1,2a]pyridin-7-amine 7 ([11C]RO6924963), N-[11C]methyl-2-(3-methylphenyl)imidazo[1,2-a]pyrimidin-7-amine 8 ([11C]RO6931643), and [18F]2-(6-fluoropyridin-3-yl)pyrrolo[2,3-b:4,5-c′]dipyridine 9 ([18F]RO6958948)) with high affinity for tau neurofibrillary tangles, excellent selectivity against Aβ plaques, and appropriate pharmacokinetic and metabolic properties in mice and non-human primates.

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plaques, sharing a similar cross β-sheet fibrillary core structure. The spreading of tau pathology in AD is well documented and follows a predictable pattern, in terms of both timing and spatial localization.3 As of today, no cure exists for AD that represents the most common reason for dementia. The increasing prevalence of AD related to aging populations, in particular in Western countries, makes it a major reason for concern for society and a topic for intensive research.4 Evidence is emerging in recent years for tau NFTs but not Aβ plaques to be indicators of clinical symptoms progression, despite the fact of Aβ pathology temporally preceding the one of tau.5 Thus, several novel therapeutic

INTRODUCTION The microtubule-associated protein tau is highly expressed in neurons, where it has an important function in the assembly and stabilization of axonal microtubules. Six isoforms of tau exist in the adult human brain, originating from a common gene by alternative splicing and varying between 352 to 441 amino acids in length.1 The native soluble forms of the tau protein exhibit predominantly a random coil conformation. By contrast, in certain types of neurological disorders, tau is encountered in protein aggregates possessing a cross β-sheet amyloid core structure.2 Most prominent among these neurological disorders is Alzheimer’s disease (AD), which is characterized by the presence of tau aggregates in the brain in form of neurofibrillary tangles (NFTs) and neuropil threads (NTs).3 The second protein aggregates present in AD consist of amyloid-β (Aβ) © 2017 American Chemical Society

Received: April 27, 2017 Published: June 27, 2017 7350

DOI: 10.1021/acs.jmedchem.7b00632 J. Med. Chem. 2017, 60, 7350−7370

Journal of Medicinal Chemistry

Article

Figure 1. Structures of PET tracers for imaging of tau aggregates reported to have entered clinical evaluation studies.

Scheme 1. Synthesis of Imidazo[1,2-a]pyridines 7 and 16−22a

a Reagents and conditions. (a) For 12, R1 = NHMe: 40% aqueous MeNH2, 160 °C, 4 h, 75%. (b) For 7, 18: 14 or 15, NaHCO3, MeOH or EtOH, reflux, 4−6 h, 22−35%. For 16, 17: 13, acetone, 65 °C, 5 h, 44−90%. (c) NaH, DMF, 30 min, then MeI, rt, 18 h, 6%. (d) LiHMDS, THF, 0 °C, 25 min, then Boc2O, rt, 1.5 h, 66%. (e) NaH, DMF, rt, 15 min, then 1-bromo-2-fluoroethane, 50 °C, 1 h, 95%. (f) TFA, DCM, rt, 3 h, 93%.

(Figure 1).10,11 More recently several novel structures of selective tau tracers were reported, the most prominent being 2 (flortaucipir, formerly known as [18F]AV-1451 and [18F]T807),12−14 3 ([18F]T808),15,16 4 ([18F]THK5351),17 5 ([11C]PBB3),18,19 and 6 ([18F]MK-6240).20,21 During running of an internal program to secure access to a selective tau PET tracer, the initial results on [18F]2 and [18F]3 were disclosed.12,13,15 The two structures were deemed as promising starting points for a PET tracer discovery program. We herein wish to report on our work that has culminated in the identification of the three novel and selective tau radiotracers [11C]7 ([11C]RO6924963),22 [11C]8 ([11C]RO6931643),23 and [18F]9 ([18F]RO6958948).24

approaches to cure or at least modify the progression of AD are focusing on tau and on preventing the spreading of aggregated tau species or clearing of existing NFTs and NTs. The development of any such new therapy would greatly benefit from the existence of a sensitive biomarker able to monitor tau pathology in the living brain. In particular, this would allow for patient selection and stratification as well as collection of longitudinal data demonstrating the efficacy of any novel antitau therapeutic agent at limiting tau pathology. Moreover, such a biomarker would represent an invaluable diagnostic tool for monitoring AD. Recently, a number of positron emission tomography (PET) tracers targeting aggregated forms of tau have been identified.6−8 Such radiotracers enable for the first time the quantitative monitoring of tau aggregates in the living human brain.9 For any novel tau PET tracer, selectivity over Aβ plaques is mandatory because of the mixed nature of pathology in AD. The first clinically evaluated radiotracer in this field was 1 ([18F]FDDNP), a ligand possessing high affinity for Aβ plaques and thus of limited use as tau aggregates biomarker

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RESULTS AND DISCUSSION Amyloid aggregates are known to possess different binding sites on the cross-β sheet fibrils surface.25−27 Caution is thus mandatory when interpreting the results of any competitive binding assay, as the addressed binding site depends on the reporter ligand that is used. Our strategy to find novel high 7351



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Scheme 2. Synthesis of Imidazo[1,2-a]pyrimidines 8 and 29−37 and Imidazo[1,2-c]pyrimidine 40a

Reagents and conditions. (a) 25: NH2Me·HCl, K2CO3, NMP, 120 °C, 2 h, 40%. 26: NHMe2·HCl, K2CO3, NMP, 120 °C, 1 h, 43%. 27: NH2cPr, K2CO3, DMF, 100 °C, 18 h, 55%. 28: 4-(2-fluoroethyl)piperidine·HCl, K2CO3, NMP, 120 °C, 12 h, 78%. (b) 8 and 29−36: α-Br-acetophenones, acetone, 60−65 °C, 5−18 h, 35−94%. Or 36: α-Br-acetophenone, pTsOH, acetone, 65 °C, 12 h, 25%. (c) 1-Bromo-2-fluoroethane, Cs2CO3, DMF, 70 °C, 1 h, 55%. (d) (TsOCH2)2, Cs2CO3, DMF, 50 °C, 2 h, 41%. (e) α-Br-acetophenone, EtOH, 80 °C, 8 h, 13%.

a

Scheme 3. Synthesis of Pyrrolo[2,3-b:4,5-c′]dipyridines 9 and 49a

Reagents and conditions: (a) [Pd(OAc)2], PPh3, Et3N, DMF, 100 °C, 3 h, ∼90% purity, 63%. (b) K2CO3, 18-crown-6, DMF, 100 °C, 3 h, 63%. (c) NaH, DMF, 0 °C to rt, then Boc2O, DMF, 0 °C to rt, 18 h, 73%. (d) [PdCl2(dppf)]·CH2Cl2, K2CO3, DMF, 90 °C, 17−18 h. 47: 10%. 49: 13%. (e) TFA, CH2Cl2, rt, 18 h, 88%. a

heteroatoms in the aromatic rings was investigated to reduce the lipophilicity, thus aiming at improving the specific to nonspecific binding ratio. The optimal range for the properties of a PET tracer for use in the central nervous system has been extensively reviewed in the past few years, and this information guided our design of novel molecules.28−30 Synthesis of Nonlabeled Compounds. The synthesis of imidazo[1,2-a]pyridines typically started from 2-amino-4chloropyridine (10) by reaction with an appropriate amine (Scheme 1). While 2,4-diaminopyridine (11) was commercially available, reaction of 10 with methylamine produced N4methyl-2,4-diaminopyridine (12). Condensation of 11 and 12 with α-bromoacetophenones 13−15 afforded imidazo[1,2a]pyridines 7 and 16−18. Direct N-alkylation of 16 to give

affinity tau NFT ligands was to build a novel medium to high throughput assay based on the displacement of [3H]3 from brain sections originating from AD donors. Ligand 3 has been described to possess high affinity of Kd = 22 nM for tau aggregates as encountered in AD.16 New chemical structures were derived from 3 and 2 following the hypothesis that related scaffolds would share a common binding site on the amyloid fibril surface. Early in the program we demonstrated 2 to indeed compete for binding with [3H]3 (vide infra). For assessing selectivity of the most promising candidates against Aβ plaques, we relied on low throughput, direct binding assays with tritiated material. Of particular importance for rapidly progressing the project was the simplification of the tricyclic aromatic core of the molecules. In addition, incorporation of 7352



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Scheme 4. Synthesis of 9H-Pyrimido[4,5-b]indole 55a

Reagents and conditions: (a) EtOH, reflux, 3 h, 70%. (b) [Pd(OAc)2(PPh3)2], NaOAc, DMA, microwave 130 °C, 4 h, 11%. (c) [PdCl2(dppf)]· CH2Cl2, K2CO3, DMF, microwave 100 °C, 1 h, 20%. a

Scheme 5. Synthesis of 5H-Pyrido[4,3-b]indole 62a

Reagents and conditions: (a) BF3·Et2O, 120 °C, 16 h, 69% (mixture of regioisomers). (b) NaH, DMF, 0 °C to rt, 30 min, then TsCl, rt, 1 h, 21%. (c) [Pd2(dba)3]·CHCl3, xantphos, Cs2CO3, THF, 90 °C, 16 h, 76%. (d) NaOH, THF/H2O, 50 °C, 20 h, then NaOH, MeOH, reflux 6 h, 25%.

a

Complete loss of the Boc protective group was observed during the Suzuki cross-coupling reaction leading to nitro derivative 49, while the synthesis of 9 under identical conditions led to a mixture of free and N-Boc protected material, thus requiring an additional cleavage step. Full consumption of starting material 45 was observed in the cross-coupling reactions leading to 47 and 49. The modest yields for these conversions were mainly originating from the formation of side products and from the poor solubility of the final compounds, which complicated their isolation. To explore structural variations of the tricyclic core of 9, one of the nitrogen atoms was shifted in the scaffold (Scheme 4). Starting from 4-chloro-5-iodopyrimidine (50) and 3-bromoaniline (51), intermediate 52 was obtained by nucleophilic aromatic substitution (Scheme 4). The intramolecular Pdcatalyzed direct arylation gave the tricyclic core of compound 5331 that was reacted under Suzuki cross-coupling conditions with boronic acid 54 to lead to the final product 55 in moderate yield. Protection of the pyrrole-NH was not required for this step. Further modification of 2 was investigated by replacing the 6fluoro-4-pyridin-3-yl moiety with unsaturated fluorinated, cyclic amines (Scheme 5). Thus, 1-acetyl-3-bromopiperidin-4-one (56) and (3-bromophenyl)hydrazine hydrochloride (57) were reacted in the presence of BF3·Et2O, leading to the formation of an inseparable mixture of regioisomers having the bromo atom in position 7 (58a) or 9 (58b) of the 5H-pyrido[4,3-b]indole scaffold. N-Tosylation to form 59 allowed for the chromatographic isolation of the desired regioisomer. Buchwald amination of 59 with 4-fluoropiperidine (60) resulted in the

the dimethylamino derivative 19 proceeded in poor yield (6%) under formation of a mixture with the monoalkylated compound 17. No attempt was made to optimize this step. Mono-N-alkylation of 16 with a fluoroethyl group was best achieved in a three-step procedure via the Boc intermediates 20 and 21, thus leading to the final product 22. Commercially available 4-chloropyrimidin-2-amine (23) or pyrimidine-2,4-diamine (24) (Scheme 2) served as starting points for the synthesis of imidazo[1,2-a]pyrimidines. Where necessary, aromatic nucleophilic substitution of 4-chloropyrimidin-2-amine (23) was used in the first step to introduce the amine substituents in position R1 to give intermediates 25−28. Condensation of 24−28 with appropriately substituted αbromoacetophenones in acetone afforded the final products 8 and 29−36. Phenol 33 was further elaborated by alkylation to the fluoroethoxy derivative 37 or it is precursor for radiofluorination, tosylate 38. Condensation of 4,6-diaminopyrimidine (39) and αbromoacetophenone in EtOH at 80 °C afforded in one step the regioisomeric imidazo[1,2-c]pyrimidine 40 (Scheme 2). The initial synthesis of the tricyclic pyrrolo[2,3-b:4,5c′]dipyridine core was achieved in two steps, beginning with a Suzuki cross-coupling of iodide 41 with boronic acid 42 leading to intermediate 43 (Scheme 3). Ring closure of 43 by intramolecular nucleophilic substitution was accompanied by concomitant loss of the Boc protective group, thus resulting in tricyclic intermediate 44. Reprotection with Boc anhydride to obtain 45 was beneficial for further transformations of this compound via Suzuki cross-coupling reaction toward both the α-fluoropyridine 47 and the corresponding nitro derivative 49, which represents the radiofluorination precursor of [18F]9. 7353



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Table 1. Results for the Determination of Affinity,a Lipophilicity (log D), Brain Lipid Membrane Binding (LIMBA log Dbrain), and Passive Membrane Permeability (PAMPA)

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Table 1. continued

a

Displacement of [3H]3 (10 nM) from fresh frozen human brain sections derived from AD cases; test concentration = 10 nM. bDetermined at pH = 7.4. cNo data determined on account of the poor affinity for tau aggregates.

tosyl protected 5H-pyrido[4,3-b]indole 61. The desired final product 62 was obtained after cleavage of the tosyl group. In Vitro Optimization of Binding Affinity and Physicochemical Properties. In order to test the affinity of novel compounds toward protein tau aggregates possessing the polymorphic form relevant for AD pathology, we set up a novel assay based on the displacement of [3H]3 from immunohistologically characterized fresh frozen human brain sections derived from AD cases. Tritiation of 3 is described in the Supporting Information and delivered the radioligand in 99% radiochemical purity and a specific activity of 1.49 TBq/mmol (40.3 Ci/mmol). Novel compounds were tested at a concentration of 10 nM (results in Table 1). As expected, both 2 and 3 demonstrated good affinity by displacing 41% and 40% of radioligand ([3H]3) binding, respectively. The affinity of 3 for tau aggregates was previously reported with Kd = 22 nM,16 thus in a comparable range as found with our new assay. In addition to the primary binding affinity assay, lipophilicity (log D) and passive cell membrane permeability (Peff in the

PAMPA assay32) were determined for all compounds and used to guide the optimization program. We complemented the standard octanol/water partition coefficient log D results with the measurement of LIMBA log Dbrain in the novel brain lipid membrane binding assay LIMBA.33,34 2 was found to be rather lipophilic, with log Doctanol = 3.28, and possessed a moderate passive permeability Peff = 1.8 × 10−6 cm s−1. By contrast, 3 had a lower log Doctanol = 2.80 and a higher Peff = 4.6 × 10−6 cm s−1. The initial goals for optimization were an increase in affinity combined with a decrease in lipophilicity to augment the specific to nonspecific binding ratio in a PET experiment and obtain a PAMPA Peff > 2 × 10−6 cm s−1 to provide high and fast brain uptake. Initial attempts to simplify the tricyclic core of 3 by replacement with a bicyclic imidazo[1,2-a]pyrimidine, flanked by an aromatic ring in position 2, led to the observation that very simple structures possessing a small amino group in position 6 of the bicyclic heteroaromatic ring system retained moderate to good affinity (29−31) (Table 1). Interestingly and 7355



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Table 2. Mouse Single-Dose PK Parameters in Plasma (iv Bolus Administration), in Vitro P-gp Efflux Ratios (ER), and Plasma Free Fractions (f u_p) of Compounds 3, 7−9, 22, 34, and 37 P-gp ER compd

iv dose [mg/kg]

terminal t1/2 [h]

total plasma Cl [mL min−1 kg−1]

Vss [L/kg]

brain/plasma total concn ratio (AUCa)

3 7 8 9 22 34 37

1.1 0.9 1.1 1.3 0.8 1.0 1.0

0.30 0.24 0.27 0.39 0.43 0.39 0.20

95 250 114 207 541 84 83

1.4 4.2 1.9 5.2 13.5 2.7 1.2

2.0 2.1 1.7 3.6 3.6 1.3 0.5

a

human 1.1 1.1 1.0 1.1 1.1 1.7

f u_p [%]

mouse

human

mouse

1.1 1.5 0.9 1.3 1.2 1.9

1.4 24 12 7 16 14 17

27 10 12 17 11 14

AUC: area under the concentration−time curve.

log D would have erroneously flagged several compounds with log D > 3 to display high nonspecific binding.29,30,34 During the course of the program, approximately 550 novel compounds originating from the structural classes discussed above were prepared and tested. In the light of the favorable in vitro properties and a reasonably good predicted chance to incorporate a 11C or 18F radionuclide, the six lead tracer molecules 7−9, 22, 34, and 37 were selected for further profiling by in vitro autoradiography of 3H-labeled material, in depth pharmacokinetic (PK) evaluation, and PET imaging in non-human primates. In Vitro Transport and in Vivo Pharmacokinetic Profile. The in vitro transport and in vivo drug pharmacokinetic profiles for the reference 3 and the six lead tracer candidates 7−9, 22, 34, and 37 are summarized in Table 2. Pharmacokinetics in mice after single intravenous (iv) bolus administration were studied at a relatively low dose (∼1 mg/ kg) to better reflect the microdosing conditions encountered in a human PET experiment, yet without compromising the quantification of the compound concentration in the biological samples. As reported in Table 2, the tested compounds showed high systemic plasma clearance in mouse in vivo studies typically exceeding liver blood flow, and short terminal plasma half-lives ranging between 0.20 > t1/2 > 0.43 h, thus being appropriate for use with a carbon-11 (isotope decay t1/2 = 20 min) or fluorine-18 (t1/2 = 110 min) label. By use of physiologically based PK modeling that integrates drug physicochemical properties, animal/human in vitro input, and in vivo PK data from various species (data not shown), compounds 7−9 were expected to undergo high systemic clearance in humans. The same model predicted a short terminal half-life of ∼2 h and a large tissue distribution, all properties that were considered appropriate for a PET tracer. Both in vitro transport and in vivo brain uptake studies in animals (methods in Supporting Information) confirmed the compounds not to be recognized by the efflux transporter Pglycoprotein (P-gp, mouse and human), to possess high passive cellular and PAMPA permeability and large distribution volume in vivo, and were therefore predicted to reach rapidly the brain in human. Free fractions (f u_p, method in Supporting Information) for the new tracer candidates in mouse and human plasma were high, in general >10%, in contrast to the reference compound 3. High f u_p was deemed to be of advantage in order to facilitate rapid in vivo clearance of the ligands from nonspecifically bound and free compartments, both contributing to confound the specific binding component in the PET images.36 Furthermore, the high f u_p values together with the lack of

to our surprise a direct analogue of 3, compound 36, had only very modest affinity (14% ligand displacement). These novel structures had an optimal log D (1.42, 2.13, and 2.33 for 29, 30, and 31, respectively), possessed excellent passive membrane permeability in the PAMPA assay (Peff ≥ 4.2 × 10−6 cm s−1), and had low molecular weight in the range of 210−238 g/mol, indicative of a high ligand efficiency LE.35 On the basis of these favorable properties, a large optimization program was started on this novel structural class. Attempts to shift one of the nitrogen atoms in the bicyclic ring to a different position led to compound 40 that had a similar affinity compared to 29. To our delight, removal of one of the ring nitrogen atoms of 29, giving imidazo[1,2-a]pyridine 16, further increased affinity (58% ligand displacement vs 40% for reference 3). Methylation of the amino group of 16 resulted in 17 and 19 which displayed similar high affinity (60% and 59% ligand displacement, respectively). All these novel imidazo[1,2-a]pyridines possessed appropriate lipophilicity (log D ≤ 2.64) in combination with excellent passive membrane permeability (Peff ≥ 4.3 × 10−6 cm s−1). Further modification of the lead structures of 29 (imidazo[1,2-a]pyrimidine) and 16 (imidazo[1,2-a]pyridine) to introduce positions for carbon-11 or fluorine-18 labeling finally led to the identification of compounds 7, 8, 22, 34, and 37. When compared to 3, these five ligands possessed comparable or better affinity for tau aggregates in combination with lower lipophilicity and thus were predicted to display a more favorable specific to nonspecific binding ratio (SB/NSB). All these structures exhibited very good passive membrane permeability in the PAMPA assay. In parallel to the work around leads 16 and 29 derived from 3, attempts were made to modify the structure and improve the properties of 2. Replacement of the 6-fluoropyridin-4-yl ring of 2 with several aliphatic amines was found to be detrimental, as exemplified by the 4-fluoropiperidine derivative 62. Introduction of an additional nitrogen atom in the tricyclic core of 2 failed to give the desired properties in the case of final compound 55 but was successful for the regioisomeric derivative 9. 9H-Dipyrido[2,3-b;3′,4′-d]pyrrole 9 maintained the high affinity (44% ligand displacement) in combination with a slight reduction of the lipophilicity (log D = 3.22). Passive membrane permeability of 9 was found to be good (PAMPA Peff = 3.8 × 10−6 cm s−1). A modest linear correlation was found between the results for log D and LIMBA log Dbrain (LIMBA log Dbrain = 0.27 + (0.42 log D); r2 = 0.32). Interestingly LIMBA log Dbrain predicted all compounds to possess low nonspecific binding (i.e., LIMBA log Dbrain < 2.0). By contrast, prediction based on 7356



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In Vitro Autoradiography of Tritiated Compounds and Selectivity Testing by Antibody Colocalization Studies. All tritiated compounds were evaluated by in vitro autoradiography using native fresh-frozen brain sections originating from late-stage AD patients (with high tau load) and healthy control subjects (devoid of tau pathology) in order to evaluate (i) tracer binding to tau aggregates, (ii) nonspecific binding (NSB), and (iii) lack of binding to other targets, in particular to Aβ plaques. Analogous experiments performed on brain sections originating from two transgenic mouse tauopathy models, Tg4510 and TauPS2APP, revealed a lack of binding of the radioligands to the forms of tau present in these animals. Results for the reference [3H]3 are included here for comparison, although extensive characterization and validation of this tool compound was done earlier in the project. The results are summarized in Figure 2. All radioligands were tested at a concentration of 3 nM. On first visual inspection of the macroscopic autoradiographies, all novel radioligands displayed a binding pattern in AD tissue sections similar to the reference [3H]3. This binding pattern in cortical tissue sections is characterized by a layered distribution and correlates with the distribution of tau aggregates (neurofibrillary tangles and neuropil threads) as visualized by immunohistochemical analysis with a tau-specific antibody (Figure 3A). Specificity of radioligand binding to tau aggregates and absence of significant off-target binding to any other CNS target were suggested by very low binding to cortical tissue sections from healthy control subjects (Figure 2, bottom row). Among the six candidates some differences in NSB levels in healthy control tissue were observed. [3H]9 displayed the lowest NSB among all tracer candidates. Specific binding (SB) to tau aggregates was assessed by quantifying the difference in binding to tau-rich cortical gray matter (GM) in AD tissue versus tau-poor cortical tissue of healthy controls. Highest SB values of 9726 fmol/mg protein were identified for [3H]7 followed by [3H]9 and [3H]8 (SB = 5905 and 5500 fmol/mg protein, respectively). Overall, SB correlated well with the affinity of the compounds as determined by the competition of [3H]3 binding (Table 1). The binding of all six radioligand candidates in advanced AD (Braak V staging3) tissue sections was also characterized by low NSB in white matter (WM) yielding excellent signal-to-noise ratios (SNR) and image contrast in vitro. The SNR was quantitatively assessed by calculating GM over WM ratios of radioligand binding to AD Braak V cortical tissue sections. All compounds possessed favorable GM/WM ratios above or equal

interaction with P-gp and high in vitro permeability were considered to be beneficial in promoting rapid brain uptake and large unbound fraction/low nonspecific binding in brain.37,38 In fact, most compounds possessed total brain to plasma AUC ratios (B/P) in rodents of >1, with the exception of 37. A B/P of 0.5 for the latter compound was deemed to be still sufficient to progress 37 into further tests. Tritiation of Lead Tracer Molecules. In order to allow for a detailed and high resolution in vitro autoradiographical characterization of the novel tracer candidates, the molecules were initially labeled with tritium. Five out of the six compounds were labeled by a hydrogen−tritium exchange reaction directed by the adjacent nitrogen-containing heterocycles. The well-known Crabtree’s catalyst ([Ir(COD)(PCy3)(Py)]PF6) was used in superstoichiometric amounts to promote the labeling and achieve high specific activity (Scheme 6). Thus, [3H]7, [3H]9, [3H]22, [3H]34, and [3H]37 were Scheme 6. Representative Tritiations As Exemplified for the Preparation of [3H]8, [3H]9, and [3H]37a

a

Reagents and conditions: (a) [3H]MeONs, Cs2CO3, toluene, rt, 24 h. (b) [3H]H2, [Ir(COD)(PCy3)(Py)]PF6, DCM/DMF, rt, 4 h.

obtained in specific activities ranging from 900 to 2000 GBq/ mmol. The position of the label was assigned based on mechanistic considerations, knowing that C−H activation and isotope exchange require the proximity of a heteroatom for complexation of the Ir atom and preferably proceed via fivemembered cyclometalated IrIII species.39 Radioligand [3H]8 was prepared in high specific activity (SA = 2600 GBq/mmol) by methylation of 32 with [3H]methylnosylate.

Figure 2. In vitro autoradiography of the reference tracer [3H]3 and all six test ligands (7−9, 22, 34, and 37) in tritiated form using native cortical tissue sections from an AD Braak V patient (top row) and a healthy control subject (bottom row). The radioligand concentration was 3 nM for all compounds. 7357



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Figure 3. Colocalization of tau mAb pS42240 (A) and Aβ mAb BAP-241 (C) immunohistochemical staining with autoradiographical tracer binding (B) of [3H]8, as a representative example. Staining and binding experiments were performed on a single fresh-frozen tissue section from an AD Braak V cortical tissue block. The detail (D) shows the autoradiography superimposed with tau mAb (red) and Aβ mAb staining (green). The black radioactive binding signal colocalizes mainly with the tau mAb staining.

Figure 4. Colocalization of tau mAb pS42240 (A) and Aβ mAb BAP-241 (C) immunohistochemical staining with microautoradiographical tracer binding (B) of [3H]8, as a representative example. Staining and binding experiments were performed on a single fresh-frozen tissue section from an AD Braak V cortical tissue block. The detail (D) shows the microautoradiographical signal superimposed with tau mAb staining (red). The black silver grain deposits (representing radioligand binding) colocalize with tau mAb staining, while areas of Aβ mAb staining (dotted circle in B) do not colocalize with microautoradiographical signals.

pS42240 and BAP-241 were used. All compounds possessed excellent selectivity for aggregated tau without any evidence of binding to Aβ plaques. Representative images of one radioligand ([3H]8) are shown in Figure 3. Radiochemical Synthesis of [11C]- and [18F]-Labeled Compounds. A radiosynthesis was established for five out of the six lead tracer candidates to introduce a fluorine-18 or a carbon-11 nuclide (Scheme 7). In the case of compound 37,

to 5, with the highest values found for [3H]34 (GM/WM = 14) and [3H]7 and [3H]9 (GM/WM = 11). High binding selectivity of the tritiated ligands for tau versus Aβ aggregates was demonstrated by colocalization experiments, assessed by autoradiography and immunohistochemistry on the same tissue section on both macroscopic and microscopic scale (Figures 3 and 4). For immunohistochemistry staining of NFTs and Aβ plaques the proprietary tau- and Aβ- specific antibodies 7358



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Scheme 7. Synthesis of Carbon-11 and Fluorine-18 Tracersa

radiochemical purity of 100%. The average concentration of carrier and precursor was 0.199 μg/mL and 0.010 μg/mL, respectively. Since many PET radiotracer programs do not have access to microwave heating for radiofluorinations, we selected to test the synthesis of [18F]9 using an easily programmed, flexible radiosynthesizer platform (Sofie ELIXYS). By use of the ELIXYS, [18F]9 was isolated in an average nondecay corrected yield of 30.8% (n = 5) in 70 min. The final product had an average specific radioactivity of 141 720 mCi/μmol (5243 GBq/μmol) and radiochemical purity of 100%. The average concentration of carrier and precursor was 0.0342 μg/mL and 0.0314 μg/mL, respectively. [11C]34 had an average nondecay corrected yield of 7.5% (n = 4) in 33 min with an average specific radioactivity of 16 848 mCi/μmol (610 GBq/μmol) and radiochemical purity of 97.7%. The average concentration of carrier and precursor was 0.059 μg/mL and 0.003 μg/mL, respectively. In the initial radiosynthesis of [11C]34, analytical HPLC showed the final product with less than 90% radiochemical purity. As was done with [11C]8, the addition of ascorbic acid in the product collection flask of the SPE formulation system increased the radiochemical and chemical purity of the final radiotracer product. Minimal change in radiochemical purity was observed in the validation 40 min stability test. [18F]37 was isolated in an average nondecay corrected yield of 25% (n = 2) in 40 min. The final product had an average specific radioactivity of 49 750 mCi/μmol (1841 GBq/μmol) and radiochemical purity of 96%. The average concentration of carrier was 0.057 μg/mL, and no residual precursor was observed. PET Imaging in Non-Human Primates. PET studies in tau-naive baboons demonstrated favorable pharmacokinetic properties for three out of five tracer candidates. [11C]7, [11C] 8, and [18F]9 were characterized by good uptake in the baboon brain with peak SUVs between 1.1 and 2.0 immediately after tracer injection and scan initiation and rapid clearance of radioactivity from the baboon brain with [18F]9 showing the fastest washout (Figure 5). All three tracer candidates were

Reagents and conditions: (a) [11C]MeI, NaH, DMF, 80 °C, 3 min; (b) [11C]MeI, 6 N NaOH, DMSO, 80 °C, 3 min; (c) [18F]KF, Kryptofix 2.2.2, DMSO, microwave 50 W, 4 min; (d) [18F]KF, Kryptofix 2.2.2, DMSO, microwave 50 W, 80 s. a

offering in principle the possibility for labeling with any of the two radioisotopes, fluorine-18 was preferred on account of the longer decay half-life, facilitating the logistics of tracer use, and lower positron emission energy, leading to higher spatial resolution of PET images. A robust radiomethylation procedure for compound 22 could not be established in the short time frame of the project, and further work to progress this candidate was thus stopped. The final product [11C]7 was isolated in an average nondecay corrected yield of 26% (n = 3) in 33 min. The final product average specific radioactivity was 15 987 mCi/μmol (592 GBq/ μmol) with a radiochemical purity of 97%. The average concentration of carrier 7 and precursor was 0.132 μg/mL and 0.009 μg/mL, respectively. Initial developmental radiosyntheses showed [11C]7 was susceptible to radiolysis during the standard semipreparative HPLC and solid phase extraction (SPE) formulation. With a change of the HPLC solvent from an acetonitrile/triethylamine buffer to an ethanol/triethylamine buffer eluent system, the radiolysis during the semipreparative HPLC was reduced dramatically. Also, a significant reduction in radiolysis was observed by including ascorbic acid during the SPE isolation and formulation procedure. No significant radiolysis was observed during the validation 40 min stability tests. The final product [11C]8 was isolated in an average nondecay corrected yield of 6.3% (n = 16) in 33 min. The final product had an average specific radioactivity of 14 080 mCi/μmol (521 GBq/μmol) and radiochemical purity of 95.5%. The average concentration of carrier product and precursor was 0.099 μg/ mL and 0.009 μg/mL, respectively. As this radiotracer product was also susceptible to radiolysis, ascorbic acid was used in the formulation to eliminate this degradation. By use of the microwave radiofluorination module, [18F]9 was isolated in an average nondecay corrected yield of 20.3% (n = 42) in 59 min. The final product had average specific radioactivity of 41 901 mCi/μmol (1550 GBq/μmol) and

Figure 5. Time−activity curves of the whole gray matter of candidate radioligands as determined in a baboon PET study.

characterized by a homogeneous distribution of the radioactivity in different brain regions, as expected in the absence of tau aggregates in the animals. Tracer candidate [11C]34 displayed moderate brain uptake (peak SUV of 1.2), but the whole brain time−activity curve slightly increased at time points later than 20 min, suggesting the generation and accumulation of a blood−brain barrier penetrating radiometabolite which counteracts the washout of the parent 7359



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compound. The time−activity curve for [18F]37 showed minimal brain uptake, followed by a steady increase of radioactivity uptake in the brain, which clearly points to the generation of a lipophilic radiometabolite that penetrates the blood−brain barrier and slowly accumulates in the brain. The unfavorable kinetic profile of [11C]34 and [18F]37 led to the exclusion of these two tracer candidates from further development.

Hz, 2H), 6.94 (d, J = 8.68 Hz, 2H), 6.31−6.36 (m, 2H), 5.60 (s, 2H), 3.77 (s, 3H). LC−HRMS (m/z): [M + H]+ calcd for C14H13N3O + H+, 240.1132; found, 240.1134; Diff 0.2 mDa. N-Methyl-2-(3-methylphenyl)imidazo[1,2-a]pyrimidin-7-amine (8). N4-Methylpyrimidine-2,4-diamine (25) (900 mg, 7.25 mmol) and 2-bromo-1-m-tolylethanone (1.62 g, 7.61 mmol) were combined with acetone (14 mL) under Ar and stirred overnight at 65 °C. The reaction mixture was cooled to rt and extracted with DCM and saturated aqueous NaHCO3. The aqueous layer was back-extracted four times with DCM adding each time a small amount of MeOH. The solid still present in the aqueous layer was filtered off. The organic layers were combined, dried over Na2SO4, filtered, combined with the filtered solid, and concentrated. The residue was absorbed on 12 g silica gel and loaded on a chromatography column for purification (50 g SiO2, DCM to DCM/MeOH, 0−5% MeOH gradient). The product was further purified by trituration with MeOH and EtOAc, then dried on the high vacuum to afford N-methyl-2-(3-methylphenyl)imidazo[1,2-a]pyrimidin-7-amine (8) (1.18 g, 65% yield) as a light yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 8.30 (d, J = 7.27 Hz, 1H), 7.79 (s, 1H), 7.68 (s, 1H), 7.63 (d, J = 7.87 Hz, 1H), 7.39 (q, J = 4.64 Hz, 1H), 7.27 (t, J = 7.60 Hz, 1H), 7.06 (d, J = 7.67 Hz, 1H), 6.26 (d, J = 7.47 Hz, 1H), 2.85 (d, J = 4.84 Hz, 3H), 2.34 (s, 3H). LC−HRMS (m/z): [M + H]+ calcd for C14H14N4 + H+, 239.1291; found, 239.1295; Diff, 0.4 mDa. 2-(6-Fluoropyridin-3-yl)-9H-dipyrido[2,3-b;3′,4′-d]pyrrole (9). A solution of 2-(6-fluoropyridin-3-yl)-dipyrido[2,3-b;3′,4′-d]pyrrole-9carboxylic acid tert-butyl ester (47) (22 mg, 60 μmol) and TFA (33.3 μL, 432 μmol) in DCM (0.5 mL) was stirred at rt overnight. After cooling to 0 °C, Et3N (70 μL, 503 μmol) was added and all volatiles were removed. The crude material was purified by preparative HPLC (YMC Triart C18 100 mm × 30 mm, 5 μm; H2O, 0.1% Et3N/ MeCN gradient) to afford 2-(6-fluoropyridin-3-yl)-9H-dipyrido[2,3b;3′,4′-d]pyrrole (9) as off-white solid (14 mg, 88%). 1H NMR (600 MHz, DMSO-d6) δ 12.43 (br s, 1H), 9.41 (s, 1H), 9.04 (d, J = 2.62 Hz, 1H), 8.76 (d, J = 8.06 Hz, 1H), 8.74 (dt, J = 2.50, 8.30 Hz, 1H), 8.52 (d, J = 5.64 Hz, 1H), 8.02 (d, J = 8.06 Hz, 1H), 7.51 (dd, J = 0.91, 5.64 Hz, 1H), 7.37 (dd, J = 2.72, 8.56 Hz, 1H). LC−HRMS (m/z): [M + H]+ calcd for C15H9FN4 + H+, 265.0884; found, 265.0888; Diff, 0.4 mDa. 4-N-Methylpyridine-2,4-diamine (12). 2-Amino-4-chloropyridine (10) (15.0 g, 117 mmol) and methanamine, 40 wt % in H2O (67.4 g, 75 mL, 868 mmol), were combined in an autoclave and stirred 4 h at 160 °C. After cooling to rt, the solution was treated with Na2CO3 (10 g) and was extracted 10 times with DCM (50 mL). The combined organic layers were dried over Na2SO4. After evaporation of the solvent, the residue was taken up in EtOAc (50 mL) and was heated to 60 °C. Then heptane (10 mL) was added. After stirring at rt for 2 h, solids were collected by filtration and washed with EtOAc/Hept 9:1. More product was obtained after adding heptane to the filtrate. 4-NMethylpyridine-2,4-diamine (12) was isolated as a light brown solid (10.8 g, 75%). 1H NMR (600 MHz, DMSO-d6) δ 7.44 (d, J = 5.84 Hz, 1H), 6.02 (br d, J = 4.73 Hz, 1H), 5.79 (dd, J = 2.12, 5.84 Hz, 1H), 5.50 (d, J = 2.01 Hz, 1H), 5.31 (s, 2H), 2.61 (d, J = 4.94 Hz, 3H). LC−HRMS (m/z): [M + H]+ calcd for C6H9N3 + H+, 124.0869; found, 124.0869; Diff, 0.0 mDa. 2-Phenylimidazo[1,2-a]pyridin-7-ylamine (16). 2,4-Diaminopyridine (11) (3.00 g, 27.5 mmol) was combined with acetone (150 mL) to give a colorless solution. 2-Bromo-1-phenylethanone (13) (8.21 g, 41.2 mmol) was added to give a white suspension. The reaction mixture was heated under Ar to 65 °C and stirred for 5.5 h. Solids were collected by filtration and washed with acetone (45 mL), then dissolved in H2O (150 mL) and treated with 25% aqueous NH4OH (135 mL). The resulting suspension was filtered and washed with H2O (45 mL). The product was dried overnight under high vacuum to yield 2-phenylimidazo[1,2-a]pyridin-7-ylamine (16) as an off-white powder (5.17 g, 90%). 1H NMR (600 MHz, DMSO-d6) δ 8.12 (d, J = 7.96 Hz, 1H), 7.95 (s, 1H), 7.85 (d, J = 7.57 Hz, 2H), 7.37 (t, J = 7.44 Hz, 2H), 7.24 (t, J = 7.35 Hz, 1H), 6.34−6.37 (m, 2H), 5.64 (br s, 2H). LC− HRMS (m/z): [M + H]+ calcd for C13H11N3 + H+, 210.1026; found, 210.1029; Diff, 0.3 mDa.

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CONCLUSIONS In summary, a lead optimization program has led to the identification of six promising PET tracer candidates for imaging of tau aggregates in AD. These novel structures possess high specificity and affinity for tau aggregates and an excellent selectivity against Aβ plaques. Further characterization of the compounds indicated the three tracers [11C]7, [11C]8, and [18F]9 to be of particular interest. In addition to the highly favorable binding properties to pathological tau aggregates, these three radioligands possess appropriate pharmacokinetic properties and good brain uptake in rodents and non-human primates, followed by fast systemic clearance. On the basis of the favorable profile, the three radiotracers were progressed into clinical validation studies in Alzheimer’s disease patients (ClinicalTrials.gov identifier: NCT02187627). The results of this work will be communicated in due course.

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EXPERIMENTAL SECTION

Chemical Synthesis of Cold Compounds. General Methods. Unless otherwise noted, all reagents and chemicals were obtained from commercial suppliers and used without further purification. Nonaqueous reactions were carried out under an inert atmosphere of Ar or N2. Microwave heating of reactions was carried out on a Biotage Initiator apparatus. Reactions were monitored by TLC (TLC plates F254, Merck) or LC−MS (liquid chromatography−mass spectrometry) analysis. Flash column chromatography was carried out using cartridges packed with silica gel (Isolute columns, Telos flash columns) on an ISCO machine or on glass columns on silica gel 60 (32−60 mesh, 60 Å). The purity of final compounds was determined by HPLC and was at least above 95%. LC−high-resolution MS spectra were recorded with an Agilent LC system consisting of an Agilent 1290 high-pressure system, a CTC PAL autosampler, and an Agilent 6520 QTOF. The separation was achieved on a Zorbax Eclipse Plus C18 1.7 μm, 2.1 mm × 50 mm column at 55 °C (A = 0.01% formic acid in water; B = 0.01% formic acid in acetonitrile) at a flow of 1 mL/ min with the following gradient: 0 min, 5% B; 0.3 min, 5% B; 4.5 min, 99% B; 5 min, 99% B. 1H NMR spectra were measured on a Bruker 300 MHz instrument, a Bruker 600 MHz instrument in a 5 mm TCI cryoprobe at 298 K, or a Bruker Avance 400 MHz. A TMS internal standard was used for experiments done in CDCl3. The deuterated DMSO-d6 solvent signal was used as the reference with 2.50 ppm. 2-(4-Methoxyphenyl)imidazo[1,2-a]pyridin-7-amine (7). A vial was charged with pyridine-2,4-diamine (11) (400 mg, 3.67 mmol), 2-bromo-1-(4-methoxyphenyl)ethanone (14) (882 mg, 3.85 mmol), NaHCO3 (329 mg, 3.92 mmol), and MeOH (3.5 mL). The reaction mixture was stirred at reflux for 4 h. After cooling to rt, the mixture was diluted with water and EtOAc, sonicated, and stirred at room temperature for 15 min. The solids were collected by filtration, then rinsed with H2O and EtOAc. The resulting pale yellow solid was dried under high vacuum to afford 2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-7-amine (7) hydrobromide. This material was suspended in 5 mL of saturated aq NaHCO3, sonicated, filtered, and washed with H2O. The residue was suspended in 5 mL of aq 2 M NaOH, sonicated, filtered, and rinsed with water. The resulting residue was put under high vacuum to afford 2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-7amine (7) as a light brown solid (310 mg, 35%). 1H NMR (300 MHz, DMSO-d6) δ 8.09 (d, J = 7.07 Hz, 1H), 7.83 (s, 1H), 7.77 (d, J = 8.68 7360



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N-Methyl-2-phenylimidazo[1,2-a]pyridin-7-amine (17). N4-Methylpyridine-2,4-diamine (12) (153 mg, 1.24 mmol) and 2-bromo-1phenylethanone (13) (247 mg, 1.24 mmol) were combined in a reaction flask, and acetone (2.5 mL) was added. The vessel was sealed and heated for 5 h at 70 °C. Solids were collected by filtration and washed with acetone. The product was purified by preparative HPLC (Gemini NX 3u, 50 mm × 4.6 mm; MeOH/5% Et3N in H2O from 20:80 to 98:2) to give N-methyl-2-phenylimidazo[1,2-a]pyridin-7amine (17) as white solid (125 mg, 44%). 1H NMR (300 MHz, DMSO-d6) δ 8.11 (d, J = 7.22 Hz, 1H), 7.95 (s, 1H), 7.86 (d, J = 7.85 Hz, 2H), 7.32−7.43 (m, 2H), 7.20−7.28 (m, 1H), 6.36 (dd, J = 2.22, 7.47 Hz, 1H), 6.23 (q, J = 4.84 Hz, 1H), 6.18 (d, J = 2.02 Hz, 1H), 2.72 (d, J = 4.84 Hz, 3H). LC−HRMS (m/z): [M + H]+ calcd for C14H13N3 + H+, 224.1182; found, 224.1183; Diff, 0.1 mDa. 4-(7-Aminoimidazo[1,2-a]pyridin-2-yl)phenol (18). A vial was charged with pyridine-2,4-diamine (11) (0.300 g, 2.75 mmol), 2bromo-1-(4-hydroxyphenyl)ethanone (15) (615 mg, 2.86 mmol), and EtOH (4.0 mL). The vial was flushed with Ar, closed, and heated at 70 °C overnight. The reaction mixture was cooled to rt and concentrated. The residue was suspended in H2O and a small amount of EtOAc. 25% aqueous NH4OH was added until neutral pH was obtained. The suspension was filtered and washed with H2O and a small amount EtOAc and finally put under high vacuum. The residue was purified by HPLC (Gemini NX 3u 50 mm × 4.6 mm; MeOH/5% Et3N in H2O from 20:80 to 98:2). Finally, trituration with EtOAc containing a few drops of MeOH and drying on the high vacuum afforded 4-(7aminoimidazo[1,2-a]pyridin-2-yl)phenol (18) as a light brown solid (134 mg, 22% yield). 1H NMR (600 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.07−8.10 (m, 1H), 7.77 (s, 1H), 7.65 (d, J = 8.07 Hz, 2H), 6.77 (d, J = 8.08 Hz, 2H), 6.32−6.37 (m, 2H), 5.62 (br s, 2H). LC−HRMS (m/ z): [M + H]+ calcd for C13H11N3O + H+, 226.0975; found, 226.0976; Diff, 0.1 mDa. N,N-Dimethyl-2-phenylimidazo[1,2-a]pyridin-7-amine (19). To a solution under N2 of 2-phenylimidazo[1,2-a]pyridin-7-amine (16) (500 mg, 2.39 mmol) in DMF (2 mL) was added NaH (60%, 172 mg, 4.30 mmol). After stirring at rt for 30 min, MeI (611 mg, 269 μL, 4.30 mmol) was added and the mixture was stirred in a closed flask for 18 h. The reaction mixture was concentrated and adsorbed on Isolute for purification by flash chromatography (Flashpack cartridge, 20 g amino modified SiO2; heptane/DCM 4:1 to DCM). N,N-Dimethyl-2phenylimidazo[1,2-a]pyridin-7-amine (19) was isolated as light brown solid (35 mg, 6%) next to N-methyl-2-phenylimidazo[1,2a]pyridin-7-amine (17) (42 mg, 8%) as brown solid. 1H NMR (300 MHz, DMSO-d6) δ 8.25 (d, J = 7.47 Hz, 1H), 8.02 (s, 1H), 7.85−7.91 (m, 2H), 7.39 (t, J = 7.57 Hz, 2H), 7.25 (t, J = 7.27 Hz, 1H), 6.69 (dd, J = 2.52, 7.57 Hz, 1H), 6.44 (d, J = 2.42 Hz, 1H), 2.97 (s, 6H). LC− HRMS (m/z): [M + H]+ calcd for C15H15N3 + H+, 238.1339; found, 238.1343; Diff, 0.4 mDa. tert-Butyl 2-Phenylimidazo[1,2-a]pyridin-7-ylcarbamate (20). 2Phenylimidazo[1,2-a]pyridin-7-amine hydrobromide (16·HBr) (720 mg, 2.36 mmol) was suspended in 5.0 mL of THF and cooled to 0 °C. LiHMDS (1 M solution in THF, 7.5 mL, 7.5 mmol) was added dropwise over a period of 25 min at 0 °C. Then Boc2O (566 mg, 2.59 mml) dissolved in 2.0 mL of THF was added over a period of 5 min. After the addition was complete, the ice bath was removed and the reaction mixture was stirred at rt for 1.5 h. The reaction mixture was quenched with 5 mL of water and extracted with DCM (30 mL) and saturated aqueous NH4Cl (15 mL). The aqueous layer was extracted with two more portions of DCM (30 mL). The organic layers were combined, dried over Na2SO4, and concentrated. The residue was purified by chromatography (40 g SiO2; DCM to 5% MeOH in DCM). tert-Butyl 2-phenylimidazo[1,2-a]pyridin-7-ylcarbamate (20) (482 mg, 66%) was obtained as a light brown solid. 1H NMR (300 MHz, DMSO-d6) δ 9.67 (s, 1H), 8.37 (d, J = 7.47 Hz, 1H), 8.21 (s, 1H), 7.92 (d, J = 7.06 Hz, 2H), 7.66 (s, 1H), 7.41 (t, J = 7.54 Hz, 2H), 7.24−7.34 (m, 1H), 6.98 (dd, J = 2.02, 7.47 Hz, 1H), 1.51 (s, 9H). LC−HRMS (m/z): [M + H]+ calcd for C18H19N3O2 + H+, 309.1477; found, 309.1486; Diff, 0.9 mDa. tert-Butyl 2-Fluoroethyl(2-phenylimidazo[1,2-a]pyridin-7-yl)carbamate (21). tert-Butyl 2-phenylimidazo[1,2-a]pyridin-7-ylcarba-

mate (20) (1.35 g, 4.36 mmol) was dissolved in 12 mL of DMF (12 mL). NaH (∼55% dispersion in mineral oil, 286 mg, 6.55 mmol) was added, and the reaction mixture was stirred at rt for 15 min. 1-Bromo2-fluoroethane (833 mg, 0.49 mL, 6.56 mmol) was added dropwise, and the reaction mixture was stirred 1 h at 50 °C. The reaction mixture was cooled to rt, diluted with H2O (10 mL), and extracted with EtOAc (100 mL). The aqueous layer was back-extracted with EtOAc (100 mL). The organic layers were washed four times with H2O (10 mL) and once with brine (10 mL). The organic layers were combined, dried over Na2SO4, and concentrated. The residue was purified by chromatography (120 g SiO2; DCM to DCM/5% MeOH). All fractions containing product were combined and concentrated. The residue was triturated with EtOAc to afford tert-butyl 2-fluoroethyl(2phenylimidazo[1,2-a]pyridin-7-yl)carbamate (21) as an off-white solid (1.05 g, 68%). The filtrate was concentrated to afford an additional portion of tert-butyl 2-fluoroethyl(2-phenylimidazo[1,2-a]pyridin-7yl)carbamate (21) (0.416 g, 27%) as a light brown solid. Total yield 95%. 1H NMR (300 MHz, DMSO-d6) δ 8.46 (d, J = 7.27 Hz, 1H), 8.36 (s, 1H), 7.92−7.98 (m, 2H), 7.41−7.49 (m, 3H), 7.29−7.36 (m, 1H), 6.86 (dd, J = 2.22, 7.27 Hz, 1H), 4.58 (td, J = 5.00, 47.40 Hz, 2H), 3.98 (td, J = 4.60, 26.20 Hz, 2H), 1.42 (s, 9H). LC−HRMS (m/ z): [M + H]+ calcd for C20H22FN3O2 + H+, 356.1769; found, 356.1774; Diff, 0.5 mDa. N-(2-Fluoroethyl)-2-phenylimidazo[1,2-a]pyridin-7-amine (22). tert-Butyl 2-fluoroethyl(2-phenylimidazo[1,2-a]pyridin-7-yl)carbamate (21) (1.38 g, 3.88 mmol) was dissolved in 8.0 mL of DCM. TFA (8.88 g, 6.0 mL, 77.9 mmol) was added dropwise, and the reaction mixture was stirred at rt for 3 h. The reaction mixture was concentrated. The residue (light brown solid) was extracted with DCM and saturated aqueous Na2CO3. The aqueous layer was back-extracted twice with DCM. The organic layers were combined, dried over Na2SO4, filtered, and concentrated. The residue was triturated with DCM to afford N(2-fluoroethyl)-2-phenylimidazo[1,2-a]pyridin-7-amine (22) (918 mg, 93% yield) as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 8.14 (d, J = 7.47 Hz, 1H), 7.97 (s, 1H), 7.79−7.92 (m, 2H), 7.33−7.42 (m, 2H), 7.18−7.31 (m, 1H), 6.45 (dd, J = 2.40, 7.50 Hz, 1H), 6.39− 6.45 (m, 1H), 6.34 (d, J = 1.82 Hz, 1H), 4.61 (td, J = 4.90, 47.60 Hz, 2H), 3.41 (qd, J = 5.05, 28.06 Hz, 2H). LC−HRMS (m/z): [M + H]+ calcd for C15H14F N3 + H+, 256.1245; found, 256.1246; Diff, 0.1 mDa. N4-Methylpyrimidine-2,4-diamine (25). To a solution of 4chloropyrimidin-2-amine (23) (2.00 g, 15.4 mmol) in NMP (12 mL) were added under an atmosphere of N2 methanamine hydrochloride (1.04 g, 15.4 mmol) and K2CO3 (4.27 g, 30.9 mmol). The reaction mixture was stirred at 120 °C for 2 h. Solvents were evaporated, and the crude yellow oil was purified by flash chromatography (50 g amino modified SiO2; DCM to DCM/ MeOH/25% aqueous NH4OH 140:10:1). The resulting oil was triturated with t-BuOMe (40 mL) and dried on the high vacuum to yield N4-methylpyrimidine-2,4-diamine (25) as light yellow solid (0.76 g, 40%). 1H NMR (300 MHz, DMSO-d6) δ 7.59 (br d, J = 5.05 Hz, 1H), 6.70 (br s, 1H), 5.83 (br s, 2H), 5.68 (d, J = 5.85 Hz, 1H), 2.66− 2.75 (m, 3H). LC−HRMS (m/z): [M + H]+ calcd for C5H8N4 + H+, 125.0822; found, 125.0821; Diff, −0.1 mDa. 2-Amino-4-(dimethylamino)pyrimidine (26). 4-Chloropyrimidin2-amine (23) (3.24 g, 25.0 mmol), dimethylamine hydrochloride (2.24 g, 27.5 mmol), and K2CO3 (10.4 g, 75.0 mmol) were combined with NMP (15 mL). The reaction mixture was stirred for 1 h at 120 °C before pouring it onto a mixture of aqueous 1 M NaOH and ice. After extraction with EtOAc (3 × 100 mL) the combined organic layers were concentrated. t-BuOMe (40 mL) was added to precipitate the product that was collected by filtration. Drying under vacuum afforded 2-amino-4-(dimethylamino)pyrimidine (26) as white powder (1.48 g, 43%). 1H NMR (300 MHz, CDCl3) δ 7.85 (d, J = 6.25 Hz, 1H), 5.88 (d, J = 6.25 Hz, 1H), 4.61 (br s, 2H), 3.04 (s, 6H). LC−HRMS (m/z): [M + H]+ calcd for C6H10N4 + H+, 139.0978; found, 139.0977; Diff, −0.1 mDa. N4-Cyclopropylpyrimidine-2,4-diamine (27). To a suspension of 4-chloropyrimidin-2-amine (23) (300 mg, 2.32 mmol) and K2CO3 (640 mg, 4.63 mmol) in DMF (3.0 mL) was added cyclopropylamine (1.21 g, 1.49 mL, 21.1 mmol), and the reaction mixture was stirred 7361



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overnight at 100 °C. The reaction mixture was cooled to rt and extracted with EtOAc and H2O, and the aqueous layer was backextracted with EtOAc. The organic layers were washed three times with H2O and once with brine. The combined organic layers were dried over Na2SO4 and concentrated. Trituration of the residue with Et2O afforded N4-cyclopropylpyrimidine-2,4-diamine (27) as off-white solid (193 mg, 55%). 1H NMR (300 MHz, CDCl3) δ 7.94 (d, J = 5.84 Hz, 1H), 6.12 (d, J = 5.84 Hz, 1H), 5.04 (br s, 1H), 4.63 (br s, 2H), 2.48−2.56 (m, 1H), 0.76−0.83 (m, 2H), 0.53−0.59 (m, 2H). LC− HRMS (m/z): [M + H]+ calcd for C7H10N4 + H+, 151.0978; found, 151.0977; Diff, −0.1 mDa. 4-[4-(2-Fluoroethyl)piperidin-1-yl]pyrimidin-2-ylamine (28). A mixture of 4-chloropyrimidin-2-amine (23) (4.0 g, 31 mmol), 4-(2fluoroethyl)piperidine hydrochloride (63) (5.7 g, 34 mmol), K2CO3 (6.4 g, 46 mmol), and NMP (12 mL) was stirred in a sealed tube 12 h at 120 °C. After cooling the reaction mixture to rt, it was poured into aqueous 1 M NaOH (120 mL) and extracted with DCM (3 × 150 mL). The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by chromatography (SiO2; 4− 6% MeOH in DCM) to afford 4-[4-(2-fluoroethyl)piperidin-1yl]pyrimidin-2-ylamine (28) as light yellow sticky solid (5.4 g, 78%). 1 H NMR (600 MHz, DMSO-d6) δ 7.72 (d, J = 6.04 Hz, 1H), 6.02 (d, J = 6.15 Hz, 1H), 5.98 (s, 2H), 4.51 (td, J = 6.00, 48.20 Hz, 2H), 4.30 (br d, J = 11.99 Hz, 2H), 2.73−2.81 (m, 2H), 1.64−1.73 (m, 3H), 1.56−1.64 (m, 2H), 1.02−1.12 (m, 2H). LC−HRMS (m/z): [M + H]+ calcd for C11H17FN4 + H+, 225.1510; found, 225.1510; Diff, 0.0 mDa. 2-Phenylimidazo[1,2-a]pyrimidin-7-amine (29). Pyrimidine-2,4diamine (24) (5.19 g, 47.1 mmol) and 2-bromo-1-phenylethanone (13) (14.1 g, 70.7 mmol) were combined with acetone (200 mL) to give a white suspension. The reaction mixture was heated to 60 °C and stirred for 5 h. The reaction mixture was filtered through sintered glass and the resulting solid washed with acetone. H2O (50 mL) and 25% aqueous NH4OH (75 mL) were added to the product. After filtration of the solid and washing with H2O, drying at 40 °C under high vacuum afforded 2-phenylimidazo[1,2-a]pyrimidin-7-amine (29) as a white powder (9.22 g, 93%). 1H NMR (600 MHz, DMSO-d6) δ 8.36 (d, J = 7.25 Hz, 1H), 7.81−7.86 (m, 3H), 7.38 (t, J = 7.48 Hz, 2H), 7.25 (t, J = 7.36 Hz, 1H), 6.80 (br s, 2H), 6.26 (d, J = 7.25 Hz, 1H). LC−HRMS (m/z): [M + H]+ calcd for C12H10N4 + H+, 211.0978; found, 211.0981; Diff, 0.3 mDa. N-Methyl-2-phenylimidazo[1,2-a]pyrimidin-7-amine (30). A yellow slurry of N4-methylpyrimidine-2,4-diamine (25) (50.0 mg, 403 μmol) and 2-bromo-1-phenylethanone (13) (120 mg, 604 μmol) in acetone (2.75 mL) was stirred at 65 °C overnight. The off-white suspension was filtered and washed with H2O (1 mL) and acetone (1 mL). The solid precipitate was suspended into H2O (1 mL) and 25% aqueous NH4OH (0.9 mL). The suspension was stirred for 10 min at rt, filtered again, and washed with H2O. High vacuum drying for 4 h finally yielded N-methyl-2-phenylimidazo[1,2-a]pyrimidin-7-amine (30) as light red solid (41 mg, 45%). 1H NMR (600 MHz, DMSOd6) δ 8.31 (d, J = 7.25 Hz, 1H), 7.83−7.86 (m, 2H), 7.82 (s, 1H), 7.35−7.43 (m, 3H), 7.25 (tt, J = 1.21, 7.35 Hz, 1H), 6.27 (d, J = 7.25 Hz, 1H), 2.85 (d, J = 4.73 Hz, 3H). LC−HRMS (m/z): [M + H]+ calcd for C13H12N4 + H+, 225.1135; found, 225.1137; Diff, 0.2 mDa. N,N-Dimethyl-2-phenylimidazo[1,2-a]pyrimidin-7-amine (31). A suspension of N4,N4-dimethylpyrimidine-2,4-diamine (26) (100 mg, 724 μmol) and 2-bromo-1-phenylethanone (13) (216 mg, 1.09 mmol) in acetone (4 mL) was stirred at 65 °C overnight. The off-white suspension was filtered and washed with acetone (5 mL). The solid was suspended in H2O (3.1 mL) and 25% aqueous NH4OH (2.5 mL). The mixture was stirred for 10 min at rt, filtered again, and washed with H2O. After high vacuum drying N,N-dimethyl-2-phenylimidazo[1,2-a]pyrimidin-7-amine (31) was obtained as light brown solid (97 mg, 56%). 1H NMR (600 MHz, CDCl3) δ 8.01 (d, J = 7.56 Hz, 1H), 7.96−7.99 (m, 2H), 7.45 (s, 1H), 7.39 (t, J = 7.25 Hz, 2H), 7.28 (t, J = 7.23 Hz, 1H), 6.34 (d, J = 7.56 Hz, 1H), 3.21 (s, 6H). LC−HRMS (m/z): [M + H]+ calcd for C14H14N4 + H+, 239.1291; found, 239.1295; Diff, 0.4 mDa.

2-(3-Methylphenyl)imidazo[1,2-a]pyrimidin-7-amine (32). A vial was charged with pyrimidine-2,4-diamine (24) (0.450 g, 4.09 mmol), 2-bromo-1-m-tolylethanone (958 mg, 4.5 mmol), and acetone (10 mL). The vial was flushed with Ar and closed. The reaction mixture was stirred at 65 °C overnight. The reaction mixture was cooled to rt, filtered, and rinsed with acetone. The residue was suspended in saturated aqueous NaHCO3 (30 mL), stirred for 10 min, filtered, then rinsed with H2O and a small amount of EtOAc. The residue was absorbed on silica gel (10 g) and loaded on a chromatography column (50 g SiO2; DCM to DCM/MeOH 9:1). Fractions containing product were combined and concentrated. The residue was dissolved under heating in EtOH (40 mL). H2O (20 mL) was added dropwise, and the mixture was concentrated until a solid started to precipitate. The resulting suspension was allowed to cool to rt, filtered, rinsed with water and a minimal amount of EtOH. Drying under high vacuum afforded 2-(3-methylphenyl)imidazo[1,2-a]pyrimidin-7-amine (32) as an off-white solid (562 mg, 61%). 1H NMR (300 MHz, DMSO-d6) δ 8.35 (d, J = 7.27 Hz, 1H), 7.80 (s, 1H), 7.67 (s, 1H), 7.62 (d, J = 7.87 Hz, 1H), 7.26 (t, J = 7.67 Hz, 1H), 7.06 (d, J = 7.47 Hz, 1H), 6.79 (s, 2H), 6.25 (d, J = 7.27 Hz, 1H), 2.29−2.39 (m, 3H). LC−HRMS (m/ z): [M + H]+ calcd for C13H12N4 + H+, 225.1135; found, 225.1138; Diff, 0.3 mDa. 4-(7-(Methylamino)imidazo[1,2-a]pyrimidin-2-yl)phenol (33). A vial was charged with N4-methylpyrimidine-2,4-diamine (25) (700 mg, 5.36 mmol), 2-bromo-1-(4-hydroxyphenyl)ethanone (15) (1.21 g, 5.62 mmol), and acetone (9 mL). The vial was flushed with Ar and closed. The reaction mixture was stirred overnight at 65 °C. The reaction mixture was filtered, rinsing with acetone. The residue was suspended in a mixture of saturated aqueous NaHCO3 (15 mL) and CH2Cl2 (5 mL) and stirred for 40 min at rt. The mixture was filtered, rinsing consecutively with H2O, EtOAc, and DCM. The residue was dried under high vacuum and then again triturated with EtOAc (5 mL) containing 0.1% of MeOH. Solids were collected and rinsed with EtOAc and few drops of MeOH. Drying under high vacuum afforded 4-(7-(methylamino)imidazo[1,2-a]pyrimidin-2-yl)phenol (33) (1.27 g, 5.02 mmol, 94%). 1H NMR (300 MHz, DMSO-d6) δ 9.59 (br s, 1H), 8.27 (d, J = 7.27 Hz, 1H), 7.63 (d, J = 8.68 Hz, 2H), 7.60 (s, 1H), 7.30 (br d, J = 4.64 Hz, 1H), 6.77 (d, J = 7.81 Hz, 2H), 6.22 (d, J = 7.27 Hz, 1H), 2.84 (d, J = 4.64 Hz, 3H). LC−HRMS (m/z): [M + H]+ calcd for C13H12N4O + H+, 241.1084; found, 241.1085; Diff, 0.1 mDa. N-Cyclopropyl-2-(4-methoxyphenyl)imidazo[1,2-a]pyrimidin-7amine (34). A vial was charged with N4-cyclopropylpyrimidine-2,4diamine (27) (700 mg, 4.66 mmol), 2-bromo-1-(4-methoxyphenyl)ethanone (14) (1.12 g, 4.89 mmol), and acetone (1 mL). The vial was flushed with Ar and closed. The reaction mixture was stirred at 65 °C overnight. The reaction mixture was extracted with DCM and saturated aqueous NaHCO3. The aqueous layer was extracted twice with DCM. The organic layers were combined, dried over Na2SO4, filtered, and concentrated. The residue was triturated with EtOAc containing a few drops of MeOH to afford N-cyclopropyl-2-(4methoxyphenyl)imidazo[1,2-a]pyrimidin-7-amine (34) (546 mg, 42% yield) as a light yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 8.33 (d, J = 7.27 Hz, 1H), 7.78 (d, J = 8.68 Hz, 2H), 7.72 (s, 1H), 7.53 (d, J = 3.03 Hz, 1H), 6.96 (d, J = 8.88 Hz, 2H), 6.26 (br d, J = 7.27 Hz, 1H), 3.78 (s, 3H), 2.68−2.81 (m, 1H), 0.67−0.84 (m, 2H), 0.41−0.54 (m, 2H). LC−HRMS (m/z): [M + H]+ calcd for C16H16N4O + H+, 281.1397; found, 281.1401; Diff, 0.4 mDa. 4-(7-(Cyclopropylamino)imidazo[1,2-a]pyrimidin-2-yl)phenol (35). A vial was charged with N4-cyclopropylpyrimidine-2,4-diamine (27) (800 mg, 5.33 mmol), 2-bromo-1-(4-hydroxyphenyl)ethanone (15) (1.15 g, 5.33 mmol), and acetone (9.0 mL). The vial was flushed with Ar and capped. The reaction mixture was stirred overnight at 65 °C. The mixture was diluted with DCM (20 mL) and saturated aqueous NaHCO3 (20 mL). The aqueous layer was extracted twice with DCM. Solids were collected by filtration, and then the combined organic layers were dried over Na2SO4 and the solvent was evaporated. The combined solid residues were triturated with EtOAc containing a few drops of MeOH to afford 4-(7-(cyclopropylamino)imidazo[1,2a]pyrimidin-2-yl)phenol (35) as a light yellow solid (501 mg, 35%).1H 7362



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NMR (300 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.31 (d, J = 7.27 Hz, 1H), 7.62−7.69 (m, 3H), 7.49 (d, J = 3.23 Hz, 1H), 6.78 (d, J = 8.68 Hz, 2H), 6.24 (br d, J = 7.47 Hz, 1H), 2.68−2.80 (m, 1H), 0.70−0.79 (m, 2H), 0.44−0.51 (m, 2H). LC−HRMS (m/z): [M + H]+ calcd for C15H14N4O + H+, 267.1241; found, 267.1246; Diff, 0.5 mDa. 7-[4-(2-Fluoroethyl)piperidin-1-yl]-2-phenylimidazo[1,2-a]pyrimidine (36). To a solution of 4-[4-(2-fluoroethyl)piperidin-1yl]pyrimidin-2-ylamine (28) (250 mg, 1.11 mmol) in acetone (8 mL) at 25 °C under N2 were added 2-bromo-1-phenylethanone (13) (332 mg, 1.67 mmol) and a catalytic amount of TsOH. The reaction mixture was heated to 65 °C for 12 h. Volatiles were evaporated under reduced pressure, and the residual mixture was extracted with DCM (3 times 150 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material thus obtained was purified by preparative HPLC (Reprosil Gold column, 250 mm × 20 mm; 5 μm/C18, reverse phase; MeOH/10 mM aqueous NH4OAc) to afford 7-[4-(2-fluoroethyl)piperidin-1-yl]-2phenylimidazo[1,2-a]pyrimidine (36) as off white solid (90 mg, 25%). 1 H NMR (400 MHz, DMSO-d6) δ 8.48 (d, J = 7.82 Hz, 1H), 7.86 (d, J = 7.83 Hz, 2H), 7.86 (s, 1H), 7.39 (t, J = 7.80 Hz, 2H), 7.26 (t, J = 7.30 Hz, 1H), 6.79 (d, J = 7.82 Hz, 1H), 4.53 (td, J = 5.90, 47.90 Hz, 2H), 4.37−4.47 (m, 2H), 2.87−3.02 (m, 2H), 1.68−1.81 (m, 3H), 1.56−1.68 (m, 2H), 1.06−1.27 (m, 2H). LC−HRMS (m/z): [M + H]+ calcd for C19H21FN4 + H+, 325.1823; found, 325.1829; Diff, 0.6 mDa. 2-(4-(2-Fluoroethoxy)phenyl)-N-methylimidazo[1,2-a]pyrimidin7-amine (37). A vial was charged with 4-(7-(methylamino)imidazo[1,2-a]pyrimidin-2-yl)phenol (33) (870 mg, 3.44 mmol), Cs2CO3 (2.24 g, 6.88 mmol), DMF (12 mL), and 1-bromo-2-fluoroethane (611 mg, 360 μL, 4.82 mmol). The reaction mixture was stirred 1 h at 70 °C. The reaction mixture was diluted with H2O and then extracted with EtOAc (100 mL). The aqueous phase was back-extracted with EtOAc (100 mL). The organic layers were washed two times with H2O (15 mL) and once with brine (15 mL). The organic layers were combined, dried over Na2SO4, and concentrated. The residue was triturated with EtOAc, rinsed with EtOAc/DCM, and dried under high vacuum. An additional purification step was done by trituration with MeOH. Drying on the high vacuum overnight afforded 2-(4-(2fluoroethoxy)phenyl)-N-methylimidazo[1,2-a]pyrimidin-7-amine (37) (570 mg, 55%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 8.29 (d, J = 7.27 Hz, 1H), 7.74−7.81 (m, 2H), 7.70 (s, 1H), 7.34 (br q, J = 4.80 Hz, 1H), 6.96−7.03 (m, 2H), 6.24 (d, J = 7.27 Hz, 1H), 4.64− 4.87 (m, 2H), 4.17−4.36 (m, 2H), 2.84 (d, J = 4.64 Hz, 3H). LC− HRMS (m/z): [M + H]+ calcd for C15H15FN4O + H+, 287.1303; found, 287.1302; Diff, −0.1 mDa. 2-[4-[7-(Methylamino)imidazo[1,2-a]pyrimidin-2-yl]phenoxy]ethyl 4-Methylbenzenesulfonate (38). A reaction vial was charged with 4-(7-(methylamino)imidazo[1,2-a]pyrimidin-2-yl)phenol (33) (335 mg, 1.39 mmol), 2-(p-tolylsulfonyloxy)ethyl 4-methylbenzenesulfonate (723 mg, 1.95 mmol), Cs2CO3 (909 mg, 2.79 mmol), and DMF (6.5 mL). The reaction mixture was stirred 2 h at 50 °C. The reaction mixture was cooled to rt, diluted with H2O (10 mL), and extracted with EtOAc (80 mL). The aqueous layer was back-extracted with EtOAc (80 mL). The organic layers were washed twice with H2O (10 mL) and once with brine (10 mL). The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by chromatography (40 g SiO2; DCM to DCM/10% MeOH). All fractions containing product were combined and concentrated. The residue was triturated with MeOH and dried on the high vacuum to afford 2-(4-(7-(methylamino)imidazo[1,2-a]pyrimidin-2-yl)phenoxy)ethyl 4-methylbenzenesulfonate (38) as a pale yellow solid (248 mg, 41%). 1H NMR (300 MHz, DMSO-d6) δ 8.28 (d, J = 7.27 Hz, 1H), 7.81 (d, J = 8.28 Hz, 2H), 7.73 (d, J = 8.68 Hz, 2H), 7.70 (s, 1H), 7.48 (d, J = 8.07 Hz, 2H), 7.34 (br q, J = 4.84 Hz, 1H), 6.86 (d, J = 8.88 Hz, 2H), 6.24 (d, J = 7.27 Hz, 1H), 4.30−4.39 (m, 2H), 4.10−4.26 (m, 2H), 2.84 (d, J = 4.84 Hz, 3H), 2.42 (s, 3H). LC−HRMS (m/z): [M + H]+ calcd for C22H22N4O4S + H+, 439.1435; found, 439.1440; Diff, 0.5 mDa. 2-Phenylimidazo[1,2-c]pyrimidin-7-ylamine (40). To a solution of pyrimidine-4,6-diamine (39) (200 mg, 1.81 mmol) in EtOH (10 mL)

was added 2-bromo-1-phenylethanone (13) (542 mg, 2.72 mmol). The reaction mixture was heated at 80 °C for 8 h. After evaporation of the solvent the crude was purified by chromatography (amino modified SiO2; 3% MeOH/DCM). 2-Phenylimidazo[1,2-c]pyrimidin7-ylamine (40) was isolated as light brown solid (50 mg, 13%). 1H NMR (600 MHz, DMSO-d6) δ 8.96 (d, J = 1.21 Hz, 1H), 8.06 (s, 1H), 7.90 (dd, J = 1.06, 8.11 Hz, 2H), 7.41 (t, J = 7.70 Hz, 2H), 7.30 (t, J = 7.52 Hz, 1H), 6.19 (s, 1H), 6.12 (s, 2H). LC−HRMS (m/z): [M + H]+ calcd for C12H10N4 + H+, 211.0978; found, 211.0983; Diff, 0.5 mDa. tert-Butyl N-[3-(2,6-Dichloro-3-pyridyl)-4-pyridyl]carbamate (43). A preheated flask under Ar was charged with tert-butyl 3-iodopyridin4-ylcarbamate (41) (4.56 g, 14.2 mmol), 2,6-dichloropyridin-3ylboronic acid (42) (5.46 g, 28.4 mmol), [Pd(OAc)2] (320 mg, 1.42 mmol), and PPh3 (371 mg, 1.41 mmol). Et3N (4.32 g, 5.94 mL, 42.7 mmol) in DMF (137 mL) was added, and the reaction mixture was stirred at 100 °C for 3 h. The solvent was evaporated almost completely. H2O was added, and the crude product suspension was extracted twice with EtOAc. The combined organic layers were washed with H2O (3×), dried over Na2SO4, and the solvent was evaporated. Trituration of the crude product with DCM afforded 1.92 g of the desired product. The DCM phase was evaporated and purified by flash chromatography (SiO2; EtOAc to heptane) to yield in total 3.39 g (∼90% purity, 63% yield) of tert-butyl N-[3-(2,6-dichloro-3pyridyl)-4-pyridyl]carbamate (43) as light yellow solid. This material was used as such in the following step. 1H NMR (300 MHz, DMSOd6) δ 9.08 (s, 1H), 8.48 (d, J = 5.85 Hz, 1H), 8.28 (s, 1H), 7.84−7.90 (m, 2H), 7.66 (d, J = 8.07 Hz, 1H), 1.42 (s, 9H), LC−HRMS (m/z): [M + H]+ calcd for C15H15Cl2N3O2 + H+, 340.0614; found, 340.0624; Diff, 1.0 mDa. 2-Chloropyrrolo[2,3-b:4,5-c′]dipyridine (44). A suspension of tertbutyl N-[3-(2,6-dichloro-3-pyridyl)-4-pyridyl]carbamate (43) (264 mg, 776 μmol), K2CO3 (215 mg, 1.55 mmol), and 18-crown-6 (226 mg, 854 μmol) in DMF (15.8 mL) was stirred at 100 °C under Ar for 3 h. H2O was added, and the product was extracted twice with EtOAc. The combined organic layers were washed twice with H2O, brine, dried over Na2SO4, and the solvent was evaporated. Trituration of the crude product with a small amount of MeOH afforded 2-chloropyrrolo[2,3-b:4,5-c′]dipyridine (44) (105 mg, 63%) as light yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 12.47 (br s, 1H), 9.39 (s, 1H), 8.68 (d, J = 8.07 Hz, 1H), 8.52 (d, J = 5.65 Hz, 1H), 7.51 (d, J = 5.46 Hz, 1H), 7.41 (d, J = 8.07 Hz, 1H). LC−HRMS (m/z): [M + H]+ calcd for C10H6ClN3 + H+, 204.0323; found, 204.0333; Diff, 1.0 mDa. 2-Chloropyrrolo[2,3-b:4,5-c′]dipyridine-9-carboxylic Acid tertButyl Ester (45). A suspension of NaH (60%, 26.5 mg, 607 μmol) in dry DMF (1.5 mL) was cooled under Ar to 0 °C, and a solution of 2-chloropyrrolo[2,3-b:4,5-c′]dipyridine (44) (103 mg, 506 μmol) in dry DMF (3.0 mL) was added. Stirring was continued at 0 °C for 10 min, then at rt for 30 min. After cooling down to 0 °C and addition of di-tert-butyl dicarbonate (132 mg, 141 μL, 1.05 mmol) in dry DMF (0.75 mL) stirring was continued at rt overnight. H2O was added, and the reaction mixture was extracted twice with EtOAc. The combined organic layers were washed twice with H2O and brine, dried over Na2SO4, filtered, and evaporated. 2-Chloropyrrolo[2,3-b:4,5-c′]dipyridine-9-carboxylic acid tert-butyl ester (45) was obtained after purification by flash chromatography (SiO2; DCM to MeOH) and drying on the high vacuum as off-white solid (113 mg, 73%). 1H NMR (300 MHz, DMSO-d6) δ 9.48 (d, J = 0.81 Hz, 1H), 8.76 (d, J = 8.17 Hz, 1H), 8.69 (d, J = 5.69 Hz, 1H), 8.11 (dd, J = 0.91, 5.75 Hz, 1H), 7.64 (d, J = 8.07 Hz, 1H), 1.70 (s, 9H). LC−HRMS (m/z): [M + H]+ calcd for C15H14ClN3O2 + H+, 303.0775; found, 303.0778; Diff, 0.3 mDa. 2-(6-Fluoropyridin-3-yl)pyrrolo[2,3-b:4,5-c′]dipyridine-9-carboxylic Acid tert-Butyl Ester (47). A reaction flask was charged under Ar with 2-chloropyrrolo[2,3-b:4,5-c′]dipyridine-9-carboxylic acid tertbutyl ester (45) (100 mg, 329 μmol), 2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (46) (147 mg, 658 μmol), K2CO3 (137 mg, 988 μmol), and [Pd(dppf)Cl2]·DCM (10.8 mg, 13.2 μmol), and the vessel was sealed. DMF (7 mL) was added via a syringe, and 7363



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the reaction mixture was stirred at 90 °C for 17 h. Solids were removed by filtration. H2O was added to the filtrate, and the mixture was extracted twice with EtOAc. The combined organic layers were washed with H2O (3×), dried over Na2SO4, and the solvent was evaporated. Trituration of the crude product with a small amount of MeOH afforded 2-(6-fluoropyridin-3-yl)pyrrolo[2,3-b:4,5-c′]dipyridine (9) as a light red solid (23 mg, 80% purity, 21%). The mother liquor was evaporated and purified by flash chromatography (SiO2; DCM to MeOH) to afford 2-(6-fluoropyridin-3-yl)pyrrolo[2,3b:4,5-c′]dipyridine-9-carboxylic acid tert-butyl ester (47) as off-white solid (12 mg, 10%). 1H NMR (300 MHz, CDCl3) δ 9.29 (d, J = 1.01 Hz, 1H), 9.01 (td, J = 0.80, 2.60 Hz, 1H), 8.71 (d, J = 5.84 Hz, 1H), 8.62 (ddd, J = 2.52, 7.66, 8.56 Hz, 1H), 8.44 (d, J = 8.06 Hz, 1H), 8.27 (dd, J = 1.01, 5.84 Hz, 1H), 7.84 (d, J = 8.26 Hz, 1H), 7.08 (ddd, J = 0.60, 3.02, 8.66 Hz, 1H), 1.81 (s, 9H). LC−HRMS (m/z): [M + H]+ calcd for C20H17FN4O2 + H+, 365.1408; found, 365.1436; Diff, 2.8 mDa. 2-(6-Nitropyridin-3-yl)pyrrolo[2,3-b:4,5-c′]dipyridine (49). 2Chloropyrrolo[2,3-b:4,5-c′]dipyridine-9-carboxylic acid tert-butyl ester (45) (285 mg, 938 μmol), 2-nitropyridine-5-boronic acid pinacol ester (48) (469 mg, 1.88 mmol), [Pd(dppf)Cl2]·DCM (34.5 mg, 42.2 μmol), and K2CO3 (389 mg, 2.81 mmol) were combined under Ar in a reaction flask. DMF (24 mL) was added, and the tube was sealed. The reaction mixture was stirred at 90 °C for 18 h. Filtration through Celite and subsequently through a small silica gel pad (neutral, mesh 32−63) was followed by rinsing with DMF and evaporation to dryness. The brown solid was dissolved in DMF (20 mL), and DMSO was added until an almost clear solution resulted. After filtration, the solvents were evaporated to almost dryness. Purification by preparative HPLC (YMC Triart C18 100 mm × 30 mm, 5 μm; H2O, 0.1% Et3N/MeCN gradient) provided 2-(6-nitropyridin-3-yl)pyrrolo[2,3-b:4,5-c′]dipyridine (49) as a yellow solid (37 mg, 13%). 1H NMR (600 MHz, DMSO-d6) δ 12.55 (br s, 1H), 9.44−9.46 (m, 2H), 8.95 (dd, J = 2.01, 8.46 Hz, 1H), 8.84 (d, J = 8.06 Hz, 1H), 8.54 (d, J = 5.64 Hz, 1H), 8.48 (d, J = 8.46 Hz, 1H), 8.20 (d, J = 8.06 Hz, 1H), 7.53 (d, J = 5.54 Hz, 1H). LC−HRMS (m/z): [M + H]+ calcd for C15H9N5O2 + H+, 292.0829; found, 292.0827; Diff, −0.2 mDa. (3-Bromophenyl)(5-iodopyrimidin-4-yl)amine (52). To a solution of 4-chloro-5-iodopyrimidine (50) (2.0 g, 8.3 mmol) in EtOH (40 mL) was added at rt dropwise 3-bromoaniline (51) (0.95 mL, 8.32 mmol). The mixture was stirred under reflux for 3 h. Solvents were evaporated, and the crude material was triturated with hexane to get (3-bromophenyl)(5-iodopyrimidin-4-yl)amine (52) as light yellow solid (2.2 g, 70%). 1H NMR (600 MHz, DMSO-d6) δ 8.98 (br s, 1H), 8.78 (s, 1H), 8.62 (s, 1H), 7.86 (t, J = 1.81 Hz, 1H), 7.60 (td, J = 1.70, 7.68 Hz, 1H), 7.31−7.37 (m, 2H). LC−HRMS (m/z): [M + H]+ calcd for C10H7BrIN3 + H+, 375.8941; found, 375.8940; Diff, −0.1 mDa. 7-Bromo-9H-pyrimido[4,5-b]indole (53). A solution of (3bromophenyl)(5-iodopyrimidin-4-yl)amine (52) (400 mg, 1.06 mmol) in DMA (8 mL) was degassed with Ar for 10 min. Dry NaOAc (130 mg, 1.60 mmol) and [Pd(OAc)2(PPh3)2] (79 mg, 0.11 mmol) were added. After repeated degassing with Ar (10 min) the vial was capped and irradiated in a microwave apparatus at 130 °C for 4 h. The reaction mixture filtered through Celite, and solvents were evaporated. Purification by chromatography over silica gel and further by trituration with DCM afforded 7-bromo-9H-pyrimido[4,5-b]indole (53) (30 mg, 11%) as light yellow solid. 1H NMR (600 MHz, DMSOd6) δ 12.47 (br s, 1H), 9.49 (s, 1H), 8.97 (s, 1H), 8.22 (d, J = 8.26 Hz, 1H), 7.73 (d, J = 1.71 Hz, 1H), 7.51 (dd, J = 1.76, 8.31 Hz, 1H). LC− HRMS (m/z): [M + H]+ calcd for C10H6BrN3 + H+, 246.9745; found, 246.9753; Diff, 0.8 mDa. 7-(6-Fluoropyridin-3-yl)-9H-pyrimido[4,5-b]indole (55). To a solution of 7-bromo-9H-pyrimido[4,5-b]indole (53) (20 mg, 0.081 mmol) and 6-fluoropyridin-3-ylboronic acid (54) (23 mg, 0.16 mmol) in DMF (2 mL) in a microwave vessel was added K2CO3 (33 mg, 0.24 mmol). The vial was purged with Ar for 10 min, and then [Pd(dppf)Cl2]·DCM (1.3 mg, 0.0020 mmol) was added and the mixture was purged again with Ar for 10 min. After sealing of the vial, the mixture was irradiated in a microwave apparatus at 100 °C for 1 h. The reaction mixture was filtered through a Celite plug. The residue

was washed with EtOAc (10 mL). The combined filtrates were washed with H2O (10 mL) and brine (10 mL), dried over Na2SO4, and the solvents were evaporated. Purification by chromatography (SiO2; 50− 60% EtOAc/hexane) afforded 7-(6-fluoropyridin-3-yl)-9H-pyrimido[4,5-b]indole (55) as light yellow solid (4.2 mg, 20%). 1H NMR (300 MHz, DMSO-d6) δ ppm 7.33 (ddd, J = 8.56, 2.92, 0.60 Hz, 1H), 7.69 (dd, J = 8.26, 1.61 Hz, 1H), 7.82 (d, J = 1.01 Hz, 1H), 8.40 (ddd, J = 8.20, 2.70 Hz, 1H), 8.38 (d, J = 8.06 Hz, 1H), 8.65 (dd, J = 1.61, 0.81 Hz, 1H), 8.96 (s, 1H), 9.50 (s, 1H), 12.50 (s, 1H). LC−HRMS (m/z): [M + H]+ calcd for C15H9FN4 + H+, 265.0884; found, 265.0885; Diff, 0.1 mDa. 7-Bromo-5-(toluene-4-sulfonyl)-5H-pyrido[4,3-b]indole (59). Step a: A solution of 1-acetyl-3-bromopiperidin-4-one (56) (3.50 g, 11.7 mmol) and (3-bromophenyl)hydrazine hydrochloride (57) (2.99 g, 13.4 mmol) in BF3·Et2O (70 mL) was heated 16 h at 120 °C. Volatilities were evaporated, and the residue was diluted with H2O (15 mL), neutralized with saturated aqueous Na2CO3, and extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, and the solvent was evaporated. Chromatography (SiO2; 2% MeOH in DCM) afforded an inseparable mixture of 7-bromo-5H-pyrido[4,3-b]indole (58a) and 9-bromo-5Hpyrido[4,3-b]indole (58b) (2.0 g, 69%). LC−HRMS (m/z): [M + H]+ calcd for C11H7BrN2 + H+, 246.9866; found, 246.9866; Diff, 0.0 mDa. Step b: The mixture of 7-bromo-5H-pyrido[4,3-b]indole (58a) and 9-bromo-5H-pyrido[4,3-b]indole (58b) (2.0 g, 8.1 mmol) was dissolved in DMF (50 mL), cooled to 0 °C, and NaH (60% 485 mg, 12.1 mmol) was added. The reaction mixture was stirred at 25 °C for 30 min, and then TsCl (2.46 g, 12.1 mmol) was added and the reaction mixture was stirred at rt for 1 h. H2O was added, and the product was extracted with EtOAc (2 × 30 mL). The combined organic layers were washed with brine, dried over Na2SO4, and the solvent was evaporated. Chromatography (SiO2; EtOAc/hexane 2:3) afforded 7-bromo-5-(toluene-4-sulfonyl)-5H-pyrido[4,3-b]indole (59) (700 mg, 21%) as light brown solid. 1H NMR (600 MHz, DMSO-d6) δ 9.45 (d, J = 0.91 Hz, 1H), 8.71 (d, J = 5.84 Hz, 1H), 8.37 (d, J = 1.51 Hz, 1H), 8.27 (d, J = 8.26 Hz, 1H), 8.17 (dd, J = 0.86, 5.79 Hz, 1H), 7.90 (d, J = 8.46 Hz, 2H), 7.73 (dd, J = 1.71, 8.26 Hz, 1H), 7.38 (d, J = 8.16 Hz, 2H), 2.28−2.33 (m, 3H). LC−HRMS (m/z): [M + H]+ calcd for C18H13BrN2O2S + H+, 400.9954; found, 400.9959; Diff, 0.5 mDa. 7-(4-Fluoropiperidin-1-yl)-5-(toluene-4-sulfonyl)-5H-pyrido[4,3b]indole (61). To a solution of 7-bromo-5-(toluene-4-sulfonyl)-5Hpyrido[4,3-b]indole (59) (50 mg, 0.12 mmol) and 4-fluoropiperidine (60) (19 mg, 0.19 mmol) in THF (7 mL) was added Cs2CO3 (60 mg, 0.19 mmol) at rt. The mixture was purged 10 min with N2 before addition of xanthphos (5.8 mg, 0.01 mmol) and [Pd2(dba)3]·CHCl3 (18.3 mg, 0.02 mmol). The reaction mixture was purged 10 min with N2, and the vial was capped and heated to 90 °C for 16 h. The mixture was filtered through Celite, and the Celite pad was washed with EtOAc (10 mL). Solvents were evaporated and the residue was purified by chromatography (SiO2; EtOAc/hexane 2:3) to give 7-(4-fluoropiperidin-1-yl)-5-(toluene-4-sulfonyl)-5H-pyrido[4,3-b]indole (61) as a light brown solid (40 mg, 76%). 1H NMR (600 MHz, DMSO-d6) δ 9.22 (d, J = 0.91 Hz, 1H), 8.54 (d, J = 5.74 Hz, 1H), 8.10 (dd, J = 0.96, 5.69 Hz, 1H), 8.02 (d, J = 8.76 Hz, 1H), 7.83 (d, J = 8.02 Hz, 2H), 7.66 (d, J = 2.12 Hz, 1H), 7.35 (dd, J = 0.60, 8.00 Hz, 2H), 7.19 (dd, J = 2.22, 8.76 Hz, 1H), 4.84−4.99 (m, 1H), 3.48−3.56 (m, 2H), 3.33− 3.37 (m, 2H), 2.28 (s, 3H), 1.99−2.09 (m, 2H), 1.82−1.89 (m, 2H). LC−HRMS (m/z): [M + H]+ calcd for C23H22FN3O2S + H+, 424.1489; found, 424.1490; Diff, 0.1 mDa. 7-(4-Fluoropiperidin-1-yl)-5H-pyrido[4,3-b]indole (62). To a solution of 7-(4-fluoropiperidin-1-yl)-5-(toluene-4-sulfonyl)-5H-pyrido[4,3-b]indole (61) (40 mg, 0.095 mmol) in THF/H2O (1:1) (10 mL) was added NaOH (19 mg, 0.47 mmol). The reaction mixture was heated to 50 °C for 16 h. More NaOH (20 mg, 0.50 mmol) and MeOH (3 mL) were added, and stirring was continued at reflux for 6 h. The solvent was evaporated, and the residue was diluted with DCM (20 mL), then washed with H2O and brine. After drying over Na2SO4 and evaporation of the solvent, the pale yellow solid was purified by column chromatography (SiO2; 0.5−1% MeOH/DCM). The product was further purified by trituration with two portions of Et2O (2 × 5 7364



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flask. The solvent and volatile byproducts were removed by vacuum transfer. The crude product was purified by preparative HPLC (XBridge C18, 5 μm, 10 mm × 250 mm) using 0.02 M H3PO4/ MeCN/H2O as eluent (gradient 1−13 min from 1:1:8 to 1:8:1, run time 15 min) at a flow rate of 6 mL/min. An amount of 460 MBq (12.4 mCi) of the desired compound was obtained with a radiochemical purity of 94% and a specific activity of 2.6 TBq/mmol (70 Ci/mmol). The identity of the labeled compound was confirmed by MS and by co-injection of the cold reference standard with the radiolabeled material. MS m/z: 238.9 [M + H]+ (10%), 240.0 [M(3H) + H]+ (12%), 242.9 [M(3H2) + H]+ (9%), 244.9 [M(3H3) + H]+ (70%). 2-(6-Fluoro[2,4-3H2]pyridin-3-yl)pyrrolo[2,3-b:4,5-c′]dipyridine ([3H]9). In a 2 mL tritiation flask, 2-(6-fluoropyridin-3-yl)pyrrolo[2,3b:4,5-c′]dipyridine (9) (2.0 mg, 7.6 μmol) and Crabtree’s catalyst (9.14 mg, 11.4 μmol) were dissolved in DCM (0.8 mL) and DMF (0.2 mL). The flask was attached to the tritium manifold 20 (RC-TRITEC) and degassed by three freeze−thaw cycles. Tritium gas was introduced, and the light orange solution was vigorously stirred for 4 h in an atmosphere of tritium at 450 mbar. The solution was cooled by liquid nitrogen and the excess tritium gas in the reaction vessel was reabsorbed on a uranium trap for waste-tritium. The solvent was lyophilized off, and labile tritium was removed by lyophilization with a 9:1 mixture of EtOH and H2O (3 × 1 mL) and toluene (2 × 1 mL). The remaining brownish oil was dissolved in EtOH (1.5 mL) and transferred on a SCX-2 cation exchanger. Remaining catalyst was eluted with MeOH (10 mL) and discarded, and the product was eluted with NH3 in MeOH (3.5 N, 10 mL), collected separately, and concentrated under reduced pressure. The crude product was purified by preparative HPLC (XBridge Prep, 5 μm, 10 mm × 250 mm) using 0.05 M KH2PO4 buffer, pH 7/MeCN/water as eluent (gradient 1−13 min from 1:1:8 to 1:8:1, run time 15 min) at a flow rate of 6 mL/min. An amount of 37 MBq (1 mCi) of the title compound was obtained with a radiochemical purity of 99% and a specific activity of 936 GBq/ mmol (25.3 Ci/mmol), determined by MS spectrometry. The compound was stored as a pH 7 buffer/DMSO solution. The identity of the labeled compound was confirmed by MS and by co-injection of the cold reference standard with the radiolabeled material. MS: m/z = 265.1 [M + H]+ (34%), 267.1 [M(3H) + H]+ (43%), 269.1 [M(3H2) + H]+ (23%). N-(2-Fluoroethyl)-2-[2,6- 3 H 2 ]phenylimidazo[1,2-a]pyridin-7amine ([3H]22). In a 2 mL tritiation flask, N-(2-fluoroethyl)-2phenylimidazo[l,2-a]pyridin-7-amine (22) (2.0 mg, 7.8 μmol) and Crabtree’s catalyst (9.6 mg, 12 μmol) were dissolved in DCM (1.0 mL). The reaction and the purification were performed in analogy to compound 9 to provide 1.5 GBq (40 mCi) of the desired compound with a radiochemical purity of 96% and a specific activity of 1.96 TBq/ mmol (52.9 Ci/mmol). The identity of the labeled compound was confirmed by MS and by co-injection of the cold reference standard with the radiolabeled material. MS m/z: 255.9 [M + H]+ (2%), 258.0 [M(3H) + H]+ (14%), 260.0 [M(3H2) + H]+ (81%), 261.9 [M(3H3) + H]+ (3%). N-Cyclopropyl-2-(4-methoxy[2,6- 3 H 2 ]phenyl)imidazo[1,2-a]pyrimidin-7-amine ([3H]34). In a 2 mL tritiation flask, N-cyclopropyl2-(4-methoxyphenyl)imidazo[1,2-a]pyrimidin-7-amine (34) (3.0 mg, 10.7 μmol) and Crabtree’s catalyst (12.9 mg, 16.1 μmol) were dissolved in DCM (1.0 mL). The reaction was performed in analogy to compound 7. After removal of the reaction vessel from the tritiation manifold, the remaining brownish oil was dissolved in DCM (45 mL) and EtOH (5 mL) and transferred to a volumetric flask. The light yellow solution was filtered over an SCX-3 cation exchanger and washed with DCM (20 mL). The filtrate was discarded, and the product was eluted with a solution of NH3 in MeOH (7 N) and DCM (1:1, 20 mL), collected separately, and concentrated under reduced pressure. The crude product was purified by preparative HPLC (XBridge Prep, 5 μm, 10 mm × 250 mm) using 0.05 M triethylammonium acetate at pH 10/MeCN/water (gradient 1−13 min from 1:1:8 to 1:8:1, run time 15 min) as eluent at a flow rate of 6 mL/min. The collected fractions were diluted with H2O (10 mL) and extracted twice with DCM (2 × 15 mL). The organic layers were

mL). 7-(4-Fluoropiperidin-1-yl)-5H-pyrido[4,3-b]indole (62) was isolated as off-white solid (6.5 mg, 25%). 1H NMR (600 MHz, DMSO-d6) δ (ppm) 11.37 (s, 1H), 9.13 (s, 1H), 8.29 (d, J = 5.54 Hz, 1H), 7.99 (d, J = 8.46 Hz, 1H), 7.35 (dd, J = 0.91, 5.54 Hz, 1H), 6.98 (s, 1H), 6.99 (d, J = 11.10 Hz, 1H), 4.81−4.94 (m, 1H), 3.41−3.47 (m, 2H), 3.20−3.25 (m, 2H), 1.98−2.07 (m, 2H), 1.81−1.88 (m, 2H). LC−HRMS (m/z): [M + H]+ calcd for C16H16FN3 + H+, 270.1401; found, 270.1402; Diff, 0.1 mDa. 4-(2-Fluoroethyl)piperidine Hydrochloride (63). A suspension of tert-butyl 4-(2-fluoroethyl)piperidine-l-carboxylate42 (1.21 g, 5.23 mmol) in HCl (4 M in dioxane, 5.23 mL, 20.9 mmol) was stirred at rt for 2 h. The solution was concentrated in vacuum and dried on high vacuum to yield 4-(2-fluoroethyl)piperidine hydrochloride (63) as white solid (906 mg, 100%). 1H NMR (600 MHz, DMSO-d6) δ 8.84 (br s, 1H), 8.57 (br s, 1H), 4.50 (td, J = 5.80, 47.50 Hz, 2H), 3.19− 3.25 (m, 2H), 2.79−2.87 (m, 2H), 1.78−1.84 (m, 2H), 1.66−1.73 (m, 1H), 1.57−1.66 (m, 2H), 1.29−1.40 (m, 2H). LC−HRMS (m/z): [M + H]+ calcd for C7H14FN + H+, 132.1183; found, 132.1182; Diff, −0.1 mDa. 3 H-Radiosynthesis. General Methods. Liquid scintillation counting for tritium compounds was accomplished using a HIDEX 300 SL and ULTIMATE GOLD cocktail (PerkinElmer Inc., Waltham, MA, USA). Precoated thin-layer chromatography sheets (TLC) were obtained from Merck KGaA (Darmstadt, Germany). Developed plates were visualized using automatic TLC linear analyzer (Berthold Technologies, Bad Wildbad, Germany). Radiochemical purity was measured using the β radioactivity HPLC detector RAMONA with internal solid scintillator (Raytest, Straubenhardt, Germany). Specific activity was determined by mass spectrometric isotopic peak intensity distribution, using 4000QTRAP system (AB Sciex GmbH, Zug, CH), flow injection mode with a CTC PAL, and an Agilent 1100 microLC pump without any separation. 2-(4-Methoxy[2,6- 3 H 2 ]phenyl)imidazo[1,2-a]pyridin-7-amine ([3H]7). In a 2 mL tritiation flask, 2-(4-methoxyphenyl)imidazo[1,2a]pyridin-7-amine (7) (2.0 mg, 8.4 μmol) and Crabtree’s catalyst (10.1 mg, 15.5 μmol) were dissolved in DCM (1.0 mL). The flask was attached to the tritium manifold (RC-TRITEC) and degassed by three freeze−thaw cycles. Tritium gas was introduced, and the light orange solution was vigorously stirred for 4 h in an atmosphere of tritium at 1050 mbar. The solution was cooled by liquid nitrogen, and the excess tritium gas in the reaction vessel was reabsorbed on a uranium-trap for waste-tritium. The solvent was lyophilized off, and labile tritium was removed by lyophilization with a 9:1 mixture of EtOH and H2O (3 × 1 mL) and toluene (2 × 1 mL). The remaining brownish oil was dissolved in DCM (25 mL) and transferred on a SCX-3 cation exchanger. Remaining catalyst was eluted with DCM (15 mL) and discarded, and the product was eluted with NH3 in MeOH (1 N, 25 mL), collected separately, and concentrated under reduced pressure. The crude product was purified by preparative HPLC (XBridge C-18 Prep, 5 μm, 10 mm × 250 mm) using 0.1 M ammonium formate, pH 9/MeCN/water as eluent (gradient 1−13 min from 1:1:8 to 1:8:1, run time 15 min) at a flow rate of 6 mL/min. An amount of 833 MBq (22.5 mCi) was obtained of the title compound with a radiochemical purity of 99% and a specific activity of 1.02 TBq/mmol (27.6 Ci/ mmol), as determined by MS spectrometry. The compound was stored as an ethanolic solution. The identity of the labeled compound was confirmed by MS and by co-injection of the cold reference standard with the radiolabeled material. MS m/z: 240.2 [M + H]+ (48%), 242.2 [M(3H) + H]+ (10%), 244.2 [M(3H2) + H]+ (40%), 246.2 [M(3H3) + H]+ (2%). 2-(3-Methylphenyl)-N-([3H3]methyl)imidazo[1,2-a]pyrimidine-7amine ([3H]8). A solution of [3H]methylnosylate in toluene (1 mL, 1.85 GBq, 0.66 μmol) was transferred to a 1.5 mL reactor and concentrated under a stream of argon. A solution of 2-(3methylphenyl)imidazo[1,2-a]pyrimidine-7-amine (32) (380 μg, 1.7 μmol) in THF (0.24 mL) and Cs2CO3 (1400 mg, 4.3 μmol) were added, and the reaction mixture was concentrated to roughly 50 μL. After rinsing with THF (50 μL), the reactor was tightly closed, and the reaction mixture was stirred for 24 h. The reaction mixture was portionwise diluted with DCM (30 mL) and transferred to a 50 mL 7365



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separated, dried over Na2SO4, and concentrated under reduced pressure. An amount of 1.30 GBq (35.1 mCi) of the title compound was obtained with a radiochemical purity of 99% and a specific activity of 1.64 TBq/mmol (44.3 Ci/mmol), determined by MS spectrometry. The compound was stored as a methanolic solution (20 mL) with additional 5 μg/mL dithiothreitol to minimize radiolytic decomposition. The identity of the labeled compound was confirmed by MS and by co-injection of the cold reference standard with the radiolabeled material. MS m/z: 280.9 [M + H]+ (9%), 282.9 [M(3H) + H]+ (27%), 284.9 [M(3H2) + H]+ (64%). 2-(4-(2-Fluoroethoxy)[2,6-3H2]phenyl)-N-methylimidazo[1,2-a]pyrimidin-7-amine ([3H]37). In a 2 mL tritiation flask, 2-(4-(2fluoroethoxy)phenyl)-N-methylimidazo[1,2-a]pyrimidin-7-amine (37) (2.0 mg, 6.8 μmol) and Crabtree’s catalyst (5.5 mg, 6.8 μmol) were dissolved in DCM (0.8 mL) and DMF (0.2 mL). The reaction and the purification were performed in analogy to compound 7 to provide 482 MBq (13 mCi) of the desired compound with a radiochemical purity of 98% and a specific activity of 2.00 TBq/mmol (54.1 Ci/mmol). The product was stored as a solution in ethanol (10 mL). The identity of the labeled compound was confirmed by MS and by co-injection of the cold reference standard with the radiolabeled material. MS m/z: 286.9 [M + H]+ (3%), 289.0 [M(3H) + H]+ (14%), 290.9 [M(3H2) + H]+ (76%), 292.9 [M(3H3) + H]+ (7%). 11 C- and 18F-Radiolabeling. General Methods for Carbon-11 and Fluorine-18 Syntheses. All chemicals and solvents were ACS or HPLC purity and purchased through Aldrich Chemical Co. or Fisher Scientific except where noted. All solid phase extraction cartridges were conditioned with 10 mL of absolute ethanol followed by 10 mL of HPLC water. The radiochemical and chemical quality control HPLC chromatograms were acquired using an Agilent 1260 Infinity system incorporating a quaternary pump, HiP ALS autosampler, and DAD UV detector with a Max-Light flow plus a Bioscan Flow-Count interface with a NaI radioactivity detector. Chromatographic data were analyzed using Agilent OpenLAB chromatography data system (revision A.04.02). The microwave radiosynthesis module (MRM) used is a custom-made semiautomated radiosynthesis device constructed and controlled as previously described.43,44 The ELIXYS (Sofie Biosciences, Inc., Culver City, CA) module is a commercially available automated multireactor radiosynthesizer.45 2-(4-[11C]Methoxyphenyl)imidazo[1,2-a]pyridin-7-amine ([11C]7). A standard gas carbon dioxide target of a General Electric (GE) Medical Systems (GEMS, Uppsala, Sweden) PETTrace cyclotron was filled with high purity nitrogen containing 0.5% oxygen. The target was irradiated with a proton beam at 60 μA for 25 min to produce approximately 2−3 Ci (74−111 GBq) of [11C]carbon dioxide. The radioactive gas was transferred to a GE FXMeI module that synthesizes 11CH3I in approximately 10 min, after which the 11CH3I was transferred by helium gas to an appropriate hot cell for radiosynthesis. The precursor, compound 18 (1 ± 0.3 mg, 4.4 μmol), was dissolved in 200 μL of dimethylformamide (DMF) and added to a vial containing sodium hydride (0.6−1.0 mg) that was subsequently sealed. Prior to the end of bombardment (EOB), the vial was placed in a lead-lined synthesis cell. After trapping of 11CH3I, the vial was heated (80 °C) for 3 min. The reaction solution was diluted with 1 mL of aqueous triethylamine/phosphoric acid buffer (pH 7.2; TEA buffer) and injected onto the semipreparative HPLC column (XBridge C-18, 10 μm, 10 mm × 150 mm), eluting with 20:80 absolute ethanol/TEA buffer (pH 7.2) at 10 mL/min (Figure 1 in Supporting Information) with the effluent monitored for radioactivity content and UV (254 nm). The product peak (tR = 5.6 min, k′ = 4.9) was collected in 50 mL of water containing approximately 250 mg of ascorbic acid. The product solution was eluted onto a conditioned Oasis SepPak Plus (Waters Corp.), and the SepPak was washed with water HPLC water (10 mL containing approximately 50 mg of ascorbic acid). The radiotracer product was eluted from the SepPak with absolute ethanol (1 mL) followed by sterile saline (10 mL) through a 0.2 μm sterile Millipore FG filter (25 mm) into a sterile product vial preloaded with sterile saline (4 mL). Aliquots were removed from the final product vial for quality control analysis. Analytical HPLC was performed to determine radiochemical and

chemical purity, specific activity (Figure 2 in Supporting Information), and chemical identity using an XBridge C-18 column (3.5 μm, 4.6 mm × 100 mm) eluted with 20:80 acetonitrile (MeCN)/TEA buffer (pH 7.2), eluted at 2 mL/min, and monitored at 254 nm. N-[11C]Methyl-2-(3-methylphenyl)imidazo[1,2-a]pyrimidine-7amine ([11C]8). 11CH3I (described above) was transferred by helium gas to an appropriate hot cell for radiosynthesis. The precursor, compound 32 (0.5 ± 0.2 mg, 2.2 μmol), was dissolved in 200 μL of dimethylsulfoxide (DMSO), and 5 μL of 6 N NaOH was added. Prior to EOB, the vial was placed in a hot cell. After addition of 11CH3I, the reaction solution is heated (80 °C) for 3 min. The reaction solution was diluted with 200 μL of aqueous triethylamine/phosphoric acid buffer (pH 7.2; TEA buffer) and injected onto the semipreparative HPLC column (XBridge C-18, 10 μm, 10 mm × 150 mm) eluted with 20:80 absolute ethanol/TEA buffer (pH 7.2) at 10 mL/min (Figure 3 in Supporting Information) with the effluent monitored for radioactivity content and UV (254 nm). The product peak (tR = 8.5 min, k′ = 7.7) was collected in 50 mL of water containing approximately 250 mg of ascorbic acid. The product solution was eluted onto a conditioned Oasis SepPak Plus (Waters Corp.), the SepPak was washed with water, HPLC water. The radiotracer product was eluted from the SepPak with absolute ethanol (1 mL) followed by sterile saline (10 mL) through a 0.2 μm sterile Millipore FG filter (25 mm) into a sterile product vial preloaded with sterile saline (4 mL). Aliquots were removed from the final bottle for quality control analysis. Analytical HPLC was performed to determine radiochemical and chemical purity, specific activity (Figure 4 in Supporting Information), and chemical identity using an XBridge C-18 column (3.5 μm, 4.6 mm × 100 mm) eluted with 25:75 acetonitrile (MeCN)/TEA buffer (pH 7.2), eluted at 2 mL/min, and monitored at 254 nm. [18F]2-(6-Fluoropyridin-3-yl)pyrrolo[2,3-b:4,5-c′]dipyridine ([18F]9) Using the Microwave Radiofluorination Module. [18O]Water (Huayi Isotopes, Jiangsu, China, approximately 2 mL) was loaded into the niobium body, high yield [18F]fluoride target of a GEMS PETTrace cyclotron. The target was irradiated with a proton beam of 55 μA for 30 min to produce approximately 1.65 Ci (61 GBq) of aqueous [18F]fluoride by the 18O(p,n)18F nuclear reaction. The microwave radiosynthesis module (MRM) was prepared with all reagents and equipment prior to EOB. At EOB, the [18F]fluoride was trapped to a Chromafix 30-PS-HCO3 SPE cartridge (ABX GmbH, Germany) previously conditioned with 1 mL of high purity water (Fluka). Under computer control, the resin was eluted with 150 μL of a potassium carbonate (K2CO3)/Kryptofix 2.2.2 stock solution (10 mg of K2CO3 and 48 mg of Kryptofix dissolved in 600 μL of 1:1 MeCN/ water) into a 5 mL reaction vial sealed with a multiport cap. After rinsing with 250 μL of MeCN, the vial was heated to 110 °C with nitrogen gas flow for 150 s. Two separate additions of 250 μL of MeCN were heated with gas flow for 150 and 180 s, respectively. After the drying sequence was complete, the 5 cc V-vial was transferred to the microwave cavity cooled with airflow. The precursor, compound 49 (0.5 mg ±0.2 mg, 1.7 μmol), in 400 μL of DMSO was added to the vial. The vial was microwave irradiated with 50 W for 240 s. The solution was diluted with 1 mL of HPLC water and 3 mL of TEA buffer (pH 7.2). The crude solution was injected onto a Xbridge C-18 column (10 μm, 10 mm × 150 mm) eluted with a solution of 15:85 MeCN/aqueous triethylamine buffer (pH 7.2) at flow rate of 15 mL/min (Figure 5 in Supporting Information). The product peak (tR = 20.7 min, k′ = 33) was collected in a reservoir of 50 mL of HPLC water. The collected fraction was pushed by nitrogen through a C-18 SepPak Plus, and the SepPak was then rinsed with HPLC water (10 mL). The radiotracer product was eluted from the cartridge with absolute ethanol (1 mL) followed by sterile saline (10 mL) through a 0.2 μm sterile Millipore FG filter (25 mm) into a sterile product vial preloaded with sterile saline (4 mL). Aliquots were removed from the final bottle for quality control analysis. Analytical HPLC was performed to determine radiochemical and chemical purity, specific activity (Figure 6 in Supporting Information), and chemical identity using an XBridge C-18 column (3.5 μm, 4.6 mm × 100 mm) eluted with 40:60 methanol/TEA buffer (pH 7.2), eluted at 1.5 mL/min, and monitored at 350 nm. 7366



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identity using an XBridge C-18 column (3.5 μm, 4.6 mm × 100 mm) eluted with 20:80 acetonitrile/TEA buffer (pH 7.2), eluted at 2 mL/ min, and monitored at 254 nm. In Vitro Biological Testing. General Considerations. All final compounds were examined for known classes of assay interference compounds (pan assay interference compounds, PAINS)46 and classified as negative. In Vitro Autoradiography. Fresh frozen human brain tissue blocks were purchased from the Banner Sun Health Research Institute (Sun City, AZ, USA). Pathological diagnosis of AD was made according to standard NIA-Reagan Institute criteria based on neuropathological data. Tissue sections (10 μm) were cut in a cryostat (Leica CM3050) at −17 °C chamber temperature and −15 °C object temperature and thaw-mounted on microscope glass slides (HistoBond, Paul Marienfeld GmbH, Lauda-Königshofen, Germany). Brain sections were incubated for 30 min in 50 mM Tris-HCl buffer at room temperature. For affinity testing of novel compounds, 10 nM [3H]3 were co-incubated with 10 nM of the novel unlabeled compound. For macroautoradiographical analysis of novel compounds, the tritiated version of the compound was incubated at 3 nM. For microautoradiographies a radioligand concentration of 30 nM was used. After incubation all sections were rinsed three times for 10 min in icecold Tris buffer and dipped three times in distilled water at 4 °C. Slidemounted brain sections were dried for at least 3 h and exposed to a Fuji imaging plate (BAS-TR 2025, Fujifilm, Dielsdorf, Switzerland) with a [3H]microscale (RPA-510, GE Healthcare, Glattbrugg, Switzerland) for 5 days. The imaging plate was scanned with 25 μm resolution in a Fujifilm high-resolution plate scanner (BAS-5000, Bucher Biotec AG, Basel, Switzerland). For microautoradiographies, the sections were dipped in NTB emulsion (Kodak) and kept for >2 weeks at 4 °C in a dark box before being developed for 2 min in Kodak developer solution, fixed for 5 min in Kodak fixer solution, and finally analyzed under the light microscope. Visualization and quantification of macroautoradiographies were performed with the MCID image analysis program (version 7; InterFocus Imaging GmbH, Mering, Germany). Total amount of radioligand bound (TB) to the brain areas of interest was expressed as fmol of bound radioligand per mg of protein. For affinity testing of novel compounds, TB was determined in gray matter (GM) regions of late-stage AD tissue sections with high tau pathology, nonspecific binding (NSB) was determined in a tau-free white matter (WM) region of the same tissue section, and specific binding (SB) was calculated as follows: SB = TB − NSB. The displacement potency of novel unlabeled test compounds was calculated according to the formula 100 − (SB test compound / SBradioligand only) × 100. For quantitative assessment of novel tritiated compounds, NSB was determined by calculating radioligand binding in GM regions of healthy control tissue sections devoid of tau pathology. SB to tau aggregates was assessed by quantifying the difference in binding to tau-rich cortical GM in AD tissue (TB) versus cortical GM of healthy controls (NSB). Signal-to-noise ratios (SNR) were quantified by calculating GM/WM ratios of radioligand binding to late-stage AD cortical tissue sections. Immunohistochemical Colocalization. Macro- and microautoradiographies were analyzed by costaining of NFTs and Aβ plaques on the same tissue section using the tau- and Aβ-specific antibodies pS42240 and BAP-241 (5 mg/mL, 2 h at room temperature). For macroautoradiographies, antibody incubation was performed after exposure and scanning of the sections. For microautoradiographies, the sections were first incubated with the antibodies before dipping in NTB emulsion and exposure. Baboon PET Imaging. Male baboons (Papio anubis), with a body weight of 22−28 kg, were used for this study. The experimental protocol was approved by the American Care and Use Committee of Johns Hopkins University. The animals were initially sedated intramuscularly with ketamine hydrochloride with restraint dosages of 7.5−10 mg/kg to achieve a light stage of anesthesia and then maintained on continuous propofol intravenous infusion at 0.3−0.4 mg kg−1 min−1 (DIPRIVAN injectable emulsion) after intubation. All tracers were delivered intravenously over 1 min as a slow bolus. The dynamic PET scanning started immediately upon initiation of the

[18F]2-(6-Fluoropyridin-3-yl)pyrrolo[2,3-b:4,5-c′]dipyridine ([18F]9) Using Standard Thermal Heating (ELIXYS). Aqueous [18F]fluoride was prepared as described above. After all chemicals and components were loaded onto the ELIXYS synthesis cassette, the [18F]fluoride ion was delivered to a 5 mL V-vial in a dose calibrator. The automated ELIXYS synthesis sequence was started with pushing the [18F]fluoride ion with nitrogen through a Chromafix 30-PS-HCO3 SPE cartridge previously preconditioned by washing with 1 mL of high purity water. [18O]Water was collected for recycling. The resin cartridge was eluted with a solution of K2CO3 (4.0 mg, 28.9 μmol) and Kryptofix (20.0 mg, 53.1 μmol) in 250 μL of 1:1 MeCN/water into the 5 mL reactor V-vial with glass stir bar in the ELIXYS reactor. The solution was dried at 110 °C under vacuum and nitrogen flow for 120 s with stirring. Two separate additions of MeCN (600 μL) were heated under vacuum and nitrogen flow for 180 and 150 s, respectively. The vial was cooled to room temperature, and a solution of the precursor compound 49 (0.5 mg, 1.7 μmol) in DMSO (500 μL) was added to the reaction vessel containing the dried [18F]fluoride. The solution was heated with stirring at 160 °C for 10 min. HPLC water (3.5 mL) was added to the reaction mixture prior to HPLC purification, formulation, and quality control as described above. [11 C]-N-Cyclopropyl-2-(4-[11 C]methoxyphenyl)imidazo[1,2-a]pyrimidin-7-amine ([11C]34). 11CH3I (described above) was transferred by helium gas to an appropriate hot cell for radiosynthesis. The precursor, compound 35 (1 ± 0.3 mg, 3.8 μmol), was dissolved in 200 μL of DMF and then added to sodium hydride (0.6−1.0 mg) and sealed. Prior to the EOB, the vial was placed in a hot cell. After addition of 11CH3I, the reaction solution is heated (80 °C) for 3 min. The reaction solution was diluted with 200 μL of aqueous triethylamine/phosphoric acid buffer (pH 7.2; TEA buffer) and injected onto the semipreparative HPLC column (XBridge C-18, 10 μm, 10 mm × 150 mm) eluted with 25:75 absolute ethanol/TEA buffer (pH 7.2) at 10 mL/min (Figure 7 in Supporting Information) with the effluent monitored for radioactivity content and UV (254 nm). The product peak (tR = 6.9 min, k′ = 5.9) was collected in 50 mL of water containing approximately 250 mg of ascorbic acid. The product solution was eluted onto a conditioned Oasis SepPak Plus (Waters Corp.), and the SepPak was washed with water, HPLC water. The radiotracer product was eluted from the SepPak with absolute ethanol (1 mL) followed by sterile saline (10 mL) through a 0.2 μm sterile Millipore FG filter (25 mm) into a sterile product vial preloaded with sterile saline (4 mL). Aliquots were removed from the final bottle for quality control analysis. Analytical HPLC was performed to determine radiochemical and chemical purity, specific activity (Figure 8 in Supporting Information), and chemical identity using an XBridge C-18 column (3.5 μm, 4.6 mm × 100 mm) eluted with 25:75 acetonitrile (MeCN)/TEA buffer (pH 7.2), eluted at 2 mL/min, and monitored at 254 nm. [ 18 F]2-(4-(2-Fluoroethoxy)phenyl)-N-methylimidazo[1,2-a]pyrimidin-7-amine ([18F]37) Using the Microwave Radiofluorination Module. Aqueous [18F]fluoride was prepared and dried as described above. After the drying sequence was complete, the 5 cc Vvial was transferred to the microwave cavity and cooled with airflow. The precursor, compound 38 (2 mg ± 0.5 mg, 4.6 μmol), in 400 μL of DMSO was added to the vial. The vial was microwave irradiated with 50 W for 80 s. The solution was diluted with 1 mL of HPLC water and was injected onto a Xbridge C-18 column (10 μm, 10 mm × 150 mm) eluted with a solution of 30:70 methanol (MeOH)/aqueous triethylamine buffer (pH 7.2) at a flow rate of 15 mL/min (Figure 9 in Supporting Information). The product peak (tR = 11.58 min, k′ = 15.4) was collected in a reservoir of 50 mL of HPLC water containing 250 mg of ascorbic acid. The collected fraction was pushed by nitrogen through an Oasis SepPak Plus, and the SepPak was rinsed with HPLC water (10 mL). The radiotracer product was eluted from the SepPak with absolute ethanol (1 mL) followed by sterile saline (10 mL) through a 0.2 μm sterile Millipore FG filter (25 mm) into a sterile product vial preloaded with sterile saline (4 mL). Aliquots were removed from the final bottle for quality control analysis. Analytical HPLC was performed to determine radiochemical and chemical purity, specific activity (Figure 10 in Supporting Information), and chemical 7367



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tracer infusion and continued for 90 min in 3D list mode. The following ranges of radiochemical and mass doses were used for the PET studies: 350−780 MBq; 0.045−1.26 μg. Each animal was positioned in an ECAT HRRT brain PET scanner (high resolution research tomography, CPS Innovations, Inc., Knoxville, TN). A 6 min transmission scan with a 37 MBq 137Cs point source was initially done for attenuation correction. PET images were acquired in full 3D mode and reconstructed with high resolution span-3 modality with the following frame schedule: four 15 s, four 0.5 min, three 1 min, two 2 min, five 4 min, 12 5 min frames (total of 30 frames in 90 min). The image reconstruction procedure consisted of histogramming of the listmode data into specified dynamic sinograms. The statistical 3D reconstruction of each frame sinogram used six iterations of the OPOSEM algorithm (16 subsets), followed by 2 mm Gaussian postsmoothing. The attenuation, dead time, and decay corrections were performed for generation of quantitative dynamic images (image volume of 256 (left-to-right) by 256 (nasion-to-inion) by 207 (neckto-cranium) voxels, voxel size of 1.22 mm by 1.22 mm by 1.22 mm). The final spatial resolution is expected to be about 2.5 mm full-width at half-maximum in three directions.47 A catheter was placed in the femoral artery to obtain arterial blood samples at various time points. Sampling of arterial blood was performed every 6 s during the first minute of the experiment and thereafter at intervals of progressively increasing duration. A total of about 42 samples were collected in each dynamic PET scan. Blood samples were immediately centrifuged, and plasma samples were assayed for radioactivity content using a γ counter that was cross-calibrated against the PET. Plasma from selected samples was analyzed by HPLC for the presence of parent compound and its radiolabeled metabolites using a general method developed previously for PET radiotracers.48 On the basis of the measurements at 0, 5, 10, 20, 30, 60, and 90 min, post-tracer injection, the metabolite ratio of each tracer at each time of arterial blood sampling was calculated using a linear interpolation method.49 Then, the total radioactivity at each measurement was distributed to authentic tracer and metabolites. The metabolite-corrected plasma activity−time curve was used as the input function for tissue tracer kinetic modeling. The individual frames from 10 min to the end of the scan were summed into one frame for purposes of MRI-to-PET coregistration and visual presentation of PET images. A standard volume of interest template50 was spatially aligned to the summed PET image of the first baseline scan of each subject. Then, VOIs were transferred to individual PET spaces according to realignment parameters between the first baseline scan and successive baseline and blocking scans for each animal. Minimal editing was allowed for individual images. VOIs were applied to individual PET frames to obtain VOI time−radioactivity curves.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (+41) 61-688-80-34. ORCID

Luca C. Gobbi: 0000-0002-0563-2491 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We sincerely thank Roland Humm and the scientists at Chembiotek for the synthesis of compounds. We acknowledge the excellent technical assistance in generating the biological data of Patricia Glaentzlin, Céline Sutter, Svenja Moes, and Jennifer Beck. We thank Christian Bartelmus for collecting HRMS spectroscopical data and Bjoern Wagner for testing compounds in the LIMBA assay. Additionally we thank the staff of the Johns Hopkins PET Radiotracer Center for their radiochemistry expertise. This study was funded by F. Hoffmann-La Roche Ltd. contract to The Johns Hopkins University (JHU). JHU faculty receive salary support through a number of sponsored research sources including DFW NIH Career Award K24 DA000412, and none receive direct funding from Roche except via sponsored JHU contracts.

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ABBREVIATIONS USED B/P, brain to plasma AUC ratio; dppf, 1,1′-bis(diphenylphosphino)ferrocene; f u_p, free fraction in plasma; GM, gray matter; Kryptofix 2.2.2, 4,7,13,16,21,24-hexaoxa-1,10diazabicyclo[8.8.8]hexacosane; LIMBA, lipid membrane binding assay; mAb, monoclonal antibody; NFT, neurofibrillary tangle; Ns, nosyl; NSB, nonspecific binding; NT, neutropil thread; PAMPA, passive cell membrane permeability assay; SB, specific binding; SPE, solid phase extraction; SUV, standardized uptake value; WM, white matter

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REFERENCES

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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.7b00632. Radiochemical synthesis procedure for [3H]3, radioactivity and ultraviolet chromatograms of [11C]7, [11C]8, [18F]9, [11C]34, and [18F]37, experimental details on the determination of drug distribution coefficients in octanol (log D), of brain lipids/water distribution coefficient (LIMBA log Dbrain), and of high throughput passive membrane permeability assay (PAMPA) values, protocols for the single dose pharmacokinetics studies in mouse, the determination of P-gp-mediated drug transport, and the determination of drug binding to plasma proteins (PDF) Molecular formula strings and some data (CSV) 7368



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