EP4658651A1 - Novel compounds for tau imaging - Google Patents

Abstract

The present disclosure provides novel compounds of the formula: (I) wherein R¹, R², R³, R⁴, and R⁵ are as described herein, methods of preparing the same, pharmaceutical compositions the same, tau imaging formuations, and methods of using the compounds for tau imaging.

Description

Novel Compounds for Tan Imaging

FIELD OF THE INVENTION

[01] The present disclosure relates to novel compounds; intermediates for preparation of these compounds; methods of using these compounds for tau imaging; compositions and formulations of these compounds for diagnostic imaging; and methods of imaging using these compounds, compositions, and formulations.

BACKGROUND

[02] Alzheimer’s disease (AD), a leading cause of dementia, develops in one percent of the population between the ages 65 and 69, and increases to 40-50% in those 95 years and older. AD patients exhibit telltale clinical symptoms that include cognitive impairment and deficits in memory function. In these patients, the presence of AD is confirmed by heavy senile plaque burden and neurofibrillary tangles (NFT) found in the cerebral cortex upon post mortem histopathological examination. The mature senile plaques consist of extracellular [3-amyloid peptides derived from enzymatic processing of amyloid precursor protein and intracellular neurofibrillary tangles (NFT), which are derived from filaments of hyperphosphorylated tau proteins. Aggregates of hyperphosphorylated tau, such as neurofibrillary tangles, are linked to the degree of cognitive impairment in Alzheimer's disease. In AD and various other tauopathies, tau aggregates appear in particular brain regions and patterns that are linked to disease risk, onset, and or progression, and these regions and patterns are known to skilled artisans. In AD patients, tau-containing tangles first appear in brain regions that are very closely linked to memory, and pathologic studies show that tangles may correlate even more strongly with cognition than plaques. Signals arising from a tau imaging agent in these regions and patterns can be used by skilled artisans to better monitor and diagnose the risk, onset and progression of the particular disease state. (See Correlation of Alzheimer disease neuropathologic changes with cognitive status: a review of the literature. Nelson PT, et al., J Neuropathol Exp Neurol. 2012 May; 71(5):362-81.) Thus, simple noninvasive methods, for detecting and/or quantitation of tau deposits in patients are desired. (See M. Maruyama et al., “Imaging of tau pathology in a tauopathy mouse model and in Alzheimer patients compared to normal controls”, Neuron, 79: 1094-1108, 2013, C. Mathis and ffl. Klunk, “Imaging Tau Deposits In Vivo: Progress in Viewing More of The Proteopathy Picture ”, Neuron, 79: 1035-10-37, 2013).

[03] Existing agents that can image tau using Positron Emission Tomography (PET) are known in the art, for example such agents are recited in W02009/102498, WO201 1/119565. Further, the compound [¹⁸F]T807 (also known as AV-1451, the structure of which is shown below), which is approved by the U.S. Food and Drug Administration (FDA), is recited in WO 2013/176698. (See also [(18)F]T807, a novel tau positron emission tomography imaging agent for Alzheimer's disease. Xia CF, et al., Alzheimer ’s Dement. 2013 Nov; 9(6):666-76)

[¹⁸F]T807

[04] However, existing tau imaging compounds have technical attributes that could be improved by the design of innovative agents, which may provide enhanced tau images, with improved tau signaling and minimal non-tau signaling or improved tau selectivity. Thus, improved methods for detecting and/or quantitation of tau in patients are eagerly sought.

[05] Current clinically validated tau tracers are limited to detection of neurofibrillary tangles found in later stage Alzheimer’s disease largely due to lack of selective binding by the tracer. (See An Autoradiographic evaluation of AV-1451 Tau PET in dementia; Lowe et al. Acta Neuropathologica Communications (2016) 4:58 and Tau PET imaging in neurodegenerative tauopathies-still a challenge; Leuzy et al. Molecular Psychiatry (2019) 24: 1112-1134). There remains a need for detection of early AD tau deposits and tau isoforms in non-AD (3R or 4R) tauopathies such as progressive supranuclear palsy (PSP), cortical basal degeneration (CBD), and Pick’s disease (PiD), atypical Alzheimer’s disease, chronic traumatic encephalopathy (CTE)and frontotemporal dementia (FTD). Novel PET tracers would be valuable in facilitating the understanding of early-AD tau and non-AD tau related neurodegenerative processes and providing better tools to improve patient stratification and earlier inclusion in clinical trials assessing novel therapeutics for these diseases. Detection of early tau accumulation could provide an endpoint for evaluation of therapeutic efficacy in early stage AD patients while identification of a PET ligand for a non-AD tauopathy (e.g.-PSP, CBD, or PiD) would be valuable for disease differentiation and staging in patients and for quantification of therapeutic efficacy. As of yet, there are no selective imaging agents for non-AD tauopathies available to facilitate a better understanding of the underlying mechanisms and progressions of these neurodegenerative diseases.

[06] There are several potential benefits of imaging tau in the brain with improved imaging agents. Enhanced tau imaging will improve diagnosis by identifying potential patients, those having high levels of tau in the brain, who may have increased chance of developing AD. Imaging with an improved tau imaging agent will also be useful to monitor tau accumulation and localization, and or progression of AD and or other tauopathies via PET, and when anti-tau drug treatments become available, tau imaging may provide an essential tool for monitoring treatment. The present disclosure provides novel compounds, compositions, formulations and methods for tau imaging. Improved technology advancing the capacity to image tau in patients is also needed to expand the clinical benefits and impact of diagnostic tau imaging. An improved imaging agent may provide better PET images with better clarity due to better tau selectivity. Improved tau imagining agents may also increase our understanding the onset and progression of dementia in general and AD and non-AD tauopathies, in particular, which in turn may lead to provide better treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[07] FIG. 1 depicts autoradiography on AD brain sections for Kd determination for Example 3 following the protocol described in Assay Example 32.

[08] FIG. 2 depicts autoradiography on AD brain sections for selectivity determination for Example 3 following the protocol described in Assay Example 33.

[09] FIG. 3 depicts autoradiography on PSP brain sections for Example 4 for the determination of binding following the protocol described in Assay Example 34.

[10] FIG. 4 depicts autoradiography on CBD brain sections for Example 4 for the determination of binding following the protocol described in Assay Example 34. DETAILED DESCRIPTION

[11] The present disclosure provides a compound of the following Formula I or a pharmaceutically acceptable salt thereof wherein: n is 0, 1, or 2;

R¹ is H, halo, C1-C3 alkyl, or C3-C6 cycloalkyl;

R² is H, halo, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, -O-(Ci-C₄ alkyl), C1-C4 alkylhalo, C2-C4 alkenylhalo,

R³ is H or F;

R⁴ is F, ¹⁸F, Ci-C₄-alkylF, Ci-C₄-alkyl¹⁸F, -O-(Ci-C₄-alkyl)F, or -O-(Ci-C₄- alkyl)¹⁸F; and

R⁵ is H, halo, or C1-C4 alkyl; provided when n is 0, R¹ is methyl, and R², R³, and R⁵ are each H, then R⁴ is not F or ¹⁸F positioned at the 3 position of the 4-membered ring.

[12] In Formula I, n may be 0, 1, or 2. If n is 0, then the ring will be a 4-membered azetidin-l-yl. If n is 1, then the ring will be a 5-membered, pyrrolidin-l-yl ring. If n is 2, then the ring will be a piperidin-l-yl. In each of these ring systems, conventional naming systems identify the nitrogen atom in the 1 position of the ring. In selected embodiments, the R⁴ substituent is attached to the 3-position on the ring. In other embodiments, when n is 2 for the piperidin-l-yl ring system R⁴ can be positioned at the 3- or 4-position on the ring.

[13] Compounds of Formula I do not include the two compounds illustrated below: [14] In other forms, the present disclosure provide a compound of Formula I above, or a pharmaceutically acceptable salt thereof wherein n is 0, 1, or 2; R¹ is H, halo, C1-C2 alkyl, or cyclopropane; R² is H, halo, -CH₃, -CH₂CH₃, -OCH₃, -CH=CH₂, -CF=CH₂, -

CH2CH2F, -CH₂CH₂ ¹⁸F, -CH2CH2CH2F, -CH2CH₂CH2¹⁸F, -OCH2CH2F, or - OCH2CH2¹⁸F; and R⁵ is H, halo, or C1-C4 alkyl.

[15] In other forms, the present disclosure provides a compound of Formula I above, or a pharmaceutically acceptable salt thereof wherein n is 0 or 1; R¹ is H, halo, C1-C2 alkyl, or cyclopropane; R² is H, halo, -CH₃, -OCH₃, each at the 3-position of the cycloalkyl ring; and R⁵ is H, halo, or -CH₃.

[16] In another form, the present disclosure provides a compound of Formula I above, or a pharmaceutically acceptable salt thereof, wherein n = 0; R¹ is H, halo, -CH₃, or -CH₂CH₃; R² is H, halo, -CH₃, -OCH₃, -CH=CH₂, -CF=CH₂,

-C=CH, or ; R³ is H or F; R⁴ is F or ¹⁸F each at the 3-position of the cycloalkyl ring; and R⁵ is H, halo, or -CH₃.

[17] In another form, the present disclosure provides a compound of Formula I above, or a pharmaceutically acceptable salt thereof, wherein R¹ is -CH₃; R² is H; R³ is H; R⁴ is F or ¹⁸F positioned at the 3-position of the cycloalkyl ring; and R5 is Cl.

[18] In another form, the present disclosure provides a compound of Formula I above, or a pharmaceutically acceptable salt thereof, wherein n is 0; R¹, R², R³, and R⁵ are each H; and R⁴ is F or ¹⁸F.

[19] In another form, the present disclosure provides a compound of Formula I above, or a pharmaceutically acceptable salt thereof, wherein n is 0; R¹ is H; R² is Cl, F or I; R³ is H; R⁴ is F or ¹⁸F; and R⁵ is H.

[20] In another form, the present disclosure provides a compound of Formula I above, or a pharmaceutically acceptable salt thereof, wherein n is 0; R¹, R³, and R⁵ are each H; R² is -CH₃, -CH2CH3, -CF=CH₂, -C=CH, F, Cl, I, or R₄ is F or ¹⁸F positioned at the 3- position of the 4-membered ring. [21] In another form, the present disclosure provides a compound of Formula I above, or a pharmaceutically acceptable salt thereof, wherein n is 0; R¹ is -CH3 or -CH2CH3; R², R³ and R⁵ are each H; and R⁴ is F or ¹⁸F positioned at the 3-position of the 4-membered ring.

[22] In another form, the present disclosure provides a compound of Formula I above, or a pharmaceutically acceptable salt thereof, where n is 2; R¹ is H; R² is H or -CH ; R3 is H; R4 is F, ¹⁸F, -C1-C3 alkylF, or -C1-C3 alkyl¹⁸F; and R⁵ is H.

[23] In some embodiments, the present disclosure provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein the compound is:

[24] In yet other forms, the present disclosure provides a pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt thereof according to Formula I above as described in the various forms above and one or more of a pharmaceutically acceptable carrier, diluent, or stabilizer. Examples of pharmaceutical compositions and processes for their preparation can be found in “Remington: The Science and Practice of Pharmacy”, Loyd, V., et al. Eds., 22nd Ed., Mack Publishing Co., 2012.

[25] In another form, the present disclosure provides a pharmaceutical composition comprising a compound according to Formula I, or a pharmaceutically acceptable salt thereof, as described in the various forms above wherein the carrier comprises ethanol, water, and a buffer suitable for injection into a patient. Preferably, the composition includes sodium chloride in an amount to provide a formulation suitable for injection into a patient.

[26] In this form, the buffer may comprise sodium chloride, sodium phosphate, or sodium ascorbate.

[27] Examples of diluents includes water for injection and saline. The diluent can be included in the pharmaceutical composition in an amount sufficient to provide a concentration of the radiolabeled embodiment of a compound of Formula I, or a pharmaceutically acceptable salt thereof, suitable to facilitate the diagnosis of a patient at risk for or suffering from dementia or AD.

[28] Examples of stabilizers, in particular radiolytic stabilizers, include ethanol, ascorbic acid, monothioglycerol, vitamin E, and cysteine.

[29] The compounds of the present disclosure are preferably formulated as pharmaceutical compositions that are administered for intravenous use in a patient, preferably in humans. Such pharmaceutical compositions and processes for preparing the compositions are known in the art. See, e.g., Remington: The Science and Practice of Pharmacy (P.P. Gerbino, 21st ed., Lippincott Williams & Wilkins, 2006). Methods of using tau imaging agents for PET imaging of tau are known to those of skill in the art. See e.g. [(18)F]T807, a novel tau positron emission tomography imaging agent for Alzheimer's disease. Xia CF, et al., Alzheimer’s Dement. 2013 Nov; 9(6):666-76.). [(18)F]T807 is also known as [18F]AV-1451.

[30] In another aspect the invention provides a pharmaceutical composition comprising a compound of Formula I, a or pharmaceutically acceptable salt thereof, for imaging tau. The tau imaging formulation is preferably formulated for use in humans. In one embodiment, the tau imaging formulation includes a compound according to Formula I, or a pharmaceutically acceptable salt thereof, formulated in 10% EtOH (v/v), 0.45% (w/v) sodium ascorbate in 0.9% sodium chloride.

[31] The present disclosure also provides methods of imaging tau comprising introducing into a patient a detectable quantity of a compound of Formula I, or a pharmaceutically acceptable salt thereof. In another form the present disclosure provides methods of imaging tau comprising introducing into a patient a pharmaceutical composition comprising a detectable quantity of a compound of Formula I, or a pharmaceutically acceptable salt thereof.

[32] The present disclosure provides a method of imaging tau comprising introducing into a mammal a detectable quantity of a pharmaceutical composition as described according to the embodiments herein, and allowing sufficient time for said pharmaceutical composition to become associated with tau; and detecting the radiolabeled compound. The preferred method for detecting the radiolabeled compound uses PET.

[33] The present disclosure provides the use of compounds of Formula I or pharmaceutically acceptable salts thereof. Further, the present disclosure also provides that the compounds (or pharmaceutically acceptable salts) of Formula I may be used, for the manufacture of a radiopharmaceutical agent for imaging tau in a patient, preferably humans.

[34] The present disclosure provides a process of making a compound according to Formula 1 with a 18F radiolabel. In certain embodiments, the present disclosure provides methods of preparing a compound of Formula 1, or a pharmaceutically acceptable salt thereof, from a precursor compound outlined herein.

[35] As used herein, “alkyl”, “Ci, C₂, C₃, C₄, C₅ or C₆ alkyl” or “Ci-C ₆ alkyl” is intended to include Ci, C₂, C₃, C₄, C5 or Ce straight chain (linear) saturated aliphatic hydrocarbon groups and C3, C₄, C5 or Ce branched saturated aliphatic hydrocarbon groups. For example, Ci-Ce alkyl is intended to include Ci, C₂, C₃, C₄, C5 and Ce alkyl groups. Examples of alkyl include, moieties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl or n-hexyl. In some embodiments, a straight chain or branched alkyl has six or fewer carbon atoms (e.g., Ci-Ce for straight chain, C3-C6 for branched chain), and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms. When the term is used with the term halogen or specifically an F atom or an 18F atom, the halogen or F(¹⁸F) atom replaces any one of the hydrogen atoms on the carbon chain. In some embodiments, the F or ¹⁸F atom is attached to the terminal carbon atom of the chain.

[36] The term “-O-(Ci-C₄ alkyl)” refers to an alkoxyl group that includes from 1 to 4 carbon atoms. Said alkyl group can be a straight or branched alkyl chain. When the term is used with the term halogen or specifically an F atom or ¹⁸F atom; the halogen or F(¹⁸F) can be attached replacing one of the hydrogen atoms attached to the carbon backbone of the chain. In some embodiments, the F atom or ¹⁸F atom is attached to the terminal carbon in a straight chain alkyl group.

[37] As used herein, the term “alkylhalo” includes saturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but where at least halogen atom replaces any one of the hydrogen atoms on the carbon chain.

[38] As used herein, the term “alkenyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term “alkenyl” includes straight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenyl groups. In some embodiments, a straight chain or branched alkenyl group has six or fewer carbon atoms in its backbone (e.g., C₂-Ce for straight chain, C3-C6 for branched chain). The term “C₂-C₄” includes alkenyl groups containing two to four carbon atoms. The term “C₂-Ce” includes alkenyl groups containing two to six carbon atoms. The term “Cs-Ce” includes alkenyl groups containing three to six carbon atoms. Depending upon the substituents attached to the two carbons of the double bond the geometry about the double bond can be described in as either a cis or trans double bond. When the term is used with a halogen or specifically an F atom or an ¹⁸F atom, the halogen or F(¹⁸F) atom can be attached replacing one of the hydrogen atoms attached to the carbon backbone of the alkenyl chain.

[39] As used herein, “C2-C6 alkenylene linker” is intended to include C2, C3, C4, C5 or Ce chain (linear or branched) divalent unsaturated aliphatic hydrocarbon groups.

[40] As used herein, the term “alkenylhalo” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but where at least halogen atom replaces any one of the hydrogen atoms on the carbon chain.

[41] As used herein, the term “alkynyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, “alkynyl” includes straight chain alkynyl groups (e.g, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl), and branched alkynyl groups. In some embodiments, a straight chain or branched alkynyl group has six or fewer carbon atoms in its backbone (e.g, C2-C6 for straight chain, C3-C6 for branched chain). The term “C2-C6” includes alkynyl groups containing two to six carbon atoms. The term “C2-C4” includes alkynyl groups containing two to four carbon atoms. The term “Cs-Ce” includes alkynyl groups containing three to six carbon atoms. As used herein, “C2-C6 alkynylene linker” is intended to include C2, C3, C4, C5 or Ce chain (linear or branched) divalent unsaturated aliphatic hydrocarbon groups. For example, C2-C6 alkenylene linker is intended to include C2, C3, C4, C5 and Ce alkenylene linker groups.

[42] As used herein, the term “cycloalkyl” refers to a saturated or partially unsaturated hydrocarbon monocyclic or polycyclic (e.g., fused, bridged, or spiro rings) system having 3 to 30 carbon atoms (e.g., C3-C12, C3-C10, or Cs-Cs). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 1,2,3,4-tetrahydronaphthalenyl, and adamantyl. In the case of polycyclic cycloalkyl, only one of the rings in the cycloalkyl needs to be non-aromatic. [43] The term “radiolabeled compound” refers to one of the compounds described below that includes an 18F atom.

[44] The term “leaving group” (LG) refers to -NO2, a trialkyl amine, alkyl sulfonate, or aryl sulfonate. Alkyl sulfonates of the present disclosure include C1-C4 alkyl sulfonate. Aryl sulfonates of the present disclosure include phenyl sulfonate, wherein the phenyl group is optionally substituted once with C1-C4 alkyl, halogen or nitro, Methanesulfonate (mesylate) and ethanesulfonate are preferred alkyl sulfonates. Benzenesulfonate, 4- methylbenzenesulfonate (tosylate), 4-bromobenzenesulfonate and 4- nitrobenzenesulfonate are preferred aryl sulfonates.

[45] The bond illustrated as indicates the point of attachment of the 4- methylpyrazolyl ring to the rest of the molecule.

[46] The term “pharmaceutically acceptable salt” as used herein refers to a salt of a compound of the invention considered to be acceptable for clinical and/or veterinary use. Examples of pharmaceutically acceptable salts and common methodology for preparing them can be found in “Handbook of Pharmaceutical Salts: Properties, Selection and Use” P. Stahl, et al., 2nd Revised Edition, Wiley-VCH, 2011 and S.M. Berge, et al., "Pharmaceutical Salts" , Journal of Pharmaceutical Sciences, 1977, 66(1), 1-19.

[47] As used herein, the term “effective amount” refers to an amount that is a dosage, which is effective in imaging tau. The attending physician, as one skilled in the art, can readily determine an effective amount by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining an effective amount or dose of a compound, a number of factors are considered, including, but not limited to whether the compound or its salt, will be administered; the co-administration of other agents, if used; the species of mammal; its size, age, and general health; the degree of involvement or the severity of the disorder; the response of the individual patient; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of other concomitant medication; and other relevant circumstances.

[48] As used herein, the term "patient" refers to a mammal. Preferably, the patient is a human or companion mammal, such as, a dog or cat; or other domesticated mammal, such as, a cow, pig, horse, sheep, rabbit, mouse, rat, and goat. [49] A treating physician, veterinarian, or other medical person will be able to determine an effective amount of the compound for treatment of a patient in need. Preferred pharmaceutical compositions can be formulated as an injectable solution. The solution can include a compound of the present disclosure in an amount effective for treating a patient in need of treatment.

[50] Novel compounds of Formula I have been discovered to be advantageous for tau imaging, preferably including human clinical imaging. Some of the preferred compounds of Formula I possess a combination of particularly useful properties for tau imaging, including high affinity for tau. In vivo, some of the preferred compounds demonstrate advantageous tissue distribution and pharmacokinetics. Ex vivo and/or in vitro, some of the compounds demonstrate high affinity binding to tau, and label tau containing tissue samples from AD brain with high selectivity with respect to A|3 and/or non-tau binding.

[51] The abbreviations used herein are defined according to Daub G.H., et al., “The Use of Acronyms in Organic Chemistry” Al dri chimica Acta, 1984, 17(1), 6-23. Other terms as used herein, the following terms have the meanings indicated: “AD” refers to Alzheimer’s disease; “Boc” or “BOC” refers to tert-butoxy carbonyl; “cat amt” refers to catalytic amount; “CT” or “CAT” refers to computer tomography; “DMAP” refers to 4- (dimethylamino)pyridine; “DMF” refers to dimethylformamide; “DMPAO” refers to (2,6-Dimethylanilino)(oxo)acetic acid; “DMSO” refers to dimethylsulfoxide; “EOS” refers to end of synthesis; “ESI” refers to electrospray ionization; “EtOH” refers to ethanol; “HPLC” refers to high performance liquid chromatography; “hr” or “h” refers to hour; “HRMS” refers to high resolution mass spectrometry; “LCMS” refers to liquid chromatography mass spectrometry; “mCT” refers to Micro-Computed Tomography; “MeOEF refers to methanol; “min” refers to minutes; “mPET” refers to Micro-Positron Emission Tomography; “MS” refers to mass spectroscopy; “OMs” refers to O-mesyl”; “OTs” refers to O-tosyl; “PBS” refers to phosphate-buffered saline; “PET” refers to Positron Emission Tomography; “PHF” refers to paired helical filament; “Prec” refers to precursor; “Prep” refers to preparations compounds; “RCP” refers to radiochemical purity; “RT” refers to room temperature; “SM” refers to starting material; “TAC” refers to Time Activity Curves; “OTMS” refers to O-trimethylsilyl; “WFI” refers to water for injection. General Chemical Synthesis

[52] The following Schemes, preparations, precursors, and examples are provided to better elucidate the practice of the present disclosure. Suitable reaction conditions for the steps of these schemes, preparations, precursors and examples are known in the art and appropriate modification of reaction conditions, including substitution of solvents and coreagents are within the ability of the skilled artisan.

[53] Furthermore, the skilled artisan will appreciate that in some circumstances, the order in which moieties are introduced is not critical. The particular order of steps required to produce the a compound of Formula I is dependent upon the particular compound being synthesized, the starting compound, and the relative lability of the substituted moieties, as is well appreciated by the skilled chemist. The skilled artisan will appreciate that not all substituents are compatible with all reaction conditions. These compounds may be protected or modified at a convenient point in the synthesis by methods well known in the art. The intermediates and final products of the present disclosure may be further purified, if desired by common techniques such as recrystallization or chromatography over solid supports such as silica gel or alumina.

[54] The compounds of the present disclosure, or salts thereof, may be prepared by a variety of procedures known in the art, some of which are illustrated in the schemes, preparations, precursors, and examples below. The specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare compounds or salts of the present disclosure. The products of each step in the chemes below can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the schemes below, all substituents unless otherwise indicated, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art.

[55] All reactions are run under a nitrogen atmosphere unless otherwise noted. Reagents, solvents, and supplies are purchased from commercial sources unless otherwise indicated. Compounds are purified using an automated Teledyne Isco Flash Chromatography System. HRMS data is obtained on a Waters QT of mass spectrometer using an electrospray ionization positive scan mode. Nominal resolution MS data are obtained on a Waters Micromass ZQ mass spectrometer using an ESI positive ionization scan mode.

EXAMPLES

Scheme 1

(5H-pyrido[l,2-a]benzimidazole ring formation)

Method A

Method A; Step 1

Copper catalyzed benzo[4,5]imidazo[l,2-a]pyridine formation

[56] The 5H-pyrido[l,2-a]benzimidazole (IV) can be prepared by reacting an appropriately substituted bromoaniline (II), a bromopyridine (III), a base such as cesium carbonate, a catalytic amount of a copper(I) iodide, and 1,10-phenanthroline in a nonpolar solvent such as a mixture of xylenes. When the reaction is complete, cool the mixture to room temperature and filter to remove any solids. Isolate the desired material using standard procedures. In some of the preparations below, X is a halogen, such as bromine or chlorine. In other preparations, X is a cyclic amine. The cyclic amine can be substituted with linker “L”, which can be a bond or alkyl or O-alkyl. A leaving group “LG”, for example, -NO2, a trialkyl amine, alkyl sulfonate, or aryl sulfonate to the cyclic amine via L to form a Precursor. Alternatively, R4 can be attached to the cyclic amine via L

Method B; Step 2

Synthesis of N-phenylpyridine-2-amines

[57] Combine an appropriately substituted aniline (V), a substituted pyridine 1 -oxide

(VI), bromotripyrrolidinophosphonium hexafluorophosphate, and a base such as diisopropylethylamine in methylene chloride is stirred at room temperature until the reaction is complete. The reaction mixture is concentrated and the residue is subjected to silica gel chromatography to isolate the desired phenylpyridine compound (VII).

Method B; Step 3

Oxidative Ring closure to form benzo[4,5]imidazo[l,2-a]pyridines:

[58] Stir a mixture of an appropriately substituted pyridine-2-amine (VII) and (di acetoxy iodo)benzene in solvent for 1 to 5 hrs monitoring the reaction to completion. Concentrate the mixture and isolate the desired benzo[4,5]imidazo[l,2-a]pyridines (VIII via chromatography.

Scheme 2

Amine Coupling

Method C

RuPhos Catalyzed Buchwald amine coupling:

[59] Combine the halobenzo[4,5]imidazo[l,2-a]pyridine (VIII), a cyclic amine (IX), a base and chloro(2-dicyclohexylphosphino-2',6'-di-i-propoxy-l,l'-biphenyl)[2-(2- aminoethylphenyl)]palladium(II), methyl -t-butyl ether adduct (Ru Phos-Pd) (cat. amt) in dioxane heat the mixture. The reaction can be monitored by LCMS. Upon completion, the reaction mixture is cooled and isolate the desired compound (XI) using standard chromatography or other purification techniques.

Method D

XPhos Catalyzed Buchwald amine coupling:

[60] A mixture of the bromobenzo[4,5]imidazo[l,2-a]pyridine (1 eq), a cyclic amine (1.3-2 eq), potassium phosphate monohydrate (7 eq), and chloro(2- dicyclohexylphosphino-2',4',6'-tri-i-propyl-l,T-biphenyl)[2-(2-aminoethyl)phenyl] palladium(II) methyl -t-butyl ether adduct (X Phos-Pd) (0.2 eq) in dioxane (0.07-0.15 M) is heated at 100 °C. The reaction is monitored by LCMS. Upon completion, the reaction mixture is cooled to room temperature and concentrated. The desired material is isolated by column chromatography (silica gel) eluting with methylene chloride:methanol gradient.

Method E

Copper catalyzed amine coupling:

[61] A mixture of the bromobenzo[4,5]imidazo[l,2-a]pyridine (1 eq), a cyclic amine (1.5 eq), L-proline (0.5 eq), copper (I) iodide (0.3 eq), and anhydrous potassium phosphate (3 eq) in dimethylsulfoxide (0.25 M) is heated at 100-110 °C in a 20 ml reaction vial. The reaction is monitored by LCMS. Upon completion, the reaction mixture is cooled to room temperature and poured into deionized water. The aqueous mixture is extracted with 90: 10 methylene chloride:methanol (5 x). The combined organic layers are washed with 50% ammonium hydroxide, dried over magnesium sulfate, filtered, and concentrated. The crude material is purified by column chromatography on silica gel using a methylene chloride:methanol gradient.

Method F Copper catalyzed amine coupling:

[62] A mixture of 2,4-dibromo-5-fluoropyridine (1 eq), the appropriate amine (1-1.4 eq), copper (I) iodide (0.1-0.15 eq), 2-((2,6-dimethylphenyl)amino)-2-oxoacetic acid (DMPAO) (0.15-0.2 eq), potassium phosphate (3 eq) and dimethylsulfoxide (0.2-0.6 M) is purged under nitrogen for 10 minutes. The reaction is heated at 100 °C for 2-24 hours. Water is added and the mixture is extracted with ethyl acetate. The combined organic layers are dried over sodium sulfate, filtered and washed with dichloromethane. The solvent is removed under vacuum. In some cases, the reaction is cooled to room temperature and added dropwise to water. The solids are isolated by filtration and washed with water. The solids are re-dissolved in 10% methanol in dichloromethane, and adsorbed onto silica gel. The crude product is purified by column chromatography on silica gel using a gradient of 5 to 65% ethyl acetate in hexanes. Scheme 3

Precursor Formation

Method G

[63] Stir a mixture of the appropriate l-(benzo[4,5]imidazo[l,2-a]pyridin-3- yl)azeti din-3 -ol (XII), where L is as defined above, methanesulfonyl chloride or p- toluenesulfonyl chloride to provide LG, which is as defined above, and a base in methylene chloride (6-25 mM). (In some cases, catalytic DMAP can be added) Upon completion, the reaction is worked up using convention neutralization and/or extraction techniques. The desired Precursor XIII can be isolated via chromatographic techniques.

Scheme 4

Fluorine-18 Radiolabeling Procedures

General Radiosynthesis Method

[64] A solution of the appropriately substituted Precursor (XIII) where L’ and LG is as defined above, is added to the reaction vessel containing the anhydrous Cryptand 2.2.2- K2CO3 [¹⁸F]fluoride and the resulting mixture is kept at 140 °C for 10 minutes followed by hydrolysis with 1 mL of IN NaOH at 65 °C for 3 minutes. After cooling to 60 °C, the crude reaction mixture is neutralized with 2 mL of 0.5N HC1 (1 mL of IN HC1 + 1 mL WFI). The reaction crude is then loaded onto a semi-preparative HPLC column for purification using isocratic elution. Preparation of Starting Materials

Preparation 1

3-Bromo-8-methylbenzo[4,5]imidazo[l,2-a]pyridine

[65] 2,4-dibromopyridine (20.0 g, 84.6 mmol), copper (I) iodide (3.22 g, 16.9 mmol), 1,10-phenanthroline (6.10 g, 33.8 mmol), cesium carbonate (110 g, 338 mmol), diatomaceous earth (16 g) and /?-xylene (170 mL) were combined. 2-bromo-4- methylaniline (10.6 mL, 84.6 mmol) was added to the resulting slurry, and nitrogen gas was bubbled through the vigorously stirred mixture for 10 minutes. The reaction mixture was heated to 135 °C for 24 hours, cooled to room temperature and filtered. The filter cake was rinsed with methylene chloride and ethyl acetate, and the combined organic filtrates were concentrated under reduced pressure over silica gel. The crude reaction product was purified by chromatography on silica gel using a gradient of 0 to 10% ethyl acetate in methylene chloride. The resulting brown solid was slurried in methylene chloride and triturate using hexanes, then isolated by filtration to provide the title compound (6.52 g, 25.0 mmol, 30% yield) as a shiny yellow solid. ES/MS m/z (⁷⁹Br/⁸¹Br) 260.92/262.92 [M+H]

[66] The compounds in Table 1 below were prepared essentially according to the procedure above for Preparation 1 and as generally described in Scheme 1; Method A (step 1).

Table 1

Preparation 24

4-Chloro-N-(4-cyclopropylphenyl)pyridin-2-amine

[67] A solution of 4-cyclopropylaniline (0.213 g, 1.6 mmol), 4-chloropyridine N-oxide (0.25 g, 1.9 mmol), and diisopropylethylamine (0.98 mL, 5.6 mmol) in dichloromethane (8 mL) was treated with bromotripyrrolidinophosphonium hexafluorophosphate (PyBrop) (1.19 g, 2.6 mmol) and stirred at room temperature overnight. The reaction mixture was purified by column chromatography on silica gel (0 to 70% ethyl acetate in hexanes) to afford the pure product as a light brown solid (0.266 g, 68%). ES/MS m/z (³⁵C1/³⁷C1) 245.1/246.9 (M+H)

Preparation 27

3-Chloro-8-cyclopropylbenzo[4,5]imidazo[l,2-a]

[68] A solution of 4-chloro-N-(4-cyclopropylphenyl)pyridin-2-amine (0.266g, 1.09 mmol) in hexafluoroisopropanol (11 mL) was treating with bis(/c/7- butylcarbonyloxy)iodobenzene (0.46 g, 1.14 mmol) and stirred at room temperature overnight. The reaction mixture was concentrated and purified by column chromatography on silica gel (0 to 60% ethyl acetate in hexanes) to afford the title compound as a light brown solid (0.22 g, 84%). ES/MS m/z (³⁵C1/³⁷C1) 243.3/245.0 (M+H)

Preparation 29

3-Chloro-8-iodobenzo[4,5]imidazo[l,2-a]pyridine

[69] The compound was prepared from 4-chloro-N-(4-iodophenyl)pyridine-2-amine in 54% yield by essentially following the procedure of Preparation 27. ES/MS m/z (³⁵C1/³⁷C1) 328.8/330.7

Preparation 30

3-Chloro-8-ethylbenzo[4,5]imidazo[l,2-a]pyridine

[70] Compound 30 was prepared essentially following Scheme 1; Method B, Steps 2 and 3. To a mixture of 4-chloropyridine /f-oxide (1.3 g, 10 mmol) and 4-ethylaniline (1.2 g, 10 mmol) in di chloromethane (50 mL) at room temperature was added diisopropylethylamine (5.2 mL, 30 mmol) and bromotripyrrolidinophosphonium hexafluorophosphate (PyBrop) (5.1 g, 11 mmol). The resulting solution was stirred at room temperature overnight, then concentrated and purified by column chromatography on silica gel (0-50% ethyl acetate in hexanes) to afford a red solid (1.4 g, 60%). This material was dissolved in hexafluoroisopropanol (40 mL) and bis(tert-butylcarbonyloxy) iodobenzene (2.56 g, 6.3 mmol) was added. The reaction was stirred at room temperature for 3-4 hours, then concentrated and purified by column chromatography on silica gel (0 to 70% ethyl acetate in hexanes) to obtain the title compound as a yellow solid (1.2 g, 5.2 mmol, 52% over 2 steps). ES/MS m/z (³⁵C1/³⁷C1) 231.1/232.9 (M+H) Preparation 31 l-(7-Bromo-8-fluorobenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol

[71] 3,7-dibromo-8-fluorobenzo[4,5]imidazo[l,2-a]pyridine (0.120 g, 0.349 mmol), 3- hydroxy azetidine hydrochloride (0.076 g, 0.698 mmol), sodium /c/V-butoxide (0.134, 1.40 mmol), chloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-l, 1 '-biphenyl) [2-(2'-amino- l,l'-biphenyl)]palladium(II) (Ruphos Pd G2) (0.027 g, 0.035 mmol) and dioxane (3mL) were combined and purged under nitrogen for 10 minutes. The mixture was heated in the microwave at 110 °C for 45 minutes and then cooled to room temperature. The solvent was removed under vacuum. The residue was purified by column chromatography on silica gel (2% to 30% methanol in methylene chloride). The selected fractions were combined and concentrated to give the title compound as a yellow solid (28 mg, 24%). ES/MS m/z 336.1(M+H)

[72] The compounds in Table 2 below were prepared essentially according to

Preparation 31 above and as generally described in Scheme 2; Method C.

Table 2

Preparation 39 l-(Benzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol Dimethylsulfoxide (110 mL) was added to a solid mixture of 3-bromo-8- methylbenzo[4,5]imidazo[l,2-a]pyridine (6.57 g, 25.2 mmol), azetidin-3-ol hydrochloride (5.52 g, 50.4 mmol), copper (I) iodide (480 mg, 2.52 mmol), DMPAO (972 mg, 5.04 mmol) and potassium phosphate tribasic (21.4 g, 101 mmol). Nitrogen gas was bubbled, with stirring, through the slurry for 15 minutes. The mixture was heated at 90 °C for 24 hours. The reaction mixture was cooled to room temperature and water (1000 mL) was slowly added with vigorous stirring. The precipitated solids were isolated by vacuum filtration and dissolved in 10% methanol in methylene chloride (500 mL). The aqueous filtrate was extracted with 10% methanol in methylene chloride (3 x 250 mL). The organic extracts were combined with the solution of isolated solids from the initial aqueous filtration, and dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting solids were dissolved in methylene chloride, adsorbed onto silica gel and purified by chromatography on silica gel using a gradient of 0 to 30% methanol in methylene chloride to afford the title compound as a grey-green solid (3.18 g, 12.6 mmol, 50% yield).HRMS (m/z): 254.1296 (M+H)

[73] The compounds in Table 3 below were prepared essentially according to Preparation 39 above and as generally described in Scheme 2; Method E.

Table 3 Preparation 49 l-(7-Bromobenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol

[74] Dimethylsulfoxide (6.7 mL) was added to a mixture of 3,7- dibromobenzo[4,5]imidazo[l,2-a]pyridine (500 mg, 1.53 mmol, 1.0 eq), azetidin-3-ol hydrochloride (336 mg, 3.06 mmol, 2.0 eq), copper (I) iodide (29.0 mg, 0.153 mmol, 0.1 eq), DMPAO (59.0 mg, 0.306 mmol, 0.2 eq) and potassium phosphate (977 mg, 4.60 mmol, 3.0 eq) and sparged with nitrogen for 10 minutes. The vial was sealed and heated at 90 °C for 20 h, then cooled and diluted with water (40 mL). The precipitate was collected by filtration and dissolved in 10% methanol in methylene chloride. This solution was combined with the organic layers obtained by extracting the aqueous filtrate with 10% methanol in methylene chloride (3 x 20 mL). The mixed organics were dried over sodium sulfate, filtered, and concentrated over silica gel. The crude product was purified by column chromatography on silica gel to afford the title compound as a brown solid (170 mg, 0.535 mmol, 35%). ES/MS m/z 319.94 (M+H)

[75] The compounds in Table 4 below were prepared essentially according to

Preparation 49 above and as generally described in Scheme 2; Method F

Table 4

Preparation 53

3-Chloro-7-(l-fluorovinyl)benzo[4,5]imidazo[l,2-a]pyridine

[76] A mixture of 3-chloro-7-iodobenzo[4,5]imidazo[l,2-a]pyridine (160 mg, 0.49 mmol), (l-fluorovinyl)(methyl)diphenylsilane (242 mg, 1.0 mmol), cesium fluoride (228 mg, 1.5 mmol), copper (I) iodide (10 mg, 0.05 mmol), and tetrakis(triphenylphosphine) palladium (0) (58 mg, 0.05 mmol) in dimethylformamide (4 mL) was stirred at room temperature for 2 days. The reaction was purified by column chromatography on silica gel to provide the title compound (25 mg, 21%).

Preparation 54

2-((l-(7-Vinylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-yl)oxy)ethanol

Step 1: preparation of l-(7-Vinylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3- ol [77] Amixture of dimethoxyethane:ethanol:water (7:2: 1 v/v, 5.0 mL) was added to 1- (7-bromobenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol (165 mg, 0.516 mmol, 1.0 eq) and potassium carbonate (213 mg, 1.55 mmol, 3.0 eq) in a screw-capped vial. Vinylboronic acid pinacol ester (130 pL, 0.773 mmol, 1.5 eq), [1,1 '-bis(di-tert- butylphosphino)ferrocene]-dichloropalladium(II) (16.8 mg, 0.026 mmol, 0.05 eq) and dimethylsulfoxide (5.0 mL) were mixed and sparged with nitrogen for 10 minutes. The vial was capped and heated at 100 °C for 16 hours. The reaction mixture was cooled to room temperature, diluted with water (40 mL), and filtered. The filter cake was dissolved in 10% methanol in methylene chloride and combined with the organic layers obtained by extracting the aqueous filtrate with methylene chloride (3 x 20 mL). The mixed organics were dried over sodium sulfate, filtered, and concentrated over silica gel. The crude product was filetered by column chromatography on silica gel (0 to 10 to 20% methanol in methylene chloride) to afford the title compound as a brown solid (75.0 mg, 0.283 mmol, 55%). ES/MS m/z 266.03 (M+H)

Step 2: preparation of 3-(3-(2-((Tetrahydro-2H-pyran-2-yl)oxy)ethoxy)azetidin-l- yl)-7-vinylbenzo[4,5]imidazo[l,2-a]pyridine

[78] Sodium hydride (60% in mineral oil, 10 mg, 0.24 mmol, 2 eq) was added to a solution of l-(7-vinylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol (31 mg, 0.12 mmol) and 2-(2 -bromoethoxy )tetrahydro-2H-pyran (50 mg, 0.24 mmol, 2 eq) in dimethylformamide (1 mL). The reaction was stirred at room temperature for 3 hours. Additional sodium hydride (60% in mineral oil, 10 mg, 0.24 mmol, 2 eq) was added and stirred at room temperature for 16 hours. The reaction was diluted with water (5 mL). The aqueous phase was extracted with dichloromethane (3 x 4 mL). The organic phase was concentrated in vacuo then placed under high vacuum to afford the crude product as a brown residue (47 mg, quantitative yield). ES/MS m/z 394.4 (M+H)

Step 3: preparation of 2-((l-(7-Vinylbenzo[4,5]imidazo[l,2-a]pyridin-3- yl)azeti din-3 -yl)oxy)ethanol

[79] /?-Toluenesulfonic acid monohydrate (23 mg, 0.12 mmol, 1 eq) was added to a solution of 3-(3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)azetidin-l-yl)-7- vinylbenzo[4,5]imidazo[l,2-a]pyridine (47 mg, 0.12 mmol) in methanol (1 mL). The reaction was stirred at room temperature for 2 hours and then concentrated in vacuo. The residue was dissolved in a 9: 1 dichloromethane:methanol mixture (6 mL) and washed with aqueous saturated sodium bicarbonate solution (5 mL). The aqueous phase was extracted with a 9: 1 dichloromethane:methanol mixture (2 x 6 mL). The organic phase was dried over sodium sulfate and concentrated in vacuo to afford the title compound as a brown residue (45 mg, quantitative yield). ES/MS m/z 310.2 (M+H)

Preparation 55 l-(7-Ethylbenzo[4,5]imidazo[l,2-a]pyri din-3 -yl)azeti din-3 -ol

[80] 2-Nitrobenzenesulfonyl chloride (175 mg, 0.792 mmol, 3.0 eq) was added to a slurry of l-(7-vinylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol (75.0 mg, 0.264 mmol, 1.0 eq) and hydrazine hydrate (65%, 101 pL, 1.32 mmol, 5.0 eq) in a mixture of methylene chloride (1.7 mL) and dimethylformamide (0.85 mL) at room temperature. The reaction mixture was stirred for 16 hours, concentrated over silica gel, and purified by column chromatography on silica gel to afford the title compound as a beige solid (28.0 mg, 0.104 mmol, 40%). ES/MS m/z 268.16 (M+H)

Preparation 56

3-(3-Fluoroazetidin-l-yl)-7-vinylbenzo[4,5]imidazo[l,2-a]pyridine

[81] A scintillation vial was charged with 7-bromo-3-(3-fluoroazetidin-l- yl)benzo[4,5]imidazo[l,2-a]pyridine (115 mg, 0.36 mmol, 1.0 eq), potassium vinyltrifluoroborate (72 mg, 0.54 mmol, 1.5 eq) and potassium carbonate (149 mg, 1.08 mmol, 3.0 eq) followed by a 7:2: 1 mixture of dimethoxyethane:ethanol:water (3.5 mL). The catalyst, [l,l'-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II), (23 mg, 0.036 mmol, 0.1 eq), was added, the reaction vial was sealed, and then heated to 80 °C for 16 hours. The reaction mixture was cooled to room temperature, concentrated over silica gel, and purified by column chromatography on silica gel (0 to 5 to 10 % methanol in methylene chloride) to afford the title compound as a light brown solid (33.0 mg, 0.124 mmol, 34%). ES/MS m/z 261.1 (M+H)

Preparation 57 l-(8-Fluoro-7-vinylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol

[82] l-(7-Bromo-8-fluorobenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol (22 mg, 0.066 mmol), vinyl pinacoloborolane (20 mg, 0.13 mmol), potassium carbonate (27 mg, 0.196 mmol), and [ l , l '-bis(di-/c/7-butylphosphino)ferrocene]dichloropalladium(II) (4.0 mg, 0.0066 mmol) were combined and purged with nitrogen for 10 minutes.

Dimethoxy ethane/ethanol/water (7/2/1, 3 mL) and dimethylsulfoxide (5 mL) were added. The reaction mixture was heated at 80 °C overnight. The reaction was partitioned between ethyl acetate and water. The organic layer was separated, washed with brine, and dried over sodium sulfate. The solid filtered and washed with dichloromethane. The solvent was removed under vacuum. Purification was performed by column chromatography on silica gel (0 to 30% methanol in methylene chloride), to obtain the title compound as a grey solid (17 mg, 91%). ES/MS m/z 284.2 (M+H)

Preparation 58 l-(2-Fluoro-7-methylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol [83] A vial was charged with 3-(3-((tert-butyldimethylsilyl)oxy)azetidin-l-yl)-2- fluoro-7-methylbenzo[4,5]imidazo[l,2-a]pyridine (0.022 g, 0.0571 mmol) and IM tetrabutlyammonium fluoride in tetrahydrofuran (1.0ml, Immol). The reaction mixture was heated to 45 °C for 4 hours. The solvent was removed under vacuum. The residue was purified by column chromatography on silica gel (0 to 20% methanol in di chloromethane). Selected fractions were concentrated to give the title compound as a white solid (11 mg, 71%). ES/MS m/z 2 \ (M+H)

Preparation 59 3-(l-(Benzo[4,5]imidazo[l,2-a]pyridin-3-yl)piperidin-4-yl)propan-l-ol

[84] A slurry of 3-bromobenzo[4,5]imidazo[l,2-a]pyridine (0.3 g, 1.21 mmol), 3- (piperidin-4-yl)propan-l-ol (260.8 mg, 1.82 mmol), (2-biphenyl)dicyclohexylphosphine (51.1 mg, 0.146 mmol), lithium bis(trimethylsilyl)amide (1 M in tetrahydrofuran, 3 mL, 2.67 mmol), and tris(dibenzylideneacetone)dipalladium(0) (55.5 mg, 60.7 mmol) was sparged with nitrogen and heated at 100 °C overnight. Purification was performed by column chromatography on silica gel to obtain the title compound as a light brown solid (53.3 mg, 14%).

Preparation of Precursors

Preparation of Pl l-(8-Methylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-yl 4- methylbenzenesulfonate

[85] A mixture of l-(8-methylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol (0.010 g, 0.036 mmol), -toluenesulfonyl chloride (13.6 mg, 0.071 mmol) and triethylamine (6 eq) in methylene chloride (6-25 mM) was stirred at room temperature. Upon completion, the reaction was treated with saturated aqueous sodium bicarbonate with vigorous stirring. The layers were separated and the aqueous layer was extracted with methylene chloride (4 times). The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The crude material was purified by chromatography on silica gel using a methylene chloride:methanol gradient to afford the title compound as an off-white solid (8 mg, 52%). LCMS (ESI+) 435.89 (M+H).

Alternative Preparation of Pl

[86] A suspension of l-(8-methylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol (3.18 g, 12.6 mmol) in di chloromethane (135 mL) was treated with triethylamine (17.5 mL, 126 mmol), stirred for 10 minutes, and /?-toluenesulfonic anhydride (12.31 g, 37.7 mmol) was added. The reaction mixture was stirred at room temperature for 22 hours. Additional /?-toluenesulfonic anhydride (1.84 g, 5.6 mmol) was added and stirred for 6 hours. The reaction mixture was concentrated, re-suspended in methylene chloride (175 mL), and treated with 1 N aqueous sodium hydroxide solution (150 mL). The biphasic mixture stirred vigorously for 90 minutes, transfered to a separatory funnel and the layers were separated. The organic layer was shaken vigorously with 1 N aqueous sodium hydroxide solution (2 x 100 mL, 1 x 150 mL), dried over magnesium sulfate, filtered, concentrated, and placed under high vacuum. A solution of the isolated solid in 10% methanol in methylene chloride was concentrated over silica gel (24 g) under reduced pressure. Purification of the title compound was performed by chromatography on silica gel using a gradient of 0 to 10% methanol in methylene chloride. The resulting solid was suspended in methylene chloride (ca. 50 mL), sonicated, and triturated with diethyl ether (750 mL). The precipitated solids were collected by filtration, rinsed with diethyl ether, and dried under vacuum to afford the title compound as a beige solid (3.21 g, 7.89 mmol, 63% yield).

[87] Precursors in Table 5 below were prepared essentially according to Preparation Pl or the Alternative Preparation following Scheme 3; Method G. Table 5

Preparation of Precursor 21 -(3-((tert-Butyldimethylsilyl)oxy)azetidin-l-yl)-7-iodobenzo[4,5]imidazo[l,2-a]pyridine Step 1 : preparation of 3-(3-((terLButyldimethylsilyl)oxy)azetidin-l-yl)-7-

(tributylstannyl)benzo[4,5]imidazo[l,2-a]pyridine

[88] A solution of 7-bromo-3-(3-((tert-butyldimethylsilyl)oxy)azetidin-l- yl)benzo[4,5]imidazo[l,2-a]pyridine (432 mg, 1.0 mmol), lithium chloride (260 mg, 6.0 mmol), bis(tributyltin) (1.74 g, 3.0 mmol), and tetrakis(triphenylphosphine)palladium(0) (120 mg, 0.1 mmol) in dioxane (20 mL) was sparged with nitrogen for 5 minute and then stirred at 100 °C for 4-5 hours. The reaction mixture was diluted with di chloromethane, filtered, and concentrated. The residue was purified by column chromatography on silica gel (0-15% methanol in di chloromethane) to obtain the title compound as a yellow solid (574 mg, 89%).

Step 2: preparation of 3-(3-((terLButyldimethylsilyl)oxy)azetidin-l-yl)-7- iodobenzo[4,5]imidazo[l,2-a]pyridine

[89] Iodine (76 mg, 0.3 mmol) in dichloromethane was added dropwise to a stirred solution of 3-(3-((tert-butyldimethylsilyl)oxy)azetidin-l-yl)-7- (tributylstannyl)benzo[4,5]imidazo[l,2-a]pyridine (190 mg, 0.3 mmol) in di chloromethane (6.0 mL) at 0 °C. After 5 minutes, the reaction was quenched with 5% aqueous sodium bisulfite solution, extracted with dichloromethane, dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on silica gel (0-20% methanol in dichloromethane) to obtain the title compound as a solid (42 mg, 29%). ES/MS m/z 480.3 (M+H) Alternative Preparation of Precursor 21 l-(7-Iodobenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-yl 4-methylbenzenesulfonate

[90] 6-7 drops of acetyl chloride were added to a solution of 3-(3-((tert- butyldimethylsilyl)oxy) azetidin-l-yl)-7-iodobenzo[4,5]imidazo[l,2-a]pyridine (42 mg, 0.088 mmol) in methanol (6 mL). The reaction was stirred at room temperature overnight, concentrated and dried under high vacuum for several hours. The residue was redissolved in di chloromethane (40 mL) and triethylamine (0.25 mL, 1.76 mmol),/?- toluenesulfonyl chloride (100 mg, 0.53 mmol), and a catalytic amount of 4-(N,N- dimethylamino)pyridine were added. The mixture was stirred at 40 °C overnight, extracted with IN sodium hydroxide, dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on silica gel (0-10% methanol in ethyl acetate), followed by a second silica gel column (0-15% methanol in di chloromethane) to afford the title compound as a yellow solid (24 mg, 52%). ES/MS m/z 520.2 (M+H)

Preparation of Precursor 22 l-(7-(l-Fluorovinyl)benzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-yl 4- methylbenzenesulfonate

[91] /?-Toluenesulfonic anhydride (273 mg, 0.84 mmol) and 4-(N,N- dimethylamino)pyridine (1 mg, 0.008 mmol) and triethylamine (0.1 mL) were added to a solution of l-(7-(l-fluorovinyl)benzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol (20 mg, 0.07 mmol) in dichloromethane (3 mL). The mixture was stirred at room temperature and monitored by LCMS until all alcohol converted to the di-tosylate product. The mixture was washed with aqueous sodium bicarbonate, and extract with 10% methanol in di chloromethane. The organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure. Potassium hydroxide (IN in water, 1 mL) and methanol (2 mL) were added. The mixture was stirred at room temperature for 1 hour until all di-tosylate was converted to desired compound. Hydrochloric acid (IN) was added slowly to adjust the pH of the mixture and washed with aqueous sodium bicarbonate. The aqueous mixture was extracted with 10% methanol in methylene chloride. The organic solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica (0-10% methanol in di chloromethane) to afford the title compound as an off-white solid (14.4 mg, 46%). ES/MS m/z 438.14 (M+H)

Preparation of Precursor 23 l-(7-Ethynylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-yl 4- methylbenzenesulfonate

Step 1 : preparation of 3-(3-((terLButyldimethylsilyl)oxy)azetidin-l-yl)-7- ((trimethylsilyl)ethynyl)benzo[4,5]imidazo[l,2-a]pyridine

[92] A mixture of 7-bromo-3-(3-((tert-butyldimethylsilyl)oxy)azetidin-l- yl)benzo[4,5]imidazo[l,2-a]pyridine (216 mg, 0.5 mmol), ethynyltrimethylsilane (0.25 mL, 1.8 mmol), tetrakis(triphenylphosphine)palladium (58 mg, 0.05 mmol), and copper (I) iodide (12 mg, 0.03 mmol) in trimethylamine and dimethylformamide (2 mL 1 : 1) was sparged with nitrogen and then stirred at 90 °C overnight. The mixture was filtered through a pad of diatomaceous earth and washed with 10% methanol in di chloromethane. The filtrate was evaporated under reduced pressure and purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to afford the title compound as a light brown solid (140 mg, 62%). ES/MS m/z 450.57 (M+H) Step 2: preparation of l-(7-((Trimethylsilyl)ethynyl)benzo[4,5]imidazo[l,2- a]pyri din-3 -yl)azeti din-3 -ol

[93] Acetyl chloride (25 uL) was added to a solution of 3-(3-((tert- butyldimethylsilyl)oxy)azetidin-l-yl)-7-((trimethylsilyl)ethynyl)benzo[4,5]imidazo[l,2- a]pyridine (140 mg, 0.31 mmol) in methanol (1 mL) at 0 °C. The reaction mixture was stirred at room temperature overnight. The mixture was diluted with aqueous sodium bicarbonate solution and extracted with 10% methanol in di chloromethane. The organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica (0-6% methanol in di chloromethane) to afford the title compound as a yellow solid (30 mg, 29%). ES/MS m/z 336.27 (M+H)

Step 3: preparation of l-(7-Ethynylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin- 3-yl 4-methylbenzenesulfonate

[94] /?-Toluenesulfonic anhydride (535 mg, 1.6 mmol), 4-(N,N- dimethylamino)pyridine (1 mg, 0.008 mmol), and triethylamine (0.1 mL) were added to a solution of l-(7-((trimethylsilyl)ethynyl)benzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3- ol (55 mg, 0.16 mmol) in dichloromethane (4 mL). The mixture was stirred at room temperature and monitored by LCMS until all the alcohol was converted to the di-tosylate product. The mixture was diluted with aqueous sodium bicarbonate and extracted with 10% methanol in di chloromethane. The organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica (0-6% methanol in dichloromethane). To the di-tosylate intermediate, potassium hydroxide (IN in water, 0.5 mL) and methanol :tetrahydrofuran (1 : 1, 2 mL) were added. The mixture was stirred at room temperature for 1 hour until all di-tosylate was converted to the desired compound. Aqueous hydrochloric acid (IN) was added slowly to adjust the pH of the mixture and washed with aqueous sodium bicarbonate. The aqueous mixture was extracted with 10% methanol in methylene chloride. The organic solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica (0-10% methanol in di chloromethane) to afford the title compound as a pale yellow solid (16.9 mg, 25%). ES/MS m/z 418.15 (M+H).

Preparation of Precursor 24 l-(7-(l-Methyl-lH-pyrazol-4-yl)benzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-yl methanesulfonate

[95] A stirring solution of l-(7-bromobenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3- ol (85mg, 0.27 mmol), l-methylpyrazole-4-boronic acid pinacol ester (112mg, 0.54 mmol), [l,r-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (18mg, 0.027 mmol) in dioxane (5 mL) and 2M aqueous sodium carbonate solution (1 mL) was sparged with nitrogen for 5 minutes and then heated at 100 °C for 3-4 hours. The reaction was diluted with 10% methanol in di chloromethane, filtered and concentrated. The residue was dried under high vacuum for several hours and then taken up in dichloromethane (50 mL). Triethylamine (0.4 mL) and methanesulfonyl chloride (154 mg, 1.35 mmol) were added at room temperature. After 1-2 hours at room temperature, the di chloromethane layer was washed with aqueous sodium bicarbonate solution, dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on silica gel (0-20% methanol in dichloromethane) to afford the title compound as a brown solid (24 mg, 22% yield over 2 steps). ES/MS m/z 397.8 (M+H)

Preparation of Precursor 25

(S)- 1 -(4-Chloro-8-methylbenzo[4,5]imidazo[ 1 ,2-a]pyri din-3 -yl)pyrrolidin-3 -yl 4- methylbenzenesulfonate (P25):

[96] To a solution of (S)-l-(8-methylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)pyrrolidin- 3-yl 4-methylbenzenesulfonate (20 mg, 0.048 mmol, 1.0 eq) in 10% methanol in methylene chloride (2 mL) was added N-chlorosuccinimide (8 mg, 0.062 mmol, 1.3 eq). The reaction was stirred at room temperature for 10 minutes then washed with IN sodium hydroxide (2 x 1 mL) and water (1 mL). The organic layer was concentrated. The material was purified on a 12 g silica gel column with a gradient running from 0% to 40% ethyl acetate in methylene chloride. The fractions containing product were concentrated to obtain a pale yellow solid (18 mg, 82% yield). ES/MS m/z ⁽³⁵C1/³⁷C1) 456.3/458.1

Preparation of Precursor 26 l-(8-methylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-yl 4-methylbenzenesulfonate

[97] Precursor 26 was prepared from l-(4-Chloro-8-methylbenzo[4,5]imidazo[l,2- a]pyri din-3 -yl)azeti din-3 -yl 4-methylbenzenesulfonate in 81% yield by following essentially the procedure for Precursor 25. ES/MS m/z ⁽³⁵C1/³⁷C1) 442.2/444.1

Preparation of the Examples

Radiolabeling Procedure

[98] Radiolabeling synthesis was performed using a GE TRACERlab FXF-N automated radiosynthesizer with starting activity in the range of 0.1 Ci - 2.4 Ci. The range of averaged synthesis time was 60 ± 10 minutes and the range of averaged decay- corrected yield was 12-54%. [99] [18F]Fluoride activity was retained on a Sep-Pak Accell Plus QMA Carbonate Plus Light Cartridge (Waters, 40 mg Sorbent per Cartridge, 40 pm Particle Size) and eluted into the reaction vessel using 0.8 mL of Cryptand 2.2.2-K2CO3 solution [Cryptand 2.2.2 (7 mg) and potassium carbonate (0.75 mg) in H2O (0.4 mL) and acetonitrile (0.4 mL)].

[100] The eluted activity was dried by heating at 70 °C under inert gas flow and vacuum for ~5 minutes. The temperature was raised to 100 °C under vacuum for 5 minutes to afford anhydrous Cryptand 2.2.2-K2CO3 [18F]fluoride. A solution of precursor [0.5 mg to 2.0 mg in DMSO (1-2 mL)] was added to the reaction vessel containing the anhydrous Cryptand 2.2.2-K2CO3 [18F]fluoride and the resulting mixture was kept at an elevated temperature (100-150 °C) for 10-20 minutes. The crude reaction was cooled (50-65 °C) and H2O (2.5-3 mL) was added for dilution. The diluted crude reaction was loaded onto a semi -preparative HPLC column for purification using isocratic elution (Agilent ZORBAX Eclipse XDB-C18 9.4 x 250 mm; flow rate of 4 mL/minutes, see Table 6 for details). The isolated fraction from the HPLC column contained the radiolabeled Example as identified in Table 6.

[101] The Examples listed in Table 6 below were prepared essentially according to Example 1 and as generally described in Scheme 4.

Table 6

Preparation of an injection ready formulation

[102] The HPLC fraction containing the radiolabeled Example prepared essentially according to the procedure for Example 1 was diluted with aqueous 0.5% sodium ascorbate (w/v) solution (30-40 mL). The diluted solution was passed through either a Sep-Pak® C18 Plus Light Cartridge (Waters 130 mg Sorbent per Cartridge, 55-105 pm Particle Size) or a Sep-Pak® Vac C18 Cartridge (Waters, 50 mg Sorbent per Cartridge, 55-105 pm Particle Size), and the retained substance was washed with water (5-10 mL). The substance was eluted off the cartridge using dehydrated alcohol, USP (0.4 to 1 mL) and combined with a solution of 0.5% sodium ascorbate (w/v) in 0.9% Sodium Chloride Injection, USP, furnishing the product (substance in 10% ethanol (v/v) 0.45% (w/v) sodium ascorbate in 0.9% Sodium Chloride Injection, USP; 4-10 mL total volume).

[103] A product sample was removed and analyzed by HPLC to determine radiochemical purity, radiochemical identity, chemical purity and specific activity. The peak(s) from the UV chromatograms and radiochromatogram were integrated for the determination of radiochemical purity (% RCP), radiochemical identity, chemical purity and specific activity. The range of RCP was 92-100%. Stability data for select Examples is provided in Table 7 below.

Table 7

Stability of [¹⁸F]-Example 5 and [¹⁸F]-Example 2.

[104] The Examples in Table 8 below were prepared essentially following Scheme 1;

Method A (step 1) Table 8 [105] The Examples listed below in Table 9 were prepared essentially following Scheme 2; Method C

Table 9

[106] The Examples in Table 10 below were prepared essentially following Scheme 2;

Method D

Table 10 [107] The Examples in Table 1 1 below were prepared essentially following Scheme 2;

Method E

Table 11 [108] The Examples in Table 12 below were prepared essentially following the procedure for Precursor 25

Table 12

Example 5

3-(3-Fluoroazetidin-l-yl)-7-iodobenzo[4,5]imidazo[l,2-a]pyridine

Step 1 : preparation of 3-(3-Fluoroazetidin-l-yl)-7-(tributylstannyl)benzo [109] The title compound was prepared from 7-bromo-3-(3-fluoroazetidin-l- yl)benzo[4,5]imidazo[l,2-a]pyridinein 46% yield using substantially the same procedure as Precursor 21, Step 1. ES/MS m/z 531.0 (M+H)

Step 2: preparation of 3-(3-Fluoroazetidin-l-yl)-7-iodobenzo[4,5]imidazo[l,2-a]pyridine

[110] The title compound was prepared from 3-(3-fluoroazetidin-l-yl)-7- (tributylstannyl)benzo[4,5]imidazo[l,2-a]pyridine in 64% yield using substantially the same procedure as Precursor 21, Step 2. ES/MS m/z 367.9 (M+H)

Example 19

7-Ethyl-3-(3-fluoroazetidin-l-yl)benzo[4,5]imidazo[l,2-a]pyridine

[111] 2-Nitrobenzenesulfonyl chloride (75.0 mg, 0.337 mmol, 3.0 eq) was added to a slurry of 3-(3-fluoroazetidin-l-yl)-7-vinylbenzo[4,5]imidazo[l,2-a]pyridine (30.0 mg, 0.112 mmol, 1.0 eq) and hydrazine hydrate (43.0 pL, 0.56 mmol, 5.0 eq) in methylene chloride (3 mL) and dimethylformamide (1 mL) at room temperature. The reaction mixture gradually cleared to an orange solution, and the solution was stirred for 16 hours. The reaction mixture was concentrated over silica gel and purified by column chromatography on silica gel (0 to 10% methanol in methylene chloride) to afford product that was contaminated with an aromatic byproduct. The yellow solid was dissolved in methylene chloride and washed with saturated aqueous sodium bicarbonate and water. The organics were dried over sodium sulfate and concentrated. The crude solid was dissolved in methylene chloride and purified by column chromatography on silica gel (0 to 10% methanol in methylene chloride) to afford the title compound as a beige solid (14.0 mg, 0.052 mmol, 46%). ES/MS m/z 270.1 (M+H) Example 21

8-Fluoro-3-(3-fluoroazetidin-l-yl)-7-vinylbenzo[4,5]imidazo[l,2-a]pyridine

[112] 7-Bromo-8-fluoro-3-(3-fluoroazetidin-l-yl)benzo[4,5]imidazo[l,2-a]pyridine (0.100 g, 0.30 mmol), vinyl pinacoloborolane (0.091 g, 0.59 mmol), potassium carbonate (0.122 g, 0.89 mmol), and di chlorofl, l’-bis(di-te/7- butylphosphino)ferrocene]palladium(II) (Pd-118) (0.019 g, 0.030 mmol) were combined. Dimethoxy ethane/ethanol/water (7/2/1, 6.0 mL) and dimethylsulfoxide (0.4 mL) were added. The reaction mixture was heated at 80 °C overnight. The reaction was partitioned between ethyl acetate and water. The organic layer was separated, washed with brine and dried over sodium sulfate. The solid was filtered and wash with dichloromethane. The solvent was removed under vacuum. Purification was performed by column chromatography on silica gel (0 to 95% ethyl acetate in methylene chloride) to obtain the title compound as brown solid (46 mg, 54%). ES/MS m/z 286.2 (M+H)

Example 22

3-(3-(2-Fluoroethoxy)azetidin-l-yl)-7-vinylbenzo[4,5]imidazo[l,2-a]pyridine

[113] To a solution of l-(7-vinylbenzo[4,5]imidazo[l,2-a]pyridin-3-yl)azetidin-3-ol (27 mg, O. lmmol) in dimethylformamide (3 mL) was added l-bromo-2-fluoroethane (38 mg, 0.3 mmol, 3 eq) followed by potassium /c/7-butoxide (34 mg, 0.3mmol, 3eq). The reaction was stirred at room temperature for 72 hours. Additional l-bromo-2-fluoroethane (63 mg, 0.5 mmol, 5 eq) was added followed by potassium Zc/V-butoxide (67 mg, 0.6 mmol, 6 eq). The reaction was stirred at room temperature for 15 minutes then cooled to 0 °C (ice bath) and water (3 mL) was added. The aqueous phase was extracted with di chloromethane (4 x 5 mL), concentrated in vacuo then purified by column chromatography on silica gel (12 g column, 0% to 30% methanol in dichloromethane) to afford the title compound as a brown solid (17 mg, 55%). ES/MS m/z 312.2 (M+H) Example 30

7-Ethynyl-3-(3-fluoroazetidin-l-yl)benzo[4,5]imidazo[l,2-a]pyridine

[114] Potassium hydroxide solution (1 N, 1 mL) was added dropwise to a solution of 3- (3-fluoroazetidin-l-yl)-7-((trimethylsilyl)ethynyl)benzo[4,5]imidazo[l,2-a]pyridine (20 mg, 0.07 mmol) in methanol: tetrahydrofuran (2 mL 1 : 1) at 0 °C. The reaction was stirred at room temperature for 1.5 hours. The reaction mixture was neutralized with hydrochloric acid (IN) and then aqueous sodium bicarbonate solution. The aqueous layer was extracted with dichloromethane (3 times). The combined organic layers were dried over magnesium sulfate and the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica (0 to 10% methanol in di chloromethane) to afford the title compound as a yellow solid (41 mg, 31% yield over 2 steps). ES/MS m/z 266.14 (M+H)

Example 17

3 -(3 -Fluoroazetidin- 1 -yl)-7-( 1 -methyl- lH-pyrazol-4-yl)benzo[4,5]imidazo[ 1 ,2-a]pyridine

[115] A stirred solution of 7-bromo-3-(3-fluoroazetidin-l-yl)benzo[4,5]imidazo[l,2- a]pyridine (48 mg, 0.15 mmol), l-methylpyrazole-4-boronic acid pinacol ester (62mg, 0.3 mmol), [l,l'-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (lOmg, 0.015 mmol) in dioxane (5 mL) and 2M aqueous sodium carbonate solution was sparged with nitrogen for 5 minutes and then heated at 100 °C for 3-4 hours. The reaction was cooled and diluted with 10% methanol in di chloromethane, filtered and concentrated. The residue was purified by column chromatography on silica gel (0-20% methanol in di chloromethane) to afford the title compound as a solid (30 mg, 62%). ES/MS m/z 1 /) (M+H) Biological Analysis

Assay Example 31 :

Ki and Kd Determination of the compound of Example 3 using tau from Alzheimer’s disease human donors.

[116] PHF (paired-helical filament) tau preparation:

[117] Purified, soluble PHF tau was isolated from AD brain tissue using a protocol modified from the procedure described by Jicha, et al. (G. A. Jicha, A. O'Donnell, C. Weaver (1999) “Hierarchical phosphorylation of recombinant tau by the paired-helical filament-associated protein kinase is dependent on cyclic AMP-dependent protein kinase” J Neurochem. 72(1):214). Briefly, AD cortex was homogenized using a handheld Kinematica Polytron, followed by high pressure batch - gas expansion using a Parr Cell disruption bomb. Crude homogenate was centrifuged at 28 kg to pellet cell debris. Soluble PHF tau was isolated from the supernatant by affinity chromatography over an Affigel-10 column on which the tau antibody MCI, which recognizes a pathological conformation of tau, has been immobilized (G. A. Jicha, R. Bowser, I. G. Kazam (1997), “Alz-50 and MC-1, a new monoclonal antibody raised to paired helical filaments, recognize conformational epitopes on recombinant tau” J Neurosci Res. 48(2): 12.)

[118] Ki determination of Example 3 (3-(3-Fluoroazetidin-l-yl)-7-methylbenzo[4,5] imidazo[ 1 ,2-a]pyridine)

[119] The IC50 is the molar concentration of competing ligand, which reduces the specific binding of a radioligand by 50%. In this competitive assay the competing ligand was the un-radiolab eled, compound of Example 3 and the radiolabeled compound or radioligand is 7-[6-(F)fluoropyridin-3-YL]-5H-pyrido[4,3-B]indole (also known as [18F]AV-1451 or T807) The binding of [18F]AV-1451 to PHF tau was determined against the un-radiolabel ed compound of Example 3 at various concentrations. The reaction mixture (200 pl) contained PHF tau (0.12 ug), [18F]AV-1451 at 0.1-0.5 nM, and the un-radiolabeled compound of Example 3 serially diluted from 316 nM to 0.01 nM; assays were performed in PBS, pH 7.4 containing 0.01% bovine serum albumin in 96 well polypropylene microplates. Nonspecific binding is defined as the binding of the radioligand in the presence of 2-[4-(2-fluoranylethyl)-l-piperidyl]pyrimido[l,2- a]benzimidazole T808/AV-680 (5 pM), a known PHF tau ligand (Zhang, J. (2012), “A highly selective and specific PET tracer for imaging of tau pathologies” J Alzheimers Dis., 31(3):601). After incubation for 1.5 h at 37°C, the bound radioactivity was harvested onto Millipore Multi Screen^HTS 96-well glass fiber FB filter plates using a Millipore Multi Screen^HTS Vacuum Manifold, followed by five washes with PBS, pH 7.4. Filters containing bound [18F]AV-1451 were assayed for radioactivity in a Wizard 2480 automatic gamma-counter [Perkin Elmer], Using these assay conditions, the total bound fraction is typically less than 10% of the added radioligand [18F]AV-1451. The IC50 is determined using an ActivityBase or XLfit model 205 (or a comparable model) in which: y=A+ (B-A)/(l+((C/x)^AD) Y= % Inhibition

X = Concentration of the cold competing ligand (nM) A= minimum Y (0%) B= maximum Y (100%) C= IC50

D= Slope factor

[120] The Ki (i.e. the equilibrium dissociation constant for binding of the unradiolabeled compound) is calculated from the IC50 value using the Cheng-Prusoff equation (Cheng Y., Prusoff W.H. (1973), "Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction" Biochem Pharmacol 22 (23):3099-3108):

Ki = IC50/(l + [L]/Kd)

[L] = the concentration of [18F]AV-1451 (typically ~0.5 nM) Ka = the dissociation constant for [18F]AV-1451 (0.57 nM).

[121] The Ki versus [18F]AV-1451 for the compound of Example 3 (3-(3- fluoroazetidin-l-yl)-7-methylbenzo[4,5]imidazo[l,2-a]pyridine) is 0.23 nM on PHF tau that is obtained from donors with Alzheimer’s disease indicating that the compound of Example 3 binds PHF tau. Therefore, PET imaging with the compound of Example 3 and examination of the imaging pattern would be useful to detect the presence of PHF tau in patients and can help confirm a diagnosis of AD or non-AD tauopathies.

[122] The compounds of the Examples disclosed herein exhibited a Ki of less than 3 nM in as evaluated according to this assay. Thus, the compounds of the Examples bind to PHF tau, and the radiolabeled compounds of the Examples would be useful in detecting PHF tau. [123] Kd determination of the compound of Example 3 (3-(3-[18F]-Fluoroazetidin-l- yl)-7-methylbenzo [4,5]imidazo[l,2-a]pyridine)

[124] The dissociation constant [Kd] for the radiolabeled compound of Example 3(18F) was determined by saturation binding, in which the total and nonspecific binding of the radioligand were measured at various radioligand concentrations. The reaction mixture (250 pl) contained PHF tau (0.15 pg), and the compound of Example 3, serially diluted from 25 nM to 0.3 nM in PBS; assays were performed in PBS containing 0.01% bovine serum albumin in 96 well polypropylene microplates. Nonspecific binding is defined as the binding of the radioligand in the presence of 2-[4-(2-fluoranylethyl)-l- piperidyl]pyrimido[l,2-a]benzimidazole (also known as T808/AV-680) (10 pM); the radioligand in this assay was the compound of Example 3. After incubation for 1.5 h at 37°C, the bound radioactivity was harvested by vacuum filtration onto Millipore

Multi ScreenHTS 96-well glass fiber FB filter plates, using a Millipore Multi ScreenHTS Vacuum Manifold, followed by five washes with PBS. Filters containing the bound compound of Example 3 were assayed for radioactivity in a Wizard 2480 automatic gamma-counter [Perkin Elmer], Using these assay conditions, the total bound fraction is typically less than 10% of the added radioligand. The total binding and nonspecific binding data were analyzed by nonlinear regression analysis using Graphpad Prism to determine the Kd for the radioligand.

[125] The Kd of the compound of Example 3 is 1.5 ± 0.2 nM on PHF tau that was obtained from donors with Alzheimer’s disease, indicating that this compound binds PHF tau with high affinity. Therefore, PET imaging with this compound and examination of the imaging pattern would be useful to detect the presence of tau in patients and could confirm a diagnosis of AD or non-AD tauopathies.

Assay Example 32

Kd Determination for the Binding of the compound of Example 3(¹⁸F) to native tau aggregates in Human AD Brain Tissue

[126] Autoradiography was employed in the Kd determination of the compound of Example 3(¹⁸F) binding to native tau-aggregates on human AD brain sections that have been characterized using anti-tau and anti-amyloid immunostaining according to methods known to the skilled artisan (See e.g. [(18)F]T807, a novel tau positron emission tomography imaging agent for Alzheimer's disease. Xia CF, et al., Alzheimer ’s Dement. 2013 Nov; 9(6):666-76), (Zhang, J. (2012), “A highly selective and specific PET tracer for imaging of tau pathologies” J Alzheimers Dis., 31(3):601). The experiment uses 15 adjacent frontal lobe sections from each of two AD brains: a tau-rich and amyloid-rich (Tau+A0+) brain as well as tau-poor and amyloid-rich (Tau-A0+) brain to define nonspecific binding. Sections were covered with 0.5 ml of 3-(3-[18F]-fluoroazetidin-l- yl)-7-methylbenzo[4,5]imidazo[l,2-a]pyridine, serially diluted from -250 nM in binding buffer (2.5% dimethylsulfoxide + 2.5% ethanol in IX PBS, pH 7.4). After a 60 min incubation at room temperature, unbound ligand was removed through successive wash cycles (2 minutes in IX PBS, 2 minutes 30% ethanol in IX PBS, 2 minutes in 70% ethanol in IX PBS, 2 minutes in IX PBS). After drying under the hood, the sections were exposed overnight to a phosphorimaging screen. The autoradiography signal recorded on the phosphorimaging screen was read using a GE Healthcare Life Sciences Typhoon FLA 7000 Phosphorimager. The signal intensity on the grey matter is measured using Fujifilm Multi Gauge software. The Kd for the compound was determined by non-linear regression analysis of the bound concentration of the compound of Example 3 versus concentration of free compound.

[127] Autoradiography from the compound of Example 3(¹⁸F) on AD brain sections for Kd determination is shown in FIG. 1. The Kd to native tau-aggregates of AD brain tissue for the compound of Example 3, determined by non-linear regression analysis, is 1.7 nM, indicating this compound binds tau. Therefore, PET imaging with the compound of Example 3 (¹⁸F) and examination of the imaging pattern, may be useful to detect the presence of tau in patients, and could confirm a diagnosis of AD or non-AD tauopathies.

Assay Example 33

Selectivity of 3-(3-[18F]-Fluoroazetidin-l-yl)-7-methylbenzo[4,5]imidazo[l,2-a]pyridine towards Tau versus 0 amyloid in AD human brain tissue.

Methods

[128] Based on the anti-tau and anti-amyloid immunostaining results of brain sections, three groups of human brain sections were selected for autoradiography experiments to determine the pathologic tau-binding selectivity of the compound of Example 3. The autoradiography of the compound of Example 3 on the three groups of human brain sections is shown in FIG. 2. Group A are pathologic tau-rich AD brain slices (labeled as Tau+Ap+), Group B are pathologic tau-poor AD brain slices (marked as Tau-Ap+), and Group Cl are Tau-AP- normal brain slices. As shown in FIG. 2, Group A human AD brain sections used are #0185, #28770, #30121, #30311, and #30461. The human AD brain sections in Group B are #33562, #32656, #33998, #35682, and #33563. The normal human brain sections in group C are #29092 and #32566. Tissue slices from the same donors were used to calculate selectivity. Autoradiography is performed for each of these three groups of brains on adjacent 10 pm sections with the amyloid tracer [18F]W372 (2- (6-fluoro-3-pyridyl)-6-methoxy-imidazo[l,2]thiazolo[2,3-c]pyridine) to quantify the P- amyloid burden. [18F]W372 is a selective amyloid binding tracer discovered by Siemens and evaluated under IND 105173 see also (US9,023,316). Sections were covered with 0.5 ml containing the compound of Example 3 in binding buffer: 2.5% dimethylsulfoxide + 2.5% ethanol in IX PBS, pH 7.4, about 20 pCi/slide and incubated for 60 minutes. Then successive washing cycles (2 min PBS, 2 min 30% EtOH/PBS, 2 min 70% EtOH/PBS, 2 min PBS) were employed to remove any unbound tracer. The sections were air dried, placed on a phosphorimaging plate (Fuji IP plate), and exposed overnight. The IP plate is read using a GE Healthcare Life Sciences Typhoon FLA 7000 Phosphorimager. The signal intensity of the grey matter is measured using Fujifilm Multi Gauge software. After subtracting the background signal (signal in the cortex region of Group C), the signal of individual sections of Group A and B were normalized with corresponding signal from autoradiography of the respective adjacent sections with [18F]W372. The calculations were based on Group B brain sections #32656 and #33998, which have undetectable tau pathology by immunohistochemistry. The normalized signal in Group B brain sections is the relative signal level of the compound of Example 3 to [18F]W372 resulting from binding to native P-amyloid aggregates. The binding level to native tau-aggregates in Group A sections was estimated by subtracting the amount of the total signal attributable to binding to P-amyloid (calculated by multiplying the total signal from [18F]W372 binding to P-amyloid in the adjacent section by the relative signal of the compound of Example 3 to [18F]W372 determined from the Group B sections). The resulting difference was then divided by the signal attributable to binding to P-amyloid to estimate the selectivity. [129] Strong signal on grey matter (cortex region) of sections in Group A (Tau+A|3+) was observed, whereas in Group B (Tau-Ap+), weak or no signal on the cortex regions of the sections was detected. No autoradiography signal was seen on the sections of the normal brains of Group C (Tau-Ap-). These results indicate that the compound of Example 3 binds to native tau aggregates of human AD brain specifically, and has weak or no interaction with native P-amyloid aggregates.

[130] Since the IHC results show that Group B brain sections #32656 and #33998 are devoid of tau protein aggregates, the normalized autoradiography signal on the cortex region of these AD brain sections is derived from the binding of the compound of Example 3 to native P-amyloid aggregates. The selectivity of the compound of Example 3 binding to native tau aggregates vs. binding native P-amyloid aggregates is reflected by the ratio of Group A (Tau+Ap+) signal to the average signal of the brain sections #32656 and #33998.

[131] The compound of Example 3 exhibits a selectivity ratio for Tau: Ap of approximately 31.4, based on 5 Tau⁺ Ap⁺ brain specimens, and 2 Tau-Ap+ brain specimens and exhibits grey matter to white matter (GM/WM) signal ratio of approximately 18.9. The autoradiography signal of the compound of Example 3 on normal brain sections is weak and even, showing little difference between grey matter and white matter, indicative of low non-specific binding. The selectivity ratio of two native tau aggregates, as compared to binding native P-amyloid aggregates in the grey matter region of human AD brains, was observed to be approximately 31 fold.

[132] The results provided in the biological assays above support the use of the compound of Example 3 as a radiolabeled Example can be used with a PET imaging probe for detecting levels of aggregated tau protein in AD patients and/or other neurodegenerative disorders, such as CTE. The results also suggest that the use of the compounds of the other Examples are useful as PET imaging probes for aggregated tau proteins to help diagnose and monitor patients with AD and other neurodegenative disorders associated with aggregated tau proteins. Example Assay 34

Autoradiography Determination for the Binding of the compound of Example 4(¹⁸F) to non-AD tau aggregates in Human PSP and CBD Brain Tissue

[133] Autoradiography was employed in the determination of the compound of Example 4(¹⁸F) binding to native tau-aggregates on human PSP and CBD brain sections that have been characterized using anti-tau immunostaining according to methods known to the skilled artisan (See e.g. [(18)F]T807, a novel tau positron emission tomography imaging agent for Alzheimer's disease. Xia CF, et al., Alzheimer ’s Dement. 2013 Nov; 9(6):666- 76), (Zhang, J. (2012), “A highly selective and specific PET tracer for imaging of tau pathologies” J Alzheimers Dis., 31(3):601). The experiment used 10 um adjacent sections from cases clinically diagnosed with AD, PSP, or CBD confirmed as tau positive by IHC employing AT8 or AT100 antibody using standard techniques or control tissue defined as amyloid and tau negative by IHC. Sections were covered with Example 4[¹⁸F] (40 pCi/ml in binding buffer (2.5% dimethylsulfoxide + 2.5% ethanol in IX PBS, pH 7.4)). After a 60 min incubation at room temperature, unbound ligand was removed through successive wash cycles (2 minutes in IX PBS, 2 minutes 30% ethanol in IX PBS, 2 minutes in 70% ethanol in IX PBS, 2 minutes in IX PBS). After drying under the hood, the sections were exposed overnight to a phosphorimaging screen. The autoradiography signal recorded on the phosphorimaging screen was read using an Amersham Typhoon Bio-Imaging System. Individual tissue samples were compared to adjacent slices with either AT8 or AT 100. A positive correlation with tau antibody indicates binding to the non-AD tau being studied (PSP or CBD).

[134] Autoradiography from the compound of Example 4(¹⁸F) on PSP brain sections (16 cases, 1-4 regions per case) for determination of binding is shown in FIG.3. PSP patient sections presented with an autoradiographic signal comparable to what was seen with ¹⁸F- labeled AT100 antibody or AT8 IHC. ARG positivity is seen in regions rich in AT8 positive tufted astrocytes, a classical neuropathological hallmark observed in PSP.

[135] Autoradiography from the compound of Example 4(¹⁸F) on CBD brain sections (5 cases, 3-6 regions per case) for determination of binding is shown in FIG. 4. Strong signal was observed in tau positive tissue from CBD patients. Note the binding in the white matter regions of the CBD patients reported to have abundant tau deposits and hence a region enriched in tau aggregates. The presence of ARG signal for the compound of Example 3, which correlates to tau positive regions of PSP and CBD human tissues indicates this compound binds non-AD tau. Both FIGs. 3 and 4 include the binding of the compound of Example 4(¹⁸F) to human AD tissue indicating strong binding to AD tau. Therefore, PET imaging with the compound of Example 4(¹⁸F) and examination of the imaging pattern, would be useful to detect the presence of AD and non-AD tau in patients, and could confirm a diagnosis of AD or non-AD tauopathies.

Claims

WHAT IS CLAIMED IS:

1. A compound or a pharmaceutically acceptable salt thereof of the formula: wherein n is 0, 1, or 2;

R¹ is H, halo, C1-C3 alkyl, or C3-C6 cycloalkyl;

R² is H, halo, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, -O-(Ci-C₄ alkyl), C1-C4 alkylhalo, C2-C4 alkenylhalo, or •

R³ is H or F;

R⁴ is F, ¹⁸F, Ci-C₄-alkylF, Ci-C₄-alkyl¹⁸F, -O-(Ci-C₄-alkyl)F, or -O-(Ci-C₄- alkyl)¹⁸F;

R⁵ is H, halo, or C1-C4 alkyl; provided when n is 0, R¹ is methyl, R² is H, R³ is H, and R⁵ is H, then R⁴ is not F or ¹⁸F positioned at the 3-position of the 4-membered ring.

2. The compound or pharmaceutically acceptable salt of claim 1, wherein n is 0, 1, or 2;

R¹ is H, halo, C1-C2 alkyl, or cyclopropyl;

R² is H, halo, -CH₃, -CH2CH3, -OCH₃, -CH=CH₂, -CF=CH₂ , -C=CH, -

R³ is H or F;

R⁴ is F, ¹⁸F, -CH₂F, -CH₂ ¹⁸F, -CH2CH2F, -CH₂CH₂ ¹⁸F, -CH2CH2CH2F, - CH2CH₂CH2¹⁸F, -OCH2CH2F, or -OCH₂CH2¹⁸F; and

R⁵ is H, halo, or C1-C4 alkyl.

3. The compound or pharmaceutically acceptable salt of claim 1 or 2, wherein n is 0 or 1;

R¹ is H, halo, C1-C2 alkyl, or cyclopropane;

R² is H, halo, -CH₃, -OCH3, -CH=CH₂, -CF=CH₂, -OCH, -OCH₂CH₂F, or

R³ is H, or F;

R⁴ is F or ¹⁸F; and

R⁵ is H, halo, or -CH3.

4. The compound or pharmaceutically acceptable salt of claim 1 or 2, wherein n is 0;

R¹ is H, halo, -CH₃, or -CH₂CH₃;

R² is H, halo,

R³ is H or F;

R⁴ is F or ¹⁸F; and

R⁵ is H, halo, or -CH3.

5. The compound or pharmaceutically acceptable salt of claim 4, wherein

R¹ is -CH₃;

R² is H;

R³ is H; and R⁵ is Cl.

6. The compound or pharmaceutically acceptable salt of claim 4, wherein n is 0;

R¹, R², R³, and R⁵ are each H; and

R⁴ is F or ¹⁸F.

7. The compound or pharmaceutically acceptable salt of claim 1, wherein n = 0;

R¹ is H;

R² is Cl, F or I;

R³ is H;

R⁴ is F or ¹⁸F; and

R⁵ is H.

8. The compound or pharmaceutically acceptable salt of claim 1, wherein n is 0;

R¹, R³, and R⁵ are each H;

R² is -CH₃, -CH2CH3, -CF=CH₂, -C=CH, F, Cl, or I; and R⁴ is F or ¹⁸F.

9. The compound or pharmaceutically acceptable salt of claim 1, wherein n is 0;

R¹ is -CH₃ or -CH₂CH₃;

R², R³ and R⁵ are each H; and

R⁴ is F or ¹⁸F.

10. The compound or pharmaceutically acceptable salt of claim 1, wherein n is 2;

R¹ is H;

R² is H or -CH₃;

R³ is H;

R⁴ is F, ¹⁸F, CI-C₃ alkylF, Ci-C₃ alkyl¹⁸F; and

R⁵ is H.

11. The compound or pharmaceutically acceptable salt of claim 1, wherein the compound is

12. A pharmaceutical composition comprising the compound or pharmaceutically acceptable salt thereof of any one of claims 1-11, and one or more pharmaceutically acceptable carrier or diluent.

13. The pharmaceutical composition of claim 12, wherein the carrier comprises ethanol, water, and a buffer suitable for injection into a patient.

14. A method of imaging aggregated tau in a mammal, the method comprising: introducing into a mammal a detectable quantity of the compound or pharmaceutically acceptable salt thereof of any one of claims 1-11, wherein said compound or pharmaceutically acceptable salt thereof has an ¹⁸F substituent; allowing sufficient time for said compound to become associated with aggregated tau; and detecting said compound.

15. The method of claim 14, wherein the mammal is a human.

16. The method of claim 14, wherein the mamal is a human and is suspected of having a neurological disease or disorder.

17. The method of claim 16, wherein the human is suspected of having Alzheimer’s Disease.

18 The method of claim 16, wherein the human is suspected of having progressive supranuclear palsy (PSP), cortical basal degeneration (CBD), Pick’s disease (PiD), atypical Alzheimer’s disease, chronic traumatic encephalopathy (CTE), and frontotemporal dementia (FTD).

19. The method of claim 16, wherein the human is suspected of having early Alzheimer’s disease tau.

20. The method of claim 16, wherein the human is suspected of having nonAlzheimer’s disease tau.

21. A compound or a pharmaceutically acceptable salt thereof of the formula: wherein n is 0, 1, or 2;

R¹ is H, halo, C1-C3 alkyl, or C3-C6 cycloalkyl;

R² is H, halo, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, -O-(Ci-C₄ alkyl), C1-C4 alkylhalo, C2-C4 alkenylhalo, •

R³ is H or F;

L is a bond, Ci-C₄-alkylF, Ci-C₄-alkyl¹⁸F, -O-(Ci-C₄-alkyl)F, or -O-(Ci- C₄-alkyl)¹⁸F;

LG is a leaving group;

R⁵ is H, halo, or Ci-C₄ alkyl; provided when n is 0, R¹ is methyl, R² is H, R³ is H, and R⁵ is H; then L is not a bond.

22. The compound or pharmaceutically acceptable salt of claim 21, wherein the LG is -NO2, a trialkyl amine, an alkyl sulfonate, an aryl sulfonate, a mesylate group, a tosylate group, a tritiate group, Cl, Br, I, or a nosylate group.

23. A compound or pharmaceutically acceptable salt of claim 18, wherein the Leaving Group is mesylate or tosylate.