Syntheses and use of potassium channel blockers in pet imaging and in therapeutics

Abstract

This disclosure relates to aminopyridine compounds of Formula (I) and (I') as positron emission tomography tracers for detecting demyelination in a subject suspected of having a neurological disorder and methods of manufacture and of uses thereof.

Description

[0001] SYNTHESES AND USE OF POTASSIUM CHANNEL BLOCKERS IN PET IMAGING AND IN THERAPEUTICS

[0002] CLAIM OF PRIORITY

[0003] This application claims priority to U.S. Patent Application Serial No. 63 / 518,164, filed on August 8, 2023, the entire contents of which are hereby incorporated by reference.

[0004] STATEMENT OF FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under Grant No. NS 114066 awarded by National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD

[0007] This disclosure relates to aminopyridine compounds as positron emission tomography tracers for detecting demyelination and methods of manufacture and of uses thereof.

[0008] BACKGROUND

[0009] 4-aminopyridine (4AP) is a potassium channel blocker used in the symptomatic treatment of multiple sclerosis (MS). Its mechanism of action involves binding from the intracellular side to voltage-gated K+(Kv) channels exposed due to demyelination, thereby blocking the aberrant efflux of K+ions and enhancing axonal conduction. Additionally, 4AP has demonstrated potential clinical utility for spinal cord injury (SCI), traumatic brain injury (TBI), and other diseases involving demyelination. Based on the mechanism of action of 4AP, it has been proposed that upregulated K+channels in demyelinated axons could be targeted for imaging demyelination using positron emission tomography (PET).

[0010] SUMMARY

[0011] The disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R, m, and n are defined herein. The disclosure also provides a method of producing a compound of Formula (I’) or a pharmaceutically acceptable salt thereof, the method comprising the steps of

[0012] (i) treating a compound of Formula (A) with a source of nucleophilic18F to provide the compound of Formula (A’)

[0013] (ii) reducing the compound of Formula (A’) to provide the compound of Formula (I) wherein each R and n are disclosed herein.

[0014] The disclosure provides a compound of Formula (F)

[0015] or a pharmaceutically acceptable salt thereof, prepared by a process comprising:

[0016] (i) treating a compound of Formula (A) with a source of nucleophilic18F to provide a compound of Formula (A’),

[0017] (ii) reducing the compound of Formula A’ to provide the compound of Formula (I), wherein each R an n are disclosed herein.

[0018] Some embodiments provide a method of imaging a tissue comprising contacting the tissue with a compound disclosed herein; and detecting a signal from the compound, thereby imaging the tissue.

[0019] Some embodiments provide a method of detecting demyelination in a subject suspected of having a neurological disorder, the method comprising administering to the subject an imaging effective amount of a compound disclosed herein; and determining the presence or absence of a signal from the compound, wherein the presence of a signal indicates a diagnosis of demyelination. Some embodiments provide a method of treating a neurological disorder in a subject suspected of having a neurological disorder, the method comprising administering to the subject an imaging effective amount of a compound disclosed herein; determining the presence or absence of a signal from the compound; and if a signal is detected, administering to the subject a therapeutically effective amount of one more agents used to treat a neurological disorder, or a combination thereof.

[0020] Some embodiments provide a kit comprising a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein; and instructions for administering the compound, the pharmaceutically acceptable salt thereof, or the pharmaceutical composition to a subject.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. Methods and materials are described herein for use in the present application; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0022] Other features and advantages of the present application will be apparent from the following detailed description and figures, and from the claims.

[0023] DESCRIPTION OF THE DRAWINGS

[0024] Fig. 1 shows a scheme of demyelination: in normally myelinated axons, sodium channels concentrate at the nodes of Ranvier (also known as myelin-sheath gaps) and potassium (K+) channels at the neighboring juxtaparanodes beneath the myelin sheath. During demyelination, K+channels become exposed, migrate through the demyelinated segment and increase in expression. This exposure of K+channels results in leakage of intracellular potassium ions, which in turn impairs propagation of electrical impulses.

[0025] Fig. 2 shows the structures of 5MeF4AP, 3F4AP, 3Me4AP, and 4AP, a comparison of (A) pKa (n=4), logD (n=4) and (B) permeability (Pe, n = 3) to an artificial membrane between 5Me3F4AP, 3F4AP, 3Me4AP and 4AP. * P < 0.05; ** P < 0.01, *** P < 0.001, **** P < 0.0001 and (C) CYP2E1 inhibition IC50 fit curves for 4AP (star), 5Me3F4AP (diamond), 3F4AP (plus), and tranylcypromine (hexagon, positive control). (D) Representative recordings at pH of 7.4 elicited from oocytes expressing the Shaker channel before (upper, black) and after (lower, colored) the blockage with 1 mM of 5Me3F4AP. Currents were recorded as the response to voltage stimulus protocol that consisted of 50 ms depolarization steps from -100 to 60 mV in increments of 10 mV. Dashed line represents the zero current value. Horizontal and vertical bars of 25 ms and 2 pA represent the time and current scale for all recordings; (E) a comparison of IC50 values to inhibition of cytochrome enzyme; (F) Relative fluorescence-time curves based on the 60-minute kinetic measurement of 4AP (star), 5Me3F4AP (diamond), 3F4AP (star), and tranylcypromine (hexagon, positive control).

[0026] Fig. 3 shows A) the chemical scheme of the radiosynthesis of [18F]5Me3F4AP; B) quality control of [18F]5Me3F4AP (radioactivity trace at the top, UV 254 nm trace in the middle, and the non- radioactive standard at the bottom); C) Representative HPLC chromatogram of the reaction crude from step 1 (radioactivity trace at the top and UV 254 nm trace at the bottom).

[0027] Fig. 4 shows the representative summed PET images captured from 0-60 minutes for [18F]5Me3F4AP and [18F]3F4AP.

[0028] Fig. 5 shows time-activity curves and terminal uptake in blood and brain for [18F]5Me3F4AP and [18F]3F4AP.

[0029] DETAILED DESCRIPTION

[0030] Compounds of Formula (I)

[0031] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein: each R is independently chosen from halogen, C1-10alkyl, [18F]C1-C3monofluoroalkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-10 membered aryl, and 5-10 membered heteroaryl; and n is 0, 1, 2, or 3; and m is 0 or 1 ; and wherein if one or more R is [18F]C1-C3monofluoroalkyl, then m is 0; if no R is [18F]C1-C3monofluoroalkyl, then m is 1.

[0032] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein: each R is independently chosen from halogen, C1-3alkyl, [18F]C1-C3monofluoroalkyl, C1-3haloalkyl, C2-3alkenyl, C2-3alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-10 membered aryl, and 5-10 membered heteroaryl; and n is 1; and m is 0 or 1 ; and wherein if one or more R is [18F]C1-C3monofluoroalkyl, then m is 0; if no R is [18F]C1-C3monofluoroalkyl, then m is 1.

[0033] In some embodiments, m is 1.

[0034] In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has only one position substituted with18F.

[0035] In some embodiments, when one or more R is |l 8F IC1-C3 monofluoroalkyl, then m is 0.

[0036] In some embodiments, when m is 0, then one or more R is [18F]C1-C3monofluoroalkyl.

[0037] In some embodiments, when m is 1, then R is not [18F]C1-C3monofluoroalkyl.

[0038] In some embodiments, when m is 1, then each R is independently chosen from halogen, C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-10 membered aryl, and 5-10 membered heteroaryl.

[0039] 18F is a fluorine radioisotope having a half-life of about 110 minutes. As such, its natural abundance is 0%. In some embodiments, the isotopic enrichment level of each18F is at least 0.0001%. In some embodiments, the isotopic enrichment level of each18F is about 0.0001% to about 5%, for example about 0.0001% to about 0.1%, about 0.0001% to about 0.001%, about 0.001% to about 0.01%, about 0.01% to about 0.1%, about 0.1% to about 1%, about 0.0001% to about 1%, about 0.0001% to about 4%, about 0.0001% to about 3%, about 0.0001% to about 2%, about 0.0001%, about 0.001%, about 0.01%, or about 0. 1%. In some embodiments, the isotopic enrichment level of each18F is about 0.0001% to about 0. 1%.

[0040] In some embodiments, n is 3.

[0041] In some embodiments, n is 2.

[0042] In some embodiments, n is i. In some embodiments, n is 0.

[0043] In some embodiments, each R is independently chosen from halogen, C1-6alkyl, [18F]C1-C3monofluoroalkyl, C1-6haloalkyl, C2-6alkenyl, C2-6alkynyl, 3-6 membered cycloalkyl, 4-6 membered heterocyclyl, phenyl, and 5-6 membered heteroaryl.

[0044] In some embodiments, each R is independently chosen from halogen, C1-3alkyl, [18F]C1-C3monofluoroalkyl, C1-3haloalkyl, C2-3alkenyl, C2-3alkynyl, 3-6 membered cycloalkyl, and 4-6 membered heterocyclyl.

[0045] In some embodiments, n is 2 and each R is independently selected from halogen and C1-6alkyl.

[0046] In some embodiments, n is 2 and each R is independently selected from halogen and [18F]CI- C3 monofluoroalkyl.

[0047] In some embodiments, n is 1 and R is halogen.

[0048] In some embodiments, n is 1 and R is C1-6alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is propyl.

[0049] In some embodiments, n is 1 and R is [18F]C1-C3monofluoroalkyl. In some embodiments, R is -CHF[18F], In some embodiments, R is -CF2[18F],

[0050] In some embodiments, n is 1 and R is C1-6haloalkyl.

[0051] In some embodiments, n is 1 and R is C2-6alkenyl.

[0052] In some embodiments, n is 1 and R is C2-6alkynyl.

[0053] In some embodiments, n is 1 and R is 3-6 membered cycloalkyl.

[0054] In some embodiments, n is 1 and R is 4-6 membered heterocyclyl.

[0055] In some embodiments, n is 1 and R is 6-10 membered aryl. In some embodiments, n is 1 and R is phenyl.

[0056] In some embodiments, n is 1 and R is 5-10 membered heteroaryl. In some embodiments, n is 1 and R is 5-6 membered heteroaryl.

[0057] In some embodiments, each R is C1-10 alkyl. In some embodiments, each R is methyl. In some embodiments, each R is ethyl. In some embodiments, each R is propyl.

[0058] In some embodiments, the compound is selected from:

[0059] or a pharmaceutically acceptable salt of any of the foregoing.

[0060] In some embodiments, the compound is:

[0061] In some embodiments, the disclosure provides a compound of Formula (I’) or a pharmaceutically acceptable salt thereof, prepared by a process comprising:

[0062] (i) treating a compound of Formula (A)

[0063] with a source of nucleophilic18F to provide a compound of Formula (A’)

[0064] (ii) reducing the compound of Formula (A’) to provide the compound of Formula (I), wherein: each R is independently chosen from halogen, C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-10 membered aryl, and 5-10 membered heteroaryl; and n is 0, 1, 2, or 3.

[0065] In some embodiments, the source of nucleophilic18F is tetrabutylammonium[18F]fluoride.

[0066] In some embodiments, the treating step is conducted in a polar aprotic solvent.

[0067] In some embodiments, the polar aprotic solvent is selected from tetrahydrofuran (THF), dimethyl sulfoxide, ethers, dimethylformamide (DMF) and hexamethylphosphorotriamide, N-methyl pyrrolidone (NMP), acetonitrile (MeCN or ACN), CS2, N-cyclohexyl -2 -pyrrolidone, dimethyl sulfoxide (DMSO).

[0068] In some embodiments, the polar aprotic solvent is DMSO.

[0069] In some embodiments, the treating step is conducted at room temperature.

[0070] In some embodiments, the reducing step is conducted with a hydrogenating agent. In some embodiments, the reducing step is conducted with a hydrogenating agent and hydrogen gas.

[0071] In some embodiments, the hydrogenating agent is palladium on carbon.

[0072] In some embodiments, the isotopic enrichment level of each18F is at least 0.0001%. In some embodiments, the isotopic enrichment level of each18F is about 0.0001% to about 5%, for example about 0.0001% to about 0.1%, about 0.0001% to about 0.001%, about 0.001% to about 0.01%, about 0.01% to about 0.1%, about 0.1% to about 1%, about 0.0001% to about 1%, about 0.0001% to about 4%, about 0.0001% to about 3%, about 0.0001% to about 2%, about 0.0001%, about 0.001%, about 0.01%, or about 0.1%. In some embodiments, the isotopic enrichment level of each18F is about 0.0001% to about 0.1%. .

[0073] In some embodiments, n is 3.

[0074] In some embodiments, n is 2.

[0075] In some embodiments, n is i.

[0076] In some embodiments, n is 0.

[0077] In some embodiments, each R is independently chosen from halogen, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, C2-6alkynyl, 3-6 membered cycloalkyl, 4-6 membered heterocyclyl, phenyl, and 5-6 membered heteroaryl.

[0078] In some embodiments, each R is independently chosen from halogen, C1-3alkyl, C1-3haloalkyl, C2-3alkenyl, C2-3alkynyl, 3-6 membered cycloalkyl, and 4-6 membered heterocyclyl.

[0079] In some embodiments, n is 2 and each R is independently selected from halogen and C1-6alkyl.

[0080] In some embodiments, n is 1 and R is halogen.

[0081] In some embodiments, n is 1 and R is C1-6alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is propyl.

[0082] In some embodiments, n is 1 and R is C1-6haloalkyl.

[0083] In some embodiments, n is 1 and R is C2-6alkenyl.

[0084] In some embodiments, n is 1 and R is C2-6alkynyl.

[0085] In some embodiments, n is 1 and R is 3-6 membered cycloalkyl.

[0086] In some embodiments, n is 1 and R is 4-6 membered heterocyclyl.

[0087] In some embodiments, n is 1 and R is 6-10 membered aryl. In some embodiments, n is 1 and R is phenyl.

[0088] In some embodiments, n is 1 and R is 5-10 membered heteroaryl. In some embodiments, n is 1 and R is 5-6 membered heteroaryl.

[0089] In some embodiments, each R is C1-10 alkyl. In some embodiments, each R is methyl. In some embodiments, each R is ethyl. In some embodiments, each R is propyl. In some embodiments, the compound of Formula (I’), or a pharmaceutically acceptable salt thereof, is selected from: or a pharmaceutically acceptable salt of any of the foregoing.

[0090] In some embodiments, the compound of Formula (I’), or a pharmaceutically acceptable salt thereof, is Pharmaceutically acceptable salts

[0091] In some embodiments, the disclosure provides a pharmaceutical composition comprising a compound of Formula of (I) or (I’), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0092] In some embodiments, a salt of a compound of this disclosure is formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group. According to another embodiment, the compound is a pharmaceutically acceptable acid addition salt.

[0093] In some embodiments, acids commonly employed to form pharmaceutically acceptable salts of the compounds of Formula (I) include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne- 1,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, P-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, naphthalene-2- sulfonate, mandelate and other salts. In one embodiment, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.

[0094] In some embodiments, bases commonly employed to form pharmaceutically acceptable salts of the compounds of Formula (I) include hydroxides of alkali metals, including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxylsubstituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N- ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(Ci-C6)-alkylamine), such as N,N- dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the Compositions and Methods of Use

[0095] The present application also provides pharmaceutical compositions comprising an effective amount of a compound of the present disclosure (e.g., Formula (I)) disclosed herein and a pharmaceutically acceptable carrier. The carrier(s) are “acceptable” in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in an amount used in the medicament.

[0096] In some embodiments, the composition comprises a mixture of compounds of Formula (I), or a pharmaceutically acceptable salts thereof.

[0097] In some embodiments, the disclosure provides a method of producing a compound of Formula

[0098] (I ) or a pharmaceutically acceptable salt thereof, the method comprising the steps of

[0099] (i) treating a compound of Formula (A) with a source of nucleophilic18F to provide the compound of Formula (A’)

[0100]

[0101] (ii) reducing the compound of Formula (A’) to provide the compound of Formula (I) wherein: each R is independently chosen from halogen, C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-10 membered aryl, and 5-10 membered heteroaryl; and n is 0, 1, 2, or 3.

[0102] In some embodiments, the isotopic enrichment level of each18F is at least 0.0001%. In some embodiments, the isotopic enrichment level of each18F is about 0.0001% to about 5%, for example about 0.0001% to about 0.1%, about 0.0001% to about 0.001%, about 0.001% to about 0.01%, about 0.01% to about 0.1%, about 0.1% to about 1%, about 0.0001% to about 1%, about 0.0001% to about 4%, about 0.0001% to about 3%, about 0.0001% to about 2%, about 0.0001%, about 0.001%, about 0.01%, or about 0.1%. In some embodiments, the isotopic enrichment level of each18F is about 0.0001% to about 0.1%.

[0103] In some embodiments, the source of nucleophilic18F is tetrabutylammonium[18F]fluoride.

[0104] In some embodiments, the treating step is conducted in a polar aprotic solvent.

[0105] In some embodiments, the polar aprotic solvent is selected from tetrahydrofuran (THF), dimethyl sulfoxide, ethers, dimethylformamide (DMF) and hexamethylphosphorotriamide, N-methyl pyrrolidone (NMP), acetonitrile (MeCN or ACN), CS2, N-cyclohexyl -2 -pyrrolidone, dimethyl sulfoxide (DMSO).

[0106] In some embodiments, the polar aprotic solvent is DMSO.

[0107] In some embodiments, the treating step is conducted at room temperature.

[0108] In some embodiments, the reducing step is conducted with a hydrogenating agent.

[0109] In some embodiments, the reducing step is conducted with a hydrogenating agent and hydrogen gas.

[0110] In some embodiments, the hydrogenating agent is palladium on carbon. In some embodiments, the isotopic enrichment level ofthe18F is about 0.0001%to about 0.1%.

[0111] In some embodiments, each R is independently chosen from halogen, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, C2-6alkynyl, 3-6 membered cycloalkyl, 4-6 membered heterocyclyl, phenyl, and 5-6 membered heteroaryl.

[0112] In some embodiments, each R is independently chosen from halogen, C1-3alkyl, C1-3haloalkyl, C2-3alkenyl, C2-3alkynyl, 3-6 membered cycloalkyl, and 4-6 membered heterocyclyl.

[0113] In some embodiments, n is 2 and each R is independently selected from halogen and C1-6alkyl.

[0114] In some embodiments, n is 1 and R is halogen. In some embodiments, n is 1 and R is flourine.

[0115] In some embodiments, n is 1 and R is C1-6alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is propyl.

[0116] In some embodiments, n is 1 and R is C1-6haloalkyl.

[0117] In some embodiments, n is 1 and R is C2-6alkenyl.

[0118] In some embodiments, n is 1 and R is C2-6alkynyl.

[0119] In some embodiments, n is 1 and R is 3-6 membered cycloalkyl.

[0120] In some embodiments, n is 1 and R is 4-6 membered heterocyclyl.

[0121] In some embodiments, n is 1 and R is 6-10 membered aryl. In some embodiments, n is 1 and R is phenyl.

[0122] In some embodiments, n is 1 and R is 5-10 membered heteroaryl. In some embodiments, n is 1 and R is 5-6 membered heteroaryl.

[0123] In some embodiments, each R is C1-10 alkyl. In some embodiments, each R is methyl. In some embodiments, each R is ethyl. In some embodiments, each R is propyl.

[0124] In some embodiments, the compound of Formula (F), or a pharmaceutically acceptable salt thereof, is selected from:

[0125] or a pharmaceutically acceptable salt of any of the foregoing.

[0126] In some embodiments, the compound of Formula (I’), or a pharmaceutically acceptable salt thereof, is

[0127] In some embodiments, the disclosure provides a method of imaging a tissue comprising: contacting the tissue with a compound of Formula (I) or (I’); and detecting a signal from the compound, thereby imaging the tissue.

[0128] In some embodiments, the tissue is CNS tissue. In some embodiments, the tissue is brain tissue. In some embodiments, the tissue is in a sample from a subject, such as a biopsy sample. In some embodiments, the tissue is in a subject.

[0129] In some embodiments, the disclosure provides a method of detecting demyelination in a subject suspected of having a neurological disorder, the method comprising administering to the subject an imaging effective amount of a compound of Formula (I) or (I’); and determining the presence or absence of a signal from the compound, wherein the presence of a signal indicates a diagnosis of demyelination. In some embodiments, the neurological disorder is a central nervous system (CNS) disorder. In some embodiments, the neurological disorder is a peripheral nervous system (PNS) disorder.

[0130] In some embodiments, the disclosure provides a method of detecting demyelination in a subject suspected of having a neurological disorder, the method comprising administering to the subject an imaging effective amount of a compound of Formula (I) or (I’); and determining the presence or absence of a signal from the compound, wherein the presence of a signal indicates a diagnosis of demyelination.

[0131] In some embodiments, the disclosure provides a method of treating a neurological disorder in a subject suspected of having a neurological disorder, the method comprising administering to the subject an imaging effective amount of a compound of Formula (I) or (I’); determining the presence or absence of a signal from the compound; and if a signal is detected, administering to the subject a therapeutically effective amount of one more agents used to treat a neurological disorder, or a combination thereof.

[0132] In some embodiments, the disclosure provides a method of treating a neurological disorder in a subject suspected of having a neurological disorder, the method comprising administering to the subject an imaging effective amount of a compound of Formula (I) or (F); determining the presence or absence of a signal from the compound; and if a signal is detected, administering to the subject a therapeutically effective amount of one more agents used to treat a neurological disorder, or a combination thereof.

[0133] In some embodiments, the neurological disorder is selected from multiple sclerosis (MS), myelin oligodendrocyte glicoprotein antibody disease (MOGAD), neuromyelitis optica, spinal cord injury, spinal cord compression, acute disseminated encephalomyelitis, optic neuromyelitis, progressive multifocal leukoencephalopathy, congenital demyelinating disease, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, metabolic disorders, multifocal motor neuropathy, leukodystrophy, adrenoleukodystrophy, Alexander's Disease, Metachromatic Leukodystrophy, vanishing white matter disease, Refsum Disease, Cockayne Syndrome, Van der Knapp Syndrome, Zellweger Syndrome, Pelizaeus-Merzbacher Disease, Krabbe Disease, Canavan Disease, traumatic brain injury, botulism intoxication, tetrodotoxin poisoning, carpal tunnel syndrome, diabetes, Epstein-Barr syndrome, amyotrophic lateral sclerosis, and other neurological diseases.

[0134] In some embodiments, the disclosure provides a kit comprising the compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein; and instructions for administering the compound, the pharmaceutically acceptable salt thereof, or the pharmaceutical composition to a subject.

[0135] Definitions

[0136] As used herein, the term “about” means “approximately” (e.g., plus or minus 10% of the indicated value).

[0137] The term “halo” or “halogen” refers to any radical of fluorine, chlorine, bromine or iodine. The term “alkyl” refers to a saturated hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, Ci-Cg alkyl indicates that the group may have from 1 to 6 (inclusive) carbon atoms in it. Any atom can be optionally substituted, e.g., by one or more substituents. Examples of alkyl groups include, without limitation, methyl, ethyl, w-propyl, isopropyl, and tert-butyl.

[0138] The term “alkenyl” refers to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon double bonds. Any atom can be optionally substituted, e.g., by one or more substituents. Alkenyl groups can include, e.g., vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double bond carbons can optionally be the point of attachment of the alkenyl substituent.

[0139] The term “cycloalkyl” refers to a fully saturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group. Any atom can be optionally substituted, e.g., by one or more substituents. A ring carbon serves as the point of attachment of a cycloalkyl group to another moiety. Cycloalkyl moieties can include groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbomyl (bicyclo[2.2.1]heptyl).

[0140] The term “heterocyclyl” refers to a fully saturated monocyclic, bicyclic, tricyclic or other polycyclic ring system having one or more constituent heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and / or form a salt), or S. The heteroatom or ring carbon can be the point of attachment of the heterocyclyl substituent to another moiety. Any atom can be optionally substituted, e.g., by one or more substituents. Heterocyclyl groups can include groups such as tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl. By way of example, a phrase such as “heterocyclic ring containing from 5-6 ring atoms”, includes (but is not limited to) tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl.

[0141] The term “aryl” refers to an aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (> 3 fused rings) hydrocarbon ring system. One or more ring atoms can be optionally substituted by one or more substituents for example. Aryl moieties include groups such as phenyl and naphthyl.

[0142] The term “heteroaryl” refers to an aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (> 3 fused rings) hydrocarbon groups having one or more heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and / or form a salt), or S. One or more ring atoms can be optionally substituted, e.g., by one or more substituents. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo [b]thienyl, benzothiazolyl, P-carbolinyl, carbazolyl, coumarinyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, and xanthenyl.

[0143] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, N=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated or synthesized as a mixture of isomers or as separated isomeric forms. In some embodiments, the compound of Formula (I) has the (R)- configuration. In some embodiments, the compound of Formula (I) has the fSj-configuration.

[0144] Compounds provided herein also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H- imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0145] As used herein, the term “cell” is meant to refer to a cell that is in vitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal.

[0146] As used herein, the term “subject” refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans. As used herein, the phrase “imaging effective amount” refers to the necessary amount of active compound or pharmaceutical agent that allows for imaging of a tissue in an animal, individual, or human to be detected by the detection method chosen. For example, a detectable quantity can be an administered amount sufficient to enable detection of binding of the labeled compound to a target of interest including, but not limited to myelin.

[0147] As used herein, the phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

[0148] As used herein the term “treating” or “treatment” refers to 1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and / or symptomatology), or 2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i. e. , reversing the pathology and / or symptomatology) .

[0149] As used herein, the term “preventing” or “prevention” of a disease, condition or disorder refers to decreasing the risk of occurrence of the disease, condition or disorder in a subject or group of subjects (e.g., a subject or group of subjects predisposed to or susceptible to the disease, condition or disorder). In some embodiments, preventing a disease, condition or disorder refers to decreasing the possibility of acquiring the disease, condition or disorder and / or its associated symptoms. In some embodiments, preventing a disease, condition or disorder refers to completely or almost completely stopping the disease, condition or disorder from occurring.

[0150] NUMBERED EMBODIMENTS

[0151] Embodiment 1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein: each R is independently chosen from halogen, C1-10alkyl, [18F]CI-C3 monofluoroalkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-10 membered aryl, and 5-10 membered heteroaryl; and n is 0, 1, 2, or 3; and m is 0 or 1 ; and wherein if one R is [18F]CI-C3 monofluoroalkyl, then m is 0; if no R is [18F]C1-C3monofluoroalkyl, then m is 1.

[0152] Embodiment 2. The compound of embodiment 1, wherein m is 1.

[0153] Embodiment 3. The compound of embodiment 1 or 2, wherein the isotopic enrichment level of each18F is about 0.0001% to about 0.1%.

[0154] Embodiment 4. The compound of embodiment 1, wherein n is 2.

[0155] Embodiment 5. The compound of embodiment 1, wherein n is 1.

[0156] Embodiment 6. The compound of any one of embodiments 1-5, wherein each R is C1-10alkyl.

[0157] Embodiment 7. The compound of any one of embodiments 1-6, wherein each R is methyl.

[0158] Embodiment 8. The compound of any one of embodiments 1-6, wherein each R is ethyl.

[0159] Embodiment 9. The compound of any one of embodiments 1-6, wherein each R is propyl.

[0160] Embodiment 10. The compound of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

[0161] or a pharmaceutically acceptable salt of any of the foregoing.

[0162] Embodiment 11. The compound of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein the compound is:

[0163] Embodiment 12. A method of producing a compound of Formula (F) or a pharmaceutically acceptable salt thereof, the method comprising the steps of

[0164] (i) treating a compound of Formula (A)

[0165] with a source of nucleophilic18F to provide the compound of Formula (A’)

[0166] (ii) reducing the compound of Formula (A’) to provide the compound of Formula (I) wherein: each R is independently chosen from halogen, C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-10 membered aryl, and 5-10 membered heteroaryl; and n is 0, 1, 2, or 3.

[0167] Embodiment 13. A compound of Formula (F) or a pharmaceutically acceptable salt thereof, prepared by a process comprising:

[0168] (i) treating a compound of Formula (A)

[0169] with a source of nucleophilic18F to provide a compound of Formula (A’)

[0170] (ii) reducing the compound of Formula (A’) to provide the compound of Formula (I), wherein: each R is independently chosen from halogen, C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-10 membered aryl, and 5-10 membered heteroaryl; and n is 0, 1, 2, or 3.

[0171] Embodiment 14. The method or compound of embodiment 12 or embodiment 13, wherein the source of nucleophilic18F is tetrabutylammonium[18F]fluoride.

[0172] Embodiment 15. The method or compound of any one of embodiments 12-14, wherein the treating step is conducted in a polar aprotic solvent.

[0173] Embodiment 16. The method or compound of any one of embodiments 12-15, wherein the polar aprotic solvent is selected from tetrahydrofuran (THF), dimethyl sulfoxide, ethers, dimethylformamide (DMF) and hexamethylphosphorotriamide, N-methyl pyrrolidone (NMP), acetonitrile, CS2, N-cyclohexyl-2 -pyrrolidone, dimethyl sulfoxide (DMSO). Embodiment 17. The method or compound of any one of embodiments 12-16, wherein the polar aprotic solvent is DMSO.

[0174] Embodiment 18. The method or compound of any one of embodiments 12-17, wherein the treating step is conducted at room temperature.

[0175] Embodiment 19. The method or compound of any one of embodiments 12-18, wherein the reducing step is conducted with a hydrogenating agent.

[0176] Embodiment 20. The method or compound of any one of embodiments 12-19, wherein the reducing step is conducted with a hydrogenating agent and hydrogen gas.

[0177] Embodiment 21. The method or compound of embodiment 20, wherein the hydrogenating agent is palladium on carbon.

[0178] Embodiment 22. The method of any one of embodiments 12-21, wherein the isotopic enrichment level of the18F is at least 0.0001%.

[0179] Embodiment 23. The method or compound of any one of embodiments 12-22, wherein the isotopic enrichment level of the18F is about 0.0001% to about 0.1%.

[0180] Embodiment 24. The method or compound of any one of embodiments 12-23, wherein each R is Ci -10 alkyl.

[0181] Embodiment 25. The method or compound of any one of embodiments 12-24, wherein each R is methyl.

[0182] Embodiment 26. The method or compound of any one of embodiments 12-24, wherein each R is ethyl.

[0183] Embodiment 27. The method or compound of any one of embodiments 12-24, wherein each

[0184] R is propyl.

[0185] Embodiment 28. The method or compound of any one of embodiments 12-23, wherein the compound of Formula (F), or a pharmaceutically acceptable salt thereof, is selected from

[0186] or a pharmaceutically acceptable salt of any of the foregoing.

[0187] Embodiment 29. The method or compound of any one of embodiments 21-23, wherein the compound of Formula (I’), or a pharmaceutically acceptable salt thereof, is

[0188] Embodiment 30. A pharmaceutical composition comprising a compound of any one of embodiments 1-11 or a compound of embodiment 13-29, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0189] Embodiment 31. A method of imaging a tissue comprising:

[0190] (a) contacting the tissue with a compound according to any one of embodiments 1-11; and

[0191] (b) detecting a signal from the compound, thereby imaging the tissue.

[0192] Embodiment 32. The method of embodiment 31, wherein the tissue is CNS tissue.

[0193] Embodiment 33. The method of embodiment 31 or 32, wherein the tissue is brain tissue. Embodiment 34. A method of detecting demyelination in a subject suspected of having a neurological disorder, the method comprising:

[0194] (a) administering to the subject an imaging effective amount of a compound according to any one of embodiments 1-11; and

[0195] (b) determining the presence or absence of a signal from the compound, wherein the presence of a signal indicates a diagnosis of demyelination.

[0196] Embodiment 35. A method of treating a neurological disorder in a subject suspected of having a neurological disorder, the method comprising:

[0197] (a) administering to the subject an imaging effective amount of a compound according to any one of embodiments 1-11;

[0198] (b) determining the presence or absence of a signal from the compound; and

[0199] (c) if a signal is detected, administering to the subject a therapeutically effective amount of one more agents used to treat a neurological disorder, or a combination thereof.

[0200] Embodiment 36. A kit comprising the compound of any of embodiments 1-11, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of embodiment 30; and instructions for administering the compound, the pharmaceutically acceptable salt thereof, or the pharmaceutical composition to a subject.

[0201] EXAMPLES

[0202] Example 1 - Synthesis of Compounds

[0203] The radiochemical synthesis of [18F]3-fluoro-5-methylpyridin-4-amine ([18F]5Me3F4AP) via a19F / 18F replacement of 3 -fluoro-5 -methyl -4-nitropyridine 1 -oxide with tetrabutylammonium [18F]fluoride ([18F]TBAF) and a subsequent palladium-on-carbon (Pd / Cj-mediated reduction process is depicted in Scheme 1.

[0204] Scheme 1. Radiochemical synthesis of [18F]5Me3F4AP.

[0205] Azeotropically drying, of the [18F]fluoride. The [18F]F‘ was first trapped on a Sep-Pak Light Accell Plus QMA carbonate Cartridge (Waters) pre-conditioned with 5 mb 50 mM potassium bicarbonate (KHCO3) and 10 mb Ultrapure water. Next, the concentrated [18F]F‘ was eluted into the reaction vessel using one of the following: 400 μL 50% MeCN in 0.075 M tetrabutylammounium bicarbonate (TABHCO3, 15 pmol) aqueous solution (ABX advanced biochemical compounds GmbH, Germany), 1.2 mL 50% MeCN in 2.5 mg / mL KHCO3 (15 pmol) aqueous solution or 1.2 mL 50% MeCN in tetraethylammonium bicarbonate (TEA-HCO3, 3 mg, 15 pmol) aqueous solution. The aqueous solution was azeotropically dried under vacuum and nitrogen flow within 14 min at 85 °C. Two aliquots of MeCN (2X500 μL) were added during the drying procedure.

[0206] Production of tetrabutylammonium f18F]fluoride (f18F]TBAF): -Fifty mCi of cyclotron produced18F" were trapped in a Sep-Pak Accell Plus QMA Plus Light Cartridge (Waters Corporation) preconditioned with 10 mL of water. The cartridge was eluted with a solution containing of 300 μL of 50 mM TBA-HCO3 in water with 5% EtOH (ABX advanced biochemical compounds GmbH) and 600 μL of acetonitrile (MeCN). The water-MeCN solution was dried azeotropically at 85 °C under with air-blowing for 7.5 min. To ensure complete dryness, two additional aliquots of MeCN (500 μL) followed by evaporation (3 min) were performed. After drying, the vial was cooled down to room temperature. The [18F]TBAF residue was dissolved in 100-400 μL of anhydrous DMSO and used for the reactions.

[0207] Radiochemical synthesis of fI8F] 3-fluoro-4-nitropyridine N-oxide from 3-fluoro-4- nitropyridine N-oxide by19F / 18F exchange'. To vials containing various amounts of 3-fluoro-4- nitropyridine N-oxide, 200 μL of [18F]TBAF solution (-2 mCi, -74 MBq) was added and allowed to react for 20 min. 10 μL of this solution was injected into HPLC (a Thermo Scientific Dionex Ultimate 3000 UHPLC) equipped with a C-18 column (XBridge, BEH, 130 A, 3.5 pm, 4.6 x 150 mm), a diode array detector set at 254 nm and a radiation detector with a gradient condition (flow 1 mL / min; solvent A: 10 mM ammonium bicarbonate (NH4HCO3), pH 8; solvent B: methanol; method: 0-2 min: 0.5% B, 2-9 min: 0.5-20% B, 9-17 min: 20% B, 17-17.5 min: 20-0.5% B, 17.5-22 min: 0.5% B) to determine the radiochemical conversion.

[0208] Table 2. Radiochemical synthesis by18F / 19F exchange

[0209] a RCY were determined by radioHPLC;bisolated RCY through an Alumina N cartridge in Fx2N synthesizer.cn = 5.

[0210] The fluoro-precursor via isotopic exchange was tested (Table 2). Manual labeling with aliquoted azeotropic [18F]fluoride ([18F]F") solution in various solvents at room temperature for 15 minutes showed that non-protic polar solvents such as DMSO, MeCN, and THF yielded moderate RCYs with tetrabutylammonium [18F]fluoride (entries 1-4). However, no product was obtained with potassium [18F]KF and kryptofix (K222) (entry 5). The low RCY was typically accompanied by the observation of precursor decomposition. This may be because KF / K222 is less nucleophilic and more basic than [18F]TBAF, leading to less reactivity and more severe precursor decomposition. When the labeling process was transferred to an automated synthesizer, the increased base ratio significantly decreased the RCY due to precursor decomposition caused by the higher amount of base required for efficient elution of the [18F]F‘ from the QMA cartridge (entry 6, elution efficiency = 71 ± 9%). Therefore, a shorter reaction time was tested to minimize decomposition. This change led to a drastic improvement in RCY (entry 7, representative traces in Fig. 3C). This also confirms that the18F / 19F exchange occurs almost immediately. Using a minimal amount of precursor, which showed no significant reduction in conversion (entries 8 vs. 7) and reacting for only 1 minute resulted in a 37 ± 9% isolated yield after quenching with the addition of one equivalent of acetic acid (AcOH) to the bicarbonate base (entry 8).

[0211] Subsequently, a palladium on carbon catalyzed hydrogenation was employed to reduce the N- oxide and nitro group in a single step, achieving a 60-90% RCY in 10 min. Starting from 0.2 mg (1.2 pmol) of precursor, we used this rapid isotope exchange labeling reaction, quenched by AcOH, followed by a one-step reduction with palladium on carbon and hydrogen. Despite some losses due to transfer and filtration of the reaction crude, this method yielded the desired [18F]5Me3F4AP with a 28 ± 7% decay corrected RCY (n = 5), >99% radiochemical purity, and a moderate molar activity of 9.25 ± 3.70 GBq / pmol. While high MA is critical for many brain tracers due to specific binding considerations31, it does not appear to be beneficial in the case of 3F4AP. In studies in rhesus macaques, adding cold 3F4AP to [18F]3F4AP doses (effectively lowering the MA) resulted in increased tracer binding in the regions of interest9. This phenomenon may be attributed to compensatory mechanism by which blocking a fraction of voltage-gated potassium channels causes more to open.

[0212] Example 2 - Alternative Synthesis of Compounds

[0213] An alternative method for the radiochemical synthesis of [18F]5Me3F4AP is shown below:

[0214] In this method, the labeling precursor 2 is synthesized by the oxidation of the amino precursor

[0215] 1 with hydrogen peroxide and sulfuric acid. Compound 2 is then purified by silica gel chromatography.

[0216] Example 3 - Basicity, lipophilicity and membrane permeability of 5Me3F4AP.

[0217] Partition coefficient determination: The octanol -water partition coefficient (logD) at pH 7.4 was determined according to our previous reported protocol33. Briefly, PBS (900 μL), 1 -octanol (900 μL). and a 10 mg / mb aqueous solution of each compound (2 μL) were added to a 2 mb HPLC vial. The compounds were partitioned between the layers via vortexing and centrifuged at 1,000 g for 1 min to allow for phase separation. A 10 μL portion was taken from each layer (autoinjector was set up to draw volume at two different heights) and analyzed by HPLC. The relative concentration in each phase was determined by integrating the area under each peak and comparing the ratio of the areas from the octanol and aqueous layers. A calibration curve was performed to ensure that the concentrations detected were within the linear range of the detector (see Figure SI in Supporting Information). This procedure was repeated four times for each compound. Determination of pATa: The pKawas determined using titration according to our previously described protocol33. A 1 mg / mL solution of 5Me3F4AP was prepared, of which 5 ml was titrated with 0.0 IM HC1 solution beyond the equivalence point. After each incremental addition of titrant, the sample was stirred and the pH reading was taken with a pH meter. The Gran plot of the titration was analyzed to obtain the pKa(see the plot in Supporting Information, Figure S3). A similar protocol was used to titrate 3F4AP and 4AP, respectively. The titration was repeated four times each for each compound.

[0218] Permeability rate determination: The permeability rates of 5Me3F4AP and 3F4AP were determined using Parallel Artificial Membrane Permeability Assay- blood-brain barrier (BBB) kit (BioAssay Systems, Hayward, USA) following the manufacturer’s protocol. Initially, solutions of each test compound were prepared in DMSO at a concentration of 10 mM. These stock solutions along with the stock solutions of control compounds (high control: promazine hydrochloride, low control: diclofenac) were then diluted with PBS (pH = 7.2) to obtain the donor solutions with a final concentration of 500 μM. At the same time, 200 μM of equilibrium standards for each compound and a DMSO blank control solution were prepared.

[0219] In the experimental setup, 300 μL of PBS was added to the desired well of the acceptor plate, and 5 μL of BBB lipid solution in dodecane was added to membranes of the donor plate. Next, 200 μL of the donor solutions of each test compound and each permeability control were added to the duplicate wells of the donor plate. The donor plate was carefully placed on the acceptor plate and incubator for 18 hours at room temperature. After incubation, UV absorption measurements were conducted using 100 μL of the resulting solutions from the acceptor plate and the equilibrium standards. UV absorption of the controls was measured by running a UV scan in the range of 200 to 500 nm. UV absorption of 5Me3F4AP and 3F4AP was measured using HPUC equipped with a UV detector and C18 column.

[0220] Measurements of these pharmacological parameters of 5Me3F4AP were taken and compared to those of its predecessors (Figure 2). As indicated in left bar graph, 5Me3F4AP demonstrates comparable basicity in comparison to 3F4AP (7.46 ± 0.01 vs. 7.37 ± 0.07). Both compounds have pVi values that are close to the physiological pH, indicating their coexistence in both protonated and neutral forms under physiological conditions. In contrast, 4AP and 3Me4AP display greater basicity (pVi values above 9), indicating that the protonated form is predominant at physiological pH.

[0221] In terms of lipophilicity, 5Me3F4AP shows an octanol / water partition coefficient value at pH 7.4 of 0.664 ± 0.005 (Figure 2), which is higher than that of 3F4AP (logD = 0.414 ± 0.002). This result indicates that both compounds preferentially partition into the octanol layer, potentially facilitating faster permeation through a lipophilic membrane like the BBB via passive diffusion. Conversely, 4AP and 3Me4AP exhibit a preference for partitioning in the water layer (logD4AP = -1.478 ± 0.014, logD3Me4AP = -1.232 ± 0.008), suggesting slower permeation rates. This trend was further validated through a parallel artificial membrane permeability assay, which demonstrated that 5Me3F4AP permeates approximately three times faster than 3F4AP (Figure 2). The compounds with lower pKaexhibited higher logD and higher membrane permeability.

[0222] Example 4 - Affinity towards K+channels.

[0223] Cut-Open Voltage Clamp Electrophysiology: Blocking potency of 5Me3F4AP was evaluated on the voltage-gated Shaker (homologous to mammalian Kv1.2) ion channel expressed in Xenopus laevis oocytes, as previously described33. Briefly, each oocyte expressing the Shaker channel was voltage-clamped in a Cut-Open Voltage Clamp (COVC) station in order to elicit K+currents in response to the voltage stimulus protocol, which entailed steps of 50 ms from -100 to 60 mV in increments of 10 mV. External and internal recording solutions for COVC were composed (in mM) of 12 KOH, 2 Ca(OH)2, 105 NMDG-MES, 20 HEPES and 120 KOH, 2 EGTA, and 20 HEPES, respectively, with pH adjusted to 7.4 with methylsufonate. For measurements achieved at pH = 9. 1 or 6.4, HEPES was replaced by 2-(cyclohexylamino)-ethanesulfonic acid or 2-(N- Morpholino)ethanesulfonic acid, respectively. Each oocyte expressing the Shaker ion channel was voltage-clamped to record K+currents, first in the absence of 5Me3F4AP, and subsequently with the addition of 5Me3F4AP, from 0.0001 to 10 mM. Relative current (Irei) was quantified as the ratio of the current in the absence to that in the presence of the indicated concentration of 5Me3F4AP. Finally, K+currents were amplified with the Oocyte Clamp Amplifier CA-1A (Dagan Corporation, Minneapolis, MN, USA) and digitized with the USB-1604-HS-2AO Multifunction Card (Measurement Computing, Norton, MA, USA). All systems were controlled with the GpatchMC64 program (Department of Anesthesiology, UCLA, Los Angeles, CA, USA) via a PC. Electrophysiology recordings were sampled at 100 kHz and filtered at 10 kHz.

[0224] Electrophysiology data analysis: Data analysis was performed as previously described33. Briefly, the half-maximal inhibitory concentration of 5Me3F4AP (IC50) was determined by fitting the Irei curve to the Hill equation at each value ofV and pH. A Hill coefficient (h) in the range of0.9<h<l. l was used. Voltage and pH dependence of IC50 was analyzed by fitting the IC50(V) at each pH with a one-step model of inhibition (Woodhull model) which allowed the determination of the fractional distance through the membrane electrical field (5) that 5Me3F4AP has to cross to reach its binding site 42. where IC50( v = 0) is the value of IC50 at V = 0 mV, F is the Faraday constant, R is the gas constant, T is the room temperature, and z is the apparent charge.

[0225] Mean values of data ± standard deviation (s.d.) are given or plotted and the number of experiments is denoted by n. Upper and lower limits of the 95% of confidence interval (CI95) are denoted as

[0226] The blocking potency at pH condition 7.4 and voltages (-100 to 60 mV) of 5Me3F4AP was evaluated by measuring the K+currents generated by Shaker voltage-gated potassium channel from I), melanogaster heterologously expressed in Xenopus laevis oocytes (Figure 2). Specifically, Figure 2 (D) shows the calculated IC50 values using the Hill equation at different values of pH. This plot shows that the IC50 of 5Me3F4AP increases with voltage and pH indicating a drop in potency. Figure 2D compares the IC50 values calculated at 40 mV using the Hill equation of the newly characterized 5Me3F4AP at different pHs with the IC50 values of the related compounds 4AP, 3F4AP and 3Me4AP.

[0227] Table 3. IC50 values of 5Me3F4AP: Hill and Woodhull parameters.

[0228] C50 values were determined at 40mV. A Hill parameter of h~l was obtained during the fitting of the data for all experimental conditions. *Data at this pH condition has been reported previously33. Example 5 - Metabolic stability towards CYP2E1 of 5Me3F4AP.

[0229] To estimate the metabolic stability of 5Me3F4AP towards CYP2E1, an in vitro investigation was conducted utilizing a competitive inhibition assay. Compounds that are good substrates of CYP2E1 result in greater reduction in the rate of formation of a fluorescent reporter than compounds that are poor substrates. In this study, we measured the reaction rates without competitor (blank) as well as in the presence of tranylcypromine (positive control), 4AP, 3F4AP and 5Me3F4AP. As illustrated in Figure 2F, the addition of tranylcypromine, a widely recognized potent substrate of CYP2E1, resulted in the most pronounced reduction in fluorogenic emission when compared to the reaction conducted without any addition of enzyme substrates (hexagon vs. circular lines). In comparison, 4AP exhibited a minor reduction in Anorogenic emission (star vs. circular lines) indicating that it is a poor substrate of CYP2E1. 3F4AP demonstrated a substantial decrease in rate (star vs. circular lines) indicating 3F4AP is a good substrate of CYP2E1, undergoing metabolism at a much faster rate than 4AP. In comparison, 5Me3F4AP demonstrated a reaction rate between 4AP and 3F4AP, bearing a higher resemblance to 3F4AP (Figure 2F, diamond vs. star and plus lines). To further quantify the inhibition potency of 5Me3F4AP towards CYP2E1, we measured the CYP2E1- mediated reaction rate in the presence of varying concentrations of 5Me3F4AP and 3F4AP and performed the dose-response fitting (Figure 2C). We also compared these results with our previous results for 4AP, 3F4AP and the positive control tranylcypromine30. This analysis showed that 5Me3F4AP has an IC50 about two times higher than 3F4AP and about 23 times lower than 4AP (Figure 2E), indicating that it is more stable than 3F4AP but not as stable as 4AP. a weaker competitive inhibitor of CYP2E1 than 3F4AP, bus stronger than 4AP. Given the previously stablished role of CYP2E 1 in the in vivo metabolism of this family of compounds and the direct correlation between inhibitory potency and CYP2E1 affinity for the substrate, this finding suggests that 5Me3F4AP may be metabolized slower than 3F4AP but not as slow as 4AP.

[0230] CYP2E1 -mediated metabolic stability assessment: The relative metabolic stability towards CYP2E1 was assessed with the competitive CYP2E1 inhibition assay utilizing the Life Technologies™ Vivid® CYP2E1 screening kit, as described in previous studies39. In this assay, Auorescence, emited by the metabolic product of a specific CYP2E1 substrate included in the kit, was measured in the absence and presence of substrate competitors. Consequently, the highest Auorescence values were obtained from the blank experiments, which lacked any competitors. As the concentration of competitors increased, or more potent competitors were introduced, the Auorogenic emission decreased accordingly. Specifically, 40 μL of 2.5X (final concentration 25 μM) solution of test compounds (4AP, 3F4AP, 5Me3F4AP, and positive control, i.e., tranylcypromine) in IX Vivid® CYP2E1 reaction buffer was added to desired wells of a falcon black / clear 384-well plate in three replicates. Afterwards, 50 μL master pre-mix 2X (40 nM) CYP2E1 BACULOSOMES® and 2X (0.6 Units / mL) Vivid® regeneration system in IX reaction buffer) was added to each well. The plate was incubated for 10 minutes at room temperature to allow the compounds to interact with the CYP2E1 in the absence of enzyme turnover. Next, the reaction was initiated by adding 10μL per well of 10X (100 μM) Vivid® substrate (2H-l-benzopyran-3-carbonitrile,7-(ethoxy-methoxy)-2-oxo-(9Cl)) and 10X (300 μM) Vivid® NADP+mixture. Immediately (in less than 2 minutes), the plate was transferred into the fluorescent plate reader and fluorescence was monitored over 60 minutes (reads in 1 -minute intervals) at 415 nm as excitation wavelength and 460 nm as emission wavelength. The obtained reads were plotted using GraphPad Prism 9.

[0231] Determination of the IC50 and Ki of 5Me3F4APto CYP2E1.

[0232] A similar Vivid® CYP2E1 assay was conducted as described above. Instead of testing a single concentration of 5Me3F4AP (final concentration 15μM), a series of concentrations (4.0 mM, 1.2 mM, 400 μM, 120 μM, 40 μM, 12 μM, 4.0 μM, 1.2 μM) were tested with three replicates for each concentration. The plate fluorescence was monitored over 60 minutes (reads in 1 -minute intervals) at 415 nm as excitation wavelength and 460 nm as emission wavelength. The reads at 60 min (recalculated by the linear trend line equation) of each concentration were used and fitted with GraphPad Prism9 dose-response-inhibition (concentration is log) curve fitting to calculate the IC.w values. Tire corresponding K,- values were calculated by and the Vi vid® EOMCC substrate concentration [S ] is 10 μM).

[0233] Table 4. IC50values and their respective confidence interval (C.I.) for CYP2E1 substrates.

[0234] * Previously reported data39. Example 6 - Animal Studies

[0235] Animals. 6-8-week-old C59BI / 6J male mice were used.

[0236] Imaging Studies and Measurement of Brain SUV ex vivo. Naive male mice were imaged on a Sedecal SuperArgus PET / CT scanner. Mice were injected with the radioligand (approximately 3.7 MBq in 200 μL solution) via a tail vein catheter and scanned for 50 mins in dynamic PET list mode (energy window: 250-700 KeV) under anesthesia (1.5% isoflurane with an oxygen flow of 2.0 L / min), followed by a CT scan (X Rays: 350 pA of current and 45kV of voltage). After completing the scan, the animals were euthanized, their blood was collected by cardiac puncture and their brains were harvested. Blood and brain samples were weighed, and the radioactive concentrations were measured using a single well gamma counter.

[0237] Imaging analysis . The images were reconstructed using a 2D ordered subset expectation maximization (OSEM) iterative reconstruction algorithm with 2 iterations and 16 subsets, including corrections for random coincident events and attenuation (using its own CT). Dynamic images were reconstructed with the following time frames 8 x 15 s, 6 x 30 s, 5 x 60 s, 4 x 300 s and remaining were 600 s frames. The obtained DICOM files were analyzed with AMIDE to generate time-activity curves with the whole brain as the region of interest.

[0238] After successfully synthesizing [18F]5Me3F4AP, we conducted PET / CT studies in naive mice to evaluate its brain penetration and kinetics. Dynamic PET images were captured from 0 to 60 minutes post-administration of the radioligands, focusing on the whole brain (n = 2 for each tracer). Representative images in Figure 4 show the average distribution and accumulation of the tracer within the brain over the 60-minute period, indicating that [18F]5Me3F4AP effectively crosses the BBB and binds to its target within the brain. Additionally, high uptake in the eyes and salivary glands was also consistent with [18F]3F4AP.

[0239] To quantitatively assess the tracer kinetics, time-activity curves (TACs) for the whole brain were generated (Fig. 5). The reproducibility was also high, with almost identical curves observed in two different animals. The TACs indicated that [18F]5Me3F4AP reached a peak standardized uptake value (SUV) of approximately 3.3 within 1 minute, similar to [18F]3F4AP. Furthermore, the washout rate of [18F]5Me3F4AP was comparable to that of [18F]3F4AP, suggesting that both tracers have similar brain penetration, binding, and clearance dynamics. In contrast, [nC]3Me4AP did not exhibit such favorable dynamics13. Terminal brain uptake was measured by gamma counting post-imaging. Results showed that at the end of the imaging period, the concentrations of [18F]5Me3F4AP in both the blood (SUV[18F]5Me3F4AP= 0.45 ± 0.07 vs. SUV[18F]3F4AP= 0.33 ± 0.01) and brain (SUV[18F]5Me3F4AP= 0.64 ± 0. 12 vs. SUV[18F]3F4AP= 0.55 ± 0.04) were also comparable to those of [18F]3F4AP and consistent with the imaging SUV data. Given that [18F]3F4AP demonstrates excellent imaging properties in anesthetized animals, the comparable kinetics and brain uptake of [18F]5Me3F4AP holds substantial promise for further investigation in animal models of disease and under awake conditions. Its potential sensitivity to lesions and metabolic stability are the crucial factors were [18F]5Me3F4AP may demonstrate superiority over [18F]3F4AP. These initial results provide a solid foundation for [18F]5Me3F4AP as a viable brain imaging tracer, with potential advantages in kinetics, brain penetration and retention.

[0240] OTHER EMBODIMENTS

[0241] It is to be understood that while the present application has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present application, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS1. A compound of formula (I)or a pharmaceutically acceptable salt thereof, wherein: each R is independently chosen from halogen, C1-10alkyl, [18F]C1-C3monofluoroalkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-10 membered aryl, and 5-10 membered heteroaryl; and n is 0, 1, 2, or 3; and m is 0 or 1 ; and wherein if one R is [18F]C1-C3monofluoroalkyl, then m is 0; if no R is [18F]C1-C3monofluoroalkyl, then m is 1.

2. The compound of claim 1, wherein m is 1.

3. The compound of claim 1 or 2, wherein the isotopic enrichment level of each18F is about 0.0001% to about 0.1%.

4. The compound of claim 1, wherein n is 2.

5. The compound of claim 1, wherein n is 1.

6. The compound of any one of claims 1-5, wherein each R is C1-10 alkyl.

7. The compound of any one of claims 1-6, wherein each R is methyl.

8. The compound of any one of claims 1-6, wherein each R is ethyl.

9. The compound of any one of claims 1-6, wherein each R is propyl.

10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:or a pharmaceutically acceptable salt of any of the foregoing.

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

12. A method of producing a compound of Formula (F)or a pharmaceutically acceptable salt thereof, the method comprising the steps of(i) treating a compound of Formula (A)with a source of nucleophilic18F to provide the compound of Formula (A’)(ii) reducing the compound of Formula (A’) to provide the compound of Formula (I) wherein: each R is independently chosen from halogen, C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-10 membered aryl, and 5-10 membered heteroaryl; and n is 0, 1, 2, or 3.

13. A compound of Formula (F)or a pharmaceutically acceptable salt thereof, prepared by a process comprising:(i) treating a compound of Formula (A)with a source of nucleophilic18F to provide a compound of Formula (A’)(ii) reducing the compound of Formula (A’) to provide the compound of Formula (I), wherein: each R is independently chosen from halogen, C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-10 membered aryl, and 5-10 membered heteroaryl; and n is 0, 1, 2, or 3.

14. The method or compound of claim 12 or 13, wherein the compound of Formula (F), or a pharmaceutically acceptable salt thereof, is selected fromor a pharmaceutically acceptable salt of any of the foregoing.

15. The method or compound of any one of claims 12-14, wherein the compound of Formula (I’), or a pharmaceutically acceptable salt thereof, is16. A pharmaceutical composition comprising a compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

17. A method of imaging a tissue comprising:(a) contacting the tissue with a compound according to any one of claims 1-15, or a pharmaceutically acceptable salt thereof; and(b) detecting a signal from the compound, thereby imaging the tissue.

18. The method of claim 17, wherein the tissue is CNS tissue.

19. The method of claim 17 or 18, wherein the tissue is brain tissue.

20. A method of detecting demyelination in a subject suspected of having a neurological disorder, the method comprising:(a) administering to the subject an imaging effective amount of a compound according to any one of claims 1-15, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 16; and(b) determining the presence or absence of a signal from the compound, wherein the presence of a signal indicates a diagnosis of demyelination.

21. A method of treating a neurological disorder in a subject suspected of having a neurological disorder, the method comprising:(a) administering to the subject an imaging effective amount of a compound according to any one of claims 1-15, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 16;(b) determining the presence or absence of a signal from the compound; and(c) if a signal is detected, administering to the subject a therapeutically effective amount of one more agents used to treat a neurological disorder, or a combination thereof.

22. A kit comprising the compound of any of claims 1-15, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 16; and instructions for administering the compound, the pharmaceutically acceptable salt thereof, or the pharmaceutical composition to a subject.

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