Methods for detecting systemic amyloidosis via binding to misfolded or aggregated proteins - Patents.com

Abstract

Provided herein are methods and compositions for determining whether a patient is afflicted with systemic amyloidosis, the methods comprising detecting the presence of a misfolded or aggregated protein in tissue of the patient, wherein the detecting comprises contacting the misfolded or aggregated transthyretin protein with a compound described herein. In some embodiments, the misfolded or aggregated protein is transthyretin.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 160,602, filed March 12, 2021, which is incorporated by reference in its entirety herein. [Background technology]

[0002] background Systemic amyloidosis is a disease that occurs when abnormal proteins called amyloids build up in organs and interfere with their normal function. Amyloids can be formed from any of many different types of proteins. Organs that can be affected include the heart, kidneys, liver, spleen, nervous system, and digestive tract. Some types of amyloidosis can lead to life-threatening organ failure. Patients may not experience signs and symptoms of amyloidosis until the condition is advanced.

[0003] Transthyretin is a protein made by the liver that helps transport thyroid hormones and vitamin A in the blood. Misfolding and aggregation of transthyretin (TTR) is associated with many conditions. Familial amyloid polyneuropathy (FAP), also called transthyretin amyloid polyneuropathy, is an inherited, progressive, rare disease caused by abnormal deposition of protein or amyloid around peripheral nerves and other tissues. FAP is caused by a mutation in the TTR gene that destabilizes the TTR tetramer. FAP was first identified in 1952, when it was observed in several Portuguese families. In some areas of northern Portugal, FAP affects approximately 1 in 500 people and occurs worldwide. In some cases, liver transplantation is indicated. There is currently no cure for FAP.

[0004] Another important disorder is a progressive systemic disorder called amyloid transthyretin (ATTR) amyloidosis.In ATTR amyloidosis, protein is deposited in the heart and / or nerves and other organs and tissues.ATTR amyloidosis is a slowly progressing condition characterized by the accumulation of abnormal deposits of protein called amyloid (amyloidosis) in organs and tissues of the body.

[0005] There is a need for safe and convenient methods for detecting systemic amyloidosis. Summary of the Invention [Means for solving the problem]

[0006] Abstract In some embodiments, a method is provided for determining whether a patient suffers from systemic amyloidosis, such as familial amyloid polyneuropathy (FAP) or transthyretin (ATTR) amyloidosis, comprising administering to the eye of the subject a compound as described herein or a pharma- ceutically acceptable salt thereof, and detecting the presence or absence of misfolded or aggregated protein. The misfolded or aggregated protein may be a misfolded or aggregated transthyretin protein (TTR). The compound may be a compound of formula I or any compound as described herein. In certain embodiments, amyloid is detected, wherein the amyloid does not include amyloid β peptide.

[0007] In some embodiments, a method is provided for diagnosing systemic amyloidosis comprising administering to an eye of a subject in need thereof a compound described herein or a pharma- ceutically acceptable salt thereof and detecting the presence or absence of misfolded or aggregated transthyretin protein.

[0008] In some embodiments, the compound has the structure: [ka] or a pharma- ceutically acceptable salt thereof is provided herein. In some embodiments, provided herein is Compound 1 for use in diagnosing systemic amyloidosis. In some embodiments, provided herein is Compound 1 for use in determining whether a patient is afflicted with systemic amyloidosis. In some embodiments, provided herein is Compound 1 for use in preparing a patient for diagnosis of systemic amyloidosis, wherein Compound 1 is administered topically to the eye of the patient.

[0009] The present disclosure provides a simple and non-invasive method for detecting systemic amyloidosis by applying an amyloid-binding compound to the ocular surface of the subject and directly detecting the fluorescence generated thereby. In ATTR and other systemic amyloidosis, amyloid formation is initiated by dissociation of TTR tetramers to form aggregation-prone monomers that self-associate to form small oligomers, amorphous aggregates, and / or fibrils. On the other hand, amyloid-β comprises a 36-43 amino acid peptide that is cleaved from the amyloid precursor protein. Both mutant and wild-type TTR form aggregates and fibrils, and the formation occurs through a complex mechanism that is poorly understood. Thus, the present disclosure surprisingly allows systemic amyloidosis to be diagnosed by surface detection of TTR amyloid by applying certain compounds to the ocular surface of the patient for its fluorescence detection. BRIEF DESCRIPTION OF THE DRAWINGS [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 illustrates the fluorescence spectra of Compound 1 with and without the presence of aggregated TTR.

[0011] [Diagram 2] FIG. 2 illustrates the relationship between fluorescence intensity and concentration of Compound 1 in the presence of aggregated TTR at a constant concentration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description definition The following description illustrates exemplary embodiments of the present technology, however, it should be recognized that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of exemplary embodiments.

[0013] As used herein, the following words, phrases and symbols are generally intended to have the meanings indicated below, except to the extent that the context in which they are used indicates otherwise.

[0014] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, which may be fully saturated, monounsaturated or polyunsaturated, and which may include divalent and polyvalent radicals, and has the specified number of carbon atoms (i.e., C1-C2). 10means 1-10 carbons) straight chain (i.e., unbranched) or branched chain, or combinations thereof. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups are those having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-).

[0015] The term "alkylene," alone or as part of another substituent, means a divalent radical derived from an alkyl, as exemplified, but not limited to, by -CH2CH2CH2CH2-, and further including those groups described below as "heteroalkylene." Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being preferred. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms.

[0016] The term "heteroalkyl", alone or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S. The nitrogen and sulfur atoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized. The heteroatoms O, N, P, and S, as well as Si, can be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, CH=CH-N(CH3)-CH3, O-CH3, -O-CH-2-CH3, and -CN. Up to two heteroatoms may be consecutive (such as, for example, -CH2-NH-OCH3). Similarly, the term "heteroalkylene," alone or as part of another substituent, means a divalent radical derived from a heteroalkyl, exemplified, but not limited to, by -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)R'- represents both -C(O)R'- and -R'C(O)-. As noted above, heteroalkyl groups, as used herein, include groups that are attached to the remainder of the molecule through a heteroatom (e.g., -C(O)R', -C(O)NR', ​​-NR'R", -OR', -SR', and / or -SOR').Where "heteroalkyl" is described followed by description of a specific heteroalkyl group (e.g., -NR'R", etc.), it is understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl group is described to add clarity. Thus, the term "heteroalkyl" should not be construed herein as excluding the specific heteroalkyl group (e.g., -NR'R", etc.), where R' and R" are each as defined below with respect to "Substituents."

[0017] The terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively. Additionally, for heterocycloalkyl, a heteroatom may occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a "heterocycloalkylene," alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0018] The term "halo" or "halogen", alone or as part of another substituent, means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" is meant to include, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0019] The term "acyl" means C(O)R, where R is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0020] The term "aryl", unless otherwise stated, refers to a polyunsaturated, aromatic, hydrocarbon substituent that may be one or more rings (preferably 1-3 rings) that are fused together (i.e., fused-ring aryl) or covalently linked. Fused-ring aryl refers to multiple rings fused together and at least one of the fused rings is an aryl ring. The term "heteroaryl" refers to an aryl group (or ring) that contains 1-4 heteroatoms selected from N, O, and S. The nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Thus, the term "heteroaryl" includes fused-ring heteroaryl groups (i.e., multiple rings fused together where at least one of the fused rings is a heteroaromatic ring). 5,6-fused-ring heteroarylene refers to two rings fused together where one ring has 5 members and the other ring has 6 members and at least one ring is a heteroaryl ring. Similarly, a 6,6-fused ring heteroarylene refers to two rings fused together, one of which has six members and the other has six members, with at least one ring being a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, one of which has six members and the other has five members, with at least one ring being a heteroaryl ring. The heteroaryl group may be bonded to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, Examples include 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "Arylene" and "heteroarylene," alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.

[0021] For brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), including alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced, for example, by an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.).

[0022] The term "oxo," as used herein, means an oxygen that is double bonded to a carbon atom.

[0023] The term "alkylsulfonyl," as used herein, refers to a moiety having the formula -S(O2)-R', where R' is an alkyl group as defined above. R' can have a specific number of carbon atoms (e.g., "C1-C4 alkylsulfonyl").

[0024] Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl" and "heteroaryl") are meant to include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0025] Substituents for the above alkyl and heteroalkyl radicals (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) may be any of the following radicals ranging in number from 0 to (2m'+1), where m' is the total number of carbon atoms in such radical: -OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R'" , -OC(O)R', -C(O)R', -COR', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R'", -NR"C(O)2R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR'", -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -CN and -NO2. R', R", R'" and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound disclosed herein includes more than one R group, for example, each of the R groups above is independently selected as are each R', R", R' and R'" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl.From the above discussion of substituents, one of skill in the art will recognize that the term "alkyl" is meant to include groups that contain carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF and -CHCF) and acyls (e.g., -C(O)CH, -C(O)CF, -C(O)CHOCH, etc.).

[0026] Similar to the substituents described for the alkyl radical, substituents on the aryl and heteroaryl groups vary in number ranging from zero to the total number of open valences on the aromatic ring system and include, for example: halogen, -OR', -NR'R", -SR', -halogen, -SiR'R"R'", -OC(O)R', -C(O)R', -COR', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R'", -NR"C(O)R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR'", -S(O)R', -S(O)R', -S(O)NR'R", -NRSO2R', -CN and -NO2, -R', -N3, wherein R', R", R'" and R"" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound disclosed herein includes more than one R group, for example, each of the R groups above is independently selected as are each R', R", R' and R'" groups when more than one of these groups is present.

[0027] Two of the substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be of the formula -TC(O)-(CRR') q-U-, where T and U are independently -NR-, -O-, -CRR'- or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -A-(CH2) r A and B may be replaced with a substituent of the formula -B-, where A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR') s -X'-(C”R'”) d -, where s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or S(O)2NR'-. The substituents R, R', R" and R'" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0028] As used herein, the term "heteroatom" or "ring heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0029] "Substituent group," as used herein, means a group selected from the following moieties: (A) -OH, -NH2, -SH, -CN, -CF3, -NO2, oxo, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (B) Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from the following: (i) oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from: *(a) oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and *(b) Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl substituted with at least one substituent selected from oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

[0030] "Size-limited substituent" or "size-limited substituent group" as used herein means a group selected from all of the substituents listed above for "substituent", where each substituted or unsubstituted alkyl is a substituted or unsubstituted C-C 20each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C4-C8 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4-8 membered heterocycloalkyl.

[0031] A "lower substituent" or "lower substituent group", as used herein, means a group selected from all of the substituents described above for "substituent", wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C5-C7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5-7 membered heterocycloalkyl.

[0032] The term "pharmaceutically acceptable salts" is meant to include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the particular substituents found on the compounds described herein. When the compounds disclosed herein contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. When the compounds disclosed herein contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, oxalic acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginate, and salts of organic acids such as glucuronic acid or galacturonic acid (see, for example, Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain compounds disclosed herein contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0033] Therefore, the compounds disclosed herein can be present as, for example, salts with pharmaceutically acceptable acids.Examples of such salts include hydrochloride, hydrobromide, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate (e.g., (+)-tartrate, (-)-tartrate or mixtures thereof, including racemic mixture), succinate, benzoate, and salts with amino acids such as glutamic acid.These salts can be prepared by methods known to those skilled in the art.

[0034] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.

[0035] Some of the compounds exist as tautomers. Tautomers are in equilibrium with each other. For example, amide-containing compounds may exist in equilibrium with imidic acid tautomers. Regardless of which tautomer is shown and regardless of the nature of the equilibrium between tautomers, it is understood by those skilled in the art that the compounds include both amide and imidic acid tautomers. Thus, the amide-containing compounds are understood to include their imidic acid tautomers. Similarly, the imidic acid-containing compounds are understood to include their amide tautomers.

[0036] Any formula or structure shown herein is also intended to represent unlabeled and isotopically labeled forms of said compounds. Isotopically labeled compounds have the structure shown by the formula shown herein, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine (e.g., 2 H (deuterium, D), 3 H (tritium), 11 C. 13 C.14 C. 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl and 125 Various isotopically labeled compounds of the present disclosure include, but are not limited to, 3 H and 14 Those incorporating a radioactive isotope such as C. Such isotopically labeled compounds may be useful in metabolism studies, reaction kinetic studies, detection or imaging techniques (e.g., positron emission tomography (PET) or single photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays), or in radioactive treatment of patients.

[0037] The present disclosure also includes "deuterated analogs" of compounds of formula I in which one to n hydrogens attached to a carbon atom are replaced with deuterium, where n is the number of hydrogens in the molecule. Such compounds exhibit increased resistance to metabolism and are therefore useful for increasing the half-life of any compound of formula I when administered to a mammal, particularly a human. See, for example, Foster, "Deuterium Isotope Effects in Studies of Drug Metabolism", Trends Pharmacol. Sci. 5(12):524-527(1984). Such compounds are synthesized by means well known in the art, for example, by using starting materials in which one or more hydrogens have been replaced by deuterium.

[0038] Therapeutic compounds of the present disclosure that are labeled or substituted with deuterium may have improved DMPK (drug metabolism and pharmacokinetic) properties related to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes (e.g., deuterium) may confer certain therapeutic benefits resulting from greater metabolic stability, e.g., increased in vivo half-life, reduced dosage requirements and / or improved therapeutic index. 18F-labeled compounds can be useful for PET or SPECT studies.Isotopically labeled compounds of the present disclosure and their prodrugs can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below, by using readily available isotopically labeled reagents instead of non-isotopically labeled reagents.It is understood that deuterium in this context is regarded as a substituent in the compounds described herein.

[0039] The concentration of such heavier isotopes, particularly deuterium, can be defined by the isotopic enrichment factor. Any atom not specifically designated as a particular isotope in the compounds of the present disclosure is meant to represent any stable isotope of that atom. Unless otherwise stated, when a position is specifically designated as "H" or "hydrogen", that position is understood to have hydrogen in its natural abundance isotopic composition. Thus, any atom specifically designated as deuterium (D) in the compounds of the present disclosure is meant to represent deuterium.

[0040] In many cases, the compounds of the present disclosure are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0041] Pharmaceutically acceptable salts, hydrates, solvates, tautomeric forms, polymorphs, and prodrugs of the compounds described herein are also provided. "Pharmaceutically acceptable" or "physiologically acceptable" refers to compounds, salts, compositions, dosage forms, and other substances that are useful in preparing pharmaceutical compositions suitable for veterinary or human pharmaceutical use.

[0042] The term "pharmaceutically acceptable salt" of a given compound refers to a salt that retains the biological effectiveness and properties of the given compound and is not biologically or otherwise undesirable. "Pharmaceutically acceptable salt" or "physiologically acceptable salt" includes, for example, salts with inorganic acids and salts with organic acids. Furthermore, when the compounds described herein are obtained as acid addition salts, the free base can be obtained by basifying a solution of the acid salt. Conversely, when the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, can be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid according to conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that can be used to prepare non-toxic pharmaceutically acceptable addition salts. Pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Salts obtained from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Similarly, pharma-ceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include, by way of example only, sodium, potassium, lithium, ammonium, calcium, and magnesium salts.Salts derived from organic bases include primary, secondary, and tertiary amines (e.g., alkylamines (i.e., NH2(alkyl)), dialkylamines (i.e., HN(alkyl)2), trialkylamines (i.e., N(alkyl)3), substituted alkylamines (i.e., NH2(substituted alkyl)), di(substituted alkyl)amines (i.e., HN(substituted alkyl)2), tri(substituted alkyl)amines (i.e., N(substituted alkyl)3), alkenylamines (i.e., NH2(alkenyl)), dialkenylamines (i.e., HN(alkenyl)2), trialkenylamines (i.e., N(alkenyl)3), substituted alkenylamines (i.e., NH2(substituted alkenyl)), di(substituted alkenyl)amines (i.e., , HN(substituted alkenyl)2), tri(substituted alkenyl)amines (i.e., N(substituted alkenyl)3, mono-, di-, or tri-cycloalkylamines (i.e., NH2(cycloalkyl), HN(cycloalkyl)2, N(cycloalkyl)3), mono-, di-, or tri-arylamines (i.e., NH2(aryl), HN(aryl)2, N(aryl)3), or mixed amines. Specific examples of suitable amines include, by way of example only, isopropylamine, trimethylamine, diethylamine, tri(isopropyl)amine, tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like.

[0043] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients can also be incorporated into the composition.

[0044] A "solvate" is formed by the interaction of a solvent and a compound. Solvates of the salts of the compounds described herein are also provided. Hydrates of the compounds described herein are also provided.

[0045] Misfolded or aggregated proteins Some systemic amyloidosis, as well as familial amyloid polyneuropathy (FAP) and especially ATTR amyloidosis, are associated with the release and / or accumulation of misfolded transthyretin protein. The misfolded or aggregated transthyretin protein can be detected by contact with a compound as described herein, where the contact causes the release of a detectable signal upon activation by light. In general, the compositions and methods described herein are useful for detecting misfolded or aggregated transthyretin protein in any tissue of a patient. However, the misfolded or aggregated transthyretin protein can accumulate in the eye of a patient. In particular, the misfolded or aggregated transthyretin protein can accumulate in the conjunctiva of the eye. Such presence of misfolded or aggregated transthyretin protein in the retina can be detected with a compound that binds to the misfolded or aggregated transthyretin protein, which binding can then be detected by means such as laser-activated fluorescent scanning of the retina.

[0046] Transthyretin ("TTR", "TTR protein" or "TBPA") is a transport protein in serum and cerebrospinal fluid that carries the thyroid hormone thyroxine (T4) and retinol-binding protein bound to retinol. The liver secretes transthyretin into the blood, and the choroid plexus secretes TTR into the cerebrospinal fluid. TTR may also be referred to as prealbumin or thyroxine-bound prealbumin.

[0047] TTR is a unique human protein synthesized in hepatocytes, retinal pigment epithelial cells, choroid plexus epithelium, pancreatic α-cells, Schwann cells, and under some conditions in neurons. In serum, it is a carrier of retinol-loaded retinol-binding protein (RBP) and a minor carrier of the thyroid hormone precursor thyroxine (T4) before it is converted by tissue deiodinases to the histologically more active triiodothyronine (T3). While a small percentage of circulating TTR carries T4, 25-50% is loaded with the RBP retinol. However, in cerebrospinal fluid, TTR synthesized in the choroid plexus is the major T4 carrier. The crystal structure shows a twofold axis of symmetry. It assembles around a central channel as a dimer of dimers that is primarily hydrophobic and contains two T4-binding sites. T4 binding at the first site induces an allosteric change that makes the second site less accessible to its natural ligand. Different parts of the protein bind T4 and RBP, both of which stabilize the tetrameric structure and reduce its tendency to dissociate.

[0048] TTR is encoded by a single gene on chromosome 18 that contains approximately 19 Kb of DNA with 4 exons contained within 7 Kb, 6 Kb of upstream (5') sequence and 6 Kb downstream that contain conventional 3' non-coding sequences that allow normal mRNA processing after transcription. The 2 Kb proximal to the promoter appear to contain all the sequences required for tissue-specific expression of the gene. Transthyretin isoform sequences include NP_000362.1. Transthyretin sequences can be variants or mutants. The variants or mutants can be in any known form, including but not limited to: G6S, C10R, L12P, M13I, D18N, D18G, D18E, A19D, V20I, R21Q, S23N, P24S, A25 S, A25T, V28M, V28S, V30M, V30A, V30G, V30L, V32A, V32G, F33I, F33L, F33V. F33C, R34G, R34T, K35N, K35T, A36D, A36P, D38A, D38V, D39V. W41L, E42G, E42D, F44Y, F44S, F44L, A45S, A45T, A45D, A45G, G47R, G47A, G47E , G47V, T49A, T49P, T49I, T49S, S50R, S50I, E51G, S52P, G53R, G53E, G53A, E54L , E54K, E54G, E54D, E54Q, L55Q, L55R, L55P, H56R, G57R, L58R, L58H, T59R, T59K , T60A, E61K, E61G, E62K, F64I, F64L, F64S, G67R, G67E, I68L, Y69H, Y69I, K70N , V71A, E72G, I73V, D74H, S77F, S77Y, Y78F, A81T, A81V, G83R, I84N, I84S, I84 T, H88R, E89Q, E89K, H90N, H90D, A91S, E92K, V93M, V94A, A97S, A97G, G101S, P1 02R, R103S, R104C, R104H, I107V, I107F, I107M, A109S, A109T, A109V, L111M, S 112I, P113T, Y114C, Y114H, Y114S, Y116S, T119M, A120S, V122A, V122I, P125S.

[0049] TTR is a non-disulfide-linked homotetramer in which the mature polypeptide monomer contains 127 amino acids after cleavage of the leader sequence. The tetramer has a Ka of 1.1×10 24 M -3 TTR is generally stable at 100-200 nm. Dissociation of TTR tetramers can lead to protein deposition and amyloid formation in tissues. Misfolded or aggregated TTR protein can be N-oligomers. TTR can be wild-type TTR or its variant or mutant.

[0050] In some embodiments, the misfolded or aggregated protein may be β2 macroglobulin, leukocyte chemotactic factor 2, apolipoprotein AIV, apolipoprotein CII, apolipoprotein CIII, or lysozyme, or a mutant or variant thereof, each of which is known in the art. The misfolded or aggregated protein may be an amyloid thereof.

[0051] Thus, according to some embodiments of the present disclosure, a method is provided for determining whether a patient is suffering from systemic amyloidosis. The method includes detecting the presence of misfolded or aggregated transthyretin protein in a tissue such as the eye of a subject by contacting the tissue of the subject with a compound described herein. The contact may be in vivo or ex vivo. The contact may be by topical administration. It is contemplated that the accumulation of the misfolded or aggregated transthyretin protein may occur in the conjunctiva of the eye. Thus, the detection may target the misfolded or aggregated transthyretin protein in the conjunctiva by topical administration to the surface of the eye. When the administration is topical, the compound may be formulated in an ophthalmologically acceptable formulation.

[0052] In another embodiment, a method is provided for preparing a patient for diagnosis of systemic amyloidosis, particularly familial amyloid polyneuropathy (FAP) or ATTR amyloidosis, comprising administering to the patient a compound that specifically binds misfolded or aggregated transthyretin protein. The compound may be administered to the patient's eye. Once the compound is administered to the patient, its binding to the misfolded or aggregated transthyretin protein is detected by any method, including the methods described herein, and the binding indicates accumulation of misfolded transthyretin protein, which is indicative of systemic amyloidosis.

[0053] Compounds that bind to misfolded or aggregated proteins The present disclosure further provides a compound that can bind to misfolded or aggregated proteins.In some embodiments, the compound can bind to misfolded or aggregated transthyretin protein.In one embodiment, the compound can be selected from the compounds described in International Publication No. WO 2011 / 072257 or U.S. Patent No. 9,551,722, which are incorporated by reference in their entirety.The synthesis of the compound can proceed by methods known in the art, including those described in the incorporated references.

[0054] In some embodiments, the present disclosure provides a compound of formula I, or a salt or solvate thereof: [ka] where EDG is R 1 -substituted or unsubstituted alkyl, R 1 -substituted or unsubstituted cycloalkyl, R 1 -substituted or unsubstituted heteroalkyl, R 1-substituted or unsubstituted heterocycloalkyl, R 1 -substituted or unsubstituted aryl, R 1 -substituted or unsubstituted heteroaryl, OR 2 NR 4 C(O)R 3 , -NR 4 R 5 , -SR 6 , or PR 7 R 8 and where R 1 is halogen, -OR 9 , -NR 10 R 11 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, R 12 -substituted or unsubstituted alkyl, R 12 -substituted or unsubstituted heteroalkyl, R 12 -substituted or unsubstituted cycloalkyl, R 12 -substituted or unsubstituted heterocycloalkyl, R 12 -substituted or unsubstituted aryl or R 12 -substituted or unsubstituted heteroaryl, where R 4 and R 5 can be combined together as necessary to produce R 12 -substituted or unsubstituted heterocycloalkyl, or R 12 - forming a substituted or unsubstituted heteroaryl; R 9 and R 10are independently hydrogen, R 12 -substituted or unsubstituted alkyl, R 12 -substituted or unsubstituted heteroalkyl, R 12 -substituted or unsubstituted cycloalkyl, R 12 -substituted or unsubstituted heterocycloalkyl, R 12 -substituted or unsubstituted aryl, or R 12 -substituted or unsubstituted heteroaryl, where R 10 and can be combined together as necessary to give R 12 -substituted or unsubstituted heterocycloalkyl, or R 12 - forming a substituted or unsubstituted heteroaryl; R 12 is halogen, -OR 13 , -NR 14 R 15 , R 16 -substituted or unsubstituted alkyl, R 16 -substituted or unsubstituted heteroalkyl, R 16 -substituted or unsubstituted cycloalkyl, R 16 -substituted or unsubstituted heterocycloalkyl, R 16 -substituted or unsubstituted aryl, or R 16 -substituted or unsubstituted heteroaryl; R 13 , R 14 and R 15 is independently hydrogen or unsubstituted alkyl; R 16 is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; Here, πCE is represented by the following formula: -L 1 -(A1 ) q -L 2 -(A 2 ) r -L 3 - or -L 1 -(A 1 ) q -L 4 -A 3 -L 2 -(A 2 ) r -L 3 - where q and r are independently 0 or 1, where at least one of q or r is 1; A 1 , A 2 and A 3 are independently 17 -substituted or unsubstituted arylene or R 17 -substituted or unsubstituted heteroarylene; L 1 , L 2 , L 3 and L 4 is independently a bond or a group of the formula: [ka] where x is an integer from 1 to 50; R 17 is halogen, -OR 18 , -NR 19 R 20 , R 21 -substituted or unsubstituted alkyl, R 21 -substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl, R 21 -substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 -substituted or unsubstituted heteroaryl; R18 , R 19 and R 20 are independently hydrogen, R 21 -substituted or unsubstituted alkyl, R 21 -substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl, R 21 -substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 -substituted or unsubstituted heteroaryl; R 21 is halogen, -OR 22 , -NR 23 R 24 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; R 22 , R 23 and R 24 is independently hydrogen or unsubstituted alkyl; where WSG is R 25 -substituted or unsubstituted alkyl, R 25 -substituted or unsubstituted heteroalkyl, R 25 -substituted or unsubstituted cycloalkyl, R 25 -substituted or unsubstituted heterocycloalkyl, R 25 -substituted or unsubstituted aryl, R 25 -substituted or unsubstituted heteroaryl; where R 25 is halogen, -OR 26 , -NR 27 R 28 , R 29 -substituted or unsubstituted alkyl, R 29-substituted or unsubstituted heteroalkyl, R 29 -substituted or unsubstituted cycloalkyl, R 29 -substituted or unsubstituted heterocycloalkyl, R 29 -substituted or unsubstituted aryl, or R 29 -substituted or unsubstituted heteroaryl; R 26 , R 27 and R 28 are independently hydrogen, R 29 -substituted or unsubstituted alkyl, R 29 -substituted or unsubstituted heteroalkyl, R 29 -substituted or unsubstituted cycloalkyl, R 29 -substituted or unsubstituted heterocycloalkyl, R 29 -substituted or unsubstituted aryl, or R 29 -substituted or unsubstituted heteroaryl, where R 27 and R 28 can be combined together as necessary to produce R 29 -substituted or unsubstituted heterocycloalkyl, or R 29 - forming a substituted or unsubstituted heteroaryl; R 29 is halogen, -OR 30 , -NR 31 R 32 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; R 30 , R 31 and R 32 is independently hydrogen or unsubstituted alkyl; A compound is provided.

[0055] In certain embodiments, the compound is [ka] or a pharma- ceutically acceptable salt thereof. [ka] or a pharma- ceutically acceptable salt thereof. It is.

[0056] Diseases and Disorders and Treatment Thereof Provided herein is a method for determining whether a patient is suffering from a disease or disorder, the method comprising detecting the presence of a misfolded or aggregated protein in the eye of the subject, wherein the detecting comprises contacting the misfolded or aggregated protein with a compound described herein. The misfolded or aggregated protein can be a misfolded or aggregated transthyretin protein. The disease or disorder can be systemic amyloidosis. The systemic amyloidosis can be familial amyloid polyneuropathy (FAP). The systemic amyloidosis can be ATTR amyloidosis. The compound can be a compound described herein. The contacting can be performed in vivo. The tissue can be an ocular tissue. The tissue can be a wrist, a spinal canal, a heart, or a lacrimal gland.

[0057] Symptoms of systemic amyloidosis may include: swelling of the ankles and / or legs, severe fatigue, weakness, shortness of breath, numbness, tingling or pain in the hands or legs, pain in the wrists (carpal tunnel syndrome), diarrhea, bloody stools, constipation, unintentional weight loss (e.g., more than 10 pounds (4.5 kilograms)), enlarged or rippled tongue, thickened skin, bruising, purplish patches around the eyes, irregular heartbeat, and / or difficulty swallowing.

[0058] The above systemic amyloidoses include familial amyloid polyneuropathy (FAP), ATTR amyloidosis, TTR cardiac amyloidosis, TTR amyloid cardiomyopathy (ATTR-CM), familial or hereditary ATTR amyloidosis (ATTRv or ATTRm), senile systemic amyloidosis (SSA or ATTRwt), AL amyloidosis, AA (serum amyloid A amyloidosis), AA amyloidosis resulting from inflammation due to infection, rheumatic diseases, autoinflammatory and autosomal dominant diseases whether acquired or hereditary. It may be an autoimmune disease, a cancer or neoplasm, fibrinogen alpha chain amyloidosis, apolipoprotein A-1 and A-2 amyloidosis, gelsolin amyloidosis, Ab2M (beta2 macroglobulin) amyloidosis, ALECT2 (leukocyte chemotactic protein 2) amyloidosis, AApoAIV (apolipoprotein AIV) amyloidosis, AApoCII (apolipoprotein CII) amyloidosis, AApoCIII (apolipoprotein CIII) amyloidosis, ALys (lysozyme) amyloidosis.

[0059] In some embodiments, the systemic amyloidosis can be familial amyloid polyneuropathy (TTR-FAP or FAP). FAP, also known as transthyretin amyloid polyneuropathy, is a rare hereditary and progressive disease caused by abnormal deposition of protein or amyloid around peripheral nerves and other tissues. FAP is caused by a mutation in the TTR gene that destabilizes the TTR tetramer. To date, more than 100 different mutations in the gene have been reported. Symptoms of FAP include peripheral neuropathy, which may appear early as abnormal sensations in the legs and feet (e.g., numbness, tingling, or burning), and progressive sensorimotor and autonomic neuropathy, including autonomic neuropathy when the nerves that control involuntary body functions (e.g., blood pressure, temperature control, and digestion) are damaged. Degeneration of motor fibers can cause progressive weakness and walking problems. One treatment for FAP is liver transplantation. Other treatments include gene therapy, immunization, dissolution of TTR aggregates, free radical scavengers, and administration of meglumine.

[0060] In some embodiments, the systemic amyloidosis is AL amyloidosis (immunoglobulin light chain amyloidosis). AL amyloidosis, the most common type of amyloidosis in developed countries, is also called primary amyloidosis. It usually affects the heart, kidneys, liver and nerves.

[0061] In some embodiments, the systemic amyloidosis is AA amyloidosis.Also known as inflammatory amyloidosis, this type is usually caused by inflammatory disease (e.g., rheumatoid arthritis).Treatment for severe inflammatory conditions can treat or prevent AA amyloidosis.It generally affects the kidney, liver and spleen.

[0062] In some embodiments, the systemic amyloidosis is localized amyloidosis. This type of amyloidosis often has a better prognosis than the types that affect multiple organ systems. Typical sites for localized amyloidosis include the bladder, skin, throat, or lungs. Accurate diagnosis is important so that treatments that affect the entire body can be avoided.

[0063] In some embodiments, the systemic amyloidosis is ATTR amyloidosis. The ATTR amyloidosis can be TTR cardiac amyloidosis, TTR amyloid cardiomyopathy (ATTR-CM), familial or hereditary ATTR amyloidosis (ATTRv or ATTRm), or senile systemic amyloidosis (ATTRwt).

[0064] ATTRm amyloidosis is a multisystem disorder with cardiovascular, peripheral and autonomic involvement, which can be difficult to diagnose due to the heterogeneity of the phenotype. ATTRm amyloidosis is a rare disease with diverse clinical manifestations determined in part by genotype. Given this complexity, diagnostic delays can occur up to 4 years from the onset of symptoms for patients with ATTRm who present with peripheral neuropathy, and up to 8 years for those who present with cardiomyopathy. Carpal tunnel syndrome (CTS) may be the initial symptom in up to 33% of patients, and there is a mean time of 4 to 6 years before other organs are clinically involved. Patients then usually develop peripheral and autonomic neuropathy, often with cardiac complications. Central nervous system (CNS) manifestations can also occur rarely, as TTR is also produced within the choroid plexus and retinal epithelium, and are more common for some disease-causing mutations with CNS predisposition, such as ATTRL12P. Additional clinical manifestations include peripheral neuropathy and autonomic neuropathy.V30M is the most common ATTR mutation, but other mutations may be underlying, including ATTRV122I and ATTRL12P, or variants or mutations as described herein.

[0065] Amyloidogenic protein variants causing the autosomal dominant clinical disorders Familial Amyloidotic Polyneuropathy (Sensorimotor and Autonomic Polyneuropathy) and Familial Amyloidotic Cardiomyopathy were found in 77 of the 127 amino acids in the TTR protein. 40 residues were found to have a single amyloidogenic mutation, whereas 15 residues have two mutations, 6 have three mutations, 5 have four mutations, and 1 has five mutations. 50 amino acids were unmutated, and 12 mutations have been described that did not result in clinically detectable amyloidosis. However, two of the involved residues had both amyloidogenic and nonamyloidogenic substitutions. The frequency of wild-type TTR amyloid deposits in the heart, carpal tunnel, and intestine increases with age and is currently thought to be related to post-synthetic (possibly oxidative) changes that may make the wild-type protein less stable, although other as yet undefined mechanisms may also be responsible. In the mutant cases, they all form tetramers that appear to be kinetically or thermodynamically unstable under physiological conditions that result in enhanced dissociation, releasing monomers that are susceptible to rapid misfolding, aggregation, and fibril formation. These observations suggest that the monomers functionally "chaperone" each other.

[0066] "Treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include one or more of the following: a) inhibiting the disease or condition (e.g., reducing one or more symptoms resulting from the disease or condition and / or reducing the severity of the disease or condition); b) improving, slowing or arresting the onset of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, preventing or slowing the progression or progression of the disease or condition, and / or preventing or slowing the spread (e.g., metastasis) of the disease or condition); and / or c) relieving the disease, i.e., causing regression of clinical symptoms (e.g., improving the disease state, providing partial or complete regression of the disease or condition, enhancing the effect of another drug therapy, slowing the progression of the disease, increasing quality of life, and / or prolonging survival).

[0067] "Prevention" or "preventing" refers to any treatment of a disease or condition that keeps the clinical symptoms of the disease or condition from occurring. In some embodiments, the methods provide for administration of a compound described herein to a patient (including a human) at risk for or with a family history of the disease or condition.

[0068] "Subject" refers to an animal, such as a mammal (including a human), that has been or will be the object of treatment, observation or experiment. The methods described herein may be useful in human therapy and / or veterinary applications. In some embodiments, the subject is a mammal. In one embodiment, the subject is a human.

[0069] The term "therapeutically effective amount" or "effective amount" of a compound described herein means an amount sufficient to effect treatment when administered to a subject to provide therapeutic benefits such as improving symptoms or slowing disease progression. For example, a therapeutically effective amount may be an amount sufficient to reduce symptoms of a disease or a state of systemic amyloidosis. The therapeutically effective amount may vary depending on the subject and the disease or condition being treated, the patient's weight and age, the severity of the disease or condition, and the mode of administration. The amount can be easily determined by those skilled in the applicable field.

[0070] The methods described herein may be applied to cell populations in vivo or ex vivo. "In vivo" means inside a living individual, such as inside an animal or human. In this context, the methods described herein may be used therapeutically in an individual. "Ex vivo" means outside a living individual. Examples of ex vivo cell populations include in vitro cell cultures and biological samples, including fluid or tissue samples obtained from an individual. Such samples may be obtained by methods well known in the art. Exemplary biological fluid samples include blood, cerebrospinal fluid, urine, and saliva. In this context, the compounds and compositions described herein may be used for a variety of purposes, including therapeutic and experimental purposes. For example, the compounds and compositions described herein may be used ex vivo to determine optimal schedules and / or dosing of the disclosed compounds for a given indication, cell type, individual, and other parameters. Information gathered from such use may be used for experimental purposes or in the clinic to set up protocols for in vivo treatment. Other ex vivo uses for which the compounds and compositions described herein may be suitable are described below or will become apparent to those skilled in the art. Selected compounds may be further characterized to determine safety or tolerated dosage in human or non-human patients. Such properties may be determined using methods commonly known to those skilled in the art.

[0071] In some embodiments, the misfolded or aggregated protein is not amyloid beta (Aβ) peptide, α-synuclein, prion peptide, huntingtin, serum amyloid A, lysozyme, amylin, immunoglobulin light chain, seminal-derived enhancer of viral infection, PAB, SEM1, protegrin-1, CsgA-R5, and CsgA-R1, superoxide dismutase, insulin, or p53. In some embodiments, the misfolded or aggregated protein does not include amyloid beta (Aβ) peptide. In some embodiments, the disease or disorder is not preeclampsia.

[0072] Detection of misfolded or aggregated proteins Provided herein is a method for the diagnosis of a disease or condition, comprising administering a compound described herein locally to the eye of the subject. The disease or disorder can be systemic amyloidosis. The systemic amyloidosis can be familial amyloid polyneuropathy (FAP). The systemic amyloidosis can be ATTR amyloidosis. The method can comprise detecting the binding of the compound to a misfolded or aggregated protein. The method can comprise detecting the binding of the compound to a misfolded or aggregated transthyretin protein. The binding can cause the emission of a detectable signal upon activation by light. The signal can be a fluorescent or infrared signal. The administration can be topical and / or localized to the conjunctiva of the eye.

[0073] Detection of misfolded or aggregated proteins can be performed with a compound that can selectively bind to misfolded or aggregated proteins. In one embodiment, the compound, when bound to the misfolded or aggregated protein, can be detected through its emitted fluorescent signal upon activation by laser light.

[0074] In situ detection of the binding of a protein-binding probe, such as a compound described herein, to misfolded or aggregated proteins in a patient's eye can be facilitated with an imaging device. The imaging device can be handheld or portable. The imaging device can include a lens and an image sensor, and optionally a laser light source. When the light source emits laser light into tissue (e.g., conjunctiva), misfolded or aggregated proteins accumulate there, and when bound to a compound that fluoresces upon binding, the accumulation can be easily detected and quantified by the lens and image sensor that collects and senses the fluorescent signal.

[0075] The method for determining whether a patient is afflicted with a disease or disorder (e.g., systemic amyloidosis) may be performed in any manner described herein or as known in the art. In some embodiments, the contacting causes emission of a detectable signal upon activation by light. In some embodiments, the detectable signal is a fluorescent signal. In some embodiments, the misfolded or aggregated protein is present in tissue as fibrils. In some embodiments, the misfolded or aggregated protein is present in tissue as plaques. In some embodiments, the method does not include determining fluorescence decay. In some embodiments, the method does not include determining fluorescence decay based on comparison to a reference.

[0076] In some embodiments, a method is provided for monitoring the response of a subject with systemic amyloidosis to treatment, the method comprising: (i) contacting the subject's eye with an effective amount of a compound described herein to form a detectable complex; and (ii) detecting the formation of the detectable complex, wherein a decrease in detectable complex compared to before the treatment indicates that the subject is responsive to the treatment. In some embodiments, the compound is applied topically to the eye.

[0077] Administration and Pharmaceutical Compositions In some embodiments, a pharmaceutical composition of the compound described herein is administered to the eye of a subject. In some embodiments, the compound is delivered to the surface of the eye. In some embodiments, the compound is administered as an eye drop. The administration can be topical.

[0078] The compounds may be effective over a wide dosage range. In some embodiments, in adult applications, dosages of 0.01-1000 mg, 0.5-100 mg, 1-50 mg, and 5-40 mg per day are examples of dosages that may be used. An exemplary dosage is 10-30 mg per day. In juvenile applications, the dosage may be the same as the adult dose or may be less. In some embodiments, an effective amount of the compound corresponds to about 50-500 mg of the compound per adult patient. The exact dosage will depend on the route of administration, the form in which the compound is administered, the patient to be treated, the weight of the patient to be treated, and the preferences and experience of the attending physician.

[0079] In some embodiments, the effective amount of the compound corresponds to about 0.01-1000 mg of compound / human patient / dosage. In some embodiments, the effective dose of the compound is 50-500 mg per dose per human. In some embodiments, the effective amount is about 0.01-100 mg, 0.01-200 mg, 0.01-300 mg, 0.01-400 mg, 0.01-500 mg, 0.01-600 mg, 0.01-700 mg, 0.01-800 mg, 0.01-900 mg, 0.01-1000 mg, 0.1-100 mg, 0.1-200 mg, 0.1-300 mg, 0.1-400 mg, 0.1-500 mg, g, 0.1~600mg, 0.1~700mg, 0.1~800mg, 0.1~900mg, 0.1~1000mg, 1~100mg, 1~200mg, 1~300mg, 1~400mg, 1~500mg, 1~600 mg, 1~700mg, 1~800mg, 1~900mg, 100~200mg, 100~300mg, 100~400mg, 100~500mg, 100~600mg, 100~700mg, 100~800mg, 10 0~900mg, 100~1000mg, 200~300mg, 200~400mg, 200~500mg, 200~600mg, 200~700mg, 200~800mg, 200~900mg, 200~1000m g, 300~400mg, 300~500mg, 300~600mg, 300~700mg, 300~800mg, 300~900mg, 300~1000mg, 400~500mg, 400~600mg, 400~70 0mg, 400-800mg, 400-900mg, 400-1000mg, 500-600mg, 500-700mg, 500-800mg, 500-900mg, 500-1000mg, 600-700mg, 600-800mg, 600-900mg, 600-1000mg, 700-800mg, 700-900mg, 700-1000mg, 800-900mg, 800-1000mg or approximately 900-1000mg.In some embodiments, the effective amount corresponds to about 50-100 mg, 50-400 mg, 50-500 mg, 100-200 mg, 100-300 mg, 100-400 mg, 100-500 mg, 200-300 mg, 200-400 mg, 200-500 mg, 300-400 mg, 300-500 mg, or 400-500 mg per dose per adult.

[0080] In some embodiments, the compound is administered in a single dose. In some embodiments, the compound is administered in multiple doses. In some embodiments, administration is about once, about twice, about three times, about four times, about five times, about six times, or more than six times per day. In some embodiments, administration is about once a month, once every two weeks, once a week, or once every two days. In other cases, the compound and another agent are administered together about once a day to about six times a day. In some embodiments, administration of the compound and agent continues for less than about seven days. In yet other cases, administration continues for more than about six days, ten days, fourteen days, twenty-eight days, two months, six months, or one year. In some embodiments, continuous administration is achieved and maintained as long as necessary.

[0081] In some embodiments, the compound is administered 1-10 times, 1-4 times, or once a day. In some embodiments, the compound is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times a day. In some embodiments, the compound is administered as drops. In some embodiments, the size of the drops administered is in the range of about 10-100 μL, about 10-90 μL, about 10-80 μL, about 10-70 μL, about 10-60 μL, about 10-50 μL, about 10-40 μL, about 10-30 μL, about 20-100 μL, about 20-90 μL, about 20-80 μL, about 20-70 μL, about 20-60 μL, about 20-50 μL, about 20-40 μL, or about 20-30 μL. An example of the present disclosure administers drops in the range of about 10 to about 30 μL. An example of the present disclosure administers drops in the range of about 10 to about 100 μL. An example of the present disclosure administers drops in the range of about 20 to about 50 μL. An example of the present disclosure administers drops in the range of about 20 to about 40 μL. An example of the present disclosure administers drops in the range of about 10 to about 60 μL. In some embodiments, the ophthalmic formulations of the present disclosure are administered several drops per administration, e.g., 1-3 drops per administration, 1-3 drops per administration, 1-4 drops per administration, 1-5 drops per administration, 1-6 drops per administration, 1-7 drops per administration, 1-8 drops per administration, 1-9 drops per administration, 1-10 drops per administration, 3-4 drops per administration, 3-5 drops per administration, 3-6 drops per administration, 3-7 drops per administration, 3-8 drops per administration, 3-9 drops per administration, 3-10 drops per administration, 5-6 drops per administration, 5-7 drops per administration, 5-8 drops per administration, 5-9 drops per administration, 5-10 drops per administration, 7-8 drops per administration, 7-9 drops per administration, or 9-10 drops per administration. In one example, the formulations of the present disclosure are administered at about 1 drop per administration and 1-6 times per day.

[0082] Pharmaceutical Compositions / Formulations In some embodiments, the compound described herein is formulated into pharmaceutical composition.In some embodiments, the pharmaceutical composition is formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliary substances that facilitate the processing of active compound into medicament-usable preparations.The suitable formulation depends on the selected route of administration. Any pharma- ceutically acceptable techniques, carriers, and excipients may be used as appropriate to formulate the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, 19th ed. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed. (Lippincott Williams & Wilkins 1999).

[0083] Provided herein is a pharmaceutical composition comprising the compound as described herein and pharma- ceutical acceptable diluent, excipient or carrier.In some cases, the compound as described herein is administered as a pharmaceutical composition, in which one or more compounds are mixed with other active ingredients, such as in combination therapy.In some cases, the pharmaceutical composition comprises one or more compounds as described herein.

[0084] Pharmaceutical composition, as used herein, refers to a mixture of the compounds described herein with other chemical components (e.g., carriers, stabilizers, diluents, dispersants, suspending agents, thickening agents, and / or excipients). In certain cases, the pharmaceutical composition facilitates administration of the compounds to an organism. In some embodiments for carrying out the methods of treatment or use provided herein, a therapeutically effective amount of one or more compounds described herein is administered in a pharmaceutical composition to a mammal having a disease or condition to be detected, diagnosed, or treated. In a specific example, the mammal is a human. In certain cases, the therapeutically effective amount varies depending on the severity of the disease, the age and relative health of the patient, the potency of the compound used, and other factors. The compounds described herein are used alone or in combination with one or more therapeutic agents as components of a mixture.

[0085] In some embodiments, the one or more compounds are formulated into an aqueous solution. In specific examples, the aqueous solution is selected from, by way of example only, physiologically compatible buffers, such as Hank's solution, Ringer's solution, aqueous acetate buffer, aqueous citrate buffer, aqueous carbonate buffer, aqueous phosphate buffer, or physiological salt buffer.

[0086] In some embodiments, the compounds described herein are formulated for ocular administration.In some embodiments, the ophthalmic formulations are liquid (in the form of solution, suspension, powder for reconstitution, sol-gel system), semi-solid (ointment and gel), solid (ophthalmic insert), and intraocular administration forms (injection, irrigation solution and implant).The compounds can be formulated, for example, for topical administration to the eye.

[0087] Provided herein is an ophthalmic formulation that comprises the compound described herein and an ophthalmologically acceptable component.Said ophthalmic formulation can be administered in any form suitable for ocular drug administration, for example, as a solution, suspension, ointment, gel, liposome dispersion, colloidal microparticle suspension, etc., or in ophthalmic insert, for example, in a biodegradable controlled release polymer matrix as needed.

[0088] By "pharmacologically acceptable" or "ophthalmologically acceptable" is meant an ingredient that is not biologically or otherwise undesirable. That is, the ingredient can be incorporated into the ophthalmic formulations of the present disclosure and administered topically to the patient's eye without causing any undesirable biological effects or interacting in a harmful manner with any of the other ingredients of the formulation composition in which it is contained. When the term "pharmacologically acceptable" is used to refer to an ingredient other than a pharmacologically active agent, it is implied that the ingredient has met the required standards of toxicological and manufacturing testing or is included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration. See, for example, Kaur et al., Drug Development and Industrial Pharmacy (2002) 28(5), 473-493.

[0089] Ophthalmic formulations can be adapted for topical administration to the eye in the form of suspension or emulsion.The ophthalmic formulations can include ophthalmologically acceptable carriers.Such carriers include, for example, water, mixtures of water, such as phosphate buffer, boric acid, sodium chloride, and sodium borate, and water-miscible solvents, such as lower alcohols, aryl alcohols, polyalkylene glycols, carboxymethylcellulose, polyvinylpyrrolidone, and isopropyl myristate.The ophthalmic formulations can also include one or more excipients, such as emulsifiers, preservatives, wetting agents, thickening agents (bodying agents). For example, the ophthalmic formulation may include polyethylene glycol 200, 300, 400 and 600, carbowax 1,000, 1,500, 4,000, 6,000 and 10,000, antimicrobial components such as quaternary ammonium compounds, phenylmercury salts, thimerosal, methyl and propyl paraben, benzyl alcohol, phenylethanol, buffering agents such as sodium borate, sodium acetate, gluconate buffer, and other agents such as sorbitan monolaurate, triethanolamine, oleate, polyoxyethylene sorbitan monopalmitylate, dioctyl sodium sulfosuccinate, monothioglycerol, thiosorbitol, and ethylenediaminetetraacetic acid. The ophthalmic formulation may be isotonic. The ophthalmic formulation may also include surfactants or stabilizers. Surfactants include Carbopol®. Stabilizers include sodium bisulfite, sodium metabisulfite and sodium thiosulfate.

[0090] The formulation may include an effective amount of a penetration enhancer that promotes the penetration of the formulation components through cell membranes, tissues, and extracellular matrices (including the cornea). The formulation may penetrate from the ocular surface to the conjunctiva. An "effective amount" of the penetration enhancer refers to a concentration sufficient to provide a measurable increase in the penetration of one or more of the formulation components through membranes, tissues, and extracellular matrices, as described immediately above. Suitable penetration enhancers include, by way of example, methylsulfonylmethane (MSM; also referred to as methylsulfone), a combination of MSM and dimethylsulfoxide (DMSO), or MSM, and in less preferred embodiments, a combination of DMSO, with MSM being particularly preferred.

[0091] Kits and Packages Also provided herein are kits and packages that include the disclosed compounds, retinal imaging devices, and, optionally, suitable packaging. In one embodiment, the kit further includes instructions for use.

[0092] The retinal imaging device may include a lens and an image sensor to detect the emitted signal (thus forming a suitable retinal scanner). In some embodiments, the retinal imaging device detects a fluorescent signal. In some embodiments, the retinal imaging device further includes a laser light source that may be used to activate the fluorescent signal. EXAMPLES

[0093] Working Example The following examples are included to clearly illustrate specific embodiments of the present disclosure. It should be recognized by those skilled in the art that the techniques disclosed in the following examples represent techniques that work well in the practice of the present disclosure and therefore can be considered to constitute specific modes for its practice. However, those skilled in the art should recognize in light of the present disclosure that many changes can be made in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the present disclosure.

[0094] Example 1: In vivo imaging of systemic amyloid deposition Three mice are bred and aged for imaging.The mice are examined for live imaging of amyloid accumulation.The compounds described herein are applied to the surface of the eye (formulation: 200 μL of 10 mg / mL solution of the compounds described herein in 20% DMSO / 80% propylene glycol), which allows visualization of TTR.

[0095] An optical imaging system modeled on a Zeiss Stemi 2000-C microscope is used. Input light is delivered near the axis of the microscope. Fluorescence excitation or far-red input is spectrally shaped with interference filters. A fluorescence emission bandpass filter is placed proximal to the integrated camera. The input source is based on a short-arc continuous xenon lamp placed adjacent to a spherical mirror that captures a portion of the energy and compresses it into a small diameter fiber optic cable (1.4 mm diameter, NA approx. 0.5). The spectral output of the lamp is approximately 1 milliwatt / cm. 2 The RIS design optical train provides a relatively uniform broadband light source from 400 to 700 nm with an output of 100 Hz. The optical train shapes the spot size of the beam, avoiding overfilling the pupil and providing efficient energy transfer of light from the illuminator to the mouse pupil. The result of the RIS design optical train is a focused narrow beam on the dilated pupil plane with an adjustable spot size ranging from 1 to 2 mm through modulation of just one optical element.

[0096] The fluorescent image of the conjunctiva is obtained and analyzed. The compounds described herein emit fluorescence upon contact with TTR protein aggregates. This indicates that such compounds can be used for in vivo retinal imaging as an indicator of systemic amyloidosis.

[0097] Example 2: TTR Aggregate Preparation Several aggregated TTR samples were prepared using a variety of conditions, as described below.

[0098] Aggregate A was prepared by resuspending TTR in 50 mM glycine buffer at a pH of about 3.6 and further incubating it at room temperature for 24 hours.

[0099] Aggregate B1 was prepared by resuspending TTR in water at a pH of about 7.4 and further incubating it for 7 days, maintained at 37° C. with agitation (200 rpm).

[0100] Aggregate B2 was prepared by resuspending TTR in water at a pH of about 7.4 and further incubating it for 14 days, maintained at 37° C. with agitation (200 rpm).

[0101] Example 3: Spectroscopic characterization of Compound I with TTR aggregates Compound 1 (4 μM) was prepared with or without aggregate B1 in 1× phosphate buffered saline (PBS). Absorption, fluorescence and excitation spectra were acquired for both samples. The excitation wavelength was optimized to be 440 nm. The emission spectra thus received were corrected against a blank solvent control to obtain the relative fluorescence intensity (RFI) as a function of wavelength. As illustrated in FIG. 1, the RFI of compound 1 was increased by about 3.2-fold in the presence of aggregate B1.

[0102] Additionally, fluorescence spectra were acquired for mixtures of compound I (4 μM) with aggregates A, B1, or B2, respectively (each at a concentration of 5 μM, at an emission wavelength of 565 nm) upon excitation at 440 nm. The increase in RFI relative to compound 1 alone was calculated and is shown in Table 1: Table 1. Increased fluorescence intensity of compound 1 in the presence of TTR aggregates. [Table 1]

[0103] Example 4: Binding affinity of Compound 1 with aggregated TTR A solution of DMSO / PBS (6 μL DMSO in 144 μL PBS, pH 7.4) was prepared as a blank control. Additionally, a 250 μM solution of compound 1 in DMSO was prepared. The following samples were then prepared in triplicate, maintaining a constant 5 μM TTR concentration in 4% DMSO / PBS: Table 2. Samples analyzed for fluorescence to determine binding constants [Table 2]

[0104] Emission spectra were acquired for each sample and corrected against a blank over the range of 445-700 nm. The RFI at λmax(em) was determined for each sample. The RFI was then plotted against increasing concentrations of compound 1 over the range of 0-10 μM using a one site specific binding algorithm. The binding affinity (K d ) was determined to be 0.3±0.1 μM, which is depicted in FIG.

[0105] Example 5: ATTR Cadaver Eye Staining The following protocol was adopted for staining formalin-fixed paraffin-embedded (FFPE) tissues: 1. Deparaffinization and Wetting of Tissue Sections: - Preheat at 60℃ for 1 hour - Slides are placed in a holder and treated with the clearing agent xylene (paraffin solvent) and a graded series of ethanol (EtOH) as follows: (i) 100% xylene - 5 min (ii) 100% xylene - 5 min (iii) 50% / 50% xylene / 100% EtOH - 3 min (iv)100% EtOH - 3 minutes (v) 95% EtOH - 3 min (vi) 70% EtOH - 3 min (vii)50% EtOH - 3 minutes (viii) Water - 2 x 3 minutes. 2. Antigen retrieval: Incubate in 99% formic acid for 5 minutes. 3. Wash with distilled water for 5 minutes. Repeat this process twice. 4. Preheat 10 mM citrate buffer (pH 6.0) to boiling. 5. Place the slides in the staining chamber containing the heated citrate buffer for 20 minutes. 6. Cool in a water bath. Wash with distilled water for 5 minutes. Repeat this process twice. 7.Equilibrate in 1x PBS for 15 min. 8. Block in 5% normal goat serum (NGS) in PBST (PBS with Tween 20) for 1 hour at room temperature (RT). 9. Incubate O / N at 4°C in primary antibody (2.5% NGS in PBST). 10. Wash tissue 3 x 10 minutes in PBST wash buffer. 11. Incubate in secondary antibody (1:500 in PBST) for 1 hour at RT - keep in the dark from this point onwards. 12. Wash tissue 3 x 10 minutes in PBST. 13. Stain with compound (60 μM) for 30 minutes at RT. - The compound is warmed to RT (approximately 30 min). - 5mg of compound is dissolved in 3.75ml DMSO - Keep in dark. - 100 μl of compound solution is diluted in 5 ml PBS. 14. Wash tissue 3 x 10 minutes in PBST. 15. Stain nuclei with Hoechst (2:1000 from 1 mg / ml dilution in PBS) for 10 minutes. 16. Wash tissue 3 x 10 minutes in PBST. 17. Mount the tissue using Prolong Glass mounting medium, secure with clips, and leave ON to dry. 18. Seal the edges with nail polish.

[0106] Example 6: Binding of Compound 1 to TTR in human tissues The Amydis retinal tracer is utilized to mark TTR aggregates in the eye for non-invasive detection using standard ocular imaging equipment, thus facilitating the diagnosis and monitoring of ATTR.

[0107] Sections from tissues with vitreous amyloid with known TTR mutations (paraffin-embedded tissue blocks) and autopsy eyes with vitreous amyloid (paraffin-embedded organs) are stained with hematoxylin and eosin (H&E) and Congo red, respectively, to mark areas with amyloidosis. Adjacent sections are co-stained with antibodies specific for Compound 1 and TTR using the optimized protocol described in Example 5. Immunofluorescence images are collected and compared. Areas of hyperfluorescence from Compound 1 are determined to correspond to areas of amyloidosis defined using Congo Red and H&E staining. It is contemplated that Compound 1 binds to TTR deposits in human tissues and undergoes increased fluorescence emission in a manner similar to the increased fluorescence in vivo in the presence of aggregated TTR, as described in the previous example. * * *

[0108] 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 this disclosure belongs.

[0109] The disclosure illustratively described herein may suitably be practiced in the absence of any element or limitation not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like, are to be read and understood broadly and without limitation. Furthermore, the terms and expressions used herein are used as terms of description and not as limitations, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure as claimed.

[0110] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0111] While the present disclosure has been described in conjunction with the above embodiments, it should be understood that the foregoing detailed description and examples are illustrative and are not intended to limit the scope of the present disclosure. Other aspects, advantages and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

Claims

1. 1. A composition for use in a method for determining whether a subject has systemic amyloidosis, comprising a compound of Formula I: 【Chemistry 6】 or a pharma- ceutically acceptable salt thereof, the method comprising administering the composition to an eye of the subject and detecting the presence or absence of misfolded or aggregated protein; Here, EDG is R １ - substituted or unsubstituted alkyl, R １ - substituted or unsubstituted cycloalkyl, R １ - substituted or unsubstituted heteroalkyl, R １ - substituted or unsubstituted heterocycloalkyl, R １ - substituted or unsubstituted aryl, R １ - substituted or unsubstituted heteroaryl, OR ２ N.R. ４ C(O)R ３ , -NR ４ R ５ , -SR ６ , or P.R. ７ R ８ and where R １ is halogen, -OR ９ , -NR １０ R １１ , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; R ２ , R ３ , R ４ , R ５ , R ６ , R ７ and R ８ are independently hydrogen, R １２ - substituted or unsubstituted alkyl, R １２ - substituted or unsubstituted heteroalkyl, R １２ - substituted or unsubstituted cycloalkyl, R １２ - substituted or unsubstituted heterocycloalkyl, R １２ - substituted or unsubstituted aryl or R １２ - substituted or unsubstituted heteroaryl, where R ４ and R ５ may be optionally joined together to form R １２ -substituted or unsubstituted heterocycloalkyl, or R １２ - forming a substituted or unsubstituted heteroaryl; R ９ and R １０ are independently hydrogen, R １２ - substituted or unsubstituted alkyl, R １２ - substituted or unsubstituted heteroalkyl, R １２ - substituted or unsubstituted cycloalkyl, R １２ - substituted or unsubstituted heterocycloalkyl, R １２ - substituted or unsubstituted aryl, or R １２ - substituted or unsubstituted heteroaryl, where R １０ and are optionally joined together to form R １２ -substituted or unsubstituted heterocycloalkyl, or R １２ - forming a substituted or unsubstituted heteroaryl; R １２ is halogen, -OR １３ , -NR １４ R １５ , R １６ - substituted or unsubstituted alkyl, R １６ - substituted or unsubstituted heteroalkyl, R １６ - substituted or unsubstituted cycloalkyl, R １６ - substituted or unsubstituted heterocycloalkyl, R １６ - substituted or unsubstituted aryl, or R １６ - substituted or unsubstituted heteroaryl; R １３ , R １４ and R １５ is independently hydrogen or unsubstituted alkyl; R １６ is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; Here, πCE is represented by the following formula: -L １ -(A １ ) ｑ -L ２ -(A ２ ) ｒ -L ３ -または-L １ -(A １ ) ｑ -L ４ -A ３ -L ２ -(A ２ ) ｒ -L ３ - where q and r are independently 0 or 1, where at least one of q or r is 1; A １ , A ２ and A ３ are independently R １７ -substituted or unsubstituted arylene or R １７ - a substituted or unsubstituted heteroarylene; L １ , L ２ , L ３ and L ４ is independently a bond or a group of the formula: 【Chemistry 7】 where x is an integer from 1 to 50; R １７ is halogen, -OR １８ , -NR １９ R ２０ , R ２１ - substituted or unsubstituted alkyl, R ２１ - substituted or unsubstituted heteroalkyl, R ２１ - substituted or unsubstituted cycloalkyl, R ２１ - substituted or unsubstituted heterocycloalkyl, R ２１ - substituted or unsubstituted aryl, or R ２１ - substituted or unsubstituted heteroaryl; R １８ , R １９ and R ２０ are independently hydrogen, R ２１ - substituted or unsubstituted alkyl, R ２１ - substituted or unsubstituted heteroalkyl, R ２１ - substituted or unsubstituted cycloalkyl, R ２１ - substituted or unsubstituted heterocycloalkyl, R ２１ - substituted or unsubstituted aryl, or R ２１ - substituted or unsubstituted heteroaryl; R ２１ is halogen, -OR ２２ , -NR ２３ R ２４ , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; R ２２ , R ２３ and R ２４ is independently hydrogen or unsubstituted alkyl; Here, WSG is R ２５ - substituted or unsubstituted alkyl, R ２５ - substituted or unsubstituted heteroalkyl, R ２５ - substituted or unsubstituted cycloalkyl, R ２５ - substituted or unsubstituted heterocycloalkyl, R ２５ - substituted or unsubstituted aryl, R ２５ - substituted or unsubstituted heteroaryl; where R ２５ is halogen, -OR ２６ , -NR ２７ R ２８ , R ２９ - substituted or unsubstituted alkyl, R ２９ - substituted or unsubstituted heteroalkyl, R ２９ - substituted or unsubstituted cycloalkyl, R ２９ - substituted or unsubstituted heterocycloalkyl, R ２９ - substituted or unsubstituted aryl, or R ２９ - substituted or unsubstituted heteroaryl; R ２６ , R ２７ and R ２８ are independently hydrogen, R ２９ - substituted or unsubstituted alkyl, R ２９ - substituted or unsubstituted heteroalkyl, R ２９ - substituted or unsubstituted cycloalkyl, R ２９ - substituted or unsubstituted heterocycloalkyl, R ２９ - substituted or unsubstituted aryl, or R ２９ - substituted or unsubstituted heteroaryl, where R ２７ and R ２８ may be optionally joined together to form R ２９ -substituted or unsubstituted heterocycloalkyl, or R ２９ - forming a substituted or unsubstituted heteroaryl; R ２９ is halogen, -OR ３０ , -NR ３１ R ３２ , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; R ３０ , R ３１ and R ３２ is independently hydrogen or unsubstituted alkyl; With the proviso that the misfolded or aggregated protein is not an amyloid beta peptide; composition.

2. The compound is 【Chemistry 8】 or a pharma- ceutically acceptable salt thereof.

3. 3. The composition of claim 1 or claim 2, wherein the misfolded or aggregated protein is transthyretin.

4. The systemic amyloidosis includes familial amyloid polyneuropathy (FAP), ATTR amyloidosis, TTR cardiac amyloidosis, TTR amyloid cardiomyopathy (ATTR-CM), familial or hereditary ATTR amyloidosis (ATTRv or ATTRm), senile systemic amyloidosis (SSA or ATTRwt), AL amyloidosis, AA (serum amyloid A amyloidosis), AA amyloidosis resulting from inflammation caused by infection, rheumatic diseases, autoinflammatory and autoimmune diseases whether acquired or hereditary, cancer, etc. or neoplasms, fibrinogen alpha chain amyloidosis, apolipoprotein A-1 and A-2 amyloidosis, gelsolin amyloidosis, Ab2M (beta 2 macroglobulin) amyloidosis, ALECT2 (leukocyte chemotactic protein 2) amyloidosis, AApoAIV (apolipoprotein AIV) amyloidosis, AApoCII (apolipoprotein CII) amyloidosis, AApoCIII (apolipoprotein CIII) amyloidosis, ALys (lysozyme) amyloidosis.

5. 20. The composition of any of the preceding claims, wherein administration is local to the ocular surface.

6. 10. The composition of any of the preceding claims, wherein said administering does not include administering directly to the interior of the eye.

7. 20. The composition of any of the preceding claims, wherein said administration does not include injection.

8. 20. The composition of any of the preceding claims, wherein contacting the compound or a pharma- ceutically acceptable salt thereof with the misfolded or aggregated protein causes emission of a detectable signal upon activation by light.

9. The composition of claim 8 , wherein the detectable signal is a fluorescent signal.

10. The composition of any one of claims 3 to 9, wherein the misfolded or aggregated transthyretin protein is wild-type.

11. The composition of any one of claims 3 to 9, wherein the misfolded or aggregated transthyretin protein is mutated.

12. 1. A composition for use in a method for diagnosing systemic amyloidosis, comprising a compound of formula I: 【Chemistry 9】 or a pharma- ceutically acceptable salt thereof, the method comprising administering the composition to an eye of a subject in need of diagnosis of systemic amyloidosis and detecting the presence or absence of misfolded or aggregated transthyretin protein; Here, EDG is R １ - substituted or unsubstituted alkyl, R １ - substituted or unsubstituted cycloalkyl, R １ - substituted or unsubstituted heteroalkyl, R １ - substituted or unsubstituted heterocycloalkyl, R １ - substituted or unsubstituted aryl, R １ - substituted or unsubstituted heteroaryl, OR ２ N.R. ４ C(O)R ３ , -NR ４ R ５ , -SR ６ , or P.R. ７ R ８ and where R １ is halogen, -OR ９ , -NR １０ R １１ , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , and R ８ are independently hydrogen, R １２ - substituted or unsubstituted alkyl, R １２ - substituted or unsubstituted heteroalkyl, R １２ - substituted or unsubstituted cycloalkyl, R １２ - substituted or unsubstituted heterocycloalkyl, R １２ - substituted or unsubstituted aryl or R １２ - substituted or unsubstituted heteroaryl, where R ４ and R ５ may be optionally joined together to form R １２ -substituted or unsubstituted heterocycloalkyl, or R １２ - forming a substituted or unsubstituted heteroaryl; R ９ and R １０ are independently hydrogen, R １２ - substituted or unsubstituted alkyl, R １２ - substituted or unsubstituted heteroalkyl, R １２ - substituted or unsubstituted cycloalkyl, R １２ - substituted or unsubstituted heterocycloalkyl, R １２ - substituted or unsubstituted aryl, or R １２ - substituted or unsubstituted heteroaryl, where R １０ and are optionally joined together to form R １２ -substituted or unsubstituted heterocycloalkyl, or R １２ - forming a substituted or unsubstituted heteroaryl; R １２ is halogen, -OR １３ , -NR １４ R １５ , R １６ - substituted or unsubstituted alkyl, R １６ - substituted or unsubstituted heteroalkyl, R １６ - substituted or unsubstituted cycloalkyl, R １６ - substituted or unsubstituted heterocycloalkyl, R １６ - substituted or unsubstituted aryl, or R １６ - substituted or unsubstituted heteroaryl; R １３ , R １４ and R １５ is independently hydrogen or unsubstituted alkyl; R １６ is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; Here, πCE is represented by the formula: -L １ -(A １ ) ｑ -L ２ -(A ２ ) ｒ -L ３ -または-L １ -(A １ ) ｑ -L ４ -A ３ -L ２ -(A ２ ) ｒ -L ３ -, where q and r are independently 0 or 1, where at least one of q or r is 1; A １ , A ２ and A ３ are independently R １７ -substituted or unsubstituted arylene or R １７ - a substituted or unsubstituted heteroarylene; L １ , L ２ , L ３ and L ４ is independently a bond or a group of the formula: 【Chemistry 10】 is a linking group having the formula where x is an integer from 1 to 50; R １７ is halogen, -OR １８ , -NR １９ R ２０ , R ２１ - substituted or unsubstituted alkyl, R ２１ - substituted or unsubstituted heteroalkyl, R ２１ - substituted or unsubstituted cycloalkyl, R ２１ - substituted or unsubstituted heterocycloalkyl, R ２１ - substituted or unsubstituted aryl, or R ２１ - substituted or unsubstituted heteroaryl; R １８ , R １９ and R ２０ are independently hydrogen, R ２１ - substituted or unsubstituted alkyl, R ２１ - substituted or unsubstituted heteroalkyl, R ２１ - substituted or unsubstituted cycloalkyl, R ２１ - substituted or unsubstituted heterocycloalkyl, R ２１ - substituted or unsubstituted aryl, or R ２１ - substituted or unsubstituted heteroaryl; R ２１ is halogen, -OR ２２ , -NR ２３ R ２４ , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; R ２２ , R ２３ and R ２４ is independently hydrogen or unsubstituted alkyl; Here, WSG is: R ２５ - substituted or unsubstituted alkyl, R ２５ - substituted or unsubstituted heteroalkyl, R ２５ - substituted or unsubstituted cycloalkyl, R ２５ - substituted or unsubstituted heterocycloalkyl, R ２５ - substituted or unsubstituted aryl, R ２５ - substituted or unsubstituted heteroaryl; where R ２５ is halogen, -OR ２６ , -NR ２７ R ２８ , R ２９ - substituted or unsubstituted alkyl, R ２９ - substituted or unsubstituted heteroalkyl, R ２９ - substituted or unsubstituted cycloalkyl, R ２９ - substituted or unsubstituted heterocycloalkyl, R ２９ - substituted or unsubstituted aryl, or R ２９ - substituted or unsubstituted heteroaryl; R ２６ , R ２７ and R ２８ are independently hydrogen, R ２９ - substituted or unsubstituted alkyl, R ２９ - substituted or unsubstituted heteroalkyl, R ２９ - substituted or unsubstituted cycloalkyl, R ２９ - substituted or unsubstituted heterocycloalkyl, R ２９ - substituted or unsubstituted aryl, or R ２９ - substituted or unsubstituted heteroaryl, where R ２７ and R ２８ may be optionally joined together to form R ２９ -substituted or unsubstituted heterocycloalkyl, or R ２９ - forming a substituted or unsubstituted heteroaryl; R ２９ is halogen, -OR ３０ , -NR ３１ R ３２ , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; R ３０ , R ３１ and R ３２ is independently hydrogen or unsubstituted alkyl; composition.

13. The compound is 【Chemistry 11】 or a pharma- ceutically acceptable salt thereof.

14. The systemic amyloidosis includes familial amyloid polyneuropathy (FAP), ATTR amyloidosis, TTR cardiac amyloidosis, TTR amyloid cardiomyopathy (ATTR-CM), familial or hereditary ATTR amyloidosis (ATTRv or ATTRm), senile systemic amyloidosis (SSA or ATTRwt), AL amyloidosis, AA (serum amyloid A amyloidosis), AA amyloidosis resulting from inflammation due to infection, rheumatic diseases, autoinflammatory and autoimmune diseases whether acquired or hereditary, cancer or The composition according to claim 12 or 13, wherein the amyloidosis is selected from the group consisting of neoplasms, fibrinogen α-chain amyloidosis, apolipoprotein A-1 and A-2 amyloidosis, gelsolin amyloidosis, Ab2M (β2 macroglobulin) amyloidosis, ALECT2 (leukocyte chemotactic protein 2) amyloidosis, AApoAIV (apolipoprotein AIV) amyloidosis, AApoCII (apolipoprotein CII) amyloidosis, AApoCIII (apolipoprotein CIII) amyloidosis, and ALys (lysozyme) amyloidosis.

15. The composition according to any one of claims 12 to 14, wherein administration is local.

16. The composition according to any one of claims 12 to 15, wherein the compound or a pharma- ceutically acceptable salt thereof reaches the conjunctiva of the eye.

17. The composition of any of claims 12 to 16, wherein said administration does not include administration to the interior of the eye.

18. 20. The composition of any of the preceding claims, wherein said administration does not include injection.

19. 1. A composition for use in a method for preparing a patient for diagnosis of systemic amyloidosis, comprising a compound of formula I: 【Chemistry 12】 and the method comprises topically administering the composition to an eye of the patient, Here, EDG is R １ - substituted or unsubstituted alkyl, R １ - substituted or unsubstituted cycloalkyl, R １ - substituted or unsubstituted heteroalkyl, R １ - substituted or unsubstituted heterocycloalkyl, R １ - substituted or unsubstituted aryl, R １ - substituted or unsubstituted heteroaryl, OR ２ N.R. ４ C(O)R ３ , -NR ４ R ５ , -SR ６ , or P.R. ７ R ８ and where R １ is halogen, -OR ９ , -NR １０ R １１ , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; R ２ , R ３ , R ４ , R ５ , R ６ , R ７ and R ８ are independently hydrogen, R １２ - substituted or unsubstituted alkyl, R １２ - substituted or unsubstituted heteroalkyl, R １２ - substituted or unsubstituted cycloalkyl, R １２ - substituted or unsubstituted heterocycloalkyl, R １２ - substituted or unsubstituted aryl or R １２ - substituted or unsubstituted heteroaryl, where R ４ and R ５ may be optionally joined together to form R １２ -substituted or unsubstituted heterocycloalkyl, or R １２ - forming a substituted or unsubstituted heteroaryl; R ９ and R １０ are independently hydrogen, R １２ - substituted or unsubstituted alkyl, R １２ - substituted or unsubstituted heteroalkyl, R １２ - substituted or unsubstituted cycloalkyl, R １２ - substituted or unsubstituted heterocycloalkyl, R １２ - substituted or unsubstituted aryl, or R １２ - substituted or unsubstituted heteroaryl, where R １０ and are optionally joined together to form R １２ -substituted or unsubstituted heterocycloalkyl, or R １２ - forming a substituted or unsubstituted heteroaryl; R １２ is halogen, -OR １３ , -NR １４ R １５ , R １６ - substituted or unsubstituted alkyl, R １６ - substituted or unsubstituted heteroalkyl, R １６ - substituted or unsubstituted cycloalkyl, R １６ - substituted or unsubstituted heterocycloalkyl, R １６ - substituted or unsubstituted aryl, or R １６ - substituted or unsubstituted heteroaryl; R １３ , R １４ and R １５ is independently hydrogen or unsubstituted alkyl; R １６ is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; Here, πCE is represented by the following formula: -L １ -(A １ ) ｑ -L ２ -(A ２ ) ｒ -L ３ -または-L １ -(A １ ) ｑ -L ４ -A ３ -L ２ -(A ２ ) ｒ -L ３ - where q and r are independently 0 or 1, where at least one of q or r is 1; A １ , A ２ and A ３ are independently R １７ -substituted or unsubstituted arylene or R １７ - a substituted or unsubstituted heteroarylene; L １ , L ２ , L ３ and L ４ is independently a bond or a group of the formula: 【Chemistry 13】 where x is an integer from 1 to 50; R １７ is halogen, -OR １８ , -NR １９ R ２０ , R ２１ - substituted or unsubstituted alkyl, R ２１ - substituted or unsubstituted heteroalkyl, R ２１ - substituted or unsubstituted cycloalkyl, R ２１ - substituted or unsubstituted heterocycloalkyl, R ２１ - substituted or unsubstituted aryl, or R ２１ - substituted or unsubstituted heteroaryl; R １８ , R １９ and R ２０ are independently hydrogen, R ２１ - substituted or unsubstituted alkyl, R ２１ - substituted or unsubstituted heteroalkyl, R ２１ - substituted or unsubstituted cycloalkyl, R ２１ - substituted or unsubstituted heterocycloalkyl, R ２１ - substituted or unsubstituted aryl, or R ２１ - substituted or unsubstituted heteroaryl; R ２１ is halogen, -OR ２２ , -NR ２３ R ２４ , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; R ２２ , R ２３ and R ２４ is independently hydrogen or unsubstituted alkyl; Here, WSG is R ２５ - substituted or unsubstituted alkyl, R ２５ - substituted or unsubstituted heteroalkyl, R ２５ - substituted or unsubstituted cycloalkyl, R ２５ - substituted or unsubstituted heterocycloalkyl, R ２５ - substituted or unsubstituted aryl, R ２５ - substituted or unsubstituted heteroaryl; where R ２５ is halogen, -OR ２６ , -NR ２７ R ２８ , R ２９ - substituted or unsubstituted alkyl, R ２９ - substituted or unsubstituted heteroalkyl, R ２９ - substituted or unsubstituted cycloalkyl, R ２９ - substituted or unsubstituted heterocycloalkyl, R ２９ - substituted or unsubstituted aryl, or R ２９ - substituted or unsubstituted heteroaryl; R ２６ , R ２７ and R ２８ are independently hydrogen, R ２９ - substituted or unsubstituted alkyl, R ２９ - substituted or unsubstituted heteroalkyl, R ２９ - substituted or unsubstituted cycloalkyl, R ２９ - substituted or unsubstituted heterocycloalkyl, R ２９ - substituted or unsubstituted aryl, or R ２９ - substituted or unsubstituted heteroaryl, where R ２７ and R ２８ may be optionally joined together to form R ２９ -substituted or unsubstituted heterocycloalkyl, or R ２９ - forming a substituted or unsubstituted heteroaryl; R ２９ is halogen, -OR ３０ , -NR ３１ R ３２ , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; R ３０ , R ３１ and R ３２ is independently hydrogen or unsubstituted alkyl; composition.

20. The composition of claim 19, wherein the method further comprises detecting binding of the compound to a misfolded or aggregated protein.

21. 21. The composition of claim 20, wherein the misfolded or aggregated protein is transthyretin.

22. 20. The composition of claim 19, wherein the compound is delivered to the conjunctiva of the eye.

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