NOVEL P62 LIGAND COMPOUND, AND COMPOSITION FOR PREVENTING, AMELIORATING OR TREATING PROTEINOPATHIES COMPRING THE SAME

Abstract

The present invention relates to a novel p62 ligand compound, a stereoisomer, hydrate, solvate or prodrug thereof, and a pharmaceutical or food composition for preventing or treating proteinopathies comprising the same as an active ingredient. The p62 ligand compound according to the present invention can be usefully used as a pharmaceutical composition for the prevention, amelioration or treatment of various proteinopathies by activating autophagy in cells and thus selectively eliminating in vivo proteins, organelles and aggregates.

Claims

1. A p62 ligand compound represented by the following Chemical Formula 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof: ##STR00030## in Chemical Formula 1, W is C6-C10 aryl; L is —O—(CH.sub.2).sub.n1—CH(OH)—, provided that O in the —O—(CH.sub.2).sub.n1—CH(OH)— is bonded to a benzene ring, where n1 is an integer of 1 to 4; m is an integer of 0 to 2; R.sub.a is R.sub.1 or —OR.sub.1, where R.sub.1 is hydrogen or —(CH.sub.2).sub.n2—R′.sub.1, R′.sub.1 is phenyl which is unsubstituted or substituted by hydroxy, halogen, C.sub.1-4 alkyl′, C.sub.1-4 alkoxy, nitro, amino, (C.sub.1-4 alkyl)amino, or di(C.sub.1-4 alkyl)amino, n2 is an integer of 1 to 6; R.sub.b is —OR.sub.2, where R.sub.2 is hydrogen or —(CH.sub.2).sub.n3—R′.sub.2, R′.sub.2 is phenyl which is unsubstituted or substituted by hydroxy, halogen, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, nitro, amino, (C.sub.1-4 alkyl)amino, or di(C.sub.1-4 alkyl)amino, n3 is an integer of 1 to 6; R.sub.c is —(CH.sub.2).sub.n4—OH, —(CH.sub.2).sub.n4—NH—C(═NH)NH.sub.2, —C(═NH)NH.sub.2, C.sub.1-6 alkyl, —CH(R.sub.3)—COO—R.sub.4, or —CH(COO—R.sub.4)—CH.sub.2CH.sub.2CH.sub.2—NH—C(═NH)NH.sub.2, —(CH.sub.2).sub.n4—O—(CH.sub.2).sub.n4—OR.sub.4, —CONH(CH.sub.2).sub.n4—OR.sub.4, —CO(CH.sub.2).sub.n5—OR.sub.4, —(CH.sub.2).sub.n5—CH(NH.sub.2)—COOR.sub.4, —(CH.sub.2).sub.n5—CONHR.sub.4, n4 is an integer of 2 to 4, n5 is an integer of 1 to 4, R.sub.3 is hydrogen or C.sub.1-4 alkyl, and R.sub.4 is hydrogen, or C.sub.1-4 alkyl.

2. The compound, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof according to claim 1, wherein the W is phenyl, and n1 is 1 or 2.

3. The compound, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof according to claim 1, wherein the R.sub.a is hydrogen or —O—(CH.sub.2).sub.n2—R′.sub.1, R′.sub.1 is phenyl that is unsubstituted or substituted by hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino or dimethylamino, and n2 is an integer of 1 to 4.

4. The compound, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof according to claim 1, wherein the R.sub.b is hydroxy, or —O—(CH.sub.2).sub.n3—R′.sub.2, R′.sub.2 is phenyl which is unsubstituted or substituted by hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino or dimethylamino, and n3 is an integer of 1 to 4.

5. The compound, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof according to claim 1, wherein the R.sub.c is —(CH.sub.2).sub.n4—OH, —(CH.sub.2).sub.n4—NH—C(═NH)NH.sub.2, —C(═NH)NH.sub.2, methyl, ethyl or isopropyl, and n4 is an integer of 2 to 3.

6. The compound, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof according to claim 1, wherein the compound of Chemical Formula 1 is selected from the group consisting of the following compounds 1) to 19: 1) (R)-1-(3,4-(bis(benzyloxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YOK-1106); 2) (R)-1-(3,4-(bis((4-chlorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-1); 3) (R)-1-(3-((4-chlorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-2); 4) (R)-1-(3-((4-chlorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-7); 5) (R)-1-(3-((4-fluorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-8); 6) (R)-1-(2-((3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YOK-1109); 7) (R)-1-(2-((3-(3,4-diphenethoxyphenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YOK-2209); 8) (R)-1-(2-((3-(3,4-bis(4-chlorobenzyl)oxy)phenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YT-9-1); 9) (R)-1-(2-((3-(3,4-bis(4-fluorobenzyl)oxy)phenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YT-9-2); 10) (R)-1-(3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)guanidine (YOK-1107); 11) (R)-1-(3-(3,4-diphenethoxyphenoxy)-2-hydroxypropyl)guanidine (YOK-2207); 12) (R)-1-(3,4-bis(benzyloxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-1104); 13) (R)-1-(3,4-diphenethoxyphenoxy)-3-(isopropylamino)propan-2-ol (YOK-2204); 14) (R)-1-(3,4-bis(3-phenylpropoxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-3304); 15) (R)-1-(3,4-bis(4-phenylbutoxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-4404); 16) (R)-1-(4-(benzyloxy)-3-phenethoxyphenoxy)-3-(isopropylamino)propan-2-ol (YOK-1204); 17) (R)-1-(4-(benzyloxy)-3-(3-phenylpropoxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-1304); 18) (R)-1-(3,4-bis((4-chlorobenzyl)oxy)phenoxy)-3-(isopropylamino)propan-2-ol (YT-4-1); and 19) (R)-1-(3,4-bis((4-fluorobenzyl)oxy)phenoxy)-3-(isopropylamino)propan-2-ol (YT-4-2).

7. A composition comprising a compound of Chemical Formula 1 according to claim 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof.

8. The composition according to claim 7, wherein the composition is a pharmaceutical composition.

9. (canceled)

10. (canceled)

11. (canceled)

12. The composition according to claim 7, wherein the composition is a food composition.

13. (canceled)

14. (canceled)

15. A method for increasing the degradation of misfolded protein aggregates, comprising administering an effective amount of the compound, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof according to claim 1 to a subject in need thereof.

16. A method for activating autophagy, comprising administering an effective amount of the compound, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof according to claim 1 to a subject in need thereof.

17. A method for preventing, ameliorating or treating proteinopathies, comprising administering the compound, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof according to claim to a subject in need thereof.

18. The method according to claim 17, wherein the proteinopathy is cancer, neurodegenerative diseases, alpha-1 antitrypsin deficiency, keratopathy, retinitis pigmentosa, type 2 diabetes, or cystic fibrosis.

19. The method according to claim 18, wherein the neurodegenerative diseases are at least one selected from the group consisting of Lyme borreliosis, fatal familial insomnia, Creutzfeldt-Jakob Disease (CJD), multiple sclerosis (MS), dementia, Alzheimer's disease, epilepsy, Parkinson's disease, stroke, Huntington's disease, Picks disease, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxias, other Poly-Q diseases, hereditary cerebral amyloid angiopathy, familial amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type 2 diabetes, injection-localized amyloidosis, beta-2 microglobulin amyloidosis, hereditary non-neuropathic amyloidosis, Alexander disease and Finnish hereditary systemic amyloidosis.

20. The method according to claim 16, wherein the method is for activating selective autophagy.

21. The composition according to claim 16, wherein the method is for increasing the autophagy activity of misfolded proteins.

22. The composition according to claim 16, wherein the method is for activating autophagy by activation of p62 proteins.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 is a schematic diagram illustrating that the proteins arginylated by N-terminal rule binds to the p62 ZZ domain and degrades intracellular substances such as proteins through intracellular autophagy activity.

[0027] FIG. 2 is an immunoblot assay result showing the effect of increasing the p62 protein oligomerization activity of p62 ligand compounds (YOK-1104, YOK-2204, YOK-3304, YOK-1204, YOK-4404, YOK-1107, YOK-1109, YOK-2207, YOK-2209, YT-4-1, YT-4-2, YT-6-1, YT-6-2, YT-6-7, YT-6-8, YT-9-1 and YT-9-2) according to the present invention.

[0028] FIG. 3 is an immunoblot assay result showing the effect of increasing the autophagy activity of p62 ligand compounds (YOK-1107, YOK-4404, 3301, 3302 and YOK-1104) according to the present invention.

[0029] FIG. 4 is an immunoblot assay result showing the effect of increasing the autophagy activity of p62 ligand compounds (YOK-1106, YOK-1107, YOK-1204, YOK-1304, YOK-2204, YOK-4404) according to the present invention,

[0030] FIG. 5 is an immunoblot assay result confirming that control compounds (1101, 1102, 1103, 1201, 1202, 1203, 1301, 1302, YTK-1105-1, 4402, 1401, 1402, 2201 and 2202) do not increase autophagy activity.

[0031] FIGS. 6a and 6b are immunofluorescence staining assay results confirming that the p62 ligand compounds (YOK-1106, YOK-1204, YOK-1504, YOK-2204, YOK-3304 and YOK-4404) according of the present invention allow the activation and oligomerization of p62 proteins, and then show the efficacy of delivering them to autophagosome and simultaneously increasing autophagy activity.

[0032] FIG. 7 is immunofluorescence staining assay results confirming that the control compounds (YOK-A-1104, YOK-G-1104, YTK-1005 and YTK-1105-1) allow the activation and oligomerization of p62 proteins and then show the efficacy of delivering them to autophagosome and simultaneously increasing autophagy activity.

[0033] FIGS. 8a and 8b, and FIGS. 9a and 9b are immunofluorescence staining assay results showing the efficacy of delivering target proteins to autophagy. This is the result showing that after treatment with the compounds, markers of target proteins, FK2 (FIGS. 8a and 8b) or HuntingtinQ103-GFP (FIGS. 9a and 9b), gradually increase in terms of the co-existence of p62 protein with intracellular puncta.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] Hereinafter, the present invention will be described in more detail.

[0035] The definition of groups used herein are described in detail. Unless otherwise indicated, each group has the following definition.

[0036] As used herein, the term “halo” includes fluoro, chloro, bromo and iodo.

[0037] As used herein, the “alkyl” refers to a linear or branched aliphatic hydrocarbon radical, and may be preferably an alkyl having 1 to 6 carbon atoms, more preferably an alkyl having 1 to 4 carbon atoms. Examples of such alkyl include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl and 2-ethylbutyl.

[0038] In one aspect of the invention, there is provided a compound represented by the following Chemical Formula 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof:

##STR00001##

[0039] in Chemical Formula 1,

[0040] W is C6-C10 aryl;

[0041] L is —O—(CH.sub.2).sub.n1—CH(OH)—, provided that O in the —O—(CH.sub.2).sub.n1—CH(OH)— is bonded to a benzene ring, where n1 is an integer of 1 to 4;

[0042] m is an integer of 0 to 2;

[0043] R.sub.a is R.sub.1 or —OR.sub.1,

[0044] where R.sub.1 is hydrogen or —(CH.sub.2).sub.n2—R′.sub.1, R′.sub.1 is phenyl which is unsubstituted or substituted by hydroxy, halogen, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, nitro, amino, (C.sub.1-4 alkyl)amino, or di(C.sub.1-4 alkyl)amino,

[0045] n2 is an integer of 1 to 6;

[0046] R.sub.b is —OR.sub.2,

[0047] where R.sub.2 is hydrogen or —(CH.sub.2).sub.n3—R′.sub.2,

[0048] R′.sub.2 is phenyl which is unsubstituted or substituted by hydroxy, halogen, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, nitro, amino, (C.sub.1-4 alkyl)amino, or di(C.sub.1-4 alkyl)amino,

[0049] n3 is an integer of 1 to 6;

[0050] R.sub.c is —(CH.sub.2).sub.n4—OH, —(CH.sub.2).sub.n4—NH—C(═NH) NH.sub.2, —C(═NH) NH.sub.2, C.sub.1-6 alkyl, —CH(R.sub.3)—COO—R.sub.4, or —CH(COO—R.sub.4)—CH.sub.2CH.sub.2CH.sub.2—NH—C(═NH) NH.sub.2, —(CH.sub.2).sub.n4—O—(CH.sub.2).sub.n4—OR.sub.4, —CONH(CH.sub.2).sub.n4—OR.sub.4, —CO(CH.sub.2).sub.n5—OR.sub.4, —(CH.sub.2).sub.n5—CH(NH.sub.2)—COOR.sub.4, —(CH.sub.2).sub.n5—CONHR.sub.4,

[0051] Preferably, the W may be phenyl.

[0052] Preferably, the n1 may be 1 or 2.

[0053] Preferably, the R.sub.a may be hydrogen or —O—(CH.sub.2).sub.n2—R′.sub.1.

[0054] Preferably, the R′.sub.1 may be phenyl that is unsubstituted or substituted by hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino or dimethylamino.

[0055] Preferably, the n2 may be an integer of 1 to 4.

[0056] Preferably, the R.sub.b may be hydroxy, or —O—(CH.sub.2).sub.n3—R′.sub.2.

[0057] Preferably, the R′.sub.2 may be phenyl which is unsubstituted or substituted by hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino or dimethylamino.

[0058] Preferably, the n3 may be an integer of 1 to 4.

[0059] Preferably, the R.sub.c may be —(CH.sub.2).sub.n4—OH, —(CH.sub.2).sub.n4—NH—C(═NH)NH.sub.2, —C(═NH)NH.sub.2, methyl, ethyl or isopropyl.

[0060] Preferably, the n4 may be an integer of 2 to 3.

[0061] Preferably, the n5 may be an integer of 1 to 3.

[0062] Preferably, the R.sub.4 may be hydrogen or methyl.

[0063] Specifically, representative examples of the compound represented by Chemical Formula 1 are as follows: [0064] 1) (R)-1-(3,4-(bis(benzyloxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YOK-1106); [0065] 2) (R)-1-(3,4-(bis((4-chlorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-1); [0066] 3) (R)-1-(3-((4-chlorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-2); [0067] 4) (R)-1-(3-((4-chlorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-7); [0068] 5) (R)-1-(3-((4-fluorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-8); [0069] 6) (R)-1-(2-((3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YOK-1109); [0070] 7) (R)-1-(2-((3-(3,4-diphenethoxyphenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YOK-2209); [0071] 8) (R)-1-(2-((3-(3,4-bis(4-chlorobenzyl)oxy)phenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YT-9-1); [0072] 9) (R)-1-(2-((3-(3,4-bis(4-fluorobenzyl)oxy)phenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YT-9-2); [0073] 10) (R)-1-(3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)guanidine (YOK-1107); [0074] 11) (R)-1-(3-(3,4-diphenethoxyphenoxy)-2-hydroxypropyl)guanidine (YOK-2207); [0075] 12) (R)-1-(3,4-bis(benzyloxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-1104); [0076] 13) (R)-1-(3,4-diphenethoxyphenoxy)-3-(isopropylamino)propan-2-ol (YOK-2204); [0077] 14) (R)-1-(3,4-bis(3-phenylpropoxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-3304); [0078] 15) (R)-1-(3,4-bis(4-phenylbutoxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-4404); [0079] 16) (R)-1-(4-(benzyloxy)-3-phenethoxyphenoxy)-3-(isopropylamino)propan-2-ol (YOK-1204); [0080] 17) (R)-1-(4-(benzyloxy)-3-(3-phenylpropoxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-1304); [0081] 18) (R)-1-(3,4-bis((4-chlorobenzyl)oxy)phenoxy)-3-(isopropylamino)propan-2-ol (YT-4-1); and [0082] 19) (R)-1-(3,4-bis((4-fluorobenzyl)oxy)phenoxy)-3-(isopropylamino)propan-2-ol (YT-4-2).

[0083] Meanwhile, the compounds of the present invention may exist in the form of a pharmaceutically acceptable salt. As the salt, an addition salt formed by pharmaceutically acceptable free acids may be useful. The term “pharmaceutically acceptable salt” used herein refers to any organic or inorganic addition salt of the compounds represented by Chemical Formulae 1 to 3, in which the adverse effect caused by the salt does not impair the beneficial effect of the compounds at a concentration exhibiting relatively non-toxic and non-harmful effective activity to a patient.

[0084] The acid addition salt may be prepared by a common method, for example, by dissolving a compound in an excess amount of aqueous acid solution and precipitating the resulting salt using a water-miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. Alternatively, an equimolar amount of a compound and an acid in water or alcohol (e.g., glycol monomethyl ether) can be heated, and subsequently, the resulting mixture can be dried by evaporating, or precipitated salts can be filtered under suction.

[0085] In this case, the free acid may be an inorganic acid or an organic acid. Examples of the inorganic acids include, but are not limited to, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid and stannic acid. Examples of the organic acids include, but are not limited to, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid.

[0086] In addition, a pharmaceutically acceptable metal salt may be prepared using a base. An alkali metal or alkaline earth metal salt may be obtained, for example, by dissolving a compound in an excess amount of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating the filtrate until dry. At this time, as the metal salts, particularly sodium, potassium or calcium salts are pharmaceutically suitable, but the present invention is not limited thereto. Also, the corresponding silver salts may be obtained by reacting an alkali metal or alkaline earth metal salt with a proper silver salt (e.g., silver nitrate).

[0087] Pharmaceutically acceptable salts of the compound of the present invention, unless otherwise indicated herein, include salts of acidic or basic groups, which may be present in the compound of Chemical Formula 1. For example, the pharmaceutically acceptable salts include sodium, calcium and potassium salts of hydroxy group, and other pharmaceutically acceptable salts of amino group, including hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate). The salts may be prepared using a salt preparation method known in the art.

[0088] Salts of the compounds of Chemical Formula 1 of the present invention are pharmaceutically acceptable salts, and can be used without particular limitation as long as they are salts of the compounds of Chemical Formula 1 which can exhibit pharmacological activities equivalent to those of the compound of Chemical Formula 1, for example, can prevent or treat neurodegenerative diseases by inducing autophagic degradation of intracellular neurodegenerative disease and tumor-associated proteins through a ligand of p62.

[0089] In addition, the compounds represented by Chemical Formula 1 according to the present invention include, but are not limited thereto, not only pharmaceutically acceptable salts thereof, but also all solvates or hydrates and all possible stereoisomers that can be prepared therefrom. All stereoisomers of the present compounds (e.g., those which may exist due to asymmetric carbons on various substituents), including enantiomeric forms and diastereomeric forms, are contemplated within the scope of this invention. Individual stereoisomers of the compounds of the present invention may, for example, be substantially free of other isomers (e.g., as a pure or substantially pure optical isomer having a specified activity), or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers. The chiral centers of the compounds of the present invention may have the S or R configuration as defined by the IUPAC 1974 Recommendations. The racemic forms can be analyzed by physical methods, such as fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography. The individual optical isomers can be obtained from the racemates by any suitable method, including without limitation, salt formation with an optically active acid followed by crystallization.

[0090] The solvate and stereoisomer of the compound represented by Chemical Formula 1 may be prepared from the compound represented by Chemical Formula 1 using methods known in the art.

[0091] Furthermore, the compound represented by Chemical Formula 1 according to the present invention may be prepared either in a crystalline form or in a non-crystalline form, When the compound is prepared in a crystalline form, it may be optionally hydrated or solvated. In the present invention, the compound of Chemical Formula 1 may not only include a stoichiometric hydrate, but also include a compound containing various amounts of water. The solvate of the compound of Chemical Formula 1 according to the present invention includes both stoichiometric solvates and non-stoichiometric solvates.

[0092] The compound of Chemical Formula 1 according to the present invention may be prepared by the same method as in Reaction Schemes 1 to 5, which are the following exemplary methods, and specific examples thereof are the same as those described in the following Examples.

##STR00002##

##STR00003##

##STR00004##

##STR00005##

##STR00006## ##STR00007##

[0093] In the preparation method of the present invention, the reactants used in Reaction Schemes may be commercially available compounds, or may be synthesized by performing one or more reactions known in the art as they are or by appropriately modifying the reactions. For example, in consideration of the presence, type and/or position of reactive functional groups and/or hetero elements contained in the skeletal structure, the reactants may be synthesized by performing one or more reactions in a series of order, but are not limited thereto.

[0094] The compound of Chemical Formula 1 according to the present invention is characterized by functioning as a ligand that binds to the ZZ domain of p62 and thus activating the function of p62. By activating the function of p62, the compound of Chemical Formula according to the present invention can activate autophagy.

[0095] Therefore, in another aspect, the present invention provides a pharmaceutical composition for autophagy activation comprising a compound of Chemical Formula 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof.

[0096] The compound of Chemical Formula 1 according to the present invention can eliminate aggregated proteins linked to misfolded protein aggregation-related diseases due to the activating action of autophagy. In addition, the compound of Chemical Formula 1 is a p62 ligand, which binds to the p62 ZZ domain and activates PB1 domain and LIR domain of p62 protein, so that it induces p62 oligomerization and aggregate formation and also increases autophagosome formation by inducing p62 aggregate formation. By the processes above, misfolded proteins can be efficiently eliminated (see FIG. 1). Such protein may be a major protein of proteinopathies, more preferably at least one selected from the group consisting of prion protein, amyloid precursor protein (APP), alpha-synuclein, superoxide dismutase 1, tau, immunoglobulin, amyloid-A, transthyretin, beta2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, islet amyloid polypeptide, ANF, gelsolin, insulin, lysozyme, fibrinogen, huntingtin, alpha-1-antitrypsin Z, crystallin, c9 open reading frame 72 (c9orf72), glial fibrillary acidic protein, cystic fibrosis transmembrane conductance regulator protein, rhodopsin, and ataxin, and other proteins with a poly-Q stretch.

[0097] Therefore, in still another aspect, the present invention provides a pharmaceutical composition for the prevention, amelioration or treatment of proteinopathies comprising a p62 ligand compound of Chemical Formula 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof.

[0098] The term “aggregation”, in accordance with the present invention, refers to the formation of oligomeric or multimeric complexes of typically one or more types of proteins, which may be accompanied by the integration of additional biomolecules, like carbohydrates, nucleic acids and lipids, into the complexes. Such aggregated proteins may form deposits in specific tissue, more preferably in nerve tissue or tissue of the brain. The extent of aggregation depends on the particular disease.

[0099] The term “proteinopathy” or “disease linked to protein aggregation” as used herein, refers to those diseases which are characterized by the presence of aggregated proteins, Examples thereof include, but are not limited to, neurodegenerative diseases, alpha-1 antitrypsin deficiency, keratopathy, retinitis pigmentosa, type 2 diabetes, cystic fibrosis, and the like.

[0100] The neurodegenerative diseases herein are preferably selected from the group consisting of Lyme borreliosis, fatal familial insomnia, Creutzfeldt-Jakob Disease (CJD), multiple sclerosis (MS), dementia, Alzheimer's disease, epilepsy, Parkinson's disease, stroke, Huntington's disease, Picks disease, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxias, other Poly-Q diseases, hereditary cerebral amyloid angiopathy, familial amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), injection-localized amyloidosis, beta-2 microglobulin amyloidosis, hereditary non-neuropathic amyloidosis, Alexander disease and Finnish hereditary systemic amyloidosis.

[0101] The dosage of the pharmaceutical composition of the present invention may vary with a broad range depending on weight, age, gender, or health condition a patient, diet, administration period, administration method, excretion and severity of disease. However, the effective dosage is generally about 1 ng to 10 mg/day and particularly about 1 ng to 1 mg/day for an adult (60 kg). As the dosage may vary depending on various conditions, it would be evident to a person skilled in the pertinent art that the dosage may be increased or decreased. Accordingly, the scope of the present invention is not limited by the aforementioned dosage in any way. As for the number of administration, the administration can be made either once or several divided times per day within a desired range, and the administration period is not particularly limited, either.

[0102] As used herein, the term “treatment” refers to all actions that alleviate or beneficially change the symptoms of various diseases linked to misfolded protein aggregation, such as proteinopathies by administering the pharmaceutical composition of the present invention.

[0103] As described above, the compound of the present invention exhibits the effects of (1) inducing p62 oligomerization and structural activation, (2) increasing p62-LC3 binding, and (3) increasing the delivery of p62 to autophagosomes, (4) activating autophagy, and finally (5) eliminating misfolded protein aggregates. Therefore, the pharmaceutical composition containing this compound as an active ingredient can be used for the prevention, amelioration or treatment of diseases linked to various misfolded protein aggregation.

[0104] For example, the composition of the present invention may further include pharmaceutically acceptable carriers, diluents or excipients. The composition can be used in the various forms such as oral dosage forms of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and injections of a sterile injectable solution, which are formulated by the conventional method according to the purpose of each of the intended use. The composition can be administered through various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal and topical administration. Examples of suitable carriers, excipients or diluents which can be included in this composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, and the like. In addition, the composition of the present invention may further include fillers, anti-coagulants, lubricants, humectants, fragrances, emulsifiers, preservatives, and the like.

[0105] A solid formulation for oral administration includes tablets, pills, powders, granules, capsules and the like, and such solid dosage forms are formulated by mixing the composition of the present invention with one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin and the like. Also, lubricants such as magnesium stearate and talc can be used in addition to simple excipients.

[0106] A liquid formulation for oral administration can be illustrated as suspensions, solutions, emulsions, syrups and the like, and can include various excipients such as humectants, sweeteners, fragrances, preservatives and the like, in addition to water and liquid paraffin which are commonly used diluents.

[0107] A formulation for parenteral administration includes sterilized aqueous solutions, non-aqueous solvents, suspension agents, emulsion agents, lyophilizing agents and suppository agents. Non-aqueous solvent and suspending agent may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a substrate for the suppository agent, Witepsol, Macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin or the like may be used. On the other hand, injections may include conventional additives such as solubilizing agents, isotonic agents, suspending agents, emulsifiers, stabilizers, or preservatives.

[0108] The formulation may be prepared according to conventional mixing, granulation or coating methods, and contains an active ingredient in an amount effective for medical treatment, specifically for the prevention, amelioration or treatment of diseases linked to misfolded protein aggregation.

[0109] In this case, the composition of the present invention is administrated in a pharmaceutically effective amount. The term “pharmaceutically effective amount” as used herein refers to an amount which is sufficient to treat the disease at a reasonable benefit/risk ratio applicable for any medical treatment, and also which is enough to not cause side effects. The level of effective amount can be determined depending on patient's health condition, disease type, severity of the disease, activity of the drug, sensitivity on the drug, administration method, administration time, administration route, excretion rate, treatment duration, combination, factors including other medicines used at the same time and other factors well-known in the medical field. The composition of the present invention may be administered as individual therapy or in combination with other therapies, and it can be administered simultaneously with or sequentially to conventional therapies, and once or multiple times. It is important to administer the minimum amount which can provide the maximum effect without the side effects in consideration of all the above factors, which can be easily determined by those skilled in the art.

[0110] For example, the dosage may be increased or decreased depending on administration route, the severity of a disease, gender, weight, age and the like, and the scope of the present invention is not limited by the aforementioned dosage in any way.

[0111] A preferred dose of the compound according to the present invention may be varied according to the condition and weight of a patient, the severity of a disease, the type of a drug, and the route and duration of administration, but it may be suitably selected by those skilled in the art.

[0112] In still another aspect, the present invention provides a method for preventing, ameliorating or treating diseases linked to the aggregation of misfolded proteins, comprising administering the pharmaceutical composition of the present invention to a subject in need thereof.

[0113] As used herein, the term “subject” refers to all animals comprising human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, or guinea pig, which have diseases linked to misfolded protein aggregation. The diseases linked to misfolded protein aggregation can be effectively prevented, ameliorated or treated by administrating the pharmaceutical composition of the present invention to the subject. In addition, since the pharmaceutical composition of the present invention functions as a p62 ligand to activate autophagy, eliminates aggregates of misfolded proteins due to the autophagy activation, and thus exhibits a prophylactic or therapeutic effect of diseases linked to these aggregated proteins, it can exhibit synergistic effects by administration in combination with existing therapeutic agents.

[0114] As used herein, the term “administration” means introduction of a prescribed amount of a substance into a patient in certain appropriate method, and the composition of the present invention can be administrated via any of the general routes as long as it can reach a target tissue. For example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration and intrarectal administration may be performed, but the present invention is not limited to these exemplified administration modes. Also, the pharmaceutical composition of the present invention can be administered using any device capable of delivering the active ingredients to target cells. Preferable administration mode and formulation are an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection, intravenous drip injection, or the like. Injectable formulations may be prepared using saline, aqueous solutions such as Ringer's solution, and non-aqueous solutions, such as vegetable oils, high fatty acid esters (e.g., ethyl oleic acid, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). The injectable preparations may include pharmaceutical carriers, including a stabilizer for preventing degeneration (e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), an emulsifier, a buffering agent for pH control, and a preservative for inhibiting microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzylalcohol, etc.).

[0115] In the other aspect, the present invention provides a food composition for the prevention or amelioration of misfolded protein-associated diseases comprising a p62 ligand compound of Chemical Formula 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof. The food composition is a health functional food and it can be used through formulation itself or be comprised in other health functional foods as an additive of health functional food. The health functional food means food having body modulating function such as prevention or amelioration of diseases, biodefense, immunity, recovery of convalescence, aging inhibition, etc., and it should be harmless to human body when taking in a long term. The mixing amount of active ingredients can be properly decided depending on purpose of use (prevention, health or therapeutic treatment).

[0116] The kind of the food is not particularly limited. Examples of food where the above substances can be added are meat, sausage, bread, chocolates, candies, snack, cookies, pizza, ramen, other noodles, gum, dairy products including ice cream, sorts of soup, beverages, tea, drinks, alcohol beverages and vitamin complex, etc., and it includes all the health functional foods in the common sense.

[0117] The food composition of the present invention can comprise common ingredients used in preparation of food or food additives, specifically, a flavoring agent; a natural sweetener such as monosaccharides like glucose, fructose, disaccharides like maltose, sucrose, and dextrin, cyclodextrin as a natural carbohydrate, or a synthetic sweetener such as saccharin, aspartame; a nutrient; vitamin; electrolyte; a coloring agent; an organic acid; a protective colloid viscosity agent; pH regulator; a stabilizer; a preservative; glycerin; alcohol; a carbonating agent which is used on carbonated drinks, etc.

[0118] In a specific embodiment of the present invention, the compounds of Examples 1 to 19, which are novel p62 ligands represented by Chemical Formula 1, were newly synthesized. In addition, in order to evaluate whether the novel p62 ligand compounds according to the present invention can increase the phenomenon of autophagy in cultured cells, HeLa cell lines, which are cell lines derived from cervical cancer patients, were treated with the novel p62 ligand compound according to the present invention and cultured, and then autophagy activity in cultured cells was confirmed by immunoblotting. As a result, it was confirmed that the level of LC3, which is a marker of autophagy activity, increased gradually according to the time of treatment with the p62 ligand compounds of the present invention, and that the p62 ligand compounds according to the present invention activated and oligomerized p62 proteins and delivered to autophagosome and simultaneously increased autophagy activity, thereby effectively eliminating misfolded protein aggregates.

EXAMPLE

[0119] Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are provided for illustrative purposes only, and the present invention is not intended to be limited by these Examples.

[0120] The compounds shown in Table 1 below were prepared by the methods according to the following Examples 1 to 19.

TABLE-US-00001 TABLE 1 Code Example No. Compound Name 1 YOK- (R)-1-(3,4-(bis(benzyloxy)phenoxy)-3-((2- 1106 hydroxyethyl)amino)propan-2-ol 2 YT- (R)-1-(3,4-(bis((4-chlorobenzyl)oxy)phenoxy)-3- 6-1 ((2-hydroxyethyl)amino)propan-2-ol 3 YT- (R)-1-(3,4-(bis((4-fluorobenzyl)oxy)phenoxy)-3- 6-2 ((2-hydroxyethyl)amino)propan-2-ol 4 YT- (R)-1-(3-((4-chlorobenzyl)oxy)phenoxy)-3-((2- 6-7 hydroxyethyl)amino)propan-2-ol 5 YT- (R)-1-(3-((4-fluorobenzyl)oxy)phenoxy)-3-((2- 6-8 hydroxyethyl)amino)propan-2-ol 6 YOK- (R)-1-(2-((3-(3,4-bis(benzyloxy)phenoxy)-2- 1109 hydroxypropyl)amino)ethyl)guanidine 7 YOK- (R)-1-(2-((3-(3,4-diphenethoxyphenoxy)-2- 2209 hydroxypropyl)amino)ethyl)guanidine 8 YT- (R)-1-(2-((3-(3,4-bis(4- 9-1 chlorobenzyl)oxy)phenoxy)-2- hydroxypropyl)amino)ethyl)guanidine 9 YT- (R)-1-(2-((3-(3,4-bis(4- 9-2 fluorobenzyl)oxy)phenoxy)-2- hydroxypropyl)amino)ethyl)guanidine 10 YOK- (R)-1-(3-(3,4-bis(benzyloxy)phenoxy)-2- 1107 hydroxypropyl)guanidine 11 YOK- (R)-1-(3-(3,4-diphenethoxyphenoxy)-2- 2207 hydroxypropyl)guanidine 12 YOK- (R)-1-(3,4-bis(benzyloxy)phenoxy)-3- 1104 (isopropylamino)propan-2-ol 13 YOK- (R)-1-(3,4-diphenethoxyphenoxy)-3- 2204 (isopropylamino)propan-2-ol 14 YOK- (R)-1-(3,4-bis(3-phenylpropoxy)phenoxy)-3- 3304 (isopropylamino)propan-2-ol 15 YOK- (R)-1-(3,4-bis(4-phenylbutoxy)phenoxy)-3- 4404 (isopropylamino)propan-2-ol 16 YOK- (R)-1-(4-(benzyloxy)-3-phenethoxyphenoxy)-3- 1204 (isopropylamino)propan-2-ol 17 YOK- (R)-1-(4-(benzyloxy)-3-(3- 1304 phenylpropoxy)phenoxy)-3- (isopropylamino)propan-2-ol 18 YT- (R)-1-(3,4-bis((4-chlorobenzyl)oxy)phenoxy)-3- 4-1 (isopropylamino)propan-2-ol 19 YT- (R)-1-(3,4-bis((4-fluorobenzyl)oxy)phenoxy)-3- 4-2 (isopropylamino)propan-2-ol

[0121] In the case of the starting materials for synthesizing the compounds of the present invention, various synthesis methods have been known, and if available on the market, the starting materials may be purchased from the providers. Examples of the reagents suppliers include Sigma-Aldrich, TCI, Wako, Kanto, Fluorchem, Acros, Alfa, Fluka, and the like, but are not limited thereto.

[0122] The compounds of the present invention can be prepared from readily available starting materials using the following general methods and procedures. As for typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvents, pressure) and the like, other process conditions may also be used unless stated otherwise. The optimal reaction state may vary depending on the specific reactants or solvent used. Such conditions can be determined by one skilled in the art by conventional optimization procedures.

[0123] Hereinafter, the preparation methods of Examples to 19 are described.

[0124] Preparation Example 1) Compounds of Examples 1, 2, 3, 12, 13, 14, 15, 18 and 19 were synthesized by the method shown in Reaction Scheme 1 below.

##STR00008##

Example 1: Preparation of (R)-1-(3,4-(bis(benzyloxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YOK-1106)

Step 1) Preparation of 3,4-bis(benzyloxy)phenol (1102)

[0125] After dichloromethane (15 ml) was added to and dissolved in 3,4-bis(benzyloxy)benzaldehyde (1101, 1.00 g, 3.0 mmol, 1 eq.), mCPBA (0.78 g, 4.5 mmol, 1.5 eq.) was added to the reaction and stirred at room temperature for 4 hours. The reaction mixture was diluted with ethyl acetate, and washed with a saturated aqueous sodium carbonate solution, and the organic layer was separated. The organic layer was washed with an aqueous sodium chloride solution, then dehydrated with anhydrous sodium sulfate and filtered under reduced pressure. The filtered solution was concentrated and then dissolved again in methanol (10 ml), and 6N NaOH was added and stirred at room temperature for 30 minutes. 4N HCl solution was added to the reaction, and further stirred for 30 minutes. The reaction mixture was diluted with ethyl acetate (50 ml), washed with brine, and then dehydrated with anhydrous sodium sulfate, and filtrated under reduced pressure. The filtered solution was concentrated and then purified by column chromatography (hexane/ethyl acetate ratio=7/3) to give 3,4-bis(benzyloxy)phenol (1102, 0.87 g, yield: 90%).

[0126] .sup.1H-NMR (CDCl.sub.3, 300 MHz): δ 7.25-7.42 (m, 10H), 6.80 (d, 1H, J=9.0 Hz), 6.48 (d, 1H, J=3.0 Hz), 6.29 (dd, 1H, J=3.0 and 9.0 Hz), 5.08 (d, 4H, J=Hz), 4.55 (s, 1H); ESIMS m/z: 307.25 [M+H].sup.+.

Step 2) Preparation of R-2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane (1103)

[0127] 3,4-Dibenzyloxyphenol (1102, 306 mg, 1.0 mmol) was diluted with ethanol (10 ml), and then KOH aqueous solution (KOH 66 mg, 1.2 mmol, 1 ml) and (R)-2-(chloromethyl)oxilrane (410 ul, 5.0 mmol) were added sequentially. The reaction mixture was stirred at room temperature or 5 hours, and then the organic solvent was removed under reduced pressure. The concentrated reaction mixture was diluted again with ethyl acetate, washed with water and then washed with brine. The extracted organic layer was dehydrated with anhydrous sodium sulfate and then filtered under reduced pressure. The filtered organic layer was concentrated and purified by column chromatography to give pure R-2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane (1103, 297 mg, yield: 82%). ESIMS m/z: 363.5 [M+H].sup.+.

[0128] Step 3) R-2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane (1103, 9 mg, 25 nmol) was diluted with anhydrous ethanol (1 ml), and then 2-aminoethan-1-ol (7.6 μL, 125 nmol) was added thereto and stirred at room temperature for 4 hours. After confirming the reaction by TLC, the reaction solvent was concentrated under reduced pressure, water was added to the concentrated reaction product, and extracted with dichloromethane (3×5 mL). The extracted organic layer was dehydrated with anhydrous sodium sulfate and then filtered under reduced pressure. The filtered organic layer was concentrated and purified by column chromatography (dichloromethane:methanol=19:1) to give (R)-1-(3,4-(bis(benzyloxy) phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YOK-1106, 9.0 mg, yield: 85%). H NMR (CDCl.sub.3, 300 MHz): δ 7.42-7.40 (m, 2H), 7.33-7.27 (m, 8H), 6.82 (d, 1H, J=9.0 Hz), 6.42 (d, 1H, J=3.0 Hz), 6.28 (dd, 1H, J=3.0 and 9.0 Hz), 5.09 (d, 1H, J=12.9 Hz), 5.06 (s, 2H), 4.93 (d, 1H, J=12.9 Hz), 4.35-4.25 (m, 1H), 3.94 (dd, 1H, J=3.6 and 9.9 Hz), 3.89-3.72 (m, 3H), 3.34 (t, 1H, J=11.4 Hz), 2.93 (brS, 2H), 2.80 (d, 1H, J=10.8 Hz); ESIMS m/z: 424.67[M+H].sup.+.

Example 2: Preparation of (R)-1-(3,4-(bis((4-chlorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-1)

[0129] (R)-1-(3,4-(Bis((4-chlorobenzyl)oxy) phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-1) was synthesized in the same manner as in the preparation method of Example 19 by using 3,4-bis((4-chlorobenzyl)oxy)benzaldehyde (1101-1) instead of 3,4-bis(benzyloxy)benzaldehyde (1101) as a starting material. H NMR (400 MHz, DMSO-d.sub.6) δ (ppm) 2.59 (m, 4H), 3.45 (s, 2H), 3.81 (m, 3H), 4.50 (b, 1H), 4.85 (b, 1H), 5.01 (s, 2H), 5.11 (s, 2H), 6.42 (dd, J=8 Hz and 2.8 Hz, 1H), 6.66 (d, J=2.8 Hz, 1H), 6.92 (d, J=8 Hz, 1H), 7.42 (s, 4H), 7.45 (s, 4H); ESI-MS Calcd m/z for C.sub.25H.sub.27Cl.sub.2NO.sub.5 [M+2].sup.+493.90 Found 492.39.

Example 3: Preparation of (R)-1-(3,4-(bis((4-fluorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-2)

[0130] (R)-1-(3,4-(Bis((4-fluorobenzyl)oxy) phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-2) was synthesized in the same manner as in as the preparation method of Example 19 by using 3,4-bis((4-fluorobenzyl)oxy)benzaldehyde (1101-2) instead of 3,4-bis(benzyloxy)benzaldehyde (1101) as a staring material. .sup.1H NMR (400 MHz, CDCl.sub.3) δ (ppm) 2.82 (m, 4H), 3.69 (t, J=5.2 Hz, 2H), 3.91 (m, 2H), 4.05 (m, 1H), 5.00 (s, 2H), 5.06 (s, 2H), 6.41 (m, 1H), 6.58 (d, J=2.8 Hz, 1H), 6.84 (d, J=8 Hz, 1H), 7.03 (m, 4H), 7.37 (m, 4H); ESI-MS Calcd m/z for C.sub.25H.sub.27F.sub.2NO.sub.5 [M+2].sup.+461.0 Found 459.49.

Example 12: Preparation of (R)-1-(3,4-bis(benzyloxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-1104)

[0131] (R)-1-(3,4-Bis(benzyloxy) phenoxy)-3-(isopropylamino)propan-2-ol (YOK-1104) was synthesized by using isopropyl amine instead of 2-aminoethan-1-ol in the preparation step 3 of Example 1. .sup.1H NMR (300 MHz, CDCl.sub.3) δ (ppm) 1.12 (d, J=6 Hz, 6H), 2.81 (m, 5H), 3.88 (d, J=3 Hz, 2H), 4.04 (m, 1H), 5.09 (d, J=15 Hz), 6.38 (dd, J=9 Hz and 3 Hz, 1H), 6.59 (s, 1H), 6.85 (d, J=9 Hz, 1H), 7.35 (m, 10H; ESI-MS Calcd m/z for C.sub.26H.sub.31NO.sub.4 [M+H].sup.+ 422.42 Found 421.54.

Example 13: Preparation of (R)-1-(3,4-diphenethoxyphenoxy)-3-(isopropylamino)propan-2-ol (YOK-2204)

[0132] (R)-1-(3,4-diphenethoxyphenoxy)-3-(isopropylamino)propan-2-ol (YOK-2204) was synthesized in the same manner as in the preparation method of Example 1, except that in step 1 of the preparation method of Example 1, 3,4-diphenethoxybenzaldehyde (2201) was used as a starting material instead of 3,4-bis(benzyloxy)benzaldehyde (1101), and in step 3, isopropylamine was used instead of 2-aminoethan-1-ol. .sup.1H NMR (300 MHz, CDCl.sub.3) δ (ppm) 1.17 (d, J=6 Hz, 6H), 2.79 (dd, J=9 Hz and 3 Hz, 1H), 2.95 (m, 2H), 3.08 (m, 4H), 3.34 (b, 2H), 3.89 (m, 2H), 4.13 (m, 5H), 6.37 (dd, J=6 Hz and 3 Hz, 1H), 6.52 (d, J=3 Hz, 1H), 6.78 (d, J=9 Hz, 1H), 7.25 (m, 10H); ESI-MS Calcd m/z for C.sub.28H.sub.35NO.sub.4 [M+H].sup.+ 451.00 Found 449.59.

Example 14: Preparation of (R)-1-(3,4-bis(3-phenylpropoxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-3304)

[0133] (R)-1-(3,4-bis(3-phenylpropoxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-3304) was synthesized in the same manner as in the preparation method of Example 1, except that in step 1 of the preparation method of Example 1, 3,4-bis(3-phenylpropoxy)benzaldehyde (3301) was used as a starting material instead of 3,4-bis(benzyloxy)benzaldehyde (1101), and step 3, isopropylamine was used instead of 2-aminoethan-1-ol. .sup.1H NMR (300 MHz, CDCl.sub.3) δ (ppm) 1.21 (d, J=6 Hz, 6H), 2.12 (m, 4H), 2.83 (t, J=9 Hz, 5H), 3.01 (m, 2H), 3.30 (b, 3H), 3.93 (m, 6H), 4.18 (m, 1H), 6.38 (dd, J=6 Hz and 3 Hz, 1H), 6.52 (d, J=3 Hz, 1H), 6.80 (d, J=9 Hz, 1H), 7.25 (m, 10H); ESI-MS Calcd m/z for C.sub.30H.sub.39NO.sub.4 [M+H].sup.+ 479.09 Found 477.65.

Example 15: Preparation of (R)-1-(3,4-bis(4-phenylbutoxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-4404)

[0134] (R)-1-(3,4-bis(4-phenylbutoxy) phenoxy)-3-(isopropylamino)propan-2-ol (YOK-4404) was synthesized in the same manner as in the preparation method of Example 1, except that in step 1 of the preparation method of Example 1, 3,4-bis(4-phenylbutoxy)benzaldehyde (4401) was used as a starting material instead of 3,4-bis(benzyloxy)benzaldehyde (1101), and in step 3, isopropylamine was used instead of 2-aminoethan-1-ol. .sup.1H NMR (300 MHz, CDCl.sub.3) δ (ppm) 1.26 (dd, J=6 Hz and 3 Hz, 8H), 1.81 (m, 7H), 2.66 (t, J=6 Hz, 4H), 2.85 (m, 1H), 3.06 (m, 2H), 3.46 (b, 2H), 3.93 (m, 6H), 4.24 (m, 1H), 6.36 (dd, J=6 Hz and 3 Hz, 1H), 6.52 (d, J=3 Hz, 1H), 6.78 (d, J=9 Hz, 1H), 7.23 (m, 10H), ESI-MS Calcd m/z for C.sub.32H.sub.43NO.sub.4 [M+H].sup.+ 505.70 Found 503.45.

Example 18: Preparation of (R)-1-(3,4-bis((4-chlorobenzyl)oxy)phenoxy)-3-(isopropylamino)propan-2-ol (YT-4-1)

[0135] (R)-1-(3,4-bis((4-chlorobenzyl)oxy)phenoxy)-3-(isopropylamino)propan-2-ol (YT-4-1) was synthesized in the same manner as in the preparation method of Example 1, except that in step 1 of the preparation method of Example 1, 3,4-bis(4-chlorobenzyloxy)benzaldehyde (1101-1) was used as a starting material instead of 3,4-bis(benzyloxy)benzaldehyde (1101), and in step 3, isopropyl amine was used instead of 2-aminoethan-1-ol. .sup.1H NMR (400 MHz, CDCl.sub.3) δ (ppm) 1.08 (d, J=6.4 Hz, 6H), 2.68 (dd, J=12 Hz and 8 Hz, 1H), 2.83 (m, 2H), 3.88 (d, J=5.2 Hz, 2H), 3.95 (m, 1H), 5.01 (s, 2H), 5.06 (s, 2H), 6.40 (dd, J=8 Hz and 4 Hz, 1H), 6.56 (d, J=2.8 Hz, 1H), 6.82 (d, J=8 Hz, 1H), 7.31 (m, 8H); ESI-MS Calcd m/z for C.sub.26H.sub.29Cl.sub.2NO.sub.4 [M+H].sup.+ 491.40 Found 490.42.

Example 19: Preparation of (R)-1-(3,4-bis((4-fluorobenzyl)oxy)phenoxy)-3-(isopropylamino)propan-2-ol (YT-4-2)

[0136] (R)-1-(3,4-bis((4-fluorobenzyl)oxy)phenoxy)-3-(isopropylamino)propan-2-ol (YT-4-2) was synthesized in the same manner as in the preparation method of Example 1, except that in step 1 of the preparation method of Example 1, 3,4-bis(4-fluorobenzyloxy)benzaldehyde (1101-2) was used as a starting material instead of 3,4-bis(benzyloxy)benzaldehyde (1101), and in step 3, isopropyl amine was used instead of 2-aminoethan-1-ol. .sup.1H NMR (400 MHz, CDCl.sub.3) δ (ppm) 1.08 (d, J=6.4 Hz, 6H), 2.68 (dd, J=12 Hz and 8 Hz, 1H), 2.83 (m, 2H), 3.89 (d, J=4 Hz, 2H), 3.95 (m, 1H), 5.00 (s, 2H), 5.05 (s, 2H), 6.40 (dd, J=8 Hz and 2.8 Hz, 1H), 6.59 (d, J=2.8 Hz, 1H), 6.83 (d, J=9.6 Hz, 1H), 7.03 (m, 4H), 7.37 (m, 4H); ESI-MS Calcd m/z for C.sub.26H.sub.29F.sub.2NO.sub.4 [M+H].sup.+ 457.90 Found 457.52.

[0137] Preparation Example 2) The compounds of Examples 6 to 11 were synthesized according to the process shown in Reaction Scheme 2 below.

##STR00009##

Example 6: Preparation of (R)-1-(2-((3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YOK-1109)

[0138] Step 1) After R-2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane (1103, 500 mg, 1.38 mmol) was dissolved in methanol (10 ml), tert-butyl 2-aminoethylcarbamate (442 mg, 2.76 mmol) was added thereto and stirred at 50° C. for 10 hours. When the reaction was completed, the reaction solvent is concentrated under reduced pressure.

[0139] Step 2) After tert-butyl (R)-(2-((3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)amino)ethyl)carbamate (1105, 600 mg, 1.15 mmol) was dissolved in methanol (3 ml), methanol/hydrochloric acid (3N, 2 ml) was added thereto, and then stirred at room temperature for 4 hours. When the reaction was completed, the reaction mixture was concentrated under reduced pressure (white solid, 1106) and used as a starting material in the next step without purification.

[0140] Step 3) (R)-1-((2-aminoethyl)amino)-3-(3,4-bis(benzyloxy)phenoxy)propan-2-ol (1106, 400 g, 0.95 mmol) was dissolved in DMF (3 ml), and then 1H-pyrazole-1-carboxamidine hydrochloride (553 mg, 3.8 mmol) and diisopropylethylamine (0.3 g, 2.4 mmol) were further added thereto. The reaction mixture was stirred at 30° C. for 12 hours, concentrated under reduced pressure and purified by high resolution liquid chromatography to synthesize (R)-1-(2-((3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YOK-1109, 22 mg) as a white solid. .sup.1H NMR (400 MHz, DMSO+D.sub.2O) δ (ppm) 3.13 (m, 4H), 3.47 (s, 2H), 3.89 (s, 2H), 5.04 (s, 2H), 5.12 (s, 2H), 6.46 (d, J=8 Hz, 1H), 6.67 (s, 1H), 6.97 (d, J=8 Hz, 1H), 7.39 (m, 10H); ESI-MS Calcd m/z for C.sub.26H.sub.32N.sub.4O.sub.4 [M+H].sup.+ 465.00 Found 464.57.

Example 7: Preparation of (R)-1-(2-((3-(3,4-diphenethoxyphenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YOK-2209)

[0141] (R)-1-(2-((3-(3,4-diphenethoxyphenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YOK-2209) was synthesized in the same manner as in the preparation method of Example 6, except that R-2-((3,4-diphenethoxyphenoxy)methyl)oxirane (2203) was used instead of R-2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane (1103) in step 1 of the preparation method of Example 6. .sup.1H NMR (400 MHz, CD.sub.3OD) δ (ppm) 3.00 (t, J=6.8 Hz, 2H), 3.06 (t, J=6.8 Hz, 2H), 3.23 (m, 1H), 3.28 (m, 3H), 3.62 (t, J=6.4 Hz, 2H), 6.46 (dd, J=8 Hz and 2.8 Hz, 1H), 6.60 (d, J=2.8 Hz, 1H), 6.85 (d, J=8 Hz, 1H), 7.25 (m, 10H); ESI-MS Calcd m/z for C.sub.28H.sub.36N.sub.4O.sub.4 [M+H].sup.+ 493.00 Found 492.62.

Example 8: Preparation of (R)-1-(2-((3-(3,4-bis(4-chlorobenzyl)oxy)phenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YT-9-1)

[0142] (R)-1-(2-((3-(3,4-bis(4-chlorobenzyl)oxy)phenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YT-9-1) was synthesized in the same manner as in the preparation method of Example 6, except that R-2-((3,4-bis(4-chlorobenzyl)oxy)phenoxy)methyl)oxirane (1103-1) was used instead of 1103 in step 1 of the preparation method of Example 6. .sup.1H NMR (400 MHz, DMSO+D.sub.2O) δ (ppm) 3.13 (m, 4H), 3.47 (t, J=6 Hz, 2H), 3.90 (m, 2H), 4.13 (m, 1H), 5.03 (s, 2H), 5.10 (s, 2H), 6.47 (dd, J=8 Hz and 2 Hz, 1H), 6.66 (d, J=2.4 Hz, 1H), 6.97 (d, J=8.8 Hz, 1H), 7.42 (s, 4H), 7.45 (s, 4H); ESI-MS Calcd m/z for C.sub.26H.sub.30Cl.sub.2N.sub.4O.sub.4 [M+H].sup.+ 532.90 Found 533.45.

Example 9: Preparation of (R)-1-(2-((3-(3,4-bis(4-fluorobenzyl)oxy)phenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YT-9-2)

[0143] (R)-1-(2-((3-(3,4-bis(4-fluorobenzyl)oxy)phenoxy)-2-hydroxypropyl)amino)ethyl)guanidine (YT-9-2) was synthesized in the same manner as in the preparation method of Example 6, except that R-2-((3,4-bis(4-fluorobenzyl)oxy)phenoxy)methyl)oxirane (1103-2) was used instead of 1103 in step 1 of the preparation method of Example 6. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ (ppm) 3.13 (m, 4H), 3.45 (t, J=6 Hz, 2H), 3.89 (m, 2H), 4.15 (m, 1H), 5.01 (s, 2H), 5.09 (s, 2H), 6.46 (dd, J=8 Hz and 2.8 Hz, 1H), 6.68 (d, J=2.8 Hz, 1H), 6.96 (d, J=9.2 Hz, 1H), 7.20 (m, 4H), 7.46 (m, 4H); ESI-MS Calcd m/z for C.sub.26H.sub.30Cl.sub.2N.sub.4O.sub.4 [M+H].sup.+ 501.00 Found 500.55.

Example 10: Preparation of (R)-1-(3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)guanidine (YOK-1107)

[0144] Step 1) After R-2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane (1103, 500 mg, 1.38 mmol) was dissolved in methanol (10 ml), ammonia/methanol (2N, 3 ml) was added thereto and stirred at 50° C. for 10 hours. When the reaction was completed, the reaction solvent was concentrated under reduced pressure.

[0145] Step 2) (R)-1-amino-3-(3,4-bis(benzyloxy)phenoxy)propan-2-ol (1107, 300 mg, 0.8 mmol) was dissolved in DMF (3 ml), and then 1H-pyrazole-1-carboxamidine hydrochloride (570 mg, 3.9 mmol) and diisopropylethylamine (0.3 g, 2.4 mmol) were added sequentially. The reaction mixture was stirred at 30° C. for 12 hours, concentrated under reduced pressure and purified by high resolution liquid chromatography to synthesize (R)-1-(3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)guanidine (YOK-1107, 23 mg). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ (ppm) 3.20 (m, 1H), 3.31 (m, 1H), 5.03 (s, 2H), 5.12 (s, 2H), 6.44 (dd, J=8.8 Hz and 2.8 Hz, 1H), 6.68 (d, J=2.8 Hz, 1H), 6.95 (d, J=8 Hz, 1H), 7.38 (m, 10H); ESI-MS Calcd m/z for C.sub.24H.sub.27N.sub.3O.sub.4 [M+H].sup.+ 422.00 Found 421.50.

Example 11: Preparation of (R)-1-(3-(3,4-diphenethoxyphenoxy)-2-hydroxypropyl)guanidine (YOK-2207)

[0146] (R)-1-(3-(3,4-diphenethoxyphenoxy)-2-hydroxypropyl)guanidine (YOK-2207, 25 mg) was synthesized in the same manner as in the preparation method of Example 10, except that R-2-((3,4-diphenoxyphenoxy)methyl)oxirane (2203) was used instead of 1103 in step 1 of the preparation method of Example 10. .sup.1H NMR (400 MHz, CD.sub.3OD) δ (ppm) 3.00 (t, J=8 Hz, 2H), 3.06 (t, J=8 Hz, 2H), 3.33 (m, 1H), 3.42 (dd, J=24 Hz and 4 Hz, 1H), 3.91 (m, 2H), 4.08 (m, 3H), 4.16 (t, J=6.4 Hz, 2H), 6.46 (dd, J=8 Hz and 2 Hz, 1H), 6.60 (d, J=2.8 Hz, 1H), 6.84 (d, J=8 Hz, 1H), 7.25 (m, 10H); ESI-MS Calcd m/z for C.sub.26H.sub.31N.sub.3O.sub.4 [M+H].sup.+ 450.00 Found 449.55.

[0147] Preparation Example 3) Compounds of Examples 4 and 5 were synthesized by the method shown in Reaction Scheme 3 below.

##STR00010##

Example 4: Preparation of (R)-1-(3-((4-chlorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-7)

[0148] Step 1) 1,3-Dihydroxybenzene (5.0 g, 45.4 mmol) was dissolved in acetonitrile (100 ml) and then K.sub.2CO.sub.3 was added thereto. Then, 1-(bromomethyl)-4-chlorobenzene (9.3 g, 45.4 mmol) was added to the reaction and then stirred at 80° C. for 16 hours. After cooling to room temperature, the reaction solvent was concentrated under reduced pressure. Water was added to the concentrated residue, followed by extraction with ethyl acetate. The organic layer was dehydrated with anhydrous sodium sulfate and filtered under reduced pressure. The filtrate was taken, concentrated under reduced pressure, and then purified by column chromatography (ethyl acetate/petroleum ether 1:15 to 1:8) to synthesize 3-((4-chlorobenzyl)oxy)phenol (2011, 2.3 g) as a light yellow oil. 1H NMR (400 MHz, CD.sub.3OD) δ (ppm) 5.03 (s, 2H), 6.35-6.43 (m, 3H), 7.04 (t, 1H), 7.45 (s, 4H), 9.41 (s, 1H).

[0149] Step 2) After 3-((4-chlorobenzyl)oxy)phenol (2011, 1.0 g, 4.2 mmol) was dissolved in ethanol (10 ml), (R)-2-(chloromethyl)oxirane (1.17 g, 12.7 mmol), purified water (1 ml), and KOH (0.24 g, 4.2 mmol) were added, and then stirred at room temperature for hours. Water (30 ml) was added to the reaction and extracted with ethyl acetate (30 ml). The aqueous layer was extracted twice more with ethyl acetate, and then the organic layer was dehydrated with anhydrous sodium sulfate and filtered under reduced pressure. The filtered solution was concentrated under reduced pressure and purified by column chromatography (ethyl acetate/petroleum ether 1:15 to 1:8) to synthesize (R)-2-((3-((4-chlorobenzyl)oxy)phenoxy)methyl)oxirane (3011, 0.85 g) as a colorless oil. .sup.1H NMR (400 MHz, CD.sub.3OD) δ (ppm) 2.74 (m, 1H), 2.90 (m, 1H), 3.34 (m, 1H), 3.91-3.95 (m, 1H), 4.18-4.22 (m, 1H), 5.01 (s, 2H), 6.52-6.59 (m, 3H), 7.16-7.26 (m, 1H), 7.35 (s, 4H).

[0150] Step 3) (R)-2-((3-((4-chlorobenzyl)oxy)phenoxy)methyl)oxirane (3011, 300 mg, 1.03 mmol) was dissolved in methanol (5 ml), and then 2-aminoethanol (189 mg, 3.09 mmol) was added thereto. The reaction mixture was stirred at 65° C. for 5 hours. After cooling to room temperature, the reaction solvent was concentrated under reduced pressure and purified by high resolution liquid chromatography to synthesize (R)-1-(3-((4-chlorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-7, 15 mg) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ (ppm) 2.83 (m, 4H), 3.69 (m, 2H), 2.96 (m, 2H), 4.08 (m, 1H), 5.00 (s, 2H), 6.55 (m, 3H), 7.18 (t, J=8 Hz, 1H), 7.35 (s, 4H); ESI-MS Calcd m/z for C.sub.18H.sub.22ClNO.sub.4 [M].sup.+352.00 Found 351.83.

Example 5: Preparation of (R)-1-(3-((4-fluorobenzyl)oxy)phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-8)

[0151] (R)-1-(3-((4-fluorobenzyl)oxy) phenoxy)-3-((2-hydroxyethyl)amino)propan-2-ol (YT-6-8, 20 mg) was synthesized in the same manner as in the preparation method of Example 4, except that 1-(bromomethyl)-4-fluorobenzene was used instead of 1-(bromomethyl)-4-chlorobenzene in step 1 of the preparation method of Example 4. .sup.1H NMR (400 MHz, CDCl.sub.3) δ (ppm) 2.03 (b, 3H), 2.83 (m, 4H), 3.69 (t, J=4.8 Hz, 2H), 3.96 (m, 2H), 4.08 (m, 1H), 4.99 (s, 2H), 6.56 (m, 3H), 7.07 (m, 2H), 7.18 (t, J=8 Hz, 1H), 7.40 (m, 2H); ESI-MS Calcd m/z for C.sub.18H.sub.22FNO.sub.4 [M+H].sup.+ 336.00 Found 335.38.

[0152] Preparation Example 4) The compounds of Examples and 17 were synthesized by the method shown Reaction Scheme 4 below.

##STR00011##

Example 16: Preparation of (R)-1-(4-(benzyloxy)-3-phenethoxyphenoxy)-3-(isopropylamino)propan-2-ol (YOK-1204)

[0153] Step 1) After 4-(benzyloxy)-3-phenethoxybenzaldehyde (1201, 332 mg, 1.0 mmol) was dis solved in dichloromethane, m-chloroperbenzoic acid (mCPBA, 260 mg, 1.5 mmol) is added thereto and then stirred at room temperature for 4 hours. An excess of ethyl acetate was added to the reaction, and then washed with saturated aqueous sodium carbonate solution, and the organic layer was separated. The organic layer was washed with an aqueous sodium chloride solution, then dehydrated with anhydrous sodium sulfate and filtered under reduced pressure.

[0154] The filtered solution was concentrated and dissolved again in methanol (10 ml), then 6N NaOH was added and stirred at room temperature for 30 minutes. 4N HCl solution was added to the reaction and then further stirred for 30 minutes. The reaction mixture was diluted with ethyl acetate (50 ml), then washed with brine, dehydrated with anhydrous sodium sulfate, and filtrated under reduced pressure. The filtered solution was concentrated and then purified by column chromatography (hexane/ethyl acetate ratio=7/3) to give 4-(benzyloxy)-3-phenoxyphenol (1202, 310 mg, yield: 90%). 1H NMR (CDCl3, 300 MHz) δ (ppm): 7.23-7.42 (m, 10H), 6.77 (d, 1H, J=9.0 Hz), 6.46 (d, 1H), 6.28 (dd, 1H, J=3.0 and 9.0 Hz), 4.98 (s, 2H), 4.58 (s, 1H), 4.20 (t, 2H, J=6 Hz), 3.14 (t, 2H, J=6 Hz).

[0155] Step 2) 4-(Benzyloxy)-3-phenethoxyphenol (1202, 320 mg, 1.0 mmol) was diluted with ethanol (5 ml), and then KOH aqueous solution (KOH 66 mg, 1.2 mmol, 1 ml) and (R)-2-(chloromethyl)oxirane (410 ul, 5.0 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 5 hours, and then the organic solvent was removed under reduced pressure. The concentrated reaction mixture was diluted again with ethyl acetate, washed with water and then with brine. The extracted organic layer was dehydrated with anhydrous sodium sulfate and then filtered under reduced pressure. The filtered organic layer was concentrated and purified by column chromatography to give pure R-2-((4-(benzyloxy)-3-phenethoxyphenoxy)methyl)oxirane (1203, 308 mg, yield: 82%). .sup.1H NMR (300 MHz, CDCl.sub.3) δ (ppm) 7.44-7.47 (m, 2H), 7.26-7.39 (m, 5H), 7.19-7.22 (m, 3H), 6.84 (d, 1H, J=6.0 Hz), 6.54 (d, 1H, J=3.0 Hz), 6.36 (dd, 1H), 5.07 (s, 2H), 4.15 (dd, 1H), 4.00 (t, 2H, J=6.0 Hz), 2.84 (t, 2H, J=9.0 Hz), 2.73 (dd, 1H), 2.10-2.19 (m, 2H); ESIMS m/z: 363.5 [M+H].sup.+.

[0156] Step 3) After R-2-((4-(benzyloxy)-3-phenethoxyphenoxy)methyl)oxirane (1203, 9.4 mg, 25 nmol) was diluted with anhydrous ethanol (1 ml), isopropylamine (10 μL, 125 nmol) was added thereto and stirred at room temperature for 4 hours. After confirming the reaction by TLC, the reaction solvent was concentrated under reduced pressure, and water was added to the concentrated reaction product and extracted with dichloromethane (3×5 mL). The extracted organic layer was dehydrated with anhydrous sodium sulfate and then filtered under reduced pressure. The filtered organic layer was concentrated and purified by column chromatography (dichloromethane:methanol=19:1) to give (R)-1-(4-(benzyloxy)-3-phenethoxyphenoxy)-3-(isopropylamino)propan-2-ol (YOK-1204, 9.3 mg, yield: 86%). .sup.1H NMR (300 MHz, CDCl.sub.3) δ (ppm) 1.15 (d, J=6 Hz, 6H), 2.71 (m, 4H), 2.80 (m, 2H), 3.14 (t, J=6 Hz, 2H), 3.91 (d, J=3 Hz, 2H), 4.09 (m, 1H), 4.21 (t, J=6 Hz, 2H), 4.99 (s, 2H), 6.35 (dd, J=6 Hz and 3 Hz, 1H), 6.54 (d, J=3 Hz, 1H), 6.82 (d, J=9 Hz, 1H), 7.26 (m, 10H); ESI-MS Calcd m/z for C.sub.27H.sub.33NO.sub.4 [M+H].sup.+ 437.00 Found 435.56.

Example 17: Preparation of (R)-1-(4-(benzyloxy)-3-(3-phenylpropoxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-1304)

[0157] (R)-1-(4-(benzyloxy)-3-(3-phenylpropoxy)phenoxy)-3-(isopropylamino)propan-2-ol (YOK-1304, 9.5 mg, yield: 85%) as a while solid was synthesized in the same manner as in the preparation method of Example 16, by using 4-(benzyloxy)-3-(3-phenylpropoxy)benzaldehyde (1301, 346 mg, 1.0 mmol) instead of 4-(benzyloxy)-3-phenethoxybenzaldehyde (1201) as a starting material in step 1 of the preparation method of Example 16. 1H NMR (300 MHz, CDCl.sub.3) δ (ppm) 1.13 (d, J=6 Hz, 6H), 2.15 (m, 2H), 2.33 (b, 3H), 2.80 (m, 5H), 3.91 (d, J=6 Hz, 2H), 4.01 (m, 3H), 5.07 (s, 2H), 6.36 (dd, J=6 Hz and 3 Hz, 1H), 6.53 (d, J=3 Hz, 1H), 6.84 (d, J=9 Hz, 1H), 7.33 (m, 10H); ESI-MS Calcd m/z for C.sub.28H.sub.35NO.sub.4 [M+H].sup.+ 451.00 Found 449.59.

[0158] Preparation Example 5) The compounds of Comparative Examples 1 and 2 were synthesized by the method shown in Reaction Scheme 5 below.

##STR00012## ##STR00013##

Comparative Example 1: Preparation of methyl (R)-(3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)glycineate (YOK-G-1104)

[0159] Methyl (R)-(3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)glycineate (YOK-G-1104) was synthesized in the same manner as in the preparation method of Example 19, except that glycine methyl ester was used instead of 2-aminoethan-1-ol in step 3 of the preparation method of Example 19. .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ (ppm) 2.55 (m, 1H), 2.81 (m, 1H), 3.51 (s, 2H), 3.66 (s, 3H), 4.05 (m, 1H), 4.20 (m, 2H), 5.16 (s, 2H), 5.18 (s, 2H), 5.37 (br s, 1H), 5.52 (br s, 1H), 6.57 (s, 1H), 6.67 (d, 1H), 6.96 (d, 1H), 7.32-7.48 (m, 10H).

Comparative Example 2: Preparation of ethyl (R)-(3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)alanineate (YOK-A-1104)

[0160] Ethyl (R)-(3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)alanineate (YOK-A-1104) was synthesized in the same manner as in the preparation method of Example 19, except that alanine ethyl ester was used instead of 2-aminoethan-1-ol in step 3 of the preparation method of Example 19. .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ (ppm) 1.21 (t, 3H), 1.27 (m, 3H), 2.56-2.81 (m, 2H), 3.56 (m, 1H), 4.05 (m, 1H), 3.95-4.20 (m, 4H), 5.16 (s, 2H), 5.18 (s, 2H), 5.37 (br s, 1H), 6.57 (s, 1H), 6.67 (d, 1H), 6.96 (d, 1H), 7.32-7.48 (m, 10H).

Experimental Example 1. Evaluation of Oligomerization Activity of p62 Protein in Cultured Cells by Immunoblotting

[0161] In order to evaluate the p62 protein oligomerization activity efficacy by the compounds (Examples 1-19), HEK293 cell line, which is human embryonic kidney-derived cell, was collected. As representative compounds among the present compounds, YOK-1104, YOK-2204, YOK-3304, YOK-1204, YOK-4404, YOK-1107, YOK-1109, YOK-2207, YOK-2209, YT-4-1, YT-4-2, YT-6-1, YT-6-2, YT-6-7, YT-6-8, YT-9-1, YT-9-2 were selected. In order to measure intracellular p62 protein activation and oligomerization according to the treatment with these selected representative compounds, the respective cells were dispensed into a pi dish. The cells were collected after further culturing for 24 hours so that the cells were completely attached to the surface of the plate. 100 ul of lysis buffer (20 mM Tris, pH 7.4), 150 mM NaCl, 1% Triton X-100, 2 mM NaF, 2 mM EDTA, 2 mM beta-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, aprotinin) were injected into each sample and the cells were lysed. Based on the measured total protein concentration, each sample was treated with test compounds at room temperature for 2 hours, and then a sample buffer was added and allowed to react at 95° C. for 10 minutes. 25 ul was taken from the samples after the reaction, and dispensed into each well of acrylamide gel, and then immunoblotting was performed. Immunoblotting showed representative results from three or more independent experiments. The results are shown in FIG. 2.

[0162] As seen in FIG. 2, when treated with the p62 ligand compounds according to the present invention, it was confirmed that the treatment with the compounds resulted in a decrease of the monomer of p62 protein and simultaneously an increase in oligomers and high-molecular aggregates.

Experimental Example 2. Evaluation of Autophagy Activity in Cultured Cells by Immunoblotting

[0163] In order to evaluate the autophagy activity efficacy by the compounds (Examples 1-19), HeLa cell line, which is a cell line derived from a cervical cancer patient, was cultured using a DMEM medium containing 10% FBS and 1% streptomycin/penicillin in an incubator with 5% carbon dioxide. As representative compounds among the present compounds, YOK-1107, YOK-4404 and YOK-1104 were selected. In order to measure autophagy activity according to the treatment with these selected representative compounds, the respective cells were dispensed into 6 well plates. Additional cultures were performed for hours so that the cells were completely attached to the surface of the plate. To find the concentration at which the respective compounds could increase the phenomenon of autophagy, the test compounds were diluted at 1, 2, 5, 10 and 20 μM and treated. After treatment with the respective compounds, the cells were cultured again in a cell incubator for 24 hours, and then the cells were collected. To extract proteins from the collected cells, 100 ul of lysis buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 2 mM NaF, 2 mM EDTA, 2 mM beta-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, aprotinin) was injected into each sample, and cells were lysed. Based on the measured total protein concentration, a sample buffer was added to each sample and allowed to react at 100° C. for 5 minutes. 5 ul was taken from the samples after the reaction, and dispensed into each well of acrylamide gel, and then immunoblotting was performed. Immunoblotting showed representative results from three or more independent experiments. The results are shown in FIG. 3.

[0164] As seen in FIG. 3, when treated with the p62 ligand compounds according to the present invention, it was confirmed that the level of LC3, a marker of macroautophagy activity, gradually increased according to the concentration of the compounds.

Experimental Example 3. Evaluation of Autophagy Activity in Cultured Cells by Immunoblotting

[0165] In order to investigate the autophagy activity efficacy by the compounds (Examples 1-19), immunoblotting was performed in the same manner as in Experimental Example 2 using LC3 as a marker. As for the difference, in order to evaluate the treatment time required for activation and the activity retention time, as the representative compounds selected among the present compounds, 5 μM of YOK-1107, YOK-1204, YOK-1304, YOK-2204, YOK-3304, YOK-were treated for 1, 3, 6, 12, 24, 48 hours. On the other hand, as the representative compounds selected among the compounds of the present invention, μM of YOK-1107, YOK-1109, YOK-2207, YOK-2209, YT-4-1, YT-4-2, YT-6-1, YT-6-2, YT-6-8, YT-6-7, YT-9-1, YT-9-2 were treated for 24 hours. Immunoblotting showed representative results from three or more independent experiments. The results are shown in FIG. 4. As seen in FIG. 4, when treated with the p62 ligand compounds according to the present invention, it was confirmed that the level of LC3, a marker of macroautophagy activity, gradually increased according to the treatment with the compounds.

Experimental Example 4. Evaluation of Autophagy Activity in Cultured Cells by Immunoblotting

[0166] In order to investigate the autophagy activity efficacy of control compounds, immunoblotting was performed in the same manner as in Experimental Example 2 by using LC3 as a marker. As the control compounds, compounds having the following structure were used.

TABLE-US-00002 TABLE 2 No. Structure Name of Compound 1101 [00014] 3,4-bis(benzyloxy)benzaldehyde 1102 [00015] 3,4-bis(benzyloxy)phenol 1103 [00016] (R)-2-((3,4-bis(benzyloxy)phenoxy) methyl)oxirane 1201 [00017] 4-(benzyloxy)-3- phenethoxybenzaldehyde 1202 [00018] 4-(benzyloxy)-3-phenethoxyphenol 1203 [00019] (R)-2-((4-(benzyloxy)-3- phenethoxyphenoxy)methyl)oxirane 1301 [00020] 4-(benzyloxy)-3-(3- phenylpropoxy)benzaldehyde 1302 [00021] 4-(benzyloxy)-3-(3- phenylpropoxy)phenol YTK- 1105- 1 [00022] 4-(3,4-bis(benzyloxy)phenyl)butan- 1-ol 4402 [00023] 3,4-bis(4-phenylbutoxy)phenol 1401 [00024] 4-(benzyloxy)-3-(4- phenylbutoxy)benzaldehyde 1402 [00025] 4-(benzyloxy)-3-(4- phenylbutoxy)phenol 2201 [00026] 3,4-diphenethoxybenzaldehyde 2202 [00027] 3,4-diphenethoxyphenol

[0167] The aforementioned compounds were confirmed by treatment at different concentrations and for different times, and immunoblotting showed representative results from three or more independent experiments. The results are shown in FIG. 5. As shown in FIG. 5, it was confirmed that the control compounds did not increase the level of LC3, a marker of macroautophagy activity, regardless of the treatment time or concentration.

Experimental Example 5. Evaluation of Autophagy Activity in Cultured Cells by Immunofluorescence Staining and Confocal Microscopy

[0168] In order to confirm the p62 protein activity and the activity level of autophagy phenomenon by the compounds (Examples 1-19), immunofluorescence staining was performed using p62 and LC3 as markers. In order to confirm the p62 activity and the activity level of autophagy phenomenon by new p62 ligands and isomers thereof in cultured cells, HeLa cell line, which is a cell line derived from cervical cancer patient, was treated with the novel p62 ligand compounds (YOK-1106, YOK-1204, YOK-1504, YOK-2204, YOK-3304, YOK-4404, YOK-1107, YOK-1109, YOK-2207, YOK-2209, YT-4-1, YT-4-2, YT-6-1, YT-9-1 and YT-9-2) and cultured. Thereafter, as markers of the autophagy phenomenon, the expression level and location of LC3 puncta and the local co-existence with p62 puncta were observed.

[0169] For immunofluorescence staining, a cover glass was placed on a 24-well plate, cells were dispensed, cultured for 24 hours, and then treated with 5 uM of the novel p62 ligands according to the present invention. For the action of the compounds, additional cultures were performed for 24 hours, and then the medium was removed. Cells were fixed using formaldehyde at room temperature. In order to prevent non-specific staining, the cells were allowed to react with a blocking solution at room temperature for 1 hour, and then treated with LC3 antibody diluted at a certain ratio using the blocking solution, and allowed to reacted at room temperature for 1 hour. The antibody-treated cells were washed three times with PBS, and goat-derived secondary antibody was diluted at a certain ratio using the blocking solution, and then allowed to react at room temperature for 30 minutes. The cells were washed again with PBS three times, and for intracellular nuclear staining, the expression level, intracellular puncta formation and intracellular coexistence level of p62 and LC3 were observed through a confocal microscopy after DAPI staining. The results are shown in FIG. 5. Immunofluorescence staining showed representative results from three or more independent experiments.

[0170] As seen in FIG. 6, it was confirmed that after treatment with p62 ligand compounds according to the present invention, the intracellular puncta of p62 protein was formed, the intracellular puncta and local coexistence of LC3 as an autophagosome marker were increased, and the intracellular puncta formation of LC3 was increased.

Experimental Example 6. Evaluation of Autophagy Activity in Cultured Cells by Immunofluorescence Staining and Confocal Microscopy

[0171] In order to confirm p62 protein activity and the activity level of autophagy phenomenon of control compounds (YOK-A-1104, YOK-G-1104, YTK-1005, YTK-1105-1), immunofluorescence staining was performed in the same manner as in Experimental Example 4 by using p62 and LC3 as markers. YOK-A-1104 and YOK-G-1104 are compounds having the following structures.

TABLE-US-00003 TABLE 3 No. Structure Name of Compound YOK-A- 1104 [00028] Ethyl (R)-(3-(3,4- bis(benzyloxy)phenoxy)-2- hydroxypropyl)alanineate YOK-G- 1104 [00029] Methyl (R)-(3-(3,4- bis(benzyloxy)phenoxy)-2- hydroxypropyl)glycineate

[0172] The compounds were treated at different concentrations, and intracellular puncta of p62 or LC3, that is, the degree to which autophagosome was formed and p62 was delivered was observed by a confocal microscopy. The results are shown in FIG. 7. Immunofluorescence staining showed representative results from three or more independent experiments.

[0173] As shown in FIG. 7, it was confirmed that regardless of the concentration treated with the control compounds, the intracellular puncta of p62 protein was formed, the intracellular puncta and local co-existence of LC3 as an autophagosome marker were increased, and the intracellular puncta formation of LC3 was not increased.

Experimental Example 7. Evaluation of the Activity of P62-Mediated Delivery of Ubiquitinylated Proteins in Cultured Cells to Autophagy by Immunofluorescence Staining and Confocal Microscopy

[0174] In order to confirm the activity level of P62-mediated delivery of ubiquitinylated proteins to autophagy in cultured cells after treatment with the compounds (Examples 1-19), immunofluorescence staining was performed in the same manner as in Experimental Example 4 by using p62 and FK2 as markers. As the compounds, YT-4-1, YT-4-2, YT-6-1, YT-6-7, YT-6-8, YOK-4404, YOK-1106, YOK-1107, YOK-1109, YOK-1204 and YOK-1204 were used. After treatment with the compounds, the intracellular puncta of p62 or FK2, that is, the extent to which p62 and ubiquitinated proteins were delivered to autophagosome, were observed by confocal microscopy. The results are shown in FIGS. 8a and 8b. Immunofluorescence staining showed representative results from three or more independent experiments.

[0175] As shown in FIGS. 8a and 8b, it was confirmed that after treatment with the p62 ligand compounds according to the present invention, intracellular punctae of FK2, markers of p62 protein and ubiquitination protein, were formed and the local co-existence of the punctae were increased.

Experimental Example 8. Evaluation of the Activity of P62-Mediated Delivery of Misfolded Huntingtin Proteins in Cultured Cells to Autophagy by Immunofluorescence Staining and Confocal Microscopy

[0176] In order to confirm the activity level of P62-mediated delivery of misfolded huntingtin (Htt-Q103), a major protein of Huntington's disease, degenerative brain disease after treatment with the compounds (Examples 1-19), immunofluorescence staining was performed in the same manner as in Experimental Example 4 by using p62 and Htt-Q103-GFP as markers. As the compounds, YOK-1106, YOK-1107, YOK-1109, YOK-1204, YOK-2204, YOK-4404, YT-4-1, YT-4-2, YT-6-1, YT-6-7 and YT-9-1 were used. After treatment with the compounds, the intracellular puncta of p62 or Htt-Q103-GFP, that is, the extent to which p62 and misfolded huntingtin proteins were delivered to autophagosome, were observed by confocal microscopy. The results are shown in FIGS. 9a and 9b. Immunofluorescence staining showed representative results from three or more independent experiments.

[0177] As shown in FIGS. 9a and 9b, after treatment with the p62 compounds according to the present invention, intracellular puncta formation of FK2, markers of p62 protein and ubiquitination protein, and an increase in the local co-existence of the punctae were confirmed.