Composition for the prevention or treatment of Charcot-Marie-Tooth disease (CMT)

Abstract

The present invention provides a pharmaceutical composition for the prevention or treatment of Charcot-Marie-Tooth disease, which is related to the peripheral nervous system. [Solution] A pharmaceutical composition for the prevention or treatment of Charcot-Marie-Tooth disease related to the peripheral nervous system, comprising as an active ingredient a compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof. TIFF2026143847000104.tif112170

Description

[Technical Field]

[0001] The present disclosure relates to a pharmaceutical composition for preventing or treating Charcot-Marie-Tooth disease associated with the peripheral nervous system, which comprises a compound represented by Chemical Formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient; a method for preventing or treating Charcot-Marie-Tooth disease associated with the peripheral nervous system using said compound; use of said compound for preventing or treating Charcot-Marie-Tooth disease associated with the peripheral nervous system; and use of said compound in the manufacture of a medicament for preventing or treating Charcot-Marie-Tooth disease associated with the peripheral nervous system. [Background Art]

[0002] Charcot-Marie-Tooth disease (CMT, HMSN, hereditary motor and sensory neuropathy) is the most common type of hereditary peripheral neuropathy, which is caused by mutations in proteins constituting nerves. To date, more than 1000 mutations from approximately 90 genes have been identified (Timmerman et al., (2014) Genes 5: 13-32). Onset of Charcot-Marie-Tooth disease causes atrophy of muscles innervated by nerves due to progressive degeneration of peripheral nerves. Accordingly, CMT patients develop symptoms such as gradual atrophy of the muscles of the feet and hands, and deformation of the limbs. CMT is genetically and clinically highly diverse and complex, and it is known that its symptoms vary greatly depending on the type of mutation, from a nearly normal condition to a condition requiring reliance on a wheelchair. It mainly onsets in the teenage years, and occurs at a rate of 1 in 2500 people (Krajewski et al., (2000) Brain 123: 1516).

[0003] CMT belongs to rare diseases such as hereditary peripheral neuropathy. However, with a prevalence of 1 in 2,500 people, there are approximately 20,000 patients in Korea and 2,800,000 patients worldwide. Currently, only limited treatments for CMT are available, including rehabilitation therapy, use of assistive devices, pain management and surgical therapy, and no therapeutic agent has been successfully developed to date. Therefore, there is a very great need for the development of CMT therapeutic agents.

[0004] For example, for CMT, the most common type of hereditary motor and sensory neuropathy, a large-scale clinical trial has been conducted on ascorbic acid, which was proven to be an essential substance for myelin formation in the peripheral nervous system through an experiment in which Schwann cells and dorsal root ganglion cells were co-cultured. However, the efficacy thereof has not been proven yet (Pareyson et al., (2011) 10(4): 3205).

Prior Art Document

Patent Document

[0005]

Patent Document 1

Non-Patent Document

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problem to be Solved by the Invention

[0007] The present disclosure provides a compound represented by Chemical Formula I, an optical isomer thereof, or The present invention provides a pharmaceutical composition for the prevention or treatment of Charcot-Marie-Tooth disease, which is associated with the peripheral nervous system, comprising a pharmaceutically acceptable salt thereof as an active ingredient.

[0008] This disclosure provides a method for preventing or treating Charcot-Marie-Tooth disease related to the peripheral nervous system, comprising administering to an individual a compound represented by the chemical formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

[0009] This disclosure provides the use of the compound represented by chemical formula I, its optical isomer, or a pharmaceutically acceptable salt thereof for the prevention or treatment of Charcot-Marie-Tooth disease relating to the peripheral nervous system.

[0010] This disclosure provides the use of a compound represented by the chemical formula I, its optical isomer, or a pharmaceutically acceptable salt thereof in the manufacture of a pharmacopoeia for the prevention or treatment of Charcot-Marie-Tooth disease relating to the peripheral nervous system. [Means for solving the problem]

[0011] This can be explained in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention cannot be said to be limited by the specific descriptions described below.

[0012] This disclosure provides a pharmaceutical composition for the prevention or treatment of Charcot-Marie-Tooth disease (CMT) associated with the peripheral nervous system (PNS), comprising as an active ingredient a compound represented by the following chemical formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

[0013] [ka]

[0014] In the aforementioned chemical formula I, L1, L2, or L3 each independently consists of a single bond or a -(C1-C2 alkyl)- can be; R1 is either -CX2H or -CX3; R2 is -NR A R B , -OR C ,

[0015] [ka]

[0016] , or

[0017] [ka]

[0018] And, {Here,

[0019] [ka]

[0020] or

[0021] [ka]

[0022] One or more H atoms are -X, -OH, -O(C1-C4 alkyl), -NR D R E, -(C1-C4 alkyl), -CF3, -CF2H, -CN, -aryl, -heteroaryl, -(C1-C4 alkyl)-aryl, or -(C1-C4 alkyl)-heteroaryl, which may be unsubstituted or substituted [in this case, one or more hydrogen atoms on -aryl, -heteroaryl, -(C1-C4 alkyl)-aryl, or -(C1-C4 alkyl)-heteroaryl are , -X, -OH, -CF3, or -CF2H, which may be unsubstituted or substituted]}; R3 is -H, -(C1-C4 alkyl), -(C1-C4 alkyl)-O(C1-C4 alkyl), -(C1-C4 alkyl)-C(=O)-O(C1-C4 alkyl), -(C3-C7 cycloalkyl), -(C2-C6 heterocycloalkyl), -aryl, -heteroaryl, -adamantyl,

[0023] [Chemical formula]

[0024] , or

[0025] [Chemical formula]

[0026] , and {wherein one or more hydrogen atoms in -(C1-C4 alkyl) may be substituted with -X or -OH , and one or more hydrogen atoms in -aryl or -heteroaryl are each independently substituted with -X, -OH, -O(C1-C4 alkyl), -OCF3, -O-aryl, -NR D R E , -(C1-C4 alkyl), -CF3, -CF2H, -C(=O)-(C1-C4 alkyl), -C(=O)-O(C1-C4 alkyl), -C(=O)-NR D R E , -S(=O)2-(C1-C4 alkyl), -aryl, -heteroaryl,

[0027] [ka]

[0028] ,

[0029] [ka]

[0030] , or

[0031] [ka]

[0032] It may also be replaced with [at this time,

[0033] [ka]

[0034] One or more H atoms are -X, -(C1-C4 alkyl), -NR D R E [May be replaced with -CF3 or -CF2H] -(C3-C7 cycloalkyl), -(C2-C6 heterocycloalkyl), -adamantyl

[0035] [ka]

[0036] , or

[0037] [ka]

[0038] One or more of the H atoms may be independently substituted with -X, -OH, or -(C1-C4 alkyl); Y1, Y2, and Y4 are independently -CH2- and -NR F -, -O-, -C(=O)-, or -S(=O)2-; Y3 is either -CH- or -N-; Z1 to Z4 are each independently N or CR Z And here, Z1 to Z4 are not N, but R if three or more are simultaneously. Z is -H, -X, or -O (C1-C4 alkyl); Z5 and Z6 are independently -CH2- or -O-; Z7 and Z8 are independently either =CH- or =N-; Z9 is -NR G -or -S-; R A and R B These are, independently, -H, -(C1-C4 alkyl), -(C1-C4 alkyl)-OH, and -(C1-C4 alkyl)-NR. D R E -aryl, -(C1-C4 alkyl)-aryl, -heteroaryl, -(C1-C4 aryl)-heteroaryl, -(C3-C7 cycloalkyl), -(C2-C6 heterocycloalkyl), or

[0039] [ka]

[0040] And {where -(C1-C4 alkyl), -(C1-C4 alkyl)-OH, or -(C1-C4 alkyl)-NR D R E One or more H's may be substituted with -X. One or more H atoms in -aryl, -(C1-C4 alkyl)-aryl, -heteroaryl, -(C1-C4 alkyl)-heteroaryl, -(C3-C7 cycloalkyl), or -(C2-C6 heterocycloalkyl) may be substituted with -X, -OH, -O(C1-C4 alkyl), -(C1-C4 alkyl), -CF3, -CF2H, or -CN.

[0041] [ka]

[0042] One or more H atoms may be substituted with -X, -OH, -O(C1-C4 alkyl), -(C1-C4 alkyl), -CF3, -CF2H, -CN, -(C2-C6 heterocycloalkyl), -aryl, -(C1-C4 alkyl)-aryl, -heteroaryl, or -heteroaryl-(C1-C4 alkyl); R C is -(C1-C4 alkyl), -aryl, -(C1-C4 alkyl)-aryl, -heteroaryl, or -(C1-C4 alkyl)-heteroaryl, where { One or more H atoms in -(C1-C4 alkyl) may be substituted with -X or -OH, and one or more H atoms in -aryl, -(C1-C4 alkyl)-aryl, -heteroaryl, or -(C1-C4 alkyl)-heteroaryl may be substituted with -X, -OH, -CF3, or -CF2H; R D and R E Each of these is independently -H, -(C1-C4 alkyl), -aryl, or -(C1-C4 alkyl)-aryl, where -(C1-C4 alkyl) One or more H atoms may be substituted with -X or -OH, and one or more H atoms in an -aryl or -(C1-C4 alkyl)-aryl group may be substituted with -X, -OH, -CF3, or -CF2H. R F -H, -(C1-C6alkyl), -(C1-C4alkyl)-OH, -(C1-C4alkyl)-O-(C1-C4alkyl), -C(=O)-(C1-C4alkyl), -C(=O)-O(C1-C4alkyl), -(C1-C4alkyl)-C(=O)-O(C1-C4alkyl), -(C1-C4alkyl)-NR D R E-S(=O)2-(C1-C4 alkyl), -aryl, -(C1-C4 alkyl)-aryl, -(C2-C4 alkenyl)-aryl, -heteroaryl, -(C1-C4 alkyl)-heteroaryl, -C(=O)-(C3-C7 cycloalkyl), -(C2-C6 heterocycloalkyl), or -(C1-C4 alkyl)-C(=O)-(C2-C6 heterocycloalkyl), {Here, -(C1-C4alkyl), -(C1-C4alkyl)-OH, -(C1-C 4alkyl)-O-(C1-C4alkyl), -C(=O)-(C1-C4alkyl), -C(=O)-O(C1-C4alkyl), -(C1-C4alkyl)-C(=O)-O(C1-C4alkyl), -(C1-C4alkyl)-NR D R E Alternatively, one or more H atoms in -S(=O)2-(C1-C4 alkyl) may be substituted with -X. One or more H atoms in -aryl, -(C1-C4 alkyl)-aryl, -(C2-C4 alkenyl)-aryl, -heteroaryl, -(C1-C4 alkyl)-heteroaryl, -C(=O)-(C3-C7 cycloalkyl), -C2-C6 heterocycloalkyl, or -(C1-C4 alkyl)-C(=O)-(C2-C6 heterocycloalkyl) may be substituted with -X, -OH, -CF3, or -CF2H. R G is -H or -(C1-C4 alkyl); Q is either an O- or a single bond;

[0043] [ka]

[0044] It is a single bond or a double bond (however,

[0045] [ka]

[0046] If it is a double bond, then Y1 is =CH-; a through e are each independent integers of 0, 1, 2, 3, or 4 (where a and b are not both 0, and c and d are not both 0); X is F, Cl, Br, or I.

[0047] Furthermore, according to a preferred embodiment of the present invention, the compound represented by chemical formula I is In the aforementioned chemical formula I, L1, L2, or L3 each independently consists of a single bond or a -(C1-C2 alkyl)- can be; R1 is either -CX2H or -CX3; R2 is -NR A R B , -OR C ,

[0048] [ka]

[0049] , or

[0050] [ka]

[0051] And, {Here,

[0052] [ka]

[0053] or

[0054] [ka]

[0055] One or more H are -X, -OH, -NR D R E, may be substituted with -(C1-C4 alkyl); R3 is -(C1-C4 alkyl), -(C3-C7 cycloalkyl), -aryl, -heteroaryl, -adamantyl

[0056] [ka]

[0057] , or

[0058] [ka]

[0059] And, {Here, one or more H in an aryl or heteroaryl group are each independently of -X -O(C1-C4 alkyl), -OCF3, -O-aryl, -NR D R E -(C1-C4 alkyl), -CF3, -S(=O)2-(C1-C4 alkyl), -aryl, -heteroaryl,

[0060] [ka]

[0061] ,

[0062] [ka]

[0063] , or

[0064] [ka]

[0065] It may also be replaced with [at this time,

[0066] [ka]

[0067] One or more H's are -NR D R E Or it may be substituted with -(C1-C4 alkyl)],

[0068] [ka]

[0069] , or

[0070] [ka]

[0071] One or more of the H atoms may be independently substituted with -(C1-C4 alkyl) groups. Y1, Y2, and Y4 are independently -CH2- and -NR F -, -O-, -C(=O)-, or -S(=O)2-; Y3 is either -CH- or -N-; Z1 to Z4 are each independently N or CR Z And here, Z1 to Z4 are not N, but R if three or more are simultaneously. Z is -H, -X, or -O (C1-C4 alkyl); Z5 and Z6 are independently -CH2- or -O-; Z7 and Z8 are independently either =CH- or =N-; Z9 is -NR G -or -S-; R A and R B These are, independently, -H, -(C1-C4 alkyl), -(C1-C4 alkyl)-OH, and -(C1-C4 alkyl)-NR. D R E-aryl, -(C1-C4 alkyl)-aryl, -(C3-C7 cycloalkyl), or

[0072] [ka]

[0073] And, {Here,

[0074] [ka]

[0075] One or more H atoms may be substituted with -X, -(C1-C4 alkyl), -CF3, -(C2-C6 heterocycloalkyl), -(C1-C4 alkyl)-aryl, -heteroaryl, or -heteroaryl-(C1-C4 alkyl); R C is -(C1-C4 alkyl) or -aryl; R D and R E Each of these is independently -H, -(C1-C4 alkyl), or -(C1-C4 alkyl)-aryl; R F -H, -(C1-C6alkyl), -(C1-C4alkyl)-OH, -(C1-C4alkyl)-O-(C1-C4alkyl), -C(=O)-(C1-C4alkyl), -C(=O)-O(C1-C4alkyl), -(C1-C4alkyl)-C(=O)-O(C1-C4alkyl), -(C1-C4alkyl)-NR D R E -S(=O)2-(C1-C4 alkyl), -aryl, -(C1-C4 alkyl)-aryl, -(C2-C4 alkenyl)-aryl, -heteroaryl, -(C1-C4 alkyl)-heteroaryl, -C(=O)-(C3-C7 cycloalkyl), -(C2-C6 heterocycloalkyl), or -(C1-C4 alkyl)-C(=O)-(C2-C6 heterocycloalkyl) {Here, -(C1-C4 alkyl), or -C(=O)-O(C1-C4 alkyl) One or more H's may be replaced by -X. -One or more H's in the aryl group may be substituted with -X; R G It is -(C1-C4 alkyl); Q is either an O- or a single bond;

[0076] [ka]

[0077] It is a single bond or a double bond (however,

[0078] [ka]

[0079] If it is a double bond, then Y1 is -CH-; a through e are each independent integers of 0, 1, 2, 3, or 4 (where a and b are not both 0, and c and d are not both 0); X is F, Cl, Br, or I.

[0080] In the pharmaceutical compositions according to this disclosure, the compound represented by chemical formula I may be the compound represented by the following chemical formula Ia:

[0081] [ka]

[0082] In the aforementioned chemical formula Ia, R2 is

[0083] [ka]

[0084] and; R3 is an aryl, where one or more H of the aryl are independently , may be replaced with -X; Y1 is -O- or -S(=O)2-; Z1 is N or CR Z And {here, R Z is -X; a and b are each independent integers of 0, 1, 2, 3, or 4 {where a and b are independent integers of 0 and 4}. b and b are both not 0. X is independently F, Cl, Br, or I.

[0085] In the pharmaceutical composition according to the present invention, the compound represented by chemical formula I is R2 is

[0086] [ka]

[0087] and; R3 is phenyl, where one or more H atoms of phenyl may be independently substituted with either F or Cl. Y1 is -O- or -S(=O)2-; Z1 is either N or CF.

[0088] In the pharmaceutical compositions according to this disclosure, the compound represented by chemical formula I may be one of the compounds listed in Table A below.

[0089] [Table 1]

[0090] [Table 2]

[0091] Table 3

[0092] Table 4

[0093] Table 5

[0094] Table 6

[0095] Table 7

[0096] Table 8

[0097] Table 9

[0098] Table 10

[0099] Table 11

[0100] Table 12

[0101] Table 13

[0102] Table 14

[0103] Table 15

[0104] Table 16

[0105] Table 17

[0106] Table 18

[0107] Table 19

[0108] Table 20

[0109] Table 21

[0110] Table 22

[0111] Table 23

[0112] [Table 24]

[0113] [Table 25]

[0114] [Table 26]

[0115] [Table 27]

[0116] [Table 28]

[0117] [Table 29]

[0118] According to embodiments of the present invention, a pharmaceutical composition comprising the compounds listed in Table A, their optical isomers, or pharmaceutically acceptable salts thereof as an active ingredient is a Charcot-Marie-Tooth compound related to the peripheral nervous system (PNS). It can prevent or treat the disease (Charcot-Marie-Tooth disease; CMT).

[0119] In the pharmaceutical composition according to the present invention, the compound represented by chemical formula I may be one of the compounds listed in Table B below.

[0120] [Table 30]

[0121] According to embodiments of the present invention, a pharmaceutical composition containing the compounds of Table B, their optical isomers, or pharmaceutically acceptable salts thereof as active ingredients can prevent or treat Charcot-Marie-Tooth disease (CMT) associated with the peripheral nervous system (PNS).

[0122] In this disclosure, the compound represented by chemical formula I may be produced by the method disclosed in Korean Patent Publication No. 10-2017-0017792, but is not limited thereto.

[0123] In this disclosure, the compound represented by chemical formula I may contain one or more chiral carbons, thereby allowing it to exist as a racemic mixture, a single enantiomer (optical isomer), a diastereomer mixture, and a single diastereomer. These isomers can be separated by conventional techniques, such as column chromatography or HPLC. Alternatively, they can be synthesized stereospecifically using optically pure starting materials and / or reagents of known sequences. Specifically, the isomers may be optical isomers (enantiomers).

[0124] In this disclosure, the term "pharmaceutically acceptable" means physiologically acceptable and administered to an individual. When this is the case, it usually means that it does not cause allergic reactions or similar reactions such as gastrointestinal disorders or dizziness.

[0125] The pharmaceutically acceptable salts according to embodiments of the present invention can be produced by conventional methods known to the ordinary technicians of the art.

[0126] Examples of pharmaceutically acceptable salts according to embodiments of the present invention include, but are not limited to, inorganic ion salts produced from calcium, potassium, sodium, magnesium, etc., inorganic acid salts produced from hydrochloric acid, nitric acid, phosphoric acid, bromate, iodic acid, perchloric acid, sulfuric acid, hydroiodic acid, etc., organic acid salts produced from acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carboxylic acid, vanillic acid, etc., sulfonate salts produced from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc., amino acid salts produced from glycine, arginine, lysine, etc., and amine salts produced from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc. In embodiments of the present invention, the salt includes hydrochloric acid, trifluoroacetic acid, citric acid, bromate, maleic acid, phosphoric acid, sulfuric acid, and tartaric acid.

[0127] In this disclosure, the term "Charcot-Marie-Tooth disease (CMT)" refers to a peripheral neurodegenerative disease characterized by dysfunction and death of peripheral nerve cells due to various genetic factors, and axonal transport defects. This refers to diseases in which transport defeat is the primary cause.

[0128] In this disclosure, Charcot-Marie-Tooth disease related to the peripheral nervous system includes CMT1, CMT2, CMT4, DSN (dejerine-sottas syndrome), CH (congenital hypomyelination), and HNPP (hereditary neuropathy with liability to One or more conditions may be selected from the group consisting of pressure parsy and GAN (giant axonal neuropathy), but this is not limited to them.

[0129] The CMT1 type may be one or more selected from the group consisting of CMT1A, CMT1B, CMT1C, CMT1D, and CMTX; the CMT2 type may be one or more selected from the group consisting of CMT2A, CMT2B, CMT2C, CMT2D, CMT2E, and CMT2F; and the CMT4 type may be one or more selected from the group consisting of CMT4A, CMT4B1, CMT4B2, CMT4C, CMT4D, CMT4E, and CMT4F.

[0130] In this disclosure, the Charcot-Marie-Tooth disease relating to the peripheral nervous system may be one or more selected from the group consisting of CMT1A, CMT2D, and CMT2F, but is not limited thereto.

[0131] According to embodiments of the present invention, the compounds of this disclosure can prevent or treat symptoms associated with the regression of the peripheral nervous system in subjects having Charcot-Marie-Tooth disease.

[0132] According to embodiments of the present invention, the compounds of this disclosure can prevent or treat symptoms associated with the dysfunction and / or death of peripheral nerve cells in subjects having Charcot-Marie-Tooth disease.

[0133] According to embodiments of the present invention, the compounds of this disclosure can prevent or treat peripheral neurodegenerative diseases caused by dysfunction and / or death of peripheral nerve cells in subjects having Charcot-Marie-Tooth disease.

[0134] In this disclosure, the term “prevention” means any action that suppresses or delays the onset of a disease by administering a compound of chemical formula I of this disclosure, its optical isomer, or a pharmaceutically acceptable salt thereof.

[0135] In this disclosure, the term “treatment” means any act that improves or beneficially alters the symptoms of an individual suspected of or exhibiting a disease by administering a compound of chemical formula I of this disclosure, its optical isomer, or a pharmaceutically acceptable salt thereof.

[0136] The compound represented by chemical formula I of this disclosure, its optical isomers, or pharmaceutically acceptable salts thereof can be usefully used for the prevention or treatment of Charcot-Marie-Tooth disease associated with the peripheral nervous system.

[0137] A pharmaceutical composition containing the compound represented by chemical formula I of this disclosure, its optical isomer, or a pharmaceutically acceptable salt thereof as an active ingredient can be usefully used for the prevention or treatment of Charcot-Marie-Tooth disease associated with the peripheral nervous system.

[0138] According to embodiments of the present invention, pharmaceutical compositions comprising the compounds of this disclosure can prevent or treat symptoms associated with peripheral nervous system regression in subjects having Charcot-Marie-Tooth disease.

[0139] According to embodiments of the present invention, pharmaceutical compositions comprising the compounds of this disclosure can prevent or treat symptoms associated with the dysfunction and / or death of peripheral nerve cells in subjects having Charcot-Marie-Tooth disease.

[0140] According to embodiments of the present invention, pharmaceutical compositions comprising the compounds of this disclosure can prevent or treat peripheral neurodegenerative diseases caused by dysfunction and / or death of peripheral nerve cells in subjects having Charcot-Marie-Tooth disease.

[0141] In this regard, in a specific embodiment of the present invention, it was confirmed that the compound represented by chemical formula I of the present disclosure, its optical isomer, or a pharmaceutically acceptable salt thereof improves and restores the migration rate of mitochondria in axons (Tables 1 and 2, Figures 1 and 2) and inhibits the expression of induced PMP22 protein (Figure 3).

[0142] Furthermore, it was confirmed that the compound represented by chemical formula I of this disclosure, its optical isomer, or a pharmaceutically acceptable salt thereof improves motor function (CRT, GST, BBT) in mice (Figures 4-8) and improves nerve conduction velocity in mice (Figures 9 and 10). In addition, it was confirmed that the compound represented by chemical formula I of this disclosure, its optical isomer, or a pharmaceutically acceptable salt thereof improves and restores axon size (Figure 11).

[0143] In other words, the compound represented by chemical formula I of the present invention, its optical isomer, or a pharmaceutically acceptable salt thereof can effectively treat or improve symptoms that appear in Charcot-Marie-Tooth disease, which is related to the peripheral nervous system, such as decreased motor nerve conduction velocity, decreased compound muscle action potential, neuronal regression, muscle weakness, paresthesia, and axonal atrophy, and can suppress or delay the onset of these symptoms. It is possible.

[0144] In a specific embodiment of the present invention, the compound represented by chemical formula I of the present disclosure, its optical isomer, or a pharmaceutically acceptable salt thereof can modulate the genetic characteristics observed in Charcot-Marie-Tooth patients to normal or near-normal levels. Thus, it can be seen that the compound represented by chemical formula I of the present disclosure, its optical isomer, or a pharmaceutically acceptable salt thereof can improve or treat the symptoms of Charcot-Marie-Tooth disease (Figures 13-15).

[0145] In a specific embodiment of the present invention, the compound represented by chemical formula I of the present disclosure, its optical isomer, or a pharmaceutically acceptable salt thereof can increase the proportion of atrophic muscle fibers and increase the cross-sectional area of ​​muscle fibers in Charcot-Marie-Tooth patients (Figures 16 and 17).

[0146] In a specific embodiment of the present invention, the compound represented by chemical formula I of the present disclosure, its optical isomer, or a pharmaceutically acceptable salt thereof can increase fully nerve-distributed neuromuscular junctions in Charcot-Marie-Tooth patients (Figures 19 and 20).

[0147] In a specific embodiment of the present invention, the compound represented by chemical formula I of the present disclosure, its optical isomer, or a pharmaceutically acceptable salt thereof can increase the axonal diameter and / or myelin thickness, reduce abnormal myelination, and increase the proportion of large-diameter axons in the sensory nerves of Charcot-Marie-Tooth patients (Figures 21-23).

[0148] In one specific embodiment of the present invention, the compound represented by chemical formula I of the present disclosure, its optical isomer, or a pharmaceutically acceptable salt thereof can increase the sensory nerve conduction velocity (SNCV) and sensory nerve action potential (SNAP) amplitude in Charcot-Marie-Tooth patients (Figures 24 and 25).

[0149] The compound represented by chemical formula I of this disclosure, its optical isomers, or pharmaceutically acceptable salts thereof may exhibit prophylactic or therapeutic effects for peripheral nervous system-related Charcot-Marie-Tooth disease at a level considered to be similar to or substantially identical to conventionally known drugs for the prophylactic or therapeutic effects of peripheral nervous system-related Charcot-Marie-Tooth disease, or at a level considered to be superior.

[0150] The pharmaceutical compositions of this disclosure may further contain, in addition to the compound represented by chemical formula I, its optical isomers, or pharmaceutically acceptable salts thereof, one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers are those commonly used in the industry and may, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidine, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, minerals, or oils. In addition to the above components, the pharmaceutical compositions of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, dispersants, stabilizers, and the like. Furthermore, the pharmaceutical composition of the present invention may be formulated using pharmaceutically acceptable carriers and excipients in the form of oral preparations such as tablets, powders, granules, pills, capsules, suspensions, emulsions, oral solutions, oils, syrups, topical preparations, suppositories, or sterile injection solutions, and manufactured in unit dose form, or manufactured in multi-volume containers. The formulations are used in formulation in the industry. The usual method, or Remington's Pharmaceutical Science (19 th It may be manufactured by the method disclosed in (ed., 1995), and may be formulated into various formulations depending on the disease or component.

[0151] Non-limiting examples of orally administered preparations using the pharmaceutical composition of the present invention include tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs. For formulating the pharmaceutical composition according to an embodiment of the present invention for oral administration, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch or sweet potato starch; lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax may be used, and sweeteners, flavoring agents, syrups and the like may also be used. Further, in the case of capsules, in addition to the aforementioned substances, liquid carriers such as fatty oils may be further used.

[0152] Non-limiting examples of parenteral administration preparations using the pharmaceutical composition according to an embodiment of the present invention include injection solutions, suppositories, powders for respiratory inhalation, aerosol sprays, ointments, topical powders, oils, creams and the like. For formulating the pharmaceutical composition according to an embodiment of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspending agents, emulsions, lyophilized preparations, external preparations and the like may be used. Examples of the non-aqueous solvents and suspending agents include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0153] The pharmaceutical composition according to an embodiment of the present invention can be administered orally or parenterally depending on the intended method, and may be applied, for example, intravenously, subcutaneously, intraperitoneally or topically. Specifically, it may be orally administered, but the administration route is not limited thereto.

[0154] The daily dose of the compound represented by chemical formula I of this disclosure, its optical isomer, or pharmaceutically acceptable salt may specifically be about 0.1 to 10,000 mg / kg, about 1 to 8,000 mg / kg, about 5 to 6,000 mg / kg, or about 10 to 4,000 mg / kg, and more specifically, about 50 to 2,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0155] The pharmaceutically effective amount and effective dosage of the pharmaceutical composition according to the embodiments of the present invention can be varied depending on the formulation method, administration method, administration time and / or route of administration of the pharmaceutical composition, and can be varied according to various factors including the type and degree of reaction to be achieved by the administration of the pharmaceutical composition, the type, age, weight, general health status, symptoms and severity of disease, sex, diet, excretion, drugs used simultaneously or at different times with the individual, and other components of the composition, as well as similar factors well known in the pharmaceutical field. A person with ordinary skill in the art can easily determine and prescribe an effective dosage for the intended treatment.

[0156] The pharmaceutical compositions according to embodiments of the present invention may be administered once a day or in several divided doses. The pharmaceutical compositions of the present invention may be administered as a single therapeutic agent or in combination with other therapeutic agents, or sequentially or simultaneously with conventional therapeutic agents. Taking all of the above factors into consideration, the amount administered should be such that the maximum effect is obtained with the minimum amount without side effects, which can be easily determined by an ordinary person skilled in the art to which the present invention belongs.

[0157] The pharmaceutical compositions according to embodiments of the present invention can exhibit excellent effects even when used alone, but may be used in combination with various methods such as hormone therapy and drug therapy to further enhance therapeutic efficiency.

[0158] This disclosure provides a method for preventing or treating Charcot-Marie-Tooth disease related to the peripheral nervous system, comprising administering to an individual a compound represented by the chemical formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

[0159] This disclosure provides a method for preventing or treating Charcot-Marie-Tooth disease related to the peripheral nervous system, comprising administering to an individual a compound of Table A, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

[0160] This disclosure provides a method for preventing or treating Charcot-Marie-Tooth disease related to the peripheral nervous system, comprising administering to an individual one of the compounds in Table B, their optical isomers, or pharmaceutically acceptable salts thereof.

[0161] The terms "Charcot-Marie-Tooth disease," "prevention," and "treatment" are as previously described.

[0162] In this disclosure, the term "administration" means introducing a given substance into an individual in an appropriate manner.

[0163] In this disclosure, the term “individual” means all animals, including humans, rats, mice, and livestock, that have or are likely to have Charcot-Marie-Tooth disease related to the peripheral nervous system, and may specifically include, but are not limited to, mammals, including humans.

[0164] A method for preventing or treating Charcot-Marie-Tooth disease related to the peripheral nervous system according to an embodiment of the present invention may involve administering a therapeutically effective amount of the compound represented by chemical formula I, its optical isomer, or a pharmaceutically acceptable salt thereof.

[0165] In this disclosure, the term “therapeutably effective dose” means a dose sufficient to treat a disease with a reasonable benefit-to-risk ratio applicable to medical treatment, without causing adverse effects, which may be determined by a person skilled in the art depending on the patient’s sex, age, weight, health status, type and severity of disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration, elimination rate, duration of treatment, drugs used in combination or concurrently with the specific composition, and other factors well known in the medical field. It is desirable that the specific therapeutically effective dose for a particular patient be applied differently depending on various factors, including the type and degree of response to be achieved, and in some cases whether other drugs are being used, as well as similar factors well known in the medical field, including the specific composition, the patient’s age, weight, general health status, sex and diet, time of administration, route of administration and elimination rate of the composition, duration of treatment, drugs used in combination with or concurrently with the specific composition, and other factors well known in the medical field.

[0166] The present invention provides a method for the prevention or treatment of Charcot-Marie-Tooth disease related to the peripheral nervous system, which includes not only treating the disease itself before the onset of symptoms, but also inhibiting or avoiding its signs by administering the compound represented by chemical formula I, its optical isomers, or pharmaceutically acceptable salts thereof. In the management of the disease, the prophylactic or therapeutic dose of a particular active ingredient varies depending on the characteristics and severity of the disease or condition, and the route through which the active ingredient is administered. The dose and frequency of dose vary depending on the age, weight, and response of the individual patient. The appropriate dosage and method of use can be easily selected by a person with ordinary knowledge of the subject, taking these factors into consideration.

[0167] Furthermore, the present invention's method for preventing or treating Charcot-Marie-Tooth disease related to the peripheral nervous system may further include administering a therapeutically effective amount of an active preparation useful for preventing or treating the disease, together with the compound represented by chemical formula I, its optical isomer, or a pharmaceutically acceptable salt thereof. By using the compound represented by chemical formula I, its optical isomer, or a pharmaceutically acceptable salt thereof together with the additional active preparation, a synergistic or additive effect can be obtained.

[0168] This disclosure provides the use of the compound represented by chemical formula I, its optical isomer, or a pharmaceutically acceptable salt thereof for the prevention or treatment of Charcot-Marie-Tooth disease relating to the peripheral nervous system.

[0169] This disclosure provides the use of the compounds in Table A, their optical isomers, or pharmaceutically acceptable salts thereof for the prevention or treatment of Charcot-Marie-Tooth disease relating to the peripheral nervous system.

[0170] This disclosure provides the use of the compounds in Table B, their optical isomers, or pharmaceutically acceptable salts thereof for the prevention or treatment of Charcot-Marie-Tooth disease relating to the peripheral nervous system.

[0171] This disclosure provides the use of a compound represented by the chemical formula I, its optical isomer, or a pharmaceutically acceptable salt thereof in the manufacture of a pharmacopoeia for the prevention or treatment of Charcot-Marie-Tooth disease relating to the peripheral nervous system.

[0172] This disclosure provides the use of the compounds in Table A, their optical isomers, or pharmaceutically acceptable salts thereof in the manufacture of agents for the prevention or treatment of Charcot-Marie-Tooth disease relating to the peripheral nervous system.

[0173] This disclosure provides the use of the compounds in Table B, their optical isomers, or pharmaceutically acceptable salts thereof in the manufacture of agents for the prevention or treatment of Charcot-Marie-Tooth disease relating to the peripheral nervous system.

[0174] The terms "Charcot-Marie-Tooth disease", "prevention" and "treatment" are as defined above.

[0175] For producing a medicament, a pharmaceutically acceptable adjuvant, diluent, carrier or the like may be mixed with the compound represented by said chemical formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, and a synergistic effect can be obtained by producing the preparation as a combined preparation together with other active agents.

[0176] The matters described in the pharmaceutical composition, therapeutic method and use of the present disclosure apply mutatis mutandis, so long as they do not contradict each other. [Advantageous Effects of Invention]

[0177] The compound represented by chemical formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the same as an active ingredient according to the present disclosure can be usefully used for preventing or treating Charcot-Marie-Tooth disease associated with the peripheral nervous system. [Brief Description of Drawings]

[0178] [Figure 1] Figure 1 shows the results of evaluating the effect of the compound of the present disclosure on mitochondrial axonal transport in HSPB1S135F CMT2F motor neurons, and shows the velocity distribution of each group (*p<0.05, **p<0.01, ***p<0.001). [Figure 2] Figure 2 shows the results of evaluating the effect of the compound of the present disclosure on mitochondrial axonal transport in GarsP234KY CMT2D motor neurons, and shows the velocity distribution of each group (##p<0.01, *p<0.05, **p<0.01). [Figure 3]Figure 3 shows the results of evaluating the effect of the compounds of this disclosure on the expression of myelin-associated protein (PMP22), indicating the degree of protein expression in each group (###p<0.001). [Figure 4] Figure 4 shows the results of a rotarod test evaluating the effects of the compounds disclosed herein in CMT1A mice (C22), indicating the latency to fall for each group (###p<0.001). [Figure 5] Figure 5 shows the results of a grip strength test evaluating the effects of the compounds of this disclosure in CMT1A mice (C22), indicating the grip strength of each group (###p<0.001, *p<0.05). [Figure 6] Figure 6 shows the results of a balance beam test evaluating the effects of the compounds of this disclosure in CMT1A mice (C22), with the number of slips for each group (###p<0.001, *p<0.05). [Figure 7] Figure 7 shows the results of a rotarod test evaluating the effects of the compounds disclosed herein in CMT2F mice (HSPB1S135F), indicating the latency to fall for each group (*p<0.05, **p<0.01). [Figure 8] Figure 8 shows the results of a grip strength test evaluating the effects of the compounds disclosed in CMT2F mice (HSPB1S135F), indicating the grip strength of each group (***p<0.001). [Figure 9] Figure 9 shows the results of nerve conduction studies evaluating the effects of the compounds disclosed in 2.5-week-old CMT1A mice (C22), with CMAP and MNCV levels for each group (###p<0.001, **p<0.01). [Figure 10] Figure 10 shows the results of nerve conduction studies evaluating the effects of the compounds disclosed in 8-month-old CMT1A mice (C22), with CMAP and MNCV levels for each group (###p<0.001, **p<0.01). [Figure 11]Figure 11 shows the results of evaluating the effect of the compounds of this disclosure on the axon size of sciatic nerve fibers, and indicates the percentage of axons included in the diameter range of each axon (##p<0.01, *p<0.05, **p<0.01). [Figure 12] Figures 12 to 15 show the results of evaluating the effects of the compounds of this disclosure in CMT1A mice (C3), and illustrate the genetic characteristics of the sciatic nerve in CMT1A mice (C3). [Figure 13] Figures 12 to 15 show the results of evaluating the effects of the compounds of this disclosure in CMT1A mice (C3), and illustrate the genetic characteristics of the sciatic nerve in CMT1A mice (C3). [Figure 14] Figures 12 to 15 show the results of evaluating the effects of the compounds of this disclosure in CMT1A mice (C3), and illustrate the genetic characteristics of the sciatic nerve in CMT1A mice (C3). [Figure 15] Figures 12 to 15 show the results of evaluating the effects of the compounds of this disclosure in CMT1A mice (C3), and illustrate the genetic characteristics of the sciatic nerve in CMT1A mice (C3). [Figure 16] Figure 16 is a diagram illustrating the effects of the compounds of this disclosure on neuromuscular junctions of muscle fiber atrophy in CMT1A mice (C3), showing the percentage of atrophic muscle fibers in each group ([###p<0.001, Veh vs WT], [***p<0.001 Veh vs Compound 43]). [Figure 17] Figure 17 is a diagram illustrating the effects of the compounds of this disclosure on muscle fiber atrophy in CMT1A mice (C3), showing the area and distribution of muscle fibers in each group ([#p<0.05, ##p<0.01, ###p<0.001, Veh vs WT], [**p<0.01, ***p<0.001 Veh vs Compound 43]). [Figure 18] Figure 18 is a diagram showing the criteria for evaluating the degree of nerve distribution at the neuromuscular junction. [Figure 19]Figures 19 and 20 are diagrams showing the results of evaluating the effects of the compounds of this disclosure on neuromuscular junctions in CMT1A mice (C3), where Figure 19 shows the degree of nerve distribution and Figure 20 shows the percentage of neuromuscular junctions that were fully nerve-distributed ([###p<0.001, Veh vs WT], [**p<0.01, ***p<0.001 Veh vs Compound 43]). [Figure 20] Figures 19 and 20 are diagrams showing the results of evaluating the effects of the compounds of this disclosure on neuromuscular junctions in CMT1A mice (C3), where Figure 19 shows the degree of nerve distribution and Figure 20 shows the percentage of neuromuscular junctions that were fully nerve-distributed ([###p<0.001, Veh vs WT], [**p<0.01, ***p<0.001 Veh vs Compound 43]). [Figure 21] Figure 21 is a diagram showing the results of evaluating the effects of the compounds of this disclosure on the sural nerve in CMT1A mice (C3) through optical microscopy. [Figure 22A] Figures 22 and 23 are diagrams showing the results of evaluating the effects of the compounds of this disclosure on the sural nerve in CMT1A mice (C3), where Figures 22A (WT), 22B (TG), and 22C (Compound 43) show the slope of the g-ratio, and Figure 23 shows the axonal diameter ([##p<0.01, ###p<0.001, Veh vs WT], [***p<0.001 Veh vs Compound 43]). [Figure 22B] Figures 22 and 23 are diagrams showing the results of evaluating the effects of the compounds of this disclosure on the sural nerve in CMT1A mice (C3), where Figures 22A (WT), 22B (TG), and 22C (Compound 43) show the slope of the g-ratio, and Figure 23 shows the axonal diameter ([##p<0.01, ###p<0.001, Veh vs WT], [***p<0.001 Veh vs Compound 43]). [Figure 22C]Figures 22 and 23 are diagrams showing the results of evaluating the effects of the compounds of this disclosure on the sural nerve in CMT1A mice (C3), where Figures 22A (WT), 22B (TG), and 22C (Compound 43) show the slope of the g-ratio, and Figure 23 shows the axonal diameter ([##p<0.01, ###p<0.001, Veh vs WT], [***p<0.001 Veh vs Compound 43]). [Figure 23] Figures 22 and 23 are diagrams showing the results of evaluating the effects of the compounds of this disclosure on the sural nerve in CMT1A mice (C3), where Figures 22A (WT), 22B (TG), and 22C (Compound 43) show the slope of the g-ratio, and Figure 23 shows the axonal diameter ([##p<0.01, ###p<0.001, Veh vs WT], [***p<0.001 Veh vs Compound 43]). [Figure 24] Figures 24 and 25 are diagrams showing the results of evaluating the effects of the compounds of this disclosure on the sural nerve in CMT1A mice (C3), where Figure 24 shows sensory nerve conduction velocity (SNCV) and Figure 25 shows sensory nerve action potential (SNAP) amplitude values ​​([###p<0.001, Veh vs WT], [**p<0.01, Veh vs Compound 43]). [Figure 25] Figures 24 and 25 are diagrams showing the results of evaluating the effects of the compounds of this disclosure on the sural nerve in CMT1A mice (C3), where Figure 24 shows sensory nerve conduction velocity (SNCV) and Figure 25 shows sensory nerve action potential (SNAP) amplitude values ​​([###p<0.001, Veh vs WT], [**p<0.01, Veh vs Compound 43]). [Modes for carrying out the invention]

[0179] The present invention will be described in more detail below with reference to examples. However, it will be obvious to those with ordinary skill in the art that these examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention.

[0180] Synthesis Example 1. Synthesis of Compound 43: N-((5-(5-(difluoromethyl)-1,3,4-oxadiazole-2-yl)pyridine-2-yl)methyl)-N-phenylthiomorpholine-4-carboxamide 1,1-dioxide [Step 1] Synthesis of N-phenylthiomorpholine-4-carboxamide 1,1-dioxide

[0181] [ka]

[0182] Aniline (3.000 g, 32.213 mmol) and N,N-diisopropylethylamine (33.439 mL, 193.278 mmol) were dissolved in methylene chloride (100 mL) at 0°C. Triphosgene (4.780 g, 16.107 mmol) was added to this solution and the mixture was stirred at the same temperature. Thiomorpholine 1,1-dioxide (4.790 g, 35.434 mmol) was added to the reaction mixture and the mixture was stirred for a further 16 hours at room temperature. Water was poured over the reaction mixture and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, water was removed with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 40 g cartridge; methanol / methylene chloride = 2%) to obtain the title compound (1.325 g, 16.2%) as a yellow solid.

[0183] [Step 2] Synthesis of methyl 6-((1,1-dioxide-N-phenylthiomorpholine-4-carboxamide)methyl)nicotinate

[0184] [ka]

[0185] The solution prepared in Step 1 by dissolving N-phenylthiomorpholine-4-carboxamide 1,1-dioxide (1,000 g, 3,932 mmol) and sodium hydride (60.00%, 0.157 g, 3,932 mmol) in N,N-dimethylformamide (10 mL) was stirred at 0°C for 1 hour, then methyl 4-(bromomethyl)-3-fluorobenzoate (0.905 g, 3,932 mmol) was added, and the mixture was stirred at room temperature for a further 2 hours. The substance was concentrated under reduced pressure to remove the solvent, and water was added to the resulting concentrate, which was then extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride aqueous solution, water was removed with anhydrous magnesium sulfate, and the mixture was filtered and concentrated under reduced pressure. The crude product was crystallized at room temperature with methanol (20 mL), filtered, and the resulting solid was washed with methanol and dried to obtain the title compound (0.816 g, 51.4%) as a brown solid.

[0186] [Step 3] Synthesis of N-((5-(hydrazinecarbonyl)pyridine-2-yl)methyl)-N-phenylthiomorpholine-4-carboxamide 1,1-dioxide

[0187] [ka]

[0188] Methyl 6-((1,1-dioxide-N-phenylthiomorpholine-4-carboxamide)methyl)nicotinate (0.816 g, 2.023 mmol) prepared in Step 2 and hydrazine monohydrate (1.910 mL, 40.451 mmol) were mixed with ethanol (10 mL) at room temperature, microwaved, and heated at 100°C for 1 hour. The temperature was then lowered to room temperature to terminate the reaction. The reaction mixture was then drained under reduced pressure to remove the solvent, and the crude product was obtained. The compound was crystallized at room temperature with methylene chloride (20 mL), filtered, and the resulting solid was washed with methylene chloride and dried to obtain the title compound (0.560 g, 68.6%) as a light brown solid.

[0189] [Step 4] Synthesis of N-((5-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)pyridine-2-yl)methyl)-N-phenylthiomorpholine-4-carboxamide 1,1-dioxide

[0190] [ka]

[0191] N-((5-(hydrazinecarbonyl)pyridine-2-yl)methyl)-N-phenylthiomorpholine-4-carboxamide 1,1-dioxide (0.260 g, 0.644 mmol) and triethylamine (0.178 mL, 1.289 mmol) prepared in Step 3 were dissolved in methylene chloride (2 mL) at room temperature. Difluoroacetic anhydride (0.087 mL, 0.580 mmol) was added to this solution and the mixture was stirred at the same temperature for 16 hours. Water was poured over the reaction mixture and extracted with methylene chloride. The mixture was then filtered through a plastic filter to remove the solid residue and aqueous layer, and the collected organic layer was concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 4 g cartridge; methanol / methylene chloride = 0% → 5%) to obtain the title compound (0.156 g, 50.3%) as a white foam.

[0192] [Step 5] Synthesis of Compound 43

[0193] [ka]

[0194] N-((5-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)pyridine-2-yl)methyl)-N-phenylthiomorpholine-4-carboxamide 1,1-dioxide (0.156 g, 0.324 mmol) and 1-methoxy-N-triethylammoniosulfonyl-methaneimidate (Burgess reagent, 0.116 g, 0.486 mmol) prepared in Step 4 were mixed in tetrahydrofuran (2 mL), heated at 150°C for 30 minutes under microwave irradiation, and then the temperature was lowered to room temperature to terminate the reaction. Water was added to the reaction mixture and extracted with methylene chloride. The two-phase mixture was then filtered through a plastic filter to remove the solid residue and aqueous layer, and the collected organic layer was concentrated under reduced pressure. The concentrate was purified and concentrated by column chromatography (SiO2, 4g cartridge; methanol / methylene chloride = 3%) to obtain the title compound (0.078g, 51.9%) as a colorless oil.

[0195] 1 HNMR (400MHz,CDCl3)δ9.23(d,1H,J=2.2Hz),8.38(dd,1H,J=8.2,2.2Hz),7.54(d,1H,J=8.2Hz),7.41-7.31(m,2H),7.19(ddd ,3H,J=6.4,3.0,1.6Hz),6.94(m,1H),5.10(s,2H),3.72(dd,4H,J=6.9,3.7Hz),2.97-2.90(m,4H);LRMS(ES)m / z464.2(M + +1).

[0196] Synthesis Example 2. Synthesis of Compound 232: N-(3-chloro-4-fluorophenyl)-N-((5-(5-(difluoromethyl)-1,3,4-oxadiazole-2-yl)pyridine-2-yl)methyl)morpholine-4-carboxamide [Step 1] Synthesis of N-(3-chloro-4-fluorophenyl)morpholine-4-carboxamide

[0197] [ka]

[0198] 3-chloro-4-fluoroaniline (0.500 g, 3.435 mmol), 1,1'-carbonyldiimidazole (0.613 g, 3.779 mmol), and triethylamine (0.575 mL, 4.122 mmol) were dissolved in acetonitrile (10 mL) at room temperature. Morpholine (0.311 mL, 3.607 mmol) was added to this solution, and the mixture was stirred at the same temperature for 18 hours. Water was poured over the reaction mixture, and it was extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride aqueous solution, water was removed with anhydrous magnesium sulfate, and then filtered and subjected to reduced pressure. The solution was concentrated. The residue was purified and concentrated by chromatography (SiO2, 12 g cartridge; methanol / dichloromethane = 0% → 5%) to obtain the title compound (0.200 g, 22.5%) as a purple solid.

[0199] [Step 2] Synthesis of methyl 6-((N-(3-chloro-4-fluorophenyl)morpholine-4-carboxamide)methyl)nicotinate

[0200] [ka]

[0201] N-(3-chloro-4-fluorophenyl)morpholine-4-carboxamide (0.200 g, 0.773 mmol) prepared in Step 1 was dissolved in N,N-dimethylformamide (5 mL) at 0°C. Sodium hydride (60.00%, 0.037 g, 0.928 mmol) was added to the solution and the mixture was stirred at the same temperature. Methyl 6-(bromomethyl)nicotinate (0.196 g, 0.850 mmol) was added to the reaction mixture and the mixture was stirred for a further 3 hours. Water was poured over the reaction mixture and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, water was removed with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified and concentrated by chromatography (SiO2, 4 g cartridge; ethyl acetate / hexane = 0% → 70%) to obtain the title compound (0.110 g, 34.9%) as brown oil.

[0202] [Step 3] Synthesis of N-(3-chloro-4-fluorophenyl)-N-((5-(hydrazinecarbonyl)pyridine-2-yl)methyl)morpholine-4-carboxamide

[0203] [ka]

[0204] The mixture of methyl 6-((N-(3-chloro-4-fluorophenyl)morpholine-4-carboxamide)methyl)nicotinate (0.110 g, 0.270 mmol) prepared in Step 2 and hydrazine monohydrate (0.262 mL, 5.394 mmol) was mixed with ethanol (5 mL) at room temperature. After heating under reflux for 18 hours, the temperature was lowered to room temperature, water was added to the reaction mixture, and it was extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride aqueous solution, water was removed with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The title compound was used without further purification (0.110 g, 100.0%, bright yellow solid).

[0205] [Step 4] Synthesis of Compound 232

[0206] [ka]

[0207] N-(3-chloro-4-fluorophenyl)-N-((5-(hydrazinecarbonyl)pyridine-2-yl)methyl)morpholine-4-carboxamide (0.110 g, 0.270 mmol) and N,N-diisopropylethylamine (0.070 mL, 0.405 mmol) prepared in Step 3 were dissolved in dichloromethane (3 mL) at 0°C. To this solution, 2,2-difluoroacetic anhydride (0.059 mL, 0.539 mmol) was added, and the mixture was stirred at room temperature for 16 hours. A saturated aqueous sodium bicarbonate solution was poured over the reaction mixture, and after extraction with dichloromethane, the two-phase mixture was filtered through a plastic filter to remove the solid residue and aqueous layer. The collected organic layer was concentrated under reduced pressure. The residue was purified and concentrated by chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 0% → 5%) to obtain the title compound (0.057 g, 45.2%) as a yellow solid.

[0208] 1 HNMR (400MHz,CDCl3)δ9.24-9.24(m,1H),8.3 6(dd,1H,J=8.2,2.2Hz),7.59(dd,1H,J=8.2,0.8Hz),7.30-7.28(m,1H),7.10-7.08(m,2H),6. 93(t,1H,J=51.6Hz),5.05(s,2H),3.54-3.52(m,4H),3.27-3.26(m,4H);LRMS(ES)m / z468.2(M + +1).

[0209] Synthesis Example 3. Synthesis of Compound 239: N-(3-chlorophenyl)-N-((5-(5-(difluoromethyl)-1,3,4-oxadiazole-2-yl)pyridine-2-yl)methyl)thiomorpholine-4-carboxamide 1,1-dioxide

[0210] [Step 1] Synthesis of N-(3-chlorophenyl)thiomorpholine-4-carboxamide 1,1-dioxide

[0211] [ka]

[0212] A solution of 1-chloro-3-isocyanatebenzene (1.000 g, 6.512 mmol) and thiomorpholine 1,1-dioxide (0.871 g, 6.447 mmol) dissolved in diethyl ether (20 mL) was stirred at room temperature for 18 hours. The precipitated solid was filtered, washed with diethyl ether, and dried to obtain the title compound (1.811 g, 96.3%) as a white solid.

[0213] [Step 2] Synthesis of methyl 6-((N-(3-chlorophenyl)-1,1-dioxide thiomorpholine-4-carboxamide)methyl)nicotinate

[0214] [ka]

[0215] N-(3-chlorophenyl)thiomorpholine-4-carboxamide 1,1-dioxide (0.200 g, 0.693 mmol) prepared in Step 1 was dissolved in N,N-dimethylformamide (5 mL) at 0°C. Sodium hydride (60.00%, 0.028 g, 0.693 mmol) was added to the solution, and the reaction mixture was stirred at the same temperature for 1 hour. Methyl 6-(bromomethyl)nicotinate (0.159 g, 0.693 mmol) was added to the reaction mixture at the same temperature, and the mixture was stirred for a further 2 hours. Water was poured over the reaction mixture and extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride aqueous solution, water was removed with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified and concentrated by chromatography (SiO2, 12 g cartridge; methanol / dichloromethane = 0% → 5%) to obtain the title compound (0.261 g, 86.0%) as brown oil.

[0216] [Step 3] Synthesis of N-(3-chlorophenyl)-N-((5-(hydrazinecarbonyl)pyridine-2-yl)methyl)thiomorpholine-4-carboxamide 1,1-dioxide

[0217] [ka]

[0218] The methyl 6-((N-(3-chlorophenyl)-1,1-dioxidethiomorpholine-4-carboxamide)methyl)nicotinate (0.261 g, 0.596 mmol) prepared in Step 2 and hydrazine monohydrate (0.290 mL, 5.958 mmol) were dissolved in ethanol (2 mL) at room temperature. The solution was stirred at 110 °C for 18 hours, after which the temperature was lowered to room temperature to terminate the reaction. The reaction mixture was concentrated under reduced pressure to remove the solvent. Water was added to the resulting concentrate, and after extraction with dichloromethane, the two-phase mixture was filtered through a plastic filter to remove the solid residue and aqueous layer. The collected organic layer was concentrated under reduced pressure. The residue was purified and concentrated by chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 5% → 15%) to obtain the title compound (0.261 g, 100.0%) as brown oil.

[0219] [Step 4] Synthesis of Compound 239

[0220] [ka]

[0221] The solution prepared in Step 3 by dissolving N-(3-chlorophenyl)-N-((5-(hydrazinecarbonyl)pyridine-2-yl)methyl)thiomorpholine-4-carboxamide 1,1-dioxide (0.261 g, 0.596 mmol), triethylamine (0.415 mL, 2.980 mmol), and 2,2-difluoroacetic anhydride (0.195 mL, 1.788 mmol) in tetrahydrofuran (2 mL) at room temperature was stirred at 80°C for 18 hours, after which the temperature was lowered to room temperature to terminate the reaction. The reaction mixture was concentrated under reduced pressure to remove the solvent, water was added to the resulting concentrate, and after extraction with dichloromethane, the two-phase mixture was filtered through a plastic filter to remove the solid residue and aqueous layer, and the collected organic layer was concentrated under reduced pressure. The residue was purified and concentrated by chromatography (SiO2, 4g cartridge; methanol / dichloromethane = 0% → 3%) to obtain the title compound (0.087g, 29.3%) as yellow foam.

[0222] 1 H NMR(400MHz,CDCl3)δ9.27(dd,1H,J=2.2,0.8Hz),8.43(dd,1H,J=8.2,2.2Hz),7.55(dd,1H,J=8.2,0.9Hz),7.31(t,1H,J=8.0Hz), 7.23(t,1H,J=2.1Hz),7.21-7.10(m,2H),7.10(t,1H),5.12(s,2H),3.75(t,4H,J=5.3Hz),3.06-2.99(m,4H);LRMS(ES)m / z498.3(M + +1).

[0223] Synthesis Example 4. Synthesis of Compound 243: N-((5-(5-(difluoromethyl)-1,3,4-oxadiazole-2-yl)pyridine-2-yl)methyl)-N-phenylmorpholine-4-carboxamide

[0224] [Step 1] Synthesis of N-phenylmorpholine-4-carboxamide

[0225] [ka]

[0226] A solution of isocyanatebenzene (1,000 g, 8.395 mmol) and morpholine (0.726 mL, 8.395 mmol) dissolved in diethyl ether (20 mL) at room temperature was stirred at room temperature for 18 hours. The precipitated solid was filtered, washed with diethyl ether, and dried to obtain the title compound (1.717 g, 99.2%) as a white solid.

[0227] [Step 2] Methyl 6-((N-phenylmorpholine-4-carboxamide)methyl) Nicotinate synthesis

[0228] [ka]

[0229] In Step 1, N-phenylmorpholine-4-carboxamide (0.200 g, 0.970 mmol) was prepared by dissolving it in N,N-dimethylformamide (5 mL) at 0°C. Sodium hydride (60.00%, 0.039 g, 0.970 mmol) was added to the solution, and the reaction mixture was stirred at the same temperature for 1 hour. Methyl 6-(bromomethyl)nicotinate (0.223 g, 0.970 mmol) was added to the reaction mixture at the same temperature, and the mixture was stirred for a further 2 hours. Water was poured over the reaction mixture, and it was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, water was removed with anhydrous magnesium sulfate, and the mixture was filtered and concentrated under reduced pressure. The residue was purified and concentrated by chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 0% → 5%) to obtain the title compound (0.294 g, 85.4%) as brown oil.

[0230] [Step 3] Synthesis of N-((5-(hydrazinecarbonyl)pyridine-2-yl)methyl)-N-phenylmorpholine-4-carboxamide

[0231] [ka]

[0232] The methyl 6-((N-phenylmorpholine-4-carboxamide)methyl)nicotinate (0.294 g, 0.828 mmol) prepared in Step 2 and hydrazine monohydrate (0.403 mL, 8.284 mmol) were dissolved in ethanol (2 mL) at room temperature. The solution was stirred at 110 °C for 18 hours, after which the temperature was lowered to room temperature to terminate the reaction. The reaction mixture was concentrated under reduced pressure to remove the solvent. Water was added to the resulting concentrate, and it was extracted with dichloromethane. The two-phase mixture was then filtered through a plastic filter to remove the solid residue and aqueous layer. The collected organic layer was concentrated under reduced pressure. The residue was purified and concentrated by chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 5% → 15%) to obtain the title compound (0.294 g, 100.0%) as brown oil.

[0233] [Step 4] Synthesis of N-((5-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)pyridine-2-yl)methyl)-N-phenylmorpholine-4-carboxamide

[0234] [ka]

[0235] The solution prepared in Step 3 by dissolving N-((5-(hydrazinecarbonyl)pyridine-2-yl)methyl)-N-phenylmorpholine-4-carboxamide (0.294 g, 0.828 mmol), triethylamine (0.577 mL, 4.142 mmol), and 2,2-difluoroacetic anhydride (0.270 mL, 2.485 mmol) in tetrahydrofuran (2 mL) at room temperature was stirred at 80°C for 18 hours, after which the temperature was lowered to room temperature to terminate the reaction. The reaction mixture was concentrated under reduced pressure to remove the solvent, water was added to the resulting concentrate, and after extraction with dichloromethane, the two-phase mixture was filtered through a plastic filter to remove the solid residue and aqueous layer, and the collected organic layer was concentrated under reduced pressure. The residue was purified and concentrated by chromatography (SiO2, 4 g cartridge; methanol / dichloromethane = 0% → 3%) to obtain the title compound (0.130 g, 44.2%) as a yellow solid.

[0236] [Step 5] Synthesis of Compound 243

[0237] [ka]

[0238] The solution of N-((5-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)pyridine-2-yl)methyl)-N-phenylmorpholine-4-carboxamide (0.130 g, 0.300 mmol) and 1-methoxy-N-triethylammoniosulfonyl-methaneimidate (Burgess reagent, 0.107 g, 0.450 mmol) prepared in Step 4 was dissolved in tetrahydrofuran (2 mL) at room temperature. The mixture was stirred at 80°C for 18 hours, after which the temperature was lowered to room temperature to terminate the reaction. The reaction mixture was concentrated under reduced pressure to remove the solvent, water was added to the resulting concentrate, and after extraction with dichloromethane, the two-phase mixture was filtered through a plastic filter to remove the solid residue and aqueous layer, and the collected organic layer was concentrated under reduced pressure. The residue was purified and concentrated by chromatography (SiO2, 4g cartridge; methanol / dichloromethane = 0% → 3%) to obtain the title compound (0.089g, 71.0%) as yellow oil.

[0239] 1 H NMR(400MHz,CDCl3)δ9.24(dd,1H,J=2.2,0.8Hz),8.37(dd,1H,J=8.2,2.2Hz),7.66(dd,1H,J=8.2,0.8Hz),7.39-7.30(m,2H),7.24-7.17(m ,2H),7.15-7.09(m,1H),7.01(t,1H,J=51.7Hz),5.15(s,2H),3.53(dd,4H,J=5.6,4.0Hz),3.28(dd,4H,J=5.6,4.0Hz);LRMS(ES)m / z416.1(M + +1).

[0240] Synthesis Example 5. Synthesis of Compound 286: N-(4-(5-(difluoromethyl)-1,3,4-oxadiazole-2-yl)-2-fluorobenzyl)-N-(3-fluorophenyl)thiomorpholine-4-carboxamide 1,1-dioxide

[0241] [Step 1] Synthesis of N-(3-fluorophenyl)thiomorpholine-4-carboxamide 1,1-dioxide

[0242] [ka]

[0243] 1-Fluoro-3-isocyanatebenzene (0.500 g, 3.647 mmol) was dissolved in diethyl ether (10 mL). Thiomorpholine 1,1-dioxide (0.493 g, 3.647 mmol) was added to this solution at 0°C and stirred at the same temperature for 1 hour, followed by stirring at room temperature for a further 4 hours. The precipitated solid was filtered, washed with diethyl ether, and dried to obtain the title compound (0.870 g, 87.6%) as a white solid.

[0244] [Step 2] Synthesis of methyl 3-fluoro-4-((N-(3-fluorophenyl)-1,1-dioxide thiomorpholine-4-carboxamide)methyl)benzoate

[0245] [ka]

[0246] The solution prepared by dissolving N-(3-fluorophenyl)thiomorpholine-4-carboxamide 1,1-dioxide (0.300 g, 1.102 mmol) and sodium hydride (60.00%, 0.048 g, 1.212 mmol) in N,N-dimethylformamide (5 mL) was stirred at 0°C for 2 hours. Methyl 4-(bromomethyl)-3-fluorobenzoate (0.299 g, 1.212 mmol) was added, and the mixture was stirred at room temperature for a further 17 hours. Then, water (2 mL) was added to the reaction mixture at room temperature, and the mixture was stirred for 10 minutes to terminate the reaction. After adding water to the reaction mixture and extracting with dichloromethane, the two-phase mixture was filtered through a plastic filter to remove the solid residue and aqueous layer, and the collected organic layer was concentrated under reduced pressure. The residue was purified and concentrated by chromatography (SiO2, 4g cartridge; ethyl acetate / hexane = 0% → 40%) to obtain the title compound (0.300g, 62.1%) as a white solid.

[0247] [Step 3] N-(2-fluoro-4-(hydrazinecarbonyl)benzyl)-N-( Synthesis of 3-fluorophenyl)thiomorpholine-4-carboxamide 1,1-dioxide

[0248] [ka]

[0249] Methyl 3-fluoro-4-((N-(3-fluorophenyl)-1,1-dioxide thiomorpholine-4-carboxamide)methyl)benzoate (0.300 s, 0.684 mmol) and hydrazine monohydrate (0.665 mL, 13.685 mmol) prepared in Step 2. mmol of the compound was mixed with ethanol (4 mL) at room temperature, microwaved, and heated at 120°C for 1 hour. The temperature was then lowered to room temperature to terminate the reaction. The reaction mixture was concentrated under reduced pressure to remove the solvent. Water was added to the concentrate, and it was extracted with dichloromethane. The two-phase mixture was then filtered through a plastic filter to remove the solid residue and aqueous layer. The collected organic layer was concentrated under reduced pressure. Diethyl ether (5 mL) and ethyl acetate (1 mL) were added to the residue and stirred at room temperature. The precipitated solid was filtered, washed with hexane, and dried to obtain the title compound (0.270 g, 90.0%) as a white solid.

[0250] [Step 4] Synthesis of Compound 286

[0251] [ka]

[0252] To a solution prepared in Step 3 by dissolving N-(2-fluoro-4-(hydrazinecarbonyl)benzyl)-N-(3-fluorophenyl)thiomorpholine-4-carboxamide 1,1-dioxide (0.100 g, 0.228 mmol) and triethylamine (0.095 mL, 0.684 mmol) in dichloromethane (4 mL) at room temperature, add 2,2-difluoroacetic anhydride (0.028 mL, 0.228 mmol) and stir at the same temperature for 17 hours. The mixture was mixed. A saturated aqueous sodium bicarbonate solution was poured into the reaction mixture, and after extraction with dichloromethane, the two-phase mixture was filtered through a plastic filter to remove the solid residue and aqueous layer, and the collected organic layer was concentrated under reduced pressure. The residue was purified and concentrated by chromatography (SiO2, 4g cartridge; ethyl acetate / hexane = 20% → 50%) to obtain the title compound (0.034g, 29.9%) as a white solid.

[0253] 1 H NMR(400MHz,CDCl3)δ7.90(dd,1H,J=8.0,1.6Hz),7.79(dd,1H,J=10.1,1.6Hz),7.68(t,1H,J =7.6Hz),7.38-7.33(m,1H),7.07-6.81(m,4H),4.95(s,2H),3.75(t,4H,J=5.2Hz),2.87(t,4H) ,J=5.2Hz);LRMS(ES)m / z499.0(M + +1).

[0254] <Experimental Example 1> Evaluation of mitochondrial axonal transport Transport assay (MATA)

[0255] This experiment aims to confirm the effects of the compounds disclosed herein on mitochondrial axonal transport.

[0256] <Experimental Example 1-1>HSPB1 S135F Mitochondrial axonal transport in CMT2F motor neurons

[0257] HSPB1S135F The effects of the compounds disclosed herein on mitochondrial movement within axons in motor neurons derived from a human iPSC-based model of CMT2F (hereinafter referred to as "CMT2F MNs") were evaluated as follows.

[0258] Wild type (WT) and HSPB1 S135F iPSCs (induced pluripotent stem cells) were provided by Samsung Medical Center (ECT11-58-37, Ewha Womans University, IRB No. 2013-10-124) and transferred to the CKD pharmacology laboratory before the experiment. They were then differentiated into motor neurons according to the following protocol. To produce embryoid bodies, the iPSC colonies were dissociated into small clumps using enzymes and cultured in a Petri dish in suspension for two days. The culture medium was supplemented with 10 μM Y27632 (Rho kinase inhibitor; Y0503, Tocris Bioscience, Bristol, UK), 20 ng / mL bFGF (PHG0024, Gibco), 10 μM SB435142 (SMAD inhibitor; S4317, Sigma), 0.2 μM LDN193189 (SMAD inhibitor; SML0559, Sigma), and penicillin / streptomycin (15140122, Gibco). On day 5, 1 μM retinoic acid (R2625, Sigma), 0.4 μg / mL ascorbic acid (A4544, Sigma), 10 ng / mL BDNF (brain-derived neurotrophic factor; SRP3014, Sigma), and 1% N2 supplement (17502048, Gibco) were added for caudalization. On day 7, for ventralization, cells were washed with SB435142 and LDN193189 (SMAD inhibitors), and 1 μM purmorphamine (sonic hedgehog agonist; SML0868, Sigma) was added. On day 17, cells were mixed with Neurobasal medium prepared on day 7, containing 10 ng / mL IGF-1 (I3769, Sigma), 10 ng / mL GDNF (G1777, Gibco), and 10 ng / mL 2% B-27. TMCells were cultured in medium supplemented with (A1486701, Gibco). Cells were cultured in a suspension in a Petri dish. After 21 days, the neurospheres were converted to an accutase solution. TM The factors were isolated using (A1110501, Gibco), plated onto poly-L-lysine / laminin-coated culture dishes or confocal dishes (211350, SPL), and supplemented with Neurobasal medium containing all previous factors, 25 μM β-mercaptoethanol (21985023, Gibco), and 25 μM glutamate (G1626, Sigma).

[0259] Differentiated nerve cells were treated with compounds 43, 232, 239, 243, and 286 (300 nM) of the present disclosure for 3 hours each. The control group cells were incubated in a medium containing 0.05% DMSO (D2650-5X 10 ml, Sigma) at a final concentration. To stain mitochondria, cells were treated with 0.01 nM Mitotracker Red CMXRos (M7512, Life Technologies, NY, USA) for 15 minutes immediately before imaging.

[0260] Mitochondria axonal migration in neuronal axons was imaged using a confocal microscope (Leica SP8; Leica microsystems, UK) equipped with a Live cell imaging chamber (Live cell instrument, Seoul, Korea) that maintains cells at 37°C in a 5% CO2 / 95% air environment. Time-lapse images were taken every second with an exposure time of 500 m / s for 1 minute, and the images were analyzed using IMARIS software (BITPLANE, Zurich, Switzerland) to calculate instantaneous velocity. Using IMARIS, each mitochondria in the neuronal axon was captured as an individual particle in each frame. To select the optimal velocity range, the instantaneous velocity of each mitochondria at each time point was plotted as a histogram at 0.5 μm / sec intervals. A section in which the instantaneous velocity of the CMT2F MNs group was approximately 30-40% lower than that of the WT MNs group was selected, and the data was then normalized to set the velocity of the WT MNs group to 1. Thus, the restorative effect of the compounds disclosed herein on mitochondrial velocity was calculated as a single relative value, and GraphPad Prism5 (GraphPad Software, Inc., USA) was used to count and plot the frequency of instantaneous velocities.

[0261] One-way ANOVA was performed using GraphPad Prism5 software, and all data were expressed as mean ± SEM.

[0262] The results of evaluating the effects of the compounds disclosed herein on mitochondrial axonal transport are shown in the table below (Figure 1).

[0263] [Table 31]

[0264] From the table above, it was confirmed that the compounds of this disclosure have the effect of improving and restoring the mitochondrial migration rate within axons.

[0265] <Experimental Example 1-2> Gars P234KY Mitochondrial axonal transport in CMT2D motor neurons

[0266] Gars P234KY The effects of the compounds disclosed herein on mitochondrial movement within axons in motor neurons derived from a human iPSC-based model of CMT2D (hereinafter referred to as "CMT2D MNs") were evaluated as follows.

[0267] Wild type (WT) and Gars P234KY iPSCs (induced pluripotent stem cells) were provided by Samsung Medical Center (IRB No. 2016-12-001) and transferred to the CKD Pharmacology Laboratory before the experiment. They were then differentiated into motor neurons according to the following protocol. To produce embryoid bodies, the iPSC colonies were dissociated into small clumps using enzymes and cultured in a suspension petri dish for two days. The culture medium was supplemented with 10 μM Y27632 (Rho kinase inhibitor; Y0503, Tocris Bioscience, Bristol, UK), 20 ng / mL bFGF (PHG0024, Gibco), 10 μM SB435142 (SMAD inhibitor; S4317, Sigma), 0.2 μM LDN193189 (SMAD inhibitor; SML0559, Sigma), and penicillin / streptomycin (15140122, Gibco). On day 5, 1 μM retinoic acid (R2625, Sigma), 0.4 μg / mL ascorbic acid (A4544, Sigma), 10 ng / mL BDNF (brain-derived neurotrophic factor; SRP3014, Sigma), and 1% N2 supplement (17502048, Gibco) were added for caudalization. On day 7, SB435142 and LDN193189 (SMAD inhibitors) were washed and 1 μM purmorphamine (Sonic) was added for ventralization. Hedgehog agonist (SML0868, Sigma) was added. On day 17, cells were incubated in Neurobasal medium [prepared on day 7, with 10 ng / mL IGF-1 (I3769, Sigma), 10 ng / mL GDNF (G1777, Gibco), and 10 ng / mL 2% B-27]. TM Cells were cultured in medium supplemented with (A1486701, Gibco). Cells were cultured in a suspension in a Petri dish. After 21 days, the neurospheres were converted to an accutase solution. TM The factors were isolated using (A1110501, Gibco), plated onto poly-L-lysine / laminin-coated culture dishes or confocal dishes (211350, SPL), and supplemented with Neurobasal medium containing all previous factors, 25 μM β-mercaptoethanol (21985023, Gibco), and 25 μM glutamate (G1626, Sigma).

[0268] Differentiated nerve cells were treated with compound 43 (20, 300, and 500 nM) of the present disclosure for 2 hours each. The control group cells were incubated in medium containing DMSO (D2650-5X 10 ml, Sigma) at a final concentration of 0.05%. To stain mitochondria, cells were treated with 0.01 nM Mitotracker Red CMXRos (M7512, Life Technologies, NY, USA) for 15 minutes immediately before imaging.

[0269] Mitochondria axonal migration in neuronal axons was imaged using a confocal microscope (Leica SP8; Leica microsystems, UK) equipped with a Live cell imaging chamber (Live cell instrument, Seoul, Korea) that maintains cells at 37°C in a 5% CO2 / 95% air environment. Time-lapse images were taken every second with an exposure time of 500 m / s for 1 minute, and the instantaneous velocity was calculated by analyzing the images with IMARIS software (BITPLANE, Zurich, Switzerland). Using IMARIS, each mitochondria in the neuronal axon was captured as an individual particle in each frame. To select a velocity range, the instantaneous velocity of each mitochondria at each time point was plotted as a histogram at 0.5 μm / sec intervals. An interval was selected in which the instantaneous velocity of the CMT2D MNs group was approximately 40% lower than that of the WT MNs group. The data was then normalized to set the velocity of the WT MNs group to 1. Thus, the recovery effect of the compounds disclosed herein on mitochondrial velocity was calculated as a single relative value, and GraphPad Prism5 (GraphPad Software, Inc., USA) was used to count and plot the frequencies of instantaneous velocities.

[0270] One-way ANOVA was performed using GraphPad Prism 5.0 software (post hoc: Dunnett's multiple comparison test), and all data were expressed as mean ± SEM.

[0271] The results of evaluating the effect of the compound of the present invention on mitochondrial axonal transport are shown in the table below (Figure 2).

[0272] [Table 32]

[0273] The above table confirms that the compounds of this disclosure exhibit a dose-dependent effect in improving and restoring the mitochondrial migration rate within axons.

[0274] <Experimental Example 2> Protein Expression Evaluation

[0275] This experiment aims to confirm the effect of the compounds disclosed herein on the expression of myelin-associated protein (PMP22) in C22 mice. 2.5-week-old wild-type (WT) and C22 mice (TG(PMP22)C22Clh) were transferred to the animal facility at the CKD Institute and kept for one week for acclimatization. The animals were given a standard diet (Central Lab Animal, Inc.) and water freely (ad libitum) and housed in a controlled environment with temperature (22±2℃), humidity (44~56%), and a 12-hour light-dark cycle. All experimental procedures were carried out at the IACUC (Institutional Animal Care and Research Center) of the CKD Laboratory Animal Center in Korea. Approved and implemented in accordance with the Use Committee (IACUC ani) mal study protocol approval number:S-17-033).

[0276] Each group was classified as shown in the table below.

[0277] [Table 33]

[0278] Compound 43 (3 mg / kg) of the present disclosure was administered twice daily for two weeks. Within 0.5 hours after the final dose, the sciatic nerve was collected and dissolved in ice-cold RIPA buffer (Cell signaling, 9803) containing a cocktail of protase inhibitors (Roche, 04693132001) and a phosphatase inhibitor (Roche, 04906837001).

[0279] The protein lysate was placed on an SDS-PAGE and transferred to an NC membrane. The membrane was blocked with 3% BSA-TBST for 1 hour, then incubated with tubulin antibody overnight at 4°C. After three washes, the membrane was incubated with secondary antibody for 1 hour and washed three more times. The membrane was visualized using ECL detection reagent (GE Healthcare, RPN2235), and the band intensity was measured using ChemiDoc. TM Measurements were taken using MP (BIO-RAD, 12003154).

[0280] All results are expressed as mean ± SEM, and statistical significance was analyzed using unpaired t-test, one-tailed t-test, and GraphPad Prism5 (GraphPad Softrage, Inc., USA).

[0281] As a result, as shown in Figure 3, we confirmed that the compounds of this disclosure have the effect of suppressing the expression of PMP22 protein induced in C22 mice.

[0282] <Experiment Example 3> Behavioral Evaluation

[0283] This experiment aims to evaluate the effectiveness of the compounds of the present invention by confirming the effects of the compounds disclosed herein on the motor function of animals.

[0284] <Experimental Example 3-1> Behavioral evaluation in a CMT1A mouse model

[0285] Male CMT1A mice (C22) aged 2.5 weeks were fed a standard diet (Central Lab Animal, Inc.) and water freely (ad libitum), and housed in a controlled environment with temperature (22±2℃), humidity (44~56%), and a 12-hour light-dark cycle. All experimental procedures were approved in accordance with the IACUC (Institutional Animal Care and Use Committee) of the Korea CKD Laboratory Animal Center. The procedure was implemented (approved number: S-17_033).

[0286] For two weeks, the Rotarod test, Balance beam test, and Grip strength test were performed once daily. Subsequently, the animals were classified into groups according to the Z-array method based on the Rotarod test, Balance beam test, Grip strength test, and body weight values, as shown in the table below.

[0287] [Table 34]

[0288] Compound 43 (0.3 and 3 mg / kg) of this disclosure was administered orally twice daily for two weeks, with behavioral testing conducted 30 minutes after administration. Behavioral testing was performed once a week in the Behavioral Assessment Room at the CKD Research Institute.

[0289] Data are expressed as mean ± SEM. Statistical significance between the compound treatment groups and vehicle groups was analyzed using an unpaired t-test for comparisons between two groups, and one-way ANOVA (post-hoc analysis using Dunnett's test) for comparisons between three or more groups. When an independent variable (e.g., duration of administration) was added, statistical significance was analyzed using two-way ANOVA (post-hoc analysis using Bonferroni's test). All statistical analyses were performed using GraphPad Prism (ver5.0).

[0290] Constant robot test (CRT)

[0291] The Rotarod test (LE8205, Panlab) was performed to assess forced motor activity and coordination function. For adaptation, all test animals underwent adaptation training five times a day at 8 rpm over a three-day course, and animals meeting the 150-180 second latency to fall category were used for additional experiments (approximately 80% of the animals met this category). Latency to fall was measured three times at a fixed speed of 8 rpm for three minutes. The Rotarod test was repeated three times per experiment, and the maximum of the three measurements was used as the test result (latency to fall).

[0292] As a result, as shown in Figure 4, it was confirmed that the compounds of this disclosure exhibit a significant effect in improving the descent delay time.

[0293] Grip strength test (GST)

[0294] One of the main symptoms of CMT is muscle degeneration due to the degeneration of nerves that control movement. Clinically, the degree of muscle degeneration is assessed by grip strength and ankle dorsiflexion. GST (BIO-GS3, BIOSEB) was performed to evaluate the function of all four legs using a grid (wire mesh). All experiments were performed by one person, and the maximum value from five consecutive measurements was used as the test result. The mouse was given a wire mesh to grasp, a weak tension was applied, and the mouse was gently pulled at an angle of approximately 15° to measure the maximum tension.

[0295] As a result, as shown in Figure 5, it was confirmed that the compounds of this disclosure exhibit a dose-dependent effect that significantly improves grip strength.

[0296] Balance beam test (BBT)

[0297] The balance beam test was performed to measure motor coordination. This measured the function of the hind limbs and observed balance. A rod (1.2 cm wide, 0.6 cm high, 1.0 m long) was fixed at a 9° angle (starting point height 45 cm, ending point height 60 cm). At the starting point, the mouse was stimulated with a 60W light, and at the ending point, a dark box without light was prepared to give the mouse a sense of security. All experimental animals were adapted to the same conditions as the experiment 30 minutes prior to evaluation. Mice were made to walk from the starting point to the ending point, and the number of slips was measured after the start. Prior to the test, the mice were trained three times a day for two days, and the results on the third day were used for grouping. Each experiment was independently evaluated by two individuals.

[0298] As a result, as shown in Figure 6, it was confirmed that the compound of the present invention has the effect of significantly reducing the number of slips.

[0299] In other words, the compounds of this disclosure significantly improved motor function (CRT, GST, BBT) in CMT1A mice, and we were able to confirm that they can be useful in the prevention and treatment of CMT.

[0300] <Experimental Example 3-2> Behavioral evaluation in the CMT2F mouse model

[0301] 7-month-old male CMT2F mouse (HSPB1 S135F The animals were fed a standard diet (Central Lab Animal, Inc.) and water freely (ad libitum), and housed in a controlled environment with a temperature of 22±2℃, humidity of 44-56%, and a 12-hour light-dark cycle. All experimental procedures were approved and carried out in accordance with the IACUC (Institutional Animal Care and Use Committee) of the Korea CKD Laboratory Animal Center (approved number: S-17_033).

[0302] The Rotarod test and grip strength test were performed once daily for two weeks. Subsequently, the animals were classified into groups according to the Z-array method based on the Rotarod test, grip strength test, and body weight values, as shown in the table below.

[0303] [Table 35]

[0304] Compound 43 (1 and 10 mg / kg) of this disclosure was administered orally twice daily for 8 weeks, with behavioral testing conducted within 1 hour after administration. Behavioral testing was performed at weeks 0, 4, and 8.

[0305] Data are expressed as mean ± SEM, and statistical significance was analyzed using two-way ANOVA (posttest, Bonfeeroni). All statistical analyses were performed using GraphPad Prism 5.0.

[0306] Constant robot test (CRT)

[0307] The RotaRod test (LE8205, Panlab) was performed to assess forced motor activity and coordination function. For adaptation, all test animals underwent a 3-day course of adaptive training at 8 rpm five times a day, and animals meeting the 150-180 second descent delay category were used for additional experiments (approximately 80% of the animals met this category). Descent delay was measured three times at a fixed speed of 8 rpm for 3 minutes. The RotaRod test was repeated three times per experiment, and the maximum of the three measurements was used as the test result (descent delay).

[0308] As a result, as shown in Figure 7, it was confirmed that the compounds of this disclosure exhibit a dose-dependent effect of significantly improving the decline delay time.

[0309] Grip strength test (GST)

[0310] One of the main symptoms of CMT is muscle degeneration due to the degeneration of nerves that control movement. Clinically, the degree of muscle degeneration is assessed by grip strength and ankle dorsiflexion. GST (BIO-GS3, BIOSEB) was performed to evaluate the function of all four legs using a grid (wire mesh). All experiments were performed by one person, and the maximum value from five consecutive measurements was used as the test result. The mouse was given a wire mesh to grasp, a weak tension was applied, and the mouse was gently pulled at an angle of approximately 15° to measure the maximum tension.

[0311] As a result, as shown in Figure 8, it was confirmed that the compounds of this disclosure exhibit a dose-dependent effect that significantly improves grip strength.

[0312] In other words, the compounds disclosed herein significantly improve motor function (CRT, GST) in CMT2F mice. We were able to improve its effectiveness and confirm its usefulness in the prevention and treatment of CMT.

[0313] <Experimental Example 4> Nerve conduction study (NCS)

[0314] This experiment aims to evaluate the effectiveness of the compounds of the present invention by confirming the effect of the compounds of the present disclosure on the nerve conduction velocity of animals.

[0315] <Experimental Example 4-1> 2.5-week-old CMT1A mouse model Two-and-a-half-week-old male CMT1A mice (C22) were fed a standard diet (Central Lab Animal, Inc.) and water freely (ad libitum), and housed in a controlled environment with temperature (22±2℃), humidity (44~56%), and a 12-hour light-dark cycle. All experimental procedures were approved and carried out in accordance with the IACUC (Institutional Animal Care and Use Committee) of the Korea CKD Laboratory Animal Center (approved number: S-17_033).

[0316] Each group was classified as shown in the table below.

[0317] [Table 36]

[0318] Compound 43 (1 mg / kg) of this disclosure was administered orally twice daily for two weeks.

[0319] Animals are exposed to isoflurane (USP Terral, Piramal) in 30% oxygen (Daehan gas) and 70% nitrogen (Daehan gas). The animal was anesthetized using Critical Care, Inc., NDC66794-017-25, and the hair on the distal back and hind limbs was completely removed by shaving. The skin was kept >32°E using an external heating device. Electrophysiological testing was performed. The oscillary nerve was recorded using the sciatic nerve, the largest nerve in the peripheral nervous system (PNS), and nerve conduction studies (NCS) were performed using the Nicolet Viking Quest. Compound motor action potential (CMAP) amplitude and motor neuron conduction velocity were measured. Neuron conduction velocity (MNCV) was measured.

[0320] Data are expressed as mean ± SEM. Statistical significance between the compound treatment group and vehicle group of the present invention was analyzed using an unpaired t-test for comparisons between two groups, and one-way ANOVA (post-hoc analysis using Dunnett's test) for comparisons between three or more groups. When an independent variable (e.g., duration of administration) was added, statistical significance was analyzed using two-way ANOVA (post-hoc analysis using Bonferroni's test). All statistical analyses were performed using GraphPad Prism (ver5.0).

[0321] As a result, as shown in Figure 9, it was confirmed that the compounds of this disclosure exhibit a significant effect in improving MNCV and CMAP.

[0322] In other words, we were able to confirm that the compounds of this disclosure improve nerve conduction velocity and can be usefully used for the prevention and treatment of CMT.

[0323] <Experimental Example 4-2> 8-month-old CMT1A mouse model

[0324] Eight-month-old male / female CMT1A mice (C22) were fed a standard diet (Central Lab Animal, Inc.) and water freely (ad libitum), and housed in a controlled environment with temperature (22±2℃), humidity (44-56%), and a 12-hour light-dark cycle. All experimental procedures were approved and carried out in accordance with the IACUC (Institutional Animal Care and Use Committee) of CKD Korea (IACUC No: S-17-033).

[0325] Each group was classified as shown in the table below.

[0326] [Table 37]

[0327] Compound 43 (1 mg / kg) of this disclosure was administered orally twice daily for 12 weeks.

[0328] Animals are exposed to isoflurane (USP Terral, Piramal) in 30% oxygen (Daehan gas) and 70% nitrogen (Daehan gas). The animal was anesthetized using Critical Care, Inc., NDC66794-017-25, and the hair on the distal back and hind limbs was completely removed by shaving. The skin was kept >32°E using an external heating device. Electrophysiological testing was performed. The oscillary nerve was recorded using the sciatic nerve, the largest nerve in the peripheral nervous system (PNS), and nerve conduction studies (NCS) were performed using the Nicolet Viking Quest. Compound motor action potential (CMAP) amplitude and motor neuron conduction velocity were measured. Neuron conduction velocity (MNCV) was measured.

[0329] Data are expressed as mean ± SEM. Statistical significance was analyzed using the unpaired Student's t-test for comparisons between two groups, and One-Way ANOVA (post-hoc analysis using Dunnett's test) for comparisons between three or more groups. All statistical analyses were performed using GraphPad Prism (ver5.0).

[0330] As a result, as shown in Figure 10, it was confirmed that the compounds of this disclosure exhibit a significant improvement in MNCV and CMAP.

[0331] In other words, we were able to confirm that the compounds of this disclosure improve nerve conduction velocity and can be usefully used for the prevention and treatment of CMT.

[0332] <Experiment Example 5> Histopathology analysis

[0333] This experiment aims to confirm the effect of the compounds disclosed herein on the axonal size of sciatic nerve fibers.

[0334] 6.5-8.5 month old male CMT2F mice (HSPB1 S135F The animals were fed a standard diet (Central Lab Animal, Inc.) and water freely (ad libitum), and were reared in a controlled environment with a temperature of 22±2℃, humidity of 44-56%, and a 12-hour light-dark cycle.

[0335] Each group was classified as shown in the table below.

[0336] [Table 38]

[0337] Compound 43 of this disclosure (1 and 10 mg / kg) was administered orally twice daily for 12 weeks. Sciatic nerves were collected 0.5 hours after the final treatment and fixed overnight in 2.5% glutaraldehyde solution (340855, Sigma). Samples were sent to the Department of Pathology at Gasan Hospital for semithin sections and toluidine blue staining (T3260, Sigma).

[0338] The fixed samples were processed using standard methods for image analysis. 0.5 μm sections were prepared and stained with toluidine blue.

[0339] Histological evaluation was performed using a light microscope. Pathological changes, including demyelination, remyelination, abnormally thin myelin, and axonal morphological changes, were examined in the sections. Finally, axonal diameter was analyzed using imageJ software.

[0340] Data are expressed as mean ± SEM. HSPB1 S135FCMT2F (Vehicle) group and HSPB1 S135F Statistical significance between the CMT2F (compound 43) groups was determined using one-way ANOVA and post-hoc analysis using Dunnett's test, comparing the WT group and HSPB1 S135F Statistical significance between the CMT2F(Vehicle) groups was analyzed using the paired T test. All statistical analyses were performed using GraphPad Prism 5.0.

[0341] The results of evaluating the effect of the compounds disclosed herein on the axonal size of sciatic nerve fibers are shown in the table below (Figure 11).

[0342] [Table 39]

[0343] From the table above, it was confirmed that the compounds of this disclosure have the effect of increasing and restoring axon size.

[0344] <Experimental Example 6> Genetic Analysis

[0345] This experiment aims to evaluate the effectiveness of the compound of the present invention by performing genetic analysis on animals.

[0346] Ten-week-old CMT1A mice (C3) were given standard food (Central Lab Animal, Inc.) and water freely (ad libitum), at a temperature of 22±2℃ and humidity of 4 The animals were reared in a controlled environment with a 4-56% exposure and a 12-hour light-dark cycle. All experimental procedures were approved and carried out in accordance with the IACUC (Institutional Animal Care and Use Committee) of the Korea CKD Laboratory Animal Center (approved number: S-17_033).

[0347] Each group was classified as shown in the table below.

[0348] [Table 40]

[0349] Vehicle was 0.5% MC (methylene chloride), and each group was administered either Vehicle or compound 43 for 8 weeks. TG or C3 refers to CMT1A mice (C3), WT (Wild type) is the group of normal mice administered only Vehicle, Vehicle (C3) or C3 group is the group of CMT1A mice (C3) administered only Vehicle, and compound 43 is the group of CMT1A mice (C3) administered compound 43. The number of mice per group was 5.

[0350] The sciatic nerve was collected 0.5 hours after the final administration of compound 43 to the vehicle, and RNA was extracted using a kit (RNeasy Kit, Qiagen, Venlo, Netherlands). RNA quantification and integrity evaluation were performed using Quant-IT RiboGreen (Invitrogen) and apeStation RNA screentape (Agilent, Santa Clara, CA, USA). The RNA was then quantified at the gene level by RNA sequencing using more than 24 million reads. From the quantified gene level, Gene Set enrichment analysis was performed to identify various biological effects (gene sets) between the WT, TG (C3), and drug administration groups (compound 43). The significance of these results was analyzed using thresholds of nominal p<0.05 and false detection rate (FDR) q<0.25. The gene sets that showed significant results were graphed using Prism9, and the results are shown in Figures 12-15.

[0351] As can be seen from Figure 12 above, when we distinguished between the WT, C3 group, and compound 43-treated group based on the gene module identification criteria (three PC values), the compound 43-treated group showed genetic characteristics closer to normal mice (WT) compared to the C3 group.

[0352] Furthermore, as can be seen from Figures 13 to 15, compound 43 was downregulated in CMT disease mice (TG, CMT1A mice (C3)) and postsynaptic dens It can be confirmed that the gene levels related to ity, postsynaptic specialization, neuromuscular junction, synaptic cleft, EGR2_SOX10 myelination, Schwann cell myelination / development, Schwann cell differentiation, lipid metabolism, and lipid biosynthesis have been increased.

[0353] In Figure 13, the group administered compound 43 showed improved myogenesis, postsynaptic density, postsynaptic specialization, neuromuscular junction, synaptic cleft, and EGR2_SOX10 compared to CMT disease mice (TG, CMT1A mice (C3)). Myelination (EGR2_SOX10 myelination), myelination / development of Schwann cells We can confirm that genes related to Schwann cell myelination / development, Schwann cell differentiation, lipid metabolism, and lipid biosynthesis are significantly regulated upward.

[0354] In particular, Figure 14 shows that the expression of individual genes included in the gene set also changes upon administration of compound 43, and Figure 15 shows that PM22 aggregates, myelination, neurotransmission, and lipid metabolism, which are thought to be associated with CMT disease, are also affected. It was confirmed that the genes were altered by the administration of compound 43, and in particular, it was found that the administration of compound 43 increased neurotransmission and myelination, and that the transcription of genes that regulate PM22 aggregation and lipid metabolism was upwardly regulated.

[0355] <Experimental Example 7> Analysis of Improvement in Muscle Fiber Atrophy

[0356] This experiment aims to evaluate the effectiveness of the compounds of the present invention by confirming the effects of the compounds of the present disclosure on muscle fiber atrophy neuromuscular junctions in CMT mice.

[0357] <Experimental Example 7-1> 10-week-old CMT1A mouse model

[0358] Ten-week-old CMT1A mice (C3) were fed a standard diet (Central Lab Animal, Inc.) and water freely (ad libitum), and housed in a controlled environment with temperature (22±2℃), humidity (44~56%), and a 12-hour light-dark cycle. All experimental procedures were approved and carried out in accordance with the IACUC (Institutional Animal Care and Use Committee) of the Korea CKD Laboratory Animal Center (approved number: S-17_033).

[0359] Each group was classified as shown in the table below.

[0360] [Table 41]

[0361] Vehicle was 0.5% MC (methylene chloride), and each group was administered either Vehicle or compound 43 for 7 weeks. TG or C3 refers to CMT1A mice (C3), WT (Wild type) is the group of normal mice administered only Vehicle, Vehicle (C3) or C3 group is the group of CMT1A mice (C3) administered only Vehicle, and compound 43 is the group of CMT1A mice (C3) administered compound 43. Each group consisted of 5 mice.

[0362] <Experimental Example 7-2> Analysis of muscle fiber atrophy and neuromuscular junction

[0363] 1) Autopsy

[0364] Seven weeks after administration of the vehicle or drug (compound 43), gastrocnemius muscles were harvested to analyze nerve distribution and muscle tissue at the neuromuscular junction. This procedure was performed 30 minutes after the last administration of the vehicle or drug.

[0365] 2) Analysis of muscle fiber atrophy

[0366] Mice were anesthetized with isoflurane and perfused systemically with physiological saline (3 mL). The gastrocnemius muscle was excised and fixed at room temperature with 10% neutral buffered formalin. The sample was cut to include the area to be evaluated, tissue processing was performed, and the sample was embedded in paraffin and cut into 4 μm thick sections using a slide microtome. The cut slides were stained with H&E and observed under a light microscope. The number of muscle fibers showing atrophy was divided by the number of normal muscle fibers to measure the percentage of muscle fibers showing atrophy, and the cross-sectional area of ​​the muscle fibers was analyzed using NIS-Elements software. The results are shown in Figures 16 and 17.

[0367] As can be seen from Figures 16 and 17, CMT1A mice (C3) (TG) had a higher proportion of atrophic muscle fibers and showed a significant decrease in muscle fiber cross-sectional area compared to normal mice (WT). However, in the group of CMT1A mice (C3) administered compound 43, the proportion of atrophic muscle fibers decreased and the muscle fiber cross-sectional area increased.

[0368] 3) Nerve distribution at the neuromuscular junction

[0369] 1) The gastrocnemius muscle was collected and frozen into 30 μm thick sections, and slides were prepared. The prepared section slides were immunostained with anti-synaptotagmin-2 and α-bungarotoxin. Subsequently, the nerve distribution level per neuromuscular junction (NMJ) was qualitatively analyzed by examining structures that stained positively with two antibodies per neuromuscular junction using a fluorescence microscope, as shown in Figure 18, and the results are shown in Figure 19.

[0370] As can be seen in Figure 19, CMT1A mice (C3) showed a decrease in fully innervated neuromuscular junctions (NMJs) in the gastrocnemius muscle compared to normal mice (WT). However, in the group of CMT1A mice (C3) administered compound 43, an increase in fully innervated NMJs was observed.

[0371] Furthermore, in section slide samples containing more than 80 neuromuscular junctions per sample, the double staining images for anti-synaptotagmin-2 and α-bungarotoxin showed positivity for both antibodies. After classifying the stained patterns into fully innervation (complete overlap), partially innervation (partial overlap), and denervation (no overlap), the percentage values ​​for each were calculated. The significance of the results and the resulting graphs were then analyzed and represented using Prism9, as shown in Figure 20.

[0372] As can be seen from Figure 20, CMT1A mice (C3) had a more than 10% reduction in fully nerve-fed neuromuscular junctions compared to normal mice (WT), and a higher proportion of partially nerve-fed and unnerve-fed neuromuscular junctions. On the other hand, in the group of CMT1A mice (C3) administered compound 43, the proportion of fully nerve-fed neuromuscular junctions increased, and the proportion of partially nerve-fed and unnerve-fed neuromuscular junctions decreased significantly.

[0373] This revealed that the compound of the present invention significantly increases nerve distribution at nerve junctions in CMT disease, and can effectively treat CMT disease, which is a peripheral nerve disease.

[0374] <Experimental Example 8> Analysis of Effects on Sensory Nerves 1

[0375] This experiment aims to evaluate the effectiveness of the compound of the present invention by confirming its effects on the sensory nerves of animals in CMT mice.

[0376] <Experimental Example 8-1> 10-week-old CMT1A mouse model

[0377] Ten-week-old CMT1A mice (C3) were fed a standard diet (Central Lab Animal, Inc.) and water freely (ad libitum), and housed in a controlled environment with temperature (22±2℃), humidity (44~56%), and a 12-hour light-dark cycle. All experimental procedures were approved and carried out in accordance with the IACUC (Institutional Animal Care and Use Committee) of the Korea CKD Laboratory Animal Center (approved number: S-17_033).

[0378] The counties were classified as shown in the table below.

[0379] [Table 42]

[0380] Vehicle was 0.5% MC (methylene chloride), and each group was administered either Vehicle or compound 43 for 8 weeks. TG or C3 refers to CMT1A mice (C3), WT (Wild type) is the group of normal mice administered only Vehicle, Vehicle (C3) or C3 group is the group of CMT1A mice (C3) administered only Vehicle, and compound 43 is the group of CMT1A mice (C3) administered compound 43. The number of mice in each group was 5.

[0381] <Experimental Example 8-2> Histopathological Evaluation

[0382] Eight weeks after administration of the vehicle or drug (compound 43), the sural nerve was collected 0.5 hours after the last drug dose and fixed overnight in a 2.5% glutaraldehyde solution. The fixed samples were subjected to standard tissue processing for image analysis, and 0.5 μm thick sections were prepared and stained with toluidine blue. Histological evaluation was performed using image files taken with a light microscope. Morphological changes in myelin, such as demyelination and remyelination, and pathological changes, including a decrease in axonal diameter, were evaluated in each sample. Axonal and myelin diameters were analyzed using imageJ software, and the g-ratio was calculated as the ratio of the internal axonal diameter to the total outer diameter. The results are shown in Figures 21-23.

[0383] Data were expressed as mean ± SEM. Statistical significance between groups was calculated using one-way ANOVA, and post-hoc tests were performed using Dunnett's test. All statistical analyses were performed using GraphPad Prism 9.0.

[0384] As can be seen in Figure 21, CMT1A mice (C3) showed a decrease in axon diameter and myelin thickness compared to WT mice, but when compound 43 was administered, both axon diameter and myelin thickness increased.

[0385] Furthermore, as can be seen from Figures 22 and 23, there was no significant difference in the proportion of demyelinated fibers between CMT1A mice (C3) and WT mice. However, significant differences were observed in the increase in the slope of the g-ratio and the decrease in axonal diameter in vehicle-treated C3 mice. Here, the g-ratio of CMT1A mice (C3) administered with compound 43 had a lower slope compared to vehicle-treated animals, which is related to the reduction in abnormal myelination caused by compound 43. In addition, compound 43 showed a tendency to increase the proportion of axons with larger diameters ([#p<0.05, ###p<0.001, Veh vs WT], [*p<0.05, **p<0.01, Veh vs compound 43]).

[0386] <Experimental Example 9> Analysis of Effects on Sensory Nerves 2

[0387] This experiment aims to evaluate the efficacy of the compounds disclosed herein by confirming their effects on sensory nerves in CMT mice.

[0388] <Experimental Example 9-1> 10-week-old CMT1A mouse model

[0389] Ten-week-old CMT1A mice were prepared in the same manner as in Experimental Example 8-1. Specifically, the mice were isolated and administered a vehicle or drug, as shown in Table 12. There were five mice in each group.

[0390] <Experimental Example 9-2> Electrophysiological Evaluation

[0391] To perform electrophysiological evaluation of sensory nerves, a stimulation cathode was placed at the tip of the mouse tail, a recording electrode was positioned 30 mm from the stimulation cathode near the body of the tail, and a ground electrode was placed on the animal's leg. Sensory nerve conduction velocity (SNCV) and sensory nerve action potential (SNAP) amplitude values ​​were obtained by stimulating the tail. The results of this study were obtained from Nicolet Viking Quest (Natsu Medical, Inc.) and are shown in Figures 24 and 25 below.

[0392] Data were expressed as mean ± SEM (standard error). Statistical significance between groups was examined using one-way ANOVA, and post-hoc tests were performed using Dunnett's test. All statistical analyses were performed using GraphPad Prism 9.0.

[0393] As can be seen from Figures 24 and 25, electrophysiological examination of sensory nerves showed that CMT1A mice (C3) exhibited a significant decrease in sensory nerve conduction velocity (SNCV) and sensory nerve action potential (SNAP) amplitude compared to WT mice. Compound 43 significantly increased both sensory nerve conduction velocity (SNCV) and sensory nerve action potential (SNAP) amplitude ([#p<0.05, ###p<0.001, Veh vs WT], [*p<0.05, **p<0.01, Veh vs Compound 43]).

[0394] This disclosure provides the following pharmaceutical compositions, methods, and uses.

[0395] Item 1. A pharmaceutical composition for the prevention or treatment of Charcot-Marie-Tooth disease (CMT) related to the peripheral nervous system (PNS), comprising as an active ingredient a compound represented by the chemical formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

[0396] Item 2. The pharmaceutical composition according to Item 1, wherein the compound represented by chemical formula I is at least one selected from the group consisting of compounds 1 to 450 listed in Table A.

[0397] Item 3. The pharmaceutical composition according to Item 1 or 2, wherein the compound represented by chemical formula I is at least one selected from the group consisting of compound 43, compound 232, compound 239, compound 243, and compound 286 as listed in Table B.

[0398] Item 4. A method for preventing or treating Charcot-Marie-Tooth disease related to the peripheral nervous system, comprising administering to an individual a compound represented by the aforementioned chemical formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, as described in Items 1 to 3.

[0399] Item 5. For the prevention or treatment of Charcot-Marie-Tooth disease relating to the peripheral nervous system, the compound represented by the aforementioned chemical formula I as described in Items 1 to 3, its optical isomer, or the Use of pharmaceutically acceptable salts.

[0400] Item 6. Use of the compound represented by the aforementioned chemical formula I, its optical isomer, or a pharmaceutically acceptable salt thereof, as described in Items 1 to 3, in the manufacture of a drug for the prevention or treatment of Charcot-Marie-Tooth disease relating to the peripheral nervous system.

[0401] Item 7. The Charcot-Marie-Tooth disease relating to the peripheral nervous system is at least one selected from the group consisting of CMT1, CMT2, CMT4, CMTX, DSN (dejerine-sottas syndrome), CH (congenital hypomyelination), HNPP (hereditary neuropathy with liability to pressure parsy), and GAN (giant axonal neuropathy), as described in any of Items 1 to 3, the method described in Item 4, or the use described in Item 5 or Item 6.

[0402] Item 8. Charcot-Marie-Tooth disease relating to the peripheral nervous system (PNS) is at least one selected from the group consisting of CMT1A, CMT2D, and CMT2F, as described in any of the pharmaceutical compositions described in Items 1 to 3, the method described in Item 4, or the use described in Item 5 or Item 6.

[0403] Item 9. The pharmaceutical composition described above is administered orally, as described in any of items 1-3, 7, and 8.

[0404] Item 10. The compounds represented by chemical formula I as described in Items 1 to 3, their optical isomers, or pharmaceutically acceptable salts thereof, which are administered orally, by any of the methods described in Items 4, 7, and 8 or by any of the methods described in Items 5 to 8.

[0405] Item 11. The composition, method, or use described herein is for the prevention or treatment of symptoms associated with the regression of the peripheral nervous system in a subject having Charcot-Marie-Tooth disease, the pharmaceutical composition described herein, the method described herein, or the use described herein, or the use described herein, or the method described herein, or the use described herein, or the method described herein, or the use described herein, or the method described herein, or the use described herein, or the method described herein, or the use described herein, or the method described herein, or the use.

[0406] Item 12. The composition, method, or use described herein is for the prevention or treatment of symptoms associated with the dysfunction and / or death of peripheral nerve cells in a subject having Charcot-Marie-Tooth disease, the pharmaceutically active composition described herein, the method

[0407] Item 13. The composition, method, or use described herein is for the prevention or treatment of peripheral neurodegenerative diseases caused by dysfunction and / or death of peripheral nerve cells in subjects having Charcot-Marie-Tooth disease, the pharmaceutical composition described herein, the method described herein, or the method described herein, or the use described herein, or the method use described herein, or the use.

[0408] Although specific parts of the present invention have been described in detail above, it will be clear to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for the prevention or treatment of Charcot-Marie-Tooth disease (CMT) related to the peripheral nervous system (PNS), comprising as an active ingredient a compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, A pharmaceutical composition wherein the Charcot-Marie-Tooth disease associated with the peripheral nervous system is at least one selected from the group consisting of CMT1, CMT2, CMT4, CMTX, DSN (dejerine-sottas syndrome), CH (congenetic hypomyelation), HNPP (hereditary neuropathy with liability to pressure palsy), and GAN (giant axonal neuropathy): Table 1

2. The pharmacochemical composition according to claim 1, wherein the Charcot-Marie-Tooth disease relating to the peripheral nervous system is one or more selected from the group consisting of CMT1A, CMT2D, and CMT2F.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered orally.

4. The use of a compound, an optical isomer thereof, or a pharmaceutically acceptable salt thereof in the manufacture of a drug for the prevention or treatment of Charcot-Marie-Tooth disease relating to the peripheral nervous system, wherein the compound has the following structure: The aforementioned Charcot-Marie-Tooth disease related to the peripheral nervous system includes CMT1, CMT2, CMT4, CMTX, DSN (dejerine-sottas syndrome), CH (congenetic hypomyelation), HNPP (hereditary neuropathy with liability to pressure palsy), and GAN (giant axonal neuropathy). At least one selected from the group consisting of athy, use: Table 2

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