Compositions and methods for treating neurodegenerative, myodegenerative and lysosomal storage disorders

Abstract

Provided herein are compositions and methods for treating or preventing a neurodegenerative disease, a myodegenerative disease, a prion disease, or a lysosomal storage disease in a subject.

Description

[0001] This application claims priority to U.S. Provisional Application No. 62 / 769,791, filed on November 20, 2018, which is hereby incorporated by reference in its entirety. Background Art

[0002] Neurodegenerative diseases include hereditary and sporadic conditions associated with progressive nervous system dysfunction. These diseases are characterized by progressive deterioration of nerve cells or nerve cell function. It is estimated that one in four Americans will develop a neurodegenerative disorder in their lifetime. However, in general, the underlying mechanisms causing the disorders are not well understood and few effective treatment options are available to prevent or treat neurodegenerative diseases.

[0003] Lysosomal storage disorders represent some of the most devastating genetic diseases, and the need to develop therapies for these disorders remains largely unmet. Some of these diseases cause damage to the central nervous system (CNS), but the underlying mechanisms of such damage are largely unknown. Although the incidence of lysosomal storage disorders is very low (less than about 1:100,000 individuals are affected, lysosomal storage disorders primarily affect children, who usually die in infancy, some months or years after birth. Other multiple children die after years of suffering from the various symptoms of their specific lysosomal storage disorders. Summary of the invention

[0004] Provided herein are compositions and methods for treating or preventing a neurodegenerative disease, a myodegenerative disease, a prion disease, or a lysosomal storage disease in a subject. Provided herein are compounds having formula I

[0005]

[0006] in

[0007] X is N or CH;

[0008] Y is unsubstituted or substituted by R 1 Substituted C 6-10 Aryl; or unsubstituted or R 1 Substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0009] R 1 Yes – (CH2) n -R 2 、-(CH2) n -C(O)-R 2 or –O(CH2) n -R 2 ;

[0010] R 2Yes –H, -CN, halogen, C 1-3 Alkyl, C 1-3 Alkoxy, phenyl, pyridyl, amino, C 1-3 Alkylamino, di-C 1-3 Alkylamino, hydroxyl C 1-3 Alkylamino, carboxyl C 1-3 Alkylamino, C 3-6 Cycloalkyl C 1-3 Alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxy C 1-3 Alkylpyrrolidinyl, carboxylpyrrolidinyl, piperidinyl, C 1-3 Alkyl piperidinyl, di-C 1-3 Alkyl piperidinyl, piperazinyl, C 1-3 Alkylpiperazine, C 1-4 Alkoxycarbonylpiperazinyl or morpholinyl;

[0011] Z is heteroaryl, heterocyclic or NR 3 R 4 ;

[0012] R 3 and R 4 Independently H, C 1-3 Alkyl, C 1-3 alkoxy or unsubstituted phenyl, and

[0013] n is an integer selected from 0 to 3,

[0014] or an isomer or a pharmaceutically acceptable salt thereof.

[0015] Also provided is a method for treating or preventing a neurodegenerative disease, a myodegenerative disease or a prion disease in a subject, the method comprising administering an effective amount of a compound having formula I to a subject suffering from the neurodegenerative disease, the myodegenerative disease or the prion disease or at risk of developing the neurodegenerative disease, the myodegenerative disease or the prion disease

[0016]

[0017] in

[0018] X is N or CH;

[0019] Y is unsubstituted or substituted by R 1 Substituted C 6-10 Aryl; or unsubstituted or R 1 Substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0020] R 1 Yes – (CH2) n -R2 、-(CH2) n -C(O)-R 2 or –O(CH2) n -R 2 ;

[0021] R 2 Yes –H, -CN, halogen, C 1-3 Alkyl, C 1-3 Alkoxy, phenyl, pyridyl, amino, C 1-3 Alkylamino, di-C 1-3 Alkylamino, hydroxyl C 1-3 Alkylamino, carboxyl C 1-3 Alkylamino, C 3-6 Cycloalkyl C 1-3 Alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxy C 1-3 Alkylpyrrolidinyl, carboxylpyrrolidinyl, piperidinyl, C 1-3 Alkyl piperidinyl, di-C 1-3 Alkyl piperidinyl, piperazinyl, C 1-3 Alkylpiperazine, C 1-4 Alkoxycarbonylpiperazinyl or morpholinyl;

[0022] Z is heteroaryl, heterocyclic or NR 3 R 4 ;

[0023] R 3 and R 4 Independently H, C 1-3 Alkyl, C 1-3 alkoxy or unsubstituted phenyl, and

[0024] n is an integer selected from 0 to 3,

[0025] or an isomer or a pharmaceutically acceptable salt thereof.

[0026] Also provided is a method for inhibiting or preventing toxic protein aggregation in neurons. The method comprises contacting the neurons with an effective amount of a compound having formula I:

[0027]

[0028] in,

[0029] X is N or CH;

[0030] Y is unsubstituted or substituted by R 1 Substituted C 6-10 Aryl; or unsubstituted or R 1 Substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0031] R 1 Yes – (CH2) n -R 2 、-(CH2) n -C(O)-R 2 or –O(CH2) n -R 2 ;

[0032] R 2 Yes –H, -CN, halogen, C 1-3 Alkyl, C 1-3 Alkoxy, phenyl, pyridyl, amino, C 1-3 Alkylamino, di-C 1-3 Alkylamino, hydroxyl C 1-3 Alkylamino, carboxyl C 1-3 Alkylamino, C 3-6 Cycloalkyl C 1-3 Alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxy C 1-3 Alkylpyrrolidinyl, carboxylpyrrolidinyl, piperidinyl, C 1-3 Alkyl piperidinyl, di-C 1-3 Alkyl piperidinyl, piperazinyl, C 1-3 Alkylpiperazine, C 1-4 Alkoxycarbonylpiperazinyl or morpholinyl;

[0033] Z is heteroaryl, heterocyclic or NR 3 R 4 ;

[0034] R 3 and R 4 Independently H, C 1-3 Alkyl, C 1-3 alkoxy or unsubstituted phenyl, and

[0035] n is an integer selected from 0 to 3,

[0036] or an isomer or a pharmaceutically acceptable salt thereof.

[0037] Also provided are methods of treating or preventing a lysosomal storage disorder (LSD) in a subject. The method comprises administering to a subject suffering from or at risk of developing a LSD an effective amount of a compound having Formula I:

[0038]

[0039] in,

[0040] X is N or CH;

[0041] Y is unsubstituted or substituted by R 1 Substituted C 6-10Aryl; or unsubstituted or R 1 Substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0042] R 1 Yes – (CH2) n -R 2 、-(CH2) n -C(O)-R 2 or –O(CH2) n -R 2 ;

[0043] R 2 Yes –H, -CN, halogen, C 1-3 Alkyl, C 1-3 Alkoxy, phenyl, pyridyl, amino, C 1-3 Alkylamino, di-C 1-3 Alkylamino, hydroxyl C 1-3 Alkylamino, carboxyl C 1-3 Alkylamino, C 3-6 Cycloalkyl C 1-3 Alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxy C 1-3 Alkylpyrrolidinyl, carboxylpyrrolidinyl, piperidinyl, C 1-3 Alkyl piperidinyl, di-C 1-3 Alkyl piperidinyl, piperazinyl, C 1-3 Alkylpiperazine, C 1-4 Alkoxycarbonylpiperazinyl or morpholinyl;

[0044] Z is heteroaryl, heterocyclic or NR 3 R 4 ;

[0045] R 3 and R 4 Independently H, C 1-3 Alkyl, C 1-3 alkoxy or unsubstituted phenyl, and

[0046] n is an integer selected from 0 to 3,

[0047] or an isomer or a pharmaceutically acceptable salt thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] This application includes the following figures. The figures are intended to illustrate certain embodiments and / or features of the compositions and methods and are intended to supplement any description of the compositions and methods. The figures do not limit the scope of the compositions and methods unless the written description explicitly indicates otherwise.

[0049] Figure 1(Left and middle panels) show that neuroprotective effects were observed in B35 cells treated with 1 μM BK41043 after 16 hours of treatment, as evidenced by a decrease in lactate dehydrogenase (LDH) and an increase in 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), respectively, as compared to controls. Figure 1 (Right panel) shows that after five hours of treatment, there was a gradual increase in cell viability via reduction of LDH at decreasing concentrations of BK40143.

[0050] Figure 2 Cell viability of B35 cells is shown 24 hours after transfection with pTau (left) or α-synuclein (right) and five hours after treatment with BK41043. Dehydrogenase (LDH) measured in the culture medium reflects cell death and was not different between control and cells expressing tau or α-synuclein with or without BK40143, indicating that this compound does not cause toxicity or cell death.

[0051] Figure 3 Shown are pTau(181) levels 24 hours after transfection (left panel, Student's t-test: unpaired, two-tailed, *p<0.05, ***p<0.01), and BK41043 reduced pTau(181) levels in pTau-transfected B35 cells (right panel, n=6, ANOVA: normal, one-way, Dunnett's multiple comparison test, p<0.05, ****p<0.0001).

[0052] Figure 4 α-synuclein levels after 24 hours transfection are shown (left panel), and BK41043 reduces α-synuclein in α-synuclein transfected B35 cells (right panel). Student's t test: unpaired, two-tailed, *p<0.05, **p<0.0, n=6). In this cell culture model, BK40143 doses ranging from 10uM-1mM appear to reduce α-synuclein levels.

[0053] Figure 5 Shown are cell viability of B35 cells after five hours of treatment with BK40197.

[0054] Figure 6 BK40143 is shown to reduce pTau(181) in Tau-expressing transgenic mice after seven days of treatment. pTau(181) levels were measured via ELISA. Student's t-test: unpaired, two-tailed, Welch's correction, *p<0.05, ***p<0.01

[0055] Using Western blot analysis, Figure 7 It was shown that treatment with BK-40143 (1.25 mg / kg, 2.5 mg / kg or 5.0 mg / kg) did not affect pTau(231) (AT180) levels in rTG4510 transgenic mice (n=4).

[0056] Figure 8 Tau (HT7) in Tau transgenic mice was shown to be significantly reduced after seven days of treatment with 1.25 mg / kg and 2.5 mg / kg of BK-41043. Student's t test: unpaired, two-tailed, Welch's correction, *p<0.05, ***p<0.01, n=4

[0057] Fig. 9 It is shown that treatment with BK-40143 (1.25 mg / kg or 2.5 mg / kg) results in in vivo inhibition of DDR1 as measured by detection of phosphorylated (active) DDR1 (pMCK10). This data indicates that BK40143 effectively inhibits DDR in vivo.

[0058] Fig.10 Shown is a decrease in pTau(191) in CamP301L mice after treatment with 2.5 mg / kg or 5 mg / kg BK-40143. Student's t test: unpaired, Welch's correction, one-tailed, *p>0.05, p=0.02, n=4.

[0059] Fig.11A It was shown that 1 mM and 100 uM BK40196 significantly reduced α-synuclein levels in transfected B35 cells.

[0060] Fig. 11B It was shown that BK40197 did not significantly reduce α-synuclein levels in transfected B35 cells.

[0061] Fig. 12A -D shows that BK40143 significantly reduces α-synuclein in A53T mice. Male and female 12-month-old A53T mice were treated with 2.5 mg / kg BK40143 ip for 21 consecutive days. Fig. 12A is an ELISA for human α-synuclein, which shows a significant 39% reduction in α-synuclein levels in BK40143 treated animals compared to DMSO treated control A53T mice. C57BL / 6J mice were used as a control and showed no detectable (ND) human α-synuclein. Fig. 12BBK40143 was shown to increase overall levels of dopamine (30%). BK40143 did increase levels of the dopamine metabolite homovanillic acid (HVA) in A53T mice, indicating more dopamine turnover, which could lead to better dopamine neurotransmission. Fig. 12C and 12D is an immunoblot for α-synuclein which reflects a 40% reduction in α-synuclein seen in the ELISA.

[0062] Fig.13 BK40143 was shown to improve the locomotor speed of A53T mice. A53T mice were tested for overall locomotor ability over a 60-minute trial in an open field test. Although the mice did not show any differences in total distance traveled or total time spent moving, their locomotor speed increased significantly with BK40143 treatment.

[0063] Fig.14A -E shows that BK40143 selectively inactivates DDR (-50%), but not Src or Abl, and reduces phosphorylated tau in the rTG4510 tauopathy mouse model. Male and female 3-month-old rTG4530 mice were treated with 1.25 mg / kg, 2.5 mg / kg, or 5 mg / kg BK40143 or DMSO ip for 7 consecutive days. Fig.14A is an immunoblot probe for activated (phosphorylated) DDR1, demonstrating that 1.25 and 2.5, but not 5 mg / kg BK40143 inactivates DDR1. Fig. 14B is an immunoblot probe for activated Src, demonstrating that BK40143 does not engage this tyrosine kinase. Fig. 14C is an immunoblot probe for activated Abl, which demonstrates that BK40143 does not engage this tyrosine kinase. Fig.14D is an immunoblot probe for phosphorylated Tau (AT8), which shows that all three doses of BK40143 reduced phosphorylated tau levels in the same mice by 41-49%, indicating that DDR inhibition is paralleled by pTau reduction. Fig.14E is an ELISA for phosphorylated Tau (AT181), which shows that 2.5 mg / kg BK40143 significantly reduced phosphorylated Tau.

[0064] Fig.15A -F shows that BK40143 significantly reduces amyloid, phosphorylated tau and inactivates DDR1 in transgenic APP mice. Male and female 7-month-old APP mice were treated with 1.25 and 2.5 mg / kg BK40143 or DMSO ip for 21 consecutive days. Fig.15Ais an immunoblot of aggregated extracellular amyloid-β (6E10), demonstrating that 1.25 and 2.5 mg / kg BK40143 significantly reduced amyloid-β plaques. Fig. 15B Immunoblot probing for phosphorylated DDR1 demonstrated that 1.25 and 2.5 mg / kg BK40143 inactivated DDR1 by 40% and 31%, respectively. Fig. 15C , detection of activated (phosphorylated Abl(245) demonstrated that BK40143 did not engage Abl. Fig.15D and 15E .By ELISA, it was found that 1.25 and 2.5 mg / kg BK40143 significantly reduced soluble human amyloid-β, but did not significantly reduce insoluble amyloid-β. Fig.15F 2.5 mg / kg BK40143 was shown to significantly reduce human phosphorylated tau (Ser396) by more than 80%.

[0065] Fig.16 It is shown that BK40143 may improve the cognitive performance of APP mice in the Morris water maze test. Measurements include the number of platform entrances (left figure), the waiting time for the first entrance (middle figure) and the distance traveled before entering the first entrance of the platform (right figure). Although there are no significant differences between the groups, 1.25mg / kg BK shows a trend of increased performance at a higher number of platform entrances and a shorter waiting time for the first entrance and a shorter distance traveled before the first entrance.

[0066] Fig.17 It is shown that BK40143 does not cause cell death in the hippocampus of APP mice. Representative 20um hippocampal sections are stained for Nissl bodies (left figure). 4x and 20x images of DMSO, 1.25mg / kg and 2.5mg / kg BK40143 are shown (left figure). The average staining intensity is quantified as the total amount of Nissl staining in all 4x images in imagej software (right figure). DETAILED DESCRIPTION

[0067] Provided herein are compositions and methods for treating or preventing a neurodegenerative disease, a myodegenerative disease, a prion disease, or a lysosomal storage disease in a subject.

[0068] Compound

[0069] In some embodiments, a class of compounds described herein includes compounds represented by Formula I:

[0070]

[0071] or an isomer or a pharmaceutically acceptable salt thereof.

[0072] In Formula I, X is N or CH.

[0073] In Formula I, Y is unsubstituted or substituted with R 1 Substituted C 6-10 aryl; or

[0074] Unsubstituted or R 1 Substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0075] In addition, in Formula I, R 1 Yes – (CH2) n -R 2 、-(CH2) n -C(O)-R 2 or –O(CH2) n -R 2 ;

[0076] In addition, in Formula I, R 2 Yes –H, -CN, halogen, C 1-3 Alkyl, C 1-3 Alkoxy, phenyl, pyridyl, amino, C 1-3 Alkylamino, di-C 1-3 Alkylamino, hydroxyl C 1-3 Alkylamino, carboxyl C 1-3 Alkylamino, C 3-6 Cycloalkyl C 1-3 Alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxy C 1-3 Alkylpyrrolidinyl, carboxylpyrrolidinyl, piperidinyl, C 1-3 Alkyl piperidinyl, di-C 1-3 Alkyl piperidinyl, piperazinyl, C 1-3 Alkylpiperazine, C 1-4 Alkoxycarbonylpiperazinyl or morpholinyl;

[0077] Z is heteroaryl, heterocyclic or NR 3 R 4 ;

[0078] In addition, in Formula I, R 3 and R 4 Independently selected from H, C 1-3 Alkyl, C 1-3 an alkoxy group or an unsubstituted phenyl group, and n is an integer selected from 0 to 3.

[0079] In some embodiments of Formula I, Y is R 1 Substituted benzyl groups:

[0080]

[0081] In some embodiments of Formula I, Y is meta-hydrocarbon. 1 Substituted benzyl groups:

[0082]

[0083] In some embodiments of Formula I, Z is NR 3 R 4 , R 3 is benzyl or H, R 4 is benzyl or H, and Y is R 1 Substituted benzyl groups:

[0084]

[0085] In some embodiments of Formula I, Z is NR 3 R 4 , R 3 is benzyl or H, R 4 is benzyl or H, and Y is meta-R 1 Substituted benzyl groups:

[0086]

[0087] In some embodiments of Formula I, Z is morpholinyl and Y is R 1 Substituted benzyl groups:

[0088]

[0089] In some embodiments of Formula I, Z is morpholinyl and Y is meta-hydrogenated R 1 Substituted benzyl groups:

[0090]

[0091] The compound of formula I is compound 1 (BK40197):

[0092]

[0093] Another compound of formula I is compound 2 (BK40193):

[0094]

[0095] In some embodiments of Formula I, the compound does not contain one or more halogen atoms. In some embodiments of Formula I, Y is 2-m-toluoyl. In some embodiments of Formula I, Z is a heterocyclyl. In some embodiments of Formula I, Z is morpholin-1-yl. In some embodiments of Formula I, R 3 is H and R 4 It is an unsubstituted phenyl group.

[0096] As used herein, the terms alkyl, alkenyl, and alkynyl include straight and branched monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. The range of these groups useful in the compounds and methods described herein includes C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Additional ranges of these groups that can be used in the compounds and methods described herein include C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl, C2-C4 alkenyl and C2-C4 alkynyl.

[0097] As used herein, the term alkoxy is an alkyl group bonded through a single, terminal ether linkage. As used herein, the term hydroxy is represented by the formula—OH.

[0098] As used herein, the term amine or amino group is represented by the formula: NR 3 R 4 Indicates that R 3 and R 4 Each may be a substituent as described herein, such as hydrogen, alkyl, cycloalkyl, halogenated alkyl, alkenyl or alkynyl as described above.

[0099] As used herein, the alkoxy, amino, alkyl, alkenyl, alkynyl or carbonyl molecules may be substituted or unsubstituted. As used herein, the term substituted includes adding an alkoxy, amino, alkyl, alkenyl, alkynyl or carbonyl group to a position attached to the main chain of the alkoxy, amino, alkyl, alkenyl, alkynyl or carbonyl group, such as replacing hydrogen by one of these molecules. Examples of substituents include, but are not limited to, hydroxyl, halogen (e.g., F, Br, Cl or I) and carboxyl. Conversely, as used herein, the term unsubstituted indicates that the alkoxy, amino, alkyl, alkenyl, alkynyl or carbonyl group has a complete complement of hydrogen, i.e., commensurate with its saturation level, without substitution, such as straight chain decane (-(CH2)9-CH3).

[0100] Heteroalkyl, heteroalkenyl, and heteroalkynyl groups are defined similarly to alkyl, alkenyl, and alkynyl groups, but may contain O, S, or N heteroatoms, or combinations thereof, within the backbone. The range of these groups useful in the compounds and methods described herein includes C1-C 20 Heteroalkyl, C2-C 20 Heteroalkenyl and C2-C 20 Additional ranges of these groups that can be used in the compounds and methods described herein include C1-C 12 Heteroalkyl, C2-C 12 Heteroalkenyl, C2-C 12C1-C6 heteroalkyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, C1-C4 heteroalkyl, C2-C4 heteroalkenyl and C2-C4 heteroalkynyl.

[0101] The terms cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclic alkyl groups having a single ring or multiple fused rings. Examples include cyclohexyl, cyclopentylethyl, and adamantyl. The range of these groups that can be used in the compounds and methods described herein includes C3-C 20 Cycloalkyl, C3-C 20 Cycloalkenyl and C3-C 20 Additional ranges of these groups that can be used in the compounds and methods described herein include C5-C 12 Cycloalkyl, C5-C 12 Cycloalkenyl, C5-C 12 cycloalkynyl, C5-C6 cycloalkyl, C5-C6 cycloalkenyl and C5-C6 cycloalkynyl.

[0102] The terms heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl are defined similarly to cycloalkyl, cycloalkenyl, and cycloalkynyl, but may contain O, S, or N heteroatoms, or combinations thereof, within the cyclic backbone. The range of these groups useful in the compounds and methods described herein includes C3-C 20 Heterocycloalkyl, C3-C 20 Heterocycloalkenyl and C3-C 20 Additional ranges of these groups that can be used in the compounds and methods described herein include C5-C 12 Heterocycloalkyl, C5-C 12 Heterocycloalkenyl, C5-C 12 C5-C6 heterocycloalkynyl, C5-C6 heterocycloalkyl, C5-C6 heterocycloalkenyl and C5-C6 heterocycloalkynyl.

[0103] Aryl molecules include, for example, cyclic hydrocarbons that incorporate one or more plane collections of six carbon atoms connected by delocalized electrons, which are numbered as if they were composed of alternating covalent single bonds and covalent double bonds. An example of an aryl molecule is benzene. Heteroaryl molecules include substitutions along the main cyclic chain of its atoms, such as O, N or S. When heteroatoms are introduced, a collection of five atoms (e.g., four carbons and one heteroatom) can produce an aromatic system. Examples of heteroaryl molecules include furans, pyrroles, thiophene, imidazoles, oxazoles, pyridines and pyrazines. Aryl and heteroaryl molecules may also include additional condensed rings, such as benzofuran, indole, benzothiophene, naphthalene, anthracene and quinoline. Unless otherwise noted, the aryl and heteroaryl molecules may be attached at any position on the ring.

[0104] Optionally, the compound of formula I is a tyrosine kinase inhibitor that inhibits one or more receptor tyrosine kinases selected from the group consisting of Abl, PDGFRα, PDGFRβ, DDR 1, DDR2, cKIT, arginase II, Src, Fyn, VEGFR and Zac. In some embodiments, the compound of formula I selectively inhibits Abl, PDGFRα, PDGFRβ, DDR 1, DDR2, cKIT, arginase II, Src, Fyn or VEGR or Zac. In some embodiments, the compound of formula I inhibits DDR 1 and / or DDR2.

[0105] As used herein, the term pharmaceutically acceptable salt refers to salts that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, etc., and are commensurate with a reasonable benefit / risk ratio within the scope of reasonable medical judgment. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds provided herein (e.g., pharmaceutically acceptable salts of nilotinib, bosutinib pazopanib, and compounds of Formula I) include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art (e.g., ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, trifluoroacetic acid, undecanoate, valerate, and the like.

[0106] The compounds described herein can be prepared in a variety of ways. The compounds can be synthesized using a variety of synthetic methods, including those provided in the examples. At least some of these methods are known in the field of synthetic organic chemistry. The compounds described herein can be prepared from readily available starting materials. Optimal reaction conditions may vary with the specific reactants or solvents used, but the conditions can be determined by those skilled in the art through routine optimization procedures.

[0107] The variation about formula I includes the addition, subtraction or movement of the multiple components as described about each compound. Similarly, when one or more chiral centers are present in a molecule, all possible chiral variants are included. In addition, compound synthesis can involve the protection and protective group removal of multiple chemical groups. The use of protection and protective group removal and the selection of appropriate protective groups can be determined by those skilled in the art. Protective group chemistry can be found in, for example, Wuts, Greene's Protective Groups in Organic Synthesis, the 5th edition, Wiley & Sons, 2014, which is incorporated herein by reference in its entirety.

[0108] The reactions that produce the compounds described herein can be carried out in a solvent that can be selected by one skilled in the art of organic synthesis. The solvent may be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions (i.e., temperature and pressure) under which the reactions are carried out. The reactions can be carried out in one solvent or in a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (eg UV-visible) or mass spectrometry, or monitoring by chromatography, such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0109] Any of the compounds described herein may be modified to enhance blood-brain barrier permeability. Optionally, one or more of the compounds described herein may be administered with an agent that enhances the blood-brain barrier permeability of the compound.

[0110] Method for treating or preventing neurodegenerative diseases, myodegenerative diseases or prion diseases

[0111] Provided herein are methods for treating or preventing neurodegenerative diseases, myodegenerative diseases or prion diseases. The neurodegenerative disease or disorder may be a neurodegenerative disease of the central nervous system. These include but are not limited to amyotrophic lateral sclerosis, Alzheimer's disease, frontotemporal dementia, TDP-43 pathology (including TDP-43 frontotemporal dementia), frontotemporal dementia associated with chromosome 17, amyloidosis, Pick's disease, Huntington's disease, mild cognitive impairment, alpha-synuclein disease (e.g., Parkinson's disease, Lewy body disease), multiple sclerosis, glial cell inclusions (including multiple system atrophy), chronic traumatic encephalopathy, Tau protein lesions, progressive supranuclear palsy and cortical basal ganglia degeneration. The neurodegenerative disease may also be secondary neurodegenerative diseases induced by traumatic brain injury, stroke or infection, such as bacterial or viral infection (e.g., HIV, herpes simplex virus (HSV)).

[0112] Muscle degenerative diseases or disorders include, but are not limited to, dystrophies (e.g., muscular dystrophy), myopathies (e.g., nematode myopathy, multinuclear / micronuclear myopathy, centronuclear myopathy, mitochondrial myopathy, metabolic myopathy, etc.), or myotonia (e.g., myotonia congenita, paramyotonia congenita, or myotonic dystrophy).

[0113] To name a few, prion diseases or disorders include, but are not limited to, Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, kuru, bovine spongiform encephalopathy, chronic wasting disease, and scrapie.

[0114] The method comprises administering to a subject having or at risk of developing the neurodegenerative disease, myodegenerative disease or prion disease an effective amount of a compound having Formula I:

[0115]

[0116] in,

[0117] X is N or CH;

[0118] Y is unsubstituted or substituted by R 1 Substituted C 6-10 Aryl; or unsubstituted or R 1 Substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0119] R 1 Yes – (CH2) n -R 2 、-(CH2)n -C(O)-R 2 or –O(CH2) n -R 2 ;

[0120] R 2 Yes –H, -CN, halogen, C 1-3 Alkyl, C 1-3 Alkoxy, phenyl, pyridyl, amino, C 1-3 Alkylamino, di-C 1-3 Alkylamino, hydroxyl C 1-3 Alkylamino, carboxyl C 1-3 Alkylamino, C 3-6 Cycloalkyl C 1-3 Alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxy C 1-3 Alkylpyrrolidinyl, carboxylpyrrolidinyl, piperidinyl, C 1-3 Alkyl piperidinyl, di-C 1-3 Alkyl piperidinyl, piperazinyl, C 1-3 Alkylpiperazine, C 1-4 Alkoxycarbonylpiperazinyl or morpholinyl;

[0121] Z is heteroaryl, heterocyclic or NR 3 R 4 ;

[0122] R 3 and R 4 Independently H, C 1-3 Alkyl, C 1-3 Alkoxy or unsubstituted phenyl,

[0123] and

[0124] n is an integer selected from 0 to 3,

[0125] or an isomer or a pharmaceutically acceptable salt thereof.

[0126] In some methods, the compound of formula I does not contain one or more halogen atoms. In some methods, in the compound of formula I, Y is 2-m-toluoyl. In some methods, in the compound of formula I, Z is a heterocyclyl. In some methods, in the compound of formula I, Z is morpholin-1-yl. In some methods, in the compound of formula I, R 3 is H and R 4 It is an unsubstituted phenyl group.

[0127] In some methods, the compound of formula I (wherein Y is 1 substituted benzyl) is administered to the subject:

[0128]

[0129] In some methods, the compound of formula I (wherein Y is meta-terminated 1 substituted benzyl) is administered to the subject:

[0130]

[0131] In some methods, the compound of Formula I (wherein Z is NR 3 R 4 , R 3 is benzyl or H, R 4 is benzyl or H, and Y is R 1 substituted benzyl) is administered to the subject:

[0132]

[0133] In some methods, the compound of formula I (wherein Z is NR 3 R 4 , R 3 is benzyl or H, R 4 is benzyl or H, and Y is meta-R 1 substituted benzyl) is administered to the subject:

[0134]

[0135] In some methods, the compound of formula I (wherein Z is morpholinyl and Y is R 1 substituted benzyl) is administered to the subject:

[0136]

[0137] In some methods, the compound of formula I (wherein Z is morpholinyl and Y is meta-terminated R 1 substituted benzyl) is administered to the subject:

[0138]

[0139] Examples of Formula I that can be used in any of the methods described herein include the following compounds:

[0140]

[0141]

[0142] The methods provided herein optionally include selecting a subject suffering from a neurodegenerative disease, a myodegenerative disease, or a prion disease, or being at risk of developing a neurodegenerative disease, a myodegenerative disease, or a prion disease. Those skilled in the art know how to diagnose a subject suffering from a neurodegenerative disease, a myodegenerative disease, or a prion disease, or being at risk of developing a neurodegenerative disease, a myodegenerative disease, or a prion disease. For example, one or more of the following tests may be used: genetic testing (e.g., identification of mutations in the TDP-43 gene) or family analysis (e.g., family history), central nervous system imaging (e.g., magnetic resonance imaging and positron emission tomography), electroencephalogram, clinical or behavioral testing (e.g., assessment of muscle weakness, tremor, gait, or memory), or laboratory testing.

[0143] The method optionally further comprises applying a second therapeutic agent to the subject. The second therapeutic agent is selected from the group consisting of levodopa, dopamine agonists, anticholinergics, cholinergics (e.g., 5-hydroxytryptamine (5-HT) inhibitors), monoamine oxidase inhibitors, COMT inhibitors, donepezil, memantine, risperidone, amantadine, rivastigmine, NMDA antagonists, acetylcholinesterase inhibitors, cholinesterase inhibitors, riluzole, antipsychotics, antidepressants, glucocorticoids (e.g., prednisone), tyrosine kinase inhibitors (e.g., nilotinib, bosutinib, imatinib, pazopanib, etc.) and tetrabenazine. The second therapeutic agent or therapy can be applied to the subject before, simultaneously, or after the administration of the compound with Formula I.

[0144] In methods wherein a tyrosine kinase inhibitor is administered as a second therapeutic agent, the tyrosine kinase inhibitor may be one that does not inhibit the tyrosine kinase receptor inhibited by the compound of Formula I or one that has reduced selectivity for a tyrosine kinase receptor as compared to the compound of Formula I.

[0145] Also provided herein is a method for inhibiting or preventing toxic protein aggregation in neurons and / or rescuing neurons from degeneration. As used herein, reference to inhibition, reduction or reduction includes, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more changes compared to control levels.

[0146] The method includes contacting neurons with an effective amount of a compound of formula I. Optionally, the compound of formula I is compound 1 or compound 2. Toxic protein aggregates optionally include one or more of amyloidogenic proteins, alpha-synuclein, tau or TDP-43. Amyloidogenic proteins refer to peptides, polypeptides or proteins with the ability to aggregate. An example of an amyloidogenic protein is beta-amyloid. The contact is performed in vivo or in vitro. The in vivo method can be used to treat subjects with toxic protein aggregates or at risk of developing toxic protein aggregates and includes administering the compound of formula I to the subject as described below. The in vitro method can be used, for example, to treat neural cells before transplantation. In such cases, the compound of formula I is generally added to the culture medium. Optionally, the target neuron is contacted with a second therapeutic agent as described above.

[0147] Methods for treating or preventing lysosomal storage disorders

[0148] Also provided is a method for treating or preventing LSD in a subject. The method comprises administering to a subject suffering from or at risk of developing LSD an effective amount of a compound having Formula I:

[0149]

[0150] in,

[0151] X is N or CH;

[0152] Y is unsubstituted or substituted by R 1 Substituted C 6-10 Aryl; or unsubstituted or R 1 Substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0153] R 1 Yes – (CH2) n -R 2 、-(CH2) n -C(O)-R 2 or –O(CH2) n -R 2 ;

[0154] R 2 Yes –H, -CN, halogen, C 1-3 Alkyl, C 1-3 Alkoxy, phenyl, pyridyl, amino, C 1-3 Alkylamino, di-C 1-3 Alkylamino, hydroxyl C 1-3 Alkylamino, carboxyl C 1-3 Alkylamino, C 3-6 Cycloalkyl C 1-3Alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxy C 1-3 Alkylpyrrolidinyl, carboxylpyrrolidinyl, piperidinyl, C 1-3 Alkyl piperidinyl, di-C 1-3 Alkyl piperidinyl, piperazinyl, C 1-3 Alkylpiperazine, C 1-4 Alkoxycarbonylpiperazinyl or morpholinyl;

[0155] Z is heteroaryl, heterocyclic or NR 3 R 4 ;

[0156] R 3 and R 4 Independently H, C 1-3 Alkyl, C 1-3 alkoxy or unsubstituted phenyl, and

[0157] n is an integer selected from 0 to 3,

[0158] or an isomer or a pharmaceutically acceptable salt thereof.

[0159] In some methods, the compound of formula I does not contain one or more halogen atoms. In some methods, in the compound of formula I, Y is 2-m-toluoyl. In some methods, in the compound of formula I, Z is a heterocyclyl. In some methods, in the compound of formula I, Z is morpholin-1-yl. In some methods, in the compound of formula I, R 3 is H and R 4 It is an unsubstituted phenyl group.

[0160] In some methods, the compound of formula I (wherein Y is 1 substituted benzyl) is administered to the subject:

[0161]

[0162] In some methods, the compound of formula I (wherein Y is meta-terminated 1 substituted benzyl) is administered to the subject:

[0163]

[0164] In some methods, the compound of Formula I (wherein Z is NR 3 R 4 , R 3 is benzyl or H, R 4 is benzyl or H, and Y is R 1 substituted benzyl) is administered to the subject:

[0165]

[0166] In some methods, the compound of formula I (wherein Z is NR 3 R 4 , R 3 is benzyl or H, R 4 is benzyl or H, and Y is meta-R 1 substituted benzyl) is administered to the subject:

[0167]

[0168] In some methods, the compound of formula I (wherein Z is morpholinyl and Y is R 1 substituted benzyl) is administered to the subject:

[0169]

[0170] In some methods, the compound of formula I (wherein Z is morpholinyl and Y is meta-terminated R 1 substituted benzyl) is administered to the subject:

[0171]

[0172] Examples of compounds of Formula I that are useful for treating or preventing LSD include the following:

[0173]

[0174]

[0175] Optionally, the compound of formula I inhibits one or more receptor tyrosine kinases selected from the group consisting of Abl, PDGFRα, PDGFRβ, DDR 1, DDR2, cKIT, arginase II, Src, Fyn, VEGFR and Zac. In some embodiments, the compound of formula I selectively inhibits Abl, PDGFRα, PDGFRβ, DDR 1, DDR2, cKIT, arginase II, Src, Fyn or VEGR or Zac. In some embodiments, the compound of formula I inhibits DDR 1 and / or DDR2. For example and not intended to be limiting, compound 1 or compound 2 can be used to inhibit DDR1 and / or DDR2. In another embodiment, the compound of formula I (e.g., compound 1 or compound 2) selectively inhibits DDR 1 or DDR2.

[0176] LSD is an inherited metabolic disorder caused by defects in lysosomal function. In most cases, LSD is caused by the lack of specific enzymes that are responsible for the degradation of lipids and glycoproteins present in lysosomes. In some cases, defective non-enzymatic lysosomal proteins or non-lysosomal proteins involved in lysosomal biosynthesis cause LSD. Progressive lysosomal accumulation of undegraded metabolites leads to systemic cellular and tissue dysfunction, and therefore leads to multi-system pathology. LSDs that can be treated or prevented using the methods provided herein include, but are not limited to, mucopolysaccharidosis type I (e.g., Hurler syndrome, Hurler-Scheie syndrome, and Scheie syndrome), mucopolysaccharidosis type I (e.g., Hunter syndrome), mucopolysaccharidosis type III (e.g., Sanfillipo syndrome A, Sanfillipo syndrome B, Sanfillipo syndrome C, and Sanfillipo syndrome D), mucopolysaccharidosis type IV (e.g., Morquio syndrome A and Morquio syndrome B), mucopolysaccharidosis type VI (e.g., Maroteaux-Lamy syndrome), mucopolysaccharidosis type VII (e.g., Sly syndrome), mucopolysaccharidosis type IX (e.g., Natowicz syndrome), pseudo-Hurler polydystrophy, Tay-Sachs, Gaucher disease, Niemann-Pick disease, fucosidosis, galactosialidosis, globoid cell leukodystrophy, G M1 Gangliosidosis, G M2 Gangliosidosis, alpha-mannosidosis, metachromatic leukodystrophy, and Pompe disease. The LSDs provided herein are examples of diseases or disorders associated with decreased lysosomal clearance.

[0177] Also provided is a method for promoting lysosomal clearance in one or more cells of a subject, the method comprising administering an effective amount of a compound of formula I to a subject suffering from a condition associated with reduced lysosomal clearance. Optionally, the compound of formula I is compound 1 or compound 2. As used throughout, lysosomal clearance is a process in which the lipids, proteins, glycoproteins or a combination thereof that accumulate are metabolized or degraded in the lysosomes of one or more cells of the subject. The reduction of lysosomal clearance means that the degradation of lipids, proteins and / or glycoproteins in the lysosomes of one or more cells of the subject is reduced, such as compared to a control, such as compared to lysosomal clearance in one or more cells of a healthy subject. Any condition associated with reduced lysosomal clearance can be treated using the methods provided herein, including but not limited to any of the LSDs stated throughout. As used herein, reference to promoting or increasing includes, as compared to control levels, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 400% or more changes. Optionally, promoting lysosomal clearance can reduce the amount of lipids, proteins, glycoproteins or their combinations in the existing aggregates in the lysosomes of one or more cells of the subject. Optionally, promoting lysosomal clearance can inhibit or prevent the formation of aggregates comprising lipids, proteins, glycoproteins or their combinations in the lysosomes of one or more cells of the subject. Optionally, as compared to a control, promoting lysosomal clearance can reduce the amount of time required for the degradation or metabolism of lipids, proteins, glycoproteins or their combinations in one or more cells of the subject.

[0178] Optionally, in the methods provided herein, an effective amount of a compound of Formula I inhibits or prevents the aggregation or accumulation of toxic substances in one or more cells of a subject, as compared to a control. As used herein, reference to decreasing, reducing or inhibiting includes changes of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, as compared to a control level. The term may include but need not include the complete elimination of toxic substances in one or more cells of a subject. Optionally, the one or more cells are brain cells, cells in one or more peripheral tissues of a subject, or a combination thereof. Optionally, the brain cells may be neurons and / or glial cells. In the methods provided herein, toxic substances that may aggregate or accumulate in cells may be one or more of lipids, proteins or glycoproteins. The toxic substances may increase cell damage and / or increase cell death in one or more cells of a subject. In the methods provided herein, the toxic substances may be in lysosomes or elsewhere in one or more cells of a subject. For example, and not intended to be limiting, LSDs characterized by accumulation of lipids in the cells of a subject include, but are not limited to, sphingolipidoses (including Gaucher's disease and Niemann-Pick disease), gangliosidoses (including Tay-Sachs disease), leukodystrophies; mucopolysaccharidoses (including Hunter syndrome and Hurler disease), glycoprotein storage disorders, mucolipidoses, and glycogen storage disease type II (Pompe disease).

[0179] The lipids and glycoproteins that accumulate in the sphingolipidoses include sphingomyelins in the brain and erythrocytes (Nieman-Pick disease); glycolipids, including ceramide trihexosylceramide, in the brain, heart, and kidneys (Fabry disease); galactocerebrosides in oligodendrocytes (Krabbe disease); glucocerebrosides in erythrocytes, spleen, and liver (Gaucher disease); GM2 gangliosides in neurons (Tay-Sachs disease and Sandhoff disease); GM1 gangliosides; and sulfatide compounds in neural tissue (metachromatic leukodystrophy).

[0180] Lysosomal storage diseases also include mucopolysaccharidoses (MPs), which have deficiencies in one or more lysosomal enzymes, such as α-L-iduronate (Hurler disease, Scheie syndrome, and Hurler Schei syndrome); idose sulfate (hunter disease) heparan sulfate (Sanfilipo type A), N-acetyl-α-D-glucosamine (Sanfilipo type B), CoA-α-aminoglucosyl-N-acetyltransferase (Sanfilipo type C), N-acetyl-α-D-aminoglucosyl-6-sulfate (Sanfilipo type D and Morquio syndrome type A), B-galactose (Morquio syndrome type B), and N-acetylgalactosamine (Maroteaus-Lamy disease), but all of these MP diseases are the result of lysosomal accumulation of heparan sulfate, dermatan sulfate, or keratan sulfate. Glycogen storage diseases (i.e., Pompe disease) are caused by the storage of sugars and phosphorylated sugars in lysosomes.

[0181] The methods provided herein optionally include selecting a subject with LSD. Those skilled in the art know how to diagnose a subject with LSD. For example, one or more of the following tests may be used: genetic testing (e.g., identification of mutations associated with LSD) or family analysis (e.g., family history, genetic testing of parents), central nervous system imaging (e.g., magnetic resonance imaging and positron emission tomography), clinical or behavioral testing (e.g., identification of assessments of mood disorders, aggression, and / or cognitive abnormalities), or laboratory testing (e.g., blood and / or urine testing for abnormal levels of metabolites or enzyme deficiencies).

[0182] The methods provided herein optionally further comprise administering to the subject an effective amount of a second therapeutic agent or therapy. The second therapeutic agent or therapy may be administered to the subject before, simultaneously with, or after the administration of the compound of Formula I. The second therapeutic agent or therapy is selected from the group consisting of an enzyme, hematopoietic stem cell, bone marrow transplantation, gene therapy, or a small molecule. For example, and not intended to be limiting, LSD associated with enzyme deficiency may be treated with an enzyme to increase the amount of the deficient enzyme in the subject. For example, using an enzyme such as imiglucerase alglucosidase alfa Taliscinase alpha Enzyme replacement therapy (ERT) with a recombinant enzyme of can be used as a second therapeutic agent to treat Gaucher disease type I. Small molecules that inhibit glycosylceramide synthase (e.g., miglustat and ilurustat) can also be used to treat Gaucher disease type I. Small molecules that act as chaperones to stabilize the deficient enzyme produced by the subject, or that reduce the amount of one or more substances that would normally be processed by the enzyme in the subject can also be used.

[0183] One or more therapeutic agents that alleviate the symptoms of LSD may also be administered. For example, anti-epileptic drugs such as gabapentin or lamotrigine can be used to prevent convulsions in a subject. Antibiotics can be used to treat bacterial infections such as pneumonia. Other agents include, but are not limited to, anti-inflammatory agents (e.g., NSAIDs and anti-inflammatory steroids) and muscle relaxants. Dialysis, physical therapy, and surgery are also contemplated herein as therapies for treating LSD.

[0184] In some methods for treating or preventing LSD, the second therapeutic agent may be a tyrosine kinase inhibitor (e.g., nilotinib, bosutinib, imatinib, pazopanib, etc.). Thus, in some embodiments, a tyrosine kinase and a compound of Formula I are administered to the subject. In methods in which a tyrosine kinase is administered as a second therapeutic agent, the tyrosine kinase may be a tyrosine kinase inhibitor that has a different selectivity for one or more receptor tyrosine kinases than the compound of Formula I.

[0185] Pharmaceutical composition

[0186] As used throughout, the term effective amount is defined as any amount necessary to produce a desired physiological response, such as inhibiting or preventing toxic protein aggregation in neurons or promoting lysosomal clearance.

[0187] Exemplary dosages for administration of any of the compounds described herein (e.g., compounds of Formula I) include dosages of about 0.5 to about 200 mg active compound / kg body weight per day, which can be administered in a single dose or in the form of individual divided doses, such as 1 to 4 times per day. Alternatively, the dosage can be about 0.5 to about 150 mg active compound / kg body weight per day, about 0.5 to 100 mg active compound / kg body weight per day, about 0.5 to about 75 mg active compound / kg body weight per day, about 0.5 to about 50 mg active compound / kg body weight per day, about 0.5 to about 25 mg active compound / kg body weight per day, about 1 to about 50 mg active compound / kg body weight per day, about 1 to about 40 mg active compound / kg body weight per day, about 1 to about 30 mg active compound / kg body weight per day, about 1 to about 30 mg active compound / kg body weight per day, about 30 mg active compound / kg body weight per day, about 20 mg active compound / kg body weight per day, about 10 mg active compound / kg body weight per day, or about 5 mg active compound / kg body weight per day.

[0188] Optionally, the dose is less than about 10 mg / kg and may be less than about 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1.25, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 mg / kg or any dose in between these amounts. The dosage may range from about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 9 mg / kg, about 0.1 mg / kg to about 8 mg / kg, about 0.1 mg / kg to about 7 mg / kg, about 0.1 mg / kg to about 6 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 4 mg / kg, about 0.1 mg / kg to about 3 mg / kg, about 0.1 mg / kg to about 2 mg / kg, about 0.1 mg / kg to about 1 mg / kg, or about 0.1 mg / kg to about 0.5 mg / kg. One skilled in the art will adjust the dosage based on the specific features of the inhibitor and the subject receiving the inhibitor as described below.

[0189] The composition may include a single unit dose of a compound of Formula I, such as about 50 mg / kg or less, 40 mg / kg or less, 30 mg / kg or less, 20 mg / kg or less, 10 mg / kg or less, about 5 mg / kg or less, about 2.5 mg / kg or less, or about 1.5 mg / kg or less of a single unit dose of Compound 1 or Compound 2, or a pharmaceutically acceptable salt thereof. Also provided is a package comprising one or more single unit doses of a compound of Formula I, such as multiple single unit doses of Compound 1 or Compound 2. The package may further include a single or multiple unit doses of one or more second therapeutic agents described herein.

[0190] The effective amount and schedule for administering one or more compounds of Formula I described herein can be determined empirically and it is within the skill of the art to perform such determinations. The dosage range for administration is large enough to produce the desired effect, wherein one or more symptoms of the disease or condition are affected (e.g., alleviated or delayed). The dosage should not be large enough to cause substantial adverse side effects, such as unwanted cross-reactions, unwanted cell death, etc. In general, the dosage will vary with the type of inhibitor, the species, age, body weight, general health, sex and diet of the subject, mode of administration and time, excretion rate, drug combination, and severity of a particular disease and can be determined by a person skilled in the art. The dosage can be adjusted by individual physicians in the case of any contraindications. The dosage can vary and can be administered once or multiple times a day.

[0191] The compounds and other agents with Formula I described herein can be provided in the form of pharmaceutical compositions. These pharmaceutical compositions include, for example, pharmaceutical compositions comprising a therapeutically effective amount of one or more compounds with Formula I and a pharmaceutical carrier. The term carrier means a compound, composition, substance or structure that helps or promotes the preparation, storage, administration, delivery, effectiveness, selectivity or any other characteristics of the compound or composition when combined with the compound or composition to achieve its intended use or purpose. For example, the carrier can be selected to minimize any degradation of the active ingredient and minimize any adverse side effects of the subject. Such pharmaceutically acceptable carriers include sterile biocompatible pharmaceutical carriers, including but not limited to saline, buffered saline, artificial cerebrospinal fluid, dextrose and water.

[0192] Depending on the intended mode of administration, the pharmaceutical composition may be in the form of a solid, semisolid or liquid dosage form, such as a tablet, suppository, pill, capsule, powder, liquid or suspension, preferably in a unit dosage form that can be used for a single administration of a precise dose. The composition will include a therapeutically effective amount of an agent described herein or a derivative thereof in combination with a pharmaceutically acceptable carrier, and may additionally include other pharmaceutical agents, medicaments, carriers or diluents. Pharmaceutically acceptable means a biologically or otherwise desirable material that can be administered to an individual along with the selected agent without causing an unacceptable biological effect or interacting in a deleterious manner with other components of the pharmaceutical composition in which the material is contained.

[0193] As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer or other materials known in the art for pharmaceutical preparations. The selection of a carrier for a composition will depend on the route of administration intended for the composition. The preparation of pharmaceutically acceptable carriers and preparations containing these materials is described in, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, edited by Loyd V. Allen et al., Pharmaceutical Press (2012).

[0194] Examples of physiologically acceptable carriers include buffers such as phosphate buffer, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as (ICI, Inc.; Bridgewater, New Jersey); polyethylene glycol (PEG), and PLURONICS TM (BASF; Florham Park, NJ).

[0195] Compositions containing agents described herein that can be used for parenteral injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Appropriate fluidity can be maintained, for example, by using a coating (such as lecithin), in the case of a dispersion by maintaining the desired particle size and by using a surfactant.

[0196] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of the action of microorganisms may be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents may also be included, for example, sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms may be induced by the use of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0197] Solid dosage forms for oral administration of the compounds described herein or their derivatives include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compound or derivative thereof described herein is mixed with at least one inert conventional excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia, (c) humectants, such as glycerol, (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, (e) solution retarders, such as paraffin, (f) absorption promoters, such as quaternary ammonium compounds, (g) wetting agents, such as cetyl alcohol and glyceryl monostearate, (h) adsorbents, such as kaolin and bentonite, and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0198] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose (milk sugar) and high molecular weight polyethylene glycols.

[0199] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other materials known in the art. It can contain an emulsifier and can also have a composition that allows it to release the one or more active compounds in a certain part of the intestinal tract in a delayed manner. Examples of spendable embedding compositions are polymeric substances and waxes. The active compound can also be in microencapsulated form, with one or more of the above-mentioned excipients when appropriate.

[0200] The compounds described herein can be incorporated into pharmaceutical compositions that allow those compounds to be immediately released or delivered to mammals. The compounds described herein can also be incorporated into pharmaceutical compositions that allow those compounds to be modified to release, such as delayed release or extended release (e.g., sustained release or controlled release) to mammals for several days, weeks, or a month or longer. Such formulations are described, for example, in U.S. Patent Nos. 5,968,895 and 6,180,608 and are otherwise known in the art. Any pharmaceutically acceptable delayed release or sustained release formulation known in the art is contemplated.

[0201] Liquid dosage forms for oral administration of the compounds described herein or their derivatives include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage form may also contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan or mixtures of these substances, etc.

[0202] Besides such inert diluents, the composition may also include additional agents such as wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, or perfuming agents.

[0203] The composition is administered in a variety of ways, depending on local or systemic treatment, and depending on the area to be treated. The composition is administered via any of several routes of administration, including oral, parenteral, intravenous, intraperitoneal, intracranial, intraspinal, intrathecal, intraventricular, intramuscular, subcutaneous, intracavitary or transdermal. The pharmaceutical composition can also be delivered locally to the area in need of treatment, for example, by surface coating or local injection. The effective dose for any of the administration methods described herein can be inferred from a dose-response curve derived from an in vitro or animal model test system.

[0204] Throughout the text, treatment (treat / treating / treatment) refers to a method of reducing or delaying one or more effects or symptoms of a neurodegenerative disease, a myodegenerative disease, a prion disease, or a lysosomal storage disease. The subject may be diagnosed with a disease or condition. Treatment may also refer to a method of reducing the underlying pathology rather than just symptoms. The effect produced by application to the subject may have, but is not limited to, the following effects: reducing one or more symptoms of the disease, reducing the severity of the disease, completely eliminating the disease, or delaying the onset or worsening of one or more symptoms. For example, when compared to the subject before treatment or when compared to a control subject or control value, if there is about 10% reduction in one or more symptoms of the disease in the subject, then the disclosed method is considered to be treatment. Therefore, the reduction may be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or any amount of reduction therebetween.

[0205] As used throughout, subject means individual. The subject may be an adult subject or a pediatric subject. Pediatric subjects include subjects whose age is between birth and eighteen years old. Therefore, pediatric subjects less than about 10 years old, five years old, two years old, one year old, six months old, three months old, one month old, one week old or one day old are also included as subjects. Preferably, the subject is a mammal, such as a primate, and more preferably a human being. Non-human primates are also subjects. The term subject includes domestic animals (such as cats, dogs, etc.), livestock (such as cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (such as ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Therefore, veterinary use and medical preparations are contemplated herein.

[0206] Materials, compositions and components that can be used for the products of the disclosed methods and compositions, can be used in conjunction with the products of the disclosed methods and compositions, can be used to prepare the products of the disclosed methods and compositions or are the products of the disclosed methods and compositions are disclosed. These and other materials are disclosed herein, and it should be understood that when the combinations, subsets, interactions, groups, etc. of these materials are disclosed, although specific references to each different individual and collective combination and arrangement of these compounds may not be explicitly disclosed, each is specifically encompassed and described herein. For example, if a method is disclosed and discussed and a variety of modifications that can be made to a variety of molecules included in the method are discussed, then unless specifically indicated to the contrary, each combination and arrangement of the method is specifically expected, and possible modifications. Similarly, any subset or combination thereof is also specifically expected and disclosed. This concept applies to all aspects of the present disclosure, including but not limited to the method steps using the disclosed compositions. Therefore, if multiple additional steps can be performed, it should be understood that each of these additional steps can be performed together with any specific method step or combination of method steps of the disclosed method, and each such combination or subset of combinations is specifically expected and should be considered disclosed.

[0207] The publications cited herein and the materials for which such publications are cited are hereby specifically incorporated by reference in their entirety.

[0208] Example

[0209] Synthesis and Characterization of Thieno[3,2-b]pyridine Derivatives

[0210] General information

[0211] Commercially available 7-chloro-2-iodothieno[3,2-b]pyridine (1), m-tolylboronic acid (2), aniline (4), m-anisidine (5), morpholine (6), reagents, catalysts and solvents were used as purchased without further purification. NMR spectra were obtained at 400 MHz ( 1 H NMR) and 100 MHz ( 13 C NMR) in deuterated solvents. The reaction products were purified by silica gel column chromatography (particle size 40-63 μm) as described below.

[0212] Synthetic methods and compound characterization

[0213] Synthesis of Thieno[3,2-b]pyridine compounds 7-10

[0214]

[0215] 7-Chloro-2-(m-tolyl)thieno[3,2-b]pyridine (3). A mixture of 7-chloro-2-iodothieno[3,2-b]pyridine (1) (500 mg, 1.69 mmol), 3-methylphenylboronic acid (2) (230 mg, 1.69 mmol), palladium(II) acetate (19 mg, 0.084 mmol), triphenylphosphine (44 mg, 0.169 mmol) and cesium carbonate (1.101 g, 3.38 mmol) in 15 mL of toluene was heated under reflux for 24 h. The reaction mixture was cooled to room temperature and partitioned between water and dichloromethane. The organic layer was washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel using hexane-ethyl acetate (8:2) as the mobile phase. Compound 3 was obtained as a colorless solid with a yield of 72% (315 mg, 1.21 mmol). f =0.2 (hexane / EtOAc, 1:1); 1 H NMR (400 MHz, chloroform-d) δ = 8.54 (d, J = 5.1 Hz, 1H), 7.73 (s, 1H), 7.55–7.52 (m, 2H), 7.34 (dd, J = 7.8, 7.8 Hz, 1H), 7.23 (m, 1H), 7.21 (d, J = 5.1 Hz, 1H), 2.42 (s, 3H); 13 C NMR (100 MHz, chloroform-d) δ = 158.2, 149.6, 148.2, 139.0, 137.6, 133.2, 133.0, 130.4, 129.2, 127.3, 123.8, 120.8, 118.6, 21.5; C 14 H 10 Anal. Calcd. for ClNS: C, 64.74; H, 3.88; N, 5.39. Found: C, 64.76; H, 4.05; N, 5.28.

[0216] General Procedure for Nucleophilic Aromatic Substitution Reactions

[0217] A 5 mL pressure vessel was charged with 7-chloro-2-(m-tolyl)thieno[3,2-b]pyridine (3) (0.3 mmol), the amine (0.6 mmol) and DMSO (1.0 mL). The pressure vessel was then placed in a 100° C. oil bath and stirred for 16 h to 4 days. 1 After H NMR analysis realizes complete conversion, the reaction mixture is extracted with EtOAc and washed with water. The combined organic layer is dried over sodium sulfate and desolventized in a vacuum. As described below, the crude product is purified by flash chromatography on silica gel using hexane-ethyl acetate as mobile phase.

[0218]

[0219] N-phenyl-2-(m-tolyl)thieno[3,2-b]pyridin-7-amine (7). According to the general procedure described above, compound 7 was obtained as a colorless solid in 94% yield (89 mg, 0.282 mmol) from 7-chloro-2-(m-tolyl)thieno[3,2-b]pyridine (78 mg, 0.3 mmol) and aniline (56 mg, 0.6 mmol) in 1 mL DMSO after 16 h at 100 °C. f =0.2 (hexane / EtOAc, 1:1); 1 H NMR (400 MHz, chloroform-d) δ = 8.38 (m, 1H), 7.70 (s, 1H), 7.57–7.50 (m, 2H), 7.43–7.38 (m, 2H), 7.37–7.23 (m, 3H), 7.21–7.17 (m, 2H), 6.90 (m, 1H), 6.15 (s, 1H), 2.43 (s, 3H); 13 C NMR (100 MHz, chloroform-d) δ = 158.5, 148.9, 146.5, 145.9, 139.4, 139.0, 133.7, 129.9, 129.7, 129.6, 129.1, 127.3, 124.8, 123.8, 122.7, 122.5, 121.6, 120.7, 102.6, 21.6; C 20 H 16 Calculated values ​​for N2S: C, 75.92; H, 5.10; N, 8.85. Found values: C, 75.71; H, 5.32; N, 9.11.

[0220]

[0221] N-(3-Methoxyphenyl)-2-(m-tolyl)thieno[3,2-b]pyridin-7-amine (8). Following the general procedure described above, compound 8 was obtained as a colorless solid in 92% yield (95 mg, 0.276 mmol) from 7-chloro-2-(m-tolyl)thieno[3,2-b]pyridine (78 mg, 0.3 mmol) and m-anisidine (74 mg, 0.6 mmol) in 1 mL DMSO after 16 h at 100 °C. f =0.2 (hexane / EtOAc, 2:1); 1H NMR (400MHz, chloroform-d) δ=8.41(d,J=5.6Hz,1H),7.70(s,1H),7.58–7.51(m,2H),7.36–7.29(m,2H),7.21(d,J=7.9Hz,1H),6.96(d,J=5.6 Hz,1H),6.87(dd,J=7.9,2.4Hz,1H),6.83(dd,J=7.8,7.7Hz,1H),6.73(dd,J=7.9,2.5Hz,1H),6.07(s,1H),3.83(s,3H),2.44(s,3H); 13 C NMR (100 MHz, chloroform-d) δ = 160.8, 158.6, 148.9, 146.5, 145.6, 140.7, 139.0, 133.7, 130.5, 130.0, 129.1, 127.3, 123.8, 121.7, 120.9, 114.5, 110.1, 108.1, 103.0, 55.5, 21.6; C 21 H 18 Calculated values ​​for N2OS: C, 72.80; H, 5.24; N, 8.09. Found values: C, 72.53; H, 5.61; N, 8.19.

[0222]

[0223] 4-(2-(m-tolyl)thieno[3,2-b]pyridin-7-yl)morpholine (9). Following the general procedure described above, compound 9 was obtained as a colorless solid in 98% yield (91 mg, 0.294 mmol) from 7-chloro-2-(m-tolyl)thieno[3,2-b]pyridine (78 mg, 0.3 mmol) and morpholine (52 mg, 0.6 mmol) in 1 mL DMSO after 4 days at 100 °C. f =0.2 (hexane / EtOAc, 1:1); 1 H NMR (400MHz, chloroform-d) δ = 8.48 (d, J = 5.4Hz, 1H), 7.69 (s, 1H), 7.59–7.52 (m, 2H), 7.34 (dd, J = 7.9, 7.8Hz, 1H) ,7.20(dd,J=7.9,2.1Hz,1H),6.64(d,J=5.4Hz,1H),4.03–3.85(m,4H),3.54–3.39(m,4H),2.43(s,3H); 13C NMR (100 MHz, chloroform-d) δ = 158.8, 153.0, 149.0, 146.6, 139.0, 133.6, 129.9, 129.1, 127.2, 123.7, 123.4, 121.4, 105.9, 66.9, 49.7, 21.6; C 18 H 18 Calculated values ​​for N2OS: C, 69.65; H, 5.85; N, 9.02. Found values: C, 69.89; H, 5.72; N, 9.38.

[0224]

[0225] 3-((2-(m-tolyl)thieno[3,2-b]pyridin-7-yl)amino)phenol (10). To a solution of N-(3-methoxyphenyl)-2-(m-tolyl)thieno[3,2-b]pyridin-7-amine (8) (69 mg, 0.2 mmol) in anhydrous dichloromethane (3 mL) was added boron tribromide (4 equiv) at -78 °C under an inert atmosphere. The mixture was stirred for 4 h and the reaction temperature was allowed to reach 0 °C. After quenching with 1M HCl, the crude reaction mixture was extracted with EtOAc and washed with water. The combined organic layers were dried over sodium sulfate and the solvent was removed in vacuo. The crude product was purified by flash chromatography on silica gel using DCM-MeOH (19: 1) as the mobile phase. Compound 10 was obtained as a colorless solid with a yield of 97% (64 mg, 0.194 mmol). f =0.4 (DCM / MeOH, 9:1); 1 H NMR (399MHz, methanol-d4) δ = 8.22 (d, J = 6.7Hz, 1H), 7.70 (s, 1H), 7.65 (s, 1H), 7.61 (dd, J = 7.5, 2.1Hz, 1H), 7.40 (d d,J=7.6,7.6Hz,1H),7.34–7.31(m,2H),6.93(d,J=6.7Hz,1H),6.88(m,1H),6.85–6.79(m,2H),2.44(s,3H); 13 C NMR (100 MHz, methanol-d4) δ = 160.1, 154.7, 153.5, 149.5, 141.1, 140.7, 139.5, 133.3, 132.4, 131.9, 131.7, 130.5, 128.2, 125.0, 117.4, 115.7, 114.9, 113.5, 102.7, 21.3; C 20 H 16Calculated values ​​for N2OS: C, 72.26; H, 4.85; N, 8.43. Found values: C, 72.29; H, 4.97; N, 8.61.

[0226] Cell culture

[0227] Rat neuroblastoma B35 cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin and incubated at 37°C and 5% CO2. For the experiment, cells were transferred to 12-well plates (Catalog No. 150628, ThermoFisher, Waltham, MA) and grown to at least 70% confluence. Transient transfection was performed for 24 hours using Fugene HD transfection reagent (Catalog No. E2311, Promega, Madison WI) with 3 μg P301L tau (Catalog No. 30145, Addgene) cDNA or 3 μg human α-synuclein cDNA. Cells were treated with 1 mM, 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM and 0.001 μM or equal 5 uL DMSO dissolved in DMSO for 5 hours. Cell culture medium is collected, and sodium-tris, EDTA, NP-40 (STEN) buffer harvested cells are used and centrifuged at 10,000xg for 20 minutes at 4 ℃, and supernatant is collected. Cell viability is determined via lactate dehydrogenase analysis (Cat. No. 88954, Thermofisher) and MTT analysis (Cat. No. V13154, Thermofisher). Protein is extracted by removing culture medium and 0.2ml 1x STEN buffer (50mM Tris (pH 7.6), 150mM NaCl, 2mM EDTA, 0.2% NP-40, 0.2% BSA, 20mM PMSF and protease mixed solution inhibitor) is added to cell layer, and incubation on ice continues 10 minutes. The bottom of the scraped hole is incubated on ice for 10 minutes. Cell lysates are collected, stored at -80 ℃, and used for additional analysis.

[0228] Drug preparation

[0229] Compound 1 (BK40197) and Compound 2 (BK40143) having molecular weights of 310.1 and 316.4 g / mol, respectively, were diluted in dimethyl sulfoxide (DMSO) to final concentrations of 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, and 0.001 μM. Drugs were stored at -80°C.

[0230] MTT assay

[0231] To measure cell viability, cells were incubated for 4 hours at 37°C and 5% CO2 with 500 μL Dulbecco's Modified Eagle's Medium (DMEM) containing 50 μL of brominated (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolyl (MTT). The culture medium was aspirated so that 125 uL of culture medium was retained. Formazan was dissolved in 250 uL DMSO. The absorbance was read at 570 nm for a blank containing 125 uL of culture medium and MTT, and 250 uL DMSO.

[0232] Lactate dehydrogenase (LDH) analysis

[0233] Cytotoxicity is quantitatively measured by evaluating the cytosolic enzyme LDH released from damaged cells into the cell culture medium after exposure to the drug for 5 hours after the initial dose. The cell culture medium is collected and aliquots are coupled with lactate and NAD+. LDH catalyzes the reaction of converting lactate into pyruvate to produce NADH. NADH reduces tetrazolium salts (INT) to red formazan products. The amount of LDH in the culture medium is proportional to the amount of formazan, which is measured at 490nm. The absorbance at 680nm measured for the background signal of the measuring instrument is subtracted from the absorbance at 490nm to calculate the LDH activity.

[0234] Cell culture transfection and treatment

[0235] To transiently transfect α-synuclein into rat neuroblastoma B35 cells, use HD transfection reagent (Promega Corporation, Madison, WI). Cells were grown in 12-well dishes and incubated with 12 μg of cDNA, 540 μg of DMEM containing 2% FBS, and 60 μl The cells were treated with 50 μl of HD transfection reagent mixture for 10 minutes. The HD transfection reagent / DNA mixture treatment lasted for 24 hours. To harvest cells after transfection, the medium was aspirated, the cells were treated with 200 ul of sodium Tris EDTA NP40 (STEN) lysis buffer, then scraped off the plate and collected into a 1.5 ml centrifuge tube.

[0236] Mouse treatment

[0237] Experiments were performed on the following mice: (a) TgAPP mice, which express the neuronal-derived human APP gene 770 isoform under the control of the mouse thymocyte antigen 1, theta (Thy1) promoter, which contains the Swedish (K670N / M671L, Dutch E693Q and Iowa D694N mutations (Davis et al. "Early-onset and robust cerebralmicrovascular accumulation of amyloid beta-protein in transgenic miceexpressing low levels of a vasculotropic Dutch / Iowa mutant form of amyloidbeta-protein precursor," The Journal of biological chemistry. 279(19):20296-20306 (2004); (b) rTg4510 mice, which express human P301L tau and have a P301L mutant variant (4R0NTau) directing human four-repeat microtubule-associated protein tau P301L) and the mouse prion protein promoter sequence (PrP or Prnp) (Santacruz et al. "Tau suppression in aneurodegenerative mouse model improves memory function," Science 309(5733):476-48 (2005); or (c) TgA53T mice, which express mutant arginine-threonine (A53T) human alpha-synuclein under the control of the prion promoter (Giasson et al. "Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein," Neuron 34(4):521-533 (2002).Mice received daily intraperitoneal (ip) injections of BK40143 (BK) (Medicinal Chemistry Program, Georgetown University), nilotinib (Nilo) (Catalog No. S1033, Selleckchem Inc., Houston, TX), bosutinib (bos) (Catalog No. S1014, Selleckchem Inc.), or a combination solution of BK + Nilo, BK + Bos, or Nilo + Bos dissolved in dimethyl sulfoxide (DMSO) (Catalog No. D128-500, Fisher Scientific, Hampton, NH) at 1.25 mg / kg, 2.5 mg / kg, or 5.0 mg / kg, or an equal dose of DMSO alone. The treatment period was 7 consecutive days or 21 consecutive days as specified in the legend.

[0238] Xmap

[0239] Xmap technology uses magnetic microspheres, which are encoded with two fluorescent dyes inside. Through the accurate combination of these two dyes, multiple proteins in the sample are measured. Each of these balls is coated with specific capture antibodies. The capture antibody is bound to the detection antibody and the reporter molecule, thereby completing the reaction on the surface of the beads. 25 μl soluble brain tissue lysates from transgenic Tg4510 mice treated with 5mg / kg, 2.5mg / kg and 1.25mg / kg BK40143 or DMSO were incubated overnight (16-20 hours) at room temperature with 25 μl detection antibody solution and 25 μl mixed bead solution, which contained the following analytes: human phosphorylated tau (181). After fully washing the plate, the sample was incubated with 25 μl streptavidin-phycoerythrin added to each well and incubated at room temperature for 30 minutes. The sample was then washed and suspended in 100ul sheath fluid. After resuspension, the sample was run with Xponent software on MAGPIX. Median fluorescence intensity (MFI) data were analyzed using a 5-parameter logistic or spline curve fitting method to calculate the analyte concentration in the samples.

[0240] Protein extraction

[0241] Brains from treated mice were homogenized in STEN lysis buffer [50 mM sodium Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.2% NP-40, 0.2% BSA, 20 mM PMSF and protease inhibitor cocktail], centrifuged at 10 000 g for 20 min at 48°C, and the supernatant containing the soluble protein fraction was collected. The supernatant was analyzed by Western blotting (WB) on SDS-NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA) and ELISA.

[0242] Pharmacokinetic studies

[0243] C57BL / 6J mice were injected once with BK ip injection. Brain and serum were collected at 2, 4, 6 or 12h (n=18 for each drug, n=3 for each dose and time point). Animals injected with vehicle (DMSO) were used for background subtraction. Drug stock solutions (approximately 1 mg / mL each) were prepared in methanol / dichloromethane (50:50). Serial dilutions of each standard were produced separately in methanol / HPLC grade water (50:50) for research. The preparation of calibration curve standards and quality samples (QC) was performed by mixing the stock solution in blank samples. Serum and brain samples were stored at -80°C and then thawed to room temperature before preparation. Thawed serum samples (20uL) were injected into tubes containing 100uL water. 500uL extraction solvent, acetonitrile / methanol (50:50) were added to the samples. The mixture was vortexed and incubated on ice for 20min to accelerate protein precipitation. After incubation, the sample was vortexed again and centrifuged at 13,000rpm for 20min at 4°C. The supernatant was then collected and transferred to a new tube, dried using a speed vac, and reconstituted in 200uL methanol / water (50:50). The mixture was briefly centrifuged again at 13,000rpm for 20min 200 at 4°C. The supernatant was then collected in a mass spectrometry sample tube cap and run in a mass spectrometer. For the brain, a small portion of the thawed brain sample from each animal was transferred to a flat-bottomed tube. 200uL methanol / water (90:10) was added, and the tissue was homogenized. Acetonitrile was then added to the mixture, thereby promoting protein precipitation. The mixture was then incubated on ice for 10min. After incubation, the sample was vortexed and centrifuged at 13,000rpm for 20min at 210 4°C. The supernatant was then collected and transferred to a new tube, dried using a Speedby Weike, and recovered in 200uL methanol / water (50:50). The mixture was centrifuged at 13,000rpm for 20min at 4°C. The supernatant was then collected in a mass spectrometry sample tube cap and run in a mass spectrometer. The sample was split on an Acquity UPLC BEH C18 1.7 μm, 2.1 × 50 mm column, which was connected to a triple quadrupole mass spectrometer (Xevo-TQ-S, Waters Corporation) operated in multiple reaction monitoring (MRM) mode. The sample cone voltage and collision energy were optimized for two analytes to obtain the maximum ion intensity for parent ions and daughter ions using the "IntelliStart" feature of MassLynx software (Waters Corporation). Instrument parameters were optimized to obtain maximum specificity and ionization sensitivity for both the parent ion [m / z = 438.25] and the daughter ion [m / z = 357.33].All MRM Q1 / Q3 ion pairs were ranked for the signal intensity of the analyte to ensure that the most intense precursor and fragment ion pairs were selected for MRM-based quantification. This approach resulted in the selection of cone voltages and collision energies that maximized the generation of each fragment ion species. The analysis was performed with a six-point to eight-point calibration curve, randomizing the sample queue and injecting a solvent blank to assess sample carryover. MRM data were processed using TargetLynx 4.1. The relative quantitative values ​​of the analytes were determined by calculating the ratio of the transition peak area of ​​the sample to the peak area normalized to the internal standard.

[0244] Tissue collection and protein extraction.

[0245] Animals were deeply anesthetized with a mixture of xylazine and ketamine (1:8), and 500 μl of whole blood was collected via cardiac puncture, centrifuged at 2000 × g to precipitate blood cells, and serum was collected. In order to wash out the remaining blood from the container and reduce contamination, animals were perfused with 25 ml 1X phosphate buffered saline (PBS) for 5 min. Brains were collected and homogenized in 1.0 ml 1x STEN buffer. Homogenized samples were centrifuged at 12,000 × g for 20 min at 4 ° C, and supernatants (soluble protein fractions) were collected and stored at -80 ° C. Insoluble proteins were extracted after removing the supernatant. Tissue aggregates were washed with 1X STEN buffer. The aggregates were resuspended in 750 ul 70% formic acid and incubated at room temperature for 30 min, followed by centrifugation at 28,000 g for 1 h at 4 ° C. Supernatants were collected as "insoluble fractions". Samples from the 70% formic acid fraction were stored at -80°C and neutralized with 1 M Tris-base (1:20) immediately before use. Protein levels were quantified using the Pierce BCA protein assay (ThermoFisher, 23225) via the manufacturer's instructions.

[0246] Immunoblot analysis

[0247] Soluble and insoluble proteins extracted from mouse brain lysates were run on SDS NuPAGE Bis-Tris gels (Cat. No. NP0301BOX, Invitrogen) and probed for phosphorylated tau with (1:1000) mouse monoclonal AT180 (Cat. No. MN1040, ThermoFisher) and (1:1000) mouse monoclonal AT8 (Cat. No. MN1020, ThermoFisher), total tau with (1:3000) mouse monoclonal Tau-5 antibody, phosphorylated DDR1 with (1:250) rabbit polyclonal MCK10 (Cat. No. PA5-64780, ThermoFisher), ubiquitin with (1:5000) rabbit polyclonal (Cat. No. PA3-16717, ThermoFisher), and IgG with (1:1000) rabbit monoclonal (Cat. No. mAb 12994, Cell Biology, Inc., MD). Atg5 was probed with (1:1000) rabbit monoclonal (Cat. No. mAb 3495, Cell Signaling), Beclin-1 was probed with (1:8000) rabbit polyclonal (Cat. No. MAB1501R, EMD Millipore, Burlington, MA). TM Imager 600 (GE Healthcare Life Sciences, Pittsburgh, PA) using Super Signal TM Blots were visualized using West Dura Extended Durable Substrate (Cat. No. 37071, ThermoFisher). Western blots were quantified by densitometry using Image J software.

[0248] Enzyme-linked immunosorbent assay (ELISA)

[0249] Human α-synuclein and p-tau ELISA was performed using 50 μl (1 μg / μl) cell lysate detected with 50 μl primary antibody (3h) and 100 μl anti-rabbit secondary antibody (30min) at RT. α-synuclein levels were measured using human specific ELISA (Invitrogen Inc., Carlsbad, CA) according to the manufacturer's protocol. Tau was measured using specific tau at serine 396 according to the manufacturer's protocol. Each sample was in duplicate.

[0250] Milliplexed ELISA for total Tau, AB 40 and AB 42(Millipore catalog number HNABTMAG60K). As stated above, Xmap technology uses magnetic microspheres that are internally encoded with two fluorescent dyes. Through the precise combination of these two dyes, multiple proteins in the sample are measured simultaneously. Each of these balls is coated with a specific capture antibody. The capture antibody binds to the detection antibody and the reporter molecule, thereby completing the reaction on the surface of the beads. All samples at baseline and 52 weeks, including placebo and resveratrol, were analyzed in parallel using the same reagents. A total of 25 μl of soluble protein was incubated at 4°C with 25 μl of a 5% PBS containing total Tau, AB 40 and AB 42 The mixed bead solution was incubated overnight. After washing, the sample was incubated with 25 μl of detection antibody solution at room temperature for 1.5 h. Streptavidin-phycoerythrin (25 μl) was added to each well containing 25 μl of detection antibody solution. The sample was then washed and suspended in 100 μl of sheath fluid. The sample was then run with Xponent software on MAGPIX. The median fluorescence intensity (MFI) data was analyzed using a 5-parameter logistic or spline curve fitting method to calculate the analyte concentration in the sample. According to the manufacturer's protocol, specific p-Tau ser396 (Invitrogen, KHB7031), human Tau thr181 (Invitrogen, KHO0631) and Aβ1–42 (Invitrogen, KHB3442) were performed on tissue-soluble extracts of midbrain lysates from 1XSTEN buffer (see above).

[0251] Behavior

[0252] Rotarod: Mice were placed on an accelerator rod (Cat. No. 76-0770, Panlab, Harvard Apparatus) equipped with an individual timer for each mouse. Mice were tested over 4 trials, 3 training sessions, and 1 test session. Mice were trained to stay on the rod at a constant 4 revolutions per minute (rpm) for at least 5 minutes, and then the speed was gradually increased to 40 rpm over 300 seconds, and the latency to fall was measured.

[0253] Open field: Mice were placed in an open field arena apparatus (25 cm x 25 cm) for 60 minutes. Animals were tracked along the arena floor by a photoelectric tube beam. During the 60-minute test, data were collected and analyzed for total distance traveled (cm), total time spent moving (sec), and speed (distance / time). The center zone was digitally defined in the software as the center of the apparatus and the center zone entrance (25 cm x 25 cm), and the center zone distance traveled (cm) and the time spent in the center zone (sec) during the 60-minute test were recorded.

[0254] Morris water maze: The water maze apparatus consists of a 4-foot-diameter pool (SanDiego Instruments) filled with water maintained at 25°C and made opaque with white paint, and is digitally divided into 4 quadrants (ANYMaze software, San Diego Instruments). Visual cues outside the maze are suspended on the wall surrounding the pool, and a hidden platform (4-inch diameter) is submerged 1 cm below the surface of the water at the center of the 'platform area'. Training consists of three trials per day for four days, followed by a probe test on the fifth day. Mice are introduced into the pool at one of three entry points (one in each non-platform quadrant), with each entry point used during the day. The position of the platform remains constant throughout the training period. Give mice 60 sec to position them on the platform, and keep them on the platform for 10 sec, then remove the mice. Mice that are not positioned on the platform within 60 sec are placed on the platform for 10 sec, then removed from the maze. During the probe test on day 5, the platform was removed and tracking software (ANYMaze) was used to record latency to find the platform, platform quadrant, swimming speed, and swimming path. This training and probe test paradigm was performed before and after treatment.

[0255] Marble burying test: The marble burying test was performed as previously described

[35] with modifications. Briefly, 20 marbles with a diameter of 15 mm were spaced 4 cm apart in five rows of four marbles each on the surface of softly packed 5-cm deep corncob litter in double-sized rat cages. Mice were left alone in the cages for 30 min. An observer who was blinded to the treatment counted the number of buried marbles. Any marble that exceeded two-thirds of its size was counted. Each mouse was assessed once before and after treatment and the data are reported as the mean ± SEM of the percentage of marbles buried by each animal. The Kuskal-Wallis test followed by the Wilcoxon post hoc test was used to determine the statistical significance of marble burying in mice treated with drug or DMSO.

[0256] Statistical analysis

[0257] All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad software Inc.). For experiments involving mice, the sample size (n) and female: male quantification used in each experiment are indicated in the legend. For experiments using cell lines, the number of independent biological replicates (N) is reported. Data are presented as mean ± SEM. When the mean value in two groups is compared, a two-tailed student's t test or Welch's t test is performed. When the mean value of multiple groups is compared, one-way analysis of variance (ANOVA), Tukey's multiple comparison post-hoc test are performed successively. Asterisks or pound symbols represent actual p-value significance (* < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001) between or within each group and are indicated in individual legends.

[0258] result

[0259] like Figure 1 As shown in (left and middle panels), neuroprotective effects were observed in B35 cells treated with 1 μM BK41043 after 16 hours of treatment, as evidenced by a decrease in LDH and an increase in MTT, respectively, as compared to controls. Figure 1 (Right panel) shows that after five hours of treatment, there was a gradual increase in cell viability via reduction of LDH at decreasing concentrations of BK40143.

[0260] Figure 2 Shown are cell viability of B35 cells after 24 hours of transfection with pTau (left panel) or α-synuclein (right panel) and five hours of treatment with BK41043.

[0261] B35 cells were grown in complete medium and transfected with cDNAs of human mutant Tau and α-synuclein using FuGene HD transfection reagent according to the manufacturer's instructions. At 24 hours, the transfection had produced significantly higher amounts of phosphorylated Tau and α-synuclein compared to non-transfected cells by ELISA. Figure 3 pTau(181) levels after 24 hours of transfection are shown (left panel), and BK41043 reduces pTau(181) levels in pTau-transfected B35 cells (right panel), returning to control levels. This occurs without any changes in LDH levels, indicating that toxicity did not increase during the experiment. Figure 4 The α-synuclein levels after 24 hours of transfection are shown (left panel), and BK41043 did not reduce α-synuclein in α-synuclein transfected B35 cells (right panel).

[0262] Figure 5Shown are cell viability of B35 cells after five hours of treatment with BK40197.

[0263] Figure 6 BK40143 is shown to reduce pTau(181) in Tau-expressing transgenic mice after seven days of treatment. pTau(181) levels were measured via ELISA.

[0264] Figure 7 It was shown that treatment with BK-40143 (1.25 mg / kg, 2.5 mg / kg or 5.0 mg / kg) did not affect pTau(231) (AT180) levels in transgenic mice.

[0265] Figure 8 It was shown that Tau (HT7) was significantly reduced in Tau transgenic mice after seven days of treatment with 1.25 mg / kg and 2.5 mg / kg of BK-41043.

[0266] Fig. 9 It was shown that treatment with BK-40143 (1.25 mg / kg or 2.5 mg / kg) resulted in in vivo inhibition of DDR1 as measured by detection of phosphorylated DDR1 (pMCK10).

[0267] Fig.10 pTau was shown to be reduced in CamP301L mice after treatment with 2.5 mg / kg or 5 mg / kg BK-40143.

[0268] As shown in Figure 11, BK40196 reduced α-synuclein at high concentrations and BK40197 did not reduce α-synuclein. 1 mM and 100 uM BK40196 significantly reduced α-synuclein levels in transfected B35 cells ( Fig.11A BK40197 did not show any ability to reduce α-synuclein levels in transfected B35 cells ( Fig. 11B ).

[0269] like Fig. 12A As shown, BK-40143 significantly reduced α-synuclein in A53T mice compared to DMSO-treated control A53T mice. C57BL / 6J mice were used as controls and showed no detectable (ND) human α-synuclein. Fig. 12B It was shown that BK-40143 did not significantly increase (approximately 30%) overall levels of dopamine, however, BK-40143 did increase levels of the dopamine metabolite homovanillic acid (HVA) in A53T mice, indicating more dopamine turnover, which may lead to better dopamine neurotransmission. Fig. 12C and Fig.12DThe immunoblot shown in for α-synuclein (ThermoFisher, MA1-12874) reflects a 40% reduction in α-synuclein visible in the ELISA.

[0270] Animal studies also showed that BK-40143 improved the movement speed of A53T mice. A53T mice were tested for overall movement capacity in an open field test over a 60-minute trial. Although the mice did not show any difference in total distance traveled or total time spent moving, their movement speed increased significantly with BK-40143 treatment ( Fig.13 This may reflect better dopamine neurotransmission, as indicated by increased HVA levels after BK ( Fig. 12B ).

[0271] BK40143 was also shown to selectively inactivate the DDR, but not Src or Abl, and reduce phosphorylated tau in the rTG4510 tauopathy mouse model. Male and female 3-month-old rTG4530 mice were treated with 1.25 mg / kg, 2.5 mg / kg, or 5 mg / kg BK40143 or DMSO ip for 7 consecutive days. Fig.14A is an immunoblot probe for activated (phosphorylated) DDR1, demonstrating that 1.25 and 2.5, but not 5 mg / kg BK40143 inactivates DDR1. Fig. 14B is an immunoblot probe for activated Src, demonstrating that BK-40143 does not engage this tyrosine kinase. Fig. 14C is an immunoblot probe for activated Abl, which demonstrates that BK-40143 does not engage this tyrosine kinase, DDR1. When probing phosphorylated Tau (AT8), Fig.14D All three doses of BK-40143 were shown to reduce phosphorylated tau levels by 41-49%. Fig.14E As shown, ELISA of phosphorylated Tau (AT181) revealed that 2.5 mg / kg BK40143 significantly reduced phosphorylated Tau.

[0272] BK40143 also significantly reduced amyloid, phosphorylated tau and inactivated DDR1. Male and female 7-month-old APP mice were treated with 1.25 and 2.5 mg / kg BK40143 or DMSO ip for 21 consecutive days. Immunoblotting of aggregated extracellular amyloid-β (6E10) demonstrated that 1.25 and 2.5 mg / kg BK40143 significantly reduced amyloid-β plaques ( Fig.15A Detection of phosphorylated DDR1 demonstrated that 1.25 and 2.5 mg / kg BK40143 inactivated DDR1 by 40% and 31%, respectively ( Fig. 15B ). Detection of activated (phosphorylated Abl(245) demonstrated that BK40143 did not engage Abl( Fig. 15C ). Fig.15D and Fig.15E It was shown that 1.25 and 2.5 mg / kg BK-40143 significantly reduced soluble human amyloid-β, but not insoluble amyloid-β, respectively, via ELISA. It was also shown that 2.5 mg / kg BK40143 significantly reduced human phosphorylated tau (Ser396) by more than 80% ( Fig.15F ).

[0273] It was also shown that BK40143 may improve the cognitive performance of APP mice in the Morris water maze test. APP mice were tested for their ability to find the target platform in the Morris water maze after treatment. Measurements included the number of platform entrances ( Fig.16 , left), the waiting time for the first entrance ( Fig.16 , middle figure) and the distance traveled before it enters the first entrance of the platform ( Fig.16 , right). Although there were no significant differences between the groups, 1.25 mg / kg BK-40143 showed a trend toward increased performance at a higher number of platform entries and a shorter wait time to the first entry and a shorter distance traveled before the first entry ( Fig.16 ).

[0274] The study also showed that BK40143 does not cause cell death in the hippocampus of APP mice. Representative 20um hippocampal sections were stained for Nissl bodies. Fig.17 4x and 20x images of DMSO, 1.25 mg / kg and 2.5 mg / kg BK40143 are shown (left). The average staining intensity was quantified in ImageJ software as the total amount of Nissl staining in all 4x images ( Fig.17 , right).

[0275] These data suggest that compounds of Formula I (eg, BK41043 or BK40197) may be useful in treating or preventing neurodegenerative disorders, muscle degenerative disorders, or lysosomal storage disorders.

Claims

1. A compound having the formula: in X is CH; R 1 is -CH3; and Z is morpholinyl or NR 3 R 4 , where R 3 H and R 4 is an unsubstituted phenyl group; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein the compound has the formula: 。 3. The compound of claim 1 or 2, wherein Z is morpholin-1-yl.

4. The compound of claim 1 or 2, wherein the compound has the formula: 。 5. The compound of claim 1 or 2, wherein the compound has the formula: 。 6. Use of a compound having the following formula in the preparation of a medicament for treating or preventing a neurodegenerative disease or a muscle degenerative disease in a subject, wherein the subject suffers from, or is at risk of developing, the neurodegenerative disease or the muscle degenerative disease of the central nervous system: in, X is CH; R 1 is -CH3; and Z is morpholinyl or NR 3 R 4 , where R 3 H and R 4 is an unsubstituted phenyl group; or a pharmaceutically acceptable salt thereof.

7. The use according to claim 6, wherein the compound has the following formula: 。 8. The method of claim 6, wherein the compound has the following formula: 。 9. The use according to any one of claims 6 to 8, wherein the compound crosses the blood-brain barrier.

10. The use according to any one of claims 6 to 8, wherein the neurodegenerative disease of the central nervous system is selected from the group consisting of amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease and mild cognitive impairment.

11. The use according to any one of claims 6 to 8, wherein the neurodegenerative disease of the central nervous system is selected from the group consisting of α-synucleinopathy and Tauopathy.

12. The use according to any one of claims 6 to 8, wherein the medicament is formulated for systemic administration.

13. The use according to any one of claims 6 to 8, wherein the medicament is formulated for oral administration.

14. The use according to any one of claims 6 to 8, wherein the drug is administered at a dose of 10 mg / kg or less.

15. The use according to any one of claims 6 to 8, wherein the drug is administered at a dose of 5 mg / kg or less.

16. The use according to any one of claims 6 to 8, wherein the drug is administered at a dose of 2.5 mg / kg or less.

17. The use according to any one of claims 6 to 8, wherein the medicament is administered daily.

18. The use according to any one of claims 6 to 8, wherein the compound is in a pharmaceutical composition.

19. The use of any one of claims 6-8, further comprising administering a second therapeutic agent to the subject.

20. The use of claim 19, wherein the second therapeutic agent is selected from the group consisting of levodopa, dopamine agonists, anticholinergics, monoamine oxidase inhibitors, COMT inhibitors, amantadine, donepezil, memantine, risperidone, rivastigmine, NMDA antagonists, acetylcholinesterase inhibitors, cholinesterase inhibitors, riluzole, antipsychotics, antidepressants and tetrabenazine.

21. Use of a composition comprising a compound having the formula: for the preparation of a medicament for inhibiting or preventing toxic protein aggregation in neurons, comprising contacting the neurons with the medicament: in, X is CH; R 1 is -CH3; and Z is morpholinyl or NR 3 R 4 , where R 3 H and R 4 is an unsubstituted phenyl group; or a pharmaceutically acceptable salt thereof, Wherein the protein is selected from the group consisting of amyloidogenic protein-β and tau.

22. The use according to claim 21, wherein the compound has the formula: 。 23. The use according to claim 21, wherein the compound has the formula: 。 24. The use according to any one of claims 21 to 23, wherein the contacting is performed in vitro.

25. The use of any one of claims 21-23, wherein the contacting is performed in vivo.

26. Use of a composition comprising a compound having the formula: for the preparation of a medicament for inhibiting or preventing toxic protein aggregation in neurons, comprising contacting the neurons with the medicament: in, X is CH; R 1 is -CH3; and Z is NR 3 R 4 , where R 3 H and R 4 is an unsubstituted phenyl group; or a pharmaceutically acceptable salt thereof, Wherein the protein is alpha-synuclein.

27. The use according to claim 26, wherein the compound has the formula: 。 28. The use according to any one of claims 26-27, wherein the contacting is performed in vitro.

29. The use of any one of claims 26-27, wherein the contacting is performed in vivo.

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