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 is a divisional application of Chinese Patent Application No. 201980087338.6, titled "Compositions and Methods for Treating Neurodegenerative, Myodegenerative, and Lysosomal Storage Disorders", filed on November 20, 2019.

[0002] This application claims the benefit of 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 of the Invention

[0003] Neurodegenerative diseases include genetic and sporadic disorders associated with progressive neurological dysfunction. These diseases are characterized by the progressive deterioration of nerve cells or nerve cell function. It is estimated that one in four Americans will develop a neurodegenerative disorder during their lifetime. However, generally speaking, the underlying mechanisms causing such disorders are not well understood and few effective treatment options are available for preventing or treating neurodegenerative diseases.

[0004] 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 low (less than about 1 in 100,000 individuals affected), lysosomal storage disorders mainly affect children, who usually die in early childhood, some within months or years after birth. Many other children die after suffering from various symptoms of their specific lysosomal storage disorder for several years. Summary of the Invention

[0005] Compositions and methods for treating or preventing neurodegenerative diseases, myodegenerative diseases, prion diseases, or lysosomal storage diseases in a subject are provided herein. Compounds having Formula I are provided herein

[0006]

[0007] wherein

[0008] X is N or CH;

[0009] Y is unsubstituted or substituted with R 1 substituted C 6-10 aryl; or unsubstituted or substituted with R 1 substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0010] R 1 is –(CH 2 ) n -R2 , -(CH2) n -C(O)-R 2 or –O(CH 2 ) n -R 2 ;

[0011] R 2 is –H, -CN, halogen, C 1-3 alkyl, C 1-3 alkoxy, phenyl, pyridyl, amino, C 1-3 alkylamino, diC 1-3 alkylamino, hydroxyC 1-3 alkylamino, carboxyC 1-3 alkylamino, C 3-6 cycloalkylC 1-3 alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxyC 1-3 alkylpyrrolidinyl, carboxypyrrolidinyl, piperidinyl, C 1-3 alkylpiperidinyl, diC 1-3 alkylpiperidinyl, piperazinyl, C 1-3 alkylpiperazinyl, C 1-4 alkoxycarbonylpiperazinyl or morpholinyl;

[0012] Z is heteroaryl, heterocyclic or NR 3 R 4 ;

[0013] R 3 and R 4 are independently H, C 1-3 alkyl, C 1-3 alkoxy or unsubstituted phenyl, and

[0014] n is an integer selected from 0 to 3,

[0015] or an isomer thereof or a pharmaceutically acceptable salt.

[0016] There is also provided a method of treating or preventing a neurodegenerative disease, a myodegenerative disease or a prion disease in a subject, the method comprising administering to a subject having or at risk of developing the neurodegenerative disease, the myodegenerative disease or the prion disease an effective amount of a compound of formula I

[0017]

[0018] wherein

[0019] X is N or CH;

[0020] Y is unsubstituted or substituted with R 1Substituted C 6-10 aryl; or unsubstituted or R- 1 substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0021] R 1 is –(CH 2 ) n -R 2 、-(CH2) n -C(O)-R 2 or –O(CH 2 ) n -R 2 ;

[0022] R 2 is –H, -CN, halogen, C 1-3 alkyl, C 1-3 alkoxy, phenyl, pyridyl, amino, C 1-3 alkylamino, di-C 1-3 alkylamino, hydroxy-C 1-3 alkylamino, carboxy-C 1-3 alkylamino, C 3-6 cycloalkyl-C 1-3 alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxy-C 1-3 alkylpyrrolidinyl, carboxypyrrolidinyl, piperidinyl, C 1-3 alkylpiperidinyl, di-C 1-3 alkylpiperidinyl, piperazinyl, C 1-3 alkylpiperazinyl, C 1-4 alkoxycarbonylpiperazinyl or morpholinyl;

[0023] Z is heteroaryl, heterocyclic or NR 3 R 4 ;

[0024] R 3 and R 4 are independently H, C 1-3 alkyl, C 1-3 alkoxy or unsubstituted phenyl, and

[0025] n is an integer selected from 0 to 3,

[0026] or an isomer thereof or a pharmaceutically acceptable salt.

[0027] 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:

[0028]

[0029] wherein,

[0030] X is N or CH;

[0031] Y is unsubstituted or substituted by R 1 aryl; or unsubstituted or substituted by R 6-10 heteroaryl, or N-methylpiperazinyl; 1 substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0032] R 1 is –(CH 2 ) n -R 2 、-(CH2) n -C(O)-R 2 or –O(CH 2 ) n -R 2 ;

[0033] R 2 is –H, -CN, halogen, C 1-3 alkyl, C 1-3 alkoxy, phenyl, pyridyl, amino, C 1-3 alkylamino, di-C 1-3 alkylamino, hydroxy-C 1-3 alkylamino, carboxy-C 1-3 alkylamino, C 3-6 cycloalkyl-C 1-3 alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxy-C 1-3 alkylpyrrolidinyl, carboxypyrrolidinyl, piperidinyl, C 1-3 alkylpiperidinyl, di-C 1-3 alkylpiperidinyl, piperazinyl, C 1-3 alkylpiperazinyl, C 1-4 alkoxycarbonylpiperazinyl or morpholinyl;

[0034] Z is heteroaryl, heterocyclic or NR 3 R 4 ;

[0035] R 3 and R 4 are independently H, C 1-3 alkyl, C 1-3 alkoxy or unsubstituted phenyl, and

[0036] n is an integer selected from 0 to 3,

[0037] or an isomer thereof or a pharmaceutically acceptable salt.

[0038] Also provided are methods for treating or preventing lysosomal storage disorders (LSDs) in a subject. The methods include administering to a subject having an LSD or at risk of developing an LSD an effective amount of a compound having Formula I:

[0039]

[0040] wherein,

[0041] X is N or CH;

[0042] Y is unsubstituted or R 1 -substituted C 6-10 aryl; or unsubstituted or R 1 -substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0043] R 1 is –(CH 2 ) n -R 2 、-(CH2) n -C(O)-R 2 or –O(CH 2 ) n -R 2 ;

[0044] R 2 is –H, -CN, halogen, C 1-3 alkyl, C 1-3 alkoxy, phenyl, pyridyl, amino, C 1-3 alkylamino, di-C 1-3 alkylamino, hydroxy-C 1-3 alkylamino, carboxy-C 1-3 alkylamino, C 3-6 cycloalkyl-C 1-3 alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxy-C 1-3 alkylpyrrolidinyl, carboxypyrrolidinyl, piperidinyl, C 1-3 alkylpiperidinyl, di-C 1-3 alkylpiperidinyl, piperazinyl, C 1-3 alkylpiperazinyl, C 1-4 alkoxycarbonylpiperazinyl or morpholinyl;

[0045] Z is heteroaryl, heterocyclic or NR 3 R 4 ;

[0046] R 3 and R 4 are independently H, C 1-3 alkyl, C 1-3 alkoxy or unsubstituted phenyl, and

[0047] n is an integer selected from 0 to 3,

[0048] or an isomer or a pharmaceutically acceptable salt thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] 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 expressly indicates otherwise.

[0050] Figure 1 (Left and middle figures) show that after 16 hours of treatment, a neuroprotective effect was observed in B35 cells treated with 1 μM BK41043, as demonstrated by a decrease in lactate dehydrogenase (LDH) and an increase in bromide (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium (MTT) compared to the control, respectively. Figure 1 (Right figure) shows a progressive increase in cell viability via a decrease in LDH at a reduced concentration of BK40143 after five hours of treatment.

[0051] Figure 2 Shows the cell viability of B35 cells after transfection with pTau (left figure) or α-synuclein (right figure) for 24 hours and treatment with BK41043 for five hours. Dehydrogenase (LDH) measured in the medium reflects cell death, which was not different between the control and cells expressing tau or a-synuclein with or without BK40143, indicating that this compound does not cause toxicity or cell death.

[0052] Figure 3 Shows the levels of pTau(181) after 24 hours of transfection (left figure, Student's t-test: unpaired, two-tailed, *p < 0.05, ***p < 0.01), and BK41043 reduces the levels of pTau(181) in pTau-transfected B35 cells (right figure, n = 6, ANOVA: general, one-way, Dunnett's multiple comparison test, p < 0.05, ****p < 0.0001).

[0053] Figure 4 Shows the levels of α-synuclein after 24 hours of transfection (left figure), and BK41043 reduces α-synuclein in α-synuclein-transfected B35 cells (right figure). Student's t-test: unpaired, two-tailed, *p < 0.05, **p < 0.0, n = 6). In this cell culture model, BK40143 doses in the range of 10 μM - 1 mM appear to reduce α-synuclein levels.

[0054] Figure 5 Shows the cell viability of B35 cells after treatment with BK40197 for five hours.

[0055] Figure 6 Shows that after seven days of treatment, BK40143 reduces pTau(181) in Tau-expressing transgenic mice. The pTau(181) levels were measured by ELISA. Student's t-test: unpaired, two-tailed, Welch's correction, *p<0.05, ***p<0.01

[0056] Using Western blot analysis, Figure 7 Shows that treatment with BK-40143 (1.25 mg / kg, 2.5 mg / kg, or 5.0 mg / kg) does not affect the pTau(231) (AT180) levels in rTG4510 transgenic mice (n = 4).

[0057] Figure 8 Shows that after seven days of treatment with 1.25 mg / kg and 2.5 mg / kg of BK-41043, Tau(HT7) in Tau transgenic mice is significantly reduced. Student's t-test: unpaired, two-tailed, Welch's correction, *p<0.05, ***p<0.01, n = 4

[0058] Figure 9 Shows that treatment with BK-40143 (1.25 mg / kg or 2.5 mg / kg) results in in vivo inhibition of DDR1, as measured by the detection of phosphorylated (active) DDR1 (pMCK10). This data indicates that BK40143 effectively inhibits DDR in vivo.

[0059] Figure 10 Shows that after treatment with 2.5 mg / kg or 5 mg / kg of BK-40143, pTau(191) in CamP301L mice is reduced. Student's t-test: unpaired, Welch's correction, one-tailed, *p>0.05, p = 0.02, n = 4.

[0060] Figure 11A Shows that 1 mM and 100 uM of BK40196 significantly reduce the levels of α-synuclein in transfected B35 cells.

[0061] Figure 11B Shows that BK40197 does not significantly reduce the levels of α-synuclein in transfected B35 cells.

[0062] Figure 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 i.p. for 21 consecutive days. Figure 12A is an ELISA for human α-synuclein, which shows a 39% significant reduction in α-synuclein levels in animals treated with BK40143 compared to DMSO-treated control A53T mice. C57BL / 6J mice were used as controls and showed no detectable (N.D.) human α-synuclein. Figure 12B shows that BK40143 increases the overall level of dopamine (30%). BK40143 does increase the level of the dopamine metabolite homovanillic acid (HVA) in A53T mice, indicating more dopamine turnover, which can lead to better dopamine neurotransmission. Figure 12C and 12D is an immunoblot of α-synuclein, which reflects a 40% reduction in α-synuclein visible in the ELISA.

[0063] Figure 13 shows that BK40143 improves the locomotor speed of A53T mice. A53T mice were tested for overall locomotor ability in an open field test over a 60-minute trial. Although the mice showed no differences in the total distance traveled or the total time spent moving, their movement speed increased significantly with BK40143 treatment.

[0064] Figure 14A -E shows that BK40143 selectively inactivates DDR (by about 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 i.p. for 7 consecutive days. Figure 14A is an immunoblot probing for activated (phosphorylated) DDR1, which demonstrates that 1.25 and 2.5 but not 5 mg / kg BK40143 inactivates DDR1. Figure 14B is an immunoblot probing for activated Src, which demonstrates that BK40143 does not engage this tyrosine kinase. Figure 14C is an immunoblot probing for activated Abl, which demonstrates that BK40143 does not engage this tyrosine kinase. Figure 14D is an immunoblot probing for phosphorylated Tau (AT8), which shows that all three doses of BK40143 reduce the phosphorylated tau level in the same mice by 41 - 49% percentage, indicating that DDR inhibition is parallel to pTau reduction. Figure 14EIt is an ELISA for phosphorylated Tau (AT181), which shows that 2.5 mg / kg of BK40143 significantly reduces phosphorylated Tau.

[0065] Figure 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 of BK40143 or DMSO i.p. for 21 consecutive days. Figure 15A It is an immunoblot of aggregated extracellular amyloid-β (6E10), which demonstrates that 1.25 and 2.5 mg / kg of BK40143 significantly reduce amyloid-β plaques. Figure 15B It is an immunoblot probing of phosphorylated DDR1, which demonstrates that 1.25 and 2.5 mg / kg of BK40143 inactivate DDR1 by 40% and 31% respectively. Figure 15C , probing for activation (phosphorylated Abl (245)), demonstrates that BK40143 does not engage Abl. Figure 15D and 15E . It was found via ELISA that 1.25 and 2.5 mg / kg of BK40143 significantly reduce soluble human amyloid-β, but do not significantly reduce insoluble amyloid-β. Figure 15F Shows that 2.5 mg / kg of BK40143 significantly reduces human phosphorylated tau (Ser396) by more than 80%.

[0066] Figure 16 Shows that BK40143 may improve the cognitive performance of APP mice in the Morris water maze test. Measurements include the number of platform entries (left figure), the latency to the first entry (middle figure), and the distance traveled before the first entry into the platform (right figure). Although there were no significant differences between the groups, 1.25 mg / kg of BK showed a trend of increased performance with a higher number of platform entries, a shorter latency to the first entry, and a shorter distance traveled before the first entry.

[0067] Figure 17 Shows that BK40143 does not cause cell death in the hippocampus of APP mice. Representative 20-μm hippocampal sections were stained for Nissl bodies (left figure). 4x and 20x images of DMSO, 1.25 mg / kg, and 2.5 mg / kg of BK40143 are shown (left figure). The average staining intensity was quantified as the total amount of Nissl staining in all 4x images in ImageJ software (right figure). Detailed Description

[0068] The present disclosure provides compositions and methods for treating or preventing neurodegenerative diseases, muscle degenerative diseases, prion diseases, or lysosomal storage diseases in a subject.

[0069] Compound

[0070] In some embodiments, a class of compounds described herein includes compounds represented by Formula I:

[0071]

[0072] or an isomer or a pharmaceutically acceptable salt thereof.

[0073] In Formula I, X is N or CH.

[0074] Additionally, in Formula I, Y is an unsubstituted or R 1 -substituted C 6-10 aryl; or

[0075] an unsubstituted or R 1 -substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0076] Additionally, in Formula I, R 1 is –(CH 2 ) n -R 2 、-(CH2) n -C(O)-R 2 or –O(CH 2 ) n -R 2 ;

[0077] Additionally, in Formula I, R 2 is –H, -CN, halogen, C 1-3 alkyl, C 1-3 alkoxy, phenyl, pyridyl, amino, C 1-3 alkylamino, diC 1-3 alkylamino, hydroxyC 1-3 alkylamino, carboxyC 1-3 alkylamino, C 3-6 cycloalkylC 1-3 alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxyC 1-3 alkylpyrrolidinyl, carboxypyrrolidinyl, piperidinyl, C 1-3 alkylpiperidinyl, diC 1-3 alkylpiperidinyl, piperazinyl, C 1-3 alkylpiperazinyl, C 1-4 alkoxycarbonylpiperazinyl or morpholinyl;

[0078] Z is heteroaryl, heterocyclic, or NR 3 R4 ;

[0079] In addition, in Formula I, R 3 and R 4 are independently selected from H, C 1-3 alkyl, C 1-3 alkoxy, or unsubstituted phenyl, and n is an integer selected from 0 to 3.

[0080] In some embodiments of Formula I, Y is benzyl substituted with R 1 :

[0081]

[0082] In some embodiments of Formula I, Y is meta-benzyl substituted with R 1 :

[0083]

[0084] 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 benzyl substituted with R 1 :

[0085]

[0086] 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-benzyl substituted with R 1 :

[0087]

[0088] In some embodiments of Formula I, Z is morpholino and Y is benzyl substituted with R 1 :

[0089]

[0090] In some embodiments of Formula I, Z is morpholino and Y is meta-benzyl substituted with R 1 :

[0091]

[0092] The compound of Formula I is compound 1 (BK40197):

[0093]

[0094] Another formula I compound is compound 2 (BK40193):

[0095]

[0096] 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 heterocyclic group. In some embodiments of formula I, Z is morpholin-1-yl. In some embodiments of formula I, R 3 is H and R 4 is an unsubstituted phenyl group.

[0097] As used herein, the terms alkyl, alkenyl, and alkynyl include straight-chain and branched-chain monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, etc. The scope of these groups useful in the compounds and methods described herein includes C 1 -C 20 alkyl, C 2 -C 20 alkenyl, and C 2 -C 20 alkynyl. Additional scope of these groups useful in the compounds and methods described herein includes C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 4 alkyl, C 2 -C 4 alkenyl, and C 2 -C 4 alkynyl.

[0098] As used herein, the term alkoxy is an alkyl group bonded through a single, terminal ether bond. As used herein, the term hydroxy is represented by the formula —OH.

[0099] As used herein, the term amine or amino is represented by the formula —NR 3 R 4 wherein R 3 and R 4 can each be a substituent as described herein, such as hydrogen, alkyl, cycloalkyl, haloalkyl, alkenyl, or alkynyl as described above.

[0100] The alkoxy, amino, alkyl, alkenyl, alkynyl or carbonyl molecules used herein may be substituted or unsubstituted. As used herein, the term "substituted" includes adding an alkoxy, amino, alkyl, alkenyl, alkynyl or carbonyl to a position attached to the backbone of the alkoxy, amino, alkyl, alkenyl, alkynyl or carbonyl, for example, replacing a 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 has a full complement of hydrogens, i.e., commensurate with its saturation level and without substitution, such as straight-chain decane (–(CH 2 ) 9 –CH 3 )。

[0101] Heteroalkyl, heteroalkenyl and heteroalkynyl are defined similarly to alkyl, alkenyl and alkynyl, but may contain O, S or N heteroatoms or combinations thereof within the backbone. The scope of these groups useful in the compounds and methods described herein includes C 1 -C 20 heteroalkyl, C 2 -C 20 heteroalkenyl and C 2 -C 20 heteroalkynyl. Additional scope of these groups useful in the compounds and methods described herein includes C 1 -C 12 heteroalkyl, C 2 -C 12 heteroalkenyl, C 2 -C 12 heteroalkynyl, C 1 -C 6 heteroalkyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, C 1 -C 4 heteroalkyl, C 2 -C 4 heteroalkenyl and C 2 -C 4 heteroalkynyl.

[0102] The terms cycloalkyl, cycloalkenyl and cycloalkynyl include cyclic alkyls having a single ring or multiple fused rings. Examples include cyclohexyl, cyclopentylethyl and adamantyl. The scope of these groups useful in the compounds and methods described herein includes C 3 -C 20 cycloalkyl, C 3 -C 20 cycloalkenyl and C 3 -C 20Cycloalkynyl. Additional ranges of these groups useful in the compounds and methods described herein include C 5 -C 12 Cycloalkyl, C 5 -C 12 Cycloalkenyl, C 5 -C 12 Cycloalkynyl, C 5 -C 6 Cycloalkyl, C 5 -C 6 Cycloalkenyl and C 5 -C 6 Cycloalkynyl.

[0103] 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. Ranges of these groups useful in the compounds and methods described herein include C 3 -C 20 Heterocycloalkyl, C 3 -C 20 Heterocycloalkenyl and C 3 -C 20 Heterocycloalkynyl. Additional ranges of these groups useful in the compounds and methods described herein include C 5 -C 12 Heterocycloalkyl, C 5 -C 12 Heterocycloalkenyl, C 5 -C 12 Heterocycloalkynyl, C 5 -C 6 Heterocycloalkyl, C 5 -C 6 Heterocycloalkenyl and C 5 -C 6 Heterocycloalkynyl.

[0104] Aryl molecules include, for example, cyclic hydrocarbons incorporating one or more planar assemblies of generally six carbon atoms joined by delocalized electrons, the planar assemblies being numbered as if they were composed of alternating single covalent bonds and double covalent bonds. An example of an aryl molecule is benzene. Heteroaryl molecules include substitutions along the main cyclic chain of their atoms, such as O, N, or S. When heteroatoms are introduced, assemblies of five atoms (e.g., four carbons and one heteroatom) can give rise to an aromatic system. Examples of heteroaryl molecules include furan, pyrrole, thiophene, imidazole, oxazole, pyridine, and pyrazine. Aryl and heteroaryl molecules may also include additional fused 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.

[0105] 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, DDR1, DDR2, cKIT, arginase II, Src, Fyn, VEGFR, and Zac. In some embodiments, the compound of Formula I selectively inhibits Abl, PDGFRα, PDGFRβ, DDR1, DDR2, cKIT, arginase II, Src, Fyn, VEGR, or Zac. In some embodiments, the compound of Formula I inhibits DDR1 and / or DDR2.

[0106] As used herein, the term pharmaceutically acceptable salt refers to salts that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio. 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 the compound 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 (such as ion exchange). Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, formates, fumarates, glucoheptanoates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactates, lactobionates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, trifluoroacetates, undecanoates, valerates, etc.

[0107] 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 art of synthetic organic chemistry. The compounds described herein can be prepared from readily available starting materials. The optimal reaction conditions can vary with the specific reactants or solvents used, but such conditions can be determined by those skilled in the art through routine optimization procedures.

[0108] Variations regarding Formula I include the addition, subtraction, or rearrangement of the various moieties described for each compound. Similarly, when one or more chiral centers are present in the molecule, all possible chiral variants are included. Additionally, the synthesis of the compounds can involve the protection and deprotection of multiple chemical groups. The use of protection and deprotection and the selection of appropriate protecting groups can be determined by those skilled in the art. Protecting group chemistry can be found, for example, in Wuts, Greene’s Protective Groups in Organic Synthesis, 5th Edition, Wiley & Sons, 2014, which is incorporated herein by reference in its entirety.

[0109] The reactions to produce the compounds described herein can be carried out in a solvent that can be selected by those skilled in the art of organic synthesis. The solvent should generally not react with the starting materials (reactants), intermediates, or products under the conditions (i.e., temperature and pressure) of the reaction. The reaction can be carried out in one solvent or a mixture of more than one solvent. The formation of the product or intermediate can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0110] Any of the compounds described herein can be modified to enhance blood-brain barrier permeability. Optionally, one or more of the compounds described herein can be administered with an agent that enhances the blood-brain barrier permeability of the compound.

[0111] Methods for treating or preventing neurodegenerative diseases, myodegenerative diseases, or prion diseases

[0112] The present invention provides 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, α-synucleinopathies (such as Parkinson's disease, Lewy body disease), multiple sclerosis, glial cell inclusions (including multiple system atrophy), chronic traumatic encephalopathy, Tau proteinopathies, progressive supranuclear palsy and corticobasal degeneration. The neurodegenerative disease may also be a secondary neurodegenerative disease induced by traumatic brain injury, stroke or infection, such as bacterial or viral infection (such as HIV, herpes simplex virus (HSV)).

[0113] Myodegenerative diseases or disorders include, but are not limited to, dystrophy (such as muscular dystrophy), myopathy (such as nemaline myopathy, multi- / minicore myopathy, central core myopathy, mitochondrial myopathy, metabolic myopathy, etc.) or myotonia (such as congenital myotonia, paramyotonia congenita or myotonic dystrophy).

[0114] By way of example, 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.

[0115] The methods include administering to a subject having the neurodegenerative disease, myodegenerative disease or prion disease or at risk of developing the neurodegenerative disease, the myodegenerative disease or the prion disease an effective amount of a compound having Formula I:

[0116]

[0117] wherein,

[0118] X is N or CH;

[0119] Y is unsubstituted or substituted by R 1 substituted C 6-10 aryl; or unsubstituted or substituted by R 1 substituted C 5-10 heteroaryl, or N-methylpiperazinyl;

[0120] R 1 is –(CH 2 ) n -R2 , -(CH2) n -C(O)-R 2 or –O(CH 2 ) n -R 2 ;

[0121] R 2 is –H, -CN, halogen, C 1-3 alkyl, C 1-3 alkoxy, phenyl, pyridyl, amino, C 1-3 alkylamino, diC 1-3 alkylamino, hydroxyC 1-3 alkylamino, carboxyC 1-3 alkylamino, C 3-6 cycloalkylC 1-3 alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxyC 1-3 alkylpyrrolidinyl, carboxypyrrolidinyl, piperidinyl, C 1-3 alkylpiperidinyl, diC 1-3 alkylpiperidinyl, piperazinyl, C 1-3 alkylpiperazinyl, C 1-4 alkoxycarbonylpiperazinyl or morpholinyl;

[0122] Z is heteroaryl, heterocyclic or NR 3 R 4 ;

[0123] R 3 and R 4 are independently H, C 1-3 alkyl, C 1-3 alkoxy or unsubstituted phenyl, 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 having formula I does not contain one or more halogen atoms. In some methods, in the compound having formula I, Y is 2-m-toluoyl. In some methods, in the compound having formula I, Z is heterocyclic. In some methods, in the compound having formula I, Z is morpholin-1-yl. In some methods, in the compound having formula I, R 3 is H and R 4 is unsubstituted phenyl.

[0127] In some methods, the compound having formula I (where Y is benzyl substituted by R 1 ) is administered to the subject:

[0128]

[0129] In some methods, a compound of formula I in which Y is a benzyl group substituted at the meta position with R 1 is administered to the subject:

[0130]

[0131] In some methods, a compound of formula I in which Z is NR 3 R 4 where R 3 is benzyl or H, R 4 is benzyl or H, and Y is a benzyl group substituted with R 1 is administered to the subject:

[0132]

[0133] In some methods, a compound of formula I in which Z is NR 3 R 4 where R 3 is benzyl or H, R 4 is benzyl or H, and Y is a benzyl group substituted at the meta position with R 1 is administered to the subject:

[0134]

[0135] In some methods, a compound of formula I in which Z is morpholino and Y is a benzyl group substituted with R 1 is administered to the subject:

[0136]

[0137] In some methods, a compound of formula I in which Z is morpholino and Y is a benzyl group substituted at the meta position with R 1 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 having a neurodegenerative disease, a myodegenerative disease, or a prion disease, or 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 having a neurodegenerative disease, a myodegenerative disease, or a prion disease, or at risk of developing a neurodegenerative disease, a myodegenerative disease, or a prion disease. For example, one or more of the following tests can be used: genetic testing (such as the identification of mutations in the TDP-43 gene) or family analysis (such as family history), central nervous system imaging (such as magnetic resonance imaging and positron emission tomography), electroencephalogram, clinical or behavioral tests (such as the assessment of muscle weakness, tremors, gait, or memory), or laboratory tests.

[0143] The methods optionally further include administering a second therapeutic agent to the subject. The second therapeutic agent is selected from the group consisting of levodopa, dopamine agonists, anticholinergic agents, cholinergic agents (such as 5-hydroxytryptamine (5-HT) inhibitors), monoamine oxidase inhibitors, COMT inhibitors, donepezil, memantine, risperidone, amantadine, rivastigmine, NMDA antagonists, acetylcholinesterase inhibitors, cholinesterase inhibitors, riluzole, antipsychotic agents, antidepressants, glucocorticoids (such as prednisone), tyrosine kinase inhibitors (such as nilotinib, bosutinib, imatinib, pazopanib, etc.), and tetrabenazine. The second therapeutic agent or therapy can be administered to the subject before, simultaneously with, or after the administration of the compound of formula I.

[0144] In a method in which a tyrosine kinase inhibitor is administered as the second therapeutic agent, the tyrosine kinase inhibitor can be a tyrosine kinase inhibitor that does not inhibit the tyrosine kinase receptor inhibited by the compound of formula I or has a reduced selectivity for the tyrosine kinase receptor compared to the compound of formula I.

[0145] The present invention also provides a method for inhibiting or preventing toxic protein aggregation in neurons and / or rescuing neurons from degeneration. As used herein, references to inhibiting, decreasing, or reducing include a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more change compared to a control level.

[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 – (CH 2 ) n -R 2 、-(CH2) n -C(O)-R 2 or –O(CH 2 ) 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, C3-6 Cycloalkyl C 1-3 Alkylamino, pyrrolidinyl, hydroxypyrrolidinyl, hydroxy C 1-3 Alkylpyrrolidinyl, carboxypyrrolidinyl, piperidinyl, C 1-3 Alkylpiperidinyl, di C 1-3 Alkylpiperidinyl, piperazinyl, C 1-3 Alkylpiperazinyl, C 1-4 Alkoxycarbonylpiperazinyl or morpholinyl;

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

[0156] R 3 and R 4 are 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 having formula I does not contain one or more halogen atoms. In some methods, in the compound having formula I, Y is 2-m-toluoyl. In some methods, in the compound having formula I, Z is heterocyclic. In some methods, in the compound having formula I, Z is morpholin-1-yl. In some methods, in the compound having formula I, R 3 is H and R 4 is unsubstituted phenyl.

[0160] In some methods, the compound having formula I (where Y is benzyl substituted by R 1 ) is administered to the subject:

[0161]

[0162] In some methods, the compound having formula I (where Y is benzyl substituted by R at the meta position 1 ) is administered to the subject:

[0163]

[0164] In some methods, the compound having formula I (where Z is NR 3 R 4 , R 3 is benzyl or H, R 4 is benzyl or H, and Y is substituted by R 1(Substituted benzyl) is administered to the subject:

[0165]

[0166] In some methods, a 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-substituted benzyl by R 1 is administered to the subject:

[0167]

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

[0169]

[0170] In some methods, a compound of formula I wherein Z is morpholino and Y is meta-substituted benzyl by R 1 is administered to the subject:

[0171]

[0172] Examples of formula I that can be used to treat or prevent LSD include the following compounds:

[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, DDR1, DDR2, cKIT, arginase II, Src, Fyn, VEGFR, and Zac. In some embodiments, the compound of formula I selectively inhibits Abl, PDGFRα, PDGFRβ, DDR1, DDR2, cKIT, arginase II, Src, Fyn, VEGR, or Zac. In some embodiments, the compound having formula I inhibits DDR1 and / or DDR2. By way of example and not limitation, Compound 1 or Compound 2 can be used to inhibit DDR1 and / or DDR2. In another embodiment, the compound having formula I (such as Compound 1 or Compound 2) selectively inhibits DDR1 or DDR2.

[0176] LSD is a hereditary metabolic disorder caused by lysosomal dysfunction. In most cases, LSD is caused by a deficiency 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 lysosome biogenesis cause LSD. Progressive lysosomal accumulation of undegraded metabolites leads to systemic cellular and tissue dysfunction and, thus, to multisystem 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 II (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, α-mannosidosis, metachromatic leukodystrophy, and Pompe disease. The LSDs provided herein are examples of diseases or disorders associated with reduced lysosomal clearance.

[0177] Also provided are methods for promoting lysosomal clearance in one or more cells of a subject, the methods comprising administering to a subject having a disorder associated with reduced lysosomal clearance an effective amount of a compound having Formula I. Optionally, the compound having Formula I is Compound 1 or Compound 2. As used throughout, lysosomal clearance is the process by which accumulated lipids, proteins, glycoproteins, or combinations thereof are metabolized or degraded in the lysosomes of one or more cells of the subject. A reduction in 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 as compared to a control, e.g., as compared to lysosomal clearance in one or more cells of a healthy subject. Any disorder associated with reduced lysosomal clearance can be treated using the methods provided herein, including but not limited to any of the LSDs recited throughout. As used herein, references to promoting or increasing include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 400%, or more as compared to a control level. Optionally, promoting lysosomal clearance reduces the amount of lipids, proteins, glycoproteins, or combinations thereof in existing aggregates in the lysosomes of one or more cells of the subject. Optionally, promoting lysosomal clearance inhibits or prevents the formation of aggregates comprising lipids, proteins, glycoproteins, or combinations thereof in the lysosomes of one or more cells of the subject. Optionally, promoting lysosomal clearance reduces the amount of time required to degrade or metabolize lipids, proteins, glycoproteins, or combinations thereof in one or more cells of the subject as compared to a control.

[0178] Optionally, in the methods provided herein, an effective amount of a compound having 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 a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to a control level. The term may or may not include the complete elimination of toxic substances in one or more cells of the subject. Optionally, the one or more cells are brain cells, cells in one or more peripheral tissues of the subject, or a combination thereof. Optionally, the brain cells may be neurons and / or glial cells. In the methods provided herein, the 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 the subject. In the methods provided herein, the toxic substances may be in lysosomes or elsewhere in one or more cells of the subject. For example, and not intended to be limiting, LSDs characterized by lipid accumulation in subject cells include, but are not limited to, sphingolipidoses (including Gaucher 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] Lipids and glycoproteins that accumulate in sphingolipidoses include sphingomyelin in the brain and red blood cells (Nieman Pick disease); glycolipids in the brain, heart, and kidney, including globotriaosylceramide (Fabry disease); galactocerebroside in oligodendrocytes (Krabbe disease); glucocerebroside in red blood cells, spleen, and liver (Gaucher disease); GM2 ganglioside in neurons (Tay-Sachs disease) and Sandhoff disease; GM1 ganglioside; and sulfatide compounds in nervous tissue (metachromatic leukodystrophy).

[0180] Lysosomal storage diseases also include mucopolysaccharidoses (MPs), which are characterized by a deficiency of one or more lysosomal enzymes, such as α-L-iduronidase (Hurler disease, Scheie syndrome, and Hurler-Scheie syndrome); iduronate sulfatase (Hunter disease), heparan sulfate (Sanfilippo type A), N-acetyl-α-D-glucosamine (Sanfilippo type B), CoA-α-glucosaminide-N-acetyltransferase (Sanfilippo type C), N-acetyl-α-D-glucosamine-6-sulfate (Sanfilippo type D and Morquio syndrome type A), β-galactosidase (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 accumulation of sugars and phosphorylated sugars in lysosomes.

[0181] The methods provided herein optionally include selecting a subject having an LSD. Those skilled in the art know how to diagnose a subject having an LSD. For example, one or more of the following tests can be used: genetic testing (e.g., identification of mutations associated with the 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., assessment for identifying mood disorders, aggression, and / or cognitive abnormalities), or laboratory testing (e.g., blood and / or urine tests for identifying abnormal levels of metabolites or enzyme deficiencies).

[0182] The methods provided herein optionally further include administering to the subject an effective amount of a second therapeutic agent or therapy. The second therapeutic agent or therapy can 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, a hematopoietic stem cell, a bone marrow transplant, gene therapy, or a small molecule. For example, and not by way of limitation, an LSD associated with an enzyme deficiency can be treated with an enzyme to increase the amount of the deficient enzyme in the subject. For example, use of recombinant enzymes such as imiglucerase or taliglucerase in enzyme replacement therapy (ERT) can be used as a second therapeutic agent to treat type I Gaucher disease. Small molecules that inhibit glucosylceramide synthase (e.g., miglustat and eliglustat) can also be used to treat type I Gaucher disease. 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 normally processed by the enzyme in the subject, can also be used.

[0183] One or more therapeutic agents that mitigate the symptoms of LSD may also be administered. For example, anti-seizure medications such as gabapentin or lamotrigine can be used to prevent seizures 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 can 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 where a tyrosine kinase is administered as the second therapeutic agent, the tyrosine kinase can be a tyrosine kinase inhibitor that has a different selectivity for one or more receptor tyrosine kinases compared to the compound of Formula I.

[0185] Pharmaceutical composition

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

[0187] Exemplary doses for administration of any of the compounds described herein (e.g., a compound of Formula I) include doses of from about 0.5 to about 200 mg of the active compound / kg body weight per day, which can be administered as a single dose or in the form of individual divided doses, such as 1 to 4 times per day. Alternatively, the dose can be from about 0.5 to about 150 mg of the active compound / kg body weight per day, from about 0.5 to 100 mg of the active compound / kg body weight per day, from about 0.5 to about 75 mg of the active compound / kg body weight per day, from about 0.5 to about 50 mg of the active compound / kg body weight per day, from about 0.5 to about 25 mg of the active compound / kg body weight per day, from about 1 to about 50 mg of the active compound / kg body weight per day, from about 1 to about 40 mg of the active compound / kg body weight per day, from about 1 to about 30 mg of the active compound / kg body weight per day, from about 1 to about 30 mg of the active compound / kg body weight per day, about 30 mg of the active compound / kg body weight per day, about 20 mg of the active compound / kg body weight per day, about 10 mg of the active compound / kg body weight per day, or about 5 mg of the 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 between these amounts. The dose 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. Those skilled in the art will adjust the dose based on the specific characteristics of the inhibitor and the subject receiving the inhibitor as described below.

[0189] The composition may comprise a single unit dose of a compound of formula I, such as a single unit dose of Compound 1 or Compound 2 of about 50 mg / kg or lower, 40 mg / kg or lower, 30 mg / kg or lower, 20 mg / kg or lower, 10 mg / kg or lower, about 5 mg / kg or lower, about 2.5 mg / kg or lower or about 1.5 mg / kg or lower, or a pharmaceutically acceptable salt thereof. Also provided is a package comprising one or more single unit doses of a compound having formula I, such as a plurality of single unit doses of Compound 1 or Compound 2. The package may further comprise a single or multiple unit doses of one or more of the 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 such determination is within the skill in the art. The dose ranges for administration are those that are large enough to produce the desired effect, where one or more symptoms of the disease or disorder are affected (e.g., alleviated or delayed). The dose should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, unwanted cell death, etc. Generally, the dose will vary with the type of inhibitor, the species, age, weight, general health, sex and diet of the subject, the mode and time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder and can be determined by those skilled in the art. The dose can be adjusted by the individual physician in the case of any contraindications. The dose may vary and may be administered in divided doses once or more times a day.

[0191] The compounds of Formula I and other agents 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 of Formula I and a pharmaceutical carrier. The term carrier means a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, effectiveness, selectivity, or any other characteristic of the compound or composition to achieve its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and minimize any adverse side effects in 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 can be in the form of a solid, semi-solid, or liquid dosage form, such as tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage forms suitable for precise dosing in a single administration. The composition will comprise a therapeutically effective amount of the agent or its derivatives described herein in combination with a pharmaceutically acceptable carrier and may additionally include other medicinal agents, pharmaceutically active agents, carriers, or diluents. Pharmaceutically acceptable means a material that is biologically or otherwise desirable and can be administered to an individual together with the selected agent without causing unacceptable biological effects or interacting in a harmful manner with other components of the pharmaceutical composition containing the material.

[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 use in pharmaceutical formulations. The choice of carrier for a composition will depend on the route of administration intended for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described, for example, in 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 buffers, citrate buffers, 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 dextrin; 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 the agents described herein for parenteral injection may comprise a physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion. 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). Adequate fluidity can be maintained, for example, by the use of coatings (such as lecithin), in the case of dispersions by maintaining the required particle size and by the use of surfactants.

[0196] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of microbial action can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. Isotonic agents such as sugars, sodium chloride, etc. may also be included. Prolonged absorption of injectable drug forms can be brought about by the use of agents that delay absorption (such as aluminum monostearate and gelatin).

[0197] Solid dosage forms for oral administration of the compounds or their derivatives described herein include capsules, tablets, pills, powders and granules. In such solid dosage forms, the compounds or their derivatives described herein are admixed with at least one inert conventional excipient (or carrier), such as sodium citrate or calcium phosphate, or (a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants, such as glycerol, (d) disintegrating agents, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, (e) solution retardants, such as paraffin, (f) absorption promoters, such as quaternary ammonium compounds, (g) wetting agents, such as cetyl alcohol and glycerol 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 forms may also contain buffering agents.

[0198] Similar types of solid compositions can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose (lactose / milk sugar) and high molecular weight polyethylene glycol.

[0199] Solid dosage forms such as tablets, sugar coated pills, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other substances known in the art. They can contain emulsifying agents and can also have a composition that causes them to release the one or more active compounds in a delayed manner in a certain part of the intestine. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in microencapsulated form, optionally with one or more of the excipients mentioned above.

[0200] The compounds described herein can be incorporated into pharmaceutical compositions that allow immediate release or delivery of those compounds to a mammal. The compounds described herein can also be incorporated into pharmaceutical compositions that allow modified release of those compounds, such as delayed release or extended release (e.g., sustained release or controlled release) to a mammal for a period of days, weeks, or a month or more. 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 or their derivatives described herein include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butanediol, dimethylformamide, oils (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] In addition to such inert diluents, the compositions may also include additional agents such as wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents or fragrances.

[0203] The compositions are administered in a variety of ways, depending on the need for local or systemic treatment and on the area to be treated. The compositions are administered via any of several routes of administration, including oral, parenteral, intravenous, intraperitoneal, intracranial, intraspinal, intrathecal, intraventricular, intramuscular, subcutaneous, intracavitary or transdermal. The pharmaceutical compositions 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 methods of administration described herein can be inferred from a dose - response curve derived from in vitro or animal model test systems.

[0204] Throughout, "treat" (treat / treating / treatment) refers to a method of alleviating 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 disorder. Treatment may also refer to a method of alleviating the underlying pathology rather than just the symptoms. The effects produced by administering to the subject may include, but are not limited to, the following effects: alleviating one or more symptoms of the disease, reducing the severity of the disease, complete ablation of the disease, or delaying the onset or worsening of one or more symptoms. For example, if there is a reduction of about 10% in one or more symptoms of the disease in a subject compared to the subject before treatment or compared to a control subject or a control value, the disclosed method is considered to be a treatment. Thus, the reduction may be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or any reduction amount therebetween.

[0205] As used throughout, "subject" means an individual. The subject may be an adult subject or a pediatric subject. Pediatric subjects include subjects between the ages of birth and eighteen years. Thus, 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. Non-human primates are also subjects. The term "subject" includes domestic animals (such as cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary uses and medical formulations are contemplated herein.

[0206] Products that are useful in the disclosed methods and compositions, products that can be used in combination with the disclosed methods and compositions, products that are useful for preparing the disclosed methods and compositions, or materials, compositions, and components that are the products of the disclosed methods and compositions are disclosed. These and other materials are disclosed herein, and it is to be understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, each and every different individual and collective combination and permutation of these compounds is specifically contemplated and described herein, even though specific mention of each may not be explicitly disclosed. For example, if a method is disclosed and discussed and various modifications that can be made to the various molecules included in the method are discussed, then each and every combination and permutation of the method, and possible modifications thereof, are specifically contemplated unless specifically indicated to the contrary. Similarly, any subset or combination thereof is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including but not limited to the method steps of using the disclosed compositions. Thus, if multiple additional steps can be performed, it is to be understood that each of these additional steps can be performed in combination with any particular method step or combination of method steps of the disclosed method, and each such combination or subset of combinations is specifically contemplated and is to be regarded as disclosed.

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

[0208] Examples

[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 in deuterated solvents at 400 MHz ( 1 1H NMR) and 100 MHz ( 13 13C NMR). As described below, the reaction products were purified by silica gel column chromatography (particle size 40 - 63 μm).

[0212] Synthesis Methods and Compound Characterization

[0213] Synthesized 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 in 72% yield (315 mg, 1.21 mmol). R f = 0.2 (hexane / EtOAc, 1:1); 1 1H 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 13C 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 C12H9ClNS: 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 Reaction

[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 an oil bath at 100 °C and stirred for 16 h to 4 days. After complete conversion was achieved based on 1 1H NMR analysis, the 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 hexane-ethyl acetate as the mobile phase as described below.

[0218]

[0219] N-Phenyl-2-(m-tolyl)thieno[3,2-b]pyridin-7-amine (7). According to the general procedure described above, after 16 h at 100 °C, compound 7 as a colorless solid was obtained 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 of DMSO in a yield of 94% (89 mg, 0.282 mmol). R f R = 0.2 (hexane / EtOAc, 1:1); 1 1H 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 13C 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 N 2 Anal. Calcd for C20H16N2S: C, 75.92; H, 5.10; N, 8.85. Found: 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). According to the general procedure described above, after 16 h at 100 °C, compound 8 as a colorless solid was obtained 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 of DMSO in a yield of 92% (95 mg, 0.276 mmol). R f R = 0.2 (hexane / EtOAc, 2:1); 11H NMR (400 MHz, chloroform-d) δ = 8.41 (d, J = 5.6 Hz, 1H), 7.70 (s, 1H), 7.58–7.51 (m, 2H), 7.36–7.29 (m, 2H), 7.21 (d, J = 7.9 Hz, 1H), 6.96 (d, J = 5.6 Hz, 1H), 6.87 (dd, J = 7.9, 2.4 Hz, 1H), 6.83 (dd, J = 7.8, 7.7 Hz, 1H), 6.73 (dd, J = 7.9, 2.5 Hz, 1H), 6.07 (s, 1H), 3.83 (s, 3H), 2.44 (s, 3H); 13 13C 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 N 2 Anal. Calcd for C19H17NOS: C, 72.80; H, 5.24; N, 8.09. Found: C, 72.53; H, 5.61; N, 8.19.

[0222]

[0223] 4-(2-(m-Tolyl)thieno[3,2-b]pyridin-7-yl)morpholine (9). According to the general procedure described above, after 4 days at 100 °C, compound 9 was obtained as a colorless solid 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 of DMSO in 98% yield (91 mg, 0.294 mmol). R f f = 0.2 (hexane / EtOAc, 1:1); 1 1H NMR (400 MHz, chloroform-d) δ = 8.48 (d, J = 5.4 Hz, 1H), 7.69 (s, 1H), 7.59–7.52 (m, 2H), 7.34 (dd, J = 7.9, 7.8 Hz, 1H), 7.20 (dd, J = 7.9, 2.1 Hz, 1H), 6.64 (d, J = 5.4 Hz, 1H), 4.03–3.85 (m, 4H), 3.54–3.39 (m, 4H), 2.43 (s, 3H); 13¹³C 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 N 2 Analytical calculated values for C, H, N, O: C, 69.65; H, 5.85; N, 9.02. Experimental 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). At -78 °C under an inert atmosphere, boron tribromide (4 equiv) was added 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). The mixture was stirred for 4 h and the reaction temperature was allowed to reach 0 °C. After quenching with 1 M 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 in 97% yield (64 mg, 0.194 mmol). R f = 0.4 (DCM / MeOH, 9:1); 1 ¹H NMR (399 MHz, methanol-d 4 ) δ = 8.22 (d, J = 6.7 Hz, 1H), 7.70 (s, 1H), 7.65 (s, 1H), 7.61 (dd, J = 7.5, 2.1 Hz, 1H), 7.40 (dd, J = 7.6, 7.6 Hz, 1H), 7.34–7.31 (m, 2H), 6.93 (d, J = 6.7 Hz, 1H), 6.88 (m, 1H), 6.85–6.79 (m, 2H), 2.44 (s, 3H); 13 ¹³C NMR (100 MHz, methanol-d 4 ) 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 16 N2 Analytical calculated values of the OS: C, 72.26; H, 4.85; N, 8.43. Experimental 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 Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin and incubated at 37 °C and 5% CO 2 For the experiments, cells were transferred to a 12-well plate (catalog number 150628, ThermoFisher, Waltham, MA) and grown to at least 70% confluence. Transient transfection was performed for 24 hours using 3 μg of P301L tau (catalog number 30145, Addgene) cDNA or 3 μg of human α-synuclein cDNA with Fugene HD transfection reagent (catalog number E2311, Promega, Madison WI). Cells were treated with 1 mM, 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, and 0.001 μM dissolved in DMSO or an equivalent of 5 μL DMSO for 5 hours. The cell culture medium was collected, and the cells were harvested using sodium-tris, EDTA, NP-40 (STEN) buffer and centrifuged at 10,000 x g at 4 °C for 20 minutes, and the supernatant was collected. Cell viability was determined via lactate dehydrogenase assay (catalog number 88954, Thermofisher) and MTT assay (catalog number V13154, Thermofisher). Proteins were extracted by removing the medium and adding 0.2 ml of 1x STEN buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.2% NP-40, 0.2% BSA, 20 mM PMSF, and protease cocktail inhibitor) to the cell layer and incubating on ice for 10 minutes. The bottom of the well was scraped and incubated on ice for an additional 10 minutes. The cell lysates were collected, stored at -80 °C, and used for additional analysis.

[0228] Drug preparation

[0229] Compound 1 (BK40197) and Compound 2 (BK40143) with 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. The drugs were stored at -80 °C.

[0230] MTT assay

[0231] To measure cell viability, cells were incubated in 5% CO at 37 °C 2 with 500 μL of Dulbecco's Modified Eagle Medium (DMEM) containing 50 μL of (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT)) for 4 hours. The medium was aspirated such that 125 μL of medium was retained. The formazan salt was dissolved in 250 μL of DMSO. Absorbance was read at 570 nm against a blank containing 125 μL of medium and MTT, and 250 μL of DMSO.

[0232] Lactate dehydrogenase (LDH) assay

[0233] Cytotoxicity was quantitatively measured by assessing the cytoplasmic enzyme LDH released into the cell culture medium 5 hours after exposure to the drug after an initial dose. The cell culture medium was collected and aliquots were coupled with lactate and NAD+. LDH catalyzes the reaction of converting lactate to pyruvate to produce NADH. NADH in turn reduces the tetrazolium salt (INT) to a red formazan product. The amount of LDH in the medium is proportional to the amount of formazan, which is measured at 490 nm. The absorbance at 680 nm measured to determine the background signal of the instrument was subtracted from the absorbance at 490 nm to calculate the LDH activity.

[0234] Cell culture transfection and treatment

[0235] To transiently transfect α-synuclein into rat neuroblastoma B35 cells, HD transfection reagent (Promega Corporation, Madison, WI) was used. Cells were grown in 12-well plates. A mixture containing 12 μg of cDNA, 540 μg of DMEM containing 2% FBS, and 60 μL of HD transfection reagent was incubated for 10 minutes. The cells were treated with 50 μL of HD transfection reagent / DNA mixture for 24 hours. After transfection, the cells were harvested, the medium was aspirated, and the cells were treated with 200 μL of sodium Tris EDTA NP40 (STEN) lysis buffer, then scraped from the plate and collected into 1.5 mL centrifuge tubes.

[0236] Mouse treatment

[0237] Experiments were conducted on the following mice: (a) TgAPP mice that express the neuronal human APP gene isoform 770 under the control of the murine thymocyte antigen 1, θ (Thy1) promoter, said gene containing the Swedish (K670N / M671L), Dutch E693Q, and Iowa D694N mutations (Davis et al., "Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch / Iowa mutant form of amyloid beta-protein precursor," The Journal of Biological Chemistry 279(19):20296 - 20306(2004); (b) rTg4510 mice that express human P301L tau and have a tet response element (TRE or tetO) and a murine prion protein promoter sequence (PrP or Prnp) that direct the expression of a P301L mutant variant of the human quadruple - repeat microtubule - associated protein tau (4R0NTau P301L) (Santacruz et al., “Tau suppression in a neurodegenerative mouse model improves memory function,” Science 309(5733):476 - 48(2005)); or (c) TgA53T mice that 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)). The mice received daily intraperitoneal (i.p.) 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 combinations of BK + Nilo, BK + Bos or Nilo + Bos in a solution 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 treatment with an equal dose of DMSO alone. As specified in the legend, the treatment period was 7 consecutive days or 21 consecutive days.

[0238] Xmap

[0239] The Xmap technology uses magnetic microspheres that are internally encoded with two fluorescent dyes. Through precise combinations of these two dyes, multiple proteins within a sample are measured. Each of these spheres is coated with a specific capture antibody. The capture antibody binds to a detection antibody and a reporter molecule, thus completing the reaction on the surface of the bead. 25 μl of soluble brain lysates from transgenic Tg4510 mice treated with 5 mg / kg, 2.5 mg / kg, and 1.25 mg / kg of BK40143 or DMSO were incubated overnight (16 - 20 hours) at room temperature with 25 μl of detection antibody solution and 25 μl of a mixed bead solution containing the following analyte: human phosphorylated tau (181). After thoroughly washing the plate, the samples were incubated with 25 μl of streptavidin - phycoerythrin added to each well and incubated at room temperature for 30 minutes. The samples were then washed and resuspended in 100 μl of sheath fluid. After resuspension, the samples were run on a MAGPIX with Xponent software. 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 cocktail inhibitor] compared to DMSO - treated mice, 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 blot (WB) and ELISA on an SDS - NuPAGE Bis–Tris gel (Invitrogen, Carlsbad, CA).

[0242] Pharmacokinetic study

[0243] C57BL / 6J mice were injected once i.p. with BK. Brains and sera were collected at 2, 4, 6, or 12 h (n = 18 for each drug, n = 3 for each dose and time point). Animals injected with vehicle (DMSO) were used for background subtraction. Stock solutions of the drugs (each approximately 1 mg / mL) were prepared in methanol / dichloromethane (50:50). Serial dilutions of each standard were generated separately in methanol / HPLC grade water (50:50) for the study. Preparation of calibration curve standards and quality control (QC) samples was performed by mixing the stock solutions into blank samples. Serum and brain samples were stored at -80 °C and then thawed to room temperature before preparation. Thawed serum samples (20 μL) were injected into tubes containing 100 μL of water. 500 μL of extraction solvent, acetonitrile / methanol (50:50), was added to the samples. The mixture was vortexed and incubated on ice for 20 min to accelerate protein precipitation. After incubation, the samples were vortexed again and centrifuged at 13,000 rpm at 4 °C for 20 min. The supernatant was then collected and transferred to a new tube, dried using a speed vac, and reconstituted in 200 μL of methanol / water (50:50). The mixture was centrifuged briefly again at 13,000 rpm at 4 °C for 20 min. The supernatant was then collected into mass spectrometry sample vials and run on a mass spectrometer. For brains, a small portion of the thawed brain sample from each animal was transferred to a flat-bottomed tube. 200 μL of methanol / water (90:10) was added and the tissue was homogenized. Acetonitrile was then added to the mixture to facilitate protein precipitation. The mixture was then incubated on ice for 10 min. After incubation, the samples were vortexed and centrifuged at 13,000 rpm at 4 °C for 20 min. The supernatant was then collected and transferred to a new tube, dried using a speed vac, and reconstituted in 200 μL of methanol / water (50:50). The mixture was centrifuged at 13,000 rpm at 4 °C for 20 min. The supernatant was then collected into mass spectrometry sample vials and run on a mass spectrometer. The samples were separated on an Acquity UPLC BEH C18 1.7 μm, 2.1 × 50 mm column, which was interfaced with a triple quadrupole mass spectrometer (Xevo-TQ-S, Waters Corporation) operating in multiple reaction monitoring (MRM) mode. Sample cone voltages and collision energies were optimized for the two analytes to obtain maximum ion intensities for the parent and daughter ions using the "IntelliStart" feature of MassLynx software (Waters Corporation). Instrument parameters were optimized to obtain maximum specificity and ionization sensitivity for the parent ion [m / z = 438.25] and the daughter ion [m / z = 357.33].The signal intensities of all MRM Q1 / Q3 ion pairs for the analyte were ranked to ensure selection of the most intense precursor and fragment ion pairs for MRM-based quantification. This approach led to the selection of cone voltages and collision energies that maximized the production of each fragment ion species. Analyses were performed with six- to eight-point calibration curves, randomizing the sample queue and injecting solvent blanks to assess sample carryover. MRM data were processed using TargetLynx 4.1. Relative quantification values for the analyte were determined by calculating the ratio of the transition peak area of the sample normalized to the peak area of 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. The blood was centrifuged at 2000×g to pellet the blood cells, and the serum was collected. To wash out remaining blood from the vessels and reduce contamination, the animals were perfused with 25 ml of 1X phosphate-buffered saline (PBS) for 5 min. Brains were collected and homogenized in 1.0 ml of 1x STEN buffer. The homogenized samples were centrifuged at 12,000x g at 4 °C for 20 min, and the supernatant (soluble protein fraction) was collected and stored at -80 °C. Insoluble proteins were extracted after removal of the supernatant. The tissue pellet was washed with 1X STEN buffer. The pellet was resuspended in 750 μl of 70% formic acid and incubated at room temperature for 30 min, followed by centrifugation at 28,000 g at 4 °C for 1 h. The supernatant was collected as the "insoluble fraction". 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 Pierce BCA Protein Assay (ThermoFisher, 23225) according to the manufacturer's instructions.

[0246] Immunoblot analysis

[0247] Soluble and insoluble proteins extracted from mouse brain lysates were run on an SDS NuPAGE Bis-Tris gel (catalog number NP0301BOX, Invitrogen) and probed for phosphorylated tau with (1:1000) mouse monoclonal AT180 (catalog number MN1040, ThermoFisher) and (1:1000) mouse monoclonal AT8 (catalog number MN1020, ThermoFisher), for total tau with (1:3000) mouse monoclonal Tau-5 antibody, for phosphorylated DDR1 with (1:250) rabbit polyclonal MCK10 (catalog number PA5-64780, ThermoFisher), for ubiquitin with (1:5000) rabbit polyclonal (catalog number PA3-16717, ThermoFisher), for Atg5 with (1:1000) rabbit monoclonal (catalog number mAb 12994, Cell Signaling, Danvers, MA), for Beclin-1 with (1:1000) rabbit monoclonal (catalog number mAb 3495, Cell Signaling), and for actin with (1:8000) rabbit polyclonal (catalog number MAB1501R, EMD Millipore, Burlington, MA). On an Amersham TM Imager 600 (GE Healthcare Life Sciences, Pittsburgh, PA), the blots were developed using Super Signal TM West Dura Extended Duration Substrate (catalog number 37071, ThermoFisher). Western blots were quantified using Image J software by densitometry.

[0248] Enzyme-linked immunosorbent assay (ELISA)

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

[0250] Total Tau, AB 40 and AB 42ELISA (Millipore catalog number HNABTMAG60K). As stated above, the Xmap technology uses magnetic microspheres that are internally encoded with two fluorescent dyes. Through the precise combination of these two dyes, multiple proteins within a sample are measured simultaneously. Each of these beads is coated with a specific capture antibody. The capture antibody binds to the detection antibody and the reporter molecule, thus completing the reaction on the surface of the bead. 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 overnight at 4 °C with 25 μl of a mixed bead solution containing total Tau, AB 40 and AB 42 . After washing, the samples were incubated with 25 μl of the detection antibody solution at room temperature for 1.5 h. Streptavidin-phycoerythrin (25 μl) was added to each well containing 25 μl of the detection antibody solution. The samples were then washed and resuspended in 100 μl of sheath fluid. The samples were then run on the MAGPIX using Xponent software. 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. 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 in 1XSTEN buffer (see above) according to the manufacturer's protocol.

[0251] Behavior

[0252] Rotarod: Mice were placed on an accelerating rod (catalog number 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 (25 cm × 25 cm) for 60 minutes. The animal was tracked along the arena floor by photoelectric beam. During the 60-minute trial, data were collected and the total distance traveled (cm), total time spent moving (sec), and speed (distance / time) were analyzed. The central zone was digitally defined in the software as the center of the apparatus and a (25 cm × 25 cm) in the central zone entrance, and the distance traveled in the central zone (cm) and the time spent in the central zone (sec) during the 60-minute trial were recorded.

[0254] Morris water maze: The water maze apparatus consisted of a 4-foot-diameter pool (San Diego Instruments) filled with water maintained at 25 °C and made opaque with white paint, and was digitally divided into 4 quadrant regions (ANYMaze software, San Diego Instruments). Extramaze visual cues were suspended on the walls surrounding the pool, and a hidden platform (4 inches in diameter) was submerged 1 cm below the surface of the water at the center of the 'platform zone'. Training consisted of three trials per day for four days, followed by a probe trial on the fifth day. Mice were introduced into the pool at one of three entry points (one in each non-platform quadrant region), with each entry point being used during the day. The position of the platform remained constant throughout the training period. Mice were given 60 sec to locate the platform and were kept on the platform for 10 sec, after which the mice were removed. Mice that did not locate the platform within 60 sec were placed on the platform for 10 sec and then removed from the maze. During the probe trial on the fifth day, the platform was removed and tracking software (ANYMaze) was used to record the latency to find the platform, the platform quadrant region, the swimming speed, and the swim path. This training and probe trial paradigm was performed before and after treatment.

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

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

[0256] Statistical analysis

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

[0258] Results

[0259] As Figure 1 (Left and middle panels) As shown, after 16 h of treatment, a neuroprotective effect was observed in B35 cells treated with 1 μM BK41043, as demonstrated respectively by a decrease in LDH and an increase in MTT as compared to the control. Figure 1 (Right panel) Shows a stepwise increase in cell viability via a decrease in LDH after 5 h of treatment at decreasing concentrations of BK40143.

[0260] Figure 2 Shows the cell viability of B35 cells after transfection with pTau (left panel) or α-synuclein (right panel) for 24 h and treatment with BK41043 for 5 h.

[0261] B35 cells were grown in complete medium and transfected with cDNA of human mutant Tau and α-synuclein using FuGene HD transfection reagent according to the manufacturer's instructions. At 24 h, ELISA revealed that the transfection had produced significantly higher amounts of phosphorylated Tau and α-synuclein compared to non-transfected cells. Figure 3 Shows the level of pTau(181) after 24 h of transfection (left panel), and BK41043 reduces the level of pTau(181) in pTau-transfected B35 cells (right panel), returning to the control level. This occurs without any change in the LDH level, indicating no increase in toxicity during the experiment. Figure 4 Shows the level of α-synuclein after 24 h of transfection (left panel), and BK41043 does not reduce the level of α-synuclein in α-synuclein-transfected B35 cells (right panel).

[0262] Figure 5Shows the cell viability of B35 cells after treatment with BK40197 for five hours.

[0263] Figure 6 Shows that after seven days of treatment, BK40143 reduces pTau(181) in Tau-expressing transgenic mice. pTau(181) levels were measured via ELISA.

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

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

[0266] Figure 9 Shows 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 DDR1 (pMCK10).

[0267] Figure 10 Shows that after treatment with 2.5 mg / kg or 5 mg / kg of BK-40143, pTau is reduced in CamP301L mice.

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

[0269] As Figure 12A shown, compared to DMSO-treated control A53T mice, BK-40143 significantly reduces α-synuclein in A53T mice. C57BL / 6J mice were used as controls and no detectable (N.D.) human α-synuclein was shown. Figure 12B Shows that BK-40143 does not significantly increase (by about 30%) the overall level of dopamine. However, BK-40143 does increase the level of the dopamine metabolite homovanillic acid (HVA) in A53T mice, indicating more dopamine turnover, which may lead to better dopamine neurotransmission. Figure 12C and Figure 12DThe immunoblotting of α-synuclein shown in (ThermoFisher, MA1-12874) reflects a 40% reduction of α-synuclein visible in ELISA.

[0270] Animal studies also showed that BK-40143 improved the locomotor speed of A53T mice. The overall locomotor ability of A53T mice was tested in an open field test for 60 minutes. Although the mice showed no differences in the total distance traveled or the total time spent moving, their locomotor speed increased significantly with BK-40143 treatment ( Figure 13 ). This may reflect better dopamine neurotransmission, as indicated by the increased HVA levels after BK ( Figure 12B ).

[0271] It was also shown that BK40143 selectively inactivated DDR, but not Src or Abl, and reduced 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 i.p. for 7 consecutive days. Figure 14A This is an immunoblotting detection of activated (phosphorylated) DDR1, which demonstrated that 1.25 and 2.5 but not 5 mg / kg BK40143 inactivated DDR1. Figure 14B This is an immunoblotting detection of activated Src, which demonstrated that BK-40143 did not engage this tyrosine kinase. Figure 14C This is an immunoblotting detection of activated Abl, which demonstrated that BK-40143 did not engage this tyrosine kinase, namely DDR1. When detecting phosphorylated Tau (AT8), Figure 14D it was shown that all three doses of BK-40143 reduced the phosphorylated tau level by 41-49% percentage. As Figure 14E shown, the 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 i.p. 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 ( Figure 15A ). Detection of phosphorylated DDR1 demonstrated that 1.25 and 2.5 mg / kg BK40143 inactivated DDR1 by 40% and 31% respectively (Figure 15B ) Detection of activated (phosphorylated) Abl(245) demonstrated that BK40143 did not engage Abl( Figure 15C ). Figure 15D and Figure 15E It was shown by ELISA that 1.25 and 2.5 mg / kg BK-40143 significantly reduced soluble human amyloid-β, but did not significantly reduce insoluble amyloid-β. It was also shown that 2.5 mg / kg BK40143 significantly reduced human phosphorylated tau (Ser396) by more than 80%( Figure 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 entries( Figure 16 , left panel), the latency to the first entry( Figure 16 , middle panel) and the distance traveled before the first entry into the platform( Figure 16 , right panel). Although there were no significant differences between groups, 1.25 mg / kg BK-40143 showed a trend towards increased performance with a higher number of platform entries and shorter latency to the first entry and shorter distance traveled before the first entry( Figure 16 ).

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

[0275] These data indicate that the compounds of formula I (e.g., BK41043 or BK40197) can be used for the treatment or prevention of neurodegenerative disorders, muscle degenerative disorders or lysosomal storage disorders.

Claims

1. A compound selected from Compound 8 or Compound 10 or a pharmaceutically acceptable salt thereof:

2. A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 2, which is in a solid dosage form.

4. The pharmaceutical composition according to claim 2, which is in liquid dosage form.

5. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of any one of claims 2 to 4 in the preparation of a medicament for treating or preventing a neurodegenerative disease, a muscle degenerative disease or a ruan disease in a subject, wherein the subject suffers from the neurodegenerative disease, the muscle degenerative disease or the ruan disease of the central nervous system, or is at risk of developing the neurodegenerative disease, the muscle degenerative disease or the ruan disease of the central nervous system.

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