USP13 inhibitors and methods of use thereof

Abstract

Novel ubiquitin-specific protease 13 (USP13) inhibitors are provided along with methods of their use. The USP13 inhibitors described herein are useful for treating and / or preventing USP13-related diseases, such as neurodegenerative diseases and cancer. Also provided are methods of inhibiting USP13 in cells using the compounds and compositions described herein.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 137,425, filed January 14, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] Ubiquitin conjugation is involved in most cellular processes and signaling pathways. Ubiquitin-specific protease (USP)-13 (USP13) is a deubiquitinase member of the cysteine-dependent protease superfamily. Specifically, USP13 is a ubiquitin-specific enzyme that cleaves ubiquitin from protein substrates to reverse ubiquitin-mediated protein degradation. Ubiquitin targets proteins to major degradation pathways, including the proteasome and lysosome. In melanoma cells, USP13 regulates the degradation of several proteins primarily through ubiquitination and deubiquitination. The role of USP13 in regulating the ubiquitination and deubiquitination cycle, initiating and controlling autophagy and protein degradation, is essential for cell homeostasis and survival. Summary of the Invention [Means for solving the problem]

[0003] Provided herein are novel ubiquitin-specific protease 13 (USP13) inhibitors, along with methods of their use. The USP13 inhibitors described herein are useful for treating and / or preventing USP13-related diseases, such as neurodegenerative diseases and cancer. Also provided are methods of inhibiting USP13 in cells using the compounds and compositions described herein.

[0004] Small molecule USP13 inhibitors have the following formula: [ka] and pharma- ceutically acceptable salts or prodrugs thereof, in which n is 0, 1, or 2 and L is S, O, or NR 7 where X is S, O, or NR 8 , or CR 5 =CR 6 and Y 1 , Y 2 , Y 3 , and Y 4 are each independently N or CR, where R is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; Y 1 , Y 2 , Y 3 , and Y 4 At least one of is N, and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; each R 2 is independently selected from the group consisting of halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; R 3 , R 4 , R 5 , and R 6are each independently selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; R 7 and R 8 are each independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

[0005] Optionally, the compound has the formula as shown below: [ka]

[0006] Optionally, the compound is [ka] is selected from the group consisting of:

[0007] The small molecule USP13 inhibitors described herein also have the following formula: [ka] and pharma- ceutically acceptable salts and prodrugs thereof, wherein AA is an amino acid or an ester thereof, Ar is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl, and AA is covalently bonded to Ar through the amino group of AA. Optionally, the amino acid or ester thereof is a natural amino acid or an ester thereof, or a non-natural amino acid or an ester thereof.

[0008] Optionally, the compound has the formula: [ka] wherein Ar is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; R 1 and R 2 are each independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; R 3 is hydrogen, halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl.

[0009] Optionally, the compound has the formula: [ka] wherein n is 0, 1, 2, 3, or 4; R4 is selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; each R 5 is independently selected from the group consisting of halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

[0010] Optionally, the compound is [ka] is selected from the group consisting of:

[0011] Also described herein are pharmaceutical compositions comprising a compound described herein and a pharma- ceutically acceptable carrier.

[0012] Further described herein are kits that contain the compounds or pharmaceutical compositions described herein.

[0013] Also provided herein is a method for treating or preventing USP13-related disease in a subject. The method for treating or preventing USP13-related disease in a subject comprises administering to the subject an effective amount of a compound or pharmaceutical composition as described herein. Optionally, the method further comprises selecting a subject with USP13-related disease.

[0014] Optionally, the USP13-related disease is a neurodegenerative disease (e.g., amyotrophic lateral sclerosis, Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia, Huntington's disease, mild cognitive impairment, alpha-synucleinopathies, tauopathies, or pathologies associated with intracellular accumulation of TDP-43). The method can further comprise selecting a subject suffering from a neurodegenerative disease. The method can further comprise administering to the subject a second therapeutic agent (e.g., levadopa, dopamine agonist, anticholinergic, monoamine oxidase inhibitor, COMT inhibitor, amantadine, rivastigmine, NMDA antagonist, cholinesterase inhibitor, riluzole, antipsychotic, antidepressant, or tetrabenazine).

[0015] Optionally, the USP13-related disease is cancer (e.g., pancreatic cancer, breast cancer, brain cancer, lung cancer, prostate cancer, colorectal cancer, thyroid cancer, kidney cancer, adrenal cancer, liver cancer, neurofibromatosis type 1, lymphoma, or leukemia). The method can further include selecting a subject suffering from cancer. The method can further include administering a second therapeutic agent (e.g., a chemotherapeutic agent or radiation) to the subject.

[0016] Also provided herein is a method of inhibiting USP13 in a cell, comprising contacting the cell with an effective amount of a compound described herein.

[0017] Also provided herein is a method for reducing alpha-synuclein levels in a cell. The method for reducing alpha-synuclein levels in a cell includes contacting the cell with an effective amount of a compound described herein. Optionally, the contacting can be performed in vitro or in vivo.

[0018] The details of one or more embodiments are set forth in the drawings and description below. Other features, objects, and advantages will become apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0019] [Figure 1A] Graphs showing data from MTT and LDH assays in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 1A), BK50118-B (Figure 1B), BK50118-C (Figure 1C), CL3-514 (Figure 1D), CL3-512 (Figure 1E), and CL3-499 (Figure 1F). N=3-4 per group. [Figure 1B] Graphs showing data from MTT and LDH assays in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 1A), BK50118-B (Figure 1B), BK50118-C (Figure 1C), CL3-514 (Figure 1D), CL3-512 (Figure 1E), and CL3-499 (Figure 1F). N=3-4 per group. [Figure 1C] Graphs showing data from MTT and LDH assays in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 1A), BK50118-B (Figure 1B), BK50118-C (Figure 1C), CL3-514 (Figure 1D), CL3-512 (Figure 1E), and CL3-499 (Figure 1F). N=3-4 per group. [Figure 1D] Graphs showing data from MTT and LDH assays in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 1A), BK50118-B (Figure 1B), BK50118-C (Figure 1C), CL3-514 (Figure 1D), CL3-512 (Figure 1E), and CL3-499 (Figure 1F). N=3-4 per group. [Figure 1E] Graphs showing data from MTT and LDH assays in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 1A), BK50118-B (Figure 1B), BK50118-C (Figure 1C), CL3-514 (Figure 1D), CL3-512 (Figure 1E), and CL3-499 (Figure 1F). N=3-4 per group. [Figure 1F]Graphs showing data from MTT and LDH assays in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 1A), BK50118-B (Figure 1B), BK50118-C (Figure 1C), CL3-514 (Figure 1D), CL3-512 (Figure 1E), and CL3-499 (Figure 1F). N=3-4 per group. [Figure 2A] Graphs showing USP13 activity by ELISA in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 2A), BK50118-B (Figure 2B), BK50118-C (Figure 2C), CL3-514 (Figure 2D), CL3-512 (Figure 2E), and CL3-499 (Figure 2F). N=3-9 per group. [Figure 2B] Graphs showing USP13 activity by ELISA in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 2A), BK50118-B (Figure 2B), BK50118-C (Figure 2C), CL3-514 (Figure 2D), CL3-512 (Figure 2E), and CL3-499 (Figure 2F). N=3-9 per group. [Figure 2C] Graphs showing USP13 activity by ELISA in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 2A), BK50118-B (Figure 2B), BK50118-C (Figure 2C), CL3-514 (Figure 2D), CL3-512 (Figure 2E), and CL3-499 (Figure 2F). N=3-9 per group. [Figure 2D] Graphs showing USP13 activity by ELISA in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 2A), BK50118-B (Figure 2B), BK50118-C (Figure 2C), CL3-514 (Figure 2D), CL3-512 (Figure 2E), and CL3-499 (Figure 2F). N=3-9 per group. [Figure 2E] Graphs showing USP13 activity by ELISA in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 2A), BK50118-B (Figure 2B), BK50118-C (Figure 2C), CL3-514 (Figure 2D), CL3-512 (Figure 2E), and CL3-499 (Figure 2F). N=3-9 per group. [Figure 2F] Graphs showing USP13 activity by ELISA in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 2A), BK50118-B (Figure 2B), BK50118-C (Figure 2C), CL3-514 (Figure 2D), CL3-512 (Figure 2E), and CL3-499 (Figure 2F). N=3-9 per group. [Figure 3A] Figure 3A shows ELISA levels of alpha-synuclein in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 3A), BK50118-B (Figure 3B), BK50118-C (Figure 3C), CL3-514 (Figure 3D), CL3-512 (Figure 3E) and CL3-499 (Figure 3F). N=3–9 per group. [Figure 3B] Figure 3A shows ELISA levels of alpha-synuclein in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 3A), BK50118-B (Figure 3B), BK50118-C (Figure 3C), CL3-514 (Figure 3D), CL3-512 (Figure 3E) and CL3-499 (Figure 3F). N=3–9 per group. [Figure 3C] Figure 3A shows ELISA levels of alpha-synuclein in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 3A), BK50118-B (Figure 3B), BK50118-C (Figure 3C), CL3-514 (Figure 3D), CL3-512 (Figure 3E) and CL3-499 (Figure 3F). N=3–9 per group. [Figure 3D] Figure 3A shows ELISA levels of alpha-synuclein in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 3A), BK50118-B (Figure 3B), BK50118-C (Figure 3C), CL3-514 (Figure 3D), CL3-512 (Figure 3E) and CL3-499 (Figure 3F). N=3–9 per group. [Figure 3E]Figure 3A shows ELISA levels of alpha-synuclein in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 3A), BK50118-B (Figure 3B), BK50118-C (Figure 3C), CL3-514 (Figure 3D), CL3-512 (Figure 3E) and CL3-499 (Figure 3F). N=3–9 per group. [Figure 3F] Figure 3A shows ELISA levels of alpha-synuclein in human SHSY5Y neuroblastoma cells treated with BK50118-A (Figure 3A), BK50118-B (Figure 3B), BK50118-C (Figure 3C), CL3-514 (Figure 3D), CL3-512 (Figure 3E) and CL3-499 (Figure 3F). N=3–9 per group. [Figure 4A] (Figure 4A) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment. Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=34 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M). [Figure 4B] (Figure 4B) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment. Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M). [Figure 4C](Figure 4C) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment. Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M). [Figure 4D](Figure 4D) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment. Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M). [Figure 4E-H](Figure 4E) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment. Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M). (Figure 4F) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment.Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M). (Figure 4G) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment. Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M).(Figure 4H) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment. Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M). [Figure 4I](Figure 4I) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment. Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M). [Figure 4J-M](Figure 4J) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment. Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M). (Figure 4K) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment.Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M). (Figure 4L) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment. Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M).(Figure 4M) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment. Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M). [Figure 4N](Figure 4N) BK50118-C significantly reduced alpha-synuclein levels but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 10 mg / Kg or 40 mg / Kg for 7 days. (A) WB of midbrain lysates showing levels of alpha-synuclein relative to actin on a 4-12% SDS-NuPAGE gel from the above mice. (B) The first blot is a WB densitometry. The second and third blots are IP alpha-synuclein (syn) or ubiquitin. ELISA shows levels of (C) alpha-synuclein and (D) ptau396 after BK50118-C treatment. Asterisks indicate statistically significant differences *p<0.05 or **p<0.01 versus corresponding vehicle. Ordinary one-way ANOVA was used for analysis. N=3-4 mice per group. DAB+Nissl staining. C significantly reduced alpha-synuclein levels in the cortex (F / H) and striatum (K / M) compared to corresponding vehicle (E / G) or (J / L), which was verified by quantification of alpha-synuclein positive cells in the cortex (I) and striatum (N). Two-tailed Student's t-test was used for analysis, * indicates p<0.05 versus vehicle. N=3-4 per group. All values ​​are presented as mean ± SD. Scale bars: 100 μm (E, F, J, K) or 200 μm (G, H, L, M). [Figure 5A-F](Figure 5A) shows that BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effects on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, which was verified by colocalization optical density (G). Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on TH levels in the striatum, which was verified by quantification of optical density (N). Asterisks indicate statistical significance *p<0.05, two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. (Figure 5B) Shows that BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effect on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, verified by colocalization optical density (G). Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on striatal TH levels, verified by quantification of optical density (N).Asterisks indicate statistical significance *p<0.05, and two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. (Figure 5C) shows that BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effects on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, which was verified by the optical density of colocalization (G). Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on TH levels in the striatum, which was verified by quantification of optical density (N). Asterisks indicate statistical significance *p<0.05, two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. (Figure 5D) Shows that BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effect on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated with intraperitoneal injection of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, which was verified by colocalization optical density (G).Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on TH levels in the striatum, which was verified by quantification of optical density (N). Asterisks indicate statistical significance *p<0.05, two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. (Figure 5E) Shows that BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effect on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, which was verified by colocalization optical density (G). Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on striatal TH levels, which was verified by quantification of optical density (N). Asterisks indicate statistical significance *p<0.05, and two-tailed Student's t-test was used for analysis. N=3–4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. (FIG. 5F) BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effects on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated with intraperitoneal injections of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days.Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, which was verified by colocalization optical density (G). Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on striatal TH levels, which was verified by quantification of optical density (N). Asterisks indicate statistical significance *p<0.05, and two-tailed Student's t-test was used for analysis. N=3–4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. [Figure 5G] (Figure 5G) shows that BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effects on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, which was verified by colocalization optical density (G). Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on TH levels in the striatum, which was verified by quantification of optical density (N). Asterisks indicate statistical significance *p<0.05, two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. [Figure 5H-M](Figure 5H) shows that BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effects on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, which was verified by colocalization optical density (G). Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on TH levels in the striatum, which was verified by quantification of optical density (N). Asterisks indicate statistical significance *p<0.05, two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. (Figure 5I) shows that BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effect on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, verified by colocalization optical density (G). Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on striatal TH levels, verified by quantification of optical density (N).Asterisks indicate statistical significance *p<0.05, and two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. (Figure 5J) shows that BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effects on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, which was verified by colocalization optical density (G). Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on striatal TH levels, which was verified by quantification of optical density (N). Asterisks indicate statistical significance *p<0.05, two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. (Figure 5K) shows that BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effect on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated with intraperitoneal injection of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, which was verified by colocalization optical density (G).Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on TH levels in the striatum, which was verified by quantification of optical density (N). Asterisks indicate statistical significance *p<0.05, two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. (Figure 5L) shows that BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effect on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, which was verified by colocalization optical density (G). Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on striatal TH levels, which was verified by quantification of optical density (N). Asterisks indicate statistical significance *p<0.05, and two-tailed Student's t-test was used for analysis. N=3–4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. (FIG. 5M) BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effects on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated with intraperitoneal injections of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days.Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, which was verified by colocalization optical density (G). Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on striatal TH levels, which was verified by quantification of optical density (N). Asterisks indicate statistical significance *p<0.05, and two-tailed Student's t-test was used for analysis. N=3–4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. [Figure 5N] (Figure 5N) shows that BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effects on tyrosine hydroxylase (TH) levels in the striatum of TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection of vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Immunochemical assays of 20 μm thick brain sections showed alpha-synuclein, ubiquitination, and DAPI staining in the striatum of TgA53T mice treated with either DMSO (A, C, E) or BK50118-C (B, D, F). BK50118-C increased alpha-synuclein ubiquitination, which was verified by colocalization optical density (G). Immunostaining showed TH and DAPI staining in vehicle DMSO (H, J, L) and BK50118-C (I, K, M). BK50118-C had minimal effect on TH levels in the striatum, which was verified by quantification of optical density (N). Asterisks indicate statistical significance *p<0.05, two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. Scale bar: 200 μm. [Figure 6A-E](Figure 6A) BK50118-C improved cell survival in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection with vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Nissl staining showed that BK50118-C significantly increased the number of neurons in the cortex B), striatum E), and substantia nigra (SN) H) compared to the corresponding vehicle A), D), G), which was verified by quantification of Nissl+ cells in the cortex C), striatum F), and SN I). Asterisks indicate statistically significant differences versus vehicle. *p<0.05 vs. vehicle. Two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. (Figure 6B) BK50118-C improved cell survival in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection with vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Nissl staining showed that BK50118-C significantly increased the number of neurons in the cortex B), striatum E), and substantia nigra (SN) H) compared to the corresponding vehicle A), D), G), which was verified by quantification of Nissl+ cells in the cortex C), striatum F), and SN I). Asterisks indicate statistically significant differences versus vehicle. *p<0.05 vs. vehicle. Two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. (Figure 6C) BK50118-C improved cell survival in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection with vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Nissl staining showed that BK50118-C significantly increased the number of neurons in the cortex B), striatum E), and substantia nigra (SN) H) compared to the corresponding vehicle A), D), G), which was verified by quantification of Nissl+ cells in the cortex C), striatum F), and SN I). Asterisks indicate statistically significant differences versus vehicle. *p<0.05 vs. vehicle. Two-tailed Student's t-test was used for analysis. N=3-4 mice per group.All values ​​are presented as mean ± SD. (Figure 6D) BK50118-C improved cell survival in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection with vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Nissl staining showed that BK50118-C significantly increased the number of neurons in the cortex B), striatum E), and substantia nigra (SN) H) compared to the corresponding vehicle A), D), G), which was verified by quantification of Nissl+ cells in the cortex C), striatum F), and SN I). Asterisks indicate statistically significant differences versus vehicle. *p<0.05 vs. vehicle. Two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. (Figure 6E) BK50118-C improved cell survival in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection with vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Nissl staining showed that BK50118-C significantly increased the number of neurons in the cortex B), striatum E), and substantia nigra (SN) H) compared to the corresponding vehicle A), D), G), which was verified by quantification of Nissl+ cells in the cortex C), striatum F), and SN I). Asterisks indicate statistically significant differences versus vehicle. *p<0.05 vs. vehicle. Two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. [Figure 6F-H](Figure 6F) BK50118-C improved cell survival in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection with vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Nissl staining showed that BK50118-C significantly increased the number of neurons in the cortex B), striatum E), and substantia nigra (SN) H) compared to the corresponding vehicle A), D), G), which was verified by quantification of Nissl+ cells in the cortex C), striatum F), and SN I). Asterisks indicate statistically significant differences versus vehicle. *p<0.05 vs. vehicle. Two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. (Figure 6G) BK50118-C improved cell survival in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection with vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Nissl staining showed that BK50118-C significantly increased the number of neurons in the cortex B), striatum E), and substantia nigra (SN) H) compared to the corresponding vehicle A), D), G), which was verified by quantification of Nissl+ cells in the cortex C), striatum F), and SN I). Asterisks indicate statistically significant differences versus vehicle. *p<0.05 vs. vehicle. Two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. (Figure 6H) BK50118-C improved cell survival in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection with vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Nissl staining showed that BK50118-C significantly increased the number of neurons in the cortex B), striatum E), and substantia nigra (SN) H) compared to the corresponding vehicle A), D), G), which was verified by quantification of Nissl+ cells in the cortex C), striatum F), and SN I). Asterisks indicate statistically significant differences versus vehicle. *p<0.05 vs. vehicle. Two-tailed Student's t-test was used for analysis. N=3-4 mice per group.All values ​​are expressed as mean ± SD. [Figure 6I] (Figure 6I) BK50118-C improved cell survival in TgA53T mice. Male and female TgA53T mice were treated by intraperitoneal injection with vehicle or BK50118-C at a daily dose of 40 mg / Kg for 7 days. Nissl staining showed that BK50118-C significantly increased the number of neurons in the cortex B), striatum E), and substantia nigra (SN) H) compared to the corresponding vehicle A), D), G), which was verified by quantification of Nissl+ cells in the cortex C), striatum F), and SN I). Asterisks indicate statistically significant differences versus vehicle. *p<0.05 vs. vehicle. Two-tailed Student's t-test was used for analysis. N=3-4 mice per group. All values ​​are presented as mean ± SD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Ubiquitin-specific protease (USP)-13 is a deubiquitinating enzyme that cleaves ubiquitin from substrates or proteins. In melanoma cells, USP13 regulates the degradation of several proteins mainly through ubiquitination and deubiquitination. The role of USP13 in regulating the ubiquitination and deubiquitination cycle, initiating and controlling autophagy and protein degradation, is essential for cell homeostasis and survival. Furthermore, USP13 is overexpressed in postmortem Alzheimer's disease (AD) and Parkinson's disease (PD) brains. The balance between ubiquitination and deubiquitination is important for the degradation of toxic proteins in neurodegenerative diseases. USP13 knockdown increases alpha-synuclein ubiquitination and clearance in PD models and regulates parkin function. USP13 knockdown increases p-tau ubiquitination and promotes p-tau and Aβ clearance in AD models. Thus, USP13 plays an important role in regulating protein clearance in neurodegeneration.

[0021] Provided herein are novel ubiquitin-specific protease 13 (USP13) inhibitors, along with methods of their use. The USP13 inhibitors described herein are useful for treating and / or preventing USP13-related diseases, such as neurodegenerative diseases and cancer. Also provided are methods of inhibiting USP13 in cells using the compounds and compositions described herein.

[0022] I. Compound The class of USP13 inhibitors described herein is represented by Formula I: [ka] and pharma- ceutically acceptable salts or prodrugs thereof.

[0023] In formula I, n is 0, 1, or 2.

[0024] Also in formula I, L is S, O, or NR 7 It is.

[0025] Additionally, in formula I, X is S, O, NR 8 , or CR 5 =CR 6 It is.

[0026] Also in formula I, Y 1 , Y 2 , Y 3 , and Y 4 are each independently N or CR, and R is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. 1 , Y 2 , Y 3 , and Y 4 At least one of Y is N. In some examples,1 , Y 2 , Y 3 and Y 4 Only one of them is N.

[0027] Further in formula I, R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl.

[0028] Also in formula I, each R 2 is independently selected from the group consisting of halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

[0029] Additionally, in formula I, R 3 , R 4 , R 5 , and R 6 are each independently selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

[0030] Further in formula I, R 7 and R 8 are each independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

[0031] In some cases, the compound of formula I is represented by structure IA: [ka] Structure IA

[0032] In structure IA, n, L, X, R 1 , R 2 , R 3 , R 4 , and R 7 is as defined above for formula I.

[0033] In some cases, the compound of formula I is represented by structure IB: [ka] Structure IB

[0034] In structure IB, n, R 1 , R 2 , R 3 , R 4 , and R 7 is as defined above for formula I. Optionally, in structure IB, n is 0. Optionally, in structure IB, R 3 , R 4 , and R 7 Each is hydrogen. Optionally, in structure IB, R 1 is aryl.

[0035] In some cases, the compound of formula I is represented by structure IC: [ka] Structural IC

[0036] In the structure IC, n, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and L are as defined above for formula I. Optionally, in structure IC, n is 0. Optionally, in structure IC, R 3 , R 4 , and R 6 are each hydrogen. Optionally, in structure IC, R 5 is halogen (e.g., fluoro, chloro, bromo, or iodo). Optionally, in structure IC, L is NH or O. Optionally, in structure IC, R 1 is benzyl or phenyl.

[0037] Examples of Formula I include the following compounds: [ka]

[0038] The class of USP13 inhibitors described herein has formula II: [ka] Formula II and pharma- ceutically acceptable salts or prodrugs thereof.

[0039] In formula II, AA is an amino acid or an ester thereof. Optionally, the amino acid or an ester thereof is a naturally occurring amino acid, also referred to herein as a natural amino acid or an ester thereof. Optionally, the naturally occurring amino acid may be selected from the group consisting of alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine, glycine, serine, tyrosine, threonine, cysteine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0040] Optionally, the amino acid or ester thereof is an unnatural amino acid, also referred to herein as a non-natural amino acid or ester thereof. As used herein, the term unnatural amino acid includes all amino acid-like compounds that are similar in structure and / or overall shape to one or more of the 20 natural amino acids. For example, the side chain of an unnatural amino acid can include a constrained ring such as an alkyl, aryl, aryl halide, vinyl halide, alkyl halide, acetyl, ketone, aziridine, nitrile, nitro, halide, acyl, keto, azide, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, thioether, epoxide, sulfone, boronic acid, boronic ester, borane, phenylboronic acid, thiol, seleno, sulfonyl, borate, boronic acid, phospho, phosphono, phosphine, heterocycle, pyridyl, naphthyl, benzophenone, cyclooctyne, thioester, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, carboxylic acid, alpha-ketocarboxylic acid, alpha- or beta-unsaturated acids and amides, glyoxylamide, or organosilane group. Suitable unnatural amino acids are described, for example, in Unnatural Amino Acids: Methods and Protocols (Methods in Molecular Biology), Volume 794, Pollegioni and Servi, eds., Springer (2012), which is incorporated by reference in its entirety, at least with respect to the description of unnatural amino acids.

[0041] The side chain of the amino acid or ester thereof may be in either the (R) or (S) configuration (or D- or L-configuration).

[0042] Also in formula II, Ar is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

[0043] Additionally, in formula II, AA is covalently bonded to Ar via the amino group of AA.

[0044] In some cases, the compound of formula II is represented by structure II-A: [ka] Structure II-A

[0045] In structure II-A, Ar is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

[0046] Further, in structure II-A, R 1 and R 2 are each independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

[0047] In addition, in structure II-A, R 3 is hydrogen, halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl.

[0048] In some cases, the compound of formula II is represented by structure II-B: [ka] Structure II-B

[0049] In structure II-B, R1 , R 2 , and R 3 is as defined above for structure II-A.

[0050] Also in structure II-B, n is 0, 1, 2, 3, or 4.

[0051] In addition, in structure II-B, R 4 is selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

[0052] Further, in structure II-B, each R 5 is independently selected from the group consisting of halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

[0053] Optionally, in structure II-B, R 1 is hydrogen. Optionally, in structure II-B, R 2 is methyl. Optionally, in structure II-B, R 3 is substituted or unsubstituted benzyl. Optionally, in structure II-B, R 4 is nitro. Optionally, in structure II-B, n is 0.

[0054] Examples of Formula II include the following compounds: [ka]

[0055] As used herein, the terms alkyl, alkenyl, and alkynyl include straight and branched chain monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. The range of these groups useful in the compounds and methods described herein includes C1-C 20 Alkyl, C2-C 20 Alkenyl, and C2-C 20 An additional scope of these groups useful in the compounds and methods described herein includes C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Includes alkynyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl, C2-C4 alkenyl, and C2-C4 alkynyl.

[0056] Heteroalkyl, heteroalkenyl, and heteroalkynyl are defined similarly to alkyl, alkenyl, and alkynyl, but can contain O, S, or N heteroatoms, or combinations thereof, in the backbone. The scope of these groups useful in the compounds and methods described herein includes C1-C 20 Heteroalkyl, C2-C 20 Heteroalkenyl, and C-C 20 An additional scope of these groups useful in the compounds and methods described herein includes C1-C 12 Heteroalkyl, C2-C 12 Heteroalkenyl, C2-C 12 Includes heteroalkynyl, C1-C6 heteroalkyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, C1-C4 heteroalkyl, C2-C4 heteroalkenyl, and C2-C4 heteroalkynyl.

[0057] The terms cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclic alkyl groups having a single cyclic ring or multiple condensed rings. Examples include cyclohexyl, cyclopentylethyl, and adamantanyl. The range of these groups useful in the compounds and methods described herein includes C3-C 20 Cycloalkyl, C3~ C20 Cycloalkenyl, and C3-C 20 An additional scope of these groups useful in the compounds and methods described herein includes C5-C 12 Cycloalkyl, C5-C 12 Cycloalkenyl, C5-C 12 Includes cycloalkynyl, C5-C6 cycloalkyl, C5-C6 cycloalkenyl, and C5-C6 cycloalkynyl.

[0058] The terms heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl are defined similarly to cycloalkyl, cycloalkenyl, and cycloalkynyl, but can contain O, S, or N heteroatoms, or combinations thereof, within the cyclic backbone. The scope of these groups useful in the compounds and methods described herein includes C3-C 20 Heterocycloalkyl, C3-C 20 Heterocycloalkenyl, and C3-C 20 An additional scope of these groups useful in the compounds and methods described herein includes C5-C 12 Heterocycloalkyl, C5-C 12 Heterocycloalkenyl, C5-C 12 Includes heterocycloalkynyl, C5-C6 heterocycloalkyl, C5-C6 heterocycloalkenyl, and C5-C6 heterocycloalkynyl.

[0059] Aryl molecules include cyclic hydrocarbons incorporating one or more planar sets of carbon atoms, usually six, connected by the same number of delocalized electrons, as if composed of alternating single and double covalent bonds. An example of an aryl molecule is benzene. Heteroaryl molecules include substituents along the main cyclic chain of atoms, such as O, N, and S. When heteroatoms are introduced, aromatic systems can be formed by sets of five atoms, for example, four carbons and one heteroatom. Examples of heteroaryl molecules include furan, pyrrole, thiophene, imadazole, oxazole, pyridine, and pyrazine. Aryl and heteroaryl molecules can also include additional fused rings, such as, for example, benzofuran, indole, benzothiophene, naphthalene, anthracene, and quinoline. Aryl and heteroaryl molecules can be attached at any position on the ring, unless otherwise noted.

[0060] The term alkoxy, as used herein, is an alkyl group bonded through a single terminal ether bond. The term aryloxy, as used herein, is an aryl group bonded through a single terminal ether bond. Similarly, the terms alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, heteroaryloxy, cycloalkyloxy, and heterocycloalkyloxy, as used herein, are alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, heteroaryloxy, cycloalkyloxy, and heterocycloalkyloxy groups, respectively, bonded through a single terminal ether bond.

[0061] The term hydroxy as used herein is represented by the formula --OH.

[0062] As used herein, the term amine or amino refers to a compound of the formula -NZ 1 Z 2 In the formula, Z 1 and Z 2Each of may be a substituent as described herein, such as hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0063] As used herein, an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl molecule may be substituted or unsubstituted. As used herein, the term substituted includes the addition of an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl group to the position attached to the backbone of an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl, such as the replacement of a hydrogen with one of these molecules. Examples of substituents include, but are not limited to, hydroxy, halogen (e.g., F, Br, Cl, or I), and carboxyl groups. Conversely, as used herein, the term unsubstituted indicates that the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl is fully supplemented with hydrogen, i.e., without substituents, to the same extent as its level of saturation, for example, straight chain decane (-(CH2)9-CH3).

[0064] II. Method for producing the compounds The compounds described herein can be prepared in a variety of ways. The compounds can be synthesized using a variety of synthetic methods. At least some of these methods are known in the field of organic synthetic chemistry. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art.

[0065] Variations of the compounds of formula I and formula II and described herein include the addition, subtraction, or movement of various components described for each compound. Similarly, when one or more chiral centers are present in a molecule, all possible chiral variants are included. In addition, the synthesis of a compound may involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of suitable protecting groups, can be determined by those skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts, Greene's Protective Groups in Organic Synthesis, 5th.Ed., Wiley&Sons, 2014, which is incorporated herein by reference in its entirety.

[0066] The reactions to produce the compounds described herein can be carried out in a solvent that can be selected by one skilled in the art of organic synthesis. The solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions, i.e., temperature and pressure, at which the reaction is carried out. The reaction can be carried out in one solvent or a mixture of two or more solvents. 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 nuclear magnetic resonance spectroscopy (e.g., 1 H-NMR or 13 The reaction may be monitored by spectroscopic means, such as by C-NMR), infrared spectroscopy (IR), spectrophotometry (e.g., UV-visible), or mass spectrometry (MS), or by chromatography, such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

[0067] Exemplary methods for synthesizing the compounds described herein are provided in Example 1 below.

[0068] III. Pharmaceutical Preparations The compounds described herein or derivatives thereof can be provided as pharmaceutical compositions.Depending on the intended mode of administration, the pharmaceutical compositions can be in solid, semi-solid or liquid form, such as tablets, suppositories, pills, capsules, powders, liquids, aerosols or suspensions, preferably in unit dosage form suitable for single administration of precise doses.The compositions comprise a therapeutically effective amount of the compounds described herein or derivatives thereof in combination with a pharma-ceutically acceptable carrier, and may further comprise other medicinal agents, pharmaceutical agents, carriers or diluents.Pharmaceutically acceptable means a biologically or otherwise undesirable substance that can be administered to an individual together with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with other components contained in the pharmaceutical composition.

[0069] As used herein, the term carrier includes any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, or other material known in the art for use in pharmaceutical formulations. The choice of carrier for use in a composition will depend on the intended route of administration of the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these substances are described, for example, in Remington: The Science and Practice of Pharmacy, 22d Edition, Loyd et al. eds., Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences (2012). 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 dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS® (BASF; Florham Park, NJ).

[0070] Compositions containing one or more compounds described herein or pharma- ceutically acceptable salts or prodrugs thereof suitable for parenteral injection may include physiologically acceptable aqueous or non-aqueous sterile solutions, dispersions, suspensions or emulsions, and sterile powders to be reconstituted into sterile injectable solutions or dispersions. Examples of suitable aqueous or non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (olive oil, etc.), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0071] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be enhanced by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example, sugars, sodium chloride, and the like, may also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0072] Solid dosage forms for oral administration of the compounds described herein or their pharma- ceutically acceptable salts or prodrugs include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or their derivatives are mixed with at least one inert excipient (or carrier), such as sodium citrate or dicalcium 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) wetting agents, such as glycerol, (d) disintegrants, such as agar, calcium carbonate, and (e) glycerol, (f) glycerol, (g) glycerol, (h) glycerol, (i) glycerol, (j) glycerol, (k) glycerol, (l ... In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0073] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose or milk sugar as well as high molecular weight polyethylene glycols and similar excipients.

[0074] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They can contain masking agents and can be of such a composition that they release the active compound(s) in a delayed manner in a certain part of the intestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. The active compound can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0075] The liquid dosage form for oral administration of the compounds described herein or their pharma- ceutically acceptable salts or prodrugs includes pharma- ceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.In addition to the active compound, the liquid dosage form may contain inert diluents, solubilizers, and emulsifiers commonly used in the art, such as water or other solvents, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, or mixtures of these substances.

[0076] Besides such inert diluents, compositions can also include additional agents, such as wetting agents, emulsifying agents, suspending agents, sweetening, flavoring, or perfuming agents.

[0077] In addition to the active compound, the suspensions may contain additional agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.

[0078] Compositions for rectal administration of the compositions described herein or pharma- ceutically acceptable salts or prodrugs thereof are optionally suppositories, which can be prepared by mixing the compound with a suitable non-irritating excipient or carrier, such as, for example, cocoa butter, polyethylene glycol, or a suppository wax, which is solid at ordinary temperatures but liquid at body temperature and thus will melt in the rectum or vaginal cavity and release the active ingredient.

[0079] The dosage form for topical administration of the compound or its derivatives described herein includes ointments, powders, sprays, inhalants, and skin patches.The compound or its derivatives described herein are mixed under sterile conditions with physiologically acceptable carriers and, if necessary, any preservatives, buffers, or propellants.Ophthalmic formulations, ointments, powders, and solutions are also contemplated within the scope of the composition.

[0080] Optionally, the compounds described herein can be included in a drug depot. The drug depot includes a physical structure that facilitates implantation and retention at a desired site (e.g., synovial joint, disc space, spinal canal, abdominal region, tissue of a patient, etc.). The drug depot can provide an optimal concentration gradient of the compound at a distance of up to about 0.1 cm to about 5 cm from the implantation site. As used herein, a depot includes, but is not limited to, a capsule, microsphere, microparticle, microcapsule, microfiber particle, nanosphere, nanoparticle, coating, matrix, wafer, pill, pellet, emulsion, liposome, micelle, gel, antibody-compound conjugate, protein-compound conjugate, or other pharmaceutical delivery composition. Materials suitable for the depot include pharma- ceutically acceptable biodegradable materials that are preferably FDA approved or GRAS materials. These materials can be polymeric or non-polymeric, as well as synthetic or natural, or a combination thereof. The depot can optionally include a drug pump.

[0081] The composition may include one or more compounds described herein and a pharma- ceutically acceptable carrier. As used herein, the term pharma- ceutically acceptable salt refers to salts of the compounds described herein or derivatives thereof that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, etc., and that are commensurate with a reasonable benefit / risk ratio and the effectiveness of the intended use, as well as the compounds described herein in zwitterionic form, where possible. The term salt refers to the relatively non-toxic inorganic and organic acid addition salts of the compounds described herein. These salts are prepared in situ during the isolation and purification of the compounds, or by separately reacting the purified compounds in free base form with an appropriate organic or inorganic acid and isolating the salts thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, methanesulfonate, and laurylsulfonate, etc. These may include cations based on alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. (See S. M. Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated by reference in its entirety for at least the compositions taught therein.)

[0082] Administration of the compounds and compositions described herein or pharma- ceutically acceptable salts thereof can be carried out for a period of time effective to treat the disorder, using a therapeutically effective amount of the compounds and compositions described herein or pharma- ceutically acceptable salts thereof described herein. Effective amounts of the compounds and compositions described herein or pharma- ceutically acceptable salts thereof described herein can be determined by one of skill in the art, and exemplary dosages for mammals include about 0.0001 to about 200 mg of active compound per kg of body weight per day, which can be administered in a single dose or in the form of individual divided doses, such as 1 to 4 times per day. Alternatively, the dosage may be from about 0.01 to about 150 mg / kg of body weight per day of the active compound, from about 0.1 to about 100 mg / kg of body weight per day of the active compound, from about 0.5 to about 75 mg / kg of body weight per day of the active compound, from about 0.5 to about 50 mg / kg of body weight per day of the active compound, from about 0.01 to about 50 mg / kg of body weight per day of the active compound, from about 0.05 to about 25 mg / kg of body weight per day of the active compound, from about 0.1 to about 25 mg / kg of body weight per day of the active compound, from about 0.5 to about 25 mg / kg of body weight per day of the active compound, The active compound per day may be about 1 to about 20 mg / kg of body weight per day, about 1 to about 10 mg / kg of body weight per day, about 20 mg / kg of body weight per day, about 10 mg / kg of body weight per day, about 5 mg / kg of body weight per day, about 2.5 mg / kg of body weight per day, about 1.0 mg / kg of body weight per day, about 0.5 mg / kg of body weight per day, or any range derivable therein. Optionally, the dosage is about 0.01 mg / kg to about 10 mg / kg of body weight per day of active compound. Optionally, the dosage is about 0.01 mg / kg to about 5 mg / kg. Optionally, the dosage is about 0.01 mg / kg to about 2.5 mg / kg.

[0083] Those skilled in the art will understand that the specific dose level and frequency of administration for any particular subject may vary, and will depend on a variety of factors, including the activity of the particular compound used, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the method and time of administration, rate of excretion, drug combination, and the severity of the particular condition.

[0084] The exact dosage to be used in the formulation also depends on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. Furthermore, depending on the route of administration, those skilled in the art will know how to determine the dose that will result in a plasma concentration for a desired level of response in the subject's cells, tissues and / or organs.

[0085] IV.How to use Provided herein is a method for treating or preventing USP13-related disease in a subject. The method comprises administering to a subject an effective amount of one or more compounds or compositions described herein, or pharma-ceutically acceptable salts or prodrugs thereof. Effective amount, when used to describe the amount of a compound in a method, refers to the amount of the compound that achieves a desired pharmacological or other biological effect. Effective amount can be, for example, the concentration of a compound at which USP13 is inhibited in vitro, as provided herein. Also contemplated is a method comprising administering to a subject an amount of one or more compounds described herein, such that an in vivo concentration in target cells in the subject that corresponds to the concentration administered in vitro is achieved.

[0086] The compounds and compositions described herein, or pharma- ceutically acceptable salts thereof, are useful for treating USP13-associated diseases in humans, including but not limited to pediatric and geriatric populations, and in animals, e.g., for veterinary use.

[0087] In some cases, the USP13-related disease is a neurodegenerative disease, such as a neurodegenerative disease of the central nervous system. Such diseases include, but are not limited to, amyotrophic lateral sclerosis, Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia, Huntington's disease, mild cognitive impairment, α-synucleinopathy, tauopathy, or pathology associated with intracellular accumulation of TDP-43.

[0088] Optionally, the USP13-related disease is cancer. As used herein, the term cancer refers to any cellular disorder in which cells proliferate more rapidly than normal tissue growth. Proliferative disorders include, but are not limited to, neoplasms, also called tumors. Neoplasms include, but are not limited to, pancreatic cancer, breast cancer, brain cancer (e.g., glioblastoma), lung cancer, prostate cancer, colorectal cancer, thyroid cancer, kidney cancer, adrenal cancer, liver cancer, neurofibromatosis type 1, and leukemia. Neoplasms can be solid neoplasms (e.g., sarcomas or carcinomas) or cancerous growths that affect the hematopoietic system (e.g., lymphomas or leukemias). Other proliferative diseases include, but are not limited to, neurofibromatosis.

[0089] Optionally, the USP13-associated disease is a myodegenerative disease, a prion disease, or a transmissible spongiform encephalopathy. In the methods provided herein, myodegenerative diseases include, but are not limited to, inclusion body myositis (IBM), spinal and bulbar muscular atrophy (SBMA), and motor neuron disease (MND). In the methods provided herein, prion diseases or transmissible spongiform encephalopathies (TSE) include, but are not limited to, Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, and kuru in humans. Prion diseases in animals include, but are not limited to, scrapie, bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD), transmissible mink encephalopathy, feline spongiform encephalopathy, and ungulate spongiform encephalopathy.

[0090] Those skilled in the art will know how to select subjects with USP13-related disorders.For example, but not limited to, those skilled in the art will know how to diagnose subjects with neurodegenerative disease or subjects at risk of developing neurodegenerative disease.For example, one or more of the following tests can be used: genetic test (e.g., identification of mutations in TDP-43 gene) or family analysis (e.g., family history), central nervous system imaging test (e.g., magnetic resonance imaging and positron emission tomography), clinical or behavioral test (e.g., evaluation of muscle weakness, tremor, or memory), and / or clinical test.

[0091] USP13 independently regulates the ubiquitination of parkin and alpha-synuclein. USP13 knockdown increases the ubiquitination and clearance of alpha-synuclein, regulating parkin function. USP13 knockdown also increases the ubiquitination of hyperphosphorylated tau (p-tau), promoting the clearance of p-tau and amyloid beta (Aβ) peptides. Neurotoxic proteins such as p-tau, Aβ, and alpha-synuclein coexist in neurodegenerative diseases and can be simultaneously degraded by inhibiting USP13. When USP13 activity is reduced, the levels of p-tau, Aβ, and / or alpha-synuclein are also reduced. Thus, the compounds described herein are also effective in reducing p-tau, Aβ, and / or alpha-synuclein levels in cells. Optionally, the compounds described herein can cross the blood-brain barrier.

[0092] The method provided herein optionally comprises administering a second therapeutic agent to the subject.For example, for treating neurodegenerative disease, the second therapeutic agent can be selected from the group consisting of levadopa, dopamine agonist, anticholinergic drug, monoamine oxidase inhibitor, COMT inhibitor, amantadine, rivastigmine, NMDA antagonist, cholinesterase inhibitor, riluzole, antipsychotic drug, antidepressant drug, and tetrabenazine.In another example, for treating cancer, the second therapeutic agent can be, for example, a chemotherapeutic agent or radiation.

[0093] The compounds described herein can increase Parkin activity in a subject. As used throughout, the increase in Parkin activity in a subject can be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase compared to a control. For example, the increase in Parkin activity can be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase compared to a subject not administered the compounds described herein or a control value.

[0094] The amount of inhibition or reduction of USP13 activity need not be complete, as it can range from a reduction to complete elimination of enzymatic activity. For example, the reduction can be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount therebetween. The inhibition or reduction of USP13 activity can be due to a reduction in mRNA expression, a reduction in protein expression, and / or a reduction in enzymatic activity of USP13.

[0095] Further provided are methods of treating or preventing a neurodegenerative disease, a muscular degenerative disease, or a prion disease in a subject, the methods comprising administering to the subject an effective amount of a compound or pharmaceutical composition described herein. The methods can optionally include selecting a subject suffering from or at risk for a neurodegenerative disease, a muscular degenerative disease, or a prion disease of the central nervous system.

[0096] Also provided is a method of treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a compound or pharmaceutical composition described herein, which can optionally include selecting a subject suffering from or at risk of cancer.

[0097] The following are exemplary modes of administration of the compositions used in the present invention. The compounds described herein can be delivered by any of a variety of routes, including injection (e.g., subcutaneous, intramuscular, intravenous, intraarterial, intraperitoneal), continuous intravenous infusion, dermal, transdermal, oral (e.g., tablet, pill, liquid medicine, edible film strip), implanted osmotic pump, suppository, or aerosol spray. Routes of administration include, but are not limited to, topical, intradermal, intrathecal, intralesional, intratumoral, intravesical, intravaginal, intraocular, intrarectal, pulmonary, intracranial, intraventricular, intraspinal, dermal, subcutaneous, intraarticular, placement in a body cavity, nasal inhalation, pulmonary inhalation, skin pressure, and electroporation.

[0098] The compounds described herein are also useful for reducing alpha-synuclein levels in cells.The method for reducing alpha-synuclein levels in cells includes contacting cells with an effective amount of one or more compounds described herein.Optionally, the contacting is performed in vivo.Optionally, the contacting is performed in vitro.

[0099] V.Kit Also provided herein is a kit (also referred to as a pharmaceutical pack) for treating or preventing a USP13-related disease (e.g., a neurodegenerative disease or cancer) in a subject. The pharmaceutical pack or kit includes one or more containers filled with the ingredients of one or more pharmaceutical compositions. The kit can include any of the compounds or compositions described herein. For example, the kit can include one or more compounds of Formula I and / or Formula II. The kit can further include one or more additional agents, such as one or more of levadopa, dopamine agonists, anticholinergics, monoamine oxidase inhibitors, COMT inhibitors, amantadine, rivastigmine, NMDA antagonists, cholinesterase inhibitors, riluzole, antipsychotics, antidepressants, tetrabenazine, or chemotherapeutic agents. The kit can include an oral formulation of any of the compounds or compositions described herein. The kit can include an intravenous formulation of any of the compounds or compositions described herein. The kit may further include instructions for using the kit (eg, instructions for treating a subject), a container, a means for administering the compound or composition (eg, a syringe), and / or a carrier.

[0100] Such container(s) can optionally be accompanied by instructions in a format prescribed by a government agency having jurisdiction over the manufacture, use, or sale of a pharmaceutical or biological product, which instructions incorporate approval by the government agency for manufacture, use, or sale for human administration. Instructions for use of the composition can also be included.

[0101] As used throughout, treat, treating, and treatment refer to a method of reducing or delaying one or more effects or symptoms of a disease or disorder, such as a neurodegenerative disease or cancer. A subject may be diagnosed with a disease or disorder. Treatment may also refer to a method of reducing background pathology rather than just symptoms. The effect of administration to a subject may have an effect that is, but is not limited to, reducing one or more symptoms of a disease or disorder, reducing the severity of a disease or disorder, eliminating a disease or disorder entirely, or delaying the onset or worsening of one or more symptoms. For example, the disclosed methods are determined to be therapeutic if there is about a 10% reduction in one or more symptoms of a disease in a subject compared to a subject prior to treatment or compared to a control subject or control value. Thus, the reduction may be about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between.

[0102] As used herein, by prevent, preventive, or prophylaxis, it is meant a method of preventing, delaying, avoiding, eliminating, forestalling, stopping, or impeding the onset, incidence, severity, or recurrence of a neurodegenerative disease or disorder.For example, the disclosed method is considered to be prophylactic if there is a reduction or delay in the onset, incidence, severity, or recurrence of neurodegeneration or one or more symptoms of neurodegeneration (e.g., tremor, weakness, memory loss, rigidity, spasticity, atrophy) in a subject susceptible to neurodegeneration compared to a control subject susceptible to neurodegeneration that has not been administered the USP13 inhibitor described herein.The disclosed method is also considered to be prophylactic if there is a reduction or delay in the onset, incidence, severity, or recurrence of neurodegeneration or one or more symptoms of neurodegeneration in a subject susceptible to neurodegeneration after administration of a USP13 inhibitor compared to the subject's progression before receiving treatment. Thus, the reduction or delay in the onset, incidence, severity, or recurrence of neurodegeneration can be about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction or any amount in between.

[0103] As used throughout, subject means an individual. Preferably, the subject is a mammal, such as a primate, more preferably a human. Non-human primates are subjects as well. The term subject includes domestic animals (such as cats, dogs, etc.), livestock animals (such as cows, horses, pigs, sheep, goats, etc.), and laboratory animals (such as ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary uses and medical preparations are contemplated herein.

[0104] Throughout this application, various publications are referenced, the disclosures of which are incorporated by reference in their entireties into this application.

[0105] The following examples are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims. EXAMPLES

[0106] The following examples are described below to illustrate the methods and results of the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the subject matter described herein that are obvious to those skilled in the art.

[0107] Example 1: Synthesis of USP-13 inhibitors General information Commercially available 7-chlorothieno[3,2-b]pyridine (1), aniline (2), 4-chloro-6-fluoroquinoline (3), benzyl alcohol (4), benzylamine (5), 4-chloro-3-nitro-2H-chromen-2-one (6), reagents and solvents were used as purchased without further purification. Methyl (S)-phenylalaninate (7) and methyl (S)-tyrosinate (8) were obtained by extraction from their HCl salts with aqueous NaHCO3 and EtOAc. NMR spectra were obtained at 400 MHz ( 1 HNMR) and 100MHz ( 13 The reaction products were purified by column chromatography on silica gel (particle size 40-63 μm) as follows:

[0108] Synthesis and characterization of compounds Compounds BK50118-A, BK50118-B, and BK50118-C were synthesized according to Scheme 1 shown below.

[0109] Scheme 1: [ka]

[0110] The general procedure for preparing compounds according to Scheme 1 is as follows: An 8 mL pressure vessel was charged with 7-chlorothieno[3,2-b]pyridine (1) (0.6 mmol), aniline (1.2 mmol), and DMSO (1.0 mL) unless otherwise stated. The pressure vessel was then placed in a 100° C. oil bath and the mixture was stirred for 24 h. 1 After complete conversion was achieved based on HNMR analysis, the reaction mixture was extracted with EtOAc. The combined organic layers were extracted with water, 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. [ka]

[0111] N-Phenylthieno[3,2-b]pyridin-7-amine (BK50118-A). Compound BK50118-A was obtained as a colorless solid in 98% yield (133 mg, 0.59 mmol) from 7-chlorothieno[3,2-b]pyridine (101 mg, 0.6 mmol) and aniline (111 mg, 1.2 mmol) in 1 mL of DMSO after 24 hours at 100° C. by following the general procedure described above. The crude product was purified using flash chromatography on silica gel using hexane / EtOAc (1:1) as the mobile phase. f = 0.3 (hexane / EtOAc, 1:1); 1 HNMR (400 MHz, chloroform-d) δ = 8.42 (d, J = 5.4 Hz, 1H), 7.63 (d, J = 5.4 Hz, 1H), 7.53 (d, J = 5.4 Hz, 1H), 7.45-7.37 (m, 2H), 7.32-7.25 (m, 2H), 7.19 (m, 1H), 6.92 (d, J = 5.4 Hz, 1H), 6.12 (s, 1H); 13 CNMR (100 MHz, chloroform-d) δ = 157.8, 148.9, 146.3, 139.4, 129.7, 128.1, 126.5, 124.9, 122.6, 120.8, 102.3. [ka]

[0112] 6-Fluoro-N-phenylquinolin-4-amine (BK50118-B). Compound BK50118-B was obtained as a colorless solid in 95% yield (135 mg, 0.57 mmol) from 4-chloro-6-fluoroquinoline (109 mg, 0.6 mmol) and aniline (111 mg, 1.2 mmol) in 1 mL of DMSO after 24 hours at 100° C. by following the general procedure described above. The crude product was purified using flash chromatography on silica gel using hexane / EtOAc (1:1) as the mobile phase. f = 0.3 (hexane / EtOAc, 1:1); 1H NMR (400 MHz, chloroform-d) δ = 8.54 (d, J = 5.2 Hz, 1H), 8.04 (dd, J = 9.9, 5.2 Hz, 1H), 7.58 (dd, J = 9.9, 2.7 Hz, 1H), 7.49-7.37 (m, 3H), 7.28 (d, J = 7.7 Hz, 2H), 7.19 (m, 1H), 7.01 (d, J = 5.2 Hz, 1H), 6.59 (s, 1H). 13 CNMR (100 MHz, chloroform-d) δ = 160.2 (d, J = 246.7 Hz), 150.3 (d, J = 2.3 Hz), 147.3 (d, J = 5.0 Hz), 146.4, 139.9, 132.8 (d, J = 9.0 Hz), 129.9, 124.9, 122.7, 120.5 (d, J = 8.5 Hz), 119.4 (d, J = 25.1 Hz), 104.1 (d, J = 23.1 Hz), 103.0; 19 FNMR (376 MHz, chloroform-d) δ = -114.04 (m). [ka]

[0113] 4-(Benzyloxy)-6-fluoroquinoline (BK50118-C). Compound BK50118-C was obtained as a colorless solid in 97% yield (147 mg, 0.58 mmol) from 4-chloro-6-fluoroquinoline (109 mg, 0.6 mmol) and benzyl alcohol (1 mL) after 16 hours at 100° C. by following the general procedure described above. The crude product was purified using flash chromatography on silica gel using hexane / EtOAc (1:1) as the mobile phase. f = 0.5 (hexane / EtOAc, 1:1); 1 HNMR (400MHz, chloroform-d) δ=8.70 (d, J=5.2Hz, 1H), 8.03 (dd, J=9.5, 5.2Hz, 1H), 7. 85(dd, J=9.5, 2.9Hz, 1H), 7.54-7.33(m, 6H), 6.80(d, J=5.2Hz, 1H), 5.27(s, 2H); 13CNMR (100MHz, chloroform-d) δ=160.8(d, J=5.1Hz), 160.2(d, J=246.7Hz), 150.6(d, J=2.5Hz), 146.4(d, J=1.2Hz), 135.5, 131.5(d, J=9.0Hz), 128.9, 128.6, 127.5, 122.2(d, J=9.6Hz), 119.9(d, J=25.6Hz), 105.9(d, J=23.4Hz), 101.7, 70.5. 19 FNMR (376 MHz, chloroform-d) δ = -113.82 (m).

[0114] Compound CL3-499 was synthesized according to Scheme 2 shown below and the procedures below.

[0115] Scheme 2: [ka] An 8 mL sealed vial was charged with 4-chloro-6-fluoroquinoline (3) (100 mg, 0.55 mmol), Pd2(dba)3 (10.1 mg, 0.01 mmol, 2 mol%), 1,3-bis(2,4,6-trimethylphenyl)imidazolium chloride (TMPCl) (11.3 mg, 0.03 mmol, 6 mol%), NaOt-Bu (169.2 mg, 1.76 mmol), benzylamine (5) (177 mg, 1.65 mmol) and dioxane (1.5 mL). The vial was then placed in a 100° C. oil bath and stirred for 56 h. The reaction was quenched with aqueous NaHCO3 and extracted with EtOAc. 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-EtOAc (8:2) as the mobile phase and recrystallized from hexane-EtOAc (1:1). [ka]

[0116] N-Benzyl-6-fluoroquinolin-4-amine (CL3-499). Compound CL3-499 was obtained as a yellow solid in 81% yield (112 mg, 0.45 mmol). f = 0.16 (hexane / EtOAc, 1:1); 1 HNMR (400 MHz, chloroform-d) δ 8.51 (d, J = 5.2 Hz, 1H), 7.99 (dd, J = 5.8 Hz, J = 5.3 Hz, 1H), 7.43-7.31 (m, 7H), 6.47 (d, J = 5.2 Hz, 1H), 5.18 (bs, 1H), 4.53 (d, J = 5.2 Hz, 2H); 13 CNMR (100 MHz, chloroform-d) δ 159.7 (d, J = 245.0 Hz), 150.4 (d, J = 2.3 Hz), 149.0 (d, J = 4.7 Hz), 145.5, 137.3, 132.4 (d, J = 8.9 Hz), 128.9, 127.9, 127.5, 119.2 (d, J = 8.6 Hz), 118.8 (d, J = 24.9 Hz), 103.7 (d, J = 22.6 Hz), 99.8, 47.6; 19 FNMR (376 MHz, chloroform-d) δ-114.97 (m).

[0117] Compounds CL3-512 and CL3-514 were synthesized according to Scheme 3 shown below. Scheme 3: [ka]

[0118] The general procedure for preparing compounds according to Scheme 3 is shown below: An 8 mL vial was charged with 4-chloro-3-nitro-2H-chromen-2-one (6), amino ester derivative (7) or (8), K2CO3 and ACN:H2O (6.8 mL, 2.4:1). The reaction mixture was stirred at 25 °C for 2 h. After complete conversion was achieved based on TLC analysis, the pH was adjusted to 7 and the reaction mixture was extracted with EtOAc. The combined organic layers were washed with water, dried over sodium sulfate and the solvent was removed in vacuo. The crude product was purified by flash chromatography on silica gel as described below. [ka]

[0119] (S)-Methyl (3-nitro-2-oxo-2H-chromen-4-yl)-phenylalaninate (CL3-512). After column purification using DCM / MeOH (9:1) as the mobile phase, compound CL3-512 was obtained as a colorless solid in 90% yield (274 mg, 0.8 mmol) from 4-chloro-3-nitro-2H-chromen-2-one (6) (200 mg, 0.89 mmol), methyl (S)-phenylalaninate (7) (146.5 mg, 0.89 mmol), and K2CO3 (246 mg, 1.78 mmol) by following the general procedure described above. R f = 0.44 (DCM / MeOH, 9:1); 1 HNMR (400MHz, methanol-d4) δ7.98(d, J=8.3Hz, 1H), 7.67(dd, J=9.1Hz, 7.2Hz, 1H), 7.37(dd, J=9.0Hz, 7.3Hz, 1H), 7.28(d , J=8.3Hz, 1H), 7.21~7.13(m, 5H), 4.57(bs, 1H), 3.34(dd, J=14.1Hz, 4.5Hz), 3.29(s, 3H), 3.22(dd, J=14.1Hz, 8.7Hz); 13 CNMR (100 MHz, methanol-d4) δ 172.1, 154.9, 151.0, 135.8, 133.8, 128.7, 128.3, 126.9, 124.5, 123.5, 117.2, 116.7, 113.1, 58.8, 53.4, 38.1. [ka]

[0120] (S)-Methyl (3-nitro-2-oxo-2H-chromen-4-yl)-tyrosinate (CL3-514). After column purification using DCM / MeOH (9:1) as the mobile phase, compound CL3-514 was obtained as a colorless solid in 42% yield (137.1 mg, 0.37 mmol) from 4-chloro-3-nitro-2H-chromen-2-one (6) (200 mg, 0.89 mmol), methyl (S)-tyrosinate (8) (161 mg, 0.89 mmol), and K2CO3 (246 mg, 1.78 mmol) by following the general procedure described above. R f = 0.21 (DCM / MeOH, 9:1); 1 HNMR (400MHz, methanol-d4) δ7.78(d, J=8.2Hz, 1H), 7.55(dd, J=8.5Hz, 8.5Hz, 1H), 7.24(dd, J=8.5Hz, 8.4)Hz, 1H), 7.18(d, J=8.2Hz, 1H), 6 .81(d, J=8.1Hz, 2H), 6.45(d, J=8.1Hz, 2H), 4.40(bs, 1H), 3.25(s, 3H), 3.16(dd, J=14.1Hz, 4.5Hz, 1H), 3.04(dd, J=14.1Hz, 8.5Hz, 1H); 13 CNMR (100 MHz, methanol-d4) δ 173.8, 156.1, 150.5, 135.9, 130.1, 126.3, 123.9, 117.4, 116.0, 114.8, 113.7, 48.4, 38.0, 29.9.

[0121] Example 2: Activity Assay Cell lines, transfection, and treatment. Human SHSY-5Y neuroblastoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (ThermoFisher Scientific; Waltham, MA) containing 10% fetal bovine serum (FBS) (ThermoFisher) and 1% penicillin-streptomycin (PenStrep, ThermoFisher) and incubated at 37 °C with atmospheric oxygen concentration (21%) containing 5% CO2. SHSY-5Y cells were cultured in DMEM containing 20% ​​FBS, 1% PenStrep in Ham's F12 (1:1) (ThermoFisher). Cells were seeded at a density adjusted to reach 70-80% confluence at the beginning of each experiment. SH-SY5Y cells were transiently transfected with human wild-type alpha-synuclein using FugeneHD transfection reagent (Promega; Madison, WI) for 24 h before adding fresh medium and drugs. Cells were treated with 1 mM, 100 μM, 10 μM, 1 μM, 0.1 μM, and 0.01 μM of BK50118-A, BK50118-B, BK50118-C, CL3-499, CL3-512, and CL3-514 dissolved in DMSO or an equal volume of DMSO for 5 h.

[0122] Cell viability was determined by lactate dehydrogenase assay (ThermoFisher) on culture medium and MTT assay (ThermoFisher) on plated cells. Using separate samples of the same treatment conditions, cells were harvested on ice by removing the medium and adding 0.2 ml 1× sodium-Tris, EDTA, NP-40 (STEN) buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.2% NP-40, 0.2% Halt protease and phosphatase inhibitor solution (ThermoFisher). Cells were detached with a cell scraper, collected in a centrifuge tube, and incubated at 4°C for 30 min with agitation. Samples were stored at -80°C and used for further analysis.

[0123] Alpha-synuclein enzyme-linked immunosorbent assay (ELISA). ELISA for total human alpha-synuclein (Millipore; Burlington, MA) was performed using a Milliplexed ELISA. Briefly, the Xmap technology uses magnetic microspheres internally coded with two fluorescent dyes. By precisely combining these two dyes, multiple proteins in a sample are measured simultaneously. Each of these spheres is coated with a specific capture antibody. The capture antibody binds to the detection antibody and a reporter molecule, completing the reaction on the bead surface.

[0124] A total of 25 μL of soluble protein was incubated with 25 μL of mixed bead solution overnight at 4°C. After washing, samples were incubated with 25 μL of detection antibody solution for 1.5 hours at room temperature. Streptavidin-phycoerythrin (25 μL) was added to each well containing 25 μL of detection antibody solution. Samples were then washed and suspended in 100 μL of sheath fluid. Samples were then run on MAGPIX using Xponent software. Median fluorescence intensity (MFI) data was analyzed using a 5-parameter logistic or spline curve fitting method to calculate analyte concentration in samples. Specific alpha-synuclein ELISA was performed on tissue soluble extracts from midbrain lysates in 1×STEN buffer. ELISAs for USP13 (MyBioSource, Cat. No. MBS9335287), human alpha-synuclein (Biolegend, Cat. No. 844101), and specific p-tau ser396 (Invitrogen, KHB7031) were performed on cell or brain tissue extracts as described above according to the manufacturer's protocols.

[0125] Western blot analysis. To extract soluble proteins from mouse midbrain lysates, tissues were isolated and homogenized in 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 inhibitors), centrifuged at 10,000 x g for 20 min at 4 °C, and the supernatant containing the soluble protein fraction was collected. Extracts were analyzed by Western blot (WB) on 4-12% SDS NuPAGE Bis-Tris gels (Invitrogen, NP0301BOX). Beta-actin (β-actin) was probed with a monoclonal antibody (Emdmillipore, MAB1501R) (1:3000). Human alpha-synuclein was probed with a monoclonal antibody (ThermoFisher, AHB0261, Rockford, IL, USA) at 1:2000. USP13 was probed with a polyclonal antibody (ThermoFisher, PA5-12014, Rockford, IL, USA) at 1:1000. Ubiquitin was probed with a polyclonal antibody (ThermoFisher, PA3-16717, Rockford, IL, USA) at 1:5000. WB was quantified by densitometry using Quantity One 4.6.3 software (Bio Rad, Hercules, CA, USA) and Image J.

[0126] Immunoprecipitation (IP). Mouse brain tissue was homogenized in 1×STEN buffer and the soluble fraction was isolated as described above. Lysates were precleared with immobilized recombinant protein A / G agarose (Santa Crutz, sc-2003, Dallas, TX, USA) and centrifuged at 2500×g for 1 min at 4°C. Supernatants were collected and quantified by protein assay, and a total of 300 μg of protein was incubated overnight at 4°C with primary anti-alpha-synuclein (1:200, Thermofisher, AHB0261) mouse antibody or anti-ubiquitin (1:100) (ThermoFisher, PA3-16717, Rockford, IL, USA) antibody in the presence of Sepharose G and IgG control containing the primary antibody. Immunoprecipitates were collected by centrifugation at 2500 × g for 3 min at 4 °C, washed five times with PBS, with a final wash step using detergent-free buffer and centrifugation at 2500 × g for 3 min, and proteins were eluted according to Pierce's instructions (Pierce #20365, Rockford, IL, USA). After IP, samples were size-fractionated by 4–12% SDS-NuPAGE and transferred to 0.45 μm nitrocellulose membranes. WB detection was then performed using horseradish peroxidase (HRP)-conjugated secondary antibodies.

[0127] Immunohistology. Animals were deeply anesthetized with a mixture of xylazine and ketamine (1:8), washed with saline for 1 min, and then perfused with 4% paraformaldehyde (PFA) for 15–20 min. Brains were quickly dissected and immediately stored in 4% PFA at 4 °C for 24 h, then transferred to 30% sucrose at 4 °C for 48 h. Brains were cut into 20 μm thick coronal sections using a cryostat microtome and stored at -20 °C.

[0128] To evaluate alpha-synuclein ubiquitination, immunohistochemistry was performed on 20-μm-thick brain sections. The antibodies used were human alpha-synuclein monoclonal antibody (ThermoFisher, AHB0261, Rockford, IL, USA) and ubiquitin polyclonal antibody (ThermoFisher, PA3-16717, Rockford, IL, USA). Optical density measurements of ubiquitin and alpha-synuclein colocalization were measured using ImageJ. Because tyrosine hydroxylase (TH) is the limiting enzyme for DA synthesis, probing TH+ neurons is useful for assessing the status of DA-generating neurons in the SN and their terminals in the striatum. Fluorescent staining of TH+ in the striatum was performed, followed by optical density measurements of striatal DA terminals. Nuclear staining with 4,6-diamidino-2-phenylindole was performed according to the manufacturer's protocol (Life Technologies, Rockford, IL, USA).

[0129] DAB staining for alpha-synuclein (ThermoFisher, AHB0261, Rockford, IL, USA) was performed on 20 μm thick mouse brain sections, and stereological counting of alpha-synuclein+ neurons counterstained with Nissl was performed.

[0130] Nissl and Silver Staining. Nissl staining was performed using FD Cresyl Violet Solution™ Regular Strength (FD NeuroTechnologies, Cat PS102-01) according to the manufacturer's instructions. Silver staining was performed using FD NeuroSilver™ (FD Neuro Technologies, Cat PK301) according to the manufacturer's instructions.

[0131] Pharmacokinetic studies. C57BL / 6 mice received a single intraperitoneal dose of BK50118-C (10 mg / kg). Brain and serum samples were collected 1, 2, 3, 4, 8, and 12 hours later (n = 3 per time point). Animals injected with vehicle (DMSO) were used for background subtraction. Stock solutions of drug (1 mg / mL) and internal standard were prepared in methanol. Intermediate solutions used for calibrators and control samples were serially diluted in methanol / water (1:1). Preparation of calibration curve standards and quality samples (QC) was performed by mixing intermediate dilutions of blank samples (brain homogenate, serum). The internal standard working solution contained deuterium-labeled BK50118-C-d7 diluted in acetonitrile (ACN) / ethyl acetate (4:1) at a concentration of 5 ng / mL. Serum and brain samples were stored at -80 °C and thawed to room temperature before preparation. Brains were homogenized in MilliQ water (1 mg brain:10 μL water). Proteins were precipitated in both brain and serum samples by mixing 25 μL of aqueous sample with 75 μL of internal standard working solution. The mixtures were centrifuged at 12,300 g for 5 min. Then, 75 μL of each supernatant and 25 μL of MilliQ water were pipetted into a 96-well PCR plate (Fisher Scientific, Dawsonville, GA, USA).

[0132] The concentration of BK50118-C in brain tissue and serum samples was measured by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS / MS). Briefly, the UHPLC-MS / MS system included an Elute HTG binary gradient UHPLC pump with an electrospray ionization (ESI) source operated in positive mode, an Elute column oven, and an EVOQ Elite triple quadrupole mass spectrometer (all from Bruker Daltonik GmbH, Bremen, Germany). Samples were injected using a PAL autosampler with a 10 μL sample loop (CTC Analytics, Zwingen, Switzerland), and samples were stored in a PAL stack cooler for six microtiter plates and operated at +6 °C. The system was controlled by Compass 2.0 / HyStar 4.0 software (Bruker), and compound screening and quantification were performed by TASQ 2.2 data acquisition and processing software (Bruker). The mass spectrometer was supplied with nitrogen and air, generated by a Genius 3045 nitrogen / air generator (Peak Scientific Instruments, Inchinnan, Scotland, UK). ESI parameters were as follows: probe gas flow rate 50, nebulizer gas flow rate 60, probe temperature +400°C, cone gas flow rate 20, cone temperature +350°C, CID gas Ar 1.5 mTorr. Mass spectra were scanned in MRM mode to probe the optimal collision energy of the test compounds and their respective precursor ions.

[0133] For chromatographic separation, a YMC-Ultra HT Hydrosphere C18 column (2.0×100 mm, particle size 2 μm) and a YMC-Hydrosphere 2.1×5 mm guard column (YMC Co. Ltd., Kyoto, Japan) were used. Mobile phase A was 10 mM ammonium formate in water, pH 4.3, and mobile phase B was 10 mM ammonium formate in 10% water, 50% acetonitrile, and 40% isopropyl alcohol, pH 4.3. The mobile phase gradient was as follows (min-A / B%): 0 min-50 / 50, 0.5 min-50 / 50, 2.0 min-20 / 80, 3 min-20 / 80, 3.1 min-50 / 50, 4.5 min-50 / 50. The flow rate was 450 μL / min, and the column temperature was set at 50° C. The sample injection volume was 10 μL.

[0134] Statistics and data analysis. All statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad software, Inc, San Diego, CA, USA). Two-tailed Student's t-test was used for comparison of the means of two groups. For comparison of the means of multiple groups, ordinary one-way analysis of variance (ANOVA) followed by Newman-Keuls or Dunnett comparison post-hoc tests was used. Asterisks indicate the significance of actual p-values ​​(*<0.05, **<0.01, ***<0.001, and ****<0.0001), and N is the number of animals per group or number of independent experiments (cell cultures). Data are expressed as mean ± SD unless otherwise stated. IC50 to USP13 levels were also estimated using GraphPad Prism version 5.0 (GraphPad software, Inc, San Diego, CA, USA).

[0135] result: Viability and activity assays were carried out on human SHSY5Y neuroblastoma cells treated with various amounts of compounds described herein.Specifically, human SHSY5Y neuroblastoma cells were treated with 1mM, 100μM, 10μM, 1μM, 0.1μM and 0.01μM of BK50118-A, BK50118-B, BK50118-C, CL3-499, CL3-512 and CL3-514 dissolved in DMSO or with an equivalent amount of DMSO for 5 hours.

[0136] The graphs in Figures 1A-1F represent MTT and LDH assays in cells treated with BK50118-A (Figure 1A), BK50118-B (Figure 1B), BK50118-C (Figure 1C), CL3-514 (Figure 1D), CL3-512 (Figure 1E) and CL3-499 (Figure 1F). N=3-4 per group. The data in Figures 1A-1F show that compounds BK50118-A, BK50118-B, BK50118-C, CL3-514, CL3-512 and CL3-499 are safe and do not cause cell death using two viability assays at concentrations up to 1 mM.

[0137] The graphs in Figures 2A-2F show USP13 activity by ELISA in cells treated with BK50118-A (Figure 2A), BK50118-B (Figure 2B), BK50118-C (Figure 2C), CL3-514 (Figure 2D), CL3-512 (Figure 2E) and CL3-499 (Figure 2F). N=3-9 per group. The data in Figures 2A-2F show that compounds BK50118-A, BK50118-B, BK50118-C, CL3-514, CL3-512 and CL3-499 significantly reduce the activity of human USP13 within the concentration range of 1 nM to 1 mM concentration.

[0138] The estimated half maximal inhibitory concentration (IC) of each compound required to inhibit USP13 activity in SHSY5Y cell line. 50 ) were determined (Table 1). Spautin-1 was also evaluated for comparative purposes. [Table 1]

[0139] The data in Table 1 show that compounds BK50118-A, BK50118-B, BK50118-C, CL3-514, CL3-512, and CL3-499 potently inhibit USP-13 activity.

[0140] The graphs in Figures 3A-3F show alpha-synuclein levels by ELISA in human SHSY5Y neuroblastoma cells treated with 1 mM, 100 μM, 10 μM, 1 μM, 0.1 μM, and 0.01 μM of BK50118-A, BK50118-B, BK50118-C, CL3-499, CL3-512, and CL3-514 dissolved in DMSO or an equal volume of DMSO for 5 h. The graphs in Figures 3A-3F show ELISA levels of alpha-synuclein in cells treated with BK50118-A (Figure 3A), BK50118-B (Figure 3B), BK50118-C (Figure 3C), CL3-514 (Figure 3D), CL3-512 (Figure 3E), and CL3-499 (Figure 3F). N=3-9 per group. The data in Figures 3A-3F show that compounds BK50118-A, BK50118-B, BK50118-C, CL3-514, CL3-512 and CL3-499 significantly reduce levels of alpha-synuclein at concentrations between 1 and 0.1 nM.

[0141] In vivo studies Because BK50118-C is a potent USP13 inhibitor, we determined the pharmacokinetics (PK) of this molecule. Wild-type C57BL6 mice were injected with 10 mg / kg BK50118-C rather than DMSO, and brain and serum were isolated at 0, 1, 2, 4, 6, 8, and 12 hours, extracted in water, and examined by mass spectrometry. Concentrations of BK50118-C were significantly higher in both serum and brain at 1 hour (T 最大 ) in the brain and serum (Table 2), with maximum concentrations (C 最大 ) The bioavailability (AUC, area under the curve) of the drug was 164.3 nM × h and 599.4 nM × h in the brain and serum, respectively. 1 / 2) was 2.32 hours in the brain and 1.84 hours in the serum. The serum:brain ratio reached 28%, indicating abundant entry of the drug into the brain.

[0142] Table 2. Pharmacological parameters of BK50118-C measured in wild type mice (C57B6) injected with 10 mg / kg deuterated BK50118-C, tissues were collected over 12 hours post-injection. [Table 2]

[0143] BK50118-C reduces alpha-synuclein, increases alpha-synuclein ubiquitination, and improves neuronal survival in mice Transgenic A53T mice carry the arginine to threonine (A53T) mutation of human alpha-synuclein under the control of the prion promoter and have abundant alpha-synuclein in the striatum as early as 3 months of age. Male and female TgA53T mice (15 months old) were treated daily with intraperitoneal injections of DMSO versus 10 mg / kg or 40 mg / kg BK50118-C for 7 days. WB of midbrain lysates showed that human alpha-synuclein was significantly reduced at the above doses (first blot in Figure 4A,B). Immunoprecipitation of alpha-synuclein protein from midbrain extracts followed by ubiquitin WB (second blot in Figure 4B) showed increased ubiquitination at 40 mg / kg BK50118-C compared to DMSO and 10 mg / kg BK50118-C. Immunoprecipitation of ubiquitin from midbrain extracts followed by alpha-synuclein WB (Figure 4B, third blot) showed no monomeric alpha-synuclein but increased ubiquitination of alpha-synuclein at 40 mg / kg BK50118-C compared to DMSO and 10 mg / kg BK50118-C. ELISA measurements of alpha-synuclein in the same extracts (Figure 4C) confirmed that human alpha-synuclein was reduced by approximately 30% (10 mg / kg) and 56% (40 mg / kg), respectively (Figure 4C).

[0144] TgA53T mice also showed elevated tauopathy status in the striatum, suggesting that tauopathy is a common feature of synucleinopathies. Therefore, tau levels were also measured. Tau levels in mice were not affected (Figure 4D). Immunostaining of 20 μm thick brain sections showed human alpha-synuclein staining in both the cortex (Figure 4E,G,I) and striatum (Figure 4J,L,N) of DMSO-treated mice. BK50118-C treatment (40 mg / kg) significantly reduced human alpha-synuclein staining in both the cortex (Figure 4F,H,I) and striatum (Figure 4K,M,I) compared to DMSO. Quantification showed that BK50118-C (40 mg / kg) significantly reduced the number of human alpha-synuclein positive neurons by 42% in the cortex and 40% in the striatum (Figure 4I,N). Nissl staining showed that BK50118-C significantly increased the number of neurons in the cortex (Fig. 6B,C), striatum (Fig. 6E,F), and substantia nigra (SN) (Fig. 6H,I) compared with DMSO (Fig. 6A,D,G), as verified by quantification of Nissl+ cells in the cortex (Fig. 6C), striatum (Fig. 6F), and SN (Fig. 6I).

[0145] BK50118-C increases ubiquitination of alpha-synuclein and has minimal effect on tyrosine hydroxylase levels in the striatum of TgA53T mice To confirm the mechanistic effect of BK50118-C on alpha-synuclein clearance, immunostaining was performed in the striatum of TgA53T mice. Human alpha-synuclein staining shows the levels of human alpha-synuclein in DMSO (Figure 5A) compared to BK50118-C (40mg / Kg) treated mice (Figure 5B). Ubiquitin staining in DMSO treated striatum (Figure 5C) showed the levels of ubiquitin compared to mice treated with 40mg / Kg BK50118-C (Figure 5D). Fusion staining of alpha-synuclein and ubiquitin in DMSO (Figure 5E) showed that alpha-synuclein colocalized with ubiquitin in mice treated with BK50118-C compared to BK50118-C (Figure 5F). The optical density of alpha-synuclein-ubiquitin staining showed a significant increase (130%) in colocalization in the striatum when mice were injected with BK50118-C compared to DMSO (Figure 5G). We also stained for tyrosine hydroxylase (TH) in the striatum of TgA53T mice. TH is the enzyme responsible for catalyzing the rate-limiting step in the synthesis of L-3,4-dihydroxyphenylalanine (L-DOPA), a precursor of dopamine (DA). Staining for TH helps to evaluate the status of DA-generating neurons in the SN and striatal terminals. Staining for tyrosine hydroxylase (TH) in the striatum of TgA53T mice showed no noticeable effect with BK50118-C (Figure 5I,K,M,N) compared to DMSO-treated mice (Figure 5H,J,L,N).

[0146] The compounds and methods of the appended claims are not limited in scope by the specific compounds and methods described herein, which are intended as illustrations of some aspects of the claims, and any compounds and methods that are functionally equivalent are within the scope of the disclosure. In addition to the modifications shown and described herein, various modifications of the compounds and methods are intended to be included within the scope of the appended claims. Furthermore, only certain representative compounds, methods, and aspects of these compounds and methods are specifically described, but other compounds and methods are intended to be included within the scope of the appended claims. Thus, the present specification may explicitly refer to a combination of steps, elements, components, or elements, but all other combinations of steps, elements, components, and elements are included, even if not explicitly described.

Claims

1. A compound of the formula: 【Chemistry 1】 or a pharma- ceutically acceptable salt or prodrug thereof, wherein: n is 0, 1 or 2; L is S, O, or NR ７ and X is S, O, NR ８ , or C.R. ５ =CR ６ and Y １ , Y ２ , Y ３ , and Y ４ are each independently N or CR, R is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; Y １ , Y ２ , Y ３ , and Y ４ At least one of is N; R １ is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; Each R ２ is independently selected from the group consisting of halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; R ３ , R ４ , R ５ , and R ６ are each independently selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; R ７ and R ８ are each independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

2. The compound has the following formula: 【Chemistry 2】 2. The compound of claim 1 having the formula:

3. The compound of claim 2 , wherein n is 0.

4. In the formula, R ３ , R ４ , and R ７ 4. The compound of claim 2 or 3, wherein each is hydrogen.

5. In the formula, R １ The compound according to any one of claims 2 to 4, wherein is aryl.

6. The compound has the following formula: 【Chemistry 3】 2. The compound of claim 1 having the formula:

7. The compound of claim 6 , wherein n is 0.

8. In the formula, R ３ , R ４ , and R ６ 8. The compound of claim 6 or 7, wherein each is hydrogen.

9. In the formula, R ５ The compound according to any one of claims 6 to 8, wherein is halogen.

10. The compound according to any one of claims 6 to 9, wherein L is NH or O.

11. In the formula, R １ The compound according to any one of claims 6 to 10, wherein is benzyl or phenyl.

12. The compound is 【Chemistry 4】 2. The compound of claim 1 selected from the group consisting of:

13. A compound of the formula: 【Chemistry 5】 or a pharma- ceutically acceptable salt or prodrug thereof, wherein: AA is an amino acid or an ester thereof; Ar is a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl; The compound, wherein AA is covalently bonded to Ar via the amino group of AA.

14. 14. The compound of claim 13, wherein the amino acid or ester thereof is a naturally occurring amino acid or ester thereof.

15. 14. The compound of claim 13, wherein the amino acid or ester thereof is an unnatural amino acid or ester thereof.

16. The compound has the formula: 【Chemistry 6】 During the ceremony, Ar is substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R １ and R ２ are each independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; R ３ is hydrogen, halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl.

17. The compound has the formula: 【Chemistry 7】 During the ceremony, n is 0, 1, 2, 3, or 4; R ４ is selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; Each R ５ is independently selected from the group consisting of halogen, cyano, trifluoromethyl, nitro, hydroxy, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

18. In the formula, R １ The compound of claim 17 , wherein is hydrogen.

19. In the formula, R ２ 19. The compound of claim 17 or 18, wherein is methyl.

20. In the formula, R ３ The compound of any one of claims 17 to 19, wherein is substituted or unsubstituted benzyl.

21. In the formula, R ４ The compound according to any one of claims 17 to 20, wherein is nitro.

22. The compound according to any one of claims 17 to 21, wherein n is 0.

23. The compound is 【Chemistry 8】 14. The compound of claim 13, selected from the group consisting of:

24. A pharmaceutical composition comprising a compound according to any one of claims 1 to 23 and a pharma- ceutically acceptable carrier.

25. A kit comprising a compound according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 24.

26. 1. A composition or pharmaceutical composition for use in treating or preventing a USP13-associated disease in a subject, comprising: A composition comprising a compound according to any one of claims 1 to 23, or a pharmaceutical composition according to claim 24.

27. The composition of claim 26, wherein the subject is selected to have a USP13-associated disease.

28. 28. The composition of claim 26 or 27, wherein the USP13-associated disease is a neurodegenerative disease.

29. The composition of claim 28, wherein the neurodegenerative disease is amyotrophic lateral sclerosis, Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia, Huntington's disease, mild cognitive impairment, α-synucleinopathy, tauopathy, or a condition associated with intracellular accumulation of TDP-43.

30. The composition described in claim 28 or 29, characterized in that the composition is administered to the subject in combination with a second therapeutic agent.

31. 31. The composition of claim 30, wherein the second therapeutic agent is selected from the group consisting of levadopa, dopamine agonists, anticholinergics, monoamine oxidase inhibitors, COMT inhibitors, amantadine, rivastigmine, NMDA antagonists, cholinesterase inhibitors, riluzole, antipsychotics, antidepressants, and tetrabenazine.

32. The composition according to any one of claims 28 to 31, wherein the subject is selected to have a neurodegenerative disease.

33. 28. The composition of claim 26 or 27, wherein the USP13-associated disease is cancer.

34. 34. The composition of claim 33, wherein the cancer is pancreatic cancer, breast cancer, brain cancer, lung cancer, prostate cancer, colorectal cancer, thyroid cancer, kidney cancer, adrenal cancer, liver cancer, neurofibromatosis type 1, lymphoma, or leukemia.

35. The composition described in claim 33 or 34, characterized in that the composition is administered to the subject in combination with a second therapeutic agent.

36. 36. The composition of claim 35, wherein the second therapeutic agent is a chemotherapeutic agent or radiation.

37. The composition of any one of claims 33 to 36, further comprising selecting a subject suffering from cancer.

38. 1. A composition for use in a method of inhibiting USP13 in a cell, comprising:

24. A composition comprising a compound according to any one of claims 1 to 23, wherein the method comprises contacting a cell with the compound.

39. 24. A composition for use in a method of reducing alpha-synuclein levels in a cell, comprising a compound according to any one of claims 1 to 23, said method comprising contacting a cell with said compound.

40. 40. The composition of claim 38 or 39, wherein the contacting is effected in vitro or in vivo.

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