Compounds for inhibiting and treating alpha-synucleinopathies, tauopathies and other diseases

Abstract

The present invention relates to compounds useful for the inhibition and treatment of alpha-synucleinopathies, tauopathies and other diseases. Disclosed is a method of treating or inhibiting a disease selected from the group consisting of alpha-synucleinopathies, tauopathies, ALS, traumatic brain injury, and ischemia-reperfusion related injury, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula: # imgabs0 # or a hydroquinone form thereof; or a solvate or hydrate thereof.

Description

[0001] This application claims and enjoys priority to U.S. Provisional Application No. 62 / 747,080, filed on October 17, 2018, entitled “2,3,5-TRIMETHYL-6-NONYLCYCLOHEXA-2,5-DIENE-1,4-DIONE FOR SUPPRESSING AND TREATING ALZHEIMER'S DISEASE AND OTHER DISORDERS” and U.S. Provisional Application No. 62 / 771,570, filed on November 26, 2018, entitled “2,3,5-TRIMETHYL-6-NONYLCYCLOHEXA-2,5-DIENE-1,4-DIONE FOR SUPPRESSING AND TREATING ALZHEIMER'S DISEASE AND OTHER DISORDERS”, the contents of which are incorporated herein by reference in their entirety for all purposes. Background of the Invention

[0002] US Publication No. 2007 / 0072943 describes certain quinone compounds and methods for treating certain mitochondrial diseases. US Publication No. 2010 / 0063161 describes the compound 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione and methods for treating pervasive developmental disorder and attention deficit hyperactivity disorder (ADHD).

[0003] Improved methods for treating or inhibiting certain diseases are needed, including α-synuclein diseases (α-synucleinopathies), tauopathies, ALS, traumatic brain injury, and ischemia-reperfusion-related injuries. Further needed are compounds with superior brain penetration and / or superior ability to partition into the brain compared to other parts of the body (e.g., plasma).

[0004] In addition, there is a need for 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione and stable polymorphs thereof in particle form that can be used in pharmaceutical compositions; preparations of pharmaceutical compositions; and methods for treating or inhibiting diseases, including methods for treating or inhibiting alpha-synuclein diseases, tauopathies, ALS, traumatic brain injury, and ischemia-reperfusion-related injuries. Summary of the Invention

[0005] In one aspect, the present invention relates to a method for treating or inhibiting a disease selected from the group consisting of alpha-synuclein disease (α-synuclein disease), tauopathy, amyotrophic lateral sclerosis (ALS), traumatic brain injury, and ischemia-reperfusion-related injury, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the formula: or its hydroquinone form; or its solvate or hydrate. In some embodiments, the compound is not a solvate or a hydrate. In some embodiments, including any of the preceding embodiments, the compound is in the quinone form. In some embodiments, including any of the preceding embodiments, the compound is in the hydroquinone form. In some embodiments, including any of the preceding embodiments, the method is used to treat or inhibit alpha-synuclein disease. In some embodiments, including any of the preceding embodiments, the alpha-synuclein disease is selected from the group consisting of: Parkinson's disease, Parkinson's disease with dementia (PDD), multiple system atrophy (MSA), frontotemporal dementia, Dementia with Lewy Bodies (DLB), Gaucher's disease (GD), neurodegeneration with brain iron deposition (NBIA), and neuroaxonaldystrophies (neuroaxonaldystrophies, PLA2G6-related neurodegenerative diseases). In some embodiments, including any of the aforementioned embodiments, the Parkinson's disease is hereditary. In some embodiments, including any of the aforementioned embodiments, the Parkinson's disease is idiopathic. In some embodiments, including any of the aforementioned embodiments, the method for suppressing or treating Parkinson's disease is that the patient has a mutation in one or more of the following genes: MAPT (microtubule-associated protein tau), PRKN (parkin), PINK1 (PINK1), LRRK2 (leucine-rich repeat kinase 2), GBA (glucocerebrosidase), SNCA (alpha synuclein), PARK7 (DJ-1), and / or UCHL1 (ubiquitin carboxyl terminal esterase L1). In some embodiments, including any of the aforementioned embodiments, the method is used to treat or suppress tau protein disease.In some embodiments, including any of the preceding embodiments, the tauopathy is selected from the group consisting of Alzheimer's disease, dementia pugilistica, Guam ALS / PD, Pick Disease, Argyrophilic grain dementia, Nieman-Pick type C, Subacute sclerosing panencephalitis (SSPE), Progressive supranuclear palsy (PSP), Multiple system atrophy (MSA), Corticobasoganlionic degeneration, Frontotemporal dementia with parkinsonism-17 (FTDP-17), Postencephalitis Parkinsonism-2 (PTDP-2), and Postencephalitis Parkinsonism-3 (PTDP-3). Parkinsonism, PEP), and autosomal recessive Parkinsonism. In some embodiments, including any of the foregoing embodiments, the method is used to treat or inhibit Alzheimer's disease. In some embodiments, including any of the foregoing embodiments, the method is used to treat or inhibit Parkinson's disease. In some embodiments, including any of the foregoing embodiments, the method is used to treat or inhibit traumatic brain injury. In some embodiments, including any of the foregoing embodiments, the method is used to treat or inhibit ischemia-reperfusion-related injury. In some embodiments, including any of the foregoing embodiments, the method is used to treat or inhibit stroke. In some embodiments, including any of the foregoing embodiments, the method is used to treat or inhibit amyotrophic lateral sclerosis (ALS). In some embodiments, including any of the foregoing embodiments, the method is used to treat the disease. In some embodiments, including any of the foregoing embodiments, the method is used to inhibit the disease. In some embodiments, including any of the foregoing embodiments, the compound is administered orally. In some embodiments, including any of the foregoing embodiments, the compound is administered intravenously.

[0006] In another aspect, the present invention relates to a method for treating or inhibiting a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, traumatic brain injury, and ischemia-reperfusion-related injury, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the formula: or its hydroquinone form; or its solvate or hydrate. In some embodiments, the compound is: Or its hydroquinone form. In some embodiments, including any of the preceding embodiments, the compound is in the quinone form. In some embodiments, including any of the preceding embodiments, the compound is in the hydroquinone form. In some embodiments, the method is used to inhibit or treat Alzheimer's disease. In some embodiments, the method is used to inhibit or treat Parkinson's disease. In some embodiments, the method for inhibiting or treating Parkinson's disease includes treating or inhibiting idiopathic Parkinson's disease. In some embodiments, the method for inhibiting or treating Parkinson's disease includes treating or inhibiting familial (ie, hereditary) Parkinson's disease. In some embodiments, the method for inhibiting or treating Parkinson's disease is that the patient has a mutation in one or more of the following genes: MAPT (microtubule-associated protein tau), PRKN (Parkin protein), PINK1 (PINK1), LRRK2 (leucine-rich repeat kinase 2), GBA (glucocerebrosidase), SNCA (alpha synuclein), PARK7 (DJ-1), and / or UCHL1 (ubiquitin carboxyl-terminal esterase L1). In some embodiments, the method is used to inhibit or treat traumatic brain injury. In some embodiments, the method is used to inhibit or treat ischemia-reperfusion-related injury. In some embodiments, the ischemia-reperfusion-related injury is a stroke. In some embodiments, the ischemia-reperfusion-related injury is ischemia-reperfusion-related retinal injury. In some embodiments, including any of the foregoing embodiments, the compound is administered orally. In some embodiments, including any of the foregoing embodiments, the compound is administered by injection. In some embodiments, including any of the foregoing embodiments, the compound is administered intravenously. In some embodiments, including any of the foregoing embodiments, the method is a method of inhibiting the disease. In some embodiments, including any of the foregoing embodiments, the method is a method of treating the disease.

[0007] In another aspect, the present invention relates to a polymorph of an anhydrate of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione, wherein the powder X-ray diffraction pattern of the polymorph comprises characteristic peaks at at least the following angular positions: 4.10, 12.12 and 16.14, wherein the angular positions can vary by ±0.2. In some embodiments, a Cu Kα1 source is used, The data is obtained using a wavelength of 23-25°C and a temperature of 23-25°C. In some embodiments, the polymorph comprises characteristic peaks at least at the following angular positions: 4.10, 11.77, 12.12, and 16.14, wherein the angular positions may vary by ±0.2. In some embodiments, the polymorph comprises characteristic peaks at least at the following angular positions: 4.10, 11.77, 12.12, 16.14, and 22.41, wherein the angular positions may vary by ±0.2. In some or any embodiments, the powder X-ray diffraction pattern of the polymorph comprises characteristic peaks at at least one of the following angular positions: 4.10, 11.77, 12.12, 16.14, and 22.41, wherein the angular positions may vary by ±0.2. In some or any embodiments, the powder X-ray diffraction pattern of the polymorph comprises characteristic peaks at at least two of the following angular positions: 4.10, 11.77, 12.12, 16.14 and 22.41, wherein the angular positions may vary by ±0.2. In some or any embodiments, the powder X-ray diffraction pattern of the polymorph comprises characteristic peaks at at least two of the following angular positions: 4.10, 11.77, 12.12 and 16.14, wherein the angular positions may vary by ±0.2. In some or any embodiments, the powder X-ray diffraction pattern of the polymorph comprises characteristic peaks at at least two of the following angular positions: 4.10, 12.12 and 16.14, wherein the angular positions may vary by ±0.2. In some embodiments, including any of the foregoing embodiments, the angular positions may vary by ±0.1. In some embodiments, including any of the foregoing embodiments, the angular positions may vary by ±0.05. In some embodiments, including any of the foregoing embodiments, the polymorph has substantially Figure 5 , 11 , 14 and 16. In some embodiments, including any of the foregoing embodiments, the polymorph has substantially Figure 7 The differential scanning calorimetry (DSC) thermogram shown. In some embodiments, including any of the foregoing embodiments, the DSC thermogram has a single endothermic peak at about 47°C to about 53°C. In some embodiments, including any of the foregoing embodiments, the DSC thermogram has a single endothermic peak at about 49°C to about 53°C. In some embodiments, including any of the foregoing embodiments, the DSC thermogram has a single endothermic peak at about 50°C to about 52°C. In some embodiments, including any of the foregoing embodiments, the DSC thermogram has a single endothermic peak at about 50.5°C. In some embodiments, including any of the foregoing embodiments, the polymorph has substantially Figure 8 In some embodiments, including any of the foregoing embodiments, the polymorph has substantially Figure 6shown 1 HNMR spectrum. In some embodiments, including any of the preceding embodiments, at least about 95 mol% of the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione is the polymorph, free of any solvent, carrier or excipient. In some embodiments, including any of the preceding embodiments, at least about 99 mol% of the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione is the polymorph, free of any solvent, carrier or excipient. In some embodiments, including any of the preceding embodiments, at least about 95% a / a of the composition is the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione, free of any solvent, carrier or excipient, as determined by HPLC. In some embodiments, including any of the foregoing embodiments, at least about 99% a / a of the composition is the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione as determined by HPLC, free of any solvent, carrier or excipient. In some embodiments, including any of the foregoing embodiments, the potency (or content) of the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione is at least about 95%. In some embodiments, including any of the foregoing embodiments, the potency (or content) of the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione is at least about 99%. In some embodiments, including any of the foregoing embodiments, the polymorph is present in the form of a plurality of particles, wherein the D90:D10 ratio of the particles is less than about 11:1. In some embodiments, including any of the foregoing embodiments, the polymorph is present as a plurality of particles, wherein the particles have a D90:D10 ratio of less than about 7:1. In some embodiments, including any of the foregoing embodiments, the polymorph is recrystallized from a solvent comprising about 75-85% IPA / water. In some embodiments, including any of the foregoing embodiments, the polymorph is recrystallized from a solvent comprising about 80-85% IPA / water. In some embodiments, including any of the foregoing embodiments, the polymorph is recrystallized from a solvent comprising about 85% IPA / water.

[0008] On the other hand, the present invention relates to a pharmaceutical composition comprising the polymorph described in the present invention, or the composition described in the present invention, and a pharmaceutically acceptable solvent, carrier or excipient; or a pharmaceutical composition prepared using the polymorph described in the present invention, or the composition described in the present invention, and a pharmaceutically acceptable solvent, carrier or excipient.

[0009] On the other hand, the present invention relates to a method for treating or inhibiting α-synucleinpathy, tauopathy, amyotrophic lateral sclerosis (ALS), traumatic brain injury, or ischemia-reperfusion related injury, which comprises administering a therapeutically effective amount of the polymorph described in the present invention or the composition described in the present invention to an individual in need thereof.

[0010] In another aspect, the present invention relates to a method for recrystallizing 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione from a composition, the method comprising: a) contacting the composition with IPA and water so that the ratio of IPA to water is about 75-87% isopropyl alcohol (IPA) / 25-13% water (v:v) at a temperature of about 40-45°C; b) cooling the mixture to about 32°C; and c) filtering the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione from the mixture. In some embodiments, step (a) comprises: a1) contacting the composition with IPA; a2) heating the mixture to about 40-45°C; and a3) adding water to the mixture so that the ratio of IPA to water is about 75-85% IPA: 25-15% water (v:v). In some embodiments, including any of the foregoing embodiments, step (a) comprises stirring to dissolve the composition. In some embodiments, including any of the foregoing embodiments, the ratio of IPA: water is about 80-85% IPA: 20-15% water (v: v). In some embodiments, including any of the foregoing embodiments, the ratio of IPA: water is about 85% IPA: 15% water (v: v). In some embodiments, including any of the foregoing embodiments, step (a3) ​​comprises returning the temperature of the mixture to about 40-45°C. In some embodiments, including any of the foregoing embodiments, the method comprises polishing and filtering the mixture after step (a). In some embodiments, including any of the foregoing embodiments, step (b) comprises cooling to about 32°C within about 2-10 hours. In some embodiments, including any of the foregoing embodiments, step (b) comprises cooling to about 32°C within about 6 hours. In some embodiments, including any of the foregoing embodiments, the method comprises step (b1) after step (b), comprising keeping the mixture at about 32°C for about 2-24 hours. In some embodiments, including any of the preceding embodiments, the method comprises a step (b1) after step (b), comprising maintaining the mixture at about 32° C. for about 6 hours. In some embodiments, including any of the preceding embodiments, if present, the method comprises a step (b2) after step (b) or (b1), comprising cooling the mixture to about 0° C. In some embodiments, including any of the preceding embodiments, step (b2) comprises cooling the mixture to about 0° C. within about 3-24 hours. In some embodiments, including any of the preceding embodiments, step (b2) further comprises maintaining the mixture at about 0° C. for about 1 hour.

[0011] In another aspect, the present invention is directed to a composition comprising 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione prepared according to the process described in the immediately preceding paragraph.

[0012] On the other hand, the present invention relates to a method for preparing a pharmaceutical composition, which comprises converting the polymorph described in any of the preceding paragraphs or the composition described in any of the preceding paragraphs into a liquid or emulsion form. In some embodiments, the pharmaceutical composition is provided as an oral solution, a liquid-filled capsule, or an injectable solution. The present invention provides a pharmaceutical composition prepared according to these methods.

[0013] In another aspect, the present invention relates to a metastable melting amorphous form of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione having an XRPD pattern substantially as shown in Fig.31 shown.

[0014] In one aspect, the present invention relates to a method for treating or inhibiting a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, traumatic brain injury, and ischemia-reperfusion-related injury, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the formula: or its hydroquinone form; or its solvate or hydrate. In some embodiments, the compound is: Or its hydroquinone form. In some embodiments, including any of the preceding embodiments, the compound is in the quinone form. In some embodiments, including any of the preceding embodiments, the compound is in the hydroquinone form. In some embodiments, the method is used to inhibit or treat Alzheimer's disease. In some embodiments, the method is used to inhibit or treat Parkinson's disease. In some embodiments, the method for inhibiting or treating Parkinson's disease includes treating or inhibiting idiopathic Parkinson's disease. In some embodiments, the method for inhibiting or treating Parkinson's disease includes treating or inhibiting familial (ie, hereditary) Parkinson's disease. In some embodiments, the method for inhibiting or treating Parkinson's disease is that the patient has a mutation in one or more of the following genes: MAPT (microtubule-associated protein tau), PRKN (Parkin protein), PINK1 (PINK1), LRRK2 (leucine-rich repeat kinase 2), GBA (glucocerebrosidase), SNCA (alpha synuclein), PARK7 (DJ-1), and / or UCHL1 (ubiquitin carboxyl-terminal esterase L1). In some embodiments, the method is used to inhibit or treat traumatic brain injury. In some embodiments, the method is used to inhibit or treat ischemia-reperfusion-related injury. In some embodiments, the ischemia-reperfusion-related injury is a stroke. In some embodiments, the ischemia-reperfusion-related injury is ischemia-reperfusion-related retinal injury. In some embodiments, including any of the foregoing embodiments, the compound is administered orally. In some embodiments, including any of the foregoing embodiments, the compound is administered by injection. In some embodiments, including any of the foregoing embodiments, the compound is administered intravenously. In some embodiments, including any of the foregoing embodiments, the method is a method of inhibiting the disease. In some embodiments, including any of the foregoing embodiments, the method is a method of treating the disease.

[0015] Any one or more compounds described in the present invention, including all the aforementioned compounds, can be used in a composition comprising a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a pharmaceutically acceptable vehicle. In some embodiments, the composition is formulated for internal use. Any one or more compounds described in the present invention, including all the aforementioned compounds, can be formulated into a unit dose formulation.

[0016] For all compounds, compositions, formulations and methods described herein, any compound in the quinone form may also be used in its reduced form (hydroquinone) when desired. That is, compounds listed herein as cyclohexadienediones (oxidized quinones) may also be used in their hydroquinone (reduced hydroquinone) form as desired.

[0017] For all compounds, compositions and formulations described herein, and all methods of using the compounds or compositions or formulations described herein, the compounds or compositions may comprise the listed components or steps, or may "consist essentially of" the listed components or steps, or may "consist of" the listed components or steps. That is, the transition phrases "comprising" or "including" may be replaced by the transition phrases "consist essentially of" or "consist essentially of." Alternatively, in some or any embodiments, the transition phrases "comprising" or "including" may be replaced by the transition phrase "consist of." When a composition is described as "consisting essentially of" the listed components, the composition comprises the listed components, and may comprise other components that do not substantially affect the disease being treated, but does not comprise any other components that substantially affect the disease being treated other than those components explicitly listed; alternatively, if the composition does comprise additional components that substantially affect the disease being treated other than the listed components, the composition does not contain sufficient concentrations or amounts of the additional components that substantially affect the disease being treated. When a method is described as "consisting essentially of" the listed steps, the method comprises the listed steps and may comprise other steps that do not substantially affect the disease being treated, but the method does not comprise any other steps other than those explicitly listed that substantially affect the disease being treated. As a non-limiting specific example, when a composition is described as "consisting essentially of" a component, the composition may additionally comprise any amount of a pharmaceutically acceptable carrier, vehicle, excipient or diluent, and other such components that do not substantially affect the disease being treated. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A Shown are the kinetics of aggregation of recombinant human alpha-synuclein (αSyn) in the presence of substoichiometric ratios of the compounds 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione ("C9"), 2,3,5-trimethyl-6-octylcyclohexa-2,5-diene-1,4-dione ("C8"), 2,3,5-trimethyl-6-heptylcyclohexa-2,5-diene-1,4-dione ("C7"), or vehicle alone.

[0019] Figure 1B The fibril content of α-synuclein (αSyn) after t=24 hours of fibrillization in the absence (vehicle) or presence of C9, C8 or C7 is shown. The fibril content was assessed based on the relative ThT fluorescence intensity (the end point value at t=45.5 hr of the mean value of the vehicle-treated α-synuclein samples was set to 100%).

[0020] Figure 2 Shown are the effects of vehicle, C9, C8, or C7 treatment on the content of Tau preformed fibrils after 94 hours of incubation.

[0021] Figure 3A Shown are nuclear and aggregated alpha-synuclein in N27 rat dopaminergic cells treated with RSL3 in the absence or presence of co-treatment with C9, C8, or C7.

[0022] Figure 3B Shown are the effects of C9, C8 or C7 treatment on RSL3-induced α-synuclein aggregation in N27 cells.

[0023] Figure 4A and Figure 4B Shown are the effects of C9 dosing on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-inhibited vertical activity (total vertical counts and vertical time, respectively) in the open field motor test in C57BL / 6 mice.

[0024] Figure 5 The XRPD diffraction pattern of the as-received material (ID 1-1) analyzed by the long scan method, ie, Pattern A, is shown.

[0025] Figure 6 The original material (ID 1-1) in MeOD is shown. 1 HNMR spectrum.

[0026] Figure 7 A free-standing DSC thermogram of the as-received material (ID-1-1) is shown.

[0027] Figure 8 TGA and DCS thermograms of the as-received material (ID-1-1) are shown.

[0028] Fig. 9A (100 μm scale bar is shown in the lower right corner) and Fig. 9B (20 μm scale bar is indicated in the lower right corner) Microscopic images of the original material (ID-1-1) at 100x and 400x magnifications are shown, respectively.

[0029] Fig.10 The DVS isotherm plot of the as-received material (ID-1-1) is shown.

[0030] Fig.11 Shown is the XRPD diffractogram of the as-received solid (ID-1-1), Form A (bottom), compared to the XRPD diffractogram of the solid after a humidification cycle in a DVS instrument (top).

[0031] Fig.12 The HPLC chromatogram of the original material (ID-1-1) is shown.

[0032] Fig.13 The DSC thermogram of ID-10-1 after heat treatment of ID 1-1 is shown.

[0033] Fig.14 Shown is the XRPD diffractogram of the as-received material (ID-1-1), Form A (bottom), compared to the XRPD diffractogram of the solid recovered from heat treatment (ID 10-1) (top).

[0034] Fig.15 The HPLC chromatogram of stable sample ID-4-1 is shown.

[0035] Fig.16 Shown are XRPD diffraction patterns of as-received sample ID-1-1 (bottom) and solid form stable sample ID-4-1 (top) after one week at 75% RH and 40°C.

[0036] Fig.17A (100 μm scale bar is shown in the lower right corner) and Fig. 17B (20 μm scale bar is indicated in the lower right corner) Microscopic images of ID-4-1 at 100-fold and 400-fold magnifications are shown, respectively.

[0037] Fig.18 A microscopic image of ID-38-1 at 25x magnification is shown, with a 500 μm scale bar indicated in the lower right corner.

[0038] Fig.19 A microscopic image of ID-38-1 at 100-fold magnification is shown, with a 100 μm scale bar indicated in the lower right corner.

[0039] Fig. 20 A microscopic image of ID-38-1 at 400 times magnification is shown, with a 20 μm scale bar indicated in the lower right corner.

[0040] Fig.21 A microscopic image of ID-38-2 at 25x magnification is shown, with a 500 μm scale bar indicated in the lower right corner.

[0041] Fig. 22 A microscopic image of ID-38-2 at 100 times magnification is shown, with a 100 μm scale bar indicated in the lower right corner.

[0042] Fig.23A and Fig. 23B A microscopic image of ID-38-2 at 400 times magnification is shown, with a 20 μm scale bar indicated in the lower right corner.

[0043] Fig.24Shown are microscopic images comparing two batches ID-38-1 (top) and ID-38-2 (bottom) at 25X magnification, with a 500 μm scale bar indicated in the lower right corner.

[0044] Fig.25 Shown are microscopic images comparing two batches ID-38-1 (top) and ID-38-2 (bottom) at 100X magnification, with a 100 μm scale bar indicated in the lower right corner.

[0045] Fig.26 Shown are microscopic images comparing two batches ID-38-1 (top) and ID-38-2 (bottom) at 400X magnification, with a 20 μm scale bar indicated in the lower right corner.

[0046] Fig. 27 and Fig.28 Particle size distributions of representative experiments for Material Example 3A, Preparation Example 1, and Preparation Example 2 are shown, respectively.

[0047] Fig.29 and Fig.30 Particle size distributions of representative experiments for Material Example 3B, Preparation Example 1, and Preparation Example 2 are shown, respectively.

[0048] Fig.31 is the XRPD diffraction pattern of liquid C9 after cooling to room temperature for 5 minutes.

[0049] Fig.32 This is the DSC thermal analysis diagram of the temperature cycling experiment under low pressure conditions. DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention provides compounds, compositions and methods for treating or inhibiting α-synuclein disease, tau disease, ALS, traumatic brain injury and ischemia-reperfusion-related injury. α-Synuclein disease is a neurodegenerative disease characterized by abnormal aggregation of α-synuclein protein aggregates in neurons, nerve fibers or glial cells. Tau disease belongs to a class of neurodegenerative diseases related to the pathological aggregation of tau protein in neurofibrillary or glial fibrillary tangles in the human brain, such as Alzheimer's disease (see, e.g., Cellular and Molecular Neurobiology (2018) 38: 965–980). As shown in the examples, the claimed compounds have been shown to reduce the aggregates of α-synuclein and the effect of reducing the aggregates of tau protein. Without wishing to be bound by theory, it may be beneficial to allow the drug to penetrate into the brain for the claimed disease, and it may be beneficial to allow the drug to be preferentially distributed to the brain compared to other tissues. For example, this may reduce off-target and side effects. Applicants surprisingly found that the claimed compounds have superior brain penetration and superior ability of the compounds to partition into the brain compared to plasma.

[0051] The present invention further provides a solid form of compound 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione, a composition of the solid form comprising higher purity and preferred characteristics (e.g., more preferred particle morphology and particle size distribution), and a method for preparing the compound and composition. As shown in more detail in the detailed description of the present invention, the experimental part and the accompanying drawings provided by the present invention, the composition has beneficial properties, such as improved purity (e.g., lower silver content), improved processing characteristics (e.g., fluidity), and improved ability to be formulated into a drug (e.g., improved grinding ability). Compared with early processes, these particles have good fluidity and morphological characteristics. The use of the particles of the present invention facilitates the production of pharmaceutical products, such as capsule filling. In addition, the use of the particles of the present invention can reduce the amount of excipients required for the production of pharmaceutical products, which has advantages in terms of cost, time and process efficiency. In fact, if the drug is viscous or not easy to flow, more excipients may be needed to improve the handling of the drug. Likewise, if the drug needs to be ground, the sticky material will have a yield loss due to the loss of grinding equipment surface, and the ground product will form more or more difficult to break agglomerates. These characteristics are undesirable in the processing of drug production and are improved in the process described in the present invention.

[0052] Unless otherwise indicated, the abbreviations used herein have their conventional meanings in the fields of chemistry and biology.

[0053] Whenever the present invention refers to "about" or "approximately" a value or parameter, it includes (and describes) variations in the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0054] Unless otherwise indicated, the terms "about" and "approximately" used in the present invention when used in conjunction with various terms, such as the temperature, dose, amount, or weight percentage of an ingredient of a composition or dosage form, refer to, for example, a temperature, dose, amount, or weight percentage recognized by a person of ordinary skill in the art to provide the same effect as that obtained from the specified temperature, dose, amount, or weight percentage. Specifically, the terms "about" and "approximately", when used in the present invention, contemplate that the temperature, dose, amount, or weight percentage, etc., is within 15%, within 10%, within 5%, within 4%, within 3%, within 2%, within 1%, or within 0.5% of the specified temperature, dose, amount, or weight percentage, etc.

[0055] As used herein, the term "a" or "an" means one or more or one or more unless the context clearly indicates otherwise.

[0056] "Subject," "individual," or "patient" refers to an individual organism, preferably a vertebrate, more preferably a mammal, and most preferably a human.

[0057] "Treating" a disease using the compounds and methods discussed herein is defined as administering one or more compounds discussed herein, with or without additional therapeutic agents, to alleviate or eliminate the disease or one or more symptoms of the disease, or to slow the progression of the disease or one or more symptoms of the disease, or to reduce the severity of the disease or one or more symptoms of the disease. "Suppressing" a disease using the compounds and methods discussed herein is defined as administering one or more compounds discussed herein, with or without additional therapeutic agents, to suppress the clinical manifestations of the disease or suppress the manifestation of adverse symptoms of the disease. The difference between treatment and suppression is that treatment occurs after the adverse symptoms of the disease or condition of the subject appear, while suppression occurs before the adverse symptoms of the disease or condition of the subject appear. Suppression can be partial, substantially complete, or complete. In some embodiments, genetic screening can be used to identify patients at risk for a disease. The compounds and methods disclosed herein can then be administered to asymptomatic patients at risk of developing clinical symptoms of the disease, thereby suppressing the appearance of any adverse symptoms.

[0058] "Therapeutic use" of the compounds discussed herein is defined as the use of one or more compounds discussed herein to treat or inhibit a disease or condition as defined herein. A "therapeutically effective amount" of a compound is an amount of the compound that, when administered to a subject, is sufficient to reduce or eliminate a disease or one or more symptoms of a disease, or to slow the progression of a disease or one or more symptoms of a disease, or to reduce the severity of a disease or one or more symptoms of a disease, or to inhibit the clinical manifestations of a disease, or to inhibit the manifestations of adverse symptoms of a disease. A therapeutically effective amount may be taken in one or more administrations.

[0059] "2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione" and "C9" are used interchangeably in the present invention.

[0060] "2,3,5-trimethyl-6-octylcyclohexa-2,5-diene-1,4-dione" and "C8" are used interchangeably in the present invention.

[0061] "2,3,5-trimethyl-6-heptylcyclohexa-2,5-diene-1,4-dione" and "C7" are used interchangeably in the present invention.

[0062] "Hydroquinone form" means the form of the compound that results when two electron reduction of the quinone ring is achieved, providing a net conversion of two oxo groups to two hydroxyl groups. For example, the quinone compound The hydroquinone form is the following compound:

[0063] "Alpha synuclein" and "α-synuclein" are used interchangeably in the present invention.

[0064] In some embodiments, the description of the compounds of the invention also includes all isotopes, and in some embodiments, partially deuterated or fully deuterated analogs of all compounds of the invention.

[0065] Ischemia-reperfusion related injury includes, but is not limited to, stroke and ischemia-reperfusion related retinal injury.

[0066] "Stroke" includes ischemic stroke (non-limiting examples include thrombotic stroke, embolic stroke), hemorrhagic stroke (non-limiting examples include intracerebral hemorrhage, subarachnoid hemorrhage), and transient ischemic attack. In some embodiments, the stroke is an ischemic stroke. In some embodiments, the stroke is a hemorrhagic stroke. In some embodiments, the stroke is a transient ischemic attack.

[0067] For all characterization data described in the claims of the present invention (e.g., XRPD peaks, DSC, TGA, particle size distribution, etc.), in some embodiments, the data are obtained by a method substantially performed as described herein (e.g., for XRPD, DSC and TGA, see Example 7 for specific methodology). "Substantially as described herein" means that a person skilled in the art will use a method recognized by a person of ordinary skill in the art to provide results substantially equivalent to the results obtained from the specified method. Pharmaceutical preparations

[0068] For the claimed crystal form, residual solvent is within the allowed range, making it very suitable for being formulated into pharmaceutical compositions. Solid forms are also easy to purify by crystallization techniques. The claimed crystal form is neither hygroscopic nor hydrate / solvate, which means that it does not require special treatment in terms of humidity exposure. In addition, the improved morphology produced by the recrystallization process makes it easier to handle during production (for example, described in more detail in the present invention). The compound of the present invention can be formulated into pharmaceutical compositions by being formulated with additives such as pharmaceutically acceptable adjuvants, pharmaceutically acceptable carriers and pharmaceutically acceptable vehicles. Suitable pharmaceutically acceptable adjuvants, carriers and vehicles include processing agents and drug delivery modifiers and reinforcing agents, for example, in some embodiments, including calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low melting point waxes, ion exchange resins, etc., and any two or more combinations thereof. Other suitable pharmaceutically acceptable excipients are described in “Remington's Pharmaceutical Sciences,” Mack Pub. Co., New Jersey (1991), and “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, Philadelphia, 20th edition (2003) and 21st edition (2005), which are incorporated herein by reference.

[0069] The pharmaceutical composition may comprise a unit dosage formulation, wherein the unit dosage is a dose sufficient to have a therapeutic effect.

[0070] The pharmaceutical composition comprising the compounds of the present invention may be in any form suitable for the intended method of administration, including solutions, suspensions or emulsions in some embodiments. Liquid carriers are generally used to prepare solutions, suspensions and emulsions. In some embodiments, the liquid carriers intended for the implementation of the present invention include water, saline, pharmaceutically acceptable organic solvents, pharmaceutically acceptable oils or fats, etc., and mixtures of two or more thereof. The liquid carrier may include other suitable pharmaceutically acceptable additives, such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickeners, viscosity regulators, stabilizers, etc. In some embodiments, suitable organic solvents include monohydric alcohols such as ethanol, and polyhydric alcohols such as glycols. In some embodiments, suitable oils include sesame oil, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, etc. For parenteral administration, the carrier may also be an oily ester, such as ethyl oleate, isopropyl myristate, etc. The composition of the present invention may also be in the form of microparticles, microcapsules, liposome encapsulation, etc., and any combination of two or more thereof.

[0071] Time-release or controlled-release delivery systems may be used, such as diffusion-controlled matrix systems or erodible systems, as described in Lee, "Diffusion-Controlled Matrix Systems", pp. 155-198 and Ron and Langer, "Erodible Systems", pp. 199-224, and "Treatise on Controlled Drug Delivery", A. Kydonieus Ed., Marcel Dekker, Inc., New York 1992. In some embodiments, the matrix may be a biodegradable material that, in some embodiments, may be spontaneously degraded in situ and in vivo by hydrolysis or enzymatic cleavage (e.g., by proteases). In some embodiments, the delivery system may be a naturally occurring or synthetic polymer or copolymer, in some embodiments in the form of a hydrogel. Exemplary polymers with cleavable bonds include polyesters, polyorthoesters, polyanhydrides, polysaccharides, poly(phosphates), polyamides, polyurethanes, poly(imidocarbonates), and poly(phosphazenes).

[0072] The compounds of the present invention can be administered enterally, orally, parenterally, sublingually, by inhalation (e.g., in the form of mist or spray), rectum or topically, as needed, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. In some embodiments, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoresis, intravenous, intraarterial, intramuscular, intraperitoneal, intranasal (e.g., through nasal mucosa), subdural, rectum, gastrointestinal tract, etc., and directly applied to specific or affected organs or tissues. Preparations for topical administration may include lotions, tinctures, creams, emulsions, ointments, sprays, gels, etc., and may be further formulated into other suitable preparations, such as sunscreens, moisturizing lotions and creams, facial gels and creams, etc. In these compositions, the active ingredient is mixed with one or more inert excipients, including, for example, water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3 diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof. Topical administration may also involve the use of transdermal administration, such as transdermal patches or iontophoresis devices. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular and intrasternal injection or infusion techniques. The compound is mixed with a pharmaceutically acceptable carrier, adjuvant and vehicle suitable for the desired route of administration. Oral administration is a preferred route of administration, and preparations suitable for oral administration are preferred preparations. Topical administration is another preferred route of administration, and preparations suitable for topical administration are preferred preparations. The compounds of the present invention can be administered in solid form, liquid form, aerosol form, or can be administered in the form of tablets, pills, powder mixtures, capsules, granules, injections, creams, solutions, suppositories, enemas, colon lavages, emulsions, dispersants, food premixes and other suitable forms. The compounds can also be administered in the form of liposome preparations. Other methods of administration are all known in the art.

[0073] Compositions for topical administration may be emulsions or sterile solutions. As solvents or vehicles, propylene glycol, polyethylene glycol, vegetable oils, particularly olive oil, or injectable organic esters may be used, in some embodiments ethyl oleate. These compositions may also contain adjuvants, particularly wetting agents, isotonic agents, emulsifiers, dispersants and stabilizers. In some embodiments, a bacterial filter may be used, sterilized in several ways by radiation or by heating. It may also be prepared in the form of a sterile solid composition, which may be dissolved in sterile water or any other injectable sterile medium when used.

[0074] Injectable preparations can be prepared according to known techniques using suitable dispersants or wetting agents and suspending agents, and in some embodiments, sterile injectable aqueous or oily suspensions are prepared. Sterile injectable preparations can also be sterile injectable solutions or suspensions in nontoxic parenteral acceptable diluents or solvents, in some embodiments, solutions in propylene glycol. Available acceptable vehicles and solvents are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media. For this reason, any gentle fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, it has been found that fatty acids (e.g., oleic acid) can be used for the preparation of injections.

[0075] Solid dosage forms for oral administration may include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound may be mixed with at least one inert diluent (e.g., sucrose, lactose or starch). Such dosage forms may also include other substances in addition to the inert diluent, such as lubricants (e.g., magnesium stearate). For capsules, tablets and pills, the dosage form may also include buffers. Tablets and pills may also be made with enteric coatings.

[0076] Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, cyclodextrins, and sweeteners, flavoring agents, and aromatics.

[0077] The compounds of the present invention can also be administered in the form of liposomes. As known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by monolayer or multilayer hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipids capable of forming liposomes can be used. In addition to the compounds of the present invention, the compositions of the present invention in liposome form may also include stabilizers, preservatives, excipients, etc. Preferred lipids are natural and synthetic phospholipids and phosphatidylcholines (lecithins). Methods for forming liposomes are all known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, NW, p. 33 et seq. (1976).

[0078] The formulations of the present invention may contain two or more compounds or compositions described herein.

[0079] The invention also provides articles of manufacture and kits for use in any of the methods described herein comprising any one or more of the compounds of the invention.

[0080] The amount of active ingredient that can be combined with a carrier material to form a single dosage form will vary depending on the host and specific mode of administration of the active ingredient being administered. However, it should be understood that the specific dosage level of any particular individual will depend on a variety of factors, including the activity of the specific compound used, the patient's age, weight, body surface area, body mass index (BMI), overall health, sex and diet; administration time, route of administration, excretion rate, or combined medication; and the type, progression and severity of a specific disease or condition. The selected drug unit dose can be manufactured and administered to provide a determined final drug concentration in the target area of ​​the body. The therapeutically effective amount for a given case can be easily determined by routine experiments and is within the skills and judgment of an ordinary clinician.

[0081] Single or multiple doses that can be used include amounts independently selected from about 0.1 mg / kg to about 600 mg / kg body weight, or about 1.0 mg / kg to about 500 mg / kg body weight, or about 1.0 mg / kg to about 400 mg / kg body weight, or about 1.0 mg / kg to about 300 mg / kg body weight, or about 1.0 mg / kg to about 200 mg / kg body weight, or about 1.0 mg / kg to about 100 mg / kg body weight, or about 1.0 mg / kg to about 50 mg / kg body weight, or about 1.0 mg / kg to about 30 mg / kg body weight, or about 1.0 mg / kg to about 10 mg / kg body weight, or about 10 mg / kg to about 600 mg / kg body weight, or about 10 mg / kg to about 500 mg / kg body weight, or about 10 mg / kg to about 1500 mg / kg body weight. kg to about 400mg / kg body weight, or about 10mg / kg to about 300mg / kg body weight, or about 10mg / kg to about 200mg / kg body weight, or about 10mg / kg to about 100mg / kg body weight, or about 50mg / kg to about 150mg / kg body weight, or about 100mg / kg to about 200mg / kg body weight, or about 150mg / kg to about 250mg / kg body weight, or about 200mg / kg to about 300mg / kg body weight, or about 250mg / kg to about 350mg / kg body weight, or about 200mg / kg to about 400mg / kg body weight, or about 300mg / kg to about 400mg / kg body weight, or about 250mg / kg to about 300mg / kg body weight, or about 300mg / kg to about 300mg / kg body weight. The compounds of the present invention can be administered in a single daily dose, or the total daily dose can be administered in divided doses twice a day, three times a day, or four times a day.

[0082] A single dose or multiple doses may be administered. In some embodiments, the dose may be administered 1, 2, 3, 4, 5, or 6 times. In some embodiments, the dose is administered once a day, twice a day, three times a day, or four times a day. In some embodiments, the dose is administered every hour, every 2 hours, every 3 hours, every 4 hours, every 6 hours, every 12 hours, or every 24 hours.

[0083] Although the compounds of the invention may be administered as the sole active drug, they may also be used in combination with one or more other drugs for treating or inhibiting the diseases described herein. In some embodiments, the compounds of the invention are administered as the sole active drug present in a therapeutically effective amount.

[0084] When additional active agents are used in combination with the compounds of the present invention, the additional active agents are generally used in therapeutic amounts as indicated in Physicians' Desk Reference (PDR) 53rd Edition (1999), or in amounts known to be therapeutically useful by those of ordinary skill in the art.

[0085] The compounds of the present invention and other therapeutically active agents or preventive agents can be administered at the recommended maximum clinical dose or at a lower dose. The dosage level of the active compound in the composition of the present invention can be changed to obtain the desired response according to the route of administration, the severity of the disease and the individual response. When administered in combination with other therapeutic agents or preventive agents, the therapeutic agent or preventive agent can be formulated into a separate composition administered at the same time or at different times, or the therapeutic agent or preventive agent can be administered as a single composition. Preparation of the compounds of the present invention

[0086] In view of the disclosure provided by the present invention, the compounds of the present invention can be prepared from readily available starting materials using general methods and procedures that are obvious to those skilled in the art. It should be understood that, given typical or preferred process conditions (i.e., reaction temperature, time, reactant molar ratio, solvent, pressure, etc.), other process conditions may also be used unless otherwise specified. The optimal reaction conditions may vary with the specific reactants or solvents used, but such conditions may be determined by conventional optimization methods by those skilled in the art. The solution of C9 is light sensitive; ideally, indoor lighting should be filtered to filter out wavelengths <450nm (amber filters). If amber lighting is not used, appropriate controls should be used to minimize the solution exposed to light, for example, using aluminum foil packaging, amber glassware.

[0087] For all compounds and methods described herein, the quinone form may also be used in its reduced (hydroquinone) form when desired. Likewise, the hydroquinone form may also be used in its oxidized (quinone) form when desired. The reduced (hydroquinone) form may be readily converted to the oxidized (quinone) form using methods known in the art. See, e.g., Air, Silica, PCT International Application No. 2006130775, filed December 7, 2006, by Miller et al. The oxidized (quinone) form may be readily converted to the reduced hydroquinone form using methods known in the art. See, e.g., Zn, AcOH, Fuchs et al EJOC 6 (2009) 833-40.

[0088] The invention is further described by the following non-limiting examples and embodiments.

[0089] Recrystallization. As shown in Examples 1A and 3A, the synthetic method used to prepare C9 results in a product having one or more undesirable characteristics, such as high silver content, stickiness of the product, and undesirable particle size and distribution. In general, for pharmaceutical uses, product solids having a narrow particle size distribution are preferred. The stickiness of the product shown in Example 3A makes the product difficult to handle, including making it difficult to grind and impossible to sieve.

[0090] Recrystallization can produce improved properties, for example, removing impurities from the product solid and generating a material with a more uniform size and distribution to improve product performance in subsequent formulation processes. As shown in the examples, a recrystallization method has been found that improves the quality of the product solid, including, for example, increased purity, higher melting point, lower silver content, better fluidity, lower viscosity, and more uniform particle size and distribution. In addition, the recrystallization method does not require seed crystals and does not have the problem of oiling out of the C9.

[0091] The recrystallization procedure generally comprises dissolving C9 in a solvent, heating the mixture to about 40-45°C to dissolve the C9, cooling the mixture to about 32°C to allow crystallization to occur, and filtering the product.

[0092] In some embodiments, the recrystallization solvent is about 75-85% IPA / water. In some embodiments, the recrystallization solvent is about 80-85% IPA / water. In some embodiments, the recrystallization solvent is about 80-87% IPA / water. In some embodiments, the recrystallization solvent is about 83-87% IPA / water. In some embodiments, the recrystallization solvent is about 85% IPA / water. In some embodiments, the ratio is determined as (v:v). In some embodiments, the ratio is determined as (weight:weight). In some embodiments, the recrystallization solvent is a combination of methanol and water. In some embodiments, the recrystallization solvent is a combination of methanol and heptane.

[0093] When the recrystallization solvent is IPA / water, in some embodiments, the method comprises dissolving the C9 in IPA (e.g., by heating the mixture to about 40-45° C.) and then adding water. After adding water, the temperature of the resulting mixture can be restored to about 40-45° C. In other embodiments, the method comprises dissolving the C9 in a mixture of IPA / water.

[0094] In some embodiments, the mixture containing dissolved C9 is subjected to polishing filtration. The polishing filtration can be performed at about 40-45°C or while maintaining the C9 solution at a desired temperature.

[0095] In some embodiments, the mixture is cooled to about 32° C. within a few hours, such as within about 2-10 hours, or about 4-8 hours, or about 6 hours. In some embodiments, the mixture is then maintained at about 32° C. for several hours to allow the C9 to crystallize, such as for about 2-24 hours, or about 4-8 hours, or about 6 hours.

[0096] In some embodiments, the mixture is then further cooled. In some embodiments, the temperature can be cooled to about -5°C to about 5°C, or about 0°C. The cooling can be performed in a single step or in multiple steps (e.g., cooling to about 24°C, then cooling to about 16°C, then cooling to about 0°C). In some embodiments, the mixture is cooled over a period of about 3-24 hours. In some embodiments, the mixture is maintained at about 0°C, for example, for at least 1 hour or about 1 hour.

[0097] The recrystallization procedure allows the purity of the C9 product to be increased. In various embodiments, the product comprises at least about 95% a / a, or at least about 96% a / a, or at least about 97% a / a, or at least about 98% a / a, or at least about 99% a / a, or at least about 99.5% a / a of the C9, as determined by HPLC, without any solvent, carrier or excipient.

[0098] The procedure can also produce a high purity of the claimed polymorph. In various embodiments, at least about 95 mol%, or at least about 96 mol%, or at least about 97 mol%, or at least about 98 mol%, or at least about 99 mol%, or at least about 99.5 mol% of the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione is a polymorph, free of any solvent, carrier or excipient.

[0099] The procedure can also result in a high titer (or content) of C9. Potency (or content) = (100% - total impurities determined by HPLC) x (100% - water content % - total residual solvent % - ignition residue %). The potency (or content) can be calculated as follows: (area purity % determined by HPLC / 100) * (100 - % wt / wt water content (KF) - % wt / wt residual solvent - % wt / wt = ignition residue (ROI)). In various embodiments, the potency (or content) of the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5%.

[0100] The procedure may also result in a narrower distribution of particle sizes. D10 represents the particle diameter corresponding to a 10% cumulative (from 0 to 100%) undersize particle size distribution (i.e., the percentage of particles less than D10 is 10%). D90 represents the particle diameter corresponding to a 90% cumulative (from 0 to 100%) undersize particle size distribution (i.e., the percentage of particles less than D90 is 90%). In some embodiments, the D90:D10 ratio of the particles is less than about 11:1. In some embodiments, the D90:D10 ratio of the particles is less than about 10:1. In some embodiments, the D90:D10 ratio of the particles is less than about 9:1. In some embodiments, the D90:D10 ratio of the particles is less than about 8:1. In some embodiments, the D90:D10 ratio of the particles is less than about 7:1. In some embodiments, the D90:D10 ratio of the particles is less than about 6:1. In some embodiments, the particles have a D90:D10 ratio of less than about 5: 1. In some embodiments, the particles have a D90:D10 ratio of less than about 4:1. Example Example 1A. Synthesis of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione (3)

[0101] At 23°C, 2,3,5-trimethylbenzene-1,4-diol (1) (1.39 kg, 9.1 mol) and ether (15 L) were added to a 50 L reactor equipped with a thermometer and a stirrer. The mixture was stirred for 30 minutes and became a clear solution. A solution of ferric chloride (5.6 kg, 34.5 mol) in water (20 L) was added dropwise over 3 hours. The reaction mixture was stirred at this temperature for another 2 hours. The organic phase was separated. The drained aqueous layer was extracted with ether (3x5 L). The combined organic phases were dried over sodium sulfate and concentrated. The residue was diluted with dichloromethane (DCM) (1 L) and then purified by silica gel chromatography (one column) to obtain the desired product 2 (1.27 kg, 95%). TLC (petroleum ether (PE) / ethyl acetate (EA) = 30 / 1). f (Compound 1) = 0.2. f (Product 2) = 0.6.

[0102] 2,3,5-Trimethyl-[1,4]benzoquinone (2) (780 g, 5.2 mol, 1.0 equiv), decanoic acid (895 g, 5.2 mol, 1.0 equiv) and acetonitrile (15 L) were added to a 50 L reactor equipped with a thermometer and a stirrer. The mixture was stirred at room temperature for 30 minutes and became a clear solution. Silver nitrate (882 g, 5.2 mol, 1.0 equiv) was added in one portion. The reaction mixture was heated to 75 °C. A solution of potassium persulfate (1.54 kg, 5.7 mol, 1.1 equiv) in water (30 L) was added dropwise over 2 hours. After the addition was complete, the reaction was stirred at 75 °C for another 3 hours. The reaction solution was cooled to room temperature. The aqueous layer was drained into 15 L of water and extracted with ethyl acetate (3 x 5 L). The combined organic phases were dried over sodium sulfate and filtered. The filtrate was concentrated to give a yellow residue, which was crystallized from hot methanol (800 mL). The solid was filtered, washed with a small amount of methanol and ether, and dried in vacuo to give 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione (3) (447 g) as a yellow crystalline agglomerate. The filtrate was concentrated and the residue was purified by flash column chromatography (petroleum ether:ethyl acetate, 100:1) to give another 155 g of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione (3) (total amount: 602 g, 42%). TLC (petroleum ether / ethyl acetate (PE / EA) = 30 / 1). f (Compound 2) = 0.5. f (Product 3) = 0.6. 1 HNMR (400 MHz, CDCl3) δ 2.46 (s, 2H), 2.02-2.01 (m, 9H), 1.35-1.26 (m, 22H), 0.88-0.86 (m, 5H). Ag = 45 ppm. Melting point 49.9.

[0103] The product had a high silver content and was a heterogeneous mixture containing fine particles and bulky compounds due to the combination of two separations, one from the MeOH trituration method and the other from column chromatography separation. Example 1B. Recrystallization of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione (C9)

[0104] Overview. The product was prepared by recrystallization from 2-propanol (IPA) / water (H2O) after NaCl pretreatment, followed by digestion, collection, and air and vacuum drying. The starting material (C9 prepared in Example 1A, 99.9 g) had a high Ag content (45 ppm), so the methyl tert-butyl ether (MTBE) solution of the C9 compound was brine washed and twice washed with water before drying and recrystallization. The MTBE solution of C9 was filtered through a 2.7 μm filter to remove any particulates present after the sodium chloride (NaCl) wash. During the solvent exchange from MTBE to the recrystallization solvent IPA, brown solids formed in the solution and required hot filtration to remove. The tan solids were probably due to the exposure of the quinone solution and were not further analyzed. The filtered bright yellow solution was then protected from light, concentrated, and the residue was dissolved in 85% IPA / H2O (486 g / 98 g) at 40°C. The heat was removed and crystals formed at 35°C. The slurry was stirred at 16°C for 48 hours, cooled to 0°C, and the solids were collected, washed, and dried in air and vacuum (-125 mmHg) to constant weight to give 82.1 g (82.1%) of fine yellow needles. The material was analyzed by NMR, UPLC, LCMS, IR, and MP.

[0105] Ag was removed by washing with NaCl. Crude C9 (99.9 g, prepared according to Example 1A) as a yellow agglomerate of a mixture of powder and crystals was dissolved in 500 mL of methyl tert-butyl ether (MTBE) and the cloudy yellow liquid with small (1-2 mm) black spots was washed with brine (100 mL, 20 wt% NaCl in H2O). The cloudy yellow organic upper layer was retained and the rag layer and clear colorless aqueous phase were discarded. The organic phase was washed with water (2 x 100 mL) and dried over anhydrous sodium sulfate (Na2SO4, 35 g). The clear yellow solution was filtered through a 55 mm Whatman Type 3 filter (6 μm) stacked on top of a 55 mm Whatman GF / D filter (2.7 μm) and the container was rinsed with MTBE (2 x 20 mL). IPA (100 mL) was added and the mixture was concentrated by rotary evaporator (125-90 mmHg, 40-25°C bath) to give a bright yellow slurry (180 g). IPA (348 g) was added and the slurry was heated to clear (35°C, 120 mmHg) for 20 minutes, then the pressure was reduced to 60 mmHg and the volume was reduced until crystals began to form (~45 min). There was no odor of MTBE in the resulting slurry. The slurry was concentrated at 35°C to a weight of 182 g, IPA (404 g) was added, heated to 40°C until dissolved, and then concentrated to give a yellow solid (35 mmHg). IPA (500 mL) was added and the mixture was stirred overnight.

[0106] Filter to remove tan solids. Brown spots were found in the IPA solution, which were removed by hot filtration (~40°C) through stacked 55 mm Whatman Type 3 (8 μm) and Whatman GF / D (2.7 μm) filters. A sticky tan residue remained on the filter, and the clear bright yellow solution was then concentrated by rotary evaporation to give a yellow solid.

[0107] Recrystallization. To the resulting yellow solid was added IPA (486 g), the vessel was heated to 40° C., and water (98 g) was added to the clear yellow solution. The heat was reduced to 21° C. over 2 hours to give a bright yellow slurry containing needles. The slurry was stirred at 16° C. for 48 hours, cooled to 0° C., and the viscous yellow slurry was filtered (Whatman #54, 150 mm Buchner). The thin yellow needles were rinsed with ice-cold 85% IPA / H2O (2×250 mL, 0° C.) and IPA (100 mL, 0° C.). The solid was pumped for 1 h, a nitrile dam was installed on a Buchner funnel, and placed under house vacuum (~100 mmHg) overnight. The yellow crystals (88.9 g) still had an IPA odor, so they were transferred to a new Buchner funnel / filter (the funnel had been clogged with C9 residue) and kept constant weight (82.1 g, 82.1%) under vacuum (100 mmHg, 6 h). The resulting crystals were transferred to a 500 mL amber Type I Schott-Duran bottle for storage at room temperature. Approximately 280 mL crystal volume (82.1 g).

[0108] CoA analysis was performed using a qualified method: water content (KF) = 0.05%; solvent content = 0.23%; HPLC purity = 100% AUC; melting point determined by DSC = 53.0°C; Ag content <2.00ppm; ignition residue = 0.02%; potency (or content) (calculated value) = 99.71%, which is consistent with type A.

[0109] The potency (or content) was calculated as follows: (area purity % / 100 determined by HPLC)*(100 - %wt / wt water content (KF) - %wt / wt residual solvent - %wt / wt = residue on ignition (ROI)). Example 2. Recrystallization screening

[0110] As shown in Examples 1A and 3A, the synthetic methods used to prepare C9 result in products having one or more undesirable properties, such as high silver content, stickiness of the product, and undesirable particle size and distribution. As described above, recrystallization can improve the properties of the product. Therefore, a variety of solvents were screened to make them suitable for recrystallization of C9.

[0111] 100 mg of C9 was placed in a test tube, solvent was added, and any changes were recorded. The sample was then briefly heated with a heat gun (to about 45-50°C), the solubility was recorded, it was allowed to cool to RT and the solubility was recorded, and then it was cooled to 0°C and the solubility was recorded. The results are shown in the table below. Table 1. Solvent screening for recrystallization

[0112] The following solvents were discarded: MeCN, THF, as C9 was too soluble at cold temperatures (cryo) and therefore unsuitable for use.

[0113] The following solvents were discarded: water was not suitable for use because C9 did not dissolve sufficiently at higher temperatures.

[0114] The following solvents were discarded: 80% EtOH because the C9 material melted before dissolving and was therefore unsuitable for use. This is unfavorable because it will form an oil suspended in the solution. Upon cooling, it may crystallize, but the crystals are usually not uniform or provide an increase in purity.

[0115] 95% EtOH was discarded because it contained MeOH and acetone, which can be problematic for the pharmaceutical industry and is therefore not suitable for use.

[0116] Methanol and ethanol are less preferred due to their low boiling points. In addition, MeOH, which precipitates long needles, is not suitable because long needle-shaped crystals are more difficult to transfer and filter than more dense particles.

[0117] It was noted that ethanol produced an almost completely soluble material when heated, but a partially soluble material when cold. IPA also exhibited similar behavior, but was soluble at elevated temperatures at 100 mg / mL and partially soluble at RT / cold. Ethanol / water mixtures were initially tested for solubility, but were abandoned due to oiling of the C9 compound at >40°C. IPA was tested after ethanol proved unsuitable due to its slightly better solubility, not oiling out at higher temperatures before being completely dissolved, and IPA being miscible with a good antisolvent (H2O). 70% IPA / H2O resulted in melting issues and oiling out, while 100% IPA was not ideally insoluble at cold temperatures. Therefore, IPA concentrations between 70-100% were tested.

[0118] Test solutions on a 1 g scale were assayed and showed good solubility (~10:1 vol:wt) for 75-85% IPA / water, fine filterable crystals, and a solution that could be heated and cooled to produce predictable melts / crystallizations. Seeding did not appear to be necessary. Hot filtration of the brown material formed during crystallization was required when the solution was exposed to room temperature for >1 h. This was only observed when the solution was exposed to light. Recovery was 92.7% fine needles, and UHPLC analysis of the supernatant was 91% a / a, with a solids content of 99%, indicating that the supernatant was free of impurities. Fine yellow needles were obtained that were easily filtered and cleaned with high recovery. UHPLC showed an improvement in UHPLC area %, and the crystals were all similar in size and flowed easily after drying. Example 3A. Synthesis of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione

[0119] A solution of compound 1 (2.0 kg, 13.15 mol, 1.0 equivalent) in dichloromethane (20 L) was stirred at 23 ° C for 30 minutes. A solution of ferric chloride (5.33 kg, 32.88 mol, 2.5 equivalent) in water (19.04 L) was added dropwise over 22 hours. The reaction mixture was stirred for another 2 hours at this temperature. HPLC showed that compound 1 was completely exhausted. The organic phase was separated. The drained water layer was extracted with dichloromethane (2 x 10 L). The combined organic phase was washed with water (2 x 20 L) and brine (2 x 10 L), dried over sodium sulfate (about 5 kg), and concentrated to give crude compound 2 (1.95 kg, crude product), which can be directly used in the next step without further purification.

[0120] A mixture of acetonitrile (25 L) of compound 2 (1.25 kg, 8.33 mol, 1.0 equivalent) and capric acid (1.44 kg, 8.33 mol, 1.0 equivalent) was stirred at room temperature for 30 minutes. Silver nitrate (353.9 g, 2.08 mol, 0.25 equivalent) was added at one time. The reaction mixture was heated to 75 ° C. A solution of potassium persulfate (2.48 kg, 9.17 mol, 1.1 equivalent) in water (100 L) was added dropwise. After the addition, the reaction was stirred at 75 ° C for another 3.5 hours. HPLC showed that compound 2 was completely exhausted. The resulting solution was cooled to room temperature. The water layer was drained into 50 L of water, which was extracted with ethyl acetate (3x10 L). The combined organic phase was washed with sodium chloride aqueous solution (5 L), dried over sodium sulfate (about 5 kg), and filtered. The filtrate was concentrated. The residue was purified by silica gel column chromatography (PE, PE / EA, 50 / 1) to obtain a crude product. After flash column chromatography (FCC) (solidified after standing), an oily product was obtained. Before solidification, it was quickly loaded into a three-necked flask. The oil was stirred and a solid was formed under stirring. The solid was ground with methanol (2 times the volume, added dropwise as the crude product after column purification) at room temperature (20-30°C) overnight (~15 hours) and then filtered. The resulting filter cake was washed with methanol (500 mL) and vacuum dried: the solid was loaded into a 20L rotary evaporator with a vacuum pump (0.5 mmHg) in a water bath (<30°C) for 7 days, 8 hours / day, to remove residual solvents: acetonitrile, methanol, dichloromethane and ethyl acetate, thereby obtaining C9 (1.28 kg, 56%) as yellow crystals. The product was spread in a stainless steel container and crushed in a mortar to obtain similar sizes. The grinding process was repeated several times. An attempt was made to sieve the solid. However, the solid could not be sieved because the product stuck to the sieve (100 mesh). Since grinding of this material is difficult, the resulting particles are large and difficult to formulate.

[0121] TLC (PE / EA=30 / 1). f (Compound 2) = 0.5. f (C9) = 0.8. LC-MS: n / a.

[0122] 1 H NMR (400MHz, CDCl3) δ2.45-2.42 (t, J = 7.4Hz, 2H), 1.99-1.94 (m, 9H), 1.24 (m, 16H).

[0123] Assay = HPLC 92.2% w / w relative to reference standard. Example 3B. Recrystallization of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione

[0124] Overview. This example summarizes the recrystallization of C9 for a 10 g engineering batch and subsequently 2.4 kg of C9 (both samples are provided by Example 3A). The material was dissolved in 2-propanol at 40°C and water was then added to give a turbid solution, which was then polished and filtered through a jacketed filter at 40°C. The resulting clear solution was slowly cooled to 32°C, at which temperature the product spontaneously crystallized as fine yellow needles. Further gradual cooling to 16°C and then to 0°C gave a slurry, which was collected by filtration and then rinsed with an 85% 2-propanol solution in water. The resulting solid was dried under vacuum at 25°C to constant weight to give the product (1.975 kg) with a recovery of 82%. The isolated solid was tested and analyzed by DSC, IR and 1 The results were characterized by H NMR.

[0125] discuss

[0126] The advantages of the process described in this example are: controlled formation of a true crystalline solid without oiling; no seeding required; increased assay / purity; no stickiness, good flowability; narrow particle size distribution; and regular shaped particles that are easy to separate and dry. Grinding of this material should be simple if necessary.

[0127] LCMS analysis of the provided C9 (prepared according to Example 3A, 92.2% purity) showed a major impurity, which was mainly manifested as a total ion count (TIC) that was not visible under UV254 or in an evaporative light scattering detector (ELSD). In the case of a high recovery of the impurity in the mother liquor, the impurity would be effectively removed from the recrystallized product.

[0128] The implemented cooling scheme produced crystals without oiling or the need for seeding, with crystallization occurring at 32°C.

[0129] Final product separation provided a wet cake which was transferred to a drying oven at 25°C without the use of a nitrile dam.

[0130] Recrystallization (2.4kg)

[0131] C9 (2400 g) and 2-propanol (15.6 L, Fisher A416) were added to a 50-liter jacketed reactor equipped with an overhead mechanical stirrer, an argon inlet, and a Teflon-coated temperature probe. The mixture was stirred at 75 rpm under argon, and the reactor was covered with aluminum foil. The reactor jacket was heated to 41° C. (internal temperature was 40° C.) and stirred for 60 minutes to form a clear solution.

[0132] Deionized water (2.5 L, Ricca 9150-5) was added to the reaction and the solution was stirred for 90 minutes. The solution was polish filtered through a P4 (10-16 um) sintered glass jacketed funnel at 40°C using positive argon pressure (8 psi) and collected in a 20 L glass carboy.

[0133] The 50 L reactor was rinsed with 2-propanol (2 L, Fisher A416) and drained to organic waste.

[0134] The clear supernatant (36°C) was re-placed into a 50-liter jacketed reactor equipped with an overhead mechanical stirrer, argon inlet, and Teflon-coated temperature probe and heated to 40°C. The solution was stirred at 75 rpm and cooled to 32°C over 6 hours and then held at 32°C for an additional 6 hours. Crystals had formed. The slurry was cooled to 24°C for 1 hour, then further cooled to 16°C for 1 hour, then cooled to 0°C for 1 hour.

[0135] Using positive argon pressure (8psi), solid is collected on P4 (10-16um) sintered glass funnel. Filter cake is rinsed with 2-propanol / water (85 / 15v / v, 2x 6L), and dried for 2 hours under argon stream to obtain 3450g yellow solid. Solid is transferred in vacuum oven, vacuum dried for 240 hours at 25 ℃. Weight is checked at intervals of about 24 hours, until constant weight is reached, to provide final product C9 (1975g, 82.2% yield) as a free-flowing yellow crystalline solid. DSC analysis of solid shows that it starts to melt at 48.66 ℃, and melting point is 50.13 ℃. HPLC analysis shows 99.4% a / a purity.

[0136] The mother liquor was concentrated under reduced pressure to obtain 400 g of a red oil. Analysis of the oil showed that there were still some products, and its main impurity peak was characterized by TIC, which did not seem to have an obvious absorbance at 254 nm. The molecular weight of the impurity seemed to be 373 (M+H=374 amu (atomic mass unit)).

[0137] CoA analysis was performed using a qualified method: water content (KF) = 0.06%; solvent content = 0.24%; HPLC purity = 100% AUC; melting point determined by DSC = 53.0°C; Ag content <2.00ppm; ignition residue = <0.01%; potency (or content) (calculated value) = 99.69%, and the crystal form is consistent with Type A. Example 4. Polymorphs of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione

[0138] Overview

[0139] The following table gives a summary of the original Form A and its melts. *BDL: below detection limit.

[0140] Characterization

[0141] The raw solid (ID 1-1 prepared according to Examples 1A and 1B) was a slightly tacky yellow powder. The container was stored in a refrigerator at 5°C. Because the material was light sensitive, an amber or foil-wrapped vial was used to protect the container and all sample bottles from light. XRPD analysis of the material (ID 1-1) showed that the material was crystalline and had high intensity peaks at 4, 12, and 16° 2θ, as well as several other less intense peaks; this crystalline form was designated Form A ( Figure 5 ). Table 2 shows a list of peaks with d spacing and intensities. Table 2. XRPD peak list of material (ID 1-1), peaks with relative intensity > 5 are reported. * The diffraction patterns in Table 2 were obtained using a CuKα1 source with a wavelength of and RigakuMiniFlex at 23-25℃ 600 obtained.

[0142] The material (ID 1-1) was dissolved in MeOD and 1 H NMR analysis ( Figure 6 ). The NMR spectrum is consistent with the structure of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione. It is also consistent with being anhydrous and not a solvate.

[0143] The DSC of material (ID 1-1) showed that it started to melt at 48.70°C ( Figure 7). Meanwhile, TGA / DSC showed a melting onset temperature consistent with that of DSC, and the associated weight loss step was 0.26% ( Figure 8 ). Table 3 shows the summary of DSC data including details of the method etc. Table 3. Peak list of DSC thermogram of material (ID 1-1)* Starting temperature (℃) Peak(℃) Normalized enthalpy (J / g) 48.70 50.45 -144.12 *DSC thermal analysis was performed using a Mettler Toledo DSC3+ in a sealed Al pan with a lid and pinhole at 10°C / min, 60mL / min N2 and ramp heating from 25-250℃.

[0144] 100 times ( Fig. 9A ) and 400 times ( Fig. 9B ) captured a microscopic image of the as-received material (ID 1-1) at a magnification of ). The material appears to have a rectangular, plate-like morphology.

[0145] Karl Fischer (KF) titration was performed on two samples of the original material (ID 1-1), the first sample was 24 mg and the second sample was 43 mg; however, no titrator results were provided for either sample. This indicates that the water content of the material is below the detection limit of the instrument (>1 ppm). This result is consistent with anhydrous material (ppm).

[0146] The original material (ID 1-1) was subjected to a humidification cycle by a dynamic vapor sorption (DVS) instrument. The mass of the material changed by 0.01% in the relative humidity range of 15% to 75%, and by 0.08% in the entire relative humidity range of 2% to 95%. The isotherm plot is shown in Fig.10 After the humidification cycle, the solid was analyzed by XRPD. The observed pattern was the same as that of the original solid (ID 1-1) (Type A); the XRPD data are shown in Fig.11 These results indicate that the material is not hygroscopic.

[0147] A sample of the original solid was dissolved to a concentration of 0.5 mg / mL and injected by HPLC for purity analysis. No impurities were observed. The chromatogram is shown in Fig.12 shown.

[0148] Heat Treatment

[0149] 15 mg of the original material (ID-1-1) was weighed and placed in a DSC pan and heat treated on a stand-alone DSC instrument by heating to 60°C at a rate of 10°C / min and then cooling to 10°C at a rate of 2°C / min. The resulting material was recovered from the pan and plated for XRPD analysis (ID-10-1). The material began to melt at 48.74°C and began to release heat at 22.58°C for recrystallization. The recovered material was shown to be Type A by XRPD. The thermal analysis diagram of the DSC treatment is shown in Figure 1. Fig.13 XRPD data are shown in Fig.14 shown.

[0150] Hot stage microscopy

[0151] The Linkam hot stage system was used to capture images of the original solid (ID-1-1) during melting. A small amount of material was placed on a microscope slide inside the hot stage, and the temperature ramp method was used to increase from 30°C to 55°C at a rate of 1°C / min. A series of images were captured at a magnification of 200 times during the ramp temperature rise. No morphological changes were observed until melting (data not shown). After melting, the hot stage was naturally cooled to room temperature, and the recrystallization of the material was monitored at a magnification of 200 times, but no solid was observed. The sample appears to be glassy. The material was treated with No. 21 in an attempt to nucleate its crystals. The slide was then removed from the hot stage and the image was captured at a magnification of 200 times. The resulting material does not seem to change (data not shown). This shows that the compound can exist in a molten liquid form (amorphous) at room temperature.

[0152] Solid form stability

[0153] The solid form stability of the material was evaluated over a week. 49.0 mg of ID-1-1 was placed in a 4 mL vial wrapped with foil and covered with a KimWipe. This vial was placed in a 20 mL vial containing a saturated sodium chloride aqueous solution. The vial was placed on a hot plate at 40° C. for 7 days, forming an atmosphere of 75% relative humidity in the system. After 7 days, the solid was sampled for HPLC analysis (ID-4-1).

[0154] The resulting chromatogram did not show any impurities in the stability sample. Fig.15 A sample of the stressed material was plated and analyzed by XRPD; the pattern observed was Type A. The XRPD data is shown in Fig.16 As shown. Microscopic images of ID-4-1 were captured at 100x and 400x magnifications, respectively. Fig.17A (100 times) and Fig. 17B (400 times) as shown.

[0155] The solid sample was heated at 55°C until melted, then cooled to room temperature for 5 minutes. The XRPD diffraction pattern showed that it was amorphous ( Fig.31 ). From the temperature cycle DSC experiment, it can be seen that at 20°C, the melt finally solidifies into type A ( Fig.32 ). This suggests that the amorphous liquid form is metastable and transforms to form A.

[0156] Solubility in simulated fluids and water

[0157] The solubility of Form A and molten solid was evaluated in aqueous solutions of fasted simulated gastric fluid (FaSSGF), fed simulated intestinal fluid (FeSSIF) and fasted simulated intestinal fluid (FaSSIF), water, and 0.5% methylcellulose + 2% Tween 80. Five 4 mL vials wrapped in foil were prepared with 11-13 mg of the original material (ID-1-1), including a 10 mm stirring bar. Five 4 mL vials wrapped in foil (containing 11-13 mg of C9) were melted on a hot plate at 70°C for 10 minutes and then cooled to room temperature for 5 minutes, including a 10 mm stirring bar.

[0158] Slurries were prepared using 2.5 mL of simulated fluid (including 0.5% methylcellulose + 2% Tween 80 in water) or raw material ID-1-1 (Type A) and melted C9 in water, respectively. Samples containing melted active pharmaceutical ingredients (API) were briefly sonicated because they appeared to contain lumpy materials. pH and solubility were evaluated at 30 minutes and 24 hours, respectively. For solubility analysis, 1 mL of each sample was pipetted into a syringe filter, the first 0.5 mL was filtered back into the source sample bottle, and the remaining 0.5 mL was filtered into an HPLC vial with a small capacity insert.

[0159] The response factors calculated from the calibration points were used to determine the concentration of the API in the simulated fluid and water. Calibration samples were prepared using virgin material (ID-1-1) in ACN. The concentration and peak area of ​​each calibration point were plotted and the response factor was calculated for solubility assessment.

[0160] The experimental design and resulting data are shown in Table 5. The remainder of each slurry was filtered and plated for XRPD analysis. All patterns observed were Type A (data not shown). Table 5. Experimental design and resulting solubility data in simulated fluids and water *BDL:Below Detection Limit #Using Phenomenex LunaPhenyl Hexyl HPLC column and improved method.

[0161] Due to the high pressure observed on the UPLC column, a different column and modified method were used to collect 24-hour data points for 0.5% methylcellulose + 2% Tween 80 in water, 24 hours of water (ID-30-4, ID-30-9 and ID-30-10). The HPLC samples of ID-30-4 and ID-30-9 (which both contained methylcellulose / Tween 80 mixtures) were diluted 10 times with ACN. These samples and a new set of calibration point injections were analyzed, new response factors were determined, and the solubility of the remaining samples was calculated (response factor: 8084.7908; R2: 0.9989). Example 5. Crystalline morphology of C9 of Example 3A and Example 3B

[0162] Overview

[0163] The material from the batches described in Examples 3A and 3B were both yellow crystalline solids visible to the naked eye.

[0164] For each batch, multiple microscopic images were taken at 3 different magnifications (25x, 10x, 400x) to avoid bias in capturing the images. The first batch ID-38-1 (Example 3B) showed mostly larger particles, ranging from about 100 μm to over 600 μm, by microscopic observation. Overall, the particles were regular, rectangular, and slightly planar in shape, and exhibited good birefringence.

[0165] The second batch, ID-38-2 (Example 3A), showed significantly smaller particles overall compared to the first batch. The observed particle range was between about 50-350 μm, and the particles tended to be more granular or irregular in shape. Unlike the first batch, where individual particles were consistently observed, the smaller particles in the second batch also showed significant agglomeration of the larger particles.

[0166] result

[0167] Microscopic examination was performed on ID-38-1 (Example 3B) at 3 different magnifications (25X, 100X, and 400X). Microscopic examination showed that this particular batch of C9 had a wide range of particle sizes.

[0168] exist Fig.18 At 25x magnification, variations in particle size and shape can be seen. Rectangular plates are the main objects observed; however, there are also some jagged two-dimensional shapes. At 100x magnification, Fig.19As shown, the length range is about 100 μm–600 μm; birefringence is observed. At 400× magnification, the particles are too large to fit within the frame, but the smooth rectangular birefringent shape is still well observed ( Fig. 20 ).

[0169] Microscopic examination was performed on ID-38-2 (Example 3A) at 3 different magnifications (25X, 100X, and 400X). Compared to ID-38-1 (Example 3B), this particular batch of C9 showed overall smaller particles, lower birefringence, and smaller agglomerated particles "stuck" to some of the larger particles observed.

[0170] exist Fig.21 In the 25x magnification, small agglomerated particles were observed. At 100x magnification, Fig. 22 As shown in Figure 2, granular particles become more visible, ranging from about 50 μm to 350 μm. Several smaller agglomerated particles are also observed. At 400x magnification, as shown in Figure 2, Fig.23A and 23B As shown, unlike the case of ID-38-1 (Example 3B), the particles were observed within the frame. Semi-transparent particles were observed to be spherical, and birefringence was also observed for many of the particles. Multiple images were captured at this magnification to demonstrate the large range of particle sizes observed, and smaller particles were observed agglomerated on some larger solids.

[0171] Batch comparison

[0172] A direct comparison of ID-38-1 (Example 3B) and ID-38-2 (Example 3A) is given below. At 25x magnification, the morphological and size differences between the two batches are quickly observed, such as Fig.24 As shown. Fig.25 At 100x magnification, more individual particles were observed for ID-38-1 compared to ID-38-02, while agglomeration of small particles onto larger particles was observed in ID-38-02. Fig.26 As shown, for ID-38-1 (Example 3B), the particles were too large to fit within the frame, but a smooth plate-like morphology was clearly observed, while for ID-38-2 (Example 3A), agglomeration of small particles onto larger solids was again observed.

[0173] Microscopic examination

[0174] Optical microscopy was performed using a Zeiss AxioScope A1 digital imaging microscope equipped with 2.5x, 10x, 20x, and 40x objectives and a polarizer. Images were captured by a built-in Axiocam 105 digital camera and processed using ZEN2 (blue version) software provided by Zeiss. Example 6. Particle size distribution of C9 of Example 3A and Example 3B

[0175] Overview. The particle size distribution of C9 from Example 3A (crude) and Example 3B (recrystallized) was determined using a Malvern 3000 Mastersizer (particle size analyzer).

[0176] Example 3A, Preparation 1. Weigh 24.4 mg of C9 sample into a vial. Add about 20 mL of water. Add 20 drops of 5% octylphenoxypolyethoxyethanol (IGEPAL) aqueous solution into the vial. Cover the solution tightly and mix gently.

[0177] Example 3A, Preparation Example 2. Weigh 25.2 mg of C9 sample into a vial. Add about 20 mL of water. Add 20 drops of 5% IGEPAL aqueous solution into the vial. Cover the solution tightly and mix gently.

[0178] Example 3B, Preparation 1. Weigh 24.7 mg of C9 sample into a vial. Add about 20 mL of water. Add 20 drops of 5% IGEPAL aqueous solution into the vial. Cover the solution tightly and mix gently.

[0179] Example 3B, Preparation Example 2. Weigh 25.2 mg of C9 sample into a vial. Add about 20 mL of water. Add 20 drops of 5% IGEPAL aqueous solution into the vial. Cover the solution tightly and mix gently.

[0180] Visual observation of PSD samples

[0181] The Example 3A sample was observed to be a very heterogeneous solid. Many large particles were observed in the sample. Both preparations contained similar heterogeneity.

[0182] Very large crystals were observed in the Example 3B sample. Visual observation indicated that very small crystals to larger crystals were present in both sample preparations. The Sample 2 preparation visually contained more smaller / finer particles than the Sample 1 preparation.

[0183] Particle size distribution

[0184] The particle size distribution was determined by laser diffraction using a Malvern 3000 Mastersizer. The settings are shown in Table 6. The same settings were used for the other samples. The results are shown in Table 7. Table 6. Malvern instrument settings for Example 3A, Preparation 1 Table 7. Particle size distribution of C9 samples

[0185] As shown in Table 7, both examples consist of fairly large particles, but the average for Example 3B is quite large: average d(v,0.5um) = 278 μm for Example 3A and average d(v,0.5um) = 433 μm for Example 3B. However, Example 3A has a broader size distribution, which may be due to partial grinding of 3A (d(v,0.1 to 0.9) = ~60 μm to 925 μm for 3A vs. d(v,0.1 to 0.9) = ~133 to 878 μm for 3B). Fig. 27 and 28 (Example 3A Preparation Examples 1 and 2) and Fig.29 and 30 (Example 3B Preparations 1 and 2) show representative examples of each analysis. Fig. 27 and Fig.29 It can be seen that the Example 3A sample has a wider particle size distribution and a bimodal characteristic compared to the Example 3B (recrystallized) sample. Compared to the Example 3A sample (the ratio of D90 and D10 is 15.5:1), the distribution range between D90 and D10 in the Example 3B sample is narrower (the ratio is 6.6:1). D10 represents the particle diameter corresponding to 10% cumulative (from 0 to 100%) of the undersize particle size distribution (i.e., the percentage of particles smaller than D10 is 10%). D90 represents the particle diameter corresponding to 90% cumulative (from 0 to 100%) of the undersize particle size distribution (i.e., the percentage of particles smaller than D90 is 90%). Representative examples of each preparation example are shown in Figure 27-30 (Example 3A Preparation Example 1, Example 3A Preparation Example 2, Example 3B Preparation Example 1, Example 3B Preparation Example 2 respectively). Figure 27-30 As shown, the Example 3A sample has a broader particle size distribution than the Example 3B (recrystallized) sample. Example 7. Screening of compounds in human skin fibroblasts from patients with Parkinson's disease (PD) and Alzheimer's disease (AD)

[0186] A preliminary screen was performed to identify compounds effective in ameliorating PD and AD. In a manner similar to that described in the following literature: Jauslin et al., Hum. Mol. Genet. 11 (24): 3055 (2002); Jauslin et al., FASEB J. 17: 1972-4 (2003); and International Patent Application WO 2004 / 003565, the samples and solvent controls were tested for their ability to rescue PD and AD fibroblasts that were exacerbated by the addition of L-buthionine-(S, R)-sulfenyl imine (BSO, a specific inhibitor of GSH synthase) and iron (e.g., ferric citrate). This specific BSO-mediated cell death can be prevented or ameliorated by administering the compounds described in the present invention.

[0187] AD experiments were performed as follows. PD experiments were performed in a similar manner; certain specific conditions are noted below in Table 1A.

[0188] MEM (amino acid and vitamin-rich medium) and medium 199 (M199) with Earle's Balanced Salts (EBS) and without phenol red were purchased from Invitrogen. Fetal bovine serum was obtained from Corning. Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were purchased from PeproTech. Penicillin-streptomycin-glutamine mixture, L-buthionine (S, R)-sulfenyl imide, ferric citrate and bovine pancreatic insulin were purchased from Sigma. Calcein AM was purchased from Anaspec. Cell culture medium was prepared by combining 450 mL MEM, 50 mL fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin. Prepare assay medium by combining 125 mL M199, 50 mL fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM glutamine, 10 μg / mL insulin, 10 ng / mL EGF, and 10 ng / mL bFGF; add MEM to bring the volume to 500 mL. Prepare 10 mM BSO and 10 mM ferric citrate solutions in water, then filter sterilize and store at -20°C.

[0189] Fill the test samples into 1.5 mL glass vials or polypropylene vials. Dilute the compounds with DMSO to obtain 1 mM stock solutions. After dissolution, store them at -20 °C.

[0190] Test samples were screened according to the following scheme:

[0191] AD or PD patient-derived fibroblasts were cultured starting from vials of approximately 500,000 cells stored in liquid nitrogen. Cells were propagated in cell culture medium and subcultured every 3 days at a ratio of 1:3 by trypsinization. After confluence, fibroblasts were harvested by trypsinization, then resuspended in assay medium and seeded in standard 96-well tissue culture plates at a final cell density of 2500 cells / 0.1 mL per well. The plates were incubated at 37°C in an atmosphere of 95% humidity and 5% CO2 for 5 hours to allow cells to attach to the culture plates, and then ferric citrate solution (aqueous solution) was added to the desired final concentration.

[0192] The test sample (1 mM DMSO solution) was diluted in a 10% DMSO aqueous solution to a final concentration of 5 μM, and then serially diluted in 10% DMSO to the desired concentration. The cells were then treated with various compound dilutions to a final DMSO concentration of 1%, and then incubated at 37°C in an atmosphere of 95% humidity and 5% CO2 for 18 hours.

[0193] The next day, BSO solution was added to the wells to achieve the desired final concentration. After 48 hours, the medium was discarded and the plate was gently inverted on a paper towel to remove residual liquid. The plate was washed once with 100 μl of PBS containing calcium and magnesium (+Ca+Mg) per well.

[0194] Then 100 microliters of Calcein AM (1 μM) in PBS + Ca + Mg was added to each well. The plates were incubated at 37°C for 30 minutes. Thereafter, fluorescence was read on a Spectramax fluorescence plate reader (excitation / emission wavelengths were 485 nm and 525 nm, respectively). The data were analyzed using a standard four-parameter curve fitting algorithm (XLFit or Prism) to determine the EC of each compound. 50 concentration.

[0195] Solvents (DMSO, water) had neither deleterious effects on the viability of cells not treated with BSO nor beneficial effects on fibroblasts treated with BSO and iron, even at the highest concentration tested (1%).

[0196] The study found that the test sample of the present invention can rescue fibroblasts from Parkinson's disease and Alzheimer's disease patients from BSO and iron-induced oxidative stress. Table 8. Rescue of PD patient fibroblasts from oxidative stress induced by BSO (125 μM) and iron (125 μM) EC 50 = concentration at which 50% maximal rescue of cell viability was observed SEM = standard error of the mean. Table 9. C9 rescues AD patient fibroblasts from BSO and iron-induced oxidative stress - = Not tested

[0197] As shown in Table 8, the C9 compound (2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione) has greater potency than the C6 and C8 analogs in rescuing PD fibroblasts from BSO and iron-mediated oxidative stress.

[0198] The C9 compound also demonstrated activity in rescuing AD patient fibroblasts from oxidative stress (Table 9). Example 8. Screening for compounds that inhibit α-synuclein aggregation

[0199] Overview: The compound 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione ("C9") and its C8 (2,3,5-trimethyl-6-octylcyclohexa-2,5-diene-1,4-dione) and C7 (2,3,5-trimethyl-6-heptylcyclohexa-2,5-diene-1,4-dione) analogs were tested as inhibitors of alpha-synuclein aggregation as determined by the presence and extent of a hysteresis phase in protein aggregation kinetics. Changes in fluorescence intensity caused by an aggregate-bound fluorophore (thioflavin T) were tracked to report changes in protein aggregation over time.

[0200] Experimental Methods: Cell-free α-synuclein aggregation assays were set up with 200 μM recombinant human α-synuclein (Proteos, Inc.) in the presence of 100 μM compound (DMSO solution from a 10 mM stock solution) or with 1% (v / v) DMSO as vehicle. All solutions were prepared in Dulbecco's phosphate buffered saline (DPBS) buffer (pH 7.4) containing 0.03% (v / v) NaN3 and 5 μM Thioflavin T (ThT) and prepared as a premix before adding protein or compound. A protein premix was then prepared and divided into 4 tubes (1 tube for 1 condition). In parallel, a premix for background determination was created under the same conditions, except that no protein was added to the solution. Each sample was loaded with compound or DMSO, vortexed for 10 seconds, and centrifuged for 3 seconds.

[0201] The protein solution + / - compound and background samples were then loaded into the wells of an optically clear 96-well plate with black walls (Corning Costar) and The plate was sealed with a 480 sealer (Roche Life Science), incubated at 70°C for 15 minutes to reach equilibrium, and then data collection was started. Every 30 minutes, data points on ThT fluorescence (excitation / emission wavelength 450 / 490 nm) were collected using a Tecan M1000 spectrometer. The plate was agitated by shaking between two fluorescence readings.

[0202] Data Analysis and Results: Fluorescence intensity data were collected for all samples over time. The endpoint fluorescence intensity unit (FIU) value of the mean of the vehicle-treated α-synuclein samples was set as 100% and all other FIU values ​​were normalized to it (see Figure 1A ). The final results were plotted as normalized ThT fluorescence (%) versus time.

[0203] Samples were compared using normalized ThT fluorescence values ​​(% FIU) at t = 24 hours (see Figure 1B ). At least 2 technical replicates were used for each condition. Statistical analysis of samples was performed by ordinary one-way ANOVA. Tukey's multiple comparison test was performed for all protein-containing samples, which showed statistical significance between vehicle-treated samples and C9-treated samples (p=0.0041), C9-treated samples and C7-treated samples (p=0.0085), and C9-treated samples and C8-treated samples (p=0.0437). All other comparisons were not statistically significant (i.e., ns), p>0.05.

[0204] like Figure 1A and Figure 1B As shown, the C9 compound has a significant inhibitory effect on α-synuclein aggregation and exhibits a stronger inhibitory effect than the C7 or C8 analogs. Example 9. Potency of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione (C9) and its structural analogs on the decomposition of Tau K18 WT preformed fibrils

[0205] Summary: The compounds 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione (C9), 2,3,5-trimethyl-6-octylcyclohexa-2,5-diene-1,4-dione (C8), and 2,3,5-trimethyl-6-heptylcyclohexa-2,5-diene-1,4-dione (C7) were tested for their ability to disaggregate preformed fibrils (PFFs) of human wild-type Tau K18 fragments as measured by the decrease in fluorescence intensity of an aggregate-bound fluorophore (thioflavin T) over time.

[0206] Experimental Methods: Preformed fibrils (PFFs) of human recombinant TauK18 WT fragments were prepared by incubating TauK18 monomers (Bio- ) and heparin sodium at a ratio of 1: 1. The mixture was incubated at 37°C for 4 days without stirring, resulting in a final concentration of 100 μM PFF.

[0207] Cell-free Tau degradation assays were set up with 10 μM Tau PFFs in the presence of 30 μM compound (DMSO solution from a 10 mM stock solution) or with 0.3% (v / v) DMSO as a solvent. All solutions were prepared in DPBS buffer (pH 7.4) containing 0.03% (v / v) NaN3 and 5 μM Thioflavin T (ThT) and prepared as a premix before adding protein or compound. The day before the assay, a protein premix was first prepared to pre-equilibrate Tau PFFs at a concentration of 10 μM at ambient temperature and atmosphere. The next day, the premixed protein solution was divided into 4 test tubes (1 test tube for 1 condition). Each sample was loaded with compound or DMSO, vortexed for 10 seconds, centrifuged for 3 seconds, and then incubated for 15 minutes at ambient temperature. At the same time, a premix for background determination was created under the same conditions, except that no protein was added to the solution.

[0208] The PFF solution + / - compound and background samples were then loaded into the wells of an optically clear 96-well plate with black walls and The cells were sealed with a 480 sealer (Roche Life Science) and incubated at 37°C for 15 minutes to reach equilibrium before data collection began. Every 30 minutes, data points on ThT fluorescence (excitation / emission wavelengths 450 / 490 nm) were collected using a Tecan M1000 spectrometer without agitation.

[0209] Data Analysis and Results: The maximum fluorescence intensity unit (FIU) value of the vehicle-treated Tau sample was set as 100%, and all other FIU values ​​were normalized relative to that. At least 2 technical replicates were used for each condition. The endpoint values ​​of fibril content for all samples measured 94 hours after the start of the experiment were reported ( Figure 2 ).

[0210] Statistical analysis of samples was performed by ordinary one-way analysis of variance (ANOVA). Dunnett's multiple comparison test was performed between vehicle-treated Tau PFF samples and compound-treated Tau PFF samples, which showed statistical significance between vehicle-treated samples and C9-treated samples (p=0.0478).

[0211] like Figure 2 As shown, the Tau fibril content of samples treated with C9 was significantly reduced at 94 hours. In contrast, the fibril content of samples treated with C7 and C8 was not significantly reduced at 94 hours. Example 10. Inhibition of RSL3-induced α-synuclein aggregation

[0212] Adopt the plasmid construct obtained from Origene (RG221446), N27 rat dopaminergic cells (purchased from EMD Millipore, SCC048) are transformed to stably overexpress the truncated α-synuclein fused with green fluorescent protein (GFP). Cells are maintained in selection medium, and the selection medium is composed of RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum (Millipore, ES-009-B), 1% (v / v) penicillin-streptomycin (Gibco, 15140-122), 1% (v / v) L-glutamine (Gibco, 25030-081) and 500 μg / mL of G418 (Gibco, 10131-027).

[0213] As described above, N27 rat dopaminergic cells overexpressing a truncated (112 amino acids) human α-synuclein-GFP fusion protein were maintained under the above normal culture conditions. One day before the experiment, cells were seeded at a density of 3,000 cells / well in a 96-well plate with an optical bottom and black walls and maintained at 37°C for 24 hours. The compound (1S,3R)-methyl 2-(2-chloroacetyl)-1-(4-(methoxycarbonyl)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (1S,3R-RSL3, described in Yang et al., Cell 156:317-331(2014)) (60 nM) and 70 nM of the compound (2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione (C9), 2,3,5-trimethyl-6-octylcyclohexa-2,5-diene-1,4-dione (C8), or 2,3,5-trimethyl-6-heptylcyclohexa-2,5-diene-1,4-dione (C7)) were co-treated with the cells and placed back in the incubator at 37°C for 24 hours to start the experiment. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed three times with Dulbecco's phosphate buffered saline (DPBS) buffer, and then incubated with 1% Triton-X 100 solution (in DPBS) at room temperature for 48 hours to selectively label aggregated α-synuclein. The cells were then washed and labeled for total α-synuclein (mouse anti-α-synuclein, 1:250, BD Biosciences) using standard immunocytochemistry (ICC) methods. The cells were incubated with the primary antibody overnight at 4°C and then washed three times with DPBS the next day. The cells were then incubated with a fluorescently conjugated secondary antibody (goat anti-mouse Alexa 647, Invitrogen) for 2 hours at room temperature. 10 nM Hoechst was also added to label nuclei and the cells were imaged on a high-content imaging platform (ThermoFisher, HSC Cellomics Arrayscan) to quantify total aggregated α-synuclein (see Figure 3A ).

[0214] Figure 3BCompound C7, C8 and C9 are shown to inhibit the aggregation of alpha synuclein induced by RSL3. Cells treated with RSL3 alone were compared with cells treated with both RSL3 and compound, and Dunnett's multiple comparison test was used to perform statistical analysis of samples by common one-way analysis of variance (ANOVA). C7 did not show any inhibition of alpha synuclein aggregation (p>0.05). C8 and C9 both showed significant inhibition of alpha synuclein aggregation (p values ​​were 0.01 and 0.0003, respectively), but C9 showed an aggregation inhibition level of about 5.45 times higher than C8. Example 11. Effectiveness of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione in the MPTP model of Parkinson's disease in mice

[0215] Overview

[0216] Treatment of animals with (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) (MPTP) (60 mg / kg) resulted in a significant depletion of dopamine in the striatum (-74%), which is consistent with literature reports and internal validation studies.

[0217] Administration of C9 (300 mg / kg) had an effect on vertical activity (vertical counts and vertical time, as Figure 4A and 4B showed a significant therapeutic effect.

[0218] Introduction

[0219] MPTP is a potent, selective nigrostriatal dopaminergic neurotoxin that produces many of the neuropathological features of Parkinson's disease (PD) in humans, non-human primates, and mice. In mice, MPTP produces nigrostriatal dopaminergic degeneration. Pharmacological drugs that increase dopaminergic function or block the neurotoxicity of MPTP can also alleviate MPTP-related motor dysfunction and have been used clinically to treat Parkinson's disease. In addition, MPTP-mediated toxicity may be related to mechanisms associated with dopamine loss in the disease, suggesting that this model may also potentially be used to identify drugs that can slow or reduce nigrostriatal dopaminergic loss.

[0220] In this study, C9 was tested in the MPTP-induced PD model. The endpoints of this study were locomotor activity parameters in the open field test.

[0221] Experimental Procedure

[0222] Animals. Species: Mouse. Strain: C57Bl / 6. Animal origin: Charles River. Age: 6 to 7 weeks. Gender: Male. Randomization: Animals were randomly assigned to treatment groups. Study blinding: The experimenters were blinded to the experimental treatments.

[0223] Housing and feeding. Acclimation / conditioning: Not less than three days. Housing: Mice were housed on a 12-hour light / dark cycle (lights on at 6:00 AM). No more than 4 mice per cage. Ventilated cage system. Diet: Standard rodent chow and water ad libitum.

[0224] Design Parameters. Route of administration: PO. Dose volume: PO: 5 mL / kg. Formulation: Prepared by Melior according to instructions for use of BioElectron. Vehicle - Sesame oil (Spectrum Chemical, NF catalog #SE130, CAS #8008-74-0). Dose level: 300 mg / kg. Dose frequency: QD, starting on Day -2 (MPTP = Day 0) until Day 7. Study duration: 11 days. Pretreatment time (up to 2 hours): For Day 0 (MPTP day): 30 minutes, pretreatment time for open field test / locomotory activity (OFA / LMA) analysis: 1 hour. Number of groups: 3. Number of animals per group: 10. Total number of animals: 30 (in this study), 42 animals in total (to ensure proper efficacy analysis of at least 10 animals / group, for all groups receiving MPTP, 4 more animals were added to mitigate the study impact if MPTP-induced mortality occurred).

[0225] MPTP treatment. MPTP was formulated in phosphate buffered saline and administered to mice at a dose of 20 mg / kg (1 mg / mL delivered to 20 mL / kg) in 3 divided doses at 2-hour intervals (final dose of MPTP = 60 mg / kg).

[0226] Spontaneous activity. On the day before (baseline) and the 7th day after the application of MPTP ((-1) day and 7th day), a fully automatic open field device (Med-Associates, Inc) was used to monitor the various aspects of the mouse motor function. The device consists of a 10.75 inch x 10.75 inch place enclosed in a soundproof box, equipped with a fan and indoor lighting. The place is equipped with 3 16-beam IR arrays on the X-axis and Y-axis for position tracking, and is equipped with a Z-axis for feeding detection.

[0227] All mice were administered a final dose of C9 or vehicle-sesame oil 1 hour before the open field test according to the dosing schedule.Key motor parameters associated with dopaminergic deficiency include rearing behavior (number of rearings / 15 min) and total distance traveled (per 15 min).

[0228] Data analysis. The data were averaged and expressed as mean ± SEM. The data were analyzed by one-way analysis of variance (ANOVA) followed by post hoc tests. A p value of less than 0.05 was considered statistically significant compared with the control group.

[0229] result

[0230] Effect of Treatment on Open Field Locomotor Test (Vertical Counts). Activity (vertical counts) in an open field apparatus was measured in animals treated with saline + vehicle, MPTP + vehicle, or MPTP + C9. Animals were treated and handled as described above. Figure 4A As shown, the vertical counts of the MPTP+vehicle treated animal group were reduced compared with the saline+vehicle treated animal group (p=0.06). After C9 treatment, the vertical counts of the MPTP-stimulated animal group returned to the vertical counts observed in the saline+vehicle control animal group (MPTP+vehicle vs. MPTP+C9, p<0.01; saline+vehicle vs. MPTP+C9, p=0.312). Data are expressed as mean+SEM, n=12-14 per group. Statistical analysis: One-way analysis of variance (ANOVA) and post hoc Tukey test for multiple comparison tests; **p<0.01; ns, indicates no statistical significance (p>0.05).

[0231] Effect of Treatment on Open Field Locomotor Test (Vertical Time). Activity (vertical time) in an open field apparatus was measured in animals treated with saline + vehicle, MPTP + vehicle, or MPTP + C9. Animals were treated and handled as described above. Figure 4B As shown, the vertical time of the animal group treated with MPTP+vehicle was reduced compared with the animal group treated with saline+vehicle (p<0.05). After C9 treatment, the vertical time of the animal group stimulated by MPTP returned to the vertical time observed in the saline+vehicle control animal group (MPTP+vehicle relative to MPTP+C9, p<0.001; saline+vehicle relative to MPTP+C9, p=0.315). Data are expressed as mean+SEM, n=12-14 per group. Statistical analysis: One-way analysis of variance (ANOVA) and post hoc Tukey test for multiple comparison tests; *, p<0.05; ***, p<0.001; ns, indicates no statistical significance (p>0.05).

[0232] Administration of C9 in the MPTP mouse model of PD showed significant improvement in locomotor activity as measured by vertical count and vertical time, two behavioral indicators reflecting dopaminergic function in the striatum (Meredith and Rademacher, J Parkinsons Dis. 1(1):19-33 (2012), and references therein). Example 12. PK characteristics of C9 after acute oral administration in male C57 mice

[0233] C9 sesame oil solution was administered orally to 4 C57BL / 6 mice at a dose of 300 mg / kg. Plasma and brain exposures were determined 8 hours after compound administration (Table 10).

[0234] Table 10. Exposure of C9 in mouse brain and plasma after oral administration

[0235] Non-limiting embodiments of the present invention include the following:

[0236] Embodiment 1. A method for treating or inhibiting a disease selected from the group consisting of: Alzheimer's disease, Parkinson's disease, traumatic brain injury, and ischemia-reperfusion-related injury, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the formula: or its hydroquinone form; or its solvate or hydrate.

[0237] Embodiment 2. The method according to embodiment 1, wherein the compound is not a solvate or a hydrate.

[0238] Embodiment 3. The method according to embodiment 1 or 2, wherein the compound is in the quinone form.

[0239] Embodiment 4. The method of Embodiment 1 or 2, wherein the compound is in the hydroquinone form.

[0240] Embodiment 5. The method according to any one of embodiments 1-4, wherein the method is used to treat or inhibit Alzheimer's disease.

[0241] Embodiment 6. The method according to any one of embodiments 1-4, wherein the method is used to treat or inhibit Parkinson's disease.

[0242] Embodiment 7. The method according to any one of embodiments 1-4, wherein the method is used to treat or inhibit traumatic brain injury.

[0243] Embodiment 8. The method according to any one of embodiments 1-4, wherein the method is used to treat or inhibit ischemia-reperfusion related injury.

[0244] Embodiment 9. The method according to any one of embodiments 1-4, wherein the method is used to treat or inhibit stroke.

[0245] Embodiment 10. The method according to any one of embodiments 1-9, wherein the method is used to treat the disease.

[0246] Embodiment 11. The method according to any one of embodiments 1-9, wherein the method is used to inhibit the disease.

[0247] Embodiment 12. The method according to any one of embodiments 1-11, wherein the compound is administered orally.

[0248] Embodiment 13. The method according to any one of embodiments 1-11, wherein the compound is administered intravenously.

[0249] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein by identification are hereby incorporated by reference in their entirety.

[0250] Although the foregoing invention has been described in detail by way of illustration and example for the purpose of clear understanding, it is obvious to those skilled in the art that some minor changes and modifications may be made. Therefore, this description and examples should not be interpreted as limiting the scope of the invention.

[0251] This application relates to the following implementation schemes: 1. A method for treating or inhibiting a disease selected from the group consisting of: α-synuclein disease (α-synuclein disease, α-synucleinpathy), tauopathy, amyotrophic lateral sclerosis (ALS), traumatic brain injury, and ischemia-reperfusion-related injury, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the formula: or its hydroquinone form; or its solvate or hydrate. 2. The method according to embodiment 1, wherein the compound is not a solvate or a hydrate. 3. The method according to embodiment 1 or 2, wherein the compound is in the quinone form. 4. The method of embodiment 1 or 2, wherein the compound is in the hydroquinone form. 5. The method according to embodiment 1, wherein the method is used to treat or inhibit α-synuclein disease. 6. The method according to embodiment 5, wherein the α-synuclein disease is selected from the group consisting of: Parkinson's Disease, Parkinson's disease dementia (PDD), multiple system atrophy (MSA), frontotemporal dementia, Dementia with Lewy Bodies (DLB), Gaucher's disease (GD), neurodegeneration with brain iron deposition (NBIA), and neuroaxonal dystrophy (PLA2G6-related neurodegenerative disease). 7. The method of embodiment 6, wherein the Parkinson's disease is hereditary. 8. The method of embodiment 6, wherein the Parkinson's disease is idiopathic. 9. The method of embodiment 1, wherein the method is used to treat or inhibit tauopathy. 10. A method according to embodiment 9, wherein the tauopathy is selected from the group consisting of: Alzheimer's disease, pugilistic dementia, Guam ALS / PD, Pick Disease, Argyrophilic grain dementia, Nieman-Pick type C, Subacute sclerosing panencephalitis (SSPE), Progressive supranuclear palsy (PSP), Multiple System Atrophy (MSA), Corticobasoganlionic degeneration, Frontotemporal dementia with Parkinsonism type 17 (FTDP-17), Postencephalitic Parkinsonism (PEP), and Autosomal recessive Parkinsonism. 11. The method according to any one of embodiments 1-4, wherein the method is used to treat or inhibit Alzheimer's disease. 12. The method according to any one of embodiments 1-4, wherein the method is used to treat or inhibit Parkinson's disease. 13. A method according to any one of embodiments 1-4, wherein the method is used to treat or inhibit traumatic brain injury. 14. A method according to any one of embodiments 1-4, wherein the method is used to treat or inhibit ischemia-reperfusion related injury. 15. The method according to any one of embodiments 1-4, wherein the method is used to treat or inhibit stroke. 16. The method according to any one of embodiments 1-4, wherein the method is used to treat or inhibit amyotrophic lateral sclerosis (ALS). 17. The method according to any one of embodiments 1-16, wherein the method is used to treat the disease. 18. The method according to any one of embodiments 1-16, wherein the method is used to inhibit the disease. 19. The method according to any one of embodiments 1-18, wherein the compound is administered orally. 20. The method according to any one of embodiments 1-18, wherein the compound is administered intravenously. 21. A polymorph of an anhydrate of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione, wherein the powder X-ray diffraction pattern of the polymorph comprises characteristic peaks at at least the following angular positions: 4.10, 12.12 and 16.14, wherein the angular positions can vary by ±0.2. 22. The polymorph according to embodiment 21, comprising characteristic peaks at at least the following angular positions: 4.10, 11.77, 12.12 and 16.14, wherein the angular positions may vary by ±0.2. 23. The polymorph according to embodiment 21, comprising characteristic peaks at at least the following angular positions: 4.10, 11.77, 12.12, 16.14 and 22.41, wherein the angular positions may vary by ±0.2. 24. The polymorph according to any one of embodiments 21-23, wherein the angular position can vary by ±0.1. 25. The polymorph according to any one of embodiments 21-23, wherein the angular position can vary by ±0.05. 26. The polymorph according to any one of embodiments 21-25, wherein the polymorph has substantially Figure 5 , 11 , 14 and 16 as shown in any one of the powder X-ray diffraction patterns. 27. The polymorph according to any one of embodiments 21-26, having substantially Figure 7 Differential Scanning Calorimetry (DSC) thermogram shown. 28. The polymorph according to any one of embodiments 21-27, wherein the DSC thermogram has a single endothermic peak at about 47°C to about 53°C. 29. The polymorph according to any one of embodiments 21-28, having substantially Figure 8 Thermogravimetric analysis (TGA) thermogram shown. 30. The polymorph according to any one of embodiments 21-29, having substantially Figure 6 shown 1 HNMR spectrum. 31. A composition comprising the polymorph of any one of embodiments 21-30, wherein at least about 95 mol% of the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione is the polymorph, free of any solvent, carrier or excipient. 32. A composition comprising the polymorph of any one of embodiments 21-30, wherein at least about 99 mol% of the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione is the polymorph, free of any solvent, carrier or excipient. 33. A composition comprising the polymorph of any one of embodiments 21-30, or the composition of embodiment 31 or 32, wherein at least about 95% a / a of the composition is 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione as determined by HPLC, without any solvent, carrier or excipient. 34. A composition comprising the polymorph of any one of embodiments 21-30, or the composition of embodiment 31 or 32, wherein at least about 99% a / a of the composition is 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione as determined by HPLC, without any solvent, carrier or excipient. 35. A composition comprising the polymorph of any one of embodiments 21-30, or the composition of any one of embodiments 31-34, wherein the potency of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione is at least about 95%. 36. A composition comprising the polymorph of any one of embodiments 21-30, or the composition of any one of embodiments 31-34, wherein the potency (or content) of the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione is at least about 99%. 37. A composition comprising the polymorph of any one of embodiments 21-30, or the composition of any one of embodiments 31-36, wherein the polymorph is present in the form of a plurality of particles, wherein the particles have a D90:D10 ratio of less than about 11:1. 38. A composition comprising the polymorph of any one of embodiments 21-30, or the composition of any one of embodiments 31-36, wherein the polymorph is present in the form of a plurality of particles, wherein the particles have a D90:D10 ratio of less than about 7:1. 39. A composition comprising the polymorph of any one of embodiments 21-30, or the composition of any one of embodiments 31-38, wherein the polymorph is recrystallized from a solvent comprising about 75-85% IPA / water. 40. A composition comprising the polymorph of any one of embodiments 21-30, or the composition of any one of embodiments 31-39, wherein the polymorph is recrystallized from a solvent comprising about 80-85% IPA / water. 41. A composition comprising the polymorph of any one of embodiments 21-30, or the composition of any one of embodiments 31-39, wherein the polymorph is recrystallized from a solvent comprising about 85% IPA / water. 42. A pharmaceutical composition comprising the polymorph described in any one of Embodiments 21-30, or the composition described in any one of Embodiments 31-41, and a pharmaceutically acceptable solvent, carrier or excipient; or a pharmaceutical composition prepared using the polymorph described in any one of Embodiments 21-30, or the composition described in any one of Embodiments 31-41, and a pharmaceutically acceptable solvent, carrier or excipient. 53. A method for treating or inhibiting α-synucleinpathy, tauopathy, amyotrophic lateral sclerosis (ALS), traumatic brain injury, or ischemia-reperfusion-related injury, comprising administering to an individual in need thereof a therapeutically effective amount of a polymorph described in any one of embodiments 21-30, or a composition described in any one of embodiments 31-42. 54. A method of recrystallizing 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione from a composition, the method comprising: a) contacting the composition with IPA and water such that the resulting IPA to water ratio is about 75-87% isopropyl alcohol (IPA) / 25-13% water (v:v) at a temperature of about 40-45°C; b) cooling the mixture to about 32° C.; and c) filtering the 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione from the mixture. 55. The method of embodiment 54, wherein step (a) comprises: a1) contacting the composition with IPA; a2) heating the mixture to about 40-45°C; and a3) Adding water to the mixture so that the ratio of IPA to water is about 75-85% IPA:25-15% water (v:v). 56. The method of embodiment 54, wherein step (a) comprises stirring to dissolve the composition. 57. The method of any one of embodiments 54-56, wherein the ratio of IPA:water is about 80-85% IPA:20-15% water (v:v). 58. The method of any one of embodiments 54-56, wherein the ratio of IPA:water is about 85% IPA:15% water (v:v). 59. The method according to any one of embodiments 54-58, wherein step (a3) ​​comprises returning the temperature of the mixture to about 40-45°C. 60. The method according to any one of embodiments 54-58, comprising performing a polishing filtration on the mixture after step (a). 61. The method of any one of embodiments 54-59, wherein step (b) comprises cooling to about 32°C in about 2-10 hours. 62. The method of any one of embodiments 54-59, wherein step (b) comprises cooling to about 32°C over about 6 hours. 63. The method according to any one of embodiments 54-61, wherein the method comprises a step (b1) after step (b), comprising maintaining the mixture at about 32°C for about 2-24 hours. 64. The method according to any one of embodiments 54-61, wherein the method comprises a step (b1) after step (b), comprising maintaining the mixture at about 32°C for about 6 hours. 65. The method according to any one of embodiments 54-63, comprising step (b) or step (b1), if present, followed by step (b2), comprising cooling the mixture to about 0°C. 66. The method according to any one of embodiments 54-63, wherein step (b2) comprises cooling the mixture to about 0°C within about 3-24 hours. 67. The method according to any one of embodiments 64-65, wherein step (b2) further comprises maintaining the mixture at about 0°C for about 1 hour. 68. A composition comprising 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione prepared according to any one of embodiments 54-67. 69. A method for preparing a pharmaceutical composition, comprising converting the polymorph of any one of embodiments 21-30 or the composition of any one of embodiments 31-42 or 68 into a liquid or emulsion form. 70. The method of embodiment 69, wherein the liquid or emulsion form is provided as an oral solution, a liquid-filled capsule, or an injectable solution. 71. A pharmaceutical composition prepared according to embodiment 69 or 70. 72. A metastable melting amorphous form of 2,3,5-trimethyl-6-nonylcyclohexa-2,5-diene-1,4-dione having an XRPD pattern substantially as follows: Fig.31 shown.

Claims

1. A method for treating or inhibiting a disease selected from the group consisting of: α-synuclein disease (α-synuclein disease, α-synucleinpathy), tauopathy, amyotrophic lateral sclerosis (ALS), traumatic brain injury, and ischemia-reperfusion-related injury, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the formula: or its hydroquinone form; or its solvate or hydrate.

2. The method of claim 1, wherein the compound is not a solvate or a hydrate.

3. The method of claim 1 or 2, wherein the compound is in the quinone form.

4. The method of claim 1 or 2, wherein the compound is in the hydroquinone form.

5. The method of claim 1, wherein the method is used to treat or inhibit alpha-synucleinopathy.

6. The method of claim 5, wherein the α-synuclein disease is selected from the group consisting of Parkinson's Disease, Parkinson's Disease Dementia (PDD), Multiple System Atrophy (MSA), Frontotemporal Dementia, Dementia with Lewy Bodies (DLB), Gaucher's disease (GD), Neurodegeneration with Brain Iron Deposits (NBIA), and Neuroaxonal Dystrophy (PLA2G6-associated neurodegenerative disease).

7. The method of claim 6, wherein the Parkinson's disease is hereditary.

8. The method of claim 6, wherein the Parkinson's disease is idiopathic.

9. The method of claim 1, wherein the method is used to treat or inhibit tauopathy.

10. The method of claim 9, wherein the tauopathy is selected from the group consisting of Alzheimer's disease, dementia pugilistica, Guam ALS / PD, Pick Disease, Argyrophilic grain dementia, Nieman-Pick type C, Subacute sclerosing panencephalitis (SSPE), Progressive supranuclear palsy (PSP), Multiple System Atrophy (MSA), Corticobasoganlionic degeneration, Frontotemporal dementia with Parkinsonism type 17 (FTDP-17), Postencephalitic Parkinsonism (PEP), and Autosomal recessive Parkinsonism.

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