ANALOGS OF PRIDOPIDINE, THEIR PREPARATION AND USE

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This invention provides a composition comprising pridopidine or pharmaceutically acceptable salt thereof and at least one of compounds 1-8: or a pharmaceutically acceptable salt thereof; and to methods of use thereof.

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Description
CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation in Part application from U.S. application Ser. No. 16/535,107 filed Aug. 8, 2019, which is a Continuation application from U.S. application Ser. No. 16/150,977 filed Oct. 3, 2018, which is a Divisional application from U.S. application Ser. No. 14/754,339 filed Jun. 29, 2015, which claims the benefit of U.S. Provisional Application No. 62/076,436, filed Nov. 6, 2014, and U.S. Provisional Application No. 62/019,337, filed Jun. 30, 2014, the entire contents of which are hereby incorporated by reference in their entirety.

Disclosures of the publications cited in this application in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as of the date of the invention described herein.

BACKGROUND OF THE INVENTION

Pridopidine (4-[3-(methylsulfonyl)phenyl]-1-propyl-piperidine) (formerly known as ACR16, Huntexil®, TV-7820), is in clinical development for treatment of HD and ALS. Processes of synthesis of pridopidine and a pharmaceutically acceptable salt thereof are disclosed in U.S. Pat. No. 7,923,459. U.S. Pat. No. 6,903,120.

Pridopidine has a selective and high affinity for the sigma-1 receptor (S1R, binding IC50 “100 nM), with low-affinity binding to additional receptors, including the dopamine D2/D3 receptors (in the micromolar range).

The S1R in endoplasmic reticulum (ER) chaperone protein implicated in cellular differentiation, neuroplasticity, neuroprotection and cognitive function in the brain. Activation of the S1R by pridopidine leads to upregulation of pathways known to promote neuronal plasticity and survival, including the AKT/Phosphoinositide kinase (PI3K) pathway and the dopamine receptor 1 (D1R).

BRIEF SUMMARY OF THE INVENTION

This invention provides an isolated compound having the structure:

or a salt thereof.

This invention also provides a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one compound 1-8:

or a pharmaceutically acceptable salt thereof, wherein the weight ratio between pridopidine and at least one of compounds 1-8 is in the range of 1:0.001 to 1:0.1.

This invention also provides a composition comprising a compound having the structure:

    • or a pharmaceutically acceptable salt thereof, wherein the composition is free of pridopidine or a salt thereof.

The invention also provides a pharmaceutical composition comprising an amount of pridopidine and at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, and Compound 7 wherein

    • a) Compound 1 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • b) Compound 2 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • c) Compound 3 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • d) Compound 4 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • e) Compound 5 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • f) Compound 6 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • g) Compound 7 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method.

This invention also provides a process for preparing Compound 1 comprising the step of oxidizing 4-hydroxy-4-(3-(methylthio)phenyl)-1-propylpiperidin-1-ium chloride with an oxidizing agent to form Compound 1.

This invention also provides a process for preparing Compound 2 comprising the steps of:

    • a) reacting 3-bromothioanisole with ethyl 3-(4-oxopiperidin-1-yl)propanoate to form 1-(3-hydroxy-3,3-bis(3-(methylthio)phenyl)propyl)-4-(3-(methylthio)phenyl)piperidin-4-ol,
    • b) dehydrating the 1-(3-hydroxy-3,3-bis(3-(methylthio)phenyl)propyl)-4-(3-(methylthio)phenyl)piperidin-4-ol formed in step a) with a dehydrating agent to obtain 1-(3,3-bis(3-(methylthio)phenyl)allyl)-4-(3-(methylthio)phenyl)-1,2,3,6-tetrahydropyridine,
    • c) oxidizing the 1-(3,3-bis(3-(methylsulfonyl)phenyl)allyl)-4-(3-(methylsulfonyl) phenyl)-1,2,3,6-tetrahydropyridine formed in step b) with an oxidizing agent to form 1-(3,3-bis(3-(methylsulfonyl)phenyl)allyl)-4-(3-(methylsulfonyl)phenyl)-1,2,3,6-tetrahydropyridine, and
    • d) hydrogenating the 1-(3,3-bis(3-(methylsulfonyl)phenyl)allyl)-4-(3-(methylsulfonyl) phenyl)-1,2,3,6-tetrahydropyridine formed in step c) with a hydrogenating agent to form Compound 2.

This invention also provides a process for preparing Compound 3 comprising the steps of:

    • a) reacting 3-bromo thiophenol and 1,4-dibromobutane to form 1,4-bis((3-bromophenyl)thio)butane,
    • b) oxidizing the 1,4-bis((3-bromophenyl)thio)butane formed in step a) with an oxidizing agent to form 1,4-bis((3-bromophenyl)sulfonyl)butane,
    • c) reacting 4-pyridinylboronic acid with the 1,4-bis((3-bromophenyl)sulfonyl)butane formed in step b) to obtain 1,4-bis((3-(pyridin-4-yl)phenyl)sulfonyl)butane,
    • d) reacting 1-iodopropane with 1,4-bis((3-(pyridin-4-yl)phenyl)sulfonyl)butane formed in step c) to form 4,4′-((butane-1,4-diyldisulfonyl)bis(3,1-phenylene))bis(1-propylpyridine-1-ium)iodide,
    • e) adding a reducing agent to 4,4′-((butane-1,4-diyldisulfonyl)bis(3,1-phenylene))bis(1-propylpyridin-1-ium)iodide formed in step d) to form 1,4-bis((3-(1-propyl-1,2,3,6-tetrahydropyridin-4-yl)phenyl)sulfonyl) butane, and
    • f) hydrogenating the 1,4-bis((3-(1-propyl-1,2,3,6-tetrahydropyridin-4-yl)phenyl)sulfonyl) butane formed in step e) with a hydrogenating agent to obtain Compound 3.

This invention also provides a process for preparing Compound 4 comprising the steps of:

    • a) epoxidizing 4-(3-(methylsulfonyl)phenyl)-1-propyl-1,2,3,6-tetrahydropyridine with an epoxidizing agent to form (1 S,6S)-6-(3-(methylsulfonyl)phenyl)-3-propyl-7-oxa-3-azabicyclo [4.1.0]heptane, and
    • b) nucleophilically opening the epoxide of the (1S,6S)-6-(3-(methylsulfonyl)phenyl)-3-propyl-7-oxa-3-azabicyclo [4.1.0]heptane of step a) with a nucleophile to obtain Compound 4.

This invention also provides a process for preparing Compound 5 comprising the step of reacting pridopidine with a peroxide to obtain Compound 5.

This invention also provides a process for preparing Compound 6 comprising the step of reacting 4-(3-(methylsulfonyl)phenyl)piperidine with 1-chloro-2-methylpentane to obtain Compound 6.

This invention also provides a process for preparing Compound 7 comprising the steps of:

    • a) dehydrating 4-hydroxy-4-(3-(methylsulfonyl)phenyl)-1-propylpiperidin-1-ium chloride with a dehydrating agent to form 4-(3-(methylthio)phenyl)-1-propyl-1,2,3,6-tetrahydropyridin-1-ium hydrogen sulfate,
    • b) oxidizing 4-(3-(methylthio)phenyl)-1-propyl-1,2,3,6-tetrahydropyridin-1-ium hydrogen sulfate of step b) with an oxidizing agent to form Compound 7. In one embodiment, the dehydrating agent is a strong acid, preferably sulphuric acid. In another embodiment, the dehydrating agent is a strong acid. In another embodiment, the dehydrating agent is sulphuric acid. In another embodiment, the oxidizing agent is a peroxide, preferably hydrogen peroxide. In another embodiment, the oxidizing agent is a peroxide. In another embodiment, the oxidizing agent is hydrogen peroxide.

This invention also provides a process for producing a pridopidine drug product comprising obtaining a pridopidine drug substance and mixing the pridopidine drug substance with suitable excipients so as to produce the pridopidine drug product, wherein the pridopidine drug substance comprises:

    • i) an amount of Compound 1 in the pridopidine drug substance that is not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
    • ii) an amount of Compound 2 in the pridopidine drug substance that is not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
    • iii) an amount of Compound 3 in the pridopidine drug substance that is not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
    • iv) an amount of Compound 4 in the pridopidine drug substance that is not more than 0.15% Compound 4, relative to the concentration of pridopidine, or
    • v) an amount of Compound 5 in the pridopidine drug substance that is not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
    • vi) an amount of Compound 6 in the pridopidine drug substance that is not more than 0.15% Compound 6, relative to the concentration of pridopidine.

This invention also provides a process for producing a pridopidine drug product for commercial sale comprising obtaining a batch of pridopidine drug product that comprises:

    • i) an amount of Compound 1 in the batch of pridopidine drug product that is not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
    • ii) an amount of Compound 2 in the batch of pridopidine drug product that is not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
    • iii) an amount of Compound 3 in the batch of pridopidine drug product that is not more than 0.15-% Compound 3, relative to the concentration of pridopidine, or
    • iv) an amount of Compound 4 in the batch of pridopidine drug product that is not more than 0.15-% Compound 4, relative to the concentration of pridopidine, or
    • v) an amount of Compound 5 in the batch of pridopidine drug product that is not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
    • vi) an amount of Compound 6 in the batch of pridopidine drug product that is not more than 0.15% Compound 6, relative to the concentration of pridopidine, and
      preparing the batch of pridopidine drug product for commercial sale.

This invention also provides a process of distributing a pridopidine drug product comprising a pridopidine drug substance comprising,

    • a) obtaining the pridopidine drug product wherein the pridopidine drug substance comprises:
      • i) an amount of Compound 1 in the pridopidine drug substance that is not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
      • ii) an amount of Compound 2 in the pridopidine drug substance that is not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
      • iii) an amount of Compound 3 in the pridopidine drug substance that is not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
      • iv) an amount of Compound 4 in the pridopidine drug substance that is not more than 0.15% Compound 4, relative to the concentration of pridopidine, or
      • v) an amount of Compound 5 in the pridopidine drug substance that is not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
      • vi) an amount of Compound 6 in the pridopidine drug substance that is not more than 0.15% Compound 6, relative to the concentration of pridopidine; and
    • b) distributing the pridopidine drug product comprising the pridopidine drug substance.

This invention also provides a process of distributing a pridopidine drug product comprising,

    • a) obtaining the pridopidine drug product that comprises:
      • i) an amount of Compound 1 in the pridopidine drug product that is not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
      • ii) an amount of Compound 2 in the pridopidine drug product that is not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
      • iii) an amount of Compound 3 in the pridopidine drug product that is not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
      • iv) an amount of Compound 4 in the pridopidine drug product that is not more than 0.15% Compound 4, relative to the concentration of pridopidine, or
      • v) an amount of Compound 5 in the pridopidine drug product that is not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
      • vi) an amount of Compound 6 in the pridopidine drug product that is not more than 0.15% Compound 6, relative to the concentration of pridopidine; and
    • b) distributing the pridopidine drug product.

This invention also provides a method of treating a subject afflicted with a neurodegenerative disease or a neurodegenerative disorder comprising administering to the subject the pharmaceutical composition.

This invention also provides a method of treating a subject afflicted with Huntington disease comprising administering to the subject the pharmaceutical composition.

This invention further provides a method of treating, slowing the progression, lessening the decline, delaying onset of symptoms, or slowing the progression of symptoms of a neurodegenerative or neurodevelopmental disease or disorder in a subject; wherein the method comprises administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof, wherein the neurodegenerative disease or disorder is selected from the group consisting from Huntington Disease, prodromal/premanifest Huntington disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease, Parkinson's Disease associate with glucocerebrosidase (GBA) deficiency, dystonia, cognitive disorder, dyskinesia, mild cognitive impairment (MCI), Alzheimer's Disease, age related memory loss, depression and anxiety, bacterial infections-induced depression, optic neuropathies including glaucoma, age-related macular degeneration (AMD), Leber's Hereditary Optic Neuropathy (LHON) and retinitis pigmentosa, mitochondrial diseases or dysfunctions (i.e. Lysosomal storage disease (LSD), leukodystrophies, vanishing white matter (NVM) disease), Wolfram disease and viral infection (i.e. COVID-19); and wherein the neurodevelopmental disease or disorder is Rett syndrome (RTT) or Fragile X Syndrome (FXS).

This invention also provides a process for validating a batch of a pharmaceutical product containing pridopidine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier for distribution comprising:

    • a) determining the amount of at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6; and
    • b) validating the batch for distribution only if
      • i) the batch is determined to have not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
      • ii) the batch is determined to have not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
      • iii) the batch is determined to have not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
      • iv) the batch is determined to have not more than 0.15% Compound 4, relative to the concentration of pridopidine, or
      • v) the batch is determined to have not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
      • vi) the batch is determined to have not more than 0.15% Compound 6, relative to the concentration of pridopidine.

This invention also provides a process for preparing a validated pharmaceutical composition comprising pridopidine comprising:

    • a) obtaining a batch of pridopidine drug substance;
    • b) determining the amount of at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6; and
    • c) preparing the pharmaceutical composition from the batch only if
      • i) the batch is determined to have not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
      • ii) the batch is determined to have not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
      • iii) the batch is determined to have not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
      • iv) the batch is determined to have not more than 0.15% Compound 4, relative to the concentration of pridopidine, or
      • v) the batch is determined to have not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
      • vi) the batch is determined to have not more than 0.15% Compound 6, relative to the concentration of pridopidine.

This invention also provides a process for preparing a pharmaceutical composition comprising pridopidine, comprising

    • a) obtaining a batch of pridopidine drug product;
    • b) performing stability testing with a sample of the batch;
    • c) determining the total amount of at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the sample of the batch after stability testing by an HPLC method; and
    • d) preparing the pharmaceutical composition from the batch after stability testing if the sample of the batch after stability testing contains:
      • i) not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
      • ii) not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
      • iii) not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
      • iv) not more than 0.15% Compound 4, relative to the concentration of pridopidine, or
      • v) not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
      • vi) not more than 0.15% Compound 6, relative to the concentration of pridopidine.

This invention also provides an isolated compound having the structure:

or a salt thereof.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1: Typical Chromatogram of the control sample 1a.

FIG. 2: Typical Chromatogram of the control sample 2b.

FIGS. 3A-3B: Synergistic effect of pridopidine and Compound 4 on BDNF Release from B104 cells. B104 neuroblastoma cells were incubated for 5 days with test compounds, and BDNF levels were assessed using in-situ ELISA. FIG. 3A: Pridopidine at a concentration of 0.00 μM and Compound 4 at a concentration of 0.001 μM. Pridopidine alone increased BDNF secretion by 13.5%. Compound 4 alone reduced BDNF secretion by −1.5%. Pridopidine and compound 4 together increased BDNF secretion by 59.1%, an effect which is greater than the added effect of both compounds administered on their own. FIG. 3B: Pridopidine at a concentration of 0.005 μM and Compound 4 at a concentration of 0.001 μM. Pridopidine alone increased BDNF secretion by 26.0%. Compound 4 alone reduced BDNF secretion by −1.5%. Pridopidine and compound 4 together increased BDNF secretion by 80.7%, an effect which is greater than the added effect of both compounds administered on their own.

FIG. 4: Synergistic effect of pridopidine and Compound 1 on BDNF Release from B104 cells. B104 neuroblastoma cells were incubated for 5 days with test compounds, and BDNF levels were assessed using in-situ ELISA. Pridopidine at a concentration of 0.01 μM alone increased BDNF secretion by 3.4%. Compound 1 at a concentration of 1 μM alone increased BDNF secretion by 12.5%. Pridopidine and compound 1 together increased BDNF secretion by 53.1%, an effect which is greater than the added effect of both compounds administered on their own.

 DETAILED DESCRIPTION OF THE INVENTION

This invention provides an isolated compound having the structure:

or a salt thereof.

In an embodiment of the present invention, the isolated compound has the structure:

or a salt thereof.

In an embodiment, the isolated compound has the structure:

or a salt thereof.

In an embodiment, the isolated compound has the structure:

or a salt thereof.

In an embodiment, the isolated compound has the structure:

or a salt thereof.

In an embodiment the isolated compound has the structure:

or a salt thereof.

This invention also provides a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and a compound which has the structure 1-8 or a pharmaceutically acceptable salt thereof:

wherein the weight ratio of the compound relative to the weight of the pridopidine in the composition is from 99:1 to 1:99. In an embodiment, the ratio of the weight of pridopidine relative to the weight of compound in the composition is from 90:10 to 10:90 or 85:15 or 15:85. In other embodiments, the weight ratio between pridopidine and at least one of compounds 1-8 is in the range of 1:0.001 to 1:0.1. In other embodiments, the weight ratio between pridopidine and at least one of compounds 1-8 is in the range of 1:0.005 to 1:0.1. In other embodiments, the weight ratio between pridopidine and at least one of compounds 1-8 is in the range of 1:0.001 to 1:0.005.

This invention also provides a composition comprising a compound having the structure:

    • or a salt thereof wherein the composition is free of pridopidine or a salt thereof.

In an embodiment, the composition comprises pridopidine or pharmaceutically acceptable salt thereof and compound 1:

or a pharmaceutically acceptable salt thereof.

In an embodiment, the composition comprises pridopidine or pharmaceutically acceptable salt thereof and compound 2:

or a pharmaceutically acceptable salt thereof.

In an embodiment, the comprises pridopidine or pharmaceutically acceptable salt thereof and compound 3.

or a pharmaceutically acceptable salt thereof.

In an embodiment, the composition comprises pridopidine or pharmaceutically acceptable salt thereof and Compound 4:

or a pharmaceutically acceptable salt thereof.

In an embodiment, the composition comprises pridopidine or pharmaceutically acceptable salt thereof and compound 5:

or a pharmaceutically acceptable salt thereof.

In an embodiment, the composition comprises pridopidine or pharmaceutically acceptable salt thereof and compound 6:

or a pharmaceutically acceptable salt thereof.

In an embodiment, the composition comprises pridopidine or pharmaceutically acceptable salt thereof and compound 7:

or a pharmaceutically acceptable salt thereof.

The invention also provides a pharmaceutical composition comprising an amount of pridopidine and at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, and Compound 7 wherein

    • a) Compound 1 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • b) Compound 2 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • c) Compound 3 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • d) Compound 4 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • e) Compound 5 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • f) Compound 6 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • g) Compound 7 is present in the pharmaceutical composition in an amount not more than 10% relative to the concentration of pridopidine, based on a determination by an HPLC method.

In an embodiment,

    • a) Compound 1 is present in the pharmaceutical composition in an amount not more than 0.15% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • b) Compound 2 is present in the pharmaceutical composition in an amount not more than 0.15% relative to the concentration of pridopidine, based on a determination by an 22 HPLC method, or
    • c) Compound 3 is present in the pharmaceutical composition in an amount not more than 0.15-% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • d) Compound 4 is present in the pharmaceutical composition in an amount not more than 0.15% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • e) Compound 5 is present in the pharmaceutical composition in an amount not more than 0.15% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • f) Compound 6 is present in the pharmaceutical composition in an amount not more than 0.15% relative to the concentration of pridopidine, based on a determination by an HPLC method.

In another embodiment,

    • a) Compound 1 is present in the pharmaceutical composition in an amount greater than 0.01%, and not more than 0.15% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • b) Compound 2 is present in the pharmaceutical composition in an amount greater than 0.01%, and not more than 0.15%, relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • c) Compound 3 is present in the pharmaceutical composition in an amount greater than 0.03%, and not more than 0.15%, relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • d) Compound 4 is present in the pharmaceutical composition in an amount greater than 0.01%, and not more than 0.15%, relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • e) Compound 5 is present in the pharmaceutical composition in an amount greater than 0.01%, and not more than 0.15%, relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • f) Compound 6 is present in the pharmaceutical composition in an amount greater than 0.01% and not more than 0.15%, relative to the concentration of pridopidine, based on a determination by an HPLC method.

In another embodiment,

    • a) Compound 1 is present in the pharmaceutical composition in an amount less than 0.04% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • b) Compound 2 is present in the pharmaceutical composition in an amount less than 0.05%, relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • c) Compound 3 is present in the pharmaceutical composition in an amount less than 0.05% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • d) Compound 4 is present in the pharmaceutical composition in an amount less than 0.04% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • e) Compound 5 is present in the pharmaceutical composition in an amount less than 0.04% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • f) Compound 6 is present in the pharmaceutical composition in an amount less than 0.04% relative to the concentration of pridopidine, based on a determination by an HPLC method.

In another embodiment,

    • a) Compound 1 is present in the pharmaceutical composition in an amount less than 0.01% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • b) Compound 2 is present in the pharmaceutical composition in an amount less than 0.01% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • c) Compound 3 is present in the pharmaceutical composition in an amount less than 0.03% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • d) Compound 4 is present in the pharmaceutical composition in an amount less than 0.01% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • e) Compound 5 is present in the pharmaceutical composition in an amount less than 0.01% relative to the concentration of pridopidine, based on a determination by an HPLC method, or
    • 1) Compound 6 is present in the pharmaceutical composition in an amount less than 0.01% N relative to the concentration of pridopidine, based on a determination by an HPLC method.

In one embodiment, at least two of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6 are present. In another embodiment, at least three of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6 are present. In another embodiment, at least four of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6 are present. In another embodiment, at least five of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6 are present. In another embodiment, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6 are present. In another embodiment, at least Compound 1 is present. In another embodiment, at least Compound 3 is present. In another embodiment, at least Compound 4 is present.

In one embodiment, the pharmaceutical composition comprises pridopidine hydrochloride salt.

In an embodiment, the pharmaceutical composition is in the form of a capsule, a tablet, a liquid solution or a liquid suspension. In another embodiment, the pharmaceutical composition is in an oral dosage unit form. In another embodiment, the pharmaceutical composition is in an oral dosage unit form, wherein the oral dosage unit is formulated as a tablet, a capsule, a pill, powder, liquid solution or as a liquid suspension.

In an embodiment, the pharmaceutical composition the oral dosage unit form comprises between 0.5-315 mg pridopidine. In another embodiment, the oral dosage unit form comprises between 0.5-10 mg pridopidine. In another embodiment, the oral dosage unit form comprises between 10-22.5 mg pridopidine. In another embodiment, the oral dosage unit form comprises between 22.5-315 mg pridopidine. In another embodiment, the oral dosage unit form comprises between 10-315 mg pridopidine. In another embodiment, the oral dosage unit form comprises between 0.5-50 ng pridopidine. In another embodiment, the oral dosage unit form comprises between 45-250 mg pridopidine. In another embodiment, the oral dosage unit form comprises between 45-135 mg pridopidine. In another embodiment, the oral dosage unit form comprises between 90-315 mg pridopidine. In another embodiment, the oral dosage unit form comprises about 22.5 mg pridopidine. In another embodiment, the oral dosage unit form comprises about 45 mg pridopidine. In another embodiment, the oral dosage unit form comprises about 67.5 mg pridopidine. In another embodiment the oral dosage unit form comprises about 90 mg pridopidine. In another embodiment, the oral unit dosage form comprises about 100 mg pridopidine. In another embodiment, the oral dosage unit form comprises about 112.5 mg pridopidine. In another embodiment, the oral dosage unit form comprises about 125 mg pridopidine. In another embodiment, the oral dosage unit form comprises about 135 mg pridopidine. In another embodiment, the oral dosage unit form comprises about 150 mg pridopidine. In another embodiment, the oral dosage unit form comprises about 180 mg pridopidine. In another embodiment, the oral dosage unit form comprises about 200 mg pridopidine. In another embodiment, the oral dosage unit form comprises about 250 mg pridopidine. In another embodiment, the oral dosage unit form comprises about 315 mg pridopidine. In another embodiment, the oral dosage unit form is prepared for once daily administration. In another embodiment, the oral dosage unit form is prepared for more than once daily administration.

This invention also provides a process for preparing Compound 1 comprising the step of oxidizing 4-hydroxy-4-(3-(methylthio)phenyl)-1-propylpiperidin-1-ium chloride with an oxidizing agent to form Compound 1. In one embodiment, the oxidizing agent is a peroxide, preferably hydrogen peroxide. In another embodiment, the oxidizing agent is a peroxide. In another embodiment, the oxidizing agent is hydrogen peroxide.

This invention also provides a process for preparing Compound 2 comprising the steps of:

    • a) reacting 3-bromothioanisole with ethyl 3-(4-oxopiperidin-1-yl)propanoate to form 1-(3-hydroxy-3,3-bis(3-(methylthio)phenyl)propyl)-4-(3-(methylthio)phenyl)piperidin-4-ol,
    • b) dehydrating the 1-(3-hydroxy-3,3-bis(3-(methylthio)phenyl)propyl)-4-(3-(methylthio)phenyl)piperidin-4-ol formed in step a) with a dehydrating agent to obtain 1-(3,3-bis(3-(methylthio)phenyl)allyl)-4-(3-(methylthio)phenyl)-1,2,3,6-tetrahydropyridine,
    • c) oxidizing the 1-(3,3-bis(3-(methylsulfonyl)phenyl)allyl)-4-(3-(methylsulfonyl) phenyl)-1,2,3,6-tetrahydropyridine formed in step b) with an oxidizing agent to form 1-(3,3-bis(3-(methylsulfonyl)phenyl)allyl)-4-(3-(methylsulfonyl)phenyl)-1,2,3,6-tetrahydropyridine, and
    • d) hydrogenating the 1-(3,3-bis(3-(methylsulfonyl)phenyl)allyl)-4-(3-(methylsulfonyl) phenyl)-1,2,3,6-tetrahydropyridine formed in step c) with a hydrogenating agent to form Compound 2.

In one embodiment, the dehydrating agent is a strong acid, preferably sulfuric acid. In one embodiment, the dehydrating agent is a strong acid. In another embodiment, the dehydration agent is sulfuric acid. In another embodiment, the oxidizing agent is a peroxide. In another embodiment, the oxidizing agent is hydrogen peroxide. In another embodiment, the hydrogenating agent is hydrogen.

This invention also provides a process for preparing Compound 3 comprising the steps of:

    • a) reacting 3-bromo thiophenol and 1,4-dibromobutane to form 1,4-bis((3-bromophenyl)thio)butane,
    • b) oxidizing the 1,4-bis((3-bromophenyl)thio)butane formed in step a) with an oxidizing agent to form 1,4-bis((3-bromophenyl)sulfonyl)butane,
    • c) reacting 4-pyridinylboronic acid with the 1,4-bis((3-bromophenyl)sulfonyl)butane formed in step b) to obtain 1,4-bis((3-(pyridin-4-yl)phenyl)sulfonyl)butane,
    • d) reacting 1-iodopropane with 1,4-bis((3-(pyridin-4-yl)phenyl)sulfonyl)butane formed in step c) to form 4,4′-((butane-1,4-diyldisulfonyl)bis(3,1-phenylene))bis(1-propylpyridin-1-ium)iodide,
    • e) adding a reducing agent to 4,4′-((butane-1,4-diyldisulfonyl)bis(3,1-phenylene))bis(1-propylpyridin-1-ium)iodide formed in step d) to form 1,4-bis((3-(1-propyl-1,2,3,6-tetrahydropyridin-4-yl)phenyl)sulfonyl) butane, and
    • f) hydrogenating the 1,4-bis((3-(1-propyl-1,2,3,6-tetrahydropyridin-4-yl)phenyl)sulfonyl) butane formed in step e) with a hydrogenating agent to obtain Compound 3.

In one embodiment, the oxidizing agent is a peroxide, preferably hydrogen peroxide. In another embodiment, the oxidizing agent is a peroxide. In another embodiment, the oxidizing agent is hydrogen peroxide. In another embodiment, the reducing agent is sodium borohydride. In another embodiment, the hydrogenating agent is hydrogen.

This invention also provides a process for preparing Compound 4 comprising the steps of.

    • a) epoxidizing 4-(3-(methylsulfonyl)phenyl)-1-propyl-1,2,3,6-tetrahydropyridine with an epoxidizing agent to form (1S,6S)-6-(3-(methylsulfonyl)phenyl)-3-propyl-7-oxa-3-azabicyclo [4.1.0]heptane, and
    • b) nucleophilically opening the epoxide of the (1S,6S)-6-(3-(methylsulfonyl)phenyl)-3-propyl-7-oxa-3-azabicyclo [4.1.0]heptane of step a) with a nucleophile to obtain Compound 4.

In one embodiment, the epoxidizing agent is sodium bromate. In another embodiment, the nucleophile is hydrogen.

This invention also provides a process for preparing Compound 5 comprising the step of reacting pridopidine with a peroxide to obtain Compound 5. In one embodiment, the peroxide is hydrogen peroxide.

This invention also provides a process for preparing Compound 6 comprising the step of reacting 4-(3-(methylsulfonyl)phenyl)piperidine with 1-chloro-2-methylpentane to obtain Compound 6.

This invention also provides a process for preparing Compound 7 comprising the steps of:

    • a) dehydrating 4-hydroxy-4-(3-(methylsulfonyl)phenyl)-1-propylpiperidin-1-ium chloride with a dehydrating agent to form 4-(3-(methylthio)phenyl)-1-propyl-1,2,3,6-tetrahydropyridin-1-ium hydrogen sulfate,
    • b) oxidizing 4-(3-(methylthio)phenyl)-1-propyl-1,2,3,6-tetrahydropyridin-1-ium hydrogen sulfate of step b) with an oxidizing agent to form Compound 7.

In one embodiment, the dehydrating agent is a strong acid, preferably sulphuric acid. In another embodiment, the dehydrating agent is a strong acid. In another embodiment, the dehydrating agent is sulphuric acid. In another embodiment, the oxidizing agent is a peroxide, preferably hydrogen peroxide. In another embodiment, the oxidizing agent is a peroxide. In another embodiment, the oxidizing agent is hydrogen peroxide.

This invention also provides a process for producing a pridopidine drug product comprising obtaining a pridopidine drug substance and mixing the pridopidine drug substance with suitable excipients so as to produce the pridopidine drug product, wherein the pridopidine drug substance comprises:

    • i) an amount of Compound 1 in the pridopidine drug substance that is not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
    • ii) an amount of Compound 2 in the pridopidine drug substance that is not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
    • iii) an amount of Compound 3 in the pridopidine drug substance that is not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
    • iv) an amount of Compound 4 in the pridopidine drug substance that is not more than 0.15-% Compound 4, relative to the concentration of pridopidine, or
    • v) an amount of Compound 5 in the pridopidine drug substance that is not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
    • vi) an amount of Compound 6 in the pridopidine drug substance that is not more than 0.15% Compound 6, relative to the concentration of pridopidine.

In one embodiment, the process further comprises determining the amount of the at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the pridopidine drug substance. In another embodiment, the process further comprises determining the amount of the at least two of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the pridopidine drug substance. In another embodiment, the process further comprises determining the amount of the at least three of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the pridopidine drug substance. In another embodiment, the process further comprises determining the amount of the at least four of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the pridopidine drug substance. In another embodiment, the process further comprises determining the amount of the at least five of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the pridopidine drug substance. In another embodiment, the process further comprises determining the amount of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the pridopidine drug substance. In another embodiment, the process further comprises subjecting a sample of the pridopidine drug substance to stability testing before the step of determining the amount of the at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the pridopidine drug substance.

This invention also provides a process for producing a pridopidine drug product for commercial sale comprising obtaining a batch of pridopidine drug product that comprises:

    • i) an amount of Compound 1 in the batch of pridopidine drug product that is not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
    • ii) an amount of Compound 2 in the batch of pridopidine drug product that is not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
    • iii) an amount of Compound 3 in the batch of pridopidine drug product that is not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
    • iv) an amount of Compound 4 in the batch of pridopidine drug product that is not more than 0.15% Compound 4, relative to the concentration of pridopidine, or
    • v) an amount of Compound 5 in the batch of pridopidine drug product that is not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
    • vi) an amount of Compound 6 in the batch of pridopidine drug product that is not more than 0.15% Compound 6, relative to the concentration of pridopidine, and
      preparing the batch of pridopidine drug product for commercial sale.

In an embodiment, the process further comprises determining the amount of the at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the batch of pridopidine drug product. In another embodiment, the process further comprises determining the amount of the at least two of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the batch of pridopidine drug product. In an embodiment, the process further comprises determining the amount of the at least three of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the batch of pridopidine drug product. In an embodiment, the process further comprises determining the amount of the at least four of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the batch of pridopidine drug product. In an embodiment, the process further comprises determining the amount of the at least five of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the batch of pridopidine drug product. In an embodiment, the process further comprises determining the amount of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the batch of pridopidine drug product. In another embodiment, the process further comprises subjecting a sample of the batch of pridopidine drug product to stability testing before determining the amount of the at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the sample of the batch of pridopidine drug product.

This invention also provides a process of distributing a pridopidine drug product comprising a pridopidine drug substance comprising,

    • a) obtaining the pridopidine drug product wherein the pridopidine drug substance comprises:
      • i) an amount of Compound 1 in the pridopidine drug substance that is not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
      • ii) an amount of Compound 2 in the pridopidine drug substance that is not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
      • iii) an amount of Compound 3 in the pridopidine drug substance that is not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
      • iv) an amount of Compound 4 in the pridopidine drug substance that is not more than 0.15% Compound 4, relative to the concentration of pridopidine, or
      • v) an amount of Compound 5 in the pridopidine drug substance that is not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
      • vi) an amount of Compound 6 in the pridopidine drug substance that is not more than 0.15% Compound 6, relative to the concentration of pridopidine; and
    • b) distributing the pridopidine drug product comprising the pridopidine drug substance.

This invention also provides a process of distributing a pridopidine drug product comprising,

    • a) obtaining the pridopidine drug product that comprises:
      • i) an amount of Compound 1 in the pridopidine drug product that is not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
      • ii) an amount of Compound 2 in the pridopidine drug product that is not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
      • iii) an amount of Compound 3 in the pridopidine drug product that is not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
      • iv) an amount of Compound 4 in the pridopidine drug product that is not more than 0.15% Compound 4, relative to the concentration of pridopidine, or
      • v) an amount of Compound 5 in the pridopidine drug product that is not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
      • vi) an amount of Compound 6 in the pridopidine drug product that is not more than 0.15% Compound 6, relative to the concentration of pridopidine; and
    • b) distributing the pridopidine drug product.

This invention also provides a method of treating a subject afflicted with a neurodegenerative disease or a neurodegenerative disorder comprising administering to the subject the pharmaceutical composition.

This invention also provides a method of treating a subject afflicted with Huntington disease comprising administering to the subject the pharmaceutical composition.

This invention provides a method of treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of a neurodegenerative or neurodevelopmental disease or disorder in a subject; wherein the method comprises administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof:

wherein the method comprises administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof, wherein the neurodegenerative disease or disorder is selected from the group consisting from Huntington Disease, prodromal/premanifest Huntington disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease, Parkinson's Disease associated with glucocerebrosidase (GBA) deficiency, dystonia, cognitive disorder, dyskinesia, mild cognitive impairment (MCI), Alzheimer's Disease, age related memory loss, depression and anxiety, bacterial infections-induced depression, optic neuropathies including glaucoma, age-related macular degeneration (AMD), Leber's Hereditary Optic Neuropathy (LHON) and retinitis pigmentosa, mitochondrial diseases or dysfunctions (i.e. Lysosomal storage disease (LSD), leukodystrophies, vanishing white matter (VWM) disease), Wolfram disease and viral infection (i.e. COVID-19); and wherein the neurodevelopmental disease or disorder is Rett syndrome or Fragile X Syndrome.

In other embodiments, the composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof for use in the methods of this invention—comprises a weight ratio between the pridopidine and at least one of compounds 1-8 in the range of 1:0.001 to 1:0.1. In other embodiments, the weight ratio between the pridopidine or a pharmaceutically acceptable salt thereof and at least one of compounds 1-8 or a pharmaceutically acceptable salt thereof is in the range of 1:0.005 to 1:0.1. In other embodiments, the weight ratio between the pridopidine or a pharmaceutically acceptable salt thereof and at least one of compounds 1-8 or a pharmaceutically acceptable salt thereof is in the range of 1:0.001 to 1:0.005.

In other embodiments, the composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof for use in the methods of this invention is administered in a daily dose of between 0.5-315 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the oral dosage unit form is administered in a daily dose of 0.5-10 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, oral dosage unit form is administered in a daily dose of 10-22.5 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the oral dosage unit form is administered in a daily dose of 22.5-315 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the oral dosage unit form is administered in a daily dose 10-315 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the oral dosage unit form is administered in a daily dose 0.5-50 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the oral dosage unit form is administered in a daily dose 22.5-315 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the oral dosage unit form the oral dosage unit form is administered in a daily dose 45-250 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the oral dosage unit form is administered in a daily dose 45-135 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the oral dosage unit form is administered in a daily dose 90-315 mg pridopidine or a pharmaceutically acceptable salt thereof.

In other embodiments, the method of treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of neurodegenerative or neurodevelopmental disease or disorder in a subject comprises administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and Compound 1 or pharmaceutically acceptable salt thereof. In other embodiment, a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and Compound 2 or pharmaceutically acceptable salt thereof. In other embodiment, a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and Compound 3 or pharmaceutically acceptable salt thereof. In other embodiment, a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and Compound 4 or pharmaceutically acceptable salt thereof. In other embodiment, a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and Compound 5 or pharmaceutically acceptable salt thereof. In other embodiment, a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and Compound 6 or pharmaceutically acceptable salt thereof. In other embodiment, a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and Compound 7 or pharmaceutically acceptable salt thereof. In other embodiment, a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and Compound 8 or pharmaceutically acceptable salt thereof.

In some embodiments this invention provides a method of treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of a neurodegenerative or neurodevelopmental disease or disorder in a subject; wherein the method comprises administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compound 1, Compound 4 pharmaceutically acceptable salt thereof or combination thereof; wherein the neurodegenerative disease or disorder is selected from the group consisting from Huntington Disease, prodromal/premanifest Huntington disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease, Parkinson's Disease associated with glucocerebrosidase (GBA) deficiency, dystonia, cognitive disorder, dyskinesia, mild cognitive impairment (MCI), Alzheimer's Disease, age related memory loss, depression and anxiety, bacterial infections-induced depression, optic neuropathies including glaucoma, age-related macular degeneration (AMD), Leber's Hereditary Optic Neuropathy (LHON) and retinitis pigmentosa, mitochondrial diseases or dysfunctions (i.e. Lysosomal storage disease (LSD), leukodystrophies, vanishing white matter (VWM) disease), Wolfram Disease, and viral infection (i.e. COVID-19); and wherein the neurodevelopmental disease or disorder is Rett syndrome or Fragile X Syndrome.

In some embodiments this invention provides a method of treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of a neurodegenerative or neurodevelopmental disease or disorder in a subject; wherein the method comprises administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and Compound 1 or pharmaceutically acceptable salt thereof; wherein the neurodegenerative disease or disorder is selected from the group consisting from Huntington Disease, prodromal/premanifest Huntington disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease, Parkinson's Disease associated with glucocerebrosidase (GBA) deficiency, dystonia, cognitive disorder, dyskinesia, mild cognitive impairment (MCI), Alzheimer's Disease, age related memory loss, depression and anxiety, bacterial infections-induced depression, optic neuropathies including glaucoma, age-related macular degeneration (AMD), Leber's Hereditary Optic Neuropathy (LHON) and retinitis pigmentosa, mitochondrial diseases or dysfunctions (i.e. Lysosomal storage disease (LSD), leukodystrophies, vanishing white matter (VWM) disease), Wolfram Disease and viral infection (i.e. COVID-19); and wherein the neurodevelopmental disease or disorder is Rett syndrome or Fragile X Syndrome.

In some embodiments this invention provides a method of treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of a neurodegenerative or neurodevelopmental disease or disorder in a subject; wherein the method comprises administering a composition comprising pridopidine, Compound 1 and Compound 4 or pharmaceutically acceptable salt thereof; wherein the neurodegenerative disease or disorder is selected from the group consisting from Huntington Disease, prodromal/premanifest Huntington disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease, Parkinson's Disease associated with glucocerebrosidase (GBA) deficiency, dystonia, cognitive disorder, dyskinesia, mild cognitive impairment (MCI), Alzheimer's Disease, age related memory loss, depression and anxiety, bacterial infections-induced depression, optic neuropathies including glaucoma, age-related macular degeneration (AMD), Leber's Hereditary Optic Neuropathy (LHON) and retinitis pigmentosa, mitochondrial diseases or dysfunctions (i.e. Lysosomal storage disease (LSD), leukodystrophies, vanishing white matter (VWM) disease), Wolfram Disease and viral infection (i.e. COVID-19); and wherein the neurodevelopmental disease or disorder is Rett syndrome or Fragile X Syndrome.

In some embodiments this invention provides a method of treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of a neurodegenerative or neurodevelopmental disease or disorder in a subject; wherein the method comprises administering a composition comprising pridopidine, Compound 4 or pharmaceutically acceptable salt thereof; wherein the neurodegenerative disease or disorder is selected from the group consisting from Huntington Disease, prodromal/premanifest Huntington disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease, Parkinson's Disease associated with glucocerebrosidase (GBA) deficiency, dystonia, cognitive disorder, dyskinesia, mild cognitive impairment (MCI), Alzheimer's Disease, age related memory loss, depression and anxiety, bacterial infections-induced depression, optic neuropathies including glaucoma, age-related macular degeneration (AMD), Leber's Hereditary Optic Neuropathy (LHON) and retinitis pigmentosa, mitochondrial diseases or dysfunctions (i.e. Lysosomal storage disease (LSD), leukodystrophies, vanishing white matter (VWM) disease), Wolfram Disease and viral infection (i.e. COVID-19); and wherein the neurodevelopmental disease or disorder is Rett syndrome or Fragile X Syndrome.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of Huntington disease comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the Huntington disease is early-stage Huntington disease. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In another embodiment the method of this invention is directed to delaying the onset of symptoms in prodromal/premanifest Huntington disease individuals which have >=36 CAG repeats in the huntingtin gene, comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In other embodiments, the methods of this invention are directed to treating or slowing the progression of symptoms of prodromal/premanifest Huntington disease.

In other embodiments, the symptoms of Huntington disease comprise decline in motor function, cognitive and functional capacity, behavioral and psychiatric symptoms and increase of Neurofilament light protein (NfL) levels. In other embodiments, the method of this invention provides maintaining, improving, or lessening the decline of motor function, cognitive and functional capacity.

In other embodiments, the methods of this invention provide maintaining, reducing, or lessening the increase of Neurofilament light protein (NfL) in biofluids (i.e. cerebrospinal fluid, blood and plasma). In other embodiments, the method of this invention provides maintaining, reducing, or lessening the increase of Neurofilament light protein (NfL) in biofluids (i.e. cerebrospinal fluid, blood and plasma) in a neurodegenerative disease including Huntington disease, ALS and Parkinson's disease patients.

Decline in “Functional Capacity” refers to patients' declined capacity to work, handle finances and domestic chores, perform activities of daily living or live independently.

In other embodiments, the methods of this invention are directed to treating, reducing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of Amyotrophic Lateral Sclerosis (ALS) comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In other embodiments, the symptoms of ALS comprise muscle atrophy and weakness, increased fatigue, problems with speech or swallowing (bulbar-onset symptoms), aspiration pneumonia, respiratory failure, behavioral disturbances, dysexecutive impairment, pseudobulbar affect (e.g. bouts of uncontrollable laughter or crying), and frontotemporal dementia, function or any combination thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof. In other embodiments, the methods of this invention provide maintaining, reducing, or lessening the increase of Neurofilament light protein (NIL) or phosphorylated Neurofilament Heavy (pNfH) in biofluids (i.e. cerebrospinal fluid, blood and plasma) in ALS patients. In another embodiment, the method of this invention provides maintaining, reducing, or lessening the increase of the extracellular domain of p75 (p75ECD) in biofluids (i.e. blood, plasma and urine) in ALS patients.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of Parkinson's disease comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of Parkinson's disease associated with glucocerebrosidase (G5BA) deficiency comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of Parkinson's disease, a disease associated with parkinsonism, or Parkinson's disease associate with glucocerebrosidase (CBA) deficiency comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In other embodiments the symptoms comprises a functional decline, cognitive decline. In certain embodiments, the functional decline of the subject is presented as a symptom selected from the group consisting of tremor, bradykinesia, rigidity, postural instability, a decline according to the Unified Parkinson's Disease Rating Scale part II (UPDRS part II), including Activities of Daily living, and a decline according to the Modified Hoehn and Yahr Staging of PD. In certain embodiments, the functional decline of the subject is presented as a decline according to the Unified Parkinson's Disease Rating Scale part II (UPDRS part II), including Activities of Daily living. In certain embodiments, the functional decline of the subject is presented as a decline according to the Modified Hoehn and Yahr Staging of PD.

In certain embodiments, the cognitive decline of the subject is presented as a symptom selected from the group consisting of intellectual impairment, thought disorder, depression, decreased motivation, decreased initiative, impaired speech, increased salvation, impaired swallowing, impaired handwriting, and increased pain sensation.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms dystonia comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the dystonia is severe dystonia. In other embodiments the symptoms of dystonia comprise involuntary limb movement or muscle contractions, twisted posture of the limbs or trunk, abnormal fixed posture of the limbs or trunk, talipes equinovarus, turning in of the leg, turning in of the arm, tremor of the hand, head, trunk or arms, dragging of the leg, torticollis, writer's cramp, or dystonia of trunk and/or extremities. In another embodiment, the dystonia is a severe dystonia.

In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

“Severe dystonia” may be determined by Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) having Rating Scale >4 for at least one body part. Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) evaluates nine body parts (eyes, mouth, speech, swallowing, neck, trunk, right arm, right leg, left arm, and left leg) by rating the severity factor and provoking factors for each part on a 5 point scale of 0 (no dystonia) to 4 (indicating the presence of dystonia at rest). The dystonia scores of the eyes, mouth and neck are assigned a weighting factor of 0.5, while the other 6 parts are assigned a weighting factor of 1.0. The score of each part is obtained by multiplying the provoking factor by the severity factor and the weighting factor, and then summing the scores of each part. The maximum score possible is 120.

Severe dystonia may be also determined by the Unified Dystonia Rating Scale (UDRS) Rating Scale having Rating Scale ≥4 for at least one body part. UDRS evaluates 14 body parts (eyes and upper face, lower face, jaw and tongue, larynx, neck, trunk, right shoulder/proximal arm, left shoulder/proximal arm, right distal arm/hand, left distal arm/hand, right proximal leg, left proximal leg, right distal leg/foot, and left distal leg/foot) by rating the severity and duration factors for each part. The severity factor for each part is rated using a 5-point scale, ranging from 0 (no dystonia) to 4 (severe dystonia). The duration factor is rating on a 5 point scale ranging from 0 (at rest/action) to 4 (submaximal/maximal). The total score is the sum of each domain (part), with the maximum being 112.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of a cognitive disorder comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In certain embodiments the cognitive disorder is mild cognitive impairment. In certain embodiments, the cognitive disorder comprises memory loss. In certain embodiments, the cognitive disorder comprises age related memory loss.

Cognitive disorder refers to impairment of cognitive function which is selected from the group consisting of global cognitive functioning, sustained cognition, memory, language, executive functioning, and attention. In another embodiment, the cognitive function is memory. In an embodiment, memory is short term memory. In another embodiment, memory is long term memory. In another embodiment, memory is working memory. In an embodiment, the subject is afflicted with a cognitive deficit. In another embodiment, the subject is prone to or predisposed to have a cognitive deficit. In an embodiment, the cognitive deficit is a memory deficit. In an embodiment, the memory deficit is a short-term memory deficit. In another embodiment, the memory deficit is memory loss. In an embodiment, the memory loss is caused by one or more of age-related changes in memory, mild cognitive impairment, dementia or depression. In an embodiment, the cognitive deficit is caused by or associated with a disease or disorder. In an embodiment, the disease or disorder is a disease or disorder associated with NMDA receptor. In another embodiment, the disease or disorder is schizophrenia or autism. In another embodiment, the disease or disorder is epilepsy or an anxiety disorder. In another embodiment, the disease or disorder is amyotrophic lateral sclerosis (ALS). In another embodiment, the disease or disorder is frontotemporal dementia (FTD). In another embodiment, the disease or disorder is mild cognitive impairment (MCI). In another embodiment, the disease or disorder is bipolar disorder. In another embodiment, the disease or disorder is Huntington disease. In another embodiment, the disease or disorder is selected from the group consisting of major depressive disorder (MDD), Parkinson's disease, Alzheimer's disease, tardive dyskinesia, depression, sickle cell anemia, stroke, chronic pain syndrome, and addiction. In another embodiment, the disease or disorder is selected from the group consisting of mild cognitive impairment, memory loss, memory deficit, a memory deficit related to brain injury or a post-stroke event, a learning deficiency, and behavioral and cognitive problems associated with brain tumors. In another embodiment, the disease or disorder is selected from the group consisting of dementia, dementia associated with Lewy Bodies, age-related cognitive decline, psychosis, attention deficit disorder (ADHD), bipolar disorder, brain injury, mood and affective disorders, Tourette's syndrome, mental retardation, progressive supranuclear palsy, Creutzfeldt-Jacob disease, corticobasal Degeneration, vascular dementia, and Pick's disease. In another embodiment, the disease or disorder is selected from the group consisting of generalized anxiety disorder (GAD), social anxiety disorder (SAD), tardive dyskinesia, depression, sickle cell anemia, chronic pain syndrome, addiction, nicotine addiction, internet addiction, cocaine addiction, tourette's syndrome, mental retardation, corticobasal degeneration, vascular dementia, Pick's disease, posttraumatic stress disorder (PTSD), obsessive compulsive disorder, panic disorder (PD), trigeminal pain, trigeminal musculoskeletal pain, phantom limb pain, irritable bowel syndrome, blepharospasm, complex regional pain syndrome, chronic low back pain, autism spectrum disorder (ASD), infantile spasm (IS).

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of dyskinesia comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

Dyskinesias are abnormal, involuntary movements which may appear as jerking, twisting or writhing of parts of the body. There are several different types of dyskinesias, which can be categorized as chorea, dystonia, myoclonus, tremor and paroxysmal tardive (late-onset type). These movement disorders include, without limitation, parkinsonism, tardive dyskinesia, chorea, dystonia, tremor, akathisia, athetosis, myoclonus or tics. In some embodiments, the dyskinesia is L-DOPA Induced Dyskinesia (LID). In some embodiments, the dyskinesia is Parkinson's disease (PD)-LID.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms lessening the decline of Alzheimer's Disease comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms age related memory loss comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of depression and anxiety comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of optic neuropathies including glaucoma, age-related macular degeneration (AMD), Leber's Hereditary Optic Neuropathy (LHON) and retinitis pigmentosa and related symptoms comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of a disease, disorder, or condition associated with mitochondrial diseases or dysfunction in a subject, or any symptom thereof comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In other embodiments, the disease, disorder, or condition associated with mitochondrial disease or dysfunction comprise: (a) a mood disorder; (b) a mitochondrial myopathy; (c) a lysosomal storage disease; (d) Frontotemporal Dementia (FTD), (e) Charcot-Marie-Tooth Disease (CMT), (t) leukodystrophies, (g) vanishing white matter (VWM) disease or a combination thereof.

In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In other embodiments, said mitochondrial myopathy is selected from MEL AS syndrome, MERRF syndrome, Leigh Disease, Alpers Syndrome, Chronic Progressive External Ophthalmoplegia (C/PEO), Diabetes mellitus and deafness (MIDD or DAD, Kearns-Sayre syndrome (KSS), Mitochondrial DNA depletion syndrome (MDS), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), Neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome, Lebers Hereditary Optic Neuropathy (LHON), Dominant Optic Atrophy (DOA), Pigmentary retinopathy, Wolfram Syndrome, Friedrich's Ataxia (FRDA), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and any combinations thereof.

In other embodiments, the lysosomal storage disease is selected from Glycogenosis Type II (Pompe Disease), Multiple Sulphatase Deficiency (MSD), Mucopolysaccharidoses (MPS), Mucolipidoses (ML) Types I-III, G(M1)-Gangliosidosis, Fabry Disease, Farber Disease, Gaucher Disease, Niernann-Pick Disease, Mucolipidoses (ML) Type IV, Cystinosis, Neuronal Ceroid-Lipofuscinoses, and any combinations thereof.

In some embodiments, the disease, disorder, condition, or symptom that is associated with mitochondrial dysfunction is vanishing white matter (VWM) disease. Vanishing White Matter Disease (VWM) is one of more than 50 conditions that affect the white matter, or myelin, of the brain known collectively as Leukodystrophies. VWM, also known as Childhood Ataxia with Central Nervous System Hypomyelination (CACH), is an extremely rare neurological condition that destroys myelin, the brain's white matter, or myelin. In doing so, it permanently affects transmission of brain signals to the rest of the body. Clinical conditions identified under VWM disease include but are not limited to: Childhood Ataxia with diffuse CNS Hypomyelination (CACH), Vanishing White Matter Leukodystrophy (VWM), Cree Leukoencephalopathy, Vanishing White Matter Leukodystrophy with Ovarian Failure, and any combinations thereof.

In further embodiments, the method for treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of symptoms mitochondrial diseases or dysfunctions, in a subject in need thereof comprise Lysosomal storage disease (LSD), leukodystrophies, vanishing white matter (VWM), poor growth, loss of muscle coordination, muscle weakness, neurological deficit, seizures, autism, autistic spectrum, autistic-like features, learning disabilities, heart disease, liver disease, kidney disease, gastrointestinal disorders, severe constipation, diabetes, increased risk of infection, thyroid dysfunction, adrenal dysfunction, autonomic dysfunction, confusion, disorientation, memory loss, poor growth, failure to thrive, poor coordination, sensory (vision, hearing) problems, reduced mental functions, disease of the organ, dementia, respiratory problems, hypoglycemia, apnea, lactic acidosis, seizures, swallowing difficulties, developmental delays, movement disorders (dystonia, muscle spasms, tremors, chorea), stroke, and brain atrophy. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a method of reducing endoplasmic reticulum (ER) stress in a subject comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment the ER stress is associated with obesity, diabetes, cancer, neurodegenerative disorders, inflammatory diseases, infectious diseases, or a combination thereof. In another embodiment, ER stress is associated with heritable forms of diabetes. In another embodiment, ER stress is associated with neurodegeneration. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of viral infection (i.e., COVID-19) comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof. In other embodiments, the viral infection, disease or disorder comprise human coronavirus, Severe acute respiratory syndrome (SARS), Middle East Respiratory Syndrome (MERS) coronavirus, SARS coronavirus 2 (SARS-CoV-2) or mutations therefrom. In other embodiments, the disease is COVID-19.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of Rett syndrome comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In other embodiments Rett syndromes symptoms comprise abnormal gait, ataxia, impaired gait initiation, a delay in acquiring purposeful hand skills or a partial or complete loss of acquired purposeful hand skills or abnormal hand movement, delayed crawling, and/or walking; decreased ability to crawl, and/or walk; or increased irritability, decreased alertness or decreased attention span. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of Fragile X Syndrome comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of Wolfram Disease comprising administering a composition comprising pidopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In other embodiments Wolfram Disease symptoms comprise urinary tract abnormalities, ataxia, loss of sense of smell, loss of gag reflex, myoclonus, peripheral neuropathy, seizures, depression, impulsive and/or aggressive behavior, psychosis, gastrointestinal problems, intellectual disability, irregular breathing, central apnea, central respiratory failure, hypogonadism in males, stomach and/or intestinal ulcers, and a tendency to bleed excessively from wounds. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

In other embodiments, the methods of this invention are directed to treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of bacterial infection-induced depression comprising administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof. In another embodiment the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or pharmaceutically acceptable salt thereof. In another embodiment, the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

This invention also provides a process for validating a batch of a pharmaceutical product containing pridopidine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier for distribution comprising:

    • a) determining the amount of at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6; and
    • b) validating the batch for distribution only if
      • i) the batch is determined to have not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
      • ii) the batch is determined to have not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
      • iii) the batch is determined to have not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
      • iv) the batch is determined to have not more than 0.15% Compound 4, relative to the concentration of pridopidine, or
      • v) the batch is determined to have not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
      • vi) the batch is determined to have not more than 0.15% Compound 6, relative to the concentration of pridopidine.

This invention also provides a process for preparing a validated pharmaceutical composition comprising pridopidine comprising:

    • a) obtaining a batch of pridopidine drug substance;
    • b) determining the amount of at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6; and
    • c) preparing the pharmaceutical composition from the batch only if
      • i) the batch is determined to have not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
      • ii) the batch is determined to have not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
      • iii) the batch is determined to have not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
      • iv) the batch is determined to have not more than 0.15% Compound 4, relative to the concentration of pridopidine, or
      • v) the batch is determined to have not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
      • vi) the batch is determined to have not more than 0.15% Compound 6, relative to the concentration of pridopidine.

This invention also provides a process for preparing a pharmaceutical composition comprising pridopidine, comprising

    • a) obtaining a batch of pridopidine drug product;
    • b) performing stability testing with a sample of the batch;
    • c) determining the total amount of at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in the sample of the batch after stability testing by an HPLC method; and
    • d) preparing the pharmaceutical composition from the batch after stability testing if the sample of the batch after stability testing contains:
      • i) not more than 0.15% Compound 1, relative to the concentration of pridopidine, or
      • ii) not more than 0.15% Compound 2, relative to the concentration of pridopidine, or
      • iii) not more than 0.15% Compound 3, relative to the concentration of pridopidine, or
      • iv) not more than 0.15% Compound 4, relative to the concentration of pridopidine, or
      • v) not more than 0.15% Compound 5, relative to the concentration of pridopidine, or
      • vi) not more than 0.15% Compound 6, relative to the concentration of pridopidine.

In an embodiment, the process further comprising step e) distributing the batch if in step d) the batch is validated for distribution.

This invention also provides an isolated compound having the structure:

or a salt thereof.

Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

For example, the elements recited in the packaging and pharmaceutical composition embodiments can be used in the method and use embodiments described herein.

TERMS

As used herein, and unless stated otherwise, each of the following terms shall have the definition set forth below.

As used herein, “pridopidine” means pridopidine base or a pharmaceutically acceptable salt thereof, including pridopidine hydrochloride. Preferably, in any embodiments of the invention as described herein, the pridopidine is in the form of its hydrochloride salt.

As used herein, “drug substance” refers to the active ingredient in a drug product or to the composition containing the active ingredient before it is formulated into in a drug product, which provides pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the body of man or animals.

As used herein, “drug product” refers to the formulated or finished dosage form containing the drug substance as well as at least one pharmaceutically acceptable carrier.

As used herein, an “isolated” compound is a compound isolated from the crude reaction mixture following an affirmative act of isolation. The act of isolation involves separating the compound from the other known components of the crude reaction mixture, with some impurities, unknown side products and residual amounts of the other known components of the crude reaction mixture permitted to remain. Purification is an example of an affirmative act of isolation.

As used herein, “stability testing” refers to tests conducted at specific time intervals and various environmental conditions (e.g., temperature and humidity) to see if and to what extent a drug product degrades over its designated shelf life time. The specific conditions and time of the tests are such that they accelerate the conditions the drug product is expected to encounter over its shelf life. For example, detailed requirements of stability testing for finished pharmaceuticals are codified in 21 C.F.R § 211.166, the entire content of which is hereby incorporated by reference.

As used herein, “about” in the context of a numerical value or range means ±10% of the numerical value or range recited.

As used herein, “approximately” in the context of a numerical value or range means ±5% of the numerical value or range recited or claimed.

As used herein, an “amount” of a compound as measured in milligrams refers to the milligrams of compound present in a preparation, regardless of the form of the preparation. An “amount of compound which is 40 mg” means the amount of the compound in a preparation is 40 mg, regardless of the form of the preparation. Thus, when in the form with a carrier, the weight of the carrier necessary to provide a dose of 40 mg compound would be greater than 40 mg due to the presence of the carrier.

As used herein, “treating” and “treatment” encompasses, e.g., inducing inhibition, regression, or stasis of a disease, disorder or condition.

The term “slowing the progression” refers to treated subjects will show less progression of the disease overtime, compared to non-treated subjects “lessening the decline” refers to the decline of a specific disease scale measured over a period of time is less (slower) with treatment than with no treatment, “delaying onset of symptoms” refers to onset of a specific symptom or symptoms of the disease occur later in time with treatment compared to no treatment, or “slowing the progression of symptoms” refers to a specific symptom or symptoms of the disease will show less progression over time with treatment, compared to no treatment.

“Administering to the subject” means the giving of, dispensing of, or application of medicines, drugs, or remedies to a subject to relieve, cure, or reduce the symptoms associated with a condition, e.g., a pathological condition.

The drug substance of the present invention, e.g., pridopidine hydrochloride, may be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. Capsules or tablets may contain suitable binders, lubricants, disintegrating agents, diluents, coloring agents, flow-inducing agents, and melting agents.

A dosage unit of the compounds used in the method of the present invention may comprise a single compound or mixtures thereof with additional therapeutic agents.

A “dose” or “dosage unit” of pridopidine as measured in milligrams refers to the milligrams of pridopidine hydrochloride present in a preparation, regardless of the form of the preparation. A dosage unit may comprise a single compound or mixtures of compounds thereof. A dosage unit can be prepared for oral dosage forms, such as tablets, capsules, pills, powders, liquid suspension, liquid solution, and granules. For example, the “dose” or “dosage unit” of pridopidine may be 22.5, 45, or 67.5 mg.

As used herein, a “pharmaceutically acceptable” component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

The subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein, including impurities. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

As used herein, “detection limit” for an analytical method used in screening or testing for the presence of a compound in a sample is a threshold under which the compound in a sample cannot be detected by the analytical method used. The detection limits of a given HPLC method for detecting an impurity in a sample containing pridopidine may vary based on the method and the impurity or impurities being detected. For example, the detection limit of the typical HPLC method for detecting Compounds 1, 2, 4, 5 and 6 is 0.01% and the detecting limit for detecting Compound 3 is 0.03%.

As used herein, “quantitation limit” for an analytical method used in screening or testing for the presence of a compound in a sample is a threshold under which the compound in a sample cannot be quantified by the analytical method used. The quantitation limits of a given HPLC method for detecting an impurity in a sample containing pridopidine may vary based on the impurity or impurities being detected. For example, the quantitation limit of the typical HPLC method for quantifying Compounds 1, 4, 5, and 6 is 0.04% and the quantitation limit for Compound 3 is 0.05%. The quantitation limit for Compound 2 is 0.05%.

A characteristic of a compound refers to any quality that a compound exhibits, e.g., peaks or retention times, as determined by 1H nuclear magnetic spectroscopy, mass spectroscopy, infrared, ultraviolet or fluorescence spectrophotometry, gas chromatography, thin layer chromatography, high performance liquid chromatography, elemental analysis, Ames test, dissolution, stability and any other quality that can be determined by an analytical method. Once the characteristics of a compound are known, the information can be used to, for example, screen or test for the presence of the compound in a sample.

As used herein, “NMT” means no more than. As used herein, “LT” means less than.

As used herein, the term “effective amount” refers to the quantity of a component that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention, i.e. a therapeutically effective amount. The specific effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.

As used herein, “preparing drug product for commercial sale” means an activity undertaken in preparation for commercial sale. Examples include, but are not limited to, coloring, coding, stamping, packaging the drug product.

It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, “20-40 mg” includes 20.0 mg, 20.1 mg, 20.2 mg, 20.3 mg, etc. up to 40.0 mg.

Pharmaceutically Acceptable Salts

The active compounds for use according to the invention may be provided in any form suitable for the intended administration. Suitable forms include pharmaceutically (i.e. physiologically) acceptable salts, and pre- or prodrug forms of the compound of the invention.

Examples of pharmaceutically acceptable addition salts include, without limitation, the non-toxic inorganic and organic acid addition salts such as the hydrochloride, the hydrobromide, the nitrate, the perchlorate, the phosphate, the sulphate, the formate, the acetate, the aconate, the ascorbate, the benzenesulphonate, the benzoate, the cinnamate, the citrate, the embonate, the enantate, the fumarate, the glutamate, the glycolate, the lactate, the maleate, the malonate, the mandelate, the methanesulphonate, the naphthalene-2-sulphonate, the phthalate, the salicylate, the sorbate, the stearate, the succinate, the tartrate, the toluene-p-sulphonate, and the like. Such salts may be formed by procedures well known and described in the art.

Pharmaceutical Compositions

While the compounds for use according to the invention may be administered in the form of the raw compound, it is preferred to introduce the active ingredients, optionally in the form of physiologically acceptable salts, in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and/or other customary pharmaceutical auxiliaries.

In an embodiment, the invention provides pharmaceutical compositions comprising the active compounds or pharmaceutically acceptable salts or derivatives thereof together with one or more pharmaceutically acceptable carriers therefore, and, optionally, other therapeutic and/or prophylactic ingredients know and used in the art. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof.

Table 1 shows the structures of Compounds 1-8.

TABLE 1 Compound 1 Potential Impurity in pridopidine. 4-(3- (methylsulfonyl) phenyl)-1- propylpiperidin-4-ol Compound 2 Potential impurity in pridopidine. 1-(3,3-bis(3- (methylsulfonyl) phenyl)propyl)-4-(3- (methylsulfonyl) phenyl)piperidone Compound 3 Potential impurity in pridopidine. 1,4-bis((3-(1- propylpiperidin-4- yl)phenyl)sulfonyl) butane Compound 4 Potential impurity in pridopidine. (3R,4S)-4-(3- (methylsulfonyl) phenyl)-1- propylpiperidin-3-ol Compound 5 Potential impurity in pridopidine. 4-(3- (methylsulfonyl) phenyl)-1- propylpiperidine 1- oxide Compound 6 1-(2-methylpentyl)- 4-(3- (methylsulfonyl) phenyl)piperidine Compound 7 4-(3- (methylsulfinyl) phenyl)-1-propyl- 1,2,3,6- tetrahydropyridine Compound 8 4-(3- (methylsulfonyl) phenyl)-1-propyl- 1,2,3,6- tetrahydropyridine

This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

EXPERIMENTAL DETAILS Examples Example 1—Preparation of Compound 1 (4-(3-(methylsulfonyl)phenyl)-1-propylpiperidin-4-ol)

To a suspension of 4-hydroxy-4-(3-(methylthio)phenyl)-1-propylpiperidin-1-ium chloride (140 g, 348 mmol) in 710 mL water were added 1.5 g sodium tungstate dihydrate, and the mixture was heated to 45° C. 102 mL of 33% H2O2 were added in 20 min at 45-55° C. The suspension dissolved after 20 mL addition. The solution was then stirred at 48-51° C. for 30 min after which HPLC showed no more starting material and two new peaks, one at RT 2.68 min (82.3%) and the other at RT 3.66 min (11.8%). After additional stirring for 2 hr and 45 min HPLC showed that the peak at RT 2.68 min decreases to 7.5% and the peak at RT 3.66 min increases to 88.5%. After another 45 min the mixture was cooled to 20° C. and into the reaction mixture were added 500 mL toluene and 150 mL. ˜5M NaOH. After stirring for 5 min the mixture was poured into B separator funnel. The solubility of the product in toluene is low. Majority of the product settled as very viscous liquid layer in the bottom. The water phase (and most of the product) was separated, toluene phase was washed successively with 5% Na2SO3 solution and with brine and dried on MgSO4. The water phase was extracted with 500 mL DCM. The organic phase was washed successively with 5% Na2SO3 solution and water and was dried on MgSO4. Both extracts were concentrated on a rotavapor. 500 mL of heptanes were added to both residues, and the suspensions were stirred at room temperature for 2 hrs. The precipitates were filtered, washed with heptane and dried. From the DCM extract were obtained 83.8 g of white powder, purity by HPLC 98.8%, 1H-NMR assay 97.9%. (From the toluene extract were obtained 13.7 g of white powder, purity by HPLC 98.0%).

NMR Identity Analysis of Compound 1

The following data in Tables 2 and 3 was determined using a sample of 78.95 mg Compound 1, a solvent of 0.55 ml DMSO-D6, 99.9 atom % D, and the instrument was a Bruker Avance III 400 MHz.

TABLE 2 Assignment of 1H NMRa,c 1H COSY HMBC Shift Assign- cross cross (ppm) Integral Multiplicity ment peaks peaks 8.07 1 t, J = 1.7 Hz H5 H1b, H3b C1, C3, C4b, C6b, C8 7.82 1 d, J = 7.9 Hz H3 H2, H5b C1, C4b, C5, C8 7.79 1 d, J = 7.9 Hz H1 H2, H5b C3, C4b, C5 7.59 1 t, J = 7.9 Hz H2 H1, H3 C3, C4, C5b, C6, C8b 5.08 1 s H17 — C8, C9, C13 3.21 3 s H18 — C6b 2.67 2 d, J = 11.5 Hz H10, H10, H12, C8b, C9b, H12 H13, H9 C10, C12, C13b 2.37 2 t, J = 11.6 Hz H10, H10, H12, C8b, C9b, H12 H13, H9 C10, C12, C13b, C14 2.28 2 t, J = 7.3 Hz H14 H15 C10, C12, C15, C16 1.97 2 dt, J = 12.5 and H9, H13 H10, H12, C9, C10, 4.1 Hz H13, H9 C12, C13 1.60 2 d, J = 12.8 Hz H9, H13 H10, H12, C8, C9, H13, H9 C13 1.46 2 hex, J = 7.5 Hz H15 H14, H16 C14, C15 0.87 3 t, J = 7.5 Hz H16 H15 C14, C15 aThe assignment is based on the coupling pattern of the signals, coupling constants and chemical shifts. bWeak signal. cSpectra is calibrated by the solvent residual peak (2.5 ppm).

TABLE 3 Assignment of 13C NMRa,b 13C Shift (ppm) Assignment 151.9 C4 140.6 C6 130.1 C3 129.0 C2 124.9 C1 123.3 C5  70.0 C8  60.2  C14  49.0 C10, C12  43.6  C18  38.0 C9, C13  19.8  C15  12.0  C16 aThe assignment is based on the chemical shifts and 1H-13C couplings extracted from HSQC and HMBC experiments. bSpectra is calibrated by a solvent peak (39.54 ppm)

Example 2—Preparation of Compound 2 (1-(3,3-bis(3-(methylsulfonyl)phenyl)propyl)-4-(3-(methylsulfonyl) phenyl)piperidine) Preparation of ethyl 3-(4-oxopiperidin-1-yl)-propanoate (Starting Material for Compound 2)

Ethanol (1550 mL) was poured into a 4 L three-necked round-bottom flask equipped with over-head stirring followed by the addition of 125 g (814 mmol, 1 eq) 4-piperidone monohydrate hydrochloride and 225 g (1628 mmol, 2 eq) potassium carbonate. Ethyl 3-chloropropanoate (111 g, 1 eq) was added and the reaction mixture was stirred for 3 h after which HPLC showed that the product reached only 10%. Another 0.5 eq of K2CO3 was added (56.2 g) and stirring continued at 24° C. After total of 45 h the product reached 86% (HPLC). Another 0.2 eq of K2CO3 was added and the reaction mixture was stirred for additional 4.5 h at 35° C. after which HPLC showed 96% of the product. The mixture was filtered through a sintered glass filter, washed with 200 ml ethanol and concentrated on vacuum to 156 g yellow colored oil that was distilled under vacuum of 2 mmHg in 156° C. bath. The main fraction distilled at 120° C. to yield 97.8 g (60%) of 99.3% (HPLC).

Preparation of 1-(3-hydroxy-3,3-bis(3-(methylthio)phenyl)propyl)-4-(3-(methylthio) phenyl)piperidin-4-ol (Compound 2, 1st Intermediate)

3-Bromothioanisole (170.3 g; 0.84 mol, 3.2 eq) and THF (700 mL) were charged to a 2 L flask, stirred under nitrogen and cooled on dry ice/acetone bath to −74° C. A solution of n-hexyllithium in hexane (2.3M; 237.4 g; 0.77 mol, 3.0 eq) was added and the reaction mixture became slightly yellowish. Stirring continued for additional 30 min at −74° C. A solution of ethyl 3-(4-oxopiperidin-1-yl)propanoate (50.2 g; 0.26 mol, leg) in THE (100 mL) was added during 1 h 15 min to the reaction mixture and the stirring continued for additional 30 min at −74° C. to give a yellow clear solution. The cooling stopped and the reaction warmed to −40° C. A solution of HCl (33%; 90 g, 0.82 mol, 3.1 eq) in water (100 mL) was added dropwise for 20 min to give a light yellow emulsion in +8° C. The light yellow organic phase was separated, washed with water (3×200 mL) and extracted twice with aqueous HCl (33% HCl 40 g/300 mL water) to give lower yellow phase (234 g). The organic upper light yellow phase was evaporated up to 159 g solution and the precipitate formed during concentration was filtered to give 19.1 g yellow sticky precipitate. The precipitate was combined with the lower yellow phase, methanol (50 mL) and THF (200 mL) were added and distilled (67° C., 248 g distilled). Heptane (200 mL was added, the two liquid phase was stirred for 20 min at 40° C. and cooled to RT. The upper heptane phase was discarded and water (200 mL) was added to the viscous yellow residue water. After stirring stopped the colorless water was decanted to leave 182 g of very viscous light yellow residue (HPLC: 82%).

Preparation of 1-(3,3-bis(3-(methylthio)phenyl)allyl)-4-(3-(methylthio)phenyl)-1,2,3,6-tetrahydropyridine (Compound 2, 2nd Intermediate)

Into the viscous light yellow residue was added 2-propanol (200 mL) and the reaction mixture was distilled at atmospheric pressure to give 200 mL of azeotropic distillate, leaving dark yellow oil into which methanol (50 mL), 2-propanol (350 mL) and conc. sulfuric acid (36.5 g, 0.35 mol. 1.35 eq) were added. The reaction mixture was heated for 26 hours (mixture temperature 81-84° C., vapor temperature 79° C.) and about 440 mL of distillate were collected. At the end the temperature reached 87° C. and the reaction mixture was foaming. After cooling was added toluene (100 ml) and water (200 mL) and the reaction mixture was heated to reflux (87° C.). The heating stopped and after cooling three phases were formed. The lower oily phase was washed with water (2×200 mL) and concentrated by vacuum distillation to give dark yellow viscous residue. Water (300 mL) was added and the mixture was refluxed then cooled to 40° C. and water phase was decanted to leave about 200 g orange turbid liquid (HPLC: 82%) which was used in the next step.

Preparation of 1-(3,3-bis(3-(methylsulfonyl)phenyl)allyl)-4-(3-(methylsulfonyl) phenyl)-1,2,3,6-tetrahydropyridine (Compound 2, 3rd Intermediate)

To the 200 g orange turbid liquid from the previous stage was added 500 mL water, sodium tungstate dihydrate (2 g, 6 mmol) and concentrated sulfuric acid (20 mL). The mixture was heated to 35° C. and 33% H2O2 was added drop-wise in 1 h during which the yellow viscous mass on the bottom of the flask dissolved slowly and the temperature rose up to 55° C. then decreased slowly to 42° C. The reaction mixture was heated to 50° C. for 2 hr and additional 32 g of 33% H2O2 were added. The reaction continued for another 4 h at 50° C. and additional 20 g of 33% H2O2 were added. After 2 h the reaction mixture was cooled down (25° C.) and alkalized to pH12 by 50% NaOH solution. Water (300 mL) was added and after 20 min of mechanical stirring was discarded. Another 200 mL of water were added, stirred mechanically for 20 min and discarded to give 158.2 g highly viscous yellow mass (HPLC: 75.4%). This mass was heated for 30 min 4 times with butanol (200 mL@95° C., 200 mL@100° C., 400 mL@100° C. and 700 mL@114° C.) and twice with acetic acid (8 mL and 250 mL@95° C.) to give light brown oil that was used in the next step (114.9 g, HPLC: 89%).

Preparation of 1-(3,3-bis(3-(methylsulfonyl)phenyl)propyl)-4-(3-(methylsulfonyl) phenyl)piperidine (Compound 2)

The light brown oil from the previous stage (114.9 g, HPLC: 89%) was added into a 2 L autoclave with 550 mL acetic acid and 10% Pd/C catalyst (25 g, 23.5 mmol). Hydrogen was introduced (120 psi) and the reaction was heated to 90° C. for 16 h. After cooling, the catalyst was filtered, washed with acetic acid (50 ml) and the clear yellowish filtrate was concentrated in vacuum to give 134 g brown viscous residue (HPLC: 82%). Water (300 ml) was added, made alkaline (40% NaOH, pH>12) and extracted with 120 mL dichloromethane that after concentration gave 77.2 g brown sticky mass (HPLC: 83%). The residue was treated with butanol (5×100 mL, 95° C.), cooled down and the butanol phase over an oily phase was filtered. A total of 74.9 g solid phase was resulted which was dissolved in 200 mL acetone and the clear yellow solution was evaporated to give 70.1 g dark yellow clear viscous residue. The residue was treated with heptane (2×100 mL, 95° C.) which was cooled and decanted. After evaporation in a rotavapor a pale yellow foamy solid was obtained (65.1 g, HPLC: 84%). The solid was dissolved in 200 mL dichloromethane, 85 g silica was added and the mixture was evaporated and loaded on 1.32 Kg silica gel column which was eluted by dichloromethane with 0.5-3.0% methanol and 0.5% triethylamine. Compound 2 was isolated to give 25.8 g, HPLC: 93.2%, 1H-NMR assay: 91.2%.

NMR Identity Analysis of Compound 2

The following data in Tables 4 and 5 was determined using a sample of 62.03 mg Compound 2, a solvent of 0.6 ml CDCl3, 99.8 atom % D, and the instrument was a Bruker Avance III 400 MHz.

TABLE 4 Assignment of 11H NMRa,c 1H Shift COSY cross HMBC cross (ppm) Integral Multiplicity Assignment peaks peaks 7.87 2 s H20 H22b, H24b C16, C21b, C22, C24 7.72- 4 m H1, H5, H22 H2, H23 C1, C3, 7.80  C5, C8, C20, C24 7.47- 6 m H2, H3, H23, H1, H3 C1, C4, C5, 7.56  H24 H20, H22 C6, C8, C16, C19, C21, C20, C22, C24b 4.33 1 t, J = H16 H15 C14b, C15, 7.1 Hz C19, C20, C24 3.05 9 s H18, H25 — — 2.94 4 d, J = H10, H12 H10, H12, C8, C9b, C10, 11.5 Hz H9, H13 C12, C13b 2.53- 1 m H8  H9, H13 C3b, C4, C5b, 2.62  C9, C13, C10b, C12b 2.24- 4 m H14, H15 H16 C10, C12, 2.34  C14, C15, C16, C19 2.02 2 t, J = H10, H12, H10, H12, C8, C9b, C10, 11.5 Hz H9, H13 C12, C13b, C14 1.72- 4 m H9, H13 H8, H10, C4b, C8, C9, 1.85  H12 C10b, C12b, C13 aThe assignment is based on the coupling pattern of the signals, coupling constants and chemical shifts. bWeak signal. cSpectra is calibrated by the solvent residual peak (7.28 ppm).

TABLE 5 Assignment of 13C NMRa,b 13C Shift (ppm) Assignment 148.0 C4  145.5 C19 141.0 C21 140.6 C6  133.2 C24 132.3 C3  129.9 C23 129.5 C2  126.7 C20 125.7 C22 125.6 C5  125.2 C1  55.9 C14 54.0 C10, C12 48.2 C16 44.51 C18 44.48 C75 42.4 C8  32.3 C9, C13 31.8 C15 aThe assignment is based on the chemical shifts and 1H-13C couplings extracted from HSQC and HMBC experiments. bSpectra is calibrated by a solvent peak (77.16 ppm)

Example 3—Preparation of Compound 3 (1,4-bis((3-(1-propylpiperidin-4-yl)phenyl)sulfonyl)butane) Preparation of 1,4-bis((3-bromophenyl)thio)butane (Compound 3, 1st Int.)

KOH (56.2 g) was added into methanol (1200 mL) in 15 min. The clear solution was cooled on water bath to 0° C. A solution of 3-bromo thiophenol (150.2 g, 0.79 mol) in methanol (200 mL) was added in 50 min keeping the temperature at 1-3° C. A solution of 1,4-dibromobutane (86.5 g; 0.40 mol) in methanol (150 ml) was added in 40 min to give a yellow turbid mixture. After additional 4 hours stirring the reaction mixture became white turbid and it was stirred for additional 20 h at 25° C. The suspension was filtered and washed with water (3×100 mL) and methanol (2×100 mL) to give 239 g wet white solid that was dried to 163.6 g (94.6% yield, HPLC: 97.9%).

Preparation of 1,4-bis((3-bromophenyl)sulfonyl)butane (Compound 3, 2nd Intermediate)

To a solution of 1,4-bis-(3-bromophenylthio)-butane (155.0 g, 0.358 mol) in acetic acid (1500 mL) was added sodium tungstate dihydrate (2.5 g, 0.0075 mol) and the suspension was heated on water-bath to 45° C. 50% H2O2 (300 mL, 5.28 mol) was added drop-wise into the reaction mixture during 3.5 h keeping the temperature at 45-55° C. The reaction mixture was kept under stirring for additional 3 h at 45° C. and 16 h at 23° C. The off white slurry was filtered, washed with water (3×200 mL) and dried on air to give 179.6 g (99% crude yield, HPLC: 92.2% product, 7.1% by product). The crude product (175 g) was added to toluene (1400 mL) and heated to >85° C. for distillation. Distillation stopped when no more water was distilled (180 mL toluene and 10 mL water). The clear reaction mixture was allowed to cool down and was filtered after overnight stirring at ambient temperature. The bright colorless crystals were washed (150 mL toluene) and dried to give 156.1 g product (86.7% yield, HPLC: product 96.0%, main by-product 3.5%).

Preparation of 1,4-bis((3-(pyridin-4-yl)phenyl)sulfonyl)butane (Compound 3 3rd Intermediate)

To a solution of 1,4-Bis-((3-bromophenyl)-sulfonyl)-butane (92.0 g, 185 mmol) and butanol (1.0 L) was added 4-pyridinylboronic acid (75.0 g, 610 mmol), potassium carbonate (172 g, 1.24 mol) and the catalyst trans-dichlorobis-(triphenylphosphine) palladium (2.0 g; 2.8 mmol). The violet suspension was heated at stirring under nitrogen to 90-95° C. within 1 h. The reaction mixture became brown and heating continued for more 4 h. Additional 4-pyridinylboronic acid (3.5 g, 28 mmol) was added and the reaction mixture heated up to 100° C. for 1 h. Heating stopped, water (600 mL) was added and the temperature dropped to 60° C. The resulting dark gray suspension was stirred overnight at ambient temperature and filtered (slowly). The filter cake was washed with water (100 mL) to give 153 g wet solid which was suspended in hot acetone (2×IL., 50° C.). The solid was then suspended with 0.51 water at 65° C. followed by 2×1 L acetone suspension. The acetone solution were combined and concentrated on a rotavapor to give 90.3 g pale yellow solid (yield: 91%, HPLC: 91.8%).

Preparation of 4,4′-((butane-1,4-diyldisulfonyl)bis(3,1-phenylene))bis(1-propylpyridin-1-ium)iodide (Compound 3 4th Intermediate)

To a solution of 1,4-Bis-((3-(pyridin-4-yl)-phenyl)-sulfonyl)-butane (85.8 g, 16 mmol) and butanol (450 mL) was added 1-iodopropane (91.7 g, 540 mmol). The stirring mixture was heated up to 90-95° C. in nitrogen atmosphere and kept at this temperature for 6 hours. The dark yellow slurry was then cooled down to room temperature and kept at this temperature for 15 h. The yellow clear solution was then decanted and butanol (300 mL) was added. The mixture was heated to 70° C. where it dissolved. Heating continued to 95° C. and light brown slurry appeared. The heating was stopped and the mixture cooled down to 10° C. The yellow cloudy liquid was decanted and a dark yellow solid mass was filtered to give 173.5 g (HPLC 84% area) which was used as is in the next step.

Preparation of 1,4-bis((3-(1-propyl-1,2,3,6-tetrahydropyridin-4-yl)phenyl)sulfonyl) butane (Compound 3, 5th Intermediate)

To the solid crude starting material (173.5 g from the previous stage) was added methanol (450 mL) and the mixture was heated to reflux to give dark yellowish red clear solution which after cooling gave two phases, the lower one weigh 150 g (HPLC: 88.4%, yield corrected to %: 131 g, 157 mmol). Methanol (400 mL) was added and the mixture was cooled (0° C.). Sodium borohydride (23.75 g, 624 mmol, 4 eq) was added and the reaction mixture was allowed to warm to RT and stirred for additional 9 h. The workup includes concentrating filtrates and precipitating from butanol and methanol, several slurries in butanol, extraction by hot butanol from water and finally active carbon treatment to the product dissolved in hot butanol to give 63.0 g (HPLC: 85%) which was used as is in the next step.

Preparation of 1,4-bis((3-(1-propylpiperidine-4yl)phenyl)sulfonyl)butane (Compound 3)

The product from the previous step (60.0 g, 51 g as HPLC is 85%, 87 mmol) was added into an autoclave with 350 mL acetic acid. A suspension of 10% Pd/C catalyst (10 g, 9.4 mmol) in water (80 mL) was added. Air was exchange to nitrogen and then hydrogen was introduced (150 psi) and the reaction was heated to 85° C. for 6 h. After cooling the catalyst was filtered, washed with acetic acid (2×30 mL) and water (2×30 mL) and concentrated under vacuum to give 98 g of slightly brownish viscous residue. The residue was dissolved with water (200 mL, filtered (to remove traces of charcoal) and washed with 50 mL water. To the slightly brownish solution was added concentrated NaOH up to pH 13 and the mixture was stirred for 30 m. The massive precipitation was filtered to give 78.1 g slightly beige wet solid. The wet solid was mixed with water (100 mL) and toluene (300 mL), heated up to 87° C. for 30 min and the dark yellow water phase was separated. The organic phase was filtered and cooled down to 30° C. After 4 h the slurry was filtered, washed with 20 mL toluene and dried to give 40.8 g off-white solid (HPLC: 74.4%). The solid was then suspended in toluene (260 mL) and water (40 mL) and heated up to 85° C. The colorless water phase was separated and the toluene phase was filtered, cooled down to 5° C. for 2 hr and filtered to give after drying 38.0 g off-white solid (HPLC: 81.5%). The solid was then crystallized twice from toluene (300 mL, heating to 90° C., cooled to 3° C., filtered, washed with 30 mL toluene, dried) to give 31.2 g, HPLC: 96.9%, 1H-NMR assay: 93.9%.

NMR Identity Analysis of Compound 3

The following data in Tables 6 and 7 was determined using a sample of 47.82 mg Compound 3, a solvent of 1.0 ml DMSO-D6, 99.9 atom % D, and the instrument was a Bruker Avance III 400 MHz.

TABLE 6 Assignment of 1H NMRa,c 1H COSY HMBC Shift cross cross (ppm) Integral Multiplicity Assignment peaks peaks  7.68- 2 m H5  H1b, H3b C1, C3, C8 7.70 7.66 2 dt, J = 7.5 and H1  H5b, H2 C3, C5 1.2 Hz 7.63 2 d, J = 7.7 Hz H3  H2, H5b C1, C5, C8 7.55 2 t, J = 7.5 Hz H2  H1, H3 C1b, C3b, C4, C6  3.30- 4 m H18 H19 C19 3.37 2.95 4 d, J = 11.5 Hz H10, H12 H10, H12, C8b H9, H13 2.61 2 t, J = 11.9 Hz H8  H9, H13 — 2.25 4 t, J = 7.2 Hz H14 H15 C10, C12, C15, C16 1.96 4 t, J = 11.9 Hz H10, H12 H10, H12, — H9, H13 1.76 4 d, J = 12.6 Hz H9, H13 H8, H9, — H10, H12, H13  1.62- 4 m H9, H13 H8, H9, C10b, C12b, 1.71 H10, H12, C9b, C13b H13  1.59- 4 m H19 H18 C19b 1.65 1.43 4 sextet, J = H15 H14, H16 C14, C16 7.3 Hz 0.86 3 t, J = 7.2 Hz H16 H15 C14, C15 aThe assignment is based on the coupling pattern of the signals, coupling constants and chemical shifts. Due to the low concentration of dissolved material some expected HMBC signals were masked by background noise. bWeak signal. cSpectra is calibrated by the solvent residual peak (2.5 ppm).

TABLE 7 Assignment of 13C NMRa ,b 13C Shift (ppm) Assignment 147.9 C6  139.2 C4  132.2 C3  129.4 C2  125.7 C5  125.2 C1  60.2 C14 53.7 C10, C12, C18 41.7 C8  32.8 C9, C13 20.7 C19 19.7 C15 11.9 C16 aThe assignment is based on the chemical shifts and 1H-13C couplings extracted from HSQC and HMBC experiments. bSpectra is calibrated by a solvent peak (39.54 ppm).

Example 4—Preparation of Compound 4 ((3R,4S)-4-(3-(methylsulfonyl)phenyl)-1-propylpiperidin-3-ol) Preparation of (1S,6S)-6-(3-(methylsulfonyl)phenyl)-3-propyl-7-oxa-3-azabicyclo [4.1.0]heptane

Into a 4 L reactor was added at room temperature Compound 8 (229 g, 820 mmol, 1 eq) and 2N sulfuric acid (1147 mL, 112 g sulfuric acid, 1.147 mol, 1.4 eq). The reaction light yellow mixture was stirred and sodium bromate (126 g, 836 mmol, 1.02 eq) was added. The mixture became yellow and the temperature dropped (endothermic dissolution). After 30 min the reaction temperature reached 35° C. and heated further to 40° C. for 6 h to give dark yellow solution with precipitate in the bottom of reactor. Toluene (2 L) and NaOH (24%, 546 g, 131 g NaOH, 3.28 mol, 4.0 eq) were added and the reaction mixture was vigorously stirred for 1 hour at 42° C. The reaction mixture was then poured into a 4 L separation funnel. The dark water phase was discarded and the dark red organic phase was washed with 1.1 L 5% sodium sulphite solution and 1 L 20% brine. The organic phase was then concentrated on a rotavapor (50° C., 90-65 mbar, finally at 45 mbar) to give 111 g dark red oil with crystals in the flask. A GC analysis (5 mg red oil dissolved in 0.6 ml toluene) showed 53% product, 29% and 5.2% unknown peaks and 0.4% Compound 8. The product goes to the reduction in the next stage.

Preparation of (3S,4R)-4-(3-(methylsulfonyl)phenyl)-1-propylpiperidin-3-ol (Compound 4)

The epoxide from the previous stage (111 g of 53% GC purity, 62.0 g, 210 mmol, 1 eq) was dissolved in ethanol (1.2 L) for 1 h. The red colored mixture was poured into 2 L Parr reactor and a solution of 10% Pd/C (14.6 g, dry) in ethanol (50 mL) was added. The mixture was reacted with hydrogen (4 bar) at 30° C. for 10 hr. Pd/C was filtered through a Celite and the filtrate was concentrated in the rotavapor to give 108 g red oil (65% product by GC). The product was added to 200 g silica gel, 0.5% triethylamine in dichloromethane were added and the mixture was concentrated and loaded on a column with 620 g silica gel. The purification was done with 0.5% triethylamine in dichloromethane to give 28 g hard residue (97.0% by GC). The residue was heated to reflux in 34 mL dichloromethane until complete dissolution to give clear red solution which was cooled slowly with parallel removal of some solvent by nitrogen flow over the solvent. The precipitation was filtered and washed with dichloromethane (5 mL) to give 20 g white solid, HPLC: 99.0%, 1H-NMR assay: 99.4%.

NMR Identity Analysis of Compound 4

The following data in Tables 8 and 9 was determined using a sample of 54.06 mg Compound 4, a solvent of 0.55 ml DMSO-D6, 99.9 atom % D, and the instrument was a Bruker Avance III 400 MHz.

TABLE 8 Assignment of 1H NMRa,c 1H COSY HMBC Shift cross cross (ppm) Integral Multiplicity Assignment peaks peaks 7.85 1 s H5  H1b, H2b, C1, C3, C8 H3b 7.75 1 d, J = 7.9 Hz H1  H2, H3b, H5b C5, C3, C2b 7.65 1 d, J = 7.7 Hz H3  H2, H1b, H5b C1, C5, C8 7.55 1 t, J = 7.6 Hz H2  H1, H3, H5b C4, C6 4.15 1 d., J = 7.5 Hz H17 H13 C12b, C13 3.78 1 d, J = 6.6 Hz H13 H12b, H17 C9b 3.18 3 s H18 — C6   2.92- 2 m H10, H12 H9, H10, C8, C10, C13b 2.76 1 dt, J = 13.0 H8  H9  C3b, C4, C5b and 3.3 Hz  2.19- 3 m H14, H9 H9, H10, C10, C12, 2.32 H15 C15, C16 2.16 1 d, J = 11.5 Hz H12 H12 C10, C14 2.00 1 t, J = 11.2 Hz H10 H9, H10 C8b, C12 1.54 1 d, J = 12.3 Hz H9  H9, H10 C13b 1.46 2 sextet, J = H15 H14, H16 C14, C16 7.3 Hz 0.88 3 t, J = 7.3 Hz H16 H15 C14, C15 aThe assignment is based on the coupling pattern of the signals, coupling constants and chemical shifts. bWeak signal. cSpectra is calibrated by the solvent residual peak (2.5 ppm). indicates data missing or illegible when filed

TABLE 9 Assignment of 13C NMRa,b 13C Shift (ppm) Assignment 145.6 C4  140.4 C6  133.3 C3  128.8 C2  126.3 C5  124.4 C1  67.8 C13 60.1 C12 59.8 C14 53.3 C10 45.6 C8  43.6 C18 25.2 C9  19.3 C15 11.9 C16 aThe assignment is based on the chemical shifts and 1H-13C couplings extracted from HSQC and HMBC experiments. bSpectra is calibrated by a solvent peak (39.54 ppm)

Example 5—Preparation of Compound 5 (4-(3-(methylsulfonyl)phenyl)-1-propylpiperidine 1-oxide)

Pridopidine (50.0 g, 178 mmol, 1 eq) was dissolved in methanol (250 mL) and 33% hydrogen peroxide (20 mL, 213 mmol, 1.2 eq). The reaction mixture was heated and kept at 40° C. for 20 h. The reaction mixture was then concentrated in a rotavapor to give 71 g light-yellow oil. Water (400 mL) was added and the suspension was extracted with isopropyl acetate (150 mL) which after separation contains unreacted pridopidine while water phase contains 91% of Compound 5 (HPLC). The product was then washed with dichloromethane (400 mL) after adjusting the water phase pH to 9 by sodium hydroxide. After phase separation the water phase was washed again with dichloromethane (200 mL) to give 100% of Compound 5 in the water phase (HPLC). The product was then extracted from the water phase into butanol (1×400 mL, 3×200 ml) and the butanol phases were combined and concentrated in a rotavapor to give 80 g yellow oil (HPLC: 100% of Compound 5). The oil was washed with water (150 mL) to remove salts and the water was extracted with butanol. The organic phases were combined and concentrated in a rotavapor to give 43 g of white solid which was suspended in MTBE for 1 hr, filtered and dried to give 33 g solid that was melted when standing on air. After high vacuum drying (2 mbar, 60° C., 2.5 h) 32.23 g pure Compound 5 were obtained (HPLC: 99.5%, 1H-NMR assay: 97.4%).

NMR Identity Analysis of Compound 5

The following data in Tables 10 and 11 was determined using a sample of 63.06 mg Compound 5, a solvent of 1.2 ml DMSO-D6, 99.9 atom % D, and the instrument was a Bruker Avarice III 400 MHz.

TABLE 10 Assignment of 1H NMRa,c 1H Shift COSY cross HMBC cross (ppm) Integral Multiplicity Assignment peaks peaks 7.81 1 bs H5 — C1, C3, C8  7.78- 1 m H1 H2 C3, C5 7.80  7.63- I m H3 H2 C1, C4b, C5, C8 7.66  7.59- 1 m H2 H1, H3 C1b, C4, C6 7.63 3.27 7 t, J = 11.2 Hz H10, H12 H9, H10, C8, C9, C13 H12, H13 3.23 3 s  H18 — C1b, C6  3.07- 7 m  H14  H15 C10, C12, C15, 3.11 C16 3.02 2 d, d = 13.1 Hz H10, H12 H9, H10, C8, C9b, C13b 2.81 1 t, J = 12.7 Hz H8 H9, H13 C3b, C4, C5b, C9, C13, C10, C12  2.39- 2 m H9, H13 H8, H9 C4, C8, C10, 2.50 H10, H12, C12, C9, C13  H13  1.79- 2 m  H15 H14, H16 C14, C16 1.89 1.64 2 d, J = 12.8 Hz H9, H13 H8b, H9, C4b, C8b, C10b, H10b, H12b, C12b  H13 0.90 3 t, J = 7.5 Hz  H16  H15 C14, C15 aThe assignment is based on the coupling pattern of the signals, coupling constants and chemical shifts. bWeak signal. cSpectra is calibrated by the solvent residual peak (2.5 ppm). indicates data missing or illegible when filed

TABLE 11 Assignment of 13C NMRa,b 13C Shift (ppm) Assignment 146.9 C4 141.0 C6 132.1 C3 129.6 C2 125.0 C1 124.9 C5 72.4  C14 63.4 C10, C12 43.5  C18 39.4 C8 27.3 C9, C13 15.1  C15 11.3  C16 aThe assignment is based on the chemical shifts and 1H-13C couplings extracted from HSQC and HMBC experiments. bSpectra is calibrated by a solvent peak (39.54 ppm)

Example 6—Preparation of Compound 6 (1-(2-methylpentyl)-4-(3-(methylsulfonyl)phenyl)piperidine)

Into a 1 L autoclave was added KI (28.4 g, 171 mmol 1 eq) and potassium carbonate (47.4 g, 343 mmol, 2 eq). 4-(3-(methylsulfonyl)phenyl)piperidine (41 g, 171 mmol, 1 eq) was dissolved in acetonitrile (420 mL) and the mixture was added into the autoclave followed by 1-chloro-2-methylpentane (25.8 mL, 188 mmol, 1.1 eq). The autoclave was closed and the reaction mixture was heated under nitrogen atmosphere to 120° C. for 30 hr. The reaction mixture was cooled down and filtered. The cake was washed with acetonitrile and the filtrate was concentrated in vacuum to give 70 g crude product with the following HPLC s: 60% of Compound 6, 1% of 4-(3-(methylsulfonyl)phenyl)piperidine and 10% of a by-product. The crude product was dissolved in toluene (350 ml) and about 20 g solid material was filtered. The toluene phase was washed with water (200 mL) and concentrated in a rotavapor to give 35.5 g (73% of product by HPLC). The residue was then dissolved in ethyl acetate (180 mL) and cooled on ice bath. Into the reaction mixture was then added 33 mL of 18% HCl solution in ethyl acetate in 1 hr and the mixture was stirred for an additional 1 h. The precipitate that was formed was then filtered, washed with ethyl acetate and dried to give 36.3 g white solid (HPLC: 94%. The product was recrystallized by dissolving in methanol (290 mL), heating to 70° C., adding ethyl acetate (400 mL) and cooling to room temperature. The precipitate was filtered, washed with ethyl acetate (60 mL) and dried in vacuum at 50° C. to give 28.3 g Compound 6 (HPLC: 99.5%, 1H-NMR assay: 99.6%).

NMR Identity Analysis of Compound 6

The following data in Tables 12 and 13 was determined using a sample of 33.93 mg Compound 6, a solvent of 8 ml DMSO-D6, 99.9 atom % D, and the instrument was a Bruker Avance III 400 MHz. Two conformers (ca 10:1) at room temperature are observed. Due to the overlap of proton signals of the major and minor conformers and relatively weak signal of the minor isomer in 21) speactra only some of the peaks of minor isomer on 1 H spectra and corresponding 1 H-1 H COSY cross peaks are given. Due to the low solubility of the material in D6-DMSO some of the expected HMBC signals are masked by background noise.

TABLE 12 Assignment of 1H NMRa,c 1H Shift COSY cross HMBC cross (ppm) Integral Multiplicity Assignment peaks peaks 9.88 1 bs NH H10, H12, — H14 7.79- 2 m H1, H5 H2, H3 C1, C3, C5, C8 7.84 7.62- 2 m H2, H3 H1, H5 C1, C4, C5, C6, 7.66 C8b 3.53- 2 m H10, H12 H10, H12 — 3.63 3.23 3 s H18 — C5b, C6 2.87- 5 m H8, H10, H12, H9, H10, C9, C12b, C13, 3.11 H14 H12, H13, C15, C16, C19b H15 2.17- 2 m H9, H13 H8, H9, — 2.34 H10, H12, H13  1.94- 3 m H9, H13, H15 H8, H9, — 2.02 H10, H12,  1.22- 3 m H19, H20 H15, H19, C20 1.45 H20, H21  1.10- 1 m H19 H15, H20 C16, C20, C21 1.21 1.02 3 d , J = 6.7 Hz H16 H15 C14, C15, C19 0.90 3 t, J = 6.S Hz H21 H20 C19, C20 Minor isomer 10.14 1 bs NH H10, H12 — 7.88 1 s H5 — — 7.75 1 d, J = S.5 Hz H1 H2 —  3.24- 4 m H10, H12 H9, H13 — 3.31  1.86- 3 m H9, H13, H15 H10, H12, — 1.84 H16 aThe assignment is based on the coupling pattern of the signals, coupling constants and chemical shifts. bWeak signal. cSpectra is calibrated by the solvent residual peak (2.5 ppm).

TABLE 13 Assignment of 13C NMRa,b 13C Shift (ppm) Assignment 145.9 C4  141.1 C6  131.9 C3  129.8 C2  125.3 C1  124.9 C5  62.5 C14 53.1 C10 51.8 C12 43.5 C18 38.5 C8  36.4 C19 29.20 and 29.24 C9, C13 27.5 C15 19.1 C20 18.0 C16 14.0 C21 aThe assignment is based on the chemical shifts and 1H-13C couplings extracted from HSQC and HMBC experiments. bSpectra is calibrated by a solvent peak (39.54 ppm)

Example 7—Preparation of Compound 7 (4-(3-(methylsulfinyl)phenyl)-1-propyl-1,2,3,6-tetrahydropyridine)

Sulfuric acid (42.23 g, 0.431 mol, 1 eq) was added to a mixture of 4-hydroxy-4-(3-(methylsulfonyl)phenyl)-1-propylpiperidin-1-ium chloride (130 g, 0.431 mo, 1 eq) and toluene (650 mL) at room temperature. The resulting two-phase solution was refluxed for 1 hour and HPLC showed that the product reached 95%. The reaction mixture was cooled down to 20° C. and the toluene phase was decanted to give viscous residue that was diluted with water (600 mL) and neutralized with 2N NaOH to pH˜4.2. Hydrogen peroxide (50%, 32.21 g, 0.474 mol, 1.1 eq) 1 was added dropwise to the water phase and the mixture was stirred at 60° C. for 1 h after which the product reached 96% (HPLC).

Toluene (600 mL) was added to the reaction mixture and made basic first with 25% NaOH (60 g) and finally with 10% Na01-1 up to pH 12. The phases were separated and the water phase was re-extracted with toluene (2×100 mL). The combined toluene phases were washed with 5% sodium sulfite (150 mL), brine (150 mL) and water (150 mL). The toluene phase was then concentrated under vacuum on a rotavapor to give 111.3 g oil (HPLC: 96.6%). Methanol (50 mL) was added to the residue and it was filtered and cooled down on ice batch. Dry HC in ethyl acetate was added up to pH 1-2 (120 mL) and 100 mL of ethyl ether were added to give two phases mixture. The mixture was seeded with the product and precipitation started. The reaction mixture was stirred on ice bath (2-5° C.) for additional 1 h, filtered and washed with 1/3 ethyl acetate/ether mixture (100 mL) to give 140 g of very hygroscopic light yellow solid that was dried on a rotavapor for 2 h and stored under nitrogen in deep freeze. The dry 4-(3-(methylsulfinyl)phenyl)-1-propyl-1,2,3,6-tetrahydropyridine-HCl is slightly yellowish solid (94.1 g, 79% yield, HPLC (254 nm): 96.3%, 1H-NMR assay: 97.5%).

NMR Identity Analysis of Compound 7

The following data in Tables 14 and 15 was determined using a sample of Compound 7, a solvent of CDCl3, and the instruments were a Bruker AMX500 and Avance III 800 MHz instrument.

TABLE 14 Assignment of 1H NMRa 1H Shift 1H Shift COSY cross (ppm) (ppm)b Integral Multiplicity Assignment peaks 2.63 2.66 2 t, 2 × 5.7 to H3 H2 H3 2.51 2.55 2 m H3 H2, H5, H6 6.10 6.13 1 tt, 2 × 3.6 to H6, H5 H3, H6 2 × 1.5 to H3 3.09 3.13 2 m H6 H3, H5 2.35 2.39 2 m H7 H8 1.51 1.54 2 m H8 H7, H9 0.86 0.89 3 t, 2 × 7.4 to H8 H9 H8 7.60 7.49 1 dt, 0.4 to H5’, 2 × 1.8 H2’ H4’, H5’, to H4’ and H6’ H6’ 7.37 7.40 1 ddd, 1.4 to H6’, 1.8 H4’ H2’, H5’, to H5’, 7.6 to H2’ H6’ 7.36 7.37 1 dt, 0.4 to H2’, 2 × 7.6 H5’ H2’ H4’, H6’ to H4’ and H6’ 7.41 7.44 1 ddd, 1.4 to H4’, 1.8 H6’ H2’, H4’, to H5’, 7.6 to H2’ H5’ 2.62 2.66 3 s H7’ — aSpectra is calibrated by the solvent residual peak (2.5 ppm). bafter addition of small amount of C6D6

TABLE 15 Assignment of 13C NMRa, 13C Shift HMBC 1H 13C Shift HMBC 1H (ppm) Assignment cross peaks (ppm) Assignment cross peaks 49.89 C2 C4, 6, 7 142.00 C1’ — 27.68 C3 C2, 4, 5 119.41 C2’ C4, 6, 3’, 4’ 133.67 C4 — 145.52 C3’ — 123.57 C5 C3 ,6, 1’ 121.51 C4’ C2’6’ 52.90 C6 C2, 4, 5, 7 128.97 C5’ — 60.04 C7 C2, 6, 8, 9 127.19 C6’ C2’, 4’, 4 20.02 C8 C7, 9 43.70 C7’ 3’ 11.72 C9 C7, 8 aSpectra is calibrated by a solvent peak (77.0 ppm)

Example 8—Analysis of the Amounts of Compounds 1, 2, 3, 4, 5 and 6 in a Sample of Pridopidine Drug Substance

Compounds 1-7 are useful to determine the purity of a pridopidine containing composition.

The procedure used for determination of assay and related substances in pridopidine HCl is a reverse phase HPLC method using X-bridge phenyl column (or equivalent) and gradient elution with UV detection at 268 nm. The mobile phase consists of a mixture of methanol and ammonium formate buffer.

Apparatus

HPLC with gradient pump column thermostat and UV-detector, Column: Waters, X-bridge Phenyl, 75×4.6 mm, 2.5 μm, or an equivalent column.

Analytical Instruction Reagents and Solutions

Solvents: Methanol, HPLC grade; Water, MilliQ-water or equivalent

Reagents Ammonium formate, purum; Ammonium hydroxide, 30% A.C.S; Formic acid, pa Ammonium formate buffer, 1.00 mM, pH 8.90-9.10. Weigh 6.3-6.4 g ammonium formate accurately into a 1000 mL volumetric flask and add 2.5 ml 30% ammonium hydroxide solution Dissolve and dilute with milliQ-water to 900 mL Measure the pH of the solution. The pH should be between 8.90 and 9.10, otherwise adjust with ammonium hydroxide or formic acid. Dilute to volume and filter through a 0.45 μm HVLP-filter.

Reference substances: Control Sample 1a: (pridopidine) (see FIG. 1, Control Sample 2b (Compound 5, Compound 1, Compound 4, pridopidine Compound 8, Compound 2, Compound 6, Compound 3)

TABLE 16 Phase Solvent Amount Mobile phase Ammonium formate buffer, 100 mM, 100 mL A pH 9.0 MilliQ-water 900 mL Mobile phase Ammonium formate buffer, 100 mM, 100 mL B pH 9.0 MilliQ-water  50 mL Methanol 850 mL Dilution Methanol 150 mL phase MilliQ-water 850 mL

TABLE 17 Analytical conditions Flow 0.8 mL/min Gradient Time (min) Mobile phase B (%)  0  50 10 100 12 100 Equilibration time 3 min. Wavelength 268 nm (bandwidth 4 nm; reference off) 190-400 nm (for information in stability studies only). Injected volume 20 μL Needle wash Set wash cycles to two. Use dilution phase in washing vial. Temperature 40°C.

TABLE 18 Approximate retention times Substance Time (min) Compound 5 1.9 Compound 1 2.4 Compound 4 3.5 Pridopidine 4.6 Compound 8 6.1 Compound 2 7.5 Compound 6 8.8 Compound 3 9.9

Blank Preparation:

Use dilution phase. Duplicate vials of blank (A and B).

Reference Preparation a (Only for Related Substances)

Use Control Sample 2b. Inject as it is.

The Control Sample 2b solution is a pridopidine solution (0.44 mg/ml free base) spiked with approximately 1% of each of the impurities: Compound 5, Compound 1, Compound 4, Compound 8, Compound 2, Compound 6 and Compound 3.

Reference Preparation B (Only for Assay) Duplicate Preparation (B1 and B2).

Weigh 43-45 mg of pridopidine reference into a 50 mL volumetric flask. Add 25 mL dilution phase and shake or sonicate at ambient temperature until the reference is dissolved. Make to volume with dilution phase. Concentration: 0.9 mg/mL pridopidine. The standard solution is stable for 48 hours when stored in daylight and in room temperature.

Reference Preparation C (Only for Related Substances) Single Preparation (C).

Dilute 1 mL of reference 1 to 100 mL with dilution phase. Dilute further 1 mL of this solution to 20 mL with dilution phase (sensitivity standard, concentration corresponding to 0.05% of sample concentration).

Sample Preparation

Duplicate preparation (sample A and B).

Weigh 43-45 mg of the sample of pridopidine into a 50 mL volumetric flask. Add 25 mL dilution phase and shake or sonicate at ambient temperature until the sample is dissolved. Make to volume with dilution phase. Concentration: 0.9 mg/mL pridopidine. The sample solution should be freshly prepared before use.

Order of Determinations

When the system is equilibrated, inject the solutions in the following order:

TABLE 19 Number of determinations/injections Solution Assay Related substances Blank A 3 (at least) 1 (at least) Blank B 1 1 Reference A  N/A 1 Reference C  N/A 1 Reference B1 5 N/A Reference B2 1 N/A Sample A 1 1 Sample B 1 1 . . . . . . . . . Reference B2 1 N/A

Calculation System Suitability For Related Substances:

R1) The Blank B should be free from interfering peaks at the retention times of Compound 5, Compound 1, Compound 4, pridopidine, Compound 8, Compound 2, Compound 6 and Compound 3.

R2) The retention time of the pridopidine peak should be 4.6±0.5 min.

R3) Compound 5, Compound 1, Compound 4, pridopidine, Compound 8, Compound 2, Compound 6 and Compound 3 in the Control Sample 2b should be possible to identify according to FIG. 2.

R4) The pridopidine peak in reference C should have a signal-to-noise ratio greater than or equal to 3.

R5) Calculate the number of theoretical plates (N) and the tailing factor (T) for the pridopidine peak in reference A. Number of theoretical plates 2: 8000 and tailing factor 0.7-1.0.

R6) Calculate the resolution between Compound 5 and Compound 1 in the Control Sample 2b, should be greater than or equal to 1.5.

R7) If the problem with split peaks Compound 1 and Compound 4 shall appear, they should be calculated as sum of each split peak.

For Assay:

A1) The Blank B should be free from interfering peak at the retention time for pridopidine.

A2) The retention time of the pridopidine peak should be 4.6±0.5 min,

A3) Calculate the RSD % of the five areas of reference B1. The RSD should be 2.0%.

A4) Calculate the assay of each injections of reference 132. The assay should be in the interval 99-101

w/w-% of the assay of the reference B1.

A5) Calculate the number of theoretical plates (N) and the tailing factor (T) for the pridopidine peak in the first injection of reference B1. Number of theoretical plates 2: 8000 and tailing factor 0.7-1.0.

A6) Calculate the deviation between the two assay determinations (Sample A and B) according to eq. 1 The deviation should be less than or equal to 2%.

Assay A - Assay B × 100 ( Assay A + Assay B ) × 0.5 ≤ 2 ⁢ % ( eq ⁢ .1 )

The analytical method description described herein will be updated to include acceptance criteria for number of theoretical plates (N) and the tailing factor (T) for pridopidine peak.

Result For Related Substances:

The content of related substances should be calculated as −% and corrected with relative response factors and reported as % according to eq. 2.


%x=area-%x×RRFx  (eq. 2)

%x percent content of an impurity ‘x’
area-%x area-% of an impurity ‘x’ calculated from the chromatogram
RRFx Relative Response Factor of an impurity ‘x’

Use following response factors:

TABLE 20 Relative response Name factor Compound 8 0.2 Compound 2 0.7

Remaining related substances will be correct for RRF 1.

For assay:

Calculate the assay of pridopidine in w/w-% using the external standard methodology (see below). Use the mean response factor obtained from the five injections of reference B1 for the calculation.

f x = c xR A xR ( eq ⁢ .3 ) A xS × f x × 100 c xS = pridopidine ⁢ ⁢ ( w ⁢ / ⁢ w ⁢ - ⁢ % ) ( eq ⁢ .4 )

fx mean response factor of pridopidine from reference solution B1
cxR concentration of pridopidine in reference solution (mg/ml)
cxS concentration of sample solution (mg/mL)
AxR area of pridopidine in each injection of reference solution B1
AxS area of pridopidine in sample chromatogram

TABLE 21 Analytical Methods for Determination of Impurities in the Drug Substance Quantitation Quantitation Detection Example Method Limit Limit Limit Parameter Number Type (wegith-%) (area-%) (area-%) Compound Example RP-HPLC 0.04 0.04 0.01 1 8 Compound Example RP-HPLC 0.03 0.05 0.01 2 8 Compound Example RP-HPLC 0.05 0.05 0.03 3 8 Compound Example RP-HPLC 0.04 0.04 0.01 4 8 Compound Example RP-HPLC 0.04 0.04 0.01 5 8 Compound Example RP-HPLC 0.04 0.04 0.01 6 8

During course of the validation the response factors for Compound 5, Compound 1, Compound 4, Compound 8, Compound 2, Compound 6 and Compound 3 has been evaluated and compared to the response factor of pridopidine. The relative response factor of the impurities are presented in Table 22:

TABLE 22 Relative Response Factors Name Relative Response Factor (α pridopidine/α Compound 5 0.91 Compound 1 1.01 Compound 4 1.02 Compound 8 0.16 Compound 2 0.65 Compound 6 1.05 Compound 3 0.99

Example 9—Specification of Pridopidine Hydrochloride Drug Substance

TABLE 23 Ret. time Resolution (tangent Name (min) method) Compound 5 1.99 N/A Compound 1 2.42 3.3 Compound 4 3.58 6.6 pridopidine 4.68 4.9 Compound 8 6.09 7.5 Compound 2 7.36 11.2 Compound 6 8.69 11.8 Compound 3 9.92 10.1

Pridopidine HCl is a white to almost white powder. The specifications of pridopidine HCl are as follows:

TABLE 24 Specification of Pridopidine Hydrochloride Drug Substance Test Acceptance Criteria Method Description White to almost white Visual inspection powder Absence of lumps Pass Visual and touching Identification IR Conforms to reference IR IR spectrum X-ray diffractogram Conforms to reference XRPD X-ray diffractogram Chloride Positive Ph. Eur. Assay, HPLC, % w/w 98.0-102.0 HPLC Related substances, HPLC, area % Compound 5 ≤0.15 HPLC Compound 1 ≤0.15 HPLC Compound 4 ≤0.15 HPLC Compound 8 ≤0.15 HPLC Compound 3 ≤0.15 HPLC Compound 2 ≤0.15 HPLC Compound 6 ≤0.15 HPLC Unknown impurities, each ≤0.10 HPLC Impurities in total ≤0.50 HPLC

Example 10—Accelerated and Long Term Stability in Pridopidine HCl Drug Product

Batches 1, 2 and 3 were manufactured according to cGMP and in scale as the expected commercial scale. Batches 4 and 5 were manufactured according to the current route of synthesis.

The stability program for each batch is detailed below in Table 25.

TABLE 25 Pridopidine HCl Stability Testing Program Batch 1 2 3 4 5 Batch size (kg) 99.7 97.2 96.6 14.9 105.4 Stability study Storage 25° C./60% RH: 0, 3, 6, 9, 12, 18, 24, 25° C./60% RH: 0, 3, 6, 9, conditions and 36, 48 and 60 months 30° C./65% RH: 12, 18, 24, 36, 48 and 60 testing 0, 3, 6, 9 and 12 months months intervals 40° C./75% RH: 0, 3 and 6 months 30° C./65% RH: 0, 3, 6, 9 and 12 months 40° C./75% RH: 0, 3 and 6 months Test Appearance, Identification, Crystal form, Appearance, Identification, parameters Assay, Impurities, Water content, Crystal form, Assay, Microbial limit Impurities, Water content, Microbial limit

Stability data for batches 1, 2, 3, 4 and 5 can be found in Tables 26-37:

TABLE 26 Stability Data of Pridopidine HCl Batch 1 Stored at 25° C./60% RH Acceptance Storage Period (Months) Parameters Criteria 0 3 6 9 12 18 24 36 48 Appearance Color and White White White White White White White White White Form powder powder powder powder powder powder powder powder powder Identitication (by IR) Complies with Conforms Conforms Conforms Conforms Conforms Conforms Conforms Conforms Conforms ref spectrum Crystal Form (by X-Ray) Complies with Conforms Conforms Conforms Conforms Conforms Conforms Not Not Not ref scheduled scheduled scheduled diffractogram Assay (by HPLC) [% 98.0-102.0 99.9 99.7 100.2 100.2 99.6 100.0 100.4 99.8 99.7 w/w] Impurities (by HPLC) [area %] Compound 1 ≤0.15 0.05 0.05 0.05 0.05 0.05 <0.05 <0.05 <0.05 <0.05 Compound 4 ≤0.15 0.09 0.09 0.09 0.09 0.09 0.08 0.08 0.09 0.08 Each (Compound 8, ≤0.15 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 Compound 3, Compound 2, Compound 6 and Compound 5) Unknown Impurities Each ≤0.10 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 Total ≤0.50 0.14 0.15 0.14 0.14 0.14 0.08 0.08 0.09 0.08 Water Content (by KF) Run and 0.03 0.03 0.02 0.01 0.02 <0.05 <0.05 0.06 <0.05 [% w/w] record Microbiological Purity [cfu/g] TAMC ≤1000 <10 Not Not Not <10 Not Not Not Not scheduled scheduled scheduled scheduled scheduled scheduled scheduled TYMC ≤10 <10 Not Not Not <10 Not Not Not Not scheduled scheduled scheduled scheduled scheduled scheduled scheduled E. Coli Absent — — — — — — — — —

TABLE 27 Stability Data of Pridopidine HCl Batch 1 Stored at 40° C./75% RH Acceptance Storage Period (Months) Parameters Criteria 0 3 6 Appearance Color and Form White powder White powder White powder Identification Complies with ref Conforms Conforms Conforms (by IR) spectrum Crystal Form Complies with ref Conforms Conforms Conforms (by X-Ray) diffractogram Assay 98.0-402.0 99.9 99.7 100.1 (by HPLC) [% w/w] Impurities (by HPLC) [area %] Compound 1 ≤0.15 0.05 0.05 0.05 Compound 4 ≤0.15 0.09 0.09 0.09 Each (Compound 8, ≤0.15 <0.05 <0.05 <0.05 Compound 3, Compound 2, Compound 6 and Compound 5 Unknown Impurities Each ≤0.10 <0.05 <0.05 <0.05 Total ≤0.50 0.14 0.14 0.14 Water Content Run and record <0.1 <0.1 <0.1 (by KF) [% w/w) Microbiological Purity [cfu/g] TAMC ≤1000 <10 Not scheduled Not scheduled TYMC ≤10 <10 Not scheduled Not scheduled E. Coli Absent — — —

TABLE 28 Stability Data of Pridopidine HCl Batch 2 Stored at 25° C./60% RH Acceptance Storage Period (Months) Parameters Criteria 0 3 6 9 12 18 24 36 48 Appearance Color and White White White White White White White White White Form powder powder powder powder powder powder powder powder powder Identification (by IR) Complies with Conforms Conforms Conforms Conforms Conforms Conforms Conforms Conforms Conforms ref spectrum Crystal Form (by X- Complies with Conforms Conforms Conforms Conforms Conforms Conforms Not Not Not Ray) ref scheduled scheduled scheduled diffractogram Assay (by HPLC) 98.0-102.0 100.1 99.9 100.4 100.3 100.2 100.1 100.4 99.7 100.5 [% w/w] Impurities (by HPLC) [area %] Compound 4 ≤0.15 0.05 0.05 0.05 0.05 0.05 <0.05 0.05 <0.05 <0.05 Each (Compound 1, ≤0.15 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 Compound 8, Compound 3, Compound 2, Compound 6 and Compound 5) Unknown Impurities ≤0.10 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 Each Total ≤0.50 0.05 0.05 0.05 0.05 0.05 <0.05 0.05 <0.05 <0.05 Water Content (by Run and 0.03 0.02 0.01 0.01 0.02 <0.05 <0.05 <0.05 <0.05 KE) [% w/w) record Microbiological Purity [cfu/g] TAMC ≤1000 <10 Not Not Not <10 Not Not Not Not scheduled scheduled scheduled scheduled scheduled scheduled scheduled TYMC ≤10 <10 Not Not Not <10 Not Not Not Not scheduled scheduled scheduled scheduled scheduled scheduled scheduled E. Coli Absent — — — — — — — — —

TABLE 29 Stability Data of Pridopidine HCl Batch 2 Stored at 40° C./75% RH Acceptance Storage Period (Months) Parameters Criteria 0 3 6 Appearance Color and Form White powder White powder White powder Identification (by IR) Complies with Conforms Conforms Conforms ref spectrum Crystal Form Complies with Conforms Conforms Conforms (by X-Ray) ref diffractogram Assay (by HPLC) 98.0-102.0 100.1 99.6 100.6 [% w/w] Impurities (by HPLC) [area %] Compound 4 ≤0.15 0.05 0.05 0.05 Each (Compound 1, ≤0.15 <0.05 <0.05 <0.05 Compound 8, Compound 3, Compound 2, Compound 6 and Compound 5) Unknown Impurities Each ≤0.10 Total ≤0.50 0.05 0.05 0.05 Water Content (by KF) Run and record <0.1 <0.1 <0.1 [% w/w) Microbiological Purity [cfu/g] TAMC ≤1000 <10 Not scheduled Not scheduled TYMC ≤10 <10 Not scheduled Not scheduled E. Coli Absent — — —

TABLE 30 Stability Data of Pridopidine HCl Batch 3 Stored at 25° C./60% RH Acceptance Storage Period (Months) Parameters Criteria 0 3 6 9 12 18 24 36 48 Appearance Color and White White White White White White White White White Form powder powder powder powder powder powder powder powder powder Identification (by Complies Conforms Conforms Conforms Conforms Conforms Conforms Conforms Conforms Conforms IR) with ref spectrum Crystal Form (by X- Complies Conforms Conforms Conforms Conforms Conforms Conforms Not Not Not Ray) with ref scheduled scheduled scheduled diffractogram Assay (by HPLC) 98.0-102.0 100.5 99.8 100.4 100.6 100.0 100.1 100.5 100.2 100.5 [% w/w] Impurities (by HPLC) [area %] Compound 4 ≤0.15 0.06 0.06 0.06 0.06 0.06 0.06 0.05 0.05 0.05 Each (Compound 1, ≤0.15 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 Compound 8, Compound 3, Compound 2, Compound 6 and Compound 5) Unknown Impurities ≤0.10 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 Each Total ≤0.50 0.06 0.06 0.06 0.06 0.06 0.06 0.05 0.05 0.05 Water Content (by Run and 0.03 0.03 0.01 0.01 0.01 <0.05 <0.05 <0.05 <0.05 KF) [% w/w) record Microbiological Purity [cfu/g] TAMC ≤1000 <10 Not Not Not <10 Not Not Not Not scheduled scheduled scheduled scheduled scheduled scheduled scheduled TYMC ≤10 <10 Not Not Not <10 Not Not Not Not scheduled scheduled scheduled scheduled scheduled scheduled scheduled E. Coli Absent — — — — — — — — —

TABLE 31 Stability Data of Pridopidine HCl Batch 3 Stored at 40° C./75% RH Acceptance Storage Period (Months) Parameters Criteria 0 3 6 Appearance Color and Form White powder White powder White powder Identification (by IR) Complies with Conforms Conforms Conforms ref spectrum Crystal Form Complies with Conforms Conforms Conforms (by X-Ray) ref diffractogram Assay (by HPLC) 98.0-102.0 100.5 99.7 100.5 [% w/w] Impurities (by HPLC) [area %] Compound 4 ≤0.15 0.06 0.06 0.06 Each (Compound 1, ≤0.15 <0.05 <0.05 <0.05 Compound 8, Compound 3, Compound 2, Compound 6 and Compound 5) Unknown Impurities Each ≤0.10 <0.05 <0.05 <0.05 Total ≤0.50 0.06 0.06 0.06 Water Content (by KF) Run and record <0.1 <0.1 <0.1 [% w/w) Microbiological Purity [cfu/g] TAMC ≤1000 <10 Not scheduled Not scheduled TYMC ≤10 <10 Not scheduled Not scheduled E. Coli Absent — — —

TABLE 32 Stability Data of Pridopidine HCl Batch 4 Stored at 25° C./60% RH Acceptance Storage Period (Months) Parameters Criteria 0 3 6 9 12 18 24 36 Appearance Color and Form White White White White White White White White powder powder powder powder powder powder powder powder Identification (by IR) Complies with Conforms Conforms Conforms Conforms Conforms Conforms Conforms Conforms ref spectrum Crystal Form (by X- Complies with Conforms Not Not Not Not Not Not Not Ray) ref scheduled scheduled scheduled scheduled scheduled scheduled scheduled diffractogram Assay by HPLC) 98.0-102.0 100.4 98.6 99.8 99.7 99.5 99.9 99.8 100.1 [% w/w] impurities (by HPLC) [area %] Compound 4 ≤0.15 0.06 0.06 0.07 0.06 0.07 0.06 0.06 0.06 Compound 3 ≤0.15 0.06 <0.05 0.06 0.08 0.06 0.07 0.06 0.05 Compound 8 ≤0.15 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Each (Compound 1, ≤0.10 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 Compound 2, Compound 6, Compound 5 and Unknown Impurities Each) Total ≤050 0.12 0.06 0.13 0.14 0.13 0.14 0.13 0.11 Water Content (by Run and record 0.06 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 KF) [% w/w) Microbiological Purity [cfu/g] TAMC ≤1000 <10 Not Not Not Not Not Not Not scheduled scheduled scheduled scheduled scheduled scheduled scheduled TYMC ≤10 <10 Not Not Not Not Not Not Not scheduled scheduled scheduled scheduled scheduled scheduled scheduled E. Coli Absent Not Not Not Not Not Not Not Not detectable scheduled scheduled scheduled scheduled scheduled scheduled scheduled

TABLE 33 Stability Data of Pridopidine HCl Batch 4 Stored at 30° C./65% RH Acceptance Storage Period (Months) Parameters Criteria 0 3 6 9 12 Appearance Color and White powder White powder White powder White powder White powder Form Identification (by Complies Conforms Conforms Conforms Conforms Conforms IR) with ref spectrum Crystal Form (by X- Complies Conforms Not scheduled Not scheduled Not scheduled Conforms Ray) with ref diffractogram Assay (by HPLC) 98.0-102.0 100.4 99.0 99.5 100.0 99.6 [% w/w] Impurities(by HPLC) [area %] Compound 4 ≤0.15 0.06 0.07 0.07 0.06 0.06 Compound 3 ≤0.15 0.06 <0.05 0.07 0.07 0.06 Compound 8 ≤0.15 <0.01 <0.01 <0.01 <0.01 <0.01 Each (Compound 1, ≤0.15 <0.05 <0.05 <0.05 <0.05 <0.05 Compound 2, Compound 6, Compound 5 and Unknown Impurities Each) Total <0.50 0.12 0.07 0.13 0.13 0.12 Water Content (by Run and <0.1 <0.1 <0.1 <0.1 <0.1 KF) [% w/w) record Microbiological Purity [cfu/g] TAMC ≤1000 <10 Not scheduled Not scheduled Not scheduled <10 TYMC ≤10 <10 Not scheduled Not scheduled Not scheduled <10 E. Coli Absent Not detectable Not scheduled Not scheduled Not scheduled Not detectable

TABLE 34 Stability Data of Pridopidine HCl Batch 4 Stored at 40° C./75% RH Acceptance Storage Period (Months) Parameters Criteria 0 3 6 Appearance Color and Form White powder White powder White powder Identification (by IR) Complies with Conforms Conforms Conforms ref spectrum Crystal Form Complies with Conforms Not scheduled Conforms (by X-Ray) ref diffractogram Assay (by HPLC) 98.0-102.0 100.4 99.5 99.7 [% w/w] Impurities (by HPLC) [area %] Compound 4 ≤0.15 0.06 0.07 0.07 Compound 3 ≤0.15 0.06 <0.05 0.07 Compound 8 ≤0.15 <0.01 <0.01 <0.01 Each (Compound 1, ≤0.15 <0.05 <0.05 <0.05 Compound 2, Compound 6, Compound 5 and Unknown Impurities Each) Total ≤0.50 0.12 0.07 0.13 Water Content (by KF) Run and record 0.06 <0.05 <0.05 [% w/w) Microbiological Purity [cfu/g] TAMC ≤1000 <10 Not scheduled <10 TYMC ≤10 <10 Not scheduled <10 E. Coli Absent Not detectable Not scheduled Not detectable

TABLE 35 Stability Data of Pridopidine HCl Batch 5 Stored at 25° C./60% RH Acceptance Storage Period (Months) Parameters Criteria 0 3 6 9 12 18 24 Appearance Color and White White White White White White White Form powder powder powder powder powder powder powder Identification (by Complies Conforms Conforms Conforms Conforms Conforms Conforms Conforms IR) with ref spectrum Crystal Form (by Complies Conforms Not Not Not Not Not Not X-Ray) with ref scheduled scheduled scheduled scheduled scheduled scheduled diffractogram Assay (by HPLC) 98.0-102 .0 99.8 100.0 99.9 99.9 99.7 100.1 100.1 [% w/w] Impurities (by HPLC) [area %] Compound 3 ≤0.15 0.10 0.09 0.07 0.09 0.11 0.11 0.07 Compound 8 ≤0.15 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Each (Compound ≤0.15 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 Compound 5, Compound 4, Compound 2, Compound 6 and Unknown Impurities Each) Total ≤0.50 0.10 0.09 0.07 0.09 0.11 0.11 0.07 Water Content (by Run and <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 KF) [% w/w) record Microbiological Purity [cfu/g] TAMC ≤1000 Not Not Not Not Not Not Not scheduled scheduled scheduled scheduled scheduled scheduled scheduled TYMC ≤10 Not Not Not Not Not Not Not scheduled scheduled scheduled scheduled scheduled scheduled scheduled E. Coli Absent Not Not Not Not Not Not Not scheduled scheduled scheduled scheduled scheduled scheduled scheduled

TABLE 36 Stability Data of Pridopidine HCl Batch 5 Stored at 30° C./65% RH Acceptance Storage Period (Months) Parameters Criteria 0 3 6 9 12 Appearance Color and White powder White powder White powder White powder White powder Form Identification (by IR) Complies with Conforms Conforms Conforms Conforms Conforms ref spectrum Crystal Form (by X- Complies with Conforms Not scheduled Not scheduled Not scheduled Conforms Ray) ref diffractogram Assay (by HPLC) 98.0-102.0 99.8 99.8 99.8 99.8 99.7 [w/w] Impurities (by HPLC) [area %] Compound 8 ≤0.15 <0.01 <0.01 <0.01 <0.01 <0.01 Compound 3 ≤0.15 0.10 0.10 0.07 0.10 0.10 Each (Compound 5, ≤0.15 <0.05 <0.05 <0.05 <0.05 <0.05 Compound 4, Compound 1, Compound 2, Compound 6 and Unknown Impurities Each) Total ≤0.50 0.10 0.10 0.07 0.10 0.10 Water Content (by Run and record <0.05 <0.05 <0.05 <0.05 <0.05 KF) [% w/w) Microbiological Purity [cfu/g] TAMC ≤1000 Not scheduled Not scheduled Not scheduled Not scheduled <10 TYMC ≤10 Not scheduled Not scheduled Not scheduled Not scheduled <10 E. Coli Absent Not scheduled Not scheduled Not scheduled Not scheduled Not detectable

TABLE 37 Stability Data of Pridopidine HCl Batch 5 Stored at 40° C./75% RH Acceptance Storage Period (Months) Parameters Criteria 0 3 6 Appearance Color and Form White powder White powder White powder Identification (by IR) Complies with Conforms Conforms Conforms ref spectrum Crystal Form Complies with Conforms Not scheduled Conforms (by X-Ray) ref diffractogram Assay (by HPLC) 98.0-102.0 99.8 99.9 99.5 [% w/w] Impurities (by HPLC) [area %] Compound 8 ≤0.15 <0.01 <0.01 <0.01 Compound 3 ≤0.15 0.10 0.10 0.06 Each (Compound 5, ≤0.15 <0.05 <0.05 <0.05 Compound 4, Compound 1, Compound 2, Compound 6 and Unknown Impurities Each) Total ≤0.50 0.10 0.10 0.06 Water Content (by KF) Run and record <0.05 <0.05 <0.05 [% w/w) Microbiological Purity [cf4/g] TAMC ≤1000 Not scheduled Not scheduled <10 TYMC ≤10 Not scheduled Not scheduled <10 E. Coli Absent Not scheduled Not scheduled Not detectable

Summary of the Results in Tables 26-37 and Conclusions: Appearance:

No significant change is observed in color or form when stored at 10° C./75% RH for up to 6 months, at 30° C./65% RH for up to 12 months or at 25° C./60% RH for up to 48 months.

Crystal Form:

No change in polymorphic form is observed when pridopidine HCl is stored at 40° C./75% RH for up to 6 months and at 30° C./65% RH for up to 12 months. X-Ray diffractograms recorded after 18 months at 25° C./60% RH showed no change. X-Ray analyses will be performed again at the end of the long term stability program (60 months).

Assay:

When pridopidine HCl is stored at 40° C./75% RH for up to 6 months, no significant change in assay is observed. Similar no significant change is observed when stored at 30° C./65% RH for up to 12 months or at 25° C./60% RH for up to 48 months.

Impurities:

No degradation of pridopidine HCl is observed when the drug substance is stored at 40° C./75% RH for up to 6 months, at 30° C./65% RH for up to 12 months or at 25° C./60% RH for up to 48 months.

Water Content

No significant change regarding water content is observed when pridopidine HCl is stored at 40° C./75% RH for up to 6 months, at 30° C./65% RH for tip to 12 months or at 25° C./60% RH for up to 48 months.

Conclusion:

No evidence of relevant changes was observed for the parameters tested at any of the storage conditions. Pridopidine HCl is considered physically and chemically stable when stored at 40° C. and 75% RH for up to 6 months, at 30° C./65% RH for up to 12 months or at 25° C. and 60% RH for up to 48 months.

Example 11—Forced Degradation Study

A forced degradation study has been performed on pridopidine HCl drug product and drug substance. The studied material was subjected to acid and base hydrolysis, thermal stress both as solid and in solution, oxidation, humidity induced stress and photolysis.

The study showed that pridopidine HCl is very stable under most of the studied conditions except for when subjected to oxidative conditions, where considerable degradation was observed. The major degradation product was Compound 5. There was also some degradation in the basic hydrolysis study but only a minor total degradation was observed with the largest degradation product being unidentified.

Mass balance was also investigated and found to be good for all studied conditions.

Summary and Conclusions of Examples 10-11

The amounts of the organic impurities remained below the accepted criteria in all the conditions tested over all time periods as shown in Example 10. Compound 5, which is the only known potential degradation product (Example 11), remained low in all the tested conditions as shown in Example 10.

Example 12—Specification of Pridopidine HCl Drug Product

As detailed in example 10, no degradation products have been detected in the pridopidine HCl in any storage conditions. In addition, no additional impurities are created during the formation of the drug product. Therefore, the same amounts of the organic impurities Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 which are controlled in the drug substance remain in the drug product, and the accepted criteria relating to the organic impurities Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 as detailed in Table 22 are relevant to the drug product.

Example 13—Batch Analysis of Pridopidine HCl Drug Substance

A number of batches of Pridopidine HCl drug substance were manufactured at various manufacturing facilities and subsequently analyzed. All batches contained the known identified impurities Compound 5, Compound 1, Compound 4, Compound 8 Compound 6 and Compound 3 in levels below the qualification limit of 0.15%.

TABLE 38 Analysis of the content of each of the impurities Compound 5, Compound 1, Compound 4, Compound 8 Compound 6 and Compound 3 available in the API batches used for tox studies Impurity/Batch No. Z Y X Compound 5 NP NP <0.05 Compound 1 NP NP 0.06 Compound 4 NP <0.05 0.06 Compound 8 0.02 NP <0.05 Compound 6 NP <0.05 <0.05 Compound 2 NP NP <0.05 Compound 3 NP <0.05 <0.05 Largest impurity 0.15 <0.05 <0.05 NP—Not performed

Example 14—Batch Analysis of Pridopidine HCl Drug Product

A number of batches of Pridopidine HCl drug product were manufactured at various manufacturing facilities and subsequently analyzed.

TABLE 39 Analysis of Pridopidine HCl Batches used for Non-Clinical and Clinical Studies Related Substances by HPLC [area %] Batch Compound Compound 4 Compound 3 Compound 6 Unknown Impurities Number 5 Compound 1 (Peak 1) Compound 8 (Peak 2) Compound 2 (Peak 3) Compound 9 Impurities in Total Acceptance ≤0.15 ≤0.15 ≤0.15 ≤0.15 ≤0.15 ≤0.15 ≤0.15 Report ≤0.10 ≤0.50 Criteria value each A — — — — — — — — — 0.51 B — — — — — — — — — 0.26 C — — <0.05 — 0.06 — <0.05 — <0.05 0.06 D — — <0.05 — <0.05 — <0.05 — <0.05 <0.05 E — — <0.05 — <0.05 — <0.05 — 0.091 0.09 F — — <0.05 — <0.05 — <0.05 — 0.071 0.07 1 — — 0.09 — ND — ND — 0.05 0.14 2 — — 0.05 — ND — ND — ND 0.05 3 — — 0.05 — ND — ND — ND 0.06 4 <0.05 <0.05 0.08 <0.05 0.07 <0.05 <0.05 <1 <0.05 0.15 5 <0.052 <0.05 <0.05 <0.05 0.10 <0.05 <0.05 <1 <0.05 0.10 G <0.05 0.06 <0.05 <0.01 0.10 <0.05 <0.05 <1 <0.05 0.15 H <0.05 0.07 <0.05 <0.01 0.08 <0.05 <0.05 <1 <0.05 0.14 I <0.05 <0.05 0.06 <0.01 0.11 <0.05 <0.05 <1 <0.05 0.17 J <0.05 <0.05 <0.05 <0.01 <0.05 <0.05 <0.05 1 <0.05 <0.05 K <0.05 0.07 <0.05 <0.01 0.08 <0.05 <0.05 <1 <0.05 0.15 Compound 9 is 4-hydroxy-4-(3-(methylsu1fonyl)phenyl)-1-propylpiperidin-1-ium chloride.

Example 15—Synergistic Effect of Pridopidine and Compound 1 or Pridopidine and Compound 4

Compound 1 and Compound 4 both display a synergistic effect with pridopidine on BDNF secretion from B104 neuroblastoma cells.

Compound 1 and Compound 4 show selective binding to the Sigma-1 Receptor (S1R, Ki=0.37 μM for compound 1 and Ki=:2.9 μM for compound 4) with no binding to the Sigma-2 receptor (S2R, Ki>100 μM for both compound 1 and 4), as shown in Table 40.

TABLE 40 Binding affinity of pridopidine, Compound 1 and Compound 4 to the Sigma-1 and Sigma-2 receptors S1R Ki S2R Ki S1R fold selectivity Compound (μM) (μM) (S2R/S1R) Pridopidine 0.057 5.45 96 Compound 1 0.37 >100 >270 Compound 4 2.9 >100 >35

In-vitro binding assays per formed at Eurofins Panlabs Taiwan, Ltd. Specific ligand binding was determined in the presence of an excess of unlabeled ligand. Inhibition constants (Ki) were calculated from in vitro binding assays using the Cheng Prusoff equation (Cheng and Prusoff 1973). Source: Johnston et al, 2019 (Johnston et al. 2019) and NC20-PHARM-2.

Thus, both Compound 1 and Compound 4 have high affinity to the S1R and no affinity (Ki >100) to the S2R.

Reductions in Brain-Derived Neurotrophic Factor (BDNF) levels play a key role in the pathogenesis of neurodegenerative disorders and its levels are reduced in neurodegenerative and neurodevelopmental disorders such as Huntington disease (HD), Parkinson's disease, Alzheimer's disease (Zuccato and Cattaneo 2009) and Rett syndrome (Katz 2014).

Pridopidine demonstrates a dose dependent increase in BDNF secretion in rat neuroblastoma cells using an in-situ ELISA assay. This effect is mediated by activation of S1R, since pharmacological inhibition of the S1R abolished pridopidine's effect (Geva, Birnberg, et al. 2016).

When assessing the effect of Compound 1 or Compound 4 with pridopidine, the applicant identified an unexpected synergistic effect. The effect was observed in a BDNF in-situ ELISA assay (Geva, Kusko, et al. 2016).

Thus, the synergistic effect on BDNF release demonstrated below is directly relevant to the therapeutic effect of pridopidine and compound 1 and compound 4.

The following data surprisingly and unexpectedly show that pridopidine together with either Compound 4 or Compound 1 demonstrates a synergistic effect on BDNF release.

Synergistic Effect of Compound 4 and Pridopidine on BDNF Release

Pridopidine alone induces an increase in BDNF release of +13.6% at a concentration of 0.001 μM and +26% at a concentration of 0.005 μM, compared to control untreated cells. Compound 4 at a concentration of 0.001 μM alone has no effect on BDNF release compared to untreated control cells (−1.5%). However, pridopidine and Compound 4 together have an unexpected synergistic effect on BDNF release.

    • Pridopidine 0.001 μM+Compound 4 at 0.001 μM induce a 59.1% increase in BDNF release compared to control untreated cells (FIG. 3A).
    • Pridopidine 0.005 μM+Compound 4 at 0.001 μM induce an 80.7% increase in BDNF release compared to control untreated cells (FIG. 3B).

The effect of pridopidine and Compound 4 together is greater than the sum of the effects of each compound individually, indicating a surprising synergistic effect on BDNF secretion. The results are shown where the values are presented as percent (%) of change compared to untreated control.

Synergistic Effect of Compound 1 and Pridopidine on BDNF Release

Pridopidine alone at a concentration of 0.01 μM induces an increase in BDNF release compared to control untreated cells of +3.4%. Compound 1 alone at a concentration of 1 μM induces a +12.5% increase in BDNF release compared to control. However, pridopidine and Compound 1 together have a synergistic effect on BDNF release (+53.1%).

    • Pridopidine (0.01 μM)+Compound 1 (1 μM) induce a 53.1% increase in BDNF release compared to control untreated cells (FIG. 4).

Again, these results indicate a surprising and unexpected synergistic effect of pridopidine and Compound 1 on BDNF secretion as their effect when administered together (+53.1%) is greater than the sum of the effects of each compound individually.

Thus, the applicant has shown that Compounds 1 and Compound 4 have selective binding affinity to the S1R, together with a surprising and unexpected synergistic effect with pridopidine on BDNF release.

REFERENCES CITED IN EXAMPLE 15

  • Cheng, Yung-Chi, and William H. Prusoff. 1973. “Relationship between the Inhibition Constant (KI) and the Concentration of Inhibitor Which Causes 50 percent Inhibition (150) of an Enzymatic Reaction.” Biochemical Pharmacology. https://doi.org/10.1016/0006-2952(73)90196-2.
  • Geva, Michal, Tal Birnberg, Brian Weiner, Andrew Lysaght, Yoonjeong Cha, Avia Merenlender-Wagner, Aric Orbach, et al. 2016. “Pridopidine Activates Neuroprotective Pathways Impaired in Huntington Disease.” Human Molecular Genetics 25 (18): 3975-87. https://doi.org/10.1093/hmg/ddw238.
  • Geva, Michal, Rebecca Kusko, Holly Soares, Kevin D Fowler, Tal Birnberg, Steve Barash, Avia Merenlender-Wagner, et al. 2016. “Pridopidine Activates Neuroprotective Pathways Impaired in Huntington Disease.” Human Molecular Genetics, July, [Epub ahead of print]. https://doi.org/10.1093/hmg/ddw238.
  • Johnston, Tom H., Michal Geva, Lilach Steiner, Aric Orbach, Spyros Papapetropoulos, Juha-Matti Savola, Ian J. Reynolds, et al. 2019. “Pridopidine, a Clinic-Ready Compound, Reduces 3,4-Dihydroxyphenylalanine-Induced Dyskinesia in Parkinsonian Macaques.” Movement Disorders, December. https://doi.org/10.1002/mds.27565.
  • Katz, D M. 2014. “Brain-Derived Neurotrophic Factor and Rett Syndrome.” Handbook of Experimental Pharmacology 220: 481-95. https://doi.org/10.1007/978-3-642-45106-5_18.
  • Zuccato, Chiara, and Elena Cattanco. 2009. “Brain-Derived Neurotrophic Factor in Neurodegenerative Diseases,” Nature Reviews Neurology 5 (6): 311-22. https://doi.org/10.1038/nrneurol.2009.54.

Claims

1. A method of treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of a neurodegenerative or neurodevelopmental disease or disorder in a subject; wherein the method comprises administering a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of Compounds 1-8 or pharmaceutically acceptable salt thereof: wherein the neurodegenerative disease or disorder is selected from the group consisting from Huntington Disease, prodromal/premanifest Huntington disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease, Parkinson's Disease associated with glucocerebrosidase (GBA) deficiency, dystonia, cognitive disorder, dyskinesia, mild cognitive impairment (MCI), Alzheimer's Disease, age related memory loss, depression and anxiety, bacterial infections-induced depression, optic neuropathies including glaucoma, age-related macular degeneration (AMD), Leber's Hereditary Optic Neuropathy (LHON) and retinitis pigmentosa, mitochondrial disease or dysfunction, Wolfram disease and a viral infection; and wherein the neurodevelopmental disease or disorder is Rett syndrome and Fragile X Syndrome.

2. The method of claim 1, wherein the method comprises treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of Huntington disease, wherein the Huntington disease is an early stage Huntington disease.

3. The method of claim 1, wherein the method comprises treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of ALS.

4. The method of claim 1, wherein the method comprises treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of dystonia, wherein the dystonia is a severe dystonia.

5. The method of claim 1, wherein the method treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of Rett Syndrome.

6. The method of claim 1, wherein the method comprises treating, slowing the progression, lessening the decline, delaying onset of symptoms or slowing the progression of symptoms of a mitochondrial disease or dysfunction, wherein the mitochondrial disease or dysfunction is Lysosomal Storage Disease (LSD), leukodystrophies or a vanishing white matter (VWM) disease.

7. The method of claim 1, wherein the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1, compound 4, pharmaceutically acceptable salt thereof or combination thereof.

8. The method of claim 1, wherein the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or pharmaceutically acceptable salt thereof.

9. The method of claim 1, wherein the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or pharmaceutically acceptable salt thereof.

10. The composition of claim 1, wherein the composition comprises pridopidine salt, wherein the salt is hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulphate, formate, acetate, aconate, ascorbate, benzenesulphonate, benzoate, cinnamate, citrate, embonate, enantate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methane-sulphonate, naphthalene-2-sulphonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate or toluene-p-sulphonate salt.

11. The method of claim 1, wherein the composition is an oral dosage unit comprising between 0.5-315 mg pridopidine.

12. The method of claim 11, wherein the oral dosage unit form comprises between 0.5-10 mg pridopidine.

13. The method of claim 11, wherein the oral dosage unit form comprises between 10-22.5 mg pridopidine.

14. The method of claim 11, wherein the oral dosage unit form comprises between 22.5-45 mg pridopidine.

15. The method of claim 11, wherein the oral dosage unit form comprises between 45-250 mg pridopidine.

16. The method of claim 15, wherein the oral dosage unit form comprises between 45-135 mg pridopidine.

17. The method of claim 11, wherein the oral dosage unit form comprises between 90-315 mg pridopidine.

18. The method of claim 1, wherein the weight ratio between the pridopidine and at least one of compounds 1-8 is in the range of 1:0.001 to 1:0.1.

19. The method of claim 18, wherein the weight ratio between the pridopidine and at least one of compounds 1-8 is in the range of 1:0.005 to 1:0.1.

20. The method of claim 19, wherein the weight ratio between the pridopidine and at least one of compounds 1-8 is in the range of 1:0.001 to 1:0.005.

21. The method of claim 11, wherein the oral dosage unit is formulated as a tablet, a capsule, a pill, powder, liquid solution or as a liquid suspension.

Patent History
Publication number: 20220023280
Type: Application
Filed: Oct 11, 2021
Publication Date: Jan 27, 2022
Applicant:
Inventors: Malle SCHMIDT (Tallinn), Malle PARI (Tallinn), Marit LAOS (Tallinn), Ants MAASALU (Tallinn), Kalle KALJUSTE (Tallinn)
Application Number: 17/498,075
Classifications
International Classification: A61K 31/451 (20060101); C07D 211/52 (20060101); C07D 211/24 (20060101); C07D 211/42 (20060101); G01N 30/88 (20060101);