WO2024242530A1 - Compound for neuroprotection, or treating diseases related to alpha-synuclein and/or neuro-inflammation - Google Patents

Abstract

New uses of a compound are disclosed. The compound of the present disclosure is useful for protecting neurons, or treating or preventing neuro-inflammation and/or α-synuclein toxicity. Thus, the compound of the present disclosure can be used for treating or preventing diseases or disorders requiring neuroprotection or caused by or related to neuro-inflammation and/or α-synuclein toxicity. The present disclosure also provides methods of using said compound or composition comprising the compound for treating or preventing such diseases or disorders.

Description

COMPOUND FOR NEUROPROTECTION, OR TREATING DISEASES RELATED TO ALPHA-SYNUCLEIN AND/OR NEURO-INFLAMMATION

This application claims priority based on Korean Patent Application No. 10-2023-0065259 filed on May 19, 2023, and all contents disclosed in the specification and drawings of the application are incorporated in this application.

The present disclosure relates to the compounds for neuroprotection, or treating diseases related to alpha-synuclein and/or neuro-inflammation, and their medical uses.

Neuroprotection refers to the relative preservation of neuronal structure and/or function. In the case of an ongoing insult (a neurodegenerative insult) the relative preservation of neuronal integrity implies a reduction in the rate of neuronal loss over time. It is a widely explored treatment option for many central nervous system (CNS) disorders including neurodegenerative diseases, stroke, traumatic brain injury, spinal cord injury, and acute management of neurotoxin consumption (e.g. methamphetamine overdoses). Neuroprotection aims to prevent or slow disease progression and secondary injuries by halting or at least slowing the loss of neurons. There are various causes of nerve damage such as decreased delivery of oxygen and glucose to the brain, energy failure, increased levels in oxidative stress, mitochondrial dysfunction, excitotoxicity, inflammatory changes, iron accumulation, and protein aggregation. Neuroprotective treatments often can be useful in treating, ameliorating or preventing diseases or disorders related to such causes.

Meanwhile, alpha-synuclein aggregates to form insoluble fibrils in pathological conditions characterized by Lewy bodies, such as Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, (pure) autonomic failure and both sporadic and familial cases with Alzheimer's disease. These disorders are known as synucleinopathies. In vitro models of synucleinopathies revealed that abnormal accumulation or aggregation of alpha-synuclein may lead to various cellular disorders including microtubule impairment, synaptic and mitochondrial dysfunctions, oxidative stress as well as dysregulation of calcium signaling, proteasomal and lysosomal pathway. Alpha-synuclein is the primary structural component of Lewy body fibrils.

These observations related to alpha-synuclein have implications for therapeutic strategies, and much recent effort is focused on developing antibodies that target extracellular α-synuclein (Alzheimers Res Ther. 2014; 6(5): 73.). There are also attempts to find existing drugs inhibiting the formation of (toxic forms of) α-synuclein in vitro and/or transforming α-synuclein-oligomers in non-toxic oligomer species and develop them as treatments for multiple system atrophy, dementia with Lewy bodies and so on (see ClinicalTrials.gov Identifier: NCT03888222 or ClinicalTrials.gov Identifier: NCT02008721).

Neuro-inflammation is inflammation of the nervous tissue. It may be initiated in response to a variety of cues, including infection, traumatic brain injury, toxic metabolites, or autoimmunity. In the central nervous system (CNS), including the brain and spinal cord, microglia are the resident innate immune cells that are activated in response to these cues. Such neuro-inflammation plays an important role in diseases or disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, peripheral neuritis, neuropathic pain, complex region pain syndrome (CRPS), and it is known that these diseases or disorders can be improved if neuro-inflammation is ameliorated.

The objective of the present disclosure is to provide new medical or pharmaceutical uses of compounds for neuroprotection, or treating diseases related to alpha-synuclein and/or neuro-inflammation.

SUMMARY

The present disclosure provides new medical or pharmaceutical uses of 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile represented by the formula (I):

or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

That is, the present disclosure provides a pharmaceutical composition for neuroprotection; treating or preventing neuro-inflammation; or treating or preventing synucleinopathy, comprising 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient, and optionally an a pharmaceutically acceptable carrier.

The present disclosure also provides methods of using said compound and compositions comprising the compound of formula (I) or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof for neuroprotection; treating or preventing neuro-inflammation; or treating or preventing synucleinopathy.

In one embodiment, the neuroprotection is for treating or preventing neurodegenerative disease (including Parkinson's disease, Alzheimer's disease, and Huntington' disease), stroke, traumatic brain injury, or spinal cord injury; and for acute management of neurotoxin consumption (e.g. methamphetamine overdoses).

In another embodiment, the synucleinopathy is any one selected from the group consisting of Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, and Alzheimer's disease with Lewy bodies.

In yet another embodiment, the present disclosure provides methods of protecting neurons; ameliorating neuro-inflammation; or modulating (e.g., inhibiting or antagonizing) toxic forms of α-synuclein, comprising contacting a sample or cell or biological system with an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof.

Also provided are methods of protecting neurons; treating or preventing neuro-inflammation; inhibiting or preventing alpha synuclein (expression); or treating or preventing synucleinopathy that includes administering a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof to a subject having or at risk from disease(s) or disorder(s) that can be treated or ameliorated by neuroprotection, or related to or caused by neuro-inflammation or abnormal alpha-synuclein. Also provided are methods of treating neuro-inflammation, Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, and/or Alzheimer's disease with Lewy bodies that include administering a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof to a subject having such disease or disorder.

3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same may achieve an excellent effect of preventing or treating neuro-inflammation and/or synucleinopathy; or inhibiting alpha synuclein expression.

In order to illustrate the manner in which the above-recited and other advantages and objects of the invention are obtained, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. Understanding that these drawings depict only typical embodiments of the invention and are not therefore to be considered to be limiting of its scope, the invention will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:

FIG. 1 is the percentage of dopaminergic neurons. Neurons of the ventral mesencephalon were lesioned with MPP+ and treated with the test compound; Lesioned control (LC) and unlesioned vehicle control (VC) were present on each plate. TH positive neurons were evaluated per total number of neurons and given as %TH neurons after the treatment. Data are shown as bar graphs with group mean + SEM (n=6). Statistical analysis: One-way ANOVA followed by Dunnett's multiple comparisons test versus LC. *p<0.05; **p<0.01; ***p<0.001.

FIG. 2 is the Number of synapses per image. Neurons of the ventral mesencephalon were lesioned with MPP+ and treated with the compound of formula (I); Lesioned control (LC) and unlesioned vehicle control (VC) were present on each plate. The number of PSD95 positive spots represent the number of synapses after the treatment. Data are shown as bar graphs with group mean + SEM (n=6). Statistical analysis: One-way ANOVA followed by Dunnett's multiple comparisons test versus LC. *p<0.05; **p<0.01; ***p<0.001.

FIG. 3 is the Number of Apoptotic dopaminergic and total neurons per image. Neurons of the ventral mesencephalon were lesioned with MPP+ and treated with the compound; Lesioned control (LC) and unlesioned vehicle control (VC) were present on each plate. The number of caspase 3 positive cells per number of TH positive neurons represent the number of apoptotic TH cells after the treatment; the number of caspase 3 positive cells per total number of neurons represent the number of apoptotic neurons after the treatment. Data are shown as bar graphs with group mean + SEM (n=6). Statistical analysis: One-way ANOVA followed by Dunnett's multiple comparisons test versus LC. *p<0.05; **p<0.01; ***p<0.001.

FIG. 4 is the effect of reference compound (RI) MK801 on neuronal read outs. (A) % TH positive neurons, (B) number of PSD95 positive synapses, (C) number of caspase 3 cells per TH neurons and (D) total number of caspase 3 positive cells after RI MK801 treatment. Data are shown as bar graphs with group mean + SEM (n=6). Statistical analysis: One-way ANOVA followed by Dunnett's multiple comparisons test versus LC. *p<0.05; **p<0.01; ***p<0.001.

FIG. 5 is the effect of test and reference items on mitochondrial health. Mitochondrial health of (A) the compound of formula (I), and (B) reference compound (RI MK801) treated neurons of the ventral mesencephalon after MPP+ lesion. Data are given as % of vehicle control (VC) and are shown as bar graphs with group mean + SEM (n=6). Statistical analysis: One-way ANOVA followed by Dunnett's multiple comparisons test versus MPP+ lesion control (LC). *p<0.05; **p<0.01; ***p<0.001.

FIG. 6 shows detergent free total alpha-synuclein in SH-SY5Y-a-syn cell extracts. Total alpha-synuclein assessed with MSD assay after the treatment. Data are given as pg alpha-synuclein per μg total protein and are shown as bar graphs with group mean + SEM (n=6). Statistical analysis: One-way ANOVA followed by Dunnett's multiple comparisons test versus vehicle control (VC). *p<0.05; **p<0.01; ***p<0.001.

FIG. 7 shows autophagy marker p62 in SH-SY5Y-alpha-syn cell extracts. P62 assessed with MSD after the treatment. Data are given as Arbitrary unit (AU) and are shown as bar graphs with group mean + SEM (n=6). Statistical analysis: One-way ANOVA followed by Dunnett's multiple comparisons test versus vehicle control (VC). *p<0.05; **p<0.01; ***p<0.001.

FIG. 8 shows autophagy marker LC3BII in SH-SY5Y-alpha-syn cell extracts. LC3BII assessed with WES after (A) the treatment. Data are given as Area under the curve (AUC) and are shown as bar graphs with group mean + SEM (n=6). Statistical analysis: One-way ANOVA followed by Dunnett's multiple comparisons test versus vehicle control (VC). *p<0.05; **p<0.01; ***p<0.001.

FIG. 9 shows effect of the compound of formula (I) on mitochondrial health. Mitochondrial health of the compound-treated SH-SY5Y-alpha-syn cells. Data are given as % of vehicle control (VC) and are shown as bar graphs with group mean + SEM (n=6). Statistical analysis: One-way ANOVA followed by Dunnett's multiple comparisons test versus vehicle control (VC). *p<0.05; **p<0.01; ***p<0.001.

FIG. 10 shows the number of synapses per image. The number of synapsin positive spots representing the number of synapses after the compound treatment in SH-SY5Y-alpha-syn cells. Data are shown as bar graphs with group mean + SEM (n=6). Statistical analysis: One-way ANOVA followed by Dunnett's multiple comparisons test versus LC. *p<0.05; **p<0.01; ***p<0.001.

Detailed Description

Compounds used as Active ingredient

As summarized above, the present disclosure provides new medical or pharmaceutical uses of 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile represented by the formula (I):

.

The compound of the present disclosure exhibits more desirable effects for the purpose of the present disclosure compared to compounds having similar structures.

In one embodiment, a solvate, a hydrate, a prodrug, and/or a stereoisomer, or a pharmaceutically acceptable salt of the compound of formula (I) may be used as active ingredient in uses according to the present disclosure.

In another embodiment, compositions comprising the compound of formula (I), or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof may be used for medical or pharmaceutical uses of the present disclosure.

The compound can protect neurons, and treat or ameliorate neuro-inflammation and/or any toxicities relate to alpha-synuclein in cells and biological systems of interest. The compound find use in a variety of therapeutic applications, including neuroprotection, or diseases or disorders related to or caused by neuro-inflammation and/or abnormal alpha-synuclein. Specifically, the compound can inhibit or prevent alpha-synuclein (expression). The compound also can protect neurons by inhibiting or preventing alpha-synuclein (expression). The compound find use in neurodegenerative disease (including Parkinson's disease, Alzheimer's disease, and Huntington' disease), stroke, traumatic brain injury, spinal cord injury, and acute management of neurotoxin consumption (e.g. methamphetamine overdoses). The compound also find use in the synucleinopathy selected from the group consisting of Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, and Alzheimer's disease with Lewy bodies.

It is understood that all variations of salts, and/or solvates, hydrates, prodrugs and/or stereoisomers of the compound of formula (I) are meant to be encompassed by the present disclosure. Accordingly, any of the compound described herein may also be referred to as a compound of formula (I) or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

Pharmaceutical Compositions

Compounds of the present disclosure may be formulated in pharmaceutical compositions. The pharmaceutical composition can include the compound of the present disclosure and at least one excipient (e.g., a pharmaceutically acceptable excipient).

The compounds described herein can find use in pharmaceutical compositions for administration to a subject in need thereof in a variety of therapeutic applications where neuroprotection is desirable. Specifically, the compounds can protect neurons by inhibiting alpha-synuclein (expression). The compounds described herein also can find use in pharmaceutical compositions for administration to a subject in need thereof in a variety of therapeutic applications where treatment and/or amelioration of neuro-inflammation and/or alpha-synuclein toxicity is desirable. The treatment and/or amelioration of alpha-synuclein toxicity may refer an inhibition or prevention of alpha-synuclein (expression).

Accordingly, in embodiments, the present disclosure provides pharmaceutical compositions comprising the compound described herein, a pharmaceutically acceptable salt thereof, or a prodrug thereof, and at least one pharmaceutically acceptable excipient. The phrase "pharmaceutically acceptable excipient," refers any ingredient other than the inventive compound described herein (for example, a vehicle capable of suspending or dissolving the active compound, or any other convenient pharmaceutically acceptable carriers, excipients or additives) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, dispensing, or dispersing agents, sweeteners, and waters of hydration. In some embodiments, the pharmaceutical composition comprises a compound as described herein, a pharmaceutically acceptable salt thereof, or a prodrug thereof in a therapeutically effective amount.

The pharmaceutical composition may be formulated according to any convenient methods, and may be prepared in various forms for oral administration such as tablets, pills, powders, nanoparticles, capsules, syrups, suspensions, emulsions and microemulsions, or in forms for non-oral administration such as preparations for intramuscular, intravenous or subcutaneous administration. The pharmaceutical compositions provided herein can be administered by a variety of routes including, but not limited to, oral (enteral) administration, parenteral (by injection) administration, rectal administration, transdermal administration, intradermal administration, intrathecal administration, subcutaneous (SC) administration, intravenous (IV) administration, intramuscular (IM) administration, and intranasal administration.

In a specific example, the pharmaceutical composition could contain a pharmaceutically allowed carrier, excipient, or additive. The pharmaceutical composition could be produced as medicine in the conventional method, and could be produced as various oral medicine such as tablet, pill, powder, capsule, syrup, emulsion, micro-emulsion, and so on, or could be produced as non-oral medicine such as muscular injection, vascular injection, or subcutaneous injection.

The term "carrier," "adjuvant," "vehicle," and "excipients" are exchangeably used herein, and the compound of this disclosure is administered with the carrier. The carrier is a solid carrier or a flowable carrier. Suitable pharmaceutical carriers include those described in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005).

If the pharmaceutical composition is produced in the form of an oral medicine, examples of the used additive or carrier could include cellulose, silicic calcium, corn starch, lactose, sucrose, dextrose, phosphoric acid calcium, stearic acid, stearic acid magnesium, stearic acid calcium, gelatin, talc, surfactant, suspension, emulsifying agent, diluting agent, and so on. If the pharmaceutical composition of this disclosure is produced in the form of an injection, the additives or carrier could include water, saline water, glucose aqueous solution, similar sugar-soluble solution, alcohol, glycol, ether (e.g., polyethylene glycol 400), oil, fatty acid, fatty acid ester, glyceride, surfactant, suspension, emulsifying agent, and so on.

In some embodiments, the pharmaceutical compositions are formulated for parenteral administration to a subject in need thereof. In some parenteral embodiments, the pharmaceutical compositions are formulated for intravenous administration to a subject in need thereof. In some parenteral embodiments, the pharmaceutical compositions are formulated for subcutaneous administration to a subject in need thereof.

Methods of the Present Disclosure

Aspects of the present disclosure include methods of protecting neurons; treating, preventing or ameliorating neuro-inflammation; and/or treating, preventing or ameliorating toxicity related to alpha-synuclein, particularly, abnormal forms of alpha-synuclein in a biological system or sample. Treating, prevention and/or ameliorating of alpha-synuclein toxicity may refer inhibiting or preventing alpha-synuclein (expression).

In some embodiments, the method includes contacting a sample with the compound of this disclosure. In some embodiments, the method includes contacting a cell or biological system with the compound of this disclosure.

Aspects of the present disclosure include methods of protecting neurons; treating, preventing or ameliorating neuro-inflammation; and/or treating, preventing or ameliorating toxicity related to alpha-synuclein using the compound described herein. Such methods may include methods of protecting neurons; treating, preventing or ameliorating neuro-inflammation; and/or treating, preventing or ameliorating abnormal reactions related to alpha-synuclein in biological systems by contacting such systems with the compound of formula (I). Biological systems may include, but are not limited to, cells, tissues, organs, bodily fluids, organisms, non-mammalian subjects, and mammalian subjects (e.g., humans, dogs, cats, mice, rats, rabbits, guinea pigs, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and, in particular, humans).

In some embodiments, the method of protecting neurons; treating, preventing or ameliorating neuro-inflammation; and/or treating, preventing or ameliorating alpha-synuclein toxicity comprises contacting a biological system or sample comprising neuro-inflammation and/or abnormal alpha-synuclein with an effective amount of any of the compound or a pharmaceutically acceptable salt thereof as described herein, or a pharmaceutical composition as described herein. In certain embodiments, the biological system or sample is in vitro. In another embodiment, the biological system or sample is in vivo. In some embodiments, the sample is a cell sample.

In some embodiments, treating, preventing and/or ameliorating alpha-synuclein toxicity and/or abnormal alpha-synuclein may refer inhibiting or preventing alpha-synuclein (expression).

Diseases or Disorders requiring Neuroprotection

The present disclosure provides a method of protecting neurons in a subject using the subject compound as therapeutic agent, and compositions including the compound. Any diseases or disorders for which neuroprotection are thought to be useful by those of ordinary skill in the art are contemplated as diseases treatable using the compound of this disclosure.

In some embodiments, the method includes administering to a subject having a disease or disorder requiring neuroprotection a therapeutically effective amount of the compound of formula (I). In some embodiments, the disease or disorder is neurodegenerative diseases (including Parkinson's disease, Alzheimer's disease, and Huntington' disease), stroke, traumatic brain injury, spinal cord injury, or neurotoxin consumption (e.g. methamphetamine overdoses). In some embodiments, the compound of formula (I) can protect neurons by reducing or inhibiting alpha-synuclein (expression).

In some embodiments, neuroprotection of the present disclosure aims to prevent or slow disease progression and secondary injuries by halting or at least slowing the loss of neurons. Thus, in some embodiments, the present disclosure provides a method of protecting neurons in a subject having neurodegenerative disease(s) using the subject compound as therapeutic agent.

In some embodiments, the method further comprises identifying a subject suffering from or at risk of a disease or disorder requiring neuroprotection.

In some embodiments, the method further comprises identifying an underlying disease or condition associated with neuroprotection.

Diseases or Disorders related to or caused by Neuro-inflammation and/or Alpha-synuclein toxicity

The present disclosure provides a method of treating, ameliorating or preventing diseases or disorders related to or caused by neuro-inflammation and/or alpha-synuclein toxicity in a subject using the subject compound as therapeutic agent, and/or compositions including the compound. Any diseases or disorders for which amelioration of neuro-inflammation and/or alpha-synuclein toxicity are thought to be useful by those of ordinary skill in the art are contemplated as diseases treatable using the compound of this disclosure.

In some embodiments, the method includes administering to a subject having a disease or disorder related to or caused by neuro-inflammation and/or alpha-synuclein toxicity a therapeutically effective amount of the compound of formula (I). In some embodiments, the disease or disorder is synucleinopathy. In some embodiments, the synucleinopathy is Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, or Alzheimer's disease with Lewy bodies. In some embodiments, the disease or disorder is a disease or disorder which can be treated by ameliorating neuro-inflammation. In some embodiments, the disease or disorder which can be treated by ameliorating neuro-inflammation is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, peripheral neuritis, neuropathic pain, or complex region pain syndrome (CRPS).

In some embodiments, the alpha-synuclein toxicity can be caused by overexpression of alpha-synuclein protein or alpha-synuclein aggregation. In this regard, treating, preventing and/or ameliorating of alpha-synuclein toxicity may refer inhibiting or preventing alpha-synuclein (expression). The present disclosure provides a method of inhibiting or preventing alpha-synuclein (expression) in a subject using the subject compound as therapeutic agent, or compositions including the compound. Any diseases or disorders for which inhibition of alpha-synuclein (expression) are thought to be useful by those of ordinary skill in the art are contemplated as diseases treatable or preventable using the compound of this disclosure.

In some embodiments, the method further comprises identifying a subject suffering from or at risk of a disease or disorder requiring amelioration of neuro-inflammation and/or alpha-synuclein toxicity.

In some embodiments, the method further comprises identifying an underlying disease or condition associated with neuro-inflammation and/or alpha-synuclein toxicity.

The amount of the aforementioned pharmaceutical composition administered is the effective amount to treat or prevent illness for an entity or patient, and could be administered orally or non-orally according to the purpose. When administered orally, the amount administered based on the active component is 0.01 to 1,000 mg per 1kg of body weight, more specifically 0.1 to 300 mg per 1kg. When administered non-orally, 0.01 to 100 mg based on the active component is administered per 1kg of body weight per day, and more specifically 0.1 to 50 mg is administered once or several times. The amount administered for a specific entity or patient can be determined based on many related factors including the patient's weight, age, sex, health status, diet, time of administration, method of administration, severity of the illness, and so on, must be understood to be able to be increased or decreased appropriately by the specialist, and the aforementioned amount of administration does not limit the scope of the present disclosure in any way. A medical doctor or veterinarian with ordinary level of knowledge in the related technological field may determine and prescribe effective amount of the pharmaceutical composition. For example, a medical doctor or veterinarian may start with the amount of the compound under the present disclosure used in a pharmaceutical composition that is lower than the amount required to achieve the desired treatment effect, and may increase the amount administered gradually until the desired effect is achieved.

Examples of suitable routes of administration include oral, rectal, transmucosal (including sublingual and buccal), intranasal, topical, transdermal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intracerebroventricular injection, direct injections to the human brain, direct intraventricular, intraperitoneal, intranasal, or intraocular injections. The compounds can also be administered in sustained or controlled release dosage forms, including nanoparticles, depot injections, osmotic pumps, electronic pumps, pills, transdermal (including electrotransport) patches, and the like, for prolonged and/or timed, pulsed administration at a predetermined rate. Sustained or controlled release dosage forms may be used to increase CNS exposure and minimize systemic exposure. It is also possible to combine at least two different routes of administration.

The compounds and compositions of this disclosure may be administered alone, in combination with a compound according to another example of the present disclosure, or in simultaneous, separate or sequential concomitant administration with at least one other therapeutic agent, for example with other pharmaceutical active ingredients described herein.

In a specific example, a pharmaceutical composition of this disclosure includes within its scope at least one of the compounds in accordance with a specific example of the effective treatment amount as effective component, or a pharmaceutical composition that is contained in combination with a pharmaceutical carrier. Arbitrarily, the compound in accordance with an embodiment of the present disclosure could be administered independently, in combination with a compound in accordance with another specific example, or simultaneously with one or more other treatment medications, for example simultaneously with an anticancer medicine (e.g., as described herein) or with an active pharmaceutical material, separately, or consecutively in conjunction.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

It is understood that the definitions provided herein are not intended to be mutually exclusive. Accordingly, some chemical moieties may fall within the definition of more than one term.

The term "alpha-synuclein (aSyn or αSyn)" used herein means a protein made of 140 amino acids that, in humans, is encoded by the SNCA gene. Alpha-synuclein is a neuronal protein that regulates synaptic vesicle trafficking and subsequent neurotransmitter release. It is abundant in the brain, while smaller amounts are found in the heart, muscle and other tissues. α-Synuclein is the major constituent of Lewy bodies as a common pathological signature of Lewy body diseases including Parkinson's disease, and Dementia with Lewy bodies. α-Synuclein exists in various forms, such as oligomers, immature filaments, and filaments, and is a cause of disease. In particular, filamentous structures are mainly found in Lewy bodies in nerve cells. These amyloid protein filaments are thermodynamically stable structures, and the process of converting toxic oligomers into amyloid structures is thought to be a product of efforts to reduce toxicity. On the other hand, oligomers affect cell membrane permeability, resulting in abnormal calcium influx and cell death. The term "alpha-synuclein" used with the terms "inhibit", "prevent", "ameliorate", "treat" and so on may mean any forms of alpha-synuclein, including oligomers, aggregates and toxic forms.

The terms "oligomer", "aggregate", "insoluble form" or "toxic form" of α-synuclein are used interchangeably herein. These terms are used to mean any toxic form of α-synuclein. Alpha-synuclein forms pathologic aggregates in various neurodegenerative diseases, and oligomeric forms of alpha-synuclein are known to be toxic, but the present disclosure is not limited to these specific forms of alpha-synuclein. The present disclosure can be used for synucleinopathy based on excessive alpha-synuclein levels as well as these toxic forms of alpha-synuclein.

The term "synucleinopathy (also called α-synucleinopathy)" used herein means a disease characterized by the (abnormal) accumulation of alpha-synuclein protein, particularly aggregates of alpha-synuclein protein in neurons, nerve fibres or glial cells. The term "synucleinopathy" used herein also means a disease characterized by specific forms, particularly, toxic forms, including oligomers or aggregates, of alpha-synuclein protein. There are three main types of synucleinopathy: Parkinson's disease with Lewy bodies, dementia with Lewy bodies, and multiple system atrophy (MSA). Other rare disorders, such as various neuroaxonal dystrophies, also have α-synuclein pathologies.

Singular articles such as "a," "an" and "the" and similar referents in the context of describing the elements are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, including the upper and lower bounds of the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (i.e., "such as") provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated.

In some embodiments, where the use of the term "about" is before a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. Where a percentage is provided with respect to an amount of a component or material in a composition, the percentage should be understood to be a percentage based on weight, unless otherwise stated or understood from the context.

Where a molecular weight is provided and not an absolute value, for example, of a polymer, then the molecular weight should be understood to be an average molecule weight, unless otherwise stated or understood from the context.

It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present disclosure remain operable. Moreover, two or more steps or actions can be conducted simultaneously.

The terms "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" are used interchangeably and refer to an excipient, diluent, carrier, or adjuvant that is useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use. The phrase "pharmaceutically acceptable excipient" includes both one and more than one such excipient, diluent, carrier, and/or adjuvant.

The term "pharmaceutical composition" is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a "pharmaceutical composition" is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (i.e., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intranasal, intramuscular, subcutaneous, and the like.

The terms "disease,"　"disorder," and "condition" are used interchangeably herein.

The term "subject" to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female　of　any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or a non-human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

As used herein, and unless otherwise specified, the terms "treat," "treating" and "treatment" contemplate an action that occurs while a subject is suffering from the specified disease,　disorder　or condition, which reduces the severity　of　the disease,　disorder　or condition, or retards or slows the progression　of　the disease,　disorder　or condition. "Treatment" or "treating" includes: (i) prevention of the disease, disorder, or condition, i.e., reducing the incidence of and/or ameliorating the effect and/or duration of a disease, disorder, or condition from occurring in subjects that may get, be exposed to and/or be predisposed to the disease, disorder or condition, but may not yet have been diagnosed as having it; or are diagnosed as having the disease, disease, or condition; or are at risk of developing such disease, disorder, or condition; (ii) inhibition of the disease, disorder, or condition, i.e., preventing or delaying the onset of a disease, disorder, or condition; arresting further development or progression of a disease, disorder, or condition in a subject already suffering from or having one or more symptoms of the disease, disorder, or condition; or reducing the risk of a disease, disorder, or condition worsening; (iii) amelioration of the disease, disorder, or condition, i.e., attenuating, relieving, reversing or eliminating the disease, disorder, or condition, or one or more of symptoms thereof.

As used herein, and unless otherwise specified, a "therapeutically effective amount"　of　a compound is an amount sufficient to provide a therapeutic benefit in the　treatment　of　a disease,　disorder　or condition, or to delay or minimize one or more symptoms associated with the disease,　disorder　or condition. A therapeutically effective amount　of　a compound means an amount　of　therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the　treatment　of　the disease,　disorder　or condition. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes　of　disease or condition, or enhances the therapeutic efficacy　of　another therapeutic agent.

As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease, disorder or condition, or one or more symptoms associated with the disease,　disorder　or condition, or prevent its recurrence. A prophylactically effective amount　of　a compound means an amount　of　a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the disease, disorder or condition. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

Exemplary Embodiments

As described herein, the text refers to various embodiments of the present compounds, compositions, and methods. The various embodiments described are meant to provide a variety of illustrative examples and should not be construed as descriptions of alternative species. Rather, it should be noted that the descriptions of various embodiments provided herein may be of overlapping scope. The embodiments discussed herein are merely illustrative and are not meant to limit the scope of the present technology.

Notwithstanding the appended claims, aspects of the present disclosure are illustrated by the following clauses.

Clause 1. A pharmaceutical composition for neuroprotection; treating or preventing neuro-inflammation; or treating or preventing synucleinopathy, comprising 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient.

Clause 2. The pharmaceutical composition according to claim 1, wherein the neuroprotection is for treating or preventing neurodegenerative disease, stroke, traumatic brain injury, spinal cord injury; and for acute management of neurotoxin consumption.

Clause 3. The pharmaceutical composition according to claim 1, wherein the synucleinopathy is any one selected from the group consisting of Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, and Alzheimer's disease with Lewy bodies.

Clause 4. A use of 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for neuroprotection; treating or preventing neuro-inflammation; or treating or preventing synucleinopathy.

Clause 5. The use of 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof according to claim 4, wherein the neuroprotection is for treating or preventing neurodegenerative disease, stroke, traumatic brain injury, spinal cord injury; and for acute management of neurotoxin consumption.

Clause 6. The use of 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof according to claim 4, wherein the synucleinopathy is any one selected from the group consisting of Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, and Alzheimer's disease with Lewy bodies.

Examples

The following examples are offered to illustrate the present disclosure and are not to be construed in any way as limiting the scope of the present technology. Any methods that are functionally equivalent are within the scope of the present technology. Various modifications of the present technology in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications fall within the scope of the appended claims.

Unless otherwise stated, all temperatures are in degrees Celsius. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviation should be allowed for.

If an abbreviation is not defined, it has its generally accepted meaning.

General Synthetic Methods

Final compounds were confirmed by high-performance liquid chromatography/ mass spectrometry (HPLC/MS) analysis and determined to be >90% pure by weight. ¹H nuclear magnetic resonance (NMR) spectra were recorded in CDCl₃ (residual internal standard CHCl₃ = δ 7.26), dimethyl sulfoxide (DMSO)-d₆ (residual internal standard CD₃SOCD₂H = δ 2.50), methanol-d₄ (residual internal standard CD₂HOD = δ 3.30), or acetone-d₆ (residual internal standard CD₃COCD₂H = δ 2.05). The chemical shifts (δ) reported are given in parts per million (ppm) and the coupling constants (J) are in Hertz (Hz). The spin multiplicities are reported as s = singlet, bs = broad singlet, bm = broad multiplet, d = doublet, t = triplet, q = quartet, p = pentuplet, dd = doublet of doublet, ddd = doublet of doublet of doublet, dt = doublet of triplet, td = triplet of doublet, tt = triplet of triplet, and m = multiplet.

HPLC-MS analysis was carried out with gradient elution. Medium pressure liquid chromatography (MPLC) was performed with silica gel columns in both the normal phase and reverse phase.

Example 1: Preparation of the compound of the present invention

Example 1.1: Production 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidine-4-yl)-2-fluorobenzonitrile (formula (I))

1.1.1. Production of 4-chloro-1-(3-methyl-4-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine

Reagents and Conditions: (a) K₂CO₃, DMAc, 80 ℃, 16 h.

To a solution of 4-chloro-1H-pyrazolo[3,4-d]pyrimidine-6-amine (0.8 g, 1 eq) and 4-(chloromethyl)-2-methyl-1-nitro-benzene (720 mg, 0.8) in DMAc (50 mL) was added K₂CO₃ (1.30 g, 2 eq). The mixture was stirred at 80 ℃ for 16hr. The reaction mixture filtered and concentrated under reduced pressure to give a residue. The crude product 4-chloro-1-[(3-methyl-4-nitro-phenyl)methyl]pyrazolo[3,4-d]pyrimidine-6-amine (800 mg, crude) was obtained as a yellow solid and used into the next step without further purification. MS: m/z = 354.9 (M+1, ESI+).

1.2.2. Production of 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidine-4-yl)-2-fluorobenzonitrile (formula (I))

Reagents and condition: (a) Pd(PPh₃)₄, K₂CO₃, dioxane/H₂O, 100 ℃, 16h; (b) Fe, NH₄Cl, EtOH/H₂O, 60 ℃, 1 h.

Stage 1: Production of 3-(6-amino-1-(3-methyl-4-nitrobenzyl)-1 H -pyrazolo[3,4- d ]pyrimidine-4-yl)-2-fluorobenzonitrile

A mixture of (3-cyano-2-fluoro-phenyl)boronic acid (434 mg, 1.2 eq), 4-chloro-1-(3-methyl-4-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine or 4-chloro-1-[(3-methyl-4-nitro-phenyl)methyl]pyrazolo[3,4-d]pyrimidine-6-amine (700 mg, 1 eq), Pd(PPh₃)₄ (253 mg, 219.63 μmol, 0.1 eq) and Na₂CO₃ (465 mg, 2 eq) in dioxane (10 mL) and water (2 mL) was degassed and purged with nitrogen for three times, and then the mixture was stirred at 110 ℃ for 16hr under nitrogen atmosphere. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0~80% Ethyl acetate/Petroleum ether gradient @ 40 mL/min). Compound 3-[6-amino-1-[(3-methyl-4-nitro-phenyl)methyl]pyrazolo[3,4-d]pyrimidine-4-yl]-2-fluoro-benzonitrile (800 mg, 90% yield) was obtained as a yellow solid. MS: m/z = 386.0 (M+1, ESI+).

Stage 2: Production of 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidine-4-yl)-2-fluorobenzonitrile (formula (I))

To a solution of 3-[6-amino-1-[(3-methyl-4-nitro-phenyl)methyl]pyrazolo[3,4-d]pyrimidine-4-yl]-2-fluoro-benzonitrile (200 mg, 1 eq) in water (4 mL) and ethanol (12 mL) were added iron dust (138 mg, 5 eq) and NH₄Cl (212 mg, 8 eq). The mixture was stirred at 60 ℃ for 1 hr. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Synergi C18 150*25mm* 10μm; mobile phase: [water(0.225%FA)-ACN];B%: 16%-46%,11min), the 89% purity compound was obtained after first purification. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 150*25mm*10μm; mobile phase: [water(0.225%FA)-ACN];B%: 18%-48%,10min). The compound of formula (I) or 3-[6-amino-1-[(4-amino-3-methyl-phenyl)methyl]pyrazolo[3,4-d]pyrimidine-4-yl]-2-fluoro-benzonitrile (111.35 mg, 59% yield, 97.78% purity) was obtained as a white solid.

¹H NMR (400 MHz, DMSO-d6) δ = 8.21 - 8.10 (m, 2H), 8.00 - 7.94 (m, 1H), 7.66 - 7.52 (m, 1H), 7.16 - 7.07 (m, 2H), 6.92 - 6.80 (m, 2H), 6.56 - 6.46 (m, 1H), 5.34 - 5.15 (m, 2H), 4.93 - 4.67 (m, 2H), 2.06 - 1.88 (m, 3H).

Example 2: Assessment of activity of the formula (I) compound

2.1 Aim of the Study

The aim of the study was to test the compound of formula (I) at 7 concentrations (1, 3, 10, 30, 100, 300, 1000 [nM], HCl salt, DMSO) in MPP+ lesioned rat TH neurons and in SH-SY5Y cells.

2.2 Study Design

The study was performed in two different setups. One was testing the compound in MPP+ lesions primary TH neurons, the other was testing the compound in SH-SY5Y cells.

(5S,10R)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-imine hydrogen maleate, Dizocilpine hydrogen maleate (Sigma, PBS (+0.1% DMSO final), 150 nM) was used as reference.

Part1: Experiment in MPP+ lesioned primary rat TH neurons

Primary TH neurons from wildtype rats were isolated and cultivated. Cells were lesioned using MPP+ at 1 concentration, and treated with the compound at 7 concentrations each, vehicle control or reference item (MK801). Cell fixation was performed at 1 time point (24 h). The project was run in two sets, n=6 technical replicates per condition.

Set 1: Immunocytochemical staining was performed to label TH, MAP2 and synapses (PSD95) for an image-based assessment of TH neurons and synapses (in %).

Set 2: Cells were stained with fixable Mitotracker stain and immunocytochemical staining for TH and Annexin V for an image-based assessment of TH neurons and mitochondrial activity (in %).

Part2: Experiment in alpha-synuclein overexpressing SH-SY5Y cells

SH-SY5Y cells (human αSyn overexpressing cells) were cultivated and differentiated with retinoic acid (RA). Cells were treated with the compound at 7 concentrations or vehicle control. The experiment was performed in either 48-well or 96-well plates for protein analysis or for mitochondrial profile and immunocytochemical staining, respectively. The experiment was planned in n=6 technical replicates per condition per assay.

Cells were harvested from 48-well plates after 48 h treatment phase. Total fractions were extracted and used for evaluation of human alpha-synuclein with MSD assay.

Autophagy was measured by evaluating levels of p62 using MSD assay, and LC3B using WES analysis.

Cells cultivated in the 96-well plates were used for staining with Mitotracker for assessment of mitochondrial activity. Cells were fixed and immunocytochemically stained for synapsin for subsequent image-based quantification of synapses.

2.3 Outcome Summary

Part1: Experiment in MPP+ lesioned primary rat TH neurons

TH positive neurons and synapses: The compound at 10-100 nM was able to rescue dopaminergic (TH positive) neurons and led to increased number of synapses compared to the lesion control.

Apoptotic cells: The compound was able to significantly reduce the number of apoptotic cells in the MPP+ lesion assay at almost all tested concentrations, except the highest dose of 1000 nM.

Mitochondrial health: The compound only showed trends towards rescued mitochondrial activity compared to the lesion control.

Part2: Experiment in alpha-synuclein overexpressing SH-SY5Y cells

Alpha-synuclein: Free total fraction was assessed with MSD assay. Due to absence of detergent this fraction represents mainly soluble monomers.

Interestingly, the compound at 100 and 1000 nM led to a reduction of these alpha-synuclein species.

Autophagy: While the treatment of the compound did not significantly affect p62 levels compared to the vehicle control. No effect on LC3B levels was observed.

Mitochondrial health: For the compound at medium concentrations the mitotracker showed trends towards increased signal, getting significant for the compound at 30 nM and 100 nM compared to the respective vehicle control.

Synapses: A significant increase in the number of synapses was detected when comparing vehicle control with 10nM the compound-treated cells.

2.4 Overview of Experimental Set-up

Experimental set-up was as follows:

Overview:

2.5 Part1: Experiment in MPP+ lesioned primary rat TH neurons

2.5.1 Euthanasia of Animals

Rat line: Sprague Dawley

Sex: Female

Provider: Envigo

Embryonic state at delivery: E8

Embryonic state at euthanasia: E13/E14

Number of animals: 6

Animals were euthanized by exposure to carbon dioxide according to SOP MET032 on day E13/E14. Sacrificed animals were transferred from the Animal Facility to the Cell Culture lab.

2.5.2 Preparation of primary rat ventral mesencephalic neurons (TH enriched)

Primary neurons from the ventral mesencephalon were prepared from time mated Sprague Dawley rat embryos at E14 according to SOP NMET135. Animals were sacrificed and embryos were dissected in Calcium and Magnesium free Hanks Balanced Salt Solution (CMF-HBSS) containing 15 mM HEPES and 10 mM NaHCO3 (cell culture tested; Sigma), pH 7.2. Embryos were decapitated, skin and skull gently removed and the mesencephalon was isolated. The mesencephalon was chopped with a sterile razor blade in Chop solution (Hibernate-E without Calcium containing 2% B-27) and digested in 2 mg/ml papain (Worthington) dissolved in Hibernate-E without Calcium for 45 minutes at 30 ℃. The mesencephalon was triturated for 15 times with a fire-polished silanized Pasteur pipette in Hibernate-E without Calcium containing 2% B-27, 0.01% DNaseI, 1 mg/ml BSA, and 1 mg/ml Ovomucoid Inhibitor. Undispersed pieces were allowed to settle by gravity for 1 min and the supernatant was centrifuged for 5 min at 228 g. The pellet was resuspended in Hibernate-E containing 2% B-27, 0.01% DNaseI, 1 mg/ml BSA, 1 mg/ml Ovomucoid Inhibitor and further diluted with Hibernate-E containing 2% B-27. After the second centrifugation step, the pellet was resuspended in Neurobasal medium containing, 2% B-27, 0.5 mM glutamine, 1% Penicillin-Streptomycin. Cells were counted in a hemacytometer and were seeded in poly-D-lysine pre-coated 96 well plates at a density of 50.000 cells/well. 4 plates were seeded in total.

Cells were cultured in TH-plating medium [DMEM/F12, 10% serum (Horse serum), 5% Glucose stock (100 mg/ml) and 1% Penicillin-Streptomycin] for 24 h at 37 ℃ and 5% CO². After priming, medium was exchanged to TH treatment medium [DMEM/F12, 1% N2, 5% Glucose stock [100 mg/ml] and 1% Penicillin-Streptomycin]. Until treatment day DIV6, no further medium exchange was required Cells were cultured at 37 ℃; 95% humidity and 5% CO², all wells were handled the same way.

2.5.3 Treatment and lesioning

On DIV6, cells were treated with the compound by addition of 10 μl of a 17x stock to 150 μl culture medium on the cells. After 1 h of pre-incubation with TIs, 10 μl of 17x MPP+ stock were added to receive the required final concentrations of the compound and MPP+ (7.5 μM; Sigma Aldrich D048-1G).

Plate1: seeded/treated twice, once for set1 and once for set2

The experiment was conducted with n=6 replicates per condition per set.

After 24 h of lesioning, primary cells were further processed as described below.

2.5.4 Staining Set1: %TH neurons and synapses

Set 1 cells were fixed after 24 h of lesioning by addition of equal volume 4% paraformaldehyde (PFA) to the medium at room temperature (RT) for 30 minutes. Fixed cells were stored in PBS at 4 ℃ until further use for immunocytochemistry (ICC).

Cells were rinsed two times with PBS and were permeabilized with 0.1% Triton X-100 in PBS for 30 minutes at RT. Next, cells were blocked for 60 min at RT with 20% horse serum, 0.1% Triton X-100 in PBS.

Then, samples were incubated with the primary antibodies described below at 4 ℃ overnight.

Next day, cells were further incubated for another 30 min at RT. After three washing steps with PBS, cells were incubated with the fluorescently labelled secondary antibodies and DAPI (nucleus) for 1.5 hours at RT in the darkness. Cells were again rinsed four times with PBS and imaged with the Cytation 5 Multimode reader (BioTek) in RFP (TH), Texas Red (MAP2) and GFP (PSD95) channels, as well as DAPI. From each well, 9 images were taken at 10x magnification.

The number of TH positive cells per MAP2 positive cells were measured using Gen05 software. PSD95 positive small spots were counted as synapses.

2.5.5 Staining Set2: Apoptotic TH neurons and mitochondrial activity

MitoTracker Red CMXRos (ThermoFischer, M7512) was used to detect active mitochondria in living cells according to SOP NMET138 with slight modifications:

Supernatant of the cultivated cells on 96-well plates was aspirated off after 24 h of lesioning.

190 μl ACSF buffer (125 mM NaCl, 2.5 mM KCl, 26.2 mM NaHCO3, 1 mM NaH2PO4, 2.5 mM CaCl2, 1.25 mM MgCl2, 25 mM Hepes, 11 mM D-Glucose, pH 7.3) were added to all wells.

Plates were incubated for 15 minutes in the incubator at 37 ℃. Then 10 μl MitoTracker Red CMXRos of 4 μM working solution were added to each well to get a final concentration of 200 nM.

Plates were incubated for 45 min in the incubator at 37 ℃ (light protected).

Supernatant was aspirated off completely and discarded. Thereafter 100 μl PBS were added and the plate was read in Cytation 5 multimode reader at 579/600 nm (+/- 10 nm).

Cells were then fixed by addition of equal volume 4% paraformaldehyde (PFA) to the PBS at room temperature (RT) for 30 minutes. Fixed cells were stored in PBS at 4 ℃ until further use for immunocytochemistry (ICC).

Cells were rinsed two times with PBS and were permeabilized with 0.1% Triton X-100 in PBS for 30 minutes at RT. Next, cells were blocked for 60 min at RT with 20% horse serum, 0.1% Triton X-100 in PBS.

Then, samples were incubated with the primary antibodies described below at 4 ℃ overnight.

Next day, cells were further incubated for another 30 min at RT. After three washing steps with PBS, cells were incubated with the fluorescently labelled secondary antibodies and DAPI (nucleus) for 1.5 hours at RT in the darkness. Cells were again rinsed four times with PBS and imaged with the Cytation 5 Multimode reader (BioTek) in RFP (TH), Texas Red TR (Mitotracker) and GFP (Caspase 3) channels, as well as DAPI. From each well, 6 images were taken at 10x magnification.

The overall number of caspase 3 positive cells as well as caspase 3 positive cells per TH positive cells were measured using Gen05 software. Additionally, the fluorescence area of TR channel was measured to quantify active mitochondria using Gen05 software (as % of VC).

2.6 Part2: Experiment in alpha-synuclein overexpressing SH-SY5Y cells

2.6.1 Cultivation and differentiation of SH-SY5Y-α-syn cells

SH-SY5Y-α-syn cells were kept in culture medium (DMEM medium, 10% FCS, 1% NEAA, 1% L-Glutamine, 100 μg/ml Gentamycin, 300 μg/ml Geneticin G-418) for ~2 days until 80-90% confluency. Next, cells were differentiated in culture medium supplemented with 10 μM retinoic acid (RA) for 5 days changing medium every 2 to 3 days. On day 5 after differentiation start (DIV1), cells were seeded into uncoated 48 well plates at a density of 1.5x10⁵ cells/well and 96 well plates at a density of 3.5x10⁴ cells/well.

2.6.2 Treatment

On DIV2, cells were treated with the compound by addition of 10 μl of a 17x stock to 160 μl culture medium on the cells, vehicle treated cells serve as control. The experiment was conducted with n=6 replicates per condition per set.

After 48 h of treatment, cells were further processed as described below.

Set1: 48 well plates for lysis

Set2: 96 well plates for ICC

2.6.3 Set1: Cell lysis

After 48 h of incubation of the compound, cells in 48-well plates were washed once with cold PBS. Thereafter 100 μl of cold Extraction buffer 1 (20 mM Tris-HCl pH 7,4; 50 mM NaCl; 1% Triton X-100; SOP NMET144) including protease and phosphatase inhibitors, were added to each well and plates were shaken for 15-20 min on an orbital shaker at medium speed.

Thereafter, the cell lysates were carefully removed from the wells and transferred into individual Eppendorf tubes for further processing.

80 μl were further processed according to SOP NMET144 into soluble and insoluble fraction, the remaining 10+μl of this total fraction were stored separately for analysis of total alpha synuclein.

Optionally samples were frozen at -80 ℃ at this step until further processing.

For processing into soluble and insoluble fraction, the 80 μl in Eppendorf tubes were centrifuged at 15.000x g for 60 minutes at 4 ℃. The supernatant (= soluble fraction) was transferred into fresh tubes and stored at -20 ℃ or -80 ℃ for long-term storage. The remaining pellet was washed once with Extraction Buffer 1 and thereafter in 40 μl Extraction Buffer 2 (20 mM Tris-HCl pH 7,4; 50 mM NaCl; 1% Triton X-100; 2% SDS) and sonicated in an ultrasound water bath for 1 min until pellet was dissolved completely (= insoluble fraction). Samples were store at -20 ℃ or -80 ℃ for long-term storage.

Small proportions of each fraction were used to determine total protein content with either UV/VIS Nanodrop measurement or BCA assay (ThermoFisher, 23225).

2.6.4 Set1: Human alpha-synuclein MSD

Human alpha-synuclein levels in the total fractions of all samples (total number of samples analysed: n=288) were determined by using a commercially available immunosorbent assay (Meso Scale Diagnostic, Cat. No. K151TGD) according to the manufacturer's protocol and plates were read on the MESO QuickPlex SQ 120 reader. Samples were applied at uniform total protein concentrations (1 μg/ml for soluble and total and 35 μg/ml for insoluble fraction) and evaluated in comparison to an adequate protein standard as pg human alpha-synuclein per μg total protein.

2.6.5 Set1: p62 levels by MSD

Soluble fractions as described under section 2.6.3 were used for the analysis (total number of samples analysed: n=90).

Samples were analysed for Total p62 (SQSTM1) with a commercially available immunosorbent assay kit from Mesoscale Diagnostics (MSD, Cat.No. K151MJD) at uniform total protein concentration of 25 μg/ml. The immune assay was carried out according to the manual and plates were read on the MESO QuickPlex SQ 120 reader. Analyte levels were evaluated according to adequate calibration curves (recombinant human SQSTM1 produced in E.Coli from Prospec, Cat.No. PRO-806) as arbitrary units (AU).

2.6.6 Set1: LC3BI/II levels by ProteinSimple WES

Automated separation and immunostaining of LC3B in the soluble fraction of all samples (n=90; section 2.6.3) were carried out using a capillary-based immunoassay, WESTM (ProteinSimple®). Samples were applied to a 25 capillary cartridge with a 2 to 220 kDa matrix, according to the manufacturer's protocol. After samples and antibodies (see table below) had been pipetted into the pre-filled assay plate purchased from the manufacturer, sample loading, separation, immunoprobing, washing, detection and quantitative data analysis was performed automatically by WESTM Western system.

The areas under the curve for 16 kDA and 18 kDA LC3B positive bands were used for the analysis. Results are presented as area under the curve (AUC).

2.6.7 Set2: Mitochondrial activity and synapse formation

MitoTracker Red CMXRos (ThermoFischer, M7512) was used to detect active mitochondria in living cells according to SOP NMET138 with slight modifications:

Supernatant of the cultivated cells on 96-well plates was aspirated off after 48 h of treatment.

190 μl ACSF buffer (125 mM NaCl, 2.5 mM KCl, 26.2 mM NaHCO₃, 1 mM NaH₂PO₄, 2.5 mM CaCl₂, 1.25 mM MgCl₂, 25 mM Hepes, 11 mM D-Glucose, pH 7.3) were added to all wells.

Plates were incubated for 15 minutes in the incubator at 37 ℃. Then 10 μl MitoTracker Red CMXRos of 4 μM working solution were added to each well to get a final concentration of 200 nM.

Plates were incubated for 45 min in the incubator at 37 ℃ (light protected).

Supernatant was aspirated off completely and discarded. Thereafter 100 μl PBS were added and the plate was read in Cytation 5 multimode reader at 579/600 nm (+/- 10 nm).

Cells were then fixed by addition of equal volume 4% paraformaldehyde (PFA) to the PBS at room temperature (RT) for 30 minutes. Fixed cells were stored in PBS at 4 ℃ until further use for immunocytochemistry (ICC).

Cells were rinsed two times with PBS and were permeabilized with 0.1% Triton X-100 in PBS for 30 minutes at RT. Next, cells were blocked for 60 min at RT with 20% horse serum, 0.1% Triton X-100 in PBS.

Then, samples were incubated with the primary antibodies described below at 4 ℃ overnight.

Next day, cells were further incubated for another 30 min at RT. After three washing steps with PBS, cells were incubated with the fluorescently labelled secondary antibodies and DAPI (nucleus) for 1.5 hours at RT in the darkness. Cells were again rinsed four times with PBS and imaged with the Cytation 5 Multimode reader (BioTek) in GFP (synapses) and Texas Red TR (Mitotracker) channels, as well as DAPI. From each well, 6 images were taken at 10x magnification.

The fluorescence area of TR channel was measured to quantify active mitochondria using Gen05 software.

Synapsin positive small spots were counted as synapses.

2.7 Statistics

Basic statistical analysis was performed using GraphPad Prism 9. Data are resented as mean ± standard error of mean (SEM) and group differences are evaluated by One-way ANOVA followed by Dunnett's multiple comparisons test versus LC or VC.

2.8. Results

2.8.1 Part1: Experiment in MPP+ lesioned primary rat TH neurons

2.8.1.1 Number of TH positive and apoptotic neurons, synapses

The ability of the compound to rescue dopaminergic neurons and synapses was tested on set1 of these experiments.

MPP+ lesioned neurons were fixed and stained for MAP2, TH and PSD95 as neuronal marker. The number of TH positive neurons in relation to total number of neurons was assessed and expressed as %TH neurons.

The lesion was able to significantly reduce all tested read outs, the reference item MK801 could at least partially rescue them (FIG. 4).

The compound of formula (I) treatment at 10-100 nM led to significant rescue of TH positive neurons compared to the vehicle treated lesion control (LC; FIG. 1). Interestingly, also the lower concentrations of the compound were able to significantly increase the number of PSD95 positive synapses compared to the lesion control (FIG. 2).

All tested concentrations of the compound of formula (I), except the highest dose of 1000 nM, were able to significantly reduce the number of apoptotic (caspase 3 positive) cells as well as TH neurons (FIG. 3 A,B).

2.8.1.2 Mitochondrial health of vM neurons

Mitochondrial health of neuronal cultures after MPP+ lesion and treatments, was assessed using mitotracker dye. The compound of formula (I) showed trends towards rescued mitochondrial activity compared to the lesion control (FIG. 5).

Also, Reference (RI MK801) led to the expected rescue of mitrotracker signal (FIG. 5 B).

2.8.2 Part2: Experiment in alpha-synuclein overexpressing SH-SY5Y cells

2.8.2.1 Total α-synuclein level

The amount of alpha-synuclein in total fractions of cell lysates was assessed by Mesoscale Discovery assay.

In addition, a detergent free total fraction was assessed with MSD assay. Due to absence of detergent this fraction represents mainly soluble monomers. Interestingly, the compound of formula (I) at 100 and 1000 nM led to a reduction of these alpha-synuclein species (FIG. 6).

2.8.2.2 Autophagy marker p62 and LC3B

When autophagy is potently inhibited, p62 accumulates in the cell. Therefore, p62 levels are reduced when autophagy is induced.

The treatment by the compound of formula (I) did not significantly affect p62 levels compared to the vehicle control (FIG. 7).

WES analysis of LC3B only showed one prominent band of 18kDa, corresponding to LC3BII, but LC3BI at 16kDa was hardly detectable, a phenomenon is often observed in this cell type. Therefore, no ratio could be calculated and only LC3BII values are given in FIG. 8.

No significant effect of the compound of formula (I) treatment on LC3BII was detected compared to the vehicle control (FIG. 8).

2.8.2.3 Mitochondrial health of SH-SY5Y-alpha-syn cells

Mitochondrial health was again assessed using mitotracker dye.

For the compound of formula (I) at medium concentrations the mitotracker showed trends towards increased signal, getting significant for the compound at 30 nM and 100 nM compared to the respective vehicle control (FIG. 9).

2.8.2.4 Number of synapses in SH-SY5Y-alpha-syn cells

The number of synapses in SH-SY5Y-alpha-syn cells was assessed by quantification of synapsin positive spots.

A significant increase in the number of synapses was detected when comparing vehicle control with 10nM the compound-treated cells (FIG. 10).

Equivalents and Incorporation By Reference

While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. Therefore, it must be understood that the embodiments described above are for illustrative purposes and do not limit the present invention.

All references, issued patents and patent applications cited within the body of the instant specification, are herein incorporated by reference in their entirety for all purposes.

Claims (6)

1. A pharmaceutical composition for neuroprotection; treating or preventing neuro-inflammation; or treating or preventing synucleinopathy, comprising 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient.
2. The pharmaceutical composition according to claim 1, wherein the neuroprotection is for treating or preventing neurodegenerative disease, stroke, traumatic brain injury, or spinal cord injury; and for acute management of neurotoxin consumption.
3. The pharmaceutical composition according to claim 1, wherein the synucleinopathy is any one selected from the group consisting of Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, and Alzheimer's disease with Lewy bodies.
4. A use of 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for neuroprotection; treating or preventing neuro-inflammation; or treating or preventing synucleinopathy.
5. The use of 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof according to claim 4, wherein the neuroprotection is for treating or preventing neurodegenerative disease, stroke, traumatic brain injury, or spinal cord injury; and for acute management of neurotoxin consumption.
6. The use of 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof according to claim 4, wherein the synucleinopathy is any one selected from the group consisting of Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, and Alzheimer's disease with Lewy bodies.

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