Therapeutic agent for neurodegenerative disease comprising compound having s1p3 receptor inverse agonist activity

Abstract

To find an effective agent for prevention and / or treatment of neurodegenerative disease and provide it as a pharmaceutical.SOLUTION: A compound with S1P3 receptor inverse agonist activity is effective as an agent for prevention and / or treatment of neurodegenerative disease. It has been found that the S1P3 receptor exhibits constitutive activity and that this constitutive activity is associated with neural inflammatory responses. Moreover, the inventors have found that a pharmaceutical containing a compound with S1P3 receptor inverse agonist activity as an active component solves the above problem.SELECTED DRAWING: None

Description

[Technical Field]

[0001] In one aspect, the present disclosure relates to a preventive and / or therapeutic agent for a neurodegenerative disease, which comprises a compound having S1P3 receptor inverse agonist activity as an active ingredient, and a treatment method using the same.

[0002] More specifically, in one aspect, the present disclosure relates to a preventive and / or therapeutic agent for a neurodegenerative disease associated with reactive astrocytes, which comprises as an active ingredient a compound having S1P3 receptor inverse agonist activity, and a therapeutic method using the same. [Background technology]

[0003] Sphingosine-1-phosphate (S1P) is a type of phospholipid produced in vivo and is a physiologically active substance that induces cell growth, differentiation, and migration by binding to receptors expressed on the cell membrane. S1P is produced by sphingokinase, and its concentration is known to increase in inflammatory conditions (bronchial asthma, autoimmune diseases, etc.).

[0004] There are five known G protein-coupled receptors (also known as GPCRs) for S1P receptors, S1P1 to S1P5. Among them, S1P3 receptors are expressed in various types of cells and control metabolic regulation, division, cell survival, and cell morphology.

[0005] Astrocytes are the main type of glial cell present in the central nervous system (brain and spinal cord) and play an important role in the central nervous system by controlling the permeability of the blood-brain barrier and blood-tumor barrier and by participating in inflammatory responses.

[0006] On the other hand, inflammation in the central nervous system (also known as neuroinflammation) is a factor that worsens neurodegenerative diseases, and it has been reported that neuroinflammation induces reactive astrocytes, that reactive astrocytes increase in neurodegenerative diseases such as Alzheimer's disease, and that reactive astrocytes induce cell death of neurons and oligodendrocytes (Non-patent document 1).

[0007] Furthermore, S1P3 receptors are highly expressed in reactive astrocytes, and their expression is enhanced by inflammatory stimuli. S1P3 receptors have been reported to enhance the inflammatory response of astrocytes by activating Ras homolog family member A (also known as RhoA) and inducing mRNA for cyclooxygenase-2 (also known as COX-2), interleukin-6 (IL-6), and vascular endothelial growth factor A (VEGFα) (Non-Patent Document 2).

[0008] It has not been known until now that the S1P3 receptor has constitutive activity and that this constitutive activity is involved in neuroinflammatory responses. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Nature, Vol.541, No.7638, pages 481-487, 2017 [Non-patent document 2] Journal of neuroinflammation, Vol.14, No.111, 2017 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present disclosure is to find an effective agent for preventing and / or treating neurodegenerative diseases and provide it as a pharmaceutical. [Means for solving the problem]

[0011] The present inventors have found that the S1P3 receptor has constitutive activity, and that constitutive activity is involved in neuroinflammatory responses. Furthermore, the present inventors have found that a pharmaceutical containing a compound having S1P3 receptor inverse agonist activity as an active ingredient can solve the above-mentioned problems.

[0012] That is, the present disclosure provides, as an embodiment thereof, for example: [1] A preventive and / or therapeutic agent for neurodegenerative diseases, comprising a compound having S1P3 receptor inverse agonist activity; [2] Provided are pharmaceutical compositions and the like containing compounds having S1P3 receptor inverse agonist activity. [Effects of the Invention]

[0013] The present disclosure is useful for the prevention and / or treatment of neurodegenerative diseases. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1a shows the luciferase activity (hereinafter referred to as RLU) of reporter cells. The vertical axis shows RLU, and the horizontal axis shows reporter cells (mock) that do not express the S1P3 receptor or reporter cells (S1P3) that express the S1P3 receptor. Figures 1b, 1c, and 1d show the results of measuring the S1P3 receptor inverse agonist activity of the compounds described in Examples 1, 2, and 3, respectively. The vertical axis shows RLU, and the horizontal axis shows the concentration of the compounds described in Examples 1, 2, and 3. [Figure 2] Figure 2 shows the relative expression level of the IL-6 gene in drug-inducible S1P3 receptor-expressing cells. The vertical axis shows the relative expression level, and the horizontal axis shows cells not treated with doxycycline (hereinafter referred to as DOX), cells treated with DOX, or cells treated with DOX and the compound described in Example 3. [Figure 3]Figure 3 shows the effect of the compound described in Example 1 on plasma neurofilament light chain (NfL) concentrations in amyotrophic lateral sclerosis (ALS) model mice. The vertical axis shows plasma NfL concentrations, and the horizontal axis shows groups administered with a vehicle or the compound described in Example 1. *p<0.05, **p<0.01 (comparison with the SOD1 vehicle-administered group: Welch's t-test). For definitions of WT1 and SOD1, see Biological Example 3 herein. [Figure 4] Figure 4a shows the results of the beam test, demonstrating the effect of the compound described in Example 1 on the motor performance decline of 5-month-old (Figure 4a, left) and 9-month-old (Figure 4a, right) multiple system atrophy (MSA) model mice. The vertical axis indicates the percentage of slips relative to the total number of steps, and the horizontal axis indicates the groups administered with the vehicle or the compound described in Example 1. *p<0.05 (compared to the WT2 vehicle-administered group: Student's t-test), #p<0.05 (compared to the PLP-SYN vehicle-administered group: Dunnett's test). For the definitions of WT2 and PLP-SYN, see Biological Example 4 herein. Figure 4b shows the results of the notch bar test, demonstrating the effect of the compound described in Example 1 on the motor performance decline of 5-month-old (Figure 4b, left) and 9-month-old (Figure 4b, right) MSA model mice. The vertical axis indicates the percentage of slips of the hind limbs, and the horizontal axis indicates the groups administered with the vehicle or the compound described in Example 1. ***p<0.001 (Student's t-test compared with the WT2 vehicle-administered group), ****p<0.0001 (Student's t-test compared with the WT2 vehicle-administered group), #p<0.05, ##p<0.01 (Dunnett's test compared with the PLP-SYN vehicle-administered group). [Figure 5]Figure 5a shows the effect of the compound described in Example 2 on the cognitive function of Alzheimer's disease (hereinafter referred to as AD) model mice. The vertical axis shows the alternation rate, and the horizontal axis shows the groups administered with the vehicle or the compound described in Example 2. **p<0.01 (comparison with the NL-GF vehicle-administered group: Welch t-test). For the definitions of WT3 and NL-GF, see Biological Example 5 herein. Figure 5b shows the effect of the compound described in Example 2 on the cognitive function of AD model mice. The vertical axis shows the novel object recognition rate, and the horizontal axis shows the groups administered with the vehicle or the compound described in Example 2. **p<0.01 (comparison with the NL-GF vehicle-administered group: Welch t-test). [Figure 6] Figure 6 shows the effect of the compound described in Example 3 on the motor function of spinal cord injury model mice. The vertical axis represents the BBB score. **p<0.01 (comparison between untreated and vehicle-administered groups: Wilcoxon rank-sum test). #p<0.05 (comparison between vehicle-administered groups and Example 3-administered groups: Wilcoxon rank-sum test). Untreated mice refer to 8-week-old female C57BL / 6J mice without spinal cord injury. [Figure 7] 7 shows the effect of the compound described in Example 3 on the deterioration of neurological symptoms in cerebral infarction model rats. The vertical axis shows the neurological symptom score, and the horizontal axis shows the groups administered with the vehicle or the compound described in Example 3. *p<0.05 (comparison with the vehicle-administered group: Steel test). [Figure 8] Figure 8a shows the effect of the compound described in Example 3 on epileptic symptoms in Alexander disease model mice. The vertical axis represents the score, and the horizontal axis represents the time after seizure onset. *p<0.05 (comparison with the vehicle-administered group: Mann-Whitney U test), $p<0.05 (comparison between the AxD vehicle-administered group and the Example 3 (3 mg / kg)-administered group: Steel test), #p<0.05 (comparison between the AxD vehicle-administered group and the Example 3 (30 mg / kg)-administered group: Steel test). For the definitions of WT4 and AxD, see Biological Example 8 herein. Figure 8b shows the effect of the compound described in Example 3 on epileptic symptoms in Alexander disease model mice. The vertical axis represents the total score, and the horizontal axis represents the groups administered with the vehicle or the compound described in Example 3. [Figure 9] 9 shows the effect of the compound described in Example 1 on the survival time of Sandhoff disease model mice. The vertical axis indicates the survival rate, and the horizontal axis indicates the survival time. For the definitions of WT5 and SD, see Biological Example 9 herein. [Figure 10] Figure 10 shows the effect of the compound described in Example 3 on the cognitive function of mucopolysaccharidosis type IIIA model mice. The vertical axis shows the novel object recognition rate, and the horizontal axis shows the groups administered with the vehicle or the compound described in Example 3. *p<0.05 (comparison with the vehicle-administered group: Mann-Whitney U test), ##p<0.01 (comparison with the vehicle-administered group: Steel test). For the definitions of WT6 and MPSIIIA, see Biological Example 10 herein. [Figure 11] Figure 11a shows the effect of the compound described in Example 2 on the increase in reactive astrocytes in the brain tissue of AD model mice. The white areas in the image indicate the anti-GFAP-positive area. Figure 11b shows the effect of the compound described in Example 2 on the increase in reactive astrocytes in the brain tissue of AD model mice. The vertical axis shows the anti-GFAP-positive area ratio, and the horizontal axis shows the groups administered with the vehicle or the compound described in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0015] [Compounds with S1P3 receptor inverse agonist activity] The compound having S1P3 receptor inverse agonist activity used in the present disclosure is not particularly limited as long as it is a substance having S1P3 inverse agonist activity. One embodiment includes a low molecular weight compound. A low molecular weight compound means a compound having a molecular weight of 1000 or less. The lower limit of the molecular weight is not particularly limited, but is, for example, 100 or more. One embodiment of the low molecular weight compound includes the following compounds:

[0016] [ka]

[0017] [ka]

[0018] [ka]

[0019] The above compounds can be used alone or in combination of two or more.

[0020] In the present disclosure, inverse agonist activity means the action of interacting with a receptor that expresses activity without being affected by a ligand, thereby attenuating its intracellular signal transduction.

[0021] In the present disclosure, a compound having S1P3 receptor inverse agonist activity means a compound having inverse agonist activity against the S1P3 receptor (also called an inverse agonist or inverse agonist agent).

[0022] In the present disclosure, the compound having S1P3 receptor inverse agonist activity is not particularly limited as long as it acts as a compound having inverse agonist activity on the S1P3 receptor.

[0023] The compounds having S1P3 receptor inverse agonist activity used in the present disclosure can be produced, for example, according to the production methods in the following [Synthesis Examples], but are not limited thereto.

[0024] Compounds having S1P3 receptor inverse agonist activity used in the present disclosure include not only those that have been discovered so far, but also those that will be discovered in the future.

[0025] Compounds having S1P3 receptor inverse agonist activity for use in the present disclosure can be selected or screened by measuring S1P3 receptor inverse agonist activity using the methods described herein, for example, in Biological Example 1.

[0026] Compounds having S1P3 receptor inverse agonist activity as used in the present disclosure include compounds that have been shown to have S1P3 receptor inverse agonist activity by the methods described herein, for example, in Biological Example 1.

[0027] In one embodiment, compounds having S1P3 receptor inverse agonist activity for use in the present disclosure can be selected or screened from a group of compounds exhibiting S1P3 receptor antagonist activity by measuring S1P3 receptor inverse agonist activity using the method described in Biological Example 1.

[0028] The compound having S1P3 receptor inverse agonist activity used in the present disclosure preferably has an EC 50 EC is 10 μmol / L or less. 50 Since the lower the concentration, the better, there is no particular lower limit, but it is, for example, 0.01 nmol / L or more.

[0029] In this disclosure, all isomers are encompassed unless otherwise specified. For example, alkyl groups, alkoxy groups, and alkylene groups include both straight-chain and branched-chain isomers. Furthermore, isomers (E, Z, cis, trans) in double bonds, rings, and fused rings; isomers due to the presence of asymmetric carbons (R, S, α, β, enantiomers, diastereomers); optically active isomers with optical rotation (D, L, d, l); polar isomers (high polarity, low polarity) obtained by chromatographic separation; equilibrium compounds; rotational isomers; mixtures of these in any proportion; and racemic mixtures are all encompassed in this disclosure. Furthermore, all isomers due to tautomerism are also encompassed in this disclosure.

[0030] In the present disclosure, constitutive activity refers to the activity that a receptor expresses without the influence of a ligand.

[0031] In the present disclosure, reactive astrocytes refer to activated astrocytes induced in conditions such as neurodegenerative diseases and neuronal trauma. Reactive astrocytes are known to express and release inflammatory cytokines such as IL-6. Therefore, one of the characteristics of reactive astrocytes is the expression of IL-6 mRNA.

[0032] The compounds having S1P3 receptor inverse agonist activity used in the present disclosure can be suitably used as astrocyte activation regulators, reactive astrocyte inhibitors, IL-6 expression inhibitors, etc. Therefore, the present disclosure encompasses astrocyte activation regulators, reactive astrocyte inhibitors, or IL-6 expression inhibitors that contain compounds having S1P3 receptor inverse agonist activity.

[0033] In this disclosure, unless otherwise specified, symbols that will be apparent to those skilled in the art are used.

[0034] [ka]

[0035] indicates bonding to the other side of the paper (i.e., α-configuration),

[0036] [ka]

[0037] represents any mixture of α- and β-configurations.

[0038] [Application to pharmaceuticals] Compounds having S1P3 receptor inverse agonist activity are useful as prophylactic and / or therapeutic agents for neurodegenerative diseases, pain, cancer, liver disease, endocrine or metabolic diseases, heart disease, respiratory diseases, etc., and are particularly useful as prophylactic and / or therapeutic agents for neurodegenerative diseases. More specifically, compounds having S1P3 receptor inverse agonist activity are useful as prophylactic and / or therapeutic agents for neurodegenerative diseases associated with reactive astrocytes.

[0039] Examples of neurodegenerative diseases or neurodegenerative diseases associated with reactive astrocytes include Alzheimer's disease (hereinafter referred to as AD), Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (hereinafter referred to as ALS), frontotemporal dementia, multiple sclerosis, Friedreich's ataxia, multiple system atrophy (hereinafter referred to as MSA), dementia with Lewy bodies, progressive supranuclear palsy, Pick's disease, Creutzfeldt-Jakob disease, spinocerebellar degeneration, Duchenne muscular dystrophy, Charcot-Marie-Tooth disease, Fabry disease, mitochondrial diseases, Prader-Willi syndrome, Rett syndrome, Krabbe disease, Batten disease, Sandhoff disease, Tay-Sachs disease, Gaucher disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, mucopolysaccharidosis type III, mucopolysaccharidosis type IV, These include mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, mucopolysaccharidosis type IX, Klippel-Feil syndrome, Niemann-Pick disease, Wilson disease, Alexander disease, Canavan disease, Lesch-Nyhan syndrome, cerebral leukodystrophy, Sturge-Weber syndrome, Machado-Joseph disease, Refsum disease, Wernicke-Korsakoff syndrome, idiopathic normal pressure hydrocephalus, autosomal dominant cerebral arteriopathy with subcortical infarction and leukoencephalopathy, creatine deficiency syndrome, Shy-Drager syndrome, SMON, spinal deformity, encephalomalacia, pontocerebellar atrophy, Moebius syndrome, Meniere's disease, basal degeneration, oculopharyngeal muscular dystrophy, spinal cord injury, cerebrovascular disorders (e.g., stroke, subarachnoid hemorrhage, cerebral hemorrhage, cerebral infarction, transient ischemic attack), and epilepsy.

[0040] In the present disclosure, neurodegenerative diseases associated with reactive astrocytes are preferably amyotrophic lateral sclerosis, multiple system atrophy, Alzheimer's disease, spinal cord injury, cerebrovascular disease, Alexander disease, Sandhoff disease, or mucopolysaccharidosis type III, more preferably amyotrophic lateral sclerosis, multiple system atrophy, or Alzheimer's disease, and even more preferably amyotrophic lateral sclerosis or multiple system atrophy.

[0041] Mucopolysaccharidosis type III is preferably mucopolysaccharidosis type IIIA.

[0042] The cerebrovascular disorder is preferably cerebral infarction.

[0043] To use a compound with S1P3 receptor inverse agonist activity for the prevention and / or treatment of the above-mentioned diseases, the active ingredient, a compound with S1P3 receptor inverse agonist activity, is typically formulated with a pharmaceutically acceptable carrier, such as various additives or solvents, and then administered systemically or locally, orally or parenterally. Here, a pharmaceutically acceptable carrier refers to a substance other than the active ingredient that is generally used in pharmaceutical formulations. A pharmaceutically acceptable carrier is preferably one that does not exhibit pharmacological activity at the dosage of the formulation, is harmless, and does not interfere with the therapeutic effect of the active ingredient. Pharmaceutically acceptable carriers can also be used for purposes such as enhancing the usefulness of the active ingredient and formulation, facilitating formulation, stabilizing quality, or improving usability. Specifically, substances such as those listed in "Dictionary of Pharmaceutical Additives," published by Yakuji Nipposha in 2000 (edited by the Japan Pharmaceutical Additives Association) can be selected appropriately depending on the purpose.

[0044] The compound having S1P3 receptor inverse agonist activity is administered to a mammal (preferably a human, more preferably a human patient) in a pharmaceutically effective amount.

[0045] The dosage of a compound having S1P3 receptor inverse agonist activity may be an effective dose, which will inevitably vary depending on age, body weight, symptoms, desired therapeutic effect, administration route, duration of treatment, etc. Generally, it is orally administered in the range of 0.1 ng to 1000 mg per patient per dose, or parenterally administered in the range of 0.01 ng to 100 mg per patient per dose, or continuously administered intravenously.

[0046] Of course, as mentioned above, the dosage varies depending on various conditions, so in some cases a smaller dosage than the above may be sufficient, and in other cases a dosage exceeding the range may be necessary.

[0047] Examples of dosage forms used for administration include oral preparations (e.g., tablets, capsules, granules, powders, oral liquids, syrups, oral jellies, etc.), oral preparations (e.g., oral tablets, oral sprays, oral semisolids, mouthwashes, etc.), injectable preparations (e.g., injectable preparations, etc.), dialysis preparations (e.g., dialysis preparations, etc.), inhalation preparations (e.g., inhalants, etc.), ophthalmic preparations (e.g., eye drops, eye ointments, etc.), otic preparations (e.g., ear drops, etc.), nasal preparations (e.g., nasal drops, etc.), rectal preparations (e.g., suppositories, rectal semisolids, enteral injections, etc.), vaginal preparations (e.g., vaginal tablets, vaginal suppositories, etc.), and dermatological preparations (e.g., solid preparations for external application, liquid preparations for external application, sprays, ointments, creams, gels, patches, etc.).

[0048] [Oral administration formulation] Oral formulations include, for example, tablets, capsules, granules, powders, oral liquids, syrups, and oral jellies. Oral formulations include rapidly disintegrating formulations, in which the release of the active ingredient from the formulation is not specifically controlled, and modified-release formulations, such as enteric-coated formulations and sustained-release formulations, in which the release is specifically controlled by a specific formulation design and manufacturing method. Enteric-coated formulations are designed to release the active ingredient primarily in the small intestine rather than in the stomach, for purposes such as preventing the active ingredient from being decomposed in the stomach or reducing the irritating effect of the active ingredient on the stomach. These formulations are typically prepared by coating with an acid-insoluble enteric base. Sustained-release formulations are formulations in which the release rate, release time, and release site of the active ingredient are controlled for purposes such as reducing the frequency of administration or reducing side effects. These formulations are typically prepared by using an appropriate sustained-release agent. Among preparations for oral administration, capsules, granules, tablets, etc. may be coated with an appropriate coating agent such as a sugar, sugar alcohol, or polymer compound for the purpose of facilitating administration or preventing decomposition of the active ingredient.

[0049] (1) Tablets Tablets are solid preparations having a certain shape that are administered orally, and include those generally called tablets, such as plain tablets, film-coated tablets, sugar-coated tablets, multi-layer tablets, and dry-coated tablets, as well as oral rapidly disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, and dissolving tablets. When plain tablets are manufactured, the following method (a), (b), or (c) is usually used: (a) Add excipients, binders, disintegrants, and other additives to the active ingredient and mix until homogeneous, then granulate using water or a solution containing a binder in an appropriate manner, add lubricants, mix, and compress; (b) The active ingredient is mixed with additives such as excipients, binders, disintegrants, etc. to form a homogeneous mixture, which is then directly compressed and molded, or the active ingredient and lubricants are added to granules prepared in advance with additives, mixed together to form a homogeneous mixture, and then compressed and molded; (c) The active ingredient is mixed with additives such as excipients and binders to form a homogeneous mixture, and the mixture is moistened with a solvent and poured into a mold to form the mixture, after which it is dried in an appropriate manner; are used. Film-coated tablets can usually be produced by coating a plain tablet with a thin coating of an appropriate coating agent such as a polymer compound. Sugar-coated tablets can usually be produced by coating a plain tablet with a coating agent containing sugars or sugar alcohols. Multilayer tablets can be produced by stacking powder particles of different compositions in layers using an appropriate method and compressing them. Dry-coated tablets can be produced by coating an inner core tablet with an outer layer of different composition. Tablets can also be made into enteric-coated or sustained-release tablets using known appropriate methods. Orally rapidly disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, and dissolving tablets are tablets that have unique functions imparted to them by appropriate selection of excipients, and can be produced in accordance with the above-mentioned tablet production methods. Furthermore, an intraorally rapidly disintegrating tablet is a tablet that can be taken by quickly dissolving or disintegrating in the mouth; a chewable tablet is a tablet that is taken by chewing; an effervescent tablet is a tablet that dissolves or disperses while rapidly effervescent in water; a dispersible tablet is a tablet that is taken by dispersing in water; and a dissolving tablet is a tablet that is taken by dissolving in water. Effervescent tablets can be produced by using appropriate acidic substances, carbonates, bicarbonates, etc. as additives.

[0050] (2) Capsules Capsules are preparations filled into capsules or encapsulated with a capsule base, and include hard capsules, soft capsules, etc. Hard capsules can be produced by blending the active ingredient with additives such as excipients to form a homogeneous mixture, or by forming it into granules or molded products using an appropriate method, and then filling the mixture directly into a capsule or by lightly molding it. Soft capsules can be produced by encapsulating the active ingredient with additives and molding it into a specific shape using an appropriate capsule base such as gelatin whose plasticity has been increased by adding glycerin, D-sorbitol, etc. Capsules can also be made into enteric-coated capsules or sustained-release capsules using appropriate known techniques, and coloring agents, preservatives, etc. can also be added to the capsule base.

[0051] (3) Granules Granules are preparations that have been granulated into granules, and include not only those generally called granules but also effervescent granules. When manufacturing granules, the following methods (a), (b), or (c) are usually used: (a) The powdered active ingredient is mixed with an excipient, binder, disintegrant, or other additive to form a homogeneous mixture, and then granulated by a suitable method; (b) Add excipients and other additives to the pre-granulated active ingredient and mix until homogeneous; (c) Add excipients and other additives to the active ingredient that has been prepared in advance into granules, mix them, and form them into granules by an appropriate method; Granules can be coated as needed, and can also be made into enteric coated granules or sustained-release granules using known appropriate methods. Effervescent granules can be produced by using appropriate acidic substances, carbonates, bicarbonates, etc. as additives. Effervescent granules refer to granules that dissolve or disperse in water while rapidly effervescent. Granules can also be made into fine granules by adjusting the particle size.

[0052] (4) Powder Powders are powdered preparations, and can usually be produced by adding excipients or other additives to the active ingredient, mixing them together, and making them homogeneous.

[0053] (5) Oral liquid Oral liquids are liquid or fluid, viscous gel-like preparations, and include not only those commonly referred to as oral liquids but also elixirs, suspensions, emulsions, lemonades, etc. Oral liquids are typically prepared by adding additives and purified water to the active ingredient, mixing to form a homogeneous solution, or emulsifying or suspending the mixture, and then filtering as necessary. Elixirs are clear, liquid oral liquids containing sweet and aromatic ethanol, typically prepared by dissolving a solid active ingredient or its extract in ethanol, purified water, flavoring agents, and sucrose, other sugars, or sweeteners, and then filtering or otherwise preparing a clear liquid. Suspensions are oral liquids in which the active ingredient is finely and homogeneously suspended, typically prepared by adding suspending agents or other additives and purified water or oil to the solid active ingredient, suspending the mixture in an appropriate manner, and homogenizing the entire mixture. An emulsion is an oral liquid preparation in which the active ingredient is finely and homogeneously emulsified, and can usually be produced by adding an emulsifier and purified water to the liquid active ingredient, emulsifying it in an appropriate manner, and making the whole homogeneous. Meanwhile, a lemonade is a clear, liquid oral preparation with a sweet and sour taste.

[0054] (6) Syrup Syrups are viscous liquid or solid preparations containing sugars or sweeteners, including syrup preparations. Syrups are typically prepared by adding an active ingredient to a solution of sucrose, other sugars, or sweeteners, or to a simple syrup, dissolving, mixing, suspending, or emulsifying the mixture, boiling the mixture as needed, and then filtering it while hot. Syrup preparations are granular or powder preparations that become syrups upon addition of water, and are sometimes referred to as dry syrups. Syrup preparations typically use sugars or sweeteners as additives and can be prepared in accordance with the manufacturing methods for the granules or powders described above.

[0055] (7) Oral jelly Oral jellies are non-flowable, molded gel-like preparations, and can usually be produced by mixing the active ingredient with additives and a polymer gel base, gelling it using an appropriate method, and molding it into a specific shape.

[0056] [Injectable preparation] (1) Injectable Injections are solutions, suspensions, emulsions, or solid sterile preparations that are administered subcutaneously, intramuscularly, or directly into body tissues or organs such as blood vessels, and are dissolved or suspended just before use. In addition to what is generally called an injection, these include freeze-dried injections, powder injections, pre-filled syringes, cartridges, infusions, implanted injections, and sustained-release injections. When manufacturing injections, the following method (a) or (b) is usually used: (a) The active ingredient, either as is or with the addition of additives, is dissolved, suspended, or emulsified in water for injection, other aqueous solvents, or non-aqueous solvents to form a homogeneous solution, which is then filled into a container for injection, sealed, and sterilized; (b) The active ingredient, either as it is or with the addition of additives, is dissolved, suspended, or emulsified in water for injection, other aqueous solvents, or non-aqueous solvents to form a homogeneous solution, which is then filtered aseptically, or the homogeneous solution is prepared aseptically and filled into a container for injection and sealed; Freeze-dried injectables are typically prepared by dissolving the active ingredient directly or with additives such as excipients in water for injection, sterile filtering, and filling into an injectable container followed by freeze-drying, or by freeze-drying in a dedicated container and then filling directly into a container. Powdered injectables are typically prepared by sterile filtering, followed by crystallization of the powder, or by adding sterilized additives to the powder, and then filling into an injectable container. Prefilled syringes are typically prepared by filling the syringe with the active ingredient directly or a solution, suspension, or emulsion prepared using the active ingredient and additives. Cartridges are injectables that are inserted into dedicated syringes and used with a drug solution. Cartridges filled with drug solutions are typically prepared by filling the cartridge with the active ingredient directly or a solution, suspension, or emulsion prepared using the active ingredient and additives. Infusion solutions are injectables typically 100 mL or more that are administered intravenously. An implantable injection refers to a solid or gel-like injection that is administered subcutaneously, intramuscularly, etc. using an implantation device or by surgery, with the aim of releasing the active ingredient over a long period of time. An implantable injection can usually be produced by using a biodegradable polymer compound and forming it into pellets, microspheres, or a gel. A sustained-release injection refers to an injection that is administered intramuscularly, etc., with the aim of releasing the active ingredient over a long period of time, and can usually be produced by dissolving or suspending the active ingredient in vegetable oil, etc., or by forming it into a suspension of microspheres using a biodegradable polymer compound.

[0057] Compounds with S1P3 receptor inverse agonist activity include: 1) complementing and / or enhancing the prophylactic and / or therapeutic effects of the compound; 2) improving the kinetics and absorption of the compound, reducing the dosage, and / or 3) Reducing the side effects of the compound For this purpose, it may be administered in combination with other drugs as a concomitant drug.

[0058] A combination of a compound having S1P3 receptor inverse agonist activity and another drug may be administered in the form of a combined preparation in which both components are combined in a single preparation, or in the form of separate preparations. Administration in these separate preparations includes simultaneous administration and staggered administration. Staggered administration may involve administering the compound having S1P3 receptor inverse agonist activity first and the other drug later, or administering the other drug first and the compound having S1P3 receptor inverse agonist activity later. The administration methods for each may be the same or different.

[0059] There are no particular limitations on the diseases for which the above-mentioned combination drug can have a preventive and / or therapeutic effect, as long as the preventive and / or therapeutic effect of the compound having S1P3 receptor inverse agonist activity is complemented and / or enhanced.

[0060] Examples of other drugs that can be used to complement and / or enhance the preventive and / or therapeutic effects of compounds having S1P3 receptor inverse agonist activity on neurodegenerative diseases or neurodegenerative diseases associated with reactive astrocytes include donepezil, rivastigmine, galantamine, memantine, levodopa, carbidopa, entacapone, pramipexole, ropinirole, rotigotine, selegiline, rasagiline, amantadine, tetrabenzadine, deutetrabenzadine, riluzole, edaravone, idebenone, and coenzyme Q10. These include Zyme Q10, L-DOPA, trihexyphenidyl, benzatropine, prednisone, deflazacort, pimozide, tacrolimus, miglustat, acetazolamide, baclofen, tizanidine, dantrolene, diazepam, clonazepam, gabapentin, thiamine, folic acid, betaine, creatine, alteplase, aspirin, clopidogrel, warfarin, rivaroxaban, apixaban, idursulfatase, baclofen, metoprolol, pregabalin, and amitriptyline. These drugs can be used alone or in combination of two or more.

[0061] Furthermore, other drugs that complement and / or enhance the preventive and / or therapeutic effects of compounds having S1P3 receptor inverse agonist activity include not only those that have been discovered so far but also those that will be discovered in the future, based on the above-mentioned mechanisms.

[0062] [Screening method] Compounds having S1P3 receptor inverse agonist activity can be screened by a method including Step A, in which the inverse agonist activity of a candidate compound against the S1P3 receptor is examined. One embodiment of the candidate compound is a low molecular weight compound. The low molecular weight compound may have a structure similar to that of the compounds described in Examples 1 to 3. In one embodiment, Step A is a step of using reporter cells expressing the S1P3 receptor to compare reporter activity in the absence of the ligand SIP and in the presence or absence of the candidate compound; specifically, this step is preferably based on Biological Example 1.

[0063] As described above, compounds having S1P3 receptor inverse agonist activity can be suitably used as prophylactic and / or therapeutic agents for neurodegenerative diseases. Therefore, prophylactic and / or therapeutic agents for neurodegenerative diseases can be screened by a method comprising step A and step B, in which candidate compounds that have been confirmed to have inverse agonist activity at the S1P3 receptor in step A are selected as prophylactic and / or therapeutic agents for neurodegenerative diseases. In step B, candidate compounds that have been confirmed to have inverse agonist activity at the S1P3 receptor are, for example, compounds that attenuate the constitutive activity of the S1P3 receptor in the absence of its ligand, S1P. Step B is also preferably a step similar to Biological Example 1.

[0064] Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0065] Furthermore, the contents of all patent and non-patent literature or references explicitly cited in this specification may be incorporated herein by reference in their entirety.

[0066] In one aspect, the present disclosure provides the following embodiments. [1] A preventive and / or therapeutic agent for neurodegenerative diseases, comprising a compound having S1P3 receptor inverse agonist activity; [2] The preventive and / or therapeutic agent according to [1], wherein the neurodegenerative disease is a neurodegenerative disease associated with reactive astrocytes. [3] The preventive and / or therapeutic agent according to [2], wherein the neurodegenerative disease associated with reactive astrocytes is a disease selected from the group consisting of amyotrophic lateral sclerosis, multiple system atrophy, Alzheimer's disease, spinal cord injury, cerebrovascular disease, Alexander disease, Sandhoff disease, and mucopolysaccharidosis type III. [4] The preventive and / or therapeutic agent according to [2], wherein the neurodegenerative disease associated with reactive astrocytes is amyotrophic lateral sclerosis, multiple system atrophy, or Alzheimer's disease. [5] Use of a compound having S1P3 receptor inverse agonist activity for the manufacture of an agent for the prevention and / or treatment of neurodegenerative diseases. [6] A method for preventing and / or treating a neurodegenerative disease, comprising administering an effective dose of a compound having S1P3 receptor inverse agonist activity to a mammal. [7] A compound having S1P3 inverse agonist activity for use in the prevention and / or treatment of neurodegenerative diseases. [8] An astrocyte activation regulator containing a compound having S1P3 receptor inverse agonist activity. [9] A pharmaceutical composition containing a compound having S1P3 receptor inverse agonist activity as an active ingredient and further containing a pharmaceutically acceptable carrier.

[10] The pharmaceutical composition according to [9], which is an agent for preventing and / or treating a neurodegenerative disease.

[11] An IL-6 expression inhibitor containing a compound having S1P3 receptor inverse agonist activity.

[12] A reactive astrocyte inhibitor containing a compound having S1P3 receptor inverse agonist activity.

[13] A method for screening a prophylactic and / or therapeutic agent for a neurodegenerative disease, comprising: step A: examining the inverse agonist activity of a candidate compound against the S1P3 receptor; and step B: selecting a candidate compound that has been found to have inverse agonist activity against the S1P3 receptor in step A as a prophylactic and / or therapeutic agent for the neurodegenerative disease. and

[14] A preventive and / or therapeutic agent for neurodegenerative diseases, an inhibitor of astrocyte activation, an inhibitor of reactive astrocytes, or an inhibitor of IL-6 expression, comprising at least one compound selected from the group consisting of:

[0067] [ka] [Example]

[0068] The invention according to the present disclosure will be specifically explained below with reference to examples, but the invention is not limited to these examples.

[0069] [Synthesis Example] The compounds having S1P3 receptor inverse agonist activity described in the present disclosure can be produced by appropriately modifying and combining known methods, such as the methods described below, methods similar thereto, the methods described in Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 3rd Edition (Richard C. Larock, John Wiley & Sons Inc, 2018), or the methods described in the Examples, but are not limited to these.

[0070] The solvent in parentheses shown in the NMR section indicates the solvent used in the measurement.

[0071] The compound names used in this specification were created using ACD / Name (registered trademark), a computer program that generally creates names according to IUPAC rules, or Chemdraw Ultra (version 12.0, Cambridge Soft), or were created according to IUPAC nomenclature.

[0072] LC-MS / ELSD was performed under the following trifluoroacetic acid (hereinafter referred to as TFA) conditions. Reversed-phase LCMS analysis was performed on a SHIMADZU LCMS-2020 with ESI. MS parameters were: mobile phase: 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B); gradient: 5% (solvent B) for 0.1 min, 5%-95% (solvent B) for 1.1 min, 95% for 0.4 min; flow rate: 1.0 mL / min; column: YMC Triart C18 Φ2.0 mm × L30 mm; wavelength: UV 220 nm, 254 nm; column temperature: 30 °C; detector: MS, ELSD; MS ionization: ESI.

[0073] Reference Example 1: (R)—N-[(E)-(5-chloro-3-fluoro-2-pyridinyl)methylene]-2-methyl-2-propanesulfinamide To a solution of 5-chloro-3-fluoropicolinaldehyde (CAS Registry Number: 214055-11-5) (6.0 g) in tetrahydrofuran (THF) (48 mL), (R)-(+)-2-methyl-2-propanesulfinamide (CAS Registry Number: 196929-78-9) (4.79 g) and titanium ethoxide (18.9 g) were added, and the reaction mixture was stirred at 60 °C overnight. The reaction mixture was filtered through Celite (registered trademark), and saturated aqueous sodium bicarbonate and ethyl acetate were added to the filtrate, which was then filtered again through Celite (registered trademark). The filtrate was extracted with ethyl acetate, and the organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate = 97:3 → 60:40) to give the title compound (7.49 g). HPLC retention time (min): 1.021; MS (ESI, Pos.): 263 (M+H) + ; 1H-NMR (CDCl3): δ8.84, 8.55-8.62, 7.61, 1.30.

[0074] Reference Example 2: (R)—N—[(R)-(4-chloro-3-fluorophenyl)(5-chloro-3-fluoro-2-pyridinyl)methyl]-2-methyl-2-propanesulfinamide A solution of the compound (5.16 g) prepared in Reference Example 1 in THF (52 mL) was cooled to -20°C, and a solution of 4-chloro-3-fluorophenylmagnesium bromide (CAS Registry Number: 170793-00-7) in THF (0.5 M, 47 mL) was added, followed by stirring at -20°C for 2 hours. Saturated sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was dried over sodium sulfate and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate = 97:3 → 70:30) to give the title compound (3.16 g). HPLC retention time (min): 1.226; MS (ESI, Pos.): 393 (M+H) + ;

[0075] Reference Example 3: (R)-1-(4-chloro-3-fluorophenyl)-1-(5-chloro-3-fluoro-2-pyridinyl)methanamine dihydrochloride To a methanol solution of the compound (7.78 g) prepared in Reference Example 2, 4N hydrogen chloride / dioxane solution (20 mL) was added and stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and azeotroped with toluene. The resulting residue was washed with hexane to give the title compound (6.36 g). 1 H-NMR (CDCl3): δ9.44, 8.35, 7.47, 7.38, 7.31, 7.22, 5.77.

[0076] Example 1: N-[(R)-(4-chloro-3-fluorophenyl)(5-chloro-3-fluoro-2-pyridinyl)methyl]-4-cyano-3-fluorobenzamide

[0077] [ka]

[0078] To a solution of the compound prepared in Reference Example 3 (3.0 g) in dichloromethane (30 mL), 4-cyano-3-fluorobenzoic acid (CAS Registry Number: 176508-81-9) (1.5 g), N,N-diisopropylethylamine (4.3 mL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (CAS Registry Number: 148893-10-1) (3.8 g) were added and stirred at room temperature for 1 hour. Water and saturated brine were added to the reaction mixture, which was then extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate = 97:3 → 70:30) to give the title compound (3.51 g). HPLC retention time (min): 1.261; MS (ESI, Pos.): 436 (M+H) + ; 1 H-NMR (CDCl3): δ8.46, 8.30, 7.70-7.78, 7.51, 7.35, 7.18, 6.49.

[0079] The compound described in Example 1 is an enantiomer of the compound of International Publication No. WO2016 / 053855A (compound ID: CYM 52718).

[0080] Example 2: 3-(5-chloro-3-fluoro-2-pyridinyl)-1-(4-chlorophenyl)-3-[(4-chlorophenyl)amino]-1-propanone

[0081] [ka]

[0082] To a solution of 5-chloro-3-fluoropicolinaldehyde (7.5 g) in methanol (30 mL), 4-chloroaniline (CAS Registry Number: 106-47-8) (6.0 g), 4'-chloroacetophenone (CAS Registry Number: 99-91-2) (7.3 g), and 10% hydrogen chloride / methanol solution (15 mL) were added and stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and tert-butyl methyl ether (100 mL) was added and stirred at 70°C for 1 hour. The mixture was cooled to room temperature and stirred for an additional 1 hour. The precipitated solid was collected by filtration to give the title compound (11.8 g). HPLC retention time (min): 1.355; MS (ESI, Pos.): 423 (M+H) + ; 1 H-NMR (DMSO-d6): δ8.46, 8.07, 7.95, 7.60, 7.06, 6.63, 5.33, 3.65-3.77.

[0083] Reference Example 4: 2-Bromo-1-(4-ethylphenyl)ethanone A solution of 4'-ethylacetophenone (CAS Registry Number: 937-30-4) (24.8 g) in THF (200 mL) was cooled to 0°C, and phenyltrimethylammonium tribromide (66 g) was added and stirred at 0°C for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate = 100:0 → 90:10) to give the title compound (41 g). HPLC retention time (min): 1.148; MS (ESI, Pos.): 227 (M+H) + ; 1 H-NMR (CDCl3): δ7.92, 7.32, 4.44, 4.12, 2.73, 1.27.

[0084] Reference Example 5: 4-[2-(4-ethylphenyl)-2-oxoethoxy]benzonitrile To a solution of the compound (41 g) produced in Reference Example 4 in N,N-dimethylformamide (DMF) (200 mL), 4-hydroxybenzonitrile (CAS Registry Number: 767-00-0) (24 g) and potassium carbonate (70 g) were added and stirred at room temperature for 3 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was washed with hexane (50 mL) / ethyl acetate (50 mL), and the solid was collected by filtration. The filtrate was purified by silica gel column chromatography (hexane:ethyl acetate = 100:0 → 0:100), and the residue after concentration under reduced pressure was washed with hexane / ethyl acetate. The title compound (30.4 g) was obtained by combining this with the solid collected by filtration. HPLC retention time (min): 1.188; MS (ESI, Pos.): 266 (M+H) + ; 1 H-NMR (CDCl3): δ7.91, 7.58, 7.34, 6.97, 5.35, 2.74, 1.28.

[0085] Reference Example 6: 4-[2-amino-2-(4-ethylphenyl)ethoxy]benzonitrile hydrochloride To a solution of the compound prepared in Reference Example 5 (16.5 g) in methanol (100 mL), ammonium formate (11.8 g), acetic acid (3.6 mL), and chloro[N-[4-(dimethylamino)phenyl]-2-pyridinecarboxamidate](pentamethylcyclopentadienyl)iridium(III) (CAS Registry Number: 1364328-83-5) (1.9 g) were added and the mixture was stirred at 60°C for 5 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. A 4N hydrogen chloride / ethyl acetate solution (47 mL) was added to a solution of the resulting residue in ethyl acetate (80 mL), and the mixture was stirred at room temperature for 15 minutes. The reaction mixture was concentrated under reduced pressure and then azeotroped with toluene. The resulting residue was washed with ethyl acetate to give the title compound (14.5 g). HPLC retention time (min): 0.893; MS (ESI, Pos.): 267 (M+H) + ; 1 H-NMR (CD3OD): δ7.71, 7.45, 7.35, 7.19, 4.76-4.80, 4.37-4.47, 2.69, 1.23.

[0086] Example 3: 5-chloro-N-[2-(4-cyanophenoxy)-1-(4-ethylphenyl)ethyl]-2-pyridinecarboxamide

[0087] [ka]

[0088] To a solution of the compound (25 g) prepared in Reference Example 6 in dichloromethane (170 mL), 5-chloro-2-pyridinecarboxylic acid (CAS Registry Number: 86873-60-1) (16 g) and N,N-diisopropylethylamine (57 mL) were added and cooled to 0°C. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (24 g) and 1-hydroxybenzotriazole monohydrate (19 g) were added and stirred at room temperature for 5 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, which was then extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate = 100:0 → 50:50). The mixture was concentrated under reduced pressure, ethyl acetate (70 mL) was added, and the mixture was stirred at 60°C for 10 minutes. Hexane (100 mL) was added, and the mixture was stirred at room temperature overnight. The precipitated solid was collected by filtration to give the title compound (17.5 g) having the following physical properties: HPLC retention time (min): 1.282; MS (ESI, Pos.): 406 (M+H) + ; 1 H-NMR (CDCl3): δ8.46-8.53, 8.15, 7.83, 7.57, 7.37, 7.22, 7.00, 5.55, 4.37-4.45, 2.65, 1.23.

[0089] [Biological Examples] Biological Example 1: S1P 3 Evaluation of receptor inverse agonist activity S1P3 receptor inverse agonist activity was measured by the following method, with reference to the membrane protein activity measurement method in Example A6 of Patent Publication No. WO2020 / 026979.

[0090] The reporter cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Nacalai Tesque) supplemented with 10% fetal calf serum (FCS) (Serana) and penicillin / streptomycin (Nacalai Tesque). Trypsin-EDTA (Nacalai Tesque, 26253-84) was used to detach the cells. They were then transferred to DMEM supplemented with 10% dialyzed FCS. The cells were then suspended and seeded into a 96-well white microplate at a density of 24,000 cells per well. The cells were cultured at 37°C in the presence of 5% carbon dioxide to ensure proper attachment of the plated cells to the microplate bottom. The cells were grown overnight. Plasmids expressing the S1P3 receptor and luciferase reporter were combined and transfected into the cells at a total dose of 100 ng per well using transfection reagent (FuGene 6, Promega, E2691). Thereafter, the cells were cultured overnight at 37°C in the presence of 5% CO2.

[0091] Immediately before the assay, a test substance solution was prepared using DMEM containing 10% dialyzed FCS and added to each well. After 6 hours of incubation at 37°C, luciferase assay reagent (Steady-Glo, Promega, E2510) was added to the wells to quantify luciferase. The amount of luciferase luminescence was then measured using a plate reader (Victor Nivo, Perkin Elmer).

[0092] The measurement results showing the concentration dependence of S1P3 receptor inverse agonist activity were approximated to the Hill equation by the nonlinear least squares method using the Python scientific calculation library Scipy. The parameters were EC 50 , Hill coefficient, curve bottom, and curve top.

[0093]

number

[0094] [result] Compared with reporter cells (mock) that did not express the S1P3 receptor, reporter cells (S1P3) that expressed the S1P3 receptor exhibited higher luciferase activity even in the absence of a ligand (Figure 1a). This result suggested that the S1P3 receptor has constitutive activity. Furthermore, the compounds described in Examples 1, 2, and 3 (hereinafter sometimes abbreviated as the disclosed compounds) attenuated the constitutive activity of the S1P3 receptor in the absence of the ligand S1P, indicating that the disclosed compounds have inverse agonist activity against the S1P3 receptor (Figures 1b to 1d).

[0095] The S1P3 receptor inverse agonist activity (EC 50 ) are shown in Table 1. (Table 1)

[0096] [Table 1]

[0097] Biological Example 2: S1P 3 Assessment of constitutive activity of receptors and S1P 3 S1P receptor inverse agonist activity 3 Evaluation of the inhibitory effect of inflammatory responses due to constitutive receptor activity To investigate the relationship between the constitutive activity of S1P3 receptors and the inflammatory response of astrocytes, we generated drug-inducible S1P3 receptor-expressing cells. Using an experimental system in which target gene expression can be reversibly regulated by doxycycline administration (Tet-on system), we constructed a vector that expresses S1P3 receptors in a doxycycline-dependent manner. We then transfected this vector into human astroglioma cells, 1321N1 cells (ECACC, 86030402), to generate cells that express S1P3 receptors in a doxycycline concentration-dependent manner (hereafter, Tet-S1P3 cells).

[0098] Next, 1.5 × 105 Cells were seeded in a 6-well dish. 24 hours after seeding, the cells were washed three times with phosphate-buffered saline (PBS, Nacalai Tesque) and then replaced with serum-free DMEM medium (Nacalai Tesque, 08459-64). Doxycycline (Takara Bio, 631311) and a compound of the present disclosure were added to the cells, and intracellular RNA was extracted 24 hours later. cDNA was prepared from the extracted RNA, and the expression level of the inflammatory cytokine IL-6 gene was measured by quantitative PCR.

[0099] [result] Induction of S1P3 receptor expression with doxycycline increased IL-6 gene expression (Figure 2). On the other hand, treatment with doxycycline and a compound of the present disclosure attenuated the increase in IL-6 gene expression (Figure 2). This attenuation was concentration-dependent of the compound of the present disclosure. Furthermore, because this experimental system was performed in the absence of S1P, which is known to be a ligand for the S1P3 receptor, it was confirmed that the S1P3 receptor enhances the expression of the IL-6 gene, a pro-inflammatory cytokine, in a ligand-independent manner.

[0100] These results indicate that the S1P3 receptor has constitutive activity, and that this constitutive activity induces inflammatory response signals in astrocytes (transition to reactive astrocytes). Furthermore, the compounds of the present disclosure, which have S1P3 receptor inverse agonist activity, were found to suppress the inflammatory response of reactive astrocytes by inhibiting the constitutive activity of the S1P3 receptor.

[0101] Biological Example 3: Efficacy evaluation using ALS model mice To evaluate ALS model mice, B6SJL-Tg(SOD1*G93A)dl1Gur / J mice (hereafter referred to as SOD1, Jackson Laboratory Japan) or their wild-type counterparts, B6SJL (hereafter referred to as WT1, Jackson Laboratory Japan), were used. Groups were uniformly divided based on baseline body weight (14 weeks of age) and the time it took to fall in the rotarod test (fall latency). From 15 weeks of age until 30 weeks of age, mice were repeatedly administered vehicle (0.5 w / v% methylcellulose solution 400, Wako Pure Chemical Industries) or the disclosed compounds at 3 mg / kg per day by oral gavage. Plasma was collected from mice at 30 weeks of age, and plasma NfL concentrations, which increase with neurodegeneration, were measured using an ultrasensitive ELISA system (Simoa®, Quanterix).

[0102] [result] Measurement of plasma NfL concentrations revealed that the plasma NfL concentration in the SOD1 vehicle-administered group was higher than that in the WT1 vehicle-administered group (Figure 3). On the other hand, the increase in plasma NfL in the SOD1 group administered with the disclosed compound was significantly suppressed (Figure 3). These results demonstrate that the disclosed compound suppresses neurodegeneration in ALS model mice.

[0103] Biological Example 4: Efficacy evaluation using MSA model mice To evaluate MSA model mice, we used oligodendrocyte-specific α-synuclein-overexpressing mice (hereinafter referred to as PLP-SYN, Motac) and their wild-type counterparts, C57 / BL / 6 (hereinafter referred to as WT2, Motac). From the age of 2 months, mice were given a vehicle (0.5 w / v% methylcellulose solution 400, Wako Pure Chemical Industries) or a compound of the present disclosure at 0.3 or 3 mg / kg by oral gavage once daily. Motor activity was evaluated at 5 and 9 months of age.

[0104] The mice were assessed for motor coordination through the beam test and notch bar test. In the beam test, the mice were guided along the end of a thin rod, and the total number of steps taken and the number of steps that missed the rod were measured.

[0105] In the notch bar test, the mice were made to walk along the edge of a notch bar with evenly spaced platforms, and the number of times they slipped their hind limbs was measured to assess their coordination ability.

[0106] [result] Results of the beam test and notch bar test at 5 and 9 months of age showed that the PLP-SYN group treated with vehicle showed significantly reduced motor ability compared to the WT2 group treated with vehicle (Figures 4a and 4b). In contrast, the PLP-SYN group treated with a compound of the present disclosure showed no reduction in motor ability (Figures 4a and 4b). These results demonstrate that the compound of the present disclosure suppresses the decline in motor ability that occurs in MSA model mice.

[0107] Biological Example 5: Efficacy evaluation using AD model mice Mice with a mutation in the amyloid-β precursor protein (also known as APP protein) (hereafter referred to as NL-GF, Riken Bio) were used as AD model mice. Three-month-old NL-GF mice and their wild-type counterparts, C57BL / 6 (hereafter referred to as WT3, Riken Bio), were administered vehicle (0.5 w / v% methylcellulose solution 400, Wako Pure Chemical Industries) or the disclosed compounds at 60 mg / kg by oral gavage once daily for three months. After three months of administration, cognitive function was assessed by the Y-maze test (hereafter referred to as YMT) and novel object recognition test (hereafter referred to as NOR).

[0108] The YMT used a Y-shaped maze consisting of three black vinyl chloride arms (10 cm x 35 cm) connected at equal angles. Mice were positioned in the center of the arms and allowed to explore freely for 8 minutes. The sequence and total number of entries into each arm were recorded from the video. Alternation behavior was defined as when a mouse entered three arms consecutively without repeating an entry, and the alternation rate was calculated using the following formula.

[0109]

number

[0110] For the NOR, an Econ cage (CLEA Japan) with a video camera mounted above was used. On Day 1, mice were placed in the Econ cage for habituation to the experimental setup and allowed to move freely for 10 minutes. On Day 2, mice were allowed to move freely for the acquisition trial, in which two objects of identical shape and color were placed in the Econ cage. The objects were placed parallel to one of the long sides of the cage, separated by a distance of 10 cm from the two adjacent inner walls. On Day 3, mice were allowed to move freely for 10 minutes. The evaluation trial began 24 hours (23–25 hours) after the acquisition trial. Exploration time for each object was measured from the video footage recorded during the evaluation trial. Total exploration time and the novel object recognition rate were calculated from the exploration time for the familiar and novel objects.

[0111]

number

[0112]

number

[0113] [result] The results of the YMT showed that the alternation rate was reduced in the NL-GF vehicle-administered group compared to the WT3 vehicle-administered group (Figure 5a). On the other hand, the group in which the compound of the present disclosure was administered to NL-GF significantly suppressed cognitive decline (Figure 5a). Furthermore, the results of the NOR showed that the novel object recognition rate was reduced in the NL-GF vehicle-administered group compared to the WT3 vehicle-administered group (Figure 5b). On the other hand, the group in which the compound of the present disclosure was administered to NL-GF significantly suppressed the decline in novel object recognition rate (Figure 5b). These results demonstrate that the compound of the present disclosure suppresses cognitive decline in AD model mice.

[0114] Biological Example 6: Efficacy evaluation using spinal cord injury model mice Spinal cord injury model mice were prepared by compressing the 10th to 12th thoracic vertebrae of 8-week-old female C57BL / 6J mice (Jackson Laboratory Japan) with a clamp (Dyadem Industrial) for 15 seconds. After preparation, the mice were given a vehicle (0.5 w / v% methylcellulose solution 400, Wako Pure Chemical Industries) or a compound of the present disclosure at 30 mg / kg by oral gavage once daily. From the day of preparation, motor function was evaluated daily using the Basso-Beattie-Bresnahan (BBB) ​​score, which can evaluate the degree of spinal cord injury.

[0115] [result] As a result of evaluating motor function using the BBB score, administration of the compound of the present disclosure significantly improved motor function compared to the vehicle group from 5 days after the creation of the spinal cord injury model mice (Figure 6). These results demonstrate that the compound of the present disclosure improves neurological symptoms in the spinal cord injury model.

[0116] Biological Example 7: Efficacy evaluation using cerebral infarction model rats A rat transient cerebral ischemia model (also known as the Koizumi model) was prepared as a cerebral infarction model rat. Under isoflurane anesthesia, the left middle aorta was occluded to induce cerebral ischemia, and the isoflurane anesthesia was discontinued and the rats were awakened. 120 minutes after cerebral ischemia, blood flow was reperfused under anesthesia again. One hour after reperfusion, vehicle (0.5 w / v% methylcellulose solution 400, Wako Pure Chemical Industries) or a compound of the present disclosure was orally administered, followed by repeated oral gavage once daily. Neurological symptoms (forelimb paralysis, hindlimb paralysis, rotational movement, lateral push, and general condition) were scored and evaluated 1, 3, and 7 days after ischemia. Forelimb paralysis was evaluated by holding the rat's tail and lifting it approximately 10 cm from the floor, and observing the degree of flexion of the right forelimb. Hindlimb paralysis was evaluated by observing the force with which the right hindlimb was returned to its original position when pulled while the rat was at rest. Rotational movement was evaluated by holding the rat's tail and observing rotational movement with the forelimbs resting on the floor. Lateral push was performed by observing the resistance when pushing the side of the rat's body on each side while the rat was at rest.

[0117] [result] As a result of administration of the compound of the present disclosure, the above neurological symptom scores were significantly reduced compared to the vehicle group 3 and 7 days after ischemia (Figure 7). These results demonstrate that the compound of the present disclosure suppresses the deterioration of neurological symptoms after cerebral ischemia-reperfusion in a cerebral infarction model rat.

[0118] Biological Example 8: Efficacy evaluation using Alexander disease model mice For the evaluation of Alexander disease model mice, FVB.129S7(Cg)-Gfaptm2Mes / Mmnc (hereafter referred to as AxD, Jackson Laboratory Japan) and its wild-type counterpart, FVB (WT4, Jackson Laboratory Japan), were used. WT4 and AxD mice were divided into groups with uniform body weight at 4 weeks of age. From 5 weeks of age, mice were repeatedly administered vehicle (0.5 w / v% methylcellulose solution 400, Wako Pure Chemical Industries) or the disclosed compounds at 3 or 30 mg / kg by oral gavage once daily.

[0119] Kainic acid-induced epilepsy was evaluated to assess epileptic seizures. Mice were administered the compound until 26 weeks of age, and kainic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) was administered intraperitoneally at 20 mg / 10 mL / kg. The animals were observed from immediately after administration until 3 hours after administration, and scored at 5-minute intervals according to the following criteria: 0: Normal 1: Grooming and exploration cease and the cat becomes immobile 2: Tension in the forelimbs and tail, stiff posture (rigidity) 3: Repeated short myoclonic jerks such as shaking of the forelimbs and head shaking 4: Forelimb clonus, rearing, falling, loss of posture. 5: Category 4 episode repeats within 5 minutes. 6: Generalized severe myoclonia or loss of postural tone 7:Death

[0120] [result] The scores increased immediately after kainic acid administration in both the AxD vehicle-administered group and the WT4 vehicle-administered group (Figure 8a). In particular, the AxD vehicle-administered group showed a significant increase in epileptic symptoms 70-80 minutes after administration compared to immediately after administration, and continued to show significantly higher scores than the WT4 vehicle-administered group (Figure 8a). On the other hand, the group treated with the disclosed compound in the AxD showed a significant suppression of the increase in scores immediately after kainic acid administration (Figure 8a). Furthermore, the group treated with the disclosed compound in the AxD showed an increase in epileptic symptoms at approximately the same time point as the AxD vehicle group, but the progression was more moderate, and the scores remained lower than those of the AxD vehicle group (Figure 8a). The total score during the observation period was highest in the AxD vehicle-administered group, and was comparable to the WT4 vehicle-administered group (Figure 8b). These results demonstrate that the compound of the present disclosure suppresses epileptic symptoms induced by kainic acid in Alexander disease model mice.

[0121] Biological Example 9: Efficacy evaluation using Sandhoff disease model mice Sandhoff disease model mice were B6;129S4-Hexbtm1Rlp / J, stock:02914 (hereafter referred to as SD, Jackson Laboratory Japan), and their wild-type counterparts, B6129SF2 / J (hereafter referred to as WT5, Jackson Laboratory Japan). WT5 and SD mice were stratified and randomized to ensure similar body weights and rotarod latency at 4 weeks of age. Starting at 5 weeks of age, mice were given repeated oral gavage once daily at 0.3 or 3 mg / kg of the disclosed compounds. The survival time of each mouse was measured.

[0122] [result] The median survival rate was calculated as 124 days for the group administered the vehicle to SD. On the other hand, for the groups administered the compound of the present disclosure to SD, the survival rate was 131 days for the 0.3 mg / kg group and 134 days for the 3 mg / kg group (Figure 9). These results demonstrate that the compound of the present disclosure extends the survival period in Sandhoff disease model mice.

[0123] Biological Example 10: Efficacy evaluation using mucopolysaccharidosis type IIIA model mice B6.Cg-Sgshmps3a / PstJ mice (hereafter referred to as MPSIIIA, Jackson Laboratory Japan) and their wild-type counterparts, C57BL / 6J (hereafter referred to as WT6, Jackson Laboratory Japan), were used as mucopolysaccharidosis IIIA model mice. WT6 and MPSIIIA mice were randomly assigned to groups stratified by body weight and sex at 5 weeks of age. From 5 weeks of age, mice were orally administered vehicle (0.5 w / v% methylcellulose solution 400, Wako Pure Chemical Industries) or a compound of the present disclosure at 3 or 30 mg / kg once daily.

[0124] NOR was performed at 30 weeks of age. For NOR, an Econ cage (CLEA Japan) with a video camera mounted above was used. The illuminance in the waiting area and near the experimental apparatus was adjusted to approximately 20 lx. To allow for habituation to the evaluation environment, mice were transferred to the evaluation room at least 1 h before the start of each trial. For habituation to the experimental apparatus on Day 1, mice were placed in the Econ cage and allowed to move freely for 10 min. For the acquisition trial on Day 2, mice were placed in the Econ cage with two objects of identical shape and color, and allowed to move freely for a similar 10 min. The two objects were placed parallel to one of the long sides of the cage, separated by a distance of 10 cm from the two adjacent inner walls. For the evaluation trial on Day 3, one of the objects used in the acquisition trial was replaced with a novel object of a different shape and color, and the mice were allowed to move freely for a similar 10 min. The evaluation trial began approximately 24 h (23–25 h) after the acquisition trial. Exploration time for each object was measured from the video footage recorded during the evaluation trial. Total exploration time and novel object recognition rate were calculated from the exploration times of the familiar and novel objects.

[0125]

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[0126]

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[0127] [result] The novel object recognition rate was significantly lower in the MPSIIIA vehicle-administered group compared to the WT6 vehicle-administered group (p=0.0452) (FIG. 10). Meanwhile, the group in which the disclosed compound was administered to MPSIIIA dose-dependently suppressed the decrease in alternation rate, and the novel object recognition rate in the group in which the disclosed compound was administered to MPSIIIA at 30 mg / kg was significantly higher than that in the MPSIIIA vehicle-administered group (p=0.0083) (FIG. 10). These results demonstrate that the disclosed compound suppresses cognitive decline in mucopolysaccharidosis IIIA model mice.

[0128] Biological Example 11: Evaluation of the inhibitory effect on the proliferation of reactive astrocytes Tissues were collected from AD model mice (NL-GF) and their wild-type counterparts (WT3) that had completed cognitive function evaluation in Biological Example 5. After isoflurane inhalation anesthesia, blood was collected from the abdominal vena cava and perfused with ice-cold PBS, followed by removal of the brain. The removed brain tissue was slowly frozen in polystyrene foam containing dry ice and stored in a -80°C freezer. The brains stored in the freezer were post-fixed by immersion in 4% paraformaldehyde phosphate buffer for 4–6 hours. After washing with PBS, they were immersed in 30% sucrose PBS solution overnight at 4°C. They were then embedded in OCT compound (Sakura Finetech Japan) and thin sagittal sections were prepared. The sections were fixed in 4% paraformaldehyde phosphate buffer and immunostained using anti-glial fibrillary acidic protein (GFAP) antibody (Thermo Fisher Scientific). The stained sections were observed under a confocal microscope (FV3000, OLYMPUS).

[0129] [result] Anti-GFAP immunostaining revealed that the anti-GFAP-positive area was increased in the NL-GF vehicle-treated group compared to the WT3 vehicle-treated group, indicating the aggregation of reactive astrocytes. On the other hand, the anti-GFAP-positive area was decreased in the NL-GF vehicle-treated group compared to the NL-GF vehicle-treated group (Figures 11a and 11b). These results demonstrate that the compounds of the present disclosure inhibit the increase in reactive astrocytes.

[0130] As described above, this disclosure has revealed that the S1P3 receptor has constitutive activity and that this constitutive activity induces inflammatory response signals in astrocytes (transition to reactive astrocytes). Because reactive astrocytes are a phenomenon observed in various neurodegenerative diseases, inhibiting the constitutive activity of the S1P3 receptor is important for the treatment of neurodegenerative diseases associated with reactive astrocytes.

[0131] Furthermore, this disclosure has revealed that compounds with various scaffolds and S1P3 receptor inverse agonist activity, such as the compounds disclosed herein, suppress the constitutive activity of the S1P3 receptor, thereby suppressing the inflammatory response caused by reactive astrocytes, and are also effective against various neurodegenerative diseases in which reactive astrocytes are involved. [Industrial Applicability]

[0132] Pharmaceuticals containing compounds having S1P3 receptor inverse agonist activity are useful as agents for the prevention and / or treatment of neurodegenerative diseases.

Claims

1. S1P ３ A preventive and / or therapeutic agent for neurodegenerative diseases, comprising a compound having receptor inverse agonist activity.

2. The preventive and / or therapeutic agent according to claim 1, wherein the neurodegenerative disease is a neurodegenerative disease associated with reactive astrocytes.

3. 3. The preventive and / or therapeutic agent according to claim 2, wherein the neurodegenerative disease associated with reactive astrocytes is a disease selected from the group consisting of amyotrophic lateral sclerosis, multiple system atrophy, Alzheimer's disease, spinal cord injury, cerebrovascular disease, Alexander disease, Sandhoff disease, and mucopolysaccharidosis type III.

4. The preventive and / or therapeutic agent according to claim 2, wherein the neurodegenerative disease associated with reactive astrocytes is amyotrophic lateral sclerosis, multiple system atrophy, or Alzheimer's disease.

5. S1P for producing a preventive and / or therapeutic agent for neurodegenerative diseases ３ Use of compounds with receptor inverse agonist activity.

6. S1P ３ A method for preventing and / or treating a neurodegenerative disease, which comprises administering to a mammal an effective dose of a compound having receptor inverse agonist activity.

7. S1P for use in the prevention and / or treatment of neurodegenerative diseases ３ A compound with inverse agonist activity.

8. S1P ３ An agent for controlling astrocyte activation, which comprises a compound having receptor inverse agonist activity.

9. S1P ３ A pharmaceutical composition comprising, as an active ingredient, a compound having receptor inverse agonist activity, and further comprising a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, which is an agent for preventing and / or treating a neurodegenerative disease.

11. S1P ３ An IL-6 expression inhibitor containing a compound having receptor inverse agonist activity.

12. S1P ３ A reactive astrocyte inhibitor containing a compound having receptor inverse agonist activity.

13. Candidate compound S1P ３ Step A: Examining the inverse agonist activity against the S1P receptor; ３ A method for screening for a prophylactic and / or therapeutic agent for neurodegenerative disease, comprising a step B of selecting a candidate compound that has been found to have inverse agonist activity against the receptor as a prophylactic and / or therapeutic agent for neurodegenerative disease.

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