WO2017171053A1 - シヌクレイノパチー治療薬 - Google Patents
シヌクレイノパチー治療薬 Download PDFInfo
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- WO2017171053A1 WO2017171053A1 PCT/JP2017/013742 JP2017013742W WO2017171053A1 WO 2017171053 A1 WO2017171053 A1 WO 2017171053A1 JP 2017013742 W JP2017013742 W JP 2017013742W WO 2017171053 A1 WO2017171053 A1 WO 2017171053A1
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- pyrazol
- phenyl
- phenoxy
- butanoic acid
- pharmaceutical composition
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- GEXLSWKCHILUAA-ZIAGYGMSSA-N C#C[C@H](CCO)C(N([C@H](Cc1ccccc1)CO1)C1=O)=O Chemical compound C#C[C@H](CCO)C(N([C@H](Cc1ccccc1)CO1)C1=O)=O GEXLSWKCHILUAA-ZIAGYGMSSA-N 0.000 description 1
- FUUGPOJEUTVLJP-UHFFFAOYSA-N CC(C)(C)c(cc1)ccc1-[n](c(-c1ccccc1)c1)nc1-c1ccccc1OCCCC(O)=O Chemical compound CC(C)(C)c(cc1)ccc1-[n](c(-c1ccccc1)c1)nc1-c1ccccc1OCCCC(O)=O FUUGPOJEUTVLJP-UHFFFAOYSA-N 0.000 description 1
- 0 CI1(*)=CC=C(c(cc2-c3ccccc3*)n[n]2-c2ccc(*)c(*)c2)C(OCCC*)=CC1 Chemical compound CI1(*)=CC=C(c(cc2-c3ccccc3*)n[n]2-c2ccc(*)c(*)c2)C(OCCC*)=CC1 0.000 description 1
- FZQJOJHCQVORFU-YLJYHZDGSA-N C[C@H](CCOCc1ccccc1)C(N([C@H](Cc1ccccc1)CO1)C1=O)=O Chemical compound C[C@H](CCOCc1ccccc1)C(N([C@H](Cc1ccccc1)CO1)C1=O)=O FZQJOJHCQVORFU-YLJYHZDGSA-N 0.000 description 1
- QKLFBGVERWJVPW-UHFFFAOYSA-N OC(CCCOc(cccc1)c1-c(cc1-c(cccc2)c2Br)n[n]1-c1ccccc1)=O Chemical compound OC(CCCOc(cccc1)c1-c(cc1-c(cccc2)c2Br)n[n]1-c1ccccc1)=O QKLFBGVERWJVPW-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/415—1,2-Diazoles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
- C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
- C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
Definitions
- the present invention relates to a therapeutic or prophylactic agent for synucleinopathy.
- the present invention further relates to a pharmaceutical composition for treating or preventing synucleinopathy comprising a 1,3,5-triphenylpyrazole derivative, and a method for treating or preventing synucleinopathy using the compound.
- ⁇ -synuclein is a protein consisting of 140 amino acid residues encoded by the SNCA gene, and is abundantly expressed at the presynaptic terminal in the brain.
- Synucleinopathy means a group of neurodegenerative diseases characterized by abnormal accumulation of ⁇ -synuclein, including accumulation in Lewy bodies and neurites in Lewy body disease, accumulation in glial cytoplasmic inclusions in multiple system atrophy, etc. It has been known. It has been reported that polyunsaturated fatty acids are involved in oligomerization of ⁇ -synuclein (Non-patent Documents 1 and 2). In Japan, where we are facing a full-fledged aging society, the number of patients suffering from synucleinopathies has increased greatly in recent years.
- Fatty acid binding protein is a low molecular weight (14-15 kDa) cytoplasmic protein and is expressed in a tissue-specific manner.
- FABP is considered to be involved in maintaining homeostasis of lipid metabolism and signal transduction using a medium chain to long chain fatty acid as a ligand.
- FABP is known to have a plurality of subtypes whose molecular structures are similar to each other.
- FABP3 is expressed in the heart and its function has not been fully elucidated, but is thought to be involved in lipid homeostasis such as lipid uptake and transport to ⁇ -oxidation system in mitochondria. .
- a compound having an inhibitory activity on FABP3 has been reported (Non-patent Document 2). It has been reported that FABP3 promotes ⁇ -synuclein aggregation (Non-patent Document 1).
- ⁇ -synuclein aggregates appear in the substantia nigra dopamine nerve in Parkinson's disease, and diffusely appear in the cerebral cortex in dementia with Lewy bodies.
- fibrous synuclein is injected into the rat striatum, aggregates propagate to the cerebral cortex in addition to the substantia nigra to form synuclein inclusions in neurons (Non-patent Document 3).
- the present invention aims to provide a pharmaceutical composition for use in the treatment or prevention of synucleinopathies. It is another object of the present invention to provide a method for treating or preventing synucleinopathies using a specific 1,3,5-triphenylpyrazole derivative.
- the inventors of the present invention have made extensive studies to achieve the above-mentioned problems.
- the 1,3,5-triphenylpyrazole derivative inhibits the aggregation of ⁇ -synuclein, and the propagation of ⁇ -synuclein aggregates has been reduced to FABP3.
- the present invention was completed by discovering that the ligand has beneficial effects such as significant suppression, improving motor dysfunction and cognitive dysfunction.
- the disclosure of this specification includes the inventions described in [1] to [20] below.
- R 1a , R 1b and R 1c are independently selected from a hydrogen atom, C 1-6 alkyl, C 1-6 alkoxy, and a halogen atom;
- R 1d is a hydrogen atom or a halogen atom;
- R 2 and R 3 are independently selected from C 1-6 alkyl, and a halogen atom;
- R 4 is selected from a hydrogen atom and C 1-6 alkyl;
- R 5 is selected from COOR 6 , CH 2 OH, and 1-tetrazolyl;
- R 6 is selected from a hydrogen atom and C 1-6 alkyl;
- n is an integer selected from 0 to 5;
- p is an integer selected from 0 to 4;
- q is 1 or 2] Or a pharmaceutically acceptable salt thereof.
- R 1a and R 1b are independently selected from a hydrogen atom, a chlorine atom, a bromine atom, methyl and methoxy.
- R 1a , R 1b , and R 6 are as defined in any one of [1] to [4]
- R 2a is a hydrogen atom or a halogen atom
- R 3a is a hydrogen atom.
- a pharmaceutically acceptable salt thereof Or a pharmaceutically acceptable salt thereof.
- the pharmaceutical composition according to any one of [1] to [4].
- R 1a , R 1b , R 1c and R 1d are independently selected from a hydrogen atom, C 1-6 alkyl, C 1-6 alkoxy, and a halogen atom;
- R 2a is a halogen atom;
- R 3a is a hydrogen atom or a halogen atom;
- R 6 is selected from a hydrogen atom and C 1-6 alkyl] Or a pharmaceutically acceptable salt thereof.
- R 1a , R 1b , R 1c and R 1d are independently selected from a hydrogen atom, C 1-6 alkyl, C 1-6 alkoxy, and a halogen atom;
- R 2 is C 1-6 alkyl or a halogen atom;
- n is an integer selected from 0 to 5;
- R 3b is a halogen atom;
- R 6 is selected from hydrogen, and C 1-6 alkyl Or a pharmaceutically acceptable salt thereof.
- a pharmaceutical composition for treating or preventing synucleinopathy comprising the compound according to any one of [10] to [17], or a pharmaceutically acceptable salt thereof.
- R 1a , R 1b and R 1c are independently selected from a hydrogen atom, C 1-6 alkyl, C 1-6 alkoxy, and a halogen atom;
- R 1d is a hydrogen atom or a halogen atom;
- R 2 and R 3 are independently selected from C 1-6 alkyl, and a halogen atom;
- R 4 is selected from a hydrogen atom and C 1-6 alkyl;
- R 5 is selected from COOR 6 , CH 2 OH, and 1-tetrazolyl;
- R 6 is selected from a hydrogen atom and C 1-6 alkyl;
- n is an integer selected from 0 to 5;
- p is an integer selected from 0 to 4;
- q is 1 or 2] Or a pharmaceutically acceptable salt thereof.
- R 1a and R 1b are independently selected from a hydrogen atom, a chlorine atom, a bromine atom, methyl and methoxy Pharmaceutical composition.
- R 1a , R 1b , and R 6 are as defined in any one of [1-1] to [1-4]
- R 2a is a hydrogen atom or a halogen atom
- R 3a Is a hydrogen atom or a halogen atom
- a pharmaceutically acceptable salt thereof according to any one of [1-1] to [1-5].
- [1-10] The pharmaceutical composition according to any one of [1-1] to [1-9], wherein the synucleinopathy is Parkinson's disease, Lewy body dementia, or multiple system atrophy.
- R 1a , R 1b , R 1c and R 1d are independently selected from a hydrogen atom, C 1-6 alkyl, C 1-6 alkoxy, and a halogen atom;
- R 2a is selected from C 1-6 alkyl and a halogen atom;
- R 3a is a hydrogen atom or a halogen atom;
- R 6 is selected from a hydrogen atom and C 1-6 alkyl] Or a pharmaceutically acceptable salt thereof.
- R 1a , R 1b , R 1c and R 1d are independently selected from a hydrogen atom, C 1-6 alkyl, C 1-6 alkoxy, and a halogen atom;
- R 2 is C 1-6 alkyl or halogen atom;
- n is an integer selected from 0 to 5;
- R 3b is a halogen atom;
- R 6 is selected from a hydrogen atom and C 1-6 alkyl] Or a pharmaceutically acceptable salt thereof.
- a pharmaceutical composition for treating or preventing synucleinopathy comprising the compound according to any one of [1-12] to [1-19], or a pharmaceutically acceptable salt thereof.
- R 1a , R 1b and R 1c are independently selected from a hydrogen atom, C 1-6 alkyl, C 1-6 alkoxy, and a halogen atom
- R 1d is a hydrogen atom or a halogen atom
- R 2 and R 3 are independently selected from C 1-6 alkyl, and a halogen atom
- R 4 is selected from a hydrogen atom and C 1-6 alkyl
- R 5 is selected from COOR 6 , CH 2 OH, and 1-tetrazolyl
- R 6 is selected from a hydrogen atom and C 1-6 alkyl
- n is an integer selected from 0 to 5
- p is an integer selected from 0 to 4
- q is 1 or 2] Or a pharmaceutically acceptable salt thereof.
- R 1a and R 1b are independently selected from a hydrogen atom, a chlorine atom, a bromine atom, methyl and methoxy.
- R 1a , R 1b , and R 6 are as defined in any of [2-1] to [2-4]
- R 2a is a hydrogen atom or a halogen atom
- R 3a Is a hydrogen atom or a halogen atom
- a pharmaceutically acceptable salt thereof according to any one of [2-1] to [2-4].
- a compound or a pharmaceutically acceptable salt thereof 4- (2- (5- (2-chlorophenyl) -1-phenyl-1H-pyrazol-3-yl) phenoxy) butanoic acid; 4- (2- (1- (4-bromophenyl) -5-phenyl-1H-pyrazol-3-yl) phenoxy) butanoic acid; 4- (2- (1- (3,4-dichlorophenyl) -5-phenyl-1H-pyrazol-3-yl) phenoxy) butanoic acid; 4- (2- (1,5-diphenyl-1H-pyrazol-3-yl) phenoxy) butanoic acid; 4- (2- (1- (4-fluorophenyl) -5-phenyl-1H-pyrazol-3-yl) phenoxy) butanoic acid; 4- (2- (1- (4-chlorophenyl) -5-phenyl-1H-pyrazol-3-yl) phenoxy) butanoic acid;
- [2-8] The method according to any one of [2-1] to [2-7], wherein the synucleinopathy is Parkinson's disease, Lewy body dementia, or multiple system atrophy.
- R 1a , R 1b , R 1c and R 1d are independently selected from a hydrogen atom, C 1-6 alkyl, C 1-6 alkoxy, and a halogen atom;
- R 2a is a halogen atom;
- R 3a is a hydrogen atom or a halogen atom;
- R 6 is selected from a hydrogen atom and C 1-6 alkyl] Or a pharmaceutically acceptable salt thereof.
- R 1a , R 1b , R 1c and R 1d are independently selected from a hydrogen atom, C 1-6 alkyl, C 1-6 alkoxy, and a halogen atom;
- R 2 is C 1-6 alkyl or a halogen atom;
- n is an integer selected from 0 to 5;
- R 3b is a halogen atom;
- R 6 is selected from hydrogen, and C 1-6 alkyl Or a pharmaceutically acceptable salt thereof.
- the present invention provides a pharmaceutical composition for use in the treatment or prevention of synucleinopathies.
- the present invention provides a compound having a therapeutic or prophylactic effect for synucleinopathies.
- FIG. 1 is a Western blot showing the results of an ⁇ -synuclein oligomer formation inhibition test using PC12 cells transfected with ⁇ -synuclein and FABP3 expression genes. It was confirmed that the compounds of Examples 1 to 3 (Ex1 to Ex3), which are FABP3 ligands, exhibited an oligomer formation inhibitory effect, whereas the compound of Comparative Example 1 (CoEx1) did not exhibit such an effect.
- FIG. 2 shows a photograph after staining of brain sections of the prefrontal cortex, dorsal hippocampus, and substantia nigra ventral tegmental area of mice subjected to the test of Test Example 4 (using the compound of Example 1).
- FIG. 5 is a graph showing the results of evaluating the motor function by the rotor rod test and the beam walking test of Test Example 5. The vertical axis of the graph of the rotor rod test result indicates the time (second, latency) until the mouse falls. In the rotor rod test, the tendency of shortening to “Latency” was confirmed in the ligand and administration groups.
- FIG. 6 is a graph showing the results of evaluating the cognitive function by the novel object recognition test of Test Example 6.
- the vertical axis shows the contact ratio between the new object and the known object (Discriminationcriindex (%)).
- Discriminationcriindex (%) the contact ratio between the new object and the known object in the ligand administration group
- FIG. 7 is a graph showing the results of evaluation of cognitive function by the novel object recognition test of Test Example 7.
- FIG. 8 is a graph showing the results of evaluating the motor function by the rotor rod test and beam walking test of Test Example 8.
- FIG. 9 is a graph showing the results of evaluating the motor function by the rotor rod test and the beam walking test of Test Example 9. The numbers shown in each bar of the rotor rod test result graph represent n in each county.
- FIG. 10 is a graph showing the results of evaluating the cognitive function by the novel object recognition test and the passive avoidance test of Test Example 9.
- the vertical axis of the graph showing the results of the passive avoidance test shows the time until the mouse that received electrical stimulation after entering the light room after entering the light room in the training trial enters the light room after entering the light room again after 24 hours. Indicates the time (seconds) (Latency).
- FIG. 11 is a graph showing the results of a dopamine neuroprotection evaluation test using the Parkinson's disease model animal of Test Example 11.
- FIG. 12 is a graph showing the results of an ⁇ -synuclein aggregation inhibition evaluation test using the Parkinson's disease model animal of Test Example 12.
- FIG. 13 shows the results of an affinity evaluation test of FABP ligand using ANS of Test Example 13.
- FIG. 14 is a diagram illustrating procedures such as a rotor rod test, a beam walking test, and a new object recognition test performed in the test example.
- FIG. 15 is a diagram showing a base sequence subjected to sequencing in Test Example 13.
- a pharmaceutical composition for the treatment or prevention of a cognitive function disease or disorder comprising as an active ingredient a compound represented by formula (I), or a pharmaceutically acceptable salt thereof.
- a pharmaceutical composition for treating or preventing a cognitive function disease or disorder which comprises a compound represented by formula (Ia) or formula (Ib) as an active ingredient.
- the compound represented by the formula (I) includes a compound represented by the formula (Ia) or the formula (Ib).
- C 1-6 alkyl means a linear, branched, cyclic or partially cyclic alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl. I-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl , 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl, and 2-ethylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, and the like, for example, C 1-4 alkyl And C 1-3 alkyl and the like are also included.
- C 1-6 alkoxy means an alkyloxy group [—O— (C 1-6 alkyl)] having an alkyl group having 1 to 6 carbon atoms already defined as the alkyl moiety, , Methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, i-butoxy, t-butoxy, n-pentoxy, 3-methylbutoxy, 2-methylbutoxy, 1-methylbutoxy, 1- Includes ethylpropoxy, n-hexyloxy, 4-methylpentoxy, 3-methylpentoxy, 2-methylpentoxy, 1-methylpentoxy, 3-ethylbutoxy, cyclopentyloxy, cyclohexyloxy, cyclopropylmethyloxy, etc. Examples thereof include C 1-4 alkoxy and C 1-3 alkoxy.
- halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
- R 1a , R 1b , R 1c and R 1d in formula (Ib) and formula (Ic) are independently a hydrogen atom, C 1-6 alkyl, C 1-6 alkoxy, And a halogen atom.
- the present invention can be carried out using the solvate.
- the compound of the present invention or a pharmaceutically acceptable salt thereof can be appropriately implemented as a mixture, solution, crystalline polymorph and the like.
- the “pharmaceutically acceptable salt” of the compound of the formula (I) is not particularly limited as long as it is a salt that can be used as a pharmaceutical product.
- Examples of the salt formed by the compound of the present invention with a base include salts with inorganic bases such as sodium, potassium, magnesium, calcium and aluminum; salts with organic bases such as methylamine, ethylamine and ethanolamine.
- the salt may be an acid addition salt.
- salts include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid and other mineral acids; and formic acid, Examples include acid addition salts with organic acid acids such as acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, and ethanesulfonic acid. .
- pharmaceutically acceptable salts include base addition salts of compounds of formula (I) which are carboxylic acids.
- a compound of formula (I), its enantiomer, its diastereomer, or a pharmaceutically acceptable salt thereof is administered as a prodrug and converted to the active compound in vivo.
- Examples of the compound included in the formula (I) include the compounds described in Examples and the following compounds.
- the synucleinopathy in the present invention means a neurodegenerative disease characterized by abnormal accumulation of ⁇ -synuclein, and includes, for example, Parkinson's disease, Lewy body dementia, or multiple system atrophy.
- the pharmaceutical compositions and methods can be used, for example, to inhibit the progression of synucleinopathies.
- the pharmaceutical composition and method of the present invention there is no particular limitation on the degree of progression, severity and pathological condition of synucleinopathy to be treated.
- Parkinson's disease severity classification Yar I to V degrees are used.
- Lewy body dementia has early, middle, and late stages, and ⁇ -synuclein is classified into brainstem dominant type, marginal type, and neocortical type from the first appearance site of inclusion bodies.
- modified Ranking Scale 1-6 is used in the severity classification of multisystem atrophy, and is classified into Shy Drager syndrome, Olive Bridge cerebellar atrophy, striatal mineral degeneration, etc., depending on the pathological condition. Any of these is a group of diseases in which ⁇ -synuclein inclusion bodies are expressed in nerve cells or glial cells, and the pharmaceutical composition and method of the present invention can be used for the treatment or prevention of diseases selected from these groups of diseases.
- Dementia is thought to develop due to neuronal cell death in the frontal cortex or hippocampus.
- the FABP3 ligand suppresses the propagation of ⁇ -synuclein aggregates in addition to suppressing the aggregation.
- neuronal cell death caused by ⁇ -synuclein aggregates in the brain is suppressed.
- the pharmaceutical composition and method of the present invention can be used particularly for the treatment and prevention of dementia with Lewy bodies.
- compositions and method of the present invention can improve motor function disorder and cognitive function disorder and suppress progression.
- Motor dysfunctions include tremors in Parkinson's disease (hand and foot tremors), immobility (movement slows), stiffness (muscles become stiff and stiff, resistance to bending and stretching of joints), posture reflexes Disability (becomes difficult to balance), Lewy body dementia, Parkinson's symptoms, and in multiple system atrophy, in addition to Parkinson's symptoms, autonomic disorders (urination disorder, erectile dysfunction, orthostatic hypotension, Cerebellar ataxia: (ataxic gait and dysarthria, limb ataxia, or cerebellar eye movement disorders).
- Lewy body dementia memory impairment, frontal / parietal dysfunction (attention disorder, visuospatial disorder, constitutional disorder, executive dysfunction, etc.), cognitive sway, hallucination .
- executive functions planning, set conversion / maintenance, problem solving, etc.
- memory functions such as working memory, procedural memory, and higher-order brain dysfunction in visuospatial functions may be seen.
- symptoms similar to Lewy body dementia such as hallucinations, visuospatial impairment, and slow thinking, may be seen.
- multisystem atrophy when dementia such as forgetfulness is seen, etc. are mentioned.
- the pharmaceutical composition of the present invention can be used in various dosage forms such as tablets, capsules, powders, granules, pills, solutions, emulsions, suspensions, solutions, spirits, syrups for oral administration.
- parenteral preparations include, for example, injections such as subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections; transdermal administration or patches , Ointments or lotions; sublingual and buccal patches for buccal administration; and aerosols for nasal administration, but not limited thereto.
- injections such as subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections; transdermal administration or patches , Ointments or lotions; sublingual and buccal patches for buccal administration; and aerosols for nasal administration, but not limited thereto.
- These preparations can be produced by known methods usually used in the preparation process.
- the pharmaceutical composition may contain various commonly used components, such as one or more pharmaceutically acceptable excipients, disintegrants, diluents, lubricants, flavoring agents, and coloring agents. , Sweeteners, flavoring agents, suspending agents, wetting agents, emulsifying agents, dispersing agents, adjuvants, preservatives, buffering agents, binders, stabilizers, coating agents and the like.
- the pharmaceutical composition of the present invention may be in a sustained or sustained release dosage form.
- the dosage of the pharmaceutical composition of the present invention can be appropriately selected depending on the administration route, the patient's body shape, age, physical condition, degree of disease, elapsed time after onset, etc.
- An effective amount and / or a prophylactically effective amount of a compound of formula (I) above may be included.
- the compound of the above formula (I) is generally 1 to 1000 mg / day / adult, for example 1 to 200 mg / day / adult, specifically 5 to 100 mg / day / adult, more specifically 10 to It can be used at a dose of 50 mg / day / adult.
- the pharmaceutical composition may be administered in a single dose or multiple doses.
- the pharmaceutical composition of the present invention contains conventionally known colorants, preservatives, flavors, flavors, coating agents, antioxidants, vitamins, amino acids, peptides, proteins, and minerals (iron, zinc, magnesium). , Iodine, etc.).
- the pharmaceutical composition of the present invention is in a form suitable for oral administration, such as granules (including dry syrup), capsules (soft capsules, hard capsules), tablets (including chewables, etc.), powders (powder). , Various solid preparations such as pills, or liquid preparations such as liquids for internal use (including liquids, suspensions and syrups).
- additives for formulation for example, excipients, lubricants, binders, disintegrants, fluidizers, dispersants, wetting agents, preservatives, thickeners, pH adjusters, colorants, Examples include flavoring agents, surfactants, and solubilizing agents.
- thickeners such as pectin, xanthan gum, and guar gum, can be mix
- thickeners such as pectin, xanthan gum, and guar gum
- it can also be set as a coating tablet using a coating agent, or it can also be set as a paste-form glue. Furthermore, even if it is a case where it prepares in another form, what is necessary is just to follow the conventional method.
- the treatment method or prevention method of the present invention can be implemented based on the above description.
- Examples of subjects to which the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered include mammals such as humans.
- Example 1 4- (2- (5- (2-Chlorophenyl) -1-phenyl-1H-pyrazol-3-yl) phenoxy) butanoic acid [First Step] (E) -3- (2-Chlorophenyl) Preparation of -1- (2-methoxyphenyl) prop-2-en-1-one
- the title compound was prepared in the same manner as in Example 1, except that benzaldehyde was used instead of 2-chlorobenzaldehyde in the first step and 4-bromophenylhydrazine was used instead of phenylhydrazine in the second step.
- the title compound was prepared in the same manner as in Example 1 except that benzaldehyde was used in place of 2-chlorobenzaldehyde in the first step and 3,4-dichlorophenylhydrazine was used in place of phenylhydrazine in the second step. .
- the title compound was prepared in the same manner as in Example 1, except that 2-bromobenzaldehyde was used instead of 2-chlorobenzaldehyde in the first step.
- the title compound was prepared in the same manner as in Example 1, except that benzaldehyde was used instead of 2-chlorobenzaldehyde in the first step and 4-isopropylphenylhydrazine was used instead of phenylhydrazine in the second step.
- the title compound was prepared in the same manner as in Example 1, except that benzaldehyde was used instead of 2-chlorobenzaldehyde in the first step and 3-chlorophenylhydrazine was used instead of phenylhydrazine in the second step.
- the title compound was prepared in the same manner as in Example 1, except that benzaldehyde was used instead of 2-chlorobenzaldehyde in the first step and 4-fluorophenylhydrazine was used instead of phenylhydrazine in the second step.
- the title compound was prepared in the same manner as in Example 1, except that benzaldehyde was used instead of 2-chlorobenzaldehyde in the first step and 4-tertbutylphenylhydrazine was used instead of phenylhydrazine in the second step. .
- the title compound was prepared in the same manner as in Example 1 except that benzaldehyde was used instead of 2-chlorobenzaldehyde and 5'-fluoro-2'-methoxyacetophenone was used instead of 2'-methoxyacetophenone in the first step.
- benzaldehyde was used instead of 2-chlorobenzaldehyde and 5'-fluoro-2'-methoxyacetophenone was used instead of 2'-methoxyacetophenone in the first step.
- the title compound was prepared in the same manner as in Example 1, except that 5'-fluoro-2'-methoxyacetophenone was used instead of 2'-methoxyacetophenone in the first step.
- Example 1 except that 5′-fluoro-2′-methoxyacetophenone was used instead of 2′-methoxyacetophenone in the first step and 4-isopropylphenylhydrazine was used instead of phenylhydrazine in the second step.
- the title compound was prepared by the procedure of
- the title compound was prepared in the same manner as in Example 1, except that 2-methylbenzaldehyde was used instead of 2-chlorobenzaldehyde in the first step.
- Test Example 1 FABP3 Ligand Activity Evaluation
- 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid)
- Fluorescent materials such as 1,8-ANS have the property of increasing fluorescence intensity in a hydrophobic environment. Utilizing this property, 1,8-ANS was competitively bound with each compound at the hydrophobic ligand binding site of FABP, and the excitation wavelength 355 nm, which is the wavelength used in the experimental system using 1,8-ANS, was measured.
- the fluorescence intensity at a wavelength of 460 nm the ligand activity of each compound against FABP was evaluated. Ethanol was adopted as a diluting solvent for the compound.
- Test Example 2 FABP4 Ligand Activity Evaluation For the evaluation of FABP4, a fluorescence displacement assay using 1,8-ANS was adopted as in FABP3.
- FABP4 was evaluated using an assay kit (FABP4 Inhibitor / Ligand Screening Assay Kit) sold by Cayman Chemical Company. Ethanol was used as a solvent for dissolving the compound, and arachidonic acid included in the kit was used as a positive control.
- the measurement procedure was the same as the FABP3 assay described above. The measurement was performed twice, and the average value was taken as the measurement result. For calculation of IC 50 , Light Stone's PC software Origin was used. The evaluation results are shown in Table 1.
- Test Example 3 ⁇ - Synuclein Oligomer Formation Inhibitory Activity Test
- PC12 cells were transfected with ⁇ -synuclein and FABP3 genes, and serum-containing DMEM ( The cells were treated with ligand (compounds of Examples 1-4, 10 ⁇ M) for 16 hours in 10% horse serum, 5% newborn calf serum, containing penicillin / streptomycin.
- ligand compounds of Examples 1-4, 10 ⁇ M
- Homogenized samples of these cells were run on a 5 to 13.5% polyacrylamide gel at 80 mA for 3 hours under native conditions, then transferred to a PVDF membrane at 70 V constant voltage for 2 hours, and subjected to Western blotting.
- MPTP Parkinson's disease model animal 1-methyl-4-phenyl-1,2,3,6-tetrahydro
- FABP3 ligand Compound of Example 3, 1.0
- the solvent (n 6)
- FABP3 ligand Compound of Example 3, 1.0 mg / kg, po
- Cognitive function was evaluated using a novel object recognition test and passive avoidance test four weeks after administration of MPTP in which cognitive impairment was observed.
- mice were placed in a bright room in a training trial, and electrical stimulation (0.3 mA, 2 seconds) was given when the mouse entered a dark room. After 24 hours, the mouse was once again placed in the light room, and the time until entering the dark room was measured. The results are shown in FIG.
- mice were fixed by perfusion, and brain sections containing a substantia nigra region were prepared at a thickness of 50 ⁇ m.
- TH tyrosine hydroxylase
- mousestar mouse monoclonal antibody 22941, 1: 1000
- a marker protein of dopamine neuron a marker protein of dopamine neuron
- a fluorescently labeled secondary antibody Alexa 594 anti-mouse IgG (Jackson ImmunoResearch) Manufactured at 1: 500
- the number of TH positive cells decreased in the solvent-administered group was significantly improved by the administration of the compounds of Examples 1 and 3, but was not improved by the compound of Comparative Example 1. That is, while the compound of Comparative Example 1 could not confirm the improvement effect on the number of dopamine neurons, the effect was significantly confirmed in the compound administration groups of Examples 1 and 3.
- mice were fixed by perfusion, and brain sections containing a substantia nigra region were prepared at a thickness of 50 ⁇ m.
- the number of TH and ⁇ -synuclein double positive cells increased in the solvent-only administration group was improved by the compound of Example 1, and an improvement trend was observed in Example 3.
- the compound of Comparative Example 1 did not improve. That is, with respect to the number of dopamine neurons in which synuclein aggregation did not occur, the compound of Comparative Example 1 could not confirm the improvement effect, but the effects of the compound administration groups of Examples 1 and 3 were significantly confirmed.
- Test Example 13 Affinity evaluation test of FABP ligand using ANS GST-FABP3 and GST-FABP4 were expressed in Escherichia coli [BL21 (DE3) strain], respectively, and affinity purification was performed using a glutathione column.
- FIG. 15 shows the base sequences of GST-FABP3 and GST-FABP4 subjected to sequencing (the first and last GGATTC and GAATTC are restriction enzyme cleavage sites).
- the purification of GST binding protein was performed using GST-purification kit (Takara Bio USA). After recovering E. coli by centrifugation, the attached Extraction buffer and aluminum oxide powder were added and crushed in a mortar. The centrifuged supernatant was added to the attached glutathione column and allowed to stand on ice for 30 minutes. Thereafter, the supernatant in the column was discarded, washed with Extraction buffer, and then eluted with an elution buffer containing glutathione. The protein concentration of the eluted product was calculated from the absorbance at 280 nm, and then used for the ANS assay.
- 1-anilinonaphthalene-8-sulfonic acid ANS, final concentration 4 mM, 10 mM KH 2 PO 4 , 40 mM KCl, pH 7.4 solution
- FABP protein final concentration 0.4 mM which bind to FABP protein and emit fluorescence
- F F 0 ⁇ ⁇ [1+ (P t + L t ) Ka ⁇ [(P t ⁇ L t ) 2 Ka 2 +2 (P t + L t ) Ka + 1] 1/2 ] / [2P t Ka] ⁇ (F 0 -F max )
- the dissociation constant Kd calculated based on the measurement results is shown in the following table.
- the Kd values in the above table are displayed as the average of three measurements ⁇ SE.
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Abstract
Description
R1dは、水素原子、またはハロゲン原子であり;
R2およびR3は、独立して、C1-6アルキル、およびハロゲン原子から選択され;
R4は、水素原子、およびC1-6アルキルから選択され;
R5は、COOR6、CH2OH、および1-テトラゾリルから選択され;
R6は、水素原子、およびC1-6アルキルから選択され;
nは、0~5から選択される整数であり;
pは、0~4から選択される整数であり;
qは、1または2である]
で表される化合物、または医薬として許容なその塩を含む、シヌクレイノパチーの治療または予防のための医薬組成物。
で表される化合物、または医薬として許容なその塩を含む、[1]~[4]のいずれかに記載の医薬組成物。
4-(2-(1-(4-ブロモフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(3,4-ジクロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1,5-ジフェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-フルオロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-クロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(5-フェニル-1-(4-イソプロピルフェニル)-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-メトキシフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(2-クロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;および
4-(2-(1-(3-クロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(5-(2-ブロモフェニル)-1-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1,5-ジフェニル-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
から選択される化合物、または医薬として許容なその塩を含有する、[1]に記載の組成物。
R2aは、ハロゲン原子であり;
R3aは、水素原子またはハロゲン原子であり;
R6は、水素原子、およびC1-6アルキルから選択される]
で表される化合物、または医薬として許容なその塩。
R2は、C1-6アルキル、またはハロゲン原子であり;
nは、0~5から選択される整数であり;
R3bは、ハロゲン原子であり;
R6は、水素原子、およびC1-6アルキルから選択される]
で表される化合物、または医薬として許容なその塩。
R1dは、水素原子、またはハロゲン原子であり;
R2およびR3は、独立して、C1-6アルキル、およびハロゲン原子から選択され;
R4は、水素原子、およびC1-6アルキルから選択され;
R5は、COOR6、CH2OH、および1-テトラゾリルから選択され;
R6は、水素原子、およびC1-6アルキルから選択され;
nは、0~5から選択される整数であり;
pは、0~4から選択される整数であり;
qは、1または2である]
で表される化合物、または医薬として許容なその塩を含む、シヌクレイノパチーの治療または予防のための医薬組成物。
で表される化合物、または医薬として許容なその塩を含む、[1-1]~[1-5]のいずれかに記載の医薬組成物。
4-(2-(1-(4-ブロモフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(3,4-ジクロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1,5-ジフェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-フルオロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-クロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(5-フェニル-1-(4-イソプロピルフェニル)-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-メトキシフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(2-クロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(3-クロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(5-(2-ブロモフェニル)-1-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1,5-ジフェニル-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
4-(2-(5-クロロフェニル-1-フェニル-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
4-(2-(5-(2-クロロフェニル)-1-(4-イソプロピルフェニル)-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
4-(2-(5-(2-メチルフェニル)-1-フェニル-1H-ピラゾール-3-イル)-フェノキシ)ブタン酸;
4-(2-(5-(2-ブロモフェニル)-1-(4-イソプロピルフェニル)-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
(S)-4-(2-(5-(2-クロロフェニル)-1-フェニル-1H-ピラゾール-3-イル)-フェノキシ)-2-メチルブタン酸;および
(R)-4-(2-(5-(2-クロロフェニル)-1-フェニル-1H-ピラゾール-3-イル)-フェノキシ)-2-メチルブタン酸;
から選択される化合物、または医薬として許容なその塩を含有する、[1-1]に記載の組成物。
R2aは、C1-6アルキル、およびハロゲン原子から選択され;
R3aは、水素原子またはハロゲン原子であり;
R6は、水素原子、およびC1-6アルキルから選択される]
で表される化合物、または医薬として許容なその塩。
R2は、C1-6アルキル、またはハロゲン原子であり;
nは、0~5から選択される整数であり;
R3bは、ハロゲン原子であり;
R6は、水素原子、およびC1-6アルキルから選択される]
で表される化合物、または医薬として許容なその塩。
R1dは、水素原子、またはハロゲン原子であり;
R2およびR3は、独立して、C1-6アルキル、およびハロゲン原子から選択され;
R4は、水素原子、およびC1-6アルキルから選択され;
R5は、COOR6、CH2OH、および1-テトラゾリルから選択され;
R6は、水素原子、およびC1-6アルキルから選択され;
nは、0~5から選択される整数であり;
pは、0~4から選択される整数であり;
qは、1または2である]
で表される化合物、または医薬として許容なその塩を対象に投与することを含む、前記方法。
で表される化合物、または医薬として許容なその塩を含む、[2-1]~[2-4]のいずれかに記載の方法。
4-(2-(5-(2-クロロフェニル)-1-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-ブロモフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(3,4-ジクロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1,5-ジフェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-フルオロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-クロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(5-フェニル-1-(4-イソプロピルフェニル)-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-メトキシフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(2-クロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(3-クロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(5-(2-ブロモフェニル)-1-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1,5-ジフェニル-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
4-(2-(5-クロロフェニル-1-フェニル-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
4-(2-(5-(2-クロロフェニル)-1-(4-イソプロピルフェニル)-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
4-(2-(5-(2-メチルフェニル)-1-フェニル-1H-ピラゾール-3-イル)-フェノキシ)ブタン酸;
4-(2-(5-(2-ブロモフェニル)-1-(4-イソプロピルフェニル)-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
(S)-4-(2-(5-(2-クロロフェニル)-1-フェニル-1H-ピラゾール-3-イル)-フェノキシ)-2-メチルブタン酸;および
(R)-4-(2-(5-(2-クロロフェニル)-1-フェニル-1H-ピラゾール-3-イル)-フェノキシ)-2-メチルブタン酸;
から選択される化合物、または医薬として許容なその塩である、[2-1]に記載の方法。
R2aは、ハロゲン原子であり;
R3aは、水素原子またはハロゲン原子であり;
R6は、水素原子、およびC1-6アルキルから選択される]
で表される化合物、または医薬として許容なその塩である、[2-1]に記載の方法。
R2は、C1-6アルキル、またはハロゲン原子であり;
nは、0~5から選択される整数であり;
R3bは、ハロゲン原子であり;
R6は、水素原子、およびC1-6アルキルから選択される]
で表される化合物、または医薬として許容なその塩である、[2-1]に記載の方法。
式(I)の化合物の「医薬として許容な塩」とは、医薬品として使用可能な塩であれば特に限定されない。本発明化合物が塩基と形成する塩としては、ナトリウム、カリウム、マグネシウム、カルシウム、アルミニウムなどの無機塩基との塩;メチルアミン、エチルアミン、エタノールアミン等の有機塩基との塩などが挙げられる。当該塩は、酸付加塩であってもよく、かかる塩としては、具体的には、塩酸、臭化水素酸、ヨウ化水素酸、硫酸、硝酸、リン酸等の鉱酸;および、ギ酸、酢酸、プロピオン酸、シュウ酸、マロン酸、コハク酸、フマル酸、マレイン酸、乳酸、リンゴ酸、酒石酸、クエン酸、メタンスルホン酸、エタンスルホン酸などの有機酸酸との酸付加塩が挙げられる。好ましくは、医薬として許容な塩として、カルボン酸である式(I)の化合物の塩基付加塩が例示される。
[第1工程](E)-3-(2-クロロフェニル)-1-(2-メトキシフェニル)プロプ-2-エン-1-オンの調製
実施例3:4-(2-(1-(3,4-ジクロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸
[第1工程](S)-4-ベンジル-3-(4-(ベンジルオキシ)ブタノイル)オキサゾリジン-2-オンの調製
1H-NMR (300 MHz, CDCl3) δ [ppm]: 8.13 (dd, 7.8, 1.8 Hz, 1H), 7.54-7.48 (m, 1H), 7.40-7.19 (m, 12H), 7.13-7.10 (m, 2H), 7.06 (s, 1H), 7.04 (td, J = 3.3, 1.2 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 4.50-4.42 (m, 1H), 4.27-4.20 (m, 1H), 4.15-4.07 (m, 1H), 4.04 (dt, J = 6.9, 1.5 Hz, 1H), 3.99 (dd, J = 9.0, 2.4 Hz, 1H), 3.90 (t, J = 8.1 Hz, 1H), 3.16 (dd, J = 13.8, 3.3 Hz, 1H), 2.71 (dd, J = 13.5, 9.6 Hz, 1H), 2.46-2.34 (m, 1H), 2.08-1.97 (m, 1H), 1.31 (d, J = 7.2 Hz, 1H)。
[第1工程](R)-4-ベンジル-3-(4-(ベンジルオキシ)ブタノイル)オキサゾリジン-2-オンの調製
[第1工程]((E)-1-(2-メトキシフェニル)-3-フェニルプロプ-2-エン-1-オンの調製
[第2工程]3-(2-(ベンジルオキシ)フェニル)-1-(2-メトキシフェニル)-5-フェニル-1H-ピラゾールの調製
FABP3リガンド活性評価に対する評価には,1,8-ANS(1-アニリノナフタレン-8-スルホン酸)を用いた蛍光ディスプレースメントアッセイを採用した。1,8-ANSのような蛍光性物質は,疎水性の環境下において,蛍光強度が増加する性質を有する。この性質を利用し,1,8-ANSをFABPの疎水性リガンド結合部位に各化合物と競合的に結合させ,1,8-ANSを用いる実験系で使用される波長である励起波長355nm,測定波長460nmでその蛍光強度を比較することにより,各化合物のFABPに対するリガンド活性を評価した。化合物の希釈溶媒はエタノールを採用した。
FABP4の評価もFABP3と同じく1,8-ANSを用いた蛍光ディスプレースメントアッセイを採用した。FABP4についてはCayman Chemical Companyから販売されているアッセイキット(FABP4 Inhibitor/Ligand Screening Assay Kit)を用いて評価した。化合物を溶解する溶媒はエタノールを用い,ポジティブコントロールにはキット付属のアラキドン酸を用いた。測定手順は上記のFABP3アッセイと同様に行った。
測定は各2回行い,その平均値を測定結果とした。IC50の算出には,Light Stone社のPCソフトOriginを用いた。評価結果を表1に示す。
既存の方法に従い(Shioda ら, J Biol Chem 2014;289:18957-18965)PC12細胞にα-シヌクレイン及びFABP3の遺伝子をトランスフェクションし、血清含有DMEM(10% ウマ血清, 5%牛新生仔血清, ペニシリン/ストレプトマイシン含有)中で、該細胞をリガンド(実施例1~4の化合物、10μM)により16時間処置した。それらの細胞をホモジナイズしたサンプルを、未変性条件で5~13.5%ポリアクリルアミドゲルで80mAで3時間泳動し、その後PVDFメンブレンに70V定電圧下で2時間転写し、ウエスタンブロッティングに供した。
無菌リン酸緩衝液生理食塩液(pH=7.4)中で、ヒトα-シヌクレインリコンビナントタンパク質(5μg/μL、rPeptide., Bogart,GA)を37℃、100rpmの条件下で7日間インキュベーションし、線維化ヒトα-シヌクレイン(α-Syn PPF 、シヌクレイン凝集体)を作製した。無菌リン酸緩衝液生理食塩液(pH=7.4)中で超音波処理後、10週齢の雄性 C57BL6 N マウス(12時間の明暗サイクル(明期9:00-21:00;暗期21:00-9:00)温度23±1℃、湿度55±5%の条件下で飼育)の右側黒質領域 (Bregma から後方3.3mm,右方1.2mm,深さ3.85mm)にα-Syn PPF を流速0.2μL/分で10μgを脳定位固定装置に脳固定後マイクロシリンジで注入した。手術24時間後、溶媒(n=3)またはFABP3リガンド(実施例1の化合物、1.0mg/kg,p.o.、n=3)を一日一回四週間投与した。各行動薬理試験終了後、マウスを還流固定し、一匹のマウスから各領域(前頭前皮質、線条体、背側海馬、黒質腹側被蓋野領域)の50μmの脳切片3枚を作製した。リン酸化α-シヌクレイン (ser129) (α-Syn S129) 抗体(Abcam, Cambridge, UK)を用い、α-シヌクレイン凝集体を免疫組織化学染色法により同定した。図2に示すように、FABP3 リガンド投与群においては、α-シヌクレイン凝集体(封入体)形成の抑制が確認された。
α-Syn PPF 注入マウスに一日一回四週間、溶媒(n=3)またはFABP3リガンド(実施例1の化合物、1.0mg/kg,p.o.、n=3)を投与し、ローターロッド試験およびビームウォーキング試験により運動機能を評価した。ローターロッド試験はマウスをローラーの上に載せ、20rpmの速度でローラーを回転させた際にマウスが落下するまでの時間(Latency)を測定した。落下するまでの時間はリガンド投与群で減少傾向が確認された。ビームウォーキング試験は細い板の上にマウスを置き歩行させ、ゴールボックスに辿り着くまでに足を踏み外した回数を測定した。結果を図5に示す。ビームウォーキング試験ではリガンド投与群において踏み外し回数の減少傾向が確認された。
α-Syn PPF 注入マウスに一日一回四週間、溶媒(n=3)またはFABP3リガンド(実施例1の化合物、1.0mg/kg,p.o.、n=3)を投与し、新規物体認識試験により、認知機能を評価した。新規物体認識試験は、訓練試行では同じ形の物体を置きマウスに記憶させ、試験試行では片方の物体を新規物体に代え、両物体に対する接触割合を評価した。結果を図6に示す。新規物体認識試験では、リガンド投与群において新規物体と既知物体の間で接触割合に大きな差があり、認知機能の改善傾向が確認された。
ドパミン神経毒である1-メチル-4-フェニル-1,2,3,6-テトラヒドロピリジン(MPTP、25mg/kg,i.p.、Sigma-Aldrich社(St Louis, MO)より購入)を10週齢の雄性 C57BL6 N マウスに一日一回五日間連続投与し、パーキンソン病モデル動物を作製した。MPTP最終投与24時間後から溶媒またはFABP3リガンド(実施例3の化合物、1.0mg/kg,p.o.、各群n=7)を一日一回二週間投与した。認知機能障害がみられるMPTP投与四週間後に新規物体認識試験により認知機能を評価した。結果を図7に示す。MPTP投与後に溶媒のみを投与した群(対象群)では認知機能障害が確認されたのに対し、リガンド投与群では障害が見られなかった。
MPTP最終投与24時間後から溶媒またはFABP3リガンド(実施例3の化合物)(1.0mg/kg,p.o.、各群n=7)を一日一回二週間投与した。運動機能障害がみられるMPTP投与三週間後にローターロッド試験およびビームウォーキング試験により、運動機能を評価した。結果を図8に示す。ローターロッド試験では溶媒のみを投与した群(対象群)でLatancy が有意に減少し、リガンド投与群で完全に回復した。ビームウォーキング試験では対象群で足を踏み外す回数が有意に上昇し、リガンド投与群で完全に回復した。両試験において、MPTP投与後に生じた運動機能障害が、リガンド投与群では回復したことが確認された。
MPTP(25mg/kg,i.p.)を10週齢の雄性C57BL6 N マウスに一日一回五日間連続投与し、パーキンソン病モデル動物を作製した。MPTP最終投与24時間後から溶媒(n=6)、FABP3リガンド(実施例1の化合物、0.1mg/kg(n=5),0.5mg/kg(n=7),または1.0mg/kg(n=6),p.o.)、FABP3リガンド(実施例3の化合物、1.0mg/kg,p.o.)および非FABP3リガンド(比較例1の化合物、1.0mg/kg,p.o.)を一日一回投与した。運動機能障害がみられるMPTP投与後三週目において、ローターロッド試験及びビームウォーキング試験により運動機能を評価した。結果を図9に示す。ローターロッド試験では溶媒のみを投与した群(対象群)で低下したLatencyは実施例1および3の化合物で有意に改善し、比較例1の化合物では改善しなかった。ビームウォーキング試験では足を踏み外す回数がMPTP投与で有意に上昇し、実施例1および3の化合物で有意に改善し、比較例1の化合物では改善しなかった。すなわち、比較例1の化合物投与群においては効果が確認できなかったのに対し、実施例1および3の化合物投与群では有意に効果が確認された。
MPTP(25mg/kg,i.p.)を10週齢の雄性C57BL6 N マウスに一日一回五日間連続投与し、パーキンソン病モデル動物を作製した。MPTP最終投与24時間後から溶媒(n=6)、FABP3リガンド(実施例1の化合物、0.1mg/kg(n=5),0.5mg/kg(n=7),または1.0mg/kg(n=6),p.o.)、FABP3リガンド(実施例3の化合物、1.0mg/kg,p.o.)および非FABP3リガンド(比較例1の化合物、1.0mg/kg,p.o.)を一日一回投与した。認知機能障害がみられるMPTP投与後四週目に新規物体認識試験および受動回避試験を用い認知機能について評価した。受動回避試験は、訓練試行では明室にマウスを入れ、暗室にマウスが入った際に電気刺激(0.3mA,2秒)を与えた。24時間後にもう一度マウスを明室にいれ、暗室に入るまでの時間を測定した。結果を図10に示す。
MPTP(25mg/kg,i.p.)を10週齢のC57BL6 Nマウスに一日一回5日間連続投与し、パーキンソン病モデル動物を作製した。MPTP最終投与24時間後から溶媒(n=6)、FABP3リガンド(実施例1の化合物、0.1mg/kg(n=5),0.3mg/kg(n=6)または1.0mg/kg(n=4),p.o.)、FABP3リガンド(実施例3の化合物、1.0mg/kg(n=4),p.o.)および非FABP3リガンド(比較例1の化合物、1.0mg/kg(n=7),p.o.)を一日一回投与した。ドパミン神経の脱落およびα-シヌクレイン多量体形成がみられるMPTP投与後4週目においてマウスを灌流固定し、50μmの厚さで黒質領域を含む脳切片を作製した。ドパミン神経のマーカー蛋白質であるチロシンヒドロキシラーゼ(TH)抗体(Immunostar社製、mouse monoclonal antibody 22941、1:1000)と反応させ、蛍光標識された二次抗体(Alexa 594 anti-mouse IgG (Jackson ImmunoResearch社製、1:500))で検出し、陽性細胞数を評価した。結果を図11に示す。
MPTP(25mg/kg,i.p.)を10週齢のC57BL6 Nマウスに一日一回5日間連続投与し、パーキンソン病モデル動物を作製した。MPTP最終投与24時間後から溶媒(n=6)、FABP3リガンド(実施例1の化合物、0.1mg/kg(n=5),0.3mg/kg(n=6)または1.0mg/kg(n=4),p.o.)、FABP3リガンド(実施例3の化合物、1.0mg/kg(n=4),p.o.)および非FABP3リガンド(比較例1の化合物、1.0mg/kg(n=7),p.o.)を一日一回投与した。ドパミン神経の脱落およびα-シヌクレイン多量体形成がみられるMPTP投与後4週目においてマウスを灌流固定し、50μmの厚さで黒質領域を含む脳切片を作製した。TH抗体(Immunostar社製、mouse monoclonal antibody 22941、1:1000)およびα-シヌクレイン抗体(Santa Cruz社製、rabbit polyclonal antibody SC-7011-R、1:200)と反応させ、蛍光標識された二次抗体(Alexa 488 anti-rabbit IgG (Jackson ImmunoResearch社製、1:500))で検出し、陽性細胞数を評価した。結果を図12に示す。
大腸菌[BL21(DE3)株]にGST-FABP3およびGST-FABP4をそれぞれ発現させたのち、グルタチオンカラムを用いてアフィニティー精製を行った。
F:ある条件における相対蛍光強度(%);
F0:リガンド非存在下における蛍光強度(=100);
Pt:FABPタンパク質濃度(=400nM);
Lt:リガンド濃度(=100,1000,2000,4000nM);
Ka:解離定数Kdの逆数(nM-1);
Fmax *:FABPがリガンドにより完全に占有された時の相対蛍光強度。
Claims (22)
- 式(I):
R1dは、水素原子、またはハロゲン原子であり;
R2およびR3は、独立して、C1-6アルキル、およびハロゲン原子から選択され;
R4は、水素原子、およびC1-6アルキルから選択され;
R5は、COOR6、CH2OH、および1-テトラゾリルから選択され;
R6は、水素原子、およびC1-6アルキルから選択され;
nは、0~5から選択される整数であり;
pは、0~4から選択される整数であり;
qは、1または2である]
で表される化合物、または医薬として許容なその塩を含む、シヌクレイノパチーの治療または予防のための医薬組成物。 - nが0または1であり、pが0または1であり、qが2である、請求項1に記載の医薬組成物。
- nが0または1であり、pが0であり、qが2である、請求項1または2に記載の医薬組成物。
- R4が水素原子であり、R5がCOOR6である、請求項1~3のいずれか1項に記載の医薬組成物。
- R1aおよびR1bが、独立して、水素原子、塩素原子、臭素原子、メチルおよびメトキシから選択される、請求項1~4のいずれか1項に記載の医薬組成物。
- R2aがC1-3アルキルまたはハロゲン原子である、請求項1~6のいずれか1項に記載の医薬組成物。
- R6が水素原子である、請求項1~7のいずれか1項に記載の医薬組成物。
- 4-(2-(5-(2-クロロフェニル)-1-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-ブロモフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(3,4-ジクロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1,5-ジフェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-フルオロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-クロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(5-フェニル-1-(4-イソプロピルフェニル)-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(4-メトキシフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(2-クロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1-(3-クロロフェニル)-5-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(5-(2-ブロモフェニル)-1-フェニル-1H-ピラゾール-3-イル)フェノキシ)ブタン酸;
4-(2-(1,5-ジフェニル-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
4-(2-(5-クロロフェニル-1-フェニル-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
4-(2-(5-(2-クロロフェニル)-1-(4-イソプロピルフェニル)-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
4-(2-(5-(2-メチルフェニル)-1-フェニル-1H-ピラゾール-3-イル)-フェノキシ)ブタン酸;
4-(2-(5-(2-ブロモフェニル)-1-(4-イソプロピルフェニル)-1H-ピラゾール-3-イル)-4-フルオロフェノキシ)ブタン酸;
(S)-4-(2-(5-(2-クロロフェニル)-1-フェニル-1H-ピラゾール-3-イル)-フェノキシ)-2-メチルブタン酸;および
(R)-4-(2-(5-(2-クロロフェニル)-1-フェニル-1H-ピラゾール-3-イル)-フェノキシ)-2-メチルブタン酸;
から選択される化合物、または医薬として許容なその塩を含有する、請求項1に記載の医薬組成物。 - シヌクレイノパチーが、パーキンソン病、レビー小体型認知症、または多系統萎縮症である、請求項1~9のいずれか1項に記載の医薬組成物。
- 経口投与用である、請求項1~10のいずれか1項に記載の医薬組成物。
- R1dは、水素原子、またはハロゲン原子である、請求項12に記載の化合物、または医薬として許容なその塩。
- R2aが塩素原子または臭素原子である、請求項12または13に記載の化合物、または医薬として許容なその塩。
- R3aが水素原子またはフッ素原子である、請求項12~14のいずれか1項に記載の化合物、または医薬として許容なその塩。
- R1dは、水素原子、またはハロゲン原子である、請求項16に記載の化合物、または医薬として許容なその塩。
- nが0または1である、請求項16または17に記載の化合物、または医薬として許容なその塩。
- R3bがフッ素原子である、請求項16~18のいずれか1項に記載の化合物、または医薬として許容なその塩。
- 請求項12~19のいずれか1項に記載の化合物、または医薬として許容なその塩を含む、シヌクレイノパチーの治療または予防のための医薬組成物。
- シヌクレイノパチーが、パーキンソン病、レビー小体型認知症、または多系統萎縮症である、請求項20に記載の医薬組成物。
- 経口投与用である、請求項20または21に記載の医薬組成物。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2022163818A1 (ja) * | 2021-01-29 | 2022-08-04 | 国立大学法人東北大学 | 認知症診断用のバイオマーカー |
WO2024063147A1 (ja) * | 2022-09-21 | 2024-03-28 | 白鳥製薬株式会社 | トリフェニルアゾール化合物 |
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