[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2019221566A1 - Composition pharmaceutique pour prévenir ou traiter une lésion cérébrale traumatique ou un accident vasculaire cérébral - Google Patents

Composition pharmaceutique pour prévenir ou traiter une lésion cérébrale traumatique ou un accident vasculaire cérébral Download PDF

Info

Publication number
WO2019221566A1
WO2019221566A1 PCT/KR2019/005969 KR2019005969W WO2019221566A1 WO 2019221566 A1 WO2019221566 A1 WO 2019221566A1 KR 2019005969 W KR2019005969 W KR 2019005969W WO 2019221566 A1 WO2019221566 A1 WO 2019221566A1
Authority
WO
WIPO (PCT)
Prior art keywords
straight
substituted
lrrk2
brain injury
traumatic brain
Prior art date
Application number
PCT/KR2019/005969
Other languages
English (en)
Korean (ko)
Inventor
최환근
고은화
고이경
박진희
이태관
김희정
이병대
부영민
배진현
배윤희
Original Assignee
재단법인 대구경북첨단의료산업진흥재단
경희대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 재단법인 대구경북첨단의료산업진흥재단, 경희대학교 산학협력단 filed Critical 재단법인 대구경북첨단의료산업진흥재단
Publication of WO2019221566A1 publication Critical patent/WO2019221566A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/326—Foods, ingredients or supplements having a functional effect on health having effect on cardiovascular health

Definitions

  • It relates to a pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke.
  • Protein kinases are enzymes that catalyze the reaction that transfers the terminal phosphate groups of adenosine triphosphate (ATP) to specific residues of the protein (tyrosine, serine, threonine), and regulates cell activity, growth and differentiation against changes in cell mediators and the environment. Is involved in the signal.
  • ATP adenosine triphosphate
  • Inappropriately high protein kinase activity is directly or indirectly associated with many diseases resulting from abnormal cellular action.
  • the disease may be caused by mutations, over-expression, or failure of the proper regulation of kinases involved in inappropriate enzyme activity, or by the production of excess or deficiency of factors involved in signal transduction upstream or downstream of cytokines or kinases.
  • selective inhibition of kinase activity can be a beneficial target of drug development for disease treatment.
  • LRRK2 leucin-rich repeat kinase-2
  • LRRK2 is a protein belonging to the leucin-rich repeat kinase family and consists of 2527 amino acid sequences with high similarity between species. It has both GTPase and Serine-threonine kinase activity.
  • the expressed LRRK2 is observed in various organs and tissues including the brain, and is known to exist in the cytoplasm or cell membrane and mitochondrial outer membrane at the cellular level.
  • Currently, research on the exact in vivo function of LRRK2 is being actively conducted, and it has 5 functionally important domains, and it is a cell through autophosphorylation and protein interaction and enzymatic activity.
  • LRRK2 is implicated in impairment of mild cognitive impairment associated with Alzheimer's disease, L-Dopa induced dyskinesia, CNS disorders associated with neuronal progenitor differentiation, cancers such as kidney cancer, breast cancer, prostate cancer, hematologic and lung cancer and acute myeloid It has been reported to be associated with leukemia, papillary kidney and thyroid carcinoma, multiple myeloma, amyotrophic lateral sclerosis, rheumatoid arthritis and ankylosing spondylitis (Patent Document 1: WO 2007/149789), to control LRRK2 activity. Effective compounds and compositions can provide therapeutic effects such as neurodegenerative diseases, CNS disorders, cancer, acute myeloid leukemia and multiple myeloma, and inflammatory diseases.
  • Traumatic Brain Injury is an external shock that affects the brain and causes damage to the brain. Brain tissues are less self-healing than other tissues. Loss and malfunction are likely. Due to the basic characteristics of neurons, the loss of neurons accompanying traumatic brain injury cannot be reversed, and the basic treatment strategy is to minimize the neuronal loss caused by primary damage and to prevent it from expanding anymore. The main focus of the treatment is to develop a therapeutic agent for a target that can directly control neuronal cell death or inhibit indirect neuronal cell death by controlling encephalitis, brain edema and excitatory toxicity.
  • the traumatic brain injury treatment currently in use is not effective in reducing the mortality rate and the incidence of sequelae, despite 20 years of advancement.
  • ischemic stroke Cerebral infarction
  • hemorrhagic stroke Cerebral hemorrhage
  • cerebrovascular burst cerebrovascular burst
  • ischemic stroke the main cause is thromboembolism of the cerebral artery due to vascular atherosclerosis, or cardiac embolism due to embolism and heart disease.
  • cerebral hemorrhage primary cerebral hemorrhage due to hypertension and subarachnoid hemorrhage due to arteriovenous malformation or aneurysm are important. to be.
  • Cerebral infarction which is caused by clogged cerebrovascular vessels, is divided into thrombosis and cerebral embolism, which causes arteriosclerosis due to hypertension, diabetes, and hyperlipidemia, resulting in thickening or stiffening of the walls of the arteries.
  • the inner wall is susceptible to wounds, so it is not smooth, and the blood is entangled and eventually clogged, and the supply of blood is reduced or stopped, resulting in a lack of oxygen and nutrient supply to brain cells, leading to disorders of the brain function.
  • Atrial fibrillation or other diseases cause blood flow in the heart, part of the blood partially stagnated, coagulate in the heart, resulting in blood clots (thrombosis), which fall off to block the cerebral blood vessels resulting in cerebral infarction. .
  • cerebral hemorrhage In the case of cerebral hemorrhage, the pressure in the cerebral blood vessels is increased due to high blood pressure, so that the walls of the small blood vessels are not able to withstand the small blood vessels.
  • Subarachnoid hemorrhage cerebral arteriovenous malformation caused by bursting is inherently present and is divided into cerebral hemorrhage caused by direct delivery to high pressure veins of arteries.
  • thrombotic drug Currently, pharmacotherapy using antithrombotics, anticoagulants, antiplatelets, and thrombolytics is used as a treatment for stroke, but currently the only approved drug, antithrombotic drug (tPA), is effective only within 4 to 5 hours after the occurrence of cerebral infarction. .
  • tPA worsens the disease in case of hemorrhagic stroke and causes side effects such as allergy. In other words, stroke is difficult to diagnose, difficult to detect early, and there is no fundamental treatment yet.
  • One object of the present invention is to provide a pharmaceutical composition for preventing or treating traumatic brain injury or stroke containing an LRRK2 inhibitor as an active ingredient.
  • Another object of the present invention to provide a health functional food composition for the prevention or improvement of traumatic brain injury or stroke containing an LRRK2 inhibitor as an active ingredient.
  • a pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke containing an LRRK2 inhibitor as an active ingredient.
  • a health functional food composition for the prevention or improvement of traumatic brain injury or stroke containing an LRRK2 inhibitor as an active ingredient.
  • a traumatic brain injury or stroke comprising the step of administering a pharmaceutical composition or health functional food composition for the prevention or treatment of traumatic brain injury or stroke containing the LRRK2 inhibitor as an active ingredient to a subject in need thereof.
  • a method of preventing or treating is provided.
  • a pharmaceutical composition or health functional food composition for the prevention or treatment of traumatic brain injury or stroke containing an LRRK2 inhibitor as an active ingredient in the prevention or treatment of traumatic brain injury or stroke Is provided.
  • a pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke contains an LRRK2 inhibitor as an active ingredient, inhibits the expression of wild-type LRRK2 overexpressing in traumatic brain injury or stroke, and due to traumatic brain injury or stroke It is effective in protecting or restoring induced damaged neurons, improving neurobehavioral or neuropathy symptoms after traumatic brain injury, and treating, alleviating or ameliorating the brain pathology caused by traumatic brain injury. It can be usefully used for the treatment of traumatic brain injury or stroke.
  • Figure 1 shows the results of the analysis of increased LRRK2 expression in the mouse brain after traumatic brain injury.
  • Figure 2 shows the results of time-phase analysis of LRRK2 expression in neurons in the mouse brain after traumatic brain injury.
  • Figure 3 shows the results of time-phase analysis of LRRK2 expression in microglia in mouse brain after traumatic brain injury.
  • Figure 4 shows the results of time-phase analysis of LRRK2 expression in astrocytes in the mouse brain after traumatic brain injury.
  • Figure 5 shows the results of LRRK2 expression analysis in traumatic brain injury tissue using additional LRRK2 antibody.
  • Figure 6 shows the results of LRRK2 expression analysis in a stroke animal model.
  • Figure 7 shows the analysis results of LRRK2 expression in neurons in animal models of stroke. Specifically, analysis of LRRK2 expression in neurons in damaged brain tissue after induction of mouse stroke was analyzed by immunofluorescence staining.
  • Figure 9 shows the results of LRRK2 expression analysis in various neurological damage animal models.
  • FIG 11 shows the analysis results of LRRK2 expression changes according to HIF-1a expression regulation and activity regulation.
  • Figure 13 shows the results of neurotoxicity according to the regulation of LRRK2 expression in a traumatic brain injury neuron model.
  • Figure 14 shows the results of neurotoxicity according to the regulation of LRRK2 activity in a traumatic brain injury neuron model.
  • FIG. 15 shows the results of neurotoxicity according to the regulation of LRRK2 activity in the traumatic brain injury neuron model. Specifically, changes in neurotoxicity following treatment with LRRK2 activity inhibitors Example 10 (FIG. A), Examples 21, and 20 (FIG. B) after scratch damage in primary cortical neurons are analyzed by LDH efflux experiments.
  • Figure 16 shows the results of neurotoxicity analysis by LRRK2 in hypoxia environment.
  • FIG. 17 shows the results of an analysis of brain injury protection effect according to inhibition of LRRK2 activity in a traumatic brain injury animal model.
  • (D) shows the results of performing tissue immunochemistry using neuronal marker (NeuN) antibody.
  • 19 shows the results of analysis of neuronal cell death according to inhibition of LRRK2 activity in a traumatic brain injury animal model.
  • FIG. 20 shows analysis of brain pathological changes according to inhibition of LRRK2 activity in a traumatic brain injury animal model.
  • One aspect of the present invention provides a pharmaceutical composition for preventing or treating traumatic brain injury or stroke containing an LRRK2 inhibitor as an active ingredient.
  • the LRK2 may be wild type.
  • the LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is characterized in that the compound represented by the following formula (1), an optical isomer thereof, or a pharmaceutically acceptable salt thereof.
  • R 1 is —H, —OH, halogen, nitro, nitrile, C 1-10 straight or branched chain alkyl, C 1-10 straight or branched chain alkoxy, C 1-5 straight or branched chain alkylamino Or straight or branched chain alkylamino of diC 1-5 ;
  • R 2 is —H, —OH, halogen, nitro, nitrile, or C 1-10 unsubstituted or substituted at least one halogen, straight or branched chain alkyl, or R 2 is R 3 and the carbon atom to which they are attached May be linked together to form an unsubstituted, substituted or fused 5 to 8 membered heterocycloalkyl comprising one or more N, or an unsubstituted or substituted 5 to 6 membered heteroaryl comprising one or more N; ,
  • substituted heterocycloalkyl and heteroaryl may be independently substituted with one or more substituents selected from the group consisting of —OH, ⁇ O, halogen and C 1-5 straight or branched chain alkyl,
  • fused 5-8 membered heterocycloalkyl is a 5-8 membered heterocycloalkyl fused with an unsubstituted C 6-10 aryl;
  • R 3 is —H, —OH, halogen, nitro, nitrile, C 1-10 straight or branched chain alkyl, C 1-10 straight or branched chain alkoxy, C 1-5 straight or branched chain alkylamino , di-C 1-5 straight or branched chain alkyl, straight or branched chain alkylsulfonyl C 6-10 arylamino, or a linear or branched alkyl sulfonylamino of C 1-5 of C 1-5 C 6- 10 arylamino, unsubstituted, substituted or fused 5 to 8 membered heterocycloalkyl containing one or more N, linked together with R 2 , or an unsubstituted or substituted 5 to 6 atom containing one or more N To form a heteroaryl,
  • substituted heterocycloalkyl and heteroaryl may be independently substituted with one or more substituents selected from the group consisting of —OH, ⁇ O, halogen and C 1-5 straight or branched chain alkyl,
  • fused 5-8 membered heterocycloalkyl is a 5-8 membered heterocycloalkyl fused with an unsubstituted C 6-10 aryl;
  • R 4 , R 5 and R 6 are independently —H, —OH, halogen, nitro, nitrile, C 1-10 straight or branched chain alkyl, C 1-10 straight or branched chain alkoxy, N and Unsubstituted or substituted 5 to 8 membered heterocycloalkyl containing one or more heteroatoms selected from the group consisting of O, unsubstituted or substituted containing 5 or more heteroatoms selected from the group consisting of N and O Heterocycloalkylcarbonyl having 8 to 8 atoms,
  • substituted heterocycloalkyl is a 6-membered heterocycloalkyl comprising straight or branched chain alkylamino of diC 1-5 or one or more N and substituted with at least one C 1-5 straight or branched chain alkyl Can be substituted,
  • R 7 , R 8 and R 9 are independently —H, —OH, halogen, nitro, nitrile, unsubstituted or substituted one or more hydroxy substituted C 1-10 straight or branched chain alkyl, or N and Unsubstituted or substituted 5 to 8 membered heterocycloalkyl comprising at least one hetero atom selected from the group consisting of O,
  • R 1 is —H, or —NH (CH 3) ;
  • R 2 is —H, halogen, or —CF 3 , or is linked with R 3 or To form;
  • R 3 is —H, —NH (CH 3) , —NH (CH 2 CH 3) , , or Or R 2 and R 3 are linked together with the carbon atoms to which they are attached or To form;
  • R 4 , R 5 and R 6 are independently -H, methoxy, halogen, , , or ego,
  • R 7 , R 8 and R 9 are independently -H, methyl, halogen, or Can be.
  • Examples of the compound represented by Formula 1 according to the present invention include the following compounds:
  • the LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is characterized in that the compound represented by the following formula (2), optical isomers thereof, or a pharmaceutically acceptable salt thereof.
  • L 1 and L 2 are independently —O—, —CH 2 —, —NH—, or —S—;
  • a 1 is unsubstituted or substituted C 3-10 cycloalkyl, wherein the substituted cycloalkyl is —OH, halogen, nitrile, nitro, C 1-5 straight or branched chain alkyl and C 1- May be substituted with one or more substituents selected from the group consisting of 5 straight or branched chain alkoxy;
  • a 2 is an unsubstituted or substituted 5 to 6 membered heteroaryl comprising one or more heteroatoms selected from the group consisting of N, O and S, wherein the substituted heteroaryl is -OH, halogen, nitrile , Nitro, may be substituted with one or more substituents selected from the group consisting of C 1-5 straight or branched alkyl and C 1-5 straight or branched alkoxy;
  • a 3 , A 4 and A 5 are independently —H, C 1-5 straight or branched alkyl or C 1-5 straight or branched alkoxy.
  • L 1 and L 2 are independently —CH 2 —, —NH—, or —S—;
  • a 1 is or ego
  • a 2 is ego
  • a 3 , A 4 and A 5 may be independently —H or methyl.
  • Examples of the compound represented by Formula 2 according to the present invention include the following compounds:
  • the LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is characterized in that the compound represented by the following formula (3), an optical isomer thereof, or a pharmaceutically acceptable salt thereof.
  • L 3 is absent, -NH-, or -O-;
  • L 4 is N, or C
  • E 1 is —H, —OH, halogen, nitrile, nitro, C 1-5 straight or branched chain alkyl, or C 1-5 straight or branched chain alkoxy;
  • E 2 is absent when L 4 is N, and when L 4 is C E 5 is C 1-5 straight or branched alkyl;
  • E 3 is -H, -OH, halogen, nitrile, nitro, C 1-5 straight or branched chain alkyl, C 1-5 straight or branched chain alkoxy, unsubstituted or substituted C 6-10 cyclo Alkyl or unsubstituted or substituted 5 to 8 membered heterocycloalkyl comprising at least one heteroatom selected from the group consisting of N and O,
  • substituted cycloalkyl and heterocycloalkyl are independently one or more substituents selected from the group consisting of —OH, halogen, C 1-3 straight or branched alkyl, and C 1-3 straight or branched alkoxy. Can be substituted;
  • E 4 is unsubstituted or substituted 6 to 8 membered heterocycloalkylcarbonyl C 1-5 straight or branched chain alkyl comprising one or more N; Or unsubstituted or substituted 6 to 8 membered heteroaryl including one or more N;
  • substituted heterocycloalkyl and heteroaryl are independently C 6-10 aryl; And an unsubstituted or substituted 6-membered heterocycloalkyl including one or more heteroatoms selected from the group consisting of N and O, and may be substituted with one or more selected from the group consisting of
  • the substituted 6-membered heterocycloalkyl may be substituted with -OH, halogen, or C 1-5 unsubstituted or substituted C 1-5 straight or branched alkyl.
  • L 3 is absent, -NH-, or -O-;
  • L 4 is N, or C
  • E 1 is -H, or methyl
  • E 2 is absent when L 4 is N, and when L 4 is C ego;
  • E 3 is -H, , or ego
  • E 4 is , , , or Can be.
  • Examples of the compound represented by Formula 3 according to the present invention include the following compounds:
  • the LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is characterized in that the compound represented by the following formula (4), optical isomers thereof, or a pharmaceutically acceptable salt thereof.
  • L 5 is N, or CH
  • G 1 is unsubstituted or substituted containing one or more of -H, -OH, halogen, nitro, nitrile, C 1-5 straight or branched chain alkyl, C 1-5 straight or branched chain alkoxy, or N 5-8 membered heteroarylamino,
  • substituted heteroaryl may be substituted with one or more selected from the group consisting of —OH, halogen and C 1-3 straight or branched alkyl;
  • G 2 is —H, —OH, halogen, nitro, nitrile, C 1-5 straight or branched chain alkyl, C 1-5 straight or branched chain alkoxy, unsubstituted or substituted C 6-10 aryl Or an unsubstituted or substituted 5 to 8 membered heterocycloalkyl comprising at least one heteroatom selected from the group consisting of N and O,
  • substituted aryl and heterocycloalkyl are independently a 6-membered unsubstituted heterocyclo comprising C 1-5 straight or branched alkyl, C 1-5 straight or branched alkoxy, and N and O. May be substituted with one or more substituents selected from the group consisting of straight or branched chain alkyl of alkyl C 1-3 ;
  • G 3 is —H, —OH, halogen, nitro, nitrile, C 1-5 straight or branched alkyl, C 1-5 straight or branched alkoxy, unsubstituted or substituted with one or more nitriles Unsubstituted or substituted 5 to 8 membered heteroaryl containing one or more of 6-10 aryl or N, said substituted heteroaryl is -OH, halogen, nitrile and C 1-3 straight or branched chain alkyl; It may be substituted with one or more substituents selected from the group consisting of.
  • G 1 is -H, or ego
  • G 2 is or ego
  • G 3 is nitrile, or Can be.
  • Examples of the compound represented by Formula 4 according to the present invention include the following compounds:
  • the LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is characterized in that the compound represented by the following formula (5), optical isomers thereof, or pharmaceutically acceptable salts thereof.
  • M 1 is C 6-10 aryl unsubstituted or substituted with one or more halogens
  • M 2 is unsubstituted 6 to 8 membered heteroaryl including one or more heteroatoms selected from the group consisting of N, O and S;
  • M 3 is an unsubstituted or substituted 6 to 8 membered heteroaryl containing one or more heteroatoms selected from the group consisting of N, O and S,
  • substituted heteroaryl is a straight or branched chain alkyl of unsubstituted 6 to 8 membered heterocycloalkyl C 1-5 comprising at least one heteroatom selected from the group consisting of halogen and N, O and S Can be substituted.
  • M 1 is or ego
  • M 3 is or Can be.
  • Examples of the compound represented by Formula 5 according to the present invention include the following compounds:
  • the Leucine Rich Repeat Kinase 2 (LRRK2) inhibitor is characterized in that the compound represented by the following formula (6), optical isomers thereof, or a pharmaceutically acceptable salt thereof.
  • Q 1 is —H, amino, C 1-5 straight or branched chain alkyl, or C 1-5 straight or branched chain alkoxy;
  • Q 2 is —H, unsubstituted C 6-10 cycloalkyl
  • Q 3 is —H, C 1-5 straight or branched alkyl, or C 1-5 straight or branched alkoxy
  • Q 4 is at least one N and is 5 membered heteroaryl substituted with one methyl.
  • Examples of the compound represented by Formula 6 according to the present invention include the following compounds:
  • the LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is characterized in that the compound represented by the following formula (7), optical isomers thereof, or a pharmaceutically acceptable salt thereof.
  • T 1 is —H, C 1-5 straight or branched chain alkyl, or unsubstituted C 3-8 cycloalkylcarbonyl.
  • Examples of the compound represented by Formula 7 according to the present invention include the following compounds:
  • the Leucine Rich Repeat Kinase 2 (LRRK2) inhibitor is characterized in that the compound represented by the following formula (8), optical isomers thereof, or a pharmaceutically acceptable salt thereof.
  • Z 1 is —H, amino, C 1-5 straight or branched chain alkyl, or C 1-5 straight or branched chain alkoxy;
  • Z 2 comprises at least one N, and is a 5-8 membered heteroaryl substituted with one C 1-5 straight or branched alkyl;
  • Z 3 is -H, or C 1-5 straight or branched alkyl, or C 1-5 straight or branched alkoxy.
  • Examples of the compound represented by Formula 8 according to the present invention include the following compounds:
  • Leucine Rich Repeat Kinase 2 (LRRK2) inhibitors according to the present invention can be used in the form of pharmaceutically acceptable salts, and salts are useful as acid addition salts formed by pharmaceutically acceptable free acids.
  • Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes.
  • Non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids and the like, and organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like.
  • Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, eye Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suve Latex, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chloride
  • the acid addition salt according to the present invention can be prepared by a conventional method, for example, a derivative of LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is dissolved in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile and the like Alternatively, the precipitate formed by adding an inorganic acid may be prepared by filtration and drying, or the solvent and excess acid may be distilled under reduced pressure and dried to crystallize in an organic solvent.
  • an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile and the like
  • the precipitate formed by adding an inorganic acid may be prepared by filtration and drying, or the solvent and excess acid may be distilled under reduced pressure and dried to crystallize in an organic solvent.
  • Bases can also be used to make pharmaceutically acceptable metal salts.
  • Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt.
  • Corresponding salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg silver nitrate).
  • the Leucine Rich Repeat Kinase 2 (LRRK2) inhibitor or a pharmaceutically acceptable salt thereof may be administered in a variety of oral and parenteral formulations for clinical administration. It may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. which are commonly used.
  • Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, troches, and the like. , Dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols.
  • Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and the like, optionally with bores such as starch, agar, alginic acid or its sodium salt, and the like. Release or boiling mixtures and / or absorbents, colorants, flavors, and sweeteners.
  • binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and the like, optionally with bores such as starch, agar, alginic acid or its sodium salt, and the like. Release or boiling mixtures and / or absorbents, colorants, flavors, and sweeteners.
  • compositions comprising the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient may be administered parenterally, and parenteral administration is subcutaneous injection, intravenous injection, intramuscular injection or chest By injection method.
  • the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof is mixed with water with a stabilizer or a buffer to prepare a parenteral dosage form in the form of a solution or suspension, which is an ampule or vial unit dosage form It can be prepared by.
  • the compositions may contain sterile and / or preservatives, stabilizers, hydrating or emulsifying accelerators, auxiliaries such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, and conventional methods of mixing, granulating It may be formulated according to the formulation or coating method.
  • the dosage to the human body of a pharmaceutical composition comprising the Leucine Rich Repeat Kinase 2 (LRRK2) inhibitor of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient is determined by the age, weight, sex, dosage form, and state of health of the patient. And the degree of disease, based on an adult patient weighing 70 Kg, typically 0.1-1000 mg / day, preferably 1-500 mg / day, and also at the discretion of the physician or pharmacist Depending on the time interval, it may be administered once a day or divided into several times.
  • LRRK2 Leucine Rich Repeat Kinase 2
  • a health functional food composition for preventing or improving traumatic brain injury or stroke containing an LRRK2 inhibitor as an active ingredient is provided.
  • LRRK2 Leucine Rich Repeat Kinase 2
  • LRRK2 Leucine Rich Repeat Kinase 2
  • a traumatic brain injury or stroke comprising the step of administering a pharmaceutical composition or health functional food composition for the prevention or treatment of traumatic brain injury or stroke containing the LRRK2 inhibitor as an active ingredient to a subject in need thereof. It provides a method of preventing or treating.
  • a pharmaceutical composition or health functional food composition for the prevention or treatment of traumatic brain injury or stroke containing an LRRK2 inhibitor as an active ingredient in the prevention or treatment of traumatic brain injury or stroke to provide.
  • a pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke contains an LRRK2 inhibitor as an active ingredient, inhibits the expression of wild-type LRRK2 overexpressing in traumatic brain injury or stroke, and due to traumatic brain injury or stroke It is effective in protecting or restoring induced damaged neurons, improving neurobehavioral or neuropathy symptoms after traumatic brain injury, and treating, alleviating or ameliorating the brain pathology caused by traumatic brain injury. It can be usefully used for the treatment of traumatic brain injury or stroke.
  • the present invention first identified the overexpression of the LRRK2 wild type in traumatic brain injury or stroke, and also for the first time that the traumatic brain injury or stroke can be treated through the above effects.
  • the experimental results related to this will be described in detail through the following experimental examples.
  • Example 11 (4- (4- (ethylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) -2-fluoro-5-methoxyphenyl) (morpholino) meta Discuss manufacturing
  • Example 12 Compounds can be prepared by the following two methods.
  • Step 3 Preparation of (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino) methanone
  • Table 1 shows the structure of the compound prepared in Example 1-23.
  • Example Chemical structure Example Chemical structure One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 -
  • the preparation method and experimental method of the material (cell, animal model, etc.) used in the experimental example of the present invention are shown below.
  • a controlled cortical impact animal model was used to construct a traumatic brain injury animal model using a mouse.
  • the production method of the animal model is as follows. After anesthetizing the mouse with 2% isoflurane and fixing it to the cerebral stereo fixation device, the skull is incised in a 4 mm circular shape at the anterior-posterior (AP) -2 mm and medial-lateral (ML) 2.0 mm positions relative to the parietal point. By exposing the brain. Afterwards, a 2 mm diameter circular impact tip was used to impact the cerebral cortex at a speed of 2 m / sec, impact duration of 300 msec, depth of 2.5 mm, and launch angle of 25 ° using an Impact One TM Stereotaxic Impactor for CCI (Leica). Was added. After restoring the skull, the mice were recovered on a 37 ⁇ 0.5 °C warm mat. As a control group, mice that had no cortical shock after cranial dissection were used.
  • mice were subjected to general anesthesia with 5% isoflurane in a mixture of 70% N 2 O and 30% O 2 , followed by incision of the skin in the center of the anterior neck and right carotid and external carotid arteries (ECA) Were isolated from surrounding tissues and nerves.
  • ECA carotid and external carotid arteries
  • the upper thyroid artery and laryngeal artery, which are the branches of the external carotid artery, are cauterized with an electrocatheter, and the pterygoid palatal artery, which is the branch of the internal carotid artery, is cauterized. After inserting about 10 mm was fixed with a thread. The skin incision was resealed and then recovered naturally under anesthesia.
  • the drug means an LRRK2 activity inhibitor (Example compound of the present invention) according to the present invention.
  • the LRRK2 activity inhibitor (Example compound of the present invention) in mice, it was dissolved in physiological saline containing 5% dimethyl sulfoxide (DMSO) and 4% Tween 80, and then treated at 50 mg / kg through intraperitoneal injection.
  • the drug (Example compound of the present invention) was treated with the same concentration every day, and the control group was treated with saline containing 5% dimethyl sulfoxide (DMSO) and 4% Tween 80.
  • RNA isolation of total RNA from brain tissue and neurons was performed using Trizol.
  • CDNA was synthesized from 2 mg total RNA using M-MLV reverse transcriptase kit (Promega) and PCR was performed using the primers described below.
  • GAPDH (F: 5'-gtcatcatctccgcc-3 ', R: 5'-gatgcctgcttcaccaccttcttg-3');
  • LRRK2 (F: 5'-agctggttctagtcgctctg-3 ', R: 5'-gtagtctgcacctcagcaac-3');
  • HIF-1a (F: 5'-ctcatcagttgccacttcc-3 ', R: 5'-tcatcttcactgtctagaccac-3');
  • IL-1b (F: 5'-tccaggatgaggacatgagcac-3 ', R: 5'-gaacgtcacacaccagcaggtta-3');
  • IL-6 (F: 5'-ccacttcacaagtcggaggctta-3 ', R: 5'-ccagtttggtagcatccatcatttc-3');
  • TNF-a (F: 5'-cttctgtctactgaacttcgg-3 ', R: 5'-caggcttgtcactcgaatttt-3').
  • Brain tissue and cells were 40 mM Tri-HCl (pH 7.4), 10 mM EDTA, 120 mM NaCl, 0.1% Nonidet P-40, 1 mM glycerophosphate, 1 mM NaF, 1 mM Na 3 VO 4 , 1 mM PMSF, and After dissolution in lysate containing protease inhibitor cocktail, the protein was separated from SDS-PAGE, and the protein was transferred to PVDF membrane. Subsequently, after treatment of the antibody for each experimental use to the membrane, the secondary antibody and the immunoactive complex were visualized on the x-ray film using an enhanced chemiluminescence (ECL) detection system.
  • ECL enhanced chemiluminescence
  • HA-HIF1alpha-pcDNA3 was purchased as Addgene (# 18949), and dominant inhibitory human HIF-1a DNA (in the form of DNA binding and transactivation domain removed) was prepared by PCR.
  • HIF-1a-DN F: 5′-ggggatccgccaccatgcgaagtaaagaatctg3 ′, R: 5′-gggcggccgctcatttgtcaaagaggctact-3 ′.
  • HIF-1a and its control siRNA were synthesized in Bioneer (Daejeon, South Korea). The base sequence for this is as follows.
  • SiHIF-1a-1 (F: 5'-cccauuccucauccgucaau-3 ', R: 5'-uggauagcgauauggucaaug-3').
  • CDNA and siRNA were transformed into primary neurons using Lipofectamine 2000 (Invitrogen).
  • a primer was prepared as follows.
  • lentiviral construct (shControl, shLRRK2-1, shLRRK2-2), 3 ug psPAX2 (Addgene), and 1.5 mg pMD2.G (Addgene) was transfected with Lipofectamine 2000 (Invitrogen) as well. After incubating the cells for 48 hours, the culture medium was collected and the virus was concentrated using Lenti-X concentrator (Clontech). Thereafter, the culture concentrate was treated with neurons with 6 mg / ml polybrene.
  • the LRRK2 promoter region (-2095 to -1-bp, based on the translation start point) was obtained by PCR using the following primers.
  • F 5'-gggtaccgctgtggttggccatgtcag-3 '
  • R 5'-gctcgagggtgcagccgggcggggact-3'.
  • the obtained PCR product was transferred to pCR2.1 using TOPO TA cloning kit (Invitrogen).
  • the reporter plasmid was prepared by transferring to pGL3 basic vector (Promega) through Kpn I and Xho I restriction enzyme digestion.
  • a quick change site-directed mutagenesis kit (Stratagene) was used to generate mutations for four HIF-1a binding predictive regions. Mutation was confirmed by automatic DNA sequencing (Macrogen, South Korea).
  • Chromatin immunoprecipitation analysis was performed using the ChIP assay kit (Millipore). After 24 hours of scratch injury to primary neurons, 100% to 500-bp DNA fragments were obtained through sonication after 1% formaldehyde / phosphate-buffered saline treatment. Chromatin was isolated by immunoprecipitation using 5 mg anti-HIF-1a (Novus) or rabbit IgG (Sigma-Aldrich). The binding to the HIF-1a binding region was confirmed by PCR using the following primers.
  • HRE3 (232-bp), (F: 5'-ggttacttggaagcaaatcc-3 ', R: 5'-gatgggtaagaggtggagg-3').
  • Nerve survival was analyzed using AlamarBlue assay (Thermo Scientific) and LDH assay (Promega). The analysis was performed according to the manufacturer's protocol. LDH assay (Lactate dehydrogenase (LDH)) was used as a marker for cell membrane damage and neuronal cell death. The amount of LDH secreted after the damage to the primary nerve and the culture after the drug and gene expression manipulation was analyzed by CytoTox 96 R Non-Radioactive Cytotoxicity Assay kit (Promage).
  • LDH assay Lacate dehydrogenase (LDH)
  • tissue sections were stained using NeuN antibody first, followed by TUNEL staining using ApopTag Plus In Situ Apoptosis Fluorescein Detection Kit.
  • Cell nuclei are stained using 4 ', 6-diamidino-2-phenylindole (DAPI), and then imaged by fluorescence microscopy (OLYMPUS DX51), and then imaged using DP2-BSW software (version 2.2) and Image Pro Plus 6.0. was analyzed.
  • mice were anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneal injection) and then perfused with PBS and 4% PFA (w / v in PBS). The brains were then treated with 4% PFA and 30% sucrose (w / v in PBS) for 24 hours, respectively. Then a 30 mm coronal incision was produced.
  • Tissue sections were placed on slides coated with poly-D-lysine and treated with 0.1% cresyl violet solution for 20 minutes. Thereafter, dehydration was performed sequentially for 2 minutes in 100, 95, 70, and 50% ethanol, followed by destaining for 2 minutes with xylene. Then, the damage area to the total damaged hemisphere area was measured in terms of percentage.
  • Balance beam test is performed 3 days and 7 days after 1 day before drug treatment.
  • the mouse is placed in the middle of a wooden circular bar (5 mm diameter, 90 cm long, 50 cm high) and then scored according to the criteria below. (0 points: If the mouse does not stay on the bar for more than 30 seconds, 1 point: If the mouse stays on the bar for 30 seconds but cannot move, 2 points: The mouse attempts to turn right or left on the bar) 3 points: if the mouse turns right or left on the bar but cannot walk later, 4 points: if the mouse moves more than one step after turning the bar and shows more than 50% foot slip, 5 points: An additional 1 point for 4 points if less than 50% of the slip of the foot is seen; an additional 1 point for 5 if no slip of the foot is seen.
  • Novel object recognition (NOR) tasks were performed on day 8 after brain injury.
  • the mouse was placed in a black wooden ceilingless box (45 x 45 cm, 25 cm high) and exposed to two old objects for 30 minutes. After 4 hours, the mouse was placed in the box containing the old object for 5 minutes, and then 1 hour later, in the box containing the new object and the old object. Then, the mouse stayed in the new object and the old object was measured for 5 minutes.
  • Discrimination index (time spent exploring the new object-time spent exploring the old object) / (total time spent exploring both objects)
  • CCI cortical shock
  • LRRK2 mRNA Figure 1A
  • protein Figure 1B
  • peaks at 2 and 6 hours after CCI The level of LRRK2 mRNA was gradually down regulated after 24 hours (FIG. 1A), but the level of LRRK2 protein remained until 72 hours after CCI (FIG. 1B).
  • brain sections were subjected to immunostaining, and it was confirmed that the immune activity against LRRK2 was increased around the abduction muscle of the cortex and hippocampal region (FIGS. 1C and 1D).
  • FIG. 5A shows an LRRK2 expression pattern similar to the results of FIGS. 1B and 1C after traumatic brain injury. After traumatic brain injury, the expression of LRRK2 was increased.
  • Figure 1 shows the results of the analysis of increased LRRK2 expression in the mouse brain after traumatic brain injury.
  • Figure 2 shows the results of time-phase analysis of LRRK2 expression in neurons in the mouse brain after traumatic brain injury.
  • Figure 3 shows the results of time-phase analysis of LRRK2 expression in microglia in mouse brain after traumatic brain injury.
  • Figure 4 shows the results of time-phase analysis of LRRK2 expression in astrocytes in the mouse brain after traumatic brain injury.
  • Figure 5 shows the results of LRRK2 expression analysis in traumatic brain injury tissue using additional LRRK2 antibody.
  • Rat local cerebral ischemia animal model (MCAo) and hypoxic primary cortical neuronal cell model were used simultaneously to analyze LRRK2 expression in stroke.
  • the expression of LRRK2 was increased in the cerebral tissue of focal cerebral ischemia, and the expression of HIF-1a expressed in the hypoxic environment was also confirmed (FIG. 6A). Further, the low-oxygen environment (1% O 2) The LRRK2 expression of mRNA and protein in primary cortical neurons was found to be increased (Fig. 6b and 6c) in.
  • Figure 6 shows the results of LRRK2 expression analysis in a stroke animal model.
  • Figure 7 shows the analysis results of LRRK2 expression in neurons in animal models of stroke. Specifically, analysis of LRRK2 expression in neurons in damaged brain tissue after induction of mouse stroke was analyzed by immunofluorescence staining.
  • Traumatic Brain Injury is known for several pathological diseases, including axon damage, excitatory toxicity, oxidative stress, hypoxic damage, and neurological inflammation.
  • TBI Traumatic Brain Injury
  • FIG. 8A LRRK2 mRNA
  • FIG. 8B protein
  • LRRK2 In primary cortical neuronal cell culture, the expression of LRRK2 was significantly increased after scratch injury. From this, it can be seen that the expression of LRRK2 is the same as that of LRRK2 in traumatic brain tissue. In addition, the simultaneous immunostaining using LRRK2 and NeuN antibody was confirmed that the LRRK2 immune activity is increased, especially in neurons (Fig. 8c).
  • Damaged neurons also release various harmful factors, such as glutamic acid and free radicals. Therefore, in order to investigate the effect of the components released from the damaged neurons, after confirming the expression of LRRK2 in primary cortical neurons after treatment with glutamic acid (FIGS. 9A and 9B) and hydrogen peroxide (FIGS. 9C and 9D), LRRK2 expression was the same as scratch damage. This increase was confirmed by immunoblot (FIGS. 9A, 9B, 9C, 9D).
  • the primary cortical neurons were treated with CoCl 2 , a hypoxic inducer, and it was confirmed that the expression of LRRK2 was increased in primary cortical neurons even during hypoxic induction (FIGS. 9E and 9F).
  • Figure 9 shows the results of LRRK2 expression analysis in various neurological damage animal models.
  • hypoxia inducible factor-1 HIF-1
  • LRRK2 expression was increased in major cerebral artery occlusion (MCAo) rat animal models. Traumatic brain injury and ischemia / stroke are known to share common pathological phenomena such as bleeding, hypoxia, nerve inflammation, and excitatory toxicity in the brain.
  • MCAo major cerebral artery occlusion
  • hypoxic inducers glutamic acid and hydrogen peroxide increased LRRK2 and HIF-1 ⁇ expression in cultured primary cortical neurons (FIG. 9).
  • hypoxic response element (HRE) was found in the promoter region of mouse LRRK2 (FIG. 12).
  • HIF-1 ⁇ was induced in control cortical shock and scratch injury traumatic brain injury animal models, respectively. As shown in Figure 10a and 10d, it was confirmed that HIF-1 ⁇ expression is increased in both traumatic brain injury animal and neuronal model.
  • LRRK2 changes in LRRK2 expression following the expression of pcDNA3-HA-HIF-1 ⁇ wild-type or dominant inhibitory mutants in primary cortical neurons, respectively.
  • LRRK2 increase was assessed at the mRNA (FIG. 11a) and protein (FIG. 11b) levels by HIF-1 ⁇ (HIF-1 ⁇ DN), and as a result, in the presence of wild type HIF-1 ⁇ (HIF-1 ⁇ WT) both before and after scratch injury. It was confirmed that LRRK2 expression was increased and LRRK2 expression was decreased in the presence of dominant inhibitory mutation HIF-1 ⁇ .
  • FIG 11 shows the analysis results of LRRK2 expression changes according to HIF-1a expression regulation and activity regulation.
  • FIG. 12C Scratch damage after transfection of the luciferase plasmid was applied to primary cortical neurons and confirmed the regulation of LRRK2 transcription by HIF-1 ⁇ through observation of increased luciferase activity (FIG. 12C). As shown in FIG. 12C, it can be seen that the amount of transcription of LRRK2 is increased by HIF-1 ⁇ .
  • the promoter region includes four putative HIF-1 ⁇ binding sites located between -1766 and -1770 (HRE1), -1418 and -1422 (HRE2), -1355 and 1359 (HRE3), and -970 and -974 .
  • HRE1 putative HIF-1 ⁇ binding sites located between -1766 and -1770
  • HRE2 -1418 and -1422
  • HRE3 -1355 and 1359
  • -970 and -974 To analyze the binding sites of HIF-1 ⁇ to the LRRK2 promoter, site-directed mutations were generated for HRE1, 2, 3 and 4, scratch damage after transfection of each HRE mutant luciferase plasmid and luciferase activity was measured. (FIG. 12B).
  • HRE3 mutation showed a dramatic decrease and the HRE4 mutation showed a moderate decrease in luciferase activity, but the HRE1 and HRE2 mutations were not seen, indicating that HIF-1 ⁇ is bound to HRE3 (FIG. 12F).
  • LRRK2 shRNA and LRRK2 kinase inhibitors were assessed for their functional consequences of increased LRRK2 expression after scratch injury.
  • Neurotoxicity was performed by monitoring alamar blue assay, LHD runoff assay, TUNEL staining and cell death marker protein expression measurement.
  • lentiviruses for transduction of shRNA LRRK2 (pLL3.7-shRNA LRRK2) were generated and infected with primary cortical neurons. Efficient downregulation of LRRK2 expression was confirmed after shRNA LRRK2 transduction (FIG. 13A).
  • shRNA LRRK2 was found to significantly reduce neurotoxicity induced by scratch damage (FIG. 13B, FIG. 13C, FIG. 13D and FIG. 13E). Particularly, referring to FIG. 13D, the neurons are markedly damaged after scratch damage in shControl, but shRNA LRRK2 is visually confirmed that neurons are maintained even after scratch damage, thereby reducing neurotoxicity.
  • Example compound according to the present invention showed neuroprotective efficacy.
  • reduction of the apoptosis markers cleaved caspase-3, cleaved PARP and p53 was also confirmed (FIG. 14, FIG. 15).
  • Figure 13 shows the results of neurotoxicity according to the regulation of LRRK2 expression in a traumatic brain injury neuron model.
  • Figure 14 shows the results of neurotoxicity according to the regulation of LRRK2 activity in a traumatic brain injury neuron model.
  • FIG. 15 shows the results of neurotoxicity according to the regulation of LRRK2 activity in the traumatic brain injury neuron model. Specifically, changes in neurotoxicity following treatment with LRRK2 activity inhibitors Example 10 (FIG. A), Examples 21, and 20 (FIG. B) after scratch damage in primary cortical neurons are analyzed by LDH efflux experiments.
  • Figure 16 shows the results of neurotoxicity analysis by LRRK2 in hypoxia environment.
  • Example 12 of the present invention In order to confirm the inhibition of LRRK2 activity after the treatment of Example 12 of the present invention in the control group and brain injury mouse experimental group, brain tissues in each experimental group were confirmed by using an immunoblot and LRRK2 activity using pS935 LRRK2 antibody. Example compounds of can be treated to effectively reduce the LRRK2 activity (Fig. 17b).
  • FIG. 17 shows the results of an analysis of brain injury protection effect according to inhibition of LRRK2 activity in a traumatic brain injury animal model.
  • LRRK2 Leucine Rich Repeat Kinase 2
  • the compound of Example 12 was used, and after treatment with the compound of Example 12, in order to confirm the recovery of brain tissue damage and neuronal protection, lesion area, nerve Cell loss and cell death marker expression were analyzed.
  • (D) shows the results of performing tissue immunochemistry using neuronal marker (NeuN) antibody.
  • 19 shows the results of analysis of neuronal cell death according to inhibition of LRRK2 activity in a traumatic brain injury animal model.
  • LRRK2 Leucine Rich Repeat Kinase 2
  • the compound of Example 12 was used, and the effect of improving the abnormal brain pathological phenomenon by the compound of Example 12 was performed.
  • astrocytes and microglia were immunostained with their respective markers, GFAP and Iba-1, and astrocytes (Figs. 20a and 20b) and microglia in the HG-11-31-01 experimental group, respectively. It was confirmed that the activity of the cells (FIGS. 20C and 20D) decreased.
  • the pharmaceutical composition for preventing or treating traumatic brain injury or stroke contains an LRRK2 inhibitor as an active ingredient, inhibits the expression of wild-type LRRK2 overexpressing in traumatic brain injury or stroke, and causes traumatic brain injury or stroke.
  • an LRRK2 inhibitor as an active ingredient, inhibits the expression of wild-type LRRK2 overexpressing in traumatic brain injury or stroke, and causes traumatic brain injury or stroke.
  • tablets were prepared by tableting according to a conventional method for producing tablets.
  • the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.
  • an injection was prepared by containing the above components in the contents shown.
  • Vitamin A Acetate 70mg
  • Vitamin E 1.0mg
  • Vitamin B6 0.5mg
  • Vitamin B12 0.2mg
  • Nicotinic Acid 1.7mg
  • composition ratio of the vitamin and mineral mixture is a composition suitable for a relatively healthy food in a preferred embodiment
  • the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method.
  • the granules may be prepared and used for preparing a health food composition according to a conventional method.
  • the resulting solution is filtered and obtained in a sterilized container, sealed sterilization and refrigerated and then stored in a healthy beverage composition Used for preparation.
  • composition ratio is a composition that is relatively suitable for a preferred beverage in a preferred embodiment
  • the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.
  • the pharmaceutical composition for preventing or treating traumatic brain injury or stroke according to the present invention can be usefully used for the treatment of traumatic brain injury or stroke.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Neurosurgery (AREA)
  • Biomedical Technology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Neurology (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne une composition pharmaceutique destinée à prévenir ou traiter une lésion cérébrale traumatique ou un accident vasculaire cérébral. La composition pharmaceutique contient un inhibiteur de LRRK2 en tant qu'ingrédient actif, et peut être utile dans le traitement d'une lésion cérébrale traumatique ou d'un accident vasculaire cérébral compte tenu de sa haute efficacité en termes d'inhibition de l'expression de LRRK2 de type sauvage surexprimé dans une lésion cérébrale traumatique ou un accident vasculaire cérébral, en termes de protection ou de réparation de lésions neuronales induites par une lésion cérébrale traumatique ou un accident vasculaire cérébral, améliorant les résultats comportementaux neurologiques ou la neuropathie survenant après une lésion cérébrale traumatique, et en termes de traitement, d'atténuation ou de réduction de pathologies causées par une lésion cérébrale traumatique.
PCT/KR2019/005969 2018-05-18 2019-05-18 Composition pharmaceutique pour prévenir ou traiter une lésion cérébrale traumatique ou un accident vasculaire cérébral WO2019221566A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020180057084A KR20190131981A (ko) 2018-05-18 2018-05-18 외상성 뇌손상 또는 뇌졸중의 예방 또는 치료용 약학적 조성물
KR10-2018-0057084 2018-05-18

Publications (1)

Publication Number Publication Date
WO2019221566A1 true WO2019221566A1 (fr) 2019-11-21

Family

ID=68540491

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2019/005969 WO2019221566A1 (fr) 2018-05-18 2019-05-18 Composition pharmaceutique pour prévenir ou traiter une lésion cérébrale traumatique ou un accident vasculaire cérébral

Country Status (2)

Country Link
KR (1) KR20190131981A (fr)
WO (1) WO2019221566A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112409337A (zh) * 2020-12-21 2021-02-26 李寒 一种取代的吡嗪衍生物及其用途
CN114010631A (zh) * 2021-11-18 2022-02-08 上海市第六人民医院 含笑内酯及其衍生物在创伤性颅脑损伤治疗中的应用
CN115819405A (zh) * 2022-12-20 2023-03-21 沪渝人工智能研究院 嘧啶氨基吡唑衍生物及其作为富亮氨酸重复激酶2抑制剂的应用
WO2023076404A1 (fr) 2021-10-27 2023-05-04 Aria Pharmaceuticals, Inc. Méthodes de traitement de lupus érythémateux disséminé

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120140643A (ko) * 2009-10-29 2012-12-31 주식회사 오스코텍 키나아제 억제제
KR20130006417A (ko) * 2009-11-13 2013-01-16 주식회사 오스코텍 키나아제 억제제
KR20150027267A (ko) * 2012-06-29 2015-03-11 화이자 인코포레이티드 LRRK2 억제제로서의 4-(치환된-아미노)-7H-피롤로[2,3-d]피리미딘
WO2017218843A1 (fr) * 2016-06-16 2017-12-21 Denali Therapeutics Inc. Pyrimidin-2-ylamino -1h-pyrazols comme inhibiteurs de lrrk2 destinés au traitement de troubles neurodégénératifs
KR20180030201A (ko) * 2015-07-23 2018-03-21 글락소스미스클라인 인털렉츄얼 프로퍼티 디벨로프먼트 리미티드 화합물

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007149789A2 (fr) 2006-06-22 2007-12-27 Applera Corporation Conversion d'amplification spécifique à une cible en séquençage universel

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120140643A (ko) * 2009-10-29 2012-12-31 주식회사 오스코텍 키나아제 억제제
KR20130006417A (ko) * 2009-11-13 2013-01-16 주식회사 오스코텍 키나아제 억제제
KR20150027267A (ko) * 2012-06-29 2015-03-11 화이자 인코포레이티드 LRRK2 억제제로서의 4-(치환된-아미노)-7H-피롤로[2,3-d]피리미딘
KR20180030201A (ko) * 2015-07-23 2018-03-21 글락소스미스클라인 인털렉츄얼 프로퍼티 디벨로프먼트 리미티드 화합물
WO2017218843A1 (fr) * 2016-06-16 2017-12-21 Denali Therapeutics Inc. Pyrimidin-2-ylamino -1h-pyrazols comme inhibiteurs de lrrk2 destinés au traitement de troubles neurodégénératifs

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112409337A (zh) * 2020-12-21 2021-02-26 李寒 一种取代的吡嗪衍生物及其用途
WO2023076404A1 (fr) 2021-10-27 2023-05-04 Aria Pharmaceuticals, Inc. Méthodes de traitement de lupus érythémateux disséminé
CN114010631A (zh) * 2021-11-18 2022-02-08 上海市第六人民医院 含笑内酯及其衍生物在创伤性颅脑损伤治疗中的应用
CN114010631B (zh) * 2021-11-18 2023-08-29 上海市第六人民医院 含笑内酯及其衍生物在创伤性颅脑损伤治疗中的应用
CN115819405A (zh) * 2022-12-20 2023-03-21 沪渝人工智能研究院 嘧啶氨基吡唑衍生物及其作为富亮氨酸重复激酶2抑制剂的应用

Also Published As

Publication number Publication date
KR20190131981A (ko) 2019-11-27

Similar Documents

Publication Publication Date Title
WO2019221566A1 (fr) Composition pharmaceutique pour prévenir ou traiter une lésion cérébrale traumatique ou un accident vasculaire cérébral
WO2018155947A1 (fr) Composition pharmaceutique comprenant un composé apte à traverser la barrière hémato-encéphalique en tant que principe actif pour prévenir ou traiter le cancer du cerveau
AU2021257373B2 (en) Pyridopyrimidinone derivatives and their use as Aryl hydrocarbon receptor modulators
WO2018139903A1 (fr) Composé pyrimidine et son utilisation pharmaceutique
WO2014061970A1 (fr) Bloqueurs de canal sodique, leur procédé de préparation et leur utilisation
WO2020171606A1 (fr) Nouveau composé hétérocyclique et utilisation associée
WO2021145655A1 (fr) Nouveau dérivé de pyrazole
WO2018088749A1 (fr) Nouveau composé de pyrimidine, son procédé de préparation et composition pharmaceutique le contenant en tant que principe actif pour la prévention ou le traitement du cancer et des maladies inflammatoires
WO2020235902A1 (fr) Dérivé de pyrimidine condensé avec un hétérocycle et son utilisation
WO2020149715A1 (fr) Dérivé de pyrrolopyridine et son utilisation dans la prévention et le traitement d'une maladie liée à la protéine kinase
WO2020149723A1 (fr) Dérivé de pyrrolopyrimidine et composition pharmaceutique pour la prévention ou le traitement d'une maladie liée à la protéine kinase le comprenant en tant que principe actif
WO2015160192A1 (fr) Composition pharmaceutique permettant de traiter et de prévenir la leucémie, contenant un dérivé de thiénopyrimidine ou un sel pharmaceutiquement acceptable de celui-ci
WO2018004065A1 (fr) Nouveau dérivé d'aryléthane et composition pharmaceutique contenant ce dérivé comme principe actif
WO2022139304A1 (fr) Nouveau composé dérivé de quinazoline en tant qu'inhibiteur de sos1, et son utilisation
EP3166945A2 (fr) Nouveaux dérivés triazolopyrimidinone ou triazolopyridinone et leur utilisation
WO2020141923A9 (fr) Composé dimère de pyrrolobenzodiazépine présentant une sécurité améliorée et son utilisation
WO2016006975A2 (fr) Nouveaux dérivés imidazotriazinone ou imidazopyrazinone et leur utilisation
WO2016006974A2 (fr) Nouveaux dérivés triazolopyrimidinone ou triazolopyridinone et leur utilisation
WO2018021826A1 (fr) Nouveau dérivé de pyrimidine-2,4-diamine et composition pharmaceutique pour la prévention ou le traitement du cancer contenant celui-ci comme ingrédient actif
WO2018044136A1 (fr) Nouveau composé, son procédé de préparation et composition pharmaceutique le contenant en tant que principe actif pour la prévention ou le traitement de maladies associées à la poly (adp-ribose) polymérase -1 (parp -1)
WO2021125800A1 (fr) Composé destiné à inhiber le facteur d'angiogenèse et son utilisation
WO2021086076A1 (fr) Composition pour la prévention ou l'inhibition de la dégénérescence axonale
WO2021096112A1 (fr) Dérivé de composé pyrrolopyrimidine, pyrrolopyridine et d'indazole, et composition pharmaceutique thérapeutique le contenant
WO2018056621A1 (fr) Nouveau dérivé de pyrimidine imidazolyle, son procédé de préparation et une composition pharmaceutique le contenant en tant que principe actif pour prévenir ou traiter le cancer
WO2020096416A1 (fr) Composé inhibant la liaison yap-tead et composition pharmaceutique pour prévenir ou traiter le cancer contenant ledit composé en tant que principe actif

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19802544

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19802544

Country of ref document: EP

Kind code of ref document: A1