KR20190131981A - Pharmaceutical composition for use in preventing or treating Traumatic Brain Injury or stroke - Google Patents

Abstract

The present invention pertains to a pharmaceutical composition for preventing or treating traumatic brain injury or stroke. The pharmaceutical composition contains an LRRK2 inhibitor as an active component, exhibits the expression of wild-type LRRK2 which is overexpressed in traumatic brain injury or stroke, protects or repairs damage to neurons induced by traumatic brain injury or stroke, alleviates neurological behavioral outcomes or neuropathy occurring after traumatic brain injury, and has an excellent effect of treating, relieving, or alleviating brain pathologies caused by traumatic brain injury, thereby being able to be usefully used for treating traumatic brain injury or stroke.

Description

Pharmaceutical composition for use in preventing or treating Traumatic Brain Injury or stroke}

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.

Inappropriately high protein kinase activity is directly or indirectly associated with many diseases resulting from abnormal cellular action. For example, 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. Can be. Thus, selective inhibition of kinase activity can be a beneficial target of drug development for disease treatment.

LRRK2 (leucin-rich repeat kinase-2) 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. As it has five functionally important domains, it is possible to regulate cell activation through autophosphorylation, protein interaction and enzyme reaction. Function regulation is expected, especially chaperone machinery, cytoskelecton arrangement, protein translational machinery, synaptic vesicle endocytosis, mitogen-activated protein kinase Mitogen-activated protein kinases signaling cascades and ubiquitin / autophage protein degradation pathways are known to be regulated by LRRK2.

In addition, LRRK2 is also implicated in 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 Compounds and compositions effective in regulating LRRK2 activity have 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). Therapeutic effects such as neurodegenerative diseases, CNS disorders, cancer, acute myeloid leukemia and multiple myeloma, and inflammatory diseases.

Traumatic Brain Injury (TBI) 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 indirect neuronal cell death by controlling encephalitis, brain edema and excitatory toxicity.

Most drugs that have been used in patients with traumatic brain injury have been used for the purpose of reducing brain edema, alleviating encephalopathy, and reducing excitatory toxicity. However, these drugs do not directly control neuronal damage and are indirect. There is a limit to neuroprotective efficacy.

Therefore, the current traumatic brain injury treatment is not effective in reducing mortality and the incidence of sequelae, despite 20 years of progress, and currently there is no treatment for targets representing pathological symptoms of traumatic brain injury. In addition, there is a need for the development of new targeted therapies for more effective traumatic brain injury treatment, overcoming existing limitations.

In addition, the stroke is largely divided into ischemic stroke (cerebral infarction) in which cerebrovascular occlusion and hemorrhagic stroke (cerebral hemorrhage) in which cerebrovascular burst. In ischemic stroke, the main cause is thromboembolism of the cerebral artery due to vascular atherosclerosis, or cardiac embolism due to embolism and heart disease.In case of cerebral hemorrhage, primary cerebral hemorrhage due to hypertension and subarachnoid hemorrhage due to arteriovenous malformation or aneurysm are important. to be.

Cerebral infarction caused by clogged cerebral blood vessels is divided into thrombosis and cerebral embolism. Cerebrothrombosis is caused by arteriosclerosis due to hypertension, diabetes, hyperlipidemia, and the walls of arteries are thickened or hardened. 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. Or 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. .

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.

Currently, anti-thrombotic, anticoagulant, antiplatelet, and thrombolytic drugs are used as a treatment for stroke, but currently the only approved drug, anti-thrombotic drug (tPA), is effective only within 4 to 5 hours after the occurrence of cerebral infarction. . However, 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.

WO 2007/149789

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.

In addition, 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.

In order to achieve the above object,

According to one aspect of the invention, there is provided a pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke containing an LRRK2 inhibitor as an active ingredient.

According to another aspect of the invention, there is provided a health functional food composition for the prevention or improvement of traumatic brain injury or stroke containing an LRRK2 inhibitor as an active ingredient.

According to another aspect of the invention, a traumatic brain injury or stroke comprising the step of administering to the subject 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 A method of preventing or treating is provided.

According to another aspect of the invention, the use of 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 according to the present invention 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 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.
(a): Traumatic Brain Injury The expression level of LRRK2 mRNA in mouse brain tissue was analyzed.
(B): Traumatic Brain Injury The expression level of LRRK2 protein in mouse brain tissue was analyzed.
(c): LRRK2 expression following traumatic brain injury was analyzed by histological immunohistochemistry.
(d): Quantification of the number of LRRK2 expressing cells following traumatic brain injury.
(e): LRRK2 expressing cell analysis site at the traumatic brain injury site is indicated.
(f): Simultaneous immunostaining with LRRK2 and neuronal markers in the indicated regions of FIG.
(g): Simultaneous immunostaining with LRRK2 and microglial markers in the indicated regions of FIG.
(h): Simultaneous immunostaining with LRRK2 and astrocyte markers in the indicated regions of FIG.
Figure 2 shows the results of time-phase analysis of LRRK2 expression in neurons in the mouse brain after traumatic brain injury.
(a): The results of immunofluorescence staining are shown.
(b): Quantify the number of LRRK2 expressing cells in neuronal marker expressing cells.
Figure 3 shows the results of time-phase analysis of LRRK2 expression in microglia in mouse brain after traumatic brain injury.
(a): The results of immunofluorescence staining are shown.
(b): Quantification of the number of LRRK2 expressing cells in microglial marker expressing cells.
Figure 4 shows the results of time-phase analysis of LRRK2 expression in astrocytes in the mouse brain after traumatic brain injury.
(a): The results of immunofluorescence staining are shown.
(b): Quantification of the number of LRRK2 expressing cells in astrocyte-expressing cells.
Figure 5 shows the results of LRRK2 expression analysis in traumatic brain injury tissue using additional LRRK2 antibody.
(a): Epitope label picture for LRRK2 antibody used.
(b): Immunoblot performed with additional LRRK2 antibody and quantification thereof.
(c): Tissue immunochemistry results using the additional LRRK2 antibody are shown.
Figure 6 shows the results of LRRK2 expression analysis in a stroke animal model.
(a): Time-phase analysis of LRRK2 expression in mouse stroke animal models.
(B): LRRK2 mRNA expression in neurons after hypoxia treatment.
(c) shows the analysis of LRRK2 protein expression in neurons after hypoxia treatment.
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.
8 shows the results of LRRK2 expression analysis in a traumatic brain injury neuron model.
(a): Analysis of the expression of LRRK2 mRNA after scratch damage to primary cortical neurons.
(b): Analysis of the expression of LRRK2 protein after scratch damage to primary cortical neurons.
(c): The results of LRRK2 expression analysis in neurons by immunofluorescence staining.
Figure 9 shows the results of LRRK2 expression analysis in various neurological damage animal models.
(a): Analysis of LRRK2 protein expression and its quantification after glutamic acid treatment in primary cortical neurons.
(b): The results of nerve cell damage analysis after glutamic acid treatment.
(c): Analysis of LRRK2 protein expression and its quantification after hydrogen peroxide treatment in primary cortical neurons.
(d): The results of neuronal cell damage analysis after hydrogen peroxide treatment.
(e): Analysis of LRRK2 protein expression and its quantification after CoCl ₂ treatment in primary cortical neurons.
(f): Neuronal damage analysis results are shown after CoCl ₂ treatment.
10 shows the results of HIF-1a expression analysis in a traumatic brain injury animal model.
(a): Analysis of HIF-1a protein expression and quantification thereof in mouse brain after traumatic brain injury.
(b): Expression of LRRK and HIF-1a in mouse brain after traumatic brain injury was analyzed by immunofluorescence staining.
(c): Results of quantification of the number of LRRK2 expressing cells in HIF-1a expressing cells.
(d): HIF-1a expression analysis and quantification results after scratch damage of primary cortical neurons.
Figure 11 shows the analysis results of LRRK2 expression changes according to HIF-1a expression regulation and activity regulation.
(a): HIF-1a wild type and dominant inhibitory expression in primary cortical neurons and subsequent LRRK2 mRNA expression analysis in scratch injury.
(B): HIF-1a wild type and dominant inhibitory expression in primary cortical neurons and subsequent LRRK2 protein expression analysis in scratch damage.
(c): The results of siHIF-1a expression and subsequent LRRK2 mRNA expression analysis in primary cortical neurons.
(D): The results of siHIF-1a expression and subsequent LRRK2 protein expression analysis in primary cortical neurons.
(e): The results of analysis of changes in LRRK2 expression after treatment with 2ME2, an inhibitor of HIF-1a expression, and scratch damage in primary cortical neurons.
12 shows the results of LRRK2 transcriptional regulation analysis by HIF-1a.
(a): HIF-1a binding candidate region schematic in the LRRK2 promoter region.
(b): Schematic of mutagenesis for HIF-1a binding candidate regions.
(c): Lucifer laser activity of the LRRK2 promoter region after scratch injury in primary cortical neurons is shown.
(d): The results of luciferase activity analysis of the LRRK2 promoter region due to scratch damage after HIF-1a wild type and dominant inhibitory expression in primary cortical neurons.
(e): The results of luciferase activity analysis on the LRRK2 promoter region due to scratch damage after siRNA HIF-1a expression in primary cortical neurons.
(f): The results of luciferase activity analysis on the LRRK2 promoter region due to scratch damage after expression of the HIF-1a binding region mutation in the LRRK2 promoter region in primary cortical neurons.
(g): Results of chromatin immunoprecipitation for the third HIF-1a binding region mutation in the LRRK2 promoter.
Figure 13 shows the results of neurotoxicity according to the regulation of LRRK2 expression in a traumatic brain injury neuron model.
(a): Analysis of LRRK2 expression according to scratch damage after LRRK2 shRNA expression in primary cortical neurons and quantification thereof are shown.
(b), (c), (d) and (e): Alama blue test results according to scratch damage after LRRK2 shRNA expression in primary cortical neurons (b), LDH efflux test results (c), and TUNEL experiments ( d, e) shows the results of nerve damage analysis.
(f): Analysis of apoptosis marker expression according to scratch damage after LRRK2 shRNA expression in primary cortical neurons.
Figure 14 shows the results of neurotoxicity according to the regulation of LRRK2 activity in a traumatic brain injury neuron model.
(a): Results of LRRK2 phosphorylation reduction analysis according to treatment with LRRK2 activity inhibitors (Example 12, Example 23) after scratch damage in primary cortical neurons.
(b), (c), (d) and (e): Alama blue experiment (b), LDH efflux experiment following treatment with LRRK2 activity inhibitor (Example 12, Example 23) after scratch damage in primary cortical neurons (c) and TUNEL experiments (d, e) show the results of nerve damage analysis.
(f): Results of apoptosis marker expression analysis following treatment with LRRK2 activity inhibitors (Example 12, Example 23) after scratch damage in primary cortical neurons.
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 through LDH efflux experiments.
Figure 16 shows the results of neurotoxicity analysis by LRRK2 in hypoxia environment.
(a): Neurotoxicity was confirmed after hypoxia treatment in primary cortical neurons and neurotoxicity change analysis according to HIF-1a shRNA expression.
(b): Neurotoxicity was confirmed after hypoxia treatment in primary cortical neurons and neurotoxicity change analysis according to LRRK2 shRNA expression.
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.
(a): Treatment of LRRK2 activity inhibitor (Example 12) and behavioral experiments.
(b): Analysis of LRRK2 phosphorylation changes according to the treatment of Example 12 in a traumatic brain injury animal model and results of quantification thereof.
(c) Balanced beam test results after Example 12 treatment in a traumatic brain injury animal model.
(d) and (e): New object recognition test results are shown after Example 12 treatment in a traumatic brain injury animal model.
18 is a result of analysis of brain defect protection efficacy according to the inhibition of LRRK2 activity in a traumatic brain injury animal model.
(a): Picture of brain defect change after Example 12 treatment in a traumatic brain injury animal model.
(b): Analytical image of brain injury site by cracil violet staining.
(c): The result of FIG. (b) was quantified.
(D) shows the results of performing tissue immunochemistry using neuronal marker (NeuN) antibody.
(e): Neuronal cell numbers before and after treatment with inhibitors of LRRK2 activity in the cerebral cortex and hippocampus.
19 shows the results of analysis of neuronal cell death according to inhibition of LRRK2 activity in a traumatic brain injury animal model.
(a): Neuronal cytotoxicity analysis results after treatment with LRRK2 activity inhibitor (Example 12) in traumatic brain injury mouse brain via TUNEL staining.
(b): The result of FIG. (a) was quantified.
(c): Results of analysis of apoptosis marker expression change after treatment with LRRK2 activity inhibitor (Example 12) in traumatic brain injury mouse brain.
20 shows analysis of brain pathological changes according to inhibition of LRRK2 activity in a traumatic brain injury animal model.
(a): In the animal model of traumatic brain injury, after the treatment of Example 12, tissue immunohistochemistry using each marker antibody was performed for analysis of microglial activity.
(b): The result of FIG. (a) was quantified.
(c): Tissue immunohistochemistry using respective marker antibodies for the analysis of astrocyte activity after the treatment of Example 12 in a traumatic brain injury animal model.
(d): The result of FIG. (c) was quantified.
(e): Result of cytokine mRNA expression analysis after Example 12 treatment in a traumatic brain injury animal model.
(f): Results of analysis of cerebrovascular barrier (MMP2, MMP9) and cerebral edema (AQP4) marker expression after Example 12 treatment in a traumatic brain injury animal model.

Hereinafter, the present invention will be described in detail.

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.

In this case, the LRK2 may be wild type.

In one example, 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.

[Formula 1]

In Chemical Formula 1,

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; ,

Wherein the 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,

Wherein said 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,

Wherein the 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,

Wherein said fused 5-8 membered heterocycloalkyl is a 5-8 membered heterocycloalkyl fused with an unsubstituted C _6-10 aryl;

또는

이고,Y is

or

ego,

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,

Wherein said 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,

Here, the substituted heterocycloalkyl may be substituted with one or more substituents selected from the group consisting of 4 to 5 membered unsubstituted heterocycloalkyl including one -OH, = 0, halogen and O.

In addition,

R ¹ is —H, or —NH (CH ₃₎ ;

또는

를 형성하고;R ² is —H, halogen, or —CF ₃ , or is linked with R ³

or

To form;

, 또는

이거나, R² 및 R³는 이들이 결합된 탄소원자와 함께 연결되어

또는

를 형성하고;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 atom to which they are attached

or

To form;

또는

이고,Y is

or

ego,

,

,

또는

이고,R ⁴ , R ⁵ and R ⁶ are independently -H, methoxy, halogen,

,

,

or

ego,

또는

일 수 있다.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:

(1) 5-chloro-N4- (2- (isopropylsulfonyl) phenyl) -N2- (2-methoxy-4- (4- (4-methylpiperazin-1-yl) piperidine-1 -Yl) phenyl) pyrimidine-2,4-diamine;

(2) N- (2- (2- (3-chloro-4-methoxyphenylamino) -5-fluoropyrimidin-4-ylamino) phenyl) methanesulfonamide;

(6) (4- (dimethylamino) piperidin-1-yl) (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) Phenyl) methanone;

(8) N4-ethyl-N2- (1- (3-fluoro-1- (oxetan-3-yl) piperidin-4-yl) -3-methyl-1H-pyrazol-4-yl) -5- (trifluoromethyl) pyrimidine-2,4-diamine;

(10) 2-[(2-methoxy-4-[[4- (4-methylpiperazin-1-yl) piperidin-1-yl] carbonyl] phenyl) amino] -5,11-dimethyl -5,11-dihydro-6H-pyrimido [4,5-b] [1,4] benzodiazepin-6-one;

(11) (4- (4- (ethylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) -2-fluoro-5-methoxyphenyl) (morpholino) methanone;

(12) (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino) methanone;

(14) 1- (5-chloro-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) -1H-pyrazol-1-yl) -2-methyl Propan-2-ol;

(16) N2- (5-chloro-1-((3S, 4R) -3-fluoro-1- (oxetan-3-yl) piperidin-4-yl) -1H-pyrazole-4- Yl) -N4-methyl-5- (trifluoromethyl) pyrimidine-2,4-diamine.

In another embodiment, 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.

[Formula 2]

In Chemical Formula 2,

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.

In addition, L ¹ and L ² are independently —CH ₂ —, —NH—, or —S—;

또는

이고;A ¹ is

or

ego;

이고;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:

(7) (S) -3- (cyclopropylthio) -7-methyl-1- (1H-pyrazol-3-yl) -6,7-dihydrothieno [3,4-c] pyridine-4 (5H) -one;

(9) 3- (cyclopentylthio) -6,6-dimethyl-1- (1H-pyrazol-3-yl) -6,7-dihydrobenzo [c] thiophen-4 (5H) -one.

In another example, 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.

[Formula 3]

In Chemical Formula 3,

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;

이고, 상기 E⁵는 C1-5의 직쇄 또는 분지쇄 알킬이고;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,

Wherein the 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;

Wherein the 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

Herein, 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.

In addition, L ³ is absent, -NH-, or -O-;

L ⁴ is N, or C;

E ¹ is -H, or methyl;

이고;E ² is absent when L ⁴ is N, and when L ⁴ is C

ego;

, 또는

이고;E ³ is -H,

, or

ego;

,

,

, 또는

일 수 있다.E ⁴ is

,

,

, or

Can be.

Examples of the compound represented by Formula 3 according to the present invention include the following compounds:

(3) (R) -3- (4- (cyclohexylamino) -1H-pyrazole [4,3-c] pyridin-3-yl) -1- (3-phenylpiperidin-1-yl) Propane-1-one;

(5) 4- (4- (6-methyl-4- (tetrahydro-2H-pyran-4-yloxy) -1H-pyrazole [4,3-c] pyridin-3-yl) pyridine-2- (1) morpholine;

(18) 3- (6- (4- (2-fluoroethyl) piperazin-1-yl) pyrimidin-4-yl) -5- (1-methylcyclopropoxy) -1H-indazole;

(20) (2S, 6R) -2,6-dimethyl-4- (6- (5- (1-methylcyclopropoxy) -1H-indazol-3-yl) pyrimidin-4-yl) morpholine .

As another example, 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.

[Formula 4]

In Chemical Formula 4,

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,

Wherein said 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,

Wherein said 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.

이고;In addition, G ¹ is -H, or

ego;

또는

이고;G ² is

or

ego;

또는

일 수 있다.G ³ is nitrile,

or

Can be.

Examples of the compound represented by Formula 4 according to the present invention include the following compounds:

(19) 6- (1-methyl-1H-pyrazol-3-ylamino) -4- (3-methyl-4- (morpholinomethyl) phenyl) -1H-pyrrolo [2,3-b] Pyridine-3-carbonitrile;

(21) 3- (4-morpholino-1H-pyrrolo [2,3-b] pyridin-3-yl) benzonitrile;

(22) 1-methyl-4- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) -1H-pyrrole-2-carbonitrile.

In another example, the LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is characterized in that the compound represented by the formula (5), optical isomers thereof, or a pharmaceutically acceptable salt thereof.

[Formula 5]

In Chemical Formula 5,

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; And

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,

Wherein the 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.

또는

이고;In addition, M ¹ is

or

ego;

이고; 및M ² is

ego; And

또는

일 수 있다.M ³ is

or

Can be.

Examples of the compound represented by Formula 5 according to the present invention include the following compounds:

(4) 2- (4-fluorobenzyloxy) -5- (1- (2-morpholinoethyl) -1H-pyrazol-4-yl) -N- (pyridin-3-yl) benzamide;

(23) 2- (benzyloxy) -5- (2-fluoropyridin-4-yl) -N- (pyridin-3-yl) benzamide.

As another example, the LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is characterized in that the compound represented by the following formula (6), optical isomers thereof, or pharmaceutically acceptable salts thereof.

[Formula 6]

In Chemical Formula 6,

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; And

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:

(13) (R) -4- (1-cyclopropylethylamino) -7- (1-methyl-1H-pyrazol-4-yl) cinnoline-3-carboxamide.

As another example, 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.

[Formula 7]

In Chemical Formula 7,

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:

(15) cyclopropyl [10,11,14,15-tetrahydro-1,16-etheno-4,8- (metheno) pyrazole [3,4-j] [1,4,7,9] Oxatriazacyclohexadecine-12 (13H) -yl] methanone.

In another example, the LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is characterized in that the compound represented by the following formula (8), optical isomers thereof, or a pharmaceutically acceptable salt thereof.

[Formula 8]

In Chemical Formula 8,

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; And

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:

(17) (4- (6-amino-5- (1-isopropyl-1H-1,2,3-triazol-4-yl) pyridin-3-yl) -2-methoxyphenyl) (morphopoly No) metanon.

LRRK2 (Leucine Rich Repeat Kinase 2) inhibitors according to the present invention can be used in the form of a pharmaceutically acceptable salt, and salts are useful as acid addition salts formed by pharmaceutically acceptable free acid. 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. It is obtained from non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids and the like, 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. Examples of 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, chlorobene Sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1 Sulfonates, naphthalene-2-sulfonates, mandelate and the like.

Acid addition salts according to the present invention can be prepared by conventional methods, 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.

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).

In the pharmaceutical composition according to the present invention, the Leucine Rich Repeat Kinase 2 (LRRK2) inhibitor or a pharmaceutically acceptable salt thereof may be administered in various dosage forms, orally and parenterally, during 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.

Pharmaceutical compositions comprising the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient can be administered parenterally, and parenteral administration is subcutaneous injection, intravenous injection, intramuscular injection or chest By injection method.

In this case, 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 formulation, and prepared as a solution or suspension, and it is an ampule or vial unit dosage form. It can be prepared by. The composition 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 valuable substances, and conventional methods of mixing, granulating It may be formulated according to the formulation or coating method.

In addition, the dosage of the pharmaceutical composition comprising the LRRK2 (Leucine Rich Repeat Kinase 2) 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 health status 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 may be administered once a day divided into several times.

According to another aspect of the present invention, a health functional food composition for preventing or improving traumatic brain injury or stroke containing an LRRK2 inhibitor as an active ingredient is provided.

Here, since the LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is the same as the LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor of the pharmaceutical composition, a detailed description thereof will be omitted.

According to another aspect of the invention, a traumatic brain injury or stroke comprising the step of administering to the subject 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 It provides a method of preventing or treating.

According to another aspect of the invention, the use of 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 according to the present invention 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 brain pathology caused by traumatic brain injury. It can be usefully used for the treatment of traumatic brain injury or stroke.

In particular, 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.

Hereinafter, the present invention will be described in detail through examples and experimental examples.

However, Examples and Experimental Examples to be described later are merely illustrative of some of the present invention, the present invention is not limited thereto.

Example 1 5-Chloro-N 4-(2- (isopropylsulfonyl) phenyl) -N 2-(2-methoxy-4- (4- (4-methylpiperazin-1-yl) piperidine Preparation of -1-yl) phenyl) pyrimidine-2,4-diamine

The title compound was prepared with reference to WO 2004080980, WO 2005016894, WO 2009102446, WO 2012019132 or WO 2015077602 patent documents.

Example 2 Preparation of N- (2- (2- (3-chloro-4-methoxyphenylamino) -5-fluoropyrimidin-4-ylamino) phenyl) methanesulfonamide

The title compound was prepared with reference to the WO 2009127642 patent document.

Example 3 (R) -3- (4- (cyclohexylamino) -1 H-pyrazole [4,3-c] pyridin-3-yl) -1- (3-phenylpiperidine-1- (1) Preparation of propane-1-one

The title compound was prepared with reference to the WO 2010106333 patent document.

Example 4 2- (4-fluorobenzyloxy) -5- (1- (2-morpholinoethyl) -1H-pyrazol-4-yl) -N- (pyridin-3-yl) benz Preparation of Amides

The title compound was prepared with reference to the WO 2011038572 patent document.

Example 5 4- (4- (6-methyl-4- (tetrahydro-2H-pyran-4-yloxy) -1H-pyrazole [4,3-c] pyridin-3-yl) pyridine- Preparation of 2-yl) morpholine

The title compound was prepared with reference to the WO 2011141756 patent document.

Example 6 (4- (dimethylamino) piperidin-1-yl) (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl Preparation of amino) phenyl) methanone

The title compound was prepared with reference to the WO 2011151360 patent.

Example 7 (S) -3- (cyclopropylthio) -7-methyl-1- (1H-pyrazol-3-yl) -6,7-dihydrothieno [3,4-c] pyridine Preparation of -4 (5H) -one

The title compound was prepared with reference to the WO 2012058193 patent document.

Example 8 N4-ethyl-N2- (1- (3-fluoro-1- (oxetan-3-yl) piperidin-4-yl) -3-methyl-1H-pyrazole-4- I) -5- (trifluoromethyl) pyrimidine-2,4-diamine

The title compound was prepared with reference to the WO 2012062783 patent document.

Example 9 3- (cyclopentylthio) -6,6-dimethyl-1- (1H-pyrazol-3-yl) -6,7-dihydrobenzo [c] thiophene-4 (5H)- Manufacture of

The title compound was prepared with reference to WO 2012118679 Patent Document.

Example 10 2-[(2-methoxy-4-[[4- (4-methylpiperazin-1-yl) piperidin-1-yl] carbonyl] phenyl) amino] -5,11 Preparation of -dimethyl-5,11-dihydro-6H-pyrimido [4,5-b] [1,4] benzodiazepin-6-one

The title compound was prepared with reference to WO 2010080712 or WO 2015176010 patent literature.

Example 11 (4- (4- (ethylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) -2-fluoro-5-methoxyphenyl) (morpholino) meta Discuss manufacturing

The title compound was prepared with reference to the WO 2011151360 patent.

Example 12 Preparation of (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino) methanone

Example 12 Compounds can be prepared by the following two methods.

<Method 1>

The title compound was prepared with reference to the WO 2011151360 patent.

<Method 2>

Step 1: Preparation of 2-chloro-5-iodo-N-methylpyrimidin-4-amine

2,4-Dichloro-5-iodopyrimidine (1.0 equiv) was dissolved in THF and then methylamine (3.5 wt% in EtOH, 1.1 equiv) was added at 0 ° C. The mixture was stirred at 0 ° C. for 2 hours, then the solvent was removed and used in the next step without further purification (yield: 100%).

Step 2: Preparation of 2-chloro-N-methyl-5- (trifluoromethyl) pyrimidin-4-amine

After filling the two-necked round-bottom flask with nitrogen, CuI (5.0 equiv) and KF (5.0 equiv) were added thereto. The mixture was heated to 150 ° C. and then stirred under reduced pressure for 2 hours. After the reaction, the temperature was lowered to room temperature and trimethyl (trifluoromethyl) silane (5.0 equiv) dissolved in DMF / NMP (1: 1) under nitrogen was added using a syringe. After reacting for 30 minutes, 2-chloro-5-iodo-N-methylpyrimidin-4-amine (1.0 equiv) dissolved in DMF / NMP (1: 1) was added using a syringe, and 50 ° C. Reaction was carried out for 18 hours. After the reaction, water was added to the reaction to form a precipitate, and the formed precipitate was removed by filtration. The organics were extracted from the filtrate obtained with EtOAc (x3). The combined organic layers were washed with brine and water remaining with Na ₂ SO ₄ was removed. The water-free mixture was purified using MPCL (EtOAc: Hex) to give the title compound as a yellowish solid (yield: 70%).

Step 3: Preparation of (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino) methanone

2-chloro-N-methyl-5- (trifluoromethyl) pyrimidin-4-amine (1.0 equiv) and (4-amino-3-methoxyphenyl) (morpholino) methanone (1.0 equiv) Was dissolved in dioxane and p-toluensulfonic acid (1.0 equiv) was added at room temperature. After stirring for 16 hours at 100 ℃, cooled to room temperature, the solvent was removed and ice water was added. The organics were extracted with EtOAc (x3). The combined organic layers were washed with brine and water remaining with Na ₂ SO ₄ was removed. The water-free mixture was purified using MPCL (EtOAc: Hex) to give the title compound as a white solid (yield: 70%).

Example 13 Preparation of (R) -4- (1-cyclopropylethylamino) -7- (1-methyl-1H-pyrazol-4-yl) cinnoline-3-carboxamide

The title compound was prepared with reference to WO 2012162254 Patent Document.

Example 14 1- (5-Chloro-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) -1H-pyrazol-1-yl) -2 Preparation of -methylpropan-2-ol

The title compound was prepared with reference to the WO 2012062783 patent document.

Example 15 Cyclopropyl [10,11,14,15-tetrahydro-1,16-etheno-4,8- (metheno) pyrazole [3,4-j] [1,4,7, 9] Preparation of Oxatrizacyclohexadecine-12 (13H) -yl] methanone

The title compound was prepared with reference to WO 2013046029 patent.

Example 16 N2- (5-Chloro-1-((3S, 4R) -3-fluoro-1- (oxetan-3-yl) piperidin-4-yl) -1H-pyrazole- Preparation of 4-yl) -N4-methyl-5- (trifluoromethyl) pyrimidine-2,4-diamine

The title compound was prepared with reference to the Journal of Medicinal Chemistry (2014), 57 (3), 921-936.

Example 17 (4- (6-Amino-5- (1-isopropyl-1H-1,2,3-triazol-4-yl) pyridin-3-yl) -2-methoxyphenyl) ( Preparation of morpholino) methanone

The title compound was prepared with reference to WO 2014106612 document.

Example 18 3- (6- (4- (2-fluoroethyl) piperazin-1-yl) pyrimidin-4-yl) -5- (1-methylcyclopropoxy) -1H-indazole Manufacture

The title compound was prepared with reference to WO 2014137723 document.

Example 19 6- (1-Methyl-1H-pyrazol-3-ylamino) -4- (3-methyl-4- (morpholinomethyl) phenyl) -1H-pyrrolo [2,3- b] Preparation of pyridine-3-carbonitrile

The title compound was prepared with reference to WO 2014170248.

Example 20 (2S, 6R) -2,6-Dimethyl-4- (6- (5- (1-methylcyclopropoxy) -1 H-indazol-3-yl) pyrimidin-4-yl) Preparation of morpholine

The title compound was prepared with reference to WO 2014137723 document.

Example 21 Preparation of 3- (4-morpholino-1H-pyrrolo [2,3-b] pyridin-3-yl) benzonitrile

The title compound was prepared with reference to WO 2015092592 document.

Example 22 Preparation of 1-methyl-4- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) -1H-pyrrole-2-carbonitrile

The title compound was prepared with reference to USA 20140005183 A1 literature.

Example 23 Preparation of 2- (benzyloxy) -5- (2-fluoropyridin-4-yl) -N- (pyridin-3-yl) benzamide

The title compound was prepared with reference to Bioorganic & Medicinal Chemistry Letters (2012), 22, 5625-5629.

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

<Preparation of Experimental Materials> and <Experiment Method>

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.

Primary Cortical Neuronal Cultures

Primary neuronal cultures were cultured using cerebral cortex taken from fetal 15-day-old ICR mice. After separation, the cerebral cortex was treated with 20 U / ml papain and 10 U / ml DNase at 37 ° C for 15 minutes in HBSS culture, followed by wiping the cortical tissue three times with MEM medium containing 10% FBS. . Isolated neurons were dispensed into 50 mg / ml poly-D-lysine-coated cell culture dishes for experimental purposes. After the overnight culture, the experiment was performed after 10-12 days in Neurobasal A culture solution containing 1% Glutamax and 2% B27 supplement.

2. Traumatic Brain Injury Neuron Model

To build a neuronal model of traumatic brain injury, the surface of the culture dish was scraped using pipette tips at regular intervals (3 mm spacing lattice) on the primary neurons cultured. After the wound the culture was replaced and further experiments were performed after a certain time interval. Intact cells were used as controls.

3. Model of Mouse Traumatic Brain Injury

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 ℃ warm mat. As a control group, mice that had no cortical shock after cranial dissection were used.

4. Stroke Animal Model Creation

Mice were subjected to general anesthesia with 5% isoflurane in a mixed gas of 70% N ₂ O and 30% O ₂ , followed by dissection 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. The upper thyroid artery and the 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. 90 minutes after the induction, the anesthetized by the same method, the probe was retreated and reperfused. All surgical procedures were performed under the microscope under the condition of maintaining isoflurane at 2%. The body temperature was used to prevent the body temperature from dropping below 37 ± 0.5 ℃.

5. Drug treatment

In the experiments of the present invention, the drug means an LRRK2 activity inhibitor (Example compound of the present invention) according to the present invention.

In order to treat 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 a concentration of 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.

6. RNA isolation and RT-PCR experiment

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'); And

TNF-a (F: 5'-cttctgtctactgaacttcgg-3 ', R: 5'-caggcttgtcactcgaatttt-3').

7. Immune Blot Analysis

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.

8. Immunofluorescence for Primary Cortical Neuronal Samples

Primary neurons cultured on coverslips were treated with 4% PFA for 20 minutes and then sequentially treated with PBS containing 0.2% Triton-X100 and 2% bovine serum albumin (BSA) for 10 minutes and 30 minutes, respectively. The coverslips were then treated with the appropriate primary and secondary antibodies, respectively. After the cover slip on the slide, the fluorescence image was obtained by lazer scanning confocal microscope (LSM700, Carl Zeiss).

9. Plasmid and siRNA transformation

HA-HIF1alpha-pcDNA3 was purchased as Addgene (# 18949), and dominant inhibitory human HIF-1a DNA (forms with DNA binding and transactivation domains removed) was prepared by PCR.

hHIF-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).

10. shRNA plasmid construction

To prepare a lentiviral vector (pLL3.7) for the mouse LRRK2 shRNA, a primer was prepared as follows.

shLRRK2-1 (F: 5'-tggcttactacttcacttcacgatattttcaagagaatatcgtgaagtagtaagccttttttc-3 'R: 5'-tgcttactacttcacgatatttttcaagagaaaatatcgtgaagtagtaagcttttttc-3');

shLRRK2-2 (F: 5'-taagttgatagtcaggctgaatttcaagagaattcagcctgactatcaacttttttttc-3 'R: 5'-tcgagaaaaaaagttgatagtcaggctgaattctcttgaaattcagcctgactatcaactta-3'); And

scramble (F: 5'-tcgctgagtacttcgaaatgtccttcaagagagacatttcgaagtactcagcgttttttc-3 'R: 5'-tcgagaaaaaacgctgagtacttcgaaatgtctctcttgaagacatttcgaagtactcagcga-3'.

11.Lenti-virus production and inoculation into neurons

In a 100 mm cell culture dish, after 24 hours of incubation of 3-4 x 10 ⁶ 293FT cells, 4.5 mg 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.

12. Mouse and Human LRRK2 Reporter Plasmid Construction and Mutation Construction

From the genome DNA obtained from mouse primary cortical neurons, the LRRK2 promoter region (-2095 ~ -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). Subsequently, 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).

13. Chromatin Immunoprecipitation Assay

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').

14. Neuronal Survival Analysis

Nerve survival was analyzed using AlamarBlue assay (Thermo Scientific) and LDH assay (Promega). 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).

15.TUNEL Dyeing

For simultaneous staining of NeuN and TUNEL, 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.

16. Immunohistochemistry

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.

17. Measuring areas of brain damage

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.

18. Balanced behavior test

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.

19. Novel object recognition test

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.

[Equation]

Discrimination index = (time spent exploring the new object-time spent exploring the old object) / (total time spent exploring both objects)

Experimental Example 1 Controlled Cortical Impact Confirmed Increased Expression of LRRK2 in Peri-Contusional Region of Mouse Brain Injured Animal Model

In brain-injured mice, the following experiment was performed to evaluate the degree of LRRK2 expression, and the results are shown in FIGS. 1, 2, 3, 4, and 5.

To study LRRK2 expression for brain injury, a mouse controlled cortical shock (CCI) animal model was applied, the most common method of generating traumatic brain injury. After CCI, tissues around the injury site of the ipsilateral cortex were collected and the levels of LRRK2 mRNA and protein were analyzed by RT-PCR and immunoblot.

Both LRRK2 mRNA (Figure 1A) and protein (Figure 1B) showed 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). In order to confirm the expression of LRRK2 in the brain, 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).

In addition, neuron, astrocyte, microglia specific markers NeuN (FIG. 1F, 2), Iba-1 (FIG. 1G, 3), GFAP (FIG. 1h, simultaneous staining of the antibody and LRRK2 antibody for Figure 4), and as a result, it was confirmed that LRRK2 expression is mainly increased in neurons.

Furthermore, to eliminate the possibility of nonspecific immune activity, three additional LRRK2 antibodies were recognized that recognize different epitopes (FIG. 5A) (FIGS. 5B and 5C), immunoblot with additional LRRK2 antibodies (FIG. 5B) and immunohistochemistry. FIG. 5C 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.

(a): Traumatic Brain Injury The expression level of LRRK2 mRNA in mouse brain tissue was analyzed.

(B): Traumatic Brain Injury The expression level of LRRK2 protein in mouse brain tissue was analyzed.

(c): LRRK2 expression following traumatic brain injury was analyzed by histological immunohistochemistry.

(d): Quantification of the number of LRRK2 expressing cells following traumatic brain injury.

(e): LRRK2 expressing cell analysis position at traumatic brain injury site.

(f): Simultaneous immunostaining with LRRK2 and neuronal markers in the indicated regions of FIG.

(g): Simultaneous immunostaining with LRRK2 and microglial markers in the indicated regions of FIG.

(h): Simultaneous immunostaining with LRRK2 and astrocyte markers in the indicated regions of FIG.

Figure 2 shows the results of time-phase analysis of LRRK2 expression in neurons in the mouse brain after traumatic brain injury.

(a): The results of immunofluorescence staining are shown.

(b): Quantify the number of LRRK2 expressing cells in neuronal marker expressing cells.

Figure 3 shows the results of time-phase analysis of LRRK2 expression in microglia in mouse brain after traumatic brain injury.

(a): The results of immunofluorescence staining are shown.

(b): Quantification of the number of LRRK2 expressing cells in microglial marker expressing cells.

Figure 4 shows the results of time-phase analysis of LRRK2 expression in astrocytes in the mouse brain after traumatic brain injury.

(a): The results of immunofluorescence staining are shown.

(b): Quantification of the number of LRRK2 expressing cells in astrocyte-expressing cells.

Figure 5 shows the results of LRRK2 expression analysis in traumatic brain injury tissue using additional LRRK2 antibody.

(a): Epitope label picture for LRRK2 antibody used.

(b): Immunoblot performed with additional LRRK2 antibody and quantification thereof.

(c): Tissue immunochemistry results using the additional LRRK2 antibody are shown.

Experimental Example 1 and the results shown in Figures 1 to 5, in brain injury, it can be seen that LRRK2 expression is mainly induced in neurons, which suggests that LRRK2 has a functional role in brain pathology.

Experimental Example 2 Confirmation of Increased LRRK2 Expression in Stroke Animal Models

In the brain injury mice, the following experiment was performed to evaluate the degree of LRRK2 expression, and the results are shown in FIGS. 6 and 7.

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 brain tissue of focal cerebral ischemia, and the expression of HIF-1a expressed in the hypoxic environment was also confirmed (FIG. 6A). In addition, it was confirmed that the expression of LRRK2 mRNA and protein in primary cortical neurons in hypoxic environment (1% O ₂ ) (Fig. 6b and 6c).

In immunofluorescence staining using LRRK2 antibody, it was confirmed that LRRK2 expression was increased during stroke. In addition, in the animal model of stroke, the neuronal marker and LRRK2 were simultaneously stained to express abundantly specific neurons of LRRK2 expression, confirming that they had nerve specificity (FIG. 7).

Figure 6 shows the results of LRRK2 expression analysis in a stroke animal model.

(a): Time-phase analysis of LRRK2 expression in mouse stroke animal models.

(B): LRRK2 mRNA expression in neurons after hypoxia treatment.

(c) shows the analysis of LRRK2 protein expression in neurons after hypoxia treatment.

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.

Experimental Example 2, the results shown in Figures 6 and 7, it was confirmed that the expression of LRRK2 increased in encephalopathy including stroke or hypoxia, suggesting that LRRK2 is associated with brain damage. In addition, the expression of a large number of LRRK2 specifically in neurons, suggesting that LRRK2 has nerve specificity.

Experimental Example 3 Confirmation of Increased LRRK2 Expression in Injured Primary Cortical Neurons

In order to evaluate the expression level of LRRK2 in traumatic brain injury, the following experiment was performed, and the results are shown in FIGS. 8 and 9.

Traumatic Brain Injury (TBI) is known for several pathological diseases, including axon damage, excitatory toxicity, oxidative stress, hypoxic damage, and neurological inflammation. To analyze the pathological cues for LRRK2 expression in TBI, the expression of LRRK2 was analyzed after inducing scratch damage, glucamine acid, hydrogen peroxide and hypoxia in cultured primary cortical neurons.

First, a scratch-damaged animal model was applied to neurons mimicking traumatic brain injury-induced pathology in vivo to investigate expression of LRRK2 after axon injury, and LRRK2 mRNA (FIG. 8A) and protein (FIG. 8B) were primary cortical neurons. It was confirmed that significantly increased after scratch damage (Figs. 8a and 8b).

In primary cortical neuronal cell culture, it was found that the expression of LRRK2 was significantly increased after scratch injury. From this, it was confirmed that the expression of LRRK2 was 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), the expression of LRRK2 is identical to that of scratch damage. This increase was confirmed by immunoblot (FIGS. 9A, 9B, 9C, 9D).

Furthermore, to mimic the hypoxic state, the primary cortical neurons were treated with CoCl ₂ , a hypoxic inducer, and it was confirmed that the expression of LRRK2 was increased in the primary cortical neurons even during hypoxic induction (FIGS. 9E and 9F).

8 shows the results of LRRK2 expression analysis in a traumatic brain injury neuron model.

(a): Analysis of the expression of LRRK2 mRNA after scratch damage to primary cortical neurons.

(b): Analysis of the expression of LRRK2 protein after scratch damage to primary cortical neurons.

(c): The results of LRRK2 expression analysis in neurons by immunofluorescence staining.

Figure 9 shows the results of LRRK2 expression analysis in various neurological damage animal models.

(a): Analysis of LRRK2 protein expression and its quantification after glutamic acid treatment in primary cortical neurons.

(b): The results of nerve cell damage analysis after glutamic acid treatment.

(c): Analysis of LRRK2 protein expression and its quantification after hydrogen peroxide treatment in primary cortical neurons.

(d): The results of neuronal cell damage analysis after hydrogen peroxide treatment.

(e): Analysis of LRRK2 protein expression and its quantification after CoCl ₂ treatment in primary cortical neurons.

(f): Neuronal damage analysis results are shown after CoCl ₂ treatment.

Experimental Example 3, the results shown in Figures 8 and 9, it was confirmed that a variety of pathological causes of neurotoxicity can induce LRRK2 expression, which is common to the upregulation of LRRK2 level, that is, increase in LRRK2 expression It suggests that a molecular mechanism exists.

Experimental Example 4 HIF-1α-dependent Regulation of LRRK2 Expression

In the traumatic brain injury animal model, the following experiment was performed to confirm the molecular mechanism of LRRK2 expression, and the results are shown in FIGS. 10 and 11.

Focusing on transcription factors associated with traumatic brain injury pathology to identify molecular mechanisms of LRRK2 expression in traumatic brain injury animal models, experiments targeting hypoxia inducible factor-1 (HIF-1) based on the following evidence Was performed.

i) 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.

ii) hypoxic inducers, glutamic acid and hydrogen peroxide increased LRRK2 and HIF-1α expression in cultured primary cortical neurons (FIG. 9).

iii) A specific sequence of hypoxic response element (HRE) was found in the promoter region of mouse LRRK2 (FIG. 12).

First, we investigated whether HIF-1α 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-1α expression is increased in both traumatic brain injury animal and neuronal model.

In addition, it was confirmed by immunostaining that LRRK2 and HIF-1α are expressed in the same cell in a traumatic brain injury animal model (FIG. 10B). The number of LRRK2 expressing cells in HIF-1α expressing cells was quantified and shown in FIG. 10C. As shown in Figure 10b and 10c, it was confirmed that the expression of LRRK2 is significantly increased in HIF-1α expressing cells.

To analyze the role of HIF-1α in LRRK2 expression, changes in LRRK2 expression following the expression of pcDNA3-HA-HIF-1α wild-type or dominant inhibitory mutations in primary cortical neurons, respectively. LRRK2 increase was assessed at the mRNA (FIG. 11a) and protein (FIG. 11b) levels by HIF-1α (HIF-1α DN), and as a result, in the presence of wild type HIF-1α (HIF-1α 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-1α.

It was also confirmed that increased expression of LRRK2 after scratch injury reduced HIF-1α dominant inhibitory mutations and siRNA HIF-1α at the mRNA (FIG. 11C) and protein (FIG. 11D) levels.

Furthermore, when treated with 2ME2, a HIF-1α inhibitor, both HIF-1α and LRRK2 expression was confirmed to decrease (FIG. 11E).

10 shows the results of HIF-1a expression analysis in a traumatic brain injury animal model.

(a): Analysis of HIF-1a protein expression and quantification thereof in mouse brain after traumatic brain injury.

(b): Expression of LRRK and HIF-1a in mouse brain after traumatic brain injury was analyzed by immunofluorescence staining.

(c): Results of quantification of the number of LRRK2 expressing cells in HIF-1a expressing cells.

(d): HIF-1a expression analysis and quantification results after scratch damage of primary cortical neurons.

Figure 11 shows the analysis results of LRRK2 expression changes according to HIF-1a expression regulation and activity regulation.

(a): HIF-1a wild type and dominant inhibitory expression in primary cortical neurons and subsequent LRRK2 mRNA expression analysis in scratch injury.

(B): HIF-1a wild type and dominant inhibitory expression in primary cortical neurons and subsequent LRRK2 protein expression analysis in scratch damage.

(c): The results of siHIF-1a expression and subsequent LRRK2 mRNA expression analysis in primary cortical neurons.

(D): The results of siHIF-1a expression and subsequent LRRK2 protein expression analysis in primary cortical neurons.

(e): The results of analysis of changes in LRRK2 expression after treatment with 2ME2, an inhibitor of HIF-1a expression, and scratch damage in primary cortical neurons.

Experimental Example 4, the results shown in Figure 10 and 11, it can be seen that HIF-1α regulates the expression of LRRK2 induced by traumatic brain injury.

Experimental Example 5 Confirmation of Transcription Regulation of LRRK2 by HIF-1α

In order to evaluate the regulation of LRRK2 transcription by HIF-1α, the following experiment was performed, and the results are shown in FIG. 12.

To analyze LRRK2 transcriptional regulation by HIF-1α, the promoter region of the mouse LRRK2 gene between nt-2094 and -1 for the LRRK2 transcription initiation site was subcloned into the luciferase report plasmid, pGL3-Basic (FIG. 12A).

Scratch damage after transfection of the luciferase plasmid was applied to primary cortical neurons and confirmed the regulation of LRRK2 transcription by HIF-1α 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-1α.

 Primary cortical neurons expressed luciferase report plasmids and HIF-1α wild-type or dominant inhibitory expression plasmids for specificity analysis of LRRK2 transcription regulation by HIF-1α, and then increased and decreased luciferase activity after scratch damage, respectively. It was confirmed (Fig. 12d). 12D, it can be seen that the amount of transcription of LRRK2 is increased by wild type or dominant mutant HIF-1α even after scratch damage.

In addition, it was confirmed that LRRK2 expression decreased as HIF-1α expression decreased through HIF-1α expression by siRNA HIF-1α (FIG. 12E).

Furthermore, the promoter region includes four putative HIF-1α binding sites located between -1766 and -1770 (HRE1), -1418 and -1422 (HRE2), -1355 and 1359 (HRE3), and -970 and -974 . To analyze the binding sites of HIF-1α 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).

As a result, the 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-1α is bound to HRE3 (FIG. 12F).

Chromatin immunoprecipitation for the third HIF-1a binding region mutation in the LRRK2 promoter showed that HIF-1α binds directly to HRE3 (FIG. 12G).

12 shows the results of LRRK2 transcriptional regulation analysis by HIF-1a.

(a): HIF-1a binding candidate region schematic in the LRRK2 promoter region.

(b): Schematic of mutagenesis for HIF-1a binding candidate regions.

(c): Lucifer laser activity of the LRRK2 promoter region after scratch injury in primary cortical neurons is shown.

(d): The results of luciferase activity analysis of the LRRK2 promoter region due to scratch damage after HIF-1a wild type and dominant inhibitory expression in primary cortical neurons.

(e): The results of luciferase activity analysis on the LRRK2 promoter region due to scratch damage after siRNA HIF-1a expression in primary cortical neurons.

(f): The results of luciferase activity analysis on the LRRK2 promoter region due to scratch damage after expression of the HIF-1a binding region mutation in the LRRK2 promoter region in primary cortical neurons.

(g): Results of chromatin immunoprecipitation for the third HIF-1a binding region mutation in the LRRK2 promoter.

The results shown in Experimental Example 5 and FIG. 12 suggest that HIF-1α-dependent LRRK 2 transcriptional regulation is possible with respect to LRRK 2 expression increased after scratch injury.

Experimental Example 6 Induction of Neurotoxicity According to Increased Expression of LRRK2 after Scratch Damage

In order to evaluate the functional results of the increased LRRK2 expression after scratch damage, the following experiment was performed, and the results are shown in FIGS. 13-15.

To investigate the functional consequences of increased LRRK2 expression after scratch injury, neurotoxicity was confirmed after inhibition of expression and activity using LRRK2 shRNA and LRRK2 kinase inhibitors, respectively, after scratch injury in primary cortical neurons. Neurotoxicity was performed by monitoring alamar blue assay, LHD runoff assay, TUNEL staining and cell death marker protein expression measurement.

First, 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).

In addition, 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.

Furthermore, the decrease of cell death markers, cleaved caspase-3, cleaved PARP and p53 increased by scratch damage was also confirmed, i.e., neuronal cell death was reduced (FIG. 13F).

In addition, to study the role of LRRK2 kinase activity on neurotoxicity induced by scratch damage, a total of five LRRK2 activity inhibitors of Examples 10, 12, 20, 21 and 23 according to the present invention were applied to damaged primary cortical neurons. It was confirmed that the Example compound according to the present invention showed neuroprotective efficacy. In addition, 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.

(a): Analysis of LRRK2 expression according to scratch damage after LRRK2 shRNA expression in primary cortical neurons and quantification thereof are shown.

(b), (c), (d) and (e): Alama blue test results according to scratch damage after LRRK2 shRNA expression in primary cortical neurons (b), LDH efflux test results (c), and TUNEL experiments ( d, e) shows the results of nerve damage analysis.

(f): Analysis of apoptosis marker expression according to scratch damage after LRRK2 shRNA expression in primary cortical neurons.

Figure 14 shows the results of neurotoxicity according to the regulation of LRRK2 activity in a traumatic brain injury neuron model.

(a): Results of LRRK2 phosphorylation reduction analysis according to treatment with LRRK2 activity inhibitors (Example 12, Example 23) after scratch damage in primary cortical neurons.

(b), (c), (d) and (e): Alama blue experiment (b), LDH efflux experiment following treatment with LRRK2 activity inhibitor (Example 12, Example 23) after scratch damage in primary cortical neurons (c) and TUNEL experiments (d, e) show the results of nerve damage analysis.

(f): Results of apoptosis marker expression analysis following treatment with LRRK2 activity inhibitors (Example 12, Example 23) after scratch damage in primary cortical neurons.

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 through LDH efflux experiments.

Experimental Example 6 and the results shown in FIGS. 13 to 15 suggest that increased LRRK2 expression after scratch injury is involved in neurotoxicity, and is a traumatic agent containing the LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor of the present invention as an active ingredient. It is demonstrated that a pharmaceutical composition for the prevention or treatment of brain injury or stroke has the effect of protecting damaged neurons.

Experimental Example 7 Evaluation of Induction of Neurotoxicity after Increasing Expression of LRRK2 after Hypoxic Injury

In order to evaluate the neurotoxic change through the suppression of increased HIF-1a and LRRK2 expression in the hypoxia neuronal model, the following experiment was performed, the results are shown in FIG.

Neurotoxic changes through inhibition of HIF-1a and LRRK2 expression were measured in hypoxic neuronal models.

After exposing the neurons to the hypoxic environment, the increased neuronal damage decreased the expression of HIF-1a and LRRK2 through HIF-1a shRNA, confirming that cell viability increased rapidly. In addition, when the expression of LRRK2 was reduced through LRRK2 shRNA, the cell survival rate also increased rapidly, and it was confirmed that neuronal damage was reduced (FIGS. 16A and 16B).

Figure 16 shows the results of neurotoxicity analysis by LRRK2 in hypoxia environment.

(a): Neurotoxicity was confirmed after hypoxia treatment in primary cortical neurons and neurotoxicity change analysis according to HIF-1a shRNA expression.

(b): Neurotoxicity was confirmed after hypoxia treatment in primary cortical neurons and neurotoxicity change analysis according to LRRK2 shRNA expression.

Experimental Example 7 and the results shown in Figure 16, it was confirmed that the expression of LRRK2 is increased in the neuronal damage induced in the hypoxic environment, which is the same as the traumatic brain injury, the neuronal damage induced in the hypoxic environment and also increased the LRRK2 expression Suggests a connection. In addition, by reducing the expression of LRRK2, it is possible to drastically increase cell survival rate, it can be seen that it is possible to prevent or recover nerve damage through the regulation of LRRK2 expression.

Experimental Example 8 Controlled Cortical Shock Induced Brain Injury Protective Effect by LRRK2 Inhibitor

 To analyze the improvement of motor and cognitive function in a traumatic brain injury animal model of a pharmaceutical composition for preventing or treating traumatic brain injury or stroke containing an LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor according to the present invention as an active ingredient Experiments were performed as shown in FIG. 17.

Balanced beam and new object recognition (NOR) tests were performed to analyze motor and cognitive improvement in traumatic brain injury animal models by inhibitors of LRRK2 activity (FIG. 17A).

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).

Subsequently, a balance beam and a new object recognition (NOR) test were performed for each experimental group, and it was confirmed that the reduction of motor and cognitive function induced by brain injury was improved by the treatment of Example 12 (FIGS. 17C and 17D). And FIG. 17E).

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.

(a): Treatment of LRRK2 activity inhibitor (Example 12) and behavioral experiments.

(b): Analysis of LRRK2 phosphorylation changes according to the treatment of Example 12 in a traumatic brain injury animal model and results of quantification thereof are shown.

(c) Balanced beam test results after Example 12 treatment in a traumatic brain injury animal model.

(d) and (e): New object recognition test results are shown after Example 12 treatment in a traumatic brain injury animal model.

Experimental Example 8 and the results shown in Figure 17, the pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke containing LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor according to the invention as an active ingredient effectively inhibits LRRK2 activity Thus, it can be seen that, after traumatic brain injury, it improves neurobehavioral outcomes.

Experimental Example 9 Evaluation of Controlled Cortical Shock Induced Brain Tissue Deficiency Recovery by LRRK2 Inhibitors

 In order to determine whether the brain tissue damage recovery and neuronal cell protection of the pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke containing LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor according to the present invention as an active ingredient Was performed, and the results are shown in FIGS. 18 and 19.

As a LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor according to the present invention, 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.

At 8 days after the injury, observation of the brain injury site confirmed a significant reduction in damage in the experimental group treated with Example 12 (FIG. 18A).

After staining the brain tissues through cresyl violet staining of the brain sections, the area of the damaged area was measured, and it was confirmed that the brain damage was reduced in the compound treated in Example 12 (FIG. 18b and 18c).

In order to measure the survival of neurons in the cerebral cortex and hippocampus, immunostaining was performed using NeuN antibody and the number of neurons in each region was measured. Was confirmed (FIGS. 18D and 18E).

In addition, apoptosis was measured at the site of injury through TUNEL staining, and apoptosis marker expression was confirmed. As a result, the cell death and apoptosis marker expression reduction was confirmed in the experimental group treated with Example 12 (FIGS. .

18 is a result of analysis of brain defect protection efficacy according to the inhibition of LRRK2 activity in a traumatic brain injury animal model.

(a): Picture of brain defect change after Example 12 treatment in a traumatic brain injury animal model.

(b): Analytical image of brain injury site by cracil violet staining.

(c): The result of FIG. (b) was quantified.

(d): Tissue immunochemistry using neuronal marker (NeuN) antibody is shown.

(e): Quantification of neuronal cell numbers before and after treatment with LRRK2 activity inhibitors in cerebral cortex and hippocampus.

19 shows the results of analysis of neuronal cell death according to inhibition of LRRK2 activity in a traumatic brain injury animal model.

(a): Neuronal cytotoxicity analysis results after treatment with LRRK2 activity inhibitor (Example 12) in traumatic brain injury mouse brain via TUNEL staining.

(b): The result of FIG. (a) was quantified.

(c): Changes in apoptosis marker expression after analysis of LRRK2 activity inhibitor (Example 12) in traumatic brain injury mouse brain.

Experimental Example 9 and the results shown in FIGS. 18 to 19 suggest that neuropathy induced by traumatic brain injury is associated with neuronal damage caused by LRRK2 activity, and according to the present invention, LRRK2 (Leucine Rich Repeat Kinase 2) The pharmaceutical composition for preventing or treating traumatic brain injury or stroke containing an inhibitor as an active ingredient demonstrates that the neuropathy induced by traumatic brain injury is improved and the nerve cell damage is restored.

Experimental Example 10 Reduction of CCI-induced Brain Pathology by LRRK2 Inhibitors

Traumatic brain injury leads to various pathological phenomena in the brain. Representatives include neuroinflammation, cerebrovascular barrier damage and cerebral edema.

To alleviate or improve the various pathologies in the brain due to such traumatic brain injury of the pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke containing LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor according to the present invention as an active ingredient In order to confirm the effect, the following experiment was performed, and the results are shown in FIG. 20.

As a LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor according to the present invention, 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.

In case of encephalitis, the activity of astrocytes and microglia was 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.

In addition, the reduction of IL-1b, IL-6 and TNF-a was confirmed by analyzing cytokine changes related to encephalitis (FIG. 20E).

Furthermore, in order to confirm the effect of improving the cerebrovascular barrier damage and cerebral edema, the results of performing the immunoblot on the respective markers MMP-2 / 9 and AQP4 were confirmed to decrease the respective markers according to Example 12 (FIG. 20F).

The results shown in Experimental Example 10 and Figure 20, suggests that the encephalopathy caused by traumatic brain injury is associated with the expression of LRRK2. Or demonstrate that a pharmaceutical composition for the prevention or treatment of stroke can treat, alleviate or ameliorate the brain pathology caused by traumatic brain injury.

Thus, 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 traumatic brain injury or stroke. Protects or repairs damaged neurons induced, improves neurobehavioral or neuropathy symptoms after traumatic brain injury, and treats, alleviates or improves brain pathology caused by traumatic brain injury. Therefore, it can be usefully used for the treatment of traumatic brain injury or stroke.

Preparation Example 1 Preparation of Powder

LRRK2 inhibitors according to the invention 2 g

Lactose 1 g

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

Preparation Example 2 Preparation of Tablet

LRRK2 inhibitors according to the invention 100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium Stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

Preparation Example 3 Preparation of Capsule

LRRK2 inhibitors according to the invention 100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium Stearate 2 mg

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

Preparation Example 4 Preparation of Injection

LRRK2 inhibitors according to the invention 100 mg

Mannitol 180 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

Distilled water 2974 mg

According to a conventional method for preparing an injection, an injection was prepared by containing the above components in the contents shown.

Preparation Example 5 Preparation of Health Food

LRRK2 inhibitors according to the invention 500ng

Vitamin mixtures Appropriate

Vitamin A Acetate 70mg

Vitamin E 1.0mg

vitamin 0.13mg

Vitamin B2 0.15mg

Vitamin B6 0.5mg

Vitamin B12 0.2mg

Vitamin c 10mg

Biotin 10mg

Nicotinic acid amide 1.7mg

Folic acid 50 mg

Calcium Pantothenate 0.5mg

Mineral mixture Appropriate

Ferrous sulfate 1.75mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium phosphate monobasic 15 mg

Dicalcium Phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health 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.

Preparation Example 6 Preparation of Health Beverage

LRRK2 inhibitors according to the invention 500ng

Citric acid 1000 mg

oligosaccharide 100 g

Plum concentrate 2 g

Taurine 1 g

Add purified water 900 ml

After mixing the above components in accordance with a conventional healthy beverage preparation method, and stirred and heated at 85 ℃ for about 1 hour, 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.

Although the 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.

Claims (23)

Pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke containing LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor as an active ingredient.
The method of claim 1,
The LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is a compound represented by Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition for preventing or treating traumatic brain injury or stroke:
[Formula 1]

(In Formula 1,
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; ,
Wherein the 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,
Wherein said 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,
Wherein the 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,
Wherein said fused 5-8 membered heterocycloalkyl is a 5-8 membered heterocycloalkyl fused with an unsubstituted C _6-10 aryl;

Y is

or

ego,
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,
Wherein said 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,
Wherein the substituted heterocycloalkyl may be substituted with one or more substituents selected from the group consisting of 4-5 membered unsubstituted heterocycloalkyl comprising one —OH, ═O, halogen and O).
The method of claim 2,
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 atom to which they are attached

or

To form;

Y is

or

ego,
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

A pharmaceutical composition for preventing or treating traumatic brain injury or stroke, characterized in that.
The method of claim 2,
Compound represented by the formula (1) is any one selected from the group of the following pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke:
(1) 5-chloro-N4- (2- (isopropylsulfonyl) phenyl) -N2- (2-methoxy-4- (4- (4-methylpiperazin-1-yl) piperidine-1 -Yl) phenyl) pyrimidine-2,4-diamine;
(2) N- (2- (2- (3-chloro-4-methoxyphenylamino) -5-fluoropyrimidin-4-ylamino) phenyl) methanesulfonamide;
(6) (4- (dimethylamino) piperidin-1-yl) (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) Phenyl) methanone;
(8) N4-ethyl-N2- (1- (3-fluoro-1- (oxetan-3-yl) piperidin-4-yl) -3-methyl-1H-pyrazol-4-yl) -5- (trifluoromethyl) pyrimidine-2,4-diamine;
(10) 2-[(2-methoxy-4-[[4- (4-methylpiperazin-1-yl) piperidin-1-yl] carbonyl] phenyl) amino] -5,11-dimethyl -5,11-dihydro-6H-pyrimido [4,5-b] [1,4] benzodiazepin-6-one;
(11) (4- (4- (ethylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) -2-fluoro-5-methoxyphenyl) (morpholino) methanone;
(12) (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino) methanone;
(14) 1- (5-chloro-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) -1H-pyrazol-1-yl) -2-methyl Propan-2-ol; And
(16) N2- (5-chloro-1-((3S, 4R) -3-fluoro-1- (oxetan-3-yl) piperidin-4-yl) -1H-pyrazole-4- Yl) -N4-methyl-5- (trifluoromethyl) pyrimidine-2,4-diamine.
The method of claim 1,
The LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is a compound represented by Formula 2, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition for preventing or treating traumatic brain injury or stroke:
[Formula 2]

(In Formula 2,
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).
The method of claim 5,
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 is independently -H or methyl pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke.
The method of claim 5,
Compound represented by the formula (2) is any one selected from the group of the following pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke:
(7) (S) -3- (cyclopropylthio) -7-methyl-1- (1H-pyrazol-3-yl) -6,7-dihydrothieno [3,4-c] pyridine-4 (5H) -one; And
(9) 3- (cyclopentylthio) -6,6-dimethyl-1- (1H-pyrazol-3-yl) -6,7-dihydrobenzo [c] thiophen-4 (5H) -one.
The method of claim 1,
The LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is a compound represented by the following formula (3), an optical isomer thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition for preventing or treating traumatic brain injury or stroke:
[Formula 3]

(In Chemical Formula 3,
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,
Wherein the 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;
Wherein the 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
Wherein the substituted 6-membered heterocycloalkyl may be substituted with —OH, halogen, or C _1-5 straight or branched chain alkyl substituted with unsubstituted or one or more halogens).
The method of claim 8,
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

A pharmaceutical composition for preventing or treating traumatic brain injury or stroke, characterized in that.
The method of claim 8,
Compound represented by the formula (3) is any one selected from the group of the following pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke:
(3) (R) -3- (4- (cyclohexylamino) -1H-pyrazole [4,3-c] pyridin-3-yl) -1- (3-phenylpiperidin-1-yl) Propane-1-one;
(5) 4- (4- (6-methyl-4- (tetrahydro-2H-pyran-4-yloxy) -1H-pyrazole [4,3-c] pyridin-3-yl) pyridine-2- (1) morpholine;
(18) 3- (6- (4- (2-fluoroethyl) piperazin-1-yl) pyrimidin-4-yl) -5- (1-methylcyclopropoxy) -1H-indazole; And
(20) (2S, 6R) -2,6-dimethyl-4- (6- (5- (1-methylcyclopropoxy) -1H-indazol-3-yl) pyrimidin-4-yl) morpholine .
The method of claim 1,
The LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is a compound represented by the following formula (4), its optical isomers, or a pharmaceutically acceptable salt thereof pharmaceutical composition for preventing or treating traumatic brain injury or stroke:
[Formula 4]

(In Formula 4,
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,
Wherein said 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,
Wherein said 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).
The method of claim 11,
G ¹ is -H, or

ego;
G ² is

or

ego;
G ³ is nitrile,

or

A pharmaceutical composition for preventing or treating traumatic brain injury or stroke, characterized in that.
The method of claim 11,
Compound represented by the formula (4) is any one selected from the group of the following pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke:
(19) 6- (1-methyl-1H-pyrazol-3-ylamino) -4- (3-methyl-4- (morpholinomethyl) phenyl) -1H-pyrrolo [2,3-b] Pyridine-3-carbonitrile;
(21) 3- (4-morpholino-1H-pyrrolo [2,3-b] pyridin-3-yl) benzonitrile; And
(22) 1-methyl-4- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) -1H-pyrrole-2-carbonitrile.
The method of claim 1,
The LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is a compound represented by Formula 5, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition for preventing or treating traumatic brain injury or stroke:
[Formula 5]

(In Chemical Formula 5,
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; And
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,
Wherein said substituted heteroaryl is a straight or branched chain alkyl of unsubstituted 6 to 8 membered heterocycloalkyl C _1-5 containing one or more heteroatoms selected from the group consisting of halogen and N, O and S May be substituted).
The method of claim 14,
M ¹ is

or

ego;
M ² is

ego; And
M ³ is

or

A pharmaceutical composition for preventing or treating traumatic brain injury or stroke, characterized in that.
The method of claim 14,
The compound represented by Formula 5 is a pharmaceutical composition for preventing or treating traumatic brain injury or stroke, characterized in that the following compounds:
(4) 2- (4-fluorobenzyloxy) -5- (1- (2-morpholinoethyl) -1H-pyrazol-4-yl) -N- (pyridin-3-yl) benzamide; And
(23) 2- (benzyloxy) -5- (2-fluoropyridin-4-yl) -N- (pyridin-3-yl) benzamide.
The method of claim 1,
The LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is a compound represented by Formula 6, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition for preventing or treating traumatic brain injury or stroke:
[Formula 6]

(In Chemical Formula 6,
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; And
Q ⁴ comprises one or more N and is a 5-membered heteroaryl substituted with one methyl).
The method of claim 17,
Compound represented by the formula (6) is a pharmaceutical composition for preventing or treating traumatic brain injury or stroke, characterized in that the following compounds:
(13) (R) -4- (1-cyclopropylethylamino) -7- (1-methyl-1H-pyrazol-4-yl) cinnoline-3-carboxamide.
The method of claim 1,
The LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is a compound represented by the following Formula 7, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition for preventing or treating traumatic brain injury or stroke:
[Formula 7]

(In Chemical Formula 7,
T ¹ is —H, C _1-5 straight or branched chain alkyl, or unsubstituted C _3-8 cycloalkylcarbonyl).
The method of claim 19,
Compound represented by the formula 7 is a pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke, characterized in that the following compounds:
(15) cyclopropyl [10,11,14,15-tetrahydro-1,16-etheno-4,8- (metheno) pyrazole [3,4-j] [1,4,7,9] Oxatriazacyclohexadecine-12 (13H) -yl] methanone.
The method of claim 1,
The LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor is a compound represented by Formula 8, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition for preventing or treating traumatic brain injury or stroke is:
[Formula 8]

(In Formula 8,
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; And
Z ³ is —H, or C _1-5 straight or branched alkyl, or C _1-5 straight or branched alkoxy).
The method of claim 21,
Compound represented by the formula (8) is a pharmaceutical composition for the prevention or treatment of traumatic brain injury or stroke, characterized in that the following compounds:
(17) (4- (6-amino-5- (1-isopropyl-1H-1,2,3-triazol-4-yl) pyridin-3-yl) -2-methoxyphenyl) (morphopoly No) metanon.
Health functional food composition for the prevention or improvement of traumatic brain injury or stroke containing LRRK2 (Leucine Rich Repeat Kinase 2) inhibitor as an active ingredient.

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