KR101990739B1 - Pharmaceutical composition for preventing or treating brain cancer comprising compounds penetrating blood-brain barrier - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating brain cancer comprising a compound capable of passing through a blood-brain barrier as an active component. The pharmaceutical composition for preventing or treating brain cancer according to the present invention efficiently passes through the blood-brain barrier of a brain to effectively inhibit LRRK2 protein, thereby being able to be usefully used for preventing or treating brain cancer.

Description

Pharmaceutical composition for preventing or treating brain cancer comprising compounds penetrating blood-brain barrier}

The present invention relates to a pharmaceutical composition for preventing or treating brain cancer containing a compound capable of passing through the blood brain barrier as an active ingredient.

Protein kinase is an enzyme that catalyzes the reaction of transferring the terminal phosphate group of adenosine triphosphate (ATP) to specific residues of protein, tyrosine, serine, or threonine. Be involved in the signal.

Improperly high protein kinase activity is directly or indirectly associated with a number of diseases resulting from abnormal cellular action. Diseases are caused, for example, by mutations, over-expression or failure of appropriate regulatory mechanisms of kinases involved in inappropriate enzymatic activity, or overproduction or deficiency of factors that participate in signaling upstream or downstream of cytokines, kinases. Can be. Therefore, selective inhibition of kinase activity can be a beneficial target for the development of new drugs for the treatment of diseases.

LRRK2 (leucin-rich repeat kinase-2) protein is a protein belonging to the leucin-rich repeat kinase family, and is composed of a sequence of 2527 amino acids with high interspecies similarity. LRRK2 protein is characterized by having both GTP hydrolase (GTPase) and serine-threonine kinase activity in one protein. The expressed LRRK2 protein has been observed in various organs and tissues including the brain, and is present in the cytoplasm or cell membrane and outer mitochondrial membrane at the cellular level.

In addition, since the LRRK2 protein has five domains that are important in function, it is expected to regulate cellular functions through autophosphorylation and protein interactions and enzymes. In particular, chaperone machinery, cytoskelecton arrangement, protein translational machinery, synaptic vesicle endocytosis, mitogen-activated protein kinase signaling process (mitogen- Activated protein kinases signaling cascades) and ubiquitin/autophage protein degradation pathways are known to be regulated by the LRRK2 protein.

The LRRK2 protein has been reported to be associated with the transfer of mild cognitive impairment associated with Alzheimer's disease, L-Dopa induced dyskinesia, and CNS disorders associated with neuronal progenitor differentiation. In addition, the G2019S mutation of the LRRK2 protein has been reported to increase the incidence of acute myelogenous leukemia (AML) as well as non-skin cancer such as kidney cancer, breast cancer, lung cancer, and prostate cancer. Specifically, the G2019S mutation of the LRRK2 protein increases the catalytic activity of the LRRK2 kinase domain. Furthermore, it has been reported that the LRRK2 protein is also associated with amyotrophic lateral sclerosis, rheumatoid arthritis, and ankylosing spondylitis (International Patent Publication WO 2011/038572).

On the other hand, treatment methods for brain tumors include surgical surgery, radiation therapy, anticancer drug therapy (chemotherapy), and hormone therapy. Among them, it has mainly relied on surgical surgery and radiation therapy, but radiation therapy in particular has a problem with side effects such as neurotoxicity and dementia. Therefore, anticancer drug therapy (chemotherapy) is attracting attention to solve such problems, but drug therapy for the treatment of brain tumors is blocked by a special protective system called'Blood-brain barrier (BBB)' to the brain. There are many drugs that cannot be delivered, and are limited due to cancer resistance to anticancer drugs.

On the other hand, the present inventor first identified through an in vivo experiment that a compound used as an inhibitor of the LRRK2 protein can be effectively used for the prevention and treatment of brain cancer by passing through the blood brain barrier (BBB) efficiently, and completed the present invention. .

International Publication Patent WO 2011/038572

An object of the present invention is to provide a pharmaceutical composition that can be usefully used in the prevention or treatment of brain cancer by efficiently passing through the blood brain barrier.

To achieve the above object,

The present invention provides a pharmaceutical composition for preventing or treating brain cancer containing an LRRK2 protein inhibitor as an active ingredient.

Furthermore, the present invention comprises a first component containing a pharmaceutically treatable amount of an LRRK2 protein inhibitor as an active ingredient; And it provides a pharmaceutical kit for the prevention or treatment of brain cancer comprising a second component containing a pharmaceutically treatable amount of avastin as an active ingredient.

The pharmaceutical composition for preventing or treating brain cancer according to the present invention contains an LRRK2 protein inhibitor as an active ingredient, and has the effect of remarkably suppressing tumor formation by easily passing through the blood brain barrier, which is difficult for general drugs to pass through.

1 is an image showing the results of confirming the expression of LRRK2 protein in cancer tissue obtained from a brain tumor patient by Western Blot.
2 is an image showing a result of confirming the correlation between phosphorylation of LRRK2 protein and malignancy of brain tumor.
3 is an image showing the results of analyzing the nucleic acid sequence encoding the LRRK2 protein expressed in brain tumor cells.
4 is an image confirming the overexpression of the LRRK2 protein (B) and the self-replicating ability of tumor cells (C) in cells infected with a lentiviral vector (A) expressing the LRRK2 protein.
5 and 6 are images showing Western Blot evaluation results confirmed when the compounds of Examples 10, 11, 12, 20, 21 and 22 were treated on NCC01 cells, which are cell lines derived from brain tumor patients.
7 and 8 are images showing Western Blot evaluation results confirmed when the compound of Example 10 was treated with various concentrations of NCC01 cells, a cell line derived from a brain tumor patient.
9 and 10 are images showing Western Blot evaluation results confirmed when the compound of Example 12 was treated with various concentrations of NCC01 cells, a cell line derived from a brain tumor patient.
11 and 12 are images showing Western Blot evaluation results confirmed as time passed by treating the compound of Example 12 at a concentration of 100 nM in NCC01 cells, a cell line derived from a brain tumor patient.
13 to 15 are images showing Western Blot evaluation results confirmed after 1 day of treatment with the compound of Example 12 in NCC01 cells in a concentration range of 50 nM to 2,000 nM.
16 is an image confirming that the compound of Example 12 induces death of brain tumor cells by inhibiting mitochondrial activity.
17 is an image confirming that the compound of Example 12 inhibits mitochondrial activity present in brain tumor stem cells.
18 is an image confirming that mitochondrial activation was suppressed by inhibiting the expression of the compound TRAP1 protein of Example 12. FIG.
19 is an image confirming that the growth of spheres formed from NCC01 (A and B) and 528NS (C and D) cell lines exposed to hypoxic conditions is inhibited by shRNA against the LRRK2 gene.
20 and 21 are images showing cell cycle FACS evaluation results confirmed after 1 day of treatment with the compounds of Examples 11, 12 and 21 at a concentration of 100 nM, respectively, in NCC01 cells.
22 is an image showing the results of observing changes in survival days when the compound of Example 12 was orally administered to a mouse model in which brain tumors were induced.
23 is an image showing the results of measuring changes in tumor size when the compound of Example 12 was orally administered to a mouse model in which brain tumors were induced.
24 is an image confirming that the effect of treating brain tumors is increased when the compound of Example 12 is treated in combination with Avastin.
25 is an image showing Western Blot evaluation results confirmed when a comparative example compound was treated with NCC01 cells, a cell line derived from a brain tumor patient.
26 is an image showing Western Blot evaluation results confirmed when the compounds of Examples 10, 11, 12, 20, and 21 were treated with 448T cells, which are cell lines derived from brain tumor patients.
FIG. 27 is an image showing Western Blot evaluation results confirmed when the compound of Example 12 was treated with various concentrations of 448T cells, a cell line derived from a brain tumor patient.
28 is an image showing the results of observing changes in survival days when the compounds of Examples 12, 20 and 21 were orally administered to a mouse model in which brain tumors were induced.
29 is an image showing Western Blot evaluation results confirmed when the compound of Comparative Example and the compounds of Examples 1, 11 and 12 were treated with 448T cells, a cell line derived from a brain tumor patient.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating brain cancer containing an LRRK2 (Leucine Rich Repeat Kinase 2) protein inhibitor as an active ingredient.

In one example, the LRRK2 protein inhibitor may be a compound represented by Formula 1 below, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

[Formula 1]

In Formula 1,

R ¹ is -H, -OH, halogen, nitro, nitrile, C _1-10 straight or branched alkyl, C _1-10 straight or branched alkoxy, C _1-5 straight or branched alkylamino, or di C _1-5 straight or branched chain alkylamino;

R ² is -H, -OH, halogen, nitro, nitrile, or unsubstituted or substituted C _1-10 straight or branched chain alkyl, or is ^{linked with R 3} to include at least one N To form an unsubstituted, substituted or fused 5- to 8-membered heterocycloalkyl, or an unsubstituted or substituted 5- to 6-membered heteroaryl containing at least one N,

Here, the substituted 5 to 8 ring heterocycloalkyl and the substituted 5 to 6 ring heteroaryl are independently selected from the group consisting of -OH, =O, halogen and C _1-5 linear or branched alkyl 1 5 to 8 ring heterocycloalkyl and 5 to 6 ring heteroaryl substituted with at least one substituent group,

Here, the fused 5 to 8 ring heterocycloalkyl is a 5 to 8 ring heterocycloalkyl in which an unsubstituted C _6-10 aryl is fused;

R ³ is -H, -OH, halogen, nitro, nitrile, C _1-10 straight or branched alkyl, C _1-10 straight or branched alkoxy, C _1-5 straight or branched alkylamino, diC _1-5 straight or branched alkylamino, C _1-5 straight or branched alkylsulfonyl C _6-10 arylamino, or C _1-5 straight or branched alkylsulfonylamino C _6-10 arylamino, or R ^{2 is} connected together to form an unsubstituted, substituted or fused 5- to 8-membered heterocycloalkyl containing at least one N,

Here, the substituted 5- to 8-membered heterocycloalkyl is independently -OH, =O, halogen and C _1-5 straight-chain or branched 5 to 8 each substituted with one or more substituents selected from the group consisting of alkyl Is a ring heterocycloalkyl,

Here, the fused 5 to 8 ring heterocycloalkyl is a 5 to 8 ring heterocycloalkyl in which an unsubstituted C _6-10 aryl is fused;

또는

이고,Y is

or

ego,

The R ⁴ , R ⁵ and R ⁶ are independently -H, -OH, halogen, nitro, nitrile, C _1-10 straight or branched alkyl, C _1-10 straight or branched alkoxy, N and O Unsubstituted or substituted 5 to 8 ring heterocycloalkyl containing one or more heteroatoms selected from the group consisting of, unsubstituted or substituted 5 containing one or more heteroatoms selected from the group consisting of N and O To 8-membered heterocycloalkylcarbonyl,

Here, the substituted 5- to 8-membered heterocycloalkyl includes diC _1-5 straight-chain or branched alkylamino or one or more N, and one or more C _1-5 straight-chain or branched-chain alkyl substituted hexagonal ring heterocycles Cycloalkyl is a substituted 5 to 8 ring heterocycloalkyl,

The R ⁷ , R ⁸ and R ⁹ are independently -H, -OH, halogen, nitro, nitrile, unsubstituted or _{straight or branched C 1-10} alkyl substituted with one or more hydroxy, or N and O It is an unsubstituted or substituted 5 to 8 ring heterocycloalkyl containing at least one hetero atom selected from the group consisting of,

Here, the substituted 5 to 8 ring heterocycloalkyl is substituted with at least one substituent selected from the group consisting of 4 to 5 ring unsubstituted heterocycloalkyl including one -OH, =O, halogen and O It is a 5 to 8 ring heterocycloalkyl.

Preferably,

R ¹ is -H, or -NH(CH ₃ );

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

To form;

, 또는

이거나, R²와 함께 연결되어

또는

를 형성하고;R ³ is -H, -NH(CH ₃ ), -NH(CH ₂ CH ₃ ),

, or

Or is connected with ^{R 2}

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

이다.R ⁷ , R ⁸ and R ⁹ are independently -H, methyl, halogen,

or

to be.

More preferably, the compound represented by Formula 1 may be any one selected from the following compound group:

(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.

In another example, the LRRK2 protein inhibitor may be a compound represented by Formula 2 below, an optical isomer 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 an unsubstituted or substituted C _3-10 cycloalkyl in, wherein the substituted cycloalkyl is a C _3- 10 by -OH, halogen, nitrile, nitro, linear or branched alkyl of _1-5 C And C _3-10 cycloalkyl substituted with one or more substituents selected from the group consisting of _{C 1-5 linear or branched alkoxy;}

A ² is an unsubstituted or substituted 5 to 6 membered heteroaryl containing at least one heteroatom selected from the group consisting of N, O and S, wherein the substituted 5 to 6 membered heteroaryl is- OH, halogen, nitrile, nitro, C _1-5 linear or branched alkyl and C _1-5 linear or branched alkoxy substituted 5 to 6 ring heteroaryl with one or more substituents selected from the group consisting of ;

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.

Preferably, 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 are independently -H or methyl.

More preferably, the compound represented by Formula 2 may be any one selected from the following compound group:

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

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

In another example, the LRRK2 protein inhibitor may be a compound represented by Formula 3 below, 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 alkyl, or C _1-5 straight or branched alkoxy;

이고, 상기 E⁵는 C1-5의 직쇄 또는 측쇄 알킬이고;E ² is ^{absent when L 4} is N, and when L ⁴ is C

And E ⁵ is C1-5 straight or branched chain alkyl;

E ³ is -H, -OH, halogen, nitrile, nitro, straight-chain or branched alkyl of C _1-5, C _1- 5 straight or branched alkoxy, cycloalkyl, unsubstituted or substituted C _6-10 alkyl, Or an unsubstituted or substituted 5 to 8 ring heterocycloalkyl containing at least one heteroatom selected from the group consisting of N and O,

Here, the substituted C _6-10 cycloalkyl and the substituted 5 to 8 ring heterocycloalkyl are independently -OH, halogen, C _1-3 linear or branched alkyl, and C _1-3 linear or branched At least one substituent selected from the group consisting of alkoxy is substituted C _6-10 cycloalkyl and 5 to 8 ring heterocycloalkyl;

E ⁴ is an unsubstituted or substituted 6 to 8 ring heterocycloalkylcarbonyl C _1-5 straight or branched alkyl containing at least one N, an unsubstituted or substituted 6 to 8 ring containing at least one N Heteroaryl,

Here, the substituted 6 to 8 ring heterocycloalkyl and the substituted 6 to 8 ring heteroaryl are independently selected from the group consisting _{of C 6-10 aryl, N and O.} Unsubstituted or substituted 6-membered heterocycloalkyl is substituted 6 to 8-membered heterocycloalkyl,

Here, the substituted 6-membered heterocycloalkyl is -OH, halogen, or a 6-membered heterocycloalkyl substituted with C _1-5 straight or branched alkyl substituted with unsubstituted or one or more halogens.

Preferably, 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

이다.E ⁴ is

,

,

, or

to be.

More preferably, the compound represented by Formula 3 may be any one selected from the following compound group:

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

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

In another example, the LRRK2 protein inhibitor may be a compound represented by Formula 4 below, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

[Formula 4]

[Formula 4]

(In Chemical Formula 4,

α is -CH=, or -N=;

G ¹ is -H, -OH, halogen, nitro, nitrile, C _1-5 straight or branched alkyl, C _1-5 straight or branched alkoxy, or unsubstituted or substituted 5 containing at least one N To 8-membered ring heteroarylamino,

Here, the substituted 5 to 8 ring heteroaryl is -OH, halogen or C _1-3 straight or branched chain alkyl substituted 5 to 8 ring heteroaryl;

G ² is -H, -OH, halogen, nitro, nitrile, C _1-5 straight or branched alkyl, C _1-5 straight or branched alkoxy, unsubstituted or substituted C _6-10 aryl, or It is an unsubstituted or substituted 5 to 8 ring heterocycloalkyl containing at least one heteroatom selected from the group consisting of N and O,

Here, the substituted C _6-10 aryl and the substituted 5 to 8 ring heterocycloalkyl are independently C _1-5 straight or branched chain alkyl, C _1-5 straight or branched alkoxy, and N and O A 6-membered unsubstituted heterocycloalkyl containing C _{1-3 is a substituted C 6-10} aryl and a 5- to 8-membered heterocycloalkyl with one or more substituents selected from the group consisting of linear or branched alkyl;

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 C _{6- 10} aryl, or an unsubstituted or substituted 5 to 10 ring heteroaryl containing one or more heteroatoms selected from the group consisting of N, O and S,

Here, the substituted 5- to 10-membered heteroaryl is a 5- to 10-membered heteroaryl substituted with one or more substituents selected from the group consisting _{of halogen, C 1-5 linear or branched alkyl and nitrile.}

Preferably,

이고;G ¹ is -H, or

ego;

또는

이고;G ² is

or

ego;

, 또는

이다.G ³ is nitrile,

, or

to be.

More preferably, the compound represented by Formula 4 may be any one selected from the following compound group:

(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.

In another example, the LRRK2 protein inhibitor may be a compound represented by Formula 5 below, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

[Formula 5]

In Chemical Formula 5,

M ¹ is an unsubstituted or substituted C _6-10 aryl with one or more halogens;

M ² is an unsubstituted 6 to 8 ring heteroaryl containing one or more heteroatoms selected from the group consisting of N, O and S; And

M ³ is an unsubstituted 6 to 8 ring heterocycloalkyl containing one or more heteroatoms selected from the group consisting of N, O and S.

Preferably, the compound represented by Formula 5 may be the following compound:

(4) 2-(4-Fluorobenzyloxy)-5-(1-(2-morpholinoethyl)-1H-pyrazol-4-yl)-N-(pyridin-3-yl)benzamide.

In another example, the LRRK2 protein inhibitor may be a compound represented by Formula 6 below, an optical isomer thereof, or a pharmaceutically acceptable salt 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 chain alkyl, or C _1-5 straight or branched chain alkoxy; And

Q ⁴ includes one or more N, and is a pentacyclic heteroaryl substituted with one methyl.

Preferably, the compound represented by Formula 6 may be the following compound:

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

In another example, the LRRK2 protein inhibitor may be a compound represented by Formula 7 below, an optical isomer 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.

Preferably, the compound represented by Formula 7 may be the following compound:

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

In another example, the LRRK2 protein inhibitor may be a compound represented by Formula 8 below, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

[Formula 8]

In Chemical Formula 8,

Z ¹ is -H, amino, C _1-5 straight or branched alkyl, or C _1-5 straight or branched alkoxy;

Z ² includes one or more N, and is a 5- to 8-membered heteroaryl substituted with _{one C 1-5 linear or branched alkyl;} And

Z ³ is -H, or C _1-5 linear or branched alkyl, or C _1-5 linear or branched alkoxy.

Preferably, the compound represented by Formula 8 may be the following compound:

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

The pharmaceutical composition according to the present invention can be used to treat brain cancer, specifically, brain cancer in which LRRK2 protein is expressed or activated. The activity of the LRRK2 protein may be exhibited by phosphorylation of the LRRK2 protein.

The LRRK2 protein inhibitor according to the present invention may be used in the form of a pharmaceutically acceptable salt, and in this case, an acid addition salt formed by a pharmaceutically acceptable free acid is useful.

Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It can be obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc., acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, and the like. Types of these pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , Sebacate, fumarate, maleate, butin-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitro benzoate, hydroxybenzoate, me Toxibenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol Rate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving a derivative of an LRRK2 protein inhibitor in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., and adding an organic acid or an inorganic acid. The precipitate can be prepared by filtration and drying, or by distilling a solvent and an excess of acid under reduced pressure and drying to crystallize under an organic solvent.

In addition, the LRRK2 protein inhibitor of the present invention can be prepared as a pharmaceutically acceptable metal salt using a base. Specifically, the alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved 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. In addition, the corresponding salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate).

In the pharmaceutical composition according to the present invention, the LRRK2 protein inhibitor, an optical isomer thereof, or a pharmaceutically acceptable salt thereof can be administered in various oral or parenteral dosage forms at the time of clinical administration. It can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Formulations for oral administration include, for example, tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, and troches. , Dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg, silica, talc, stearic acid and magnesium or calcium salts thereof and/or polyethylene glycol). Tablets may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, and in some cases disintegrants such as starch, agar, alginic acid or sodium salt thereof, or It may contain boiling mixtures, absorbents, colorants, flavoring agents, and sweetening agents.

The pharmaceutical composition containing the LRRK2 protein inhibitor according to the present invention, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, and parenteral administration may be administered by subcutaneous injection, intravenous injection, intramuscular injection, or It can be administered via intrathoracic injection.

At this time, in order to formulate a formulation for parenteral administration, the LRRK2 protein inhibitor, its optical isomer, or a pharmaceutically acceptable salt thereof is mixed in water together with a stabilizer or buffer to prepare a solution or suspension, and it is prepared in an ampoule or vial unit. It can be prepared in a dosage form. The composition may be sterilized and additionally contain adjuvants such as preservatives, stabilizers, hydrating agents or emulsifying accelerators, salts and buffers for controlling osmotic pressure, and other therapeutically useful substances, and mixing, granulating or It can be formulated according to the coating method.

In addition, the dosage to the human body of the pharmaceutical composition containing the LRRK2 protein inhibitor of the present invention, its optical isomer, or a pharmaceutically acceptable salt thereof as an active ingredient is the patient's age, weight, sex, dosage form, health status, and It may vary depending on the degree of disease, and based on an adult patient weighing 70 Kg, it is generally 0.1-1000 mg/day, preferably 1-500 mg/day, and according to the judgment of a doctor or pharmacist. It may be dividedly administered once or several times a day at regular time intervals.

Furthermore, the present invention comprises a first component containing a pharmaceutically treatable amount of an LRRK2 protein inhibitor as an active ingredient; And it provides a pharmaceutical kit for the prevention or treatment of brain cancer comprising a second component containing a pharmaceutically treatable amount of avastin as an active ingredient.

In the pharmaceutical kit of the present invention, for the prevention or treatment of brain cancer, the first component and the second component may be administered sequentially or simultaneously. When the first component and the second component are sequentially administered, the second component may be administered before and after 30 minutes to 180 minutes, 60 minutes to 120 minutes, and 80 minutes to 100 minutes of the administration of the first component. According to an embodiment of the present invention, the second component may be administered 90 minutes after the administration of the first component.

When the first component and the second component included in the pharmaceutical kit are administered in combination, anticancer activity against brain cancer can be increased. The administration may be administered to a mammal, specifically to a human.

The present invention was the first to find out that the LRRK2 protein inhibitor has the effect of remarkably suppressing tumor formation by easily passing through the blood brain gate, which is difficult for general drugs to pass through, and the experimental results related to this will be described in detail through the experimental examples to be described later. do.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples to be described later.

However, the examples and experimental examples to be described later are merely illustrative of the present invention, and the present invention is not limited thereto.

<Example 1> 5-Chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(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)-1H-pyrazole[4,3-c]pyridin-3-yl)-1-(3-phenylpiperidin-1- 1) Preparation of propan-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 amide

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- 2-day) Preparation of 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 document.

<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- Preparation of one)-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 on

The title compound was prepared with reference to the 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 Paddy manufacturing

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

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

12-1. Preparation of (3-methoxy-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)phenyl)(morpholino)methanone-1

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

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

Step 1. After dissolving 2,4-dichloro-5-iodopyrimidine (1.0 equivalent) in THF, methylamine (3.5 wt% in EtOH, 1.1 equivalent) was added at 0°C. After the mixture was stirred at 0° C. for 2 hours, the solvent was removed and used in the next step without further purification (yield: 100%).

Step 2. After filling the two-necked round-bottom flask with nitrogen gas, CuI (5.0 equivalent) and KF (5.0 equivalent) were added. The mixture was heated to 150° C. and then stirred for 2 hours under reduced pressure. After the reaction, the temperature was lowered to room temperature, and trimethyl(trifluoromethyl)silane (5.0 equivalents) dissolved in DMF/NMP (1:1) was added using a syringe under nitrogen. After reacting for 30 minutes, 2-chloro-5-iodo-N-methylpyrimidin-4-amine (1.0 equivalent) dissolved in DMF/NMP (1:1) was added using a syringe, and 50°C It was reacted at for 18 hours. After the reaction, water was added to the reactant to form a precipitate, and the formed precipitate was removed by filtration. Organics were extracted from the obtained filtrate using EtOAc (x3). The collected organic layer was washed with brine, and water remaining with _{Na 2} SO _{4 was removed.} The mixture from which water was removed was purified using MPCL (EtOAc:Hex) to obtain the target compound as a yellowish solid (yield: 70%).

Step 3. 2-Chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (1.0 equivalent) and (4-amino-3-methoxyphenyl)(morpholino)methanone ( 1.0 equivalent) was dissolved in dioxane, and p-toluensulfonic acid (1.0 equivalent) was added at room temperature. The mixture was stirred at 100° C. for 16 hours, cooled to room temperature, and then the solvent was removed and ice water was added. The organics were extracted with EtOAc (x3). The collected organic layer was washed with brine, and water remaining with _{Na 2} SO _{4 was removed.} The mixture from which water was removed was purified using MPCL (EtOAc:Hex) to obtain the target 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 the 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 oxatriazacyclohexadecin-12(13H)-yl]methanone

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

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

The title compound was prepared with reference to 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.

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

The title compound was prepared with reference to WO 2014137723.

*<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)-1H-indazol-3-yl)pyrimidin-4-yl) Preparation of morpholine

The title compound was prepared with reference to WO 2014137723.

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

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

<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.

Table 1 below shows the structure of the compound prepared in Example 1-22.

<Comparative Example> 2-methyl-2-[3-methyl-4-[[4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl]amino]pyrazol-1-yl] Propane Nitrile

The title compound was prepared with reference to WO 2012062783.

<Experimental Example 1> Confirmation of the expression of LRRK2 protein in cancer stem cells of brain tumor patients

In order to confirm the possibility of the LRRK2 protein as a target protein for the treatment of brain tumors, Western Blot was performed using cancer tissues obtained from 19 brain tumor patients to confirm the expression of the LRRK2 protein.

Specifically, after obtaining cancer tissue from a brain tumor patient, Western Blot was performed to confirm the expression of the LRRK2 protein, and the results are shown in FIG. 1.

As shown in FIG. 1, it was confirmed that LRRK2 protein was expressed in cancer tissues obtained from brain tumor patients, which increased as the degree of malignancy of the brain tumor increased. Therefore, from the above, it was found that the LRRK2 protein can be used as a target for the treatment of brain tumors.

<Experimental Example 2> Confirmation of changes in prognosis of brain tumor patients according to phosphorylation of LRRK2 protein

LRRK2 protein exhibits its activity by being phosphorylated. Accordingly, changes in the prognosis of brain tumor patients due to phosphorylation of LRRK2 protein in brain tumor cells were confirmed.

Specifically, tissue samples were obtained from about 200 brain tumor stage 3 and stage 4 patients, and the expression of phosphorylated LRRK2 protein in the obtained tissue samples was confirmed through immunostaining.

As a result, as shown in FIG. 2, phosphorylation of the LRRK2 protein increased as the malignancy of the brain tumor increased, that is, as the brain tumor progressed from stage 3 to stage 4. Therefore, it was found from the above that the phosphorylated LRRK2 protein can be used as a target protein for the treatment of brain tumors with high malignancy.

<Experimental Example 3> Confirmation of correlation between mutation of LRRK2 protein and brain tumor

According to a conventional study, various carcinomas can occur due to mutations in the LRRK2 protein, and it has been reported that Parkinson's disease, which is known to be related to the LRRK2 protein, is also caused by mutations in the LRRK2 protein. Accordingly, it was confirmed whether the LRRK2 protein, which was confirmed to be overexpressed in brain tumor cells in the above experimental example, was a mutation thereof. Specifically, sequencing was performed using brain tumor cells obtained from 5 patients, and at this time, primers as shown in Table 2 were used.

As a result, as shown in FIG. 3, the nucleic acid sequence encoding the LRRK2 protein expressed in brain tumor cells did not have specific mutations such as those found mainly in Parkinson's disease, except for the conventionally known single nucleotide polymorphism (SNP). . Therefore, it was confirmed from the above that, unlike Parkinson's disease and other types of cancer, in the case of brain tumors, cancer occurs due to overexpression of the normal LRRK2 protein.

<Experimental Example 4> Confirmation of tumor formation ability by overexpression of LRRK2 protein

As confirmed in Experimental Example 3, in order to determine whether a brain tumor is caused by overexpression of the LRRK2 protein, a lentiviral vector containing a polynucleotide encoding the LRRK2 protein (pLenti CMV GFP DEST (736-1), Addgene #19732) was prepared and then infected with 528NS, 464T (Samsung Seoul Hospital, Korea) and ex-vivo cell lines, respectively, to confirm the formation of tumors. At this time, a lentiviral vector not expressing the LRRK2 protein was infected as a control.

As a result, as shown in FIG. 4, the size of GSC (glioma stem cell) spheres was increased in 528NS, 464T, or ex-vivo cell lines infected with lentivirus expressing LRRK2 protein compared to the control group. From this, it was found that tumors were formed by overexpression of the LRRK2 protein.

<Experimental Example 5> Evaluation of phosphorylation inhibitory activity of the compound against LRRK2 protein (in vitro)-(1)

To evaluate drug efficacy in vitro, a Western Blot experiment was performed using NCC01 cells, a cell line derived from a brain tumor patient.

More specifically, ^{7.5x10 5} to ^{1.0x10 6} NCC01 cells were treated with the compounds prepared in Examples 10, 11, 12, 20, 21 and 22 at a concentration of 100 or 1,000 nM, respectively, and samples after 1 day elapsed. Was collected and evaluated for phosphorylation inhibitory activity on LRRK2 protein through Western Blot. The group that was not treated with the compound of the example was used as the untreated group (Vehicle). The results are shown in FIGS. 5 and 6.

5 and 6, the compounds of Examples 10, 11, 12, 20, 21 and 22 according to the present invention are phosphorylated against LRRK2 protein in NCC01 cells, which are cell lines derived from brain tumor patients, compared to the untreated group (Vehicle). Was significantly suppressed.

Therefore, it was confirmed that the compound according to the present invention inhibits phosphorylation of the LRRK2 protein expressed in a cell line derived from a brain tumor patient.

<Experimental Example 6> Evaluation of phosphorylation inhibitory activity against LRRK2 protein according to the concentration of the compound (in vitro)-(1)

The experiment was performed similarly to the evaluation of the phosphorylation inhibitory activity for the LRRK2 protein of <Experimental Example 1>, but the experiment was performed to confirm whether the compound of the Example according to the present invention has excellent efficacy in a specific concentration range.

More specifically, ^{7.5x10 5} to ^{1.0x10 6} NCC01 cells were treated with the compounds prepared in Examples 10 and 12 at concentrations of 0.5, 1, 5, 10, 50 and 100 nM, and samples after 1 day elapsed. Was collected and evaluated for phosphorylation inhibitory activity on LRRK2 protein through Western Blot. The group that was not treated with the compound of the example was used as the untreated group (Vehicle). The results are shown in Figs. 7 to 10.

As shown in Figures 7 to 10, the compound of Example 10 was detected phosphorylated LRRK2 protein even at a concentration of 100 nM, but the compound of Example 12 most inhibited phosphorylation of the LRRK2 protein at concentrations of 50 and 100 nM. .

From this, it was confirmed that the compound according to the present invention remarkably inhibited phosphorylation of the LRRK2 protein even at a low concentration of nM.

<Experimental Example 7> Evaluation of phosphorylation inhibitory activity for LRRK2 protein according to the treatment time of the compound (in vitro)

The experiment was performed similarly to the evaluation of the phosphorylation inhibitory activity on the LRRK2 protein of <Experimental Example 1>, but the experiment was performed to confirm how the activity changes over time after treatment of the Example compound according to the present invention. .

More specifically, ^{7.5x10 5} to ^{1.0x10 6} NCC01 cells were treated with the compound prepared in Example 12 at a concentration of 100 nM, and after 1, 4, and 8 days elapsed, samples were collected and Western Blot was performed. Through the LRRK2 protein was evaluated for inhibitory activity of phosphorylation. The group not treated with the Example compound was used as the untreated group (Vehicle). The results are shown in Figs. 11 and 12.

As shown in FIGS. 11 and 12, the phosphorylation level of the LRRK2 protein was rapidly decreased from 1 day after the compound of Example 12 was treated on NCC01 cells, a cell line derived from a brain tumor patient, and this continued even after a period of time.

From this, it was confirmed that the compounds of Examples according to the present invention inhibit phosphorylation of LRRK2 protein continuously for a long period of time.

<Experimental Example 8> Evaluation of changes in signaling pathways in cells due to inhibition of phosphorylation of LRRK2 protein (in vitro)

An experiment was performed to confirm the effect of the compounds of the examples according to the present invention on the intracellular signal transduction pathway for cancer stem cell growth.

More specifically, ^{1.0×10 6} NCC01 cells were treated with the compound prepared in Example 12 at concentrations of 50, 100, 500, 1,000 and 2,000 nM, and after 1 day, samples were collected and used in Western Blot. Expression of NESTIN protein, a cancer stem cell marker involved in the intracellular signal transduction pathway, was confirmed. The group that was not treated with the compound of the example was used as the untreated group (Vehicle). The results are shown in Figs. 13 to 15.

13 to 15, phosphorylation of the LRRK2 protein was inhibited in NCC01 cells by the compound of Example 12, which decreased depending on the concentration of the treated compound.

From this, the compound of Example 12 according to the present invention inhibits signal transduction by the LRRK2 protein in cells derived from brain tumor patients, and thereby inhibits cell growth by reducing the expression of NESTIN protein involved in tumor stem cell growth. Was confirmed.

<Experimental Example 9> Confirmation of mitochondrial function change through inhibition of LRRK2 protein activity

The LRRK2 protein is located in the outer membrane of mitochondria, and it has been reported to be involved in mitochondrial fragmentation and regulation of membrane potential. Therefore, an experiment was conducted to determine how the compound that inhibits the LRRK2 protein prepared in the above example affects mitochondrial function.

Specifically, the NCC01 cell line was treated with 1 μM of the compound of Example 12 and 1 day later, TMRE (tetramethylrhodamine ethylester) dye stained on the activated mitochondria was added to confirm the change in mitochondrial function. At this time, the group not treated with the compound of the example was used as the untreated group (control). The stained results were photographed under a microscope and shown in FIG. 16.

As shown in FIG. 16, the cells of the control group maintained the mitochondrial activity present in the cells, so that the TMRE dye was clearly colored. However, in the cells treated with the compound of Example 12, it was confirmed that not only the degree of color development of the TMRE dye was reduced, but also the fragmentation of the nuclei present in the cells occurred. From this, it was found that the compound of Example 12 according to the present invention had an effect on the death of brain tumor cells by inhibiting mitochondrial activity.

<Experimental Example 10> Evaluation of phosphorylation inhibitory activity against LRRK2 protein in brain tumor stem cell lines

An experiment was carried out to confirm whether apoptosis of stem cells is induced by inhibiting phosphorylation of LRRK2 protein in tumor stem cells known to be associated with tumor metastasis.

Specifically, the compound prepared in Example 12 was added to the NCC01 cell line, which is a brain tumor stem cell line, at a concentration of 0.1 or 1 μM, and after 1 day elapsed, As described in <Experimental Example 9>, mitochondria were stained to confirm their functional change. At this time, the group that was not treated with the compound of the example was used as the untreated group (Vehicle). The stained results were photographed under a microscope and shown in FIG. 17.

As shown in FIG. 17, the mitochondrial activity present in brain tumor stem cells was suppressed in the group treated with the compound of Example 12 compared to the control group. Therefore, it was confirmed from the above that the compound according to the present invention can inhibit the metastasis of the tumor by inducing the death of brain tumor stem cells by inhibiting mitochondrial activity.

<Experimental Example 11> Evaluation of TRAP1 expression inhibition activity by LRRK2 protein inhibitor in brain tumor stem cell line

Inhibition of mitochondrial activity in brain tumor stem cell lines of the inhibitor of the LKKR2 protein according to the present invention identified in <Experimental Example 10> changes the expression of the TNF receptor associated protein 1 (TRAP1) protein, which is known to be involved in the regulation of mitochondrial function We did an experiment to see if we were involved in

Specifically, the compound prepared in Example 12 was added to the NCC01 cell line, which is a brain tumor stem cell line, at a concentration of 100 or 1,000 nM, and after 1 day, changes in the expression of the TRAP1 protein were confirmed through the Western Blot method. At this time, the group not treated with the compound of the example was used as the untreated group (Vehicle).

As a result, as shown in FIG. 18, the expression of the TRAP1 protein was suppressed by the compound of Example 12. From this, it was found that the compound according to the present invention inhibits the mitochondrial activity by inhibiting the expression of the TRAP1 protein.

<Experimental Example 12> Confirmation of changes in LRRK2 protein expression under hypoxic conditions

It has been known through conventional studies that hypoxic conditions are important in the function of GSC. Therefore, the changes in the expression of the LRRK2 protein under hypoxic conditions and the size of the GSC sphere were confirmed when the expression was suppressed.

Specifically, after exposing the NCC01 and 528NS cell lines to hypoxic conditions, two types of shRNA for the LRRK2 gene were treated therein, and then the size change of the sphere was confirmed. At this time, shRNA (shNT) against lucirupase was used as a control.

As a result, as shown in FIG. 19, the sphere size of the NCC01 (A and B) and 528NS (C and D) cell lines exposed to hypoxic conditions increased compared to the control, but this is the shRNA for the LRRK2 gene, LRRK2 shRNA-1 and Inhibited by LRRK2 shRNA-2. From this, it was found that the growth of GSCs produced under hypoxic conditions was inhibited by inhibiting the expression of the LRRK2 protein.

<Experimental Example 13> Evaluation of apoptosis induction activity through inhibition of phosphorylation of LRRK2 protein (in vitro)

In order to evaluate whether or not to induce apoptosis of brain tumor cells by inhibiting phosphorylation of LRRK2 protein, an experiment was performed using fluorescence-activated cell sorting (FACS).

More specifically, ^{7.5x10 5} to ^{1.0x10 6} NCC01 cells were treated with the compounds prepared in Examples 11, 12 and 21 at a concentration of 100 nM, respectively, and after 1 day elapsed, samples were collected and FACS was performed. By doing so, the apoptosis inducing activity was evaluated. The group that was not treated with the compound of the example was used as the untreated group (Control). The results are shown in Figs. 20 and 21.

20 and 21, the compounds of Examples 11, 12 and 21 induced apoptosis of NCC01 cells. In particular, the compound of Example 12 showed about 3 times higher apoptosis rate than the compounds of Examples 11 and 21. From this, it was found that the compound of Example 12 according to the present invention has a remarkably excellent effect of inducing apoptosis compared to the compounds of other examples.

<Experimental Example 14> Evaluation of the growth inhibitory activity of the compound against brain tumor cells in an animal model-(1)

Experiments were performed to confirm whether the compounds of the examples according to the present invention exhibit growth inhibitory activity against brain tumor cells in an animal model.

More specifically, in the Balb/c-nude mouse model, NCC01, a cell line derived from a brain tumor patient ^{, was injected into the brain so as to be 1.0x10 4} cells, and after 3 days, the compound prepared in Example 12 was 10 mg/kg or 60 It was administered orally at mg/kg. After administration of the compound, the survival rate of the mouse according to the survival period and the size of the tumor generated in the mouse were measured using MRI. The group that was not treated with the compound of the example was used as the untreated group (Vehicle). The results are shown in Figs. 22 and 23.

As shown in FIG. 22, in the untreated group, the average survival time of mice was 54 days, whereas in the group administered orally at 10 mg/kg of the compound of Example 12, the average survival day was 71 days, In the group administered orally with the compound at 60 mg/kg, all subjects survived up to 100 days (Vehicle vs. 10mg/kg: p=0.032, Vehicle vs. 60mg/kg: p<0.001).

In addition, as shown in Figure 23, in the untreated group not treated with the compound, a tumor was formed at the location where the NCC01 cells were injected, but in the group to which the compound of Example 12 was orally administered 10 mg/kg or 60 mg/kg, the tumor was No formation was observed.

From this, it was found that the compounds of Examples according to the present invention remarkably inhibit the formation of tumors derived from brain tumor cells in an animal model.

<Experimental Example 15> Evaluation of the effect of treating brain tumors by combination treatment of a compound and avastin

When the compound of Examples according to the present invention was treated in combination with Avastin, which is prescribed as a brain tumor treatment drug, an experiment was performed to confirm its effect in an animal model.

More specifically, an orthotopic mouse model was prepared using a 5-week-old Balb/c-nude mouse. At this time, U87MG cells (ATCC, USA), which is a brain tumor cell line, were injected into the left side 2.2 mm, the rear side 0.5 mm, and the depth 3.5 mm based on the bregma using a stereotaxic device. Three days after the injection of the cells, the mice were divided into 4 groups of 5 mice each (vehicle, avastin administered group, or the compound of Example 12 and avastin administered group), and the drug was administered. The compound of Example 12 was orally administered at a dose of 30 mg/kg, and Avastin was injected intraperitoneally at a dose of 10 mg/kg. Thereafter, the survival period was measured when the mouse showed tumor formation findings such as a decrease of 20% or more of the total body weight or exhibiting abnormal behavior. As a result, the survival graph shown using the statistical program is shown in FIG. 24.

As a result, as shown in FIG. 24, when the compound of Example 12 was treated in combination with Avastin, it was found that the effect of treatment on brain tumors by Avastin was further significantly increased.

<Experimental Example 16> Evaluation of the phosphorylation inhibitory activity of the compound of Comparative Example on LRRK2 protein (in vitro)

To evaluate the drug efficacy in vitro of the comparative compound, a compound known as an LRRK2 protein inhibitor, a Western Blot experiment was performed using NCC01 cells, a cell line derived from a brain tumor patient.

More specifically, 7.5x10 ⁵ to 1.0x10 ⁶ cells were treated with the compound prepared in Comparative Example at a concentration of 0.1 or 1.0 μM, respectively, and after 1 day, samples were collected and phosphorylated for LRRK2 protein through Western Blot. Inhibitory activity was evaluated. The group that was not treated with the compound of the example was used as the untreated group (Vehicle). The results are shown in FIG. 25.

As shown in FIG. 25, it was confirmed that the comparative compound according to the present invention was known to inhibit the LRRK2 protein, but did not inhibit the phosphorylation of the LRRK2 protein.

<Experimental Example 17> Evaluation of the phosphorylation inhibitory activity of the compound against LRRK2 protein (in vitro)-(2)

To evaluate drug efficacy in vitro, a Western blot experiment was performed using 448T, a cell line derived from a brain tumor patient. Experiments were carried out in the same conditions and methods as in Experimental Example 5, except that 448T cells (Samsung Seoul Hospital, Korea) were used instead of NCC01 cells, and the compounds prepared in Examples 10, 11, 12, 20 and 21 were treated. Became.

As a result, as shown in FIG. 26, the compounds of Examples 10, 11, 12, 20 and 21 according to the present invention showed phosphorylation of LRRK2 protein in 448T cells, which are cell lines derived from brain tumor patients, compared to the untreated group (Vehicle). Remarkably suppressed.

Therefore, it was reconfirmed that the compound according to the present invention inhibits phosphorylation of LRRK2 protein expressed in cell lines derived from brain tumor patients.

<Experimental Example 18> Evaluation of phosphorylation inhibitory activity on LRRK2 protein according to the concentration of the compound (in vitro)-(2)

An experiment was performed to confirm whether the compound of the example according to the present invention has excellent efficacy in a specific concentration range for 448T cells. Experiments were carried out in the same conditions and methods as in Experimental Example 6, except that 448T cells were used instead of NCC01 cells, and the compounds prepared in Example 12 were treated at concentrations of 50, 100, 500, 1,000, and 2,000 nM.

As a result, as shown in FIG. 27, the compound of Example 12 inhibited most of the phosphorylation of the LRRK2 protein at a concentration of 50 nM.

From this, it was reconfirmed that the compound according to the present invention remarkably inhibits phosphorylation of LRRK2 protein even at a low concentration of nM.

<Experimental Example 19> Evaluation of the growth inhibitory activity of the compound against brain tumor cells in an animal model-(2)

Experiments were performed to confirm whether the compounds of the examples according to the present invention exhibit growth inhibitory activity against brain tumor cells in an animal model. Experiments were carried out in the same conditions and methods as in Experimental Example 14, except that 448T cells were used instead of NCC01 cells, and the compounds prepared in Examples 12, 20 and 21 were orally administered at 60 mg/kg.

As a result, as shown in 28, in the untreated group not treated with the compound, a tumor was formed at the location where 448T cells were injected, but in the group to which the compounds prepared in Examples 12, 20 and 21 were orally administered, tumor formation. Was not observed.

From this, it was reconfirmed that the compounds of Examples according to the present invention markedly inhibit the formation of tumors derived from brain tumor cells in an animal model.

<Experimental Example 20> Evaluation of phosphorylation inhibitory activity of compounds of Examples and Comparative Examples on LRRK2 protein (in vitro)

An experiment was performed to evaluate the drug efficacy of the comparative compound, a compound known as an LRRK2 protein inhibitor, in vitro. Experiments were carried out in the same conditions and methods as in Experimental Example 16, except that 448T cells were used instead of NCC01 cells, and the compounds prepared in Examples 1, 11 and 12 were treated.

As a result, as shown in FIG. 29, it was confirmed that the compound of Comparative Example was known to inhibit the LRRK2 protein, but unlike the compounds of Examples 1, 11 and 12 according to the present invention, it was not possible to inhibit phosphorylation of the LRRK2 protein.

<Formulation Example 1> Preparation of pharmaceutical formulation

1-1. Preparation of powder

500 mg of LRRK2 protein inhibitor of the present invention

100 mg lactose

Talc 10 mg

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

1-2. Manufacture of tablets

500 mg of LRRK2 protein inhibitor of the present invention

Corn starch 100 mg

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet preparation method.

1-3. Preparation of capsules

500 mg of LRRK2 protein inhibitor of the present invention

Corn starch 100 mg

100 mg lactose

2 mg of magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.

1-4. Preparation of injections

500 mg of LRRK2 protein inhibitor of the present invention

Appropriate amount of sterile distilled water for injection

proper amount of pH adjuster

It is prepared with the above ingredients per ampoule (2 ml) according to a conventional injection preparation method.

1-5. Preparation of liquid

LRRK2 protein inhibitor of the present invention 100 mg

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to the usual preparation method of liquid preparation, add and dissolve each component in purified water, add lemon scent, mix the above ingredients, add purified water, add purified water, adjust the total to 100 ml, and fill in a brown bottle. The liquid is prepared by sterilization.

<110> Daegu-Gyeongbuk Medical Innovation Foundation NATIONAL CANCER CENTER SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> Pharmaceutical composition for preventing or treating brain cancer comprising compounds penetrating blood-brain barrier <130> 2018P-06-016 <150> KR 10-2017-0024812 <151> 2017-02-24 <160> 35 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F1_For <400> 1 gagcagcgga cgttcatg 18 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F1_rev <400> 2 tatgagctgg gaaatggcca 20 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F1_For_2 <400> 3 gcgggttgga agcaggtg 18 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F1_rev_2 <400> 4 tcagcatgga gagcatcact 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F2_For <400> 5 agtacccaga gaatgcagca 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F2_rev <400> 6 gctcccaata gaagcaagca 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F3_For <400> 7 gaactggaca tctgctggc 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F3_rev <400> 8 ctcgtgagtg cattctggtg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F3_For_2 <400> 9 cataggaact ggacatctgc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F3_rev_2 <400> 10 cattctggtg aagctccagc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F4_For <400> 11 ctcaggcagc gatggaaatt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F4_rev <400> 12 gacagccaat ctcaggagga 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F5_For <400> 13 gctatgcctt tcttgcctcc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F5_rev <400> 14 gcagtgctgg gtcttgaaaa 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F6_For <400> 15 cctgtgattc tcgttggcac 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F6_rev <400> 16 aggctgctcg gtaaactgat 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F7_For <400> 17 ttcctgggtt gctggagatt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F7_rev <400> 18 tcccagacac aatccagctt 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F8_For <400> 19 acttctgccc aggtctttga 20 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F8_rev <400> 20 tgtgagtacc ctttccatgt ga 22 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F1_500F <400> 21 gatctcctcc taacttcag 19 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F1_700r <400> 22 caatgcagca catgaagc 18 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F2_500F <400> 23 gaagtggctg aaagtggctg 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F2_700r <400> 24 gaatatcatt cttgaaacac 20 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F3_500F <400> 25 catcagcttg ctcttaagg 19 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F3_700r <400> 26 gaggctgttc cttcttcc 18 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F5_500F <400> 27 cttataaccg aatgaaac 18 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F5_700r <400> 28 ctatagaatt cctcacgac 19 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F7_500F <400> 29 ggatcgcctg cttcagcag 19 <210> 30 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F7_700r <400> 30 cattctacag cagtactg 18 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F8_500F <400> 31 gctgctcctt tgaagatac 19 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LRRK2_F8_700r <400> 32 gtctaccacc actgttatg 19 <210> 33 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> LRRK2 shRNA-1 <400> 33 ccggccacaa attcaacgga aagaactcga gttctttccg ttgaatttgt ggttttt 57 <210> 34 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> LRRK2 shRNA-2 <400> 34 ccggcccaaa ttggtggaac tcttactcga gtaagagttc caccaatttg ggttttt 57 <210> 35 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> luciferase shRNA <400> 35 ccggcgctga gtacttcgaa atgtcctcga ggacatttcg aagtactcag cgttttt 57

Claims (5)

A pharmaceutical composition for preventing or treating stage 4 brain cancer, which comprises, as an active ingredient, any one compound selected from the following group of compounds, or a pharmaceutically acceptable salt thereof:
(21) 3- (4-Morpholino-lH-pyrrolo [2,3-b] pyridin-3-yl) benzonitrile; And
(22) l-Methyl-4- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) -lH-pyrrole-2-carbonitrile.
delete delete A first component comprising as an active ingredient any compound selected from the group consisting of the following compounds in a pharmaceutically therapeutically acceptable amount, or a pharmaceutically acceptable salt thereof:
(21) 3- (4-Morpholino-lH-pyrrolo [2,3-b] pyridin-3-yl) benzonitrile; And
(22) l-Methyl-4- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) -lH-pyrrole-2-carbonitrile; And

A second component comprising as an active ingredient a pharmaceutically therapeutically effective amount of avastin; Or a pharmaceutically acceptable salt thereof.
5. The method of claim 4,
Wherein the first component and the second component are administered sequentially or concurrently.

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