KR101992059B1 - 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 containing a compound capable of penetrating a blood-brain barrier as an active component. The pharmaceutical composition for preventing or treating brain cancer according to the present invention efficiently penetrates 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

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating brain cancer containing, as an active ingredient, a compound capable of passing through the blood brain barrier,

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

Protein kinase is an enzyme that catalyzes the transfer of terminal phosphate group of adenosine triphosphate (ATP) to tyrosine, serine or threonine, which is a specific residue of protein. It regulates cell activity and growth and differentiation of cell medium and environment ≪ / RTI >

Inappropriately high protein kinase activity is directly or indirectly related to a number of diseases resulting from abnormal cellular function. For example, failure of the appropriate regulatory mechanism of the kinase involved in mutation, over-expression or inappropriate enzyme activity, or the excessive production or deficiency of factors involved in signal transduction of cytokines, upstream or downstream of cytokines, . Thus, selective inhibition of kinase activity can be a beneficial target for the development of new drugs for the treatment of disease.

The leucine-rich repeat kinase-2 (LRRK2) protein belongs to the leucine-rich repeat kinase family and is composed of 2527 amino acid sequences with high interspecies similarity. The LRRK2 protein is characterized by having both GTPase (GTPase) and serine-threonine kinase (GTPase) activity in a single protein. The expressed LRRK2 protein is found in various organs and tissues, including the brain, and is present in the cytoplasm or cell membrane and mitochondrial outer membrane at the cellular level.

In addition, the LRRK2 protein has five functionally important domains and is expected to regulate cell function through autophosphorylation, protein interaction, and enzyme action. In particular, chaperone machinery, cytoskelecton arrangement, protein translational machinery, synaptic vesicle endocytosis, mitogen-activated protein kinase signaling processes, activated protein kinases signaling cascades and ubiquitin / autophage protein degradation pathways are regulated by LRRK2 protein.

It has been reported that the LRRK2 protein is associated with CNS disorders associated with transposition of mild cognitive impairment associated with Alzheimer ' s disease, L-dopa induced dyskinesia, and neuronal progenitor differentiation. In addition, the G2019S mutation of the LRRK2 protein has been reported to increase the incidence of non-skin cancer such as acute myelogenous leukemia (AML) as well as kidney cancer, breast cancer, lung cancer, prostate cancer and the like. Specifically, the G2019S mutation of the LRRK2 protein increases the catalytic activity of the LRRK2 kinase domain. Furthermore, the LRRK2 protein has also been implicated in amyotrophic lateral sclerosis, rheumatoid arthritis, and ankylosis (International Patent Publication No. WO 2011/038572).

On the other hand, treatment methods of brain tumors include surgical operations, radiation therapy, chemotherapy (chemotherapy), and hormone therapy. Among them, mainly surgical surgery and radiation therapy have been relied on, especially radiation therapy has neurotoxicity and side effects such as dementia is accompanied. Therefore, anti-cancer drug therapy (chemotherapy) has been attracting attention in order to solve such a problem. However, the drug therapy for the treatment of brain tumor is blocked by the special protection system called the blood-brain barrier (BBB) There are many drugs that can not be delivered, and they are limited due to the resistance of cancer to anticancer drugs.

Meanwhile, the inventors of the present invention firstly confirmed in vivo experiments that a compound used as an inhibitor of LRRK2 protein can be effectively used for the prevention and treatment of brain cancer by efficiently passing through the blood brain barrier (BBB), and completed the present invention .

International Publication No. WO 2011/038572

It is an object of the present invention to provide a pharmaceutical composition which can be effectively used to prevent or treat brain cancer by efficiently passing through the blood brain barrier.

In order 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.

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 an effect of easily inhibiting tumor formation through a blood brain barrier which is difficult for general drugs to pass through.

FIG. 1 is an image showing the result of Western blotting the expression of LRRK2 protein in cancer tissues obtained from brain tumor patients.
2 is an image showing the result of confirming the correlation between phosphorylation of LRRK2 protein and malignancy of brain tumor.
3 is an image showing the result of analyzing a nucleic acid sequence encoding LRRK2 protein expressed in brain tumor cells.
FIG. 4 is an image showing overexpression (B) of LRRK2 protein and self-replication ability (C) of tumor cells in a cell infected with a lentivirus vector (A) expressing the LRRK2 protein.
FIGS. 5 and 6 are images showing Western blot results obtained when the compound of Example 1 was treated with NCC01 cells derived from a brain tumor patient. FIG.
FIG. 7 is an image showing that 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 LRRK2 gene.
FIGS. 8 and 9 are images showing cell cycle FACS evaluation results obtained after 1 day of treatment with 100 nM of each of the compounds of Example 1 in NCC01 cells. FIG.
FIG. 10 is an image showing the result of evaluation of a Western Blot, which is confirmed when a compound of the present invention is treated with NCC01 cells, a cell line derived from a brain tumor patient.
FIG. 11 is an image showing the Western blot evaluation result confirmed when the compound of Example 1 was treated with 448T cells derived from a brain tumor patient. FIG.
12 is an image showing the result of observing the survival day change when the compound of Example 1 was orally administered to a mouse model in which a brain tumor was induced.

Hereinafter, the present invention will be described in detail.

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

The LRRK2 protein inhibitor may be a compound represented by the following formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

(In the formula 1,

a is -CH =, or -N =;

G ¹ is -H, -OH, a halogen, a, nitrile, unsubstituted or substituted 5, including straight or branched chain alkyl, straight or branched chain alkoxy, or one or more of the N C _1-5 of C _1-5 Day Lt; / RTI > is heteroarylamino,

Wherein the heteroaryl of the substituted 5- to 8-membered ring is -OH, heteroaryl of a 5 to 8-membered ring substituted with halogen or C _1-3 linear or branched alkyl;

G ² is selected from the group consisting of -H, -OH, halogen, nitro, nitrile, C _1-5 linear or branched alkyl, C _1-5 straight or branched alkoxy, unsubstituted or substituted C _6-10 aryl, Lt; / RTI > is a heterocycloalkyl of an unsubstituted or substituted 5 to 8-membered ring comprising at least one heteroatom selected from the group consisting of N, < RTI ID = 0.0 &

Wherein said substituted C _6-10 aryl and substituted 5-to 8-membered heterocycloalkyl are independently C _1-5 straight or branched chain alkyl, C _1-5 straight or branched alkoxy, and N and O Wherein at least one substituent selected from the group consisting of straight chain or branched chain alkyl of unsubstituted heterocycloalkyl C _1-3 is substituted C _6-10 aryl and heterocycloalkyl of 5 to 8-membered ring;

G ³ is selected from the group consisting of -H, -OH, halogen, nitro, nitrile, C _1-5 linear or branched alkyl, C _1-5 straight or branched alkoxy, unsubstituted or substituted C _6- from ₁₀ aryl, or N, O and beach, including a hetero atom at least one member selected from the group consisting of S ring or a substituted 5 to 10, each ring heteroaryl,

Wherein said heteroaryl of said substituted 5- to 10-membered ring is heteroaryl of 5 to 10 ring members substituted with at least one substituent selected from the group consisting of halogen, C _1-5 straight or branched chain 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.

The compound represented by Formula 1 may be a compound represented by the following formula.

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 demonstrated 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. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful.

Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid and the like, aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, And organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like can be obtained. Examples of such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluene sulfonate, chlorobenzene Sulfonates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, and the like, as well as sulfonates such as benzyl sulfonate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, -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 the 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 may be filtered and dried, or the solvent and excess acid may be distilled off under reduced pressure, followed by drying and crystallization in an organic solvent.

In addition, the LRRK2 protein inhibitor of the present invention can be made 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 amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding salt can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (for example, 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 may be administered orally or parenterally in various formulations at the time of clinical administration. In the case of formulation, Such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like.

Examples of formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules, elixirs and troches, (E.g., silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycols), such as, for example, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine. Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, and may optionally contain disintegrating agents such as starch, agar, alginic acid or its sodium salt, Boiling mixtures, absorbents, coloring agents, flavoring agents, and sweetening agents.

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

In order to formulate the formulation for parenteral administration, the LRRK2 protein inhibitor, its optical isomer, or a pharmaceutically acceptable salt thereof is mixed with water or a stabilizer or a buffer to prepare a solution or suspension, which is then mixed with an ampule or vial unit May be prepared in dosage forms. The composition may be sterilized and further contain adjuvants such as preservatives, stabilizers, wettable or emulsifying accelerators, salts for controlling osmotic pressure and buffers, and other therapeutically useful substances, and may be mixed, granulated, Can be formulated according to the coating method.

The dosage of the pharmaceutical composition comprising the LRRK2 protein inhibitor of the present invention, its optical isomer, or a pharmaceutically acceptable salt thereof as an active ingredient, on the human body depends on the age, body weight, sex, dosage form, It is usually 0.1-1000 mg / day, preferably 1-500 mg / day, based on adult patients weighing 70 Kg, and may be varied according to the judgment of the physician or pharmacist. And may be administered once or several times a day at a predetermined time interval.

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 demonstrated by phosphorylation of the LRRK2 protein.

The present invention has for the first time demonstrated that LRRK2 protein inhibitor has an effect of easily inhibiting tumor formation through a blood brain barrier which is difficult for general drugs to pass through, and the experimental results related thereto are described in detail do.

Hereinafter, the present invention will be described in detail with reference to the following Examples and Experimental Examples.

It should be noted that the following examples and experimental examples are only illustrative of the present invention, but the present invention is not limited thereto.

Example 1 Synthesis of 3- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) benzonitrile

The title compound was prepared by reference to WO 2014/001973.

Comparative Example 1 Synthesis of 2-methyl-2- [3-methyl-4 - [[4- (methylamino) -5- (trifluoromethyl) pyrimidin- ] Propanenitrile

The title compound was prepared by reference to WO < RTI ID = 0.0 >

EXPERIMENTAL EXAMPLE 1 Expression of LRRK2 Protein in Cancer Stem Cells of Brain Tumor Patients

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

Specifically, cancer tissues were obtained from brain tumor patients, Western blot was performed to confirm the expression of LRRK2 protein, and the results are shown in FIG.

As shown in FIG. 1, the expression of LRRK2 protein in cancer tissues obtained from brain tumor patients was confirmed, and the expression level of cancer increased with increasing malignancy of brain tumor. Thus, it can be seen from the above that the LRRK2 protein can be used as a target for the treatment of brain tumors.

<Experimental Example 2> Determination of prognosis of brain tumor patients by phosphorylation of LRRK2 protein

The LRRK2 protein exhibits its activity by being phosphorylated. Thus, we confirmed the prognosis of brain tumor patients due to phosphorylation of LRRK2 protein in brain tumor cells.

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

As a result, as shown in FIG. 2, phosphorylation of LRRK2 protein increased as the malignancy of the brain tumor increased, that is, the brain tumor progressed from the third stage to the fourth stage. Thus, it can be seen from the above that the phosphorylated LRRK2 protein can be used as a target protein for the treatment of a highly malignant brain tumor.

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

Previous studies have shown that a variety of carcinomas can arise from mutations in the LRRK2 protein and that Parkinson's disease, which is associated with the LRRK2 protein, is also caused by mutations in the LRRK2 protein. Thus, it was confirmed that the LRRK2 protein, which was confirmed to be overexpressed in brain tumor cells in the above experimental example, is a mutant thereof. Specifically, sequencing was performed using brain tumor cells obtained from five patients, wherein primers as described in Table 1 below were used.

As a result, as shown in Fig. 3, the nucleic acid sequence coding for the LRRK2 protein expressed in brain tumor cells had no specific mutation such as that which is mainly found in Parkinson's disease except for the known single nucleotide polymorphism (SNP) . Therefore, from the above, it was confirmed that, in contrast to Parkinson's disease or other types of cancer, brain tumors are over-expressed by normal LRRK2 protein, resulting in cancer.

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

In order to confirm whether brain tumors were caused by overexpression of the LRRK2 protein, as confirmed in Experimental Example 3, lentiviral vectors containing the polynucleotides encoding the LRRK2 protein (pLenti CMV GFP DEST (736-1), Addgene # 19732), which were then infected with 528NS, 464T (Samsung Medical Center, Korea) and ex-vivo cell lines, respectively, to confirm tumor formation. At this time, a lentivirus vector that did not express the LRRK2 protein was infected as a control.

As a result, as shown in FIG. 4, the size of the GSC (glioma stem cell) sphere was increased in 528NS, 464T or ex-vivo cell line infected with lentivirus expressing the LRRK2 protein as compared with the control group. From these results, it was found that tumor overgrowth of LRRK2 protein was formed.

Experimental Example 5 Evaluation of phosphorylation inhibitory activity of LRRK2 protein of a compound (in vitro)

Western blot experiments were performed using NCC01 cells, a brain tumor cell line, for the evaluation of drug efficacy in vitro.

More specifically, 7.5x10 &lt; ⁵ &gt; to 1.0x10 &lt; ⁶ &gt; NCC01 cells were treated with the compound of Example 1 at a concentration of 100 or 1,000 nM, respectively. After 1 day, samples were collected and inhibited phosphorylation of LRRK2 protein through Western Blot Activity. The group without treatment of the compounds of the Examples was used as a vehicle. The results are shown in FIGS. 5 and 6. FIG.

As shown in FIGS. 5 and 6, the compound of Example 1 according to the present invention significantly inhibited the phosphorylation of LRRK2 protein in NCC01 cells, a cell line derived from a brain tumor patient, compared with no treatment group.

Thus, it was confirmed that the compound of the present invention inhibits phosphorylation of LRRK2 protein expressed in brain tumor-derived cell lines.

<Experimental Example 6> Determination of LRRK2 protein expression under hypoxic conditions

Previous studies have shown that hypoxic conditions are important in the function of GSC. Therefore, when the expression of LRRK2 protein and the expression of LRRK2 protein were suppressed under hypoxic condition, the size of GSC sphere was confirmed.

Specifically, NCC01 and 528NS cell lines were exposed to hypoxic conditions, followed by treatment with two shRNAs against the LRRK2 gene, and then the size of the sphere was observed. At this time, shRNA (shNT) against luciferase was used as a control.

As a result, as shown in Fig. 7, the sphere size of the NCC01 (A and B) and 528NS (C and D) cell lines exposed to hypoxic conditions was increased as compared with the control, but the shRNA for LRRK2 gene, LRRK2 shRNA- LRRK2 shRNA-2. From this, it was found that GSC produced under hypoxic condition was inhibited by inhibition of LRRK2 protein expression.

<Experimental Example 7> Evaluation of inducing activity of apoptosis through inhibition of phosphorylation of LRRK2 protein (in vitro)

Experiments were performed using fluorescence-activated cell sorting (FACS) to evaluate whether or not inducing apoptosis of brain tumor cells by inhibiting phosphorylation of LRRK2 protein.

More specifically, 7.5x10 &lt; ⁵ &gt; to 1.0x10 &lt; ⁶ &gt; NCC01 cells were treated with the compound of Example 1 at a concentration of 100 nM, and after 1 day, samples were collected and subjected to FACS to evaluate the apoptosis inducing activity Respectively. The groups not treated with the compounds of the Examples were used as the control group. The results are shown in Figs. 8 and 9.

As shown in FIGS. 8 and 9, it can be seen that the compound of Example 1 induces apoptosis of NCC01 cells.

Experimental Example 8 Evaluation of Phosphorylation Inhibitory Activity of LRRK2 Protein of Comparative Example Compounds (in vitro)

Western blot experiments were carried out using NCC01 cells derived from brain tumor patients to evaluate the drug efficacy of the comparative compounds, which are known compounds as LRRK2 protein inhibitors, in vitro.

More specifically, 7.5 × 10 ⁵ to 1.0 × 10 ⁶ cells were treated with the compounds prepared in the comparative examples at a concentration of 0.1 or 1.0 μM, respectively. One day later, samples were collected and phosphorylated for LRRK2 protein Inhibitory activity. The group without treatment of the compounds of the Examples was used as a vehicle. The results are shown in Fig.

As shown in FIG. 10, the comparative compound according to the present invention is known to inhibit the LRRK2 protein, but it is confirmed that the phosphorylation of the LRRK2 protein is not inhibited.

Experimental Example 9 Evaluation of phosphorylation inhibitory activity of LRRK2 protein of a compound (in vitro)

Western blot experiments were performed using 448T, a brain tumor cell line, to evaluate drug efficacy in vitro. Experiments were carried out in the same manner as in Experimental Example 5 except that 448T cells (Samsung Medical Center, Korea) were used instead of NCC01 cells and the compound of Example 1 was treated.

As a result, as shown in FIG. 11, the compound of Example 1 according to the present invention significantly inhibited phosphorylation of LRRK2 protein in 448T cells, a cell line derived from a brain tumor patient, compared to the no treatment group.

Accordingly, it was confirmed that the compound according to the present invention inhibits phosphorylation of LRRK2 protein expressed in brain tumor-derived cell lines.

<Experimental Example 10> Evaluation of growth inhibitory activity of compounds on brain tumor cells in animal models

Experiments were conducted to confirm that the compound of Example 1 according to the present invention exhibited growth inhibitory activity against brain tumor cells in an animal model.

More specifically, 448T, a brain tumor-derived cell line, was injected into a brain of a Balb / c-nude mouse model so as to be 1.0 × 10 ^4. After 3 days, the compound of Example 1 was orally administered at 60 mg / kg. The survival rate of the mice after administration of the compounds was determined, and the results are shown in Fig.

As shown in Fig. 12, the survival rate of mice was significantly increased in the group treated with the compound as compared with the control group.

&Lt; Formulation Example 1 > Preparation of pharmaceutical preparation

1-1. Manufacture of Powder

500 mg of the LRRK2 protein inhibitor of the present invention

Lactose 100 mg

10 mg of talc

The above components are mixed and filled in airtight bags to prepare powders.

1-2. Manufacture of tablets

500 mg of the LRRK2 protein inhibitor of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-3. Manufacture of capsules

500 mg of the LRRK2 protein inhibitor of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

1-4. Injection preparation

500 mg of the LRRK2 protein inhibitor of the present invention

Sterile sterilized water for injection

pH adjuster

(2 ml) per ampoule in accordance with the usual injection method.

1-5. Manufacture of liquid agent

100 mg of the LRRK2 protein inhibitor of the present invention

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, 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          penetrating blood-brain barrier <130> 2018P-08-019 <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> RNA <213> Artificial Sequence <220> <223> LRRK2 shRNA-1 <400> 33 ccggccacaa attcaacgga aagaactcga gttctttccg ttgaatttgt ggttttt 57 <210> 34 <211> 57 <212> RNA <213> Artificial Sequence <220> <223> LRRK2 shRNA-2 <400> 34 ccggcccaaa ttggtggaac tcttactcga gtaagagttc caccaatttg ggttttt 57 <210> 35 <211> 57 <212> RNA <213> Artificial Sequence <220> <223> luciferase shRNA <400> 35 ccggcgctga gtacttcgaa atgtcctcga ggacatttcg aagtactcag cgttttt 57

Claims (4)

A pharmaceutical composition for the prophylaxis or treatment of stage 4 brain cancer, comprising a compound represented by the following formula: or a pharmaceutically acceptable salt thereof as an active ingredient:

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