KR101948555B1 - Pharmaceutical composition for preventing or treating cancer comprising receptor tyrosine kinases inhibitor - Google Patents

Abstract

The present invention relates to a receptor tyrosine kinase activity inhibitory compound represented by the general formulas (1) and (2), wherein the compound represented by the above formula (1) or (2) inhibits the growth of cancer cells through inhibition of kinase activity, It has been confirmed that apoptosis killing effect of cancer cells is improved when they are used in combination with an anticancer agent or radiotherapy. Accordingly, the compounds represented by Formula 1 or 2 are useful as pharmaceutical compositions for preventing or treating cancer, May be provided as a composition for enhancing the effect of the anticancer therapy.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising a receptor tyrosine kinase inhibitor as an active ingredient,

The present invention relates to a pharmaceutical composition for preventing or treating cancer, a composition for enhancing anticancer activity, or a health food for cancer prevention or improvement, which comprises a receptor tyrosine kinase activity inhibitory compound as an active ingredient.

Receptor tyrosine kinases (RTKs) are transmembrane proteins that penetrate cell membranes and are one of the most important receptor groups responsible for cell signaling systems that regulate cell proliferation, cell migration, cell cycle and cell differentiation , And human RTKs are classified into about 20. Platelet Derived Growth Factor Receptor (PDGFR) can be activated by ligand platelet-derived growth factor (PDGF), mitogen-activated protein kinases (MAPK) The phosphotidyl inositol 3-kinase (PI3K) and STAT3 (signal transducer and activator of transcription 3) can activate the downstream signaling pathway. Activation of PDGFR, including mutations, is observed in GISTs, Glioblastoma multiforme, and chronic myeloid leukemia. An internal tandem duplication (IDT) mutation in the juxtamembrane domain of FLT3 (FMS-like tyrosine kinase 3) gene in approximately 30% of patients with acute myeloid leukemia (AML) FLT3 inhibition may be helpful in the treatment of acute myelogenous leukemia and acute lymphoblastic leukemia as it is known in about 3% of patients with acute lymphoblastic leukemia (ALL), and those with these mutations are known to have poor prognosis .

c-Kit can be activated by ligand stem cell factor (SCF), and point mutants such as D816V or V560G are constantly activated without ligand to resist cell proliferation, cell survival and apoptosis Lt; / RTI > In particular, c-Kit induces mastocytosis by activating mast cells and increases expression in various organs (skin, liver, gastrointestinal tract, reproductive tract, etc.) to induce autoimmune diseases, leukemia, gastrointestinal stromal tumors gastrointestinal stromal tumor (GIST), small cell lung cancer, testicular cancer, and polymorphic glioblastoma. Patients with about 70-80% GISTs have a gain-of-function mutation in c-Kit. It has been reported that the increase of SCF by nicotine leads to the rapid growth and metastasis of non-small cell lung cancer due to activation of c-Kit of non-small cell lung cancer.

Cyclin-depenedent kinase (CDK) is a serine / threonine protein kinase that is crucial to the cell cycle. These complexes consist of two catalytic small molecules, cyclin-dependent kinase (CDK) and regulatory small molecule, cyclin. Each CDK is complexed with a specific cyclin to activate it. The progression of each cell cycle can be controlled by specific CDK / cyclin complexes. In particular, CDK1 / cyclin B1, CDK2 / cycle E, and CDK2 / cyclin A are important factors in cell cycle progression.

In clinical trials, a number of RTK inhibitors showed initial clinical responses, but were identified as transient responses and identified problems with rapid resistance. In addition, the RTK inhibitor may cause a D816V mutation in the KIT, a D842V mutation in the PDGFR, and a D835V mutation in the FLT3, and these mutations may interfere with the binding of the RTK inhibitor to the target.

Therefore, RTK activation and rapid cell cycle progression are involved in the regulation of abnormal cells in various cancer species. Therefore, the development of inhibitors targeting RTK activity and CDK1 / cyclin B and CDK2 / cyclin A and CDK5 / p35 complex kinase activity Is required.

Korean Patent Publication No. 2008-0047607 (Published May 29, 2008)

The present invention provides a composition containing, as an active ingredient, a compound capable of effectively inhibiting the activity of a receptor tyrosine kinase (RTK) in various cancer species as an anticancer therapeutic agent, and can be used in combination with other anticancer agents or radiotherapy, And to provide a composition for promoting therapeutic effect.

상기 화학식 1에서,
X는 질소 및 탄소에서 선택된 어느 하나일 수 있다.
본 발명은 하기 화학식 1과 같이 표시되는 화합물 또는 이의 약제학적으로 이용가능한 염을 유효성분으로 함유하는 암 예방 또는 치료용 약학조성물을 제공한다.The present invention provides a compound represented by the following Chemical Formula 1 or a pharmaceutically usable salt thereof.
[Chemical Formula 1]

In Formula 1,
X may be any one selected from nitrogen and carbon.
The present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient, a compound represented by Formula 1 below or a pharmaceutically usable salt thereof.

[Chemical Formula 1]

In Formula 1,

X may be any one selected from nitrogen and carbon.

The present invention provides a composition for promoting an anti-cancer effect comprising a compound represented by Formula 1 or a pharmaceutically usable salt thereof as an active ingredient.

The present invention also provides a health food for preventing or ameliorating cancer, comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

According to the present invention, it has been confirmed that the compound represented by Formula 1 inhibits the growth of cancer cells through inhibition of kinase activity, and when cancer treatment is combined with conventional anticancer drugs or radiation therapy, apoptosis killing effect of cancer cells is improved The compound represented by Formula 1 can be provided as a pharmaceutical composition for preventing or treating cancer or as a composition for enhancing the effect of chemotherapy of chemotherapy and radiation therapy.

FIG. 1 shows the inhibition of the phosphorylation of STAT5 (signal transducer and activator of transcription 5), a substrate protein of FLT3, which is a downstream signal transduction system when 10 μM of Compound 11 (Formula 1) and Compound 12 (Formula 2) were treated with human leukemia MV4-11 cell line. .
FIG. 2 shows the results of apoptosis-induced apoptosis when 10 μM of Compound 11 (Formula 1) and 12 (Formula 2) were treated with human leukemia MV4-11 cell line.
FIG. 3 is a result of confirming that cell proliferation is inhibited when 2 μM of Compound 11 (Formula 1) and 2 μM of anticancer agent cisplatin 5 μM are used in combination with human leukemia MV4-11 cell line.
FIG. 4 shows the results of confirming that cell death was increased when the compound 11 (Formula 1) and the compound 12 (Formula 2) were applied to a human leukemia MV4-11 cell line in combination with 10 μM radiation 1 Gy irradiation.

상기 화학식 1에서,
X는 질소 및 탄소에서 선택된 어느 하나일 수 있다.
본 발명은 하기 화학식 1과 같이 표시되는 화합물 또는 이의 약제학적으로 이용가능한 염을 유효성분으로 함유하는 암 예방 또는 치료용 약학조성물을 제공할 수 있다.The present invention provides a compound represented by the following general formula (1) or a pharmaceutically usable salt thereof.
[Chemical Formula 1]

In Formula 1,
X may be any one selected from nitrogen and carbon.
The present invention can provide a pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient, a compound represented by Formula 1 below or a pharmaceutically usable salt thereof.

[Chemical Formula 1]

In Formula 1, X may be any one selected from nitrogen and carbon.

More specifically, the compound represented by Formula 1 may inhibit receptor tyrosine kinase activity.

Said cancer is selected from the group consisting of lung cancer, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, breast cancer, gastric cancer, liver cancer, colon cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, Cancer, cancer of the bladder, cancer of the esophagus, cancer of the thyroid, cancer of the bladder, kidney cancer, blood cancer and rectal cancer, and the leukemia is selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia and chronic lymphocytic leukemia Can be selected from the group.

According to one embodiment of the present invention, compounds 1 and 2, which are represented by Formula 1, exhibit the effect of inhibiting 23 kinds of kinase activity as shown in Table 2. In particular, cKit V560G), FLT1, FLT3, FLT3 (D835Y), FLT3 (ITD) and FLT4 kinase.

As shown in Table 4, Compound 1 and Compound 2 showed excellent proliferation inhibitory effects in lung cancer, breast cancer, colon cancer, and leukemia cells, while CCD18- The cell survival rate of Co - normal cells was not inhibited at the same concentration, so that selective cancer cell proliferation inhibitory effect was confirmed.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating cancer, which comprises the compound represented by Formula 1 or a pharmaceutically utilizable salt thereof as an active ingredient, may be administered orally, parenterally, , Pills, capsules, suppositories, gels, suspensions, emulsions, drops, or liquid preparations can be used.

In another embodiment of the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient, disintegrant, sweetener, coating, swelling agent, lubricant, lubricant, flavoring agent, antioxidant, buffer, A dispersant, a surfactant, a binder, and a lubricant.

Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

According to one embodiment of the present invention, the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, ≪ / RTI > can be administered to the subject in a conventional manner.

The preferred dosage of the compound represented by Formula 1 may vary depending on the condition and body weight of the subject, the type and degree of disease, the drug form, the administration route and the period, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dose may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

The present invention can provide a composition for promoting an anti-cancer effect comprising, as an active ingredient, a compound represented by Formula 1 below or a pharmaceutically usable salt thereof.

[Chemical Formula 1]

In Formula 1, X may be any one selected from nitrogen and carbon.

The anticancer effect enhancing composition may be treated with an anticancer agent or radiation.

The anticancer agent is selected from the group consisting of cisplatin, 5-fluorouracil, Paclitaxel, Doxorubicin, Daunorubicin, Vinblastine, Vincristine, Actinomycin D, Teniposide, Etoposide, cyclophosphamide, epirubicin, adriamycin, daunomycin, and mitomycin, And C (mitomycin-C).

The composition may comprise 1 to 99 parts by weight of an anticancer agent and 1 to 99 parts by weight of a compound represented by the general formula (1), based on 100 parts by weight of the total composition.

The composition may be used for the treatment and / or prophylaxis of lung cancer, leukemia, breast cancer, stomach cancer, liver cancer, colon cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, breast cancer, larynx cancer, prostate cancer, bladder cancer, And rectal cancer, the anticancer effect of the present invention can be enhanced.

The leukemia may be selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia and chronic lymphocytic leukemia.

According to another embodiment of the present invention, MV4-11 leukemia cells were treated with 2 μM of Compound 1 and 2 and 5 μM of known anti-cancer drug cisplatin (Cisplatin; 5 μM) for 48 hours and assayed by MTT As shown in FIG. 3, the cell survival rate of the experimental group treated with cisplatin and compound 1 was more than 40% higher than that of the control group treated with cisplatin alone, as shown in FIG. 3, and the combination of cisplatin and compound 2 And 60% more in the experimental group.

In addition, the cultured human blood cancer cell MV4-11 cell line was treated with 10 μM of Compound 1 and Compound 2 and 1 Gy of radiation, and cultured for 48 hours. Using FACSort flow cytometer (Becton dickinson, USA), apoptosis-induced cells As shown in FIG. 4, it was confirmed that apoptosis-induced apoptosis in MV4-11 human blood cancer cell line was 2.1 times and 1.8 times higher than that in the radiation alone group, respectively.

From the results, it was confirmed that the compound 1 and the compound 2 of the present invention can be combined with the conventional anticancer drug or radiation therapy to increase the anticancer effect.

The present invention also provides a health food for preventing or ameliorating cancer, which comprises, as an active ingredient, a compound represented by Formula 1 below or a pharmaceutically usable salt thereof.

[Chemical Formula 1]

In Formula 1, X may be any one selected from nitrogen and carbon.

Wherein the cancer is selected from the group consisting of lung cancer, leukemia, breast cancer, stomach cancer, liver cancer, colon cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, breast cancer, brain cancer, larynx cancer, prostate cancer, bladder cancer, And rectal cancer.

The leukemia may be selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia and chronic lymphocytic leukemia.

The health food is used together with food or food additives in addition to the compound represented by Formula 1 or its pharmaceutically usable salt, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.

The effective dose of the compound contained in the above-mentioned health food may be used in accordance with the effective dose of the therapeutic agent, but may be less than the above range for health and hygiene purposes or for long-term intake for health control purposes, It is clear that the component can be used in an amount of more than the above range since there is no problem in terms of safety.

There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Reference example > Substance And apparatus

The melting point of the synthesized compound was determined using a Kruess M5000 Melting Point apparatus and the value was not corrected. Proton NMR spectra were recorded on an Avance-300 (Bruker) at 300 MHz. Chemical shifts were recorded in ppm and Me4Si was used as a reference standard.

The mass spectra were recorded on a JEOL, JMS-600W VG Trio-2 GC-MS.

The reaction products were purified using clean column chromatography on silica gel 60 (230-400 mesh, Merck), precoated silica gel 60 F254 (precoated silica gel 60 F254, E. Merch, Mumbai, India ) Using thin layer chromatography. The spots were stained with phosphomolybdic acid (PMA) or Hanessian's solution and visualized with UV light (254 nm).

< Example  1> Compound Synthesis

The compound shown in Table 1 was synthesized by following the procedure of Scheme 1 below.

[Reaction Scheme 1]

compound R ₁
R ₂
Vehicle (Vehicle) = DMSO One
(3a)
Br

2
(3b)
Br

3
(3c)
Br

4
(3d)
Br

5
(3e)
Br

6
(3f)
F

7
(3g)
F

8
(3h)
F

9
(3i)
F

10
(3j)
F

11
(3k)

12
(3l)

One. Azide ( Azide ) synthesis

Bromophenacyl bromide (1.0 eq) or 2-bromo-4'-fluoroacetophenone was dissolved in acetone of 0.2 M, Sodium azide (3.0 eq) was added. After stirring and reaction at room temperature for 16 hours, the mixture was filtered and concentrated in vacuo to afford the azide compound (1a-b) (93% yield).

2. Isothiocyanate ( Isothiocyanate ) synthesis

Dichloromethane (dichloromethane, DCM) in the amine solution of aniline (Aniline), p - anisidine (p -Anisidine), 2,5- dimethoxy aniline (2,5-Dimethoxyaniline), 1- naphthylamine (1 Amino-4-methoxyphenyl ethyl sulfone) was dissolved in 1.0 eq each, 1.2 eq of thiophosgene was treated, and the mixture was stirred for 4 hours. After the reaction was complete, the mixture was diluted with K ₂ CO ₃ saturated solution and extracted with DCM at each time.

The combined organic layers (organic layers) is over anhydrous MgSO ₄ , Filtered, and concentrated in vacuo to give isothiocyanate compound (2a-1).

3. Isoxazole ( Isooxazole ) synthesis

1.0 eq of the azide compound (1a-b) synthesized in the above manner and an isothiocyanate compound (2a-1; 1.0 eq) dissolved in 0.2M dioxane were mixed and stirred. Then, triphenylphosphine _{a; (triphenylphosphine, PPh 3 1.0 eq} ) was added.

The mixture was stirred for 4 hours while heating to 90 DEG C, and after the reaction was completed, the temperature was cooled to room temperature, and the solvent was removed by applying pressure.

After removal of the solvent, the residue was purified by flash column chromatography on silica gel using ethylacetate (EtOAc) / hexane (1:10) as the mobile phase to give the title compound as an eluent (3a-j, 32% yield) .

4. Cross coupling

The synthesized 3e compound, 3-pyridinylboronic acid (1.5 eq) or phenylboronic acid (1.5 eq) and K ₂ CO ₃ Dissolved Pd (dppf) Cl ₂ (0.5 mol%) was added to a mixed solution of 0.2M DMF and the mixture was stirred at 90 ° C for 10 hours and then extracted with ethyl acetate (ethyl acetate) and water for each hour.

The combined organic layers were dried under anhydrous MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by clean column chromatography on silica gel using an EtOAc / hexane (1: 5) mobile phase to give the following compounds 11 (3k) and 12 (3l) as eluants (3k and 3l, ).

[Compound 11]

N- (5- ( Ethylsulfonyl )-2- Methoxyphenyl ) -5- (4- (pyridin-3-yl) phenyl) Oxazole -2- Amine  [N- (5- (ethylsulfonyl) -2-methoxyphenyl) -5- (4- (pyridin-3-yl) phenyl) oxazol-2-amine; Compound (3k)]

White ^{solid, mp = 183-185 ℃, 1 H} NMR (300 MHz, DMSO-d 6) δ 10.52 (s, 1H), 9.86 (br s, 1H), 8.68-9.02 (m, 1H), 7.41-7.88 (m, 9H), 7.08-7.38 (m, 1H), 3.83-4.20 (m, 3H), 3.24 (br d, J = 7.70 Hz, 2H), 1.15 (br t, J = 7.34 Hz, 3H); MS (FAB) m / z 436 (MH ^&lt; ⁺ & gt ^; ).

[Compound 12]

5- (biphenyl-4-yl) -N- (5- ( Ethylsulfonyl )-2- Methoxyphenyl ) Oxazole -2- Amine  [5- (biphenyl-4-yl) -N- (5- (ethylsulfonyl) -2-methoxyphenyl) oxazol-2-amine; Compound ( 3l)]

Yellow ^{solid, mp = 203.0-203.2 ℃, 1 H} NMR (300 MHz, DMSO-d 6) δ 10.52 (s, 1H), 9.81 (s, 1H), 8.70-8.84 (m, 1H), 7.18-7.85 ( m, 11H), 3.90-4.06 (m, 3H), 3.14-3.28 (m, 2H), 1.05-1.18 (m, 3H); MS (FAB) m / z 435 (MH ^&lt; ⁺ & gt ^; ).

< Example  2> Confirmation of inhibition of kinase activity

Compound 11 and compound 12 synthesized in the same manner as in Example 1 were confirmed to have an inhibitory effect on RTK kinase activity.

Various kinase activity inhibitory effects of Compound 11 and Compound 12 at 0.1 μM concentrations were measured using the kinase profiling service of Eurofins (UK) and Reaction Biology (USA), and are shown in Table 2 below.

Kinase Compound 11 (0.1 [mu] M) Active% Compound 12 (0.1 [mu] M) Active% Abl 68 72 ARK5 73 71 CDK1 / cyclinB 78 77 CDK5 / p35 75 72 cKit (h) 74 78 cKit ( D816H ) (h) 64 88 cKit ( V560G ) (h) 41 57 Flt1 (h) 18 28 Flt3 ( D835Y ) (h) 6 11 Flt3 ( ITD ) (h) 8 15 Flt3 (h) 8 20 Flt4 (h) 2 One PDGFRα (h) 78 77 PDGFR? (h) 79 84

As shown in Table 2, Compound 11 and Compound 12 showed 23 kinds of kinase activity inhibitory effects. In particular, cKit (V560G), FLT1, FLT3, FLT3 (D835Y) and FLT3 (ITD) , And showed very excellent inhibitory effect on FLT4 kinase.

In addition, the half-maximal inhibitory concentration (IC ₅₀ ) of the compound 11 and compound 12 was confirmed as shown in Table 3 with respect to the kinase exhibiting the above-mentioned significant inhibitory effect.

IC ₅₀ Compound 11
( μM ) Compound 12
( μM ) FLT3 0.025 0.008 FLT3 (D835Y) 0.009 0.007 FLT3 (ITD) 0.004 0.016

As shown in Table 3, Compound 11 and Compound 12 showed excellent inhibitory effect at 50% inhibition concentration of nM for D835Y and ITD mutant FLT3 kinase in FLT3 and FLT3 genes.

< Example  3> Various human body Cancer cell  Check the effect on

The inhibitory effect of the compound 11 and the compound 12 synthesized in Example 1 on the cancer cell proliferation inhibitory effect on the cell growth rate of various cancer cell lines was confirmed.

First, the human lung cancer cell line H1299, the human breast cancer cell line MDA-MB231, the human colorectal cancer cell line HCT116, the human acute leukemia cell line MV4-11, and the human colon cancer cell line CCD18-Co purchased from the American Type Culture Collection (ATCC) (Dulbecco's modified Eagle's medium, WELGENE) supplemented with 10% fetal bovine serum (FBS, WELGENE, Korea), 100 ug / ml streptomycin and 100 unit / ml penicillin (WELGENE) or MEM (MV4-11; WELGENE) medium at 5% CO ₂ and 37 ° C, respectively.

One. STAT5  Identification of inhibition of phosphorylation

Cultured MV4-11 leukemia cells were treated with Compound 11 and Compound 12 and confirmed by Western blotting to inhibit phosphorylation of STAT5, a lower signaling system of FLT3.

After treating compound 11 or compound 12 at a concentration of 0.1, 1, 2, 5 or 10 μM for 1 hour, cells were obtained and a cell lysate was obtained by a method commonly used in the art. The cell lysate was separated by performing electrophoresis using 9% gradient SDS-PAGE, and transferred to a nitrocellulose membrane (BioRad, Hercules, CA, USA). The membranes were blocked with 5% skim milk and incubated with anti-phospho-STAT5 antibody (Santa Cruz Biotechnology, USA) as a primary antibody and anti-beta actin (β-actin, Santa Cruz Biotechnology ) Was reacted for 1 hour. Thereafter, horseradish peroxidase-conjugated anti-mouse IgG antibody (Santa Cruz Biotechnology) was incubated and visualized using an ECL system (GE, USA).

As a result, it was confirmed that the phosphorylation of STAT5, a substrate protein of FLT3 kinase, was suppressed from 1 hour in MV4-11 leukemia cells treated with Compound 11 or Compound 12 as shown in Fig.

2. Cancer cell line  Confirm survival inhibition effect

Various cell lines cultured by the above method were dispensed into 96-well plates at a number of 3 × 10 ³ , and then cultured for 24 hours. Then, the compound 11 and compound 12 at a concentration of 10 μM synthesized in the same manner as in Example 1 were treated respectively, Lt; / RTI &gt; 0.5 mg / mL MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) solution was then added to each well and further incubated at 37 ° C for 3 hours. After the culture was completed, the supernatant was removed, and the formazan crystals formed were dissolved in DMSO. The resultant solution was measured at 590 nm using a spectrophotometer (Lab Systems, USA), and the control group was treated with DMSO.

The results are shown in Table 4 as relative growth rates relative to 100% survival rate.

Experimental group DMSO (control) Compound 11 Compound 12 MV4-11
Blood cancer cells 100 ± 10.78 15.15 + 0.26 14.69 ± 0.82 DLD-1
Colon cancer cells 100 ± 2.31 45.15 + 0.26 44.69 + - 0.82 H460
Lung cancer cell 100 ± 5.25 76.40 ± 2.21 58.61. + -. 3.42 MDA-MB231
Breast cancer cell 100 ± 4.70 72.53 + - 3.84 45.91 + - 4.22 CCD18-Co
Colonic normal cell 100 ± 1.25 98.20 + - 4.85 95.52 ± 1.22

As shown in Table 4, the compounds 11 and 12 according to the present invention showed excellent proliferation inhibitory effects in lung cancer, breast cancer, colon cancer and leukemia cells, and the cell viability of CCD18-Co normal cells was not inhibited at the same concentration It was confirmed that there was a selective cancer cell proliferation inhibiting effect.

In addition, the compounds synthesized in Example 1 were treated with MV4-11, which is an acute leukemia cell, and derivatives 2 to 12 synthesized in Example 1, and the inhibitory effect on cell survival was confirmed.

Handle 3 × 10 After ³ Conger channel dispensed in 96-well plates of cultured for 24 hours, and then the compound 2 to compound 12 synthesized in Example 1 of 10 μM and each cell was cultured for 48 hours. A 0.5 mg / mL MTT solution was then added to each well and further incubated at 37 [deg.] C for 3 hours. After incubation, the supernatant was removed and the formazan crystals formed were dissolved in DMSO and measured at 590 nm using a spectrophotometer.

The control group was treated with DMSO and the cell survival rate was expressed as a relative value to the survival rate of 100% as shown in Table 5 below.

Experimental group Cell survival rate % ) DMSO 100 ± 4.34 Compound 2 32.43 ± 1.77 Compound 3 59.25 + 2.09 Compound 4 44.16 ± 1.49 Compound 5 29.30 ± 1.34 Compound 6 30.89 ± 1.00 Compound 7 34.30 + - 0.80 Compound 8 34.58 ± 1.19 Compound 9 65.75 ± 2.11 Compound 10 79.53 + - 4.04 Compound 11 29.39 + - 0.53 Compound 12 34.11 + 1.19

Meanwhile, cleavage of caspase-3 (caspase-3) involved in apoptosis and PARP (poly-ADP ribose polymerase) involved in DNA repair was confirmed by Western blotting. At this time, proteins and cleaved PARP proteins were detected with caspase-3 antibody (Cell signaling technology, Inc. USA) and PARP-1 antibody (PARP-1, Cell signaling technology, Inc. USA).

As a result, expression of caspase-3, a gene involved in apoptosis-induced apoptosis in MV4-11 leukemia cells treated with Compound 11 and Compound 12, and expression of PARP (poly-ADP ribose polymerase) It was confirmed that the cutting was increased.

< Example  4> Confirmation of anticancer effect by combination treatment

1. Identification of anticancer effect by combination treatment with anticancer agent

MV4-11 leukemia cells cultured as in Example 3 were treated with 2 μM of Compound 11 or Cisplatin (5 μM), a known anticancer drug of 12 μM and 5 μM, for 48 hours, Survival rate was analyzed.

As a result, as shown in FIG. 3, the cell proliferation inhibitory effect of the test group treated with cisplatin and compound 1 was more than 40%, compared with the control group treated with cisplatin, which was an anticancer agent, %.

From the above results, it was confirmed that the compound 11 and the compound 12 according to the present invention can be used as an anticancer agent adjuvant which can enhance the therapeutic effect of the anticancer drug.

2. Confirmation of anticancer effect by radiation irradiation treatment

The cultured human blood cancer cell MV4-11 cell line was treated with 10 μM of Compound 11 and Compound 12 and 1 Gy of irradiation, followed by incubation for 48 hours. Then, annexin V and propidium iodide (PI) After staining and analysis using a FACSort flow cytometer (Becton dickinson, USA), total apoptosis was determined by summing up early and late apoptotic cells.

As a result, apoptosis-induced apoptosis in the MV4-11 human blood cancer cell line was induced 2.1-fold and 1.8-fold, respectively, as compared with the radiation alone group, as shown in FIG.

From the above results, it was confirmed that the compound 11 and the compound 12 of the present invention can be used as a radiotherapy adjuvant which can enhance the radiation treatment effect.

Hereinafter, formulation examples of the composition containing Compound 11 or 12 according to the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

&Lt; Prescription Example 1 > Prescription Example of Pharmaceutical Composition

1. Manufacturing of powder

20 mg of the compound, 100 mg of lactose and 10 mg of talc were mixed and filled in an airtight container to prepare a powder.

2. Preparation of tablets

20 mg of the compound, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed and tableted according to a conventional preparation method.

3. Preparation of capsules

10 mg of the compound, 100 mg of the corn starch, 100 mg of the lactose and 2 mg of the magnesium stearate were mixed, and the above components were mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

4. Preparation of injections

10 mg of the compound, sterile distilled water suitable amount for injection, and pH adjuster were mixed, and the contents were adjusted to the above contents in the amount of 2 ml per ampoule according to the usual injection preparation method.

5. Manufacture of Ointment

After mixing 10 mg of the compound, 250 mg of PEG-4000, 650 mg of PEG-400, 10 mg of white petrolatum, 1.44 mg of methyl p-hydroxybenzoate, 0.18 mg of propyl p-hydroxybenzoate and the remaining amount of purified water, An ointment agent was prepared.

&Lt; Prescription Example 2 >

1. Manufacture of health food

Compound 1 mg Vitamin A acetate 70, Vitamin E 1.0 mg Vitamin B 1 0.13 mg Vitamin B 2 0.15 mg Vitamin B 6 0.5 mg Vitamin B 12 0.2 비 Vitamin C 10 mg Biotin 10 (Ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium phosphate monobasic 15 mg, dibasic calcium phosphate 55 mg, nicotinate amide 1.7 mg, folic acid 50 mg, calcium pantothenate 0.5 mg) Mg of calcium citrate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride) were mixed to prepare a granule, and a health food was prepared according to a conventional method.

2. Manufacture of health drinks

1 mg of the compound, 1000 mg of citric acid, 100 g of the oligosaccharide, 2 g of the plum concentrate, 1 g of taurine and purified water were added to make a total of 900 mL, and the above components were mixed according to a conventional health drink manufacturing method, The solution was filtered and sterilized in a sterilized 2 L container, and then refrigerated.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (14)

delete A pharmaceutical composition for preventing or treating leukemia comprising a compound represented by the following formula (1) or a pharmaceutically usable salt thereof as an active ingredient.
[Chemical Formula 1]

In Formula 1,
X is any one selected from nitrogen and carbon. [Claim 2] The pharmaceutical composition for preventing or treating leukemia according to claim 2, wherein the compound represented by Formula 1 inhibits receptor tyrosine kinase activity. delete The pharmaceutical composition according to claim 2, wherein the leukemia is selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia. 1. A composition for promoting an anti-cancer effect of leukemia comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]

In Formula 1,
X is any one selected from nitrogen and carbon. [Claim 7] The composition according to claim 6, wherein the leukemia anti-cancer effect enhancing composition is treated with radiation. delete delete delete [Claim 7] The composition according to claim 6, wherein the leukemia is selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia and chronic lymphocytic leukemia. A health food for preventing or ameliorating leukemia, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]

In Formula 1,
X is any one selected from nitrogen and carbon. delete [Claim 12] The health food for preventing or ameliorating leukemia according to claim 12, wherein the leukemia is selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia and chronic lymphocytic leukemia.

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