KR101688978B1 - Novel 2-Aminothiazole Derivatives and Composition for Treating Cancer Comprising the Same - Google Patents

Abstract

The present invention relates to novel 2-aminothiazole derivatives and their use for anti-cancer. The 2-aminothiazole derivative of the present invention inhibits the growth and proliferation of various cancer cells. The anticancer activity induces apoptosis (apoptosis) through activation of caspase-3 in cancer cells, - arrest in the G1-phase. The 2-aminothiazole derivatives of the present invention can be very usefully developed as anticancer drugs.

Description

[0001] The present invention relates to a novel 2-aminothiazole derivative and a composition for anticancer comprising the same,

The present invention relates to a novel 2-aminothiazole derivative, a pharmaceutically acceptable salt thereof and an anticancer composition containing the same as an active ingredient.

Cancer, also called malignant neoplasm, causes major health problems globally as a disease that causes a group of cells to develop uncontrolled growth and infiltration and sometimes metastasis. Cancer is a disease caused by uncontrolled cell proliferation due to defects in cell cycle regulation. Cancer is also considered a disease due to uncontrollable cell proliferation in which survival signals are continuously generated to suppress apoptosis. Thus, modulation of cell cycle and apoptosis is considered an effective cancer therapy [Blank, M .; Shiloh: Programs for cell death. Cell Cycle 6: 686-695, 2007). However, it takes years to develop a cancer-treating drug to actually benefit the patient. This period is a very long time and the development period of new drugs needs to be shortened in order for patients to quickly benefit from new technologies and drug development ideas.

Combinatorial chemistry has been widely used as a technology and tool from biology and chemistry to promote the discovery and development of new drugs [1, 2].

Thiazole derivatives exist in many natural products and compounds and have various pharmacological activities such as anticancer activity, antiviral activity, antibiotic activity, antifungal activity and anti-inflammatory activity [Conrath, U .; Pieterse, CM; Mauch-Mani, B. Trends. Plant Sci. 2002, 7, 210]. Among them, 2-aminothiazole derivatives have antitumor activity through kinase inhibition [3-5].

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The inventors searched for a strong anticancer agent from a compound library using a cell-based high-throughput screening system (HTS) as a process of searching for an anticancer agent. Based on the fact that many aminothiazoles have anticancer activity and can be easily synthesized at a high yield based on the facts known in the prior art [6-11], the method disclosed in the conventional literature [12] 2-aminothiazole derivatives were prepared by changing the position of the 2nd amino group and the 5th substituent in a high-speed parallel format. From the synthesized compound library, the inhibitory activity against cancer cells and the cell cycle arrest were examined. And a novel aminothiazole compound having an activity of inducing apoptosis were searched and confirmed to complete the present invention.

It is therefore an object of the present invention to provide novel 2-aminothiazole derivatives or pharmaceutically acceptable salts thereof.

Another object of the present invention is to provide an anticancer composition comprising the novel 2-aminothiazole derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention relates to a novel 2-aminothiazole derivative represented by the following general formula (1) or a pharmaceutically acceptable salt thereof:

R ₁ is phenyl, halogen-substituted phenyl, alkyl having 1 to 4 carbon atoms, alkyl having 1 to 4 carbon atoms substituted with phenyl, and R ₂ is alkyl having 1 to 9 carbon atoms, Lt; / RTI > alkyl of 1 to 4 carbon atoms, or a furyl substituted alkenyl of 1 to 4 carbon atoms.

The term "alkyl" used in the above formula (1) means a straight-chain or branched-chain saturated aliphatic hydrocarbon.

The term "halogen" refers to a halogen group element and includes, for example, fluoro, chloro, bromo and iodo, preferably fluoro, chloro or bromo.

The term "furyl" means a furan substituent which is a heterocyclic organic compound consisting of a five-membered aromatic ring consisting of one oxygen atom and four carbon atoms.

The term "phenyl" means a substituent having the formula C ₆ H ₅ - and wherein six carbon atoms form a cyclic ring structure.

The term "substituted" means that the substitution can occur at one or more positions and, unless otherwise stated, the substitution at each substitution site is independently selected from the specified optional substitutions.

According to a preferred embodiment of the present invention, the derivatives of the present invention are those wherein R ₁ is phenyl, fluorophenyl, CH ₂ -phenyl or methyl, R ₂ is propyl, n-nonyl, CH 2-furyl or ₂ -CH ₂ CH ₂ = CH ₂ is 2-furyl.

The compound of the present invention has cancer cell growth inhibitory activity. As demonstrated in the following examples, the compounds of the present invention induce apoptosis (apoptosis) through caspase-3 activation in cancer cells and inhibit cancer cells in the G1-phase of the cell cycle arrest. Therefore, the compounds of the present invention can be usefully used for anticancer applications.

According to another aspect of the present invention, there is provided an anticancer composition comprising the 2-aminothiazole derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

R ₁ is phenyl, halogen-substituted phenyl, alkyl having 1 to 4 carbon atoms, alkyl having 1 to 4 carbon atoms substituted with phenyl, and R ₂ is alkyl having 1 to 9 carbon atoms, Lt; / RTI > alkyl of 1 to 4 carbon atoms, or a furyl substituted alkenyl of 1 to 4 carbon atoms.

According to a preferred embodiment, the derivative is R ₁ is selected from phenyl, fluorinated phenyl, CH ₂ - phenyl, or methyl, R ₂ is n- propyl (propyl), n- octyl (nonyl), CH ₂ -CH ₂ is a 2-furyl or CH ₂ = CH ₂ -2- furyl derivatives.

According to a more preferred embodiment of the present invention, the derivative is R ₁ is phenyl and R ₂ is CH ₂ -CH ₂ -2-furyl or CH ₂ = CH ₂ -2-furyl.

According to a more preferred embodiment of the present invention, the derivative is R ₁ is para-fluorophenyl and R ₂ is CH ₂ -CH ₂ -2-furyl or CH ₂ = CH ₂ -2-furyl.

According to a more preferred embodiment of the present invention, the derivative is R ₁ is CH ₂ -phenyl and R ₂ is CH ₂ -CH ₂ -2-furyl.

According to a more preferred embodiment of the present invention, the derivative is R ₁ is methyl and R ₂ is CH ₂ -CH ₂ -2-furyl or CH ₂ = CH ₂ -2-furyl.

As used herein, the term "IC ₅₀ " refers to the concentration of a particular compound required to inhibit 50% of the specific activity measured.

The term "pharmaceutically acceptable salts" refers to conventional acid-addition salts or base-addition salts formed from suitable non-toxic organic or inorganic acids, or non-toxic organic or inorganic bases, possessing the biological effectiveness and properties of the compounds of the present invention . Exemplary acid-addition salts include acid addition salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, And acid-addition salts derived from organic acids such as formic acid, malic acid, lactic acid, fumaric acid, and the like. Exemplary base-addition salts include base-addition salts derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides, such as tetramethylammonium hydroxide. To obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of a compound, chemically modifying a pharmaceutical compound (i.e., drug) with a salt is a technique well known to pharmaceutical chemists. For this, reference can be made to the following references: [H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457].

The term "pharmaceutically acceptable" means that, for example, pharmaceutically acceptable carriers, excipients, etc., are pharmacologically acceptable and substantially non-toxic to a patient to whom a particular compound is administered.

According to a preferred embodiment of the present invention, the composition of the present invention can be produced in the form of a pharmaceutical composition for the treatment or prevention of cancer.

According to another preferred embodiment of the present invention, the cancer to be treated or prevented in the present invention is not limited to a specific cancer, and examples thereof include breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, Ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer Cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer, bone cancer, neural cancer, squamous cell carcinoma and the like. More preferred are lung cancer, uterine cancer, skin cancer, neural cancer, colon cancer or squamous cell carcinoma.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier in addition to a 2-aminothiazole derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the oral dosage amount of the pharmaceutical composition of the present invention is preferably 0.001-1000 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by local application to the skin, intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, .

The concentration of the 2-aminothiazole derivative of the active ingredient contained in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, and the like, and is not limited to a specific range of concentration.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The following schemes and the related discussion describe a general methodology for the preparation of the novel compounds of the present invention.

Scheme 1

A 2-aminothiazole derivative is prepared by varying the 2-amino-position and the 5-position substituent of the 2-aminothiazole core in a high-speed parallel format. Specific synthesis methods thereof can be found in the following references: [Helal, C. J .; Sanner, M. A .; Cooper, C. B .; Gant, T .; Adam, M .; Lucas, J. C .; Kang, Z .; Kupchinsky, S. Ahlijanian, M. K .; Tate, B .; Menniti, F. S .; Kelly, K .; Peterson, M. Bioorg. Med. Chem. Lett. 2004, 14, 5521].

The change in position 2 of the 2-amino-1,3-thiazole core can be achieved through the condensation reaction of thiourea with chloroaldehyde, while thiourea and chloroaldehyde are commercially available , Or may be obtained using NSC under proline catalyst [Halland, N .; Braunton, A .; Bachmann, S .; Marigo, M .; Jorgensen, K. A. J. Am. Chem. Soc. 2004, 126, 4790]. Changes in the acylamino moiety can be achieved by acylating various carboxylic acids in Scheme 1 above.

The present invention relates to novel 2-aminothiazole derivatives and their use for anti-cancer. The 2-aminothiazole derivative of the present invention inhibits the growth and proliferation of various cancer cells. The anticancer activity induces apoptosis (apoptosis) through activation of caspase-3 in cancer cells, - arrest in the G1-phase. The 2-aminothiazole derivatives of the present invention can be very usefully developed as anticancer drugs.

1 is a graph showing the effect of the compound H154 (a compound synthesized by the method of the following Example 1-1 and 3- (furan-2-yl) -N- (5-phenylthiazol-2-yl) propanamide] Dependent growth and time-dependent growth inhibition. Panel A shows the results of MTT cell proliferation assay and cell growth of 10 - 500 nM H154 treated for 24 hours. Panel B is the result of measuring the proliferation inhibitory effect in cells treated with 50 nM of H154 for a certain period of time.
Figure 2 shows the result that compound H154 induces apoptosis in HrpH2 cells. Panel A was obtained by treating cells with 100 nM of H154 for 72 hours and then staining with DAPI (10 μg / ml of PI, 100 μg / ml of Rnase A). This shows a typical form of apoptosis such as a reduction in the volume of cells after the compound treatment and the appearance of an apoptotic body. Panel B shows the results of measuring the nucleosomal DNA fragmentation in a time- and dose-dependent manner. The cells were treated with 100 nM of H154 for the indicated time. Total genomic DNA was prepared, separated on 1.5% agarose gel, and visualized by staining ethidium bromide (EtBr).
Figure 3 shows the results of analysis of caspase activity using Ac-DEVD-AMC as a substrate based on the fact that caspase-3 activation is associated with H154 induced apoptosis.
Figure 4 shows the results of G1 / S arrest in H154 treated HepG2 cells. Cells were treated with H154 for a certain period of time, and the cells were stained with PI. DNA content was analyzed by flow cytometry at each time point.
FIG. 5 shows the results of analysis of protein levels of p53L, p21 and cdk2 by Western blotting at the designated time points after treatment of cells with 100 nM of H154.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  1. Compound library ( chemical library )

In order to prepare a library of compounds having 2-amino-1,3-thiazole cores, the change in position 5 of the building block proceeded by the reaction of thiourea and chloroaldehyde. The thiourea and chloroaldehyde are either commercially available or can be obtained using NCS in the presence of a proline catalyst. In addition, DCC and HOBT were treated in ethanol to adjust the range of various carboxylic acids to acylate the amino moiety at position 2.

Example  1-1. 3- (furan-2-yl) -N- (5- Phenyl thiazole -2 days) Propanamide Preparation of [Compound 1c] Step 1: 5- Phenyl thiazole -2- Amine  Produce

A mixture of 2-chloro-2-phenylacetaldehyde (1.7 g, 11.28 mmol) and thiourea (0.86 g, 11.28 mmol) dissolved in ethanol (5 ml) was stirred at 70-80 ° C overnight. The reaction mixture was diluted with water, and then an aqueous ammonia solution was added to make a basic solution. The reaction mixture was extracted with dichloromethane. The separated organic layer was washed with anhydrous magnesium sulfate The moisture was removed. The reaction was concentrated under reduced pressure to remove the solvent and then separated by chromatography. The purified product was recrystallized from hexane-ethyl acetate to give pale yellow crystalline 5-phenylthiazol-2-amine (907.5 mg, 45.6%). ^{mp (206.7-207 ℃) 1 H NMR} (600 MHz, CDCl3) δ 7.47 - 7.18 (m, 6H), 4.96 (s, 2H). ¹³ C NMR (151 MHz, CDCl 3) 隆 166.79, 134.35, 132.14, 129.30, 128.90, 127.05, 125.64, 77.22, 77.01, 76.80, 0.00.

Step 2: 3- (Furan-2-yl) -N- (5- Phenyl thiazole -2 days) Propanamide  Produce

(150 mg, 1.07 mmol), DCC (220 mg, 1.07 mmol) and HOBT (14.4 mg, 0.107 mmol) were successively dissolved in anhydrous dichloromethane and treated under nitrogen atmosphere with 1-2 Lt; / RTI > 5-Phenylthiazol-2-amine (188.5 mg, 1.07 mmol) was added and the mixture was stirred for two days, and then the reaction was terminated by using dichloromethane and distilled water. The separated organic layer was dehydrated with anhydrous magnesium sulfate, concentrated under reduced pressure to remove the solvent, and then separated by chromatography. The purified product was recrystallized from ethanol to obtain white solid (3- (furan-2-yl) -N- (5-phenylthiazol-2-yl) propanamide ( Compound 1c ) (74.8 mg, 23.5%). ^{mp (225.5-229.0 ℃) 1 H NMR} (600 MHz, CDCl3) δ 7.64 (s, 1H), 7.57 - 7.52 (m, 2H), 7.41 (t, J = 7.7, 2H), 7.35 - 7.29 (m, 3H), 6.32-6.29 (t, 1H), 6.11 (d, J = 3.2,1H), 3.17 (t, J = 7.6,2H), 2.97-2.90 (t, 2H). ¹³ C NMR (151 MHz, CDCl3) 灌 169.73, 158.60, 153.56, 141.46, 133.01, 131.78, 131.47, 129.09, 128.27, 127.90, 126.13, 110.38, 105.87, 34.86, 23.69.

Examples 1-2. Preparation of compounds 1a, 1b, and 1d

Butyric acid, decanoic acid and 2-furanacrylic acid were used instead of 3- (furan-2-yl) propionic acid in a similar manner to the step 2 of Example 1-1. 5-phenylthiazole-2-amine (synthesized by the method of Example 1, Step 1) to synthesize the compounds 1a, 1b, and 1d, respectively.

Examples 1-3. Preparation of compounds 3a, 3b and 3c

Similarly to the step 1 of Example 1-1 above, using 2-chloro-3-phenylpropanal instead of 2-chloro-2-phenylacetaldehyde as a starting material, 5- Benzylthiazol-2-amine. The obtained 5-benzylthiazol-2-amine was reacted with 3- (furan-2-yl) propionic acid, butyric acid, and decanoic acid in a similar manner to the step 2 of Example 1-1 to obtain the compounds 3a, 3b, 3c Respectively.

Examples 1-4. Preparation of compounds 4a, 4b, 4c and 4d

Similar to the procedure of Step 1 of Example 1-1 above using 2-chloropropanal instead of 2-chloro-2-phenylacetaldehyde as starting material, 5-methylthiazol-2-amine Were synthesized. The synthesized 5-methylthiazol-2-amine was reacted with 3- (furan-2-yl) propionic acid, butyric acid, decanoic acid and 2-furanacrylic acid respectively in the same manner as in the step 2 of Example 1-1 Compounds 4a, 4b, 4c and 4d were synthesized, respectively.

Examples 1-5. Preparation of compound 2c

(5-bromo-2-thiazolyl) - (2-methylthiazol-2-yl) -methanone obtained by reacting 3- (2-furyl) propionic acid with 2-amino-5-bromothiazole monohydrobromide according to the method of Step 2 of Example 1-1. A solution of 2-furanpropanamide (600 mg, 1.99 mmole), 4-fluorophenylboronic acid (0.42 g, 2.99 mmole) and anhydrous sodium carbonate (1.05 g, 9.95 mmole) in DMF (18 ml) And the mixture was stirred for 1 hour and 30 minutes. After stirring, it was kept under nitrogen condition for 5 minutes or more. Tetrakis (triphenylphosphine) -palladium (0.68 g, 0.588 mmole) was added thereto, and the mixture was stirred at 80 ° C for 1 day. The reaction was extracted with dichloromethane and distilled water. The separated organic layer was dehydrated with anhydrous magnesium sulfate. The reaction mixture was concentrated under reduced pressure to remove the solvent and then separated by column chromatography. The purified product was recrystallized from hexane-ethyl acetate to give 336 mg (54%) of a white crystalline compound 2c. ^{1 H NMR (300 MHz, CDCl3} ) δ10.68 (s, 1H), 7.52 (m, 2H), 7.31 (d, 1H), 7.09 (t, 2H), 6.29 (t, 1H), 6.10 (d, 1H), 3.14 (t, 2H), 2.87 (t, 2H)

Examples 1-6. Preparation of compounds 2a, 2b, 2d

Amino-5-bromothiazole was used in place of 5-phenylthiazol-2-amine [synthesized by the step 1 of Example 1-1], and 3- ( Compounds 2a, 2b and 2d were prepared by reacting butyric acid, decanoic acid, and 2-furanacrylic acid, respectively, in place of furan-2-ylpropionic acid in a similar manner to the synthesis of compound 2c of Example 1-5 Were synthesized.

The compounds synthesized in Examples 1-1 to 1-5 are as follows.

The spectroscopic data of the synthesized compounds are shown in Table 2 below.

Example  2. Cell culture

Human hepatoma cell HepG2 cells were purchased from the American Type Culture Collection (ATCC) and were cultured in RPMI 1640 medium containing 2 mM L-glutamine and 4.5 g / L glucose (GIBCO BRL, Grand Island, NY) The growth was maintained in the DMEM medium supplemented with fetal bovine serum (GIBCO BRL) in an incubator under an atmosphere of humidified air at 37 ° C, 5% CO ₂ -95%.

Example  3. Cytotoxicity test of compound of compound library

Compounds were dissolved in DMSO and made into aliquots and stored at -20 ° C until use. To evaluate the effect of compounds on HepG2 cells, cells (2 X 10 ⁴ cells / well) were inoculated into 96-well plates. The following day, different concentrations of compound were added to a final volume of 200 [mu] l medium / well. To the control wells were added the same volume of DMSO as the added compound. The plates 5% CO ₂ - atmosphere under the supplement was incubated at 37 ℃ for 24 hours. Then, the cultured medium was removed and replaced with 150 μl of 0.5 μg / ml MTT (Sigma-Aldrich Chemical Co., USA). Plates were again incubated at 37 [deg.] C for 4 hours. The reduced MTT dye was dissolved in 150 mu l per well of DMSO. The optical density (OD) was measured at 570 nm using an ELX800 Universal Microplate reader (Bio-Tek Co., CA, USA).

The percentage inhibition was calculated as follows:

% Inhibition = (1 - OD _test / OD _control ) X 100%.

Additional cytotoxicity tests were performed on the following human cancer cell lines using the SRB (sulforhodamine B) method (12) of the Korea Research Institute of Chemical Technology: A549 (non-small cell lung cancer), SK-OV-3 (adenocarcinoma, ascites), SK-MEL-2 (malignant melanoma, femoral skin metastasis), XF498 (central nervous system tumor), HCT15 (colorectal adenocarcinoma), and A431 (squamous cell carcinoma). Doxorubicin was used as a positive control.

Example 4. DNA Fragmentation Analysis

Cells grown at a concentration of 2 x 10 ⁶ cells / ml in a 100 mm dish were exposed to the compounds at various concentrations and at various times. Genomic DNA was prepared using the Wizard Genomic DNA Purification Kit (Promega, Madison, Wis.). DNA was precipitated using isopropanol, separated on 1.5% agarose gel, and visualized by UV irradiation after ethidium bromide (EtBr) staining.

Example 5. Morphological analysis

Cells placed on sterile microscope slides were treated with compounds and cells were washed with PBS and fixed with paraformaldehyde for 30 minutes. Cells were stained with DAPI solution (10 μg / ml PI, 100 μg / ml Rnase A) for 10 minutes. The morphology of the cells was examined using a Leitz phase contrast microscope or a Zeiss live cell imaging fluorescence microscope.

Example  6. Flow cytometry

After diluting the cells in the growth phase, 2 X 10 ⁶ cells were inoculated into 10 ml of medium in a 100 mm culture dish. The cells were exposed to 100 nM of compound for various times and centrifuged at 800 g for 5 minutes. The pellet was mixed with a 1: 1 (v / v) mixture of PBS and 0.2 M Na ₂ HPO4 with 0.1 M citric acid (pH 7.5) and fixed in ice-cold ethanol at 4 ° C for 1 hour. Cells were washed twice with PBS and resuspended in 1 mL of staining solution containing 50 μg / mL PI with 100 μg / mL DNase-free RNase A. The cell suspension was incubated at room temperature for 1 hour and 10,000 cells were analyzed on a FACScalibur flow cytometer (Becton-Dickinson).

Example 7. Western blotting analysis

Cells were lysed in a suspension of aPRO-PREP ^TM protein extraction solution (Intron Biotechnology Inc, Korea) and incubated on ice for 1 hour. Cell lysates were centrifuged and protein content was determined using commercially available kits (BioRad, Mississauga, Canada). After SDS-PAGE, 75 μg of proteins were transferred to an immobilon nitrocellulose membrane (Millipore) at 200 ° C for 3 hours at 4 ° C. The blots were probed using polyclonal rabbit p53 antibody, polyclonal mouse p21 antibody, beta-actin antibody (Cell Signaling Technology, Beverly, MA) and polyclonal rabbit Cdk2 antibody (Santa Cruz, CA). Immunoconjugates were detected using anti-mouse or anti-rabbit peroxidase-conjugated secondary immunoglobulin G antibody (Boehouringer Mannheim, Mannheim, Germany) and electrochemiluminescence Western Blotting Detection Reagents (Amersham, Piscataway, NJ) Respectively.

Example 8. Analysis of caspase activity

Caspase-3 activity was estimated from the release of fluorescent AMC after degradation of the peptide substrate specific for caspase-3 (Ac-DEVD-AMC). The substrate was added to the cell lysate in assay buffer (50 mM HEPES, 100 mM NaCl, 0.1% CHAPS, 10 mM dithiothoureitol, 1 mM EDTA, 10% glycerol, pH 7.4) and incubated at 37 ° C for 3 hours. The degradation of the peptide substrate was monitored by detecting excitation and emission at 380 nm and 460 nm, respectively. Results are expressed as percent change in activity compared to untreated control.

Example  9. Statistical Analysis

All values were expressed as mean ± SD compared to the control. Significant differences were estimated using Student's t-test. Statistical significance was defined as P <0.05. The statistical software program Graphpad prism 5.1 (Graphpad software, Inc, USA) was used for data analysis.

Example 10. Screening of Compounds Having Cell Proliferation Inhibitory Effect from Compound Libraries

High throughput screening (HTS) using a library of compounds is one method of finding biologically active compounds. Thus, the experiments of the present invention examined the cytotoxicity of HepG2 cells using the MTT assay for hundreds of aminothiazole compounds prepared in high-speed parallel format.

(5-phenylthiazol-2-yl) propanamide [Compound synthesized according to the method of Example 1-1, referred to herein as "H154" ] Showed a significant inhibitory effect on cell proliferation (IC ₅₀ value of 0.040 μM). To examine the time dependence, cell proliferation inhibition was tested using MTT assay after treatment of HepG2 cells with 50 nM of H154 for 72 hours. As shown in Figure 1, H154 inhibited cell growth to a significant extent in a time and dose dependent manner.

The cytotoxic effect of various cancer cells was examined. The cytotoxicity test was carried out using the compound H154 and the known cytotoxic substance doxorubicin as a positive control, using the SRB (sulforhodamine B) method currently adopted in the screening method of NCI's in vitro cancer drug. The rate of inhibition of cell proliferation was estimated after continuous exposure of the test material to the six human tumor cell lines A549, SK-OV-3, SK-MEL-2, XF498, HCT15 and A431 for 48 hours.

As shown in the following Table 3, the compound H154 exhibited an antiproliferative effect within the range of the nano-molar concentration, and about 170-fold higher than that of doxorubicin for uterine cancer cells (SK-OV-3) and colorectal cancer cells And 11 times higher anticancer efficacy.

Table 4 below shows the results of cytotoxicity measurement of the various 2-aminothiazole derivatives of the present invention against Hep3B cells by IC ₅₀ values. The IC ₅₀ values measured in Table 2 are the average values of at least two independent measurements taking standard deviation into account. Each IC ₅₀ value was determined from the dose-response curves at six points that were repeated three times.

Example  11. Within liver cancer cells BFA On by Apoptosis  Judo

To test the cytotoxicity of H154, 100 nM of compound was added to the subconfluent cultures of HepG2 cells and cell morphology and DNA damage were examined at various concentrations and durations. As shown in Fig. 2, a DNA ladder was clearly generated by the H154 treatment. Nucleosomal DNA laddering was observed in H154 treated cells, confirming that a group of cells were undergoing apoptotic cell death in a dose- and time-dependent manner.

Example 12. Activation of caspase-3 by H154

The level of caspase-3 in cells exposed to H154 was determined to determine if compound induced apoptosis was associated with activation of caspase. Western blotting data confirmed that levels of pro-caspase-3 were retained in H154-treated cells without change. However, expression levels of caspase active subunits increased in a time-dependent manner. To quantitate the proteolytic activity of caspase-3 in cells, a cell lysate equivalent to a protein from a cell treated with the compound H154 was assayed for their activity by quantitative detection of a fluorimetric tetrapeptide substrate Respectively. As shown in Fig. 3, caspase-3 activity in the cells was increased in a time-dependent manner.

Example 13. Induction of Gl phase termination in HepG2 cells by compound H154

Cell cycle analysis was performed to investigate the mechanism of antiproliferative properties of the compound H154 in HepG2 cells. Analysis of cell cycle distribution by flow cytometry revealed G1 cell cycle arrest (see FIG. 4). This indicates that cell accumulation occurs in the G1-phase in a time-dependent manner. The cells treated with the compound were found to have 51.9%, 64.8%, 66.8% and 76.8% of the cells in the G1- group at 12 hours, 24 hours, 48 hours and 72 hours respectively, whereas the compounds were treated with DMSO The control cells were 42.7% in the G1-group.

Example 14. Effect of Compound H154 on Cell Cycle Regulator

The tumor suppressor gene p53 plays an important role in cell cycle arrest and induction of apoptosis (12). Therefore, protein expression of p53 and p21 was evaluated. As shown in FIG. 5, the amount of p53 and p21 protein was increased in a time-dependent manner by compound H154. The effect of compound H154 on the expression level of the Cdk2 protein, which allows the cells to go from the G1 stage to the S stage, was evaluated. However, H154 treatment did not result in any change in Cdk2 expression levels (FIG. 5).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (6)

delete delete 1. An anticancer composition for liver cancer, ovarian cancer, or colorectal cancer comprising 2-aminothiazole derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]

In Formula 1,
R ₁ is phenyl and R ₂ is CH ₂ -CH ₂ -2-furyl;
R ₁ is phenyl and R ₂ is CH = CH-2-furyl;
R ₁ is para-fluorophenyl and R ₂ is CH ₂ -CH ₂ -2-furyl;
R ₁ is para-fluorophenyl and R ₂ is CH = CH-2-furyl; or
R ₁ is CH ₂ -phenyl and R ₂ is CH ₂ -CH ₂ -2-furyl.
delete The composition according to claim 3, wherein the anticancer composition is a pharmaceutical composition for treating or preventing liver cancer, ovarian cancer, or colon cancer. delete

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