KR100844131B1 - Anticancer drug comprising rhodanine derivatives or their pharmaceutically acceptable salts - Google Patents

Abstract

A rhodanine-based compound or a pharmaceutically acceptable salt thereof is provided to show excellent inhibitory activity on CDC25B phosphatase acting on cell cycle decisively, thereby showing high anticancer activity on cancer cells. A rhodanine-based compound is represented by a formula(1), wherein each Ar1 and Ar2 is independently phenyl or pyridine, which may be substituted by at least two substituents selected from the group consisting of C1-4 alkyl, halogen, C1-7 alkoxy, C3-4 alkenyloxy, C2-4 acyl, cyano or nitro. An anti-cancer agent composition comprises the rhodanine-based compound or a pharmaceutically acceptable salt thereof as an effective ingredient.

Description

Anticancer agent containing a rhodanine-based compound or a pharmaceutically acceptable salt thereof

The present invention relates to a rhodanine-based compound of Formula 1 or a pharmaceutically acceptable salt thereof, and an anticancer agent containing the same as an active ingredient, which has an excellent inhibitory activity against CDC25B dephosphatase which acts critically in the cell division cycle. .

[Formula 1]

Wherein Ar ₁ and Ar ₂ are independently selected from C ₅ ~ C ₁₀ aromatic ring or heteroaromatic ring, wherein Ar ₁ and Ar ₂ is C ₁ ~ C ₄ alkyl group, halogen atom, C ₁ ~ C ₇ alkoxy group It may be substituted with one or two or more substituents selected from C ₃ ~ C ₄ alkenyloxy group, C ₂ ~ C ₄ acyl group, cyan group or nitro group.

Reports of rhodanine-based compounds similar to Formula 1 of the present invention have been filed in PCT with two patents known as Pin1-modulating compounds (WO 2004093803 A2, WO 2004028535 A1). The compounds of the above patents contain some of the chemical structure of ( 1H -pyrazol-4-ylmethylene) rhodanine, but the 1-position of the pyrazole is substituted with methyl and the substituent is located at the 5-position of the pyrazole. It is different from the compound of the present invention. Another similar chemical structure is that the compound of Japanese Patent (JP 55029804) has a pyrazole structure linked to the 5-position of thiazole, which is a pyrazolone structure, which is different from the compound of Formula 1 of the present invention. It is known to be useful as a dye which inhibits development.

CDC25B is a very important dephosphatase that determines the progression to the G2 / M cycle in cell division. When CDC25B activity is inhibited, cell division is inhibited and eventually leads to cell death, resulting in anticancer activity. CDC25B can be used for gastric cancer (Jpn. J. Cancer Res. 1997, 88, 9947), colon cancer (Exp. Cell Res. 1998, 240, 236; Lab. Invest. 2001, 81, 465), lung cancer (Cancer Res. 1998, 58, 4082), brain cancer and laryngeal cancer (Cancer Res. 1997, 57, 2366), and breast cancer (Cancer statistics in Japan 1997 Foundation for Promotion of Cancer Research, Tokyo, Jpn.) Mammary hyperplasia of CDC25B-overexpressed mice (Oncogene 1999, 18, 4561) was observed, and breast cancer was very easy when treated with Casinogen (9,10-dimethyl-1,2-benzanthracene). Has been reported. Therefore, CDC25B is an important target in the development of anticancer drugs, and it can be said that there is a great possibility of developing a new anticancer agent that inhibits CDC25B and has low cytotoxicity against specific cells.

Therefore, the present inventors have studied to develop a compound having a selective inhibitory activity against CDC25B, and as a result, 5- (3-aryl- 1H -pyrazol-4-ylmethylene) rhodanine derivatives have excellent inhibitory activity against CDC25B. The present invention has been completed by confirming that it has selectivity for other similar dephosphoryases and at the same time confirming anticancer effects on various cancer cells.

Accordingly, an object of the present invention is to provide a novel rhodanine-based compound or a pharmaceutically acceptable salt thereof having excellent inhibitory activity on CDC25B and high anticancer effect against cancer cells.

In another aspect, the present invention is to provide a pharmaceutical composition for inhibiting the anti-cancer or dephosphorase (CDC25B) activity containing a new rhodanine-based compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention relates to a new rhodanine-based compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof, and also an anticancer agent and dephosphorylation enzyme containing the new rhodanine-based compound or a pharmaceutically acceptable salt thereof as an active ingredient. (CDC25B) relates to a pharmaceutical composition for inhibiting activity.

[Formula 1]

Wherein Ar ₁ and Ar ₂ are independently selected from C ₅ to C ₁₀ aromatic rings or heteroaromatic rings.

Referring to the present invention in more detail as follows.

The present invention is characterized in that the rhodanine-based compound represented by Formula 1 has an inhibitory activity against CDC25B dephosphoryase, which plays a crucial role in determining the progression of G2 / M in the cell division cycle. In addition, it exhibits selectivity to other dephosphorylase enzymes in the human body and also has the effect of destroying human cancer cells in experiments using nude mice.

Ar ₁ and Ar ₂ in the rhodanine-based compound represented by the formula (1) is independently selected from C ₅ ~ C ₁₀ aromatic ring or hetero aromatic ring, the hetero aromatic ring is selected from nitrogen, oxygen or sulfur in the aromatic ring It means an aromatic ring containing a hetero element. More preferably, Ar ₁ and Ar ₂ are independently selected from phenyl or pyridine, Ar ₁ and Ar ₂ are C ₁ ~ C ₄ alkyl group, halogen atom, C ₁ ~ C ₇ alkoxy group, C ₃ ~ C ₄ It may be substituted with one or two or more substituents selected from alkenyloxy group, C ₂ to C ₄ acyl group, cyan group or nitro group. Among the substituents, the C ₁ to C ₇ alkoxy group may include a C ₃ to C ₆ cycloalkyl group.

More preferably, the rhodanine-based compound of Formula 1 is selected from the compounds of Formula 1-1.

[Formula 1-1]

Wherein X ₁ to X ₃ are independently hydrogen, C ₁ C ₄ alkyl group, halogen atom, C ₁ ~ C ₇ alkoxy group, C ₃ C ₄ alkenyloxy group, C ₂ ~ C ₄ acyl group, a substituent selected from a cyan group or a nitro group, Ar ₂ is the same as defined above.

In Formula 1-1, X ₁ to X ₃ are independently selected from isopropyl group, tertiary butyl group, bromine, iodine, methoxy group, ethoxy group, propyloxy group, allyloxy group or nitro group desirable.

The rhodanine-based compound represented by Formula 1 includes a compound having the following structure.

In addition, the rhodanine-based compound represented by Formula 1 according to the present invention can form a pharmaceutically acceptable salt by a conventional method in the art. Forming salts of pharmaceutically acceptable acids thereof with organic or inorganic acids such as, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, fumaric acid, lactic acid, maleic acid, succinic acid and tartaric acid, or sodium It can be reacted with alkali metal ions such as potassium, to form their metal salts, or with ammonium ions to form another form of pharmaceutically acceptable salt.

In addition, the pharmaceutical composition for inhibiting the anticancer agent or dephosphorylase (CDC25B) activity according to the present invention contains the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient and is a conventional non-toxic pharmaceutical composition An acceptable carrier, adjuvant, excipient, and the like may be added to form a conventional formulation in the pharmaceutical field, for example, oral administration such as tablets, capsules, troches, solutions, suspensions, and the like. In addition, the dosage of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and the weight is 70 kg. It is generally 10-400 mg / day based on adult patients, and may be divided once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.

On the other hand, the method for producing a rhodanine-based compound represented by Formula 1 can be described by dividing into the steps of Scheme 1 and Scheme 2. However, the method for preparing the compound of Formula 1 is not limited to the following scheme and can be variously prepared using other known organic synthesis reactions.

Scheme 1

In Scheme 1, Ar ₁ and Ar ₂ are independently selected from C ₅ ~ C ₁₀ aromatic ring or heteroaromatic ring, more preferably selected from phenyl or pyridine, wherein Ar ₁ and Ar ₂ is C ₁ ~ It may be substituted with one or two or more substituents selected from C ₄ alkyl group, halogen atom, C ₁ ~ C ₇ alkoxy group, C ₃ ~ C ₄ alkenyloxy group, C ₂ ~ C ₄ acyl group, cyan group or nitro group.

Referring to the preparation method according to Scheme 1, the ketone compound represented by Formula 2 and the hydrazine compound represented by Formula 3 are dissolved in a suitable solvent and reacted with a catalyst to synthesize a hydrazone derivative represented by Formula 4 do. Here, the solvent is mainly alcohols such as methanol or ethanol, but water or other organic solvents may be used, and a small amount of acetic acid is good as a catalyst, but the same result can be obtained by reacting with a weak acid such as benzoic acid as a catalyst. The compound represented by Formula 4 thus obtained is 1,3-diaryl-pyrazole-4 represented by Formula 5 using anhydrous dimethylformamide (DMF) as a solvent and phosphorusoxy chloride (POCl ₃ ). Obtain a carboxaldehyde derivative. The same result can be obtained by using a reagent such as thionyl chloride or oxalyl chloride instead of phosphorusoxy chloride in the reaction.

Scheme 2

Ar ₁ and Ar ₂ in Scheme ₂ are as defined in Scheme 1, and pyrazolcarboxaldehyde of Formula 5 obtained from Scheme 1 and rhodanine are reacted with a catalyst under an appropriate solvent to obtain a compound of Formula 1. In Scheme 2, the solvent is benzene or toluene, and other organic solvents may be used. The catalyst is preferably a mixture of acetic acid and piperidine, but salts of weak acids such as acetic acid and benzoic acid, pyridine, amines, and aniline. The same result can be obtained by using a weak base alone.

In order to improve the performance of the compound of Formula 1, inorganic salts such as sodium, potassium, and calcium, or organic salts of amines and anilines can be converted. To this end, compound 1 is dissolved in water or organic solvents such as alcohol, ether, and the like. Salts can be obtained by adding a base containing calcium, amines and anilines and evaporating the solvent. In addition, when Ar ₁ or Ar ₂ is a heteroaromatic ring containing a nitrogen atom, salts such as hydrochloric acid and sulfuric acid may be prepared. Such salt making methods are generally well known.

The present invention as described above will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Preparation of Compound of Formula 1

Preparation Example 1 Preparation of Phenylhydrazone of Acetophenone

Acetophenone (6.0 g, 50.0 mmol) was stirred at room temperature was placed with phenyl hydrazine (5.0 mL, 50.0 mmol), glacial acetic acid (137.00 μ L, 2.50 mmol) as a catalyst was dissolved in 50 mL of absolute ethanol for 3 hours. After the reaction was completed, the solid formed by adding ice water was filtered and washed with distilled water (20 mL × 3 times) and n-hexane (20 mL × 1 times). Drying the filtered solid well gives 7.0 g (66.6%) of the desired product.

Preparation Example 2 Preparation of 1,3-diphenyl-1hydro-pyrazole-4-carboxyaldehyde

Phosphorus oxychloride (3.73 mL, 40.00 mmol) was added to 5 mL of anhydrous dimethylformamide and stirred at 0 ° C. for 1 hour. Phenylhydrazone (4.2 g, 20.00 mmol) of acetophenone dissolved in 10 mL of anhydrous dimethylformamide was slowly added dropwise, and the temperature was raised to 70-80 ° C. and stirred for 6 hours. The temperature was cooled to 0 ° C. with ice water, slowly added dropwise with 30% aqueous sodium hydroxide solution to pH 7-8, and the resulting solid was filtered and washed with water (10 mL × 3 times). Drying of the filtered solid yields 4.0 g (80.5%) of the desired product.

¹ H NMR (200 MHz, CDCl 3) δ 7.37-7.84 (m, 9H), 8.56 (s, 1H), 10.06 (s, 1H).

Example 1-1 5- (1,3-diphenyl-1 H -Pyrazol-4ylmethylene) -2-thiooxothiazolidin-4-one (5- (1,3-Diphenyl-1) H -pyrazol-4-ylmethylene) -2-thioxothiazolidin-4-one) (compound 11)

1,3-diphenyl -1 H - carbazol-4-aldehyde (1,3-Diphenyl-1 H -pyrazole -4- carbaldehyde) (250 mg, 1.0 mmol) was dissolved in 20 mL of glacial acetic acid (glacial AcOH) After rhodanine (133 mg, 1.0 mmol) and sodium acetate (98 mg. 1.2 mmol) was added and refluxed for 24 hours. After the reaction was completed, the solid formed by adding ice water was filtered. Washed three times with distilled water and once with n-hexane (n-hexane). The yellow solid was dried to give 300 mg of the desired 5- (1,3-diphenyl-1 H -pyrazol-4ylmethylene) 2-thiooxothiazolidin-4-one.

¹ H NMR (200 MHz, CDCl ₃ ) δ 7.27 (s, 1H), 7.49-7.83 (m, 10H), 8.55 (s, 1H).

Example 1-2 5- [3- (3-cyclopentyloxy-4-methoxyphenyl) -1-phenyl-1 H -Pyrazol-4ylmethylene) -2- Thioxothiazolidine -4-one (5- [3- (3- cyclopentyloxy -4- methoxyphenyl ) -1-phenyl-1 H -pyrazol -4- ylmethylene ]-2- thioxothiazolidin -4-one) (compound 12)

3- (3-cyclopentyloxy-4-methoxyphenyl) -1-phenyl-1 H -pyrazole-4-carbaaldehyde (3- (3-cyclopentyloxy-4-methoxy-phenyl) -1-phenyl-1 H- pyrazol-4-carbaldehyde) (362 mg, 1.0 mmol) was dissolved in 20 mL of glacial acOH, followed by rhodanine (133 mg, 1.0 mmol) and sodium acetate (98 mg. 1.2 mmol) was added and refluxed for 24 hours. After the reaction was completed, the solid formed by adding ice water was filtered. Washed three times with distilled water and once with n-hexane (n-hexane). The yellow solid was dried to give 300 mg (63%) of the title compound.

¹ H NMR (200 MHz, CDCl ₃ ) δ 3.83 (s, 3H), 4.86 (m, 1H), 7.12-8.06 (m, 9H), 8.73 (s, 1H), 13.74 (br, 1H, NH).

[ Example  1-3] 5- [3- (3,5- Dibromo -4- Propyloxy - Phenyl )-One- Phenyl -1H- Pyrazole -4- Ilmethylene ]-2- Thioxothiazolidine 4-one (5- [3- (3,5- Dibromo -4- propyloxy -phenyl) -1-phenyl-1H-pyrazol-4-ylmethylene] -2-thioxo-thiazolidin-4-one) (Compound 13)

3- (3,5-Dibromo-4-propyloxy-phenyl) -1-phenyl-1H-pyrazole-4-carbaaldehyde (3- (3,5-Dibromo-4-propyloxy-phenyl) -1 -phenyl-1H-pyrazol-4-carbaldehyde) (0.5 g, 1.07 mmol) was dissolved in 20 ml of toluene, and a small amount of rhodanine (133 mg, 1.07 mmol), acetic acid (AcOH) and piperidine was added. Water was removed and refluxed using a stark trap. The resulting solid was filtered with ethyl ether and the solid was dried in a desiccator to give the title compound (495 mg, 80%) as yellow.

¹ H NMR (200 MHz, CDCl ₃ ) δ 1.12 (t, 3H, J = 7.3 Hz), 1.84-1.95 (m, 2H), 4.06 (t, 2H, J = 6.3 Hz), 7.40 (s, 1H) , 7.49 (d, 1H, J = 7.3 Hz), 7.61 (t, 2H, J = 7.5 Hz), 7.97 (s, 2H), 8.08 (d, 2H, J = 8.1 Hz), 8.79 (s, 1H) .

Example 1-4 5- [3- (3,5-Dibromo-4-allyloxy-phenyl) -1-phenyl-1H-pyrazol-4-ylmethylene] -2-thiooxothiazoli Din-4-one (5- [3- (3,5-Dibromo-4-allyloxy-phenyl) -1-phenyl-1H-pyrazol-4-ylmethylene] -2-thioxo-thiazolidin-4-one) (Compound 14)

3- (3,5-Dibromo-4-allyloxy-phenyl) -1-phenyl-1H-pyrazole-4-carbaaldehyde (3- (3,5-Dibromo-4-allyloxy-phenyl-1- phenyl-1H-pyrazol-4-carbaldehyde) (0.5 g, 1.08 mmol) was dissolved in 20 ml of toluene, and a small amount of rhodanine (143 mg, 1.08 mmol), acetic acid (AcOH) and piperidine was added. Water was removed using a dean stark trap and refluxed. The resulting solid was filtered through ethyl ether, and the solid was dried in a desiccator to give a yellow title compound (511). mg, 82%).

¹ H NMR (200 MHz, CDCl ₃ ) δ 4.64 (d, 2H, J = 5.7 Hz), 6.12-6.29 (m, 1H), 5.37 (d, 2H, J = 10.2 Hz), 5.55 (d, 2H, J = 17.1 Hz), 7.35-8.10 (m, 8H), 8.78 (s, 1H).

Example 1-5 5- [3- (3,5-bis-cyclopropylmethyloxy-phenyl) -1-phenyl-1H-pyrazol-4-ylmethylene] -2-thiooxothiazolidine- 4-one (5- [3- (3,5-Bis-cyclopropylmethyloxy-phenyl) -1-pyridin-2-yl-1H-pyrazol-4-ylmethylene] -2-thioxo-thiazolidin-4-one) (compound 15)

3- (3,5-bis-cyclopropylmethyloxy-phenyl) -1-phenyl-1H-pyrazole-4-carbaaldehyde (3- (3,5-Bis-cyclopropylmethyloxy-phenyl) -1-phenyl-1H -pyrazol-4-carbaldehyde) (0.5 g, 1.28 mmol) was dissolved in 20 ml of toluene, and a small amount of rhodanine (170 mg, 1.28 mmol), acetic acid (AcOH) and piperidine was added. stark trap) was used to remove water and reflux. The resulting solid was filtered with ethyl ether and the solid was dried in a desiccator to give the title compound (550 mg, 2.5 mmol, 85%) as yellow.

¹ H NMR (200 MHz, CDCl ₃ ) δ 0.38 (t, 2H, J = 4.2 Hz), 0.64 (t, 2H, J = 4.2 Hz), 1.20-1.30 (m, 1H), 3.92 (d, 4H, J = 7.0 Hz), 6.63-8.38 (m, 7H), 8.78 (s, 1H), 13.8 (br, 1H NH).

[ Example  1-6] 5- [3- (3- Ethoxy -4-isopropyl- Phenyl )-One- Phenyl -1H- Pyrazole -4- Ilmethylene ] -2-T Ohxothiazoli Din-4-one (5- [3- (3- Ethoxy -4-isopropyl-phenyl) -1-phenyl-1H-pyrazol-4-ylmethylene] -2-thioxo-thiazolidin-4-one) (Compound 16)

3- (3-ethoxy-4-isopropyl-phenyl) -1-phenyl-1H-pyrazole-4-carbaaldehyde (3- (3-Ethoxy-4-isopropyl-phenyl) -1-phenyl-1H- Dissolve pyrazol-4-carbaldehyde) (0.5 g, 1.50 mmol) in 20 ml of toluene, add small amount of rhodanine (200 mg, 1.50 mmol), acetic acid (AcOH) and piperidine, and then dean stark trap) to remove water and reflux. The resulting solid was filtered with ethyl ether and the solid was dried in a desiccator to give a yellow title compound (560 mg, 83%).

¹ H NMR (200 MHz, CDCl ₃ ) δ 1.27 (d, 6H, J = 6.9 Hz), 1.44 (t, 3H, J = 6.7 Hz), 1.60-1.65 (m, 1H), 4.17 (q, 2H, J = 6.9 Hz), 7.17 (d, 1H, J = 8.1 Hz), 7.45-7.65 (m, 6H), 8.09 (d, 2H, J = 8.1 Hz), 8.74 (s, 1H), 13.76 (br, 1H, NH).

[ Example  1-7] 5- [3- (3- Bromo -4- Ethoxyphenyl ) -1- (3- Nitrophenyl ) -1H- Pyrazole -4- Methyl Ren] -2- Thioxothiazolidine -4-one (5- [3- (3- Bromo -4- ethoxyphenyl ) -1- (3- nitrophenyl ) -1H-pyrazol-4-ylmethylene] -2-thioxo-thiazolidin-4-one) (compound 17)

3- (3-bromo-4-ethoxyphenyl) -1- (3-nitrophenyl) -1H-pyrazole-4-carbaaldehyde (3- (3-Bromo-4-ethoxy-phenyl) -1- (3-nitro-phenyl) -1H-pyrazole-4-carbaldehyde) (0.5 g, 1.20 mmol) was dissolved in 20 ml of toluene, rhodanine (160 mg, 1.20 mmol), acetic acid (AcOH) and piperidine After the addition of a small amount of water was removed by reflux using a Dean stark trap (dean stark trap). The resulting solid was filtered with ethyl ether and the solid was dried in a desiccator to give a yellow title compound (0.53 g, 83%).

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.42 (t, 3H, J = 6.9 Hz), 4.22 (q, 2H, J = 6.9 Hz), 7.32-7.90 (m, 5H), 8.24 (d, 1H, J = 8.4 Hz), 8.52 (d, 1H, J = 8.1 Hz), 8.86 (s, 1H), 8.95 (s, 1H), 13.77 (br, 1H, NH).

Example 1-8 5- [3- (4-ethoxy-3-iodophenyl) -1-pyridin-2-yl-1 H-pyrazol-4-ylmethylene] -2- Thioxothiazolidine 4-one (5- [3- (4- Ethoxy -3- iodo -phenyl) -1- pyridin -2- yl  -1H-pyrazol-4-ylmethylene] -2-thioxo-thiazolidin-4-one) (compound 18)

3- (4-ethoxy-3-iodophenyl) -1-pyridin-2-yl-1H-pyrazole-4-carbaaldehyde (3- (4-Ethoxy-3-iodo-phenyl) -1-pyridin -2-yl-1H-pyrazole-4-carbaldehyde) (0.52 g, 1.24 mmol) was dissolved in 20 ml of toluene and a small amount of rhodanine (148 mg, 1.114 mmol), acetic acid (AcOH) and piperidine was added. Then water was removed and refluxed using a dean stark trap. The resulting solid was filtered with ethyl ether and the solid was dried in a desiccator to give a yellow title compound (574 mg, 84%).

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.42 (t, 3H, J = 6.9 Hz), 4.22 (q, 2H, J = 6.9 Hz), 7.23-8.09 (m, 7H), 8.58 (t, 1H, J = 5.4 Hz), 8.77 (s, 1H), 13.84 (br, 1H, NH).

Example 1-9 5- [3- (4-ethoxy-3-iodophenyl) -1-pyridin-2-yl-1 H-pyrazol-4-ylmethylene] -2- Thioxothiazolidine 4-one hydrochloride (5- [3- (4- Ethoxy -3- iodo -phenyl) -1-pyridin-2-yl-1H-pyrazol-4-ylmethylene] -2-thioxo-thiazolidin-4-one HCl  salt) (compound 19)

Compound 18 (20 mg, 0.037 mmol) prepared in Example 1-8 was added to 1 mL of MeOH, and HCl (3.0 M in MeOH, 37 μl) was added at 0 ° C., and the mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure and dried to afford the title compound.

TABLE 1

[ Example  2] Formulation Example

On the other hand, the compound represented by Formula 1 according to the present invention can be formulated in various forms according to the purpose. The following illustrates some formulation methods containing the compound represented by Formula 1 according to the present invention as an active ingredient, but the present invention is not limited thereto.

Formulation 1 : tablet (direct pressure)

After sifting 5.0 mg of the active ingredient, 14.1 mg of lactose, 0.8 mg of crospovidone USNF and 0.1 mg of magnesium stearate were mixed and pressed to form a tablet.

Formulation 2 : Tablet (Wet Granulation)

After sifting 5.0 mg of the active ingredient, 16.0 mg of lactose and 4.0 mg of starch were mixed. 0.3 mg of polysorbate 80 was dissolved in pure water and then an appropriate amount of this solution was added and then atomized. After drying, the fine particles were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The granules were pressed into tablets.

Formulation 3 : Powders and Capsules

5.0 mg of the active ingredient was sieved, followed by mixing with 14.8 mg of lactose, 10.0 mg of polyvinyl pyrrolidone, and 0.2 mg of magnesium stearate. No. solid the mixture using a suitable device. Filled in 5 gelatin capsules.

Example 3 CDC25B Enzyme Assay (Enzyme assay)

A portion corresponding to the catalytic domain (aa 378-566) of CDC25B was combined with GST to make a fusion protein, which was then expressed and produced in E. coli and used as an enzyme source. Enzyme assay was performed by adjusting the final reaction volume per well to 200 μl on a 96-well plate. To the reaction, 170 μl of buffer (30 mM Tris buffer, pH 8.5, 75 mM NaCl, 0.67 mM EDTA, 1 mM DTT), 20 μl of CDC25B enzyme (0.2 μg) and 10 μl of test compound dissolved in DMSO were added. Was adjusted to 1 μg / mL CDC25B enzyme, 20 μM FDP, and cultured for 1 hour at room temperature. At this time, FDP and DTT were added to the buffer just before the assay, so that the assay was always performed in the fresh state. After 1 hour of incubation, the fluorescence generated by the enzyme reaction was measured at 485 nm (excitation) and 538 nm (emission).

The test compound was first treated with a final concentration of 10 μM, followed by primary screening, and then, a compound having excellent inhibitory activity was selected to obtain an IC ₅₀ value. Compounds screened by primary screening were generally treated with final concentrations of 1, 2, 5, and 10 μM to obtain% inhibition rates. In the case of), the concentration of the compound was increased to 20 μM, and the percent inhibition rate was obtained to obtain an IC ₅₀ value.

IC ₅₀ values for cdc25B are listed in Table 2 below for the compounds synthesized in Example 1.

TABLE 2

Example 4 Cell cytotoxicity assay

Cancer Cell Culture

The cells used to measure anticancer activity are Hela, HCT116. These cancer cells all used tumor cell lines derived from humans. These cells were all cultured in a 37 ° C. constant temperature and 5% CO ₂ , incubator using RPMI 1640 culture supplemented with 5% fetal bovine serum as culture. Cells were passaged once every 3 to 4 days, and 0.25% trypsin and 3 mM trans-1,2-diaminocyclohexane-N, N, in PBS (phosphate buffered saline) solution were used to separate the cells from the adherent surface. A solution in which N, N-tetraacetic acid (CDTA) was dissolved was used.

Activity measurement

In 1989, the US National Cancer Institute used the sulforhodamine B assay method developed to measure the in vitro anticancer activity of drugs. That is, in order to use the passaged cells for experiments, cells were separated from the implant using trypsin-EDTA solution, and the plate (96 well microplate, manufactured by Falcon) was dispensed so that the number of cells per well was 2 × 10 ^3. It was. The aliquoted cells were incubated for 24 hours in a CO ₂ incubator and attached to the bottom, followed by removal of the culture solution with an aspirator, and diluting the compound solutions diluted in the culture solution with a log dose of 6 concentrations. Into each well containing 100 mL in 3 multiples, and further incubated for 48 hours. Dimethyl sulfoxide (DMSO) was used as needed to dissolve the compound. In addition, the diluted compound solution was filtered through a 0.22 mL filter prior to addition to the cells to maintain the sterility of the experiment. After incubating the cells with the drug for 48 hours, the culture solution of each well was removed, and 100 mL of 10% trichloroacetic acid (TCA) was added thereto and left at 4 ° C. for 1 hour to fix the cells on the bottom of the plate. After fixing the cells, the plate was washed 5 to 6 times with water to completely remove the remaining TCA solution, and dried at room temperature to ensure no remaining water. Completely dried plates were stained for 30 minutes by adding dye solution dissolved in 0.4% SRB to 100 mL of 1% acetic acid solution per well, and washed again 5 to 6 times with 1% acetic acid solution to remove SRB that did not bind to cells. Removed. The stained cell plates are then dried at room temperature and then shaken for 10 minutes with a titer plate shaker by adding 100 mL of 10 mM unbuffered trisma base solution per well. After elution, the absorbance at 520 nm was measured using a microplate reader. In order to calculate the effect of the drug on cancer cells, the cell number (Tz) when the drug was added, the cell number (C) and the concentration of the drug when incubated for 48 hours with the drug-free medium were added. The cell number (T) at the time of incubation was measured and calculated by the following equation.

Using the data regression of the LOTUS program from the calculated values, the degree to which the drug inhibits the growth of cancer cells is shown in Table 3 as% inhibition.

TABLE 3

In addition, as a result of toxicity test on the normal cells of the compounds synthesized in Example 1 almost did not cause toxicity and as a result of the selectivity test for dephosphorylation other than CDC25B it was confirmed that the selectivity to other dephosphorase.

The rhodanine-based compound according to the present invention, namely 5- (1,3-diaryl- 1H -pyrazol-4-ylmethylene) rhodanine derivative, exhibits an excellent inhibitory activity against the CDC25B enzyme, and cancer cells, that is, Hela, Since it shows cytotoxicity against HCT116, it is evident that there is anticancer activity based on CDC25B enzyme inhibition. CDC25B is a very important dephosphorylation enzyme that determines the progression of the G2 / M cycle in cell division. When it is inhibited, cell division is inhibited and eventually leads to cell death, thereby acting as an anticancer activity. CDC25B is overexpressed in tumors such as gastric cancer, lung cancer, brain cancer and laryngeal cancer, and breast cancer, and patients with large CDC25B overexpression have low survival rate. Judging.

Therefore, the rhodanine-based compound according to the present invention can be used as a low toxicity anticancer agent with less side effects.

Claims (7)

A rhodanine-based compound of Formula 1 or a pharmaceutically acceptable salt thereof. [Formula 1]

Wherein Ar ₁ and Ar ₂ are independently selected from phenyl or pyridine, and Ar ₁ and Ar ₂ are C ₁ to C ₄ alkyl group, halogen atom, C ₁ to C ₇ alkoxy group, C ₃ to C ₄ alkenyloxy group It may be substituted with one or two or more substituents selected from C ₂ to C ₄ acyl group, cyan group or nitro group. delete The method of claim 1, The compound of formula 1 is a rhodanine-based compound or a pharmaceutically acceptable salt thereof, characterized in that the compound of formula 1-1. [Formula 1-1]

Wherein X ₁ to X ₃ are independently hydrogen, C ₁ to C ₄ alkyl group, halogen atom, C ₁ to C ₇ alkoxy group, C ₃ to C ₄ alkenyloxy group, C ₂ to C ₄ acyl group, cyan group or And a substituent selected from a nitro group, Ar ₂ is as defined in claim 1. The method of claim 3, wherein X ₁ to X ₃ is independently a rhodanine compound, characterized in that selected from hydrogen, isopropyl group, tertiary butyl group, bromine, iodine, methoxy group, ethoxy group, propyloxy group, allyloxy group or nitro group Or a pharmaceutically acceptable salt thereof. The method of claim 4, wherein The rhodanine-based compound of Formula 1 is selected from the compound of the following structure rhodanine-based compound or a pharmaceutically acceptable salt thereof.

An anticancer composition comprising the rhodanine-based compound of any one of claims 1, 3 to 5, or a pharmaceutically acceptable salt thereof as an active ingredient. delete