KR101473965B1 - Composition containing sorafenib and imidazopyridine derivative for preventing or treating pancreatic cancer - Google Patents

Abstract

The present invention relates to a composition containing sorafenib and an imidazopyridine derivative as active ingredients for preventing or treating pancreatic cancer. The sorafenib and an imidazopyridine derivative of the present invention simultaneously block Ras/Raf/MEK/ERK signaling pathway and PI3K/Akt/mTOR signaling pathway to thereby have an excellent effect of inhibiting metastasis and proliferation of cancer cells. Thus, the sorafenib and imidazopyridine derivative can be valuably used to treat cancer diseases including pancreatic cancer.

Description

[0001] The present invention relates to a composition for preventing or treating pancreatic cancer, which comprises sorapenib and an imidazopyridine derivative as active ingredients,

The present invention relates to a composition for preventing or treating pancreatic cancer containing sorafenib and an imidazopyridine derivative as an active ingredient.

Pancreatic cancer is less common than other cancers, but it is known to have the highest mortality rate in cancer patients over a period of time. Pancreatic cancer is a cancer that is difficult to diagnose early. It is characterized by the fact that it easily migrates to surrounding organs or lymph nodes, and its prognosis is known to be worse than other cancers. Despite several efforts to treat pancreatic cancer, the 5 - year survival rate after the onset of pancreatic cancer has not improved over the past 30 years, and mortality from pancreatic cancer continues to rise, making it the fifth leading cause of cancer deaths. According to the data released by the Ministry of Health and Welfare Cancer Registration Division, the mortality rate within one year after the onset of pancreatic cancer was found to be about 80% of the patients with pancreatic cancer, and the survival rate of pancreatic cancer patients was found to be very low. Because pancreatic cancer has a very high transfusion rate, it is often missed.

Chemotherapy and radiation therapy have been used for treating pancreatic cancer, but the therapeutic effect is very low. Gemcitabine, the most commonly used anticancer drug in pancreatic cancer, has been identified as a pyrimidine antagonist, with a remission rate of 6-11% in Phase II studies. Gemcitabine has been used in combination with oxalate or other drugs such as 5-fluorouracil (5-FU) for the treatment of patients with pancreatic cancer. However, this gemcitabine was not found to have a significant effect on the survival rate of pancreatic cancer. Thus, there is a need to develop new drugs that can treat pancreatic cancer and to develop new dosing therapies that can be used in combination with other drugs.

On the other hand, sorafenib (generic name: Nexavar, Nexavar) is an anticancer drug developed by Bayer and Onyx Pharmaceuticals. It is an anticancer drug approved by the US FDA for the treatment of kidney cancer and liver cancer. Sorapanib is a multiple kinase inhibitor that blocks the activity of kinase proteins such as VEGFR, PDGFR, and Raf as target therapeutics. The main target of sorafenib is Raf-1, which is also known to inhibit the Ras / Raf / MEK / ERK signaling cascade. In addition, sorapenib has been reported to inhibit the proliferation of cancer cells by targeting also receptor tyrosine kinases such as EFG1, 2, 3, PDGFR, and FGFR, but the response rate is very low at 2-3% There is a problem that drug resistance and tolerance may occur when administered over a long period of time.

Recently, it has been reported that PI3K / Akt / mTOR signaling is commonly expressed in the body of patients suffering from various types of cancer. The PI3K / Akt / mTOR signal is known to play an important role in cell growth and survival. However, studies on the relationship between PI3K / Akt / mTOR signal and cancer disease are limited.

DISCLOSURE OF THE INVENTION The present inventors resolved the above problems and found that when sorafenib and imidazopyridine derivatives were administered to Ras / Raf / MEK / ERK signaling pathway and PI3K / Akt / mTOR It has been confirmed that the effect of suppressing the proliferation and metastasis of cancer cells is excellent by blocking the signal path simultaneously, thus completing the present invention.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating pancreatic cancer containing sorapenib and an imidazopyridine derivative as an active ingredient.

Another object of the present invention relates to a food composition for preventing or ameliorating pancreatic cancer containing sorapenib and an imidazopyridine derivative as an active ingredient.

In order to solve the above problems, the present invention provides a composition for preventing or treating pancreatic cancer, which comprises as an active ingredient, sorapanib represented by the following formula (1) and imidazopyridine derivative represented by the following formula (2).

The composition comprises a pharmaceutical composition or a food composition.

In Formula 2, X is carbon or nitrogen,

R ₁ is hydrogen, a C ₁ -C ₆ alkyl group; An acetyl group; Pyrazole group; A morpholine group; - (C = O) O- ( C 1- -C 6 alkyl); Cyano; Tetrazole group; An oxadiazole group; Or a C ₆ to C ₃₀ aryl group substituted with a cyano group or a C ₆ to C ₃₀ heteroaryl group,

R ₂ is a C ₆ to C ₃₀ aryl group or a C ₆ to C ₃₀ heteroaryl group which is substituted or unsubstituted with at least one member selected from the group consisting of an amine group, an arylsulfonamide group and a morpholinosulfonyl group .

로 이루어진 군으로부터 선택된 1종 이상이고, Preferably, R < ₁ >

And at least one member selected from the group consisting of

로 이루어진 군으로부터 선택된 1종 이상이다. R ₂ is

≪ / RTI >

More preferably, the imidazopyridine derivative of Formula 2 is at least one selected from the group consisting of the following compounds:

1) N- (5- (3- (5-methyl-1,2,4-oxadiazol-3-yl) imidazo [1,2- a] pyridin- Sulfonamide;

2) N- (5- (3-Cyanoimidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

3) N- (5- (3-morpholinoimidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

4) N- (5- (3-acetylimidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

5) N- (5- (3-acetylimidazo [1,2-a] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide;

6) 5- (3-morpholinoimidazo [1,2-a] pyridin-6-yl) -3- (morpholinosulfonyl) pyridin-2-amine;

7) 3,5-Difluoro-N- (5- (3- (5-methyl-1,2,4-oxadiazol- Yl) pyridin-3-yl) benzenesulfonamide;

8) 2,4-Difluoro-N- (5- (3- (5-methyl-1,2,4-oxadiazol-3- yl) imidazo [ Yl) pyridin-3-yl) benzenesulfonamide;

9) N- (5- (3- (3-cyanophenyl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

10) N- (5- (3- (2H-tetrazol-5-yl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

11) N- (5- (3- (Pyridin-3-yl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

12) N- (5- (3- (Pyridin-4-yl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

13) 3- (imidazo [1,2-a] pyridin-6-yl) aniline;

14) 5- (Imidazo [l, 2-a] pyridin-6-yl) pyridin-3-amine;

15) N- (5- (imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

16) ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate;

17) 6- (6-Amino-5- (morpholinosulfonyl) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carbonitrile;

18) N- (5- (3- (1H-pyrazol-4-yl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

19) N- (5- (3-Cyanoimidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) -2,4-difluorobenzenesulfonamide;

20) N- (5- (3- (2H-tetrazol-5-yl) imidazo [1,2- a] pyridin-6-yl) pyridin- Sulfonamide;

21) N- (5- (3-Acetylimidazo [1,2-a] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide;

22) N- (5- (Imidazo [1,2-a] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide; And

23) Ethyl 6- (5- (2,4-difluorophenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate.
Most preferably, the imazapyridine derivative of formula 2 is ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate.

delete

Soraphenib of Formula 1 and imidazopyridine derivative of Formula 2 according to the present invention may form pharmaceutically acceptable salts. Such pharmaceutically acceptable salts include those acids which form non-toxic acid addition salts containing a pharmaceutically acceptable anion such as, for example, inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid and the like; And organic acids such as tartaric acid, formic acid, citric acid, acetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, lactic acid, malonic acid, malic acid, maleic acid, salicylic acid, succinic acid, oxalic acid, propionic acid, ; Acid addition salts formed with sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and the like, and sorbates of the formula 1 according to the present invention, including free carboxy substituents, And imidazopyridine derivatives of formula (2) may include the above acid addition salts and salts of sodium, calcium and ammonium.

In addition, pharmaceutically acceptable base addition salts such as alkali metal or alkaline earth metal salts formed by lithium, sodium, potassium, calcium, magnesium and the like; Amino acid salts such as lysine, arginine and guanidine; Organic salts such as dicyclohexylamine, N-methyl-D-glucamine, tris (hydroxymethyl) methylamine, diethanolamine, choline, triethylamine and the like.

"Sorapenib" represented by Formula 1, which is one of the active ingredients of the present invention, is a multi-kinase inhibitor targeting VEGFR, PDGFR, and Raf, and is excellent in blocking the Ras / Raf / MEK / ERK signal pathway.

The "imidazopyridine derivative " represented by Formula 2, which is one of the active ingredients of the present invention, has an excellent PI3K inhibitory activity and thus has an effect of blocking the PI3K / Akt / mTOR signaling pathway involved in the proliferation and metastasis of cancer cells great.

The imidazopyridine derivative can be synthesized by a method described in a publication (Design and Synthesis of Imidazopyridine Analogues as Phosphoinositide 3-Kinase Signaling and Angiogenesis, 2011 American Chemical Society, dx.doi.org/10.1021/jm101582z Chem., J. Med. Chem., 2011, 54, 2455-466, Hong Soon Sun et al., 5).

The sorapenib and imidazopyridine derivatives of the present invention are effective in inhibiting the proliferation and metastasis of cancer cells by simultaneously blocking the Ras / Raf / MEK / ERK signal pathway and the PI3K / Akt / mTOR signal pathway, Can be usefully used for the treatment of diseases.

The composition of the present invention may contain at least one known active ingredient having anticancer activity together with sorapenib of formula (1) and imidazopyridine derivative of formula (2).

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included 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. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. 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. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For a desired effect, the daily dose of sorapenib of Formula 1 and the imidazopyridine derivative of Formula 2 of the present invention may be administered in an amount of 0.01 mg / kg to 100 mg / kg per day, It may be administered several times.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the anticancer effect.

In the present invention, the term "health functional food" refers to a food having a biological control function such as prevention and improvement of disease, bio-defense, immunity, recovery after disease and aging inhibition.

The composition of the present invention can be added to a health functional food for the purpose of anti-cancer effect. When sorapanib of formula 1 of the present invention and imidazopyridine derivative of formula 2 are used as food additives, sorapanib of formula 1 and imidazopyridine derivative of formula 2 may be added as they are or used together with other food or food ingredients 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 the intended use (prevention, health or therapeutic treatment). Generally, sorapenib of formula (I) or imidazopyridine derivative of formula (II) is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The sorapenib and imidazopyridine derivatives of the present invention are effective in inhibiting the proliferation and metastasis of cancer cells by simultaneously blocking the Ras / Raf / MEK / ERK signal pathway and the PI3K / Akt / mTOR signal pathway, Can be usefully used for the treatment of diseases.

1 is a graph showing the results of measurement of cytotoxicity of pancreatic cancer cells treated with sorapenib and imidazopyridine derivatives of the present invention in combination.
FIG. 2 is a graph showing the inhibitory effect of signal transduction pathway protein on pancreatic cancer cells treated with sorapenib and imidazopyridine derivatives of the present invention.
FIG. 3 is a graph showing the results of measurement of DNA fragmentation degree in pancreatic cancer cells treated with sorapenib and imidazopyridine derivatives of the present invention. FIG.
FIG. 4 is a graph showing the inhibitory effect of the sorapenib and imidazopyridine derivatives of the present invention on the expression of the apoptosis-related protein in pancreatic cancer cells treated with the combination.
FIG. 5 is a graph showing cell cycle changes of pancreatic cancer cells treated with sorapenib and imidazopyridine derivatives according to the present invention.
6 is a graph showing the inhibitory effect of angiogenic factors VEGF and HIF-Ia on pancreatic cancer cells treated with sorapanib and imidazopyridine derivatives of the present invention.
7 is a graph showing the angiogenesis inhibitory effect of vascular endothelial cells in pancreatic cancer cells treated with the combination of sorapenib and imidazopyridine derivatives of the present invention.
8 is a graph showing the angiogenesis inhibitory effect in the mouse epidermis treated with the combination of sorapenib and imidazopyridine derivative of the present invention.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the present invention to the precise form disclosed. It will be obvious to you.

[Example 1: Preparation and preparation of sorapanib and imidazopyridine derivative of the present invention]

1. Preparation of sorafenib

Sorapenib was purchased from LC Laboratories Inc. (USA) and Sorafenib p-Toluenesulfonate Salt was dissolved in DMSO at the time of administration.

2. Preparation of imidazopyridine derivatives

The imidazopyridine derivative as an active ingredient of the present invention can be prepared by the method described in the publication of Design and Synthesis of Imidazopyridine Analogues as Phosphoinositide 3-Kinase Signaling and Angiogenesis, 2011 American Chemical Society, dx.doi. org / 10.1021 / jm101582z | J. Med. Chem., 2011, 54, 2455-466, Hong Soon Sun et al., 5). In the examples of the present invention, ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate as an imidazopyridine derivative for measuring anticancer activity (Hereinafter referred to as "HS-173") was used and dissolved in DMSO in the experiment.

[Experimental Example 1: Measurement of growth inhibitory activity of pancreatic cancer cells by combination treatment of sorapenib and imidazopyridine derivative of the present invention]

To investigate the growth inhibitory activity of pancreatic cancer cells by the combination treatment of sorapenib and imidazopyridine derivative (HS-173) of the present invention, three pancreatic cancer cell lines, Panc-1, Miapaca-2 and Aspc- Cytotoxicity assays were performed.

1. Preparation of pancreatic cancer cell line

Pancreatic cancer cell lines Panc-1, Miapaca-2 and Aspc-1 were purchased from the American Type Culture Collection (Manassas, Va., USA). The medium was supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (Dulbeccos modified Eagles medium) supplemented with penicillin / streptomycin was used. The cells were cultured in an incubator maintained at 37 ° C and 5% CO ₂ .

2. Measurement of pancreatic cancer cell growth inhibitory activity

To investigate the pancreatic cancer cell growth inhibitory activity by the combination treatment of sorapenib and imidazopyridine derivative of the present invention, sorapenib and ethyl 6- (5- (phenylsulfonamido) pyridin-3- 1-yl) imidazo [1,2-a] pyridine-3-carboxylate in the pancreatic cancer cells was measured by a conventionally known MTT assay.

Specifically, the three pancreatic cancer cell lines Panc-1, Miapaca-2 and Aspc-1 cells prepared in 1 above were treated with 10 μM sorafenib and 0, 0.1, 1, 5 or 10 μM ethyl 6- (5 - (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate in combination at various concentrations for 48 hours (experimental group). Thereafter, MTT analysis was performed on each reaction culture. A combination treatment of sorapenib with ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- ), The combination index (CI) values were calculated using the CalcuSyn software (Biosoft) using the measured values obtained from the MTT analysis (CI value <1; synergy effect (synergistic effect), and the results are shown in Fig.

As shown in FIG. 1, the combination of sorapenib and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- One experimental group increased the cytotoxicity of three pancreatic cancer cell lines, Aspc-1, Miapaca-2 and Panc-1 cell lines, in particular, significantly increased the cytotoxicity of Panc-1 cell line.

CI values for each experimental group were determined by combining 10 μM sorapenib and 1 μM ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- In combination with 10 μM sorapenib and 5 μM imidazopyridine derivatives, the highest concentration (Combination index = 0.666) was obtained in the Miapaca-2 cell line Respectively.

Therefore, the sorapenib and imidazopyridine derivatives of the present invention are excellent in the effect of increasing the cytotoxicity of pancreatic cancer cells, so that they are excellent in inhibiting proliferation and metastasis of pancreatic cancer cells.

[Experimental Example 2: Ras / Raf / MEK / ERK signaling pathway and PI3K / Akt / mTOR signaling pathway blocking effect by the combination treatment of sorapenib and imidazopyridine derivative of the present invention]

To examine the Ras / Raf / MEK / ERK signaling pathway and PI3K / Akt / mTOR signaling pathway blocking effect by the combination treatment of sorapenib and imidazopyridine derivative (HS-173) of the present invention, Western blot analysis and immunofluorescence analysis of proteins, phospho-AKT, mTOR, P70S6K, ERK, MEK, were performed.

Specifically, 10 μM sorapenib and 1 μM ethyl 6- (5- (phenylsulfonamido) pyridine prepared in Example 1 above were added to the pancreatic cancer cell line Panc-1, Miapaca-2 cell line prepared in Experimental Example 1, -3-yl) imidazo [1,2-a] pyridine-3-carboxylate for 6 hours alone (alone treatment group) or combination treatment (experimental group), phospho-AKT, mTOR, P70S6K, Western blotting was performed on MEK. Immunofluorescence analysis of phospho-AKT, mTOR, P70S6K and MEK expressed in the Panc-1 cell line was performed to further confirm the results, and the results are shown in FIG.

2, the combination of sorapenib and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate of the present invention One experimental group significantly reduced phospho-AKT, mTOR, P70S6K, ERK and MEK protein levels compared to the single treatment group.

Thus, the combination of sorapanib and imidazopyridine derivatives of the present invention blocks both the Ras / Raf / MEK / ERK signaling pathway and the PI3K / Akt / mTOR signaling pathway involved in the proliferation and metastasis of cancer cells, The effect of preventing or treating is excellent. It is also understood that the superior signal pathway blocking effect of the sorapenib and the imidazopyridine derivative is due to the synergistic effect of the combination treatment.

[Experimental Example 3: induction of apoptosis of pancreatic cancer cells by the combination treatment of sorapanib and imidazopyridine derivative of the present invention]

In order to investigate the apoptosis inducing action of pancreatic cancer cell by the combination treatment of sorapenib and imidazopyridine derivative (HS-173) of the present invention, DNA fragmentation and apoptosis-associated protein of pancreatic cancer cell line were analyzed by TUNEL analysis and Western blot analysis .

1. DNA fragmentation measurement

In order to measure the DNA fragmentation of the pancreatic cancer cell line treated with the sorapenib and the imidazopyridine derivative of the present invention, the following procedure was performed.

Specifically, the pancreatic cancer cells prepared in Experimental Example 1 (1) were intraperitoneally injected with sorapenib and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a ] Pyridine-3-carboxylate were treated with either 1 μM or 10 μM each alone or in combination, and analyzed using a TUNEL kit (Chemicon, Temecula, CA, USA) to confirm the degree of apoptosis. Cells were fixed with acetic acid: ethanol mixture (1: 2) and peroxidase was removed with H ₂ O ₂ . After reaction with TdT enzyme at 37 ° C for 1 hour, Anti-Digoxigenenin Conjugate (Ab) was reacted at 4 ° C for one day. After washing with PBS, the cells were reacted with DAB substrate solution for 2-5 minutes. After confirming the expression level, the cells were mounted with a cover slide and photographed with a microscope. The results are shown in Fig.

3, the sorapenib of the present invention and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- One experimental group showed higher percentage of TUNEL positive cells than the single treatment group.

Therefore, the sorapenib and imidazopyridine derivatives of the present invention are excellent in inducing DNA fragmentation in pancreatic cancer cells, and thus, they are effective in inducing apoptosis of pancreatic cancer cells.

2. Apoptosis-related protein measurement

In order to measure the apoptosis-associated protein of the pancreatic cancer cell line treated with the combination of sorapenib and imidazopyridine derivative of the present invention, the following procedure was performed.

Specifically, the pancreatic cancer cells prepared in Experimental Example 1 (1) were intraperitoneally injected with sorapenib and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a ] Pyridine-3-carboxylate was treated alone or in combination at a concentration of 1 [mu] M and 10 [mu] M, and then caspase-3 (caspase-3), caspase- caspase-9) and PARP. The results are shown in Fig.

4, the sorapenib of the present invention and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- One experimental group significantly increased the expression of caspase-3, caspase-9 and truncated PARP than the single treatment group administered with each of them.

Therefore, the sorapenib and imidazopyridine derivatives of the present invention induce apoptosis-related protein expression in pancreatic cancer cells, leading to apoptosis of pancreatic cancer cells.

[Experimental example 4: induction of cell cycle change by combination treatment of sorapanib and imidazopyridine derivative of the present invention]

FACS analysis and western blot analysis were performed to examine cell cycle changes by the combination treatment of sorapanib and imidazopyridine derivative (HS-173) of the present invention.

Specifically, the pancreatic cancer cells prepared in Experimental Example 1 (1) were intraperitoneally injected with sorapenib and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a ] Pyridine-3-carboxylate were treated with 1 μM and 10 μM, respectively, or in combination with 70% ethanol and fixed at 4 ° C. After washing with PBS, 50 g / mL PI (propidium iodide ) And 100 g / mL RNase A were added and treated at room temperature for 1 hour. Thereafter, a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif.) Was used to analyze the percentage of each phase of the cell cycle (sub-G ₁ , G ₀ / G ₁ , S, G ₂ / M) CA) was used to confirm the cell cycle of the cells. The measured data were analyzed with FlowJo software (Tree Star, Inc.). Western blot was further performed to confirm the expression of proteins involved in the cell cycle. The results are shown in Fig.

5, the sorapenib of the present invention and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- One experimental group synergistically changed the G2 / M phase to an arrest state and increased the expression of proteins involved in the G2 / M phase arrest, namely p-cdc2 and p-cdc25c.

Therefore, the sorapenib and imidazopyridine derivatives of the present invention change the cell cycle in the pancreatic cancer cell to a state of G2 / M termination, so that it is shown that the effect of suppressing proliferation of pancreatic cancer cells is excellent.

[Experimental example 5: Effect of the present invention on inhibition of angiogenesis by combination treatment of sorapenib and imidazopyridine derivative]

In order to examine the angiogenesis inhibitory effect of the combination treatment of sorapenib and imidazopyridine derivative (HS-173) of the present invention, cell experiments and animal experiments were performed as follows.

1. Inhibitory effect of VEGF and HIF-1α on angiogenesis

The inhibitory effect on the formation of VEGF HIF-1 alpha, which is a protein that plays a representative role in angiogenesis by the combination treatment of sorapanib and imidazopyridine derivative of the present invention, was confirmed.

Specifically, hypoxia induced by treatment with CoCl ₂ was induced in the pancreatic cancer cells prepared in Experimental Example 1, and then sorafenib and ethyl 6- (5- (phenylsulfonamido) pyridine prepared in Example 1 -3-yl) imidazo [1, 2-a] pyridine-3-carboxylate were used alone or in combination at concentrations of 1 [mu] M and 10 [mu] M, and the levels of VEGF and HIF- . The results are shown in Fig.

6, the sorapenib of the present invention and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- One experimental group significantly inhibited the production of VEGF and HIF-1α compared to the single treatment group.

Therefore, it can be seen that the sorapenib and imidazopyridine derivatives of the present invention are excellent in the effect of inhibiting the angiogenesis, because they are excellent in the effect of inhibiting the production of the proteins VEGF and HIF-1? Involved in angiogenesis .

2. Anti-angiogenic effect of vascular endothelial cells (HUVEC)

Tube formation assay was performed to examine the angiogenesis inhibitory effect of vascular endothelial cells by the combination treatment of sorapanib and imidazopyridine derivative of the present invention.

Specifically, Matrigel (BD Bioscience) was slowly dissolved at 4 占 폚, coated on a 48-well plate, and then vascular endothelial cells were cultured at 37 占 폚 for 30 minutes to 1 hour Gel state. To the cultured vascular endothelial cells (HUVEC) were added sorapenib and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- carboxylate 0.1 μM, and then alone or in combination treatment at a concentration of 10 μM, vascular endothelial growth factor; and redispersed with that contains (vascular endothelial growth factor hereinafter, VEGF) medium dispensed in 5 × 10 ⁴ cells And then cultured for 16 hours. After completion of the incubation, the vascular endothelial cells (HUVEC) were observed for vascularization using an optical microscope, and the results are shown in Fig.

7, the sorapenib of the present invention and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- One experimental group significantly inhibited the angiogenesis of vascular endothelial cells (HUVEC) compared to the single treatment group.

Accordingly, it can be seen that the sorapenib and the imidazopyridine derivative of the present invention are excellent in the effect of inhibiting angiogenesis of vascular endothelial cells by the combination treatment.

3. Angiogenesis inhibition test confirmed by animal experiment

In order to confirm that the angiogenesis inhibitory effect by the combination treatment of sorapenib and imidazopyridine derivative of the present invention is effective in vivo, the animal experiment was performed as follows.

Specifically, Matrigel inducing angiogenesis was slowly dissolved for 24 hours at 4 ° C, VEGF (50 ng / ml) was mixed with Matrigel (BD Bioscience) as a positive control, and as an experimental group, 10 μM sorapenib and 1 μM ethyl 6- (5- (phenylsulfanamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate were each used alone or in combination VEGF and matrigel were mixed. Each matrigel was injected thinly into the dorsal epidermis of 6-7 weeks old male mice (positive control and experimental group matrigel) to induce angiogenesis. After 5 to 7 days, only the matrigel plug was cut and made into blocks. The blood was stained again by H & E staining, and the resultant blood vessels were observed. The results are shown in FIG.

As shown in FIG. 8, the sorapenib of the present invention and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- One experimental group significantly inhibited the angiogenesis of the mouse epidermis compared to the single treatment group.

Therefore, it can be seen that the sorapenib and imidazopyridine derivatives of the present invention are excellent in the effect of inhibiting the proliferation and metastasis of cancer cells by angiogenesis, because they are excellent in the effect of inhibiting angiogenesis in animal experiments.

Hereinafter, pharmaceutical compositions and food composition preparations for the prevention or treatment of pancreatic cancer containing the sorapenib and imidazopyridine derivatives of the present invention will be described, but the present invention is not to be construed as limiting the present invention, but will be specifically described.

[Preparation Example 1: Preparation of pharmaceutical preparation]

1. Manufacturing of powder

Soraphenib of Formula 1 and 20 mg of imidazopyridine derivative of Formula 2

Lactose 100 mg

Talc 10 mg

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

2. Preparation of tablets

Soraphenib of Formula 1 and 10 mg of imidazopyridine derivative of Formula 2

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

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

3. Preparation of capsules

Soraphenib of Formula 1 and 10 mg of imidazopyridine derivative of Formula 2

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

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

4. Preparation of injections

Soraphenib of Formula 1 and 10 mg of imidazopyridine derivative of Formula 2

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ .2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

5. Manufacture of liquids

Soraphenib of Formula 1 and 20 mg of imidazopyridine derivative of Formula 2

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

[Formulation example 2. Preparation of food preparation]

1. Manufacture of health food

100 mg of sorapenib of formula (1) and imidazopyridine derivative of formula (2)

Vitamin mixture quantity

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2. Manufacture of health drinks

100 mg of sorapenib of formula (1) and imidazopyridine derivative of formula (2)

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 liter container, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (9)

(Sorbenib) represented by the following formula (1) and ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2- a] pyridine- PHARMACEUTICAL COMPOSITION FOR THE PREVENTION OR TREATMENT OF Pancreatic Cancer,
[Chemical Formula 1]

delete delete delete The pharmaceutical composition for preventing or treating pancreatic cancer according to claim 1, wherein the composition simultaneously blocks the Ras / Raf / MEK / ERK signal pathway and the PI3K (phosophoinositide 3-kinase) / Akt / mTOR signaling pathway .
(5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate which is an imidazopyridine derivative represented by the following formula A food composition for preventing or improving pancreatic cancer, the composition comprising:
[Chemical Formula 1]

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