KR20190118287A - Composition for preventing or treating colon cancer comprising 1-(4-(3-chloro-4(3-fluorobenzyloxy) phenylamino)quinazolin-6-yl)urea - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating, or treating colorectal cancer, comprising 1-(4-(3-chloro-4(3-fluorobenzyloxy)phenylamino)quinazoline-6-yl)urea represented by chemical formula 1 or a pharmaceutically acceptable salt thereof. The pharmaceutical composition for preventing or treating colorectal cancer, a method for increasing apoptosis in colorectal cancer cells, a method for preventing or treating colorectal cancer, and a functional health food composition for preventing or alleviating colorectal cancer according to the present invention comprise 1-(4-(3-chloro-4(3-fluorobenzyloxy)phenylamino)quinazoline-6-yl)urea or a pharmaceutically acceptable salt thereof and thus can be beneficially used in the prevention, alleviation, or treatment of colorectal cancer.

Description

Composition for preventing or treating colorectal cancer comprising 1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea {COMPOSITION FOR PREVENTING OR TREATING COLON CANCER COMPRISING 1 -(4- (3-CHLORO-4 (3-FLUOROBENZYLOXY) PHENYLAMINO) QUINAZOLIN-6-YL) UREA}

The present invention relates to a composition for preventing, improving or treating colon cancer comprising 1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea as an active ingredient. will be.

Cancer is one of the incurable diseases that humanity has to solve, and huge capital is invested in the development to cure it all over the world.In Korea, it is the number one disease death cause and more than 100,000 people a year It is diagnosed and about 60,000 or more die.

Cancer is classified into blood cancer and solid cancer, and it occurs in almost every part of the body such as lung cancer, stomach cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer, and skin cancer.

Among them, colon cancer is an incurable disease that causes 1 million patients worldwide and 530,000 deaths annually.

Recently, the incidence rate is increasing in Asia due to westernized eating habits, and related factors include excessive animal fats, sugars, alcohol consumption and fiber, antioxidant vitamins, and insufficient intake of vegetables or fruits. In particular, eating a lot of animal fats and meat, unlike intake of fiber foods such as vegetables and grains, the amount of stool is small and the contents take a long time to pass through the large intestine. In addition, the excretion of bile acids and sterols increases, and the composition of the bacterial species present in the large intestine causes changes in the types of bacteria that chemically change these substances. Therefore, a lot of carcinogens are generated, and the time that the carcinogen stays in contact with the colon is long, so colon cancer frequently occurs.

In particular, colorectal cancer is increasing rapidly at the third place, accounting for 12.7% of all cancers in Korea due to changes in dietary patterns and aging.

In the case of colorectal cancer, a typical solid cancer cell in which a large number of cells grow in the form of agglomerates around blood vessels is one of the cancers in which treatment methods are extremely difficult to cure. That is, it is not easy to completely remove colon cancer cells because the drug does not penetrate properly to the center of the agglomerated colon cancer cells.

Currently, there are few methods for treating colorectal cancer with drugs. Surgical treatment such as surgery or radiation therapy is the only limited method for treating colorectal cancer, but it is difficult to cure colorectal cancer.

Current anticancer agents effective against colorectal cancer include fluoropyrimidine-based drugs such as 5-F oil, UFT, and capecitabine (trade name: Zeloda), irinotecan (trademark: Gamputo), and oxaliplatin. In addition, the market for colorectal cancer drugs is expected to grow rapidly from $ 900 million to $ 7.7 billion in 2017 in the seven major pharmaceutical markets in the US and Europe. The constant use of such drugs has always been a problem of increased resistance and side effects, and it is time to study the development and diagnosis of new therapeutic agents.

Against this background, the present inventors conducted research to invent anti-cancer compositions that effectively act against colorectal cancer, and thus, 1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin- 6-day) urea has been found to be effective in increasing cell death due to apoptosis in colorectal cancer cell lines, and came to complete the composition for the prevention, improvement or treatment of cancer using the same.

 Ahmed Elkamhawy, et al., Targeting EGFR / HER2 tyrosine kinases with a new potent series of 6-substituted 4-anilinoquinazoline hybrids: Design, synthesis, kinase assay, cell-based assay, and molecular docking., Bioorganic & Medicinal Chemistry Letters, Volume 25, Issue 22, Pages 5147-5154, 2015.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer that can enhance the anticancer effect by selectively inducing apoptosis of colorectal cancer cells without affecting normal cells.

In addition, another object of the present invention to provide a health functional food composition for preventing or improving colorectal cancer.

In order to achieve the above object, the present invention is 1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea represented by the following formula (1) or a medicament thereof It provides a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising a salt acceptable as an active ingredient.

[Formula 1]

In addition, the present invention provides a 1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof It provides a method for enhancing colorectal cancer cell death, which is administered to colon cancer cells of a mammal.

In addition, the present invention provides a 1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof Provided is a method of preventing or treating colorectal cancer, comprising administering to a subject.

In addition, the present invention provides 1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof It provides a health functional food composition for preventing or improving colorectal cancer comprising as an active ingredient.

Pharmaceutical composition for preventing or treating colorectal cancer according to the present invention, a method for promoting colon cancer cell death, a method for preventing or treating colorectal cancer and a dietary supplement for preventing or improving colorectal cancer is 1- (4- (3-chloro-4 By containing (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea or a pharmaceutically acceptable salt thereof, it can be usefully used for the prevention, amelioration or treatment of colorectal cancer.

1 is a graph showing the results of performing the WST-1 assay to investigate the effect of 8c of the present invention on cell growth.
2 to 7 are graphs showing the results of performing the WST-1 assay to investigate the effect of the compounds of Formulas 3 to 8 on cell growth.
FIG. 8 is a cell viability graph showing the extent to which 8c affects cell growth of colorectal cancer.
9 is a photograph showing the appearance of colony forming ability is inhibited when 8c is administered to colon cancer cells.
10 is a Western blot showing that the expression of apoptosis-related protein is increased when 8c is treated in the colorectal cancer cell line (HT-29, HCT116).
11 is a graph showing a significant increase in apoptosis in colorectal cancer cell lines administered 8c.
12 is a western blotting result confirming that DR5 (Death receptor5) known to be associated with apoptosis is significantly increased when 8c is administered to colon cancer cells.
FIG. 13 shows the results of a significant increase in DR5 (Death receptor5), which is known to be associated with apoptosis upon administration of 8c to colorectal cancer cells, at the RNA level.
14 is a western blot result showing that the reduction of DR5 inhibits the death of colorectal cancer cells induced by 8c when inhibiting the expression of DR5 in colorectal cancer cell lines (HT-29, HCT116).
Figure 15 is a graph showing the results of performing WST-1 assay after treatment with 8c after knocking down DR5 in colorectal cancer cell lines (HT-29, HCT116).
FIG. 16 is a graph showing that administration of 8c induces death of colorectal cancer cells and that reduction of DR5 inhibits the death of colorectal cancer cells induced by 8c.
FIG. 17 shows a significant increase in Nrf2, which is known to be associated with apoptosis, when 8c is administered to colorectal cancer cells.
FIG. 18 is a result showing at the RNA level that 8c administration to colorectal cancer cells significantly increased Nrf2, which is known to be associated with apoptosis.
19 is a western blot result showing that the reduction of Nrf2 inhibits the death of colorectal cancer cells induced by 8c when inhibiting the expression of Nrf2 in colorectal cancer cell lines (HT-29, HCT116).
20 is a graph showing the results of performing WST-1 assay after treatment with 8c after knocking down Nrf2 in colorectal cancer cell lines (HT-29, HCT116).
FIG. 21 is a graph showing that administration of 8c induces death of colorectal cancer cells, and that reduction of Nrf2 inhibits death of colorectal cancer cells induced by 8c.
22 is a graph showing the expression level of Nrf2 at the RNA level in colorectal cancer cells knocked down DR5.
Figure 23 is a graph showing the expression level of Nrf2 according to the administration of 8c in colorectal cancer cells knocked down DR5.
24 is a graph showing the expression level of DR5 at the RNA level in colorectal cancer cells knocked down Nrf2.
25 is a graph showing the expression level of Keap1 according to the administration of 8c in colorectal cancer cells knocked down DR5.
26 is a graph showing the expression level of HO-1 according to the administration of 8c in colorectal cancer cells knocked down DR5.
27 is a result of Western blot experiment to confirm whether the ER Stress related factor is increased by 8c.
FIG. 28 is a graph showing the extent to which CHOP reduction affects cell viability in colorectal cancer cells (HT-29, HCT116) administered 8c.
29 is a graph showing that apoptosis decreases when CHOP is decreased in the colorectal cancer cell line administered 8c.
Figure 30 shows the result of measuring the ROS generated after the administration of 8c to colorectal cancer cell lines (HT-29, HCT116) to confirm that 8c increases ROS.
31 shows that apoptosis-related factors (DR5, Nrf2) and ER stress-related factors (p-IRE1α, p-PERK, p-elF2α, CHOP, GRP94, Bip) are increased when ROS is increased in colorectal cancer cells. Western blot results.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

The inventors of the present invention, while studying how to selectively induce apoptosis in colorectal cancer cells, one of the derivatives of lapatinib known as a therapeutic agent for breast cancer, 1- (4- (3-chloro-4 (3-fluorine) When lobenzyloxy) phenylamino) quinazolin-6-yl) urea was administered to colon cancer cells, it was confirmed that apoptosis of colon cancer cells was increased, thereby completing the present invention.

Therefore, the present invention provides a pharmaceutical composition for preventing or treating colorectal cancer, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

The compound represented by Formula 1 is 1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea [1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea], hereinafter referred to as '8c'.

8c represented by Chemical Formula 1 of the present invention exhibits anticancer efficacy of inhibiting proliferation of colon cancer cells and inducing apoptosis, and can be used stably without seriously stimulating normal cells or causing harmful effects. There is this.

Accordingly, the 8c may be usefully used as an active ingredient of the pharmaceutical composition for preventing or treating colorectal cancer.

In particular, the 8c is a poly (ADP-ribose) polymerase [(Poly (ADP-)) and the activation of caspase 3 (caspase 3), caspase 8 (caspase 9) and caspase 9 (color) Ribose) polymerase, PARP] is characterized by inducing apoptosis of colorectal cancer cells by inducing cleavage.

According to one embodiment of the present invention, it is confirmed that apoptosis increases when the 8c is administered to colon cancer cells.

The compound represented by Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and the salt may be used in the form of either a pharmaceutically acceptable basic salt or an acid salt. The basic salt may be used in the form of organic salt or inorganic salt, and may be sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aluminum salt, ammonium salt or triethyl. It may be selected from the group consisting of aluminum salts and pyridinium salts, but is not limited thereto.

In addition, acid salts are useful acid addition salts formed by free acid. The inorganic acid and organic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, and methanesulfonic acid may be used as the organic acid. , Benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glyconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid Etc. can be used. Preferably, hydrochloric acid may be used as the inorganic acid, and methanesulfonic acid may be used as the organic acid.

In addition, the compound represented by Formula 1 of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.

The addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound of Formula 1 in a water miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile and adding an excess of organic base It can be prepared by adding an aqueous base solution of an inorganic base and then precipitating or crystallizing it. Alternatively, the solvent or excess base may be evaporated and dried in this mixture to obtain an addition salt, or the precipitated salt may be prepared by suction filtration.

8c of the present invention is N- [3-chloro-4-[(3-fluorophenyl) methoxy] phenyl] -6- [5-[(2-methylsulfonylethylamino) methyl represented by the following formula (2): ] -2-furyl] quinazolin-4-amine is a compound made by modifying the quinazoline core moiety. The compound of Formula 2 is lapatinib known as a breast cancer treatment agent.

[Formula 2]

In the present invention, "cancer" refers to a disease associated with cell death control, and refers to a disease caused by overproliferation of cells when a balance of normal apoptosis is broken. These abnormal hyperproliferative cells can sometimes invade surrounding tissues and organs to form masses and cause destruction or deformation of normal structures in the body, commonly referred to as cancer.

In general, the term "tumor" refers to a mass grown abnormally by autonomous overgrowth of body tissues, and the tumor may be classified into a benign tumor and a malignant tumor. Malignant tumors grow much faster than benign tumors and invade surrounding tissues, resulting in metastasis, which is life threatening. Such malignant tumors are commonly referred to as 'cancer'.

"Colon cancer" in the present invention refers to a malignant tumor consisting of cancer cells generated in the large intestine. Most colorectal cancers are adenocarcinomas that occur in the colon's mucous membranes, including lymphomas, sarcomas, squamous cell carcinomas, and metastatic lesions of other cancers.

In the present invention, "prophylaxis" means any action that inhibits or delays the invention of cancer by administering the pharmaceutical composition of the present invention to a subject, and "treatment" means cancer by administering the pharmaceutical composition of the present invention to a subject It means all the actions that improve or benefit the condition of righteousness.

When the pharmaceutical composition of the present invention is treated to colon cancer cells, caspase 3, caspase 8 and caspa in cancer cells by the action of the 8c or a pharmaceutically acceptable salt thereof of the present invention. By activating cas 9 and cleavage of poly (ADP-Ribose) polymerase [(Poly (ADP-Ribose) Polymerase, PARP]), colon cancer can be effectively prevented, improved or treated. .

In addition, the action of 8c or a pharmaceutically acceptable salt thereof induces the activity of DR5 (death receptor 5) and Nrf2 (Nuclear factor erythroid-derived 2-related factor 2) in cancer cells, thereby effectively preventing colon cancer, Improve or cure.

In the pharmaceutical composition of the present invention, the 8c or a pharmaceutically acceptable salt thereof may be contained in an amount of preferably 0.1 to 500 μM, more preferably 1 to 100 μM, based on the total volume of the pharmaceutical composition. It may be contained as. The use of the 8c or a pharmaceutically acceptable salt thereof in an appropriate amount within the above range has the advantage that the economic and anti-cancer effect according to the 8c is sufficiently exhibited to be more suitable to achieve the object of the present invention.

In addition, the pharmaceutical composition may further contain one or more active ingredients exhibiting the same or similar functions in addition to the 8c.

The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent.

In the present invention, the "pharmaceutically acceptable" means that it is commonly used in the pharmaceutical field, without stimulating the organism upon administration thereof, without inhibiting the biological activity and properties of the compound to be administered.

In the present invention, the type of the carrier is not particularly limited and any carrier can be used as long as it is commonly used in the art. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. Can be. These may be used alone or in combination of two or more thereof.

In addition, the pharmaceutical composition of the present invention may be used by adding other pharmaceutically acceptable additives, such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, if necessary, fillers, extenders, wetting agents, disintegrants, dispersants, surfactants , Binders or lubricants may be additionally added and used.

The pharmaceutical compositions of the present invention can be formulated and used in a variety of formulations suitable for oral or parenteral administration.

Non-limiting examples of the formulation for oral administration include troches, lozenges, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs, and the like. Can be mentioned.

In order to formulate the pharmaceutical composition for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose (Cellulose) or gelatin (Gelatin) and the like; Excipients such as Dicalcium phosphate and the like; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearte, sodium stearyl fumarate, or polyethylene glycol wax can be used, and sweeteners, fragrances, and syrups can also be used. Can be. Furthermore, in the case of a capsule, a liquid carrier such as fatty oil may be additionally used in addition to the above-mentioned materials.

Non-limiting examples of the parenteral preparations include injection solutions, suppositories, respiratory inhalation powders, spray aerosols, ointments, application powders, oils, creams, and the like.

In order to formulate the pharmaceutical composition for parenteral administration, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, external preparations, and the like may be used, and as the non-aqueous solvents and suspensions, propylene glycol, polyethylene Glycols, vegetable oils such as olive oil, injectable esters such as ethyloleate and the like can be used.

Further, more specifically, when the pharmaceutical composition of the present invention is formulated as an injection solution, the composition of the present invention is mixed with water with a stabilizer or buffer to prepare a solution or suspension, which may be used as an ampoule or vial. It may be formulated for unit administration. In addition, when the pharmaceutical composition of the present invention is formulated with an aerosol, a propellant or the like may be combined with the additive to disperse the dispersed concentrate or the wet powder.

In addition, when the pharmaceutical composition of the present invention is formulated into an ointment, a cream, or the like, animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide It may be formulated using such as carrier.

A pharmaceutically effective amount, effective dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the mode of administration, the time of administration and / or the route of administration, and the likeness of the reaction to be achieved by the administration of the pharmaceutical composition. The type and extent of the subject, the type, age, weight, general state of health, condition or extent of the disease, sex, diet, excretion, components of the drug or other composition used concurrently or simultaneously with the subject, Various factors and similar factors well known in the medical arts can be varied, and one of ordinary skill in the art can easily determine and prescribe a dosage effective for the desired treatment.

The dosage for the more preferred effect of the pharmaceutical composition of the present invention is preferably 0.001 mg / kg to 1,000 mg / kg, more preferably 0.01 mg / kg to 50 mg / kg, most preferably 0.1 per day. mg / kg to 5 mg / kg. At this time, the concentration of 8c in the pharmaceutical composition is preferably 0.1 to 500 μM, preferably 1 to 100 μM. Administration of the pharmaceutical composition of the present invention may be administered once a day, may be divided into several times. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The route of administration and mode of administration of the pharmaceutical composition of the present invention may be independent of each other, and are not particularly limited in the way, and may be any route of administration and mode of administration as long as the pharmaceutical composition can reach the desired site. Can follow. The pharmaceutical composition may be administered by oral or parenteral administration.

The parenteral administration method may be administered by, for example, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine epidural injection, cerebrovascular injection, or intrathoracic injection, A method of applying, spraying or inhaling the composition to a diseased site may also be used, but is not limited thereto.

The pharmaceutical composition of the present invention may be preferably administered orally or by injection, more preferably orally.

In addition, the pharmaceutical composition may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

In addition, the pharmaceutical compositions of the present invention may be administered for human or animal use.

The present invention also provides a method for enhancing colon cancer cell death, characterized by administering 8c or a pharmaceutically acceptable salt thereof to colorectal cancer cells of a mammal.

The present invention also provides a method of preventing or treating colorectal cancer comprising administering 8c or a pharmaceutically acceptable salt thereof to a subject.

In the present invention, "mammal" or "individual" refers to all animals including mammals including rats, rabbits, dogs, cats, cattle, sheep, pigs and other domestic animals, humans and the like.

In the method for preventing or treating colorectal cancer of the present invention, the dosage, route of administration, mode of administration, etc. of the 8c or a pharmaceutically acceptable salt thereof is as described above in connection with the pharmaceutical composition of the present invention.

The present invention also provides a dietary supplement for preventing or improving colorectal cancer comprising 8c or a pharmaceutically acceptable salt thereof as an active ingredient.

"Improvement" in the present invention means any action that at least reduces the parameters associated with the condition being treated, for example the extent of symptoms.

When the health functional food composition of the present invention is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method.

The kind of the food is not particularly limited, and includes all foods in a general sense. Non-limiting examples of foods to which the substance can be added include, but are not limited to, meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, Tea, a drink, an alcoholic beverage, or a vitamin complex.

When the health functional food composition of the present invention is a beverage composition, it may contain various flavors or natural carbohydrates and the like as an additional ingredient, as in a conventional beverage. Non-limiting examples of the natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin, cyclodextrin; Synthetic sweeteners such as saccharin and aspartame; and the like. The proportion of the additional components added above may be appropriately determined by the choice of those skilled in the art.

The dietary supplement composition of the present invention is a variety of nutritional agents, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginsam and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, Alcohols, carbonating agents used in carbonated drinks, and the like. The health functional food composition of the present invention may contain the pulp for the production of natural fruit juice, fruit drink or vegetable drink. These components can be used independently or can be used in combination of 2 or more type. The proportion of such additives may also be appropriately selected by those skilled in the art.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

EXAMPLE

Production Example

Ahmed Elkamhawy et al. (Targeting EGFR / HER2 tyrosine kinases with a new potent series of 6-substituted 4-anilinoquinazoline hybrids: Design, synthesis, kinase assay, cell-based assay, and molecular docking., Bioorganic & Medicinal Chemistry Letters, Volume 25, Issue 22, Pages 5147-5154, 2015.), 8c (1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) Urea). The 8c was obtained by modifying the quinazoline core portion of lapatinib represented by the formula (2).

Experimental Example 1 Effect of 8c on Cell Growth in Colorectal Cancer Cells

1-1. Inhibition of cancer cell growth in colorectal cancer cell line-WST-1 assay

WST-1 assay was performed to investigate the effect of 8c represented by Formula 1 on cell growth in colorectal cancer cell lines and normal colon cells.

First, seeding colon cancer cells (HT29, colo205, DLD-1, HCT116) and colon normal cells (CCD-18Co) in 96well, respectively, and then dissolved 8c in dimethyl sulfoxide (DMSO) the next day. , 10, 25, 50, 100 μM).

After 24 hours, the reaction was carried out for 4 hours after the addition of WST-1 solution. The absorbance at 450 nm was measured, and the number of living cells was measured.

1 is a graph showing the results of performing the WST-1 assay to investigate the effect of 8c of the present invention on cell growth.

Looking at Figure 1, it can be seen that 8c inhibits the growth of colon cancer cells concentration-dependently induces death.

In addition, to compare with the experimental results of 8c, as another derivative of lapatinib, 8d compound represented by the following formula (3), 10b compound represented by the formula (4), 10c compound represented by the formula (5), The effect of the 10d compound, 11a compound represented by the following formula (7), and 11c compound represented by the following formula (8) on cell growth was investigated in the same manner as in 8c.

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

2 to 7 are graphs showing the results of performing the WST-1 assay to investigate the effect of the compounds of Formulas 3 to 8 on cell growth.

1 to 7, 8c of the present invention and 11a of Formula 7 significantly inhibited cell growth of the colorectal cancer cell line, but in the case of 11a, it was confirmed that also induces the death of normal colon cells.

1-1-1. Inhibition of Cell Proliferation of 8c in Colorectal Cancer Cell Lines

First, K-RAS wild-type colorectal cancer cells (HT-29), mutant colorectal cancer cells (HCT116), and colonic normal cells (CCD-18Co) were treated with 10% fetal calf serum and 1% in Dulbecco's Modification of Eagle's Medium (DMEM) medium. Penicillin-streptomycin was added and cultured at 37 ° C. and 5% CO2 constant temperature. Colorectal cancer cells of 8 × 10 ⁴ were suspended in 200 μl of cell culture medium, seeded uniformly in 96-well microplate, and cultured overnight. Then, 8c was dissolved in dimethyl sulfoxide (DMSO) and added to each well at various concentrations (0, 20, 40, 60, 80, 100 μM) and incubated for 24 hours.

In addition, after the reaction for 3 hours in the WST-1 solution was measured by absorbance at 450 nm to measure the number of living cells is shown in Figure 8.

8 is a cell viability graph showing the extent to which 8c affects cell growth of colorectal cancer. Referring to FIG. 8, in the normal colon cells, 8c had little effect, but in K-RAS wild-type and mutant colon cancer cells, 8c was found to inhibit cell growth in a concentration-dependent manner.

1-1-2. Of 8c colon cancer cell colony formation-colony formation assay

In addition, colon cancer cells (HT-29, HCT116) were seeded on a soft agar plate, and then cultured overnight. Then, 40 μM of 8c was added to each plate and incubated for 14 days. When colonies were formed, staining was performed to check the number of colonies.

9 is a photograph showing the appearance of colonies formed in the experiment. Referring to FIG. 9, it can be seen that the experimental group to which 8c is added significantly reduces the colony forming ability.

1-2. Protein level confirmed apoptosis in colorectal cancer cell line treated with 8c-Western blot

In order to determine whether cell growth inhibited by 8c is caused by apoptosis, the expression level of apoptosis-related molecules was examined.

Specifically, experiments were performed to compare the expression of apoptosis-related proteins when 8c was treated in the colorectal cancer cell lines (HT-29, HCT116). To this end, first, the colon cancer cell lines (HT-29, HCT116) were seeded in 60 dishes, and then treated with 8c (40 μM) the next day. After 4 hours, the protein was collected by lysis and western blotting was performed. In addition, changes in expression were confirmed using markers related to cell death (PARP, Caspase) and are shown in FIG. 10.

10 is a Western blot showing that expression of apoptosis-related proteins is increased when 8c is treated in a colorectal cancer cell line (HT-29, HCT116). Referring to FIG. 10, cell death such as c-Caspase 3 (Cleaved Caspase-3), c-Caspase 8 (Cleaved Caspase-8), c-Caspase 9 (Cleaved Caspase-9), and c-PARP (Cleaved PARP). It was confirmed that the expression of the proteins involved in.

Through these results, it was confirmed at the cellular level that apoptosis increases when 8c is administered to colon cancer cells.

1-3. Increased apoptosis in colorectal cancer cell line treated with 8c-Flow cytometry

The purpose of this study was to determine whether 8c affects cell death in colorectal cancer cell lines (HT-29, HCT116).

To this end, first, the colon cancer cell lines (HT-29, HCT116) were seeded in 60 dishes, and then treated with 8c (40 μM) the next day. After 4 hours, the cells were harvested. Annexin V-FITC, PI (propidium iodide) stained and killed by using a flow cytometry machine to measure the number of cells are shown in FIG.

11 is a graph showing a significant increase in apoptosis in colorectal cancer cell lines administered 8c.

Experimental Example 2. Identification of apoptosis regulatory molecule (DR5) induced by 8c in colorectal cancer cells

2-1. Death receptor expression in colon cancer cell line treated with 8c-Western blotting and PCR

2-1-1. western blotting

The aim of this study was to determine whether 8c influences the expression of signal transduction molecule (DR5 protein), which is known to be involved in apoptosis in colorectal cancer cell lines (HT-29, HCT116).

To this end, the colon cancer cell lines (HT-29, HCT116) were seeded in 60 dishes, and then treated with 8c (40 μM) the next day. After 4 hours, the cells were harvested and the protein was collected by lysis, followed by western blotting.

12, it was confirmed at the protein level that a significant increase in death receptor5 (DR5), which is known to be associated with apoptosis, was observed when 8c was administered to colon cancer cells.

2-1-2. PCR

In addition, we investigated whether 8c affects the expression of a signaling molecule (mRNA of DR5) that is known to be involved in apoptosis in colorectal cancer cell lines (HT-29, HCT116).

To this end, after seeding the colorectal cancer cell lines (HT-29, HCT116) at 60πdish, the next day was treated with 8c (40 μM), cells were harvested after 24 hours. After RNA extraction, cDNA was synthesized through PCR, and shown in FIG. 13 after PCR.

13, it was confirmed at the RNA level that 8c administration of colorectal cancer cells significantly increased DR5 (Death receptor5), which is known to be associated with cell death.

2-2. Western blotting

The purpose of this study was to examine whether the phenomenon of increasing DR5 in colorectal cancer cells is associated with apoptosis induced by 8c.

To this end, knockdown of DR5 using DR5 siRNA in colorectal cancer cell lines (HT-29, HCT116) was performed after 8c treatment, followed by western blot.

14 is a western blot result showing that the reduction of DR5 inhibits the death of colorectal cancer cells induced by 8c when inhibiting the expression of DR5 in colorectal cancer cell lines (HT-29, HCT116). Referring to FIG. 14, when 8c is administered to the colorectal cancer cell lines (HT-29, HCT116), expression of cleaved PARP associated with apoptosis is significantly increased, but when 8c is administered after 8c is administered, the 8c is suppressed by 8c. It can be seen that the expression of induced Cleaved PARP is reduced.

2-3. WST-1 assay

In addition, Figure 15 is a graph showing the results of performing WST-1 assay after treatment with 8c after knocking down DR5 in colon cancer cell lines (HT-29, HCT116). Looking at Figure 15, it can be seen that the reduction of DR5 inhibits the death of colorectal cancer cells induced by 8c.

2-4. Flow cytometry

In colon cancer cells, we examined the effect of DR5 on apoptosis induced by 8c.

To this end, the colon cancer cell lines (HT-29, HCT116) were seeded in 60 dishes, and then treated with 8c (40 μM) the next day. After harvesting the cells after 24 hours, double staining with Annexin V-FITC, PI (propidium iodide), and measuring the number of living cells using a flow cytometry machine are shown in Figure 16.

FIG. 16 is a graph showing that administration of 8c induces death of colorectal cancer cells and that reduction of DR5 inhibits the death of colorectal cancer cells induced by 8c.

These results demonstrate that an increase in DR5 by 8c induces apoptosis of colon cancer cells.

Experimental Example 3. Confirmation of apoptosis regulatory molecule (Nrf2) induced by 8c in colorectal cancer cells

3-1. Nrf2 increase in colorectal cancer cell line treated with 8c-Western blotting and PCR

3-1-1. western blotting

The aim of this study was to determine whether 8c influences the expression of the signal transduction molecule (Nrf2), which is known to be involved in apoptosis in colorectal cancer cell lines (HT-29, HCT116).

To this end, the colon cancer cell lines (HT-29, HCT116) were seeded in 60 dishes, and then treated with 8c (40 μM) the next day. After 4 hours, the cells were harvested and the protein was collected by lysis, followed by western blotting.

17, it was confirmed at the protein level that a significant increase in Nrf2, which is known to be associated with apoptosis when administered 8c to colon cancer cells.

3-1-2. PCR

In addition, we investigated whether 8c influences the expression of signal transduction molecule (Nrf2 mRNA), which is known to be involved in apoptosis in colorectal cancer cell lines (HT-29, HCT116).

To this end, after seeding the colorectal cancer cell lines (HT-29, HCT116) at 60πdish, the next day was treated with 8c (40 μM), cells were harvested after 24 hours. After cell lysis with TriZol, RNA was isolated using chloroform and isopropanol, and cDNA was synthesized by PCR. Realtime PCR was performed using the synthesized cDNA and Nrf2 TaqMan probe, and the results are shown in FIG. 18.

18, it was confirmed at the RNA level that 8c administration to colon cancer cells significantly increased Nrf2, which is known to be associated with apoptosis.

3-2. Western blotting

The purpose of this study was to examine whether the increase in Nrf2 in colorectal cancer cells is associated with apoptosis induced by 8c.

To this end, Nrf2 was knocked down using Nrf2 siRNA in colorectal cancer cell lines (HT-29, HCT116), followed by 8c treatment, followed by western blot.

19 is a western blot result showing that the reduction of Nrf2 inhibits the death of colorectal cancer cells induced by 8c when inhibiting the expression of Nrf2 in colorectal cancer cell lines (HT-29, HCT116). 19, the expression of Cleaved PARP associated with apoptosis was significantly increased when 8c was administered to the colorectal cancer cell lines (HT-29, HCT116), but when 8c was administered after 8c was administered after suppressing the expression of Nrf2. It can be seen that the expression of induced Cleaved PARP is significantly reduced.

3-3. WST-1 assay

In addition, Figure 20 is a graph showing the results of performing the WST-1 assay after treatment with 8c after knocking down Nrf2 in colorectal cancer cell lines (HT-29, HCT116). Looking at Figure 20, it can be seen that the reduction of Nrf2 inhibits the death of colorectal cancer cells induced by 8c.

3-4. Flow cytometry

In colon cancer cells, we examined the effect of Nrf2 on apoptosis induced by 8c.

To this end, the colon cancer cell lines (HT-29, HCT116) were seeded in 60 dishes, and then treated with 8c (40 μM) the next day. After harvesting the cells after 24 hours, double staining with Annexin V-FITC, PI (propidium iodide), and measuring the number of living cells using a flow cytometry machine are shown in Figure 21.

FIG. 21 is a graph showing that administration of 8c induces death of colorectal cancer cells, and that a decrease in Nrf2 inhibits the death of colorectal cancer cells induced by 8c.

These results demonstrate that the increase of Nrf2 by 8c induces apoptosis of colon cancer cells.

Experimental Example 4 Relationship between DR5 and Nrf2 Increased by 8c in Colorectal Cancer Cells

The relationship between DR5 and Nrf2 controlled by 8c was attempted.

To this end, DR5 and / or Nrf2 were knocked down in colorectal cancer cell line (HT-29). Colorectal cancer cells knocked down the DR5 and / or Nrf2 were seeded in a 60π dish, and the cells were harvested after 24 hours. After cell lysis with TriZol, RNA was isolated using chloroform and isopropanol, and cDNA was synthesized by PCR. Then, Realtime PCR was performed using the synthesized cDNA and DR5, Nrf2, Keap1 or HO-1 TaqMan probe, and the results are shown in FIGS. 22 to 26.

22 is a graph showing the expression level of Nrf2 at the RNA level in colorectal cancer cells knocked down DR5. Looking at Figure 22, it can be seen that the expression of Nrf2 is reduced when knocking down DR5 in colorectal cancer cells.

Figure 23 is a graph showing the expression level of Nrf2 according to the administration of 8c in colorectal cancer cells knocked down DR5. Referring to FIG. 23, when knocking down DR5 in colorectal cancer cells, expression of Nrf2 increased by 8c may be decreased.

24 is a graph showing the expression level of DR5 at the RNA level in colorectal cancer cells knocked down Nrf2. Looking at Figure 24, when knocking down Nrf2 in colorectal cancer cells it can be seen that there is no significant change in the mRNA level of DR5.

25 is a graph showing the expression level of Keap1 according to the administration of 8c in colorectal cancer cells knocked down DR5. Referring to FIG. 25, when knocking down DR5 in colorectal cancer cells, expression of Keap1 that degrades Nrf2 may be increased.

26 is a graph showing the expression level of HO-1 according to the administration of 8c in colorectal cancer cells knocked down DR5. When knocked down DR5 in colorectal cancer cells, mRNA expression of HO-1 located below Nrf2 was also reduced.

Experimental Example 5. Confirmation of ROS and ER stress increase by 8c in colorectal cancer cells

DR5 is known to be increased by ROS-ER stress. Thus, we tried to determine whether the increase of DR5 by 8c is due to ROS-ER stress.

For this purpose, we specifically examined whether ER stress-related factors increase by 8c administration in colorectal cancer cells.

5-1. Western blot

First, the colorectal cancer cell lines (HT-29, HCT116) were seeded in 60 dishes, and then treated with 8c (40 μM) the next day. After 4 hours, the cells were harvested and the protein was collected by western blotting, followed by lysis.

27 is a result of Western blot experiment to confirm whether the ER Stress related factor is increased by 8c.

Referring to FIG. 27, it can be seen that p-IRE1α, p-PERK, p-elF2α, ATF4, CHOP, GRP94, etc., which are factors related to ER Stress, are increased by 8c.

5-2. WST-1 assay

The purpose of this study was to determine the effect of 8c treatment after knocking down CHOP, the final transcription factor of ER stress, in colon cancer cell lines (HT-29, HCT116).

First, the colorectal cancer cell lines (HT-29, HCT116) with or without knocking down CHOP were seeded in 60 dishes and then treated with 8c (40 μM) the next day. After 24 hours, the reaction was carried out for 2 hours in the WST-1 solution, and the absorbance was measured at 450 nm. The number of living cells was measured and shown in FIG. 28.

FIG. 28 is a graph showing the extent to which CHOP reduction affects cell viability in colorectal cancer cells (HT-29, HCT116) administered 8c.

Referring to FIG. 28, treatment of 8c after knocking down CHOP in colorectal cancer cells (HT-29, HCT116) reduced cell death that was increased by 8c.

5-3. Flow cytometry

The aim of this study was to determine whether the reduction of CHOP affects cell death in 8c-administered colorectal cancer cell lines (HT-29, HCT116).

To this end, the CHOP was knocked down using siCHOP in colon cancer cell lines (HT-29, HCT116). Colorectal cancer cells knocked down or non-knockdown colon cancer cells were seeded in 60 dishes and treated with 8c (40 μM) after 18 hours. After 24 hours, cells were harvested with Trypsin-EDTA. Stained with Annexin V-FITC, PI (propidium iodide) and the number of dead cells was measured using a flow cytometry machine is shown in Figure 29.

29 is a graph showing that apoptosis is reduced when CHOP is decreased in the colorectal cancer cell line administered 8c.

5-4. Flow cytometry

ER stress is known to be associated with ROS, and ROS is known to be associated with Nrf2. In addition, it was confirmed through the above experiment that Nrf2 is increased when 8c is administered to colon cancer cells.

Accordingly, to determine whether ROS is induced when 8c is administered to colon cancer cells.

Colon cancer cells (HT-29, HCT116) were seeded in 6-well plate and treated with 8c the next day. After 24 hours, 10 μM of DCF-DA was added, stained for 10 minutes, cells were removed with trypsin, released again with PBS, and measured by flow cytometry.

Figure 30 shows the results of measuring the ROS generated after the administration of 8c to colorectal cancer cell lines (HT-29, HCT116) to confirm whether 8c increases ROS. 30, it was confirmed that ROS is significantly increased by administering 8c to colon cancer cells.

5-5. Western blotting

The purpose of this study was to investigate the effect of ROS increased by the administration of 8c in colorectal cancer cells on the expression of signaling factors (DR5, Nrf2) and ER stress related factors associated with cell death.

To this end, the colon cancer cell lines (HT-29, HCT116) was treated with H ₂ O ₂ and / or ROS inhibitor NAC, and then western blot.

Colon cancer cell lines (HT-29, HCT116) were seeded in 96 well plates. The next day, NAC (0 or 1 μM) and / or H ₂ O ₂ (0 or 300 μM) was administered and reacted for 1 hour. Thereafter, the cells were harvested and the protein was collected by lysis, followed by western blotting, as shown in FIG. 31.

31 shows that apoptosis-related factors (DR5, Nrf2) and ER stress-related factors (p-IRE1α, p-PERK, p-elF2α, CHOP, GRP94, Bip) are increased when ROS is increased in colorectal cancer cells. Western blot results.

Based on the above results, ROS is increased when 8c is administered to colon cancer cells, and the increased ROS is associated with apoptosis-related factors (DR5, Nrf2) and ER stress-related factors (p-IRE1α, p-PERK, p-elF2α, Increasing CHOP, GRP94, Bip) induces apoptosis of colorectal cancer.

Hereinafter, the present invention will be described in more detail with reference to preferred examples. However, these examples are intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Claims (11)

1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient Pharmaceutical composition for preventing or treating colorectal cancer.
[Formula 1]

The method of claim 1,
1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof is caspase 3 (caspase) 3) to induce the activation of caspase 8 and caspase 9 and to induce cleavage of poly (ADP-Ribose) polymerase (PARP). Characterized in that the composition. The method of claim 1,
1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea represented by Formula 1 or a pharmaceutically acceptable salt thereof is DR5 (death receptor 5 And anti-cancer activity through induction of the activity of Nrf2 (Nuclear factor erythroid-derived 2-related factor 2). The method of claim 1,
The pharmaceutically acceptable salt is a composition, characterized in that the basic salt or acid salt. The method of claim 4, wherein
The basic salt is any one selected from the group consisting of sodium salts, potassium salts, calcium salts, lithium salts, magnesium salts, cesium salts, aluminum salts, ammonium salts, triethylaminium salts and pyridinium salts Composition. The method of claim 4, wherein
The acid salt is one inorganic acid selected from the group consisting of hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid and phosphoric acid, or citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid. , By one organic acid selected from the group consisting of malonic acid, glutaric acid, acetic acid, glyconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid and aspartic acid A composition characterized in that the acid addition salt formed. The method of claim 1,
A composition comprising a pharmaceutically acceptable carrier, excipient or diluent further. Mammals other than humans include 1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea or a pharmaceutically acceptable salt thereof represented by Formula 1 below A method for enhancing colon cancer cell death, comprising administering to colon cancer cells.
[Formula 1]

1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea or a pharmaceutically acceptable salt thereof represented by Formula 1 below A method of preventing or treating colorectal cancer comprising the step of administering.
[Formula 1]

1- (4- (3-chloro-4 (3-fluorobenzyloxy) phenylamino) quinazolin-6-yl) urea represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient Health functional food composition for preventing or improving colon cancer.
[Formula 1]

The method of claim 10,
A composition characterized in that it further comprises food acceptable additives, excipients or flavoring agents.

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