KR102563834B1 - Novel compound for inducing apoptosis and composition for anticancer comprising the same - Google Patents

Abstract

The present invention relates to a novel compound that induces apoptosis and an anticancer composition containing the same, and more particularly, the novel compound activates caspase to induce apoptosis in cancer cells, thereby inducing prostate cancer, breast cancer, lung cancer, and colon cancer. It relates to a novel compound that induces apoptosis that can prevent, treat, and improve cancer diseases such as cancer and skin cancer, and an anticancer composition comprising the same.

Description

Novel compound inducing apoptosis and anti-cancer composition comprising the same

The present invention relates to a novel compound that induces apoptosis and an anti-cancer composition comprising the same.

There are three processes by which cells die. Among them, when cells damaged by external problems die due to external factors, it is called necrosis. On the other hand, when a cell is infected, damaged, or at the end of its lifespan, cell suicide often regulated by the cell itself occurs, which is called apoptosis. When a cell eats itself and dies, it is called autophagy. During this process, DNA is cut, and organelles and cytoplasmic components within the cell are fragmented. Protein or nucleic acid fragments released at this time are absorbed and recycled by other cells. In other words, it is also called programmed cell death, and the cell commits suicide through a special program (suicide signal) that is designed to cause the cell to die.

Programmed apoptosis is an essential factor in the development and differentiation of organisms. For example, a fetus in the womb develops a spatula-shaped hand, then takes the shape of a finger as the cells between the fingers die. Another example is when a tadpole loses its tail in the process of becoming a frog. This implies that if this program does not work properly, it can cause structural abnormalities in animals. In addition, if there is an abnormality in the part that causes apoptosis or if the cell has immunity to a substance that causes apoptosis and suicide does not occur, cancer may occur.

There are two types of apoptosis: type-I (apoptosis) and type-II (autophagic cell death). In general, type-I apoptosis progresses through the process of condensation of chromatin, formation of DNA fragments, and generation of small cell fragments (apoptotic bodies). The process by apoptosis is characterized by not inducing inflammation due to phagocytosis of surrounding cells.

Normally, proteins such as p53 or pRb correct DNA when DNA mutations occur, but when this method does not work, apoptosis is the last resort. In other words, if there is a problem or a mutation in the DNA constituting proteins such as p53 or pRb, even if there is a problem with the cell, it does not commit suicide and continues to grow, and eventually becomes a cancer cell.

Intrinsic pathways occur when cells sense that their DNA is in serious trouble. Once the mitochondrial membrane is cleaved, the self-destruct proteins attached to the membrane are exposed to the cytoplasm, and one of the self-destruct proteins, Cytochrome C, binds to APAF-1 to form an apoptosome, and procaspase ( It binds to the end of pro-caspase-9 and activates caspase-9, followed by activation of caspase-3 and caspase-7 involved in the execution of apoptosis.

In addition, death receptors in the Extrinsic Pathway exist on the cell surface, and function to recognize and accept substances that induce death in cells and transmit signals into cells. For example, the FAS receptor is a membrane protein with a lot of cysteine on the outside and a death domain inside the cell. When FAS ligand, which can activate these receptors, binds to them, the structure of the receptor changes and the death domain is activated, which activates the inactive pro-caspase to produce caspases. When caspase-3 and caspase-7 are activated, cells begin to degrade in response to substances that induce cell death through apoptosis.

Since cancer cells are actively differentiated, the effect of apoptosis is much greater than that of normal cells, so attempts to kill cancer cells by inducing apoptosis are being actively studied in the development of anticancer drugs. Therefore, substances activating caspase enzymes have utility as anticancer agents.

Accordingly, the inventors of the present invention made diligent efforts to find a substance capable of inhibiting the proliferation of cancer cells by activating caspase enzyme, which is a protein involved in the induction of apoptosis, and promoting apoptosis function. A derivative compound was synthesized. The present invention was completed by confirming that the 'compound' is a structure having appropriate molecular weight and flexibility that a drug should have, and promotes apoptosis of cancer cells by activating 'caspase' enzyme.

Susan Elmore; Apoptosis: A Review of Programmed Cell Death Toxicol Pathol; 35(4): 495-516 (2007)

In order to solve the above problems, the present invention is a novel compound capable of inhibiting the proliferation of cancer cells by activating the caspase enzyme, which is a protein involved in inducing apoptosis, and inducing apoptosis, and an anticancer compound comprising the same. It is an object thereof to provide a composition.

The object of the present invention is not limited to the object mentioned above. The objects of the present invention will become more apparent from the description that follows, and will be realized by means and combinations thereof set forth in the claims.

The novel compound according to the present invention activates the caspase enzyme to induce apoptosis of cancer cells and inhibits the proliferation of cancer cells, thereby having the advantage of being applicable to drugs and foods capable of preventing, improving or treating cancer diseases.

Specifically, the present invention provides Compound 1 represented by Formula 1 below, or a pharmaceutically or food-acceptable salt thereof.

[Formula 1]

In Formula 1, R ₁ and R ₂ are each independently selected from the group consisting of hydrogen, halogen, and lower alkyl having 1 to 10 carbon atoms;

R ₃ , R ₄ and R ₅ are each independently selected from the group consisting of hydrogen, halogen, lower alkyl having 1 to 10 carbon atoms, and lower alkyl having 1 to 10 carbon atoms substituted with 1 to 3 halogen atoms;

R ₆ is selected from the group consisting of hydrogen, a hydroxyl group and lower alkyl having 1 to 10 carbon atoms, and n is an integer of 0 to 5.

In the present invention, the term 'halogen' means chloro, fluoro, bromo and iodine.

A pharmaceutically or food-acceptable salt of the compound represented by Formula 1 may be prepared by a conventional method in the art. A pharmaceutically or food-acceptable salt should have low toxicity to the human body and not adversely affect the biological activity and physicochemical properties of the parent compound.

In the present invention, the pharmaceutically or food-wise acceptable salt is not particularly limited as long as it is pharmaceutically- or food-wise acceptable, but may preferably be an inorganic acid salt, an organic acid salt, or a metal salt. Examples of preferred inorganic acid salts may be hydrochloride, phosphate, sulfate, or bisulfate salts. Examples of preferred organic acid salts may include malate, maleate, citrate, fumarate, besylate, camsylate, or edisyl salt. Examples of preferred metal salts include: may be a calcium salt, sodium salt, magnesium salt, strontium salt or potassium salt.

As one specific example of the present invention, compound 1 represented by Formula 1 can be synthesized by coupling a piperazine derivative and various compounds, examples of which are compounds 2, 4, 6, 8, 10, and 12 am. In addition, compounds of compounds 3, 5, 7, 9, 11, and 13 are further exemplified:

Compound 2 represented by Formula 2 below;

[Formula 2]

Compound 3 represented by Formula 3 below;

[Formula 3]

Compound 4 represented by Formula 4 below;

[Formula 4]

Compound 5 represented by Formula 5:

[Formula 5]

Compound 6 represented by Formula 6:

[Formula 6]

Compound 7 represented by Formula 7:

[Formula 7]

Compound 8 represented by Formula 8:

[Formula 8]

Compound 9 represented by Formula 9:

[Formula 9]

Compound 10 represented by Formula 10:

[Formula 10]

Compound 11 represented by Formula 11:

[Formula 11]

Compound 12 represented by Formula 12:

[Formula 12]

Compound 13 represented by Formula 13:

[Formula 13]

On the other hand, the anti-cancer composition of the present invention may include the compound 1. Preferably, the anti-cancer composition of the present invention may be one selected from the group consisting of the compounds 2 to 13.

The anti-cancer composition may include one or more pharmaceutically acceptable carriers, diluents or excipients. Examples of the carrier, diluent or excipient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In addition, the anti-cancer composition may include a second anti-cancer agent or anti-cancer adjuvant. The second anticancer agent or anticancer adjuvant includes interferon, interleukin-2, paclitaxel, vincristine, vinblastin, doxorubicin, etoposide (etoposide), irinotecan hydrochloride, cisplatin, amsacrine, cytosine arabinoside, fluoro uracil and taxol. Not limited.

In addition, the anti-cancer composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. there is. Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like. A solid preparation for oral administration may be prepared by mixing the hydroxymethoxychalcone compound with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

At this time, the anti-cancer composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is the patient's health condition, Depending on the type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field can The anti-cancer composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art. For example, the dosage may increase or decrease depending on the route of administration, severity of disease, sex, weight, age, etc., so the dosage is not limited to the scope of the present invention in any way.

A preferred dosage of the anti-cancer composition varies depending on the condition and body weight of the patient, the severity of the disease, the type of drug, the route and duration of administration, but can be appropriately selected by those skilled in the art.

The anti-cancer composition may induce apoptosis by activating caspase. The anti-cancer composition can prevent or treat cancer diseases by inhibiting the proliferation of cancer cells by activating caspase enzyme, which is a protein involved in the induction of apoptosis, to promote apoptosis of cancer cells. The caspase is derived from cysteine aspartate protease, and is a protein having the character of an apoptosis executor. When a cell receives internal and external stimuli, it sends an apoptosis signal, induces the activation of caspase-8 and caspase-9, activates the inactive procaspase-3 into caspase-3, cleaves PARP, and finally undergoes apoptosis. can cause

In addition, apoptosis promoting proteins and apoptosis inhibitory proteins may play an important role in the induction of apoptosis in cancer cells. These are present inside and outside the mitochondria and can be increased or decreased by stimulation of factors activated by various apoptotic signals. Bid exists in the cytoplasm in an inactive state and is cleaved into truncated Bid (tBid) by caspase-8, which can affect the activation of Bcl-2 family proteins by increasing mitochondrial membrane permeability. The expression of Bcl-2 and Bcl-xL proteins decreased when apoptosis was induced, and the expression of Bax and Bad proteins increased. Expression of these molecules increases the permeability of the mitochondrial membrane and releases cytochrome c, which activates caspase-9 and activates caspase-3, which can finally cleavage PARP.

The cancer disease may be selected from the group consisting of prostate cancer, endometrial cancer, cervical cancer, liver cancer, rectal cancer, breast cancer, colon cancer, skin cancer, lung cancer, ovarian cancer, melanoma, pancreatic cancer, polycystic cancer, and neuroblastoma. Preferably, it may be selected from the group consisting of prostate cancer, breast cancer, lung cancer, colon cancer, and skin cancer.

The anti-cancer composition can effectively inhibit the growth of cancer cells by causing apoptosis through the activation of caspase enzyme in cancer cells and exhibiting a cell death effect. In addition, cancer can be prevented, improved, or treated by effectively inhibiting the proliferation of cancer cells. In addition, the anti-cancer composition may be effective in treating patients who are not responsive to treatment with other anti-cancer agents or have developed resistance to these other anti-cancer agents.

In addition, the present invention can provide a food for the prevention or improvement of cancer comprising an anti-cancer composition containing the compound 1, preferably one selected from the group consisting of the compounds 2 to 13.

The food may be prepared in the form of tea, juice, or drink using the compound 1 as an active ingredient for drinking, or may be ingested after being granulated, encapsulated, or powdered. In addition, it can be prepared in the form of a composition by mixing the compound of the present invention with a known active ingredient known to have an effect of preventing and improving cancer diseases.

The above foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled food, jam, marmalade, etc.), fish, meat and their processed foods (e.g. ham, sausage corn beef, etc.), breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort food, frozen It can be prepared by adding food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.) and the like.

In addition, the present invention can provide a method for inhibiting proliferation or inducing apoptosis of cancer cells by administering an anti-cancer composition containing Compound 1 to a subject to activate caspases present in organs in the subject. The method can prevent, improve, or treat cancer diseases by inducing apoptosis of cancer cells and inhibiting the proliferation of cancer cells by activating the caspase present in the organ of the subject with the anti-cancer composition.

In addition, the present invention can provide a method for preventing or treating cancer using the anticancer composition containing the compound 1. The treatment method may include administering the anti-cancer composition into the human body in a pharmaceutically effective amount. The anti-cancer composition may be administered parenterally, subcutaneously, intraperitoneally, intrapulmonaryly, and intranasally, and may be administered by a suitable method including intralesional administration if necessary for local treatment. Injectables may include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Preferred modes of administration may include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and drip injection.

The dosage of the anti-cancer composition may vary depending on various factors, and the route of administration may also vary depending on various factors such as the patient's age, administration period, weight, severity of the disease, whether the patient is conscious, the type of concomitant drug, etc. A variety of parenteral routes of administration can be used. In addition, it is also possible to administer each separately simultaneously or serially or separately over time.

In the present invention, the term "subject" refers to monkeys, cows, horses, pigs, sheep, dogs, cats, and rats, including humans, whose symptoms can be improved by administering the anticancer composition or anticancer adjuvant of the present invention. , mice, chimpanzees, and other mammals.

In the present invention, the term "administration" means introducing a predetermined substance into a subject by any suitable method, and the administration route of the anticancer composition or anticancer adjuvant of the present invention is any general route as long as it can reach the target tissue. It can be administered orally or parenterally through.

As used herein, the term “prevention” refers to inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed with the disease or condition, but is predisposed to the disease or condition. As used herein, the term “treatment” refers to (a) inhibiting the development of a disease or condition, (b) alleviating a disease or condition, and (c) elimination of a disease or condition in a subject.

The novel compound according to the present invention activates caspase enzyme to induce apoptosis of cancer cells and inhibit the proliferation of cancer cells, thereby preventing, improving or treating cancer diseases such as prostate cancer, breast cancer, lung cancer, colon cancer and skin cancer. There are advantages applicable to drugs and foods.

The effects of the present invention are not limited to the effects mentioned above. It should be understood that the effects of the present invention include all effects that can be inferred from the following description.

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited by the following examples.

Preparation Example 1: Preparation method of Compound A-1:

[Scheme 1]

Reactor 4.8g 3-bromo-5-(trifluromethyl)aniline, 4-methylimidazole

1.97g, K ₂ CO ₃ 3.04g, CuI 0.57g, Ethylenediamine 0.18g, and DMSO 20ml were added, and the inside was substituted with nitrogen bubbling, followed by a nitrogen balloon. After heating the reactant to 150 ℃ reacted for 12 hours. When the reaction was completed, the reactant was cooled to 45-50 ° C, and then 20 ml of 14% ammonia water was added and stirred for 1 hour. The reactant was cooled to room temperature, 30ml of EA and 30ml of H ₂ O were added, and the layers were separated after stirring for 30 minutes. 30 ml of EA was added to the lower aqueous layer, and the layers were separated after stirring for 30 minutes. The organic layer was collected, passed through a short silica gel column to remove green-blue Cu salt, and washed with 30 ml of EA. After concentrating the filtrate under reduced pressure at 35-40 ° C, 20 ml of EA was added to the residue to dissolve it, and then 40 ml of n-hexane was slowly added to crystallize. After stirring at room temperature for 1 hour, it was filtered and washed twice with 10 ml of n-hexane to obtain 2.44 g of 3-(trifluoromethyl)-5-(4-methyl-1H-imidazol-1-yl)benzeneamine as an off-white solid. (Yield = 50%)

^1H -NMR (400 MHz, DMSO-d6) δ 8.08 (d, J=1.3Hz, 1H), 7.37 (s, 1H), 6.97 (s, 1H), 6.92 (t, J=1.8Hz, 1H) , 6.80 (s, 1H), 5.88 (s, 2H), 2.15 (s, CH3, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 151.2, 138.8, 135.1, 131.6, 120.8, 114.5, 108.2, 108.1, 103.6, 103.5, 13.9

9 g of 4,6-Dichloride-2-methylpyrimidine and 300 ml of THF were added to the reactor and stirred at room temperature to make a clear solution. 12.1 g of 3-(trifluoromethyl)-5-(4-methyl-1H-imidazol-1-yl)benzeneamine was added thereto, and the reactant was cooled to 0-5°C. 17 g of sodium t-butoxide was slowly added at once. The reactant exothermed at 3-10 °C, and the reactant exhibited a yellow-light brown suspension. While gradually raising the temperature at room temperature, the mixture was stirred for 24 hours. Upon completion of the reaction, the mixture was concentrated under reduced pressure at 40°C to obtain a brown oily solid. 500ml of EA was added, stirred at room temperature for 1 hour, filtered, and washed twice with 50ml of EA to remove insoluble matter. 1000 ml of EA and 1 g of charcoal were added and heated to reflux for 1 hour. After filtering with a Celite pad, washing with EA 100ml x 2 times, and concentrating under reduced pressure at 40℃, an ivory-yellow solid was obtained. After adding 100ml of EA and stirring at room temperature for 1 hour, the solid was filtered and washed 3 times with 25ml of EA. Compound A-1(6-chloro-2-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)pyrimidin as a white solid by drying in a vacuum oven at 40℃ -4-amine) 14.3 g was obtained. (Yield = 77.5%) The purity measured by HPLC is 97.9%.

¹ H NMR (400 MHz, DMSO-d6) δ 10.16 (bs, NH), 8.14 (s, 2H), 7.91 (s, 1H), 7.56 (s, 1H), 7.42, (s, 1H), 6.64 ( s, 1H), 2.44 (s, CH3, 3H), 2.14 (s, CH3, 3H)

Preparation Example 2: Preparation method of Compound A-2:

[Scheme 2]

4.8 g of 5-bromo-2-(trifluromethyl)aniline, 2.46 g of 2-methylimidazole, 5.53 g of K ₂ CO ₃ , 0.57 g of CuI, 0.18 g of ethylenediamine, and 20 ml of DMSO were added to the reactor. After heating the reactant temperature to 150 ℃ reacted for 12 hours. When the reaction was completed, the reactant was cooled to 20 to 30 ° C, and then 20 ml of 14% ammonia water was added and stirred for 1 hour. When ammonia water is added, heat of about 10 ° C is observed. The reactant was cooled to room temperature, 30 ml of EA and 30 ml of H ₂ O were added, and the layers were separated after stirring for 30 minutes. The upper organic layer was collected, passed through a short silica gel column to remove the green-blue Cu salt, and washed twice with 30ml of EA. The filtrate was separated into layers again, and the upper organic layer was collected, and 2.5 g of neutral charcoal was added thereto, followed by stirring at room temperature for 1 hour. After filtering with a Celite pad, washing with EA 50ml x 2 times, and concentrating under reduced pressure at 40℃, a brown oily liquid was obtained. A silica gel column was run under 100% EA eluent to obtain 2.5 g of 2-(trifluoromethyl)-5-(2-methyl-1H-imidazol-1-yl)benzenamine as a light yellow solid. (Yield = 51.7%)

¹ H NMR (400 MHz, DMSO-d6) δ 7.23 (m, 1H), 6.87 (m, 1H), 6.85 (m, 1H), 6.75 (m, 2H), 1.98 (bs, NH2), 2.24 (s , CH3, 3H)

1.88 g of 4,6-Dichloride-2-methylpyrimidine and 65 ml of THF were added to the reactor and stirred at room temperature to make a clear solution. Here, 2.5 g of 2-(trifluoromethyl)-5-(2-methyl-1H-imidazol-1-yl)benzenamine was dissolved in 20 ml of THF, and the reactant was cooled to 0 to 5°C. 3.5 g of sodium t-butoxide was slowly added at once. The reactant exothermed at 3-10 °C, and the reactant exhibited a yellow-light brown suspension. While gradually raising the temperature at room temperature, the mixture was stirred for 24 hours. Upon completion of the reaction, the mixture was concentrated under reduced pressure at 40°C to obtain a brown oily solid. 200ml of EA was added, stirred at room temperature for 30 minutes, filtered, and washed twice with 50ml of EA to remove insoluble matter. 2.5 g of neutral charcoal was added and heated to reflux for 1 hour. After filtering with a Celite pad, washing with EA 50ml x 2 times, and concentrating under reduced pressure at 40℃, an ivory-yellow solid was obtained. After adding 30ml of EA and stirring at room temperature for 1 hour, the solid was filtered and washed twice with 10ml of EA. Compound A-2(6-chloro-N-(2-(trifluoromethyl)-5-(2-methyl-1H-imidazol-1-yl)phenyl)-2-methylpyrimidin- 4-amine) 1.77 g was obtained. (Yield = 37.9%) The purity measured by HPLC is 98.1%.

^1H NMR (400 MHz, DMSO-d6) δ 10.24 (bs, NH), 8.08 (s, 1H), 7.99 (s, 1H), 7.44 (s, 1H), 7.38(s, 1H), 6.92(s , 1H), 6.66(s, 1H), 2.35(s, CH3, 3H)

Preparation Example 3: Preparation method of Compound A-3:

[Scheme 3]

5.82 g of 4-methylimidazole and 100 ml of NMP were added to the reactor and stirred at room temperature to make a clear solution. Here, 5 g of 3-fluoronitrobenzene and 23.1 g of Cs ₂ CO ₃ were added. After heating the reactant temperature to 120 ° C., the reaction was performed for 6 hours. When the reaction was completed, the reactant was cooled to 20-30 ° C., 300 ml of EA and 300 ml of H ₂ O were added, and the layers were separated after stirring for 30 minutes. The upper organic layer was collected, 200 ml of H ₂ O was added, and the layers were separated after stirring for 30 minutes. The organic layer was treated with anhydrous MgSO ₄ , dried, filtered, and washed twice with 15 ml of EA. The filtrate is a clear red-brown solution. 4g of 10% Pd/C was added and reacted at room temperature for 12 hours under hydrogen balloon pressure. When the reaction was completed, the reactant was filtered through a Celite pad, washed twice with 50 ml of EA, and then concentrated under reduced pressure at 40° C. to obtain a brown oily liquid. 2.1 g of 3-(4-methyl-1H-imidazol-1-ylbenzenamine was obtained as a light brown oil by running a silica gel column under 100% EA eluent. (Yield = 34.2%)

¹ H NMR (400 MHz, DMSO-d6) δ 7.12 (d, 1H), 7.09 (t, 1H), 6.85 (d, 1H), 6.58-6.55 (m, 1H), 6.49 (t, 1H), 6.45 ~6.42 (m, 1H), 5.37 (bs, NH), 2.21 (s, CH3, 3H)

2.17 g of 4,6-Dichloride-2-methylpyrimidine and 55 ml of THF were added to the reactor and stirred at room temperature to make a clear solution. Here, 2.1 g of 3-(4-methyl-1H-imidazol-1-ylbenzenamine was dissolved in 10 ml of THF and added, and the reactant was cooled to 0-5 ° C. 4.1 g of sodium t-butoxide was slowly added at once. Exotherm was generated at 3~10℃, and the reactant showed a yellow~light brown suspension. The temperature was gradually raised at room temperature and stirred for 24 hours. When the reaction was completed, it was concentrated under reduced pressure at 40℃ to obtain a brown oily solid. EA 100ml was added, stirred at room temperature for 30 minutes, filtered, washed 25ml EA x 2 times to remove insoluble matter, added 2g neutral charcoal, heated to reflux for 1 hour, filtered with a Celite pad, and washed 2 times 25ml EA Then, it was concentrated under reduced pressure at 40°C to obtain a light brown solid. After adding 10ml of EA, stirring at room temperature for 1 hour, the solid was filtered, washed twice with 5ml of EA, and dried in a vacuum oven at 40°C to obtain compound A- as a white solid. 1.71 g of 3(6-chloro-2-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)phenyl)pyrimidin-4-amine) was obtained (yield = 47.1%) measured by HPLC. One purity is 93.7%.

¹ H NMR (400 MHz, DMSO-d6) δ 9.98 (bs, NH), 7.83 (s, 1H), 7.53 (s, 1H), 7.45 (s, 1H), 7.26 (s, 1H), 7.07 (m , 1H), 6.88(s, 1H), 6.64(s, 1H), 2.32(s, CH3, 3H)

Example 1: Method for preparing compound 2:

[Scheme 4]

1 g of (6-chloro-2-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)pyrimidin-4-amine) [Compound A-1] in the reactor , 35ml of EA and 20ml of DMSO were added and stirred at room temperature to make a clear solution. Here, 2 g of 1-(2-Hydroxyethyl)piperazine was diluted in 10 ml of EA, added to the reaction mixture, and washed with 5 ml of EA. The reaction was heated to reflux and stirred for 12 hours. When the reaction was completed, the reactant was cooled to room temperature, and then 50ml of H ₂ O was slowly added at once. After adding 30ml of THF and 30ml of EA, stirring for 30 minutes to separate the layers, the upper organic layer was collected. The organic layer was treated with anhydrous MgSO ₄ , dried, filtered, and washed twice with 10 ml of EA. The filtrate was concentrated under reduced pressure at 40°C to obtain a light yellowish oily. After adding 8ml of toluene and stirring at room temperature for 30 minutes, the resulting solid was filtered, washed twice with 5ml of toluene, and dried in a vacuum oven at 40°C to obtain 20.5g of the compound as a white solid. (Yield = 40%) The purity measured by the HPLC method is 99.1%.

Confirmation of manufacture of compound 2

^1H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.16 (t, J=1.96Hz, 1H), 8.11 (d, J=1.57Hz, 1H), 7.95 (m, 1H), 7.41(m, 1H), 7.39 (t, J=1.17Hz, 1H), 5.79 (s, 1H), 3.52~3.44 (m, 6H), 2.45~2.37 (m, 6H), 2.31 (s, CH3, 3H), 2.14 (s, CH3, 3H)

Examples 2-4: Preparation of compounds 3-5

As shown in the chemical reaction formula below, (6-chloro-2-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)pyrimidin-4 of Preparation Example 1 -amine) The target compounds of Examples 2 to 4 were synthesized through a coupling reaction between [Compound A-1] and various piperazine derivative compounds.

Example 2: Method for preparing compound 3:

[Scheme 5]

Yield = 76.5%, purity measured by HPLC method is 99.0%.

Manufacturing confirmation of compound 3

^1H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.16 (t, J=1.96Hz, 1H), 8.11 (d, J=1.56Hz, 1H), 7.96 (s, 1H), 7.41(s, 1H), 7.40 (t, J=1.18Hz, 1H), 5.79 (s, 1H), 4.58 (t, J=5.48Hz, 1H), 3.51 (t, J=6.26, 5.87Hz, 2H) ), 3.47~3.44 (m, 6H), 3.39~3.36 (m, 2H), 2.50~2.43 (m, 6H), 2.31 (s, CH3, 3H), 2.14 (d, J=0.79Hz, CH3, 3H )

Example 3: Method for preparing compound 4:

[Scheme 6]

Yield = 92.3%, purity measured by HPLC method is 99.2%.

Manufacturing confirmation of compound 4

^1H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.16 (t, J=1.96Hz, 1H), 8.11 (d, J=1.56Hz, 1H), 7.96 (s, 1H), 7.41(s, 1H), 7.40 (t, J=1.18Hz, 1H), 5.80 (s, 1H), 4.41 (bs, OH), 3.47~3.44 (m, 4H), 3.43~3.40 (t, J= 6.65, 6.26Hz, 2H), 2.39~2.323 (m, 6H), 2.31 (s, CH3, 3H), 2.14 (d, J=1.18Hz, CH3, 3H), 1.57 (p, 2H)

Example 4: Method for preparing compound 5:

[Scheme 7]

Yield = 88.7%, purity measured by HPLC method is 99.2%.

Manufacturing confirmation of compound 5

^1H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.16 (t, J=1.96, 1.56Hz, 1H), 8.11 (d, J=1.56Hz, 1H), 7.96 (s, 1H) ), 7.42(s, 1H), 7.40 (t, J=1.18Hz, 1H), 5.80 (s, 1H), 3.46 (t, J=4.70, 5.08Hz, 4H), 2.33~2.32 (m, 4H) , 2.31 (s, CH3, 3H), 2.17 (s, CH3, 3H), 2.14 (d, J=1.18Hz, CH3, 3H)

Example 5: Method for preparing compound 6:

[Scheme 8]

진공 오븐에서 건조하여 white solid로 화합물 60.41g을 얻었다. (수율=82%)HPLC method로 측정한 순도는 98.8 이다.6-chloro-N-(2-(trifluoromethyl)-5-(2-methyl-1H-imidazol-1-yl)phenyl)-2-methylpyrimidin-4-amine [Compound A-2] 0.4 g, EA 14ml and 8ml of DMSO were added and stirred at room temperature to make a clear solution. Here, 0.78 g of 1-(2-Hydroxyethyl)piperazine was diluted in 5ml of EA, added to the reaction mixture, and washed with 2ml of EA. The reaction was heated to reflux and stirred for 12 hours. When the reaction was completed, the reactant was cooled to room temperature, and then 20ml of H ₂ O was slowly added at once. After adding 12ml of THF and 12ml of EA, the mixture was stirred for 30 minutes to separate the layers, and the upper organic layer was collected. The organic layer was treated with anhydrous MgSO ₄ , dried, filtered, and washed twice with 10 ml of EA. The filtrate was concentrated under reduced pressure at 40°C to obtain a light yellowish oily. After adding 3.5ml of Toluene, stirring at room temperature for 30 minutes, filtering the generated solid, washing 2 times with 5ml of Toluene, and 40

After drying in a vacuum oven, 60.41 g of the compound was obtained as a white solid. (Yield = 82%) The purity measured by the HPLC method is 98.8.

Manufacturing confirmation of compound 6

^1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.08 (t, J=1.96Hz, 1H), 8.01 (s, 1H), 7.35 (d, J=1.57Hz, 1H), 7.25 (s, 1H), 6.91 (d, J=1.57Hz, 1H), 5.78 (s, 1H), 4.40 (t, J=5.48Hz, 1H), 3.51~3.44 (m, 6H), 2.48~2.42 (m, 4H), 2.38 (t, J=6.26Hz, 2H), 2.34 (s, CH3, 3H), 2.28 (s, CH3, 3H)

Examples 6-8: Preparation of compounds 7-9

As shown in the chemical reaction formula below, 6-chloro-N-(2-(trifluoromethyl)-5-(2-methyl-1H-imidazol-1-yl)phenyl)-2-methylpyrimidin-4-amine of Preparation Example 2 The target compounds of Examples 6 to 8 were synthesized through a coupling reaction between [Compound A-2] and various piperazine derivative compounds.

Example 6: Method for preparing compound 7:

[Scheme 9]

Yield = 85.3%, purity measured by HPLC method is 94.5%.

Manufacturing confirmation of compound 7

^1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.08 (t, J=1.96, 1.57Hz, 1H), 8.01 (s, 1H), 7.35 (d, J=1.57Hz, 1H) ), 7.25 (s, 1H), 6.91 (d, J=1.57Hz, 1H), 5.78 (s, 1H), 4.58 (bs, OH), 3.51 (t, J=5.87Hz, 2H), 3.45~3.43 (m, 6H), 3.39~3.36 (m, 2H), 2.49~2.43 (m, 6H), 2.30 (s, CH3, 3H), 2.28 (s, CH3, 3H)

Example 7: Method for preparing compound 8:

[Scheme 10]

Yield = 84.6%, purity measured by HPLC method is 99.2%.

Manufacturing confirmation of compound 8

^1H NMR (400 MHz, DMSO-d6) δ 9.56 (s, 1H), 8.08 (d, J=1.96Hz, 1H), 8.01 (s, 1H), 7.35 (d, J=1.57Hz, 1H), 7.25 (s, 1H), 6.91 (d, J=1.63Hz, 1H), 5.78 (s, 1H), 4.41 (bs, OH), 3.47~3.44 (m, 4H), 3.43~3.40 (t, J= 6.26Hz, 2H), 2.38~2.36 (m, 4H), 2.34 (s, CH3, 3H), 2.32~2.30 (m, 2H), 2.28 (s, CH3, 3H), 1.56 (p, 2H)

Example 8: Preparation of compound 9:

[Scheme 11]

Yield = 88.7%, purity measured by HPLC method is 98.9%.

Manufacturing confirmation of compound 9

^1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.08 (t, J=1.96Hz, 1H), 8.01 (s, 1H), 7.35 (d, J=1.57Hz, 1H), 7.26 (s, 1H), 6.91 (d, J=1.57Hz, 1H), 5.78 (s, 1H), 3.46 (t, J=4.70, 5.09Hz, 4H), 2.34 (s, CH3, 3H), 2.32 (t, J=5.09Hz, 4H), 2.28 (s, CH3, 3H), 2.17 (s, CH3, 3H)

Example 9: Method for preparing compound 10:

[Scheme 12]

0.4 g of 6-chloro-2-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)phenyl)pyrimidin-4-amine [Compound A-3], 14 ml of EA, and 8 ml of DMSO were added to the reactor. It was added and stirred at room temperature to make a clear solution. Here, 0.95 g of 1-(2-Hydroxyethyl)piperazine was diluted in 5 ml of EA, added to the reaction mixture, and washed with 2 ml of EA. The reaction was heated to reflux and stirred for 12 hours. When the reaction was completed, the reactant was cooled to room temperature, and then 20ml of H ₂ O was slowly added at once. After adding 12ml of THF and 12ml of EA, the mixture was stirred for 30 minutes to separate the layers, and the upper organic layer was collected. The organic layer was treated with anhydrous MgSO ₄ , dried, filtered, and washed twice with 10 ml of EA. The filtrate was concentrated under reduced pressure at 40°C to obtain a light yellowish oily. A silica gel column was run under 100% EA eluent to obtain 100.13 g of the compound as a yellow oily solid. (Yield = 25.0%) The purity measured by HPLC is 98.4%.

Confirmation of manufacture of compound 10

^1H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H), 7.84 (t, J=1.96Hz, 1H), 7.50~7.47 (m, 1H), 7.35 (t, J=7.82, 8.22Hz , 1H), 7.23 (d, J=1.17Hz, 1H), 6.92~6.89 (m, 1H), 6.87 (d, J=1.17Hz, 1H), 5.79 (s, 1H), 4.41 (bs, OH) , 3.49 (t, J=6.26Hz, 2H), 3.44~3.424 (m, 4H), 2.44~2.41 (m, 4H), 2.38 (t, J=6.26Hz, 2H), 2.31 (s, CH3, 3H) ), 2.25 (s, CH3, 3H)

Examples 10-12: Preparation of compounds 11-13

As shown in the chemical reaction formula below, 6-chloro-2-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)phenyl)pyrimidin-4-amine [Compound A-3 ] and various piperazine derivative compounds, the target compounds of Examples 10 to 12 were synthesized.

Example 10: Preparation of compound 11:

[Scheme 13]

Yield = 22.4%, purity measured by HPLC method is 99.5%.

Confirmation of manufacture of compound 11

^1H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 1H), 7.83 (t, J=1.96Hz, 1H), 7.50~7.47 (m, 1H), 7.35 (t, J=7.82, 8.22Hz , 1H), 7.23 (d, J=1.56Hz, 1H), 6.92~6.90 (m, 1H), 6.87 (d, J=1.56Hz, 1H), 5.78 (s, 1H), 4.58 (bs, OH) , 3.50 (t, J=5.86Hz, 2H), 3.44~3.42 (m, 6H), 3.39~3.36 (m, 2H), 2.48~2.43 (m, 6H), 2.31 (s, CH3, 3H), 2.26 (s, CH3, 3H)

Example 11: Preparation of compound 12:

[Scheme 14]

Yield = 55.5%, purity measured by HPLC method is 96.4%.

Manufacturing confirmation of compound 12

^1H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 1H), 7.84 (t, J=1.96Hz, 1H), 7.50~7.47 (m, 1H), 7.35 (t, J=7.83, 8.21Hz , 1H), 7.23 (d, J=1.56Hz, 1H), 6.92~6.90 (m, 1H), 6.87 (d, J=1.17Hz, 1H), 5.78 (s, 1H), 4.42 (bs, OH) , 3.44~3.39 (m, 6H), 2.38~2.32 (m, 6H), 2.31 (s, CH3, 3H), 2.26 (s, CH3, 3H), 1.56 (p, 2H)

Example 12: Preparation of compound 13:

[Scheme 15]

Yield = 75.5%, purity measured by HPLC method is 95.9%.

Confirmation of manufacture of compound 13

^1H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 1H), 7.84 (t, J=1.96Hz, 1H), 7.50~7.47 (m, 1H), 7.35 (t, J=7.83, 8.21Hz , 1H), 7.23 (d, J=1.17Hz, 1H), 6.92~6.90 (m, 1H), 6.87 (d, J=1.56Hz, 1H), 5.79 (s, 1H), 3.44 (t, J= 4.70, 5.08Hz, 4H), 2.33~2.30 (m, 4H), 2.31 (s, CH3, 3H), 2.26 (s, CH3, 3H), 2.16 (s, CH3, 3H)

Experimental Example 1: Confirmation of cancer cell proliferation inhibitory ability

As a representative compound according to the present invention, the inhibitory activity of the compound 2 synthesized in Example 1 against prostate cancer, breast cancer, and colorectal cancer was measured.

[Ingredients]

The human cancer cell lines used in this experiment were prepared for prostate cancer, breast cancer, and colorectal cancer as shown in Table 1 below. And 10% FBS was added using RPMI, 37, 5% CO ₂ It was subcultured and used in an incubator.

prostate cancer breast cancer colorectal cancer LNCaP PC-3 RC-58T MCF-7 SW480 HT-29

In addition, DMEM medium supplemented with 100 units/mL of Antibiotic Antimycotic and 10% fetal bovine serum (FBS) was used. Cancer cells were subcultured with 0.25% trypsin-EDTA solution 2 to 3 times a week in a 37 °C, 5% CO ₂ incubator (HERA cell 150, Heraeus, Hanau, Germany), and discarded when the passage number was 10 or more. The cells were cultured again and tested.

[Test method]

Cancer cell proliferation inhibitory ability was measured using the SRB (sulforhodaime B) method. The SRB method is a method for confirming the degree of apoptosis by expressing the total amount of protein in living cells as absorbance. Only viable cells are attached to the well plate by TCA (trichloroacetic acid), and basic amino acid residues in the protein of these cells combine with SRB and dissolve in Tris buffer, which shows absorbance.

Cancer cell lines grown in monolayer were treated with 0.25% trypsin-EDTA solution to make single cells, diluted with culture medium to a final concentration of 1×10 ⁵ cells/mL, and then dispensed into 24 well plates at 37°C, 5% CO ₂ After culturing in an incubator for 24 hours, Compound 2 was added at each concentration and reacted for 24 hours, and the degree of cancer cell proliferation was measured at 540 nm by the sulforhodamine B (SRB, Sigma-Aldrich Co.) method. The effect of Compound 2 on the proliferation of human cancer cells was confirmed through SRB assay.

Experimental Example 1-1: Evaluation of apoptosis efficacy in prostate cancer cells

The effect of Compound 2 prepared in Example 1 on the death of human prostate cancer cells was confirmed through SRB assay, and the results are shown in Table 2.

SRB (sulforhodaime B) assay measurement result division prostate cancer LNCaP PC-3 RC-58T reference drug Sorafenib 18.39 22.67 20.59 Imatinib 36.79 66.86 54.37 Poziotinib 3.99 12.61 5.64 Dasatinib 29.09 54.54 48.14 Nilotinib 61.97 30.43 24.89 Example 1 compound 2 2.64 7.97 6.09

According to the results of Table 2, it was confirmed that Compound 2 prepared in Example 1 had an improved apoptosis effect in all prostate cancer cell lines compared to Poziotinib, which had the highest activity among reference drugs for prostate cancer cell lines. In particular, it was found that the cancer cell growth inhibitory effect on LNCaP was very good.

Experimental Example 1-2: Evaluation of apoptosis efficacy in breast cancer cells

The effect of Compound 2 prepared in Example 1 on the death of human breast cancer cells was confirmed through SRB assay, and the results are shown in Table 3.

SRB (sulforhodaime B) assay measurement result division breast cancer MCF-7 reference drug Sorafenib 32.94 Imatinib 51.81 Poziotinib 7.89 Dasatinib 40.21 Nilotinib 80.94 Example 1 compound 2 6.29

According to the results of Table 3, it was confirmed that Compound 2 prepared in Example 1 had an improved apoptosis effect in breast cancer cell lines compared to Poziotinib, which had the highest activity among reference drugs for breast cancer cell lines.

Experimental Example 1-3: Evaluation of apoptosis efficacy in colon cancer cells

The effect of Compound 2 prepared in Example 1 on the death of human colorectal cancer cells was confirmed through SRB assay, and the results are shown in Table 4.

SRB (sulforhodaime B) assay measurement result division colorectal cancer SW-480 HT-29 reference drug Sorafenib 32.00 40.58 Imatinib 65.88 71.87 Poziotinib 67.65 7.06 Dasatinib 90.19 77.79 Nilotinib 42.51 94.57 Example 1 compound 2 9.41 6.46

According to the results of Table 4, it was confirmed that Compound 2 prepared in Example 1 had an improved apoptosis effect in all colon cancer cell lines compared to Poziotinib, which had the highest activity among reference drugs for colon cancer cell lines. In particular, it was found that the cancer cell growth inhibitory action against HT-29 was very good.

Experimental Example 1-4: Evaluation of apoptosis efficacy in lung cancer cells

The effect of compound 2 prepared in Example 1 on the death of human lung cancer cells was confirmed through SRB assay, and the results are shown in Table 5.

SRB (sulforhodaime B) assay measurement result division lung cancer A549 H1975 PC9 reference drug Sorafenib 27 36 10 Imatinib 96 71 41 Poziotinib 47 12 3 Dasatinib 60 50 20 Nilotinib 128 116 51 Example 1 compound 2 61 59 5

According to the results of Table 5, compared to Poziotinib (excluding the A549 result), which has the highest activity among reference drugs for lung cancer cell lines, Compound 2 prepared in Example 1 has an improved apoptosis effect in lung cancer cell lines, especially PC9 cells. confirmed that there is

In addition, the effect of compounds 2 to 13 prepared in Examples 1 to 12 on the death of human lung cancer cells was confirmed through SRB assay, and the results are shown in Table 6.

SRB (sulforhodaime B) assay measurement result division Lung cancer PC9 compound 2 5.00 compound 3 6.04 compound 4 1.46 compound 5 10.90 compound 6 4.99 compound 7 9.08 compound 8 5.93 compound 9 9.00 compound 10 20.66 compound 11 26.45 compound 12 20.66 compound 13 18.60

According to the results of Table 6, it was confirmed that Compound 4 prepared in Example 3 had an enhanced apoptotic effect on lung cancer cell line PC9 cells.

Experimental Example 1-4: Evaluation of apoptosis efficacy in normal cells

The effect of Compound 2 prepared in Example 1 on the death of human normal cells was confirmed through SRB assay, and the results are shown in Table 7.

SRB (sulforhodaime B) assay measurement result division lung normal cells Venous endothelial normal cells WI-38 HUVEC reference drug Sorafenib 7.85 5.23 Imatinib 46.55 33.84 Poziotinib 21.71 10.94 Dasatinib 25.57 13.78 Nilotinib >100 >100 Example 1 compound 2 32.64 28.99

According to the results of Table 7, Compound 2 prepared in Example 1 showed better apoptosis results than the synthetic anticancer drug (reference drug) used as a control drug for human normal cells. Therefore, it was confirmed that Compound 2 prepared in Example 1 had an excellent apoptosis effect on cancer cells and an excellent apoptosis inhibitory effect on normal cells.

Claims (6)

A compound represented by Formula 1 or a pharmaceutically acceptable salt thereof:
[Formula 1]

In Formula 1, R ₁ and R ₂ are each independently selected from the group consisting of hydrogen, halogen, and lower alkyl having 1 to 10 carbon atoms;
R ₃ , R ₄ and R ₅ are each independently selected from the group consisting of hydrogen, halogen, lower alkyl having 1 to 10 carbon atoms, and lower alkyl having 1 to 10 carbon atoms substituted with 1 to 3 halogen atoms;
R ₆ is selected from the group consisting of hydrogen, a hydroxyl group and lower alkyl having 1 to 10 carbon atoms, and n is an integer of 0 to 5. At least one compound selected from the group consisting of Formulas 2 to 13 or a pharmaceutically acceptable salt thereof:
[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

An anti-cancer composition comprising the compound of claim 1.
Anti-cancer composition comprising the compound of claim 2:
According to claim 3,
The anti-cancer composition is to induce apoptosis by activating caspase.
According to claim 3,
The cancer composition for anticancer is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colon cancer and skin cancer.