KR20240020636A - Novel compound and composition for preventing or treating HER2 positive cancer comprising the same - Google Patents

Abstract

The present invention relates to a novel compound and a composition containing the same for preventing or treating HER2-positive cancer. The novel compound of the present invention can suppress the level of HER2 by inhibiting the protein-protein interaction of ELF3 and MED23, and thereby has a significant anticancer effect. Moreover, even in cases of resistance to trastuzumab, which is previously used clinically as a cancer treatment, treatment with the new compound of the present invention showed a remarkable level of anticancer effect. Therefore, it is expected that the new compound of the present invention can be widely used in the field of cancer treatment.

Description

Novel compound and composition for preventing or treating HER2 positive cancer comprising the same}

It relates to a novel compound and a composition containing the same for preventing or treating HER2-positive cancer.

Protein-protein interaction (PPI) is an essential phenomenon in the human body and is not properly regulated in patients with disease. Accordingly, efforts to develop drugs targeting these PPIs have continued, and a number of antibody drugs have been developed, but only a few small molecule PPI modulators have been developed due to the difficulty in identifying small-sized ligands. Small molecule modulators can solve the problems of antibody treatments, such as low cell internalization, low oral bioavailability, and high immunogenicity, so research on them is needed.

HER2, a member of the HER family, plays a key role in various tumorigenic processes by regulating cell survival, proliferation and differentiation. HER2 overexpression was first observed in breast cancer and was evaluated as a classic therapeutic marker for breast cancer, but today many reports show its association with cancer, suggesting a negative prognostic role in gastrointestinal cancer, especially gastric cancer. For HER2-overexpressing cancer subtypes, chemotherapy is usually administered with trastuzumab (TZMB), an antibody drug that targets the extracellular domain of the HER2 protein. However, in the case of trastuzumab, although it is a clinically successful drug, there is a problem in that treatment becomes difficult when resistance develops (Cancer Res (2008) 68 (5): 1471-1477). Therefore, there is a need to develop small molecule drugs that can solve these problems.

One aspect is to provide a compound represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[Formula 1]

R ₁ is hydrogen;

R ₂ and R ₃ are independently hydrogen or a hydroxy group;

R ₄ is p-methoxynaphthalenyl, , or ego;

where A ₁ is nitrogen, oxygen or sulfur;

A ₂ is halogen or C _1-3 alkoxy.

Another aspect is to provide a compound represented by the following formula (2), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[Formula 2]

R ₅ to R ₇ are independently hydrogen or a hydroxy group;

R ₈ is p -Methoxynaphthalenyl ( p -methoxynaphthalenyl), 1-Methoxynaphthalenyl (1-Methoxynaphthalenyl), , or ego;

R ₉ is C _1-3 alkyl;

where A ₁ is nitrogen, oxygen or sulfur;

A ₂ is halogen, or C _1-3 alkoxy

Another aspect is to provide a composition for inhibiting protein-protein interaction, comprising the compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

Another aspect is to provide a pharmaceutical composition for preventing or treating cancer, comprising the compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

Another aspect is to provide a health functional food composition for preventing or improving cancer, comprising the compound, a stereoisomer thereof, or a foodologically acceptable salt thereof as an active ingredient.

However, the problem to be solved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

One aspect provides a compound represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[Formula 1]

R ₁ is hydrogen;

R ₂ and R ₃ are independently hydrogen or a hydroxy group;

R ₄ is p-methoxynaphthalenyl, , or ego;

where A ₁ is nitrogen, oxygen or sulfur;

A ₂ is halogen or C _1-3 alkoxy.

In this specification, the “halogen” is a fluorine, chlorine, bromine, or iodine atom.

In the present specification, the “C _1-3 alkoxy” is an OR group in which R is C _1-3 in -OR form. For example, but not limited thereto, it may be methoxy, ethoxy, propoxy, or 1-methylethoxy.

The compound may be any one selected from the group consisting of the following compounds:

3-Furan-2-yl-1-(3-hydroxyphenyl)-propenone;

1-(3-Hydroxyphenyl)-3-(4-methoxyphenyl)-propenone);

1-(3-Hydroxyphenyl)-3-(4-methoxynaphthalen-1-yl)-propenone);

3-(4-Chlorophenyl)-1-(3-hydroxyphenyl)-propenone (3-(4-Chlorophenyl)-1-(3-hydroxyphenyl)-propenone);

3-(4-Hydroxyphenyl)-1-(3-hydroxyphenyl)-propenone);

1-(2-Hydroxyphenyl)-3-(4-methoxyphenyl)-propenone);

1-(2-Hydroxyphenyl)-3-(4-methoxynaphthalen-1-yl)-propenone);

3-Furan-2-yl-1-(2-hydroxyphenyl)-propenone;

3-(4-Hydroxyphenyl)-1-(2-hydroxyphenyl)-propenone);

3-(4-Chlorophenyl)-1-(2-hydroxyphenyl)-propenone); and

1-(2-Hydroxyphenyl)-3-thiophen-2-yl-propenone.

Another aspect provides a compound represented by the following formula (2), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[Formula 2]

R ₅ to R ₇ are independently hydrogen or a hydroxy group;

R ₈ is p -Methoxynaphthalenyl ( p -methoxynaphthalenyl), 1-Methoxynaphthalenyl (1-Methoxynaphthalenyl), , or ego;

R ₉ is C _1-3 alkyl;

where A ₁ is nitrogen, oxygen or sulfur;

A ₂ is halogen or C _1-3 alkoxy.

The compound of Formula 2 may be any one selected from the group consisting of the following compounds:

1-[5-furan-2-yl-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-Furan-2-yl-3 -(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);

1-[3-(3-hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-(3-Hydroxyphenyl) -5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);

1-[3-(3-hydroxyphenyl)-5-(4-methoxynaphthalen-1-yl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-( 3-Hydroxyphenyl)-5-(4-methoxynaphthalen-1-yl)-4,5-dihydropyrazol-1-yl]-ethanone);

1-[5-(4-Chlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-(4-Chlorophenyl)- 3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);

1-[5-(4-hydroxyphenyl)-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-(4-Hydroxyphenyl) -3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);

1-[5-furan-2-yl-3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-Furan-2-yl-3 -(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);

1-[3-(2-hydroxyphenyl)-5-(4-methoxynaphthalen-1-yl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-( 2-Hydroxyphenyl)-5-(4-methoxynaphthalen-1-yl)-4,5-dihydropyrazol-1-yl]-ethanone);

1-[5-(4-hydroxyphenyl)-3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-(4-Hydroxyphenyl) -3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);

1-[5-(4-Chlorophenyl)-3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-(4-Chlorophenyl)- 3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);

1-[3-(2-Hydroxyphenyl)-5-thiophen-2-yl-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-(2-Hydroxyphenyl)- 5-thiophen-2-yl-4,5-dihydropyrazol-1-yl]-ethanone);

1-[3-(2-hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-(2-Hydroxyphenyl) -5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);

1-[3-(3-hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-propan-1-one (1-[3-(3 -Hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-propan-1-one);

1-[3-(3-hydroxyphenyl)-5-thiophen-2-yl-4,5-dihydropyrazol-1-yl]-propan-1-one (1-[3-(3- Hydroxyphenyl)-5-thiophen-2-yl-4,5-dihydropyrazol-1-yl]-propan-1-one);

1-[5-(4-chlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-propan-1-one (1-[5-(4- Chlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-propan-1-one).

As used herein, the term "stereoisomer" refers to molecules that have the same chemical formula and the same bonding order between constituent atoms, but have different three-dimensional structures, and are divided into enantiomers and diastereomers. In addition, the stereochemical isomeric form of the pyridone derivative compound according to one embodiment defines all possible compounds that the compound of Formula 1 may have. Unless otherwise stated or indicated, the chemical name of a compound refers to a mixture of all possible stereochemical isomeric forms, which mixture includes all diastereomers and enantiomers of the basic molecular structure. In particular, the stereogenic center can have the R- or S-configuration and the substituents on the divalent cyclic (partially) saturated radical can have the cis- or trans-configuration. Compounds containing a double bond may have E or Z-stereochemistry at the double bond. Stereochemical isomeric forms of the compound represented by Formula 1 are intended to be included within the scope of the invention.

Another aspect provides a composition for inhibiting protein-protein interaction, comprising a compound represented by Formula 1 or Formula 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. .

As used herein, the term “protein-protein interaction” refers to an established high molecular weight relationship between two or more protein molecules as a result of biochemical events mediated by interactions involving electrostatic forces, hydrogen bonding, and hydrophobic effects. It means physical contact of singularity. This is most often observed in certain biomolecular contexts, which refers to physical contact with molecular bonds between chains that occur in cells or living organisms. Various reactions within cells proceed by protein-protein interactions, and abnormal protein-protein interactions can be the starting point of various diseases such as Creutzfeldt-Jakob disease and Alzheimer's disease.

As used herein, the term “cancer” refers to a condition in which problems occur in the normal division, differentiation, and death control functions of cells, causing abnormal hyperproliferation, infiltrating surrounding tissues and organs, forming lumps, and destroying or deforming existing structures. it means.

In addition, but not limited thereto, the above cancers include cervical cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, colon cancer, bone cancer, skin cancer, head cancer, cervical cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, and rectal cancer. , brain tumor, bladder cancer, blood cancer, stomach cancer, perianal cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, Renal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma And it may be any one selected from the group consisting of pituitary adenoma.

As used herein, the term “prevention” refers to any action that suppresses a disease or delays its onset by administering a composition. The term “treatment” refers to any action that improves or beneficially changes the symptoms of a disease by administering a composition.

As used herein, the term "pharmaceutically acceptable" means that the benefit/risk ratio is reasonable and is suitable for use in contact with the tissue of a subject (e.g., a human) without excessive toxicity, irritation, allergic reaction, or other problems or complications, and sound medical judgment. It means a composition within the scope of.

As used herein, the term “pharmaceutically acceptable salt” refers to an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, as well as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids.

These pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodine. Ide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, methylbenzoate Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol It may include, but is not limited to, nitrate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandelate.

Additionally, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically appropriate to prepare sodium, potassium, or calcium salts as metal salts. The corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

The pharmaceutical composition may further include a carrier, excipient, or diluent. Carriers, excipients and diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, not determined. It may contain quality cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

The pharmaceutical composition may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

In the pharmaceutical composition, the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule. The solid preparation may further include excipients. Excipients may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. Additionally, the solid preparation may further include a lubricant such as magnesium stearate or talc. In the pharmaceutical composition, the liquid preparation for oral administration may be a suspension, oral solution, emulsion, or syrup. The liquid formulation may contain water or liquid paraffin. The liquid formulation may contain excipients such as wetting agents, sweeteners, flavoring agents, or preservatives. In the pharmaceutical composition, preparations for parenteral administration may be sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried products, and suppositories. Non-aqueous solvents or suspensions may contain vegetable oil or ester. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The base of the suppository may be witepsol, macrogol, tween 61, cacao, laurel, or glycerogelatin.

The preferred dosage of the pharmaceutical composition varies depending on the individual's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, the compound, its stereoisomer, derivative, solvate, or pharmaceutically acceptable salt may be administered in an amount, for example, from about 0.0001 mg/kg to about 100 mg/kg, or from about 0.001 mg/kg to about 100 mg/kg. The amount can be divided into 1 to 24 times per day, 1 to 7 times per 2 days to 1 week, or 1 to 24 times per month to 12 months. In the pharmaceutical composition, the compound, its stereoisomer, or a pharmaceutically acceptable salt may be included in an amount of about 0.0001% to about 10% by weight, or about 0.001% by weight to about 1% by weight, based on the total weight of the composition.

The administration method may be oral or parenteral administration. Methods of administration may be, for example, oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal. The composition can be administered systemically or topically, alone or in combination with other pharmaceutically active compounds.

Another aspect provides a method of preventing or treating cancer, comprising administering the pharmaceutical composition according to one aspect to a subject. Details of the pharmaceutical composition, administration, prevention and treatment are as described above.

The subject may be a mammal, for example, a human, cow, horse, pig, dog, sheep, goat, or cat, or may be a mammal other than a human. The individual may be suffering from cancer or may be at high risk of suffering from cancer.

The administration method may be oral or parenteral administration. Methods of administration may be, for example, oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal. The pharmaceutical composition can be administered systemically or topically, alone or in combination with other pharmaceutically active compounds.

The dosage of the composition may range, for example, from about 0.0001 mg/kg to about 100 mg/kg, or from about 0.001 mg/kg to about 100 mg/kg. The administration may be divided into 1 to 24 times per day, 1 to 7 times per 2 days to 1 week, or 1 to 24 times per month to 12 months.

Another aspect provides a use of a composition comprising a compound according to one aspect, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof for the prevention or treatment of cancer.

Another aspect provides a health functional food composition comprising the compound according to one aspect, a stereoisomer thereof, or a foodologically acceptable salt thereof.

As used herein, the term “improvement” refers to any action that at least reduces the severity of a parameter associated with an abnormal condition, such as a symptom. At this time, the health functional food composition can be used simultaneously or separately with a drug for treatment before or after the onset of the disease in order to prevent or improve cancer.

The health functional food can be prepared by encapsulating, powdering, or suspending a compound according to one aspect, a stereoisomer thereof, or a food-acceptable salt thereof, and using it as a functional food or adding it to various foods. The above foods include, for example, meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamins. It is a combination medicine, functional food and health food.

The novel compound of the present invention can suppress the level of HER2 by inhibiting the protein-protein interaction of ELF3 and MED23, and thereby has a significant anticancer effect. Moreover, even in cases of resistance to trastuzumab, which is previously used clinically as a cancer treatment, treatment with the new compound of the present invention showed a remarkable level of anticancer effect. Therefore, it is expected that the new compound of the present invention can be widely used in the field of cancer treatment.

Figure 1 shows the results of in silico- based structural analysis to predict the hotspot of the ELF3-MED23 complex. Figure 1a shows the protein binding model of MED23WT and ELF3WT prepared using the Phyre2 server, and Figure 1b shows the MED23 protein. A diagram showing the 3D docking of the ELF3 _137-144 peptide, Figure 1c is a diagram showing the docking of wrenchnolol, adamanolol, gefitinib, and canertinib, which are known to have ELF3-MED23 PPI inhibitory activity, and Figure 1d is a diagram showing the docking of wrenchnolol, adamanolol, gefitinib, and canertinib, which are known to have ELF3-MED23 PPI inhibitory activity. This is a diagram showing a 2D interaction diagram of gefitinib docking generated by //proteins.plus), and Figure 1e is a diagram showing hydrogen bonding and p-stacking involved in the interaction between MED23 and the compound.
Figure 2 shows a 2D diagram of canertinib, showing that canertinib can p-interact with F399 of MED23.
Figure 3 shows the results of analyzing key residues involved in the ELF3-MED23 protein-protein interaction hotspot. Figure 3a shows the effect of each MED23 mutation on the binding level of gal4-ELF3 and MED23 confirmed by SEAP analysis. 3b is a diagram showing split luciferase analysis using a biosensor, and FIGS. 3c and 3d are diagrams showing the binding levels of ELF3 and MED23 _391-582 fragments in images (FIG. 3c) and quantification (FIG. 3d). Figure 3e is a diagram showing the interaction between several full-length MED23 mutants and ELF3 using GST pull-down analysis, Figure 3f is a diagram showing the transcriptional activity level of HER2, and Figure 3g is a diagram showing the expression level of HER2.
Figure 4 shows the results of analyzing small molecules that can affect the protein-protein interaction of ELF3-MED23. Figure 4a is a diagram showing the small molecule design strategy, and Figure 4b shows the ELF3-MED23 protein-protein interaction of the prepared small molecule. This is a diagram confirming the inhibitory effect and cell survival rate, and Figure 4c is a diagram confirming the inhibitory effect and cell survival rate again by varying the concentration of a small molecule compound showing an ELF3-MED23 protein-protein interaction inhibitory effect of more than 65%, and Figure 4d is a diagram confirming the inhibitory effect and cell survival rate. This diagram shows the docking poses of the 25 designed small molecule compounds.
Figure 5 shows the results of selecting small molecule compounds that significantly affect the protein-protein interaction of ELF3-MED23, and Figure 5a shows the results of analyzing the docking poses of gefitinib and candidate compounds overlapping, with hydrogen bond donors in red; A diagram showing the hydrogen bond receptor visualized in blue, Figure 5b is a diagram showing the docking pose diagram of the candidate compound, and Figure 5c shows the binding of the candidate compound to MED23 wild type through hydrogen bonding through LC-MS/MS analysis. Figure 5d is a diagram confirming the binding affinity of candidate compounds to various MED23 fragments through LC-MS/MS analysis, and Figures 5e and 5f show Nluc-ELF3 ^WT and Cluc-MED23 _391-462 fragments. A diagram showing images and quantitative data by luciferase biosensor analysis performed using luciferase biosensor, Figure 5g is a diagram showing fluorescence polarization according to dose-dependent treatment of candidate compounds or unlabeled ELF3 protein, Figure 5h is a diagram showing the IC50 value and Ki value of the candidate compound, Figure 5i is a diagram showing the protein-protein interaction inhibition effect of the candidate compound by immunoprecipitation analysis, and Figure 5j is a diagram showing the HER2 according to dose-dependent treatment of the candidate compound. This is a diagram showing the mRNA expression level, and Figure 5k is a diagram showing the expression level of HER2 and HER2-related downstream signaling molecules according to dose-dependent treatment of the candidate compound.
Figure 6 shows the results of biochemical confirmation of the anticancer activity of the candidate compound. Figure 6a is a diagram confirming the expression levels of HER2, ELF3, and MED23 in various gastric cancer cell lines, and Figure 6b shows the candidate compound in stomach cancer cell lines showing different HER2 levels. is a diagram confirming the cell viability after treatment, Figure 6c is a diagram confirming the cell survival rate after treating NCI-N87 cells with candidate compounds and comparison compounds, and Figure 6d is a diagram confirming the cell viability after treating NCI-N87 cells with candidate compounds and comparison compounds, and Figure 6d is a diagram confirming the cell viability after treating NCI-N87 cells This is a diagram showing cell viability after treatment with candidate compounds (NIH 3T3, and CHO-K1).
Figure 7 shows the results of confirming the anticancer effect of the candidate compound in vitro and in vivo . Figures 7A and 7B are diagrams showing the overall survival rate (Figure 7A) and progression-free survival (Figure 7B) of 1,065 gastric cancer patients, and Figure 7C. is a diagram showing tumor regression after treating NCI-N87 xenograft mice with a candidate compound, Figure 7D is a diagram showing tumors obtained from mice in the vehicle-treated group and the candidate material-treated group, and Figure 7E is a diagram showing the tumor Figure 7F and Figure 7G are diagrams confirming the cell cycle after performing IHC staining for HER2 and Ki75, and Figure 7I shows apoptosis when treating the candidate compounds. This is a diagram confirming the effect, and Figure 7h is a diagram confirming the levels of apoptosis-promoting markers and anti-apoptosis markers caused by treatment with the candidate compound.
Figure 8 shows the results of pharmacological evaluation of the candidate compound. Figure 8a shows the physicochemical properties (e.g. solubility, Log P, and permeability, etc.) of the candidate compound, and Figure 8b shows the genotoxicity of the candidate compound ( Figure 8c is a diagram showing cardiac toxicity, and Figure 8d is a diagram showing the intravenous and oral route pharmacokinetic profiles of the candidate compound.
Figure 9 shows the results confirming the resistance-overcoming effect of the candidate compound, and Figures 9a to 9c show the candidate compounds for parental cells (BT474 and NCI-N87) and trastuzumab resistance-acquired cells (BT474 TR, NCI-N87 TR, and JIMT-1). This is a diagram confirming the dose-dependent therapeutic effect of the compound and trastuzumab, and Figures 9d to 9f show trastzumab-sensitive and trastzumab-resistant cells treated with trastzumab and the candidate compound at different doses, and then HER2 and its major downstream signals. This is a diagram confirming the molecular level, and Figures 9g to gi are diagrams confirming the survival and proliferation of trastuzumab-sensitive and trastuzumab-resistant cells while treating the candidate compound and trastuzumab in a dose-dependent manner.
Figure 10 is a diagram confirming the change in body weight of JIMT-1 xenograft mice treated with trastuzumab or a candidate compound.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

As used herein, the singular forms include the plural forms unless the context clearly indicates otherwise. Additionally, when used herein, “comprise, include” and/or “comprising, including” refer to the mentioned shapes, numbers, steps, operations, members, elements and/or groups thereof. It specifies the presence and does not exclude the presence or addition of one or more other shapes, numbers, operations, members, elements and/or groups.

Manufacturing example

The chemicals and reagents used were mainly obtained from Aldrich Chemical Co., while others were obtained from companies such as TCI. Melting point was measured without correction using a Barnstead Electrothermal melting point apparatus, Manual MELTEMP (model number: 1202D). Chromatographic separation was monitored by thin-layer chromatography using commercially available pre-coated Merck Kieselgel 60 F254 plates (0.25 mm) and detected by visualization at UV 254 and 365 nm. Silica gel column chromatography was performed using Merck Kieselgel 60 (0.040-0.063 mm). All solvents used in chromatography were used as is without distillation. Purity was evaluated by HPLC (Shimadzu LC-20AD) analysis, and the evaluation conditions were as follows: column, SunFire C18 (4.6 mm × 150 mm, 5 mm); Mobile phase conditions: When water is A and acetonitrile is B, following a linear gradient, 50-70% B in 0-15 minutes, 70% B in 15-20 minutes, 100% B in 20-25 minutes, and 25- 50% B in 30 min, flow rate; 1.0 mL/min; Detection, diode array detector (Shimadzu Spd-M20A). The purity of the compound was expressed in percentage (%), and the retention time was expressed in minutes. NMR spectra were recorded with a Varian AS 400 (1H NMR at 400 MHz and 13C NMR at 100 MHz). Chemical shift (δ) was expressed in ppm, and coupling constant (J) was expressed in Hz.

Preparation Example 1. Method for preparing chalcone analogue candidate compounds

1-1. General preparation method of chalcone analogues

50% NaOH (4.0 equivalent) was added to the reaction mixture of acetophenone derivative and benzaldehyde derivative (1.0 equivalent) dissolved in EtOH (10 mL). The reaction mixture was stirred at room temperature (24 hours) and then water was added. The solid formed was filtered, washed with water and dried under vacuum. The solid was purified by silica gel column chromatography (eluent: ethyl acetate: n-hexane) to obtain the desired product.

1-2. Methods for preparing candidate compounds

① Production of 3-Furan-2-yl-1-(3-hydroxyphenyl)-propenone

The reaction was performed according to the general method of Preparation Example 1-1 using 3-hydroxyacetophenone (1.00 g, 7.34 mmol), furfural (0.61 mL, 7.34 mmol), and 50% NaOH (2.35 mL, 29.35 mmol). Purified with eluent (ethyl acetate: n-hexane = 1:3), the desired brown solid compound (compound No. 1) was obtained (Formula 3 below, 1.45 g, 92.1%).

[Formula 3]

Melting point: 155 - 156 °C; R _f 0.54 (ethyl acetate: n -hexane = 1:1); HPLC: R _T 4.46 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 6.37 (d, J = 4.8 Hz, 1H), 6.57 (s, 1H), 6.91 (ddd, J = 8.0, 1.2, 0.8 Hz, 1H), 7.15 (dd, J = 8.0, 8.0 Hz, 1H), 7.25 (d, J = 15.6 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 8.8 Hz, 1H), 9.04 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz): 112.5, 115.0, 115.9, 120.2, 120.4, 129.4, 130.2, 139.2, 144.8, 151.4, 157.6, 189.6 ppm.

② Production of 1-(3-hydroxyphenyl)-3-(4-methoxyphenyl)-propenone (1-(3-Hydroxyphenyl)-3-(4-methoxyphenyl)-propenone)

The reaction was performed according to the general method of Preparation Example 1-1 above using 3-hydroxyacetophenone (1.00 g, 7.34 mmol), 4-methoxy benzaldehyde (0.89 mL, 7.34 mmol), and 50% NaOH (2.35 mL, 29.35 mmol). did. By purifying with eluent (ethyl acetate: n-hexane = 1:3), the desired yellow solid compound (compound No. 2) was obtained (Formula 4 below, 2.40 g, 98.9%)

[Formula 4]

③ 1-(3-Hydroxyphenyl)-3-(4-methoxynaphthalen-1-yl)-propenone (1-(3-Hydroxyphenyl)-3-(4-methoxynaphthalen-1-yl)-propenone) manufacture of

According to the general method of Preparation Example 1-1 above using 3-hydroxyacetophenone (1.00 g, 7.34 mmol), 4-methoxy-1-naphthaldehyde (1.37 g, 7.34 mmol) and 50% NaOH (2.35 mL, 29.35 mmol) The reaction was carried out. Purified with eluent (ethyl acetate: n-hexane = 1:3), the desired orange solid compound (compound No. 3) was obtained (Formula 5 below, 1.56 g, 69.8%).

[Formula 5]

Melting point: 189 - 190 °C; R _f 0.63 (ethyl acetate: n -hexane = 1:1); HPLC: R _T 10.34 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 3.99 (s, 3H), 4.73 (s, 1H), 6.81 (d, J = 8.4 Hz, 1H), 7.03 (ddd, J = 8.0, 2.4, 0.8 Hz, 1H), 7.26 (dd, J = 8.0, 5.6 Hz, 1H), 7.44-7.49 (m, 4H), 7.53 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.51 (d, J = 15.2 Hz, 1H), 8.98 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 55.8, 103.9, 115.3, 119.7, 120.4, 122.4, 122.5, 122.7, 123.1, 123.9, 124.6, 125.3, 126.2, 127.5, 137.9 , 157.7, 190.4 ppm.

④ Production of 3-(4-Chlorophenyl)-1-(3-hydroxyphenyl)-propenone

The reaction was performed according to the general method of Preparation Example 1-1 above using 3-hydroxyacetophenone (1.00 g, 7.34 mmol), 4-chloro benzaldehyde (1.03 g, 7.34 mmol), and 50% NaOH (2.35 mL, 29.35 mmol) did. By purifying with eluent (ethyl acetate: n-hexane = 1:3), the desired pale yellow solid compound (compound No. 4) was obtained (Formula 6 below, 0.99 g, 52.6%).

[Formula 6]

Melting point: 160 - 162 °C; R _f 0.76 (ethyl acetate: n -hexane = 1:1); HPLC: R _T 8.35 min (purity: 99.9%)

¹ H-NMR (CDCl ₃ , 400 MHz) 7.01 (ddd, J = 8.4, 0.8, 0.8 Hz, 1H), 7.24 (d, J = 15.6 Hz, 1H), 7.30 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 8.8 Hz, 2H) ), 7.41 (dd, J = 8.4, 2.4 Hz, 1H), 7.48 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 16.0 Hz, 1H), 9.02 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 115.2, 119.5, 120.4, 122.7, 129.1, 129.5, 129.6, 133.4, 136.1, 139.2, 142.8, 157.7, 190.1 ppm.

⑤ Production of 3-(4-hydroxyphenyl)-1-(3-hydroxyphenyl)-propenone (3-(4-Hydroxyphenyl)-1-(3-hydroxyphenyl)-propenone)

The reaction was performed according to the general method of Preparation Example 1-1 above using 3-hydroxyacetophenone (1.00 g, 7.34 mmol), 4-hydroxy benzaldehyde (0.90 g, 7.34 mmol), and 50% NaOH (2.35 mL, 29.35 mmol) did. By purifying with eluent (ethyl acetate: n-hexane = 1:3), the desired yellow solid compound (compound No. 5) was obtained (Formula 7 below, 1.38 g, 78.5%)

[Formula 7]

Melting point: 199 - 200 °C; R _f 0.45 (ethyl acetate : n -hexane = 1 : 1); HPLC: R _T 2.80 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 6.80 (d, J = 8.4 Hz, 2H), 6.98 (ddd, J = 8.0, 2.4, 0.8 Hz, 1H), 7.23 (dd, J = 8.0, 8.0 Hz, 1H), 7.25 (d, J = 15.6 Hz) , 1H), 7.37-7.41 (m, 2H), 7.42 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 15.6 Hz, 1H), 8.80 (s, 1H), 9.20 (s, 1H) ; ¹³ C-NMR (CDCl ₃ , 100 MHz) 115.3, 116.2, 119.2, 119.6, 120.0, 126.4, 129.5, 130.4, 140.0, 145.0, 157.6, 160.1, 190.7 ppm.

⑥ Production of 1-(2-Hydroxyphenyl)-3-(4-methoxyphenyl)-propenone

The reaction was performed according to the general method of Preparation Example 1-1 above using 2-hydroxyacetophenone (1.00 mL, 6.50 mmol), 4-methoxy benzaldehyde (0.78 mL, 6.50 mmol) and 50% NaOH (2.08 mL, 26.00 mmol) did. By purifying with eluent (ethyl acetate: n-hexane = 1:3), the desired yellow solid compound (compound No. 6) was obtained (Formula 8 below, 0.62 g, 37.4%).

[Formula 8]

Melting point: 92 - 93 ℃; R _f 0.45 (ethyl acetate: n -hexane = 1:1); HPLC: R _T 14.91 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 3.87 (s, 3H), 6.94 (ddd, J = 8.8, 8.4, 1.2 Hz, 1H), 6.60 (d, J = 8.8 Hz, ２H), 7.03 (dd, J = 8.0, 1.2 Hz, 2H), 7.49 (ddd, J = 8.8, 8.4, 1.2 Hz, 1H), 7.55 (d, J = 15.6 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 7.91 (d, J = 15.6 Hz, 2H) , 7.93 (dd, J = 8.8 1.6 Hz, 1H), 12.93 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 55.7, 114.7, 117.8, 118.8, 119.0, 120.3, 127.6, 129.7, 130.8, 136.4, 145.6, 162.3, 163.8, 193.9 ppm.

⑦ 1-(2-Hydroxyphenyl)-3-(4-methoxynaphthalen-1-yl)-propenone) manufacture of

According to the general method of Preparation Example 1-1 above using 2-hydroxyacetophenone (1.00 mL, 6.50 mmol), 4-methoxy-1-naphthaldehyde (1.21 mL, 6.50 mmol) and 50% NaOH (2.08 mL, 26.00 mmol) The reaction was carried out. By purifying with eluent (ethyl acetate: n-hexane = 1:3), the desired orange solid compound (compound No. 7) was obtained (Formula 9 below, 0.57 g, 28.9%).

[Formula 9]

Melting point: 158 - 160 ℃; R _f 0.57 (ethyl acetate: n -hexane = 1:1); HPLC: R _T 20.25 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 4.08 (s, 3H), 6.89 (d, J = 8.0 Hz, 1H), 6.95 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 7.51 (ddd , J = 8.8, 8.4, 0.8 Hz, 1H), 7.55 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.64 (ddd, J = 8.8, 8.0, 1.2 Hz, 1H), 7.69 (d, J = 15.4 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 8.8 Hz, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.76 (d, J = 15.4 Hz, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 56.0, 104.0, 118.9, 119.0, 120.1, 120.4, 123.0, 123.3, 124.5, 125.9, 126.0, 126.8, 128.0, 129.8, 133.1 , 136.4, 142.6, 158.4, 163.9, 193.8 ppm.

⑧ Production of 3-Furan-2-yl-1-(2-hydroxyphenyl)-propenone

The reaction was performed according to the general method of Preparation Example 1-1 using 2-hydroxyacetophenone (1.00 mL, 6.50 mmol), furfural (0.53 mL, 6.50 mmol), and 50% NaOH (2.08 mL, 26.00 mmol). By purifying with eluent (ethyl acetate: n-hexane = 1:3), the desired yellow solid compound (compound No. 8) was obtained (Formula 10 below, 0.78 g, 56.1%).

[Formula 10]

Melting point: 108 - 109 °C; R _f 0.78 (ethyl acetate: n -hexane = 1:1); HPLC: R _T 10.43 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 6.54 (dd, J = 8.4, 0.8 Hz, 1H), 6.78 (s, 1H), 6.94 (ddd, J = 8.0, 1.4, 0.8 Hz, 1H), 7.02 (dd, J = 8.4, 0.8 Hz, 1H) , 7.49 (ddd, J = 8.0, 8.4, 0.8 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 15.2 Hz, 1H), 7.68 (d, J = 15.2 Hz, 1H), 7.92 (dd, J = 8.0, 1.6 Hz, 1H), 12.88 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 113.1, 117.3, 117.9, 118.8, 119.1, 120.3, 129.9, 131.4, 136.5, 145.6, 151.8, 136.8, 193.6 ppm.

⑨ Production of 3-(4-hydroxyphenyl)-1-(2-hydroxyphenyl)-propenone (3-(4-Hydroxyphenyl)-1-(2-hydroxyphenyl)-propenone)

The reaction was performed according to the general method of Preparation Example 1-1 above using 2-hydroxyacetophenone (1.00 mL, 6.50 mmol), 4-hydroxy benzaldehyde (0.80 mL, 6.50 mmol), and 50% NaOH (2.08 mL, 26.00 mmol) did. By purifying with eluent (ethyl acetate: n-hexane = 1:3), the desired yellow solid compound (compound No. 9) was obtained (Formula 11 below, 0.46 g, 29.8%).

[Formula 11]

Melting point: 165 - 166 °C; R _f 0.80 (ethyl acetate : n -hexane = 1 : 1); HPLC: R _T 6.93 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 6.75 (d, J = 8.8 Hz, 2H), 6.79 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 6.83 (dd, J = 8.0, 1.2 Hz, 1H), 7.33 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.37 (d, J = 15.2 Hz, 1H), 7.40 (d, J = 8.8, 2H), 7.72 (d, J = 15.2, 1H), 7.79 (dd, J = 8.0, 1.2, 1H), 9.40 (s, 1H), 12.84 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 116.3, 117.8, 118.0, 118.8, 119.0, 120.3, 127.9, 129.8, 131.0, 136.4, 145.4, 158.3, 163.8, 193.9 ppm.

⑩ Production of 3-(4-Chlorophenyl)-1-(2-hydroxyphenyl)-propenone

The reaction was performed according to the general method of Preparation Example 1-1 using 2-hydroxyacetophenone (1.00 mL, 6.50 mmol), 4-chlorobenzaldehyde (0.91 mL, 6.50 mmol), and 50% NaOH (2.08 mL, 26.00 mmol). . By purifying with eluent (ethyl acetate: n-hexane = 1:3), the desired yellow solid compound (compound No. 10) was obtained (Formula 12 below, 1.28 g, 76.2%).

[Formula 12]

Melting point: 154 - 155 °C; R _f 0.78 (ethyl acetate: n -hexane = 1:1); HPLC: R _T 16.86 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 6.95 (ddd, J = 8.8, 8.4, 0.8 Hz, 1H), 7.04 (dd, J = 8.4, 1.2 Hz, 1H), 7.40 (d, J = 8.4, Hz, 2H), 7.51 (ddd, J = 8.8 , 8.4, 1.6 Hz, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 15.6 Hz, 1H), 7.87 (d, J = 15.6 Hz, 1H), 7.91 (dd, J = 8.0, 1.6 Hz, 1H), 12.74 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 118.9, 119.1, 120.2, 120.8, 129.6, 129.8, 130.0, 133.3, 136.8, 137.1, 144.2, 163.9, 193.7 ppm.

⑪ Production of 1-(2-Hydroxyphenyl)-3-thiophen-2-yl-propenone

Reaction was carried out according to the general method of Preparation Example 1-1 above using 2-hydroxyacetophenone (1.00 mL, 6.50 mmol), thiophene-2-carboxaldehyde (0.60 mL, 6.50 mmol), and 50% NaOH (2.08 mL, 26.00 mmol). carried out. By purifying with eluent (ethyl acetate: n-hexane = 1:3), the desired orange solid compound (compound No. 11) was obtained (Formula 13 below, 0.86 g, 57.5%).

[Formula 13]

Melting point: 101 - 102 °C; R _f 0.85 (ethyl acetate: n -hexane = 1:1); HPLC: R _T 12.35 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 6.95 (ddd, J = 8.0, 8.4, 1.2 Hz, 1H), 7.03 (dd, J = 8.4, 1.2 Hz, 1H), 7.12 (d, J = 8.4, Hz, 1H), 7.41 (dd, J = 8.4 Hz, 1H), 7.44 (d, J = 15.2 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.50 (ddd, J = 8.4, 8.4, 1.2 Hz, 1H) 7.89 (dd, J = 8.0, 1.6 Hz, 1H), 8.04 (d, J = 15.2 Hz, 1H), 12.84 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 118.8, 119.1, 120.2, 128.7, 129.8, 132.9, 136.6, 138.1, 140.4, 163.8, 193.4 ppm.

Manufacturing example 2. N -Acetylpyrazoline analogues ( N -acetylpyrazoline analogues) Preparation of candidate compounds

2-1. General manufacturing method

A reaction mixture of chalcone analogue and hydrazine·H2O (65%) (4.0 equivalents) dissolved in AcOH (10 mL) was refluxed for 2 hours and then cooled to room temperature. The reaction mixture was poured onto ice and kept at room temperature overnight. The solid formed was filtered and washed with water. The solid was dried under vacuum and purified by silica gel column chromatography (eluent: MeOH: chloroform) to obtain the product.

2-2. Preparation of candidate compounds

① 1-[5-furan-2-yl-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-Furan-2-yl- Preparation of 3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone)

The reaction was performed according to the general method of Preparation Example 2-1 using compound No. 1 (0.28 g, 1.31 mmol) and hydrazine H ₂ O (65%) (0.25 mL, 5.24 mmol) prepared above. By purifying with eluent (MeOH: chloroform = 1:15), the desired beige solid compound (compound No. 12) was obtained (Formula 14 below, 0.40 g, 11.5%).

[Formula 14]

Melting point: 163 - 164 °C; R _f 0.45 (MeOH:chloroform = 1:9); HPLC: R _T 2.95 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 2.27 (s, 3H), 3.29 (dd, J = 17.6, 4.8 Hz, 1H), 3.51 (dd, J = 17.6, 12.0 Hz, 1H), 5.55 (dd, J = 11.6, 4.4 Hz, 1H), 6.22 (d, J = 8.4 Hz, 2H), 6.83 (ddd, J = 8.8, 8.8, 1.2 Hz, 1H), 7.09 - 7.17 (m, 3H), 7.23 (d, J = 2.4 Hz, 1H), 9.27 ( s, 1H): ¹³ C-NMR (CDCl ₃ , 100 MHz) 21.9, 53.2, 107.2, 110.5, 113.4, 117.8, 117.9, 129.7, 132.3, 141.9, 152.3, 154.5, 157.6, 168.6 ppm.

② 1-[3-(3-hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-(3-Hydroxyphenyl) )-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone) Preparation

The reaction was performed according to the general method of Preparation Example 2-1 using compound No. 2 (0.30 g, 1.18 mmol) and hydrazine H2O (65%) (0.23 mL, 4.72 mmol) prepared above. By purifying with eluent (MeOH: chloroform = 1:15), the desired pale orange solid compound (compound No. 13) was obtained (Formula 15 below, (0.18 g, 48.1%).

[Formula 15]

Melting point: 209 - 210 °C; R _f 0.19 (MeOH:chloroform = 1:9); HPLC: R _T 3.58 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 2.28 (s, 3H), 3.01 (dd, J = 17.6, 4.4 Hz, 1H), 3.60 (dd, J = 17.6, 11.6 Hz, 1H), 3.67 (s, 3H), 5.41 (dd, J = 12.0, 4.8 Hz, 1H), 6.73 (d, J = 8.8 Hz, 2H), 6.83 (ddd, J = 8.0, 1.6, 1.6 Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H), 7.08 - 7.13 ( m, 2H), 7.15 (d, J = 8.0 Hz, 1H), 8.89 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 21.9, 42.4, 55.2, 59.3, 113.4, 114.1, 117.8, 117.8, 126.8, 129.7, 132.5, 134.1, 154.2, 157.5, 158.9, 1 68.6 ppm.

③ 1-[3-(3-hydroxyphenyl)-5-(4-methoxynaphthalen-1-yl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[3- Preparation of (3-Hydroxyphenyl)-5-(4-methoxynaphthalen-1-yl)-4,5-dihydropyrazol-1-yl]-ethanone)

The reaction was performed according to the general method of Preparation Example 2-1 using compound No. 3 (0.10 g, 0.34 mmol) and hydrazine H ₂ O (65%) (0.06 mL, 1.31 mmol) prepared above. By purifying with eluent (MeOH: chloroform = 1:15), the desired yellow solid compound (compound No. 14) was obtained (Formula 16 below, (46 mg, 29.1%).

[Formula 16]

Melting point: 280 - 282 °C; R _f 0.16 (MeOH:chloroform = 1:9); HPLC: R _T 6.33 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 2.37 (s, 3H), 2.95 (dd, J = 17.2, 4.4 Hz, 1H), 3.75 (dd, J = 17.2, 11.6 Hz, 1H), 3.83 (s, 3H), 6.06 (dd, J = 11.6, 4.4 Hz, 1H), 6.59 (d, J = 8.0 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 7.03 (d, J = 8.0 Hz) , 2H), 7.09 - 7.10 (m, 1H), 7.37 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.45 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.89 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 21.9, 42.2, 55.4, 103.1, 113.3, 117.7, 122.6, 122.9, 124.9, 126.1, 126.7, 127.9, 129.5, 130.3, 136.4, 154.8, 154.9, 157.4, 168.7 ppm.

④ 1-[5-(4-Chlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-(4-Chlorophenyl) Preparation of -3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone)

The reaction was performed according to the general method of Preparation Example 2-1 using compound No. 4 (0.30 g, 1.16 mmol) and hydrazine H O (65%) (0.22 mL, 4.64 mmol) prepared above. It was purified with eluent (MeOH: chloroform = 1:15) to obtain a yellow solid candidate compound (compound No. 15, hereinafter referred to as YK1, formula 17 below, 0.29 g, 97.5%).

[Formula 17]

Melting point: 170 - 172℃; Rf 0.15 (MeOH : chloroform = 1 : 9); HPLC: RT 3.40 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 2.21 (s, 3H), 2.91 (dd, J = 17.6, 4.4 Hz, 1H), 4.57 (dd, J = 17.6, 12.4 Hz, 1H), 5.35 (dd, J = 11.6, 4.8 Hz, 1H), 6.75 (dd, J = 8.0, 2.4 Hz, 1H), 6.84 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 8.0 Hz, 2H), 7.06 (dd, J = 8.0, 2.4 Hz, 1H) , 7.10 (d, J = 8.0 Hz, 2H), 8.91 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 22.0, 42.2, 55.3, 113.6, 118.1, 118.1, 124.6, 124.7, 126.8, 129.8, 132.4, 144.4, 154.4, 157.8, 169.0 ppm .

⑤ 1-[5-(4-hydroxyphenyl)-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-(4-Hydroxyphenyl )-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone) Preparation

The reaction was performed according to the general method of Preparation Example 2-1 using compound No. 5 (0.30 g, 1.25 mmol) and hydrazine H ₂ O (65%) (0.24 mL, 5.00 mmol) prepared above. By purifying with eluent (MeOH: chloroform = 1:15), the desired yellow solid compound (compound No. 16) was obtained (Formula 18 below, 0.19 g, 51.6%).

[Formula 18]

Melting point: 220 - 221 °C; R _f 0.13 (MeOH:chloroform = 1:9); HPLC: R _T 2.32 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 2.19 (s, 3H), 2.92 (dd, J = 8.8, 1.2 Hz, 1H), 3.50 (dd, J = 18.0, 12.0 Hz, 1H), 5.30 (dd, J = 15.6, 4.4 Hz, 1H), 6.58 (d, J = 8.4 Hz, 2H), 6.74 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 6.86 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 8.0 Hz, 2H) , 7.07 - 7.09 (m, 1H), 8.55 (s, 1H), 8.87 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 21.8, 42.3, 59.2, 113.2, 115.5, 117.6, 117.6, 126.6, 129.5, 132.5, 132.6, 154.0, 156.5, 157.4, 168.3 ppm .

⑥ 1-[5-furan-2-yl-3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-Furan-2-yl- Preparation of 3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone)

The reaction was performed according to the general method of Preparation Example 2-1 using compound No. 8 (0.80 g, 3.73 mmol) and hydrazine H ₂ O (65%) (0.74 mL, 14.9 mmol) prepared above. By purifying with eluent (MeOH: chloroform = 1:15), the desired pale pink solid compound (compound No. 17) was obtained (Formula 19 below, 0.91 g, 90.3%).

[Formula 19]

Melting point: 132 - 133 °C; R _f 0.10 (MeOH:chloroform = 1:9); HPLC: R _T 5.92 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 2.36 (s, 3H), 3.58 (dd, J = 17.6, 4.8 Hz, 1H), 3.69 (dd, J = 17.6, 3.2 Hz, 1H), 5.67 (dd, J = 11.6, 4.8 Hz, 1H), 6.32 - 6.37 (m, 2H), 6.96 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 7.31 ( ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.37 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 10.19 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 22.3, 38.8, 52.1, 108.4, 110.9, 115.3, 117.3, 120.0, 128.6, 132.5, 142.4, 151.5, 156.7, 157.9, 168.1 ppm .

⑦ 1-[3-(2-hydroxyphenyl)-5-(4-methoxynaphthalen-1-yl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[3- Preparation of (2-Hydroxyphenyl)-5-(4-methoxynaphthalen-1-yl)-4,5-dihydropyrazol-1-yl]-ethanone)

The reaction was performed according to the general method of Preparation Example 2-1 using compound No. 7 (0.30 g, 3.94 mmol) and hydrazine H ₂ O (65%) (0.19 mL, 3.94 mmol) prepared above. By purifying with eluent (MeOH: chloroform = 1:15), the desired yellow solid compound (compound No. 18 hereafter, YK2) was obtained (Formula 20 below, 91 mg, 21.8%).

[Formula 20]

Melting point: 203 - 204 °C; R _f 0.17 (MeOH:chloroform = 1:9); HPLC: R _T 11.52 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 2.50 (s, 3H), 3.27 (dd, J = 17.6, 4.4 Hz, 1H), 3.97 (s, 3H), 4.04 (dd, J = 19.2, 8.8 Hz, 1H), 6.22 (dd, J = 11.6, 4.8 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 6.87 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.06 (d, J = 8.8 Hz, 1H), 7.14 (ddd, J = 8.0, 8.0, 1.2 Hz, 2H), 7.33 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.52 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 7.60 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 8.34 (d, J = 8.4 Hz, 1H), 10.35 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 22.9, 43.4, 56.3, 103.9, 116.0, 117.8, 120.5, 122.7, 123.2, 124.0, 125.9, 127.2, 127.7, 128.0, 129.2, 131.1, 133.0, 156.1, 157.9, 158.5 , 168.6 ppm.

⑧ 1-[5-(4-hydroxyphenyl)-3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-(4-Hydroxyphenyl) )-3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone) Preparation

The reaction was performed according to the general method of Preparation Example 2-1 using compound No. 9 (0.30 g, 1.25 mmol) and hydrazine H ₂ O (65%) (0.24 mL, 4.99 mmol) prepared above. By purifying with eluent (MeOH: chloroform = 1:15), the desired yellow solid compound (compound No. 19) was obtained (Formula 21 below, 0.22 g, 60.4%).

[Formula 21]

Melting point: 260 - 262 °C; R _f 0.20 (MeOH:chloroform = 1:9); HPLC: R _T 3.64 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 2.20 (s, 3H), 3.13 (dd, J = 18.0, 4.4 Hz, 1H), 3.68 (dd, J = 18.0, 12.0 Hz, 1H), 5.33 (dd, J = 11.6, 4.4 Hz, 1H), 6.63 (d, J = 8.0 Hz, 2H), 6.78 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 6.89 (d, J = 8.0 Hz, 2H) 7.09 (d, J = 8.0 Hz, 1H), 7.18 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 8.72 (s, 1H), 10.12 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 21.8, 42.5, 57.7, 115.0, 115.5, 116.6, 119.4, 126.5, 128.3, 131.7, 131.8, 156.2, 156.7, 157.3, 167.1 ppm .

⑨ 1-[5-(4-Chlorophenyl)-3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-(4-Chlorophenyl) Preparation of -3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone)

The reaction was performed according to the general method of Preparation Example 2-1 using compound No. 10 (0.30 g, 1.16 mmol) and hydrazine H ₂ O (65%) (0.22 mL, 4.64 mmol) prepared above. By purifying with eluent (MeOH: chloroform = 1:15), the desired yellow solid compound (compound No. 20) was obtained (Formula 22 below, 0.34 g, 94.4%).

[Formula 22]

Melting point: 143 - 144 °C; R _f 0.42 (MeOH:chloroform = 1:9); HPLC: R _T 9.46 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 2.39 (s, 3H), 3.27 (dd, J = 17.6, 4.8 Hz, 1H), 3.87 (dd, J = 18.0, 12.0 Hz, 1H), 5.53 (dd, J = 6.8, 4.8 Hz, 1H), 6.94 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 7.19 (d, J = 8.8 Hz, 2H), 7.20 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.37 (ddd, J = 8.0, 8.0, 0.8 Hz, 2H), 10.20 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 22.3, 42.8, 58.1, 115.2, 117.4, 120.0, 127.3, 128.6, 129.4, 132.7, 134.0, 139.9, 156.4, 158.0, 168.0 ppm .

⑩ 1-[3-(2-Hydroxyphenyl)-5-thiophen-2-yl-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-(2-Hydroxyphenyl) Preparation of -5-thiophen-2-yl-4,5-dihydropyrazol-1-yl]-ethanone)

The reaction was performed according to the general method of Preparation Example 2-1 using compound No. 11 (0.30 g, 1.25 mmol) and hydrazine H ₂ O (65%) (0.24 mL, 4.99 mmol) prepared above. By purifying with eluent (MeOH: chloroform = 1:15), the desired yellow solid compound (compound No. 21) was obtained (Formula 23 below, 0.17 g, 48.9%).

[Formula 23]

Melting point: 120 - 122 °C; R _f 0.76 (MeOH:chloroform = 1:9); HPLC: R _T 6.37 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 2.37 (s, 3H), 3.49 (dd, J =17.6, 4.0 Hz, 1H), 3.83 (dd, J = 17.6, 11.2 Hz, 1H), 5.89 (dd, J = 11.6, 4.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 2H), 7.05 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 6.8 Hz, 1H), 7.27 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H) , 7.37 (ddd, J = 8.8, 8.4, 1.2 Hz, 1H), 10.20 (s, 2H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 22.3, 42.6, 54.0, 115.3, 117.4, 120.0, 125.2, 125.3, 127.1, 128.6, 132.6, 143.7, 156.5, 158.0, 168.1 ppm .

⑪ 1-[3-(2-hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-(2-Hydroxyphenyl) )-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone) Preparation

The reaction was performed according to the general method of Preparation Example 2-1 using Compound 6 (0.30 g, 1.18 mmol) and hydrazine H ₂ O (65%) (0.23 mL, 4.72 mmol) prepared above. By purifying with eluent (MeOH: chloroform = 1:15), the desired pale yellow solid compound (compound No. 22) was obtained (Formula 24 below, 0.34 g, 93.9%).

[Formula 24]

Melting point: 139 - 140 °C; R _f 0.24 (MeOH:chloroform = 1:9); HPLC: R _T 6.87 min (purity: 99.9%).

¹ H-NMR (CDCl ₃ , 400 MHz) 2.37 (s, 3H), 3.30 (dd, J = 17.6, 4.4 Hz, 1H), 3.78 (s, 3H), 3.82 (dd, J = 18.0, 6.0 Hz, 1H), 5.53 (dd, J = 11.6, 4.8 Hz, 1H), 6.86 (d, J = 8.8 Hz, 2H), 6.94 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 8.8 Hz, 2H), 7.23 (d, J = 8.0 Hz, 1H), 7.36 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 10.29 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 22.3, 42.3, 55.5, 58.2, 114.6, 115.4, 117.3, 120.0, 127.2, 128.6, 132.5, 133.6, 156.6, 157.9, 159.5, 1 68.0 ppm.

Manufacturing example 3. N -Propionylpyrazoline analogues ( N -propionylpyrazoline analogues) Preparation of candidate compounds.

3-1. General manufacturing method

The reaction mixture of chalcone analog and hydrazine·H ₂ O (65%) (4.0 equivalents) dissolved in propionic acid (10 mL) was refluxed (2 hours) and then cooled to room temperature. The reaction mixture was poured onto ice and kept at room temperature overnight. The solid formed was filtered and washed with water. The solid was dried under vacuum and purified by silica gel column chromatography (elute: MeOH: chloroform) to obtain the desired product.

3-2. Preparation of candidate compounds

① 1-[3-(3-hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-propan-1-one (1-[3-( Preparation of 3-Hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-propan-1-one)

The reaction was performed according to the general method of Preparation Example 3-1 using Compound 6 (0.30 g, 1.18 mmol) and hydrazine H ₂ O (65%) (0.23 mL, 4.72 mmol) prepared above. Purified with eluent (MeOH: chloroform = 1:15), the desired white solid compound (compound No. 23) was obtained (Formula 25 below, 93.2 mg, 24.4%).

[Formula 25]

Melting point: 233 - 234 °C; R _f 0.19 (MeOH:chloroform = 1:9); HPLC: R _T 5.59 min (purity: 98.6%).

¹ H-NMR (CDCl ₃ , 400 MHz) 1.05 (t, J = 7.6, 3H), 2.38 (s, 3H), 2.66 (dd, J = 14.8, 4.6 Hz, 2H), 2.99 (dd, J = 17.6, 4.8 Hz, 1H), 3.57 (dd, J = 18.0, 12.0 Hz, 1H), 5.38 (dd, J = 11.6, 4.8 Hz, 1H), 6.71 (d, J = 8.8 Hz, 2H), 6.80 (ddd, J = 8.0, 1.6, 1.2 Hz, 1H) ), 7.03 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 8.8 Hz, 2H), 7.14 (ddd, J = 8.0, 1.6, 1.2 Hz, 1H), 8.84 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 8.9, 27.5, 55.2, 59.4, 113.4, 114.1, 117.7, 117.8, 126.9, 129.6, 132.7, 134.4, 153.8, 157.5, 158.8, 17 2.0 ppm.

② 1-[3-(3-hydroxyphenyl)-5-thiophen-2-yl-4,5-dihydropyrazol-1-yl]-propan-1-one (1-[3-(3 Preparation of -Hydroxyphenyl)-5-thiophen-2-yl-4,5-dihydropyrazol-1-yl]-propan-1-one)

The reaction was performed according to the general method of Preparation Example 3-1 using Compound 11 (0.30 g, 1.30 mmol) and hydrazine H ₂ O (65%) (0.25 mL, 5.21 mmol) prepared above. Purified with eluent (MeOH: chloroform = 1:15), the desired orange solid compound (compound No. 24) was obtained (Formula 26 below, 63.6 mg, 16.3%).

[Formula 26]

Melting point: 183 - 184 °C; R _f 0.36 (MeOH:chloroform = 1:9); HPLC: R _T 5.21 min (purity: 98.1%).

¹ H-NMR (CDCl ₃ , 400 MHz) 1.08 (t, J = 7.6 Hz, 3H), 2.63 - 2.70 (m, 2H), 3.19 (dd, J = 17.6, 4.0 Hz, 1H), 5.33 (s, 1H), 5.77 (dd, J = 11.2, 4.0 Hz, 1H), 6.82 (d, J = 8.0 Hz, 2H), 6.90 - 6.91 (m, 1H), 7.10 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H) , 9.17 (s, 1H); ¹³ C-NMR (CDCl ₃ , 100 MHz) 8.5, 26.9, 41.4, 49.0, 54.7, 112.7, 117.2, 123.7, 123.9, 126.2, 129.2, 131.8, 144.3, 153.4, 157.1, 171 .2 ppm.

③ 1-[5-(4-chlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-propan-1-one (1-[5-(4 Preparation of -Chlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-propan-1-one)

The reaction was performed according to the general method of Preparation Example 3-1 using Compound 4 (0.30 g, 1.16 mmol) and hydrazine H ₂ O (65%) (0.22 mL, 4.64 mmol) prepared above. By purifying with eluent (MeOH: chloroform = 1:15), the desired white solid compound (compound No. 25) was obtained (Formula 27 below, 0.12 g, 31.8%).

[Formula 27]

Melting point: 206 - 207 °C; R _f 0.26 (MeOH:chloroform = 1:9); HPLC: R _T 10.74 min (purity: 99.6%).

¹ H-NMR (CDCl ₃ , 400 MHz) 1.02 (t, J = 7.6, Hz, 3H), 2.64 (dd, J = 14.8, 7.6 Hz, 2H), 2.93 (dd, J = 17.6, 4.8 Hz, 1H), 3.58 (dd, J = 17.6, 1.2 Hz, 1H), 5.36 (dd, J = 12.4, 4.8 Hz, 1H), 6.78 (ddd, J = 8.0, 1.2, 1.2 Hz, 1H), 7.02 (d, J = 8.4 Hz, 2H), 7.03 - 7.06 (m, 1H), 7.10 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 8.89 (s, 1H); ¹³ C-NMR (CDCL ₃ , 100 MHz) 8.8, 27.4, 59.3, 113.3, 117.6, 117.7, 127.0, 128.8, 129.6, 132.2, 133.0, 140.6, 153.7, 157.5, 172.0 ppm.

Example

1. Experimental method and preparation

1-1. Western blot analysis

For Western blot analysis, cells were seeded in 6-well plates and then lysed in 1X RIPA lysis buffer (Cell Signaling, USA) containing 1% 0.1M PMSF and 1% 100X protease inhibitor cocktail solution (Genedepot, USA). . Total protein levels were standardized using Pierce™ BCA protein assay kit (Thermo Fisher Scientific, USA) and microplate reader (Tecan Group Ltd., Switzerland). 20 μg of protein was loaded onto a 10–15% acrylamide gel for SDS-PAGE and then transferred to a 0.2 μm PVDF membrane (Pall Life Science, USA). Membranes were blocked by treatment with 5% skim milk or 5% BSA, and then incubated with primary antibodies (purchased from Thermo Fisher, Cell Signaling, Santa Cruz, Novus, or MBL) for 3 hours at room temperature or overnight at 4°C. cultured. Blots were washed three times with Tris-buffered saline-0.1% Tween20 (1X TBST) and incubated with HRP-conjugated secondary antibodies (GeneTex, USA). Protein bands were detected using ECL solution reagent (GE Healthcare, USA) and LAS-3000 (Fuji Photo Film Co., Ltd., Japan), and captured images were captured using Multi-Gauge software (Fuji Photo Film Co. Ltd.). ) was evaluated. Cell lysate preparation for the following analyzes was performed in the same manner as for Western blot analysis.

1-2. Immunoprecipitation assay

NCI-N87 cells, a gastric cancer cell line, were seeded in a 100 mm cell culture dish and then cultured until they reached 60-70% confluence. Cultured cells were transfected with p3Xflag-myc-CMV26-empty or p3Xflag-myc-CMV26-MED23 ^WT plasmid using Lipofectamine® 2000 (Invitrogen, USA). After transfection, 12 hours later, YK1 (5 μM) was treated and cultured for 12 hours. 750 μg of cell extract was incubated with 20 μL of FLAG-agarose beads (Sigma Aldrich, USA) slurry at 4°C for 4 hours on a rotator, and then the samples were centrifuged at 12000 rpm for 1 minute to sediment the beads. I ordered it. After removing the supernatant, the remaining beads were washed three times with 200 μL of lysis buffer (RIPA). After removing the final supernatant, the beads were eluted with 2X sample buffer by boiling at 98°C for 5 minutes. Immunoprecipitated proteins were loaded, separated by SDS-PA, and analyzed by Western blot.

1-3. Luciferase promoter analysis

HEK293 cells were plated in 6-well plates and then incubated with 1 μg of pNeuLite (Addgene) alone or 0.5 μg of pcDNA3.1-flag-ELF3 ^WT (provided by Dr. Seung Bae Rho, National Cancer Center) and p3Xflag-myc-CMV26- Transformed with MED23 mutant plasmid. To normalize the luciferase activity of each sample, 0.5 μg of β-galactosidase expression plasmid (provided by Dr. Eun-Sook Hwang, Ewha Womans University) was added to all sample groups. All transfections were performed using Lipofectamine® 2000 Transfection Reagent (Invitrogen, USA). After 24 hours, firefly luciferase and β-galactosidase (β-galactosidase) activities were measured using the Luciferase Assay System (Promega) and Galacto-Light Plus β -Galactosidase Reporter Gene Assay System (Invitrogen). Evaluation was performed using an Infinite M200 PRO Microplate reader (Tecan Group Ltd., Switzerland).

1-4. Protein-small molecule interaction analysis

HEK293 cells were seeded in 100 mm ³ dishes and incubated until 70-80% confluency was reached. Cells were transfected with p3Xflag-myc-CMV26-MED23 ^WT and p3Xflag-myc-CMV26-MED23 ^D400A/H449G plasmids for 24 hours and lysed to obtain protein extracts. To isolate the candidate compound (YK1) bound to MED23, the entire lysate was incubated with YK1 for 4 hours and finally pulled down using FLAG-agarose beads (Sigma Aldrich, USA). The precipitated beads were washed three or more times to completely remove unbound compounds. To isolate candidate compounds from the precipitated MED23, the sample (50 μL) was extracted with 1 mL of acetonitrile and then centrifuged at 13,000 rpm at 4°C for 10 minutes. Each sample was aliquoted from the upper layer (1000 μL) into a new tube and evaporated to dryness under nitrogen gas at 37°C. For analysis of YK1, the residue was dissolved in 50 μL of ACN and vortexed for 10 min. Each sample was mixed with an internal reference material (a material similar to the candidate material but with low binding affinity for MED23, hereafter YK2) contained in ACN at a concentration of 5000 μg/mL, and then mixed with 450 μL by sonication and vortex mixing. Extracted with ACN. The candidate material combined with MED23 was finally analyzed using an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, USA).

Example 2. Confirmation of the structure of the ELF3-MED23 complex

To obtain in-depth structural insights into the binding modules of ELF3 and MED23, we first built a homology model of the ELF3 protein using the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2). Based on the already known structure of MED23 (PDB ID: 6H02), the automated protein-protein docking server ClusPro was applied to confirm the protein interaction (Figure 1a). Residues 291-582 of MED23 in the final model are shown in green, and the TAD domain of ELF3 is shown in magenta. S137 to E144 (8aa) of ELF3, which was previously identified as a residue required for interaction, is shown in yellow. For specific verification, ELF3 _137-144 peptide was prepared as a ligand and docking study was performed on the binding interface of the predicted complex (Figure 1b). As a result, it was confirmed that five hydrophobic residues (W138, I139, I140, L142, and L143) of ELF3 make hydrophobic contact with K406, D400, E405, H449, P446, and M396 of MED23. Among the five major hydrophobic residues of ELF3, the indole portion of W138 was confirmed to have a hydrogen bond with D400 and a fairly strong π-π interaction with F399 of MED23. Additionally, residues I139 and I140 of ELF3 are predicted to create additional hydrogen bonds with E405 and H449 of MED23, incorporating several hydrophobic interactions between residues D400, H449, E405, M396, P446, and K406 of ELF3 and MED23 ( It is expected to consolidate.

To specifically identify the MED23 residue that plays a critical role in the interaction with ELF3, adamanolol, wrenchnolol, canertinib, and gefitinib, which are known to inhibit protein-protein interaction (PPI) between ELF3 and MED23, were used. Docking studies were performed. As a result, as shown in Figures 1c to 1e, it was confirmed that all compounds used in the experiment and ELF3 _137-144 formed hydrogen bonds with D400 and H449 of MED23. In addition, among the above compounds, ELF3 _137-144 and canertinib, which were confirmed to have superior PPI inhibitory activity than gefitnib, were confirmed to form additional π-interactions with F399 of MED23 (Figure 2).

Based on the above results, it is expected that the D400, H449, and F399 residues of MED23 form hydrogen bonds (D400, H449) and π-stacking (F399) with ELF3, thereby serving as hotspots for EL3-MED23 PPI, and additional An experiment was conducted.

Example 3. Identification of key residues interacting in the ELF3-MED23 PPI hotspot

3-1. SEAP analysis

To confirm that D400 and H449 residues actually play a key role in ELF3-MED23 protein interaction, SEAP analysis was performed. Specifically, 293T kidney cells were seeded in a 96-well microplate (SPL, Korea) at a density of 5 x 10 ³ cells/well and cultured overnight. The cells were then simultaneously transfected using 100ng of each plasmid and transfection optimized medium (Welgene, Korea). The plasmid used was a SEAP reporter gene (pG5IL2SX) and a GAL4-ELF3 expression vector (pBJ GAL4-ELF3). For transfection, cells were incubated with WelFectQ (Welgene, Korea) for 3 hours, and then candidate compounds or MED23 protein constructs (pcDNA_MED23 ^{WT, K397A, D400A, H449G, and D400A/H449G} ) were added. Treated cells were incubated for 12 hours. The candidate compound was placed in an incubator under 5% CO ₂ and 37°C conditions, and then the plate was incubated at 65°C for 3 hours to inactivate all enzymes except SEAP (Secreted Alkaline Phosphatase). A mixture of 100 μL of substrate solution made from 1.19 μL of 0.1 M 4-methylumbelliferyl phosphate (Sigma, USA), 25 μL of inactivation medium, and 75 μL of double distilled water was added, followed by 98.81 μL of 2 M diethanolamine (pH 10, Sigma, USA). . Incubation was performed overnight at 37°C. Then, the fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 440 nm using a fluorometer (SPECTRAmax GEMINIEM, Molecular Devices, USA).

As a result, MED23 ^WT induced an increase in SEAP activity, but MED23 ^D400A , MED23 ^H449G , and MED23 ^D400A/H449G mutants, which do not have the ability to form hydrogen bonds with ELF3, did not induce an increase in SEAP activity (Figure 3a). In addition, even when alanine was introduced into the K397 residue, which was predicted to be a PPI non-contributing residue (MED23 ^K397A ), there was no significant change compared to the wild type.

3-2. Split luciferase complementation assay

The biosensor used in the analysis was prepared using the In-Fusion® HD Cloning Kit with firefly (Photinus pyralis). Luciferase was split into two fragments (Nluc and Cluc). Full-length ELF3 (wild type) and MED23 mutants were cloned into HindIII and Kpnl sites and Notl and Kpnl sites of p3Xflag -myc-CMV26, respectively. The full-length cDNA of human ELF3 (accession number NM_001114309.1) was fused with the N-terminal fragment of split luciferase (Nluc), and the full-length cDNA of MED23 (accession number NM_001270521.1) was fused to the C-terminal fragment of split luciferase (Cluc). -fused with the extremities.

The intensity of bioluminescence produced complementary by ELF3-MED23 binding was measured using the above method (Figure 3b). As a result, co-transfection of Nluc-ELF3 and Cluc-MED23 ^391-582WT or K397A significantly increased luciferase activity, but no such changes were observed with Cluc-MED23 ^391-582 D400A and H449G constructs (Figures 3c and 3d).

3-3. Pull-down analysis

HEK293 cells were seeded in 100 mm cell culture dishes and cultured until they reached 70-80% confluence. Labeled GST-tagged ELF3 constructs were transfected using Lipofectamine® 2000 (Invitrogen, USA). 1000 μg of cell extract was incubated with 20 μL of Glutathione Sepharose™ beads (GE Healthcare, UK) for 1 hour at 4°C on a rotator. Beads were washed three times with 200 μL of ice-cold 1X PBS. After removing the final supernatant, the beads were eluted with elution buffer (20mM Glutathione, 100mM Tris-HCl (pH 8.0), 120mM NaCl, 10% glycerol). The precipitated proteins were loaded, separated by SDS-PAGE, and analyzed by Western blot.

As a result, similar to what was confirmed in Examples 3-1 and 3-2, the interaction of ELF3 and MED23 was significantly reduced in D400A and H449G, but in the case of K397A, no significant change was confirmed (Figure 3e) . It was confirmed that successful interaction between MED23 and ELF3 (wild type and K397A) can upregulate the expression level of HER2 along with an increase in the transcriptional activity of ELF3 (Figures 3f and 3g).

Through the above results, it was confirmed that the interaction with EFL3 through hydrogen bond formation between the D400 and H449 residues of MED23 plays an important role in the protein-protein interaction of ELF3-MED23 that activates HER2 gene transcription.

Example 4. Confirmation of the effect of small molecule compounds on key residues of ELF3-MED23 PPI and their dominant anti-proliferative effect on HER2-positive gastric cancer cell lines.

4-1. Selection of candidate compounds

In order to embody the above findings, we designed and synthesized a minor compound that can affect ELF3-MED23 PPI. Specifically, non-essential side chains were removed from gefitnib, and then a compound capable of having both hydrogen bond donor and acceptor moieties for each of the D400 and H449 residues of MED23 was designed (Figure 4a). Specifically, chalcones and pyrazolines were selected as the skeleton, and 25 compounds (11 chalcones and 14 pyrazoline base compounds) were prepared. The ELF3-MED23 PPI inhibitory activity of all the compounds prepared above was measured through SEAP analysis, and compounds that showed more than 65% inhibition were further tested at various concentrations (3, 5, and 10 μM) to confirm the initial screening data. (Figure 4b, Figure 4c).

By performing reporter gene analysis and cell survival tests in parallel, it was confirmed that the inhibition of ELF3-MED23 PPI was not due to the cytotoxicity of the compound, but rather the inhibition of the interaction itself. To confirm this specifically, in silico docking analysis was performed on the identified hotspot of MED23. Docking analysis was performed using the protein-protein docking model for the ELF3-MED23 interaction obtained through the ClusPro web server. Before performing the docking analysis, amino acids 137-144 of ELF3 were extracted as an internal ligand, the ELF3 protein was removed from the model, and then a protomol was prepared using amino acids 137-144 of ELF3. The 3D structure of the candidate compound YK1 was generated using the SKETCH module of the SYBYL program (Tripos Inc., St. Louis, USA). Docking of all molecules was performed using Surflex -Dock (Tripos International, 2012) linked to Sybyl -X 2.3, and the results are shown in Table 1 below.

Compounds Interacting residues ^a by
in silico study SEAP assay SEAP assay Inhibition
(%) ^b Inhibition
(%) ^b Peptide
(8 a.a.) D400, E405, H449 53 53 Gefitinib D400, H449 85 85 One D400, H449 86 86 2 H449 41 41 3 D400, H449 70 70 4 D400, H449 80 80 5 F399, K406, H449 60 60 6 D400, H449 90 90 7 D400 80 80 8 H449 50 50 9 F399, Y403, E405, H449 50 50 10 D400 80 80 11 H449 50 50 12 H449 58 58 13 H449 58 58 14 F399, W429 14 14 15 D400, H449 95 95 16 F399, W429, P446 37 37 17 - 23 23 18 - 0 0 19 F399, Y403 39 39 20 H449 45 45 21 H449 57 57 22 D400 63 63 23 D400, H449 87 87 24 - 34 34 25 F399, H449 42 42

As a result, it was confirmed that the compound predicted to form hydrogen bonds with both D400 and H449 residues had excellent ELF3-MED23 PPI inhibitory activity (Figure 4d). Compounds 1 to 22 were treated with HCT15 (HER2-positive colon cancer cell line), NCI-N87 (HER2-positive gastrointestinal cancer cell line), T47D (HER2-negative breast cancer cell line), and HeLa (HER2-negative cervical cancer cell line) for 72 hours. As a result of measuring the anti-proliferative effect, it was confirmed that there was a generally strong anti-proliferative effect in the NCI-N87 cell line (Table 2 below).

Comp# HCT15 (+) NCI-N87 (+) T47D (-) HeLa (-) One 29.66±0.14 13.50±0.29 8.48±0.02 19.50±0.04 2 8.51±0.01 10.65±0.31 18.89±0.20 9.85±0.37 3 7.64±0.09 3.45±0.05 1.21±0.02 9.73±0.04 4 11.96±0.17 4.82±0.10 17.57±0.13 25.64±0.09 5 8.97±0.17 17.82±0.17 9.04±0.07 30.02±0.18 6 >50 44.88±0.80 >50 >50 7 14.18±0.05 11.36±0.12 15.56±0.15 16.13±0.08 8 >50 >50 >50 42.19±0.01 9 >50 >50 27.89±0.06 >50 10 14.36±0.12 14.38±0.06 8.96±0.02 5.54±0.21 11 >50 47.76±0.22 >50 44.31±0.11 12 >50 >50 >50 >50 13 >50 >50 >50 >50 14 19.47±0.41 42.35±0.86 5.57±0.26 0.69±0.00 15 7.66±0.04 1.56±0.14 9.99±0.20 16.92±0.10 16 >50 >50 >50 >50 17 >50 >50 >50 >50 18 19.70±0.05 >50 29.28±0.27 6.69±0.40 19 >50 >50 >50 >50 20 >50 >50 >50 >50 21 >50 >50 >50 >50 22 40.29±0.51 >50 >50 >50

Among the compounds, the candidate compound showing the best PPI inhibitory activity and the best antiproliferative effect in HER2-positive gastric cancer cell lines was selected (compound No. 15, hereinafter referred to as YK1). The selected candidate compounds showed 95% PPI inhibition at 10 μM, IC50 (μM) = 1.18 ± 0.34, and cancer cell proliferation inhibition effects of IC50 (μM) = 1.56 ± 0.14 in the NCI-N87 cell line.

4-2. Confirm the effect of selected candidate compounds

When the docking poses of the selected candidate compounds and gefitinib were overlapped, it was confirmed that they had similar binding directions within the hotspot of ELF3-MED23 PPI (Figure 5a). Instead of the nitrogen atom in the quinazoline ring of gefitinib, the oxygen atom in the methoxy group of the pyrazoline ring of the candidate compound serves as a hydrogen bond acceptor for H449 of MED23. In addition, the hydrogen atom of the hydroxy group in the phenyl ring of the candidate compound acts as a hydrogen bond donor, like the quinazoline ring of gefitinib. However, unlike gefitinib, the candidate compound was confirmed to form a hydrogen bond with the oxygen in the backbone of the D400 residue, forming an additional p-interaction with F399 of MED23. Moreover, YK1 is predicted to form hydrophobic interactions with MED23 residues F399, E405, and P446, similar to the ELF3 _137-144 peptide (Figure 5b).

In order to specifically confirm the effect of the above candidate substances, LC-MS/MS-based quantitative analysis was performed. Specifically, RNA was extracted from cells using FavorPrep™ Tri-RNA Reagent (FAVORGEN Biotech Corp., Taiwan) and PrimeScript™ RT Reagent Kit (Takara Bio Inc., Japan) according to the manufacturer's instructions, and complementary DNA was extracted. used to synthesize DNA, cDNA). Quantitative analysis of genes was performed using the SensiFAST™ SYBR No-ROX kit (Bioline, Canada). PCR was performed using a CFX96 ^TM real-time PCR detection system (Bio-Rad, USA) according to the following protocol: 95°C for 2 min, 27 cycles at 95°C for 10 s, 56°C for 10 s, and 72°C for 20 s. It was performed using . Relative amounts of mRNA were determined using the ㅿㅿCt method and normalized to GAPDH or ACTIN. As a result, it was confirmed that the candidate substance directly binds to the D400 and H449 residues of MED23 through hydrogen bond formation. The candidate material could bind to FLAG-tagged wild-type MED23, but could not bind to the double mutant MED23 ^D400A/H449G (Figure 5c). However, among the fragments of MED23, it specifically interacted with MED23 _391-625 , a fragment containing D400 and H449 residues (FIG. 5d).

Even when measuring the change in bioluminescence intensity, it was confirmed that the activity of luciferase, which was significantly increased by treatment with MED23 _391-462 , was significantly reduced in a dose-dependent manner by treatment with the candidate substance (Figures 5e and 5f ).

Through the above results, it was confirmed that the candidate material can specifically bind to MED23 _391-462 and thereby inhibit the PPI of ELF3-MED23 to a significant level.

Example 5. Confirmation of HER2 inhibition effect of candidate compound

5-1. Confirmation of HER expression level

Fluorescence Polarization Assay was performed to determine whether the candidate compound whose ELF3-MED23 PPI inhibitory effect was confirmed above could inhibit the expression of HER2. Specifically, various concentrations of (His) ₆ -MED23 _391-582 and fluorescein isothiocyanate (FITC) labeled ELF3 _129-145 peptides (10 nM) were incubated in assay buffer (30mM NaCl and 5mM β-mer). After mixing in 20mM sodium phosphate (pH 8.0) containing captoethanol (β-mercaptoethanol), the K _d value was determined. The samples were adjusted to a final volume of 50 μL and incubated for 30 minutes at room temperature. Determination of K _d value was performed with Prism 6.0 (GraphPad Software, USA) using the least-squares non-linear fit method. The FP signal was measured using an Infinite F200 PRO microplate reader (Tecan Group Ltd., Switzerland) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm in millipolarization (mP). For displacement analysis, graded concentrations of YK1 and unlabeled-ELF3 _137--144 peptides were mixed with 80 nM (His)6-MED23 _391-582 and 10 nM FITC-ELF3 _129-145 . The effect of compound YK1 was confirmed by measuring the level of reduction in FP signal using unlabeled peptide as a positive control. A 4-parameter logistic equation was used to determine the IC ₅₀ of each compound. This was performed using the Table Curve 2D program (SPSS Inc.). The Ki value was calculated based on the Cheng-Prusoff equation (K _i = IC ₅₀ /1+([Ligand]/K _d )).

As a result, the candidate compound was confirmed to significantly reduce the FP signal by inducing the release of FITC-labeled ELF3 _129-145 peptide from MED23 _391-582 , which reconfirmed that it can directly inhibit PPI (Figure 5g) . In addition, when the PPI inhibitory activity (4.27 ± 0.25 μM) of the candidate compound was calculated according to the above Ki value, it was confirmed to be superior to the inhibitory activity (0.78 ± 0.05 μM) of the unlabeled ELF3 _137-144 peptide (Figure 5h) )

In order to confirm whether the PPI inhibitory effect described above is specific to ELF3-MED23, when the PPI inhibitory effect on MED23 and ELK1 and IRF7, known as partner transcription factors, was confirmed, it was confirmed that PPI was not inhibited by the candidate compound ( Figure 5i). It was confirmed that the candidate compound can significantly downregulate the expression levels of the HER2 gene and protein in a dose-dependent manner through ELF3-MED23-specific PPI inhibition (Figures 5j and 5k).

Through the above results, it was confirmed that the candidate compound can specifically inhibit protein-protein interaction in ELF3-MED23, thereby suppressing the expression level of HER2.

5-2. Confirmation of kinase inhibition effect

The kinase inhibitory activity of the candidate compounds at 10 μM and 25 μM was confirmed through Eurofins kinase profiling service. The inhibitory effect on c-RAF, EGFR, ErbB2, ErbB4 and PI3 kinases was analyzed according to the Kinase Profiler Service Assay protocol.

As a result, unlike gefitinib, the candidate compound did not show significant inhibitory activity against HER family kinases (EGFR, ErbB2, and ErbB4) or AKT/MAPK pathway-related kinases (PI3 kinase and c-RAF) (Table 3 below) ).

Kinase In vitro kinase inhibitory activity (%) at the concentration of 10 μM 25 μM c-RAF 3 10 EGFR 0 0 ErbB2 2 0 ErbB4 0 0 PI3 Kinase (p110a/p85a) 3 17

The above results show that the transcriptional downregulation of HER2 induced by the candidate compound was specifically triggered by ELF3-MED23 PPI inhibition, resulting in attenuation of the HER2-related signaling cascade.

5-3. Confirmation of anti-proliferative effect

The antiproliferative effect of the candidate compound due to HER2 inhibition was confirmed. Specifically, to confirm the anticancer effect of the candidate compound, cancer cells were seeded at 10 ⁴ cells/well in a 96-well cell culture plate for 20 hours. Cells were starved in serum-free medium for 4 hours, then replaced with medium supplemented with the indicated concentration of compounds and cultured in an incubator at 5% CO ₂ and 37°C for 72 hours. Cell viability was confirmed by applying 5 μL of EZ-cytoX to each well and then checking the absorbance at 450 nm. For measurement, an ELISA Microplate Reader (VersaMax, Molecular Devices) was used.

As a result, it was confirmed that the candidate compound significantly inhibited cell growth of NCI-N87 (FIGS. 6A to 6C). The above results confirmed that the antiproliferative activity of the candidate compound was caused by regulating the expression of HER2.

Example 6. Confirmation of anticancer effect of candidate compounds

6-1. Confirmation of anti-cancer effect through data analysis

To determine whether the candidate compounds have anticancer effects against HER2-positive gastric cancer, Kaplan-Meier survival analysis was performed using a publicly available dataset of 1,065 gastric cancer patients ( www.kmplot.com/gastric/ ). . The results showed that there was a significant negative correlation between HER2 mRNA levels and overall survival (OS) or progression-free survival (PFS) (FIGS. 7A and 7B).

6-2. Confirmation of anticancer effect on xenograft mice

To confirm the anticancer effect, xenograft mice were prepared. NCI-N87 and JIMT-1 cells (5 x 10 ⁶ cells) dissolved in 100 μL of 1 Korea) was injected subcutaneously into the right flank of the mouse. When tumors reached a volume of 90–100 mm ³ , mice were randomly separated into each designated group. Drug administration was repeated 6 times intravenously for NCI-N87 xenografts and 8 times intraperitoneally for JIMT-1 xenografts, all at 3-day intervals. The candidate compound (YK1) and trastuzumab (TZMB) were prepared at the same concentration of 4 mg/kg and mixed with a mixture of DMAC/Tween80/saline (5:10:85) and saline, respectively. , was injected. After drug injection, changes in tumor size were monitored until the average tumor size in the control group reached 2000-2500 mm ³ . Mice were sacrificed 28 days after the first drug injection for NCI-N87 and 46 days for JIMT-1 xenografts, then tumors were excised from each mouse and relative tumor size was determined. The length (L) and width (W) of the tumor were measured with calipers, and the tumor volume was calculated using the formula (LXW ² ) / 2.

As a result, it was confirmed that when 4 mg/kg of the candidate compound was administered intravenously (IV) to NCI-N87 xenograft mice, the tumor volume was significantly reduced and tumor growth was delayed (FIGS. 7C and 7D).

To perform IHC analysis, the following experiment was performed. Tumors obtained from a xenograft mouse model were used to prepare paraffin-embedded block sections for immunohistochemistry (IHC). HER2 and Ki-67 primary antibodies were incubated overnight at 4°C, then washed three times with 1X PBS, incubated with secondary antibodies, and stained with DAB solution (Dako, Carpinteria, USA). The control group was stained with hematoxylin, and IHC staining was evaluated under a light microscope at 200X magnification.

As a result, it was confirmed that HER2 was reduced to a 1.8-fold level compared to the group treated with the candidate compound (Figure 7e, upper panel), and Ki67, a proliferation marker, was confirmed to be significantly reduced (Figure 7e, lower panel).

6-3. Check cell cycle and apoptosis effects

Since HER2 overexpression is related to cell cycle and apoptosis, experiments were conducted to determine the effect of candidate compounds on these. Specifically, 5 x 10 ⁵ NCI-N87 cells were inoculated into a 60 mm dish and then cultured until reaching 70-80% confluence. The candidate compound YK1 was treated with different concentrations and then incubated for 8 hours. The cells were treated with trypsin and then washed with cold PBS (pH 7.4). For cell cycle analysis, the cells were fixed with 70% ethanol, incubated at 4°C for 30 minutes, and then stained using FxCycle ^™ PI/RNase staining solution (Invitrogen ^™ , USA). Cell cycle analysis was performed using a fluorescence-activated cell sorting (FACS) instrument (BD Biosciences, USA).

As a result, time- and dose-dependent treatment with the candidate compounds resulted in a significant increase in the cell fraction in G1. Specifically, it was confirmed that it not only reduced the S and G2/M phases, but also promoted overall G1 arrest (Figures 7f and 7g).

Additionally, cells for apoptosis analysis were prepared in the same manner as the cells prepared in the cell cycle analysis method. Cells were trypsinized and washed with 1X PBS, and then each sample was resuspended using FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen ^™ ). Apoptosis was analyzed using a FACS machine.

As a result, the candidate compound can significantly promote apoptosis in a dose-dependent manner (Figure 7h), inducing an increase in pro-apoptotic markers c-PARP and c-caspase 7, and anti-apoptotic marker bcl- It was confirmed that a decrease in 2 was induced (Figure 7i).

6-4. Check physical and chemical characteristics

The candidate compound was confirmed to have a reasonable level of solubility (103.2 ± 0.6 μg/ml at pH 7) and permeability (FIGS. 8A to 8C). This means that the candidate compound meets general drug-likeness criteria and, when confirmed PK profile, the candidate compound has a long half-life (T1/2 (IV), 9.74 ± 2.76 h; T1/2 (PO), 10.9 ± 11.00 h). It was confirmed that it had a decent level of bioavailability (Ft, 25.1%) (Figure 8d).

Through the above results, it was confirmed that the candidate compound has a remarkable level of anticancer effect and has physicochemical properties suitable for use as an anticancer agent.

Example 7. Confirmation of resistance avoidance effect of candidate compound

7-1. Confirm resistance evasion effect

It was determined whether the candidate compound could overcome trastuzumab resistance. Specifically, the prepared compounds Nos. 1 to 22 were used for JIMT-1 (ER-/PR-/HER2+ subtype, congenital TZMB resistance) and TZMB-refractory BT474 (BT-TR, ER+/PR+/HER2+ subtype, acquired resistance). and TZMB-refractory NCI-N87 (NCI-N87 TR; HER2+ subtype, acquired resistance) group, and then the effect was confirmed.

While treatment with TZMB is effective only on TZMB-sensitive cells, treatment with the candidate compound was confirmed to have a significant anticancer effect not only on TZMB-sensitive cells but also on TZMB-resistant cells (Figures 9a to 9f ). It was confirmed that the expression of HER2 and downstream signals such as phosphorylated AKT and MAPK could also be significantly downregulated. In contrast, no such changes were observed when TZMB-resistant cells were treated with TZMB (Figures 9D to 9F).

7-2. Confirmation of resistance avoidance effect through clone analysis

In order to reconfirm the above results, clonal analysis was performed. Specifically, cells were seeded in 6-well culture plates at a density of 2000 cells/well and then incubated for 14 days with or without TZMB (10 or 20 μg/mL) and YK1 (0.5 or 1 μM). The incubated cells were fixed with 100% methanol for 1 hour and then stained with 200 μL of crystal violet solution per well. Cells were washed with tap water, then images were taken with a ChemiDoc bio-image analyzer (Bio-Rad) and quantified with ImageJ software (NIH, Bethesda, MD, USA). After fixation, all above steps were performed at room temperature.

As a result, as confirmed in Example 7-1, the candidate compound was effective in TZMB resistance, but it was confirmed that TZMB did not have this effect (FIGS. 9g to 9i).

7-3. Confirmation of resistance evasion effect in xenograft mice

JIMT-1 xenograft mice were treated with 4 mg/kg of the candidate compound and TZMB, respectively, and the resistance evasion effect was confirmed in vivo . As a result, it was confirmed that when treated with the candidate compound, the level of HER2 was significantly reduced, and the growth and volume of the tumor were significantly reduced (FIGS. 9J to 9M). In addition, Ki67, a proliferation marker, was also confirmed to be significantly reduced in the candidate compound treatment group compared to the TZMB treatment group (FIG. 9n). Meanwhile, the body weights of mice were generally similar throughout the experiment (Figure 10).

Through the above results, it was confirmed that the candidate compound can be effectively used even in cells resistant to TZMB, which is currently used clinically.

Claims (14)

A compound represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
[Formula 1]

R ₁ is hydrogen;
R ₂ and R ₃ are independently hydrogen or a hydroxy group;
R ₄ is p-methoxynaphthalenyl, , or ego;
where A ₁ is nitrogen, oxygen or sulfur;
A ₂ is halogen or C _1-3 alkoxy.
In claim 1,
The compound of Formula 1 is a compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that any one selected from the group consisting of the following compounds:
3-Furan-2-yl-1-(3-hydroxyphenyl)-propenone;
1-(3-Hydroxyphenyl)-3-(4-methoxyphenyl)-propenone);
1-(3-Hydroxyphenyl)-3-(4-methoxynaphthalen-1-yl)-propenone);
3-(4-Chlorophenyl)-1-(3-hydroxyphenyl)-propenone (3-(4-Chlorophenyl)-1-(3-hydroxyphenyl)-propenone);
3-(4-Hydroxyphenyl)-1-(3-hydroxyphenyl)-propenone);
1-(2-Hydroxyphenyl)-3-(4-methoxyphenyl)-propenone);
1-(2-Hydroxyphenyl)-3-(4-methoxynaphthalen-1-yl)-propenone);
3-Furan-2-yl-1-(2-hydroxyphenyl)-propenone;
3-(4-Hydroxyphenyl)-1-(2-hydroxyphenyl)-propenone);
3-(4-Chlorophenyl)-1-(2-hydroxyphenyl)-propenone); and
1-(2-Hydroxyphenyl)-3-thiophen-2-yl-propenone.
A compound represented by the following formula (2), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
[Formula 2]

R ₅ to R ₇ are independently hydrogen or a hydroxy group;
R ₈ is p -Methoxynaphthalenyl ( p -methoxynaphthalenyl), 1-Methoxynaphthalenyl (1-Methoxynaphthalenyl), , or ego;
R ₉ is C _1-3 alkyl;
where A ₁ is nitrogen, oxygen or sulfur;
A ₂ is halogen or C _1-3 alkoxy.
In claim 3,
The compound of Formula 2 is a compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that any one selected from the group consisting of the following compounds:
1-[5-furan-2-yl-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-Furan-2-yl-3 -(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);
1-[3-(3-hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-(3-Hydroxyphenyl) -5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);
1-[3-(3-hydroxyphenyl)-5-(4-methoxynaphthalen-1-yl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-( 3-Hydroxyphenyl)-5-(4-methoxynaphthalen-1-yl)-4,5-dihydropyrazol-1-yl]-ethanone);
1-[5-(4-Chlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-(4-Chlorophenyl)- 3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);
1-[5-(4-hydroxyphenyl)-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-(4-Hydroxyphenyl) -3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);
1-[5-furan-2-yl-3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-Furan-2-yl-3 -(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);
1-[3-(2-hydroxyphenyl)-5-(4-methoxynaphthalen-1-yl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-( 2-Hydroxyphenyl)-5-(4-methoxynaphthalen-1-yl)-4,5-dihydropyrazol-1-yl]-ethanone);
1-[5-(4-hydroxyphenyl)-3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-(4-Hydroxyphenyl) -3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);
1-[5-(4-Chlorophenyl)-3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[5-(4-Chlorophenyl)- 3-(2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);
1-[3-(2-Hydroxyphenyl)-5-thiophen-2-yl-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-(2-Hydroxyphenyl)- 5-thiophen-2-yl-4,5-dihydropyrazol-1-yl]-ethanone);
1-[3-(2-hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone (1-[3-(2-Hydroxyphenyl) -5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-ethanone);
1-[3-(3-hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-propan-1-one (1-[3-(3 -Hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-propan-1-one);
1-[3-(3-hydroxyphenyl)-5-thiophen-2-yl-4,5-dihydropyrazol-1-yl]-propan-1-one (1-[3-(3- Hydroxyphenyl)-5-thiophen-2-yl-4,5-dihydropyrazol-1-yl]-propan-1-one);
1-[5-(4-chlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-propan-1-one (1-[5-(4- Chlorophenyl)-3-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl]-propan-1-one).
A composition for inhibiting protein-protein interaction, comprising a compound represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

R ₁ is hydrogen;
R ₂ and R ₃ are independently hydrogen or a hydroxy group;
R ₄ is p-methoxynaphthalenyl, , or ego;
where A ₁ is nitrogen, oxygen or sulfur;
A ₂ is halogen or C _1-3 alkoxy.
A composition for inhibiting protein-protein interaction, comprising a compound represented by the following formula (2), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 2]

R ₅ to R ₇ are independently hydrogen or a hydroxy group;
R ₈ is p -Methoxynaphthalenyl ( p -methoxynaphthalenyl), 1-Methoxynaphthalenyl (1-Methoxynaphthalenyl), , or ego;
R ₉ is C _1-3 alkyl;
where A ₁ is nitrogen, oxygen or sulfur;
A ₂ is halogen or C _1-3 alkoxy.
According to claim 5 or claim 6,
A composition for inhibiting protein-protein interaction, wherein the protein-protein interaction is the interaction of ELF3 and MED23.
A pharmaceutical composition for preventing or treating cancer, comprising the compound of any one of claims 1 to 4, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
In claim 8,
The above cancers include cervical cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, colon cancer, bone cancer, skin cancer, head cancer, cervical cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, brain tumor, bladder cancer, and blood cancer. , stomach cancer, perianal cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer. , from the group consisting of penile cancer, prostate cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. A pharmaceutical composition that is any one selected.
In claim 8,
A pharmaceutical composition wherein the cancer overexpresses HER2.
In claim 8,
A pharmaceutical composition, wherein the cancer has resistance to trastuzumab.
In claim 8,
A pharmaceutical composition, wherein the composition inhibits the level of HER2.
In claim 8,
The composition is a pharmaceutical composition that induces apoptosis of cancer cells.
A health functional food composition for preventing or improving cancer, comprising the compound of any one of claims 1 to 4, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.