WO2023080714A1 - Composition for prevention, amelioration or treatment of cancer that overcomes resistance to proteasome inhibitor - Google Patents

Abstract

The present invention relates to a composition for the prevention, amelioration or treatment of cancer, comprising a novel compound that inhibits the deubiquitinase (DUB) activity of a 19S regulatory particle (RP) in a proteasome consisting of a 20S catalytic core particle (CP) and the 19S RP. By inhibiting the DUB activity of the 19S RP of the proteasome by compounds of the present invention, ubiquitinated proteins to be degraded are accumulated in cancer cells, and apoptosis of the cancer cells is caused by endoplasmic reticulum stress.

Description

Composition for preventing, improving or treating cancer that overcomes resistance to proteasome inhibitors

The present invention relates to a novel composition for preventing, improving or treating cancer.

The ubiquitin proteasome system is an important control mechanism in cell growth and division, cell cycle, intracellular signal transduction, and apoptosis. Through this control mechanism, proteins acting as substrates are degraded by the proteasome. do. That is, multiple ubiquitin protein chains are covalently bonded to substrates, and they are recognized and degraded by the 26S proteasome composed of 20S CP (catalytic core particle) and 19S RP (regulatory particle). 19S has deubiquitinic activity, which is involved in discriminating between long and short multiubiquitin chains, particularly in cell cycle progression, differentiation, DNA replication and repair, transcription, protein quality control, immune response and cellular It is known to be involved in several processes, including self-destruction.

The ubiquitin-proteasome system has been reported to affect various types of cancer, neurodegenerative diseases, metabolic disorders, viral diseases, heart diseases, and aging-related diseases, and inhibition of proteasome activity leads to cancer cell suicide and cancer cell death. Known to inhibit the proliferation of , interest in and development of proteasome inhibitors (PIs) as anticancer agents are greatly increasing. In particular, the treatment of patients with multiple myeloma, a blood cancer that occurs in plasma cells circulating in the body, depends on the development of new therapeutic agents such as proteasome inhibitors (PI).

Bortezomib, a 20S proteasome inhibitor recently approved by the US FDA, is a good example for confirming the effectiveness of a proteasome inhibitor (PI) anticancer drug. However, cancers resistant to this drug have been reported. Although many chemicals target enzymes in the UB-pathway, ubistatin has been identified as an inhibitor that blocks the interaction of ubiquitin with proteins by directly binding to K48-linked polyubiquitin. In addition, it has been demonstrated that ubistatin can prevent cell cycle progression by inhibiting proteasome-dependent degradation of cell cycle components. However, the cell membrane impermeability of ubistatin is a major limitation in its development into applications including therapeutic agents and intracellular probes. Thus, there is still a need for development of ubiquitin-pathway inhibitors that improve the above limitations.

One object of the present invention is to provide a composition for preventing, improving or treating cancer.

In order to achieve the above object, one aspect of the present invention provides a composition for preventing or treating cancer comprising a compound represented by Formula 1 or Formula 2 as an active ingredient.

[Formula 1]

(In Formula 1,

R ₁ is hydrogen or halogen;

R ₂ is a heteroalkyl or heterocycloalkyl containing at least one selected from the group consisting of O and N;

R ₃ is hydrogen, halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy or amine;

Y ₁ , Y ₂ and Y ₃ are each independently selected from the group consisting of S and C)

[Formula 2]

(In Formula 2,

R' ₁ is hydrogen or halogen;

R′ ₂ is a heteroalkyl or heterocycloalkyl including at least one selected from the group consisting of O and N;

R' ₃ and R' ₄ are hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, or amine;

Y' ₁ is carbon or nitrogen)..

The compound of the present invention inhibits the activity of the deubiquitination enzyme DUB (deubiquitinase) of 19S RP, so that ubiquitinated proteins to be degraded are accumulated in cancer cells and cancer cells are killed (apoptosis) by endoplasmic reticulum stress.

Furthermore, since the compound of the present invention targets the 19S RP of the proteasome, the specific target site is different from that of conventional drugs such as bortezomib, which targets the 20S CP of the proteasome. It can be usefully used as an alternative or combination anticancer agent to overcome resistance to existing drugs.

1 is a result showing that the survival rate of cancer cells is reduced in a concentration-dependent manner by the compounds of the present invention. 1, 19SI-BR-1, 19SI-BR-3, 19SI-BR-11, and 19SI-BR-12 refer to compounds having chemical structures of Formulas 3, 5, 13, and 14, respectively.

2 is a result showing that ubiquitinated proteins are accumulated in cells in a concentration-dependent manner by the compounds according to the present invention. In FIG. 2, 19SI-BR-1, 19SI-BR-3, 19SI-BR-11, and 19SI-BR-12 refer to compounds having chemical structures of Formulas 3, 5, 13, and 14, respectively.

Figure 3 is a result showing that the compounds according to the present invention have cell proliferation inhibitory effects on various carcinomas. In FIG. 3, 19SI-BR-1, 19SI-BR-3, 19SI-BR-11, and 19SI-BR-12 refer to compounds having chemical structures of Formulas 3, 5, 13, and 14, respectively.

Figure 4 is a result showing that the compound having the chemical structure of Formula 13 inhibits the proliferation of various cancer cells, promotes protein ubiquitination, and induces endoplasmic reticulum stress. A compound having a chemical structure.

5 is a result showing that endoplasmic reticulum stress is induced in various cancer cells in a concentration-dependent manner by a compound having a chemical structure of Formula 13, and 19SI-BR-11 in FIG. 5 means a compound having a chemical structure of Formula 13 .

6(A) is a result showing that the compound having the chemical structure of Formula 13 inhibits the deubiquitin activity of the 19S proteasome in a concentration-dependent manner. Figure 6 (B) is a result showing that the compound of Formula 13 does not inhibit the proteolytic activity of the 20S proteasome, and in Figure 6, 19SI-BR-11 means a compound having the chemical structure of Formula 13. .

7 is a result showing that cell proliferation is not inhibited by bortezomib in a bortezomib-resistant cancer cell line, whereas cell proliferation is inhibited by bortezomib in wild-type cancer cell lines.

8 is a result showing that the compounds of the present invention exhibit cell proliferation inhibitory effects in wild-type cancer cells and bortezomib-resistant cell lines, and 19SI-BR-1 to 19SI-BR-19 in FIG. A compound having a chemical structure.

9 is a result showing that proteins ubiquitinated by the compounds according to the present invention are accumulated in bortezomib-resistant cancer cells in a concentration-dependent manner. 9, 19SI-BR-11 means a compound having the chemical structure of Chemical Formula 13.

One aspect of the present invention provides a compound represented by Formula 1 or Formula 2 below.

[Formula 1]

In Formula 1,

R ₁ is hydrogen or halogen;

R ₂ is a heteroalkyl or heterocycloalkyl containing at least one selected from the group consisting of O and N;

R ₃ is hydrogen, halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy or amine;

Y ₁ , Y ₂ and Y ₃ are each independently any one selected from the group consisting of S and C, and specifically, at least one of Y ₁ , Y ₂ and Y ₃ may be S.

[Formula 2]

In Formula 2,

R' ₁ is hydrogen or halogen;

R′ ₂ is a heteroalkyl or heterocycloalkyl including at least one selected from the group consisting of O and N;

R' ₃ and R' ₄ are hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, or amine;

Y' ₁ is carbon or nitrogen.

In a specific embodiment of the present invention, the compound represented by Formula 1 may be a compound represented by Formulas 3 to 5, Formulas 7 to 9, and Formula 11 below.

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 11]

In addition, in a specific embodiment of the present invention, the compound represented by Formula 2 may be a compound represented by Formulas 13 to 15 and Formula 18 below.

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 18]

The compound of Formula 1 or Formula 2 may inhibit the activity of 19S RP (regulatory particle).

The compound inhibits the activity of the deubiquitination enzyme DUB (deubiquitinase), which is an activity of 19S RP, so that ubiquitinated proteins to be degraded accumulate in cancer cells and cause endoplasmic reticulum stress to kill cancer cells. The 19S RP is a component of the ubiquitin proteasome system, and the system is an important regulatory mechanism in cell growth and division, cell cycle, intracellular signal transduction, and apoptosis. This regulatory mechanism is a process in which multiple ubiquitin protein chains covalently bind to substrate proteins, and ubiquitinated substrate proteins are recognized and degraded by the 26S proteasome composed of 20S CP (catalytic core particle) and 19S RP (regulatory particle). depends on 19S RP has deubiquitinase activity, which is involved in discriminating between long and short multi-ubiquitin chains, especially in cell cycle progression, differentiation, DNA replication and repair, transcription, protein quality control, and immune response. And it is known to be related to several processes including apoptosis. The ubiquitin-proteasome system has been reported to affect the induction of several types of cancer, neurodegenerative diseases, metabolic disorders, viral diseases, heart diseases and aging-related diseases, and inhibition of proteasome activity leads to cancer cell suicide and It is known to inhibit the proliferation of cancer cells. In particular, treatment of patients with multiple myeloma, a hematological cancer arising from plasma cells circulating in the body, relies on treatment with a proteasome inhibitor (PI).

In a specific example of the present invention, it was confirmed that ubiquitinated proteins were accumulated in cancer cells in a concentration-dependent manner by the compounds of the present invention to induce endoplasmic reticulum stress (see FIGS. 2 and 4). In addition, it was confirmed that endoplasmic reticulum stress was induced in a concentration-dependent manner by the compound of the present invention in a luminescence assay for endoplasmic reticulum stress (see FIG. 5). In addition, it was confirmed that the compound of the present invention inhibits the deubiquitination enzyme activity of 19S RP (see (A) in FIG. 6).

The compound may not inhibit the activity of 20S CP (catalytic core particle). The compound may have an excellent anticancer effect in anticancer treatment targeting the ubiquitin proteasome system when used simultaneously or separately with other anticancer agents that inhibit the proteasome activity or proteolytic enzyme activity of 20S CP.

In a specific example of the present invention, it was confirmed that the compound of the present invention inhibits the deubiquitin activity of 19S RP in a concentration-dependent manner, but does not affect the proteasome activity of 20S CP regardless of the concentration (Fig. See (B) in 6).

Therefore, the compounds of the present invention can be used as an active ingredient of a composition for preventing, improving or treating cancer, and thus another aspect of the present invention provides a composition for preventing or treating cancer containing the compound as an active ingredient. do.

Cancer, which is a disease to be treated in the present invention, generally refers to the physiological state of mammals characterized by abnormal cell growth, and abnormally overproliferates due to problems in the control function of normal cell division, differentiation, and death. It refers to a condition infiltrating tissues and organs to form lumps and destroying or transforming existing structures.

The cancers include colorectal cancer, lung cancer, myeloma, hematological cancer, breast cancer, head or neck cancer, uterine cancer, cervical cancer, ovarian cancer, lung cancer, bladder cancer, esophageal cancer, semothelioma, neuroblastoma, testicular cancer, lymphoma, leukemia, stomach cancer, liver cancer, and skin cancer. , brain cancer, neuroma, laryngeal cancer, prostate cancer, thyroid cancer, kidney cancer, pancreatic cancer and rectal cancer, specifically non small cell lung cancer, large cell lung carcinoma, multiple myeloma, neuroma , glioblastoma, breast cancer, stomach cancer, prostate cancer, liver cancer, colorectal cancer, and the like. In particular, it may be multiple myeloma, which is a tumor of malignant plasma cells in the bone marrow, but is not limited thereto, and any cancer in a physiological state generally characterized by abnormal cell growth may be included without limitation.

In a specific embodiment of the present invention, the compound of the present invention is used to treat cervical cancer (Hela), breast cancer (MDA-MB-231), gastric cancer (AGS, NUGC3), liver cancer (HepG2, Hep3B), and kidney cancer (Caki-1, 786). -O), pancreatic cancer (Mia-Paca), prostate cancer (PC3), colorectal cancer (WiDr, SW480, HT29, HCT116), myeloma (IM-9, RPMI-8226), neuroma (U87MG, T89), lung cancer (H1229) , H460, A549), it was confirmed that the effect of inhibiting cancer cell proliferation was excellent (see FIG. 3).

In the present invention, the cancer may be a cancer that is resistant to 20S CP (catalytic core particle) inhibitors.

The 20S CP (catalytic core particle) inhibitor may be bortezomib, carfilzomib, ixazomib, etc., but is not limited thereto, and inhibits the activity of 20S CP to inhibit the ubiquitin proteasome system It can be included without limitation.

Cancer resistant to the 20S CP inhibitor exhibits extremely low sensitivity to cancer therapy or drugs for cancer treatment, such as chemotherapy using the 20S CP inhibitor, especially anticancer drug therapy, and symptoms are improved or alleviated by the therapy , cancer that does not show any remission or cure symptoms. The resistant cancer may have resistance to a specific treatment from the beginning, or may not show resistance at first, but may develop due to a long-term treatment and no longer show sensitivity to the same treatment due to genetic mutations in cancer cells. For example, the cancer resistant to the 20S CP inhibitor may be a cancer that does not significantly affect the survival rate of cancer cells regardless of the concentration of the 20S CP inhibitor, and specifically, hematological cancer, lung cancer or multiple myeloma resistant to the 20S CP inhibitor. can be

Since the compound of the present invention inhibits the activity of 19S RP without affecting the activity of 20S CP, it can exhibit excellent anticancer effects by inhibiting the ubiquitin-proteasome system even in cancers resistant to 20S CP inhibitors. .

In a specific embodiment of the present invention, it was confirmed that the compounds of the present invention could kill cancer cells in a concentration-dependent manner in bortezomib-resistant myeloma cells and bortezomib-resistant lung cancer cells (see FIGS. 7 and 8, Tables 3 and 4). ). In addition, it was confirmed that ubiquitin protein was accumulated in bortezomib-resistant cancer cells treated with the compound of the present invention to induce endoplasmic reticulum stress (see FIG. 9).

The prevention means any action that suppresses or delays the occurrence or progression of cancer, the improvement means any action that improves the patient's health condition, such as enhancing immunity, and the treatment means not only killing cancer cells but also cancer cells. Reduction of growth, reduction of proliferation, reduction of recurrence, alleviation to some extent of one or more symptoms associated with cancer, reduction of immune system anticancer activity and immune memory formation against cancer cells, or reduction of invasion of cancer cells, etc. It means all effects that do not, but are not limited thereto.

The composition may be a pharmaceutical composition.

The pharmaceutical composition may further include an active ingredient exhibiting anticancer activity in addition to the above compounds. In particular, it may be Lenalidomide, Dexamethasone, Cyclophosphamide, Daratumumab, etc., which are used for the treatment of multiple myeloma, but are not limited thereto, and are active ingredients that exhibit anticancer effects, It may be included without limitation as long as it does not inhibit the anticancer effect of the compound of the present invention.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier or additive in addition to the active ingredient.

The meaning of 'pharmaceutically acceptable' means that it does not inhibit the activity of the active ingredient and does not have toxicity more than is adaptable to the subject of application (prescription). The carrier is defined as a compound that facilitates the addition of the compound into cells or tissues.

The pharmaceutical composition may be administered by mixing with any convenient carrier, etc., and such a dosage form may be a single or repeated dosage form. The composition may be a solid formulation or a liquid formulation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, and suppositories. Solid formulations may include carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, and the like, but are not limited thereto. Liquid preparations include solutions, suspensions, and emulsions such as water and propylene glycol solutions, but are not limited thereto, and may be prepared by adding appropriate colorants, flavoring agents, stabilizers, viscosifiers, and the like. For example, a powder may be prepared by simply mixing an appropriate pharmaceutically acceptable carrier such as lactose, starch, or microcrystalline cellulose in addition to genipin, which is an active ingredient of the present invention. Granules are prepared by mixing the active ingredient of the present invention with a suitable pharmaceutically acceptable carrier and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone and hydroxypropylcellulose, and then adding a solvent such as water, ethanol, isopropanol, etc. It can be prepared using a wet granulation method using a dry granulation method using a compressive force. In addition, tablets may be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate and then tableting them using a tableting machine. When formulating the pharmaceutical composition as an injection, it may be prepared according to a conventional method for preparing an injection known in the art. When formulated as an injection, it may be dispersed in a sterile medium so that it can be used as it is when administered to a patient, or may be administered after dispersing in an appropriate concentration by adding distilled water for injection.

Depending on the disease to be treated and the condition of the subject, the composition may be an injection (eg, intravenous injection, intramuscular injection, intraperitoneal injection, infusion, subcutaneous injection, implant), inhalant, oral agent, intranasal agent, vaginal agent, It may be administered as a rectal agent, a sublingual agent, a transdermal agent, a topical agent, and the like, but is not limited thereto. Depending on the route of administration, it can be formulated into an appropriate dosage unit formulation containing a pharmaceutically acceptable carrier, excipients, and vehicles that are commonly used and non-toxic.

The composition may be administered at about 0.0001 mg/kg to about 10 g/kg daily, and at a daily dosage of about 0.001 mg/kg to about 1 g/kg. The therapeutically effective amount or effective dose of the pharmaceutical composition may vary depending on the formulation method, administration method, administration time and/or administration route of the pharmaceutical composition, and the type of response to be achieved by administration of the composition Various factors, including degree, type of subject to be administered, age, weight, general health condition, symptom or severity of disease, sex, diet, excretion, drugs used simultaneously or at different times in the subject, or components of other compositions And it may vary according to similar factors well known in the medical field, and those skilled in the art can easily determine and prescribe an effective dosage for the desired treatment. In addition, if necessary, for convenience, the total daily dose may be divided and administered several times during the day. The term “treatment-effective amount” refers to a side effect or anticancer drug resistance caused by anticancer treatment in a patient with cancer, improvement of a condition (eg, one or more symptoms), delay of progression of a disease, etc. means an amount sufficient to produce the desired effect, including

The composition may be a health functional food composition.

The health functional food refers to food manufactured and processed in the form of tablets, capsules, pills, liquids, powders and granules using raw materials or ingredients having useful functionalities for the human body. Here, 'functionality' means obtaining useful effects for health purposes, such as adjusting nutrients for the structure and function of the human body or physiological functions. In addition to the active ingredient, the health functional food composition contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, It may further contain glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like. The ratio of the components to be added is not very important, but is generally selected from the range of 0.01 to 0.1 parts by weight, based on 100 parts by weight of the health functional food composition of the present invention, by a method commonly used in the art. It can be manufactured, and during manufacture, it can be manufactured by adding raw materials and components commonly added in the conventional technical field.

In addition, the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food. There is no particular limitation on the type of health functional food. Examples of foods to which the functional food composition may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, There are tea drinks, alcoholic beverages and vitamin complexes, and includes all health foods in a conventional sense.

The health functional food composition of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of using food as a raw material and free from side effects that may occur when taking drugs for a long time, and has excellent portability. The health functional food of the present invention can be consumed as an adjuvant to enhance the effectiveness of anticancer drugs.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[Example 1]

As shown in Table 1 below, 19 compounds having chemical structures of Formulas 3 to 21 were synthesized and prepared by requesting the Innovative New Drug Library Research Center of Dongguk University.

Formula 3	formula 4
Formula 5	formula 6
Formula 7	Formula 8
Formula 9	Formula 10
Formula 11	Formula 12
Formula 13	Formula 14
Formula 15	Formula 16
Formula 17	Formula 18
Formula 19	Formula 20
Formula 21

[Example 2]

Proliferative inhibitory effect on cancer cells

Among the compounds prepared in Example 1, it was confirmed whether four compounds of Formula 3, Formula 5, Formula 13, and Formula 14 had an effect of reducing the viability of colon cancer cells.

Specifically, 0.16, 0.31, 0.63, 1.25, and 2.5 μM of the four compounds of Formula 3, Formula 5, Formula 13, and Formula 14 were added to the human colon cancer cell line HCT116 wild type in DMEM medium at a concentration of 5% fetal bovine serum. After melting as much as possible, each cell was treated for 72 hours. Thereafter, cells were fixed using 10% formalin, and nuclei were stained using methylene blue solution. Then, the cell viability was measured by dissolving the dye using 0.5% hydrochloric acid solution and measuring the absorbance.

As a result, as shown in FIG. 1, the effect of reducing the viability of cancer cells in a concentration-dependent manner by the four compounds was confirmed.

[Example 3]

Accumulation effect of ubiquitination of proteins in cancer cells

The effect of the four compounds having chemical structures of Formula 3, Formula 5, Formula 13, and Formula 14 prepared in Example 1 on the accumulation of ubiquitin protein was confirmed.

Specifically, the compounds of Formula 3, Formula 5, and Formula 14 prepared in Example 1 were prepared in DMEM medium containing 5% fetal calf serum for culturing the human colorectal cancer cell line HCT116 at concentrations of 1, 2, and 4 μM, respectively. The compound of Formula 13 was dissolved to a concentration of 4 and 8 μM, and then incubated for 6 hours. Thereafter, the cells were lysed using RIPA lysis buffer to extract proteins, and the same amount of proteins were separated by polyacrylamide gel electrophoresis. After transferring the separated protein to a fluorinated polyvinylidene membrane, non-specific binding was blocked with 5% skim milk. The membrane was agitated overnight at 4°C in a refrigerator using ubiquitin antibody, and the next day, the secondary antibody of the rabbit species, which is the extraction species of the primary antibody, was stirred at room temperature for 1 hour on the membrane, and then Western blotting was performed using Merck's Luminata Immobilon Crescendo Western HRP substrate reagent. A blot was performed.

As a result, as shown in FIG. 2, it was confirmed that ubiquitinated proteins were accumulated in a concentration-dependent manner by the four compounds.

[Example 4]

Confirmation of anticancer effect on various cancer cells

[4-1] Confirmation of proliferation inhibitory effect on various cancer cells

Among the compounds prepared in Example 1, for liver cancer (HepG2), pancreatic cancer (Mia-Paca-2), colon cancer (HCT116), myeloma (IM-9, RPMI-8226), and lung cancer (H1229, H460, A549) Representatively, the compounds of Formula 3, Formula 5, or Formula 13 were treated, and the cell viability of each cancer cell was measured in the same manner as in Example 2, and then the GI50 value was calculated.

As a result, as shown in FIG. 3, it was confirmed that the compounds of the present invention have an effect of inhibiting cell proliferation against various types of cancer cells.

Among them, the compound of formula 13 (19SI-BR-11), which was confirmed to be most effective, was used for cervical cancer (Hela), breast cancer (MDA-MB-231), gastric cancer (AGS, NUGC3), liver cancer (HepG2, Hep3B), kidney cancer (Caki-1, 786-O), pancreatic cancer (Mia-Paca), prostate cancer (PC3), colorectal cancer (WiDr, SW480, HT29, HCT116), myeloma (IM-9, RPMI-8226) After treating and culturing cells of various carcinomas such as , neuroma (U87MG, T89) and lung cancer (H1229, H460, A549), cell viability was measured in the same manner as in Example 2, and the GI50 value was calculated. The amount of ubiquitinated protein in each cell was measured in the same manner as in Example 3. In addition, in order to confirm whether endoplasmic reticulum stress is induced by accumulation of ubiquitinated proteins, the amounts of CHOP and GRP78, which are endoplasmic reticulum stress marker proteins, were measured in the same manner as in Example 3 above.

As a result, as shown in FIG. 4, the compound of Formula 13 inhibits cell proliferation and increases the expression of CHOP and GRP78 in various types of cancer cells other than colorectal cancer cells, thereby promoting ubiquitination of intracellular proteins. was found to induce endoplasmic reticulum stress.

[4-2] Endoplasmic reticulum stress induction effect in various cancer cells

Whether the compound of Formula 13 prepared in Example 1 induces endoplasmic reticulum stress was reconfirmed using a reporter assay system.

Specifically, viruses capable of expressing luciferase in response to endoplasmic reticulum stress were stably expressed in human colon cancer cell line HCT116, human gastric cancer cell line HT29, human cervical cancer cell line HeLa, and human lung cancer cell line H1703. Thereafter, the compound of Formula 13 was dissolved in a DMEM medium containing 5% fetal bovine serum concentration in which each cell was cultured, to a final concentration of 0.3125, 0.625, 1.25, 2.5, 5, and 10 μM, and then each cell was treated for 12 hours. . Thereafter, luminescence was measured using Promega's Dual luciferase Reporter assay system.

As a result, as shown in FIG. 5 , it was confirmed that the compound having the chemical structure of Formula 13 induces endoplasmic reticulum stress in a concentration-dependent manner in various types of cancer cells other than colon cancer cells.

[Example 5]

19S proteasome activity inhibitory effect

The effect of the compound of Formula 13 prepared in Example 1 on the deubiquitin activity of the 19S proteasome and the proteolytic activity of the 20S proteasome was confirmed.

Specifically, 10 ng/10 μl of recombinant 19S proteasome protein was reacted with 1.25, 2.5, 5, and 10 μM of the compound of Formula 13 or 100 μM of the comparative drug NEM, respectively, at room temperature for 30 minutes, followed by RnD Ub-AML (Ub-Aminoluciferin) was reacted at room temperature for 30 minutes with a final concentration of 1 μM. Thereafter, 10 μl of each reaction solution was transferred to a half-well white plate, and 20 μl of Promega's Luciferin detection reagent was added to each well to react, and luminescence was measured. The activity of the 20S proteasome was determined by lysing HCT116 cells with RIPA buffer, extracting intracellular proteins, and then adding the compound of Formula 13 to 0.5 μg of protein at concentrations of 1.25, 2.5, 5, and 10 μM, or the comparative drug Borte. Zomib was treated at a final concentration of 100 nM, reacted at room temperature for 10 minutes, and then 10 μl of proteasome-Glo assay substrate from promega was added and reacted again at room temperature for 30 minutes. Thereafter, 10 μl of each reaction solution was transferred to a half-well white plate, and 20 μl of Promega's Luciferin detection reagent was added to each well to react, and luminescence was measured.

As a result, as shown in FIG. 6 , it was confirmed that the compound of Formula 13 inhibited only the DUB (deubiquitinase) activity of 19S RP and had no effect on 20S CP, the target of bortezomib.

[Example 6]

Effect of overcoming bortezomib resistance

[6-1] Construction of bortezomib-resistant cell lines

In order to confirm whether the 19 compounds prepared in Example 1 exhibit therapeutic effects on bortezomib-resistant cancer, first, bortezomib-resistant cell lines were constructed.

Specifically, the human colorectal cancer cell line HCT116 was treated with bortezomib twice a week at gradually increasing concentrations, and during subculture, it was divided into two cell culture dishes, one to maintain the concentration and cell viability, and one to increase the concentration. treated for a long period of time. After bortezomib treatment, dead cells were removed by PBS washing and centrifugation, and surviving cells, that is, bortezomib-resistant cells, were cultured at a concentration twice higher than the previous bortezomib treatment concentration in a new culture vessel, showing resistance to bortezomib. A cell line was established.

As a result, as shown in FIG. 7, the growth of the wild-type HCT116 cell line was inhibited by bortezomib, whereas the bortezomib-resistant HCT116 cell line constructed as described above was no longer affected by bortezomib despite treatment with high concentrations of bortezomib. It was confirmed that proliferation was not inhibited.

[6-2] Confirmation of cancer cell proliferation inhibitory effect in bortezomib-resistant cancer cell lines

In order to confirm whether the 19 compounds prepared in Example 1 exhibit therapeutic effects on bortezomib-resistant cancer, bortezomib-resistant cell lines were treated with the 19 compounds, and then cancer cell proliferation inhibitory effects were confirmed.

Specifically, to wild-type HCT116 cells and bortezomib-resistant cells constructed in Example [6-1], bortezomib was added in DMEM medium at a concentration of 5% fetal bovine serum twice from a final concentration of 1 μM to 0.0039 μM. serial dilution. In addition, the compounds of formulas 3 to 21 were diluted from 20 μM to 0.078125 μM in DMEM medium with 5% fetal bovine serum concentration, and treated for 72 hours. Thereafter, cells were fixed using 10% formalin, and nuclei were stained using methylene blue solution. Then, the dye was dissolved in 0.5% hydrochloric acid solution, and the absorbance was measured.

As a result, as shown in FIG. 8, the survival rate of cancer cells was decreased by 13 compounds having chemical structures of Formulas 3 to 9, Formula 11, Formula 13 to 16, and Formula 18, and 11 types of compounds among them The effect of reducing the survival rate of bortezomib-resistant cell lines was confirmed by

In addition, the efficacy of inhibiting the viability of wild-type human colorectal cancer cells and bortezomib-resistant cells by the compound of the present invention was comparatively analyzed by IC ₅₀ value, and the ratio of bortezomib-resistant cell line IC ₅₀ / wild-type cell line IC ₅₀ was calculated and the results are as follows. Table 2 shows. In a resistant cell line in which the cell viability inhibitory activity of Bortezomib is reduced by about 40 times, 11 compounds having chemical structures of Formulas 3 to 5, Formulas 7 to 9, Formula 11, Formulas 13 to 15, and Formula 18 are As the apoptotic activity was maintained, it was confirmed that the bortezomib resistance overcoming activity was exhibited.

compound	Wild-type HCT116 cell viability IC 50 (μM)	Bortezomib-resistant HCT116 cell viability IC 50 (μM)	Bortezomib-resistant IC 50 / wild-type IC 50 ratio
Formula 3	0.22 ± 0.02	0.33 ± 0.01	1.5
formula 4	1.97 ± 0.14	3.45 ± 0.08	1.75
Formula 5	0.59 ± 0.01	0.64±0	1.08
formula 6	13.61 ± 0.39	>20	-
Formula 7	9.53 ± 0.63	12.96 ± 0.25	1.35
Formula 8	9.9 ± 0.26	18.19 ± 4.25	1.83
Formula 9	10.52 ± 0.31	15.59 ± 1.78	1.48
Formula 10	>20	>20	-
Formula 11	7.96 ± 0.58	10.12 ± 0.44	1.27
Formula 12	>20	>20	-
Formula 13	3.12 ± 0.25	4.9 ± 0.32	1.57
Formula 14	1.95 ±0.01	2.12 ± 0.05	1.08
Formula 15	11.93 ± 0.8	17.86±0.23	1.49
Formula 16	10.49 ± 0.5	>20	-
Formula 17	>20	>20	-
Formula 18	3.07 ± 0.64	4.39±2.22	1.42
Formula 19	>20	>20	-
Formula 20	>20	>20	-
Formula 21	>20	>20	-
Bortezomib	5.6 nM	201 nM	39.41

In addition, bortezomib-resistant myeloma cells RPMI-8226 and IM-9 were constructed in the same manner as in Example [6-1], and the viability inhibitory effect of wild-type human myeloma cells and bortezomib-resistant cells was compared and analyzed by IC ₅₀ value did Specifically, the results of calculating the ratio of IC ₅₀ of the bortezomib-resistant cell line/IC ₅₀ of the wild-type cell line are shown in Table 3 (RPMI-8226) and Table 4 (IM-9). In resistant myeloma cell lines in which the cell viability inhibitory activity of bortezomib is reduced by about 3 to 15 times, the compound having the chemical structure of Formula 13 maintained or exhibited better cell killing activity. It was confirmed that it exhibits Zomib resistance overcoming activity.

compound	Wild type RPMI-8226 cell viability IC50 (μM)	Bortezomib-resistant RPMI-8226 cell viability IC50 (μM)	Bortezomib-resistant IC50/wild-type IC50 ratio
Formula 13	1.37 ± 0.01	1.63 ± 0.06	1.19
Bortezomib	11.89 ± 0.27 (nM)	185.69 ± 6.33 (nM)	15.62

compound	Wild-type IM-9 cell viability IC50 (μM)	Bortezomib-resistant IM-9 cell viability IC50 (μM)	Bortezomib-resistant IC50/wild-type IC50 ratio
Formula 13	1.71 ± 0.02	1.49 ± 0.04	0.87
Bortezomib	11.58 ± 0.06 (nM)	42.23 ± 0.81 (nM)	3.65

[6-3] Effect of ubiquitin protein accumulation in bortezomib-resistant cancer cell lines

Representatively, among the 19 compounds prepared in Example 1, in a DMEM medium with a concentration of 5% fetal calf serum in which wild-type HCT116 cells and bortezomib-resistant cells constructed in Example [6-1] were cultured, The compounds were dissolved at concentrations of 2.5, 5, and 10 μM, respectively, and then incubated for 6 hours. Afterwards, Western blotting was performed in the same manner as in Example 3 to measure ubiquitinated proteins.

As a result, as shown in FIG. 9, ubiquitinated proteins were accumulated in a concentration-dependent manner not only in wild-type cells but also in bortezomib-resistant cancer cells by the compound having the chemical structure of Formula 13, indicating bortezomib resistance-overcoming activity. confirmed.

From this, it was found that the compounds of the present invention can be used for the treatment of bortezomib-resistant cancer.

In the above, preferred embodiments of the present invention have been described by way of example, but the scope of the present invention is not limited to the specific embodiments as described above, and those skilled in the art are within the scope described in the claims of the present invention. will be able to change accordingly.

Claims (10)

1. A pharmaceutical composition for preventing or treating cancer comprising a compound represented by Formula 1 or Formula 2 below as an active ingredient:

   [Formula 1]

   

   (In Formula 1,

   R ₁ is hydrogen or halogen;

   R ₂ is a heteroalkyl or heterocycloalkyl containing at least one selected from the group consisting of O and N;

   R ₃ is hydrogen, halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy or amine;

   Y ₁ , Y ₂ and Y ₃ are each independently selected from the group consisting of S and C)

   [Formula 2]

   

   (In Formula 2,

   R' ₁ is hydrogen or halogen;

   R′ ₂ is a heteroalkyl or heterocycloalkyl including at least one selected from the group consisting of O and N;

   R' ₃ and R' ₄ are each independently hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, or amine;

   Y' ₁ is carbon or nitrogen).
2. The method of claim 1,

   The compound represented by Formula 1 is any one compound selected from among compounds represented by Formulas 3 to 5, Formulas 7 to 9, and Formula 11. A pharmaceutical composition for preventing or treating cancer:

   [Formula 3]

   

   [Formula 4]

   

   [Formula 5]

   

   [Formula 7]

   

   [Formula 8]

   

   [Formula 9]

   

   [Formula 11]

   
3. The method of claim 1,

   A pharmaceutical composition for preventing or treating cancer, wherein the compound represented by Formula 2 is any one compound selected from compounds represented by Formulas 13 to 15 and Formula 18:

   [Formula 13]

   

   [Formula 14]

   

   [Formula 15]

   

   [Formula 18]

   
4. According to any one of claims 1 to 3,

   The compound of Formula 1 or Formula 2 is a pharmaceutical composition for preventing or treating cancer that inhibits the activity of 19S RP (regulatory particle).
5. According to any one of claims 1 to 3,

   The cancer is a pharmaceutical composition for preventing or treating cancer, which is a cancer that exhibits resistance to 20S CP (catalytic core particle) inhibitors.
6. The method of claim 5,

   Wherein the 20S CP inhibitor is bortezomib, ixazomib or carfilzomib, a pharmaceutical composition for preventing or treating cancer.
7. The method of claim 1,

   A pharmaceutical composition for preventing or treating cancer, further comprising at least one agent selected from the group consisting of lenalidomide, dexamethasone, and daratumumab.
8. The method of claim 1,

   The cancer is any one or more selected from the group consisting of lung cancer, myeloma, cervical cancer, breast cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, colon cancer and neuroma, a pharmaceutical composition for preventing or treating cancer.
9. The method of claim 1,

   The cancer is a pharmaceutical composition for the prevention or treatment of multiple myeloma cancer.
10. A health functional food composition for preventing or improving cancer comprising a compound represented by Formula 1 or Formula 2 as an active ingredient:

    [Formula 1]

    

    (In Formula 1,

    R ₁ is hydrogen or halogen;

    R ₂ is a heteroalkyl or heterocycloalkyl containing at least one selected from the group consisting of O and N;

    R ₃ is hydrogen, halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy or amine;

    Y ₁ , Y ₂ and Y ₃ are each independently selected from the group consisting of S and C)

    [Formula 2]

    

    (In Formula 2,

    R' ₁ is hydrogen or halogen;

    R′ ₂ is a heteroalkyl or heterocycloalkyl including at least one selected from the group consisting of O and N;

    R' ₃ and R' ₄ are each independently hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, or amine;

    Y' ₁ is carbon or nitrogen).

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