KR20230037853A - Composition for preventing or treating pancreatic cancer - Google Patents

Abstract

The present invention relates to a composition for preventing or treating pancreatic cancer containing the compound represented by chemical formula 1 as an active ingredient. In the present invention, the compound is a NUAK1 inhibitor, which blocks a PI3K signaling pathway to have an excellent effect of inhibiting the proliferation and metastasis of cancer cells. Therefore, it is possible to be usefully used for treating cancer diseases including pancreatic cancer.

Description

Composition for preventing or treating pancreatic cancer {Composition for preventing or treating pancreatic cancer}

The present invention relates to a composition for preventing or treating pancreatic cancer comprising the compound represented by Formula 1 as an active ingredient.

Pancreatic cancer has a very poor prognosis and is a representative malignant cancer with the 7th highest mortality rate among cancer patients. Until now, pancreatic cancer is difficult to diagnose early because the initial symptoms are vague, and it is characterized by easy metastasis to nearby organs or lymph nodes, so most patients are in stage 3 or 4 at the time of diagnosis. The treatment of pancreatic cancer is largely divided into three types: surgical resection, radiation therapy, and chemotherapy, and among them, surgical resection is the most effective treatment.

However, it is known that only patients who are detected early can undergo surgery, and even among patients who have undergone surgery, recovery is slow, making it difficult to treat with general chemotherapy or radiation therapy, and the incidence of metastasis and complications is high.

For patients diagnosed with stage 3-4, chemotherapy is the main treatment, and the most commonly used drug for treatment over the past 20 years is gemcitabine. Along with gemcitabine, combination therapy with various drugs is used in pancreatic cancer, but the effect is not only insignificant, but also causes resistance and does not affect the significant increase in survival rate of pancreatic cancer patients. Therefore, pancreatic cancer patients need a new treatment to replace it.

NUAK1 is a protein highly expressed in various cancer tissues, and is particularly related to migration and invasion of cancer cells. Recently, a lot of research on NUAK1 has been conducted, and accordingly, the development of NUAK1 inhibitors has also progressed. However, among the NUAK1 inhibitors developed so far, there are no FDA-approved drugs, and there are no drugs that show anticancer effects in pancreatic cancer.

Korean Patent Publication No. 10-2014-0006048 (published on January 15, 2014)

An object of the present invention is to provide a composition for preventing and treating pancreatic cancer comprising a NUAK1 inhibitor excellent in preventing and treating pancreatic cancer as an active ingredient.

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

In addition, the present invention provides a reagent composition for inhibiting PI3K signaling comprising the compound represented by Formula 1 as an active ingredient.

The composition for preventing or treating pancreatic cancer according to the present invention has an excellent effect of inhibiting the proliferation and metastasis of cancer cells by blocking a signal transduction pathway, and thus can be usefully used in the treatment of cancer diseases including pancreatic cancer.

1 is a graph showing the results of measuring cell viability through an MTT test of pancreatic cancer cells treated with the compound of Formula 1 of the present invention.
Figure 2 is an image showing the effect of inhibiting the expression of signaling pathway proteins in pancreatic cancer cells by treatment with the compound of Formula 1 of the present invention.
3 is a view showing the effect of inhibiting cell migration ability in pancreatic cancer cells by treatment with the compound of Formula 1 of the present invention.
4 is a view showing the effect of inhibiting the penetration ability of pancreatic cancer cells by treatment with the compound of Formula 1 of the present invention.
5 is a graph showing photographs and weights of tumors obtained through animal experiments using pancreatic cancer cell lines treated with the compound of Formula 1 of the present invention.
6 and 7 are results of immunohistochemical staining of tumors obtained through animal experiments using pancreatic cancer cell lines treated with the compound of Formula 1 of the present invention.
8 shows the results of immunohistochemical staining and TUNEL testing on tumors obtained through animal experiments using pancreatic cancer cell lines treated with the compound of Formula 1 of the present invention.

Hereinafter, in order to explain the present invention in more detail, preferred embodiments according to the present invention will be described in more detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms.

In order to meet the demand in the industry, the applicant of the present application developed a drug called KI-301670 that targets NUAK1 while researching a new anti-cancer drug with excellent therapeutic effects for pancreatic cancer, and administered the drug to maximize the therapeutic effect of pancreatic cancer. . It was confirmed that the NUAK1 inhibitor (KI-301670) suppressed the survival, proliferation and metastasis of cancer cells by inhibiting the interaction with the PI3K signaling protein and significantly increased the apoptosis effect.

Accordingly, the applicant of the present application intends to propose a technique capable of providing a NUAK1 inhibitor having excellent treatment effects for pancreatic cancer, unlike existing NUAK1 inhibitors, below.

The present invention provides a pharmaceutical composition for preventing or treating pancreatic cancer comprising a compound represented by Formula 1 below as an active ingredient.

[Formula 1]

The compound may be included at a concentration of 0.1 to 10 μM, 0.1 to 7 μM or 1 to 5 μM.

The compound is an inhibitor targeting NUAK1, and can inhibit the proliferation and metastasis of cancer cells by blocking the PI3K signaling pathway.

In addition, the compound may exhibit anticancer effects by inhibiting the proliferation and metastasis of cancer cells by reducing the expression of at least one of phospho-AKT, P70S6K and GSK3β.

When the composition of the present invention is a pharmaceutical composition, it may contain a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described active ingredients for administration. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods. . Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc., in addition to the active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It may be prepared by adding various excipients, for example, wetting agents, sweeteners, aromatics, and preservatives, in addition to liquids and liquid paraffin for oral use. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and tablets. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrosol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

A suitable dose of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the drug type, and the time, but can be appropriately selected by a person skilled in the art, and the daily dose of the composition is preferably 0.001 mg/kg to 50 mg/kg or 1 mg/kg to 50 mg/kg, and may be divided and administered once a day to several times as needed.

In addition, the present invention provides a reagent composition for inhibiting PI3K signaling comprising a compound represented by Formula 1 below as an active ingredient.

[Formula 1]

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Example 1> Preparation of the compound of Formula 1

1.1) 2-chloro-4-((S)-tetrahydrofuran-2-yl)methylamino-5-

Preparation of trifluoromethylpyrimidine

After putting 2,4-dichloro-5-trifluoromethylpyrimidine (0.3 g, 1.38 mmol) in a round bottom flask and dissolving it in tetrahydrofuran (20 ml), (S)-tetrahydrofurfurylamine (0.14 g , 0.1.38 mmol) and triethylamine (0.39 ml, 2.76 mmol) were added and stirred at 0 °C for 2 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure, water was added to the reaction mixture, extracted with dichloromethane, dried over anhydrous magnesium sulfate, the solvent was removed by distillation under reduced pressure, and column chromatography (ethyl acetate: hexane, 1: 3) to obtain the compound (0.171 g, 44%); 1H NMR (400 MHz, CDCl3) δ 1.56-1.65 (m, 1H), 1.91-1.98 (m, 2H), 2.03-2.11 (m, 1H), 3.48-3.54 (m, 1H), 3.77-3.93 (m , 3H), 4.09-4.15 (m, 1H), 6.02 (s, 1H), 8.26 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 25.80, 28.64, 44.75, 68.24, 76.61, 105.89 (q, J = 32.51 Hz), 123.55 (q, J = 271.38 Hz), 155.02 (q, J = 5.14 Hz), 159.19 , 163.56.

1.2) 4-((S)-tetrahydrofuran-2-yl)methylamino-5-trifluoro

Preparation of Romethyl-2-[4-{2-(pyrrolidin-1-yl)ethoxy}phenylamino]pyrimidine

In a round bottom flask, 2-chloro-4-((S)-tetrahydrofuran-2-yl)methylamino-5-trifluoromethylpyrimidine (0.1 g, 0.355 mmol) and 4-(2-(pyrroly) After dissolving din-1-yl)ethoxy)aniline (0.073 g, 0.355 mmol) in 2-methoxyethanol (9 ml), hydrochloric acid (0.04 ml of 4M dioxane solution) was added and stirred at 110 °C for 24 hours. . After the reaction was completed, the solvent was removed by distillation under reduced pressure, a saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, extracted with dichloromethane, dried over anhydrous magnesium sulfate, the solvent was removed by distillation under reduced pressure, and column chromatography (methanol: dichloromethane) was performed. Phosphorus, 2:3) gave the compound (0.105 g, 66%); 1H NMR (400 MHz, CDCl3) δ 1.55-1.63 (m, 1H), 1.79-1.82 (m, 4H), 1.86-1.92 (m, 2H), 1.93-2.02 (m, 1H), 2.61-2.64 (m , 4H), 2.90 (t, J = 5.99 Hz, 2H), 3.48-3.54 (m, 1H), 3.69-3.75 (m, 1H), 3.75-3.80 (m, 1H), 3.85-3.91 (m, 1H) ), 4.10 (t, J = 5.99 Hz, 3H), 5.58 (s, 1H), 6.89 (d, J = 9.0 Hz, 2H), 7.43 (d, J = 8.99 Hz, 2H), 7.89 (s, 1H) ), 8.12 (s, 1H);13C NMR (100 MHz, CDCl3) δ 23.47, 25.91, 28.69, 44.41, 54.69, 55.13, 67.37, 68.29, 77.21, 114.72, 122.22, 125.03 Hz (q, 9.4 Hz) 132.35, 154.71 (q, J = 4.95 Hz), 155.01, 159.05, 161.21.

In the examples of the present invention, the compound of Formula 1 (KI-301670) was used to measure anticancer activity, and was dissolved in DMSO during experiments, and dissolved in DMSO: PEG400: DW = 1: 4: 5 ratio during animal experiments. used

<Experimental Example 1> Measuring cell viability through MTT experiment of pancreatic cancer cells

In order to examine the viability of pancreatic cancer cells treated with the compound of Formula 1 of the present invention, cell viability was evaluated for two pancreatic cancer cell lines, MIA PaCa-2 and AsPC-1 cell lines.

1. Preparation of pancreatic cancer cell lines

MIA PaCa-2 and AsPC-1 cell lines, which are pancreatic cancer cell lines, were purchased from the American Type Culture Collection (ATCC Manassas, VA) and cultured in DMEM and RPMI medium containing 10% Fetal Bovine SerμM (FBS) and 1% Antibiotics with 5% CO2 and It is grown in an incubator at 37°C and humidity is maintained.

2. Measurement of pancreatic cancer cell viability

In order to examine the viability of pancreatic cancer cells according to the treatment with the compound of Formula 1 of the present invention, the cell viability of the pancreatic cancer cells treated with the compound of Formula 1 prepared in Example 1 was measured by a conventionally known MTT test method.

Specifically, the two pancreatic cancer cell lines prepared in 1 above, MIA PaCa-2 and AsPC-1 cell lines, were treated with the compound of Formula 1 at a concentration of 0-10 μM for 48 hours and then treated for 48 hours. Thereafter, MTT assays were performed for each reaction culture. In order to compare the evaluation results of pancreatic cancer cell viability according to the treatment with the compound of Formula 1, the numerical values of the measured values calculated through the MTT test method were analyzed, and the results are shown in FIG. 1 .

As shown in FIG. 1, the experimental group treated with the compound of Formula 1 of the present invention significantly inhibited the cell viability of two pancreatic cancer cell lines, MIA PaCa-2 and AsPC-1 cell lines.

Therefore, since treatment with the compound of Formula 1 of the present invention has an excellent effect of inhibiting the viability of pancreatic cancer cells, it can be seen that the composition according to the present invention has an excellent effect of inhibiting the proliferation of pancreatic cancer cells.

<Experimental Example 2. Inhibiting the Expression of Signal Transduction Pathway Proteins in Pancreatic Cancer Cells>

In order to examine the effect of blocking the PI3K signaling pathway by treatment with the compound of Formula 1 of the present invention, Western blotting was performed for proteins in the signaling pathway, ie, phospho-AKT, P70S6K, and GSK3β.

As shown in Figure 2, it was confirmed that the group treated with the compound of Formula 1 of the present invention significantly reduced the levels of phospho-AKT, P70S6K, and GSK3β proteins.

Therefore, since treatment with the compound of Formula 1 of the present invention blocks the PI3K signaling pathway involved in proliferation and metastasis of cancer cells, it can be seen that the composition according to the present invention has an excellent effect in preventing or treating cancer.

<Experimental Example 3. Inhibition of cell migration ability in pancreatic cancer cells>

In order to examine the cell migration ability by treatment with the compound of Formula 1 of the present invention, migration assay was performed.

Specifically, when the pancreatic cancer cell lines MIA PaCa-2 and AsPC-1 cells prepared in Experimental Example 1 grew to about 90% on the plate, they were treated with 0-2 μM compound of Formula 1 prepared in Example 1, and then migrated. The assay was performed to confirm the ability of the cells to migrate. The results are shown in Figure 3.

As shown in Figure 3, unlike the experimental group treated with the compound of Formula 1 of the present invention, cell migration ability was significantly inhibited, it was confirmed that the wounds of the control cells were healed up to 100% within 24 hours.

Therefore, since treatment with the compound of Formula 1 of the present invention inhibits cell migration ability in pancreatic cancer cells, it can be seen that the composition according to the present invention has an excellent effect of inhibiting metastasis of pancreatic cancer cells.

<Experimental Example 4. Effect of Inhibiting Penetration Ability of Pancreatic Cancer Cells>

In order to examine the cell penetration ability by the treatment of the compound of Formula 1 of the present invention, invasion assay was performed.

Specifically, MIA PaCa-2 and AsPC-1 cells, which are pancreatic cancer cell lines prepared in Experimental Example 1, were treated with 0-2 μM compound of Formula 1 prepared in Example 1 for 24 hours, and then invasion assay was performed. . These results are shown in FIG. 4 .

As shown in Figure 4, the cell penetration ability of the experimental group treated with the compound of formula 1 of the present invention was significantly inhibited compared to the control group. Specifically, it was confirmed that as the concentration of the compound of Formula 1 increased, the number of pancreatic cancer cells significantly decreased.

Therefore, since treatment with the compound of Formula 1 of the present invention inhibits cell penetration ability in pancreatic cancer cells, it can be seen that the composition according to the present invention has an excellent effect of inhibiting metastasis of pancreatic cancer cells.

<Experimental Example 5. Cell cycle arrest effect in pancreatic cancer cells>

In order to examine the cell cycle arrest ability by treatment with the compound of Formula 1 of the present invention, a cell cycle assay was performed.

Specifically, MIA PaCa-2 and AsPC-1 cells, which are pancreatic cancer cell lines prepared in Experimental Example 1-1, were treated with 0-2 μM compound of Formula 1 prepared in Example 1 for 24 hours, followed by cell cycle analysis was performed. These results are shown in FIG. 5 .

As shown in Figure 5, when the compound of Formula 1 of the present invention was treated, the cell cycle arrest ability significantly increased, and it was confirmed that the ratio of G0-G1 cells increased as the concentration of the compound of Formula 1 increased. .

Therefore, since treatment with the compound of Formula 1 of the present invention increases the ability of pancreatic cancer cells to arrest the cell cycle, it can be seen that the composition according to the present invention has an excellent effect of stopping the pancreatic cancer cell cycle.

<Experimental Example 6. Tumor growth inhibitory effect>

In order to confirm whether the tumor suppression effect by treatment with the compound of Formula 1 of the present invention is effective in vivo, animal experiments were performed as follows.

Specifically, in order to confirm the effect of inhibiting tumor growth when treated with the compound of Formula 1, the tumor size was confirmed in vivo. Specifically, the pancreatic cancer cell line MIA PaCa-2 was inoculated into 5-week-old male BALB/c nude mice at a cell count of 5×10 6 in 200 µL of PBS. When the size of the tumor reached 50 mm 3 or more, 7 rats were divided into 3 experimental groups, and the compound of Formula 1 (10, 30 mg/kg) was intraperitoneally injected every day for 21 days. In addition, the body weight was measured once every 3 days, and the size and weight of the tumor were measured by sacrifice on the last day, and the results are shown in FIG. 6 .

As shown in Figure 6, it was confirmed through animal experiments that the treatment with the compound of Formula 1 effectively inhibited tumor growth compared to the control group.

Therefore, it can be seen that the composition containing the compound of Formula 1 of the present invention has an excellent effect of effectively inhibiting tumor growth even in animal models.

<Experimental Example 7. Tumor inhibitory effect>

It is a diagram showing cancer metastasized to other organs obtained through animal experiments using pancreatic cancer cell lines treated with the compound of Formula 1 of the present invention through immunohistochemical staining.

Specifically, in order to confirm whether the treatment with the compound of Formula 1 has a tumor suppression effect, a paraffin block was made and hematoxylin-eosin staining was performed, and the results are shown in FIG. 7 .

As shown in Figure 7, it was confirmed that the treatment with the compound of Formula 1 of the present invention effectively inhibited tumor growth compared to the control group.

Therefore, it can be seen that the composition according to the present invention has an excellent effect of effectively inhibiting tumors.

<Experimental Example 8. Selective inhibitory effect of genes>

In order to confirm the selective inhibitory effect of the gene by treatment with the compound of Formula 1 of the present invention, immunohistochemical staining and TUNEL experiments were performed as follows.

For immunohistochemical staining, phospho-NUAK1, AKT, and Ki67 antibodies were diluted at a ratio of 1:30 and treated for one day, followed by treatment with a biotinylated secondary antibody diluted at a ratio of 1:60 for 1 hour and ABC (Avidin- Immunohistochemical staining was performed using a Biotin complex kit.

In order to reduce the non-specific reaction similarly to the TUNEL test method, TUNEL staining was performed by incubating the TdT enzyme at 37° C. for 3 hours and then reacting with the secondary antibody for 1 hour at room temperature. This result is shown in FIG. 8 .

As shown in Figure 8, in the immunohistochemical analysis through animal experiments, it was confirmed that the case of treatment with the compound of Formula 1 effectively inhibited the downstream signaling pathway of PI3K compared to the control group, and in the TUNEL analysis, the compound of Formula 1 It was confirmed that the treatment effectively induced cell death compared to the control group.

Therefore, it can be seen that the composition according to the present invention has an excellent effect of effectively inhibiting a specific signal transduction system and inducing cell death in immunohistochemical analysis and TUNEL analysis.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (5)

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

According to claim 1,
The concentration of the compound is a pharmaceutical composition for preventing or treating pancreatic cancer, characterized in that 0.1 to 10 μM. According to claim 1,
The compound is an inhibitor targeting NUAK1, and a pharmaceutical composition for preventing or treating pancreatic cancer, characterized in that it inhibits the proliferation and metastasis of cancer cells by blocking the PI3K signaling pathway. According to claim 1,
The compound is a pharmaceutical composition for preventing or treating pancreatic cancer, characterized in that the suppression of proliferation and metastasis of cancer cells by reducing the expression of at least one of phospho-AKT, P70S6K and GSK3β. A reagent composition for inhibiting PI3K signaling comprising a compound represented by Formula 1 below as an active ingredient:
[Formula 1]

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