KR20160028057A - Pyrimidine derivatives having anti-cancer effect, combination therapeutic effect with radiation, and anti-diabetic effect, and PPAR activity, and medical use thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition containing as an active ingredient a pyrimidine derivative having an anticancer effect, a radiotherapeutic effect and a diabetic therapeutic effect, wherein the pyrimidine derivative is a PPAR-γ ligand and has anticancer activity against various cancers In addition, since it has selective cytotoxicity against cancer cells, it can act as an anticancer agent that can minimize side effects. In addition, it can act as a radiotherapy sensitizer in radiation therapy for cancer, and can also be useful as a therapeutic agent for diabetes.

Description

(Pyrimidine derivatives having an anti-cancer effect, a combination of a therapeutic effect with radiation and an anti-diabetic effect, and a PPAR activity, and a medical use thereof)

The present invention relates to a purine derivative having an anticancer effect, a radiotherapeutic effect and a therapeutic effect on diabetes, and a medical use thereof.

Recently, the incidence of cancer is rapidly increasing due to rapid development of industry, change of global ecosystem and dietary life, but it is still known as a refractory disease because the mechanism of cancer development is unclear. The currently used anticancer drugs can be broadly divided into biological agents such as enzymes and vaccines, pure synthetic drugs, and medicines derived from natural products. Anticancer drugs have various pharmacological actions depending on the type of cancer, and toxic side effects vary widely And it is a problem in cancer treatment because it appears. In addition, the anticancer drug effectively inhibits the growth of cancer cells, but it may sometimes show toxicity to normal cells. Therefore, many scholars have made efforts to develop anticancer drugs that are more effective and can maximize toxicity to normal cells.

Lung cancer is the second most common cause of gastric cancer, and the mortality rate from lung cancer among cancer deaths is the highest since 2000 due to poor prognosis. Lung cancer is classified into small cell lung cancer and non - small cell lung cancer histologically. Chemotherapy and radiotherapy are recommended for small cell lung cancer. Curative resection for non - small cell lung cancer is the best method. Lung cancer has difficulty in local control of tumor and microscopic metastatic cancer cells that are difficult to detect by imaging. Therefore, the 5-year survival rate of less than 10% in radiotherapy alone is not good because it may cause recurrence in remote organs after radiation-limited radiation therapy. Therefore, in order to prevent recurrence in these remote organs, chemotherapy is combined with radiation therapy in various ways, and it is found that this combination therapy is superior to radiation alone therapy.

On the other hand, peroxisome proliferator activated receptors (PPARs) are nuclear receptors belonging to the steroid / thyroid hormone / retinoid receptor superfamily, which are transcription factors whose activity is regulated by several kinds of ligands. It is also a key regulator of glucose and lipid metabolism, and has been shown to regulate cell division, differentiation, and cell death in various tissues. Activation of PPAR is known to have various anticancer activities.

The therapeutic agent for thiazolidinediones diabetes, including troglitazone (TGZ), ciglitazone, rosiglitazone or pioglitazone, is a synthetic PPAR gamma agonist and TGZ is also used for the treatment of human colon cancer, (Nat Med. 4 (9): 1046-52, 1998; Proc Natl Acad Sci U S A. 95 (15). In addition, it has been shown to have a cytotoxic effect on various cancers such as breast cancer, liver cancer, lung cancer, kidney cancer and prostate cancer ): 8806-11,1998).

It is known that PPAR [beta] / [delta] activity is able to attenuate lung cancer and L165041, a high-affinity synthetic PPAR [beta] / delta ligand, inhibits cellular proliferation of human lung cancer and eliminates PPAR [ It is known to aggravate lung cancer in transgenic mice. In addition, PPARγ expression was decreased in patients with poor prognosis (J Clin Oncol. 25 (12): 1476-81, 2007), and the activity of PPAR-γ by endogenous agonists and synthetic agonists decreased in lung cancer cells (Biochem Biophys Res Commun. 270 (2): 400-5, 2000). Treatment of non-small cell lung cancer with PPAR-γ active substance has been reported to induce apoptosis and differentiation (Cancer Res. 60 (4): 1129-38, 2000). It has also been reported that ciglitazone inhibits tumors derived from A-549 in nude mice (J Huazhong Univ Sci Technol Med Sci. 26 (1): 36-39, 2006).

Patients receiving thiazolidinediones, a PPAR-γ agonist for the treatment of diabetes, were significantly less likely to develop lung cancer (J Clin Oncol. 25 (12): 1476-81, 2007) (Clin Cancer Res. 14 (20): 6478-86, 2008; J Investig Med. 56 (2): 528-33, 2008).

In addition to PPAR-γ-dependent pathways, PPAR-γ ligands have been reported to have anti-cancer activity through pathways that are independent of PPAR-γ, and PPAR-γ ligands are known to have independent pathways and association with lung cancer (Prostagladins & other Lipid Mediators 94: 104-111, 2011).

It is an object of the present invention to provide a pharmaceutical composition for treating cancer comprising as an active ingredient a pyrimidine derivative which can act as a PPAR-gamma ligand and has an anticancer effect, a radiotherapeutic effect and a diabetic therapeutic effect.

It is still another object of the present invention to provide a pharmaceutical composition for a radiation-sensitive sensitizer for a cancer containing the pyrimidine derivative as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of diabetes containing the pyrimidine derivative as an active ingredient.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for treating cancer comprising as an active ingredient a pyrimidine derivative represented by the following formula (1), a pharmaceutically acceptable salt thereof or a solvate thereof.

[Chemical Formula 1]

The present invention also provides a pharmaceutical composition for radiotherapy sensitizing cancer, which comprises the pyrimidine derivative represented by the above formula (1), a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for the treatment of diabetes comprising the pyrimidine derivative represented by the above formula (1), a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient.

The pyrimidine derivatives according to the present invention are PPAR-gamma ligands and have anticancer activity against various cancers as well as selective cytotoxicity against cancer cells, so they can act as anticancer agents that can minimize side effects. In addition, it can act as a radiotherapy sensitizer in radiation therapy for cancer, and can also be useful as a therapeutic agent for diabetes.

FIG. 1 is a photograph showing the results of inducing differentiation into adipocytes for 10 hours at a concentration of 10 μM of the pyrimidine derivative CB13 according to the present invention and then determining the degree of induction of differentiation by Oil-Red-O staining method .
FIG. 2 shows the result of measuring the absorbance at 510 nm after extracting Oil-Red-O from isopropanol in the stained 3T3-L1 cells of FIG.
Figure 3 shows that the pyrimidine derivatives CB13 0, 5, 10, 20, 30 and 40 μM according to the present invention were added to arsenic lung cancer cell lines A549 and H460 and MRC5 normal human lung cells and cultured in WST-1 Cell survival rate.
FIG. 4 shows the result of measurement of sub-G1 phase cells by flow cytometry after 30 μM of the pyrimidine derivative CB13 according to the present invention was cultured in H460 and A549 cell lines for 48 hours.
FIG. 5 is a graph showing the results of analysis of apoptosis by CB13 by flow cytometry after annexin V-FIPC staining after incubating 30 μM of the pyrimidine derivative CB13 according to the present invention in H460 and A549 cell lines for 48 hours.
FIG. 6 shows the result of SDS-PAGE separation and Western blotting of proteins extracted from cells cultured by treating 30 μM of the pyrimidine derivative CB13 according to the present invention for 24 hrs on A549 and H460 cells.
FIG. 7 is a graph showing the cell survival rates of lung cancer cell lines after treatment with lung cancer cell lines H460 and A549 with CB13 10, 20 and 30 μM alone or with 1 and 2 Gy radiation, by WST-1 method to be.
FIG. 8 is a photograph showing the result of separating 3T3-L1 cells by treatment with CB13 at a concentration of 10 .mu.M and then extracting RNA by agarose gel electrophoresis and staining with Et-Br. FIG.
FIG. 9 shows the results of analysis of glucose migration from adipocytes into cells according to CB13 treatment.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for treating cancer comprising as an active ingredient a pyrimidine derivative represented by the following general formula (1), or a pharmaceutically acceptable salt or solvate thereof:

[Chemical Formula 1]

In the above formula, R may be any one selected from C1 to C4 alkyl, C1 to C4 alkoxy, halogen or hydroxy.

The pyrimidine derivative may be C1 to C4 alkyl.

The pyrimidine derivative may be 1-benzyl-5- (4-methylphenyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) -dione.

The cancer may specifically be selected from the group consisting of bladder cancer, stomach cancer, colon cancer, esophageal cancer, pancreatic cancer, lung cancer, non-small cell lung cancer, colon cancer, bone cancer, skin cancer, skin or intraocular melanoma, uterine cancer, rectum cancer, Renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brain stem, lung cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, liver cancer, liver cancer, Glioma, pituitary adenoma, and the like. More specifically, the cancer is lung cancer, breast cancer, colorectal cancer, or white blood cell cancer. More specifically, the cancer is non-small cell lung cancer.

The present invention also provides a pharmaceutical composition for radiation-sensitive sensitization of cancer comprising, as an active ingredient, a pyrimidine derivative represented by the following formula (1), a pharmaceutically acceptable salt thereof or a solvate thereof:

[Chemical Formula 1]

In the above formula, R may be any one selected from C1 to C4 alkyl, C1 to C4 alkoxy, halogen or hydroxy.

The pyrimidine derivative may be C1 to C4 alkyl.

The pyrimidine derivative may be 1-benzyl-5- (4-methylphenyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) -dione.

The cancer may specifically be selected from the group consisting of bladder cancer, stomach cancer, colon cancer, esophageal cancer, pancreatic cancer, lung cancer, non-small cell lung cancer, colon cancer, bone cancer, skin cancer, skin or intraocular melanoma, uterine cancer, rectum cancer, Renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brain stem, lung cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, liver cancer, liver cancer, Glioma, pituitary adenoma, and the like. More specifically, the cancer is lung cancer, breast cancer, colorectal cancer, or white blood cell cancer. More specifically, the cancer is non-small cell lung cancer.

The present invention also provides a pharmaceutical composition for the treatment of diabetes comprising as an active ingredient a pyrimidine derivative represented by the following formula (1), a pharmaceutically acceptable salt thereof or a solvate thereof:

[Chemical Formula 1]

In the above formula, R may be any one selected from C1 to C4 alkyl, C1 to C4 alkoxy, halogen or hydroxy.

The pyrimidine derivative may be C1 to C4 alkyl.

The pyrimidine derivative may be 1-benzyl-5- (4-methylphenyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) -dione.

In addition, the pyrimidine derivatives have an effective therapeutic effect on insulin-resistant diabetes such as diabetes mellitus, visceral obesity, nonalcoholic fatty disease, arthritis, and type 2 diabetes.

The pharmaceutical compositions may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions.

Examples of the carrier, excipient or diluent which can be used in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The amount of the pyrimidine derivative which is an active ingredient of the pharmaceutical composition according to the present invention may vary depending on the age, sex, weight and disease of the patient, but it is preferably 0.001 to 1000 mg / kg, preferably 0.01 to 100 mg / kg per day It may be administered once or several times.

Further, the dosage of the pyrimidine derivative according to the present invention may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intratracheal, intrauterine or intracerebroventricular injections.

The pharmaceutical composition may contain the pyrimidine derivative in an amount of about 0.0001 to 10% by weight, preferably 0.001 to 1% by weight, based on the total weight of the total pharmaceutical composition.

In addition, the pyrimidine derivative according to the present invention may be utilized as a health food in addition to a pharmaceutical composition. The health food may be provided in the form of a powder, a granule, a tablet, a capsule, a syrup or a beverage. The health food is used together with other food or food additives other than the purine derivative according to the present invention, Can be appropriately used. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.

The effective dose of the compound contained in the health food may be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range for the purpose of health and hygiene or long-term intake for the purpose of health control, Since the active ingredient has no problem in terms of safety, it can be used in an amount exceeding the above range.

There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.

Hereinafter, in order to facilitate understanding of the present invention, experimental examples will be described in detail. It should be understood, however, that the following examples are illustrative only and are not intended to limit the scope of the present invention. Experimental examples of the present invention are provided to enable those skilled in the art to more fully understand the present invention.

The following reference example is provided to provide a reference example commonly applied to each experimental example according to the present invention.

< Reference Example  1> Chemicals

Propidium iodide (PI); Cyclosporin A (CsA); Nacetylcysteine (NAC) with antioxidants; Quercetin and 4, 5-dihydroxy-1, 3-benzenedisulfonic acid (tiron) were purchased from Sigma-Aldrich (St. Louis, Mo.); Ciglitazone; A selective PPAR-gamma antagonist and GW9662 were purchased from Cayman (Ann Arbor, MI).

CB13 (1-benzyl-5- (4-methylphenyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) -dione), a candidate PPAR agonist, was purchased from Chembridge chemical .

< Reference Example  2> Cell culture and chemical treatment

Human non-small cell lung cancer cell lines H406 and A549 purchased from American type culture collection (ATCC, Manassas, Va.) Were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin (Gibco BRL Life Technologies , Gaithersburg, MD, USA) in Dulbecco's modified Eagle's medium (DMEM) medium at 37 ° C and 5% CO ₂ .

Ciglitazone 10 mM and 50 mM of CB13 were each dissolved in dimethylsulfoxide (DMSO) to prepare a stock and added to the culture medium at various concentrations of Siglitazone 10 μM or CB13. In addition, the PPAR-γ ligands were treated with cells in a culture medium containing 0.2% fetal bovine serum (FBS).

< Experimental Example  1> PPAR -γ Ligand  Verification analysis

Whether CB13 has PPAR-γ ligand activity To test for its ability to induce adipocyte differentiation, 3T3-L1 cells were treated with PPAR-γ antagonist GW9662, siglitazone and a novel PPAR-γ agonist, respectively, .

3T3-L1 cells were plated in Dulbecco's modified Eagle's medium (DMEM) medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin per well in a 6-well plate ⁴ cells and cultured for 48 hours until the cells were grown to 80%. (Sigma-Aldrich), 5 μg / mL insulin (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX, Sigma-Aldrich), 10% fetal calf serum (FBS), 1 μM dexamethasone Aldrich) and incubated for 48 hours.

Then, 3T3-L1 cells were treated with 10 μM of CB13 and 10 μM of siglitazone as a positive control, respectively, and 10 μM of GW9662 and 10 μM of CB13 were treated together to induce differentiation. After induction of differentiation, cells were replaced with DMEM medium supplemented with 10% fetal bovine serum (FBS) and 5 μg / mL insulin every 48 hours.

After 3 days of differentiation of 3T3-L1 cells, each cell was stained with Oil-Red-O staining method (Lin et al., 2007; Ramirez-Zacarias et al., 1992) The cells were analyzed with a microscope (Olympus, Tokyo, Japan) and dissolved in oil-red-O stained with isopropyl alcohol. Absorbance was measured at 510 nm.

As shown in Fig. 1, differentiation was confirmed in CB13 and 3T3-L1 cells treated with siglitazone, whereas inhibition of differentiation was observed in 3T3-L1 cells treated with CB13, a PPAR-γ antagonist, GW9662. Also, referring to the results of the absorbance analysis of FIG. 2, it was confirmed that CB13 exhibited excellent effects in inducing adipocyte differentiation, which is a characteristic of the PPAR-γ ligand. Therefore, it was confirmed that CB13 was a PPAR-gamma ligand.

< Experimental Example  2> Cancer cell growth inhibition efficacy analysis

In order to measure the inhibitory effect of the compound CB13, which was proved by the PPAR-γ ligand, in the human arsenic lung cancer cell lines A549 and H460 and MRC5, which is a normal human lung cell, the inhibitory effect of CB13 on WST-1 (4- [3 - (4-iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolio] -1,3-benzenedisulfonate) analysis.

For WST-1 analysis, each cell was subcultured in a 96-well plate at 5 × 10 ³ / well and incubated overnight. Ciglitazone 10 μM and CB13 0. 5 10. 10. 20, 30 and 40 μM Respectively, and cultured for 24 hours, 48 hours, and 72 hours. After washing each cell, 10 μl of WST-1 solution was added to each well, incubated at 37 ° C. for 1 hour, and then measured at 450 nm to confirm cell viability by CB13 treatment.

As a result, as shown in Fig. 3, the inhibitory effect on the growth of human arsenic lung cancer cell line was excellent in the concentration and time-dependent of PPAR-γ ligand CB13, and the cell growth inhibitory effect was not observed in normal lung cells other than cancer cells. Therefore, it was confirmed that CB13 selectively inhibits cell growth only in cancer cell lines.

< Experimental Example  3> of the pyrimidine derivative Apoptosis  Association analysis

The apoptosis inducing effect of PPAR-y ligand was confirmed by flow cytometry and western blot analysis.

One. sub - G1 group Cell number  On change CB13 The impact of

Sub-G ₁ in order to determine the apoptotic cell death induced by CB13 Cells were counted. First, H460 and A549 cells were inoculated at 3.0 × 10 ⁵ cells per 60 mm plate, cultured for 24 hours, and treated with 30 μM of CB13 for 48 hours. Each cell was then harvested and fixed with cold 70% ethanol overnight at -20 ° C, washed twice with Dulbecco's PBS (DPBS) and then incubated with PI (50 μg / mL; Sigma-Aldrich) and RNase A (Sigma-Aldrich) at room temperature for 30 minutes.

The sub-G ₁ of the stained cells Cell numbers were measured using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and the data were analyzed using CellQuest Pro software (Becton Dickinson).

As a result, sub-G ₁ H460 and A549 cells in the experimental group treated with PPAR-γ ligand for 48 hours were increased by 2 to 4 times as compared with the control group not treated with CB13, as shown in FIG.

From the above results, it can be suggested that CB13 is apoptosis through the apoptosis of H460 and A549 cells.

2. CB13  By treatment Arsenic  Cell death confirmation

An annexin V assay was performed to confirm apoptosis for accurate confirmation of the above results. First, H460 and A549 cells were inoculated at 3.0 × 10 ⁵ cells per 60 mm plate, cultured for 24 hours, treated with 30 μM of CB13, cultured for 40 hours, recovered and harvested using Annexin V-FITC Apoptosis Detection kit (BioVision , CA, USA), and the number of apoptotic cells in the CB13-treated H460 and A549 experimental groups was determined by FACSCalibur system after 5 minutes of dark staining with annexin V / PI as described by the manufacturer.

As a result, as shown in FIG. 5, increase in annexin V-positive cells was confirmed in the cells treated with CB13, which is a PPAR-γ ligand, and CB13 was found to induce apoptotic cell death through apoptosis mechanism.

3. Arsenic  Identification of apoptosis related proteins

Through these experiments, it was found that the apoptosis mechanism of the pyrimidine derivative, CB13, is achieved through apoptosis. Therefore, in order to clarify the mechanism of the apoptosis-related protein change and the apoptosis reaction in the cell during apoptosis, .

Human non-apoptotic lung cancer cells, H460 and A549, were treated with 30 μM of CB13 for 48 hours, then trypsinized, and the cells were recovered and resuspended in RIPA buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM PMSF and 1 mM DTT) in a buffer solution containing 50 mM NaCl, 1 mM EGTA, 1% Triton X-100, 50 mM NaF, 10 mM Na3VO4, 10 g / mL aprotinin, 10 g / mL leupeptin, (Bio-Rad Laboratories, Hercules, Calif.) After the cells were lysed on ice for 20 min. Protein samples for which the concentration was measured were dispensed at 20 μg / lane and electrophoresed on SDS-PAGE. After the SDS-PAGE protein was transferred to a polyvinylidene fluoride (PVDF) membrane (NEN; PerkinElmer, Wellesley, Mass.), The membrane was blocked with blocking buffer (1 × TBS, 0.1% Tween- The cells were incubated overnight at 4 ° C, reacted with secondary antibody (HRP) conjugated with horseradish peroxidase (HRP) for 1 hour, and incubated with ECL or ECL- plus (Amersham Biosciences) system and sensitized to film.

As a result, as shown in FIG. 6, caspase-3, caspase-7 and caspase-PARP, which were cleaved in CB13-treated cells, were increased compared with the control group without treatment.

From the above results, it was confirmed that the antiproliferative effect of the PPAR-γ agonist, CB13, was induced by inducing apoptosis.

< Experimental Example  4> Measurement of radiation sensitivity

H460 and A549 cells, which are lung cancer cell lines, were suspended in 500-mm cell culture plates, and then cultured for 24 hours. After confirming that the cells stably adhered to the floor, they were treated with 0, 10, 20 and 30 μM CB13 and cultured for 24 hours. After 24 hours, γ-irradiation (3.2 Gy / min, Gammacell 3000; Atomic Energy of Canada, Ltd., Mississauga, ON, Canada) was examined at 1 and 2 Gy intensity. After 48 hours of incubation, the cell viability was confirmed by the WST-1 method.

As a result, as shown in FIG. 7, the efficacy of CB13 treated with radiation irradiation as a radiation sensitizer was confirmed by the cell survival rate of lung cancer cell lines H460 and A549. As a result, when the maximum concentration of CB13 30 μM was treated alone, H460 cells The survival rate of H560 cells was 41% and the survival rate of A549 cells was 34% when 30μM CB13 was treated with irradiation. From the above results, it was confirmed that CB13 has an effect as a very high radiation sensitizer.

< Experimental Example  5> Identification of adipocyte differentiation mechanism

In the previous experiment, it was found that the compound of CB13 induces adipocyte differentiation as a PPAR-γ ligand. Thus, RT-PCR experiment was carried out in order to clarify the change of the gene involved in differentiation induction in the adipocyte differentiation process .

That is, 3T3-L1 cells were treated with 10 μM of CB13 for 3 hours to induce differentiation, and Trizol ^™ was treated to extract RNA. CDNA was prepared from reverse transcriptase from the obtained RNA, and various primers related to adipocyte differentiation were prepared based on the synthesized cDNA to perform PCR. The expression levels of PPAR-γ, C / EBPα, and aP2, which are adipocyte differentiation markers, and adiponectin, which is a gene related to GLUT4 and anti-diabetic activity, After isolating the genes using agarose gel electrophoresis, the expression level of each gene was stained with Et-Br and confirmed by UV.

As a result, the expression of PPAR-γ, C / EBPα, and aP2, which are known as differentiation markers, was increased as shown in FIG. 8, and the expression of GLUT4, which transports glucose into cells through insulin signal transduction, Respectively. In addition, the expression of adiponectin, an adipocine secreted from adipocytes, was significantly increased.

Adiponectin is significantly reduced in individuals with visceral obesity and insulin resistance (nonalcoholic fatty disease, arthritis, type 2 diabetes). In other words, the insulin resistance is inversely related to the blood level of adiponectin. Therefore, it was confirmed that the CB13 compound has an improvement effect on type 2 diabetes because the CB13 compound increases the expression of adiponectin having such a characteristic.

< Experimental Example  6> in adipocytes Intracellular  Glucose transport analysis

3T3-L1 cells were plated in 96-well plates and treated with 10 μM CB13 over time to differentiate. As a control group, an insulin-treated group and an insulin-treated group were prepared, and the degree of accumulation of 2-deoxyglucose in the cells was measured to analyze glucose transport into the cells.

As a result, as shown in FIG. 9, when CB13 was treated at a concentration of 10 .mu.M, no significant difference was observed in the absence of insulin. However, CB13 actively migrated glucose into the cells under the conditions of insulin treatment, compared with the control group. These results confirm that CB13 increases insulin sensitivity by increasing insulin-induced glucose transport.

< Experimental Example  7> Insulin resistance improvement effect

3T3-L1 adipocytes were treated with dexamethasone for 48 hours to produce an insulin resistance model. Then, CB13 was treated for 24 hours to treat Trizol ^™ , total RNA was extracted, and RT-PCR was performed using primers of PPAR-γ, C / EBPα, GLUT4, adiponectin and GAPDH to analyze the expression level of each gene Respectively.

As a result, it was confirmed that the expression level of reduced PPPAR-γ, C / EBPα, GLUT4 and adiponectin genes in the dexamethasone-induced insulin resistance model was restored when treated with 10 μM CB11. This suggests that the CB13 compound can improve insulin resistance and regain insulin sensitivity.

Hereinafter, formulation examples of the composition containing CB13 according to the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

< Prescription example  1 > Prescription example

&Lt; Prescription Example 1-1 > Preparation of powder

20 mg of CB13, 100 mg of lactose, and 10 mg of talc were mixed and filled in airtight bags to prepare powders.

&Lt; Prescription Example 1-2 > Preparation of tablets

20 mg of CB13, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed and tableted according to a conventional preparation method.

&Lt; Prescription Example 1-3 > Preparation of capsules

10 mg of CB13, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed, and the above components were mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

&Lt; Prescription Example 1-4 > Preparation of injection

CB13 (10 mg), sterile distilled water suitable amount for injection, and pH adjuster were mixed, and the contents were adjusted to the above contents in the amount of 2 ml per ampoule according to the usual injection preparation method.

&Lt; Prescription Example 1-5 > Preparation of ointment preparation

An ointment was prepared according to a conventional ointment preparation method after mixing 10 mg of CB13, 250 mg of PEG-4000, 650 mg of PEG-400, 10 mg of white petrolatum, 1.44 mg of methyl p-oxybenzoate, 0.18 mg of p- .

< Prescription example  2> Health supplements

&Lt; Prescription Example 2-1 > Preparation of health food

1, vitamin B 2 0.1 mg, vitamin B 6 0.5 mg, vitamin B 12 0.2 쨉 g, vitamin C 10 mg, biotin 10 &lt; RTI ID = 0.0 &gt; (Ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium phosphate monobasic 15 mg, dibasic calcium phosphate 55 mg, nicotinate amide 1.7 mg, folic acid 50 mg, calcium pantothenate 0.5 mg) Mg of calcium citrate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride) were mixed to prepare a granule, and a health food was prepared according to a conventional method.

&Lt; Prescription Example 2-2 > Preparation of health drink

1 mg of CB13, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of a plum concentrate, 1 g of taurine and purified water were added to make a total of 900 ml, and the above components were mixed according to a conventional health drink manufacturing method, The solution was filtered and sterilized in a sterilized 2 L container, and then refrigerated.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (11)

A pharmaceutical composition for treating cancer comprising, as an active ingredient, a pyrimidine derivative represented by the following formula (1), or a pharmaceutically acceptable salt or solvate thereof:
[Chemical Formula 1]

In the above formula, R may be any one selected from C1 to C4 alkyl, C1 to C4 alkoxy, halogen or hydroxy. The pharmaceutical composition according to claim 1, wherein the pyrimidine derivative is C1 to C4 alkyl. The cancer therapeutic agent according to claim 1, wherein the pyrimidine derivative is 1-benzyl-5- (4-methylphenyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) &Lt; / RTI &gt; The pharmaceutical composition according to claim 1, wherein the cancer is selected from lung cancer, breast cancer, colon cancer or white blood cancer. A pharmaceutical composition for radiation-sensitive sensitization of cancer comprising a pyrimidine derivative represented by the following formula (1), a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient:
[Chemical Formula 1]

In the above formula, R may be any one selected from C1 to C4 alkyl, C1 to C4 alkoxy, halogen or hydroxy. The pharmaceutical composition according to claim 5, wherein the pyrimidine derivative is C1 to C4 alkyl. [Claim 6] The method according to claim 5, wherein the pyrimidine derivative is 1-benzyl-5- (4-methylphenyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) A pharmaceutical composition for use in radiation therapy for a Korean patient. The pharmaceutical composition according to claim 5, wherein the cancer is selected from lung cancer, breast cancer, colorectal cancer, or white blood cancer. A pharmaceutical composition for the treatment of diabetes comprising as an active ingredient a pyrimidine derivative represented by the following formula (1), or a pharmaceutically acceptable salt or solvate thereof:
[Chemical Formula 1]

In the above formula, R may be any one selected from C1 to C4 alkyl, C1 to C4 alkoxy, halogen or hydroxy. The pharmaceutical composition according to claim 9, wherein the pyrimidine derivative is C1 to C4 alkyl. The diabetes mellitus treatment according to claim 9, wherein the pyrimidine derivative is 1-benzyl-5- (4-methylphenyl) pyrido [2,3-d] pyrimidine-2,4 (1H, 3H) &Lt; / RTI &gt;

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