KR20070030653A - Pharmaceutical composition comprising 1-furan-2-yl-3-pyridin-2-yl-propenone for treating or preventing angiogenesis-related disease and cancer disease - Google Patents

Abstract

The present invention is 1-furan-2-yl-3-pyridin-2-yl-propenone represented by the formula (1) to inhibit angiogenesis and cancer growth (1-furan-2-yl-3-pyridin-2- yl-propenone (FPP-3), and the compound of the present invention is excellent in inhibiting angiogenesis and inhibiting cancer cell growth in chorionic ureteric model. In addition to inhibiting the metastasis of cancer cells, it can be used as a pharmaceutical composition for the prevention and treatment of diseases caused by cancer diseases and angiogenesis.

1-furan-2-yl-3-pyridin-2-yl-propenone, FPP-3, angiogenesis, cancer, pharmaceutical composition

Description

Pharmaceutical composition comprising 1-furan-2-yl-3- for the prevention or treatment of diseases and cancer diseases caused by angiogenesis containing 1-furan-2-yl-3-pyridin-2-yl-propenone pyridin-2-yl-propenone for treating or preventing angiogenesis-related disease and cancer disease}

1 shows the angiogenesis inhibitory effect of FPP-3 in C6 glioblastoma cells,

2a to 2f show the angiogenesis inhibitory effect of FPP-3 in HUVEC cells,

Figure 3 shows the changes in the volume of tumors following FPP-3 administration in a non-thymus nude mouse model,

Figure 4 shows the cancer cell migration inhibitory effect of FPP-3 in HT-1080 cells,

5A to 5D show HT-1080 cell infiltration ability of the control group, 10 μM FPP-3 treatment group, 20 μM FPP-3 treatment group, and 30 μM FPP-3 treatment group, respectively,

6a and 6b show the effects of MMP secretion of FPP-3 in HT-1080 cells,

7 is Inhibit the VEGF secretion effect of FPP-3 in HT-1080 cells,

Figure 8 shows the results of cancer cell toxicity test of FPP-3.

The present invention relates to a composition containing 1-furan-2-yl-3-pyridin-2-yl-propenone having antiangiogenic activity and growth inhibitory activity of cancer.

Angiogenesis is the process by which new capillaries are formed from existing microvascular vessels. Angiogenesis normally occurs during embryonic development, tissue regeneration and wound healing, as well as the development of the corpus luteum, a change in the genital system of women periodically, and in these cases is strictly controlled (Folkman J et al., Int . Rev. Exp. Pathol., 16, pp207-248, 1976).

In adults, vascular endothelial cells grow very slowly and do not divide relatively well compared to other types of cells. The angiogenesis process is generally caused by stimulation of angiogenesis factors, resulting in the formation of lumen due to the degradation of the basal membrane, proliferation, proliferation, and differentiation of vascular endothelial cells due to proteases. Consists of being generated.

However, there are diseases caused by angiogenesis that is not controlled autonomously and grows pathologically. Diseases related to angiogenesis in pathological conditions include hemangioma, angiofibroma, angioplasty and cardiovascular diseases such as atherosclerosis, angiogenesis, and edema sclerosis. Glaucoma, diabetic retinopathy, neovascularized corneal disease, spot degeneration, pterygium, retinal degeneration, posterior capsular fibrosis, granular conjunctivitis, and the like. Chronic inflammatory diseases such as arthritis, psoriasis, capillary dilatation, purulent granulomas, seborrheic dermatitis, dermatological diseases such as acne, Alzheimer's and obesity are also associated with angiogenesis, and cancer growth and metastasis necessarily depends on angiogenesis (D'Amato RJ et al., Ophthalmology, 102 (9), pp1261-1262, 1995; Arbiser JL, J. Am. Acad. Dermatol., 34 (3), pp486-497, 1996; O'Brien KD et al. Circulation, 93 (4), pp 672-682, 1996; Hanahan D et al., Cell, 86 , pp 353-364, 1996).

Angiogenesis, in particular, plays an important role in the growth and metastasis of cancer cells. Tumors are supplied with nutrients and oxygen for growth and proliferation through neovascularization, and new blood vessels that penetrate the tumor allow metastases to metastasize by giving metastatic cancer cells the chance to enter the blood circulation (Folkman and Tyler, 1999). Cancer Invasion and metastasis , Biologic mechanisms and Therapy (SB Day ed.) Raven press, New York, pp 94-103, 1977; Polverini PJ, Crit. Rev. Oral. Biol. Med. , 6 (3), pp230-247, 1995). The main cause of death of cancer patients is metastasis, and the current metastasis of cancer does not contribute to the survival of cancer patients.

Arthritis, a representative disease of inflammatory diseases, is caused by autoimmune abnormalities, but as the disease progresses, chronic inflammation in the synovial cavity between joints induces angiogenesis and destroys cartilage. In other words, synovial cells and vascular endothelial cells proliferate in the synovial cavity with the help of inflammation-inducing cytokines, forming joint pannus, a layer of connective tissue that develops in the cartilage as the angiogenesis progresses, destroying cartilage that acts as a cushion. (Koch AE et al., Arthritis. Rheum., 29, pp471-479, 1986; Stupack DG et al., Braz J. Med. Biol. Rcs., 32 (5), pp578-581, 1999; Koch AE , Atrhritis. Rheum., 41 ( 6), pp951-962, 1998).

Many ocular diseases, which cause millions of blindness worldwide each year, are also caused by angiogenesis (Jeffrey MI et al., J. Clin . Invest ., 103, pp1231-1236, 1999). For example, diseases such as macular degeneration, diabetic retinopathy, retinopathy of premature infants, neovascular glaucoma and corneal disease caused by neovascularization are the diseases caused by neovascularization (Adamis AP). et al., Angiogenesis, 3, pp 9-14, 1999). Among them, diabetic retinopathy is a complication of diabetes, in which capillaries in the retina invade the vitreous body and eventually become blind.

Psoriasis, which is characterized by red spots and skin, is also a chronic proliferative disease of the skin. It does not heal and involves pain and malformations. In normal cases, keratinocytes proliferate once a month, whereas psoriasis patients proliferate at least once a week. This rapid proliferation requires a large supply of blood and angiogenesis is bound to occur (Folkman J, J. Invest . Dermatol ., 59, pp 40-48, 1972).

Since angiogenesis inhibitors can be applied as a therapeutic agent for such various angiogenesis-related diseases, studies are being actively conducted to treat the above diseases by inhibiting angiogenesis. Such angiogenesis inhibitors usually require long-term administration to patients, so they should be of low toxicity and be orally available for use as the ideal therapeutic. Therefore, there is a demand for the development of a drug having low toxicity as an angiogenesis inhibitor.

1-furan-2-yl-3-pyridin-2-yl-propenone inhibits NF-κB activity resulting in nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α, Tumor Necrosis Factor) (Lee ES et al., Biol. Pharm. Bull . , 27 (5), pp617-620, 2004) reported that anti-inflammatory activity is inhibited by inhibiting the production of -α) . There is no teaching or disclosure about having anti-neoplastic and anti-cancer activity.

Thus, the present inventors have completed the present invention by confirming that 1-furan-2-yl-3-pyridin-2-yl-propenone of the present invention has excellent antiangiogenic and cancer cell growth inhibitory effects.

The present invention is directed to diseases and cancers caused by angiogenesis containing 1-furan-2-yl-3-pyridin-2-yl-propenone (1-furan-2-yl-3-pyridin-2-yl-propenone) It is to provide a pharmaceutical composition for the prevention and treatment of diseases.

In order to achieve the above object, the present invention provides a 1-furan-2-yl-3-pyridin-2-yl-propenone (1-furan-2-yl-3-pyridin-2-yl represented by the following formula (1) It provides a pharmaceutical composition for the prevention and treatment of diseases caused by angiogenesis, containing -propenone) as an active ingredient, and containing a pharmaceutically acceptable carrier or excipient.

In addition, the present invention is effective to 1-furan-2-yl-3-pyridin-2-yl-propenone (1-furan-2-yl-3-pyridin-2-yl-propenone) represented by the formula (1) It provides a pharmaceutical composition for the prevention and treatment of cancer diseases, including as a component, and containing a pharmaceutically acceptable carrier or excipient.

The angiogenesis-related diseases include rheumatoid arthritis, osteoarthritis, septic arthritis, psoriasis, corneal ulcer, age-related macular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, immature retinopathy, ophthalmic inflammation, cone cornea, shogren Syndrome, myopia ophthalmic tumor, corneal transplant rejection, abnormal wound union, bone disease, proteinuria, abdominal aortic aneurysm, degenerative cartilage loss due to traumatic joint injury, demyelination of the nervous system, cirrhosis, renal glomerular disease, immature rupture of the embryonic membrane, Inflammatory bowel disease, periodontal membrane disease, arteriosclerosis, restenosis, inflammatory diseases of the central nervous system, Alzheimer's disease, skin aging or cancer infiltration and metastasis.

The cancer diseases include lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal muscle cancer, colon cancer, breast cancer, and fallopian tube carcinoma. , Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis cancer, prostate cancer , Chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma or pituitary adenoma have.

Hereinafter, the present invention will be described in detail.

The 1-furan-2-yl-3-pyridin-2-yl-propenone compound of the present invention can be obtained as follows.

C ₁ An aldehyde compound, preferably on a solution in which a lower alcohol solvent of C ₄ to C ₄ is dissolved in a strong base such as potassium hydroxide, sodium hydroxide, and the like, preferably sodium hydroxide in a temperature of 10 to 30 ° C. and nitrogen in a low alcohol solvent, preferably ethanol. After stirring 54.85 mM 2-pyridinecarboxaldehyde and ketone compound, preferably 49.86 mM 2-acetylfuran at 10 to 30 ° C. for 2 to 5 hours, the mixture was added with water at a ratio of 5 to 10 times by weight. The water soluble fraction can be extracted with dichloromethane to obtain a water soluble fraction and a dichloromethane soluble fraction. The organic layer dichloromethane soluble fraction is washed with water and saturated sodium chloride solvent and dried over sodium sulfate. 2: collection of the solvent is dried under reduced pressure and the residue was subjected to silica gel column chromatography, and can perform (SiO ₂ column chromatography), ethyl acetate: n- mixed solvent, preferably ethyl acetate in hexane: 1 n- hexane Column chromatography may be performed with a mixed solvent to separate and identify the compound represented by Chemical Formula 1.

The 1-furan-2-yl-3-pyridin-2-yl-propenone compound of the present invention obtained by the above method is angiogenesis such as vascular endothelial growth factor and fibroblast growth factor in chicken chorion model Inhibition of angiogenesis was increased by the treatment of the inducer, and the angiogenesis was also inhibited by implanting cancer cells into the chorionic ureter, and the volume increase of gliomas was also suppressed according to the above results. It was confirmed that it can be usefully used as a composition for prevention and treatment.

In addition, the 1-furan-2-yl-3-pyridin-2-yl-propenone compound of the present invention concentration-dependently inhibits angiogenesis by human umbilical vein endothelial cells (HUVEC) and HT-1080 human fibroblastoma cells Inhibition of tumor volume in the non-thymus nude mouse tumor model transplanted with, inhibits cancer cell metastasis by inhibiting cancer cell migration and cancer cell invasion in HT-1080 human fibroblastoma cells, and activity of MMP and VEGF And inhibiting expression.

Therefore, the present invention comprises a 1-furan-2-yl-3-pyridin-2-yl-propenone compound obtained by the above process as an active ingredient, and due to angiogenesis containing a pharmaceutically acceptable carrier or excipient Provided are pharmaceutical compositions effective for the treatment and prevention of diseases and cancer diseases.

The application amount and application method of the pharmaceutical composition comprising the 1-furan-2-yl-3-pyridin-2-yl-propenone compound represented by Formula 1 may vary depending on the formulation and the purpose of use.

A pharmaceutical composition for the treatment and prevention of diseases and cancer diseases due to angiogenesis containing a compound of the present invention comprises 0.1 to 50% by weight of the compound relative to the total weight of the composition.

In addition, the composition comprising the compound of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions.

Carriers, excipients or diluents which may be included in the compositions comprising the compounds of the invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The compositions comprising the compounds according to the invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions according to conventional methods. Can be used.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid form may include at least one excipient such as starch, calcium carbonate, water Prepare a mixture of sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of the compound of the present invention may vary depending on the age, sex, and weight of the patient, but the amount of 0.1 to 100 mg / kg may be administered once to several times daily. The dosage of the compound can also be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition may be administered to various mammals such as mice, mice, livestock, humans, and the like. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Hereinafter, the present invention will be described in detail by Examples, Formulation Examples and Experimental Examples.

However, the following Examples, Formulation Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples, Formulation Examples and Experimental Examples.

Example  1.1- Furan -2-yl-3-pyridin-2-yl- Propenon  Produce

2-pyridinecarboxaldehyde (5.2 mL, 54.85 mM) was added to 34 mg of solid sodium hydroxide solution dissolved in ethanol under nitrogen at 25 ° C. for reaction. After dissolving it, 2-acetylfuran (2-acetylfuran, 5 ml, 49.86 mmol) was stirred at a temperature of 25 ° C. for 3 hours, and then 80 ml of water was added to the mixture, followed by 120 ml of dichloromethane (CH ₂ Cl ₂ ) and the organic layer was washed with 80 mL × 2 water and saturated 80 mL sodium chloride (NaCl) solvent and dried over sodium sulfate (Na ₂ SO ₄ ). The solvent was dried under reduced pressure, and the residue was purified by silica gel column chromatography (purification solvent EtOAc: n-hexane = 1: 2, v: v) to give 1-furan-2- of yellow crystals having the following physical properties of 3.84 g. Il-3-pyridin-2-yl-propenone (yield 38.7%) was obtained (hereinafter referred to as FPP-3).

TLC (EtOAc: n-Hexane = 1: 2, v: v), R _f = 0.167

¹ H-NMR (250 MHz, CDCl ₃ ): δ 8.70 (ddd, J = 4.8, 1.7, 0.9 Hz, 1 H, pyridine H-6), 7.97 (d, J = 15.4 Hz, 1 H, -CH = CH-CO), 7.84 (d, J = 15.4 Hz, 1 H, -CH = CH-CO-), 7.75 (dt, J = 7.7, 1.8 Hz, 1 H, pyridine H-4), 7.68 (dd, J = 1.7, 0.7 Hz, 1 H, furan H-5), 7.48 (dt, J = 7.8, 1.2 Hz, 1 H, pyridine H-3), 7.42 (dd, J = 3.6, 0.7 Hz, 1 H, Furan H-3), 7.31 (ddd, J = 7.6, 4.8, 1.1 Hz, 1 H, pyridine H-5), 6.61 (dd, J = 3.6, 1.7 Hz, 1 H, furan H-4)

Reference Example  1. Analysis of test materials, reagents and instruments

Yoo Jung Ran was purchased from Yeungnam University ranch (Gyeongsan, Korea) and used as normal human hepatocyte Chang cell, human glioblastoma cell A172 cell, human neuroblastoma cell SK-N-SH, human HT-1376 cells, which are bladder cancer cells, HT-29 cells, which are human colon cancer cells, HepG2 cells, which are human liver cancer cells, and HT-1080 cells, which are human fibroblastoma cells, are ATCC (Rockville, MD, USA) Purchased from Human umbilical vein endothelial cells (HUVECs) were also purchased from Clonetics (San Diego, Calif.).

Five-week-old athymic nude mice were purchased from Orient Co., Ltd, Seoul, Korea.

2-pyridinecarboxaldehyde, 2-acetylfuran and cortisone acetate were purchased from Aldrich Chemical Co., St. Louis, Mo., USA Fibroblast growth factor (FGF) was purchased from Invitrogen, USA, and vascular endothelial growth factor (VEGF) was purchased from R & D systems, Minneapolis, MN, USA. Tetrac and XT199 were obtained from Albany College of Pharmacy, and whatman filter discs were purchased from Whatman, UK.

(Finnigan LCQ Advantage

) LC/MS/MS 분광계(spectrometry)를 엑스칼리버(Xcalibur)

프로그램을 이용하여 분석하였다. TLC(Thin-layer chromatography)와 컬럼 크로마토그래피(column chromatography)는 머크사 (Merck)의 실리카겔 키젤겔 (Silicagel Kieselgel) 60 F₂₅₄ (230 ~ 240 mesh)을 사용하였다.For structural characterization of the synthesized materials, the Bruker AMX 250 MHz model was used to obtain ¹ H-NMR spectra, and LC / Mass Spectrometry was used for Pinigan LCQ Advantage.

Finnigan LCQ Advantage

) LC / MS / MS spectrometry with Xcalibur

Analyzed using the program. Thin-layer chromatography and column chromatography used Merck silica gel Kieselgel 60 F ₂₅₄ (230 ~ 240 mesh).

Reference Example  2. Cell Culture

Human cancer cell lines such as HT-29 colorectal cancer cells, HT-1376 bladder cancer cells, Hep2G hepatocellular carcinoma cells, SK-N-SH neuronal cells, A172 glial cancer cells, HT-1080 fibroblasts, and normal hepatocytes Chang, the US Type Culture Collection (ATCC) , Rockville, Mass.) Was used. The cells were cultured with Eagle's minimum essential medium (MEM) containing 10% fetal bovine serum (GIBCO), 200 IU / ml penicillin, 200 (g / ml streptomycin, 1 mM sodium pyruvate). Chemical CO.) Medium, a humidified incubator at 37 ℃, 5% CO2 / 95% air condition, the culture medium was replaced once every day. After growing confluent (confluent), Subcultures were trypsinized with 0.25% trypsin-EDTA solution.

HUVEC cells, on the other hand, were cultured in a 0.2% gelatin coated flask. The HUVEC cells were cultured in EBM-2 (Endothelial Cell Basal Medium-2, Clonetics, San Diego, Calif.) Medium. The EBM-2 medium is fetal bovine serum (FBS), hydrocortisone (hydrocortisone), human basic fibroblast growth factor (hFGF-B), vascular endothelial growth factor (VEGF), R3-IGF-1 (human) recombinant insulin-like growth factor), ascorbic acid, human epidermal growth factor (hEGF), GA-1000 and heparin. In this experimental example, HUVEC cells between the first and sixth passages were used.

Experimental Example  1. of chicken Chorionic  analysis ( CAM assay )through FPP -3 inhibitory of angiogenesis

In vivo test phase in In order to confirm the antiangiogenic effects in vivo , CAM (CAM, chorioallantoic membrane) analysis was performed (Nguyen M et al., Microvascular Res . , 47, pp 31-40, 1994). Chicken eggs were incubated at 37 ° C and 55% RH, and the first small hole was drilled using a hypodermic needle (Green Cross Medical Industry, Korea) on the air sac on day 10. A second hole was drilled in the flat part of the fertilized egg for the window.

By venting air through the hole in the vesicle area, the first hole, the chorioallantoic membrane (CAM) is separated from the egg of the fertilized egg, which is then transferred to a grinding wheel (Multipro 395JA, Dremel, Mexico). I cut and made a window. Next, 3 mg / ml of cortisone acetate was dried on a Whatman filter disk (whatman, USA) and fibroblast proliferation factor (FGF) was 15 ng / CAM. Vascular endothelial growth factor (VEGF) was soaked at a concentration of 20 ng / CAM.

The filter disc was placed on the blood vessel through the prepared window, and the compound FPP-3 of Example 1 of the present invention was dissolved in dimethyl sulfoxide (DMSO), diluted with phosphate buffer solution (PBS), and the concentration (100 ng, 1 ㎍). , 5 μg, 10 μg, 20 μg). After 3 days of drug treatment, the CAM part on the filter disc was removed and washed with phosphate buffer solution, followed by a stereo microscope (Stemi SV6 stereomicroscope, Carl Zeiss, Germany) and Image-Pro Plus software (Media Cybernetics; Silver Spring, MD, USA) was used to measure the number of vessel branches and analyze the data (see Table 1).

Treatment Branch points ± SEM % of inhibition ± SEM PBS 37.0 ± 8.8 VEGF (20 ng / CAM) 83.0 ± 13.0 VEGF + FPP-3 (1 μg / CAM) 62.8 ± 10.2 30.0 ± 13.6 VEGF + FPP-3 (5 μg / CAM) 52.6 ± 6.9 34.4 ± 9.1 VEGF + FPP-3 (10 μg / CAM) 40.8 ± 9.6 36.3 ± 12.8 FGF (15 ng / CAM) 72.5 ± 6.2 FGF + FPP-3 (100 ng / CAM) 45.0 ± 3.7 41.2 ± 4.8 FGF + FPP-3 (1 μg / CAM) 44.6 ± 6.3 41.7 ± 4.8 FGF + FPP-3 (5 μg / CAM) 37.6 ± 4.3 50.9 ± 5.6 FGF + FPP-3 (10 μg / CAM) 31.9 ± 5.4 58.4 ± 7.0

As shown in Table 1, the increase in angiogenesis following treatment of angiogenesis inducers such as vascular endothelial growth factor and fibroblast growth factor was shown to be decreased by the treatment of FPP-3.

Experimental Example  2. of chickens transplanted with cancer cells Chorionic  Using the model FPP -3 Antiangiogenic effect and

According to the method of gu (Gu JW et al., Cancer , 103 (2), pp422-431, 2000) in the fertilized eggs on the 10th day of cultivation in the same manner as in Experiment 1 and made a window through the C6 glioblastoma Cells were inoculated with 1 × 10 ⁶ cell counts / 25 ml on chorionic viscera (CAM). After 2 hours of inoculation, 10 μg / CAM of XT199 and Tetrac were treated as positive controls, and the FPP-3 compound of Example 1 was treated with concentrations (5, 10 μg / CAM) at sites where cancer cells were inoculated. After culturing for 7 days, the entire chorionic viscera where tumor tissue was formed was removed and washed with phosphate buffer solution, followed by stereomicroscopy (Stemi SV6 stereomicroscope, Carl Zeiss, Germany) and Image-Pro Plus software (Media Cybernetics; Silver Spring, MD, USA) was taken to measure the number of vascular branches and to analyze the data (see FIG. 1). In addition, the tumor tissue was weighed after storage and stored in 10% formalin or liquid nitrogen for RNA isolation and morphological studies.

Treatment Branch points ± SEM % of inhibition ± SEM C6 (1 × 10 ⁶ cells / CAM) 187.0 ± 11.9 C6 + XT199 (10 μg) 146.1 ± 10.3 21.8 ± 5.5 C6 + Tetrac (10 μg) 181.7 ± 13.1 2.9 ± 7.0 C6 + FPP-3 (5 μg) 111.5 ± 12.2 40.4 ± 6.5 C6 + FPP-3 (10 μg) 118.3 ± 11.7 36.8 ± 6.3

Treatment Ave. tumor weight (mg) ± SEM % of inhibition ± SEM C6 (4 × 10 ⁶ cells / CAM) 99.0 ± 7.8 C6 + FPP-3 (5 μg) 72.1 ± 6.7 26.4 ± 6.8

As shown in Table 2, the neovascularization activated in the chorionic villus into which the cancer cells were transplanted was inhibited by the treatment of FPP-3, and in accordance with the above results, the increase in the volume of the gliomas was also suppressed.

Experimental Example  3. HUVEC  In the cell FPP -3 inhibitory effect on angiogenesis

To investigate the effect of FPP-3 on angiogenesis in HUVEC cells, HUVEC cells were cultured in 96-well plates. 40 μl of Matrigel (BD biosciences, Bedford, Mass.) Was coated on each well of a 96-well plate, and then used after storing the 96-well plate at 37 ° C. for 30 minutes.

HUVEC cells were suspended in EBM-2 (Endothelial Cell Basal Medium-2, Clonetics, San Diego, Calif.) Medium containing 2% fetal bovine serum. These HUVEC cells were allowed to inject 1 × 10 ⁴ cells into each well coated with the matrigel. Next, different concentrations of FPP-3 were added to each well and stored for 7 hours. The HUVEC cells thus treated were taken with a digital camera connected with an inverted microscope, and photographs thereof are shown in FIGS. 2A to 2F.

Figure 2a is a picture showing the HUVEC cells not treated with FPP-3 as a control, Figure 2b is a picture showing HUVEC cells treated with 100 nM FPP-3, Figure 2c is treated with 500 nM FPP-3 Figure 2d is a photograph showing the HUVEC cells, Figure 2d is a photograph showing the HUVEC cells treated with 1μM FPP-3, Figure 2e is a photograph showing the HUVEC cells treated with 5μM FPP-3, Figure 2f is 10μM FPP A photograph showing HUVEC cells treated with -3.

First, referring to FIG. 2A, it can be observed that many blood vessels are formed in a control. Here, it can be seen that as the concentration of FPP-3 increases, the amount of blood vessels formed decreases. Accordingly, it can be confirmed that FPP-3 inhibits angiogenesis of HUVEC cells.

Experimental Example  4. Transplanting Cancer Cells Non-thymus  Using a nude mouse model FPP -3 Angiogenesis memory Effect

Five-week-old athymic nude mice were allowed to feed and water freely in a light-controlled environment with a temperature of 21 ± 1 ° C and a light / dark cycle repeated every 12 hours. These non-thymus nude mice were placed in the above environment for at least 2 days and then used for the experiment. The animal experiments described below were performed in accordance with the ethical regulations of the Korea National Institutes of Health.

100 μl of MEM medium containing 5 × 10 ⁶ HT-1080 cells The volume of tumors produced 10 days after subcutaneous injection into the right flank of non-thymus nude mice was measured and then measured twice a week. Using a calipers (calipers) to measure the small diameter and large diameter of the generated tumor was calculated the volume of the tumor based on the following equation (1).

V = 1/2 (L × W ² )

Where V represents the volume of the tumor, L represents the large diameter, and W represents the small diameter.

Since the tumor volume generated in the non-thymus nude mouse was 200 mm based on Equation 1 above, daily change of the tumor volume after oral administration of the compound of Example 1, FPP-3, to the non-thymus nude mouse Was measured. Here, 1 mg of FPP-3 per kg of non-thymus nude mouse was administered for 20 days.

Hereinafter, a description will be given with reference to FIG. 3. Figure 3 is a graph showing the change in volume of tumors following FPP-3 administration in a non-thymus nude mouse model.

Here, the horizontal axis represents Days After Drug Treatment based on the date of the first FPP-3 administration, and the vertical axis represents Tumor Volume Increase (㎣) measured in the non-thymus nude mouse model.

Experimental results of non-thymus nude mice orally administered with FPP-3 daily were expressed as FPP-3, and experimental results of non-thymus nude mice not orally administered with FPP-3 under the same conditions were indicated as controls. Here, five non-thymus nude mouse models were tested in each group to reduce the experimental error.

As a result, as shown in FIG. 3, it was observed that the tumors of the non-thymus nude mice administered FPP-3 daily significantly reduced the volume increase compared to the tumors of the control.

Experimental Example  5. FPP -3 cancer cell migration ( migration ) Inhibitory effect

HT-1080 cancer cells cultured in 90% confluent were pretreated with 25 μg / ml mitomycin C for 30 minutes. After treatment with mitomycin C, the culture plate was wound with a tip to produce a 1 mm wide strip (Mi-Sung Kim et al.). al . , Cancer Research , 63, 5454-5461, 2003).

Thereafter, HT-1080 cells of the culture plate were washed with Hanks balanced salt solution (HBSS), and then cancer cell migration was observed over time.

The results are shown in FIG. 4 is a photograph showing the cell migration inhibitory effect of FPP-3.

In FIG. 4, the horizontal axis represents the progress of 0, 6, 12, and 24 hours (Hr). Here, the upper picture is a control that observed the cell movement after the same treatment to the HT-1080 cells, the lower picture is a control of 30μM FPP-3 treated cells under the same conditions as the control It observed and is the photograph which expanded at 100 magnification, respectively.

It was found that the HT-1080 cells cultured in the culture plate were wounded to form a 1 mm wide string, and a large number of HT-1080 cells were cultured on the upper and lower sides of the 1 mm wide string (0 hr). Here, as 6 hours, 12 hours, and 24 hours elapsed, it was found that in the control group, the row of 1 mm width disappeared and the HT-1080 cells were cultured by moving in the row of 1 mm width. However, in the case of HT-1080 cells treated with FPP-3, it was found that even after 24 hours, the cells hardly migrated to the 1 mm wide row. Thus, it can be seen that FPP-3 significantly inhibits the migration of cancer cells.

Experimental Example  6. FPP -3 inhibits cancer cell metastasis

To investigate the inhibitory effect of FPP-3 on cancer cell metastasis, in Experiments were carried out using HT-1080 cells in vitro (Mi-Sung Kim et al. al ., Cancer Research , 63, 5454-5461, 2003 Sang-Oh Yoon et al ., The journal of Biological chemistry , 276, 20085-20092, 2001; Sonia Zorzet et al., The journal of pharmacology and experimental therapeutics , 295, 927-933, 2000). The culture plate used here was a 24-well plate (Corning Costar, Cambridge, Mass.) Containing a polycarbonate filter with multiple 8 mm size pores. The bottom surface of this polycarbonate filter was coated with 20 l of collagen type 1 (type I collagen) at a concentration of 0.5 mg / ml, and the top surface was coated with 20 l of Matrigel (BD biosciences, Bedford, MA) at a concentration of 1.5 mg / ml. Here, the lower region of the polycarbonate filter was filled with medium containing 10% FBS, and HT-1080 cells were seeded on top of the polycarbonate filter. The inoculated HT-1080 cells were incubated at 37 ° C. for 18 hours, and then the cells infiltrated on the lower surface of the polycarbonate filter were fixed with methanol, and hematoxylin and eosin. Stained. The results are shown in Figures 5a to 5d.

5a to 5d are micrographs of the lower surface of the polycarbonate filter at 400 magnification to confirm the cancer cell metastasis inhibiting effect of FPP-3, and a control group, a 10 μM FPP-3 treatment group, a 20 μM FPP-3 treatment group, and It is a photograph showing the cancer cell infiltration ability of the 30μM FPP-3 treatment group.

Herein, when FPP-3 was treated, cancer cells that penetrated into the lower surface of the polycarbonate filter were significantly reduced compared to the control group, and thus FPP-3 reduced the metastasis of cancer cells in a concentration-dependent manner.

Experimental Example  7. In cancer cells FPP -3 MMP  Secretory effect

In order to know the effect of FPP-3 on matrix metalloproteinase (MMP) secretion in cancer cells, 1 × 10 ⁶ HT-1080 cells were injected into each well of a 6-well plate. Thus injected HT-1080 cells were incubated in MEM medium containing 10% fetal bovine serum (FBS) for 12 hours to allow HT-1080 cells to adhere to the plate. Next, MEM medium containing fetal bovine serum (FBS) was removed and washed with MEM medium without serum. These HT-1080 cells were again incubated for 24 hours in MEM medium containing no serum. After culturing HT-1080 cells for 24 hours, supernatants were obtained from 6-well plates, respectively, and gelatin zymography was performed (Herron et. al . , J. Biol . Chem . , 261, 2814-2818, 1986).

Briefly describing the gelatin zymography process, the supernatant obtained from the 6-well plate was subjected to electrophoresis on a 10% sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) gel containing gelatin. It was. After electrophoresis, the SDS-PAGE gel was washed twice in buffer (50 mM Tris-HCl, pH7.5, 100 mM NaCl, 2.5% Triton X-100) to remove SDS. Subsequently, SDS-PAGE gels were incubated in culture buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 10 mM CaCl _{2 ,} 0.02% NaN ₃ ) at 37 ° C. This SDS-PAGE gel was stained with 0.25% Coomassie brilliant blue R250, Sigma Chemical Co., St. Louis, Mo. and then destained.

In addition, in order to examine the effect of FPP-3 on TPA-induced MMP activation, TPA was treated with 12 ng / ml when treated with FPP-3 as described above.

These experimental results are shown in Figures 6a and 6b.

6a and 6b show the effect of suppressing the MMP secretion of FPP-3 in HT-1080 cells, each lane in Figure 6a (lane) in the concentration of FPP-3 treated to each HT-1080 cells for 24 hours ( μM) shows the activity of MMP-2 and MMP-9.

Here, MMP-2 and MMP-9 have a function of gelatin degrading enzymes that degrade gelatin contained in SDS-PAGE. Accordingly, a band can be observed where MMP-2 and MMP-9 are present. Referring to FIG. 6A, when FPP-3 is treated from 0 μM to 30 μM, it can be observed that the band becomes blurred as the concentration of the treated FPP-3 increases. Accordingly, it can be seen that the higher the concentration of FPP-3 treated, the lower the enzyme activity of MMP-2 and MMP-9 secreted from HT-1080 cells.

Next, referring to FIG. 6B, the first lane is the result when TPA and FPP-3 are not treated, and the second lane is the result when TPA 12 ng / ml is treated and FPP-3 is not treated. The third lane is the result of treatment with 12ng / ml TPA and 20μM FPP-3, and the fourth lane is the result of treatment with 12ng / ml TPA and 30μM FPP-3. Here, a relatively large MMP-9 protein is present on the upper side of the SDS-PAGE gel, and underneath are the PRO-MMP-2 protein and the ACTIVE-MMP-2 protein, which are precursors of the MMP-2 protein. .

Here, MMP-2 and MMP-9 are gelatin degrading enzymes, which decompose the gelatin contained in the SDS-PAGE gel to form a band as shown in FIG. 6B. Expression of these MMP-2, MMP-9 is increased in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). Accordingly, since the first lane was not stimulated with TPA on HT-1080 cells, almost no band was observed in MMP-9 and active-MMP-2, and in pro-MMP-2, the precursor of MMP-2. Was observed. The second lane shows the results of treatment of HT-1080 cells with only TPA, and dark bands can be observed in MMP-9 and MMP-2. The third and fourth lanes show the results of treatment with FPP-3 at concentrations of 20 μM and 30 μM, respectively, in the presence of TPA. The higher the concentration of FPP-3, the higher the band amplitude of MMP-2 and MMP-9. It can be seen that the following is blurred.

Accordingly, it can be seen that FPP-3 reduces the secretion amount of MMP-2 and MMP-9 activated by TPA in HT-1080 cells.

Experimental Example  8. In cancer cells FPP -3 VEGF  Secretory effect

To investigate the effect of FPP-3 on the secretion of VEGF (Vascular endothelial growth factor) in cancer cells, HT-1080 cells were tested. This VEGF is a cytokine that is secreted by various types of cancer cells and promotes angiogenesis.

HT-1080 cells were cultured in 24-well plates for 24 hours in the presence of different concentrations of FPP-3 in medium without serum. Here HT-1080 cells used as a control (control) was not treated with FPP-3. After incubating the HT-1080 cells for 24 hours, only the upper medium was collected from each well. The collected supernatant was used to measure the concentration of VEGF released to the outside of the cell. Here, the concentration of the discharged VEGF was measured using a Quantikine human VEGF ELISA kit (R & D system, Minneapolis, MN). The VEGF concentration was measured according to the usage of the Quantikine human VEGF ELISA kit.

7 is The VEGF secretion inhibitory effect of FPP-3 in HT-1080 cells, the horizontal axis represents the concentration of FPP-3 (μM), the vertical axis represents the concentration of VEGF (ng / ㎖). Here, the control (control) shows the experimental results of the HT-1080 cells not treated with FPP-3.

Referring to the drawings, it can be seen that the most secreted VEGF in the control group, the concentration of VEGF secreted from HT-1080 cells decreases as the concentration of FPP-3 is gradually increased to the concentration of 1, 10, 20μM You can check it. Accordingly, it can be seen that FPP-3 reduces the amount of VEGF secreted from HT-1080 cells, which are cancer cells.

Experimental Example  9. FPP -3 cytotoxicity experiment

In order to measure cell viability, each cell of Reference Example 2 was cultured for 4 to 5 days and dispensed into a 24-well plate at a density of 5 × 10 ⁴ cells / well, and the medium dose of each well was adjusted to 1 ml. Thereafter, FPP-3 was treated at 2, 10, 25, 50, 100, and 250 μM concentrations for 48 hours, and then 100 μl of MTT (3- (4,5-dimethylthiazol-2-yl) -2 , 5-diphenyl tetrazolium bromide; 5 g MTT / Lin H ₂ O) was added and the cells were further incubated for 4 hours. Thereafter, 200 μl of dimethyl sulfoxide (DMSO) was added to the wells containing the respective cells and mixed by pipette to dissolve the reduced MTT crystals. Relative cell viability was determined by scanning with a microplate reader (Molecular Devices, Menlo Park, CA) with a 540 nm filter (see Table 4).

As shown in FIG. 8, the FPP-3 compound showed no toxicity at low concentrations while toxicity at various concentrations of human cancer cells. In addition, as shown in Table 4, the IC ₅₀ was 50 μM or more, and the cytotoxicity against the Chang cells, which were normal cells, was not large.

IC ₅₀ Chang > 50 HT29 50 HT-1376 25 A172 25 SK-N-SH 18 HepG2 18 HT-1080 20

Hereinafter, an example of the preparation of a pharmaceutical composition comprising the FPP-3 compound of the present invention will be described, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation example  One. Powder  Produce

FPP-3 300 mg

Lactose 100 mg

Talc 10 mg

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

FPP-3 50 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components was prepared by tableting according to the conventional manufacturing method of the tablet.

Formulation Example 3 Preparation of Capsule

FPP-3 50 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

According to a conventional capsule preparation method, the above ingredients were mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

FPP-3 50 mg

Appropriate sterile distilled water for injection

pH adjuster

According to the conventional method for preparing an injection, the amount of the above ingredient was prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

FPP-3 100 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution was prepared by sterilization.

As described above, the 1-furan-2-yl-3-pyridin-2-yl-propenone compound of the present invention inhibits angiogenesis, inhibits cancer cell growth, and non-thymus nude mouse tumor model in choriocapillary model. In addition to inhibiting the growth of cancer in cancer cells in the cancer metastasis because it can be used as a pharmaceutical composition for the prevention and treatment of diseases and cancer diseases due to angiogenesis containing it as an active ingredient.

Claims (4)

To include 1-furan-2-yl-3-pyridin-2-yl-propenone (1-furan-2-yl-3-pyridin-2-yl-propenone) represented by the formula (1) as an active ingredient, A pharmaceutical composition for the prevention or treatment of diseases caused by angiogenesis, containing a pharmaceutically acceptable carrier or excipient. [Formula 1]

According to claim 1, The disease caused by angiogenesis is rheumatoid arthritis, osteoarthritis, sepsis arthritis, psoriasis, corneal ulcer, age-related macular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, immature retinopathy, ophthalmic inflammation , Cone cornea, Sjogren's syndrome, myopic ocular tumor, corneal transplant rejection, abnormal wound union, bone disease, proteinuria, abdominal aortic aneurysm, degenerative cartilage loss due to traumatic joint injury, demyelination of the nervous system, cirrhosis, renal glomerular disease, Prevention of diseases caused by at least one angiogenesis selected from the group consisting of immature rupture of the embryonic membrane, inflammatory bowel disease, periodontal disease, arteriosclerosis, restenosis, inflammatory diseases of the central nervous system, Alzheimer's disease, skin aging and cancer infiltration and metastasis Pharmaceutical composition for treatment. 1-furan-2-yl-3-pyridin-2-yl-propenone (1-furan-2-yl-3-pyridin-2-yl-propenone) as an active ingredient, a pharmaceutically acceptable carrier Or a pharmaceutical composition for the prevention or treatment of cancer diseases containing excipients. The cancer disease according to claim 3, wherein the cancer disease is lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal muscle cancer, colon cancer , Breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, Penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, renal or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem nerve A pharmaceutical composition for the prophylaxis or treatment of one or more cancer diseases selected from the group consisting of glioma and pituitary adenoma.

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