KR20060033112A - Anti-cancer and anti-cancer aid composition containing flavonoid derivatives for preventing and treating cancer disease - Google Patents

Abstract

The present invention relates to an anticancer agent and an anticancer adjuvant composition separable from natural substances, and in particular, the composition comprising the flavonoid family compounds of the present invention inhibits the activity of P-glycoprotein (P-gp) overexpressed in cancer cells. Since it increases cytotoxicity against cancer cells having multi-drug resistance (MDR) against anticancer drugs, it can be used in medicines as anticancer agents and anticancer agents.

Flavonoids, glycoproteins, multidrug resistance, anticancer agents, anticancer agents, pharmaceuticals

Description

Anti-cancer and anti-cancer aid composition containing flavonoid derivatives for preventing and treating cancer disease             

1 is a graph showing the effect on the accumulation of DNM (donomycin) in MCF-7 / ADR cancer cells of the example compounds of the present invention (each data point is mean ± SD (n = 3), * p <0.001 );

2 is a graph showing the effect of DNM excretion from MCF-7 / ADR cancer cells of the example compounds of the present invention (each data point is mean ± SD (n = 3), * p <0.01, ** p <0.001).

The present invention relates to an anticancer and anticancer composition for preventing and treating cancer diseases, including the flavonoid derivative compounds.

Cancer is one of the incurable diseases that humanity has to solve, and huge capital is invested in the development to cure it all over the world.In Korea, it is the number one disease death cause and more than 100,000 people a year It is diagnosed and about 60,000 or more die. Carcinogens, which cause cancer, are smoking, ultraviolet rays, chemicals, foods, and other environmental factors. However, various causes are not only difficult to develop a therapeutic agent, but also effective effects vary depending on the site of occurrence. Currently, the substances used as therapeutic agents are extremely toxic and cannot selectively remove only cancer cells. Therefore, there is an urgent need for the development of low-toxic and effective anti-cancer agents to prevent cancer after treatment. .

Cancer is characterized by "uncontrolled cell growth," which results in the formation of cell masses called tumors that infiltrate surrounding tissues and, in severe cases, metastasize to other organs of the body. Academia is also called neoplasia. Cancer is an intractable chronic disease that, even if treated with surgery, radiation, and chemotherapy, in many cases does not heal fundamentally, suffers the patient, and ultimately leads to death. There are many factors that cause cancer, but they can be divided into internal and external factors. It is not known exactly how normal cells are transformed into cancer cells, but at least 80-90% are known to be influenced by external factors such as environmental factors. Internal factors include genetic factors, immunological factors, and external factors include chemicals, radiation, and viruses. Genes involved in the development of cancer include oncogenes and tumor suppressor genes, which occur when the balance between them is broken down by internal or external factors described above.

Cancer is classified into blood cancer and solid cancer, and it occurs in almost every part of the body such as lung cancer, stomach cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer, and skin cancer. Among the methods used to treat these malignancies, chemotherapy agents, except surgery or radiation therapy, are collectively called anticancer agents, and most of them show anticancer activity by inhibiting the synthesis of nucleic acids. Chemotherapeutic agents are broadly classified into antimetabolites, alkylating agents, antimitotic drugs, and hormones, and folates that inhibit metabolic processes necessary for the proliferation of cancer cells. Derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine), pyrimidine derivatives (5-fluorouracil, Cytarabine), etc.Introducing alkyl groups to guanine of DNA to modify the structure of DNA and chain-cutting Alkylating agents that exert the effect include nitrogen mustard compounds (chlorambucil, cyclophosphamide), ethyleneimine compounds (thiotepa), alkylsulfonate compounds (busulfan), nitrosourea compounds (carmustine), triazene compounds (dacarbazine) There is. Mitosis inhibitors that inhibit mitosis by blocking mitosis as anti-mitotic drugs include plant drugs such as actinomycin D, doxorubicin, bleomycin, and anticancer drugs such as mitomycin, vincristine, and vinblastine. Alkaloids, taxoids including mitosis inhibitors including taxane rings, and the like. In addition, hormones such as corticosteroids and progesterone and platinum-containing compounds such as cisplatin are used as anticancer agents.

The biggest problem with chemotherapeutic agents is drug resistance, which, despite the initial successful response by anticancer agents, is a major factor that eventually causes treatment to fail. In connection with the problem of overcoming these side effects, efforts have recently been made to find the active ingredient in natural products that are used in the private sector.

One of the main reasons why cancer treatment fails in the human body is due to the phenomenon of multidrug resistance (MDR), which causes cancer cells to be resistant to anticancer drugs with different structures or mechanisms of action. P-glycoprotein, It is closely related to the overexpression of a transport protein called P-gp). P-gp is an ATP-dependent transport protein that releases various types of fat-soluble substances, including anticancer drugs, to increase resistance by decreasing the concentration of therapeutic drugs in cancer cells (Juranka et al., FASEB J. , 3 , pp2583). -2592, 1989; Fuqua et al., Cancer Res. , 47 , pp 2103-2106, 1987; Endicott et al., Ann. Rev. Biochem., 58 , pp137-171, 1989; Gottesman et al., Ann. Rev Biochem , 62 , pp 385-427, 1993). P-gp, on the other hand, is also present in many normal organs and also detoxifies toxic substances exposed to living bodies (Theibaut et al., Proc. Natl. Acad. Sci., 84 , pp7735-7738, 1987). Lum et al., Hematol.Oncol.Clin.North Am., 9, pp319-336, 1995; MF Fromm, Int. J. Clin. Pharmacol. Ther. , 38 , pp69-74, 2000; Stenkampf et al, Cancer Res. , 48, pp 3025-3030, 1988).

It is now known that high intakes of fruits or vegetables reduce the risk of cancer (JD Potter, Cancer Lett., 114 , pp7-9, 1997; MJ Wargovich, Cancer Lett., 114 , pp11-17, 1997), Flavonoids abundant in tea, fruits and vegetables have been reported to be involved in the regulation of P-gp activity in human cancer cells and mouse liver cells (Ikegawa et al., Cancer lett. , 177, pp89-93). , 2002; Scambia et al., Cancer Chemother.Pharmacol., 34 , pp 459-464, 1994; Chieli et al., Life Sci., 57 , pp1741-1751, 1995). It has also been reported that flavonoids affect the release of dimethylbenz [α] anthracene, benzo [α] pyrene and adriamycin by P-gp (Yeh et al., Cancer Res. , 52 , pp6692-). 6695, 1992; Phang et al., Cancer Res., 53 , pp5977-5981, 1993; Critchfield et al., Biochem.Pharmacol ., 48 , pp1437-1445, 1994), organic isosomes found in various edible plants It has been reported that isothiocyanates inhibit the production of chemical carcinogens in animal models to prevent cancer induction (Talalay et al., Biochem. Soc. Trans., 24 , pp806-810, 1996; Zhang et al., Cancer Res. ( Suppl ) , 54, pp 1976-1981, 1994).

Derivative compounds of the present invention possible is separated as ginger (Zingiber zerumbet (L.) JESmith. ), Which ginger (Zingiberaceae) as a displacement member (Zingiber) in plants belonging to the plant, is widely distributed in Southeast Asia and remedies Has been used. Examples of the use of creeper plants as a folk remedy include the fact that the roots of various creeper plants in Malaysia include stomach pain, motion sickness, vomiting, epilepsy, sore throat, cough, bruises, wounds, dissolution, eye disease, liver disease, rheumatism, myalgia, ringworm ( Ringworm), asthma, fever, malignancy and boils. In addition, the components of the genus Citrus plants are curcuminoids (Ruby et al., Cancer Lett., 94 , pp79-83, 1995; Ishida et al., Bioorg. Med. Chem., 10 , pp3481-3487, 2002; Vimala et al., Br. J. Cancer, 80 , pp110-116, 1999), flavonoid glycosides (Nakatani et al., Agric. Biol. Chem. , 55 , pp455-460, 1991; Murakami et al., Phytochemistry, 31 , pp2689-2693, 1992), sesquiterpenoids (Murakami et al., Carcino genesis, 23 , pp795-802, 2002) and Polyphenols (Murakami et al., Phytochemistry, 31 , pp2689-2693, 1992) and the like have been reported.

Ginger root has been used in Indonesia as a traditional medicine for treating abdominal pain, diarrhea, hookworm, pain, rheumatism and uterine disease (Ozaki et al., Chem. Pharm. Bull., 39 , pp2353-2356, 1991). In addition, studies on activity include antioxidant activity (Masuda et al., Chem. Lett., 1 , pp 189-192, 1993; Jitoe et al., Tetrahedron Lett., 35 , pp981-984, 1994), anti-inflammatory action ( Ozaki et al., Chem. Pharm. Bull., 39 , pp2353-2356, 1991; Jeenapongsa et al., J. Ethnopharmacol. , 87 , pp143-148, 2003), insecticidal action (Nugroho et al., Phytochemistry , 41 , pp129-132, 1996) and uterine relaxation (Kanjanapothi et al., Planta Med. , 53 , pp329-332, 1987), and studies on the components of the roots of ginger have been reported in Curcuminoids have been reported (Masuda and Jitoe, Phytochemistry, 39 , pp459-461, 1995; Tuntiwachwuttikul et al., Phytochemistry, 20 , 1164-1165, 1981; Masuda et al., Chem. Lett., 1 , pp 189-192, 1993; Jitoe et al., Tetrahedron Lett., 35 , pp981-984, 1994).

However, despite continued studies on the substances isolated from ginger, flavonoid derivatives isolated from Zingiber zerumbet exhibit specific activity against cancer cells that are resistant to conventional anticancer agents by exhibiting P-gp activity inhibition. No teaching or suggestion has been made of its use as an anticancer agent and as a chemosensitizer.

Accordingly, the present inventors have completed the present invention by confirming that the flavonoid derivatives of the present invention that can be separated from ginger exhibit excellent anticancer effects as a result of studying cancer drug treatment agents of herbal origin, which are widely used in folk medicine.

An object of the present invention is to provide an anticancer and anticancer adjuvant composition for the prevention and treatment of cancer diseases comprising the flavonoid derivative compound of the present invention.

In order to achieve the above object, the present invention is an adjuvant for anticancer and anticancer agent for the prevention and treatment of cancer diseases comprising a flavonoid derivative compound of the general formula (I) or a pharmacologically acceptable salt thereof isolated from ginger as an active ingredient To provide a composition.

(I)

(I)

Where

R ₁ , R ₂ and R ₃ are each independently a hydrogen atom, a C1-C3 lower alkyl group,

R ₄ is a C1-C3 lower alkyl group or a rhamnopyranosyl group in which the 1 ′, 2 ′ and 3 ′ position carbon atoms are each independently substituted with one or more hydroxy, methoxy, ethoxy or acetyl groups.

Among the compounds of the general formula (I), preferred compounds are those in which R ₁ to R ₃ are each independently -H or a methyl group, and R ₄ Methoxy or a group of compounds in which the 1 ', 2' and 3 'position carbon atoms are each independently substituted with one or more hydroxy or rhamnopyranosyl groups substituted with an acetyl group.

More preferably, camphorol-3- O -methyl ether (4), camphorol-3,4'- O -dimethyl ether (5) and camphorol-3,4 ', 7- O -trimethyl ether (3), para-hydroxybenzaldehyde (1) and vanillin (2), camphorol-3- O- (2,4- O -diacetyl-alpha-L-ramnopyranoside) (7), Camperol-3- O- (3,4- O -diacetyl-alpha-L-ramnopyranoside) (8), Camperol-3- O- (4- O -acetyl-alpha-L -Ramnopyranoside) (6) compounds.

The cancer diseases include general cancer diseases, preferably gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer , Anal leiomyoma, 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 Cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, renal or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, One or more diseases selected from brain stem glioma, pituitary adenoma, and the like, and particularly all cancer diseases caused by cancer cells that are multidrug resistant to existing anticancer agents.

Hereinafter, the present invention will be described in more detail by way of example, but is not limited thereto.

Flavonoid derivative compounds of the present invention can be obtained from the ginger ( Zingiber zerumbet (L.) JESmith.) Non-polar solvent extract through the following process:

For example, first, the ginger of the present invention is dried, the roots are chopped, and then about 1 to 20 times, preferably about 3 to 10 times, water C ₁ to C ₄ of the sample weight (g). A lower alcohol or a mixed solvent thereof, preferably methanol, at an extraction temperature of 20 ° C. to 100 ° C., preferably 50 ° C. to 70 ° C. for about 1 to 10 days, preferably about 2 to 5 hours. After recovering the supernatant using cold needle, hot water extraction, ultrasonic extraction, reflux cooling extraction, more preferably reflux cooling extraction method, repeat the above procedure 2 to 7 times to collect the supernatant and concentrated under reduced pressure to ginger ginger Solvent soluble extracts can be obtained.

In addition, the non-polar solvent soluble extract of the present invention, after suspending the polar solvent soluble extract in distilled water, the suspension is about 1 to 100 times, preferably about 1 to 5 times the volume of hexane, ethyl acetate, methylene chloride, chloroform and The same nonpolar solvent can be added to extract the nonpolar solvent soluble layer 1 to 10 times, preferably 2 to 5 times, to obtain a nonpolar soluble fraction. It is also possible to further carry out conventional fractionation processes (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis., 3rd Ed. , Pp 6-7, 1998).

More specifically, the fractions having high activity in the ginger non-polar solvent soluble extract obtained by the above process are taken, the fractions having similar R _f values are collected on TLC to form a single fraction, and dried to obtain a purified fraction of purified form. Several fractions obtained by performing silica gel column chromatography using methylene chloride, chloroform, hexane, ethyl acetate, methanol, distilled water or a mixed solvent thereof, preferably using a methylene chloride: methanol mixed solvent. The flavonoid family compounds of the present invention can be obtained by modifying the recrystallization method or Sephadex column chromatographic method conventional in the art, and derivative compounds other than the flavonoid derivative compounds separated from the separation process are replaced with the basic mother core. Substituent The manufacture is possible by a usual substituent in the part synthesis or total synthesis by the manufacturing process, such as well-known alkylation, acetylation in the art. Herbert O. House: Modern Synthetic Reactions , 2nd Ed ., The Benjamin / Cummings Publishing Co., 1972).

The compounds of the present invention represented by the general formula (I) may be prepared with pharmaceutically acceptable salts and solvates according to conventional methods in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and acid or alcohol (eg, glycol monomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid, and the like.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the above general formula (I) include salts of acidic or basic groups which may be present in compounds of general formula (I) unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts, and methods or processes for preparing salts known in the art It can be prepared through.

In order to find out the anticancer effect and the effect as an anticancer adjuvant for cancer cells having multidrug resistance of the compounds of the general formula (I) of the present invention obtained by the above method, representative P-glycoprotein (P-glycoprotein, P-gp) As a result of experiments on the accumulation and excretion of donomycin (DNM), a conventional anticancer agent with verapamil, known as an inhibitor, in cancer cells, the compounds of the present invention are excellent anticancer agents for resistant cancer cells. It was confirmed to exhibit a sensitizing effect.

Accordingly, the present invention also provides an anti-cancer sensitizer (chemosensitizer) due to P-gp activity inhibitory activity comprising the compound of formula (I).

 The present invention also provides a P-gp inhibitor comprising the above general formula (I) compound.

The anticancer and anticancer composition for preventing and treating cancer diseases of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition.

Pharmaceutical compositions comprising the compounds of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compounds of the invention may also be used in the form of their pharmaceutically acceptable salts, and may also be used alone or in other pharmaceutically active compounds, such as anticancer agents well known in the art, namely adriamycin. combination with existing anticancer agents such as adriamycin, cyclophosphamide, 5-FU, amsacrine, danoomycin, taxol, etc. It can be used as an anticancer adjuvant for inhibiting the growth of cancer cells having multi-drug resistance (MDR).

Therefore, the present invention is a compound of the general formula (I) and other anticancer agents used in conventional anticancer therapy, preferably folate derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine), pyrimidine derivatives (5-fluorouracil, Antimetabolites such as Cytarabine); Alkylating agents such as nitrogen mustard compounds (chlorambucil, cyclophosphamide), ethyleneimine compounds (thiotepa), alkylsulfonate compounds (busulfan), nitrosourea compounds (carmustine), triazene compounds (dacarbazine) ); Mitosis inhibitors such as actinomycin D, anti-cancer anticancer agents such as doxorubicin, bleomycin, mitomycin, plant alkaloids such as vincristine and vinblastine, and mitosis inhibitors including taxane rings antimitotic drugs); Hormones such as corticosteroids and progesterone; Platinum-containing anticancer agents such as cisplatin; More preferably, at least one anticancer agent selected from adriamycin, cyclophosphamide, 5-FU, amsacrine, and taxol, and a combination ratio capable of maximizing anticancer activity, eg For example, the present invention provides an anticancer composition in which the ratio of the existing anticancer agent and the general formula (I) compound is combined in a combination ratio of 1: 0.1 to 10 times.

The pharmaceutical compositions comprising the compounds according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the compound 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. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate 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.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

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

The present invention will be described in more detail based on the following examples and experimental examples, but the present invention is not limited thereto.

Example 1 Preparation of Flavonoid Derivatives of the Invention

1-1. ginger( Zingiber zerumbet (L.) J.E.Smith.) Preparation of Crude Extracts

Ginger ( Zingiber cassumunar Roxb.) Was collected and dried in Surabaya, Indonesia, and 500 g of the sample obtained by cutting the roots was placed in 3 l of 100% methanol, and extracted three times at room temperature in an extraction container. Extracted. This process was repeated twice to collect the supernatant, and then concentrated under reduced pressure to give 69.6 g of crude ginger extract.

1-2. Preparation of Ginger Nonpolar Solvent Soluble Fraction

69 g of the crude crude extract obtained in step 1-1 was suspended in 0.5 L of distilled water, and then dissolved by adding 1 L of hexane, which was then dried in vacuo by separating only the components dissolved in the hexane layer. This process was repeated five times to give 16.6 g of hexane fraction. 1 L of chloroform was added to the remaining aqueous layer, and only the components dissolved in the chloroform layer were separated and dried under vacuum. This procedure was repeated five times to give 12.0 g of a chloroform soluble fraction. The same procedure as described above gave 3.3 g of n-butanol fraction and 11.4 g of aqueous layer.

Example 2. Camperol-3- O Preparation of Methyl Ether (Compound 4)

12 g of the chloroform soluble fraction of Example 1 was subjected to silica gel column chromatography (silica gel 60, 230 to 400 mesh, Merck) using a methylene chloride: methanol mixed solvent concentration gradient (1: 0 to 0: 1). Divided into 11 fractions. Recrystallization with methanol from the sixth fraction (0.55 g) separated from this first column yielded 80 mg of camphorol-3- O -methylether (hereinafter referred to as "Compound 4") having the following physical properties.

¹ H-NMR (200 MHz, CDCl ₃ ) δ ppm : 3.87 (s), 6.26 (d, J = 1.8), 6.50 (d, J = 1.8), 7.02 (d, J = 8.8), 8.03 (d, J = 8.8);

¹³ C-NMR (50 MHz, CDCl ₃ ) δ ppm : see Table 1.

Example 3. Camperol-3, 4'- O Preparation of Dimethyl Ether (Compound 5)

12 g of the chloroform soluble fraction of Example 1 was subjected to silica gel column chromatography (silica gel 60, 230 to 400 mesh, Merck) using a methylene chloride: methanol mixed solvent concentration gradient (1: 0 to 0: 1). Divided into 11 fractions. From the sixth fraction (0.55 g) separated from this first column, 62 mg of camphorol-3,4'- O -dimethylether (hereinafter referred to as "Compound 5") having the following physical properties were determined by recrystallization with methanol. Obtained.

¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm : 3.19 (s), 3.88 (s), 6.26 (d, J = 2.0), 6.50 (d, J = 2.0), 7.11 (d, J = 9.2), 8.09 (d, J = 9.2);

¹³ C-NMR (100 MHz, acetone-d ₆ ,) δ ppm : see Table 1.

Example 4. Camperol-3, 4 ', 7- O Preparation of Trimethyl Ether (Compound 3)

12 g of the chloroform soluble fraction of Example 1 was subjected to silica gel column chromatography (silica gel 60, 230 to 400 mesh, Merck) using a methylene chloride: methanol mixed solvent concentration gradient (1: 0 to 0: 1). Divided into 11 fractions. Take fraction 4 (0.60 g) from this first column and use silica gel column chromatography (silica gel 60, 230 to 400 mesh, Merck) with a hexane: ethyl acetate mixed solvent gradient (15: 1 to 1: 1). 26.5 mg of camphorol-3,4 ', 7- O -trimethyl ether (hereinafter referred to as "Compound 3") having the following physical properties were separated by the recrystallization method using acetone. .

¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm : 3.86 (s), 3.87 (s), 3.89 (s), 6.34 (d, J = 2.0), 6.43 (d, J = 2.0), 7.01 (d, J = 9.0), 8.07 (d, J = 9.0);

¹³ C-NMR (100 MHz, CDCl ₃ ) δ ppm : see Table 1.

Example 5. Camperol-3- O -(4- O -Acetyl-alpha-L-lamnopyranoside) (Compound 6)

12 g of the chloroform soluble fraction of Example 1 was subjected to silica gel column chromatography (silica gel 60, 230 to 400 mesh, Merck) using a methylene chloride: methanol mixed solvent concentration gradient (1: 0 to 0: 1). Divided into 11 fractions. Fraction 9 (0.43 g) from this first column was subjected to camphorol-3- O- (4- O ) having the following physical properties using Sephadex LH-20 column chromatography (chloroform-methanol 1: 3). 168 mg of -acetyl-alpha-L-rhamnopyranoside (hereinafter referred to as "Compound 6") was isolated.

¹ H-NMR (400 MHz, acetone- d ₆ ) δ ppm : 0.80 (d, J = 6.4), 2.01 (s), 3.44 (dq, J = 6.4, 9.6), 3.91 (dd, J = 3.4, 9.6) , 4.29 (dd, J = 3.4, 1.2), 4.87 (t, J = 9.6), 5.55 (d, J = 1.2), 6.28 (s), 6.47 (s), 7.05 (d, J = 8.6), 7.84 (d, J = 8.6).

¹³ C-NMR (100 MHz, acetone- d ₆ ) δ ppm : see Table 1.

Example 6. Camperol-3- O -(2,4- O Preparation of -Diacetyl-alpha-L-lamnopyranoside (Compound 7)

Chloroform soluble fraction of Example 1 12 g was subjected to silica gel column chromatography (silica gel 60, 230 to 400 mesh, Merck) using a methylene chloride: methanol mixed solvent concentration gradient (1: 0 to 0: 1) and divided into 11 fractions. Fractions 5 (2.3 g) and 8 (0.15 g) from the first column were combined, and then silica gel column chromatography (9: 1 to 1: 1, finally methanol) was mixed with a hexane: ethyl acetate mixture. Silica gel 60, 230-400 mesh, Merck) was subjected to camphorol-3- O- (2,4- O -diacetyl-alpha-L-lamnopyranoside (hereinafter "compound") having the following physical properties: 791) was obtained.

¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm : 0.80 (d, J = 6.0), 1.94 (s), 1.99 (s), 3.47 (dq, J = 6.4, 9.2), 4.40 (brs), 5.05 ( t, J = 10.4), 5.16 (dd, J = 3.2, 10.4), 5.43 (d, J = 1.2), 6.25 (d, J = 2.0), 6.45 (d, J = 2.0), 7.03 (d, J = 8.8), 7.83 (d, J = 8.8).

¹³ C-NMR (100 MHz, CDCl ₃ ) δ ppm : see Table 1.

Example 7. Camperol-3- O -(3,4- O Preparation of -Diacetyl-alpha-L-lamnopyranoside (Compound 8)

12 g of the chloroform soluble fraction of Example 1 was subjected to silica gel column chromatography (silica gel 60, 230 to 400 mesh, Merck) using a methylene chloride: methanol mixed solvent concentration gradient (1: 0 to 0: 1). Divided into 11 fractions. Fractions 5 (2.3 g) and 8 (0.15 g) from the first column were combined, and then silica gel column chromatography (9: 1 to 1: 1, finally methanol) was mixed with a hexane: ethyl acetate mixture. Silica gel 60, 230 to 400 mesh, Merck Co., Ltd. was carried out to obtain camperol-3- O- (3,4- O -diacetyl-alpha-L-lamnopyranoside (hereinafter "compound") having the following physical properties: 873) 373 mg were obtained.

¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm : 0.81 (d, J = 6.0), 1.99 (s), 2.07 (s), 3.38 (dq, J = 6.4, 9.6), 4.05 (dd, J = 7.2 , 14.4), 4.77 (t, J = 9.6), 5.48 (dd, J = 1.2, 3.6), 5.61 (s), 6.27 (d, J = 2.0), 6.47 (d, J = 2.0), 7.06 (d , J = 8.4), 7.86 (d, J = 8.8).

¹³ C-NMR (100 MHz, CDCl ₃ ) δ ppm : see Table 1.

¹³ C NMR Data of Compound 3-8 location Compound 3 ^a Compound 4 ^b Compound 5 ^b Compound 6 ^b Compound 7 ^a Compound 8 ^a 2 156.1 156.1 156.4 158.1 157.9 158.0 3 139.0 138.5 139.4 135.6 134.6 134.0 4 179.0 178.8 179.4 179.2 178.4 178.3 4a 106.2 105.2 105.9 106.0 105.1 105.0 5 161.9 162.5 163.1 163.2 162.5 162.5 6 98.0 99.5 99.4 99.8 98.9 99.0 7 165.6 164.1 164.8 165.1 164.4 164.4 8 92.3 93.8 94.5 94.8 94.0 94.0 8a 156.9 157.1 157.8 158.7 157.4 157.3 One' 123.0 122.0 123.7 122.6 121.6 121.5 2 '/ 6' 130.4 131.3 130.9 131.8 131.0 131.0 3 '/ 5' 114.3 116.5 114.9 116.5 115.7 115.7 4' 162.0 160.0 162.6 161.1 160.4 160.5 One" 102.5 101.4 98.5 2" 71.7 68.5 71.6 3 " 70.0 71.5 67.3 4" 74.5 70.3 73.7 5 " 69.2 68.5 68.4 6 " 17.9 16.8 16.9 2 "-O C OCH ₃ 169.5 2 "-OCO C H ₃ 20.1 3 "-O C OCH ₃ 169.9 3 "-OCO C H ₃ 20.2 4 "-O C OCH ₃ 171.0 169.6 169.9 4 "-OCO C H ₃ 21.3 20.0 20.2 3-OCH ₃ 60.3 60.3 7-OCH ₃ 55.9 4'-OCH ₃ 56.0 55.9 ^a CDCl _3, ^b acetone- d - ₆

Experimental Example 1. Anticancer and anticancer sensitization experiment

In order to examine the effects on the P-gp activity of the compounds of Examples 2 to 7 P-gp is overexpressed and cytotoxicity, anticancer agent accumulation and in human breast cancer cells (MCF-7 / ADR) resistant to various anticancer agents Ejection experiments were performed. (Skehan et al., J. Natl. Cancer. Inst ., 82 , pp 1107-1112, 1990; Critchfield et al., Biochem. Pharmacol., 48 , pp 1437-1445, 1994; Harker et al., Cancer Res. , 45 , pp 4091-4096, 1985; Yeh et al., Cancer Res. , 52 , pp6692-6695, 1992; Zhang et al., J. Pharmacol.Exp.Ther., 304 , pp 1258-1267, 2004).

1-1. Reagents and Instruments

Cell culture media RPMI 1640, trypsin-EDTA (0.25% trypsin-1 mM EDTA) and penicillin (10,000 units / ml) -streptomycin (10,000 μg / ml) were obtained from Invitrogen (Calsbad, USA). Fetal bovine serum (FBS) was purchased from Hyclone (South Logan, USA). Hanks' balanced salts without sodium (HBSS), dounomycin (DNM), verapamil, DMSO, sulforhodamine B (SRB), sodium bicarbonate, L-glutamine and trichloroacetic acid Trichloroacetic acid (TCA) was purchased from Sigma-Aldrich, St. Louis, USA. NaCl, KCl, MgCl2 are from Duksan pure chemical, Ansan, Korea, CaCl ₂ is from Showa chemical, Tokyo, Japan, and acetic acid is Daejung, Siheung, Korea It was purchased from the company. HEPES (N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid), Triton X-100 and Tris base were purchased from USB (Cleveland, USA), Microscint TM40, scintillation cocktail was purchased from Packard Instrument Co. Inc., Meriden, USA, and [ ³ H] -DNM (1-5 Ci / mmol) was Perkin-Elmer Inc., Wellesley. , USA). 6 well plates and 96 well plates were purchased from BD biosciences (Bedford, USA) and used. Instruments include cell culture (3158, Forma Scientific Inc., Marietta, USA), radiometer (Topcount NXT, Packard Instrument Co. Inc., Meriden USA), orbital shake (SLOS-20, SLB, Seoul, Korea), Inverted Microscope, Axiovert 200, Carl Zeiss, Oberkochen, Germany, and an ELISA Reader (3550, Bio-Rad, Hercules, USA).

1-2. Cell culture conditions

MCF-7 / ADR cells, which are multi-drug resistant human breast cancer cells (P-gp overexpressing cells), were treated with 10% FBS, 10 mM HEPES, 24 mM hydrogen carbonate in a 37 ° C. cell incubator with 5% CO ₂ and 95% air. Cultured in RPMI 1640 medium containing sodium, 2 mM L-glutamine and antibiotics (100 units / ml penicillin-100 μg / ml streptomycin).

1-3. Cytotoxicity experiment

MCF-7 / ADR cells were placed in 96 well plates containing 5,000 revisions per well, and then incubated at 37 ° C. for 24 hours to allow cells to adhere to the plates. Samples of donomycin (DNM) in the range of 9 × 10 ⁻⁸ M to 7.2 × 10 ⁻⁵ M and verapamil (positive control), which are representative P-gp inhibitors at 100 μM, and the sample of Examples 2-7 Put into each well. At this time, instead of the example compounds, a group containing only DMSO solvent was used as a control. After 2 hours, the culture medium (DNM ± Example Compound or Verapamil) was removed and the cells washed twice with HBSS and fresh medium was added. After 3 days, the cytotoxicity was examined by SRB staining. The method is briefly described as follows. Cells were fixed with 10% TCA for 1 hour, washed 5 times with water and dried. SRB reagent (0.4% w / v in 1% acetic acid) was added to each well and washed 4 times with 1% acetic acid after 1 hour. After the plate was dried, the dye bound to the protein was dissolved in 10 mM Tris base for 1 hour to measure the absorbance at 515 nm, and then the IC ₅₀ value was obtained. The IC ₅₀ value refers to the concentration of B that can reduce the size of response A to 50% using a drug called B, assuming that the size of any response A is 100%. Determines that the significance as if the IC ₅₀ value as in Example and then to the compound treated cells by statistical analysis the IC ₅₀ values obtained by Student's test (student's t-test) p value in the control group of 0.05 or less (p <0.05) Statistical It was.

As a result, as shown in Table 2, the remaining five example compounds except Example 6 compound (EA198H-9-34-K1) are as much as Verapamil, a representative P-gp inhibitor, in human breast cancer cells that are resistant to anticancer agents. Although it did not inhibit the activity of P-gp, the value was statistically significant compared to the control and showed almost the same result as the IC ₅₀ value of verapamil.

IC ₅₀ value obtained after incubating the sample with donomycin for 2 hours sample IC ₅₀ (μM) DNM (Voice Control) 37.127 ± 1.054 DNM + Verapamil (positive control) 6.936 ± 0.590 * DNM + Compound 3 15.492 ± 1.642 * DNM + Compound 4 14.557 ± 1.442 * DNM + Compound 5 10.930 ± 1.085 * DNM + Compound 6 35.420 ± 1.402 DNM + Compound 7 19.052 ± 1.662 * DNM + Compound 8 13.831 ± 0.639 * Each data point represents the mean ± S.D. (n = 3) * p <0.0001 compared with control (DNM only)

1-4. [ ³ H] -Donomycin Intracellular Accumulation Impact

Example To study the effect of compounds on P-gp activity, MCF-7 / ADR cells were placed in 6 well plates at 150,000 / well and cultured for 72 hours. In each well, 100 μM concentration of verapamil or cytotoxicity experiments, which were determined to be potential P-gp inhibitors and uptake buffer containing 0.05 μM concentration of [ ³ H] -DNM 1 ml (137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl ₂ , 1.2 mM MgCl ₂ , 10 mM HEPS, pH 7.4) was added and incubated for 2 hours. After removing the input buffer of each well and adding ice-cold stop solution (137 mM NaCl, 14 mM Tris, pH 7.4) to terminate the [ ³ H] -DNM uptake, 1 ml of lysis buffer (1% Triton X-100) was added to each well, shaken for 1 hour at 150 rpm, and 100 µl was taken to measure radioactivity using a radiometer (LSC). The control result and the input result obtained after the treatment of the cells with the example compounds were statistically treated by the student's t-test to determine that the p value was statistically significant (p <0.05). It was.

As a result of the anticancer drug accumulation into human breast cancer cells (MCF-7 / ADR), in the cells treated with Compound 4 and Compound 5 compared with the control group, the accumulation of DNM was increased by Verapamil-treated cells, and Compound 7 and Compound 8 were higher than Verapamil. However, it significantly increased intracellular accumulation of DNM compared to the control. In contrast, Compound 3 did not increase intracellular accumulation of anticancer agents (see FIG. 1).

1-5. From cells ³ H] -donomycin in extracellular excretion Impact

In addition, the effect of the example compounds on the release of DNM from the cells to confirm once again whether or not the increase of the accumulation of DNM in human breast cancer cells inhibited the release of DNM introduced into the cells, such as The experiment was conducted by performing the process.

Put MCF-7 / ADR cells in 6 well plates at 150,000 / well, incubate for 72 hours, and add 1 ml of input buffer containing 0.05 uM of [ ³ H] -DNM to each well. Incubated for 1 hour at to accumulate DNM into cells. Then, after removing the input buffer containing [3H] -DNM and washing the cells, each well containing 100 μM concentrations of Verapamil or cytotoxicity experiments containing the Example compounds that were considered to be a potential P-gp inhibitor After adding the buffer, the cells were incubated for 1 hour. On the other hand, the control group received a drug-free input buffer containing no these substances. After 1 hour, the cells were washed twice with ice-cold stop solution, 1 ml of lysis buffer was added to each well, shaken at 150 rpm for 1 hour, and 100 µl was taken. Radioactivity was measured by (LSC). Since the measured value is the amount of DNM remaining in the cell, the cells were cultured with 0.05 uM of [ ³ H] -DNM for 1 hour immediately after incubating the cells with the example compound for 1 hour with the amount of DNM introduced into the cell. After culturing the cells, the amount of [ ³ H] -DNM released from the cells was measured for 1 hour by subtracting the remaining amount in the cells.

 As a result, in the cells treated with Compound 4 and Compound 5, DNM emission was reduced as much as Verapamil treated cells, and Compound 7 and Compound 8 significantly inhibited the release of DNM as compared to the control group, although not as much as Verapamil. In contrast, Compound 3 did not reduce the release of anticancer drugs from the cells. These results were consistent with the results of the anticancer drug accumulation into the cancer cells (see FIG. 2).

Based on the above results, Compound 4 and Compound 5 did not lower the IC ₅₀ value of DNM as well as Verapamil, a well-known P-gp inhibitor in cytotoxicity experiments. It is considered to be. Therefore, it is expected that the development of anti-cancer sensitizer (chemosensitizer) through the inhibition of P-gp activity is very high. In addition, Compound 7 and Compound 8 also showed almost the same efficacy as verapamil, it was confirmed that there is potential for development as a P-gp inhibitor derived from natural products.

Hereinafter, an example of the preparation of a pharmaceutical composition comprising the compounds 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 1 Preparation of Powder

Compound 3 20 mg

Lactose 100 mg

Talc 10 mg

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

Formulation Example 2 Preparation of Tablet

Compound 3 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

Compound 4 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

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

Formulation Example 4 Preparation of Injection

Compound 5 10 mg

Donomycin 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

_{Na 2 HPO 4 · 12H 2 O} 26 mg

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

Formulation Example 5 Preparation of Liquid

Compound 8 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

As described above, the compounds of formula (I) isolated from the ginger extract of the present invention inhibit the P-glycoprotein (P-gp) activity to increase the cytotoxicity and accumulation of cancer drugs into cancer cells, thereby increasing the anticancer effect Inhibit the growth of cancer. Therefore, the composition containing the compound isolated from the ginger extract of the present invention can be usefully used as an anticancer and anticancer adjuvant composition for the prevention and treatment of cancer diseases.

Claims (8)

An anticancer and anticancer adjuvant composition for preventing and treating cancer diseases comprising the flavonoid derivative compound of Formula (I) or a pharmacologically acceptable salt thereof as an active ingredient:

(I) Where R ₁ , R ₂ and R ₃ are each independently a hydrogen atom, a C1-C3 lower alkyl group, R ₄ is a C1-C3 lower alkyl group or a rhamnopyranosyl group in which the 1 ′, 2 ′ and 3 ′ position carbon atoms are each independently substituted with one or more hydroxy, methoxy, ethoxy or acetyl groups. A compound according to claim 1, wherein in the definition of general formula (I), R ₁ to R ₃ are each independently —H or a methyl group, and R ₄ is A composition wherein the methoxy or 1 ', 2' and 3 'position carbon atoms are each independently a group of compounds substituted with one or more hydroxy or rhamnopyranosyl groups substituted with an acetyl group. The compound of formula (I) according to claim 2, wherein the compound of formula (I) is camperol-3- O -methylether, camphorol-3,4'- O -dimethylether, camphorol-3,4 ', 7- O -trimethyl ether, camphorol-3- O- (2,4- O -diacetyl-alpha-L-ramnopyranoside, camphorol-3- O- (3,4- O -diacetyl- Alpha-L-rhamnopyranoside), camphorol-3- O- (4- O -acetyl-alpha-L-rhamnopyranoside) compound. The cancer disease according to any one of claims 1 to 3, wherein the cancer disease is gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye melanoma, uterine cancer, ovary Cancer, rectal cancer, anal muscle 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, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, A composition comprising one or more diseases selected from spinal cord tumors, brainstem glioma, pituitary adenoma, and the like. The composition according to any one of claims 1 to 3, wherein the cancer disease is any cancer disease caused by cancer cells having multidrug resistance to existing anticancer agents.  An anticancer composition in combination with the general formula (I) compound of claim 1 and at least one anticancer agent selected from cyclophosphamide, 5-FU, amsacrine, donomycin, and taxol.  An anticancer agent due to P-gp activity inhibitory activity comprising the compound of formula (I) according to claim 1. Inhibitor of P-gp activity containing the compound of general formula (I) of Claim 1.

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