KR100597535B1 - Anti-cancer aid composition containing phenyl ring derivatives for preventing and treating cancer disease - Google Patents

Abstract

The present invention relates to an anticancer adjuvant composition which can be separated from a natural substance. Specifically, the composition comprising the phenylpropane-based compound 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 as a drug as an anticancer agent.

Phenylpropane, glycoprotein, multidrug resistance, anticancer adjuvant, anticancer sensitizer, medicine

Description

Anti-cancer aid composition containing phenyl ring 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 the mean ± SD (n = 3), * p value < 0.05, ** p value <0.01, *** p value <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 adjuvant composition for preventing and treating cancer diseases including a phenyl ring derivative compound.

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 can be separated into ginger ( Zingiber cassumunar Roxb.), Which is a genus of Zingiber belonging to the Zingiberaceae plant, widely distributed in Southeast Asia and has been used as a folk remedy . 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 diseases, 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 , pp 459-461, 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 continuous studies on the substances isolated from ginger, these phenyl ring derivatives isolated from the ginger family ( Zingiber cassumunar Roxb.) Exhibited a P-gp activity inhibitory effect, thereby preventing cancer cells resistant to existing anticancer drugs. No teaching or suggestion has been made of its use as a chemosensitizer that specifically acts on.

Accordingly, the present inventors have completed the present invention by confirming that the phenyl ring derivatives of the present invention which can be separated from ginger exhibit excellent anticancer sensitizing 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 adjuvant composition for preventing and treating cancer diseases comprising the phenyl ring derivative compound of the present invention.

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

(I)

(I)

Where

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

(Ia) 기이며, 여기에서 R' 는 하나 이상의 히드록시, 또는 C1 내지 C4 알콕시기가 임의로 치환가능한 치환기이다. R ₄ is a C2 to C6 ethylene group or

(Ia) group wherein R 'is one or more hydroxy, or a substituent wherein a C1 to C4 alkoxy group is optionally substituted.

Among the compounds of the general formula (I), preferred compounds are those in which R ₁ to R ₃ are each independently a hydrogen atom or a methyl group in the definition of general formula (I), and R ₄ is an ethylene group or R 'is an ethoxy group. A group of compounds substituted with formula (Ia).

More preferably, ( E ) -4- (3,4-dimethoxyphenyl) but-1,3-diene (1), ( E ) -4- (2,4,5-trimethoxyphenyl) but- 1,3-diene (2), (±) -trans-3- (3,4-dimethoxyphenyl) -4-[( E ) -3,4-dimethoxystyryl] cyclohexe-1-cene ( 3) compounds.

In addition, the compounds include Alpinia katsumadai Hayata, Alpinia officinarum Hance, Alpinia oxyphylla Miq, Amomum cardamomum Linn., Amomum villosum Lour., Curcuma aromatica Salisb .), Curcuma longa Linn., Curcuma zedoaria Rosc., Zingiber mioga (Thunb. Rosc.) And Ginger ( Zingiber cassumunar Roxb.) Plants, preferably obtained separately from ginger have.

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.

Phenyl ring derivative compounds of the present invention can be obtained from the ginger ( Zingiber cassumunar Roxb.) Non-polar solvent extract through the following process:

For example, the ginger of the present invention ( Zingiber cassumunar Roxb.) Is first dried, the roots are chopped , and then about 1 to 20 times the sample weight (g), preferably about 3 to 10 times the water, C ₁ to C ₄ lower alcohols or mixed solvents thereof, preferably methanol, about 1 hour to 10 days at an extraction temperature of 20 ° C. to 100 ° C., preferably 50 ° C. to 70 ° C., preferably about 2 The supernatant was recovered by using cold soaking, hot water extraction, ultrasonic extraction, reflux cooling extraction, and more preferably reflux cooling extraction for a period of time to 5 hours, and then the above procedure was repeated 2 to 7 times to collect the supernatant. Concentration under reduced pressure may yield a ginger polar solvent soluble extract.

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. Silica gel column chromatography was carried out using methylene chloride, chloroform, hexane, ethyl acetate, methanol, distilled water or a mixed solvent thereof, preferably using a methylene chloride: methanol mixed solvent, and then several The fractions are subjected to recrystallization or Sephadex column chromatography, which are conventional in the art, to obtain the flavonoid compounds of the present invention. Derivative compounds other than the phenyl ring derivative compounds separated from the separation process are separated into the basic mother core. Substituted substituents Conventional substituents can be prepared by partial synthesis or total synthesis through manufacturing processes known in the art such as alkylation and acetylation. 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. Equivalent molar amounts of the compound and acid or alcohol (eg, glycol monomethyl ether) 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 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 investigate the anticancer effect and anticancer effect on 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), an existing anticancer agent with verapamil, known as an inhibitor, as a positive control, the compounds of the present invention are excellent anticancer drugs against 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 adjuvant 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, taxol and anticancer activity It provides an anticancer composition that can be maximized in combination ratio, for example, preferably in a ratio of 1: 0.1 to 10 times the ratio of the existing anticancer agent and the general formula (I) compound.

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 include at least one excipient such as starch, calcium carbonate, sucrose, or the like in the compound. ) 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 is described in more detail based on the following Reference Examples, Examples and Experimental Examples, but the present invention is not limited thereto.

Reference Example 1. Determination of Structure of Compound

The purity of each component was evaluated using analytical TLC for the compounds isolated from the examples below. In the state identified as a single active ingredient, various spectroscopic IR (Bio-Rad Laboratories, USA), Mass (JEOL, Japane), ¹ H-NMR (Varian, CA), ¹³ C-NMR, DEPT, HSQC, HMBC and the like were carried out to determine the structure of the compound as follows.

Example 1. Isolation and Identification of Compounds

 1-1. ginger( Zingiber cassumunar  Roxb.) Preparation of Crude Extracts

Ginger ( Zingiber cassumunar Roxb.) From Surabaya, Indonesia, was collected and dried, and the roots were chopped . 500 g of the sample was placed in 10 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 obtain 50.0 g of crude ginger extract.

   1-2. ginger( Zingiber cassumunar  Roxb.) Preparation of nonpolar solvent soluble fraction

50.0 g of the crude crude extract obtained in step 1-1 was suspended in 1 L of distilled water, and then dissolved by adding 1 L of hexane, and then vacuum-dried by separating only the components dissolved in the hexane layer in the fractionated filter. This process was repeated five times to give 14.3 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 process was repeated five times to give 13.0 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. E Preparation of) -4- (3,4-dimethoxyphenyl) but-1,3-diene (Compound 1)

13.0 g of the most active chloroform fraction of the solvent fraction obtained in Example 1 was subjected to silica gel column chromatography (silica gel 60, 230 to 400 mesh) using a methylene chloride: methanol mixed solvent (100: 1 → 10: 1). , Merck), and separated into 13 fractions, and 1.8 g of the first fraction of these fractions was again subjected to silica gel column chromatography (silica gel 60, using hexane: ethyl acetate mixed solvent (100: 0 → 0: 100)). 230 to 400 mesh, Merck). From fraction 1 therefrom, 143 mg of ( E ) -4- (3,4-dimethoxyphenyl) but-1,3-diene (designated compound 1) having the following physical properties was isolated.

Appearance: pale yellow gum

Molecular Structure: C ₁₂ H ₁₄ O ₂

TLC phase: R _f = 0.5 (hexane: ethyl acetate = 2: 1), colored brown with 10% (v / v) sulfuric acid reagent.

UV (CHCl ₃ ) λ _max (log ε) 293 (4.3), 238.5 (4.1) nm

IR (liquid film) ν _max 2925, 2853, 1597, 1514, 1463, 1419, 1266, 1233, 1138, 1026 cm ^-1

EIMS m / z (%) [M] ⁺ 190 (100), 159 (75), 144 (40), 115 (60), 103 (20), 51 (15)

¹ H NMR (200 MHz, CDCl ₃ ) δ ppm : 3.88 (3H, s), 3.91 (3H, s), 5.12 (1H, br d, J = 9.8 Hz), 5.29 (1H, dd, J = 16.0, 2.0 Hz), 6.45 (1H, d, J = 16.0 Hz), 6.50 (1H, dt, J = 16.0, 9.8 Hz), 6.67 (1H, dd, J = 16.0, 9.8 Hz), 6.82 (1H, d, J = 8.0 Hz), 6.94 (1H, dd, J = 8.0, 1.6 Hz), 6.96 (1H, d, J = 1.6 Hz);

¹³ C NMR (50 MHz, CDCl ₃ ) δ ppm : 56.0, 56.1, 108.9, 111.4, 116.9, 120.1, 128.1, 130.5, 132.8, 137.5, 149.1, 149.3.

 Example 3. E Preparation of) -4- (2,4,5-trimethoxyphenyl) but-1,3-diene (Compound 2)

13.0 g of the chloroform fraction having the highest activity among the solvent fractions obtained in Example 2 was subjected to silica gel column chromatography (silica gel 60, 230 to 400 mesh, using methylene chloride: methanol mixed solvent (100: 1 → 10: 1)). Merck), and separated into 13 fractions, and 1.8 g of fraction 1 of these fractions was again subjected to silica gel column chromatography (silica gel 60, 230) using a hexane: ethyl acetate mixed solvent (100: 0 → 0: 100). To 400 mesh, Merck). In the second fraction therefrom, 60 mg of ( E ) -4- (2,4,5-trimethoxyphenyl) but-1,3-diene (named Compound 2) having the following physical properties was isolated.

Appearance: pale yellow solid

Molecular Structure: C ₁₃ H ₁₆ O ₃

TLC phase: R _f = 0.4 (hexane: ethyl acetate = 2: 1), brown color with 10% (v / v) sulfate reagent.

UV (CHCl ₃ ) λ _max (log δ) 281.5 (4.28), 238.5 (4.26) nm

IR (liquid film) ν _max 2926, 2853, 1607, 1510, 1463, 1401, 1317, 1208, 1034 cm ^-1

EIMS m / z (%) [M] ⁺ 220 (100), 189 (90), 174 (25), 145 (30), 115 (10), 91 (15)

¹ H NMR (200 MHz, CDCl ₃ ) δ ppm : 3.84 (3H, s), 3.88 (3H, s), 3.90 (3H, s), 5.11 (1H, dd, J = 9.8, 1.1 Hz), 5.27 (1H , dd, J = 16.5, 1.1 Hz), 6.50 (1H, s), 6.52 (1H, dt, J = 16.5, 9.8 Hz), 6.68 (1H, dd, J = 16.0, 9.8 Hz), 6.86 (1H, d, J = 16.0 Hz), 7.01 (1H, s);

¹³ C NMR (50 MHz, acetone-d ₆ ,) δ ppm: 56.3, 56.7, 56.9, 97.9, 109.6, 116.2, 118.2, 127.4, 128.3, 138.2, 143.6, 149.8, 151.9.

Example 4. (±) -trans-3- (3,4-dimethoxyphenyl) -4-[( E ) -3,4-dimethoxystyryl] cyclohexen-1-(compound 3)

13.0 g of the chloroform fraction having the highest activity among the solvent fractions obtained in Example 1 was subjected to silica gel column chromatography (silica gel 60, 230 to 400 mesh, using methylene chloride: methanol mixed solvent (100: 1 → 10: 1)). Merck), and separated into 13 fractions, and 1.8 g of fraction 1 was again purified by silica gel column chromatography (100: 0 → 0: 100) using silica gel column chromatography (silica gel 60, 230). To 400 mesh, Merck). 650 g of the combined fractions from Nos. 9 to 10 were again subjected to silica gel column chromatography (silica gel 60, 230 to 400 mesh, Merck) using a hexane: acetone mixed solvent (70: 1 to 0: 100). In the third fraction thus obtained, (±) -trans-3- (3,4-dimethoxyphenyl) -4-[( E ) -3,4-dimethoxystyryl] cyclo having the following physical properties 186 mg of hex-1-cene (designated compound 3) were isolated.

Appearance: pale yellow solid

Molecular Structure: C ₂₄ H ₂₈ O ₄

TLC phase: R _f = 0.5 (hexane: acetone = 3: 2), purple color with 10% (v / v) sulfuric acid reagent.

[α] ²⁵ _D 0.00˚ (acetone, c 0.17)

UV (CHCl ₃ ) λ _max (log ε) 272 (3.9), 239 (3.8) nm

IR (liquid film) ν _max 1650, 1541, 1515, 1458, 1262, 1138, 1028 cm ^-1

EIMS m / z (%) [M] ⁺ 380 (100), 191 (70), 159 (95), 115 (30)

¹ H NMR (200 MHz, CDCl ₃ ) δ ppm : 1.66 (2H, m), 1.93 (1H, dd, J = 12.8, 3.2 Hz), 1.66 (2H, m), 2.22 (1H, m), 2.35 (1H , br d, J = 8.4 Hz), 3.18 (1H, dd, J = 8.4, 2.6 Hz), 3.82 (3H, s), 3.85 (3H, s), 3.86 (3H, s), 3.87 (3H, s ), 5.68 (1H, dd, J = 10.0, 2.6 Hz), 5.90 (1H, br d, J = 10.0 Hz), 6.02 (1H, dd, J = 16.0, 6.8 Hz), 6.10 (1H, d, J = 16.0 Hz), 6.70 (1H, d, J = 2.0 Hz), 6.73 (1H, dd, J = 9.1, 2.0 Hz), 6.77 (2H, d, J = 9.1 Hz), 6.78 (1H, dd, J = 9.1, 2.4 Hz), 6.81 (1H, broad singlet);

¹³ C NMR (50 MHz, acetone-d ₆ ,) δ ppm : 24.7, 28.0, 45.6, 48.2, 56.0, 56.0, 56.1, 56.1, 108.9, 111.0, 111.3, 111.9, 119.0, 120.6, 127.8, 129.1, 130.5, 131.2 , 132.4, 137.8, 147.5, 148.5, 148.8, 149.1

Experimental Example 1. Anticancer and anticancer sensitization experiment

In order to examine the effect on the P-gp activity of the compounds of the above example, cytotoxicity, anticancer accumulation and excretion experiments were performed in human breast cancer cells (MCF-7 / ADR) that are overexpressed and resistant to various anticancer drugs. (Skehan et al., J. Nat'l .. Cancer. Inst ., 82 , pp 1107-1112, 1990; Critchfield et al., Biochem. Pharmacol., 48 , pp 1437-1445, 1994; Harker et al. , Cancer Res. , 45 , pp4091-4096, 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, MgCl ₂ are from Duksan pure chemical, Ansan, Korea, CaCl ₂ is from Showa chemical, Tokyo, Japan, and acetic acid is Daejung, Siheung , Korea). 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 [3H] -DNM (1-5 Ci / mmol) was purchased from 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) ranging from 9 × 10 ⁻⁸ M to 7.2 × 10 ⁻⁵ M concentrations, and verapamil (positive control), a representative P-gp inhibitor at 100 μM concentration, and sample of sample compounds were added to each well. Put it. 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 media 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 of the experiment, the compound of Example 3 (EA204C-6-10-K3), as shown in Table 1, decreased the IC ₅₀ value in MCF-7 / ADR cells more than Verapamil, which is known as a representative P-gp inhibitor. It is thought that there is potential for development as a natural-derived P-gp inhibitor which is superior to the -gp inhibitor.

IC ₅₀ value after incubation of donomycin and sample for 2 hours sample IC ₅₀ value of DNM (μＭ) DNM (Voice Control) 37.127 ± 1.054 DNM + Verapamil (positive control) 6.936 ± 0.590 * DNM + Compound 1 9.134 ± 0.096 * DNM + Compound 2 14.462 ± 0.406 * DNM + Compound 3 4.307 ± 0.401 ** Each data point represents the mean ± S.D. (n = 3) * p <0.0001, ** p <0.00001 compared with control (DNM only)

1-4. [ ³ H] -Danomycin 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. Each well was then treated with 100 μM concentrations of verapamil or cytotoxicity, which was determined to be a potential P-gp inhibitor and uptake buffer containing 0.05 μM [3H] -DNM. 1 ml of 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. Then remove the input buffer of each well, add ice-cold stop solution (137 mM NaCl, 14 mM Tris, pH 7.4) to terminate the [ ³ H] -DNM input, and then dissolve buffer ( 1 ml of lysis buffer) (1% Triton X-100) was added to each well, shaken at 150 rpm for 1 hour, and then 100 µl was taken to measure radioactivity using a radiometer (LSC). The result of the input of the control group and the uptake result obtained after treating 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). .

As a result of the anticancer drug accumulation into human breast cancer cells (MCF-7 / ADR), the cells treated with Compound 3 (EA204C-6-10-K3) increased the accumulation of DNM in cells treated with verapamil compared to the control group. Compound 1 (EA204C-6-7-K1) and Compound 2 (EA204C-6-7-K2) also increased intracellular accumulation of DNM compared to the control, almost equivalent to verapamil (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 of the process was performed.

MCF-7 / ADR cells were placed in 6 well plates at 150,000 / well and incubated for 72 hours, followed by 1 mL of input buffer containing 0.05 uM of [ ³ H] -DNM in each well at 37 ° C. Incubate for 1 hour to accumulate DNM into cells. Then, after removing the input buffer containing [ ³ H] -DNM and washing the cells, each well containing example compounds containing 100 μM concentrations of verapamil or cytotoxicity experiments which were determined to be a potential P-gp inhibitor Add buffer and incubate for 1 hour. On the other hand, the control group was given a drug-free uptake buffer that does not contain 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, the cells treated with Compound 3 (EA204C-6-10-K3) had a lower emission of DNM than the cells treated with Verapamil, and Compound 1 (EA204C-6-7-K1) was almost equivalent to Verapamil. Compared to the control group, it showed a result of significantly suppressing the emission of DNM (see FIG. 2).

Based on the above experimental results, Compound 3 (EA204C-6-10-K3) is far superior to P-gp activity inhibitory effect than verapamil, which is known as a representative P-gp inhibitor until now, and anticancer effect through inhibition of P-gp activity It is expected to be very likely to be developed as a chemosensitizer. In addition, compound 1 (EA204C-6-7-K1) and compound 2 (EA204C-6-7-K2) also show almost the same effect as verapamil, and thus, may be developed 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 1 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 2 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 3 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 3 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 1 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 P-glycoprotein (P-gp) activity and inhibit the production of cancer by accumulation of cancer in cancer cells of cytotoxic and anticancer agents . Therefore, the composition comprising the compound of the present invention isolated from the ginger extract of the present invention can be usefully used as an anticancer agent composition for the prevention and treatment of cancer diseases.

Claims (8)

delete delete delete delete delete delete ( E ) -4- (3,4-dimethoxyphenyl) but-1,3-diene (1), ( E ) -4- (2,4,5-trimethoxyphenyl) but-1,3- Phenyl of diene (2) or (±) -trans-3- (3,4-dimethoxyphenyl) -4-[( E ) -3,4-dimethoxystyryl] cyclohex-1-cene (3) An anticancer agent due to P-glycoprotein activity inhibitory activity, including a ring derivative compound. delete

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