WO2005056001A1

WO2005056001A1 - A use of khellactone derivatives and the composition comprising the same

Info

Publication number: WO2005056001A1
Application number: PCT/KR2004/003137
Authority: WO
Inventors: Hyeong Kyu Lee; Byung Sun Min; Ok Kyoung Kwon; Doo Young Kim; Jung Hee Kim; Joong Ku Lee; Hyouk Joung; Seog Chang Kim
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2003-12-10
Filing date: 2004-12-01
Publication date: 2005-06-23
Also published as: KR20050056487A

Abstract

The present invention is related to novel use of khellactone derivative compounds having anticancer activity and a pharmaceutical composition comprising the same. The present invention provides a pharmaceutical composition for preventing and treating the cancer disease comprising lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer or cancer of the anal region, stomach cancer, colon cancer, breast cancer, gynecologic tumors, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, sarcomas of soft tissues, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphonas, cancer of the bladder, cancer of the kidney or ureter, or neoplasms of the central nervous system, therefore, those compounds can be useful in treating or preventing cancer disease as a anti-cancer agent, health care food, functional health food, or special nutrient food.

Description

Description A USE OF KHELLACTONE DERIVATIVES AND THE COMPOSITION COMPRISING THE SAME Technical Field

[1] The present invention relates to a use of khellactone derivative compounds isolated from Angelica purpuraefolia and the composition comprising the same having anticancer activity. Background Art

[2] Cancer is characterized that cell cluster called as tumor caused by abnormal and uncontrolled cell growth is formed, permeated into neighboring tissue and severed to be transferred to other organ, which is called as neoplasia. Over than 20 million peoples per year are suffered with cancer in the world and among them 6 million people per year were died from the disease. The origin of cancer is classified into internal factor e.g, genetic factor, immunological factor etc and external factor e.g, various chemical substances, radioactive ray, virus etc. Cancer may occur when the balance between oncogene and tumor suppressor genes is collapsed by above explained factors.

[3] Accordingly, there have been needed to develop effective anti-cancer drugs which show potent treating and preventing activity of cancer and low toxicity till now.

[4] There have been reported on the studies to develop anti-cancer drugs, for example, a method for screening cell cytotoxic substances directly acting on cancer cell, a method for screening an immuno-modulating substance, a method for screening an inhibitors of cancer cell metastasis, and a method for screening an inhibitor of an- giogenesis and so on. To use in the prevention and treatment of cancer disease, the substance should satisfy various requirements for examples, a preventing or postponing activity of the metastasis pathway converting normal cell into cancer cell by effectively blocking various carcinogenic mechanisms, a recovering activity from cancer, low or no toxicity such as long-term or acute toxicity, ease use in administration, and low cost etc.

[5] Recently, it have been needed to develop more potent cancer preventive agents and those agents can be useful in preventing cancer disease as well as other involved diseases. Accordingly, the development of cancer preventive agents has been extensively focused on natural product material in the field medicine as well as food chemistry field till now. There have been several reports on the development of phar- macologically active substance showing preventing effect on cancer diseases which was isolated from natural product or food, for example, beta -carotene (Suda, Carcinogenesis, 7, pp711-715, 1986), seseline-type coumarin (Nishino, Carcinogenesis ϋ , pp 1557- 1561, 1990), curcurnin isolated from Curcuma longa (Rao, Carcinogenesis 14. pp2219-2225, 1993), myristicin isolated from parsley(Zheng, Carcinogenesis 13, ppl921-1923, 1992), epigallocatechin-3-gallic acid (Katiyar, Nutrition and cancer 18. pp73, 1992) and so on.

[6] According to recently published literature (Annual reports of Medicinal Chemistry), it has been reported that about 60% newly approved anti-cancer agents in FDA from 1983 to 1994 were developed by isolating the agents from natural product or modifying its structure by semi-synthesis or total synthesis based on the agents. Among the 93 species of anticancer drugs, 6 species of the drugs were derived from a microorganism such as an interferon, interleukin-2 etc, 15 species from natural resources such as paclitaxel, vincristine, vinblastine, doxorubicin etc, and 25 species were developed being modifying the substance isolated from natural resource by semi- synthesis such as etoposide, irinotecan HC1 etc, 14 species by total-synthesis such as cytosine arabinoside, fluorouracil etc.

[7] As described above, there have been lots of reports on the substances showing potent preventing or treating effect on cancer disease isolated from natural resource, especially plant resource. Since the plant extract shows various advantages such as low toxicity due to long-term administration, ease production of materials, relatively stable storage against external environment etc, the development of an anti-cancer agent from plant extract has been highlighted till now.

[8] However, there has been not reported or disclosed on the preventing or treating activity of khellactone derivative compounds showing potent anticancer effect in any of above cited literatures, the disclosures of which are incorporated herein by reference.

[9] To investigate the anti-cancer effect of khellactone derivatives compound of the present invention, the inventors of present invention have intensively carried out in vitro experiment concerning the inhibition effect on the mouse solid cancer cell line such as LLC, HL-60, K562 and human solid cancer cell line such as SK-OV-3. As a result of the investigation, the inventors finally completed the present invention by confirming that the khellactone derivative compounds of the present invention strongly inhibited the cancer cell line and it can be useful as a potent anti-cancer agent. Disclosure [10] Thus, the present invention provides The present invention also provides a pharmaceutical composition comprising khellactone derivatives compound represented by the following general foπmla (I), or the pharmacologically acceptable salt thereof as an active ingredient in an effective amount to treat and prevent cancer diseases.

(I)

[11] wherein [12] R and R is at least one selected from the group consisting of hydrogen atom, C -C 1 2 1 lower alkyl, alkoxy group, C -C lower alkyl oxy group, propanoyl group, 2-methyl 3 1 3 propanoyl group, ketone group, 2,3-ethoxy-methyl butanoyl group, gucoside group, sulfate group, acetyl group, benzoyl group, angeloyl group, tigloyl group, 2,7-dimethyl-5-oxo-2,6-octadienol group, 4-hydroxycinnamoyl group, methanesulfonyl group, p-toluene sulfonyl group, trifluoroacetyl group, citryl group, maleyl group, succinyl group, oxalyl group, benzoyl group, tartaryl group, fiπnaryl group, manderyl group, propionic group, latinic group, glycolic group, glyconic group, galacturonic group, glutamic group, gluraryl group, glucuronic group, aspartic group, ascorbic group, carbonic group, vanilic group, and hydroiodic group independently.

[13] The compounds of general formula (I) comprise khellactone derivatives of which R , R substitutes are independently hydrogen atom, C -C lower alkyl or C -C lower 1 2 1 3 1 3 alkyl oxy group, preferably hydrogen atom , C -C lower alkyl oxy group , more 1 3 preferably, which can be represented by following chemical foπmla (I a) isolated from Angelica purpuraefolia Chung and 9,10-diacetylkhellactone.

(la) [14] 1)R , R = H : Khellactone 1 2

[15] 2)R , R = COCH : 9,10-diacetylkhellactone 1 2 3

[16] The present invention also provides a use of a compound represented by general foπmla (I), the pharmaceutically acceptable salt or the isomer thereof, for the manufacture for treating or preventing cancer disease in a mammal including human in need thereof.

[17] It is the other object of the present invention to provide a method of treating or preventing cancer disease in a mammal comprising administering to said mammal an effective amount of khellactone derivatives compoundrepresented by general foπmla (I), or the pharmacologically acceptable salt thereof, together with a pharmaceutically acceptable carrier thereof.

[18] Above described cancer disease defined herein means preferably solid cancer or blood cancer and the like .

[19] The khellactone derivative compounds of the present invention can be prepared by following procedures.

[20] The inventive khellactone compounds isolated from the extract of Angelica pur- pur aefolia can be prepared by following procedures.

[21] R)r example, Angelica purpuraefolia is dried, cut, crushed and mixed with 2 to 10-fold, preferably, about 3 to 5-fold volume of distilled water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol and the like, or the mixtures thereof, preferably, the mixture of approximately ratio of 1:0.1 to 1:10, more preferably, with methanol, and then is subjected to the extraction, reflux extraction, or ultra-sonication extraction at the temperature ranging from R.T to 100°C for the period ranging from 5 hours to 1 day and repeated 2 to 7 times, preferably 5 times, consecutively. The residue is filtered to obtain the supernatant to be concentrated with rotary evaporator, and then concentrated under redxed pressure to obtain crude extract of Angelica purpuraefolia .

[22] Also, non-polar solvent soluble extract of present invention can be prepared by following procedure, for example, the crude extract prepared by above described step, is suspended in water, and then is mixed with 1 to 100-fold, preferably, 1 to 5-fold volume of non-polar solvent such as ethyl acetate, chloroform, hexane and the like and fractionated 1 to 10 times, preferably 2 to 5 times, the non-polar solvent soluble layer is collected to obtain non-polar solvent soluble extract of the present invention. Also, above described procedures may be modified or subjected to further step to fractionate or isolate more potent fractions or compounds by conventional procedure well-known in the art, for example, the procedure disclosed in the literature (Harborne J. B. Phy- tochemical methods: A guide to modern techniques of plant analysis, 3 Ed. pp6-7, 1998).

[23] More specifically, above described non-polar solvent soluble extract, preferably, hexane soluble fraction showing potent pharmacological activity is subjected to adsorption column chromatography. The column is eluted with a stepwise application of solvent mixture containing linear gradient, i.e., starting with 100% hexane, followed by increased ratio of EtOAc in hexane (0% ~ 100%) to give 5 - 8 sub-fractions. Then, the fraction is subjected to further column chromatography repeatedly in order to obtain khellactone (compound la) of the present invention.

[24] Alternatively, The inventive khellactone compounds of the present invention may be also synthesized by the conventional synthetic method in accordance with the using method well known in the art (Herbert O. House, Modern Synthetic Reactions, 2" Ed., The Benjamin/Curnmings Publishing Co., 1972).

[25] Moreover, the ester, ether or salt of khellactone compounds may be prepared by conventional method for synthesizing ester or ether well known in the art.

[26] R)r example, the ether or ester derivative of khellactone may be prepared by following procedure: khellactone is dissolved in excessive inert organic solvent such as methylenchloride, chloroform etc, and reacted with reacting substance such as acetic acid in the presence of catalysts such as DCC (1,3-dicyclohexylcarboimide) and base such as 4-dimethyl amino pyridine in the temperature ranging from -10 to 5 °C, preferably at 0°C . The resulting product is extracted with organic solvent and purified by silica gel column chromatography to obtain the ester or ether of khellactone compound.

[27] The inventive compounds represented by general formula (I) can be transformed into their pharmaceutically acceptable salt and solvates by the conventional method well known in the art. Ibr the salts, acid-addition salt thereof formed by a pharmaceutically acceptable free acid thereof is useful and can be prepared by the conventional method. Ibr example, after dissolving the compound in the excess amount of acid solution, the salts are precipitated by the water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile to prepare acid addition salt thereof and further the mixture of equivalent amount of compound and diluted acid with water or alcohol such as glycol monomethylether, can be heated and subsequently dried by evaporation or filtrated under reduced pressure to obtain dried salt form thereof.

[28] As a free acid of above-described method, organic acid or inorganic acid can be used. Ibr example, organic acid such as methansulfonic acid, p-toluensulfonic acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonylic acid, vanillic acid, hydroiodic acid and the like, and inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used herein.

[29] Further, the pharmaceutically acceptable metal salt form of inventive compounds may be prepared by using base. The alkali metal or alkali-earth metal salt thereof can be prepared by the conventional method, for example, after dissolving the compound in the excess amount of alkali metal hydroxide or alkali-earth metal hydroxide solution, the insoluble salts are filtered and remaining filtrate is subjected to evaporation and drying to obtain the metal salt thereof. As a metal salt of the present invention, sodium, potassium or calcium salt are pharmaceutically suitable and the corresponding silver salt can be prepared by reacting alkali metal salt or alkali-earth metal salt with suitable silver salt such as silver nitrate.

[30] The pharmaceutically acceptable salt of the compound represented by general foπmla (I) comprise all the acidic or basic salt which may be present at the compounds, if it does not indicated specifically herein. Ibr example, the pharmaceutically acceptable salt of the present invention comprise the salt of hydroxyl group such as the sodium, calcium and potassium salt thereof; the salt of amino group such as the hydrogen bromide salt, sulfuric acid salt, hydrogen sulfuric acid salt, phosphate salt, hydrogen phosphate salt, dihydrophosphate salt, acetate salt, succinate salt, citrate salt, tartarate salt, lactate salt, mandelate salt, methanesulfonate(mesylate) salt and ? - toluenesulfonate (tosylate) salt etc, which can be prepared by the conventional method well known in the art.

[31] To investigate the effect of the khellactone derivative compounds on the cancer disease, in vitro and in vivo experiment concerning the inhibition effect on the mouse solid cancer cell line (LLC), human leukemia cancer cell lines (HL-60, K562) and human solid cancer cell line (SK-ON-3) were performed. As a result of the investigation, it is confirmed that the khellactone derivative compounds of the present invention showed potent inhibiting activity of the cancer cell line and did not show any toxic activity or clinical change.

[32] The present invention also provides a pharmaceutical composition comprising an efficient amount of the compound represented by general formula (I) or the pharmaceutically acceptable salt thereof as an active ingredient in amount effective to treat or prevent cancer diseases together with pharmaceutically acceptable carriers or diluents .

[33] The pharmaceutical composition of the present invention can contain about 0.01 ~ 50 % by weight of the above extract based on the total weight of the composition.

[34] The compound of formula (I) according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents. Ibr example, the compounds of the present invention can be dissolved in oils, propylene glycol or other solvents which are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. Ibr topical administration, the compounds of the present invention can be formulated in the form of ointments and creams.

[35] The pharmaceutical compositions comprising the compound of the present invention can be treat and prevent various cancer disease, for example , lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer or cancer of the anal region, stomach cancer, colon cancer, breast cancer, gynecologic tumors (e.g, uterine sarcomas, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina or carcinoma of the vulva), Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system (eg, cancer of the thyroid, parathyroid or adrenal glands), sarcomas of soft tissues, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphonas, cancer of the bladder, cancer of the kidney or ureter (e.g, renal cell carcinoma, carcinoma of the renal pelvis), or neoplasms of the central nervous system (e.g, primary CΝS lymphoma, spinal axis tumors, brain stem gliomas or pituitary adenomas), preferably solid cancer such as lung cancer or blood cancer. [36] The compound of the present invention has potent anti-cancer activity, and the pharmaceutical composition of the present invention thus may be employed to treat or prevent the cancer disease.

[37] Hereinafter, the following foπmlation methods and excipients are merely exemplary and in no way limit the invention.

[38] The compounds of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

[39] The compounds of the present invention may be formulated into preparations for injections by dissolving, suspending, or eimlsifying them in aqueous solvents such as normal saline, 5% Dextrose, or non-aqueous solvent such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol. The foπmlation may include conventional additives such as solubilizers, isotonic agents, suspending agents, eimlsifying agents, stabilizers and preservatives.

[40] The desirable dose of the inventive compounds varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging 0.0001 - 100 mg/kg, preferably 0.001 - 100 mg/kg by weight/day of the inventive compounds of the present invention. The dose may be administered in single or divided into several times per day. In terms of composition, the compounds should be present between 0.0001 to 10% by weight, preferably 0.0001 to 1% by weight based on the total weight of the composition.

[41] The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intrathecal, epidural or intracerebroventricular injection.

[42] The khellactone derivative compounds of the present invention also can be used as a main component or additive and aiding agent in the preparation of various functional health food and health care food.

[43] Accordingly, it is the other object of the present invention to provide a functional health food comprising khellactone derivative compoundsrepresented by the following general formula (I), or the pharmacologically acceptable salt thereof for aiding cancer chemotherapy.

[44] The term 'a functional health food' defined herein means the functional food having enhanced functionality such as physical functionality or physiological functionality by adding the compound of the present invention to conventional food to prevent or improve cancer disease in human or mammal.

[45] It is the other object of the present invention to provide a health care food or special nutritional food comprising khellactone derivatives compound represented by the following general foπmla (I), or the pharmacologically acceptable salt thereof, together with a sitologically acceptable additive for the prevention and alleviation of cancer disease.

[46] The term 'a health care food' defined herein means the food containing the compound of the present invention showing no specific intended effect but general intended effect in a small amount of quantity as a form of additive or in a whole amount of quantity as a form of capsule, pill, tablet etc.

[47] The term 'a sitologically acceptable additive' defined herein means any substance the intended use which results or may reasonably be expected to result-directly or indirectly-in its becoming a component or otherwise affecting the characteristics of any food' for example, thickening agent, maturing agent, bleaching agent, se- questerants, humectant, anticaking agent, clarifying agents, curing agent, eimlsifier, stabilizer, thickner, bases and acid, foaming agents, nutrients, coloring agent, flavoring agent, sweetner, preservative agent, antioxidant, etc, which shall be explained in detail as follows.

[48] If a substance is added to a food for a specific purpose in that food, it is referred to as a direct additive and indirect food additives are those that become part of the food in trace amounts due to its packaging, storage or other handling

[49] The term 'special nutritional food' defined herein means the food containing the compound of the present invention showing specific intended effect by supplementing a substitute food such as a baby food or nutrient meal used in the baby or patient in need of special nutrient or ingredients as a form of food.

[50] Above described health foods can be contained in food, health beverage, dietary therapy etc, and may be used as a form of powder, granule, tablet, chewing tablet, capsule, beverage etc for preventing or improving cancer disease.

[51] Also, above described compounds can be added to food or beverage for prevention and improvement of cancer disease. The amount of above described compound in food or beverage as a functional health food or health care food may generally range from about 0.01 to 100 w/w % of total weight of food for functional health food composition. In particular, although the preferable amount of the compound of the present invention in the functional health food, health care food or special nutrient food may be varied in accordance to the intended purpose of each food, it is preferably used in general to use as a additive in the amount of the compound of the present invention ranging from about 0.01 to 5% in food such as noodles and the like, from 40 to 100% in health care food on the ratio of 100% of the food composition.

[52] Providing that the health beverage composition of present invention contains above described compound as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional stgar such as dextrin, cyclodextrin; and stgar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be usefiil favorably. The amount of above described natural carbohydrate isgenerally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100ml of present beverage composition.

[53] The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese, chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition. Examples of addable food comprising aforementioned compound therein are various food, beverage, gum, vitamin complex, health improving food and the like.

[54] The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner. Description Of Drawings [55] The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

[56] Eg 1 shows the anti-cancer effect on LLC solid cancer cell line of the groups treated with saline solution as a negative control, adriamycin as a positive control and khellactone(10, 20mg/kg) in animal model. Mode for Invention

[57] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[58] The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

[59] EXAMPLES

[60] The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

[61] Example 1. Preparation of the methanol extract of Angelica purpuraefolia

[62] 1kg of dried Angelica purpureaolia distributed in Korea was cut into about 1mm of small pieces, mixed with 4 L of methanol and extracted for 8 hours at 70°C. Above extraction steps were repeated five times to collect supernatant and the supernatant was concentrated under redxed pressure to obtain 65g of methanol extract (yield: 6.5%).

[63] Example 2. Preparation of polar solvent and non-polar solvent soluble extract

[64] 65g of MeOH extract prepared in Example 1 was suspended with 1 L of distilled water and 1 L of chloroform was added thereto. The solution was subjected to frac- tionation with chloroform layer and water layer five times and then the collected hexane layer was dried to obtain 34g of chloroform fraction. 1 L of ethylacetate was added to remaining water layer and then the solution was subjected to fractionation with ethylacetate layer and water layer. The fractionation was repeated five times to collect lg of ethylacetate soluble fraction.

[65] Example 3. Preparation of khellactone derivative compound

[66] 3-1. Preparation of Khellactone

[67] 34g of chloroform soluble fraction prepared in Example 2 was subjected to Silica gel column chromatography to isolate khellactone derivative compounds. [68] 34g of chloroform soluble fraction was loaded onto the Silica gel column and the column was eluted with a stepwise application of solvent mixture containing linear gradient of hexane:acetone (50:1 ?> 1:1) and chloroform:methanol (9:1 ?>? 4:1) to give 9 sub-fractions of chloroform soluble fraction. Among the 9 fractions, the 7 fraction was further loaded onto the Silica gel column and the column was eluted with a stepwise application of solvent mixture containing linear gradient of hexane:chloroform (2:1 ?> 1:1) to give 6 sub-fractions. Among the 6 fractions, the 4 fraction was further loaded onto the Silica gel column and the column was eluted with reverse phase C-18 column chromatography with an eluting solvent mixture of 50% acetonitrile to give khellactone showing following physicochemical properties.

[69] Khellactone:C H O_ (MW:262^ 11. 14. 5.

[70] IR: 3580, 3400, 1780, 1680 cm^"1;

[71] Specific rotation: [ alpha ] +110 (c=0.91, MeOH); D

[72] ^λ H-NMR (300MHz, CDC1 ): delta 7.67 (1H, d, 7=9.3, H-4), 7.34 (1H, d, 7=8.7, H- 3 5), 6.81 (1H, d, 7=8.7, H-6), 6.27 (1H, d, 7=9.3, H-3), 5.23 (1H, d, 7=5.1, H-4'), 3.89 (1H, d, 7=5.1, H-3'), 1.48 (3H, CH ), 1.43 (1H, CH ). 3 3

[73] 3-2. Preparation of diacetyl khellactone

[74] 262mg of khellactone (lmM) prepared from above step was dissolved in 20ml of methylene chloride, mixed with 144mg of acetic acid (2.4mM), 494mg of 1,3-dicyclohexylcarbodiimde (DCC) and 98mg of 4-DMAP (dimethyl amino pyridine, 0.8mM) and reacted at 0 ° C for 2 hours. Appropriated amount of distilled water was added thereto and the suspension was extracted with ethyl acetate to obtain EtOAc soluble layer. The layer was fiirther subjected to silica gel column chromatography with a solvent mixture of hexane: EtOAc (4:1) as an eluting solvent to isolate 250mg of 9,10-O-diacetylkhellactone.

[75] 3-3. Preparation of khellactone ester and ether derivatives

[76] Excepting that methansulfonic acid, p-toluensulfonic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonylic acid, vanillic acid and hydroiodic acid were used as a reacting substance in stead of acetic acid, various esters or ethers of khellactone derivative were synthesized t hrotgh similar procedure to the disclosed in Example 3-2.

[77] Experimental Example 1. Inhibition Effect on tumor cell growth using in vitro model [78] To determine the inhibiting activity of the khellactone derivative compounds for the cancer cell growth, following experiment was performed.

[79] 1-1. Cell culture

[80] LLC (Lewis lung carcinoma) cell line (mouse lung cancer cell line, ATCC Co. USA) and HL-60 (human blood cancer cell line, ATTC Co., USA) were cultivated in RPMI 1640 culture medium containing 100 units/ml of penicillin-streptomycin (GIBCO) and 10% PBS (Fetal bovine serum, GIBCO) at 37 ° C in 5% CO condition. 2 The subculture of cell line was performed once for 3 - 4 days.

[81] 1-2. Determination of inhibition effect on cancer cell

[82] Each LLC and HL-60 cell line was poured into 96 well plates and various concentrations of sample ranging from 80?g /ml to 1 ?g /ml were added thereto. 0.5% DMSO was used as a negative control and 2mg/kg of adriamycin was used as a positive control. The plates were cultivated for 48 hours and the culture was dissolved in lysis buffer. The reaction mixture containing MTT (3-(4,5-demethylthiazol)-2,5-diphenyltetrazolium bromide, Sigma Co., USA ) was added thereto and cultivated at 37 ° C for 50 minutes. The plates were subjected to centriftgation in the speed of 1000 rpm for 10 mins and remaining medium was removed gently. The absorbance of supernatant was determined by microplate reader (Benchmark, BIO-RAD , Japan ) at 610 nm (Scott. G., 7. Clin. Microbial., 36, pp362-366, 1998).

[83] IC values of khellactone for both LLC and HL-60 cell line showed about 20.0 ?g 50 /ml. [84] Experimental Example 2. Inhibition Effect on tumor cell growth using in vivo mouse model

[85] To determine the inhibiting activity of the khellactone derivative compounds the cancer cell growth, following experiment was performed.

[86] lour weeks aged BDF-1 female mice weighing about 15g (Daehan Biolink Co.) were used and 25 mice were divided into five groups. After the acclimation period for 1 week, sub-cultured LLC (Lewis lung carcinoma) cell lines (mouse lung cancer cell line, ATCC Co. USA) were floated to reach at the cell concentration of about 1,000,000 cells/ml as a cancer initiator and each 0.1 ml of cell was transplanted to the left thigh region of healthy mice in order to giving rise to cancer. After the day of transplantation of solid cancer, each mouse was treated with 0.1 ml of physiological saline solution as a negative control, 2mg/kg of adriamycin as a positive control, lOmg/kg of khellactone and 25mg/kg of khellactone as test groups respectively. [87] After the tumor transplantation, the length and volume of tumor were estimated from 14 day the end of transplantation once a 2 days and the estimated values of tumor growth were transformed into percentage (%) according to the literature (Yatsunami J., Int. J. Oncol, 17, ppl 151-1156, 2000).

[88] At the result, adriamycin showed 56.6% of inhibiting effect, while khellactone showed 58.7% in lOmg/kg treatment group and 60.1% in 25mg/kg treatment group ( See Table 1). The khellactone treatment group did not showed any change of appearance such as weight loss and toxicity in lOmg/kg and 25mg/kg treatment groups, respectively ( See Table 2).

[89] [Table 1] [90]

[91] [92]

[93] Experimental Example 3. Toxicity test [94] Methods [95] The acute toxicity tests for the test compounds on six weeks aged SPF SD rats were performed by following procedure. [96] Rur groups consisting of 2 rats was administrated orally with lOOmg/kg of khellactone compound and observed for 2 weeks. [97] Results

[98] There were no treatment-related effects on mortality, clinical signs, body weight changes and gross findings in any group or either gender. The miniimm LD value in 50 oral administration was more than 100g/kg These results suggested that the test compounds prepared in the present invention were potent with safe. [99] Hereinafter, the foπmlating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows. [100] Preparation of powder

[101] Khellactone 50mg

[102] Lactose lOOmg

[103] Talc lOmg

[104] Powder preparation was prepared by mixing above components and filling sealed package. [105] Preparation of tablet

[106] Khellactone 50mg

[107] Corn Starch lOOmg

[108] Lactose lOOmg

[109] Magnesium Stearate 2mg

[110] Tablet preparation was prepared by mixing above components and entabletting

[111] Preparation of capsule

[112] Khellactone 50mg

[113] Corn starch lOOmg

[114] Lactose lOOmg

[115] Magnesium Stearate 2mg

[116] Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method. [117] Preparation of injection

[118] Khellactone 50mg

[119] Distilled water for injection optiimm amount

[120] PH controller optiimm amount

[121] Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2

[122] ml ample and sterilizing by conventional injection preparation method. [123] Preparation of liquid

[124] Khellactone 0.1~80g

[125] Stgar 5~10g

[126] Citric acid 0.05-0.3%

[127] Caramel 0.005-0.02%

[128] Vitamin C 0.1-1%

[129] Distilled water 79-94%

[130] CO gas 0.5-0.82% 2

[131] liquid preparation was prepared by dissolving active component, filling all the components and sterilizing by conventional liquid preparation method.

[132] Preparation of health care food

[133] Khellactone lOOOmg

[134] Vitamin mixture optiimm amount

[135] Vitamin A acetate 70 m g

[136] Vitamin E l.Omg

[137] Vitamin B 0.13mg

[138] Vitamin B 0.15mg 2

[139] Vitamin B 0.5mg 6

[140] Vitamin B 0.2 m g 12

[141] Vitamin C lOmg

[142] Biotin 10 m g

[143] Amide nicotinic acid 1.7mg

[144] Iblic acid 50 m g

[145] Calcium pantothenic acid 0.5mg

[146] Mneral mixture optiimm amount

[147] Feπous sulfate 1.75mg

[148] Zinc oxide 0.82mg

[149] Magnesium carbonate 25.3mg

[150] Monopotassium phosphate 15mg

[151] Dicalcium phosphate 55mg

[152] Potassium citrate 90mg

[153] Calcium carbonate lOOmg

[154] Magnesium chloride 24.8mg [155] The above-mentioned vitamin and mineral mixture may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention. [156] Preparation of health beverage

[157] Khellactone lOOOmg

[158] Citric acid lOOOmg

[159] Oligosaccharide lOOg

[160] Apricot concentration 2g

[161] Taurine lg

[162] Distilled water 900 ml

[163] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 °C for 1 hour, filtered and then filling all the components in 1000 ml ample and sterilizing by conventional health beverage preparation method. [164] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims. Industrial Applicability [165] As described in the present invention, the khellactone derivative compounds of the present invention showed potent inhibiting effect on the cancer cell and no toxicity, therefore, those compounds can be useful in treating or preventing cancer disease as a anti-cancer agent, health care food, functional health food, or special nutrient food.

Claims

[1] 1. A pharmaceutical composition comprising khellactone derivative compound srepresented by the following general formula (I), or the pharmacologically acceptable salt thereof as an active ingredient in an effective amount to treat and prevent cancer diseases:

(I) wherein R and R is at least one selected from the group consisting of hydrogen 1 2 atom, C -C lower alkyl, alkoxy group, C -C lower alkyl oxy group, 1 3 1 3 propanoyl group, 2-methyl propanoyl group, ketone group, 2,3-ethoxy-methyl butanoyl group, gucoside group, sulfate group, acetyl group, benzoyl group, angeloyl group, tigloyl group, 2,7-dimethyl-5-oxo-2,6-octadienol group, 4-hydroxycinnamoyl group, methanesulfonyl group, p-toluene sulfonyl group, trifluoroacetyl group, citryl group, maleyl group, succinyl group, oxalyl group, benzoyl group, tartaryl group, fumaryl group, manderyl group, propionic group, latinic group, glycolic group, glyconic group, galacturonic group, glutamic group, gluraryl group, glucuronic group, aspartic group, ascorbic group, carbonic group, vanilic group, and hydroiodic group independently.

[2] 2. The composition according to claim 1, wherein said compound is khellactone or 9,10-diacetylkhellactone.

[3] 3. The composition according to claim 1, wherein said cancer disease is solid cancer or blood cancer.

[4] 4. A use of a compound represented by the following general foπmla (I) as set forth in claim 1, the pharmaceutically acceptable salt or the isomer thereof, for the manufacture for treating or preventing cancer disease in a mammal including human in need thereof.

[5] 5. A functional health food comprising khellactone derivative compounds represented by general foπmla (I) as set forth in claim 1, or the pharmacologically acceptable salt thereof for aiding cancer chemotherapy.

[6] 6. A health care food comprising khellactone derivatives compound represented by general foπmla (I) as set forth in claim 1, or the pharmacologically acceptable salt thereof, together with a sitologically acceptable additive for the prevention and alleviation of cancer disease.

[7] 7. A special nutritional food comprising khellactone derivatives compound represented by general foπmla (I) as set forth in claim 1, or the pharmacologically acceptable salt thereof, together with a sitologically acceptable additive for the prevention and alleviation of cancer disease.