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WO2005116042A1 - Treatment and prevention of cancer with new ginsenoside derivatives - Google Patents

Treatment and prevention of cancer with new ginsenoside derivatives Download PDF

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WO2005116042A1
WO2005116042A1 PCT/KR2005/001006 KR2005001006W WO2005116042A1 WO 2005116042 A1 WO2005116042 A1 WO 2005116042A1 KR 2005001006 W KR2005001006 W KR 2005001006W WO 2005116042 A1 WO2005116042 A1 WO 2005116042A1
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dammarane
glucopyranosyl
ginsenoside
diol
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Myung Hwan Park
Tae Yoon Park
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/207—Cyclohexane rings not substituted by nitrogen atoms, e.g. kasugamycins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane

Definitions

  • the present invention relates to ginsenoside derivatives effective in prevention and treatment of cancers. More specifically, the present invention relates to ginsenoside derivatives with novel structures effective in prevention and treatment of cancers, methods of their preparation, and a pharmaceutical composition comprising the above novel compounds as active ingredients.
  • angiogenesis inhibitor as an anticancer drug.
  • the agent can block both growth and metastasis of any cancer because angiogenesis is an essential step for growth and metastasis of cancer.
  • drug resistance which is normally induced by the heterogenecity and genetic instability of cancer cells, because an angiogenesis inhibitor is not targeted to aneuploid cancer cells but targeted to attack diploid endothelial cells.
  • its efficacy is not limited to certain specific kinds of cancer but is applicable to all kinds of cancer which require angiogenesis.
  • ginseng has been known for thousand years as a plant which contains a non-toxic tonic to improve homeostasis of human, has therapeutic effects in many diseases, and is capable of extending extend human life expectancy.
  • the effect of expanding life expectancy of ginseng is due to prevention of aging by its antioxidant activity (Korean Biochem. J., 12(1), 33 (1979)) and there have been proposed hypotheses and relevant experimental evidences that can be achieved by the inhibition and prevention of development of cancer (Ann. NY Acad.
  • Anticancer active ingredients in ginseng are known as ginsenosides Rg3, Rh2, Rhl, and Compound K, an enteric microbial metabolite of ginsenoside and the like (J. Korean Med. Sci., l ⁇ (Suppl), S28 (2001); J. Ginseng Res., 28, 1 (2004)).
  • ginsenoside Rg5 which is contained only a very small amount in red ginseng has a strong anticancer effect, and this effect is attained by blocking the Gl/S transient step of cell cycle (Anticancer Res., 17, 1067 (1997)).
  • the precise mechanisms of antimetastasis and anticancer activities are elucidated by means of inhibition of penetration, inhibition of adsorption and inhibition of angiogenesis (Biol. Pharm. Bull., 18, 1197 (1995)).
  • ginsenoside Rg3 is now commercially available in China as “Rg3 Shenyi Jiaonang", an anticancer drug for inhibition of metastasis in liver and lung cancers.
  • an object of the present invention is to provide a novel ginsenoside derivative.
  • Another object of the present invention is to provide a method for preparing a novel ginsenoside derivative by hydrogenation of the double bond in ⁇ 20 -ginsenoside and is to provide a method for preparing ⁇ 20 -ginsenoside. Further, still another object of the present invention is to provide a pharmaceutical composition comprising a novel ginsenoside derivative which is effective in prevention and treatment of cancer and inhibition of metastasis of cancer.
  • the present invention relates to a ginsenoside derivative of the following formula 1,
  • R 1 is H, a C -C 2 acyl group, a glucose group, or a glucosyl(l ⁇ 2)glucose group
  • R 2 is H, a hydroxyl group, a C2-C24 O-acyl group, O- glucose group, or a rhamnosyl(l ⁇ 2)glucose group
  • R3 is H, or a C 2 -C 24 acyl group.
  • the compound of the above formula 1 can have at least one asymmetric center, which can be mirror image isomer or a partial stereoisomer. Therefore, the present invention also relates to all the isomers of a compound of the above formula 1 and the mixtures of the isomers thereof.
  • the compound of the above formula 1 of the present invention can exist as a solvate (e.g., hydrate).
  • preferred examples of compounds are as follows: 3-0-[ ⁇ -D-glucopyranosyl(l ⁇ 2)- ⁇ -D-glucopyranosyl] dammarane-3 ⁇ ,12 ⁇ - diol (Compound 1) ; 3-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,12 ⁇ -diol (Compound 2) ; 6-0-[ ⁇ -L-rharnnopyranosyl(l ⁇ 2)- ⁇ -D-glucopyranosyl] dammarane-
  • the present invention also relates to a method of manufacturing a ginsenoside derivative of the above formula 1.
  • the ginsenoside derivative of the above formula 1 is, as shown in the following reaction scheme 1, manufactured by hydrogenation of compounds of the following formulas 2a or 2b or a mixture of these compounds.
  • Ri, R 2 , 3 are the same as defined in the above formula 1.
  • the solvent to be used in the hydrogenation of a double bond of ⁇ 20 - ginsenoside represented by the formula 2a or 2b according to the above reaction scheme 1 is an organic solvent selected from a lower alkanol having carbon atoms of
  • the solvent can be selected from the group consisting of methanol, ethanol, or ethylacetate, or a mixed solvent of the above organic solvent and water.
  • a reaction catalyst palladium-charcoal (Pd/C) is used in general but other metals such as platinum or rhodium can be also used.
  • the catalyst is used in the range of about 1 to 20 wt. % with reference to the weight of reactants. The temperature and pressure conditions in the above hydrogenation are determined depending on the device to be used.
  • the temperature is in the range of from room temperature to 40 °C
  • the hydrogen pressure is from atmospheric pressure to 50 psi
  • the hydrogenation is performed for 3 to 30 hrs.
  • Compounds, wherein Ri, R 2 and/or R3 is an acyl group can be synthesized by reacting a compound, wherein Ri, R and/or R 3 is H or a hydroxyl group, with an appropriate acylating agent in the presence of a base.
  • a base used can be an organic base such as pyridine, triethylamine, and the like, or an inorganic base such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, potassium t-butoxide and the like.
  • An acylating agent to be used is a C 2 -C 24 carboxylic acid anhydride or acid chloride.
  • the above reaction can be performed using an excessive amount of an acylating agent without using a solvent but it is preferable to perform the reaction in the presence of an organic solvent.
  • Compounds, wherein Ri and/ or R 2 is H or a glucose group, were synthesized using glucosyl(l— >2)glucose group by modifying the already known method (Yakhakhoeji 35, 432 (1991)).
  • the compound wherein Ri and/ or R 2 is a glucosyl(l ⁇ 2)glucose group was manufactured by heating it along with sodium hydroxide in butanol solution and then purified via silica gel colum chroma tography .
  • Compounds, wherein and/ or R3 is H and R 2 is a hydroxyl group were synthesized using compound R 2 is O-glucose group by modifying the already known method (Chem. Pharm. Bull. 35, 1653 (1987)). That is, the compound wherein Ri and/ or R3 is H and R is a hydroxyl group was manufactured by heating it along with sodium hydroxide in butanol solution and then purified via silica gel column chromatography.
  • the products can be separated by the conventional separation/ purification method.
  • the solvent to be used can be a single solvent or a mixture of more than two solvents selected from the group consisting of dichloromethane, chloroform, methanol, ethanol, water, ethylacetate and the like.
  • the compounds represented by the above formula 2a or 2b being used as starting materials in the above reaction scheme 1 were manufactured according to the already known method (Yakhakhoeji 39, 85 (1995), Arch. Pharm. Res. 5, 551 (1996), Planta Medica 62, 86 (1996), Phytochemistry 44, 931 (1997)).
  • the compounds represented by the above formula 2a or 2b were manufactured via acid hydrolysis of a respective compound or a mixture of at least two compounds selected from the group consisting of ginsenosides Ral, Ra2, Ra3, Rbl, Rb2, Rb3, Re, Rd isolated from ginseng, or a protopanaxadiol ginsenoside mixture, ginsenoside Rgl or Re.
  • ginsenosides can be easily isolated and purified by any one with a skill in the art via column chromatography according to the method disclosed in references on active ingredients of ginseng.
  • ginseng extract was extracted in an aqueous alkali solution using butanol to remove protopanaxatriol ginsenoside and neutralized the aqueous layer with acid and then extracted with butanol.
  • an acid hydrolysis for manufacturing compounds represented by the above formula 2a or 2b there has been known a method of heating in 50% acetic acid solution.
  • ginsenosides Ral Heating of a respective compound or a mixture of at least two compounds selected from the group consisting of ginsenosides Ral, Ra2, Ra3, Rbl, Rb2, Rb3, Re, Rd isolated from ginseng, or a protopanaxadiol ginsenoside mixture, ginsenosides Rgl or Re, respectively, at 50 °C in 50% acetic acid solution would lead to production of prosapogenin, a hydrolysate of a sugar at C20 position, and at the same time production of a dehydrogenated compound ( ⁇ 20 -Ginsenoside).
  • the above-mentioned acid analysis method is a method of manufacturing prosapogenin producing more than 70% of prosapogenin and thus it is not considered economical as a method for manufacturing ⁇ 20 -ginsenosides.
  • a O-C ⁇ alcohol is used, and preferably a single or mixed solvent selected from methanol, ethanol and propanol.
  • An organic acid is selected from the group consisting of citric acid, tartaric acid, succinic acid, lactic acid, malic acid, acetic acid, and formic acid.
  • the organic acid is used with a concentration in the range of 0.1 to 30 vol.%, preferably 5 to 20 vol.%.
  • the reaction is performed at 50 to 100 °C, preferably at a boiling temperature for the reactants, for about 10 to 72 hrs. Upon completion of the reaction, the reaction mixture is extracted by using a low grade aliphatic alcohol, washed with an alkali solution and then concentrated.
  • the ginsenoside derivatives of the above formula 1 according to the present invention are highly effective in inhibition of cancer cell growth, inhibition of angiogenesis, inhibition of metastasis of cancer cells and cancer treatment and are thus useful as a therapeutic agent for prevention and treatment of various tumor- related diseases. Therefore, the present invention relates to a pharmaceutical composition comprising ginsenoside derivatives of the above formula 1 according to the present invention and their pharmaceutically acceptable salts as active ingredients.
  • the above pharmaceutical composition can be formulated in various forms via the conventional pharmaceutical formulation methods by adding non- toxic and pharmaceutically acceptable carriers, fortifiers, and excipients to the above-mentioned compounds of the above formula 1.
  • the pharmaceutical composition of the present invention can be formulated in the forms of soft capsules, hard capsules, pills, suspensions, granules, tablets and the like by adding additives, lubricants, adhesives, sweeteners, flavours, preservatives, and the like, which are conventionally added for administration purposes in pharmaceutical fields.
  • Injectionable preparations can be prepared in suspension or liquid by adding an emulsifier, an additive, a preservative and the like.
  • the dosage of the compounds of the present invention is preferably 0.1 to 10 mg/kg of body weight of a person, more preferably 0.3 to 4 mg/kg, and can be administered in aliquots 1 to 6 times per day at regular intervals of time after consultation with a physician or a pharmacist.
  • Fig. 1 is a result of analysis by high performance liquid chromatography (HPLC) of ⁇ 20 -ginsenoside mixture obtained by acid hydrolysis methods according to Reference Examples 1 and 2 and Comparative Reference Example 2.
  • HPLC high performance liquid chromatography
  • Example 1 Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl Fifty two grams of protopanaxadiol ginsenoside mixture and 60g of citric acid were dissolved in 700 mL of 99.8% ethanol and heated for 25 hrs at 90 °C and then ethanol was removed under reduced pressure. The resulting concentrate was dispersed in a solution, where 60 g of sodium hydroxide was dissolved in 300 mL of water, and extraction was performed twice with 600 mL of water-saturated butanol.
  • the resulting butanol layers were combined and washed twice with 300 mL of water and obtained 21.2 g of a product by concentration under reduced pressure.
  • the resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1 ⁇ 5:1 ⁇ 4:1) containing 2% water in 300 g silica gel and finally obtained 12.2 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
  • Example 2 Preparation of 3-0-[ ⁇ -D-glucopyranosyl(l- ⁇ 2)- ⁇ -D- glucopyranosyl] dammarane-3 ⁇ ,12 ⁇ -diol (Compound 1) Twelve grams of a mixture of ginsenoside Rg5 and ginsenoside Rkl prepared in example 1 was dissolved in 150 mL of methanol, added with 400 mg of 10% palladium-charcoal (Pd-C) and was allowed to react at room temperature by shaking for 16 hrs under hydrogen gas pressure of 35 psi using Parr reactor. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure.
  • Pd-C palladium-charcoal
  • the resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of chloroform : methanol (6:1 ⁇ 5:1 ⁇ 4:1) containing 1% water in 200 g silica gel and finally obtained 11 g of compound 1. m.p.
  • Example 3 Preparation of 3-O- ⁇ -D-glucopyranosyl dammaranne-3 ⁇ ,12 ⁇ -diol (Compound 2) and dammarane-3 ⁇ ,12 ⁇ -diol (Compound 5)
  • Two grams of 3-0-[ ⁇ -D-glucopyranosyl(l ⁇ 2)- ⁇ -D- glucopyranosyl]dammarane-3 ⁇ ,12 ⁇ -diol prepared in example 1 was dissolved in 25 mL of pyridine and 25 mL of acetic anhydride and reacted at 60 °C for 24 hrs and the solvent was removed under reduced pressure.
  • the resulting concentrate was dissolved in 100 mL of butanol, added with 5 g of sodium hydroxide and then stirred at 85 °C for 12 hrs.
  • the reactants were added to 100 mL of water and then butanol layer was separated.
  • the water layer was extracted twice with 100 mL of butanol and the resulting butanol layers were combined with the initially collected butanol layer.
  • the butanol layer was washed twice with 50 mL of water and then concentrated under reduced pressure.
  • Example 4 Preparation of a Mixture of Ginsenoside F4 and Ginsenoside Rg6 Twenty grams of ginsenosie Re and 20 g of citric acid were dissolved in 200 mL of 99.8% ethanol, heated at 85 °C for 24 hrs and the ethanol was removed under reduced pressure. The resulting concentrate was dispersed in 500 mL of butanol and then washed with a solution, wherein 20 g of sodium hydroxide is dissolved in 150 mL of water, washed twice with 150 mL of water and the butanol layer was concentrated under reduced pressure to obtain 16.2 g of a concentrate.
  • the resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (8:1- ⁇ 7.T ⁇ 6:1 ⁇ 5:1 ⁇ 4:1) containing 2% water in 340 g silica gel and finally obtained 5.8 g of a mixture of ginsenoside F4 and ginsenoside Rg6.
  • Example 5 Preparation of 6-0-[ ⁇ -L-rhamnopyranosyl(l ⁇ 2)- ⁇ -D- glucopyranosyljdammarane -3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 5)
  • 4.2 g of the mixture of ginsenoside F4and ginsenoside Rg6 prepared in example 4 was dissolved in 240 mL of methanol, added with 800 mg of 10% palladium-charcoal and then reacted by using Parr reactor under 15 psi of hydrogen gas pressure with shaking for 16 hrs. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure.
  • Example 6 Preparation of a Mixture of Ginsenoside Rk3 and Ginsenoside Rh4 Twenty grams of ginsenoside Rgl and 20 g of citric acid were dissolved in
  • Example 7 Preparation of 6-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 4) 5.7 g of the mixture of ginsenoside Rk3and ginsenoside Rh4 prepared in example was dissolved in 150 mL of ethanol and 130 mL of ethylacetate, added with 800 mg of 10% palladium-charcoal and then reacted by using Parr reactor under 25 psi of hydrogen gas pressure by shaking for 6 hrs. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure.
  • Compound 4 6-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol
  • the resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of chloroform : methanol (8:1 ⁇ 7:1- ⁇ 6:1 ⁇ 5:1) containing 1% water in 120 g silica gel and finally obtained 5.4 g of a target compound.
  • Example 8 Preparation of dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 6) 0.8 g of compound 4 prepared in example 7 was dissolved in 80 mL of butanol, added with 3.5 g of sodium hydroxide and stirred at 85 °C for 32 hrs. The reactants were added to 30 mL of butanol, washed three times with 40 mL of water and the butanol layer was concentrated under reduced pressure. The resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (50:1 ⁇ 40:1 ⁇ 30:1 ⁇ 20:1) in 30 g silica gel and finally obtained 0.53 g of target compound 6. m.p.
  • Example 9 Preparation of 3-0-[ ⁇ -D-glucopyranosyl(l— >2)- ⁇ -D- glucopyranosyl]dammarane-3 ⁇ ,12 ⁇ -diol (Compound 1) 0.15 g of ginsenoside Rg5 prepared according to the method in a reference (Arch. Pharm. Res. 19, 551 (1996)) was dissolved in 15 mL of ethanol, added with 30 mg of 5% palladium-charcoal and reacted at 40 °C by shaking using Parr reactor for 6 hrs under 45 psi of hydrogen gas pressure. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and finally obtained 0.14 g of target compound 1.
  • Example 10 Preparation of 3-O- ⁇ -D-gIucopyranosyl dammarane-3 ⁇ ,12 ⁇ -diol (Compound 2) 0.2 g of ginsenoside Rh3 prepared according to the method in a reference (Yakhakhoeji 39, 85 (1995)) was dissolved in 20 mL of ethanol, added with 50 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 40 psi of hydrogen gas pressure. The catalyst was removed by filtering through cellite and the mixture was concentrated under reduced pressure and finally obtained 0.18 g of target compound 2.
  • Example 11 Preparation of 6-0-[ ⁇ -L-rhamnopyranosyl(l ⁇ 2)- ⁇ -D- glucopyranosyl]dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 3)
  • 0.11 g of ginsenoside F4 prepared according to the method in a reference (Planta Medica 56, 298 (1990)) was dissolved in 10 mL of ethanol, added with 20 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 40 psi of hydrogen gas pressure. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and finally obtained 0.1 g of target compound 3.
  • Example 12 Preparation of 6-0-[ ⁇ -L-rhamnopyranosyl(l ⁇ 2)- ⁇ -D- glucopyranosyl]dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 3) 0.11 g of ginsenoside Rg6 in the amount of 0.11 g prepared according to the method in a reference (Phytochemistry 44, 931 (1997)) was dissolved in 10 mL of methanol, added with 20 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 40 psi of hydrogen gas pressure. The catalyst was removed by filtering through cellite and the mixture was concentrated under reduced pressure and finally obtained 0.1 g of target compound 3.
  • Example 13 Preparation of 6-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 4) 0.16 g of ginsenoside Rh4 in the amount of 0.16 g prepared according to the method in a reference (Planta Medica 62, 86 (1996)) was dissolved in 15 mL of methanol, added with 20 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 30 psi of hydrogen gas pressure. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and finally obtained 0.13 g of target compound 4.
  • Compound 4 6-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol
  • Example 14 Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl
  • Ten grams of a protopanaxadiol ginsenoside mixture and 10 g of tartaric acid were dissolved in 100 mL of 99.8% ethanol, heated at 90 °C for 12 hrs and ethanol was removed under reduced pressure.
  • the resulting concentrate was dispersed in 300 mL of butanol and then washed with a solution, wherein 20 g of sodium hydroxide is dissolved in 150 mL of water, washed twice with 150 mL of water and the butanol layer was concentrated under reduced pressure to obtain 3.94 g of a concentrate.
  • the concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1 ⁇ 5:1 ⁇ 4:1) containing 2% water in 100 g silica gel and finally obtained 2.25 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
  • Example 15 Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl
  • Ten grams of a protopanaxadiol ginsenoside mixture and 20 g of fumaric acid were dissolved in 100 mL of 99.8% methanol, heated at 90 °C for 72 hrs and methanol was removed under reduced pressure.
  • the resulting concentrate was dispersed in 300 mL of butanol and then washed with a solution, wherein 15 g of sodium hydroxide is dissolved in 150 mL of water, washed twice with 150 mL of water and the butanol layer was concentrated under reduced pressure to obtain 3.84 g of a concentrate.
  • the concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1 ⁇ 5:1 ⁇ 4:1) containing 2% water in 100 g silica gel and finally obtained 2.2 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
  • Example 16 Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl
  • Ten grams of a protopanaxadiol ginsenoside mixture and 25 g of maleic acid were dissolved in 100 mL of 99.8% methanol, heated at 90 °C for 72 hrs and methanol was removed under reduced pressure.
  • the resulting concentrate was dispersed in 300 mL of butanol and then washed with a solution, wherein 15 g of sodium hydroxide is dissolved in 150 mL of water, washed twice with 150 mL of water and the butanol layer was concentrated under reduced pressure to obtain 3.8 g of a concentrate.
  • the concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1— »5:1 ⁇ 4:1) containing 2% water in 100 g silica gel and finally obtained 2.28 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
  • Example 17 Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl
  • the resulting concentrate was dispersed in 300 mL of butanol and then washed with a solution, wherein 5 g of sodium hydroxide is dissolved in 150 mL of water, washed twice with 150 mL of water and the butanol layer was concentrated under reduced pressure to obtain 4.1 g of a concentrate.
  • the concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1 ⁇ 5:1— >4.T) containing 2% water in 100 g silica gel and finally obtained 2.38 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
  • Example 18 Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl
  • the resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1 ⁇ 5:1 ⁇ 4.T) containing 2% water in 100 g silica gel and finally obtained 2.48 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
  • Example 19 Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl
  • the resulting concentrate was dispersed in 300 mL of butanol and then washed twice with 150 mL of water and the butanol layer was concentrated under reduced pressure to obtain 3.88 g of a concentrate.
  • the concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1 ⁇ 5:1— * 4:1) containing 2% water in 100 g silica gel and finally obtained 2.24 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
  • Example 20 Preparation of 3-0-[ ⁇ -D-glucopyranosyl(l ⁇ 2)- ⁇ -D-glucopyranosyl] dammarane-3 ⁇ ,12 ⁇ -diol octaacetate (Compound 7) Two hundred milligrams of 3-0-[ ⁇ -D-glucopyranosyl(l ⁇ 2)- ⁇ -D- glucopyranosyl]dammarane-3 ⁇ ,12 ⁇ -diol (Compound 1) prepared in example 2 was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride. After keeping it at room temperature for 24 hrs, it was concentrated under reduced pressure.
  • the concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate in 20 g silica gel and obtained target compound 7.
  • Example 21 Preparation of 3-0-[ ⁇ -D-glucopyranosyl(l- ⁇ 2)- ⁇ -D-glucopyranosyl] dammarane-3 ⁇ ,12 ⁇ -diol octapalmitate (Compound 8) Two hundred milligrams of 3-0-[ ⁇ -D-glucopyranosyl(l ⁇ 2)- ⁇ -D- glucopyranosyl]dammarane-3 ⁇ ,12 ⁇ -diol (Compound 1) prepared in example 2 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. It was then added with 1 g of palmitic acid chloride and kept at room temperature for 24 hrs.
  • Example 22 Preparation of 3-0-[ ⁇ -D-glucopyranosyl(l ⁇ 2)- ⁇ -D-glucopyranosyl] dammarane-3 ⁇ ,12 ⁇ -diol octaundecanoate (Compound 9) Two hundred milligrams of 3-0-[ ⁇ -D-glucopyranosyl(l ⁇ 2)- ⁇ -D- glucopyranosyl]dammarane-3 ⁇ ,12 ⁇ -diol (Compound 1) prepared in example 2 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. It was then added with 1 g of undecanoic acid chloride and kept at room temperature for 24 hrs.
  • Example 23 Preparation of 3-0-[ ⁇ -D-glucopyranosyl(l ⁇ 2)- ⁇ -D-glucopyranosyl] dammarane-3 ⁇ ,12 ⁇ -diol octabutyrate (Compound 10) Two hundred milligrams of 3-0-[ ⁇ -D-glucopyranosyl(l— >2)- ⁇ -D- glucopyranosyl]dammarane-3 ⁇ ,12 ⁇ -diol (Compound 1) prepared in example 2 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. It was then added with 0.5 g of butyric acid chloride and kept at room temperature for 24 hrs.
  • Example 24 Preparation of 3-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,12 ⁇ -diol pentaacetate (Compound 11) Two hundred milligrams of 3-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,12 ⁇ - diol (Compound 2) prepared in example 2 was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride and cooled down. After keeping at room temperature for 24 hrs, it was concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (3:1) in 20 g silica gel and obtained target compound 11.
  • Example 25 Preparation of 3-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,12 ⁇ -diol pentabutyrate (Compound 12)
  • One hundred fifty milligrams of 3-O- ⁇ -D-glucopyranosyl dammarane- 3 ⁇ ,12 ⁇ -diol (Compound 2) prepared in example 3 was dissolved in 2 mL of pyridine and then added with 0.5 mL of triethylamine and cooled down. Then, it was added with 0.3 g of butyric acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (5:1) in 20 g silica gel and obtained target compound 12.
  • Example 26 Preparation of 3-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,12 ⁇ -diol pentapalmitate (Compound 13)
  • One hundred fifty milligrams of 3-O- ⁇ -D-glucopyranosyl dammarane- 3 ⁇ ,12 ⁇ -diol (Compound 2) prepared in example 3 was dissolved in 3 mL of pyridine and then added with 0.5 mL of triethylamine and cooled down. Then, it was added with 0.4 g of palmitic acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (5:1) in 20 g silica gel and obtained target compound 13.
  • Example 27 Preparation of 6-0-[ ⁇ -L-rhamnopyranosyl(l— >2)- ⁇ -D-glucopyranosyl] dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol octaacetate (Compound 14) Two hundred milligrams of 6-0-[ ⁇ -L-rhamnopyranosyl(l— ->2)- ⁇ -D- glucopyranosyl]dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 3) prepared in example 5 was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride. After keeping at room temperature for 24 hrs, it was concentrated under reduced pressure.
  • Example 28 Preparation of 6-0-[ ⁇ -L-rhamnopyranosyl(l- ⁇ 2)- ⁇ -D-glucopyranosyl] dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol octabutyrate (Compound 15)
  • One hundred fifty milligrams of 6-0-[ ⁇ -L-rhamnopyranosyl(l ⁇ 2)- ⁇ -D- glucopyranosyl]dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 3) prepared in example 5 was dissolved in 2 mL of pyridine and then added with 0.5 mL of triethylamine and cooled down.
  • Example 29 Preparation of 6-0-[ ⁇ -L-rhamnopyranosyl(l— »2)- ⁇ -D- glucopyranosyl]dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol octapalmitate (Compound 16)
  • One hundred fifty milligrams of 6-0-[ ⁇ -L-rhamnopyranosyl(l— >2)- ⁇ -D- glucopyranosyl]dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 3) prepared in example 5 was dissolved in 2 mL of pyridine and then added with 0.5 mL of triethylamine and cooled down.
  • Example 30 Preparation of 6-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol hexaacetate (Compound 17)
  • Two hundred milligrams of 6-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ - triol (Compound 4) prepared in example 7 was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride. After keeping at room temperature for 24 hrs, it was concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (3:1) in 20 g silica gel and obtained target compound 17.
  • Example 31 Preparation of 6-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol hexabutyrate (Compound 18)
  • 6-O- ⁇ -D-glucopyranosyl dammarane- 3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 4) prepared in example 7 was dissolved in 2 mL of pyridine and then added with 0.5 mL of triethylamine and cooled down. Then, it was added with 0.3 g of butyric acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (5:1) in 20 g silica gel and obtained target compound 18.
  • Example 32 Preparation of 6-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ , 6 ⁇ ,12 ⁇ -triol hexapalmitate (Compound 19)
  • 6-O- ⁇ -D-glucopyranosyl dammarane- 3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 4) prepared in example 7 was dissolved in 2 mL of pyridine and then added with 0.5 mL of triethylamine and cooled down. Then, it was added with 0.45 g of palmitic acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (5:1) in 20 g silica gel and obtained target compound 19.
  • Example 33 Preparation of dammarane-3 ⁇ ,12 ⁇ -diol dipalmitate (Compound 20) Two hundred milligrams of dammarane-3 ⁇ ,12 ⁇ -diol (Compound 5) prepared in example 3 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. Then, it was added with 1 g of palmitic acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (6:1) in 30 g silica gel and obtained target compound 20.
  • Example 34 Preparation of dammarane-3 ⁇ ,12 ⁇ -diol diacetate (Compound 21) Two hundred milligrams of dammarane-3 ⁇ ,12 ⁇ -diol (Compound 5) prepared in example 3 was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride. After keeping at room temperature for 24 hrs, it was concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (6:1) in 20 g silica gel and obtained target compound 21.
  • Example 35 Preparation of dammarane-3 ⁇ , 12 ⁇ -diol dibutyrate (Compound 22) Two hundred milligrams of dammarane-3 ⁇ ,12 ⁇ -diol (Compound 5) prepared in example 3 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. Then, it was added with 1 g of butyric acid chloride and placed at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (6:1) in 20 g silica gel and obtained target compound 22.
  • Example 37 Preparation of dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol tributyrate (Compound 24) Two hundred milligrams of dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 6) prepared in example 8 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. Then, it was added with 1 g of butyric acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (6:1) in 20 g silica gel and obtained target compound 24.
  • Example 38 Preparation of dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol triacetate (Compound 25) Two hundred milligrams of dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol (Compound 6) prepared in example 8 was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride. After keeping at room temperature for 24 hrs, it was concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (6:1) in 20 g silica gel and obtained target compound 25.
  • Example 39 Preparation of 3-0-[ ⁇ -D-glucopyranosyl(l— >2)- ⁇ -D-glucopyranosyl] dammarane-3 ⁇ ,12 ⁇ -diol octaacetate (Compound 7)
  • a mixture of ginsenoside Rg5 and ginsenoside Rkl in the amount of 0.3 g prepared in example 1 was dissolved in 3 mL of pyridine, added with 3 mL of acetic anhydride and reacted at room temperature by stirring for 24 hrs. It was then concentrated under reduced pressure and a mixture octaacetate of ginsenoside Rg5 and ginsenoside Rkl was produced.
  • Example 40 Preparation of 3-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,12 ⁇ -diol pentaacetate (Compound 11) 0.5 g of ginsenoside Rh3 prepared according to the method in a reference (Yakhakhoeji 39, 85 (1995)) was dissolved in 2 mL of pyridine and 2 mL of acetic anhydride. After stirring at room temperature for 16 hrs, the mixture was concentrated under reduced pressure to obtain ginsenoside Rh3 pentaacetate.
  • the ginsenoside Rh3 pentaacetate was dissolved in 10 mL of ethanol, added with 50 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 40 psi of hydrogen gas pressure.
  • the catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and then passed through a silica gel column chromatography in 20 g silica gel, solvent of dichloromethane and ethylacetate (3:1) to obtain 0.1 g of target compound 11.
  • Example 41 Preparation of 6-0-[ ⁇ -L-rhamnopyranosyl(l ⁇ 2)- ⁇ -D-glucopyranosyl] dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol octaacetate (Compound 14) 0.1 g of ginsenoside prepared according to the method in a reference (Planta Medica 56, 298 (1990)) was dissolved in 2 mL of pyridine and 2 mL of acetic anhydride. After stirring at room temperature for 16 hrs, the mixture was concentrated under reduced pressure to obtain ginsenoside F4 octaacetate.
  • the ginsenoside F4 octaacetate was dissolved in 5 mL of ethanol and 5 mL of ethylacetate, added with 20 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 40 psi of hydrogen gas pressure.
  • the catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and then passed through a silica gel column chromatography in 20 g silica gel, solvent of dichloromethane and ethylacetate (4:1) to obtain target compound 14.
  • Example 42 Preparation of 6-O- ⁇ -D-glucopyranosyl dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol hexaacetate (Compound 17) O.lg of ginsenoside Rh4 prepared according to the method in a reference
  • ginsenoside Rh4 hexaacetate was dissolved in 5 mL of ethanol and 5 mL of ethylacetate, added with 20 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 30 psi of hydrogen gas pressure.
  • the catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and then passed through a silica gel column chromatography in 20 g silica gel, solvent of dichloromethane and ethylacetate (4:1) to obtain target compound 17.
  • Example 43 Preparation of dammarane-3 ⁇ ,6 ⁇ ,12 ⁇ -triol triacetate (Compound 25) 150 mg of dammarane-20(22),24-dien-3 ⁇ ,6 ⁇ ,12 ⁇ -triol prepared according to the method in a reference (Planta Medica 62, 86 (1996)) was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride. After stirring at room temperature for 24 hrs, the mixture was concentrated under reduced pressure to obtain dammarane-20(22), 24-dien-3 ⁇ ,6 ⁇ ,12 ⁇ -triol triacetate.
  • the rhamnopyranosyl- 20(22),24-dien-3 ⁇ ,6 ⁇ ,12 ⁇ -triol triacetate was dissolved in 5 mL of ethanol and 5 mL of ethylacetate, added with 20 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 30 psi of hydrogen gas pressure.
  • the catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and then passed through a silica gel column chromatography in 20 g silica gel, solvent of dichloromethane and ethylacetate (6:1) to obtain target compound 25.
  • each preparation example represents 3-0-[ ⁇ -D-glucopyranosyl(l— >2)- ⁇ -D-glucopyranosyl] dammarane-3 ⁇ ,12 ⁇ -diol (Compound 1) prepared in the example 2 and can be replaced with other compounds with an amount having equal effect depending on the situations.
  • Preparation Example 1 Preparation of Tablets Five grams of an effective compound (Compound 1), 15 g of lactose, 2.5 g of carboxy methyl cellulose calcium and 2.5 g of crystalline cellulose were well mixed and formulated into 50 mg of tablets according to the conventional tableting method.
  • Preparation Example 2 Preparation of Granules Five grams of an effective compound (Compound 1), 15 g of lactose, 2.5 g of carboxy methyl cellulose sodium and 2.5 g of crystalline cellulose were well mixed and formulated into granules according to the conventional wet granulation method.
  • Preparation Example 3 Preparation of Hard Capsules Five grams of an effective compound (Compound 1), 10 g of lactose, 2.5 g of carboxy methyl cellulose calcium and 7.5 g of crystalline cellulose were well mixed and formulated into 50 mg of hard capsules according to the conventional manufacturing method.
  • Preparation Example 4 Preparation of Soft Capsules Five grams of an effective compound (Compound 1), 42 g of soybean oil, 1.5 g of soybean lecithin and 1.5 g of yellow wax were well mixed and formulated into 100 mg of soft capsules according to the conventional manufacturing method.
  • Preparation Example 5 Preparation of Injections Five grams of an effective compound (Compound 1) was dissolved by applying heat in the presence of 5 g of polyethyleneglycol, 5 mL of ethanol, and 400 mL of phosphate saline solution. After cooling down, it was added with phosphorylated physiological saline solution to the final volume of 500 mL and then filled into 1 mL vials to manufacture injection preparations according to the conventional method.
  • the following reference examples and comparative reference examples show examples of acid hydrolysis method for manufacturing ⁇ 20 -ginsenoside represented by the above formula 2a or 2b.
  • the reference examples 1 and 2 show examples of acid hydrolysis method performed under the condition of a mixed solution of an organic acid and aliphatic alcohol while comparative reference examples 1 and 2 show the conventional acid hydrolysis method.
  • Reference Example 1 Preparation of a Mixture of Ginsenoside Rg5 andGinsenoside Rkl
  • One gram of protopanaxadiol ginsenoside mixture and I g of citric acid were dissolved in 10 mL of 99.8% ethanol and heated at 85 °C for 12hrs.
  • the reactants were diluted in 200 mL of butanol and washed with a solution, wherein 2 g of sodium hydroxide is dissolved in 60 mL of water.
  • the butanol layer was washed twice with 60 mL of water and then concentrate under reduced pressure to obtain 0.4 g of target product.
  • Reference Example 2 Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl
  • One gram of protopanaxadiol ginsenoside mixture and I g of citric acid were dissolved in 10 mL of 95% ethanol and heated at 85 °C for 12hrs.
  • the reactants were diluted in 200 mL of butanol and washed with a solution, wherein 2 g of sodium hydroxide is dissolved in 60 mL of water.
  • the butanol layer was washed twice with 60 mL of water and then concentrate under reduced pressure to obtain 0.41 g of target product.
  • Comparative Reference Example 1 Preparation of a Mixture of protopanaxadiol Ginsenoside Two hundred fifty grams of 70% ethanol extraction powder of Panax ginseng was dissolved in 1,000 mL of water and added with 50 g of sodium hydroxide and extracted with 4 times with water-saturated butanol 1,200 mL in order toremove protopanaxatriol ginsenoside mixture. After cooling down, the water layer was neutralized by adding 200 mL of cooled 6N HC1 and extracted four times with water saturated butanol 1,200 mL and the butanol layer was concentrated under reduced pressure. The concentrate was dissolved in 300 mL of methanol and filtered. The filtrate was concentrated under reduced pressure to obtain 42 g of protopanaxadiol ginsenoside mixture.
  • Comparative Reference Example 2 Preparation of Ginsenoside Rg5 and Ginsenoside Rkl
  • One gram of protopanaxadiol ginsenoside mixture and 1 g of citric acid were dissolved in 10 mL of water and heated at 85 °C for 12hrs.
  • the reactants were diluted in 200 mL of butanol and washed with a solution, wherein 2 g of sodium hydroxide is dissolved in 60 mL of water.
  • the butanol layer was washed twice with 60 mL of water and then concentrate under reduced pressure to obtain 0.41 g of target product.
  • PI represents 20S-ginsenoside Rg3
  • P2 represents 20R-ginsenoside Rg3
  • P3 represents ginsenoside Rkl
  • P4 represents ginsenoside Rg5, respectively.
  • ginsenoside Rg3(Pl+P2) and ginsenoside Rkl and Ginsenoside Rg5 are generated in about 1:1 ratio in terms of their peak area.
  • ginsenoside Rg3(Pl+P2) and ginsenoside Rkl and Ginsenoside Rg5 are generated in about 1:1 ratio in terms of their peak area.
  • protopanaxadiol ginsenoside mixture is hydrolyzed in 95% ethanol or 99.8% ethanol solution by organic acid according to the method in comparative reference examples
  • ginsenoside Rg3 are generated only a small amount while ginsenoside Rkl and ginsenoside Rg5 as ⁇ 20 -ginsenoside are produced more than 90%. Further, in order to investigate the effects of ginsenoside derivatives represented by the formula 1 of the present invention on prevention and treatment of cancer as well as inhibition of metastasis, their biological activities were measured by using the procedures shown in the following experimental examples 1 - 5.
  • the compounds of the present invention have shown 2 to 3 times more improved anticancer activities as compared to those of the conventional ginsenosides in lung cancer cells (A549), ovary cancer cells (SK-OV-3), melanoma cells (SK-MEL-2), brain tumor cells (XF498), reactal cancer cells (HCT15).
  • 3-0- [ ⁇ -D-glucopyranosy 1(1 - ⁇ 2)- ⁇ -D-glucopy ranosyl] dammarane- 3 ⁇ ,12 ⁇ -diol (Compound 1) shows 11 times stronger antitumor activity as compared to that of ginsenoside Rg3 currently used for the treatment of lung and liver cancers.
  • mice Female nude mice Balb/c (8 weeks old, 18 g of body weight, 8 mice per group) were hypodermically transplanted with caner cells at the concentration of lxlO 7 cell/mL with 0.3 mL. Starting from the next day (Day 1) after the transplantation till one day prior to termination of the experiment, the above mice were orally administered with a specimen and a solvent (control group: 0.2% Tween-80 solution) 0.2 mL per 20 g of body weight of a mouse once per day to a total of 13 times.
  • a solvent control group: 0.2% Tween-80 solution
  • Adriamycin a positive control substance
  • the size of tumors were measured a total of 6 times.
  • Experimental Dosage Vol. of Tumor Cells (mm 3 ) As shown in Table 2, the compounds of the present invention showed superior anticancer activity when orally administered in the amount of 3 and 10 mg/kg.
  • Experimental Example 3 Effect on Inhibition of Metastasis of Cancer Cells After culturing B16F10 melanoma cells, the cells were separated using trypsin-EDTA and diluted with 0.85% saline solution to obtain a 2.5 ⁇ 10 6 cells/mL. Thus prepared cancer cell suspension was injected to female S.P.F.
  • mice C57BL/6 mice (6 mice per group) into the caudal veins with 0.2 mL per mouse and then the mice were administered with 0.2 mL of a drug in 0.2% Tween-80 solution per 20 g of body weight of a mouse once per day starting from 4 hours after the above caudal vein injection.
  • Adriamycin a positive control substance
  • autopsies were performed for the mice.
  • the cancer-transinfected lungs were ablated and then fixed in neutral formaline solution and photographed.
  • the cancer cell colonies shown in the resulting photographs were counted by naked eyes and the results are shown in Table 3.
  • the compound of the present invention showed superior inhibitory activity against metastasis of cancer cells when orally administered in dose of 3 and 10 mg/kg.
  • compound 1 had more potent anti-metastasis activities as compared to that 2 mg/kg of adriamycin, ip injection.
  • Each group was respectively adminsitered orally with 3-0-[ ⁇ -D-glucopyranosyl(l- ⁇ 2)- ⁇ -D-glucopyranosyl]dammarane-3 ⁇ ,12 ⁇ - diol (Compound 1) in dose of 1 mg/kg, 3 mg/kg, 10 mg/kg, which was dissolved in Tween-80 solution, once per day starting from the day of a week prior to the treatment with DMBA(100 ⁇ g/50 ⁇ Laceton/ mouse) until the termination of the experiment, and then treated with 0.1 mL of croton oil 3 times a week from the day of after the DMBA treatment until the termination of the experiment.
  • Papilloma developed on skin was observed and measured once a week during the entire 16 weeks of experimental period, and those papilloma consecutively observed to be greater than 1 mm in diameter were used for measurement. The results are shown in Table 5.
  • mice were orally administered with 3-0-[ ⁇ -D- glucopyranosyl(l ⁇ 2)- ⁇ -D-glucopyranosyl] dammarane-3 ⁇ ,12 ⁇ -diol (Compound 1) of the present invention in dose of 1 mg/kg, 3 mg/kg, 10 mg/kg, respectively, the number of papilloma, weight of tumor, tumor incidence were drastically decreased and the latent period of tumor development was also greatly delayed. Therefore, the compounds of the present invention show excellent cancer-preventive effects as well as anticancer activities.
  • mice per group were orally administered with compounds 1, 2, 3, 4, 5 and 6, respectively, which were dissolved or suspended in carboxy methyl cellulose, in dose of 2,000 mg/kg once per day for one week. The mice were observed for two weeks after last administration and no dead mice were observed.
  • ginsenoside derivatives of the present invention of the formula 1 are shown to have excellent activities of angiogenesis inhibition and metastasis inhibition as well as anticancer activities, and thus they are very useful as a cancer preventive agent as well as a therapeutic agent for treating cancers or preventing metastasis. Further, ginsenoside derivatives of the present invention of the formula 1 did not show any acute toxicities in animal experiments and are thus expected to be safely used as an effective pharmaceutical drug and a nutraceutical.

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Abstract

The present invention relates to ginsenoside derivatives effective in prevention and treatment of cancer. More specifically, the present invention relates to ginsenoside derivatives with novel structures effective in prevention and treatment of cancers, method of their preparation, and a pharmaceutical composition comprising the above novel compounds as active ingredients.

Description

TREATMENT AND PREVENTION OF CANCER WITH NEW GINSENOSIDE DERIVATIVES
Technical Field The present invention relates to ginsenoside derivatives effective in prevention and treatment of cancers. More specifically, the present invention relates to ginsenoside derivatives with novel structures effective in prevention and treatment of cancers, methods of their preparation, and a pharmaceutical composition comprising the above novel compounds as active ingredients.
Background of Invention According to the recent report from WHO, the global death toll dying of cancers is expected to reach 10 million people in 2020 from 6 million at present and the number of new cancer patients will be increased to 15 million people in 2020 from 10 million people at present. Besides, more than 12% of the total global death is ascribed to cancers, and in advanced countries for example, cancer has been ranked next to heart disease, the most deadly cause of death. In order to conquer cancer, one of most incurable diseases, there have been conducted active researches to develop effective anticancer agents and many of them are currently at clinical use. However, those anticancer drugs which are already in commercial market have a few disadvantages. First, they do not have selectivity but often cause serious adverse effects on kidney, heart and bone marrow because they were originally developed as cytotoxic agents, inhibitors of synthesis of DNA or RNA, or inhibitors of protein synthesis. Second, those drugs are targeted to cancer cells and thus the anticancer efficacy of the drugs are greatly reduced due to the frequent occurrence of drug-resistant cells resulted from heterogenecity or genetic instability of cancer cells. Recently, there have been performed active researches to develop anticancer drugs with a new concept by taking advantage of the recent highly advanced molecular biology. Of them, researches on the development of anticancer drugs via inhibition of angiogenesis or inhibitory drugs against metastasis and development of inhibitors of phosphorylation of growth factor receptors highly expressed on cancer- specific cells have been very competitive. There are many advantages of an angiogenesis inhibitor as an anticancer drug. First, the agent can block both growth and metastasis of any cancer because angiogenesis is an essential step for growth and metastasis of cancer. Second, it does not cause drug resistance, which is normally induced by the heterogenecity and genetic instability of cancer cells, because an angiogenesis inhibitor is not targeted to aneuploid cancer cells but targeted to attack diploid endothelial cells. Third, its efficacy is not limited to certain specific kinds of cancer but is applicable to all kinds of cancer which require angiogenesis. Lastly, it is expected to greatly reduce side effects, which are often observed in the conventional cancer drugs, because angiogenesis is a very rare phenomenon to normal adults except those with special occasions such as wound healing or menstruation in women. On the other hand, ginseng has been known for thousand years as a plant which contains a non-toxic tonic to improve homeostasis of human, has therapeutic effects in many diseases, and is capable of extending extend human life expectancy. The effect of expanding life expectancy of ginseng is due to prevention of aging by its antioxidant activity (Korean Biochem. J., 12(1), 33 (1979)) and there have been proposed hypotheses and relevant experimental evidences that can be achieved by the inhibition and prevention of development of cancer (Ann. NY Acad. Sci., 889, 157 (1999); Cancer Epidemiol. Biomarkers Prev., 4, 401 (1995); The Lancet Oncology, 2, 49 (2001)). Anticancer active ingredients in ginseng are known as ginsenosides Rg3, Rh2, Rhl, and Compound K, an enteric microbial metabolite of ginsenoside and the like (J. Korean Med. Sci., lό(Suppl), S28 (2001); J. Ginseng Res., 28, 1 (2004)). Recently, it was disclosed that ginsenoside Rg5 which is contained only a very small amount in red ginseng has a strong anticancer effect, and this effect is attained by blocking the Gl/S transient step of cell cycle (Anticancer Res., 17, 1067 (1997)). The precise mechanisms of antimetastasis and anticancer activities are elucidated by means of inhibition of penetration, inhibition of adsorption and inhibition of angiogenesis (Biol. Pharm. Bull., 18, 1197 (1995)). Further, ginsenoside Rg3 is now commercially available in China as "Rg3 Shenyi Jiaonang", an anticancer drug for inhibition of metastasis in liver and lung cancers.
The inventors of the present invention, in the course of developing a therapeutic cancer agent, synthesized a novel ginsenoside derivative by hydrogenation of the double bond in Δ20-Ginsenoside, which is contained in a very small amount in red ginseng, and by comparison with the conventional ginsenosides, the above novel ginsenoside derivative were shown to have much improved effects in inhibition of cancer cell growth, inhibition of angiogenesis, inhibition of metastasis and therapeutic anticancer effects thereby completing the present invention. Therefore, an object of the present invention is to provide a novel ginsenoside derivative. Further, another object of the present invention is to provide a method for preparing a novel ginsenoside derivative by hydrogenation of the double bond in Δ20-ginsenoside and is to provide a method for preparing Δ20-ginsenoside. Further, still another object of the present invention is to provide a pharmaceutical composition comprising a novel ginsenoside derivative which is effective in prevention and treatment of cancer and inhibition of metastasis of cancer.
Detailed Description of Invention The present invention relates to a ginsenoside derivative of the following formula 1,
Figure imgf000005_0001
wherein R1 is H, a C -C2 acyl group, a glucose group, or a glucosyl(l→2)glucose group; R2 is H, a hydroxyl group, a C2-C24 O-acyl group, O- glucose group, or a rhamnosyl(l→2)glucose group; R3 is H, or a C2-C24 acyl group. Further, the compound of the above formula 1 can have at least one asymmetric center, which can be mirror image isomer or a partial stereoisomer. Therefore, the present invention also relates to all the isomers of a compound of the above formula 1 and the mixtures of the isomers thereof. In addition, the compound of the above formula 1 of the present invention can exist as a solvate (e.g., hydrate). Of the compounds of the above formula 1 of the present invention, preferred examples of compounds are as follows: 3-0-[β-D-glucopyranosyl(l→2)-β-D-glucopyranosyl] dammarane-3β,12β- diol (Compound 1) ; 3-O-β-D-glucopyranosyl dammarane-3β,12β-diol (Compound 2) ; 6-0-[α-L-rharnnopyranosyl(l→2)-β-D-glucopyranosyl] dammarane-
3β,6α,12β-triol (Compound 3) ; 6-O-β-D-glucopyranosyl dammarane-3β,6α,12β-triol (Compound 4) ; dammarane-3β,12β-diol (Compound 5) ; dammarane-3β,6α,12β-triol (Compound 6) ; 3-0-[β-D-glucopyranosyl(l— >2)-β-D-glucopyranosyl]dammarane-3β,12β-diol octaacetate (Compound 7) ; 3-0-[β-D-glucopyranosyl(l-→2)-β-D-glucopyranosyl]dammarane-3β,12β-diol octapalmitate (Compound 8) ; 3-0-[β-D-glucopyranosyl(l→2)-β-D-glucopyranosyl]dammarane-3β,12β-diol octaundecanoate (Compound 9) ; 3-0-[β-D-glucopyranosyl(l→2)-β-D-glucopyranosyl]dammarane-3β,12β-diol octabutyrate (Compound 10) ; 3-O-β-D-glucopyranosyl dammarane-3β,12β-diol pentaacetate (Compound 11) ; 3-O-β-D-glucopyranosyl dammarane-3β,12β-diol pentabutyrate (Compound 12) ; 3-O-β-D-glucopyranosyl dammarane-3β,12β-diol pentapalmitate
(Compound 13) ; 6-0-[α-L-rhamnopyranosyl(l→2)-β-D-glucopyranosyl]dammarane-
3β,6α,12β-triol octaacetate (Compound 14) ; 6-0-[α-L-rhamnopyranosyl(l→2)-β-D-glucopyranosyl]dammarane- 3β,6α,12β-triol octabutyrate (Compound 15) ; 6-0-[α-L-rhamnopyranosyl(l→2)-β-D-glucopyranosyl]dammarane- 3β,6α,12β-triol octapalmitate (Compound 16) ; 6-O-β-D-glucopyranosyl dammarane-3β,6α,12β-triol hexaacetate
(Compound 17) ; 6-O-β-D-glucopyranosyl dammarane-3β,6α,12β-triol hexabutyrate
(Compound 18) ; 6-O-β-D-glucopyranosyl dammarane-3β, 6α,12β-triol hexapalmitate
(Compound 19) ; dammarane-3β,12β-diol dipalmitate (Compound 20) ; dammarane-3β,12β-diol diacetate (Compound 21) ; dammarane-3β, 12β-diol dibutyrate (Compound 22) ; dammarane-3β, 6α, 12β-triol dipalmitate (Compound 23) ; dammarane-3β,6α,12β-triol tributyrate (Compound 24) ; and dammarane-3β,6α,12β-triol triacetate (Compound 25). The present invention also relates to a method of manufacturing a ginsenoside derivative of the above formula 1. The ginsenoside derivative of the above formula 1 is, as shown in the following reaction scheme 1, manufactured by hydrogenation of compounds of the following formulas 2a or 2b or a mixture of these compounds.
[Reaction Scheme 1]
Hydrogenation
Figure imgf000008_0002
Figure imgf000008_0001
In the above reaction scheme 1, Ri, R2, 3 are the same as defined in the above formula 1. The solvent to be used in the hydrogenation of a double bond of Δ20- ginsenoside represented by the formula 2a or 2b according to the above reaction scheme 1 is an organic solvent selected from a lower alkanol having carbon atoms of
1 to 6 and ethylacetate or a mixed solvent consisting of the above organic solvent and water. More preferably, the solvent can be selected from the group consisting of methanol, ethanol, or ethylacetate, or a mixed solvent of the above organic solvent and water. As a reaction catalyst, palladium-charcoal (Pd/C) is used in general but other metals such as platinum or rhodium can be also used. The catalyst is used in the range of about 1 to 20 wt. % with reference to the weight of reactants. The temperature and pressure conditions in the above hydrogenation are determined depending on the device to be used. Preferably, the temperature is in the range of from room temperature to 40 °C, the hydrogen pressure is from atmospheric pressure to 50 psi, and the hydrogenation is performed for 3 to 30 hrs. Compounds, wherein Ri, R2 and/or R3 is an acyl group, can be synthesized by reacting a compound, wherein Ri, R and/or R3 is H or a hydroxyl group, with an appropriate acylating agent in the presence of a base. A base used can be an organic base such as pyridine, triethylamine, and the like, or an inorganic base such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, potassium t-butoxide and the like. An acylating agent to be used is a C2-C24 carboxylic acid anhydride or acid chloride. The above reaction can be performed using an excessive amount of an acylating agent without using a solvent but it is preferable to perform the reaction in the presence of an organic solvent. Compounds, wherein Ri and/ or R2 is H or a glucose group, were synthesized using glucosyl(l— >2)glucose group by modifying the already known method (Yakhakhoeji 35, 432 (1991)). That is, the compound wherein Ri and/ or R2 is a glucosyl(l→2)glucose group was manufactured by heating it along with sodium hydroxide in butanol solution and then purified via silica gel colum chroma tography . Compounds, wherein
Figure imgf000010_0001
and/ or R3 is H and R2 is a hydroxyl group, were synthesized using compound R2 is O-glucose group by modifying the already known method (Chem. Pharm. Bull. 35, 1653 (1987)). That is, the compound wherein Ri and/ or R3 is H and R is a hydroxyl group was manufactured by heating it along with sodium hydroxide in butanol solution and then purified via silica gel column chromatography. When it is necessary to perform purification after completion of the reaction, the products can be separated by the conventional separation/ purification method. When the products are separated via silica gel column chromatography, the solvent to be used can be a single solvent or a mixture of more than two solvents selected from the group consisting of dichloromethane, chloroform, methanol, ethanol, water, ethylacetate and the like. The compounds represented by the above formula 2a or 2b being used as starting materials in the above reaction scheme 1 were manufactured according to the already known method (Yakhakhoeji 39, 85 (1995), Arch. Pharm. Res. 5, 551 (1996), Planta Medica 62, 86 (1996), Phytochemistry 44, 931 (1997)). The compounds represented by the above formula 2a or 2b were manufactured via acid hydrolysis of a respective compound or a mixture of at least two compounds selected from the group consisting of ginsenosides Ral, Ra2, Ra3, Rbl, Rb2, Rb3, Re, Rd isolated from ginseng, or a protopanaxadiol ginsenoside mixture, ginsenoside Rgl or Re. The above ginsenosides can be easily isolated and purified by any one with a skill in the art via column chromatography according to the method disclosed in references on active ingredients of ginseng. As for protopanaxadiol ginsenoside mixture, ginseng extract was extracted in an aqueous alkali solution using butanol to remove protopanaxatriol ginsenoside and neutralized the aqueous layer with acid and then extracted with butanol. As a method of an acid hydrolysis for manufacturing compounds represented by the above formula 2a or 2b, there has been known a method of heating in 50% acetic acid solution. That is, heating of a respective compound or a mixture of at least two compounds selected from the group consisting of ginsenosides Ral, Ra2, Ra3, Rbl, Rb2, Rb3, Re, Rd isolated from ginseng, or a protopanaxadiol ginsenoside mixture, ginsenosides Rgl or Re, respectively, at 50 °C in 50% acetic acid solution would lead to production of prosapogenin, a hydrolysate of a sugar at C20 position, and at the same time production of a dehydrogenated compound (Δ20-Ginsenoside). However, the above-mentioned acid analysis method is a method of manufacturing prosapogenin producing more than 70% of prosapogenin and thus it is not considered economical as a method for manufacturing Δ20-ginsenosides. In the present invention, on the other hand, when a respective compound or a mixture of at least two compounds selected from the group consisting of ginsenosides Ral, Ra2, Ra3, Rbl, Rb2, Rb3, Re, Rd isolated from ginseng, or a protopanaxadiol ginsenoside mixture, ginsenoside Rgl or Re, respectively, were heated in a mixed solution of an organic acid and a lower aliphatic alcohol would lead to production of Δ20-ginsenosides represented by the above formula 2a or 2b with more than 90% of yield. As a low grade aliphatic alcohol, a O-Cό alcohol is used, and preferably a single or mixed solvent selected from methanol, ethanol and propanol. An organic acid is selected from the group consisting of citric acid, tartaric acid, succinic acid, lactic acid, malic acid, acetic acid, and formic acid. The organic acid is used with a concentration in the range of 0.1 to 30 vol.%, preferably 5 to 20 vol.%. The reaction is performed at 50 to 100 °C, preferably at a boiling temperature for the reactants, for about 10 to 72 hrs. Upon completion of the reaction, the reaction mixture is extracted by using a low grade aliphatic alcohol, washed with an alkali solution and then concentrated. Thus obtained residue is purified through silica gel column chromatography to obtain compounds of the above formula 2a or 2b or their mixture in high purity. Examples of the solvent used in the above chromatography are dichloromethane, chloroform, ethanol, methanol and water or a mixture of these.
The ginsenoside derivatives of the above formula 1 according to the present invention are highly effective in inhibition of cancer cell growth, inhibition of angiogenesis, inhibition of metastasis of cancer cells and cancer treatment and are thus useful as a therapeutic agent for prevention and treatment of various tumor- related diseases. Therefore, the present invention relates to a pharmaceutical composition comprising ginsenoside derivatives of the above formula 1 according to the present invention and their pharmaceutically acceptable salts as active ingredients. The above pharmaceutical composition can be formulated in various forms via the conventional pharmaceutical formulation methods by adding non- toxic and pharmaceutically acceptable carriers, fortifiers, and excipients to the above-mentioned compounds of the above formula 1. For example, the pharmaceutical composition of the present invention can be formulated in the forms of soft capsules, hard capsules, pills, suspensions, granules, tablets and the like by adding additives, lubricants, adhesives, sweeteners, flavours, preservatives, and the like, which are conventionally added for administration purposes in pharmaceutical fields. Injectionable preparations can be prepared in suspension or liquid by adding an emulsifier, an additive, a preservative and the like. The dosage of the compounds of the present invention is preferably 0.1 to 10 mg/kg of body weight of a person, more preferably 0.3 to 4 mg/kg, and can be administered in aliquots 1 to 6 times per day at regular intervals of time after consultation with a physician or a pharmacist.
Brief Description of Drawings Fig. 1 is a result of analysis by high performance liquid chromatography (HPLC) of Δ 20-ginsenoside mixture obtained by acid hydrolysis methods according to Reference Examples 1 and 2 and Comparative Reference Example 2. (PI: 20S- Ginsenoside, P2: 20R-Ginsenoside, P3: Ginsenoside Rkl, P4: Ginsenoside Rg5)
Examples The present invention will be described in greater detail with reference to the following examples, however, they should not be construed as limiting the scope of this invention. Example 1 : Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl Fifty two grams of protopanaxadiol ginsenoside mixture and 60g of citric acid were dissolved in 700 mL of 99.8% ethanol and heated for 25 hrs at 90 °C and then ethanol was removed under reduced pressure. The resulting concentrate was dispersed in a solution, where 60 g of sodium hydroxide was dissolved in 300 mL of water, and extraction was performed twice with 600 mL of water-saturated butanol. The resulting butanol layers were combined and washed twice with 300 mL of water and obtained 21.2 g of a product by concentration under reduced pressure. The resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1→5:1→4:1) containing 2% water in 300 g silica gel and finally obtained 12.2 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
Example 2 : Preparation of 3-0-[β-D-glucopyranosyl(l-→2)-β-D- glucopyranosyl] dammarane-3β,12β-diol (Compound 1) Twelve grams of a mixture of ginsenoside Rg5 and ginsenoside Rkl prepared in example 1 was dissolved in 150 mL of methanol, added with 400 mg of 10% palladium-charcoal (Pd-C) and was allowed to react at room temperature by shaking for 16 hrs under hydrogen gas pressure of 35 psi using Parr reactor. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure. The resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of chloroform : methanol (6:1→5:1→4:1) containing 1% water in 200 g silica gel and finally obtained 11 g of compound 1. m.p. 250-253 °C; 13C-NMR(Pyridine-d5, δppm) 106.0, 105.1, 88.9, 83.4, 78.3, 78.2, 78.0, 77.9, 77.1, 72.7, 72.7, 71.6, 71.6, 62.8, 62.72, 56.4, 51.0, 50.9, 50.8, 49.5(49.2), 44.0, 40.1, 39.7, 39.3, 38.0, 37.0, 35.3, 34.9(34.5), 33.6, 32.6(32.5), 28.1, 28.1, 26.7, 26.8(25.9), 22.9, 22.7, 20.4, 18.4, 17.2, 16.5,((16.5), 15.8(15.7), 13.8.
Example 3 : Preparation of 3-O-β-D-glucopyranosyl dammaranne-3β,12β-diol (Compound 2) and dammarane-3β,12β-diol (Compound 5) Two grams of 3-0-[β-D-glucopyranosyl(l→2)-β-D- glucopyranosyl]dammarane-3β,12β-diol prepared in example 1 was dissolved in 25 mL of pyridine and 25 mL of acetic anhydride and reacted at 60 °C for 24 hrs and the solvent was removed under reduced pressure. The resulting concentrate was dissolved in 100 mL of butanol, added with 5 g of sodium hydroxide and then stirred at 85 °C for 12 hrs. The reactants were added to 100 mL of water and then butanol layer was separated. The water layer was extracted twice with 100 mL of butanol and the resulting butanol layers were combined with the initially collected butanol layer. The butanol layer was washed twice with 50 mL of water and then concentrated under reduced pressure. The resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane: methanol (30:1→20:1→15:1→10.T→5.T→4:1→3:1) containing 1% water in 100 g silica gel and finally obtained 300 mg of Compound 2, 550 mg of Compound 5 and 210 mg of a starting material (Compound 1). Compound 2: m.p. 219-221 °C; 13C-NMR(Pyridine-d5, δppm) 106.8, 88.9, 78.6, 78.2, 75.7, 72.7, 71.6, 62.9, 62.7, 56.4, 51.0, 50.9, 50.9, 49.5(49.3), 44.0, 40.1, 39.8, 39.3, 38.1, 37.1, 35.3, 34.9(34.5), 33.6, 32.6(32.5), 28.2, 28.1, 26.7, 26.4(25.9), 22.9, 22.8, 20.4, 18.51, 17.3, 16.5(16.5), 15.8(15.8), 13.4. Compound 5: m.p. 178-180 °C; 13C-NMR(Pyridine-d5, δppm) 78.9, 78.6, 78.2, 75.7, 72.7, 71.6, 62.9, 62.7, 56.4, 51.0, 50.9, 50.8, 49.50(49.26), 44.0, 40.1, 39.7, 39.3, 38.0, 37.0, 35.3, 34.9(34.5), 33.6, 32.64(32.54), 28.1, 28.1, 26.7, 26.3(25.9), 22.9, 22.7, 20.4, 18.4, 17.2, 16.5(16.52), 15.82(15.7), 14.8.
Example 4 : Preparation of a Mixture of Ginsenoside F4 and Ginsenoside Rg6 Twenty grams of ginsenosie Re and 20 g of citric acid were dissolved in 200 mL of 99.8% ethanol, heated at 85 °C for 24 hrs and the ethanol was removed under reduced pressure. The resulting concentrate was dispersed in 500 mL of butanol and then washed with a solution, wherein 20 g of sodium hydroxide is dissolved in 150 mL of water, washed twice with 150 mL of water and the butanol layer was concentrated under reduced pressure to obtain 16.2 g of a concentrate. The resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (8:1-→7.T→6:1→5:1→4:1) containing 2% water in 340 g silica gel and finally obtained 5.8 g of a mixture of ginsenoside F4 and ginsenoside Rg6.
Example 5 : Preparation of 6-0-[α-L-rhamnopyranosyl(l→2)-β-D- glucopyranosyljdammarane -3β,6α,12β-triol (Compound 5) 4.2 g of the mixture of ginsenoside F4and ginsenoside Rg6 prepared in example 4 was dissolved in 240 mL of methanol, added with 800 mg of 10% palladium-charcoal and then reacted by using Parr reactor under 15 psi of hydrogen gas pressure with shaking for 16 hrs. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure. The resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of chloroform : methanol (8:1→7:1→6:1→5:1) containing 1% water in 200 g silica gel and finally obtained 4.5 g of target compound 5. m.p. 182-184 °C; 13C-NMR(Pyridine-d5, δppm): 102.0, 101.8, 79.5, 78.6, 78.4, 78.3, 74.4, 74.2, 72.6, 72.4, 72.3, 69.6, 63.3, 60.9, 52.0, 51.0, 50.7, 50.1, 46.2, 41.5, 40.1, 40.0, 39.6, 38.8, 37.7(37.1), 35.6, 32.5, 32.2, 30.6, 30.1, 29.8, 29.4, 27.8, 27.1(26.4), 22.7, 22.5, 18.8, 18.4(17.8), 17.7, 17.6, 17.3, 17.0
Example 6 : Preparation of a Mixture of Ginsenoside Rk3 and Ginsenoside Rh4 Twenty grams of ginsenoside Rgl and 20 g of citric acid were dissolved in
200 mL of 99.8% ethanol, heated at 85 °C for 18 hrs and the ethanol was removed under reduced pressure. The resulting concentrate was dispersed in 500 mL of butanol, washed twice with 150 mL of water and then concentrated under reduced pressure to obtain 16.1 g of a concentrate. The resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (8:1→7:1— >6:1— >5:1→4:1) containing 2% water in 340 g silica gel and finally obtained 8.8 g of a mixture of ginsenoside Rk3and ginsenoside Rh4. Example 7 : Preparation of 6-O-β-D-glucopyranosyl dammarane-3β,6α,12β-triol (Compound 4) 5.7 g of the mixture of ginsenoside Rk3and ginsenoside Rh4 prepared in example was dissolved in 150 mL of ethanol and 130 mL of ethylacetate, added with 800 mg of 10% palladium-charcoal and then reacted by using Parr reactor under 25 psi of hydrogen gas pressure by shaking for 6 hrs. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure. The resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of chloroform : methanol (8:1→7:1-→6:1→5:1) containing 1% water in 120 g silica gel and finally obtained 5.4 g of a target compound. m.p. 168-171 °C; i3C-NMR(Pyridine-d5, δppm): 105.0, 79.0, 78.3, 77.6, 74.9, 74.4, 72.6,
63.1, 60.9, 52.0, 51.0, 50.7, 50.1, 46.2, 41.5, 40.1, 40.0, 39.6, 38.8, 37.7(37.0), 32.6, 32.5,
32.2, 30.6, 30.1, 29.8, 29.4, 27.8, 27.1(26.6), 22.7, 22.5, 18.4(17.8), 17.7, 17.6, 17.3, 17.0
Example 8 : Preparation of dammarane-3β,6α,12β-triol (Compound 6) 0.8 g of compound 4 prepared in example 7 was dissolved in 80 mL of butanol, added with 3.5 g of sodium hydroxide and stirred at 85 °C for 32 hrs. The reactants were added to 30 mL of butanol, washed three times with 40 mL of water and the butanol layer was concentrated under reduced pressure. The resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (50:1→40:1→30:1→20:1) in 30 g silica gel and finally obtained 0.53 g of target compound 6. m.p. 153-155 °C; 13C-NMR(Pyridine-d5, δppm) 78.3, 73.6, 60.9, 52.0, 51.0, 50.7, 50.1, 46.2, 41.5, 40.1, 40.0, 39.6, 38.8, 37.7(37.0), 32.6, 32.5, 32.2, 30.6, 30.1, 29.8, 29.4, 27.8, 27.1(26.6), 22.7, 22.5, 18.4(17.8), 17.7, 17.6, 17.3, 17.0
Example 9 : Preparation of 3-0-[β-D-glucopyranosyl(l— >2)-β-D- glucopyranosyl]dammarane-3β,12β-diol (Compound 1) 0.15 g of ginsenoside Rg5 prepared according to the method in a reference (Arch. Pharm. Res. 19, 551 (1996)) was dissolved in 15 mL of ethanol, added with 30 mg of 5% palladium-charcoal and reacted at 40 °C by shaking using Parr reactor for 6 hrs under 45 psi of hydrogen gas pressure. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and finally obtained 0.14 g of target compound 1.
Example 10 : Preparation of 3-O-β-D-gIucopyranosyl dammarane-3β,12β-diol (Compound 2) 0.2 g of ginsenoside Rh3 prepared according to the method in a reference (Yakhakhoeji 39, 85 (1995)) was dissolved in 20 mL of ethanol, added with 50 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 40 psi of hydrogen gas pressure. The catalyst was removed by filtering through cellite and the mixture was concentrated under reduced pressure and finally obtained 0.18 g of target compound 2.
Example 11 : Preparation of 6-0-[α-L-rhamnopyranosyl(l→2)-β-D- glucopyranosyl]dammarane-3β,6α,12β-triol (Compound 3) 0.11 g of ginsenoside F4 prepared according to the method in a reference (Planta Medica 56, 298 (1990)) was dissolved in 10 mL of ethanol, added with 20 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 40 psi of hydrogen gas pressure. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and finally obtained 0.1 g of target compound 3.
Example 12 : Preparation of 6-0-[α-L-rhamnopyranosyl(l→2)-β-D- glucopyranosyl]dammarane-3β,6α,12β-triol (Compound 3) 0.11 g of ginsenoside Rg6 in the amount of 0.11 g prepared according to the method in a reference (Phytochemistry 44, 931 (1997)) was dissolved in 10 mL of methanol, added with 20 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 40 psi of hydrogen gas pressure. The catalyst was removed by filtering through cellite and the mixture was concentrated under reduced pressure and finally obtained 0.1 g of target compound 3.
Example 13 : Preparation of 6-O-β-D-glucopyranosyl dammarane-3β,6α,12β-triol (Compound 4) 0.16 g of ginsenoside Rh4 in the amount of 0.16 g prepared according to the method in a reference (Planta Medica 62, 86 (1996)) was dissolved in 15 mL of methanol, added with 20 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 30 psi of hydrogen gas pressure. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and finally obtained 0.13 g of target compound 4. Example 14 : Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl Ten grams of a protopanaxadiol ginsenoside mixture and 10 g of tartaric acid were dissolved in 100 mL of 99.8% ethanol, heated at 90 °C for 12 hrs and ethanol was removed under reduced pressure. The resulting concentrate was dispersed in 300 mL of butanol and then washed with a solution, wherein 20 g of sodium hydroxide is dissolved in 150 mL of water, washed twice with 150 mL of water and the butanol layer was concentrated under reduced pressure to obtain 3.94 g of a concentrate. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1→5:1→4:1) containing 2% water in 100 g silica gel and finally obtained 2.25 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
Example 15 : Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl Ten grams of a protopanaxadiol ginsenoside mixture and 20 g of fumaric acid were dissolved in 100 mL of 99.8% methanol, heated at 90 °C for 72 hrs and methanol was removed under reduced pressure. The resulting concentrate was dispersed in 300 mL of butanol and then washed with a solution, wherein 15 g of sodium hydroxide is dissolved in 150 mL of water, washed twice with 150 mL of water and the butanol layer was concentrated under reduced pressure to obtain 3.84 g of a concentrate. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1→5:1→4:1) containing 2% water in 100 g silica gel and finally obtained 2.2 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
Example 16 : Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl Ten grams of a protopanaxadiol ginsenoside mixture and 25 g of maleic acid were dissolved in 100 mL of 99.8% methanol, heated at 90 °C for 72 hrs and methanol was removed under reduced pressure. The resulting concentrate was dispersed in 300 mL of butanol and then washed with a solution, wherein 15 g of sodium hydroxide is dissolved in 150 mL of water, washed twice with 150 mL of water and the butanol layer was concentrated under reduced pressure to obtain 3.8 g of a concentrate. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1— »5:1→4:1) containing 2% water in 100 g silica gel and finally obtained 2.28 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
Example 17 : Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl Ten grams of a protopanaxadiol-based ginsenoside mixture and 5 g of lactic acid were dissolved in 100 mL of 99.8% ethanol, heated at 90 °C for 48 hrs and ethanol was removed under reduced pressure. The resulting concentrate was dispersed in 300 mL of butanol and then washed with a solution, wherein 5 g of sodium hydroxide is dissolved in 150 mL of water, washed twice with 150 mL of water and the butanol layer was concentrated under reduced pressure to obtain 4.1 g of a concentrate. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1→5:1— >4.T) containing 2% water in 100 g silica gel and finally obtained 2.38 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
Example 18 : Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl Ten grams of a protopanaxadiolginsenoside mixture and 30 g of glacial acetic acid were dissolved in 100 mL of 99.8% ethanol, heated at 90 °C for 24 hrs and ethanol was removed under reduced pressure. The resulting concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1→5:1→4.T) containing 2% water in 100 g silica gel and finally obtained 2.48 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl.
Example 19 : Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl Ten grams of a protopanaxadiol ginsenoside mixture and 20 g of formic acid were dissolved in 100 mL of 99.8% ethanol, heated at 90 °C for 22 hrs and ethanol was removed under reduced pressure. The resulting concentrate was dispersed in 300 mL of butanol and then washed twice with 150 mL of water and the butanol layer was concentrated under reduced pressure to obtain 3.88 g of a concentrate. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane : methanol (6:1→5:1— *4:1) containing 2% water in 100 g silica gel and finally obtained 2.24 g of a mixture of ginsenoside Rg5 and ginsenoside Rkl. Example 20 : Preparation of 3-0-[β-D-glucopyranosyl(l→2)-β-D-glucopyranosyl] dammarane-3β,12β-diol octaacetate (Compound 7) Two hundred milligrams of 3-0-[β-D-glucopyranosyl(l→2)-β-D- glucopyranosyl]dammarane-3β,12β-diol (Compound 1) prepared in example 2 was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride. After keeping it at room temperature for 24 hrs, it was concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate in 20 g silica gel and obtained target compound 7. Η-NMR(400 MHZ, CDC13) δ 4.70(1H, d, J=8.0 Hz), 4.45(1H, d, J=7.6Hz), 3.80(1H, dd, 1=8.5, 7.6Hz), 3.77(1H, br. s.), 3.08(1H, dd, J=11.4, 4.0Hz), 2.10, 2.07(3Hχ2, s), 2.02(3Hχ2, s), 2.01, 1.98(3Hχ2, s), 1.03(3H, s), 1.02(3H, s), 0.96(3H, d), 0.95(3H, d), 0,90(3H, d), 0.87(3H, s), 0.81(3H, s).
Example 21 : Preparation of 3-0-[β-D-glucopyranosyl(l-→2)-β-D-glucopyranosyl] dammarane-3β,12β-diol octapalmitate (Compound 8) Two hundred milligrams of 3-0-[β-D-glucopyranosyl(l→2)-β-D- glucopyranosyl]dammarane-3β,12β-diol (Compound 1) prepared in example 2 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. It was then added with 1 g of palmitic acid chloride and kept at room temperature for 24 hrs. Then, it was added with 50 mL of water and extracted twice with 50 mL of dichloromethane and concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate in 30 g silica gel and obtained target compound 8.
Example 22. Preparation of 3-0-[β-D-glucopyranosyl(l→2)-β-D-glucopyranosyl] dammarane-3β,12β-diol octaundecanoate (Compound 9) Two hundred milligrams of 3-0-[β-D-glucopyranosyl(l→2)-β-D- glucopyranosyl]dammarane-3β,12β-diol (Compound 1) prepared in example 2 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. It was then added with 1 g of undecanoic acid chloride and kept at room temperature for 24 hrs. Then, it was added with 50 mL of water, extracted twice with 50 mL of dichloromethane and concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (3:1) in 30 g silica gel and obtained target compound 9.
Example 23 : Preparation of 3-0-[β-D-glucopyranosyl(l→2)-β-D-glucopyranosyl] dammarane-3β,12β-diol octabutyrate (Compound 10) Two hundred milligrams of 3-0-[β-D-glucopyranosyl(l— >2)-β-D- glucopyranosyl]dammarane-3β,12β-diol (Compound 1) prepared in example 2 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. It was then added with 0.5 g of butyric acid chloride and kept at room temperature for 24 hrs. Then, it was added with 50 mL of water, extracted twice with 50 mL of dichloromethane and concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (3:1) in 30 g silica gel and obtained target compound 10.
Example 24 : Preparation of 3-O-β-D-glucopyranosyl dammarane-3β,12β-diol pentaacetate (Compound 11) Two hundred milligrams of 3-O-β-D-glucopyranosyl dammarane-3β,12β- diol (Compound 2) prepared in example 2 was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride and cooled down. After keeping at room temperature for 24 hrs, it was concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (3:1) in 20 g silica gel and obtained target compound 11.
Η-NMR(400 MHZ, CDC13) δ 4.45(1H, d, J=7.6Hz), 3.77(1H, br.s.), 3.08(lH,. dd, 1=11.4, 4.0Hz), 2.10(3H, s), 2.08(3H, s), 2.03(3H, s), 2.01 (3H, s), 1.99(3H, s)> 1.03(3H, s), 1.02(3H, s), 0.96(3H, d), 0.95(3H, d), 0,90(3H, d), 0.87(3H, s), 0.81 (3H, s).
Example 25 : Preparation of 3-O-β-D-glucopyranosyl dammarane-3β,12β-diol pentabutyrate (Compound 12) One hundred fifty milligrams of 3-O-β-D-glucopyranosyl dammarane- 3β,12β-diol (Compound 2) prepared in example 3 was dissolved in 2 mL of pyridine and then added with 0.5 mL of triethylamine and cooled down. Then, it was added with 0.3 g of butyric acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (5:1) in 20 g silica gel and obtained target compound 12.
Example 26 : Preparation of 3-O-β-D-glucopyranosyl dammarane-3β,12β-diol pentapalmitate (Compound 13) One hundred fifty milligrams of 3-O-β-D-glucopyranosyl dammarane- 3β,12β-diol (Compound 2) prepared in example 3 was dissolved in 3 mL of pyridine and then added with 0.5 mL of triethylamine and cooled down. Then, it was added with 0.4 g of palmitic acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (5:1) in 20 g silica gel and obtained target compound 13.
Example 27 : Preparation of 6-0-[α-L-rhamnopyranosyl(l— >2)-β-D-glucopyranosyl] dammarane-3β,6α,12β-triol octaacetate (Compound 14) Two hundred milligrams of 6-0-[α-L-rhamnopyranosyl(l— ->2)-β-D- glucopyranosyl]dammarane-3β,6α,12β-triol (Compound 3) prepared in example 5 was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride. After keeping at room temperature for 24 hrs, it was concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (3:1) in 20 g silica gel column and obtained target compound 14. Example 28 : Preparation of 6-0-[α-L-rhamnopyranosyl(l-→2)-β-D-glucopyranosyl] dammarane-3β,6α,12β-triol octabutyrate (Compound 15) One hundred fifty milligrams of 6-0-[α-L-rhamnopyranosyl(l→2)-β-D- glucopyranosyl]dammarane-3β,6α,12β-triol (Compound 3) prepared in example 5 was dissolved in 2 mL of pyridine and then added with 0.5 mL of triethylamine and cooled down. Then, it was added with 0.3 g of butyric acid chloride and placed at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (5:1) in 20 g silica gel and obtained atarget compound 15.
Example 29 : Preparation of 6-0-[α-L-rhamnopyranosyl(l— »2)-β-D- glucopyranosyl]dammarane-3β,6α,12β-triol octapalmitate (Compound 16) One hundred fifty milligrams of 6-0-[α-L-rhamnopyranosyl(l— >2)-β-D- glucopyranosyl]dammarane-3β,6α,12β-triol (Compound 3) prepared in example 5 was dissolved in 2 mL of pyridine and then added with 0.5 mL of triethylamine and cooled down. Then, it was added with 0.5 g of palmitic acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (5:1) in 20 g silica gel and obtained target compound 16. Example 30 : Preparation of 6-O-β-D-glucopyranosyl dammarane-3β,6α,12β-triol hexaacetate (Compound 17) Two hundred milligrams of 6-O-β-D-glucopyranosyl dammarane-3β,6α,12β- triol (Compound 4) prepared in example 7 was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride. After keeping at room temperature for 24 hrs, it was concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (3:1) in 20 g silica gel and obtained target compound 17.
Example 31 : Preparation of 6-O-β-D-glucopyranosyl dammarane-3β,6α,12β-triol hexabutyrate (Compound 18) One hundred fifty milligrams of 6-O-β-D-glucopyranosyl dammarane- 3β,6α,12β-triol (Compound 4) prepared in example 7 was dissolved in 2 mL of pyridine and then added with 0.5 mL of triethylamine and cooled down. Then, it was added with 0.3 g of butyric acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (5:1) in 20 g silica gel and obtained target compound 18.
Example 32 : Preparation of 6-O-β-D-glucopyranosyl dammarane-3β, 6α,12β-triol hexapalmitate (Compound 19) One hundred fifty milligrams of 6-O-β-D-glucopyranosyl dammarane- 3β,6α,12β-triol (Compound 4) prepared in example 7 was dissolved in 2 mL of pyridine and then added with 0.5 mL of triethylamine and cooled down. Then, it was added with 0.45 g of palmitic acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (5:1) in 20 g silica gel and obtained target compound 19.
Example 33 : Preparation of dammarane-3β,12β-diol dipalmitate (Compound 20) Two hundred milligrams of dammarane-3β,12β-diol (Compound 5) prepared in example 3 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. Then, it was added with 1 g of palmitic acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (6:1) in 30 g silica gel and obtained target compound 20.
Example 34. Preparation of dammarane-3β,12β-diol diacetate (Compound 21) Two hundred milligrams of dammarane-3β,12β-diol (Compound 5) prepared in example 3 was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride. After keeping at room temperature for 24 hrs, it was concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (6:1) in 20 g silica gel and obtained target compound 21.
Example 35 : Preparation of dammarane-3β, 12β-diol dibutyrate (Compound 22) Two hundred milligrams of dammarane-3β,12β-diol (Compound 5) prepared in example 3 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. Then, it was added with 1 g of butyric acid chloride and placed at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (6:1) in 20 g silica gel and obtained target compound 22.
Example 36 : Preparation of dammarane-3β, 6α, 12β-triol tripalmitate (Compound
23) Two hundred milligrams of dammarane-3β,12β-diol (Compound 5) prepared in example 8 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. Then, it was added with 1 g of palmitic acid chloride and placed at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (6:1) in 30 g silica gel and obtained target compound 23. Example 37 : Preparation of dammarane-3β,6α,12β-triol tributyrate (Compound 24) Two hundred milligrams of dammarane-3β,6α,12β-triol (Compound 6) prepared in example 8 was dissolved in 2 mL of pyridine and then added with 1 mL of triethylamine and cooled down. Then, it was added with 1 g of butyric acid chloride and kept at room temperature for 24 hrs. After adding 50 mL of water, it was extracted twice using 50 mL of dichloromethane and then concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (6:1) in 20 g silica gel and obtained target compound 24.
Example 38 : Preparation of dammarane-3β,6α,12β-triol triacetate (Compound 25) Two hundred milligrams of dammarane-3β,6α,12β-triol (Compound 6) prepared in example 8 was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride. After keeping at room temperature for 24 hrs, it was concentrated under reduced pressure. The concentrate was passed through a silica gel column chromatography using a mixed solvent of dichloromethane and ethylacetate (6:1) in 20 g silica gel and obtained target compound 25.
Example 39 : Preparation of 3-0-[β-D-glucopyranosyl(l— >2)-β-D-glucopyranosyl] dammarane-3β,12β-diol octaacetate (Compound 7) A mixture of ginsenoside Rg5 and ginsenoside Rkl in the amount of 0.3 g prepared in example 1 was dissolved in 3 mL of pyridine, added with 3 mL of acetic anhydride and reacted at room temperature by stirring for 24 hrs. It was then concentrated under reduced pressure and a mixture octaacetate of ginsenoside Rg5 and ginsenoside Rkl was produced. Thus concentrate obtained was dissolved in 15 mL of ethylacetate and 10 mL of ethanol, added with 100 mg of 5% palladium- charcoal and reacted at room temperature using Parr reactor by shaking for 16 hrs under 30 psi of hydrogen pressure. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure. The resulting concentrate was passed through a silica gel column chromatography in 20 g silica gel and finally obtained target compound 7.
Example 40 : Preparation of 3-O-β-D-glucopyranosyl dammarane-3β,12β-diol pentaacetate (Compound 11) 0.5 g of ginsenoside Rh3 prepared according to the method in a reference (Yakhakhoeji 39, 85 (1995)) was dissolved in 2 mL of pyridine and 2 mL of acetic anhydride. After stirring at room temperature for 16 hrs, the mixture was concentrated under reduced pressure to obtain ginsenoside Rh3 pentaacetate. The ginsenoside Rh3 pentaacetate was dissolved in 10 mL of ethanol, added with 50 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 40 psi of hydrogen gas pressure. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and then passed through a silica gel column chromatography in 20 g silica gel, solvent of dichloromethane and ethylacetate (3:1) to obtain 0.1 g of target compound 11.
Example 41 : Preparation of 6-0-[α-L-rhamnopyranosyl(l→2)-β-D-glucopyranosyl] dammarane-3β,6α,12β-triol octaacetate (Compound 14) 0.1 g of ginsenoside prepared according to the method in a reference (Planta Medica 56, 298 (1990)) was dissolved in 2 mL of pyridine and 2 mL of acetic anhydride. After stirring at room temperature for 16 hrs, the mixture was concentrated under reduced pressure to obtain ginsenoside F4 octaacetate. The ginsenoside F4 octaacetate was dissolved in 5 mL of ethanol and 5 mL of ethylacetate, added with 20 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 40 psi of hydrogen gas pressure. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and then passed through a silica gel column chromatography in 20 g silica gel, solvent of dichloromethane and ethylacetate (4:1) to obtain target compound 14.
Example 42. Preparation of 6-O-β-D-glucopyranosyl dammarane-3β,6α,12β-triol hexaacetate (Compound 17) O.lg of ginsenoside Rh4 prepared according to the method in a reference
(Planta Medica 62, 86 (1996)) was dissolved in 2 mL of pyridine and 2 mL of acetic anhydride. After stirring at room temperature for 16 hrs, the mixture was concentrated under reduced pressure to obtain ginsenoside Rh4 hexaacetate. The ginsenoside Rh4 hexaacetate was dissolved in 5 mL of ethanol and 5 mL of ethylacetate, added with 20 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 30 psi of hydrogen gas pressure. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and then passed through a silica gel column chromatography in 20 g silica gel, solvent of dichloromethane and ethylacetate (4:1) to obtain target compound 17.
0.1
Example 43. Preparation of dammarane-3β,6α,12β-triol triacetate (Compound 25) 150 mg of dammarane-20(22),24-dien-3β,6α,12β-triol prepared according to the method in a reference (Planta Medica 62, 86 (1996)) was dissolved in 2 mL of pyridine and then added with 2 mL of acetic anhydride. After stirring at room temperature for 24 hrs, the mixture was concentrated under reduced pressure to obtain dammarane-20(22), 24-dien-3β,6α,12β-triol triacetate. The rhamnopyranosyl- 20(22),24-dien-3β,6α,12β-triol triacetate was dissolved in 5 mL of ethanol and 5 mL of ethylacetate, added with 20 mg of 10% palladium-charcoal and reacted at 30 °C by shaking using Parr reactor for 10 hrs under 30 psi of hydrogen gas pressure. The catalyst was removed by filtering through celite and the mixture was concentrated under reduced pressure and then passed through a silica gel column chromatography in 20 g silica gel, solvent of dichloromethane and ethylacetate (6:1) to obtain target compound 25.
The following preparation examples are given for the purpose of explaining general pharmaceutical compositions according to the present invention. The active ingredients in each preparation example represents 3-0-[β-D-glucopyranosyl(l— >2)- β-D-glucopyranosyl] dammarane-3β,12β-diol (Compound 1) prepared in the example 2 and can be replaced with other compounds with an amount having equal effect depending on the situations. Preparation Example 1 : Preparation of Tablets Five grams of an effective compound (Compound 1), 15 g of lactose, 2.5 g of carboxy methyl cellulose calcium and 2.5 g of crystalline cellulose were well mixed and formulated into 50 mg of tablets according to the conventional tableting method.
Preparation Example 2 : Preparation of Granules Five grams of an effective compound (Compound 1), 15 g of lactose, 2.5 g of carboxy methyl cellulose sodium and 2.5 g of crystalline cellulose were well mixed and formulated into granules according to the conventional wet granulation method.
Preparation Example 3 : Preparation of Hard Capsules Five grams of an effective compound (Compound 1), 10 g of lactose, 2.5 g of carboxy methyl cellulose calcium and 7.5 g of crystalline cellulose were well mixed and formulated into 50 mg of hard capsules according to the conventional manufacturing method.
Preparation Example 4 : Preparation of Soft Capsules Five grams of an effective compound (Compound 1), 42 g of soybean oil, 1.5 g of soybean lecithin and 1.5 g of yellow wax were well mixed and formulated into 100 mg of soft capsules according to the conventional manufacturing method.
Preparation Example 5 : Preparation of Injections Five grams of an effective compound (Compound 1) was dissolved by applying heat in the presence of 5 g of polyethyleneglycol, 5 mL of ethanol, and 400 mL of phosphate saline solution. After cooling down, it was added with phosphorylated physiological saline solution to the final volume of 500 mL and then filled into 1 mL vials to manufacture injection preparations according to the conventional method.
The following reference examples and comparative reference examples show examples of acid hydrolysis method for manufacturing Δ20-ginsenoside represented by the above formula 2a or 2b. The reference examples 1 and 2 show examples of acid hydrolysis method performed under the condition of a mixed solution of an organic acid and aliphatic alcohol while comparative reference examples 1 and 2 show the conventional acid hydrolysis method.
Reference Example 1 : Preparation of a Mixture of Ginsenoside Rg5 andGinsenoside Rkl One gram of protopanaxadiol ginsenoside mixture and I g of citric acid were dissolved in 10 mL of 99.8% ethanol and heated at 85 °C for 12hrs. The reactants were diluted in 200 mL of butanol and washed with a solution, wherein 2 g of sodium hydroxide is dissolved in 60 mL of water. The butanol layer was washed twice with 60 mL of water and then concentrate under reduced pressure to obtain 0.4 g of target product.
Reference Example 2 : Preparation of a Mixture of Ginsenoside Rg5 and Ginsenoside Rkl One gram of protopanaxadiol ginsenoside mixture and I g of citric acid were dissolved in 10 mL of 95% ethanol and heated at 85 °C for 12hrs. The reactants were diluted in 200 mL of butanol and washed with a solution, wherein 2 g of sodium hydroxide is dissolved in 60 mL of water. The butanol layer was washed twice with 60 mL of water and then concentrate under reduced pressure to obtain 0.41 g of target product.
Comparative Reference Example 1 : Preparation of a Mixture of protopanaxadiol Ginsenoside Two hundred fifty grams of 70% ethanol extraction powder of Panax ginseng was dissolved in 1,000 mL of water and added with 50 g of sodium hydroxide and extracted with 4 times with water-saturated butanol 1,200 mL in order toremove protopanaxatriol ginsenoside mixture. After cooling down, the water layer was neutralized by adding 200 mL of cooled 6N HC1 and extracted four times with water saturated butanol 1,200 mL and the butanol layer was concentrated under reduced pressure. The concentrate was dissolved in 300 mL of methanol and filtered. The filtrate was concentrated under reduced pressure to obtain 42 g of protopanaxadiol ginsenoside mixture.
Comparative Reference Example 2 : Preparation of Ginsenoside Rg5 and Ginsenoside Rkl One gram of protopanaxadiol ginsenoside mixture and 1 g of citric acid were dissolved in 10 mL of water and heated at 85 °C for 12hrs. The reactants were diluted in 200 mL of butanol and washed with a solution, wherein 2 g of sodium hydroxide is dissolved in 60 mL of water. The butanol layer was washed twice with 60 mL of water and then concentrate under reduced pressure to obtain 0.41 g of target product.
The specimens obtained in the reference examples 1 and 2 and comparative reference example 2 were analyzed by HPLC: Column; Capcell pak MG C18 (4.6x250 cm), detection; at 203 nm and gradient elution solvents; (A) 10% acetonitrile/ water, (B) 90% acetonitrile/ water. The results are shown in Fig. 1. In Fig. 1, PI represents 20S-ginsenoside Rg3, P2 represents 20R-ginsenoside Rg3, P3 represents ginsenoside Rkl, and P4 represents ginsenoside Rg5, respectively. When protopanaxadiol ginsenoside mixture is hydrolyzed in an organic aqueous solution according to the method in comparative reference example 2, ginsenoside Rg3(Pl+P2) and ginsenoside Rkl and Ginsenoside Rg5 are generated in about 1:1 ratio in terms of their peak area.. On the other hand, when protopanaxadiol ginsenoside mixture is hydrolyzed in 95% ethanol or 99.8% ethanol solution by organic acid according to the method in comparative reference examples
1 and 2, ginsenoside Rg3 are generated only a small amount while ginsenoside Rkl and ginsenoside Rg5 as Δ20-ginsenoside are produced more than 90%. Further, in order to investigate the effects of ginsenoside derivatives represented by the formula 1 of the present invention on prevention and treatment of cancer as well as inhibition of metastasis, their biological activities were measured by using the procedures shown in the following experimental examples 1 - 5.
Experimental Example 1 : Effect on In Vitro Inhibition of Cancer Cell Growth The inhibitory effect of ginsenoside derivatives on cancer cell growth were measured by the sulforodamin B method (J. National Cancer Institute, 82, 1107 (1990)). The 50% inhibition concentration of cell growth (ED50) is shown in Table 1.
Table 1
Figure imgf000040_0001
As shown in Table 1, the compounds of the present invention have shown 2 to 3 times more improved anticancer activities as compared to those of the conventional ginsenosides in lung cancer cells (A549), ovary cancer cells (SK-OV-3), melanoma cells (SK-MEL-2), brain tumor cells (XF498), reactal cancer cells (HCT15). In particular, 3-0- [β-D-glucopyranosy 1(1 -→2)-β-D-glucopy ranosyl] dammarane- 3β,12β-diol (Compound 1) shows 11 times stronger antitumor activity as compared to that of ginsenoside Rg3 currently used for the treatment of lung and liver cancers.
Experimental Example 2 : Effect on Tumor Inhibition in a nude mouse Xenograft Model using human cancer cell line HT-1080(Fibrosarcoma) Female nude mice Balb/c (8 weeks old, 18 g of body weight, 8 mice per group) were hypodermically transplanted with caner cells at the concentration of lxlO7 cell/mL with 0.3 mL. Starting from the next day (Day 1) after the transplantation till one day prior to termination of the experiment, the above mice were orally administered with a specimen and a solvent (control group: 0.2% Tween-80 solution) 0.2 mL per 20 g of body weight of a mouse once per day to a total of 13 times. Adriamycin, a positive control substance, was intraperitoneally injected 2 mg/kg once every two days. During the period of from the 7th day after the transplantation to the day of termination of the experiment, the size of tumors were measured a total of 6 times. The size of tumors were measured in 3 different directions using a caliper and calculated using the following equation 1. The results are shown in Table 2. [Equation 1] Vol. of Tumor = (length x width x height)/ 2
Table 2 Inhibitory Effect on Tumor Cell Growth by Oral Administration
Experimental Dosage Vol. of Tumor Cells (mm3)
Figure imgf000042_0001
As shown in Table 2, the compounds of the present invention showed superior anticancer activity when orally administered in the amount of 3 and 10 mg/kg. Experimental Example 3 : Effect on Inhibition of Metastasis of Cancer Cells After culturing B16F10 melanoma cells, the cells were separated using trypsin-EDTA and diluted with 0.85% saline solution to obtain a 2.5χ 106cells/mL. Thus prepared cancer cell suspension was injected to female S.P.F. C57BL/6 mice (6 mice per group) into the caudal veins with 0.2 mL per mouse and then the mice were administered with 0.2 mL of a drug in 0.2% Tween-80 solution per 20 g of body weight of a mouse once per day starting from 4 hours after the above caudal vein injection. Adriamycin, a positive control substance, was intraperitoneally injected 2 mg/kg once every two days. On the 14th day after the cancer cell transplantation, autopsies were performed for the mice. The cancer-transinfected lungs were ablated and then fixed in neutral formaline solution and photographed. The cancer cell colonies shown in the resulting photographs were counted by naked eyes and the results are shown in Table 3.
Table 3
Figure imgf000043_0001
As shown in Table 3, the compound of the present invention showed superior inhibitory activity against metastasis of cancer cells when orally administered in dose of 3 and 10 mg/kg. In particular, compound 1 had more potent anti-metastasis activities as compared to that 2 mg/kg of adriamycin, ip injection.
Experimental Example 4 : Effect on Inhibition of Angiogenesis 3-0-[β-D-glucopyranosyl(l→2)-β-D-glucopyranosyl] dammarane-3β,12β- diol (Compound 1) and ginsenoside Rg3 were dissolved in 1% carboxymethylcellulose saline solution. On the 4th day after initiation of hatching, Chick embryo chorioallantoic membrane was administered with a drug (14 eggs per each group) and cultured at 37 °C for 48 hrs and calculated those which were prevented from angiogenesis. The results are shown in Table 4.
Table 4
Figure imgf000044_0001
As shown in Table 4, 3-0-[β-D-glucopyranosyl(l→2)-β-D- glucopyranosyl]dammarane-3β,12β-diol (Compound 1) showed 10 times more effective inhibitory activity against angiogenesis as compared to that of conventional ginsenoside Rg3.
Experimental Example 5 : Effect on Inhibition of Cancers induced by Carcinogens (Cancer Preventive Effect) In order to develop a cancer, carcinogen 7, 12-dimethylbenz(a)anthracene (DMBA) was tropically applied once to the backskin and then croton oil as a promoter was applied 3 times a week as a second step protocol. Swiss albino mice with body weight of about 24 g were used (30 mice per group). The control group was treated with DMBA (100μg/50μL acetone/ mouse) and then 0.1 mL of croton oil was applied 3 times a week starting from 2 weeks after the treatment until the termination of the experiment. Three groups were prepared. Each group was respectively adminsitered orally with 3-0-[β-D-glucopyranosyl(l-→2)-β-D-glucopyranosyl]dammarane-3β,12β- diol (Compound 1) in dose of 1 mg/kg, 3 mg/kg, 10 mg/kg, which was dissolved in Tween-80 solution, once per day starting from the day of a week prior to the treatment with DMBA(100μg/50μLaceton/ mouse) until the termination of the experiment, and then treated with 0.1 mL of croton oil 3 times a week from the day of after the DMBA treatment until the termination of the experiment. Papilloma developed on skin was observed and measured once a week during the entire 16 weeks of experimental period, and those papilloma consecutively observed to be greater than 1 mm in diameter were used for measurement. The results are shown in Table 5.
Table 5
Figure imgf000045_0001
Figure imgf000046_0001
As shown in Table 5, when mice were orally administered with 3-0-[β-D- glucopyranosyl(l→2)-β-D-glucopyranosyl] dammarane-3β,12β-diol (Compound 1) of the present invention in dose of 1 mg/kg, 3 mg/kg, 10 mg/kg, respectively, the number of papilloma, weight of tumor, tumor incidence were drastically decreased and the latent period of tumor development was also greatly delayed. Therefore, the compounds of the present invention show excellent cancer-preventive effects as well as anticancer activities.
Experimental Example 6 : Acute Toxicity Test 20 ICR mice per group were orally administered with compounds 1, 2, 3, 4, 5 and 6, respectively, which were dissolved or suspended in carboxy methyl cellulose, in dose of 2,000 mg/kg once per day for one week. The mice were observed for two weeks after last administration and no dead mice were observed.
Industrial Applicability The ginsenoside derivatives of the present invention of the formula 1 are shown to have excellent activities of angiogenesis inhibition and metastasis inhibition as well as anticancer activities, and thus they are very useful as a cancer preventive agent as well as a therapeutic agent for treating cancers or preventing metastasis. Further, ginsenoside derivatives of the present invention of the formula 1 did not show any acute toxicities in animal experiments and are thus expected to be safely used as an effective pharmaceutical drug and a nutraceutical.

Claims

Claims
1. A ginsenoside derivative of the following formula 1,
Figure imgf000048_0001
wherein Ri is H, a C2-C2 acyl group, a glucose group, or a glucosyl(l→2)glucose group; R2 is H, a hydroxyl group, a C2-C2 O-acyl group, O- glucose group, or a rhamnosyl(l→2)glucose group; R3 is H, or a C2-C24 acyl group.
2. In claim 1, said ginsenoside derivative of the formula 1 is an isomer or a mixture of isomers.
3. In claim 1 or claim 2, said ginsenoside derivative of the formula 1 is selected from the group consisting of: 3-0-[β-D-glucopyranosyl(l→2)-β-D-glucopyranosyl]dammarane-3β,12β-diol
(Compound 1) ; 3-O-β-D-glucopyranosyl dammarane-3β,12β-diol (Compound 2) ; 6-0-[α-L-rhamnopyranosyl(l-→2)-β-D-glucopyranosyl]dammarane-
3β,6α,12β-triol (Compound 3) ; 6-O-β-D-glucopyranosyl dammarane-3β,6α,12β-triol (Compound 4) ; dammarane-3β,12β-diol (Compound 5) ; rhamnopyranosyl-3β,6α,12β-triol (Compound 6) ; 3-0-[β-D-glucopyranosyl(l→2)-β-D-glucopyranosyl] dammarane-3β,12β- diol octaacetate (Compound 7) ; 3-0-[β-D-glucopyranosyl(l— »2)-β-D-glucopyranosyl] dammarane-3β,12β- diol octapalmitate (Compound 8) ; 3-0-[β-D-glucopyranosyl(l→2)-β-D-glucopyranosyl]dammarane-3β,12β-diol octaundecanoate (Compound 9) ; 3-0-[β-D-glucopyranosyl(l→2)-β-D-glucopyranosyl]dammarane-3β,12β-diol octabutyrate (Compound 10) ; 3-O-β-D-glucopyranosyl dammarane-3β,12β-diol pentaacetate (Compound
11) ; 3-O-β-D-glucopyranosyl dammarane-3β,12β-diol pentabutyrate (Compound 12) ; 3-O-β-D-glucopyranosyl dammarane-3β,12β-diol pentapalmitate
(Compound 13) ; 6-0-[α-L-rhamnopyranosyl(l-→2)-β-D-glucopyranosyl]dammarane- 3β,6α,12β-triol octaacetate (Compound 14) ; 6-0-[α-L-rhamnopyranosyl(l→2)-β-D-glucopyranosyl]dammarane-
3β,6α,12β-triol octabutyrate (Compound 15) ; 6-0-[α-L-rhamnopyranosyl(l→2)-β-D-glucopyranosyl]darnmarane- 3β,6α,12β-triol octapalmitate (Compound 16) ; 6-O-β-D-glucopyranosyl dammarane-3β,6α,12β-triol hexaacetate
(Compound 17) ; 6-O-β-D-glucopyranosyl dammarane-3β,6α,12β-triol hexabutyrate
(Compound 18) ; 6-O-β-D-glucopyranosyl dammarane-3β, 6α,12β-triol hexapalmitate (Compound 19) ; dammarane-3β,12β-diol dipalmitate (Compound 20) ; dammarane-3β,12β-diol diacetate (Compound 21) ; dammarane-3β, 12β-diol dibutyrate (Compound 22) ; dammarane-3β, 6α, 12β-triol dipalmitate (Compound 23) ; dammarane-3β,6α,12β-triol t ibutyrate (Compound 24) ; and dammarane-3β,6α,12β-triol triacetate (Compound 25).
4. A method of preparing ginsenoside derivative of the following formula 1 by hydrogenation of compounds of the following formula 2a or 2b or a mixture thereof,
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000051_0002
wherein
Figure imgf000051_0003
is H, a C2-C2 acyl group, a glucose group, or a glucosyl(l— >2)glucose group; R2 is H, a hydroxyl group, a C2-C2 O-acyl group, O- glucose group, or a rhamnosyl(l— »2)glucose group; R3 is H, or a C2-C2 acyl group.
5. In claim 4, said hydrogenation is performed in a solvent consisting of an organic solvent selected from the group consisting of ethanol, methanol, and ethylacetate, and water, at a pressure from an atmosphere to 50 psi at room temperature to 40 °C using a catalyst such as palladium or platinum
6. In claim 4, the compound represented by the formula 1, wherein said Ri, R2 or Rj are respectively an acyl group, is manufactured by acylation of a compound of the formula 1, wherein said Ri, R2 or R3 are respectively H or a hydroxyl group, with a C2-C carboxylic acid anhydride or an acid chloride compound.
7. In claim 4, the compounds of the above formula 2a or formula 2b or a mixture of these compounds thereof are manufactured by hydrolyzing ginsenosides Ral, Ra2, Ra3, Rbl, Rb2, Re, Rd, Rgl, Re, or a mixture of these compounds or a protopanaxa diol ginsenoside by an organic acid.
8. In claim 7, said hydrolysis reaction is performed in a methanol or ethanol by using an organic acid selected from the group consisting of citric acid, tartaric acid, lactic acid, maleic acid, fumaric acid, formic acid and acetic acid with a concentration of about 0.1 to 30 vol.% at 50 to 100 °C.
9. A pharmaceutical composition effective in inhibition of cancer metastasis, prevention and treatment of- cancer comprising ginsenoside derivatives of the following formula 1 as active ingredients,
Figure imgf000053_0001
wherein Rr is H, a C2-C24 acyl group, a glucose group, or a glucosyl(l→2)glucose group; R2 is H, a hydroxyl group, a C2-C2 O-acyl group, O- glucose group, or a rhamnosyl(l→2)glucose group; R3 is H, or a C2-C24 acyl group.
10. In claim 9, said ginsenoside derivative is an isomer or a mixture of isomers.
11. In claim 9 or claim 10, said pharmaceutical composition is formulated in the form of tablets, granules, hard capsules, soft capsules or injections.
PCT/KR2005/001006 2004-05-29 2005-04-07 Treatment and prevention of cancer with new ginsenoside derivatives Ceased WO2005116042A1 (en)

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AU2007281038B2 (en) * 2006-08-03 2012-11-15 Oncology Research International Limited Methods and compositions for inhibiting angiogenesis
US8440632B2 (en) 2008-01-24 2013-05-14 Raptor Therapeutics Inc. Protopanaxadiol-type ginsenoside compositions and uses thereof
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CN116098911A (en) * 2023-02-23 2023-05-12 大连医科大学 Application of a kind of ginsenoside Rg6 in the preparation of anti-cisplatin-resistant ovarian cancer drug
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