Compounds

专利摘要:
The present invention relates to a compound represented by the following formula (1) or an optically active substance thereof or salts thereof. Formula 1 In the above formula, the bond represented by the dotted line in the five-membered ring means that the 5-membered ring is a cyclopentene ring having a double bond, or it may be any of saturated cyclopentane rings, and in the case of the cyclopentene ring X is OH, Y is = O, Z is H, and in the case of the cyclopentane ring, X is = O, Y is OH, Z is OH, and R is a residue from which a SH group is removed from a compound containing an SH group.
公开号:KR20000075824A
申请号:KR1019997007898
申请日:1998-02-26
公开日:2000-12-26
发明作者:고야마노부토；에노키다츠지；이카이가츠시게；우하강；오노기히로무；도미나가다카나리；니시야마에이지；하기야미치오；사가와히로아키；죠노히데토；가토이쿠노신
申请人:이치로 스즈키；타카라 스조 캄파니., 리미티드.；
IPC主号:

专利说明:

Compound {COMPOUNDS}
[2] Conventionally, drugs used in clinical therapies include alkylating agents, metabolic deactivators, anticancer agents such as plant alkaloids, antibiotics, immune promoters, immunomodulators, and the like, but these drug therapies are not yet completed.
[3] Of these, among prostaglandins derived from natural products, since prostaglandins A and J having α, β-unsaturated carbonyl in the 5-membered ring inhibit DNA synthesis, it has been reported that this may be a highly stable anticancer agent. Various derivatives of have been synthesized (see Japanese Patent Laid-Open No. 62-96438).
[1] The present invention relates to a compound having a physiological activity such as anticancer action useful in the pharmaceutical field and a method for producing the compound.
[20] 1 is a diagram showing a mass spectrum of CM1.
[21] 2 shows a ¹ H-NMR spectrum of CM1.
[22] 3 shows the UV absorption spectrum of CM1.
[23] 4 shows a ¹ H-NMR spectrum of CM2.
[24] 5 shows ¹³ C-NMR spectra of CM1.
[25] 6 shows the IR absorption spectrum of a CM.
[26] 7 is a diagram illustrating a relationship between a holding time and an absorbance.
[27] 8 is a diagram illustrating a ¹ H-NMR spectrum of GM.
[28] 9 is a diagram illustrating ¹³ C-NMR spectra of GM.
[29] 10 is a diagram showing a mass spectrum of GM.
[30] 11 shows the UV absorption spectrum of GM.
[31] 12 shows the IR absorption spectrum of GM.
[32] 13 is a diagram showing an elution pattern of one example of a cyclopentanone thio derivative.
[33] Fig. 14 shows the relationship between the reaction time and the absorbance at 215 nm when L-cysteine is used.
[34] It is a figure which shows the relationship between reaction time and the absorbance at 215 nm in the case of using glutathione.
[35] 16 is a diagram illustrating a chromatogram of an example of a reactant of the present invention.
[36] FIG. 17 is a diagram showing the mass spectrum of the peak at 2.99 minutes in FIG. 4.
[37] 18 is a diagram showing a UV absorption spectrum of a reaction solution immediately after dissolution.
[38] Fig. 19 shows the UV absorption spectrum of the reaction solution after 50 minutes of reaction.
[39] It is a figure which shows the UV absorption spectrum of the reaction liquid immediately after melt | dissolution in the case of using glutathione.
[40] It is a figure which shows the UV absorption spectrum of the reaction liquid after 50 minutes of reactions when glutathione is used.
[41] FIG. 22 is a ¹³ C-NMR spectrum of a reactant. FIG.
[42] Fig. 23 is a diagram showing the relationship between the holding time and the absorbance.
[43] FIG. 24 is a diagram showing a ¹ H-NMR spectrum of GD. FIG.
[44] Fig. 25 shows the ¹³ C-NMR spectra of GD.
[45] Fig. 26 shows the mass spectrum of GD.
[46] Fig. 27 shows the IR absorption spectrum of GD.
[47] Fig. 28 shows cancer cell proliferation inhibitory activity of CM.
[48] 29 shows cancer cell proliferation inhibitory activity of CM.
[49] It is a figure which shows the relationship between GM amount and tumor necrosis factor production amount.
[50] Fig. 31 is a graph showing the relationship between GM amount and foot edema growth rate.
[51] Fig. 32 shows the relationship between the GM concentration and the NO ₂ ⁻ concentration in the culture solution.
[52] Fig. 33 shows the relationship between the incubation time and the number of living cells.
[53] 34 shows the effect of GM on proliferation of Jurkat cells.
[54] 35 shows the effect of GM on the proliferation of Molt-3 cells.
[55] Fig. 36 shows the expression of Fas antigen in malt-3 cells.
[56] Fig. 37 shows the expression of Pas antigen in Jurkat cells.
[57] Fig. 38 shows changes in the proportion of Pas antigen expressing cells.
[58] Fig. 39 shows the relationship between the GM dose and the blood glucose level.
[59] Fig. 40 shows the relationship between the GM dose and the serum insulin level.
[60] Fig. 41 shows the relationship between the GM dose and the serum total cholesterol level.
[61] Fig. 42 shows the relationship between the GM dose and the serum triglyceride level.
[62] Fig. 43 shows the relationship between the GM dose and the serum free fatty acid value.
[63] Fig. 44 shows the relationship between GM concentration and cell viability.
[64] Fig. 45 shows the relationship between the GM concentration and the p24 production amount.
[65] 46 is a diagram showing the action of inhibiting delayed-type hypersensitivity reaction of GM.
[66] Fig. 47 shows the three-dimensional structure of CD and (-)-cyclopentenone of p-dimethylaminobenzoyl derivative of (-)-cyclopentenone.
[67] Fig. 48 shows the three-dimensional structure of CD and (+)-cyclopentenone of p-dimethylaminobenzoyl derivative of (+)-cyclopentenone.
[68] Hereinafter, the present invention will be described in detail.
[69] The cyclopentenone represented by the general formula (4) used in the present invention includes both isomers and trans isomers in which the three-dimensional arrangement of the hydroxyl groups at the 4 and 5 positions is cis. In the present invention, cis-cyclopentenone may be used, trans-cyclopentenone may be used, or a mixture of cis-cyclopentenone and trans-cyclopentenone may be used. Moreover, you may use these optically active bodies.
[70] Cis-cyclopentenone is prepared by chemical synthesis (Helvetica Chimica Acta, Vol. 55, pages 2838-2844 (1972)). Trans-cyclopentenone is also obtained by chemical synthesis [Carbo hydrate Res., Vol. 247, pp. 217-222 (1993)], uronic acid such as glucuronic acid, glucuronolactone, and the like. Uronic acid derivatives or these contents are produced by heat treatment (see PCT / JP97 / 03052). In the present invention, these heat treated products containing cyclopentenone, partial purified products thereof and purified products can also be used.
[71] For example, by using D-glucuronic acid as uronic acid, a 1% solution thereof is heat treated at 121 ° C. for 4 hours, whereby cyclopentenone is produced in the heat treated product. The cyclopentenone in this heat treated product is extracted with a solvent, and the extract is concentrated. The concentrate was then separated by silica gel column chromatography, the eluted cyclopentenone fraction was concentrated, the cyclopentenone was extracted from the concentrate with chloroform, and the extract concentrate was subjected to normal phase column chromatography. The cyclopentenone in the heat treated product is isolated.
[72] The physical properties of cyclopentenone are shown below. In addition, the mass spectrometry of cyclopentenone was performed using the DX302 mass spectrometer (made by Nippon Denshi Co., Ltd.). In addition, the measurement of the NMR spectrum using the heavy chloroform solvent used JNM-A500 (made by Nippon Denshi Corporation). Specific light intensity is DIP-370 type photometer (manufactured by Nippon Bunko Co., Ltd.), UV absorption spectrum is UV-2500 spectrophotometer (manufactured by Shimatsu Corporation), and infrared absorption spectrum (IR) is FTIR-8000 infrared. It measured using the spectrophotometer (made by Shimadzu Corporation), respectively.
[73] MS m / z 115 [M + H] ⁺
[74] ¹ H-NMR (CDCl ₃ )
[75] δ 4.20 (1H, d, J = 2.4 Hz, 5-H), 4.83 (1H, m, 4-H), 6.30 (1H, dd, J = 1.2, 6.1 Hz, 2-H), 7.48 (1H , dd, J = 2.1, 6.1 Hz, 3-H)
[76] However, the chemical displacement value of ¹ H-NMR was expressed on the basis of the chemical displacement value of CHCl ₃ as 7.26 ppm.
[77] Radiance: [α] _D ²⁰ 0 ° (c 1.3, water)
[78] UV: lambda max 215 nm (water)
[79] IR (KBr method): It has absorption at 3400, 1715, 1630, 1115, 1060, 1025 cm <^-1> .
[80] By optically separating the isolated cyclopentenone, (-)-4,5-dihydroxy-2-cyclopenten-l-one and (+)-4,5-dihydroxy-2-cyclopenten-l- You can get on. Naturally, the cyclopentenone obtained by the synthesis method can also be optically divided.
[81] For example, cyclopentenone is dissolved in ethanol. Hexane / ethanol (94/6) is further added to this ethanol solution to form a cyclopentenone solution. The sample solution was subjected to HPLC, for example, using a Chiral Pack AS (manufactured by Daicel Chemical Industries, Ltd.) column with a column temperature of 40 ° C. and a mobile phase of hexane / ethanol (94/6). Can split optically.
[82] The luminous intensity of the divided (-)-trans-4,5-dihydroxy-2-cyclopenten-l-one [hereinafter referred to as (-)-cyclopentenone] is [α] _D ²⁰ -105 ° ( c 0.30, ethanol), and the photoluminescence of (+)-trans-4,5-dihydroxy-2-cyclopenten-l-one [hereinafter referred to as (+)-cyclopentenone] is [α] _D ²⁰ + 104 ° (c 0.53, ethanol). In addition, the optical density was measured using the above-mentioned DIP-370 type photometer (made by Nippon Bunco Co., Ltd.).
[83] Next, (-)-cyclopentenone and (+)-cyclopentenone were respectively described by mass spectrometry, structural analysis by nuclear magnetic resonance (NMR), measurement of UV absorption spectrum, and measurement of infrared absorption spectrum. Perform in accordance with As a result, both optically active agents show the same results as cyclopentenone before optical splitting.
[84] The optically divided (-)-cyclopentenone and (+)-cyclopentenone were each used as p-dimethylaminobenzoyl derivatives and cyclic dichroism using a J-720 type cyclic dichroic dispersion system (manufactured by Nippon Bunco Co., Ltd.). Spectra (CD) were measured and the results applied to the dibenzoate chiral rule [J. Am. Chem. Soc., Vol. 91, pp. 3989-3991 (1969)].
[85] The stereostructure of CD and (-)-cyclopentenone of the p-dimethylaminobenzoyl derivative of (-)-cyclopentenone is shown in FIG. In the figure, the vertical axis represents molar cyclic dichroism, and the horizontal axis represents wavelength (nm). In addition, the three-dimensional structure is represented by the following formula (5).
[86]
[87] The stereostructure of CD and (+)-cyclopentenone of the p-dimethylaminobenzoyl derivative of (+)-cyclopentenone is shown in FIG. In the figure, the vertical axis represents molar dichroism and the horizontal axis represents wavelength (nm). In addition, the three-dimensional structure is represented by the following formula (6).
[88]
[89] As shown in FIG. 47, FIG. 48 and Formula 5 and Formula 6, (-)-cyclopentenone is (-)-(4R, 5S) -trans-4,5-dihydroxy-2-cyclopentene-1- While on, (+)-cyclopentenone is (+)-(4S, 5R) -trans-4,5-dihydroxy-2-cyclopenten-l-one.
[90] As described above, the cyclopentenone or the optically active substance thereof used in the present invention may be produced by any method. That is, it may be manufactured by the method disclosed in this specification, may be synthesize | combined by the chemical synthesis method, and the trans | transformer, the cis body, these mixtures, and these optically active agents of cyclopentenone are also used for this invention.
[91] Salts of cyclopentenone or its optically active agent include pharmaceutically acceptable salts, which can be converted by known methods.
[92] By reacting cyclopentenone, its optically active substance and / or these salts with the SH group-containing compound, a compound represented by the formula (1) of the present invention is produced in the reaction solution.
[93] In addition, by reacting cyclopentenone, its optically active substance and / or these salts with an SH group-containing compound such as an SH group-containing amino acid or a derivative thereof under acidic conditions, the compound represented by the formula (2) in the reaction solution (hereinafter, cyclo Called pentenone thio derivatives).
[94] There is no restriction | limiting in any SH group containing compound, For example, methane thiol, butane thiol, mercaptoethanol, SH group containing amino acid, SH group containing amino acid derivative etc. are mentioned. Cysteine, homocysteine, etc. are mentioned as an example of SH group containing amino acid.
[95] Examples of SH group-containing amino acid derivatives include derivatives of such amino acids, such as cysteine derivatives, cysteine-containing peptides and cysteine derivative-containing peptides. The cysteine-containing peptide may be one in which the cysteine is a constituent in the peptide, and there is no particular limitation. Cysteine-containing peptides of the present invention include low molecules such as oligopeptides (e.g. glutathione) to polymers such as proteins. In addition, a cysteine or homocysteine-containing peptide can also be used as the cysteine or homocysteine-containing peptide of the present invention under the conditions of becoming a cysteine or homocysteine-containing peptide during the reaction, for example by combining a reduction treatment. Cysteine-containing peptides also include cysteine-containing peptides containing sugars, lipids and the like. Moreover, salts, acid anhydrides, esters, etc. of the above-mentioned various substances may be sufficient. As described above, the cyclopentenone reacts with the SH group-containing compound under acidic acid to form a cyclopentenone thio derivative.
[96] As a means for purifying and isolating the cyclopentenone thio derivative or the optically active substance produced by reacting a cyclopentenone, its optically active substance and / or these salts with an SH group-containing compound such as an SH group-containing amino acid or a derivative thereof, Well-known purification means, such as a chemical method and a physical method, may be used, and conventionally well-known purification methods, such as a gel filtration method, the fractionation method by a molecular weight fractionation membrane, the solvent extraction method, the classification method, the various chromatography methods using ion exchange resin, etc. In combination, the cyclopentenone thio derivative or its optically active substance and salts thereof in the reaction product can be purified and isolated. For example, by reacting cyclopentenone and cysteine at pH 4, 60 ° C. for 16 hours, a cyclopentenone thio derivative represented by the following formula (7) is produced in the reaction solution, and by performing normal phase column chromatography of the reaction product containing this derivative. The cyclopentenone thio derivative can be purified and isolated.
[97]
[98] In addition, for example, by reacting cyclopentenone and glutathione under acidic conditions, a cyclopentenone thio derivative represented by the following formula (8) is produced in the reaction solution, and the reaction product containing the derivative is treated by reverse phase column chromatography or the like. Cyclopentenone thio derivatives can be purified and isolated.
[99]
[100] By reacting cyclopentenone, its optically active substance and / or these salts with an SH group-containing compound such as an SH group-containing amino acid or a derivative thereof in neutral conditions, the compound represented by the formula (3) in the reaction solution (hereinafter cyclopentanone) Termed thio derivatives).
[101] There is no restriction | limiting in particular in the said SH group containing compound, The SH group containing compound mentioned above is mentioned as an example. It is preferable to perform the said reaction of cyclopentenone, its optically active substance, and / or these salts, and SH group containing compound at neutral pH.
[102] As a cyclopentenone, its optically active substance and / or these salts, and the cyclopentanone thio derivative produced | generated by making SH group containing compound react, the optically active substance, these salts, and the purification and isolation means of these, well-known chemical methods, physical methods, etc. Cyclopenta in the reaction product may be used by combining conventionally known purification methods such as gel filtration, fractionation by molecular weight fractionation membranes, solvent extraction, fractionation, and chromatographic methods using ion exchange resins. The non-thio derivative, its optically active substance, and these salts can be refine | purified and isolated.
[103] For example, by reacting cyclopentenone and cysteine at pH 7, 37 ° C. for 30 minutes, a cyclopentanone thio derivative represented by the following formula (9) is produced in the reaction solution, and reverse phase column chromatography of the reaction product containing the derivative is performed. Thereby, the cyclopentanone thio derivative can be purified and isolated.
[104]
[105] Further, for example, by reacting cyclopentenone and glutathione under neutral conditions, a cyclopentanone thio derivative represented by the following formula (10) is produced in the reaction solution, and reverse phase column chromatography or the like of the reaction product containing the derivative is performed. Cyclopentanone thio derivatives can be purified and isolated.
[106]
[107] Separation of optically active compounds of the compounds of the present invention can be accomplished by mechanical cleavage of the racemic mixture, prior crystallization, crystallization as diastereomer salts or clathrate compounds, dynamic cleavage using enzymes or microorganisms, chromatography By division by
[108] Separation by chromatography includes gas chromatography, liquid chromatography, thin layer chromatography, and the like, and a chiral stationary phase suitable for each may be used.
[109] As optical separation by liquid chromatography, a method using a chiral stationary phase, a method using a chiral eluent, separation as a diastereomeric salt, and the like can be used.
[110] As the chiral stationary phase, an amide stationary phase, a urea stationary stationary phase, a ligand exchange type stationary phase, a polysaccharide stationary phase and a polysaccharide derivative stationary phase, a protein stationary phase, a polymethacrylic acid ester stationary phase, a polymethacrylamide stationary phase, or the like can be used.
[111] Hexane, alcohol, water (buffer), etc. can be used as an eluent, and it can use suitably in combination with the said stationary phase.
[112] As salts of the compounds of the present invention or optically active agents thereof, there are pharmaceutically acceptable salts, which can be converted by known methods.
[113] Compounds of the present invention or their optically active agents or salts thereof have anticancer activity, cancer cell proliferation inhibitory activity, apoptosis-inducing activity, topoisomerase II inhibitory activity, cancer cell differentiation inducing activity, antirheumatic activity, chronic arthritis rheumatoid inhibition Action, fas antigen generation induction activity, antibacterial activity, antiviral activity, liver function improvement activity, heat shock protein induction activity, normalizing blood components, cancer immune enhancing activity, anti-inflammatory activity, tumor necrosis factor generation inhibitory activity, nitric oxide Physiological activities such as production inhibitory activity, immunomodulatory activity such as delayed type hypersensitivity inhibitory activity, lymphocyte weakening reaction inhibitory activity, mixed lymphocyte reaction inhibitory activity, IgE production inhibitory activity, carrageenan edema inhibitory activity, At least one or more compounds selected from the compounds of the present invention or their optically active agents or salts thereof by activity Drugs containing the compound as an active ingredient include, for example, drugs that act on the biological defense mechanisms, such as agents that act on the antibody-producing mechanism, drugs that act on carbohydrate metabolism, such as anti-inflammatory agents, anti-allergic agents, antirheumatic agents, interferon inducing agents, and the like. It is useful as an antidiabetic agent, a medicine acting on a hospital organism, such as an antibacterial agent, an antiviral agent, and the like. Therefore, the medicament obtained in the present invention is a medicament for diseases exhibiting susceptibility to the compound of the present invention or the optically active substance or salts thereof, such as cancer, viral diseases, rheumatism, diabetes, allergies, autoimmune diseases, inflammation and the like. It is very useful as a medicament or a preventive medicament.
[114] Compounds of the present invention or optically active agents or salts thereof include, for example, human promyelocytic leukemia cells HL-60, human acute lymphoid leukemia blasts MOLT-3, lung cancer cells A-549, SV40 transgenic lung cells WI-38VA13, liver cancer It has a cell proliferation inhibitory effect and anticancer activity on cancer cells such as cell Hep G2, colon cancer cell HCT 116, human colon cancer cell SW480, human colon cancer cell WiDr, gastric cancer cell AGS, myeloma cells, etc. These salts can be used as an active ingredient of an anticancer agent. In addition, these compounds have apoptosis-inducing action on cancer cells. The mechanism of inhibiting cancer cell proliferation of the compound of the present invention or its optically active agent or salts thereof is not limited to the present invention, and for example, apoptosis-inducing action, and phase isomerase II inhibitory action on cancer cells are also included in the present invention. It is involved in anticancer action.
[115] An anticancer agent can be prepared by using a compound of the present invention having an anticancer action, or an optically active substance thereof or at least one or more compounds selected from these salts as an active ingredient, which is combined with a known pharmaceutical carrier. The preparation of an anticancer agent generally comprises combining a compound of the present invention or at least one compound thereof selected from an optically active agent or salts thereof with a pharmaceutically acceptable liquid or solid carrier, and furthermore, a solvent, a dispersant, Emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants and the like may be added to form solids such as tablets, granules, powders, powders, capsules, and the like, and usually liquids such as liquids, suspensions, and emulsions. It may also be added to a suitable carrier to form a dry product that can be liquidized before use.
[116] The pharmaceutical carrier can be selected according to the above dosage forms and formulations, and in the case of oral preparations, for example, starch, lactose, sugar, mannitol, carboxymethyl cellulose, corn starch, inorganic salts and the like are used. Moreover, in preparation of an oral preparation, you may mix | blend a binder, a disintegrating agent, surfactant, a lubricating agent, a fluidity promoter, a mating agent, a coloring agent, a fragrance | flavor, etc. further.
[117] On the other hand, in the case of parenteral agents, a compound of the present invention, an optically active substance thereof, or at least one compound selected from these salts, which are the active ingredient of the present invention, according to a conventional method, may be used for injection distilled water, physiological saline, aqueous glucose solution, It is prepared by dissolving or suspending in diluents such as vegetable oil, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, and the like, if necessary, by adding a fungicide, a stabilizer, an isotonicity agent, a painless agent and the like.
[118] The anticancer agent of the present invention is administered by a suitable route of administration depending on the form of the preparation. There is no restriction | limiting in particular in the administration method, It can administer by internal use, external use, and injection. Injectables can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, and the like and suppositories include suppositories.
[119] The dosage as an anticancer agent is appropriately set according to the form of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient, and although not constant, the amount of cyclopentenone and / or the optically active substance contained in the preparation is generally From 0.1 μg to 200 mg / kg per adult. Of course, since the dosage varies depending on various conditions, an amount smaller than the dosage may be sufficient, or it may be necessary to exceed the above range. In addition to oral administration as it is, the medicament of the present invention may be added to any food or drink and consumed daily.
[120] The compound of the present invention or its optically active agent or salt thereof has anticancer activity, but at low concentrations, the compound of the present invention or its optically active agent or salt thereof is an agent for inducing differentiation of cancer cells (decancer). It is also useful as. A cancer cell differentiation inducing agent comprising the compound of the present invention or at least one compound selected from the optically active agent or these salts as an active ingredient can be formulated in accordance with the anticancer agent, and can be administered by a method according to the anticancer agent.
[121] The dosage of the cancer cell differentiation inducing agent is appropriately set according to the type of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient. The amount of at least one compound selected from is from 0.1 μg to 100 mg / kg per adult per day. Of course, since the dosage varies according to various conditions, an amount smaller than the dosage may be sufficient, or it may be necessary beyond the above range. In addition to oral administration as it is, the medicament of the present invention may be added to any food or drink and consumed daily.
[122] The cancer cell differentiation inducing agent can be used in a method for inducing cancer cell differentiation. In other words, cancer cells can be differentiated by using the compound of the present invention, or an optically active substance thereof or at least one compound selected from these salts as an active ingredient, and the method can be used for screening of differentiation inducing mechanisms and screening agents for differentiation of cancer cells. Useful)
[123] The compound of the present invention or the optically active substance thereof and salts thereof have an antimicrobial effect, and an antimicrobial agent can be prepared by formulating at least one or more compounds selected from these compounds as an active ingredient and in combination with a known pharmaceutical carrier. The said agent can be formulated according to the said anticancer agent, and can be administered by the method according to an anticancer agent.
[124] The dosage as an antimicrobial agent is appropriately set according to the form of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient, and the amount thereof is not constant, but generally the compound of the present invention or the optically active substance or salt thereof contained in the preparation. The amount of at least one compound selected from is from 10 μg to 20 mg / kg per adult per day. Of course, since the dosage varies according to various conditions, an amount smaller than the dosage may be sufficient, or it may be necessary beyond the above range.
[125] In addition to oral administration as it is, the medicament of the present invention may be added to any food or drink and consumed daily. Moreover, you may use the compound of this invention, its optically active substance, or these salts as a raw material of antimicrobial food-drinks. Moreover, you may use in combination with other antimicrobial substances, such as ethanol, glycine, sodium acetate, ascorbic acid, glycerol fatty acid ester, salt, and EDTA.
[126] The antimicrobial agent of this invention can be used as an antiseptic agent which improves the shelf life of a food or a drink by using as an active ingredient the compound of this invention, its optically active substance, or at least 1 sort (s) selected from these salts. In addition, a compound selected from these compounds can be added to the food or beverage to be used in a method for preventing the corruption of the food or beverage. The amount of the compound of the present invention or the optically active substance or salt thereof added to the food or beverage depends on the type of food or beverage, and may be added in an appropriate amount depending on the purpose.
[127] As a use method of the antimicrobial agent of this invention, the method of adding to food or a drink by a suitable method is performed. Although a conventional method is added during the food or beverage manufacturing process, a method of immersing the food in a solution containing the compound of the present invention or an optically active substance thereof and their salts for a certain time can also be used. In addition, the addition method and the immersion method can also be used together.
[128] By using the antimicrobial agent of this invention as an antiseptic agent, the shelf life of a food or a drink can be improved further. Moreover, in frozen foods, frozen desserts, etc., the proliferation of the contaminated microorganisms can be suppressed in the processing process before freezing, and the hygienic very preferable result can be obtained. The antimicrobial agent of the present invention has an effect on both gram positive bacteria and gram negative bacteria, such as drug resistant bacteria such as methicillin resistant Staphylococcus aureus, Salmonella, Enterotoxin producing Staphylococcus aureus, vomiting Bacillus cereus, It is also very effective against food poisoning bacteria such as diarrhea Bacillus cereus and intestinal hemorrhagic Escherichia coli O-157. Moreover, it is also effective for fire hydrants. Also, bacteria caused by bacterial diseases, such as Legionella pneumophila, which is caused by veterans disease, Vibrio parahaemolyticus, which is caused by food poisoning, and Helicobacter, which is caused by ulcers Helicobacter pylori (Helicobacter pylori), Campylobacter jejuni, a gastroenteritis bacterium, such as Legionella pneumophila (ATCC 33153), Vibrio parahaemolyticus (ATCC 17802), Helicobacter pylori (NCTC 1l637), Campylobacter jejuni (ATCC 29428) It has an effect and is also effective for microorganisms such as yeast and mold. Further, the antibacterial agent of the present invention can be used to sterilize clothes, needle sheets, etc., and the target bacteria can be removed and sterilized by spraying the antimicrobial agent of the present invention or wiping with the antimicrobial agent of the present invention. For example, by adding to the cooling water of a building, a veteran disease can be prevented.
[129] The antimicrobial agent of the present invention shows antibacterial activity against caries or periodontal bacteria, and can provide an oral agent containing the antimicrobial agent of the present invention. The shape of the mouthwash may be a known shape such as liquid or paste. As a mouthwash, a toothpaste is illustrated. The toothpaste may be a liquid, or may be a paste or a powder, and may be in the form of a known toothpaste. The content of the compound of the present invention in the toothpaste, the optically active substance thereof, or the salt of the toothpaste in the toothpaste is not particularly limited and may be contained in an effective concentration against caries or periodontal disease. In the toothpaste, known additives such as wetting agents, surfactants, binders, flavorings, sweeteners and the like can be added.
[130] The antimicrobial cosmetics can be provided by using the antimicrobial agent of this invention. Examples of the antimicrobial cosmetics include makeup cosmetics such as creams, emulsions, lotions, face washes, packs, basic cosmetics, lipsticks, foundations, etc. containing an effective amount of a compound of the present invention or at least one compound selected from the optically active substance or salts thereof, It can be prepared in the form of bath soap, soap, and the like. It is also effective for hair, and can be used in the form of hair products such as hair tonics, hair liquids, hair setting lotions, hair blowers, hair creams, hair coatings, and hair care products such as shampoos, rinses, and hair treatments. Can be. What is necessary is just to determine the compounding quantity to cosmetics suitably according to the antimicrobial activity. As another component of the cosmetic, a compound usually formulated into a cosmetic can be used. The antimicrobial cosmetics act effectively on the causative agent of atopic dermatitis, and have a remarkable effect on the improvement and prevention of atopic dermatitis.
[131] A bath agent can be provided by using the antimicrobial agent of this invention. The bath agent of the present invention can be prepared in the form of an effective amount of a compound of the present invention, an optically active agent or at least one compound selected from these salts, powder baths, granule baths, solid baths, liquid baths and the like. The compounding quantity to bath agent can be suitably determined according to the desired antimicrobial activity. The other components of the bath may be used in combination with the bath. The bath agent of the present invention acts effectively on the causative agent of atopic dermatitis, and has a remarkable effect on the improvement and prevention of atopic dermatitis. It is also effective for the removal of pathogens from baths.
[132] In addition, a food or beverage containing the compound of the present invention or at least one compound selected from the optically active agent or salts thereof is very useful for improving and / or preventing food poisoning, gastroenteritis, and the like.
[133] The apoptosis inducing agent of the present invention may be formulated in accordance with the anticancer agent by using the compound of the present invention having an apoptosis-inducing compound or an optically active agent thereof or at least one or more compounds selected from these salts as active ingredients. It may be administered by a standard method.
[134] Dosages as apoptosis inducing agents are appropriately set according to the type of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient to which it is applied, but is not constant, but the compound of the present invention or the optical thereof generally contained in the preparation The amount of the active agent or at least one compound selected from these salts is from 0.1 μg to 100 mg / kg per adult per day. Of course, since the dose varies depending on various conditions, an amount smaller than the dose may be sufficient, or it may be necessary beyond the range. In addition to oral administration as it is, the medicament of the present invention may be added to any food or drink and consumed daily.
[135] In addition, apoptosis is thought to be a deadly planned death from the gene of the cell itself, unlike necrosis, which is a pathological cell death. That is, genes that program apoptosis are activated due to some external or internal factors, and program gene gene proteins are biosynthesized based on these genes, and the cells themselves are decomposed by the generated program yarn proteins. It is thought to lead to death.
[136] The apoptosis inducing agent of the present invention can express such apoptosis into desired tissues and cells, and it is very useful to be able to exclude unnecessary path cells from the living body in its natural form.
[137] Apoptosis inducing agent of the present invention can be used as a method of inducing apoptosis. That is, by using the compound of the present invention or an optically active substance or at least one compound selected from these salts as an active ingredient, apoptosis can be induced, and the method can be used for elucidation of apoptosis inducing mechanisms, apoptosis inducing agents and cells. It is useful for selection of extinction inhibitors.
[138] The compound of the present invention or its optically active agent or salt thereof has an anti-rheumatic effect, and an anti-rheumatic agent can be prepared by formulating at least one compound selected from these compounds as an active ingredient and combining it with a known pharmaceutical carrier. Can be. The medicine can be formulated in accordance with the anticancer agent and can be administered by a method in accordance with the anticancer agent.
[139] The dosage of the antirheumatic agent of the present invention is appropriately set according to the indication, the form of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient, but is not constant, but the compound of the present invention generally contained in the preparation or The amount of the optically active agent or one or more compounds selected from these salts is from 0.1 μg to 200 mg / kg per adult. Of course, since the dose varies depending on various conditions, an amount smaller than the dose may be sufficient, or it may be necessary beyond the range. In addition to oral administration as it is, the medicament of the present invention may be added to any food or drink and consumed daily. Moreover, you may use the compound of this invention, its optically active substance, and these salts as a raw material of the pharmaceutical of this invention.
[140] Compounds of the present invention or optically active agents or salts thereof have anti-inflammatory action against arthritis, inhibit carrageenan edema, inhibit tumor necrosis factor production, enhance interleukin-10 production, inhibit nitric oxide production, induce pas antigen production And various physiological activities such as immunomodulatory action such as delayed type hypersensitivity inhibitory action, lymphocyte weakening reaction inhibitory action, mixed lymphocyte reaction inhibitory action, and IgE production inhibitory action. Anti-inflammatory or anti-inflammatory agents, tumor necrosis factor production inhibitors or tumor necrosis factor production inhibitors, interleukin-10 production enhancers, immunomodulators, nitric oxide production inhibitors, PAS antigen generation inducers, immunomodulators, comprising at least one compound selected as an active ingredient, IgE production inhibitors, delayed-type hypersensitivity inhibitors, anti-allergic agents Such medicines can be formulated in accordance with the anti-rheumatic agent, and can be administered by the method according to the medicine.
[141] The dosage of these preparations is appropriately set according to the indication, the preparation form of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient, and is not constant, but generally the compound of the present invention or the optically active agent contained in the preparation or The amount of at least one compound selected from these salts is between 0.1 μg and 200 mg / kg per adult. Of course, since the dosage varies depending on various conditions, an amount smaller than the dosage may be sufficient, or it may be necessary beyond the range. For example, as an anti-inflammatory agent or tumor necrosis factor production inhibitor, the amount of the compound of the present invention or an optically active agent thereof or one or more compounds selected from these salts contained in the preparation is suitably 10 pg to 50 mg / kg per adult, and nitrogen monoxide. As the production inhibitor, the amount of the compound of the present invention or the optically active agent or one or more compounds selected from these salts contained in the formulation is suitably 0.1 µg to 20 mg / kg per adult, and the amount of the active ingredient in the formulation, depending on the purpose of use. Can be adjusted.
[142] In addition to oral administration as it is, the medicament of the present invention may be added to any food or drink and consumed daily.
[143] Rheumatoid is an autoimmune disease in which damage occurs to periosteal cells and chondrocytes. The anti-allergic agent of the present invention is also useful as an agent for treating autoimmune diseases.
[144] It is considered that the compound of the present invention or the optically active substance thereof and salts thereof directly cause inflammation in organ-specific autoimmune diseases and inflammatory diseases such as chronic rheumatoid arthritis. Inhibition of tumor necrosis factor production enhances the production of interleukin-10, a Th1 inhibitory cytokine. Thus, symptoms of inflammation, such as long-term autoimmune rheumatoid, especially chronic articular rheumatoid symptoms, are improved, such that the inflammatory markers of C-reactive protein (CRP), rheumatoid factor (RF), and erythrocyte sedimentation rate (bleeding) In addition, the symptom of merger such as difficulty walking is remarkably improved.
[145] Tumor necrosis factor has been found as a factor inducing hemorrhagic necrosis at the tumor site, but is now recognized as a cytokine widely associated with inflammation-based biological defense and immune mechanisms. The destruction of the mechanism for regulating the production of tumor necrosis factor causes various problems in the host, and the production of excessive or unregulated tumor necrosis factor is caused by chronic arthritis rheumatoid, rheumatoid myelitis, deformable arthrosis, gouty arthritis, sepsis, plaque Secondary to fever and myalgia, infection or malignancy due to hematological shock, endotoxin shock, Gram-negative sepsis, toxic shock syndrome, cerebral malaria, chronic pneumonia, graft-to-host response, allograft rejection, infections such as influenza Cachexia, secondary cachexia to human AIDS, AIDS, AIDS-related syndrome, keloid formation, ulcerative colitis, multiple sclerosis, autoimmune diabetes and systemic lupus erythematosus Is involved in most of these diseases, including autoimmune diseases [Molecular Medicine, Vol. 33, pp. 1010-1020, 1182-118]. P. 9 (1996). Tumor necrosis factor production inhibitors of the present invention are usefully used in the treatment of a condition mediated or worsened by tumor necrosis factor. In addition, the present invention provides a method for regulating tumor necrosis factor production using at least one or more compounds selected from the compounds of the present invention, the optically active agents thereof, and these salts as active ingredients. In addition, according to the present invention, a food or drink for improving the condition of a disease containing a compound of the present invention or at least one or more compounds selected from the optically active substance or salts thereof and mediated or worsened by tumor necrosis factor, or disease Preventive foods or drinks are provided.
[146] Nitric oxide (hereinafter abbreviated as NO) is the body of endothelial cell-derived vascular smooth muscle relaxation factor (EDRF) [Nature, Vol. 327, pp. 524-526 (1987)]. The present invention provides a medicament for the treatment of a disease or a medicament for the prevention of a disease that requires suppression of NO production, which contains the compound of the present invention or an optically active substance thereof or at least one or more compounds selected from these salts as an active ingredient. In the present invention, a disease requiring NO inhibition is not particularly limited, and for example, systemic blood pressure lowering, blood pressure response lowering, autoimmune disease, inflammation, arthritis, for example, by toxic shock or treatment with some kind of cytokines, Rheumatoid arthritis, diabetes mellitus, inflammatory bowel disease, vascular insufficiency, pathogenic vasodilation, tissue damage, cardiovascular ischemia, hypersensitivity, cerebral ischemia, diseases involving angiogenesis, cancer, and the like. The diseases described in 504524, Unexamined-Japanese-Patent No. 9-505288, Unexamined-Japanese-Patent No. 8-501069, Unexamined-Japanese-Patent No. 8-512318, and Unexamined-Japanese-Patent No. 6-508849 are mentioned.
[147] The NO production inhibitor containing the compound of the present invention or an optically active substance thereof or at least one or more compounds selected from these salts as an active ingredient is useful for the study of NO production mechanism, the study of NO mechanism of action, and also involved in the NO production mechanism. It can also be used for screening.
[148] The compound of the present invention, or an optically active substance thereof or salts thereof exhibits NO production inhibitory action on NO producing cells. For example, when endotoxin (liposaccharide: LPS) is added to a macrophage macrophage cell line, inducible NO synthase (NOS) is expressed and NO is secreted in the medium, but the compound of the present invention or an optically active substance thereof The action of LPS in the presence of salts inhibits NO production. In the case of inducing NO production by LPS treatment, cell viability is lowered by NO cell disruption activity. However, the addition of the compound of the present invention or its optically active agent or these salts during LPS treatment results in a decrease in NO production. The hindrance for the decrease is reduced.
[149] Angiogenesis is essential for the augmentation of solid cancers, but defective endothelial growth factor / vascular permeability hyperactivity factor (VEGF) plays an important role in this process. VECF is induced by NO in various cancer cells. The compound of the present invention, the optically active substance thereof, and the salt thereof inhibit NO production of cancer cells, thereby inhibiting NO production. As a result, angiogenesis around cancer tissues is inhibited. Administration of a compound of the present invention, an optically active substance thereof, or salts thereof to a mouse in which a cancer cell is implanted subcutaneously to form a solid tumor results in insufficient formation of peripheral blood vessels of cancer tissues, thereby eliminating cancer.
[150] Nitrosoamine is a group of compounds in which a nitroso group is added to secondary amines, and hundreds of them are known, and most of them show carcinogenicity to animals by damaging DNA. Nitrosoamines are also deeply involved in human carcinogenesis and are usually produced by the reaction of nitrite and amines in the stomach. NO reacts with amines to produce nitrosoamines even under physiological conditions with a neutral pH. In addition, NO production is increasing in patients with hepatic insect repellent infection and liver cirrhosis who have a strong relationship with cancer. Therefore, the administration of the compound of the present invention or its optically active agent or these salts prevents the increase of NO production, thereby preventing carcinogenesis of the high-risk group in particular. As described above, the compound of the present invention, or an optically active substance thereof, or a salt thereof exhibits anticancer action at two stages: inhibition of carcinogenesis and inhibition of angiogenesis in cancer tissues.
[151] NO also causes edema characteristic of inflammatory lesions, namely vascular permeable hyperactivity (Maeda et al., Japanese Journal of Cancer Research, Vol. 85, pp. 331-334 (l994)). Promotes the biosynthesis of prostaglandins, an inflammatory mediator (Salvemini et al., Proceedings of National Academy of Sciences. USA, Vol. 90, pp. 7240-7244 (1993). On the other hand, NO rapidly reacts with superoxide radicals to yield peroxy nitrite, and it is also considered that peroxy nitrite causes inflammatory cells, tissue disorders.
[152] When activated immune cells enter the organs and release cytokines, NO production is induced. Insulin dependent diabetes mellitus is a disease caused by the specific destruction of Langerhans β cells, and is believed to be destroyed by NO. In addition, the articular fluid in the lesion of a patient with chronic arthritis, rheumatoid arthritis, gouty arthritis, and Behcet's disease contains a higher concentration of NO than the joint fluid of a normal joint of the same patient or a joint of an ordinary person. Administration of the compound of the present invention, the optically active agent thereof, or these salts to these patients suppresses NO production in the lesion and improves symptoms.
[153] During cerebral ischemia and after reperfusion, NO production is increased, resulting in damage to brain tissue. When cerebral ischemia is administered to a patient by administering a compound of the present invention, an optically active substance thereof, or a salt thereof, damage to brain tissue is alleviated, thereby improving the progress after treatment.
[154] Cell surface antigens called pars antigens (APO-1 antigen, CD95) have attracted attention as molecules inducing apoptosis [Cell, Vol. 66, pp. 233-243 (1991); J. Exp. Med., Vol. 169, pages 1747-1756 (1989); J. Biol. Chem. 267, pp. 10709-10715 (1992); J. Immunology, Vol. 184, pp. 1274-1279 (1992).
[155] PAS antigen is expressed in immune system cells, such as thymic cells, T cells, cytotoxic T cells, B cells, or NK cells. In the invasion of foreign nonmagnetic antigens, the immune system causes an immune response and excludes nonmagnetic antigens. However, self tolerance is established without showing an immune response to self antigens. This is because lymphocyte lineage cells with self-response are excluded by cell death due to apoptosis upon receiving a negative selection of clone removal. However, when these cells do not undergo apoptosis due to any abnormality in the living body such as genetic defects of the parse antigen, for example, self-reactive T cells accumulate at the periphery. In normal living organisms, self-tolerance is established for B cells, which are immune cells, and these self-reactive B cells usually die by apoptosis. However, self-reactive B cells may cause abnormalities such as genetic defects of the PAS antigen. In the absence of apoptosis by vi), self-reactive B cells accumulate at the periphery. Moreover, in the case of articular rheumatism, the above-described abnormalities of the autoreactive lymphocytes and abnormalities of turn-over of synovial cells become part of the pathogenesis.
[156] The pars antigen-generating inducer comprising as an active ingredient a compound of the present invention or at least one compound selected from the optically active agent or salts thereof, is an active biological component cell that could not be excluded from the living body due to abnormalities in self-reactive lymphocytes and turnover. It is useful for inducing apoptosis and can be used for the method of inducing pas antigen production. In addition, a compound containing the compound of the present invention or at least one or more compounds selected from these optically active agents or these salts as an active ingredient is usefully used as a prophylactic or therapeutic agent for diseases involving pars antigen production abnormality. In the present invention, there are no particular limitations on the disease involving abnormality in parsing antigen generation, and examples thereof include autoimmune diseases caused by autoreactive T cells, autoreactive B cells, arthritis rheumatoid, and the like, and WO97 / 0965 publication. It includes the disease described.
[157] Compounds of the present invention or optically active agents thereof, or salts thereof may enhance interleukin-10 production, inhibit delayed-type hypersensitivity, inhibit lymphocyte weakening, inhibit mixed lymphocytes, inhibit IgE production, inhibit carrageenan edema, and the like. An immunomodulatory agent having an immunomodulatory action of the present invention or an optically active compound or at least one compound selected from these salts as an active ingredient is an agent for the treatment or prevention of diseases caused by abnormalities of these immune systems and immune factors. useful.
[158] That is, Th1 is activated by the production | generation reduction of interleukin-10, and autoimmune inflammation of Th1 advantage is caused. This inflammation is involved in organ specific autoimmune diseases such as nephritis and hepatitis, and diseases such as graft rejection and allergic contact dermatitis. The immunomodulatory agent of the present invention is useful for the treatment or prevention of these diseases by enhancing the production of interleukin-10 and inhibiting the activity of Th1.
[159] In addition, the lymphocyte weakening reaction is a reaction in which a mitogen binds to a receptor on the surface of lymphocytes, activates lymphocytes, and promotes division and proliferation. Mixed lymphocyte reaction is a reaction in which lymphocytes obtained from allogeneic animals are mixed and cultured to induce activation of lymphocytes due to mismatches in major tissue-compatible antigens, thereby promoting lymphocyte division and proliferation. The immunomodulatory agents inhibit these reactions and are particularly useful for the treatment or prevention of autoimmune diseases caused by abnormal hyperplasia of lymphocytes, such as chronic nephritis, chronic colitis, type I diabetes, chronic arthritis rheumatoid, etc. It is also useful in suppressing rejection.
[160] Carrageenan foot edema model is injected subcutaneously to the foot and spinal carrageenan inflammatory inducer to induce inflammatory cells such as macrophages, neutrophils, vascular permeability is enhanced by the inflammatory factors generated from these cells It is a reaction that causes edema. The edema inhibitory action of the immunomodulatory agent is useful for the treatment or prevention of diseases, such as chronic arthritis rheumatoid, which require hypervascular control of vascular permeability.
[161] In allergic diseases typified by asthma or atopic dermatitis, the release of chemical mediators from mast cells plays a large role in allergic reactions. This response is caused by the binding and crosslinking of IgE to receptors on the cell membrane, and the immunomodulatory agents of the present invention inhibit the production of IgE and are mediated or worsened by IgE production, such as allergic diseases caused by IgE, For example, it is very useful for improving symptoms and / or preventing diseases such as bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, urticaria, anaphylactic shock and the like. The immunomodulators of the present invention also inhibit delayed hypersensitivity reactions and are associated with delayed hypersensitivity reactions such as contact hypersensitivity, allergic contact dermatitis, bacterial allergies, fungal allergies, viral allergies, drug allergies, thyroiditis, allergies It is useful in the treatment and prevention of sexual encephalitis.
[162] Recently, from the results of pathology studies of diabetes, normal fat cells play an important role for normal insulin action in the whole body, and normal fat cells are required to smoothly proceed sugar metabolism [Jikken Igaku, 14, 61-68 (1996).
[163] The compounds of the present invention or their optically active agents or salts thereof have the ability to induce differentiation of progenitor cells, such as fibroblasts, and induce differentiation of the cells into adipocytes. Therefore, by ingesting the compound of the present invention or a compound selected from the optically active substance or salts thereof, normal fat cells are increased to improve the symptoms of diabetes.
[164] The compound of the present invention or its optically active agent or salt thereof has a hypoglycemic action, and can be used in the preparation of a therapeutic or prophylactic agent for diabetics comprising at least one compound selected from the compound of the present invention or its optically active agent or salts thereof as an active ingredient. Can be.
[165] That is, when the compound of the present invention, or an optically active substance thereof or at least one or more compounds selected from these salts is used as an active ingredient, it is formulated in combination with a known pharmaceutical carrier to prepare a therapeutic or preventive agent for diabetes. The agent can be formulated in accordance with the anticancer agent and can be administered by the method according to the above medicine.
[166] Dosage as a therapeutic or prophylactic agent for diabetes is appropriately set according to the form of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient, and is not constant, but generally the compound of the present invention or the optically active agent contained in the preparation or The amount of at least one compound selected from salts is from 10 pg to 200 mg / kg per adult. Of course, since the dose varies depending on various conditions, an amount smaller than the above dose may be sufficient, or it may be necessary beyond the range. In addition to oral administration as it is, the medicament of the present invention may be added to any food or drink and consumed daily.
[167] In addition, the compound of the present invention or the optically active substance thereof and salts thereof may be used as a raw material for food or drink for improving or preventing diabetes. By ingesting the compound of the present invention, the optically active substance thereof or these salt-containing substances, diabetes is improved and the amount of diabetes is reduced. In addition, complications such as decreased sexual function are significantly improved. Moreover, hyperlipidemia is improved.
[168] The compound of the present invention or the optically active agent or salt thereof has a hyperlipidemic action, that is, lowering total cholesterol in serum, lowering serum triglyceride, lowering serum free fatty acid, and having the action of the compound of the present invention or the optically active agent By using at least one compound selected from these salts as an active ingredient and formulating it in combination with a known pharmaceutical carrier, an antihyperlipidemic agent or an antihyperlipidemic agent can be prepared. The preparation of the preparation may be carried out in accordance with the diabetic therapeutic or preventive agent, and may be administered by a method in accordance with the diabetic therapeutic or preventive agent. Moreover, you may use the compound of this invention, its optically active substance, and these salts as a raw material of the food-drinks for hyperlipidemia improvement or prevention. By ingesting the compound of the present invention, or an optically active substance thereof or the contents of these salts, hyperlipidemia is improved and the amount of lipids in the blood is reduced.
[169] In addition, when the compound of the present invention having the ability to induce differentiation of progenitor cells into adipocytes, or an optically active substance thereof or salts thereof, is prepared as an active ingredient and is combined with a known pharmaceutical carrier, the adipocytes are converted into adipocytes. Differentiation inducers can be prepared. The agent may be prepared in accordance with the diabetic therapeutic agent or prophylactic agent and can be administered by a method according to the diabetic therapeutic agent or prophylactic agent.
[170] In addition, the compound of the present invention, or an optically active substance thereof and salts thereof exhibit a tumor necrosis factor production inhibitory action, and cause insulin-independent diabetes mellitus caused by tumor necrosis factor [Nature, Vol. 389, 610-614 pages (1997)]. It is useful for treatment and prevention.
[171] The compound of the present invention or the optically active agent or salt thereof has an antiviral action, and an antiviral agent can be prepared by formulating at least one compound selected from these compounds as an active ingredient and combining it with a known pharmaceutical carrier. have. An antiviral agent can be formulated according to the said anticancer agent, and can be administered by the method according to the said medicine.
[172] The dosage of the antiviral agent is appropriately set according to the form of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient, but is not constant, but is generally determined from the compound of the present invention or the optically active agent or salt thereof contained in the preparation. The amount of one or more substances selected is between 0.1 μg and 20 mg / kg per adult. Of course, since the dose varies depending on various conditions, an amount smaller than the dose may be sufficient, or it may be necessary beyond the range. In addition to oral administration as it is, the medicament of the present invention may be added to any food or drink and consumed daily. Moreover, you may use the compound of this invention, its optically active substance, or these salts as a raw material of antiviral food-drinks.
[173] The compounds of the present invention or their optically active agents or salts thereof exhibit antiviral activity against DNA viruses, RNA viruses, retroviruses and viroids.
[174] Therefore, agricultural and horticultural agents such as antiviral agents for humans, antiviral agents for non-human animals such as livestock, poultry, aquaculture animals such as fish, shrimp, and the like, which are effective against virus diseases such as fish and shrimp, and plant antiviral agents such as petals and vegetables. It can be used as an antiviral agent for useful organisms, such as an antiviral agent effective for a virus virus of a crop.
[175] Examples of DNA viruses that infect animals include pox virus, herpes virus, adenovirus, hepatitis virus, papilloma virus, polyoma virus, Epstein-Barr virus and baculo virus. Examples thereof include cauliflower virus. Examples of RNA viruses that infect animals include rota virus, rubella virus, Japanese encephalitis virus, dengue virus, Newcastle disease virus, measles virus, mumps virus, distemper virus, influenza virus, bullous oralitis virus, human polio virus And hepatitis A virus and hepatitis C virus. Examples of RNA viruses infected with plants include tobacco mosaic virus, varicose dwarf virus, rice rot virus, and tobacco bleeding. ) Viruses. Examples of retroviruses include adult T cell leukemia virus, human immunodeficiency virus, and viroids include, for example, potato spindle tuba viroids.
[176] The compounds of the present invention or their optically active agents or salts thereof are effective for the treatment and prophylaxis of viral diseases in non-human mammals, birds such as chickens and turkeys, cold blooded animals such as fish, and these compounds are effective against viruses of It shows antiviral activity against. Sciruid herpes virus type 1, cavlid herpes type 1, lagomorph herpes virus type 1, phasianid herpes virus type 1, facyanide herpes virus type 2, turkey Herpes Virus Type 1, Anatid Herpes Virus Type 1, Catfish Herpes Virus Type 1, Equid Herpes Virus Type 3, Bovid Herpes Virus Type 1, Bobid Herpes Virus 3 Type, bovid herpes virus type 4, swine herpes virus type 1, swine herpes virus type 2, murid herpes virus type 1, cebid herpes virus type 1, cevid herpes virus type 2, tupalid (tupalid) herpes virus type 1, canine herpes virus type 1, feline herpes virus type 1, Equid herpes virus type 1, e De herpes virus type 2.
[177] By a well-known method in veterinary surgery and breeding, such as injecting an antiviral agent of the present invention into birds, or adding it to feed or beverages, a compound of the avian viral diseases such as Marek's disease is used in the present invention, Prevented and / or treated. In addition, the compounds used in the present invention can be added directly to a pool, a water tank, a holding tank or a water or seawater in a breeding area, or a compound used in the present invention can be mixed in a feed to produce a herpes virus such as a small catfish virus. , Virus, such as herpes virus, Solomon, nerka virus, etc., in fish, tanks, holding tanks, or fish living in small areas in breeding areas, such as infectious hematopoietic necrosis of salmon roe, herpes virus infection Or infectious pancreatic necrosis, tiger trout viral hemorrhagic sepsis, carp spring virus, lymphocytic disease of various fish, viral red blood cell necrosis of marine fish and cattle And viral pancreatic necrosis such as defense baby fish, and goofy disease such as cock suit. Rooms can be and / or treated. In addition, the precise regulation in administering the compound used for this invention and the antiviral agent of this invention depends on each subject to be treated, the kind of treatment, and the judgment of a breeder.
[178] Animals other than humans to which the antiviral agent of the present invention is administered, the improvement of survival rate, growth rate, egg laying rate, etc. is remarkable by maintaining the health.
[179] The compound of the present invention or the optically active substance or salt thereof used in the present invention exhibits strong antiviral action by inhibiting the synthesis of these viral proteins and also inhibiting the synthesis of the viral genome. It also selectively kills these virus infected cells.
[180] For example, even in a human acquired immunodeficiency virus (hereinafter abbreviated as HIV) infection patient, not all CD4-positive cells are infected with HIV, but only some cells. The antiviral agent of the present invention selectively kills infected cells while inhibiting HIV proliferation of the infected cells, inducing viral resistance to uninfected cells, and allowing the removal of HIV from the cells.
[181] The compound of the present invention or its optically active agent or salt thereof has a liver function-improving effect and a heat shock protein inducing action, and a liver function comprising the compound of the present invention or its optically active agent or at least one compound selected from these salts as an active ingredient An improving agent or a heat shock protein inducing agent can be formulated in accordance with the antiviral agent, and can be administered by a method according to the antiviral agent.
[182] Dosage as a liver function improving agent or heat shock protein inducing agent is appropriately set depending on the type of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient, but is not constant, but the compound of the present invention generally contained in the preparation or The amount of the optically active agent or one or more compounds selected from these salts is between 0.1 μg and 20 mg / kg per adult. Of course, since the dose varies depending on various conditions, an amount smaller than the dose may be sufficient, or it may be necessary beyond the range. In addition to oral administration as it is, the medicament of the present invention may be added to any food or drink and consumed daily. In addition, the compound of the present invention, or an optically active substance thereof, or at least one compound selected from these salts may be used as a raw material for food and drink for improving liver function and food and drink for inducing heat shock protein.
[183] Ingestion of the compound of the present invention, the optically active substance thereof, or salts thereof improves liver function impairment and normalizes GOT and GPT values.
[184] In addition, the compound of the present invention or the optically active substance or salt thereof has heat shock protein inducing activity such as 70 kDa (HSP70), RNA virus such as hepatitis virus, AIDS virus, influenza virus, bullous oralitis virus, herpes virus, Has antiviral action against DNA viruses. In addition, heat shock proteins are involved in cancer immunity, and these compounds are also effective in cancer immunity. It also has biological defenses such as anti-inflammatory. By ingesting the compound of the present invention, the optically active substance thereof, or these salts, viral diseases such as cold disease caused by influenza virus can be prevented and treated.
[185] In addition, heat shock protein is a generic term for proteins inducing synthesis when cells or individuals undergo rapid temperature changes of about 5 to 10 ° C. above the normal temperature, and are widely distributed from prokaryotic substances to higher eukaryotes. As heat shock proteins of eukaryotes, HSP90, HSP70, ubiquitous kitchen, HSP26 and the like are known. Among them, HSP70 is a kind of molecular chaperone, which binds to an incomplete or incompletely folded protein and helps to form a three-dimensional structure. The amino acid sequence of the heat shock protein is well preserved during evolution and HSP70 is homologous to the E. coli DnaK protein. There are about 10 HSP70 genes in humans, but some of them are constitutively expressed and some are induced by various stimuli. In addition to heat shock, the synthesis of heat shock proteins is also induced by cellular disorders such as various chemicals and oxidative stress.
[186] C. Amici et al., Journal of Virology, Vol. 68, pp. 6890-6899 (1994), reported the infection of Sendai virus in the presence of prostaglandin A ₁ with α, β-unsaturated carbonyl. It has been reported that culturing animal cells induces the synthesis of HSP70 and HSP90, and inhibits the synthesis of viral proteins while the synthesis of HSP70 is induced. In addition, A. Rossi et al., The Journal of Biological Chemistry, Vol. 271, pp. 32192-32196 (1996), suggests that 2-cyclopenten-l-one can synthesize HSP70 like prostaglandin A ₁ . Induction and inhibit the synthesis of bullous oralitis virus protein.
[187] The HSP70 inducibility by the compounds of the present invention can be seen as 10 μM and peaks at 20 to 30 μM, but this compares with 2-cyclopenten-l-one requiring a concentration of several hundred μM to induce HSP70. It can be said that very high inductive capacity.
[188] Since the compound of the present invention or the optically active substance or salt thereof has such a high heat shock protein inducing action, it exhibits antiviral activity against DNA virus, RNA virus, retrovirus and viroid. Examples of these viruses and viroids include the viruses and viroids described above.
[189] The compound of the present invention, or an optically active substance thereof, or a salt thereof has a proliferation inhibitory activity against cancer cells transformed with a cancer gene, and has an action of preventing carcinogenesis by the cancer gene.
[190] For example, the papilloma virus is a DNA virus of the papovaviridae and papillomavirus. As the papillomavirus (HPV) of humans, for example, HPV16 is known to cause cervical cancer. The compound of the present invention or its optically active agent and salts thereof have a proliferation inhibitory effect on cells cancerized by cancer gene E7 of HPV16 type, and the compound of the present invention or at least one compound selected from the optically active agent or salts thereof. As an active ingredient, a virus carcinogenic cell proliferation inhibitor can be provided, and carcinogenesis by a cancer gene can be prevented.
[191] In addition, the compound of the present invention or the optically active agent or salt thereof has a two-step carcinogenic action by an initiator and an accelerator, and at least one compound selected from these compounds can be used as an active ingredient to provide a chemocancer inhibitor. .
[192] Therefore, it is possible to provide a food for preventing cancer or a beverage for preventing cancer, which contains the compound of the present invention or an optically active substance thereof or at least one compound selected from these salts.
[193] The compound of the present invention or the optically active agent or salt thereof has an inhibitory effect on IgE production and a delayed type hypersensitivity reaction, and is formulated in combination with a known pharmaceutical carrier using at least one compound selected from these compounds as an active ingredient. If it is anti-allergic agent can be prepared. Preparation of the said formulation can be formulated according to the said anticancer agent. In addition, the anti-allergic agent of the present invention is administered by a suitable route of administration depending on the form of the preparation. There is no restriction | limiting in particular also in the administration method, It can administer by content, external use, and injection. For example, tablets, pills, granules, powders, solutions, suspensions, syrups, capsules can be administered orally. Injections can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermal, and the like. Ointments, creams and the like can be administered transdermally. Suppositories can be administered rectally. In addition, aqueous or non-aqueous eye drops can be prepared, and examples of the formulation administered to the eye by eye drops include eye ointments, coating liquids, spraying agents, and inserts. Also for inhalation, solutions or suspensions of active ingredients with conventional pharmaceutical excipients are used, for example as aerosol sprays for inhalation. The dry powdered active ingredient may also be administered by an inhaler or other device that allows direct contact with the lungs.
[194] The dosage as an anti-allergic agent is appropriately set according to the form of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient administering the same, but is not constant, but generally the compound of the present invention or optical activity contained in the preparation The amount of at least one compound selected from the sieve or these salts is 10 pg to 50 mg / kg per adult per day. Of course, since the dose varies depending on various conditions, an amount smaller than the dose may be sufficient, or it may be necessary beyond the range. In addition to oral administration as it is, the medicament of the present invention may be added to any food or drink and consumed daily.
[195] IgE production inhibitor and delayed-type hypersensitivity inhibitor can be prepared as an active ingredient from the compound of the present invention or an optically active substance thereof or one or more compounds selected from these salts. These preparations can be formulated according to the antiallergic agent described above, and can be administered by the method according to the antiallergic agent.
[196] In addition, the compound of the present invention, the optically active substance thereof, and these salts may be used as raw materials for foods for antiallergic or beverages for antiallergic. By ingesting the compound of the present invention or the optically active substance or salts thereof, the symptoms of the disease due to IgE production and delayed hypersensitivity are remarkably improved, and the prevention effect of the disease is also excellent.
[197] Antiallergic agents of the present invention inhibit the production of IgE and are mediated or worsened by IgE production, such as allergic diseases caused by IgE, such as bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, urticaria It is very useful for improving symptoms and / or preventing diseases such as anaphylactic shock. It also inhibits delayed-type hypersensitivity and treats diseases involving delayed-type hypersensitivity, such as contact hypersensitivity, allergic contact dermatitis, bacterial allergy, fungal allergy, viral allergy, drug allergy, thyroiditis, allergic encephalitis, It is useful for prevention.
[198] Compounds of the present invention or optically active agents or salts thereof include anticancer activity, cancer cell proliferation inhibitory activity, apoptosis-inducing activity, phase isomerase II inhibitory activity, cancer cell differentiation inducing activity, antirheumatic activity, chronic arthritis rheumatoid activity, pars Antigen generation inducing activity, antibacterial activity, antiviral activity, liver function improving activity, heat shock protein inducing activity, blood component normalizing activity, cancer immune enhancing activity, anti-inflammatory activity, tumor necrosis factor production inhibitory activity, nitric oxide production inhibitory activity, It has physiological activities such as immunomodulatory activity such as delayed type hypersensitivity inhibitory activity, lymphocyte weakening reaction inhibitory activity, mixed lymphocyte reaction inhibitory activity, IgE production inhibitory activity, carrageenan edema inhibitory activity and the like. Food containing a compound or an optically active substance thereof or at least one compound selected from these salts The beverage is useful as a functional food or functional beverage having various physiological activities described above.
[199] In the production of the food or beverage of the present invention, a heat treatment product containing cyclopentenone, a partially purified cyclopentenone derived from the heat treatment product, and a purified cyclopentenone and SH group-containing compound are used during the production process. The compound of this invention produced | generated can also be used.
[200] That is, the compound of the present invention, or an optically active substance thereof, or a salt thereof, which is a reactant of a heat treatment product containing cyclopentenone, a raw material selected from partially purified cyclopentenone and purified cyclopentenone from the heat treatment product, and a SH group-containing compound Foods or beverages which contain, dilute and / or add at least one compound selected from are included in the food or beverage of the present invention.
[201] The production method of the food or drink of the present invention is not particularly limited, but examples thereof include cooking, processing, and production by a food or beverage production method that is generally used, and the present invention has an effective amount of physiological action on the produced food or drink. What is necessary is just to contain the compound of this, its optically active substance, and at least 1 sort (s) of compound chosen from these salts.
[202] In the production of foods or beverages, the heat treatment may be performed at any step, the cyclopentenone may be contained in the heat treatment product and reacted with the SH group-containing compound, and a heat treatment product containing the compound of the present invention may be added. You may also do it. Moreover, you may add food or a drink, or its raw material to the heat processing material containing the compound of this invention, and may dilute the compound of this invention in the heat processing material. In addition, you may add once or several times. Therefore, it is possible to easily prepare foods or beverages with new physiological action.
[203] As a food or drink of this invention, the compound of this invention which has a physiological function, such as anticancer effect, antibacterial effect, apoptosis induction, antiviral effect, liver function improvement effect, or at least 1 sort (s) chosen from these optically active agents or these salts. If the above compound is contained, added, and / or diluted, the shape thereof is not particularly limited, and such shapes include orally ingestible forms such as tablets, granules, capsules, gels, and sols.
[204] The food or beverage of the present invention contains a compound of the present invention having a physiological activity or an optically active substance or salt thereof, and has various physiological activities, anticancer action, antibacterial action, apoptosis induction action, antiviral action, Symptoms of diseases exhibiting susceptibility to compounds of the present invention, such as carcinogenesis prevention, cancer suppression effects, viral diseases, diabetes, rheumatism, allergies, autoimmune diseases, or optically active agents or salts thereof by ingesting them by improving liver function. It is a health food or drink which has an improvement effect, a preventive effect, a liver function improvement effect, etc., It is a food or drink useful for maintaining homeostasis of a living body, especially gastrointestinal health. In addition, due to its antimicrobial activity, the food or beverage is very preservable.
[205] No toxicity is recognized even when the compound of the present invention or its optically active substance or salt thereof is administered an effective amount of these physiological activities. For example, in the case of oral administration, death of the compound represented by Formula 7, Formula 8, Formula 9, or Formula 10, or any one of these optically active agents or salts thereof to the rat once at 1000 mg / kg was not recognized. .
[206] As mentioned above, the compound of this invention, its optically active substance, or these salts are compounds which are very useful in the field of medicine, food, a drink, etc. according to various physiological functions. This compound of the present invention is a reaction product of a cyclopentenone with an SH group-containing compound such as an SH group-containing amino acid or a derivative thereof, such as a cystine-containing amino acid derivative, and is also used in foods and beverages. It is included in the present invention.
[4] An object of the present invention is to develop a compound having a physiological action such as anticancer action, to provide a method for producing the compound and a medicament containing the compound.
[5] MEANS TO SOLVE THE PROBLEM The present inventors earnestly examined in order to achieve such an objective, and, as a result, the compound represented by following General formula (1, hereafter simply called "the compound of this invention") is represented by the following general formula (4), 4, 5- dihydroxy-2 It is produced by reacting cyclopenten-l-one (hereinafter simply referred to as "cyclopentenone") with an SH group-containing compound, and the compound of the present invention has various strong physiological activities and susceptibility to the compound. The invention has been found to be useful for the treatment and / or prevention of the diseases indicated.
[6] Briefly describing the present invention, a first object of the present invention is to provide a compound represented by the following formula (1) or an optically active agent or salts thereof.
[7]
[8] In the above formula, the bond indicated by the dotted line in the 5-membered ring means that the 5-membered ring is a cyclopentene ring having a double bond, or it may exist as any of the saturated cyclopentane rings. And in the case of a cyclopentene ring, X is OH, Y is = O and Z is H. On the other hand, in the case of a cyclopentane ring, X is = 0, Y is OH, and Z is OH. In addition, R is a residue which removed SH group from the SH group containing compound.
[9] As a specific example of the 1st objective of this invention, the compound represented by following formula (2), its optically active substance, or these salts are mentioned.
[10]
[11] In the above formula, R is a residue obtained by removing the SH group from the SH group-containing compound.
[12] As another form of 1st object of this invention, the compound represented by following General formula (3), its optically active substance, or these salts is mentioned.
[13]
[14] In the above formula, R is a residue obtained by removing the SH group from the SH group-containing compound.
[15] A second object of the present invention is to react a compound selected from 4,5-dihydroxy-2-cyclopenten-l-one represented by the following formula (4) or an optically active agent thereof or salts thereof with a SH group-containing compound The present invention relates to a compound represented by the formula (1) of the first object of the present invention, an optically active substance thereof, or a method for producing these salts.
[16]
[17] In preferred embodiments of the first and second objects of the present invention, the SH group containing compound is an SH group containing amino acid or derivative thereof.
[18] A third object of the present invention relates to a medicament comprising the compound represented by the formula (1) of the first object of the present invention, or an optically active substance thereof or at least one compound selected from these salts as an active ingredient.
[19] In a preferred embodiment of the third object of the present invention, the medicament is a biological defense agent (e.g., an immunomodulator, an antiallergic agent, an antirheumatic agent), an antidiabetic agent, an anticancer agent, an apoptosis inducing agent, and an anti-hospital agent. Microbial agents (eg antiviral agents, antibacterial agents).
[207] Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to these Examples at all. In addition,% in an Example means weight%.
[208] Comparative Example 1
[209] 10 g of D-glucuronic acid (manufactured by Sigma, G 5269) was dissolved in 1 L of water, heated at 121 ° C. for 4 hours, and then concentrated under reduced pressure until it became about 10 ml. 40 ml of the upper layer of the butyl acetate: acetic acid: water = 3: 2: 2 mixed solution was added thereto and mixed, and the supernatant obtained by centrifugation was concentrated to about 10 ml under reduced pressure.
[210] The extract was applied to silica gel BW-300SP for column chromatography (2 x 28 cm, manufactured by Fujisilician Chemical Co., Ltd.), and 0.2 kg / of the upper layer of butyl acetate: acetic acid: water = 3: 2: 2 was used as an eluent as an eluent for 0.2 kg / Pressurization was carried out at cm 2, and separation was carried out at a flow rate of 5 ml per minute. The fractions were separated to 10 ml per fraction, and a portion of each fraction was analyzed by thin layer chromatography. As a result, fractions 61 to 80 contained high-purity cyclopentenone. These fractions were combined, concentrated under reduced pressure, extracted with 40 ml of chloroform, and the extract was concentrated under reduced pressure to yield 100 mg of cyclopentenone.
[211] This fraction was separated by normal phase HPLC using a Palpack type S column (manufactured by Takara Shuzo Co., Ltd.), and detected by ultraviolet absorption at 215 nm. The purity was 98%.
[212] 113.9 mg of the cyclopentenone was dissolved in 2.85 mL of ethanol. 3.85 ml of hexane / ethanol (94/6) was further added to this ethanol solution to prepare a 17 mg / ml cyclopentenone solution. This solution was filtered through a 0.5 µm filter to obtain an optical split HPLC sample solution.
[213] The sample solution was subjected to optical split HPLC under the following conditions, and the fractions of the (-)-cyclopentenone of the front peak and the (+)-cyclopentenone of the rear peak were collected and dried under reduced pressure, respectively, and the (-)-cyclophene. 43.2 mg of tenones and 43.0 mg of (+)-cyclopentenone were obtained, respectively.
[214] Optical Split HPLC Conditions
[215] Column: Chiralpak AS (manufactured by Daicel Chemical Industries) 2.0 cm × 25.0 cm
[216] Column temperature: 40 ℃
[217] Mobile phase: Hexane / ethanol (94/6)
[218] Flow rate: 4.0 ml / min
[219] Detection: UV 210 nm
[220] Sample injection volume: 150 μl (2.55 mg)
[221] The obtained (-)-cyclopentenone and (+)-cyclopentenone both contained about 1% of enantiomer, and thus were optically divided under the above conditions. As a result, 37.7 mg of the (-)-cyclopentenone of the preceding peak and 27.7 mg of 37.4 mg of the (+)-cyclopentenone without the 19.7 mg of the enantiomer were added. (+)-Cyclopentenone which does not contain the enantiomer of was obtained, respectively. Moreover, the elution time of optical split HPLC of (-)-cyclopentenone and (+)-cyclopentenone was 33 minutes and 40 minutes, respectively.
[222] Example 1
[223] (1) 100 µl of 1M aqueous cyclopentenone solution and 100 µl of 1M L-cysteine hydrochloride (manufactured by Nakarai Tesque, 103-13) adjusted to pH 4 with NaOH were mixed and allowed to react at 60 ° C for 16 hours. The reaction was filtered through a 0.5 μm Cosmo Nice filter (manufactured by Nakarai Tesque, 440-84), followed by 0.5% aqueous solution of 0.1% trifluoroacetic acid (manufactured by Nakarai Tesque, 349-01) as a mobile phase. At a flow rate of ml, HPLC was performed using a capsule pack column C ₁₈ SG120 (6 mm × 250 mm: manufactured by Shiseido), and the absorbance of 210 nm was detected. As a result, major peaks were observed at 19.1 and 19.5 minutes. The mixture was concentrated to dryness under reduced pressure to isolate two diastereomers, CM1 (19.1 min) and CM2 (19.5 min).
[224] (2) Structure of cyclopentenone and L-cysteine reactant
[225] Mass spectrometry of CM1 and CM2 isolated in Example 1- (1) was performed using a DX302 mass spectrometer (manufactured by Nippon Denshi Co., Ltd.). Furthermore, it dissolved in 0.1 N DCl aqueous solution and analyzed the structure by nuclear magnetic resonance (NMR). The nuclear magnetic resonance apparatus used JNM-A500 (made by Nippon Denshi Co., Ltd.). Ultraviolet (UV) absorption spectra were measured using a UV-2500 spectrophotometer (manufactured by Shimadzu Corporation). The results are shown below.
[226] CM1
[227] FAB-MS m / z 218 [M + H] ⁺
[228] Glycerol was used as the matrix.
[229] ¹ H-NMR
[230] δ2.32 (1H, dd, J = 20.0, 1.5 Hz, 5-H), 2.89 (1H, dd, J = 20.0, 6.0 Hz, 5-H), 3.01 (1H, dd, J = 15.0, 7.0 Hz , 1'-H), 3.09 (1H, dd, J = 15.0, 4.5 Hz, 1'-H), 4.01 (1H, m, 4-H), 4.16 (1H, dd, J = 7.0, 4.5 Hz, 2'-H), 6.49 (1H, d, J = 3.0 Hz, 3-H)
[231] UV: λ _max nm 250 nm (water)
[232] CM2
[233] FAB-MS m / z 218 [M + H] ⁺
[234] Glycerol was used as the matrix.
[235] ¹ H-NMR
[236] δ2.31 (1H, dd, J = 20.0, 1.5 Hz, 5-H), 2.87 (1H, dd, J = 20.0, 6.0 Hz, 5-H), 3.01 (1H, dd, J = 15.0, 6.5 Hz , 1'-H), 3.11 (1H, dd, J = 15.0, 4.5 Hz, 1'-H), 4.00 (1H, m, 4-H), 4.20 (1H, dd, J = 6.5, 4.5 Hz, 2'-H), 6.46 (1H, d, J = 3.0 Hz, 3-H)
[237] However, the chemical displacement value for ¹ H-NMR was expressed based on the chemical displacement value of HOD as 4.65 ppm.
[238] Furthermore, CM1 was dissolved in 0.1N DCl heavy aqueous solution, and ¹³ C-NMR spectrum was measured using JNM-A500.
[239] ¹³ C-NMR
[240] 30.3 (1'-C), 39.4 (4-C), 42.0 (5-C), 53.4 (2'-C), 132.8 (3-C), 154.6 (2-C), 171.1 (3 ' -C), 205.5 (1-C)
[241] However, the chemical displacement value of ¹³ C-NMR was expressed on the basis of the chemical displacement value of dioxane as 67.4 ppm.
[242] In addition, the attribution number of the peak of ¹ H-NMR and ¹³ C-NMR is as shown in the following formula (7).
[243] Formula 7
[244]
[245] Infrared absorption (IR) spectra of an equivalent mixture of CM1 and CM2 (called CM) were also measured using an FTIR-8000 PC infrared spectrophotometer (manufactured by Shimadzu Corporation). The results are shown.
[246] IR
[247] ν ^KBr _max cm ^-1 , 3000, 1705, 1625, 1201
[248] It carried out according to the diffuse reflection method.
[249] From these values, it became clear that either one of CM1 and CM2 is a structure represented by the formula (8), and the other is a structure represented by the formula (9).
[250] Formula 8
[251]
[252] Formula 9
[253]
[254] The mass spectrum of CM1 in FIG. 1, its ¹ H-NMR spectrum in FIG. 2, the UV absorption spectrum of CM1 in FIG. 3, the ¹ H-NMR spectrum of CM2 in FIG. 4, and the ¹³ C-NMR spectrum of CM1 in FIG. 6 shows the IR absorption spectrum of the CM. In Figure 1, the axis of abscissas is m / z value, and the axis of ordinates is relative intensity (%). 2, 4 and 5, the horizontal axis represents chemical displacement (ppm) and the vertical axis represents signal intensity. In FIG. 3, the horizontal axis represents wavelength (nm) and the vertical axis represents absorbance. 6, the horizontal axis represents wave number (cm ⁻¹ ) and the vertical axis transmittance (%).
[255] Example 2
[256] (1) 100 µl of a 1M aqueous solution of cyclopentenone and 500 µl of an aqueous 200 mM glutathione (reduced form: Nakarai Tesque Company: 170-10) (pH 3.0) were mixed and reacted at 60 ° C for 5 hours. The reaction solution was filtered through a 0.5 μm Cosmo Nice filter and separated by HPLC under the following conditions.
[257] Column: TSK Gel ODS-80Ts (5 μm), 20 mm × 25 cm
[258] Mobile phase: A 0.1% TFA aqueous solution
[259] B 0.1% TFA / 50% acetonitrile solution
[260] Flow rate: 7.5 ml / min
[261] Gradient: Mobile phase A for 10 minutes A → B = 1: 1 for mobile phase A for 55 minutes
[262] → Mobile phase B from 15: mobile phase A: B = 1: 1
[263] Detection: absorbance at 220 nm
[264] 200 µl of the reaction solution was added to HPLC, and the peaks of the holding time of 35.7 minutes and 36.1 minutes were separated, and concentrated to dryness under reduced pressure, respectively, to isolate two diastereomers, GM1 and GM2. The yield of GM1 was 2.5 mg, and the yield of GM2 was 3.0 mg.
[265] This chromatogram is shown in FIG. That is, FIG. 7 is a diagram showing the relationship between the holding time and the absorbance, where the horizontal axis is the holding time (minutes) and the vertical axis is the absorbance at 220 nm.
[266] (2) The structures of the equivalent mixtures (abbreviated as GM) of GM1 and GM2 prepared in Example 2- (1) were analyzed. Nuclear magnetic resonance (NMR) spectra are JNM-A500 (manufactured by Nippon Denshi Corporation), mass spectra (MS) are DX302 mass spectrometers (manufactured by Nippon Denshi Corporation), and ultraviolet (UV) absorption spectra are UV-2500 spectrophotometers And infrared absorption (IR) spectra were measured using an FTIR-8000PC infrared spectrophotometer (manufactured by Shimadzu Corporation). The results are shown below.
[267] ¹ H-NMR
[268] δ 2.09 (2H, m, 5'-H), 2.28 (1H, dd, J = 13.0, 20.0 Hz, 5-H), 2.44 (2H, m, 4'-H), 2.78 (1H, dd, J = 8.5, 14.0, 1'-H), 2.85 or 2.89 (1H, dd, J = 3.0, 6.0 Hz, 5-H), 2.92 or 2.95 (1H, dd, J = 1.0, 5.5 Hz, 1'- H), 3.86 (2H, S, 9'-H), 3.95 (2H, m, 4-H, 6'-H), 4.46 (1H, m, 2'-H), 6.47 or 6.49 (1H, d , J = 3.0 Hz, 3-H)
[269] The sample was dissolved in 0.1N DCl heavy aqueous solution and the chemical displacement value of HOD was expressed as 4.65 ppm.
[270] ¹³ C-NMR
[271] δ 26.3 (5'-C), 31.7 (4'-C), 31.9 or 32.1 (1'-C), 39.3 (4-C), 41.9 (9'-C), 42.2 or 42.3 (5-C) ), 53.3 (6'-C), 54.1 (2'-C), 133.5 (3-C), 154.4 (2-C), near 173 (3'-C, 7'-C, 8'-C, 10'-C), 205.8 (1-C)
[272] The sample was dissolved in 0.1 N DCl aqueous solution and the chemical displacement value of dioxane was expressed as 67.4 ppm.
[273] In addition, peaks attributable to the number of the ^{1 H-NMR, 13 C-} NMR shown in formula (10).
[274] Formula 10
[275]
[276] FAB-MS
[277] m / z 404 (M + H) ⁺ , 426 (M + Na) ⁺
[278] Glycerol was used as the matrix.
[279] UV λ _max , 251 nm (water)
[280] IR ν ^KBr _max , cm ^-1 2949, 1710, 1660, 1539, 1404, 1203
[281] It was performed according to the diffuse reflection method.
[282] The results are shown in FIGS. 8 to 12. That is, FIG. 8 is a diagram showing ¹ H-NMR spectra of GM, where the horizontal axis represents chemical displacement (ppm) and the vertical axis represents signal intensity. Fig. 9 shows the ¹³ C-NMR spectrum of GM, where the axis of abscissas is chemical displacement (ppm) and the axis of ordinates is intensity of signal. Fig. 10 shows the mass spectrum of GM, where the horizontal axis represents m / z value and the vertical axis represents relative intensity (%). Fig. 11 shows the UV absorption spectrum of GM, where the horizontal axis represents wavelength (nm) and the vertical axis represents absorbance. Fig. 12 shows the IR absorption spectrum of GM, where the horizontal axis represents wave number (cm ⁻¹ ) and the vertical axis represents transmittance (%).
[283] From the above results, GM1 and GM2 are diastereomers of 2-hydroxy-4-glutathione-S-yl-2-cyclopenten-l-one, one of which is represented by the formula (11), and the other is represented by the formula (12). It became clear that it was a structure shown in.
[284]
[285]
[286] Example 3
[287] (1) Cyclopentenone and L-cysteine hydrochloride (manufactured by Nakarai Tesque, 103-13) were dissolved in phosphate buffered saline (pH 7.2) so as to have a concentration of 10 mM, and reacted at 37 ° C for 30 minutes. The reaction was filtered through a 0.5 μm Cosmo Nice filter (manufactured by Nakarai Tesque, 440-84), followed by aqueous solution of 0.1% trifluoroacetic acid (trade name, 349-01 manufactured by Nakarai Tesque) as a mobile phase. At a flow rate of 1 ml per minute, HPLC was performed using TSK-Gel ODS 80Ts (4.6 mm x 250 mm; manufactured by Tosoh Corp.) and detected at an absorbance of 210 nm. Peaks of cyclopentenone and L-cysteine were detected. Decreased and major peaks emerged at 4.0 and 4.3 minutes.
[288] The results are shown in FIG. That is, FIG. 13 is a graph showing the relationship between the elution time and the absorbance at 210 nm, with the horizontal axis representing the elution time (minutes) and the vertical axis representing the absorbance at 210 nm.
[289] In Fig. 13, 3.1 minutes is the elution position of L-cysteine, and 4.9 minutes is the elution position of cyclopentenone.
[290] (2) Cyclopentenone and L-cysteine hydrochloride were dissolved in phosphate buffered saline (pH 7.2) so that their concentrations were 100 μM, and the change in absorbance at 215 nm at 37 ° C. over time was measured by UV-2200 spectrophotometer. It measured using (the brand name manufactured by Shimadzu Corporation). As a result, the absorbance decreased with the reaction time and became substantially constant after 500 seconds. The results are shown in FIG. That is, FIG. 14 is a diagram showing the relationship between the reaction time and the absorbance at 215 nm when L-cysteine is used. The horizontal axis represents the reaction time (seconds) and the vertical axis represents the absorbance at 215 nm.
[291] The same operation was then performed using glutathione (reduced form, sold by Nakarai Tesque, 170-10) instead of L-cysteine hydrochloride. As a result, the absorbance at 215 nm was lowered with the reaction time, and was almost constant from 2000 seconds. Done The results are shown in FIG. In other words, Fig. 15 is a graph showing the relationship between the reaction time in the case of using glutathione and the absorbance at 215 nm, where the horizontal axis represents the reaction time (seconds) and the vertical axis represents the absorbance at 215 nm.
[292] (3) Cyclopentenone and L-cysteine hydrochloride were dissolved in phosphate buffered saline (pH 7.2) so as to have a concentration of 10 mM and reacted at 37 ° C for 30 minutes. The reaction was filtered through a 0.5 μm Cosmo Nice filter, followed by TSK-Gel ODS 80Ts (4.6 mm × 250 mm; manufactured by Tosoh Corp.) at a flow rate of 1 ml per minute using a 0.1% acetic acid aqueous solution as a mobile phase. High liquid chromatography mass spectrometry). Mass spectrometry was performed in positive ion mode. As a result, a signal of m / z = 236.1 [M + H] ⁺ was observed, and the molecular weight of the substance newly produced by the reaction was 235.
[293] The results are shown in FIGS. 16 and 17. That is, FIG. 16 is a chromatogram of a reaction product of one example of the present invention described above, where the horizontal axis represents retention time (minutes) and the vertical axis represents total ionic strength (relative value). FIG. 17 is a diagram showing the mass spectrum of the fraction eluted at 2.99 minutes in FIG. 16, where the horizontal axis represents m / z and the vertical axis represents ionic strength (relative value). Moreover, also in the mass spectrum of the fraction eluting at 3.15 minutes and 3.24 minutes of FIG. 16, the signal of m / z = 236.1 [M + H] ⁺ was observed.
[294] (4) Cyclopentenone and L-cysteine hydrochloride were dissolved in phosphate buffered saline (pH 7.2) so as to have a concentration of 100 μM and reacted at 37 ° C. for 50 minutes. Ultraviolet absorption spectra before and after the reaction were measured using a UV-2200 spectrophotometer (manufactured by Shimadzu Corporation).
[295] The results are shown in FIGS. 18 and 19. That is, FIG. 18 is a figure which shows the ultraviolet absorption spectrum of the reaction liquid immediately after melt | dissolution, and FIG. 19 is a figure which shows the ultraviolet absorption spectrum of the reaction liquid after reacting for 50 minutes. 18 and 19, the horizontal axis represents wavelength (nm) and the vertical axis represents absorbance.
[296] The same operation was performed using glutathione instead of L-cysteine hydrochloride.
[297] The results are shown in FIGS. 20 and 21. That is, FIG. 20 is a diagram showing an ultraviolet absorption spectrum of the reaction solution immediately after dissolution using glutathione, and FIG. 21 is a diagram showing an ultraviolet absorption spectrum of the reaction solution after reacting for 50 minutes using glutathione. 20 and 21, the horizontal axis represents wavelength (nm) and the vertical axis represents absorbance.
[298] (5) 60 µl of 1.4 M cyclopentenone heavy aqueous solution, 80 µl of 1 M L-cysteine aqueous solution and 160 µl of heavy water prepared in phosphate buffered saline (pH 7.2) were mixed and reacted at 37 ° C for 3 hours. ¹³ C-nuclear magnetic resonance (NMR) spectra of the reaction solution were measured using JNM-A500 (manufactured by Nippon Denshi Co., Ltd.). The results are shown below.
[299] ¹³ C-NMR
[300] δ33 (1'-C), 43 (4-C and 5-C), 55 (2'-C), 75 and 78 (3-C), 80 and 81 (2-C) ), Near 173 (3'-C), near 215 and near 217 (1-C)
[301] In addition, the peak number of the peak in ¹³ C-NMR is as shown in the following formula (13).
[302]
[303] ¹³ C-NMR spectrum of this reaction is shown in FIG. 22. In Figure 22, the axis of abscissas is chemical displacement (ppm), and the axis of ordinates is intensity of signal.
[304] Since the product in this reaction is a mixture of four types of diastereomers, up to four signals can be seen for each carbon atom, and the chemical structure of the reactants produced in the reaction solution is cyclopentanone represented by the formula (9). Thio derivatives.
[305] From the above results, the chemical structure of the reactant having a dissolution time of 4.0 to 4.3 minutes in Fig. 13 produced by the reaction between cyclopentenone and cysteine is a cyclopentanone thio derivative represented by the formula (9), and the dissolution time is 4.0. The reaction product was peaked for 4.3 minutes to obtain a cyclopentanone thio derivative represented by the formula (9) (hereinafter referred to as CD).
[306] Example 4
[307] (1) 60 μl of 1.4 M aqueous solution of cyclopentenone, 600 μl of 200 mM glutathione (reduced form, sold by Nakarai Tesque, 170-10) (adjusted to pH 7.0 with NaOH) and 1340 μl of phosphate buffered saline (pH 7.2) The mixture was reacted at 37 ° C for 1 hour. 700 µl of the reaction solution was filtered through a 0.5 µm Cosmo Nice filter to separate a reaction product of cyclopentenone and glutathione (hereinafter referred to as GD1) under HPLC under the following conditions.
[308] Column: TSK gel ODS-80Ts (5 μm), 20 mm × 25 cm (trade name manufactured by Tosoh Corporation)
[309] Mobile phase: A 0.1% trifluoroacetic acid [TFA, Merck trade name, 8262] aqueous solution
[310] B aqueous solution of 0.1% TFA / 50% acetonitrile (trade name, 004-30, manufactured by Nakarai Tesque)
[311] Flow rate: 9 ml / min
[312] Gradient: Mobile phase A for 40 minutes → Mobile phase A to B for 40 minutes → Mobile phase B after
[313] Detection: absorbance at 220 nm
[314] 500 µl of the reaction solution was placed in HPLC, and a peak of 27.7 minutes of holding time was sorted and lyophilized to isolate 5 mg of GD.
[315] This chromatogram is shown in FIG. That is, FIG. 23 is a diagram showing the relationship between the holding time and the absorbance, where the horizontal axis is the holding time (minutes) and the vertical axis is the absorbance at 220 nm.
[316] (2) The structure of GD1 prepared in Example 4- (1) was analyzed. The nuclear magnetic resonance (NMR) spectrum is JNM-A500 (manufactured by Nippon Denshi), the mass spectrum (MS) is DX302 mass spectrometer (manufactured by Nippon Denshi), and the ultraviolet (UV) absorption spectrum is UV-2500 spectrophotometer (SHIMA). Infrared absorption (IR) spectra were measured using an FTIR-8000PC infrared spectrophotometer (manufactured by Shimadzu Corporation). The results are shown below.
[317] ¹ H-NMR
[318] δ2.07 (2H, m, 5'-H), 2.17 (1H, dt, J = 20.0, 11.0 Hz, 5-H), 2.45 (2H, m, 4'-H), 2.88 (1H, m, 1'-H), 2.95 (1H, m, 5-H), 3.08 (1H, m, 1'-H), 3.19 (1H, m, 4-H), 3.76 (1H, m, 3-H) , 3.86 (3H, m, 6'-H, 9'-H), 4.09 (1H, d, J = 10.5 Hz, 2-H), 4.49 (1H, m, 2'-H)
[319] The sample was dissolved in heavy water and the chemical displacement of HOD was expressed at 4.65 ppm.
[320] ¹³ C-NMR
[321] δ 26.4 (5'-C), 31.7 (4'-C), 33.3 (1'-C), 41.9 (9'-C), 42.4 or 42.6 (4-C), 42.8 (5-C), 53.4 (6'-C), 54.1 (2'-C), 79.4 or 79.6 (3-C), 81.1 (2-C), near 173 (3'-C, 7'-C, 8'-C, 10'-C), 214.9 (1-C)
[322] The sample was dissolved in heavy water and the chemical displacement value of dioxane was 67.4 ppm.
[323] Further, the peak attributable to the number of the ^{1 H-NMR, 13 C-} NMR shown in formula (14).
[324]
[325] FAB-MS
[326] m / z 422 [M + H] ⁺
[327] Glycerol was used as the matrix.
[328] UV
[329] Terminal absorption
[330] IR
[331] ν ^KBr _max cm ^-1 3275, 1749, 1654, 1541, 1203, 1145
[332] It was performed according to the diffuse reflection method.
[333] The results are shown in FIGS. 24 to 27. That is, FIG. 24 shows a ¹ H-NMR spectrum of GD, where the horizontal axis represents chemical displacement (ppm) and the vertical axis represents signal intensity. Fig. 25 shows the ¹³ C-NMR spectrum of GD, where the axis of abscissas is chemical displacement (ppm), and the axis of ordinates is intensity of signal. Fig. 26 shows the mass spectrum of GD, where the axis of abscissas is m / z value, and the axis of ordinates is relative intensity (%). Fig. 27 shows the IR absorption spectrum of GD, where the horizontal axis represents wave number (cm ⁻¹ ) and the vertical axis represents transmittance (%).
[334] From the above result, it became clear that GD is 2, 3- dihydroxy-4- glutathione-S-yl- cyclopentanone shown by the said General formula (10). In addition, GD is a mixture of four diastereomers, and each diastereomer was separated from GD.
[335] Example 5
[336] (1) 0.5 ml of 0.2 mM CM, 0.8 mM CM, or 0.5 ml of RPMI1640 medium containing 4.5 ml of 10% fetal bovine serum, each containing 2.5 × 10 ⁵ promyeloid leukemia cells HL-60 cells (ATCC CCL-240), or Live cells were measured after incubation for 24 hours or 48 hours at 37 ° C. in the presence of 5% carbon dioxide by addition of 1.6 mM CM.
[337] As a result, CM showed strong cancer cell proliferation inhibitory activity. In addition, apoptotic bodies were formed in the cells at each cell proliferation inhibitory concentration.
[338] The results are shown in FIG. 28 is a diagram showing the relationship between the concentration of the added sample, that is, the culture medium of CM, and the number of living cells. The horizontal axis represents the concentration of CM (μM), and the vertical axis represents the number of live cells contained in 5 ml of culture medium (× It represents the 10 ^5/5 ㎖). In FIG. 28, □ indicates that the culture was performed for 24 hours after the addition of CM, and ■ indicates the culture for 48 hours after the addition of CM. In addition, (triangle | delta) mark shows that the sample was added and cultured for 24 hours, and ▲ mark shows that the sample was added and cultured for 48 hours, respectively.
[339] (2) 10 μl of water as an 8, 40, 200 or 1000 μM GM aqueous solution or control was added to each well of a 96 well microtiter plate. HL-60 (ATCC CCL-240) is suspended in RPMI164O medium containing 10% fetal bovine serum at 5 x lO ⁴ / ml, poured into each well of the microtiter plate by 90 μl, and 5% CO ₂ hours were incubated at 37 degreeC for 48 hours. 10 μl of 5 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT; manufactured by Sigma) phosphate buffered saline solution was added for 4 hours. After further incubation, the growth state of the cells was observed under a microscope. Further, 100 µl of 0.04N HCl-containing 2-propanol was added and stirred well, and the absorbance at 590 nm was measured.
[340] As a result, cell proliferation was not seen in the 40 μM GM addition zone (final concentration 4.0 μM). Thus, it became clear that GM completely inhibited the proliferation of HL-60 cells at a concentration of 4.0 μM. In addition, apoptotic bodies were formed in the cells at each cell proliferation inhibitory concentration.
[341] (3) 100, 200, or 400 μM of GM or CM aqueous solution or 10 μl of water as a control was added to each well of a 96 well microtiter plate, and in the same manner as in Example 5- (2), HL-60 Proliferation inhibitory activity was measured. However, the extent of cell proliferation was expressed as the ratio of the absorbance of the water addition zone at 590 nm to the absorbance of the sample addition zone at 590 nm (%). The results are shown in Table 1.
[342] GMCM 40 μM3.22.8 20 μM1.816.1 10 μM14.848.9
[343] (4) HL-60 cells (ATCC CCL240) incubated at 37 ° C with RPMI1640 medium (manufactured by Nisshi Corp.) containing 10% fetal bovine serum (manufactured by Gibco), treated at 56 ° C for 30 minutes. The medium was suspended to 2.5 × 10 ⁵ pieces / 4.5 ml. To this suspension 0.5 ml of a 100 μm GM aqueous solution was added (final concentration 10 μM) and incubated for 24 hours, 48 hours and 72 hours at 37 ° C. in the presence of 5% carbon dioxide gas.
[344] The cultured cells were stained with Trypan Blue to count the number of viable and dead cells, resulting in a significant reduction in viable cell number and cell viability in the 10 μM GM addition zone compared to the control water addition zone. In addition, the cultured cells were observed under an optical microscope to confirm the condensation of the nucleus, the shrinking of the cells, and the formation of apoptotic bodies. Furthermore, DNA was extracted from the cells and subjected to agarose gel electrophoresis. As a result, fragmentation of DNA was recognized in chromatin units. In addition, these phenomena were not observed in the control water addition zone.
[345] The results are shown in FIG. 29. In other words, Fig. 29 is a diagram showing the relationship between the incubation time when 10 μM is added to the culture medium of HL-60 and the number of living cells in the culture solution, where the horizontal axis represents the culture time (hours) and the vertical axis represents the number of live cells in the culture solution It represents a ⁽⁵ × lO gae / 5 ㎖). In Fig. 29, Indicates a GM addition of 10 µM, and Indicates a control example of water addition.
[346] (5) 10 μl of 4.12, 12.3, 37.0, 111, 333 or 1000 μM GD aqueous solution, or 10 μl of water as a control was added to each well of a 96 well microtiter plate. Suspend HL-60 (ATCC CCL-240) in RPMI1640 medium containing 10% fetal bovine serum to 5 × 10 ⁴ / ml, pour 90 μl into each well of the microtiter plate, and add 5% CO ₂ hours were incubated at 37 degreeC for 48 hours. 10 μl of 5 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT; manufactured by Sigma) phosphate buffered saline solution is added to After further incubation, the growth state of the cells was observed under a microscope. Further, 100 µl of 0.04 NHCl-containing 2-propanol was added and stirred well, and the absorbance at 590 nm was measured.
[347] As a result, cell proliferation was not seen in the 37.0 μM GD addition zone (final concentration 3.70 μM). Thus, it was evident that GD completely inhibited the proliferation of HL-60 cells at a concentration of 3.70 μm. In addition, apoptotic bodies were formed in the cells at each cell proliferation inhibitory concentration.
[348] (6) Cyclopentenone became 55 mM and L-cysteine hydrochloride became 80 mM in phosphate buffered saline (pH 7.2), and the mixture was reacted at 37 ° C. for 15 minutes at pH 7.2. A portion of the reaction solution was analyzed by reverse phase HPLC using a TSK gel ODS-80Ts (4.6 mm × 250 mm, manufactured by Tosoh Corporation) column with 0.1% trifluoroacetic acid aqueous solution as a mobile phase and a flow rate of 1 ml per minute. As a result, cyclopentenone was not detected. This reaction solution was a solution containing 55 mM of CD.
[349] As in Example 5- (4), cancer cell proliferation inhibitory activity of 100, 200 or 400 μM GD or CD aqueous solution was measured, respectively. However, the degree of cell proliferation was expressed by the ratio (%) of absorbance at 590 nm of the sample addition region: 590 nm of the hydrophobic content of the control example.
[350] The results are shown in Table 2.
[351] GDCD 40 μM2.15.3 20 μM5.856.2 10 μM47.289.0
[352] As shown in Table 2, GD and CD showed cancer cell proliferation inhibitory activity. In addition, apoptotic bodies were formed in the cells at each cell proliferation inhibitory concentration.
[353] As described above, as shown in Example 5, each compound exhibited cancer cell proliferation inhibitory activity and apoptosis-inducing activity. In addition, various diastereomers showed the same effect.
[354] Example 6
[355] GM was diluted to predetermined concentration using physiological saline, and the following test was done.
[356] (1) Meth A cells (2 × 10 ⁶ cells / mouse) were injected subcutaneously into the abdomen of 8-week old female BALB / c mice (body weight about 20 g). Thereafter, GM (5 mg / kg / day) was subcutaneously injected at the same spot continuously for 5 days. The control group was injected subcutaneously with the same physiological saline solution. Two weeks later, the cancer tissue formed in the abdomen of the mouse was removed and its weight was measured. Compared with the control group, the cancer proliferation inhibitory effect was remarkably recognized in the GM administration group. Similar results were obtained for CM, CD, and GD.
[357] (2) Mouse Leukemia P-388 (1.1 × 10 ⁶ cells / mouse) was injected intraperitoneally into 7-week-old female DBA / 2 mice (body weight about 20 g). Thereafter, GM, CM, GD or CD (10 mg / kg / day) was intraperitoneally injected for 5 consecutive days. In contrast, the control group was intraperitoneally injected with physiological saline. In two groups of 8 mice each, survival numbers, average days of survival, and life span of mice were calculated. In each sample administration group, the mean survival days were extended compared to the control group, and a noticeable proliferation effect was recognized. In addition, the system using the Sarcoma-180 / ICR mice, IMC / CDF1 mice and EAC / DDY mice, respectively showed the same life-saving effect.
[358] As described above, as shown in Example 6, GM, CM, GD or CD showed an anticancer effect. Similar results were also obtained for each diastereomer.
[359] Example 7
[360] Bacillus subtilis IFO3021 was incubated overnight in susceptible bouillon medium (manufactured by Nissisa Corporation) (seed culture). The absorbance at 600 nm was measured and the number of viable cells was calculated from a calibration curve indicating the relationship between the number of viable cells produced in advance and the absorbance at 600 nm. Seed cultures were diluted to 1 × 10 ⁶ / ml in fresh susceptible bouillon media and 180 μl aliquots were added to each well of a 96 well microtiter plate. 20 [mu] l of 4 mg / ml and 2 mg / ml aqueous solutions of GM or water were added to each well, followed by incubation at 37 [deg.] C. overnight (main culture). On the other hand, a part of the seed culture solution was diluted with sterile water, applied to a sensitive bouillon agar plate medium and incubated overnight at 37 ° C, and the colonies were counted to determine the correct viable count.
[361] The culture solution of each well was diluted with sterile water and applied to the susceptible bouillon agar plate medium and incubated overnight at 37 ° C., and the colonies were counted to measure viable cell count.
[362] As a result, the viable cell count in the 4 mg / ml GM addition zone (final concentration 400 μg / ml) was 4.4 × 10 ⁷ cells / ml, compared with 1.3 × 10 ⁸ cells / ml in the water addition zone. Therefore, GM showed a growth inhibitory effect against the test bacteria at 400 µg / ml. Similar results were obtained for CM, GD, CD or their diastereomers and GM diastereomers. In addition, these compounds showed the same antimicrobial activity against other bacteria.
[363] Example 8
[364] (1) Intraperitoneal administration of LPS (liposaccharide: manufactured by Sigma Co., Ltd.) using 8-week-old female CDF1-based mice (prepared for 1 week with a 20-week-old mouse purchased from Japan SL Corporation) (10 μg / mouse) and an endotoxin shock model was made. GM was orally administered at doses of 100 and 1000 mg / kg, respectively, 30 minutes prior to LPS administration. One hour after LPS administration, the blood was collected from mice to separate serum, and the amount of tumor necrosis factor-α in serum was measured by a commercial ELISA kit (manufactured by Endogen). The results are shown in Table 3 below. That is, tumor necrosis factor production was significantly suppressed in the GM 1000 mg / kg administration group compared to the control group administered with distilled water.
[365] groupDose mg / kgMouse numbersSerum tumor necrosis factor (ng / ml mean ± SE Control 42.32 ± 0.15 GM dosage00051.16 ± 0.26 10052.22 ± 0.26
[366] (2) 2 ml of paraffin oil (manufactured by Cosmo Bio Co., Ltd.) was administered intraperitoneally of 8-week-old female CDF1-based mice to induce peritoneal macrophages (MØ). One week after the administration of paraffin oil, 4 ml of RPMI-1640 medium (manufactured by Gibco) was injected into the abdominal cavity of the mouse, massaged well, and the cells were recovered to obtain the abdominal cells.
[367] Peritoneal cells were washed twice in RPMI-1640 medium and then suspended in RPMI-1640 medium containing 10% fetal bovine serum (FCS: manufactured by Hycron) to bring the cell concentration to 1 × 10 ⁶ cells / ml. Adjusted. 1 ml of the prepared cell solution was seeded in a 24-well plate, and incubated for 2 hours in a 37 ° C CO ₂ incubator. After incubation, the non-adhesive cells contained in the supernatant were removed, and the adherent cells were used as intraperitoneal MØ.
[368] Add 800 μl of RPMI-1640 medium containing 10% FCS to each well of the plate, then add 100 μl of GM 1, 10, 100, 1000 μM dissolved in physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) 37 Incubated for 1 hour in a C0 ₂ CO ₂ incubator.
[369] After incubation, 100 µl of 100 ng / ml LPS (manufactured by Sigma Co., Ltd.) was added thereto and incubated for 24 hours. After the completion of the culture, the culture supernatant was recovered and the amount of generated tumor necrosis factor -α was quantified using a commercial ELISA kit (manufactured by Endzen).
[370] The results are shown in FIG. That is, FIG. 30 is a diagram showing the relationship between the GM amount and the tumor necrosis factor production amount, the vertical axis represents the tumor necrosis factor production amount (pg / ml), and the horizontal axis represents the GM concentration (μM). The bar graph also shows a control of GM no addition.
[371] GM significantly inhibited tumor necrosis factor production from LPS-induced mouse peritoneal macrophages at concentrations above 10 μM.
[372] As mentioned above, as shown in Example 8, GM showed a tumor necrosis factor production inhibitory effect. In addition, the same results were obtained for CM, GD, CD, each of these diastereomers, and each of the diastereomers of GM.
[373] Example 9
[374] A carrageenan-induced foot edema model, a chronic articular rheumatoid animal model, was constructed using a 6-week-old female Lewis rat (5 weeks old, approximately 130 g of body weight purchased from Seak-Yoshito Misa, 1 week preliminary). The test drug was evaluated.
[375] Rats fasted 18 hours before the start of the experiment were orally administered GM prepared to 10, 100 mg / ml in distilled water (manufactured by Otsuka Pharmaceutical Co., Ltd.) at a dose of 10 ml / kg.
[376] 0.5 hours after administration of the test drug, carrageenan (manufactured by Otsuka Pharmaceutical Co., Ltd.) suspended in physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) and adjusted to a concentration of 1% was injected into the foot foot of the right foot at 100 μl / rat, and foot foot edema was obtained. Caused. Three hours after the injection of carrageenan, the volume of the rat's right foot was measured with a volumetric device (manufactured by Ugobasil). In addition, the measured value calculated and displayed the increase rate from the right foot volume of each rat measured before carrageenan administration.
[377] The results are shown in FIG. That is, FIG. 31 is a diagram showing the relationship between the GM amount and the foot edema increase rate, the vertical axis represents the increase rate (%), and the horizontal axis represents the GM dose (mg / kg).
[378] GM showed significant inhibitory activity from doses of 100 mg / kg or more.
[379] In addition, the same results were obtained for CM, GD, CD or their diastereomers and GM diastereomers.
[380] Example 10
[381] The NO production inhibitory activity and cytotoxicity inhibitory activity of GM were measured using mouse macrophage line RAW264.7 cells (ATCC TIB 71) and LPS as follows.
[382] 2 mM L-glutamine (trade name manufactured by Life Technology Oriental, Inc.) without phenol red containing 5 ml of 10% fetal bovine serum (manufactured by Gibcosa) containing 1.5 × 10 ⁶ RAW264.7 cells -149) Dulbecco modified Eagle's medium (trade name, manufactured by Life Technologies Oriental, 11054-020) was incubated for 12 hours at 37 ° C in the presence of 5% carbon dioxide in a 6-well tissue culture plate. 50 μl of 50 μg / ml LPS (trade name, manufactured by Sigma, L-2012) was added, and 50 μl of 250 μM GM or 100 μM GM was added to each well, followed by further incubation for 12 hours. The measurement of NO ₂ ⁻ and live cell number generated by oxidation in the medium was performed. In addition, the zone without addition of LPS and the zone without addition of GM were set as controls.
[383] For measurement of NO ₂ ⁻ , 100 μl of the culture supernatant was separated from each well, and 10 μl of 50 μg / ml 2,3-diaminonaphthalene (trade name, 341-07021, manufactured by Dojindo Chemical Research Institute, Inc.) (0.62). N hydrochloric acid solution) was added and left at room temperature for 15 minutes, and 5 µl of 2.8 N aqueous sodium hydroxide solution was added to the resulting naphthalenetriazole fluorescence to obtain Titertec Fluoroscan II (available from Dainippon Pharmaceutical Co., Ltd.). Fluorescence measurement was carried out using an excitation wavelength of 355 nm and a measurement wavelength of 460 nm. The experiments were carried out a total of two times, and the control value of the LPS non-addition was subtracted from this average value and compared with each zone relative to the value of the LPS addition zone.
[384] As a result, GM suppressed NO production induced in RAW264.7 cells by LPS, and further suppressed cell damage to RAW264.7 cells by LPS.
[385] The results are shown in FIGS. 32 and 33. Fig. 32 shows the relationship between the GM concentration and the NO ₂ ⁻ concentration in the culture medium, and the vertical axis represents the relative value (%) of the NO ₂ ⁻ concentration. Figure 33 is a graph showing the relationship between GM presence bottoms can the incubation time and the production cell, the horizontal axis represents incubation time (hour) and the vertical axis represents the raw number of cells (× 10 ^5/5 ㎖) contained in the 5 ㎖ culture. In Fig. 33, Mark indicates no LPS addition, o mark indicates LPS addition, o mark indicates 2.5 μM GM addition, △ indicates 2.5 μM GM + LPS addition, and ■ indicates 1 μM GM + LPS addition. Display.
[386] As mentioned above, GM showed the inhibitory effect of NO production. Similar results were also obtained for each diastereomer of CM, GD, CD or each of these diastereomers and GM.
[387] Example 11
[388] 10% fetal bovine serum (FBS, product name manufactured by Gibcos, 26140-) was obtained from DSEK cells (cell lines preserved in Saitama Medical University Second Medical Center), a fibroblast line established from the synovial membrane of patients with chronic rheumatoid rheumatism. 079) incubated with Iscob modified Dulbecco's medium (IMDM, trade name manufactured by Gibco, 12440-053) in the presence of 5% carbon dioxide gas until fused at 37 ° C, and trypsin-EDTA ( Cells were collected by peeling under the trade name 25300-054 manufactured by Gibco Corporation. The cells were suspended in the medium to 25000 cells / ml, and 100 µl was poured into each well of a 96 well microtiter plate. After 5 days of culture, the medium was discarded at the time of almost fusion and the medium containing 2.5, 5, 10, 20 or 30 μM GM was added. After 24 hours, 48 hours, or 72 hours of incubation, 10 μl of premixed WST-1 (trade name, manufactured by Takara Shuzo, MK400) was added and reacted at 37 ° C. for 4 hours and absorbance at 450 nm (A ₄₅₀ ). Was obtained by subtracting the absorbance at 650 nm (A ₆₅₀ ) at.
[389] The results are shown in Table 4.
[390] Concentration (μM)A ₄₅₀ -A ₆₅₀24 hours48 hours 00.8461.270 2.50.7681.133 50.6210.942 100.4200.486 200.2380.185 300.2410.196
[391] Cell proliferation was inhibited in the region containing 5 μM or more of GM for 24 hours and 48 hours of culture, and cell death was observed in the region in which 10 μM of GM was added. Almost no live cells were observed in the Old Testament containing more than 20 μM of GM.
[392] As described above, GM showed apoptosis-inducing action and proliferation inhibitory action on synovial cells. Similar results were also obtained for each diastereomer of CM, GD, CD or each of these diastereomers and GM.
[393] Example 12
[394] (1) RPMI1640 medium containing 10% fetal bovine serum (FCS, manufactured by BioWittaker) containing Jurkat cells (ATCC TIB-152) and Molt-3 cells (ATCC CRL-1552), human T-cell leukemia cell lines (Manufactured by Gibco BRL), incubated at 37 ° C. in the presence of 5% CO ₂ and suspended the cells at 5 × 10 ⁵ cells / ml in the medium containing GM of O, 5, 10 or 20 μM. And incubated for 24 hours. Cell proliferation was performed by MTT method (Mosmann et al., J. Immunol. Methods, Vol. 65, pp. 55-63 (1983)], measured cell proliferation with absorbance at 560 nm.
[395] As a result, the cell proliferation was inhibited in both cell lines by about 50% in the 10 μM GM addition zone and 75% or more in the 20 μM GM addition zone as compared to the control addition. The addition of GM of 5 μM or less did not significantly affect the proliferation of cells.
[396] As mentioned above, GM suppressed the proliferation of Jurkat cells and Molt-3 cells which are T cell leukemia cell lines in a concentration dependent manner.
[397] The results are shown in FIGS. 34 and 35. That is, Figure 34 is a diagram showing the effect of GM on the proliferation of Jurkat cells, Figure 35 is a diagram showing the effect of GM on the proliferation of Molt-3 cells. 34 and 35, the horizontal axis represents GM concentration (μM), and the vertical axis represents absorbance at 560 nm.
[398] (2) The effect of GM on pars antigen expression (production induction) in Jurkat cells and Molt-3 cells was measured as follows. RPMI1640 medium containing 10% FCS containing 0, 1, 5, 10 or 20 μM of cyclopentenone or GM was used to prepare 5 × 10 ⁵ / ml Jurkat cells or Molt-3 cells at 37 ° C. in the presence of 5% CO _2. Incubated for 24 hours in Munker, R. [Ann. Hematol. Vol. 70, pp. 15-17 (1995)] was subjected to two-stage immunostaining using an anti-pas antibody (produced by Boehringer Ingelheim).
[399] Fluorescence intensities of 1 × 10 ⁴ stained cells were measured using a flow cytometer (Orthocytron: manufactured by Ortho Diagnotic Systems), and the ratio was calculated using cells expressing a certain level of fluorescence intensity as Pas antigen expressing cells. .
[400] As a result, in both cell lines, when GM was added, the ratio of pars antigen-expressing cells increased in a concentration-dependent manner in the range of 1 to 10 µM, and was approximately the same as 10 µM when 20 µM was added.
[401] The results are shown in FIGS. 36 and 37. That is, FIG. 36 shows Molt-3 and FIG. 37 shows the expression of the pas antigen in Jurkat cells. 36 and 37, the horizontal axis represents the concentration of GM (μM), and the vertical axis represents the percentage of pars antigen-expressing cells (%), and the phage antigen production-inducing action by GM was recognized.
[402] (3) 10 μM of GM was added to incubate Molt-3 cells for 1, 3, 6, 12 or 24 hours under the same conditions as in Example 12- (2), and in the same manner as in Example 12- (2). The percentage of Pas antigen expressing cells was measured.
[403] As a result, when 10 μM GM was added, the percentage of pars antigen-expressing cells rose after 12 hours of culture and further increased at 24 hours.
[404] The results are shown in FIG. That is, FIG. 38 is a diagram showing the change in the ratio of Pas antigen-expressing cells when cultured by adding 10 μM GM to Molt-3 cells, wherein the horizontal axis represents the culture time (hours), and the vertical axis represents the percentage of Pas antigen-expressing cells. (%).
[405] As described above, as described in Example 12, pars antigen production-inducing activity by GM was confirmed. Similar results were also obtained for each diastereomer of CM, GD, CD or each of these diastereomers and GM.
[406] Example 13
[407] By purchasing the KK-A ^y mice (male, 4 weeks old) from Japan creatinine captured, and then bred until 10 weeks of age, by administering GM 21 ilgan oral, and reviewed glucose, the influence to have on serum insulin and lipid. GM used doses of 30, 100 and 300 mg / kg. Blood glucose levels were lowered in each group of GM administration (FIG. 39). Serum insulin levels were lowered in each group of GM administration (FIG. 40). For serum lipids, serum total cholesterol was lowered in the GM-administered group (FIG. 41), serum triglycerides were lowered in the GM-administered group (FIG. 42), and serum free fatty acids were lowered in the GM-administered group (FIG. 43).
[408] That is, FIG. 39 is a diagram showing the relationship between the GM dose and the blood glucose level, in which the vertical axis represents serum glucose level (mg / dl), and the horizontal axis represents GM dose (mg / kg). Fig. 40 is a diagram showing the relationship between the GM dose and the serum insulin level, in which the vertical axis represents the serum insulin level (μU / ml), and the horizontal axis represents the GM dosage (mg / kg). Fig. 41 is a diagram showing the relationship between GM dose and serum total cholesterol level, in which the vertical axis represents serum total cholesterol level (mg / dl), and the horizontal axis represents GM dose (mg / kg). Fig. 42 is a diagram showing the relationship between GM dosage and serum triglyceride value, wherein the vertical axis represents serum triglyceride value (mg / dl), and the horizontal axis represents GM dosage (mg / kg). Fig. 43 shows the relationship between the GM dose and the serum free fatty acid value, wherein the vertical axis represents the serum free fatty acid value (μEq / L) and the horizontal axis represents the GM dose (mg / kg). In the figure, * means significant difference p <0.05 for the GM no-administered group and ** means p <0.01 by multiple comparison tests of turkeys.
[409] Animals were divided into four groups, each group was 10 animals, and four groups were administered with physiological saline (5 ml / kg) or GM of 30, 100 and 300 mg / 5 ml / kg. Each test substance was orally administered once a day for 21 days, and on the final administration day, anesthetized with ether 4 hours after the test substance administration, and blood was collected from the lower abdominal vein. Serum insulin was measured by enzyme-immunoassay (commercial kit: Glyzem insulin-EIATEST, manufactured by Wako Pure Chemical Industries, Ltd.). Sugars, triglycerides and free fatty acids in serum were measured using an automatic analyzer (type 7070: manufactured by Hitachi, Ltd.) by the hexonase-G6PDH method, the GPODAOS method, and the ACS ACD method, respectively. Serum total cholesterol value was measured using the automatic analyzer (type 7070: manufactured by Hitachi, Ltd.) by the cholesterol-oxidase-DAOS method.
[410] From the above results, it was confirmed that GM has an effect of lowering blood sugar, insulin and lipids. Similar results were also obtained for each diastereomer of CM, GD, CD or each of these diastereomers and GM.
[411] Example 14
[412] (1) ⁵ ml of RPMI1640 medium containing 10% fetal bovine serum containing 2 x 10 ⁵ cells / ml HL-60 (ATCC CCL-240) was added to each well of a 6 well plate, 37 ° C., 5% CO ₂ After incubation for 24 hours in the presence of GM, GM was added so that the final concentration was 0, 0.5, 1.0, 3.O, 5.0, 10.0, 20.0 μM, followed by further 8 hours of incubation.
[413] After the incubation, the cell number was measured, the cells were collected by centrifugation, washed with PBS, and GM treated cells were prepared. Furthermore, after heat-processing at 45 degreeC for 10 minutes, the same cultured cell was also prepared.
[414] Using these treated cells, SDS-PAGE was carried out according to the method described in “Molecular Cloning” (Cold Spring Harb or Laboratory Press (1989)) The treated cells were 2.5 × 10 ⁶ cells / ml. Suspend in SDS-PAGE sample buffer as much as possible, treat this cell suspension at 100 ° C. for 10 minutes, and then apply 5 μl each to two SDS-PAGE gels (5% accumulation gel, 10% separation gel), followed by electrophoresis. One gel was dyed with Coomassie Brilliant Blue R250, and the other gel was a polyvinylidene difluoride transfer membrane (ImmobilonTM: manufactured by Millipore). # IPVH000-10] The membrane was blocked with Block Ace (Dainippon Co., Ltd. Cat. # UK-B25) overnight at 4 ° C.
[415] Monoclonal antibody HSP 72/73 (Ab-1), which specifically reacts with a heat shock protein of 70 kDa induced by the blocked membrane [trade name Cat. # HSP01], followed by washing with TBS containing 0.05% Tween20 and further washing with TBS. Subsequently, peroxidase conjugated secondary antibody HRP-Rabbit Anti-Mouse IgG (H + L) (trade name Cat. Manufactured by ZYMED Laboratories, Inc.). # 61-6520] was washed in the same manner as the previous operation. Thus, the membranes reacted with the primary antibody and the secondary antibody were RENAISSANCE ^™ [chemiluminor reagent manufactured by Dupont NEN Corporation. # NEL-100] was reacted and then exposed to x-ray film to detect the induction of heat shock protein of 70 kDa.
[416] As a result, the induction of a heat shock protein of 70 kDa was recognized by the addition of GM. The strengths and weaknesses of the induction are shown in Table 5. In Table 5, + indicates the strength of induction, and more + means that induction is stronger. In addition,-means that induction is not recognized, ± means that induction is slightly.
[417] Processed cellsInduction of Heat Shock Protein 10 minutes heat treatment at 45 ℃+++ 0 μM GM- 0.5 μM GM± 1.0 μM GM± 3.0 μM GM+ 5.0 μM GM+ 10 μM GM++ 20 μM GM++
[418] As shown in Table 5, it is evident that GM has inducibility of HSP70.
[419] (2) GM was added to the cultured cells so that the final concentration was 0, 10, 20, 30, 40, 50 μM, and the expression of HSP70 was measured by the method described in Example 14- (1).
[420] As a result, the induction of the heat shock protein of 70 kDa by CM was confirmed. The strengths and weaknesses of the induction are shown in Table 6. In Table 6, + indicates the strength of induction, and the more +, the stronger the induction. In addition,-means that induction is not recognized, ± means that induction is slightly.
[421] Processed cellsInduction of Heat Shock Protein 10 minutes heat treatment at 45 ℃+++ 0 μM CM- 5 μM CM- 10 μM CM+ 20 μM CM++ 30 μM CM++ 40 μM CM+ 50 μM CM+
[422] As described above, in Example 14, GM and CM showed heat shock protein induction. Similar results were also obtained for each diastereomer of GD, CD, or each of these diastereomers, GM, and CM.
[423] Example 15
[424] CEM-SS cells infected with 2 × 10 ⁵ / ml CEM-SS cells (ATCC CCL-119) and HIV-1 _IIIB (more than 90% of cells are infected with HIV-1 _IIIB : abbreviated as CEM-3B) 0, 4, 8, 16 μM of GM was added and cultured for 1 day, and cell viability was calculated by counting live and dead cells. As a result, when GM was added so as to be 0, 4, and 8 µM, viability of CEM-SS cells did not decrease, whereas CEM-3B cells significantly reduced survival depending on the concentration of GM added. Moreover, the survival rate of CEM-SS cells also decreased with the addition of 16 µM, but the survival rate of CEM-3B cells significantly decreased. In other words, GM showed anti-HIV action.
[425] The results are shown in FIG. 44. That is, Fig. 44 is a diagram showing the relationship between the added GM concentration and the cell viability, where the horizontal axis represents the GM concentration (μM) added, and the vertical axis represents the cell survival rate (%) after culturing for one day. The □ mark is the result when using CEM-SS, and the ● mark is the result when using CEM-3B cells.
[426] (2) The concentration of p24 antigen contained in the culture supernatant after culturing CEM-3B cells of Example 15- (1) for 1 day was measured. As a result, the concentration of p24 antigen decreased with the added GM concentration, and anti-HIV activity was recognized. The results are shown in FIG. That is, FIG. 45 is a diagram showing the relationship between the added GM concentration and the p24 production amount in the culture supernatant, the horizontal axis represents GM addition amount (μM), and the vertical axis represents p24 production amount (ng / ml).
[427] As described above, as shown in Example 15, GM selectively showed killer cell effects on HIV-infected cells and antiviral action on HIV. In addition, the same results were obtained for CM, GD, CD, each of these diastereomers, and each of the diastereomers of GM.
[428] Example 16
[429] (1) Control plasmid pcD2-Y [Mol. Cel1. Biol. 7, pp. 2745-2752 (1987)] and plasmid pcD2-16E7 [Jpn. J. Cancer Res. Vol. 82, pp. 1340-1343 (1991)] were transformed with E. coli HB101, incubated with L-broth medium, plasmid was extracted from the collected cells, and the cesium chloride density gradient was obtained. Purification by ultracentrifugation yielded a vector plasmid for gene introduction.
[430] NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% calf serum at 37 ° C., 5% CO ₂ conditions.
[431] 10 μg of the purified plasmid was introduced into NIH3T3 cells using cationic liposomes (TransIT LT-1, manufactured by Takara Shuzo Co., Ltd.), and cells were treated at 37 ° C., 5% CO ₂ , and 0.4 mg / g of G418 (GIBC0). Two weeks were selected from Dulbecco's modified Eagle's medium containing 10% calf serum, and the colonies obtained were cloned to obtain control vectors into which NIH3T3 cells were introduced, and NIH3T3 cells cancerized by HPV16 type E7. Established.
[432] Cell lines into which the control vector was introduced were NIH3T3 / Y-1, NIH3T3 / Y-2, NIH3T3 / Y-3, NIH3T3 / Y-4, NIH3T3 / Y-5, NIH3T3 / Y-6, NIH3T3 / Y-7, NIH3T3 / Y-8 and NIH3T3 / Y-9.
[433] E7 introduced cell lines were NIH3T3 / E7-1, NIH3T3 / E7-2, NIH3T3 / E7-3, NIH3T3 / E7-4, NIH3T3 / E7-5, NIH3T3 / E7-6, NIH3T3 / E7-7, NIH3T3 / E7-8 and NIH3T3 / E7-9.
[434] (2) The NIH3T3 cells, the cell line into which the control vector was introduced, and the cell line into which the E7 were introduced were increased to 50 to 70% in Dulbecco's modified Eagle's medium containing 10% calf serum with a 100 mm tissue culture plate and washed with PBS. The cells were then detached with 0.25% trypsin-EDTA solution and suspended in 5 ml of Dulbecco's modified Eagle's medium containing 10% calf serum.
[435] A portion of the suspension was taken and cell density was calculated with a Neubauer-type hemocytometer. Based on the numbers obtained, the cells were diluted with Dulbecco's modified Eagle's medium containing 10% calf serum to reach 200 cells / plate in a 60 mm diameter tissue culture plate, and culture was started in 3 ml of medium. After 24 hours from the start of the culture, GM was added to 5 μM. After 24 hours, GM was added to 5 μM in exchange for fresh medium.
[436] Thereafter, the medium was changed every 2-3 days, and GM was added to 5 μM. As a control experimental zone, plates without GM were prepared and cultured identically. The culture was performed three times in succession. After 9 days of incubation, colonies were stained with Gimja solution (GIBCO) by fixing with methanol.
[437] Moreover, it evaluated using NIH3T3, NIH3T3 / Y-1, and NIH3T3 / E7-2.
[438] Table 7 shows the results of counting the stained colonies. The cells into which E7 was introduced had a higher sensitivity to GM than control cells, and GM selectively acted on cancer gene transformed cells. Similar results were also obtained for each diastereomer of CM, GD, CD or each of these diastereomers and GM.
[439] cellNumber of colonies (mean ± SD) contrastGM treatment NIH3T391.7 ± 11.979.0 ± 2.6 NIH3T3 / Y-183.3 ± 8.472.0 ± 9.5 NIH3T3 / E7-267.3 ± 3.213.3 ± 3.2
[440] Example 17
[441] (1) 2 μl of phase isomerase II (trade name, manufactured by TopoGEN, 2 units / μl), 10-fold concentration buffer [0.5 M Tris-HCl (pH 8.0), 1.2 M KCl, 0.1 M ₂ μl of MgCl ₂ , 5 mM adenosine triphosphate, 5 mM dithiothreitol], 2 μl of 0.1% bovine serum albumin (manufactured by Takara Shuzo), 11 μl of distilled water and control distilled water or samples (50, 100, 200, 2 µl of 500, 1000 or 2500 µM GM) was mixed, and 1 µl of 0.25 µg / µl pBR322 DNA (produced by Takara Shuzo Co., Ltd.) was added and reacted at 37 ° C. After reacting for 30 minutes, 2 µl of an aqueous solution of 1% sodium dodecyl sulfate, 50% glycerol and 0.02% bromophenol blue was added to stop the reaction.
[442] To a 1% agar gel made using agar L03 (manufactured by Takara Shuzo) and TAE buffer [40 mM Tris, 5 mM sodium acetate, 1 mM disodium ethylenediaminetetraacetate (EDTA), adjusted to pH 7.8 with acetic acid]. 20 μl of the reaction solution was added and electrophoresis was performed in TAE buffer. After electrophoresis, the gel was immersed in an aqueous solution of 1 μg / ml ethidium bromide and irradiated with ultraviolet light to observe the DNA electrophoresis pattern. In addition, in the hydro addition control, DNA is completely changed from the ultra-helix to the relaxed form, but when the phase isomerase II activity is inhibited, the change from the ultra-helix to the relaxed form is partially or completely inhibited.
[443] The results are shown in Table 8.
[444] Concentration in the reaction solution (μM)Inhibitory activity 0- 5- 10+ 20++ 50++ 100+++ 250+++
[445] In the hydrogenated control example, the DNA was completely changed from the ultra helix to the relaxed form, but when the GM concentration was 10 μM or more, the change of the DNA from the helix to the relaxed form was partially or completely inhibited, and the GM phase isomerase II Inhibitory activity was confirmed. In Table 8,-completely changes from the super spiral to the relaxed, + represents a moderate change, + + represents the state where most super spirals remain, and + + + indicates that the super spiral is not reduced at all.
[446] (2) GM phase isomerase I inhibitory activity was measured in the same manner as in Example 17- (1). However, instead of phase isomerase II, phase isomerase I (manufactured by Topogen, 0.01 units / μl), 100 mM Tris-HCl (pH 7.9), 10 mM EDTA, 1 mM as a 10-fold concentration buffer Spermidine, 50% glycerol was used. In addition, GM was added as a sample so that the final concentration might be 1 mM.
[447] As a result, phase isomerase I was inhibited at 1 mM GM.
[448] As described above, the phase isomerase II, which is temporarily expressed only in the dividing phase but is expressed highly through the whole cell cycle due to cancer, and the phase isomerase I whose expression amount and activity are increased by the cancer. , GM showed inhibitory activity. In addition, the same results were obtained for CM, GD, CD, each of these diastereomers, and each of the diastereomers of GM.
[449] Example 18
[450] C57BL / 6 mice (female, 5 weeks old, weighing about 20 g) were purchased from Japanese SLC, pre-cultured for 1 week, and used for the experiment. A positive red blood cell (Shimizu-jiken gyro), an antigen causing a delayed-type hypersensitivity reaction, was washed three times with physiological saline (Otsuka Pharmaceutical) and prepared at 1 × 10 ⁹ cells / ml, and 200 μl was injected into the abdominal cavity of the mouse. The antigen was sensitized by injection.
[451] After 5 days from sensitization, 40 μl of the same prepared antigen was injected into the right foot foot to cause antigen and cause foot edema. GM was administered 30 mg / kg or 300 mg / kg intraperitoneally once a day for 3 days to 5 mice of group 1 once a day from antigen sensitization day.
[452] Two days after antigen induction, the volume of the right foot of the mouse was measured by a foot edema measuring device (Ugobasil Co., Ltd.), which was used as an index of delayed type hypersensitivity reaction. The measurement was expressed by calculating the increase rate from the volume of the right foot of the mouse measured before antigen induction.
[453] The results are shown in FIG. 46. That is, FIG. 46 is a diagram showing the delayed-type hypersensitivity action of GM, with the vertical axis representing the increase rate (%) and the horizontal axis representing the GM dose (mg / kg).
[454] GM showed a significant delayed-type hypersensitivity inhibitory effect at the dose of 30, 300 mg / kg. In addition, the same results were obtained for CM, GD, CD, each of these diastereomers, and each of the diastereomers of GM.
[455] Example 19
[456] To five five-week-old Wister male rats (SLC, Japan), 100 μl of 0.01% physiological saline solution of egg white albumin (Sigma) and Alum [brand name: Eject allum ( Imject Alum); Pierce Corporation] 100 µl was sensitized by intraperitoneal administration, and blood was collected from the abdominal vein 14 days later.
[457] The collected blood was centrifuged (2000 rpm, 5 minutes), plasma was separated, and antigen-specific IgE amount was measured by rat manual skin anaphylaxis (PCA) reaction for 48 hours.
[458] In other words, plasma doubling dilution ranged from 4 to 64 times with physiological saline and injected 0.1 ml into the back of the back of the 7-week-old Wister male rats with hair. 48 hours after intradermal injection, 1 ml of a mixture of 0.05% egg white albumin and 0.5% Evans blue (manufactured by Nacalai Tesque) was injected from the tail vein. Thirty minutes after injection into the tail vein, rats were headed and bleeded to observe blue spots appearing behind their backs, spots of 5 mm or more in diameter were made positive, and the highest dilution was expressed as the appropriate IgE concentration.
[459] The GM-administered group received intraperitoneal administration of 3 mg / kg or 30 mg / kg of GM once daily for 3 days from the antigen sensitization day. In the control group, distilled water was administered intraperitoneally as well. The results are shown in Table 9.
[460] IgE titer Control64 GM 3 mg / kg / day32 GM 30 mg / kg / day8
[461] Elevation of antigen-specific IgE by egg white albumin sensitization was inhibited by administration of GM in a dose dependent manner. The same IgE production inhibitory activity was recognized also for CM, GD, CD, each of these diastereomers, and each of the diastereomers of GM.
[462] Example 20
[463] Injection
[464] (1) An injection was made by adding 1% of CM to physiological saline solution.
[465] (2) GM and glycyrrhizic acid were added to physiological saline (same as above) at 0.5% and 0.1% concentrations to make injections.
[466] Example 21
[467] refine
[468] (1) A tablet containing 100 mg of CD and an appropriate amount of microcrystalline cellulose was prepared and coated with sugar to prepare a tablet.
[469] (2) A tablet containing 0.1 mg of GD, 10 mg of dipotassium glycylisate and a small amount of microcrystalline cellulose was prepared and coated with sugar to make a tablet.
[470] Example 22
[471] Ointment
[472] GM 1 g
[473] 99 g of absorption ointment (Japanese Pharmacy Defense)
[474] GM was first kneaded sufficiently with a small amount of absorbent ointment, and then the remaining absorbent ointment was gradually added and kneaded until homogeneous to form an ointment.
[475] This ointment is applied to the affected area 4-5 times a day.
[476] Effects of the Invention
[477] Anti-cancer action, cancer cell proliferation inhibiting action, cancer cell differentiation inducing action, apoptosis inducing action, antibacterial action, antiviral action, liver function improving action, tumor necrosis factor production inhibitory action, NO production inhibitory action, anti-rheumatic effect etc. Provided are a compound of the present invention having various physiological activities, an optically active substance thereof, and a salt thereof, and a medicament containing at least one or more compounds selected from these compounds as an active ingredient. The medicament is useful as a therapeutic or prophylactic agent for diseases exhibiting susceptibility to these compounds, and in particular, biological defense agents such as antiallergic agents, antirheumatic agents, diabetes treatment agents, anticancer agents, anti-pathogenic microorganisms such as antiviral agents, antimicrobials, immunomodulators, etc. It is useful as.
[478] In addition, according to the present invention, an appropriate amount of a compound of the present invention having a physiological activity, an optically active substance thereof, or a salt thereof can be contained in a food or a beverage. According to the present invention, according to the various physiological activities, anticancer action, differentiation inducing action, abnormal cell proliferation inhibiting action, apoptosis-inducing action, antiviral action, antibacterial action, liver function improving action, immunomodulatory action and the like of these compounds, The food or beverage provided is a health food or beverage having a homeostatis function of living body such as carcinogenic effect, anticancer effect, viral disease prevention effect, antibacterial effect, and apoptosis inducing effect, and according to the present invention, Contained foods or beverages are provided that are functional substances useful for maintaining health.

权利要求:
Claims (28)
[1" claim-type="Currently amended] The compound represented by following General formula (1) or its optically active substance, or these salts.
Formula 1

In the above formula, the bond represented by the dotted line in the five-membered ring means that the 5-membered ring is a cyclopentene ring having a double bond, or it may be any of saturated cyclopentane rings, and in the case of the cyclopentene ring X is OH, Y is = O, Z is H, and in the case of the cyclopentane ring, X is = O, Y is OH, Z is OH, and R is a residue from which a SH group is removed from a compound containing an SH group.
[2" claim-type="Currently amended] According to claim 1, wherein the compound is a compound represented by the formula (2) or an optically active agent or salts thereof.
Formula 2

In the above formula, R is a residue in which the SH group is removed from the compound containing an SH group.
[3" claim-type="Currently amended] The compound according to claim 1, or an optically active agent or salt thereof, which is a compound represented by the following formula (3).
Formula 3

In the above formula, R is a residue in which the SH group is removed from the compound containing an SH group.
[4" claim-type="Currently amended] The compound according to any one of claims 1 to 3, or an optically active agent or salt thereof, wherein the compound containing an SH group is an amino acid containing a SH group or a derivative thereof.
[5" claim-type="Currently amended] The compound according to claim 4, or an optically active agent or salt thereof, wherein the compound containing an SH group is cysteine or glutathione.
[6" claim-type="Currently amended] 4,5-dihydroxy-2-cyclopenten-l-one represented by the following formula (4) or an optically active substance or a compound selected from these salts is reacted with a compound containing an SH group The compound represented by these, its optically active substance, or the manufacturing method of these salts.
Formula 1

Formula 4

In the above formula, the bond represented by the dotted line in the 5-membered ring means that the 5-membered ring is a cyclopentene ring having a double bond, or it may be any of saturated cyclopentane rings, and in the case of a cyclopentene ring X is OH, Y is = O, Z is H, and in the case of the cyclopentane ring, X is = O, Y is OH, Z is OH, and R is a residue from which a SH group is removed from a compound containing an SH group.
[7" claim-type="Currently amended] The compound represented by the following formula (1) is a compound represented by the following formula (2), or an optically active agent or a method for producing these salts.
Formula 2

In the above formula, R is a residue in which the SH group is removed from the compound containing an SH group.
[8" claim-type="Currently amended] 8. A compound according to claim 7, wherein the reaction is carried out under acidic conditions, or an optically active substance or a method for producing these salts.
[9" claim-type="Currently amended] The compound represented by the following formula (1) is a compound represented by the following formula (3), or an optically active agent or a method for producing these salts.
Formula 3

In the above formula, R is a residue in which the SH group is removed from the compound containing an SH group.
[10" claim-type="Currently amended] The method according to claim 9, wherein the reaction is carried out under neutral conditions, or an optically active compound or a salt thereof.
[11" claim-type="Currently amended] The compound according to any one of claims 6 to 10, wherein the compound containing an SH group is an amino acid containing an SH group or a derivative thereof, or an optically active substance or a method for producing these salts.
[12" claim-type="Currently amended] The compound or a method for producing an optically active agent or salt thereof according to claim 11, wherein the compound containing an SH group is cysteine or glutathione.
[13" claim-type="Currently amended] A pharmaceutical preparation comprising as an active ingredient a compound represented by the following formula (1), an optically active substance thereof, or at least one compound selected from these salts.
Formula 1

In the above formula, the bond represented by the dotted line in the 5-membered ring means that the 5-membered ring is a cyclopentene ring having a double bond, or it may be any of saturated cyclopentane rings, and in the case of a cyclopentene ring X is OH, Y is = O, Z is H, and in the case of the cyclopentane ring, X is = O, Y is OH, Z is OH, and R is a residue from which a SH group is removed from a compound containing an SH group.
[14" claim-type="Currently amended] The pharmaceutical preparation according to claim 13, wherein the preparation contains as an active ingredient a compound represented by the following formula (2), or an optically active substance thereof or one or more compounds selected from these salts.
Formula 2

In the above formula, R is a residue in which the SH group is removed from the compound containing an SH group.
[15" claim-type="Currently amended] The pharmaceutical preparation according to claim 13, wherein the preparation contains, as an active ingredient, a compound represented by the following formula (3) or an optically active substance thereof or one or more compounds selected from these salts.
Formula 3

In the above formula, R is a residue in which the SH group is removed from the compound containing an SH group.
[16" claim-type="Currently amended] The pharmaceutical formulation according to any one of claims 13 to 15, wherein the compound containing an SH group is an amino acid containing a SH group or a derivative thereof.
[17" claim-type="Currently amended] The pharmaceutical formulation according to claim 16, wherein the compound containing an SH group is cysteine or glutathione.
[18" claim-type="Currently amended] The pharmaceutical formulation according to any one of claims 13 to 17, wherein the formulation is a biological defense agent.
[19" claim-type="Currently amended] The pharmaceutical preparation according to claim 18, wherein the biological defense agent is an antiallergic agent.
[20" claim-type="Currently amended] The pharmaceutical preparation according to claim 18, wherein the biological defense agent is an antirheumatic agent.
[21" claim-type="Currently amended] The pharmaceutical preparation according to any one of claims 13 to 17, wherein the preparation is a diabetes treatment.
[22" claim-type="Currently amended] The pharmaceutical formulation according to any one of claims 13 to 17, wherein the formulation is an anticancer agent.
[23" claim-type="Currently amended] The pharmaceutical preparation according to any one of claims 13 to 17, wherein the preparation is an apoptosis inducing agent.
[24" claim-type="Currently amended] The pharmaceutical preparation according to any one of claims 13 to 17, wherein the preparation is an anti-pathogenic microbial agent.
[25" claim-type="Currently amended] The pharmaceutical preparation according to claim 24, wherein the anti-pathogenic microbial agent is an antibacterial agent.
[26" claim-type="Currently amended] The pharmaceutical preparation according to claim 24, wherein the anti-pathogenic microbial agent is an antiviral agent.
[27" claim-type="Currently amended] The pharmaceutical formulation according to claim 26, wherein the virus is human acquired immunodeficiency virus or hepatitis C virus.
[28" claim-type="Currently amended] The pharmaceutical preparation according to claim 26, wherein the antiviral agent is a human antiviral agent or an antiviral agent for animals except for humans or an antiviral agent for plants.

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同族专利:

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公开号 | 公开日

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AT359999T|2007-05-15|

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WO1998039291A1|1998-09-11|

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CN100391940C|2008-06-04|

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JP3753438B2|2006-03-08|

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EP0984001B1|2007-04-18|

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CA2278938A1|1998-09-11|

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EA001888B1|2001-10-22|

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EA199900798A1|2000-04-24|

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AU6117598A|1998-09-22|

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EP0984001A4|2004-12-08|

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US6177592B1|2001-01-23|

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EP0984001A1|2000-03-08|

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KR100570936B1|2006-04-13|

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DE69837597T2|2007-12-27|

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CN1248243A|2000-03-22|

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DE69837597D1|2007-05-31|

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AU739628B2|2001-10-18|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1997-03-05|Priority to JP97-65616

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1997-03-05|Priority to JP6561697

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1997-04-30|Priority to JP12469697

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1997-04-30|Priority to JP97-124696

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1997-06-16|Priority to JP17278697

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1997-06-16|Priority to JP97-172786

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1997-06-16|Priority to JP97-172782

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1997-06-16|Priority to JP17278297

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1997-08-08|Priority to JP97-225533

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1997-08-08|Priority to JP22553397

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1997-08-25|Priority to JP24168097

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1997-08-25|Priority to JP97-241680

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1997-10-01|Priority to JP97-283204

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1997-10-01|Priority to JP28320497

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1997-12-12|Priority to JP97-362273

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1997-12-12|Priority to JP36227397

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1997-12-16|Priority to JP36328197

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1997-12-16|Priority to JP97-363281

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1998-02-26|Application filed by 이치로 스즈키, 타카라 스조 캄파니., 리미티드.

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1998-02-26|Priority to PCT/JP1998/000815

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2000-12-26|Publication of KR20000075824A

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2006-04-13|Application granted

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2006-04-13|Publication of KR100570936B1

优先权:

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申请号 | 申请日 | 专利标题

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JP97-65616|1997-03-05|

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JP6561697|1997-03-05|

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JP12469697|1997-04-30|

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JP97-124696|1997-04-30|

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JP17278697|1997-06-16|

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JP97-172786|1997-06-16|

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JP97-172782|1997-06-16|

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JP17278297|1997-06-16|

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JP97-225533|1997-08-08|

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JP22553397|1997-08-08|

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JP97-241680|1997-08-25|

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JP24168097|1997-08-25|

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JP28320497|1997-10-01|

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JP97-283204|1997-10-01|

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JP36227397|1997-12-12|

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JP97-362273|1997-12-12|

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JP97-363281|1997-12-16|

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JP36328197|1997-12-16|

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PCT/JP1998/000815|WO1998039291A1|1997-03-05|1998-02-26|Compounds|