JPH0441157B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention comprises the following general formula [] [In the formula, R ₁ represents hydrogen, an alkyl group having 1 to 10 carbon atoms, or an alkylcarbonyl group having 1 to 6 carbon atoms, R ₂ represents hydrogen or halogen, and R ₃
represents hydrogen, an alkyl group having 1 to 6 carbon atoms, or an alkylcarbonyl group having 1 to 6 carbon atoms, R ₄ represents hydrogen or an alkyl group having 1 to 6 carbon atoms,
R ₅ represents hydrogen or an alkylcarbonyl group having 1 to 6 carbon atoms; X is a carbamoyl group;
-4 hydroxyalkyl group, or -COOR ₆ (R ₆
represents hydrogen or an alkyl group having 1 to 4 carbon atoms), and Y represents a carbamoyl group and 1 to 4 carbon atoms.
4, or -COOR ₇ (R ₇ represents hydrogen, an alkyl group having 1 to 10 carbon atoms, or a benzyl group). However, R ₁ , R ₂ , R ₃ , R ₄
and when R ₅ is simultaneously hydrogen, X is a carbamoyl group, and Y is a carboxyl group, and when R ₁ is a methyl group and R ₂ , R ₃ , R ₄ and R ₅ are simultaneously hydrogen and X
except when is a carbamoyl group and Y is a carboxyl group. ] This relates to a compound represented by: The present inventors conducted a search for new physiologically active substances produced by microorganisms, and discovered that microorganisms belonging to the genus Penicillium newly isolated from soil contained
It was discovered that a physiologically active substance that strongly inhibits aminopeptidase B activity, that is, a water-soluble amphoteric peptide substance named OF4949 by the present inventors, is accumulated. As a result of further detailed research, the present inventors found that OF4949
Two substances with more similar properties OF4949− and OF4949−
was successfully isolated and purified. It was confirmed from physicochemical and biological properties that OF4949- and OF4949- are new physiologically active substances with the same structure except for the presence of a different substituent at one position, and furthermore, they have the following structural formula. Also confirmed. OF4949- and/or OF4949-
is called OF4949. The present inventors synthesized derivatives of these compounds,
As a result of further research, the present invention was completed by confirming that these derivatives have aminopeptidase inhibitory activity and also have an immune-enhancing effect on living organisms. In recent years, immunomodulatory therapy has been attempted for autoimmune diseases such as nephritis, rheumatoid arthritis, and systemic lupus erythematosus, or malignant tumors, and various drugs have been developed. For example, it has been reported that levamisole is effective to some extent when applied as an immunotherapeutic agent for cancer or a therapeutic agent for autoimmune diseases such as rheumatoid arthritis. Various other immunomodulators have been developed, but many of these drugs have problems such as side effects that occur when used for a long period of time, and there is currently no satisfactory therapeutic agent. The OF4949 derivative according to the present invention exhibits a remarkable effect of enhancing cell-mediated immunity as indicated by delayed-type hypersensitivity obtained by inoculating sheep red blood cells as an antigen into mouse footpads. That is, OF4949 derivatives have an immune-enhancing effect and enhance the immune capacity of living bodies, so they can be used as immunotherapeutic agents for malignant tumors and the like. Also, OF4949 derivatives are anti-aminopeptidase B
Due to its activity, it inhibits the production of bradykinin, exhibits anti-inflammatory effects, and can be used as a therapeutic agent for various diseases. A representative strain producing OF4949, which is a raw material compound for the compound of the present invention, was isolated from soil in Kyoto Prefecture by the present inventors, and its mycological properties are as follows. (1) Growth status on various media (24℃, 2 weeks) Malt extract agar Growth is slow and the diameter of colonies is 2.0 to 2.5 cm in 2 weeks. The surface of the village is dark green or blue-green, and the center of the village is slightly raised. The area around the colony is flat and forms a velvety mycelium. The greenery in the village is 1 to 2 mm wide.
And white. The underside becomes partially yellow, but no pigment is secreted into the agar. Potato, glucose agar Growth is slow, and the diameter of the colony is 2.5 to 3.0 cm in two weeks.
The center of the village is white or gray-green. The surrounding area is yellow to yellow-green and forms a velvety mycelium.
Radial grooves can be seen on the surface of the village. The underside is pale yellow to yellow-orange, slightly folded, and no pigment is secreted into the agar. Tuapetsk agar Growth is extremely slow and conidia do not adhere well. The diameter of the colony is 1.5 to 2.0 cm in two weeks. The surface forms pale yellow to yellow-orange velvety mycelium. It may be partially white or gray. The back side is not colored and no pigment is secreted into the agar. Tuapetsk agar with added Constaplica Growth is slow and the diameter of the colony is 2.0 to 2.5 cm in two weeks. The surface forms yellow-green to dark green velvety mycelium, and the center of the colony may form folds.
The greenery in the village is 1-2 mm wide and white. The back side is not colored and no pigment is secreted into the agar. Wort agar Growth is good and the diameter of the colony reaches 3.0-4.0 cm in two weeks. The center of the village is slightly raised and has a dark green or blue-green velvety color, and the surrounding area is yellow or yellow-green. It forms many folds on both the front and back sides, and the back side is yellow-orange to yellowish brown, but no pigment is secreted into the agar. (2) Morphology The hyphae are colorless and have septate walls, and most penicillas are biwhorled, but occasionally there are monowhorled ones. The conidia are oval in shape, 3.0 μm to 4.0 μm long, 2.5 μm to 3.0 μm wide, and the size of the conidial chain ranges from 50 μm to 80 μm. The stalks are comb-shaped or spear-shaped, growing in parallel clusters of 4 to 8 pieces, length 10.0 μm to 13.0 μm, width.
It is 2.0 μm to 2.5 μm. The basal sycamores grow in clusters of 2 to 8 in a compact state or in a somewhat open state,
The length is 10 μm to 13 μm, and the width is 2.5 μm to 3.0 μm. Conidiophores are 50 μm to 80 μm long and wide.
Most of them are 2.5 μm to 3.0 μm in diameter and protrude from aerial or vegetative hyphae without branching, but branched ones can also be seen. (3) Growth conditions PH: Grows in the range of 2 to 12, and the optimal growth PH is 3.
~6. Temperature: Can grow in the range of 6°C to 33°C, and the optimum range is 18°C to 38°C. As a result of the above observations, this bacterium was identified as Penicillium rugulosum. The mycological properties of Penicillium rugulosum are described by Raper & Thom.
Co-authored, 1968, “A Manual of the Penicilia” published by Hafner Publishers.
Manual of the Penicillia, Domsh & Gams, 1972, Longman's Fungi in Agricultural Soils.
Agricultural Soils)” and “Journal of General and Applied Soils” by Abe.
Microbiology (Journal of General
and Applied Microbiology)” Vol. 2, p. 1, 1956. The present inventors have identified this bacterium as “Penicillium rugrosum OF4949” based on OF4949 productivity.
(Penicillium rugulosum OF4949)”.This bacterium was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, as part of the Microbiological Research Institute No. 203 (FERM BP
-203) has been deposited under the accession number. As for the bacteria used in the production of OF4949, which is the raw material compound of the compound of the present invention, the deposited bacteria mentioned above are representative strains, and in addition to these, any strain belonging to the genus Penicillium that produces OF4949 can be used. In addition, artificial mutant strains obtained by irradiating these strains with ultraviolet rays or treatment with mutagenic agents used for mutagenesis of microorganisms such as nitrosoguanidine, and naturally occurring mutant strains. Some strains that produce OF4949 have also been found, and these mutant strains can also be used in the present invention. For the culture used in the production of OF4949, which is the raw material compound for the compound of the present invention, the medium may be either liquid or solid, but shaking culture or aerated agitation culture using a liquid medium is usually used. Any medium may be used as long as it is suitable for mold growth and can produce OF4949. That is, as carbon sources, sugars such as glucose, fructose, maltose, sucrose, lactose, dextrin, starch, glycerin, and sorbitol, and vegetable oils and fats such as soybean oil are used. Examples of nitrogen sources include peptone, yeast extract, meat extract, soybean flour, cottonseed flour, cornstarch, malt extract, amino acids such as glutamic acid, aspartic acid, tyrosine, and phenylalanine, and their salts, and ammonium urea. Salts, nitrates, etc. are used. Others include magnesium phosphate, calcium,
Micronutrients such as inorganic salts such as sodium, potassium, iron, and manganese, and vitamins such as vitamin B ₁ and calcium pantothenate can be added as appropriate. Liquid culture is preferred for large-scale culture. Culture conditions such as medium PH and culture temperature are as follows:
It may be changed as appropriate within the scope of producing OF4949, but
For liquid shaking or aeration agitation culture, pH4~
7. Cultivation at a culture temperature of 25°C to 30°C and a culture period of 2 to 10 days is appropriate. OF4949 is accumulated in the culture fluid thus obtained. OF4949 exists in both culture filtrate and bacterial cells, and can be collected from either. To extract the target substance from the culture filtrate and bacterial cells,
Based on the properties of OF4949, for example, adsorption chromatography using activated carbon, nonionic adsorption resin, ion exchange chromatography using ion exchange resin, partition chromatography using cellulose, etc., reverse phase partition chromatography using alkyl group-bonded silica gel, etc. Conventional means for separating and purifying organic substances from microbial cultures, such as gel filtration using graphite and gel filtration carriers, are used in appropriate combinations. More specifically, culture filtrate, acetone extract of bacterial cells, or this substance is added to a column packed with an adsorbent such as activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.) or Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation). After adsorption through the containing liquid, it is eluted using a mixed solvent of acid, alkali, water, methanol, ethanol, acetone, etc. alone or in combination. Furthermore, this eluate was subjected to Dowex 50W (H ⁺ type) (manufactured by Dow Chemical Company).
Strongly basic cation exchange resin such as DOWEX 1×2 (OH ^- type) (manufactured by Dow Chemical Company)
It is adsorbed onto a column packed with a strongly acidic anion exchange resin such as , and eluted using an acid, alkali, or salt solution. Further, impurities can also be removed by passing through a Dowex 1×2 (C1 ^- type). From the solution obtained in this way, Diamondion HP was prepared as described above.
-20 etc., followed by desalting by elution with a solvent, and then concentration under reduced pressure to obtain the target product as a yellowish brown crude substance. Next, this crude material was transferred to crystalline cellulose Avicel (manufactured by Funakoshi Co., Ltd.), alkyl group-bonded silica gel Ricroprep RP-8 (manufactured by Merck & Co., Ltd.), and cartridge column C ₁₈ .
(manufactured by Waters), acid,
By developing with a mixed solvent such as alkali, water, buffer, methanol, acetonitrile, propanol, n-butanol, etc. alone or in combination, the desired product can be obtained as a single product. OF4949− and OF4949− are new
It can lead to OF4949 derivatives. The structures of typical OF4949 derivatives included in the present invention are shown in Table 5, and their infrared absorption spectra and proton NMR spectra are shown in Table 6. Among these, compounds that can form salts are shown in Table 5. Regarding carboxylic acids, metal salts or salts with organic bases and salts with mineral acids or organic acids are included in the present invention as long as they are pharmacologically acceptable. [Table] [Table] [Table] [Table] [Table] [Table] [Table] The compound represented by the general formula [] is OF4949
- or OF4949- as a raw material, it can be produced by the following method or a combination thereof. (1) A compound in which R ₁ in the general formula [] is a lower alkyl group can be obtained by using a compound in the general formula [] in which R ₁ is hydrogen as a raw material and using a known method of alkylating the phenolic hydroxyl group. I can do it. The alkylating agent used in the alkylation reaction is an alkyl halide, an alkyl sulfate, an alkyl P-toluenesulfonic acid ester, a diazoparaffin, or in the presence of an acid catalyst or a dehydrating agent such as N,N'-dicyclohexylcarbodiimide. alcoholic beverages, etc. Reaction conditions vary depending on the type of raw material and alkylating agent used, and the reaction temperature, reaction time, and solvent combination are selected depending on the target compound. In some cases, the reaction may be improved by adding a base. For example, in the case of alkylation with an alkyl halide, a methyl halide is used in an example where the target is a compound having a methyl group in _R1 , and a butyl halide is used in an example where the target is a compound with a butyl group in _R1 . , dimethyl sulfoxide, N,N-dimethylformamide, 1,2-dimethoxyethane, tetrahydrofuran, etc. are used as a solvent, and sodium amide, potassium carbonate, triethylamine, sodium hydroxide, barium oxide, silver oxide, sodium hydride, etc. are used as a base. You can choose from and use them. The reaction temperature is usually room temperature to 60°C, but preferably room temperature. The reaction time varies depending on the raw materials, alkylating agent, reaction temperature, and type of solvent, but
Usually, the reaction is completed in several tens of minutes to several tens of hours.
After completion of the reaction, the target product is isolated and purified from the reaction mixture by known means such as concentration, solvent extraction, dissolution, crystallization, chromatography such as ion exchange, adsorption, and distribution. (2) Compounds in which R ₁ in the general formula [] is a lower acyl group can be obtained by using a known acylation method using a compound in which R ₁ in the general formula [] is hydrogen as a raw material. . As the acylation method, for example, N,
Reaction with carboxylic acids in the presence of dehydration such as N'-dicyclohexylcarbodiimide, reaction with acid halides in the presence of chlorine groups such as pyridine, dimethylaniline, tetramethylurea, metallic magnesium, sulfuric acid, p-toluene Examples include reaction with an acid anhydride in the presence of a catalyst such as sulfonic acid, zinc chloride, sodium acetate, or pyridine. Reaction conditions vary depending on the type of raw materials and acylating agent used, and depending on the target compound.
The combination of reaction temperature, reaction time, and solvent is selected. For example, a compound with an acetyl group in R ₁ is
It can be obtained by using acetic anhydride as a catalyst such as sulfuric acid, p-toluenesulfonic acid, zinc oxide, sodium acetate, or pyridine. The reaction temperature is usually room temperature to 60°C, but preferably room temperature. The reaction time varies depending on the raw materials, catalyst, reaction temperature, and type of solvent, but the reaction is usually completed in several tens of minutes to several tens of hours. After the reaction is complete, the target product is extracted from the reaction mixture by concentration, solvent extraction,
It is obtained by isolation and purification by known means such as dissolution, crystallization, chromatography such as ion exchange, adsorption, and distribution. (3) A compound in which R ₂ in the general formula [] is a halogen can be obtained by a known method of halogenating an aromatic compound using a compound in the general formula [] in which R ₂ is hydrogen as a raw material. Examples of the halogenation method include a method of reacting with a simple halogen in the presence of a catalyst such as iron, Lewis acid, or iodine, N-bromosuccinimide, oxalyl chloride, sulfuryl chloride, tert-butyl hypobromite, A method of reacting with a halogenating agent such as tert-butyl hypochlorite is used. Reaction conditions vary depending on the type of raw material and halogenating agent used, and the combination of reaction temperature, reaction time, and solvent is selected depending on the target compound. For example, a compound with bromine in _R2 is water,
It is obtained by treatment with N-bromosuccinimide in a polar solvent such as alcohol.
The reaction temperature is usually 0° to 80°C, but preferably room temperature. Although the reaction time varies depending on the raw materials, halogenating agent, reaction temperature, and type of solvent, the reaction is usually completed in several tens of minutes to several tens of hours. After completion of the reaction, the target product is isolated and purified from the reaction mixture by known means such as concentration, solvent extraction, dissolution, crystallization, chromatography such as ion exchange, adsorption, and distribution. (4) A compound in which R ₃ in the general formula [] is a lower acyl group can be obtained by a known method of acylating an amino group using a compound in which R ₃ in the general formula [] is hydrogen as a raw material. . Acylation methods include, for example, methods of condensing carboxylic acids using carbodiimides, silicon tetrachloride/anhydrous pyridine, titanium chloride, etc. as condensing agents, tertiary amines, sodium hydroxide, sodium acetate, alkali metal carbonates, etc. , a method of reacting with an acid halide in the presence of a salt such as pyridine, and a method of reacting with an acid anhydride in the presence of a reaction accelerator such as sulfuric acid or sodium acetate, if necessary. Reaction conditions vary depending on the type of raw materials and acylating agent used, and depending on the target compound.
The combination of reaction temperature, reaction time, and solvent is selected. For example, a compound with a formyl group in R ₃ is
By the formic acid-acetic anhydride treatment, a compound having an acetyl group at _R3 can be obtained by treatment with an aqueous acetic anhydride solution, acetic anhydride-methanol, acetic anhydride-pyridine, acetic anhydride-sodium acetate, or the like. The reaction temperature is usually 0 to 60°C, but preferably room temperature. The reaction time depends on the raw materials, reaction temperature,
Although it varies depending on the type of solvent, the reaction usually completes in several tens of minutes to several tens of hours. After completion of the reaction, the target product is isolated and purified from the reaction mixture by known means such as concentration, solvent extraction, dissolution, crystallization, chromatography such as ion exchange, adsorption, and distribution. (5) Compounds in which R ₃ and/or R ₄ of the general formula [] are lower alkyl groups are obtained by using a compound in which R ₃ and R ₄ of the general formula [] are both hydrogen, and using known N-alkylation. can be obtained by law. Examples of the N-alkylation method include reaction with an alkyl halide, reaction of an alkyl with an ester of sulfuric acid or sulfonic acid such as dimethyl sulfate, or a reductive alkylation reaction using an aldehyde. Reaction conditions vary depending on the type of raw materials and alkylating agent used, and the combination of reaction temperature, reaction time, and solvent is selected depending on the target compound. For example, a compound in which one of R ₃ or R ₄ is a lower alkyl group can be obtained by appropriately adjusting the amount of alkylating agent, reaction temperature, and reaction time; however, by increasing these conditions, R Compounds are now obtained in which both ₃ and R ₄ are lower alkyl groups. For example, when obtaining an N-methyl compound and an N-ethyl compound by a reductive alkylation method, for example, an aqueous formalin solution and acetaldehyde are added as aldehydes and reacted,
Next, reduction with lithium aluminum hydride, sodium cyanoborohydride, sodium borohydride, etc. is recommended. Particularly preferred is reduction with sodium cyanoborohydride. As the solvent, for example, water, alcohol, acetonitrile, etc. can be used. The reaction temperature is usually 0 to 60°C, preferably room temperature. The reaction time varies depending on the raw materials and the aldehyde used, but as the reaction time increases, a compound in which one of R ₃ or R ₄ is a lower alkyl group is initially produced, and then both R ₃ and R ₄ are a lower alkyl group. The reaction is completed when an appropriate amount of the desired compound is produced. Usually, the reaction is completed in several tens of minutes to several tens of hours. After the reaction is complete, the target product is extracted from the reaction mixture by concentration, solvent extraction, dissolution,
It is obtained by isolation and purification by known means such as crystallization, chromatography such as ion exchange, adsorption, and distribution. (6) A compound in which R ₅ in the general formula [] is a lower acyl group can be obtained by using a compound in the general formula [] in which R ₅ is hydrogen as a raw material and acylating the hydroxyl group by known means. Acylation methods include, for example, reaction with an acid halide in the presence of a base such as pyridine, dimethylaniline, tetramethylurea, metallic magnesium, sulfuric acid, p-toluenesulfonic acid, zinc chloride, sodium acetate, pyridine, etc. Examples include reactions with acid anhydrides in the presence of catalysts such as. Reaction conditions vary depending on the type of raw materials and acylating agent used, and depending on the target compound.
The combination of reaction temperature, reaction time, and solvent is selected. For example, a compound with an acetyl group at _R5 is
It can be obtained by treating acetic anhydride with sulfuric acid, acetic anhydride with p-toluenesulfonic acid, acetic anhydride with zinc chloride, acetic anhydride with sodium acetate, or acetic anhydride with pyridine, and acetic anhydride with pyridine is particularly preferred. The reaction temperature is usually 0 to 100°C, preferably room temperature to 60°C. The reaction time is based on the raw materials,
Although it varies depending on the reagent and reaction temperature, the reaction usually completes in several tens of minutes to several tens of hours. After the reaction is complete,
The target product can be isolated and purified from the reaction mixture by known means such as concentration, solvent extraction, dissolution, crystallization, chromatography such as ion exchange, adsorption, and distribution. (7) A compound in which X in the general formula [] is a carboxylic acid can be obtained, for example, by using a compound in which the X in the general formula [] is a carboxylic acid amide as a raw material and converting the carboxylic acid amide into a carboxylic acid by a known method. be able to. These methods include, for example, hydrolysis with an acid or alkali, hydrolysis using sodium peroxide, or nitrosation of an acid amide and decomposing it into a carboxylic acid. As the nitroning agent, nitrite, alkyl nitrite, nitrosyl chloride, nitrogen dioxide, etc. are used. Reaction conditions vary depending on the raw materials and reagents used, and the combination of reaction temperature, reaction time, and solvent is selected depending on the target compound. For example, in the case of a compound in which X is a carboxylic acid amide and Y is a carboxylic acid in the general formula [], it is preferably 1 to 2 N
By heating in hydrochloric acid at 100° C. for 2 to 8 hours, a compound of the general formula [] in which X and Y are carboxylic acids can be obtained. After the reaction is complete, the target product is concentrated from the reaction mixture.
Solvent extraction, dissolution, crystallization, ion exchange/adsorption/
It is obtained by isolation and purification by known means such as chromatography such as partitioning. (8) A compound in which X in the general formula [] is a carboxylic acid ester can be obtained, for example, by using a compound in the general formula [] in which X is a carboxylic acid as a raw material and using a known esterification reaction. . Esterification reactions include mineral acids such as sulfuric acid and hydrochloric acid, organic acids such as aromatic sulfonic acids, Lewis acids such as boron fluoride etherate, dehydration reactions with alcohols catalyzed by cation exchange resins, diazomethane, etc. reaction with an O-alkylating agent, reaction with dialkyl sulfate or alkyl halide, etc. Reaction conditions vary depending on the raw materials and esterification reagent used, and the combination of reaction temperature, reaction time, and solvent is selected depending on the target compound.
For example, a compound having a methyl ester in It can be obtained by using an ion exchange resin or the like. The reaction temperature is 0 to 60°C, preferably room temperature. The reaction time depends on the raw material,
Although it depends on the type of catalyst and the reaction temperature, the reaction is usually completed in several tens of minutes to several tens of hours. After the reaction is complete, the target product is concentrated from the reaction mixture.
Solvent extraction, dissolution, crystallization, ion exchange/adsorption/
It is obtained by isolation and purification by known means such as chromatography such as partitioning. (9) Compounds in which X in the general formula [] is a hydroxymethyl group are prepared by using a compound in which the X in the general formula [] is a carboxylic ester as a raw material, and using a reducing agent that does not affect moieties other than the carboxylic ester. It can be obtained by reduction. As the reducing agent, lithium alkoxyaluminum hydride, sodium borohydride, sodium borohydride-aluminum chloride, lithium bromide, lithium borohydride, diborane, etc. are used, and lithium borohydride is particularly preferred. Reaction conditions vary depending on the type of raw materials and reducing agent used, and the combination of reaction temperature, reaction time, and solvent is selected depending on the target compound. Ethyl ether, tetrahydrofuran, dioxane, etc. are used as the solvent. The reaction temperature varies depending on the raw materials, solvent, reducing agent, etc., but
℃ to the boiling point of the solvent. The reaction time varies depending on the reducing agent and reaction temperature, but the reaction is usually completed in several tens of minutes to several tens of hours. After the reaction is complete, the target product is concentrated from the reaction mixture.
Solvent extraction, dissolution, crystallization, ion exchange/adsorption/
It is obtained by isolation and purification by known means such as chromatography such as partitioning. (10) A compound in which Y in the general formula [] is a carboxylic acid ester can be obtained by using a known esterification reaction using a compound in the general formula [] in which Y is a carboxylic acid as a raw material. Esterification reactions include mineral acids such as sulfuric acid and hydrochloric acid, organic acids such as aromatic sulfonic acids, Lewis acids such as boron fluoride etherate, dehydration reactions with alcohols catalyzed by cation exchange resins, diazomethane, etc. reaction with an O-alkylating agent, reaction with dialkyl sulfate or alkyl halide, etc. Reaction conditions vary depending on the raw materials and esterification reagent used, and the combination of reaction temperature, reaction time, and solvent is selected depending on the target compound.
For example, a compound having a methyl ester in Y in the general formula [] is prepared in a methanol solvent, a compound having a benzyl ester in Y is prepared in a benzyl alcohol solvent, and a mineral acid such as sulfuric acid, hydrochloric acid, or an aromatic sulfonic acid is used as a catalyst. It can be obtained by using an organic acid, a Lewis acid such as boron fluoride etherate, a cation exchange resin, or the like. The reaction temperature is usually room temperature to 100°C. The reaction time varies depending on the raw materials, the alcohol used, the type of catalyst, and the reaction temperature, but the reaction is usually completed in several tens of minutes to several tens of hours. After the reaction is complete, the target product is concentrated from the reaction mixture.
Solvent extraction, dissolution, crystallization, ion exchange/adsorption/
It is obtained by isolation and purification by known means such as chromatography such as partitioning. (11) Compounds in which Y in the general formula [] is a carboxylic acid amide can be obtained by using a compound in which the Y in the general formula [] is a carboxylic acid ester as a raw material and using a known method to convert the carboxylic ester into a carboxylic acid amide. You can get it by twisting it. For example, by treating a compound in which Y is a carboxylic acid ester with concentrated aqueous ammonia or liquid ammonia, a compound in which Y is a compound amide can be obtained. At this time, the reaction is promoted by using ammonium chloride, sodium methoxide, sodium amide, butyllithylium, or the like as a catalyst. Reaction conditions vary depending on the raw materials and esterification reagent used, and the combination of reaction temperature, reaction time, and solvent is selected depending on the target compound.
The reaction temperature is usually -70°C to room temperature. The reaction time varies depending on the raw materials, solvent, type of catalyst, and reaction temperature, but the reaction is usually completed in several tens of minutes to several tens of hours. After the reaction is complete, the target product is concentrated from the reaction mixture.
Solvent extraction, dissolution, crystallization, ion exchange/adsorption/
It is obtained by isolation and purification by known means such as chromatography such as partitioning. (12) Compounds in which Y in the general formula [] is a hydroxymethyl group are prepared by using a compound in which Y in the general formula [] is a carboxylic acid ester as a raw material, and using a reducing agent that does not affect moieties other than the carboxylic acid ester. It can be obtained by reduction. As the reducing agent, lithium alkoxyaluminum hydride, sodium borohydride, sodium borohydride-aluminum chloride, lithium bromide, lithium borohydride, siborane, etc. are used, and lithium borohydride is particularly preferred. Reaction conditions vary depending on the type of raw materials and reducing agent used, and the combination of reaction temperature, reaction time, and solvent is selected depending on the target compound. Ethyl ether, titrahydrofuran, dioxane, etc. are used as the solvent. The reaction temperature varies depending on the raw materials, solvent, reducing agent, etc., but
℃ to the boiling point of the solvent. The reaction time varies depending on the reducing agent and reaction temperature, but the reaction is usually completed in several tens of minutes to several tens of hours. After the reaction is complete, the target product is concentrated from the reaction mixture.
Solvent extraction, dissolution, crystallization, ion exchange/adsorption/
It is obtained by isolation and purification by known means such as chromatography such as partitioning. (13) The compound represented by the general formula [] is
It can also be obtained by a combination of two or more of the production methods (1) to (12), or by eliminating one or more substituents of the obtained compound by a known method. You can also get it. Also (1)~
In the production method of (12), the functional group of the compound represented by the general formula [] may be protected with a known protecting group before the reaction and deprotected after the reaction depending on the target compound. For example, a compound represented by the general formula []
Using a compound in which R ₃ and R ₄ are hydrogen and R ₇ is hydrogen or a lower alkyl group as a raw material, by introducing an amino protecting group into the amino group by a known method, P Compounds in which is a protecting group for an amino group and R ₇ is hydrogen or a lower alkyl group can be obtained. This compound is alkylated by a known method for alkylating the phenolic hydroxyl group as described in the production method (1), and if necessary, deprotecting the amino group and/or hydrolyzing the ester. , a compound of the general formula [] in which R ₁ ' is a lower alkyl group, R ₃ ' is hydrogen or a protecting group for an amino group, and R ₇ ' is hydrogen or a lower alkyl group can be obtained. ) (In the formula, P represents hydrogen or a protecting group for an amino group, R ₇ represents hydrogen or an alkyl group having 1 to 10 carbon atoms, R ₁ ′ represents an alkyl group having 1 to 10 carbon atoms, and R ₃ ′ represents hydrogen or a protecting group for an amino group, and R ₇ ' represents hydrogen or an alkyl group having 1 to 4 carbon atoms.) The protecting group for an amino group used here is a urethane-type protecting group such as a benzyloxycarbonyl group. Known protective groups such as acyl type protective groups such as , formyl group, etc. can be used. Further, the method for deprotecting the protecting group for the amino group varies depending on the protecting group used, and a known deprotecting method for each protecting group can be used. For example, benzyloxycarbonyl groups can be easily deprotected by catalytic reduction, and formyl groups can be easily deprotected by acid or alkali. In addition, for hydrolysis of esters, a mixed solvent of water and a water-soluble organic solvent such as methanol, ethanol, N,N-dimethylformamide, etc. and water is used as a solvent in the presence of a basic catalyst such as sodium hydroxide or potassium carbonate. This can be done by For example, OF4949- was converted to N- with formic acid-acetic anhydride.
After formylation, as a solvent, for example, N,N
- using dimethylformamide as base,
For example, using potassium carbonate and reacting with an alkyl halide, the resulting compound is 4N
- A compound in which R ₁ ′ of the general formula [] is a lower alkyl group and R ₃ ′ and R ₇ ′ are hydrogen by simultaneously hydrolyzing the N-formyl group and the alkyl ester with an aqueous solution of sodium hydroxide compound is obtained. In the above method, when iodomethyl is used as the alkyl halide, compound 13 (OF4949-) can be obtained from OF4949-. After completion of the reaction, the target product is isolated and purified from the reaction mixture by known means such as concentration, solvent extraction, dissolution, crystallization, chromatography such as ion exchange, adsorption, and distribution. [Biological Properties] The OF4949 derivative according to the present invention has a physiological activity that significantly inhibits aminopeptidase B activity of Ehrlichi cancer cells. The method for measuring aminopeptidase B is described by Hops et al. [VKHops, KKMakinen, GGGlenner,
Archives of Biochemistry and Biophysics
144 557 (1966)].
That is, a mixture of 0.1 ml of 3mM arginine-β-naphthylamide (manufactured by Sigma) dissolved in Hank's solution (manufactured by Nitsusui Pharmaceutical Co., Ltd.) and 0.7 ml of Hank's solution containing the specimen was heated at 37°C for 3 minutes. Add 0.2 ml of Ehrlichi cancer cell suspension prepared to a concentration of 2.5 x ¹⁰ cells/ml to Hank's solution, and incubate at 37°C.
After reacting for 30 minutes, the concentration of garnet was reduced to 0.3 mg/ml.
Contains GBC (ortho-aminoazotoluene diazonium salt) (manufactured by Sigma) and 1.0M containing Tween20 (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 3%.
After adding 3 ml of acetate buffer (PH4.2) and leaving it at room temperature for 15 minutes, the absorbance of the supernatant at 525 nm a
was measured. At the same time, the absorbance b of a control using only Hank's solution containing no specimen was measured, and aminopeptidase B
The inhibition rate was calculated by b-a/b×100. The Ehrlichi cancer cells used above were of the ddY strain and were maintained in the peritoneal cavity of 4-6 week old (male) mice, and 7 days after transplantation of 2 x 10 ⁶ cells into the peritoneal cavity of the mouse. Ascites fluid was collected on day 10. To obtain the cell suspension, ascites fluid containing Ehrlichi cancer cells is treated with Tris-ammonium chloride solution (PH7.2) to remove red blood cells, washed three times with Hank's salt solution, and adjusted to the desired cell number with Hank's salt solution. This is what I did. This is a homogeneous cell population with a living cell count of 96% or more of Ehrlich cancer cells. The inhibition rates at several concentrations of OF4949 derivatives were determined by the above test method, and a 50% inhibitory concentration was assigned to each. Table 7 shows the results of tests on representative compounds of the present invention. [Table] This substance also produces delayed-type hypersensitivity, which can be obtained by inoculating sheep red blood cells as an antigen into the footpads of mice.
It showed an effect of enhancing cell-mediated immunity using Type Hyper-sensitivity (abbreviated as DTH) as an indicator. That is, using sheep red blood cells as an antigen, 1 x ¹⁰⁵ cells were transferred to _CDF1 system
Eight 10-week-old female mice in one group were sensitized by arrhythmia injection.
Immediately after sensitization, OF4949 derivatives dissolved in sterile water were injected intraperitoneally at concentrations of 5, 50, and 500 μg/Kg, and 4 days later, sheep red blood cells were injected into the footpad of the left hind paw.
10 ⁸ pieces were subcutaneously injected, and 24 hours later, the degree of swelling observed in the footpad (thickness of the footpad) was determined by measuring with a caliper. The results are shown in Table 8. The degree of swelling in mice administered with OF4949 derivatives was significantly enhanced compared to controls. This indicates that OF4949 derivatives have an enhancing effect on the establishment of cell-mediated immunity. [Table] All of the compounds of the present invention have low toxicity,
Can be used as a medicine. Table 9 shows the results of an acute toxicity test of representative compounds of the present invention when administered to ICR mice at 300 mg/Kg intraperitoneally. [Table] When the compound of the present invention is administered as a medicament, the compound of the present invention may be administered as it is or in a pharmaceutically acceptable non-toxic and inert carrier, for example, from 0.1% to 99.5%.
It is preferably administered to animals including humans as a pharmaceutical composition containing 0.5% to 90%. As carriers, one or more solid, semisolid, or liquid diluents, fillers, and other formulation auxiliaries are used. Preferably, the pharmaceutical composition is administered in dosage unit form. The pharmaceutical composition of the present invention can be administered by oral administration, intratissue administration, local administration, (transdermal administration, etc.)
Or it can be administered rectally. Of course, it is administered in a dosage form suitable for these administration methods. For example, injections are particularly preferred. It is desirable to adjust the dose of an immunomodulator after considering the patient's condition such as age, weight, route of administration, nature and severity of the disease, etc., but usually,
The amount of the active ingredient of the present invention for adults is generally in the range of 0.1 to 1000 mg per day. In some cases, a lower dose than this may be sufficient, and in other cases, a higher dose may be required. When administering a large amount, it is desirable to divide the dose into several times a day. Oral administration is carried out in solid or liquid dosage units, such as powders, powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, sublingual tablets, and other dosage forms. be able to. Powders are prepared by comminuting the active substance to a suitable fineness. Powders are prepared by bringing the active substance to a suitable fineness and then mixing it with similarly finely divided pharmaceutical carriers, such as starch, edible carbohydrates such as mannitol, and the like. Flavoring agents, preservatives, dispersants, coloring agents, fragrances, and other substances may be mixed as necessary. Capsules are manufactured by first filling powdered powders or powders as described above, or granules as described in the section on tablets, into capsule shells such as gelatin capsules. Ru. Lubricants and flow agents, such as colloidal silica, talc, magnesium stearate,
Mix something like calcium stearate and solid polyethylene glycol into a powder,
A filling operation can also be carried out afterwards. Addition of disintegrants or solubilizers, such as carboxymethylcellulose, calcium carboxymethylcellulose, low-substituted hydroxypropylcellulose, calcium carbonate, sodium carbonate, can improve the effectiveness of the drug when the capsule is ingested. . Alternatively, the fine powder of this product can be suspended and dispersed in vegetable oil, polyethylene glycol, glycerin, or a surfactant, and then wrapped in a gelatin sheet to form a soft capsule. Tablets are manufactured by preparing a powder mixture, granulating or slugging it, adding a disintegrant or lubricant, and then tableting. Powder mixtures are prepared by mixing suitably powdered materials with the diluents and bases mentioned above, and optionally binders (e.g. sodium carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, polyvinyl alcohol, etc.) and dissolution retarders. (e.g. paraffin), reabsorbents (e.g. quaternary salts) and/or adsorbents (e.g. bentonite, kaolin, dicalcium phosphate, etc.) may also be used in combination. The powder mixture can first be moistened with a binder such as syrup, starch paste, gum arabic, cellulose solution or polymeric substance solution and then forced through a sieve to form granules. Instead of granulating the powder in this way, it is also possible to first run it through a tablet press and then crush the resulting imperfectly formed slugs into granules. The granules thus produced can be prevented from sticking to each other by adding stearic acid, stearate salts, talc, mineral oil, etc. as lubricants. The mixture thus lubricated is then compressed into tablets. Alternatively, the drug may be mixed with a fluid inert carrier and then directly compressed into tablets without going through the granulation or slugging steps as described above. Transparent or translucent protective coatings such as occlusive coatings of silica, coatings of sugar or polymeric materials, and polishing coatings of wax may also be used. Other oral dosage forms, such as solutions, syrups,
Elixirs and the like can also be in dosage unit form so that a fixed amount of the drug contains a fixed amount of the drug. Syrups are prepared by dissolving the compound in a suitable flavored aqueous solution, and elixirs are prepared by using a non-toxic alcoholic carrier.
Suspensions are formulated by dispersing the compound in a non-toxic carrier. Solubilizers and emulsifiers (e.g. ethoxylated isostearyl alcohol water,
polyoxyethylene sorbitol esters),
Preservatives, flavoring agents (eg, peppermint oil, saccharin), and others may also be added as desired. If desired, dosage unit formulations for oral administration may be microencapsulated. The formulation can also be coated or embedded in polymers, waxes, etc. to prolong the action time and provide sustained release. Parenteral administration may be carried out using liquid dosage unit forms, such as solutions or suspensions, intended for subcutaneous, intramuscular or intravenous injection.
These are prepared by suspending or dissolving a given amount of the compound in a non-toxic liquid carrier compatible with the purpose of injection, such as an aqueous or oily vehicle, and then sterilizing the suspension or solution. Ru. Alternatively, a fixed amount of the compound may be placed in a vial, and then the vial and its contents may be sterilized and sealed. Extra vials or carriers may be provided with the powdered or lyophilized active ingredient for dissolution or mixing immediately prior to administration. Nontoxic salts or salt solutions may be added to make the injection solution isotonic. Furthermore, stabilizers, preservatives, emulsifiers, and the like can also be used in combination. Rectal administration is carried out by using suppositories containing the compound in water-soluble or water-soluble solids of low melting point, such as polyethylene glycol, cocoa butter, higher esters (e.g. palmitin myristyl ester), and mixtures thereof. sell. In addition to the active ingredient according to the present invention, other drugs such as cytosine arabinoside, adriamycin, or mitomycin may be added to the preparation of the compound of the present invention, or may be used in combination. Reference Example 1 Preparation of OF4949 Penicillium rugulosum OF4949 (Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Institute of Microbial Technology, accession number FAIKEN JOKYO NO. 203) was cultured on a malt extract agar slant medium for 7 days. From this slant culture, 500 ml of a medium containing 2% glucose, 0.5% polypeptone, 0.1% yeast extract, 0.05% potassium phosphate, and 0.05% magnesium sulfate was dispensed into 2-volume Erlenmeyer flasks. Sterilized at 120℃ for 20 minutes, inoculated and incubated at 27℃ with shaking at 190 revolutions per minute.
Cultured for 1 day. This culture was placed in 30 stainless steel acid baths 15 containing 20 previously sterilized medium containing 2% glucose, 0.5% polypeptone, 0.1% yeast extract, 0.05% potassium phosphate, and 0.05% magnesium sulfate. Culture was started by inoculating one seed per group. Culture is carried out at an aeration rate of 20 per minute.
It was carried out at 27° C. at a rotation speed of 300 revolutions per minute, and an antifoaming agent was added as necessary. After culturing for 4 days, the culture was taken out, the bacterial cells were filtered, and the culture filtrate was separated, yielding a compressed capacity of 6.5 bacterial cells and 300 culture filtrate. Reference Example 2 Preparation of OF4949 The bacterial cells obtained in Reference Example 1 were mixed with 50% acetone water24
The acetone was removed by extracting the extract 72 three times and concentrating the extract 72 to a volume of 30% under reduced pressure. Concentrate 30
It was adsorbed on an activated carbon column together with 300ml of the culture filtrate obtained in Reference Example 1, and washed thoroughly with 80ml of water.
It was eluted with a PH2 hydrochloric acid aqueous solution of 150 containing 50% acetone. Aminopeptidase inhibitory activity fraction 80
After concentrating to 30% under reduced pressure and removing acetone,
8 concentrated solution was applied to a 1.2 column of strongly basic anion exchange resin Dowex 1×2 (C1 ¹ type) and eluted with water. Next, correct the eluate 80 to pH 3,
Strongly acidic cation exchange resin Dowex 50W (H ⁺
After washing with 10% water adsorbed on a 2.0 column (type 2.0), it was eluted with 1.0N aqueous ammonia 100%. After neutralizing active area 60 with 2N hydrochloric acid, it was adsorbed on nonionic adsorption resin Diaion HP-202.
After washing with 20 ml of water, it was eluted with 50% methanol water 20 ml. The obtained active area was concentrated under reduced pressure to obtain the crude substance.
28.8g was obtained. Reference Example 3 Preparation of OF4949 28.8 g of the crude material obtained in Reference Example 2 was sprinkled on 30 g of crystalline cellulose Avicel, and after sufficiently drying under reduced pressure, it was packed in the upper part of Avicel column 1. Developing solvent 1N ammonia water: n-butanol = 15:85
When developed with 12, an active area mainly containing OF4949- was separated, and an active area mainly containing OF4949- was then separated. Each active area was neutralized with 1NHC1, concentrated under reduced pressure, redissolved in water, and adsorbed onto 500 ml of Diaion HP-20. 2/50 after washing
By eluting with % methanol water and concentrating the active area under reduced pressure, 3.1 g of coarse powder of OF4949- and
4.1 g of coarse powder of OF4949- was obtained. Reference Example 4 Preparation of OF4949 OF4949-coarse powder obtained in Reference Example 3
Dissolve 3.1 g in 15 ml of water and use high-performance liquid chromatography (System 500, Waters) for large-scale preparative separation, column: cartridge column (C ₁₈ , Waters), developing solvent: 0.1M citrate buffer. Reversed phase partition chromatography was performed under conditions of liquid:acetonitrile=90:10, and the aminopeptidase B inhibitory activity was assayed. Adsorb the active area in 200ml of Diaion HP-20, wash with water, 1
It was eluted with 50% methanol water. By collecting the active area, concentrating it under reduced pressure, and freeze-drying it,
455 mg of OF4949 was obtained. OF4949- coarse powder 4.1 obtained in Reference Example 3
Similarly, for g, C ₁₈ reverse phase partition chromatography was performed using the developing solvent 0.1M citrate buffer: acetonitrile = 95.5, and then the diamond ion
Perform HP-20 column chromatography,
OF4949−671 mg was obtained. Example 1 Production of Compound 2 223.7 mg of OF4949- was dissolved in 10 ml of 1N hydrochloric acid, sealed in a tube, and heated at 100°C for 7 hours. The reaction solution was concentrated to dryness, the residue was dissolved in 10 ml of water, and using high performance liquid chromatography, column: Nucleosil.
30C ₁₈ 2.0cm×25.0cm, developing solution: 0.1M citrate buffer-acetonitrile was used to obtain a fraction containing compound 2. This fraction was adsorbed onto 250 ml of a strongly acidic cation exchange resin Gwex 50W (H ⁺ type), washed with water, and eluted with 1N aqueous ammonia. The fraction containing compound 2 was concentrated to dryness, and the residue was dissolved in 5 ml of water.
It was adsorbed onto a Nucleosil 30C ₁₈ 2.0cm x 25.0cm column and eluted with water. The fraction containing Compound 2 was concentrated to dryness to obtain 78.8 mg of Compound 2 as a colorless powder. Example 2 Production of Compound 10 50mg of OF4949 was added to 0.1M phosphate buffer (PH
7.0) Dissolve in 5ml, add 100μ of formalin,
After stirring at room temperature for 10 minutes, 30 mg of sodium cyanoborohydride was added, and the mixture was further stirred at room temperature for 1 hour. The reaction solution was analyzed using high performance liquid chromatography.
Column: Nucleosil 30C ₁₈ 2.0 cm x 25.0 cm, developing solution: Developed with 0.1 M citrate buffer (PH5.7)-acetonitrile to obtain a fraction containing compound 10. Concentrate this fraction and transfer the concentrated solution to Diaion HP-20.
(20 ml), washed with water, and eluted with 50% methanol water. The fraction containing Compound 2 was concentrated to dryness to obtain 24.6 mg of Compound 10 as a colorless powder. Example 3 Production of Compound 12 60mg of OF4949 was added to 0.1M phosphate buffer (PH
7.0) 5 ml of acetaldehyde was added, and the mixture was stirred at room temperature for 10 minutes. After that, 30 mg of sodium cyanoborohydride was added, and the mixture was further stirred at room temperature for 1 hour. The reaction solution was analyzed using high performance liquid chromatography.
Column: Nucleosil 30C ₁₈ 2.0 cm x 25.0 cm, developing solution: Developed with 0.1 M citrate buffer (PH5.7)-acetonitrile to obtain a fraction containing compound 12. Concentrate this fraction and transfer the concentrated solution to Diaion HP-20.
(20ml), washed with water, and eluted with 50% methanol water. The fraction containing Compound 12 was concentrated to dryness to obtain 31.4 mg of Compound 12 as a colorless powder. Example 4 Production of compounds 7, 23 and 13 267 mg of compound 6, 20 ml of N,N-dimethylformamide, 200 mg of potassium carbonate, iodomethyl
200μ was added and stirred at room temperature for 24 hours. After neutralizing the reaction solution, using high performance liquid chromatography, column: Nucleosil 30C ₁₈ 2.0cm x 25.0cm,
Developing solution: 0.1M citrate buffer (PH5.7)-acetonitrile was used to obtain fractions containing compounds 7 and 23. Each fraction was concentrated, and the concentrated solution was adsorbed onto 20 ml of Amberlite x AD-2 (manufactured by Organo), washed with water, and eluted with methanol. Each fraction was concentrated to dryness to obtain a colorless powder of compound 7.
52.3 mg of compound 23 and 137.2 mg of colorless powder of compound 23 were obtained. In addition, 42.2 mg of powder of compound 23 was added with 20 mg of 4NNaOH aqueous solution.
ml and stirred at room temperature for 30 minutes, neutralized with 2N hydrochloric acid, adsorbed on 20 ml of Diaion HP-20, washed with water, and eluted with 50% methanol water. Colorless powder of compound 13 was obtained by concentrating the obtained fractions to dryness.
39.0 mg was obtained. Example 5 Production of compounds 14, 1, 3 and 19 OF4949-67.8 mg and 10 ml of 0.2N hydrochloric acid methanol
was added, stirred overnight at room temperature, and then concentrated to dryness.
Dissolve the residue in 10 ml of water, use high performance liquid chromatography, column: Nucleosil 30C ₁₈ 2.0 cm x
25.0cm, developing solution: 0.1M citrate buffer (PH5.7)
-Development with acetonitrile gave a fraction containing compound 14. This fraction was concentrated, and the concentrated liquid was adsorbed onto 20 ml of Amberlite x AD-2 (manufactured by Organo).
After washing with water, 30.6 mg of colorless powder of Compound 14 was obtained by elution with methanol water. In the same manner, compound 1 (74.3 mg) was obtained from OF4949- (91 mg), compound 3 (55.5 mg) consisting of compound 2 (90.4 mg), compound 19 (22.3 mg) from compound 18 (33.8 mg), I got each. Further, 11 mg of OF4949 was suspended in 10 ml of an equal volume mixture of methanol and ether, passed through diazomethane, and after the crystals were dissolved, the suspension was concentrated to dryness. Water the residue 0.5
ml and purified in the same manner using high performance liquid chromatography and Amberlite x AD-2 to obtain 3.6 mg of colorless powder of Compound 14. Compound 19 was added to compound 18 (10.5 mg) in the same manner.
(1.9 mg) was obtained. Example 6 Production of Compound 15 To 200 mg of OF4949, 10 ml of N,N-dimethylformamide and 10 ml of 2N butanol hydrochloride were added and stirred at room temperature overnight. The reaction solution was dissolved in 200 ml of water, adsorbed on 20 ml of xAD-2, washed with water, and eluted with 50% methanol water.
The fraction containing Compound 15 was concentrated to dryness to obtain 72.9 mg of Compound 15 as a colorless powder. Example 7 Production of Compound 16 200 mg of OF4949- was taken, 10 ml of 2N hydrochloric acid-benzyl alcohol was added, and the mixture was stirred at room temperature overnight.
Add hexane-chloroform (1:1) mixture to the reaction solution.
Add 50ml, filter and dry the resulting precipitate to give 196.2
mg of filtrate was obtained. The filtrate was dissolved in 1 ml of dimethyl sulfoxide, and using high performance liquid chromatography, column: Nucleosil 30C ₁₈ 2.0 cm x 25.0
cm, developing solution: 0.1M citrate buffer (PH5.7) −
It was developed with acetonitrile to obtain a fraction containing compound 16. This fraction was concentrated, and the concentrated solution was adsorbed onto Amberlite x AD-220ml, washed with water, and eluted with methanol. The fraction containing Compound 16 was concentrated to dryness to obtain 19.1 mg of Compound 16 as a colorless powder. Alternatively, 10 ml of benzyl alcohol, 20 ml of benzene, and 100 mg of p-toluenesulfonic acid were added to 200 mg of OF49490, and the mixture was reduced for 4 hours. 100 mg of an ether-hexane (1:1) mixture was added to the reaction solution, and the resulting precipitate was filtered and dried to obtain 343.7 mg of filtrate. The filtrate is dimethyl sulfoxide 2
ml, and using high performance liquid chromatography, column: Nucleosil 30C ₁₈ 2.0cm x 25.0cm,
Developing solution: Developed with 0.1M citrate buffer (PH5.7)-acetonitrile to obtain a fraction containing compound 16. The fraction was concentrated and Amberlite×AD−
20 ml, washed with water, eluted with methanol, and concentrated to dryness to obtain 83.8 mg of colorless powder of Compound 16. Example 8 Production of Compound 17 102 mg of Compound 14 was taken, 5 ml of concentrated aqueous ammonia was added, and the mixture was left stirring at room temperature overnight. After neutralizing the reaction solution with 6N hydrochloric acid, using high performance liquid chromatography, column: Nucleosil 30C ₁₈ 2.0cm x 25.0cm,
Developing solution: Developed with 0.1M citrate buffer (PH5.7)-acetonitrile to obtain a fraction containing compound 17. Concentrate the fraction and use the concentrated liquid as a diamond ion
The compound was adsorbed onto 20ml of HP-20, washed with water, eluted with 50% methanol, and concentrated to dryness.
48.3 mg of colorless powder of 17 was obtained. Compound 17 (30.8 mg) was obtained from Compound 19 (59.6 mg) in the same manner. Example 9 Production of compound 18 1.0 g of OF4949 was dissolved in 20 ml of 1N hydrochloric acid,
The tube was sealed and heated at 100°C for 7 hours. The reaction solution was concentrated to dryness, and the residue was dissolved in 10 ml of water. Using preparative high performance liquid chromatography, column: Preppack-500/C ₁₈ (manufactured by Waters), developing solution:
Developed with 0.1M citrate buffer-acetonitrile,
A fraction containing compound 18 was obtained. Concentrate the fraction,
Adjust the concentrated solution to PH3 with 6N hydrochloric acid and make it Diaion HP-
After adsorption with 70 ml of 20 mL of water, it was eluted with 50% methanol and concentrated. The resulting crystals were filtered to obtain 345.3 mg of Compound 18 in the form of colorless columnar crystals. Regarding the melting point, it does not show a clear melting point up to 300℃. Elemental analysis value (as C ₂₃ H ₂₅ N ₃ O ₉・2H ₂ O) Actual value C: 53.01 H: 5.83 N: 8.09 Calculated value C: 52.77 H: 5.58 N: 8.03 Example 10 Production of compound 20 Compound 19 59.6 Add 3 ml of concentrated ammonia water to mg,
Stir overnight at room temperature. Dilute the reaction solution with 3 ml of water,
Using high performance liquid chromatography, column: Nucleosil 30C ₁₈ 2.0cm x 25.0cm, developing solution: 0.1M
It was developed with citrate buffer-acetonitrile to obtain a fraction containing compound 20. The fraction was concentrated, and the concentrated solution was adsorbed on 20 ml of Diaion HP-20, washed with water, eluted with 50% methanol, and concentrated to dryness to obtain 9.2 mg of Compound 20 as a colorless powder. Example 11 Production of Compounds 21, 4 and 5 40 ml of tetrahydrofuran was added to 462.2 mg of Compound 19, 500 mg of lithium borohydride was added, and the mixture was refluxed for 6 hours. 2N hydrochloric acid and methanol were added to the reaction mixture to consume excess lithium borohydride, and the mixture was concentrated to dryness. Dissolve the residue in 10 ml of water, use high performance liquid chromatography, column: Nucleosil 30C ₁₈ 2.0 cm x
25.0 cm, developing solution: 0.1M citrate buffer-acetonitrile was used to obtain a fraction containing compound 21. Concentrate the fraction and use the concentrated liquid as a diamond ion
The compound was adsorbed onto 20ml of HP-20, washed with water, eluted with 50% methanol, and concentrated to dryness.
233.6 mg of colorless powder of 21 was obtained. Compound 4 was extracted from compound 1 (37 mg) in the same manner.
(6.1 mg) was added to compound 5 (12.0 mg) from compound 3 (40 mg).
mg) respectively. Example 12 Production of Compounds 22 and 6 5 ml of 98% formic acid was added to 100 mg of OF4949, and 5 ml of acetic anhydride was added dropwise while stirring under ice cooling. After the dropwise addition, the mixture was stirred for 30 minutes under water cooling, and then stirred for 1 hour at room temperature. 10 ml of water was added to the reaction solution, stirred for 10 minutes, and then concentrated. The concentrated solution was transferred using high performance liquid chromatography, column: Nucleosil 30C ₁₈ 2.0cm x 25.0cm,
Developing solution: Developed with 0.1M citrate buffer-acetonitrile to obtain a fraction containing compound 22. Concentrate the fraction and use the concentrated liquid as Diaion HP-20.
After adsorption in 20 ml of water, the mixture was washed with water, eluted with 50% methanol, concentrated, and the resulting crystals were filtered to obtain 42.2 mg of colorless microneedle crystals of Compound 22. Regarding the melting point, it does not show a clear melting point up to 300℃. Elemental analysis values (as C ₂₄ H ₂₆ N ₄ O ₉ ) Measured value C: 55.87 H: 5.08 N: 10.94 Calculated value C: 56.03 H: 5.09 N: 10.89 Compound 6 (24.1 mg) was obtained. Example 13 Production of Compound 24 10 ml of N,N-dimethylformamide, 100 mg of potassium carbonate, and 200 μl of n-butyl bromide were added to 134.0 mg of Compound 22, and the mixture was stirred at room temperature overnight. Neutralize the reaction solution and use high performance liquid chromatography.
Column: Nucleosil 30C ₁₈ 2.0 cm x 25.0 cm, developing solution: Developed with 0.1 M citrate buffer-acetonitrile to obtain a fraction containing compound 24. Concentrate the fraction and transfer the concentrated solution to Amberlite x AD-2 20
ml, washed with water, eluted with methanol water,
Colorless powder of compound 24 by concentration to dryness 59.0
I got mg. Example 14 Production of Compounds 25 and 8 200 mg of OF4949- was dissolved in 20 ml of water, and 10 ml of acetic anhydride was added dropwise with stirring under ice cooling. Ice cold 15
After stirring for several minutes, the mixture was stirred at room temperature overnight. Add the reaction solution to 100% water.
ml, adsorbed on Diaion HP-20 (20 ml), washed with water, eluted with 50% methanol water, and concentrated to dryness to obtain a colorless powder of compound 25 (156.9 ml).
I got mg. Compound 8 (44.9 mg) was obtained from OF4949- (50 mg) in the same manner. Example 15 Production of Compounds 26 and 9 10 ml of methanol and 5 ml of acetic anhydride were added to 100 mg of OF4949-, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated to dryness, and the residue was dissolved in 7 ml of water-dimethyl sulfoxide mixture. Using high performance liquid chromatography, column: Nucleosil 30C ₁₈ 2.0 cm x 25.0 cm,
Developing solution: 0.1M citrate buffer-acetonitrile was used to obtain a fraction containing compound 26. Concentrate the fraction and add the concentrated solution to Amberlite x AD2 20
ml, washed with water, eluted with 50% methanol water, and concentrated to dryness to obtain a colorless powder of compound 26.
Obtained 57.0 mg. Compound 9 (23.3 mg) was obtained from OF4949- (50 mg) in the same manner. Example 16 Production of Compound 27 30.6 mg of Compound 14, 1 ml of pyridine, and acetic anhydride
100μ was added and stirred at room temperature for 4 hours. Add 2 ml of water to the reaction solution, and using high performance liquid chromatography, column: Nucleosil 30C ₁₈ 2.0 cm x 25.0 cm,
Developing solution: Developing with 0.1M citrate buffer-acetonitrile to obtain a fraction containing compound 27. Concentrate the fraction and add the concentrated solution to Amberlite x AD2 20
After washing with water, perform gradient elution of metal from water, and concentrate the fraction containing compound 27. Filter the resulting crystals to obtain colorless crystals of compound 27.
Obtained 12.9 mg. Example 17 Production of compounds 28 and 11 OF4949- 200 mg was added to 0.1 M phosphate buffer (PH
7.0) Dissolved in 10ml, added 40μ of acetaldehyde, and stirred at room temperature for 10 minutes. Next, 100 mg of sodium cyanoborohydride was added, and the mixture was further heated for 20 min at room temperature.
After stirring for a minute, the mixture was concentrated to dryness. Dissolve the residue in water, use high performance liquid chromatography, column: Nucleosil 30C ₁₈ 2.0cm x
25.0cm, developing solution: 0.1M citrate buffer (PH5.7)
-Development with acetonitrile gave a fraction containing compound 28. The fraction was concentrated, the concentrated solution was adsorbed on 20 ml of Diaion HP-20, washed with water, eluted with 50% methanol, and concentrated to dryness.
128.9 mg of colorless powder of 28 was obtained. Compound from OF4949− (50 mg) in the same manner.
11 (28.6 mg) was obtained. Example 18 Production of Compounds 29 and 30 200 mg of OF4949- was dissolved in 20 ml of water, 250 mg of N-bromosuccinimide was added, and the mixture was stirred at room temperature for 1 hour. A precipitate will form in the reaction solution, so add a few drops of 4N ammonia water to dissolve it and use high performance liquid chromatography, column: Nucleosil 30C ₁₈ 2.0cm.
×25.0 cm, developing solution: 0.1M citrate buffer-acetonitrile was used to obtain a fraction containing compound 29. The fraction was concentrated, the concentrated solution was adsorbed on 20 ml of Amberlite x AD2, washed with water, eluted with 50% methanol, concentrated, and the resulting crystals were filtered to obtain 75.8 mg of Compound 29 as colorless columnar crystals. Obtained. Regarding the melting point, it does not show a clear melting point up to 300℃. Elemental analysis value (as C ₂₃ H ₂₅ N ₄ O ₈ Br・H ₂ O) Actual value C: 46.91 H: 4.93 N: 9.57 Calculated value C: 47.35 H: 4.67 N: 9.60 Compound 18 (41.0 mg ) from compound 30
(38.6 mg) was obtained. Example 19 Production of Compound 31 20 ml of N,N-dimethylformamide, 200 mg of potassium carbonate, and 500 μl of n-butyl bromide were added to 278.7 mg of Compound 6, and the mixture was stirred at room temperature overnight. Neutralize the reaction solution and apply high performance liquid chromatography to column: Nucleosil 30C ₁₈ 2.0cm x 25.0cm.
Developing solution: 0.1M citrate buffer-acetonitrile was used to obtain an n-butylated fraction. By concentrating the fraction and filtering the resulting precipitate
52.0 mg of powder was obtained. 2 ml of 4N caustic soda aqueous solution was added to 52.0 mg of the powder, stirred at room temperature for 2 hours to neutralize the reaction solution, and purified again by high performance liquid chromatography to obtain a fraction containing Compound 31. The fraction was concentrated, the concentrated solution was adsorbed on 20 ml of Amberlite x AD2, washed with water, eluted with 50% methanol, concentrated, and the resulting crystals were filtered to obtain compound 31 as colorless microneedle crystals at 25.5 ml. I got mg. Regarding the melting point, it does not show a clear melting point up to 300℃. Elemental analysis value (as C ₂₆ H ₃₂ N ₄ O ₆ ) Actual value C: 58.55 H: 6.36 N: 10.49 Calculated value C: 59.08 H: 6.10 N: 10.60 Example 20 Production of compound 32 OF4949- 300 mg, sodium hydrogen carbonate 100
mg was dissolved in 10 ml of water, 5 ml of ether and 200 μl of carbobenzooxychloride were added, and the mixture was stirred at room temperature.
After 2 hours, the pH was adjusted to 3 with 2N hydrochloric acid, and the resulting precipitate was collected by filtration and dried to obtain 365.0 mg of colorless powder. To 365.0 mg of the powder, 10 ml of acetic anhydride and 100 mg of sodium acetate were added, and after stirring at room temperature for 3 hours, the reaction solution was poured into ice water, and the resulting precipitate was collected by filtration and dried to obtain 383.9 mg of colorless powder. Dissolve 104.2 mg of the powder in 10 ml of a mixture of methanol-acetic acid-water (8:2:1),
25 mg of 10% palladium-carbon was added, and hydrogen gas was passed through the mixture with stirring. After 3 hours, the reaction solution was filtered, and the filtrate was concentrated to dryness. The residue was dissolved in 1 ml of methanol and developed using high performance liquid chromatography using a column: Nucleosil 30C ₁₈ 2.0 cm x 25.0 cm and a developing solution: 0.1 M citrate buffer - acetonitrile to obtain a fraction containing compound 32. Ta. The fraction was concentrated, adsorbed on 20 ml of Amberlite x AD2, washed with water, eluted with 50% methanol, and concentrated to dryness to obtain 22 mg of compound 32 as a colorless powder.

Claims (1)

[Claims] First-order general formula [] [In the formula, R ₁ represents hydrogen, an alkyl group having 1 to 10 carbon atoms, or an alkylcarbonyl group having 1 to 6 carbon atoms, R ₂ represents hydrogen or halogen, and R ₃
represents hydrogen, an alkyl group having 1 to 6 carbon atoms, or an alkylcarbonyl group having 1 to 6 carbon atoms, R ₄ represents hydrogen or an alkyl group having 1 to 6 carbon atoms,
R ₅ represents hydrogen or an alkylcarbonyl group having 1 to 6 carbon atoms; X is a carbamoyl group;
-4 hydroxyalkyl group, or -COOR ₆ (R ₆
represents hydrogen or an alkyl group having 1 to 4 carbon atoms), and Y represents a carbamoyl group and 1 to 4 carbon atoms.
4, or _-COOR7 ( _R7 represents hydrogen, an alkyl group having 1 to 10 carbon atoms, or a benzyl group). However, R ₁ , R ₂ , R ₃ ,
When R ₄ and R ₅ are simultaneously hydrogen, X is a carbamoyl group, and Y is a carboxyl group, and R ₁
Except when is a methyl group, R ₂ , R ₃ , R ₄ and R ₅ are all hydrogen, X is a carbamoyl group, and Y is a carboxyl group. ] A compound represented by or a salt thereof. 2nd order general formula [] [In the formula, R ₁ represents hydrogen, an alkyl group having 1 to 10 carbon atoms, or an alkylcarbonyl group having 1 to 6 carbon atoms, R ₂ represents hydrogen or halogen, and R ₃
represents hydrogen, an alkyl group having 1 to 6 carbon atoms, or an alkylcarbonyl group having 1 to 6 carbon atoms, R ₄ represents hydrogen or an alkyl group having 1 to 6 carbon atoms,
R ₅ represents hydrogen or an alkylcarbonyl group having 1 to 6 carbon atoms; X is a carbamoyl group;
-4 hydroxyalkyl group, or -COOR ₆ (R ₆
represents hydrogen or an alkyl group having 1 to 4 carbon atoms), and Y represents a carbamoyl group and 1 to 4 carbon atoms.
4, or _-COOR7 ( _R7 represents hydrogen, an alkyl group having 1 to 10 carbon atoms, or a benzyl group). However, R ₁ , R ₂ , R ₃ ,
When R ₄ and R ₅ are simultaneously hydrogen, X is a carbamoyl group, and Y is a carboxyl group, and R ₁
Except when is a methyl group, R ₂ , R ₃ , R ₄ and R ₅ are all hydrogen, X is a carbamoyl group, and Y is a carboxyl group. ] An immune enhancer whose main ingredient is a compound represented by: or a salt thereof.