JP3784091B2 - Compound TAN-2294 and its derivatives, production method and use - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention contains a novel compound TAN-2294A or / and B (hereinafter sometimes abbreviated as TAN-2294) having a suppressive action on interleukin 1 production and a derivative thereof, a production method thereof, and TAN-2294 or a derivative thereof It is related with the therapeutic / preventive agent of the disease in which interleukin 1 production inhibitory effect is effective.
[0002]
[Prior art]
Interleukin 1 (hereinafter also abbreviated as IL-1) is a cytokine produced and secreted by various in vivo cells including stimulated monocytes and macrophages, and is an important mediator in immune and inflammatory reactions. It is thought that there is. These factors are immune system cells (eg T cells, B cells), inflammatory cells (monocytes, polynuclear leukocytes), connective tissue cells (eg synovial cells, fibroblasts, chondrocytes), central nervous system cells ( It works on the hypothalamus, astrocytes), hepatocytes, vascular endothelial cells, etc., and promotes each cell function. As a result, differentiation and proliferation of T and B lymphocytes, enhanced production of other lymphokines such as interleukin 2 (IL-2) and colony stimulating factor (CSF), production of collagenase in connective tissue, acute phase protein in liver And various physiological effects such as promotion of bone resorption are expressed [M.Martin et al., Trends in Pharmacological Sciences, Vol. 9, pp. 171-177 (1988)] . Recently, it has been found that it is also involved in inducing production of inflammatory cytokines such as interleukin 6 (IL-6), interleukin 8 (IL-8), and tumor necrosis factor (TNF). Inflammation, Vol. 11, 117-126 (1991)]. IL-1 is known to be involved in heat generation [C. A. Dinarello, Blood, Vol. 77, pp. 1627-1652 (1991)]. Diseases thought to be caused by overproduction of IL-1 include rheumatoid arthritis, osteoporosis, sepsis, inflammatory bowel disease, insulin-dependent diabetes, arteriosclerosis, dryness, asthma, alcoholic hepatitis, etc. -1 dependent disease is known to be myeloid leukemia, and IL-1 is also known to play an important role in transplant rejection of tissues and organs [New England Journal of Medicine, Vol. 328, pp. 106-113 (1993)]. From these, if a substance that suppresses IL-1 production is found, a drug for the treatment and prevention of the above-mentioned diseases and IL-1-dependent tumors caused by excessive IL-1 production, a fever suppressant, In addition, it seems to lead to the development of immunosuppressants and the like during organ transplantation. So far, it has been known that steroid drugs suppress the production of IL-1 [Cellular Immunology, Vol. 69, pages 235-247 (1982)], but it is used in terms of side effects. Limited. Accordingly, there are expectations for non-steroidal IL-1 production inhibitors.
[0003]
Iludol is known as a compound having the same carbon skeleton as the compound of the present invention [TC McMorris et al., Journal of the American Chemical Society (Journal of the American Chemical Society), 89, 4562-4563 (1967)]. M. Anchel et al. Reported that Ildol was an inert compound [Proceedings of the National Academy of Sciences of the United States of America ( Proceedings of the National Academy of Sciences of the United States of America), 36, 300-305 (1950)], IL-1 production inhibitory action has not been reported.
[0004]
[Problems to be solved by the invention]
The present invention seeks to provide a substance that suppresses IL-1 production, which is a therapeutic and prophylactic agent and organ for diseases and IL-1-dependent tumors caused by excessive IL-1 production. It seems to lead to the development of immunosuppressive agents at the time of transplantation.
[0005]
[Means for Solving the Problems]
In view of the present situation, the present inventors have conducted research from a new point of view. As a result, among a large number of microorganisms separated from mushrooms, certain microorganisms produce new substances, and the microorganisms are basidiomycetes. Knowing that it belongs to the phylum and that the active substance that suppresses IL-1 production can be accumulated in the medium by culturing the microorganism in an appropriate medium, the active substance is isolated, and this is called TAN-2294 It was to be. The antibiotic is a neutral substance and exhibits fat solubility. The present inventors have confirmed that these compounds are novel substances from the physicochemical and biological properties of these compounds, and completed the present invention.
[0006]
That is, the present invention
(1) General formula [I]
[Chemical 2]
(Wherein R¹Is a formyl group or an optionally substituted hydroxymethyl group, R²Represents an optionally substituted hydroxyl group. Or a salt thereof,
(2) In the general formula [I], R¹Is an optionally substituted hydroxymethyl group, R²Is a hydroxyl group which may be substituted, the compound or a salt thereof according to (1),
(3) In the general formula [I], R¹Is a formyl group, R²Is a hydroxyl group which may be substituted, the compound or a salt thereof according to (1),
(4) In the general formula [I], R¹Is a hydroxymethyl group, R²Is a hydroxyl group, the compound TAN-2294A according to (1),
(5) In the general formula [I], R¹Is a formyl group, R²Is a hydroxyl group, the compound TAN-2294B according to (1),
(6) A microorganism belonging to Basidiomycotina and capable of producing the compounds TAN-2294A and / or B of (3) and (4) is cultured in a medium, and TAN-2294A or / and B is A process for producing a compound TAN-2294A or / and B, characterized in that the product is accumulated and collected;
(7) The production method according to (6), wherein the microorganism is basidiomycete FL-61524 strain,
(8) A medicament comprising the compound or salt thereof according to (1) and
(9) It relates to an interleukin 1 production inhibitor comprising the compound according to (1) or a salt thereof.
[0007]
R above¹An optionally substituted hydroxymethyl group and R²Examples of the substituent in the hydroxyl group which may be substituted include a hydrocarbon group, a heterocyclic group, an acyl group, a carboxyl group which may be esterified, and a carbamoyl group which may be substituted.
Examples of the hydrocarbon group include hydrocarbon groups having 1 to 20 carbon atoms. Among them, preferred examples are hydrocarbon groups having 1 to 10 carbon atoms. Particularly preferred examples include an alkyl group, alkenyl group, alkynyl group, aryl group, aralkyl group, cycloalkyl group, cycloalkyl-alkyl group and the like.
[0008]
Preferred examples of the alkyl group include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1-methylbutyl, 2-methylbutyl, 1, 2-dimethylpropyl, 1-ethylpropyl, hexyl, isohexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl -1-methylpropyl, 1-ethyl-2-methylpropyl, heptyl, isoheptyl, octyl, isooctyl, nonyl, isono Decyl, isodecyl, undecyl, isoundecyl, dodecyl, isododecyl, tridecyl, isotridecyl, tetradecyl, isotetradecyl, hexadecyl, isohexadecyl, octadecyl, etc., among them methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec Particularly preferred are -butyl, tert-butyl, pentyl, hexyl, octyl, decyl, dodecyl, tetradecyl and the like.
Preferred examples of the alkenyl group include vinyl, allyl, isopropenyl, 1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 1-methyl-2-propenyl, 2 -Methyl-1-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl -1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1 -Methyl-1-pentenyl, 2-methyl-1-pentenyl, 4-methyl-3-pentenyl, 2-ethyl-1-butenyl, 2-heptenyl, 2-octenyl, 2-nonenyl, 2-decenyl, 2-undecenyl 2-dodecenyl 2-tridecenyl, 2-tetradecenyl, 9-tetradecenyl, 2-hexadecenyl, 9-hexadecenyl, 2-octadecenyl, 9-octadecenyl, 9,12-octadienyl, 9,12,15-octatrienyl, among others Vinyl, allyl, isopropenyl, 2-methyl-1-propenyl and the like are particularly preferable.
Preferred examples of the alkynyl group include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 1-pentynyl, 2-pentynyl, 3- Pentynyl, 4-pentynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 2-heptynyl, 2-octynyl, 2-nonynyl, 2-decynyl, 2-undecynyl, 2-dodecynyl, 2-tridecynyl, 2-tetradecynyl, 9-tetradecynyl, 2-hexadecynyl, 9-hexadecynyl, 2-octadecynyl, 9-octadecynyl, 9,12-octadienyl, 9,12,15-octatrienyl and the like can be mentioned, among which ethynyl, 1-propynyl, 2-propynyl and the like are particularly preferable.
[0009]
Preferable examples of the aryl group include aryl groups having 6 to 12 carbon atoms, such as phenyl, tolyl, o-xylyl, m-xylyl, p-xylyl, biphenyl, etc. Among them, phenyl and tolyl are particularly preferable.
Preferable examples of the aralkyl group include benzyl, phenethyl, 3-phenylpropyl, benzhydryl, triphenylmethyl and the like, and among them, benzyl, phenethyl, benzhydryl and the like are particularly preferable.
Preferable examples of the cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.
Preferred examples of the cycloalkyl-alkyl group include cyclopropylmethyl, cyclopropylethyl, cyclopropylpropyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cycloheptylmethyl, and the like. Cyclopropylmethyl, cyclopropylethyl, cyclopentylmethyl, cyclohexylmethyl and the like are particularly preferable.
Examples of the heterocyclic group include non-aromatic heterocyclic groups such as oxiranyl, oxetanyl, tetrahydrofuryl, tetrahydropyranyl, dioxanyl, oxolanyl and the like.
Examples of the acyl group include an organic acid-derived acyl group, and among them, an organic carboxylic acid, an organic sulfonic acid, or an amino acid-derived acyl group is preferable.
Examples of the acyl group derived from the organic carboxylic acid include a chain or cyclic group having 1 to 20 carbon atoms, or an acyl group derived from an aromatic carboxylic acid. Among them, preferred examples include acyl groups having 1 to 10 carbon atoms, such as alkanoyl group, alkenoyl group, alkinoyl group, aroyl group, cycloalkyl-alkanoyl group and the like.
[0010]
Preferred examples of the alkanoyl group include, for example, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, isopentanoyl, hexanoyl, isohexanoyl, 2-methylpentanoyl, 3-methylpentanoyl and 2,2-dimethylbutano Noyl, 3,3-dimethylbutanoyl, 2,3-dimethylbutanoyl, 2-ethylbutanoyl, heptanoyl, isoheptanoyl, octanoyl, isooctanoyl, nonoyl, isononoyl, decanoyl, isodecanoyl, undecanoyl, isoundecanoyl, dodecanoyl, isododecanoyl , Tridecanoyl, isotridecanoyl, tetradecanoyl, isotetradecanoyl, hexadecanoyl, isohexadecanoyl, octadecanoyl, etc. , Propionyl, butyryl, isobutyryl, pentanoyl, isopentanoyl, hexanoyl, iso-hexanoyl, octanoyl, decanoyl, dodecanoyl, such as tetradecanoyl is particularly preferred.
Preferred examples of the alkenoyl group include propenoyl, 2-butenoyl, 3-butenoyl, 2-methyl-2-propenoyl, 2-pentenoyl, 3-pentenoyl, 4-pentenoyl, 2-methyl-2-butenoyl and 3- Methyl-2-butenoyl, 2-hexenoyl, 3-hexenoyl, 4-hexenoyl, 5-hexenoyl, 2,4-hexadienoyl, 4-methyl-3-pentenoyl, 2-heptenoyl, 2-octenoyl, 2-nonenoyl, 2- Decenoyl, 2-undecenoyl, 2-dodecenoyl, 2-tridecenoyl, 2-tetradecenoyl, 9-tetradecenoyl, 2-hexadecenoyl, 9-hexadecenoyl, 2-octadecenoyl, 9-octadecenoyl, 9,12-octadenoyl, 9,12,15- Such as octatrienoyl, propenoyl, 2-but Noyl, 3-butenoyl, 2-methyl-2-propenoyl, 3-methyl-2-butenoyl, 2-hexenoyl, 3-hexenoyl, 4-hexenoyl, 5-hexenoyl, 2,4-hexadienoyl, 3,5-hexadienoyl, Particularly preferred are 9-hexadecenoyl, 9-octadecenoyl, 9,12-octadienoyl, 9,12,15-octatrienoyl and the like.
Preferred examples of the alkinoyl group include propinoyl, 2-butinoyl, 3-butinoyl, 2-pentinoyl, 3-pentinoyl, 4-pentinoyl, 2-hexinoyl, 3-hexinoyl, 4-hexinoyl, 5-hexinoyl, 2- Heptinoyl, 2-octinoyl, 2-noninoyl, 2-decinoyl, 2-undecinoyl, 2-dodecinoyl, 2-tridecinoyl, 2-tetradecinoyl, 9-tetradecinoyl, 2-hexadecinoyl, 9-hexadecinoyl, 2-octadecinoyl, 9-octadecinoyl, 9,12-octadinoinoyl, 9,12,15-octatriinoyl and the like can be mentioned, among which propinoyl, 2-butinoyl, 3-butinoyl and the like are particularly preferable.
Preferable examples of the aroyl group include an aroyl group having 6 to 13 carbon atoms such as benzoyl, 1-naphthalenecarbonyl, 2-naphthalenecarbonyl, biphenylcarbonyl, etc. Among them, phenyl, tolyl and the like are particularly preferable.
Preferred examples of the cycloalkyl-acanoyl group include, for example, cyclopropanecarbonyl, cyclopropylacetyl, 2-cyclopropylpropionyl, 3-cyclopropylpropionyl, cyclobutanecarbonyl, cyclopentanecarbonyl, cyclohexanecarbonyl, cyclohexylacetyl, 2-cyclohexylpropionyl. Examples include 3-cyclohexylpropionyl, cycloheptanecarbonyl, etc. Among them, cyclopropanecarbonyl, cyclobutanecarbonyl, cyclopentanecarbonyl, cyclohexanecarbonyl and the like are particularly preferable.
[0011]
Examples of acyl groups derived from organic sulfonic acids include chain or cyclic such as methanesulfonyl, ethanesulfonyl, propanesulfonyl, butanesulfonyl, cyclohexanesulfonyl, octanesulfonyl, decanesulfonyl, dodecanesulfonyl, benzenesulfonyl, toluenesulfonyl, trifluoromethanesulfonyl, etc. Or the acyl group derived from aromatic sulfonic acid is mentioned. Of these, methanesulfonyl, ethanesulfonyl, octanesulfonyl, dodecanesulfonyl, benzenesulfonyl, toluenesulfonyl, trifluoromethanesulfonyl and the like are particularly preferable.
Examples of the acyl group derived from an amino acid include an acyl group derived from a natural type or non-natural type amino acid. Examples of acyl groups derived from natural and unnatural amino acids include glycyl, L- and D-alanyl, L- and D-valyl, L- and D-leucyl, L- and D-isoleucil, L- and D -Prolyl, L- and D-hydroxyprolyl, L- and D-phenylalanyl, L- and D-tryptophanyl, L- and D-seryl, L- and D-threonyl, L- and D-tyrosyl, L -And D-asparagyl, L- and D-asparaginyl, L- and D-glutamyl, L- and D-glutaminyl, L- and D-methionyl, L- and D-cysteinyl, L- and D-cystyl, L -And D-lysyl, L- and D-arginyl, L- and D-histidyl and the like, among others glycyl, L- and D-hydroxyprolyl, L- and D-seryl, L- and D-threonyl Such as L- and D- tyrosyl is particularly preferred.
[0012]
Examples of the carboxyl group that may be esterified include a carboxyl group derived from carbonic acid, an alkoxycarbonyl group, an alkenyloxycarbonyl group, an aralkyloxycarbonyl group, and the like, such as a carboxyl group, a methoxycarbonyl group, an ethoxycarbonyl group, and a propoxy group. Examples thereof include a carbonyl group, an n-butoxycarbonyl group, a tert-butoxycarbonyl group, a vinyloxycarbonyl group, and a benzyloxycarbonyl group.
Examples of the optionally substituted carbamoyl group include a carbamoyl group, a lower alkyl group and an N-mono or disubstituted carbamoyl group (eg, N-methylcarbamoyl group, N, N-dimethylcarbamoyl group, N-ethylcarbamoyl group). ), Nitrogen-containing heterocyclic substituted carbonyl groups (eg, morpholinocarbonyl group, piperidinocarbonyl group, piperazinocarbonyl group, pyrrolidinocarbonyl group, pyrrolinocarbonyl group, imidazinocarbonyl group, etc.) .
The lower alkyl group, which is a substituent of the alkoxycarbonyl group, alkenyloxycarbonyl group, aralkyloxycarbonyl group, carbamoyl group, may be further substituted with a hydroxyl group, a carboxyl group, a halogen group, an alkoxy group, an alkyl group or an aryl group. It may be substituted with an amino group, acyloxy group, nitro group, nitrile group or heterocyclic group which may be mono- or di-substituted with a group or an alkyl group.
[0013]
The hydrocarbon group, heterocyclic group, and acyl group are 1 to 5 suitable substituents such as a hydroxyl group, a carboxyl group, a halogen group, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, and an aralkyl group. A thiol group that may be substituted with an alkoxy group, an alkyl or aryl group, a sulfinyl group that may be substituted with an alkyl or aryl group, a sulfonyl group that may be substituted with an alkyl or aryl group, Alternatively, it may be substituted with an optionally substituted amino group, acyloxy group, nitro group, nitrile group, heterocyclic group, or the like.
Since the compound of the present invention has four asymmetric carbons in the skeleton, 16 types of isomers can exist, and each of these isomers and mixtures thereof are also included in the present invention. Further, when the substituent has an asymmetric carbon, a stereoisomer is similarly produced, and each of these isomers and a mixture thereof are also included in the present invention.
When the compound of the present invention has an acidic group or basic group as a substituent, a salt with an appropriate base or acid can be formed. The type of salt is preferably a pharmacologically acceptable salt, for example, a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid. Etc. Examples of the salt with an inorganic base include an alkali metal salt (eg, sodium salt, potassium salt, etc.), an alkaline earth metal salt (eg, calcium salt, magnesium salt, etc.), or an ammonium salt. Examples of the salt with an organic base include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, dicyclohexylamine and the like. Examples of the salt with an inorganic acid include salts with hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid and the like. Examples of the salt with an organic acid include salts with formic acid, acetic acid, oxalic acid, fumaric acid, maleic acid, succinic acid, citric acid, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, and the like. Examples of salts with basic amino acids include salts with arginine, lysine, ornithine, and examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, and the like.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Any microorganism may be used as long as it is a microorganism belonging to Basidiomycotina and capable of producing TAN-2294 as a microorganism producing TAN-2294 used in the production method of the present invention. As an example, basidiomycete FL-61524 strain isolated from mushrooms collected in Hiroshima can be mentioned.
The mycological properties of the FL-61524 strain are shown below.
(A) Growth state in each medium
(1) Malt extract agar medium
Growth was moderate, and the diameter of the colonies after 2 weeks at 24 ° C. was 53 mm. The surface is flat, felt-like hyphae spread radially, and form a white powdery settlement in the center. The outer edge is regularly trimmed. White to grayish white is exhibited from the center to the middle, and the periphery is ivory. A light yellowish brown color is exhibited from the center to the middle of the back surface, and the periphery is yellowish white. Production of soluble pigment is not observed.
(2) Potato / glucose agar medium
Growth was moderate, colony diameter after 2 weeks at 24 ° C. was 55 mm. The surface is flat, and the felt-like mycelium spreads radially, forming a white powdery settlement around the periphery. The outer edge is regularly trimmed. From gray to yellowish white to grayish white from the center to the middle, and from yellowish white to grayish white at the periphery. Ivory color is exhibited from the center to the middle of the back surface, and the periphery is grayish white.
It grows at any of pH 3 to pH 12, the growth temperature range is 7 ° C. to 35 ° C., and the optimum temperature is 24 ° C. to 30 ° C. It does not grow at 37 ° C.
Production of soluble pigment is not observed.
(3) Czapek agar medium
It does not grow on this medium.
(4) Oatmeal agar medium
Growth was moderate and the diameter of the colonies after 2 weeks at 24 ° C. was 50 mm. The surface is flat and consists of felt-like hyphae, forming a powdery settlement from the center to the periphery. The outer edge is bordered somewhat irregularly. Grayish white from the center to the periphery. A light yellowish brown to yellowish grayish white is exhibited from the center to the periphery of the back surface. Production of soluble pigment is not observed.
(B) Morphological features
Mycelium: The diameter is 1.2 to 1.8 μm. Slight concatenation is permitted.
A large number of egg-shaped thick spore-like organs are formed from the hyphae.
The size is 4.0 to 6.0 × 5.7 to 7.7 μm.
When the above properties were collated with Shunichi Udagawa and Keisuke Tsuji, “Fungus Encyclopedia (below)” (1978, Kodansha Scientific), it was determined that this strain belongs to Basidiomycotina. Therefore, this strain was identified as basidiomycete FL-61524.
[0015]
The FL-61524 strain was deposited with the Foundation Fermentation Research Institute (IFO) on June 14, 1995 under the accession number IFO32701. On August 2, 1995, the Institute of Industrial Technology, Ministry of International Trade and Industry Deposited at the Technical Research Institute (NIBH) under the accession number FERM P-15084.
As in the case of other actinomycetes, the TAN-2294-producing bacteria belonging to the Basidiomycete subfamily should be mutated by, for example, irradiation with ultraviolet rays, X-rays, radiation, etc., single spore separation, various mutation treatments, and other means. Even if it is such a mutant strain or a naturally obtained mutant strain, it is substantially different from the above taxonomic characteristics and has the property of producing the compound. Can all be used in the method of the present invention.
The medium used for culturing the compound-producing bacterium of the present invention may be liquid or solid as long as it contains a nutrient source that can be used by the bacterium, but it is more appropriate to use a liquid medium when treating in large quantities. The medium is appropriately mixed with a carbon source, a nitrogen source, an inorganic substance, and a trace nutrient source that can be assimilated by the compound-producing bacteria. Examples of the carbon source include glucose, lactose, sucrose, maltose, dextrin, starch, soluble starch, glycerin, mannitol, sorbitol, fats and oils (eg, soybean oil, lard oil, chicken oil, etc.), n-paraffin, etc. Examples of the nitrogen source include meat extract, yeast extract, dry yeast, soybean flour, corn steep liquor, peptone, polypeptone, cottonseed flour, molasses, urea, ammonium salts (eg, ammonium sulfate, ammonium chloride, ammonium nitrate, Ammonium acetate).
In addition, salts containing sodium, potassium, calcium, magnesium, etc., salts containing metals such as iron, manganese, zinc, cobalt and nickel, salts such as phosphoric acid and boric acid, and salts of organic acids such as acetic acid and propionic acid You may use suitably. In addition, amino acids (eg, glutamic acid, aspartic acid, alanine, lysine, methionine, proline, etc.), peptides (eg, dipeptide, tripeptide, etc.), vitamins (eg, B1, B2, nicotinic acid, B12, C, etc.), Nucleic acids (eg, purines, pyrimidines, derivatives thereof, etc.) may be included. Of course, an inorganic or organic acid or alkali, a buffering agent or the like may be added for the purpose of adjusting the pH of the medium, or an appropriate amount of oils or fats, a surfactant or the like may be added for the purpose of defoaming. In the liquid culture, the pH of the medium is preferably near neutral, for example, pH 4.5 to 8, particularly preferably pH 5 to 7. The culture temperature is preferably about 24 ° C. to 30 ° C., and the culture time is preferably about 7 to 20 days, particularly preferably about 10 to 18 days.
The quantification of TAN-2294 produced with the progress of culture was performed using the inhibitory activity of IL-1 production in THP-1 cells according to Test Example 1 described later as an index. Usually, the production of TAN-2294 reaches the maximum after 12 to 16 days of culture.
[0016]
A method for collecting the target compound TAN-2294 from the culture is described below.
Since this compound is a fat-soluble substance and exhibits neutrality, a general means utilizing this property may be employed. Moreover, since this compound is mainly contained in the filtrate, the following purification method is employed.
First, after adjusting the culture solution to pH 1.5 to 12, preferably pH 3 to 9, a filter aid is added and the cells are removed by filtration or centrifugation. The obtained filtrate is mixed with an organic solvent immiscible with water, such as hexane, toluene, diethyl ether, dichloromethane, dichloroethane, ethyl acetate, methyl isobutyl ketone, chloroform, butanol, and the like, added for 10 minutes to 10 hours, preferably 30 minutes to 2 The crude material containing TAN-2294 is obtained by mixing with stirring for a period of time and concentrating the resulting organic solvent layer. At this time, an appropriate salt (eg, sodium chloride, ammonium chloride, sodium sulfate, ammonium sulfate, sodium hydrogen carbonate, sodium carbonate, potassium hydrogen carbonate, potassium carbonate, etc.) may be added in advance to the filtrate.
Various chromatographic methods are advantageously used to further purify the crude material to obtain pure TAN-2294A and B. Carriers used in the chromatography method include silica gel, crystalline cellulose, activated carbon, adsorptive resins such as Diaion HP-20 and SP-207 (Mitsubishi Kasei), Amberlite XAD-I or II (Rohm and Haas) Such as Biogel P (manufactured by Bio-Rad, USA), Sephadex LH-20 (manufactured by Pharmacia, Sweden), etc., which are usually performed by column chromatography. It is. The elution of the active substance from the carrier varies depending on the type of the carrier, but a suitable organic solvent such as dichloroethane, toluene, ethyl acetate, hexane, acetone, 2-propanol, methanol alone or a mixed solvent, or miscible with water. A mixed solvent of an organic solvent such as acetone, acetonitrile, methanol and the like and an aqueous solution such as water, dilute alkaline water, dilute acid water, and buffer solution can be used.
TAN-2294A and B can also be purified by preparative high performance liquid chromatography (HPLC). As the carrier, octadecylsilane (ODS) and silica gels are advantageously used. For example, in the case of ODS, methanol or acetonitrile and water, or a mixed solution of an aqueous solution containing salts or acids is advantageously used. The eluate containing the active substance is concentrated to dryness or lyophilized to give pure TAN-2294 as a colorless or yellow oil. Further, compound TAN-2294A is a suitable organic solvent such as toluene, ethyl acetate, hexane, diethyl ether, diisopropyl ether, acetone, 2-propanol, methanol, etc., or a mixed solvent, preferably ethyl acetate, hexane, diethyl ether, diisopropyl ether. It can be obtained as a white or yellow solid or powder by pulverizing from a mixed solvent such as 0 to 30 ° C.
[0017]
Compounds TAN-2294A and B are neutral fat-soluble substances, and as physicochemical properties, they are positive for iodine, phosphomolybdic acid and sulfuric acid in the color reaction, and against Ehrlich, Dragendorf and Sakaguchi reagents. Negative. The behavior in silica gel thin layer chromatography (TLC) is shown in [Table 1].
[Table 1]
[0018]
The physicochemical properties of the compounds TAN-2294A and B obtained in Example 2 described later are shown below.
TAN-2294A
(1) Appearance: White powder
(2) Molecular weight: m / z 250 (M⁺ ), 232 (M⁺-H2O) (from EI mass spectrum)
(3) Elemental analysis value: C₁₅H_{twenty two}O_Three・ 0.2H₂As O
Actual value; C, 70.71; H, 8.89 (%)
Calculated value; C, 70.95; H, 8.89 (%)
(4) Molecular formula: C₁₅H_{twenty two}O_Three
(5) Ultraviolet absorption (UV) spectrum: in methanol (Fig. 1)
Maximum 256 nm (E1% 335)
(6) Infrared absorption (IR) spectrum: in KBr tablet (Fig. 2)
Shows main absorption (wave number, cm^-1)
3430, 2950, 2860, 1730, 1660, 1460, 1390, 1100, 1050
(7)¹³C nuclear magnetic resonance (NMR) spectrum: 75 MHz in deuterated methanol (Fig. 3)
δppm; 198.5 (Q), 150.6 (Q), 150.5 (Q), 74.9 (CH), 61.2 (CH2), 60.7 (CH2), 53.3 (CH), 47.9 (CH2), 47.2 (CH), 42.3 (CH2 ), 41.4 (CH2), 37.4 (Q), 29.9 (CH3), 27.3 (CH3), 20.6 (CH3).
(8)¹1 H NMR spectrum: 300 MHz in deuterated methanol (Fig. 4)
δppm; 4.47 (1H, dd, J = 16.1, 1.2 Hz), 4.38 (1H, dd, J = 16.1, 1.2 Hz), 4.18 (1H, brd, J = 7.9 Hz), 2.82 (1H, d, J = 16.9 Hz), 2.69 (1H, d, J = 16.9 Hz), 2.49 (1H, m), 2.44 (1H, m), 1.85 (1H, dd, J = 12.0, 6.9 Hz), 1.55 (1H, m) , 1.52 (1H, m), 1.26 (1H, dd, J = 12.0, 9.9 Hz), 1.17 (3H, s), 1.14 (3H, s), 1.02 (3H, s).
[0019]
TAN-2294B
(1) Appearance: colorless oil
(2) Molecular formula: C₁₅H₂₀O_Three
(3) Ultraviolet absorption (UV) spectrum: in methanol
Maximum 252 nm (E1% 318)
(4) Infrared absorption (IR) spectrum: in KBr tablet (Fig. 5)
Shows main absorption (wave number, cm^-1)
3470, 2950, 2870, 1750, 1680, 1460, 1390, 1140, 1060
(Five)¹³C NMR spectrum: 75 MHz, in deuterated chloroform (Fig. 6)
δppm; 194.3 (Q), 190.1 (CH), 170.0 (Q), 136.1 (Q), 72.9 (CH), 61.6 (CH2), 51.2 (CH), 46.7 (CH2), 46.0 (CH), 41.4 (CH2 ), 40.4 (Q), 38.5 (Q), 29.5 (CH3), 27.1 (CH3), 20.0 (CH3).
(6)¹1 H NMR spectrum: 300 MHz in deuterated chloroform (Fig. 7)
δppm; 9.98 (1H, s), 4.44 (1H, d, J = 7.6 Hz), 2.00 (1H, d, J = 17.2 Hz), 2.93 (1H, d, J = 17.2 Hz), 2.53 (2H, m ), 1.96 (1H, ddd, J = 12.5, 6.9, 1.6 Hz), 1.60 (1H, ddd, J = 12.7, 7.6, 1.7 Hz), 1.53 (1H, dd, J = 12.7, 9.3 Hz), 1.27 ( 1H, m), 1.24 (3H, s), 1.15 (3H, s), 1.01 (3H, s).
From the physicochemical and biological properties described above, TAN-2294A and B were found to be novel compounds.
[0020]
Next, a method for producing the compound of the present invention will be described.
Among the compounds represented by the general formula [I], the compound in which the substituent in the optionally substituted hydroxymethyl group or the optionally substituted hydroxyl group is a hydrocarbon group or a heterocyclic group is represented by the general formula [ R] of the compounds represented by I]¹Is a hydroxymethyl group, or R²Is produced by reacting with a compound having a leaving group in the presence of a base using a compound in which is a hydroxyl group (hereinafter sometimes abbreviated as compound [II]) or a salt thereof as a raw material. Examples of the base include alkali metal hydrides or alkaline earth metal hydrides (eg, sodium hydride, potassium hydride, calcium hydride, etc.), alkali metal alkoxides (eg, sodium methoxide, sodium ethoxy). , Potassium tert-butoxide, etc.), inorganic bases (eg, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide), organic bases (eg, pyridine, 2,4,6-trimethylpyridine) , Aromatic bases such as picoline, 4-dimethylaminopyridine, 2,6-lutidine, diazabicycloundecene, tertiary amines such as triethylamine, diisobutylethylamine, dimethylaniline), alkali metals (eg, sodium, Potassium), alkyl lithiums (eg, methyl lithium, Butyllithium such like), or alkali metal amides (e.g., lithium diisopropylamide, lithium bis trimethylsilyl amide, sodium bis trimethylsilyl amide) and the like. Of these, liquid ones are also used as solvents. Compounds having a leaving group include halides such as iodide (eg, methyl iodide, ethyl iodide, propyl iodide, isopropyl iodide, butyl iodide, isobutyl iodide, pentyl iodide, iodoacetic acid, iodoacetamide). , Iodoacetonitrile, methyl iodoacetate, tert-butyl iodoacetate, etc.), bromides (eg, ethyl bromide, propyl bromide, isopropyl bromide, butyl bromide, isobutyl bromide, pentyl bromide, allyl bromide, bromide) Benzyl, tert-butyl bromide, triphenylmethyl bromide), chlorides (eg methoxymethyl chloride, methoxyethoxymethyl chloride, methylthiomethyl chloride, tetrahydropyranyl chloride, tetrahydrofuryl chloride, etc.), sulfonate esters ( For example, methyl p-toluenesulfonate, ethyl p-toluenesulfonate P-toluenesulfonate esters, methanesulfonate esters such as methyl methanesulfonate, butyl methanesulfonate, benzenesulfonate esters such as methyl benzenesulfonate, butylbenzenesulfonate, ethyl trifluoromethanesulfonate, etc. Trifluoromethanesulfonic acid esters, etc.), sulfate esters (eg, dimethyl sulfate, diethyl sulfate, etc.) or epoxides (eg, propylene oxide, glycidol, etc.). Of these, halides and sulfonic acid esters can be obtained from the corresponding alcohols by known methods. Solvents include amides (eg, dimethylformamide, dimethylacetamide, etc.), ethers (eg, tetrahydrofuran, diethyl ether, dioxane, dimethoxyethane, etc.), sulfoxides (eg, dimethylsulfoxide, etc.), alcohols (eg, methanol) , Ethanol, 1-propanol, 2-propanol, tert-butanol, etc., nitriles (eg, acetonitrile, etc.), ketones (eg, acetone, methyl isobutyl ketone, etc.), esters (eg, ethyl formate, ethyl acetate, Tert-butyl acetate, etc.), hydrocarbons (eg, hexane, benzene, toluene, etc.), amines (eg, liquid ammonia, methylamine, etc.), or a mixed solvent thereof. Of these, dimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane, dimethoxyethane and the like are preferable. These are particularly preferably anhydrous solvents. The base is used in an amount of 1 to 50 equivalents, preferably 1 to 5 equivalents, the compound having a leaving group is used in an amount of 1 to 10 equivalents, preferably 1 to 5 equivalents, and the reaction temperature is -70 ° C to 150 ° C, preferably 0 ° C. The reaction time is 80 ° C. and 2 minutes to 5 days, preferably 10 minutes to 2 days.
[0021]
The compound in which the substituent in the optionally substituted hydroxymethyl group or the optionally substituted hydroxyl group is a hydrocarbon group or a heterocyclic group is obtained from the compound [II] or a salt thereof, for example, in the presence of an acid catalyst. It is produced by subjecting to acetalization or ketalization reaction. The reaction is carried out by reacting the starting compound or a salt thereof in the presence of a suitable acid catalyst, for example, enol ethers (eg, dihydropyran, dihydrofuran, 2-methoxypropene, 2-ethoxypropene, ethyl vinyl ether, isopropyl vinyl ether, glucar which may be protected). , Optionally protected galactals), acetals (eg, 1,1-dimethoxyethane, 1,1-dimethoxypropane, 1,1-diethoxyethane, 1,1-diethoxypropane, 2,2) -Dimethoxypropane, 2,2-diethoxypropane, 2,2-diethoxybutane, etc.). Preferred examples of the acid used include, for example, haloacetic acid (eg, trifluoroacetic acid, etc.), inorganic acid (eg, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc.), organic carboxylic acid (eg, acetic acid, Citric acid, tartaric acid, oxalic acid, etc.), Lewis acid (eg, zinc-acetic acid, boron trifluoride ether complex, etc.), organic sulfonic acid (eg, benzenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, etc.), etc. Among them, hydrochloric acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, camphorsulfonic acid, boron trifluoride ether complex and the like are particularly preferable. Solvents include ethers (eg, tetrahydrofuran, diethyl ether, dioxane, dimethoxyethane, etc.), nitriles (eg, acetonitrile, etc.), esters (eg, ethyl formate, ethyl acetate, tert-butyl acetate, etc.), halogenated Examples include hydrocarbons (eg, dichloromethane, chloroform, etc.), amides (eg, dimethylformamide, dimethylacetamide, etc.), sulfoxides (eg, dimethyl sulfoxide, etc.), and mixed solvents thereof. The acid is used in an amount of 1 to 20 equivalents, preferably 1 to 5 equivalents, and the reaction temperature is -20 ° C to 150 ° C, preferably 0 ° C to 100 ° C. The reaction time is 10 minutes to 7 days, preferably 20 minutes to 2 days.
[0022]
Of the compounds represented by the general formula [I], the compound [II] or a salt thereof may be used for the optionally substituted hydroxymethyl group or the compound in which the substituent in the optionally substituted hydroxyl group is an acyl group. Is produced by subjecting to acylation reaction in the presence of a base. As the acylating agent used in this reaction, for example, an organic acid that induces an acyl group or a reactive derivative thereof is used.
For example, when an organic carboxylic acid is used, it can be acylated by reacting with an appropriate condensing agent. Examples of condensing agents include diimides such as dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, or the above condensing agent and pentachlorophenol, 2,4,5-trichlorophenol, 2 , 4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxy-5-norbornene-2,3-dicarboximide, N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxybenztriazole, etc. A mixture of these is used. Alternatively, a mixture of azodicarboxylic acid esters (eg, diethyl azodicarboxylate) and trialkylphosphine (eg, triphenylphosphine, tributylphosphine) is used.
Alternatively, when reactive derivatives of organic acids are used, examples of these reactive derivatives include acid anhydrides, acid halides, acid active esters, acid active amides, and acid active thioesters. Acid anhydrides are, for example, symmetrical acid anhydrides such as acetic anhydride, propionic anhydride, and methanesulfonic anhydride, or mixed acid anhydrides such as acetic anhydride, propionic anhydride, benzoic anhydride, and trichloroacetic anhydride. Etc. Examples of the acid halide include acid chloride, acid bromide and the like, for example, acetyl chloride, acetyl bromide, propionyl chloride, butyryl chloride, isobutyryl chloride, valeryl chloride, isovaleryl chloride, methanesulfonyl chloride, benzene. Examples include sulfonyl chloride and toluenesulfonyl chloride. Acid active esters include, for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxy-5-norbornene-2,3-dicarboxyl. Examples thereof include esters with imide, N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxybenzotriazole and the like. Examples of the acid active amides include amides with pyrazole, dimethylpyrazole, imidazole, 4-substituted imidazole, benzotriazole and the like. Examples of the acid-active thioesters include thioesters with heterocyclic thiols such as 2-pyridylthiol and 2-benzothiazolylthiol.
About 1 to 50 equivalents, preferably about 1 to 10 equivalents, of the acylating agent and the condensing agent such as an organic acid for deriving an acyl group or a reactive derivative thereof are used. These acylation reactions are advantageously performed in the presence of a base in a solvent that does not affect the reaction. Solvents that do not affect the reaction include, for example, the solvents and halogenated hydrocarbons used in the production of the compound in which the substituent in the above-mentioned optionally substituted hydroxyl group is a hydrocarbon group, such as dichloromethane, chloroform, 1, 2-Dichloroethane or the like is used, and dichloromethane is particularly preferable. These are particularly preferably anhydrous solvents. As the base, for example, a base used in the production of a compound in which the substituent in the optionally substituted hydroxyl group is a hydrocarbon group is used. The base is used in an amount of 1 to 50 equivalents, preferably 1 to 5 equivalents, the reaction temperature is -70 ° C to 150 ° C, preferably 0 ° C to 80 ° C, the reaction time is 2 minutes to 5 days, preferably 10 minutes to 2 days. It is.
[0023]
Of the compounds represented by the general formula [I], the compound in which the substituent in the optionally substituted hydroxymethyl group or the optionally substituted hydroxyl group is an optionally esterified carboxyl group is the compound [ II] or a salt thereof as a raw material and subjected to a carboxylation reaction in the presence of a base. As the carboxylating agent used in this reaction, for example, a reactive derivative of carbonic acid that induces a carboxyl group is used.
Examples of carbonic acid reactive derivatives include phosgene, oxalyl chloride, carbonates (eg, dimethyl carbonate, diethyl carbonate, diisopropyl carbonate, etc.), haloformates (eg, methyl chloroformate, ethyl chloroformate, chloroformate 1 -Chloroethyl, chloroformic acid 2,2,2-trichloroethyl, propyl chloroformate, isopropyl chloroformate, butyl chloroformate, isobutyl chloroformate, tert-butyl chloroformate, etc.).
[0024]
Among the compounds represented by the general formula [I], a compound in which the optionally substituted hydroxymethyl group or the optionally substituted hydroxyl group is a substituted carbamoyl group is represented by the general formula: Of the compounds represented by [I], R¹Hydroxyl group in the hydroxymethyl group of R, or R²A compound in which at least one of the hydroxyl groups represented by formula (I) is a hydroxyl group substituted with an active esterified carboxyl group or an active amidated carbonyl group (hereinafter sometimes abbreviated as compound [III]) And a carbamoylation reaction, that is, by reacting with a primary or secondary amine in the presence of a base. Examples of the active esterified carboxyl group in compound [III] include 1-chloroethoxycarbonyl, 1-chloropropoxycarbonyl, 2,2,2-trichloroethoxycarbonyl and the like, which are produced by the above carboxylation reaction and the like. Is done. Examples of the active amidated carbonyl group in the compound [III] include an imidazinocarbonyl group. For example, the compound [II] is used as a raw material and reacted with 1,1′-carbonyldiimidazole or the like. Manufactured. This reaction is advantageously carried out in a solvent that does not affect the reaction. For example, the solvent used in the acylation reaction is used, and dichloroethane, chloroform, benzene, toluene and the like are particularly preferable. These are particularly preferably anhydrous solvents. The active amidating agent such as 1,1′-carbonyldiimidazole is used in an amount of 1 to 50 equivalents, preferably 1 to 20 equivalents, the reaction temperature is 0 ° C. to 150 ° C., preferably 20 ° C. to 100 ° C., and the reaction time is 2 Min to 72 hours, preferably 10 minutes to 48 hours.
[0025]
The carboxylation reaction and carbamoylation reaction are advantageously performed in a solvent that does not affect the reaction. As the solvent that does not affect the reaction, for example, the solvent used in the above acylation reaction and the like are used, and among them, dichloromethane, chloroform and the like are preferable. These are particularly preferably anhydrous solvents. Examples of the base include bases used in the production of a compound in which the substituent at the hydroxyl group which may be substituted is a hydrocarbon group, among which pyridine, triethylamine, diisopropylethylamine, diazabicycloundecene and the like are used. Organic bases are preferred. The base is used in an amount of 1 to 30 equivalents, preferably 1 to 5 equivalents, the reaction temperature is −50 ° C. to 150 ° C., preferably −20 ° C. to 80 ° C., and the reaction time is 2 minutes to 48 hours, preferably 10 minutes to 24. It's time.
As described above, when the target product is obtained in a free state, it may be converted into a salt according to a conventional method. When the target product is obtained as a salt, the free product or other product may be converted according to a conventional method. It can also be converted to the salt. The compound thus obtained or a salt thereof can be isolated and purified from the reaction solution by a known means such as phase transfer, concentration, solvent extraction, fractional distillation, crystallization, recrystallization, chromatography and the like.
[0026]
Next, the biological activity of the compound represented by the general formula [I] or a salt thereof will be described.
Test Example 1 Suppression of IL-1 production in THP-1 cells derived from human monocytes
Human monocyte-derived cell line, THP-1 [International Journal of Cancer, Vol. 26, pages 171 to 176 (1980)] cultured in RPMI-1640 medium containing 10% FCS (fetal calf serum) did. THP-1 cells are seeded so as to be 5 × 10 5 / ml, lipopolysaccharide (hereinafter abbreviated as LPS, final concentration 10 μg / ml) is added, the test compound is added simultaneously with stimulating IL-1 production, and 8 hours 5% CO at 37 ° C₂Cultured in the presence. IL-1 was extracted by the method of Endres et al. [Clinical Immunology and Immunopathology, Vol. 49, pp. 424-438 (1988)], and the production amounts of IL-1α and IL-1β Was obtained using a commercially available EIA kit (CAYMAN, USA). As shown in Table 2, these compounds suppressed the production of IL-1α and IL-1β.
[Table 2]
[0027]
Test Example 2 Suppression of IL-1 transcription on THP-1 cells
LPS (final concentration 100 μg / ml) was added to THP-1 cells, and simultaneously, the test compound was added so that the final concentration was 5 μg / ml. Moreover, the chemical | medical agent non-processing and what added the methanol used for the solvent were used as control. Cells cultured for 3 hours were washed twice with physiological saline and used for RNA preparation. Isogen (Nippon Gene) was used for RNA extraction. Reverse transcription reaction and PCR were performed using the obtained total RNA (1 μg) and an RNA-PCR kit (Takara Shuzo). In addition, IL-1α, IL-1β and β-actin specific primers used for PCR were purchased from Clontech (USA). The DNA obtained by PCR was subjected to agarose electrophoresis, photographed, and analyzed with a densitometer. The transcriptional repression of IL-1α and IL-1β of Compound 1 was corrected using β-actin mRNA as an internal standard. As shown in [Table 3], these compounds were found to suppress IL-1α and β production at the transcriptional level.
[Table 3]
[0028]
Test Example 3 Antipyretic effect on rats
LPS (Sigma; physiological saline solution) 1.25 μg / kg was intravenously administered to SD rats (7-week-old male Jcl: 6 mice per group), and rectal temperature at 1 hour intervals from 3 to 6 hours after administration. Was measured. TAN-2294A (suspended in 5% gum arabic solution) was orally administered 1 hour before LPS administration. The effect of the drug was evaluated by performing a significant difference test (T-test) using the difference in body temperature from the non-treated control group to the sample administration group as an index. As a result, TAN-2294A was 50 mg / kg at the time of oral administration, and the fever body temperature 5 hours after LPS administration was significantly lowered.
[0029]
Test Example 4 Acute toxicity test using mice
Compound TAN-2294A (Compound 1A) of the present invention had an LD50 value of 100 to 200 mg / kg when administered intraperitoneally in an acute toxicity test using mice.
The above results indicate that the compound TAN-2294 derivatives and salts thereof of the present invention are useful as interleukin 1 production inhibitors.
[0030]
Regarding IL-1 production inhibitors containing TAN-2294, derivatives thereof and salts thereof as active ingredients, diseases caused by excessive IL-1 production (eg, rheumatoid arthritis, osteoporosis, etc.) and IL-1-dependent tumors It can be used as a therapeutic agent and a preventive agent, and an immunosuppressive agent for organ transplantation. The compound of the general formula [I] or a salt thereof is also useful as a research reagent for elucidating the IL-1 production mechanism.
The IL-1 production inhibitor of the present invention is used for the prevention and treatment of the above-mentioned diseases and the like. Examples of target animals include warm-blooded mammals, and examples thereof include mice, cats, cows, sheep, goats and pigs. , Rabbits and humans.
The administration can be carried out either parenterally or orally. Parenteral administration includes, for example, injection and suppositories, and oral administration includes tablets and capsules.
These preparations can be produced by a method known per se generally used in the preparation process.
[0031]
For example, the compound TAN-2294 derivatives and salts thereof of the present invention are dispersants (eg, Tween 80 (Atlas Powder, USA), HCO 60 (Nikko Chemicals), polyethylene glycol, carboxymethylcellulose, alginic acid. Sodium), preservatives (eg, methylparaben, benzyl alcohol, chlorobutanol, etc.), isotonic agents (eg, sodium chloride, glycerin, sorbitol, glucose, etc.), etc. for aqueous injections, or olive oil, sesame oil, peanut oil These can be dissolved, suspended or emulsified in vegetable oils such as cottonseed oil and corn oil, propylene glycol and the like, and then formed into oily injections to obtain injections.
For example, in order to obtain a preparation for oral administration, according to a method known per se, the compound of the general formula [I] of the present invention or a salt thereof is used, for example, as an excipient (eg, lactose, sucrose, starch, etc.), a disintegrant (eg, Add starch, calcium carbonate, etc.), binder (eg, starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, etc.) or lubricant (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.) And then, if necessary, coated by a method known per se for the purpose of taste masking, direct solubility or persistence, to give an oral preparation. Examples of the coating agent include hydroxypropylmethylcellulose, ethylcellulose, hydroxymethylcellulose, polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, Eudragit (Rohm, Germany, Methacrylic acid / acrylic acid copolymer) and pigments such as titanium oxide and bengara are used.
[0032]
For example, in order to obtain an external preparation, the compound of general formula [I] of the present invention or a salt thereof can be made into a solid, semi-solid or liquid external administration according to a method known per se. For example, as the solid, the compound of the general formula [I] of the present invention or a salt thereof is used as it is, or an excipient (eg, glycol, mannitol, starch, microcrystalline cellulose, etc.), a thickener (eg, Natural rubbers, cellulose derivatives, acrylic acid polymers, etc.) are added and mixed to obtain a powdery composition. The liquid form is almost the same as in the case of injections, and is an oily or aqueous suspension. In the case of a semi-solid form, an aqueous or oily gel or an ointment is preferred. In addition, these are all pH regulators (eg, carbonic acid, phosphoric acid, citric acid, hydrochloric acid, sodium hydroxide, etc.), preservatives (eg, paraoxybenzoates, chlorobutanol, benzalkonium chloride, etc.), etc. May be added.
For example, to make a suppository, the compound of general formula [I] of the present invention or a salt thereof can be converted into an oily or aqueous solid, semi-solid or liquid suppository according to a method known per se. Examples of the oily base used in the composition include glycerides of higher fatty acids [eg, cocoa butter, witepsol (manufactured by Dynamite Nobel), etc.] or vegetable oils (eg, sesame oil, soybean oil, cottonseed oil, etc.). . Examples of the aqueous base include polyethylene glycols and propylene glycol. Examples of the aqueous gel base include natural rubbers, cellulose derivatives, vinyl polymers, and acrylic acid polymers.
[0033]
The dose is appropriately selected in consideration of the administration method and the purpose of treatment, but the point is to restore the normal value to a subject whose amount is higher than the amount of IL-1 in a normal state. May be administered. For example, in the case of humans, the average value of IL-1β in normal plasma is about 45 pg / ml, whereas the amount of IL-1β in plasma of rheumatoid arthritis patients is about 98 pg / ml, which is more than doubled. Overproduction has been observed [Lancet II, 706-709, 1988], and such normal values can be administered to the target. Specifically, for example, when used by injection, the compound of general formula [I] or a pharmacologically acceptable salt thereof is orally administered at a dose of about 0.1 μg to 50 mg per day per kg of body weight. When used as, it is preferable to administer about 0.1 μg to 100 mg per day. The IL-1 production inhibitor of the present invention is used as an appropriate preparation in consideration of the administration subject, target disease, and the like.
When administering the preparation of the present invention, it may be administered once a day, or intermittently, for example, a method of administering about once a week. Further, it may be administered as a sustained-release preparation. Examples of the sustained release preparation include microcapsules and embedding agents. In particular, it is preferable to exert the effect of the active ingredient over a long period of time by embedding subcutaneously what is formed into a sustained-release agent.
[0034]
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereby. In the present specification, the percentage (%) in the medium and the aqueous solution represents weight / volume percentage unless otherwise specified. Moreover, the mixing ratio of the solvent indicates the volume ratio unless otherwise specified.¹³C and¹1 H nuclear magnetic resonance (NMR) spectrum was measured with an AC-300 spectrum meter (Bruker, Germany) using tetramethylsilane as an internal standard, and all δ values were shown in ppm. Q is quaternary carbon, s is singlet, d is doublet, t is triplet, q is quartet, m is multiplet, dd is double doublet, ddd is double doublet, dq is double quartet, br means wide The coupling constant (J) is shown in Hz.
[0035]
Example 1
Basidiomycete FL-61524 strain cultured on slanted agar medium 2% glucose, 3% maltose, 1% corn steep liquor, 1.5% raw soybean flour, 0.5% polypeptone, yeast extract 0 in a 200 ml Erlenmeyer flask Five seeds (pH 6.0) of 40 ml containing 0.3% and 0.3% sodium chloride were inoculated and cultured on a reciprocating shaker at 24 ° C. for 120 hours to obtain a seed culture solution. In a 200 ml Erlenmeyer flask, 2.0 ml of this seed culture solution was mixed with 0.5% glucose, 5.0% mannitol, 1.0% succinic acid, 0.2% corn steep liquor, 0.1% potassium dihydrogen phosphate. And 40 ml of a main medium (pH 5.6) containing 0.03% of magnesium sulfate, and cultured on a reciprocating shaker at 24 ° C. for 14 days to obtain a culture solution.
The obtained culture solution and flask washing solution (3.9 liters in total) were adjusted to pH 7 and filtered through a high flow supercell (Johns Manville, USA). The obtained filtrate (3.5 liters) was adjusted to pH 7, ethyl acetate (1.8 liters) was added, and the mixture was stirred and mixed for 30 minutes. The resulting aqueous layer was extracted with ethyl acetate (1.8 liter). The organic layers were combined, washed with water (1.8 L), dried over anhydrous sodium sulfate, and concentrated to dryness to give a brown oil (870 mg). The obtained oil (855 mg) was subjected to column chromatography on silica gel 60 (30 ml, Kieselgel 60, 70-230 mesh, Art. 7734, manufactured by A-Merck, Germany), and hexane: ethyl acetate (9: 1). ), (7: 3), (5: 5), (3: 7) and ethyl acetate (90 ml of each solvent), and fractionated into 30 ml fractions. Fractions Nos. 6 to 8 and Fraction No. 3 were concentrated and dried to give yellow oily substances (589 mg and 41 mg) containing compounds 1A and 1B, respectively. A yellow oily substance (587 mg) containing Compound 1A was divided into two portions, each with reverse phase preparative HPLC (carrier: ODS, YMC-Pack, S-363-15, manufactured by YMC, mobile phase: 37% acetonitrile / 0 0.01M phosphate buffer, pH 6.3). Fractions with an elution volume of 640-780 ml were collected, concentrated to about 100 ml, corrected to pH 7, and extracted twice with ethyl acetate (50 ml). The organic layers were combined, washed with water (50 ml), dehydrated over anhydrous sodium sulfate, concentrated to dryness, and compound 1A (TAN-2294A, R¹= Hydroxymethyl group, R²= Hydroxyl) was obtained as a pale yellow oil (438 mg). A yellow oily substance (40 mg) containing Compound 1B was subjected to reverse phase preparative HPLC (carrier: ODS, YMC-Pack, D-ODS-5, mobile phase: 43% acetonitrile / 0.05% aqueous trifluoroacetic acid). did. Fractions having an elution volume of 380 to 460 ml were collected, concentrated to about 50 ml, adjusted to pH 7, and extracted twice with ethyl acetate (30 ml). The organic layers were combined, washed with water (30 ml), dried over anhydrous sodium sulfate, concentrated to dryness, and compound 1B (TAN-2294B, R¹= Formyl group, R²= Hydroxyl) colorless oil (15 mg) was obtained.
[0036]
Example 2
Basidiomycete FL-61524 strain cultured on slant agar medium 2% glucose, 3% maltose, 1% corn steep liquor, 1.5% raw soybean flour, 0.5% polypeptone, yeast extract 0 in a 2000 ml Sakaguchi flask Two 500 ml seed media (pH 6.0) containing 0.3% and 0.3% sodium chloride were inoculated and cultured on a reciprocating shaker at 24 ° C. for 120 hours to obtain a seed culture solution. 1000 ml of this seed culture solution in a 200 liter stainless steel tank was glucose 0.5%, mannitol 5.0%, succinic acid 1.0%, corn steep liquor 0.2%, potassium dihydrogen phosphate 0 Transplanted to 120 liters of main medium (pH 5.6) containing 0.1% and magnesium sulfate 0.03%, 24 ° C., aeration 120 liters / minute (0-18 hours 84 liters / minute), stirring 180 rotations / minute For 14 days under conditions of 1 kg / cm @ 2 of internal pressure to obtain a main culture solution.
The obtained culture liquid (115 liters) was filtered with Radiolite 600 (manufactured by Showa Chemical Industry Co., Ltd.). The obtained filtrate (110 liters) was adjusted to pH 7, ethyl acetate (100 liters) was added, and the mixture was stirred and mixed for 30 minutes. The obtained organic layer was washed with water (50 liters), concentrated to about 4 liters, dried over anhydrous sodium sulfate, and concentrated to dryness to give a brown oil (32.9 g). The resulting oil (32.9 g) was subjected to column chromatography on silica gel 60 (1 liter, Kieselgel 60, 70-230 mesh, Art. 7734), hexane: ethyl acetate (9: 1, 3 liters), Elution was carried out in the same order (8: 2, 3 liters) and (7: 3, 9 liters), and fractionated into 1 liter fractions. Fractions Nos. 7 to 11 were collected and concentrated to dryness, and ethyl acetate and diethyl ether were added to make a powder at 4 ° C. The obtained powder was washed with ethyl acetate-hexane to give a white powder of compound 1A (7.06 g). ) The mother liquor and the washing solution were combined and concentrated to dryness to obtain a yellow oily substance (8.25 g, purity about 70%) containing Compound 1A.
[0037]
Example 3
The oil of Compound 1A obtained in Example 2 (122 mg, purity about 70%) was dissolved by adding pyridine and acetic anhydride (4: 1, 1.0 ml), and stirred at room temperature for 1 hour. The reaction solution was concentrated to dryness and dissolved by adding ethyl acetate (20 ml). The organic layer was washed twice with 0.05N hydrochloric acid, twice with 2% aqueous sodium hydrogen carbonate, water, and saturated brine (10 ml each). After washing with, dried over anhydrous sodium sulfate, concentrated under reduced pressure and dried to obtain an oily substance (141 mg). This was subjected to column chromatography on silica gel 60 (Kieselgel 60, Art. 7734, 10 ml) and eluted sequentially with hexane (30 ml), hexane: ethyl acetate (90:10, 30 ml) and the same (85:15, 60 ml). And fractionated into 10 ml portions. Fractions Nos. 7 to 10 were collected and concentrated to dryness to obtain a colorless oily substance (99 mg) of Compound 2A.
Compound 2A (R¹= Acetoxymethyl group, R²= Acetoxy group)
Elemental analysis value: C₁₉H₂₆O_Five As measured value; C, 68.17; H, 8.03 (%)
Calculated value; C, 68.24; H, 7.84 (%)
¹³C NMR spectrum: 75 MHz in deuterated chloroform,
δppm; 194.2 (Q), 170.7 (Q), 170.4 (Q), 153.3 (Q), 137.0 (Q), 74.4 (CH), 61.1 (CH2), 59.4 (CH2), 49.1 (CH), 46.3 (CH2 ), 46.1 (CH2), 41.2 (CH2), 40.7 (CH2), 37.0 (Q), 29.3 (CH3), 26.8 (CH3), 20.9 (CH3), 20.8 (CH3), 20.2 (CH3).
[0038]
Example 4
Compound 1A oil obtained in Example 2 (100 mg, purity about 70%) was dissolved in tetrahydrofuran (3.0 ml), triethylamine (61 μl) and benzoyl chloride (34 μl) were added, and the mixture was stirred at room temperature. Triethylamine (30 μl) and benzoyl chloride (17 μl) were added 75 minutes and 115 minutes after the start of the reaction, respectively. Further, 3 hours after the start of the reaction, the mixture was stirred at 50 ° C. for 2 hours. The reaction mixture was concentrated, diluted with ethyl acetate (20 ml), and the organic layer was washed twice with saturated aqueous sodium hydrogen carbonate, twice with water, and once with saturated brine (10 ml each), and then anhydrous. The extract was dried over sodium sulfate, concentrated under reduced pressure, and dried to obtain an oily substance (171 mg). This was subjected to column chromatography on silica gel 60 (Kieselgel 60, Art. 7734, 10 ml) and eluted successively with hexane (30 ml), hexane: ethyl acetate (90:10, 60 ml) and the same (85:15, 60 ml). And fractionated into 10 ml portions. Fractions Nos. 9-12 were collected and concentrated to dryness to give a colorless oil (55 mg) of compound 3A.
Compound 3A (R¹= Benzoyloxymethyl group, R²= Hydroxyl)
Elemental analysis value: C_{twenty two}H₂₆O_Four・ 0.5H₂As O Measured value; C, 72.70; H, 7.49 (%)
Calculated value; C, 72.77; H, 7.31 (%)
¹³C NMR spectrum: 75 MHz in deuterated chloroform,
δppm; 195.3 (Q), 167.2 (Q), 154.3 (Q), 140.0 (Q), 133.6 (CH), 133.3 (CH), 130.2 (CH), 130.0 (2CH), 129.8 (Q), 128.5 (CH ), 128.4 (2CH), 73.8 (CH), 61.2 (CH2), 59.5 (CH2), 51.9 (CH), 46.9 (CH2), 46.4 (CH2), 41.3 (CH2), 40.7 (CH2), 37.2 (Q ), 29.5 (CH3), 27.0 (CH3), 20.3 (CH3).
[0039]
Formulation Example 1
All ingredients of the formulation shown below are mixed uniformly and filled into gelatin capsules to produce capsules containing 10 mg of compound 3A per capsule.
Compound 3A 10mg
Lactose 100mg
Corn starch 40mg
Magnesium stearate 10mg
1 capsule 160mg
[0040]
Formulation Example 2
Compound 1A and magnesium stearate are granulated with an aqueous solution of soluble starch, dried, admixed with lactose and corn starch, and the mixture is compression molded to produce tablets containing 10 mg of Compound 1A per tablet.
Compound 1A 10mg
Lactose 65mg
Corn starch 30mg
Soluble starch 35mg
Magnesium stearate 20mg
1 tablet 160mg
[0041]
Formulation Example 3
Compound 1A is dissolved in physiological saline containing 30% (w / v) polyethylene glycol 400 to prepare a 0.05% solution of Compound 1A. After sterile filtration, 30 ml is dispensed into each vial, and 15 mg per vial is obtained. An injection containing Compound 1A is prepared.
[0042]
Formulation Example 4
Compound 1A and sodium carboxymethylcellulose are mixed uniformly in a mortar, and a solution of saccharin sodium dissolved in purified water is added little by little and mixed well. 1 ml of 10% sodium benzoate aqueous solution is added, the whole amount is made up to 100 ml with purified water, and a suspension containing 10 mg of compound 1A per 100 ml is produced.
Compound 1A 0.01 g
Sodium carboxymethylcellulose 2.0g
Saccharin sodium 0.04g
Sodium benzoate 0.1g
100ml total volume
[0043]
【The invention's effect】
Since the compound represented by the general formula [1] or a salt thereof, for example, Compound 1A has an IL-1 production inhibitory action, it treats and prevents diseases caused by excessive IL-1 production and IL-1-dependent tumors. It can be used as an immunosuppressant for drugs or organ transplantation.
[0044]
[Brief description of the drawings]
FIG. 1 UV spectrum of TAN-2294A
FIG. 2 IR spectrum of TAN-2294A
[Fig. 3] TAN-2294A¹³C NMR spectrum
[Fig. 4] TAN-2294A¹H NMR spectrum
FIG. 5: IR spectrum of TAN-2294B
FIG. 6: TAN-2294B¹³C NMR spectrum
FIG. 7: TAN-2294B¹H NMR spectrum

Claims (5)

Formula [I]
In, R ¹ is a hydroxymethyl group, R ² is hydroxyl der Ru of compound TAN-2294A. Formula [I]
In, R ¹ is a formyl group, R ² is hydroxyl der Ru of compound TAN-2294B. Of a compound TAN-2294A or / and B characterized by culturing basidiomycete FL-61524 strain having accession number FERM P-15084 in a medium, producing and accumulating TAN-2294A or / and B, and collecting the same Manufacturing method. A pharmaceutical agent comprising the claims 1 or 2 A compound according. Interleukin 1 production inhibitor comprising the claims 1 or 2 A compound according.