JPS6318598B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to lactoyl tetrapeptide, a novel compound with biological activity. The lactoyl tetrapeptide of the present invention is represented by the following formula (). Lactoyl tetrapeptide (1) of the present invention can be produced by the method shown by the following formula. (1) Synthesis method (2) Fermentation method (In the formula, R ¹ is a hydroxy group, a protected hydroxy group or a protected hydrazino group (-
NHNHY, Y means amino protecting group),
R ^2a means an amino protecting group, R ³ means a hydroxy group or a protected hydroxy group, R ⁴ means a protected hydroxy group, and R ⁵ means a hydroxy protecting group. ) Next, each of the above groups will be explained. Regarding Amino Protecting Groups in a R ^2a Amino protecting groups include common protecting groups used in the field of amino acid and peptide chemistry. Suitable examples include acyl groups such as organic acyl groups, such as alkoxycarbonyl (eg methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, t-
pentoxycarbonyl, etc.), substituted or unsubstituted aralkoxycarbonyl (e.g. benzyloxycarbonyl, P-nitrobenzyloxycarbonyl, P-phenylazobenzyloxycarbonyl, P-(P′-methoxy-phenylazo)benzyloxycarbonyl) , P-chlorobenzyloxycarbonyl, P-bromobenzyloxycarbonyl, α-naphthylmethoxycarbonyl, P-biphenylisopropoxycarbonyl, etc.). b About the amino-protecting group in Y As a preferable example of the amino-protecting group in the protected hydrazino group (-NHNHY, where Y means an amino-protecting group), the example of the amino-protecting group in R ^2a above can be cited as is. c Regarding protected hydroxy groups in R ¹ , R ³ and R ⁴ Groups with protected hydroxy groups in R ¹ R ³ and R ⁴ --COR ¹ , --COR ³ and --COR ⁴ are respectively protected carboxy groups Examples of such protected carboxy groups include esterified carboxy groups such as esters with aliphatic hydrocarbon hydroxy compounds and esters with hydroxy compounds containing aromatic groups, etc. . Suitable examples of such esterified carboxy groups include, for example, alkyl (e.g. methyl,
Ethyl, propyl, isopropyl, butyl, t-
butyl, etc.) esters, aralkyl (eg, benzyl, phenethyl, etc.) esters, and the like. As mentioned above, preferable examples of the protected hydroxy groups in R ¹ , R ³ and R ⁴ include alkoxy groups, aralkyloxy groups and the like. Regarding the hydroxy protecting group in d R ⁵ Suitable examples of the hydroxy protecting group include acyl groups such as alkanoyl (e.g. formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, bromoacetyl, dichloroacetyl, trifluoroacetyl, etc.) . Pharmaceutically acceptable salts of lactoyltetrapeptide () with inorganic or organic bases (e.g., sodium salts, potassium salts, calcium salts, ammonium salts, ethanolamine salts,
triethylamine salt, dicyclohexylamine salt, etc.), acid addition salts with inorganic or organic acids (e.g.
acetate, lactate, maleate, fumarate,
oxalate, citrate, methanesulfonate, hydrochloride, sulfate, nitrate, phosphate, etc.). Next, a method for producing lactoyl tetrapeptide () will be explained. (1) Method 1: Compound () + Compound () → Compound (-1) This method relates to a method for producing Compound (-1) by reacting Compound () or its salt with Compound (). This method can be performed as described below. That is, one method is to first activate the carboxy group in the compound () by a conventional method, and then react the compound (). Here, examples of the method for activating the carboxy group include an acid halide method, an azide method, an acid anhydride method, a mixed acid anhydride method, and an activated ester method. Another method is to directly react the compound () or its salt with the compound () or its salt in the presence of a commonly used condensing agent such as N,N-dicyclohexylcarbodiimide. The method for activating the carboxy group and the condensing agent described above are appropriately selected depending on the type of compound () and the carboxyl group-protecting group of () and the reaction conditions (eg, type of solvent, reaction temperature, etc.). This reaction is usually carried out in a solvent such as methylene chloride, chloroform, tetrahydrofuran, dioxane, ethyl acetate, methanol, ethanol, water, etc. at a temperature ranging from cooling to room temperature. Further, when the reaction is carried out in the presence of a condensing agent, it is carried out under anhydrous conditions and mild conditions. (2) Method 2: Compound (-1) → Compound () This method converts compound (-1) into R ^2a , R ¹ ,
The present invention relates to a method for producing compound () by subjecting R ³ .R ⁴ and R ⁵ to an elimination reaction of protecting groups at amino groups, carboxy groups, and hydroxy groups. Method 2-1: Elimination of the amino protecting group on R ^2a and Y This elimination reaction of the amino protecting group on R ^2a and Y is carried out by a conventional method. That is, it is carried out by a catalytic reduction method, a liquid ammonia-alkali metal method, a method using an acid, a zinc-acid method, a base method, a hydrazine method, etc. 1 Catalytic Reduction Method This method is applied when the amino protecting group in R ^2a and Y is a protecting group that can be removed by catalytic reduction. Preferred examples of such amino protecting groups include substituted or unsubstituted aralkoxycarbonyl (e.g. benzyloxycarbonyl, P
-Phenylazobenzyloxycarbonyl, P-
(P'-methoxyphenylazo)-benzyloxycarbonyl, P-nitrobenzyloxycarbonyl, etc.). This catalytic reduction method is carried out in a conventional manner. Suitable catalysts used for catalytic reduction include platinum catalysts (e.g., platinum plates, platinum sponges, platinum black, colloidal platinum, platinum oxide, platinum wires, etc.), palladium catalysts (e.g., palladium sponges, palladium black, palladium oxide, colloids) palladium, etc.), nickel catalysts (e.g., reduced nickel, nickel oxide, Raney nickel, etc.), cobalt catalysts (e.g., reduced cobalt, Raney cobalt, etc.), iron catalysts (e.g., reduced iron, Raney iron, etc.), copper catalysts (e.g., reduced copper,
Raney copper, Ullmann copper, etc.). This reduction reaction is usually carried out in a solvent. Examples of solvents include water, methanol, ethanol, propanol, ethyl acetate, tetrahydrofuran,
Examples include dioxane, N,N-dimethylformamide, acetic acid, and a mixed solvent of these solvents and water. Moreover, this reduction reaction is preferably carried out in the presence of an acid such as acetic acid. This reaction is preferably carried out under mild conditions such as cooling to heating. In this method, the protected hydroxy groups in R ³ and R ⁴ are substituted or unsubstituted aralkyl ester-type esters, e.g.
benzyl ester, P-nitrobenzyl ester, P-chlorophenyl ester, P-phenylazobenzyl ester, etc.), such protecting groups are removed at the same time. Method using acid -1 Method using trifluoroacetic acid or formic acid This method is applied when the amino protecting group in R ^2a and Y is a protecting group that can be removed by treatment with trifluoroacetic acid or formic acid. . Suitable examples of such amino protecting groups include branched alkoxycarbonyl (e.g., t-butoxycarbonyl, t-pentoxycarbonyl, etc.),
Examples include substituted or unsubstituted aralkoxycarbonyl (eg, P-methoxybenzyloxycarbonyl, etc.). This reaction is carried out in the presence of acetic acid or formic acid, usually in a solvent such as methylene chloride, chloroform, acetic acid, or water. Further, good results are often obtained when the reaction is advanced by adding anisole. Trifluoroacetic acid and formic acid are also used as solvents. This reaction is usually carried out at a temperature ranging from cooling to room temperature. In this method, if the protected hydroxy group in R ³ and R ⁴ is, for example, a branched alkyl (such as t-butyl ester), such a protecting group is also removed simultaneously in this reaction. do. - Method using dihydrochloric acid or P-toluenesulfonic acid This method is applied when the amino protecting group in R ^2a and Y is a protecting group that can be removed by treatment with hydrochloric acid or P-toluenesulfonic acid. Preferred examples of such amino-protecting groups include, in addition to the groups exemplified in -1 above, substituted or unsubstituted branched alkoxycarbonyl (e.g., t-butoxycarbonyl, 1-(P
-biphenyl)-1-methylethoxycarbonyl, etc.). This reaction is carried out in a solvent such as methylene chloride, chloroform, or tetrahydrofuran in the presence of hydrochloric acid or P-toluenesulfonic acid. Also, good results are often obtained by adding anisole. This reaction is carried out between cooling and room temperature. In this method, the protected hydroxy groups in R ³ and R ⁴ are, for example, branched alkyl esters (e.g. t-butyl esters etc.),
In the case of substituted or unsubstituted aralkyl esters (eg, diphenylmethyl ester, P-methoxybenzyl ester, etc.), such protecting groups are simultaneously eliminated during this reaction. Method 2-2: Removal of the protecting group from the protected hydroxy group in R ¹ , R ³ and R ⁴ This salt is removed by conventional methods such as hydrolysis and reduction. (1) Hydrolysis Here, hydrolysis refers to solvolysis such as hydrolysis, acidolysis, alcohololysis, aminolysis, and hydrazinolysis. Hydrolysis is usually carried out in the presence of an acid or a base, such as inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, acidic acid, acetic acid, trifluoroacetic acid, propionic acid, benzenesulfonic acid, etc. ,
In addition to organic acids such as P-toluenesulfonic acid, acidic ion exchange resins can also be used. Bases include, for example, alkali metal or alkaline earth metal hydroxides, carbonates or hydrogen carbonates (e.g.
sodium hydroxide, potassium carbonate, sodium bicarbonate, calcium hydroxide, magnesium hydroxide, etc.), inorganic bases such as ammonium hydroxide, alkoxides or phenoxides of alkali metals or alkaline earth metals (e.g., sodium methoxide, sodium ethoxide) , lithium phenoxide, etc.) mono-, di- or trialkylamines (e.g. methylamine, ethylamine, propylamine, isopropylamine, butylamine, N,N-dimethyl-1,3-propanediamine, trimethylamine, triethylamine, etc.), non- Substituted or mono(or di)substituted arylamines (e.g., aniline, N-methylaniline, N,N-dimethylaniline, etc.), heterocyclic compounds (e.g., pyrrolidine, morpholine, N-
Methylmorpholine, N-methylpiperidine, N,
Examples include organic salts such as N-dimethylpiperazine, pyridine, etc.), hydrazines (eg, hydrazine, methylhydrazine, ethylhydrazine, etc.), as well as basic ion exchange resins. The reaction temperature for this hydrolysis is usually carried out under mild conditions such as cooling or heating.
This reaction is usually carried out in a solvent such as water, methanol, ethanol, propanol, dimethylformamide, tetrahydrofuran, dioxane,
Hydrophilic solvents such as dimethyl sulfoxide or mixed solvents thereof are often used, but when the acid or base exemplified above is liquid, it can also be used as a solvent. (2) Reduction This reduction method is carried out by conventional methods such as chemical reduction in addition to the catalytic reduction method described in Method 2-1, 1) above. The catalytic reduction method is as described in Method 2-1, 1) above. Examples of reducing agents used in the chemical reduction method include metals such as tin, zinc, and iron, or chromium chloride,
Metal compounds such as chromium acetate, hydrochloric acid, hydrobromic acid,
Acetic acid, acetic acid, propionic acid, trifluoroacetic acid,
Examples include combinations with inorganic or organic acids such as P-toluenesulfonic acid. This chemical reduction is usually carried out in a solvent, and water, methanol, ethanol, propanol, a mixed solvent of these solvents and other organic solvents, etc. are frequently used. The above-mentioned protective group elimination reactions include the following cases depending on the applied method, reaction conditions, etc. That is, if all the protecting groups in the protected hydroxy groups in R ³ and R ⁴ , the amino protecting group in R ^2a and the hydroxy protecting group in R ⁵ are removed simultaneously, the protection in R ³ and R ⁴ protecting group in hydroxy, amino protecting group in R ^2a and
When the hydroxy protecting groups at R ⁵ are removed sequentially. Method 2-3: Elimination of hydrazino group The protected hydrazino group represented by the group -NHNHY was obtained by first subjecting compound (-1) to the elimination reaction (method 2-1) of the amino protecting group in Y (this reaction By R ¹ -NHNHY -NHNH ₂
After this reaction, the hydrazino group is eliminated. This reaction is carried out by a conventional method, and the compound (-
1) with commonly used oxidizing agents (groups - CONHNH ₂
This is done by treating COOH with an oxidizing agent that can convert COOH to COOH. Examples of such oxidizing agents include halogens such as iodine, periodic acid or its salts (e.g. sodium salts, calcium salts, etc.), perhalogen acids such as perchloric acid, N-halimides such as N-succinimide, and tetrachloride. Examples include lead, hydrogen peroxide or a salt thereof (for example, nickel peroxide, etc.), metal oxides such as mercury oxide and manganese dioxide, copper compounds (for example, copper acetate, copper sulfate, etc.). This reaction is usually carried out in a solvent such as water, acetic acid, methanol, ethanol, tetrahydrofuran, dioxane, etc., and these solvents are appropriately selected depending on the type of oxidizing agent used. This reaction is carried out under cooling to about room temperature or under reflux. Method 2-4: Elimination of hydroxy protecting group at R ⁵ This reaction can be carried out by a conventional method, for example, the method described in the explanation of method 2-2 above. Hydrolysis carried out in the presence of acetic acid is preferred. [2] Fermentation method: Bacteria belonging to the genus Streptomyces - → Compound (a) This method uses FR, which belongs to the genus Streptomyces.
-900156 This is done by culturing substance-producing bacteria in a medium. Streptomyces olibaseogriseus
SP C-353 Streptomyces Olivaceogriseus
SP C-353 is a strain newly isolated from soil collected in Kochi Prefecture, and is an American type culture strain.
It has been deposited in the National Institute of Industrial Science and Technology as ATCC No. 31427, and has been deposited in the National Institute of Microbiology, Agency of Industrial Science and Technology as No. 5360. This Streptomyces olibaseogriseus SP C-353 has the following mycological properties. 1 Morphological properties At 30℃ for 10 days on each of the following plate culture substrates: seuucrose/nitrate agar, glycerin-asparagine agar, yeast-malt extract agar, oatmeal agar, powder-mineral salt agar, and Bennett's agar. The results of observation under a microscope after growing for ~14 days are as follows. (1) Branching method of spore-forming hyphae: Simple branching (2) Morphology of spore-forming hyphae: Loop to spiral type (closed spiral, lupus) (3) Number of spores: 5-20 (4) Surface structure of spores and size: smooth 0.6 to 1.2 x 1.0 to 1.9 microns (5) Presence or absence of sporangium and flagellar spores: Not observed (6) Spore settlement position: On aerial hyphae (7) Presence or absence of sclerotia formation: Not observed 2. Properties on various media The following observations on various media were made after culturing at 30°C for 10 to 14 days. (1) Seuucrose-nitrate agar medium AM: none or very faint, powdered VG: pale yellow, small colony SP: none (2) Glucose-asparagine agar medium AM: light gray to greenish gray, powdered VG: pale yellow ~Pale and color, small colony SP: None (3) Glycerin-asparagine agar AM: Pale, powdery, light gray VG: Pale yellow and color, small colony SP: None or solid (4) Starch - Inorganic salt Agar medium AM: olive gray, powdery VG: grayish yellow and colored, small colony SP: none (5) Tyrosine agar medium AM: light olive gray, pale, powdery VG: yellow and colored, small colony SP: light and colored (6) Nutrient agar medium AM: None VG: Pale yellow, flat SP: None (7) Yeast-malt extract agar AM: Green-gray, powdery VG: Yellow and colored, wrinkled colonies SP: None (8) Oatmeal agar AM: light gray to greenish gray, powdery VG: colorless, small colony SP: none (9) Peptone-yeast-iron agar AM: none VG: colorless to pale yellow, slightly wrinkled growth SP: light Cutlet color (10) Glucose-peptone-gelatin AM: white, pale powder VG: colorless to pale yellow, wrinkled colony SP: cutlet color (11) Milk AM: white, very pale powder VG: colorless, surface Annular growth SP: None Note AM: Aerial hyphae VG: Vegetative hyphae growth SP: Soluble pigment 3 Biological and physiological properties (1) Growth temperature range (on Bennett's agar slant) 15-40℃, optimum temperature 28 ℃ (2) Hydrolysis of starch (starch - inorganic salt agar medium) Weakly hydrolyzed (3) Liquefaction of gelatin (bulcose - peptone agar) Negative (4) Coagulation of milk/peptonization coagulation: Negative Peptonization: Negative (5) Production of melanin-like pigment (peptone - yeast -
(on iron agar, tyrosine agar, tyrosine yeast) Positive (6) Utilization of various carbon sources (Pridham-Gotzlieb basic agar) L-arabinose - D-xylose ± D-glucose + D-fructose + D-galactose + seuucrose + Glycerin + Inositol + Lactose + L-Rhamnose - Maltose + Raffinose - D-Mannitol + D-Mannose + Salicin - Note +: Frequently used. ±: Usage is questionable. -: Not used. Regarding bacteria with the above-mentioned mycological properties, Barzei's Manual of Determinative
Bacteriology 8th edition, ISP report (E.B. Shearling and D. Gottlieb;
International Journal of Systematic Bacteriology, Volume 18, Page 69,
Volume 19, page 391, 1969,
22, p. 265, 1972), we found that Streptomyces eurithermus is a relatively similar bacterium to this bacterium.
eurthermus) and Streptomyces galbus (Okami). However, the ATCC31427 strain differs from these bacteria in the following points, for example: (1) Streptomyces eurothermus (Okami): cannot assimilate inositol Assimilation of rhamnose and raffinose appears to be unknown. Loops are not formed. Straight to curved aerial hyphae are sometimes observed. (2) Streptomyces galvus Open spirals are usually formed. It produces a yellow to yellow-green pigment.It cannot assimilate sucrose.From the above observation results, strain ATCC31427 is judged to be a new bacterial species belonging to the genus Streptomyces. Seus Nobu SP C-353
(Streptomyces olivaceogriseus Nov.SP C-
353). Streptomyces violaceus No.
6724: Streptomyces violaceus No. 6724 is a strain newly isolated from soil collected on Ishigaki Island, Okinawa Prefecture, and has been deposited with the American Type Culture Collection as ATCC No. 31481, and the National Institute of Microbial Technology, Agency of Industrial Science and Technology. It has been deposited as Microtechnical Research Institute No. 5361. This Streptomyces violaceus No.
Strain 6724 exhibits the following mycological properties. 1 Morphological properties On plate culture media of sucrose/nitrate agar, glycerin-asparagine agar, yeast-malt extract agar, starch-mineral salt agar, and oatmeal agar at 30°C for 10 days.
The results of observation under a microscope after growing for ~14 days are as follows. (1) Branching method of spore-forming hyphae: Simple branching (2) Morphology of spore-forming hyphae: Spiral, forming many open spirals with 3 to 4 turns, and only a few incomplete loops. Not observed. (3) Spore surface structure: spiny (spine-like) (4) Spore size: 0.3 to 0.7 x 0.6 to 1.1 microns (5) Number of spores: 10 to 50 (6) Spore settlement Location: On aerial hyphae (7) Presence or absence of sporangia and flagellated spores: Not observed (8) Presence or absence of sclerotia formation: Not observed (9) Division of vegetative hyphae: Not observed 2 Properties on various media and Physiological Properties The following observations on various media are after culturing at 30°C for 10 to 14 days. (1) Eucrose-nitrate agar medium AM: purplish white, powdered VG: purple, small colony SP: purple (2) Glucose-asparagine agar medium AM: purplish white, powdered VG: yellowish red, small colony SP : Peach color (3) Glycerin-asparagine agar medium AM: Peach color white, powdery VG: Yellowish red, small colonies, slightly wrinkled growth SP: Peach color - red (4) Starch - Inorganic salt agar Medium AM: whitish red, short cotton-like VG: yellowish red, slightly wrinkled growth SP: red (5) Tyrosine agar medium AM: None VG: red, wrinkled colony SP: none to trace (6) Nutrient agar Medium AM: None or very light powdery VG: Colorless to purple, flat SP: Reddish-purple (7) Yeast-malt extract agar medium AM: Peach-colored to purple peach-colored, short cotton-like VG: red and colored to Purple and colored, wrinkled colony SP: Red-purple (8) Oatmeal agar medium AM: Peach color - purple peach color, powdery VG: colorless to purplish peach color, small colony SP: peach color - purple (9) Milk AM: pale powdery VG: red, growth on surface SP: red and colored (10) Glucose-peptone-gelatin medium AM: white, pale powdery VG: red, wrinkled colony SP: slightly Katsu color (11) Peptone-yeast-iron agar medium AM: None VG: Colorless, wrinkled colony SP: Katsu color Note: AM: Aerial hyphae VG: Growth of vegetative hyphae SP: Soluble pigment The back side of the colony of this strain Both the color and the soluble dye diffused into the agar medium change with pH. In other words, when a 0.05N aqueous sodium hydroxide solution is added dropwise, the color changes from purple to violet;
When 0.05N hydrochloric acid is added dropwise, the color becomes peach-colored to red. 3 Biological and physiological properties (1) Growth temperature range (on Bennett's agar slant) 15℃ - 40℃, optimum temperature 28℃ (2) Hydrolysis of starch (starch-inorganic salt agar medium) Positive (3) Liquefaction of gelatin (glucose-peptone gelatin spike) Negative (4) Coagulation of milk. Peptonization Coagulation: Positive, Peptonization: Negative (5) Production of melanin-like pigment Positive (Peptone-Yeast-Iron Agar, Tyrosine-Yeast Extract Broth) Very weak on tyrosine agar4 Availability of various carbon sources (Pridham- Gottlieb basic agar culture medium) L-arabinose + D-xylose + L-rhamnose + D-glucose + D-fructose + D-mannose + D-galactose + seuclose + lactose + maltose + raffinose + inulin ± cellulose - chitin - glycerin + D - Mannitol + Salicin + Inositol + Sodium acetate - Sodium citrate + Sodium succinate + Note +: Frequently used. ±: Usage is questionable. -: Not used. From the above results, this bacterium is a species of the genus Streptomyces, and its characteristics can be summarized as follows. The morphology of aerial hyphae is simple branching and spiral sections. The surface of the spore is spiny. The color of the surface of the bacterial flora is peach-colored to purple.The growth is yellow-red to reddish and the color changes depending on the pH. It is chromogenic. It produces a peach-colored to purple diffusible pigment, but this pigment also changes depending on the pH. It mostly utilizes carbon sources. Regarding bacteria with such mycological characteristics, Barzei's Manual of Determinative
Bacteriology 8th Edition, ISP Report {EB Shiring and D. Gottlieb; International Journal of Systematic Bacteriology (EBShiring
and D. Gottlieb: International journal of
Systematic Bacteriology) Vol. 18, p. 69, same no.
279 pages, 1968, Volume 19, page 391, 1969: Volume 22
(Vol. 265, 1972)} Based on the book The Actinomycetes, Vol. 1, written by SAWaksman, etc., it was found that Streptococcus aeruginosa is a bacterial species with properties very similar to this bacterium. Streptomyces violaceus, Streptomyces purpurascens
Purpurascens), Streptomyces roseovicolaceus, and Streptomyces violarus.As a result of a more detailed study of these strains and this No. 6724 strain, No. 6724 strains are Streptomyces
It showed properties that most closely matched those of Violaceus.
Therefore, this strain No. 6724 was identified as Streptomyces violaceus. Production of the FR-900156 substance is carried out by culturing FR-900156 substance-producing bacteria belonging to the genus Streptomyces, such as Streptomyces olivaceogriseus and Streptomyces violaceus, in a medium. The culture method is basically similar to that of general microorganisms, but deep culture using a liquid medium is usually advantageous. As the medium used for culture, a synthetic medium, a semi-synthetic medium, or a natural medium can be used, and the composition of the medium can be a normal medium as it is. That is, as carbon sources, for example, glucose, mannose, glycerin, molasses, starch, liquefied starch, etc. are used, and as nitrogen sources, meat extract, casein hydrolyzate, peptone, gluten meal, corn meal, cotton meal, soybean flour, etc. are used. Corn steep liquor, dried yeast, ammonium phosphate, ammonium sulfate, urea, etc. are used. In addition, inorganic compounds such as metal phosphates and chlorides such as disodium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, ferrous sulfate, magnesium sulfate, copper sulfate, zinc sulfate, manganese chloride, magnesium chloride, etc. Salt is also added if necessary. Also,
When foaming is significant during culturing, antifoaming agents such as vegetable oils such as soybean oil and linseed oil, higher alcohols such as octadecanol, and silicon compounds may be added as appropriate. The appropriate culture temperature is around 30°C. Good results are often obtained if the culture is carried out appropriately as the culture volume increases. The appropriate culture time for the main culture is about 50 to 100 hours. The culture conditions described above are applied by selecting optimal conditions depending on the characteristics of the production strain used. The FR accumulated in the culture in this way
Since the 900156 substance is mainly contained in the culture solution, after removing the bacterial cells from the culture by centrifugation or filtration, it is separated from the filtrate by conventional methods used in the production of general antibiotics. It is collected and refined. i.e. vacuum concentration, freeze drying, solvent extraction,
Liquid exchange, treatment with resins such as anion exchange resins, enteric ion exchange resins, nonionic adsorption resins, treatment with adsorbents such as activated carbon, silicic acid, silica gel, alumina, crystallization, recrystallization, etc. The FR-900156 substance can be separated from the filtrate by using them alone or in combination in any order, or repeatedly.
It is collected and refined. The FR-900156 substance accumulated in the culture medium can also be isolated in free form or in salt form. In the case of isolation in the form of a salt, the solution obtained by filtering the culture solution or its concentrate may be used as an alkali metal compound (e.g., sodium hydroxide, potassium hydroxide, etc.) or an alkaline earth metal compound (e.g., calcium hydroxide). ，
(e.g. magnesium hydroxide, etc.), inorganic bases such as ammonia, organic bases such as ethanolamine, triethylamine, dicyclohexylamine, etc.; , acetic acid, P-toluenesulfonic acid, citric acid, oxalic acid, etc.), the corresponding salts of the FR-900156 substances can be obtained. The salt of FR-900156 substance thus obtained can also be converted into free FR-900156 substance by conventional methods. The FR-900156 substance obtained in this way has the following physical and chemical properties (the following data are based on Example 10).
(This is the data obtained in ). 1. Elemental analysis As a result of qualitative analysis, it was found that the elements of carbon, hydrogen, nitrogen and oxygen were included. 2. Molecular weight Yass spectrometry (field, desorption method) M=519 (basic peak: M+1=520) 3. Melting point 143-148℃ (decomposition) ) 4 Specific rotation [α] ²⁵ _D = -27.1 (C = 0.4 water) 5 Ultraviolet absorption spectrum Terminal absorption 6 Infrared absorption spectrum (KBr) 1050, 1130, 1235, 1340, 1400, 1450,
1535, 1660, 1735, 2950, 2980, 3080, 3350 ^-1 7 Nuclear magnetic resonance absorption spectrum As shown in the drawing (Solvent: D ₂ O, Internal standard: Sodium 3-(trimethylsilyl)propionate 8 Thin layer chromatography Yi:

[Table] 9 Solubility in solvents Soluble in water and slightly soluble in methanol. Insoluble in ethanol, acetone, ethyl acetate, benzene, hexane, and chloroform. 10 Color reaction positive: ninhydrin, potassium permanganate,
Reactions with sulfuric acid Negative: Dragendorff reaction, Ehrlich reaction 11 Basic, acidic, neutral distinction Amphoteric 12 Color of substance White 13 Amino acid analysis molar ratio Glycine 1.00 Glutamic acid 1.06 Alanine 1.04 α,β-diaminopimelic acid 1.03 (above) The molar ratio was calculated assuming glycine as 1) 14 Molecular formula C ₂₀ H ₃₃ N ₅ O _11As a result of measuring the physical and chemical properties of the product obtained in Example 11, the melting point was 147-153℃ (decomposition), and other physical and chemical properties The properties were the same as above. As a result of the above physical and chemical properties and separate research,
The chemical structural formula of FR-900156 substance was identified as below. [D-lactoyl-L-alanyl-r-D-glutamyl-(L)-mesodiaminopimeryl(L)-glycine] Lactoyl tetrapeptide of the present invention ()
contains one or more stereoisomers based on the asymmetric carbon atoms in the molecule. The starting materials () and () each include novel compounds, which can be produced, for example, by the method shown in the following diagram:
In addition, in detail, by the method described in Production Examples 1 to 48,
can be manufactured. (In the formula, 2 ^2b represents an amino protecting group, R ^2a and R ³ ′ represent a protected hydroxy group, and R ¹ , R ³ and R ⁴ each have the same meaning as above.) Definition of R ^2b and R ³ ′ and Preferred examples are the same as R ^2a and R ³ , respectively. Lactoyl tetrapeptide of this invention ()
and its pharmaceutically acceptable salts have immune response-promoting effects (cellular immune activity, humoral antibody production promoting effect), reticuloendothelial system-enhancing effects, interferon production activity, rejuvenation activity, infection prevention effects, and anticancer effects. It has various activities and is useful as a medicine. Next, an example of a pharmacological experiment on lactoyltetrapeptide () of the present invention will be shown. The compound used in the following experiments is represented by the following formula. 1 Effect of promoting cell-mediated immunity and humoral antibody production A water-in-oil suspension was prepared by mixing the same volume of Freunds Lncomplete Adjuvent (FIA) with physiological saline containing ovalbumin. This 0.1ml
Administer to both left and right hind footpads. 0.1 ml of the suspension contained 500 μg of ovalbumin, and the test drug was used by dissolving the ovalbumin in physiological saline.
A skin reaction was induced on the 14th day, and blood was collected on the 16th day.

【table】

〔Method〕

(1) Animals Test 1: BALB/C male mouse (13 weeks old) Test 2: BALB/C female mouse BALB/C (9 weeks old)
(2) Tissue Culture Medium Roswell Park Memorial Institute
(RPMI)-1640 complete medium was used. The medium used contains penicillin G (100 units/ml), streptomycin sulfate (100 μg/ml) and 10% fetal bovine serum. (3) Preparation of splenocyte suspension The spleen was aseptically removed from the mouse, washed with Hanks solution, and then teased in a medium. The splenocytes were suspended in the medium to give a splenic suspension of 8×10 ⁶ cells/ml. (4) Culture conditions Pour 0.1 ml of the above splenocyte suspension and 0.1 ml of the test compound solution at the specified concentration into each hole of a Microtest tissue culture plate (8 x 12 holes) (manufactured by Falcon Plastics) for 3 holes each. In a carbon dioxide gas furan vessel (5% carbon dioxide, 95% air), 37℃
The cells were cultured for 48 hours. For controls, 0.1 ml of medium was used instead of the test compound. (5) Incorporation of [3H]thymidine 24 hours before the end of culture, 20μ of 10μ-Cyurin (μCi)/ml of methyl [3H]-thymidine (2.0Ci/mmol) was added to each hole. After the culture was completed, the plate was cooled on ice to stop the reaction, and the plate was passed through Watmann GF83 paper. Cells on paper were washed with saline followed by 5% trichloroacetic acid. The paper was dried, placed in a toluene scintillator, and the amount of [3H]thymidine incorporated into DNA was measured. (6) Acceleration rate Acceleration rate = [3H]thymidine incorporation in treated culture (
net cpm)/[3H]thymidine incorporation in control culture (net cpm)

【table】 D L D 〓

Claims (1)

[Scope of Claims] A compound represented by the following formula or a pharmaceutically acceptable salt thereof, 2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is a compound represented by the following formula: 3. The compound according to claim 1, which is a compound represented by the following formula, or a pharmaceutically acceptable salt thereof 4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is a compound represented by the following formula: 5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is a compound represented by the following formula: 6. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is a compound represented by the following formula: 7. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is a compound represented by the following formula: 8. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is a compound represented by the following formula: 9. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is a compound represented by the following formula: 10 formula (In the formula, R ¹ is a hydroxy group, a protected hydroxy group or a protected hydrazino group (-
NHNHY, Y means amino protecting group),
R ^2a means an amino protecting group, R ³ means a hydroxy group or a protected hydroxy group, R ⁴ means a protected hydroxy group, and R ⁵ means a hydroxy protecting group. ) is subjected to a protective group elimination reaction to form the formula A formula characterized in that it yields a compound represented by A method for producing the compound shown in 11 FR belonging to the genus Streptomyces -
A method for producing FR-900156 substance, which comprises culturing 900156 substance-producing bacteria in a medium, and separating and collecting FR-900156 substance from the obtained culture.