Peptides, processes for their preparation and pharmaceutical compositions containing them.

Description: TECHNICAL FIELD [Detailed description of the invention]

The present invention relates to novel peptides. In particular, the present invention relates to novel peptides having pharmacological activity and pharmaceutically acceptable salts thereof, methods for producing them, novel intermediates for synthesizing such peptides, and pharmaceutical compositions containing the above. is there. The novel peptide of the present invention is represented by the following formula (I).<img file="JPH0613552B2_D0001.tif" />[In the formula, R¹Is hydroxy or lower alkanoyloxy, R²Is a carboxy or phenyl lower alkoxycarbonyl, R³Is hydrogen or methyl, R⁴Is a carboxy or a group represented by the formula: CONHNHY (where Y means lower alkoxycarbonyl), R⁵Means hydrogen or lower alkoxycarbonyl, respectively] Details and preferred examples of the various definitions described above and below are as follows. Here, the term "lower" means a group having 1 to 6 carbon atoms unless otherwise specified. Preferred examples of the phenyl lower alkoxycarbonyl include benzyloxycarbonyl, and preferred examples of the lower alkoxycarbonyl include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, tertiary butoxycarbonyl and the like. Examples of the pharmaceutically acceptable salt of the novel peptide represented by the above formula (I) include alkali metal salts (for example, sodium salt, potassium salt, etc.), alkaline earth metal salts (for example, calcium salt, etc.), amonium salt, and ethanolamine. Salts with inorganic or organic bases such as salts, triethylamine salts, dicyclohexylamine salts, etc., as well as organic acids such as methanesulfonates, hydrochlorides, sulfates, nitrates, phosphates and acid addition salts with inorganic acids. Is also included. The compound (I) of the present invention can be synthesized by various methods, and the details thereof are as follows. (1) Method 1 Acylation<img file="JPH0613552B2_D0002.tif" /><img file="JPH0613552B2_D0003.tif" />(2) Method 2: Removal of protecting group<img file="JPH0613552B2_D0004.tif" />[In the above formula, R¹_aIs a lower alkanoyloxy, R²_aIs a phenyl lower alkoxycarbonyl, R⁴_aIs a group represented by the formula: CONHNHY (where Y means lower alkoxycarbonyl), R⁵_aMeans lower alkoxycarbonyl, respectively, R³_aMeans the same meanings as above] (1) Method 1: Acylation In this method, compound (II) is reacted with compound (III) to form compound (I).^a) Is related to the method of manufacturing. The compound (II) used in this reaction can be used as an activated organic acid, i.e. a reactive acid derivative, such reactive derivatives of the organic acid include acid halides, acid azides, acid anhydrides, activities. Examples thereof include amides and active esters. This reaction is usually carried out in water, alcohol (eg methanol, ethanol, propanol, etc.), acetone, ethyl ether, dioxane, acetonitrile, ethyl acetate, N, N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dichloromethane, chloroform, etc. or pyridine, N. -It is carried out in a usual solvent such as methylmorpholin, N-methylpyrrolidin, or a mixed solvent thereof. This reaction involves alkali metals (eg sodium, potassium, etc.), alkaline earth metals (calcium, etc.), alkali or alkali earth metal hydrides (eg sodium hydride, calcium hydride, etc.), alkali metals or alkaline earth. Metal hydroxides (eg sodium hydroxide, potassium hydroxide, calcium hydroxide, etc.), alkali metal or alkaline earth metal carbonates or bicarbonates (eg sodium carbonate, potassium carbonate, sodium hydrogencarbonate, lithium carbonate, etc.) ), Alcoxides of alkali metals or alkaline earth metals (eg sodium ethoxide, lithium methoxide, magnesium methoxide, etc.), trialkylamines (eg triethylamine, etc.), pyridine, bicyclodiaza compounds (eg 1,5-diazabicyclo [3,, Good results are often obtained when performed in the presence of organic or inorganic bases such as 4,0] nonen-5,1,5-diazabicyclo [5,4,0] undecene-5, etc.). Among the above bases, liquid ones can also be used as a solvent. This reaction temperature is not particularly limited, but is preferably cooled or carried out at room temperature. (2) Method 2: Elimination reaction of protecting group Compound (I^a) Compound (I^b) This method is a compound (I^a) Is subjected to the elimination reaction of the carboxy protecting chamber (ie, phenyl lower alkoxycarbonyl, the same below) and the amino protecting group (ie, the lower alkoxycarbonyl, the same below) in the protected carboxy group to the compound (I).^b) Is related to the method of manufacturing. Method 2-1 Amino in a protected amino group Elimination reaction of protecting groups The elimination reaction of the amino protecting group is a usual method such as a catalytic reduction method, a method using liquid ammonia-alkali metal, a method using an acid, a method using zinc and an acid, a method using a base, and a method using hydrazine. Is applied. Next, each of the above methods will be described below. i) Contact reduction method Suitable catalysts used for this catalytic reduction include platinum catalysts (eg platinum plate, platinum sponge, platinum black, colloidal platinum, platinum oxide or platinum wire, etc.) and palladium catalysts (eg palladium sponge, palladium black, colloidal palladium, palladium). -Barium sulfate, palladium-Barium carbonate, etc.), nickel catalyst (eg reduced nickel, nickel oxide, Raney nickel, etc.), cobalt catalyst (eg reduced cobalt, Raney cobalt, etc.), iron catalyst (eg reduced iron, Raney iron, etc.), copper Examples thereof include ordinary catalysts such as catalysts (for example, reduced copper, Raney copper, Ulmann copper, etc.). The reduction is usually carried out in a solvent. Suitable solvents used include, for example, water, methanol, ethanol, propanol, ethyl acetate, tetrahydrofuran, dioxane, N, N-dimethylformamide, acetic acid, as well as water and alcohols (eg methanol, ethanol, etc.), tetrahydrofuran, dioxane. Alternatively, a mixed solution with ethyl acetate or the like, and other ordinary organic solvents or mixtures thereof are used. In addition, good results are often obtained when the reduction reaction is carried out in the presence of an acid such as acetic acid. The reduction reaction is preferably carried out under relatively mild conditions such as cooling or heating. ii) Method using acid ii) -1 Method using trifluoroacetic acid or formic acid: This reaction is usually carried out in a solvent such as methylene chloride, chloroform, acetic acid or water in the presence of trifluoroacetic acid or formic acid, and particularly good results are often obtained when anisole is added. Trifluoroacetic acid and formic acid can also be used as a solvent. This reaction is usually carried out under water cooling or room temperature. ii) -2 Method using hydrochloric acid or p-toluenesulfonic acid This reaction is carried out in the presence of an inorganic or organic strong acid such as hydrochloric acid, p-toluenesulfonic acid, etc. in a solvent such as ethyl acetate, methylene chloride, chloroform, tetrahydrofuran, and good results are obtained when an appropriate amount of anisole is added. Is often done. This reaction is carried out at ice cooling to room temperature. ii) -3 Method using hydrogen bromide This reaction is usually carried out in the presence of hydrogen bromide in a solvent such as ethyl acetate, acetic acid, trifluoroacetic acid. iii) Liquid ammonia-method using alkali metal This reaction involves compounds (I) in liquid ammonia.^a) Is dissolved and an alkali metal is added therein, and it is usually carried out at a low temperature, for example, from -78 ° C to the boiling point of liquid ammonia. iv) Method using hydrazine Preferred examples of the hydrazine compound include hydrazine, methylhydrazine, phenylhydrazine and the like, and preferred examples of the amine compound include hydroxylamine, dialkylaminoalkylamine (for example, N, N-dimethylaminopropylamine and the like) and the like. Be done. This reaction is usually carried out in a solvent such as water, alcohol (eg methanol, ethanol, etc.), tetrahydrofuran, dioxane, etc. under room temperature to reflux conditions, with a hydrazine compound or amine compound and compound (I).^a) Is reacted. v) How to use metallic zinc-acid This method uses metallic zinc and compounds (I) in the presence of weak acids such as formic acid and acetic acid.^a) Is reacted. This reaction is usually carried out in a solvent such as methylene chloride, chloroform, tetrahydrofuran, ethyl acetate, alcohol (eg methanol, ethanol, etc.), dimethylformamide and the like. Then, the weak acid as described above is added to the solvent, and it is usually carried out at about -10 ° C to room temperature. vi) How to use bases This reaction is usually carried out at room temperature or ice-cooled in the presence of a base. Suitable examples of bases used are alkali metal hydroxides, alkaline earth metals hydroxide, or equivalent carbonates or bicarbonates (eg, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, etc. Inorganic bases such as sodium hydrogen carbonate, calcium hydroxide, magnesium hydroxide, ammonium hydroxide, etc., alkoxyside, phenoxide (eg, sodium ethoxide, sodium methoxide, lithium phenoxide, etc.) of the above metals, mono, g, etc. Or trialkylamines (eg methylamines, ethylamines, propylamines, isopropylamines, butylamines, N, N-dimethyl-1,3-propanediamines, trimethylamines, triethylamines, etc.), unsubstituted or mono- or di-substituted arylamines. (Eg aniline, N-methylaniline, N, N-dimethylaniline, etc.), heterocyclic bases (eg, pyrrolidine, morpholin, N-methylmorpholin, N-methylpiperidin, N, N-dimethylpiperazine, pyridine, etc.), etc. Examples thereof include organic bases such as amines and basic ion exchange resins. This method is preferably carried out under geometrically mild conditions such as cooling or heating and is usually water, alcohol (eg methanol, ethanol, propanol, etc.), N, N-dimethylformamide, tetrahydrofuran, dioxane, dimethyl sulfoxide. Alternatively, it is carried out in a hydrophilic solvent that does not adversely affect the reaction, such as a mixed solvent thereof. Method 2-2: Elimination reaction of carboxy protecting group in protected carboxy The elimination reaction of the protecting group in the protected carboxy is carried out by a usual method such as hydrolysis or reduction. This will be described in detail below. i) Hydrolysis Hydrolysis is used interchangeably with sorbolisis, which includes, for example, acidorisis, alcoholysis, aminolysis, hydroxynorisis, and the like. Hydrolysis is preferably carried out in the presence of an acid or base. Such acids include inorganic acids (eg, hydrochloric acid, odorous acid, sulfuric acid, etc.), organic acids (eg, formic acid, acetic acid, trifluoroacetic acid, propionic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.), acidic ion exchanges. Examples include resins. Bases include hydroxides of alkali metals or alkaline earth metals or corresponding carbonates or bicarbonates (eg sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, sodium hydrogen carbonate, sodium hydroxide). , Calcium hydroxide, magnesium hydroxide, etc.), inorganic bases such as annium hydroxide, alkoxyside or phenoxide of the above metals (eg, sodium ethoxide, sodium methoxide, lithium phenoxide, etc.), mono, di or trialkylamine Amines such as (eg, methylamine, ethylamine, propylamine, isopropylamine, butylamine, N, N-dimethyl-1,3-propanediamine, trimethylamine, triethylamine, etc.), unsubstituted-or mono-, di-substituted arylamine (For example, aniline, N-methylaniline, N, N-dimethylaniline, etc.), heterocyclic bases (for example, pyrrolidine, morpholin, N-methylmorpholin, N-methylpiperidin, N, N-dimethylpiperazine, pyridine, etc.), hydrazine Examples include organic bases such as hydrazine, methylhydrazine, ethylhydrazine, etc., basic ion exchange resins, and the like. Hydrolysis is usually carried out under somewhat mild conditions such as cooling or heating in a solvent that does not adversely affect the reaction. Examples of such a solvent include hydrophilic solvents such as water, alcohol (for example, methanol, ethanol, propanol, etc.), acetone N, N-dimethylformamide tetrahydrofuran, dioxane, dimethyl sulfoxide, etc., or a mixed solvent thereof. In some cases, a hydrophobic solvent such as benzene or diethyl ether may be used. Of the above-mentioned acids and bases, liquid ones can also be used as a solvent. ii) Reduction The reduction includes chemical reduction and catalytic reduction and is carried out by a conventional method. Suitable reducing agents used for chemical reduction include metals (eg tin, zinc, iron, etc.) or the above metals and / or the metal compounds (eg chromium chloride, chromium acetate, etc.) and organic or inorganic acids (eg formic acid, etc.). Combination with acetic acid, propionic acid, trifluoroacetic acid, p-trienesulfonic acid, hydrochloric acid, formic acid, etc.) can be mentioned. As an appropriate catalyst used for catalytic reduction, those exemplified in the description of the reduction method of Method 1 can be mentioned as they are. The reduction reaction is carried out under mild conditions such as cooling or heating in a solvent. Examples of the solvent include water, alcohol (for example, methanol, ethanol, propanol, etc.) and other organic solvents or a mixed solvent thereof. Further, the above-mentioned liquid acid used in chemical reduction can also be used as a solvent. Further, suitable solvents used in catalytic reduction include the above-mentioned solvents and other ordinary solvents such as diethyl ether, dioxane, tetrahydrofuran and the like, or a mixed solvent thereof. The elimination reaction of the protecting group is selected or applied in combination depending on the type of protecting group such as the carboxy protecting group and the amino protecting group to be eliminated. Method 2-3 Elimination of hydrazino groups This method is generally represented by the formula: CONHNHY (Y in the formula means lower alkoxycarbonyl) and the protected carbazoyl group is a compound (I).^a) Is first subjected to the reaction of Method 2-1 above to remove the amino protecting group (ie Y), and the formula: -CONHNH₂After obtaining a compound having a group represented by, a compound having a -COOH group can be obtained by subjecting it to the reaction of this method. The reaction of this method is the formula: -CONHNH₂A compound (I) that is an oxidizing agent capable of oxidizing the group represented by the formula COOH to the group represented by the formula COOH.^a) Is processed according to the conventional method. Preferred examples of such an oxidizing agent include halogens such as iodide and bromine, perioic acids or salts thereof (for example, sodium salts, potassium salts, etc.), perhalogenic acids such as perchloric acid, N-bromosulfuric acid imide and the like. N-haloimide, etc., tetraacetate, hydrogen peroxide or a salt thereof (nickel peroxide), mercury oxide, manganese dioxide, nickel peroxide, copper compounds (eg, copper acetate, copper sulfate, etc.), etc. Be done. This reaction is usually carried out in a solvent such as water, acetic acid, methanol, ethanol, tetrahydrofuran, dioxane and the like and a mixed solvent thereof, and these solvents are appropriately selected depending on the type of oxidizing agent used. This reaction is carried out under water cooling, room temperature or reflux. The starting compounds (II) and (III) are novel and can be synthesized by the following methods.<img file="JPH0613552B2_D0005.tif" /><img file="JPH0613552B2_D0006.tif" />(In the formula, R¹_a, R²_a, R³, R⁴_aAnd R⁵_aHas the same meaning as above) (1) Method 1^s:: In this method, compound (II) can be synthesized from compounds (IV) and (V) by a method substantially similar to that of Method 1. (2) Method 2^s:: In this method, compound (III) can be obtained by subjecting compound (II) to an elimination reaction of a protecting group in a manner substantially similar to that of Method 2. The target compound (I) and the starting compounds (II) and (III) produced by the above-mentioned various methods each contain one or more stereoisomers due to asymmetric carbon atoms in the molecule. And all of these isomers are included within the scope of the present invention. The novel peptide (I) of the present invention and a pharmaceutically acceptable salt thereof have an immune response-promoting effect (cell-mediated immunity activity, humoral antibody production-promoting effect), a reticuloendotheliogenic effect, a juvenile activity, and an infection-preventing effect. Has. Therefore, the novel peptide (I) and its medicinal salts are useful, for example, in the treatment of infectious diseases caused by pathogenic microorganisms, especially Gram-negative, Gram-positive and fungi. Among the novel peptides (I)), the pharmacological test results are shown for the representative compounds. 1. Reticuloendothelial enhancement (reagent): (1) Carbon suspension Rotring drafting ink (170 mg carbon / ml) was diluted to 1/5 with 1% gelatin-containing salt water. (2) 0.1% aqueous sodium hydrogen carbonate solution Design of experiments: Administer 0.01 ml of the above carbon turbid solution per 1 g of body weight from the tail vein of DDY mice (5-week-old male). Blood is collected from the ocular plexus using a heparin-washed capillary pipette over time, and 50 μl of the blood is mixed well with 0.1% of sodium carbonate solution to hemolyze. This lysed blood is colorimetrically quantified at 660 nm and the concentration of carbon particles in the blood is measured. The carbon disappearance rate constant K is calculated from the following equation.<img file="JPH0613552B2_D0007.tif" />T₁, T₂Is the time of blood collection, C₁, C₂Is T₁, T₂The carbon concentration in the blood at the time is shown respectively.

[Effect test of test compound on carbon clearance] An aqueous solution of the given drug was subcutaneously administered to the mice. After 24 hours, the carbon clearance capacity was measured. The K values of the control group and the treatment group were compared. The test results are shown in Table 1.<img file="JPH0613552B2_D0008.tif" />2. Infection prevention effect In order to investigate the preventive effect on mouse experimental infections, the test compound was dissolved in sterile saline to give each predetermined concentration. ICR male mice (4 weeks old) were used and 10 mice per group were used. Escherichia coli 22 was cultured overnight on trypticase soy agar medium at 37 ° C, turbid in sterile saline, and the viable cell count was 9.0 × 10.⁷CFU / ml solution was used. 0.2 ml of this suspension was administered intravaginally to mice. Twenty-four hours prior to this, each of the 10 mice was administered with each predetermined concentration of the test compound intravaginally. The survival rate was calculated based on the number of surviving animals on the 4th day after injection, and it was as shown in Table 2.<img file="JPH0613552B2_D0009.tif" />The pharmaceutical composition of the present invention can be used in various preparations such as solid, semi-solid or liquid, and these are administered externally, internally or parenterally in addition to the active substance obtained by the present invention. It contains an inorganic or organic carrier or auxiliary agent suitable for the above. The active ingredient is blended with non-toxic and non-toxic tablets, granules, capsules, suppositories, liquids, emulsions, suspensions and other carriers to make them suitable for administration. Carriers that can be used include water, glucose, lactose, acacia gum, gelatin, mannitol, starch paste, magnesium trisilicate, talc, cornstarch, keratin, colloidal silica, potato starch, urea, or solid, semi-solid or liquid. Other carriers and the like suitable for the formulation can be used, and auxiliary agents, stabilizers, bulking agents, pigments, fragrances and the like can also be blended. Further, the pharmaceutical composition may contain a preservative or an antibacterial agent in order to maintain the activity of the active ingredient. The active target substance should contain an amount sufficient to exert an appropriate therapeutic effect on the process and situation of the disease. When applying this composition to the human body, intravenous injection, intramuscular injection or oral administration is adopted. The effective dose for the treatment of the target substance of the present invention varies depending on the age and symptoms of the patient, but the usual dose for humans and animals is 2 to 100 mg / kg per day, and the average dose per dose is 2 to 100 mg / kg. Appropriate amounts are 50 mg, 100 mg, 250 mg and 300 mg. Next, an example of the present invention will be shown. In the following examples, the starting substance and the target substance are represented by using the following symbols. Lac: Lactoyl Ala: Alanil Glu: Glutamil Gly: Glycyl DAP: α, ε-diaminopimeryl Z: benzyloxycarbonyl Boc: Tertiary butoxycarbonyl Bzl: benzyl Su: N-hydroxysuccinimide Ac: Acetyl Example 1 (1) Process 1<img file="JPH0613552B2_D0010.tif" /><img file="JPH0613552B2_D0011.tif" />In a mixed aqueous solution of D-Lac (OAc) -L-Ala-γ-D-Glu (α-OBz1)-(L) -meso DAP- (L) -GlyOH (1) (960 mg) and triethylamine (300 mg), D-Lac (OAc) -L-Ala-γ-D-Glu (α-OBz1)-(L) -Boc- (D) -Mezo DAP- (D) -NHNHBoc- (L) -GlyOH (1.28g) D-Lac (OAc) -L-Ala-γ-D synthesized by stirring N-hydroxysuccinimide (170 mg) and bis (trimethylsilyl) acetamide (310 mg) in a dioxane (40 ml) solvent at room temperature overnight. -Glu (α-OBz1)-(L) -Boc- (D) -Mezo DAP- (D) -NHNHBoc- (L) -GlyOSu (2) was added. Triethylamine was added to this mixed solution, and the mixture was stirred at room temperature for 8 hours while maintaining the pH at 7 to 8. After dioxane in the reaction mixture was distilled off, the obtained solution was washed with ethyl acetate, adjusted to pH 2 by adding 5% hydrochloric acid, and extracted with n-butanol (100 ml). Ether is added to the residue obtained by concentrating the extract to make it powdery. D-Lac (OAc) -L-Ala-γ-D-Glu (α-OBz1)-(L)-[D-Lac (OAc) -L-Ala-γ-D-Glu (α-OBz1)-(L) -Boc- (D) -Mezo DAP- (D) -NHNHBoc- (L) -Gly]-(D) -Mezo DAP-( L)-GlyOH (3) (1.78 g) was obtained. IR (Nujoru): 3600-2200,3260,1720,1640cm^-1(2) Process 2<img file="JPH0613552B2_D0012.tif" />D-Lac (OAc) -L-Ala-γ-D-Glu (α-OBz1)-(L)-[D-Lac (OAc) -L-Ala-γ-D-Glu (α-OBz1)-( L) -Boc- (D) -Mezo DAP- (D) -NHNHBoc- (L) -Gly]-(D) -Mezo DAP- (L) -GlyOH (3) (1.20g), acetic acid (40ml) In the solution, hydrogenated at room temperature at 2.0 atm in the presence of 10% palladium black (240 mg) for 3.5 hours. After removing the catalyst by filtration, the filtrate was evaporated, the residue was dissolved in a 50% aqueous methanol solution (20 ml), a 10% aqueous potassium carbonate solution was added, and the mixture was stirred at room temperature for 2.5 hours while maintaining the pH at 9. The solution was concentrated to about 10 ml and IN hydrochloric acid (10 ml) was added. A solution of perioic acid (260 mg) in water (3 ml)) was added dropwise to this mixed solution, and the mixture was stirred for 30 minutes under cooling in an ice bath, and the excess reagent was decomposed by adding sodium hydrogen sulfite. After adjusting the pH to 2 by adding 1N sodium hydroxide to the obtained solution, the solution was concentrated to about 10 m, passed through a column packed with HP20 resin (20 ml), and eluted with a 50% aqueous methanol solution. Freeze-dry the eluate and D-Lac-L-Ala-γ-D-Glu (α-OH)-(L)-[D-Lac-L-Ala-γ-D-Glu (α-OH)- (L) -Mezo DAP- (D) -Gly]-(D) -Mezo DAP- (L) -GlyOH (4) (0.49 g) was obtained. IR (Nujoru): 3600-2200,1720,1640cm^-1NMR (D₂O), δ (ppm): 1.3-1.5 (12H, m), 3.88 (1H, t, J = 6Hz), 4.00 (4H, s), 4.2-4.5 (1H, m) Example 2 (1) Process 1<img file="JPH0613552B2_D0013.tif" /><img file="JPH0613552B2_D0014.tif" />D-Lac (OAc) -L-Ala-γ-D-Glu (α-OBz1)-(L)-[D-Lac (OAc) -L-Ala-γ- D-Glu (α-OBz1)-(L) -Boc- (D) -Mezo DAP-(D) -NHNHBoc- (L)-(D) Ala]-(D) -Mezo DAP- (L) -D -Manufactured Ala (3). IR (nujor): 3250,1720,1640cm^-1(2) Process 2<img file="JPH0613552B2_D0015.tif" />D-Lac-L-Ala-γ-D-Glu (α-OH)-(L)-[D-Lac-L-Ala-γ-D-Glu (α-)) in the same manner as in Step 2 of Example 1. OH)-(L) -Mezo DAP- (L) -D-Ala]-(D) -Mezo DAP- (L) -D-Ala OH (4) was produced. IR (Nujoru): 3600-2200,1720,1660-1630cm^-1NMR (D₂O), δ (ppm): 1.2-1.5 (18H, m), 3.82 (1H, t, J = 6Hz), 4.1-4.5 (11H, m)