JPH0414120B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel muramyl peptide derivative that has an immune function-enhancing effect and an excellent antitumor effect, and more specifically, a novel muramyl peptide derivative with the general formula () [In the above formula, R ¹ is a lower alkyl group, R ² is a hydrogen atom or a lower alkyl group, n is an integer of 1 to 6, and R ³ is a carbon atom number of 2 to 50 which may be substituted with a hydroxyl group means an alkylcarbonyl group. ] The present invention relates to a muramyl peptide derivative represented by: As a result of intensive studies on compounds with excellent adjuvant activity and antitumor activity and weak pyrogenicity, the present inventor found that the compound represented by the general formula () has excellent adjuvant activity and antitumor activity. The present invention was completed based on the discovery that it is non-pyrogenic and has few side effects. In contrast to conventional muramyl peptide derivatives, the compound of the present invention is characterized by the N-alkylation of the alanyl group in its chemical structure, which allows the above-mentioned effects to be expressed. . The effects of the compounds of the present invention are as follows [] Adjuvant activity (cell-mediated immunity test) Monoazobenzene arsonate-N-acetyl-L-tyrosine (hereinafter referred to as ABA-Tyr).
The induction of a delayed allergic reaction was investigated using the skin reaction of guinea pigs as an indicator. That is, 50 μg of ABA-Tyr and the sample were mixed with Freund's incomplete adjuvant (hereinafter referred to as FIA).
A water-in-oil emulsion was administered to the footpads of a group of four Hartley guinea pigs.
After 24 weeks, a skin reaction was performed with 100 μg of azobenzene arsonate-bacterial α-amylase (ABA-B-α-A) dissolved in saline.
Induration in the skin was measured after hours. (Table-
1)

[Table] [] Antitumor activity Fibrosarcoma cells (MethA) induced with methylcholanthrein from BALB/C male mice 2×10 ⁵
cells were inoculated intradermally into syngeneic BALB/C mice,
After 7 days, a PBS solution containing 100 μg of the specimen was administered into the tumor 7 times approximately every other day, and the weight of the sarcoma tumor was determined 4 weeks later. (Table-2)

[Table] Percentage when pyrogenic substance test The test was conducted according to the Japanese Pharmacopoeia Pyrogenic Substance Test Method (10th revised Japanese Pharmacopoeia Manual B-206, 1981). As a result, the compound-3 of the present invention was
No pyrogenic effect was observed at the dose of 0.375 mg/Kg. As described above, the compound of the present invention has excellent adjuvant activity and antitumor activity, and is not pyrogenic, so it can be used as a highly safe antitumor agent in clinical applications and is effective in inducing immune responses in living organisms. It can be used for many diseases that require it. Compounds 1 to 5 of the present invention and control compounds 1 and 2 shown in Tables 1 and 2 are as follows. Compound of the present invention-1: N〓-(N-acetylmuramyl-N-methyl-L-alanyl-D-isoglutaminyl)-N〓-acetyl-L-lysine Compound of the present invention-2: N〓-(N-acetyl Muramyl-N-methyl-L-alanyl-D-isoglutaminyl)-N〓-decanoyl-L-lysine Invention compound-3: N〓-(N-acetylmuramyl-N-methyl-L-alanyl-D- isoglutaminyl)-N〓-stearoyl-L-lysine Compound of the present invention-4: N〓-(N-acetylmuramyl-N-methyl-L-alanyl-D-isoglutaminyl)-N〓-(2-tetradecylhexadeca Noyl)-L-lysine Compound of the present invention-5: N〓-(N-acetylmuramyl-N-methyl-L-alanyl-D-isoglutaminyl)-N〓-(3-hydroxy-2-docosylhexacosanoyl )-L-lysine control compound-1: N-acetylmuramyl-L-alanyl-D-isoglutamine control compound-2: N〓-(N-acetylmuramyl-
L-alanyl-D-isoglutaminyl)-N〓-
Nocaldomicoloyl-L-lysine methyl ester The compound of the present invention can be produced according to the following reaction steps. (In the formula, the substituents R, R ¹ , R ² and R ³ are as defined above.) That is, the compound of the present invention () can be prepared by the method (a) of reacting the compound represented by the formula () with the muramyl peptide () method) and muramyl peptide ()
It can be produced by employing a method (method b) in which a compound represented by the formula () is reacted with the compound represented by the formula (). Condensation reaction when method a is adopted, i.e. ()+
For the reaction of () → () and the condensation reaction when method b is adopted, that is, the reaction of () + () → (), the condensation method commonly used in the field of peptide synthetic chemistry can be usually adopted. . Examples include the carbodiimide method, active ester method, Eintop method, and acid anhydride method, and these methods may be appropriately selected and employed. For example, active esters of the compound represented by formula () (Active esters include P-nitrophenyl ester, pentachlorophenyl ester, N
-hydroxysuccinimide ester, 1-hydroxybenzotriazole ester, N-hydroxy-5-norbornene-2,3-dicarboximide ester, and the like. ) and the compound represented by the formula (), for example, acetonitrile, tetrahydrofuran, N,N-dimethylformamide,
0 to 60 in a single or mixed solvent such as chloroform or water
C., preferably about 25 to 40.degree. C., for about 1 to 2 days. In this condensation reaction, an organic base such as triethylamine, N-methylmorpholine, N-ethylmorpholine or 1-
Hydroxybenzotriazole can also be present. For example, a compound represented by the formula () may be N,N-
dissolved in dimethylformamide, tetrahydrofuran or acetonitrile or a mixture thereof;
In addition to this, P-nitrophenol, pentachlorophenol, N-hydroxysuccinimide, 1-hydroxybenzotriazole, N-hydroxy-
One type of 5-norbornene-2,3-dicarboximide and dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)
- Carbodiimide or its hydrochloride and usually room temperature to approx.
The active ester may be reacted at 60°C for several hours to about 2 days, and the compound represented by the formula () may be condensed. In this condensation reaction, the condensation conditions of formulas () and () can be applied in almost the same way. The raw material compounds used in the production of the compounds of the present invention, ie, formulas (), (), and (), can be produced according to the following method. In other words, to produce a compound represented by formula (), use the formula (In the formula, R ¹ , R ² , and n are as defined above. Z is a protecting group for an amino group that is commonly used in peptide synthesis chemistry, such as benzyloxy, which may have a substituent such as a halogen atom or a nitro group. This can be achieved by condensing N-acetylmuramic acid with a compound represented by carbonyl group, and then removing the protecting group by employing appropriate protecting group removal conditions as necessary. For this condensation reaction, a condensation method frequently used in the field of peptide synthetic chemistry can be employed. For example, the carbodiimide method, the Eintop method,
Examples include an active ester method and an acid anhydride method, which can be appropriately selected and employed. During the condensation reaction of this formula () and N-acetylmuramic acid, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, etc. may be present alone or in combination. In addition, the hydroxyl groups at the 4th and 6th positions of the sugar of N-acetylmuramic acid are replaced with an arylidene group, such as benzylidene, P-methoxybenzylidene, etc., and the hydroxyl group at the 1st position of the sugar is replaced with an aralkyl group, such as one halogen atom, nitro group, or methoxy group. It is preferable to use a compound protected with a benzyl group or the like, which may be substituted as described above, since side reactions in the condensation reaction can be reduced. At this time, after the condensation reaction, the protecting group may be removed if necessary. To prepare a compound represented by the formula (), use the formula (In the formula, R ¹ is as defined above. .Y is a carboxyl group protecting group frequently used in the field of ordinary peptide synthesis chemistry, such as a methyl group,
Examples include ethyl group, t-butyl group, and benzyl group. ) After removing the protecting group Y of the carboxyl group by adopting appropriate protecting group removal conditions, the compound represented by the formula (In the formula, R ² , n, and Z are as defined above.) are condensed, and the thus obtained compound is then removed from the protecting group X using appropriate protecting group removal conditions. This condensation reaction can be carried out by appropriately selecting the above-mentioned condensation method that is commonly used in peptide synthesis chemistry. Also, for removing the protecting group For example, acetic acid, etc.) treatment method is adopted. To prepare a compound represented by the formula (), use the formula (In the formula, R ¹ and X are as defined above.) (In the formula, Y is as defined above.) This can be achieved by condensing a compound represented by the above formula. As the condensation method, the aforementioned condensation method frequently used in the field of peptide synthetic chemistry can be employed. To produce the starting compound (), the compound represented by the formula () is removed by removing the protecting group X of the amino group using appropriate protecting group removal conditions, and the resulting compound is combined with N-acetylmuramic acid. This is achieved by condensation and, if necessary, removing the protecting group by employing appropriate protecting group removal conditions. During this condensation reaction, substantially the same conditions as in the condensation reaction of formula () and N-acetylmuramic acid described above can be employed. To produce the starting compound (), use the formula (In the formula, R ² , n, and X are as defined above.) After condensing the compound represented by the formula () with the compound represented by This is achieved by eliminating X.
The condensation reaction may be carried out by appropriately selecting the above-mentioned condensation methods that are commonly used in the field of peptide synthetic chemistry. Example 1 t-butyloxycarbonyl-N-methyl-L
- Dissolve 4.00 g of alanine in 25 ml of tetrahydrofuran, N-hydroxy-5-norbornene-2,
Add 3.88 g of 3-dicarboximide. 4.47 g of dicyclohexylcarbodiimide was added under ice-cooling and stirring, and after 30 minutes, the mixture was gradually warmed to room temperature and reacted for 5 hours. After filtering off the precipitated dicyclohexyl urea,
Concentrate under reduced pressure and dissolve the residue in 30 ml of N,N-dimethylformamide. To this is added 5.93 g of D-isoglutamine benzyl ester hydrochloride. Add 6.06 ml of triethylamine under ice-cooling and stirring, and after 30 minutes,
Gradually return to room temperature and react overnight. The reaction solution was concentrated under reduced pressure, and 200 ml of ethyl acetate was added to the residue.
% citric acid aqueous solution, water, 5% sodium bicarbonate aqueous solution, and water. Ethyl acetate was distilled off under reduced pressure, and the residue was purified by chromatography on silica gel, eluting with chloroform-ethyl acetate (8:1) and then with ethyl acetate.
Colorless oil t-butyloxycarbonyl-N-
7.56 g of methyl-L-alanyl-D-isoglutamine benzyl ester are obtained. [α] ²⁵ _D −6.2° (C=1.7, methanol). Elemental analysis value C ₂₁ H ₃₁ N ₃ O ₆ Calculated value (%) C 59.84, H 7.41, N 9.97 Actual value (%) C 59.76, H 7.43, N 9.87 Dissolve 7.32 g of the obtained above compound in 200 ml of tetrahydrofuran. Then, hydrogenolysis is carried out at room temperature in a hydrogen stream in the presence of palladium on carbon. After the reaction, the catalyst is filtered off and the filtrate is concentrated under reduced pressure. Add 10 ml of ethyl acetate to the residue, collect the precipitated powder by filtration, and add t-butyloxycarbonyl-N-methyl-
4.14 g of L-alanyl-D-isoglutamine are obtained. Melting point 107-111℃. [α] ²⁵ _D −6.5° (C=2.3, methanol). Elemental analysis value C ₁₄ H ₂₅ N ₃ O ₆・3/4H ₂ O Calculated value (%) C 48.76, H 7.75, N
12.18 Actual value (%) C 48.64, H 7.62, N
12.03 3.21 g of the above compound obtained above is dissolved in 50 ml of tetrahydrofuran and 1.91 g of N-hydroxy-5-norbornene-2,3-dicarboximide is added. Add 2.20 g of dicyclohexylcarbodiimide under ice-cooling and stirring, and after 30 minutes, gradually return to room temperature.
React for 5 hours. After the precipitated dicyclohexylurea was filtered off, the mixture was concentrated under reduced pressure, and the residue was dissolved in 25 ml of N,N-dimethylformamide. To this was added 4.33 g of N-benzyloxycarbonyl-L-lysine benzyl ester hydrochloride. Add 2.98 ml of triethylamine under ice-cooling and stirring, and after 30 minutes, gradually return to room temperature and react overnight. The reaction solution was concentrated under reduced pressure, 200 ml of ethyl acetate was added to the residue, and the mixture was washed successively with a 5% aqueous citric acid solution, water, a 5% aqueous sodium bicarbonate solution, and water. Ethyl acetate was distilled off under reduced pressure, and the residue was recrystallized from ethyl acetate-ether to give N〓-(t-butyloxycarbonyl-N-methyl-L-alanyl-D-isoglutaminyl)-N〓
4.62 g of -benzyloxycarbonyl-L-lysine benzyl ester are obtained. Melting point 87-88℃. [α] ²⁵ _D
−20.9° (C=0.7, methanol). Elemental analysis value C ₃₅ H ₄₉ N ₅ O Calculated value (%) as ₉・1/4H ₂ O C 61.07, H 7.25, N
10.17 Actual value (%) C 60.96, H 7.21, N
10.42 3.50 g of the above compound obtained was dissolved in dichloromethane 6
10 ml of trifluoroacetic acid under ice-cooling and stirring.
Add. After 5 minutes, the temperature was gradually returned to room temperature and the reaction was continued for 1 hour. Concentrate the reaction solution under reduced pressure and add 6.5N to the residue.
Add a solution of 2 ml of hydrochloric acid/dioxane in 20 ml of ether. The precipitated powder was collected by filtration and dissolved in 35 ml of N,N-dimethylformamide. 1-α under ice-cooling and stirring
-O-benzyl-4,6-O-benzylidene-N
-Acetylmuramic acid 2.17g, 4-dimethylaminopyridine 0.90g, 1-hydroxybenzotriazole 0.32g, dicyclohexylcarbodiimide
Add 1.00 g, and after 30 minutes, gradually return to room temperature and react overnight. After filtering off the precipitated dicyclohexyl urea, it is concentrated under reduced pressure, and 200 ml of ethyl acetate is added to the residue, which is washed successively with a 5% aqueous citric acid solution, water, a 5% aqueous sodium bicarbonate solution, and water. Ethyl acetate was distilled off under reduced pressure, the residue was subjected to silica gel chromatography, and chloroform-ethyl acetate (1:
N〓-(1-α-
O-benzyl-4,6-O-benzylidene-N-
Acetylmuramyl-N-methyl-L-alanyl-
3.42 g of D-isoglutaminyl)-N-benzyloxycarbonyl-L-lysine benzyl ester are obtained. Melting point 152-153℃. [α] ²⁵ _D +35.2° (C=0.6,
tetrahydrofuran). Elemental analysis value C ₅₅ H ₆₈ N ₆ O ₁₄・1/2H ₂ O Calculated value (%) C 63.14, H 6.65, N 8.03 Actual value (%) C 63.20, H 6.54, N 8.07 Obtained above compound 3.00 g was dissolved in 50 ml of acetic acid, and hydrogenolyzed at room temperature in a hydrogen stream in the presence of palladium on carbon. After the reaction, the catalyst is filtered off and the filtrate is concentrated under reduced pressure. The residue was purified by Sephadex G-15 gel chromatography, eluting with n-butanol-acetic acid-water (4:1:5, upper layer). After concentrating the target fraction under reduced pressure and lyophilizing it, N〓-(N-acetylmuramyl-
1.48 g of N-methyl-L-alanyl-D-isoglutaminyl)-L-lysine are obtained. Melting point 124-126℃
(Disassembly). [α] ²⁵ _D +8.5° (C=0.9, water, 2 days later). Elemental analysis value C ₂₆ H ₄₆ N ₆ O ₁₂ .CH ₃ CO ₂ H・1 3/4H ₂ O Calculated value (%) C 46.31, H 7.43, N
11.57 Actual value (%) C 46.28, H 7.48, N
11.81 0.50 g of the obtained above compound was dissolved in 10 ml of N,N-dimethylformamide, and 0.21 g of N-hydroxy-5-norbornene-2,3-dicarboximide active ester of acetic acid, N-methyl Add 0.17 ml of morpholine. After 30 minutes, gradually return to room temperature and react overnight. Concentrate the reaction solution under reduced pressure,
Ether is added to the residue and the precipitated powder is collected by filtration. This was subjected to silica gel chromatography and chloroform-methanol-acetic acid (4:1:
Elute and purify with 0.1). The desired fraction is concentrated under reduced pressure, ether is added to the residue, and the precipitated powder is collected by filtration. This is dissolved in water and passed through a strongly acidic ion exchange resin (H type). By freeze-drying the passed liquid
N〓-(N-acetylmuramyl-N-methyl-L-
0.38 g of alanyl-D-isoglutaminyl)-N-acetyl-L-lysine is obtained. Melting point 142-145℃
(Disassembly). [α] ²⁵ _D +17.7° (C=0.7, N,N-dimethylformamide-water (50:2), after 1 day). Elemental analysis value C ₂₈ H ₄₈ N ₆ O Calculated value (%) as ₁₃・H ₂ O C 48.41, H 7.25, N
12.10 Actual value (%) C 48.50, H 7.05, N
12.17 Example 2 N〓-(N-acetylmuramyl-N-methyl-L
By reacting 0.30 g of N-hydroxysucciimide active ester of n-decanoic acid with 0.30 g of N-hydroxysuccinimide (alanyl-D-isoglutaminyl)-L-lysine in substantially the same manner as in Example 1, N〓(N-acetylmuramyl- N-methyl-L-alanyl-D-isoglutaminyl)-N-decanoyl-L-lysine 0.23g
get. Melting point 137-140℃ (decomposition). [α] ²⁵ _D +13.7°
(C=0.4, N,N-dimethylformamide-water (50:2), after 1 day). Elemental analysis value C ₃₆ H ₆₄ N ₆ O ₁₃ .1 Calculated value (%) as 1/2H ₂ O C 52.99, H 8.28, N
10.29 Actual value (%) C 53.07, H 7.98, N
10.38 Example 3 N〓-(N-acetylmuramyl-N-methyl-L
-alanyl-D-isoglutaminyl)-L-lysine 0.40g and N-hydroxy-5-stearic acid.
By reacting 0.31 g of norbornene-2,3-dicarboximide active ester in substantially the same manner as in Example 1, N〓-(N-acetylmuramyl-N-
methyl-L-alanyl-D-isoglutaminyl)
0.34 g of -N-stearoyl-L-lysine is obtained.
Melting point 160-161℃ (decomposition). [α] ²⁵ _D +9.9° (C=0.6
,
N,N-dimethylformamide-water (50:2),
1 day later). Elemental analysis value C ₄₄ H ₈₀ N ₆ O ₁₃・1 1/2H ₂ O Calculated value (%) C 56.94, H 9.01, N 9.05 Actual value (%) C 56.82, H 8.92, N 9.35 120 mg of the above compound was dissolved in N , N-dimethylformamide, treated with diazomethane, concentrated under reduced pressure, ether was added to the residue, and the precipitated powder was collected by filtration. This was purified by silica gel chromatography, eluted with chloroform-methanol (9:1), and recrystallized from methanol-ether. 80 mg of -alanyl-D-isoglutaminyl)-N-stearoyl-L-lysine methyl ester are obtained. Melting point 121-124°C (decomposition).
[α] ²⁵ _D +5.4° (C=0.4, N,N-dimethylformamide-water (50:2), after 2 days). Elemental analysis value C ₄₅ H ₈₂ N ₆ O ₁₃・1.5H ₂ O Calculated value (%) C 57.36, H 9.09, N 8.92 Actual value (%) C 57.47, H 8.93, N 8.79 Example 4 t-Butyloxy carbonyl-N-methyl-L
2.28 g of -alanyl-D-isoglutamine benzyl ester is dissolved in 5 ml of dichloromethane, and 5 ml of trifluoroacetic acid is added while stirring under ice cooling. 5 minutes later,
Gradually return to room temperature and react for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was mixed with 1.10% of 5N hydrochloric acid/dioxane.
Add 30 ml of ether solution. The precipitated powder was collected by filtration and mixed with N,N-dimethylformamide 7.
Dissolve in ml. Under ice cooling and stirring, 2.32 g of 1-α-O-benzyl-4,6,O-benzylidene-N-acetylmuramic acid, 4-dimethylaminopyridine.
0.96g, 1-hydroxybenzotriazole 0.33
g and 1.07 g of dicyclohexylcarbodiimide were added, and after 30 minutes, the mixture was gradually warmed to room temperature and reacted overnight. After filtering off the precipitated dicyclohexyl urea, it was concentrated under reduced pressure, and 50 ml of chloroform was added to the residue.
% citric acid aqueous solution, water, 5% sodium bicarbonate aqueous solution, and water. Chloroform was concentrated under reduced pressure, and the residue was subjected to silica gel chromatography. Chloroform-ethyl acetate (1:1),
It was then purified by elution with chloroform-methanol (20:1) to give 1-α-O-benzyl-4,6-O-benzylidene-N-acetylmuramyl-N-methyl-L-alanyl-D- 2.14 g of isoglutamine benzyl ester is obtained. melting point 86
~90℃. [α] ²⁵ _D +37.9° (C=0.6, methanol). Elemental analysis value C ₄₁ H ₅₀ N ₄ O Calculated value (%) as ₁₁ C 63.55, H 6.50, N 7.23 Actual value (%) C 63.84, H 6.72, N 7.18 Dissolve 1.98 g of the obtained above compound in 15 ml of acetic acid. Then, hydrogenolysis is carried out at room temperature in a hydrogen stream in the presence of palladium on carbon. After the reaction, the catalyst is filtered off and the filtrate is concentrated under reduced pressure. The residue was subjected to silica gel chromatography and n-butanol-water-
Purify by eluting with acetic acid (10:1:1). The desired fraction is concentrated under reduced pressure, ether is added to the residue, and the precipitated powder is collected by filtration. This is dissolved in water and passed through a strongly acidic ion exchange resin (H type). By freeze-drying the solution, 1.05 g of N-acetylmuramyl-N-methyl-L-alanyl-D-isoglutamine is obtained. Melting point 116-118°C (decomposition). [α] ²⁵ _D +17.6° (
C
= 0.7, Wednesday, 1 day later). Elemental analysis value C ₂₀ H ₃₄ N ₄ O Calculated value (%) as ₁₁・1/2H ₂ O C 46.60, H 6.84, N
10.87 Actual value (%) C 46.34, H 6.56, N
10.71 177 mg of the obtained above compound was dissolved in 1 ml of N,N-dimethylformamide, and N-hydroxy-5
-Norbornene-2,3-dicarboximide
Add 62.7mg. Add 72.2 mg of dicyclohexylcarbodiimide under ice-cooling and stirring, and after 30 minutes, gradually warm to room temperature and react overnight. After filtering off the precipitated dicyclohexyl urea, it is concentrated under reduced pressure, ether is added to the residue, and the precipitated powder is collected by filtration. This was dissolved in a mixture of 6 ml of tetrahydrofuran and 2 ml of N,N-dimethylformamide, and stirred under ice-cooling.
-tetradecylhexadecanoyl)-L-lysine
Add 170mg and 64.0μ of N-methylmorpholine.
After 30 minutes, gradually return to room temperature, and after reacting overnight,
React at 40°C for 7 hours. After removing insoluble matter by filtration, it is concentrated under reduced pressure, water is added to the residue, and the precipitated powder is collected by filtration. This was subjected to silica gel chromatography and eluted with chloroform-methanol (9:1). The target fraction was concentrated under reduced pressure, 3 ml of 0.01N hydrochloric acid was added to the residue, insoluble materials were collected by filtration, washed with water, and recrystallized from methanol-acetonitrile to obtain N〓-(N-acetylmuramyl-N- Methyl-
L-alanyl-D-isoglutaminyl)-N〓-
54 mg of (2-tetradecylhexadecanoyl)-L-lysine is obtained. Melting point 170-171℃ (decomposition). [α] ²⁵ _D
+10.7° (C=0.3, N,N-dimethylformamide-water (50:2), after 2 days). Elemental analysis value C ₅₆ H ₁₀₄ N ₆ O ₁₃・2 As 1/2H ₂ O Calculated value (%) C 60.34, H 9.85, N 7.54 Actual value (%) C 60.52, H 9.67, N 7.43 N〓-(2 -tetradecylhexadecanoyl)-
L-lysine was prepared by the method shown below. N〓-benzyloxycarbonyl-L-lysine-benzyl ester tosylate 580mg was added to N,N-
Dissolve 0.11 ml of N-methylmorpholine and 400 mg of 2-tetradecylhexadecanoic acid in 4 ml of tetrahydrofuran under ice-cooling and stirring in 6 ml of dimethylformamide.N-Hydroxy-5-norbornene-
Add 190 mg of 2,3-dicarboximide and 220 mg of dicyclohexylcarbodiimide. 30 minutes later,
Gradually return to room temperature, react overnight, and then incubate at 40℃ for 20 minutes.
Time reacts. After removing the precipitated dicyclohexylurea by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography and eluted with chloroform to obtain N〓-benzyloxycarbonyl-N〓-(2-tetra 510 mg of L-lysine benzyl ester (decylhexadecanoyl) are obtained. Melting point 100-101℃. [α] ²⁵ _D +1.2° (C=0.5, chloroform). Elemental analysis value C ₅₁ H ₈₄ N ₂ O ₅・1/4H ₂ O Calculated value (%) C 75.65, H 10.52, N
3.46 Actual value (%) C 75.70, H 10.68, N
3.59 350 mg of the above compound obtained above is dissolved in 40 ml of tetrahydrofuran and subjected to hydrolysis in a hydrogen stream in the presence of palladium black. After reaction, methanol
40 ml was added, the catalyst was filtered off, and the filtrate was concentrated under reduced pressure. The residue was recrystallized from methanol-ether to obtain 187 mg of N-(2-tetradecylhexadecanoyl)-L-lysine. Melting point 175~
177°C, [α] ²⁵ _D −3.4° (C=0.2, methanol). Elemental analysis value C ₃₆ H ₇₂ N ₂ O ₃・1/2H ₂ O Calculated value (%) C 73.29, H 12.47, N
4.75 Actual value (%) C 73.13, H 12.29, N
4.51 Example 5 106 mg of N-t-butyloxycarbonyl-L-lysine was dissolved in 0.5 ml of water, and N-hydroxy-5-norbornene- of 3-hydroxy-2-docosylhexacosanoic acid was dissolved in 0.5 ml of water. A solution of 316 mg of 2,3-dicarboximide active ester in 5 ml of tetrahydrofuran and 36.0 µ of N-methylmorpholine are added. After 30 minutes, the mixture was gradually returned to room temperature and reacted for 1 hour, and then at 40°C overnight. The reaction solution was concentrated under reduced pressure, 50 ml of chloroform was added to the residue, and the mixture was washed successively with a 5% aqueous citric acid solution and water. Chloroform was distilled off under reduced pressure, the residue was subjected to silica gel chromatography, and chloroform-methanol (9:
By elution and purification with step 1), N〓-t-butyloxycarbonyl-N〓-(3-hydroxy-
2-docosylhexacosanoyl)-L-lysine 172
Get mg. Melting point 71-73℃. 160 mg of the obtained above compound was added with 55 mg of dichloromethane.
ml of trifluoroacetic acid under ice-cooling and stirring.
Add. After 5 minutes, the temperature was gradually returned to room temperature and the reaction was continued for 1 hour. The reaction solution was concentrated under reduced pressure, ether was added to the residue, and the precipitated powder was collected by filtration. This is N,N
-N-acetylmuramyl-N-methyl-L-alanyl-D-
N-hydroxy-5-norbornene-2,3-dicarboximide active ester of isoglutamine
Add 134 mg and 37.0μ of N-methylmorpholine.
After 30 minutes, the mixture was gradually returned to room temperature, reacted for 1 hour, and then reacted at 40°C overnight. Concentrate the reaction solution under reduced pressure,
Water is added to the residue and the precipitated powder is collected by filtration. Dissolve this in 50ml of 10% water/tetrahydrofuran,
Add strong acidic ion exchange resin (H type) and incubate at room temperature for 1
Stir for an hour. After filtering off the resin, the resin was concentrated under reduced pressure and the residue was recrystallized from tetrahydrofuran-acetonitrile to give N〓-(N-acetylmuramyl-N-methyl-L-alanyl-D-isoglutaminyl)-N〓-( 82 mg of 3-hydroxy-2-docosylhexacosanoyl)-L-lysine are obtained. Melting point 177-178°C (decomposition). [α] ²⁵ _D +11.7° (C=0.7,
N,N-dimethylformamide-water (50:21, 1
days after). Elemental analysis value C ₇₄ H ₁₄₀ N ₆ O ₁₄・2H ₂ O Calculated value (%) C 64.48, H 10.56, N
6.10 Actual value (%) C 64.53, H 10.30, N
5.80

【table】

Claims (1)

[Claims] 1. General formula A muramyl peptide derivative represented by [in the above formula, R ¹ is a lower alkyl group, R ²
represents a hydrogen atom or a lower alkyl group, n represents an integer of 1 to 6, and R ³ represents an alkylcarbonyl group having 2 to 50 carbon atoms which may be substituted with a hydroxyl group. ]