JPH0363560B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention provides novel muramyl tripeptide derivatives, more specifically, the general formula () (In the formula, R ₁ and R ₂ each represent a benzyl group, R ₃ and R ₄ each represent a hydrogen atom or both together represent a benzylidene group, Ac represents an acetyl group, and n is 6 to 28 , L means L body, D
means D body. ) The invention relates to a muramyl tripeptide fatty acid amide derivative represented by:

The compound () of the present invention has the following general formula () having excellent adjuvant activity and microbial infection preventive/therapeutic effects. (In the formula, n is the same as above.) It can be easily led to the muramyl tripeptide derivative represented by the above formula, and is therefore an extremely useful compound as an intermediate for the production of the compound of the above general formula ().

Conventionally, it has been known that compound () can be synthesized by the method described below (Japanese Patent Application Laid-Open No. 188996
Publication No.).

(In the formula, n is the same as above.) However, the above method is not satisfactory as an industrial production method, although it may be a laboratory production method.

That is, when using this method, it is inevitable that various by-products are mixed into the compound (), and it is not industrially easy to separate and remove these by-products from the compound (). The contamination of impurities into the compound () is not only due to the occurrence of impurities in the reaction process of () + () → (), but also due to the unavoidable contamination of impurities into the raw material () itself, and its isolation and purification. This is due to the fact that it is extremely difficult.

Therefore, in order to produce a highly pure compound of formula (), it is important to first thoroughly purify the raw material (), but such purification is not easy. More specifically, the raw material compound () is purified by column chromatography, which is commonly used, but the raw material compound () itself and various impurities mixed therein are structurally being complex and having many polar groups such as hydroxyl groups, amino groups, carboxyl groups, and amide groups in its own molecules;
Alternatively, it is difficult to separate and purify them because they may be structurally similar to each other.

In addition, if such impurities are to be sufficiently separated and removed, a large amount of column chromatography base material is required, elution takes a long time, and the purification yield is expected to decrease, which is not desirable from an industrial viewpoint. do not have.

Therefore, as a result of intensive study on an industrially advantageous manufacturing method for compound () as an alternative to the conventional method, the present inventors found that the manufacturing method via the compound of the present invention represented by the above general formula () is suitable for manufacturing compound (). The present invention has been completed based on the discovery that the present invention is extremely useful.

That is, the compound of the present invention represented by the general formula () has less polarity than the conventional starting material compound (), and is highly fat-soluble itself, so it can be easily subjected to silica gel column chromatography using a common elution solvent. By purification by recrystallization and/or recrystallization, highly pure products can be easily obtained in high yield. Furthermore, by treating the compound () of the present invention obtained here using commonly known means, a highly purified compound () can be easily obtained. These points are great advantages in terms of both production scale and purity from the viewpoint of industrial production methods.

The compound () of the present invention is advantageously produced, for example, by the method described below.

(In the formula, R ₁ , R ₂ , R ₃ , R ₄ and n are the same as above.) That is, the compound of the present invention represented by the general formula () is the acetylmuramic acid derivative represented by the general formula (). It can be easily produced by a method of condensing with an alkanoyl tripeptide derivative represented by the general formula (). For the condensation reaction, methods generally used for peptide synthesis, such as a carbodiimide method, an active ester method, and an acid anhydride method, can be appropriately employed.

For example, first, compound () is dissolved in a solvent such as chloroform, methylene chloride, tetrahydrofuran, etc. alone or in a mixture, and N-hydroxysuccinimide, 1-hydroxybenzotriazole, N-hydroxy-5-norbornene-2, 3
- a dicarbodiimide or pentachlorophenol and as a coupling agent, for example, N,
Add N′-dicyclohexylcarbodiimide,
The reaction is usually carried out at about 0 to 60°C for several hours to more than ten hours to obtain an active ester form of the compound (). However, especially when substituents R ₃ and R ₄ in compound () are hydrogen atoms, transesterification (e.g., exchange of pentachlorophenol with trichloroacetic ester) without using a coupling reagent may be used. Preferably, an active ester is produced. Next, the compound () of the present invention can be easily produced by condensing the active ester form of the compound () thus obtained with the compound (). The condensation reaction is carried out in a solvent commonly used for peptide synthesis, such as N,N-dimethylformamide, tetrahydrofuran, chloroform, acetonitrile, etc., alone or in combination, at about 0 to 80°C, preferably about 5 to 35°C. This can be achieved by stirring for several tens of minutes to more than ten hours. Also, in this reaction,
If necessary, an organic base such as triethylamine, N-methylmorpholine, etc. may be appropriately added.

On the other hand, the compound of the present invention () can be obtained by reacting an acetylmuramic acid dipeptide derivative with an alkanoyl-L-lysine ester, or an amino acid derivative of acetylmuramic acid with a D-isoglutaminyl-alkanoyl-L-lysine ester, in addition to the above-mentioned method. It can also be easily produced by the method of

The thus obtained compound of the present invention () can be easily converted into compound () by removing substituents R ₁ , R ₂ , R ₃ and R ₄ by a known method as required.
can be obtained.

That is, the substituent R ₁ can be dissolved in a suitable solvent (e.g. acetic acid, methanol or N,N -dimethylformamide) at room temperature and under normal pressure for several hours to several days. Substituents R ₂ and R ₃
When R ₄ is a substituted or unsubstituted benzyl group, it can be eliminated by hydrogenation in the same manner as above. In addition, when the substituents R ₃ and R ₄ are lower alkylidene groups, by hydrolysis in the presence of an acid (for example, acetic acid or hydrochloric acid), or by hydrogenation or acid hydration when they are substituted or unsubstituted benzylidene groups. It can be easily removed either by decomposition.
Therefore, by using these methods alone or in combination, it is possible to easily derive the compound () from the compound () of the present invention, and it is also easy to purify it to obtain a highly pure product. I can do it.

Hereinafter, the present invention will be further explained in detail with reference to Examples and Reference Examples, but these are not intended to limit the present invention.

Example 1 1.35 g of 1-α-O-benzyl-4,6-O-benzylidene-N-acetylmuramic acid was added to 100 ml of tetrahydrofuran with 0.33 g of N-hydroxysuccinimide and 0.60 g of N,N'-dicyclohexylcarbodiimide. Dissolve and stir under ice cooling for 30 minutes and then at room temperature for 5 hours. After filtering off the precipitated crystals, the filtrate is concentrated under reduced pressure. The active ester obtained in this way was treated with N,N-dimethylformamide.
Dissolve in 80 ml and cool on ice, N〓-L-alanyl-D-
Add 1.75 g of isoglutaminyl-N-stearoyl-L-lysine benzyl ester and 0.30 g of N-methylmorpholine. Stir overnight while gradually returning to room temperature. The crystals precipitated in the reaction solution are collected by filtration and washed with methanol. 2.60 g of crude crystals are obtained. This was dissolved in a chloroform-methanol mixture and purified by chromatography by filtration using 10 g of silica gel.
After concentration under reduced pressure, recrystallization from chloroform-methanol-ether gave N〓-(1-α-O-benzyl-
4,6-O-benzylidene-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N〓
-stearoyl-L-lysine benzyl ester
Obtain 1.97g. Melting point 255-259℃ (decomposed) Elemental analysis value C ₆₄ H ₉₄ N ₆ O ₁₃・H ₂ O Calculated value (%) C65.61, H8.25, N7.16 Actual value (%) C65.24, H8 .08, N7.38 Example 2 1.54 g of 1-α-O-benzyl-N-acetylmuramic acid pentachlorophenyl ester and N〓-L
-Alanyl-D-isoglutaminyl-N-stearoyl-L-lysine benzyl ester (140 g) was suspended in N,N-dimethylformamide (1.40 ml),
Stir under ice cooling. Gradually return to room temperature and react overnight, then add 30 ml of ether and collect the crystals by filtration. Coarse crystal 2.07
get g. Dissolved in chloroform-methanol,
Chromatography is performed by filtration using 20 g of silica gel. Next, it was crystallized from chloroform-methanol ether to give N〓-(1-α-O-benzyl-N
1.05 g of -acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-stearoyl-L-lysine benzyl ester are obtained. Melting point 241-244℃
(Disassembly).

Elemental analysis value C ₅₇ H ₉₀ N ₆ O ₁₃・H ₂ O Calculated value (%) C63.08, H8.54, N7.74 Actual value (%) C63.23, H8.29, N7.72 Example 3 1-α-O-benzyl-4,6-O-nzylidene-N-acetylmuramyl-L-alanyl-D-
0.1g of isoglutamine to 0.04g of N-hydroxy-5-norbornene-2,3-dicarboximide
and N,N'-dicyclohexylcarbodiimide
With 0.04g, 20ml of tetrahydrofuran, N,
Stir in a mixture of 20 ml of N-dimethylformamide under ice cooling. After 5 hours, N〓-triacontanoyl-L
- Add 0.1 g of lysine benzyl ester, gradually warm to room temperature, and react overnight. Filter the precipitated crystals and wash thoroughly with methanol. Recrystallized from N,N-dimethylformamide-tetrahydrofuran,
N〓-(1-α-O-benzyl-4,6-O-benzylidene-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N〓-tricontanoyl-L-lysine benzyl ester is obtained. melting point
246-253℃.

Example 4 In Example 1, N〓-L-alanyl-D-
Instead of isoglutaminyl-N〓-stearoyl-L-lysine benzyl ester, N〓-L-alanyl-D-isoglutaminyl-N〓-lauroyl-L-lysine benzyl ester was used and reacted in the same manner to obtain N〓-(1 -α-O-benzyl-4,6-
O-benzylidene-N-acetylmuramyl-L-
Alanyl-D-isoglutaminyl)-N-lauroyl-L-lysine benzyl ester is obtained. melting point
257-259℃.

Elemental analysis value C ₅₈ H ₈₂ N ₆ O ₁₃・1/2H ₂ O Calculated value (%) C64.49, H7.66, N7.96 Actual value (%) C64.34, H7.51, N8.03 Example 5 In Example 1, N〓-L-alanyl-D-
N〓-L-alanyl-D-isoglutaminyl-N〓-octanoyl-L-lysine benzyl ester was used instead of isoglutaminyl-N〓-stearoyl-L-lysine benzyl ester, and reacted in the same manner to obtain N〓-(1- α-O-benzyl-4,6-
O-benzylidene-N-acetylmuramyl-L-
Alanyl-D-isoglutaminyl)-N-octanoyl-L-lysine benzyl ester is obtained. Melting point 256-260℃.

Elemental analysis value C ₅₄ H ₇₄ N ₆ O Calculated value as ₁₃・1/2H ₂ O (%) C63.33, H7.29, N8.21 Actual value (%) C63.27, H7.38, N8.16 Example 6 In Example 2, N-L-alanyl-D-isoglutaminyl-N-octanoyl-L-lysine was used instead of N-L-alanyl-D-isoglutaminyl-N-stearoyl-L-lysine benzyl ester. A similar reaction is performed using benzyl ester to obtain N-(1-α-O-benzyl-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-octanoyl-L-lysine benzyl ester. Melting point 239-241℃.

Elemental analysis value C ₄₇ H ₇₀ N ₆ O ₁₃・1/2H ₂ O Calculated value (%) C60.30, H7.64, N8.98 Actual value (%) C60.32, H7.61, N9.13 Reference Example 1 N〓-(1-α-O-
Benzyl-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N〓-stearoyl-
0.7 g of L-lysine benzyl ester, 20 ml of acetic acid
Hydrolysis is carried out in a hydrogen stream for 24 hours in the presence of 0.7 g of 5% palladium on carbon. After the reaction, the catalyst is removed by filtration, the filtrate is concentrated under reduced pressure, ether is added, and the precipitated crystals are collected by filtration.

Recrystallization from DMF-ether gives N-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-stearoyl-L-lysine. This product had IR, melting point, and optical rotation that matched the standard, and was a single spot on TLC at a loading of 50-100 gamma. (Silica gel plate, developing solvent;
Butanol:acetic acid:water 4:1:5 upper layer, color development; iodine 30 minutes).

Reference Example 2 N〓-(1-α-O-
Suspend 1.3 g of benzyl-4,6-O-benzylidene-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-stearoyl-L-lysine benzyl ester in 18 ml of acetic acid and 10 ml of water,
Heat and stir at ℃ for 2 hours. Add 4 ml of ethylene dichloride to dissolve the insoluble crystals, then heat and stir for an additional 30 minutes. Concentrate under reduced pressure, add ether, and collect the precipitated crystals by filtration. Recrystallized from methanol-chloroform to give N〓-(1-α-O-benzyl-
0.8 g of N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-stearoyl-L-lysine benzyl ester is obtained. This product is treated in the same manner as in Reference Example 1 to obtain N-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-stearoyl-L-lysine. This product was identified in the same manner as in Reference Example 1, and its
TLC was single spot under the same conditions.

Reference Example 3 N〓-(1-α-O-
0.5 g of benzyl-4,6-O-benzylidene-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-stearoyl-L-lysine benzyl ester in 20 ml of acetic acid in the presence of 0.5 g of 5% palladium on carbon. , Hydrolysis is carried out for 36 hours in a hydrogen stream. After the reaction, the catalyst is removed by filtration, the filtrate is concentrated under reduced pressure, and ether is added to crystallize to obtain 0.4 g of crude crystals. Recrystallized from DMF-ether and N〓-(N-
Acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-stearoyl-L-lysine is obtained.

This product was identified as in Reference Example 1, and its TLC was a single spot under the same conditions.

Reference example 4 (1) N ⁶ -stearoyl-L-lysine benzyl ester 88.6 g of N ⁶ -stearoyl-L-lysine, para-toluenesulfonic acid monohydrate in 600 ml of benzene
Add 53.1g and 111.6g of benzyl alcohol,
The mixture was heated to reflux for about 22 hours with azeotropic dehydration using a Dean-Starck apparatus. After the reaction, the solvent was distilled off under reduced pressure and the residue was diluted with isopropyl ether.
2.5 was added, the precipitated crystals were collected by filtration, and N ⁶ −
A para-toluenesulfonate salt of stearoyl-L-lysine benzyl ester was obtained. This was suspended in chloroform 1, 600 ml of 5% sodium bicarbonate water was added and stirred, and then the chloroform layer was separated. Next, add a chloroform layer to 5%
After washing with aqueous sodium hydrogen carbonate and saturated brine, it was dried over anhydrous sodium sulfate. The solvent was distilled off to obtain 72.5 g of crystals of the title compound.

Melting point: 78.0-80.0°C NMR (90MHz, CD ₃ OD) δ; 0.88 (3H, t) 5.16 (2H, s) 7.37 (5H, s) (2) N ² -(tertiary butoxycarbonyl-L-alanyl -D-isoglutaminyl) -N ⁶ -stearoyl-L-lysine benzyl ester tertiary butoxycarbonyl-L-alanyl-
62.3 g of D-isoglutamine was dissolved in 400 ml of tetrahydrofuran, and 42.5 g of dicyclohexylcarbodiimide and 29.2 g of 1-hydroxybenzotriazole were added all at once while stirring under ice cooling. After stirring for 30 minutes, the mixture was gradually warmed to room temperature and stirred for 5 hours. Next, a solution of 103.6 g of N ⁶ -stearoyl-L-lysine benzyl ester dissolved in 1.6 g of tetrahydrofuran was added to the above reaction system. after that,
After standing overnight, the precipitated crystals were collected by filtration. The obtained crystals were slurried washed with methanol 2 and dried to obtain 145.9 g of white crystals of the title compound.

Melting point: 178-180℃ NMR (500MHz, CDCl ₃ + CD ₃ OD) δ; 0.89 (3H, t, J = 6.8Hz) 1.34 (3H, d, J = 7.1Hz) 1.45 (9H, s) 5.16.5.20 ( 2H, ABq, J=11.9Hz) 7.32-7.39 (5H, m) (3) N ² -L-alanyl-D-isoglutaminyl-N ⁶ -stearoyl-L-lysine benzyl ester N ² -(tertiary butoxycarbonyl 20 ml of trifluoroacetic acid was added to 2.0 g of -L-alanyl-D-isoglutaminyl)-N ⁶ -stearoyl-L-lysine benzyl ester and stirred for 30 minutes under ice cooling, and then stirred for 1 hour at room temperature. After the reaction was completed, the mixture was concentrated under reduced pressure and ice water was added. The mixture was then made alkaline with aqueous ammonia, and the precipitated crystals were collected by filtration. Then, the slurry is washed with water and the melting point is 190-193.
Obtained 1.6 g of colorless powder at ℃.

Claims (1)

[Claims] 1. General formula (In the formula, R ₁ and R ₂ each represent a benzyl group, R ₃ and R ₄ each represent a hydrogen atom or both together represent a benzylidene group, Ac represents an acetyl group, and n is 6 to 28 , L means L field, D
means D body. ) Muramyl tripeptide derivatives shown by