JPS58172399A - Muramyl tripeptide derivative - Google Patents

Abstract

NEW MATERIAL:The muramyltripeptide derivative of formula I [R1 is lower alkyl or (substituted)benzyl; R2 is (substituted)benzyl; R3 and R4 are H or (substituted)benzyl, or together form lower alkylidene or (substituted)benzylidene; n is 6-28]. EXAMPLE:N<alpha>-( 1-alpha-O- Benzyl-4,6-O- benzylidene-N- acetylmuramyl-L- alanyl-D- isoglutaminyl)-N<epsilon>-stearoyl-L-lysine benzyl ester. USE:An intermediate for the preparation of a high-purity compound of formula IIhaving adjuvant activity and activities to prevent and remedy the microbism. PROCESS:For example, the acetylmuramic acid derivative of formula III is converted to active ester, and condensed with the alkanoyltripeptide derivative of formula IV to obtain the objective compound of formula I .

Description

Detailed Description of the Invention More specifically, general formula (I) (wherein, R represents a lower alkyl group or a substituted or unsubstituted benzyl group, R represents a substituted or unsubstituted benzyl group, and R4 represents a substituted or unsubstituted benzyl group. Each of the same or different hydrogen atoms, a substituted or unsubstituted benzyl group, or both mean a lower alkylidene group or a substituted or unsubstituted benzylidene group combined to form a ring, and ru means an integer from 6 to 28.)
The present invention relates to a muramyl tripeptide fatty acid amide derivative represented by:

The compound (1) of the present invention can be easily converted into a muramyl-ripeptide derivative represented by the following general formula (II) (wherein ? & is the same as above), which has excellent adjuvant activity and microbial infection preventive/therapeutic effects. Therefore, it is an extremely useful compound as an intermediate for the production of the compound of the above general formula (II).

Conventionally, it has been known that compound Ql) can be synthesized by the method described below (Japanese Unexamined Patent Publication No. 188996/1983).

(In the formula, L is the same as above.) However, the above method is a laboratory production method, that is, when this method is used, compound (1) is unavoidably contaminated with various by-products, and these Separation and removal of by-products from compound (1) is not easy industrially. In addition, the compound (n
) The mixing of impurities into (1) + (IV) → (It
) In addition to the generation of impurities in the reaction process, raw materials @
) itself is unavoidable from being contaminated with impurities, and its isolation and purification is extremely difficult.

Therefore, in order to produce a highly pure ambassador compound, it is important to first sufficiently purify the raw material (Iff), but such purification is not easy. For more details.

The raw material compound (2) is purified by commonly used column chromatography, but the raw material compound (6) itself and various impurities mixed therein are structurally complex. and having many polar groups such as hydroxyl groups, amino groups, carboxyl groups, and amide groups in its own molecule.

Or because they are likely to be structurally similar to each other.

Separation and purification of these substances is not easy.

In addition, in order to sufficiently separate and remove such impurities,
This method is not preferable from an industrial point of view because it requires a large amount of column chromatography base, requires a long time for elution, and is expected to reduce the purification yield.

Therefore, the present inventors developed a compound (It
) As a result of intensive studies on industrially advantageous manufacturing methods, it was discovered that the manufacturing method via the compound of the present invention represented by the above general formula (1) is extremely useful for manufacturing compound (1)9. completed.

That is, the compound of the present invention represented by general formula (I).

Since it is less polar than the conventional raw material compound (1) and is highly fat-soluble, it can easily be purified to high purity by simple silica gel column chromatography using a normal elution solvent and/or purification by recrystallization. can be obtained in high yield. Furthermore, the compound of the present invention (I
) can be easily obtained by treating the compound (It) with conventionally known methods. These points are great advantages in terms of both production scale and purity from the viewpoint of industrial production methods.

Compound (1) of the invention is advantageously prepared by the method described below, for example.

(In the formula, + R1! R2+ R3+ R4 and R are the same as above.) That is, the compound of the present invention represented by the general formula (I).

It is easily produced by a method of condensing an acetylmuramic acid derivative represented by general formula (A) and an alkanoyl tripeptide derivative represented by general formula (4). : I can do it. 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 (7) is dissolved in a solvent such as chloroform, methylene chloride, tetrahydrofuran, etc. alone or in a mixture, and N-hydroxysuccinimide, l-hydroxybenzotriazole, N-hydroxy-5-norbornene-2 , 3-dicarbodiimide or pentachlorophenol and as coupling reagents.

Add N,N'-dicyclohexylcarbodiimide.

Usually react at about 0 to 60°C for several hours to more than ten hours,
It is an active ester compound disclosed in the patent publication. However, compound (2)
In particular, when substituents R1 and R4 are hydrogen atoms,
Preferably, the active ester thereof is prepared by transesterification (for example, exchange of pentachlorophenol with trichloroacetic ester) without the use of a force or a Bulling reagent.

Next, the compound (1) of the present invention can be easily produced by condensing the active ester form of the compound (1) thus obtained with the compound (6). The condensation reaction is carried out in a solvent 9 commonly used for peptide synthesis, such as N,N-dimethylformamide, tetrahydrofuran, chloroform, acetonitrile, etc., alone or in combination, at about θ to about 80°C, preferably about 5 to 85°C. This is achieved by stirring for several tens of minutes to more than ten hours. In addition, in this reaction, 9, if necessary, an organic base 1, for example.

Triethylamine, N-methylmorpholine, etc. may be added as appropriate.

On the other hand, 9 compounds of the present invention (1) were obtained by methods other than those described above.

Acetylmuramic acid dipeptide derivatives and alkanoyl-
L-lysine ester or amino acid derivative of acetylmuramic acid and D-isoglutaminyl-alkamyl-L-
It can also be easily produced by a method of reacting with lysine ester.

Compound (n) can be easily obtained from the thus obtained compound (1) of the present invention by removing the substituents R, , R, , R3 and R4 by a known method, if necessary.

That is, the substituent R3 can be dissolved in a suitable solvent (e.g. acetic acid, methanol or N,N- dimethylformamide).

It can also be easily desorbed by hydrogenation at room temperature and under normal pressure for several hours to several days. When the substituents R2, R3 and R4 are substituted or unsubstituted benzyl groups, they can be eliminated by hydrogenation in the same manner as above. In addition, when the substituents R1 and R4 are lower alkylidene groups, by hydrolysis in the presence of an acid (for example, acetic acid or jJi#), or by hydrogenation or acid hydrolysis 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, compound Q[) can be easily derived from the compound (I) of the present invention, and its purification is also easy and a highly pure product can be obtained. 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-α-0-benzyl-4,6-0-benzinodene-N-acetylmuramic acid 1,359 was converted to N-hydroxysuccinimide 0.389. The mixture was dissolved in 100% of tetrahydro7ran along with 0.60% of N,N'-dicyclohexylcarbodiimide, and stirred for 80 minutes under water cooling and then for 6 hours at room temperature. After filtering out the precipitated crystals, the filtrate is concentrated under reduced pressure. The active ester obtained in this way is N,N
-Dissolved in dimethylformamide 80- and cooled with water.

N″-L-alanyl-D-isoglutaminyl-1-stearoyl-L-lysine benzyl ester 1.759 and N
- Add 0.809 methylmorpholine. Stir overnight while gradually returning to room temperature.

The crystals precipitated in the reaction solution are collected by filtration and washed with methanol. 2.609 crude crystals are obtained. This was dissolved in a chloroform-methanol mixture and purified by chromatography by filtration using silica gel 109.

Decompression 11 Compression 1. Recrystallized from chloroform-methanol-ether to give N-(1-α-0-penzyl-4,6-0
-Benzylidene-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-stearoyl-L-lysine benzyl ester 1.979 is obtained. Melting point 255
~259°C (decomposed).

Elemental analysis value Calculated value as CB4H, N, 0.3 H, O (G 65.51. H8, 25, N 7.1
6 Actual measurement value (%) C65, 24, H8, 0B, Nfu,
88 Example 2 1-α-〇-benzyl-N-acetylmuramic acid pentachlorophenyl ester 1.549 and N"-L-alanyl-D-isoglutaminyl-N6-styaroyl-L-
140 g of lysine benzyl ester is suspended in 1.40@t of N,N-dimethylformamide and stirred under water cooling.

Gradually return to room temperature and react overnight, then add ether 80 and collect crystals by filtration. Crude crystals 2.079 are obtained. Dissolve in chloroform-methanol and perform chromatography by filtration using silica gel 209. Next.

Chloroform-methanol ether crystallizes, N'
-(1-α-〇-benzyl-N-acetylmuramyl-L
-alanyl-D-isoglutaminyl)-N-stearoyl-L-lysine benzyl ester 1. Q Get 59. Melting point 241-244°C (decomposed).

Elemental analysis value Cl17HQON6013 Calculated value (equity) assuming 20. H8,54,N 7.
74 actual measurement value (4) C63, 28, H8, 29, N 7
．． 72 Example 8 1-α-0-benzyl-4,6-0-benzylidene-N
-Acetylmuramyl-L-alanyl-D-isoglutamine 0.1g N-hydroxy-5-norbornene-43
-Dicarboximide 0.049 and N,N'-dicyclohexylcarbodiimide (LO49) are stirred in a mixture of 20 d of tetrahydro7ran and 20 d of N,N-dimethylformamide under ice cooling. 5 hours later N+!
Add 0.1 g of acontanoyl L-lysine benzyl ester, gradually warm to room temperature, and react overnight. Filter the precipitated crystals and wash thoroughly with methanol. N, N-
Recrystallized from dimethylformamide-tetrahydrofuran to give N″-(1-α-0-benzyl-4,6-0-benzylidene-H-acetylmuramyl-L-alanyl-D-
Isoglutaminyl)-N-)liacontanoyl-L-lysine benzyl ester is obtained. Melting point 246-258°C.

Example 4 In Example 1, instead of N''-L-alanyl-D-isoglutaminyl-N-stearoyl-L-lysine benzyl ester, N-L-alanyl-D-isoglutaminyl-N-lauroyl- Using L-lysine benzyl ester.

Similarly, N“-(i-α-0-benzyl-4,6
-0-benzylidene-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N6-lauroyl-L
-Lysine benzyl ester is obtained. Melting point 257-25
9℃.

Elemental analysis value Cl1lH82N801114H20 Calculated value (support) 1 G 64.49. H7,66
, N 7.96 actual value (a) Cfs4J4. H7
,51,N 8.08 Example 5 In Example 1, N"-L-alanyl-D-isoglutaminyl ester was substituted for N"-L-alanyl-D-isoglutaminiroot-stearoyl-L-lysine benzyl ester. Mini-N-octanoyl-L-lysine benzyl ester.

Similarly, N″-(1-α-0-benzyl-+, e
-o-benzylidene-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N'-octanoyl-
L-lysine benzyl ester is obtained. Melting point 256~2
60℃.

Elemental analysis value C54H? Calculated value (%) as 4Nl101m+H,O C63,1,H7,29,N 8.
21 actual measurement value (a) C63, 27, H7, 38, N 8
．． 16 Example 6 In Example 2, N-L-alanyl-D-isoglutamin-N-octanoyl-L was substituted for N-L-alanyl-D-isoglutaminyl-N-stearoyl-L-lysine benzyl ester. -Lysine benzyl ester is used and reacted in the same manner to obtain N''=(l-α-0-benzyl-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-octanoyl-L-lysine benzyl ester. Melting point: 239-241°C.

Elemental analysis value C4tHtoNaO+* ' Calculated value as +HtO ((a) C60,80,H7,64,N
8.98 Actual value (1) c 60.32. H7,6
1, N 9. ls Reference Example 1 N"-(1-α-〇-benzyl-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N'!-stearoyl-L-lysine benzyl ester obtained in Example 2 0.79 of.

Hydrolysis is carried out for 24 hours in a stream of hydrogen in the presence of 0.7 g of 5% para-1-dim carbon in 20% acetic acid. After repulsion, the catalyst is removed by filtration, the filtrate is concentrated under reduced pressure, ether is added, and the precipitated crystals are collected by filtration.

Recrystallized from DMF-ether to give N''-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-1
-stearoyl-L-lysine is obtained. This product is an IR
, melting point, and optical rotation are consistent with the standard product, and TLC shows that it is 50γ~1
It was a single spot with a load of 00γ. (Silica gel plate, developing solvent; butanol:acetic acid:water 4:1=
5 Upper layer, coloration; iodine 30 minutes).

Reference Example 2 N-(1-α-〇-benzyl-4.6-0-penzylideneto-4cetylmuramyl-L obtained in Example 1)
-Alanyl-D-isoglutaminyl)-N-stearoyl-1,-IJ ginbenzyl ester (1.3 g) was added to 1.3 g of acetic acid.
8ml, suspended in 10d of water, heated and stirred at 90°C for 2 hours. Ethylene dichloride 4- is added and after dissolving the insoluble crystals -1, the mixture is further heated and stirred for 30 minutes. Concentrate under reduced pressure, add ether, and collect the precipitated crystals by filtration.

Recrystallized from methanol-chloroform to obtain N“-(1-
α-0-benzyl-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-stearoyl-L-
Lysine benzyl ester 0.89 is obtained. By treating this product in the same manner as in Reference Example 1, 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 A-conditions.

Reference Example 8 N''-(1-α-0-benzyl-4,6-0-benzylidene-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-stearoyl-L- obtained in Example 1 Lysine benzyl ester 0.59 was hydrogenolyzed in 20% acetic acid in the presence of 0.5% palladium on carbon for 86 hours in a hydrogen stream. After the reaction, the catalyst was filtered off and the filtrate was concentrated under reduced pressure. , add ether and crystallize to obtain 0.49 crude crystals.Recrystallize from DMF-ether to obtain N
"-(N-acetylmuramyl-shi-alanyl-D-isoglutaminyl)-N-stearyl-L-lysine is obtained.

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

Claims (1)

Claims: General formula (wherein R3 is a lower alkyl group or a substituted or unsubstituted benzyl group, R2 is a substituted or unsubstituted benzyl group, R3 and R4 are the same or different hydrogen atoms, substituted or means an unsubstituted benzyl group, a lower alkylidene group in which both are bonded to form a ring, or a substituted or unsubstituted benzylidene group, and ru means an integer from 6 to 28.)
A muramyl tripeptide derivative represented by