JPS59172497A - Novel muramyl peptide derivative - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (Ala is alanine; R is H, alkyl, or aralkyl; n is an integer 1-6; A1 and A2 are H or 2-30C fatty acid residue). EXAMPLE:N<epsilon>-(6-O-Stearoyl-N-acetylmuramyl-alanyl-D-isoglutaminy l)-lysine. USE:A preventing and treating agent for microbial infection and an antitumor agent. PREPARATION:The protecting group (Z) in a compound expressed by formula II(Z is a protecting group of the amino group) is eliminated, and if necessary the resultant compound is then condensed with an active substance of a compound expressed by the formula A3-OH (A3 has the same meaning as A1 and A2).

Description

[Detailed description of the invention] The present invention has effects on preventing and treating microbial infections.

The present invention relates to a novel muramyl tipebutide derivative which is also recognized to have an excellent anticancer effect, and more specifically to a peptide derivative of muramyl represented by the general formula (1).

(In the above formula, Ala means alanine, R is a hydrogen atom,
an alkyl group or an aralkyl group, n is an integer of 1 to 6 +
AI and A2 each have a hydrogen atom or a carbon atom number of 2 to 80
refers to a saturated or unsaturated fatty acid residue which may have branching. ) N-acetylmuramyl-L-alanyl-D-isoglutamine is known as an immune adjuvant substance, but from a therapeutic and practical standpoint, it is not necessarily satisfactory in terms of microbial infection prevention activity, antitumor activity, etc. Not a kimono.

Therefore, the present inventors have determined that the activity of preventing microbial infection.

As a result of intensive studies on compounds having antitumor activity, the present invention was completed by discovering that the compound represented by general formula (1) has excellent microbial infection prevention activity and antitumor activity.

The compounds obtained according to the present invention can be used for many diseases, for example, as agents for preventing and treating microbial infections, anticancer agents, and the like.

The effects of the compounds of the present invention are as follows.

(1) Microbial infection prevention effect The infection prevention effect of the compound of the present invention was determined by the following method.

The compound of the present invention was 500 μg, p, a, 5 (pH 7
, 4) Prepared to become a solution. 0.0 of the prepared drug solution.
2- was subcutaneously administered to 5TD-ddY mice (5 weeks old),
Then, 24 hours later, E. coli E77156 was detected in Table 1.
Subcutaneous infection was carried out using the inoculum amount shown in . Efficacy was determined from the mouse survival rate (%) 7 days after infection. As shown in Table 2, the compound of the present invention exhibited excellent infection prevention effects.

Table I E, Protective effect on coli infected mice (2
) Antitumor activity BALB/C male mouse fibrosarcoma cells induced with tilcholanthrene (Meth A) 2 X 10
5 pieces and the sample were suspended in PBS pH 7.4, and the same type of B
It was administered intradermally to ALB/G mice, and the inhibitory effect on fibrosarcoma growth was examined 4 weeks later. (Table 2) Number of mice with completely suppressed tumor growth/Number of mice used Percentage when the tumor weight of the untreated group is taken as 100 Compound of the present invention Compound-1 of the present invention: N'-(6-0-stearoyl- N-acetylmuramyl-alanyl-D-isoglutaminyl)-lysine Compound of the present invention-2 = l-acetyl-N6-(6-0-stearoyl-N-acetylmuramyl-alanyl-D-isoglutaminyl)-
Lysine compound of the present invention -8-stearoyl-N'-(N-acetylmuramyl-alanyl-D-isoglutaminyl)-lysine methyl ester Compound of the present invention-4 = 1-acetyl-N'-(6-0-stearoyl-N- acetylmuramyl-alanyl-D-isoglutaminyl)-
Lysine methyl ester The compound (1) of the present invention is, for example, 2
Formula () (In the formula, R and n are as defined above, and 2. z is a protecting group for an amine group that is commonly used in peptide synthesis. It may have a substituent such as a halogen atom or a nitro group. By removing the protecting group (Z) of the compound represented by the formula A3-0H(l[) (in the formula r A3
means a saturated or unsaturated fatty acid residue which may have branches of 2 to 30 carbon atoms. ) can be obtained by condensing an active form 9 of the compound represented by, for example, an active ester form, an acid anhydride, etc. It can also be obtained by condensing an active form 1 of the compound represented by formula (1), such as an acid chloride form or an active ester form, with formula (It) and then removing the protective group.

When condensing the active ester of formula (II) with formula (II).

It is preferable to carry out the condensation reaction in the presence of an organic base 2 such as triethylamine, N-methylmorpholine, N-ethylmorpholine, N,N-dimethylaminopyridine, etc. and 1-hydroxybenzotriazole. Furthermore, the formula () obtained in this way (in the formula + A3+ R and n are as defined above) and the formula (
The activated form 9 of II) can also be obtained by condensing active esters, acid anhydrides, etc.

Raw material compound used for producing compound (I) of the present invention.

That is, the compound represented by formula (n) can be produced according to the following method.

That is, it can be achieved by condensing N-acetylmuramic acid with the formula (wherein R, Z and n are as defined above). This condensation reaction is generally carried out by the carbodiimide method, the Aintop method, which is often used in peptide synthesis.
An active ester method, an arsenic anhydride method, etc. can be adopted.

To prepare a compound represented by formula (V), the formula (wherein Y
is an amino group protecting group 9 that is often used in ordinary peptide synthesis.
Examples include t-butoxycarbonyl group and P-methoxybenzyloxycarbonyl group. ) is condensed with the formula (wherein R and 2 are as defined above).

The compound thus obtained can then be obtained by removing the protecting group Y using appropriate conditions for removing the protecting group.

For the condensation reaction of formulas 0) and (b)), the above-mentioned condensation method that is commonly used in peptide synthesis can be adopted, and for the elimination of Y, a method of trifluoroacetic acid or hydrochloric acid/tetrahydro 7 run treatment can be adopted. .

In addition, during the condensation reaction of formula (V) with N-acetylmuramic acid, the hydroxyl groups at the 4th and 6th positions of the sugar of N-acetylmuramic acid are replaced with allylidene groups 9 such as benzylidene, P-methoxybenzylidene, etc., and the hydroxyl group at the 1st position of the sugar It is preferable to use an aralkyl group 2 protected by a benzyl group or the like, which may be substituted with one or more of a halogen atom, a diF 2 group, or a methoxy group, since side reactions in the condensation reaction can be reduced.

The following description will be given with reference to examples and reference examples.

Example 1 90% of tetrahydrofuran is added to 8.959% of 11penzyloxybenzylbenzyl ester p-)luenesulfonate. While cooling with water and stirring, add 1.65 i of N-methylmorpholine, 4.76 g of t-butyloxycarbonyl-alanyl-D-isoglutamine, 1.9 (l) of N-hydroxysuccinimide and 8.4 (l) of dicyclohexylcarbodiimide. , 30 minutes later, return to room temperature (return to room temperature). Because a gel-like substance precipitates in the reaction solution,
i,N,N-dimethylformamide 20- is further added and the reaction is stirred overnight. The precipitated dicyclohexylurea is filtered off, and the filtrate is concentrated under reduced pressure. Add 100frLt of water to the residue and collect the precipitated crystals by filtration.2 Then, add 5% citric acid solution,
After washing sequentially with water, 5% sodium bicarbonate solution, and water.

By recrystallizing from N,N-dimethylformamide-ether solution,
6-(t-Butyloxycarbonyl-alanyl-D-isoglutaminel)-1-nodine benzyl ester 7.1(
get l. Melting point 175-177°C (decomposed). [α 5-
14.5° (C-1,6°N, N-dimethylformamide).

Elemental analysis value Cs a H47N509 calculated value (2))
060.97. H7,07,N 10.46 Analysis value (%) C61,12,H6,98,N 10.
52 The above compound 5.609 is suspended in 30 m of dichloromethane, and 80 m of trifluoroacetic acid is added while stirring while cooling with water. 5
After a few minutes, the mixture was returned to room temperature, reacted for 1 hour, and then concentrated to dryness under reduced pressure. The residue was dissolved in 45 d of N,N-dimethylformamide and neutralized by adding N-methylmorpholine while stirring while cooling with water. 1-α-0-benzyl-4 obtained as shown below is added to this solution.
, 6-0-benzylidene-N-acetylmuramic acid N
- Add 35- of a solution of hydroxysuccinimide ester in N,N-dimethylformamide.

(3.58 parts of 1-α-0-benzyl-'1,6-0-benzylidene-N-acetylmuramic acid and 0.96 parts of N-hydroxysuccinimide were dissolved in tetrahydrofuran, and dicyclohexylcarbodiimide was dissolved under water-cooling and stirring. 1.72
Add 9. After 300 minutes, the mixture was returned to room temperature, and after stirring for 5 hours, the precipitated dicyclohexyl urea was filtered off. The filtrate was concentrated under reduced pressure and the resulting residue was converted to 1-α-0-benzyl-4,6-0
-Used as N-hydroxysuccinimide ester of benzylidene-N-acetylmuramic acid) After 80 minutes, the mixture was returned to room temperature and reacted overnight. The gel precipitated in the reaction solution was collected by filtration and washed with ether and 95% ethanol to obtain 7.3'l of l-benzyloxycarbonyl-r-(x-α-〇-benzyl-4.6 -0-benzylidene-N-acetylmuramyl-
Alanyl-D-isoglutaminyl)-lysine benzyl ester is obtained. Melting point 236-239°C (decomposed).

Add 80% of 60% acetic acid solution to 7.8'l of the above compound and add 1
Heat on a boiling water bath for an hour. After cooling.

150 ml of water is added to the residue obtained by concentration under reduced pressure, and the precipitated crystals are collected by filtration. It was then recrystallized from N,N-dimethylformamide-ethyl acetate to yield 5.619 as white crystals.
11penzyloxycarbonylN'-(1-α-0
-benzyl-N-acetylmuramyl-alanyl-D-isoglutaminyl)-lysine benzyl ester is obtained. Melting point 222-225°C (decomposition). [α), +55.9°CG-0,9,N,N-dimethylformamide).

Elemental analysis value Calculated value (%) as C47H62Ns Ot 4 C60,87, H6,68, N 8.99 Analysis value (%> 060.19. H6,66, N 9
．． 28 0.509 of the above compound was dissolved in 2 otnl of acetic acid.

The reduction reaction is carried out in a hydrogen stream in the presence of palladium black. The catalyst was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was subjected to Sephadex LH-20 gel filtration chromatography. H6-(N-acetylmuramyl-alanyl-
0.219 of D-isoglutaminyl)-lysine is obtained.

Example 2 By reacting in substantially the same manner as in Example 1, 11penzyloxycarbonyluridine methyl ester hydrochloride 11.
629 and t-butyloxycarbonyl-alanyl-D-
From isoglutamine 9.839, 11penzyloxycarbonyl-NE-(t-butyloxycarbonyl-D-isoglutaminyl)-lysine methyl ester 10.919 is obtained as white crystals. Melting point 124-126°C (decomposed). [α]Ll-13.6° (C-0,6°N, N-dimethylformamide).

Elemental analysis value C2a H4s Ns Calculated value (%) as 09 C56,65,H7,30,N 11.80
Analysis value (%) C56,61, H7,16, N 11
．． 62 From the above compound 10.099 and 7.29 g of 1-α-0-benzyl-4,6-0-benzylidene-N-acetylmuramic acid, 11penzyloxycarbonyl-N5-(1-α-0 -benzyl-4,6-0
-benzylidene-N-a7tylmuramyl-alanyl-D
-isoglutaminyl)-lysine methyl ester 18.1
Get 02. Melting point 235-237°C (decomposition). [α)
, +59.9° (0-0,4,N,N-dimethylformamide).

Elemental analysis value C4aH62NaO1r Calculated value (support) as H20) C59,74,H6,68,N 8.
71 Analysis value (%) G 59.44. H6,39
,N 8.74 From the above compound 18.102, 11penzyloxycarbonylN6-(1-α-
0-Benzyl-N-acetylmuramyl-alanyl-D-
isoglutaminyl)-lysine methyl ester 6.779
get. Melting point 198-199°C (decomposed). (α)D
+64.6° (c-0,5,N,N-dimethylformamide).

Direct elemental analysis Calculated value as C4t Hs Na Ot 4 (support)) G 57. Ll, H6,81,
N 9.78 analysis value same) C57,18, H6,98
, N 9.65 From 1.50 g of the above compound, N5-(N-acetylmuramyl-alanyl-D-isoglutaminyl)-lysine methyl ester o,69 is obtained as a white powder. Melting point 88-90'C (decomposed).

[α), +22.9° (C-1,1, water, 2 days later).

Elemental analysis value C25H4sNaOtr GHa';OO
H・4H20 and shite calculation value (%) C47,79,H
7,30,N 11.94 analysis value) C47,62
, H7,21,N 11.73 Example 8 N'-(N-acetylmuramyl-alanyl-D-isoglutaminyl)-lysine methyl ester 0.649 with N,
Dissolve in 1511 L/N-dimethylformamide. Under water-cooled stirring, N-hydroxy-5-norbornene-2,3-dicarboximide active ester of stearic acid 0
Add 0.11 g of 49 g of N-methylmorpholine. 30 minutes later.

Gradually return to room temperature and stir overnight for reaction. The reaction solution was concentrated under reduced pressure, ether was added to the residue, and the precipitated crystals were collected by filtration. This was purified by subjecting it to silica gel chromatography, eluting with chloroform-methanol (9:1), and then recrystallizing from methanol-ether.
Stearoyl N'-(N-acetylmuramyl-alanyl-D-isoglutaminyl)-lysine methyl ester 0
．． Get 409. Melting point 187-5 188.5°C (decomposed). [α]. +18.8° (C knee 1.2°N, N-dimethylformamide-820-5
0/2.

2 days later).

Elemental analysis value Calculated value (%) as G4aHsoNs013/2H20 C56,19,H9,03,N 8.
97 analysis value (%) C56,35,H8,74,N
9.01 Example 4 f-benzyloxycarbonyl-N6-(1-α-0-
Benzyl-N-acetylmuramyl-alanyl-D-ingluminyl)-lysine benzyl ester 5.007 to pyridine 400 ml, N,N-dimethylformamide 5
Add 0 tnl and cool to -15 to -10°C. Stirred stearoyl chloride 6.489 chloroform solution 1
Add 0- gradually. After 10 minutes, a chloroform solution of 6.48 parts of stearoyl chloride (10 parts) is added.

After 10 minutes, 100 methanol was added and the reaction solution was concentrated under reduced pressure. Add 500ml of water to the residue and collect the precipitate by filtration. After drying.

After dissolving in 200% of chloroform and adding diazomethane-ether solution, the mixture was stirred and reacted for 30 minutes.

Add acetic acid until the yellow color of the reaction solution disappears. After concentrating the reaction solution under reduced pressure, the resulting residue was purified by silica gel chromatography and eluted with chloroform-metal (9:1), and then recrystallized from chloroform-ether to give 11-penzyloxy. Power Ruboni Lou NE
-(1-α-0-benzyl-6-0-stearoyl-N
-acetylmuramyl-alanyl-D-isoglutaminyl)-lysine benzyl ester 2.819 is obtained. Melting point 2
00-202°C (decomposition).

[α), +35.2° (C-0,6, chloroform) 0
Elemental analysis value: Calculated value (%) as c6sH9aNs01s/4H20 C64,49,H8,07,N 6.
94 Analysis value (%) G 64.26. H8,08
, N 7.20 2.50 liters of the above compound was dissolved in acetic acid 150 fn
Dissolved in t.

The reduction reaction is carried out in a hydrogen stream in the presence of palladium black.

The catalyst was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was subjected to Sephadex LH-20 gel filtration chromatography. 0.
N6- (6-0-stearoyl-
1.61 g of N-acetylmuramyl-alanyl-D-isoglutaminyl)-lysine are obtained. Melting point 185-187°
C (decomposition). [α], →-21.8° (C-0.5° water, 3 days later) Elemental analysis value C45HtsNeO1s・CH#OOH・
H2O and shite calculation value (%) G 56.00. H
8,77, N 8.71 Analysis value (%) C55,99
,H8,76,N 8.76Example 5 By reacting in substantially the same manner as in Example 4, 11penzyloxycarbonyl-N" (1-α-〇-benzyl-N
-acetylmuramyl-alanyl-D-inglutaminyl)-lysine methyl ester 1.509(l-penzyloxycarbonyl)'-NE-(1-(1-0-he>cyl-6-0-stearoyl-N -acetylmuramyl-alanyl-D-inglutaminyl)-lysine methyl ester is obtained, melting point 204-205°C (min #).

(α) +44.41' (G -0.9, ri 1:1
1: Iform).

Elemental analysis value C5cnH92N60ts4) Calculated value (%) as L+O C62,46,H8,26,N 7
．． 40 analysis value C%) c 62.41. H8,2
2, N 7. +4 The above compound 1.189 was subjected to catalytic reduction reaction.

N'-(6-0-stearoyl-N-acetylmuramyl-alanyl-D-isoglutaminyl)- as a white powder
0.45 g of lysine methyl ester is obtained. Melting point 174~
176.5°C (decomposition).

[α) p+zo, o' (co, e, water, 2 days later) 0
Elemental analysis value C44Hs o Ns O+ s ・GH
, +C0OH-H2O calculated value (%) C56,
42, H8, 85, N 8.58 analysis value (support)) c
56.21. H8,74,N 8.45 Example 6 N'-(6-o-stearoyl-N-acetylmuramyl-alanyl-D-isoglutaminyl)-lysine 0.80
Add N,N-dimethylformamide 60- to the solution.

N-Hydroxy-5-norbornene 2 in acetic acid under stirring under water cooling
．． 8-dicarboximide active ester 0.209. N
-Methylmorpholine 0.09- was added and the temperature was returned to room temperature after 30 minutes.

Stir for 2 days. This is because the reaction is not progressing sufficiently.

The reaction solution was cooled with water and further mixed with N-hydroxy-5-fulbornene 2,3-dicarboximide active ester of acetic acid.
209. Add 0.09% of N-methylmorboline, gradually warm to room temperature, and react for 2 days. The reaction solution was concentrated under reduced pressure, and ether 100- was added to the residue. The precipitate is collected by filtration, dissolved in warm water at about 40°C, and passed through a strongly acidic ion exchange resin Dowex 50 (H"). The solution is freeze-dried to form a white powder, l-acetyl-Ne-( 6-0
-stearoyl-N-acetylmuramyl-alanyl-D
-isoglutaminyl)-lysine 0.747 is obtained. Melting point 191.5-193°C (decomposed). [α)D+25.3°
(c-o, 5. Acetic acid: H2O-50' 2+ after 2 days).

Elemental analysis value Calculated value as C45HaoNso1rH20 Jin C57,06,H8,7,9,N 8.87Analysis value (%) C56,81,H8,48,N 9.0
5 Example 7 1-Acetyl-N'-(6-0-stearoyl-N-acetylmuramyl-alanyl-D-inglutaminyl)-
Dissolve lysine O, aO in 10-methanol and add diazomethane-ether solution. After 30 minutes, add 9 petroleum ether.

The precipitated crystals are collected by filtration. Then, recrystallization from methanol-ether-n-hexane yields 1-acetyl-N'-(6-0-stearoyl-N-acetylmuramylalanyl-D-isoglutaminyl)-lysine methyl ester 0.219. Melting point 202.5-208.
5°C (decomposition). [α]. +25.3° (G-0,6,
Acetic acid-water-50: 2.2 days later).

Elemental analysis value Calculated value (%) as C45HszNaO14・1+H20 c 56.95. H8,88,
N 8.66 analysis value) C57,12, H8,64
, N 8.88 Reference Example Preparation of active ester of fatty acid Dissolve fatty acid LOmmole in 10-tetrahydrofuran, and add dicyclohexylcarbodiimide 2-
06Tn9 and N-hydroxy-5-norbornene-2
, 8-dicarboximide 179WI9. 30
After a few minutes, the mixture was gradually returned to room temperature and reacted for about 5 hours. The precipitated dicyclohexyl urea is removed and the filtrate is concentrated. After adding dry ether and filtering off insoluble matter, the filtrate was concentrated to dryness under reduced pressure to obtain the fatty acid N-hydroxy-5-norbornene-2,
White crystals of 8-dicarbogyshiimide active ester are obtained.

Claims (1)

[Claims] Muramyl represented by the general formula is a peptide derivative (Al in the above formula
a means alanine, R is a hydrogen atom, an alkyl group or an aralkyl group, n is an integer of 1 to 6 + Al and A2
means a saturated or unsaturated fatty acid residue which may each have a hydrogen atom or a branch of 2 to 30 carbon atoms. )