JPH0124798B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the general formula () The present invention relates to a novel muramyl dipeptide derivative represented by: In the above formula, Acyl represents an acyl group having 2 to 6 carbon atoms,
X is L-alanine residue, Y is -NH(CH ₂ ) _o -
O-A ₂ or

[Formula] means a group, R ₁ is a carboxyl group, n is an integer of 1 to 6, and A ₁ and A ₂ are the same or different intermediate to higher mycoloyl groups. The present inventors have discovered that N-acetylmuramyl-L-alanyl-D is the smallest structural unit for expression of adjuvant activity in the cell walls of Mycobacterium tuberculosis, BCG, other mycobacteria, and cell-parasitic bacteria, which is useful as an immune adjuvant substance. - Focusing on isoglutamine (hereinafter referred to as muramyl dipeptide), we investigated its tumor cytotoxic activity and antitumor activity, which are mainly related to cell-mediated immunity. However, this muramyl dipeptide did not exhibit cytotoxic activity against mouse mastocytoma P815-X2 cells, which is closely related to antitumor activity. As a result of further investigation into the reason for this, the present inventors found that it is necessary for the adjuvant substance to have appropriate hydrophilicity and lipophilicity in order to express cytotoxic activity and antitumor activity. I thought about it. Therefore, the present inventors have conducted intensive studies on compounds that increase the lipophilicity of muramyl dipeptide and exhibit tumor cytotoxic activity, which is closely related to antitumor activity, and have strong promise as immunotherapeutic agents for human cancer in the future. As a result, it was discovered that the compound of the general formula () can be prepared into a uniform suspension in phosphate buffer-physiological saline, has high lipophilicity, and exhibits excellent tumor cytotoxic activity. completed. The cytotoxic activity for the compounds of this invention is shown in the table below. Γ Cytotoxic Activity Test Samples were suspended in phosphate buffer-physiological saline and control muramyl dipeptide was dissolved, along with 2 x 10 ⁴ mastocytoma P815-X2 tumor cells and C57.
It was administered intraperitoneally to BL/6J mice and measured by the method of Brunner et al. (Immunology 18 , 501-515, 1970).

[Table] Invention compound-1: 6-O-mycomycoloyl-N
-Acetylmuramyl-L-alanyl-D-isoglutamine 2-mycomycholoyloxyethylamide Compound of the present invention-2:N〓-(6-O-mycomycholoyl-N-acetylmuramyl-L-alanyl-D
−Isoglutaminyl)−N〓−Micomycoloyl−
L-Lysine The compound of the present invention can be produced according to the following reaction steps. That is, the compound of the present invention has 6-
It is produced by condensing O-acylmuramyl dipeptide with a compound represented by the formula (). For this condensation reaction, methods generally used for peptide synthesis, such as the carbodiimide method, the Eintop method (Proteins, Nucleic Acids, Enzymes Vol. 16 No. 1 P56, 1971), the active ester method, and the acid anhydride method, can be employed.
For example, a compound represented by the formula () is dissolved in N,N-dimethylformamide, tetrahydrofuran, or a mixture thereof, and N-hydroxysuccinimide, 1-hydroxybenzotriazole, N-hydroxy-5-norbornene-
2,3-dicarboximide, pentachlorophenol, etc. and carbodiimide (dicyclohexylcarbodiimide or 1-ethyl-3-(3-
(dimethylaminopropyl)-carbodiimide (WSCI) or its hydrochloride) and usually at room temperature to about 60℃
The active ester may be reacted for about 1 to 2 days, and the compound represented by the formula () may be condensed thereto. The condensation reaction is carried out at 0 to 60°C in a solvent commonly used for peptide synthesis, such as chloroform, dimethyl sulfoxide, water, tetrahydrofuran, dimethylformamide, etc. alone or in combination.
This is preferably achieved by stirring at about 25-40°C for about 1-2 days. Note that the raw material compound used for producing the compound of the present invention, that is, the compound represented by the formula (), can be produced according to the following method. That is, to produce a compound represented by the formula (), the formula ZHN−(CH ₂ ) _o −OW () or the formula

[Formula] (In the formula, R ₁ is a carboxyl group, n is an integer of 1 to 6, and Z is a benzyloxycarbonyl group that may have a substituent such as a halogen atom, a nitro group, or a lower alkoxy group at the para position, Reaction of a protecting group for an amino group frequently used in peptide synthesis such as t-butoxycarbonyl group (W means an activated form such as methanesulfonyl group, p-toluenesulfonyl group or halogen atom) and mycolic acid or its reaction The active substance is reacted with the active substance in a solvent such as N,N-dimethylformamide (DMF) or hexamethylphosphoramide (HMPA) at room temperature to about 150°C for 15 minutes to 24 hours to cause condensation. This condensation reaction is carried out in a less polar solvent such as benzene as a reaction solvent.
A method of refluxing for 1 to 24 hours in the presence of Crown 6 may also be adopted. Next, the compound of formula () is produced by removing the protecting group using appropriate removing conditions for the protecting group. For example, a catalytic reduction method or a hydrobromic acid-acetic acid treatment method is suitable for removing a benzyloxycarbonyl group, and a t-butoxycarbonyl group or a p-
In order to remove the methoxybenzyloxycarbonyl group, it is desirable to treat with trifluoroacetic acid or hydrochloric acid/tetrahydrofuran. Mycolic acid, which plays an important role as a structural unit of the compound of the present invention, can be obtained by hydrolyzing whole cells, cell walls, bound lipids, etc. of various bacteria, and then purifying it by column chromatography using activated alumina, silicic acid, etc. It can be manufactured. Mycolic acids are originally defined as higher fatty acids having a long-chain branched alkyl group on the α-carbon and a hydroxyl group on the β-carbon (Asselineau.J; The Bacterial Lipids,
Hermann Paris (1966), mycolic acids produced by the above-mentioned method are generally obtained as a mixture of several types. Of course, it is possible to perform more rigorous purification and separation to provide a complete single compound or a pure synthetic product for the production of the target compound of the present invention. However, from the viewpoint of biological activity, which is the objective of the present invention, it is not necessary to completely purify mycolic acid, and it is considered that it is sufficient to use a mixture of several types of mycolic acid. In general, high-grade mycolic acids are found in Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, and other mycobacterial genera (e.g., Mycobacterium phlei, Mycobacterium phlei,
Mycobacterium smegmatis) is a higher fatty acid with a total of about 70 to 90 carbon atoms, having a branched alkyl group with 22 to 24 carbon atoms on the α carbon and a hydroxyl group on the β carbon (hereinafter referred to as mycomycolic acid). . In addition, examples of intermediate mycolic acids include nocardomicolic acid, corinonomycolic acid, and Arthrobacter mycolic acid, which have a branched alkyl group having about 8 to 16 carbon atoms at the α-carbon, and a branched alkyl group having about 8 to 16 carbon atoms at the β-carbon. It is a higher fatty acid with a total number of carbon atoms of about 28 to 70 and has a hydroxyl group. Bacteria that obtain nocardidomycolic acid include bacteria of the genus Nocardia (e.g. Nocardia asteroides,
Nocardia ruba, Nocardia polychromogens,
Nocardia brasiliensis, etc.), but the bacteria that obtain corinomicolic acid include Corynebacterium and Arthrobacter, such as
Corynebacterium diphteriae,
Corynebacterium pseudotuberculosis, C.
xerosis, C.renale, Arthrobacter simplex, A.
flavescens etc. Therefore, the term mycolic acid used in this specification refers to a mycolic acid having a total of about 28 to 90 carbon atoms, which has a branched alkyl group with about 8 to 24 carbon atoms on the α-carbon and a hydroxyl group on the β-carbon. It means a single higher fatty acid or a mixture of higher fatty acids. Representative examples of mycolic acids used for carrying out the present invention are as follows. Whole cells of Nocardia asteroides 131 were subjected to alkaline hydrolysis to produce methyl ester using a conventional method, and then purified with silicic acid and column chromatography, followed by hydrolysis to release and obtain intermediate mycolic acid. Ta. The average molecular formula of the intermediate mycolic acid obtained was C ₅₁ H ₉₇ O _3.6 as determined by acid titration and elemental analysis. Mycobacterium tuberculosis strain
Aoyama B) wax fraction is alkaline hydrolyzed,
This was then subjected to activated alumina column chromatography. The average molecular formula of the obtained mycomycolic acid was C ₈₀ H ₁₅₈ O _3.5 as determined by acid titration and elemental analysis. The present invention will be explained below with reference to Examples. Example 1 10g of 2-bromoethylamine hydrobromide
6.90 g of benzyloxycarbonyl chloride and
Add 10 ml of 4N sodium hydroxide solution dropwise over about 1 hour. If the reaction solution becomes cloudy, add dioxane.
Add 50ml. After further stirring at room temperature for about 2 hours, the reaction solution was concentrated under reduced pressure to distill off dioxane.
The precipitated oil was extracted with ethyl acetate, washed with water, and dried over anhydrous sodium sulfate. When the syrup obtained by distilling off ethyl acetate is cooled on ice, 9.22 g of crystals of benzyloxycarbonylaminoethyl bromide are obtained. Melting point 45-46℃. Potassium mycomycolate 1.03g and benzyloxycarbonylaminoethyl bromide 0.43g and
18−Dissolve 0.10 g of Crown6 in 30 ml of benzene,
Heat and reflux for about 3 hours. Concentrate under reduced pressure, add methanol to the residue, and collect the precipitated crystals by filtration.
1 g of the obtained crude crystals was subjected to silica gel chromatography, and the desired fractions eluted with benzene-ethyl acetate (10:1) were collected and concentrated under reduced pressure. Recrystallization of the residue from tetrahydrofuran-methanol yields 0.60 g of benzyloxycarbonylaminoethyl micomycholoyl ester. Melting point 40-42℃. Dissolve 0.55 g of the above compound in 20 ml of tetrahydrofuran, add 0.45 ml of 1N hydrochloric acid solution, and react for about 5 hours in a hydrogen stream in the presence of palladium black. After the catalyst is then filtered off, the tetrahydrofuran is distilled off. The residue was recrystallized from benzene-acetone-methanol to give 0.49 g of mycomycolic acid 2-aminoethyl ester hydrochloride (-
a) is obtained. Melting point 61-65℃. Mycomycoloylmuramyl peptide () 100mg
77 mg of (-a) and 8 mg of N-hydroxysuccinimide were dissolved in 4 ml of tetrahydrofuran and stirred under ice cooling. Add 15 mg of dicyclohexylcarbodiimide and 0.01 ml of N-methylmorpholine dissolved in 1 ml of tetrahydrofuran. After 30 minutes, return to room temperature and stir overnight. The reaction solution is concentrated and methanol is added to obtain crude crystals. Then, it is recrystallized from benzene-acetone-methanol to obtain 95 mg of 6-O-mycomycholoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine-2-mycomycholoyloxyethylamide (-a). melting point 65
~77℃. [α] ²⁵ ° _D +13.6° (C=0.5, tetrahydrofuran-water=50:1, after 24 hours). Elemental analysis value C ₁₈₁ H ₃₄₉ O ₁₆ N ₅ Calculated value (%) C 76.21, H 12.36, N 2.46 Analysis value (%) C 76.04, H 12.17, N 2.52 Example 2 Mycomycolic acid 0.5 g and N-hydroxysuccinimide Dissolve 56 mg in 10 ml of tetrahydrofuran, and add 100 mg of dicyclohexylcarbodiimide while stirring on ice. After 30 minutes, return to room temperature and react overnight. After removing the precipitated dicyclohexyl urea by filtration, methanol is added to the filtrate to obtain 0.5 g of mycomycolic acid N-hydroxysuccinyl ester. Dissolve 0.5 g of this in 10 ml of tetrahydrofuran, and add 180 mg of N-t-butoxycarbonyl-L-lysine hydrochloride dissolved in 4 ml of N,N-dimethylformamide and 0.1 ml of N-methylmorpholine while stirring on ice. . After 30 minutes, the mixture was returned to room temperature and reacted with stirring for 5 days. The reaction solution was concentrated under reduced pressure, and the residue was subjected to silica gel chromatography. The target fractions eluted with chloroform-methanol (7:1) were collected and concentrated under reduced pressure, and the residue was recrystallized from tetrahydrofuran-methanol.
Butoxycarbonyl-N〓-mycomycoloyl-L
- Obtain 0.36 g of lysine. Melting point 50-52℃. Elemental analysis value C ₉₁ H ₁₇₈ O _6.5 N ₂・H ₂ O Calculated value (%) C 76.82, H 12.78, N 1.97 Analysis value (%) C 76.63, H 12.30, N 2.18 Add 0.32 g of the above compound to 9 ml of dichloromethane. Dissolve and add 9 ml of trifluoroacetic acid while stirring on ice. After 5 minutes, the temperature was returned to room temperature and the reaction was stirred for 1 hour. After the reaction, it was concentrated to dryness under reduced pressure to obtain N-mycomycholoyl-L-lysine trifluoroacetate (-b).
Obtain 0.27g of. Melting point 59-61℃. Elemental analysis value C ₈₆ H ₁₇₀ O _4.5 N ₂・CF ₃ COOH・1/2H ₂ O Calculated value (%) C 74.03, H 12.17, N 1.96 Analysis value (%) C 73.90, H 11.78, N 2.18 Micomicoil unevenness Milpeptide () 198mg
and 5 ml of 18.6 mg of N-hydroxysuccinimide
of tetrahydrofuran and stir while cooling on ice.
It was then dissolved in 2 ml of tetrahydrofuran. Add 28 mg of dicyclohexylcarbodiimide. After 30 minutes, return to room temperature and stir overnight. The precipitated dicyclohexylurea was filtered off, and the filtrate was stirred under ice cooling. Next, 126 mg of (-b) and 0.02 ml of N-methylmorpholine were added, and after 30 minutes, the mixture was returned to room temperature and reacted with stirring overnight. Concentrate under reduced pressure, add methanol to the residue, and subject the precipitated crystals to silica gel chromatography. Chloroform: Methanol:
The target fraction eluted in the lower layer of the 8:3:1 water mixture is collected and concentrated to dryness under reduced pressure. The residue was again subjected to silica gel chromatography and eluted with chloroform:methanol:acetic acid=95:5:3. The desired fractions were collected and concentrated under reduced pressure, and methanol was added to the residue to give N〓-(6-O-mycomycoloyl-N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N〓-mycomycoloyl-L-lysine. (−
Obtain 48 mg of b). Melting point 135-142℃. [α]25゜D+
14.7゜(C=0.5, tetrahydrofuran: water=50:
1, 24 hours later). Elemental analysis value C ₁₈₅ H ₃₅₆ O ₁₇ N ₆・3H ₂ O Calculated value (%) C 74.27, H 12.22, N 2.81 Analysis value (%) C 74.03, H 11.77, N 3.47

Claims (1)

[Claims] 1. General formula A muramyl dipeptide derivative represented by In the above formula, Acyl represents an acyl group having 2 to 6 carbon atoms,
X is L-alanine residue, Y is -NH(CH ₂ ) _o -
It means O-A ₂ or [Formula], R ₁ is a carboxyl group, n is an integer of 1 to 6, and A ₁ and A ₂ are the same or different intermediate to higher mycoloyl groups.