JPS6033119B2 - Novel muramyl dipeptide derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel muramyl dipeptide derivative which is expected to have a strong anti-sun effect, and more specifically to a derivative of general formula (1) (where Y represents a mycolic acid residue and GIy represents glycine). , jsoG1n means isoglutamine.

) 6-0-myco0il-N-acetylmuramyl dipeptide transfer derivative. In recent years, as a promising method for preventing or treating cancer, the immune system is restored by administering an appropriate immune adjuvant substance to the body, which is damaged due to physiological or pathological causes. Strategies have been considered to artificially enhance the immune response of humans, especially the ability of cell-mediated immune responses, which are thought to be involved in the elimination of non-self cells such as sitz bath cells.

Conventionally, Mycobacterium tuberculosis, BCG has been used as an immune adjuvant substance.
It is already known that the cell walls of other mycobacteria and cell-parasitic bacteria are useful, but the present inventors investigated the constituent components that exert adjuvant activity in these bacterial cell walls, and found that ovalbuminin in guinea pigs N-acetylmuramyl-L-alanyl- is the minimum unit that participates in the expression of adjuvant activity against protein antigens.
The required structure of D-isoglutamine was clarified and announced earlier. (12th Peptide Chemistry Symposium and 48th Japanese Bacteriological Meeting). Subsequently, Kotani (Osaka University School of Dentistry) similarly investigated the constituent components that exert adjuvant activity in the cell extremities of plant pathogenic bacteria. revealed that the structure of is necessary for the expression of adjuvant activity (Clinical Science, Vol. 11, No. 9,
089-1097, 1973 hand). Muramyl dipeptide was the minimum unit for expressing adjuvant activity based on the measurement results of blood antibody levels and delayed allergic reactions, but it is closely related to anti-tumor activity and cell-mediated immunity is mainly involved. Cytotoxic activity test against mouse mastocytoma-P815 1×2 cells (Cell-mediated
dc
4 leukemia) was negative. 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 anti-tumor activity. I thought about it.

This is consistent with the oil-in-water emulsion used in cytotoxic activity tests and antitumor activity tests.
et) and when preparing a phosphate-buffered saline suspension, muramyl dipeptide itself cannot form a good emulsion, or it cannot be homogeneously suspended because it dissolves in phosphate-buffered saline. We considered that the problem is deeply related to the physical properties of adjuvant active substances.

On the other hand, it is known that mycolic acid, which is a long-chain branched fatty acid, exists as a characteristic component in cell walls and fractions thereof of BCG and Mycobacterium tuberculosis, which have anti-tumor activity. Based on this viewpoint, the present inventors conducted extensive studies on muramyl dipeptides having appropriate hydrophilicity and lipophilicity, and as a result, discovered a novel muramyl dibeptide domicolic acid ester represented by formula (1). The invention has been completed.

The compound of formula (1) is used during the formation of the emulsion and the preparation of the homogeneous phosphate buffered saline suspension as described above.
This product completely eliminates the defects of muramyl dipeptide. Therefore, if the compound of formula (1) has cytotoxic activity, which is thought to be played mainly by cell-mediated immunity, and also has antitumor activity against syngeneic metastatic fin cancer, it can be used as a human bran immunotherapeutic agent. This is something to look forward to. The anti-tumor activity test results for the compound of formula (1) are as follows. ■ Cytotoxic activity The compound of the present invention is suspended in phosphate buffered saline,
Muramyl dipeptide was dissolved in phosphate-buffered saline and added to each mastocytoma P815 1x sleeve pupil wound cell.
The method of Br et al. (lmm dishology 18, 50
1-515, 1970).

The results are shown in Table 1 below, where the compound of the present invention was administered in the form of a suspension in phosphate buffered saline.
It showed strong adjuvant activity in the appearance of lymphocytes exhibiting pupil cytotoxic activity in the o lineage.

Table-1 Cytotoxic activity * Mastocytoma P815 1 x 2 types Only 1 x 1 cell ■ Anti-tumor activity Sample 1 suspended or dissolved in phosphate buffered saline
00 evening (compound of the present invention: suspended state, muramyl dipeptide; dissolved state) was mixed with 1 x 1 MHI milk Patoma and administered intradermally to syngeneic C3 day/He mice. We investigated the growth-inhibiting effect of .

As shown in Table 2, the compound of the present invention exhibited anti-tumor activity that was not observed in muramyl dipeptide. Table-2 *Number of mice with complete tumor growth inhibition/Number of mice used*
* Phosphate-buffered saline only As is clear from the above results, the target compound of the present invention has cytotoxic activity and antitumor activity that are not observed in muramyl dipeptide, and is effective against BCG and other mycobacteria. It can be fully expected to be used as an immunotherapeutic agent for cancer, similar to the bacterial cells or their cell wall fractions.

Further, the object compound of the present invention has the following properties. That is, (1) it is a compound that can be synthesized with a simpler structure than conventional bacterial cells and cell wall fractions, and can be obtained as a highly pure homogeneous component. ■As mentioned above, it is possible to prepare a homogeneous suspension of a compound alone in phosphate buffered saline, and unlike bacterial cell wall fractions, intravenous administration to living organisms is possible; It is expected that side effects such as tissue damage at the injection site will not occur due to intradermal or intramuscular administration of oil-in-water emulsions, which has been carried out in et al. ＠While conventional immunoadjuvant substances have immunogenicity in themselves, as seen in Freund's complete adjuvant, there is no possibility that the target compound of the present invention has immunogenicity by itself. few. Taking the above points into consideration, the target compound of the present invention is worthy of great attention as a future immunotherapeutic agent for human cancer.

In order to produce the target compound of the present invention, N-acetylmuramic acid with the 1-position hydroxyl group protected with an appropriate protecting group is used as a raw material,
If necessary, it can be produced by protecting the carboxyl group and activating the 6-position hydroxyl group, then reacting with mycolic acid, then with glycyl-D-isoglutamine, and finally removing the protecting group. That is, the reaction formula is as follows.
(In the formula, Z is a pennzyl group which may be substituted with a halogen atom, a nitro group or a lower alkoxy group, X is a protecting group for a carboxyl group such as a tertiary butyl group or a diphenylmethyl group, and Y is a mycoloyl group. ) protection reaction of the carboxyl group of the compound of formula (0) (i.e. (n)→(m
) is not necessarily essential, but preferably has a suitable protecting group in order to allow the subsequent esterification reaction to proceed more efficiently.

This protecting group introduction reaction can be carried out by conventional means. Formula (m)
The reaction for producing the compound of formula (W) from the compound of formula (W), that is, the activation reaction of the 6-hydroxyl group, may be selected as appropriate.
The compound m) may be dissolved in a solvent having a deoxidizing effect and reacted with valatorenesulfonyl chloride, methanesulfonyl chloride, or the like.

The reaction between the compound of formula (W) and mycolic acid (alkali metal salt) is usually carried out using a suitable solvent (e.g. dimethylformamide,
(polar solvent such as dimethyl sulfoxide).

The reaction is preferably carried out by heating to 100 to 140°C, but if a cyclic compound such as 18-Crown-6 is reacted in the presence of a polyether compound, it can be carried out at a low temperature in the presence of a nonpolar solvent such as benzene. . The protecting group of the carboxyl group of the compound of formula (V) thus obtained was removed ((V)→
(M)) is reacted with glycyl-D-isoglutamine using a suitable condensing agent.

This reaction is usually carried out in the presence of a suitable solvent (for example, a nonpolar solvent such as ethyl acetate, benzene, dioxane, or tetrahydrofuran), and it proceeds quickly by pouring the reaction solution. It can also be promoted by Finally, the protecting group is removed to obtain the target product, and the protecting group can be removed using a conventional method, such as catalytic reduction in the presence of a catalyst such as palladium charcoal or platinum, or a hydrobromic acid-acetic acid solution. This is carried out by methods such as processing.

Mycolic acid, one of the raw materials used in the present invention, plays an important role as a moiety that binds to muramyl dipeptide and imparts appropriate lipophilicity. There are things you can do.

That is, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, and other mycobacteria (e.g., MyC.
phlej, MyC. baCteri mountain ms
It is produced by hydrolyzing the whole cells of A.megmatis) and atypical mycobacteria, or Rho D, bound lipids, etc., and purifying it by column chromatography using activated alumina, sulfuric acid, etc. The mycolic acids produced in this way have the molecular structures disclosed in "Tuberculosis No. 5, No. 11, p. 431 (1975)", such as Q-mycolic acid, Q'-mycolic acid,
It is usually obtained as a mixture of several types of 8-mycolic acid, Q-smegmicolic acid, Q-cansamicolic acid, methoxymycolic acid, and the like.

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, but From this point of view, it seems that there is not much difference depending on the role played by mycolic acid residues. That is, the mycolic acid used in the present invention is Asselino (pseudo-selineau J.;
me axial cteriaILipids, Hennann
Parisl 966), a higher fatty acid with a total number of carbon atoms of 70 to 90 having a long mirror-branched alkyl group at the Q1 carbon and a hydroxyl group at the 81 carbon may be used, and single or mixtures of these may be used. Both are used. An example of mycolic acid used for carrying out the present invention is as follows.
Mycobacterium tuberculosis
A wax fraction of sVain Ao maB) was obtained by alkaline hydrolysis and subsequent activated alumina column chromatography.

The average molecular formula of the obtained mycolic acid was C8 rainbow side 03.5 based on acid titration and elemental analysis. Reference Example Synthesis of t-butoxycarbonylglycyl-D-isoglutamine benzyl ester Dissolve 1.0 ml of t-butoxycarbonylglycine in 20 parts of tetrahydrofuran, add N-hydroxysuccinimide 657 part 9 and Add 1.176 ml of N.N-dicyclohexylcarbodiimide.

Next, a mixture of 1.555 g of D-isoglutamine benzyl ester hydrochloride and 0.8 g of triethylamine was added to 30 g of ice-cooled tetrahydrofuran under ice-cooling, and the mixture was allowed to stand overnight at room temperature. continue. The resulting triethylamine hydrochloride and N·N'-dicyclohexylurea are removed, and then the solvent is distilled off. The residue was dissolved in ethyl acetate and diluted with 0. - Wash sequentially with hydrochloric acid, water, and 5% aqueous sodium carbonate solution. After drying the ethyl acetate layer over anhydrous sodium sulfate, the solvent was distilled off. The residue was recrystallized from ethanol-ether to obtain 1.93 g of t-butoxycarbonylglycyl-D-isoglutamine benzyl ester. Melting point 111-11〆0. [Q] Yoju 3.4o (
C=1. methanol). Elemental analysis value C, Rainbow 270
Calculated value (%) as 6N3 Group C. 00th 6.92, NIO. Iso analysis value (%) C spots. 33rd 6.91, NIO. Example 44 1.0 ml of penzyl N-acetyl-Q muramid was dissolved in 10 ml of tetrahydrofuran, 0.8 ml of diphenyldiazomethane was added thereto, and the mixture was stirred at room temperature for 30 minutes.

After distilling off the solvent, the residue is crystallized by adding hexane. The crude crystals obtained here were recrystallized from ethyl acetate-hexane to give 1
．． 1-Q-○-benzyl-N-acetylmuramic acid diphenylmethyl ester (m) was obtained after 3 days, and this was recrystallized again from ethyl acetate-hexane to obtain a pure product. Melting point 1
55-156q0. [Q] Volume + 12r (C = 1. chloroform). Elemental analysis value C3, day 3508N calculated value (%) C67.74 day 6.42, N2.55 analysis value (%) C67.62, day 6.5 or more N2.521-Q
-0-Besodyl-N-acetylmuramic acid diphenylmethyl ester (0.3 mm) was dissolved in pyridine (3 mm), and to this solution was added 1.2 mm of tosyl chloride under ice cooling.

After cooling on ice for 1 hour, pour into water and extract with ethyl acetate.

The ethyl acetate layer was washed with 0.3N caustic soda solution, 1N hydrochloric acid solution and water, and dried over magnesium sulfate. Remove the droplets under reduced pressure and purify the pickles using silica gel column chromatography. When the solvent is completely removed from the benzene-ethyl acetate eluate, 11Q-benzyl-610-tosilyl-N-acetylmuramic acid diphenylmethyl ester (W) of 0.34 ml is obtained. Melting point Iso~7zu○. [Q] Customer = 8
4.40 (C=0.5 chloroform). Elemental analysis value C
Calculated value (%) as 38 days 4,0,oNS C64.8
Fuday 5.87, NI. 99S4.56 analysis value (%) C
64.6 & Sun 5.92, NI. 93S4.31 potassium mycolate 0.38 1.8-C (W) 0.33 1.8-C
Add 0.02 ml of Rown-6 to a solution of 10% benzene and reflux for 3 hours.

The solvent was distilled off under reduced pressure and the residue was washed with acetone. The insoluble material is subjected to silica gel column chromatography. The fraction eluted in benzene-ethyl monoacetate (10:1) is treated with an ethereal solution of diazomethane at room temperature. Methysterification of excess mycolic acid facilitates chromatographic purification of the target product. The solvent was distilled off under reduced pressure and the residue was subjected to silica gel chromatography again. After eluting methyl mycolate with benzene, the benzene-ethyl acetate (10:1) eluate was collected. After the solvent was removed, the residue was recrystallized from acetone to obtain 1-Q-benzyl-610-mycoloyl-N-acetylmuramic acid diphenylmethyl ester (V) with a yield of 0.32 hours. Melting point payment~5
70. [Q] Customer +32.60 (C = 0. Fuku. Roholm). Elemental analysis value C,. , Sun, 9, 0, o. Randomly calculated value (%) C78.07, HII. 27, NO. 82
Analysis value (%) C783LHII. 48 NO. 85 (V
Dissolve 0.65 g of anisole and 1 g of anisole in 20 g of chloroform, and add 5 g of trifluoroacetic acid under ice cooling. 3
After the rice bran is heated, acetone is added to the reaction bran and the rice bran is removed under reduced pressure.

The residue was washed with ethanol and then dissolved in 10 ships of tetrahydrofuran. To this solution were added 73 N-hydroxysuccinimide, 72 N-N'-dicyclohexylcarbodiimide, a solution of 1.2 mmol of triethylamine dissolved in 0.2 M of tetrahydrofuran, and t-butoxycarbonyl obtained by the method described in the reference example. Each of the glycyl-isoglutamine benzyl ester hydrochloride 237 and 9 obtained by treating the glycyl-isoglutamine benzyl ester with hydrogen chloride and acetic acid was added to the ice-cold eel paste, and further stirred overnight at room temperature. continue. wealth.

triethylamine hydrochloride and N·N'-disic.
Remove hexylurea. The solvent is removed under reduced pressure, the ethanol-soluble portion is removed, and the residue is subjected to silica gel chromatography. The eluted fraction of benzene-acetone (3:1) (excluding the initial eluted fraction) was collected, and after the solvent was removed, the residue was recrystallized from benzene-methanol. Milu glycyl-D-isoglutamine benzyl ester (W) 0.28
I got 4 nights. Melting point: 148-150 pm○. [Q] Color 513
6.7 (C=0.5 chloroform). Elemental analysis value C,
．． 2nd, 960, 3. is the calculated value assuming 4・CH30 days (
%) C73.3ku HII. 03N3.03 analysis value (%
) C73.27, HIO. 83N2.87 (blood) 0.
A solution prepared by dissolving 23 ml of tetrahydrofuran in 40 ml of tetrahydrofuran is subjected to a hydrogenation reaction at room temperature in the presence of palladium black.

Claims (1)

[Claims] 1 Formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, Y represents mycolic acid residue, Gly represents glycine,
isoGln means isoglutamine. ) 6-0-mycoloyl-N-acylmuramyl dipeptide derivative.