JPS6033120B2 - 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 the general formula (1) (wherein Y represents a mycolic acid residue and Ser represents serine). , isoG1n is enriched with isoglutamine.

) relating to the shown N-acetylmuramyl dipeptide derivatives. In recent years, a promising method for preventing or treating cancer (malignant tumors) has been to restore the immune surveillance mechanism of the body that has been damaged due to physiological or pathological causes by administering an appropriate immune adjuvant substance to the body. Measures have been considered to artificially increase the body's immune response, particularly the cell-mediated immune response capability, which is thought to be related to the elimination of non-self cells such as pupillary cells.

As an immune adjuvant substance, Mycobacterium tuberculosis, BCG
Although it is already known that the cell walls of other mycobacteria and cell-parasitic bacteria are useful, the present inventors investigated the constituent components that exert the adjuvant activity exhibited by these bacterial cell walls, and determined that the cell walls of these bacterial cells exhibit adjuvant activity. The technical unit involved in this process is N-acetylmuramyl-L-alanyl-isoglutamine (hereinafter referred to as muramyl dipvetide).

), and then synthesized various derivatives of muramyl dipeptide and evaluated their adjuvant activity and oxidation activity. As a result, muramyl dibeptide domicolic acid ester, in which mycolic acid, an ultrahigher fatty acid, is ester-linked to the 6-hydroxyl group of the muramic acid skeleton, is closely related to anti-tumor activity, and cell-mediated immunity is mainly involved in mice. We found positive results in both the cytotoxic activity test against mastocytoma P815-x cysts and the antitumor activity test using mouse hepatoma MH-1 I, and we would like to present the proc.
eedingoftheJapan Academy.
, 53 63-601977).

However, these muramyl dibeptide domicolic acid esters have been shown to have clinical effects against cancer.
A chemical addictive agent was orally administered to guinea pig strain-2 (guinea pig strain-2) to induce carcinogenesis, and 2 x 1 liver cancer cells were transplanted into syngeneic guinea pigs. In a tumor wound activity test using a tumor type, the activity was weak, and it was difficult to see whether it would directly lead to clinical effects or not.

Furthermore, when these muramyl dibeptide domicolic acid esters were intravenously injected into rabbits in accordance with the Japanese Pharmacopoeia's pyrogen test, fever may be observed, suggesting the possibility of clinical side effects. . Therefore, as a result of intensive studies aimed at enhancing the anti-tumor activity of muramyl dibeptide domicolic acid ester in a guinea pig hawking system and weakening its pyrogenicity, the present inventor found that the amino acid composition of muramyl dibeptide We synthesized a compound of formula (1) in which L-alanine was changed to L-serine, and examined its antitumor activity in a guinea pig tumor system (line-10) and its pyrogenicity in rabbits. It was found that the invention met the requirements and the present invention was completed. The results of investigating the antitumor activity and pyrogenicity of the compounds of the present invention are as follows.

■ Antitumor activity test A frame medicine shortener (jetyl nitrosamine) was orally administered to guinea pigs (stwin-2), and 2×
One tumor cell was intradermally transplanted into a syngeneic guinea pig, and 7 to 8 days after transplantation, when the seedling had 10 to 11 ribs,
A paste of 300 grams of sample and 150 grams of trehalose dimycolate was made into a paste with mineral oil (Drachiol 6 VR) in the hawk face, and phosphate buffered saline containing 0.2% Tween-80 was added to form an oil-in-water emulsion. administered.

Four weeks after administration, the number of guinea pigs whose tumor had completely regressed was divided by the number of guinea pigs used, and the value was multiplied by 100 to calculate the antitumor activity (%).
). As shown in Table 1, the compound of the present invention has stronger antitumor activity than 610-mycomycoloyl-N-acetylmuramyl-L-alanyl-D-isoglutamine, which is one of the muramyl dibeptide domicolic acid esters. Indicated.

Table-1 * 6-0-Mycomycoloyl-N-acetelmuramyl-L-seryl-D-incultamine※※ 6-○-Nocaldomycoloyl-N-Ryocetelmuramyl-L-seryl-
D-incultamine※※※ 6-0-earthmicoloinone-N-acetylmuramyl-L-seryl-D-incultamine■ Pyrogenic substance test test method is Japanese Pharmacopoeia Pyrogenic Substances Testing Method (9th revision) Japanese Pharmacopoeia Manual B-176,
(1976).

Specifically, three rabbits were used as one group, and the sample was suspended in distilled water for injection and injected into the rabbit ear vein. Body temperature measurement after injection was performed three times at 1-hour intervals after injection, and 2 or 3 test animals (rabbits) with a temperature increase of 0.6o or more after injection were
When the wings were released, it was determined to be positive for pyrogenic substances. The results are shown in Table 2.
As shown, 6-0-nocardomicoloyl-N-acetylmuramyl-L-alanyl-D-isoglutamine, which is one of the muramyl dibeptide domicolic acid esters, was positive, but the compound of the present invention was negative.

Table 2 As described above, the compounds of the present invention are BCG and BCG, which are currently recognized to have clinical effects as immunotherapeutic agents for various cancers.
A guinea pig line whose CG cell wall exhibits strong anti-tumor activity.
1 It shows strong anti-tumor activity in the pupil field system and does not have the pyrogenicity observed with muramyl dibebutide domicolic acid ester. It holds great promise as an immunotherapeutic drug for algae.

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,
It can be produced by protecting the carboxyl group if necessary and activating the 6-position hydroxyl group, then reacting with mycolic acid, then with L-seryl-D-isoglutamine, and finally removing the protecting group.

That is, the reaction formula is as follows. (In the formula, Z means a penzyl group which may be substituted with a halogen atom, a nitro group or a lower alkoxy group, X means a tertiary butyl group, a protecting group for the carboxyl group of a diphenylmethyl group, and Y means a mycoloyl group. ) The protection reaction of the carpoxyl group of the compound of formula (b) (i.e., (0) → (m)) is not necessarily essential, but it is suitable for the subsequent esterification reaction to proceed more efficiently. It is preferable to have a protective group.

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 balatroluenesulfonyl chloride, methanesulfonyl chloride, etc.

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,
(sacrificial solvent such as dimethyl sulfoxide).

The reaction is preferably carried out by heating to 100 to 140°C, but if the reaction is carried out in the presence of a cyclic polyether compound such as 18-Crown-6, 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 is removed ((V)→(
W)) is reacted with L-seryl-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, tetrahydrofuran, etc.), and rapidly proceeds by swirling the reaction solution. After the technique, the protecting group is removed to obtain the target product, and the protecting group can be removed using the usual methods, such as catalytic reduction in the presence of a catalyst such as palladium charcoal, or treatment with a hydrobromic acid monoacetic acid solution. It is carried out by the method etc.

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. can be manufactured.

That is, it can be produced by hydrolyzing whole cells, cell walls, and bound components of various bacteria and then purifying them by column chromatography using activated alumina, citric acid, or the like. Mycolic acids are originally defined as higher fatty acids having a long branched alkyl group on the Q-carbon and a hydroxyl group on the B-carbon (Fuselineau J: The
Mother cterial Lipi ship, HermannPa
risl966), 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, in view of the role played by mycolic acid residues in terms of biological activity, which is the subject matter of the present invention, complete purification of mycolic acid is not required, and use in a mixture of several types is sufficient. It is believed that there is. In general, high-grade mycolic acids are found in Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, and other mycobacterial genera (e.g., Mycobactenum phlej, Mycobacterium
It is an 8-carbon fatty acid with a branched alkyl group having 22 to 24 carbon atoms on the Q carbon (hereinafter referred to as mycomycolic acid).
.

In addition, examples of intermediate mycolic acids include nocardomicolic acid, cholynomicolic acid, and Arthrobacter mycolic acid, which have a branched alkyl group having about 8 to 1 carbon atoms on the Q-carbon, and 3 Approximately 28 total carbon atoms with hydroxyl groups on carbon
~70 higher fatty acids.

/Bacteria that obtain cardomiclic acid include bacteria of the genus Nocardia (for example, Nwardia asteroides,
NMardia mbra, NMardia poly
Chromoge ship S, Nmardiabrasili
ens;
ae, Corynebacterium mpsedotu
rc mountain osis, C. xerosls, C. re
nale, ~tmo broadside simplex, A.
Examples include navesce ships.

Therefore, the term mycolic acid used in this specification refers to a compound having a total of 28 to 9 carbon atoms and a branched alkyl group having about 8 to 24 carbon atoms on the first carbon and a hydroxyl group on the 8th carbon. It means a single or a mixture of higher fatty acids. Representative examples of the mycolic acids used to carry out the present invention are as follows.

0/Cardia asteroides 131 bacteria (NMard
IAasteroi ship s131) was subjected to alkaline hydrolysis to obtain methyl ester using a conventional method, and then purified using citric acid and column chromatography, followed by hydrolysis and release to obtain intermediate mycolic acid.

The average molecular formula of the intermediate mycolic acid obtained was C.9703 P as determined by acid titration and elemental analysis. 0 Arthrobacter para8in ship ATCC-15
590), Nocardia asteroides 131
Intermediate mycolic acid was obtained by processing in the same manner as in the case of bacteria.

The average molecular formula of this intermediate mycolic acid was C3 rainbow 7403.5 based on acid titration and elemental analysis. 0 Mycobacterium tuberculosis
The wax fraction of standard B) was subjected to alkaline hydrolysis and then subjected to activated alumina column chromatography.

The average molecular formula of the obtained mycomycolic acid was determined by acid titration and elemental analysis to be C8oH shelf Q. It was 5. Reference Example Synthesis of t-butoxycarbonyl-0-penzyl-L-seryl-D-isoglutamine benzyl ester Dissolve 2.0 g of t-butoxycarbonyl-0-benzyl-L-serine in 40 g of tetrahydrofuran, and cool with an ice lamp. Add 0.78 g of N-hydroxysuccinimide and 1.39 g of dicyclohexylcarbodiimide.

Next, a mixture of 1.84 g of D-isoglutamine benzyl ester hydrochloride and 0.95 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. Triethylamine hydrochloride and dicyclohexylurea are removed. The oak liquor is concentrated under reduced pressure, and the residue is dissolved in acetic acid. The organic layer was reduced to 0. state hydrochloric acid,
Wash sequentially with a solution of water, 5% sodium bicarbonate, and water. After drying the acetic acid layer over anhydrous sodium sulfate, the solution is evaporated. The residue was recrystallized from acetic acid and petroleum ether to obtain 3.13% of t-butoxyalbonyl-0-penzyl-L-seryl-D-isoglutamine benzyl ester. Melting point 77-78qo. [Q] Color 55. is (C=,.stop methanol). Elemental analysis value C27th Calculated value (%) as 3507N3 C63.1 Arrival date 6.80N8.18 Analysis value (%) C63.3 Day 6.93N8.2 Example 1
1.0 parts of penzyl N-acetyl-Q muramid was dissolved in 10 parts of tetrahydrofuran, 0.8 parts of diphenyldiazomethane was added thereto, and the mixture was heated 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, and 1
．． 1-Q-0-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
55-156°C. [Q] Customer 112〆 (C = 1.0 Ku. Roholm). Calculated value (%) as elemental analysis value C3, day 3508N C67.74 day 642, N2.55 analysis value (%)
) C67.62, 65 LiN2.521-Q-0-benzyl-N-acetyl muramic acid diphenylmethyl ester 0.3 ml was dissolved in 3 parts of pyridine, and to this was added 1.3 ml of toluenesulfonyl chloride under ice cooling. Add 2 evenings.

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. The solvent is removed under reduced pressure and the residue is purified by silica gel column chromatography. When the solvent is completely distilled off from the benzene-ethyl acetate eluate, 0. 1-Q-0-benzyl-6-tosy-N-acetylmuramic acid diphenylmethyl ester (N) of milk powder is obtained with a total melting point of ~7%. [Q] Color 2 = Hammer.
4o (C=0.5, chloroform). Elemental analysis value C3
Calculated value (%) as 8th 4,0,oNS C rat. 8th day 5.87, NI. 99S4.56 Analysis value (%) C rat.
8 entry into Japan 592, NI. 93S4.31 Potassium mycolate 0. Madarayu (W)0. Spot and 18-Crown 6 0
．． The solution was added to a solution of benzene l0wZ and refluxed for 3 hours.

Remove the solvent under reduced pressure and wash the cash register with acetone. The insoluble material is subjected to silica gel column chromatography. The benzene-ethyl acetate (10:1) effluent section 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 eluate of benzene monoethyl acetate (10:1) was collected. After evaporation of the solvent, the residue was recrystallized from acetone to obtain 1-Q-0-benzyl-6-10-mycomycoloyl-N-acetylmuramic acid diphenylmethyl ester (V) of 0.32%. Nezumi point payment~
57℃. [Q] Customer +32. Pi (C=0.5 chloroform). Elemental analysis value C,. Calculated values as H, m○, and 5N (
%) C7807, HII. 27, NO. 82 analysis value (
%) C783 HII. 7iNO. 851-O-0-benzyl-6-0-mycomycoloyl-N-acetylmuramic acid diphenylmethyl ester (Va) was added to a solution of 0.5 ml of anisole and 0.2 M of anisole dissolved in 20 ml of dichloromethane under ice-cooling. Add 3 drops of fluoroacetic acid and light for 3 minutes.

After evaporating the solvent under reduced pressure, the residue was subjected to silica gel chromatography, and after removing anisole and diphenylmethanol with benzene-acetic acid (5:1), the chloroform-methanol (5:1) eluted fraction was collected. When the solvent is removed under reduced pressure, 1-Q-0-penzyl-610-micomicoloyl-N-acetylmuramic acid (Wa) is obtained. To this was added a mixture of 0.15 parts of 0-benzy-L-seryl-D-isoglutamine benzyl ester hydrochloride and 0.05 parts of triethylamine in 10 parts of tetrahydrofuran, and then cooled in a refrigerator at -10°C. Add 50 parts of N-hydroxysuccinimide and 6 parts of hexylcarposiimide and leave to stand for 1 hour.

After further incubation overnight at room temperature, insoluble triethylamine hydrochloride and dicyclohexyl urea were removed. The solvent was removed under reduced pressure and the resulting residue was subjected to silica gel chromatography. The solvent of the chloroform-methanol (30:1) elution fraction was distilled off under reduced pressure, and the residue was recrystallized from benzene-methanol. Ru N - Acetylmuramyl Ru 0 -
Penzyl-L-seryl-D-isoglutamine benzyl ester (skin a) is obtained. Melting point 1 to 16 is 0. [o
] 85132.10 (C=0.5, chloroform). Elemental analysis value C side daily 60,4. Calculated value (%) assuming 4
C74.39HIO. 7 tons N2.89 analysis value (%) C
74.41, HIO. 59N2.870.2 Evening (Wa
) was dissolved in 20% of tetrahydrofuran and subjected to a 20% hydrolysis reaction in the presence of palladium black.

The residue obtained by removing the solvent under reduced pressure was recrystallized from tetrahydrofuran-methanol to obtain the desired compound 6 at 0.14 methanol.
10-Mycomycoloyl-N-acetylmuramyl-L-seryl-D-isoglutamine (la) was obtained. Melting point 114
~120qo (decomp). [Q]. 135. 〆(
C=1.0 tetrahydrofuran-water (50:1), 4
after a short time). Calculated value (%) as elemental analysis value C9 Rainbow, Europe○, 4.5N4 C71.3 entry into Japan 11.39N3.36
Analysis value (%) C71.03HII. 33N3.42 Example 2 1-Q-0 manufactured in the same manner as the previous stage of Example 1
-Pendyl-6-0-nocardomicoloyl-N-acetylmuramic acid diphenylmethylester (Vb) 0.5
To a solution of 2 and 0.2 of anisole dissolved in 20 parts of dichloromethane, add 3' of trifluoroacetic acid under ice cooling.
Perform rope worship for 0 minutes.

The residue obtained by distilling off the solvent under reduced pressure was subjected to silica gel chromatography, and anisole and diphenylmethanol were eluted with benzene-acetic acid (5:1), and the chloroform-methanol (5:1) solvent fraction was collected. When the solvent is distilled off under reduced pressure, 1-Q-0-penzyl-610-nocaldomicoloyl-N-acetylmuramic acid (Wb) is obtained. To this, 0-benzyl-L-○-isoglutamine benzyl ester hydrochloride 0.2 and triethylamine 0.0
A solution prepared by mixing 10% of tetrahydrofuran with 10% of tetrahydrofuran was added, and then cooled in an 11mm bathtub, 66% of N-hydroxysuccinimide and 91M of dicyclohexylcarbodiimide were added, and the mixture was heated for 1 hour. do.

After further incubation at room temperature overnight, insoluble triethylamiso hydrochloride and dicyclohexylurea were removed. The residue obtained by distilling off the solvent under pressure is subjected to silica gel chromatography. The solvent of the chloroform-methanol (30:1) elution fraction was removed under reduced pressure, and the residue was recrystallized from methanol-acetone-water. 11Q-0-Benzy-6-10 Nocaldomicoloyl-N-acetylmuramyl-0-Benzy-L-seryl-D-isoglutamine benzyl ester (skin b) is obtained. Melting point 167-16
9℃. [Q] 85140. H (C=1.0 chloroform). Calculated value as elemental analysis value C9, day, illusion, 48N4 (
%) C71.4 & day 9.50N367 analysis value (%)
C71.69 day 9.32, N3.510.29 (skin b
) is dissolved in 20% of tetrahydrolane and subjected to a hydrolysis reaction in the presence of palladium black.

The solvent was removed under reduced pressure, and the resulting residue was recrystallized from methanol-acetone-water to obtain 6-0-nocardicoloyl-N-acetylmuramyl-L-seryl-D-isoglutamine. Melting point 125-130 qo (decomposed). [Q] 85-32.20 (C=1.u tetrahydrofuran-water (50:1), after 4 anchor hours). Calculated value (%) as elemental analysis value C7 Rainbow, 20, 4bu N4・Gender○ C
658fuHIO. 21, N4.39 analysis value (%) C6
5.62, HIO. 33N4. False Example 3 1-Q-0-Benzylene L610-Arthromycoloyl-N-acetylmuramic acid diphenyl ester (Vc) 332 and anisole 1. $M
To a solution dissolved in 130 parts of dichloromethane, 20 parts of trifluoroacetic acid is added under ice-cooling, and the mixture is incubated for 30 minutes.

Claims (1)

[Claims] 1 Formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, Y is a mycolic acid residue, Ser is serine, i
soGln means isoglutamine. ) N-acylmuramyl dipeptide derivative represented by: 2
6-0-mycomycholoyl-N-acetylmuramyl-L
The compound according to claim 1, which is -seryl-D-isoglutamine. 3. The compound according to claim 1, which is 6-0-nocardomicoloyl-N-acetylmuramyl-L-seryl-D-isoglutamine. 4. The compound according to claim 1, which is 6-0-arthromycoloyl-N-acetylmuramyl-L-seryl-D-isogutamine.