CH619469A5 - Process for the preparation of the esters of N-acetyl-muramyl-L-alanyl-D-glutamic acid and of N-acetyl-muramyl-L-alanyl-D-isoglutamine - Google Patents

Abstract

These compounds which correspond to the formula I where R1 = -NH2 or -OCnH2n+1 (n = 1, 2 or 3), R2 = -OCpH2p+1 and p = 0, 1, 2 or 3, p NOTEQUAL 0 when R1 = -NH2, are prepared by peptide linkage starting with the two amino acids or even the dipeptide L-Ala-D-Glu or L-Ala-D-iso-Gln. The compounds obtained are useful as anti-infectious agents or immune adjuvants. <IMAGE>

Description

The invention relates to the preparation of monoesters in position a, 40 or of methyl, ethyl and propyl diesters of 2- (2-acetamido-2-deoxy-3-0-D-glucopyrannosyl) -D-propionyl-L -alanyl-D-glutamic or methyl, ethyl and propyl esters of 2- (2-acetamido-2-deoxy-3-0-D-glucopyran-nosyl) -D-propionyl-L-alanyl-D-isoglutamine ; these compounds are soluble in water, effective as immunological adjuvants to promote immune responses, or even as anti-infective agents.

These compounds correspond to the following general developed formula:

nh - coch.

h-c - ch - co - nh - ch - co

3 I

CHn

(D

- nh - ch - horn,

(ch2) 2

horn

2

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in which Ri is - 0CnH2n +1 or - NH2, with n = 1, 2 or 3, R2 is - OCpH2p + 1, with p = 0, 1, 2 or 3, p being not equal to 0 when Ri is - NH2.

A preferred group of compounds to be obtained according to the invention consists of the mono- (in position a) or dimethyl esters of 2- (2-acetamido-2-deoxy-3-0-D-glucopyrannosyl) -D acid -propionyl-L-alanyl-D-glutamic acid, and 2- (2-acetamido-2-deoxy-3-0-D-glucopyrannosyl) -D-propionyl-L-alanyl-D-iso-glutamine, c ' that is, the compounds for which, in the formula (I) indicated above, Ri is - OCnH2n + i with N = 1 and p = 0 or 1, or Ri is —NH2etp = 1.

We will designate these, below, respectively by the abbreviations:

Mur-NAc-L-Ala-D-Glu-a-OCH3, Mur-NAc-L-Ala-D-Glu (OCH3) 2, Mur-NAc-L-Ala-D-iso-Gln (OCH3).

The methods according to the invention are defined in claims 1 and 2.

To prepare the compounds of formula I, it is therefore possible, firstly, to synthesize an adequate derivative of the fragment corresponding to the peptide chain and to that of the fragment designated by the abbreviation Mur-NAc, the functional groups not having to react being protected beforehand, then, in a second step, the two derivatives of these fragments are coupled. Protective groups are finally eliminated, freeing up previously blocked functions.

It is also possible to carry out the synthesis of these compounds by carrying out the separate coupling of a derivative of Wall-NAc with a derivative of L-alanine, then coupling the resulting product with the appropriate derivative of glutamic acid or iso-glutamine, according to the methods generally used in peptide synthesis.

The drug compositions containing said agents serve in particular to reinforce the action of weak immunogeies or else for the treatment of infectious diseases. The compositions containing said agent can be used for immunization or for the treatment of humans and warm-blooded animals to prevent or cure bacterial, viral and parasitic infections, or to combat various tissue antigens of normal or pathological origin .

One of the advantages of the new products lies in the fact that it is not necessary to have recourse to particular administration media on which the manifestation of their pharmacological activity, in particular the adjuvant action, and the excipients to which is led to associate them only for the purpose of facilitating the use of these products. In particular, it is not necessary, when these products are injected, to use for this purpose a composition containing an oily phase.

In addition, these compounds, whether used for their adjuvant action or their anti-infectious action, can be administered orally or parenterally, and in particular by injection.

The state of the art is illustrated by French patents NK 2248025 and 2292486 which describe muramyl derivatives useful as an adjuvant of immunity.

However, unexpectedly, it has been found that the compounds of formula I with adjuvant property also have anti-infectious properties, which does not appear anywhere in the previous publications.

A product obtained according to the invention can be used, in particular Mur-NAc-L-Ala-D-Glu-a-OCH3, Mur-NAc-L-Ala-D-G1u (OCH3) 2 or Mur-NAc -L-Ala-D-iso-Gln (OCH3), in combination with a pharmaceutically acceptable vehicle. Particularly preferred compositions of this type consist of injectable solutions containing an effective dose of a product according to the invention. Advantageously, sterile solutions in an aqueous phase, preferably isotonic, are used for this purpose, such as isotonic saline or gluco-

sées. This is of course not limiting; you can also use a simple solution in distilled water.

These adjuvanting medicinal compositions can be presented also in various forms, using for this purpose excipients suitable for the chosen mode of administration. For example, compositions in the form of cachets, tablets or capsules will be used for oral administration, aerosols or gels for application to the mucous membranes.

To allow the extemporaneous preparation of adjuvant medicinal compositions, the adjuvant agent can also be in lyophilized form.

An advantageous pharmaceutical form comprises unit doses of approximately 100 to 800 μg of the adjuvant product of formula I and preferably of approximately 400 g.

The compounds of formula I can be combined with an immunogenic agent, in particular with a weak vaccinating antigen.

According to another aspect, the products of formula I and in particular the Mur-NAc-L-Ala-D-Glu-a-OCH3, the Mur-NAc-L-Ala-D-Glu (OCH3) 2 or the Mur-NAc -L-Ala-D-iso-Gln (OCH) 3, are useful as anti-infective agents. These products have indeed shown, when administered alone, that is to say without vaccinating composition, in particular without weak immunogenic agent, that they manifested anti-infectious properties of preventive or even curative type. In other words, the anti-infectious properties are noted when these products are administered at the same time as the contamination is carried out, or even after it.

These anti-infectious properties are quite unexpected, given what was previously known about the activity of compounds capable of increasing the resistance of the host.

Thus, we know that we can increase non-specific resistance to infection by previously injecting various immunostimulants of bacterial origin, such as certain strains of Corynebacterium, mycobacteria and their cord factor, or lipopolyosides (LPS) extracted from bacteria. Gram negative. However, this protection only manifests itself if certain time intervals are observed between the administration of these immunostimulatory agents and the time of contamination. It has thus been shown experimentally that the best percentages of survival in mice infected with Klebsiella are observed when the administration of the immunostimulant is carried out approximately 14 days before for BCG, approximately 7 days before for corynebacteria, 6 to 48 hours before for the LPS. In all cases, immunostimulation with these agents must precede infection. It is well known for example that, if LPS is injected at the same time as or after the bacterial inoculum, it produces a negative reaction which tends to decrease the resistance of the host which can succumb after the administration of a bacterial strain even little virulent. On the other hand, administered under good conditions, these treatments considerably stimulate non-specific immunity even with regard to strains made resistant to antibiotics by mutation or by transfer of plasmids. However, it is difficult or even impossible to use these treatments because of the side effects observed after the administration of large doses of corynebacteria or BCG, and especially because of the toxic effect, in humans, of LPS which represents the toxic antigen of Gram negative bacteria.

Based on the results obtained with the aid of adjuvants of bacterial origin, and in particular the experiments made with LPS, it was therefore quite surprising to find that the synthetic adjuvants indicated above exhibited, in addition to their adjuvant properties implemented within the framework of preventive immune treatments, an anti-infectious activity which manifests itself in a preventive, or even curative, manner without being associated with vaccinating antigens.

These products are still devoid of mitogenic activity (absence of blastic transformation of lymphocytes). They are not

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not antigenic; in fact, they do not trigger a delayed sensitivity reaction in guinea pigs sensitized beforehand with the aid of Freund's complete adjuvant. They have no hyper-thermizing action in rabbits for doses much higher than those for which their anti-infectious action is manifested. They are negative on the Limulus test and their injection does not cause the death of adrenalectomized mice while they are made extremely sensitive to the lethal effect of endotoxins by this operation. These results show that these compounds are completely devoid of endotoxic nature. They have the advantage of being active as anti-infective agents in the absence of an oily phase, whether the administration is done parenterally or orally, while adjuvants such as LPS are only active. than parenterally.

An important advantage of the anti-infectious use of the compounds designated above is the possibility of combating pathogenic germs which have become antibiotic-resistant after treatment with traditional antibiotic means.

It should also be noted that the curative anti-infectious activity of these compounds is all the more remarkable and unexpected as the experiments show that they are devoid of bactericidal or bacteriostatic activity in vitro.

As for the compositions intended to favor the immune responses, the medicinal compositions containing the abovementioned agents, and used in anti-infectious therapy, can take very varied forms, since the properties of the product are manifested, that the administration takes place orally or parenterally. The drug compositions may in particular be in the form of injectable solutions (in particular in the form of isotonic aqueous solutions), oral solutions, cachets, capsules, aerosols, gels, etc.

Other characteristics will appear during the description of the examples of preparation of products, as well as tests highlighting the pharmacological properties of these products.

In the remainder of this presentation, the abbreviations used have the following meanings:

Wall-NAc

2-acetamido-2-deoxy-3-0- (D-2-propionyl) -D-gluco-

pyrannosis

Ala alanine

Glu glutamic acid iso-Gin isoglutamine

4,6-O-bzi

4,6-O-benzylidene

ß-bzl

ß-benzyl

BOC

t-butyloxycarbonyl

OBzl benzyl ester

OSu succinimide ester

Bzl

benzyl ether

Example of synthesis of the methyl a-ester of 2- (2-acetamido-2-deoxy-3-0-D-glucopyranose) -D-propionyl-L-alanyl-D-glutamic a) a-Benzyl ester of t -butyloxycarbonyl-L-alanyl-D-glutamic (I)

4 g (14 mmol) of the succinimide ester of BOC-L-alanine, prepared as described by Schnabel E. [“Justus Liebig's Ann. Chem. ”, 702, 188 (1967)], are dissolved in 15 ml of tetrahydrofuran. This solution is added to an aqueous solution (25 ml) of 3.3 g (14 mmol) of the y-benzyl ester of D-glutamic acid, obtained by following the method of Guttmann S. and Boissenas RA [« Helv. Chim. Acta ”, 41.1864 (1958)], and 1.4 g (14 mmol) of potassium bicarbonate. After one night, the pH is adjusted to 8.5 and the reaction mixture is extracted with ethyl acetate. The aqueous phase is acidified, cold, to pH 3.5 with a 4N hydrochloric acid solution, then it is extracted with ethyl acetate. The organic phase is then washed with water, dried and concentrated. The product is crystallized from an ethyl acetate / petroleum ether mixture. 4.77 g of product are obtained,

or a yield of 87.8%. Its physical constants are: m.p. 68-70 ° C.

[oc] d25 = —12 ° (methanol).

Analysis for C20H28O7N2 (408.45):

Calculated: C 58.81 H 6.9 N 6.85%

Found: C58.7 H 6.4 N6.8%

b) Ci-methyl ester, benzyl y-ester of t-butyloxycarbonyl-L-alanyl-D-glutamic acid (II)

408 mg of (I) (1 mmol) are dissolved in 100 ml of anhydrous methanol. An ethereal solution of diazomethane (approximately 10 mmol) is added over 15 min at 0 ° C. After 2 h at ordinary temperature, the reaction mixture is concentrated to dryness and taken up in 25 ml of ethyl acetate. The organic phase is washed successively with a 10% citric acid solution, with water, with a 1M solution of sodium bicarbonate, and with water until neutral pH. The ethyl acetate phase is dried over MgSO4, filtered and concentrated. An uncrystallizable oil is obtained (385 mg, ie a yield of 91%).

c) Hydrochloride of methyl a-ester, benzyl y-ester of L-alanyl-D-glutamic acid (III)

385 mg of (II) (0.91 mmol) are treated with 3 ml of a solution N of hydrochloric acid in glacial acetic acid for 30 min. The reaction mixture is concentrated to dryness and the oil obtained dried (330 mg, ie a yield of 100%).

d) a-Methyl ester, benzyl y-ester of 2- (benzyl-2-acetamido-4,6-0-benzylidene-2-deoxy-3-0-aD-glucopyrannosyl) -D-propionyl-L-alanyl- D-glutamic (IV)

473 mg (1 mmol) benzyl-2-acetamido-4,6-benzylidene-3-0- (D-carboxyethyl) -2-deoxy-aD-glucopyrannoside, prepared as described by Flowers and Jeanloz RW ["J . Org. Chem. ”, 28, 2983 (1963)], are dissolved in 5 ml of dimethylformamide cooled to -15 ° C. 0.11 ml (1 mmol) of N-methylmorpholine and 0.13 ml (1 mmol) of isobutyl chlorocarbonate are successively added.

To this reaction mixture is added a solution in 5 ml of dimethylformamide, previously cooled to -15 ° C, of 330 mg (0.9 mmol) of (III) and 0.1 ml (0.9 mmol) of N-methylmorpholine .

After overnight at -15 ° C, 1 ml of a 2.5N potassium bicarbonate solution is added. After 30 min, the product is precipitated by adding 40 ml of distilled water, filtered and dried. 667 mg of product are obtained, ie a yield of 95.5%.

e) a-2- (2-acetamido-2-deoxy-3-0-D-gluco-pyrannose) -D-propionyl-L-alanyl-D-glutamic methyl ester (V)

305 mg of (IV) (0.39 mmol) are hydrogenated for 15 h in solution in 50 ml of glacial acetic acid, in the presence of 300 mg of 5% palladium on carbon. After filtration of the catalyst, then concentration of acetic acid to dryness, the product is precipitated in methanol / acetone / ether, then centrifuged. 140 mg is obtained, ie a yield of 70%. The product is purified by chromatography on a column (2 x 10 cm) filled with an ion exchange resin, sold under the name AG1X-2 by the company Biorad (acetate form). It is eluted with a 0.2M solution of acetic acid, the interesting fractions are combined and lyophilized. 117 mg are recovered, ie a yield of 83.5%, of a product the rotary power of which is [oc] d25 = + 39 ° (methanol). The product is finally obtained after passage through a column (2 x 80 cm), filled with ion exchanger marketed under the name of Sephadex G.15 by the company Pharmacia Upsala (elution with 0.2M acetic acid) and lyophilization of the interesting fractions. Finally, 94 mg of product are obtained.

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(V), ie a yield of 80%. The rotary power remains [ol] d25 = + 39 ° (methanol).

Analysis for C20H33O12N3, IH2O (525.50):

Calculated: C45.7 H 6.7 N7.99%

Found: C 44.93 H 6.32 N7.91%

Example of synthesis of the methyl diester of 2- (2-acetamido-2-deoxy-3-0-D-glucopyranose) -D-propionyl-L-alanyl-D-glutamic acid

First, we prepare the Mur-NAc-L-Ala-D-Glu as follows:

a) Preparation of the benzyl diester of BOC-L-alanyl-D-glutamic acid (A)

2.3 g (8 mmol) of succinimide ester of t-butyloxy-carbonyl-L-alaline, the amine function of which is protected by the t-butyloxycarbonyl group (BOC-L-Ala-OSu), are added with stirring to a solution in dimethylformamide of 4.5 g (9 mmol) of p-toluenesulfonate of the benzyl diester of D-glutamic acid and 1 ml (9 mmol) of N-methylmorpholine. The reaction mixture is left at room temperature for 12 h. It is then concentrated to dryness. The dry compound is taken up in 50 ml of ethyl acetate and washed successively with a 10% citric acid solution, with water, with an IN sodium bicarbonate solution, finally with water. The ethyl acetate phase is dried over MgSC> 4, filtered and concentrated. By crystallizing from an ethyl acetate / hexane mixture, 2.50 g (67.5%) of the desired product is obtained, the physical constants of which are: mp 105-106 ° C.

aD25 = + 7.3 °.

Analysis for C27H34O7N2 (498.5):

Calculated: C65 H 6.9 N5.6%

Found: C 64.85 H 7.0 N5.5%

b) Preparation of the benzylic diester of 2- (benzyl-2-acetamido-4,6 benzylidene-2-deoxy-3-0-D-glucopyrannosyl) -D-propionyl- (0-benzyl) -L-alanyl- acid D-glutamic (B)

500 mg (1 mmol) of compound (A) are treated with 5 ml of a solution of IN hydrochloric acid in glacial acetic acid. After 30 min, the reaction mixture is concentrated to dryness. The oil obtained is taken up in 25 ml * of an acetonitrile / dimethylformamide mixture (2/1, v / v). The mixture is cooled to 0 ° C and 0.141 ml (1 mmol) of triethylamine is added. The prepared solution is poured with stirring at 0 ° C into a suspension prepared for 1.5 h and formed of 472 mg (1 mmol) of benzyl-2acetamido-4,6-0-benzylidene-3-0- (DI-carboxyethyl ) -2-deoxy-PD-glucopyrannoside and 0.141 ml (1 mmol) of triethylamine in 25 ml of the acetonitrile / dimethylformamide mixture (2/1, v / v).

The mixture is left for 12 h at room temperature; it is then concentrated and the residue is precipitated in a 10% citric acid solution. The precipitate is filtered, washed with copious amounts of water and dried. 800 mg (94%) of the desired product are obtained, the constants of which are:

M.p. 198-199 ° C.

ocd25 = + 4.92 ° (dimethylformamide). •

After recrystallization from ethanol, the melting point is established at 220 ° C.

Analysis for C47H53O12N3 (851.96):

Calculated: C 66.26 H 6.27 N 4.93%

Found: C 66.34 H 6.45 N 4.92%

c) Preparation of 2- (2-acetamido-2-deoxy-3-0-D-gluco-pyrannosyl) -D-propionyl-L-alanyl-D-glutamic acid (C) or Mur-NAc-L -Ala-D-Glu

700 mg (0.8 mmol) of compound (B) are treated with 40 ml of a 60% acetic acid solution in a boiling water bath for 1 h. The reaction mixture is then concentrated to dryness (then dried over MgSO4). The residue is taken up in 1 ml of a chloroform / methanol mixture (3/3, v / v) and placed on a silica column (35 g) previously equilibrated with the same mixture of solvent. The product-containing fractions are collected and concentrated to dryness (their homogeneity is tested by thin layer chromatography on silica gel in the same mixture of solvents). 185 mg (30%) of derivative are obtained.

76 mg of this derivative are dissolved in 15 ml of glacial acetic acid, then subjected to hydrogenation in the presence of 5% palladium on carbon. After filtration, the mixture is concentrated to dryness and precipitated in a methanol / acetone / ether mixture. 45 mg (92%) of the desired product are thus obtained, the constants of which are:

M.p. 150-155 ° C.

ocD25 = + 33 ° (glacial acetic acid).

Analysis for Ci9H3i0i2N3lH20 (511.48):

Calculated: C44.6 H 8.2 N6.5%

Found: C44.7 H 8.1 N6.4%

In a second step, the Mur-NAc-L-Ala-D-Glu previously prepared is esterified.

100 mg (0.2 mmol) of Mur-NAc-L-Ala-D-Glu are dissolved in 10 ml of absolute methanol. In 15 min, 10 ml of an ethereal solution of diazomethane (approximately 0.7 mmol / ml) are added. After 90 min, a drop of acetic acid is added and the reaction mixture is concentrated to dryness. The residue obtained is purified on a silica gel column (1 x 16 cm), with the chloroform / methanol mixture (6/2, v / v) as solvent. The pure fractions are combined and concentrated. The product is precipitated from a methanol / acetone / ether mixture. 83 mg of product are obtained (yield 80%)

whose constants are:

M.p. 137-142 ° C.

otD25 = + 31.6 ° (glacial acetic acid).

Analysis for C21H35O12N3 (521.53):

Calculated: C 48.36 H 6.76 N 8.57%

Found: C48.0 H 7.0 N8.2%

Example of synthesis of the methyl ester of 2- (2-acetamido-2-deoxy-3-O-D-glucopyranose) -D-propionyl-L-alanyl-D-isoglutamine

For the preparation of this product, use is made of Mur-NAc-L-Ala-D-iso-Gln obtained in the manner described in French patent application No. 74.22909.

100 mg (0.2 mmol) of Mur-NAc-L-Ala-D-iso-Gln are treated as described above for the esterification of Mur-NAc-L-Ala-D-Glu. The purification is carried out using the chloroform / methanol mixture (5/5, v / v). 80 mg of product are obtained (80% yield) the constants of which are:

P.F. 20PC.

<xd25 = + 44.2 ° (glacial acetic acid).

Analysis for C20H34O11N4 (506.52):

Calculated: C 47.52 H 6.76 NI 1.06%

Found: C47 H 6.5 N10.3% '

Pharmacological properties 1. Toxicity

The toxicity of the products of formula I was studied by parenteral administration on mice and rabbits. It has been found that the toxic doses are of an order of magnitude much higher than that of the doses at which these products manifest their activity. Thus, these products are well tolerated in mice at doses equal to or greater than 100 mg / kg of animal, and in rabbits for doses equal to or greater than 5 mg / kg of animal.

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2. Adjuvant nature of Mur-NAc-L-Ala-D-Glu- <x-0Cff3, Mur-NAc-L-Ala-D-Glu (OCHj) 2 and Mur-NAc-L-Ala-D- iso-Gln (OCH3) in aqueous phase

In the series of tests, the results of which are given below, the influence of the active principle of formula I on the level of anti-albumin antibodies was studied under the following conditions.

Groups of 8 two-month-old Swiss mice receive, by subcutaneous (SC) or oral (PO) injection, 0.5 mg of antigen consisting of bovine serum albumin (BSA) with or without the substance tested in isotonic saline. This high dose of antigen, because it is at the limit of the paralyzing dose vis-à-vis the immune response, therefore results in a weak or no response to the antigen alone in the controls; it therefore constitutes a severe criterion for demonstrating the activity of an adjuvant substance. 30 d later, the mice receive, by the same route of administration, a booster containing 0.1 mg of the same antigen.

The level of antibody is determined by passive haemagglutination using red blood cells treated with formalin and coated with the antigen studied according to the method described by A.A. Hirata and M. W. Brandiss [«J. Immunol. ”, 100, 641-648 5 (1968)]. The samples are taken 14, 28, 34 and 36 days after the first injection.

By way of comparison, mice receive, in place of the compounds of formula I, either lipopolysaccharides (LPS) 10 (extract of S. enteritidis by the water / phenol method), or the adjuvant designated under the name WSA and described by Adam et al. ["Infect. Immun. " (1973), 7, 855-861], Control mice receive only the antigen.

The results of these tests are given in Table 1. The antibody titers express the maximum serum dilution agglutinating a given quantity of sheep red cells.

Table 1

Witnesses (BSA) Administration Antibody titer

14th day 28th day 34th day 36th day

SC <3 3 3 20

BSA + LPS (100 | * g) SC <3 6 50 1310

BSA + WSA (300 ng) SC <3 <3 <3 6

BSA + Mur-NAc-L-Ala-D-Glu-oi-OCHa (100 | ig) SC 12 12 200 400

BSA + Mur-NAc-L-Ala-D-Glu-oc-OCH3 (10 (xg) SC 6 6 200 400

BSA + Mur-NAc-L-Ala-D-Glu-a-OCH3 (2000ng) PO 6 6 50 200

BSA + Mur-NAc-L-Ala-D-iso-Gln- (OCH3) SC 12 12 100 800

(100 ng)

BSA + Mur-NAc-L-Ala-D-Glu (OCH3) 2 (100 | ig) SC 50 50 400 1800

BSA dose: 0.5 mg / animal.

These results show that the products of formula I, administered in isotonic saline solution, cause a significant increase in the levels of antibodies formed, and this even in the case where the adjuvant is administered orally. The active ingredients according to the invention generate responses of the same nature as those obtained with LPS, but it should be noted that, unlike the latter, they are devoid of toxicity.

3. Adjuvant nature of Mur-NAc-L-Ala-D-Glu-a-OCHs, Mur-NAc-L-Ala-D-Glu (OCH3) 2 and Mur-NAc-L-Ala-D-iso -Gln (OCH3) in the presence of an oily phase

In these tests, the increase in the level of antibodies specific for a given antigen is followed when the latter is injected,

with or without the adjuvant compound of formula I, in a water in oil emulsion.

The tests are carried out on batches of 6 Hartley guinea pigs of 350 g. The administration is made by intradermal injection into the plantar pad of each of the hind legs. The ovalbumin (constituting the antigen) at a rate of 1 mg or 0.5 mg is prepared in 0.1 ml of an emulsion of saline isotonic solution, in an oily phase constituted either by the incomplete adjuvant of Freund (FIA ), or by the complete adjuvant (FCA) formed by the FIA to which is added 0.1 mg of whole cells of Mycobacterium smegmatis. The compound according to the invention is administered in an amount of 0.1 mg added to the emulsion containing the FIA.

18 d after this immunization, we look for possible delayed hypersensitivity reactions to the antigen by injecting intradermally 0.01 mg or 5 (ovalbumin ig on the flank of 45 animals, and we observe, 48 After the reaction at the injection site, the diameter in millimeters of the reaction thus caused is measured.

21 days after the injection, the animals are bled. On the serum collected, the specific antibody content of oval-so bumina is measured by precipitation of the antibody / antigen complex in the equivalence zone. The quantity of protein nitrogen contained in this precipitate is evaluated according to the Folin method. The average values of the antibody contents are indicated in the table of results. These values express the quantity, in micro-55 grams, of nitrogen precipitable by the antigen, per milliliter of serum. In some tests, the level of antibodies was also determined by passive hemagglutination (PHA) according to the method described above.

The results of these tests are reported in Table 2.

60 (Table at the top of the next page)

These results show that the compounds obtained according to the invention, administered in the presence of an oily phase, promote the increase in the level of antibodies formed, and that they induce a “delayed hypersensitivity reaction with respect to the same antigen.

4. Anti-infective character

The following tests illustrate the anti-infectious properties

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Table 2

Composition of the emulsion containing the antigen

Antibody titer

Skin test

- (diameter

Precipitation

Aggluti in mm)

(Hg / ml)

nation

Ovalbumin (1 mg) + FIA

<500

-

0

Ovalbumin (1 mg) + FCA (100 Hg)

3200

-

10 + 1.5

Ovalbumin (1 mg) + FIA + Mur-NAc-L-Ala-D-Glu-a-OCH3 (100 Hg)

2600

-

6 + 3

Ovalbumin (0.5 mg) + FIA

<500

900

0

Ovalbumin (0.5 mg) + FCA (100 Hg)

2100

3600

13.5

Ovalbumin (0.5 mg) + FIA + Mur-NAc-L-Ala-D-iso-Gln (OCHj) (100 Hg)

2100

3000

12

Ovalbumin 0.5 mg + FIA + Mur-NAc-L-Ala-D-Glu (OCH3) 2 (100 Hg)

950

2600

6.2

Mur-NAc-L-Ala-D-Glu-a-OCH3, Mur-NAc-L-Ala-D-Glu (OCH3) 2 and Mur-NAc-L-Ala-D-iso-Gln (OCH3 ).

In preliminary tests, an experimental mode was established making it possible to demonstrate the antiinfectious nature of the products. It has thus been shown that a dose of 104 Klebsiella pneumoniae, injected intramuscularly into mice, results in the progressive death of a significant proportion, if not all, of the animals in the week following inoculation. After 8 days, the survival of the animals is definitively acquired.

The survival of groups of mice inoculated under the conditions indicated above and treated with the compounds considered was followed.

For comparison, batches of mice were treated with BCG and LPS. The latter, as we know, is an extremely active immunostimulant when administered 24 hours before infection.

For these tests, hybrid mice (C57B1 / 6 x AKR) F1 used at the Pasteur Institute were used, from strains from the breeding of C.N.R.S. in Orleans. The endotoxin or LPS was extracted by the phenol / water method from Salmonella enteritidis variety Danysz (No. 5629 of the Pasteur Institute). BCG comes from the strain Mycobacterium tuberculosis var. bovis (N ° 1173 P2

from the Pasteur Institute), grown on Sauton medium and killed by a 2% phenol solution.

Infection with Klebsiella pneumoniae, strain of capsu-25 type 2, biotype d, is made from a culture of 16 h in medium for pneumococci (No. 53515, Institut Pasteur). The preparations injected before or at the time of infection are always diluted in pyrogen-free physiological solution, at a rate of 0.2 ml for parenteral administration and 0.5 ml for oral administration, the controls receiving the solution. only.

In the trials, the results of which are given in Tables 3 and 4, the influence of the treatment was determined by varying the modes, the doses and the time of administration of the product studied. The percentage of protection expresses the difference in the percentages of survivors in the group of animals treated compared to the corresponding control group.

The results show that the products studied exhibit anti-infective activity, whether administered parenterally or orally. LPS, on the other hand, is inactive by the oral route, even at very high doses (100 μg of LPS represents 10,000 times the anti-infectious dose by parenteral route).

In addition, the results are the same, whether the products are administered 24 h or only 1 h before the infective injection, by intramuscular route.

Table 3

. Anti-infectious protection against intramuscular inoculation of IO4 K. pneumoniae

Treatment 24 hours before infection

Mode of Number Number of animals% treatment of animals surviving on the protection day treated

Witnesses 24 12 8 2

LPS (Hg) 24 24 22 22 83

Witnesses 24 11 9 6

BCG (100 Hg) 24 24 24 21 63

Witnesses 24 15 11 7

IV Mur-NAc-L-Ala-D-Glu-a-OCHs (100 Hg) 24 24 24 23 67

Witnesses 24 13 9 4

Mur-NAc-L-Ala-D-Gln-OCH3 (100 jig) 24 21 13 11 29

Witnesses 24 13 9 4

Mur-NAc-L-Ala-D-Glu- (OCH3) 2 (100 Hg) 24 21 19 15 46

619,469

Table (continued)

Processing mode

Number of animals treated

Number of animals surviving to the day

% of protection per os

Witnesses 12 6

Mur-NAc-L-Ala-D-Glu-a-OCHî (2000 iig) 12 11

Witnesses 12 6

Mur-NAc-L-Ala-D-Glu- (OCH3) 2 (2000 Hg) 12 10

LPS controls (100 Hg)

24 24

14 13

4 9

10 10

40.5

50

Table 4

Anti-infectious protection against intramuscular inoculation of IO4 K. pneumoniae

Treatment 1 hour after infection

Mode of Number Number of animals% treatment of animals surviving on the protection day treated

Witnesses

Wall-NAc-L-Ala-D-Glu-a-OCH3 (100 Hg)

IV Witnesses

Wall-NAc-L-Ala-D-Glu- (OCH3) 2 (100 Hg)

BCG controls (100 Hg)

16 6 2 1

16 16 16 14 88

8 6 5 1

8 8 8 8 88

16 10 6 1

16 14 13 10 50

In another series of tests, the anti-inflammatory properties were studied. In these tests, also carried out on mice, the infectious products of formula I vis-à-vis an infection treatments are carried out 24 h before inoculation . The modes and the very violent ones caused by the intravenous injection of 103 K. tire- 40 doses are indicated in table 5 in which are reported the moniae. results of these tests.

Table 5

Anti-infectious protection against an intravenous inoculation of 103 K. pneumoniae Treatment 24 h before infection

Mode of Number Number of animals% treatment of animals surviving on the protection day treated

IV

Wall-NAc-L-Ala-D-Glu-a-OCH3 (100 Hg)

16

16

16

12

65

IV

Wall-NAc-L-Ala-D-Glu- (OCH3) 2 (100 Hg)

16

15

12

10

52.5

IV

LPS (1 Hg)

16

16

16

16

90

IV

BCG (100 Hg)

16

16

16

16

90

per os

BCG (2000 Hg)

16

3

0

The results show significant protection in the absence of oily phase, as well as agents for the treatment of mice treated with the products according to the invention. infectious diseases, devoid of toxicity, manageable

Thanks to the invention, a new agent is thus available, parenterally or orally, and active even for the adjuvant genes of immunity soluble in water and active even in antibiotic-resistant pathogens.

R

Claims (5)

619,469 2 . CLAIMS '1. Process for the preparation of 2- (2- CH20H acetamido-2-deoxy-3-0-D-glucopyrannosyl) -D-propionyl-L-alanyl-D-glutamic acid and 2- (2-acetamido-2-deoxy-3-0-D-glucopyrannosyl) -D -propionyl-L-alanyl-D-isoglutamine corresponding to the formula: HO H, OH NH - COCH, (i) h3C -CO-NH-CH-CO-NH-CH- COR L (CH2) 2 COR ^ 1 D in which Ri is - OCnH2n + 1, with n = 1, 2 or 3, or —NH2, R2 is —OCpH2p + i, with p = 0, 1, 2 or 3, p not being equal to 0 when Ri est - NH2, characterized in that a suitable derivative of N-acetylmuramic acid is coupled, the hydroxyl groups being blocked, via the carboxyl group, with the appropriate peptide chain, the groups of which possibly free reagents are blocked with the exception of the amine used for the coupling, then, this being carried out, the blocking groups are removed. 2. Process for the preparation of a compound of formula I, characterized in that a first coupling of acetylmuramic acid, whose hydroxyl groups are blocked, is carried out with L-alanine, the only free functional group of which is l amine used for coupling, which releases the terminal carboxyl function of the compound formed, which is carried out a second coupling with the chosen derivative of glutamic acid or iso-glutamine, and which is eliminated finally blocking groups. 3. Method according to claim 1, characterized in that the coupling is carried out by means of esters comprising reactive functional groups or by means of NN'-dicyclohexyl-carbodiimide. 4. Method according to claim 2, characterized in that the coupling is carried out by means of esters comprising reactive functional ch2oh hjoh groups or by means of NN'-dicyclohexylcarbodi-imide. 5. Method according to claim 1, characterized in that the peptide chain coupled to N-acetylmuramic acid is one of the following chains: L-alanyl-D-glutamic a-methylester, L-alanyl-D-glutamic dimethylester, L-alanyl-D-isoglutaminemethylester. 35