GB1573126A - Immunising and anti-infectious adjuvant agents comprising peptide derivatives of muramic acid - Google Patents

Description

(54) IMMUNISING AND ANTI-INFECTIOUS ADJUVANT AGENTS COMPRISING PEPTIDE DERIVATIVES OF MURAMIC ACID (71) We, AGENCE NATIONALE DE VALORISATION DE LA RECHERCHE (ANVAR), a French body corporate, of 13, rue Madeleine Michelis, 92522, Neuilly-Sur-Seine, France, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The invention relates to water-soluble agents which are effective as immunological adjuvants for promoting immunising responses, or also as anti-infectious agents.

The invention also relates to the medicinal compositions which contain these agents, as well as to the processes for their preparation.

The agents according to the invention are the methyl, ethyl and propyl mono-esters in the zz-position, or the methyl, ethyl and propyl diesters of 2 - (2 - acetamido - 2 deoxy - 3 - 0 - D - glucopyranosyl) - D - propionyl - L - alanyl - D - glutamic acid, or also the methyl, ethyl and propyl esters of 2 - (2 - acetamido - 2 - deoxy - 3 - 0 D - glucopyranosyl) - D - propionyl - L - alanyl - D - isoglutamine.

The compounds correspond to the general formula developed below:

in which R1 is --06H,,,, or -NH2, wherein n is 1, 2 or 3; R2 is OC H2p+" wherein p is 0, 1, 2 or 3, p being other than 0 when R1 is -NH2.

A preferred group of compounds according to the invention is constituted by the monomethyl (in the a-position) or dimethyl ester of the 2 - (2 - acetamido - 2 - deoxy 3 - O - D - glucopyranosyl) - D - propionyl - L - alanyl - D - glutamic acid and the methyl ester of the 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) - D propionyl - L - alanyl - D - isoglutamine, i.e. the compounds for which, in the general formula indicated above, R1 is OCH2n+1 with n=1 and p=O or 1, or R1 is -NH2 and p=1.

These compounds will subsequently be denoted by the abbreviations Mur- NAc-L-Ala-D-Glu-rr- OCHs Mur-NAc-L-Ala-D- Glu(OCH,), and Mur- NAc - L - Ala - D - iso - Gln(OCHs).

In order to prepare the compounds according to the invention, one can, for example, synthesise in a first stage, an equivalent derivative of the fragment corresponding to the peptide chain and to that of the fragment denoted by the abbreviation Mur-NAc, the functional groups which must not react being first protected, then in a second stage, the coupling of these two derivatives of these fragments is effected. The protecting groups are finally removed, liberating the functions which were previously blocked.

It is also possible for example to effect the synthesis of these compounds by effecting the separate coupling of a derivative of the Mur-NAc with a derivative of the L-alanine, then coupling the resulting product with the equivalent derivative of the glutamic acid or the isoglutamine, according to the processes generally used in peptide synthesis.

The invention also relates to the medicinal compositions containing the said agents and serving especially to increase the action of the weak immunising substances or also to the treatment of infectious diseases. More particularly, the invention concerns compositions containing the said agent which can be used for the immunisation or for the treatment of warm-blooded animals other than of the species homo sapiens so as to prevent or cure bacterial, viral and parasitic infections, or to fight against various organic tissue antigens of normal or pathological origin. The invention therefor includes a process which comprises treating a warm-blooded animal other than of the species homo sapiens with a compound of the general formula given above.

One of the interests of the new products according to the invention is in the fact that it is not necessary to use particular media for their administration on which the manifestation of their pharmacological activity would depend, especially the adjuvant action, and the vehicles-with which one is led to associate them have only the object 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.

Further, these compounds, which may be used for their adjuvant or anti-infectious action, may be administered orally or parenterally, and especially by injection. ~~~ The invention relates in particular to medicinal adjuvant compositions of immunity comprising a product of the invention, especially Mur - NAc - L - Ala - D - Glu - a OCH3, Mur - NAc - L - Ala - D - Glu - (OCH3)2 or Mur - NAc - L - Ala - D - iso- Gln(OCH3), in association with a pharmaceutically acceptable vehicle. Compositions of this type which are particularly preferred are constituted by injectable solutions containing the product of the invention. Sterile solutions in an aqueous, preferably isotonic, phase, such as saline isotonic solutions or isotonic solutions treated with glucose, are advantageously used for this purpose. This is of course not restrictive; a simple solution in distilled water can also be used.

The adjuvant medicinal compositions of the invention may also be presented in various forms, by using for this purpose vehicles suitable for the selected method of administration. For example, compositions will be used in the form of cachets, compressed tablets or gelatine-coated pills, for oral administration, and aerosols or gels for the application to mucous membranes.

The adjuvant agent may also be in lyophilised form so as to permit the extemporaneous preparation of the adjuvant medicinal compositions.

A pharmaceutically preferred form comprises unit doses of about 100 to 800 yg of the adjuvant product according to the invention, and more preferably of about 400 Xug.

The invention also includes medicinal compositions in which the products of the invention are associated with an immunising agent, especially a weak vaccinating antigen.

According to another aspect, the invention also concerns medicinal compositions containing the products of the invention and especially the Mur - NAc - L - Ala Glu - a - OCH3, the Mur - NAc - L - Ala - D - Glu(OCH3)2 or the Mur - NAc - L- Ala - D - iso - Gln(OCHss), and useful as anti-infectious agents. These products in fact, when they are administered alone, i.e. without vaccinating composition, especially without a weak immunising agent, have shown that they manifest anti-infectious properties of the preventive or even curative type. In other words, the anti-infectious properties are found when these products are administered at the same time that the contamination is accomplished, or even subsequently to this.

These anti-infectious properties are quite unexpected taking into account that one knew beforehand of the activity of the compounds capable of increasing the resistance of the host.

Thus, it is known that one can increase the non-specific resistance to an infection by previously injecting different immunostimulants of bacterial origin, such as certain strains of Corynebacterium, Mycobacteria and their "cord factor", or lipopolyosides (LPS) extracts of gram-negative bacteria. This protection is only manisted, however, on condition of respecting certain intervals of time between the administration of these immunostimulant agents and the moment of the contamination. Thus one has been able to show experimentally that the better percentages of survival in the case of mice infected with Klebsiella are observed when the administration of the immunostimulant is effected about 14 days beforehand for the BCG, about 7 days before for the Corynebacteria, 6 to 48 hours before for the LPS. In all these cases, the immunostimulation by means of these agents must precede the infection. For example, it is well known that, if the LPS is injected at the same time as the inoculum bacteria or after, it produces a "negative reaction" which has a tendency to diminish the resistance of the host who can succumb after the administration of a bacterial strain of even little virulence. On the other hand, when administered under good conditions, these treatments stimulate considerably the non-specific immunity even with regard to strains rendered resistant to antibiotics by mutation or by transfer of plasmides. However, it is difficult or even impossible to use these treatments on account of secondary effects observed after the administration of strong doses of Corynebacteria or of BCG, and above all on account of the toxic effect to man, of the LPS which represents the toxic antigen of the gram-negative bacteria.

On the basis of the results obtained by means of the adjuvant agents of bacterial origin, and especially the tests made with the LPS, it was therefore quite surprising to find that the above-mentioned synthetic adjuvants, according to the invention, show in addition to their adjuvant properties used within the compass of preventive immunising treatments, an anti-infectious activity which is manifested in a preventive, or even curative way, without them being associated with vaccine antigens. These products also have no mitogen activity (absence of blastic transformation of the lymphocytes).

They are not antigenic; in fact, they do not release any retarded sensibility reaction in the case of the guinea-pig previously sensitised by means of the Freund complete adjuvant. They have no hyperthermising action with the rabbit for doses much greater than those for which their anti-infectious action is shown. They are negative to the Limulus test and their injection does not cause the death of suprarenalectomised mice although these are rendered extremely sensitive to the lethal effect of the endotoxins by this operation. The Limulus test is described by R. J. Chic and S. M. Wolff in J. Infect. Dis. 1973, 128, 349. These results show that these compounds are completely deprived of endotoxic character. They have the advantage of being active considered as anti-infectious agents in the absence of an oily phase, whether the administration is made parenterally or orally, although the adjuvants such as the LPS are only active administered parenterally.

An important advantage of the anti-infectious use according to the invention of the compounds denoted above is the possibility of action against the pathogenic germs which have become resistant to antibiotics following treatments by the traditional antibiotic methods.

It must also be indicated that the anti-infectious curative activity of these compounds is all the more remarkable and unexpected since the tests show that they have no bactericidal or bacteriostatic activity in vitro.

As for the compositions intended to promote the immunising responses, the medicinal compositions containing the above-mentioned methyl esters, and used in anti-infectious therapeutics, can take very varied forms since the properties of the products are shown when the administration is oral or parenteral. The medicinal compositions in particular could be for example in the form of injectable solutions (especially in the form of isotonic aqueous solutions) drinkable solutions, cachets, gelatinecoated pills, aerosols or gels.

Other characteristics of the invention will appear during the description of examples of preparation of products according to the invention, as well as tests which reveal the pharmacological properties of these products.

In the course of this account, the abbreviations used have the following meanings: Mur-NAc: 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) D - ol propionic acid Ala: alanyl Glu: glutamic acid iso-Gln: isoglutamine BOC: t-butyloxycarbonyl OBzl: benzyl ester OSu: succinimide ester Bzl: benzyl ether Example of synthesis of 2 - (2 - actamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) D - propionyl - L- alanyl - D - glutamic acid a - methyl ester a) t - butyloxycarbonyl - L - alanyl - D - glutamic acid Y - benzyl ester (I) 4 g (14 mmoles) of the succinimide ester of BOC - L - alanine, prepared in the manner described by E. Schnabel (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 mmoles) of the 7-benzyl ester of D-glutamic acid, obtained by following the method of S. Gutanann and R. A. Boissenas (Helv. Chim. Acta, 41, 1864 (1958)), and 1,4 g (14 mmoles) 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 in the cold, to pH 3.5 with a 4 N solution of hydrochloric acid, then it is extracted with ethyl acetate. The organic phase is then washed with water, dried and concentrated. The product is crystallised from an ethyl acetate-petrol ether mixture 4,77 g of product are obtained, i.e. a yield of 87.8%. Its physical constants are: M.p.

68--700C [am22 - 120 (methanol).

The elementary analysis gives: C2oH2SO7NI (408.45) 0% H% N% calculated: ' 58.81 6.9 6.85 found : 58.7 6.4 6.8 b) t - butyloxycarbonyl - L - alanyl - D - glutamic acid a - methyl ester, ty - benzyl ester (II) 408 mg of (I) (1 mmole) are solubilised in 100 ml of anhydrous methanol. A solution of diazomethane (about 10 mmoles) in diethyl ether is added in 15 minutes at OOC. After 2 hours 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% by weight solution of citric acid, water, a 1 M solution of sodium bicarbonate, and water until a neutral pH is obtained. The ethyl acetate phase is dried over MgSO4, filtered and concentrated. An uncrystallisable oil is obtained (385 mg, i.e. a yield of 91%).

c) The hydrochloride of L - alanyl - D - glutamic acid a - methyl ester, y - benzyl ester (Ill) 385 mg of (I1) (0.91 mmole) are treated with 3 ml of an N solution of hydrochloric acid in glacial acetic acid for 30 minutes. The reaction mixture is concentrated to dryness and the oil obtained is dried (330 mg, i.e. a yield of 100%).

d) 2 - (benzyl - 2 - acetamido - 4,6 - 0 - benzytidene - 2 - deoxy - 3 - 0 - a glucopyranosyl) - D - propionyl - L - alanyl - D - glutamic acid a - methyl ester, y - benzyl ester (IV) 473 mg (1 mmole) of benzyl - 2 - acetamido - 4,6 - benzylidene - 3 - 0 - (Dcarboxyethyl) - 2 - deoxy - a - D - glucopyranoside, prepared in the way described by Flowers and R. W. Jeanloz (J. Org. Chem. 28, 2983 (t1963)), are dissolved in 5 ml of dimethylformamide cooled to - 1500. 0.11 ml (1 mmole) of N-methylmorpholine and 0.13 ml (1 mmole) of isobutyl chlorocarbonate are successively added.

To this reaction mixture is added a solution of 330 mg (0.09 mmole) of (III) and 0.1 ml (0.9 mmole) of N-methylmorpholine in 5 ml of dimethylformamide, previously cooled to - 1500.

After one night at --1S"C, 1 ml of 2.5 N solution of potassium bicarbonate is added. After 30 minutes, the product is precipitated by addition of 40 ml of distilled water, filtered off and dried. 667 mg of product are obtained, i.e. a yield of 95.5%.

e) 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) - D - propionyl alanyl - D - glutamic acid a - methyl ester (V) 305 mg of (IV) (0.39 mmole) are hydrogenated for 15 hours in solution in 50 ml of glacial acetic acid, in the presence of 300 mg of 5% by weight palladium on charcoal. After fi!tration of the catalyst, then concentration to dryness of the acetic acid, the product is precipitated from methanol-acetone-diethyl ether, then centrifuged.

140 mg are obtained, i.e. a yield of 70%. The product is purified by chromatography on a column (2X10 cm) filled with an ion-exchanger resin, commercialised under the name AG1X-2 by the BIORAD Company (acetate form). It is eluted with a 0.2 M solution of acetic acid, the fractions containing the product are united and lyophilised.

117 mg are recovered, i.e. a yield of 83.5%, of a product of which the rotatory power is [aj n25 = + 390 (methanol). The product is finally obtained after passage over a column (2X80 cm), filled with ion-exchanger sold under the Trade Mark SEPHA DEX G. 15 by PHARMACIA UPSALA (elution with acetic acid 0.2 M) and lyophilisation of the fractions of interest. At the end 94 mg of product (V) are obtained, i.e. a yield of 80%. The rotatory power remains at [rr]25=+39" (methanol), and the elementary analysis thereof is: C20H,3O12N,, lH2O (525.50) C% H% N% calculated: 45.7 6.7 7.99 found : 44.93 6.32 7.91 Example of synthesis of 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranose) D - propionyl - L-alanyl - D - glutamic acid dimethyl ester In a first stage, the Mur - NAc - L - Ala - D - Glu is prepared in the following way: a) Preparation of the benzyl diester of BOC - L - alanyl - D - glutamic acid (A) 2.3 g (8 mmoles) of succinimide ester of t - butyloxy - carbonyl - L - alanine, 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 cf 4.5 g (9 mmoles) of the p-toluinesulphonate of the benzyl diester of D-glutamic acid and 1 ml (9 mmoles) of N-methylmorpholine. The reaction mixture is left for 12 hours at the ambient temperature. It is then concentrated to dryness. The dry compound is taken up in 50 ml of ethyl acetate and washed successively with a 10% by weight solution of citric acid, water, with a solution of 1 N sodium bicarbonate, and finally with water.

The ethyl acetate phase is dried over MgSO4, filtered and concentrated. On crystallising from a mixture of ethyl acetate and n-hexane, 2.50 g (67.5%) are obtained of the desired product of which the physical constants are: M.P. 105-1060C.

[fr] 25= +7 3O The elementary analysis of this product is: C27H3lO7N2 (498.5) C% H% N% calculated: 65 6.9 5.6 found 64.85 7.0 5.5 b) Preparation of the benzyl diester of 2 - (benzyl - 2 - acetamido - 4,6 - 0 - benzyf- idene - 2 - deoxy - 3-0 - D - glucopyranosyl) - D - propionyl - L - alanyl glutamic acid (B) 500 mg (l mmole) of compound (A) are treated with 5 ml of a 1 N solution of hydrochloric acid in glacial acetic acid. After 30 minutes, the reaction mixture is concentrated to dryness. The oil obtained is taken up in 25 ml of an acetonitriledimethylformamide mixture (2:1, v/v). The mixture is cooled to o0C and 0.141 ml (1 mmole) of triethylamine is added. The solution prepared is poured with stirring at 0 C into a suspension prepared 1.5 hours before and formed from 472 mg (1 mmole) of benzyl - 2 - acetamido - 4,6 - 0 - benzylidene - 3 - 0 - (D - 1 - carboxyethyl) - 2-deoxy - - D - glucopyranoside and 0.141 ml (1 mmole) of triethylamine in 25 ml cf the acetonitrile-dimethylformamide mixture (2:1, v/v).

The mixture is left for 12 hours at the ambient temperature; it is then concenprated and the residue is precipitated in a 10% by weight solution of citric acid. The precipitate is filtered off, washed copiously with water and dried. 800 mg (94%) are obtained of the desired product the constants of which are: M.p. 198--199"C [uy],25=4.92" (dimethylformamide) After recrystallisation from ethanol, the melting point is established at 2200 C.

The elementary analysis of this product is: C4,Hs3Ol2N3 (851.96) C% H / , N% calculated: 66.26 6.27 4.93 found : 66.34 6.45 4.92 c) Preparation of the dimethyl ester of 2 - (2 - acetamido - 2 - deoxy - 3 - 0 glucopyranosyl) - D - propionyl - L - alanyl - D - glutamic acid (C) or Mur NAc - L - Ala - D - Glu(ocHs)s 700 mg (0.8 mmole) of the compound (B) are treated with 40 ml of a 60% by weight solution of acetic acid on a boiling water-bath for 1 hour. 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 (1:1, v/v) and placed on a silica column (35 g) previously equilibrated with the same solvent mixture. The fractions containing the product are collected and concentrated to dryness (their homogeneity is tested by chromatography on a thin layer of 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 a hydrogenation in the presence of 5% by weight palladium on charcoal.

After filtration, the mixture is concentrated to dryness and precipitated in a methanolacetone-diethyl ether mixture. 45 mg (92%) of the desired product are thus obtained, of which the constants are: M.p. 150-1550C [ ]B25=+33O (glacial acetic acid) The elementary analysis of this product is C1H,1O12N31H2O (511.48) C% H% N% calculated: 44.6 8.2 6.5 found 44.7 8.1 6.4 In the second stage, the previously prepared Mur - NAc - L - Ala - D - Glu is esterified.

100 mg (0.2 mmole) of Mur - NAc - L - Ala - D - Glu are dissolved in 10 ml of absolute methanol. In 15 minutes, 10 ml of a solution of diazomethane in diethyl ether (about 0.7 mmole per ml) are added. After 90 minutes, a drop of acetic acid is added and the reaction mixture is concentrated to dryness. The residue obtained is purified on a column of silica gel (1 X 16 cm), with for solvent the mixture chloroformmethanol (3:1, v/v). The pure fractions are united and concentrated. The product is precipitated from a methanol-acetone-diethyl ether mixture. 83 mg of product (yield 809) are obtained, of which the constants are: M.p. 137-1420C [a]D25=+31.6 (glacial acetic acid) The elementary analysis is as follows: C21H,O12N3 (521.53) C% H% N% calculated: 48.36 6.76 8.57 found 48.0 7.0 8.2 Example of synthesis of 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) D - propionyl - L - alanvl - D - isoglutamine methyl ester For the preparation of this product, the Mur - NAc - L - Ala - D - iso - Gln obtained in the way described in the U.K. Patent No. 1,496,332 is used.

100 mg (0.2 mmole) of Mur - NAc - L - Ala - D - iso - Gln are treated in the way previously described for the esterification of the Mur - NAc - L - Ala - D - Glu.

The purification is effected by means of the chloroform-methanol mixture (1:1, v/v).

80 mg of product are obtained (80% of yield) of which the constants are: M.p. 2010C [a]D25=444.2 (glacial acetic acid) The elementary analysis of this product is: C20H34OltN4 (506.52) C% H% N% calculated: 47.52 6.76 11.06 found 47 6.5 10.3 PHARMACOLOGICAL PROPERTIES 1) Toxicity The toxicity of the products according to the invention has been studied by parenteral administration to mice and rabbits. It was found that the toxic doses are of an order of magnitude much greater than that of the doses at which these products show their activity. Thus, these products are well tolerated by mice at doses equal to or greater than 100 mg/kg of animal, and by rabbits at doses equal to or greater than 5 mg/kg of animal.

2) Adjuvant character of the Mur - NAc - L - Ala - D - Glu - a - SOCH8, of the Mur - NAc - L - Ala - D - Glu(OCHs) and of the Mur - NAc - L - Ala iso - Gln(OCH,) in aqueous phase In the series of tests of which the results are indicated hereafter, the influence of the active principle according to the invention on the proportion of the anti-albumin antibody has been studied under the following conditions.

Groups of 8 Swiss mice aged two months receive by subcutaneous injection (SC) or orally (PO) 0.5 mg of antigen constituted by the bovine serum albumin (BSA) with or without the substance tested in an isotonic saline solution. This large dose of antigen, since it is situated at the limit of the paralysing dose with respect to the immunising response, on account of this fact has a weak response or no response to the antigen alone in the case of the controls; it therefore constitutes a severe criterion for showing the acivity of an adjuvant substance. Thirty days later, the mice receive, by the same method of administration, a further dose containing 0.1 mg of the same antigen.

The proportion of antibody is determined by passive hemagglutination by using the red blood corpuscles of sheep treated with formalin and recovered from the antigen studied according to the method described by A. A. Hirata and M. W. Brandiss (J.

Immunol., 100, 641-648, 1968). The taking of the blood occurred 14, 28, 34 and 36 days after the first injection.

By way of comparison, mice receive, instead of the product according to the invention, either lipopolysaccharides (LPS) (extract of S.Enteritidis by the waterphenol method), or the adjuvant denoted by the name "WSA" and described by Adam et al. [Infect. Immun. (1973) 7, 855-861]. The control mice only receive the antigen.

The results of these tests are given in the following Table. The proportions of antibody express the maximum serum dilution which agglutinates a given quantity of red blood corpuscles of sheep.

TABLE 1

Proportion of antibody 14th 78th 34th 36th Administration day day day day BSA controls S.C. < 3 3 3 30 BSA r LPS (100us) S.C. < 3 6 50 1310 BSA + WSA (300at) S.C. < 3 < 3 < 3 6 BSA + Mur-NAc-L-Ala-D-Glu-a-OCH, (100rug) S.C. 12 12 200 400 BSA s Mur-NAc-L-Al a-D-(3lu-a-OCH, (101lug) S.C. 6 6 200 400 BSA + Mur-NAc-L-Al a-D-Glu-a-OCH, (2000 g) P.O. .6 6 50 200 BSA + Mur-NAc-L-Al a-D-i so-Gln(OCH,) (100g) S.C. 1 12 12 100 800 BSA + Mur-NAc-L-Al a-D-Glu-(OCH3)2 (100crag) S.C. 50 50 400 1800 Dose BSA 0.5 mgi'animal.

These results show that the products of the invention, administered in isotonic saline solution, cause a large increase of the proportion of antibody formed, even in the case where the adjuvant is orally administered.

The active principles according to the invention engender responses which may be considered in a general way as comparable to those that are obtained with the "LPS", but it must be remarked that, contrary ro the latter, they have no toxicity.

3) Adjuvant character of the Mur - NAc - L - Ala - D - Glu - a - OCH,, of antigen, per ml of serum. In some cases, the antibody level has been determined too by passive hemagglutination (PHA) as above indicated.

The results of these tests are reported in the following Table 2.

TABLE 2

Serum antibody Precipi Composition of the emulsion tation Cutaneous test containing the antigen Wgiml) Agglutination (diameter in mm) ovalbumin (1 mg)t FIA < 500 - 0 ovalbumin (1 mg) + FCA (100 ag) 3200 - 10 + 1.5 ovalbumin (1 mg) + FIA + Mur-NAc-L Ala-D-Glu-a-OCH, (100 eg) 2600 - 6 j 3 ovalbumin (0.5 mg) + FIA 4500 900 0 ovalbumin (0.5 mg) + FCA (100 sg) 2100 3600 13.5 ovalbumin (0.5 mg) + FIA + Mur-NAc L-Ala-D-iso-Gln(OCH (100 llg) 2100 3000 12 ovalbumin (0.5 mg) + FIA + Mur NAc-L-Ala-D-Glu(OCH,)2 (100 pg) 950 2600 6.2 These results show that the compounds of the invention, administered in an oily emulsion, have an influence on the proportion of antibody formed in response to the injection of antigen, and that it induces a reaction of retarded hypersensitivity with respect to the same antigen.

4) Anti-infectious character The following tests illustrate the anti-infectious properties of the Mur - NAc Ala - D - Glu - a - SOCH2, of the Mur - NAc - L - Ala - D - Glll(OCH2)2 and of the Mur - NAc - L - Ala - D - iso - Gln(OCH2).

In the preliminary tests, an experimental method was- established permitting the anti-infectious character of the products to be shown. It has thus been shown that a dose of 104 Klebsiella pneumoniae, injected intramuscularly in mice, produced the progressive decrease of a large part, if not the whole, of the animals in the week follow ing the inoculation. After eight days, the survival of the animals is finally ascertained.

The survival of groups of mice inoculated under the conditions indicated above and treated by means of the methyl ester considered was followed.

By way of comparison, batches of mice have been treated with BCG and LPS.

This latter, as is known, is an extremely active immunostimulant when it is administered 24 hours before the infection.

For these tests, hybrid mice (C57B1/6 X AKR)F1, reared at the PASTEUR INSTITUTE, from strains coming from the breeding of the C.NoR.S. at Orleans, were used. The endotoxin or LPS was extracted by the phenol-water method from Salmonella ententidis variety Danysz (No. 5629, PASTEUR INSTITUTE). The BCG comes from the strain Mycobacteeium tuberculosis var. bovis (No. 1173 P2 of the PASTEUR INSTITUTE), cultivated on Sauton medium and killed by a solution of 2% by weight of phenol.

The infection by Kiebsiella pneumoniae, strain of capsular type 2, biotype d, is made from a culture of 16 hours in a medium for Pneumococcus (No. 53515, PASTEUR INSTITUTE). The preparations injected before or at the moment of the infection are always diluted in apyrogenic physiological solution, at the rate of 0.2 ml for parenteral administration and 0.5 mi for oral administration, the controls receiving the solution alone.

In the tests of which the results are reported in the Tables 3 and 4, the influence of the treatment by varying the methods, the doses and the time of administration of the products studied has been determined. The percentage of protection expresses the difference of the percentages of survivors in the group of treated animals with respect to the corresponding control group.

The results show that the products studied have an anti-infectious activity, whether they are administered parenterally or orally. On the contrary, the LPS is inactive when given orally, even for the very large doses (100 g of LPS represent 10000 times the anti-infectious dose taken parenterally) .

In addition, the results are the same if the products are administered 24 hours or only 1 hour before the infectant injection, administered intramuscularly.

TABLE 3 Anti-infectious protection with respect to an intramuscular inoculation of 104 K pneumoniae Treatment 24 hours before the infection

Number Numberof animals Method of surviving on day of animals r % of treatment treated 3 5 8 protection Control 24 12 8 2 LPS 1 ag 24 24 22 22 83 Control 24 11 9 6 BCG 100 ag 24 24 24 21 63 Control 24 15 11 7 I.V. Mur-NAc-L-Ala-D-Glu- a-OCH3 100 llg 24 24 24 23 67 Control 24 13 9 4 Mur-NAc-UM a-D-Gln OCH3 100 gg 24 21 13 11 29 Control 24 13 9 4 Mur-NAc-L-Ala-D-Glu- (OCH,)2 100 g 24 21 19 15 46 Control 12 6 4 2 Mur-NAc-L-Al a-D-Glu a-OCH3 2000 g 12 11 9 6 40.5 Control 12 6 5 2 per os Mur-NAc-L-Al a-D-Glu (OCH,)2 2000 g 12 10 9 8 50 Control 24 14 10 7 LPS 100 lug 24 13 10 8 TABLE 4 Anti-infectious protection with respect to an intramuscular inoculation of 104 K. pneurnoniae Treatment 1 hour after the infection

Number Number of animals of surviving on day Method of animals Cc of treatment treated 3 5 8 protection Control 16 6 2 1 Mur-NAc-L-Ala-D-Glu &alpha;-OCH3 100 g 16 16 16 14 88 Control 8 6 5 1 I.V.

Mur-NAc-L-Ala-D-Glu (OCH,)2 100 ttg 8 8 8 8 88 Control 16 10 6 1 BCG 100 g 16 14 13 10 50 In another series of tests. the anti-infectious properties of the products were studied with respect to a very violent infection caused by the intravenous injection (and not intramuscular) of 103 K. pneumoniae.

In these tests, also effected on mice, the treatments are effected 24 hours before the inoculation. The methods and the doses are indicated in Table 5 in which the results of these tests are reported.

TABLE 5 Anti-infectious protection with respect to an intravenous inoculation of 103 K. pneumoniae Treatment 24 hours before the infection

Number Number of animals of surviving on day Method of animals % of treatment treated 3 5 8 protection Mur-NAc-L-Al a-D-Glu I.V. a-OCH, 100 g 16 16 16 12 65 Mur-NAc-L-Al a-D-Glu- I.V. (OCH,)2 100 g 16 15 I 12 10 52.5 I.V. LPS 1 ltg 16 16 16 16 90 I.V. BCG 100,ag 16 16 16 16 90 peros BCG 2000 ,ag 16 3 0 The results show a significant protection in the case of mice treated by means of the products according to the invention.

Thus, owing to the invention, there is provided a new adjuvant agent of immunity soluble in water and active even in the absence of an oily phase, as well as agents for the treatment of infectious diseases, which have no toxicity and can be administered parenterally or orally and are active even for the antibiotic-resistant pathogenic genes.

WHAT WE CLAIM IS: 1. Esters of 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) propionyl - L - alanyl - D - glutamic acid and of 2 - (2 - acetamido - 2 - deoxy - 30 - D - glucopyranosyl) - D - propionyl - L - alanyl - D - isoglutamine corresponding to the formula

in which Rl is --06H,,,,, wherein n is 1, 2 or 3, or -NH2; R2 is -OC,H22, wherein p is 0, 1, 2 or 3, p being other than 0 when Rl is -NH2.

2. The a-methyl ester of 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) - D - propionyl - L - alanyl - D - glutamic acid, corresponding to the abridged formula Mur - NAc - L - Ala - D - Glu - a - SOCH2.

3. The dimethyl diester of 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) - D - propionyl - L - alanyl - D - glutamic acid corresponding to the abridged formula Mur - NAc - L - Ala - D - Glu - (OCH2)2.

4. The methyl ester of the 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) - D - propionyl - L - alanyl - D - isoglutanune corresponding to the abridged formula Mur - NAc - L - Ala - D - iso - Gln(OCH3).

5. A process for the preparation of a compound according to claim 1, in which a suitable derivative of the (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) D - propionic acid or an ester thereof, the hydroxyl groups of which are blocked, is coupled with the peptide chain or a salt thereof previously prepared and of which the z carboxyl function is also blocked, and when the coupling is effected, the blocking groups are removed.

6. A process according to claim 5, in which the coupling with the peptide chain is replaced by two consecutive couplings, in the first with L-alanine or a salt thereof, then to the ester of glutamic acid or of the isoglutamine or a salt thereof.

7. A medicinal composition comprising a compound according to any one of claims 1 to 4 and a pharmaceutically acceptable vehicle.

8. A composition according to claim 7, in the form of an injectable solution.

9. A composition according to claim 8 wherein the solution constituted is a saline isotonic solution.

10. A composition according to claim 7 in a form for oral administration, or in a form to facilitate its application to mucous membranes.

11. A composition according to any one of the claims 7 to 10 which additionally comprises an immunising agent.

12. A medicinal composition in unit dosage form containing 100 aag to 800 ug of a compound as claimed in any of claims 1 to 4.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (14)

**WARNING** start of CLMS field may overlap end of DESC **. soluble in water and active even in the absence of an oily phase, as well as agents for the treatment of infectious diseases, which have no toxicity and can be administered parenterally or orally and are active even for the antibiotic-resistant pathogenic genes. WHAT WE CLAIM IS:

1. Esters of 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) propionyl - L - alanyl - D - glutamic acid and of 2 - (2 - acetamido - 2 - deoxy - 30 - D - glucopyranosyl) - D - propionyl - L - alanyl - D - isoglutamine corresponding to the formula

in which Rl is --06H,,,,, wherein n is 1, 2 or 3, or -NH2; R2 is -OC,H22, wherein p is 0, 1, 2 or 3, p being other than 0 when Rl is -NH2.

2. The a-methyl ester of 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) - D - propionyl - L - alanyl - D - glutamic acid, corresponding to the abridged formula Mur - NAc - L - Ala - D - Glu - a - SOCH2.

3. The dimethyl diester of 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) - D - propionyl - L - alanyl - D - glutamic acid corresponding to the abridged formula Mur - NAc - L - Ala - D - Glu - (OCH2)2.

4. The methyl ester of the 2 - (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) - D - propionyl - L - alanyl - D - isoglutanune corresponding to the abridged formula Mur - NAc - L - Ala - D - iso - Gln(OCH3).

5. A process for the preparation of a compound according to claim 1, in which a suitable derivative of the (2 - acetamido - 2 - deoxy - 3 - 0 - D - glucopyranosyl) D - propionic acid or an ester thereof, the hydroxyl groups of which are blocked, is coupled with the peptide chain or a salt thereof previously prepared and of which the z carboxyl function is also blocked, and when the coupling is effected, the blocking groups are removed.

6. A process according to claim 5, in which the coupling with the peptide chain is replaced by two consecutive couplings, in the first with L-alanine or a salt thereof, then to the ester of glutamic acid or of the isoglutamine or a salt thereof.

7. A medicinal composition comprising a compound according to any one of claims 1 to 4 and a pharmaceutically acceptable vehicle.

8. A composition according to claim 7, in the form of an injectable solution.

9. A composition according to claim 8 wherein the solution constituted is a saline isotonic solution.

10. A composition according to claim 7 in a form for oral administration, or in a form to facilitate its application to mucous membranes.

11. A composition according to any one of the claims 7 to 10 which additionally comprises an immunising agent.

12. A medicinal composition in unit dosage form containing 100 aag to 800 ug of a compound as claimed in any of claims 1 to 4.

13. A medicinal composition in unit dosage form containing about 400 jag of a

compound as claimed in any of claims 1 to 4.

14. Process which comprises treating a warm blooded animal other than of the species homo sapiens with a compound as claimed in any of claims 1 to 4.