CH619470A5 - Process for the preparation of 2-(2-acetamido-2-deoxy-3-O-D-glucopyranosyl)-D-propionyl-L-seryl-D-iso glutamine and 2-(2-acetamido-2-deoxy-3-O-D-glucopyranosyl)-D-propionyl-L-seryl-D-glu tamic acid - Google Patents

Abstract

The compounds indicated are obtained by peptide linkage starting with the optionally esterified or amidated chain L-seryl-D-(iso)glutamine and an N-acetylmuramic acid derivative, any group which should not react being temporarily blocked. A variant consists in coupling, using the same reaction, L-serine with N-acetylmuramic acid and then with D-isoglutamine or D-glutamic acid, and the esters or amides thereof. The water-soluble compounds thus obtained are non-toxic potent immunological adjuvants.

Description

The invention relates to the preparation of 2- (2-acetamido-2-deoxy-3-0-D-glucopyrannosyl) -D-propionyl-L-seryl-D-iso-glutamine corresponding to the formula:

ch20h h, oh nh-coch,

©

h, g - ch - co - nh - ch - co - nh - ch - conh,

ch2oh

AT/

L

i

(cn2) 2

c00h

"sr d

hereinafter designated by the abbreviation Mur-NAc-L-Ser-D-iso-Gln, and 2- (2-acetamido-2-deoxy-3-0-D-glucopyrannosyl) -D-propionyl acid -L-seryl-D-glutamic corresponding to the formula:

ch20h h, oh

(not)

h3C

nh-cochj ch - co - nh - ch - co - nh - ch - cooh

(çh2) 2

ch2oh

-v-

c00h hereinafter designated by the abbreviation Mur-NAc-L-Ser-D-Glu, as well as their mono- and diester derivatives, for example methyl, ethyl or propyl groups, and their amide derivatives which may be substituted on the nitrogen of the amide, for example by methyl residues. These compounds are water soluble and effective as immunological adjuvants for stimulating immune responses in a host. They serve in particular to strengthen the action of weak immunogens. More particularly, said agents can be used for the immunization of humans and warm-blooded animals against bacterial infections,

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619,470

viral and parasitic, and against different tissue antigens of normal or pathological origin.

The very particular advantage of the products obtained according to the invention lies in the fact that it is not necessary to have recourse to particular administration media, on which the manifestation of the adjuvant action would depend, and the excipients we are led to associate them only for the purpose of facilitating the use of these products. In particular, it is not necessary,

when one of these products is injected, use for this purpose a composition containing an oily phase.

In addition, the adjuvants obtained according to the invention can be administered orally or parenterally, in particular by injection, scarification, or by application to mucous membranes.

These medicinal compositions may contain one of the agents obtained according to the invention and in particular Mur-NAc-L-Ser-D-iso-Gln in combination with a pharmaceutically acceptable vehicle. Particularly preferred compositions of this type consist of injectable solutions containing an effective dose of a product obtained according to the invention. Advantageously, sterile solutions in an aqueous phase, preferably isotonic, are used for this purpose, such as saline or glucose isotonic solutions. This is of course not limiting; you can also use a simple solution in distilled water. It is also possible to use injection media containing an oily phase, and in particular water-in-oil emulsions. Such emulsions are obtained in particular with metabolizable vegetable oils, as described in French patent application No. 75.04003.

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

The adjuvants obtained according to the invention can also be in lyophilized form to allow the extem-porane preparation of injectable solutions, or for scarification.

An advantageous pharmaceutical form for the extemporaneous preparation of injectable solutions comprises a dose of approximately 2 mg of an adjuvant product according to the invention, in the lyophilized state, in association with approximately 10 mg of lactose as an excipient.

A product obtained according to the invention, and in particular the Mur-NAc-L-Ser-D-iso-Gln, can also be an immunogenic agent, in particular to a weak vaccinating antigen.

The methods according to the invention are defined in the claims. According to one of these methods, the peptide chain L-Ser-D-iso-Gln, L-Ser-D-Glu, is attached to N-acetylmuramic acid, or to a corresponding esterified or amidated chain from which the reactive groups, with the exception of the amine group intended to react with N-acetylmuramic acid. After fixing, the protected functions are released.

According to the second method, the two amino acids forming the peptide chain are successively attached. First, an adequate derivative of L-serine is attached to the derivative of N-acetylmuramic acid, then, on the product obtained, the derivative of D-glutamic acid or isoglutamine is attached to L-serine. by forming a peptide bond.

Other characteristics will appear during the description of an example of preparation of the Mur-NAc-L-Ser-D-iso-Gln according to the invention, as well as tests demonstrating the pharmacological properties of this product.

The state of the art is illustrated by French patents N05 2248025 and 2292486 which generally describe muramyl derivatives effective as adjuvants of immunity.

The nature of the first amino acid in the substitution chain is not specified. However, by using, in accordance with the invention, derivatives comprising a 1-seryl group, remarkable adjuvants are obtained, which was not suggested by the cited patents.

Example of synthesis of Mur-NAc-L-Ser-D-iso-Gln

This compound, whose name according to the official nomenclature is 2- (2-acetamido-2-deoxy-3-0-D-glucopyrannosyl) -D-propionyl-L-seryl-D-isoglutamine, is prepared in several stages. First, the peptide chain is synthesized, and then, this chain is attached to the muramyl residue. During this synthesis, the functions which should not enter into reaction are protected by protection or blocking groups which are then eliminated.

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

Glu glutamic acid iso-Gin isoglutamine

Ser serine

4,6-O-bzi 4,6-O-benzylidene

ß-bzl ß-benzyl

BOC t-butyloxycarbonyl

OBzl benzyl ester

OSu succinimide ester

Bzl benzyl ether

BOC-L-Ser (Bzl) -D-iso-Gln-OBzl (I)

1.48 g (5 mmol) BOC-L-Ser (Bzl) [E. Wünsch, A. Zwick, "Chem. Berg. ”, 97 (1964) 2497] is added with stirring to a solution in dimethylformamide of 1.64 g (6 mmol) of the hydrochloride of the benzyl ester of D-isoglutamine [P. Lefrancier, E. Bricas, “Bull. Soc. Chim. France ”, (1969) 3561] and 0.66 ml (6 mmol) of N-methylmorpholine. 1.24 g (6 mmol) of NN'-dicyclohexylcarbodiimide are added, and the reaction mixture is left at room temperature for 12 h. It is then concentrated and taken up in 50 ml of ethyl acetate and the organic phase is washed successively with a 10% citric acid solution, with water, with a solution N of sodium bicarbonate, then with 1 water to neutral pH. The ethyl acetate phase is dried over MgSO4, then concentrated. The product is crystallized from the ether / petroleum ether mixture. 1.40 g of product are obtained, ie a yield of 55%.

Mp 65-66 ° C.

ccd25 = + 5.8 ° (absolute methanol).

Analysis for C27H35O7N3 (513.6):

Calculated: C63, W H 6.9 N 8.2%

Found: C63.2 H 7.1 N7.97%

L-Ser (Bzl) -D-iso-Gln-OBzl (II) hydrochloride

1.3 g (2 mmol) of (I) are treated for 30 min with 5 ml of an approximately N solution of hydrochloric acid in acetic acid. The reaction mixture is concentrated to dryness and the product crystallized from a methanol / ether mixture. 765 mg of product is obtained, ie a yield of 85%.

Mp 158-161 ° C.

<xd25 = + 8.2 ° (absolute methanol).

Wall-NAc ($ -bzl-4,6-bzi) -L-Ser (Bzl) -D-iso-Gln-OBzl (III)

A solution of 471.43 mg (1 mmol) of Mur-NAc (ß-bzl-4,6-bzi) in the acetonitrile / dimethylformamide mixture (2/1, v / v -10 ml) containing 0.141 ml (1 mmol ) of triethylamine is poured into a suspension, maintained at 0 ° C., of N-ethyl-5-phenylisoxazolium-3'-sulfonate (253 mg -1 mmol) (reagent D of Woodward, Aldrich) in 10 ml of acetonitrile.

s

10

15

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25

30

35

40

45

50

55

60

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619,470

4

The mixture is stirred at 0 ° C. until a clear solution is obtained (approximately 90 minutes), then 450 mg (1 mmol) of (II) dissolved in 10 ml of the acetonitrile / dimethyl-formamide mixture are added. (2/1, v / v) containing 0.141 ml (1 mmol) of triethylamine. After stirring overnight at room temperature, the solvents are evaporated in vacuo and the product precipitated from the dimethylformamide / water mixture. 818 mg of product are obtained,

or a yield of 94.3%.

Mp 180-186 ° C.

<xd25 = —17 ° (NN'-dimethylformamide). io

Wall-NAc-L-Ser-D-iso-Gln (IV)

768 mg of the compound (III) (0.88 mmol) are treated with 30 ml of a 60% acetic acid solution for 1 h at 100 ° C.

After cooling the solution, the acetic acid is evaporated off under vacuum and the residue is chromatographed on a column of silica gel (35 g) (chloroform / methanol, 6/2, v / v). The pure fractions are combined, evaporated under vacuum, giving a chromatographically pure residue (plate of silica gel / chloroform / methanol, 6/2, v / v). 287 mg of product are obtained, i.e. a 20

42% yield.

This product, taken up in 20 ml of glacial acetic acid, is then hydrogenated for 10 h in the presence of palladium on carbon. After filtration of the catalyst and concentration of the reaction mixture to dryness, the product is obtained by precipitation in an ethanol / acetone / ether mixture. 140 mg of product are obtained, ie a yield of 74%. The rotary power is aD25 = + 47.1 ° (glacial acetic acid).

The product is chromatographically homogeneous on silica gel plates in n-butanol / pyridine / acid mixtures 30

acetic / water (30/20/6/24, v / v) and n-butanol / acetic acid / water (4/1/5, v / v - upper phase).

Analysis for C19H32O12N4 (508.5):

Calculated: C 44.88 H 6.34 NI 1.02% 3J

Found: C44.8 H 6.3 Nll, l%

Pharmacological properties

I. Determination of the safety of Mur-NAc-L-Ser-D-iso-Gln

The safety, or on the contrary the toxicity of the product according to 40

the invention is studied by intravenous injection in mice two months old. By gradually increasing the doses administered, it is determined that for which the mortality of the mice of the same batch is established at 50% (LD50).

For the product studied, it has been shown that the LD 50 is greater than 1 g / kg of animal, which is much higher than the doses for which this product manifests its adjuvant properties.

2. Adjuvant nature in aqueous phase

In the series of tests, the results of which are given below, the influence of Mur-NAc-L-Ser-D-iso-Gln on the level of anti-albumin antibodies was studied under the following conditions.

Groups of 8 two-month-old Swiss mice receive, by subcutaneous injection, 0.5 mg of antigen consisting of bovine serum albumin (BSA) and 0.1 mg of the test substance in saline solution isotonic. This high dose of antigen, because it is at the limit of the paralyzing dose vis-à-vis the immune response, therefore causes a weak or zero 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 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 AA Hirata and MW Brandiss ("J. Immunol.", 100, 641-648 , 1968). The samples are taken 14, 28, 34 and 36 days after the first injection.

For comparison, mice receive, instead of Mur-NAc-L-Ser-D-iso-Gln, either lipopolysaccharides (LPS) (extract of S. enteritidis by the water / phenol method), or adjuvant designated as 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.

Table 1

Antibody titer

14th day

28th day

34 = d

36th day

Witness (BSA)

<3

<3

<3

<3

BSA + LPS (100 ng)

<3

6

50

1310

BSA + WSA (300 | ig)

<3

<3

<3

6

BSA + Mur-NAc-L-Ser-D-iso-Gln (100 (J.g)

12

25

1600

1600

These tests were repeated by limiting the dose of antigen from the first injection to 0.05 mg, the other conditions being identical elsewhere. This lower dose of antigen by itself gives a positive response in the controls who received the antigen without adjuvant. It therefore makes it possible to highlight a substance which inhibits the humoral immune response. In addition, an adjuvant substance, under these conditions, always raises the level of antibodies compared to that of the controls.

In these tests, the determination of the antibody level was carried out simultaneously by passive hemagglutination (PA) and by the method of fixing the antigen by the serum (ABC), as described by P. Minden and S. Farr ( "Handbook of Exp. Immunol." P 463, Weir DM ed., Blackwell Scient. Pub. Oxford and Edinburgh, 1967).

The results of these tests are shown in Table 2.

Finally, patent table)

These results show that the Mur-NAc-L-Ser-D-iso-Gln, administered in saline isotonic solation, causes a significant increase in the level of antibodies formed, whereas, under the same conditions, the WSA is practically inactive. .

In the second series of trials, for which the dose of antigen, at the time of the first injection, is low, the presence of WSA even seems to inhibit the formation of antibodies, while the 65 Mur-NAc-L-Ser- D-iso-Gln, on the contrary, promotes the appearance of antibodies.

The Wall-NAc-L-Ser-D-iso-Gln can be considered, in general, as comparable to LPS for its capacity to stimulate

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the format of the antibodies, but, unlike LPS, they can be used in particular to promote immu-

it is devoid of toxicity. nisation of the host (human or animal patients) with regard to

Adjuvant compositions are thus obtained devoid of infections of bacterial or viral origin, of antigens of toxicity and which can be used to increase the efficiency of tumors, etc. They are also effective for the manufacture of vaccines of bacterial or viral origin, in particular when these 5 sera containing antibodies active against these antigens,

are weakly immunogenic.

Table 2

Antibody titer

14th day

28th day

34th day

36th day

42nd day

PA

ABC

PA

ABC

PA

ABC

PA

ABC

PA

BSA (witness)

<3

<20

<3

<20

6

<20

50

25

25

BSA + LPS

<3

<20

25

<20

400

300

3200

500

800

BSA + WSA (100 ng)

<3

<20

<3

<20

6

<20

3

<20

3

BSA + WSA (300 ng)

<3

<20

<3

<20

<3

<20

<3

<20

<3

BSA + Wall-NAc-L-Ser-D-iso-Gln (100 (ig)

<3

<20

3

<20

50

<20

800

230

260

R

at

Claims (4)

619,470 2 1. Process for the preparation of 2- (2-acetamido-2-deoxy-3-0-D-glucopyrannosyl) -D-pyrannosyl) -D-propionyl-L-seryl-D-isoglutamine, 2- acid (2-acetamido-2-deoxy-3-0-D-gluco-pyrannosyl) -D-propionyl-L-seryl-D-glutamic acid and their esters or amides, characterized in that the peptide chain is fixed L-seryl-D-isoglutamine, L-seryl-D-glutamic, or a corresponding esterified or amidated chain, the reactive groups of these chains being blocked with the exception of the amine group which must react, with an adequate derivative of the acid N-acetylmuramic, whose hydroxyl groups are also protected by blocking groups, and which, once the fixing is done, the blocking groups are removed. 2. Process for the preparation of the compounds listed in claim 1, characterized in that one successively fixes on the derivative of N-acetylmuramic acid, L-serine, then D-isoglutamine, D-glutamic acid , or their corresponding esterified or amidated derivative, by blocking the reactive groups prior to each fixation, with the exception of those which are to be involved in this peptide bond fixation, then, after the fixation has been achieved, the blocking groups are eliminated. 3. Method according to claim 1, characterized in that the preparation 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 preparation is carried out by means of esters comprising 15 reactive functional groups or by means of NN'-dicyclohexyl-carbodiimide.