CZ238794A3 - Complexes of peptidoglycan monomer with trivalent metals, process of their preparation and use - Google Patents

Abstract

2133087 9320105 PCTABS00027 The invention relates to complexes of the peptidoglycan monomer (PGM) of formula (I) with trivalent metals, such as e.g. aluminum, lanthanum, vanadium and rhodium (except iron and bismuth), to a process for their preparation as well as to their use as immunomodulators in pharmaceutical preparations. According to this invention the complexation reaction of the peptidoglycan monomer is performed in an aqueous solution in the presence of salts of trivalent metals upon correction of the pH of the solution with alkalies, the concentration, and the addition of an organic solvent which is miscible with water and precipitates the product.

Description

The present invention relates to novel trivalent-metal peptidoglycan monomer (PGM) complexes, processes for their preparation and their use as immunomodulators in pharmaceutical compositions.

BACKGROUND OF THE INVENTION

It is known that in submersive cultures of Brevibacterium divarucatum (NRRL 2311) and Micrococcus glutaoiicus (ATCC 13287) in the presence of a cell wall biosynthesis inhibitor, fragments of peptidoglycan are secreted from which, after incubation with lysozyme and fractionation on monomeric molecular sieves, (L) -D-alanyl-alanine (Jug. Patent 40,472, AT 362,740) Carbohydr Res., 100 (1982), 320-325), of Formula I

CH-N1K

AND

CONH ₂

CO-NH-CH-CO-NH-CH-CONH,

I I

CH} (CH, H

AND

CO - NH - CH - CO - NH - CH - CO - NH - CH - COOH

I ^'1 fCJd ₂₎ CH CH _3} which exhibits immunostimulatory and antimetastatic activity (Z. Immun. Forsch. 155 (1979), 312-318; Eur. J. Cancer Oncol. (1983), 618-686). Our earlier observations have shown that, under precisely defined PGM conditions, they form complexes with divalent metals (Jug. Pat. Application P-1982/86) of highly complex structure, identified by analytical and spectral data as essentially a chelate structure, which is also charac for some amino acids or peptides (Acta Pharm. Jug. 38 (1988), 310; J. Srb. Chem. Soc. 55, (5) (1990), 265-269). The observations made by M. Tonkovič et al. (Inorganic Chim. Acta 161 (1989), 81-85) showed that PGM forms a complex with a completely different structure in aqueous solution and excess Fe (III) ions and without adjusting the pH of the solution. While continuing our work on this problem, further research into the formation of PGN complexes with other trivalent metals under different conditions was conducted.

SUMMARY OF THE INVENTION

Surprisingly, it has now been found that some trivalent metals provide the formation of new complexes with PGM in solution at a specific pH. Thus, potentiometric titration shows these effects for aluminum (Al (III)), rhodium ((III)), lanthanum (La (III)), and vanadium (V (III)), whereas these effects were not observed for iron / Fe ( And bismuth (Bi (III)). (Fig. 1 - 4).

In the appended FIG. 1-4 denotes A 0.01 M PGM (25 ml), C denotes A + B, while B denotes 0.005 M Al ^{3 +} in Figs. 1, 0.005 M Rh ^{3 +} In Fig. 2, 0.005 M La ³⁺ in Fig. 1. 3, and 0.005 MV ³⁺ in FIG. 4.

Potentiometric titrations revealed that the complexes formed had different stability constants, significantly increased for the aluminum (PGM-A1 (III) -K) and vanadium (PGM-V (III)) complex compared to the values for divalent metals (Table 2). This implies the possibility of far better pharmacokinetic characteristics of the trivalent metal complexes. It is also apparent that trivalent metals form two different complexes at different pH values in solution (Table 1). Only complexes formed at higher pH values are the subject of this invention.

There was also a significant difference between the preparation of our trivalent metal complexes and the observations of M. Tomkovič et al. In particular, under the conditions described in this invention, Fe (III) and Bi (III) do not form any complexes with PGM.

Calculation:

[Lo]. _and [L] ή = [Ho]

^K 1 - D For or <1 (1 - Ó) [L] K ¹ ~ ň-i 2> n> 1 i 2- d i [Lj K _s = Κι X Ki

TABLE 1 - Overview of Complexes p ions of trivalent metals Metal ion Salt Log K-j, Log K ₂ Log Kg pH for iol. Al ³⁺ A1 (NO ₃ ) ₃ X 9 H ₂ 0 6.28 6.15 12.43 4.2 La ³⁺ LaCl ₃ x 7 H ₂ O 3.13 2.53 5.66 7.5 Rh ^{3 +} Rhci ₃ X 3 H ₂ 0 - - 8.0 V ^{3 +} VC1 ₃ 5.89 5.58 11.47 5.0

TABLE 7 - Comparison with other metal complexes and PGM

Metal ion Log Log K ₂ Log Kg Al ³⁺ 6.28 6.15 12.43 La ³⁺ 3.13 2.53 5.66 _v 3 + 5.89 5.58 11.47 Cu ²⁺ 4.35 3.95 8.3 0 La ² * 2.64 - - La ²⁺ 2.50 - - La ^{2 +} 2.38 - - La ²⁺ 3.37 - -

Another object of the present invention is a process for the preparation of trivalent metal peptidoglycan monomer complexes such as aluminum, lanthanum, vanadium and rhodium (except iron and bismuth), wherein the peptidoglycan monomer is reacted in aqueous solution with the trivalent metal salt to adjust pH a solution of alkali and wherein the product is isolated from the obtained reduced volume solution at a pH of about 4.2 for the aluminum salt, about 7.5 for the lanthanum salt, about 8.0 for the rhodium salt, and about 5.0 for the vanadium salt, followed by the addition of organic a water-miscible solvent but which does not dissolve the complex, and wherein the end product is obtained by filtration isolation.

Hydrolysis of such isolated complexes with 6 N hydrochloric acid provides alanine, glutamic acid and diaminopimelic acid in a 3: 1: 1 ratio for amino acid analysis, demonstrating that the peptide composition remains complexed in accordance with PGM. In this post, sugar ingredients were found,

Ί Which were partially decomposed during hydrolysis. C NMR spectra also confirm the presence of all the fragments that make up PGM. However, some signals are significantly shifted (Table 3), confirming the metal-molecule coordination bond

PGM.

TABLE 3 - Characteristic shifts of ¹³ C NMR spectra for rhodium-lanthanum PGM complexes

Molecular residue PGM * PGM-Rh (III) -K PGM-La (III) -K N-acetyl-glucosaminyl ^C1 100.4 101.2 101.2 ^C 4 70.45 71.2 71.1 ^c 5 75.35 74.4 74.3 N-acetyl- muramoyl ^ lbeta 95.1 no signal 85.9 ^c lalfa 90.2 81.1 81.0 ^C 3 79.55 78.7 78.7 meso-diaminopimelyl ^c alpha ^(L) 52.75 51.8 51.8 ^c beta 30.6 31.1 31.4 C gamma 20.75 19.4 19.0 ^C beta ' 30.7 31.0 31.0 ^c alpha '< ^D > 52.75 54.7 54.6 D-alanine- C 1-4 carbonyl 175.7 180.6 181.5

* B. Klaič Carbohydr. Res. 110 (1982) 32-325

In PGM-Rh (III) -K, for example, the signals of the carbon atom of the terminal alanine, the carbon atoms of the diaminopimelic acid residue, are significantly shifted, indicating the coordinating bond of the amino groups and the new spatial arrangement of the residues in the molecule. The beta-anomeric conformation carbon atom signal disappeared in the muramine residue. This is evidenced by the formation of a coordination bond, forming such a configuration of the complex, which is not possible arrangement OH ^- group in position muramínového residue in β position. In contrast, in the PGM-La (III) -K, signals for the alpha and beta anomer are present but shifted to lower values by approximately and 35 ppm), leading to the conclusion that lanthanum binds with this rest of the molecule to form a new complex, , different from the configuration of the respective Me (II) ppm (90 coordinate configuration of the ions).

The increased stability of the complex in an alkaline environment, as compared to PGM as such, a biological approach to utilization, for example, stable at pH 8, is also highly relevant to the complex. The rhodium complex is while PGM decomposes completely in an alkaline environment. Importantly, there are enzymes in the blood serum that break down PGM very quickly into sugar and peptide components. The ions of some metals, especially magnesium, play a distinct role in this. In the absence of magnesium, said enzymes are not effective. {J. Tomasic et al. J. Chromatogr. 440 (1938), 405-414. The enzyme is not effective against the complex where the metal bonding site is already occupied and the molecule cannot decompose for a long time. The presence of metal significantly affects not only stability and biotransformation, but also distribution and excretion, as well as other properties of the molecule. Biological assays of some of the trivalent metal complexes of PGM were performed using the standard method of PFC testing in CBA mice (female). Observations were performed using standard peptidoglycan monomer (PGM) and saline (FO) as controls. The results are shown in Table 4 and show the same efficacy of the investigated complexes and standard PGM in stimulating the recipient's immunological response.

TABLE 4. Immunomodulatory with trivalent metals activity of PGM complexes Action % PFC spleen weight,% FO 100 ALIGN! 100 ALIGN! PGM 180 153 PGM-Rh (III) -K 178 120 PGM-A1 (III) -K 167 120 PGM-1A (III) -K 163 140

To date, PGM complexes with trivalent metals, with the exception of iron (III), have not been described for peptidoglycan structures to date.

The invention is illustrated by the following Examples.

Example 1. Preparation of a peptidoglycan monomer complex with aluminum

The peptidoglycan monomer (504.50 mg, 0.50 mmol) was dissolved in 25 mL of water and stirred for one hour after addition of Al (NO ₃ ) ₃ x 9 H ₂ O (93.75 mg, 0.25 mmol). The pH of the solution was then adjusted to 4.2 by adding 0.1 N NaOH and stirred for an additional hour. The mixture was concentrated by evaporation under reduced pressure to a volume of about 2 ml, and 50 ml of acetone was added dropwise from the separator over about 1/2 hour. A white precipitate was obtained, which was collected by filtration, stripped thoroughly, washed with ethanol and dried under reduced pressure to constant weight. Yield 460 mg.

IR ^(KBR cm ^-1): 3400-3260, 3070, 2970, 2920, 1650, 1535, 1380,

1350, 1040. 595

M.p. Melting point = 200 DEG C. (dec.).

^u _max (H ₂ O) (nm) = 192, A ^1% = 608.42

Analysis of metals by atomic absorption spectrometry.

Example 2. Preparation of a peptidoglycan monomer complex with rhodium

Following the procedure described in Example 1, except that rhodium chloride trihydrate (RhCl ₃ x 3 H ₂ O, 65.83 mg, 0.25 mmol) is used instead of aluminum nitrate, aqueous sodium hydroxide is added up to pH 8.0 and the solution is stirred for an additional two hours. Yield 490 mg.

IR ^(KBR cm ^-1): 3370-3270, 3070, 2980, 2915, 1650, 1535, 1450,

1370, 1315, 1230, 1160, 1105, 1065, 1040, 535.

- v

B. t.

Melting point = 200 DEG C. (dec.).

^uv max ( ^H2O >< ^nm ) = 192.440 (shoulder); A ^1% (192 nm) = 608.42

Example 3. Preparation of a peptidoglycan monomer complex with lanthanum

Following the procedure described in Example 1, with the only exceptions that LaCl-x 7 H ₂ O (92.85 mg, 0.25 mmol) was used instead of aluminum nitrate and aqueous sodium hydroxide was added up to pH 7.5 . Yield 479 mg,

IR ^(KBR cm ^-1): 3400-3240, 3060, 2980, 2915, 1650, 1550, 1425,

1410, 1370, 1315, 1245, 1160, 1110, 1070-1040, 600.

M.p. Melting point = 200 DEG C. (dec.).

^UV max ( ^H 2 O) ^{= 190; α1%} ( ¹⁹ ° = 576.47).

Example 4. Preparation of a complex of peptidoglycan monomer with vanadium

Following the procedure described in Example 1, with the only exceptions that VCl ₃ (39.33 mg, 0.25 mmol) was used instead of aluminum nitrate and aqueous sodium hydroxide was added up to pH 5.0. Yield 455 mg.

IR ^(KBR cm ^-1): 3400-3260, 2940, 1670-1650, 1550, 1460,

1380, 1320, 1260-1235, 1120, 1062, 1050, 625 ^UV max ( ^H 2 °) ^{= 194;} ( ^{194 nm} ) ⁼ 294.54.

Claims (8)

Complexes of peptidoglycan monomer (PGM, Formula I) H. OH (1 ') CO-Nrh CH · CO-NH-CH · CONlh CH, (CHd, CO-NH-CH-CO-NM-CH-CO-NH-CH-COOH; ÍCH ^ AND CH-NH, AND I conb ₂ ch. ch ₃ with trivalent metals such as iron and bismuth. aluminum, lanthanum, vanadium and rhodium, The peptidoglycan monomer complex (PGM, Formula I) is claimed in claim 1, with aluminum. The peptidoglycan monomer complex (PGM, Formula I) is claimed in claim 1, with lanthanum. The peptidoglycan monomer complex (PGM, Formula I) is claimed in claim 1, with rhodium. The peptidoglycan monomer complex (PGM, Formula I) is claimed in claim 1, with vadaad. A process for the preparation of peptidoglycan monomer complexes as claimed in claim 1, wherein the peptidoglycan monomer is reacted in aqueous solution with a salt of the respective trivalent metal to adjust the pH of the solution with alkali and wherein the product is isolated from the obtained reduced volume solution at pH about 4.2 for the aluminum salt, about 7.5 for the lanthanum salt, about 8.0 for the rhodium salt, and about 5.0 for the vanadium salt, followed by the addition of a water miscible organic solvent that does not dissolve the complex, and wherein the final the product is obtained by isolation by filtration. Use of trivalent metal peptidoglycan monomer complexes as claimed in claim 1, characterized by the preparation of an immunomodulatory pharmaceutical composition. Use of trivalent metal peptidoglycan monomer complexes as claimed in claim 1, characterized in that they are used as immunomodulators in pharmaceutical preparations.