HU177118B - Process for producing metallocomplexes of olygo- and polygalacturonic acid-dinitrates - Google Patents

Description

The present invention relates to novel complexes of essential metal ions of oligo- and polygalacturonic acid dinitrates. The invention also relates to the preparation of pharmaceutical compositions containing the novel metal complexes.

It is known that complexes of oligogalacturonic acids and medium molecular weight polygalacturonic acids with essential metal ions such as iron, copper, magnesium, cobalt, manganese, zinc, chromium, molybdenum / or nickel ions can be used very advantageously in human and in veterinary medicine for the prevention and treatment of trace element deficiency disorders (Hungarian Patent No. 172,831). However, these compounds have the disadvantage that, due to their unfavorable solubility characteristics, they cannot be converted to parenteral pharmaceutical formulations and are therefore not suitable for use in situations such as life-threatening conditions, coma, indigestion and absorption, physiological anemia in neonates, etc. should be delivered at high speed into the body.

Parenteral substitutions of essential elements have been attempted so far with a variety of metal compounds and metal complexes, but without any finding of suitable agents (Brit. Med. J. 1, 881 (1893), RR McCury, "Parenteral writer The2 rapy in writer Deficiency, "Halberg et al., Acad. Press, London-New York (1970 / p. 537).

Inorganic metal salts such as magnesium sulfate, magnesium chloride, calcium chloride, potassium chloride, iron (III) chloride, etc., as well as metal complexes with low stability (less than 3.0 pK) use of metal acetates and metal formates) is severely limited by the high toxicity of these compounds due to the rapid and high release of metal ions to precipitate transport proteins such as apotransferrin and consequently fever and respiratory paralysis. Intravenous iron (II) and magnesium (II) aquiones also cause narcosis.

Highly stable (relatively pK> 6) metal complexes, such as iron (III) ethylenedianne tetraacetate, iron (in) citrate, magnesium citrate and the like, have the disadvantage that these compounds, namely because of their high stability - they are unable to transfer sufficient amounts of metal ions to the proper transport proteins. These complexes are excreted unchanged and at high speed by the kidneys and are therefore only marginally utilized (W. Forth, K. Pfleger, R. Rohns and W. Rummel: "Dér Einfluss von Complexbildnem auf die Verteilung intra veninene"). verabreichten Eisens ím Organismus', Radionucleide in Organismen und ihrer Umwelt, Deutschland177118

-Usterreich-Schweiz, Ges. Für Biophys. and Strahlenbiol, Vienna, 14-16 September 1964).

In addition to the stability of the complex, the molecular activity of the metal complexes is significantly influenced by the molecular weight and particle weight as well. High molecular weight complexes such as polysaccharide-stabilized colloidal iron oxide hydrates (such as iron (III) hydroxide dextran (Imferon, Imferdex, Myofer, Impozil), colloidal iron (III) oxide dextrin (Pigdex, Astrafer, Ferrigen) or iron (III) hydroxide saccharate (Ferriverin)] by intravenous injection or infusion, there is no significant iron release even if the amount of iron administered is several hundred times the physiological iron concentration in plasma (J. Clin. Path, 21). A further disadvantage of these compounds is that they have undesirable side effects such as liver and spleen swelling, hemosiderosis (J. Exp. Med. 109, 197 (1959), J. Foot. Clin. Med. 73/2/1968). , 181 (1969), Arch, Path. Foot. Med. 103, 21 (1979)), inflammatory infiltration, edema, swelling and local tissue necrosis (Path. Vet. 4, 58, 1967).

In contrast, low molecular weight formulations such as iron (III) gluconate, iron (II) gluconate lactobionate, iron (II) lactobionate, iron (II) oxalate and iron (II) lactate are both administered intramuscularly and subcutaneously. they rapidly enter the blood vessels and saturate apotransferrin, apolactoferrin, serum albumin and gamma globulin. This protein saturation leads to adverse side effects such as fever, vomiting, diarrhea, dizziness, and headache (RR Mc. Cury, "The Therapeutic in the Writer's Deficiency", Halberg et al., Acad. Press, London-New York, 1970, 537). side). A further disadvantage is the presence of much higher levels of metal ions in the blood when added to low molecular weight formulations, which is readily available to strong chelators produced by infectious bacteria. Thus, the bacteria entering the body through the infection can easily obtain the metal ions essential for their growth, thereby rapidly multiplying in the body and destroying the host (Adv. Chem. Ser. 162, 55 (1977)).

Some known metal complexes, such as sodium iron (III) gluconate and iron (III) dextran, also exhibit carcinogenic side effects (Brit. J. Cancer 22, 521/1968/15, 838/1961/21). 448 (1967) and 22, 116 (1968), British Med. J. 1, 1593 (1964) and 1, 1800 (1962), J. Am. Med. Ass. 182, 1334 (1962), which further limits the applicability of these formulations.

A further disadvantage of formulations made by stabilizing colloidal iron (III) hydroxide with a polysaccharide is that they are not uniform, their chemical composition is undefined, and their iron content can vary considerably depending on the conditions of preparation. These formulations are thus unsuitable for inducing a predetermined and reproducible biological effect (Chain 1947, II, 49, British Med. J. 1954, I 984, Scand. J. Haem. S. 32, 21, 1977).

It is an object of the present invention to provide novel metal complexes which are free from the disadvantages of known metal complexes, i.e. they are chemically and biologically well defined, water soluble, of medium stability and, when administered parenterally to living organisms, effectively transfer their essential metal ion content. however, without causing adverse side effects or toxic symptoms. In its experiments, it has been found that the oligo- or polygalacturonic acid dinitrate metal complexes of formula (I) fully meet the above requirements.

The present invention therefore relates to a process for the preparation of oligonuclear or polygalacturonic acid dinitrate metal complexes of the formula I wherein n is an integer from 10 to 150,

M represents an alkali metal ion, an alkaline earth metal ion and / or a transition metal ion having a 3d path, provided that the metal complex contains only an alkali metal ion or an alkali metal ion and at least one additional non-alkaline metal ion and . is an integer equal to the valence or oxidation number of the metal ion.

As used herein and in the claims, the term "metal ion" includes positive metal ions such as [Mo (O) ₂ f and VO ^{2 +]} .

According to the present invention comprising the formula (II), oligo- or polygalacturonic dinitrate formula - wherein n is as defined above - or an alkaline aqueous medium a complex of one or more M ^{z +} metal ions of formula a salt or formed oligo- or polygalacturonic dinitrate with a lower stability constant (e.g., acetate), or addition of alkali metal ions in addition to the alkali metal hydroxide.

As mentioned above, the oligo- or polygalacturonic acid dinitrate metal complexes of the present invention each contain an alkali metal ion. As stated above, alkali metal ions may be incorporated into the molecule in the form of their salts (such as their chlorides, carbonates, bicarbonates, etc.), low stability constants or hydroxides. Alkali metal hydrolyzing alkali metal compounds such as alkali metal hydroxides, carbonates and bicarbonates are particularly preferred for the introduction of alkali metal ions. In some cases, ions other than alkali metal ions are not included in the oligo- or polygalacturonic acid dinitrate. However, complexes which have at least one other metal ion of the formula M ^{z +} (e.g., iron, copper, cobalt, manganese, zinc, chromium) are also more preferred than mono-metal complexes (containing only alkali metal ions). and / or nickel ions). The metal ions can be incorporated into the oligo- or polygalacturonic acid dinitrate in a single step or in separate operations. Preferably, the initial oligo- or polygalactonic acid is first neutralized with an alkaline hydrolyzing alkali metal compound (e.g. metal reagents.

By the process of the present invention, metal complexes suitable for a variety of medical purposes can be prepared by appropriate selection of the quantity and quality of the metal ions incorporated. Polymeric complexes, particularly those containing iron, copper, cobalt, manganese, zinc, molybdenum and potassium, are very useful for the prevention and treatment of anemic conditions, since all essential elements of the treatment are delivered in a single composition. The multi-metal complexes are also very useful for the prevention of myocardial infarction and atherosclerosis, for stimulating wound healing, and for geriatric purposes. Besides kizárlólag alkali metals or alkali metal complexes containing only one additional metal is preferably used in the treatment of conditions where only one kind of metal ions (e.g., iron) from the body must be taken to θ _2.

The compounds of formula (I) may be converted into pharmaceutical compositions using conventional pharmaceutical carriers, diluents and / or excipients. ₂₅ The pharmaceutical preparations for oral administration (such as tablets, capsules or solutions for oral administration) may be, but is preferably parenteral administration (e.g., intravenously, intramuscularly or subcutaneously administered intra- ₃₀ injections or infusions) forming these active compounds. These pharmaceutical compositions may contain, in addition to the active compounds of the formula I, other therapeutic substances, for example vitamins.

Note that, depending on the preparation conditions, some of the saccharide moieties ₄₀ may only have one nitro group attached to the polysaccharide chain of the compounds of formula (II). In this specification and in the claims, all dinitrates (I) and (II) compounds of the formula which are average per saccharide unit

They contain from 1.5 to 2 nitro groups. The average number of nitro csopor- casing ₄₅ is not significantly influenced within these limits, the solubility characteristics of the starting material and the resulting complex will not exert any measurable effect on the complexing capacity.

The following biological tests were performed with the oligogalacturonic acid dinitrate potassium (I)-iron (III) complex (compound of the formula I, n = 10, M ^{z +} = K ⁺ and Fe ³⁺ , iron content: 8.6%). The results are described. This metal complex is hereinafter referred to as the "A" complex, the comparator being iron (III) gluconate, well known in the medical art, hereinafter referred to as the "B" complex.

The incorporation of the complexes into red blood cells was studied in rats. Stock solutions of the following compositions were used:

Aqueous solution of complex A (iron content: 1.72 mg / ml), total 20 ml, and aqueous solution of complex B (iron content: 1.72 mg / ml), total 20 ml.

^S9 FeCl ₃ with mCi activity was dissolved in 1.3 mL of 0.1 N aqueous hydrochloric acid and the volume was adjusted to 4 mL with distilled water. 0.6-0.6 ml of this radioactive stock solution was added to the stock solutions described above.

The experiments were performed on male white rats (stock culture of Horizon KTSZ). Between 10 and 10 animals were included in each experiment. The animals were injected intravenously with 0.2 ml / 100 g of stock solutions containing the labeled iron atoms described above. One week after treatment, the animals were bled under ether narcosis, and the erythrocytes were separated from the plasma and the radioactivity in the erythrocyte suspension and in a 1 ml aliquot of plasma was determined. The observed results are reported in Tables 1 and 2. The figures in the tables are average values for two measurements. The data in the tables show that the incorporation characteristics of the iron content of complex A are more favorable than those of complex B

Table 1

Plasma radioactivity 1 week after treatment

Treatment Specimen number cpm / ml plasma A complex 10,179 ± 167 B complex 10 45 ± 30

Table 2

Radioactivity in red blood cell samples 1 week after treatment

Treatment Sample- song cpm / ml erythrocyte suspension cpm / 100 mg hemoglobin % Incorporation into hemoglobin Complex "A" 10 29464 18401 63.14 ± 10822 ± 4316 ± 12.48 B complex 10 24401 17530 60.32 ± 4277 ± 6182 ± 9.12

The acute toxicity of Complex A was investigated in rats. Administration of Complex A at doses of 0.86, 1.72, 3.44 and 6.88 mg / kg of iron, respectively, showed no toxic symptoms or side effects. The animals' weight gain 5 was normal compared to controls.

The following examples illustrate the invention without limiting it.

Example 1

Preparation of Oligogalacturonic Acid Dinitrate

Into a 250 ml three-necked round-bottom flask equipped with a magnetic stirrer, a funnel, a thermometer and a calcium chloride tube was charged with 100 ml of fuming nitric acid (specific gravity: 1.52 g / ml). The reaction vessel was cooled to 0 ° C and then 10 g of decagalacturonic acid (compound of formula II, n = 10) previously dried in vacuo over phosphorus pentoxide (10 g) was added slowly to the acid under vigorous stirring (15-20 minutes). . After the addition, the mixture was stirred at 0 ° C for 2 hours and then poured onto 200 g of ice while stirring. The precipitated pale yellow powder was isolated by centrifugation, washed with 500 ml of distilled water and dissolved in 200 ml of acetone with vigorous stirring. The acetone solution is poured into 1000 ml of distilled water with stirring. The precipitated fibrous product is filtered off, washed with distilled water until the pH of the wash liquor reaches 3 and rinsed with a little alcohol. The product was dried in vacuo over phosphorus pentoxide at 35-40 ° C. Decagalacturonic acid dinitrate was obtained in 80% yield.

Repeat the above purification procedure to obtain a completely pure product in 72% yield.

Analysis: Calculated: C, 27.1; H, 2.27;

N, 10.53; 0, 60.1; Found: C, 27.9; H, 3.07;

N = 10.0%, O = 59.0%. 45 [AJJ, ⁹ · ⁵ = 296.15 (c = 0.388 g / 100 ml acetone). Acid number: 3.4 meq / g.

Infrared yellow: 3458, 3451 (very strong, H-bonded -OH), 3000, 2928 and 50

2982, 2950 (aliphatic CH), 1776.2, exceeding 1774.5 (aliphatic -COOH), 1660 and 1654 (asymmetric -ONO ₂ ), 1384 and 1384 (β-OH deformation vibration), 1281 (symmetric -ONO) ₂ ), 1144 (CC, CO, C-H vibration vibration), 1060 (COH) deformation vibration), 1026 and 1022 (γ COH and RBC vibration), 998 and 986 [αϊ 1-4) 0 and RBC vibration, 840 (C-ONO ₂ valence vibration), 744 and 748 (β-OCO deformation and ring-breathing vibration), 666 and 684 (γ-OH deformation vibration) cm ^{1, respectively} .

Free decagalacturonic acid dinitrate is relatively easily degraded. Therefore, this compound is converted to its sodium salt and stored and used in the following reactions.

Other oligogalacturonic acids can also be converted to the corresponding dinitro compounds using the above procedure.

In the above process starting from polygalacturonic acid (compound of formula II, n = 136). Polygalacturonic acid dinitrate is obtained in a yield of 70-80%.

Example 2

Preparation of Metal Complexes of Oligogalacturonic Acid and Polygalacturonic Acid Dinitrates To a solution of oligogalacturonic acid or polygalacturonic acid dinitrate sodium salt (g = 10 or 136), previously dried in vacuo at 30-40 ° C in 30 ml distilled water, add 0.1 ml of distilled water Aqueous sodium hydroxide solution was added. The solids dissolve and the resulting solution has a pH of about 8. To the solution is added 60 mL of 0.1 M aqueous metal salt solution (100% excess based on metal) and the reaction mixture is diluted with dilute aqueous sodium hydroxide Adjust to table 3. The resulting solution is dialyzed against distilled water until the reaction of the cations and anions in question is negative. After dialysis, the solution is freeze-dried and the resulting metal complex is analyzed. The metal complexes are obtained in a yield of 96-98%.

By the above procedure, the metal complexes listed in Tables 3 and 4 are prepared. The metal complexes listed in Table 3 are characterized by their analytical data and the metal complexes listed in Table 4 are characterized by infrared spectroscopic data.

Table 3

Analytical data for decagalacturonic acid dinitrate metal complexes

Metal ion (s) Production pH Metal Analysis data N Calculated,% found,% So C H SHE K * 7.0 12.9 - 23.7 9.2 1.7 52.6 12.8 - 23.3 9.0 3.2 51.7 So* 7.0 8.0 8.0 25.0 9.7 1.8 55.5 7.9 7.9 23.8 7.7 3.0 57.6

Continuation of Table 3

Metal ion (s) Production pH Metal Analysis data Well C N Calculated,% found,% Η O Mg ² *, Na * 10.0 4.4 26.0 10.1 1.8 57.7 4.0 2.0 22.2 7.6 4.2 59.9 Ca ² *, Na * 10.0 7.3 25.3 9.8 1.8 56.1 7.0 0.2 25.5 8.1 2.5 56.7 Fe ² *, Na * 5.0 9.5 24.6 9.6 1.7 54.6 9.0 0.6 23.2 6.8 4.2 56.2 Fe ³ *, Na * 10.0 6.6 «25.4 9.9 1.8 56.4 8.6 0.2 23.4 6.0 2.8 59.0 Cu ² *, Na * 5.6 10.7 24.3 9.4 1.7 53.9 9.4 1.0 23.0 7.5 3.4 55.7 Cu ² *, Na * 7.0 10.0 24.5 9.5 1.7 54.3 10.5 1.6 24.0 8.0 4.8 51.0 Mn ² *, Well * 7.0 9.4 24.6 9.6 1.8 54.7 7.0 1.9 22.0 8.2 2.0 58.9 Zn ² *, Na * 7.0 11.0 24.2 9.4 1.7 53.7 9.0 1.8 21.3 7.2 3.2 57.5 Cr ³ *, Na * 5.0 6.1 25.5 9.9 1.8 56.6 6.0 0.1 20.4 6.2 2.0 65.3 Fe ³ *, Cu ² *, CO ² *, Na * X 25.1 6.3 2.0 51.0

^x Fe: 8.5%, Cu: 1.2%, Co: 0.26%

Table 4

Infrared spectroscopic data of metal complexes of oligonucleotides and polygalacturonic acid dinitrates

Group (1) (2) Metals in the complex (9) (10) 3) (4) (5) (6) (7) (8) H-bridge 3427 3428 3380 3350 3490 3600 3426 3350 3330 3580 knit — OH 3429 3440 3434 3436 3340 3432 3420 Aliphatic C-H 2983 2930 2944 2980 2940 2940 2936 2990 2980 2980 2943 2940 2939 2943 2940 2934 2940 anti Symmetric 1651 1650 1657 1658 1656 1660 1645 1656 1656 1660 -0N0 ₂ 1660 1653 1662 1660 1655 1657 1662 anti Symmetric 1600 1600 1610 1600 1628 1650 1625 1614 1616 1620 COC 1600 1600 1650 I650 1615 1610 1620 Symmetrical 1412 1399 1424 1434 1435 1443 1410 1423 1428 1420 COO 1423 1400 1440 1409 1407 1415 1409 deformation 1386 1360 1384 1384 1375 1384 1380 1384 1385 1380 / 3-OH 1386 1384 1386 1385 1385 1385 1385

Continuation of Table 4

Metals in the complex

Group (1) (2) (3) (4) deformation / 3 COH 1339 1340 1324 1322 1322 Symmetrical -ONO2 1284 1283 1282 1282 1282 C0 (0H) CC valence vibrations 1140 1143 1148 1145 1148 deformation C-OH 1054 1056 1058 1055 1066 Deformation γ-C-OH 1022 1022 1019 1025 1023 Frame and o (1-4) 0 995 980 ·,. . 968 970 970 C-ONO ₂ 850 847 844 843 842 β-OCO 748 747 748 745 748 deformation γ-ΟΗ 684 678 688 680 680

(5) (6) (?) (8) (9) (10) 1330 1325 1350 1314 1316 1310 1335 1325 1335 1330 1318 1320 1280 1280 1280 1282 1283 1278 1282 1283 1282 1282 1282 1282 1135 1145 1145 1145 1148 1144 1145 1152 1145 1144 1144 1146 1055 1060 1045 1050 1055 1060 1063 1065 1062 1062 1060 1060 1025 1025 1025 1033 1033 1023 1022 1027 1022 1022 1021 1019 955 955 956 960 970 970 955 955 960 970 965 970 840 840 836 842 842 838 837 840 836 842 843 845 745 750 748 746 746 744 745 747 746 747 748 745 670 680 635 680 680 682 668 668 680 683 682 686

Explanation for Table 4:

(1) Decagalacturonic acid dinitrate sodium complex 35 (2) Polygalactone dinitrate t-potassium complex (3) Decagalacturonic acid dinitrate calcium and sodium complex (4) Decagalacturonic acid dinitrate complex with magnesium 40 Complex of polygalacturonic acid dinitrate with magnesium and sodium (lower data) (5) Complex of decagalacturonic acid dinitrate with iron (III) and sodium (upper data), polygalacturonic acid dinitrate with iron (HI) and sodium (complex) lower data) (6) Decagalacturonic acid dinitrate chromium (III> complex with sodium and sodium (upper data), polygalacturonic acid dinitrate chromium (in) and sodium complex (lower data) ₅₀ (7) Decagalacturonic acid dinitrate copper (II) ) and sodium (upper data), polygalacturonic acid dinitrate complex with copper (II) and sodium (lower data) (8) Decagalacturonic acid dinitrate with cobalt and sodium mmol complex (upper data), polygalacturonic acid dinitrate complex with cobalt and sodium (lower data) (9) Decagalacturonic acid dinitrate with manganese and sodium complex (upper data), polygalacturonic acid dinitrate manganese (data) Zinc and sodium complex of decagalacturonic acid dinitrate (upper data), polygalacturonic acid dinitrate zinc and sodium complex (lower data)

Table 5

Hyperfine coupling

Metal ion (s) constants (in G)

A _o THE THE, Μη ² *, Well * -91.76 - - VO ² *, Well * (1) 111.4 196.1 73.6 (2) 108.5 187.8 68.9 Cu ² *, Na * -154.45 22.246

Spectroscopic rupture factor go 8 "to 8,

2,003

1,9366 1,993

Line width (in G)

2,3184 2,070

The EPR spectroscopic data of some polygalacturonic acid dinitrate metal complexes are summarized in Table 5.

From the data listed in Table 5, it can be seen that manganese (II) ions and sodium ions in hydrated form are arranged in an outer spherical complex while copper (II) ions are directly attached to polygalacturonate dinitrate anions in an inner spherical arrangement , The vanadyl ions are linked in both types (1) and (2).

Stability of the decagalactyronic acid dinitrate complex with copper (II) and sodium as determined by Orion type metal selective electrode

In a pH 4.5 medium containing 1 M NaNO ₃ (sodium nitrate provides practically constant ionic strength) pK = 3.4, thus forming a moderately stable complex.

Example 3

Preparation of injection preparation containing decagalacturonic acid dinitrate potassium (I) iron (IH) complex

Prepared as described in Example 2

Dissolve 2.6 g of decagalacturonic acid dinitrate potassium (I) - iron (III) complex in 100 ml of double-distilled water and place the resulting solution, containing 1.72 mg Fe / ml, in a 100 ml aluminum-stoppered rubber stopper. The vial was sealed and steam sterilized at 110 ° C for 30 minutes. A liquid for injection is obtained from which the volume of solution required for treatment is withdrawn by injection syringe.

Patent claims:

Claims (2)

Patent claims: 1. A process for preparing ^five steps in the oligo- or polygalacturonic dinitrate-metal complexes of general formula (I) - wherein n is an integer between 10 and 150 M an alkali metal ion, alkaline earth metal ions and / or transition metal ion in a 3d-court, provided that the metal complex contains only an alkali metal ion or an alkali metal ^10- ion and at least one other non-alkali metal ion and z represents an integer equal to the valence or oxidation number of the metal ion, characterized in that general oligo- or polygalacturonic dinitrate 15 - wherein n is as defined above - or an alkaline aqueous medium of less than one or more M ^{z +} metal ions of formula a salt or formed oligo- or polygalacturonic dinitrate complex of stabi20 Bending constant complex, or alkali metal ions entering in addition to those listed with an alkali metal hydroxide 2. Improvement of the process according to claim 1 for the preparation of pharmaceutical compositions, preferably injectable preparations, characterized in that a complex of the formula I - wherein η, M and z are as defined in claim 1 The compounds of the present invention are formulated into a pharmaceutical composition, preferably an injection composition, using conventional pharmaceutical diluents, carriers and / or excipients, optionally in the presence of other biologically active compounds which do not show synergistic effects. Figure 2 Responsible for publishing: Director of the Economic and Legal Publishing House 824394 - Zrínyi Nyomda, Budapest