Salts of 2-deoxy-2-amino (or 2-methylamino)-d-glucose with n - greenexpo acid having antimicrobial activity


(57) Abstract:

Claimed synthetic biologically active compounds belonging to the class of derivatives of amino sugars. Salts of 2-deoxy-2-amino (or 2-methylamino)-D-glucose with N-greenexpo acid, of General formula given in the text of the description, where R = -H or-CH3possessing antimicrobial activity. Proposed chemical and structural formulas, examples of the synthesis and the results of biological tests on models of bacterial DNA - and RNA-containing viruses. table 4.

The invention relates to medicine, namely to synthetic set of biologically active dietary compounds belonging to the class of derivatives of amino sugars.

Salts of 2-deoxy-2-amino (or 2-methylamino)-D-glucose with N-greenexpo acid of General formula

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where R= -H or-CH3< / BR>
Both of the proposed compounds have a strong effect against several pathogens: DNA and RNA genome viruses, and also able to resolve the resistance of gram-positive and gram-negative bacteria to antibiotics of different classes.

The main component (cation) of the claimed compounds is 2-deoxy-2-amino (or 2-methylamino)-D-glucose is Asia instead of one or more alcoholic hydroxyl groups, amino - or aminoalkyl group.

The largest group of natural amino sugars, which have important biological value, is 2-deoxy-2-amino-hexalogy.

They are structural components of glycoproteins, mucopolysaccharides, aminopolysaccharide and certain antibiotics (aminoglycosides).

The greatest biological importance 2-deoxy-2-amino-D-glucose (chitosamine) and 2-deoxy-2-methylamino-D-glucose (methylhistamine) [1]

It is known that both of these amino sugars participate in key stages of cellular metabolism. So, fructose-6-phosphate interaction with glutamine enters 2-deoxy-2-amino-glucose-6-phosphate. This connection, interacting with acetylcoenzyme And turns into a 2-deoxy-2-acetylaminofluorene-6-phosphate, which isomerizes 2-deoxy-2-acetylamino-glucose-1-phosphate, and further, through a series of stages, forms N-acetylmuramyl and N-acetylneuraminic acid, which are inducers of education interleukin-2 (T-cell growth factor), which is a key factor in the immune response during infection by micro-organisms [2,3]

Both these amino sugar directly participate in the synthesis of glutamylcysteine, which is associated with a number of promotional is s), that violates the integrity of the membranes of the bacteria and, therefore, its viability [4]

The most important biological significance of natural amino(alkylamino)of hexoses has been the subject of numerous studies on the establishment of pharmacological preparations on their basis. Well known, for example, salts of N-methylcytosine with polyene macrolide antibiotic amphotericin b (infopyramid), and 5-methyluracil (amiloretic), widely used in medical practice [5]

The second biologically active component (anion) is criconematina acid.

In 1972 Fryer and Grunberg first discovered that certain derivatives of acridone have strong antiviral properties [6]

In the future, Inglot, A. D. et al. showed that the sodium salt of N-greenexpo acid has the interferon-inducing activity in mice [7]

Further research helped to create on the basis of greenexpo acid highly active drugs: Caledon and its structural analogue of neovir [8-11]

Work conducted in 1991-1994 year, has revealed a very interesting feature: N-criconematina acid and /or its salts, when combined, espresso change their properties, in some cases, acquiring new types of pharmacological activity, is not peculiar to the components, taken separately [12]

Interesting attempt of the authors of the International patent application N PCT/EN 94/00032 [13] to obtain a compound of greenexpo acid with 1-deoxy-1-methylaminoethanol. The authors of the patent N-methyl-N-/, -glyukopiranozil/ammonium-2/-acridin-9-one-10-Il/acetate called them cold with their data interferonogenna, anti-viral including anti-HIV, anti-parasitic, antipredatory, radioprotective activity, formulas

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This connection is the closest structural analogue of the described compounds.

Proposed by the authors of the prototype connection is identical to the claimed compounds their acridinium fragment, but dramatically different structure of the carbohydrate component, as used 1-deoxy-1-methylamine - hexose. It is known that the amino sugar 1-deoxy-1-methylaminorex in nature do not occur, which is highly unstable in solution, relative to the highly toxic and very expensive. The synthesis of these compounds from natural hexoses (glucose, galactose) includes a number of labor-intensive stages that occur with low yields and in tough conditions. In addition, the way Metabox chemical compounds derivatives of natural 2-deoxy-2-amino (or 2-alkylamino)-D-glucose, with pronounced antimicrobial activity against DNA and RNA genome of the virus, as well as having the ability to eliminate the resistance of gram-positive and gram-negative bacteria to antibiotics of different classes and with lower toxicity and higher stability in solution and less labor intensive synthesis than the prototype.

The essence of the invention.

The problem is solved by the synthesis of new chemical compounds, namely salts of 2-deoxy-2-amino (or 2-methyl-amino)-D-glucose, the General formula

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where R= -H or-CH3< / BR>
It is known that one of the fragments of the molecule: 2-deoxy-2-amino-D-glucose and 2-deoxy-2-methylamino-D-glucose currently produced industrially, gidrolizu chitin (shells of crabs and other crustaceans) in alkaline conditions, with the release of the final products more than 80% [16]

Physico-chemical properties of these amino sugars are well studied [17, 18]

2-Deoxy-2-amino-D-glucose and its N-methyl analogue approved for use in medicine, where they found wide application [5]

The second fragment of the claimed compounds (anion): N-locusts is>/BR>The synthesis of the claimed compounds are obtained by salt formation due to a significantly different acid-base properties. It is known that such compounds due to mild acid-base properties are usually highly unstable in solutions, so the manufacture of dosage forms based on them was considered impossible. In the process carried out by the applicants studies unexpectedly revealed that the used connection 2-deoxy-2-amino(or 2-methylamine)-D-glucose with N-greenexpo acid allowed us to obtain stable during prolonged storage (>2 years) aqueous solutions of the inventive salts, with all the pharmacological properties of the prototype and, in addition, significantly higher antiviral activity, as well as new property: able to eliminate the resistance of gram-positive and gram-negative bacteria to antibiotics of different classes.

When this was developed by applicants synthesis methods are more economical than that of the prototype.

The invention is illustrated by two examples of synthesis and four tables of biological tests.

Example 1. The method of synthesis of salts of 2-deoxy-2-amino-D-glucose with N-greenexpo acid.

Table 2. Protective effect of the proposed drug, comedone and prototype models of smallpox infection in cotton rats.

Table 3. The influence of the proposed drugs and prototype on the course of pneumococcal pneumonia in white mice.

Table 4. The influence of the proposed drugs and prototype for alisasis in mice.

The NMR spectra of H1and C13measured on a spectrometer Bruker-200 and the supporting structure of the obtained compounds can optionally be provided on request examination.

Example 1. 100 g of N-greenexpo acid are dissolved in 480 ml of hot absolute ethyl alcohol, in small portions under vigorous stirring contribute 71,15 g 2-deoxy-2-amino-D-glucose. The obtained pale-yellow solution is refluxed for 20 minutes, cooled to 5oC, filtered. The filter cake was washed with 150 ml of cold absolute ethanol, dried in a vacuum at a temperature of 65-70oC. Get 157,5 g (yield 92% ) of light yellow crystalline precipitate salts of 2-deoxy-2-amino-D-glitch is giving does not require recrystallization.

Salt of 2-deoxy-2-amino-D-glucose with N-acrimoniously acid has no distinct melting point above 130oC slowly charred.

To characterize the purity of the synthesized compounds is the most convenient method UV-spectroscopy. Studies have shown that the purity of the compounds 99.5% of the ratio of the specific extinction E1%1is 2001.

Molecular weight: 432, 43

Brutto-formula: C21H24N2O8< / BR>
Elemental analysis:

Calculated with 58.33 C H 5,59 N 6,48

Found, C 58,37; H 5,62; N, 6.42 PER

These NMR (characteristic signals) H1 DMSO d6:

7,3-8,4 m D. the area of the signals of the aromatic heterocyclic system acridone (8 H);

4,9 memorial plaques (s, 2H) signal of the protons of the N-medienkombination;

4,45 M. D. (d, 1H) signal H1 proton of amino sugar;

3,1 M. D. (d, 2H) signal H6 protons of the amino sugar;

C13 DMSO (d6):

176,75 171,18 M. D. carbonyl and carboxyl carbon atoms of the N-th greenexpo acid;

93,92 M. D. carbon atom in the amino sugar with polyacetylenes hydroxyl group;

50,32 M. D. carbon atom in the amino sugar that is associated with the amino group.

LD 50 (intravenous, white mouse) is 620 mg/kg

Example 2. 150 g of N-AK for 15 min make 115 g of 2-deoxy-2-methylamino-D-glucose. The resulting mixture was refluxed for 30 min and then cooled to a temperature of 0 to 2oC. this forms the bottom layer of thick dark yellow oily product. The upper liquid colorless layer practically does not contain the reaction product, decanted, and the oily residue is washed 2 times with absolute ethanol and 1 times with ethyl acetate, then dried in vacuum at a temperature not exceeding 90oC. Obtain 235 g (yield 89%) of a yellow, transparent, glassy, solid, hygroscopic product salt of 2-deoxy-2-methylamino-D-glucose with N-greenexpo acid. The compound obtained is recrystallized from absolute ethanol.

Melting point above 85oC slowly liquefies.

The specific extinction coefficient E1%1cm1941

Brutto-formula C22H26N2O8< / BR>
Molecular weight: 446,46

Elemental analysis:

Calculated C 59,19 H by 5.87; N 6,27

Found, C 59,11; H 5,85 N 6,23

These NMR (characteristic signals) H1 DMSO d6

7,2-8,4 m D. the area of the signals of the aromatic heterocyclic system acridone (8H);

4,85 m DV (s, 2H) signal of the protons of the N-medienkombination;

4,85 m D. (d, 1H) signal H1 proton is urbanely and carboxyl carbon atoms of the N-th greenexpo acid;

116,47 M. D. carbon atom in the amino sugar with polyacetylenes hydroxyl group;

50,55 M. D. carbon atom in the amino sugar that is associated with the amino group;

33,04 M. D. carbon atom in the N-CH3group.

LD 50 (intravenous, white mouse) is 580 mg/kg

Industrial applicability.

In the experiments for the determination of the biological activity used 10% sterile aqueous solutions of drugs: compounds salts of 2-deoxy-2-amino-D-glucose(1) and salts of 2-deoxy-2-methyl-amino-D-glucose (2), and the Comparators used Caledon in the form of a 12.5% aqueous solution of production NIF "Medical" and cycloferon (prototype) in the form of a 12.5% aqueous solution produced by LLC "Polysan". Both drugs comparison were acquired in veterinary pharmacy network in St. Petersburg.

Study of antiviral activity of the claimed compounds: (1) salts of 2-deoxy-2-amino-D-glucose and (2) salts of 2-deoxy-2-methylamino-D-glucose was carried out in experimental encephalomyelitis of horses (VAL).

The experiments were performed on weinbrenner white mice-males weighing 16-18g. Pathogen VAL was administered at a dose of 6 and 60 LD50 Drugs (1) and (2) was applied subcutaneously at a dose of 50 mg/kg, preventive (-4 h to Sarai is Vania used Caledon and cold. The effectiveness of antiviral drugs was assessed by the level of the death of the mice. The results of the experiment are presented in table.1

As follows from the presented data, products (1) and (2) have a pronounced protective effect in experimental VAL, particularly for prophylactic purpose in this case they are much superior to Comparators Caledon and cold.

The influence of the proposed drugs on the course of experimental smallpox infection.

The experiments were conducted on experimental orthopoxvirus infection. In experiments were used of male rats weighing 50-70 g, the virus was administered intranasally. Infected animals was a constant dose of the virus, equal 10LD50.

The drugs were injected rats intramuscularly and subcutaneously at a dose of 50 mg/kg for 4 h before and on the second day after infection of laboratory animals.

Drugs (1) and (2) used in the form 10-percentage of sterile solutions. In each experiment as the reference drug used ribamidil (virazole), which was given to rats orally at a dose of 100 mg/kg for standard medical scheme. The data obtained are presented in table. 2.

From the presented data we can conclude that the AOR 80% of infected cotton rats.

The study of protective properties of the proposed drugs and prototype in experimental pneumonia.

Use of male mice weighing 18-20g. Animals were infected intranasally pneumococcus serotype 1 (strain 395, in a dose -5106microbial cells in the mouse.

1. Drugs (1), (2) and the comparison drug cycloferon was administered at a dose of 50 mg/kg subcutaneously. Treatment by medical scheme: (-4; +48; +120).

2. Drugs used in the same doses, but in combination with gentamicin. Antibiotic therapy was started after 1 day. after infection of mice with pneumococcal, the drug was administered intramuscularly within 8 days at a dose of 5 mg/kg

Set (PL. 3) that claimed the drugs alone, practically no influence on the course of pneumococcal infection in mice, but in combination with gentamicin significantly increase the protective action of this antibiotic. The drug cycloferon (prototype) had no effect alone or in combination with the antibiotic.

Evaluation of the activity of the claimed compounds and the prototype model of acute alisasis white mice.

White mice weighing 16-18 g were infected intraperitoneally with Escherichia coli strain 60 on the URS of treatment for medical scheme: (-48; -24; +2).

2. Drugs used in the same doses, but in combination with intramuscular antibiotic ampicillin dose of 5 mg/mouse.

Set (PL. 4) that the claimed drugs, taken individually, have an impact on the course of infection in mice, reducing the case fatality rate up to 60-70% Microbiological study of the colonization of the internal organs of the experimental and control groups show that preventive and therapeutic effect of the claimed preparations contribute to the rehabilitation of the spleen and kidney from Escherichia coli.

In combination with ampicillin drugs (1) and (2) significantly increase the protective action of this antibiotic, reducing mortality up to 20% of the Drug cycloferon (prototype) had no effect alone or in combination with the antibiotic.

Thus, from the examples presented and the results of biological tests, it follows that the advantages of the claimed medicinal products: salts of 2-deoxy-2-amino-D-glucose with N-greenexpo acid, and salts of 2-deoxy-2-methylamino-D-glucose with N-greenexpo acid are:

the emergence of strong new properties that are absent in both of the components, taken separately, such as the ability to eliminate re/BR> a wide range of pharmacological activity and the absence of any side effects with long term use;

the solutions proposed drug is extremely stable during prolonged storage (observation period 2 years) and have a pH close to physiological (7,1-7,5);

getting these medicines are easily implemented in an industrial environment, as 2-deoxy-2-amino-D-glucose, 2-deoxy-2-methylamino-D-glucose and N-criconematina acid produced on the number of existing pharmaceutical production;

metabolic pathways of 2-deoxy-2-amino-D-sexualidad studied in detail and found that none of the products of metabolism of these compounds is not toxic, and N-criconematina acid is excreted in unchanged form.

Thus, the claimed compounds are new and non-obvious and industrially applicable, i.e., meet all the requirements of the invention.

Salts of 2-deoxy-2-amino(or 2-methylamino)-D-glucose with N-greenexpo acid of General formula

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< / BR>
where R is H or-CH3,

possessing antimicrobial activity.


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