RU2368609C1 - Silver salt of n-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5h-pyrimidinesulphone)-n'-isonicotinoylhydrazide, which exhibits immunotropic, antimicrobial, antifungal (antimycotic) activity and medicinal agent based on said salt - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a new silver salt of N-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulphone)-N'-isonicotinoylhydrazide, which exhibits immunotropic, antimicrobial, antifungal (antimycotic) activity, obtained by adding silver nitrate to an aqueous suspension of N-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulphone)- N'-isonicotinoylhydrazide, thoroughly mixed to a homogeneous state, in molar ratio of hydrazide to silver nitrate from 1:1 to 1:4 in the absence of light until a bulky residue is obtained, which is later separated. Usually N-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulphone)-N'-isonicotinoylhydrazide in fine dispersed state is used to obtain the silver salt. Normally, the salt is extracted through filtration, repeated washing with bidistilled water and drying at temperature 50-80°C. The invention also relates to a medicinal agent, containing the silver salt of N-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulphone)-N'-isonicotinoylhydrazide as active substance, obtained using the method described above, or a mixture of N-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulphone)-N'-isonicotinoylhydrazide and its silver salt in ratio of 1:1 to 4:1, possibly in form of tablets, capsules, ointment, suppositories.

EFFECT: more effective application.

4 cl, 3 tbl, 9 ex

Description

The invention relates to medicine, namely to the creation of medicines based on new biologically active substances, in particular to the silver salt N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulfone) -N'-isonicotinoyl hydrazide, exhibiting immunotropic, antimicrobial, antifungal (antimycotic) activity, the method of its production and a drug based on it.

According to the WHO, at present, the first characteristic of the state of public health in the world is a decrease in immunoreactivity: according to various sources, up to 50-70% of people have immunity disorders. And the second feature arising from the first is an increase in the frequency of diseases caused by opportunistic microbiota. In this regard, recently, drugs that have a dual effect - immunomodulatory and antimicrobial, are of particular relevance.

Known immunomodulator N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulfone) -N'-isonicotinoyl hydrazide hydrate (isophone) (RF patent No. 2141322), possessing antimycobacterial properties not only in relation to mycobacteria leprosy, but also tuberculosis, as well as other diseases that occur against the background of immunodeficiency. An isophone is prepared by boiling equimolecular amounts of isonicotinic acid hydrazide and 6-methyluracil-5-sulfochloride in anhydrous acetonitrile. The disadvantage of the isofone immunomodulator with antimycobacterial activity is its low biological activity, due to poor solubility in water, which is confirmed by an experiment on animals (rabbits), which shows that about 50% of the isofone is unchanged in vital products. Along with the name "IZOPHON", the immunomodulator has the widespread trade name "KRISTAFON".

Domestic immunomodulatory drugs with antimycobacterial activity, cristafon and isophon normalize the immune status of patients with leprosy, tuberculosis, chronic non-specific pneumonia and chlamydia, including the body in the fight against the disease, as well as penetrating the cells, stimulate its function and bring the active principle to the pathogen, destroy it . They have an effect on sexually transmitted pathogens (chlamydia, Trichomonas, ureaplasma infections). They are well tolerated and do not show side effects. Typically, these drugs are introduced into the human body in tablet form in a sufficiently large dose. With local administration of substances, it is necessary to increase the effectiveness of the drug either by introducing other drugs that have a synergistic effect, or by modifying the substance isophone and cristaphone.

Closest to the invention is the modification of isophone-disodium salt of isophone (isodinafon, RF patent No. 2191015), exhibiting antimicrobial and immunotropic activity. Isodinafon is prepared by reacting equimolar amounts of isophone and sodium hydroxide in water, followed by precipitation of the reaction product with ethanol. The drug as an active substance contains isodinafon in an amount of 0.01-2 g and can be used in the form of capsules, tablets, injections and ointments. The use of isodinafon as a drug can reduce the dose of the drug used, thereby reducing the cost of treatment. However, a compound of this kind, despite the advantages in the solubility of the drug in water, easily decomposes into two parts according to the hydrazine moiety: sulfopyrimidine derivative (III), isonicotinic acid derivative (IV), and other compounds.

Accordingly, one should expect a decrease in the activity of the drug isodinafon and the manifestation of the biological activity of its decay products. In addition, this modification of the isophone has a fairly narrow spectrum of bactericidal action. So, isodinafon showed bactericidal antimicrobial activity in the case of B. cereus, and bacteriostatic antimicrobial activity in the case of St. Aureus.

The problem to which the invention is directed is to obtain a new compound based on derivatives of pyrimidone and isonicotinic acid hydrazide, which exhibits enhanced immunotropic, antimicrobial, antifungal (antimycotic) activity in comparison with cristaphone and isophone, and the creation of a drug based on it.

The problem is solved by a group of inventions, united by a single inventive concept, namely: a method for producing a new compound - silver salt of cristaphone and a medicine based on it.

The new compound is the silver salt of N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulfone) -N'-isonicotinoyl hydrazide (cristaphone), which shows immunotropic, antimicrobial, antifungal (antimycotic) activity obtained by adding to a pre-intensively mixed to a homogeneous aqueous suspension of N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulfone) -N'-isonicotinoyl hydrazide silver nitrate with a molar the ratio of hydrazide to silver nitrate from 1: 1 to 1: 4 without access of light to obtain volume homo gene precipitate, which is then isolated.

N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulfone) -N'-isonicotinoyl hydrazide is used in a finely divided state, the resulting salt being isolated by filtration, washing repeatedly with distilled water and drying under temperature 50-80 ° С.

The problem is also solved by a drug having immunotropic, antimicrobial, antifungal activity, containing as an active substance the silver salt of N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulfone) - N'-isonicotinoyl hydrazide or a mixture of N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulfone) -N'-isonicotinoyl hydrazide and its silver salt in a ratio of from 1: 1 to 4 :one.

The drug can be made in the form of tablets, capsules, ointments, suppositories.

According to the invention, the silver salt of cristaphone may be a compound of formula I

or a compound of formula (II)

or a mixture of compounds (I) and (II).

The essence of the proposed invention lies in the fact that to enhance the immunotropic, antimicrobial, antifungal (antimycotic) activity due to the additional action of silver, it is proposed to use a substance that is a silver salt of cristaphone.

Silver has a powerful bactericidal, immunomodulatory, astringent, analgesic properties and has a stimulating effect on the blood-forming organs. Most widely used in medicine is the ability of silver to have a bactericidal effect in small doses, and only silver ions have activity. Bactericidal activity is already apparent at a concentration of 0.0001%. The spectrum of action of silver preparations is much wider than many antibiotics and sulfonamides. It includes both gram-positive and gram-negative bacteria (including antibiotic-resistant strains), viruses and fungi. Thus, based on the above properties, silver exhibits an oligodynamic effect.

Introducing silver into the body is difficult, because its ions directly at the injection site form sparingly soluble complexes with negatively charged proteins and a number of functional groups of enzymes, and therefore there is an acute problem of choosing an effective dosage form.

Lactim-lactam (prototropic) tautomerism is characteristic of a cristaphone in an aqueous solution, and, in fact, the cristaphone, when passing into a solution, exists in two tautomeric forms - lactam (keto form) and lactim (enol form).

The lactim enol form acquires an aromatic character and the hydroxyl group in the pyrimidine ring acquires the properties of a phenolic hydroxyl. The interaction of a suspension of cristaphone in water with silver nitrate AgNO ₃ forms a silver salt (isomers).

Наиболее вероятно образование следующих структур серебряной соли кристафона (I), (II) или их смеси.

The formation of the following structures of the silver salt of cristaphone (I), (II) or a mixture thereof is most likely.

A silver ion is present in the silver salt molecule of cristaphone, which can extremely slowly dissociate in a lipophilic medium, releasing silver ions, which ensures a prolonged effect of the preparations. The critical factor for the bactericidal action in the wound is the required local concentration and the possibility of achieving this level by systemic administration or local application. If local blood supply is compromised, for example, with limb ischemia, tissue edema or thermal coagulation, as well as with scab and / or lipoderma sclerosis, the chances of systemic therapy to achieve therapeutic concentrations are significantly reduced, while silver salts can provide the necessary concentration due to long-term preservation in lipid tissues (Belikov VG "Modern synthetic and natural medicines", Pyatigorsk, 2003), (The use of silver preparations in medicine, Novosibirsk, 19 94 g, p. 106).

In addition, the bactericidal action of both silver ions and silver nanoclusters is known, capable of forming from the silver salt of cristaphone in the human body. It is known that silver nanoclusters are specific nanoreactors that act similarly to some enzymes. Probably, due to this function, an increase in immunotropic activity occurs.

On the other hand, although the mechanism of the bactericidal action is not yet known exactly, it is believed that silver ions released from the lipophilic compound into the lipophilic medium inhibit a specific enzyme that is involved in the metabolism of many types of bacteria, viruses and fungi.

The resulting compound - silver salts of Christophone - can be used as an independent drug, and in various proportions with Christophone to enhance the effect of the latter.

The invention is illustrated by the following examples.

Example 1. A method of obtaining a silver salt of N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinesulfone) -N'-isonicotinoyl hydrazide (cristaphone).

An aqueous suspension containing 2 g (0.0061 mol) of cristaphone is pre-treated with ultrasound at a frequency of 22-44 kHz until a homogeneous dispersion is added, silver nitrate 4.2 g (0.0244 mol) is added. Then, without access to light, the reaction mixture is sonicated to obtain a homogeneous volumetric precipitate. The precipitate obtained is filtered, washed repeatedly with double-distilled water and dried in a vacuum oven (100-200 mm Hg) at a temperature of 50-80 ° C. Get a powder of white or white with a grayish tint. In the resulting mixture, 24.88 wt.% Of silver is found, which corresponds to 99.8 purity of silver salt. The amount of silver was determined by titration of ammonium thiocyanate (Folhard's method) in dilute HNO ₃ (HF X) medium. As an indicator, a solution of iron-ammonium alum is used. Titration is stopped after the appearance of a color of the thiocyanate complex of iron that does not disappear with vigorous shaking of the solution.

In the PMR spectrum of the silver salt of cristaphone after treatment in an AgNO ₃ solution, a proton shift δ of the pyridine ring from 7.65 (cristaphone) to 7.8 (silver salt of cristaphone) ppm (further ppm), a shift of triplets of CH ₃ group from 2.3-2.5 (cristaphone) to 2.0 (silver salt of cristaphone) ppm, the absence of changes in the chemical shifts of protons in the hydrazide group, fundamental changes in the part of the spectrum of the pyrimidine fragment: the absorption intensity in the silver salt of cristaphone decreased by half compared to cristaphone, silver in the PMR spectrum hydrochloric salt kristafona marked with new doublet δ = 10,7; 10.8 ppm, and a new singlet appears in the region of the phenolic group δ = 5.2 ppm.

The structure of the cristaphone silver salt was determined by comparing the IR spectra of the cristaphone and its silver salt. The analysis of the IR spectra was carried out on two reference bands in the region of 3307 cm ^-1 and 1743 cm ^-1 (СО in position 2), characterizing the carbonyl group in the pyrimidine cycle of the keto-form of the cristaphone, as well as the group of intense bands in the region of 3200-3000 cm ^{- 1} (NH at position 3). In the IR spectrum of the silver salt of cristaphone, the band 1743 cm ^-1 disappears, and the absorption intensity of the group of bands 3200-3000 cm ^-1 sharply decreases, which indicates the formation of the silver salt of cristaphone.

Example 2. A method for producing silver salt of cristaphone.

An aqueous suspension containing 1 g (0.003 mol) of cristaphone is preliminarily mixed thoroughly on a mechanical stirrer 3000 rpm until a uniform dispersion is added, silver nitrate 1.03 g (0.006 mol) is added. Then, without access to light, the reaction mixture is stirred on a mechanical stirrer until a homogeneous volumetric precipitate is obtained. The precipitate obtained is filtered, washed repeatedly with double-distilled water and dried in a vacuum oven (100-200 mm Hg) at a temperature of 50-80 ° C. Get a powder of white or white with a grayish tint.

In the resulting mixture, 24.78 wt.% Of silver is found, which corresponds to 99.4 purity of silver salt.

Analysis of the PMR and IR spectra of the reaction products in examples 1 and 2 showed the identity of both products and their correspondence to the silver salt of cristaphone.

Example 3. A method of obtaining a silver salt of cristaphone.

An aqueous suspension containing 1 g (0.003 mol) of cristaphone is preliminarily mixed thoroughly on a mechanical stirrer 1000-3000 rpm until a uniform dispersion is added, silver nitrate 0.516 g (0.003 mol) is added. Then, without access to light, the reaction mixture is stirred on a mechanical stirrer until a homogeneous volumetric precipitate is obtained. The precipitate obtained is filtered, washed repeatedly with double-distilled water and dried in a vacuum oven (100-200 mm Hg) at a temperature of 50-80 ° C. Get a powder of white or white with a grayish tint.

In the resulting mixture, 24.73 wt.% Of silver is found, which corresponds to 99.2 purity of silver salt.

Analysis of the PMR and IR spectra of the reaction products in examples 1, 2 and 3 showed the identity of all products and their correspondence to the silver salt of cristaphone.

Examples 4-5. Preparation of mixtures of cristaphone and its silver salt.

For preclinical studies, mixtures of pure cristaphone and silver salt of cristaphone with a purity of 98.3-99.5 were used with mass ratios of 50:50 (wt.%) (Example 4), 80:20 (wt.%) (Example 5).

Example 6. The manufacture of suppositories with silver salt of cristaphone, cristaphone and their mixtures.

From cristaphone, silver salt of cristaphone (examples 1-3) and their mixtures taken in the ratio specified in examples 4-5, suppositories based on polyethylene oxide and Vitepsol based in weight of 1.5 g were made. Each suppository contains 0.2 g of substance (cristaphone, its silver salt or a mixture thereof).

For the manufacture of suppositories in a base warmed up in a water bath (up to 60-70 ° C for a polyethylene oxide base and up to 40-60 ° C for a Vitepsol base) in an amount of 13 g, a cristaphone (or its silver salt or a mixture of them) pre-ground in a mortar is introduced in an amount of 2 g. The resulting mixture was thoroughly and vigorously stirred until a homogeneous suspension. The resulting suspension is poured into the nests of the mold for the manufacture of suppositories, after solidification, the suppositories are removed from the mold and packaged.

Example 7. The results of preclinical trials of preparations containing cristaphone, silver salt of cristaphone and their mixture for the presence of antimicrobial and antifungal activity in in vitro experiments.

The determination of the antimicrobial and antifungal action of the substances proposed according to the invention and their combinations was carried out according to the standard procedure approved by the Ministry of Health of the State Pharmacopoeia, M., "Medicine", 1990, issue. 2, “General methods of analysis. Medicinal plant material. "

Tests conducted in relation to:

S.aureus - museum (control strain) number 906, obtained from the GISK named after Tarasevich;

E.coli M-17 - museum (control strain) number B-8208, obtained from the Tarasevich GISK;

Ps.aeruginosae - isolate isolated from the patient;

C.albicans is an isolate isolated from a patient.

The results of the in vitro test on S.aureus, E.coli, Ps. Aeruginosae, Candida albicams determine the antimicrobial, antifungal (antimycotic) activity of the silver salt of cristaphone and its mixture with cristaphone are summarized in table 1.

Table 1 Preparations S.aureus E.coli Ps.Aerugino sae Candida albicams Christafon ++ Silver salt of Christophone +++ +++ +++ + Christaphone: Silver salt of Christophone 1: 1 +++ +++ +++ + Christaphone: Christophon's Silver Salt 4: 1 ++ +++ +++

As can be seen from table 1, the silver salt of cristaphone has a pronounced antimicrobial effect in relation to all tested cultures and a small antifungal effect in contrast to cristaphone, which is active only against S.aureus.

Example 8. The results of preclinical studies on the effectiveness of drugs containing silver salt of cristaphone in the treatment of bacterial vaginitis in rats in comparison with the well-known drug Betadine, vaginal suppositories.

As pathogens for infection, S.aureus (type strain 7M (NNIEM, N.Novgorod)) and E.coli (strain 782-2) were selected as one of the most common causative agents of bacterial vaginitis. The study of the microbiocidal activity of the preparations was carried out by the method of serial dilutions according to the previously described recommendations (Medical Microbiology, 1999; Encyclopedia of Clinical Laboratory Tests, 1997; Laboratory Research Methods in the Clinic, 1987).

Table 2 presents the results of changes in the cytological picture of vaginal smears of rats in points. When cytological examination adhered to the following criteria when scoring: 1 - single bacterial cells; 2 - few cells; 3 - a moderate number of cells; 4 - a lot of cells.

table 2 A drug Microflora, M ± SEM 5 days after the use of the drug 10 days after using the drug Silver salt of Christophone 1.7 ± 0.78 2.4 ± 1.32 Christaphone: Christophon's silver salt 1: 1 2.3 ± 0.97 2.7 ± 1.08 Christaphone: Christophon's silver salt 4: 1 3 ± 0.55 4 ± 1.56 Christafon 3 ± 1.23 3.3 ± 2.23 Control No. 1 (without treatment) 4 ± 0.56 4 ± 0.25 Control No. 2 (Betadine) 3.4 ± 0.88 3.3 ± 0.76

From the results obtained, it can be seen that for all preparations containing the silver salt of cristaphone, the effect is greater than in the absence of treatment or in the treatment with Betadine.

Example 9. The results of microbiological studies.

In microbiological studies, the most pronounced decrease in S.aureus was observed when using a drug containing a mixture of cristaphone and its silver salt in the vaginal cavity of rats after invaginal administration of drugs.

Table 3 A drug Decrease in the number of CFU of S.aureus strain 7M (in the number of times relative to the initial level of infection) The decrease in the number of CFU E. coli strain 782-2 (the number of times relative to the initial level of infection) 5 days after the use of the drug 10 days after using the drug 5 days after the use of the drug 10 days after using the drug Silver salt of Christophone 4.58 ± 1.12 6.92 ± 2.94 9.69 ± 1.8 25.6 ± 2.6 Christaphone: Christophon's silver salt 1: 1 7.41 ± 2.34 6.67 ± 4.44 6.68 ± 4.29 4.93 ± 1.37 Christaphone: Christophon's silver salt 4: 1 3.12 ± 2.3 5.93 ± 2.35 3.3 ± 2.02 4.07 ± 0.19 Christafon 2.0 ± 0.14 1.93 ± 1.2 0.65 ± 0.72 0.65 ± 0.8 Placebo 1.55 ± 0.92 2.03 ± 1.98 1.22 ± 1.36 0.59 ± 0.2 Betadine 4.81 ± 3.6 6.98 ± 0.73 4.41 ± 3.4 12.7 ± 8.9

During the experiment, in rats of the control (without treatment) group, increased vaginal discharge was observed on days 3-7 of the experiment. During the observation period the animals of the experimental groups remained mobile, looked healthy outwardly. Clinical examination data indicate the absence of a generalized septic systemic reaction of the body during invaginal infection. As for the pH of the vaginal environment with vaginitis, and the normal acidity of the vaginal environment in rats is close to neutral pH = 7 [Larsen et al., 1977], in the control group, acidification of the medium was observed during the development of bacterial vaginitis (pH = 6). In the experimental groups, however, a reliable return of the pH to normal values was observed. In the control series of experiments without treatment, the vaginal mucosa is represented by loose connective tissue covered with non-keratinizing flat epithelium having an uneven basement membrane due to shallow acanthosis. In the mucous membrane, a moderately pronounced inflammatory infiltrate was observed, which was diffusely distributed in its own plate. When treating animals with preparations containing the silver salt of cristaphone and its mixture with cristaphone, there were no pathological changes in the structure of the vagina; no edema of the underlying connective tissue was detected.

The silver salt of cristaphone in the form of a powder or in the form of a powder in capsule form, where the auxiliary substance is lactose (in vitro results) and vaginal suppositories (preclinical results), has close immunomodulating, antimicrobial, antifungal (antimycotic) activity. These properties will also appear in preparations in the form of vaginal and external ointments, as well as a general immunomodulating and antimicrobial effect when administered orally in the form of hard gelatin capsules and tablets by analogy with cristaphone and isophone, further enhanced by the presence of a silver atom in the molecule ( including in relation to mycobacterium tuberculosis and leprosy).

Examples of formulations of other forms of the drug are presented below. Capsules: the active principle of cristaphone, its silver salt and mixtures thereof, taken in the ratio specified in examples 4-5 to 100 mg, 200 mg, 500 mg, in the first two cases, lactose was used as a filler.

Tablets: the active principle of cristafon, argocristone, and mixtures thereof, taken in the ratio specified in examples 4-5-200 mg, excipients: starch - 30 mg, talc - 5 mg, lactose - 15 mg.

Ointment: the active principle of cristafon, its silver salt and their mixtures, taken in the ratio specified in examples 4-5 - 3 g, base: emulsion wax - 20 g, liquid paraffin - 20 g, petrolatum - 10 g, POE 400 - 10 g, distilled water - 37 g.

Given the wide spectrum of antimicrobial activity of the silver salt of cristaphone and its immunotropic properties, it can be recommended for use in medical practice by specialists - gynecologists, venereologists and urologists - for the treatment of sexually transmitted diseases, for the treatment of vaginitis, vaginosis of various nature, skin diseases caused by a decrease immunity and the development of pathogenic microflora, as well as for the treatment of fungal diseases of various localization.

Claims (4)

1. The silver salt of N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulfone) -N'-isonicotinoyl hydrazide, exhibiting immunotropic, antimicrobial, antifungal (antimycotic) activity, obtained by adding to the pre-intensively mixed to a homogeneous aqueous suspension of N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulfone) -N'-isonicotinoyl hydrazide silver nitrate in a molar ratio of hydrazide to nitrate silver from 1: 1 to 1: 4 without access of light to obtain a volumetric homogeneous precipitate, which they emit it. 2. The silver salt according to claim 1, for the preparation of which N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulfone) -N'-isonicotinoyl hydrazide is used in a finely divided state with the selection of the obtained salt by filtration, repeated washing with double-distilled water and drying at a temperature of 50-80 ° C. 3. A drug having immunotropic, antimicrobial, antifungal activity, containing as an active substance the silver salt of N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulfone) -N ' -isonicotinoyl hydrazide according to any one of claims 1 and 2, or a mixture of N- (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrimidinesulfone) -N'-isonicotinoyl hydrazide and its silver salt in the ratio from 1: 1 to 4: 1. 4. The tool according to claim 3 in the form of tablets, capsules, ointments, suppositories.