WO2009091291A1 - N-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5h-pyrimidinesulfone)-isonicotinoyl hydrazide silver salt - Google Patents

Abstract

The invention relates to the novel N-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5h-pyrimidinesulfone)-isonicotinoyl hydrazide silver salt exhibiting immunotropic, antimicrobial, antifungal (antimicotic) activity. The method for producing said salt involves adding N-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5h-pyrimidinesulfone)-isonicotinoyl hydrazide silver nitrate into an intensively agitated to a homogeneous form aqueous suspension with a mole ratio between hydrazide and silver nitrate ranging from 1:1 to 1:4 in darkness until a volume homogeneous deposit, filtering said deposit, washing it in bidistilled water and drying the deposit at a temperature of 50-80°C.

Description

 Silver salt of N- (6-methyl-2,4-dioxo-l, 2,3,4-tetrahydro-5H-pyrimidinecylphone) -N'-isonicotinoyl hydrazide.

Technical field

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

State of the art

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 with a dual effect - immunomodulatory and antimicrobial have become particularly relevant.

Known immunomodulator N- (6-methyl-2,4-dioxo-l, 2,3,4-tetrahydro-5H-pyrimidinecylphone) -N ^' -isoconoylhydrazide hydrate (isophone) (RF patent N ° 2141322), which has anti-mycobacterial properties only in relation to mycobacteria of 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-methylyl-acyl-5-cyphlochloride in anhydrous acetonitrile. The disadvantage of the isofone immunomodulator with antimycobacterial activity is the low biological activity due to poor solubility in water, as evidenced by an experiment on animals (rabbits), in which It is estimated that about 50% of the isophone remains unchanged in the waste products. Along with the name "ISOFOH", the immunomodulator has the widespread trade name "KPISTAFOH".

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 into the cell, stimulate its function and convey the active principle to the pathogen destroy him. They affect sexually transmitted pathogens, chlamydia, Trichomonas, and 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 a modification of the isophone — the isophone disodium salt (isodinafon, RF patent N ° 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

производное изоникотиновои кислоты

isonicotinic acid derivative

и другие соединения.

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. Thus, in the case of B. serus, isodinafon showed bactericidal antimicrobial activity, in the case of St. Aureus - bacteriostatic antimicrobial activity.

Disclosure of invention

The problem to which the invention is directed, is to obtain a new compound based on derivatives of pyrimidone and isonicotinic acid hydrazide, exhibiting enhanced immunotropic, antimicrobial, antifungal (antimycotic) activity in comparison with cristaphone and isophone, and the creation of a drug on its basis facilities. The problem is solved by a group of inventions, united by a single inventive concept, namely: the development of a new compound - cristaphone silver salt, a method for producing this compound and the creation of a medicine based on it.

A new compound was obtained — the silver salt of N- (6-methyl-2,4-dioxol, 2,3,4-tetrahydro-5H-pyrimidine cyclone) -N'-isonicotinoyl hydrazide (cristaphone) by its interaction with silver nitrate when they are dispersed mixtures in a molar ratio of 50:50 mol% to 20:80 mol% in water, followed by washing, filtering and drying, exhibiting immunotropic, antimicrobial, antifungal (antimycotic) activity. 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 problem is achieved by the method of obtaining silver salt of cristaphone, which consists in the fact that silver nitrate is added to an aqueous suspension of cristaphone, intensively mixed until homogeneous, with a molar ratio of cristaphone to silver nitrate from 1: 1 to 1: 4, then the reaction mixture without light stirred until a homogeneous bulk Nogo precipitate, the resulting precipitate was filtered, washed several times with double distilled water and dried at a temperature of 50-80 ⁰ C. For the preparation of an aqueous suspension according Images eteniyu use krista- background in finely dispersed state.

The problem is also solved by the creation of a drug having immunotropic, antimicrobial, antifungal (antimycotic) activity, containing an active substance, which, according to the invention, contains silver as an active substance cristaphone salt or a mixture of cristaphone and its silver salt in a ratio of 1: 1 to 4: 1. The drug can be made in the form of tablets, capsules, ointments, suppositories.

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, the problem of choosing an effective dosage form is acute.

Cristaphone in aqueous solution is characterized by lactim-lactam (prototropic) tautomerism, and, in fact, cristaphone, when passing into 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).

Lactim (enol-form) Christophon Christaphon's silver salt

Most likely, the formation of the following structures of the Christophon silver salt (I), (II), or a mixture thereof.

(AND) 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 (V. Belikov “Modern synthetic and natural medicines)), Pyatigorsk, 2003), (The use of silver drugs in medicine, Novosibirsk, 199 4 g, p. 106) In addition, the bactericidal action of both silver ions and silver nano-clusters, capable of forming from the silver salt of cristaphone in the human body, is known. It is known that silver nano-clusters are specific nano-reactors 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.

Examples of carrying out the invention Example 1. A method for producing the silver salt of N- (6-methyl-2,4-dioxol, 2,3,4-tetrahydro-5-pyrimidine-cyclone) -N'-isonicotinoylhydrazide (cristaphone).

In an aqueous suspension containing 2 g (0.0061 mol) of cristaphone, they are preliminarily 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 bidistilled water and dried in a vacuum oven (100-200 mmHg) at a temperature of 50-80 ⁰ C. A white or white powder with a grayish tint is obtained.

In the resulting mixture is found on May 24.88. % silver, which corresponds to 99.8 purity of silver salt. The amount of silver was determined by titration of ammonium thiocyanate (Folhard's method) in diluted HNO ₃ (HF X). As an indicator, a solution of iron-ammonium alum is used. Titration is terminated after the appearance of a colorless solution of the thiocyanate complex of iron that disappears with vigorous shaking.

In the PMR spectrum of the silver salt of cristaphone after treatment in an AgNO ₃ solution, a proton shift of δ of the pyridine ring from 7.65 (cristaphone) to 7.8 (silver salt of cristaphone) ppm (hereinafter referred to as ppm), a shift of triplets of CH ₃ group c 2.3-2.5 (cristaphone) to 2.0 (silver salt of cristaphone) ppm, the absence of changes in 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 two compared to cristaphone, in the PMR spectrum, silver In the cristaphone salt, a new doublet is noted with 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. Analysis of infrared spectra was carried out for two reference bands in the 3307 cm ^"and 1743 ^cm" '(CO at position 2) characterizing the carbonyl group in the pyrimidine ring kristafona keto form as well as the group of strong bands in the region 3200-3000 cm ^{" 1} (NH at position 3). In the IR spectrum of the silver salt of cristapho the band 1743 cm ^"1 disappears, and the absorption intensity of the group of bands 3200-

3000 cm ^"1 decreases sharply, indicating the formation of a 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 pre-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 many times with double-distilled water and dried in a vacuum oven (100-200 mm Hg) at a temperature of 50-80 ⁰ C. A white or white powder with a grayish tint is obtained.

In the resulting mixture is found on May 24.78. % silver, 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 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 from 1000 to 3000 rpm to a uniform dispersion, 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 many times with double-distilled water and dried in a vacuum oven (100-200 mm Hg) at a temperature of 50-80 ⁰ C. A white or white powder with a grayish tint is obtained.

In the resulting mixture is found May 24.73. % silver, 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 (mass%) (example 4), 80:20 (mass%) (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 ⁰ C for a polyethylene oxide base and up to 40-60 ⁰ C for a Vitepsol base) in the amount of 13 g, a cristaphone (or its silver salt, or a mixture of them), previously 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 experiments in vitro.

The antimicrobial and antifungal action of the substances and their combinations proposed by the invention was determined according to the standard methodology approved by the Ministry of Health of the State Pharmacopoeia, M., “Medicine”, 1990, issue 2, “General methods of analysis. Medicinal vegetable raw materials. " 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 GISK named after Tarasevich;

Рs.аеrugiпосае - isolate isolated from the patient; C.albisaps - isolate isolated from the patient.

The results of the ip-vitro test on S. aureus, E. coli, Ps. Arugius, Sapdida albicam determine the antimicrobial, antifungal (antimycotic) activity of the silver salt of cristaphone and its mixture with cristaphone are summarized in table 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 slight 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.

S. aires (type 7M strain (NNIEM, N. Novgorod)) and E. coli (strain 782-2), as one of the most common causative agents of bacterial vaginitis, were selected as pathogens for infection. 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- moderate number of cells; 4- many cells.

From the results obtained, it is 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.

Table 3 presents the results of microbiological studies in the treatment of bacterial vaginitis in rats in comparison with the known drug Betadine.

From the obtained results it is seen that during microbiological studies, the most pronounced stable decrease in the number of S. aureus and E. coli in the vaginal tissue of rats after invaginal administration of drugs was observed when using silver salt of cristaphone, a mixture of silver salt of cristaphone and cristaphone in a ratio of 1: 1 and the control drug Betadine. 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 in vaginitis, and the normal acidity of the vaginal environment in rats is close to neutral pH = 7 [Larsep et al., 1977], in the control group, acidification of the medium was noted during the development of bacterial vaginitis (pH = 6). In the experimental groups, however, a reliable return of the pH value 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 the lamina propria. When treating animals with preparations containing the silver salt of the cristaphone and its mixture with cristaphone, no pathological changes in the structure of the vagina were noted, no phenomena of edema of the underlying connective tissue were 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 (ip-vitro results) and vaginal suppositories (preclinical test results), has close immunomodulating, antimicrobial, antifungal (antimycotic) activity. These properties 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 the mycobacteria of 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 cristaphone, the silver salt of cristaphone and their mixtures, taken in the ratio specified in examples 4-5-200 mg, fillers: 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 in immunity and the development of pathogenic microflora, as well as for the treatment of fungal diseases of various localization.

Table 1

Таблица 3table 2

Table 3

Claims

Claim 1. The silver salt of N- (6-methyl-2,4-dioxo-1,2,3, 4-tetrahydro-5H-pyrimidinecylphone) -NH-isonicotinoyl hydrazide (cristaphone), obtained by its interaction with silver nitrate when dispersing their mixture in a molar ratio of 50:50 mol% to 20:80 mol% in water, followed by washing, filtering and drying, exhibiting immunotropic, antimicrobial, antifungal (antimycotic) activity. 2. The silver salt of Christophone according to claim 1, characterized in that it is a compound of the formula I

or a compound of formula II

or a mixture of compounds I and P. 3. A method for producing a silver salt of cristaphone according to claim 1 or claim 2, which consists in adding silver nitrate in a molar ratio of cristaphone to silver nitrate from 1: 1 to 1: 4 to an aqueous suspension of cristaphone, intensively mixed until homogeneous. then, without access to light, the reaction mixture is stirred until a homogeneous bulk precipitate is obtained, the resulting precipitate is filtered, washed repeatedly with bidistilled water and dried at a temperature of 50-80 ° C. 4. The method according to p. 3, characterized in that for the preparation of an aqueous suspension using cristaphone in a finely dispersed state. 5. A drug having immunotropic, antimicrobial, antifungal activity, containing an active substance, characterized in that as an active substance it contains a silver salt of the cristaphone according to any one of paragraphs 1, 2 or a mixture of cristaphone and its silver salt in the ratio from 1: 1 to 4: 1. 6. The tool according to p. 5, characterized in that it is made in the form of tablets, capsules, ointments, suppositories.