RU2525911C1 - Fungicidal preparation - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: preparation can be applied for elimination of fungi and in treatment of diseases, caused by fungi, as well as for the prevention of damage by fungi to different materials and agricultural products. The fungicidal preparation represents an associate of 5-[3,5-dichloro-2-hydroxybenzylidine)amino]-4-hydroxy-1H-pyrimidine-2-one with 1,2,3,4,5-pentahydroxy-6-methylaminohexane and corresponds to the following formula:

. Compounds were obtained in the crystalline form, and their structure is proved by spectra of proton magnetic resonance in dimethylsulphoxide.

EFFECT: preparation has a wide spectrum of action and high solubility, which increases efficiency of its application in the form of solutions.

2 tbl, 6 ex

Description

The invention relates to means for destroying fungi and can be used to treat diseases caused by fungi, as well as to prevent spoilage of various materials and agricultural products by mushrooms.

A very serious problem of modern medicine, veterinary medicine and plant growing is the treatment of fungal, as well as bacterial and viral diseases, many of which are extremely difficult to treat. This is due to the lack of effectiveness of known drugs, as well as the rapid variability of microbes, leading to the emergence of resistant forms, Fidel P.L. Jr, Vazquez J.A., Sobel J.D. Candida glabrata: review of epidemiology, pathogenesis and clinical disease with comparison to C. albicans 1999, 1: 80-96. White T. Antifungal drug resistance in Candida albicans ASM News 8: 427-433.

The above is relevant for veterinary medicine and industry, as there is a spoilage of products associated with the development and spread of microorganisms.

A number of drugs for the treatment of fungal diseases are known: nystatin, amphotericin B, fluconazole and terbinafine (Encyclopedia of Radar Medicines - 2009. Moscow, p. 928). Each of them has significant drawbacks. Fluconazole mainly exhibits a fungistatic effect and practically does not have fungicidal properties [Pharmaceutical microbiology. Ed. by W. B. Hugo and A. D. Rassel Blackwell Scientific Publications, Oxford, 1987, 511 p.]. Fluconazole can also be used to prevent spoiling of plant and agricultural products by fungi. The use of fluconazole in archiving for paper processing is also known.

The main disadvantage of nystatin is its low activity against multicellular fungi.

Amphotericin B is an active antifungal drug, but it is very toxic.

A fungicidal agent is known, characterized in that it is a salt of 2,4-dioxo-5- (2-hydroxy-3,5-dichlorobenzylidien) amino-1,3-pyrimidine:

or its dimer:

where X is selected from the series: Na ⁺ , K ⁺ , Li ⁺ , NH ₄ ⁺ , RU 2448960 C1.

This technical solution is made as a prototype of the present invention.

The drug has a pronounced antifungal activity of a wide spectrum of action.

However, this substance is very poorly soluble both in the aquatic environment and in fats. The solubility of the known drug in water is not more than 0.002%, and in fats - not more than 0.001% (octanol, which is a generally accepted model of lipids). The low solubility of the fungicidal prototype is due to the fact that this substance has large particle sizes: length - 30 μm, cross section - about 300 nm, which does not contribute to the dissolution of the substance in the aquatic environment or in fats. Solutions with this concentration of active substance are not effective enough. At the same time, the task of increasing the efficiency of using a fungicidal preparation in the form of solutions for use in medicine and veterinary medicine in the form of inhalations and injections and for processing various materials and agricultural products is extremely urgent.

The objective of the present invention is to provide a broad-spectrum fungicidal agent with a higher solubility and thereby increase the efficiency of its use in the form of solutions.

According to the invention, the problem is solved by synthesis of a fungicidal agent, which is an associate of the salt of 5- [3,5-dichloro-2-hydroxybenzylidene) amino] -4-hydroxy-1H-pyrimidin-2-one with 1,2,3,4 , 5-pentahydroxy-6-methylaminohexane.

The applicant has not identified sources containing information on technical solutions identical to the present invention, which allows us to conclude that it meets the criterion of "Novelty."

The length of 70% of the particles of the claimed substance does not exceed 10 μm, which provides an important new property of the object (technical result): increased solubility in water to concentrations of 0.3-0.4%, and in oil solutions - up to 0.25-0.3 % (more than 100 times), which can significantly increase the efficiency of the use of drug solutions.

These circumstances prove, in the applicant’s opinion, that the claimed technical solution meets the patentability condition “Inventive step”.

A 0.1% solution of the claimed substance was prepared as follows.

0.04 g of sodium hydroxide and 10 ml of 96% ethanol were placed in the flask. The mixture was heated until sodium hydroxide was completely dissolved. To the resulting solution was added portionwise 0.127 g of 5-aminouracil at a temperature of 40 ° C. Then the resulting mass was heated in a boiling water bath until complete dissolution of aminouracil. In addition, 0.191 g of 3,5-dichlorosalicylic aldehyde was dissolved in 40 ml of 96% ethanol. The resulting solution was added dropwise with stirring to a solution of 5-aminouracil sodium salt. After the formation of an orange-red precipitate, the reaction mass was continued to be heated for 1.5 h; after the reaction mass was cooled to room temperature, the precipitate was filtered off, washed with 10 ml of 96% ethanol, and dried. The resulting precipitate was transferred to a flask containing a pre-prepared solution of 0.195 1,2,3,4,5-pentahydroxy-6-methylaminohexane in 300 ml of water. The reaction mixture was stirred at t = 40 ° C until the precipitate was completely dissolved. The resulting solution was cooled to room temperature.

Obtaining solutions of the claimed substance of higher concentration is similar.

To influence the fungi for medical, veterinary and agricultural purposes, water and oil solutions of the drug with a concentration of 0.1% were used.

To prevent damage to fungi of various materials, aqueous solutions with a concentration of 0.3% were used.

Acceptable solvents and transporters, in addition to water, can be an isotonic sodium chloride solution, Ringer's solution, etc., while benzyl alcohol or any suitable substance can serve as a stabilizer, and fluorocarbons can be a absorption activator to increase bioavailability. To create an oil solution of the drug, you need to use non-volatile vegetable oils, which are usually used to create oil solutions or suspensions.

Any neutral, non-volatile oil, including synthetic mono- and diglycerides, fatty acids, is suitable for this purpose. To create injectable preparations, oleic acid and glycerides, olive or castor oils, especially their polyoxyethylated derivatives, can be used. Long chain alcohols or other similar substances may also be included in the composition of oil solutions and suspensions as stabilizers and detergents. If the dosage form is in the form of an aqueous suspension for inhalation or nasal sprays, then emulsifiers and substances giving the preparation a sweet taste, pleasant smell and color can be added to the active substance.

Example 1

Treatment of respiratory diseases

The studies were carried out on outbred white mice (females, 6-8 weeks old) obtained from the Rappolovo nursery (Leningrad Region), which were kept under observation for two weeks on a standard diet in regulated vivarium conditions. The selection of animals in the experimental groups was carried out by random sampling.

To reproduce aspergillus pneumonia, white outbred mice received a single intraperitoneal solution of cyclophosphamide (150 mg / kg). After 3 days, the mice were injected once intraperitoneally with a solution of hydrocortisone (250 mg / kg). One day after the administration of hydrocortisone, mice were infected intranasally under ether anesthesia with a suspension of Aspergillus niger (approximately 10 ⁷ cells / ml).

On the 5th day after infection, 5 animals from each group were taken to study microbial seeding of the lungs (euthanasia in rodents was performed with an overdose of ether). Hemogenates were prepared from lung tissues, in which the titer of Aspergillus fungi was determined. The remaining mice were monitored for 14 days after infection to account for mortality.

In the experiment, the following groups of animals infected with Aspergillus niger were formed.

1. Control groups: K1, K2, K3; animals received an aerosol of isotonic sodium chloride solution.

2. Groups E1, E2, E3; animals received an aqueous solution of 0.1% of the claimed drug in the form of inhalation.

3. Groups P1, P2; the animals received an aqueous solution of the fungicidal prototype in the form of inhalations in concentrations of 0.002% and 0.001%, respectively).

Animals were placed in aerosol chambers (12 L) for a period of 30 minutes, during which 500 ml of air passed through the lungs.

An OMRON U1 ultrasonic atomizer (Japan) was used as an aerosol generator. To maintain a constant concentration of aerosol in the chamber volume, a pulsed spray mode was used using a vortex pneumatic generator to obtain a stable aerosol with a particle size of 3 μm. The animals were placed in immobilization containers and exposed to the obtained aerosol for 30 minutes once a day for 3 days, starting from 1 day after infection.

A series of serial dilutions were prepared from samples of hemogenized lung tissue, followed by plating on Saburo's medium. The results are shown in table 1.

Table 1 Groups of animals The number of colonies (in different dilutions) 10 ^-2 10 ^-4 10 ^-6 K1 75 2 - K2 64 2 - K3 48 - - E1 four - - E2 four - - T3 2 - - P1 26 one - P2 37 2 -

The above results confirm the effective antifungal effect of the claimed fungicidal agent.

Example 2

Treatment of fungal skin disease - versicolor (tinea versicolor, pityriasis versicolor)

The disease is caused by the fungus Pityrosporum orbiculare - Malassezia furfur (mycelial form).

For the study were prepared: oil (olive oil) 0.1% solution of the claimed drug and aqueous 0.002% solution of the prototype substance. Olive oil was used as a control. The study involved 3 groups of patients. Each group included 2 patients with this pathology. The duration of treatment was 7 days. The drug was applied with a cotton swab 2 times a day - in the morning and in the evening.

The criterion of mycological cure was the absence of yeast and mycelial forms of fungi of the genus Malassezia, and the criterion of complete cure was the absence of clinical symptoms and fungi of the genus Malassezia in skin flakes obtained from the skin surface at the site of resolved lesions.

The results of the study are shown in table 2.

table 2 A drug Mycological cure Complete healing one 2 3 The control Absent Absent

one 2 3 Claimed drug Yeast and mycelial forms of fungi of the genus Malassezia are absent Reached Prototype A slight decrease in the yeast and mycelial forms of fungi of the genus Malassezia Absent

Thus, a pronounced positive effect is achieved only when using the claimed drug.

Example 3

Treatment of candidiasis vulvovaginitis

In the experiment, people with candidal vulvovaginitis caused by fungi of the genus Candida were treated with aqueous solutions: 1) the claimed drug - 0.1% solution (3 people), 2) prototype means - 0.002% solution (3 people).

Treatment in the form of double (morning and evening) douching was carried out for 7 days. The effectiveness of therapy was evaluated according to the clinical picture and the results of mycological examination (sowing on Saburo medium).

The results of the study are shown in table 3.

Table 3 A drug Mycological cure Complete healing Claimed A tenfold decrease in the number of mushrooms sown on a solid medium. Reached Prototype The decrease in the number of mushrooms sown on a dense medium by 1.7 times. Not reached

Thus, the use of the claimed drug allows to achieve complete cure of candidiasis infection, while in patients receiving an aqueous solution of the prototype substance, a certain decrease in the contamination of the fungus was recorded while maintaining the main complaints and manifestations of the infection.

Example 4

Treatment of fungal infections of plants

The study was conducted on tuber begonia (Begonia tuberosa hybridum) infected with powdery mildew. Powdery mildew affects many plants, including trees and shrubs. Powdery mildew affects chrysanthemums, begonias, roses, while on the green parts of plants appears white, sometimes darkening, plaque. The disease is transmitted by spores through the air.

The test plants were infected with spores of the fungus (Leveillula taurica) when kept until white plaque appeared on the leaves. Three leaves on each plant were infected. After plaque, the plants of the control group (three plants) were isolated from three plants that received therapy in the form of a spray treatment, which is a 0.1% aqueous solution of the claimed drug. Processing was carried out once a day for 7 days. After completion of the treatment, the observation of the plants was continued for another three weeks. During the observation, the control plants recorded an increase in the amount of plaque, which is the mycelium of the fungus, and the number of affected leaves increased on average to 5-7, while the plaque covered the stems of the plant. In plants treated with the test substance, the growth and spread of the fungus to other parts of the plant did not occur. When flushing from a leaf and sowing on a nutrient medium, fungal growth was not recorded.

Example 5

Treatment of fungal infections of animals

The drug was used in the treatment of dermatomycosis in cats (two animals) caused by a fungus of the genus Microsporum and characterized by the appearance of small rounded bald patches on the muzzle and ears. The drug in the form of an aqueous 0.1% solution was applied to the affected area 3 times a day for 6 days.

As a result of treatment, the affected areas ceased to increase, the disappearance of manifestations of inflammation was recorded. Seeding swabs from the affected area after treatment was not performed on growth media.

Thus, the use of the claimed drug was effective for the treatment of dermatomycosis caused by the Microsporum fungus.

Example 6

Protection of building wood from fungal infections

To test the drug, boards of 20x40x2 cm in size were used. Boards were divided into three groups. Group 1: boards impregnated with water 0.3% of the claimed drug. The impregnation was carried out by applying the solution twice with a spray with intermediate drying. Group 2: the boards are saturated with an aqueous 0.02% solution of the prototype drug. The impregnation was carried out by applying the solution twice with a spray with intermediate drying. Group 3 (control): the boards are saturated with distilled water. The impregnation was carried out by applying the water twice with a spray with intermediate drying.

The boards dried after processing were soaked for two hours in water, after which cultures of Aspergillus niger and Penicillium notatum fungi were applied to them at different ends of one side. Boards were placed for 5 days in thermostats at a temperature of 30 ° C in a humid environment. After incubation, the boards were visually inspected and culture media were plated from their surface (Saburo).

As a result, it was found that on the boards of the control group or treated with an aqueous 0.02% solution of the prototype substance, it is possible to detect the growth of seeded test microbes, which then gave growth on a nutrient medium. On the boards impregnated with a 0.3% solution of the declared fungicidal agent, the growth of test microbes was not registered, and control seeding did not give growth on a nutrient medium.

Claims (1)

A fungicidal agent, which is an associate of the salt of 5- [3,5-dichloro-2-hydroxybenzylidene) amino] -4-hydroxy-1H-pyrimidin-2-one with 1,2,3,4,5-pentahydroxy-6-methylaminohexane