Antifungal agent

Abstract

The present invention relates to fungicides and can be used for treating diseases caused by fungi and for preventing fungal damage to various materials and agricultural products. What is claimed is an anti-fungal infection agent in the form of a salt of 2,4-dioxo-5-arylamino-6-methyl-1,3-pyrimidine, wherein R is 4-NO2, 2-OH-5-NO2, 2-OH-3,5-Cl2, 5-Br-4-OH-3-OCH3, 2-OH-5-Cl, 2,4-Cl2, 3,5-Br2-2-OH. The result is an anti-fungal infection agent with a broad spectrum of activity and high solubility, making it more effective when used in the form of a solution.

Description

[0001] Cross-reference to related applications

[0002] This application is the national phase in China of International Patent Application No. PCT / RU2016 / 000499, filed on August 1, 2016, which was published as WO 2017 / 023193A1 on February 9, 2017, and claims the priority of Russian Patent Application No. RU2015132513, filed on August 4, 2015, all of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to antifungal agents and can be used for treating diseases caused by fungi, as well as in agriculture and veterinary medicine, and for preventing fungal damage to various materials.

[0004] The treatment of fungal infections remains an unsolved and serious problem for modern medicine, veterinary medicine, crop production, and industry. This is due to the insufficient efficacy of known agents and the high variability of microorganisms that lead to the emergence of stable forms (see 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 agent resistance in Сandida albicans ASM News 8:427 - 433). Background Art

[0005] A number of agents for the treatment of fungal diseases are known in the prior art: nystatin, amphotericin B, fluconazole, terbinafine (Dixon DM, Walsh TJ. Antifungal Agent, available at World Wide Web ncbi.nlm.nih.gov / books / NBK8263 / ?report=reader location).

[0006] They each have significant drawbacks. Fluconazole mainly shows an inhibitory effect on fungi and, in practice, does not show fungicidal properties. [Pharmaceutical microbiology. Edited by W.B. Hugo and A.D. Rassel Blackwell Scientific Publications, Oxford, 1987, 511p]. The foregoing makes it difficult to use these agents to treat people with a weakened immune system. Fluconazole can also be used to prevent fungal damage to plants and agricultural products. It is also known to use fluconazole in archival preservation for paper treatment.

[0007] The main drawback of nystatin is its low activity against multicellular fungi.

[0008] Amphotericin B is an active antifungal agent; however, it is highly toxic and causes several serious side effects.

[0009] All of these agents have been used for many years and various bacteria have developed resistance to them (see A. Kanafani1 J.R. Perfect2 Resistance to Antifungal Agents: Mechanisms and Clinical Impact Clinical Infectious Diseases 2008:46 120 - 126).

[0010] A fungicide as an association compound of 5 - [(3,5 - dichloro - 2 - hydroxybenzylidene)amino] - 4 - hydroxy - 1H - pyrimidin - 2 - one salt and 1,2,3,4,5 - pentahydroxy - 6 - methylaminohexane is known in the prior art:

[0011]

[0012] where X = Na, K, NH4 + , RU 2525911С1, publ. 20.08.2014.

[0013] This technical solution is adopted as the prototype of the present invention.

[0014] This agent shows significant broad - spectrum antifungal activity.

[0015] However, this substance is poorly soluble in both aqueous media and fats. The known preparations have a solubility in water of no more than 0.4%, and in oil solutions it is at most 0.3%. The insufficient solubility of the prototype fungicides makes it impossible to obtain a stable solution suitable for practical applications. At the same time, the problem of increasing the effectiveness of the use of fungicides in the form of solutions for applications in medicine and veterinary medicine in the form of inhalants and injectables and for the treatment of various materials and agricultural products is extremely relevant. Summary of the Invention

[0016] The object of the present invention is to provide an effective antifungal agent with a broad spectrum of activity and high solubility.

[0017] According to the present invention, the above object is achieved by the synthesis of fungicides of 2,4-dioxo-5-arylamino-6-methyl-1,3-pyrimidines,

[0018]

[0019] R is 4-NO2, 2-OH-5-NO2, 2-OH-3,5-Cl2, 5-Br-4-OH-3-OCH3, 2-OH-5-Cl, 2,4-Cl2, 3,5-Br2-2-OH.

[0020] The values of R are given in Table 1.

[0021] The applicant is not aware of any information source containing information on the same technical solution, which allows to infer that the claimed invention complies with the criterion of "novelty" ("N").

[0022] By implementing the claimed technical solution, a technical result is achieved, which consists in providing an effective antifungal agent with a broad spectrum of activity and high solubility.

[0023] The applicant has not found any information source containing data on the influence of the distinguishing features of the present invention on the technical results achieved due to their implementation.

[0024] The above situation allows to infer that the claimed technical solution meets the criterion of "inventive step" (IS). Brief Description of the Drawings

[0025] Hereinafter, without referring to the drawings, the present invention is explained by a detailed description of the embodiments of its implementation. Detailed Description of the Invention

[0026] The structure of the obtained compounds is proven by proton magnetic resonance spectroscopy and IR spectroscopy.

[0027] The desired salts of 2,4-dioxo-5-arylideneamino-6-methyl-1,3-pyrimidines are prepared by reacting the sodium salt of 5-amino-6-methyluracil with aromatic aldehydes. As the solvent, a 3:1 ethanol-water mixture is used. The product is obtained in a yield of 80% above the theoretical value. The individual characteristics of the target compound are demonstrated by thin-layer chromatography: the eluent system - chloroform-acetone 3:1. The melting temperature of the product exceeds 300 °C.

[0028] Example 1: Synthesis of sodium 2,4-dioxo-5-(4-nitrobenzylidene)amino-6-methyl-1,3-pyrimidine (I-1).

[0029] 0.5 g of 5-amino-6-methyluracil and 0.5 ml of a 50% sodium hydroxide solution were placed in a flask. The mixture was heated with stirring until the initial materials were completely dissolved. At the same time, 0.18 ml of ethanol was dissolved in 0.6 g of 4-nitrobenzaldehyde and added to the solution of 5-amino-6-methyluracil, while a wine-red precipitate began to form in the flask. Then the reaction mixture was stirred for 30 minutes and cooled. The resulting precipitate was filtered off, washed with ethanol and dried. The product yield was 83% of the theoretical value.

[0030] Example 2: Synthesis of sodium 2,4-dioxo-5-(2-hydroxy-3,5-dichlorobenzylidene)amino-6-methyl-1,3-pyrimidine (I-3).

[0031] 1.41 g of 5-amino-6-methyluracil and 0.4 g of sodium hydroxide in 10 ml of water were placed in a reaction flask. The mixture was heated until the original uracil was completely dissolved. At the same time, 1.91 g of 3,5-dichlorosalicylaldehyde was dissolved in 30 ml of ethanol, and the resulting solution was added dropwise with stirring to the solution of the sodium salt of 5-amino-6-methyluracil. The reaction mass was heated with stirring for 1 hour, then cooled to room temperature, and the formed precipitate was filtered off, washed with ethanol and dried. The yield of the desired product was 2.86 g (85%).

[0032] Example 3: Treatment of respiratory diseases.

[0033] This study was conducted on hybrid white mice (female, 6 - 8 weeks) in Rappolovo (Leningrad region), with a quarantine period of 2 weeks.

[0034] To reproduce aspergillus pneumonia in mice, to induce immunodeficiency, cyclophosphamide solution (150 mg / kg) was intraperitoneally injected once. Three days later, hydrocortisone (250 mg / kg) was intraperitoneally injected into the mice once. One day after the hydrocortisone administration, the mice were infected intranasally with a suspension of Aspergillus niger (about 3×10 7 cells / ml) under ether anesthesia.

[0035] On the 5th day after infection, 5 animals from each group were taken to study the microbial infection of the lungs (euthanasia of rodents was performed using an overdose of ether). Homogenates were prepared from the lung tissues in which the titers of fungi of the Aspergillus genus were determined. Observations of the remaining mice were made within 14 days after infection for counting the number of deaths.

[0036] The test solution was injected in the aerosol chamber.

[0037] During the experiment, the following groups of animals were formed:

[0038] 1 - Spray control (K), 3 identical groups (K1, K2, K3). The animals were infected with Aspergillus and received an aerosol of isotonic sodium chloride solution.

[0039] 2 - The conjugate of 5 - [(3,5 - dichloro - 2 - hydroxybenzylidene)amino] - 4 - hydroxy - 1H - pyrimidin - 2 - one and 1,2,3,4,5 - pentahydroxy - 6 - methylaminohexane, 3 identical groups (E1 - 3). The animals were infected with Aspergillus and received an aerosol of the 0.1% studied preparation.

[0040] 3. Aqueous solution of Substance I - 1

[0041] 4. Aqueous solution of Substance I - 2

[0042] 5. Aqueous solution of Substance I - 3

[0043] 6. Aqueous solution of Substance I - 4

[0044] 7. Aqueous solution of Substance I - 5

[0045] 8. Aqueous solution of Substance I - 6

[0046] 9. Aqueous solution of Substance I - 7.

[0047] Experimental setup.

[0048] The animals were placed in the aerosol chamber (12 l) for 30 minutes, during which 500 ml of air was passed through the lungs.

[0049] As the aerosol generator, the ultrasonic nebulizer OMRON U1 (Japan) was used. In order to maintain a constant concentration of the aerosol of the preparation in the chamber, a vortex pneumatic generator in the pulsed spray mode was used to obtain a stable aerosol with a particle size of 3 μm. The animals were placed in an immobilization container and exposed to the obtained aerosol for 30 minutes starting from the first day after infection, once a day for 3 consecutive days.

[0050] Results.

[0051] From the samples of homogenized lung tissue, several serial dilutions were prepared and then inoculated onto Saburo medium. The results are shown in Table 2.

[0052] Therefore, under the conditions used, the most effective antifungal agent was Substance I-3.

[0053] Example 4: Candidiasis in mammals caused by Candida albicans.

[0054] Rabbits were intradermally inoculated with a suspension of a 48-hour Candida albicans culture at a dose of 10,000 fungal cells in 500 μl of 0.9% sodium chloride solution. After 7 - 10 days, some of the animals showed ulcers at the injection site; they were used for the experiment.

[0055] For this study, a 0.1% oil (olive oil) solution of the salt of 5-[3,5-dichloro-2-hydroxybenzylidene)amino]-4-hydroxy-1H-pyrimidin-2-one associated with 1,2,3,4,5-pentahydroxy-6-methylaminohexane and Substance I-3 were prepared.

[0056] Sterile water was used as the control substance.

[0057] The duration of treatment was 7 days, starting from the first day after the ulcer. The preparations were applied with a cotton swab, twice a day - in the morning and in the evening.

[0058] The criteria for mycological cure were the absence of yeast and hyphal forms of Candida albicans, and the criteria for complete cure were scar formation of the lesion by day 7.

[0059] The results are shown in Table 3.

[0060] Therefore, a positive effect was achieved only in the case of using Substance I-3.

[0061] Example 5: Treatment of fungal infections in plants.

[0062] This study was conducted on tuberous begonias (Begonia tuberosa hybridum) infected with Powdery mildew. Powdery mildew affects many plants including trees and shrubs. Powdery mildew affects chrysanthemums, begonias and roses; it appears white in color and sometimes blackens the exudates on the green parts of the plants. Plant diseases are spread by air via spores.

[0063] The test plants were infected with spores of the fungus (Leveillula taurica) and kept until white exudates appeared on the leaves. Three leaves on each plant were infected. After the exudates appeared, the control group of plants (3 plants) were isolated from 3 plants treated with a spray of an aqueous solution of Substance I-3. The treatment was carried out once a day for 3 days and 7 days. After the treatment was completed, the observation of the plants continued for 3 more weeks. During the observation period, an increase in the amount of exudates representing the fungal mycelium was recorded in the control plants and the number of affected leaves increased on average to 7 - 8, while the exudates also covered the stems of the plants. In the plants treated with the substance under study, no increase in the fungus or spread to other parts of the plant was recorded. When rinsed from the leaves and inoculated on a nutrient medium, no growth of the fungus was recorded.

[0064] Industrial applicability

[0065] The present invention is implemented using conventional materials and equipment, which, in the opinion of the applicant, results in the invention meeting the "Industrial Applicability" ("IA") patentability criterion.

[0066] Table 1 Characteristics of the target compounds of General Formula 1

[0067]

[0068] Table 2 Results of preparing serial dilutions and subsequent inoculation onto Saburo medium.

[0069]

[0070] Table 3 Results of treating candidiasis in mammals

[0071]

Claims

1. A compound of formula (I): wherein R is 2-OH-3,5-Cl2, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, which is the sodium salt of the compound of formula (I).

3. A composition comprising the compound according to claim 1 and a carrier, wherein the carrier is water.

4. A pharmaceutical dosage form comprising the compound according to claim 1 and a pharmaceutically acceptable carrier, wherein the dosage form is in the form of an aerosol or a spray, and the carrier is water.

5. A pharmaceutical dosage form comprising the compound according to claim 1 and a pharmaceutically acceptable carrier, wherein the dosage form is in the form of an aqueous solution.

6. Use of the compound according to claim 1 in the preparation of a medicament for preventing or inhibiting the growth of fungi in plants, wherein the fungus is Leveillula taurica.

7. Use of the composition according to claim 3 in the preparation of a medicament for preventing or inhibiting the growth of fungi in plants, wherein the fungus is Leveillula taurica.

8. Use of the compound according to claim 1 in the preparation of a medicament for treating fungal infections in a subject in need thereof, wherein the fungal infection is aspergillosis pneumonia or candidiasis in mammals caused by Candida albicans.

9. Use of the composition according to claim 3 in the preparation of a medicament for treating fungal infections in a subject in need thereof, wherein the fungal infection is aspergillosis pneumonia or candidiasis in mammals caused by Candida albicans.

10. Use of the dosage form according to claim 4 or 5 in the preparation of a medicament for treating fungal infections in a subject in need thereof, wherein the fungal infection is aspergillosis pneumonia or candidiasis in mammals caused by Candida albicans.

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