RU2702224C2 - Phenyl formamidine derivatives, having antimycobacterial activity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine and aims at preventing and treating infectious diseases caused by mycobacterial infections. One embodiment is compound N'-hydroxy-N-(4-isopentyl-2-methylphenyl)formimidamide, characterized by the following structural formula:

.

Other embodiments are use of said compound for treating and/or preventing an infectious disease in a subject caused by a mycobacterial infection, and use of said compound to produce a pharmaceutical composition for treating and/or preventing an infectious disease caused by a mycobacterial infection. Also provided is a pharmaceutical composition for treating and/or preventing an infectious disease caused by a mycobacterial infection, comprising an effective amount of said compound and at least one pharmaceutically acceptable excipient.

EFFECT: using the group of inventions enables providing more effective prevention and treatment of diseases associated with the mycobacterial infection, particularly tuberculosis.

15 cl

Description

Derivatives of phenyl formamidine with antimycobacterial activity

Technical field

This invention relates to the chemistry of organic compounds, pharmacology, medicine, veterinary medicine and relates to the prevention and treatment of diseases caused by mycobacterial infections, in particular tuberculosis, using a new class of compounds.

State of the art

Currently, tuberculosis (TB) is the leading infectious cause of death in the world [1]. Most deaths are associated with high sensitivity of HIV-infected patients to TB; in the presence of double HIV / TB infection, deaths are recorded in a quarter of cases.

Treatment of mycobacterial infections is difficult. This is due to pathogen features such as a low growth rate of pathogens, the ability to transition to a resting state, the presence of a highly hydrophobic cell surface of Mycobacterium tuberculosis, which serves as a kind of physical barrier, intracellular localization of pathogens, and a high concentration of M. tuberculosis cells in the affected organ. Currently, the main method of treatment for TB patients is complex chemotherapy with antimicrobial agents, aimed at suppressing the multiplication of TB mycobacteria or deep damage to the function of the microbial cell, leading to its death. Existing anti-TB drugs have different efficacy, toxicity and different mechanisms of action, however, each has serious side effects (hepatotoxicity, nephrotoxicity, ototoxicity, etc.).

The situation is aggravated due to the fact that the emergence and spread of new drugs after some time is regularly accompanied by the sequential emergence of forms of mycobacteria resistant to them; new stability is often “layered” on the previous one, resulting in a multiple character. Quite often, de novo infection with strains that have already acquired multidrug resistance is carried out. In this regard, the emergence of drug-resistant strains of M. tuberculosis, including multiple (MDR - multiple drug resistance), extensive (XDR - extensive drug resistance) and absolute (TDR - total drug resistance) forms of resistance, leads to the preservation of high morbidity and mortality rates from TB in the world, despite significant efforts to combat this disease.

It becomes obvious that one of the most important tasks in the fight against TB and other diseases associated with mycobacterial infections is the development of new generation drugs for the treatment of mycobacterial infections.

Disclosure of invention

The present invention is the development and creation of new anti-infective agents with antimycobacterial activity, promising for use in clinical practice for the treatment of tuberculosis and other diseases associated with mycobacterial infections.

The technical result of the invention is the development and production of new chemical compounds with increased activity and effectiveness in inhibiting mycobacteria, in particular Mycobacterium tuberculosis, including resistant strains of Mycobacterium tuberculosis. The compounds of the invention are promising for use in the treatment of diseases associated with mycobacterial infection, in particular in the treatment of tuberculosis, including resistant forms of tuberculosis.

The specified technical result is achieved through the development and creation of compounds of General formula I and / or General formula II:

or their Z-stereoisomer, a pharmaceutically acceptable salt, solvate and / or hydrate, where:

R is independently selected and is C _4-14 methyl branched alkyl, partially or fully halogenated C _4-14 methyl branched, C _4-14 methyl branched alkenyl, partially or fully halogenated C _4-14 methyl branched alkenyl, with R being located in the para or meta position relative to the nitrogen atom of the hydroxyformimidamidine moiety;

R ′ is independently selected and is C _4-14 methyl branched alkyl, partially or fully halogenated C _4-14 methyl branched alkyl, C _4-14 methyl branched alkenyl or partially or fully halogenated C _4-14 methyl branched alkenyl, wherein R 'is located in the para- or meta-position relative to the nitrogen atom of the hydroxyformimidamidine fragment.

A separate subclass of compounds of interest includes compounds of the invention selected from the group:

N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide;

N'-hydroxy-N- (4-isohexyl-2-methylphenyl) formimidamide;

N'-hydroxy-N- (4-isoheptyl-2-methylphenyl) formimidamide.

This invention also relates to the use of the compounds of the invention for the treatment and / or prevention of an infectious disease in a subject caused by a mycobacterial infection.

In particular embodiments of the invention, the infectious disease is caused by Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium marinum, Mycobactérium kansasii, Mycobacterium avium or other microorganisms included in the Mycobacterium tuberculosis complex (Mycobacterium tuberculium avcobcomplex).

In particular embodiments, the disease is tuberculosis.

In some particular embodiments, the tuberculosis is resistant.

In particular embodiments, the subject is a mammal or a bird. In particular embodiments, the mammal is a human.

This invention also relates to the use of the compounds of the invention for the preparation of a pharmaceutical composition for the treatment and / or prevention of an infectious disease caused by a mycobacterial infection.

In addition, the invention provides pharmaceutical compositions for treating and / or preventing an infectious disease caused by a mycobacterial infection, comprising an effective amount of at least one compound of the invention and at least one pharmaceutically acceptable excipient. In particular embodiments of the invention, the infectious disease is caused by Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium marinum, Mycobactérium kansasii, Mycobacterium avium or other microorganisms included in the Mycobacterium tuberculosis complex (Mycobacterium tuberculium avcobcomplex). In some particular embodiments, the disease is tuberculosis. In some other particular embodiments, the tuberculosis is in a drug resistant form.

In particular embodiments, the pharmaceutically acceptable excipient is a carrier, excipient and / or solvent.

In particular embodiments of the invention, the pharmaceutical composition is characterized in that it has a bacteriostatic effect.

In particular embodiments of the invention, the pharmaceutical composition is characterized in that it has a bactericidal effect.

The present invention also relates to a method for treating and / or preventing infectious diseases, comprising administering (as monotherapy or in combination with one or more agents) a therapeutically effective amount of a compound of the invention to a human or animal organism in need of treatment and / or prevention of such diseases. "Administration" of a compound of the present invention to an organism includes the delivery to a recipient of a compound described in the present invention of a prodrug, or other pharmacologically acceptable derivative of such a compound, using any acceptable formulations or routes of administration to the body that are well known in the art.

The invention also includes the preparation of compounds of general formula I and general formula II.

Definitions (terms)

For a better understanding of the present invention, the following are some of the terms used in the present description of the invention.

In the description of the present invention, the terms “includes” and “including” are interpreted as meaning “includes, inter alia,”. These terms are not intended to be construed as “consists of only”.

The term “alkyl” as used herein refers to branched alkyls, namely methyl branched alkyl groups, for example 3-methylbutyl, 3-methylpentyl, 3-methylhexyl, etc., preferably 3-methylbutyl and 3,6-dimethylheptyl. In addition, “alkyl” may be either substituted or unsubstituted. The term “alkyl” as used herein also refers to groups typically having four to fourteen carbon atoms. For example, the term —C _4-14 -alkyl means isobutyl, isopentyl, isohexyl, isoheptyl, etc. Similar conventions apply to another general term, such as “alkenyl,” where the position of one or more double bonds is defined at any carbon atom C _4-14 , preferably 3-methylbut-1-enyl, 3,6-dimethylhept-2-enyl , 3,6-dimethylhept-2,5-dienyl.

The term "partially or fully halogenated alkyl" includes branched saturated hydrocarbon chains in which one or more hydrogen atoms are replaced by halogen. Examples of haloalkyl groups include, but are not limited to, the following groups: 3-methyl-4,4,4-trifluorobutyl, 5,5,5-trifluoro-4- (trifluoromethyl) pentyl, 4,4,4-trifluoro-3- ( trifluoromethyl) butyl and the like.

The term "halogen" by itself or in part of another term refers to an atom of fluorine, chlorine, bromine or iodine.

The term “solvate” refers to an association or complex of one or more solvent molecules and a compound of the invention. Examples of solvate forming solvents include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

The term "hydrate" refers to a complex wherein the solvent molecule is water.

The compounds of the present invention may exist in free form or, if desired, in the form of a pharmaceutically acceptable salt or other derivative. As used herein, the term “pharmaceutically acceptable salt” refers to those salts which, within the framework of a medical opinion, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, etc., and correspond to a reasonable balance of benefits and risk. Pharmaceutically acceptable salts of amines, carboxylic acids, phosphonates and other types of compounds are well known in medicine. Salts can be prepared in situ during the isolation or purification of the compounds of the invention, and can also be prepared separately by reacting the free base of the compound of the invention with a suitable acid. An example of pharmaceutically acceptable, non-toxic acid salts is the amino group formed by inorganic acids such as hydrochloric, hydrobromic, phosphoric and sulfuric acids, or organic acids such as acetic, oxalic, maleic, tartaric, succinic or malonic acids, or obtained by other methods used in this field, for example, by ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, dodecyl sulfate, heptofluorosulfonate, glucose gluconate, formate glucose phosphate heptane, hexanate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pepate, pepate Ulfat, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undekanat, valerate, and the like.

The term “patient” encompasses all types of mammals, preferably humans, and also includes birds.

The terms “treatment”, “therapy” cover the treatment of pathological conditions in mammals, preferably in humans, and include: a) blocking (stopping) the course of the disease, b) alleviating the severity of the disease, i.e. induction of disease regression.

The term "prevention", "prevention", "preventive therapy" covers the elimination of risk factors, as well as the preventive treatment of subclinical stages of the disease in humans, aimed at reducing the likelihood of the clinical stages of the disease. Patients for prophylactic therapy are selected on the basis of factors that, based on known data, entail an increase in the risk of clinical stages of the disease compared with the general population. Preventive therapy includes a) primary prevention and b) secondary prevention. Primary prophylaxis is defined as prophylactic treatment in patients whose clinical stage of the disease has not yet begun. Secondary prophylaxis is the prevention of the repeated onset of the same or similar clinical condition of the disease.

The term "risk reduction" covers therapy that reduces the incidence of the clinical stage of the disease. Examples of reducing the risk of disease are primary and secondary disease prevention.

Method for the therapeutic use of compounds

The subject of the invention also relates to a method for treating and / or preventing diseases caused by mycobacterial infection, comprising administering to a patient a compound of the invention. In particular, to a method for treating and / or preventing diseases caused by Mycobacterium tuberculosis.

In preferred cases, the subject of this invention relates to a method for the treatment and / or prevention of tuberculosis, including resistant forms of tuberculosis.

The subject of this invention also includes the administration to a subject in need of appropriate treatment, a therapeutically effective amount of a compound of general formula (I) and / or general formula (II). By a therapeutically effective amount is meant an amount of one or more of the described compounds, administered or delivered to a patient, in which the patient is most likely to exhibit the desired response to treatment (prophylaxis). The exact amount required can vary from subject to subject, depending on the age, body weight and general condition of the patient, the severity of the disease, the method of administration of the drug, combined treatment with other drugs, etc.

The substance or pharmaceutical composition containing the substance can be introduced into the patient's body in any quantity and by any route of administration effective for treating or preventing a disease.

After mixing the drug with a specific suitable pharmaceutically acceptable carrier in the desired dosage, the compositions comprising the essence of the invention can be administered orally, parenterally, topically, and the like to the human or other animals. The introduction can be carried out both once and several times a day, week (or any other time interval), or from time to time. In addition, the compound can be introduced into the patient’s body daily for a certain period of days, and then a period follows without a substance.

In the case where the compound of the present invention is used as part of a combination therapy regimen, a dose of each of the components of the combination therapy is administered during the required treatment period. The compounds that make up the combination therapy can be administered into the patient's body both at a time, in the form of a dosage containing all the components, and in the form of individual dosages of the components.

Pharmaceutical Compositions

The invention also relates to pharmaceutical compositions that contain at least one of the compounds described herein (or a prodrug, a pharmaceutically acceptable salt or other pharmaceutically acceptable derivative) and one or more pharmaceutically acceptable carriers, adjuvants, solvents and / or excipients, such that can be introduced into the patient’s body together with the compound that is the essence of this invention, and which do not destroy the pharmacological activity of this compound, and I are non-toxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

The pharmaceutical compositions of this invention comprise the compounds of this invention together with pharmaceutically acceptable carriers, which may include any solvents, diluents, dispersions or suspensions, surfactants, isotonic agents, thickeners and emulsifiers, preservatives, astringents, lubricants materials, etc., suitable for a particular dosage form. Materials that may serve as pharmaceutically acceptable carriers include, but are not limited to, mono- and oligosaccharides, as well as their derivatives; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut, cottonseed, safrole, sesame, olive, corn and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic solution, Ringer's solution; ethyl alcohol and phosphate buffers. Also in the composition of the composition may be other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as dyes, release fluids, film-forming agents, sweeteners, flavors and fragrances, preservatives and antioxidants.

The subject of the present invention is also dosage forms — a class of pharmaceutical compositions whose composition is optimized for a particular route of administration into the body in a therapeutically effective dose, for example, for administration to the body orally, topically, pulmonally, for example, as an inhaled spray, or intravascularly, intranasally , subcutaneously, intramuscularly, as well as by the infusion method, in recommended dosages.

The dosage form of the present invention may contain a compound of the formula described herein or a pharmaceutically acceptable salt thereof, and an additional preparation, for example, selected from the following: an antitumor preparation, an antiviral drug, an anti-inflammatory drug, and any pharmaceutically acceptable carrier, adjuvant or solvent. Dosage forms of the present invention may contain formulations prepared using liposome or microencapsulation methods, methods for preparing nanoforms of the preparation, and other examples known in the pharmaceutical art.

In preparing the composition, for example, in tablet form, the active principle is mixed with one or more pharmaceutical excipients, such as gelatin, starch, lactose, magnesium stearate, talc, silica, gum arabic, mannitol, microcrystalline cellulose, hypromellose or the like.

Tablets may be coated with sucrose, a cellulosic derivative, or other suitable coating materials. Tablets can be prepared in various ways, such as direct compression, dry or wet granulation, or hot fusion.

A pharmaceutical composition in the form of a gelatin capsule can be prepared by mixing the active principle with a solvent and filling the mixture with soft or hard capsules.

Detailed Disclosure of Invention

Overview of the methods for producing compounds of the invention

The compounds of the present invention can be prepared using the synthetic methods described below. The listed methods are not exhaustive and allow the introduction of reasonable modifications. These reactions should be carried out using suitable solvents and materials. When implementing these general procedures for the synthesis of specific substances, it is necessary to take into account the functional groups present in the substances and their influence on the course of the reaction. To obtain some substances, it is necessary to change the order of the stages or give preference to one of several alternative synthesis schemes. It should be understood that these and all examples cited in the application materials are not limiting and are provided merely to illustrate the present invention.

Preparation of Compounds of the Invention

Scheme 1 shows the preparation of compounds of the invention.

Scheme 1. General scheme for the preparation of derivatives of N-hydroxy-N '- (phenyl) -formamidine.

Preparation of Compound (E) -N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide

Scheme 2. General scheme for obtaining the compound (E) -N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide

The stage of obtaining 2-methyl-4- (3-methylbut-1-enyl) nitrobenzene (2)

In a three-necked round-bottom flask equipped with a magnetic stirrer, a thermometer and a dropping funnel, 250 ml of toluene and 39 g of isobutyltriphenylphosphonium bromide are placed. A solution funnel of 11 g of potassium tert-butoxide in 60 ml of tetrahydrofuran is placed in a dropping funnel and added to the flask with stirring for 15 minutes; after addition, the solution is stirred for 2 hours and cooled to -40 ° C. Then add a solution of 13 g of nitrobenzaldehyde (compound 1) in 50 ml of toluene with stirring, maintaining the temperature minus 40 ° C. After the addition of the aldehyde, cooling was removed and the mixture was stirred at room temperature for 3 hours. Then, 100 ml of a saturated solution of ammonium chloride were added, the organic layer was separated, washed with 100 ml of water, dried and evaporated. The residue was purified on a silica gel column to give 15 g of nitroolefin (compound 2; 93%).

The stage of obtaining 4-isopentyl-2-methylaniline (3)

150 ml of methanol, 15 g of olefin (compound 2) and 1.5 g of 10% palladium on activated carbon are placed in a round bottom flask equipped with a magnetic stirrer. The mixture was stirred at 1 atm of hydrogen for 36 hours, after which the catalyst was filtered off. The filtrates were evaporated, the residue was purified on a silica gel column to obtain 11.4 g of amine (compound 3; 88%).

The step of obtaining N '- (4-isopentyl-2-methylphenyl) -N, N-dimethylformimidamide (4)

In a round bottom flask equipped with a reflux condenser and a magnetic stirrer, 60 ml of dry toluene, 11.4 g of amine (compound 3) and 10.3 ml of dimethylformamide dimethyl acetal are placed. The mixture was boiled for 4 hours, cooled, the solvent was evaporated, and 11.4 g of N, N-dimethylformimidamide (compound 4) were obtained, which was used in the next step without further purification.

The step of obtaining (E) -N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide

In a round bottom flask equipped with a magnetic stirrer, place 500 ml of methanol; 11.4 g of N, N-dimethylformimidamide (compound 4) and 5.33 g of hydroxylamine hydrochloride. The mixture was stirred at room temperature for 2 hours, then the solvent was evaporated. The residue was diluted with 100 ml of diethyl ether and the precipitate was filtered off. The filtrate was washed with water, dried and the solvent was evaporated. The residue was diluted with petroleum ether, the resulting crystals were filtered off and 10.3 g of hydroxyformimidamide were obtained (compound 5; 73% based on compound 5).

¹ H and ¹³ C NMR spectra for compound 5 were recorded on an AVANCE 500 spectrometer with an operating frequency of 500 and 125 MHz for ¹ H and ¹³ C nuclei, respectively, at a sample temperature of 293 K for solutions in CDCl ₃ . As an internal standard, the residual solvent signal δ CDCl ₃ - 7.26 ppm was used. ( ¹ N), 77.16 ppm. ( ¹³ C):

¹ H NMR (500 MHz, CDCl ₃ ) δ: 7.36 (1H CHN), 7.08 - 6.96 (3H, H-3, H-5, NH), 6.93 (J = 8.5 Hz, 1H, H-6), 6.43 (1H, OH), 2.54 (2H, H-1 '), 2.26 (3H, H-3), 1.58 (1H, H-3'), 1.47 (2H, H-2 '), 0.93 (J = 6.5 Hz, 6H, H-4 ').

¹³ C NMR (125 MHz, CDCl ₃ ) δ 140.90 (CN), 137.83 (C-1), 135.43 (C-4), 131.24 (C-3), 127.13 (C-5), 125.81 (C-2) 115.23 (C-6), 41.13 (C-2 '), 33.11 (C-1'), 27.80 (C-3 '), 22.67 (C-4'), 17.47 (C-2).

Characterization of the biological activity of the compounds

For compounds that are the subject of the present invention, their effectiveness as compounds having antimycobacterial activity has been studied. Studies have been conducted, in particular, in relation to the compound N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide with the chemical formula:

Assessing the effect of the compounds of the invention on typical and cell wall deficient (CWD) mycobacterium tuberculosis.

Evaluation of the effect of the compounds of the invention on a typical strain of M. tuberculosis and cell wall deficient (CWD) strain M. tuberculosis was carried out in accordance with recommendations for standardized methods of microbiological studies in the detection, diagnosis and treatment of tuberculosis. Strain M. tuberculosis H37Rv grown on Levenshtein-Jensen medium (2-week growth). Cell wall deficient (CWD) strain of M. tuberculosis grown on Myccell DW.

The bacterial mass was removed into a sterile mortar and homogenized in a sterile 0.9% sodium chloride solution. The suspension was transferred by syringe into a sterile tube and settled for 15 min to precipitate conglomerates of cells. The supernatant (2-3 ml) was transferred via a syringe into a special sterile tube and, using the turbidity standard (5 units), the cell concentration was aligned to the standard (Standard Turbidity Sample named after Tarasevich OSO 42-28-10). 1.5 ml of the suspension was diluted in 13.5 ml of isotonic solution (1:10 dilution).

0.22 ml of the resulting suspension of mycobacteria was added to sterile eppendorfs K1 (control - 0.9% aqueous solution of sodium chloride) and K2 (control 0.9% aqueous solution of sodium chloride with 1% DMSO). 0.22 ml of a 0.9% aqueous solution of sodium chloride was added to the selected volume of the suspension in eppendorf K 1, and 0.2 ml of a 0.9% aqueous solution of sodium chloride with DMSO was added to eppendorf K2 (so that the final DMSO concentration was 1%). For test samples, 0.12 ml were taken from the suspension into sterile eppendorfs. 0.12 ml of a solution of N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide were added to the selected volume so that the final concentration of the compound was 25 μg / ml. The mixtures were incubated for 12 h at 37 ° C, then the mixture was suspended on a vortex and 0.2 ml were sown on Levenshtein-Jensen medium (for M. tuberculosis strain H37Rv) or Myccell DW medium (for cell wall deficient (CWD) strain M. tuberculosis) in 10 ml glass tubes. The tubes were kept for 4 days in a horizontal position at 37 ° C, then transferred to a tripod and incubated in an upright state for 21 days at 37 ° C. Mycobacteria growth was assessed visually by coating the surface of the medium with colonies of mycobacteria.

The use of N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide at a concentration of 25 μg / ml leads to complete growth retardation of both the H37Rv strain of M. tuberculosis and the cell wall deficient (CWD) strain of M. tuberculosis. In control tubes (K1 and K2), a continuous growth of mycobacteria was observed.

Determination of MIC _100% (100% minimum inhibitory concentration) of the compounds of the invention.

Determination of MIC _100% (100% minimum inhibitory concentration) of the compounds of the invention was carried out in accordance with a previously developed and applied method [2].

A suspension of M. tuberculosis strain H37Rv in Dubo broth [500 (500) H37Rv / 180 μl / well], a suspension of M.avium in Dubo broth [500 (500) M.avium / 180 µl / well] was added to the wells of a 96-well plate. or a suspension of M. tuberculosis strain CN-40 [250 (two hundred and fifty) CN-40/180 μl / well].

The plates were incubated in a CO ₂ incubator for 24 hours, after which 20 μl of various concentrations of N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide or 20 μl of Dubo broth (control) were added to the wells and incubated for 12 14 days.

For MIC, _{100% was} taken as the minimum concentration of the test substance, which completely suppresses the growth of mycobacteria by the 12-14 day of incubation, which manifests itself in the absence of a visible sediment of mycobacteria at the bottom of the well.

Thus, the inhibition or optimal blocking of the growth of the reference strain and resistant strains of M. tuberculosis (in vitro testing using specific strains) by using the compounds of the invention was investigated. As a result of the study, it was found that the MIC of _100% against both the M. tuberculosis strain H37Rv and the M. tuberculosis strain CN-40 (isoniazid-resistant strain) lies in the range 7.1–9.5 μg / ml. MIC _100% against Mycobacterium avium in the range of 9.5 ÷ 12.7 μg / ml.

Assessment of cellular toxicity of compounds

Assessment of cellular toxicity of compounds was carried out in accordance with existing standard protocols [3,4].

The MTT test was carried out, in particular, with N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide at concentrations from 1 to 50 μM on a HEK293T cell culture. One day after sieving, cells were incubated with the compounds of the invention for 1 day. Next, the ability of cells to restore MTT was determined spectrophotometrically. Based on the data obtained, the nature of the relationship between the concentration of the inhibitor and the metabolic activity of the cells was revealed.

Methodology:

1) HEK 293T cells are seeded in a 96-well plate at a concentration of 100,000 cells / ml, 100 μl per well.

2) After 24 hours, after the cells are attached, the test substance is added to the medium. The concentration of the substance: 50, 35, 20 and 5 μm in 2 replicates. This leaves holes for control samples:

a. Wells without cells, passing all stages of the experiment (Blank);

b. Wells with cells passing all stages of the experiment. Instead of the test substance, a solvent (DMSO) is added.

3) After incubation with the test substance, remove the medium from the wells. Add 90 μl PBS + 10 μl MTT (5 mg / ml). The culture plate is returned to the CO ₂ incubator for 2 hours.

4) Remove 80 μl of PBS with MTT. Add 100 μl of DMSO to dissolve the crystals of formazan.

5) Read the optical absorption at 570 nm.

As a result of the assessment of cytotoxicity in cell cultures (MTT test on HEK293 cells), it was found that the compounds of the invention show no toxicity, in particular, N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide does not show toxicity concentration of 50 μm.

Evaluation of the effect of compounds on phagocytosed pulmonary macrophages strain M. tuberculosis H37Rv

Evaluation of the effect of the compounds of the invention, in particular N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide, on phagocytosed pulmonary macrophages (Mph) M. tuberculosis strain H37Rv (by inclusion of 3H-uracil by mycobacteria) was carried out in accordance with technique [5]. The experiments were carried out in three repetitions.

The content of 3H-uracil incorporated by mycobacteria expressed in CPM (counts per minute) (mean ± SD):

Mph + H37Rv (control, without compound of the invention): 76263 ± 5011;

H37Rv (control for inclusion of uracil by extracellular H37Rv): 1561 ± 211;

Mph + H37Rv + compound (40 μg / ml.): 15188 ± 1603;

Mph + H37Rv + compound (10 μg / ml.): 19574 ± 2894;

Mph + H37Rv + compound (2.5 μg / ml.): 57348 ± 6451.

The results obtained indicate effective inhibition of the growth of mycobacteria in pulmonary macrophages using the compounds of the invention and, in particular, using N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide.

Thus, as a result of the studies, it was unexpectedly found that the compounds of the invention of general formula (I) and general formula (II) are characterized by high antimycobacterial activity, in particular with respect to M. tuberculosis, and are promising for use in the treatment of various infectious diseases caused by (associated) mycobacterial infections, including tuberculosis and its resistant forms.

Although the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are for the purpose of illustrating the present invention and should not be construed as in any way limiting the scope of the invention. It should be understood that various modifications are possible without departing from the gist of the present invention.

List of literature, which is included in the present description of the invention by reference:

1. Global tuberculosis report 2016. (2016) World Health Organization, 21st edition, P1-214.

2. In Vitro Activity of 3-Triazeneindoles against Mycobacterium tuberculosis and Mycobacterium avium. Nikonenko BV, Kornienko A, Majorov K, Ivanov P, Kondratieva T, Korotetskaya M, Apt AS, Salina E, Velezheva V. Antimicrob Agents Chemother. 2016 Sep 23; 60 (10): 6422-6424. PMID: 27503657.

3. MTT Cell Proliferation Assay Instruction Manual (ATCC Kit # 30-1010K).

4. Cell sensitivity assays: the MTT assay. van Meerloo J, Kaspers GJ, Cloos J. Methods Mol Biol. 2011; 731: P.237-245.

5. Lyadova I, Yeremeev V, Majorov K, Nikonenko B, Khaidukov S, Kondratieva T, Kobets N, Apt A. An ex vivo study of T lymphocytes recovered from the lungs of I / Stice infection with and susceptible to Mycobacterium tuberculosis. Infect Immun. 1998; 66 (10): 4981-8.

Claims (16)

1. The compound N'-hydroxy-N- (4-isopentyl-2-methylphenyl) formimidamide, characterized by the following structural formula:

. 2. The use of a compound according to claim 1 for the treatment and / or prevention of an infectious disease in a subject caused by mycobacterial infection. 3. The use according to claim 2, characterized in that the infectious disease is caused by Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium marinum, Mycobacterium kansasii or Mycobacterium avium. 4. The use according to claim 2, characterized in that the disease is tuberculosis. 5. The use according to claim 4, characterized in that the tuberculosis has a resistant form. 6. The use according to claim 2, characterized in that the subject is a mammal or bird. 7. The use according to claim 6, characterized in that the mammal is a human. 8. The use of a compound according to claim 1 for preparing a pharmaceutical composition for treating and / or preventing an infectious disease caused by a mycobacterial infection. 9. A pharmaceutical composition for treating and / or preventing an infectious disease caused by a mycobacterial infection, comprising an effective amount of a compound according to claim 1 and at least one pharmaceutically acceptable excipient. 10. The pharmaceutical composition according to claim 9, characterized in that the infectious disease is caused by Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium marinum, Mycobacterium kansasii or Mycobacterium avium. 11. The pharmaceutical composition according to claim 9, characterized in that the disease is tuberculosis. 12. The pharmaceutical composition according to claim 11, characterized in that the tuberculosis has a resistant form. 13. The pharmaceutical composition according to claim 9, characterized in that the pharmaceutically acceptable excipient is a carrier, excipient and / or solvent. 14. The pharmaceutical composition according to claim 9, characterized in that it has a bactericidal effect. 15. The pharmaceutical composition according to claim 9, characterized in that it has a bacteriostatic effect.