RU2495036C1 - Biologically active substances suppressing pathogenic bacteria, and method for inhibiting type iii secretion in pathogenic bacteria - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to indolyl-substituted derivatives of thiadiazinones prepared from oxamic acid thiohydrazide of general formula:

, wherein R represents H; R¹ represents pyridinyl; phenyl substituted by alkyl C1-C5, Hal, CF₃; R² represents H; alkyl C1-C5; -CH₂COOR⁴; benzyl substituted by Hal, OR⁴; benzoyl substituted by Hal, OR⁴, while R⁴ represents unsubstituted alkyl C1-C4.

EFFECT: there are prepared new compound which can find application in medicine for developing the therapeutic agent possessing pathogenic bacteria inhibitory activity.

2 cl, 2 dwg, 2 tbl, 13 ex

Description

Technical field

The invention relates to an indolyl-substituted derivative of thiadiazinones derived from thiohydrazides of oxamic acids of the general formula

which can be used to suppress pathogenic bacteria, in particular, affect the type III secretion system in pathogens.

State of the art

According to the WHO, almost 60% of deaths are associated with chronic diseases, which are based on chronic inflammation induced by pathogenic microorganisms. The general trend is a significant decrease in the effectiveness of antibiotics used in clinical practice against chronic infections. At the moment, effective antibiotics that have a pronounced therapeutic effect on a chronic infection do not exist on the drug market. This is due to the fact that in chronic infections, an evolutionarily developed adaptation of the pathogen takes place, aimed at long-term survival in the macroorganism, including a change in metabolic activity. This leads to a decrease in the sensitivity of the microorganism to antibacterial drugs, effective in the acute stage of the infectious process.

No less acute is the problem of the development of antibiotic resistance to the antibacterial drugs used. The formation of antibiotic resistance by microorganisms is associated with a number of negative consequences for both individual patients and society as a whole. With the resistance of the causative agent of the infectious disease to the main antibiotics, the probability of failure of the empirical treatment of a particular patient increases sharply. At the same time, it is quite obvious that each specific disease caused by a resistant microorganism is a manifestation of the process of formation and spread of resistance in the microbial population. The situation is complicated by the fact that large pharmaceutical companies have significantly reduced the development of antibiotics, because the growth of antibiotic resistance limits the possibility of using the drug over time and, accordingly, reduces the profit from sales.

If we evaluate the economic side of the problem under consideration, then the main motivating factor in the development of new antibacterial drugs at present is multidrug resistance. However, the need for drugs for the treatment of chronic and recurrent infections covers a much larger number of patients who do not receive the necessary effective treatment, which determines broad prospects in the development and implementation of new drugs.

In connection with the foregoing, the necessity of choosing a different drug search strategy based on the identification of fundamentally new targets for bacterial pathogens is quite obvious.

A detailed study and understanding of the mechanisms of interaction of pathogens with the host organism and the identification of the most vulnerable links in the chain of dynamic events of the manifestation of pathogenetic potential can be the basis for the effective selection of targets for the action of new generation drugs aimed at suppressing virulence, the so-called antivirulent drugs. Such drugs should have a number of advantages compared to the antibiotics currently in use. Firstly, the effect on the virulent properties of the pathogen, which are important when interacting in the pathogen-host system without suppressing viability, allows us to predict the absence of selective pressure and the selection of resistant bacteria strains. Secondly, drugs acting on specific targets of pathogenic bacteria will not be toxic to eukaryotic cells and normal human microflora, which is a serious problem when using antibiotics. Indeed, the microbial cells that make up the microbiota of a healthy person outnumber human cells by tens of times, colonizing various organs and contributing to the implementation of many vital processes, the role of the microbiota in which is only beginning to be realized. The microbiota is a dynamic state, and a violation of its population composition under the action of antibiotics leads to a loss of symbiotic benefit and the development of infections caused by opportunistic pathogens.

The leading role in the realization of the virulent properties of pathogenic bacteria belongs to the type III secretion system, through which protein virulence factors are transported to the macroorganism cell. The process is carried out by secretion and export of effector molecules through membranes into the intercellular space or directly into the cytoplasm of the host cell, which leads to a change in its normal physiological state and promotes adhesion, invasion and intracellular reproduction of the pathogen. In addition, through the secretion system, a range of adaptive mechanisms are realized, aimed at long-term survival in the host organism, related to their ability to inactivate the body's defense reactions.

Compounds affecting the virulent properties of pathogenic bacteria are known in the art. These compounds inhibit the secretory functions of certain gram-negative bacteria, such as Yersinia pseudotuberculosis, Salmonella enterica, Pseudomonas aeruginosa, pathogenic strains of E. coli, Chlamydia spp, and do not cause the development of resistance to drugs based on them. These compounds include hydrazones obtained on the basis of hydrazides of benzoic and pyridinecarboxylic acids

("Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle" FEBS Letters, 581 (2007), 587-595 / Low molecular weight inhibitors of the Yersinia type III secretion system block the Chlamydia pneumoniae infection cycle;

"Small-Molecule Inhibitors Specifically Targeting Type III Secretion" Infection and Immunity, 2005, p.3104-3114, Vol.73, No.5 / Low molecular weight inhibitors specifically acting on type III secretion system;

“A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis” PNAS, 26, 2006, vol. 103, No. 39, 14566-14571 / Small molecule inhibitors of type III secretion system inhibit various stages of the intracellular cycle of development of Chlamydia trachomatis).

However, the disadvantage of these compounds is their significant toxicity to eukaryotic cells, which fundamentally limits the possibility of their further development as drugs.

Disclosure of invention

The objective of the invention is to provide compounds that suppress the secretory function of pathogenic bacteria that are not toxic to normal microflora and host cells, reducing the risk of resistance.

The technical result is achieved by obtaining biologically active compounds with high selective inhibitory activity against pathogenic bacteria, including those causing chronic diseases. Moreover, the compounds obtained are non-toxic to human and animal cells, they are readily soluble in organic solvents, and are also characterized by specific activity under the conditions of biological systems of exposure to pathogenic bacteria and reduce the risk of resistance of pathogenic bacteria.

The technical result is achieved due to the fact that the claimed biologically active compounds that suppress pathogenic bacteria are indolyl-substituted derivatives of thiadiazinones derived from thiohydrazides of oxamic acids of the general formula

Where

R represents H;

R ¹ represents pyridinyl, phenyl substituted with alkyl C1-C5, Hal, CF ₃ ;

R ² represents H, alkyl C1-C5, -CH ₂ COOR ⁴ , benzyl, substituted Hal, OR ⁴ , benzoyl, substituted Hal, OR ⁴ , and

R ⁴ is unsubstituted C1-C4 alkyl.

A method of inhibiting type III secretion in pathogenic bacteria is to expose the bacteria to an effective amount of a compound of the claimed compound.

The secretion system was detected in taxonomically distant microorganisms (pathogenic bacteria) - pathogens of especially dangerous infections, such as Yersinia, Brucella, and pathogens of socially significant infections, such as Chlamydia, Salmonella, Shigella, etc. The type III secretion system, called the conservative, is absolutely necessary for the development of an acute infectious process, and the chronization of the infection fundamentally depends on its functioning. Therefore, type III secretion inhibitors in pathogens will have a directed effect on the mechanisms causing the process of chronic infection, without affecting the viability of bacteria, actually “disarming” the pathogen. Unlike antibiotics, the action of such specific inhibitors will not lead to the development of genetically determined resistance. In addition, the secretion system III is present only in pathogenic bacteria; therefore, its inhibitors are not toxic to normal human microflora and host cells, i.e. characterized by selectivity.

Examples of the synthesis of compounds

Substituted thiadiazines are prepared based on thiohydrazides of oxamic acids of the general formula (I), which are prepared by reacting the corresponding chloroacetamides with a pre-prepared solution of elemental sulfur with morpholine and then reacting the monothiooxamides with hydrazine hydrate. The thiohydrazides thus obtained are condensed with the corresponding indolecarbaldehydes, reduced with sodium borohydride in methanol and cyclized with monochloracetic acid under conditions of alkaline catalysis to form the corresponding thiadiazine. Substituted thiadiazines based on derivatives of thiohydrazides of oxamic acids can be synthesized according to the following scheme:

General method for producing chloroacetamides

To a solution (0.1 mol) of the corresponding aniline in 100 ml of DMF was added, with cooling, 8.75 ml (0.11 mol) of chloroacetyl chloride, making sure that the temperature did not exceed 20 ° C. After completion of the addition, the solution was stirred at room temperature for another 2 hours. Then the reaction mixture was poured into 600 ml of cold water and the precipitate was filtered off. Washed it on the filter with water and dried in air.

General procedure for the preparation of thiohydrazides

A solution of 0.15 mol of elemental sulfur in 50 ml of DMF was prepared and 0.06 mol of morpholine was added. The resulting mixture was stirred for 20-30 minutes and a solution of 0.05 mol of the corresponding chloroacetamide in a minimum amount of DMF was added to it with cooling, making sure that the temperature did not exceed 15 ° C. The resulting mixture was stirred for 3-6 hours (control by TLC). After completion of the reaction, the reaction mixture was poured into water, the substance was extracted with ethyl acetate, the organic layer was passed through a column of silica gel, evaporated on a rotary evaporator, then the obtained residue was dissolved in a small amount (5-6 ml) of DMF and 6 ml (0.12 mol) was added hydrazine hydrate and stirred at room temperature for 2 hours. Then it was poured into water and acidified with diluted hydrochloric acid to pH 7. The precipitate was filtered off and dried in air.

General Procedure for the Preparation of N-Indolyl-Substituted Thiohydrazides

To 10 mmol of the corresponding oxamic acid thiohydrazide in 5 ml of methanol, 11 mmol of the corresponding aldehyde was added and refluxed for 5 minutes. The reaction mixture was cooled. Under ice-cooling (0-5 ° C.) and stirring, 55 mg (1.5 mmol) of sodium borohydride was added in small portions. The reaction mixture was left for 2 hours while cooling and stirring, then 25 ml of water was added and neutralized with dilute hydrochloric acid to a neutral medium. The precipitate formed was filtered off and, if necessary, recrystallized from isopropanol.

General procedure for the preparation of indolyl substituted thiadiazinones

1 mmol of the corresponding N'-indolyl-substituted oxamic acid thiohydrazide was added to a solution of 1.2 mmol of chloroacetic (or bromoacetic) acid in 5 ml of isopropanol. A catalytic amount of ammonium acetate was added to the reaction mixture and boiled for 2-5 hours (control by thin layer chromatogram). After completion of the reaction, the reaction mixture was poured into 50 ml of water and the substance was extracted with ethyl acetate, the organic layer was washed with water 2 × 50 ml. Then the organic layer was separated, dried over sodium sulfate and the solvent was removed on a rotary evaporator. The residue was recrystallized from a minimum amount of ethanol.

Obtaining starting thiohydrazides

N-chloroacetyl-2-aminopyridine (1)

Yield: N-chloroacetyl-2-aminopyridine (1) 11.9 g (70%). Mp 140-141 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 4.20 (s, 2H, CH ₂ Cl); 7.41-7.45 (m, 2H, Ar); 8.07-8.11 (m, 2H, arom); 10.31 (s, 1H, NH). Found (%): C 49.20, H 4.03, N 16.21. Calculated (%): C 49.28, H 4.14, N 16.42. Mass spectrum, m / z: 170.

2-Hydrazino-N-pyridin-2-yl-2-thioxo-acetamide (2)

Yield: 2-hydrazino-N-pyridin-2-yl-2-thioxo-acetamide (2) 1.2 g (60%). Mp 164-165 ° C. NMR ¹ H DMSO _d6 (δ, ppm, J, Hz): 7.40-7.43 (m, 2H, Ar); 8.07-8.10 (m, 2H, arom); 10.59 (s, 1H, NH). Found (%): C 42.76, H 4.01, N 28.44. Calculated (%): C 42.85, H 4.11, N 28.55. Mass spectrum, m / z: 196.

N-chloroacetyl-4- (trifluoromethyl) aniline (3)

The output of chloroacetamide (3) 19 g, 80%. Mp 115-116 ° C. NMR ¹ H DMSO _d6 (δ, ppm, J, Hz): 4.20 (s, 2H, Ar-CH ₂ -Cl); 7.07 (d, 2H, arom); 7.21 (d, 2H, arom). Found (%): C 45.38, H 2.81, N 5.96. Calculated (%): C 45.49, H 2.97, N 5.89. Mass spectrum, m / z: 237.

2-Hydrazino-2-thioxo-N- [4- (trifluoromethyl) phenyl] acetamide (4)

The yield of thiohydrazide (4) is 6.4 g, 60%. Mp 154-155 ° C. NMR ¹ H DMSO _d6 (δ, ppm, J, Hz): 7.08 (d, 2H, arom); 7.24 (d, 2H, arom); 10.59 (s, 1H, NH). Found (%): C 41.16, H 3.13, N 15.83. Calculated (%): C 41.06, H 3.06, N 15.96. Mass spectrum, m / z: 263.

N-chloroacetyl-2,4-difluoroaniline (5)

The yield of chloroacetamide (5) is 87%. Mp 101-102 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 4.20 (s, 2H, ArCH ₂ Cl); 7.10-7.25 (m, 2H, Ar); 7.53 (m, 1H, Ar); 10.25 (s, 1H, Ar-NH-CO). Found (%): C 46.62, H 2.99, N 6.84. Calculated (%): C 46.74, H 2.94, N 6.81. Mass spectrum, m / z: 205.

2- [Hydrazino- (thioxo) -acetylamino] -2,4-difluoroaniline (6)

Yield: (6) 8.1 g, 70%. Mp 154-156 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 7.10-7.26 (m, 2H, Ar); 7.50 (m, 1H, Ar); 10.30 (s, 1H, Ar-NH-CO). Found (%): C 41.40, H 3.18, N 18.25. Calculated (%): C 41.56, H 3.05, N 18.17. Mass spectrum, m / z: 231.

N-chloroacetyl-2-methylaniline (7)

The yield of chloroacetamide (7) 15.5 g, 85%. Mp 124-126 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 1.74 (s, 3H, ArCH ₃ ); 4.20 (s, 2H, CH ₂ Cl); 7.07 (m, 1H, Ar); 7.21 (m, 1H, Ar); 7.41 (m, 1H, Ar); 7.63 (m, 1H, Ar); 10.57 (s, 1H, Ar-NH-CH ₂ Cl). Found (%): C 58.86, H 5.47, N 7.64. Calculated (%): C 58.87, H 5.49, N 7.63. Mass spectrum, m / z: 183.

2- [Hydrazino- (thioxo) -acetylamino] -2-methylaniline (8)

The yield of thiohydrazide (8) is 10.4 g, 60%. Mp 154-157 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 1.74 (s, 3H, ArCH ₃ ); 7.07-7.61 (m, 4H, Ar); 10.32 (s, 1H, Ar-NH-CO). Found: C 51.41, H 5.53, N 20.40. Calculated: C 51.66, H 5.30, N 20.08. Mass spectrum, m / z: 209.

N-chloroacetyl-4-fluoroaniline (9)

Yield (9) 80%. Mp 110-112 ° C. NMR ¹ H DMSO _d6 (δ, ppm, J, Hz): 4.20 (s, 2H, ArCH ₂ Cl); 7.21 (m, 2H, Ar); 7.61 (m, 2H, Ar); 10.21 (s, 1H, Ar-NH-CO). Found (%): C 51.17, H 3.73, N 7.44. Calculated (%): C 51.22, H 3.76, N 7.47. Mass spectrum, m / z: 187.

2- [Hydrazino- (thioxo) -acetylamino] -4-fluoroaniline (10)

Yield: (10) 6.4 g, 60%. Mp 154-155 ° C. NMR ¹ H DMSO _d6 (δ, ppm, J, Hz): 7.22 (m, 2H, Ar); 7.63 (m, 2H, Ar); 10.17 (s, 1H, Ar-NH-CO). Found (%): C 45.01, H 3.85, N 19.80. Calculated (%): C 45.06, H 3.78, N 19.71. Mass spectrum, m / z: 213.

Brief Description of the Figures

Figure 1 shows the most characteristic morphological changes in the intracellular development of chlamydial infection in vitro under the influence of the studied compounds on the example of compound CL-79.

In figure 2. analysis of the specific activity of the selected compounds in relation to the III transport system of chlamydia is shown.

Examples of the performance of the claimed method

Example 1

(CL-79)

2- [2- (1H-indol-3-ylmethyl) hydrazino] -2-thioxo-N- [2- (trifluoromethyl) phenyl] acetamide (11)

As starting compounds, thiohydrazide 4 and indole-3-carbaldehyde were used.

The yield of hydrazide (9) is 255 mg, 75%. Mp 238-239 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 5.32 (s, 1H, -NH-CH ₂ -Indole); 7.07-7.77 (m, 8H, Ar); 7.92 (s, 1H, (CH) Indole); 10.12 (s, 1H, Ar-NH-CO); 10.92 (s, 1H, (NH) Indole). Found (%): C 59.54, H 4.31, N 16.22. Calculated (%): C 59.64, H 4.42, N 16.36. Mass spectrum, m / z: 341.

4- (1H-indol-3-ylmethyl) -5-oxo-N- [2- (trifluoromethyl) phenyl] -5,6-dihydro-4H-1,3,4-thiodiazine-2-carboxamide (CL-79 )

Yield (CL-79) 250 mg, 75%. Mp > 250 ° C with decomp. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 4.02 (s, 2H, -S-CH ₂ ); 5.36 (s, 1H, -NH-CH ₂ -Indole); 7.07-7.79 (m, 8H, Ar); 7.90 (s, 1H, (CH) Indole); 10.10 (s, 1H, Ar-NH-CO); 10.94 (s, 1H, (NH) Indole). Found (%): C 59.60, H 4.02, N 14.60. Calculated (%): C 59.68, H 3.95, N 14.65. Mass spectrum, m / z: 387.

Example 2

(CL-80)

2- (2 - {[1- (4-Fluorobenzyl) -1H-indol-3-yl] methyl} hydrazino) -2-thioxo-N- [2- (trifluoromethyl) phenyl] acetamide (13)

As starting compounds, thiohydrazide 4 and N- (4-fluorobenzyl) -indole-3-carbaldehyde were used.

The yield of hydrazide (13) is 269 mg, 60%. Mp 279-280 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 4.32 (d, 2H, -NH-CH ₂ -Indole); 4.98 (s, 2H, -NH _{Indole-CH 2} -Ar); 5.32 (s, 1H, NH); 6.77-8.18 (m, 13H, Ar); 10.14 (s, 1H, Ar-NH-CO); 10.92 (s, 1H, CS-NH). Found (%): C 63.92, H 4.57, N 12.38. Calculated (%): C 63.99, H 4.47, N 12.44. Mass spectrum, m / z: 449.

4 - {[1- (4-Fluorobenzyl) -1H-indol-3-yl] methyl} -5-oxo-N- [2- (trifluoromethyl) phenyl] -5,6-dihydro-4H-1,3, 4-thiadiazine-2-carboxamide (CL-80)

Yield (CL-80) 367 mg, 75%. Mp > 250 ° C with decomp. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 4.05 (s, 2H, -S-CH ₂ ); 4.98 (s, 2H, -NH _{Indole-CH 2} -Ar); 5.36 (s, 1H, -NH-CH ₂ -Indole); 7.07-7.79 (m, 8H, Ar); 7.90 (s, 1H, (CH) Indole); 10.10 (s, 1H, Ar-NH-CO); 10.94 (s, 1H, (NH) Indole). Found (%): C 63.60, H 4.19, N 11.37. Calculated (%): C 63.66, H 4.11, N 11.42. Mass spectrum, m / z: 489.

Example 3

(CL-81)

2- {2 - [(1-Methyl-1H-indol-3-yl) methyl] hydrazino} -2-thioxo-N- [2- (trifluoromethyl) phenyl] acetamide (15)

As starting compounds, thiohydrazide 4 and N-methyl-indole-3-carbaldehyde were used.

The yield of hydrazide (15) 220 mg, 62%. Mp 253-254 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 3.61 (s, 3H, CH ₃ ); 4.35 (d, 2H, -NH-CH ₂ -Indole); 4.90 (s, 2H, -NH _{Indole-CH 2} -Ar); 5.33 (s, 1H, NH); 6.79-8.10 (m, 9H, Ar); 10.12 (s, 1H, Ar-NH-CO); 10.89 (s, 1H, CS-NH). Found (%): C 60.61, H 4.85, N 15.66. Calculated (%): C 60.66, H 4.81, N 15.72. Mass spectrum, m / z: 355.

4 - [(1-Methyl-1H-indol-3-yl) methyl] -5-oxo-N- [2- (trifluoromethyl) phenyl] -5,6-dihydro-4H-1,3,4-thiadiazine- 2-carboxamide (CL-81)

Yield (CL-81) 316 mg, 75%. Mp > 250 ° C with decomp. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 3.62 (s, 3H, CH ₃ ); 4.02 (s, 2H, -S-CH ₂ ); 5.31 (s, 1H, -NH-CH ₂ -Indole); 6.89-7.71 (m, 8H, Ar); 7.92 (s, 1H, (CH) Indole); 10.11 (s, 1H, Ar-NH-CO). Found (%): C 60.52, H 4.36, N 14.10. Calculated (%): C 60.59, H 4.32, N 14.13. Mass spectrum, m / z: 395.

Example 4

(CL-82)

2- [2- (1H-indol-3-ylmethyl) hydrazino] -N- (2-methylphenyl) -2-thioxoacetamide (17)

As starting compounds, thiohydrazide 8 and indole-3-carbaldehyde were used.

The yield of hydrazide (17) is 179 mg, 52%. Mp 251-252 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 2.23 (s, 3H, CH ₃ ) 4.34 (d, 2H, -NH-CH ₂ -Indole); 5.32 (s, 1H, NH) 6.79-8.11 (m, 9H, Ar); 10.11 (s, 1H, Ar-NH-CO); 10.87 (s, 1H, CS-NH). Found (%): C 63.74, H 5.39, N 16.50. Calculated (%): C 63.88, H 5.36, N 16.55. Mass spectrum, m / z: 337.

4- (1H-indol-3-ylmethyl) -N- (2-methylphenyl) -5-oxo-5,6-dihydro-4H-1,3,4-thiadiazine-2-carboxamide (CL-82)

Yield (CL-82) 324 mg, 86%. Mp > 250 ° C with decomp. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 2.23 (s, 3H, CH ₃ ); 4.08 (s, 2H, -S-CH ₂ ); 5.33 (s, 1H, NH); 5.38 (s, 1H, -NH-CH ₂ -Indole); 6.77-8.09 (m, 9H, Ar); 10.11 (s, 1H, Ar-NH-CO). Found (%): C 63.32, H 4.81, N 14.72. Calculated (%): C 63.47, H 4.79, N 14.80. Mass spectrum, m / z: 377.

Example 5

(CL-83)

2- (2 - {[1- (4-Methoxybenzyl) -1H-indol-3-yl] methyl} hydrazino) -2-thioxo-N- [2,4-difluorophenyl] acetamide (19)

As starting compounds, thiohydrazide 6 and N- (4-methoxybenzyl-indole-3-carbaldehyde) were used.

The yield of hydrazide (19) is 388 mg, 81%. Mp 281-282 ° C. NMR ¹ H CDCl ₃ (6, ppm, J, Hz): 3.87 (s, 3H, CH ₃ ); 4.36 (d, 2H, -NH-CH ₂ -Indole); 4.87 (s, 2H, -NH _{Indole-CH 2} -Ar); 5.30 (s, 1H, NH); 6.82-8.23 (m, 12H, Ar); 10.05 (s, 1H, Ar-NH-CO); 10.84 (s, 1H, CS-NH). Found (%): C 62.42, H 4.69, N 11.52. Calculated (%): C 62.49, H 4.61, N 11.66. Mass spectrum, m / z: 479.

4 - {[1- (4-Methoxybenzyl) -1H-indol-3-yl] methyl} -5-oxo-N- [2,4-difluorophenyl] -5,6-dihydro-4H-1,3,4 -thiadiazine-2-carboxamide

Yield (CL-83) 436 mg, 86%. Mp > 300 ° C with decomp. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 3.87 (s, 3H, CH ₃ ); 4.05 (s, 2H, -S-CH ₂ ); 4.86 (s, 2H, -NH _{Indole-CH 2} -Ar); 5.31 (s, 1H, -NH-CH ₂ -Indole); 6.82-8.24 (m, 12H, Ar); 10.06 (s, 1H, Ar-M / -CO). Found (%): C 62.20, H 4.32, N 10.61. Calculated (%): C 62.30, H 4.26, N 10.76. Mass spectrum, m / z: 519.

Example 6

(CL-84)

2- (2 - {[1- (4-Fluorobenzyl) -1H-indol-3-yl] methyl} hydrazino) -2-thioxo-N- [4-fluorophenyl] acetamide

As starting compounds, thiohydrazide 10 and N- (4-fluorobenzyl) -indole-3-carbaldehyde were used.

The yield of hydrazide (21) 328 mg, 72%. Mp 254-255 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 4.32 (d, 2H, -NH-CH ₂ -Indole); 4.98 (s, 2H, -NH _{Indole-CH 2} -Ar); 5.32 (s, 1H, NH); 7.13-8.20 (m, 13H, Ar); 10.11 (s, 1H, Ar-NH-CO); 10.82 (s, 1H, CS-NH). Found (%): C 63.90, H 4.53, N 12.40. Calculated (%): C 63.99, H 4.47, N 12.44. Mass spectrum, m / z: 449.

4 - {[1- (4-Fluorobenzyl) -1H-indol-3-yl] methyl} -5-oxo-N- [4-fluorophenyl] -5,6-dihydro-4H-1,3,4-thiadiazine -2-carboxamide

Yield (CL-84) 316 mg, 75%. Mp > 250 ° C with decomp. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 4.05 (s, 2H, -S-CH ₂ ); 4.32 (d, 2H, -NH-CH ₂ -Indole); 4.97 (s, 2H, -NH _{Indole-CH 2} -Ar); 7.13-8.22 (m, 13H, Ar); 10.13 (s, 1H, Ar-NH-CO). Found (%): C 63.56, H 4.18, N 11.34. Calculated (%): C 63.66, H 4.11, N 11.42. Mass spectrum, m / z: 489.

Example 7

(CL-85)

N- (2,4-Difluorophenyl) -2- (2 - {[1- (4-fluorobenzoyl) -1H-indol-3-yl] methyl} hydrazino) -2-thioxoacetamide

As starting compounds, thiohydrazide 6 and N- (4-fluorobenzyl) -indole-3-carbaldehyde were used.

The yield of hydrazide (23) 414 mg, 86%. Mp > 300 ° C with decomp. NMR ¹ H CDCl ₃ (5, ppm, J, Hz): 4.12 (d, 2H, -NH-CH ₂ -Indole); 5.38 (s, 1H, NH); 6.92-8.32 (m, 12H, Ar); 10.11 (s, 1H, Ar-NH-CO); 10.98 (s, 1H, CS-NH). Found (%): C 59.61, H 3.59, N 11.52. Calculated (%): C 59.75, H 3.55, N 11.61. Mass spectrum, m / z: 481.

N- (2,4-difluorophenyl) -4 - {[1- (4-fluorobenzoyl) -1H-indol-3-yl] methyl} -5-oxo-5,6-dihydro-4H-1,3,4 -thiadiazine-2-carboxamide

Yield (CL-85) 316 mg, 75%. Mp > 300 ° C with decomp. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 4.02 (s, 2H, -S-CH ₂ ); 4.45 (d, 2H, -NH-CH ₂ -Indole); 5.38 (s, 1H, NH); 6.92-8.32 (m, 12H, Ar); 10.11 (s, 1H, Ar-NH-CO). Found (%): C 59.70, H 3.31, N 10.68. Calculated (%): C 59.77, H 3.28, N 10.72. Mass spectrum, m / z: 521.

Example 8

(CL-86)

N- (2,4-difluorophenyl) -2- (2 - {[1- (4-methoxybenzoyl) -1H-indol-3-yl] methyl} hydrazino) -2-thioxoacetamide

As starting compounds, thiohydrazide 6 and N- (4-fluorobenzoyl) -indole-3-carbaldehyde were used.

The yield of hydrazide (25) is 409 mg, 83%. Mp > 300 ° C with decomp. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 4.03 (d, 2H, -NH-CH ₂ -Indole); 5.30 (s, 1H, NH); 6.98-8.21 (m, 12H, Ar); 10.09 (s, 1H, Ar-NH-CO); 10.92 (s, 1H, CS-NH). Found (%): C 60.61, H 4.14, N 11.23. Calculated (%): C 60.72, H 4.08, N 11.33. Mass spectrum, m / z: 493.

N- (2,4-difluorophenyl) -4 - {[1- (4-methoxybenzoyl) -1H-indol-3-yl] methyl} -5-oxo-5,6-dihydro-4H-1,3,4 -thiadiazine-2-carboxamide

Yield (CL-86) 442 mg, 83%. Mp > 300 ° C with decomp. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 3.87 (s, 3H, CH ₃ ); 4.05 (s, 2H, -S-CH ₂ ); 4.03 (d, 2H, -NH-CH ₂ -Indole); 5.30 (s, 1H, NH); 6.98-8.21 (m, 12H, Ar); 10.09 (s, 1H, Ar-NH-CO); 10.92. Found (%): C 60.52, H 3.81, N 10.31. Calculated (%): C 60.67, H 3.77, N 10.48. Mass spectrum, m / z: 533.

Example 9

(CL-87)

Methyl {3 - [(2- {2 - [(2,4-difluorophenyl) amino] -2-oxoethanethioyl} hydrazino) methyl] -1H-indol-1-yl} acetate

As starting compounds, thiohydrazide 6 and methyl (3-formyl-1H-indol-1-yl) acetate were used.

Yield of hydrazide (27) 336 mg, 78%. Mp 247-248 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 3.61 (s, 3H, CH ₃ ); 4.28 (d, 2H, -NH-CH ₂ -Indole); 4.45 (s, 2H, -NH _{Indole-CH 2} -COOMe); 5.36 (s, 1H, NH); 6.82-7.96 (m, 8H, Ar); 10.12 (s, 1H, Ar-NH-CO); 10.78 (s, 1H, CS-NH). Found (%): C 55.42, H 4.25, N 12.85. Calculated (%): C 55.55, H 4.20, N 12.96. Mass spectrum, m / z: 431.

Methyl {3 - [(2 - {[(2,4-difluorophenyl) amino] carbonyl} -5-oxo-5,6-dihydro-4H-1,3,4-thiadiazin-4-yl) methyl] -1 N-indol-1-yl} acetate (CL-87)

Yield (CL-87) 316 mg, 75%. Mp > 250 ° C with decomp. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 3.62 (s, 3H, CH ₃ ); 4.04 (s, 2H, -S-CH ₂ ); 4.27 (d, 2H, -NH-CH ₂ -Indole); 4.46 (s, 2H, -NH _{Indole-CH 2} -COOMe); 6.80-7.92 (m, 8H, Ar); 10.14 (s, 1H, Ar-NH-CO). Found (%): C 55.85, H 3.88, N 11.72. Calculated (%): C 55.93, H 3.84, N 11.86. Mass spectrum, m / z: 471.

Example 10

(CL-88)

2- {2 - [(1-Methyl-1H-indol-3-yl) methyl] hydrazino} -N-pyridin-2-yl-2-thioxoacetamide

As starting compounds, thiohydrazide 2 and N-methyl-indole-3-carbaldehyde were used.

The yield of hydrazide (29) is 139 mg, 44%. Mp 279-280 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 3.62 (s, 3H, CH ₃ ); 4.34 (d, 2H, -NH-CH ₂ -Indole); 5.37 (s, 1H, NH); 6.67-8.30 (m, 9H, Ar); 10.27 (s, 1H, Ar-NH-CO); 10.82 (s, 1H, CS-NH). Found (%): C 60.12, H 5.11, N 20.53. Calculated (%): C 60.16, H 5.05, N 20.63. Mass spectrum, m / z: 338.

4 - [(1-Methyl-1H-indol-3-yl) methyl] -5-oxo-N-pyridin-2-yl-5,6-dihydro-4H-1,3,4-thiadiazine-2-carboxamide (CL-88)

Yield (CL-88) 223 mg, 59%. Mp > 300 ° C with decomp. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 3.62 (s, 3H, CH ₃ ); 4.12 (s, 2H, -S-CH ₂ ); 4.45 (d, 2H, -1H-CH ₂ -Indole); 6.74-8.32 (m, 9H, Ar); 10.29 (s, 1H, Ar-NH-CO). Found (%): C 60.10, H 4.58, N 18.41. Calculated (%): C 60.14, H 4.52, N 18.46. Mass spectrum, m / z: 378.

Biological Examples

The following methods were used to determine the toxicity and biological activity of indolyl-substituted thiadiazinone derivatives derived from oxaminic acid thiohydrazides.

1. Methods for determining the cytotoxic effect

A) To determine the toxicity to eukaryotic cells, we used the method of staining cells with methylene blue (standard method) with subsequent spectrometric consideration of the results. The work was carried out in the format of 96-well plates.

In the daily monolayer of McCoy cells (a hybrid line of human synovial cells and murine fibroblasts), the culture medium was replaced with fresh medium and various doses of the studied chemical compounds were introduced. Cells were incubated for 24 hours in a CO ₂ incubator at 37 ° C. After 24 hours, culture medium was taken from the wells and cells were washed with 0.1 mM phosphate buffered saline (PBS). Cells were fixed with chilled methanol (20 μl) for 15 min at 4 ° C. 40 μl of 0.5% methylene blue was added to the fixed cells and incubated for 20 min at room temperature. After incubation, methylene blue was taken from the wells and FSB cells were washed 4 times. 100 μl of 5% sodium dodecyl sulfate (SDS) in PBS was added to the wells and incubated for 1 hour at room temperature until the cells were completely lysed. The number of living cells was determined spectrophotometrically at a wavelength of 540 nm on a MultiscanEX photometer.

B) A method aimed at determining the metabolic activity of the cell - MTT test (Niks M., Otto M. Towards an optimized MTT assay. // J Immunol. 1900. V.130, No. 1, p. 149-151), based restoration of the colorless tetrazolium salt (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide, MTT) with mitochondrial and cytoplasmic dehydrogenases of living metabolically active cells with the formation of blue formazan crystals, the amount of which is measured spectrophotometrically.

The method was performed in the format of a 96-well culture plate. In the daily monolayer of McCoy cells, the culture medium was replaced with fresh medium and various doses of the studied chemical compounds were introduced. Cells were incubated for 24 hours in a CO ₂ incubator at 37 ° C. 4 hours before the end of the experiment, 1:10 of the volume of the culture medium of 10 × MTT solution (5 mg / ml) was added. Incubated for 4 hours at 37 ° C 5% CO ₂ . The culture medium was selected, FSB was washed once. 100 μl of isopropanol (propanol-2) was added to each well. Incubated at room temperature for 30 minutes. The optical density at a wavelength of 540 nm was evaluated on a Multiscan EX photometer. The substrate absorption was evaluated at 405 nm.

1. The method of suspension infection of eukaryotic cells

The method of suspension infection of eukaryotic cells with chlamydia allows you to standardize the conduct of biological experiments in a screening format on 96-well plates. To obtain a cell suspension, the daily McCoy monolayer of cells was used, which was treated with a trypsin and versene solution (1: 3 ratio, respectively) to detach cells from the surface of the vial. The vial was placed in a thermostat for 5 minutes. Then a trypsin and versene solution was taken and the cells suspended in the culture medium (RPMI 1640 with 10% fetal serum).

To obtain a monolayer from the prepared cell suspension, cells were counted in a Goryaev chamber at the rate of 1.5x10 ⁵ cells / ml Cells were infected with the strain Bu-434 Chlamydia trachomatis serovar L2 in the ratio bacterium: cell 1: 1 in the required volume of the transport medium (RPMI medium with 5% fetal serum, 25 mM glucose solution, 5 μg / ml amphotericin and 4 μg / ml gentamicin) that provides 80-90% infection of cells. The finished suspension was added to the wells of 96- or 24-well plates in a volume of 100 μl or 1000 μl, respectively. To precipitate cells and stimulate the interaction of chlamydia with them, the plates were centrifuged at 3000 rpm for 1 hour at 25 ° C. After that, the tablet was placed in an incubator for 48 hours at 37 ° C.

2. Determination of the effect of indolyl-substituted derivatives of thiadiazinones obtained on the basis of thiohydrazides of oxaminic acids on the viability of chlamydia

The studied chemical compounds at different concentrations were introduced into the cell culture simultaneously with the pathogen to assess their effect on intracellular reproduction of chlamydia. The effect was evaluated by direct immunofluorescence.

Immunofluorescence methods are aimed at identifying objects containing a certain antigen, and are based on the treatment of drugs with appropriate antibodies labeled with fluorochrome, followed by microscopy in ultraviolet light.

In this work, we used the direct immunofluorescence method (standard method), which allows semi-quantitative monitoring of the development of chlamydial infection using monoclonal antibodies to the species-specific protein antigen of C.trachomatis.

The work was carried out on McCoy cell lines infected by suspension infection with Vi-434 C.trachomatis serovar L2 in the format of 96-well plates or 24-well with glasses. The studied chemical compounds were added in different doses directly upon infection of the cells. After 48 hours, a supernatant was taken from the wells and cells were fixed. When working with 96-well plates, fixation was performed with icy 72 ° ethanol, followed by placement of the plate for 30-40 minutes at -20 ° C. When working with 24-well plates, the glasses were washed in a 0.1 mM FSB solution and dried. After this, the cells were fixed with acetone for 15 min at room temperature. 30-50 μl of FITC-labeled monoclonal antibodies to the protein antigen C.trachomatis (HlaMonoScrin-2, LLC NIARMEDIC PLUS) were applied to fixed cells and incubated for 30 min in a humid chamber at 37 ° C. After incubation, the cells were washed 2 times with a solution of FSB. The drug was completely dried. In the format of 24-well plates, the thus prepared glasses were mounted on a glass slide using mounting liquid (glycerin in the FSB). Finished preparations were examined in a luminescent microscope.

3. Determination of the effect of indolyl-substituted derivatives of thiadiazinones and oxaminic acids derived from thiohydrazides on the III transport system of chlamydia

The influence of the studied compounds on the III transport system (TCC) of chlamydia was assessed based on the detection of translocation of the IncA C. trachomatis effector protein on the intracellular chlamydial inclusion membrane. The IncA protein was detected using the indirect immunofluorescence method (standard method) using commercial antibodies to the IncA C. trachomatis TTC effector protein (IncA-specific polyclonal rabbit serum, Innovagen, Sweden), followed by detection of FITC-labeled anti-rabbit antibodies.

For this, the daily monolayer was infected to obtain 80-90% of infected cells, centrifuged at 3000 rpm for 30 min at 25 ° C. 8 hours after infection (the start time of translocation of the effector protein to the inclusion membrane), different doses of the studied chemical compounds were added. After 24 hours, culture medium was taken from the wells, the glasses were washed in a 0.1 mM FSB solution and dried. After this, the cells were fixed with acetone for 15 min at room temperature. 50 μl of primary anti IncA antibodies were applied to the fixed cells and incubated for 30 min at 37 ° C. Then the glasses were washed with the indicated FSB solution and antibodies labeled with FITC were applied. Incubated for 30 min in a humid chamber at 37 ° C. After incubation, the glasses with cells were washed 2 times with a solution of FSB. The drug was completely dried. Glasses thus prepared were mounted on a glass slide using a mounting liquid. Finished preparations were examined in a luminescent microscope. At the same time, monoclonal antibodies to the MOMP protein of the outer membrane of the cell wall were stained, which allowed us to evaluate the development of chlamydial infection.

Example 11

The effect of compounds of the class of indolyl-substituted derivatives of thiadiazinones derived from oxoamino acids thiohydrazides on the viability of eukaryotic cells

The compounds are characterized by a high degree of solubility in dimethylformamide (DMF), do not form a precipitate during storage and when transferred to a transport medium.

Toxicity analysis of 10 test compounds and 1 prototype compound related to hydrazones prepared on the basis of benzoic hydrazides (INP0400) in vitro when eukaryotic cells are added to the culture medium at doses of 12.5, 25 and 50 μM and then incubated for 24 hours at 37 ° C, carried out by methods 1.A and B, revealed the absence of a negative effect on the viability of eukaryotic cells of compounds of the class of indolyl-substituted derivatives of thiadiazinones obtained on the basis of oxoamine thiohydrazides lot. The results are presented in table 1.

The smallest cytotoxic effect according to the results of the two tests used was shown for compounds CL-79 and CL-82. Even at the highest concentration of 50 μM, these compounds caused no more than 1-5% cell death and reduced metabolic activity in no more than 10-14% of the cells. The other 8 compounds also showed satisfactory cytotoxicity results, which suggests that the selected compounds are low toxic. On the contrary, the prototype compound LHC-540 showed pronounced toxicity to eukaryotic cells, which limits the possibility of its further development as a therapeutic agent and indicates the obvious advantages of the compounds claimed in this patent.

Example 12

The effect of compounds of the class of indolyl-substituted derivatives of thiadiazinones obtained on the basis of thiohydrazides of oxaminic acids on the development of chlamydial infection

The ability of the ten compounds shown in examples 1-10 to inhibit the intracellular life cycle of chlamydia in vitro was evaluated using methods 2-3, the results of which are presented in table 2.

The best effect was exerted by compounds CL-79, CL-83, CL-88, suppressing intracellular accumulation of chlamydia by 98-99% at a concentration of 50 μM under the conditions of adding compounds to the transport medium simultaneously with infectious material (strain C.trachomatis L2) and subsequent cultivation within 48 hours. It was shown that under these conditions, infected cells in the analyzed monolayer are practically absent.

Compounds CL-81, CL-82 and CL-84 quite effectively suppressed the development of chlamydia at a dose of 50 μM. An analysis of the activity of these compounds at a dose of 50 μM under similar experimental conditions revealed a pronounced decrease in the size of inclusions and a reduction in their number by 88, 85, and 93%. Compounds CL-80, CL-85, CL-86, and CL-87 showed an inhibitory effect at a concentration of 50 μM, inhibiting the development of infection by 78, 50, 38, and 48%, respectively.

Figure 1 shows the most characteristic morphological changes in the intracellular development of chlamydial infection in vitro under the influence of the studied compounds on the example of compound CL-79. In the control preparation, without adding a compound, typical intracellular chlamydial inclusions corresponding to the duration of infection were observed. At a concentration of compound CL-79 of 12.5 μM, a decrease in the number of inclusions in the monolayer was observed. The detected inclusions were significantly smaller when compared with the control drug. At a compound concentration of 25 μM, the growth of fine inclusions was observed. At a concentration of 50 μM, chlamydial inclusions were not detected. Thus, the use of different concentrations of compound CL-79 showed a dose-dependent effect of the suppression of infection in cell culture.

Example 13

Characterization of the effect of compounds of the class of indolyl-substituted thiadiazinone derivatives derived from oxamic acids thiohydrazides on the III transport system of chlamydia

A study of the specific activity of indolyl-substituted thiadiazinone derivatives derived from oxaminic acid thiohydrazides with respect to inhibition of the function of the third transport system (TTC) was carried out according to method 4.

When immunochemical detection of the IncA effector protein on the surface of chlamydial inclusions in the control preparations, inclusions corresponding to the size of the infection period are detected. In the case of the action of the drug on the TTC, specific staining should not be detected.

The characterization of the effect of compounds CL-79, CL-83 and CL-88 on TTC showed that the introduction of these compounds 8 hours after infection inhibited the translocation of the IncA effector protein.

Another test for the specificity of the compounds with respect to TTC is related to the fact that the IncA chlamydial protein translocated by TTC is involved in the fusion of individual inclusions developing inside the cell. When the infected cells were stained with antibodies to the MOMP protein in the experimental format: infection - after 8 hours, the compound was introduced - incubation up to 24 hours after infection, large inclusions were observed in the control cells, one in each cell, and in the case of the effect of the compound on the TTC small non-fused inclusions in the cytoplasm of the cell.

A specific TTS inhibitory effect has been shown for compounds CL-79, CL-83 and CL-88. Figure 2 shows the inhibition of translocation of C. trachomatis IncA effector protein, characteristic of these compounds, by the example of the action of compound CL-79. In the control preparation, typical inclusions corresponding in size to 30 hours of infection were observed when stained with antibodies to the MOMP protein. If 50 μM of compound CL-79 was added 8 hours after infection and subsequent cultivation for 22 hours, small and medium-sized inclusions, several in one cell, were observed in the monolayer. When stained with antibodies to the IncA protein, typical inclusions were also detected in the control preparation, and no inclusions were detected in the case of the action of the chemical compound.

The tests performed indicate that biologically active compounds, indolyl-substituted thiadiazinone derivatives derived from oxamic acid thiohydrazides, exhibit specific activity against TTC of C. trachomatis, inhibiting the translocation of the IncA effector protein and thereby hindering the process of homotypic fusion of primary inclusions. The obtained compounds inhibit the type III secretion system in pathogenic bacteria, reducing the risk of drug resistance as a result of selection of resistant mutants, as well as exerting a directed effect on the mechanisms that determine the process of chronic infection.

The presented synthesis examples as well as biological examples confirm the industrial applicability of the claimed invention.

The foregoing allows us to conclude that the technical task posed is solved.

Claims (2)

1. Biologically active compounds that suppress pathogenic bacteria, which are indolyl-substituted derivatives of thiadiazinones, obtained on the basis of thiohydrazides of oxaminic acids of the general formula

where R represents H;
R ¹ represents pyridinyl; phenyl substituted with C1-C5 alkyl, Hal, CF ₃ ;
R ² represents H; alkyl C1-C5; -CH2COOR ⁴ ; benzyl substituted with Hal, OR ⁴ ; benzoyl substituted with Hal, OR ⁴ , and
R ⁴ is unsubstituted C1-C4 alkyl. 2. A method of inhibiting the secretion of type III in pathogenic bacteria, which consists in exposing the bacteria to an effective amount of a compound according to claim 1.

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