RU2675240C1 - Pyranoindoles with anti-tuberculosis activity - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to compounds, namely, pyranoindoles of general formula 1, where R¹ means H, HO, CH₃, C₂H₅, C₃H₇, AcO, Cl, CF₃, CF₃O, NO₂, MeO; R² means H, CH₃, C₂H₅, C₃H₇, Ac, CH₂Ph, CH₂Ph-4-CN, CH₂Ph-4-OMe; R³ means H, NHCH₃, NHC₃H₅; R⁴ means CN, CONH₂, COOC₂H₅, NHCOPh, with the exception of the compounds 2-oxo-2,5-dihydropyrano[3,2-b]indole-3-carbonitrile, 2-oxo-2,5-dihydropyrano[3,2-b]indole-3-ethoxycarbonyl and 2-oxo-2,5-dihydropyrano[3,2-b]indole-3-urea.

1.

EFFECT: new compounds have been obtained that can be used in medicine as anti-tuberculosis drugs.

4 cl, 2 tbl, 32 ex

Description

The invention relates to new biologically active compounds derived from pyranoindoles of the general formula (I), which are active against tuberculosis mycobacteria, the method for their preparation and their use as anti-tuberculosis drugs.

Since 1998, when the complete genome sequence of mycobacterium tuberculosis became available, genomics has provided a valuable resource for discovering new drugs by catalyzing genetic manipulations and generating modern powerful technologies, including transcriptomics, proteomics, comparative and structural genomics. These post-genomic advances significantly influenced our understanding of the biology of tuberculosis and contributed to attempts at targeted drug development, identification and verification. However, many of the tasks that initially seemed very important and provided a lot of information for using genetic methods in the development of in vitro drugs, such as replacing a gene or saturating mutagenesis, did not actually produce the desired result due to genetic redundancy or metabolism. As a result, it became clear that standard microbiological and pharmacological tests are much more reliable and better for success in terms of creating new anti-infective agents.

The discovery of new anti-tuberculosis drugs can follow two main routes: from drug to target, or from target to drug. All existing drugs and candidates in clinical trials were obtained on the way from “drug to target” with high-performance screening against whole cells, which emphasizes the importance of this approach. However, postgenomic tools now support this traditional route to developing new drugs.

Although it looks more elegant in terms of applied biology, the target-to-drug approach was unsatisfactory, mainly because of the change in the ability of the compound to inhibit the purified enzyme compared to the study of activity against whole cells (minimal inhibitory concentration) an obstacle to rational drug design. Promising enzyme inhibitors are generally poorly active against the bacterium itself, and this is probably due to the complex mycobacterial cell wall that prevents the cell from absorbing the compound, to the effect of efflux pumps or inactivating the compound as a result of intracellular metabolism. In addition, the level of penetration of the compound into the cell alone does not always explain the lack of correlation between IC 50 and MIC. For example, the level of protein inhibition required to induce a cid effect is another important issue, and can vary widely.

Some of the clinically most advanced candidates for treating tuberculosis are molecules that were originally developed to treat other infectious diseases, but were redirected to tuberculosis. These include compounds from the group of rifamycin, fluoroquinolone, oxazolidinone.

The classic approach to the development of new anti-TB drugs

Screening libraries of chemical compounds with the definition of MICs against live mycobacteria has now presented several promising candidates for anti-TB drugs: Nitroimidazoles (PA-824 and Delamanide), Bedaquiline (TMC207), the recently identified imidazopyridine amide compound Q203, diethylamine SQ109 and benzothiazinone BTZ043.

The successes of the post-genomic era include the detection of targets and the mechanism of action of bicyclic nitroimidazoles, PA-824 and delamanide. Both nitroimidazole are highly active against Mtb under aerobic conditions and against non-dividing and hypoxic bacteria. Genome sequencing microarrays were used to detect mutations associated with resistance to PA-824. It is interesting to note that mutations affecting specific nitroreductase confer resistance to both RA-824 and delamanid. Nitroreductase has been shown to activate both of these nitroimidazoles, resulting in intracellular release of nitric oxide, which is thought to kill bacteria. A profiled analysis of gene expression after exposure to PA-824 showed activation of genes involved in cell wall synthesis as well as respiration, suggesting that RA-824 possibly inhibits both pathways.

In 2005, using the classic phenotypic approach, a very strong molecule, TMC207 or Bedaquiline, was discovered, an innovative, recently approved drug that offers significant hope for treating MDR-TB cases. A genetic study of spontaneously acquired TMS-207 resistant mutants showed that only 15 out of 53 mutants have atpE mutations, indicating that the drug has either additional targets or resistance mechanisms, such as forced drug withdrawal. The discovery of bedaquiline, which affects energy metabolism in general and the inhibition of ATP synthase, in particular, makes these targets extremely interesting from the point of view of drug development specifically affecting them.

Recently, the importance of ATP synthesis as the main target for a TB drug with the discovery of a new class of imidazopyridine amide compounds that block the growth of mycobacteria by exposing the BC1 cytochrome complex to the respiratory chain has been further confirmed. Using phenotypic screening on infected macrophages, a number of compounds were selected and chemically optimized with access to Q203, an inhibitor with active nanomolar concentrations and with promising efficacy in murine TB models. Spontaneous resistant mutants indicate a single amino acid substitution in the cytochrome subunit of the bc1 complex.

The main target of SQ109, which inhibits the synthesis of mycolic acids, has only recently been identified as MmpL3, a transmembrane carrier of trehalose monomicolate. Initially, researchers were unable to obtain spontaneous resistant mutants to SQ109. However, using ethylenediamine analogs, resistant mutants were generated and they showed cross-resistance to SQ109 and thus mutations in the mmpL3 gene were detected.

Benzothiazinones, a new class of sulfur-containing heterocyclic compounds, are extremely potent against Mycobacterium tuberculosis, showing low nanomolar bactericidal activity in vitro and in models of intracellular infections. Genetic and biochemical studies, as well as transcriptomic and proteomic assays, have identified the DprE1 subunit as the vital decaprenylphosphoryl-D-ribose 2'-epimerase enzyme as a target. Inhibition of DprE1 stops the synthesis of arabinogalactan, which ultimately leads to cell lysis. It seems that DprE1 also seems to be an extremely promising target for potential anti-TB drugs.

Rational drug design

Despite the considerable efforts expended, a targeted approach based on the search for compounds affecting known targets in the tuberculous cell was very limitedly successful in terms of the discovery of new anti-TB drugs. Some of the most promising results obtained identified in this way are given below.

Ethionamide is an approved second-line drug, but it has significant side effects. Its target is enoyl-ACP reductase, Inha, the same target as for isoniazid, and its mechanism of action is based on the inhibition of the synthesis of mycolic acids. Ethionamide is a prodrug, it is activated by mono-oxygenase, and its expression is suppressed by EthR. The activation and, in turn, the effectiveness of this drug is limited by the low design features of ethionamide. Drug-like EthR inhibitors, such as BDM31343, have been developed using its crystalline structure as a matrix, and new ethionamide derivatives show better activity in vitro and better in animal experiments.

The glyoxylate pathway is an important metabolic pathway for the physiology of mycobacteria, as it enhances its activity in the absence of glycolysis to maintain the tricarboxylic acid cycle during the persistent phase of the infection. Mammals lack two main enzymes, isocytratliase and malate synthase, which makes these enzymes promising and specific as antibacterial targets for drugs. The enzyme structure was used in drug design and powerful selective phenyl-diketosteroid acid malate synthase inhibitors that are active in the mouse TB model have been developed. This chemically substantiates the glyoxylate pathway as a promising new target for the search for an anti-TB drug.

Screening for natural compounds

Natural compounds, in particular streptomycin and rifampicin, have played a significant role in the fight against tuberculosis. Despite the predominance of natural antibiotics to fight other bacterial infections, it was not possible to detect new natural compounds active on mycobacteria until 2012. The mechanism of action of pyridomycin, an anti-tuberculosis natural compound found for the first time since 1953, was recently deciphered. After selection of pyridomycin-resistant mutants, Inha were identified being a target for isoniazid as a target and for pyridomycin. As isoniazid is a prodrug, which requires its activation using KatG catalase peroxidase, inactivation of pyridimycin is most often associated with mutations in the katG gene. Isoniazid-resistant clinical isolates carrying mutations in katG remain susceptible to pyridomycin, which makes this natural product a promising compound for the fight against drug resistant strains and an additional study of libraries of natural products, but using new approaches, is promising.

Thus, it can be unequivocally argued that only an integrated approach to the directed search for new generation anti-TB drugs has a chance of success. This research area should include screening for full-cell forms of mycobacteria, screening for dormant forms and a detailed genetic study of both resistant mutant strains, and sequencing of sensitive strains that have been treated with potential drugs.

This problem was solved by the synthesis of pyranindole derivatives corresponding to the general structural formula 1.

The compound of General formula 1.

1

one

Where

R ¹ is H, HO, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , AcO, Cl, CF ₃ , CF ₃ O, NO ₂ , MeO;

R ² is H, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , Ac, CH ₂ Ph, CH ₂ Ph-4-CN, CH ₂ Ph-4-OMe;

R ³ means H, NHCH ₃ , NHC ₃ H ₅ ;

R ⁴ means CN, CONH ₂ , COOC ₂ H ₅ , NHCOPh.

Compounds 2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile, 2-oxo-2,5-dihydropyrano [3,2-b] indole-3-ethoxycarbonyl and 2-oxo-2 , 5-dihydropyrano [3,2-b] indole-3-carbamide previously described in the literature (N. S. Masterova, S. Yu. Ryabova, L. M. Alekseeva, M. I. Evstratova, S. S. Kiselev, V. G. Granik, Izv. Akkad. Nauk, Ser. Chem. 2010, 3, 623), but their biological activity was not detected.

Compounds can be obtained and used in crystalline form.

We first discovered that pyranoindoles have anti-tuberculosis activity and we synthesized a series of compounds containing various substituents in this scaffold in order to identify the positions of the molecule by which it is possible to obtain derivatives in the future with the preservation or, ideally, with an increase in the level of antimicrobial activity. For this, we introduced substituents both on the indole nitrogen atom, on the indole ring. Moreover, taking into account the electron-acceptor nature of the substituent in the pyran ring, we obtained derivatives that also contain electron-acceptor substituents in this position, amide and carbonyl. For introduction into the indole core, we selected a series of substituents that differ greatly in properties, alkyl substituents of various sizes, nitro group, halogen, trifluoromethyl group, in order to study not only their effect on anti-tuberculosis activity, but also to understand the effect of lipophilicity of the final compounds on the desired activity .

In order to obtain a library of new original derivatives of pyranoindole, we have developed an effective eight-step method for their synthesis based on commercially available reagents. This method was used to generate all of the target 35 pyranoindoles. Each synthesis experiment was repeated twice with a quantitative coefficient 5. The total yield of the target pyranoindoles is 30–40% and the purity of the products obtained is> 99%. The structure of the obtained pyranoindole samples is shown in table 1.

Table 1. The structure of synthesized and investigated pyranoindoles.

number Assumed Library Connection Cipher Structural formula 11126066

11126067

11526065

11526066

11526108

11526109

11526110

11626056

11626057

11626058

11626062

11626210

11626211

11626212

11626213

11626214

11626215

11626216

11626217

11626218

11626219

11626220

11626223

11626224

11626225

11626226

11626227

11626229

11626230

11626231

11626232

11626233

So, the well-known Vilsmeier reaction, which leads to the introduction of the formyl group in the third position of indole, was proposed as the first stage of the process.

The next stage is the introduction of a protective group at the indole nitrogen atom. This process was implemented by the traditional method using an excess of acetic anhydride and a catalytic amount of triethylamine. This method of introducing acetyl protection proved to be good on the studied system.

The process of mild oxidation of the formyl group was carried out using chloroperbenzoic acid.

The introduction of the piperidine fragment in the second position of the indole was proposed to be carried out using dimethoxymethylpiperidine. The next stage, indole acetylation at the third position, was carried out by the classical method using acetyl chloride. The reaction went well with a yield close to quantitative. The interaction of malondinitrile with the formyl group of indole must be carried out in the presence of some basic agent. Therefore, we investigated reactions using sodium hydroxide, sodium hydride, piperidine, triethylamine and pyridine. The best yield was obtained using piperidine and triethylamine. The yield in this reaction reached 90%. The closure of the pyran ring is a key and limiting stage in the synthesis of target pyranoindoles.

General procedure for the synthesis of pyranoindoles

In vitro experiments showed a high activity of pyranoindole representatives against tuberculosis mycobacteria with low cytotoxicity of compounds.

An object of the invention is a method for preventing or treating a tuberculosis infection, comprising administering or applying to a subject a compound of formula I or pharmaceutical compositions based thereon in an effective amount. A method of treatment using the compounds of the invention, pharmaceutical compositions or drugs based on them is also effective against strains resistant to currently existing drugs.

This work was supported by the Ministry of Education and Science of the Russian Federation (Agreement No. 14.616.21.0065; unique identification number RFMEFI61616X0065)

The following are examples of the preparation of compounds of the invention.

Synthesis of pyranoindoles.

Stage a).

At a temperature of 5 ^{° C} was dropwise added 12 mmol POCl ₃ to 50 mmol of DMF, the solution was stirred for 30 min and to it was added in one portion 5-R ¹ -1H-indole (10 mmol). The reaction mass was stirred for 12 hours at room temperature and poured into 200 ml of ice water. The mixture was stirred for 2 hours, the precipitate was filtered, washed with water and dried to constant weight.

Stage b).

A mixture of 5-R ¹ -1H-indole-3-carbaldehyde (10 mmol), acetic anhydride (70 mmol) and triethylamine (3 mmol) was refluxed for 3 hours. The reaction mixture was cooled with acetic anhydride and the triethylamine was removed in vacuo. Water was added to the residue, the mixture was stirred for 1 h, the precipitate was filtered, washed with water and dried to constant weight.

Stage c).

Meta-chloroperbenzoic acid (m-CPBC) (15 mmol) was added to a solution of 5-R ¹ -1-acetyl-1H-indole-3-carbaldehyde (10 mmol) in 50 ml of methylene chloride. The solution was stirred at room temperature for 20 hours, 100 ml of water was added to the reaction mass, stirred for 30 minutes, the aqueous layer was separated, the organic phase was washed with an aqueous solution of NaHCO ₃ , water and dried with Na ₂ SO ₄ . Methylene chloride was removed in vacuo, the residue was triturated with ether. The precipitated crystals were filtered, washed with ether and dried to constant weight.

Stage d).

To a solution of 5-R ¹ -1-acetyl-1H-indol-3-yl format (10 mmol) in 40 ml of benzene were added 1- (dimethoxymethyl) piperidine (20 mmol) and piperidine (1.1 mmol). The mixture was stirred for 30 minutes at room temperature and then refluxed for 3 hours. The reaction mixture was evaporated in vacuo, 30 ml of methanol, triethylamine (10 mmol) were added to the residue and refluxed for 2 hours. Volatiles were removed in vacuo and the residue was triturated with ether. The precipitated crystals were filtered, washed with ether and dried to constant weight.

Stage e).

A suspension of (2E, Z) -5-R ¹ -2- (piperidin-1-ylmethylene-1,2-dihydro-3H-indol-3-one (10 mmol) in 40 ml of DMF was added dropwise acetyl chloride (30 mmol) The reaction mass was stirred for 2 hours at room temperature and evaporated in vacuo, 30 ml of water was added to the residue and the mixture was stirred for 2 hours, the resulting crystals were filtered, washed with water and dried to constant weight.

Stage e).

Malononitrile (12.5 mmol) and triethylamine (1 mmol) were added to a suspension of 2-formyl-5-R ¹ -1H-indol-3-yl acetate (10 mmol) in 100 ml of benzene. The reaction mass was stirred for 4 hours at room temperature, the crystals were filtered, washed with ether and dried to constant weight.

Stage g).

To a boiling suspension of 2- (2,2-dicyanovinyl) -5-R ¹ -1H-indol-3-yl acetate (10 mmol) in 250 ml of acetonitrile, 30 ml of concentrated hydrochloric acid were added dropwise with stirring. The reaction mass was refluxed for 3 hours and concentrated in vacuo to a volume of 70 ml. The crystals were filtered, washed with a mixture of acetonitrile-water (1: 1) with water to a pH of ~ 6.5, methanol and dried to constant weight. The resulting 8-R ¹ -2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitriles were crystallized from acetonitrile or a mixture of acetonitrile-DMF.

Stage h).

To a suspension of 8-R ¹ -2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile (1 mmol) and cesium carbonate (1.25 mmol) in 5 ml of dry DMF was added the corresponding alkyl halide (1.5 mmol ) The reaction mass was stirred for 3 hours at room temperature, 0.3 ml (5 mmol) of acetic acid and 10 ml of water were added to it. The precipitated crystals were filtered, washed with a mixture of DMF-water (1: 1), water and dried to constant weight. The resulting 8-R ¹ -5-R ² -2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitriles crystallized from acetonitrile.

Example 1

2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11126066

The gross formula is C ₁₂ H ₆ N ₂ O ₂ ; Mp (speed 10 ^o C / min) 299-302 ^o C; Mass spectrum (EU) ESI-MS / MS - 210; IR spectrum, cm ^-1 2610, 2100, 1746, 1234; ¹ H NMR (DMSO-d ₆ ) σ: 9.88 (1H, s, NH), 8.69 (1H, s, CH), 7.80 (1H, d, CH), 7.43 (1H, d, CH), 7.28 (1H, t, CH); 7.16 (1H, t, CH) ppm; Elemental analysis%: C 68.62, H 2.84, N 13.35. Calculated%: C 68.57, H 2.88, N 13.33.

Example 2

Ethyl 2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carboxylate 11126067

The gross formula is C ₁₄ H ₁₁ N ₂ O ₄ ; M.p. (speed 10 ^o C / min) 212-214 ^o C; Mass spectrum (EU) ESI-MS / MS - 472; IR spectrum, cm ^-1 2614, 1789, 1740, 1231; ¹ H NMR (DMSO-d ₆ ) σ: 9.89 (1H, s, NH), 8.71 (1H, s, CH), 7.84 (1H, d, CH), 7.47 (1H, d, CH), 7.30 (1H, t, CH), 7.16 (1H, t, CH), 4.25 (2H, q, CH ₂ ), 1.30 (3H, t, CH ₃ ) ppm; Elemental analysis%: C 65.41, H 4.29, N 5.44. Calculated%: C 65.37, H 4.31, N 5.44.

Example 3

4-methylamino-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11526065

The gross formula is C ₁₃ H ₉ N ₃ O ₂ ; Mp (speed 10 ^o C / min) 256-259 ^o C; Mass spectrum (EU) ESI-MS / MS - 239; IR spectrum, cm ^-1 2610, 2578, 2100, 1741; ¹ H NMR (DMSO-d ₆ ) σ: 8.69 (1H, s, CH), 7.80 (1H, d, CH), 7.43 (1H, d, CH), 7.28 (1H, t, CH), 7.16 (1H , t, CH), 3.11 (3H, s, CH ₃ ) ppm; Elemental analysis%: C 65.25, H 3.74, N 17.49. Calculated%: C 65.27, H 3.79, N 17.56.

Example 4

4-cyclopropylamino-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11526066

The gross formula is C ₁₅ H ₁₁ N ₃ O ₂ ; M.p. (speed 10 ^o C / min) 224-226 ^o C; Mass spectrum (EU) ESI-MS / MS - 265; IR spectrum, cm ^-1 2613, 2569, 2107, 1749; ¹ H NMR (DMSO-d ₆ ) σ: 8.69 (1H, s, CH), 7.80 (1H, d, CH), 7.43 (1H, d, CH), 7.28 (1H, t, CH), 7.16 (1H , t, CH), 3.40 (1H, s, NHCH), 1.06 (4H, m, CH ₂ CH ₂ ) ppm; Elemental analysis%: C 67.64, H 4.15, N 15.79. Calculated%: C 67.62, H 4.18, N15.84.

Example 5

8-fluoro-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11526108

The gross formula is C ₁₂ H ₅ FN ₂ O ₂ ; Mp (speed 10 ^o C / min) 303-305 ^o C; Mass Spectrum (EU)

ESI-MS / MS - 228; IR spectrum, cm^-one2567, 2112, 1748;^oneH NMR (DMSO-d₆) σ: 9.89 (1H, s, NH), 8.68 (1H, s, CH), 7.80 (1H, s, CH), 7.28 (1H, d, CH), 7.21 (1H, d, CH) ppm . Elemental analysis%: C 63.11, H 2.19, N 12.31. Calculated%: C 63.17, H 2.21, N 12.28.

Example 6

8-isopropyl-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11526109

The gross formula is C ₁₅ H ₁₂ N ₂ O ₂ ; Mp 277-280 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 252; IR spectrum, cm ^-1 2574, 2110, 1750; ¹ H NMR (DMSO-d ₆ ) σ: 9.89 (1H, s, NH), 8.68 (1H, s, CH), 7.80 (1H, s, CH), 7.28 (1H, d, CH), 7.21 (1H , d, CH), 3.06 (1H, m, CH), 1.34 (6H, m, C (CH ₃ ) ₂ ) ppm. Elemental analysis%: C 71.45, H 4.76, N 11.08. Calculated%: C 71.42; H 4.79; N 11.10.

Example 7

8-ethyl-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11526110

The gross formula is C ₁₄ H ₁₀ N ₂ O ₂ ; Mp 273-275 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 252; IR spectrum, cm ^-1 2568, 2107, 1744; ¹ H NMR (DMSO-d ₆ ) σ: 9.87 (1H, s, NH), 8.67 (1H, s, CH), 7.84 (1H, s, CH), 7.31 (1H, d, CH), 7.20 (1H, d, CH), 2.64 (2H, q, CH ₂ ), 1.35 (3H, t, CH ₃ ) ppm. Elemental analysis%: C 70.57, H 4.20, N 11.72. Calculated%: C 70.58, H 4.23, N 11.76.

Example 8

5,8-dimethyl-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626056

The gross formula is C ₁₄ H ₁₀ N ₂ O ₂ ; Mp 299-302 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 238; IR spectrum, cm ^-1 2568, 2107, 1744; ¹ H NMR (DMSO-d ₆ ) σ: 8.64 (1H, s, CH), 7.81 (1H, s, CH), 7.33 (1H, d, CH), 7.22 (1H, d, CH), 3.79 (3H , s, NCH ₃ ), 2.45 (3H, s, C-CH ₃ ) ppm. Elemental analysis%: C 70.59, H 4.25, N 11.80. Calculated%: C 70.58, H 4.23, N 11.76.

Example 9

8-hydroxy-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626057

The gross formula is C ₁₂ H ₆ N ₂ O ₃ ; Mp 329-333 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 226; IR spectrum, cm ^-1 2563, 2479, 2110, 1746; ¹ H NMR (DMSO-d ₆ ) σ: 9.63 (1H, s, NH), 8.60 (1H, s, CH), 7.80 (1H, s, CH), 7.24 (1H, d, CH), 7.16 (1H, d, CH) ppm. Elemental analysis%: C 63.73, H 2.64, N 12.37. Calculated%: C 63.72, H 2.67, N 12.38.

Example 10

5-acetyl-3-cyano-2-oxo-2,5-dihydropyrano [3,2-b] indol-8-yl acetate 11626058

Gross formula C ₁₆ H ₁₀ N ₂ O ₅ ; Mp 286-288 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 310; IR spectrum, cm ^-1 2113, 1794, 1787, 1746; ¹ H NMR (DMSO-d ₆ ) σ: 8.79 (1H, d, CH), 8.18 (1h, d, CH), 7.86 (1H, d, CH), 7.31 (1H, s, CH), 2.51 (3H , s, CH ₃ ), 2.26 (3H, s, CH ₃ ) ppm. Elemental analysis%: C 61.87, H 3.22, N 9.08. Calculated%: C 61.94, H 3.25, N 9.03.

Example 11

8-trifluoromethoxy-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626062

The gross formula is C ₁₃ H ₅ FN ₂ O ₃ ; Mp 288-290 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 294; IR spectrum, cm ^-1 2597, 2110, 1787, 1245; ¹ H NMR (DMSO-d ₆ ) σ: 9.88 (1H, s, NH), 8.69 (1H, s, CH), 7.72 (1H, s, CH), 7.53 (1H, d, CH), 7.35 (1H , d, CH) ppm. Elemental analysis%: C 53.00, H 1.15, N 9.55. Calculated%: C 53.08, H 1.17, N 9.52.

Example 12

N- (5-acetyl-2-oxo-2,5-dihydropyrano [3,2-b] indol-3-yl) benzamide 11626210

Gross formula C ₂₀ H ₁₄ N ₂ O ₄ ; Mp 241-243 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 346; IR spectrum, cm ^-1 2578, 1787, 1779, 1746; ¹ H NMR (DMSO-d ₆ ) σ: 9.50 (1H, s, NH), 8.38 (1H, s, CH), 7.74-7.20 (9H, m, 9 CH), 2.54 (3H, s, CH ₃ ) ppm Elemental analysis%: C 69.29, H 4.02, N 8.13. Calculated%: C 69.36, H 4.07, N 8.09.

Example 13

N- (2-oxo-2,5-dihydropyrano [3,2-b] indol-3-yl) benzamide 11626211

Gross formula C ₂₀ H ₁₄ N ₂ O ₄ ; Mp 331-332 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 346; IR spectrum, cm ^-1 2572, 2555, 1780, 1747; ¹ H NMR (DMSO-d ₆ ) σ: 9.37 (1H, s, NH), 8.37 (1H, s, CH), 7.80-7.20 (9H, m, 9 CH) ppm. Elemental analysis%: C 70.92, H 3.93, N 9.16. Calculated%: C 71.05, H 3.97, N 9.21.

Example 14

8-methoxy-5-methyl-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626212

The gross formula is C ₁₄ H ₁₀ N ₂ O ₃ ; Mp 308-311 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 254; IR spectrum, cm ^-1 2113, 1780, 1231, 984; ¹ H NMR (DMSO-d ₆ ) σ: 8.66 (1H, s, CH), 7.33 (1H, s, CH), 6.92 (1H, d, CH), 6.63 (1H, d, CH), 3.85 (3H , s, OCH ₃ ), 3.79 (3H, s, NCH ₃ ) ppm. Elemental analysis%: C 66.11, H 3.94, N 10.99. Calculated%: C 66.14, H 3.96, N 11.02.

Example 15

8-chloro-5-methyl-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626213

Gross formula is C ₁₃ H ₇ ClN ₂ O ₂ ; Mp 308-311 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 258; IR spectrum, cm ^-1 2111, 1746, 1231, 971; ¹ H NMR (DMSO-d ₆ ) σ: 8.66 (1H, s, CH), 7.39 (1H, s, CH), 7.22 (1H, d, CH), 7.18 (1H, d, CH), 3.79 (3H , s, CH ₃ ) ppm. Elemental analysis%: C 60.28, H 2.72, N 10.84. Calculated%: C 60.37, H 2.73, N 10.83.

Example 16

8-nitro-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626214

Gross formula C ₁₂ H ₅ N ₃ O ₄ ; Mp 310-313 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 210; IR spectrum, cm ^-1 2569, 2109, 1741, 1239; ¹ H NMR (DMSO-d ₆ ) σ: 9.88 (1H, s, NH), 8.85 (1H, s, CH), 8.69 (1H, s, CH), 8.13 (1H, d, CH), 7.48 (1H , d, CH) ppm. Elemental analysis%: C56.45, H 1.92, N 16.46. Calculated%: C 56.48; H 1.97; N, 16.47.

Example 17

5-ethyl-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626215

The gross formula is C ₁₄ H ₁₀ N ₂ O ₂ ; Mp 303-305 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 238; IR spectrum, cm ^-1 2108, 1749, 1233; ¹ H NMR (DMSO-d ₆ ) σ: 8.61 (1H, s, CH), 7.43 (1H, d, CH), 7.35 (1H, d, CH), 7.26 (1H, t, CH), 7.18 (1H , t, CH), 4.06 (2H, q, CH ₂ ), 1.43 (3H, t, CH ₃ ) ppm. Elemental analysis%: C 70.52, H 4.20, N 11.80. Calculated%: C 70.58, H 4.23, N 11.76.

Example 18

5-ethyl-8-methyl-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626216

Gross formula C ₁₅ H ₁₂ N ₂ O ₂ : So pl. 307-310 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 252, IR spectrum, cm ^-1 2113, 1750, 1236. ¹ H NMR (DMSO-d ₆ ) σ: 8.61 (1H, s, CH), 7.22 (1H, s, CH), 7.02 (1H, d, CH), 6.74 (1H, d, CH), 4.06 (2H, q, CH ₂ ), 2.45 (3H, t, CH ₃ ), 1.43 (3H, s, CH ₃ ) ppm. Elemental analysis%: C 71.42, H 4.80, N 11.94. Calculated%: C 71.42, H 4.79, N 11.10

Example 19

5-allyl-8-methyl-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626217

Gross formula C ₁₆ H ₁₂ N ₂ O ₂ ; Mp 300-302 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 264; IR spectrum, cm ^-1 2110, 1743, 1230, 972; ¹ H NMR (DMSO-d ₆ ) σ: 8.59 (1H, s, CH), 7.36 (1H, s, CH), 7.04 (1H, d, CH), 6.88 (1H, d, CH), 6.70 (1H , m, CH), 5.28-5.22 (2H, m, = CH ₂ ), 4.78 (2H, m, CH ₂ ), 2.45 (3H, s, CH ₃ ) ppm. Elemental analysis%: C 72.63, H 4.57, N 10.63. Calculated%: C 72.72, H 4.58, N 10.60.

Example 20

5-allyl-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626218

The gross formula is C ₁₅ H ₁₀ N ₂ O ₂ ; Mp 317-320 ^o C (speed 10 ^o C / min); Mass Spectra (EU) ESI-MS / MS - 250; IR spectrum, cm ^-1 2114, 1750, 1227, 985; ¹ H NMR (DMSO-d ₆ ) σ: 8.59 (1H, d, CH), 7.45 (1H, d, CH), 7.31 (1H, d, CH), 7.17 (1H, t, CH), 7.48 (1H , t, CH), 6.70 (1H, m, CH), 5.28 (2H, m, = CH ₂ ), 4.78 (2H, m, CH ₂ ) ppm. Elemental analysis%: C 72.07, H 4.00, N 11.14. Calculated%: C 71.99, H 4.03, N 11.19.

Example 21

2-oxo-5-prop-2-yn-1-yl-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626219

The gross formula is C ₁₅ H ₈ N ₂ O ₂ ; Mp 321-323 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 248; IR spectrum, cm ^-1 2167, 2110, 1738, 1221; ¹ H NMR (DMSO-d ₆ ) σ: 8.84 (1H, s, CH), 7.47 (1H, s, CH), 7.44 (1H, s, CH), 7.27 (1H, d, CH), 7.09 (1H , d CH), 4.82 (1H, s, CCH), 2.72 (2H, s, CH ₂ ) ppm. Elemental analysis%: C 72.55, H 3.29, N 11.36. Calculated%: C 72.58, H 3.35; N, 11.28.

Example 22

8-methyl-2-oxo-5-prop-2-yn-1-yl-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626220

Gross formula C ₁₆ H ₁₀ N ₂ O ₂ ; Mp 324-327 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 262; IR spectrum, cm ^-1 2163, 2111, 1747, 1232; ¹ H NMR (DMSO-d ₆ ) σ: 8.84 (1H, s, CH), 7.32 (1H, s, CH), 7.06 (1H, d, CH), 6.84 (1H, d, CH), 4.82 (2H , s, CH ₂ ), 2.72 (3H, s, CH ₃ ), 2.45 (1H, s, CH) ppm. Elemental analysis%: C 73.25, H 3.79, N 10.74. Calculated%: C 73.27, H 3.84, N 10.68.

Example 23

methyl (3-cyano-2-oxopyrano [3,2-b] indol-5 (2H) -yl) acetate 11626223

Gross formula C ₁₅ H ₁₀ N ₂ O ₄ ; Mp 314-316 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 282; IR spectrum, cm ^-1 2110, 1785, 1740, 1238; ¹ H NMR (DMSO-d ₆ ) σ: 8.78 (1H, s, CH), 7.52 (1H, s, CH), 7.51 (1H, s, CH), 7.34 (1H, d, CH), 7.13 (1H , d, CH), 4.96 (2H, s, CH ₂ ), 3.60 (3H, s, CH ₃ ) ppm. Elemental analysis%: C 63.76, H 3.54, N 10.01. Calculated%: C 63.83, H 3.57, N 9.92.

Example 24

methyl (3-cyano-8-methyl-2-oxopyrano [3,2-b] indol-5 (2H) -yl) acetate 11626224

Gross formula C ₁₆ H ₁₂ N ₂ O ₄ ; Mp 320-322 ^o C (speed 10 ^o C / min); Mass spectrum (EU) ESI-MS / MS - 296; IR spectrum, cm ^-1 2111, 1780, 1747, 1241; ¹ H NMR (DMSO-d ₆ ) σ: 8.78 (1H, s, CH), 7.38 (1H, s, CH), 7.11 (1H, d, CH), 6.90 (1H, d, CH), 4.96 (2H , s, CH ₂ ), 3.60 (3H, s, OCH ₃ ), 2.45 (3H, s, CH ₃ ) ppm. Elemental analysis%: C 64.79, H 4.00, N 9.42. Calculated%: C 64.86, H 4.08, N 9.45.

Example 25

5- (cyanomethyl) -2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626225

The gross formula is C ₁₄ H ₇ N ₃ O ₂ ; Mp 300-303 ^o C (speed 10 ^o C / min); Mass spectra (EU) ESI-MS / MS - 249; IR spectrum, cm ^-1 2122, 2111, 1740, 1233; ¹ H NMR (DMSO-d ₆ ) σ: 8.85 (1H, s, CH), 7.68 (1H, d, CH), 7.39 (1H, d, CH), 7.20 (1H, 7, CH), 7.56 (1H 7, CH), 5.47 (2H, s, CH ₂ ) ppm. Elemental analysis%: C 67.39, H 2.78, N 16.95. Calculated%: C 67.47, H 2.83, N 16.86.

Example 26

5-hexyl-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626226

Gross formula C ₁₈ H ₁₈ N ₂ O ₂ ; Mp 275-277 ^o C (speed 10 ^o C / min); Mass spectra (EU) ESI-MS / MS - 294; IR spectrum, cm ^-1 2112, 1740, 1231, 993; ¹ H NMR (DMSO-d ₆ ) σ: 8.66 (1H, s, CH), 7.47 (1H, d, CH), 7.29 (1H, d, CH), 7.13 (1H, t, CH), 7.46 (1H , t, CH), 4.13 (2H, t, CH ₂ ), 1.87-1.25 (8H, m, 4CH ₂ ), 0.88 (3H, t, CH ₃ ) ppm. Elemental analysis%: C 73.39, H 6.17, N 9.45. Calculated%: C 73.45, H 6.16, N 9.52.

Example 27

methyl 2- (3-cyano-2-oxopyrano [3,2-b] indol-5 (2H) -yl) propionate 11626227

Gross formula C ₁₆ H ₁₂ N ₂ O ₄ ; Mp 328-330 ^o C (speed 10 ^o C / min); Mass spectra (EU) ESI-MS / MS - 296; IR spectrum, cm ^-1 2111, 1781, 1751, 1240; ¹ H NMR (DMSO-d ₆ ) σ: 8.78 (1H, s, CH), 7.49 (1H, d, CH), 7.32 (1H, d, CH), 7.14 (1H, t, CH), 7.47 (1H , t, CH), 5.25 (1H, q, CH), 3.64 (3H, s, OCH ₃ ), 1.88 (3H, d, CH ₃ ) ppm. Elemental analysis%: C 64.60, H 4.02, N 9.53. Calculated%: C 64.68, H 4.08, N 9.45.

Example 28

[(3-aminocarbonyl) -2-oxopyrano [3,2-b] indole-5 (2H) -yl] acetic acid 11626229

The gross formula is C ₁₄ H ₁₀ N ₂ O ₅ ; Mp 335-338 ^o C (speed 10 ^o C / min); Mass spectra (EU) ESI-MS / MS - 286; IR spectrum, cm ^-1 2587, 1793, 1747, 1226. ¹ H NMR (DMSO-d ₆ ) σ: 8.90 (2H, br s, NH ₂ ), 8.50 (1H, s, CH), 7.57 (1H, d , CH), 7.32 (1H, t, CH), 7.24 (1H, d, CH), 7.11 (1H, t, CH), 4.82 (2H, s, CH ₂ ) ppm. Elemental analysis%: C 57.69, H 3.46, N 9.77. Calculated%: C 58.75, H 3.52, N 9.79.

Example 29

methyl 2- (3-cyano-2-oxopyrano [3,2-b] indol-5 (2H) -yl) butanoate 11626230

Gross formula C ₁₇ H ₁₄ N ₂ O ₄ ; Mp 301-303 ^o C (speed 10 ^o C / min); Mass Spectra (EU) ESI-MS / MS - 310; IR spectrum, cm ^-1 2113, 1786, 1751, 1222; ¹ H NMR (DMSO-d ₆ ) σ: 8.80 (1H, s, CH), 7.47 (1H, d, CH), 7.40 (1H, d, CH), 7.29 (1H, t, CH), 7.11 (1H , t, CH), 4.94 (1H, q, CH), 3.83 (3H, s, OCH ₃ ), 2.17 (2H, m, CH ₂ ), 1.17 (3H, t, CH ₃ ) ppm. Elemental analysis%: C 65.74, H 4.51, N 9.08. Calculated%: C 65.80, H 4.55, N 9.03.

Example 30

5- (4-cyanobenzyl) -2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626231

Gross formula C ₂₀ H ₁₁ N ₃ O ₂ ; Mp 297-300 ^o C (speed 10 ^o C / min); Mass spectra (EU) ESI-MS / MS - 325; IR spectrum, cm ^-1 2132, 2110, 1749, 1221. ¹ H NMR (DMSO-d ₆ ) σ: 8.67 (1H, s, CH), 7.53-7.14 (8H, m, 8CH), 5.52 (2H, s , CH ₂ ) ppm. Elemental analysis%: C 73.79, H 3.43, N 12.98. Calculated%: C 73.84, H 3.41, N 12.92.

Example 31

2- (3-cyano-2-oxopyrano [3,2-b] indole-5 (2H) -yl) -N- (4-methoxyphenyl) acetamide 11626232

The gross formula is C ₂₁ H ₁₅ N ₃ O ₄ . Mp 312-315 ^o C (speed 10 ^o C / min); Mass spectra (EU) ESI-MS / MS - 373; IR spectrum, cm ^-1 2110, 1777, 1749, 1228; ¹ H NMR (DMSO-d ₆ ) σ: 9.24 (1H, br s, NH), 8.62 (1H, s, CH), 7.53-6.82 (8H, m, 8CH), 4.17 (2H, s, CH ₂ ), 3.75 (3H, s, CH ₃ ) Elemental analysis%: C 67.52, H 3.98, N 11.31. Calculated%: C 67.56, H 4.05, N 11.25.

Example 32

5-benzyl-2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile 11626233

Gross formula C ₁₉ H ₁₂ N ₂ O ₂ ; Mp 298-301 ^o C (speed 10 ^o C / min); Mass Spectra (EU) ESI-MS / MS - 300; IR spectrum, cm ^-1 2110, 1752, 1229; ¹ H NMR (DMSO-d ₆ ) σ: 8.67 (1H, s, CH), 7.51-7.00 (9H, m, 9CH), 5.52 (2H, s, CH ₂ ) ppm. Elemental analysis%: C 75.91, H 4.02, N 9.35. Calculated%: C 75.99, H 4.03, N 9.33.

Determination of cytotoxicity and anti-tuberculosis activity

To determine the cytotoxicity of 35 synthesized compounds, A549 lung epithelium cells and HepG2 human hepatocellular carcinoma cells were seeded in 96-well plates at a concentration of 10 ⁴ cells / ml in DMEM (without phenol red) supplemented with 10% fetal calf serum (FCS); cell-containing medium was used as a 100% inhibition control. Cells were incubated in the presence of compounds in 5% CO2 in a humidified incubator at 37 ° C for 3 days, resazurin was added and cell viability was determined by staining after 6 hours.

The anti-tuberculosis activity of the synthesized compounds was determined by assessing the minimum inhibitory concentration (MIC) using the resazurin method according to the protocol developed previously [Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F. Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2002; 46: 2720-2]. The resazurin indicator (7-hydroxy-3Hphenoxazin-3-one-10-oxide) is a weakly fluorescent dye of blue color (Bueno et al., 2002), which, when reduced, turns into pink, highly fluorescent resofurin. Since the metabolic activity of cells correlates with the restoration of resazurin to resofurin (accompanying cell respiration), a color change from blue to pink can be easily detected fluorimetrically. Due to its high sensitivity, the assessment of cell viability even of such a slowly growing organism as M. tuberculosis can be carried out in just 7-8 days. The formulation of testing the activity of various chemical compounds against resting M. tuberculosis cells in a 96-well plate format using resazurin microanalysis (PEMA) allows rapid high-throughput testing of the anti-tuberculosis activity of the compounds.

The data obtained are shown in table 2.

Protocol for assessing cell viability using resazurin microanalysis in a 96-well plate format

A culture of M. tuberculosis H37Rv was inoculated into each well of the plate (final concentration of 3 x 10 ⁴ cells / ml) containing 100 μl of 7H9 medium. Each of the test compounds at a concentration of 25 μg / ml (maximum concentration of test compounds) was added to the upper row of the plate wells, then seven consecutive two-fold dilutions for each of the test compounds were made vertically down the plate (25; 12.5; 6.2; 3.1; 1.5; 0.7; 0.35 μg / ml), thus the effect of eight different concentrations of each compound on M. tuberculosis growth was studied. DMSO (1%) and rifampicin (0.5 μg / ml) were used as negative and positive controls, respectively. The plate was incubated for 7 days in static mode at 37 ° C, then 10 μl of a sterile 0.1% resazurin solution was added to each well (to a final concentration of 0.025% w / v) and incubated for another 16-18 hours (night) under similar conditions, after which the fluorescence level of resazurin metabolite resofurin (excitation and emission wavelengths of 560 and 590 nm, respectively) was measured on a TECAN Infinite M200 plate reader (LifeSciences). These time parameters were selected empirically, based on the minimum time for which a stable and reproducible result of resofurin fluorescence detection is achievable, and in accordance with the previously published description of resazurin microanalysis [Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F. Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2002; 46: 2720-2].

Average fluorescence values and standard deviations were determined based on 3 independent changes in fluorescence for each of the studied concentrations of the tested compounds, the compound was considered active at this concentration if the average fluorescence of the test sample exceeded the average fluorescence of the negative control by an amount equal to three times the standard deviation for the average (from three measurements) fluorescence values of the negative control.

The data obtained are shown in table 2.

Protocol for assessing cell viability of non-replicating cells of strain Mycobacterium tuberculosis ss18b using a resazurin microanalysis in the format of a 96-well plate

It was found that all the original pyranoindole derivatives listed in Table 1 were highly M. tuberculosis active against non-replicating forms of streptomycin-depleted M. tuberculosis ss18b strain. The ss18b strain of Mycobacterium tuberculosis is known for being able to pass into replicating in the absence of streptomycin. The cause of this phenotypic manifestation is the insertion of the cytosine residue in 16S ribosomal rRNA (in the site responsible for streptomycin resistance) (Honore´, N., G. Marchal, and ST Cole. 1995. Novel mutation in 16S rRNA associated with streptomycin dependence in Mycobacterium dependence tuberculosis. Antimicrob. Agents Chemother. 39: 769–770). It is important that, in the absence of streptomycin, cells of strain 18b, although they did not have the ability to divide, did not lose their viability even for several weeks. Their growth ability quickly recovered when streptomycin was introduced into the growth medium (Hashimoto, T. 1955. Experimental studies on the mechanism of infection and immunity in tuberculosis from the analytical standpoint of streptomycin-dependent tubercle bacilli. 1. Isolation and biological characteristics of a streptomycin -dependent mutant, and effect of streptomycin administration on its pathogenicity in guinea pigs. Kekkaku 30: 4–8; 45–46).

Strain 18b was actively used in the development of in vivo resting models in mice and guinea pigs to further develop vaccines (Kashino, SS, P. Ovendale, A. Izzo, and A. Campos-Neto. 2006. Unique model of dormant infection for tuberculosis vaccine development. Clin. Vaccine Immunol. 13: 1014-1021; Kashino, SS, DR Napolitano, Z. Skobe, and A. Campos-Neto. 2008. Guinea pig model of Mycobacterium tuberculosis latent / dormant infection. Microbes Infect. 10: 1469 –1476). If the animals received injections of streptomycin, cells of strain 18b multiplied in the lungs and spleen of the animals, and vice versa, in the absence of streptomycin, they passed into an indivisible state close to the resting state. Thus, cells of strain 18b (streptomicyn-starved (ss) 18b) that do not divide in the absence of streptomycin are an in vivo and in vitro model of mycobacterial resting (Sala C, Dhar N, Hartkoorn RC, Zhang M, Ha YH, Schneider P, Cole ST. Simple model for testing drugs against nonreplicating Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2010, 54, 4150-8).

Interestingly, the non-dividing M. tuberculosis cells in the ss18b in vitro model had extremely low sensitivity to antibiotics such as isoniazid and benzothiazinone cell wall enzyme inhibitors active against dividing cells of strain 18b (in the presence of streptomycin). The activity of rifampicin, moxifloxacin, and bedaquiline against dividing and non-dividing cells of strain 18b was similar in value (Sala C, Dhar N, Hartkoorn RC, Zhang M, Ha YH, Schneider P, Cole ST. Simple model for testing drugs against nonreplicating Mycobacterium tuberculosis Antimicrob Agents Chemother. 2010, 54, 4150-8). Similar activity values for various antibiotics were obtained for non-dividing cells of strain 18b in vivo in a model of chronic tuberculosis in mice (Sala C, Dhar N, Hartkoorn RC, Zhang M, Ha YH, Schneider P, Cole ST. Simple model for testing drugs against nonreplicating Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2010, 54, 4150-8). A culture of M. tuberculosis 18b (non-replicating culture, 14 days incubation in the absence of streptomycin) was inoculated into each well of the plate (final concentration of 3 x 10 ⁴ cells / ml) containing 100 μl of 7H9 medium. Each of the tested compounds at a concentration of 10 μg / ml (maximum concentration of the tested compounds) was added to the upper row of the plate wells, then seven consecutive two-fold dilutions for each of the tested compounds were made vertically down the plate (5.0; 2.5; 1, 25; 0.625; 0.31; 0.16 μg / ml), thus the effect of eight different concentrations of each compound on M. tuberculosis growth was studied. DMSO (1%) and rifampicin (0.5 μg / ml) were used as negative and positive controls, respectively. The plate was incubated for 7 days in static mode at 37 ° C, then 10 μl of a sterile 0.1% resazurin solution was added to each well (to a final concentration of 0.025% w / v) and incubated for another 16-18 hours (night) under similar conditions, after which the fluorescence level of resazurin metabolite resofurin (excitation and emission wavelengths of 560 and 590 nm, respectively) was measured on a TECAN Infinite M200 plate reader (LifeSciences). These time parameters were selected empirically, based on the minimum time for which a stable and reproducible result of resofurin fluorescence detection is achievable, and in accordance with the previously published description of resazurin microanalysis [Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F. Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2002; 46: 2720-2].

Average fluorescence values and standard deviations were determined based on 3 independent changes in fluorescence for each of the studied concentrations of the tested compounds, the compound was considered active at this concentration if the average fluorescence of the test sample exceeded the average fluorescence of the negative control by an amount equal to three times the standard deviation for the average (from three measurements) fluorescence values of the negative control.

The data obtained are shown in table 2.

Protocol for assessing cell viability of non-replicating cells of Mycobacterium tuberculosis strain under potassium deficiency

A study of selected compounds of 2-thiopyridine leaders in vitro was conducted on a latent tuberculosis model in conditions of potassium deficiency [Salina E, Ryabova O, Kaprelyants A, Makarov V. New 2-thiopyridines as potential candidates for killing both actively growing and dormant Mycobacterium tuberculosis cells. Antimicrob Agents Chemother 2014; 58: 55-60] and using the statistical method of "most probable numbers" (LF) [de Man JC. The probability of most probable numbers. J Appl Microbiol 1975; 1: 67-78]. Briefly, 2 x 10 ⁷ resting “uncultured” mycobacteria (CFU = 0) were treated with 25 μM of each of the most active compounds for 7 days. Then, to determine the number of potentially reactive resting cells, 10-fold serial dilutions of cultures of Mycobacterium tuberculosis in a medium of normal composition (containing potassium) were prepared in triplicate in 48-well plates. Plates were incubated statically at 37 ° C for 30 days, wells with visible bacterial growth were considered positive. The number of reactivated bacteria was calculated using the standard statistical method [de Man JC. The probability of most probable numbers. J Appl Microbiol 1975; 1: 67-78]. The number of reactivated resting “uncultured” bacteria (LFV values) after treatment with the compounds was compared with the number of reactivated cells of the control sample untreated with the compounds, and the bactericidal effect of each compound against the resting cells of M. tuberculosis was calculated as the ratio of these two values.

The data obtained are shown in table 2.

Connection number Cytotoxicity
IC ₅₀
mcg / ml MIC ₉₉ , µg / mL
H37rv MIC ₉₉ , µg / mL
ss18b LOG bactericidal effect against dormant non-cultured bacteria A549
lungs Hepg2
liver one. 11126066 64 > 25 0.78 0.78 2.97-3.22 2. 11126067 > 100 > 25 25 25 NT 3. 11526065 > 100 > 100 25 > 25 NT four. 11526066 > 100 > 100 25 > 25 NT 5. 11526067 32 8 25 25 NT 6. 11526108 32 8 1,6 3.2 2.91-3.22 7. 11526109 32 8 1,5 1,5 2.97-3.22 8. 11526110 32 8 1,6 1,6 2.91-3.22 9. 11626056 > 100 > 100 0.3 0.6 3.91-4.14 10. 11626057 32 8 25 > 25 NT eleven. 11626058 > 100 > 100 25 25 NT 12. 11626059 64 32 6.2 6.2 NT 13. 11626060 64 32 6.2 12.5 NT fourteen. 11626062 32 8 3.6 3.6 NT fifteen. 11626210 > 100 > 100 25 25 NT 16. 11626211 > 100 > 100 25 > 25 NT 17. 11626212 > 100 > 100 12.5 12.5 2.94-4.00 eighteen. 11626213 > 100 > 100 25 25 NT 19. 11626214 > 100 > 100 25 > 25 NT twenty. 11626215 32 16 6.2 6.2 NT 21. 11626216 > 100 > 100 25 25 2.94-4.00 22. 11626217 > 100 > 100 25 > 25 NT 23. 11626218 > 100 > 100 25 > 25 NT 24. 11626219 > 100 > 100 25 25 NT 25. 11626220 > 100 > 100 25 25 NT 26. 11626223 > 100 > 100 6.2 12.5 NT 27. 11626224 64 32 3,1 3,1 NT 28. 11626225 > 100 > 100 25 25 NT 29. 11626226 > 100 > 100 25 > 25 NT thirty. 11626227 > 100 > 100 25 > 25 NT 31. 11626229 > 100 > 100 25 25 NT 32. 11626230 32 8 12.5 12.5 NT 33. 11626231 32 16 6.2 6.2 NT 34. 11626232 64 32 25 > 25 NT 35. 11626233 > 100 > 100 25 25 NT

Claims (16)

1. The compound of General formula 1

one, Where R ¹ is H, HO, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , AcO, Cl, CF ₃ , CF ₃ O, NO ₂ , MeO; R ² is H, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , Ac, CH ₂ Ph, CH ₂ Ph-4-CN, CH ₂ Ph-4-OMe; R ³ means H, NHCH ₃ , NHC ₃ H ₅ ; R ⁴ means CN, CONH ₂ , COOC ₂ H ₅ , NHCOPh, excluding compounds 2-oxo-2,5-dihydropyrano [3,2-b] indole-3-carbonitrile, 2-oxo-2,5-dihydropyrano [3,2-b] indole-3-ethoxycarbonyl and 2-oxo -2,5-dihydropyrano [3,2-b] indole-3-carbamide.
2. The compound according to formula (1) where R ¹ = R ² = CH ₃ , R ³ = H, R ⁴ = CN.
3. The use of compounds according to formula (1) according to claim 1

                                                                                               one, Where R ¹ is H, HO, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , AcO, Cl, CF ₃ , CF ₃ O, NO ₂ , MeO; R ² is H, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , Ac, CH ₂ Ph, CH ₂ Ph-4-CN, CH ₂ Ph-4-OMe; R ³ means H, NHCH ₃ , NHC ₃ H ₅ ; R ⁴ means CN, CONH ₂ , COOC ₂ H ₅ , NHCOPh, for the prevention and / or treatment of tuberculosis infection in humans.
4. The use of a compound according to claim 2 for the prevention and / or treatment of human tuberculosis infection caused by Mycobacterium tuberculosis.