RU2477127C1 - Preparing quaternised usnic acid derivatives and their biological properties - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmaceutics and concerns gram-positive pathogen growth inhibitors representing (+) and (-)-enantiomers of usnic acid derivatives containing a quaternised nitrogen atom. The inhibitors possess high biological activity.

EFFECT: invention provides effective action on mycobacteria and thereby increased clinical effectiveness in tuberculosis.

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Description

FIELD OF THE INVENTION

The invention relates to pharmaceutical chemistry and relates to derivatives of usnic acid containing a quaternized nitrogen atom. These compounds can be used as substances active against pathogenic gram-positive bacteria, including mycobacteria.

State of the art

Improving the effectiveness of treatment for tuberculosis patients is the most important social task in Russia and the world. Each year, these diseases claim more than 2 million lives. Improving the results of treatment for tuberculosis depends on the development of modern pharmaceuticals, the main one being the development of new technological approaches to the creation of new drugs by targeting the important pathogens of the tuberculosis pathogen - Mycobacterium tuberculosis. One of the reasons for the difficulty in treating tuberculosis is the massive emergence of multiple drug resistance (MDR or MDR - Multiple Drug Resistance) in the causative agent of tuberculosis, both to traditional anti-TB drugs of the so-called first row (especially to rifampicin and isoniazid), and to second-line drugs (fluoroquinolones and aminoglycosides) (Edlin et al. An outbreak of multidrug-resistant tuberculosis among hospitalized patients with the acquired immunodeficiency syndrome. N Engl J Med: 1992; 326 (23): 1514-1521; Fischl et al., An outbreak of tuberculosis caused by multiple-drug-resistant tubercle bacilli among patients with HIV infection. Ann Intern Med: 1992; 117 (3): 177 -183). Among resistant strains, strains with extensive drug resistance are particularly dangerous (XDR or XDR - Extensively Drug-Resistant); they are resistant to 4-9 drugs of the first and second row. Therefore, overcoming the problems facing phthisiology with the advent of MDR is a task of paramount importance (Tomioka N. Prospects for the development of new antituberculous drugs putting our hopes on new drug targets. Kekkaku. 2010; 85 (11): 815-22, Shi R, Sugawara I. Development of new anti-tuberculosis drug candidates. Tohoku J Exp Med. 2010; 221 (2): 97-106; Koul A, Arnoult E, Lounis N, Guillemont J, Andries K The challenge of new drug discovery for tuberculosis. Nature. 2011; 469 (7331): 483-90). The need is formulated to develop new candidates for drugs, first of all, semi-synthetic, based on natural substances of new classes, which showed in previous studies a new mechanism of action and have been tested on safety for humans. Semisynthetic derivatives of usnic acid belong to this class of compounds. The disadvantage of natural usnic acid, as a previously used anti-TB drug, is its relatively low efficiency compared to existing first-line drugs and low water solubility. The proposed semi-synthetic quaternized derivatives of usnic acid are soluble and more effective.

Problem to be solved: Search for antibacterial agents of a new mechanism of action, including anti-TB drugs, active against multidrug-resistant strains.

The compounds disclosed in the present invention are derivatives of (R) - and (S) -usnic acid containing a quaternized nitrogen atom, the synthesis of compounds 3-6 is described in [A.A. Tazetdinova, O.A. Luzina, M.P. Polovinka, N.F. Salakhutdinov and G.A. Tolstikov. Amino-derivatives of usninic acid. Chemistry of Natural Compounds, 2009, 45, 800], compounds 7 and 8 are new, not described previously.

Disclosure of invention

The objective of the invention is the use as antibacterial (including anti-TB) agents for the first time synthesized derivatives of usnic acid.

(S) -Unicovic acid 1 was isolated from lichen Cladonia stellaris, (R) -usnic acid 2 from a mixture of Usnea lichens according to the procedure [N.F. Salakhutdinov, M.P. Polovinka, M.Yu. Panchenko, Pat. RF No. 2317076 C1; Bull. Fig. 2008, No. 5]. Compounds 3 and 4 can be prepared by reacting compounds 1 and 2 with diethylaminoethylamine, respectively. Compounds 5 and 6 can be prepared by reacting compounds 3 and 4 with methyl iodide, respectively. Both reactions are carried out according to previously proposed methods [A.A. Tazetdinova, O.A. Luzina, M.P. Polovinka, N.F. Salakhutdinov and G.A. Tolstikov. Amino-derivatives of usninic acid. Chemistry of Natural Compounds, 2009, 45, 800] according to the scheme:

Compound 7 can be prepared by reacting, respectively, compound 1 with dimethylaminoethylamine. Compound 8 can be prepared by reacting compound 7 with methyl iodide. Both reactions are carried out according to the methods described below in accordance with the scheme:

The invention is illustrated by the following examples:

Example 1

Synthesis of (E) -6-acetyl-2- (1- (2- (diethylamino) ethylidene) -7,9-dihydroxy-8,9b-dimethyldibenzo [b, d] furan-1,3 (2H, 9bH) - dion 3.4

1.1 mmol of diethylaminoethylamine was added to 1 mmol of compound 1 or 2 and the mixture was dissolved in 12 ml of ethyl alcohol. Boiled in a water bath for 3 hours. Cooled, added 10 ml of distilled water. A light precipitate precipitated, the precipitate was filtered off, washed with water, and dried in air. Compounds 3 or 4 were isolated, respectively, in 88% yield.

(R, E) -6-acetyl-2- (1- (2- (diethylamino) ethylidene) -7,9-dihydroxy-8,9b-dimethyldibenzo [b, d] furan-1,3 (2H, 9bH) dione 3. Yield 88%, mp 68 ° C. ¹ H NMR (CDCl ₃ , δ, ppm, J Hz): 1.16 (6H, t, J = 7.1, 3H-19 and 3H- 21), 1.73 (3H, s, H-15), 2.13 (3H, s, H-10), 2.67 (6H, s, 3H-12 and 3H-14), 2.69 (4H; q, J = 7.1, 2H-18 and 2H-20), 2.86 (2H, t, J = 6.0, H-17), 3.66 (2H, d.t, J = 5.7, J = 6.0, H-16), 5.82 (1H, s , H-4), 12.01 (1H, br s, OH-9), 13.36 and 13.39 (1H, s and 1H, s, NH and OH-7). ¹³ C NMR (CDCl ₃ , δ, ppm) .): 7.3 (C-10), 11.5 (C-19 and C-21), 18.7 (C-12), 31.8 (C-14), 31.9 (C-15), 42.3 (C-16), 46.7 (C-18 and C-20), 50.8 (C-17), 59.0 (C-9b), 102.5 (C-4), 101.2 (C-6), 105.0 (C-2), 105.1 (C-9a ), 107.7 (C-8), 155.8 (C-5a), 158.2 (C-9), 163.7 (C-7), 173.7 (C-11), 174.3 (C-4a), 191.5 (C-3) , 197.9 (C-1), 200.6 (C-13) IR spectrum (ν, cm ^-1):. 841, 1061, 1287, 1370, 1555, 1627, 1701, 2926, 3387. In Deno: m / z 442.2110 [M] + C ₂₄ H ₃₀ N ₂ O _{6 Calculated: M = 442.2098 [α] D} + 366 ° (c 0.390; CHCl _3)...

(S, E) -6-acetyl-2- (1- (2- (diethylamino) ethylidene) -7,9-dihydroxy-8,9b-dimethyldibenzo [b, d] furan-1,3 (2H, 9bH) -dione 4. The spectral data are the same as for compound 3 [α] _D -370 ° (c 0.340; CHCl ₃ ).

Example 2

Synthesis of (E) -2- (1- (6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-1,9b-dihydrodibenzo [b, d] furan-2 (3H) - ylidene) ethylamino) -N, N-diethyl-N-methylethanammonium iodides 5.6

To 1.1 mmol of amines 3 or 4 in 10 ml of methylene chloride was added 3 mmol of methyl iodide. The reaction mixture was kept for 3 days, a yellowish precipitate formed. The resulting precipitate was filtered off, washed with chloroform, and then dried in air. Compounds 5 or 6 were isolated, respectively.

(R, E) -2- (1- (6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-1,9b-dihydrodibenzo [b, d] furan-2 (3H) -ilidene) ethylamino) -N, N-diethyl-N-methylethanammonium iodide 5. Yield 64%. so pl. 125 ° C. ¹ H NMR (DMSO-d ₆ , δ, ppm, J Hz): 1.22 (6H, t, J = 7.1, 3H-19 and 3H-21), 1.60 (3H, s, H-15), 1.90 (3H, s, H-10), 2.57 (3H, s, H-12), 2.64 (3H, s, H-14), 3.05 (3H, s, H-22), 3.38 (4H, q, J = 7.1, 2H-18 and 2H-20), 3.59 (2H, t, J = 6.0, H-17), 4.04 (2H, d.t, J = 5.7 and J-6.0, H-16), 5.83 (1H, s, H-4), 12.06 (1H, s, OH-9), 12.97 and 13.32 (1H, s, NH and 1H, s, OH-7). ¹³ C NMR (CDCl ₃ , δ, ppm): 7.6 (C-10), 7.7 (C-19 and C-21), 18.6 (C-12), 31.2 and 31.8 (C-14 and C- 15), 37.2 (C-16), 47.1 (C-22), 56.4, 56.6, 57.5 (C-17, C-18, C-20), 59.0 (C-9b), 100.1 (C-6), 102.4 (C-4), 102.4 (C-2), 105.4 (C-9a), 106.6 (C-8), 155.8 (C-5a), 157.6 (C-9), 162.6 (C-7), 173.3 (C-11), 176.2 (C-4a), 189.0 (C-3), 197.7 (C-1), 201.1 (C-13). IR spectrum (ν, cm ^-1 ): 846, 1060, 1288, 1369, 1556, 1629, 1700, 2976, 3440. Elemental analysis: Found,%: C 52.48, H 6.13, N 4.78, I 21.00. Calculated,%: C 51.38, H 5.69, N 4.79, I 21.71. C ₂₅ H ₃₃ IO ₆ N ₂ . [α] _D + 198 ° (c 0.164; CH ₃ OH).

(S, E) -2- (1- (6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-1,9b-dihydrodibenzo [b, d] furan-2 (3H) -ilidene) ethylamino) -N, N-diethyl-N-methylethanammonium iodide 6. The spectral data are the same as for compound 5. [α] _D -201 ° (c 0.146; CH ₃ OH).

Example 3

Synthesis of (R, E) -6-acetyl-2- (1- (2- (dimethylamino) ethylidene) -7,9-dihydroxy-8,9b-dimethyldibenzo [b, d] furan-1,3 (2H, 9bH ) -dione 7

1.1 mmol of dimethylaminoethylamine was added to 1 mmol of compound 1 and the mixture was dissolved in 12 ml of ethyl alcohol. Boiled in a water bath for 3 hours. Cooled, added 10 ml of distilled water. A light precipitate precipitated, the precipitate was filtered off, washed with water, and dried in air. Compound 7 was isolated in 86% yield.

(R, E) -6-acetyl-2- (1- (2- (dimethylamino) ethylidene) -7,9-dihydroxy-8,9b-dimethyldibenzo [b, d] furan-1,3 (2H, 9bH) dione 7. Yield 86%, mp 84 ° C. ¹ H NMR (CDCl ₃ , δ, ppm, J Hz): 1.67 (3H, s, H-15), 2.06 (3H, s , H-10), 2.31 (6H, s, 3H-18 and 3H-19), 2.61 and 2.64 (6H, s, 3H-12 and 3H-14), 2.60 (2H, t, J = 6.0, H- 17), 3.50 (2H, dt, J = 5.7, J = 6.0, H-16), 5.75 (1H, s, H-4), 12.02 (1H, br.s, OH-9), 13.33 ( 1H, br.s and 1H, br.s, NH and OH-7) ¹³ C NMR (CDCl ₃ , δ, ppm): 7.0 (C-10), 18.3 (C-12), 30.8 ( C-14), 31.5 (C-15), 41.6 (C-16), 44.9 (C-18 and C-19), 56.7 (C-17), 59.0 (C-9b), 102.1 (C-4) , 100.9 (C-6), 101.2 (C-2), 104.7 (C-9a), 107.4 (C-8), 155.5 (C-5a), 157.9 (C-9), 163.0 (C-7), 173.5 (C-11), 174.0 (C-4a), 192.1 (C-3), 197.5 (C-1), 200.2 (C-13). IR spectrum (ν, cm ^-1 ): 1059, 1288, 1371 , 1556, 1626, 1701, 2773, 2922, 3437. Found: m / z 414.1796 [M] + C ₂₂ H ₂₆ O ₆ N ₂ Calcd. techniques, _{are: M = 414.1791 [α] D} + 290 ° (c 0.685; CHCl _3)..

Example 4

Synthesis of (R, E) -2- (1- (6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-1,9b-dihydrodibenzo [b, d] furan-2 (3H ) -ilidene) ethylamino) -N, N-dimethyl-N-methylethanammonium iodide 8

To 1.1 mmol of amine 7 in 10 ml of methylene chloride, 3 mmol of methyl iodide was added. The reaction mixture was kept for 3 days, a white precipitate formed. The resulting precipitate was filtered off, washed with chloroform, and then dried in air. Compound 8 was isolated in 88% yield.

(R, E) -2- (1- (6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-1,9b-dihydrodibenzo [b, d] furan-2 (3H) -ilidene) ethylamino) -N, N-dimethyl-N-methylethanammonium iodide 8. Yield 88%. so pl. 240 ° C. ¹ H NMR (DMSO-d ₆ , δ, ppm, J Hz): 1.67 (3H, s, H-15), 1.98 (3H, s, H-10), 2.64 (3H, s, H- 12), 2.66 (3H, s, H-14), 3.17 (9H, s, 3H-18, 3H-19, 3H-20), 3.69 (2H, t, J = 6.0, H-17), 4.08 ( 2H, dt, J = 5.5 and J = 6.0, H-16), 5.93 (1H, s, H-4), 12.15 (1H, s, OH-9), 13.07 (1H, t, J = 5.5 , NH), 13.41 (1H, s, OH-7). ¹³ C NMR (CDCl ₃ , δ, ppm): 7.5 (C-10), 18.5 (C-12), 31.1 and 31.6 (C-14 and C-15), 37.6 (C-16), 52.8 , (C-18, C-19, C-20), 56.5 (C-17), 62.8 (C-9b), 100.9 (C-6), 102.2 (C-2), 102.4 (C-4), 105.0 (C-9a), 106.4 (C-8), 155.8 (C-5a), 157.6 (C-9), 162.6 (C-7), 173.2 (C-11), 176.0 (C-4a), 188.9 (C-3), 197.6 (C-1), 201.0 (C-13). IR spectrum (ν, cm ^-1 ): 1055, 1191, 1288, 1373, 1556, 1628, 1699, 2787, 2954, 3439. Elemental analysis: Found,%: C 45.33, H 5.06, N 3.70, I 20.45. Calculated,%: C 49.65, H 5.25, N 5.03, I 22.81. C ₂₃ H ₂₉ IO ₆ N ₂ . [α] _D + 203 ° (c 0.310; CH ₃ OH).

Example 5

The antibacterial activity of substance 5 against pathogenic gram-positive microorganisms, determined by the method of determining the minimum inhibitory concentrations in a liquid medium.

A solution of the test compound was prepared at a concentration of 1000 μg / ml in DMSO, then diluted with water to a concentration of 128 μg / ml.

The inoculum was prepared using a daily culture of gram-positive microorganisms Staphylococcus, Streptococcus and Enterococcus grown on a solid nutrient medium corresponding to each type of microorganism. A suspension of microorganisms was prepared in a sterile isotonic sodium chloride solution, bringing the inoculum density to 0.5 according to the MacFarland standard (1.5 × 10 ⁸ CFU / ml). Then, the resulting inoculum was diluted to a concentration of 5 × 10 ⁵ CFU / ml of Mueller-Hinton broth.

We used a micromethod for determining the minimum inhibitory concentration (MIC) by serial dilution in Muller-Hinton broth using 96-well sterile plates.

MIC was taken as the minimum concentration of the studied compounds, providing complete suppression of the visible growth of the studied strains of microorganisms.

Bacterial strains MIC in μg / ml Staphylococcus aureus 25923 ATCC 16.0 Staphylococcus epidermidis 12228 ATCC 32.0 Streptococcus pneumoniae 49619 ATCC 64.0 Enterococcus faecalis 29212 ATCC 32.0

The substance is active against all tested gram-positive microorganisms: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis.

Example 6

Antibacterial activity 5 against anaerobic gram-positive microorganisms.

To determine the activity of the substance, the paper disc method was used. The method consists in determining the size of the zone of inhibition of growth of the strain, seeded with a lawn on an agar medium, around paper disks containing a substance in various concentrations.

Bacterial strains MIC in mcg / disk Lactobacillus casei 20011 6.0 Lactobacillus rhamnosus 421-2 5.0 Bifidobacterium bifidum 791wt 5.5

Representatives of the genera Lactobacillus and Bifidobacterium are sensitive to 5.0-6.0 μg / disk of the substance.

Example 7

Antibacterial activity of the substance 5, 6, 7, 8 in the test system Mycobacterium smegmatis ATCC-607.

Mycobacterium smegmatis is a model test system used in the first stage of screening for potential anti-TB drugs (K.E. A. Lougheed, S. A. Osborne, B. Saxty, et al. Effective inhibitors of the essential kinase PknB and their potential as anti -mycobacterial agents. Tuberculosis (Edinb. 2011 July; 91 (4): 277-286). Determination of the activity of the substance was carried out by the method of paper disks. The method consists in determining the size of the zone of inhibition of growth of the strain, seeded with a lawn on an agar medium, around paper disks containing a substance in various concentrations.

Strain Mycobacterium smegmatis ATCC-607 Substance 5 6 8 MIC in mcg / disk 5.8 6.5 5.7

The strain Mycobacterium smegmatis ATCC-607 is sensitive to 5.8 μg / disk of substance 5, to 6.0 μg / disk of substance 6, to 5.7 μg / disk of substance 8.

Example 8

Determination of acute toxicity of a substance 5.

The determination of a semi-lethal dose of a substance (LD ₅₀ ) was carried out using the linear regression equation. The substance, dissolved in DMSO at various concentrations, was administered to 0.3 ml Balb / C inbred mice orally through a tube. Within 30 days, the dynamics of animal death was observed. Based on the data obtained, a linear regression equation was constructed and a semi-lethal dose of the substance was determined. LD ₅₀ = 975 mg / kg.

Claims (1)

Growth inhibitor of pathogenic gram-positive microorganisms, derivatives of usnic acid, which are selected from the group (R, E) -2- (1- (6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-1 , 9b-dihydrodibenzo [b, d] furan-2 (3H) -ilidene) ethylamino) -N, N-diethyl-N-methylethanammonium iodide 5 and (S, E) -2- (1- (6-acetyl-7 , 9-dihydroxy-8,9b-dimethyl-1,3-dioxo-1,9b-dihydrodibenzo [b, d] furan-2 (3H) -ilidene) ethylamino) -N, N-diethyl-N-methylethanammonium iodide 6 , (R, E) -2- (1- (6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-1,9b-dihydrodibenzo [b, d] furan-2 (3H ) -ilidene) ethylamino) -N, N-dimethyl-N-methylethanammonium iodide 8.