Furilidene furanone derivatives of usnic acid as new antituberculosis agents

FIELD: medicine.

SUBSTANCE: invention concerns Mycobacterium tuberculosis growth inhibitors representing (+) and (-)-enantiomers of derivatives of usnic acid containing a furilidene furanone fragment, namely (10R,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(2-furanylmethylidene)-5,16-dioxatetracyclo[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 4a and (10S,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(2-furanylmethylidene)-5,16-dioxatetracyclo[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 4b

EFFECT: inhibitors possess the high antimicrobial activity.

2 tbl, 7 ex

The technical field to which the invention relates.

The invention relates to pharmaceutical chemistry and relates to derivatives of usninovoy acid containing hurricanranna fragment. These compounds may be used as substances active against Mycobacterium tuberculosis.

The level of technology

Improving the efficiency of treatment of tuberculosis patients is the most important social problem in Russia and in the world. Every year these diseases claim more than 2 million lives. The improvement of results of treatment of tuberculosis depends on the development of modern pharmaceuticals, the main which is to develop new technological approaches to the development of new drugs by acting on targets that are important for the activity of the causative agent of TB, Mycobacterium tuberculosis. One of the reasons for the difficulties of TB treatment is the mass emergence of multidrug resistance (MDR-TB or MDR - Multiple Drug Resistance) is the causative agent of tuberculosis, as traditional anti-TB drugs, the so-called first row (especially to rifampicin and isoniazid)and second-line drugs (fluoroquinolones and aminoglycosides) (Zhang Y, Yew WW. Mechanisms of drug resistance in Mycobacterium tuberculosis. // Int J Tuberc Lung Dis. 2009 Nov; 13(11). P. 1320-30; Chiang CY, Centis R, Migliori GB. Drug-resistant tuberculosis: past, present, future. Respirology. 2010 Apr; 15(3). P. 413-432). Among sustainable stammo is particularly dangerous strains with extensively drug-resistant (XDR or XDR Extensively Drug-Resistant); they are resistant to 4-9 drugs first and second series. Therefore, overcoming challenges Phthisiology in connection with the emergence of MDR is of paramount importance (Tomioka H. 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. V. 469(7331): P. 483-90; Prozorov, A. A., Zaichikov M. C., Danilenko Century. N. Mutants of Mycobacterium tuberculosis and multidrug resistance: a story of the emergence, genetic and molecular mechanisms of resistance, emerging issues / Genetics. 2012. So 48. No. 1. C. 5-20; Butler MS, Cooper MA. Antibiotics in the clinical pipeline in 2011. J Antibiot (Tokyo). 2011 Jun; 64(6). P. 413-25).

Formulated the need for development of new candidate drugs, primarily synthetic, on the basis of natural substances new classes that have been shown in previous studies, a new mechanism of action and past research on the safety. Natural usnic acid was previously used as a drug, but its disadvantages are the relatively low efficiency compared to existing first-line drugs and low solubility. Previously described quaternion derivatives of usninovoy acid with a modification to the ICA With molecules manifesting greater than native uninove acid, antibacterial activity against several bacteria, including Mycobacterium smegmatis, model test system used in the first stage of screening potential anti-tuberculosis drugs (K. E. A. Lougheed, S. A. Osborne, C. Saxty, et al. Effective inhibitors of the essential kinase PknB and their potential as anti-mycobacterial agents // Tuberculosis (Edinb). 2011. V. 91(4): P. 277-286). The results may indicate the prospects of synthetic modifications usanovich acids. However, based on literature data about the important role of triketone slice ring With usninovoy acid in the mechanism of penetration of the drug into the cell (L. Guo, Q. Shi, J. Fang, et al Review of usnic acid and Usnea barbata toxicity // Journal of environmental science and health. Part C, Environmental carcinogenesis &ecotoxicology reviews. 2008. V. 26. P. 317-338), we can assume that the perspective of semi-synthetic derivatives of usninovoy acid will be compounds containing the modified fragment, not touching her triketone system.

The proposed semi-synthetic derivatives of usninovoy acid containing hurricanranna fragment in a loop And usninovoy acid is more effective than quaternion derivative itself usnic acid against Mycobacterium smegmatis and Mycobacterium tuberculosis, and have bactericidal action.

Solved problem: the search for anti-tuberculosis drugs, the new fur is ISM actions including active against strains with multidrug resistance.

Compounds disclosed in the present invention are derived from (R)- and (S)-usninovoy acid containing hurricanranna fragment.

Disclosure of inventions

The objective of the invention is the use as anti-tuberculosis drugs, semi-synthetic derivatives of usninovoy acid is bactericidal against Mycobacterium tuberculosis.

To achieve this goal, we conducted a series of chemical modifications, the synthesis of the target compounds described in the article (Sokolov, A. N., Luzina O. A., Chernikov A. C., Salakhutdinov N. F. Synthesis of euronav based on usninovoy acid // Chemistry of natural compounds. 2012. No. 3. S. 350-355). As a parent compounds were taken as R-(+)-usnic acid 1A, obtained by extraction of the mixture of lichens of the genus Usnea, and S-(-)-usnic acid 1B, obtained by extraction of lichen Cladonia Stellaris methodology (N. F. Salakhutdinov, M. P. Spouse, M. Y. Panchenko, RF patent №2317076 C1; BI 2008, No. 5). Bromination of usninovoy acid 1A, b with bromine in the presence of Hydrobromic acid gives the derivative 2A, b, the treatment of which with potassium acetate leads to intramolecular cyclization with the formation of compounds with furninova fragment 3A, b. The next step was to obtain the target compounds with anti-TB the th activity by reaction of the compound 3A, b furfural.

The invention is illustrated by the following examples.

Example 1.

The interaction of usninovoy acid 1A, b with a twofold excess of bromine

To 1 mmol of usninovoy acid 1A (or 1B) (344 mg) was added to the complex bromooctane (2 mmol bromide (0.10 ml) was dissolved in 14 ml of dioxane), a few drops of HBr and left for 7 days at room temperature. After concentration of the reaction mixture on a rotary evaporator was chromatographically the resulting residue on silica gel (60-200µ), eluent - CH₂Cl₂.

(R)-2-acetyl-6-(2-bromoacetyl)-3,7,9-trihydroxy-8,9 b-dimethylbenzo[b,d]furan-1(9bH)-2A. Exit 283 mg (67%). So pl. 97-100°C. [α]_D+349 (0.5; CHCl₃). NMR¹H (CDCl₃, δ, ppm, J Hz): 1.75 (3H, s, H-15), 2.08 (3H, s, H-10), 2.64 (3H, s, H-12), 4.52 (2H, DD, J=12.4, J=14.0, H-14), 6.00 (1H, s, H-4), 11.17 (1H, s, OH-9), 12.68 (1H, s, OH-7), 18.81 (1H, , OH-3). NMR¹³With (CDCl₃, δ, M. D.): 7.5 (C-10), 27.7 (C-12), 31.9 (C-15), 34.5 (C-14), 61.6 (C-9b), 98.7 (C-4), 99.0 (C-6), 104.3 (C-9a), 105.1 (C-2), 109.6 (C-8), 154.3 (C-5a), 158.4 (C-9), 164.1 (C-7), 178.5 (C-4a), 191.5 (C-3), 192.7 (C-13), 197.7 (C-1), 201.7 (C-11). IR spectrum, ν, cm^-1): 842, 1140, 1292, 1458, 1628, 3013, 3497. Found: m/z 421.9976 [M]⁺C₁₈H₁₅O₇Br. Calculated: M=421.9996.

(S)-2-acetyl-6-(2-bromoacetyl)-3,7,9-trihydroxy-8,9 b-dimethylbenzo[b,d]furan-1(9bH)-2B. [α]_D-342 (0.5; CHCl₃).

Example 2.

The interaction of compounds 2A, b with potassium acetate

To a solution of 1 mmol of compound 2A (b) (423 mg) in 25 ml of acetone was added 150 mg (1.5 mmol) of potassium acetate and heated the reaction mixture for 2 hours. Then diluted with water (up to ~50-60 ml), acidified with HCl (1:4) to pH=3-4. Was extracted with CH₂Cl₂(3×10 ml), dried over calcined MgSO₄, solvent was removed and chromatographically the residue on a column of silica gel, eluent - CH₂Cl₂.

(10R)-8,13-dihydroxy-7,10-dimethyl-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 3A. The output 308 mg (90%). So pl. 202-203 °C. [α]_D+397 (0.5; CHCl₃). NMR¹H (CDCl₃, δ, M. D., J Hz): 1.73 (3H, s, H-15), 2.13 (3H, s, H-10), 2.64 (3H, s, H-12), 4.66 (2H, s, H-14), 6.02 (1H, s, H-4), 11.27 (1H, s, OH-9), at 18.82 (1H, s, OH-3). NMR¹³C (CDCl₃, δ, M. D.): 6.91 (C-10), 27.80 (C-12), 31.91 (C-15), 58.71 (C-9b), 75.70 (C-14), 99.01 (C-4), 100.61 (C-9a), 105.10 (C-6), 105.7 (C-2), 107.10 (C-8), 149.13 (C-5a), 159.80 (C-9), 173.81 (C-7), 179.61 (C-4a), 191.61 (C-3), 194.10 (C-13), 197.90 (C-1), 201.71 (C-11). Found: m/z 342.0736 [M]⁺C₁₈H₁₄O₇. Calculated: M=342.0734.

(10S)-8,13-dihydroxy-7,10-dimethyl-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 3b. [α]_D-390 (0.5; CHCl₃).

Example 3.

The interaction of compounds 3A, b with furfural

To a solution of 1 mmol of compound 3A (3b) (342 mg) in 24 ml of MeOH was added 1.1 mmol of furfural (105 mg), 1 ml of 50% aqueous solution of KOH and heated for 1.5 hours. Then the reaction mixture was diluted with water (up to ~50-60 ml), acidified with HCl (1:4) to pH=3-4. Was extracted with CH₂Cl₂(3×10 ml), dried over calcined MgSO₄, solvent was removed and chromatographical is whether the residue on a column of silica gel, eluent - CH₂Cl₂.

(10R,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(2-foranimation)-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 4A. Yield 335 mg (65%). So pl. 188-192°C. [α]_D+365 (0.3; CHCl₃). NMR¹H (CDCl₃, δ, M. D.): 1.76 (3H, s, H-15), 2.29 (3H, s, H-10), 2.65 (3H, s, H-12), 6.05 (1H, s, H-4), 6.59 (1H, s, H-16), 6.83 (1H, m, H-19), 7.07 (1H, m, H-18), 7.58 (1H, m, H-20), 11.38 (1H, , OH-9), 18.83 (1H, s, OH-3). NMR¹³With (CDCl₃, δ, M. D.): 7.41 (C-10), 27.89 (C-12), 31.96 (C-15), 58.72 (C-9b), 99.13 (C-4), at 101.30 (C-6), 100.84 (C-19), 105.13 (C-2), 105.80 (C-8), 108.19 (C-9a), 113.01 (C-18), 116.69 (C-16), 145.03 (C-20), 145.70 (C-14), 148.65 (C-17), 149.67 (C-5a), 159.21 (C-9), 165.30 (C-7), 178.78 (C-4a), 179.63 (C-13), 191.64 (C-3), 197.89 (C-1), 201.81 (C-11). Found: m/z 420.0839 [M]⁺C₂₃H₁₆O₈. Calculated: M=420.0840.

(10S,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(2-foranimation)-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 4B. [α]_D-355 (0.3; CHCl₃).

Example 4.

Activity 4A and 4B on Mycobacterium smegmatis 155 mc²and Mycobacterium tuberculosis H37Rv.

Mycobacterium smegmatis is a model test system used in the first stage of screening potential anti-tuberculosis drugs (K. E. A. Lougheed, S. A. Osborne, C. 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).

The determination of the activity of the substances was carried out using paper disks. The method consists in determining the size of the zone of inhibition of growth of strain, sown grass field and risovannoy environment, around the paper discs containing substance in various concentrations.

Name	Formula	M. smegmatis 155 mc2	M. tuberculosis H37Rv
Conc., µg/disk	Area, mm	MIC, mcg/ml
4B		4.2	7.5	5 mcg/ml MIC (100)
2.5 ág/ml MIC (90)
4A		4.2	8

Strain M. smegmatis 155 mc²sensitive to 4.2 µg/disk substances 4A and 4B.

Strain M. tuberculosis H37Rv sensitive to 5 µg/disc substance 4B.

Example 5.

Determination of the bactericidal concentration of the compound 4B against M. tuberculosis.

To study the bactericidal activity of compound 4b in relation to sensitive laboratory strain M. tuberculosis H37Rv and clinical strains of MDR MS-115 was held reseeding washed precipitation cultures, inkubirovanija connection with the 4B in concentrations which was not registered with the growth of culture. The tested concentrations 4B, starting with 25 μg/ml, had a bactericidal effect on the culture of M. tuberculosis, which was reflected in the lack of culture growth when re-sowing rainfall washed in fresh culture medium. Thus, IBC compound 4B was 25 µg/ml, and 4B has bactericidal activity against susceptible laboratory strain M. tuberculosis H37Rv and clinical strains of MDR.

Example 6.

Cytotoxic activity of the compounds on the intact mouse macrophages after 5 hours after making

Tested compounds 4A and 4B in the following concentrations (µg/ml):

- 50, 10, 5 (MIC) and 2.5 (MIC)

Control: macrophages without adding compounds represent a monolayer of well-spread fusiform cells without foreign inclusions in the cytoplasm.

Adding 4A and 4B at a concentration of 50 μg/ml were recorded beginning of the death of Matt expressed in the loss of flatness of the cells. At concentrations of 10, 5 and 2.5 μg/ml of the compounds had no pronounced effect on the macrophages, and the picture did not differ from the control.

The effect of compounds on intact MF after 5 days after application

4A and 4B moderately toxic, because during incubation with them lysis of macrophages exceeds spontaneous, in concentrations of 2.5 μg/ml, 5 μg/ml and 10 μg/ml of adding substances to the macrophages in these concentrations them lysis exceeds spontaneous approximately 20%.

Example 7.

Determination of acute toxicity of the substance 4B.

Research conducted on inbred mice male BDF_1.Determination of sub-lethal doses of a substance (LD₅₀) was carried out using the linear regression equation. The substance was ground in a mortar with 1% starch gel and was administered in different doses orally, one dose using a syringe with a special metal probe. Within 15 days was observed dynamics of the state and behavior of animals. On the basis of the obtained data was constructed a linear regression equation and determined the half-lethal dose of the substance. Dose, characterizing toxicity, were as follows:

LD₅₀=1866 (1620÷2113) mg/kg

MTD=1417 mg/kg

Thus, substances 4A, 4B have a pronounced antimycobacterial effect, which makes them promising for use as a drug.

Inhibitors of p is a hundred Mycobacterium tuberculosis, derivatives usninovoy acid, representing (10R,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(2-foranimation)-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 4A and (10S,4Z)-8,13-dihydroxy-7,10-dimethyl-4-(2-foranimation)-5,16-doxytetracycline[7.7.0.0^2.6.0^10.15]hexadeca-1,6,8,12,14-pentaen-3,11-dione 4B.