RU2464033C1 - Usnic acid and its oxidated derivative as influenza virus reproduction inhibitors - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and concerns influenza virus reproduction inhibitors - the A/California/07/09 (H1N1)v strain representing (R)-usnic acid (1) and an oxidated

derivative of (S)-usnic acid (2):

.

EFFECT: inhibitors possess high inhibitory activity.

4 ex, 1 tbl

Description

The invention relates to medicine and pharmaceuticals, specifically to biologically active substances that can be used as inhibitors of the reproduction of influenza virus.

Influenza is a widespread respiratory infection worldwide. It causes annual epidemics that quickly spread from country to country, involving in severe cases (pandemics) a significant part of the human population of the globe. It is also responsible for 20,000-40000 deaths in the USA per year [Ghendon Y // World Health Statistic 1992, Q 45, P.Zob-311]. Despite the successes achieved in the field of chemotherapy, vaccine prophylaxis and influenza immunology, it remains a difficult to control infection due to high genetic variability and various long-term complications after the acute stage, leading to “hidden” or secondary mortality caused not by the influenza virus itself, but by virus-induced secondary processes [Zambon M. // J. Antimicrob. Chemother. 1999, V.44B, P.3-9].

Vaccination against influenza is an effective anti-epidemic agent, however, due to the constant change in the antigenic properties of the pathogen, constant monitoring and development of new vaccine strains corresponding to those circulating in the human population in each specific epidemic season is required.

Chemoprophylaxis and chemotherapy for influenza are used along with vaccination to prevent and treat the disease. Currently, a wide range of pathogenetic, immunomodulatory, general strengthening drugs is available for these purposes along with specific anti-influenza therapy. The latter drugs are represented by chemical compounds of two groups that differ in the mechanism of action and targets in the life cycle of the influenza virus. The drugs of the first group - rimantadine (α-methyl-1-adamantylmethylamine hydrochloride) and amantadine (1-aminoadamantane) block the influenza virus M2 protein, which plays the role of an ion channel in the viral membrane, thereby interfering with the process of hemagglutinin cleavage and fusion of the viral membrane and lysosomal [Scholtissek S., Quack G., Klenk HD, Webster RG // Antiviral Res. 1998, V.37, P.83-95]. The drugs of the second group are aimed at inhibiting viral neuraminidase, an enzyme necessary for the normal budding of viral particles and the manifestation of the infectious properties of the influenza virus. This group of compounds includes zanamivir (5- (acetylamino) -4 - [(aminoiminomethyl) amino] -2,6-anhydro-3,4,5-tridesoxy-D-glycero-D-galactonon-2-enonic acid) , oseltamivir ((3R, 4R, 5S) -ethyl-4-acetamido-5-amino-3- (pentan-3-yloxy) cyclohex-1-enecarboxylate) [Woodhead M., Lavanchy D., Johnston S., Colman P., Fleming D. // Int. J. Clin. Pract. 2000 V. 54 (9), P.604-610] and peramivir ((1S, 2S, 3S, 4R) -3 - [(1S) -1-acetamido-2-ethyl-butyl] -4- (diaminomethylidenamino) -2-hydroxy-cyclopentane-1-carboxylic acid) [O'Malley R. // Clin. Nurse Spec. 2010, V.24 (2), P.51-53]. Both groups of compounds have their drawbacks. In relation to the group of adamantane derivatives, one can note a relatively high toxicity, a narrow spectrum of action (the drugs are active against influenza A, but not against influenza B) and the rapid formation of virus resistance to drugs. Neuraminidase inhibitors are characterized by slightly lower clinical efficacy and high cost of synthesis, which makes these drugs less accessible for widespread use. All of the above indicates the need for the search and development of effective and cheap anti-influenza drugs of the broadest possible spectrum of action.

Ribavirin is another drug sometimes used in the treatment of influenza. Being an analog of nucleosides, ribavirin is effective in subtoxic concentrations, and its systemic use causes side reactions, in particular, anemia and teratogenic effect when used during pregnancy. Mostly, ribavirin in combination with interferon is used in the treatment of hepatitis, however, its effect is indicated for local (inhalation) use in the case of PC-virus and early stages of influenza infection.

However, in cases of human atypical pneumonia (SARS) and, later, bird flu subtype H5N1, ribavirin was used intravenously in the treatment of severe cases of the disease. After using ribavirin, cases of relief of the disease and complete recovery of patients were noted.

Ribavirin is a drug of a complex mechanism of action. It affects the replication of the influenza virus in two ways - directly by interfering with the polymerase processes during transcription and replication of the viral genome, and indirectly by inhibiting the cellular enzyme inosine monophosphate dehydrogenase and thereby depleting the cell pool of GTP necessary for building viral RNA. Since the main target of the drug is not a viral, but a cellular enzyme, influenza virus strains resistant to ribavirin are extremely rare or not detected at all. At the same time, significant side effects and low activity allow recommending it only in severe conditions of patients with influenza (De Clercq E. // Nat Rev Dmg Discov. 2006; 5: 1015-25; Beigel J, Bray M. // Antiviral Research 2008 ; 78: 91-102).

The objective of the invention is to expand the range of inhibitors of reproduction of influenza virus.

The problem is solved by the use of (R) -usnic acid of formula 1 and an oxidized derivative of (S) -usnic acid of formula 2:

as inhibitors of the reproduction of influenza virus.

Usnic acid is a unique and affordable domestic plant metabolite. Optically active usnic acid with opposite rotation angles and high optical purity is distinguished from lichens of various species in sufficient quantities. Both enantiomers have a range of bioactive properties. The antibacterial, insecticidal and fungicidal properties of usnic acid are most widely studied, but data on its antiviral activity are also known. Thus, commercially available (S) -usnic acid inhibits the cytopathic effect of herpes simplex virus type 1 [Perry N.B., Benn M.H., Brennan N.J., Burgess E.J., Ellis G., Galloway D.J., Lorimer S.D., Tangney R.S. // Lichenologist 1999, V.31, P.627-636]. The complex preparation Zn-usnic acid was tested against papillomavirus and showed the effect of preventing the multiplication of the virus up to 6 months after therapy [Scirpa P., Scambia G., Masciullo V., Battaglia F., Foti E., Lopez R., Villa P. , Malecore M., Mancuso S. // Minerva Ginecol. 1999, V.51, P.255-260]. (S) -Unic acid at concentrations of 1.0 µg / ml showed an inhibitory effect on Epstein-Barr virus replication, while (R) -usnic acid turned out to be less active, its effective concentration was 5.0 µg / ml [Yamamoto Y., Miura Y ., Kimoshita Y., Higuchi M., Yamada Y., Murakami A., Ohigashi H., Koshimizu K. // Chem. Pharm. Bull. 1995, V.43, P.1388-1390]. The biological activity of usnic acid and its derivatives in relation to the influenza virus is not described in the literature.

(R) -Unicovic acid 1 was isolated from lichen Cladonia stellaris, (S) -usnic acid 3 from a mixture of Usnea lichens according to the procedure [N.F. Salakhutdinov, M.P. Polovinka, M.Yu. Panchenko, Pat. RF №2317076 C1; Bull. Fig. 2008, No. 5]. Compounds 2 and 4 can be obtained by oxidizing compounds 3 and 1, respectively, with various organic peracids according to a previously proposed procedure [Sokolov, DN; Luzina, O.A .; Polovinka, MP; Korchagina, DV; Gatilov, Yu.V .; Salakhutdinov, NF Chemistry of Natural Compounds, 2010, 46, 616] according to the scheme:

Studies of the biological activity of compounds 1 to 4 , carried out in relation to the influenza virus, showed their high efficiency as inhibitors of the reproduction of this virus. As a result of studies, it was found that compounds 1 and 2 more actively affect the reproduction of the influenza virus, which is confirmed by the data below. Note that biological modifications are reversed with modifications of usnic acid isomers. (R) -Unicinic acid 1 exhibits greater antiviral activity than its (S) -enantiomer 3 , while from the pair of enantiomeric derivatives 2 and 4, the (S) -enantiomer - compound 2 has a significantly higher activity.

The obtained quantitative indicators of inhibition confirm the high degree of suppression of influenza virus replication in the MDCK cell culture with compounds 1 and 2 , which, for example, exceeds the same indicator in the reference standard, ribavirin, by 2–3 times.

The invention is illustrated by the following examples.

Example 1

Synthesis of (1aR, 4aS, 4bS, 9aS) -1a, 6-diacetyl-4,4a, 7-trihydroxy-3,4b-dimethyl-1aH-benzo [b] oxireno [2,3-h] benzofuran-2,5 (4aH, 4bH) dione 2

To a solution of compound 3 (1 mmol) in 5 ml is added 3 ml of a solution of peracetic acid (1 mmol / ml) in chloroform and left under stirring at room temperature for one day. After one day, the reaction mixture was washed with water, dried over MgSO ₄ , the solvent was removed and chromatographed on a column of silica gel, eluent chloroform. Compound 2 was isolated in 41% yield.

(1aR, 4aS, 4bS, 9aS) -1a, 6-diacetyl-4,4a, 7-trihydroxy-3,4b-dimethyl-1aH-benzo [b] oxireno [2,3-h] benzofuran-2,5 ( 4aH, 4bH) -dione 2. mp. 145 ° C (from CHCl ₃ ). [α] _D + 36.8 ° (c 0.199; CHCl ₃ ). ¹ H NMR (CDCl ₃ , δ ppm): 1.47 (3H, s, H-13), 1.78 (3H, s, H-12), 2.40 (3H, s, H-11), 2.63 (3H , s, H-15), 3.51 (1H, s, OH-4a), 5.85 (1H, s, H-8), 11.28 (1H, s, OH-4), 18.81 (1H, s, OH-7 ) ¹³ C NMR (CDCl ₃ , δ ppm): 7.70 (C-12), 24.22 (C-13), 27.95 (C-15), 28.49 (C-11), 59.88 (C-4b), 65.67 (C-1a), 75.49 (C-4a), 95.26 (C-9a), 99.21 (C-8), 106.53 (C-6), 114.95 (C-3), 162.09 (C-4), 173.97 ( C-8a), 185.81 (C-2), 191.53 (C-7), 194.46 (C-5), 198.00 (C-10), 202.15 (C-14). IR spectrum (KBr, ν, cm ^-1 ): 841, 1115, 1123, 1465, 1646, 1688, 1719, 2708, 3311. Mass spectrum, m / z (I _rel ,%): 376.1 (M ⁺ 36.27 ), 182.1 (43.78), 167.1 (100.00), 43.0 (48.46).

Example 2

Synthesis of (1aS, 4aR, 4bR, 9aR) -1a, 6-diacetyl-4,4a, 7-trihydroxy-3,4b-dimethyl-1aH-benzo [b] oxireno [2,3-h] benzofuran-2,5 (4aH, 4bH) dione 4.

To a solution of compound 1 (1 mmol) in 5 ml, 3 ml of a solution of peracetic acid (1 mmol / ml) in chloroform was added and left under stirring at room temperature for one day. After one day, the reaction mixture was washed with water, dried over MgSO ₄ , the solvent was removed and chromatographed on a column of silica gel, eluent chloroform. Compound 4 was isolated in 41% yield.

(1aS, 4aR, 4bR, 9aR) -1a, 6-diacetyl-4,4a, 7-trihydroxy-3,4b-dimethyl-1aH-benzo [b] oxireno [2,3-h] benzofuran-2,5 ( 4aH, 4bH) dione 4. The spectral data are the same as for compound 2, [α] _D -36.2 ° (c 0.199; CHCl ₃ ).

Example 3

Drug toxicity studies

The toxicity of the products has been previously studied in relation to MDCK cells. MDCK cells were seeded in 96-well plates and cultured at 37 ° C in MEM supplemented with 10% cattle serum in an atmosphere of 5% CO ₂ (in a Sanyo-175 gas flow incubator) to a monolayer state. From the studied preparations, a mother solution of a concentration of 10 mg / ml in dimethyl sulfoxide was prepared, after which a series of two dilutions of the preparations in MEM medium from 1000 to 3.75 μg / ml was prepared. Dissolved preparations were added to the wells of the plates and incubated for 2 days at 37 ° C. After this period, the cells were washed 2 times for 5 minutes with phosphate-buffered saline, and the number of living cells was estimated using microtetrazolium test (MTT). For this purpose, 100 μl of a solution (5 mg / ml) of 3- (4,5-dimethylthiazolyl-2) 2,5-diphenyltetrazolium bromide (ICN Biochemicals Inc., Aurora, Ohio) in physiological saline was added to the wells of the plates. Cells were incubated at 37 ° C in an atmosphere of 5% CO ₂ for 2 hours and washed 5 minutes with phosphate-buffered saline. The precipitate was dissolved in 100 μl per well of DMSO, after which the absorbance in the wells of the plates was measured on a Victor 1420 multifunction reader (Perkin Elmer, Finland) at a wavelength of 535 nm. According to the test results, a 50% cytotoxic dose (CTD ₅₀ ) was determined for each product, i.e. the concentration of the drug, causing the death of 50% of the cells in the culture.

Example 4

Antiviral activity of drugs

The antiviral activity of the preparations was determined on MDCK cells in 96-well cell culture plates. The compounds were dissolved in a cell support medium, introduced into the wells of the cell monolayer panels and incubated for 1 hour at 36 ° C. in an atmosphere of 5% CO ₂ .

From a virus-containing liquid (strain A / California / 07/09 (H1N1) v), a series of ten-fold dilutions from 10 ^-1 to 10 ^{-7 was} prepared, added to drug wells and incubated at 36 ° C for 48 hours in an atmosphere of 5% CO ₂ . At the end of the incubation period, an aliquot of the culture fluid was mixed with an equal volume of 1% chicken red blood cells in separate round-bottom plates. Analysis was carried out after 60 minutes of incubation at 20 ° C. For the titer of the virus in control and experiment, the reciprocal of the decimal logarithm of the highest dilution of the original virus, capable of inducing a positive hemagglutination reaction in the well, was expressed in the amount of 50% of infectious doses (ID ₅₀ ). Virus-inhibitory effect of the studied compounds was evaluated by reducing the titer of the virus in the experiment compared with the control. Based on the data obtained, a 50% inhibitory dose of ED _{50 was} calculated, i.e. the concentration of the drug, which reduces the level of viral replication by half (by 0.3 lg ID ₅₀ ), and the selectivity index, or chemotherapeutic index (SI), which is the ratio of CTD ₅₀ to ED ₅₀ .

In the process of studying the inhibition of reproduction of the influenza virus by compounds 1-4 and the reference standard (ribavirin), we obtained the following results:

Compound Formula CTD ₅₀ μg / ml ED ₅₀ μg / ml SI one

82,4 2,3 36 2

125 3.3 38 3

133 15,2 9 four

58 eleven 5.3 5 Ribavirin > 1000 6.8 > 147

Thus, compounds 1 and 2 have high biological activity, which manifests itself in the fact that they effectively inhibit the replication of influenza virus in cells, and can be used in pure form or as a basis for new highly effective dosage forms for treating influenza.

Claims (1)

The use of (R-) - usnic acid of formula 1:

and an oxidized derivative of (S) -usnic acid of formula 2:

as inhibitors of the reproduction of influenza virus (strain A / California / 07/09 (H1N1) v).