RU2530554C1 - Using 1,7,7-trimethylbicyclo[2,2,1]heptan-2-ylidene-aminoethanol as influenza virus reproduction inhibitor - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to use of 1,7,7-trimethylbicyclo[2,2,1]heptan-2-ylidene-aminoethanol of formula I

as an influenza virus reproduction inhibitor.

EFFECT: improved effectiveness of inhibiting replication of the influenza virus and low toxicity.

1 tbl, 3 ex

Description

The invention relates to chemistry and medicine, namely to medicines, specifically to the known compound 1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene-aminoethanol of the formula I (including its spatial isomers, including optically active forms ):

which revealed a new biological activity, which consists in inhibiting the reproduction of the influenza virus.

Influenza viruses pose a serious threat to human health due to the ease of transmission through the upper respiratory tract. The development of new drugs for the treatment and prevention of viral infections is one of the urgent tasks of modern pharmacology and medical chemistry, since globalization, population mobility and the nature of the transmission of viral infections contribute to their active spread around the world. The epidemic of bird flu H5N1 (1997-2006) and then the 2009 pandemic flu caused by the swine virus A (H1N1) pdm09 made it necessary to revise the state of the problem with the development of antiviral drugs [Kiselev O.I. Chemotherapy and flu chemotherapy. St. Petersburg, Rostock, 2012].

Antiviral agents for treating influenza are an extremely limited group of drugs, with drug resistance being known for most of them. The creation of antiviral drugs is the immediate prospect of the development of medical science in the field of the development of means for treating and preventing viral infections [M. Eropkin, V. Zarubaev The current state of development of new antiviral drugs against influenza and SARS // Pharmaceutical Bulletin, 2012, No. 1, p. 68]. Due to the features of the organization of the genome (lack of a mechanism for correcting replication errors) and the short life cycle, the influenza virus has a high mutation rate. As a result, the antigenic structure of the virus is highly susceptible to changes as a result of the selective pressure of the host’s immune system. In addition, the use of chemotherapy is perceived by the virus as a selection factor, as a result of which the formation of resistant strains also occurs. These two processes lead to the appearance of variants of viruses that can avoid both the activity of neutralizing antibodies and thereby escape the body’s immune response and overcome the effects of chemotherapeutic drugs, initially aimed at a certain stage of virus reproduction. Moreover, each type of virus has its own mechanism of adaptation to a chemical preparation [Ison M.G. Antivirals and resistance: influenza virus // Current Opinion in Virology. 2011, V. 1, P.563-573].

Inhibition of the reproduction of influenza virus can be at different stages of its life cycle, this is the basis for the classification of antiviral agents according to the mechanism of action.

Known neuraminidase inhibitors registered in Russia: Oseltamivir (Tamiflu) and Zanamivir (Relenza), as well as those used in the USA: Peramivir (Rapiakta) and Laninamivir (Inavir), which act at the stage of budding newly synthesized influenza virions from the cell membrane, blocking the removal of particles viral progeny from the surface of cells [Ison MG. Clinical use of approved influenza antivirals: therapy and prophylaxis. // Influenza Other Respi Viruses. 2013; 7 Suppi 1: 7-13]. The practice of using neuraminidase inhibitors in the treatment of influenza has shown that the high effectiveness of this group of drugs is limited by the early stage of infection.

Antiviral drugs of direct action on the replication of influenza viruses are known, for example, the drug Remantadine (α-methyl-1-adamantylmethylamine hydrochloride) and Amantadine (1-aminoadamantane) [Davies, W.L .; Grunert, R.R .; Haff, R.F .; McGahen, J.W .; Neumayer, E.M .; Paulshock, M .; Watts, J.C .; Wood, T.R .; Hermann, E.C .; Hoffmann, C.E. Antiviral Activity of 1-Adamantanamine (Amantadine) // Science, 1964, V.144, P.862]. These compounds block the influenza virus M2 protein, thereby interfering with the process of hemagglutinin cleavage and fusion of virus membranes and lysosomal vacuoles [Scholtissek C., Quack G., Klenk H. D., Webster R. G. // Antiviral Res. 1998, V.37, P.83-95]. The mechanism of action of these drugs has been studied quite fully [Cady S.D., Schmidt-Rohr K., Wang J., Soto C.S., DeGrado W.F., Hong M.H. Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers // Nature. 2010. Vol. 463. P.689-692]. The tetramer M2 protein is an ion channel that functions as a proton pump. It is known that these drugs irreversibly inhibit the M2 protein and thereby stop the flow of protons through the virion membranes, which is necessary to lower the pH, break down hemagglutinin and realize its function as a fusion factor of the viral and cell membranes. Remantadine blocks the activity of ion channels and thereby disrupts the process of "undressing" the virus.

However, the specific localization of the binding site of remantadine to the M2 protein has not been unambiguously clarified. An analysis of this complex using nuclear magnetic resonance revealed four equivalent binding sites accessible from the side of the lipid bilayer between the transmembrane regions of the M2 tetramer. Moreover, remantadine inhibits the ion channel allosterically, i.e. not directly contacting the active center, but stabilizing the closed state of M2 [Cross TA, Dong H, Sharma M, Busath DD, Zhou HX. M2 protein from influenza A: from multiple structures to biophysical and functional insights. Curr Opin Virol. 2012; 2: 128-33].

An alternative model involves direct blocking of the ionic pore by the remantadine molecule, which is coordinated by the hydrophobic adamantyl region with the amino acid of the series at the 31 position of the protein. The replacement of this amino acid traditionally leads to the development of drug resistance of influenza viruses [Cady SD, Schmidt-Rohr K, Wang J, Soto CS, Degrade WF, Hong M. Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers. Nature. 2010; 463: 689-92]. The first model, therefore, gives greater, compared with the second, freedom for the development of inhibitors of the viral M2 protein due to less severe steric restrictions.

Adamantane preparations are significantly cheaper and easier to manufacture than commercially available neuraminidase inhibitors, which makes them more affordable for the treatment and prevention of influenza in the population. However, as a result of the widespread use of adamantane preparations (remantadine and amantadip), their antiviral properties against influenza A viruses have been significantly lost. The loss of activity is mainly associated with a mutation in the transmembrane domain of the influenza virus M2 protein.

Interest in compounds acting on the M2 protein has been especially enhanced in connection with the discovery of amino spiro [5.5] undecans and spiro piperidines [Duque M.D., Torres E., Valverde E., et. al. Inhibitors of the M2 channel of influenza A virus // Recent Advances in Pharmaceutical Sciences, 2011, P.35-64]. Adamantanes as a class of frame compounds were the starting point in the construction of a large class of antiviral drugs [Zoidis G., Kolocouris N., Naesens L., Clercq E.D. Design and synthesis of 1,2-annulated adamantane piperidines with anti-influenza virus activity // Bioorganic & Medicinal Chemistry, 2009, N.17, P.1534]. In addition to the well-known remantadine, a fairly broad class of compounds has a comparable antiviral activity.

Known means based on frame compounds - bananins - replication inhibitors of SARS coronavirus, which are closest to classical adamantanes [Tanner J.A., Zheng B.J., Zhou. J., et. al. The Adamantane-Derived Bananins Are Potent Inhibitors of the Helicase Activities and Replication of SARS Coronavirus // Chemistry & Biology, 2005, V.12, P.303].

Frame compounds remain attractive as the basis for the design of antiviral drugs. So, it is known a tool based on deuteiforin (2- (1'-aminoethyl) bicyclo [2.2.1] heptane, which is one of the most interesting drugs based on natural bicyclic framework compounds - bornanes [Patent RU 2448692 C2, published on 04/27/2012] .

Known agents synthesized based on a natural pinan skeleton, which are inhibitors of the influenza virus [Zhao X, Li C, Zeng S, Hu W. Discovery of highly potent agents against influenza A virus // Eur J Med Chem. 2011; 46: 52-7], exceeding the virus-inhibiting effect of the reference drug amantadine by 240 times when tested on an amantadine-sensitive strain of the virus. However, the authors of the work do not provide data on the toxicity of the studied compounds, and therefore it is impossible to talk about a high chemotherapeutic index of the synthesized substances.

Closest to the claimed compound, the prototype is deutiforin, which is a pharmaceutical salt of 2- (1'-aminoethyl) bicyclo [2.2.1] heptane of the general formula II:

A disadvantage of the known compound is its low antiviral activity.

The objective of the invention is to identify a new effective inhibitor of reproduction of influenza virus, which can be synthesized from available reagents.

Effect: increasing the efficiency of suppressing reproduction of the influenza virus and expanding the range of inhibitors of reproduction of the influenza virus to overcome the drug resistance of modern viral strains.

The problem is solved by using the known compound, which is 1,7,7-trimethylbicyclo [2.2.1] heptane-2-yldea-aminoethanol of the formula I (including its spatial isomers, including optically active forms):

which revealed a new biological activity, consisting in the inhibitory effect on the reproduction of influenza virus.

Compound I is known and described in [Gavrilov K., Tsarev V., Zheglov S., et. al., N-Bidentate Phosphites with a Chiral Ketimine Fragment, Their Application in Enantioselective Allylic Substitution and Comparison with Phosphine Analogues // Synthesis -2007, V.11, P.1717-1723].

The proposed compound can be obtained as follows.

Camphor and aminoethanol are used as the starting material for the synthesis of the target compound I. As a result of the interaction of camphor with an amino alcohol, 1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene-aminoethanol of the formula I are obtained. The reaction is carried out under conditions of azeotropic distillation of water using anhydrous ZnCl ₂ as a catalyst.

Studies of the biological activity of compound I in relation to the influenza virus have shown the extremely high effectiveness of this substance as an inhibitor of the reproduction of this virus.

The obtained quantitative indicators of inhibition confirm the high degree of suppression of the replication of influenza virus in the MDCK cell culture by compound I, which is 120 times or more higher than that of the reference standards amantadine and remantadine. The use of adamantane derivatives as comparison preparations is due to the similarity of structures: the presence of frame structural fragments in both compound I and comparison standards.

The invention is illustrated by the following examples:

Example 1

Preparation of 1,7,7-trimethylbicyclo [2.2.1] heptan-2-ylidene-aminoethanol.

To a solution of 10 g camphor (0.065 mol) in toluene (30 ml) add 8 ml of aminoethanol (0.13 mol) in 20 ml of toluene and 0.1 g of anhydrous ZnCl ₂ . The reaction is carried out with constant boiling of the reaction mass for 28 hours in a Soxhlet apparatus filled with calcined 4A molecular sieves. Control over the reaction was carried out by GLC and HMS. The reaction mixture was washed with saturated Nad solution, extracted with CH ₂ Cl ₂ , dried with Na ₂ SO ₄ , and the solvent was removed. Compound I is purified by vacuum distillation, b.p. 112-114 ° C at 2 mm Hg, isolated in 82% yield. The NMR spectrum is the same as published previously [Gavrilov K., Tsarev V., Zheglov S., Korlyukov A., Antipin M., Davankov V. P, N-Bidentate Phosphites with a Chiral Ketimine Fragment, Their Application in Enantioselective Allylic Substitution and Comparison with Phosphine Analogues // Synthesis - 2007, - V.11, P.1717-1723]. Mp 66-67 ° C.

=30.5 (CHCl₃, с 0.82). Найдено: m/z 195.1599 [M]⁺ C₂₆H₄₄N₂. Вычислено: М=195.1603. $${[\alpha ]}_D^{22}$$

= 30.5 (CHCl ₃ , s 0.82). Found: m / z 195.1599 [M] ⁺ C ₂₆ H ₄₄ N ₂ . Calculated: M = 195.1603.

Example 2

The study of the toxicity of the claimed compound I.

Product toxicity has been studied for MDCK cells. MDCK cells were seeded in 96-well plates and cultured at 37 ° C in MEM medium supplemented with 10% cattle serum in an atmosphere of 5% CO ₂ (in a Sanyo-175 gas flow incubator) to a monolayer state. A stock solution of a concentration of 10 mg / ml in dimethyl sulfoxide was prepared from test compound I, after which a series of two dilutions of preparations in MEM medium from 1000 to 3.75 μg / ml was prepared. The dissolved preparation was 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 evaluated 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 optical density in the wells of the plates was measured using 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 compound that causes the death of 50% of the cells in the culture. The results are shown in the table.

Example 3

A study of the antiviral activity of compound I. The antiviral activity of the preparation was determined on MDCK cells in 96-well cell culture plates. The compound was 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) pdm09) a series of ten-fold dilutions of 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, 100 μl of 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, 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 taken and expressed in the amount of 50% of the 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, that is, the concentration of the drug that halves the level of viral replication (by 0.3 lg ID ₅₀ ), and the chemotherapeutic index, or selectivity index (SI), which is the ratio of CTD ₅₀ to ED ₅₀ .

In the study of the inhibition of reproduction of influenza virus by compound I and reference standards (amantadine, remantadine and deuteriforin), the results are shown in the table.

Table Compound Formula CTD ₅₀ μg / ml EC ₅₀ μg / ml SI I

447 0.7 639 Amantadine

42.9 9.7 four Remantadine

60 12 5 Datiforin

176 29th 6 Camphor

500 250 2

The table shows that compound I exhibits a pronounced antiviral activity along with low toxicity. The chemotherapeutic index of compound I exceeds that of comparison preparations by 100 times or more.

The advantage of this compound is its activity against the rimantadine-resistant strain of the influenza virus A / California / 07/09 (H1N1) pdm09, which indicates the promise of its use in the treatment of modern epidemiologically relevant viruses, the vast majority of which are resistant to remantadine.

Claims (1)

The use of 1,7,7-trimethylbicyclo [2.2.1] heptan-2-ylidene-aminoethanol of the formula I:

as an inhibitor of the reproduction of influenza virus.