RU2554934C1 - IMINE COMPOUNDS OF CAMPHOR - EFFECTIVE INHIBITORS OF REPRODUCTION OF FLU VIRUS (A/California/07/09 (H1N1)pdm09 STRAIN) - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to the new imine compounds of camphor with the generalized formula I

displaying the properties of inhibitors of reproduction of flu virus (A/California/07/09 (H1N1) pdm09 strain). In the formula I n = 2-4, R means hydroxy, methoxy, dimethylamine group or morpholinyl. These compunds can be used as antiviral preparations for flu treatment.

EFFECT: improvement of strain properties.

1 tbl, 7 ex

Description

The invention relates to medicine and pharmaceuticals, specifically to biologically active substances, which are imino derivatives of camphor of the general formula I:

where n = 2-4, R is hydroxy, methoxy, dimethylamino group or morpholinyl,

which can be used as inhibitors of the reproduction of influenza virus (strain A / California / 07/09 (H1N1) pdm09) and can be used in medicine, virology and pharmacology.

Today, medicine has at its disposal a relatively small list of highly specific antiviral agents (chemotherapy drugs) that have received international recognition. 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. Influenza A virus causes infectious diseases of the upper respiratory tract. Every year, flu epidemics in the world lead to 3.5 million cases of serious illness and 300-500 thousand deaths. 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. New epidemic strains of influenza A virus occur every 1-2 years as a result of point mutations in two surface glycoproteins - hemagglutinin (HA) and neuraminidase (NA) [Eropkin M.Yu., Zarubaev V.V. The current state of development of new antiviral drugs against influenza and SARS // Pharmaceutical Bulletin - 2012. No. 1. - C 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 from the body's immune response, and overcome the effect of chemotherapy 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 Suppl 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 Remantadin (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 inhibiting the process of hemagglutinin cleavage and fusion of the 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 factor in the fusion of viral and cell membranes. Remantadine blocks the activity of ion channels and thereby disrupts the process of "undressing" the virus.

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 amantadine), 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. In addition to the well-known remantadine, a fairly broad class of compounds has a comparable antiviral activity. So, it is known an agent based on deutiforin (2- (1′-aminoethyl) bicyclo [2.2.1] heptane, which is one of the most interesting drugs based on natural bicyclic framework compounds - bornans [Patent RU 2448692 C2, op. 27.04.2012] and introduced into medical practice. Symmetric camphor-based diimines, separated by an aliphatic chain, have a relatively high activity (selectivity index from 40 to 89) [RF Patent 2520967].

Closest to the claimed compound (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 the expansion of a new class of effective inhibitors of reproduction of the influenza virus, which can be synthesized from available natural reagents in one stage and in high yield.

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 new compounds of the general formula I having pronounced properties of inhibitors of reproduction of the influenza virus.

where n = 2-4, R is hydroxy, methoxy, dimethylamino group or morpholinyl.

Compounds of general formula 1, after conducting in-depth pharmacological studies, can be used both in pure form and as a component of new low-toxic highly effective antiviral dosage forms.

Studies of the biological activity of compounds 1 in relation to the influenza virus (strain A / California / 07/09 (H1N1) pdm09) showed their high efficiency as inhibitors of the reproduction of this virus. Camphor, being a bicyclic terpenoid with a rigid skeleton type, available in both dextrorotatory and levorotatory enantiomers, did not show antiviral activity in the studies. The obtained quantitative indicators of inhibition confirm the high degree of suppression of the replication of influenza virus in the MDCK cell culture by compounds 1, which is 15 times or more higher than that of the reference standards amantadine and rimantadine. The use of adamantane derivatives as comparison preparations is due to the presence of hard structural fragments both in the compounds we obtained and in these preparations. The synthesis of compounds Ia, b (examples 1, 2) was carried out using a Soxhlet apparatus; at the same time, calcined molecular sieves were placed in a Soxhlet cartridge, as a desiccant, and anhydrous ZnCl _{2 was} used as a catalyst; By azeotropic distillation of the solvent, the resulting water was removed from the reaction mixture. The synthesis of compounds Ic, d, e (examples 3,4,5) was carried out without solvent, ZnCl _{2 was} also used as a catalyst; purification of the reaction mixtures was carried out by vacuum distillation and, if necessary, additionally column chromatography. The progress of reactions was monitored by sampling and analysis of the chromato-mass spectra.

The specific rotation [α] ₅₈₉ values were determined on a PolAAr 3005 spectrometer. ¹ H and ¹³ C NMR spectra were recorded on Bruker AV-400 ( ¹ H: 400.13 MHz, ¹³ C: 100.61 MHz) and AV-600 ( ¹ H: 600.30 MHz) spectrometers ¹³ C: 150.95 MHz). As internal standard the residual signal of the solvent - chloroform (δ _H 7.24, δ ppm _to 76.90). The assignment of signals in the NMR spectra was carried out using standard one-dimensional and two-dimensional experiments (COSY, HETCOR, COLOC, NMVS, HSQC). The numbering of atoms in compounds is given for assigning signals in the NMR spectra and does not coincide with the numbering of atoms in the nomenclature name. High-resolution mass spectra were recorded on a DFS ThermoScientific spectrometer in the full scan mode in the range m / z 0-500, electron impact ionization of 70 eV with direct injection of the sample. The reaction products were separated by column chromatography on silica gel (60-200 µ, Masherey-Nagel). Chromatography-mass spectra were recorded on an Agilent 7890 A gas chromatograph with an Agilent 5975C quadrupole mass spectrometer as a detector, an HP-5MS quartz column of 30,000 × 0.25 mm, and a carrier gas was helium. The specific rotation is expressed in (deg · ml) · (g · dm) ^-1 , the concentration of the solution (g) · (100 ml) ^-1 . The solvents were dried and distilled before use.

The invention is illustrated by the following examples.

Example 1

Preparation of 1,7,7-trimethylbicyclo [2.2.1] heptan-2-ylideneaminopropan-1-ol Ia

A suspension of 5 g (33 mmol) of camphor and 8 ml (100 mmol) of 3-aminopropan-1-ol in toluene was placed in a Soxhlet apparatus. An azeotropic distillation of the reaction mixture was carried out, while calcined molecular sieves were placed in a Soxhlet cartridge, as a desiccant, and ZnCl _{2 was} used as a catalyst. Then a saturated NaCl solution was added, extracted with sulfuric ether, dried over Na ₂ SO ₄ . The mass of the resulting mixture 6.5 g, was purified by vacuum distillation. Received 4.8 g of compound Ia. Bp = 109 ° C (10 mm). Yield 74%.

3 - (((1R, 4R) -1,7,7-trimethylbicyclo [2.2.1] heptan-2-ylidene) amino) propan-1-ol.

(CHCl₃, c=0.92). Найдено: m/z 209.1776 [M]⁺ C₁₃H₂₃ON. Вычислено: M=209.1774 ¹ H NMR spectrum (600 MHz, CDCl ₃ , δ, ppm, J / Hz): 0.69 (3H, s, Me-9), 0.87 (3H, s, Me-10), 0.87 (3H, s, Me -8), 1.15 (1H, dts, ² J = 12.3, J _{4n, 5n} = 9.3, J _{4n, 5n} = 4.2, H-4n), 1.28 (1H, ddd, ² J = 12.3, J _{5n, 4n} = 9.3, J _{5n, 4k} = 4.5, H-5n), 1.61 (1H, ddd, ² J = J _{5k, 4k} = 12.3, J _{5k, 4n} = 4.2, Η-5k), 1.71-1.79 (2Η, m, H-12), 1.75 (1H, d, ² J = 16.9, H-2n), 1.81 (1H, ddddd, ² J = J _{4k, 5k} = 12.3, J _{4k, 5n} = J _{4k, 3} = 4.5, J _{4k, 2k} = 3.2, H-4k), 1.91 (1H, dd, J _3.2k = J _3.4k = 4.5, H-3), 2.28 (1H, ddd, ² J = 16.9, J _{2k, 3} = 4.5, J _{2k, 4k} = 3.2, H-2k), 3.28-3.39 (2H, m, H-11), 3.78-3.85 (2H, m, H-13). NMR ¹³ C (150 MHz, CDCl _3, δ, ppm): 182.80 with (C-1), 63.98 m (C-13), with 53.56 (C-6), 51.97 m (C-11), 46.99 s (C-7), 43.65 d (C-3), 35.08 t (C-2), 31.74 t (C-12), 31.71 t (C-5), 27.20 t (C-4), 19.36 k (Me-9), 18.75 k (Me-10), 11.01 k (Me-8). $${\left[\alpha \right]}_D^{25}=35$$

(CHCl ₃ , c = 0.92). Found: m / z 209.1776 [M] ⁺ C ₁₃ H ₂₃ ON. Calculated: M = 209.1774

Example 2

Preparation of 1,7,7-trimethylbicyclo [2.2.1] heptan-2-ylideneaminoprentan-1-ol Ib

A suspension of 3 g (19.7 mmol) of camphor and 4 g (40 mmol) of 5-amino-1-pentanol in toluene was placed in a Soxhlet apparatus. Next, the reaction was carried out as described in example 1. The mass of the resulting mixture 2.9 g, was purified by column chromatography, 40 g of SiO ₂ , eluent hexane / ethyl acetate (100: 0 → 0: 100) + methanol (1%). Obtained 2.2 g of compound Ib. Yield 48%

5 - (((1R, 4R) -1,7,7-trimethylbicyclo [2.2.1] heptan-2-ylidene) amino) pentan-1-ol.

(CHCl₃, с=0.82). Найдено: m/z 237.2090 [М]⁺ С₁₅ Н₂₇ ON. Вычислено: М=237.2087. ¹ H NMR spectrum (400 MHz, CDCl ₃ , ppm, J / Hz): 0.70 (3H, s, Me-9), 0.87 (3H, s, Me-10), 0.92 (3H, s, Me -8), 1.15 (1Н, ddd, ² J = 12.3, J _{4н, 5н} = 9.3, J _{4н, 5к} = 4.2, Н-4н), 1.28 (3Н, m, Н-5н, 2Н-12), 1.51 -1.65 (5Н, m, 2Н-13, 2Н-14, Н-5к), 1.77 (1Н, d, ² J = 16.9, Н-2н), 1.76-1.84 (1Н, m, Н-4к), 1.88 (1H, dd, J _3.2k = J _3.4k = 4.5, H-3), 2.28 (1H, ddd, ² J = 16.9, J _{2k, 3} = 4.5, J _{2k, 4k} = 3.2, Η-2k ), 3.09-3.23 (2Η, m, H-11), 3.57-3.62 (2H, m, H-15). ¹³ C NMR (150 MHz, CDCl _3, δ, ppm): 182.10 s (C-1), 61.81 m (C-15), 53.34 s (C-6), 51.89 m (C-11) , 46.70 s (S-7), 43.53 d (S-3), 35.28 t (S-2), 32.12 t (S-14), 31.91 t (S-5), 29.59 t (S-12), 27.20 t (C-4), 23.50 t (C-13), 19.30 k (Me-9), 18.69 k (Me-10), 11.15 k (Me-8). $${\left[\alpha \right]}_D^{25}=21$$

(CHCl ₃ , c = 0.82). Found: m / z 237.2090 [M] ⁺ C ₁₅ H ₂₇ ON. Calculated: M = 237.2087.

Example 3

Preparation of 3-methoxy-1,7,7-trimethylbicyclo [2.2.1] heptan-2-ylidene) propan-1-amine I s

The reaction mixture containing 2 g (0.013 mol) of camphor, 1.2 ml (0.013 mol) of 3-methoxypropylamine and a catalytic amount of ZnCl ₂ was refluxed for 12 hours. The mixture was treated with a saturated solution of NaCl, extracted with diethyl ether, the mass of the resulting mixture was 2.3 g. It was purified by vacuum distillation Tkip = 105 ° C (10 mmHg), additionally purified by column chromatography on 20 g of SiO ₂ , eluent hexane / ethyl acetate (100: 0 → 0: 100) + methanol (1%). Received 1 g of compound 1 s, yield 36%.

(CHCl₃, с=0.84). Найдено: m/z 223.1934 [М]⁺ C₁₄H₂₅ON. Вычислено: М=223.1931.3-methoxy-N - ((1R, 4R) -1,7,7-trimethylbicyclo [2.2.1] heptan-2-yl-iden) propan-1-amine. ¹ H NMR (400 MHz, CDCl ₃ , δ, ppm, J / Hz): 0.70 (3H, s, Me-9), 0.87 (3H, s, Me-10), 0.91 (3H, s, Me-8), 1.14 (1H, ddd, ² J = 12.3, J _{4N, 5N} = 9.3, J _{4N, 5K} = 4.2, H-4 H), 1.28 (1H, ddd, ² J = 12.3, J _{5N, with 4N} = 9.3, J _{5n, 4k} = 4.5, H-5n), 1.60 (1H, ddd, ² J = J _{5k, 4k} = 12.3, J _{5k, 4k} = 4.2, Η-5k), 1.74-1.89 (5Η, m , H-4k, H-2n, H-12, H-3), 2.30 (1H, ddd, ² J = 16.9, J _{2k, 3} = 4.5, J _{2k, 4k} = 3.2, H-2k), 3.26 ( 3H, s, OMe-14), 3.16-3.28 (2H, m, H-11), 3.34 (2H, m, H-13). ¹³ C NMR (100 MHz, CDCl ₃ , δ, ppm): 181.78 s (C-1), 70.41 t (C-13), 58.23 k (OMe-14), 53.28 s (C-6), 48.54 t (C-11), 46.63 s (C-7), 43.65 d (C-3), 35.21 t (C-2), 32.04 t (C-5), 30.29 t (C-12), 27.30 t (C-4), 19.29 k (Me-9), 18.75 k (Me-10), 11.21 k (Me-8). $${\left[\alpha \right]}_D^{23}=21$$

(CHCl ₃ , c = 0.84). Found: m / z 223.1934 [M] ⁺ C ₁₄ H ₂₅ ON. Calculated: M = 223.1931.

Example 4

Preparation of 3-morpholine-1,7,7-trimethylbicyclo [2.2.1] heptan-2-ylidene) propan-1-amine Id

The reaction mixture, containing 2.5 g (0.016 mol) of camphor, 1.3 g (0.016 mol) of N-3-aminopropylmorpholine and a catalytic amount of ZnCl ₂ , was refluxed for 10 hours. The mixture was treated with a saturated NaCl solution, extracted with diethyl ether, the mass of the resulting mixture was 3.47 g. It was purified by vacuum distillation, bp = 145 ° C (6 mmHg), further purified by column chromatography on 20 g of SiO ₂ , eluent hexane / ethyl acetate (100: 0 → 0: 100) + methanol (1%).

Obtained 2.2 g of compound Id, yield 49%.

(CHCl₃, с=0.9). Найдено: m/z 278.2350 [М]⁺ C₁₇H₃₀ON₂. Вычислено: М-278.2353.3-morpholin-N - ((1R, 4R) -1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene) propan-1-amine. ¹ H NMR (400 MHz, CDCl ₃ , δ, ppm, J / Hz): 0.70 (3H, s, Me-9), 0.88 (3H, s, Me-10), 0.91 (3H, s, Me-8), 1.14 (1H, ddd, ² J = 12.3, J _{4n, 5n} = 9.3, J _{4n, 5k} = 4.2, H-4n), 1.29 (1H, ddd, ² J = 12.3, J _{5n, 4n} = 9.3, J _{5n, 4k} = 4.5, H-5n), 1.62 (1H, ddd, ² J = J _{5k, 4k} = 12.3, J _{5k, 4n} = 4.2, H-5k), 1.70-1.85 (3H, m , 1H-4k, 2H-12), 1.80 (1H, d, ² J = 16.9, H-2n), 1.88 (1H, dd, J _3.2k = J _3.4k = 4.5, H-3), 2.25 (3Н, m, Н-2к, 2Н-13), 2.35-2.42 (4Н, m, 2Н-14, 2Н-17), 3.13-3.27 (2Н, m, Н-11), 3.61-3.71 (4Н, m, 2H-15, 2H-16). ¹³ C NMR (100 MHz, CDCl _3, δ, ppm): 181.63 s (C-1), 66.84 m (C-15, C-16), 56.64 m (C-14, C-17), 53.91 t (C-11), 52.87 s (C-6), 49.92 t (C-13), 46.66 s (C-7), 43.68 d (C-3), 35.23 t (C-2), 32.05 t (C-5), 27.32 t (C-12), 27.21 t (C-4), 19.35 k (C-9), 18.77 k (C-10), 11.24 k (C-8). $${\left[\alpha \right]}_D^{25}=\mathrm{eighteen}$$

(CHCl ₃ , c = 0.9). Found: m / z 278.2350 [M] ⁺ C ₁₇ H ₃₀ ON ₂ . Calculated: M-278.2353.

Example 5

Preparation of Ν1, Ν1-dimethyl-N3-1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene) propane-1,3-diamine Ie

A reaction mixture containing 3 g (0.019 mole) of camphor, 2.4 g (0.023 mol) Ν, Ν-dimethylpropane-1,3-diamine and a catalytic amount of ZnCl _2, was refluxed 8 hours. The mixture was treated with a saturated solution of NaCl, extracted with diethyl ether, the mass of the resulting mixture was 3.8 g. Purified by vacuum distillation, bkp = 120-126 ° C (6 mmHg), 3.1 g of compound Ie was obtained, 68% yield.

N1, N1-dimethyl-N3 - ((1S, 4R) -1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene) propane-1,3-diamine.

¹ H NMR (600 MHz, CDCl ₃ , δ, ppm, J / Hz): 0.69 (3H, s, Me-9), 0.86 (3H, s, Me-10), 0.90 (3H, s, Me-8), 1.12 (1H, ddd, ² J = 12.3, J _{4n, 5n} = 9.3, J _{4n, 5k} = 4.2, H-4n), 1.28 (1H, ddd, ² J = 12.3, J _{5n, 4n} = 9.3, J _{5n, 4k} = 4.5, H-5n), 1.59 (1H, ddd, ² J = J _{5k, 4k} = 12.3, J _{5k, 4k} = 4.2, Η-5k), 1.66-1.74 (2H, m , H-12), 1.78 (1H, d, ² J = 16.9, H-2n), 1.78 (1H, ddddd, ² J = J _{4k, 5k} = 12.3, J _{4k, 5n} = J _{4k, 3} = 4.5, J _{4k, 2k} = 3.2, H-4k), 1.86 (1H, dd, J _3.2k = J _3.4k = 4.5, H-3), 2.15 (6H, s, Me-14 and Me-15), 2.20-2.24 (2H, m, H-13), 2.28 (1H, ddd, 2J = 16.9, J _{2k, 3} = 4.5, J _{2k, 4k} = 3.2, H-2k), 3.15 and 3.20 (both 1H, dt , ² _{J = 12.2, J 11,12 = 7.2} , H-11). ¹³ C NMR (150 MHz, CDCl ₃ , δ, ppm): 181.54 s (C-1), 57.67 t (C-13), 53.27 s (C-6), 50.18 t (C-11), 46.68 s (S-7), 45.37 k (Me-14 and Me-15), 43.66 d (S-3), 35.24 t (S-2), 32.03 t (S-5), 28.53 t (S-12 ), 27.33 t (C-4), 19.36 k (Me-9), 18.81 k (Me-10), 11.28 k (Me-8).

Example 6

The study of the toxicity of compounds 1A-e

Product toxicity has been studied for 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 compounds 1a-e, a mother solution of a concentration of 10 mg / ml in dimethyl sulfoxide was prepared, after which a series of twofold dilutions of the 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 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 (CTD50) 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 7

The study of antiviral activity of drugs

The antiviral activity of the preparations was determined on MDCK cells in 96-well cell culture plates. Compounds were dissolved in 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 fluid (strain A / California / 07/09 (H1N1) pdm09) a series of ten-fold dilutions from 10 ^-1 to 10 ^{-7 was} prepared, added to the wells with the preparations 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 largest 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 infectious doses (ID ₅₀ ). The virus-inhibiting effect of the test compounds was evaluated by the decrease in virus titer 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, halving 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 process of studying the inhibition of reproduction of influenza virus by compounds of the general formula I and reference standards (amantadine, remantadine and deuteriforin), the results are shown in table 1.

From table 1 it is seen that compounds 1A-e show a pronounced antiviral activity along with low toxicity. The chemotherapeutic index of compounds exceeds that in comparison preparations by 15 or more times. The advantage of these substances is their activity against the rimantadine-resistant strain of the influenza virus A / California / 07/09 (H1N1) pdm09, which indicates the promise of their use in the treatment of modern epidemiologically relevant viruses, the vast majority of which are resistant to remantadine.

Claims (1)

Imino derivatives of camphor of the general formula I:

,
where n = 2-4, R is hydroxy, methoxy, dimethylamino group or morpholinyl, showing the properties of inhibitors of reproduction of influenza virus (strain A / California / 07/09 (H1N1) pdm09).