RU2607451C1 - IMINE DERIVATIVES OF CAMPHOR, CONTAINING AROMATIC OR HETEROAROMATIC FRAGMENT - INHIBITORS OF REPRODUCTION OF FLU VIRUS (STRAIN A/CALIFORNIA/07/09(H1N1)pdm09) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and pharmaceutics, namely to use of camphor imine derivatives of general formula I, where n=0, 1, 2; X=CH or N, R – hydroxy, alkoxy group, as inhibitors of reproduction of flu virus (strain A/California/07/09(H1N1)pdm09).

EFFECT: said imine compounds exhibit evident antiviral activity along with low toxicity.

1 cl, 1 tbl, 10 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 = 0, 1, 2; X-C or N; R-hydroxy, alkoxy group,

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.

Influenza is an acute infectious disease of the respiratory tract caused by an RNA-containing virus that has high epidemiological and clinical significance with a high frequency of complications among people at high risk.

Influenza viruses and other acute respiratory viral infections (SARS) cause massive outbreaks of disease, which take on an epidemic almost every year. From 27 to 41 million cases of these diseases are registered annually, in particular, from 5 to 15% of the Russian population is ill with the flu annually. Influenza and SARS remain virtually uncontrolled diseases due to the high variability of the antigenic structure of circulating influenza viruses and the heterogeneity of the SARS pathogens. In addition, influenza viruses and other acute respiratory viral infections are able to change their properties and pathogenicity. The latest example of such changes is the H1N1 influenza pathogen that circulates during the 2009–2010 epidemic season, called “swine flu.”

Antiviral agents for treating influenza are a very limited group of drugs, with drug resistance being known for most of them. Medicines for non-specific prophylaxis and therapy of influenza and other acute respiratory viral infections can be divided into two main classes.

1. Non-selective antiviral agents include interferons and inducers of endogenous interferon. Among the most famous drugs are domestic remedies Arbidol, Kagocel, Ingavirin, as well as Tiloron generics. However, the effectiveness of popular non-selective agents is not sufficiently supported by clinical data. Therefore, for the treatment and prevention of pandemic influenza, WHO recommends the use of chemotherapy drugs etiotropic action.

2. Selective antiviral agents (etiotropic effects) that have a direct direct effect on the reproduction of the virus. The reproduction of influenza virus can be inhibited at different stages of its life cycle, and this is the basis for the classification of antiviral and influenza drugs according to the mechanism of action (Eropkin M.Yu., Zarubaev V.V., 2012).

Antiviral drugs that act in the late, final stage of viral reproduction. These are inhibitors of neuraminidase, an enzyme that promotes the exit of the virus from the cell at the end of reproduction, and its inhibition leads to aggregation of viruses on the inner surface of the cell and slows down further spread (Ison M.G., 2013). Of the drugs of this class registered in Russia, these include: oseltamivir (Tamiflu) and zanamivir (Relenza). Peramivir (Rapiakta) and laninamivir (Inavir), which also act at the stage of budding newly synthesized influenza virions from the cell membrane, blocking the cleavage of particles of viral offspring from the cell surface, are registered and used in the United States. 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 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 a tool 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 - bornanes [Patent RU 2448692 C2, op. 27.04.2012] and introduced into medical practice. Symmetric camphor-based diimines separated by an aliphatic chain have relatively high activity (selectivity index 40 to 89) [Patent RF 2520967]. A derivative of camphor and aminoethanol having high antiviral activity is known [RF Patent 2530554].

Closest to the claimed compound, the prototype, is deuteriforin, 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 = 0, 1, 2; X-C or N; R-hydroxy, alkoxy group.

Compounds of the general formula 1, after conducting in-depth pharmacological studies, can be used both in pure form or as a component of new low-toxic highly effective protoviral 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 obtained and in these preparations. Acute toxicity in vivo was studied for compounds Ic, Id, If. It was shown that the LD50 of compounds Id, If exceeds 2000 mg per kg, and compounds Ic 1200 mg per kg, which confirms the low toxicity of these compounds.

The synthesis of compounds was carried out without solvent; ZnCl _{2 was} 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.

Spectral studies were performed at the Chemical Service Center for Collective Use of the SB RAS. 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 spectrometers ( ¹ H: 400.13 MHz, ¹³ C: 100.61 MHz), DRX-500 ( ¹ H: 500.13 MHz) ¹³ C: 125.76 MHz) and AV-600 ( ¹ H: 600.30 MHz, ¹³ C: 150.95 MHz). The residual signals of the solvent, chloroform (δ _H 7.24, δ _C 76.90 ppm), were used as the internal standard. 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 Thermo Scientific spectrometer in the full scan mode in the m / z range 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 μm, 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. Specific rotation is expressed in (deg⋅ml) ⋅ (g⋅dm) ^-1 , solution concentration (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-ylidene) amino-phenol Ia.

The reaction mixture containing 1 g (0.0066 mol) of camphor, 0.7 g (0.0066 mol) of 4-aminophenol was heated to 220 ° C in a stream of argon. The mixture was cooled, dissolved in chloroform, treated with saturated NaCl solution, the mass of the resulting mixture was 1.3 g. The target product was purified by column chromatography on 20 g of SiO ₂ , eluent hexane / ethyl acetate (100: 0 → 0: 100). 0.52 g of compound Ia was obtained. Yield 33%. Mp = 148-150 ° C.

=35 (CHCl₃, с=0.92). Найдено: m/z 209.1776 [М]⁺C₁₃H₂₃ON. Вычислено: М=209.1774. ¹ H NMR (600 MHz, CDCl ₃ , δ, ppm, J / Hz): 0.82 (3H, s, Me-9), 0.93 (3H, s, Me-10), 1.09 (3H, s, Me -8), 1.18-1.24 (1H, m, Н-4н), 1.45-1.52 (1H, m, Н-5н), 1.72-1.78 (1H, m, Н-5к), 1.77 (1H, d, ² J = 18.0, Н-2н), 1.81-1.88 (2Н, m, Н-4к, Н-3), 2.21 (1Н, ddd, ² J = 18.0, J _2к , ₃ = 4.8, J _{2к, 4к} = 2.9 , H-2k), 6.55 (2H, d, J _12.13 = 8.8, H-12.16), 6.62 (2H, d, J _13.12 = 8.8, H-13.15), 8.05 (1H, S.S., OH-14). ¹³ C NMR (150 MHz, CDCl ₃ , δ, ppm): 186.77 s (C-1), 152.92 s (C-14), 142.87 s (C-11), 120.82 d (C-12.16 ), 115.91 d (C-13.15), 54.19 s (C-6), 47.16 s (C-7), 43.61 d (C-3), 36.56 t (C-2), 31.93 t (C-5 ), 27.25 t (С-4), 19.46 k (С-9), 18.85 k (С-10), 11.16 k (С-8).

= 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-phenyl-N-1,7,7-trimethylbicyclo [2.2.1] heptan-2-ylidene) methanamine Ib.

The reaction mixture containing 1 g (g (0.0066 mol) of camphor, 1.4 g (0.0132 mol) of benzylamine and a catalytic amount of ZnCl ₂ was refluxed for 4 hours. The mixture was treated with saturated NaCl solution, then extracted with diethyl ether, the weight of the resulting mixture was 1.2 g. Purified by column chromatography on 20 g of SiO ₂ , eluent hexane / ethyl acetate (100: 0 → 0: 100), 1.1 g of compound Ib was isolated, yield 70%.

=-12 (CHCl₃, c=0.9). Найдено: m/z 241.1822 [M]⁺C₁₇H₂₃N. Вычислено: M=241.1825.The spectral data are consistent with those presented in the literature (Philip C. Bulman Page, Corinne Limousin, Victor L. Murrell. Asymmetric Electrophilic Animation of Various Carbon Nucleophiles with Enantiomerically Pure Chiral NH Oxaziridines Derived from Camphor and Fenchone. // J. Org. Chem. - 2002. - V. 67. - P. 7787-7796).

= -12 (CHCl ₃ , c = 0.9). Found: m / z 241.1822 [M] ⁺ C ₁₇ H ₂₃ N. Calculated: M = 241.1825.

Example 3

Preparation of 1- (4-methoxy-phenyl-N-1,7,7-trimethylbicyclo [2.2.1] heptan-2-ylidene) methanamine Ic.

The reaction mixture containing 2 g (0.013 mol) of camphor, 3 g (0.022 mol) of 3-methoxypropylamine and a catalytic amount of ZnCl ₂ was refluxed for 12 hours. The mixture was treated with saturated NaCl solution, then with water, extracted with diethyl ether, the mass of the resulting mixture 2.3. g. Purified by vacuum distillation bp = 105 ° C (10 mmHg). Received 1.9 g of compound Ic, yield 54%.

=-5.6 (CHCl₃, с=0.82). HRMS: m/z calcd. for C₁₈H₂₅ON: 271.1931. Found: 271.1928. ¹ H NMR spectrum (400 MHz, CDCl ₃ , δ, ppm, J / Hz): 0.74 (3H, s, Me-9), 0.92 (3H, s, Me-10), 1.00 (3H, s, Me -8), 1.19 (1H, ddd, ² J = 12.3, J _{4n, 5n} = 9.3, J _{4n, 5k} = 4.2, H-4n), 1.39 (1H, ddd, ² J = 12.3, J _{5n, 4n} = 9.3, J _5n , _4k = 4.5, H-5n), 1.67 (1H, ddd, ² J = J _{5k, 4k} = 12.3, J _{5k, 4n} = 4.2, H-5k), 1.80-1.91 (1H, m, H-4k), 1.88 (1H, d, ² J = 16.8, H-2n), 1.93 (1H, dd, J _3.2k = J _3.4k = 4.5, H-3), 2.38 (1H, ddd, ² J = 16.8, J _2k , ₃ = 4.5, J _{2k, 4k} = 3.2, H-2k), 3.77 (3H, s, Me-18), 4.35 and 4.41 (both 1H, d, ² J = 14.6, H -11), 6.82 (2H, d, J _14.13 = 8.8, H-14.16), 7.17 (2H, d, J _13.14 = 8.8, H-13.17). ¹³ C NMR (100 MHz, CDCl ₃ , δ, ppm): 183.06 s (C-1), 158.03 s (C-15), 132.53 s (C-12), 128.32 d (C-13, C -17), 113.54 d (S-14, S-16), 55.11 k (S-18), 54.88 t (S-11), 53.72 s (S-6), 46.95 s (S-7), 43.73 d (C-3), 35.66 t (C-2), 32.06 t (C-5), 27.33 t (C-4), 19.49 k (C-9), 18.85 k (C-10), 11.33 k (C -8).

= -5.6 (CHCl ₃ , s = 0.82). HRMS: m / z calcd. for C ₁₈ H ₂₅ ON: 271.1931. Found: 271.1928.

Example 4

Preparation of 1- (pyridin-3-yl) -N - ((1R, 4R) -1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene) methanamine 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 saturated NaCl solution, extracted with diethyl ether, the mass of the resulting mixture 3.47 g Purified by vacuum distillation bp = 145 ° C (6 mmHg). Obtained 2.2 g of compound Id, yield 49%.

=20 (CHCl₃, с=0.86). Найдено: m/z 242.1776 [М]⁺C₁₆H₂₂N₂. Вычислено: М=242.1778. ¹ H NMR (600 MHz, CDCl ₃ , δ, ppm, J / Hz): 0.71 (3H, s, Me-9), 0.90 (3H, s, Me-10), 0.97 (3H, s, Me-8), 1.17 (1H, ddd, ² J = 12.3, J _{4n, 5n} = 9.3, J _{4n, 5k} = 4.2, H-4n), 1.35 (1H, ddd, ² J = 12.3, J _{5n, 4n} = 9.3, J _{5n, 4k} = 4.5, H-5n), 1.66 (1H, ddd, ² J = J _{5k, 4k} = 12.3, J _{5k, 4n} = 4.2, H-5k), 1.83 (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, d, ² J = 16.9, H-2n), 1.93 (1H, dd, J _3.2k = J _3.4k = 4.5, N-3), 2.36 (1H, ddd, ² J = 16.9, J _{2k, 3} = 4.5, J _2k , _4k = 3.2, H-2k ), 4.36 and 4.42 (both 1Н, d, ² J = 15.4, Н-11), 7.19 (1Н, ddd, J ₁₅ , ₁₆ = 7.8, J ₁₅ , ₁₄ = 4.8, J ₁₅ , ₁₃ = 0.6, H- 15), 7.60 (1H, ddd, J ₁₆ , ₁₅ = 7.8, J _16.13 = 2.2, J _16.14 = 1.7, H-16), 8.41 (1H, dd, J _14.15 = 4.8, J ₁₄ , ₁₆ = 1.7, H-14), 8.49 (1H, dd, J _13.16 = 2.2, J _13.15 = 0.6, H-13). ¹³ C NMR (150 MHz, CDCl ₃ , δ, ppm): 184.35 s (C-1), 148.88 d (C-13), 147.66 d (C-14), 135.83 s (C-12), 134.99 d (S-16), 123.14 d (S-15), 53.91 s (S-6), 52.87 t (S-11), 47.00 s (S-7), 43.67 d (S-3), 35.75 t (S-2), 31.98 t (S-5), 27.24 t (S-4), 19.43 k (S-9), 18.76 k (S-10), 11.19 k (S-8).

= 20 (CHCl ₃ , c = 0.86). Found: m / z 242.1776 [M] ⁺ C ₁₆ H ₂₂ N ₂ . Calculated: M = 242.1778.

Example 5

Preparation of 2-phenyl-N- (1,7,7-trimethylbicyclo o [2.2.1] heptane-2-ylidene) ethanamine Ie.

The reaction mixture containing 2 g (0.013 mol) of camphor, 3 g (0.024 mol) of phenythylamine and a catalytic amount of ZnCl ₂ was refluxed for 10 hours. The mixture was treated with saturated NaCl solution, then with water, extracted with diethyl ether, the mass of the resulting mixture was 2.7 g. Purified by vacuum distillation bp = 118-120 ° C (10 mmHg). Received 2.1 g of compound Ie, yield 72%.

=29 (CHCl₃, с=0.9). Найдено: m/z 255.1986 [М]⁺C₁₈H₂₅N. Вычислено: М=255.1982. ¹ H NMR (600 MHz, CDCl ₃ , δ, ppm, J / Hz): 0.58 (3H, s, Me-9), 0.86 (3H, s, Me-10), 0.94 (3H, s, Me-8), 0.97 (1H, ddd, ² J = 12.3, J _{4n, 5n} = 9.3, J _{4n, 5k} = 4.2, H-4n), 1.25 (1H, ddd, ² J = 12.3, J _{5n, 4n} = 9.3, J _{5n, 4k} = 4.5, H-5n), 1.54 (1H, d, ² J = 16.9, H-2n), 1.58 (1H, ddd, ² J = J _5k , _4k = 12.3, J _{5k, 4n} = 4.2, H-5k), 1.74 (1H, ddddd, ² J = J _4k , _5k = 12.3, J _4k , _5n = J _{4k, 3} = 4.5, J _4k , _2k = 3.2, H-4k), 1.79 (1H, dd, J _3.2k = J _3.4k = 4.5, H-3), 2.15 (1H, ddd, ² J = 16.9, J _{2k, 3} = 4.5, J _{2k, 4k} = 3.2, H-2k ), 2.85-2.83 (2Н, m, Н-12), 3.18 and 3.25 (both 1Н, dt, ² J = 11.9, J _11,12 = 7.3, H-11), 7.13-7.16 (1Н, m, Н -16), 7.16-7.19 (2Н, m, Н-14), 7.21-7.25 (2Н, m, Н-15). ¹³ C NMR (150 MHz, CDCl ₃ , δ, ppm): 182.26 s (C-1), 140.54 s (C-13), 128.91 d (C-14), 128.02 d (C-15), 125.73 d (S-16), 53.95 t (S-11), 53.40 s (S-6), 46.66 s (S-7), 43.57 d (S-3), 36.88 t (S-12), 35.28 t (S-2), 32.04 t (S-5), 27.20 t (S-4), 19.26 k (S-9), 18.79 k (S-10), 11.35 k (S-8).

= 29 (CHCl ₃ , c = 0.9). Found: m / z 255.1986 [M] ⁺ C ₁₈ H ₂₅ N. Calculated: M = 255.1982.

Example 6

Preparation of 2 (3,4-dimethoxyphenyl) -N- (1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene) ethanamine If.

The reaction mixture containing 3 g (0.019 mol) of camphor, 1.3 g (0.016 mol) of 3,4-dimethoxyphenethylamine and a catalytic amount of ZnCl ₂ was refluxed for 7 hours. The mixture was treated with saturated NaCl solution, extracted with diethyl ether, the mass of the resulting mixture was 6.1 g. Purified by vacuum distillation bp = 165-169 ° C (10 mm Hg). Received 4.3 g of compound If, yield 73%.

¹ H NMR (500 MHz, CDCl ₃ , δ, ppm J / Hz): 0.57 (3H, s, Me-9), 0.84 (3H, s, Me-10), 0.92 (3H, s, Me- 8), 0.99 (1H, ddd, ² J = 12.3, J _{4n, 5n} = 9.3, J _{4n, 5k} = 4.2, H-4n), 1.23 (1H, ddd, ² J = 12.3, J _{5n, 4n} = 9.3 , J _{5n, 4k} = 4.5, H-5n), 1.57 (1H, d, ² J = 16.9, H-2n), 1.58 (1H, ddd, ² J = J _{5k, 4k} = 12.3, J _{5k, 4n} = 4.2, H-5k), 1.73 (1H, ddddd, ² J = J _4k , _5k = 12.3, J _{4k, 5n} = J _{4k, 3} = 4.5, J _4k , _2k = 3.2, H-4k), 1.79 (1H , dd, J _3.2k = J _3.4k = 4.5, H-3), 2.14 (1H, ddd, ² J = 16.9, J _2k , ₃ = 4.5, J _2k , _4k = 3.2, H-2k), 2.77-2.87 (2H, m, H-12), 3.38 and 3.45 (each 1H, dt, ² J = 12.0, J _11.12 = 7.2, H-11), 3.79 (3H, s, O Me -19) 3.81 (3H, s, O Me -20); 6.68-6.78 (3H, m, H-14, H-17, H-18). ¹³ C NMR (125 MHz, CDCl ₃ , δ, ppm): 182.01 s (C-1), 148.50 s (C-16), 147.15 s (C-15), 133.33 s (C-13), 120.68 d (C-18), 112.46 d (C-14), 111.15 d (C-17), 55.82 k (C-19), 55.61 k (C-20), 54.04 t (C-11), 53.33 s (C-6), 46.60 s (C-7), 43.59 d (C-3), 36.44 t (C-12), 35.26 t (C-2), 32.04 t (C-5), 27.21 t (C -4), 19.21 k (C-9), 18.74 k (C-10), 11.32 k (C-8).

Example 7

The study of the cytotoxicity of compounds Ia-f.

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 Ia-e, a mother solution of a concentration of 10 mg / ml in dimethyl sulfoxide was prepared, after which a series of double 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 min 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 h and washed 5 min 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 compound that causes the death of 50% of the cells in the culture. The results are shown in the table.

Example 8

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 h 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 h 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. The results were taken into account after 60 min 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 ). 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 reproduction (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 the table.

Example 9

Studies of the acute toxicity of compound Ic were performed on outbred mice with runoff CD-1 SPF status. Each group consisted of 5-6 mice. The test substance in doses of 2500 mg / kg, 1250 mg / kg, 625 mg / kg, 312.5 mg / kg, 156.25 mg / kg was administered in a volume of 1 ml intragastrically once in the form of a suspension, the carrier was aqueous 0.5% carboxymethyl cellulose solution. After administration, animals were monitored for 14 days. Fixed mortality and serious condition. According to the results of the study, LD ₅₀ was 1218 mg / kg (per os, mice, females).

Example 10

To evaluate the acute toxicity of the synthesized products (Id, If) in vivo, white outbred mice (weight 16-20 g) were used. In each study group, 6 animals were taken. The test compounds were dissolved in physiological phosphate buffer (pH 7.2) and administered to animals intraperitoneally once in a volume of 0.2-0.5 ml. The dose range of the compounds was 200-2000 mg / kg of animal weight. Animal mortality was recorded within 3 days after administration. Based on mortality data, a 50% lethal dose, i.e. dose causing the death of 50% of the animals in the group. It has been shown that the LD50 of Id, If compounds exceeds 2000 mg / kg.

The table shows that compounds Ia-f exhibit 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 (4)

The use of imino derivatives of camphor of the general formula I

, where n = 0, 1, 2; X = CH or N, R is hydroxy, alkoxy, as inhibitors of the reproduction of influenza virus (strain A / California / 07/09 (H1N1) pdm09).