RU2520967C1 - SYMMETRIC DIIMINES BASED ON CAMPHOR - INHIBITORS OF REPRODUCTION OF INFLUENZA VIRUS (STRAIN A/California/07/09 (H1N1)pdm09) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to new symmetrical diimines based on camphor of general formula 1a-f, which are inhibitors of influenza virus reproduction (strain A/California/07/09 (H1N1) pdm09). In the general formula 1a-f

The compounds along with pronounced antiviral activity against the said influenza virus have low toxicity.

EFFECT: chemotherapeutic index exceeds that of the known reference preparations three or more times.

1 tbl, 8 ex

Description

The invention relates to medicine and pharmaceuticals, specifically to biologically active substances, symmetric camphor-based dimines of formula 1a-f, which can be used as inhibitors of the reproduction of influenza virus (strain A / California / 07/09 (H1N1) pdm09).

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].

As practice has shown, the most promising source for the development of antiviral drugs are natural compounds [Advances in Antiviral Drug Design. Vol. 5. Ed. by E. De Clercq. Publ. by Elsever, 2007]. Among the new drugs approved for clinical use in the United States in recent decades, about half are natural substances and their chemically modified derivatives. Antiviral drugs for treating influenza are an extremely limited group of drugs, and drug resistance is 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. - 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 the body’s immune response and overcome the effects of chemotherapeutic drugs, initially aimed at a certain stage of virus reproduction. The complexity of the problem lies in the fact that each type of virus has its own mechanism of adaptation to a chemical drug [Ison M.G. Antivirals and resistance: influenza virus // Current Opinion in Virology. - 2011. - V.1. P. 563-573]. It is known that it is possible to inhibit the reproduction of influenza virus at different stages of its life cycle.

The basis of modern influenza chemotherapy is the use of several classes of compounds whose action is aimed at suppressing the replication of the influenza virus (see Fig. 1). These are neuraminidase inhibitors - Oseltamivir (Tamiflu) [Jong MD, Thanh T.T., Khanh T.N., Hien VM, Smith GJD, Chau NV, Cam BV, Qui PT, Ha DQ, Malik Peiris JS, Hien TT, Farrar J. Oseltamivir Resistance during Treatment of Influenza A (H5N1) Infection // N. Engl. J. Med. 2005. - V 25. P.2667-72.] And Zanamivir (Relenza) [Collins 1 P.J., Hairel L.F., Lin Y.P., Liu J., Russell R.J., Walker P.A., Skehel J.J., Martin S.R., Hay A.J. Gamblin S.J. Crystal structures of oseltamivir-resistant influenza virus pas-aminidase mutants // Nature 2008. - V.453 - P.1258.], They 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. 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. Ribavirin is a broad-spectrum antiviral drug with high inhibitory activity against DNA and mainly RNA viruses [Leyssen P., Clercq ED, Neyts J. The Anti-Yellow Fever Virus Activity of Ribavirin Is Independent of Error-Prone Replication // Mol. Pharmacol - 2006. - V.69. - P.1461]. Being an analog of nucleosides, ribaverine is effective in subtoxic concentrations and its systemic use causes side reactions, in particular anemia and teratogenic effect when consumed during pregnancy. In Russia, ribaverine is not used in clinical practice in the treatment of influenza.

Rimantadine (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, which plays the role of an ion channel in the viral membrane, thereby inhibiting the process of hemagglutinin cleavage and fusion of the virus 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].

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]. Closest compounds to classical adamantanes are scaffold banana inhibitors of SARS coronavirus [Tanner J.A., Zheng B.J., Zhou J., Watt R.M., Jiang J.Q., Wong K.L., Lin Y.P., Lu L.Y., He M.L., Kung H.F., Kesel A. 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, deutiforin - (2- (G-aminoethyl) bicyclo [2.2.1] heptane, is one of the most interesting drugs based on natural bicyclic framework compounds - bornans [RF Patent No. 2448692. Kiselev OI, Tandura S.N ., Deeva EG Pharmaceutical salts of aminobicyclo [2.2.1] heptanes as inhibitors of the transcription factor NF-KB with antiviral activity and their use]. It is important to emphasize that this compound is actually of natural origin, and borneol and isobornoleol are widely known in folk medicine as an anti-inflammatory medium dstva.

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

The problem is solved by new compounds of the formula la-f:

possessing the properties of inhibitors of reproduction of the influenza virus.

Studies of the biological activity of compounds 1a-f in relation to the influenza virus (strain A / California / 07/09 (H1N1) pdm09) showed their high effectiveness as inhibitors of the reproduction of this virus. As a result of studies, it was found that compounds 1a, b, and 1d most actively affect the reproduction of the influenza virus, which is confirmed by the data below (Example 8). Camphor is a bicyclic terpenoid with a rigid skeleton type, available in both dextrorotatory and levorotatory enantiomers.

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 1a-f, which is 5 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 most active are symmetric diimines having an aliphatic linker. It should be noted that the length of the aliphatic chain primarily affects the toxicity of the compounds. So, compound 1d is the least toxic, having in its core -C ₁₂ H ₂₄ a linker.

The inventive compounds are prepared as follows:

As starting compounds for the synthesis of the target substances, (+) - (1R) camphor and diamines of various structures are used. A method for the interaction of camphor with 1,2-diaminethane, which leads to the formation of diastereometrically pure diimine, the restoration of which produces a stereoselective exo-exo diamine, is described in literature [Caselli, A., Giovenzanna, G. B., Palmisano, G., Sisti , M., Pilati, T., Synthesis of C ₂ -syrnmetrical diamine based on (1R) - (+) - camphor and application to oxidative aryl coupling of naphthols // Tetrahedron: Asymmetry - 2003 - V.14 - P.1451- 1454]. As a result of the interaction of camphor with aliphatic diamines in one stage, the claimed diimins 1a-d are obtained. The reaction is carried out under conditions of azeotropic distillation of water using boron trifluoride as an etherate. The degree of conversion is controlled by sampling and analysis by GLC, HMS. Compounds le, f are prepared by reacting camphor with aromatic diamines in tetraethoxysilane, used as a dehydrating agent, as described in [Love B.E., Ren JJ Synthesis of Sterically Hidered Imines // Org. Chem. - 1993 - V.58 - P.5556-5557]. Compounds 1a-f were purified by column chromatography; all obtained substances were not previously described in the literature.

The invention is illustrated by the following examples.

Example 1

Obtaining N ¹ , N ⁶ -bis (1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene) hexane-1,6-diamine (1a)

To a solution of 3 g of (1R) - (+) camphor (19.7 mmol) in toluene (30 ml) was added 1,6-diaminohexane (9.8 mmol), 0.14 ml of BF ₃ · Et ₂ O in 5 ml of toluene. The reaction is carried out at a constant boil for 28 hours, using a Dean-Stark nozzle, water is azeotropically removed. Control over the reaction was carried out by GLC and HMS. The reaction mixture was washed with saturated NaCl, extracted with CH ₂ Cl ₂ , dried with Na ₂ SO ₄ , and the solvent was removed. Diimine 1a was purified by column chromatography, eluent hexane / ethyl acetate (100: 0 → 0: 100) + methanol (1%), isolated in 46% yield.

(CHCl₃, с 1.0). Найдено: m/z 384.3499 [М]⁺ C₂6H₄₄N₂. Вычислено: M=384.3503. ¹ H NMR spectrum (CDCl ₃ , δ, ppm): 0.70 s and 0.87 s (C ⁸ H ₃ , C ^{8 '} H ₃ and C ⁹ H ₃ , C ^9' H ₃ ), 0.91 s (C ¹⁰ N ₃ , C ^{10 '} H ₃ ), 1.13 ddd (H ⁴ⁿ , H ^4n' , ² J 12.5, J _{4n, 5n} 9.3, J _{4n, 5k} 4.1 Hz), 1.24-1.32 m (2H ¹³ , 2H ¹³ , H ⁵ⁿ , H ^5'n ), 1.50-1.57 m (2Н ¹² , 2Н ^{12 '} ), 1.60 ddd (H ^5k , H ^5k' , ² J 13.0, J _{5k, 4k} 12.2, J _5k , _4m 4.1 Hz), 1.77 d (H ²ⁿ , H ^2'n , ² J 16.8 Hz), 1.75-1.83 m (H ^4k , H ^4'k ), 1.87 dd (H ³ , H ^{3 '} , J _3.2k = J ₃ , _4k = 4.3 Hz), 2.27 ddd (H ^2k , H ^2'k , ² J 16.8, J _{2k, 3} 4.3, J _{2k, 4k} 3.2 Hz), 3.08-3.2 m (2H ¹¹ , 2H ^{11 '} ). ¹³ C NMR Spectrum (CDCl ₃ ), δ, ppm: 180.99 s (C ¹ , C ^{1 '} ), 35.211 (C ² , C ^2' ), 43.71 d (C ³ , C3 ^' ), 27.371 (C ⁴ , С ^{4 '} ), 32.09 t (С ⁵ , С ^5' ), 53.23 s (С ⁶ , С ^{6 '} ), 46.66 s (С ⁷ , С ^7' ), 19.40 k and 18.83 k (С ⁸ , С ^{8 '} and C ⁹ , C ^9' ), 11.31 k (C ¹⁰ , C ^{10 '} ), 52.191 (C ¹¹ , C ^11' ), 30.441 (C ¹² , C ^{12 '} ), 27.311 (C ¹³ , C ^13' ) $${[\alpha ]}_D^{22}=\mathrm{eighteen}$$

(CHCl ₃ , s 1.0). Found: m / z 384.3499 [M] ⁺ C ₂ 6H ₄₄ N ₂ . Calculated: M = 384.3503.

Example 2

Preparation of N ¹ , N ⁷ bis (1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene) heptane-1,7-diamine (1b).

To a solution of 3 g of (1R) - (+) camphor (19.7 mmol) in toluene (30 ml) was added 1,7-diaminoheptane (9.8 mmol), 0.14 ml of BF ₃ · Et ₂ O in 5 ml of toluene. The reaction is carried out as described in example 1, keeping the reaction mixture for 32 hours, compound (1b) is isolated in 65% yield.

(СНС1₃, c=0.66). Найдено: m/z 398.3656 [М]⁺ C₂₇H₄₆N₂. Вычислено: M=398.3653. ¹ H NMR spectrum (CDCl ₃ , δ, ppm): 0.70 s and 0.87 s (C ⁸ H ₃ , C ^{8 '} H ₃ and C ⁹ H ₃ , C ^9' H ₃ ), 0.91 s (C ¹⁰ H ₃ , C ^{10 '} H ₃ ), 1.13 m (H ⁴ⁿ , H ^4n' ), 1.18-1.32 m (2H ¹³ , 2H ¹³ , H ⁵ⁿ , H ^5'n , 2H ¹⁴ ), 1.50-1.57 m (2H ¹² , 2H ^{12 '} ), 1.60 m (H ^5k , H ^5k' ), 1.77 d (H ²ⁿ , H ^2'n ), 1.75-1.83 m (H ^4k , H ^4'k ), 1.87 m (H ³ , H ^{3 '} ), 2.27 m (H ^2k , H ^2'k ), 3.08-3.2 m (2H ¹¹ , 2H ^11' ). ¹³ C NMR Spectrum (CDCl ₃ ), δ, ppm: 180.97 s (С ¹ , С ^{1 '} ), 35.211 (С ² , С ^2' ), 43.73 d (С ³ , С ^{3 '} ), 27.381 ( C ⁴ , C ^{4 '} ), 32.101 (C ⁵ , C ^5' ), 53.23 s (C ⁶ , C ^{6 '} ), 46.67 s (C ⁷ , C ^7' ), 18.83 k (C ⁸ , C ^{8 '} ) , 19.40 k (С ⁹ , С ^{9 '} ), 11.32 k (С ¹⁰ , С ^10' ), 52.26 t (С ¹¹ , С ^{11 '} ), 30.391 (С ¹² , С ^12' ), 27.401 (С ¹³ , С ^{13 '} ), 29.281 (C ¹⁴ ). $${[\alpha ]}_D^{25}=20.7$$

(СНС1 ₃ , c = 0.66). Found: m / z 398.3656 [M] ⁺ C ₂₇ H ₄₆ N ₂ . Calculated: M = 398.3653.

Example 3

Preparation of N ¹ , N ⁸ bis (1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene) octane-18-diamine (1c).

To a solution of 3 g of (1R) - (+) camphor (19.7 mmol) in toluene (30 ml) was added 1,8-diamino octane (9.8 mmol), 0.14 ml of BF ₃ · Et ₂ O in 5 ml of toluene. The reaction is carried out as described in example 1, keeping the reaction mixture for 35 hours, isolate the compound (1c) with a yield of 67%.

(CHCl₃, c=0.92). Найдено: m/z 412.3813 [М]⁺ C₂₈H₄₈N₂. Вычислено: M=412.3812. ¹ H NMR spectrum (CDCl ₃ , δ, ppm): 0.69 s and 0.86 s (C ⁹ H ₃ , C ^{9 '} H ₃ and C ⁸ H ₃ , C ^8' H ₃ ), 0.91 s (C ¹⁰ N ₃ , C ^{10 '} H ₃ ), 1.13 dvd (H ⁴ⁿ , H ^4n' , ² J 12.3, J _{4n, 5n} 9.3, J _{4n, 5k} 4.2 Hz), 1.19-1.30 m (2H ¹³ , 2H ¹³ , 2H ¹⁴ , 2Н ¹⁴ ), 1.29 ddd (Н ^5н , Н ^{5н '} , ² J 13, J _{5н, 4н} 9.3, J _{5н, 4k} 4.4 Hz), 1.47-1.56 m (2Н ¹² , 2Н ^12' ), 1.59 ddd (H ^5k , H ^{5k ' 2} J 13.0, J _{5k, 4k} 12.2, J _{5k, 4n} 4.2 Hz),), 1.77 d (H ²ⁿ , H ^2'n , ² J 16.7 Hz), 1.74-1.82 m (H ^4k , H ^4'k ), 1.86 dd (H ³ , H ^{3 '} J _3,2k = J _3,4k = 4.4 Hz), 2.27 ddd (H ^2k , H ^2'k , ² J 16.7, J _{2k, 3} 4.4, J _{2k, 4k} 3.2 Hz), 3.12 dt (H ^11a , H ^11'a , ² J 12.1, J _11a , ₁₂ 7.3 Hz), 3.16 dt (H ^llв , Н ^11'в , ² J 12.1, J _{11в , 12} 7.3 Hz) ¹³ C NMR spectrum (CDCl ₃ ), δ, ppm: 180.00 s (С ¹ , С ^{1 '} ), 35.191 (С ² , С ^2' ), 43.71 d (С ³ , С ^{3 '),} 27.35 t (C ^4, C ^4'), 32.07 t (C ^5, C ^5), 53.23 s (C ^6, C ^{6 '),} 46.66 s (C ^7, C ^7'), 19.38 k 18.81 k (C ^8, C ^{8 ',} and C ^9, C ^9'), 11.29 k (C ^10, C ^{10 '),} 52.22 t (C ^11, C ^11'), 30.37 t (C ^12, C ^{12 ')} , 27.33 t (С ¹³ , С ^{13 '} ), 29.32 t (С ¹⁴ , С ^14' ). $${[\alpha ]}_D^{25}=-32.0$$

(CHCl ₃ , c = 0.92). Found: m / z 412.3813 [M] ⁺ C ₂₈ H ₄₈ N ₂ . Calculated: M = 412.3812.

Example 4

Preparation of N ¹ , N ¹² bis (1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene) dodecane-1,12-diamine (1d).

To a solution of 3 g of (1R) - (+) camphor (19.7 mmol) in toluene (30 ml), 1.12-diaminododecane (9.8 mmol), 0.14 ml of BF ₃ · Et ₂ O in 5 ml of toluene are added. The reaction is carried out as described in example 1, keeping the reaction mixture for 48 hours, isolated in 41% yield.

(CHCl₃, с=0.8). Найдено: m/z 468.4435 [М]⁺ C₃₂H₅₆N₂. Вычислено: M=468.4435. ¹ H NMR spectrum (CDCl ₃ , δ, ppm): 0.71 s and 0.88 s (C ⁸ H ₃ , C ^{8 '} H ₃ and C ⁹ H ₃ , C ^9' H ₃ ), 0.92 s (C ¹⁰ H ₃ , C ^{10 '} H ₃ ), 1.15 ddd (H ⁴ⁿ , H ^4n' , ² J 12.2, J _{4n, 5n} 9.3, J _{4n, 5k} 4.2 Hz), 1.18-1.28 m (2H ¹³ , 2H ^{13 '} , 2H ¹⁴ , 2H ^{14 '} 2H ¹⁵ , 2H ^15' , 2H ¹⁶ , 2H ^{16 '} ), 1.30 ddd (H ⁵ⁿ , H ^5n' , ² J 12.8, J _{5k, 4k} 9.3, J _5h , _4k 4.3 Hz), 1.49- 1.57 m (2H ¹² , 2H ^{12 '} ), 1.61 ddd (H ^5k , H ^5k' , ² J 12.8, J _{5k, 4k} 12.2, J _{5m, 4H} 4.1 Hz), 1.78 d (H ²ⁿ , H ^2'n , ² J 16.8 Hz), 1.76-1.84 m (H ^4k , H ^4'k ), 1.88 dd (H ³ , H ^{3 '} , J _3.2k = J _3.4k = 4.4 Hz), 2.28 ddd (H ^2k , H ^2'k , ² J 16.8, J _{2k, 3} 4.3, J _{2k, 4k} 3.2 Hz), 3.13 dt (H ^11a , H ^{11'a 2} J 12.1, J _{11a, 12} 7.3 Hz), 3.17 d. t (Н ^11в , H ^n'в , ² J 12.1, J _11в.12 7.3 Hz). ¹³ C NMR Spectrum (CDCl ₃ ), δ, ppm: 180.98 s (C ¹ , C ^{1 '} ), 35.23 t (C ² , C ^2' ), 43.75 d (C ³ , C ^{3 '} ), 27.401 (C ⁴ , C ^{4 '} ), 32.11 t (C ⁵ , C ^5' ), 53.25 s (C ⁶ , C ^{6 '} ), 46.68 s (C ⁷ , C ^7' ), 18.85 k (C ⁸ , C ^{8 '} ) 19.41 k (C ⁹ , C ^9' ), 11.33 k (C ¹⁰ , C ^{10 '} ), 52.261 (C ¹¹ , C ^11' ), 30.421 (C ¹² , C ^{12 '} ), 27.411 (C ¹³ , C ^{13 '} ), 29.38 t (C ¹⁴ , C ^14' ), 29.46 t (C ¹⁵ , C ^{15 '} ), 29.49 t (C ¹⁶ , C ^16' ). $${[\alpha ]}_D^{25}=-32.7$$

(CHCl ₃ , c = 0.8). Found: m / z 468.4435 [M] ⁺ C ₃₂ H ₅₆ N ₂ . Calculated: M = 468.4435.

Example 5

Preparation of N, N'-4,4'-methylenebis (N- (1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene) aniline (1e).

To 3 g (19.8 mmol) of camphor, 1.95 g (9.9 mmol) of 4,4'-methylendibenzenamine and 0.1 ml conc. H ₂ SO ₄ , 4.1 ml (19.8 mmol) of Si (OEt) ₄ , is boiled for 10 hours in an argon atmosphere. The residual alcohol that was formed from tetraethoxysilane was removed by distillation on a rotary evaporator, saturated NaHSO ₃ solution was added, and extracted with ether. The organic layer was dried with MgSO ₄ , and the solvent was removed. Next, 4 ml of 1 M KOH and 20 ml of EtOH are added to the reaction mixture. It is stirred for 20 minutes, then the silicates formed are filtered off, the organic layer is washed with saturated NaCl, extracted with ether, dried with Na ₂ SO ₄ . Sulfur ether was removed to give 3.8 g of a reaction mixture. Column chromatography on 40 g of SiO ₂ , eluent hexane / ethyl acetate (100: 0 → 0: 100) + methanol (1%) gives diimine (1e) in 41% yield.

(CHCl₃, c=0.6). Т_пл=129-130°C. Найдено: m/z 466.3343 [М]⁺ C₃₃H₄₂N₂. Вычислено: M=466.3347 ¹ H NMR spectrum (CDCl ₃ , δ, ppm): 0.83 s and 0.94 s (C ⁸ H ₃ , C ^{8 '} H ₃ and C ⁹ H ₃ , C ^9' H ₃ ), 1.06 s (C ¹⁰ N ₃ , C ^{10 '} H ₃ ), 1.17-1.25 m (H ⁴ⁿ , H ^4'n ) m 1.52-1.46 (H ⁵ⁿ , H ^5'n ), m 1.70-1.77 (H ²ⁿ , H ^2'n , H ^5k , H ^{5k '} ) m 1.81-1.88 (H ^4k , H ^4k , H ³ ), m 2.15-2.22 (H ^2k , H ^2k' ), s 3.87 (2H ¹⁷ ), m 6.63 (n ^{13.15,13 ', 15'} ), m 7.04 (H ^{12.16.16'16 '} ). ¹³ C NMR spectrum (CDCl ₃ ), δ, ppm: 184.43 s (C ¹ , C ^{1 '} ), 36.141 (C ² , C ^2' ), 43.71 d (C ³ , C ^{3 '} ), 27.341 ( C ⁴ , C ^{4 '} ), 31.971 (C ⁵ , C ^5' ), 53.81 s (C ⁶ , C ^{6 '} ), 46.99 s (C ⁷ , C ^7' ), 18.93 k (C ⁸ , C ^{8 '} ) 19.45 k (C ⁹ , C ^{9 '} ), 11.12 k (C ¹⁰ , C ^10' ), 150.01 s (C ¹¹ , C ^{11 '} ), 119.37 d (C ¹² , C ^12' ), 129.24 d (C ¹³ , C ^{13 '} ), 137.86 s (C ¹⁴ , C ^14' ), 129.24 d (C ¹⁵ , C ^{15 '} ), 119.37 d (C ¹⁶ , C ^16' ) 40.58 t (C ¹⁷ ), $${[\alpha ]}_D^{\mathrm{thirty}}=\mathrm{29th}$$

(CHCl ₃ , c = 0.6). _Mp = 129-130 ° C. Found: m / z 466.3343 [M] ⁺ C ₃₃ H ₄₂ N ₂ . Calculated: M = 466.3347

Example 6

Preparation of N- (1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidene) -4- (4- (1,7,7-trimethylbicyclo [2.2.1] heptane-2-ylidelamino) phenoxy) aniline (1f).

To 3 g (19.8 mmol) of camphor, 1.95 g (9.9 mmol) of 4,4'-oxydibenzenamine and 0.1 ml conc. H ₂ SO ₄ , 4.1 ml (19.8 mmol) of Si (OEt) ₄ , is boiled for 10 hours in an argon atmosphere. Further reaction processing is carried out as described in Example 5. Compound (1f) is isolated in 46% yield.

(CHCl₃, c=0.56). Т_плав=165С*. Найдено: m/z 468.3138 [M]⁺ $$C_{32}{H}_{40}{N}_2^{+}$$

. Вычислено: M=468.3135. ¹ H NMR spectrum (CDCl ₃ , δ, ppm): 0.83 s and 0.94 s (C ⁸ H ₃ , C ^{8 '} H ₃ and C ⁹ H ₃ , C ^9' H ₃ ), 1.07 s (C ¹⁰ N ₃ , C ^{10 '} H ₃ ), 1.19-1.26 m (H ⁴ⁿ , H ^4'n ) m 1.53-1.46 (H ⁵ⁿ , H ^5'n ), m 1.70-1.80 (H ²ⁿ , H ^2'n , H ^5k , H ^5k ) m 1.81-1.92 (H ^4k , H ^4k , H ³ ), m 2.18-2.25 (H ^2k , H ^2k ), m 6.68 (H ^{13.15.13 ', 15'} ), m 6.89 ( H, ^{12.16.12 ', 16'} ). ¹³ C NMR spectrum (CDCl ₃ ), δ, ppm: 184.94 s (C ¹ , C ^{1 '} ), 36.141 (C ² , C ^2' ), 43.70 d (C ³ , C ^{3 '} ), 27.31 t (C ⁴ , C ^{4 '} ), 31.93 t (C ⁵ , C ^5' ), 53.86 s (C ⁶ , C ^{6 '} ), 46.98 s (C ⁷ , C'), 18.89 k (C ⁸ , C ^{8 '} ), 19.43 k (С ⁹ , С ^{9 '} ), 11.10 k (С ¹⁰ , С ^10' ), 147.29 s (С ¹¹ , С ^{11 '} ), 120.53 d (С ¹² , С ^12' ), 118.98 d (С ¹³ , C ^{13 '} ), 153.36 s (C ¹⁴ , C ^14' ), 118.98 d (C ¹⁵ , C ^{15 '} ), 120.53 d (C ¹⁶ , C ^16' ). $${[\alpha ]}_D^{31}=30.3$$

(CHCl ₃ , c = 0.56). T _melt = 165c *. Found: m / z 468.3138 [M] ⁺ $$C_{32}{H}_{40}{N}_2^{+}$$

. Calculated: M = 468.3135.

Example 7

The study of the toxicity of drugs.

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. 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 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 drug, causing the death of 50% of the cells in culture. The results are shown in the table.

Example 8

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 drug wells 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. Analysis was performed after 60 min 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 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, 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 la-f compounds and reference standards (amantadine and rimantadine), the results are shown in the table.

Thus, the claimed compounds exhibit pronounced antiviral activity along with low toxicity. The chemotherapeutic index of compounds (1a-d) exceeds that of comparison preparations by three or more times. The advantage of these compounds is their activity against rimantadine-resistant strain of the influenza virus A / California / 07/09 (HlNl) pdm09, which indicates the promise of their use in the treatment of modern epidemiologically relevant viruses, the vast majority of which are resistant to rimantadine.

Table. Compound Formula CTD ₅₀ μg / ml EC ₅₀ μg / ml SI 1a

517 5.8 89 1b

> 500 9.1 55 1c

161 10.0 16 1d

1039 21.1 49 1e

> 500 164 3 1f

> 500 175 3 Amantadine 42.9 9.7 four Rimantadine 60 12 5

Claims (1)

Symmetric camphor-based diimines of formula 1a-f,

showing the properties of inhibitors of reproduction of influenza virus (strain A / California / 07/09 (H1N1) pdm09).