RU2424800C1 - N-{3,5-dioxo-4-azatetracyclo[5.3.2.02,6.08,10]dodec-11-en-4-yl}-4-hydroxybenzamide, antiviral medication, inhibiting replication of various species of orthopoxviruses - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of organic chemistry and pharmaceutic industry and deals with novel compound, namely N-{3,5-dioxo-4-azatetracyclo[5.3.2.0^2,6.0^8,10]dodec-11-en-4-yl}-4-hydroxybenzamide of formula

demonstrating high antiviral activity with respect to various species of orthopoxviruses and can be applied in medicine and veterinary for treatment of wide spectrum of orthopoxviral infections.

EFFECT: invention ensures high treatment efficiency.

2 tbl, 3 ex

Description

The invention relates to the field of pharmaceutical industry and relates to a new biologically active chemical compound of the formula I:

possessing high antiviral activity, which will make it possible to use it in medicine and veterinary medicine for the treatment and prevention of diseases caused by viruses pathogenic for humans and animals, in particular orthopoxviruses.

The most dangerous for the human population due to the extremely high pathogenicity, diversity and epidemiological indicators are viruses of the genus Orthopoxviruses (family Poxviridae, genus Orthopoxvirus). In recent years, the problem of smallpox has become unexpectedly acute and urgent: since the population has not been vaccinated since 1980, a significant layer of the population has appeared without immunity or with weakened immunity against smallpox. There are no guarantees for the complete disappearance of the viruses that cause this disease, and there is also the possibility of unauthorized leakage of viral material from its official storage and, possibly, unofficial storage. Moreover, in the context of the constant growth of categories of the population with immunodeficiency conditions, the very idea of vaccination with the smallpox vaccine virus becomes problematic, and the solution of the problem of post-vaccination complications, as well as outbreaks of diseases caused by other orthopoxviruses pathogenic for humans, with the help of highly effective chemotherapeutic drugs is of great importance. The pathogenicity of monkeypox viruses and cow smallpox viruses of the same genus Orthopoxvirus and capable of causing fatal disease in humans is well known. So, recently in Africa, especially in areas with a high percentage of HIV-infected people, there have been outbreaks of monkeypox (mortality reaches 10-20%). The wide spread of monkeypox virus through meadow dogs in the United States in 2003 also shows the completely unusual epidemic potential of orthopoxviruses. In addition, there is a real possibility of selection of new variants of orthopoxviruses in natural conditions, as well as the creation of new genetic variants of orthopoxviruses with unusual properties. This probability has been demonstrated in the laboratory for a low-virulent strain of mouse pox virus, which, after genetic manipulation, has become pathogenic for these animals.

Currently, there are virtually no effective means of specific prophylaxis and treatment of diseases caused by viruses of the genus orthopoxviruses, therefore, the search and creation of highly effective antiviral drugs with high activity, low toxicity and long-term action against orthopoxviruses is an important task to ensure the safety of human health and life.

The drug metisazon is also well known (also available under the name Kemoviran; Marboran; Mariron) [Quenelle BC, Keith KA, Kern ER // Antiviral Res. 2006; 71 (1): 24-30], which has a low prophylactic activity, against variola virus. Also known is cidofovir (Vistide ^® ), which is currently undergoing research as a preventive or therapeutic drug, but has not yet been completed [Jesus DM, Costa LT, Goncalves DL, Achete CA, Attias M, Moussatché N, Damaso CR // J Virol. 2009; 83 (22): 11477-11490].

There are currently practically no drugs on the pharmaceutical market that are active against smallpox viruses; in this regard, the development of such substances can be an important step in ensuring the pharmacological safety of people's lives.

Adamantane derivatives are known to be potential sources of substances with antiviral properties. In particular, vaccinia virus inhibitors were identified in the series of adamantoyl-1-hydrazines [Guzhova SV, Danilenko GI, Korobchenko LV, Denisova LV, Andreeva OT, Boreko E.I. , Danilenko V.F., Baklan V.F. Inhibitors of the vaccine virus in the series of adamantoyl-1-hydrazines // Fiziol. active in-va. Kiev: Naukova Dumka. 1986, issue 18. P.24]. The isomeric 1- (aminobenzoyl) aminoadamantanes, along with the action on various types of viruses, exhibit moderate activity against the vaccinia virus [Votyakov V.I., Andreeva O.T., Kitsara M.S. et al. // In: Physiologically Active Substances. Kiev. 1978. Issue 10. P.88]. Published evidence of a marked inhibitory effect on the propagation of vaccinia viruses of substituted aminoacetyladamantylamines, aminoalkoxyacetyladamantylamines and thromantadine [May G., Peteri D. // Arzneimitt. -Forsch. 1973. Bd. 23. No. 5. S.718]. A wide range of hydroxy-, nitrogen-containing and sulfur-containing functional derivatives of the adamantane series showed a weak and moderate degree of activity against the vaccinia virus [Klimochkin Yu.N., Leonova MV, Korzhev IR, Moiseev IK, Vladyko G.V. ., Korobchenko L.V., Boreko E.I., Nikolaeva S.N. // Chem.-farm. Journal. 1992.No.7-8. S.58; Klimochkin Yu.N., Moiseev I.K., Vladyko G.V., Korobchenko L.V., Boreko E.I. // Chem.-farm. Journal. 1991.V.25. No. 7. P.46; Klimochkin Yu.N., Moiseev I.K., Abramov O.V., Vladyko G.V., Korobchenko L.V., Boreko E.I. // Chem.-farm. g. 1991.V.25. No. 7. P.49].

The disadvantage of the above analogues is the low activity of the compounds against the vaccinia virus.

Among the adamantane derivatives, the biologically active compound 1-adamantanoylhydrazone α-azidomethyl (1-adamantyl) ketone is known to have antiviral activity against the vaccinia virus [SU 1568481, IPC C07D 253/06, publ. 2006]. The specified compound has not been studied for antiviral activity against cow pox virus, monkeypox virus and others. The disadvantages of this compound include insufficiently high efficacy against vaccinia virus.

Known 3-phenyl-6-R ₁ -7-R ₂ -1,2,4-benzotriazines (compound SK-29 and its derivatives) having antiviral activity, in particular against orthopoxviruses pathogenic for humans [RU 2252218, IPC C07D 253/10, A61K 31/53, publ. 05/20/2005]. These chemical compounds have insufficient antiviral activity and a low selectivity index for orthopoxviruses. The low antiviral activity of these compounds stimulated the search for new drugs.

The closest analogue (prototype) is a chemical compound - 4-trifluoromethyl-N- (3,3a, 4,4a, 5,5a, 6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop

[f] isoindole-2 (1H) -yl) benzamide (ST-246), which has antiviral activity against orthopoxviruses with a high selectivity index [US 20060235051, IPC A61K 31/4439, C07D 403/02, published October 19, 2006 ; Bailey T.R., Rippin S.R., Opsitnick E., et al, // J. Med Chem. 2007; 50 (7): 1442-1444; Yang G., Pevear D.C., Davies M.H., et al, J. Virol. 2005; 79 (20): 13139-13149].

The study of the prototype showed that drug-resistant ST-246 orthopoxviruses are extremely easy to produce. This is one of the significant disadvantages of the prototype. Another important circumstance is that the experience of using various antiviral agents shows: (a) the need to use a combination of several drugs to increase the effectiveness of the treatment of viral infection; (b) the ease of the emergence of new variants of viruses that are resistant to the action of a single drug. This led to the fact that for the treatment of a number of viral diseases, for example HIV infection, 3-5 different drugs are used simultaneously. Moreover, the consistent use of several such mixtures is recommended to achieve the maximum therapeutic effect and to prevent the emergence of new drug-resistant variants of pathogenic viruses. It is possible to overcome this shortcoming only by creating new highly effective antiviral drugs - analogues for the complex treatment of orthopoxvirus infections with several antiviral drugs.

The objective of the invention is the creation of a new chemical compound having a simple production technology with a high selectivity index and antiviral effect against various types of orthopoxviruses pathogenic for humans, domestic and laboratory animals.

The problem is solved by a new biologically active organic compound - N- {3,5-dioxo-4-azatetracyclo [5.3.2.0 ^2.6 .0 ^8.10 ] dodec-11-en-4-yl} -hydroxybenzamide, of the formula I:

exhibiting antiviral activity against various types of orthopoxviruses, which can be used in medical practice as a drug for the treatment of orthopoxvirus infections in humans, laboratory and domestic animals.

Compound I is prepared by reacting n-hydroxybenzoic acid hydrazide with 4.4a, 5.5a, 6,6a-hexahydro-4,6-etheno-1H-cycloprop [f] isobenzofuran-1,3 (3aH) -dione in acetic acid at boiling for 10 hours according to the following scheme:

The invention is illustrated by the following examples.

Example 1. Obtaining N- {3,5-dioxo-4-azatetracyclo [5.3.2.0 ^2.6 .0 ^8.10 ] dodec-11-en-4-yl} -4-hydroxybenzamide. A solution of 1.9 g (10 mmol) of 4.4a, 5.5a, 6,6a-hexahydro-4,6-etheno-1H-cycloprop [f] isobenzo-furan-1,3 (3aH) -dione and 1.52 g (10 mmol) of para-hydroxybenzoic acid hydrazide is boiled in 30 ml of acetic acid for 10 hours. The solvent is distilled off in a vacuum of a water-jet pump. 50 ml of water are added to the residue. The precipitate formed is filtered off, washed on the filter with water (3 times 15 ml), dried in air to a constant weight. 2.91 g (85%) of the substance are obtained in the form of white crystals; so pl. 219 ° C. ¹ H NMR spectrum (400 MHz, J / Hz, CDCl ₃ ), δ, ppm: 0.05 (br s, 1H); 0.26 (dd, J = 5.8, 7.0, 1H); 1.17 (m, 2H); 3.20 (br.s, 2H); 3.28 (m, 2H); 5.75 (dd, J = 3.9, 5.1, 2H); 6.85 (d, J = 8.7, 2H); 7.75 (d, J = 8.7, 2H); 10.28 (s, 1H); 10.54 (s, 1H); 10.80 (s, 1H). ¹³ C NMR spectrum (400 MHz, CDCl ₃ ), δ, ppm: 5.14, 10.23, 33.72, 34.03, 43.91, 44.36, 116.16, 122.48, 128.25, 128.55, 130.80, 130.90, 162.14. 162.30, 164.64, 165.30, 176.16. IR spectrum (KBr), cm ^-1 : 597, 736, 849, 1174, 1194, 1218, 1236, 1283, 1497, 1593, 1610, 1668, 1715, 1781, 2961, 3013, 3353, 3536. Calculated,% : C, 63.15; H, 5.30; N, 8.18. C ₁₈ H ₁₆ N ₂ O · H ₂ O. Found,%: C, 63.79, 63.50; H, 5.35, 5.32; N, 8.38, 8.27.

The proposed compound I differs from those described in the prototype [US 20060235051, IPC A61K 31/4439, C07D 403/02, published October 19, 2006; Bailey T.R., Rippin S.R., Opsitnick E., et al, // J. Med Chem. 2007; 50 (7): 1442-1444; Yang G., Pevear D.C., Davies M.H., et al, J. Virol. 2005; 79 (20): 13139-13149] in that, being polycyclic, it contains a triply acylated hydrazine molecule in the molecule, one of the nitrogen atoms of which is part of a heterocyclic isoindole structure, and the other is bound to an n-hydroxy-substituted benzoyl residue.

The method of obtaining the claimed substance is simple to perform, the starting materials are available, the yield is 85% of theoretically calculated. The proposed compound is stable in air, readily soluble in chloroform, dimethylformamide and dimethyl sulfoxide, slightly soluble in aliphatic alcohols, water, practically insoluble in ether, hexane.

Example 2. The study of the antiviral activity of the claimed compounds.

The following methodology was used to evaluate the antiviral activity of the compounds. A Vero cell culture was grown in the wells of flat-bottomed 96-well plates. Serial dilutions of the test compounds and the corresponding virus were added to the culture medium. After incubation for 3-5 days, the cell monolayer was stained with a neutral red vital dye, after removing the dye and washing the excess dye, a lysis solution was added and the amount of dye included in the cell monolayer was taken into account on a spectrophotometer at a wavelength of 490 nm. As controls, we used the wells of the plate into which the virus was not introduced (control of toxicity of compounds), into which the virus was added without compounds (virus control), and the wells into which neither virus nor compounds were added (control of cell culture). This technique is based on the ability of the tested compounds to prevent the reproduction of the virus and its spread from cell to cell, and therefore the cells do not die and retain the ability to phagocytize neutral red. The results were analyzed using the SoftMax program (version 4.0, Molecular Devices, Memo Park, USA).

As the studied viruses, a cow-smallpox virus (Grishak strain) and smallpox vaccine virus, a strain of LIVP used to vaccinate the population, were pathogenic for humans. In addition to the orthopoxviruses pathogenic for humans, the ectromelia virus (mouse pox) and strain K-1 were used to evaluate antiviral activity.

Analysis of the obtained data clearly shows that the claimed compound (I) and the prototype compound (ST-246) have a high antiviral effect, in particular against orthopoxviruses pathogenic for humans.

As can be seen from the data in table 1, the studied compounds (the claimed compound I and prototype ST-246) effectively inhibit the reproduction of orthopoxviruses in cell culture: they inhibit the reproduction of three types of orthopoxviruses at concentrations of 0.005-0.224 and 0.002-0.003 μg / ml, respectively, whose values are significantly superior to the analogue drug [RU 2252218, IPC C07D 253/10, A61K 31/53, publ. 05/20/2005] based on derivatives of benzotriazines, the active concentration of which is 0.49-2.23 μg / ml.

Table 1. Comparison of the activity of the claimed compound (I), ST-246 (prototype) and SK-29 (analogue) A drug Toxicity for Vero cell culture (IC ₅₀ , μg / ml) Inhibitory activity (IC ₅₀ , μg / ml) against viruses Smallpox Virus Virus Selectivity Index Vaccine vaccines Smallpox cows Mouse pox The inventive compound (I) > 100 0.005 0.224 0.144 > 20,000 ST-246, prototype > 100 0.003 0.002 0.003 > 30000 SK-29 analog > 300 0.5 2.23 0.49 134 to 600

Thus, the claimed compound N- {3,5-dioxo-4-azatetracyclo [5.3.2.0 ^2,6 .0 ^8,10 ] dodec-11-en-4-yl} -4-hydroxybenzamide of the formula I exhibits high antiviral activity in relation to various types of orthopoxviruses.

Example 3. Evaluation of the antiviral activity of the claimed compound (I) against viruses: human adenovirus of the 5th serotype, echovirus of the 12th type, West Nile virus and the herpes simplex virus of the 2nd type.

Evaluation of the antiviral activity of the studied compounds was carried out on cell cultures AD293 (human adenovirus of the 5th serotype and echovirus of the 12th type) and Vero (West Nile virus and herpes simplex virus of the 2nd type) in 96-well plates. IC _{50 of} compounds in relation to the above viruses was determined by recording the inhibition of the virus-induced cytopathic effect in the presence of various concentrations of the studied drugs (final concentrations in the wells of the microplate with a cell monolayer of 80, 40, 20, 10 and 5 μg / ml). After the test compounds were added to the cells at the above concentrations, cells were infected with various viruses. For this, infectious doses of each virus were used, causing a 90% cytopathic effect three days after infection. After the cells were incubated in tablets for 72 hours at 37 ° C in an atmosphere containing 5% CO ₂ , the cells were fixed with a 5% glutaraldehyde solution. The cytopathic effect was visualized by staining the cells with 0.1% crystal violet solution and was recorded spectrophotometrically at 570 nm. IC _{50 was} calculated according to G. Yang et al., 2005.

The results presented in table 2 showed that compound (I) does not actually inhibit the replication of type 12 echovirus, human serotype 5 adenovirus, West Nile virus, and type 2 herpes simplex virus. Compound (I) was used in subtoxic concentrations for this type of cell, which also further confirms its non-toxicity for AD293 cells. The lack of activity of compound (I) against two types of DNA-containing viruses and two types of RNA-containing viruses suggests that the mechanism of action of compound (I) is not related to the type of nucleic acid.

Table 2. Antiviral activity of compound (I) in vitro A drug Inhibitory activity (IC ₅₀ , μg / ml) against viruses 5th Serotype Human Adenovirus Echovirus type 12 {pcs. Lev4) West Nile Virus (pc. Eg101) Herpes simplex virus type 2 (pcs. MS) Compound (I) > 80.0 > 80.0 > 80.0 > 80.0 ST-246, prototype > 40.0 > 40.0 > 80.0 > 80.0

For the claimed compound (I), the IC ₅₀ for the vaccinia virus is 0.005 μg / ml (Table 1), which shows the high specificity of the action of the compound (I) with respect to orthopoxviruses and indicates a high degree of selectivity of the claimed compound for this type of virus. The selectivity index was at least 20,000 for the vaccinia virus (example 3).

Thus, the claimed compound (I) with low toxicity to mammalian cells still has a pronounced selectivity for three types of orthopoxviruses and does not inhibit the replication of other RNA and DNA viruses.

Claims (1)

N- {3,5-Dioxo-4-azetetracyclo [5.3.2.0 ^2.6 .0 ^8.10 ] dodec-11-en-4-yl} -4-hydroxybenzamide of the formula I:

exhibiting antiviral activity against various types of orthopoxviruses pathogenic for humans and animals.