RU2487876C1 - Agent showing antiviral activity on dna-viruses - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: agent refers to an agent representing one of N-substituted 1,4-diazabicyclo[2.2.2.]octane derivatives of general formula (I) (compounds 1-3),

wherein: wherein R=1,4-substituted but-2-ine for (1), 1,5-substituted pentane for (2), 1,4-dimethylphenyl for (3) showing antiviral activity on DNA-viruses.

EFFECT: agent can find application in medicine as an active ingredient for developing therapeutic dosage forms used for treating viral diseases.

1 cl, 7 dwg, 3 tbl, 7 ex

Description

The invention relates to the field of chemistry and biomedicine, and in particular to agents exhibiting antiviral activity against DNA viruses.

Viral diseases pose a common threat to public health worldwide. Viruses containing the genome in the form of DNA cause a number of serious animal diseases (domestic and wild) and are among the most dangerous for humans. DNA viruses have the ability to transform the cells that they infect. So, papillomaviruses are the etiological factor of cervical tumors [Adams, M. et. al., Vaccine, 2007, 25 (16), 3007-3013], hepatitis B virus is associated with liver tumors [Chu, CM., J Gastroenterol Hepatol., 2000, E25-30], Epstein-Barr virus is associated with cancer nasopharynx and Burkitt’s lymphoma [Moormann, AM. et al., Curr Opin Infect Dis., 2011, 24 (5), 435-41], and herpes simplex virus type 8 is associated with Kaposi’s sarcoma [Cai, Q et al., Adv Virus Res. 2010, 78, 87-142].

The danger of diseases caused by DNA viruses makes the development of new antiviral drugs and new tools and methods for their inactivation one of the most urgent tasks of today.

At present, physical methods (exposure to ultraviolet light, ionizing radiation, heating under elevated pressure) and a number of chemical compounds (formalin, secondary ethyleneimine, a number of detergents) are used as means of virus inactivation, which differ in the mechanism of action and, therefore, the scope of application, toxicity, virus inactivation efficiency and cost (De Benedictis, P. et al. Zoonoses Public Health. 2007, 54 (2), 51-68.).

The spectrum of compounds that allow inactivation of the virus, including for the preparation of whole-virion vaccines, is limited to several compounds: formaldehyde, binary ethyleneimine (Hulskotte EGJ, Vaccine, 1997, 15, 1839-1845), caprylate (Korneyeva M et.al., Biologicals , 2002, 30, 153-162), oxidized polyamines (Bachrach U., Amino Acids, 2007, 33 (2), 267-272), irradiation with long-wave UV light in the presence of compounds that increase the photosensitivity of the virus (Hanson CV, et. al., J. gen. Virol., 1978, 40, 345-358; Specht, KG Photochem Photobiol. 1994, 59, 506-514). It is known that treatment of the virus with such compounds leads to a partial destruction of the viral antigenic determinants, and thereby to a decrease in the immunogenic properties of vaccines, and selective destruction by the formalin of the antigenic determinants of the surface virus glycoproteins can induce the development of an unbalanced immune response (Kasermann F., et al., Antiviral Res., 2001, 52, 33-41).

The closest to the claimed tool prototype is a tool that is a derivative of 1,4-diazabicyclo [2.2.2.] Octane of the General formula Dxn, with high ribonuclease activity [N. Koval'ov, et al., Nucleosides, Nucleotides and Nucleic Acids. 2004, 32, 981-985.] Figure 1 shows the structural formula of the compounds Dxn, where n = 1, 4, 6 or 12, x is the position in the benzene ring - ortho, meta or steam. However, in the available literature there is no data on the antiviral activity of these compounds against DNA viruses.

The objective of the invention is to obtain an effective, low toxicity agent with antiviral activity against DNA viruses.

The problem is solved by using the known derivatives of N-substituted 1,4-diazabicyclo [2.2.2.] Octane of the general formula (I):

where R = 1,4-substituted but-2-in for (1), 1,5-substituted pentane for (2), 1,4-dimethylphenyl for (3), which revealed a new biological activity, which consists in antiviral effect against DNA viruses.

The figure 2 presents the structural formulas of the compounds (1), (2), (3).

Known compounds (1-3), of general formula (I) were prepared according to the general scheme shown in Figure 3. The process for preparing the claimed known compounds involves the reaction of commercially available 1,4-diazabicyclo [2.2.2] octane (D) with dodecyl chloride to form monosubstituted 1,4-diazabicyclo- [2.2.2] octane (DR) as described in (Katritzky AR; Rao MSCJ Heterocyclic Chem. 1991, 28, 1115), which further reacts with commercially available dihalogen derivatives of various hydrocarbons. As a result of the reaction in high yields, the product [D (R)] ₂ L is formed containing two N-substituted residues of 1,4-diazabicyclo [2.2.2] octane linked by various linkers (butyn-2 in case (1), pentane - in case (2), benzene - in case (3)).

A comparative analysis of the claimed known compounds with widely used compounds, such as formalin, UV light or monomeric or binary ethyleneimine (Hulskotte E.G. J., Vaccine, 1997, 15, 1839-1845), showed that the proposed compounds have the following advantages:

1) The inventive known compounds have antiviral activity against DNA viruses, which allows us to consider them as promising disinfecting agents for use in veterinary medicine and healthcare.

2) The claimed known compounds are low toxic to humans and do not require special precautions (gas mask, gloves, work under the hood) when working with them. In addition, these compounds are storage stable in the form of concentrated aqueous solutions or solutions in aprotic solvents such as dimethyl sulfoxide.

3) The claimed compounds have membrane -olytic activity, which allows us to consider them as promising agents against DNA viruses. By “membrane -olytic” activity is meant “the ability of a compound to disintegrate lipid membranes, including the shells of virions.”

A search by the sources of scientific, technical and patent literature showed that the antiviral activity against DNA viruses for derivatives of N-substituted 1,4-diazabicyclo [2.2.2.] Octane of the general formula [D (R)] ₂ L in known sources not described.

The invention is illustrated by the following examples.

Example 1. Obtaining 1,4-bis- (4-dodecyl-1,4-diazoniabicyclo [2.2.2] octyl-1-methyl) but-2-in tetrachloride (1)

The synthesis scheme of compound (1) is shown in Figure 4. To a solution of 1-dodecyl-4-aza-1-azoniabicyclo [2.2.2] octane chloride (4) (349 mg, 1.1 mmol) in 20 ml of acetonitrile was added dropwise solution 1 , 4-dichlorobut-2-in (5) (61.5 mg, 0.5 mmol) in 5 ml of acetonitrile. After stirring for 24 hours, the precipitate formed was filtered off, washed with acetonitrile (3 × 15 ml) and dried in vacuo. The product is a hygroscopic yellow crystalline powder. The mass of the obtained compound (1) 296 mg (yield 78%).

¹ H NMR spectrum, δ, ppm (J, Hz): 0.89 (br t, 6H, C H ₃ (CH ₂ ) ₁₁ N ⁺ , J 6.6), 1.27 (m, 36 H, CH ₃ (C H ₂ ) ₉ CH ₂ CH ₂ N ⁺ ) ; 1.73 (m, 4H, CH ₃ (CH ₂ ) ₉ C H ₂ CH ₂ N ⁺ ); 3.55 (m, 4H, CH ₃ (CH ₂ ) ₁₀ C H ₂ N ⁺ ); 4.15 (m, 24H, ⁺ N (C H ₂ C H ₂ ) ₃ N ⁺ ); 5.09 (s, 4H, CHC H ₂ N ⁺ ). ¹³ C NMR spectrum, δ, ppm: 13.73 ( C H ₃ (CH ₂ ) ₁₁ N ⁺ ); 21.27 (CH ₃ (CH ₂ ) ₉ C H ₂ CH ₂ N ⁺ ); 21.88 (CH ₃ C H ₂ (CH ₂ ) ₁₀ N ⁺ ); 25.43, 28.27, 28.50, 28.56, 28.74, 28.82, 31.10 (CH ₃ (CH ₂ ) ₂ ( C H ₂ ) ₈ CH ₂ N ⁺ ); 53.22 (CH ₃ (CH ₂ ) ₁₀ C H ₂ N ⁺ ); 50.31 and 50.97 ( ⁺ N ( C H ₂ C H ₂ ) ₃ N); 63.62 (C C H ₂ N ⁺ ); 80.16 ( C CH ₂ N ⁺ ).

IR spectrum: 850.7, 1061, 1113, 1173, 1468, 1636, 2058 (small), 2853, 2923, 3417.

Example 2. Obtaining 1,5-bis- (4-dodecyl-1,4-diazoniabicyclo [2.2.2] octan-1-yl) pentane dichromide dibromide (2)

The synthesis scheme of compound (2) is shown in Figure 5. To a solution of 1-dodecyl-4-aza-1-azoniabicyclo [2.2.2] octane chloride (4) (349 mg, 1.1 mmol) in 10 ml of dimethylformamide was added dropwise 1, 5-dibromide pentane (6) (115 mg, 0.5 mmol). After stirring for 24 hours at 80 ° C, the precipitated precipitate of compound (2) was filtered off, washed with acetonitrile (3 times 15 ml) and dried in vacuum. A hygroscopic fine crystalline substance is obtained. The mass of the obtained compound (2) 247 mg (yield 57%).

¹ H NMR spectrum, δ, ppm (J, Hz): 0.93 (br t, 6H, C H ₃ (CH ₂ ) ₁₁ N ⁺ , J 6.6), 1.32-1.45 (m, 36 H, CH ₃ (C H ₂ ) ₉ CH ₂ CH ₂ N ⁺ ); 1.32 (m, 2H, ⁺ NCH ₂ CH ₂ C H ₂ CH ₂ CH ₂ N ⁺ ); 1.88 (m, 4H, CH ₃ (CH ₂ ) ₉ C H ₂ CH ₂ N ⁺ ); 1.98 (m, 4H, ⁺ NCH ₂ C H ₂ CH ₂ C H ₂ CH ₂ N ⁺ ); 3.62 (m, 4H, CH ₃ (CH ₂ ) ₁₀ C H ₂ N ⁺ ); 3.75 (m, 4H, CH ₂ C H ₂ N ⁺ ); 4.07-4.12 (m, 24H, ⁺ N (C H ₂ C H ₂ ) ₃ N ⁺ ). ¹³ C NMR spectrum, δ, ppm: 12.95 ( C H ₃ (CH ₂ ) ₁₁ N ⁺ ); 21.27 (CH ₃ (CH ₂ ) ₉ C H ₂ CH ₂ N ⁺ ); 21.64 (CH ₃ C H ₂ (CH ₂ ) ₁₀ N ⁺ ); 22.21, 25.63, 28.66, 28.95, 29.10, 29.21, 31.54 (CH ₃ (CH ₂ ) ₂ ( C H ₂ ) ₈ CH ₂ N ⁺ ); 50.81 and 51.03 ( ⁺ N ( C H ² C H ² ) ³ N); 64.71 (C C H ₂ N ⁺ ).

Example 3. Obtaining 1,4-bis - [(4-dodecylazonium-1-azoniabicyclo [2.2.2] octyl) methyl] benzene tetrachloride (3 Dp12)

The synthesis scheme of compound (3) is shown in Figure 6. To a solution of n-bis (chloromethyl) benzene (7) in 5 ml of acetonitrile (175 mg, 1 mmol) was added a solution of 1-dodecyl-4-aza-1-azoniabicyclo chloride [2.2 .2] octane (4) (349 mg, 2.1 mmol) in 20 ml of acetonitrile. After stirring for 30 h at 24 ° С, the precipitated precipitate of compound (3) was filtered off, washed with acetonitrile (3 times 15 ml) and dried in vacuum. A hygroscopic fine crystalline substance is obtained. The mass of the obtained compound (3) 526 mg (yield 65%).

¹ H NMR spectrum, δ, ppm (J, Hz): 0.86 (br t, 6H, C H ₃ (CH ₂ ) ₁₁ N ⁺ , J 6.3), 1.28-1.41 (m, 36H, CH ₃ (C H ₂ ) ₉ CH ₂ CH ₂ N ⁺ ); 1.83 (m, 4H, CH ₃ (CH ₂ ) ₉ C H ₂ CH ₂ N ⁺ ); 3.71 (m, 4H, CH ₃ (CH ₂ ) ₁₀ C H ₂ N ⁺ ); 4.18-4.22 (m, 24H, ⁺ N (C H ₂ C H ₂ ) ₃ N ⁺ ), 7.96 (br.s, 4H, N ^arom ). ¹³ C NMR spectrum, δ, ppm: 14.55 ( C H ₃ (CH ₂ ) ₁₁ N ⁺ ); 22.61 (CH ₃ (CH ₂ ) ₉ C H ₂ CH ₂ N ⁺ ); 22.61 (CH ₃ C H ₂ (CH ₂ ) ₁₀ N ⁺ ); 23.29, 26.62, 29.68, 30.01, 30.08, 32.62, 31.10 (CH ₃ (CH ₂ ) ₂ ( C H ₂ ) ₈ CH ₂ N ⁺ ); 51.80 and 52.14 ( ⁺ N ( C H ₂ C H ₂ ) ₃ N); 66.08 (CH ₃ (CH ₂ ) ₁₀ C H ₂ N ⁺ ); 68.98 (C C H ₂ N ⁺ ); 129.37, 135.01 (6C ^benzene ).

Example 4. Inactivation of vaccinia virus compounds (1),

(2), (3).

The antiviral activity of compounds (1), (2), (3) was studied on the model of smallpox vaccine virus strain LI VP.

Used vaccinia virus (BOB) with an infectious titer of 10 ⁵ plaque-forming units (PFU) of virus-containing material. Infectious activity of the virus was determined by counting PFU on CV-1 cells (monkey kidney cells). The method is based on the ability of the virus to form single sections of lysed cells (plaques) on the cell monolayer after infection of one cell with a certain number of viral particles, which are minimally necessary for infection of one cell. Upon infection of the cell monolayer by successive dilutions of the virus and subsequent staining of living cells with the “gentian violet” dye, the number of lysed plaques is visualized, which is a measure of the amount of virus. Inactivation of vaccinia virus by compounds (1), (2), (3) was carried out at 37 ° C in 50 mM Tris-HCl buffer, pH 7.0, containing 0.2 M KCl and 2 mM EDTA, for 24 hours at 37 ° C at concentrations compounds (1), (2), (3) from 0.04 μm to 1 mm.

Compounds (1), (2), (3) effectively inactivate BOB, and the level of virus inactivation increases with increasing concentration of compounds at which the virus was inactivated. So, incubation of the virus for 24 hours at 37 ° C with compound (1) at a concentration of 0.05 mM, with compound (2) - 0.01 mM, with compound (3) - 0.025 reduces the titer of the virus by approximately 1 order of magnitude compared to control. With an increase in the concentration of compounds (1), (2), (3) to 0.1, 0.06, and 0.5 mM, respectively, complete inactivation of BOB is observed. Table 1 presents data on the antiviral effect of compounds (1), (2), (3).

Table 1 Connection Name Vaccinia virus Concentrations of compounds used to inactivate the virus Virus titer upon incubation for 18 hours at 37 ° C, log (PFU / ml) (one) 0.1 mm <1 (2) 0.06 mm <1 (3) 0.5 mm <1 the control - 5

The results obtained clearly indicate a high antiviral activity of compounds (1), (2), (3).

Example 5. The study of the membrane -olytic activity of compounds (1), (2), (3).

The membranolytic activity of the compounds was investigated in experiments with sheep erythrocytes. The ability of compounds to cause lysis of erythrocyte membranes was evaluated after incubation of sheep erythrocytes at a concentration of 15 × 10 ⁶ in the presence of one of the compounds (1), (2), (3) in the concentration range from 0.001 to 2 mM for 120 minutes at 37 ° C. After incubation, lysis efficiency was assessed by the change in phosphate buffer staining measured on a spectrophotometer at a wavelength of 550 nm. Membraneolytic activity of an equal volume of distilled water was taken as a positive control for 100%. As a negative control, 100 mM phosphate buffer was used, in the presence of which, under the indicated conditions, the efficiency of lysis of erythrocyte membranes did not exceed 2%.

Compounds (1), (2), (3) caused lysis of erythrocyte membranes, and its effectiveness increased with an increase in the concentration of compounds at which the erythrocyte suspension was incubated. So, incubation of the virus for 2 hours at 37 ° C with compound (1) at a concentration of 0.1 mM, with compounds (2), (3) - 0.05 mM led to complete lysis of erythrocyte membranes, comparable to lysis of red blood cells, incubated in the presence of distilled water. A decrease in the concentration of compounds to 0.001 mM in case (1), 0.005 mM in case (2), and 0.001 mM in case (3) led to a complete loss of membraneolytic activity by these compounds. Table 2 presents the values of the efficiency of lysis of erythrocyte membranes by compounds (1), (2), (3).

table 2 Connection Name Membrane activity of compounds The concentration of compounds at which lysis of erythrocyte membranes is observed, mm The efficiency of lysis during incubation for 2 hours at 37 ° C,% (one) 0.1 one hundred (2) 0.05 one hundred (3) 0.05 one hundred Phosphate buffer one hundred 2 Distilled water - one hundred

Thus, it was demonstrated that compounds (1), (2), (3) have a high membraneolytic activity.

Example 6. The study of the morphology of viral particles in the process of inactivation using compound (1).

The study of the morphology of viral particles after incubation with compound (1) was carried out using electron microscopy by negative contrast. The studied samples were sorbed onto support electron microscopic grids coated with a formar film for 30 seconds, the remainder of the liquid was removed, the grid was dried and placed in a contrasting solution (2% solution of phosphoric-tungsten acid in distilled water) for 30 sec, the remainder of the solution was removed. the mesh was dried. Samples (4-6 grids) were studied in a Jem 1400 transmission electron microscope at magnifications from 10,000 to 200,000.

Electron microscopic examination of the morphology of the viral particles inactivated by compound (1) revealed virions with a damaged lipid membrane, a change in the thickness of the lipid layer was also observed in a small part of the viral particles (Fig. 7A), control is shown in Fig. 7B. The degree of damage to the structure of virions directly depended on the concentration of compound (1). As the concentration of the compound increased from 0.05 to 0.5 mM, damage to their structure occurred. Based on the results of electron microscopic studies, we can conclude that compound (1) disintegrates the lipid shells of virions, that is, has membrane -olytic activity, which indirectly proves the possibility of inactivation of DNA viruses.

Example 7. The effect of compounds (1), (2), (3) on viability

cell CV-1

CV-1 cells (African green monkey kidney cells) were cultured in DMEM containing 5% fetal calf serum, antibiotics (100 units / ml penicillin and 0.1 mg / ml streptomycin) and antimycotic amphotericin (0.25 μg / ml) in an atmosphere of 5% CO ₂ at 37 ° C.

Cell viability after incubation with compounds (1), (2), (3) was determined using the MTT test, which is based on the ability of living cells to convert tetrazole-based compounds (MTT) into brightly colored formazan crystals, which allows spectrophotometric estimation of the number of living cells in the drug. For this, cells were plated in 96-well plates (15 × 10 ³ cells per well). After 48 hours, the medium was changed in the wells, and an equal volume of an aqueous solution of compound (1), (2), or (3) was added to the cells to a final concentration in the medium of 10 ^-7 to 10 ^-3 M. Cells were incubated during the day under the same conditions. At the end of incubation without changing the medium, MTT solution (5 mg / ml) in phosphate-buffered saline was added to the cells to a concentration of 0.5 mg / ml and incubated for 3 h under the same conditions. The medium was removed, 100 μl of dimethyl sulfoxide, in which the formazan crystals formed in the cells were dissolved, were added to the cells, and the optical density was measured on a multichannel spectrophotometer at wavelengths of 570 and 630 nm, where A ₅₇₀ is the absorption of formazan and A ₆₃₀ is the background of the cells.

From the experimental data, the IC ₅₀ value was calculated, the concentration of compounds at which 50% cell death was observed. The IC ₅₀ values of compounds (1), (2) and (3) for CV-1 cells are shown in table 3.

Table 3 Compound IC ₅₀ mm (one) 0.035 (2) 0.015 (3) 0.02

From the above data it is seen that the treatment of cells with compounds (1), (2) or (3) causes their effective death only at concentrations of compounds higher than 0.05 μM, which indicates a low toxicity of the compounds.

Thus, the given examples unambiguously indicate a high membrane -olytic and antiviral activity of compounds (1), (2), (3), which makes it possible to use them as active components for the development of dosage forms of drugs intended for the treatment of viral diseases.

Claims (1)

The agent is an N-substituted 1,4-diazabicyclo [2.2.2.] Octane derivative of the general formula (I):

,
where R = 1,4-substituted but-2-in for (1), 1,5-substituted pentane for (2), 1,4-dimethylphenyl for (3), showing antiviral activity against DNA viruses .

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