RU2340614C2 - 2-r-4-(allyloxymethyl)-6-nitro-1,2,4-triasolo[5,1-c]-1,2,4-triazin-7(4h)-ons and 2-r-4-(propargyloxymethyl)-6-nitro-1,2,4-triasolo[5,1-c]-1,2,4-triazin7(4h)-ons, possessing anti-viral activity - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: 2-R-4-(allyloxymethyl)-6-nitro-1,2,4-triazolo [5,1-c]-1,2,4-triazin-7(4H)-ons of general formula (1)

and 2-R-4-(propargyloxymethyl)-6-nitro-1,2,4-triasolo[5,1-c]-1,2,4-triazin-7(4H)-ons of general formula (2)

.

EFFECT: anti-viral activity of compound.

1 cl, 3 tbl, 5 ex, 2 dwg

Description

1. The technical field to which the invention relates.

The invention relates to the field of biologically active compounds and relates to the development of new derivatives of 1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -ones having antiviral effect, intended for the treatment and prevention of infectious diseases of animals and humans of viral nature.

2. The level of technology

The urgency of the problem of antiviral therapy, especially in conditions of rapid mutation of viruses, the identification of new pathogens of dangerous and slow viral infections causes a constant need for new drugs that would have high activity, prolonged action and low toxicity.

One of the methods for creating effective antiviral agents is the creation of acyclic nucleosides - structural analogues of natural nucleosides containing acyclic structures as a fragment simulating the ribofuranoside cycle. Thus, the antiviral effect of zovirax, ganciclovir, famciclovir, acyclic structural analogues of the natural nucleoside guanosine, used to treat diseases caused by herpes viruses, cytomegaloviruses, adenoviruses, etc., is described. (The Register of Medicinal Products of Russia. Encyclopedia of Medicines. Edited by G.L. Vyshkovsky. Issue 11. M: Radar: 2004, 1504 pp .; Von A. Kleemann, J. Engel. Pharmazeutishe Wirkstoffe. Synthesen, Patente, Anwerendungen // New York, Stutgart - Georg Thieme, Verlag, 1982. 1040 S .; PC Vartanyan, Synthesis of Essential Medicines./ M.: Medical News Agency, 2004. - 845 p.)

Among the acyclic nucleosides that exhibited an antiviral effect, compounds were identified that contain a terminal multiple bond in the acyclic fragment (M.S. Novikov, A.A. Ozerov, A.K. Brel, E.I. Boreko et al. Synthesis and antiviral activity 1 - [2- (allyloxy) ethoxymethyl] - and 1- [1,3-di (allyloxy) -2-propoxymethyl] pyrimidines. / Pharmaceutical Chemistry Journal, 1993, No. 4, p. 26-28; Ozerov A.A ., Brel A.K., Ozerov T.P., Borenko E.I. et al. / 1-Allylokimethyluracil: improved synthesis and activity against an acyclovirresistant strain of the herpes virus // Chem. Zh., 1993, No. 1, pp. 42-43; Ozerov A.A., Brel A.K. New acetylene acyclonucleosides. Synthesis of 1 (-propargyloxymethyl) derivatives of uracil // HGS, 1993, No. 6, p.797- 799; Lee K.-H., Wu YS., Hall IH, Antitumor Agents. 25. Synthesis and Antitumor Activity of Uracil and Thymine α-Methylene-γ-lactones and Related Derivatives // J. Med. Chem., 1977, Vol.20, No. 7, p. 911-914). The disadvantages of antiviral acyclic nucleosides include low metabolic stability, short-term clearance, lack of effect on cells with the cytopathic consequences of a viral infection; as a result, the need to take drugs in high dosages, side effects on hematopoiesis and, in particular, on bone marrow, relatively rapid development of resistance with prolonged and frequent use due to the appearance of mutations in the genes of thymidine kinases (Virology. T.2. Translation, ed. N.V. Kaverina, L.L. Kiseleva / M .: Mir. 1989, 494 p.). The disadvantages of abnormal nucleosides used in therapeutic practice should also be added to the high cost of drugs, due to both the high cost of raw materials and the complexity of the technological scheme of production.

Modern drugs with high antiviral activity are remantadine, adaptromin, thromantadine (Russian Drug Register. Encyclopedia of Medicines. Edited by G.L. Vyshkovsky. Issue 11. M .: RLS: 2004, 1504 p.). Remantadine and adapromine are widely used in medical practice as a prophylactic during influenza epidemics, as well as in the early stages of treatment for type A influenza. In addition, adapromine is used to treat type B influenza. Thromantadine has a different spectrum of activity: it is active against HSV -1 and is used to treat herpes (Ershov F.I., Chizhov N.P., Tazulakhova E.B. Antiviral drugs (reference) / St. Petersburg. 1993, 104 p.). The disadvantages of these drugs include their ability to suppress the reproduction of viruses only at an early stage of infection, which severely limits the practical applicability of these drugs.

Thus, the search for new antiviral agents for influenza viruses of type A, B, respiratory syncytial virus and herpes simplex virus is an urgent task.

On the other hand, there is evidence of the antiviral effect of 6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -ones, coupled with low toxicity of the compounds (synthesis and antiviral activity of 6-nitro-7-oxo-4,7-dihydroazolo [5.1-s] [1,2,4] triazines V.L. Rusinov, E.N. Ulomsky, O.N. Chupakhin, M .M. Zubairov, A.B.Kapustin, N.I. Mitin et al. / Chemical-Pharmaceutical Journal. No. 9, 1990, pp. 41-44).

The antiviral effect of 4-alkylthio-5,7-dihydro-7-oxo-1,2,4-triazolo [4,3-d] -1,2,4-triazines is also known (Cristesku C. Derivati de 5-oxo- 5,6-dihidro-s-triazolo (4,3-d) - (as-triazins si procedeu pentru prepararea lor. Pat CPP Cl. 12 (C07d 55/10), No. 56269, 03/13/1974).

The most widely used prototype is the antiviral drug remantadine, which is active against a wide range of influenza viruses.

3. Disclosure of invention

The technical result of the invention is the discovery of new chemical compounds of triazolo-1,2,4-triazine derivatives having antiviral effects, in particular with respect to influenza virus, respiratory syncytial infection, herpes and avian influenza virus. The specified technical result is achieved by the fact that according to the invention proposed new compounds - 4- (allyloxymethyl) -2-R-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazine- 7 (4H) -ones of the general formula (1) and 4- (propargyloxymethyl) -2-R-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -ones of the general formula (2) containing, as a fragment of R, an H atom or a methyl group; or methylthio group.

4- (Allyloxymethyl) -2-R-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7 (4H) -ones of the general formula (1) and 4 - (propargyloxymethyl) -2-R-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -ones of the general formula (2) are obtained from 2 -R-6-nitro-1,2,4-triazolo [5,1-c] -1,2,4-triazin-7 (4H) -ones (3) by alkylation with allyloxymethyl acetate (4) or propargyloxymethyl acetate (5 ) in accordance with scheme 1.

1.1: R = H, 1.2: R = CH ₃ , 1.3: R = SCH ₃ ; 2.1: R = H, 2.2: R = CH ₃ , 2.3: R = SCH ₃ .

The structure of the claimed compounds (1.1 ÷ 1.3 and 2.1 ÷ 2.3) is established on the basis of elemental analysis of IR and NMR spectroscopy and mass spectrometry.

Section 4 provides examples of the synthesis and physicochemical characteristics of the claimed compounds.

4. Examples of the synthesis of the claimed compounds

Example 1

4-allyloxymethyl-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7-one (1.1).

Suspension 0.182 g (0.001 mol) of 6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7-one in 1.35 ml (0.001 mol) of allyloxymethyl acetate (4) boiled in a round bottom flask under reflux for 5 minutes. After cooling, the precipitate formed was recrystallized from ethanol.

Exit, %; T _PL , ° C. Found,%, C, H, N. Gross formula. Calculated,%, C, H, N; ¹ H NMR spectrum (DMSO-d6), δ, ppm

58; 45; 38.14, 3.17, 33.30. C ₈ H ₈ N ₆ O ₄ . 38.10, 3.20, 33.32.

4.24 m. (2H, O-CH ₂ ), 5.33 m. (2H, C = CH ₂ ), 5.64 s. (2H, N-CH ₂ ), 5.82 m. (1H, C = CH), 8.34 s. (1H, H (2))

4-allyloxymethyl-2-methyl-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7-one (1.2)

Suspension 0.196 g (0.001 mol) of 2-methyl-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7-one in 1.35 ml (0.001 mol) allyloxymethyl acetate (4) was boiled in a round bottom flask under reflux for 5 minutes. After cooling, the precipitate formed was recrystallized from ethanol.

Exit, %; T _PL , ° C. Found,%, C, H, N. Gross formula. Calculated,%, C, H, N; ¹ H NMR spectrum (DMSO-d6), δ, ppm

63; 36; 40.64, 3.81, 31.54. C ₉ H ₁₀ N ₆ O ₄ . 40.61, 3.79, 31.57.

2.51 s (3H, C-CH ₃ ), 4.20 m. (2H, O-CH ₂ ), 5.30 m. (2H, C = CH ₂ ), 5.67 s. (2H, N-CH ₂ ), 5.87 m. (1H, C = CH)

4-allyloxymethyl-2-methylthio-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7-one (1.3)

A suspension of 0.228 g (0.001 mol) of 2-methylthio-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7-one in 1.35 ml (0.001 mol) allyloxymethyl acetate (4) was boiled in a round bottom flask under reflux for 5 minutes. After cooling, the precipitate formed was recrystallized from ethanol.

Exit, %; T _PL , ° C. Found,%, C, H, N. Gross formula. Calculated,%, C, H, N; ¹ H NMR spectrum (DMSO-d6), δ, ppm

62; 48; 36.27, 3.40, 28.16. C ₉ H ₁₀ N ₆ O ₄ S. 36.24, 3.38, 28.17.

2.68 s. (3H, C-CH ₃ ), 4.22 m. (2H, O-CH ₂ ), 5.25 m. (2H, C = CH ₂ ), 5.61 s. (2H, N-CH ₂ ), 5.88 m. (1H, C-CH).

Example 2

4-Propargyloxymethyl-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7-one (2.1)

A suspension of 0.182 g (0.001 mol) of 6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7-one in 1.30 ml (0.001 mol) of propargyloxymethyl acetate was boiled in a round bottom flask with reflux condenser 5 min. After cooling, the precipitate formed was recrystallized from ethanol.

Exit, %; T _PL , ° C. Found,%, C, H, N. Gross formula. Calculated,%, C, H, N; ¹ H NMR spectrum, δ, ppm

56; 59; 38.80, 2.61, 33.63. C ₈ H ₆ N ₆ O ₄ . 38.41, 2.42, 33.59.

3.25 t. (1H, C≡CH), 4.35 (2H, d, O-CH ₂ ), 5.73 (2H, s, N-CH ₂ ), 8.45 (1H, s ., H (2))

4-Propargyloxymethyl-2-methyl-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7-one 2.2

Suspension 0.195 g (0.001 mol) of 2-methyl-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7-one in 1.30 ml (0.001 mol) propargyloxymethyl acetate was boiled in a round bottom flask under reflux for 5 minutes. After cooling, the precipitate formed was recrystallized from ethanol.

Exit, %; _Mp, ° C. Found,%, C, H, N. Gross formula. Calculated,%, C, H, N; ¹ H NMR spectrum, δ, ppm

67; 65; 40.73, 3.21, 31.85. C ₉ H ₈ N ₆ O ₄ . 40.92, 3.05, 31.81.

2.50 s. (3H, C-CH ₃ ), 3.25 t. (1H, C≡CH), 4.35 d. (2H, O-CH ₂ ), 5.75 s. (2H, N-CH ₂ )

4-Propargyloxymethyl-2-methylthio-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7-one (2.3)

Suspension 0.228 g (0.001 mol) of 2-methyl-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7-one in 1.30 ml (0.001 mol) propargyloxymethyl acetate was boiled in a round bottom flask under reflux for 5 minutes. After cooling, the precipitate formed was recrystallized from ethanol.

Exit, %; T _PL , ° C. Found,%, C, H, N. Gross formula. Calculated,%, C, H, N; ¹ H NMR spectrum, δ, ppm

58; 72 36.51 2.71 28.36. C ₉ H ₈ N ₆ O ₄ S. 36.49 2.72 28.37.

2.54 s (3H, C-CH ₃ ), 3.22 t. (1H, CHCH), 4.34 d. (2H, O-CH ₂ ), 5.77 s. (2H, N-CH ₂ )

Presence in 4- (allyloxymethyl) -2-R-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -ones (1.1-1.3) terminal groups proved by ¹ H NMR spectra based on chem. shifts and multiplicities of resonant signals. So, in the ¹ H NMR spectra of 1,2,4-triazolo [5,1-s] -1,2,4-triazines (1.1-1.3), the allyl fragment appears as a characteristic ABX system.

So, in the spectrum of compound 1.3, the protons of the terminal group —C = CH _{2 are} represented by a two-proton multiplet with chem. a shift in the region of 5.20-5.30 ppm. At the same time, the proton -CH = C- appears as a multiplet in the region of 5.80-5.90 ppm. Figure 1 represents the NMR spectrum ¹ H 2-methylthio-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -one (1.3).

In the IR spectra of the claimed compounds (1.1-1.3) there are absorption bands in the region of 1730-1760 cm ^-1 corresponding to stretching vibrations of the carbonyl group and absorption bands in the regions of 1330-1560 and 1540-1570 cm ^-1 corresponding to stretching vibrations of the nitro group.

NMR spectrum analysis ¹ H 4- (propargyloxymethyl) -2-R-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -ones (2.1 -2.3) allows you to uniquely establish the structure of the compounds obtained. So, in the spectrum of the compound (6.1, R = H) a signal with chem. a shift of 3.18 ppm, presented in the form of a triplet ( ³ J = 2.4 Hz), belongs to the proton of the acetylene group.

Figure 2 is a ¹ H NMR spectrum of 6-nitro-4-propargyloxymethyl-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazine (2.1)

In addition, evidence of the presence of an acetylene fragment in the structure of 2-R-6-nitro-4-propargyloxymethyl-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazines (2.1- 2.3) is the presence in the IR spectra of these compounds of the band in the region of 2110-2130 cm ^-1 , corresponding to stretching vibrations of the triple bond.

5. Determination of the biological effect of the claimed compounds

Example 1. Determination of antiviral activity of compounds against influenza viruses

Influenza viruses of type A (H1N1, H3N2, H5N1 and H0N1 (remantadine-resistant) strains) and type B were used in the work.

Determination of the antiviral activity of the compounds in the relations of the influenza virus was carried out on the HAO model on 71-beam polystyrene panels. The compounds in the studied concentrations were dissolved in the HAO medium and added to the wells of the panels with HAO fragments, whereupon the virus was added in dilutions and incubated at 33-34 ° C for 48 (for type A flu) and 72 (for type B flu) hours. The inhibitory effect on the reproduction of viruses of the studied compounds was evaluated by the reaction of GA with the addition of 1% chicken red blood cells to the culture fluid.

The effectiveness of the compounds was evaluated by reducing the infectious activity of the virus in the experiment compared with the control - neutralization index (log ID ₅₀ ). With an IN value of up to 1.0 lg ID _{50, the} drug was considered inactive, with an ID of 1.0 and 2.0 lg, ID ₅₀ was weakly active, and with an ID above 2.0 lg ID ₅₀ was active.

The virus titer was calculated by the method of Reed and Mancha. The controls during the experiment were: the control of the CAO, the control of the virus, the control of the drug, the antiviral drug - remantadine.

Table 1 shows the antiviral activity of the claimed compounds (1) and (2) in relation to influenza viruses of type A and B. The claimed compounds (1.1) and (1.3) have comparable activity with remantadine in relation to influenza A and exceed the effect of remantadine in against influenza virus type B. The claimed compound (1.2) has an antiviral effect against influenza viruses type A, comparable to the action of remantadine and exceeds the action of remantadine against influenza virus type B. As for compounds 2.1-2.2 (especially from rinses against compound 2.2), upon further study, they showed activity against remant-resistant strains (H0N1) and significantly reduced the reproduction of influenza B. Remantadin is inactive against influenza virus type B.

From the data given in table 2, it can be seen that compounds 1.1-1.3 showed an antiviral effect exceeding that of remantadine against type B influenza virus and type A influenza viruses (strains A / H0N1 (Mong) and A / H0N1 (PR )).

Example 2. Determination of antiviral activity of compounds against respiratory syncytial virus

Respiratory syncytial virus (Long strain) was used in the work.

The main experience in testing the inhibitory effect of the drug against PC viruses included the following steps:

1. The introduction into test tubes with cell culture of the studied concentrations of drugs.

2. Contact of the drug with tissue culture cells for 1 hour at room temperature.

3. At the end of the contact, add the drug to the test tube with the virus.

The antiviral activity of the compounds was determined by the neutralization index, the assessment of which is similar to the evaluation of indicators when studying antiviral activity against influenza viruses.

The controls during the experiment were the control of cell culture, drug control, virus control.

Table 3 shows data on the antiviral activity of the claimed compounds (2) in relation to the respiratory syncytial virus. Remantadine is inactive against respiratory syncytial virus.

Example 3. Determination of antiviral activity of compounds against herpes simplex virus

Herpes simplex virus, an EU strain, was used in the work.

The study of the antiviral activity of the compounds against herpes simplex virus was carried out on 96-well polystyrene microplates on the Vero and Hep-2 cell lines. The results were evaluated after 48 hours of cultivation of infected cells according to the degree of cytopathic effect caused by the virus:

"+++" - rounding of all cells, "+" - changing the morphology of cells, "+/-" - granularity of the cytoplasm, local rounding of cells, "-" - cells are not changed. The controls during the experiment were: virus control, cell control.

Table 3 shows the antiviral activity of the claimed compounds (2) against herpes simplex virus. Remantadine is inactive against herpes simplex virus.

Table 1 Influenza activity of the claimed compounds

No. p / p Compound Activity against influenza viruses R R ' Type A Type B 1.1 H

active active 1.2 CH ₃

active weakly active 1.3 SCH ₃

active active 2.1 N

active active 2.2 CH ₃

active active 2.3 SCH ₃

active active remantadine active inactive

table 2 Influenza activity of the claimed compounds (1) Connection No. The concentration of the drug, mcg / ml Decrease in the infectious titer of virus strains, lg (EID ₅₀ ) A / H3N2 AT A / H5N1 A / H0N1 (Mong) A / H0N1 (PR) 1.1 100 (200) 2.5 (2.5) 4.0 (4.0) 3.5 (3.5) 4.0 (4.0) 4,5 (4,5) 1.2 100 (200) 0.5 (2.0) 1.0 (2.5) 1.5 (3.0) 2.5 (3.0) 2.5 (4.0) 1.3 100 (200) 2.0 (2.5) 2.5 (3.0) 2.5 (3.0) 4.0 (4.0) 2.5 (4.0) Remantadine 100 (200) 2.5 (2.5) 0,0 (0,5) 3.0 (3.0) 0,0 (1,5) 1.0 (1.5) Original virus titer 3,5 4,5 4.0 5,0 5.5

Table 3 The activity of the claimed compounds (2) against respiratory syncytial virus and herpes simplex virus

No. p / p Compound Respiratory syncytial virus Herpes simplex virus R Protection Index,% Titer reduction, lg 2.1 N 56 ≥1.7 ++ 2.2 CH ₃ 34 ≥1.1 ++ 2.3 SCH ₃ 22 ≥1.0 +++

Claims (1)

2-R-4- (allyloxymethyl) -6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -ones of the general formula (1) and 2 -R-4- (propargyloxymethyl) -6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -ones of the general formula (2)

where R: H, CH ₃ , SCH ₃ , possessing antiviral action.

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