4-amino-3-arylamino-6-arylpyrazolo[3,4-d]pyrimidine derivatives, methods for preparing and applying them as antiviral agents

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to 4-amino-3-arylamino-6-arylpyrazolol[3.4-d]-pyrimidine derivatives showing antiviral activity. In formula I: the groups A and B independently represent phenyl, naphthyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, pyrazolyl, triazinyl, imidazolyl, furanyl, thienyl, and in each of these groups one to three hydrogen atoms can be independently substituted by the radical R1; R1 can be NO2, CN, CONR22, COOR2, CHO, CONH2, halogen, saturated or unsaturated, linear or branched alkyl with a number of atoms in the chain 1 to 7, saturated or unsaturated, linear or branched alkanole with a number of atoms in the chain 1 to 8, OR2, SR2, NR22, SO2NR32, di- or trifluoromethyl, phenyl; R2 represents hydrogen, CF3, and linear or branched alkyl with a number of atoms in the chain 1 to 7; the radical R3 represents H, benzyl, or linear or branched alkyl with a number of atoms in the chain 1 to 7; the radicals R4 and R5 represent hydrogen.

EFFECT: developing the method for preparing the compound of formula (I) and applying the compounds of the present invention as a biological agent exhibiting antiviral activity, eg for treating picornavirus infections.

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The prior art inventions

The present invention relates to derivatives of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]-pyrimidine, methods for their preparation and their use as antiviral agents, preferably for the treatment picornavirus infections.

The picornaviruses, in particular enteroviruses and rhinoviruses are responsible for a wide range of human diseases. The enteroviruses are more than 60 different pathogenic human serotypes (Melnick J: Fields et al., editors. Virology. Philadelphia: Lippincott-Raven Publishers; 1996, 655-712). Infection of enterovirus, ECHO virus, Coxsackie virus group a and group b are often characterized by non-specific fever and cause diseases of the upper respiratory tract, which are often impossible to distinguish from rhinoviral infections. More severe clinical picture, which may also have an epidemic include hemorrhagic conjunctivitis, herpangina, viral disease of the mouth and extremities, aseptic meningitis, encephalitis and acute myocarditis. The problem in this case is that different types of viruses can lead to the manifestation of the same symptoms or one type of virus can cause very different clinical pictures. Thanks to the introduction of modern and sensitive methods of viral diagnostics, sustainable enterovirus RNA and proteins of viruses which you can identify in connection with chronic diseases, such as type II diabetes, polymyositis, and most of all chronic myocarditis. Chronic enteroviral infections are also observed in patients with agammaglobulinemia and appear in this case in the form of chronic enteroviral meningoencephalitis. As additional symptoms are often observed dermatomyositis or polymyositis.

Rhinoviruses include about 100 serotypes. Rhinoviral infections cause more than half of all respiratory diseases of the upper respiratory tract (Couch RB in: Fields VM et al., editors: Fields Virology, 3rdedition. Lippincott-Raven, Philadelphia, 1996, 713-35). With an average period of illness of about 10 days, these colds, which mainly take the harmless nature, cause amounting to millions of outpatient visits and loss of work and hours of study. Possible emerging complications are inflammation of the middle ear, sinusitis, exacerbation of asthma and cystic fibrosis, as well as infectious diseases of the lower respiratory tract, mainly in young children, elderly patients and patients with weakened immune systems. Because of the huge diversity of types, prevention by vaccination is currently not possible. Due to associated with these diseases, loss of working time, visits to doctors and medicines, Reno and enterovirus the s annually cause enormous costs. Data viral infections to date have been symptomatic treatment, since there is no specific virus therapy (Rotbart: Antiviral Res 2002, 53(2), 83-98). In addition, often without the need for antibiotics. Therefore, it is necessary to develop new virustatic.

The results of an intensive search for possible methods of treatment of enteroviral and rhinoviral infections summarized Rotbart in 2002 in a review article (Rotbart: Antiviral Res 2002, 53(2), 83-98). For example, ribavirin inhibits the enzyme in the host cell, inosine 5'-monophosphate (MR)-dehydrogenase. By de-activating this key for the synthesis of purine nucleotides enzyme can inhibit the replication of picornaviruses in vitro and in vivo. In addition, ribavirin directly integrated into the genome of poliovirus and thus additionally acts as a mutagen for RNA viruses (Crotty S et al.: Nat Med 2000, 6(12),1375-9). Due to serious side effects, these compounds are not used for the treatment of infectious diseases caused by Rhino - and enteroviruses.

Specific targets for the inhibition of synthesis of viral RNA is itself genome, the viral RNA-dependent RNA polymerase and other viral proteins required for replicative complex. For a long time, guanidine, thiosemicarbazones, benzimidazole, deeper damali and flavones were known as inhibitors of polymerases of different picornaviruses in cell culture. Thus was achieved variable success in vivo. Derivatives enviroxime regarded as the most promising candidate with a wide protivoavariynoy and protivoleprosnoe activity. Enviroxime blocks the synthesis of plus-strand RNA by binding to the viral protein 3A, which is required for the formation of RNA intermediates during playback virus (Heinz VA and Vance LM: J Virol, 1995, 69(7), 4189-97). In clinical studies were observed average therapeutic effects or their absence, poor pharmacokinetics, and adverse effects (Miller FD et al.: Antimicrob Agents Chemother, 1985, 27(1), 102-6). To date there is no clinical data more new derivatives with the best indicators of bioavailability and tolerability.

Proteiny inhibitor AG-7088 was developed on the basis of information about the fine structure and function of the viral protease 2C. In the culture of cells in the nanomolar concentration range, AG 7088 effective against 48 types of rhinovirus and Coxsackie virus group A21 and group B3, enterovirus 70 and echo-virus 11 (Pattick AK et al.: Antimicrobila Agents Chemother, 1999, 43(10), 2444-50). Final data from the clinical trials are still unknown.

Thanks to the elucidation of the molecular structure of the viral capsid, were obtained prerequisites for purposeful development of the blocker capsid, "WIN substances" (Diana GD: Curr Med Chem 2003, 2, 1-12). They will ybiraut adsorption and/or "strip" Rhino - and enteroviruses. Some of the WIN substances are highly specific action only against the individual genera or viral types of picornaviruses. Other derivatives inhibit replication as Rhino-and enteroviruses. To WIN substances belong, for example, eildon, disoxaril and pirodavir. These compounds exhibit very good antiviral activity in cell culture. Poor solubility (eildon), low bioavailability (eildon and disoxaril), rapid destruction in the process of metabolism and excretion from the body (disoxaril and WIN 54954), as well as side effects such as skin rash (WIN 54954), made the clinical application impossible. Great hopes were pinned on pleconaril, another inhibitor of the capsid. Pleconaril has a very good bioavailability when administered orally and, after binding to hydrophobic "pocket" viral capsid, inhibits the penetration of Reno-, ECHO and Coxsackie viruses (Pevear DC et al.: Antimicrob Agents Chemother 1999, 43(9), 2109-15; McKinlay MA et al.: Annu Rev Environ 1992, 46, 635-54). Therefore, it is potentially effective against a broad spectrum of viral diseases, from the common cold to viral meningitis or myocarditis. Observed resistance to rhinovirus, enterovirus 71 and Coxsackie virus group B3 (Ledford RM et al.: J Virol 2004, 78(7), 3663-74; Groarke JM et al.: J Infect Dis 1999, 179(6), 1538-41). Clinical studies of children and adults with enteroviral mining the volume (Abzug MJ et al.: Pediatr Infect Dis J, 2003, 22, 335-41) and respiratory infections caused by rhinoviruses (Hayden FG et al.: Antivir Ther, 2002, 7, 53-65; Hayden FG et al.: Clin Infect Dis, 2003, 36, 1523-32) moved in a positive direction. However, confirmed therapeutic effect was insufficient for registration pleconaril (Picovir, Viropharma, USA) as a tool for the treatment of rhinoviral infections in the United States. In March 2002 an application was rejected by Management under the control over products and medicines (FDA) due to a low success therapy for simultaneously observed side effects.

Pyrazolopyrimidine also been described as CRF antagonists (e.g., EP 674642 and EP 691128), which, for example, inhibit adenosine kinase (EP 496617 or US 4,904,666), the xanthine oxygenase (J. Heterocyc. Chem. 19,1565,1982) or other enzyme systems (US 2,965,643 and US 3,600,389).

Thus, the development of high-performance virustatic for the treatment of Rhino - enterovirus diseases remains an important task of antiviral research. New connections must be well-tolerated and able to overcome the existing types of resistance, such as, for pleconaril.

Description of the invention

The aim of the present invention is to develop new compounds that can be used as antiviral agents against enteroviruses and rhinoviruses and deprived of weeks the mistakes, inherent in the existing prior art, in particular problems with sustainability and intolerance in respect of the relevant drugs, and describes the production and use of these compounds.

According to the present invention, this problem is solved by using a special way of substituted derivatives of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]pyrimidine of the General formula I, including their pharmaceutically acceptable salt,

where

group a and b represent, independently of one another, phenyl, naphthyl, pyridyl, hinely, pyrazinyl, pyrimidyl, pyrazolyl, triazinyl, imidazolyl, furanyl, thienyl, and in each of these groups, independently from each other, from one to three hydrogen atoms may be substituted by the radical R1defined hereinafter,

the radical R1can be NO2, CN, CONR22, COOR2, Cho, CONH2, halogen, saturated or unsaturated, linear or branched aliphatic radical with the number of atoms in the chain from 1 to 7, nasypany or unsaturated, linear or branched alkanol with the number of atoms in the chain from 1 to 8, OR2, SR2, NR22, SO2NR32, di - or trifluoromethyl, phenyl,

the radicals R2, R3, R4, R5represent, independently of one another, H, a saturated or nenasi the military, halogenated or dehalogenating, linear or branched aliphatic radical with the number of atoms in the chain from 1 to 7, benzyl, phenyl or naphthyl, saturated or unsaturated, mono - or polyheterocyclic with heteroatoms N, S, O, and each of the just mentioned groups independently may be replaced by fluorine, chlorine, bromine, trifluoromethyl, alkyl, alkoxy group, cyano group, nitro group, amino group, aminoalkyl, group C(O)-alkyl, C(O)O-alkyl, benzyl, phenyl or naphthyl.

Dependent claims explain the preferential embodiments of a special way of substituted derivatives of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]-pyrimidine and methods for their production and possible ways of their application, without limiting them present invention.

In the preferred embodiment, the present invention relates to compounds of General formula (I)selected from the group of 6-phenylaminopropyl[3,4-d]pyrimidines, including:

4-amino-6-phenyl-3-(tri-R1)phenylaminopropyl[3,4-d]pyrimidine,

4-amino-6-(tri-R1)phenyl-3-phenylaminopropyl[3,4-d]pyrimidine,

1-alkyl-4-amino-6-phenyl-3-(tri-R1)phenylaminopropyl[3,4-d]pyrimidine,

4-amino-1,6-di(tri-R1)phenyl-3-phenylaminopropyl[3,4-d]pyrimidine,

4-amino-6-phenyl-3-(tri-R1)phenylalkylamines[3,4-d]pyrimidine,

1-alkyl-4-amino-6-FeNi is-3-(tri-R 1)phenylalkylamines[3,4-d]pyrimidine.

It is preferable if the present invention also includes a 6-phenylaminopropyl[3,4,d]pyrimidines of General formula (I), including:

4-amino-3-(3-forfinal)amino-6-phenylpyrazol[3,4-d]pyrimidine,

4-amino-3-(3-forfinal)amino-6-(4-chlorophenyl)pyrazolo[3,4-d]pyrimidine,

4-amino-3-(3-chloro)amino-6-phenylpyrazol[3,4-d]pyrimidine,

4-amino-3-(3-methoxy)amino-6-phenylpyrazol[3,4-d]pyrimidine,

4-amino-3-(4-forfinal)amino-6-phenylpyrazol[3,4-d]pyrimidine,

4-amino-3-(4-forfinal)amino-6-(4-chlorophenyl)pyrazolo[3,4-d]pyrimidine,

4-amino-3-(4-chlorophenyl)amino-6-phenylpyrazol[3,4-d]pyrimidine,

4-amino-3-(3-forfinal)amino-1-methyl-6-phenylpyrazol[3,4,d]pyrimidine,

4-amino-1-benzyl-3-(3-forfinal)amino-6-phenylpyrazol[3,4,d]pyrimidine.

Unexpectedly, the compounds of this invention exhibit strong antiviral activity against picornaviruses, in particular against entero - and rhinoviruses, in nano - or micromolar concentration range.

Therefore, the invented pharmaceutical preparations containing the compound of formula (I)are particularly suitable for the treatment of humans and animals respiratory infections, aseptic meningitis, encephalitis, herpangina, etc. that may be caused by picornaviruses, in particular entero - and rhinoviruses.

The following is a detailed explanation of the invention pic what edstam methods of synthesis, separate derivatives of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]pyrimidine of the General formula (I), and their useful effect and use against picornavirus infections.

In Fig. 1 shows the General scheme of synthesis invented pyrazolo[3,4-d]pyrimidine 1, and at the first stage, it involves the condensation of [bis(methylthio)methylene]malononitrile 2 arylamine 3 in alcohol with getting aryl derivatives 4. Each of the latter can be extracted and cleaned for further reactions or be used directly in subsequent reactions without purification (dorectory synthesis). The next stage is the interaction of the aryl derivative 4 with hydrazine or hydrazine derivatives. The reaction is boiling within 1-4 hours and gives pyrazole 5 high yield. A decisive stage of the synthesis of pyrazolo[3,4-d]pyrimidine 1 is condensed pyrazole 5 arylamidine 6 in the presence of acetic acid, triperoxonane acid or sodium acetate.

Alternatywny method of synthesis is taking place in one reaction vessel, the reaction malononitrile with arylisocyanate in the presence of sodium hydride and subsequent processing of the reaction mixture by methyliodide or dimethylsulfate. In the described process produces large quantities of enamines. In this case, the condensation of pyrazole 5 Allami the ins 6 in the presence of acid, such as acetic acid, triperoxonane acid, or their salts (acetates) is also a decisive stage receiving pyrazolo[3,4-d]pyrimidine 1.

In the following examples of individual compounds of General formula (I)are preferably used against picornavirus infections (without limiting them present invention), and invented compounds can be obtained in the form of a solution or suspension in a pharmaceutically acceptable aqueous, organic or aqueous-organic medium for local or parenterale administration by intravenous, subcutaneous or intramuscular injection or intranasal, or enter into the composition of the tablets, capsules or aqueous suppositories.

The presented compounds of formula (I) can be used in dosages of from 0.1 to 1000 mg/kg body weight.

1. Obtaining and analysis of derivatives of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]pyrimidine

The structure of the invented compounds was established by synthesis, elemental analysis, NMR spectroscopy and mass spectroscopy.

Educt

5-Amino-4-cyano-3-arinaminpathy were synthesized according to the method shown in figure 1, and described Tominaga Y, et al. (J. Heterocycl. Chem., 1990, 27, 775-779). According to the prior art, arylamidine were synthesized from the corresponding compounds, t is relating to the source of cyanide (Boere, RT et al.: J. Were Obtained. Chem., 1987, 331, 161-167; Garigipati RS: Tetrahedron Lett., 1990, 31, 1969-1978; About Dann et al.: Justus Liebigs Ann. Chem., 1982, 1836-1839).

Example 1

4-amino-3-phenylamino-6-phenylpyrazol[3,4-d]pyrimidine

3.0 g (17,24 mmol) hydrate benzamidine hydrochloride and 2.2 g (23,0 mmol) of sodium acetate is added to 2.3 g (11.5 mmol) of 5-amino-4-cyano-3-phenylaminopropyl under stirring. The reaction mixture is heated at 220°C for 30 minutes the resulting material was treated with 50 ml of water, filtered and washed with 20 ml of cold methanol and 20 ml of cold ether. The product was then purified by crystallization from a mixture of DMF/water.

Solid light-yellow crystalline substance. Yield 57%. TPL 253-5°C. Rf(chloroform - methanol; 10/1) to 0.8 (silica gel 60).

MS m/z 302 (M+).

1H NMR (DMSO-d6) δ 12,38 (1H, s, NH(9)), 8,32-at 8.36 (2H, q, CH(18), SN(19)), 8,23 (1H, user. with, NH(7)), to 7.67 (2H, d, CH(2), CH(6)), of 7.48 (2H, user. s, NH2), 7,42 (3H, m, CH(20), SN(21), SN(22)), for 7.12 (2H, d, CH(3), CH(5)) and 6,98 (1H, m, CH(4)) ppm

13With NMR (DMSO-d6) δ 161,0 ((11)), 156,2 ((12)), 153,0 ((10)), 144,2 ((8)), 138,3 ((17)), 136,0 ((1)), 130,3 ((4)), 129,8 ((22)), 128,8 (3), (5)), USD 128.0 and 127,7 (18), (19)), To 120.4 (4)), 120,2 (2), (6)), 88,7 ((13)) ppm

Calculated for C17H14N6: C, 67,54; N, Of 4.67; N, 27,80

Found: C, 67,61; N, 4,82; N, 27,79

Example 2

4-Amino-3-(3-forfinal)amino-6-phenylpyrazol[3,4-d]pyrimidine

Obtaining carried out by the method described in example 1.

Light is Ellie solid crystalline substance. Yield 46%. TPL 267-9°C. Rf(chloroform - methanol; 10/1) - 0,85 (silica gel 60). MS m/z 320 (M+).

1H NMR (DMSO-d6) δ was 12.61 (1H, s, NH(9)), 8,35 is 8.38 (2H, q, CH(18), SN(19)), 8,64 (1H, user. with, NH(7)), 7,46 (2H, user. s, NH2), 7,3-7,52 (6N, m, CH(2), CH(4), CH(6), CH(20), SN(21), SN(22)), 6,60 (1H, t, CH(5)) ppm

13With NMR (DMSO-d6) δ to 166.2 and 161,2 ((3)), 162,2 ((11)), 161,8 ((10)), 156,1 ((12)), 144,3 ((1)), 143,4 ((8)), 130,0 ((17)), 129,8 ((5)), 128,5 ((22)), 127,0 (18), (19)), 112,5 ((6)), 105,5 and 105,8 ((2)), 102,6 and 103,9 ((4)), 89,39 ((13)) ppm

Calculated for C17H14FN6: C, 63,74; H, 4.09 To; N, 26,24

Found: C, 63,60; H, Was 4.02; N, 27,99

Example 3

4-Amino-3-(3-were)amino-6-phenylpyrazol[3,4-d]pyrimidine

Obtaining carried out by the method described in Example 1.

Almost white, crystalline solid substance. The yield was 73%. TPL 246-8°C. Rf(chloroform - methanol; 10/1) - 0,90 (silica gel 60). MC m/z 316 (M+).

1H NMR (DMSO-d6) δ 12,38 (1H, s, NH(9)), 8,32-at 8.36 (2H, q, CH(18), SN(19)), 8,23 (1H, user. with, NH(7)), 7,42-to 7.67 (6N, m, NH2CH(6), CH(20), SN(21), SN(22)), 7,21-7,29 (2H, m, CH(2)CH(5)) and 6.42 per (1H, d, CH(4)), 2,17 (3H, s, CH3) ppm

Calculated for C18H16N6: C, 68,34; N, 5,10; N, 26,56

Found: C, 68,43; N, 5,16; N, 26,71

Example 4

4-Amino-3-(4-were)amino-6-phenylpyrazol[3,4-d]pyrimidine

Obtaining carried out by the method described in Example 1.

Physico-chemical parameters are as follows:

almost white, crystalline solid substances is O. Yield 43%. TPL 266-8°C.

Rf(chloroform - methanol; 10/1) - 0,85 (silica gel 60). MC m/z 316 (M+).

1H NMR (DMSO-d6) δ 12,38 (1H, s, NH(9)), 8,33 is 8.38 (2H, q, CH(18), SN(19)), 8,15 (1H, user. with, NH(7)), 7,60 (2H, d, CH(2), CH(6)), of 7.48 (2H, user. s, NH2), 7,42 (3H, m, CH(20), SN(21), SN(22)), at 6.84 (2H, d, CH(3), CH(5)) and of 2.34 (3H, s, CH3) ppm

13With NMR (DMSO-d6) δ 161,0 ((11)), 156,2 ((12)), 153,0 ((10)), 144,2 ((8)), 138,3 ((17)), 136,0 ((1)), 130,3 ((4)), 129,8 ((22)), USD 128.0 and 127,7 (18), (19)), 123,8 (3), (5)), 118,2 (2)From(6)), 88,9 ((13)), 20,8(CH3) ppm

Calculated for C18H16N6: C, 68,34; N, 5,10; N, 26,56

Found: C, 68,38; N, 5,07; N, 26,47

Example 5

4-Amino-3-(4-bromophenyl)amino-6-phenylpyrazol[3,4-d]pyrimidine

of 1.87 g (10,7 mmol) hydrate benzamidine hydrochloride and 0,89 g (of 10.7 mmol) of sodium acetate are added to 1.0 g (3.6 mmol) of 5-amino-4-cyano-3-(4-bromophenyl)aminopyrazole in 20 ml of acetic acid under stirring. The resulting mixture was boiled for 4 h, treated with 50 ml of water, filtered and washed with 20 ml of cold methanol and 20 ml of cold ether. The crude product is purified by crystallization from ethanol. Physico-chemical parameters are as follows:

yellow, crystalline solid. Yield 38%. TPL 272-4°C. Rf(chloroform - methanol; 10/1) to 0.9 (silica gel 60).

MC m/z 381 (M+).

1H NMR (DMSO-d6) δ to 12.44 (1H, s, NH(9)), 8,33 is 8.38 (2H, q, CH(18), SN(19)), to 8.12 (1H, user. with, NH(7)), 7,40-7,53 (7H, m, NH2CH(3), CH(5), CH(20), SN(21), SN(22)), ,10 (2H, d, SN(2), CH(6)) ppm

Calculated for C17H13BrN6: C, 53,65; N, 3,44; N, 22,04

Found: C, 5,80; N, 3,48; N, 21,95

Example 6

4-Amino-3-(4-forfinal)amino-6-phenylpyrazol[3,4-d]pyrimidine

Obtaining carried out by the method described in Example 5, the solvent used triperoxonane acid. Crystallization of the final product is carried out from a mixture of ethanol/DMF. Physico-chemical parameters are as follows:

white-yellow, crystalline solid. Yield 58%. TPL 259-263°C.

Rf(chloroform - methanol; 10/1) to 0.8 (silica gel 60).

MS m/z 320 (M+).

1H NMR (DMSO-d6) δ 12,69 (1H, s, NH(9)), 8,33-to 8.41 (4H, m, CH(2), CH(6), SN(18), SN(19)), 8,18 (1H, user. with, NH(7)), 7,58-65 (5H, m, NH2, SN(20), SN(21), SN(22)), 7,27-7,31 (2H, m, CH(3), CH(5)) ppm

Calculated for C17H14FN6: C, 63,74; H, 4.09 To; N, 26,24

Found: C, 63,57; N, 4,07; N, 26,33

2. The use of invented derivatives of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]pyrimidine as antiviral agents

2.1 tolerance of the compounds of the just described Examples 1 to 6 in the cell culture

1×104HeLa cells (DSMZ, ACC 57) were sown in wells for microanalysis in 0.2 ml of culture medium RPMI 1640. Titration microplates were incubated without the test compound according to the standard (at 37°C, 5% CO2and about 95% relative humidity) for 48 h at a physiologically the ski conditions getting subconfluent monolayers. After that, the monolayers were added serially diluted test substance and incubated under physiological conditions within 72 hours after the time of incubation was measured by the extinction of all holes titration microplate at 660 nm using a reader for microplates (Sunrise, ASAP) after fixation with glutaraldehyde and painting methylene blue, and determine the values of the SS50using program analysis "Magellan". Because pre-incubation of HeLa cells by itself leads to the formation subconfluent continuous layer of cells, cytolysis during subsequent incubation with the test substance is crucial for the analysis.

The GMK cells were sown in tetralonia the microplate and pre-incubated in 5% CO2, 37°C and humidity of 95% in the incubator for 48 h to obtain a continuous layer of cells (Schmidtke M et al.: J Virol Meth, 2001, 95(1-2), 133-143). Then deleted the environment and added substances in various concentrations (100 ál/well/concentration, dilution factor 2) in a nutrient medium. For definitions of each of the reference values used 100 μl of medium (six control samples of cells not exposed to the treatment). At 72 h after addition of substances and incubation, the cells were stained with crystal violet in methanol. After extras the work of the dye was measured by optical density (OD) of each individual wells in tablet photometer company Dynatech (550/630 nm) and compared with a mean of control samples of cells. The mean value of control samples was taken as 100%. On average curves dose-effect was calculated 50% cytotoxic concentration (CC50), using the linear interpolation method.

Examplesthe 50% cytotoxic concentration (µg/ml)
HeLa cellscells GMK
139.6>50
245.7>50
327.7't been tested
4>50>50
58.542.9
644.3>50

2.2 Antiviral effect of the compounds of the above examples 1-6 in cell culture

Test for inhibition of cytopathic effect (CPE) with the international standard line of Coxsackie virus group B3 Nancy (CVB3 Nancy), human rhinovirus 2 and 8 (HRV2 and HRV14) in HeLa cells/p>

Replication of the viruses used in this test leads to the complete destruction of the host cells, a strong cytopathic effect (CPE). The addition of antiviral agents (100 ál/well/concentration, dilution factor 2) specific inhibition of virus-induced CPE (Schmidtke, M. et al.: J Virol Meth, 2001, 95(1-2), 133-143). In this test, the raw and processed substances compact solid layers of cells infected by the virus in dosage leading to complete CPE in untreated control virus samples after 24 h (CVB3 Nancy) or 72 h (HRV2 and HRV8) after infection. At this point in time still adhesive cells were fixed and stained with a solution of crystal violet in formalin. Inhibition of virus-induced CPE was quantitatively determined photometrically by the reading device for Dynatech microplate after elution of the dye. The antiviral effect was calculated by comparing the optical densities of the processed substances and raw cells infected with viruses, taking the mean optical density of the control samples of cells as 100%. On average curves dose-effect to calculate the 50% inhibitory concentration. As a control substance used pleconaril. The results obtained with the substances described in the examples shown in the following table.

Examples50% inhibitory concentration (mcg/ml)CVB3 NancyHRV2HRV8Pleconarilineffective0.011.310.002ineffectiveineffective20.0014.14.630.021.11.040.081.82.250.040.71.060.030.92.0

The reaction suppressing belascoaran (PRT) with a substance from example 1 and Coxsackievirus groups B1, B2, B4, B5, B6 (CVB1, CVB2, CVB4, CVB5, CVB6)

To conduct this test, the CD is s solid layers of HeLa cells by the age of 2-3 days infected in 12-hole tablet for tissue culture 50-80 plaque-forming units (PFU) (Schmidtke M et al.: J Virol Meth, 2001, 95(1-2), 133-143). Two uninfected wells tablet was used as a control cell sample (SS). After adsorption of the virus at 37°C for 1 h, vaccinated supernatant was removed. Infected cells were covered with a control medium containing 0.4% agar without (virus control samples) or with a substance in non-cytotoxic concentrations (dilution factor 2, two samples (concentration) and incubated at 37°C for 48 hours After fixation and staining tablets crystal violet in formalin, removed agar and washed tablets running water. The number of virus-induced plaques were counted on a viewing table with lights, and then to calculate the proportional reduction in the number of plaques caused by the substance. Spent three identical test and averaged curve dose-effect was calculated concentration causing a 50%decrease in the number of plaques (IC50). The results obtained with the substance of example 1, shown in the following table.

Virusesthe 50% cytotoxic concentration in HeLa cells (SS50) in mcg/ml50% inhibitory concentration in HeLa cells (IC50) in mcg/mlThe coefficient of selectivity (SI) = SS50:IC 50
CVB139.612.73.1
CVB239.60.3132
CVB439.67.15.6
CVB539.62.814.1
CVB639.62.615.3

2.3 Acute and subacute toxicity of the compounds of examples 2 and 4 for mice

Acute toxicity of the compounds from examples 2 and 4 were determined for mice aged 4-5 weeks (without defining line). 1-2 drops TWIN-80 was added to 1% aqueous solution of carboxymethyl cellulose, and the resulting mixture is used to obtain a suspension of a substance. 1500, 2000, 2500, 3000, 4000 or 5000 mg/kg of the compounds from examples 2 and 4 were administered orally 5 mice, each number once. Over the next three days defined the General health of the mice, the changes in their weight, rectal temperature and survival rate.

Survived all the animals, which were injected concentration of the substance up to 3000 mg/kg, if the substance is from examples 2 and 4 were injected once (see table below). Did not change any of the overall health status of the mice, or rectal temperature or body weight.

50% lethal dose for two of the investigated substances amounted to about 3500 mg/kg (calculated according Kärber in Mayer et al. Virologische Arbeitsmethoden. (Virological working methods) Gustav-Fischer-Verlag, Jena, 1973). After administration of a dosage of 5000 mg/kg animals died within 3-5 hours

Concentration (mg/kg)The number of victims/survivors of mice
Example 2Example 4
15000/50/5
20000/50/5
25000/50/5
30000/50/5
40003/54/5
50005/55/5

Based on the obtained results, the substances of examples 2 and 4 can be regarded as a well-tolerated after a single oral administration.

Under Troy toxicity of the same substances (examples 2 and 4) was determined for mice aged 4 weeks (without defining line). 1-2 drops TWIN-80 was added to 1% aqueous solution of carboxymethyl cellulose, and the resulting mixture is used to obtain suspensions of substances. 100 mg/kg of the compounds from examples 2 and 4 were administered orally to each of the 7 mice once daily for 5 days. The mice were observed for 10 days. Each day was determined overall health, weight changes, changes in rectal temperature and survival rate. At the end of the test investigated the morphological changes of the spleen, lungs and liver after taking slices.

Processing substances has not had any impact on the overall health or body temperature. The body weight of mice that were injected substance, the period of observation grew up in the same way as the weight of the control animals, which substance is not administered. None of the animals died.

Thus, the substances of examples 2 and 4 at a concentration of 100 mg/kg can be considered as a well-tolerated after their five oral administration.

Position labels

1 - pyrazolo[3,4-d]pyrimidine

2 - [bis(methylthio)methylene]malononitrile

3 - arylamino

4 - aryl derivative

5 - pyrazole

6 - arylamides

1. Derivative of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]pyrimidine of the General formula I

where:
group a and b represent independently of each other phenyl, the Teal, pyridyl, hinely, pyrazinyl, pyrimidyl, pyrazolyl, triazinyl, imidazolyl, furanyl, thienyl, and in each of the just mentioned groups independently from each other from one to three hydrogen atoms may be substituted by the radical R1defined hereinafter,
the radicals R1can be NO2, CN, CONR22, COOR2CHO, CONH2, halogen, saturated or unsaturated, linear or branched aliphatic radical with the number of atoms in the chain from 1 to 7 saturated or unsaturated, linear or branched alkanol with the number of atoms in the chain from 1 to 8, OR2, SR2, NR22, SO2NR32, di - or trifluoromethyl, phenyl,
the radical R2represents hydrogen, CF3and linear or branched alkyl with the number of atoms in the chain from 1 to 7; the radical R3represents H, benzyl or a linear or branched alkyl with the number of atoms in the chain from 1 to 7; the radicals R4and R5represent hydrogen.

2. Derivative of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]pyrimidine according to claim 1, which represents a 4-amino-6-phenyl-3-(tri-Rl)phenylaminopropyl[3,4-d]pyrimidine of the General formula I, the radicals R1in groups a and b are independently from each other CONH2CN, halogen, NO2or CF3.

3. Derivative of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]pyrimidine according to claim 1, PR is dostavljaust a 1-R 3-4-amino-6-phenyl-3-(tri-R1)phenyl-aminopyrazole[3,4-d]pyrimidine of the General formula I, the radicals R1in groups a and b are independently from each other CONH2CN, halogen, NO2or CF3.

4. Derivative of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]pyrimidine according to claim 1, selected from the group of 6-phenylaminopropyl[3,4-d]pyrimidines, including:
4-amino-6-phenyl-3-(tri-R1)phenylaminopropyl[3,4-d]pyrimidine,
4-amino-6-(tri-Rl)phenyl-3-phenylaminopropyl[3,4-d]pyrimidine,
1-alkyl-4-amino-6-phenyl-3-(tri-R1)phenylaminopropyl[3,4-d]pyrimidine,
4-amino-1,6-di(tri-Rl)phenyl-3-phenylaminopropyl[3,4-d]pyrimidine.

5. Derivative of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]pyrimidine according to claim 1, selected from the group of 6-phenylaminopropyl[3,4-d]pyrimidines, where the radicals R1in groups a and b are independently from each other halogen.

6. Derivative of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]pyrimidine according to claim 1, selected from the group of 6-phenylaminopropyl[3,4-d]pyrimidines, including:
4-amino-3-(3-chlorophenyl)amino-6-phenylpyrazol[3,4-d]pyrimidine,
4-amino-3-(3-methoxyphenyl)amino-6-phenylpyrazol[3,4-d]pyrimidine,
4-amino-3-(4-forfinal)amino-6-phenylpyrazol[3,4-d]pyrimidine,
4-amino-3-(4-forfinal)amino-6-(4-chlorophenyl)pyrazolo[3,4-d]pyrimidine,
4-amino-3-(4-chlorophenyl)amino-6-phenylpyrazol[3,4-d]pyrimidine,
4-amino-3-(3-forfinal)amino-1-methyl-phenylpyrazol[3,4-d]pyrimidine,
4-amino-1-benzyl-3-(3-forfinal)amino-6-phenylpyrazol[3,4-d]pyrimidine.

7. A method of obtaining a derivative of 4-amino-3-arylamino-6-arylpyrazole[3,4-d]pyrimidine according to claim 1, in which the corresponding 3-substituted amino-4-cyano-5-aminopyrazole condense with the corresponding arylamidine in the presence of an acid selected from acetic acid, triperoxonane acid or their salts, such as acetates.

8. The use of derivative 4-amino-3-arylamino-6-arylpyrazole[3,4-d]pyrimidine according to claims 1 to 6, or their pharmaceutically acceptable salts as biological agents with antiviral activity.

9. The use of claim 8, where they are used in prophylactic and therapeutic programs against viral infections, for example for the treatment of picornavirus infections.



 

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Polycyclic compound // 2451685

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12 cl, 3 dwg, 7 tbl, 3 ex

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19 cl, 7 tbl, 18 ex

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19 cl, 3 dwg, 11 tbl, 12 ex

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2 cl, 3 tbl, 5 ex

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53 cl, 7 dwg, 8 ex

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