DE102012004736A1 - New 4-amino-3-phenylamino-6-phenylpyrazolo(3,4-d)pyrimidine derivatives useful as a medicament for prophylactic- or therapeutic treatment of viral infections, preferably picornavirus infections and rhinovirus infections - Google Patents

Abstract

4-Amino-3-phenylamino-6-phenylpyrazolo[3,4-d]pyrimidine derivatives (I), where at least one hydrogen atom in at least one of the phenyl groups A and B1, is replaced by a substituent R11 exhibiting a Hammett constant of greater than 0.23, and each further hydrogen atom in each phenyl groups A and B1, is optionally replaced by a group R1, are new. 4-Amino-3-phenylamino-6-phenylpyrazolo[3,4-d]pyrimidine derivatives of formula (I) or their salts or prodrugs, where at least one hydrogen atom in at least one of the phenyl groups A and B1, is replaced by a substituent R11 exhibiting a Hammett constant of greater than 0.23, and each further hydrogen atom in each of the phenyl groups A and B1, is optionally replaced by a group R1, are new. R1 : aliphatic group with 1-7 atoms or alkanol group with 1-8 atoms (both optionally saturated and optionally branched), halo, NO 2, CN, CON(R2) 2, COR2, COOR2, OR2, SR2, N(R2) 2, SO 2N(R2) 2, CX3, CR2(X) 2, OC(X) 3, OCR2(X) 2or phenyl; R2 : aliphatic group with 1-7 atoms (optionally saturated, optionally halogenated and optionally branched), mono- or poly-heterocyclyl (optionally saturated and contains N, S or O), H, benzyl, phenyl or naphthyl (all optionally substituted by F, Cl, Br, CF 3, alkyl, alkoxy, CN, NO 2, NH 2, aminoalkyl, C(O)-alkyl, C(O)O-alkyl, benzyl, phenyl or naphthyl); X : F, Cl, Br or I; and R11 : NO 2, CN, CF 3, CCl 3, CBr 3, OCF 3, OCCl 3, OCBr 3, CHF 2, CHCl 2, CHBr 2, OCHCl 2, CHO, COOH, COCH 3, COC 2H 5, COOCH 3or COOC 2H 5, preferably CF 3or OCF 3. Independent claims are also included for: (1) a pharmaceutical composition comprising (I); (2) preparing the pharmaceutical composition, comprising formulating (I) with a carrier; and (3) preparing (I). [Image] ACTIVITY : Virucide. The antiviral activity of (I) was tested against 20 clinical coxsackievirus b3 patient isolates. The results showed that 4-amino-3-(4-trifluoromethylphenyl)amino-6-phenylpyrazolo[3,4-d]pyrimidine exhibited mean IC50 value of 4.97+- 4.05 mu m. MECHANISM OF ACTION : None given.

Description

The invention relates to novel 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives and their use as antiviral agents, preferably for the treatment of picornavirus infections.

Picornaviruses, especially enteroviruses and rhinoviruses, are responsible for a wide range of human diseases. The enteroviruses include more than 60 different human pathogenic serotypes ( Melnick J in: Fields B et al., Editors. Virology. Philadelphia: Lippincott-Raven Publishers; 1996, 655-712 ). Enterovirus, echovirus, coxsackievirus A and B infections are often associated with nonspecific fever and cause upper respiratory tract disorders, which are often indistinguishable from rhinovirus infections. Haemorrhagic conjunctivitis, herpangina, hand-foot-and-mouth disease, aseptic meningitis, encephalitis, and acute myocarditis are among the more serious conditions that can also be epidemic. Different virus types can cause the same symptoms or a virus type very different clinical pictures. With the introduction of modern and sensitive methods in virus diagnostics succeeded in the detection of persistent enteroviral RNA and virus proteins in connection with chronic diseases such. As type II diabetes, poliomyositis and especially chronic myocarditis. Persistent enterovirus infections also occur in patients with agammaglobulinemia, manifesting as persistent enterovirus meningoencephalitis. Dermatomyositis or polymyositis often appeared as side effects. The rhinoviruses include about 100 serotypes. Rhinovirus infections cause more than half of all respiratory diseases of the upper respiratory tract in humans ( Couch RB in: Fields BM et al., Editors: Fields Virology, 3rd edition. Lippincott Raven, Philadelphia, 1996, 713-35 ). With a mean illness duration of approx. 10 days these usually harmless running colds lead annually to millions of physician visits, work and school failures. Complications may include otitis media, sinusitis, exacerbation of asthma and cystic fibrosis, and lower respiratory tract infections, especially in infants, the elderly, and immunocompromised patients. Because of the variety of types, vaccine prophylaxis is currently not possible. Due to the absence of work, visits to doctors and medicines associated with these diseases, rhino and enteroviruses cause enormous costs each year. The treatment of these viral infections is so far symptom-related, since no virus-specific therapeutics are available ( Redbeard HA: Antiviral Res 2002, 53 (2), 83-98 ). In addition, antibiotics are often prescribed useless. The development of new antivirals is therefore essential.

The results of the intensive search for treatment options for enterovirus and rhinovirus infections were summarized in a review by Rotbart in 2002 ( Redbeard HA: Antiviral Res 2002, 53 (2), 83-98 ). For example, ribavirin inhibits a host cell enzyme, inosine 5'-monophosphate (IMP) dehydrogenase. By eliminating this key enzyme for the synthesis of purine nucleotides, the replication of picornaviruses in vitro and in vivo can be inhibited. In addition, ribavirin should be incorporated directly into the genome of polioviruses and thereby additionally act as mutagen for RNA viruses ( Crotty S et al .: Nat Med, 2000, 6 (12), 1375-9 ). Because of severe side effects, these compounds are not used to treat rhinosome and enterovirus infections. Specific targets for inhibiting viral RNA synthesis are the genome itself, the viral RNA-dependent RNA polymerase and other viral proteins necessary for the replication complex. Guanidines, thiosemicarbazones, benzimidazoles, dipyridamoles and flavones have long been recognized as inhibitors of the polymerases of various picomaviruses known in cell culture. Very different successes were thus achieved in vivo. The most promising candidates with broad anti-enterovirus and anti-rhinovirus activity are enviroxime derivatives.

Enviroxime prevents the synthesis of plush strand RNA by binding to the viral protein 3A, which is necessary for the formation of RNA intermediates in virus replication ( Heinz BA and Vance LM: J Virol, 1995, 69 (7), 4189-97 ). In clinical trials, moderate or no therapeutic effects, poor pharmacokinetics, and undesirable side effects have been reported ( Miller FD et al .: Antimicrob Agents Chemother, 1985, 27 (1), 102-6 ). There are no clinical data on newer derivatives with better bioavailability and tolerability.

Based on the knowledge of the fine structure and function of the viral protease 2C, the protease inhibitor AG 7088 was developed. AG 7088 acts in cell culture in the nanomolar concentration range against 48 rhinovirus types as well as coxsackievirus A21, 63, enterovirus 70 and echovirus 11 ( Pattick AK et al .: Antimicrobila Agents Chemother, 1999, 43 (10), 2444-50 ). So far, the final data from the clinical trials are not known.

With the elucidation of the molecular structure of the viral capsids, the preconditions for a targeted design of capsid blockers, the "WIN substances", were created ( Diana GD: Curr Med Chem 2003, 2, 1-12 ). They prevent adsorption and / or uncoating of rhino and enteroviruses. Some of the WIN substances are highly specific only to single genera or virus types of picornaviruses. Other derivatives inhibit the proliferation of rhino and enteroviruses. The WIN substances include z. Arildone, disoxaril and pirodavir. These compounds showed very good antiviral effects in cell culture. Poor solubility (arildone), low bioavailability (arildone and disoxaril), rapid metabolism and excretion (disoxaril and WIN 54954) and side effects such as rash (WIN 54954) made clinical use impossible. Great hopes were placed on Pleconaril, another capsid blocker. Pleconaril has a very good oral bioavailability and, after binding to the hydrophobic pocket in the viral capsid, inhibits the penetration of rhino-, echo- and coxsackieviruses ( Pevear DC et al .: Antimicrob Agents Chemother 1999, 43 (9), 2109-15 ; McKinlay MA et al .: Annu Rev Microbiol 1992, 46, 635-54 ). This makes it potentially effective against a wide range of viral diseases, from the common cold to viral meningitis or myocarditis. Resistance has been observed in rhinoviruses, enterovirus 71 and coxsackievirus B3 ( Ledford RM et al .: J Virol 2004, 78 (7), 3663-74 ; Groark JM et al .: J Infect Dis 1999, 179 (6), 1538-41 ). Clinical studies in children and adults with enterovirus meningitis ( Deduction MJ et al .: Pediatr Infect Dis J, 2003, 22, 335-41 ) and rhinovirus-induced respiratory infections ( Hayden FG et al .: Antivir Ther, 2002, 7, 53-65 ; Hayden FG et al .: Clin Infect Dis, 2003, 36, 1523-32 ) went positively. However, the proven therapeutic effect was insufficient for the approval of pleconaril (Picovir, Viropharma, USA) for the treatment of rhinovirus infections in the United States. In March 2002, a request was rejected by the Food and Drug Administration (FDA) for an unfavorable benefit-risk assessment.

Pyrazolopyrimidines are also described as CRF antagonists (corticotropin-releasing factor antagonists) (eg. EP 674 642 and EP 691 128 ) which, for example, adenosine kinase ( EP 496 617 or US 4,904,666 ), xanthine oxigenase ( J. Heterocyc. Chem. 19, 1565, 1982 ) or other enzyme systems ( US 2,965,643 and US 3,600,389 ) inhibit.

Thus, an urgent task of antiviral research continues to be the development of highly effective antivirals for the treatment of rhino and enterovirus diseases. The new compounds should be well tolerated and existing resistances, e.g. B. over Pleconaril overcome.

WO 00/43394 A discloses substituted pyrazolo [3,4-d] pyrimidine derivatives and their use as antiviral agents.

Also EP 2 049 540 discloses 4-amino-3-arylamino-6-arylpyrazolo [3,4-d] pyrimidine derivatives and their use as antiviral agents.

The object of the invention is to provide further pyrazolo [3,4-d] pyrimidine derivatives and their preparation and use, which can be used as antiviral agents against enteroviruses and rhinoviruses and the stated disadvantages of the prior art, for example in terms of stability and bioavailability of Avoid substances.

wobei zumindest ein Wasserstoffatom in zumindest einer der Phenylgruppen A und B durch einen Substituenten R^H ersetzt ist, der eine Hammett-Konstante σ_p größer als 0,23 aufweist,
wobei jedes weitere Wasserstoffatom in jeder der Phenylgruppen A und B unabhängig voneinander ersetzt sein kann durch einen Rest R¹, wobei

• – jedes R¹ unabhängig ein Halogen, ein gesättigtes oder ungesättigtes, lineares oder verzweigtes aliphatisches Radikal mit 1–7 Kettengliedern, ein gesättigtes oder ungesättigtes, lineares oder verzweigtes alkanoles Radikal mit 1–8 Kettengliedern, NO₂, CN, CONR² ₂, COR², COOR², OR², SR², NR² ₂, SO₂NR² ₂, CX₃, CR²X₂, OCX₃, OCR²X₂, oder Phenyl sein kann;
• – jedes R² unabhängig Wasserstoff, ein gesättigtes oder ungesättigtes, halogeniertes oder nicht halogeniertes, lineares oder verzweigtes aliphatisches Radikal mit 1–7 Kettengliedern, Benzyl, Phenyl oder Naphtyl, ein gesättigter oder ungesättigter, Mono- oder Polyheterozyklus mit den Heteroatomen N, S oder O ist, wobei jede der vorstehenden genannten Gruppen unabhängig mit Fluor, Chlor, Brom, Trifluormethyl, Alkyl, Alkoxy, Cyano, Nitro, Amino, Aminoalkyl, C(O)-Alkyl, C(O)O-Alkyl, Benzyl, Phenyl oder Naphtyl substituiert sein kann; und
• – X unabhängig F, Cl, Br, oder I ist.

This object is achieved by specifically substituted 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives of general formula I, including their pharmaceutically acceptable salt compounds and their prodrugs,

wherein at least one hydrogen atom in at least one of the phenyl groups A and B is replaced by a substituent R ^H having a Hammett's constant σ _p greater than 0.23,
wherein each additional hydrogen atom in each of the phenyl groups A and B can be replaced independently of one another by a radical R ¹ , where

• Each R ^{1 is} independently a halogen, a saturated or unsaturated, linear or branched aliphatic radical of 1-7 chain members, a saturated or unsaturated, linear or branched alkanoles radical of 1-8 chain members, NO ₂ , CN, CONR ² ₂ , COR ² , COOR ² , OR ² , SR ² , NR ² ₂ , SO ₂ NR ² ₂ , CX ₃ , CR ² X ₂ , OCX ₃ , OCR ² X ₂ , or phenyl;
• - Each R ^{2 is} independently hydrogen, a saturated or unsaturated, halogenated or non-halogenated, linear or branched aliphatic radical having 1-7 chain members, benzyl, phenyl or naphthyl, a saturated or unsaturated, mono- or polyheterocycle having the heteroatoms N, S or O, wherein each of the foregoing groups is independently fluoro, chloro, bromo, trifluoromethyl, alkyl, alkoxy, cyano, nitro, amino, aminoalkyl, C (O) alkyl, C (O) O-alkyl, benzyl, phenyl or Naphtyl may be substituted; and
• - X is independently F, Cl, Br, or I.

In the dependent claims advantageous embodiments of the specifically substituted 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives and applications are carried out without the invention being limited thereto.

Advantageously, 4-amino-3-phenylamino-6-phenytpyrazol [3,4-d] pyrimidine derivatives of general formula I, wherein at least one hydrogen atom in at least one of the phenyl groups A and B is replaced by a substituent R ^H , selected from NO ₂ , CN, CF ₃ , CCl ₃ , CBr ₃ , OCF ₃ , OCCl ₃ , OCBr ₃ , CHF ₂ , CHCl ₂ , CHBr ₂ , OCHCl ₂ , CHO, COOH, COMe, COEt, COOMe or COOEt, preferably CF ₃ or OCF ₃ . In one or both of the phenyl groups A and B, one, two or three hydrogen atoms are advantageously replaced by a substituent R ^H. In a specific embodiment, exactly one hydrogen atom in one of the phenyl groups A and B is replaced by a substituent R ^H. The substituent R ^H may be located in the para position of the phenyl ring A or B.

In addition to R ^H , each of the phenyl groups A and B may independently carry further radicals R ¹ . Preferably, the phenyl groups A and B independently carry no, one, two or three further radicals R ¹ , preferably no or one further radical R ¹

Suitable alkyls in connection with the invention are in particular linear or branched C _1-7 -alkyls, for example methyl, ethyl, n-propyl, i-propyl and butyl. The same applies to alkanols, alkylamines and alkylamides in connection with the invention.

wobei jeder Substituent R^A, R^B unabhängig Wasserstoff, ein Halogen, ein gesättigtes oder ungesättigtes, lineares oder verzweigtes aliphatisches Radikal mit 1–7 Kettengliedern, ein gesättigtes oder ungesättigtes, lineares oder verzweigtes alkanoles Radikal mit 1–8 Kettengliedern, NO₂, CN, CONR² ₂, COR², COOR², OR², SR², NR² ₂, SO₂NR² ₂, CX₃, CR²X₂, OCX₃, OCR²X₂, oder Phenyl sein kann; und R² und X wie oben definiert sind; wobei zumindest einer der Substituenten R^A, R^B eine Hammett-Konstante σ_p größer 0,23 aufweist.In particular, 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives of general formula II are included in the invention

wherein each substituent R ^A , R ^{B is} independently hydrogen, a halogen, a saturated or unsaturated, linear or branched aliphatic radical having 1-7 chain members, a saturated or unsaturated, linear or branched alkanoles radical having 1-8 chain members, NO ₂ , CN , CONR ² ₂ , COR ² , COOR ² , OR ² , SR ² , NR ² ₂ , SO ₂ NR ² ₂ , CX ₃ , CR ² X ₂ , OCX ₃ , OCR ² X ₂ , or phenyl; and R ² and X are as defined above; wherein at least one of the substituents R ^A , R ^{B has} a Hammett constant σ _p greater than 0.23.

wobei R^H ausgewählt ist aus NO₂, CN, CF₃, CCl₃, CBr₃, OCF₃, OCCl₃, OCBr₃, CHF₂, CHCl₂, CHBr₂, OCHCl₂, CHO, COOH, COMe, COEt, COOMe oder COOEt ist, bevorzugt CF₃ oder OCF₃.The invention comprises in particular 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives of the general formula IIa

wherein R ^{H is} selected from NO ₂ , CN, CF ₃ , CCl ₃ , CBr ₃ , OCF ₃ , OCCl ₃ , OCBr ₃ , CHF ₂ , CHCl ₂ , CHBr ₂ , OCHCl ₂ , CHO, COOH, COMe, COEt, COOMe or COOEt, preferably CF ₃ or OCF ₃ .

Also included are pharmaceutical compositions containing a 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivative according to general formulas I, II, IIa or IIb. Such pharmaceutical compositions may contain other substances, for example pharmaceutically acceptable excipients and carriers. In a particular aspect, the pharmaceutical compositions may comprise other active substances, in particular antiviral agents, in particular active substances against picorniaviruses.

It has surprisingly been found that the 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives according to the invention have a markedly improved stability in liver microsomes over the substances of the prior art. In addition, studies on the pharmacokinetics in the mouse have shown that the 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives according to the invention have a significantly better bioavailability than prior art substances. At the same time, the compounds of the present invention show a strong antiviral activity against picornaviruses, in particular entero- and rhinoviruses in the nano- or micromolar concentration range.

Therefore, the pharmaceutical preparations containing a compound of the formulas I, II, IIa or IIb are particularly suitable for the treatment of respiratory infections, aseptic meningitis, encephalitis, herpangina, etc., in humans and animals, which are particularly susceptible to picornaviruses. and rhinoviruses.

The 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives are characterized in that they bear at least one substituent R ^H on one or both phenyl groups, which has a Hammett constant σ _p greater as 0.23. This value 0.23 corresponds to the Hammett constant σ _{p of} the bromine, which shows the highest Hammett constant for the para position among the halogens.

The determination of Hammett's constants for various substituents is based on the ionization constant of benzoic acid according to the Hammett equation σ _x = logK _X - logK _H where K _{H is} the ionization constant for benzoic acid in water at 25 ° C and K _{X is} the corresponding constant for a meta- or para-substituted benzoic acid. A method for determining the Hammett's constant for various substituents in meta- (σ _m ) and para-position (σ _p ) and already determined values of a variety of substituents can be found in the publication of Hansch et al., "A Survey of Hammett Substituent Constants and Resonance and Field Parameters," Chem. Rev. 1991, 97, 165-195 , incorporated herein by reference in their entirety. Of importance for the present invention is in each case exclusively the value σ for the para position (σ _p ), regardless of which position the at least one substituent R ^{H is} ultimately located.

Examples of the invention are compounds of Table 1, including their pharmaceutically acceptable salt compounds.

CRCV-340-Prodrug Also included are pro-drugs (prodrugs) of the compounds, especially those characterized by a substituent on the pyrazole heteroatom in position 1. It has been found that such compounds are converted in vivo to the 1H pyrazole compound. By way of example, mention may be made of compounds in which the pyrazole heteroatom in position 1 is substituted by an imino (phenyl) methyl substituent, for example 1- [imino (phenyl) methyl] -4-amino-3- (4-trifluoromethyl-phenyl ) amino-6-phenylpyrazolo [3,4-d] pyrimidine, also designated by the IUPAC name 1-benzylcarboximidoyl-6-phenyl-3-N- [4- (trifluoromethyl) phenyl] -1H-pyrazolo [3,4- d] pyrimidine-3,4-diamine. This is a by-product of the Preparation of the above-identified compound CRCV-340 resulting from the reaction of the compound CRCV-340 with an excess of benzamidine in the reaction mixture and having the formula:

CRCV-340 prodrug

It has been found that these compounds (e.g., CRCV-340 prodrug) are very readily converted to the target compound in vivo, such that essentially only the final drug (eg, CRCV-340) can be detected in serum ,

The invention will be explained in more detail below with reference to synthetic methods, specific 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives of the general formula (I) and their action and application against Picornavirusinfektionen.

shows a general scheme for the synthesis of pyrazolo [3,4-d] pyrimidine 1 and includes in the first step the condensation of [bis (methylthio) methylene] malononitrile 2 with phenylamines 3 in alcohol to give phenyl derivatives 4. The latter can each be isolated and purified for further reactions or used directly without purification for subsequent reaction ("one-pot" reaction). The next step is the interaction of the phenyl derivative 4 with hydrazine or hydrazine derivatives. The reaction proceeds with boiling for 1 to 4 hours and gives a high yield of pyrazole 5. The final step of the synthesis of pyrazolo [3,4-d] pyrimidine 1 consists in the condensation of pyrazole 5 with phenylamidines 6 in the presence of acetic acid , Trifluoroacetic acid or sodium acetate.

The compounds can be advantageously obtained by reacting the pyrazole (5) in the last step of the synthesis with corresponding benzamidine hydrochloride in the presence of an excess of sodium acetate at 200-220 ° C. in the absence of solvent.

Alternatively, the compounds can be obtained by reacting the pyrazole (5) in the last step of the synthesis with corresponding benzonitrile (in large excess) under microwave irradiation and in the presence of potassium tert-butoxide.

An alternative synthetic method relies on the one-pot reaction of malononitrile with aryl isothiocyanates in the presence of a base, especially sodium hydride, and subsequent treatment of the reaction mixture with an alkylating agent such as iodomethyl or dimethyl sulfate. This large quantities of an enamine are formed, which can be reacted with hydrazine to 4-cyano-3,5-diamino-pyrazole compounds.

• a) die Reaktion eines Phenylamins mit Dimethylthiocarbamoylchlorid unter Bildung eines Isothiocyanats,
• b) die Reaktion des organischen Isothiocyanats mit Malonitril und einer Base unter Bildung eines Enamin-Zwischenprodukts und anschließende Alkylierung des Enamins,
• c) die Reaktion des Produkts aus Schritt b) mit Hydrazin unter Bildung einer 4-Cyano-3,5-dimethylamino-pyrazol-Verbindung und
• d) die Reaktion des Produkts aus Schritt c) mit einem Benzamidin unter Bildung des 4-Amino-3-phenylamino-6-phenylpyrazolo[3,4-d]pyrimidin-Derivats.

The present invention therefore more particularly relates to a process for the preparation of 4-amino-3-phenylamino-6-phenylpyrazola [3,4-d] pyrimidine derivatives of general formula (I) comprising

• a) the reaction of a phenylamine with dimethylthiocarbamoyl chloride to form an isothiocyanate,
• b) the reaction of the organic isothiocyanate with malonitrile and a base to form an enamine intermediate and subsequent alkylation of the enamine,
• c) the reaction of the product of step b) with hydrazine to form a 4-cyano-3,5-dimethylamino-pyrazole compound and
• d) the reaction of the product of step c) with a benzamidine to form the 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivative.

• a) Reaktion eines Phenylamins mit Dimethylthiocarbamoylchlorid unter Bildung eines Isothiocyanats,
• b) Reaktion des Isothiocyanats mit Malonitril und einer Base unter Bildung eines Enamin-Zwischenprodukts und anschließende Alkylierung des Enamins und
• c) Reaktion des Produkts aus Schritt b) mit Hydrazin unter Bildung der 4-Cyano-3,5-diamino-pyrazol-Verbindung umfasst.

The process described is also suitable for the preparation of 4-cyano-3,5-diamino-pyrazole compounds omitting step d), the process comprising the steps

• a) reaction of a phenylamine with dimethylthiocarbamoyl chloride to form an isothiocyanate,
• b) reaction of the isothiocyanate with malonitrile and a base to form an enamine intermediate and subsequent alkylation of the enamine and
• c) reacting the product of step b) with hydrazine to form the 4-cyano-3,5-diamino-pyrazole compound.

The two aforementioned methods can be advantageously designed by isolating the isothiocyanate before the reaction in step b). It has been found that a reaction without prior isolation of the isocyanate is feasible, but that the yield of the product in the following step b) decreases from 70 to 80% to 30 to 40%. It has also been found to be useful if the isothiocyanate is isolated by vacuum distillation. Therefore, since the obtained product crystallizes relatively easily, transfer lines used in an industrial purification apparatus should be appropriately heated to prevent crystallization therein.

The process can be further advantageously characterized by isolating the alkylated enamine obtained in step b) by crystallization. It has been shown that the enamine can be advantageously purified by crystallization from water and subsequent recrystallization from ethanol, wherein the yield of the product does not fall below 70%. Such purification is therefore preferred within the scope of the described process. In the alkylation step of the enamine intermediate obtained, the use of bases such as sodium hydride, triethylamine, potassium carbonate or potassium tert-butylate has been found to be particularly advantageous. In particular, in laboratory-scale approaches, sodium hydride is a suitable base because the by-products of the alkylation can be separated relatively easily. A disadvantage of sodium hydride, however, is that its use is problematic on a larger scale, since in its reaction with protic substances, hydrogen is released and the resulting gas must be handled safely. For this reason, bases whose reaction with the enamine does not lead to the formation of hydrogen are preferred in the context of the present invention.

The alkylation of the enamine can be carried out with the aid of an alkylamide, in particular an alkyl iodide or alkyl bromide, or with the aid of an alkyl sulfate. The use of alkyl sulfates over alkyl halides, such as in particular alkyl iodides, is preferred since these are generally more expensive than the corresponding sulfates and, moreover, the high vapor pressure of, for example, methyl iodide in combination with its toxicity is problematic.

The method can be further advantageously characterized in that the hydrazine in step c) in an amount of 4 to 1 Äqualenten, preferably 1.2 to 1 Äqualenten, based on the product of step b) is used. A slight excess of hydrazine over the product of step b) has the advantage that the residual hydrazine content in the product obtained is substantially lower and can be adjusted to about 10 ppm or less. This is important for later use as a medicinal product, since hydrazine derivatives usually have a high toxicity and therefore must be removed as completely as possible from the product.

In the preparation of the 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyridine derivative as the end product, the benzamidine usually reacts in the form of the free base. This may conveniently be prepared in situ from a salt precursor by adding to the salt a suitable base. The bases used may be sodium acetate, DBU, ammonium acetate and lithium amide, as well as other bases which have a pKa value suitable for the deprotonation of the benzamidine salt.

It has been found to be advantageous when a free amidine is used. Under a free amidine is understood in the context of the present invention, an amidine, which is not in the form of a salt. Furthermore, it has been found to be advantageous if the amidine is used in an amount of 1.9 to 1.0 Äqualenten, based on the 4-cyano-3,5-diaminopyrazolvorprodukt. When using larger excesses of benzamidine, the amount of unwanted by-products, which are also difficult to remove from the desired product in the following purification steps, increases significantly. If the amount of aqualene in benzamidine is less than 1.0, these impurities can be reduced significantly, but the conversion can no longer be carried to completion, so that the overall yield in these cases is also significantly reduced.

Furthermore, it has been found that the amount of solvent used for the reaction affects the yield of the desired product. It has proven to be particularly advantageous if, in step d), a solvent is used in an amount of 1 to 3 parts, in particular about 2 parts is (here is the reference size to use). With regard to the solvent, the present invention is not subject to any relevant limitations. However, it has been found that the use of ethanol, DMSO, diphenyl ether, DMF or butanol with regard to the yield to be achieved are associated with advantages, in particular diphenyl ether and butanol, of which butanol is most preferred, since it is less with the Remove product.

In addition, the reaction temperature in the reaction in step d) plays an important role. It has been shown that a reaction temperature in step d) between 70 and 130 ° C, in particular between 80 and 100 ° C, is associated with particularly favorable yields. If a reaction temperature below 70 ° C. is selected, the reaction can not be carried to completion or the reaction times to completion are very long. At reaction temperatures of more than 130 ° C, however, the amount of undesired by-products increases.

Furthermore, it has been shown that the reaction time in step d) should be expediently between 12 and 24 hours, in particular between 16 and 20 hours. At reaction times of less than 12 hours, the reaction usually does not proceed completely, while at reaction times of more than 24 hours, the costs of the process increase disproportionately without a significantly improved conversion would be observed.

The final product obtained can then be purified by recrystallization. Any solvent suitable for this purpose can be used for the recrystallization, as long as it has a sufficient solubility in hot form, but a low solubility in the cold form for the product. Examples of solvents which may be mentioned at this point are 1-butanol, toluene, ethanol, dichloromethane and ethyl acetate. For the recrystallization in particular a mixture of THF and toluene as solvent has proven to be particularly expedient, since this mixture is able to provide the product in purities of> 99% and yields of 60 to 70% by a single recrystallization.

Alternatively, the process described above can also be carried out by the condensation of the pyrazole obtained from step c) with benzamidines in the presence of acid such as acetic acid or trichloroacetic acid or their salts in acetate in the final synthesis step d) for the preparation of pyrazolo [3,4-] pyrimidine derivatives (1) are reacted.

The following examples list specific compounds of the general formula (I) which are preferably suitable for applications against picornavirus infections, the compounds being in a solution or suspension in a pharmaceutically acceptable aqueous, organic or aqueous-organic medium for the local or parenteral Application by intravenous, subcutaneous or intramuscular injection or for intranasal administration or in the form of a tablet, capsule or aqueous suspension with conventional carrier for oral administration or as a suppository.

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

Preparation and analysis of 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives

The structure of the compounds of the invention was determined by the nature of the synthesis, elemental analysis, NMR and mass spectroscopy.

Starting materials:

The 5-amino-4-cyano-3-phenylaminopyrazoles were prepared according to the method described in shown methods and after the description of Tominaga Y et al. (J.Heterocycl.Chem., 1990, 27, 775-779 ), synthesized. Arylamidines are synthesized according to the known state of the art from the corresponding cyan starting compounds ( Boere, RT et al .: J. Organomet. Chem., 1987, 331, 161-167 ; Garigipati RS: Tetrahedron Lett., 1990, 31, 1969-1978 ; Then O et al .: Justus Liebigs Ann. Chem., 1982, 1836-1839 ).

Reference Example 1: 4-Amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine

(3-N, 6-diphenyl-2H-pyrazolo [3,4-d] pyrimidine-3,4-diamine)

Hellgelber, fester kristalliner Stoff. Ausbeute 57%. mp 253–5°C. R_f(Chloroform – Methanol; 10/1) – 0.8 (Silikagel 60). MS m/z 302 (M⁺).With stirring, to 2.3 g (11.5 mmol) of 5-amino-4-cyano-3-phenylaminopyrazole are added 3.0 g (17.24 mmol) of benzamidine hydrochloride hydrate and 2.2 g (23.0 mmol) of sodium acetate added. The reaction mixture is heated at 220 ° C for 30 minutes. The resulting material is treated with 50 ml of water, filtered and washed with 20 ml of cold methanol and 20 ml of cold ester. The product is purified by crystallization from DMF / water.

Pale yellow, solid crystalline substance. Yield 57%. mp 253-5 ° C. R _f (chloroform-methanol; 10/1) - 0.8 (silica gel 60). MS m / z 302 (M ⁺ ).

Reference Example 2: 4-Amino-3- (4-chlorophenyl) amino-6-phenylpyrazolo [3,4-d] pyrimidine

(3-N- (4-chlorophenyl) -6-phenyl-2H-pyrazolo [3,4-d] pyrimidine-3,4-diamine)

Reference Example 3: 4-Amino-3- (3,4-difluorophenyl) amino-6-phenylpyrazolo [3,4-d] pyrimidine (3-N- (3,4-difluorophenyl) -6-phenyl-2H-pyrazolo [ 3,4-d] pyrimidine-3,4-diamine)

Example 1: 4-Amino-3- (4-trifluoromethylphenyl) amino-6-phenylpyrazolo [3,4-d] pyrimidine (CRCV-340)

(6-phenyl-3-N- [4- (trifluoromethyl) phenyl] -2H-pyrazolo [3,4-d] pyrimidine-3,4-diamine)

The preparation was carried out as described in Reference Example 1, using the appropriately substituted precursor compounds.

Example 2: 4-Amino-3-phenylamino-6- [4- (trifluoromethoxy) -phenyl] pyrazolo [3,4-d] pyrimidine (MS-112)

(3-N-phenyl-6- [4- (trifluoromethoxy) phenyl] -2H-pyrazolo [3,4-d] pyrimidine-3,4-diamine)

The preparation was carried out as described in Reference Example 1, using the appropriately substituted precursor compounds.

Preparation of CRCV-340 "one pot reaction" of malononitrile with aryl isothiocyanates in the presence of a base

In a vessel, 4-trifluoromethylaniline and dimethylthiocarbamoyl chloride were reacted in toluene. After completion of the reaction, the formed salt (dimethyl ammonium chloride) was filtered off and the reaction solution was evaporated to dryness. Finally, the resulting product was purified by vacuum distillation. As the product, a material crystallizing at room temperature was obtained.

In the next step malonitrile was deprotonated by addition of sodium hydride (60%) in DMF solution. To this solution was added 4-trifluoromethylphenyl isothiocyanate dissolved in DMF at room temperature. The resulting enamine intermediate was then reacted by adding methyl iodide to the desired enamine. The isolation was achieved by crystallization of the enamine from water with the addition of seed crystals and by double recrystallization from ethanol. The yield was 70 to 80%.

The enamine obtained was dissolved in ethanol and reacted by adding different amounts of hydrazine hydrate and then refluxing the reaction mixture. Subsequently, the product obtained was crystallized by addition of water and recrystallized from mixtures of water and ethanol. The yield was 60%.

Table 2 shows the results of reactions with different amounts of hydrazine, the residual amounts of hydrazine being indicated in the obtained recrystallized product. It turns out that the use of a Äqualentmenge of hydrazine of 1.05, based on the starting material with a high yield with high purity and low residual hydrazine content of only 1 ppm can be obtained. Table 2: Hydrazine equivalents for the 3rd stage synthesis of CRCV 340 entry Equivalent amount of hydrazine Purity [% -a / a] Yield [%] Residual hydrazine [ppm] 1 4.00 > 99.95 93.6 22 2 1.05 > 99.95 92.6 1

In the final step, the resulting pyrazole was reacted with benzamidine hydrochloride to form the pyrazolo [3,4-d] pyrimidine. The reactions were carried out at various temperatures in the range of 140 ° C to 170 ° C and overpressure in diphenyl ether and 1-butanol and with various bases, with purities in the range of up to about 79% could be achieved (see Table 3). Table 3: Screening of different bases for deprotonation entry solvent base Temperature [° C] Sales [% -a / a] Purity [% -a / a] Pollution at RRT = 1.31 [% -a / a] Pollution at RRT = 0.64 [% -a / a] 1 Diphenyl ether Sodium acetate 140 31.6 19.7 1.6 <0.05 2 Diphenyl ether DBU 140 89.7 74.8 0.7 1.0 3 Diphenyl ether NH ₄ OAc 140 38.1 7.9 2.5 <0.05 4 Diphenyl ether LiNH ₂ 140 91.5 49.1 12.4 5.6 5 Diphenyl ether Sodium acetate 170 81.0 60.0 1.7 0.7 6 Diphenyl ether DBU 170 95.2 79.0 0.3 4.4 7 Diphenyl ether NH ₄ OAc 170 81.8 60.0 1.0 1.0 8th Diphenyl ether LiNH ₂ 170 98.3 39.4 2.0 34.8 9 butanol Sodium acetate 140 42.6 29.6 0.4 <0.05 10 butanol DBU 140 87.2 72.6 0.6 1.0 11 butanol NH ₄ OAc 140 24.7 15.2 0.04 <0.05 12 butanol LiNH ₂ 140 79.6 52.2 0.4 9.1 13 butanol Sodium acetate 170 74.9 61.6 0.5 0.7 14 butanol DBU 170 88.2 73.9 0.6 3.0 15 butanol NH ₄ OAc 170 66.7 53.3 0.4 0.4 16 Bitanol LiNH ₂ 170 88.9 52.2 0.4 18.5

The main impurities were determined by RP-HPLC. The RRT (relative retention time relative to CRCV 340) 1.31 has a mass of 473.

In a subsequent experiment, the influence of the amount of solvent on the reaction is examined using 1-butanol as solvent and the benzamidine as the hydrochloride in a Equivalent amount of 2 to 2.5 based on the 4-cyano-3,5-diamino-pyrazole compound was used. The results of these investigations are shown in Table 4. Table 4: Screening of different amounts of solvent. entry Solvent quantity [volume] Temperature [° C] Sales [% -a / a] Purity [% -a / a] Pollution at RRT = 1.31 [% -a / a] Pollution at RRT = 0.64 [% -a / a] 1 2 90 82.9 79.5 1.00 <0.05 2 4 90 76.9 72.7 1.40 0.12 3 6 90 81.4 74.9 2.40 0.26

In a further experiment, the influence of the equivalent amount of benzamidine hydrochloride on the reaction was investigated. The results of these investigations are shown in Table 5. Table 5: Screening of different benzamidine equivalent amounts. solvent Equivalent benzamidine Temperature [° C] Sales [% -a / a] Purity [% -a / a] Pollution at RRT = 1.31 [% -a / a] Pollution at RRT = 0.64 [% -a / a] butanol 1.00 90 76,90 72.70 1.40 0.12 butanol 1.25 90 89,80 80.70 2.90 0.27 butanol 1.50 90 94,90 81.50 4.40 0.35 butanol 2.00 90 97.60 83,00 3.90 0.44

In these studies, it has been found that an equalenment amount of benzamidine in the range of 1 to 1.25 gives a conversion in the range of about 90%, at the same time the content of impurities is relatively low.

In a final experiment, the optimized reaction conditions were tested with 1-butanol as solvent. For this purpose, the benzamidine was used as the free base. After cooling the reaction mixture of the reaction with benzamidine to 0-5 ° C, the crude product obtained was washed with 2 volumes of butanol and dried at 60 ° C / 50 mbar. The yield was 55 to 65% with a purity of more than 95%. The results of these investigations are shown in Table 6: TABLE 6 Reaction under Optimized Reaction Conditions Amount of solvent Equivalent benzamidine Temperature [1 ° C] Purity [% -a / a] Pollution at RRT = 1.31 [% -a / a] Pollution at RRT = 0.64 [% -a / a] 2 1.05 90 97.4 1.22 <0.05 2 1.05 90 95.17 1.78 <0.05 2 1.10 90 96,87 1.88 <0.05 2 1.15 90 95.89 2.54 <0.05

Under these optimized reaction conditions, it turns out that an amount of 1.05 to 1.15 equivalents of benzamidine (relative to CRCV-340), two units of butanol as a solvent, a reaction temperature of 90 ° C and a reaction time of 18 hours one Content of impurities at 1.31 RRT or 0.64 RRT of ≤ 1.5% and <0.05%, respectively. Subsequent recrystallization of the resulting products in a mixture of 6 units of THF and 7 units of toluene afforded the product with a purity in the range of greater than 99% and a content of impurities at RRT = 1.31 and RRT = 0.64 of <0 , 37 or <0.05 (Table 7). Table 7: Purity before and after recrystallization of the product obtained. solvent Rough crude product [% -a / a] Pollution at RRT = 1.31 [% -a / a] Pollution at RRT = 0.64 [% -a / a] Purity of final product [% -a / a] Pollution at RRT = 1.31 [% -a / a] Pollution at RRT = 0.64 [% -a / a] THF / toluene 6: 7 97.49 1.65 <0.05 99.62 0.37 <0.05 THF / Taluol 6: 7 95.17 1.17 <0.05 99.48 0.32 <0.05

ADMET studies on the metabolism of OBR 5-340 in vitro

Objective: To examine absorption (absorption), distribution, biochemical degradation (metabolism), secretion (excretion) and toxicity (abbreviated to ADMET) of OBR 5-340 in vitro, as well as in vitro studies for plasma protein binding and stability in plasma and liver microsomes.

Investigations: The in vitro tests performed with CRCV-340 and the data obtained are summarized in Table 8 in comparison to Ref-2. Table 8: Summary of results from the ADMET studies for CRCV-340 compared to Ref-2. Test parameters Ref-2 CRCV-340 Binding to plasma protein 99.5% 96.7% Release of plasma protein 61.0% 122.3% plasma stability 117.0% 100.0% Stability in liver microsomes 32.0% 112.0%

Summary: Substance CRCV-340 was characterized by a better stability in liver microsomes as well as a stronger release of plasma protein compared to Ref-2.

Investigation of the pharmacokinetics of CRCV-340 in the mouse

Objective: To collect pharmacokinetic data on CRCV-340.

Investigations: The substance and the reference substance were each administered once to the mice in a concentration of 100 mg / kg body weight, in 0.5 ml of a 10% Cremophor solution per os. After 0.5, 1, 2, 3, 4, 5, 6 and 7 h, the serum was recovered and determined by HPLC analysis, the concentrations of CRCV-340, Ref-2 and Ref-3 in the plasma of the mice.

The pharmacokinetic parameters of the substances were calculated with the computer program ESTRIP on the basis of the substance concentrations in the blood plasma determined by means of HPLC (Table 9). Table 9: Pharmacokinetic data after single intragastric administration of 100 mg / kg CRCV-340, Ref-2 and Ref-3 in mice. substance C _max T _max MRI T _1/2 K _el CL V _d AUC _0-t AUC _{0 -∞} C _max / AUC _0-t (Ng / ml) (H) (H) (H) (h ^-1 ) (ml × h / kg) (Ml / kg) (ng × ml / h) (ng × ml / h) (h ^-1 ) Ref-2 1,295.4 1.0 2.14 2.96 0.23 36.6 156.7 2,354.5 2,732.1 0.55 Ref-3 424.5 0.5 2.44 1.47 0.55 91.8 168.1 1085.7 1,089.4 0.39 CRCV-340 1,254.4 3.0 3.54 3.54 0.18 10.4 52.7 6,418.3 9,598.5 0.20 C _max maximum concentration in the blood
T _max Time to reach the maximum concentration in the blood
MRI mean residence time in the blood (mean residence time)
T _1/2 half-life
K _el elimination constant
CL clearance
V _d volume of distribution
AUC area under the curve (area under curve)
C _max / AUC absorption rate from stomach to blood

Result: The pharmacokinetic studies in the mouse showed a significantly better bioavailability for CRCV-340 than for Ref-2 and Ref-3.

Acute toxicity studies of CRCV-340 in the mouse

Objective: To determine the 50% lethal dose of CRCV-340

Investigations: The acute toxicity of the substance CRCV-340 was tested in 19.5-20.5 g mice given per os 40, 60, 80 or 120 mg substance / mouse (5 mice per substance and concentration) , This corresponds to a dose of about 2g, 3g, 4g or 6g per kilogram of body weight. The toxic effects observed in the result correlated with the administered substance dose. At the 40 and 60 mg / mouse doses, co-ordination disorders and hyperactivity were observed as side effects up to 3 h after substance administration. After application of the two higher dosages, respiratory problems, aggressive behavior and hyperkinesia were added. For CRCV-340, an LD ₅₀ of 3120 mg / kg was determined (Table 10). Table 10: Results of the acute toxicity study of the substance CRCV-340 after a single peroral administration in the mouse dose Dead / survivors 72 h after application of Ref-2 CRCV-340 40 mg / mouse 0/5 1.5 60 mg / mouse 1.5 1.5 80 mg / mouse 3.5 3.5 120 mg / mouse 4.5 5.5 LD ₅₀ 78.6 mg / mouse (3930 mg / kg) 62.4 mg / mouse (3120 mg / kg)

Result: The survival data obtained confirm a very good tolerability of both test substances.

Long-term toxicity studies in the mouse

Objective: To determine the subacute toxicity in the mouse to avoid possible side effects. For example, the general condition, weight, internal organs, and metabolism that could occur with subcutaneous administration of CRCV-340 over 28 days.

Investigations: For this, approximately 20 g male and female mice were given 12.5, 50 or 200 mg / kg of the substance CRCV-340 in a cremophor formulation or the control group Cremophor once daily for 28 days intragastrically administered via a probe. Throughout the experimental period, the condition of the coat and mucous membranes, excreta and general condition were evaluated. The logging of the weight took place on days 7, 14, 21 and 28. Approx. 24 hours after the last substance administration, the orientation of the animals was examined. The serum analyzed the following biochemical parameters: protein content, urea, creatinine, the serum activity of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase. The hemoglobin concentration, hematocrit, the number of red and white blood cells and blood platelets were determined in the blood smear. At the end of the experiment, the mice were sacrificed and dissected under anesthesia, the state of the internal organs was estimated macroscopically, the organ weight was determined and specimens were placed in formalin for subsequent histological examinations.

Summary of results: The results show a very good compatibility of the substance concentrations examined. Neither the general condition of the animals, their fur, the excreta nor the orientation ability or the mobility changed as a result of the substance giving. Also in terms of body weight dynamics, the CRCV-340-treated animals did not differ from the control group. There were no differences between the substance-treated and vehicle-treated experimental groups in the blood smear and the biochemical blood parameters. The same applies to the macroscopic examination of the internal organs (liver, kidney, heart, lungs, spleen, pancreas and testes), the organ weights and the results of the histological examinations of the heart, lung, liver, spleen, thymus and pancreas , Kidney, glandular, testicular, gastric and intestinal tissue samples (results not shown).

Determination of antiviral spectrum against rhinoviruses

Aim: To determine the antiviral activity spectrum of CRCV-340 against 50 different human rhinovirus serotypes and CVB3 patient isolates.

Investigations: The 50 HRV serotypes and 20 clinical CVB3 isolates were propagated in HeLa, HeLa Wis and / or LF cells, their titer determined and serotyped by serotyping. Subsequently, zpE (cytopathic effect) inhibition tests with the HRV and plaque reduction tests with the CVB3 isolates were established. In the corresponding antiviral tests, dose-response studies with Ref-3 and CRCV-340 were performed. Tables 11 and 12 provide an overview of the determined 50% inhibitory concentrations. TABLE 11 Overview of the spectrum of action of Ref-3 and CRCV-340 against HRV. In summary, the means and standard deviations were separated for all 50 serotypes tested as well as for the 45 pleconaril-sensitive and 5 pleconaril-resistant serotypes. substance IC ₅₀ [μM] all HRV (n = 50) IC ₅₀ [μM] pleconaril-sensitive HRV (n = 45) IC ₅₀ [μM] pleconaril-resistant HRV (n = 5) Number of sensitive HRV mean SD mean S. D mean SD Ref-3 16,11 11.94 18.52 10.88 0.07 0.01 23 CRCV-340 25.73 21,49 26,09 18,98 22,62 40.69 49 Table 12: Overview of the spectrum of action of Ref-3 and CRCV-340 against the clinical CVB3 isolates. In summary, the mean and standard deviation were separated for all 20 isolates tested, as well as for the 19 pleconaril-sensitive isolates and the one pleconaril-resistant serotype. substance IC ₅₀ [μM] all CVB3 (n = 20) IC ₅₀ [μM] pleconaril-sensitive CVB3 (n = 19) IC ₅₀ [μM] pleconaril-resistant CVB3 (n = 1) Number of sensitive CVB3 mean SD mean S. D mean SD Ref-3 0.68 0.90 0.71 0.91 0.13 20 CRCV-340 4.97 4.05 5.16 4.10 1.39 18 * * maximum tested concentration 13.5 μM

Summary of results: The spectrum of activity of CRCV-340 compared to HRV was significantly broader compared to Ref-3.

Investigations on the antiviral effect in vivo in the mouse model of CVB3-induced chronic myocarditis

Objective: To confirm the antiviral effects of CRCV-340 in the mouse model

Investigations: The effect of CRCV-340 was investigated in the model of CVB 3-induced myocarditis in 8-week-old male NMRI mice. In addition, CVB3 31-1-93 or CVB3 H3 infected animals received 100 mg / kg of the substance in 50% or 20% PEG-400 in 1% CMC in water once or twice daily (Placebo) administered over 7 days. Parameters for evaluating the therapeutic effect included changes in body weight, general condition, virus titers in cardiac and pancreatic tissue, and histopathological changes in the heart and pancreas. Mock-infected, placebo-treated or CRCV-340-treated animals served as a negative control and infected, placebo- or pleconaril-treated animals as a positive control in the course of infection.

Summary of results: In contrast to placebo and pleconaril, CRCV-340 was shown to mediate CVB3 31-1-93-induced chronic myocarditis in NMRI mice. Clinically and statistically significant effects were observed for all investigated parameters (body weight, general condition, virus titers in cardiac and pancreatic tissue on day 7 p.i, histopathology in the heart and pancreas on day 7 and 21 p.i.).

Studies on the in vivo efficacy of CRCV-340 in the lethal mouse model compared to the reference substance pleconaril

Objective: To verify the antiviral effects of CRCV-340 in the lethal mouse model

Investigations: The effect of the development candidate CRCV-340 was investigated after testing the infection dose in the model of CVB 3-induced myocarditis in 6-7 week old male BALB / c mice. To this end, CVB3 31-1-93 and CVB3 H3-infected animals were given 100 mg / kg twice daily of the substance in 20% PEG-400 in 1% CMC in water (placebo) for 7 days. Changes in body weight, general condition and lethality defined by the surrogate marker 25% body weight loss served as parameters to evaluate the therapeutic effect. Mock-infected, placebo-treated or CRCV-340-treated animals were the negative control and infected, placebo or pleconaril-treated animals were the positive control in the course of infection.

RESULTS: In contrast to placebo and pleconaril, CRCV-340 was active in the lethal BALB / c mouse model after infection with CVB3 31-1-93. Clinically and statistically significant effects were observed for all investigated parameters (body weight, general condition and lethality).

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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Claims (15)

4-Amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives of the general formula I

or a pharmaceutically acceptable salt or prodrug thereof, wherein at least one hydrogen atom in at least one of the phenyl groups A and B is replaced by a substituent R ^H having a Hammett's constant σ _p greater than 0.23, each further hydrogen atom in each of the phenyl groups A and B may be independently replaced by a group R ¹ , wherein each R ^{1 is} independently a halogen, a saturated or unsaturated, linear or branched aliphatic radical of 1-7 chain members, a saturated or unsaturated, linear or branched alkanoles Radical with 1-8 chain members, NO ₂ , CN, CONR ² ₂ , COR ² , COOR ² , OR ² , SR ² , NR ² ₂ , SO ₂ NR ² ₂ , CX ₃ , CR ² X ₂ , OCX ₃ , OCR ² X ₂ , or phenyl; each R ^{2 is} independently hydrogen, a saturated or unsaturated, halogenated or non-halogenated, linear or branched aliphatic radical of 1-7 chain members, benzyl, phenyl or naphthyl, a saturated or unsaturated, mono- or polyheterocycle having the heteroatoms N, S or O. wherein each of the foregoing groups is independently fluoro, chloro, bromo, trifluoromethyl, alkyl, alkoxy, cyano, nitro, amino, aminoalkyl, C (O) -alkyl, C (O) O-alkyl, benzyl, phenyl or naphthyl may be substituted; and X is independently F, Cl, Br, or I. 4-Amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives according to claim 1, characterized in that the at least one substituent R ^{H is} independently NO ₂ , CN, CF ₃ , CCl ₃ , CBr ₃ , OCF ₃ , OCCl ₃ , OCBr ₃ , CHF ₂ , CHCl ₂ , CHBr ₂ , OCHCl ₂ , CHO, COOH, COMe, COEt, COOMe, or COOEt, preferably CF ₃ or OCF ₃ . 4-Amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives according to Claim 1 of the general formula II

wherein each substituent R ^A , R ^{B is} independently hydrogen, a halogen, a saturated or unsaturated, linear or branched aliphatic radical having 1-7 chain members, a saturated or unsaturated, linear or branched alkanoles radical having 1-8 chain members, NO ₂ , CN , CONR ² ₂ , COR ² , COOR ² , OR ² , SR ² , NR ² ₂ , SO ₂ NR ² ₂ , CX ₃ , CR ² X ₂ , OCX ₃ , OCR ² X ₂ , or phenyl; wherein R ² and X are as defined in claim 1; characterized in that at least one of the substituents R ^A , R ^{B has} a Hammett constant σ _p greater than 0.23. 4-Amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives according to one of claims 1 or 2 of general formula IIa

characterized in that R ^{H is} NO ₂ , CN, CF ₃ , CCl ₃ , CBr ₃ , OCF ₃ , OCCl ₃ , OCBr ₃ , CHF ₂ , CHCl ₂ , CHBr ₂ , OCHCl ₂ , CHO, COOH, COMe, COEt, COOMe or COOEt, preferably CF ₃ or OCF ₃ . 4-Amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives according to claim 4 of the formula

A pharmaceutical composition containing a 4-amino-3-phenylamino-6-phenylpyrazolo] 3,4-d] pyrimidine derivative according to any one of the preceding claims. A pharmaceutical composition according to claim 6, further comprising a pharmaceutically acceptable carrier. A pharmaceutical composition according to claim 6 or 7, further comprising one or more further active ingredients. A pharmaceutical composition according to claim 8, wherein the one or more further active agents are antiviral agents, preferably antipornaviral agents. A process for producing a 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivative according to any one of claims 1 to 5, which comprises a) reacting a phenylamine with dimethylthiocarbamoyl chloride to form an isothiocyanate, b) reacting the organic isothiocyanate with malononitrile and a base to form an enamine intermediate and subsequent alkylation of the enamine, c) reaction of the product of step b) with hydrazine to form a 4-cyano-3,5-diamino-pyrazole compound and d) reaction the product of step c) with a benzamidine to form the pyrazolo [3,4-d] -pyrimidins, wherein at least one hydrogen atom in at least one of the phenyl groups a and B is replaced by a substituent R ^a which a Hemmet constant σP of > 0.23. A process for producing a 4-cyano-3-phenylamino, 5-aminopyrazole compound comprising a) reaction of an organic primary amine with dimethylthiocarbamoyl chloride to form an isothiocyanate, b) reaction of the isothiocyanate with malonitrile and a base to form an enamine intermediate and subsequent alkylation of the enamine and c) reaction of the product of step b) with hydrazine to form the 4-cyano-3,5-diamino-pyrazole compound. A process for the preparation of a pharmaceutical composition comprising formulating a 4-amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivative according to any one of claims 1-5 with a pharmaceutically acceptable carrier. 4-Amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives according to any one of claims 1-5 for use as a medicament. 4-Amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives according to any one of claims 1-5 for use in the prophylactic or therapeutic treatment of viral infections. 4-Amino-3-phenylamino-6-phenylpyrazolo [3,4-d] pyrimidine derivatives according to claim 12, characterized in that the viral infections are picornavirus infections.