Molecules inhibiting a metabolic pathway involving the syk protein tyrosine kinase and method for identifying said molecules

Abstract

The present invention relates to the C-13 molecule (methyl 2-{5-[(3-benzyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}-benzoate) and to organic molecules functionally equivalent to the C-13 molecule, capable of inhibiting the binding of an antibody or antibody fragment with the human Syk protein tyrosine kinase, to the use of these molecules for the production of medicaments for the prevention or treatment of diseases dependent on metabolic pathways involving Syk, and also to a method for identifying such molecules.

1 . A compound which binds with Syk tyrosine kinase protein on a three-dimensional cavity comprising the Arginine residue situated in position 68 and the two glutamic acid residues situated in positions 121 and 155 of human Syk protein, the sequence of which is set forth in SEQ ID No. 1, and capable of inhibiting by at least 10% in vitro binding of
 (i) antibody fragment G4G11 (SEQ ID No. 2), or   (ii) an antibody or antibody fragment which binds with human Syk tyrosine kinase protein on an epitope comprising at least one of residues 65 to 74 of the amino acid sequence of human Syk tyrosine kinase protein represented by the sequence SEQ ID No. 1, or   (iii) an antibody or antibody fragment which binds with human Syk tyrosine kinase protein and inhibiting by at least 10% the binding of antibody fragments G4G11 with human Syk tyrosine kinase protein (SEQ ID No. 1),   
       with human Syk tyrosine kinase protein or with any of the variants thereof in animals, for the prevention or treatment of a condition dependent on a metabolic pathway involving Syk in humans or animals, where said compound (C-13) has the formula 
       
         
           
           
               
               
           
         
       
       or an organic compound functionally equivalent to molecule C-13 having a molecular weight between 50 and 2500 Dalton, selected from compounds having any of the following formulae: 
       
         
           
           
               
               
           
         
       
       where
 R1 is an unsubstituted or substituted aromatic group, or an unsubstituted or substituted heterocycle comprising at least one S, O or N atom; 
 R2 is an unsubstituted or substituted aromatic group, an unsubstituted or substituted heterocycle, an unsaturated or saturated carbon chain comprising an amine group, an unsaturated or saturated carbon chain comprising an unsubstituted or substituted aromatic group or an unsaturated or saturated carbon chain comprising an unsubstituted or substituted heterocycle comprising at least one S, O or N atom; 
 R3 is an unsubstituted or substituted phenyl, 2-pyridinyl, 3-pyridinyl or 4-pyridinyl group; 
 
       or 
       
         
           
           
               
               
           
         
       
       where
 n=0 or 1; n′=0 or 1; 
 R4 is an unsaturated or saturated carbon chain comprising 1 to 5 carbon atoms, unsubstituted or substituted with an aromatic group; 
 R5 is an unsubstituted or substituted aromatic group or an unsubstituted or substituted amine group; 
 R6 is a hydrogen atom, an alkoxy group, an alkyl group or a halogen; 
 R7 is a hydrogen atom, an alkoxy group, an alkyl group or a halogen; 
 R8 is a hydrogen atom, an alkoxy group, an alkyl group or a halogen; 
 
       or 
       
         
           
           
               
               
           
         
       
       where
 m=0, 1 or 2; 
 R9 is a hydrogen atom and R10 is an unsubstituted or substituted phenyl group, or R9 and R10 are part of the same unsubstituted or substituted heterocycle, or 
 R9 and R10 are part of the same unsubstituted or substituted aromatic group; 
 R11 is a hydrogen atom, an alkoxy group or an alkyl group; 
 R12 is a hydrogen atom, an alkoxy group or an alkyl group; 
 R13 is a hydrogen atom or an alkyl or alkoxy group; 
 R14 is a hydrogen atom or an alkyl or alkoxy group; 
 
       
         
           
           
               
               
           
         
       
       or 
       where
 A is an oxygen or sulphur atom; 
 R15 is an unsaturated or saturated carbon chain comprising 1, 2 or 3 carbon atoms, unsubstituted or substituted by an unsubstituted or substituted aromatic group, an unsubstituted or substituted heterocycle or an amine group belonging to unsubstituted or substituted heterocycle; 
 R16 is a hydrogen atom, a halogen or an alkoxy group; 
 R17 is a hydrogen atom, an alkoxy group or an acetoxy group; 
 
       or 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       or a stereo-isomer, racemate or pharmacologically acceptable salt of C-13 or said equivalent compound. 
     
     
         2 . A compound according to  claim 1 , wherein said compound binds with Syk tyrosine kinase protein at a site located outside the catalytic domain thereof. 
     
     
         3 . A compound according to  claim 1 , wherein said antibody or antibody fragment binds with human Syk tyrosine kinase protein on an epitope comprising at least 5 of residues 65 to 74 of the amino acid sequence of human Syk tyrosine kinase protein illustrated by SEQ ID No. 1. 
     
     
         4 . A compound according to  claim 1 , wherein said three-dimensional cavity further comprises the Serine residue situated in position 9, the Glutamine residue situated in position 43, the Phenylalanine residue situated in position 51, the Isoleucine residue situated in position 66, the Glutamate residues situated in position 67 and 69, the Leucine residue situated in position 70, the Asparagine residue situated in position 71, the Glycine residue situated in position 72, the Threonine residue situated in position 73, the Tyrosine residue situated in position 74 and the Alanine residue situated in position 75 of human Syk protein, the sequence of which is illustrated by SEQ ID No. 1. 
     
     
         5 . A compound according to  claim 1  wherein said compound is used for the prevention or treatment of type I hypersensitivity reactions. 
     
     
         6 . A compound according to  claim 5 , wherein said compound inhibits IgE-dependent mast cell degranulation. 
     
     
         7 . A compound according to  claim 6 , wherein said compound is capable of inhibiting, by 50% in vitro, mast cell degranulation at a concentration (IC50) between 1 ng/ml and 1 mg/ml. 
     
     
         8 . A compound according to  claim 5 , wherein said the metabolic pathway involving Syk is a mast cell or basophil activation pathway. 
     
     
         9 . Molecule A compound according to  claim 5 , wherein said condition is allergic asthma, allergic conjunctivitis, allergic rhinitis, anaphylaxis, angioedema, urticaria, eosinophilia or an allergy to an antibiotic. 
     
     
         10 . A compound according to  claim 5 , wherein said compound has no effect on the metabolic pathways involving human Syk protein (SEQ ID No. 1) other than those giving rise to mast cell degranulation and/or type I hypersensitivity reactions. 
     
     
         11 . A compound according to  claim 10 , wherein said compound has no effect on the antibody response which follows immunisation by a thymus-dependent antigen or on neutrophil-dependent Syk recruitment. 
     
     
         12 . A compound according to  claim 1 , wherein said metabolic pathway involving Syk is a B lymphocyte, T lymphocyte, neutrophil, eosinophil, NK cell, platelet, erythrocyte, osteoclast, epithelial cell or cancer cell activation pathway. 
     
     
         13 . A compound according to  claim 12 , wherein said condition is rheumatoid arthritis, an autoimmune disease, inflammation or cancer. 
     
     
         14 . A compound according to  claim 1 , wherein said compound is used in combination with a further therapeutic molecule. 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . A compound according to  claim 1 , wherein said compound is intended to be administered by the oral, sublingual, nasal, ocular, local, intravenous, intraperitoneal, subcutaneous route, by aerosol or by inhalation. 
     
     
         18 . A compound according to  claim 1 , wherein said compound is intended to be administered to adult, child or newborn human patients. 
     
     
         19 . A compound according to  claim 1 , wherein said compound is intended to be administered at doses between 0.01 mg/kg and 200 mg/kg. 
     
     
         20 . A compound according to  claim 19 , wherein said equivalent compound is selected from compounds having any of the following formulae:
 formula (I) where
 R1 is
 a phenyl group, unsubstituted or substituted by an F or Cl atom, a methyl or ethyl group, an N,N-dimethyl-sulphonamide or two groups selected from the methyl, ethyl, hydroxy, methoxy or ethoxy groups, 
 or a group 
 
   
       
         
           
           
               
               
           
         
         
           
             a furan group unsubstituted or substituted by a methyl, ethyl, hydroxyl, methoxy or ethoxy group, 
             a thiophene group unsubstituted or substituted by a methyl, ethyl, hydroxy, methoxy or ethoxy group; 
           
           R2
 a group 
 
         
       
       
         
           
           
               
               
           
         
         
           
             where R21 and R22 are carbon atoms each belonging to an alkyl chain comprising 1, 2 or 3 carbon atoms, or both belonging with the nitrogen atom with which they are bound to the same unsaturated or saturated heterocycle also comprising an oxygen atom or a second nitrogen atom, 
             or a group 
           
         
       
       
         
           
           
               
               
           
         
         
           
             where R23 and R24 are carbon atoms each belonging to an alkyl chain comprising 1, 2 or 3 carbon atoms, or both belonging with the nitrogen atom with which they are bound to the same unsaturated or saturated heterocycle also comprising an oxygen atom or a second nitrogen atom, 
             or a group 
           
         
       
       
         
           
           
               
               
           
         
         
           and R3 is
 a non-substituted 2-pyridinyl, 3-pyridinyl or 4-pyridinyl group, 
 a phenyl group unsubstituted or substituted by a benzoxy group, and/or by a hydroxyl group, and/or by a methyl group, and/or by an ethyl group, and/or by a propyl group, and/or by one or two Br, F or Cl atoms, and/or by one to three hydroxyl, methoxy or ethoxy groups. 
 
         
       
       or
 formula (II) where
 R4 is an unsaturated or saturated carbon chain comprising 1, 2 or 3 carbon atoms; 
 R5 is a phenyl group or a secondary amine group unsubstitued or substituted by an unsubstituted or substituted phenyl group, or by a group 
 
 
       
         
           
           
               
               
           
         
         
           R6 is a hydrogen or chlorine atom or a methyl, ethyl, hydroxyl, methoxy or ethoxy group; 
           R7 is a hydrogen or chlorine atom or a methyl, ethyl, hydroxy, methoxy or ethoxy group; 
           R8 is a hydrogen or chlorine atom or a methyl, ethyl, hydroxy, methoxy or ethoxy group. 
         
       
       or
 the formula (III) where
 R9 is a hydrogen atom and the group R10 is an unsubstituted or substituted phenyl group, or the groups R9 and R10 belong to the same unsubstituted or substituted heterocycle comprising 2 nitrogen atoms and 4 carbon atoms; 
 R11 is a hydrogen atom or methyl, ethyl, hydroxy, methoxy or ethoxy group; 
 R12 is a hydrogen atom or methyl, ethyl, hydroxy, methoxy or ethoxy group; 
 R13 is a hydrogen atom or methyl, ethyl, hydroxy, methoxy or ethoxy group; 
 
 R14 is a hydrogen atom or methyl, ethyl, hydroxy, methoxy or ethoxy group; 
 
       or
 formula (IV) where
 A is an Oxygen or Sulphur atom; 
 R15 is a group 
 
 
       
         
           
           
               
               
           
         
         
           R16 is a hydrogen or chlorine atom or a methyl, ethyl, hydroxy, methoxy or ethoxy group; 
           R17 is a methyl, ethyl, hydroxy, methoxy, ethoxy, acetoxy, methoxycarhonyl or ethoxycarbonyl group. 
         
       
     
     
         21 . A compound according to  claim 20 , wherein said equivalent compound is selected from the following: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         22 . A compound according to  claim 1 , wherein the in vitro affinity of said compound for Syk protein is less than 25 μM. 
     
     
         23 . A composition comprising a compound according to  claim 1  and a pharmacologically acceptable excipient for the prevention or treatment of a condition dependent on a metabolic pathway involving Syk in humans or animals. 
     
     
         24 . (canceled) 
     
     
         25 . A method for identifying an organic compound having a molecular weight between 50 and 2500 Dalton binding with Syk tyrosine kinase protein and capable of inhibiting by at least 10% in vitro binding of
 (i) antibody fragment G4G11 (SEQ ID No. 2), or   (ii) an antibody or antibody fragment which binds with human Syk tyrosine kinase protein on an epitope comprising at least one of residues 65 to 74 of the amino acid sequence of human Syk tyrosine kinase protein represented by the sequence SEQ ID No. 1, or   (iii) an antibody or antibody fragment which binds with human Syk tyrosine kinase protein and inhibiting by at least 10% the binding of antibody fragment G4G11 with human Syk tyrosine   kinase protein (SEQ ID No. 1),   
       with human Syk tyrosine kinase protein or with any of the variants thereof in animals, comprising at least the following steps:
 a) screening, from a bank of candidate organic compounds having a molecular weight between 50 and 2500 Da, those liable to bind with Syk protein on the three-dimensional binding cavity on the Syk protein of a compound selected from the molecules having formula C-13, I, II, III, IV or 1 to 87 as illustrated above; 
 b) selecting from the compounds identified in a) those capable of inhibiting by at least 10% in vitro the binding of the antibody or antibody fragment (i) or (ii) with Syk protein. 
 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . (canceled)