Affinity small molecules for the EPO receptor

Abstract

Compounds are provided that complex with the modulating domain of erythropoietin receptor (EPO-R) for use with EPO-R to determine the presence of EPO-R, the ability of other molecules to bind to the modulating domain in competitive assays and to induce a signal by EPO-R into a cell when bound by the subject compounds in a physiological environment. The compounds are characterized by having a six-membered heterocyclic ring comprising at least one nitrogen atom and include substituted triazolopyrimidine, pyridazinone, pyridine and piperidine.

What is claimed is:  
     
         1 . A combination comprising a polypeptide comprising the modulating sequence of the erythropoietin receptor and a non-peptide organic molecule of from 12 to 36 atoms other than hydrogen, from 9 to 20 carbon atoms, and from 4 to 12 of the heteroatoms chalcogen, nitrogen, halogen, and metal ion of Groups I and II of the periodic chart, and of the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 X is of from 1 to 7 atoms other than hydrogen and is oxygen, sulfur bonded to 0 to 2 oxygen atoms, amino and alkyl substituted amino;  
 n is 0 or 1;  
 R 1  is hydrogen or an organic group of from 1 to 12 carbon atoms and from 0 to 6 heteroatoms, which are chalcogen, nitrogen, and halogen consisting of an aliphatic group of from 1 to 6 carbon atoms having from 0 to 2 sites of unsaturation, non-oxo-carbonyl and the nitrogen and sulfur derivatives thereof, alicyclic having from 0 to 2 sites of unsaturation, aryl, heterocyclic and combinations thereof, where the cyclic structures may have from 1 to 2 rings;  
 R 2  is hydrogen, a heterofunctionality having nitrogen and/or chalcogen bonded to annular carbon, a heterofunctionality having nitrogen and/or chalcogen bonded to annular carbon to which is substituted with an organic group of from 1 to 10 carbon atoms, aryl, alkaryl, aralkyl and aralkenyl of from 5 to 10 carbon atoms, aroyl of from 6 to 10 carbon atoms, or an organic group bonded through a carbon atom of from 1 to 12 carbon atoms having from 1 to 4, as described above for R 1 ;  
 R 3  is hydrogen or an organic group of from 1 to 10 carbon atoms and from 0 to 4 chalcogen and nitrogen heteroatoms;  
 R 4  is hydrogen or alkyl and substituted alkyl of from 1 to 6 carbon atoms, where the substituents are oxy, amino and halo;  
 with the proviso that R 3  and R4 can be taken together to form a ring with the annular atoms to which they are attached of from 4 to 10 annular atoms and forming from 1 to 2 rings, where the annular atoms are unsubstituted or substituted with halo, alkyl of from 1 to 3 carbon atoms, oxy of from 0 to 3 carbon atoms, thio of from 0 to 3 carbon atoms and amino of from 0 to 4 carbon atoms;  
                     
 wherein:  
 p is 0, 1 or 2; and  
 R 5  is a group having from 1 to 3 atoms other than hydrogen and is oxy, thio, amino, nitro, cyano, and alkyl;  
                     
 wherein:  
 Y is O, S(O) m ,, wherein m is 0, 1 or 2, amino or CH 2 ;  
 R 6  is H or alkyl of from 1-3 carbon atoms;  
 R 7  is hydrogen, or a group of from 0 to 3 atoms other than hydrogen, and is oxy, thio amino, nitro, cyano, and alkyl;  
 V is an aryl group having 6 annular members comprising 0 to 2 nitrogen atoms and the remainder carbon atoms  
 U is a substituent group of from 0 to 5 atoms other than hydrogen, and is oxy, thio amino, nitro, cyano, halo, and alkyl; and  
 u is 0 to 3; and  
 (4) diazolohexahydroquinoline  
                     
 wherein:  
 Y is oxygen, sulfur, NH, (alkyl of from 1 to 3 carbon atoms, H) or H 2    
 R 7  is hydrogen or an organic group of from 1 to 12 carbon atoms and 0 to 4 heteroatoms;  
 R 8  is hydrogen, an aliphatic group of from 1 to 6 carbon atoms or a heterocycle of from 5 to 6 annular members and from 1 to 2 heteroannular members that are oxygen, nitrogen or sulfur; and  
 R 9 , R 10 , R 13 , R 14 , R 15  and R 16  are the same or different and are hydrogen or an organic radical of from 1 to 12 carbon atoms or a heterosusbtituent of from 1 to 3 heteroatoms;  
 R 11  and R 12  are the same or different and are hydrogen or an organic group of from 1 to 12 carbon atoms.  
 
     
     
         2 . A combination according to  claim 1 , wherein said polypeptide and said non-peptide organic molecule are complexed at the modulating domain of EPO-R.  
     
     
         3 . A combination according to  claim 2 , wherein said polypeptide is EPO-R bound to a cellular membrane.  
     
     
         4 . A combination comprising a polypeptide comprising the modulating domain sequence of the erythropoietin receptor and a non-peptide organic molecule of from 12 to 36 atoms other than hydrogen, from 9 to 20 carbon atoms, and from 4 to 12 of the heteroatoms chalcogen, nitrogen, halogen, and metal ion of Groups I and II of the periodic chart, and of the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 X is of from 1 to 3 atoms other than hydrogen and is oxygen, sulfur bonded to 0 to 2 oxygen atoms, amino and alkyl substituted amino;  
 n is 0 or 1;  
 R 1  is a lower alkyl group of 1 to 3 carbon atoms or an organic group having a six annular membered aromatic group having from 0 to 3 substituents, where the substituents are halo, lower alkyl of from 1 to 3 carbon atoms, nitro, trihalomethyl, and is either directly bonded to X or bonded through a linking group of from 1 to 4 carbon, nitrogen, or chalcogen atoms in the chain, being particularly carbon and nitrogen, and there being from 0 to 2 heteroatoms in the chain, where heteroatoms are bonded solely to carbon and hydrogen, or alpha-acetamidinyl having from 0 to 1 N—OH;  
 R 2  is hydrogen, amino of 0 to 3 carbon atoms, oxy of from 0 to 3 carbon atoms, a heterofunctionality having nitrogen or chalcogen bonded to annular carbon to which is substituted an organic group of from 1 to 10 carbon atoms and from 0 to 3 heteroatoms;  
 R 3  is hydrogen or an organic group of from 1 to 10 carbon atoms and from 0 to 4 chalcogen and nitrogen heteroatoms;  
 R 4  is hydrogen, alkyl or substituted alkyl of from 1 to 6 carbon atoms, where the substituents are oxy, amino and halo;  
 with the proviso that R 3  and R 4  can be taken together to form a ring with the annular atoms to which they are attached of from 4 to 10 annular atoms and forming from 1 to 2 rings, where the annular atoms are unsubstituted or substituted with halo, alkyl of from 1 to 3 carbon atoms, oxy of from 0 to 3 carbon atoms, thio of from 0 to 3 carbon atoms and amino of from 0 to 4 carbon atoms.  
 
     
     
         5 . A combination according to  claim 4 , wherein R 3  is hydrogen or an organic group of from 1 to 8 carbon atoms and 0 to 4 chalcogen, nitrogen and halo heteroatoms.  
     
     
         6 . A combination according to  claim 5 , wherein R 3  is cyclopropylmethylamino.  
     
     
         7 . A combination according to  claim 5 , wherein R 3  is H.  
     
     
         8 . A combination according to  claim 4 , wherein R 1  is a six annular membered aromatic group having from 0 to 3 substituents, where the substituents are halo, lower alkyl of from 1 to 3 carbon atoms, nitro, trihalomethyl, and is either directly bonded to X or bonded through a linking group of from 1 to 4 carbon, nitrogen, or chalcogen atoms in the chain.  
     
     
         9 . A combination according to  claim 4 , wherein R 4  is methyl.  
     
     
         10 . A combination according to  claim 4 , wherein R 4  is H.  
     
     
         11 . A combination comprising a polypeptide comprising the modulating sequence of the erythropoietin receptor and a non-peptide organic molecule of from 12 to 36 atoms other than hydrogen, from 9 to 20 carbon atoms, and from 4 to 12 of the heteroatoms chalcogen, nitrogen, halogen, and metal ion of Groups I and II of the periodic chart, and of the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 Y is O, S(O) m ,, wherein m is 0, 1 or 2, amino or CH 2 ;  
 R 6  is H or alkyl of from 1-3 carbon atoms;  
 R 7  is hydrogen, or a group of from 0 to 3 atoms other than hydrogen, and is oxy, thio amino, nitro, cyano, and alkyl;  
 V is a phenyl group;  
 U is oxy, thio amino, nitro, cyano, halo, and alkyl and from 0 to 3 atoms other than hydrogen; and u is 0 to 3.  
 
     
     
         12 . A combination according to  claim 11 , wherein Y is SO 2 , V is phenyl, R 7  is Cl and u is 0.  
     
     
         13 . A combination comprising a polypeptide comprising the modulating domain sequence of the erythropoietin receptor and a non-peptide organic molecule of from 12 to 36 atoms other than hydrogen, from 9 to 20 carbon atoms, and from 4 to 12 of the heteroatoms chalcogen, nitrogen, halogen, and metal ion of Groups I and II of the periodic chart, and of the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 X is of from 1 to 3 atoms other than hydrogen and is oxygen, sulfur bonded to 0 to 2 oxygen atoms, amino and alkyl substituted amino;  
 n is 0 or 1;  
 R 1  is alkyl of from 1 to 3 carbon atoms, substituted phenyl having from 0 to 3 substituents that are CH 3 , Cl, NO 2 , and CF 3  and bonded directly to an annular carbon atom or through a linking group of from 1 to 3 carbon and nitrogen atoms in the chain or N-hydroxyamidinyl;  
 R 2  is CH 3 , NH 2 , OH, and aroylamido of from 7 to 8 carbon atoms having from 0 to 2 susbtituents that are CH 3 , Cl, NO 2 , and CF 3 ;  
 R 3  is cycloalkylalkyl of from 4 to 8 carbon atoms, having from 3 to 4 annular atoms, H or carboxy;  
 R 4  is H, lower alkyl of from 1 to 3 carbon atoms or alkoxymethyl of from 2 to 4 carbon atoms;  
 with the proviso that R 3  and R 4  may be taken together to define 1,2-dimethylene-alpha-halo, alpha-CH 3 -halobenzene, where halo is F or Cl.  
 
     
     
         14 . A method for modulating the activity of EPO-R present as a cell membrane component comprising: forming a complex by bringing together the members of the combination of  claim 13  under complex forming conditions, where said polypeptide is EPO-R.  
     
     
         15 . A method for modulating the activity of EPO-R comprising: 
 forming a complex by bringing together the members of the combination of  claim 11  under complex forming conditions, where said polypeptide is EPO-R.    
     
     
         16 . A compound according to  claim 13  and a pharmaceutically acceptable vehicle.  
     
     
         17 . A compound according to  claim 11  and a pharmaceutically acceptable vehicle.  
     
     
         18 . A method of determining the binding affinity of a test compound to the modulating domain of EPO-R, said method comprising: 
 adding said test compound to a combination according to  claim 1  and determining the amount of complex of said combination in the presence of said test compound as compared to the absence of said test compound.    
     
     
         19 . A method of inducing a physiological response of EPO-R in a host, said method comprising: 
 administering to said host a physiologically effective amount of a non-peptide organic molecule of from 12 to 36 atoms other than hydrogen, from 9 to 20 carbon atoms, and from 4 to 12 of the heteroatoms chalcogen, nitrogen, halogen, and metal ion of Groups I and II of the periodic chart, and of the formula:                           wherein:     X is of from 1 to 3 atoms other than hydrogen and is oxygen, sulfur bonded to 0 to 2 oxygen atoms, amino and alkyl substituted amino;     n is 0 or 1;     R 1  is alkyl of from 1 to 3 carbon atoms, substituted phenyl having from 0 to 3 substituents that are CH 3 , Cl, NO 2 , and CF 3  and bonded directly to an annular carbon atom or through a linking group of from 1 to 3 carbon and nitrogen atoms in the chain, N-hydroxyamidinyl;     R 2  is CH 3 , NH 2 , OH, and aroylamido of from 7 to 8 carbon atoms having from 0 to 2 susbtituents that are CH 3 , Cl, NO 2 , and CF 3 ;     R 3  is cycloalkylalkyl of from 4 to 8 carbon atoms, having from 3 to 4 annular atoms, H or carboxy;     R 4  is H, lower alkyl of from 1 to 3 carbon atoms or alkoxymethyl of from 2 to 4 carbon atoms;     with the proviso that R 3  and R 4  may be taken together to define 1,2-dimethylene-alpha-halo, alpha-CH 3 -halobenzene, where halo is F or Cl; or                           wherein:     X is of from 1 to 3 atoms other than hydrogen and is oxygen, sulfur bonded to 0 to 2 oxygen atoms, amino and alkyl substituted amino;     n is 0 or 1;     Y is O, S(O) m ,, wherein m is 0, 1 or 2, amino or CH 2 ;     R 6  is H or alkyl of from 1-3 carbon atoms;     R 7  is hydrogen, or a group of from 0 to 3 atoms other than hydrogen, and is oxy, thio amino, nitro, cyano, and alkyl;     V is a phenyl group;     U is oxy, thio amino, nitro, cyano, halo, and alkyl and from 0 to 3 atoms other than hydrogen; and     u is 0 to 3.    
     
     
         20 . A method according to  claim 19 , wherein said non-peptide organic molecule is of formula 1.  
     
     
         21 . A method according to  claim 20 , wherein X is amino, R 2  is o-methyl, p-chlorophenyl-1, R 2  is H, R 3  is cyclopropylmethylamino and R 4  is methyl.  
     
     
         22 . A method of modulating the response to a stimulus of hematopoietic or neuronal cells influenced by the binding of EPO to EPO-R, said method comprising: 
 contacting said cells with an effective amount to modulate said response of a non-peptide organic molecule of from 12 to 36 atoms other than hydrogen, from 9 to 20 carbon atoms, and from 4 to 12 of the heteroatoms chalcogen, nitrogen, halogen, and metal ion of Groups I and II of the periodic chart, and of the formula:                           wherein:     R 1  is alkyl of from 1 to 3 carbon atoms, substituted phenyl having from 0 to 3 substituents that are CH 3 , Cl, NO 2 , and CF 3  and bonded directly to an annular carbon atom or through a linking group of from 1 to 3 carbon and nitrogen atoms in the chain, N-hydroxyamidinyl;     R 2  is CH 3 , NH 2 , OH, and aroylamido of from 7 to 8 carbon atoms having from 0 to 2 susbtituents that are CH 3 , Cl, NO 2 , and CF 3 ;     R 3  is cycloalkylalkyl of from 4 to 8 carbon atoms, having from 3 to 4 annular atoms, H or carboxy;     R 4  is H, lower alkyl of from 1 to 3 carbon atoms or alkoxymethyl of from 2 to 4 carbon atoms;     with the proviso that R 3  and R 4  may be taken together to define 1,2-dimethylene-alpha-halo, alpha-CH 3 -halobenzene, where halo is F or Cl; or                           wherein:     Y is O, S(O) m ,, wherein m is 0, 1 or 2, amino or CH 2 ;     R 6  is H or alkyl of from 1-3 carbon atoms;     R 7  is hydrogen, or a group of from 0 to 3 atoms other than hydrogen, and is oxy, thio amino, nitro, cyano, and alkyl;     V is a phenyl group;     U is oxy, thio amino, nitro, cyano, halo, and alkyl and from 0 to 3 atoms other than hydrogen; and     u is 0 to 3.