GPR 17 Agonists and Screening Assay

Abstract

The present invention is related to a method of determining a test compound's ability to modify the biological activity of a GPR17. Said method comprises, among others, the step of contacting the test compound with a GPR17, or a functional GPR17 fragment in the presence of a suitable amount of a GPR17 agonist of formula I.

1 .- 7 . (canceled) 
     
     
         8 . A method of treating, alleviating or preventing of a GPR17 mediated disease, optionally selected from ischemic diseases, stroke or multiple sclerosis, in a patient in need thereof comprising the steps of
 (a) identifying a compound that modulates GPR17 biological activity by
 (i) contacting a compound with a GPR17, or a functional GPR17 fragment in the presence of a suitable amount of a GPR17 agonist of formula I 
   
       
         
           
           
               
               
           
         
         
           or a salt thereof, 
           (ii) measuring the biological activity of said GPR17 or said functional GPR17 fragment after addition of said compound, and 
           (iii) comparing the biological activity measured in step (ii) with the biological activity of said GPR17 or said functional fragment thereof in the presence of said GPR 17 agonist of formula I without the addition of said compound to identify whether the compound is one that modulates GPR17 activity, 
           wherein in formula I 
           R1 and R2 are independently selected from the group comprising hydrogen, halogen, hydroxy, formyl, oxime, cyano, nitro, amino, NR6R7, carboxy, carbamoyl, (C 1 -C 8 )alkyl, (C 1 -C 8 ) all yloxy, (C 1 -C 8 )alkylthio, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkloxy, (C 3 -C 8 )cycloalkylamino, aryl, heteroaryl, aryloxy, heteroaryloxy, halogen, trifluoromethyl, (C 1 -C 8 )alkylcarbonyl, (C 1 -C 8 ) alkylaminocarbonyl, di(C 1 -C 8 )alkylaminocarbonyl, arylcarbonyl, heteroarylcarbonyl, aryl(C 1 -C 8 )alkyl, heteroaryl(C 1 -C 8 )alkyl, aryl(C 1 -C 8 )alkyloxy, heteroaryl(C 1 -C 8 )alkyloxy, aryl(C 1 -C 8 )alkylcarbonyl, heteroaryl(C 1 -C 8 )alkylcarbonyl, aryl(C 1 -C 8 )alkyloxycarbonyl, heteroaryl(C 1 -C 8 )alkyloxycarbonyl, (C 1 -C 8 )alkyloxycarbonyl, (C 1 -C 8 )alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, sulfamoyl, sulfonylamino, (C 1 -C 8 )alkylaminosulfonyl, di(C 1 -C 8 )alkylaminosulfonyl, arylsulfonylamino, heteroarylsulfonylamino and (C 1 -C 8 )alkylsulfonylamino; wherein each alkyl, alkenyl alkynyl or cycloalkyl may be unsubstituted or substituted with one or more residues selected from among hydroxyl, (C 1 -C 5 )alkyloxy, (C 1 -C 3 )alkyloxy(C 1 -C 3 )alkyloxy, halogen, and NR6R7; and wherein each aryl or heteroaryl can be unsubstituted or substituted with one or more residues selected from among hydroxyl, (C 1 -C 5 )alkyloxy, halogen, (C 1 -C 5 )alkyl, (C 3 -C 8 )cycloalkyl, carboxy, NR6R7, cyano, trifluormethyl and nitro; 
           R3 is selected from hydrogen, a group —(CH 2 ) m CH 2 —COOH, OH, NH, and (C 1 -C 5 )alkyl which is optionally substituted with one or more halogens, one or two hydroxyl groups or (C 1 -C 3 )alkoxy; 
           R4 is selected from hydrogen and fluoro; 
           R5 is selected from hydrogen, halogen, (C 1 -C 3 )alkyl, (C 1 -C 3 )alkyloxy, (C 1 -C 3 )alkylthio, (C 2 -C 4 )alkenvl, (C 2 -C 4 )alkynyl, and NR6R7; 
           R6 and R7 are independently selected from hydrogen, (C 1 -C 6 )alkyl, (C 3 -C 7 )cycloalkyl, phenyl, heteroaryl, phenyl(C 1 -C 6 )alkyl, heteroaryl(C 1 -C 6 )alkyl, (C 1 -C 6 )alkylsulfonyl, phenylsulfonyl, heteroarylsulfonyl, (C 1 -C 6 )alkylcarbonyl, (C 1 -C 6 )alkoxycarbonyl, aminocarbonyl, (C 1 -C 6 )alkylaminocarbonyl, phenylcarbonyl, and heteroarylcarbonyl; wherein each alkyl may be unsubstituted or substituted with one or more residues selected from among hydroxyl, (C 1 -C 3 )alkyloxy, phenyl, halo, carboxy, and NR8R9; and wherein R6 and R7 may form a 5- to 7-membered cycle; and wherein phenyl or heteroaryl can be unsubstituted or substituted with one or more residues selected from among hydroxyl, (C 1 -C 3 )alkyloxy, halogen, (C 1 -C 3 )alkyl, carboxy, NR8R9, cyano, trifluormethyl and nitro; 
           R8 and R9 are independently selected from among hydrogen and (C1-C3)alkyl; 
           n and m are independently 0, 1 or 2; 
           and 
         
         (b) administering said GPR17 modulating compound to the patient, optionally together with one or more pharmaceutically acceptable excipients. 
       
     
     
         9 . The method according to  claim 8 , wherein the GPR17 mediated disease is multiple sclerosis. 
     
     
         10 . A screening assay for the identification of GPR 17 modulators among a multitude of compounds, said screening assay comprising
 (a) cells or membrane fractions expressing GPR17 or a functional GPR17 fragment and   (b) a suitable amount of a GPR17 agonist of formula I   
       
         
           
           
               
               
           
         
         wherein in formula I 
         R1 and R2 are independently selected from the group comprising hydrogen, halogen, hydroxy, formyl, oxime, cyano, nitro, amino, NR6R7, carboxy, carbamoyl, (C 1 -C 8 )alkyl, (C 1 -C 8 ) alkyloxy, (C 1 -C 8 )alkylthio, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyloxy, (C 3 -C 8 )cycloalkylamino, aryl, heteroaryl, aryloxy, heteroaryloxy, halogen, trifluoromethyl, (C 1 -C 8 )alkylcarbonyl, (C 1 -C 8 ) alkylaminocarbonyl, di(C 1 -C 8 )alkylaminocarbonyl, arylcarbonyl, heteroarylcarbonyl, aryl(C 1 -C 8 )alkyl, heteroaryl(C 1 -C 8 )alkyl, aryl(C 1 -C 8 )alkyloxy, heteroaryl(C 1 -C 8 )alkyloxy, aryl(C 1 -C 8 )alkylcarbonyl, heteroaryl(C 1 -C 8 )alkylcarbonyl, aryl(C 1 -C 8 )alkyloxycarbonyl, heteroaryl(C 1 -C 8 )alkyloxycarbonyl, (C 1 -C 8 )alkyloxycarbonyl, (C 1 -C 8 )alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, sulfamoyl, sulfonylamino, (C 1 -C 8 )alkylaminosulfonyl, di(C 1 -C 8 )alkylaminosulfonyl, arylsulfonylamino, heteroarylsulfonylamino and (C 1 -C 8 )alkylsulfonylamino; wherein each alkyl, alkenyl, alkynyl or cycloalkyl may be unsubstituted or substituted with one or more residues selected from among hydroxyl, (C 1 -C 5 )alkyloxy, (C 1 -C 3 )alkyloxy(C 1 -C 3 )alkyloxy, halogen, and NR6R7; and wherein each aryl or heteroaryl can be unsubstituted or substituted with one or more residues selected from among hydroxyl, (C 1 -C 5 )alkyloxy, halogen, (C 1 -C 5 )alkyl, (C 3 -C 8 )cycloalkyl, carboxy, NR6R7, cyano, trifluormethyl and nitro; 
         R3 is selected from hydrogen, a group —(CH 2 ) m CH 2 —COOH, OH, NH, and (C1-C5)alkyl which is optionally substituted with one or more halogens, one or two hydroxyl groups or methoxy; 
         R4 is independently selected from hydrogen and fluoro; 
         R5 is selected from hydrogen, halogen, (C 1 -C 3 )alkyl, (C 1 -C 3 )alkyloxy, (C 1 -C 3 )alkylthio, (C 2 -C 4 )alkenyl, (C 2 -C 4 )alkynyl, and NR6R7; 
         R6 and R7 are independently selected from hydrogen,(C 1 -C 6 )alkyl, (C 3 -C 7 )cycloalkyl, phenyl, heteroaryl, phenyl(C 1 -C 6 )alkyl, heteroaryl(C 1 -C 6 )alkyl, (C 1 -C 6 )alkylsulfonyl, phenylsulfonyl, heteroarylsulfonyl,(C 1 -C 6 )alkylcarbonyl,(C 1 -C 6 )alkoxycarbonyl, aminocarbonyl, (C 1 -C 6 )alkylaminocarbonyl, phenylcarbonyl, and heteroarylcarbonyl; wherein each alkyl may be unsubstituted or substituted with one or more residues selected from among hydroxyl, (C 1 -C 3 )alkyloxy, phenyl, halo, carboxy, and NR8R9; and wherein R6 and R7 may form a 5- to 7-membered cycle; and wherein phenyl or heteroaryl can be unsubstituted or substituted with one or more residues selected from among hydroxyl, (C 1 -C 3 )alkyloxy, halogen, (C 1 -C 3 )alkyl, carboxy, NR8R9, cyano, trifluormethyl and nitro; 
         R8 and R9 are independently selected from among hydrogen and (C 1 -C 3 )alkyl; 
         n and m are independently 0, 1 or 2, or 
         a salt thereof, 
         and wherein, optionally, the GPR agonist of formula I is as defined in claim  2 . 
       
     
     
         11 . The screening assay according to  claim 10  further comprising means for determining the GPR17 activity,
 wherein, optionally, said means for determining the GPR17 activity is selected from one or more of the group consisting of: 
 a. means for determining the release of calcium from intracellular calcium stores associated with GPR17 activation, said means optionally comprising a cell membrane permeable indicator which binds to calcium released within the cell thereby providing a measurable signal, preferably fluorescence or luminescence; 
 b. means for determining GTP Y S binding to heterotrimeric G proteins, wherein said heterotrimeric G protein is optionally  35 SGTP Y S; 
 c. means for determining the inhibition of cAMP formation or its increase associated with GPR17 activation, said means optionally comprising a stimulator of the adenylyl cyclase and a suitable cAMP indicator system; 
 d. means for determining the increase in inositolphosphates (IP), quantified as IP1, said means optionally comprising a suitable IP 1 detector system; and 
 e. means for determining the recruitment of cytoplasmic β-arrestin proteins, optionally by detection of the B-arrestin translocation to the receptor using fluorescence or bioluminescence resonance energy transfer. 
 
     
     
         12 . The screening assay according to  claim 11  (c) wherein said stimulator of the adenylyl cyclase is forskolin, and wherein said cAMP indicator system is a competitive immunoassay, optionally providing a fluorescence signal. 
     
     
         13 . (canceled) 
     
     
         14 . A compound selected from
 3-(2-carboxyethyl)-4,6-dibromo-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-4,6-dichloro-(1-carboxyethyl)-indole-2-carboxylic acid,   3-(2-carboxyethyl)-6-bromo-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-6-iodo-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-4,6-diiodo-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-4,6-diphenyl-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-6-phenyl-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-6,7-dichloro-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-6-chloro-7-fluoro-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-6-bromo-2-carboxy-7-fluoro-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-6-benzyl-2-carboxy-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-4,6-dihydroxy-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl-6-(4-fluorophenyl)-1H-indol-2-carboxylic acid,   3-(2-carboxyethyl)-6-furanyl-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-6-thienyl-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-7-fluoro-6-phenyl-1H-indole-2-carboxylic acid,   3-(2-carboxyethyl)-6-(4-fluorophenyl)-7-fluoro-1H-indol-2-carboxylic acid,   3 -(2-carboxyethyl)-6-furanyl-7-fluoro-1H-indole-2-carboxylic acid,   3 -(2-carboxyethyl)-6-thienyl-7-fluoro-1H-indole-2-carboxylic acid,   and salts of any of the foregoing.   
     
     
         15 . The method according to  claim 8 , wherein in formula I
 R1 is selected from fluoro, chloro, bromo, iodo, (C 1 -C 3 )alkyl, (C 1 -C 3 )alkoxy, phenyl, phenyl(C 1 -C 3 )alkyl, phenyl(C 1 -C 3 )alkoxy, phenylcarbonyl, (C 5 -C 6 )heteroaryl, (C 5 -C 6 )heteroarylcarbonyl, (C 5 -C 6 )heteroaryl(C 1 -C 3 )alkyl and (C 5 -C 6 )heteroaryl(C 1 -C 3 )alkoxy, wherein the alkyl and alkoxy groups are optionally substituted with one or more halogens, (C 1 -C 3 )alkoxy, or hydroxyl, and wherein the phenyl and (C 5 -C 6 )heteroaryl groups can be substituted with one or more halogens, (C 1 -C 3 )alkoxy, (C 1 -C 3 )alkyl, NR6R7,or hydroxyl;   R2 is selected from hydrogen, fluoro, chloro, bromo, iodo, (C 1 -C 3 )alkyl, (C 1 -C 3 )alkoxy, and phenyl, wherein the alkyl and alkoxy groups are optionally substituted with one or more halogens, (C 1 -C 3 )alkoxy, or hydroxyl, and wherein the phenyl group can be substituted with one or more halogens, (C 1 -C 3 )alkoxy, (C 1 -C 3 )alkyl, or hydroxyl;   R3 is selected from hydrogen and a group —(CH 2 ) m CH 2 —COOH;   R4 is hydrogen or fluoro, and is preferably hydrogen;   R5 is selected from hydrogen, halogen, methyl or methoxy, and preferably represents fluoro or hydrogen, and particularly hydrogen;   n and m are independently 0, 1 or 2.   
     
     
         16 . The method according to  claim 8 , wherein in formula I
 R1 is selected from the group consisting of methyl, methoxy, hydroxy, CF 3 , chloro, fluoro, bromo, iodo, thienyl, furanyl, pyridyl, and phenyl which is optionally substituted with halogen, methyl or methoxy;   R2 is selected from the group consisting of hydrogen, methyl, methoxy, hydroxyl, CF 3 , chloro, fluoro, bromo, iodo and phenyl;   R3 is hydrogen, carboxymethyl, or carboxyethyl;   R4 and R5 are both hydrogen; and   n is 1.   
     
     
         17 . The method according to  claim 8  wherein the GPR 17 agonist of formula I is selected from the group consisting of
 3 -(2-carboxyethyl)-4,6-dichloro-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-4,6-dichloro-(1 -carboxyethyl)-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-4,6-dimethyl-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-6-chloro-1H-indole-2-carboxylic acid, 
 3-(2-carboxyethyl)-4,6-difluoro-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-4,6-dibromo-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-6-bromo-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-6-iodo-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-4,6-diiodo-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-4,6-dichloro-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-4,6-diphenyl-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-6-phenyl-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-6,7-dichloro-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-6-chloro-7-fluoro-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-6-bromo-2-carboxy-7-fluoro)-1H-indole-2-carboxylic acid, 
 3 -(2-carboxyethyl)-4,6-dimethoxy-1H-indole-2-carboxylic acid, 
 3-(2-carboxyethyl)-6-phenoxy-1H-indole-2-carboxylic acid, 
 3-(2-carboxyethyl)-6-benzyl-2-carboxy-1H-indole-2-carboxylic acid, 
 3-(2-carboxyethyl)-4,6-dihydroxy-1H-indole-2-carboxylic acid, 
 3-(2-carboxyethyl)-6-(4-fluorophenyl)-1H-indol-2-carboxylic acid, 
 3-(2-carboxyethyl)-6-furanyl-1H-indole-2-carboxylic acid, 
 3-(2-carboxyethyl)-6-thienyl-1H-indole-2-carboxylic acid, 
 3-(2-carboxyethyl)-7-fluoro-6-phenyl-1H-indole-2-carboxylic acid, 
 3-(2-carboxyethyl)-6-(4-fluorophenyl)-7-fluoro-1H-indo1-2-carboxylic acid, 
 3-(2-carboxyethyl)-6-furanyl-7-fluoro-1H-indole-2-carboxylic acid, 
 3-(2-carboxyethyl)-6-thienyl-7-fluoro-1H-indole-2-carboxylic acid, 
 or a salt of any of the foregoing. 
 
     
     
         18 . The method according to  claim 8 , wherein the biological activity of GPR17 is determined by measuring one or more of the following parameters:  35 SGTP Y S binding, the inhibition of cAMP formation, the release of calcium from intracellular calcium stores, the increase in inositolphosphates (IP), and/or the recruitment of cytoplasmic β-arrestin proteins. 
     
     
         19 . The method according to  claim 8 , wherein the activity of GPR17 is measured in a transfected cell line or membrane fragments thereof, which is preferably selected from transfected CHO cells, astrocytoma cells, COST cells or HEK293 cells and/or in naturally expressing tissue or cells and membrane fragments thereof. 
     
     
         20 . The method according to  claim 8  wherein step (a) comprises the additional steps of
 (iv) selecting one or more analogs of the GPR 17 modulating compound(s) identified in steps (i) to (iii), 
 (v) subjecting the one or more GPR 17 modulating compound analogs of step (iv) to the method and/or assay of steps (i)-(iii) thereby determining the GPR 17 modulating properties of said GPR 17 modulating compound analogs, and 
 (vi) optionally repeating steps (iv)-(v) one or more times by producing further chemical analogs of the GPR 17 modulating compound(s) thereby identifying one or more GPR17 modulating compound(s) with improved GPR17 modulating properties.