Quinolinone pyrimidines compositions as mutant-isocitrate dehydrogenase inhibitors

Abstract

The invention relates to methods of reducing the level of 2-hydroxyglutarate in cells by administration of an inhibitor of mutant isocitrate dehydrogenase-1.

1 . A method of reducing the production of 2-hydroxyglutarate (2-HG) in a cell producing 2-HG (“2-HG producing cell”),
 wherein the method comprises the step of contacting the 2-HG producing cell with an inhibitor of mutant isocitrate dehydrogenase-1 protein (mt-IDH1), 
 wherein the inhibitor of mt-IDH1 has an IC 50  value of less than 1 micromolar against HCT116 cells harboring mt-IDH1 (“HCT116 mt-IDH1 cells”) selected from the group consisting of HCT116 isogenic IDH1-R132H cells and HCT116 isogenic IDH1-R132C cells; 
 wherein the IC 50  value of the inhibitor of mt-IDH1 is determined by a cellular 2-HG assay using HCT116 mt-IDH1 cells according to the following steps:
 a. culturing the HCT116 mt-IDH1 cells in a growth media comprising McCoy's 5A, 10% fetal bovine serum, 1× antibiotic-antimycotic solution, and 0.3 mg/mL G418 in 5% CO 2  in an incubator at 37° C.; 
 b. trypsinizing and resuspending the HCT116 mt-IDH1 cells in an assay media comprising McCoy's 5A with no L-glutamine, 10% fetal bovine serum, 1× antibiotic-antimycotic solution, and 0.3 mg/mL G418; 
 c. obtaining an aliquot of 10,000 cells/100 μL and transferring the aliquot to each well of a clear 96-well tissue culture plate; 
 d. incubating the cells in the culture plate in 5% CO 2  at 37° C. in an incubator overnight; 
 e. obtaining an aliquot of 50 μL of an assay media comprising the inhibitor of mt-IDH1, and adding the aliquot to each well of the clear 96-well tissue culture plate and maintaining the culture plate in 5% CO 2  at 37° C. in an incubator for 24 hours; 
 f. removing the media from each well of the clear 96-well tissue culture plate and adding 150 μL of a methanol/water mixture (80/20 v/v) to each well; 
 g. maintaining the plate at −80° C. freezer for 12 hours; 
 h. obtaining an aliquot of 125 μL of extracted methanol/water mixture and analyzing the aliquot by a high-throughput-mass spectrometry to determine the cellular 2-HG level; and 
 i. determining the IC 50  value of the inhibitor of mt-IDH1 for the HCT116 mt-IDH1 cells. 
 
 
     
     
         2 . The method of  claim 1 , wherein 2-HG neomorphic activity of the 2-HG producing cells is reduced after contacting the 2-HG producing cells with the inhibitor of mt-IDH1. 
     
     
         3 . The method of  claim 2 , wherein the 2-HG neomorphic activity is R-2-HG neomorphic activity. 
     
     
         4 . The method of  claim 1 , wherein the 2-HG producing cells comprise a mutation in IDH1 at residues 97 or 100. 
     
     
         5 . The method of  claim 4 , wherein the 2-HG producing cells comprise a mutation in IDH1 selected from the group consisting of: G97D and R100Q. 
     
     
         6 . The method of  claim 1 , wherein the 2-HG producing cells comprise a mutation in IDH1 at residue 132. 
     
     
         7 . The method of  claim 6 , wherein the 2-HG producing cells comprise a mutation in IDH1 selected from the group consisting of R132H and R132C. 
     
     
         8 . The method of  claim 6 , wherein the 2-HG producing cells comprise a mutation in IDH1 selected from the group consisting of R132S, R132G, R132L, and R132V. 
     
     
         9 . The method of  claim 1 , wherein the 2-HG producing cells comprise a mutation in mt isocitrate dehydrogenase-2 (IDH2) at residues 140 or 172. 
     
     
         10 . The method of  claim 9 , wherein the 2-HG producing cells comprise a mutation in IDH2 selected from the group consisting of R140Q, R140G, R172K, R172M, R172S, R172G, and R172W 
     
     
         11 . The method of  claim 1 , wherein the 2-HG producing cells are obtained from a biological sample from a human patient. 
     
     
         12 . The method of  claim 1 , wherein the 2-HG producing cells are in a patient diagnosed with acute myeloid leukemia (AML). 
     
     
         13 . The method of  claim 12 , wherein the level of 2-HG is reduced in the patient as compared to a level of 2-HG in the patient prior to administration of the inhibitor of mt-IDH1. 
     
     
         14 . The method of  claim 13 , wherein the level of 2-HG in the patient is reduced following administration of the inhibitor of mt-IDH1 as compared to a level of 2-HG in the patient prior to administration of the inhibitor of mt-IDH1. 
     
     
         15 . A method of reducing the production of 2-hydroxyglutarate (2-HG) in a cell producing 2-HG (“2-HG producing cell”) wherein the 2-HG producing cell harbors a mutant IDH1 (mt-IDH1) selected from the group consisting of R132H, R132C, R132S, R132G, R132L, and R132V,
 wherein the method comprises the step of contacting the 2-HG producing cell with an inhibitor of mutant isocitrate dehydrogenase-1 protein (mt-IDH1), 
 wherein the inhibitor of mt-IDH1 has an IC 50  value of less than 1 micromolar against HCT116 cells harboring mt-IDH1 (“HCT116 mt-IDH1 cells”) selected from the group consisting of HCT116 isogenic IDH1-R132H cells and HCT116 isogenic IDH1-R132C cells; 
 wherein the IC 50  value of the inhibitor of mt-IDH1 is determined by a cellular 2-HG assay using HCT116 mt-IDH1 cells according to the following steps:
 a. culturing the HCT116 mt-IDH1 cells in a growth media comprising McCoy's 5A, 10% fetal bovine serum, 1× antibiotic-antimycotic solution, and 0.3 mg/mL G418 in 5% CO 2  in an incubator at 37° C.; 
 b. trypsinizing and resuspending the HCT116 mt-IDH1 cells in an assay media comprising McCoy's 5A with no L-glutamine, 10% fetal bovine serum, 1× antibiotic-antimycotic solution, and 0.3 mg/mL G418; 
 c. obtaining an aliquot of 10,000 cells/100 μL and transferring the aliquot to each well of a clear 96-well tissue culture plate; 
 d. incubating the cells in the culture plate in 5% CO 2  at 37° C. in an incubator overnight; 
 e. obtaining an aliquot of 50 μL of an assay media comprising the inhibitor of mt-IDH1, and adding the aliquot to each well of the clear 96-well tissue culture plate and maintaining the culture plate in 5% CO 2  at 37° C. in an incubator for 24 hours; 
 f. removing the media from each well of the clear 96-well tissue culture plate and adding 150 μL of a methanol/water mixture (80/20 v/v) to each well; 
 g. maintaining the plate at −80° C. freezer for 12 hours; 
 h. obtaining an aliquot of 125 μL of extracted methanol/water mixture and analyzing the aliquot by a high-throughput-mass spectrometry to determine the cellular 2-HG level; and 
 i. determining the IC 50  value of the inhibitor of mt-IDH1 for the HCT116 mt-IDH1 cells. 
 
 
     
     
         16 . The method of  claim 15 , wherein the 2-HG producing cell comprises a mutation in IDH1 selected from the group consisting of R132S, R132G, R132L, and R132V. 
     
     
         17 . A method of reducing the level of 2-hydroxyglutarate (2-HG) in a patient diagnosed with acute myeloid leukemia (AML) having a mutation at residue 132 of IDH1 as compared to a level of 2-HG in the patient prior to administration of the inhibitor of mt-IDH1, wherein the method comprises the step of administering to the patient a therapeutically effective amount an inhibitor of mutant isocitrate dehydrogenase 1 protein (mt-IDH1),
 wherein the inhibitor of mt-IDH1 has an IC 50  value of less than 1 micromolar against HCT116 cells harboring mt-IDH1 (“HCT116 mt-IDH1 cells”) selected from the group consisting of HCT116 isogenic IDH1-R132H cells and HCT116 isogenic IDH1-R132C cells;   wherein the IC 50  value of the inhibitor of mt-IDH1 is determined by a cellular 2-HG assay using HCT116 mt-IDH1 cells according to the following steps:
 a. culturing the HCT116 mt-IDH1 cells in a growth media comprising McCoy's 5A, 10% fetal bovine serum, 1× antibiotic-antimycotic solution, and 0.3 mg/mL G418 in 5% CO 2  in an incubator at 37° C.; 
 b. trypsinizing and resuspending the HCT116 mt-IDH1 cells in an assay media comprising McCoy's 5A with no L-glutamine, 10% fetal bovine serum, 1× antibiotic-antimycotic solution, and 0.3 mg/mL G418; 
 c. obtaining an aliquot of 10,000 cells/100 μL and transferring the aliquot to each well of a clear 96-well tissue culture plate; 
 d. incubating the cells in the culture plate in 5% CO 2  at 37° C. in an incubator overnight; 
 e. obtaining an aliquot of 50 μL of an assay media comprising the inhibitor of mt-IDH1, and adding the aliquot to each well of the clear 96-well tissue culture plate and maintaining the culture plate in 5% CO 2  at 37° C. in an incubator for 24 hours; 
 f. removing the media from each well of the clear 96-well tissue culture plate and adding 150 μL of a methanol/water mixture (80/20 v/v) to each well; 
 g. maintaining the plate at −80° C. freezer for 12 hours; 
 h. obtaining an aliquot of 125 μL of extracted methanol/water mixture and analyzing the aliquot by a high-throughput-mass spectrometry to determine the cellular 2-HG level; and 
 i. determining the IC 50  value of the inhibitor of mt-IDH1 for the HCT116 mt-IDH1 cells. 
   
     
     
         18 . The method of  claim 17 , wherein the mutation at residue 132 of IDH1 is selected from the group consisting of R132H and R132C. 
     
     
         19 . The method of  claim 17 , wherein the mutation at residue 132 of IDH1 is selected from the group consisting of R132S, R132G, R132L, and R132V. 
     
     
         20 . The method of  claim 17 , wherein the patient has an IDH1 mutation selected from the group consisting of: R132H, R132C, R132S, R132G, R132L, and R132V.