Fgfr gatekeeper mutant gene and drug targeting same

Abstract

The present inventors successfully identified novel gatekeeper mutations for FGFR. Further, they discovered that mutant FGFR having the mutations demonstrate resistance to known FGFR inhibitors such as AZD4547, and at the same time demonstrate sensitivity to specific compounds. Mutant polypeptides having the mutations may be used as biomarkers in cancer treatment by FGFR inhibitors to prevent the development of side effects in therapy by conventional FGFR inhibitors, and to control the therapeutic mode for receiving the best therapeutic effect, thus making individualized treatment possible.

1 . A pharmaceutical composition for cancer treatment comprising as an active ingredient the compound represented by Formula (I) below or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition for cancer treatment is characterized in being used by administering it to a patient expressing an FGFR mutant polypeptide comprising a substitution of valine to phenylalanine at the 7 th  amino acid from the N terminus and/or a substitution of valine to leucine at the 5 th  amino acid from the N terminus in the partial amino acid sequence described in SEQ ID NO: 53 or 54 in an FGFR polypeptide, or having a polynucleotide encoding said mutant polypeptide: 
       
         
           
           
               
               
           
         
         wherein R 1 , R 2 , R 3 , and R 4  each independently represents the group listed below: 
         R 1  represents hydrogen, hydroxy, halogen, cyano, nitro, C 1-4  haloalkyl, C 1-6  alkyl, C2-6 alkenyl, C 2-6  alkynyl, C 3-7  cycloalkyl, C 6-10  aryl C 1-4  alkyl, —OR 5 , —NR 6 R 7 , —(CR 8 R 9 ) n Z 1 , —C(O)NR 12 R 13 , —SR 14 , —SOR 15 , —SO 2 R 16 , —NR 17 SO 2 R 18 , COOH, C 6-10  aryl which is optionally substituted by one or more groups independently selected from group P, 5- to 10-membered heteroaryl or 3- to 10-membered heterocyclyl which is optionally substituted by one or more groups independently selected from group Q, —COR 19 , —COOR 20 , —OC(O)R 21 , —NR 22 C(O)R 23 , —NR 24 C(S)R 25 , —C(S)NR 26 R 27 , —SO 2 NR 28 R 29 , —OSO 2 R 30 , —SO 3 R 31 , or —Si(R 32 ) 3 ; 
         R 2  represents hydrogen, hydroxy, halogen, cyano, nitro, C 1-4  haloalkyl, C 1-6  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 3-7  cycloalkyl, C 6-10  aryl C 1-4  alkyl, —OR 5 , —NR 6 R 7 , —(CR 8 R 9 ) n Z 1 , —C(O)NR 12 R 13 , —SR 14 , —SOR 15 , —SO 2 R 16 , —NR 17 SO 2 R 18 , COOH, C 6-10  aryl which is optionally substituted by one or more groups independently selected from group P, 5- to 10-membered heteroaryl or 3- to 10-membered heterocyclyl which is optionally substituted by one or more groups independently selected from group Q, —COR 19 , —COOR 20 , —OC(O)R 21 , —NR 22 C(O)R 23 , —NR 24 C(S)R 25 , —C(S)NR 26 R 27 , —SO 2 NR 28 R 29 , —OSO 2 R 30 , —SO 3 R 31 , or —Si(R 32 ) 3 ; or R 1  and R 2 , together with an atom linked thereto, form 3- to 10-membered heterocyclyl or 5- to 10-membered heteroaryl, wherein the heterocyclyl or heteroaryl is optionally substituted by halogen; 
         R 3  represents methyl; 
         R 4  represents hydrogen; 
         A is indole; 
         R 5  represents C 1-5  alkyl, C 3-7  cycloalkyl, C 3-7  cycloalkyl C 1-3  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 1-4  haloalkyl, C 1-3  alkoxy C 1-4  alkyl, C 1-3  alkoxy C 1-4  alkoxy C 1-4  alkyl, C 1-4  aminoalkyl, C 1-4  alkylamino C 1-4  alkyl, di(C 1-4  alkyl)amino C 1-4  alkyl, C 6-10  aryl, C 6-10  aryl C 1-3  alkyl, or 3- to 10-membered heterocyclyl C 1-3  alkyl, 3- to 10-membered heterocyclyl, 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl C 1-3  alkyl, C 1-6  monohydroxy alkyl, C 1-6  dihydroxy alkyl, or C 1-6  trihydroxy alkyl which is optionally substituted by one or more groups independently selected from group Q; 
         R 6  and R 7 , which can be the same or different, each represents hydrogen, C 1-4  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 1-4  haloalkyl, C 1-3  alkoxy C 1-4  alkyl, C 6-10  aryl C 1-3  alkyl, 3- to 10-membered heterocyclyl C 1-3  alkyl, 5- to 10-membered heteroaryl C 1-3  alkyl, C 1-6  monohydroxy alkyl, C 1-6  dihydroxy alkyl, C 1-6  trihydroxy alkyl, 3- to 10-membered heterocyclyl, C 1-4  aminoalkyl, C 1-4  alkylamino C 1-4  alkyl, di(C 1-4  alkyl)amino C 1-4  alkyl, or cyano(C 1-3  alkyl); or alternatively R 6  and R 7 , together with a nitrogen atom linked thereto, form 3- to 10-membered heterocyclyl or 5- to 10-membered heteroaryl; 
         n represents 1 to 3; 
         R 8  and R 9 , which can be the same or different, each represents hydrogen, C 1-4  alkyl, or halogen; 
         or alternatively R 8  and R 9 , together with a carbon atom linked thereto, form a cycloaliphatic ring; 
         Z 1  represents hydrogen, NR 10 R 11 , —OH, or 3- to 10-membered heterocyclyl or 5- to 10-membered heteroaryl which is optionally substituted by one or more groups independently selected from group Q; 
         R 10  and R 11 , which can be the same or different, each represents C 1-4  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 1-4  haloalkyl, C 1-3  alkoxy C 1-4  alkyl, cyano(C 1-3  alkyl), or C 1-3  alkylsulfonyl C 1-4  alkyl; 
         or alternatively R 10  and R 11 , together with a nitrogen atom linked thereto, form 3- to 10-membered heterocyclyl or 5- to 10-membered heteroaryl; 
         R 12  and R 13 , which can be the same or different, each represents hydrogen, C 1-4  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 1-4  haloalkyl, C 1-3  alkoxy C 1-4  alkyl, C 6-10  aryl, 5- to 10-membered heteroaryl, 3- to 10-membered heterocyclyl, C 6-10  aryl C 1-4  alkyl, 3- to 10-membered heterocyclyl C 1-3  alkyl, 5- to 10-membered heteroaryl C 1-3  alkyl, cyano(C 1-3  alkyl), C 1-3  alkylsulfonyl C 1-4  alkyl, 3- to 10-membered cycloaliphatic ring, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl; or alternatively R 12  and R 13 , together with a nitrogen atom linked thereto, form 3- to 10-membered heterocyclyl or 5- to 10-membered heteroaryl which is optionally substituted by one or more groups independently selected from group Q; 
         R 14  represents C 1-4  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 1-4  haloalkyl, C 6-10  aryl which is optionally substituted by one or more groups independently selected from group P, or 5- to 10-membered heteroaryl or 3- to 10-membered heterocyclyl which is optionally substituted by one or more groups independently selected from group Q; 
         R 15  represents C 1-4  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 1-4  haloalkyl, C 6-10  aryl which is optionally substituted by one or more groups independently selected from group P, or 5- to 10-membered heteroaryl or 3- to 10-membered heterocyclyl which is optionally substituted by one or more groups independently selected from group Q; 
         R 16  represents C 1-4  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 1-4  haloalkyl, C 6-10  aryl which is optionally substituted by one or more groups independently selected from group P, or 5- to 10-membered heteroaryl or 3- to 10-membered heterocyclyl which is optionally substituted by one or more groups independently selected from group Q; 
         R 17  represents hydrogen or C 1-4  alkyl; 
         R 18  represents C 1-4  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 1-4  haloalkyl, C 6-10  aryl which is optionally substituted by one or more groups independently selected from group P, or 5- to 10-membered heteroaryl or 3- to 10-membered heterocyclyl which is optionally substituted by one or more groups independently selected from group Q; 
         R 19  represents hydrogen, C 1-4  alkyl, C 3-7  cycloalkyl, haloalkyl, C 6-10  aryl, or 5- to 10-membered heteroaryl or 3- to 10-membered heterocyclyl which is optionally substituted by one or more groups independently selected from group Q; 
         R 29  represents C 1-4  alkyl, C 3-7  cycloalkyl, C 1-4  haloalkyl, C 6-10  aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl; 
         R 21  represents C 1-4  alkyl, C 3-7  cycloalkyl, C 1-4  haloalkyl, C 6-10  aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl; 
         R 22  represents hydrogen, C 1-4  alkyl, or C 1-4  haloalkyl; 
         R 23  represents hydrogen, C 1-4  alkyl, C 3-7  cycloalkyl, C 1-4  haloalkyl, C 6-10  aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl; 
         R 24  represents hydrogen, C 1-4  alkyl, or C 1-4  haloalkyl; 
         R 25  represents C 1-4  alkyl, C 3-7  cycloalkyl, C 1-4  haloalkyl, C 6-10  aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl; 
         R 26  and R 27 , which can be the same or different, each represents hydrogen, C 1-4  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 1-4  haloalkyl, C 1-3  alkoxyl C 1-4  alkyl, C 6-10  aryl, 5- to 10-membered heteroaryl, 3- to 10-membered heterocyclyl, C 6-10  aryl C 1-4  alkyl, 3- to 10-membered heterocyclyl C 1-3  alkyl, 5- to 10-membered heteroaryl C 1-3  alkyl, cyano(C 1-3  alkyl), C 1-3  alkylsulfonyl C 1-4  alkyl, or 3- to 10-membered cycloaliphatic ring; or alternatively R 26  and R 27 , together with a nitrogen atom linked thereto, form 3- to 10-membered heterocyclyl or 5- to 10-membered heteroaryl; 
         R 28  and R 29 , which can be the same or different, each represents hydrogen, C 1-4  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 1-4  haloalkyl, C 1-3  alkoxyl C 1-4  alkyl, C 6-10  aryl, 5- to 10-membered heteroaryl, 3- to 10-membered heterocyclyl, C 6-10  aryl C 1-4  alkyl, 3- to 10-membered heterocyclyl C 1-3  alkyl, 5- to 10-membered heteroaryl C 1-3  alkyl, cyano(C 1-3  alkyl), C 1-3  alkylsulfonyl C 1-4  alkyl, or 3- to 10-membered cycloaliphatic ring; or alternatively R 28  and R 29 , together with a nitrogen atom linked thereto, form 3- to 10-membered heterocyclyl or 5- to 10-membered heteroaryl; 
         R 30  represents C1-4 alkyl, C 3-7  cycloalkyl, C 1-4  haloalkyl, C 6-10  aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl; 
         R 31 represents C 1-4  alkyl, C 3-7  cycloalkyl, C 1-4  haloalkyl, C 6-10  aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl; 
         R 32  represents C 1-4  alkyl or C 6-10  aryl; 
         <group P> 
         halogen, C 1-4  alkyl, C 1-4  haloalkyl, —OH, C 1-3  alkoxy, C 1-3  haloalkoxy, 3- to 10-membered heterocyclylamino, —SO 2 R 16 , —CN, —NO 2 , and 3- to 10-membered heterocyclyl; 
         <group Q> 
         halogen, C 1-4  alkyl, C 1-4  haloalkyl, —OH, C 1-3  alkoxy, C 1-6  monohydroxy alkyl, C1_6 dihydroxy alkyl, C 1-6  trihydroxy alkyl, 3- to 10-membered heterocyclyl amine, —SO 2 R 16 , —CN, —NO 2 , C 3-7  cycloalkyl, —COR 19 , and 3- to 10-membered heterocyclyl which is optionally substituted by C 1-4  alkyl. 
       
     
     
         2 . The pharmaceutical composition of  claim 1 , comprising as an active ingredient the compound represented by the formula below or a pharmaceutically acceptable salt thereof: 
       
         
           
           
               
               
           
         
       
     
     
         3 . An FGFR mutant polypeptide comprising a substitution of valine to phenylalanine at the 7 th  amino acid from the N terminus and/or a substitution of valine to leucine at the 5 th  amino acid from the N terminus in the partial amino acid sequence described in SEQ ID NO: 53 or 54 in an FGFR polypeptide. 
     
     
         4 . A polynucleotide encoding the mutant polypeptide of  claim 3 . 
     
     
         5 . A vector comprising the polynucleotide of  claim 4 . 
     
     
         6 . A recombinant cell comprising the vector of  claim 5 . 
     
     
         7 . An antibody, or antigen-binding fragment thereof, that specifically binds to the mutant polypeptide of  claim 3 . 
     
     
         8 . A pair of oligonucleotide primers or an oligonucleotide probe(s) comprising an oligonucleotide(s) specifically hybridizing to a polynucleotide encoding the mutant polypeptide of  claim 3  for detecting or amplifying the polynucleotide. 
     
     
         9 . An oligonucleotide that binds to an mRNA polynucleotide encoding the mutant polypeptide of  claim 3  and has an activity to inhibit translation of the mRNA polynucleotide into protein. 
     
     
         10 . The oligonucleotide of  claim 9 , which is an siRNA that cleaves the mRNA polynucleotide. 
     
     
         11 . A method for detecting an FGFR mutant polypeptide, which comprises the step of detecting the mutant polypeptide in a sample isolated from a subject by using an antibody, or antigen-binding fragment thereof, that specifically binds to the mutant polypeptide of  claim 3 . 
     
     
         12 . A method for detecting a polynucleotide encoding an FGFR mutant polypeptide, which comprises the step of detecting a polynucleotide encoding the mutant polypeptide in a sample isolated from a subject by using a pair of oligonucleotide primers or an oligonucleotide probe(s) comprising an oligonucleotide(s) specifically hybridizing to a polynucleotide encoding the mutant polypeptide of  claim 3  for detecting or amplifying the polynucleotide. 
     
     
         13 . A kit for detecting a polynucleotide encoding an FGFR mutant polypeptide, which comprises a pair of oligonucleotide primers or an oligonucleotide probe(s) comprising an oligonucleotide(s) specifically hybridizing to a polynucleotide encoding the mutant polypeptide of  claim 3  for detecting or amplifying the polynucleotide. 
     
     
         14 . A kit for detecting an FGFR mutant polypeptide, which comprises an antibody or antigen-binding fragment thereof that specifically binds to the mutant polypeptide of  claim 3 . 
     
     
         15 . A method for treating cancer, which comprises determining the presence or absence of the mutant polypeptide of  claim 3  or a polynucleotide encoding the mutant polypeptide in a sample isolated from a subject and administering the pharmaceutical composition of  claim 1  or  2  to the subject when the mutant polypeptide or the polynucleotide is detected. 
     
     
         16 . The method of  claim 15 , wherein the cancer is bladder cancer, brain tumor, head and neck squamous cell carcinoma, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, skin melanoma, endometrial cancer, breast cancer, prostate cancer, colon cancer, esophageal cancer, gastric cancer, bile duct cancer, biliary tract cancer, or liver cancer. 
     
     
         17 . A method for selecting a patient to which the pharmaceutical composition of  claim 1  or  2  is applicable, which comprises the steps of:
 (a) determining the presence or absence of the mutant polypeptide of  claim 3  in a sample isolated from a subject; and 
 (b) selecting a subject confirmed to have the mutant polypeptide as a patient to which the pharmaceutical composition is applicable. 
 
     
     
         18 . A method for selecting a patient to which the pharmaceutical composition of  claim 1  or  2  is applicable, which comprises the steps of:
 (a) determining the presence or absence of a polynucleotide encoding the mutant polypeptide of  claim 3  in a sample isolated from a subject; and 
 (b) selecting a subject confirmed to have a polynucleotide encoding the mutant polypeptide as a patient to which the pharmaceutical composition is applicable. 
 
     
     
         19 . The method of  claim 18 , wherein the cancer is bladder cancer, brain tumor, head and neck squamous cell carcinoma, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, skin melanoma, endometrial cancer, breast cancer, prostate cancer, colon cancer, esophageal cancer, gastric cancer, bile duct cancer, biliary tract cancer, or liver cancer. 
     
     
         20 . The compound defined in  claim 1  or  2  or a pharmaceutically acceptable salt thereof for use in cancer treatment in a patient who expresses the mutant polypeptide of  claim 3  or has a polynucleotide encoding the mutant polypeptide. 
     
     
         21 . The compound of  claim 20  or a pharmaceutically acceptable salt thereof, wherein the cancer is bladder cancer, brain tumor, head and neck squamous cell cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, skin melanoma, endometrial cancer, breast cancer, prostate cancer, colon cancer, esophageal cancer, gastric cancer, bile duct cancer, biliary tract cancer, or liver cancer. 
     
     
         22 . A pharmaceutical composition for cancer treatment comprising as an active ingredient the compound represented by the formula below or a pharmaceutically acceptable salt thereof, wherein the cancer expresses an FGFR mutant polypeptide comprising a substitution of valine to phenylalanine at the 7 th  amino acid from the N terminus and/or a substitution of valine to leucine at the 5 th  amino acid from the N terminus in the partial amino acid sequence described in SEQ ID NO: 53 or 54 in an FGFR polypeptide: 
       
         
           
           
               
               
           
         
       
     
     
         23 . A pharmaceutical composition for cancer treatment comprising as an active ingredient a substance that inhibits the function or expression of an FGFR mutant polypeptide comprising a substitution of valine to phenylalanine at the 7 th  amino acid from the N terminus and/or a substitution of valine to leucine at the 5 th  amino acid from the N terminus in the partial amino acid sequence described in SEQ ID NO: 53 or 54 in an FGFR polypeptide.