Inhibitors of protein methyltransferase dot1l and methods of use thereof

Abstract

The present invention relates to DOT1L inhibitors and methods of identifying, designing, or optimizing them. The present invention also relates to crystals of DOT1L-inhibitor complexes, the crystal structures thereof, and the use of the crystal structures. Also disclosed are pharmaceutical compositions containing these DOT1L inhibitors and methods of treating disorders in which DOT1-mediated protein methylation plays a part, such as cancer and neurological disorders, by administering these compounds and pharmaceutical compositions to subjects in need thereof.

1 . A compound of Formula (I) below or a pharmaceutically acceptable salt or ester thereof: 
       
         
           
           
               
               
           
         
         wherein, 
         Nuc is adenosine-like moiety or an analog or a derivative thereof, 
         T is —CH 2 -L 1 -L 2 -L 3 -, with L 3  connected to R 9  wherein: 
         L 1  is N(Y), S, SO, or SO 2 ; 
         L 2  is CO or absent when L 1  is N(Y), or L 2  is absent when L 1  is S, SO, or SO 2 , in which Y is H, R d , SO 2 R d , or COR d  when L 2  is absent, or Y is H or R d  when L 2  is CO, R d  being C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl, and R d  being optionally substituted with one or more substituents selected from the group consisting of C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, halo, carboxyl, cyano, C 1 -C 6  alkoxyl, C 1 -C 6  alkylsulfonyl, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 8-membered heterocycloalkyl, 5 to 6-membered heteroaryl, OR d′ , OCOR d′ , and N(R d′ ) 2 , and with C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl further optionally substituted with C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, halo, hydroxyl, carboxyl, C(O)OH, C(O)O—C 1 -C 6  alkyl, OC(O)—C 1 -C 6  alkyl, cyano, C 1 -C 6  alkoxyl, amino, mono-C 1 -C 6  alkylamino, di-C 1 -C 6  alkylamino, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl, each R d′  independently being H, C 1 -C 6  alkyl, silyl, C 1 -C 6  alkyl-C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 5 to 6-membered heteroaryl, aralkyl, or heteroaralkyl; 
         L 3  is —(CR 4 R 5 ) n -unsubstituted or substituted C 3 -C 8  cycloalkyl-(CR 6 R 7 ) m —, with (CR 6 R 7 ) m  connected to R 9 ; 
         each of R 4 , R 5 , R 6  and R 7 , independently, is H, halo, hydroxyl, carboxyl, cyano, or R S2 , R S2  being amino, C 1 -C 6  alkoxyl, C 1 -C 6  alkyl, C 2 -C 6  alkenyl, or C 2 -C 6  alkynyl, and each R S2  being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C 1 -C 6  alkoxyl, amino, mono-C 1 -C 6  alkylamino, di-C 1 -C 6  alkylamino, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl; or two geminal R 4  and R 5  or two geminal R 6  and R 7  taken together are ethylene, propylene or butylene; 
         m is 0, 1, or 2; 
         n is 0, 1, or 2; 
         Nuc-T is capable of binding within the SAM binding pocket of human DOT1L which comprises amino acid residues 135-241 of SEQ ID NO: 1; and 
         R 9  is a group such that R 9  induces a conformational adaptation in human DOT1L, wherein the conformational adaptation is the formation of a hydrophobic pocket domain which comprises amino acid residues 139-312 of SEQ ID NO: 1. 
       
     
     
         2 . The compound of  claim 1  wherein the R 9  is a group such that R 9  induces a residence time of the compound greater than 20 seconds in a complex formed of the compound and human DOT1L. 
     
     
         3 . The compound of  claim 1 , wherein the SAM binding pocket of human DOT1L is characterized by the crystallography coordinates of one or more human DOT1L amino acids Val135, Thr139, Asp161, Gly163, Gln168, Glu 186, Asp222, Phe223, and Asn241, according to Table S1 or S2. 
     
     
         4 . The compound of  claim 1 , wherein the hydrophobic pocket domain of human DOT1L is characterized by the crystallography coordinates of human DOT1L amino acids Leu143, Met147, Phe239, and Tyr 312, according to Table S1 or S2. 
     
     
         5 . The compound of  claim 1 , wherein the binding affinity (K i ) of the compound to human DOT1L is not greater than 50 μM. 
     
     
         6 . The compound of  claim 1 , wherein R 9  comprises C 6 -C 10  aryl or 5 to 10-membered heteroaryl optionally substituted with one or more substituents selected from the group consisting of unsubstituted or substituted t-butyl, CF 3 , cyclohexyl, C 6 -C 10  aryl, and 5 to 10-membered heteroaryl. 
     
     
         7 . The compound of  claim 6 , wherein the one or more substituents are t-butyl. 
     
     
         8 . The compound of  claim 6 , wherein R 9  is selected from the group consisting of 
       
         
           
           
               
               
           
         
       
     
     
         9 . The compound of  claim 1 , being of formula (II): 
       
         
           
           
               
               
           
         
       
       wherein,
 A is O or CH 2 ; 
 each of G and J, independently, is H, halo, C(O)OH, C(O)O—C 1 -C 6  alkyl or OR a , R a  being H, C 1 -C 6  alkyl, C(O)—C 1 -C 6  alkyl, or silyl, wherein C(O)O—C 1 -C 6  alkyl, C 1 -C 6  alkyl or C(O)—C 1 -C 6  alkyl is optionally substituted with one or more substituents selected from the group consisting of halo, cyano hydroxyl, carboxyl, C 1 -C 6  alkoxyl, amino, mono-C 1 -C 6  alkylamino, di-C 1 -C 6  alkylamino, and C 3 -C 8  cycloalkyl; 
 each X independently is N or CR x , in which R x  is H, halo, hydroxyl, carboxyl, cyano, or R S1 , R S1  being amino, C 1 -C 6  alkoxyl, C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl, and R S1  being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C 1 -C 6  alkoxyl, amino, mono-C 1 -C 6  alkylamino, di-C 1 -C 6  alkylamino, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl; 
 each of R 1  and R 2 , independently is H, halo, hydroxyl, carboxyl, cyano, or R S2 , R S2  being amino, C 1 -C 6  alkoxyl, C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, or C 3 -C 8  cycloalkyl, and each R S2  being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C 1 -C 6  alkoxyl, amino, mono-C 1 -C 6  alkylamino, di-C 1 -C 6  alkylamino, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl; 
 R 8  is H, halo or R S3 , R S3  being C 1 -C 6  alkyl, C 2 -C 6  alkenyl, or C 2 -C 6  alkynyl, and R S3  being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano amino, C 1 -C 6  alkoxyl, mono-C 1 -C 6  alkylamino, di-C 1 -C 6  alkylamino, and C 3 -C 8  cycloalkyl; and 
 Q is H, NH 2 , NHR b , NR b R c , R b , ═O, OH, or OR b , in which each of R b  and R c  independently is C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 7-membered heterocycloalkyl, 5 to 10-membered heteroaryl, or -M 1 -T 1  in which M 1  is a bond or C 1 -C 6  alkyl linker optionally substituted with halo, cyano, hydroxyl or C 1 -C 6  alkoxyl and T 1  is C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, or 5 to 10-membered heteroaryl, or R b  and R c , together with the N atom to which they attach, form 4 to 7-membered heterocycloalkyl having 0 or 1 additional heteroatoms to the N atom optionally substituted with C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, halo, hydroxyl, carboxyl, C(O)OH, C(O)O—C 1 -C 6  alkyl, OC(O)—C 1 -C 6  alkyl, cyano, C 1 -C 6  alkoxyl, amino, mono-C 1 -C 6  alkylamino, di-C 1 -C 6  alkylamino, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl, and each of R b , R c , and T 1  is optionally substituted with one or more substituents selected from the group consisting of C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, halo, hydroxyl, carboxyl, cyano, C 1 -C 6  alkoxyl, amino, mono-C 1 -C 6  alkylamino, di-C 1 -C 6  alkylamino, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl. 
 
     
     
         10 . (canceled) 
     
     
         11 . The compound of  claim 9 , wherein R 9  is 
       
         
           
           
               
               
           
         
       
       in which:
 each of R e , R f , R g , and R h , independently is -M 2 -T 2 , in which M 2  is a bond, SO 2 , SO, S, CO, CO 2 , O, O—C 1 -C 4  alkyl linker, C 1 -C 4  alkyl linker, NH, or N(R t ), R t  being C 1 -C 6  alkyl, and T 2  is H, halo, or R S4 , R S4  being C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 8-membered heterocycloalkyl, or 5 to 10-membered heteroaryl, and each of O—C 1 -C 4  alkyl linker, C 1 -C 4  alkyl linker, R t , and R S4  being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, C 1 -C 6  alkoxyl, amino, mono-C 1 -C 6  alkylamino, di-C 1 -C 6  alkylamino, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl, R i  is H or C 1 -C 6  alkyl optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C 1 -C 6  alkoxyl, amino, mono-C 1 -C 6  alkylamino, di-C 1 -C 6  alkylamino, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl, 
 D is O, NR j , or CR j R k , each of R j  and R k  independently being H or C 1 -C 6  alkyl, or R j  and R k  taken together, with the carbon atom to which they are attached, form a C 3 -C 10  cycloalkyl ring, and 
 E is -M 3 -T 3 , M 3  being a bond or C 1 -C 6  alkyl linker optionally substituted with halo or cyano, T 3  being C 3 -C 14  carbocycle or 4 to 14-membered heterocycle, and T 3  being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, thiol, carboxyl, cyano, nitro, C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, C 1 -C 6  alkoxyl, C 1 -C 6  haloalkyl, C 1 -C 6  haloalkoxyl, C 1 -C 6  alkylthio, C 1 -C 6  alkylsulfonyl, C 1 -C 6  haloalkylsulfonyl, C 1 -C 6  alkylcarbonyl, C 1 -C 6  alkoxycarbonyl, oxo, amino, mono-C 1 -C 6  alkylamino, di-C 1 -C 6  alkylamino, C 3 -C 8  cycloalkyl, C 4 -C 12  alkylcycloalkyl, C 6 -C 10  aryl, C 6 -C 10  aryloxyl, C 7 -C 14  alkylaryl, C 6 -C 10  aminoaryloxyl, C 6 -C 10  arylthio, 4 to 6-membered heterocycloalkyl optionally substituted with halo, C 1 -C 4  alkyl, C 1 -C 4  haloalkyl, 5 to 6-membered heteroaryl optionally substituted with halo, C 1 -C 4  alkyl, and C 1 -C 6  alkyl that is substituted with hydroxy, halo, C 1 -C 6  alkoxycarbonyl, C 3 -C 8  cycloalkyl, C 6 -C 10  aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl optionally further substituted with halo, hydroxyl, or C 1 -C 6  alkoxyl. 
 
     
     
         12 . The compound of  claim 13 , wherein the compound is of formula (IIIb): 
       
         
           
           
               
               
           
         
         wherein R 3  is H, halo, hydroxyl, carboxyl, cyano, or R S2 , and q is 0, 1, 2, 3, or 4. 
       
     
     
         13 . (canceled) 
     
     
         14 . The compound of  claim 12 , wherein the compound is of formula (IIIb) and R 9  is 
       
         
           
           
               
               
           
         
       
     
     
         15 . (canceled) 
     
     
         16 . A method of identifying the compound of  claim 1  as a binding compound of protein DOT1L, the method comprising:
 computationally identifying a binding compound that binds to DOT1L using the atomic coordinates of Leu143, Met147, Phe239, and Tyr 312 according to Table S1 or S2. 
 
     
     
         17 . (canceled) 
     
     
         18 . A method of identifying the compound of  claim 1  as a drug candidate for the treatment of a disease, the method comprising:
 a) using the atomic coordinates set forth in Table S1 or S2 to form a three-dimensional structure of DOT1L; 
 b) selecting a test compound having the best fit with the structure of DOT1L; and 
 c) assaying the ability of the test compound to modulate DOT1L activity, 
 
       wherein a test compound that modulates DOT1L activity is considered a drug candidate for treating a disease. 
     
     
         19 . The compound of  claim 1 , wherein the compound molecular dimensions compatible with the shape of a hydrophobic pocket domain of DOT1L characterized by the crystallography coordinates of human DOT1L amino acids Leu143, Met147, Phe239, and Tyr 312, according to Table S1 or S2, wherein the compound has a biochemical IC 50  for DOT1L of less than 100 nM. 
     
     
         20 . A computer readable medium comprising the atomic coordinates of one or more DOT1L-Compound A2, DOT1L-Compound C1, DOT1L-Compound C118 and DOT1L-Compound D16. 
     
     
         21 .- 24 . (canceled) 
     
     
         25 . A method for designing, identifying, and/or optimizing a candidate DOT1L inhibitor compound or complex that interacts with all or a part of a hydrophobic pocket domain which comprises amino acid residues 139-312 of SEQ ID NO: 1 and the SAM binding pocket of human DOT1L which comprises amino acid residues 135-241 of SEQ ID NO: 1, the method comprising:
 (a) producing the structure coordinates of the hydrophobic binding pocket of DOT1L in silico comprising the means for generating three-dimensional structural information from the coordinates;   (b) designing, selecting and/or optimizing the candidate DOT1L inhibitor compound or complex by performing a fitting operation between the candidate DOT1L inhibitor compound or complex and the three-dimensional structural information of all or part of the hydrophobic binding pocket or DOT1L protein; and   (c) optionally employing computerized and/or reiterative steps.   
     
     
         26 . A pharmaceutical composition comprising the compound of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         27 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a DOT1L inhibitor designed, identified, and/or optimized by the method of  claim 15 . 
     
     
         28 . A method for treating cancer comprising administrating a therapeutically effective amount of the compound of  claim 1  to a subject in need thereof. 
     
     
         29 .- 32 . (canceled)