Bioorthogonal Turn-on Probes

Abstract

This present application relates to fluorescent tetrazine-containing compounds consisting of a single pi-system. For example, a compound of Formula (I): F-L-Tz or a salt thereof, wherein: F is a fluorophore, L is a conjugated linker, and Tz is a substituted or unsubstituted tetrazine; wherein the linker bridges the Tz and F moieties in a single conjugated pi-system. Also provided herein are methods of using the compounds provided herein for biomedical imaging.

1 . A compound of Formula (I):
   F-L-Tz   or a salt thereof,   wherein:   F is a fluorophore selected from the group consisting of:   
       
         
           
           
               
               
           
         
       
       wherein:
 R 1  and R 2  are selected from the group consisting of: H, (C 1 -C 6 )alkyl, (C 1 -C 6 )haloalkyl, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkynyl, (C 1 -C 6 )alkoxy, —COR 10 , —CO 2 R 10 , —SOR 12 , —SO 2 R 12 , —NR 10 R 11 , —NO 2 , (C 3 -C 10 )carbocyclyl, (C 6 -C 10 )aryl, 4-10 membered heterocyclyl, and 5-10 membered heteroaryl, each of which is independently substituted or unsubstituted, and wherein if both R 1  and R 2  are present, no more than one of R 1  and R 2  is H; 
 R 3 , R 4 , R 5 , R 6 , and R 7  are independently selected from H, (C 1 -C 6 )alkyl, (C 1 -C 6 )haloalkyl, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkynyl, (C 1 -C 6 )alkoxy, -SO3H, (C 3 -C 10 )carbocyclyl, (C 6 -C 10 )aryl, 4-10 membered heterocyclyl, 5-10 membered heteroaryl, each of which is independently substituted or unsubstituted, and a reactive moiety; 
 R 8  and R 9  are independently selected from halogen, (C 1 -C 6 )alkyl, (C 2 -C 6 )alkynyl, —CO 2 R 10 , (C 1 -C 6 )alkoxy, O(4 membered heterocyclyl), —O—(C 1 -C 6 )alkyl-O(nPEG), each of which is independently substituted or unsubstituted; 
 PEG is a polyethylene glycol polymer; 
 each R 10  and R 11  are independently selected from H and (C 1 -C 6 )alkyl; and 
 each R 12  is independently a (C 6 -C 10 )aryl; 
 L is a conjugated linker; and 
 Tz is a substituted or unsubstituted tetrazine; 
 wherein the linker bridges the Tz and F moieties in a single conjugated pi-system. 
 
     
     
         2 . The compound of  claim 1 , or a salt thereof, wherein the single conjugated pi-system is non-coplanar. 
     
     
         3 . The compound of  claim 2 , or a salt thereof, wherein steric factors enforce a twist in the L-Tz bond to F and originate from substituents on either the linker or on the fluorophore. 
     
     
         4 . The compound of  claim 1 , or a salt thereof, wherein the L-Tz moiety is oriented with respect to the F moiety such that the tetrazine transition dipole is either collinear with or parallel to the transition dipole of the fluorophore. 
     
     
         5 .- 6 . (canceled) 
     
     
         7 . The method of  claim 1 , or a salt thereof, wherein R 1  and R 2  are independently selected from methyl, ethyl, isopropyl, and tert-butyl. 
     
     
         8 . The method of  claim 1 , or a salt thereof, wherein R 3 , R 4 , R 5 , R 6 , and R 7  are independently selected from H, methyl, ethyl, 
       
         
           
           
               
               
           
         
       
     
     
         9 .- 26 . (canceled) 
     
     
         27 . The compound of  claim 1 , or a salt thereof, wherein the linker is an aromatic linker. 
     
     
         28 . The compound of  claim 27 , or a salt thereof, wherein the aromatic linker is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         wherein: 
         R 1a , R 2a , R 3a , and R 4a  are selected from the group consisting of: H, (C 1 -C 6 )alkyl, (C 1 -C 6 )haloalkyl, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkynyl, (C 1 -C 6 )alkoxy, —COR 13 , —CO 2 R 13 , —SOR 15 , —SO 2 R 15 , —NR 13 R 14 , —NO 2 , (C 3 -C 10 )carbocyclyl, (C 6 -C 10 )aryl, 4-10 membered heterocyclyl, and 5-10 membered heteroaryl, each of which is independently substituted or unsubstituted. 
       
     
     
         29 . The compound of  claim 1 , or a salt thereof, wherein Tz is a moiety having the structure: 
       
         
           
           
               
               
           
         
         wherein: 
         R 1b  is selected from the group consisting of: H, (C 1 -C 6 )alkyl, (C 1 -C 6 )haloalkyl, (C 6 -C 10 )aryl, and 5-10 membered heteroaryl, each of which is independently substituted or unsubstituted. 
       
     
     
         30 . The compound of  claim 29 , or a salt thereof, wherein the Tz moiety is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         31 . The compound of  claim 1 , wherein the compound of Formula (I) is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       or a salt thereof. 
     
     
         32 .- 35 . (canceled) 
     
     
         36 . A method of imaging a subject, the method comprising:
 a) administering to the subject an effective amount of a dienophile conjugated to one or more of a small molecule therapeutic agent, antibody, nanoparticle, polymer, and mixtures thereof;   b) administering to the subject an effective amount of a compound of  claim 1 , or a salt thereof; and   c) imaging the subject.   
     
     
         37 . The compound of  claim 1 , or a salt thereof, wherein F is: 
       
         
           
           
               
               
           
         
       
     
     
         38 . The compound of  claim 37 , or a salt thereof, wherein R 1  and R 2  are each methyl. 
     
     
         39 . The compound of  claim 37 , or a salt thereof, wherein R 3 , R 4 , R 5 , R 6 , and R 7  are independently selected from H, methyl, and ethyl. 
     
     
         40 . The compound of  claim 37 , or a salt thereof, wherein L is: 
       
         
           
           
               
               
           
         
       
       wherein R 1a , R 2a , R 3a , and R 4a  are each independently selected from the group consisting of H and (C 1 -C 6 )alkyl. 
     
     
         41 . The compound of  claim 37 , or a salt thereof, wherein Tz is a moiety having the structure: 
       
         
           
           
               
               
           
         
       
       wherein R 1b  is selected from the group consisting of H and (C 1 -C 6 )alkyl. 
     
     
         42 . A compound, which is: 
       
         
           
           
               
               
           
         
       
       or a salt thereof. 
     
     
         43 . A pharmaceutical composition comprising the compound of  claim 42 , or a salt thereof, and a pharmaceutically acceptable excipient. 
     
     
         44 . A method of imaging a subject, the method comprising:
 a) administering to the subject an effective amount of a dienophile conjugated to one or more of a small molecule therapeutic agent, antibody, nanoparticle, polymer, and mixtures thereof;   b) administering to the subject an effective amount of the compound of  claim 42 , or a salt thereof; and   c) imaging the subject.