Carbopyronone Compounds Useful as Diagnostic Adjuvants

Abstract

Described herein are fluorescent compounds and methods and comprising these compounds. The compounds disclosed herein are carbopyronine reagents that fluoresce in the red portion of the UV/VIS spectrum and provide bright fluorescence intensity, uniform cell staining, and good retention within live cells as well as low toxicity toward cells.

1 . A compound of structural formula (I): 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein 
         R 12  and R 13  are each independently H, alkyl, substituted alkyl, -L-R x , or -L-S c ; 
         R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 14 , R 15 , and R 16  are each independently H, halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate, hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol, substituted alkylthiol, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, -L-R x , or -L-S c ; or 
         R 7  taken together with R 14  are part of an optionally substituted 5- or 6-membered ring; 
         R 8  taken together with R 15  are part of an optionally substituted 5- or 6-membered ring; 
         R 9  taken together with R 11  are part of an optionally substituted 5- or 6-membered ring; 
         R 10  taken together with R 16  are part of an optionally substituted 5- or 6-membered ring; 
         L is a linker; 
         R x  is a reactive group; and 
         S c  is a conjugated substance. 
       
     
     
         2 - 6 . (canceled) 
     
     
         7 . The compound according to  claim 1 , wherein R 12  and R 13  are each methyl. 
     
     
         8 . The compound according to  claim 1 , wherein R 3 , R 4 , R 5  and R 6  are each independently H or halo. 
     
     
         9 . (canceled) 
     
     
         10 . The compound according to  claim 1 , wherein R 2  is carboxyl, -L-R x , or -L-S c . 
     
     
         11 . The compound of  claim 1 , wherein R 11  and R 14  are each independently alkyl, substituted alkyl, sulfoalkyl, sulfo, -L-R x , or -L-S c . 
     
     
         12 - 15 . (canceled) 
     
     
         16 . The compound according to  claim 1 , wherein R X  is an acrylamide, a carboxylic acid, an activated ester of a carboxylic acid, an acyl azide, an acyl halide, hydroxy, an aldehyde, an alkyl halide, a sulfonate, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carbodiimide, a diazoalkane, an epoxide, a glycol, a haloacetamide, a halomethyl, a halotriazine, a hydrazine, a hydroxylamine, an imido ester, an iodoacetamide, an isothiocyanate, a ketone, a maleimide, a sulfonyl halide, a thiol group, a succinimidyl ester (SE), a sulfodichlorophenyl (SDP) ester, a sulfotetrafluorophenyl (STP) ester, a tetrafluorophenyl (TFP) ester, a pentafluorophenyl (PFP) ester or a cyclooctyne-amine. 
     
     
         17 - 18 . (canceled) 
     
     
         19 . The compound according to  claim 1 , wherein S c  is an amino acid, a peptide, a protein, a monosaccharide, a polysaccharide, an ion-complexing moiety, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a drug, a toxin, a tyramide, a lipid, a phospholipid, a lipoprotein, a lipopolysaccharide, a liposome, a lipophilic polymer, a PEG group, a non-biological organic polymer, a polymeric microparticle, an animal cell, a plant cell, a bacterium, a yeast, or a virus. 
     
     
         20 - 24 . (canceled) 
     
     
         25 . A method for tracking cell proliferation, differentiation, and/or function, the method comprising the steps of:
 a) incubating a mixture of cells and a compound of structural formula (I):   
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein 
         R 12  and R 13  are each independently H, alkyl, substituted alkyl, -L-R x , or -L-S c ; 
         R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 14 , R 15 , and R 16  are each independently H, halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate, hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol, substituted alkylthiol, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, -L-R x , or -L-S c ; or 
         R 7  taken together with R 14  are part of an optionally substituted 5- or 6-membered ring; 
         R 8  taken together with R 15  are part of an optionally substituted 5- or 6-membered ring; 
         R 9  taken together with R 11  are part of an optionally substituted 5- or 6-membered ring; 
         R 10  taken together with R 16  are part of an optionally substituted 5- or 6-membered ring; 
         L is a linker; 
         R x  is a reactive group; and 
         S c  is a conjugated substance; 
         b) providing a stimulus to the mixture to elicit a fluorescent signal; and 
         c) analyzing the stimulated mixture. 
       
     
     
         26 - 28 . (canceled) 
     
     
         29 . The method according to  claim 25 , wherein the
 compound is selected from the group consisting of   
       
         
           
           
               
               
           
         
       
       and amine-reactive forms thereof. 
     
     
         30 . The method according to  claim 25 , further comprising a second compound excitable at a different wavelength that the cell-tracking compound of structural formula (I). 
     
     
         31 . The method according to  claim 30 , wherein the second compound is CFDA-SE or GFP. 
     
     
         32 . The method according to  claim 25 , wherein R X  is an acrylamide, a carboxylic acid, an activated ester of a carboxylic acid, an acyl azide, an acyl halide, hydroxy, an aldehyde, an alkyl halide, a sulfonate, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carbodiimide, a diazoalkane, an epoxide, a glycol, a haloacetamide, a halomethyl, a halotriazine, a hydrazine, a hydroxylamine, an imido ester, an iodoacetamide, an isothiocyanate, a ketone, a maleimide, a sulfonyl halide, or a thiol group. 
     
     
         33 . (canceled) 
     
     
         34 . The method according to  claim 32 , wherein R X  is a succinimidyl ester (SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester, nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine. 
     
     
         35 . The method according to  claim 25 , wherein step a) is conducted for approximately 20 minutes. 
     
     
         36 . The method according to  claim 25 , wherein step b) and step c) are carried out concurrently. 
     
     
         37 . The method according to  claim 25 , wherein step c) involves flow cytometry. 
     
     
         38 . A kit for tracking cell proliferation, differentiation, and/or function, the kit comprising:
 a) a compound of  claim 1 ;   b) an organic solvent; and   c) a desiccant.   
     
     
         39 - 46 . (canceled) 
     
     
         47 . A composition for tracking cell proliferation, differentiation, and/or function, the compositions comprising:
 (a) one or more of the compounds of  claim 1 ; and   b) a carrier,   wherein the one or more of the compounds are present in an amount effective to track cell proliferation, differentiation, and/or function.   
     
     
         48 . A composition for tracking cell proliferation, differentiation, and/or function, the compositions comprising:
 (a) one or more of the compounds of  claim 1 ; and   (b) an analyte,   
       wherein the one or more of the compounds are present in an amount effective to track cell proliferation, differentiation, and/or function. 
     
     
         49 - 52 . (canceled)