Derivatives of 1,2-dihydro-7-hydroxyquinolines containing fused rings

Abstract

The present invention describes novel dyes, including coumarins, rhodamines, and rhodols that incorporate additional fused aromatic rings. The dyes of the invention absorb at a longer wavelength than structurally similar dyes that do not possess the fused aromatic rings. Many of the dyes of the invention are useful fluorescent dyes. The invention includes chemically reactive dyes, dye-conjugates, and the use of such dyes in staining samples and detecting ligands or other analytes.

1 .- 14 . (canceled) 
     
     
         15 . A kit for staining a sample, wherein the kit comprises a solution comprising a buffer and a compound of formula: 
       
         
           
           
               
               
           
         
         wherein G is a 5- or 6-membered aromatic or heteroaromatic fused ring, where said ring may be fused to one or two additional fused aromatic or heteroaromatic rings, and where said ring is optionally substituted one or more times by sulfonic acid, carboxylic acid, or C 1 -C 6  alkyl or alkoxy that is optionally substituted by carboxylic acid, sulfonic acid, or halogen; or by an aryl or heteroaryl ring that is optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, cyano, halogen, azido, carboxylic acid, sulfonic acid, or halomethyl; 
         R 3  and R 4  are each independently H, a C 1 -C 6  alkyl optionally substituted by carboxylic acid, sulfonic acid, amino, hydroxy, or halogen; an aromatic or heteroaromatic ring optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl; or R 3  and R 4 , taken in combination, form a 5- or 6-membered ring that optionally contains 1 or 2 heteroatoms; 
         R 5  is H, methyl, carboxymethyl, or a C 2 -C 6  alkyl optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen; an aryl or heteroaryl ring optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl; or R 4  taken in combination with R 5 , or R 5  taken in combination with R 6 , forms a 5- or 6-membered alicyclic ring; 
         R 6  is H, cyano, halogen, carboxylic acid, sulfonic acid, a C 1 -C 6  alkyl or C 1 -C 6  alkoxy optionally substituted by carboxylic acid, sulfonic acid, or halogen; an aryl or heteroaryl ring optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, cyano, halogen, azido, carboxylic acid, sulfonic acid, or halomethyl; 
         R 21 , R 23 , R 24 , and R 25  are each independently hydrogen, cyano, nitro, halogen, carboxylic acid, sulfonic acid, -L-R x  or -L-S C , C 1 -C 6  alkyl, where said alkyl is optionally substituted by carboxylic acid, sulfonic acid, or halogen, or an aromatic or heteroaromatic ring, where said aromatic or heteroaromatic ring is optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl; 
         J is O or NR 37 R 38 , where R 37  and R 38  are independently H, -L-R x  or -L-S C , or a C 1 -C 6  alkyl, where said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen; and 
         Q is N or CR 28 , wherein R 28  is H, F, CN, a carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6  alcohol; a C 1 -C 6  alkyl optionally substituted one or more times by carboxylic acid, sulfonic acid, amino, or halogen; or R 28  has the formula 
       
       
         
           
           
               
               
           
         
         where R 30 , R 31 , R 32 , R 33  and R 34  are independently H, F, Cl, Br, I, -L-R x , -L-S C , sulfonic acid, carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino, C 1 -C 18  alkyl, C 1 -C 18  alkoxy, C 1 -C 18  alkylthio, C 1 -C 18  alkanoylamino, C 1 -C 18  alkylaminocarbonyl, C 2 -C 36  dialkylaminocarbonyl, C 1 -C 18  alkyloxycarbonyl, or C 7 -C 18  arylcarboxamido, wherein said alkyl or aryl portions of said R 30 , R 31 , R 32 , R 33  and R 34  are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a carboxylic acid ester of a C 1 -C 6  alcohol, sulfonic acid, amino, C 1 -C 6  alkylamino, C 2 -C 6  dialkylamino or C 1 -C 6  alkoxy; or a pair of adjacent substituents R 31  and R 32 , R 32  and R 33  or R 33  and R 34 , when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid. 
       
     
     
         16 . A method of staining a biological sample, comprising:
 combining a dye solution comprising a compound of formula:   
       
         
           
           
               
               
           
         
         wherein G is a 5- or 6-membered aromatic or heteroaromatic fused ring, where said ring may be fused to one or two additional fused aromatic or heteroaromatic rings, and where said ring is optionally substituted one or more times by sulfonic acid, carboxylic acid, or C 1 -C 6  alkyl or alkoxy that is optionally substituted by carboxylic acid, sulfonic acid, or halogen; or by an aryl or heteroaryl ring that is optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, cyano, halogen, azido, carboxylic acid, sulfonic acid, or halomethyl; 
         R 3  and R 4  are each independently H, a C 1 -C 6  alkyl optionally substituted by carboxylic acid, sulfonic acid, amino, hydroxy, or halogen; an aromatic or heteroaromatic ring optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl; or R 3  and R 4 , taken in combination, form a 5- or 6-membered ring that optionally contains 1 or 2 heteroatoms; 
         R 5  is H, methyl, carboxymethyl, or a C 2 -C 6  alkyl optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen; an aryl or heteroaryl ring optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl; or R 4  taken in combination with R 5 , or R 5  taken in combination with R 6 , forms a 5- or 6-membered alicyclic ring; 
         R 6  is H, cyano, halogen, carboxylic acid, sulfonic acid, a C 1 -C 6  alkyl or C 1 -C 6  alkoxy optionally substituted by carboxylic acid, sulfonic acid, or halogen; an aryl or heteroaryl ring optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, cyano, halogen, azido, carboxylic acid, sulfonic acid, or halomethyl; 
         R 21 , R 23 , R 24 , and R 25  are each independently hydrogen, cyano, nitro, halogen, carboxylic acid, sulfonic acid, -L-R x  or -L-S C , C 1 -C 6  alkyl, where said alkyl is optionally substituted by carboxylic acid, sulfonic acid, or halogen, or an aromatic or heteroaromatic ring, where said aromatic or heteroaromatic ring is optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl; 
         J is O or NR 37 R 38 , where R 37  and R 38  are independently H, -L-R x  or -L-S C , or a C 1 -C 6  alkyl, where said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen; and 
         Q is N or CR 28 , wherein R 28  is H, F, CN, a carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6  alcohol; a C 1 -C 6  alkyl optionally substituted one or more times by carboxylic acid, sulfonic acid, amino, or halogen; or R 28  has the formula 
       
       
         
           
           
               
               
           
         
         where R 30 , R 31 , R 32 , R 33  and R 34  are independently H, F, Cl, Br, I, -L-R x , -L-S C , sulfonic acid, carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino, C 1 -C 18  alkyl, C 1 -C 18  alkoxy, C 1 -C 18  alkylthio, C 1 -C 18  alkanoylamino, C 1 -C 18  alkylaminocarbonyl, C 2 -C 36  dialkylaminocarbonyl, C 1 -C 18  alkyloxycarbonyl, or C 7 -C 18  arylcarboxamido, wherein said alkyl or aryl portions of said R 30 , R 31 , R 32 , R 33  and R 34  are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a carboxylic acid ester of a C 1 -C 6  alcohol, sulfonic acid, amino, C 1 -C 6  alkylamino, C 2 -C 6  dialkylamino or C 1 -C 6  alkoxy; or a pair of adjacent substituents R 31  and R 32 , R 32  and R 33  or R 33  and R 34 , when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid; 
         with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions. 
       
     
     
         17 . The method according to  claim 16 , further comprising:
 combining the sample with an additional detection reagent that has spectral properties that are detectably different from said optical response; and/or   determining a characteristic of the sample by comparing the optical response with a standard response parameter; and/or   wherein the sample is immobilized in or on a solid or semi-solid matrix that is a membrane, an electrophoretic gel, a silicon chip, a microwell plate, or a microfluidic chip.   
     
     
         18 . The method according to  claim 16 , wherein the sample comprises cells; and/or further comprising tracing the temporal or spatial location of the optical response with the sample. 
     
     
         19 . The method according to  claim 16 , wherein for said compound S c  is an amino acid, a peptide, a protein, a polysaccharide, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid polymer, an ion-complexing moiety, a lipid, or a non-biological organic polymer or polymeric microparticle, that is optionally bound to one or more additional fluorophores that are the same or different. 
     
     
         20 . The method according to  claim 16 , wherein for said compound, R 28  is an -L-S c , and S c  is an ion-complexing moiety that is a BAPTA or an APTRA. 
     
     
         21 . A method of staining a biological sample, comprising:
 combining a dye solution comprising a compound of formula:   
       
         
           
           
               
               
           
         
         wherein G is a 5- or 6-membered aromatic or heteroaromatic fused ring, where said ring may be fused to one or two additional fused aromatic or heteroaromatic rings, and where said ring is optionally substituted one or more times by sulfonic acid, carboxylic acid, or C 1 -C 6  alkyl or alkoxy that is optionally substituted by carboxylic acid, sulfonic acid, or halogen; or by an aryl or heteroaryl ring that is optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, cyano, halogen, azido, carboxylic acid, sulfonic acid, or halomethyl; 
         R 3  and R 4  are each independently H, a C 1 -C 6  alkyl optionally substituted by carboxylic acid, sulfonic acid, amino, hydroxy, or halogen; an aromatic or heteroaromatic ring optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl; or R 3  and R 4 , taken in combination, form a 5- or 6-membered ring that optionally contains 1 or 2 heteroatoms; 
         R 5  is H, methyl, carboxymethyl, or a C 2 -C 6  alkyl optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen; an aryl or heteroaryl ring optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl; or R 4  taken in combination with R 5 , or R 5  taken in combination with R 6 , forms a 5- or 6-membered alicyclic ring; 
         R 6  is H, cyano, halogen, carboxylic acid, sulfonic acid, a C 1 -C 6  alkyl or C 1 -C 6  alkoxy optionally substituted by carboxylic acid, sulfonic acid, or halogen; an aryl or heteroaryl ring optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, cyano, halogen, azido, carboxylic acid, sulfonic acid, or halomethyl; 
         R 20 , and R 2 ′ are each independently hydrogen, cyano, nitro, halogen, carboxylic acid, sulfonic acid, -L-R x  or -L-S C , C 1 -C 6  alkyl, where said alkyl is optionally substituted by carboxylic acid, sulfonic acid, or halogen, or an aromatic or heteroaromatic ring, where said aromatic or heteroaromatic ring is optionally substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl; 
         J is O or NR 37 R 38 , where R 37  and R 38  are independently H, -L-R x  or -L-S C , or a C 1 -C 6  alkyl, where said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen; and 
         Q is N or CR 28 , wherein R 28  is H, F, CN, a carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6  alcohol; a C 1 -C 6  alkyl optionally substituted one or more times by carboxylic acid, sulfonic acid, amino, or halogen; or R 28  has the formula 
       
       
         
           
           
               
               
           
         
         where R 30 , R 31 , R 32 , R 33  and R 34  are independently H, F, Cl, Br, I, -L-R x , -L-S C , sulfonic acid, carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino, C 1 -C 18  alkyl, C 1 -C 18  alkoxy, C 1 -C 18  alkylthio, C 1 -C 18  alkanoylamino, C 1 -C 18  alkylaminocarbonyl, C 2 -C 36  dialkylaminocarbonyl, C 1 -C 18  alkyloxycarbonyl, or C 7 -C 18  arylcarboxamido, wherein said alkyl or aryl portions of said R 30 , R 31 , R 32 , R 33  and R 34  are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a carboxylic acid ester of a C 1 -C 6  alcohol, sulfonic acid, amino, C 1 -C 6  alkylamino, C 2 -C 6  dialkylamino or C 1 -C 6  alkoxy; or a pair of adjacent substituents R 31  and R 32 , R 32  and R 33  or R 33  and R 34 , when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid; 
         with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions. 
       
     
     
         22 . The method according to  claim 21 , further comprising:
 combining the sample with an additional detection reagent that has spectral properties that are detectably different from said optical response; and/or   determining a characteristic of the sample by comparing the optical response with a standard response parameter; and/or   wherein the sample is immobilized in or on a solid or semi-solid matrix that is a membrane, an electrophoretic gel, a silicon chip, a microwell plate, or a microfluidic chip.   
     
     
         23 . The method according to  claim 21 , wherein the sample comprises cells; and/or further comprising tracing the temporal or spatial location of the optical response with the sample. 
     
     
         24 . The method according to  claim 21 , wherein for said compound S c  is an amino acid, a peptide, a protein, a polysaccharide, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid polymer, an ion-complexing moiety, a lipid, or a non-biological organic polymer or polymeric microparticle, that is optionally bound to one or more additional fluorophores that are the same or different. 
     
     
         25 . The method according to  claim 21 , wherein for said compound, R 28  is an -L-S c , and S c  is an ion-complexing moiety that is a BAPTA or an APTRA.