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.

We claim: 
     
         1 . A compound of the formula: 
       
         
           
           
               
               
           
         
         wherein 
         R 1  is hydrogen or sulfonic acid; 
         R 2  is selected from the group consisting of hydrogen, cyano, halogen, carboxylic acid, sulfonic acid, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, aryl, and heteroaryl, wherein said alkyl or alkoxy is optionally substituted by carboxylic acid, sulfonic acid, or halogen and said aryl or heteroaryl 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 methyl; 
         R 5  is selected from the group consisting of hydrogen, methyl, carboxymethyl, C 2 -C 6  alkyl, aryl, heteroaryl, wherein said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen and said aryl or heteroaryl 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; 
         R 6  and R 7  are both hydrogen; 
         one of X and E is O, S, or NR 8 , and the other is absent;
 wherein R 8  is independently selected from the group consisting of hydrogen, methyl, carboxymethyl, C 2 -C 6  alkyl, -L-R x  and -L-S c , wherein said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen; 
 
         Y is independently selected from the group consisting of H, OH, NH 2 , NO, —(CO)—R 9 , —(CO)—O—R 10 , wherein R 9  and R 10  are independently H, C 1 -C 6  alkyl, or a substituted or unsubstituted aryl or heteroaryl ring system having 1-2 rings; and 
         Z is OH;
 wherein L is a covalent linkage; 
 R x  is a reactive group; and 
 S c  is a conjugated substance. 
 
       
     
     
         2 . A compound of the formula: 
       
         
           
           
               
               
           
         
         wherein 
         R 1  and R 41  are independently hydrogen or sulfonic acid; 
         R 2  and R 42  are independently selected from the group consisting of hydrogen, cyano, halogen, carboxylic acid, sulfonic acid, C 1 -C 6  alkyl or alkoxy, aryl, heteroaryl, -L-R x  and -L-S c , wherein said alkyl or alkoxy is optionally substituted by carboxylic acid, sulfonic acid, or halogen and said aryl or heteroaryl 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 , R 4 , R 43 , and R 44  are each methyl; 
         R 5  and R 45  are independently selected from the group consisting of hydrogen, methyl, carboxymethyl, C 1 -C 6  alkyl, aryl, heteroaryl, -L-R x  and -L-S c  wherein said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen and said aryl or heteroaryl 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; 
         R 6  and R 46  are both hydrogen; 
         one of E and X is O, S, or NR 8 ; the other is absent; and one of E′ and X′ is O, S, or NR 8 ; the other is absent;
 wherein R 8  is independently selected from the group consisting of hydrogen, methyl, carboxymethyl, C 2 -C 6  alkyl, -L-R x  and -L-S c  wherein said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen; and 
 
         Q is CR 28 , wherein R 28  is of the formula 
       
       
         
           
           
               
               
           
         
         wherein R 30 , R 31 , R 32 , R 33 , and R 34  are independently selected from the group consisting of hydrogen, F, Cl, Br, I, 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, C 7 -C 18  arylcarboxamido, -L-R x  and -L-S c , 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 substituents selected from the group consisting of F, Cl, Br, I, hydroxy, carboxylic acid, a carboxylic acid ester of a C 1 -C 6  alcohol, amino, C 1 -C 6  alkylamino, C 1 -C 6  dialkylamino and C 1 -C 6  alkoxy; or a pair of adjacent R 31 , R 32 , R 33 , and R 34  substituents, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid; and 
         wherein L is a covalent linkage; 
         R x  is a reactive group; and 
         S c  is a conjugated substance. 
       
     
     
         3 . The compound according to  claim 2 , wherein said L is independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O, P, and S. 
     
     
         4 . The compound according to  claim 2 , wherein said R x  is independently selected from the group consisting of an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, and a thiol group. 
     
     
         5 . The compound according to  claim 2 , wherein said S c  is independently selected from the group consisting of an amino acid, a peptide, a protein, a tyramine, a carbohydrate, a metal chelating moiety, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a polymer, a polymeric microparticle, a biological cell, and a virus. 
     
     
         6 . The compound according to  claim 2 , wherein one of R 30 , R 31 , R 32 , R 33 , and R 34  is -L-R x  or -L-S c . 
     
     
         7 . The compound according to  claim 2 , having the formula: 
       
         
           
           
               
               
           
         
         wherein 
         R 5  and R 45  are independently methyl or ethyl; 
         R 30  is sulfonic acid or carboxylic acid; 
         R 31  and R 34  are independently hydrogen, F, or Cl; one of R 32  and R 33  is H, F, or Cl, and the other of R 32  and R 33  is -L-R x  or -L-S c ,
 wherein L is a covalent linkage of the formula —S(CH 2 ) a COO(CH 2 ) b — or the formula S(CH 2 ) a CONH(CH 2 ) b —, wherein a is an integer between 0 and 10, and b is an integer between 0 and 10 provided that a and b are not both 0; 
 wherein R x , where present, is a carboxylic acid, an activated ester of a carboxylic acid, a haloacetamide, a hydrazine, an isothiocyanate, a maleimide group, or a reactive platinum complex; and 
 wherein S c , where present, is an amino acid, a peptide, a protein, an ion-complexing moiety, a nucleoside, a nucleotide, an oligonucleotide, lectin or a nucleic acid. 
 
       
     
     
         8 . The compound according to  claim 7 , wherein R x  is independently a maleimide group or a succinimidyl ester of a carboxylic acid. 
     
     
         9 . The compound according to  claim 7 , wherein S c  is independently an antibody or antibody fragment or a BAPTA or APTRA ion-complexing moiety. 
     
     
         10 . A method of staining a biological sample, comprising:
 combining a dye solution comprising a compound of the formula as described in  claim 2  with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions.   
     
     
         11 . The method according to  claim 10 , 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.   
     
     
         12 . The method according to  claim 10 , wherein the sample comprises cells; and/or further comprising tracing the temporal or spatial location of the optical response with the sample. 
     
     
         13 . The method according to  claim 10 , 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. 
     
     
         14 . The method according to  claim 10 , 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.