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

• US 6,716,979 B2
• Filed: 08/04/2001
• Issued: 04/06/2004
• Est. Priority Date: 08/04/2000
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Diwu, Zhenjun, Haugland, Richard P., Gee, Kyle R., Liu, Jiaxing
• Primary Examiner(s): POWERS, FIONA
• Application Number:
  US09/922,333
• Publication Number:
  US 20020059684A1
• Time in Patent Office: 976 Days
• Field of Search: 544/99, 546/28, 546/36, 436/172, 436/800, 435/6, 435/40.5
• US Class Current: 544/99
• CPC Class Codes: C07D 471/04 Ortho-condensed systems
• CPC Class Codes: C07D 491/04 Ortho-condensed systems
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C07D 495/14 Ortho-condensed systems
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

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.

Claims

1. A compound of the formula:

wherein R¹, R², R⁶, R⁴¹, R⁴², and R⁴⁶ are independently selected from the group consisting of hydrogen, cyano, halogen, carboxylic acid, sulfonic acid C₁-C₆ alkyl, C₁-C₆ 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 C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, cyano, halogen, azido, carboxylic acid, sulfonic acid, or halomethyl;

or R¹ in combination with R², or R⁴¹ in combination with R⁴², or both, forms a fused aromatic or heteroaromatic ring that is optionally sulfonated one or more times, or said ring is substituted by —

L—

R_x or —

L—

S_C;

R³, R⁴, R⁴³, and R⁴⁴ are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl, an aromatic or heteroaromatic ring, L—

R_x or —

L—

S_C, wherein said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, hydroxy, or halogen and said aromatic or heteroaromatic ring that is optionally substituted one or more times by C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl;

or R² in combination with R³, R⁴² in combination with R⁴³, or R³ in combination with R⁴, or R⁴³ in combination with R⁴⁴, or any combination thereof, forms a 5- or 6-membered alicyclic ring;

R⁵ and R⁴⁵ are independently selected from the group consisting of hydrogen, methyl, carboxymethyl, C₂-C₆ alkyl, 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 C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl;

or R⁴ in combination with R⁵, or R⁵ in combination with R⁶, or R⁴⁴ in combination with R⁴⁵, or R⁴⁵ in combination with R⁴⁶, or any combination thereof, forms a 5- or 6-membered alicyclic ring;

wherein one of said E, E′

, X′ and

X is O, S, or NR⁸ provided that E and X or E′ and

X′

are not both present;

wherein R⁸ is independently selected from the group consisting of hydrogen, methyl, carboxymethyl, C₂-C₆ alkyl, —

L—

R_x and —

L—

S_C, wherein said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen;

Q is N or CR²⁸, wherein R²⁸ is independently selected from the group consisting of hydrogen, F, CN, carboxylic acid, a carboxylic acid ester of a C₁-C₆ alcohol, a C₁-C₆ alkyl, —

L—

R_x and —

L—

S_C, wherein said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen;

or R²⁸ has a formula

where R³⁰, R³¹, R³², R³³ and R³⁴ are independently selected from the group consisting of hydrogen, F, Cl, Br, I, sulfonic acid, carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino, or C₁-C₁₈ alkyl, C₁-C₁₈ alkoxy, C₁-C₁₈ alkylthio, C₁-C₁₈ alkanoylamino, C₁-C₁₈ alkylaminocarbonyl, C₂-C₃₆ dialkylaminocarbonyl, C₁-C₁₈ alkyloxycarbonyl, or C₇-C₁₈ arylcarboxamido, —

L—

R_x and —

L—

S_C, wherein said alkyl or aryl portions of said R³⁰, R³¹, R³², R³³ and R³⁴ 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 C₁-C₆ alcohol, sulfonic acid, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino and C₂-C₆ alkoxy;

or a pair of adjacent R³⁰, R³¹, R³², R³³ and R³⁴ 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.

2. The compound according to claim 1, wherein Q is CR²⁶ and R²⁸ has the formula

3. The compound according to claim 2, wherein one of R⁵, R³⁰, R³¹, R³², R³³, R³⁴, and R⁴⁵ is —

L—

R_x or —

L—

S_C.

4. The compound according to claim 2, wherein

said R³, R⁴, R⁴³, and R⁴⁴ are each methyl;

each R¹ and R⁴¹ is independently H or sulfonic acid; and

R⁶ and R⁴⁸ are H.

5. The compound according to claim 2, wherein said compound is substituted one or more times by sulfonic acid.

6. The compound according to claim 1, wherein one of said R¹, R^1′

, R², R^2′

, R³, R⁴, R⁵, R⁶, R²⁸, R³⁰, R³¹, R³², R³³, R³⁴, R⁴¹, R⁴², R⁴³, R⁴⁴, R⁴⁵, and R⁴⁸ is —

L—

R_x or —

L—

S_C.

7. The compound according to claim 6, wherein each L is independently a single covalent bond, or L is a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O, P, and S.

8. The compound according to claim 6, 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, a perfluorobenzamido, an azidopefluorobenzamido group, and a thiol group.

9. The compound according to claim 8, wherein R_X is independently selected from the group of a phosphoramidite, a succinimidyl ester of a carboxylic acid, a haloacetamide, a hydrazine, an isothiocyanate, a maleimide group, a perfluorobenzamido, an azidoperfluorobenzamido group, or a reactive platinum complex.

10. The compound according to claim 6, wherein S_C is independently selected from the group consisting of an amino acid, a peptide, a protein, a tyramine, a cabohydrate, an ion-complexing 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, or a virus.

11. The compound according to claim 10, wherein S_C is an amino acid, a peptide, a protein, an ion-complexing moiety, a nucleoside, a nucleotide, an oligonucleotide, and a nucleic acid.

12. The compound according to claim 6, wherein said compound comprises a formula:

wherein R³, R⁴, R⁵, R⁴³, R⁴⁴, and R⁴⁵ are independently methyl or ethyl;

R³⁰ is sulfonic acid or carboxylic acid;

R⁹¹ and R⁹⁴ are independently H, F, or Cl;

one of R³² and R³³ is H, F, or Cl, and the other of R³² and R³³ is —

L—

R_x or —

L—

S_C,

wherein said L is a covalent linkage comprising —

S(CH₂)_aCOO(CH₂)_b—

or —

S(CH₂)_aCONH(CH₂)_b—

wherein a is an integer between 0 and 10, and b is an integer between 0 and 10; and

wherein R_x, is selected from the group consisting of a carboxylic acid, an activated ester of a carboxylic acid, a haloacetamide, a hydrazine, an isothiocyanate, a maleimide group, and a reactive platinum complex; and

wherein S_c, is selected from the group consisting of an amino acid, a peptide, a protein, an ion-complexing moiety, a nucleoside, a nucleotide, an oligonucleotide, or a nucleic acid.

13. The compound according to claim 12, wherein R_x is a maleimide group or is a succinimidyl ester of a carboxylic acid.

14. The compound according to claim 12, wherein S_c is a peptide or a protein.

15. The compound according to claim 14, wherein S_c is an antibody or antibody fragment or a lectin.

16. The compound according to claim 12, wherein S_c is a nucleotide or an oligonucleotide.

17. The compound according to claim 12, wherein S_c is a BAPTA or APTRA ion-complexing moiety.

18. A method of staining a sample, said method comprising steps:

combining a solution with said sample, wherein said solution comprises a compound having formula

 

wherein

R¹, R², R⁶, R⁴¹, R⁴², and R⁴⁶ are independently selected from the group consisting of hydrogen, cyano, halogen, carboxylic acid, sulfonic acid C₁-C₆ alkyl, C₁-C₆ 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 C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, cyano, halogen, azido, carboxylic acid, sulfonic acid, or halomethyl;

or R¹ in combination with R², or R⁴¹ in combination with R⁴², or both, forms a fused aromatic or heteroaromatic ring that is optionally sulfonated one or more times or said ring is substituted by —

L—

R_x;

or —

L—

S_C;

R³, R⁴, R⁴³, and R⁴⁴ are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl, an aromatic or heteroaromatic ring, L—

R_x and —

L—

S_C, wherein said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, hydroxy, or halogen and said aromatic or heteroaromatic ring that is optionally substituted one or more times by C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl;

or R² in combination with R³, R⁴² in combination with R⁴³, or R³ in combination with R⁴, or R⁴³ in combination with R⁴⁴, or any combination thereof, forms a 5- or 6-membered alicyclic ring;

R⁵ and R⁴⁵ are independently selected from the group consisting of hydrogen, methyl, carboxymethyl, C₂-C₆ 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 C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl;

or R⁴ in combination with R⁵, or R⁵ in combination with R⁶, or R⁴⁴ in combination with R⁴⁵, or R⁴⁵ in combination with R⁴⁶, or any combination thereof, forms a 5- or 6-membered alicyclic ring;

wherein one of E, E′

, and X′ and

X is O, S, or NR⁸, provided that E and X or E′ and

X′

are not both present;

wherein R⁸ is independently selected from the group consisting hydrogen, methyl, carboxymethyl, C₂-C₆ alkyl, —

L—

R_x and —

L—

S_C, wherein said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen;

Q is N or CR²⁸, wherein R²⁸ is independently selected from the group consisting of hydrogen, F, CN, carboxylic acid, a carboxylic acid ester of a C₁-C₆ alcohol, C₁-C₆ alkyl, —

L—

R_x and —

L—

S_C, wherein said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen;

or R²⁸ comprises a formula

 

wherein R³⁰, R³¹, R³², R³³ and R³⁴ are independently selected from the group consisting of hydrogen, F, Cl, Br, I, sulfonic acid, carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino, C₁-C₁₈ alkyl, C₁-C₁₈ alkoxy, C₁-C₁₈ alkylthio, C₁-C₁₈ alkanoylamino, C₁-C₁₈ alkylaminocarbonyl, C₂-C₃₆ dialkylaminocarbonyl, C₁-C₁₈ alkyloxycarbonyl, C₇-C₁₈ arylcarboxamido, —

L—

R_x and —

L—

S_C, wherein said alkyl or aryl portions of said R³⁰, R³¹, R³², R³³ and R³⁴ 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 C₁-C₆ alcohol, sulfonic acid, amino, C₁-C₆ alkylamino, C₂-C₆ dialkylamino and C₁-C₆ alkoxy;

or a pair of adjacent R³⁰, and R³¹, R³², R³³ and R³⁴, subsituents 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;

b) illuminating said sample with a suitable light wavelength to yield a detectable optical response.

19. The method according to claim 18, wherein said method further comprises combining said sample with an additional detection reagent.

20. The method according to claim 18, wherein said sample comprises cells, growth medium, tissue, proteins, peptides, or biological fluids.

21. The method according to claim 18, wherein said sample is immobilized in or on a solid or semi-solid matrix that is a membrane, an electrophoretic gel, a silicon chip, a glass slide, a microwell plate, or a microfluidic chip.

22. The method according to claim 18, wherein at least one of said R²⁸, R³⁰, R³¹, R³², R³³, and R³⁴ is —

L—

R_x or —

L—

S_C;

R_x is selected from the group consisting of a carboxylic acid, an activated ester of a carboxylic acid, an amine, an azide, a hydrazine, a haloacetamide, an alkyl halide, an isothiocyanate, and a maleimide group; and

S_c is selected from the group consisting of 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, wherein said Sc is optionally bound to one or more additional fluorophores.

23. The method according to claim 22, wherein R²⁸ is an —

L—

S_C, and S_C is an ion-complexing moiety that is a BAPTA or an APTRA.

24. The method according claim 22, wherein at least one of said R²⁸, R³⁰, R³¹, R³², R³³, and R³⁴, is —

L—

S_C, and S_C is a nucleoside, a nucleotide, an oligonucleotide, or a nucleic acid polymer.

25. A kit for staining a sample, wherein said kit comprises

a solution comprising a buffer and a compound having formula

wherein R¹, R², R⁵, R⁴¹, R⁴² and R⁴⁶ are independently selected from the group consisting of hydrogen, cyano, halogen, carboxylic acid, sulfonic acid C₁-C₆ alkyl, C₁-C₆ 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 C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, cyano, halogen, azido, carboxylic acid, sulfonic acid, or halomethyl;

or R¹ in combination with R², or R⁴¹ in combination with R⁴², or both, forms a fused aromatic or heteroaromatic ring that is optionally sulfonated one or more times, or said ring is substituted by —

L—

R_x or —

L—

S_C;

R³, R⁴, R⁴³, and R⁴⁴ are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl, an aromatic ring, a heteroaromatic ring, L—

R_x and —

L—

S_C, wherein said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, hydroxy, or halogen and said aromatic or heteroaromatic ring is optionally substituted one or more times by C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl;

or R² in combination with R³, or R⁴² in combination with R⁴³, or R³ in combination with R⁴, or R⁴³ in combination with R⁴⁴, or any combination thereof, forms a 5- or 6-membered alicyclic ring;

R⁵ and R⁴⁵ are independently selected from the group consisting of hydrogen, methyl, carboxymethyl, C₂-C₆ 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 C₁-C₆ alkyl, C₁-C ₆ perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl;

or R⁴ in combination with R⁵, or R⁵ in combination with R⁶, or R⁴⁴ in combination with R⁴⁵, or R⁴⁵ in combination with R⁴⁶, or any combination thereof, forms a 5- or 6-membered alicyclic ring;

wherein one of said E, E′

, X′ and

X is O, S, or NR⁸, provided that E and X or E′ and

X′

are not both present;

wherein R⁸ is independently selected from the group consisting of hydrogen, methyl, carboxymethyl, C₂-C₆ 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 N or CR²⁸, wherein R²⁸ is independently selected from the group consisting of hydrogen, F, CN, carboxylic acid, a carboxylic acid ester of a C₁-C₆ alcohol, a C₁-C₆ alkyl, —

L—

R_x and —

L—

S_C, wherein said alkyl is optionally substituted by carboxylic acid, sulfonic acid, amino, or halogen;

or R²⁸ comprises a formula

wherein R³⁰, R³¹, R³², R³³ and R³⁴ are independently selected from the group consisting of hydrogen, F, Cl, Br, I, sulfonic acid, carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazine, C₁-C₁₈ alkyl, C₁-C₁₈ alkoxy, C₁-C₁₈ alkylthio, C₁-C₁₆, alkanoylamino, C₁-C₁₈ alkylaminocarbonyl, C₂-C₃₆ dialkylaminocarbonyl, C₁-C₁₈ alkyloxycarbonyl, C₇-C₁₈ arylcarboxarmido, —

L—

R_x and —

L—

S_C, wherein said alkyl or aryl portions of said R³⁰, R³¹, R³², R³³ and R³⁴ 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 C₁-C₆ alcohol, sulfonic acid, amino, C₁-C₆ alkylamino, C₂-C₆ dialkylamino and C₁-C₆ alkoxy;

or a pair of adjacent R³⁰, R³¹, R³², R³³ and R³⁴ 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.

26. The kit according to claim 25, wherein said kit further comprises an additional detection reagent, a purification medium, or standards.

27. The kit according to claim 25, wherein at least one of said R¹, R², R³, R⁴, R⁵, R⁶, R²⁶, R³⁰, R³¹, R³², R³³, R³⁴, R⁴³, R⁴⁴, R⁴⁵ and R⁴⁶ is L—

R_x wherein said R_x is independently selected from the group consisting of a carboxylic acid, an activated ester of a carboxylic acid, an amine, an azide, a hydrazine, a haloacetamide, an alkyl halide, an isothiocyanate, and a maleimide group.

28. The kit according to claim 27, wherein at least one of said R³¹, R³², R³³, or R³⁴ is L—

R_x and R³⁰ is carboxylic acid or sulfonic acid.

29. The kit according to claim 25, wherein at least one of said R¹, R², R³, R⁴, R⁵, R⁶, R²⁶, R³⁰, R³¹, R³², R³³, R³⁴, R⁴³, R⁴⁴, R⁴⁵ and R⁴⁶ is L—

Sc, wherein said Sc is independently selected from the group consisting of an amino acid, a peptide, a protein, an antibody, an antibody fragment, a carbohydrate, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid polymer, an ion-complexing moiety, a lipid, a non-biological organic polymer and polymeric microparticle.

30. The kit according to claim 29, wherein said Sc is an antibody or fragment thereof.