Benzazolylcoumarin-based ion indicators for heavy metals

• US 5,459,276 A
• Filed: 05/20/1994
• Issued: 10/17/1995
• Est. Priority Date: 05/20/1994
• Status: Expired due to Term

• Current Assignee: United States Navy (U.S. Department Of Defense)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Hoyland, Brian M., Haugland, Richard P., Kuhn, Michael A.
• Primary Examiner(s): Gerstl, Robert
• Application Number:
  US08/246,847
• Time in Patent Office: 515 Days
• Field of Search: 548/159, 548/217, 548/304.7, 436/74, 436/800
• US Class Current: 548/159
• CPC Class Codes: C07D 417/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: G01N 31/22 using chemical indicators G01N31/02 takes precedence
• CPC Class Codes: G01N 33/84 involving inorganic compounds or pH
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

The indicator compounds of the present invention are substituted or unsubstituted 5'"'"'-nitro-BAPTA chelators that contain a benzazolyl-coumarin substructure, and the pharmaceutically acceptable non-toxic salts and esters thereof. These compounds are useful for the detection and quantification of polycationic metal ions, particularly Zn²⁺, Pb²⁺, Ba²⁺, Cd²⁺, Hg²⁺ and La³⁺.

The compounds of the invention have the structure: ##STR1## where m=2 or 3 and X can be S, O, or C(CH₃)₂ ; optionally substituted by substituents that alter the binding affinity of the indicator, shift the spectral properties of the indicator, or act as a reactive site for the preparation of a variety of conjugates.

Claims

1. A compound of the formula:

##STR3## wherein m is either 2 or 3;

R¹ is H, an alkyl group having 1-5 carbons, a benzyl, an alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group, a t-butyldimethylsilyl ester, or a pharmaceutically acceptable salt;

R², R³, R⁴ and R⁵ are independently H or methyl;

R⁶ is H, CN, CH₃, CF₃ or CONH₂ ;

X is O, S, or C(CH₃)₂ ;

Y is H, an alkyl having 1-18 carbons, --NO₂, --NH₂, --NH(C═

O)(CH₂)_n CH₃, --CF₃, F, Cl, Br, I, --OR⁸, --CO₂ R⁹, or --OCH₂ CO₂ R⁹, where n=0-16;

R⁸ is H, an alkyl group having 1-18 carbons, a benzyl (C₆ H₅ CH₂ --), and alpha-acyloxyalkyl, acetate, or a t-butyldimethylsilyl ether; and

R⁹ is H, an alkyl group having 1-17 carbons, a benzyl (C₆ H₅ CH₂ --) an alpha-acyloxyalkyl or other pharmaceutically acceptable esterifying group, a t-butyldimethylsilyl ester, or a pharmaceutically acceptable salt;

or Y is a covalently attached conjugant; and

Z is one of H, an alkyl having 1-18 carbons, --NH₂, --NH(C═

O)(CH₂)_n CH₃, --CF₃, F, Cl, Br, I, --OR⁸, --CO₂ R⁹, or --OCH₂ CO₂ R⁹ ;

or Z is a covalently attached conjugant.

2. A compound as claimed in claim 1, wherein one of Y and Z is a covalently attached conjugant.

3. A compound as claimed in claim 2, wherein said covalently attached conjugant is a polymer film, a polymer microparticle, a dextran, or glass.

4. A compound as claimed in claim 1, wherein m=2.

5. A compound as claimed in claim 1, wherein R¹ and R⁹ are acetoxymethyl esters, or R¹ is an acetoxymethyl ester and Y is H.

6. A compound as claimed in claim 1, wherein X is O or S.

7. A compound as claimed in claim 1, wherein R², R³, R⁴ and R⁵ are each H.

8. A compound as claimed in claim 1, wherein R⁶ is H or CN.

9. A method of detecting a polycationic metal ion in a sample, wherein said metal ion is Cd²⁺, Hg²⁺, Zn²⁺, La³⁺, Ba²⁺, Tb³⁺, Pb²⁺, Bi³⁺, Co²⁺ or Al³⁺, comprising:

a) adding to said sample, in an amount sufficient to generate a detectable fluorescent response to said metal ion, a compound according to the formula ##STR4## wherein m is either 2 or 3;

R¹ is H, an alkyl group having 1-5 carbons, a benzyl, an alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group, a t-butyldimethylsilyl ester, or a pharmaceutically acceptable salt;

R², R³, R⁴ and R⁵ are independently H or methyl;

R⁶ is H, CN, CH₃, CF₃ or CONH₂ ;

X is O, S, or C(CH₃)₂ ;

Y is H, an alkyl having 1-18 carbons, --NO₂, --NH₂, --NH(C═

O)(CH₂)_n CH₃, --CF₃, F, Cl, Br, I, --OR⁸, --CO₂ R⁹, or --OCH₂ CO₂ R⁹, where n=0-16;

R⁸ is H, an alkyl group having 1-18 carbons, a benzyl (C₆ H₅ CH₂ --), an alpha-acyloxyalkyl, acetate, or a t-butyldimethylsilyl ether; and

R⁹ is H, an alkyl group having 1-17 carbons, a benzyl (C₆ H₅ CH₂ --) an alpha-acyloxyalkyl or other pharmaceutically acceptable esterifying group, a t-butyldimethylsilyl ester, or a pharmaceutically acceptable salt;

or Y is a covalently attached conjugant; and

Z is one of H, an alkyl having 1-18 carbons, --NH₂, --NH(C═

O)(CH₂)_n CH₃, --CF₃, F, Cl, Br, I, --OR⁸, --CO₂ R⁹, or --OCH₂ CO₂ R⁹ ;

or Z is a covalently attached conjugant. b) illuminating said sample to generate an absorbance or fluorescence response; and

c) observing said absorbance or fluorescence response.

10. A method, as claimed in claim 9, further comprising the step of determining the concentration of said metal ion.

11. A method, as claimed in claim 9, wherein the step of observing further comprises quantifying said absorbance or fluorescence response.

12. A method, as claimed in claim 11, wherein the step of observing is performed using a fluorometer, fluorescence microscope, laser scanner, or flow cytometer.

13. A method, as claimed in claim 9, wherein said metal ion is Zn²⁺, Pb²⁺, Ba²⁺, Cd²⁺, Hg²⁺ and La³⁺.

14. A method, as claimed in claim 13, wherein said metal ion is detected in the presence of physiological levels of Ca²⁺ or Mg²⁺.

15. A method, as claimed in claim 9, wherein said compound is present as part of a fiber optic probe.

16. A method, as claimed in claim 9, wherein said sample further comprises living cells or biological fluids.