Fluorescent indicator dyes for calcium working at long wavelengths

• US 5,049,673 A
• Filed: 10/30/1987
• Issued: 09/17/1991
• Est. Priority Date: 10/30/1987
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Minta, Akwasi, Tsien, Roger Y.
• Primary Examiner(s): Raymond, Richard L.
• Assistant Examiner(s): COVINGTON, RAYMOND K
• Application Number:
  US07/115,921
• Time in Patent Office: 1,418 Days
• Field of Search: 549/223, 549/225, 549/227, 549/26, 549/27, 549/472, 549/476, 549/50, 549/60, 549/65, 549/21, 549/415, 549/417, 549/28, 549/2, 546/107, 546/265, 546/255, 546/256, 546/261, 546/269, 546/272, 546/274, 546/285, 546/296, 546/300, 546/301, 546/302, 562/403, 562/441, 260/351, 260/501.11, 260/501.13, 548/528, 548/517, 548/518, 548/525, 548/527, 548/541
• US Class Current: 546/107
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: G01N 33/84 involving inorganic compounds or pH

Abstract

The present invention discloses a new class of calcium specific fluorescent indicator dyes having visible excitation and emission wavelengths. The new fluorescent indicator dyes combine at least one tricyclic chromophore with a tetracarboxylate parent CA²⁺ chelating compound having the octacoordinate pattern of liganding groups characteristic of BAPTA to give a rhodamine-like or fluorescein-like fluorophore. Binding of calcium²⁺ increases the fluorescence of the new compounds by up to 40-fold. The calcium²⁺ dissociation constants are in the range 0.37-2.3 microM, so that the new indicators give better resolution of high [CA²⁺ ] levels than were previously obtainable with predecessor compounds such as quin-2 or fluo-2. The visible excitation wavelengths of the new compounds are more convenient for fluorescent microscopy and flow cytometry than the UV required by previous indicators.

Claims

1. A chemical compound having the general formula:

##STR14## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein;

E¹ and E² are independently H, CH₃, C₂ H₅, CH₂ OH, COOH, or CH₂ COOH, or E¹ and E² together are --(CH₂)_m --V--CH₂)_n -- where m and n are independently 1 or 2 and V is selected from the group consisting of --CH₂ --, --O--, --NH--, --NMe--, --S--, and --S--S--;

W is H, OH, or COOH;

X is H, Me, COOH, F, Cl, Br, I, or NO₂ ;

Y is --O--, --NMe--, --S--, --CH₂ --, --CMe₂ --, --CF₂ --, ##STR15## or a direct sigma bond making a five-membered central ring;

Z¹, Z², Z³, and Z⁴ are independently H, F, Cl, Br, I, or Me, and Q¹, Q² equal R₁ R₂ N^-, ##STR16## or HO^-, O.sup.═

or R₁ R₂ N--, O.sup.═

, where R¹ and R₂ are independently selected from the group consisting of H, Me, and Et;

or Z¹, Q¹, Z³ together are ##STR17## and Z², Q², Z⁴ together are ##STR18##

2. The compound of claim 1 wherein said tetraacetic acid esters are alpha-acyloxyalkyl esters.

3. The compound of claim 1 wherein said alpha-acyloxyalkyl esters are acetoxymethyl esters.

4. A chemical compound having the general formula:

##STR19## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein;

E¹ and E² are independently H, CH₃, C₂ H₅, CH₂ OH, COOH, or CH₂ COOH, or E¹ and E² together are --(CH₂)_m --V--CH₂)_n -- where m and n are independently 1 or 2 and V is selected from the group consisting of --CH₂ --, --O--, --NH--, --NMe--, --S--, and --S--S--;

W is H, OH, or COOH;

X is H, Me, COOH, F, Cl, Br, I, or NO₂ ;

Y is --O--, --NMe--, --S--, --CH₂ --, --CMe₂ --, --CF₂ --, ##STR20## or a direct sigma bond making five-membered central ring;

Z¹, Z², Z³, and Z⁴ are independently H, F, Cl, Br, I, or Me, and Q¹, Q² equal R₁ R₂ N^-, ##STR21## or HO^-, O.sup.═

or R₁ R₂ N^-, O.sup.═

, where R¹ and R₂ are independently selected from the group consisting of H, Me, and Et;

or Z¹, Q¹, Z³ together are ##STR22## and Z², Q², Z⁴ together are ##STR23##

5. The compound of claim 4 wherein said tetraacetic acid esters are alpha-acyloxyalkyl esters.

6. The compound of claim 4 wherein said alpha-acyloxyalkyl esters are acetoxymethyl esters.

7. A fluorescent, calcium binding compound comprised of (9-(6-hydroxy-4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy) phenyl)-6-dimethylamino-3H-xanthen-3-ylidene)dimethylammonium and the pharmaceutically acceptable non-toxic salts and esters thereof.

8. A fluorescent, calcium binding compound comprised of (9-(4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy)phenyl)-6-dimethylamino-3H-xanthen-3-ylidene)dimethylammonium and the pharmaceutically acceptable non-toxic salts and esters thereof.

9. A fluorescent, calcium binding compound comprised of 9-(6-hydroxy-4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy) phenyl)-6-hydroxy-3H-xanthen-3-one and the pharmaceutically acceptable non-toxic salts and esters thereof.

10. A fluorescent, calcium binding compound comprised of 9-(4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy)phenyl)-6-hydroxy-3H-xanthen-3-one and the pharmaceutically acceptable non-toxic salts and esters thereof.

11. A fluorescent, calcium binding compound comprised of 9-(4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy)phenyl)-2,7-dichloro-6-hydroxy-3H-xanthen-3-one and the pharmaceutically acceptable non-toxic salts and esters thereof.