Fluorescent indicator dyes for calcium ions

• US 4,603,209 A
• Filed: 09/07/1984
• Issued: 07/29/1986
• Est. Priority Date: 09/07/1984
• 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: Tsien, Roger Y., Grynkiewicz, Grzegorz
• Primary Examiner(s): Hoke, Veronica P.
• Application Number:
  US06/648,649
• Time in Patent Office: 690 Days
• Field of Search: 8/648, 427/64, 427/152, 436/800, 546/81, 546/171, 546/89, 546/64, 546/70, 546/153, 546/158, 546/49, 546/56, 549/451, 549/444, 548/490, 548/471, 548/418, 548/215, 548/236, 260/465 D, 260/465 E, 562/426, 562/431, 562/452
• US Class Current: 548/236
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: C07D 209/08 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
• CPC Class Codes: C07D 215/40 attached in position 8
• CPC Class Codes: C07D 307/79 with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
• CPC Class Codes: C07D 413/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

The present invention discloses a new class of highly fluorescent indicator dyes that are specific for calcium ions. The new fluorescent indicator dyes combine a stilbene-type fluorophore with a tetracarboxylate parent Ca²⁺ chelating compound having the octacoordinate pattern of liganding groups characteristic of EGTA and BAPTA. Preferred forms contain extra heterocyclic bridges to reinforce the ethylenic bond of the stilbene and to reduce hydrophobicity. Compared to their widely used predecessor, "quin2", the new dyes offer up to thirty-fold brighter fluorescence, major changes in wavelength (not just intensity) upon Ca²⁺ binding, slightly lower affinities for Ca²⁺, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca²⁺ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca²⁺, make the dyes useful indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.

The present invention also discloses an improved method for synthesizing alpha-acyloxyalkyl bromides wherein the bromides so synthesized are free of contaminating bis(1-bromoalkyl)ether. The improved method is exemplified herein in the synthesis of acetoxymethyl bromide, a compound useful in preparing the acetoxymethyl esters disclosed herein as novel Ca²⁺ specific fluorescent indicators.

Claims

1. A chemical compound having the general formula:

##STR9## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein;

R¹ is selected from the group comprised of --H, --CH₃, --F, --Cl, and --Br;

R² is selected from the group comprised of --H, --CH₃, --F, --Cl, --Br, and C₀ -C₄ alkoxy;

R³ or R^3a are independently H or a polar, electron withdrawing function selected from the group comprised of --CO₂ H, --CO₂ R⁴, --CONR⁵ R⁶, --SO₃ H, --SO₂ NR⁵ R⁶, --SO₂ CF₃, --COCH₃, and --CN, or

R³ and R^3a together are --(CO)NR⁵ (CO)--;

R⁴ is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group;

R⁵ and R⁶ are independently --H, methyl, or ethyl;

Z is an aromatic or heteroaromatic system coupled to the 5 position of one 2-aminophenoxy ring through a trans ethylenic linkage, wherein said ethylenic linkage may itself be part of an aromatic heteroaromatic ring system;

W is H and X is H;

or

W and X together are O, or NR⁵ ;

Y is H;

Y'"'"' is H unless A is NR⁵, O, or S;

or

Y and Y'"'"' together are NR⁵ and A is C;

A and A'"'"' are independently C, N, O, or S;

D is O, N, NH, or N alkyl;

Q is a double bond when D is N and a single bond otherwise; and

R⁷ is H and R⁸ is H or

R⁷ and R⁸, together with heteroatom D, the carbon adjacent R⁷, and the phenyl ring adjacent to R⁸, form a quinoline ring system D is N and Q is a double bond.

2. The compound of claim 1 wherein said tetraacetic acid esters include alpha-acyloxyalkyl ester groups.

3. The compound of claim 2, wherein said alpha-acyloxyalkyl ester groups are acetoxymethyl ester groups.

4. The compound of claim 1 wherein Z is a benzene ring and W, X, Y and Y'"'"' are --H.

5. The compound of claim 1 wherein W and X together are oxygen, and together with the ethylenic bridge and one 2-aminophenoxy ring constitute a substituted benzofuran.

6. The compound of claim 1 wherein A is carbon and Y and Y'"'"' together are nitrogen, and together with the ethylenic bridge and ring Z constitute a substituted indole.

7. A bis(2-aminophenoxy)ethane-N,N,N'"'"',N'"'"'-tetraacetic acid having the general formula:

##STR10## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein;

R¹ is selected from the group comprised of --H, --CH₃, --F, --Cl, and --Br;

R² is selected from the group comprised of --H, --CH₃, --F, --Cl, --Br, and C₀ -C₄ alkoxy;

R³ or R^3a are independently H or a polar, electron withdrawing function selected from the group comprised of --CO₂ H, --CO₂ R⁴, --CONR⁵ R⁶, --SO₃ H, --SO₂ NR⁵ R⁶, --SO₂ CF₃, --COCH₃, and --CN, or

R³ and R^3a together are --(CO)NR⁵ (CO)--;

R⁴ is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group;

R⁵ and R⁶ are independently --H, methyl, or ethyl;

Z is an aromatic or heteroaromatic system coupled to the 5 position of one 2-aminophenoxy ring through a trans ethylenic linkage, wherein said ethylenic linkage may itself be part of an aromatic heteroaromatic ring system;

W is H and X is H;

or

W and X together are O, or NR⁵ ;

Y is H;

Y'"'"' is H unless A is NR⁵, O, or S;

or

Y and Y'"'"' together are NR⁵ and A is C; and

A and A'"'"' are independently C, N, O or S.

8. The compound of claim 7 wherein said tetraacetic acid esters include alpha-acyloxyalkyl esters.

9. The compound of claim 8, wherein said alpha-acyloxyalkyl ester groups are acetoxymethyl ester groups.

10. The compound of claim 7 wherein Z is a benzene ring and W, X, Y and Y'"'"' are --H.

11. The compound of claim 7 wherein W and X together are oxygen, and together with the ethylenic bridge and one 2-aminophenoxy ring constitute a substituted benzofuran.

12. The compound of claim 7 wherein A is carbon and Y and Y'"'"' together are nitrogen, and together with the ethylenic bridge and ring Z constitute a substituted indole.

13. A chemical compound having the general formula:

##STR11## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein;

R³ or R^3a are independently H of a polar, electron withdrawing function selected from the group comprised of --CO₂ H, --CO₂ R⁴, --CONR⁵ R⁶, --SO₃ H, --SO₂ NR⁵ R⁶, --SO₂ CF₃, --COCH₃, and --CN, or

R³ and R^3a together are --(CO)NR⁵ (CO)--;

R⁴ is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group;

R⁵ and R⁶ are independently --H, methyl, or ethyl;

Z is an aromatic or heteroraromatic system coupled to the 5 position of one 2-aminophenoxy ring through a trans ethylenic linkage, wherein said ethylenic linkage may itself be part of an aromatic heteroaromatic ring system;

W is H and X is H;

or

W and X together are O, or NR⁵ ;

Y is H;

Y'"'"' is H unless A is NR⁵, O, or S;

or

Y and Y'"'"' together are NR⁵ and A is C;

A and A'"'"' are independently C, N, O, or S;

D is O, N, NH, or N alkyl;

Q is a double bond when D is N and a single bond otherwise; and

R⁷ is H and R⁸ is H or

R⁷ and R⁸, together with heteroatom D, the carbon adjacent R⁷, and the phenyl ring adjacent to R⁸, form a quinoline ring system D is N and Q is a double bond.

14. The compound of claim 13 wherein said tetraacetic acid esters include alpha-acyloxyalkyl esters.

15. The compound of claim 14, wherein said alpha-acyloxyalkyl ester groups are acetoxymethyl ester groups.

16. The compound of claim 13 wherein Z is a benzene ring and W, X, Y and Y'"'"' are --H.

17. The compound of claim 13 wherein W and X together are oxygen, and together with the ethylenic bridge and one 2-aminophenoxy ring constitute a substituted benzofuran.

18. The compound of claim 13 wherein A is carbon and Y and Y'"'"' together are nitrogen, and together with the ethylenic bridge and ring Z constitute a substituted indole.

19. A bis(2-aminophenoxy)ethane-N,N,N'"'"', N'"'"'-tetraacetic acid having the general formula:

##STR12## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein;

R³ or R^3a are independently H or a polar, electron withdrawing function selected from the group comprised of --CO₂ H, --CO₂ R⁴, --CONR⁵ R⁶, --SO₃ H, --SO₂ NR⁵ R⁶, --SO₂ CF₃, --COCH₃, and --CN, or

R³ and R^3a together are --(CO)NR⁵ (CO)--;

R⁴ is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group;

R⁵ and R⁶ are independently --H, methyl, or ethyl;

Z is an aromatic or heteroaromatic system coupled to the 5 position of one 2-aminophenoxy ring through a trans ethylenic linkage, wherein said ethylenic linkage may itself be part of an aromatic heteroaromatic ring system;

W is H and X is H;

or

W and X together are O, or NR⁵ ;

Y is H;

Y'"'"' is H unless A is NR⁵, O, or S;

or

Y and Y'"'"' together are NR⁵ and A is C; and

A and A'"'"' are independently C, N, O, or S.

20. The compound of claim 19 wherein said tetraacetic acid esters include alpha-acyloxyalkyl esters.

21. The compound of claim 20, wherein said alpha-acyloxyalkyl ester groups are acetoxymethyl esters.

22. The compound of claim 19 wherein Z is a benzene ring and W, X, Y and Y'"'"' are --H.

23. The compound of claim 19 wherein W and X together are oxygen, and together with the ethylenic bridge and one 2-aminophenoxy ring constitute a substituted benzofuran.

24. The compound of claim 19 wherein A is carbon and Y and Y'"'"' together are nitrogen, and together with the ethylenic bridge and ring Z constitute a substituted indole.

25. A fluorescent, calcium binding compound comprised of 1-(2-amino-5-[(2-(4-carboxyphenyl)-E-ethenyl-1-]phenoxy)-2-(2'"'"'-amino-5'"'"'-methylphenoxy) ethane-N,N,N'"'"',N'"'"'-tetraacetic acid and the pharmaceutically acceptable non-toxic salts and esters thereof

26. A fluorescent, calcium binding compound comprised of 1-(2-amino-5-[(2-(4-N,N-dimethylsulfonamidophenyl)-E-ethenyl-1-]phenoxy), 2-(2'"'"'-amino-5'"'"'-methylphenoxy)ethane-N,N,N'"'"',N'"'"'-tetraacetic acid and the pharmaceutically acceptable non-toxic salts and esters thereof.

27. A fluorescent, calcium binding compound comprised of 1-(2-amino-5-[6-carboxyindolyl-2]-phenoxy)-2-(2'"'"'-amino-5'"'"'-methylphenoxy)ethane-N, N,N'"'"',N'"'"'-tetraacetic acid and the pharmaceutically acceptable non-toxic salts and esters thereof.

28. A fluorescent, calcium binding compound comprised of 1-(2-(4-carboxyphenyl)-6-amino benzofuranoxy-5-)-2-(2'"'"'-amino-5'"'"'-methylphenoxy)ethane-N, N,N'"'"',N'"'"'-tetraacetic acid and the pharmaceutically acceptable non-toxic salts and esters thereof.

29. A fluorescent, calcium binding compound comprised of 1-(2-(4-cyanophenyl)-6-aminobenzofuranoxy-5-)-2-(2'"'"'-amino-5'"'"'-methylphenoxy)ethane-N, N,N'"'"',N'"'"'-tetraacetic acid and the pharmaceutically acceptable non-toxic salts and esters thereof.

30. A fluorescent, calcium binding compound comprised of 1-(2-(5'"'"'-carboxyoxazol-2'"'"'-yl)-6-aminobenzofuranoxy-5-)-2-(2'"'"'-amino-5'"'"'-methylphenoxy-ethane-N, N,N'"'"',N'"'"'-tetraacetic acid and the pharmaceutically acceptable non-toxic salts and esters thereof.