Fluorescent indicator dyes for calcium ions
First Claim
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 R3a 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, orR3 and R3a 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;
orW and X together are O, or NR5 ;
Y is H;
Y'"'"' is H unless A is NR5, O, or S;
orY 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 orR7 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.
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Accused Products
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.
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Citations
30 Claims
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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 R3a 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 R3a 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;
orW and X together are O, or NR5 ; Y is H;
Y'"'"' is H unless A is NR5, O, or S;
orY 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. - View Dependent Claims (2, 3, 4, 5, 6)
- ##STR9## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein;
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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 R3a 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 R3a 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;
orW and X together are O, or NR5 ; Y is H;
Y'"'"' is H unless A is NR5, O, or S;
orY and Y'"'"' together are NR5 and A is C; and A and A'"'"' are independently C, N, O or S. - View Dependent Claims (8, 9, 10, 11, 12)
- ##STR10## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein;
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13. A chemical compound having the general formula:
- ##STR11## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein;
R3 or R3a 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 R3a 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;
orW and X together are O, or NR5 ; Y is H;
Y'"'"' is H unless A is NR5, O, or S;
orY 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. - View Dependent Claims (14, 15, 16, 17, 18)
- ##STR11## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein;
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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 R3a 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 R3a 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;
orW and X together are O, or NR5 ; Y is H;
Y'"'"' is H unless A is NR5, O, or S;
orY and Y'"'"' together are NR5 and A is C; and A and A'"'"' are independently C, N, O, or S. - View Dependent Claims (20, 21, 22, 23, 24)
- ##STR12## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein;
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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
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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.
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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.
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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.
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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.
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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.
Specification