US4603209A
Expired / Lapsed
US
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Fluorescent indicator dyes for calcium ions
Assignee: University of California
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Filing: 1984-09-07
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Granted: 1986-07-29
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Expires: 2004-09-07
Abstract
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.
Inventors
- 👤 Roger Y. Tsien
- 👤 Grzegorz Grynkiewicz
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 17:39
CPC Classifications
C; C07; C07D; C07D307/00; C07D307/77; C07D307/78; C07D307/79; A; A61; A61P; A61P5/00; C07D209/00; C07D209/02; C07D209/04; C07D209/08; C07D215/00; C07D215/02; C07D215/16; C07D215/38; C07D215/40; C07D413/00; C07D413/02; C07D413/04; Y; Y10; Y10S; Y10S436/00; Y10S436/80
Claims Analysis
Total Claims: 40
Claim 1
Independent Claim
304 words
A chemical compound having the general formula: ##STR9## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein: R 1 is selected from the group comprised of --H, --CH 3 , --F, --Cl, and --Br; R 2 is selected from the group comprised of --H, --CH 3 , --F, --Cl, --Br, and C 0 -C 4 alkoxy; R 3 or R 3a are independently H or a polar, electron withdrawing function selected from the group comprised of --CO 2 H, --CO 2 R 4 , --CONR 5 R 6 , --SO 3 H, --SO 2 NR 5 R 6 , --SO 2 CF 3 , --COCH 3 , and --CN, or R 3 and R 3a together are --(CO)NR 5 (CO)--; R 4 is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group; R 5 and R 6 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 NR 5 ; Y is H; Y' is H unless A is NR 5 , O, or S; or Y and Y' together are NR 5 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 R 7 is H and R 8 is H or R 7 and R 8 , together with heteroatom D, the carbon adjacent R 7 , and the phenyl ring adjacent to R 8 , form a quinoline ring system D is N and Q is a double bond.
Claim 2
Dependent Claim
304 words
A chemical compound having the general formula: ##STR9## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein: R 1 is selected from the group comprised of --H, --CH 3 , --F, --Cl, and --Br; R 2 is selected from the group comprised of --H, --CH 3 , --F, --Cl, --Br, and C 0 -C 4 alkoxy; R 3 or R 3a are independently H or a polar, electron withdrawing function selected from the group comprised of --CO 2 H, --CO 2 R 4 , --CONR 5 R 6 , --SO 3 H, --SO 2 NR 5 R 6 , --SO 2 CF 3 , --COCH 3 , and --CN, or R 3 and R 3a together are --(CO)NR 5 (CO)--; R 4 is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group; R 5 and R 6 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 NR 5 ; Y is H; Y' is H unless A is NR 5 , O, or S; or Y and Y' together are NR 5 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 R 7 is H and R 8 is H or R 7 and R 8 , together with heteroatom D, the carbon adjacent R 7 , and the phenyl ring adjacent to R 8 , form a quinoline ring system D is N and Q is a double bond.
Claim 3
Dependent Claim
14 words
The compound of claim 1 wherein said tetraacetic acid esters include alpha-acyloxyalkyl ester groups.
Claim 4
Dependent Claim
14 words
The compound of claim 2, wherein said alpha-acyloxyalkyl ester groups are acetoxymethyl ester groups.
Claim 5
Dependent Claim
19 words
The compound of claim 1 wherein Z is a benzene ring and W, X, Y and Y' are --H.
Claim 6
Dependent Claim
26 words
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.
Claim 7
Dependent Claim
29 words
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.
Claim 8
Dependent Claim
233 words
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: R 1 is selected from the group comprised of --H, --CH 3 , --F, --Cl, and --Br; R 2 is selected from the group comprised of --H, --CH 3 , --F, --Cl, --Br, and C 0 -C 4 alkoxy; R 3 or R 3a are independently H or a polar, electron withdrawing function selected from the group comprised of --CO 2 H, --CO 2 R 4 , --CONR 5 R 6 , --SO 3 H, --SO 2 NR 5 R 6 , --SO 2 CF 3 , --COCH 3 , and --CN, or R 3 and R 3a together are --(CO)NR 5 (CO)--; R 4 is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group; R 5 and R 6 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 NR 5 ; Y is H; Y' is H unless A is NR 5 , O, or S; or Y and Y' together are NR 5 and A is C; and A and A' are independently C, N, O or S.
Claim 9
Dependent Claim
233 words
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: R 1 is selected from the group comprised of --H, --CH 3 , --F, --Cl, and --Br; R 2 is selected from the group comprised of --H, --CH 3 , --F, --Cl, --Br, and C 0 -C 4 alkoxy; R 3 or R 3a are independently H or a polar, electron withdrawing function selected from the group comprised of --CO 2 H, --CO 2 R 4 , --CONR 5 R 6 , --SO 3 H, --SO 2 NR 5 R 6 , --SO 2 CF 3 , --COCH 3 , and --CN, or R 3 and R 3a together are --(CO)NR 5 (CO)--; R 4 is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group; R 5 and R 6 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 NR 5 ; Y is H; Y' is H unless A is NR 5 , O, or S; or Y and Y' together are NR 5 and A is C; and A and A' are independently C, N, O or S.
Claim 10
Dependent Claim
13 words
The compound of claim 7 wherein said tetraacetic acid esters include alpha-acyloxyalkyl esters.
Claim 11
Dependent Claim
14 words
The compound of claim 8, wherein said alpha-acyloxyalkyl ester groups are acetoxymethyl ester groups.
Claim 12
Dependent Claim
19 words
The compound of claim 7 wherein Z is a benzene ring and W, X, Y and Y' are --H.
Claim 13
Dependent Claim
26 words
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.
Claim 14
Dependent Claim
29 words
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.
Claim 15
Dependent Claim
265 words
A chemical compound having the general formula: ##STR11## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein: R 3 or R 3a are independently H of a polar, electron withdrawing function selected from the group comprised of --CO 2 H, --CO 2 R 4 , --CONR 5 R 6 , --SO 3 H, --SO 2 NR 5 R 6 , --SO 2 CF 3 , --COCH 3 , and --CN, or R 3 and R 3a together are --(CO)NR 5 (CO)--; R 4 is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group; R 5 and R 6 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 NR 5 ; Y is H; Y' is H unless A is NR 5 , O, or S; or Y and Y' together are NR 5 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 R 7 is H and R 8 is H or R 7 and R 8 , together with heteroatom D, the carbon adjacent R 7 , and the phenyl ring adjacent to R 8 , form a quinoline ring system D is N and Q is a double bond.
Claim 16
Dependent Claim
265 words
A chemical compound having the general formula: ##STR11## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein: R 3 or R 3a are independently H of a polar, electron withdrawing function selected from the group comprised of --CO 2 H, --CO 2 R 4 , --CONR 5 R 6 , --SO 3 H, --SO 2 NR 5 R 6 , --SO 2 CF 3 , --COCH 3 , and --CN, or R 3 and R 3a together are --(CO)NR 5 (CO)--; R 4 is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group; R 5 and R 6 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 NR 5 ; Y is H; Y' is H unless A is NR 5 , O, or S; or Y and Y' together are NR 5 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 R 7 is H and R 8 is H or R 7 and R 8 , together with heteroatom D, the carbon adjacent R 7 , and the phenyl ring adjacent to R 8 , form a quinoline ring system D is N and Q is a double bond.
Claim 17
Dependent Claim
13 words
The compound of claim 13 wherein said tetraacetic acid esters include alpha-acyloxyalkyl esters.
Claim 18
Dependent Claim
14 words
The compound of claim 14, wherein said alpha-acyloxyalkyl ester groups are acetoxymethyl ester groups.
Claim 19
Dependent Claim
19 words
The compound of claim 13 wherein Z is a benzene ring and W, X, Y and Y' are --H.
Claim 20
Dependent Claim
26 words
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.
Claim 21
Dependent Claim
29 words
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.
Claim 22
Dependent Claim
195 words
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: R 3 or R 3a are independently H or a polar, electron withdrawing function selected from the group comprised of --CO 2 H, --CO 2 R 4 , --CONR 5 R 6 , --SO 3 H, --SO 2 NR 5 R 6 , --SO 2 CF 3 , --COCH 3 , and --CN, or R 3 and R 3a together are --(CO)NR 5 (CO)--; R 4 is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group; R 5 and R 6 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 NR 5 ; Y is H; Y' is H unless A is NR 5 , O, or S; or Y and Y' together are NR 5 and A is C; and A and A' are independently C, N, O, or S.
Claim 23
Dependent Claim
195 words
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: R 3 or R 3a are independently H or a polar, electron withdrawing function selected from the group comprised of --CO 2 H, --CO 2 R 4 , --CONR 5 R 6 , --SO 3 H, --SO 2 NR 5 R 6 , --SO 2 CF 3 , --COCH 3 , and --CN, or R 3 and R 3a together are --(CO)NR 5 (CO)--; R 4 is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group; R 5 and R 6 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 NR 5 ; Y is H; Y' is H unless A is NR 5 , O, or S; or Y and Y' together are NR 5 and A is C; and A and A' are independently C, N, O, or S.
Claim 24
Dependent Claim
13 words
The compound of claim 19 wherein said tetraacetic acid esters include alpha-acyloxyalkyl esters.
Claim 25
Dependent Claim
13 words
The compound of claim 20, wherein said alpha-acyloxyalkyl ester groups are acetoxymethyl esters.
Claim 26
Dependent Claim
19 words
The compound of claim 19 wherein Z is a benzene ring and W, X, Y and Y' are --H.
Claim 27
Dependent Claim
26 words
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.
Claim 28
Dependent Claim
29 words
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.
Claim 29
Dependent Claim
19 words
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
Claim 30
Dependent Claim
19 words
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
Claim 31
Dependent Claim
19 words
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.
Claim 32
Dependent Claim
19 words
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.
Claim 33
Dependent Claim
19 words
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.
Claim 34
Dependent Claim
19 words
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.
Claim 35
Dependent Claim
20 words
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.
Claim 36
Dependent Claim
20 words
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.
Claim 37
Dependent Claim
19 words
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.
Claim 38
Dependent Claim
19 words
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.
Claim 39
Dependent Claim
19 words
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.
Claim 40
Dependent Claim
19 words
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.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-07-28 | 1990-01-27 | 1990-07-29 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-07-28 | 1994-01-27 | 1994-07-29 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-07-28 | 1998-01-27 | 1998-07-29 | $7700.00 | Window Closed / Lapsed |