US5405975A
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Fluorescent ion-selective diaryldiaza crown ether conjugates
Assignee: Molecular Probes Inc
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Filing: 1993-03-29
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Granted: 1995-04-11
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Expires: 2013-03-29
Abstract
Abstract This invention describes novel sensors for ions that are based on the combination of xanthylium-based dyes with metal-binding N,N'-diaryldiaza crown ethers. These sensors are primarily useful for detection and quantitation of alkali-metal ions in aqueous solution. Binding of the ion results in a change in the fluorescence properties of the indicating dye that can be correlated with the ion concentration. Methods are provided for attaching reactive groups on these sensors for conjugation to dyes, lipids and polymers and for enhancing entry of the indicators into living cells.
Inventors
- 👤 Michael A. Kuhn
- 👤 Richard P. Haugland
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 12:06
CPC Classifications
C; C07; C07D; C07D273/00; C09; C09K; C09K11/00; C09K11/06; G; G01; G01N; G01N31/00; G01N31/22; G01N33/00; G01N33/48; G01N33/50; G01N33/84
Claims Analysis
Total Claims: 19
Claim 1
Independent Claim
617 words
A fluorescent ion-selective compound having the formula: ##STR5## where Y P is --(CH 2 CH 2 --O) j --CH 2 CH 2 -- and Y Q is --(CH 2 CH 2 --O) k --CH 2 CH 2 --; j and k are independently 1 or 2; where A is a substituted aryl of the formula: ##STR6## where at least one of R p , R q , R r , R s and R t is a first fluorophore according to the formula: (R a ) n (R b ) n' --FLUOR, wherein n=0 or 1; n'=0 or 1; R a is --OCH 2 R 3 --, --OR 3' --, --SR 3 --, --SR 3' --, --NH(C═O)CH 2 R 3 --, --NH(C═O)R 3' --, --(C═O)NHCH 2 R 3 --, --(C═O)NHR 3' --, --NHSO 2 R 3 --, --NHSO 2 R 3' --, --NH(C═O)NHCH 2 R 3 --, --NH(C═O)NHR 3' --, --NH(C═S)NHCH 2 R 3 --, or --NH(C═S)NHR 3' --, where R 3 is (CH 2 ) m and m=1-6, and R 3' is phenylene (--C 6 H 4 --), carboxyphenylene (--C 6 H 3 COOH--) or sulfophenylene (--C 6 H 3 SO 3 H--) or the pharmaceutically acceptable salt or ester thereof; R b is --NH--, --NH(C═ O)--, --NH(C═S)--, --S--, --O--, --(C═O)--; or --CH 2 -- or --(C═O)CH 2 --; and --FLUOR is a pyronine, xanthene, fluorescein, rhodamine rosamine, rhodol, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof; where the remaining A substituents R p , R q , R r , R s , and R t , which may be the same or different, are independently H, CH 3 , NO 2 , CF 3 , F, Cl, Br, I, --OR 5 , --(C═O)OR 5 , or --OCH 2 (C═O)OR 5 , where R 5 is an alkyl group having 1-6 carbons, a benzyl (C 6 H 5 CH 2 --), an acetoxymethyl ester or a pharmaceutically acceptable esterifying group, or a pharmaceutically acceptable salt; where B is a substituted aryl of the formula: ##STR7## where at least one of R p' , R q' , R r' , R s' and R t' has the formula: (R a' ) n (R b' ) n' --X wherein n=0 or 1; n'=0 or 1; R a' is --OCH 2 R 3 --, --OR 3' --, --SR 3 --, --SR 3' --, --NH(C═O)CH 2 R 3 --, --NH(C═O)R 3' --, --(C═O)NHCH 2 R 3 --, --(C═O)NHR 3' , --NHSO 2 R 3 --, --NHSO 2 R 3' --, --NH(C═O)NHCH 2 R 3 --, --NH(C═O)NHR 3' --, --NH(C═S)NHCH 2 R 3 --, or --NH(C═S)NHR 3' --, where R 3 is (CH 2 ) m and m=1-6, and R 3' is phenylene (--C 6 H 4 --), carboxyphenylene (--C 6 H 3 COOH--) or sulfophenylene (--C 6 H 3 SO 3 H--) or the pharmaceutically acceptable salt or ester thereof; R b' is --NH-- , --NH(C═O)--, --NH(C═S), --S--, --O--, --(C═O)'; or --CH 2 -- or --(C═O)CH 2 --; and --X is second fluorophore --FLUOR' that is a pyronine, xanthene, fluorescein, rhodamine, rosamine, rhodol, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof; where the remaining B substituents R p' , R q' , R r' , R s' or R t' , which may be the same or different, are independently H, CH 3 , NO 2 , CF 3 , F, Cl, Br, I, --OR 5 , --(C═O)OR 5 , or --OCH 2 (C═O)OR 5 , where R 5 is an alkyl group having 1-6 carbons, a benzyl (C 6 H 5 CH 2 --), an acetoxymethyl ester or a pharmaceutically acceptable esterifying group, or a pharmaceutically acceptable salt.
Claim 2
Dependent Claim
617 words
A fluorescent ion-selective compound having the formula: ##STR5## where Y P is --(CH 2 CH 2 --O) j --CH 2 CH 2 -- and Y Q is --(CH 2 CH 2 --O) k --CH 2 CH 2 --; j and k are independently 1 or 2; where A is a substituted aryl of the formula: ##STR6## where at least one of R p , R q , R r , R s and R t is a first fluorophore according to the formula: (R a ) n (R b ) n' --FLUOR, wherein n=0 or 1; n'=0 or 1; R a is --OCH 2 R 3 --, --OR 3' --, --SR 3 --, --SR 3' --, --NH(C═O)CH 2 R 3 --, --NH(C═O)R 3' --, --(C═O)NHCH 2 R 3 --, --(C═O)NHR 3' --, --NHSO 2 R 3 --, --NHSO 2 R 3' --, --NH(C═O)NHCH 2 R 3 --, --NH(C═O)NHR 3' --, --NH(C═S)NHCH 2 R 3 --, or --NH(C═S)NHR 3' --, where R 3 is (CH 2 ) m and m=1-6, and R 3' is phenylene (--C 6 H 4 --), carboxyphenylene (--C 6 H 3 COOH--) or sulfophenylene (--C 6 H 3 SO 3 H--) or the pharmaceutically acceptable salt or ester thereof; R b is --NH--, --NH(C═ O)--, --NH(C═S)--, --S--, --O--, --(C═O)--; or --CH 2 -- or --(C═O)CH 2 --; and --FLUOR is a pyronine, xanthene, fluorescein, rhodamine rosamine, rhodol, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof; where the remaining A substituents R p , R q , R r , R s , and R t , which may be the same or different, are independently H, CH 3 , NO 2 , CF 3 , F, Cl, Br, I, --OR 5 , --(C═O)OR 5 , or --OCH 2 (C═O)OR 5 , where R 5 is an alkyl group having 1-6 carbons, a benzyl (C 6 H 5 CH 2 --), an acetoxymethyl ester or a pharmaceutically acceptable esterifying group, or a pharmaceutically acceptable salt; where B is a substituted aryl of the formula: ##STR7## where at least one of R p' , R q' , R r' , R s' and R t' has the formula: (R a' ) n (R b' ) n' --X wherein n=0 or 1; n'=0 or 1; R a' is --OCH 2 R 3 --, --OR 3' --, --SR 3 --, --SR 3' --, --NH(C═O)CH 2 R 3 --, --NH(C═O)R 3' --, --(C═O)NHCH 2 R 3 --, --(C═O)NHR 3' , --NHSO 2 R 3 --, --NHSO 2 R 3' --, --NH(C═O)NHCH 2 R 3 --, --NH(C═O)NHR 3' --, --NH(C═S)NHCH 2 R 3 --, or --NH(C═S)NHR 3' --, where R 3 is (CH 2 ) m and m=1-6, and R 3' is phenylene (--C 6 H 4 --), carboxyphenylene (--C 6 H 3 COOH--) or sulfophenylene (--C 6 H 3 SO 3 H--) or the pharmaceutically acceptable salt or ester thereof; R b' is --NH-- , --NH(C═O)--, --NH(C═S), --S--, --O--, --(C═O)'; or --CH 2 -- or --(C═O)CH 2 --; and --X is second fluorophore --FLUOR' that is a pyronine, xanthene, fluorescein, rhodamine, rosamine, rhodol, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof; where the remaining B substituents R p' , R q' , R r' , R s' or R t' , which may be the same or different, are independently H, CH 3 , NO 2 , CF 3 , F, Cl, Br, I, --OR 5 , --(C═O)OR 5 , or --OCH 2 (C═O)OR 5 , where R 5 is an alkyl group having 1-6 carbons, a benzyl (C 6 H 5 CH 2 --), an acetoxymethyl ester or a pharmaceutically acceptable esterifying group, or a pharmaceutically acceptable salt.
Claim 3
Dependent Claim
49 words
A compound as in claim 1 where R p , R q , R r , R s or R t is (R a ) n (R b ) n' --FLUOR, where FLUOR is a fluorescein, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof.
Claim 4
Dependent Claim
19 words
A compound as in claim 1 where R r is (R a ) n (R b ) n' --FLUOR.
Claim 5
Dependent Claim
10 words
A compound as in claim 1 where j=2 and k=1.
Claim 6
Dependent Claim
10 words
A compound as in claim 1 where j=2 and k=2.
Claim 7
Dependent Claim
38 words
A compound as in claim 1 where X is --FLUOR', where --FLUOR and --FLUOR', which may be the same or different, are independently a fluorescein, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof.
Claim 8
Dependent Claim
474 words
A fluorescent ion-selective compound having the formula: ##STR8## where Y P is --(CH 2 CH 2 --O) j --CH 2 CH 2 -- and Y Q is --(CH 2 CH 2 --O) k --CH 2 CH 2 --; j and k are independently 1 or 2; --FLUOR is a pyronine, xanthene, fluorescein, rhodamine, rosamine, rhodol, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof that is linked to substituted aryl A by a first linkage that is --(R a ) n (R b ) n' --, wherein n=0 or 1; n'=0 or 1; R a is --OCH 2 R 3 --, --OR 3' --, --SR 3 --, --SR 3' --, --NH(C═O)CH 2 R 3 --, --NH(C═O)R 3' --, --(C═O)NHCH 2 R 3 --, --(C═O)NHR 3' --, --NHSO 2 R 3 --, --NHSO 2 R 3' --, --NH(C═O)NHCH 2 R 3 --, --NH(C═O)NHR 3' --, --NH(C═S)NHCH 2 R 3 --, or --NH(C═S)NHR 3' --, where R 3 is (CH 2 ) m and m=1-6, and R 3' is phenylene (--C 6 H 4 -- ), carboxyphenylene (--C 6 H 3 COOH--) or sulfophenylene (--C 6 H 3 SO 3 H--) or the pharmaceutically acceptable salt or ester thereof; R b is --NH--, --NH(C═O)--, --NH(C═S)--, --S--, --O--, --(C═O)--; or --CH 2 -- or --(C═O)CH 2 --; and --FLUOR', which is the same as or different from --FLUOR, is a pyronine, xanthene, fluorescein, rhodamine, rosamine, rhodol, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof that is linked to substituted aryl B by a second linkage that is --(R a' ) n (R b' ) n' --, wherein n=0 or 1; n'=0 or 1; R a' is --OCH 2 R 3 --, --OR 3' --, --SR 3 --, --SR 3' --, --NH(C═O)CH 2 R 3 --, --NH(C═O)R 3' --, --(C═O)NHCH 2 R 3 --, --(C═O)NHR 3' , --NHSO 2 R 3 --, --NHSO 2 R 3' --, --NH(C═O)NHCH 2 R 3 --, --NH(C═O)NHR 3' --, --NH(C═S)NHCH 2 R 3 --, or --NH(C═S)NHR 3' --, where R 3 is (CH 2 ) m and m=1-6, and R 3' is phenylene (--C 6 H 4 --), carboxyphenylene (--C 6 H 3 COOH--) or sulfophenylene (--C 6 H 3 SO 3 H--) or the pharmaceutically acceptable salt or ester thereof; R b' is --NH--, --NH(C═O)--, --NH(C═S), --S--, --O--, --(C═O)--; or --CH 2 -- or --(C═O)CH 2 --; and the remaining A and B substituents, which may be the same or different, are independently H, CH 3 , NO 2 , CF 3 , F, Cl, Br, I, --OR 5 , --(C═O)OR 5 , or --OCH 2 (C═O)OR 5 , where R 5 is an alkyl group having 1-6 carbons, a benzyl (C 6 H 5 CH 2 --), an acetoxymethyl ester or a pharmaceutically acceptable esterifying group, or a pharmaceutically acceptable salt.
Claim 9
Dependent Claim
474 words
A fluorescent ion-selective compound having the formula: ##STR8## where Y P is --(CH 2 CH 2 --O) j --CH 2 CH 2 -- and Y Q is --(CH 2 CH 2 --O) k --CH 2 CH 2 --; j and k are independently 1 or 2; --FLUOR is a pyronine, xanthene, fluorescein, rhodamine, rosamine, rhodol, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof that is linked to substituted aryl A by a first linkage that is --(R a ) n (R b ) n' --, wherein n=0 or 1; n'=0 or 1; R a is --OCH 2 R 3 --, --OR 3' --, --SR 3 --, --SR 3' --, --NH(C═O)CH 2 R 3 --, --NH(C═O)R 3' --, --(C═O)NHCH 2 R 3 --, --(C═O)NHR 3' --, --NHSO 2 R 3 --, --NHSO 2 R 3' --, --NH(C═O)NHCH 2 R 3 --, --NH(C═O)NHR 3' --, --NH(C═S)NHCH 2 R 3 --, or --NH(C═S)NHR 3' --, where R 3 is (CH 2 ) m and m=1-6, and R 3' is phenylene (--C 6 H 4 -- ), carboxyphenylene (--C 6 H 3 COOH--) or sulfophenylene (--C 6 H 3 SO 3 H--) or the pharmaceutically acceptable salt or ester thereof; R b is --NH--, --NH(C═O)--, --NH(C═S)--, --S--, --O--, --(C═O)--; or --CH 2 -- or --(C═O)CH 2 --; and --FLUOR', which is the same as or different from --FLUOR, is a pyronine, xanthene, fluorescein, rhodamine, rosamine, rhodol, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof that is linked to substituted aryl B by a second linkage that is --(R a' ) n (R b' ) n' --, wherein n=0 or 1; n'=0 or 1; R a' is --OCH 2 R 3 --, --OR 3' --, --SR 3 --, --SR 3' --, --NH(C═O)CH 2 R 3 --, --NH(C═O)R 3' --, --(C═O)NHCH 2 R 3 --, --(C═O)NHR 3' , --NHSO 2 R 3 --, --NHSO 2 R 3' --, --NH(C═O)NHCH 2 R 3 --, --NH(C═O)NHR 3' --, --NH(C═S)NHCH 2 R 3 --, or --NH(C═S)NHR 3' --, where R 3 is (CH 2 ) m and m=1-6, and R 3' is phenylene (--C 6 H 4 --), carboxyphenylene (--C 6 H 3 COOH--) or sulfophenylene (--C 6 H 3 SO 3 H--) or the pharmaceutically acceptable salt or ester thereof; R b' is --NH--, --NH(C═O)--, --NH(C═S), --S--, --O--, --(C═O)--; or --CH 2 -- or --(C═O)CH 2 --; and the remaining A and B substituents, which may be the same or different, are independently H, CH 3 , NO 2 , CF 3 , F, Cl, Br, I, --OR 5 , --(C═O)OR 5 , or --OCH 2 (C═O)OR 5 , where R 5 is an alkyl group having 1-6 carbons, a benzyl (C 6 H 5 CH 2 --), an acetoxymethyl ester or a pharmaceutically acceptable esterifying group, or a pharmaceutically acceptable salt.
Claim 10
Dependent Claim
30 words
A compound as in claim 7 where --FLUOR and --FLUOR', which are the same or different, are independently a rhodamine, rosamine, rhodol, or a pharmaceutically acceptable salt or ester thereof.
Claim 11
Dependent Claim
26 words
A compound as in claim 7 where --FLUOR and --FLUOR' are the same and are each linked to the substituted aryls para to a crown nitrogen.
Claim 12
Dependent Claim
10 words
A compound as in claim 7 where j=2 and k=1.
Claim 13
Dependent Claim
51 words
A compound as in claim 10 where FLUOR and FLUOR' are both fluoresceins or a pharmaceutically acceptable salt or ester thereof, that are each linked to a substituted aryl para to a crown nitrogen; and where the remaining A substituents and remaining B substituents are independently H or --OCH 3 .
Claim 14
Dependent Claim
51 words
A compound as in claim 10 where FLUOR and FLUOR' are both rhodamines or a pharmaceutically acceptable salt or ester thereof, that are each linked to a substituted aryl para to a crown nitrogen; and where the remaining A substituents and remaining B substituents are independently H or --OCH 3 .
Claim 15
Dependent Claim
10 words
A compound as in claim 7 where j=2 and k=2.
Claim 16
Dependent Claim
52 words
A compound as in claim 13 where FLUOR and FLUOR' are both fluoresceins or a pharmaceutically acceptable salt or ester thereof, each of which are linked to a substituted aryl para to a crown nitrogen; and where the remaining A substituents and remaining B substituents are independently H or --OCH 3 .
Claim 17
Dependent Claim
51 words
A compound as in claim 10 where FLUOR and FLUOR' are both rhodamines or a pharmaceutically acceptable salt or ester thereof, that are each linked to a substituted aryl para to a crown nitrogen; and where: the remaining A substituents and remaining B substituents are independently H or --OCH 3 .
Claim 18
Dependent Claim
85 words
A fluorescent ion-selective compound comprising N,N'-diaryldiazacrown ether that is a DA15C5 or DA18C6 containing two aryl residues, wherein one aryl residue attached to the crown ether is covalently linked to a pyronine, xanthene, fluorescein, rhodamine, rosamine, rhodol, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof, and the other aryl residue attached to the crown ether is covalently linked to an additional pyronine, xanthene, fluorescein, rhodamine, rosamine, rhodol, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof.
Claim 19
Dependent Claim
85 words
A fluorescent ion-selective compound comprising N,N'-diaryldiazacrown ether that is a DA15C5 or DA18C6 containing two aryl residues, wherein one aryl residue attached to the crown ether is covalently linked to a pyronine, xanthene, fluorescein, rhodamine, rosamine, rhodol, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof, and the other aryl residue attached to the crown ether is covalently linked to an additional pyronine, xanthene, fluorescein, rhodamine, rosamine, rhodol, benzofluorescein, dibenzofluorescein, seminaphthofluorescein, or naphthofluorescein, or a pharmaceutically acceptable salt or ester thereof.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1998-04-10 | 1998-10-10 | 1999-04-11 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2002-04-10 | 2002-10-10 | 2003-04-11 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2006-04-10 | 2006-10-10 | 2007-04-11 | $7700.00 | Window Closed / Lapsed |