US 6,130,101 AGranted

Sulfonated xanthene derivatives

AssigneeMolecular Probes, Inc.

InventorsFei Mao, Wai-Yee Leung, Richard P. Haugland

Life of this patent

Estimating the term…

Abstract

The present invention describes xanthene dyes, including rhodamines, rhodols and fluoresceins that are substituted one or more times by a sulfonic acid or a salt of a sulfonic acid. The dyes of the invention, including chemically reactive dyes and dye-conjugates are useful as fluorescent probes, particularly in biological samples.

Claims73

Dependent claims

A compound having the formula ##STR51## or the formula ##STR52## wherein R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are independently H, F, Cl, Br, I, CN;

or C.sub.1 -C.sub.18 alkyl, or C.sub.1 -C.sub.18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol;
or --SO.sub.3 X where X is H or a counterion
R.sup.1 and R.sup.6 are H;
or R.sup.1 taken in combination with R.sup.2, or R.sup.5 taken in combination with R.sup.6, or both, form a fused aromatic six membered ring that is optionally substituted one or more times by --SO.sub.3 X;
A is NR.sup.8 R.sup.9,
R.sup.8 and R.sup.9 are independently H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 carboxyalkyl, C.sub.1 -C.sub.6 sulfoalkyl, a salt of C.sub.1 -C.sub.6 carboxyalkyl, or a salt of C.sub.1 -C.sub.6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.8 in combination with R.sup.9 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.8 in combination with R.sup.2, or R.sup.9 in combination with R.sup.3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C.sub.1 -C.sub.6 alkyls or --CH.sub.2 SO.sub.3 X moieties;
C is OR.sup.17 or NR.sup.18 R.sup.19 ;
where R.sup.17 is H, or C.sub.1 -C.sub.18 alkyl;
where R.sup.18 and R.sup.19 are independently H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 carboxyalkyl, C.sub.1 -C.sub.6 sulfoalkyl, a salt of C.sub.1 -C.sub.6 carboxyalkyl, or a salt of C.sub.1 -C.sub.6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.18 in combination with R.sup.19 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.18 in combination with R.sup.4, or R.sup.19 in combination with R.sup.5, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C.sub.1 -C.sub.6 alkyls or --CH.sub.2 SO.sub.3 X moieties;
B is O or N.sup.+ R.sup.18 R.sup.19 ;
R.sup.10 is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol;
or R.sup.10 is a saturated or unsaturated C.sub.1 -C.sub.18 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol, --SO.sub.3 X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;
or R.sup.10 has the formula ##STR53## where R.sup.12, R.sup.13, R.sup.14, R.sup.15 and R.sup.16 are independently H, F, Cl, Br, I, --SO.sub.3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino;
or C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 alkoxy, C.sub.1 -C.sub.18 alkylthio, C.sub.1 -C.sub.18 alkanoylamino, C.sub.1 -C.sub.18 alkylaminocarbonyl, C.sub.2 -C.sub.36 dialkylaminocarbonyl, C.sub.1 -C.sub.18 alkyloxycarbonyl, or C.sub.6 -C.sub.18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol, --SO.sub.3 X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;
or one pair of adjacent substituents R.sup.13 and R.sup.14, R.sup.14 and R.sup.15 or R.sup.15 and R.sup.16, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid;
and
R.sup.11 is H, hydroxy, CN or a C.sub.1 -C.sub.6 alkoxy;
or R.sup.10 in combination with R.sup.11 forms a 5-membered spirolactone ring or a 5-membered spirosultone ring;
or R.sup.11 in combination with R.sup.12 forms a 5- or 6-membered spirolactone ring or a 5- or 6-membered spirosultone ring that is optionally and independently substituted by H, F or CH.sub.3 ;
or R.sup.10 when taken in combination with R.sup.11 is a carbonyl oxygen;
provided that either at least one of R.sup.2, R.sup.3, R.sup.4, and R.sup.5 is --SO.sub.3 X;
or R.sup.8 in combination with R.sup.2, or R.sup.9 in combination with R.sup.3, or R.sup.18 in combination with R.sup.4, or R.sup.19 in combination with R.sup.5, form a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by at least one --CH.sub.2 SO.sub.3 X moiety.

23 more elements

A compound having the formula ##STR56## or the formula ##STR57## wherein R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are independently H, F, Cl, Br, I, CN;

or C.sub.1 -C.sub.18 alkyl, or C.sub.1 -C.sub.18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol;
or --L-R.sub.x ;
or --SO.sub.3 X where X is H or a counterion
R.sup.1 and R.sup.6 are H;
or R.sup.1 taken in combination with R.sup.2, or R.sup.5 taken in combination with R.sup.6, or both, form a fused aromatic six membered ring that is optionally substituted one or more times by --SO.sub.3 X;
A is NR.sup.8 R.sup.9,
R.sup.8 and R.sup.9 are independently H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 carboxyalkyl, C.sub.1 -C.sub.6 sulfoalkyl, a salt of C.sub.1 -C.sub.6 carboxyalkyl, or a salt of C.sub.1 -C.sub.6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.8 in combination with R.sup.9 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or --L-R.sub.x ;
or R.sup.8 in combination with R.sup.2, or R.sup.9 in combination with R.sup.3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C.sub.1 -C.sub.6 alkyls or --CH.sub.2 SO.sub.3 X moieties;
C is OR.sup.17 or NR.sup.18 R.sup.19 ;
where R.sup.17 is H, or C.sub.1 -C.sub.18 alkyl;
or --L-R.sub.x ;
where R.sup.18 and R.sup.19 are independently H, C.sub.1 -C.sub.6 alkyl, C.sup.1 -C.sup.6 carboxyalkyl, C.sub.1 -C.sub.6 sulfoalkyl, a salt of C.sub.1 -C.sub.6 carboxyalkyl, or a salt of C.sub.1 -C.sub.6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.18 in combination with R.sup.19 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or --L-R.sub.x ;
or R.sup.18 in combination with R.sup.4, or R.sup.19 in combination with R.sup.5, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C.sub.1 -C.sub.6 alkyls or --CH.sub.2 SO.sub.3 X moieties;
B is O or N.sup.+ R.sup.18 R.sup.19 ;
R.sup.10 is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol;
or R.sup.10 is a saturated or unsaturated C.sub.1 -C.sub.18 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol, --SO.sub.3 X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;
or --L-R.sub.x ;
or R.sup.10 has the formula ##STR58## where R.sup.12, R.sup.13, R.sup.14, R.sup.15 and R.sup.16 are independently H, F, Cl, Br, I, --SO.sub.3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino;
or --L-R.sub.x ;
or C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 alkoxy, C.sub.1 -C.sub.18 alkylthio, C.sub.1 -C.sub.18 alkanoylamino, C.sub.1 -C.sub.18 alkylaminocarbonyl, C.sub.2 -C.sub.36 dialkylaminocarbonyl, C.sub.1 -C.sub.18 alkyloxycarbonyl, or C.sub.6 -C.sub.18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol, --SO.sub.3 X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;
or one pair of adjacent substituents R.sup.13 and R.sup.14, R.sup.14 and R.sup.15 or R.sup.15 and R.sup.16, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid;
and
R.sup.11 is H, hydroxy, CN or a C.sub.1 -C.sub.6 alkoxy;
or R.sup.10 in combination with R.sup.11 forms a 5-membered spirolactone ring or a 5-membered spirosultone ring;
or R.sup.11 in combination with R.sup.12 forms a 5- or 6-membered spirolactone ring or a 5- or 6-membered spirosultone ring that is optionally and independently substituted by H, F or CH.sub.3 ;
or R.sup.10 when taken in combination with R.sup.11 is a carbonyl oxygen;
for all --L-R.sub.x, each L is a covalent linkage;
and R.sub.x is a reactive group;
provided that either at least one of R.sup.2, R.sup.3, R.sup.4, and R.sup.5 is --SO.sub.3 X;
or R.sup.8 in combination with R.sup.2, or R.sup.9 in combination with R.sup.3, or R.sup.18 in combination with R.sup.4, or R.sup.19 in combination with R.sup.5, form a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by at least one --CH.sub.2 SO.sub.3 X moiety;
and that at least one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.8, R.sup.9, R.sup.10, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, R.sup.18, and R.sup.19 is --L-R.sub.x.

32 more elements

A compound having the formula ##STR61## or the formula ##STR62## wherein R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are independently H, F, Cl, Br, I, CN;

or C.sub.1 -C.sub.18 alkyl, or C.sub.1 -C.sub.18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol;
or --L-S.sub.c ;
or --SO.sub.3 X where X is H or a counterion
R.sup.1 and R.sup.6 are H;
or R.sup.1 taken in combination with R.sup.2, or R.sup.5 taken in combination with R.sup.6, or both, form a fused aromatic six membered ring that is optionally substituted one or more times by --SO.sub.3 X;
A is OR.sup.7 or NR.sup.8 R.sup.9,
R.sup.7 is H, C.sub.1 -C.sub.18 alkyl;
or --L-S.sub.c ;
R.sup.8 and R.sup.9 are independently H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 carboxyalkyl, C.sub.1 -C.sub.6 sulfoalkyl, a salt of C.sub.1 -C.sub.6 carboxyalkyl, or a salt of C.sub.1 -C.sub.6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.9 in combination with R.sup.9 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or --L-S.sub.c ;
or R.sup.8 in combination with R.sup.2, or R.sup.9 in combination with R.sup.3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C.sub.1 -C.sub.6 alkyls or --CH.sub.2 SO.sub.3 X moieties;
C is OR.sup.17 or NR.sup.18 R.sup.19 ;
where R.sup.17 is H, or C.sub.1 -C.sub.18 alkyl;
or --L-S.sub.c ;
where R.sup.18 and R.sup.19 are independently H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 carboxyalkyl, C.sub.1 -C.sub.6 sulfoalkyl, a salt of C.sub.1 -C.sub.6 carboxyalkyl, or a salt of C.sub.1 -C.sub.6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.18 in combination with R.sup.19 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or --L-S.sub.c ;
or R.sup.18 in combination with R.sup.4, or R.sup.19 in combination with R.sup.5, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C.sub.1 -C.sub.6 alkyls or --CH.sub.2 SO.sub.3 X moieties;
B is O or N.sup.+ R.sup.18 R.sup.19 ;
R.sup.10 is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol;
or R.sup.10 is a saturated or unsaturated C.sub.1 -C.sub.18 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol, --SO.sub.3 X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;
or --L-S.sub.c ;
or R.sup.10 has the formula ##STR63## where R.sup.12, R.sup.13, R.sup.14, R.sup.15 and R.sup.16 are independently H, F, Cl, Br, I, --SO.sub.3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino;
or --L-S.sub.c ;
or C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 alkoxy, C.sub.1 -C.sub.18 alkylthio, C.sub.1 -C.sub.18 alkanoylamino, C.sub.1 -C.sub.18 alkylaminocarbonyl, C.sub.2 -C.sub.36 dialkylaminocarbonyl, C.sub.1 -C.sub.18 alkyloxycarbonyl, or C.sub.6 -C.sub.18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol, --SO.sub.3 X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;
or one pair of adjacent substituents R.sup.13 and R.sup.14, R.sup.14 and R.sup.15 or R.sup.15 and R.sup.16, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid;
and
R.sup.11 is H, hydroxy, CN or a C.sub.1 -C.sub.6 alkoxy;
or R.sup.10 in combination with R.sup.11 forms a 5-membered spirolactone ring or a 5-membered spirosultone ring;
or R.sup.11 in combination with R.sup.12 forms a 5- or 6-membered spirolactone ring or a 5- or 6-membered spirosultone ring that is optionally and independently substituted by H, F or CH.sub.3 ;
or R.sup.10 when taken in combination with R.sup.11 is a carbonyl oxygen;
for all --L-S.sub.c, each L is a covalent linkage;
and S.sub.c is a conjugated substance;
provided that either at least one of R.sup.2, R.sup.3, R.sup.4, and R.sup.5 is --SO.sub.3 X;
or R.sup.8 in combination with R.sup.2, or R.sup.9 in combination with R.sup.3, or R.sup.18 in combination with R.sup.4, or R.sup.19 in combination with R.sup.5, form a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by at least one --CH.sub.2 SO.sub.3 X moiety;
and that at least one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.8, R.sup.9, R.sup.10, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.18, and R.sup.19 is --L-S.sub.c.

34 more elements

A method of staining a biological sample, comprising:

combining a dye solution comprising a compound of the formula ##STR66## or the formula ##STR67## wherein R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are independently H, F, Cl, Br, I, CN;
or C.sub.1 -C.sub.18 alkyl, or C.sub.1 -C.sub.18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol;
or --L-R.sub.x ;
--L-S.sub.c ;
or --SO.sub.3 X where X is H or a counterion;
R.sup.1 and R.sup.6 are H;
or R.sup.1 taken in combination with R.sup.2, or R.sup.5 taken in combination with R.sup.6, or both, form a fused aromatic six membered ring that is optionally substituted one or more times by --SO.sub.3 X;
A is OR.sup.7 or NR.sup.8 R.sup.9,
R.sup.7 is H, C.sub.1 -C.sub.18 alkyl;
or --L-R.sub.x ;
or --L-S.sub.c ;
R.sup.8 and R.sup.9 are independently H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 carboxyalkyl, C.sub.1 -C.sub.6 sulfoalkyl, a salt of C.sub.1 -C.sub.6 carboxyalkyl, or a salt of C.sub.1 -C.sub.6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.8 in combination with R.sup.9 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or --L-R.sub.x ;
or --L-S.sub.c ;
or R.sup.8 in combination with R.sup.2, or R.sup.9 in combination with R.sup.3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C.sub.1 -C.sub.6 alkyls or --CH.sub.2 SO.sub.3 X moieties;
C is OR.sup.17 or NR.sup.18 R.sup.19 ;
where R.sup.17 is H, or C.sub.1 -C.sub.18 alkyl;
or --L-R.sub.x ;
or --L-S.sub.c ;
where R.sup.18 and R.sup.19 are independently H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 carboxyalkyl, C.sub.1 -C.sub.6 sulfoalkyl, a salt of C.sub.1 -C.sub.6 carboxyalkyl, or a salt of C.sub.1 -C.sub.6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.18 in combination with R.sup.19 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or --L-R.sub.x ;
or --L-S.sub.c ;
or R.sup.18 in combination with R.sup.4, or R.sup.19 in combination with R.sup.5, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C.sub.1 -C.sub.6 alkyls or --CH.sub.2 SO.sub.3 X moieties;
B is O or N.sup.+ R.sup.18 R.sup.19 ;
R.sup.10 is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol;
or R.sup.10 is a saturated or unsaturated C.sub.1 -C.sub.18 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol, --SO.sub.3 X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;
or --L-R.sub.x ;
or --L-S.sub.c ;
or R.sup.10 has the formula ##STR68## where R.sup.12, R.sup.13, R.sup.14, R.sup.15 and R.sup.16 are independently H, F, Cl, Br, I, --SO.sub.3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino;
or --L-R.sub.x ;
or --L-S.sub.c ;
or C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 alkoxy, C.sub.1 -C.sub.18 alkylthio, C.sub.1 -C.sub.18 alkanoylamino, C.sub.1 -C.sub.18 alkylaminocarbonyl, C.sub.2 -C.sub.36 dialkylaminocarbonyl, C.sub.1 -C.sub.18 alkyloxycarbonyl, or C.sub.6 -C.sub.18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol, --SO.sub.3 X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;
or one pair of adjacent substituents R.sup.13 and R.sup.14, R.sup.14 and R.sup.15 or R.sup.15 and R.sup.16, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid;
and
R.sup.11 is H, hydroxy, CN or a C.sub.1 -C.sub.6 alkoxy;
or R.sup.10 in combination with R.sup.11 forms a 5-membered spirolactone ring or a 5-membered spirosultone ring;
or R.sup.11 in combination with R.sup.12 forms a 5- or 6-membered spirolactone ring or a 5- or 6-membered spirosultone ring that is optionally and independently substituted by H, F or CH.sub.3 ;
or R.sup.10 when taken in combination with R.sup.11 is a carbonyl oxygen;
each L is a covalent linkage;
each R.sub.x is a reactive group;
and each S.sub.c is a conjugated substance;
provided that either at least one of R.sup.2, R.sup.3, R.sup.4, and R.sup.5 is --SO.sub.3 X;
or R.sup.8 in combination with R.sup.2, R.sup.9 in combination with R.sup.3, or R.sup.18 in combination with R.sup.4, or R.sup.19 in combination with R.sup.5, form a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by at least one --CH.sub.2 SO.sub.3 X moiety;
with a biological sample in a concentration sufficient to yield a detectable optical response under the desired conditions.

43 more elements

A compound having the formula ##STR70## or the formula ##STR71## wherein R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are independently H, F, Cl, Br, I, CN;

or C.sub.1 -C.sub.18 alkyl, or C.sub.1 -C.sub.18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol;
or --L-R.sub.x ;
or --SO.sub.3 X where X is H or a counterion
R.sup.1 and R.sup.6 are H;
or R.sup.1 taken in combination with R.sup.2, or R.sup.5 taken in combination with R.sup.6, or both, form a fused aromatic six membered ring that is optionally substituted one or more times by --SO.sub.3 X;
A is OR.sup.7,
R.sup.7 is H, C.sub.1 -C.sub.18 alkyl;
or --L-R.sub.x ;
C is OR.sup.17 ;
where R.sup.17 is H, or C.sub.1 -C.sub.18 alkyl;
or --L-R.sub.x ;
B is O;
R.sup.10 has the formula ##STR72## where R.sup.12, R.sup.13, R.sup.14, R.sup.15 and R.sup.16 are independently H, F, Cl, Br, I, --SO.sub.3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino;
or --L-R.sub.x ;
or C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 alkoxy, C.sub.1 -C.sub.18 alkylthio, C.sub.1 -C.sub.18 is alkanoylamino, C.sub.1 -C.sub.18 alkylaminocarbonyl, C.sub.2 -C.sub.36 dialkylaminocarbonyl, C.sub.1 -C.sub.18 alkyloxycarbonyl, or C.sub.6 -C.sub.18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol, --SO.sub.3 X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;
or one pair of adjacent substituents R.sup.13 and R.sup.14, R.sup.14 and R.sup.15 or R.sup.15 and R.sup.6, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid;
and
R.sup.11 is H, hydroxy, CN or a C.sub.1 -C.sub.6 alkoxy;
or R.sup.10 in combination with R.sup.11 forms a 5-membered spirolactone ring or a 5-membered spirosultone ring;
or R.sup.11 in combination with R.sup.12 forms a 5- or 6-membered spirolactone ring or a 5- or 6-membered spirosultone ring that is optionally and independently substituted by H, F or CH.sub.3 ;
or R.sup.10 when taken in combination with R.sup.11 is a carbonyl oxygen;
for all --L-R.sub.x, each L is a covalent linkage;
and R.sub.x is a reactive group;
provided that at least two of R.sup.2, R.sup.3, R.sup.4, and R.sup.5 is --SO.sub.3 X;
and that at least one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17 is --L-R.sub.x.

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Dependencies are inferred from the claim text.

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Abstract

A compound having the formula ##STR51## or the formula ##STR52## wherein R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are independently H, F, Cl, Br, I, CN;

or C.sub.1 -C.sub.18 alkyl, or C.sub.1 -C.sub.18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol;
or --SO.sub.3 X where X is H or a counterion
R.sup.1 and R.sup.6 are H;
or R.sup.1 taken in combination with R.sup.2, or R.sup.5 taken in combination with R.sup.6, or both, form a fused aromatic six membered ring that is optionally substituted one or more times by --SO.sub.3 X;
A is NR.sup.8 R.sup.9,
R.sup.8 and R.sup.9 are independently H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 carboxyalkyl, C.sub.1 -C.sub.6 sulfoalkyl, a salt of C.sub.1 -C.sub.6 carboxyalkyl, or a salt of C.sub.1 -C.sub.6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.8 in combination with R.sup.9 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.8 in combination with R.sup.2, or R.sup.9 in combination with R.sup.3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C.sub.1 -C.sub.6 alkyls or --CH.sub.2 SO.sub.3 X moieties;
C is OR.sup.17 or NR.sup.18 R.sup.19 ;
where R.sup.17 is H, or C.sub.1 -C.sub.18 alkyl;
where R.sup.18 and R.sup.19 are independently H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 carboxyalkyl, C.sub.1 -C.sub.6 sulfoalkyl, a salt of C.sub.1 -C.sub.6 carboxyalkyl, or a salt of C.sub.1 -C.sub.6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.18 in combination with R.sup.19 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alkyl;
or R.sup.18 in combination with R.sup.4, or R.sup.19 in combination with R.sup.5, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C.sub.1 -C.sub.6 alkyls or --CH.sub.2 SO.sub.3 X moieties;
B is O or N.sup.+ R.sup.18 R.sup.19 ;
R.sup.10 is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol;
or R.sup.10 is a saturated or unsaturated C.sub.1 -C.sub.18 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol, --SO.sub.3 X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;
or R.sup.10 has the formula ##STR53## where R.sup.12, R.sup.13, R.sup.14, R.sup.15 and R.sup.16 are independently H, F, Cl, Br, I, --SO.sub.3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino;
or C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 alkoxy, C.sub.1 -C.sub.18 alkylthio, C.sub.1 -C.sub.18 alkanoylamino, C.sub.1 -C.sub.18 alkylaminocarbonyl, C.sub.2 -C.sub.36 dialkylaminocarbonyl, C.sub.1 -C.sub.18 alkyloxycarbonyl, or C.sub.6 -C.sub.18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C.sub.1 -C.sub.6 alcohol, --SO.sub.3 X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;
or one pair of adjacent substituents R.sup.13 and R.sup.14, R.sup.14 and R.sup.15 or R.sup.15 and R.sup.16, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid;
and
R.sup.11 is H, hydroxy, CN or a C.sub.1 -C.sub.6 alkoxy;
or R.sup.10 in combination with R.sup.11 forms a 5-membered spirolactone ring or a 5-membered spirosultone ring;
or R.sup.11 in combination with R.sup.12 forms a 5- or 6-membered spirolactone ring or a 5- or 6-membered spirosultone ring that is optionally and independently substituted by H, F or CH.sub.3 ;
or R.sup.10 when taken in combination with R.sup.11 is a carbonyl oxygen;
provided that either at least one of R.sup.2, R.sup.3, R.sup.4, and R.sup.5 is --SO.sub.3 X;
or R.sup.8 in combination with R.sup.2, or R.sup.9 in combination with R.sup.3, or R.sup.18 in combination with R.sup.4, or R.sup.19 in combination with R.sup.5, form a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by at least one --CH.sub.2 SO.sub.3 X moiety.

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