Agent for the detection of esterolytic and/or proteolytic enzymes and with substrates suitable for this purpose

Claim 2

An agent for the detection of at least one of esterolytic and proteolytic enzymes, comprising: a carrier containing a detection principle effective to detect at least one of esterase and pro-tease and a buffer, said detection principle comprising a combination of a substituted diazonium salt and an ester, the reactivity of said combination being mutually coordinated by appropriate selection of the substituents in said salt, such that a sufficiently rapid coupling takes place with an alcohol component liberated from the ester but no reaction with other components of a test solution occurs, said diazonium salt being a compound of the formula (1): (I) in which: R1 is a lower alkyl, lower alkoxy, lower alkylthio, N-morpholino, N-thiomorpholino, N-pyrroli-dino, N-piperazino, N-alkylated N-piperazino or N-piperidino radical or is a halogen or hydrogen atom, R3 is a lower alkyl, lower alkoxy, aryloxy, lower alkylthio, alkylamino, dialkylamino, hydroxyl, N-morpholino, N-thiomorpholino, N-pyrrolidino, N-piperazino, N'-alkylated N-piperazino, N-piperidino or phenylamino radical or a phenyl radical unsub-stituted or substituted with a lower alkyl or lower alkoxy radical or is a hydrogen or halogen atom, R2, R4 and R5, which may be the same or different, are lower alkyl, lower alkoxy or lower alkylthio radicals or halogen or hydrogen atoms, and X is a stabilising anion; and said ester is selected from the group consisting of: i) a compound of the formula (II): (II) in which: R'1, R'2, R'3 and R'4, which may be the same or different, are hydrogen or halogen atoms or lower alkyl, lower alkoxy, aryl, aralkyl, aralkoxy, hydroxyl, carboxy, carboxy lower alkoxy, aralkoxy-carbonyl, aralkoxycarbonyl lower alkoxy, nitro or acylamino radicals or in which two adjacent substi-tuents represent an unsubstituted or halogen-substi-tuted fused benzene ring, X' is a sulphur atom or an imino group unsub-stituted or substituted by a lower alkyl, aryl, aralkyl or acyl radical, A is a residue of an amino acid or of a peptide, and B is a nitrogen protective group in peptide chemistry or derived therefrom; and ii) a compound of the formula (III): (III) in which: R"1, R"2, R"3 and R"4, which may be the same or different, are hydrogen atoms or lower alkyl, lower alkoxy, alkylamino or dialkylamino radicals or in which two adjacent substitutents can also represent a fused benzene ring, A is a residue of an amino acid or of a peptide, and B is a nitrogen protective group in peptide chemistry or derived therefrom.

Claim 3

A diagnostic agent according to claim 1, wherein said ester is a compound of formula (II).

Claim 4

A diagnostic agent according to claim 1, wherein said ester is a compound of formula (III).

Claim 5

A diagnostic agent according to claim 1, 2 or 3, which additionally contains at least one additional adjuvant selected from the group consisting of wetting agents, stabilisers, activators, film formers, galenical additives and structure formers.

Claim 6

A diagnostic agent according to claim 1, 2 or 3, wherein said carrier comprises an absorbent support.

Claim 7

A diagnostic agent according to claim 1, 2 or 3, wherein said carrier comprises a film layer.

Claim 8

A diagnostic agent according to claim 1, 2 or 3, wherein said carrier comprises a powder mixture.

Claim 9

A diagnostic agent according to claim 1, 2 or 3, wherein said carrier comprises a lyophilisate.

Claim 10

A diagnostic agent according to claim 1, 2 or 3, wherein said carrier comprises a liquid vehicle.

Claim 11

A diagnostic agent according to claim 1, 2 or 3, wherein said carrier comprises a reagent tablet.

Claim 12

A diagnostic agent according to claim 1, 2 or 3, wherein said carrier comprises a polyvinyl ester or polyamide film layer.

Claim 13

A diagnostic agent according to claim 1, 2 or 3, wherein said carrier comprises a filter paper.

Claim 14

A diagnostic agent according to claim 1, 2 or 3, wherein said buffer is effective to establish a pH of 7 to 9.

Claim 15

A diagnoatic agent according to claim 1, 2 or 3, wherein in said compound (I) at least one of R1 and R3 is selected from the group consisting of N-thiomorpholino, N-pyrrolidino, N-piperazino, N'-alkylated-piperazino and N-piperidino.

Claim 16

A diagnostic agent according to claim 1, 2 or 3, wherein in said compound (I), R1 is lower alkyl, lower alkoxy, lower alkylthio, halogen or hydrogen, R3 is hydrogen, halogen, lower alkyl, lower alkoxy, aryloxy, lower alkylthio, lower alkylamino, di-lower-alkylamino, hydroxyl, phenylamino or phenyl unsubstituted or substituted by lower alkyl or lower alkoxy.

Claim 17

A diagnostic agent for the detection of at least one of esterolytic and proteolytic enzymes present in leukocytes in body fluids comprising a carrier containing: a) a buffer effective to establish a pH of from 6 to 10, b) a detection principle effective to detect at least one of esterase and protease, comprising a com-bination of a substituted diazonium salt and an ester, the reactivity of said combination being mutually coordinated by selection of the substituents in said salt such that a sufficiently rapid coupling takes place with an alcohol component liberated from the ester and interfering reactions with, other components of-the test do not occur, said combination being present in an amount to produce a perceptible colouration or colour change by coupling of said salt with alcohol liberated from said ester, said diazonium salt being a compound of formula (I):- (I) in which R1 is lower alkyl of 1 to 3 carbon atoms, lower alkoxy of 1 to 3 carbon atoms, lower alkylthio of 1 to 3 carbon atoms N-morpholino, N-thiomorpholino, N-pyrrolidino, N-piperazino, N'-alkylated N-piperazino in which the alkyl has 1 to 3 carbon atoms, N-piperidino, hydrogen, chlorine or bromine, R3 is lower alkyl of 1 to 3 carbon atoms, lower alkoxy of 1 to 3 carbon atoms, phenyloxy, naphthyloxy, lower alkylthio of 1 to 3 carbon atoms, alkylamino of 1 to 3 carbon atoms, dialkylamino in which each alkyl has 1 to 3 carbon atoms, hydroxyl, N-morpho-lino, N-thiomorpholino, N-pyrrolidino, N-piperazino, N'-alkylated N-piperazino in which the alkyl has 1 to 3 carbon atoms, N-piperidino, phenylamino, phenyl unsubstituted or substituted by lower alkyl or lower alkoxy of 1 to 3 carbon atoms, hydrogen, chlorine or bromine, R2, R4 and R5, which may be the same or different, are lower alkyl of 1 to 3 carbon atoms, lower alkoxy of 1 to 3 carbon atoms, lower alkylthio of 1 to 3 carbon atoms, hydrogen, chlorine or bromine and X is a stabilising anion selected from tetrafluoroborate, tetrachlorozincate and perchlorate anion, said ester being a compound selected from the group consisting of i) a compound of formula (II):- (II) in which R'1, R'2, R'3 and R'4, which may be the same or different are selected from hydrogen, chlorine, bromine, lower alkyl of 1 to 3 carbon atoms, lower alkoxy of 1 to 3 carbon atoms, phenyl, naphthyl, benzyl, phenyl-lower-alkyloxy in which said alkyl contains 1 to 3 carbon atoms, naphthyl-lower-alkoxy in which said alkyl contains 1 to 3 carbon atoms, hydroxyl, carboxy, carboxy lower alkoxy in which said alkoxy contains 1 to 3 carbon atoms, aralkoxy carbonyl and aralkoxy carbonyl lower alkoxy in which said aralkoxy is phenyl-lower-alkyloxy or naphthyl lower-alkyloxy, in which said alkyl and alkoxy contains 1 to 3 carbon atoms, nitro or acylamino of up to 3 carbon atoms, or two adjacent substituents represent an unsubstituted or halogen-substituted fused benzene ring, X' is a sulphur atom or an imino group unsubstituted or substituted by lower alkyl of 1 to 3 carbon atoms, phenyl, naphthyl, benzyl, aliphatic acyl of 1 to 3 carbon atoms, benzoyl or naphthoyl, A is a residue of an L-amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine and tyrosine, any hydroxyl therein being free or acetylated, or of a di-, tri-, tetra- or pentapeptide based on amino acids of said group, and B is a nitrogen protective group in peptide chemistry or is derived therefrom, and ii) a compound of formula (III):- (III) in which R"1, R"2, R"3 and R"4, which may be the same or different, are selected from hydrogen, lower alkyl of 1 to 3 carbon atoms, lower alkoxy of 1 to 3 carbon atoms, alkylamino of 1 to 3 carbon atoms, dialkylamino in which each alkyl has 1 to 3 carbon atoms, or in which two adjacent substituents can also represent a fused benzene ring, and A and B are as defined in i) above, and wherein c) said agent having a sensitivity for leukocytes in a concentration of 75 to 5000 leukocytes/µl.

Claim 18

A diagnostic agent for the detection of at least one of esterolytic and proteolytic enzymes present in leukocytes in body fluids comprising a carrier containing: a) a buffer effective to establish a pH of from 6 to 10, b) a detection principle effective to detect at least one of esterase and protease, comprising a com-bination of a substituted diazonium salt and an ester, the reactivity of said combination being mutually coordinated by selection of the substituents in said salt such that a sufficiently rapid coupling takes place with an alcohol component liberated from the ester and interfering reactions with, other components of-the test do not occur, said combination being present in an amount to produce a perceptible colouration or colour change by coupling of said salt with alcohol liberated from said ester, said diazonium salt being a compound of formula (I):- (I) in which R1 is lower alkyl of 1 to 3 carbon atoms, lower alkoxy of 1 to 3 carbon atoms, lower alkylthio of 1 to 3 carbon atoms N-morpholino, N-thiomorpholino, N-pyrrolidino, N-piperazino, N'-alkylated N-piperazino in which the alkyl has 1 to 3 carbon atoms, N-piperidino, hydrogen, chlorine or bromine, R3 is lower alkyl of 1 to 3 carbon atoms, lower alkoxy of 1 to 3 carbon atoms, phenyloxy, naphthyloxy, lower alkylthio of 1 to 3 carbon atoms, alkylamino of 1 to 3 carbon atoms, dialkylamino in which each alkyl has 1 to 3 carbon atoms, hydroxyl, N-morpho-lino, N-thiomorpholino, N-pyrrolidino, N-piperazino, N'-alkylated N-piperazino in which the alkyl has 1 to 3 carbon atoms, N-piperidino, phenylamino, phenyl unsubstituted or substituted by lower alkyl or lower alkoxy of 1 to 3 carbon atoms, hydrogen, chlorine or bromine, R2, R4 and R5, which may be the same or different, are lower alkyl of 1 to 3 carbon atoms, lower alkoxy of 1 to 3 carbon atoms, lower alkylthio of 1 to 3 carbon atoms, hydrogen, chlorine or bromine and X is a stabilising anion selected from tetrafluoroborate, tetrachlorozincate and perchlorate anion, said ester being a compound selected from the group consisting of i) a compound of formula (II):- (II) in which R'1, R'2, R'3 and R'4, which may be the same or different are selected from hydrogen, chlorine, bromine, lower alkyl of 1 to 3 carbon atoms, lower alkoxy of 1 to 3 carbon atoms, phenyl, naphthyl, benzyl, phenyl-lower-alkyloxy in which said alkyl contains 1 to 3 carbon atoms, naphthyl-lower-alkoxy in which said alkyl contains 1 to 3 carbon atoms, hydroxyl, carboxy, carboxy lower alkoxy in which said alkoxy contains 1 to 3 carbon atoms, aralkoxy carbonyl and aralkoxy carbonyl lower alkoxy in which said aralkoxy is phenyl-lower-alkyloxy or naphthyl lower-alkyloxy, in which said alkyl and alkoxy contains 1 to 3 carbon atoms, nitro or acylamino of up to 3 carbon atoms, or two adjacent substituents represent an unsubstituted or halogen-substituted fused benzene ring, X' is a sulphur atom or an imino group unsubstituted or substituted by lower alkyl of 1 to 3 carbon atoms, phenyl, naphthyl, benzyl, aliphatic acyl of 1 to 3 carbon atoms, benzoyl or naphthoyl, A is a residue of an L-amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine and tyrosine, any hydroxyl therein being free or acetylated, or of a di-, tri-, tetra- or pentapeptide based on amino acids of said group, and B is a nitrogen protective group in peptide chemistry or is derived therefrom, and ii) a compound of formula (III):- (III) in which R"1, R"2, R"3 and R"4, which may be the same or different, are selected from hydrogen, lower alkyl of 1 to 3 carbon atoms, lower alkoxy of 1 to 3 carbon atoms, alkylamino of 1 to 3 carbon atoms, dialkylamino in which each alkyl has 1 to 3 carbon atoms, or in which two adjacent substituents can also represent a fused benzene ring, and A and B are as defined in i) above, and wherein c) said agent having a sensitivity for leukocytes in a concentration of 75 to 5000 leukocytes/µl.

Claim 19

A diagnostic agent according to claim 16, wherein said buffer is effective to establish a pH of 7 to 9.

Claim 20

A diagnostic agent according to claim 16 or 17, wherein said carrier comprises an absorbent strip.

Claim 21

A diagnostic agent according to claim 16 or 17, wherein said carrier comprises a filter paper.

Claim 22

A diagnostic agent according to claim 16 or 17, wherein said diazonium salt and said ester are introduced to said carrier from respective impregnation solutions, said salt and ester being present in said respective solutions in a concentration of 10-4 mol/l to 10-1 mol/l.

Claim 23

An agent according to claim 1, 2 or 3, wherein said diazonium salt is selected from the group consisting of 2,4-dimethoxybenzenediazonium tetrafluoroborate, 4-methoxynaphthalene-1-diazonium tetrafluoroborate, 2,5-dimethoxy-4-dimethylaminobenzenediazonium tetrafluoro-borate, 4-diemthylaminobenzenediazonium tetrafluoro-borate, 2-methoxy-4-(N-pyrrolidino)-benzenediazonium tetrafluoroborate, 2-methoxy-4-(N-piperidino)-benzene-diazonium tetrafluoroborate, 2,6-dimethoxy-4-(N-morpho-lino)-benzenediazonium tetrafluoroborate, 4-methoxy-2-(N-morpholino)-benzenediazonium tetrafluoroborate, 2-methoxy-4-[N-(N'-methyl)-piperazino]-benzenediazonium tetrafluoroborate, 2-methoxy-4-(N-thiomorpholino)-benzenediazonium tetrafluoroborate, and 2-methoxy-4-(N-morpholino)-benzenediazonium tetrachlorozincate and said ester is selected from the group consisting of 3-[N-benzyloxycarbonyl-L-alanyloxy]-indole, 3-[N-benzyloxycarbonyl-L-alanyloxy]-methylindole, 3-[N-benzyloxycarbonyl-L-alanyloxy]-7-methylindole, 3-[N-benzyloxycarbonyl-L-alanyloxy]-4-chloroindole, 3-[N-benzyloxycarbonyl-L-alanyloxy]-5-bromoindole, 3[N-(toluene-4'-sulphonyl)-L-alanyloxy]-indole, 3-[N-(toluene-4'-sulphonyl)-L-alanyloxy]-5-methoxyindole, 3-[N-(4'-acetamidobenzenesulphonyl)-L-alanyloxy]-indole, 3-[N-(4'-methoxytosyl)-L-alanyloxy]-indole, [N-(toluene-4'-sulphonyl)-L-alanyloxy]-benzene, 1-[N-(toluene-4'-sulphonyl)-L-alanyloxy]-naphthalene, 1-[N-(toluene-4'-sulphonyl)-L-alanyloxy]-3-methoxybenzene, 1-[N-(toluene-4'-sulphonyl)-L-alanyloxy]-3,5-dimethoxybenzene, 3-[N-(toluene-4'-sulphonyl)-L-alanyloxy]-1-methoxynaphthalene, 1-[N-(toluene-4'-sulphonyl)-L-alanyloxy]-4-isopropoxy-naphthalene, 1-[N-(toluene-4'-sulphonyl)-L-alanyloxy]-4-pentoxynaphthalene, 1-[N-(toluene-4'-sulphonyl)-L-alanyloxy]-3-dimethylaminobenzene, 1-[N-(toluene-4'-sulphonyl)-L-alanyloxy]-3-diethylaminobenzene, 1-[N-(toluene-4'-sulphonyl)-L-alanyloxy]-3-methyl-5-methoxy-benzene, and 2-[N-(toluene-4'-sulphonyl)-L-alanyloxy]-4-methoxynaphthalene.

Claim 24

A method for detecting the presence of leukocytes in body fluids comprising contacting a sample of a body fluid with an agent as defined in claim 16 or 17, and evaluating any colouration or colour change developed as an indication of the leukocyte content of the sample.

Claim 25

A method for detecting the presence of leukocytes in body fluids comprising contacting a sample of a body fluid with an agent as defined in claim 1, 2 or 3, and evaluating any colouration or colour change developed as an indication of the leukocyte content of the sample.

Claim 26

A method for detecting the presence of leukocytes in body fluids comprising contacting a sample of a body fluid with a diagnostic agent as defined in claim 16 or 17, and evaluating any colouration or colour change developed as an indication of the leukocyte content of the sample.