# US 5,707,844 A · Immunoassay reagents and methods for detecting brequinar and analogs

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- Number: US 5,707,844 A
- Document ID: US5707844A
- Status: Granted
- Kind: A
- Application number: 08/372,472
- Filed: 1995-01-13
- Granted: 1998-01-13
- Earliest claimed: 1993-06-08
- Assignees: The DuPont Merck Pharmaceutical Company
- Inventors: Douglas Guy Batt; William Galbraith; Paul Moore Simon
- Web page: https://patentgrep.com/patent/US5707844A

## CPC

- C07D 401/04: directly linked by a ring-member-to-ring-member bond
- A61K 49/0043: {Fluorescein, used in vivo}
- A61K 49/0052: {Small organic molecules (oligomers, polymers, dendrimers A61K49/0054)}
- C07D 215/52: with aryl radicals attached in position 2
- C07D 405/04: directly linked by a ring-member-to-ring-member bond
- C07D 409/04: directly linked by a ring-member-to-ring-member bond
- C07K 16/44: against material not provided for elsewhere {, e.g. haptens, metals, DNA, RNA, amino acids}
- Y10S 436/80: Fluorescent dyes, e.g. rhodamine
- Y10S 530/807: Hapten conjugated with peptide or protein
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The present invention is directed to immunoassay reagents including specific antibodies to quinoline carboxylic acids, such as brequinar; novel quinoline carboxylic acid haptens useful as standards in immunoassays or for conjugating to large molecular weight carriers as immunogens or conjugating to detectable fluorescent moieties as tracer compounds. The present invention also relates to immunoassays for detecting quinoline carboxylic acids in clinical samples, and finally to kits containing said reagents.

## Claims

### Claim 1

An immunogen comprised of a suitable high molecular weight carrier molecule, selected from the group consisting of proteins, polysaccharides, and latex particles, conjugated to a hapten of the Formula (I): ##STR10## wherein: R is ##STR11## X is O, S, NR.sup.7 or CH.dbd.N; R.sup.1 is alkyl of 5-12 carbon atoms, alkenyl of 5-12 carbon atoms, cycloalkyl of 3-7 carbon atoms, cycloalkylalkyl of 5-12 carbon atoms, phenyl substituted with 0-3 Y, or benzyl substituted with 0-3 Y;
R.sup.2 is phenyl substituted with 0-3 Y, or benzyl substituted with 0-3 Y;
R.sup.3 is H, alkoxy of 1-3 carbon atoms, alkylthio of 1-3 carbon atoms, or alkyl of 1-2 carbon atoms;
R.sup.4 is COOH;
R.sup.5 is H, F, Cl, Br, I, CH.sub.3, CF.sub.3, S(O).sub.n R.sup.8 or ethyl;
R.sup.6 is (CR.sup.9 R.sup.10).sub.p CH.sub.2 NH.sub.2 ;
R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are independently H or alkyl of 1-3 carbon atoms;
Y is selected independently at each occurrence from the group consisting of: H, F, Cl, Br, alkyl of 1-5 carbon atoms, NO.sub.2, alkoxy of 1-5 carbon atoms, alkylthio of 1-5 carbon atoms, OH, CF.sub.3 and NH.sub.2 ;
m and n are independently 0, 1 or 2; and
p is 0-12;
or a suitable salt form thereof.

### Claim 2 (depends on 1)

An immunogen of claim 1 wherein the hapten is selected from the group consisting of:
2-(2'-Fluoro-1,1'-biphenyl-4-yl)-3-methyl-6-(2-aminoethyl)-4-quinolinecarbo xylic acid;
2-(2'-Fluoro-1,1'-biphenyl-4-yl)-3-methyl-6-(2-aminoethyl)-4-quinolinecarbo xylic acid, sodium salt;
2-(2'-Fluoro-4'-hydroxy-1,1'-biphenyl-4-yl)-3-methyl-6-(2-aminoethyl)-4-qui nolinecarboxylic acid; and
2-(2'-Fluoro-4'-hydroxy-1,1'-biphenyl-4-yl)-3-methyl-6-(2-aminoethyl)-4-qui nolinecarboxylic acid, sodium salt.

### Claim 3 (depends on 1)

An immunogen of claim 1 wherein the protein molecule is selected from the group consisting of bovine serum albumin, ovalbumin, and keyhole limpet hemocyanin.

### Claim 4 (depends on 2)

An immunogen of claim 2 wherein the protein molecule is selected from the group consisting of bovine serum albumin, ovalbumin, and keyhole limpet hemocyanin.

### Claim 5

A tracer compound comprised of a hapten of Formula (I): ##STR12## wherein: R is ##STR13## X is O, S, NR.sup.7 or CH.dbd.N; R.sup.1 is alkyl of 5-12 carbon atoms, alkenyl of 5-12 carbon atoms, cycloalkyl of 3-7 carbon atoms, cycloalkylalkyl of 5-12 carbon atoms, phenyl substituted with 0-3 Y, or benzyl substituted with 0-3 Y;
R.sup.2 is phenyl substituted with 0-3 Y or benzyl substituted with 0-3 Y;
R.sup.3 is H, alkoxy of 1-3 carbon atoms, alkylthio of 1-3 carbon atoms, or alkyl of 1-2 carbon atoms;
R.sup.4 is COOH;
R.sup.5 is H, F, Cl, Br, I, CH.sub.3, CF.sub.3, S(O).sub.n R.sup.8 or ethyl;
R.sup.6 is (CR.sup.9 R.sup.10).sub.p CH.sub.2 NH.sub.2 ;
R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are independently H or alkyl of 1-3 carbon atoms;
Y is selected independently at each occurrence from the group consisting of: H, F, Cl, Br, alkyl of 1-5 carbon atoms, NO.sub.2, alkoxy of 1-5 carbon atoms, alkylthio of 1-5 carbon atoms, OH, CF.sub.3 and NH.sub.2 ;
m and n are independently 0, 1 or 2; and
p is 0-12;
or a suitable salt thereof;
conjugated to a detectable fluorescent moiety.

### Claim 6 (depends on 5)

A tracer compound of claim 5 wherein:
R is para-phenylene;
R.sup.1 is cycloalkyl of 3-7 carbon atoms; phenyl optionally substituted with hydroxyl; phenyl substituted with one halogen, alkyl of 1-5 carbon atoms or CF.sub.3 and also optionally with one hydroxyl; phenoxy; or phenoxy substituted with one halogen or alkyl of 1-5 carbon atoms;
R.sup.3 is H, alkyl of 1-2 carbon atoms or methylthio;
R.sup.5 is H, halogen, CH.sub.3 or CF.sub.3 ; and
p is 1-12;
or a suitable salt thereof.

### Claim 7 (depends on 6)

A tracer compound of claim 6 wherein:
R.sup.1 is cyclohexyl, phenyl, hydroxyphenyl, fluorophenyl, fluorohydroxyphenyl, or methylphenyl;
R.sup.5 is hydrogen; and
R.sup.6 is (CH.sub.2).sub.p NH.sub.2, and is located at the 6-position of the quinoline ring; and
p is 2-8.

### Claim 8 (depends on 7)

A tracer compound of claim 7 selected from the group consisting of:
2-(2'-Fluoro-1,1'-biphenyl-4-yl)-3-methyl-6-(2-aminoethyl)-4-quinolinecarbo xylic acid;
2-(2'-Fluoro-1,1'-biphenyl-4-yl)-3-methyl-6-(2-aminoethyl)-4-quinolinecarbo xylic acid, sodium salt;
2-(2'-Fluoro-4'-hydroxy-1,1'-biphenyl-4-yl)-3-methyl-6-(2-aminoethyl)-4-qui nolinecarboxylic acid; and
2-(2'-Fluoro-4'-hydroxy-1,1'-biphenyl-4-yl)-3-methyl-6-(2-aminoethyl)-4-qui nolinecarboxylic acid, sodium salt.

### Claim 9 (depends on 5)

A tracer compound of claim 5 wherein said detectable fluorescent moiety is selected from the group consisting of chemiluminescent molecules, luminescent molecules and enzyme molecules.

### Claim 10 (depends on 6)

A tracer compound of claim 6 wherein said detectable fluorescent moiety is selected from the group consisting of chemiluminescent molecules, luminescent molecules and enzyme molecules.

### Claim 11 (depends on 7)

A tracer compound of claim 7 wherein said detectable fluorescent moiety is selected from the group consisting of chemiluminescent molecules, luminescent molecules and enzyme molecules.

### Claim 12 (depends on 8)

A tracer compound of claim 8 wherein said detectable fluorescent moiety is selected from the group consisting of chemiluminescent molecules, luminescent molecules and enzyme molecules.

### Claim 13 (depends on 9)

A tracer compound of claim 9 wherein said luminescent molecules are fluorescein molecules or analogs thereof.

### Claim 14 (depends on 10)

A tracer compound of claim 10 wherein said luminescent molecules are fluorescein molecules or analogs thereof.

### Claim 15 (depends on 11)

A tracer compound of claim 11 wherein said luminescent molecules are fluorescein molecules or analogs thereof.

### Claim 16 (depends on 12)

A tracer compound of claim 12 wherein said luminescent molecules are fluorescein molecules or analogs thereof.

### Claim 17 (depends on 13)

A tracer compound of claim 13 wherein said fluorescein molecules or analogs thereof are selected from the group consisting of aminomethylfluoresceins, carboxyfluoresceins, and fluoresceinamines.

### Claim 18 (depends on 13)

A tracer compound of claim 13 wherein said fluorescein molecules or analogs thereof are selected from the group consisting of aminomethylfluoresceins, carboxyfluoresceins, and fluoresceinamines.

### Claim 19 (depends on 13)

A tracer compound of claim 13 wherein said fluorescein molecules or analogs thereof are selected from the group consisting of aminomethylfluoresceins, carboxyfluoresceins, and fluoresceinamines.

### Claim 20 (depends on 13)

A tracer compound of claim 13 wherein said fluorescein molecules or analogs thereof are selected from the group consisting of aminomethylfluoresceins, carboxyfluoresceins, and fluoresceinamines.