CA2062240A1 - Labeled hydantoin derivatives for immunoassays - Google Patents
Labeled hydantoin derivatives for immunoassaysInfo
- Publication number
- CA2062240A1 CA2062240A1 CA002062240A CA2062240A CA2062240A1 CA 2062240 A1 CA2062240 A1 CA 2062240A1 CA 002062240 A CA002062240 A CA 002062240A CA 2062240 A CA2062240 A CA 2062240A CA 2062240 A1 CA2062240 A1 CA 2062240A1
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- Prior art keywords
- hydantoin
- label
- labeled
- linking
- groups
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/948—Sedatives, e.g. cannabinoids, barbiturates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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Abstract
LABELED HYDANTOIN DERIVATIVES FOR IMMUNOASSAYS
Abstract of the Disclosure The invention is directed to labeled hydantoin derivatives comprising:
(A) a label, of the type used in immunoassays, having an amine or sulfhydryl group;
(B) a hydantoin nucleus and (C) a linking chain linking the 3-position of the hydantoin nucleus to the label through a carbonyl bridge.
Abstract of the Disclosure The invention is directed to labeled hydantoin derivatives comprising:
(A) a label, of the type used in immunoassays, having an amine or sulfhydryl group;
(B) a hydantoin nucleus and (C) a linking chain linking the 3-position of the hydantoin nucleus to the label through a carbonyl bridge.
Description
LABELED HYDANI`OIN DERIVATIVES FOR IMMUNOASSAYS
~he~ :
This invention relates to clinical chemistry.
More specifically the invention relates to immunoassays in which labeled hydantoin derivatives are used.
Back~round of the Invention Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as ---analytical techniques in clinical chemistry. Because 10 of the specificity of the reactions, they are particu- ~-larly advantageous in quantifying biological analytes that are present in very low concentration in biological fluids. Such analytes (called ligands herein) include, for example, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
In competitive binding immunoassays, a labeled ligand, including immunocompetent derivatives and analogs of the ligand is placed in competition with unlabeled ligand for reaction with a fixed amount of the appropriate binding material (called a receptor herein). Unknown con~entrations of the ligand can be determined from the measured signal of either the bound or unbound (i.e. free) labeled ligand. The reaction proceeds as follows:
ligand + labeled ligand + receptor -~
ligand-receptor + labeled ligand-receptor.
Conventional labels include radioactive tag9, enzymes, chromophores, fluorophores, stable free radicals, and enzyme cofactors, inhibitors and allosteric effectors.
Consistent with the foregoing an immunoassay for a hydantoin deriyative, such as phenytoin, in serum can be based on competition of an enzyme labeled derivative of a hydantoin (referred to hereafter as 2062240 :
LDH) with the hydantoin in the patient serum for immobilized antibody binding sites.
Specific requirements for the LDH include: 1) at least 85% of the LDH can be bound by excess immobilized phenytoin antibody; 2) affinity of the LDH
for immobilized antibody is such that competition of a fixed amount of LDH with hydantoin occurs in a therapeutically relevant phenytoin concentration range; ~
and 3) stability of the LDH against hydrolysis of its enzyme label under storage conditions. Requirements imposed on the hydantoin derivative include: 1) accessibility of the derivative to the immobilized antibody following conjugation with the enzyme label;
~he~ :
This invention relates to clinical chemistry.
More specifically the invention relates to immunoassays in which labeled hydantoin derivatives are used.
Back~round of the Invention Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as ---analytical techniques in clinical chemistry. Because 10 of the specificity of the reactions, they are particu- ~-larly advantageous in quantifying biological analytes that are present in very low concentration in biological fluids. Such analytes (called ligands herein) include, for example, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
In competitive binding immunoassays, a labeled ligand, including immunocompetent derivatives and analogs of the ligand is placed in competition with unlabeled ligand for reaction with a fixed amount of the appropriate binding material (called a receptor herein). Unknown con~entrations of the ligand can be determined from the measured signal of either the bound or unbound (i.e. free) labeled ligand. The reaction proceeds as follows:
ligand + labeled ligand + receptor -~
ligand-receptor + labeled ligand-receptor.
Conventional labels include radioactive tag9, enzymes, chromophores, fluorophores, stable free radicals, and enzyme cofactors, inhibitors and allosteric effectors.
Consistent with the foregoing an immunoassay for a hydantoin deriyative, such as phenytoin, in serum can be based on competition of an enzyme labeled derivative of a hydantoin (referred to hereafter as 2062240 :
LDH) with the hydantoin in the patient serum for immobilized antibody binding sites.
Specific requirements for the LDH include: 1) at least 85% of the LDH can be bound by excess immobilized phenytoin antibody; 2) affinity of the LDH
for immobilized antibody is such that competition of a fixed amount of LDH with hydantoin occurs in a therapeutically relevant phenytoin concentration range; ~
and 3) stability of the LDH against hydrolysis of its enzyme label under storage conditions. Requirements imposed on the hydantoin derivative include: 1) accessibility of the derivative to the immobilized antibody following conjugation with the enzyme label;
2) specific recognition of the derivative by the antibody to the hydantoin; and 3) sufficient reactivity of the derivative with the enzyme label, either directly or following activation of the enzyme or derivative, under conditions that do not adversely affect enzyme activity.
Glucose oxidase (GOD) and alkaline phosphatase (ALP) enzyme labels coupled to hydantoin derivatives shown in structure I and disclosed in U.S.
Patent 4,752,568, especially phenytoin derivatives, gave adequate enzyme labeled phenytoin derivatives for conducting effective competitive immunoassays in the desired format:
~0 HN~N-CH2)nC
OH
O
- . . : . .
.;.. -.. .. . .. , . :
. ~ . . , -: - , . :
Glucose oxidase (GOD) and alkaline phosphatase (ALP) enzyme labels coupled to hydantoin derivatives shown in structure I and disclosed in U.S.
Patent 4,752,568, especially phenytoin derivatives, gave adequate enzyme labeled phenytoin derivatives for conducting effective competitive immunoassays in the desired format:
~0 HN~N-CH2)nC
OH
O
- . . : . .
.;.. -.. .. . .. , . :
. ~ . . , -: - , . :
Hydantoin derivatives of structure I were unsatisfactory for conducting competitive immunoassays when the enzyme horseradish peroxidase (HRP) was used as the label. The coupling reactions between such derivatives and HRP were both slow and incomplete.
Moreover previous phenytoin-HRP labels were bound very -weakly so that much higher concentrations of label or antibody binding sites would be re~uired to give a readable signal.
It would be highly desirable to provide new labeled hydantoin derivatives (a)that do react with HRP, and other enzymes such as GOD and ALP, faster and more completely, than prior art hydantoin derivatives (b) to form covalent bonds with such enzymes without adverse effect on enzyme activity, and (c) that are more readily recognized and tightly bound by antibodies to hydantoins.
Summary of the Invention The present invention provides labeled hydantoin derivatives having:
(i) a label, of the type used in immunoassays, having an amine or sulfhydryl group;
(ii) a hydantoin nucleus and (iii) a linking chain linking the 3-position of the hydantoin nucleus to the label through a carbonyl bridge; wherein the linking chain has about 5 to about 40 atoms consisting of (1) C1 to C1o alkylene groups, (2) phenylene groups, and (3) 5 to 7 membered heterocyclic rings (e.g., a 1,4-piperazinylene, 2,5-dimethyl-1,4-piperazinylene-1,3-imidazolidinylene, and 1,3-hexahydrodiazepinylene group) joined to the linking group through ring nitrogen atoms bonded to each other through chemical groups selected from (a) esters, : -, , ~
- . : : ~ , ; . . .
: , , ~
.. . .. .
,. . , ~ - . : . .
Moreover previous phenytoin-HRP labels were bound very -weakly so that much higher concentrations of label or antibody binding sites would be re~uired to give a readable signal.
It would be highly desirable to provide new labeled hydantoin derivatives (a)that do react with HRP, and other enzymes such as GOD and ALP, faster and more completely, than prior art hydantoin derivatives (b) to form covalent bonds with such enzymes without adverse effect on enzyme activity, and (c) that are more readily recognized and tightly bound by antibodies to hydantoins.
Summary of the Invention The present invention provides labeled hydantoin derivatives having:
(i) a label, of the type used in immunoassays, having an amine or sulfhydryl group;
(ii) a hydantoin nucleus and (iii) a linking chain linking the 3-position of the hydantoin nucleus to the label through a carbonyl bridge; wherein the linking chain has about 5 to about 40 atoms consisting of (1) C1 to C1o alkylene groups, (2) phenylene groups, and (3) 5 to 7 membered heterocyclic rings (e.g., a 1,4-piperazinylene, 2,5-dimethyl-1,4-piperazinylene-1,3-imidazolidinylene, and 1,3-hexahydrodiazepinylene group) joined to the linking group through ring nitrogen atoms bonded to each other through chemical groups selected from (a) esters, : -, , ~
- . : : ~ , ; . . .
: , , ~
.. . .. .
,. . , ~ - . : . .
o Il ( - cz - ) including thioesters , where Z is O or S; (b) o Il :
(-CNH-) amides, , (c) hetero atoms selected from - _ O-, -S-, and -NR-; wherein R represents C1 to C6 alkyl (e.g. methyl, ethyl, propyl, butyl etc.); and (d) carbonyl, with the proviso that the linking group is other than a derivative of a saturated or unsaturated monocarboxylic acid having from to 2 to 12 carbon atoms.
The above labeled hydantoin derivatives includes those that conform to the structure:
(I) HN ~ N-R2 { -N' ~N-C-R~ ~ C Z R /'C R ~ C~ L b I
wherein R1 each independently represents hydrogen, alkyl of 1 to 10 carbon atoms , unsubstituted or substituted phenyl;
R2, R4, R5, and R6, each independently represents C1 to C1o alkylene or such alkylene groups interrupted with ester groups, amide groups, -O-, -S-, and -NR-;
R3 represents C1 to C3 alkylene;
Z represents -O-, -S-, and -NR-, wherein R
- - ~
- - . .
- : . . . -_5_ 2062240 represents hydrogen or C1 to C6 alkyl, e.g., methyl propyl and hexyl;
m is 0, 1, or 2;
n is 0, 1, or 2; and the total number of atoms comprised in m, n and R2 is 5 to 40;
LABEL is an enzyme;
and further provided that (i) at least one of the Rl groups is substituted or unsubstituted phenyl;
(ii) one of R4, R5, and R6 can be phenylene;
(iii) the bracketed components of structure I can appear therein in any order and (iv~ the linking group is other than a derivative of a saturated or unsaturated monocarboxylic acid having from to 2 to 12 carbon atoms.
Consistent with the above definitions Rl can represent hydrogen methyl, propyl, hexyl, decyl, unsubstituted phenyl or phenyl substituted with alkyl of about 1 to 6 carbon atoms, nitro, halogen, cyano, and alkoxy of about 1 to ~ carbon atoms;
R~, R4, R5, and R6, can each independently represent alkylene selected from ethylene, butylene, pentylene, octylene, or such alkylene interrupted with ester groups, amide groups, -O-, -S-, and -NR-;
R3 represents methylene, ethylene, or trimethylene;
Z represents -NR-, wherein R can represent hydrogen methyl propyl or hexyl; and hABEL represents an enzyme.
At least 85~ of the labeled hydantoin derivatives can be bound by immobilized hydantoin antibodies, particularly phenytoin antibodies, at antibody concentrations of interest. The labeled hydantoin derivatives with extended linkers, ., ... ~ ........ , , . . ~ . .
- ~ ' . ~ , , .
,: ~. .. . . ' , particularly those having amide bonds in the linking chain are similarly bound by all the immobilized antibody types tested in reducing this invention ~o practice. Also the derivatives having the amide in the extended linker are very stable against hydrolysis.
The novel labeled hydantoin derivatives used in the immunoassay of the present invention were prepared from novel hydantoin derivatives of copending U. S. Patent Application No. filed on even date herewith in the name of The novel hydantoin derivatives comprise:
(a) an active ester group,such as a succinimidoxycarbonyl;
(b) a hydantoin nucleus and (c) a linking chain linking the active ester group to the hydantoin nucleus;
wherein the linking chain is as previously defined.
More specifically, the preferred new hydantoin active esters of this invention are those conforming to the structure:
H N~N R 2{C-N' ~N - C - R ~EC - Z - R 5 - Z C - R~C - - N' 'R 7 wherein R1, R2, R3, R4, R5, R6, Z, m, and n and the provisos related thereto are as previously defined, and R7 is an ethylene or o-phenylene group.
.. ~ ~ , . ................................. .
. - : : . ,. , : .
.. ~. . ~ . . . . .
Several advantages are realized by use of the above hydantoin derivatives. First, it was found that the active esters of these hydantoin derivatives having short linking chains between the hydantoin nucleus and the active ester group were sufficiently reactive with HRP to prepare an acceptable enzyme label for use with -some immobilized antibodies. Derivatives with longer linker groups (R2 plus the bracketed groups) of 8 to 20 atoms between the active ester group and the hydantoin nucleus gave labels that could be bound by all immobilized antibodies tested. Linking chains in which each Z is -NR- which with the adjacent carbonyl forms an amide group, are particularly useful in that :
hydantoin derivatives containing such chains are more resistant to hydrolysis than chains wherein Z is -O- or -S- so that with the adjacent carbonyl it forms an ester group.
preparation of Hydantoin Derivative~
The described hydantoin derivatives can be made according to the following preparations in which phenytoin derivatives, a subclass of hydantoin compounds, are made.
1. Preparation of HD 1, 5,5-Diphenyl-3-{4-[2-(3-succinimidoxycarbonylpropionyloxy)ethylamino-carbonyl]butyl)hydantoin.
Step 1: preparation of 5,5-Diphenyl-3-[4-(2-hydroxyethylamino-carbonyl)butyl]-hydantoin.
Part A: First the Acid Chloride is prepared.
A mixture of 3-(4-carboxybutyl)-S,5-diphenyl-2,4-imidazolidinedione (3.52 g, 0.01 mole) thionyl chloride (20 mL), N,N-dimethylformamide (2 drops) and chloroform (50 mL) was stirred at room temperature for . .
.. .,. ., . ............... . . : . ..................... .
- - .... . . , .- -. ~ : - . -:
3 hours. The solvent was removed on a rotary evaporator in vacuo, and this product was used directly in the next Part B.
Part B: The Acid Chloride is reacted with Ethanolamine.
The acid chloride in chloroform (50 mL) was added dropwise over 15 minutes to a mixture of ethanolamine (1.2 g, 0.02 mole) and triethylamine (2.4 g, 0.024 mole) in chloroform (100 mL). The mixture was then heated to 60C for 2 hours and stirred to room temperature for 1 hour. The solution was then washed with 5% hydrochloric acid (2 x 100 mL), washed with saturated sodium bicarbonate solution (100 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent removed on a rotary evaporator. The filtrate was then chromatographed using an aluminum oxide column to give material (3.0 g) showing one spot on TLC. This material was used directly in the next preparation.
Step 2: preparation of 3-l4-[2-(3-Carboxy-propionyloxy)ethylaminocarbonyl]-butyl}-5,5-diphenylhydantoin.
The hydroxy compound of Step 1 (3.0 g, 0.0075 mole), succinic anhydride (1.0 g, 0.01 mole), and dimethylaminopyridine (0.9 g, 0.0075 mole) in chloroform (100 mL) were heated at 50-60C for 4 hours and allowed to cool to room temperature over the weekend.
Dichloromethane (300 mL) was added, and the mixture was washed with 5~ hydrochloric acid solution (3 x 100 mL), washed with saturated sodium chloride solution (100 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent removed to give a material that gave one spot on TLC.
Step 3: preparation of HD 1: 5,5-Diphenyl-3-.
: :: :
g {4-[2-(3-succinimidoxycarbonylpro-pionyloxy)ethylaminocarbonyl]butyl}-hydantoin.
~' ' .
` >=
o N
o N H ~
~0 - .
0~0 ~N~O
O=~
A mixture of the acid from Step 2 (3.0 g, 0.006 mole), N,N'-dicyclohexylcarbodiimide (1.5 g, 0.007 mole), and N-hydroxysuccinimide (0.7 g, 0.006 mole) in chloroform (80 mL) was stirred at room temperature for 20 hours. The mixture was filtered, and the filtrate was concentrated on a rotary evaporator in vacuo. The residue was then chromatographed using silica to give 1.3 g (40% yield).
Anal. calc. for C30H32N40g: C, 60.8; H, 5.44; N, 9.45.
Found: C 59.6; H, 5.51, N, 8.91 2. Preparation of HD 2: 5,5-Diphenyl-3-{4-[4-(3-succinimidoxycarbonylpropionyl)-l-piperazinyl-carbonyl]butyl}-2,4-imidazolidinedione Step 1: preparation of 5,5-Diphenyl-3-(1-piperazinylcarbonylbutyl)hydantoin.
Part A: First, 3-[4-(4-Benzyloxycarbonyl-piperazinylcarbonyl)butyl]-5,5-diphenyl-2,4-imidazolidinedione was prepared.
The acid chloride prepared as described in the preparation of HD 1, supra, Part A (.01 mole) was added dropwise over 15 minutes to a mixture of benzyl 1-piperaz-.necarboxylate (2.4 g, 0.011 mole) and triethylamine (2.0 g, 0.02 mole) in chloroform (50 mL).
This mixture was stirred at room temperature overnight, and dichloromethane (300 mL) was added. The mixture was washed with 5% hydrochloric acid (2 x 100 mL), washed with dilute sodium carbonate solution (100 mL), washed with saturated sodium chloride solution (100 mL), dried over anhydrous magnesium sulfate solution, filtered, and the solvent removed on a rotary evaporator in vacuo. The filtrate was then chromatographed to give an oil, 4.3 g (78~ yield) which was used directly in the next step.
Part B: The protected amine of Part A (4.8 g, 0.008 mole) and 30-35% hydrogen bromide acetic acid solution (25 mL) was allowed to stir at room temperature for 1.5 hours. This mixture was then poured into diethyl ether (1 L), and the oil which separated was triturated with fresh portions of ether (3 x 1 L). The oil was dissolved in 10~ aqueou5 sodium hydroxide solution (pH=14) and the aqueous solution extracted with dichloromethane (4 x 100 mL). The combined organic solution was washed with saturated sodium chloride solution (150 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent removed in a rotary evaporator in vacuo. The filtrate solidifies to give a white solid (2.6 g, 77% yield). This - . . . - , .
- : ~
: - :
:
-11- 2062240 :
material was used directly in the next step.
Step 2: preparation of 3-{4-[4-(3-Carboxy-propionyl)-l-piperazinylcarbonyl~-butyl~-5,5-diphenyl-2,4-imidazoli-dinedione.
A mixture of the amine from Preparation 7 (2.1 g, 0.005 mole) and succinic anhydride (0.54 g, 0.0054 mole) in chloroform (15 mL) was heated for 30 minutes at 50-60C and allowed to stand at ambient temperature for 20 hours. Dichloromethane (150 mL) was added, and the mixture was washed with 5% hydrochloric acid (2 x 100 mL), saturated sodium chloride solution (100 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent removed on a rotary evaporator in vacuo to give a white solid, 2.5 g (95%) which was used directly in the next step.
Step 3: preparation of HD 2: 5,5-Diphenyl-3-{4-[4-(3-succinimidoxycarbonyl-propionyl)-l-piperazinylcarbonyl]-butyl}-2,4-imidazolidinedione.
, '':,' . . . : - ., :
,' N H
O N
~N~
I\,N~,O
N~O
O <~
A mixture of the acid from step 2 (1.56 g, 0.003 mole), N,N'-dicyclohexylcarbodiimide (0.64 g, O.003 mole), and N-hydroxysuccinimide (0.36 g, 0.003 mole) in chloroform (40 mL) was stirred at room temperature over the weekend. The mixture was filtered and the solvent removed from the filtrate on a rotary evaporator in vacuo to give 1.9 g (100~ yield). The solid was chromatographed, and the product fraction was dissolved in dichloromethane (200 mL), washed with dilute sodium carbonate solution (2 x 100 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent removed on a rotary evaporator to give a white solid which gives one spot on TLC. Anal. calc. for C32H3sNsOg: C, 62,23; H, 5.71; N, 11.34. Found: C, 59.07; H, 5.40; N, 10.45.
3. Preparation of HD 3, 5,5-Diphenyl-3-{4-[6-(3-. . - :
''" ' :: ' ' ' ' 2062240 ~
succinimidoxycarbonylpropionamido)hexylamino-carbonyl]butyl}-2,4-imidazolidinedione.
Step 1: preparation of 3-14-(6-Aminohexyl-aminocarbonyl)butyll-5,5-diphenyl-2,4-imidazolidinedione.
Part A: preparation of 3-[4-(6-Benzyloxy-carbonylaminohexylaminocarbonyl)butyl]-5,5-diphenyl-2,4-imidazolidinedione.
The acid chloride prepared as an intermediate in the preparation of HD 1 was treated with N-benzyl-ox~carbonyl-1,6-hexanediamine by the procedures described in step 1 in the preparation of HD 2, to give 7.5 g, 85% yield, of the protected amine.
Part B: The protected amine of Part A was treated with hydrobromic acid-acetic acid by the procedures of Step 1, Part B in the preparation of HD
2, to give the free amine which was used in step 3 without purification.
Step 3: preparation of 3-{4-[6-(3-Carboxy-propionamido)hexylaminocarbonyl]-butyl)-5,5-diphenyl-2,4-imida-zolidinedione.
This compound was prepared using the same procedures as step 2 of the HD 2 preparation. Anal.
Calc. for C30H3gN406: C, 65.44; H. 6.96; N, 10.17.
Found: C, 63.26, H, 7.01; N, 9.39.
Step 4: preparation of HD 3: 5,5-Diphenyl-3-l4-[6-(3-succinimidoxycarbonyl-propionamido)hexylaminocarbonyl]-butyl}-2,4-imidazolidinedione.
.: . -, . . , ~
;'~,'.
~N H
O N
o N~
NH o 0~0 N~O
¢J
This material was prepared using the procedures of step 3 in the preparation of HD 2 to give 2.6 g (80% yield~, mpt 133-134C. Anal. Calc. for C34H41NsOg: C, 63.05; H, 6.38; N. 10.81. Found: C, 62.91; H, 6.41; N, 10.69.
We have prepared new labeled hydantoin derivatives from the above described hydantoin derivatives that are useful in competitive immunoassays for drugs within the hydantoin group, particularly phenytoin. The labels are those commonly used in immunoassays having an amine or sulfhydryl group commonly used with analytes or analyte analogs in competitive immunoassays such as enzymes, visible dyes, - . . .
. " ~
. . : . ~ .
, . ~ , . .
leuco dyes, fluorescent dyes, radioactive materials, etc.
Preferred labels are enzymes such as alkaline phosphatase (ALP), glucose oxidase (GOD) and horseradish peroxidase (HRP), and the most preferred is HRP.
The labeled hydantoin derivatives are prepared with a new process comprising the steps of:
1) contacting a label having a nucleophilic group thereon such as an amine or sulfhydryl group, with an excess of a hydantoin active ester derivative described hereinbefore. Preferably both the hydantoin derivative and the label are dissolved in a water miscible organic solvent such as N,N-dimethylformamide, or mixture of solvent and water (buffered) before mixing together; and 2) removing the unused active ester and condensation by-products, preferably by dialysis.
The examples provided hereinafter illustrate the preparation of the new labeled hydantoin derivatives of this invention. The labeled derivatives were prepared using the phenytoin embodiments of the hydantoin derivatives prepared above, HD 1, HD 2 and HD 3.
~mDle 1 Preparation of an Amine Enriched HRP labeled hydantoin HD 1 (containing an extended linker, label AHRP-HD 1, Label A).
HD 1 was dissolved in 1.452 mL dry DMF
containing 10 mM 4'-hydroxyacetanilide (DMF 4'-HA).
Amine-enriched HRP was prepared as described in USSN 540,428, filed June 18, 1990 in the name of Susan J. Danielson and Donald P. Specht, entitled ~Amine-Enriched Proteins~, the contents of which are . . - - , - , -.
.. , ,.. . :: :
: . . . ~ -:. - . . :
206224~
expressly incorporated herein by reference. Briefly, dry HRP was dissolved in 0.1 M MES buffer, pH 5.5, to achieve a final concentration of 2.5 x 10-6 l (100 mg) in 10 mL of buffer (MES = 2-(N-morpholino)-ethanesulfonic acid). The protein concentration wasdetermined by A403 measurement using the conversion factor A403 1 mg/mL = 2.24. The HRP solution was combined with 1.5 x 10-3 mol (275 mg) of L-lysine ~~
monohydrochloride dissolved in 10 mL of 0.1 M MES
buffer at pH 5.5. A solution of freshly prepared 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 5 x 10-4 mol, 960 mL) in MES buffer was added. The container was capped and mixed overnight at room temperature. The reaction was dialyzed against 0.02 M MOPS buffer at pH 7.0 (3 L at 10C). The dialysis buffer was changed 3X. MOPS = 3-(N-morpholino)propanesulfonic acid.
Prior to reaction, a sample of the amine- -enriched HRP was exchanged from MOPS buffer into 0.1 M
EPPS buffer, pH 8.0, using 30,000 NMWL (nominal molecular weight limit cutoff) Centricells centrifugal ultrafilters. This sample was then diluted to obtain a solution with a final concentration of 10 mg/mL.
The amine-enriched HRP (AHRP) (1 mL) was combined with 500 ~L of a 10 mM solution of 4'-hydroxyacetanilide in dimethylformamide (DMF 4'-HA) with vortex mixing and was then placed in a 42C water bath. An HD 1 solution prepared by dissolving 21 mg of HD 1 in 1.452 mL of dry DMF 4'-HA solution (500 ~L) was added to the AHRP with vortex mixing so that the molar ratio of phenytoin/HRP was 50/1. The reaction was incubated for 1 hour in a 42C water bath with gentle shaking.
The reaction was placed in Spectrapor #2 ... . :. , . i . . . .: .
., : .:
2a62240 dialysis tubing along with an additional 0.5 mL of DMF
4'-HA/0.1 M EPPS (1:1) used to rinse the reaction container.
me reaction mixture was dialyzed as follows:
a) 1 L DMF 4'-HA/0.1 M EPPS. pH 8.0, (1:1) at 42C for 1 hr;
b) Dialysis condition a) was repeated 1 time c) 2 L 0.1 M EPPS, pH 8.0, containing 0.1%
BSA at 8C for 15 hrs d) 2 L of 0.1 M EPPS, pH 8.0, at 8C for 3 hours;
e ) 3 L of 0.04 M tris(hydroxymethyl)-aminomethane hydrochloride (Tris HCl)/0.15 M NaCl, PH
7.5, at 8C for 3 hours; and f) Dialysis condition e) was repeated 1 time for 3 hrs;
Following dialysis, merthiolate was added to 0.02~ as a preservative, and the AHRP-HD 1 was stored refrigerated.
mDliLz Preparation of an Amine Enriched HRP labeled HD 2 (containing an extended linker with an amide bond, label AHRP-HD 2, Label B).
HD 2 (15.5 mg) was dissolved in 1.031 mL dry DMF containing 10 mM 4'-hydroxyacetanilide (DMF 4'-HA).
A solution of AHRP (10 mg/mL, 1 mL in 0.1 M
EPPS, pH 8.0), prepared as described in Example 1, was combined with 500 ~L of DMF 4'-HA with vortex mixing and was then placed in a 42C water bath. The HD 2 solution described above (500 ~L) was added to the AHRP
with vortex mixing so that the molar ratio was 50/1.
The reaction was incubated for 1 hour in a 42C water bath with gentle shaking.
The reaction product was placed in .
:
.. . : .. . - :
.
, .. .
,, , . : . , . ~.
20~2240 Spectrapor #2 dialysis tubing and dialyzed as follows:
a) 1 L DMF 4'-HA/O.1 M EPPS, pH 8.0 (1:1) at 42C for 1 hr;
b) Dialysis condition a) was repeated 1 time;
c) 1.5 L 0.1 M EPPS, pH 8.0, containing 0.1% bovine serum albumin (BSA) at 8C for 1.5 hr;
d) 1.5 L 0.1 M EPPS, pH 8.0, at 8C for 18 hrs;
e) 1.5 L 0.04 M Tris HCl/0.15M NaCl, pH
7.5, at 8C for 2 hrs; and f ) Dialysis condition e) was repeated 1 time for 4 hrs.
Following dialysis, 0.02% merthiolate was added as a preservative. The labeled hydantoin derivative was stored in a refrigerator.
E~im~ l Preparation of AHRP-HD 3 (containing an extended linker with an amide bond label AHRP-HD 3, Label C).
HD 3 (9.2 mg) was dissolved in 1 mL dry DMF
containing 10 mM 4'-hydroxyacetanilide (DMF 4'-HA).
A solution of AHRP, prepared as described in Example 1, was dialyzed into 0.1 M EPPS buffer, pH 8Ø
The final concentration was determined to be 5.71 mg/ml.
The AHRP (1 mL) was combined with 500 ~L of DMF 4'-HA with vortex mixing and was then placed in a 42C water bath. The HD 3 solution described above (500 ~L) was added to the AHRP dropwise with vortex mixing so that the molar ratio of HD 3/AHRP was 50/1.
The reaction was incubated for 1 hour in a 42C water bath with gentle shaking. The reaction was placed in Spectrapor #2 dialysis tubing along with an additional 0.5 mL of DMF 4'-HA/0.1 M EPPS (1:1) used to rinse the - : . . . ..
- , , : ~ :: :. .:~ , : - - , -. -~ . : . . -reaction container.
The reaction was dialyzed as follows:
a) 1 L DMF 4'-HA/0.1 M EPPS, pH 8.0, (1:1) at 42C for 1 hour;
b) dialysis condition a) was repeated 1 time;
c) 1.5 L 0.1 M EPPS, pH 8.0, containing 0.1% BSA at 5C for 15 hours;
d) 1.5 L 0.1 M EPPS, pH 8.0, for 8 hours;
e) 2 L 0.02 M 3-morpholinopropanesulfonic acid (MOPS), pH 7Ø at 5C for 13 hours; and f) Dialysis condition e) was repeated 2 times.
Following dialysis, merthiolate was added to a concentration of 0.02% as a preservative, and the label was stored refrigerated.
Utility exam~les The following examples demonstrate the immunocompetence of the labels prepared in examples 1-3, supra.Exam~le 4 Immunocompetency of label AHRP-HD 1 (Label A).
In this example, the ability of several immobilized antibodies (DilAsg, DilAsg, DilAsl4, DilAsl6, and DilAs2l) to bind AHRP-HD 1 (label A) from Example 1) is examined.
(a) Polymer bead samples, each sample having one of the above-identified types of antibodies covalently bound thereto were prepared using methods and materials as described in USSN 081,206 filed August 3, 1987 ~published EPA 88 307172.2).
(b) The ability of the immobilized antibodies to bind AHRP-HD 1 (label A) was determined as follows:
.
'~ :, ' `: : , ~
:~ .
, ' Each of the various antibody beads were serially diluted with PBS containing 1% BSA to give concentrations between 500 and 0.50 nM antibody binding sites. The bead dilutions were mixed with equal volumes of the label at 10 x 10-11 M. Following a 1 hour incubation, the beads were pelleted by centrifugation. A sample (100 ~L) of the supernatant was mixed with 100 ~L of substrate (o-phenylenedi- '~
amine/H202). The rates of color development at 450 nm were compared with those of standards to calculate the amount of phenytoin-HRP label remaining in solution.
The amount of label bound to immobilized antibody at the highest antibody concentration tested (250 nM
binding sites) is reported.
~ Label Bound at 2,5,Q nM Antibody Bindin~ Sites Label A
(Extended Linker) DilAsg DilAsg 94 DilAS14 DilAs16 96 DilAs21 These results show that these antibody 52-2 recognize very well AHRP-HD 1 Labels (label A).
~m~le 5 Hydrolytic stability of AHRP-HD 2 (label B) This example illustrates the hydrolytic stability of a labeled phenytoin hydantoin derivative of the invention having an amide bond in the linking chain between the label and the phenytoin nucleus AHRP-HD 2, (label B).
Beads having immobilized Kallestad antibodies were prepared as described in USSN 081,206, filed August 3, 1987 (published EPA 88 307172.2).
AHRP-HD 2 (label B) was diluted to 1 x 10-10 .: : : . . ~ . ~. . , M in PBS containing 1% BSA adjusted to pH 7.3 or 8.5.
The label was incubated at room temperature for 6 days.
The label was tested for binding by immobilized antibody after 2 days and 6 days as follows:
Xallestad 52-2 antibody beads were serially diluted with PBS containing 1% BSA to give -concentrations between 500 and 0.50 nM antibody binding sites. The bead dilutions were mixed with equal volumes of labels at 10 x 10-11 M. Following a 1 hour incubation, the beads were pelleted by centrifugation.
A sample (100 uL) of the supernatant was mixed with 100 uL of substrate (o-phenylenediamine/H2O2). The rates were compared with those of standards to calculate the amount of label remaining in solution. The amount of -label bound to immobilized antibody at the highest antibody concentration tested (250 nM binding sites) is reported.
% La~el Bo~n~ at 250 n~ Antibodv ~i~din~ Site~
Label B
(Amide Bond) P~_7.3 H 8.5 0 days 100 --2 days 98 99 6 days 99 100 The results show that binding of AHRP-HD 2 (label B) containing the amide bond in the linkin~
chain showed no degradation over this time period.
This indicates that label B will resist degradation due to hydrolysis. Such hydrolysis could cause a shift in the assay response with time.
The invention has been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and 2~6~24D
scope of the invention.
- - : . . : , . . . . . .. .
(-CNH-) amides, , (c) hetero atoms selected from - _ O-, -S-, and -NR-; wherein R represents C1 to C6 alkyl (e.g. methyl, ethyl, propyl, butyl etc.); and (d) carbonyl, with the proviso that the linking group is other than a derivative of a saturated or unsaturated monocarboxylic acid having from to 2 to 12 carbon atoms.
The above labeled hydantoin derivatives includes those that conform to the structure:
(I) HN ~ N-R2 { -N' ~N-C-R~ ~ C Z R /'C R ~ C~ L b I
wherein R1 each independently represents hydrogen, alkyl of 1 to 10 carbon atoms , unsubstituted or substituted phenyl;
R2, R4, R5, and R6, each independently represents C1 to C1o alkylene or such alkylene groups interrupted with ester groups, amide groups, -O-, -S-, and -NR-;
R3 represents C1 to C3 alkylene;
Z represents -O-, -S-, and -NR-, wherein R
- - ~
- - . .
- : . . . -_5_ 2062240 represents hydrogen or C1 to C6 alkyl, e.g., methyl propyl and hexyl;
m is 0, 1, or 2;
n is 0, 1, or 2; and the total number of atoms comprised in m, n and R2 is 5 to 40;
LABEL is an enzyme;
and further provided that (i) at least one of the Rl groups is substituted or unsubstituted phenyl;
(ii) one of R4, R5, and R6 can be phenylene;
(iii) the bracketed components of structure I can appear therein in any order and (iv~ the linking group is other than a derivative of a saturated or unsaturated monocarboxylic acid having from to 2 to 12 carbon atoms.
Consistent with the above definitions Rl can represent hydrogen methyl, propyl, hexyl, decyl, unsubstituted phenyl or phenyl substituted with alkyl of about 1 to 6 carbon atoms, nitro, halogen, cyano, and alkoxy of about 1 to ~ carbon atoms;
R~, R4, R5, and R6, can each independently represent alkylene selected from ethylene, butylene, pentylene, octylene, or such alkylene interrupted with ester groups, amide groups, -O-, -S-, and -NR-;
R3 represents methylene, ethylene, or trimethylene;
Z represents -NR-, wherein R can represent hydrogen methyl propyl or hexyl; and hABEL represents an enzyme.
At least 85~ of the labeled hydantoin derivatives can be bound by immobilized hydantoin antibodies, particularly phenytoin antibodies, at antibody concentrations of interest. The labeled hydantoin derivatives with extended linkers, ., ... ~ ........ , , . . ~ . .
- ~ ' . ~ , , .
,: ~. .. . . ' , particularly those having amide bonds in the linking chain are similarly bound by all the immobilized antibody types tested in reducing this invention ~o practice. Also the derivatives having the amide in the extended linker are very stable against hydrolysis.
The novel labeled hydantoin derivatives used in the immunoassay of the present invention were prepared from novel hydantoin derivatives of copending U. S. Patent Application No. filed on even date herewith in the name of The novel hydantoin derivatives comprise:
(a) an active ester group,such as a succinimidoxycarbonyl;
(b) a hydantoin nucleus and (c) a linking chain linking the active ester group to the hydantoin nucleus;
wherein the linking chain is as previously defined.
More specifically, the preferred new hydantoin active esters of this invention are those conforming to the structure:
H N~N R 2{C-N' ~N - C - R ~EC - Z - R 5 - Z C - R~C - - N' 'R 7 wherein R1, R2, R3, R4, R5, R6, Z, m, and n and the provisos related thereto are as previously defined, and R7 is an ethylene or o-phenylene group.
.. ~ ~ , . ................................. .
. - : : . ,. , : .
.. ~. . ~ . . . . .
Several advantages are realized by use of the above hydantoin derivatives. First, it was found that the active esters of these hydantoin derivatives having short linking chains between the hydantoin nucleus and the active ester group were sufficiently reactive with HRP to prepare an acceptable enzyme label for use with -some immobilized antibodies. Derivatives with longer linker groups (R2 plus the bracketed groups) of 8 to 20 atoms between the active ester group and the hydantoin nucleus gave labels that could be bound by all immobilized antibodies tested. Linking chains in which each Z is -NR- which with the adjacent carbonyl forms an amide group, are particularly useful in that :
hydantoin derivatives containing such chains are more resistant to hydrolysis than chains wherein Z is -O- or -S- so that with the adjacent carbonyl it forms an ester group.
preparation of Hydantoin Derivative~
The described hydantoin derivatives can be made according to the following preparations in which phenytoin derivatives, a subclass of hydantoin compounds, are made.
1. Preparation of HD 1, 5,5-Diphenyl-3-{4-[2-(3-succinimidoxycarbonylpropionyloxy)ethylamino-carbonyl]butyl)hydantoin.
Step 1: preparation of 5,5-Diphenyl-3-[4-(2-hydroxyethylamino-carbonyl)butyl]-hydantoin.
Part A: First the Acid Chloride is prepared.
A mixture of 3-(4-carboxybutyl)-S,5-diphenyl-2,4-imidazolidinedione (3.52 g, 0.01 mole) thionyl chloride (20 mL), N,N-dimethylformamide (2 drops) and chloroform (50 mL) was stirred at room temperature for . .
.. .,. ., . ............... . . : . ..................... .
- - .... . . , .- -. ~ : - . -:
3 hours. The solvent was removed on a rotary evaporator in vacuo, and this product was used directly in the next Part B.
Part B: The Acid Chloride is reacted with Ethanolamine.
The acid chloride in chloroform (50 mL) was added dropwise over 15 minutes to a mixture of ethanolamine (1.2 g, 0.02 mole) and triethylamine (2.4 g, 0.024 mole) in chloroform (100 mL). The mixture was then heated to 60C for 2 hours and stirred to room temperature for 1 hour. The solution was then washed with 5% hydrochloric acid (2 x 100 mL), washed with saturated sodium bicarbonate solution (100 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent removed on a rotary evaporator. The filtrate was then chromatographed using an aluminum oxide column to give material (3.0 g) showing one spot on TLC. This material was used directly in the next preparation.
Step 2: preparation of 3-l4-[2-(3-Carboxy-propionyloxy)ethylaminocarbonyl]-butyl}-5,5-diphenylhydantoin.
The hydroxy compound of Step 1 (3.0 g, 0.0075 mole), succinic anhydride (1.0 g, 0.01 mole), and dimethylaminopyridine (0.9 g, 0.0075 mole) in chloroform (100 mL) were heated at 50-60C for 4 hours and allowed to cool to room temperature over the weekend.
Dichloromethane (300 mL) was added, and the mixture was washed with 5~ hydrochloric acid solution (3 x 100 mL), washed with saturated sodium chloride solution (100 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent removed to give a material that gave one spot on TLC.
Step 3: preparation of HD 1: 5,5-Diphenyl-3-.
: :: :
g {4-[2-(3-succinimidoxycarbonylpro-pionyloxy)ethylaminocarbonyl]butyl}-hydantoin.
~' ' .
` >=
o N
o N H ~
~0 - .
0~0 ~N~O
O=~
A mixture of the acid from Step 2 (3.0 g, 0.006 mole), N,N'-dicyclohexylcarbodiimide (1.5 g, 0.007 mole), and N-hydroxysuccinimide (0.7 g, 0.006 mole) in chloroform (80 mL) was stirred at room temperature for 20 hours. The mixture was filtered, and the filtrate was concentrated on a rotary evaporator in vacuo. The residue was then chromatographed using silica to give 1.3 g (40% yield).
Anal. calc. for C30H32N40g: C, 60.8; H, 5.44; N, 9.45.
Found: C 59.6; H, 5.51, N, 8.91 2. Preparation of HD 2: 5,5-Diphenyl-3-{4-[4-(3-succinimidoxycarbonylpropionyl)-l-piperazinyl-carbonyl]butyl}-2,4-imidazolidinedione Step 1: preparation of 5,5-Diphenyl-3-(1-piperazinylcarbonylbutyl)hydantoin.
Part A: First, 3-[4-(4-Benzyloxycarbonyl-piperazinylcarbonyl)butyl]-5,5-diphenyl-2,4-imidazolidinedione was prepared.
The acid chloride prepared as described in the preparation of HD 1, supra, Part A (.01 mole) was added dropwise over 15 minutes to a mixture of benzyl 1-piperaz-.necarboxylate (2.4 g, 0.011 mole) and triethylamine (2.0 g, 0.02 mole) in chloroform (50 mL).
This mixture was stirred at room temperature overnight, and dichloromethane (300 mL) was added. The mixture was washed with 5% hydrochloric acid (2 x 100 mL), washed with dilute sodium carbonate solution (100 mL), washed with saturated sodium chloride solution (100 mL), dried over anhydrous magnesium sulfate solution, filtered, and the solvent removed on a rotary evaporator in vacuo. The filtrate was then chromatographed to give an oil, 4.3 g (78~ yield) which was used directly in the next step.
Part B: The protected amine of Part A (4.8 g, 0.008 mole) and 30-35% hydrogen bromide acetic acid solution (25 mL) was allowed to stir at room temperature for 1.5 hours. This mixture was then poured into diethyl ether (1 L), and the oil which separated was triturated with fresh portions of ether (3 x 1 L). The oil was dissolved in 10~ aqueou5 sodium hydroxide solution (pH=14) and the aqueous solution extracted with dichloromethane (4 x 100 mL). The combined organic solution was washed with saturated sodium chloride solution (150 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent removed in a rotary evaporator in vacuo. The filtrate solidifies to give a white solid (2.6 g, 77% yield). This - . . . - , .
- : ~
: - :
:
-11- 2062240 :
material was used directly in the next step.
Step 2: preparation of 3-{4-[4-(3-Carboxy-propionyl)-l-piperazinylcarbonyl~-butyl~-5,5-diphenyl-2,4-imidazoli-dinedione.
A mixture of the amine from Preparation 7 (2.1 g, 0.005 mole) and succinic anhydride (0.54 g, 0.0054 mole) in chloroform (15 mL) was heated for 30 minutes at 50-60C and allowed to stand at ambient temperature for 20 hours. Dichloromethane (150 mL) was added, and the mixture was washed with 5% hydrochloric acid (2 x 100 mL), saturated sodium chloride solution (100 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent removed on a rotary evaporator in vacuo to give a white solid, 2.5 g (95%) which was used directly in the next step.
Step 3: preparation of HD 2: 5,5-Diphenyl-3-{4-[4-(3-succinimidoxycarbonyl-propionyl)-l-piperazinylcarbonyl]-butyl}-2,4-imidazolidinedione.
, '':,' . . . : - ., :
,' N H
O N
~N~
I\,N~,O
N~O
O <~
A mixture of the acid from step 2 (1.56 g, 0.003 mole), N,N'-dicyclohexylcarbodiimide (0.64 g, O.003 mole), and N-hydroxysuccinimide (0.36 g, 0.003 mole) in chloroform (40 mL) was stirred at room temperature over the weekend. The mixture was filtered and the solvent removed from the filtrate on a rotary evaporator in vacuo to give 1.9 g (100~ yield). The solid was chromatographed, and the product fraction was dissolved in dichloromethane (200 mL), washed with dilute sodium carbonate solution (2 x 100 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent removed on a rotary evaporator to give a white solid which gives one spot on TLC. Anal. calc. for C32H3sNsOg: C, 62,23; H, 5.71; N, 11.34. Found: C, 59.07; H, 5.40; N, 10.45.
3. Preparation of HD 3, 5,5-Diphenyl-3-{4-[6-(3-. . - :
''" ' :: ' ' ' ' 2062240 ~
succinimidoxycarbonylpropionamido)hexylamino-carbonyl]butyl}-2,4-imidazolidinedione.
Step 1: preparation of 3-14-(6-Aminohexyl-aminocarbonyl)butyll-5,5-diphenyl-2,4-imidazolidinedione.
Part A: preparation of 3-[4-(6-Benzyloxy-carbonylaminohexylaminocarbonyl)butyl]-5,5-diphenyl-2,4-imidazolidinedione.
The acid chloride prepared as an intermediate in the preparation of HD 1 was treated with N-benzyl-ox~carbonyl-1,6-hexanediamine by the procedures described in step 1 in the preparation of HD 2, to give 7.5 g, 85% yield, of the protected amine.
Part B: The protected amine of Part A was treated with hydrobromic acid-acetic acid by the procedures of Step 1, Part B in the preparation of HD
2, to give the free amine which was used in step 3 without purification.
Step 3: preparation of 3-{4-[6-(3-Carboxy-propionamido)hexylaminocarbonyl]-butyl)-5,5-diphenyl-2,4-imida-zolidinedione.
This compound was prepared using the same procedures as step 2 of the HD 2 preparation. Anal.
Calc. for C30H3gN406: C, 65.44; H. 6.96; N, 10.17.
Found: C, 63.26, H, 7.01; N, 9.39.
Step 4: preparation of HD 3: 5,5-Diphenyl-3-l4-[6-(3-succinimidoxycarbonyl-propionamido)hexylaminocarbonyl]-butyl}-2,4-imidazolidinedione.
.: . -, . . , ~
;'~,'.
~N H
O N
o N~
NH o 0~0 N~O
¢J
This material was prepared using the procedures of step 3 in the preparation of HD 2 to give 2.6 g (80% yield~, mpt 133-134C. Anal. Calc. for C34H41NsOg: C, 63.05; H, 6.38; N. 10.81. Found: C, 62.91; H, 6.41; N, 10.69.
We have prepared new labeled hydantoin derivatives from the above described hydantoin derivatives that are useful in competitive immunoassays for drugs within the hydantoin group, particularly phenytoin. The labels are those commonly used in immunoassays having an amine or sulfhydryl group commonly used with analytes or analyte analogs in competitive immunoassays such as enzymes, visible dyes, - . . .
. " ~
. . : . ~ .
, . ~ , . .
leuco dyes, fluorescent dyes, radioactive materials, etc.
Preferred labels are enzymes such as alkaline phosphatase (ALP), glucose oxidase (GOD) and horseradish peroxidase (HRP), and the most preferred is HRP.
The labeled hydantoin derivatives are prepared with a new process comprising the steps of:
1) contacting a label having a nucleophilic group thereon such as an amine or sulfhydryl group, with an excess of a hydantoin active ester derivative described hereinbefore. Preferably both the hydantoin derivative and the label are dissolved in a water miscible organic solvent such as N,N-dimethylformamide, or mixture of solvent and water (buffered) before mixing together; and 2) removing the unused active ester and condensation by-products, preferably by dialysis.
The examples provided hereinafter illustrate the preparation of the new labeled hydantoin derivatives of this invention. The labeled derivatives were prepared using the phenytoin embodiments of the hydantoin derivatives prepared above, HD 1, HD 2 and HD 3.
~mDle 1 Preparation of an Amine Enriched HRP labeled hydantoin HD 1 (containing an extended linker, label AHRP-HD 1, Label A).
HD 1 was dissolved in 1.452 mL dry DMF
containing 10 mM 4'-hydroxyacetanilide (DMF 4'-HA).
Amine-enriched HRP was prepared as described in USSN 540,428, filed June 18, 1990 in the name of Susan J. Danielson and Donald P. Specht, entitled ~Amine-Enriched Proteins~, the contents of which are . . - - , - , -.
.. , ,.. . :: :
: . . . ~ -:. - . . :
206224~
expressly incorporated herein by reference. Briefly, dry HRP was dissolved in 0.1 M MES buffer, pH 5.5, to achieve a final concentration of 2.5 x 10-6 l (100 mg) in 10 mL of buffer (MES = 2-(N-morpholino)-ethanesulfonic acid). The protein concentration wasdetermined by A403 measurement using the conversion factor A403 1 mg/mL = 2.24. The HRP solution was combined with 1.5 x 10-3 mol (275 mg) of L-lysine ~~
monohydrochloride dissolved in 10 mL of 0.1 M MES
buffer at pH 5.5. A solution of freshly prepared 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 5 x 10-4 mol, 960 mL) in MES buffer was added. The container was capped and mixed overnight at room temperature. The reaction was dialyzed against 0.02 M MOPS buffer at pH 7.0 (3 L at 10C). The dialysis buffer was changed 3X. MOPS = 3-(N-morpholino)propanesulfonic acid.
Prior to reaction, a sample of the amine- -enriched HRP was exchanged from MOPS buffer into 0.1 M
EPPS buffer, pH 8.0, using 30,000 NMWL (nominal molecular weight limit cutoff) Centricells centrifugal ultrafilters. This sample was then diluted to obtain a solution with a final concentration of 10 mg/mL.
The amine-enriched HRP (AHRP) (1 mL) was combined with 500 ~L of a 10 mM solution of 4'-hydroxyacetanilide in dimethylformamide (DMF 4'-HA) with vortex mixing and was then placed in a 42C water bath. An HD 1 solution prepared by dissolving 21 mg of HD 1 in 1.452 mL of dry DMF 4'-HA solution (500 ~L) was added to the AHRP with vortex mixing so that the molar ratio of phenytoin/HRP was 50/1. The reaction was incubated for 1 hour in a 42C water bath with gentle shaking.
The reaction was placed in Spectrapor #2 ... . :. , . i . . . .: .
., : .:
2a62240 dialysis tubing along with an additional 0.5 mL of DMF
4'-HA/0.1 M EPPS (1:1) used to rinse the reaction container.
me reaction mixture was dialyzed as follows:
a) 1 L DMF 4'-HA/0.1 M EPPS. pH 8.0, (1:1) at 42C for 1 hr;
b) Dialysis condition a) was repeated 1 time c) 2 L 0.1 M EPPS, pH 8.0, containing 0.1%
BSA at 8C for 15 hrs d) 2 L of 0.1 M EPPS, pH 8.0, at 8C for 3 hours;
e ) 3 L of 0.04 M tris(hydroxymethyl)-aminomethane hydrochloride (Tris HCl)/0.15 M NaCl, PH
7.5, at 8C for 3 hours; and f) Dialysis condition e) was repeated 1 time for 3 hrs;
Following dialysis, merthiolate was added to 0.02~ as a preservative, and the AHRP-HD 1 was stored refrigerated.
mDliLz Preparation of an Amine Enriched HRP labeled HD 2 (containing an extended linker with an amide bond, label AHRP-HD 2, Label B).
HD 2 (15.5 mg) was dissolved in 1.031 mL dry DMF containing 10 mM 4'-hydroxyacetanilide (DMF 4'-HA).
A solution of AHRP (10 mg/mL, 1 mL in 0.1 M
EPPS, pH 8.0), prepared as described in Example 1, was combined with 500 ~L of DMF 4'-HA with vortex mixing and was then placed in a 42C water bath. The HD 2 solution described above (500 ~L) was added to the AHRP
with vortex mixing so that the molar ratio was 50/1.
The reaction was incubated for 1 hour in a 42C water bath with gentle shaking.
The reaction product was placed in .
:
.. . : .. . - :
.
, .. .
,, , . : . , . ~.
20~2240 Spectrapor #2 dialysis tubing and dialyzed as follows:
a) 1 L DMF 4'-HA/O.1 M EPPS, pH 8.0 (1:1) at 42C for 1 hr;
b) Dialysis condition a) was repeated 1 time;
c) 1.5 L 0.1 M EPPS, pH 8.0, containing 0.1% bovine serum albumin (BSA) at 8C for 1.5 hr;
d) 1.5 L 0.1 M EPPS, pH 8.0, at 8C for 18 hrs;
e) 1.5 L 0.04 M Tris HCl/0.15M NaCl, pH
7.5, at 8C for 2 hrs; and f ) Dialysis condition e) was repeated 1 time for 4 hrs.
Following dialysis, 0.02% merthiolate was added as a preservative. The labeled hydantoin derivative was stored in a refrigerator.
E~im~ l Preparation of AHRP-HD 3 (containing an extended linker with an amide bond label AHRP-HD 3, Label C).
HD 3 (9.2 mg) was dissolved in 1 mL dry DMF
containing 10 mM 4'-hydroxyacetanilide (DMF 4'-HA).
A solution of AHRP, prepared as described in Example 1, was dialyzed into 0.1 M EPPS buffer, pH 8Ø
The final concentration was determined to be 5.71 mg/ml.
The AHRP (1 mL) was combined with 500 ~L of DMF 4'-HA with vortex mixing and was then placed in a 42C water bath. The HD 3 solution described above (500 ~L) was added to the AHRP dropwise with vortex mixing so that the molar ratio of HD 3/AHRP was 50/1.
The reaction was incubated for 1 hour in a 42C water bath with gentle shaking. The reaction was placed in Spectrapor #2 dialysis tubing along with an additional 0.5 mL of DMF 4'-HA/0.1 M EPPS (1:1) used to rinse the - : . . . ..
- , , : ~ :: :. .:~ , : - - , -. -~ . : . . -reaction container.
The reaction was dialyzed as follows:
a) 1 L DMF 4'-HA/0.1 M EPPS, pH 8.0, (1:1) at 42C for 1 hour;
b) dialysis condition a) was repeated 1 time;
c) 1.5 L 0.1 M EPPS, pH 8.0, containing 0.1% BSA at 5C for 15 hours;
d) 1.5 L 0.1 M EPPS, pH 8.0, for 8 hours;
e) 2 L 0.02 M 3-morpholinopropanesulfonic acid (MOPS), pH 7Ø at 5C for 13 hours; and f) Dialysis condition e) was repeated 2 times.
Following dialysis, merthiolate was added to a concentration of 0.02% as a preservative, and the label was stored refrigerated.
Utility exam~les The following examples demonstrate the immunocompetence of the labels prepared in examples 1-3, supra.Exam~le 4 Immunocompetency of label AHRP-HD 1 (Label A).
In this example, the ability of several immobilized antibodies (DilAsg, DilAsg, DilAsl4, DilAsl6, and DilAs2l) to bind AHRP-HD 1 (label A) from Example 1) is examined.
(a) Polymer bead samples, each sample having one of the above-identified types of antibodies covalently bound thereto were prepared using methods and materials as described in USSN 081,206 filed August 3, 1987 ~published EPA 88 307172.2).
(b) The ability of the immobilized antibodies to bind AHRP-HD 1 (label A) was determined as follows:
.
'~ :, ' `: : , ~
:~ .
, ' Each of the various antibody beads were serially diluted with PBS containing 1% BSA to give concentrations between 500 and 0.50 nM antibody binding sites. The bead dilutions were mixed with equal volumes of the label at 10 x 10-11 M. Following a 1 hour incubation, the beads were pelleted by centrifugation. A sample (100 ~L) of the supernatant was mixed with 100 ~L of substrate (o-phenylenedi- '~
amine/H202). The rates of color development at 450 nm were compared with those of standards to calculate the amount of phenytoin-HRP label remaining in solution.
The amount of label bound to immobilized antibody at the highest antibody concentration tested (250 nM
binding sites) is reported.
~ Label Bound at 2,5,Q nM Antibody Bindin~ Sites Label A
(Extended Linker) DilAsg DilAsg 94 DilAS14 DilAs16 96 DilAs21 These results show that these antibody 52-2 recognize very well AHRP-HD 1 Labels (label A).
~m~le 5 Hydrolytic stability of AHRP-HD 2 (label B) This example illustrates the hydrolytic stability of a labeled phenytoin hydantoin derivative of the invention having an amide bond in the linking chain between the label and the phenytoin nucleus AHRP-HD 2, (label B).
Beads having immobilized Kallestad antibodies were prepared as described in USSN 081,206, filed August 3, 1987 (published EPA 88 307172.2).
AHRP-HD 2 (label B) was diluted to 1 x 10-10 .: : : . . ~ . ~. . , M in PBS containing 1% BSA adjusted to pH 7.3 or 8.5.
The label was incubated at room temperature for 6 days.
The label was tested for binding by immobilized antibody after 2 days and 6 days as follows:
Xallestad 52-2 antibody beads were serially diluted with PBS containing 1% BSA to give -concentrations between 500 and 0.50 nM antibody binding sites. The bead dilutions were mixed with equal volumes of labels at 10 x 10-11 M. Following a 1 hour incubation, the beads were pelleted by centrifugation.
A sample (100 uL) of the supernatant was mixed with 100 uL of substrate (o-phenylenediamine/H2O2). The rates were compared with those of standards to calculate the amount of label remaining in solution. The amount of -label bound to immobilized antibody at the highest antibody concentration tested (250 nM binding sites) is reported.
% La~el Bo~n~ at 250 n~ Antibodv ~i~din~ Site~
Label B
(Amide Bond) P~_7.3 H 8.5 0 days 100 --2 days 98 99 6 days 99 100 The results show that binding of AHRP-HD 2 (label B) containing the amide bond in the linkin~
chain showed no degradation over this time period.
This indicates that label B will resist degradation due to hydrolysis. Such hydrolysis could cause a shift in the assay response with time.
The invention has been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and 2~6~24D
scope of the invention.
- - : . . : , . . . . . .. .
Claims (9)
1. Labeled hydantoin derivatives comprising:
(A) a label, of the type used in immunoassays, having an amine or sulfhydryl group;
(B) a hydantoin nucleus and (C) a linking chain linking the 3-position of the hydantoin nucleus to the label through a carbonyl bridge; wherein the linking chain has about 5 to about 40 atoms consisting of (1) C1 to C10 alkylene groups, (2) phenylene groups, and (3) 5 to 7 membered heterocyclic rings joined to the linking group through ring nitrogen atoms bonded to each other through chemical groups selected from (a) esters, including thioesters , where Z is O or S;
(b) amides, , (c) hetero atoms selected from -O-, -S-, and -NR-; wherein R represents C1 to C6 alkyl; and (d) carbonyl, with the proviso that the linking group is other than a derivative of a saturated or unsaturated monocarboxylic acid having from to 2 to 12 carbon atoms.
(A) a label, of the type used in immunoassays, having an amine or sulfhydryl group;
(B) a hydantoin nucleus and (C) a linking chain linking the 3-position of the hydantoin nucleus to the label through a carbonyl bridge; wherein the linking chain has about 5 to about 40 atoms consisting of (1) C1 to C10 alkylene groups, (2) phenylene groups, and (3) 5 to 7 membered heterocyclic rings joined to the linking group through ring nitrogen atoms bonded to each other through chemical groups selected from (a) esters, including thioesters , where Z is O or S;
(b) amides, , (c) hetero atoms selected from -O-, -S-, and -NR-; wherein R represents C1 to C6 alkyl; and (d) carbonyl, with the proviso that the linking group is other than a derivative of a saturated or unsaturated monocarboxylic acid having from to 2 to 12 carbon atoms.
2. The labeled hydantoin derivatives of claim 1 wherein the linking chain includes a heterocyclic group selected from 1,4-piperazinylene, 2,5-dimethyl-1,4-piperazinylene, 1,3-imidazolidinylene, and 1,3-hexahydrodiazepinylene.
3. The labeled hydantoin derivatives of claim 1 conforming to the structure:
(I) wherein R1 each independently represents hydrogen, alkyl of 1 to 10 carbon atoms, unsubstituted or substituted phenyl;
R2, R4, R5, and R6, each independently represents C1 to C10 alkylene or such alkylene groups interrupted with ester groups, amide groups, -O-, -S-, and -NR-;
R3 represents C1 to C3 alkylene;
Z represents -O-, -S-, and -NR-, wherein R
represents hydrogen or C1 to C6 alkyl;
m is 0, 1, or 2;
n is 0, 1, or 2; and the total number of atoms comprised in m, n and R2 is 5 to 40;
LABEL is an enzyme;
and further provided that (i) at least one of the R1 groups is substituted or unsubstituted phenyl;
(ii) one of R4, R5, and R6 can be phenylene;
(iii) the bracketed components of structure I can appear therein in any order and (iv) the linking group is other than only a derivative of a saturated or unsaturated monocarboxylic acid having from to 2 to 12 carbon atoms.
(I) wherein R1 each independently represents hydrogen, alkyl of 1 to 10 carbon atoms, unsubstituted or substituted phenyl;
R2, R4, R5, and R6, each independently represents C1 to C10 alkylene or such alkylene groups interrupted with ester groups, amide groups, -O-, -S-, and -NR-;
R3 represents C1 to C3 alkylene;
Z represents -O-, -S-, and -NR-, wherein R
represents hydrogen or C1 to C6 alkyl;
m is 0, 1, or 2;
n is 0, 1, or 2; and the total number of atoms comprised in m, n and R2 is 5 to 40;
LABEL is an enzyme;
and further provided that (i) at least one of the R1 groups is substituted or unsubstituted phenyl;
(ii) one of R4, R5, and R6 can be phenylene;
(iii) the bracketed components of structure I can appear therein in any order and (iv) the linking group is other than only a derivative of a saturated or unsaturated monocarboxylic acid having from to 2 to 12 carbon atoms.
4. The labeled hydantoin derivative of claim 3 wherein:
R1 represents hydrogen, methyl, propyl, hexyl, decyl, unsubstituted phenyl or phenyl substituted with alkyl of about 1 to 6 carbon atoms, nitro, halogen, cyano, and alkoxy of about 1 to 6 carbon atoms;
R2, R4, R5, and R6, each independently represent alkylene selected from ethylene, trimethylene, butylene, pentylene, octylene or such alkylene interrupted with ester groups, amide groups, O-, -S-, and -NR-;
R3 represents methylene, ethylene, or trimethylene;
Z represents -NR-, wherein R represents hydrogen methyl, propyl, or hexyl; and LABEL represents an enzyme.
R1 represents hydrogen, methyl, propyl, hexyl, decyl, unsubstituted phenyl or phenyl substituted with alkyl of about 1 to 6 carbon atoms, nitro, halogen, cyano, and alkoxy of about 1 to 6 carbon atoms;
R2, R4, R5, and R6, each independently represent alkylene selected from ethylene, trimethylene, butylene, pentylene, octylene or such alkylene interrupted with ester groups, amide groups, O-, -S-, and -NR-;
R3 represents methylene, ethylene, or trimethylene;
Z represents -NR-, wherein R represents hydrogen methyl, propyl, or hexyl; and LABEL represents an enzyme.
5. The labeled hydantoin derivative of claim 1 wherein the label is selected from horseradish peroxidase (HRP) or amine enriched horseradish peroxidase (AHRP); the hydantoin is phenytoin and the linking group is selected from:
tetramethylenecarbonyliminohexamethylene-iminocarbonylethylenecarbonyl, tetramethylenecarbonyl-1,4-piperazinylene-carbonylethylenecarbonyl, and tetramethylenecarbonyliminoethyleneoxy-carbonylethylenecarbonyl.
tetramethylenecarbonyliminohexamethylene-iminocarbonylethylenecarbonyl, tetramethylenecarbonyl-1,4-piperazinylene-carbonylethylenecarbonyl, and tetramethylenecarbonyliminoethyleneoxy-carbonylethylenecarbonyl.
6. A method of making the labeled hydantoin derivatives of any one of the preceding claims comprising the steps of:
(1) contacting a label having an amine or sulfhydryl group, with an excess of a hydantoin derivative comprising:
(a) an active ester group,such as succinimidoxycarbonyl;
(b) a hydantoin nucleus and (c) a linking chain linking the active ester group to the hydantoin nucleus;
wherein the linking chain is defined in claim 1 or 2, and (2) removing the unused active ester and condensation by-products, preferably by dialysis.
(1) contacting a label having an amine or sulfhydryl group, with an excess of a hydantoin derivative comprising:
(a) an active ester group,such as succinimidoxycarbonyl;
(b) a hydantoin nucleus and (c) a linking chain linking the active ester group to the hydantoin nucleus;
wherein the linking chain is defined in claim 1 or 2, and (2) removing the unused active ester and condensation by-products, preferably by dialysis.
7. The method of claim 6 wherein the hydantoin derivative and the label are dissolved in a water-miscible organic solvent or mixture of solvent and water before being contacted together.
8. A labeled hydantoin derivative having a linking chain of 4 to 18 atoms.
9. A labeled hydantoin derivative according to claims 2, 3, 4, or 5 wherein Z is -NR- .
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US71233091A | 1991-06-07 | 1991-06-07 | |
| US712,330 | 1991-06-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2062240A1 true CA2062240A1 (en) | 1992-12-08 |
Family
ID=24861668
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002062240A Abandoned CA2062240A1 (en) | 1991-06-07 | 1992-03-03 | Labeled hydantoin derivatives for immunoassays |
Country Status (2)
| Country | Link |
|---|---|
| US (1) | US5298403A (en) |
| CA (1) | CA2062240A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100361933B1 (en) * | 1993-09-08 | 2003-02-14 | 라 졸라 파마슈티칼 컴파니 | Chemically defined nonpolymeric bonds form the platform molecule and its conjugate |
| US6458953B1 (en) * | 1998-12-09 | 2002-10-01 | La Jolla Pharmaceutical Company | Valency platform molecules comprising carbamate linkages |
| JP2003535208A (en) * | 2000-06-08 | 2003-11-25 | ラ ホヤ ファーマシューティカル カンパニー | Multivalent platform molecules containing high molecular weight polyethylene oxide |
| NZ511705A (en) * | 2001-05-14 | 2004-03-26 | Horticulture & Food Res Inst | Methods and rapid immunoassay device for detecting progesterone and other steroids |
| WO2008054827A2 (en) * | 2006-11-03 | 2008-05-08 | Pharmacyclics, Inc. | Bruton's tyrosine kinase activity probe and method of using |
| US8133743B2 (en) * | 2007-10-19 | 2012-03-13 | Roche Diagnostics Operations, Inc. | Phenobarbital derivatives useful in immunoassay |
| CN109212200A (en) * | 2017-06-30 | 2019-01-15 | 北京维德维康生物技术有限公司 | A kind of Nicarbazin haptens, artificial antigen and the preparation method and application thereof |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3905871A (en) * | 1971-05-14 | 1975-09-16 | Syva Co | Lactam conjugates to enzymes |
| US4065354A (en) * | 1974-10-10 | 1977-12-27 | Syva Company | Lysozyme conjugates for enzyme immunoassays |
| US4092479A (en) * | 1976-04-05 | 1978-05-30 | Baxter Travenol Laboratories, Inc. | Labeled 5,5-diphenylhydantoin derivatives for radioimmunoassay |
| US4213964A (en) * | 1978-12-07 | 1980-07-22 | Miles Laboratories, Inc. | Diphenylhydantoin antibodies |
| US4656252A (en) * | 1980-01-24 | 1987-04-07 | Giese Roger W | Amidobiotin compounds useful in a avidin-biotin multiple layering process |
| US4348524A (en) * | 1980-03-28 | 1982-09-07 | Ciba-Geigy Corporation | Amide derivatives of polyalkylpiperidines |
| GB8400653D0 (en) * | 1984-01-11 | 1984-02-15 | Beecham Group Plc | Conjugates |
| US4752568A (en) * | 1986-01-13 | 1988-06-21 | Eastman Kodak Company | Labeled hydantoin conjugate and its use in analytical element and immunoassays |
| US5002883A (en) * | 1987-10-30 | 1991-03-26 | Abbott Laboratories | Covalent attachment of antibodies and antigens to solid phases using extended length heterobifunctional coupling agents |
| US5024834A (en) * | 1988-07-12 | 1991-06-18 | Cetus Corporation | Thioether linked immunotoxin conjugates |
-
1992
- 1992-03-03 CA CA002062240A patent/CA2062240A1/en not_active Abandoned
- 1992-03-16 US US07/851,439 patent/US5298403A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| US5298403A (en) | 1994-03-29 |
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| Date | Code | Title | Description |
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| EEER | Examination request | ||
| FZDE | Discontinued |