CA2062239A1 - Hydantoin derivatives for competitive enzyme immunoassays - Google Patents

Abstract

HYDANTOIN DERIVATIVES FOR COMPETITIVE ENZYME
IMMUNOASSAYS
Abstract of the Disclosure The invention is directed to new hydantoin derivatives comprising:
(A) an active ester group;
(B) a hydantoin nucleus and (C) a linking chain linking the 3-position of the hydantoin nucleus to the active ester group.

Description

HYDANTOIN DERIVATIVES FOR COMPETITIVE ENZYME
IMMUNOASSAYS
Field of the Invention This invention relates to clinical chemistry.
More specifically the invention relates to hydantoin derivatives for use in immunoassays.
Background of the Invention Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as analytical techniques in clinical chemistry. Because 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 concentrations of the ligand can be determined from the measured signal of either the bound or unbound (i.e. free) labeled ligand. The reaction 5 proceeds as follows:
ligand + labeled ligand + receptor -~ligand-receptor + labeled ligand-receptor.
Conventional labels include radioactive tags, enzymes, chromophores, fluorophores, stable free radicals, and enzyme cofactors, inhibitors and allosteric effectors.
Consistent with the foregoing an immunoassay for a hydantoin derivative, such as phenytoin, in serum can be based on competition of an enzyme labeled ' .

derivative of a hydantoin (referred to hereafter as LDH) with the hydantoin in the patient serum for immobilized antibody binding sites.
Specific requirements for the LDH include~
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, ~ 25 gave adequate enzyme labeled phenytoin derivatives for ; conducting effective competitive immunoassays in the desired format:

: - , , . , :-- , . ~ .

Q

R ~O
HN~N-CH2)nC
ll OH
O
I.
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 required to give a readable signal.
It would be highly desirable to provide new 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 novel hydantoin derivatives comprising:
(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 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-piperazin-ylene-1,3-imidazolidinylene, and 1,3-hexahydrodiazepinylene group) joined to the linkinggroup through ring nitrogen atoms bonded to each other through chemical groups selected from (a) esters, O
Il ., including thioesters , where Z is O or S; (b) O
Il .
( - C N H - ) amides, , (c) hetero atoms selected from -O-, -S-, and -NR R represents Cl 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.
More specifically, the preferred new hydantoin active esters of this invention are those conforming to the structure:

~ ~01 ~ ,o, ~01 0 ~0 Il .
:.
whereln R1 each independently represents hydrogen, alkyl of 1 to 10 carbon atoms , unsubstituted or 20622~9 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;
R7 is an ethylene or o-phenylene group;
Z represents -O-, -S-, and -NR-, wherein R
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;
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 a 20 derivative of a saturated or unsaturated monocarboxylic -acid having from to 2 to 12 carbon atoms.
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 20~22~9 .

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.
n~ OF TH~ IN~ION
The described hydantoin derivatives can be made according to the following preparations in which phenytoin derivatives, a species of hydantoin compounds, are made.
Example 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)-5,5-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 ; 30 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-{4-[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-{4-[2-(3-succinimidoxycarbonylpro pionyloxy)ethylaminocarbonyl]butyl}-hydantoin.

.- . . - , , . , .;. . - . . . .
... , . - . : . : :

20622~9 ~, N H

O N

o NH

~0 ~ 1 ~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 C30H32N409: C, 60.8; H, 5.44; N, 9.45.
Found: C 59.6; H, 5.51, N, 8.91 Example 2: Preparation of HD 2: 5,5-Diphenyl-3-{4-[4-(3-succinimidoxycarbonylpropionyl)-1-piperazinyl-carbonyl]butyl}-2,4-imidazolidinedione Step 1: preparation of 5,5-Diphenyl-3-(1-piperazinylcarbonylbutyl)hydantoin.
Part A: First, 3-[4-(4-Benzyloxycarbonyl-r ..
,: ......... ~ , . .

:........... . . , - ~ ~ .

.... .

2~22~9 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 l.01 mole) was added dropwise over 15 minutes to a mixture of benzyl l-piperazinecarboxylate (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% a~iueous 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 material was used directly in the next step.
Step 2: preparation of 3-{4-[4-(3-Carboxy-propionyl)-l-piperazinylcarbonyl]-2~62239 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.

.. . .

, ,.~...........

- : ~
:

20~2239 OE~ >_o ~.
o N

~N~
~,N~O

N ~
O--~J ' A mixture of the acid from step 2 (1.56 g, 0.003 mole), N,N'-dicyclohexylcarbodiimide (0.64 g, 0.003 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.
Example 3: Preparation of HD 3, 5,5-Diphenyl-3-{4-[6-~' . ' . ' ,~ , - - ' ' . ' 20~2239 (3-succinimidoxycarbonylpropionamido)hexylamino-carbonyl]butyl)-2,4-imidazolidinedione.
Step 1: preparation of 3-[4-(6-Aminohexyl-aminocarbonyl)butyl]-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-oxycarbonyl-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.
25 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-{4-[6-(3-succinimidoxycarbonyl-propionamido)hexylaminocarbonyl]-butyl}-2,4-imidazolidinedione.

.. ~ : ., .

2062239 :

~,.

~N H ~ .

o N

o N H :

N~ o ~1 N~O -:
O=~

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 C34H4lNsOg: 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, . . : . .. : ........ . - , :

20622~9 leuco dyes, fluorescent dyes, radioactive materials, etc.
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 scope of the invention.

; , , :. , . ~ :

: . .: ~ ;
- : , .

~ . ~ : ~ -: . :

Claims (7)

1. New hydantoin derivatives comprising:
(A) an active ester group;
(B) a hydantoin nucleus and (C) a linking chain linking the 3-position of the hydantoin nucleus to the active ester group; 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 hydantoins of claim 1 wherein the linking chain includes a heterocyclic group selected from 1,4-piperazinylene, 2,5-dimethyl-1,4-piper-azinylene, 1,3-imidazolidinylene, and 1,3-hexa-hydrodiazepinylene.

3. The hydantoin of claim 1 wherein the acive ester group is succinimidoxycarbonyl.

4. The hydantoins of claim 1 conforming to the structure:
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;
R7 is an ethylene or o-phenylene;
Z represents -O-, -S-, and -NR-, wherein R
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;
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 a derivative of a saturated or unsaturated monocarboxylic acid having from to 2 to 12 carbon atoms.

5. The hydantoin derivatives of claim 4 according to structure I wherein R1 represents phenyl;
R2 is butylene;
R3, R4, R5 and R6 represent ethylene or hexylene.

6. Hydantoin derivatives according to any one of the preceding claims wherein the active ester group is succinimidoxycarbonyl.

7. Hydantoin derivative according to claim 3, structure I selected from 5,5-Diphenyl-3-{4-[4-(3-succinimidoxy-carbonylpropionyl)-1-piperazinylcarbonyl]butyl}-2,4-imidazolidinedione;
5,5-Diphenyl-3-{4-[2-(3-succinimidoxy-carbonylpropionyloxy)ethylaminocarbonyl]butyl)-2,4-imidazolidinedione and 5,5-Diphenyl-3-{4-[6-(3-succinimidoxy-carbonylpropionamido)hexylaminocarbonyl]butyl}-2,4-imidazolidinedione.