Note: Descriptions are shown in the official language in which they were submitted.
2062239
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 reaction5 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
' .
2062239
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:
: - , , . , :-
- , . ~ .
2062239
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
.. . . . . . . . . . .
, . ~ :. -, . , . ,, .. . - , ,, , . , , -. - -
2062239
--4--
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
.
-6-
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
.;. .. . ~ , . , . ., . , . ... , . . ~ . . , .:
" ~
2062239
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
--10--
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 :
-13-
~,.
~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
-14-
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.
; , , :. , . ~ :
: . .: ~ ;
- : , .
~ . ~ : ~ -: . :
