Competition binding assay for detecting P2YADP receptor ligands

Details Competition binding assay for detecting P2YADP receptor ligands Competition binding assay for detecting P2YADP receptor ligands

2000-01-06

2002-09-24

Ceperley, Mary E. (Department: 1641)

Chemistry: analytical and immunological testing

Biospecific ligand binding assay

Utilizing isolate of tissue or organ as binding agent

C435S007100, C436S501000, C436S804000, C436S815000, C544S254000

Reexamination Certificate

active

06455322

ABSTRACT:

It is known that ADP (adenosine-5′-diphosphate) plays a pivotal role in terms of platelet function by causing adhesion, degranulation, shape change and aggregation of platelets via interaction with cell surface P2 receptors. It is the P2Y
ADP
receptor (formerly known as P
2T
) that is primarily involved in mediating platelet aggregation, which is an as yet uncloned G-protein linked receptor. The pharmacological characteristics of this receptor have been described, for example, in the references by Humphries et al., Br. J. Pharmacology, (1994), 113, 1057-1063, and Fagura et al., Br. J. Pharmacology, (1998), 124, 157-164.
Compounds having antagonist activity at the P2Y
ADP
receptor are known, for example, from WO 98/28300, WO 97/03084, WO 94/18216 and EP-A-508 687 and are useful as anti-thrombotic agents in the treatment or prophylaxis of diseases such as unstable angina, coronary angioplasty (PTCA) and myocardial infarction.
It would be desirable to identify further compounds with binding activity at the P2Y
ADP
receptor, and also to identify further tissues/cell lines containing this receptor.
In accordance with the present invention, there is therefore provided a competition binding assay which comprises contacting a P2Y
ADP
receptor, preferably a human P2Y
ADP
receptor, with a P2Y
ADP
receptor radioligand and a candidate P2Y
ADP
receptor ligand, and measuring bound radioactivity.
The assay according to the present invention is very conveniently carried out on multi-well microtitre plates, thereby enabling a fast, simple and reproducible way of screening large numbers of potential P2Y
ADP
receptor ligands.
In the context of the present specification, the term “P2Y
ADP
receptor ligand”, unless otherwise indicated, defines a ligand, e.g. an agonist or antagonist, of the P2Y
ADP
receptor other than a naturally-occurring ligand. The ligand may, for example, be a chemical compound, or a salt or solvate thereof.
The radioligand used in the assay is a substance which binds to the P2Y
ADP
receptor and which may be synthesised containing one or more radioactive atoms. Examples of substances which when radiolabelled may be used as the radioligand include:
(a) Compounds of formula (I)
 wherein
X is OH or NHR
3
;
R
1
is C
1-6
-alkyl, C
3-8
-cycloalkyl or a phenyl group, each group being optionally substituted by one or more halogen atoms and/or OR
4
, NR
4
R
5
, C
1-6
-thioalkyl and/or C
1-6
-alkyl (itself optionally substituted by one or more halogen atoms); R
2
is C
1-8
-alkyl or C
2-8
-alkenyl each of which is optionally substituted by one or more halogen atoms and/or OR
4
, NR
4
R
5
, C
1-6
-thioalkyl, C
3-8
-cycloalkyl, aryl and/or C
1-6
-alkyl groups; or R
2
is a C
3-8
-cycloalkyl group optionally substituted by one or more halogen atoms and/or OR
4
, NR
4
R
5
, C
1-6
-thioalkyl, phenyl and/or C
1-6
-alkyl groups; the optional phenyl substituent being further optionally substituted by one or more halogen atoms and/or NO
2
, C(O)R
4
, OR
4
, NR
4
R
5
, C
1-6
-thioalkyl and/or C
1-6
-alkyl groups;
R
3
is hydrogen or C
1-6
-alkyl substituted by one or more hydroxy and/or phenyl groups and optionally by one or more halogen atoms, wherein the phenyl group is substituted by one or more hydroxy groups and optionally substituted by one or more halogen atoms and/or NO
2
, C(O)R
4
, OR
4
, NR
4
R
5
, C
1-6
-thioalkyl and/or C
1-6
-alkyl groups, or R
3
is a C
1-6
-alkyl group substituted by a C(O)NR
4
R
5
or a COOH group and optionally by one or more halogen atoms and/or OR
4
, C(NH)NR
4
R
5
, C(O)NR
4
R
5
, phenyl and/or C
1-6
-alkyl groups, wherein the alkyl group is optionally substituted by one or more hydroxy and/or phenyl groups and wherein the phenyl group is optionally substituted as defined above for R
3
; or
R
3
is a lactam ring of formula (i):
 wherein Q is a (CH
2
)
m
moiety wherein m is 1, 2 or 3, Z is O, C(O) or CH
2
;
R
4
and R
5
each independently represent hydrogen, phenyl or a C
1-6
-alkyl wherein the alkyl group is optionally substituted by one or more phenyl groups; and salts and solvates thereof;
(b) Compounds of formula (II)
 wherein
B is O or CH
2
;
X is selected from NR
1
R
2
, SR
1
and C
1
-C
7
alkyl;
Y is selected from NR
1
R
2
, SR
1
and C
1
-C
7
alkyl;
R
1
and R
2
is each and independently selected from H, or C
1
-C
7
alkyl optionally substituted upon or within the alkyl chain by one or more of O, S, N or halogen;
R
3
and R
4
are both hydrogen, or R
3
and R
4
together form a bond;
A is COOH, C(O)NH(CH
2
)
p
COOH, C(O)N[(CH
2
)
q
COOH]
2
, C(O)NHCH(COOH)(CH
2
)
r
COOH or 5-tetrazolyl, wherein p, q and r is each and independently 1, 2 or 3; and salts and solvates thereof;
(c) Compounds of formula (I)
 wherein
R
1
and R
2
independently represent hydrogen or halogen;
R
3
and R
4
independently represent phenyl, or C
1
-C
6
alkyl optionally substituted by one or more substituents selected from OR
5
, C
1
-C
6
alkylthio, NR
6
R
7
, phenyl, COOR
8
and halogen;
R
5
, R
6
, R
7
and R
8
independently represent hydrogen or C
1
-C
6
alkyl;
X represents an acidic moiety; and salts and solvates thereof;
(d) Compounds of formula (IV)
 wherein
Q represents CR
1
R
2
;
R represents O or CR
3
R
4
;
W represents O or CH
2
;
R
1
, R
2
, R
3
and R
4
independently represent hydrogen or halogen;
X represents S(O)
n
R
5
, C
1
-C
6
alkyl, C
1
-C
6
alkoxy, C
1
-C
6
acylamino, CONR
6
R
7
, NR
8
R
9
, halogen, a 5- or 6-membered S containing heterocycle, or phenyl optionally substituted by C
1
-C
6
alkyl;
n represents 0, 1 or 2;
R
5
represents aryl or C
1
-C
6
alkyl optionally substituted by one or more substituents selected from hydroxy, C
1
-C
6
alkoxy, halogen and aryl;
R
6, R
7
, R
8
and R
9
independently represent hydrogen or C
1
-C
6
alkyl;
Y represents NH
2
or C
1
-C
6
alkoxy;
Z represents an acidic moiety;
in addition, when R represents CR
3
R
4
, then —Q—Z may also represent hydroxy or —OP(O)(OH)
2
, provided that:
i) when R is O, W is O, X is Cl, Y is NH
2
and Z is —P(O)(OH)
2
, then CR
1
R
2
does not represent CH
2
; and
ii) when R is O, W is O, X is SCH
3
, Y is NH
2
and Z is —P(O)(OH)
2
, then —CR
1
R
2
does not represent CH
2
, CF
2
or CCl
2
; and salts and solvates thereof;
(e) Compounds of formula (V)
 wherein
R
1
is a C
1-6
alkyl, C
3-8
-cycloalkyl or a phenyl group, each group being optionally substituted by one or more substituents selected from halogen, OR
8
, NR
9
R
10
, SR
11
or C
1-6
alkyl (itself optionally substituted by one or more halogen atoms);
R
2
is C
1-8
alkyl optionally substituted by one or more substituents selected from halogen, OR
8
, NR
9
R
10
, SR
11
, C
3-8
-cycloalkyl, aryl (optionally substituted by one or more alkyl groups and/or halogen atoms), or C
1-6
-alkyl; or R
2
is a C
3-8
-cycloalkyl group optionally substituted by one or more substituents selected from halogen, OR
8
, NR
9
R
10
, SR
11
, C
1-6
-alkyl or phenyl (the latter two being optionally substituted by one or more substituents selected from halogen, NO
2
, C(O)R
8
, OR
8
, SR
11
, NR
12
R
13
, phenyl and C
1-6
-alkyl which is optionally substituted by one or more halogen atoms);
one of R
3
or R
4
is hydroxy and the other is hydrogen, hydroxy or NR
9
R
10
;
R
5
is (CH
2
)
n
NR
14
R
15
where n is 0 to 6 and R
14
and R
15
are independently hydrogen, C
1-6
-alkyl or phenyl; or R
5
is CONR
16
R
17
where R
16
is hydrogen or C
1-6
-alkyl, and R
17
is C
1-6
-alkyl or C
3-6
-cycloalkyl each of which is substituted by NR
18
R
19
and optionally substituted by phenyl, or R
17
is C
1-6
-alkyl or C
3-6
-cycloalkyl substituted by phenyl which is substituted by NR
18
R
19
where R
18
and R
19
are independently hydrogen, C
1-6
-alkyl or phenyl; or R
17
is a 5- to 8-membered saturated heterocycle containing one or more nitrogen atoms and optionally substituted on nitrogen by hydrogen, C
1-6
-alkyl or phenyl;
or R
16
and R
17
together with the nitrogen atom to which they are attached form a 5- to 8-membered ring which is substituted by NR
18
R
19
as defined above; or
R
1

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