U.S. patent application number 11/225379 was filed with the patent office on 2006-05-25 for thiazolopyrimidines and their use as modulators of chemokine receptor activity.
Invention is credited to Roger Bonnert.
Application Number | 20060111569 11/225379 |
Document ID | / |
Family ID | 20283779 |
Filed Date | 2006-05-25 |
United States Patent
Application |
20060111569 |
Kind Code |
A1 |
Bonnert; Roger |
May 25, 2006 |
Thiazolopyrimidines and their use as modulators of chemokine
receptor activity
Abstract
The invention provides certain thiazolopyrimidine compounds of
formula (I) or a pharmaceutically acceptable salt or solvate there:
in which: A is a group of formula (a) or (b): processes and
intermediates used in their preparation, pharmaceutical
compositions containing them and their use in therapy.
Inventors: |
Bonnert; Roger; (Charnwood,
GB) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
P.O BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
20283779 |
Appl. No.: |
11/225379 |
Filed: |
September 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10474610 |
Oct 9, 2003 |
6949643 |
|
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PCT/SE02/00731 |
Apr 12, 2002 |
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11225379 |
Sep 12, 2005 |
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Current U.S.
Class: |
544/255 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 19/02 20180101; A61P 29/00 20180101; C07D 513/04 20130101;
C07D 487/04 20130101; C07D 471/04 20130101; A61P 17/06 20180101;
A61P 11/00 20180101 |
Class at
Publication: |
544/255 |
International
Class: |
C07D 239/00 20060101
C07D239/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 12, 2001 |
SE |
0101322-6 |
Claims
1. A compound of formula (I) or a pharmaceutically acceptable salt
or solvate thereof: ##STR12## in which: A is a group of formula (a)
or (b): ##STR13## R.sup.1 represents a C.sub.3-C.sub.7 carbocyclic,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.6 alkenyl or C.sub.2-C.sub.6
alkynyl group, each of which may be optionally substituted by one
or more substituent groups independently selected from halogen
atoms, --OR.sup.4, --NR.sup.5R.sup.6, --CONR.sup.5R.sup.6,
--COOR.sup.7, --NR.sup.8COR.sup.9, --SR.sup.10, --SO.sub.2R.sup.10,
--SO.sub.2NR.sup.5R.sup.6, --NR.sup.8SO.sub.2R.sup.9, an aryl or
heteroaryl group, which last two may themselves be optionally
substituted by one or more substituents independently selected from
halogen atoms, cyano, nitro, --OR.sup.4, --NR.sup.5R.sup.6,
--CONR.sup.5R.sup.6, --COOR.sup.7, --NR.sup.8COR.sup.9,
--SR.sup.10, --SO.sub.2R.sup.10, --SO.sub.2NR.sup.5R.sup.6,
--NR.sup.8SO.sub.2R.sup.9, C.sub.1-C.sub.6 alkyl or trifluoromethyl
groups; R.sup.2 and R.sup.3 each independently represent a hydrogen
atom, or a C.sub.3-C.sub.7 carbocyclic, C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.6 alkenyl or C.sub.2-C.sub.6 alkynyl group, the
latter four groups may be optionally substituted by one or more
substituent groups independently selected from: (a) halogen atoms,
--OR.sup.4, --NR.sup.5R.sup.6, --CONR.sup.5R.sup.6, --COOR.sup.7,
--NR.sup.8COR.sup.9, --SR.sup.10, --SO.sub.2R.sup.10,
--SO.sub.2NR.sup.5R.sup.6, --NR.sup.8SO.sub.2R.sup.9; (b) a 3-8
membered ring optionally containing one or more atoms selected from
O, S, NR.sup.8 and itself optionally substituted by
C.sub.1-C.sub.3-alkyl or halogen; or (c) an aryl group or
heteroaryl group each of which may be optionally substituted by one
or more substituents independently selected from halogen atoms,
cyano, nitro, --OR.sup.4, --NR.sup.5R.sup.6, --CONR.sup.5R.sup.6,
--NR.sup.8SO.sub.2R.sup.9, --SO.sub.2NR.sup.5R.sup.6,
--NR.sup.8SO.sub.2R.sup.9, C.sub.1-C.sub.6 alkyl and
trifluoromethyl groups; R.sup.4 represents hydrogen,
C.sub.1-C.sub.6 alkyl or a phenyl group the latter two of which may
be optionally substituted by one or more substituent groups
independently selected from halogen atoms, phenyl, --OR.sup.11 and
--NR.sup.12R.sup.13 R.sup.5 and R.sup.6 independently represent a
hydrogen atom or a C.sub.1-C.sub.6 alkyl or phenyl group the latter
two of which may be optionally substituted by one or more
substituent groups independently selected from halogen atoms,
phenyl, --OR.sup.14 and --NR.sup.15R.sup.16, --CONR.sup.15R.sup.16,
--NR.sup.15COR.sup.16, --SONR.sup.15R.sup.16,
NR.sup.15SO.sub.2R.sup.16 or R.sup.5 and R.sup.6 together with the
nitrogen atom to which they are attached form a 4-to 7-membered
saturated heterocyclic ring system optionally containing a further
heteroatom selected from oxygen and nitrogen atoms, which ring
system may be optionally substituted by one or more substituent
groups independently selected from phenyl, --OR.sup.14,
--COOR.sup.14, --NR.sup.15R.sup.16, --CONR.sup.15R.sup.16,
--NR.sup.15COR.sup.16, --SONR.sup.15R.sup.16,
NR.sup.15SO.sub.2R.sup.16 or C.sub.1-C.sub.6 alkyl, itself
optionally substituted by one or more substituents independently
selected from halogen atoms and --NR.sup.15R.sup.16 and --OR.sup.17
groups; R.sup.10 represents a C.sub.1-C.sub.6-alkyl or a phenyl
group, either of which may be optionally substituted by one or more
substituent groups independently selected from halogen atoms,
phenyl, --OR.sup.17 and --NR.sup.15R.sup.16, X is CH or CCN, Y is N
or CR.sup.18, and each of R.sup.7, R.sup.8, R.sup.9, R.sup.11,
R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17 and
R.sup.18 independently represents a hydrogen atom or a
C.sub.1-C.sub.6, alkyl, or a phenyl group.
2. A compound according to claim 1, wherein R represents an
optionally substituted benzyl group.
3. A compound according to claim I or claim 2, wherein one of
R.sup.2 and R.sup.3 is hydrogen and the other is C.sub.1- C.sub.8
alkyl substituted by hydroxy and one or more methyl or ethyl
groups.
4. A compound according to any one of claims 1 to 3 in which A is a
group of formula (a).
5. A compound according to any one of claims 1 to 3 in which A is a
group of formula (b).
6. A compound according to claim 5 in which X is CH and Y is N.
7. A compound according to claim 1 selected from:
-[[(2,3-difluorophenyl)methyl]thio]-7-[[(1R)-2-hydroxy-1-methylethyl]amin-
o]-thiazolo[4,5-d]pyrimidine-2(3H)-thione,
2-[[(2,3-difluorophenyl)methyl]thio]4[[(1R)-2-hydroxy-1-methylethyl]amino-
]-7(8H)-pteridinethione, and pharmaceutically acceptable salts
thereof.
8. A process for the preparation of a compound of formula (I) which
comprises: (a) treatment of a compound of formula (IIA): ##STR14##
where R.sup.1, R.sup.2 and R.sup.3 are as defined in formula (I) or
are protected derivatives thereof and L is a leaving group with a
metal hydrosulphide, or (b) treatment of a compound of formula
(IIB): ##STR15## where R.sup.1, R.sup.2 and R.sup.3 are as defined
in formula (I) or are protected derivatives thereof with a thiating
agent, and optionally thereafter process (a) or (b) and in any
order: removing any protecting groups forming a pharmaceutically
acceptable salt.
9. A pharmaceutical composition comprising a compound of formula
(I), or a pharmaceutically acceptable salt or solvate thereof, as
claimed in any one of claims 1 to 7 in association with a
pharmaceutically acceptable adjuvant, diluent or carrier.
10. A process for the preparation of a pharmaceutical composition
as claimed in claim 9 which comprises mixing a compound of formula
(I), or a pharmaceutically acceptable salt or solvate thereof, as
claimed in any one of claims 1 to 7 with a pharmaceutically
acceptable adjuvant, diluent or carrier.
11. A compound of formula (I), or a pharmaceutically-acceptable
salt or solvate thereof, as claimed in any one of claims 1 to 7 for
use in therapy.
12. Use of a compound of formula (I), or a pharmaceutically
acceptable salt or solvate thereof, as claimed in any one of claims
1 to 7 in the manufacture of a medicament for use in therapy.
13. A method of treating a chemokine mediated disease wherein the
chemokine binds to one or more chemokine, receptors, which
comprises administering to a patient a therapeutically effective
amount of a compound of formula (I), or a pharmaceutically
acceptable salt or solvate thereof, as claimed in any one of claims
1 to 7.
14. A method according to claim 13 in which the chemokine receptor
belongs to the CXC chemokine receptor subfamily.
15. A method according to claim 13 or 14 in which the chemokine
receptor is the CXCR2 receptor.
16. A method of treating an inflammatory disease in a patient
suffering from, or at risk of, said disease, which comprises
administering to the patient a therapeutically effective amount of
a compound of formula (I), or a pharmaceutically acceptable salt or
solvate thereof, as claimed in any one of claims 1 to 7.
17. A method according to claim 16, wherein the disease is
psoriasis, rheumatoid arthritis or COPD.
18. A method according to claim 16, wherein the disease is
rheumatoid arthritis.
Description
[0001] The present invention relates to certain heterocyclic
compounds, processes and intermediates used in their preparation,
pharmaceutical compositions containing them and their use in
therapy.
[0002] Chemokines play an important role in immune and inflammatory
responses in various diseases and disorders, including asthma and
allergic diseases, as well as autoimmune pathologies such as
rheumatoid arthritis and atherosclerosis. These small secreted
molecules are a growing superfamily of 8-14 kDa proteins
characterised by a conserved four cysteine motif. At the present
time, the chemokine superfamily comprises three groups. exhibiting
characteristic structural motifs, the Cys-X-Cys (C--X--C), Cys-Cys
(C--C)) and Cys-X.sub.3-Cys (C--X.sub.3--C) families. The C--X--C
and C--C families have sequence similarity and are distinguished
from one another on the basis of a single amino acid insertion
between the NH-proximal pair of cysteine residues. The
C--X.sub.3--C family is distinguished from the other two families
on the basis of having a triple amino acid insertion between the
NH-proximal pair of cysteine residues.
[0003] The C--X--C chemokines include several potent
chemoattractants and activators of neutrophils such as
interleukin-8 (IL-8) and neutrophil-activating peptide 2
.(NAP-2).
[0004] The C--C chemokines include potent chemoattractants of
monocytes and lymphocytes but not neutrophils. Examples include
human monocyte chemotactic proteins 1-3 (MCP-1, MCP-2 and MCP-3),
RANTES (Regulated on Activation, Normal T. Expressed and Secreted),
eotaxin and the macrophage inflammatory proteins 1.alpha. and
1.beta. (MIP- 1.alpha. and MIP-1.beta.).
[0005] The C--X.sub.3--C chemokine (also known as fractalkine) is a
potent chemoattractant and activator of microglia in the central
nervous system (CNS) as well as of monocytes, T cells, NK cells and
mast cells.
[0006] Studies have demonstrated that the actions of the chemokines
are mediated by subfamilies of G protein-coupled receptors, among
which are the receptors designated CCR1, CCR2, CCR2A, CCR2B, CCR3,
CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11 (for the C--C
family); CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5 (for the C--X--C
family) and CX.sub.3CR1 for the C--X.sub.3--C family. These
receptors represent good targets for drug development since agents
which modulate these receptors would be useful in the treatment of
disorders and diseases such as those mentioned above.
[0007] The present invention therefore provides compounds of
formula (I) and pharmaceutically acceptable salts or solvates
thereof: ##STR1## in which:
[0008] A is a group of formula (a) or (b): ##STR2##
[0009] R.sup.1 represents a C.sub.3-C.sub.7 carbocyclic,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.6 alkenyl or C.sub.2-C.sub.6
alkynyl group, each of which may be optionally substituted by one
or more substituent groups independently selected from halogen
atoms, --OR.sup.4, --NR.sup.5R.sup.6, --CONR.sup.5R.sup.6,
--COOR.sup.7, --NR.sup.8COR.sup.9, --SR.sup.10, --SO.sub.2R.sup.10,
--SO.sub.2NR.sup.5R.sup.6, --NR.sup.8SO.sub.2R.sup.9, an aryl or
heteroaryl group, which last two may themselves be optionally
substituted by one or more substituents independently selected from
halogen atoms, cyano, nitro, --OR.sup.4, --NR.sup.5R.sup.6,
--CONR.sup.5R.sup.6, --COOR.sup.7, -NR.sup.8COR.sup.9, --SR.sup.10,
--SO.sub.2R.sup.10, --SO.sub.2NR.sup.5R.sup.6,
--NR.sup.8SO.sub.2R.sup.9, C.sub.1-C.sub.6 alkyl or trifluoromethyl
groups;
[0010] R.sup.2 and R.sup.3 each independently represent a hydrogen
atom, or a C.sub.3-C.sub.7 carbocyclic, C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.6 alkenyl or C.sub.2-C.sub.6 alkynyl group, the
latter four groups may be optionally substituted by one or more
substituent groups independently selected from:
(a) halogen atoms, --OR.sup.4, --NR.sup.5R.sup.6,
--CONR.sup.5R.sup.6, --COOR.sup.7, --NR.sup.8COR.sup.9,
--SR.sup.10, --SO.sub.2R.sup.10, --SO.sub.2NR.sup.5R.sup.6,
--NR.sup.8SO.sub.2R.sup.9;
(b) a 3-8 membered ring optionally containing one or more
atoms-selected from O, S, NR.sup.8 and itself optionally
substituted by C.sub.1-C.sub.3-alkyl or halogen; or
[0011] (c) an aryl group or heteroaryl group each of which may be
optionally substituted by one or more substituents independently
selected from halogen atoms, cyano, nitro, --OR.sup.4,
--NR.sup.5R.sup.6, --CONR.sup.5R.sup.6, --NR.sup.8COR.sup.9,
--SO.sub.2NR.sup.5R.sup.6, --NR.sub.8SO.sub.2R.sup.9,
C.sub.1-C.sub.6 alkyl and trifluoromethyl groups;
[0012] R.sup.4 represents hydrogen, C.sub.1-C.sub.6 alkyl or a
phenyl group the latter two of which may be optionally substituted
by one or more substituent groups independently selected from is
halogen atoms, phenyl, --OR.sup.11 and --NR.sup.12R.sup.13
[0013] R.sup.5 and R.sup.6 independently represent a hydrogen atom
or a C.sub.1-C.sub.6 alkyl or phenyl group the latter two of which
may be optionally substituted by one or more substituent groups
independently selected from halogen atoms, phenyl, --OR.sup.14 and
--NR.sup.15R.sup.16, --CONR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--SONR.sup.15SR.sup.16, NR.sup.15SO.sub.2R.sup.16
or
[0014] R.sup.5 and R.sup.6 together with the nitrogen atom to which
they are attached form a 4- to 7-membered saturated heterocyclic
ring system optionally containing a further heteroatom selected
from oxygen and nitrogen atoms, which ring system may be optionally
substituted by one or more substituent groups independently
selected from phenyl, --OR.sup.1, --COOR.sup.14,
--NR.sup.15R.sup.16, --CONR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--SONR.sup.15R.sup.16, NR.sup.15SO.sub.2R.sup.16 or C.sub.1-C.sub.6
alkyl, itself optionally substituted by one or more substituents
independently selected from halogen atoms and --NR.sup.15R.sup.16
and --OR.sup.17 groups;
[0015] R.sup.10 represents a C.sub.1-C.sub.6-alkyl or a phenyl
group, either of which may be optionally substituted by one or more
substituent groups independently selected from halogen atoms,
phenyl, --OR.sup.17 and --NR.sup.15R.sup.16,
[0016] X is CH or CCN,
[0017] Y is N or CR.sup.18, and
[0018] each of R.sup.7 , R.sup.8, R.sup.9, R.sup.11, R.sup.12,
R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17 and R.sup.18
independently represents a hydrogen atom or a C.sub.1-C.sub.6,
alkyl, or a phenyl group.
[0019] In the context of the present specification, unless
otherwise indicated, an alkyl or alkenyl group or an alkly or
alkenyl moiety in a substituent group may be linear or
branched.
[0020] Aryl groups include phenyl and naphthyl. Heteroaryl is
defined as a 5- or 6-membered aromatic ring optionally containing
one or more heteroatoms selected from N, S, O. Examples include
pyridine, pyrimidine, thiazole, oxazole, pyrazole, imidazole,
furan.
[0021] Certain compounds of formula (I) are capable of existing in
stereoisomeric forms. It will be understood that the invention
encompasses all geometric and optical isomers of the compounds of
formula (I) and mixtures thereof including racemates. Tautomers and
mixtures thereof also form an aspect of the present invention.
[0022] Suitably the group R.sup.1 represents a C.sub.3-C.sub.7
carbocyclic, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.6 alkenyl or
C.sub.2-C.sub.6 alkynyl group, each of which may be may be
optionally substituted by one or more substituent groups
independently selected from halogen atoms, --OR.sup.4,
--NR.sup.5R.sup.6, --CONR.sup.5R.sup.6, --COOR.sup.7,
--NR.sup.8COR.sup.9, --SR.sup.10, --SO.sub.2R.sup.10,
--SO.sub.2NR.sup.5R.sup.6, --NR.sup.8SO.sub.2R.sup.9, an aryl or
heteroaryl group both of which can be optionally substituted by one
or more substituents independently selected from halogen atoms,
cyano, nitro, --OR.sup.4, --NR.sup.5R.sup.6, --CONR.sup.5R.sup.6,
--COOR.sup.7, --NR.sup.8COR.sup.10, --SR.sup.10,
--SO.sub.2R.sup.10, --SO.sub.2NR.sup.5R.sup.6,
--NRSO.sub.2R.sup.10, C.sub.1-C.sub.6 alkyl or trifluoromethyl
groups. Particularly advantageous compounds of formula (I) are
those in which R.sup.1 represents an optionally substituted benzyl
group. More preferably R.sup.1 represents benzyl or benzyl
substituted by one or more C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxy, or halogen atoms, in particular benzyl substituted by two
fluoro atoms.
[0023] Preferably one of R.sup.2 and R.sup.3 is hydrogen and the
other is C.sub.1-C.sub.8 alkyl substituted by hydroxy and one or
more methyl or ethyl groups. More preferably one of R.sup.2 and
R.sup.3 is hydrogen and the other is CH(CH.sub.3)CH.sub.2OH,
CH(Et)CH.sub.2OH, C(CH.sub.3).sub.2CH.sub.2OH or
CH(CH.sub.2OH).sub.2. When one of R.sup.2 and R.sup.3 is hydrogen
and the other is CH(CH.sub.3)CH.sub.2OH or CH(Et)CH.sub.2OH the
resulting compounds of formula (1) are preferably in the form of
the (R) isomer. Most preferably one of R.sup.2 and R.sup.3 is
hydrogen and the other is CH(CH.sub.3)CH.sub.2OH.
[0024] Suitably in formula (b) X represents CH or CCN and Y is N or
CR.sup.18. Preferably X is CH and Y is N.
[0025] Particularly preferred compounds of the invention
include:
5-[[(2,3-difluorophenyl)metbyl]thio]-7-[[(1R)-2-hydroxy-1-methylethyl]am-
ino]-thiazolo[4,5-d]pyrimidine-2(3H)-thione,
2-[[(2,3-difluorophenyl)methyl]thio]4-[[(1R)-2-hydroxy--1-methylethyl]am-
ino]-7(8H)-pteridinethione,
and pharmaceutically acceptable salts thereof.
[0026] According to the invention there is also provided a process
for the preparation of a compound of formula (I) which comprises:
(a) treatment of a compound of formula (IIA): ##STR3## where
R.sup.1, R.sup.2 and R.sup.3 are as defined in formula (I) or are
protected derivatives thereof and L is a leaving group with a metal
hydrosulphide, or (b) treatment of a compound of formula (IIB):
##STR4## where R.sup.1, R.sup.2 and R.sup.3 are as defined in
formula (I) or are protected derivatives thereof with a thiating
agent, and optionally thereafter process (a) or (b) and in any
order: [0027] removing any protecting groups [0028] forming a
pharmaceutically acceptable salt.
[0029] The reaction of compounds of formula (IIA) with a metal
hydrosulphide may be carried out in a solvent such as DMSO at a
temperature between 0.degree. C. and 100.degree. C. Suitable
leaving groups L include halogen, especially chloro or bromo, and a
suitable metal bydrosulphide is sodium hydrosuphide.
[0030] The reaction of compounds (IIB) with a thiating agent may be
carried out in a solvent such as dioxan at reflux. A suitable
thiating agent is Lawesson's reagent.
[0031] Compounds of formula (IIA) where R.sup.1, R.sup.2 and
R.sup.3 are as defined in formula (I) and L is a leaving group may
be prepared from compounds of formula (IIA) where R.sup.1, R.sup.2
and R.sup.3 are as defined above and L is NH.sub.2 by treatment
with sodium nitrite and aqueous mineral acid at a temperature
between 0.degree. C. and room temperature. Suitable acids include
hydrochloric acid.
[0032] Compounds of formula (IIA) where R.sup.1, R.sup.2 and
R.sup.3 are as defined in formula (I) and L is NH.sub.2 may be
prepared by treatment of a compound of formula (III): ##STR5##
where R.sup.1 is as defined in formula (I) and L is a leaving group
such as chlorine with an amine HNR.sup.2R.sup.3 where R.sup.2 and
R.sup.3 are as defined in formula (I). The reaction may be carried
out in a solvent such as N-methyl-pyrrolidine at a temperature
between 0.degree. C. and 150.degree. C.
[0033] Compounds of formula (III) where R.sup.1 is as defined in
formula (I) and L is a halogen may be prepared by treating a
compound of formula (III) where R.sup.1 is as defined in formula
(I) and L is a hydroxyl group with a halogenating agent such as
phosphorous oxychloride. The reaction may be carried out in the
presence of dimethylaniline at reflux.
[0034] Compounds of formula (III) where R.sup.1 is as defined in
formula (I) and L is a hydroxyl group may be formed by treatment of
a compound of formula (IV) with a compound of formula R.sup.1X
where R.sup.1 is as defined above and X is a leaving group such as
bromide in the presence of a base such as potassium tert-butoxide
in an inert solvent such as DMSO at ambient temperature.
##STR6##
[0035] Compounds of formula (IIB) where R.sup.1, R.sup.2 and
R.sup.3 are as defined in formula (I) may be prepared by treatment
of a compound of formula (V): ##STR7## where R.sup.1 is as defined
in formula (I) and L is a leaving group such as bromo with an amine
HNR.sup.2R.sup.3 where R.sup.2 and R.sup.3 are as defined in
formula (I). The reaction may-be carried out in a solvent such as
N-methyl-pyrrolidine at a temperature between 0.degree. C. and
150.degree. C.
[0036] Compounds of formula (V) where R.sup.1 is as defined in
formula (I) and L is a leaving group such as bromo may be prepared
by treating a compound of formula (V) where R.sup.1 is as defined
above and L is NH.sub.2 with a diazotizing agent such as isoamyl
nitrite in the presence of a halogenating agent such as bromoform.
The reaction may be performed in a solvent such as DMSO at a
temperature between 0.degree. C. and 150.degree. C.
[0037] Compounds of formula (V) where R.sup.1 is as defined in
formula (I) and L is NH.sub.2 may be prepared by treatment of a
compound of formula (VI): ##STR8## where R.sup.1 and L are as
defined above with ethyl glyoxylate in the presence of a base such
as sodium methoxide in a solvent such as methanol at room
temperature.
[0038] Compounds of formula (VI) where R.sup.1 is as defined in
formula (I) and L is NH.sub.2 may be prepared by treating a
compound of formula (VII) where R.sup.1 and L are as defined above
with a reducing agent such as sodium hydrosulphite. The reaction
may be carried out in a solvent such as water at reflux.
##STR9##
[0039] Compounds of formula (VII) where R.sup.1 is as defined in
formula (I) and L is NH.sub.2 may be prepared by treating a
compound of formula (VIII) where R.sup.1 and L are as defined above
with a nitrosating agent such as sodium nitrite. The reaction may
be performed in a solvent such as aqueous acetic acid at a
temperature between 0.degree. C. and 100.degree. C. ##STR10##
[0040] Compounds of formula (VIII) where R.sup.1 is as defined in
formula (I) and L is NH.sub.2 may be prepared by treating a
compound of formula (IX) with a compound of formula R.sup.1X where
R.sup.1 is as defined above and X is a leaving group such as
bromide in the presence of a base such as potassium tert-butoxide.
The reaction may be performed in a solvent such as DMSO at room
temperature. ##STR11##
[0041] Compounds of formula (IV) or (IX) are either commercially
available or are well known in the literature.
[0042] It will be appreciated by those skilled in-the art that in
the processes of the present invention certain functional groups
such as hydroxyl or amino groups in the starting reagents or
intermediate compounds may need to be protected by protecting
groups. Thus, the preparation of the compounds of formula (I) may
involve, at an appropriate stage, the removal of one or more
protecting groups. The protection and deprotection of functional
groups is fully described in `Protective Groups in Organic
Chemistry`, edited by J. W. F. is McOmie, Plenum Press (1973), and
`Protective Groups in Organic Synthesis`, 2nd edition, T. W. Greene
& P. G. M. Wuts, Wiley-Interscience (1991).
[0043] The compound is of formula (I) above may be converted to a
pharmaceutically acceptable salt or solvate thereof, preferably a
basic addition salt such as sodium, potassium, calcium, aluminium,
lithium, magnesium, zinc, benzathine, chloroprocaine, choline,
diethanolamine, ethanol amine, ethyldiamine, meglumine,
tromethamine or procaine, or an acid addition salt such as a
hydrochloride, hydrobromide, phosphate, acetate, fumarate, maleate,
tartrate, citrate, oxalate, methanesulphonate or
p-toluenesulphonate.
[0044] The compounds of formula (I) have activity as
pharmaceuticals, in particular as modulators of chemokine receptor
(especially CXCR2) activity, and may be used in the treatment
(therapeutic or prophylactic) of conditions/diseases in human and
non-human animals which are exacerbated or caused by excessive or
unregulated production of cbemokines. Examples of such
conditions/diseases include: [0045] (1) (the respiratory tract)
obstructive airways diseases including chronic obstructive
pulmonary disease (COPD); asthma, such as bronchial, allergic,
intrinsic, extrinsic and dust asthma, particularly chronic or
inveterate, asthma (e.g. late asthma and airways
hyper-responsiveness); bronchitis; acute, allergic, atrophic
rhinitis and chronic rhinitis including rhinitis caseosa,
hypertrophic rhinitis, rhinitis purulenta, rhinitis sicca and
rhinitis medicamnentosa; membranous rhinitis including croupous,
fibrinous and pseudomembranous rhinitis and scrofoulous rhinitis;
seasonal rhinitis including rhinitis nervosa (hay fever) and
vasomotor rhinitis; sarcoidosis, farmer's lung and related
diseases, fibroid lung and idiopathic interstitial pneumonia;
[0046] (2) (bone and joints) rheumatoid arthritis, seronegative
spondyloarthropathies (including ankylosing spondylitis, psoriatic
arthritis and Reiter's disease), Behcet's disease, Sjogren's
syndrome and systemic sclerosis; [0047] (3) (skin) psoriasis,
atopical dermatitis, contact dermatitis and other eczmatous
dermitides, seborrhoetic dermatitis, Lichen planus, Pemphigus,
bullous Pemphigus, Epidermolysis bullosa, urticaria, angiodermas,
vasculitides, erythemas, cutaneous eosinophilias, uveitis, Alopecia
areata and vernal conjunctivitis; [0048] (4) (gastrointestinal
tract) Coeliac disease, proctitis, eosinopilic gastro-enteritis,
mastocytosis, Crohn's disease, ulcerative colitis, food-related
allergies which have effects remote from the gut, e.g., migraine,
rhinitis and eczema; [0049] (5) (central and peripheral nervous
system) Neurodegenerative diseases and dementia disorders, e.g.
Alzheimer's disease, amyotrophic lateral sclerosis and other motor
neuron diseases, Creutzfeldt-Jacob's disease and other prion
diseases, HIV encephalopathy (AIDS dementia complex), Huntington's
disease, frontotemporal dementia, Lewy body dementia and vascular
dementia; polyneuropathies, e.g.. Guillain-Barre syndrome, chronic
inflammatory demyelinating polyradiculoneuropathy, multifocal motor
neuropathy, plexopathies; CNS demyelination, e.g. multiple
sclerosis, acute disseminated/haemorrhagic encephalomyelitis, and
subacute sclerosing panencephalitis; neuromuscular disorders, e.g.
myasthenia gravis and Lambert-Eaton syndrome; spinal diorders, e.g.
tropical spastic paraparesis, and stiff-man syndrome:
paraneoplastic syndromes, e.g. cerebellar degeneration and
encephalomyelitis; CNS trauma; migraine; and stroke. [0050] (6)
(other tissues and systemic disease) atherosclerosis, Acquired
Immunodeficiency Syndrome (AIDS), lupus erythematosus, systemic
lupus, erytbematosus, Hashimoto's thyroiditis, type I diabetes,
neiphrotic syndrome, eosinophilia fascitis, hyper IgE syndrome,
lepromatous leprosy, and idiopathic thrombocytopenia pupura;
post-operative adhesions, and sepsis. [0051] (7) (allograft
rejection) acute and chronic following, for example,
transplantation of kidney, heart, liver, lung, bone marrow, skin
and comea; and chronic graft versus host disease; [0052] (8)
Cancers, especially non-small cell lung cancer (NSCLC), malignant
melanoma, prostate cancer and squamous sarcoma, and tumour
metastasis; [0053] (9) Diseases in which angiogenesis is associated
with raised CXCR2 chemokine levels (e.g. NSCLC, diabetic
retinopathy). [0054] (10) Cystic fibrosis, re-perfusion injury in
the heart, brain, peripheral limbs and other organs. [0055] (11)
Burn wounds & chronic skin ulcers [0056] (12) Reproductive
Diseases (e.g. Disorders of ovulation, menstruation and
implantation, Pre-term labour, Endometriosis)
[0057] Thus, the present invention provides a compound of formula
(I), or a pharmaceutically-acceptable salt or solvate thereof, as
hereinbefore defined for use in therapy.
[0058] Preferably the compounds of the invention are used to treat
diseases in which the chemokine receptor belongs to the CXC
chemokine receptor subfamily, more preferably the target chemokine
receptor is the CXCR2 receptor,
[0059] Particular conditions which can be treated with the
compounds of the invention are, psoriasis, rheumatoid arthritis and
diseases in which angiogenesis is associated with raised CXCR2
chemokine levels, and COPD. It is preferred that the compounds of
the invention are used to treat rheumatoid arthritis.
[0060] As a further aspect of the present invention, certain
compounds of formula (I) may have utility as antagonists of the
CX3CR1 receptor. Such compounds are expected to be particularly
useful in the treatment of disorders within the central and
peripheral nervous system and other conditions characterized by an
activation of microglia and/or infiltration of leukocytes (e.g.
stroke/ischemia and head trauma).
[0061] In a further aspect, the present invention provides the use
of a compound of formula (I), or a pharmaceutically acceptable salt
or solvate thereof, as hereinbefore defined in the manufacture of a
medicament for use in therapy.
[0062] In a still further aspect, the present invention provides
the use of a compound of formula (I), or a pharmaceutically
acceptable salt or solvate thereof, as hereinbefore defined in the
manufacture of a medicament for the treatment of human diseases or
conditions in which modulation of chemokine receptor activity is
beneficial.
[0063] In the context of the present specification, the term
"therapy" also includes "prophylaxis" unless there are specific
indications to the contrary. The terms "therapeutic" and
"therapeutically" should be construed accordingly.
[0064] The invention still further provides a method of treating a
chemokine mediated disease wherein the chemokine binds to a
chemokine (especially CXCR2) receptor, which comprises
administering to a patient a therapeutically effective amount of a
compound of formula (I), or a pharmaceutically acceptable salt or
solvate thereof, as hereinbefore defined.
[0065] The invention also provides a method of treating an
inflammatory disease, especially psoriasis, in a patient suffering
from, or at risk of, said disease, which comprises administering to
the patient a therapeutically effective amount of a compound of
formula (I), or a pharmaceutically acceptable salt or solvate
thereof, as hereinbefore defined.
[0066] For the above-mentioned therapeutic uses the dosage
administered will, of course, vary with the compound employed, the
mode of administration, the treatment desired and the disorder
indicated.
[0067] The compounds of formula (I) and pharmaceutically acceptable
salts and solvates thereof may be used on their own but will
generally be administered in the form of a pharmaceutical
composition in which the formula (I) compound/salt/solvate (active
ingredient) is in association with a pharmaceutically acceptable
adjuvant, diluent or carrier. Depending on the mode of
administration, the pharmaceutical composition will preferably
comprise from 0.05 to 99% w (per cent by weight), more preferably
from 0.05 to 80% w, still more preferably from 0.10 to 70% w, and
even more preferably from 0.10 to 50% w, of active ingredient, all
percentages by weight being based on total composition.
[0068] The present invention also provides a pharmaceutical
composition comprising a compound of formula (I), or a
pharmaceutically acceptable salt or solvate thereof, as
hereinbefore defined, in association with a pharmaceutically
acceptable adjuvant, diluent or carrier.
[0069] The invention further provides a process for the preparation
of a pharmaceutical composition of the invention which comprises
mixing a compound of formula (I), or a pharmaceutically acceptable
salt or solvate thereof, as hereinbefore defined, with a
pharmaceutically acceptable adjuvant, diluent or carrier.
[0070] The pharmaceutical compositions may be administered
topically (e.g. to the lung and/or airways or to the skin) in the
form of solutions, suspensions, heptafluoroalkane aerosols and dry
powder formulations; or systemically, e.g. by oral administration
in the form of tablets, capsules, syrups, powders or granules, or
by parenteral administration in the form of solutions or
suspensions, or by subcutaneous administration or by rectal
administration in the form of suppositories or transdermally.
Preferably the compounds of the invention are administered
orally.
[0071] The invention will now be further illustrated by reference
to the following examples. In the examples the Nuclear Magnetic
Resonance (NMR) spectra were measured on a Varian Unity Inova 300
or 400 MHz spectrometer and the Mass Spectrometry (MS) spectra
measured on a Finnigan Mat SSQ7000 or Micromass Platform
spectrometer. Where necessary, the reactions were performed under
an inert atmosphere of either nitrogen or argon. Chromatography was
generally performed using Matrex Silica 60.RTM. (35-70 micron) or
Prolabo Silica gel 60.RTM. (35-70 micron) suitable for flash silica
gel chromatography. High pressure liquid chromatography
purification was performed using either a Waters Micromass LCZ with
a Waters 600 pump controller, Waters 2487 detector and Gilson FC024
fraction collector or a Waters Delta Prep 4000. The abbreviations
m.p. and DMSO used in the examples stand for melting point and
dimethyl sulphoxide respectively.
EXAMPLES
Example 1
5-[[(2,3-difluorophenyl)methyl]thio]-7-[[(1R)-2-hydroxy-1-methylethyl]amin-
o]-thiazolo 4,5-d]pyrimidine-2(3H)-thione
[0072] a)
2-Amino-5-[[(2,3-difluorophenyl)methyl]thio]thiazolo[4,5-d]pyri-
midin-7(4H)-one
[0073] Potassium 1-butoxide solution (0.45 ml of 1M solution in
tetrahydrofuran) was added to a stirred solution of
2-amino-5,6-dihydro-5-thioxo-thiazolo[4,5-d]pyrimidin-7(4H)-one
(0.09 g) [Cited: Indian J. Chem., Sect. B (1989), 28B(11),
964-5.]and 2,3-difluorobenzyl bromide in dimethyl sulphoxide (2
ml). After stirring for 3 days, the reaction mixture was poured
onto water to give the subtitled compound, isolated by
filtration.
MS (APCI) 327 (M+H.sup.+, 100%).
[0074] (b)
7-Chloro-5-[[(2,3-difluorophenyl)methyl]thio]thiazolo[4,5-dipyrimidin-2-a-
mine
[0075] The product from example 1, step (a) (0.89 g), phosphorus
oxychloride (12 ml) and N,N-dimethylaniline (1.2 ml) were heated at
reflux for 2 hours. The cooled reaction mixture was poured onto ice
water and stirred for 2 hours. Chromatography (SiO.sub.2,
methanol/dichloromethane as eluant) gave the subtitled
compound.
m.p. 217-218.5.degree. C.
MS (APCI) 346(M+H, 100%).
[0076] (c)
(2R)-2-[[2-amino-5-[[(2,3-difluorophenyl)methyl]thio]thiazolo[4,5-d]pyrim-
idin-7-yl[amino]-1-propanol,
[0077] The product from example 1, step (b) (70.0 g) and
(R)-1-amino-propan-2-ol (32 ml) in N-methylpyrrolidinone (600 ml)
and Hunigs base (71 ml) was heated at 110.degree. C. for 16 hours.
The mixture was poured into water (5 L) and the crude product
collected at by filtration. This solid was purified by
recrystallisation from acetonitrile (2.5 L) to give 65 g of the
subtitled compound.
MS (APCI) 384 (M+H, 100%).
[0078] (d)
(2R)-2-[[2-chloro-5-[[(2,3-difluorophenyl)methyl]thio]thiazoio[4,5-d]pyri-
nidin-7-yl]amino]-1-propanol,
[0079] The product from example 1, step (c) (0.5 g) was dissolved
in concentrated hydrochloric acid (18 ml). To this solution was
added a mixture of acetonitile (8 ml) and water (16 ml),
maintaining the temperature below 20.degree. C. The solution was
then cooled in an ice bath and a solution of sodium nitrite (0.135
g) in water (0.5 ml) added. Upon completion of addition the mixture
was allowed to stir for a further 1 hour. The solid was then,
collected by filtration, washed well with water and dried at
40.degree. C. in vacuo to give the subtitled compound (0.43 g).
MS (APCI) 403 (M+H, 100%).
[0080] (e)
5-[[(2,3-difluorophenyl)methyl]thio]-7-[[(1R)-2-hydroxy-1-methylethyl]ami-
no]-thiazolo[4,5-d]pyrimidine-2(3H)-thione,
[0081] A mixture of the product from example 1, step (d) (150 mg)
and sodium hydrosulfide (150 mg) in DMSO (5 ml) was stirred at room
temperature for 30 mins. The mixture was poured into water (100 ml)
and the pH adjusted to 7 and the product collected by filtration
and purified by chromatography (SiO.sub.2, ethyl
acetate/dichloromethane as eluant) to give the title compound (67
mg).
MS (APCI) 401 (M+H, 100%).
[0082] NMR .delta.H (d.sub.6-DMSO) 14.06 (1H, s), 7.75 (1H, d),
7.45-7.11 (3H, m), 4.75 (1H, bs), 4.41 (2H, m), 4.21 (1H, bs),
3.46-3.32 (2H, m), 1.09 (3H, d).
Example 2
2-[[(2,3-Difluorophenyl]methyl]thio]4-[[(1R)-2-hydroxy-1-methylethyl]amino-
]-7(8H)-pteridinone
[0083] a)
2-[[(2,3-Difluorophenyl)methyl]thio]-4,6-pyrimidinediamine
4,6-diamino-2-pyrimidinethiol (7.3 g) was dissolved in DMSO (100
ml) at room temperature under an atmosphere of nitrogen. Potassium
tert-butoxide (1M in THF, 48.3 ml) was added followed by
2,3-difluorobenzyl-bromide (10.0 g). The mixture was stirred for 2
hours at room temperature. The reaction mixture was then
partitioned between ethyl acetate and ammonium chloride. The
organic phase was washed with ammonium chloride (3.times.) and
brine, then dried over magnesium sulphate and evaporated to give
the subtitled product as a white solid (12.2 g)
MS: ADCI (+ve) 269 (M+1)
[0084] b)
2-[[(2,3-Difluorophenyl)methyl]thio]-5-nitroso4,6-pyrimidinedia-
mine
[0085] The product of example 2, step (a) (2.5 g) was dissolved in
acetic acid (150 ml) and the solution cooled to 5.degree. C. A
solution of sodium nitrite (625 mg) in water (50 ml) was added
dropwise resulting in a dark blue colouration. The reaction was
stirred at room temperature for 30 minutes during which time a pink
solid precipitated from solution. This was isolated by filtration
and washed with water, then dried at 50.degree. C. to give the
sub-titled product as a blue solid (4.14 g)
MS: ADCI (+ve) 298 (M+1)
[0086] .sup.1H NMR: .delta. (DMSO) 4.44 (s,2H), 7.13-7.54 (m,3H),
8.13 (s,1H), 8.51 (s,1H), 9.10 (s,1H), 10.18 (s,1H).
[0087] c)
2-[[(2,3-Difluorophenyl)methyl]thio]-4,5,6-pyrimidinetriamine
[0088] To a suspension of the product of example 2, step (b) (2 g)
in boiling water (40 ml) was added Na.sub.2S.sub.2O.sub.4 (5.4 g)
portion-wise. The suspension was allowed to cool and then 50%
sulphuric acid was added slowly and then the mixture was cooled to
0.degree. C. The solid was isolated by filtration and washed with
cold water, then dried over P.sub.2O.sub.5 at 50.degree. C. to give
the sub-titled product as a yellow solid.
MS: ADCI (+ve) 284 (M+1)
[0089] .sup.1H NMR: .delta. (DMSO) 4.33 (s,2H), 6.42 (brs,3H),
7.10-7.48 (m,3H)
[0090] d)
4-amino-2-[[(2,3-difluorophenyl)methyl]thio]-7(8H)-pteridinone
[0091] The product of example 2, step (c) (100 mg) was dissolved in
a solution of sodium (0.05 g) in methanol (5 ml). This was left to
stir for 15 min at room temperature, then ethyl glyoxalate (134
.mu.l) was added to the mixture which was left to stir for 12 hr at
room temperature. Water (5 ml) was added, then concentrated
hydrochloric acid was slowly added to acidify the solution to
.about.pH5 whereupon a solid precipitated which was isolated by
filtration and dried over P.sub.2O.sub.5 at 50.degree. C. to yield
a pale yellow solid (44.5 mg).
MS: ADCI (+ve) 322 (M+1)
[0092] .sup.1H NMR: .delta. (DMSO) 4.18 (s,2H), 7.11-7.58 (m,3H),
7.84 (s,1H), 12.69 (bs,1H)
[0093] e)
4-bromo-2-[[(2,3-difluorophenyl)methyl]thio]-7(8H)-pteridinone
[0094] The product of example 2,-step (d) (6.0 g) was suspended in
DMSO (90 ml) and bromoform (60 ml) was added and the mixture was
heated to 100.degree. C. Isopentylnitrite (25 ml) was added and the
mixture stirred for 5 min. The mixture was quickly cooled in an ice
bath then evaporated to leave an oil. This was repeated three
times. Acetonitrile (200 ml) was added and the solid which
separated was removed by filtration. The solvent was evaporated and
the residue was purified by flash chromatography, eluting with
dichloromethane and then 5% ethyl acetate in dichloromethane to
give a yellow solid which was slurried with ether then collected.
The solid was washed with ether and dried to give the subtitled
compound as a colourless solid (8.74 g).
MS: APCI (-ve) 382/4 (M-H), 382 (100%)
[0095] .sup.1H NMR: .delta. (DMSO) 4.47 (s, 2H), 7.13-7.55 (m,3H),
8.14 (s,1H), 13.33 (bs,1H)
[0096] f)
2-[[(2,3-difluorophenyl)methyl]thio]4-[[(1R)-2-hydroxy-1-methyl-
ethyl]amino]-7(8H)-pteridinone
[0097] The product of example 2, step (e) (8.7 g) was dissolved in
N-methylpyrrolidinone (40 ml) and Hunigs base (7.9 ml) was added
followed by D-alaninol (2.7 ml). The mixture was stirred at
100.degree. C. for 15 mins. The cooled solution was poured onto
water, (11), and acidified with dilute hydrochloric acid. The solid
which separated was collected, washed with water and air dried.
Crystallisation from acetonitrile afforded the title compound as a
pale yellow solid (7.4 g).
m.p. 215-217.degree. C.
MS: APCI (+ve) 380 (M+H, 100%)
[0098] .sup.1NMR: .delta. (DMSO) 1.14 (d, 3H), 3.48 (m, 2H), 4.31
(m, 1H), 4.45 (dd, 2H) 4.82 (t, 1H) 7.15 (m, 1H), 7.33 (m, 1H),
7.47 (t, 1H), 7.76 (d, 1H), 7.83 (d,1H), 12.70 (s, 1H).
[0099] g)
2-[[(2,3-Difluorophenyl)methyl]thio-4-[[(1R)-2-hydroxy-1-methyl-
ethyl]amino-7(8H)-pteridinethione
[0100] The product of example 2, step (f) (0.50 g) and Lawessons
reagent (1.06 g) were stirred in dioxan (10 mL) and heated under
reflux for 30 mins. Water (5 mL) was added and heating continued
for 10 mins. The cooled mixture was diluted with water to give a
suspension. The solid was collected, washed with water and dried.
Purification by flash chromatography over silica using
dichloromethane/acetonitrile (5-10%) as eluant afforded the title
compound 0.225 g.
mp 232-233.degree. C.
MS: APCI 396 (M+H, 100%)
[0101] .sup.1H NMR: 3 (DMSO) 1.15(d, 3H), 3.47 (m, 2H), 4.30 (m,
1H), 4.46 (q, 2H), 4.82 (bm, 1H), 7.16 (m, 1H), 7.34 (m, 1H), 7.53
(t, 1H), 8.10 (d, 1H), 8.21 (s,1H), 14.45 (s, 1H).
Pharmacological Data
Ligand Binding Assay
[0102] [.sup.125I]IL-8 (human, recombinant) was purchased from
Amersham, U.K. with a specific activity of 2,000 Ci/mmol. All other
chemicals were of analytical grade. High levels of hrCXCR2 were
expressed in HEK 293 cells (human embryo kidney 293 cells ECACC No.
85120602) (Lee et al. (1992) J. Biol. Chem. 267 pp 16283-16291).
hrCXCR2 cDNA was amplified and cloned from human neutrophil mRNA.
The DNA was cloned into PCRScript (Stratagene) and clones were
identified using DNA. The coding sequence was sub-cloned into the
eukaryotic expression vector RcCMV (Invitrogen). Plasmid DNA was
prepared using Quiagen Megaprep 2500 and transfected into HEK 293
cells using Lipofectamine reagent (Gibco BRL). Cells of the highest
expressing clone were harvested in phosphate-buffered saline
containing 0.2% (w/v) ethylenediaminetetraacetic acid (EDTA) and
centrifuged (200 g, 5 min.). The cell pellet was resuspended in ice
cold homogenisation buffer [10 mM HEPES (pH 7.4), 1 mM
dithiothreitol, 1 mM EDTA and a panel of protease inhibitors (1 mM
phenyl methyl sulphonyl fluoride, 2; g/ml soybean trypsin
inhibitor, 3 mM benzamidine, 0.5 .mu.g/ml leupeptin and 100
.mu.g/ml bacitracin)] and the cells left to swell for 10 minutes.
The cell preparation was disrupted using a hand held glass
mortar/PTFE pestle homogeniser and cell membranes harvested by
centrifugation (45 minutes, 100,000 g, 4.degree. C). The membrane
preparation was stored at -70.degree. C. in homogenisation buffer
supplemented with Tyrode's salt solution (137 mM NaCl, 2.7 mM KCl,
0.4 mnM NaH.sub.2PO.sub.4), 0.1%(w/v) gelatin and 10%(v/v)
glycerol.
[0103] All assays were performed in a 96-well MultiScreen 0.45
.mu.m filtration plates (Millipore, U.K.). Each assay contained
.about.50 pM [.sup.125L]IL-8 and membranes (equivalent to
.about.200,000 cells) in assay buffer [Tyrode's salt solution
supplemented with 19 mM HEPES (pH 7.4), 1.8 mM CaCl.sub.2, 1 mM
MgCl.sub.2, 0.125 mg/ml bacitracin and 0.1%(w/v) gelatin]. In
addition, a compound of formula (I) according to the Examples was
pre-dissolved in DMSO and added to reach a final concentration of
1%(v/v) DMSO. The assay was initiated with the addition of
membranes and after 1.5 hours at room temperature the membranes
were harvested by filtration using a Millipore MultiScreen vacuum
manifold and washed twice with assay buffer (without bacitracin).
The backing plate was removed from the MultiScreen plate assembly,
the filters dried at room temperature, punched out and then counted
on a Cobra .gamma.-counter.
[0104] The compounds of formula (I) according to the Examples were
found to have IC.sub.50 values of less than (<) 10 .mu.M.
Intracellular Calcium Mobilisation Assay
[0105] Human neutrophils were prepared from EDTA-treated peripheral
blood, as previously described (Baly et al. (1997) Methods in
Enzymology 287 pp 70-72), in storage buffer [Tyrode's salt solution
(137 mM NaCl, 2.7 mM KCl, 0.4 mM NaH.sub.2PO.sub.4) supplemented
with 5.7 mM glucose and 10 mM HEPES (pH 7.4)].
[0106] The chemokine GRO.alpha. (human, recombinant) was purchased
from R&D Systems (Abingdon, U.K.). All other chemicals were of
analytical grade. Changes in intracellular free calcium were
measured fluorometrically by loading neutrophils with the calcium
sensitive fluorescent dye, fluo-3, as described previously (Merritt
et al. (1990) Biochem. J. 269, pp 513-519). Cells were loaded for 1
hour at 37.degree. C. in loading buffer (storage buffer with
0.1%(w/v) gelatin) containing 5 .mu.M fluo-3 AM ester, washed with
loading buffer and then resuspended in Tyrode's salt solution
supplemented with 5.7 mM glucose, 0.1%(w/v) bovine serum albumin
(BSA), 1.8 mM CaCl.sub.2 and 1 mM MgCl.sub.2. The cells were
pipetted into black walled, clear bottom, 96 well micro plates
(Costar, Boston, U.S.A.) and centrifuged (200 g, 5 minutes, room
temperature).
[0107] A compound of formula (I) according to the Examples was
pre-dissolved in DMSO and added to a final concentration of
0.1%(v/v) DMSO. Assays were initiated by the addition of an
A.sub.50 concentration of GRO.alpha. and the transient increase in
fluo-3 fluorescence (.lamda..sub.Ex=490 nm and .lamda..sub.Em=520
nm) monitored using a FLIPR (Fluorometric Imaging Plate Reader,
Molecular Devices, Sunnyvale, U.S.A.).
[0108] The compounds of formula (I) according to the Examples were
tested and found to be antagonists of the CXCR2 receptor in human
neutrophils.
* * * * *