U.S. patent application number 10/935248 was filed with the patent office on 2005-02-03 for anti-inflammatory indole derivatives.
This patent application is currently assigned to AstraZeneca AB. Invention is credited to Faull, Allan W., Kettle, Jason.
Application Number | 20050026975 10/935248 |
Document ID | / |
Family ID | 10847082 |
Filed Date | 2005-02-03 |
United States Patent
Application |
20050026975 |
Kind Code |
A1 |
Faull, Allan W. ; et
al. |
February 3, 2005 |
Anti-inflammatory indole derivatives
Abstract
Therapeutic compounds of formula (I) wherein X is CH.sub.2 or
SO.sub.2; R.sup.1 is an optionally substituted aryl or heteroaryl
ring; R.sup.2 and R.sup.3 are various specified groups, R.sup.4 is
a group NHCOR.sup.15, NHSO.sub.2R.sup.15 or OCONR.sup.16R.sup.17
where R.sup.15 is optionally substituted alkyl, optionally
substituted aryl or optionally substituted heteroaryl and R.sup.16
and R.sup.17 are independently selected from hydrogen, optionally
substituted alkyl, optionally substituted aryl and optionally
substituted heteroaryl, with the proviso that at least one of
R.sup.16 or R.sup.17 is other than hydrogen, or R.sup.16 and
R.sup.17 together with the nitrogen atom to which they are attached
from an optionally substituted heterocyclic ring which optionally
contains further heteroatoms; and R.sup.5, R.sup.6 and R.sup.7 are
independently selected from hydrogen, a functional group or an
optionally substituted hydrocarbyl groups or optionally substituted
heterocyclic groups; and further provided that when R.sup.4 is a
group NHCOR.sup.15, R.sup.15 is substituted alkyl, optionally
substituted aryl or optionally substituted heteroaryl; as well as
pharmaceutical compositions containing them are described and
claimed. These compounds and compositions are useful in the
treatment of disease mediated by monocyte chemoattractant protein-1
or RANTES (Regulated Upon Activation, Normal T-cell Expressed and
Secreted), such as inflammatory disease. 1
Inventors: |
Faull, Allan W.; (Cheshire,
GB) ; Kettle, Jason; (Cheshire, GB) |
Correspondence
Address: |
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
AstraZeneca AB
Sodertalje
SE
|
Family ID: |
10847082 |
Appl. No.: |
10/935248 |
Filed: |
September 7, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10935248 |
Sep 7, 2004 |
|
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09889515 |
Oct 10, 2001 |
|
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09889515 |
Oct 10, 2001 |
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PCT/GB00/00260 |
Jan 31, 2000 |
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Current U.S.
Class: |
514/378 ;
514/381; 514/414; 514/419; 548/243; 548/254; 548/465; 548/495 |
Current CPC
Class: |
C04B 35/632 20130101;
A61P 43/00 20180101; C07D 403/12 20130101; C07D 409/14 20130101;
A61P 29/00 20180101; C07D 401/12 20130101; C07D 209/42
20130101 |
Class at
Publication: |
514/378 ;
514/381; 514/414; 514/419; 548/243; 548/465; 548/495; 548/254 |
International
Class: |
A61K 031/42; A61K
031/41; A61K 031/405; C07D 413/02; C07D 43/02 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 5, 1999 |
GB |
9902459.8 |
Claims
1. A compound of formula (I) 29X is CH.sub.2 or SO.sub.2 R.sup.1 is
an optionally substituted aryl or heteroaryl ring; R.sup.2 is
carboxy, cyano, --C(O)CH.sub.2OH, --CONHR.sup.8,
--SO.sub.2NHR.sup.9, tetrazol-5-yl, SO.sub.3H, or a group of
formula (VI) 30where R.sup.8 is selected from hydrogen, alkyl,
aryl, cyano, hydroxy, --SO.sub.2R.sup.12 where R.sup.12 is alkyl,
aryl, heteroaryl, or haloalkyl, or R.sup.8 is a group
--(CHR.sup.13).sub.r--COOH where r is an integer of 1-3 and each
R.sup.13 group is independently selected from hydrogen or alkyl;
R.sup.9 is hydrogen, alkyl, optionally substituted aryl such as
optionally substituted phenyl or optionally subtituted heteroaryl
such as 5 or 6 membered heteroaryl groups, or a group COR.sup.14
where R.sup.14 is alkyl, aryl, heteroaryl or haloalkyl; R.sup.10
and R.sup.11 are independently selected from hydrogen or alkyl,
particularly C.sub.1-4alkyl; R.sup.3 is hydrogen a functional
group, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl, optionally substituted
aryl, optionally substituted heterocyclyl, optionally substituted
alkoxy, optionally substituted aralkyl, optionally substituted
aralkyloxy, optionally substituted cycloalkyl; R.sup.4 is a group
NHCOR.sup.15, NHSO.sub.2R.sup.15 or OCONR.sup.16R.sup.17 where
R.sup.15 is optionally substituted alkyl, optionally substituted
aryl or optionally substituted heteroaryl and R.sup.16 and R.sup.17
are independently selected from hydrogen, optionally substituted
alkyl, optionally substituted aryl and optionally substituted
heteroaryl, with the proviso that at least one of R.sup.16 or
R.sup.17 is other than hydrogen, or R.sup.16 and R.sup.17 together
with the nitrogen atom to which they are attached form an
optionally substituted heterocyclic ring which optionally contains
further heteroatoms; and R.sup.5, R.sup.6 and R.sup.7 are
independently selected from hydrogen, a functional group or an
optionally substituted hydrocarbyl groups or optionally substituted
heterocyclic groups; and further provided that when R.sup.4 is a
group NHCOR.sup.15, R.sup.15 is substituted alkyl, optionally
substituted aryl or optionally substituted heteroaryl.
2. A compound according to claim 1 wherein a group R.sup.15,
R.sup.16 and R.sup.17 as they appear in the definition of R.sup.4,
is substituted by at least one functional group, or an aryl or
heterocyclyl groups, either of which may themselves be substituted
by one or more functional groups or further aryl or heterocyclyl
groups.
3. A compound according to any one of the preceding claims wherein
R.sup.4 is a group NHCOR.sup.15 or NHSO.sub.2R.sup.15 and R.sup.15
is a substituted alkyl group or an optionally subsituted
hetercyclyl or optionally substituted phenyl group.
4. A compound according to claim 3 wherein R.sup.15 is alkyl
substituted by a group of formula NR.sup.19R.sup.20 where R.sup.19
and R.sup.20 are independently selected from hydrogen or optionally
substituted hydrocarbyl, or R.sup.19 and R.sup.20 together form an
optionally substituted ring which optionally contains further
heteroatoms such as S(O).sub.m, oxygen and nitrogen, n is an
integer of 1 or 2, m is 1 or 2.
5. A compound according to any one of the preceding claims where
R.sup.2 is carboxy.
6. A compound according to any one of the preceding claims wherein
R.sup.1 is 3,4-dichlorophenyl, 3-fluoro-4-chlorophenyl,
3-chloro-4-fluorophenyl or 2,3-dichloropyrid-5-yl.
7. A compound according to any one of the preceding claims where X
is CH.sub.2.
8. A process for preparing a compound according to claim 1 which
process comprises either (a) where R.sup.4 is NHCOR.sup.15or
NHSO.sub.2R.sup.15, reacting a compound of formula (VII) 31where X,
R.sup.1, R.sup.3, R.sup.5, R.sup.6 and R.sup.7 are as defined in
claim 1, and R.sup.2 is a group R.sup.2 as defined in relation to
formula (I) or a protected form thereof, with a compound of formula
(VIII) Z-R.sup.22 (VIII) where Z is a leaving group and R.sup.22 is
a group COR.sup.15' or SO.sub.2R.sup.15' where R.sup.15' is group
R.sup.15 as defined in relation to formula (I) or a precursor
thereof; or (b) where R.sup.4 is a group OCONR.sup.16R.sup.17,
reacting a compound of formula (VIIA) 32where X, R.sup.2', R1,
R.sup.3, R.sup.5, R.sup.6 and R.sup.7 are as defined claim 1 and
R.sup.2 is a group R.sup.2 as defined in claim 1 or a protected
form thereof, with a compound of formula (VIIIA)
Z-CONR.sup.16R.sup.17 (VIIIA) where Z, R.sup.16 and R.sup.17 are as
defined above; and thereafter if desired or necessary: (i)
converting a precursor group R.sup.15' to a group R.sup.15 and/or
converting a group R.sup.15 to a different such group; (ii)
deprotecting a group R.sup.2' to a group R.sup.2.
9. A pharmaceutical composition comprising a compound according to
any one of claims 1 to 7 in combination with a pharmaceutically
acceptable carrier.
10. A compound according to any one of claims 1 to 7 for use in the
preparation of a medicament for use in the treatment of disease
mediated by monocyte chemoattractant protein-1 or RANTES Regulated
upon Activation, Normal T-cell Expressed and Secreted), such as
inflammatory disease.
Description
[0001] The present invention relates to chemical compounds, to
their production as well as to pharmaceutical compositions
containing them as well as to their use in therapy, in particular
of inflammatory disease.
[0002] MCP-1 is a member of the chemokine family of
pro-inflammatory cytokines which mediate leukocyte chemotaxis and
activation. MCP-1 is a C--C chemokine which is one of the most
potent and selective T-cell and monocyte chemoattractant and
activating agents known. MCP-1 has been implicated in the
pathophysiology of a large number of inflammatory diseases
including rheumatoid arthritis, glomerular nephritides, lung
fibrosis, restenosis (International Patent Application WO
94/09128), alveolitis (Jones et al., 1992, J. Immunol., 149, 2147)
and asthma. Other disease areas where MCP-1 is thought to play a
part in their pathology are atherosclerosis (e.g. Koch et al.,
1992, J. Clin. Invest., 90, 772-779), psoriasis (Deleuran et al.,
1996, J. Dermatological Science, 13, 228-236), delayed-type
hypersensitivity reactions of the skin, inflammatory bowel disease
(Grimm et al., 1996, J. Leukocyle Biol., 59, 804-812), multiple
sclerosis and brain trauma (Berman et al, 1996, J. Immunol., 156,
3017-3023). An MCP-1 inhibitor may also be useful to treat stroke,
reperfusion injury, ischemia, myocardial infarction and transplant
rejection.
[0003] MCP-1 acts through the MCP-1 receptor (also known as the
CCR2 receptor). MCP-2 and MCP-3 may also act, at least in part,
through the MCP-1 receptor. Therefore in this specification, when
reference is made to "inhibition or antagonism of MCP-1" or "MCP-1
mediated effects" this includes inhibition or antagonism of MCP-2
and/or MCP-3 mediated effects when MCP-2 and/or MCP-3 are acting
through the MCP-1 receptor.
[0004] Copending International Patent Application Nos.
PCT/GB98/02340 and PCT/GB98/02341 describe and claim groups of
compounds based upon the indole ring structure which arc inhibitors
of MCP-1 and therefore have applications in therapy.
[0005] The use of certain indole derivatives as NMDA antagonists is
described is U.S. Pat. No. 5,051,442, WO93 12780, EP-483881. Other
indoles and their use as inhibitors of leukotriene biosynthesis is
described in for example, EP-A-275-667.
[0006] The applicants have found a particular substitution on the
indole ring produces advantageous results when used therapeutically
as inhibitors of MCP-1.
[0007] According to the present invention there is provided a
compound of formula (I) 2
[0008] X is CH.sub.2 or SO.sub.2
[0009] R.sup.1 is an optionally substituted aryl or heteroaryl
ring;
[0010] R.sup.2 is carboxy, cyano, --C(O)CH.sub.2OH, --CONHR.sup.8,
--SO.sub.2NHR.sup.9, tetrazol-5-yl, SO.sub.3H, or a group of
formula (VI) 3
[0011] where R.sup.8 is selected from hydrogen, alkyl, aryl, cyano,
hydroxy, --SO.sub.2R.sup.12 where R.sup.12 is alkyl, aryl,
heteroaryl, or haloalkyl, or R.sup.8is a group
--(CHR.sup.13).sub.r--COOH where r is an integer of 1-3 and each
R.sup.13 group is independently selected from hydrogen or alkyl;
R.sup.9 is hydrogen, alkyl, optionally substituted aryl such as
optionally substituted phenyl or optionally subtituted heteroaryl
such as 5 or 6 membered heteroaryl groups, or a group COR.sup.14
where R.sup.13 is alkyl, aryl, heteroaryl or haloalkyl; R.sup.10
and R.sup.11 are independently selected from hydrogen or alkyl,
particularly C.sub.1-4 alkyl;
[0012] R.sup.3 is hydrogen, a functional group, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted heterocyclyl, optionally substituted alkoxy, optionally
substituted aralkyl, optionally substituted aralkyloxy, optionally
substituted cycloalkyl;
[0013] R.sup.4 is a group NHCOR.sup.15, NHSO.sub.2R.sup.15 or
OCONR.sup.16R.sup.17 where R.sup.15 is optionally substituted
alkyl, optionally substituted aryl or optionally substituted
heteroaryl and R.sup.16 and R.sup.17 are independently selected
from hydrogen, optionally substituted alkyl, optionally substituted
aryl and optionally substituted heteroaryl, with the proviso that
at least one of R.sup.16 or R.sup.17 is other than hydrogen, or
R.sup.16 and R.sup.17 together with the nitrogen atom to which they
are attached form an optionally substituted heterocyclic ring which
optionally contains further heteroatoms; and
[0014] R.sup.5, R.sup.6 and R.sup.7 are independently selected from
hydrogen, a functional group or an optionally substituted
hydrocarbyl groups or optionally substituted heterocyclic
groups.
[0015] Suitably, where R.sup.4 is a group NHCOR.sup.15, R.sup.15 is
substituted alkyl, optionally substituted aryl or optionally
substituted heteroaryl.
[0016] Compounds of formula (I) are inhibitors of monocyte
chemoattractant protein-1. In addition, they appear to inhibit
RANTES (Regulated upon Activation, Normal Tcell Expressed and
Secreted), induced chemotaxis. RANTES is another chemokine from the
same family as MCP-1, with a similar biological profile, but acting
though the CCR1 receptor. As a result, these compounds can be used
to treat disease mediated by these agents, in particular
inflammatory disease.
[0017] In this specification the term `alkyl` when used either
alone or as a suffix includes straight chained, branched
structures. These groups may contain up to 10, preferably up to 6
and more preferably up to 4 carbon atoms. Similarly the terms
"alkenyl" and "alkynyl" refer to unsaturated straight or branched
structures containing for example from 2 to 10, preferably from 2
to 6 carbon atoms. Cyclic moieties such as cycloalkyl, cycloalkenyl
and cycloalkynyl are similar in nature but have at least 3 carbon
atoms. Terms such as "alkoxy" comprise alkyl groups as is
understood in the art.
[0018] The term "halo" includes fluoro, chloro, bromo and iodo.
References to aryl groups include aromatic carbocylic groups such
as phenyl and naphthyl. The term "heterocyclyl" includes aromatic
or non-aromatic rings, for example containing from 4 to 20,
suitably from 5 to 8 ring atoms, at least one of which is a
heteroatom such as oxygen, sulphur or nitrogen. Examples of such
groups include furyl, thienyl, pyrrolyl, pyrrolidinyl, imidazolyl,
triazolyl, thiazolyl, tetrazolyl, oxazolyl, isoxazolyl, pyrazolyl,
pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl,
quinolinyl, isoquinolinyl, quinoxalinyl, benzothiazolyl,
benzoxazolyl, benzothienyl or benzofuryl.
[0019] "Heteroaryl" refers to those groups described above which
have an aromatic character. The term "aralkyl" refers to aryl
substituted alkyl groups such as benzyl.
[0020] Other expressions used in the specification include
"hydrocarbyl" which refers to any structure comprising carbon and
hydrogen atoms. For example, these may be alkyl, alkenyl, alkynyl,
aryl, heterocyclyl, alkoxy, aralkyl, cycloalkyl, cycloalkenyl or
cycloalkynyl.
[0021] The term "functional group" refers to reactive substituents.
They may comprise electron-donating or electron-withdrawing.
Examples of such groups include halo, cyano, nitro,
C(O).sub.nR.sup.18, OR.sup.18, S(O).sub.nR.sup.18,
NR.sup.19R.sup.20, C(O)NR.sup.19R.sup.20, OC(O)NR.sup.19R.sup.20,
--NR.sup.19C(O).sub.nR.sup.18, --NR.sup.18CONR.sup.19R.sup.20,
--N.dbd.CR.sup.19R.sup.20, S(O).sub.mNR.sup.19R.sup.20 or
--NR.sup.19S(O).sub.mR.sup.18 where R.sup.18, R.sup.19 and R.sup.20
are independently selected from hydrogen or optionally substituted
hydrocarbyl, or R.sup.19 and R.sup.20 together form an optionally
substituted ring which optionally contains further heteroatoms such
as S(O).sub.m, oxygen and nitrogen, n is an integer of 1 or 2, m is
1 or 2.
[0022] Suitable optional substituents for hydrocarbyl groups
R.sup.18, R.sup.19 and R.sup.20 include halo, perhaloalkyl such as
trifluoromethyl, mercapto, hydroxy, carboxy, alkoxy, heteroaryl,
heteroaryloxy, alkenyloxy, alkynyloxy, alkoxyalkoxy, aryloxy (where
the aryl group may be substituted by halo, nitro, or hydroxy),
cyano, nitro, amino, mono- or di-alkyl amino, oximino or
S(O).sub.nR.sup.x where n is as defined above and R.sup.x is alkyl
such as C.sub.1-4 alkyl.
[0023] Suitable substituents for these hydrocarbyl or heterocylic
groups include those listed above for R.sup.18, R.sup.19 and
R.sup.20.
[0024] Suitably R.sup.1 is an optionally substituted phenyl,
pyridyl, naphthyl, furyl or thienyl ring, and in particular is a
substituted phenyl or pyridyl ring.
[0025] Suitable optional substitutents for R.sup.1 in formula (I)
include alkyl, alkenyl, alkynyl, halo, haloalkyl including
perhaloalkyl such as trifluoromethyl, mercapto, alkoxy, haloalkoxy,
alkenyloxy, alkynyloxy, hydroxyalkoxy, alkoxyalkoxy, alkanoyl,
alkanoyloxy, cyano, nitro, amino, mono- or di-alkyl amino, oximino,
sulphonamido, carbamoyl, mono or dialkylcarbamoyl or
S(O).sub.mR.sup.21 where m is as defined above and R.sup.21 is
hydrocarbyl.
[0026] Particular examples of substituents R.sup.5, R.sup.6 and
R.sup.7, and where appropriate also R.sup.4 include hydrogen,
hydroxy, halo, optionally substituted alkyl such as aralkyl,
carboxyalkyl or the amide derivative thereof; alkoxy; aryloxy;
aralkyloxy; or an amino group which is optionally substituted with
alkyl, aryl or aralkyl. A specific functional group which is
suitable for R.sup.4, R.sup.5, R.sup.6 and/or R.sup.7 is a group of
sub-formula (IV). 4
[0027] Particular examples of groups R.sup.5, R.sup.6 and R.sup.7
are hydrogen, hydroxy, halo or alkoxy. In particular R.sup.6 and
R.sup.7 are hydrogen. R.sup.5 may be hydrogen but in addition is
suitably a small subsitutent such as hydroxy, halo or methoxy.
[0028] Particular substituents for R.sup.1 include trifluoromethyl,
C.sub.1-4alkyl, halo, trifluoromethoxy, C.sub.1-4alkoxy,
C.sub.1-4alkanoyl, C.sub.1-4alkanoyloxy, nitro, carbamoyl,
C.sub.1-4alkoxycarbonyl, C.sub.1-4alkylsulphanyl,
C.sub.1-4alkylsulphinyl- , C.sub.1-4alkylsulphonyl, sulphonamido,
carbamoylC.sub.1-4alkyl,
N--(C.sub.1-4alkyl)carbamoylC.sub.1-4alkyl,
N--(C.sub.1-4alkyl).sub.2carb- amoyl-C.sub.1-4alkyl,
hydroxyC.sub.1-4alkyl or C.sub.1-4alkoxyC.sub.1-4alk- yl.
[0029] Additionally or alternatively, two such substituents
together may form a divalent radical of the formula
--O(CH.sub.2).sub.1-4O-- attached to adjacent carbon atoms on the
R.sup.1 ring.
[0030] Preferred substitutents for R.sup.1 are one or more
non-polar substituents such as halo.
[0031] In particular, R.sup.1 is substituted by one or more halo
groups, in particular chlorine. A particular example of an R.sup.1
group is 3,4-dichlorophenyl, 3-fluoro-4-chlorophenyl,
3-chloro-4-fluorophenyl or 2,3-dichloropyrid-5-yl.
[0032] Examples of groups R.sup.2 include carboxy; cyano;
tetrazol-5-yl; SO.sub.3H; --CONHR.sup.8 where R.sup.8 is selected
from cyano, hydroxy, --SO.sub.2R.sup.12 where R.sup.12 is alkyl
such as C.sub.1-4alkyl, aryl such as phenyl, heteroaryl or
trifluoromethyl, or R.sup.8 is a group --(CHR.sup.10).sub.r--COOH
where r is an integer of 1-3 and each R.sup.10 group is
independently selected from hydrogen or alkyl such as C.sub.1-4
alkyl; or R.sup.2 is a group --SO.sub.2NHR.sup.9 where R.sup.9 is
an optionally substituted phenyl or an optionally substituted 5 or
6 membered heteroaryl group, or a group COR.sup.14 where R.sup.14
is alkyl such as C.sub.1-4alkyl, aryl such as phenyl, heteroaryl or
trifluoromethyl, or R.sup.2 is a group of formula (VI) 5
[0033] where R.sup.10 and R.sup.11 are independently selected from
hydrogen or alkyl, particularly C.sub.1-4 alkyl.
[0034] Preferably R.sup.2 is carboxy or a pharmaceutically
acceptable salt or ester thereof.
[0035] Suitable groups R.sup.3 include hydrogen, fluoro, chloro,
bromo, iodo, methyl, cyano, trifluoromethyl, hydroxymethyl,
alkoxyalkyl such as C.sub.1-4alkoxymethyl, methoxy, benzyloxy,
carboxyalkoxy such as carboxymethoxy, methylsulphanyl,
methylsulphinyl, methylsulphonyl or carboxyC.sub.3-6cycloalkyl,
--(CHR.sup.22).sub.r--NR.sup.23R.sup.24 (where r is 0-2, each
R.sup.22 is independently hydrogen or alkyl, in particular
C.sub.1-4alkyl, R.sup.23 and R.sup.24 are independently selected
from H and C.sub.1-4alkyl or R.sup.23 and R.sup.24 together with
the nitrogen to which they are attached form a 5 or 6 membered ring
optionally containing one further heteroatom selected from O, N, S,
S(O) or SO.sub.2. Suitably R.sup.23 and R.sup.24 together form a
heterocylic ring such as morpholino or piperazinyl.
[0036] Other such groups R.sup.3 include optionally substituted
aryl groups, such as optionally substituted phenyl or naphthyl
group. Suitable substituents for phenyl groups R.sup.3 include one
or more groups selected from chlorine, fluorine, methyl,
trifluoromethyl, trifluoromethoxy, amino, formyl, phenyl, methoxy,
phenoxy or phenyl.
[0037] R.sup.3 may comprise a range of substituents as listed
above, in particular, hydrogen or a small substituent group such as
C.sub.1-4alkyl in particular methyl, or trifluoromethyl, and is
preferably hydrogen.
[0038] Suitable optional substitutents for the group R.sup.15,
R.sup.16 and R.sup.17 as they appear in the definition of R.sup.4,
include functional groups as hereinbefore defined, as well as aryl
or heterocyclyl groups, either of which may themselves be
substituted by one or more functional groups or further aryl or
heterocyclyl groups.
[0039] Particular examples of substituents for groups R.sup.15,
R.sup.16 and R.sup.17 include one or more groups selected from halo
such as chloro; hydroxy, cyano; amino, mono- or di-alkylamino;
C.sub.1-4 alkoxy; carboxy; sulphonamido; CONH.sub.2; alkylamido
where the alkyl moiety is optionally substituted for example with a
functional groups such as carboxy; morpholino; pyridyl;
pyrimidinyl; phenyl optionally substituted by halo such as chloro,
hydroxy, alkoxy such as methoxy, carbamoyl, acyl such as acetyl, or
hydroxyalkyl where the alkyl group suitably includes at least two
carbon atoms, such as hydroxyethyl. Other examples of subsitutents
for phenyl groups R.sup.15 is alkanoylamino group such as
methoylamino.
[0040] Where R.sup.15, R.sup.16 and/or R.sup.17 is a heterocyclyl
group, or where R.sup.16 and R.sup.17 together form an optionally
substituted heterocyclic ring, these may be substituted by
functional groups such as halo or hydroxy, or by alkyl groups such
as methyl or ethyl, or alkenyl or alkynyl groups any of which may
be subsituted, for example with hydroxy, as well as with further
heteroaryl groups such as pyridyl. Particular examples of
heterocyclic groups R.sup.15, R.sup.16 and/or R.sup.17 are
optionally substituted thiophenyl, optionally substituted
imidazolyl, optionally subtituted pyridyl.
[0041] Thus thiophenyl groups R.sup.15, R.sup.16 and/or R.sup.17
may comprise pyridyl-thiophenyl, whilst an example of a substituted
imidazolyl group for R.sup.15, R.sup.16 and/or R.sup.17 is
methylimidazolyl and halopyridyl in particular chloropyridyl is an
example of a substituted pyridyl moiety for these groups.
[0042] Particular examples of R.sup.15 include alkyl in particular
methyl optionally substituted by a functional groups or, in
particular, a heterocyclyl group where the heterocyclyl group may
be optionally substituted by a functional group such as halo or
hydroxy or by an alkyl group such as methyl. Preferably, R.sup.15
is a substituted alkyl group. Where the substitutent is a
functional group, it is preferably a group of formula
NR.sup.19R.sup.20 where R.sup.19 and R.sup.20 are as defined above.
Thus examples of substituted alkyl groups R.sup.15 include
morpholinomethyl or alkyl such as methyl substituted with a
substituted alkyl amino group wherein the substitutents include
carboxy, alkanoyl, phenyl or alkyl sulphonyl.
[0043] Other examples of R.sup.15 are heterocylcyl groups which are
optionally subsitituted for example by alkyl such as methyl,
functional groups such as chloro or heterocycyl groups such as
pyridyl.
[0044] Particular examples of R.sup.16 and R.sup.17 are alkyl such
as methyl.
[0045] X is CH.sub.2 or SO.sub.2 and is preferably CH.sub.2.
[0046] Suitable pharmaceutically acceptable salts of compounds of
formula (I) include acid addition salts such as methanesulfonate,
fumarate, hydrochloride, hydrobromide, citrate, maleate and salts
formed with phosphoric and sulphuric acid. In another aspect
suitable salts are base salts such as an alkali metal salt for
example sodium, an alkaline earth metal salt for example calcium or
magnesium, an organic amine salt for example triethylamine,
morpholine, N-methylpiperidine, N-ethylpiperidine, procaine,
dibenzylamine, N,N-dibenzylethylamine or amino acids for example
lysine. There may be more than one cation or anion depending on the
number of charged functions and the valency of the cations or
anions. A preferred pharmaceutically acceptable salt is a sodium
salt.
[0047] An in vivo hydrolysable ester of a compound of the formula
(I) containing carboxy or hydroxy group is, for example, a
pharmaceutically acceptable ester which is hydrolysed in the human
or animal body to produce the parent acid or alcohol.
[0048] Suitable pharmaceutically acceptable esters for carboxy
include alkyl esters, such as C.sub.1-6 alkyl esters for example,
ethyl esters, C.sub.1-6alkoxymethyl esters for example
methoxymethyl, C.sub.1-6alkanoyloxymethyl esters for example
pivaloyloxymethyl, phthalidyl esters,
C.sub.3-8cycloalkoxy-carbonyloxyC.sub.1-6alkyl esters for example
1-cyclohexylcarbonyloxyethyl; 1,3-dioxolen-2-onylmethyl esters for
example 5-methyl-1,3-dioxolen-2-onylmethyl; and
C.sub.1-6alkoxycarbonyloxyethyl esters for example
1-methoxycarbonyloxyethyl and may be formed at any carboxy group in
the compounds of this invention.
[0049] Suitable pharmaceutically acceptable esters of compounds of
formula (I) are in vivo hydrolysable ester of a compound of the
formula (I) containing a hydroxy group includes inorganic esters
such as phosphate esters and .alpha.-acyloxyalkyl ethers and
related compounds which as a result of the in vivo hydrolysis of
the ester breakdown to give the parent hydroxy group. Examples of
.alpha.-acyloxyalkyl ethers include acetoxymethoxy and
2,2-dimethylpropionyloxymethoxy. A selection of in vivo
hydrolysable ester forming groups for hydroxy include alkanoyl,
benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl,
alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl
and N-(dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates),
dialkylaminoacetyl and carboxyacetyl.
[0050] Esters which are not in vivo hydrolysable are useful as
intermediates in the production of the compounds of formula (I) and
therefore these form a further aspect of the invention.
[0051] Thus examples of compounds of formula (I) include the
following:
1TABLE 1 6 Compd No. R.sup.3 R.sup.4 R.sup.5 R.sup.6 R.sup.a
R.sup.b 1 H 7 H H H H 2 H 8 H H Cl Cl 3 H 9 H H Cl Cl 4 H 10 H H Cl
Cl 5 H 11 H H Cl Cl 6 H 12 H H Cl Cl 7 H 13 H H Cl Cl 8 H
NHC(O)CH.sub.2NHCH.sub.2COOH H H Cl Cl 9 H 14 H H Cl Cl 10 H
OC(O)N(CH.sub.3).sub.2 H H Cl Cl 11 H 15 H H Cl Cl 12 H 16 H H Cl
Cl 13 H 17 H H Cl Cl 14 H NHC(O)CH.sub.2N(CH.sub.3)CH- .sub.2COOH H
H Cl Cl 15 H 18 H H Cl Cl where * indicates the point of attachment
of the group to the indole ring.
[0052] Compounds of formula (I) are suitably prepared by methods
such as those described in International Patent Application Nos.
PCT/GB98/02340 and PCT/GB98/02341.
[0053] In particular compounds of formula (I) where R.sup.4 is
NHCOR.sup.15 or NHSO.sub.2R.sup.15 can be prepared by reacting a
compound of formula (VII) 19
[0054] where X, R.sup.1, R.sup.3, R.sup.5, R.sup.6 and R.sup.7 are
as defined in relation to formula (I). R.sup.2 is a group R.sup.2
as defined in relation to formula (I) or a protected form thereof,
with a compound of formula (VIII)
Z-R.sup.22 (VIII)
[0055] where Z is a leaving group and R.sup.22 is a group
COR.sup.15' or SO.sub.2R.sup.15' where R.sup.15' is group R.sup.15
as defined in relation to formula (I) or a precursor thereof;
[0056] and thereafter if desired or necessary:
[0057] (i) converting a precursor group R.sup.15' to a group
R.sup.15 and/or converting a group R.sup.15 to a different such
group;
[0058] (ii) deprotecting a group R.sup.2' to a group R.sup.2.
[0059] Suitable leaving groups Z include halo such as chloro.
[0060] The reaction is suitably effected in an organic solvent such
as dichloromethane or tetrahydrofuran in the presence of a base
such as triethylamine or pyridine. Moderate temperatures, for
example from 0.degree. to 50.degree. C. and conveniently ambient
temperature, are employed in the reaction.
[0061] Compounds of formula (I) where R.sup.4 is a group
OCONR.sup.16R.sup.17 may be prepared by a broadly similar method by
reacting a compound of formula (VIIA) 20
[0062] where X, R.sup.2', R1, R.sup.3, R.sup.5, R.sup.6 and R.sup.7
are as defined in relation to formula (I), R.sup.2 is a group
R.sup.2 as defined in relation to formula (I) or a protected form
thereof, with a compound of formula (VIIIA)
Z-CONR.sup.16R.sup.17 (VIIIA)
[0063] where Z, R.sup.16 and R.sup.17 are as defined above.
[0064] Compounds of formula (VIIA) can be prepared by reacting a
compound of formula (IX) 21
[0065] where R.sup.3, R.sup.5, R.sup.6 and R.sup.7 are as defined
in relation to formula (I) and R.sup.2' is as defined in relation
to formula (VII) and R.sup.40 is a protecting group; with compound
of formula (X)
R.sup.1--X-Z.sup.1 (X)
[0066] where R.sup.1 and X are as defined in relation to formula
(I) and Z.sup.1 is a leaving group, and thereafter, removing the
protecting group R.sup.40.
[0067] Suitable leaving groups for Z include halide such as
chloride, bromide or iodide, as well as mesylate or tosylate. The
reaction is suitably effected in an organic solvent such as
dimethylformamide (DMF) tetrahydrofuran (THF) or DCM in the
presence of a base such as sodium hydride, sodium hydroxide,
potassium carbonate. Optionally the reaction is effected in the
presence of a suitable phase transfer catalyst. The choice of base
and solvent is interdependent to a certain extent in that certain
solvents are compatible with some bases only as is understood in
the art. For example, sodium hydride may preferably be used with
dimethylformamide or tetrahydrofuran and sodium hydroxide is
preferably used with dichloromethane and a phase transfer
catalyst.
[0068] The reaction can be carried out at moderate temperatures,
for example from 0 to 50.degree. C. and conveniently at about
ambient temperature.
[0069] Preferably, R.sup.2' is an ester group in the compound of
formula IX and this may be subsequently converted to an acid or to
another ester or salt, by conventional methods later in the
process. For example, when X is a group SO.sub.2 and R.sup.2 is a
methyl ester of carboxy, it may be converted to the corresponding
carboxylic acid by reaction with lithium iodide in dry pyridine or
DMF.
[0070] Suitable protecting groups R.sup.40 include acetyl or
benzyl. The reaction conditions employed will be variable depending
upon the nature of the protecting group R.sup.40 and would be
apparent to a skilled person. Acetyl groups may be removed by
reaction with a strong base such as sodium methoxide, whereas
benzyl groups may be removed by hydrogenation for example in the
presence of a catalyst such as a palladium catalyst.
[0071] Compounds of formula (IX) may be prepared by cyclisation of
a compound of formula (XII) 22
[0072] where R.sup.5, R.sup.6, R.sup.7 and R.sup.40 are as defined
above and R.sup.42 and R.sup.43 represent a combination of moieties
which can cyclise to form an appropriately substituted pyrrole
ring. For example, one of R.sup.42 and R.sup.43 can be a group of
formula --CH.dbd.C(R.sup.44)N.sub.3 where R.sup.44 is a group
R.sup.2 as defined above, or a protected form thereof, and the
other may be hydrogen. Cyclisation to form a compound of formula
(XII) may then be effected by heating for example under reflux in
an organic solvent, in particular a high boiling aprotic solvent
such as xylene or toluene.
[0073] Alternatively, one of R.sup.42 and R.sup.43 may be nitro and
the other may be a group of formula --CH.sub.2C(O)R.sup.2 where
R.sup.2' is as defined above in relation to formula (VII). These
compounds will cyclise in the presence of a catalyst such as
palladium on carbon in the presence of hydrogen. The reaction may
be effected at moderate temperatures for example of from 0 to
80.degree. C., conveniently at about ambient temperature.
[0074] Thus examples of compounds of formula (XII) include
compounds of formula (XIII) and (XIV) 23
[0075] Compounds of formula (XIII) where R.sup.3 is hydrogen may be
prepared for example by reacting a compound of formula (XV) 24
[0076] with a compound of formula (XVI)
N.sub.3CH.sub.2R.sup.2' (XVI)
[0077] where R.sup.5, R.sup.6, R.sup.7, and R.sup.2' are as defined
hereinbefore. The reaction may be effected in an organic solvent
such as ethanol at low temperatures of from -20 to 0.degree. C.,
suitably at about 0.degree. C. The reaction is suitably effected in
the presence of a base such as an alkoxide, in particular an
ethoxide, for example potassium ethoxide.
[0078] Compounds of formula (XVI) are suitably prepared by reacting
a compound of formula (XVII)
R.sub.47CH.sub.2R.sup.2' (XVII)
[0079] where R.sup.2' is defined above and R.sup.47 is a leaving
group such as halide and in particular bromide, with an azide salt,
such as an alkali metal azide salt in particular sodium azide.
[0080] Compounds of formula (XIV) may be prepared by reacting a
compound of formula (XVIII) 25
[0081] where R.sup.5, R.sup.6, R.sup.7, R.sup.3, R.sup.40 and
R.sup.2' are as defined above, with a compound of formula (XIX)
26
[0082] where R.sup.2' is as defined above and R.sup.48 leaving
group such as hydroxy. Examples of compounds of formula (XVI) are
oxalates such as diethyloxalate. The reaction is suitably effected
in the presence of a base such as sodium hydride in an organic
solvent such as THF. Moderate temperatures of from 0.degree. to
40.degree. C. and conveniently ambient temperature is employed.
[0083] Compounds of formula (VII) are suitably prepared using a
reaction analogous to that between compounds (IX) and (X), where in
place of the compound of formula (IX), a compound of formula (IXA)
is employed 27
[0084] where R.sup.2', R.sup.3, R.sup.5, R.sup.6 and R.sup.7 are as
defined above. Such compounds may be obtained by reduction of the
corresponding nitro compound of formula (XX) 28
[0085] where R.sup.2', R.sup.3, R.sup.5, R.sup.6 and R.sup.7 are as
defined above.
[0086] Compounds of formula (X), (XVI), (XV), (XVII), (XVIII),
(XIX) and (XX) are either known compounds or they may be prepared
from known compounds by conventional literature methods.
[0087] According to a further aspect of the invention there is
provided a compound of the formula (I) as defined herein, or a
pharmaceutically acceptable salt or an in vivo hydrolysable ester
thereof, for use in a method of treatment of the human or animal
body by therapy. In particular, the compounds are used in methods
of treatment of inflammatory disease.
[0088] According to a further aspect of the present invention there
is provided a method for antagonising an MCP-1 mediated effect in a
warm blooded animal, such as man, in need of such treatment, which
comprises administering to said animal an effective amount of a
compound of formula (I), or a pharmaceutically acceptable salt, or
an in vivo hydrolysable ester thereof.
[0089] The invention also provides a pharmaceutical composition
comprising a compound of formula (I) as defined herein, or a
pharmaceutically acceptable salt, or an in vivo hydrolysable ester
thereof, in combination with a pharmaceutically acceptable diluent
or carrier.
[0090] The compositions of the invention may be in a form suitable
for oral use (for example as tablets, lozenges, hard or soft
capsules, aqueous or oily suspensions, emulsions, dispersible
powders or granules, syrups or elixirs), for topical use (for
example as creams, ointments, gels, or aqueous or oily solutions or
suspensions), for administration by inhalation (for example as a
finely divided powder or a liquid aerosol), for administration by
insufflation (for example as a finely divided powder) or for
parenteral administration (for example as a sterile aqueous or oily
solution for intravenous, subcutaneous, intramuscular or
intramuscular dosing or as a suppository for rectal dosing).
[0091] The compositions of the invention may be obtained by
conventional procedures using conventional pharmaceutical
excipients, well known in the art. Thus, compositions intended for
oral use may contain, for example, one or more colouring,
sweetening, flavouring and/or preservative agents.
[0092] Suitable pharmaceutically acceptable excipients for a tablet
formulation include, for example, inert diluents such as lactose,
sodium carbonate, calcium phosphate or calcium carbonate,
granulating and disintegrating agents such as corn starch or
algenic acid; binding agents such as starch; lubricating agents
such as magnesium stearate, stearic acid or talc; preservative
agents such as ethyl or propyl p-hydroxybenzoate, and
anti-oxidants, such as ascorbic acid. Tablet formulations may be
uncoated or coated either to modify their disintegration and the
subsequent absorption of the active ingredient within the
gastrointestinal track, or to improve their stability and/or
appearance, in either case, using conventional coating agents and
procedures well known in the art.
[0093] Compositions for oral use may be in the form of hard gelatin
capsules in which the active ingredient is mixed with an inert
solid diluent, for example, calcium carbonate, calcium phosphate or
kaolin, or as soft gelatin capsules in which the active ingredient
is mixed with water or an oil such as peanut oil, liquid paraffin,
or olive oil.
[0094] Aqueous suspensions generally contain the active ingredient
in finely powdered form together with one or more suspending
agents, such as sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellul- ose, sodium alginate,
polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents such as lecithin or condensation products of an
alkylene oxide with fatty acids (for example polyoxyethylene
stearate), or condensation products of ethylene oxide with long
chain aliphatic alcohols, for example heptadecaethyleneoxycetan-
ol, or condensation products of ethylene oxide with partial esters
derived from fatty acids and a hexitol such as polyoxyethylene
sorbitol monooleate, or condensation products of ethylene oxide
with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions may also contain one or more
preservatives (such as ethyl or propyl p-hydroxybenzoate,
anti-oxidants (such as ascorbic acid), colouring agents, flavouring
agents, and/or sweetening agents (such as sucrose, saccharine or
aspartame).
[0095] Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil (such as arachis oil, olive oil,
sesame oil or coconut oil) or in a mineral oil (such as liquid
paraffin). The oily suspensions may also contain a thickening agent
such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set out above, and flavouring agents may be added to
provide a palatable oral preparation. These compositions may be
preserved by the addition of an anti-oxidant such as ascorbic
acid.
[0096] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water generally contain
the active ingredient together with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients such as sweetening,
flavouring and colouring agents, may also be present.
[0097] The pharmaceutical compositions of the invention may also be
in the form of oil-in-water emulsions. The oily phase may be a
vegetable oil, such as olive oil or arachis oil, or a mineral oil,
such as for example liquid paraffin or a mixture of any of these.
Suitable emulsifying agents may be, for example,
naturally-occurring gums such as gum acacia or gum tragacanth,
naturally-occurring phosphatides such as soya bean, lecithin, an
esters or partial esters derived from fatty acids and hexitol
anhydrides (for example sorbitan monooleate) and condensation
products of the said partial esters with ethylene oxide such as
polyoxyethylene sorbitan monooleate. The emulsions may also contain
sweetening, flavouring and preservative agents.
[0098] Syrups and elixirs may be formulated with sweetening agents
such as glycerol, propylene glycol, sorbitol, aspartame or sucrose,
and may also contain a demulcent, preservative, flavouring and/or
colouring agent.
[0099] The pharmaceutical compositions may also be in the form of a
sterile injectable aqueous or oily suspension, which may be
formulated according to known procedures using one or more of the
appropriate dispersing or wetting agents and suspending agents,
which have been mentioned above. A sterile injectable preparation
may also be a sterile injectable solution or suspension in a
non-toxic parenterally-acceptable diluent or solvent, for example a
solution in 1,3-butanediol.
[0100] Suppository formulations may be prepared by mixing the
active ingredient with a suitable non-irritating excipient which is
solid at ordinary temperatures but liquid at the rectal temperature
and will therefore melt in the rectum to release the drug. Suitable
excipients include, for example, cocoa butter and polyethylene
glycols.
[0101] Topical formulations, such as creams, ointments, gels and
aqueous or oily solutions or suspensions, may generally be obtained
by formulating an active ingredient with a conventional, topically
acceptable, vehicle or diluent using conventional procedure well
known in the art.
[0102] Compositions for administration by insulation may be in the
form of a finely divided powder containing particles of average
diameter of, for example, 30.mu. or much less, the powder itself
comprising either active ingredient alone or diluted with one or
more physiologically acceptable carriers such as lactose. The
powder for insufflation is then conveniently retained in a capsule
containing, for example, 1 to 50 mg of active ingredient for use
with a turbo-inhaler device, such as is used for insufflation of
the known agent sodium cromoglycate.
[0103] Compositions for administration by inhalation may be in the
form of a conventional pressurised aerosol arranged to dispense the
active ingredient either as an aerosol containing finely divided
solid or liquid droplets. Conventional aerosol propellants such as
volatile fluorinated hydrocarbons or hydrocarbons may be used and
the aerosol device is conveniently arranged to dispense a metered
quantity of active ingredient.
[0104] For further information on Formulation the reader is
referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal
Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon
Press 1990.
[0105] The amount of active ingredient that is combined with one or
more excipients to produce a single dosage form will necessarily
vary depending upon the host treated and the particular route of
administration. For example, a formulation intended for oral
administration to humans will generally contain, for example, from
0.5 mg to 2 g of active agent compounded with an appropriate and
convenient amount of excipients which may vary from about 5 to
about 98 percent by weight of the total composition. Dosage unit
forms will generally contain about 1 mg to about 500 mg of an
active ingredient. For further information on Routes of
Administration and Dosage Regimes the reader is referred to Chapter
25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin
Hansch; Chairman of Editorial Board), Pergamon Press 1990.
[0106] The size of the dose for therapeutic or prophylactic
purposes of a compound of the Formula I will naturally vary
according to the nature and severity of the conditions, the age and
sex of the animal or patient and the route of administration,
according to well known principles of medicine. As mentioned above,
compounds of the Formula I are useful in treating diseases or
medical conditions which are due alone or in part to the effects of
farnesylation of rats.
[0107] In using a compound of the Formula I for therapeutic or
prophylactic purposes it will generally be administered so that a
daily dose in the range, for example, 0.5 mg to 75 mg per kg body
weight is received, given if required in divided doses. In general
lower doses will be administered when a parenteral route is
employed. Thus, for example, for intravenous administration, a dose
in the range, for example, 0.5 mg to 30 mg per kg body weight will
generally be used. Similarly, for administration by inhalation, a
dose in the range, for example, 0.5 mg to 25 mg per kg body weight
will be used. Oral administration is however preferred.
[0108] A further aspect of the invention comprises the use of a
compound of formula (I) as defined above in the preparation of a
medicament for the treatment of inflammatory disease.
[0109] The invention is further illustrated, but not limited by the
following Examples in which the following general procedures were
used unless stated otherwise.
[0110] Preparation 1
[0111] Ethyl N-(3,4-dichlorobenzyl)-4-nitroindole-2-carboxylate
[0112] Ethyl 4-nitroindole-2-carboxylate (26 g) [prepared according
to S. M. Parmerter et. al J. Amer. Chem. Soc., 1958, 80, 4621],
3,4-dichlorobenzyl chloride (16 ml), potassium carbonate (17 g) and
potassium iodide (2 g) in DMF (250 ml) were stirred at 60.degree.
C. for 2 hours. The reaction was concentrated in vacuo and the
residue partitioned between water and dichloromethane. Iso-hexane
was added to the combined organic extracts resulting in
crystallisation of the product as yellow needles (39 g, 89%) NMR d
(CD.sub.3SOCD.sub.3) 1.30 (t, 3H), 4.32 (q, 2H), 5.93 (s, 2H), 6.88
(dd, 1H), 7.18 (d, 1H), 7.52 (d, 1H), 7.56 (dd, 1H), 7.78 (s, 1H),
8.17 (m, 2H), M/z (+)395 (MH.sup.+), 393.
[0113] Preparation 2
[0114] Ethyl N-benzyl-4-aminoindole-2-carboxylate
[0115] A mixture of ethyl 4-nitroindole-2-carboxylate (8.2 g),
anhydrous potassium carbonate (6.0 g) and benzyl bromide (4.3 ml)
in DMF (100 ml) was stirred at 50-60.degree. C. for 2 hours. The
solvent was evaporated in vacuo and the residue partitioned between
dichloromethane and water (250 ml each); the organic layer was
separated, dried (MgSO.sub.4) and evaporated to give a yellow solid
(12 g), which was dissolved in a mixture of tetrahydrofuran/ethanol
(200 ml, 1:1) and stirred while adding a solution of sodium
dithionite (26 g) in water (50 ml). The mixture was stirred for 1
hour at 25.degree. C. and partitioned between dichloromethane and
water (200 ml each), the organic layer was washed with water (100
ml) and dried (MgSO.sub.4). Combined organic extracts were
concentrated in vacuo and the residue purified by column
chromatography using dichloromethane as eluent to give a the
product as a brown solid (1.4 g. 14%); NMR d (CD.sub.3SOCD.sub.3)
1.28 (t, 3H), 4.27 (q, 2H), 5.57 (s, 2H), 5.73 (s, 2H), 6.22 (d,
1H), 6.62 (d, 1H), 6.95-7.05 (m, 3H), 7.15-7.30 (m, 3H), 7.60 (s,
1H).
[0116] Preparation 3
[0117] Ethyl N-(3,4-dichlorobenzyl)-4-nitroindole-2-carboxylate
[0118] Sodium hydroxide (3M, 20 ml) was added to a vigorously
stirred solution of ethyl 4-nitroindole-2-carboxylate (4 g),
3,4-dichlorobenzyl chloride (4.73 ml) and tetra-n-butylammonium
hydrogensulphate (0.2 g) in dichloromethane (60 ml). The reaction
was stirred for 48 hours then partitioned between 2M HCl and
dichloromethane. Combined organic extracts were dried (MgSO.sub.4)
and concentrated in vacuo and the residue purified by column
chromatography using iso-hexane: 20% ethyl acetate as eluent to
give the product as a yellow crystalline solid (5.26 g, 78%); NMR
.delta. (CD.sub.3SOCD.sub.3) 1.3 (t, 3H), 4.3 (q, 2H), 5.95 (s,
2H), 6.9 (m, 1H), 7.6 (m, 4H), 8.2 (t, 2H); M/z (+) 393.3
(M.sup.+).
[0119] Ethyl N-(3,4-dichlorobenzyl)-4-aminoindole-2-carboxylate
[0120] A solution of ethyl
N-(3,4-dichlorobenzyl)-4-nitroindole-2-carboxyl- ate (2.41 g) in
tetrahydrofuran (100 ml) was stirred in the presence of titanium
trichloride (1.5% aqueous solution. 50 ml) at room temperature
overnight. The reaction was treated with 40% sodium hydroxide
solution and extracted with 5% methanol in dichloromethane.
Combined organic extracts were dried (MgSO.sub.4) and concentrated
in vacuo to Live the product as a brown solid (1.98 g, 89%); NMR d
(CD.sub.3SOCD.sub.3) 1.3 (t, 3H), 4.2 (q, 2H), 5.7 (s, 4H), 6.2 (d,
1H), 6.6 (d, 1H), 7.0 (m, 2H), 7.25 (m, 1H), 7.5 (d, 1H), 7.6 (m,
1H); M/z (+) 363.3 (MH.sup.+).
[0121] Preparation 4
[0122] Ethyl
4-chloroacetamido-N-(3,4-dichlorobenzyl)indole-2-carboxylate
[0123] Ethyl 4-amino-N-(3,4-dichlorobenzyl)indole-2-carboxylate
(2.03 g), chloroacetyl chloride (0.5 ml) and triethylamine (4.0 ml)
were stirred in dichloromethane (50 ml) for 16 hours. The reaction
was washed with water, dried (MgSO.sub.4) and concentrated in
vacuo. The residue was triturated with toluene to give the product
as a pale grey solid (1.61 g, 65%); NMR d (CD.sub.3SOCD.sub.3) 1.28
(t, 3H), 4.30 (q, 2H), 4.40 (s, 2H), 5.81 (s, 2H), 6.88 (dd, 1H),
7.30 (m, 3H), 7.50 (d, 1H), 7.76 (s, 1H), 7.78 (d, 1H), 10.19 (brs,
1H); M/z (-) 439 (M.sup.+), 437.
EXAMPLE 1
[0124] Compound 2
[0125] Ethyl
4-chloroacetamido-N-(3,4-dichlorobenzyl)indole-2-carboxylate (0.15
g) and morpholine (2.0 ml) were dissolved in methoxyethanol (5.0
ml) and the reaction stirred for 72 hours. The reaction was then
poured into water (100 ml) and the resulting solid filtered and
dried in vacuo. The solid was dissolved in THF (2.5 ml) and
methanol (2.5 ml), and to this was added NaOH (3M, 2.0 ml). The
reaction was stirred for 16 hours, then concentrated. The residue
was dissolved in water, and precipitated by dropwise addition of
acetic acid. The resulting solid was filtered and dried in vacuo to
give the title compound as a white solid (0.1 g, 63%, 2 steps); NMR
d (CD.sub.3SOCD.sub.3) 2.58 (t, 4H), 3.29 (s, 2H), 3.65 (t, 4H),
5.82 (s, 2H), 6.90 (dd, 1H), 7.30 (m, 3H), 7.52 (m, 2H), 7.72 (d,
1H), 9.80 (s, 1H); M/z (-) 462 (M.sup.+), 460, 418.
EXAMPLE 2
[0126] The procedure described in Example 1 above was repeated
using the appropriate amines. Thus were obtained the compounds
described below.
[0127] Compound 3
[0128] 49% yield, 2 steps; NMR d (CD.sub.3SOCD.sub.3) 2.27 (s, 3H),
2.54 (t, 4H), 2.62 (t, 4H), 3.22 (s, 2H), 5.84 (s, 2H), 6.95 (dd,
1H), 7.22 (m, 2H), 7.33 (s, 1H), 7.41 (s, 1H), 7.50 (d, 1H), 7.72
(d, 1H), 9.75 (s, 1H); M/z (-) 475 (M.sup.+), 473, 429, 109.
[0129] Compound 6
[0130] 14% yield, 2 steps; M/z (-) 510 (M.sup.-), 508, 464.
EXAMPLE 3
[0131] Di-ester of Compound 8
[0132] Ethyl
4-chloroacetamido-N-(3,4-dichlorobenzyl)indole-2-carboxylate (0.4
g), glycine methyl ester hydrochloride (0.57 g) and triethylamine
(1.25 ml) were dissolved in methoxyethanol (4.0 ml) and the
reaction heated at 100.degree. C. for 6 hours. The reaction was
cooled and partitioned between water and ethyl acetate. Combined
organic extracts were dried (MgSO.sub.4) and concentrated and the
residue purified by chromatography using toluene ethyl acetate
(1:1) as eluent to give the product, ethyl
4-[(N-(methoxycarbonylmethyl)-glycyl)amino]-N-(3,4-dichlor-
obenzyl)indole-2-carboxylate, as a pale yellow solid (0.17 g, 38%);
NMR d (CD.sub.3SOCD.sub.3) 1.28 (t, 3H), 3.44 (s, 2H), 3.50 (s,
2H), 3.63 (s, 3H), 4.28 (q, 2H), 5.82 (s, 2H), 6.88 (dd, 1H),
7.10-7.30 (m, 4H), 7.50 (d, 1H), 7.69 (s, 1H), 7.80 (dd, 1H), 10.00
(brs, 1H); M/z (+) 494, 492 (M.sup.+).
EXAMPLE 4
[0133] Di-ester of Compound 11
[0134] Methanesulphonyl chloride (0.1 ml) was added to stirred
solution of ethyl
4-[(N-(methoxycarbonylmethyl)glycyl)amino]-N-(3,4-dichlorobenzyl)in-
dole-2-carboxylate (0.33 g) and triethylamine (0.47 ml) in
dichloromethane (4.0 ml) and the reaction stirred for 3 hours. The
reaction was poured into water and extracted with ethyl acetate.
Combined organic extracts were dried (MgSO.sub.4) and concentrated
and the residue triturated with ether, filtered and dried in vacuo
to give the product as a white solid (0.24 g, 63%); NMR d
(CD.sub.3SOCD.sub.3) 1.27 (t, 3H), 3.10 (s, 3 H), 3.67 (s, 3H),
4.20 (s, 2H), 4.28 (q+s, 2H+2H), 5.82 (s, 2H), 6.87 (dd, 1H), 7.28
(m, 3H), 7.50 (d, 1H), 7.80 (m, 2H), 10.00 (brs, 1H); M/z (+) 572,
570 (M.sup.+).
EXAMPLE 5
[0135] The procedure described in the Example 4 above was repeated
using the appropriate acid chloride. Thus was obtained the compound
described below.
[0136] Di-ester of Compound 12
[0137] 64% yield; M/z (-) 534 (M.sup.+), 532.
EXAMPLE 6
[0138] Di-ester of Compound 14
[0139] Sarcosine ethyl ester hydrochloride (1.23 g) and potassium
carbonate (1.11 g) were added to a solution of ethyl
4-chloroacetamido-N-(3,4-dichlorobenzyl)indole-2-carboxylate (700
mg) in acetone (25 ml), stirred and heated at 65.degree. C.
overnight. The reaction was partitioned between water (50 ml) and
ethyl acetate (50 ml), extracted with ethyl acetate (2.times.50
ml), and dried (MgSO.sub.4). The combined organic extracts were
concentrated in vacuo, and the residue purified by column
chromatography using 30% ethyl acetate:toluene as eluent, to afford
the product as a yellow solid (768 mg, 92%); NMR d
(CD.sub.3SOCD.sub.3) 1.21 (t, 3H), 1.28 (t, 3H), 2.45 (s, 3H), 3.42
(s, 2H), 3.53 (s, 2H), 4.16 (q, 2H), 4.30 (q, 2H), 5.81 (s, 2H),
6.88 (d, 1H), 7.27 (m, 2H), 7.52 (d, 1H), 7.67 (s, 1H), 7.84 (d,
1H), 9.95 (s, 1H), M/z (+) 520.3 (MH.sup.+)
EXAMPLE 7
[0140] The procedure described in Example 6 above was repeated
using the appropriate amine. Thus was obtained the compound
described below.
[0141] Diester of Compound 13
[0142] 93% yield; NMR d (CD.sub.3SOCD.sub.3) 1.15 (t, 3H), 1.28 (t,
3H), 3.52 (s, 3H), 3.57 (s, 3H), 3.87 (s, 2H), 4.10 (q, 2H), 4.31
(q, 2H),5.83 (s, 2H), 6.90 (d, 1H), 7.15-7.44 (m, 8H), 7.53 (d,
1H), 7.67 (s, 1H), 7.83 (d, 1H); M/z (+) 596.5 (MH.sup.+).
EXAMPLE 8
[0143] Di-ester of Compound 15
[0144] A solution of methyl iodide (0.026 ml) in DMF (2 ml) was
added to a solution of sodium hydride (15 mg, 60% in mineral oil)
and ethyl
4-[(N-benzyl-N-ethoxycarbonylmethyl)glycyl]amino-N-(3,4-dichlorobenzyl)in-
dole-2-carboxylate (the diester of Compound 13) ( 200 mg) in DMF (4
ml), and stirred under an atmosphere of argon at ambient
temperature for 4 hours. The reaction was quenched with water (50
ml) and extracted with ethyl acetate (3.times.50 ml), and the
combined organic extracts were dried (MgSO.sub.4), and concentrated
in vacuo to afford the product as a pale brown oil (93 mg, 45%);
NMR d (CD.sub.3SOCD.sub.3) 1.05 (t, 3H), 1.30 (t, 3H), 3.21 (s,
2H), 3.28 (s, 3H), 3.41 (s, 2H), 3.70 (s, 2H), 3.93 (q, 2H), 4.30
(q, 2H), 5.84 (s, 2H), 6.90 (d, 1H), 7.01 (d, 1H), 7.07-7.40 (m,
8H), 7.48-7.64 (m, 2H); M/z (+) 610.5 (MH.sup.+).
EXAMPLE 9
[0145] Compound 8
[0146] Ethyl
4-[(N-(methoxycarbonylmethyl)glycyl)amino]-N-(3,4-dichloroben-
zyl)indole-2-carboxylate (0.15 g) was dissolved in THF/methanol
(1:1) (10 ml) and sodium hydroxide (2M, 2.5 ml) was added and the
reaction stirred for 16 hours. The reaction was then concentrated
in vacuo and the residue dissolved in water. The solution was
acidified by dropwise addition of acetic acid, resulting in the
precipitation of a white solid which was filtered, washed with
water and dried in vacuo to give the desired end product as a white
solid (108 mg, 79%); NMR d (CD.sub.3SOCD.sub.3) 3.40 (s, 2H), 3.64
(s, 2H), 5.82 (s, 2H), 6.92 (dd, 1H), 7.20-7.38 (m, 3H), 7.50 (d,
1H), 7.62 (s, 1H), 7.78 (d, 1H), 10.15 (brs, 1H).
EXAMPLE 10
[0147] The procedure described in Example 9 above was repeated
using the appropriate ester. Thus were obtained the compounds
described below.
[0148] Compound 11
[0149] 79% yield; NMR d (CD.sub.3SOCD.sub.3) 3.10 (s, 3H), 4.02 (s,
2H), 4.20 (s, 2H), 5.83 (s, 2H), 6.88 (dd, 1H), 7.25 (m, 3H), 7.50
(d, 1H), 7.75 (s, 1H), 7.80 (d, 1H), 10.49 (brs, 1H); M/z (-) 528
(M.sup.+), 526, 360, 358, 289, 253, 217.
[0150] Compound 12
[0151] 78% yield; NMR d (CD.sub.3SOCD.sub.3) 2.00 (d, 3H), 4.03 (s,
1H), 4.20 (s, 1H), 4.23 (s, 1H), 4.40 (s, 1H), 5.82 (s, 2H), 6.88
(m, 1H), 7.25 (m, 3H), 7.52 (dd, 1H), 7.76 (m, 2H), 10.13 (brs,
1H); M/z (-) 492 (M.sup.+), 490, 324, 253, 224.
[0152] Compound 14
[0153] 60% yield; NMR d (CD.sub.3SOCD.sub.3) 2.46 (s, 31H), 3.38
(s, 2H), 3.42 (s, 2H), 5.88 (s, 2H), 6.92 (d, 1H), 7.20 (m, 2H),
7.31 (s, 1H), 7.50 (m, 2H), 7.82 (d, 1H), M/z (-) 462.2
(M-H.sup.+).
[0154] Compound 15
[0155] 15% yield; NMR d (CD.sub.3SOCD.sub.3) 3.21 (s, 2H), 3.31 (s,
3H), 3.40 (s, 2H), 3.69 (s, 2H), 5.83 (s, 2H), 6.90 (d, 2H), 6.98
(d, 2H), 7.15 (m, 6H), 7.27 (t, 1H), 7.39 (s, 1H), 7.53 (m, 2H);
M/z (-) 554.3 (M-H.sup.+).
[0156] Compound 13
[0157] 25% yield; NMR d (CD.sub.3SOCD.sub.3) 3.44 (s, 2H), 3.46 (s,
2H), 3.85 (s, 2H), 5.91 (s, 2H), 6.87 (m, 1H), 7.13-7.36 (m, 6H),
7.40 (m, 2H), 7.53 (m, 2H), 7.78 (d, 1H), M/z (-) 538.2
(M-H.sup.+), 253.2.
EXAMPLE 11
[0158]
N-Benzyl-4-(2-(pyrid-2-yl)thiophene-5-sulphonyl))aminoindole-2-carb-
oxylic Acid (Compound 1)
[0159] To a solution of ethyl N-benzyl-4-aminoindole-2-carboxylate
(140 mg) and pyridine (0.08 ml) in dichloromethane (10 ml) at
20.degree. C. was added 2-(pyrid-2-yl)thiophene-5-sulphonyl
chloride (140 mg) and thc reaction stirred for 2 hours. The mixture
was washed with HCl (2M, 10 ml), the organic layer was concentrated
in vacuo and the residue purified by chromatography on silica using
ethyl acetate as eluent, to give a yellow solid which was dissolved
in ethanol (50 ml) at 60.degree. C. and treated with NaOH (2M, 4.0
ml) with stirring for 2 hours. The solvent was evaporated in vacuo,
the residue dissolved in water (50 ml) and filtered. The clear
yellow filtrate was acidified with 2N HCl and extracted with
dichloromethane/methanol (9:1, 100 ml). The organic layer was dried
(MgSO.sub.4) and evaporated to give a pale brown solid, which was
triturated with ether to give the product as an off white powder
(150 mg, 63%, 2 steps); NMR d (CD.sub.3SOCD.sub.3) 5.87 (s, 2H),
6.9-7.1 (m, 9H), 7.30 (dd, 2H), 7.43 (d, 1H), 7.63 (d, 1H), 7.81
(dd, 1H), 7.96 (d, 1H), 8.50 (d, 1H); M/z (-) 488 (M-H.sup.+).
EXAMPLE 12
[0160] The procedure described in Example 11 above was repeated
using the appropriate aminoindole and sulphonyl chloride. Thus were
obtained the compounds described below.
[0161]
4-(4-Acetylaminobenzenesulphonyl)amino-N-(3,4-dichlorobenzyl)indole-
-2-carboxylic Acid (Compound 4)
[0162] 66% yield (2 steps); NMR d (CD.sub.3SOCD.sub.3) 2.00 (s,
3H), 5.75 (s, 2H), 6.80 (dd, 1H), 6.92 (d, 1H), 7.12 (dd, 1H), 7.22
(m, 2H), 7.48 (d, 1H), 7.56 (s, 1H,), 7.66 (s, 4H), 10.24 (brs,
1H), 10.45 (brs, 1H); M/z (-) 532 (M-H.sup.+), 530.
[0163]
N-(3,4-Dichlorobenzyl)-4-(2-(pyrid-2-yl)thiophene-5-sulphonyl))amin-
oindole-2-carboxylic Acid (Compound 5)
[0164] 69% yield (2 steps); NMR d (CD.sub.3SOCD.sub.3) 5.80 (s,
2H), 6.80 (dd, 1H), 7.0-7.5 (m, 8H), 7.68 (d, 1H), 7.83 (dd, 1H),
7.92 (d, 1H), 8.48 (dd, 1H); M/z (-) 558 (M-H.sup.+), 556.
[0165]
N-(3,4-Dichlorobenzyl)-4-(1-methylimidazole-4-sulphonyl)aminoindole-
-2-carboxylic Acid (Compound 7)
[0166] 66% yield (2 steps); NMR d (CD.sub.3SOCD.sub.3) 3.60 (s,
3H)) 5.78 (s, 2H), 6.86 (dd, 1H),7.04(1H, d), 7.15 (dd, 1H), 7.20
(d, 1H), 7.30 (d, 1H) 7.50 (d, 1H), 7.68 (m, 2H), 7.75 (s, 1H),
10.20 (brs, 1H); M/z (-) 479 (M-H.sup.+), 477.
[0167]
N-(3,4-Dichlorobenzyl)-4-(2-chloropyridyl-5-sulphonyl)aminoindole-2-
carboxylic Acid
[0168] (Compound 9)
[0169] 30% yield (2 steps); NMR d (CD.sub.3SOCD.sub.3) 5.85 (s,
2H), 6.83 (d, 1H), 6.93 (dd, 1H), 7.03 (dd, 1H), 7.15 (d, 1H), 7.20
(s, 1H), 7.26 (s, 1H), 7.46 (d, 1H), 7.60 (d, 1H), 8.05 (dd, 1H),
8.62 (d, 1H); M/z (-)512(M-H.sup.+),510, 508.
EXAMPLE 13
Methyl
N-(3,4-dichlorobenzyl)-4-(dimethylcarbamyloxy)indole-2-carboxylate
(Methyl Ester of Compound 10)
[0170] Dimethylcarbamyl chloride (83 mg) was added to a stirred
solution of methyl
N-(3,4-dichlorobenzyl)-4-hydroxyindole-2-carboxylate (150 mg),
triethylamine (65 mg),and DMAP (5 mg) in dichloromethane. The
reaction was stirred for 16 hours at room temperature under an
atmosphere of nitrogen. The reaction was washed with hydrochloric
acid (2M, 70 ml), saturated aqueous sodium hydrogencarbonate
solution, water and saturated sodium chloride solution. Combined
organic extracts were dried (MgSO.sub.4), concentrated in vacuo and
the residue purified by column chromatography using 60% ethyl
acetate:iso-hexane as eluent to give the product as a colourless
gum (132 mg, 74%); NMR d (CD.sub.3SOCD.sub.3) 2.94 (s, 3H), 3.12
(s, 3H), 3.81 (s, 3H), 5.82 (s, 2H), 6.91 (m, 2H), 7.21 (s, 1H),
7.27-7.36 (m, 2H), 7.46 (d, 1H), 7.52 (d, 1H); M/z (+) 421
(MH.sup.+).
EXAMPLE 14
N-(3,4-Dichlorobenzyl)-4-(dimethylcarbamyloxy)indole-2-carboxylic
Acid (Compound 10)
[0171] Desesterifiation of the compound of Example 13 using the
method described in Example 9 above yielded Compound 10.
[0172] 93% yield; NMR d (CD.sub.3SOCD.sub.3) 2.94 (s, 3H), 3.11 (s,
3H), 5.91 (s, 2H), 6.82 (d, 1H), 6.94-7.03 (m, 2H), 7.18 (t, 1H),
7.29-7.39 (m, 2H), 7.50 (d, 1H); M/z (-) 405 (M-H.sup.+).
EXAMPLE 15
Biological Assays for hMCP-1 Antagonists
[0173] The following biological test methods, data and Examples
serve to illustrate the present invention.
[0174] Abbreviations:
[0175] ATCC American Type Culture Collection, Rockville, USA.
[0176] BCA Bicinchroninic acid, (used, with copper sulphate, to
assay protein)
[0177] BSA Bovine Serum Albumin
[0178] DMEM Dulbecco's modified Eagle's medium
[0179] EGTA Ethylenebis(oxyethylenenitrilo)tetraacetic acid
[0180] FCS Foetal calf serum
[0181] HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulphonic
acid])
[0182] HBSS Hank's Balanced Salt Solution
[0183] hMCP-1 Human Monocyte Chemoattractant Protein-1
[0184] PBS Phosphate buffered saline
[0185] PCR Polymerase chain reaction
[0186] AMPLITAQ.TM., available from Perkin-Elmer Cetus, is used as
the source of thermostable DNA polymerase.
[0187] Binding Buffer is 50 mM HEPES, 1 mM CaCl.sub.2, 5 mM
MgCl.sub.2, 0.5% foetal calf serum, adjusted to pH 7.2 with 1 M
NaOH.
[0188] Non-Essential Amino Acids (100.times. concentrate) is:
L-Alanine, 890 mg/l; L-Asparagine, 1320 mg/l; L-Aspartic acid, 1330
mg/l; L-Glutamic acid, 1470 mg/l; Glycine, 750 mg/l; L-Proline,
1150 mg/l and; L-Serine, 1050 mg/l.
[0189] Hypoxanthinc and Thymidine Supplement (50.times.
concentrate) is: hypoxanthine, 680 mg/l and; thymidine, 194
mg/l.
[0190] Penicillin-Streptomycin is: Penicillin G (sodium salt); 5000
units/ml. Streptomycin sulphate, 5000 .mu.g/ml.
[0191] Human monocytic cell line THP-1 cells are available from
ATCC, accession number ATCC TIB-202.
[0192] Hank's Balanced Salt Solution (HBSS) was obtained from
Gibco; see Proc. Soc. Exp. Biol. Med., 1949, 71, 196.
[0193] Synthetic cell culture medium, RPMI 1640 was obtained from
Gibco; it contains inorganic salts [Ca(NO.sub.3).sub.2.4H.sub.2O
100 mg/l; KCl 400 mg/l; MgSO.sub.1.7H.sub.2O 100 mg/l; NaCl 6000
mg/l; NaHCO.sub.3 2000 mg/l & Na.sub.2HPO.sub.4 (anhyd) 800
mg/l], D-Glucose 2000 mg/l, reduced glutathione 1 mg/l, amino acids
and vitamins.
[0194] FURA-2/AM is
1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-
-(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid
pentaacetoxymethyl ester and was obtained from Molecular Probes,
Eugene, Oreg., USA.
[0195] Blood Sedimentation Buffer contains 8.5 g/l NaCl and 10 g/l
hydroxyethyl cellulose.
[0196] Lysis Buffer is 0.15M NH.sub.4Cl.sup.-, 10 mM KHCO.sub.3, 1
mM EDTA
[0197] Whole Cell Binding Buffer is 50 mM HEPES, 1 mM CaCl.sub.2, 5
mM MgCl.sub.2, 0.5% BSA, 0.01% NaN.sub.3, adjusted to pH 7.2 with
1M NaOH.
[0198] Wash buffer is 50 mM HEPES, 1 mM CaCl.sub.2, 5 mM
MgCl.sub.2, 0.5% heat inactivated FCS, 0.5M NaCl adjusted to pH7.2
with 1M NaOH.
[0199] General molecular biology procedures can be followed from
any of the methods described in "Molecular Cloning--A Laboratory
Manual" Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring
Harbor Laboratory, 1989).
[0200] i) Cloning and Expression of hMCP-1 Receptor
[0201] The MCP-1 receptor B (CCR2B) cDNA was cloned by PCR from
THP-1 cell RNA using suitable oligonucleotide primers based on the
published MCP-1 receptor sequences (Charo et al., 1994, Proc. Natl.
Acad. Sci. USA, 91, 2752). The resulting PCR products were cloned
into vector PCR-II.TM. (InVitrogen, San Diego, Calif.). Error free
CCR2B cDNA was subcloned as a Hind III-Not I fragment into the
eukaryotic expression vector pCDNA3 (InVitrogen) to generate
pCDNA3/CC-CKR2A and pCDNA3/CCR2B respectively.
[0202] Linearised pCDNA3/CCR2B DNA was transfected into CHO-K1
cells by calcium phosphate precipitation (Wigler et al., 1979,
Cell, 16, 777). Transfected cells were selected by the addition of
Geneticin Sulphate (G418, Gibco BRL) at 1 mg/ml, 24 hours after the
cells had been transfected. Preparation of RNA and Northern
blotting were carried out as described previously (Needham et al,
1995, Prot. Express. Purific., 6, 134). CHO-K1 clone 7 (CHO-CCR2B)
was identified as the highest MCP-1 receptor B expressor.
[0203] ii) Preparation of Membrane Fragments
[0204] CHO-CCR2B cells were grown in DMEM supplemented with 10%
foetal calf serum. 2 mM glutamine, 1.times. Non-Essential Amino
Acids, 1.times. Hypoxanthine and Thymidine Supplement and
Penicillin-Streptomycin (at 50 .mu.g streptomycin/ml. Gibco BRL).
Membrane fragments were prepared using cell lysis/differential
centrifugation methods as described previously (Siciliano et al.,
1990, J. Biol. Chem., 265, 19658). Protein concentration was
estimated by BCA protein assay (Pierce, Rockford, Ill.) according
to the manufacturer's instructions.
[0205] iii) Assay
[0206] .sup.125I MCP-1 was prepared using Bolton and Hunter
conjugation (Bolton et al., 1973, Biochem. J., 133, 529; Amersham
International plc]. Equilibrium binding assays were carried out
using the method of Ernst et al., 1994, J. Immunol., 152, 3541.
Briefly, varying amounts of .sup.125I-labeled MCP-1 were added to 7
.mu.g of purified CHO-CCR2B cell membranes in 100 .mu.l of Binding
Buffer. After 1 hour incubation at room temperature the binding
reaction mixtures were filtered and washed 5 times through a plate
washer (Brandel MLR-96T Cell Harvester) using ice cold Binding
Buffer. Filter mats (Brandel GF/B) were pre-soaked for 60 minutes
in 0.3% polyethylenimine prior to use. Following filtration
individual filters were separated into 3.5 ml tubes (Sarstedt No.
55.484) and bound .sup.125I-labeled MCP-1 was determined (LKB 1277
Gammamaster). Cold competition studies were performed as above
using 100 pM .sup.125I-labeled MCP-1 in the presence of varying
concentrations of unlabelled MCP-1. Non-specific binding was
determined by the inclusion of a 200-fold molar excess of
unlabelled MCP-1 in the reaction.
[0207] Ligand binding studies with membrane fragments prepared from
CHO-CCR2B cells showed that the CCR2B receptor was present at a
concentration of 0.2 pmoles/mg of membrane protein and bound MCP-1
selectively and with high affinity (IC.sub.50=110 pM, K.sub.d=120
pM). Binding to these membranes was completely reversible and
reached equilibrium after 45 minutes at room temperature, and there
was a linear relationship between MCP-1 binding and CHO-CCR2B cell
membrane concentration when using MCP-1 at concentrations between
100 pM and 500 pM.
[0208] Test compounds dissolved in DMSO (5 .mu.l) were tested in
competition with 100 pM labelled MCP-1 over a concentration range
(0.01-50 .mu.M) in duplicate using eight point dose-response curves
and IC.sub.50 concentrations were calculated.
[0209] Compounds tested of the present invention had IC.sub.50
values of 50 .mu.M or less in the hMCP-1 receptor binding assay
described herein. For example Compound 2 in Table 1 showed
IC.sub.50 of 1.17 .mu.M in hMCP-1.
[0210] b) MCP-1 Mediated Calcium Flux in THP-1 Cells
[0211] The human monocytic cell line THP-1 was grown in a synthetic
cell culture medium RPMI 1640 supplemented with 10% foetal calf
serum, 6 mM glutamine and Penicillin-Streptomycin (at 50 .mu.g
streptomycin/ml, Gibco BRL). THP-1 cells were washed in HBSS
(lacking Ca.sup.2+ and Mg.sup.2+)+1 mg/ml BSA and resuspended in
the same buffer at a density of 3.times.10.sup.6 cells/ml. The
cells were then loaded with 1 mM FURA-2/AM for 30 min at 37.degree.
C., washed twice in HBSS, and resuspended at 1.times.10.sup.6
cells/ml. THP-1 cell suspension (0.9 ml) was added to a 5 ml
disposable cuvette containing a magnetic stirrer bar and 2.1 ml of
prewarmed (37.degree. C.) HBSS containing 1 mg/ml BSA, 1 mM
MgCl.sub.2 and 2 mM CaCl.sub.2. The cuvette was placed in a
fluorescence spectrophotometer (Perkin Elmer, Norwalk, Conn.) and
preincubated for 4 min at 37.degree. C. with stirring. Fluorescence
was recorded over 70 sec and cells were stimulated by addition of
hMCP-1 to the cuvette after 10 sec. [Ca.sup.2+]i was measured by
excitation at 340 nm and 380 nm alternately and subsequent
measurement of the intensity of the fluorescence emission at 510
nm. The ratio of the intensities of the emitted fluorescent light
following excitation at 340 nm and 380 nm, (R), was calculated and
displayed to give and estimate of cytoplasmic [Ca.sup.2+] according
to the equation:- 1 [ Ca 2 + ] i = K d ( R - R min ) ( R max - R )
( Sf2 / Sb2 )
[0212] where the K.sub.d for FURA-2 Ca.sup.2+ complex at 37.degree.
C. was taken to be 224 nm. R.sub.max is the maximal fluorescence
ratio determined after addition of 10 mM lonomycin, R.sub.min is
the minimal ratio determined by the subsequent addition of a
Ca.sup.2+ free solution containing 5 mM EGTA, and Sf2/Sb2 is the
ratio of fluorescence values at 380 nm excitation determined at
R.sub.min and R.sub.max, respectively.
[0213] Stimulation of THP-1 cells with hMCP-1 induced a rapid,
transient rise in [Ca .sup.2+].sub.i in a specific and dose
dependent manner. Dose response curves indicated an approximate
EC.sub.50 of 2 nm. Test compounds dissolved in DMSO (10 .mu.l) were
assayed for inhibition of calcium release by adding them to the
cell suspension 10 sec prior to ligand addition and measuring the
reduction in the transient rise in [Ca.sup.2+]i. Test compounds
were also checked for lack of agonist activity by addition in place
of hMCP-1.
[0214] c) hMCP-1 and RANTES Mediated Chemotaxis.
[0215] In vitro chemotaxis assays were performed using the human
monocytic cell line THP-1. Cell migration through polycarbonate
membranes was measured by enumerating those passing through either
directly by Coulter counting or indirectly by use of a
colourimetric viability assay measuring the cleavage of a
tetrazolium salt by the mitochondrial respiratory chain (Scudiero
D. A. et al. 1988, Cancer Res., 48, 4827-4833).
[0216] Chemoattractants were introduced into a 96-well microtitre
plate which forms the lower well of a chemotaxis chamber fitted
with a PVP-free 5 .mu.m poresize polycarbonate adhesive framed
filter membrane (NeuroProbe MB series, Cabin John, Md. 20818, USA)
according to the manufacturer's instructions. The chemoattractant
was diluted as appropriate in synthetic cell culture medium, RPMI
1640 (Gibco) or supplemented with 2 mM glutamine and 0.5% BSA, or
alternatively with HBSS with Ca.sup.2- and Mg.sup.2+ without Phenol
Red (Gibco) plus 0.1% BSA. Each dilution was degassed under vacuum
for 30 min and was placed (400 .mu.l) in the lower wells of the
chamber and THP-1 cells (5.times.10.sup.5 in 100 .mu.l RPMI
1640+0.5% BSA) were incubated in each well of the upper chamber.
For the inhibition of chemotaxis the chemoattractant was kept at a
constant submaximal concentration determined previously (1 nM
MCP-1) and added to the lower well together with the test compounds
dissolved in DMSO (final DMSO concentration <0.05% v/v) at
varying concentrations. The chamber was incubated for 2 h at
37.degree. C. under 5% CO.sub.2. The medium was removed from the
upper wells which were then washed out with 200 .mu.l physiological
saline before opening the chamber, wiping dry the membrane surface
and centrifuging the 96-well plate at 600 g for 5 min to harvest
the cells. Supernatant (150 .mu.l) was aspirated and 10 .mu.l of
cell proliferation reagent, WST-1,
{4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1-
,3-phenyl disulfonate} plus an electron coupling reagent
(Boehringer Mannheim, Cat. no. 1644 807) was added back to the
wells. The plate was incubated at 37.degree. C. for 3 h and the
absorbance of the soluble formazan product was read on a microtitre
plate reader at 450 nm. The data was input into a spreadsheet,
corrected for any random migration in the absence of
chemoattractant and the average absorbance values, standard error
of the mean, and significance tests were calculated. hMCP-1 induced
concentration dependent cell migration with a characteristic
biphasic response, maximal 0.5-1.0 nm.
[0217] In an alternative form of the above assay, fluorescently
tagged cells can be used in order to assist in end point detection.
In this case, the THP-1 cells used are fluorescently tagged by
incubation in the presence of 5 mM Calcein AM (Glycine.
N,N'-[[3',6'-bis(acetyloxy)-3-oxosp-
iro[isobenzofuran-1(3H),9'-[9H]xanthene]-2',7'-diyl]bis(methylene)]bis[N-[-
2-[(acetyloxy)methoxy]-2-oxoethyl]]-bis[(acetyloxy)methyl]ester:
Molecular Probes) for 45 minutes in the dark. Cells are harvested
by centrifugation and resuspended in HBSS (without Phenol Red) with
Ca.sup.2+, Mg.sup.2+ and 0.1% BSA. 50 .mu.l (2.times.105 cells) of
the cell suspension are placed on the filter above each well and,
as above, the unit is incubated at 37.degree. C. for 2 hours under
5% CO.sub.2. At the end of the incubation, cells are washed off the
upper face of the filter with phosphate buffered saline, the filter
removed from the plate and the number of cells attracted to either
the underside of the filter or the lower well estimated by reading
fluorescence at 485 nm excitation, 538 nm emission wavelengths
(fmax, Molecular Devices). The data was input into a spreadsheet,
corrected for any random migration in the absence of
chemoattractant and the average fluorescence values, standard error
of the mean, percentage inhibition and IC.sub.50 of compounds under
test and significance tests can be calculated. In addition to MCP-1
induced chemotaxis, this alternative form of the assay was also
used to measure inhibition of RANTES (2 nM) induced chemotaxis.
[0218] d) Binding to Human Peripheral Blood Mononuclear Cells
(PBMCs)
[0219] i) Preparation of Human PBMCs
[0220] Fresh human blood (200 ml) was obtained from volunteer
donors, collected into sodium citrate anticoagulant to give a final
concentration of 0.38%. The blood was mixed with Sedimentation
Buffer and incubated at 37.degree. C. for 20 minutes. The
supernatant was collected and centrifuged at 1700 rpm for 5 minutes
(Sorvall RT6000D). The pellet obtained was resuspended in 20 ml
RPMI/BSA (1 mg/ml) and 4.times.5 mls of cells were carefully
layered over 4.times.5 mls of Lymphoprep (Nycomed) in 15 ml
centrifuge tubes. Tubes were spun at 1700 rpm for 30 minutes
(Sorvall RT6000D) and the resultant layer of cells was removed and
transferred to 50 ml Falcon tubes. The cells were washed twice in
Lysis Buffer to remove any remaining red blood cells followed by 2
washes in RPMI/BSA. Cells were resuspended in 5 mls of Binding
Buffer. Cell number was measured on a Coulter counter and
additional binding buffer was added to give a final concentration
of 1.25.times.10.sup.7 PBMCs /ml.
[0221] ii) Assay
[0222] [.sup.125I]MCP-1 was prepared using Bolton and Hunter
conjugation (Bolton et al. 1973, Biochem. J., 133, 529; Amersham
International plc]. Equilibrium binding assays were carried out
using the method of Ernst et al., 1994, J. Immunol., 152, 3541.
Briefly, 50 .mu.l of .sup.125I-labeled MCP-1 (final concentration
100 pM) was added to 40 .mu.l (5.times.10.sup.5 cells) of cell
suspension in a 96 well plate. Compounds. diluted in Whole Cell
Binding Buffer from a stock solution of 10 mM in DMSO were added in
a final volume of 5 .mu.l to maintain a constant DMSO concentration
in the assay of 5%. Total binding was determined in the absence of
compound. Non-specific binding was defined by the addition of 5
.mu.l cold MCP-1 to give a final assay concentration of 100 nM.
Assay wells were made up to a final volume of 100 .mu.l with Whole
Cell Binding Buffer and the plates sealed. Following incubation at
37.degree. C. for 60 minutes the binding reaction mixtures were
filtered and washed for 10 seconds using ice cold Wash Buffer using
a plate washer (Brandel MLR-96T Cell Harvester). Filter mats
(Brandel GF/B) were pre-soaked for 60 minutes in 0.3%
polyethylenimine plus 0.2% BSA prior to use. Following filtration
individual filters were separated into 3.5 ml tubes (Sarstedt No.
55.484) and bound .sup.125I-labeled MCP-1 was determined (LKB 1277
Gammamaster).
[0223] Test compound potency was determined by assay in duplicate
using six point dose-response curves and IC.sub.50 concentrations
were determined.
[0224] Compound No. 13 in Table 1 showed 94% inhibition at 20
.mu.m.
[0225] No physiologically unacceptable toxicity was observed at the
effective dose for compounds tested of the present invention.
EXAMPLE 16
Pharmaceutical Compositions
[0226] The following Example illustrates, but is not intended to
limit, pharmaceutical dosage forms of the invention as defined
herein (the active ingredient being termed "Compound X"), for
therapeutic or prophylactic use in humans:
2 (a) Tablet I mg/tablet Compound X. 100 Lactose Ph. Eur 182.75
Croscarmellose sodium 12.0 Maize starch paste (5% w/v paste) 2.25
Magnesium stearate 3.0 (b) Tablet II mg/tablet Compound X 50
Lactose Ph. Eur 223.75 Croscarmellose sodium 6.0 Maize starch 15.0
Polyvinylpyrrolidone (5% w/v paste) 2.25 Magnesium stearate 3.0 (c)
Tablet III mg/tablet Compound X 1.0 Lactose Ph. Eur 93.25
Croscarmellose sodium 4.0 Maize starch paste (5% w/v paste) 0.75
Magnesium stearate 1.0 (d) Capsule mg/capsule Compound X 10 Lactose
Ph. Eur 488.5 Magnesium 1.5 (e) Injection I (50 mg/ml) Compound X
5.0% w/v 1M Sodium hydroxide solution 15.0% v/v 0.1M Hydrochloric
acid to adjust pH to 7.6 Polyethylene glycol 400 4.5% w/v Water for
injection to 100% (f) Injection II (10 mg/ml) Compound X 1.0% w/v
Sodium phosphate BP 3.6% w/v 0.1M Sodium hydroxide solution 15.0%
v/v Water for injection to 100% (g) Injection III (1 mg/ml.
buffered to pH6) Compound X 0.1% w/v Sodium phosphate BP 2.26% w/v
Citric acid 0.38% wlv Polyethylene glycol 400 3.5% w/v Water for
injection to 100% (h) Aerosol I mg/ml Compound X 10.0 Sorbitan
trioleate 13.5 Trichlorofluoromethane 910.0 Dichlorodifluoromethane
490.0 (i) Aerosol II mg/ml Compound X 0.2 Sorbitan trioleate 0.27
Trichlorofluoromethane 70.0 Dichlorodifluoromethane 280.0
Dichlorotetrafluoroethane 1094.0 (j) Aerosol III mg/ml Compound X
2.5 Sorbitan trioleate 3.38 Trichlorofluoromethane 67.5
Dichlorodifluoromethane 1086.0 Dichlorotetrafluoroethane 191.6 (k)
Aerosol IV mg/ml Compound X 2.5 Soya lecithin 2.7
Trichlorofluoromethane 67.5 Dichlorodifluoromethane 1086.0
Dichlorotetrafluoroethane 191.6 (l) Ointment ml Compound X 40 mg
Ethanol 300 .mu.l Water 300 .mu.l 1-Dodecylazacycloheptan-2-one 50
.mu.l Propylene glycol to 1 ml Note: Compound X in the above
formulation may comprise a compound illustrated in Examples. The
above formulations may be obtained by conventional procedures well
known in the pharmaceutical art. The tablets (a)-(c) may be enteric
coated by conventional means, for # example to provide a coating of
cellulose acetate phthalate. The aerosol formulations (h)-(k) may
be used in conjunction with standard, metered dose aerosol
dispensers, and the suspending agents sorbitan trioleate and soya
lecithin may be replaced by an alternative # suspending agent such
as sorbitan monooleate, sorbitan sesquioleate, polysorbate 80,
polyglycerol oleate or oleic acid.
* * * * *