Indole-3-acetic acid antagonists of the prostaglandin D2 Receptor CRTH2

Article abstract of DOI:10.1021/jm050519b

Prostaglandin D₂ (PGD₂) acting at the CRTH2 receptor (chemoattractant receptor-homologous molecule expressed on Th2 cells) has been linked with a variety of allergic and other inflammatory diseases. We describe a family of indole-1-s

Full text of DOI:10.1021/jm050519b

6174
J. Med. Chem. 2005, 48, 6174-6177
PGD2 on leukocyte activation are thought to be medi-
ated by an action on CRTH2 (chemoattractant receptor-
homologous molecule expressed on Th2 cells), which is
expressed by Th2 lymphocytes, eosinophils, and baso-
phils.5 It seems clear that the effect of PGD2 on the
activation of Th2 lymphocytes and eosinophils is medi-
ated through CRTH2 because the selective CRTH2
agonists 13,14-dihydro-15-keto-PGD2 (DK-PGD2) (2) and
15R-methyl-PGD2 (3) can elicit this response and the
effects of PGD2 are blocked by an anti-CRTH2 anti-
body.5a,6 In contrast, the selective DP agonist BW245C
(4) does not promote migration of Th2 lymphocytes or
eosinophils.5a,7 On the basis of this evidence, antagoniz-
ing PGD2 at the CRTH2 receptor should be an attractive
approach to treat the inflammatory component of Th2-
dependent allergic diseases such as asthma, allergic
rhinitis, and atopic dermatitis.
Indomethacin (5) is a nonsteroidal antiinflammatory
drug (NSAID) that achieves analgesic and antipyretic
activity through the inhibition of cylcooxygenases. It
was previously shown to bind to the human CRTH2
receptor with values ranging from 25 nM to 8 µM and
to act in an agonistic fashion in vitro with a potency of
approximately 15-50 nM.8 Sulindac sulfide (6), another
NSAID that has a similar spatial arrangement of acetic
acid and aromatic groups, was also shown to bind with
moderate affinity to the human CRTH2 receptor (Ki )
3.5 µM)8b and more recently to act agonistically on
murine CRTH2.9 More recent studies describe a range
of indolylacetic acid analogue type ligands for human
CRTH2; however, no pharmacokinetic and only limited
human cellular activity data were presented for selec-
tive antagonist compounds.10 L-888,607 (7), which also
possesses a fluoroindolecarboxylic acid template, has
been reported to be a potent (hCRTH2 binding Ki ) 0.8
( 0.4 nM) and orally bioavailable CRTH2 agonist.10d On
the basis of the earlier observations around indometha-
cin and sulindac sulfide,8 we hypothesized that it is
possible to discover novel molecules based on indole
acetic acid structures that show antagonistic activity at
the human CRTH2 receptor.
Indole-3-acetic Acid Antagonists of the
Prostaglandin D2 Receptor CRTH2
Richard E. Armer,*,† Mark R. Ashton,‡
Edward A. Boyd,‡ Chris J. Brennan,‡
Frederick A. Brookfield,‡ Lucien Gazi,†
Shaˆn L. Gyles,† Philip A. Hay,‡ Michael G. Hunter,†
David Middlemiss,# Mark Whittaker,‡
Luzheng Xue,† and Roy Pettipher†
Oxagen Ltd., 91 Milton Park, Abingdon, Oxfordshire, OX14
4RY, U.K., Evotec OAI, 151 Milton Park, Abingdon,
Oxfordshire, OX14 4SD, U.K., and XaviaPharm, 61 Thorley
Hill, Bishop’s Stortford, Hertfordshire, CM23 3NF, U.K.
Received June 3, 2005
Abstract: Prostaglandin D2 (PGD2) acting at the CRTH2
receptor (chemoattractant receptor-homologous molecule ex-
pressed on Th2 cells) has been linked with a variety of allergic
and other inflammatory diseases. We describe a family of
indole-1-sulfonyl-3-acetic acids that are potent and selective
CRTH2 antagonists that possess good oral bioavailability. The
compounds may serve as novel starting points for the develop-
ment of treatments of inflammatory disease such as asthma,
allergic rhinitis, and atopic dermatitis.
Prostaglandin D2 (PGD2) (1) is an eicosanoid, a class
of chemical mediator synthesized by cells in response
to local tissue damage and hormonal and immunological
stimuli. Eicosanoids bind to specific cell surface recep-
tors on a wide variety of tissues throughout the body
and mediate various effects in these tissues. PGD2 is
the major prostanoid produced by mast cells and is also
synthesized by macrophages and Th2 lymphocytes. It
was detected in high concentrations in the airways of
asthmatic patients challenged with antigen,1 and instil-
lation of PGD2 into airways can provoke many features
of the asthmatic response including bronchoconstriction2
and eosinophil accumulation.3
The potential of PGD2 to induce inflammatory re-
sponses was confirmed by the use of transgenic mice
overexpressing human PGD2 synthase. These mice
exhibit exaggerated eosinophilic lung inflammation and
Th2 cytokine production in response to antigen.4
The first PGD2 specific receptor to be discovered was
the DP receptor, which is linked to elevation of the
intracellular levels of cAMP. However, the effects of
Our founding hypothesis was that the carboxylic acid
group was important for binding human CRTH2 be-
cause the endogenous agonist, indomethacin, and sulin-
dac sulfide contain this moiety and the human CRTH2
receptor is rich in basic residues in the transmembrane
region. Preliminary studies indicated that the (5-fluoro-
2-methyl-1H-indolyl-3-yl)acetic acid template (with a
substitution pattern similar to that found in sulindac
sulfide (6) and L-888,607 (7)) was readily synthetically
accessible and that small SAR probing compound li-
braries derived from this template retained moderate
* To whom correspondence should be addressed. Phone: +44 1235
443300. Fax: +44 1235 443301. E-mail: r.armer@oxagen.co.uk.
† Oxagen Ltd.
‡ Evotec OAI.
# D.M. of XaviaPharm as an independent consultant.
10.1021/jm050519b CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/10/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6175
Table 1. Assay Data for the Indole N-Sulfonyl Compounds
Scheme 1a
a Reagents and conditions: (a) ethyl levulinate, DCE/TFA (6:
4), 43%; (b) RSO2Cl, KOtBu, 18-crown-6, THF; (c) LiOH, H2O, THF.
binding potency with good microsomal metabolic stabil-
ity. As a consequence, the (5-fluoro-2-methyl-1H-indolyl-
3-yl)acetic acid core was kept constant for the remainder
of the study. The most significant improvements in
hCRTH2 binding were achieved with modification of the
indole N-group. In particular, N-sulfonyl analogues were
found to be particularly potent and selective compounds
in ligand binding experiments. The indole N-sulfonyl
analogue 10a (R ) 4-chlorophenyl) was prepared using
the conditions described in Scheme 1.
(5-Fluoro-2-methyl-1H-indol-3-yl)acetic acid ethyl es-
ter (9) was obtained via a Fischer indole synthesis using
4-fluorophenylhydrazine hydrochloride (8) and ethyl
levulinate.11 The indole N-sulfonyl group was introduced
from reaction with the corresponding sulfonyl chloride
with potassium tert-butoxide and 18-crown-6. Hydroly-
sis was achieved using lithium hydroxide in aqueous
tetrahydrofuran to give the free acid.
10a displayed potent binding to hCRTH2 (Ki ) 29
nM) with excellent selectivity over the DP receptor
(membranes from Chinese hamster ovary (CHO) cells
expressing recombinant human CRTH2 or DP were
used in these experiments). With binding of 10a to
hCRTH2 established, it was pivotal to ascertain that
the interaction was resulting in agonism or the desired
antagonism of the receptor. This was determined through
measurement of the compound’s ability to inhibit PGD2-
stimulated Ca2+ flux in intact CHO cells expressing
human CRTH2; IC50 values for this assay are shown in
Table 1. 10a on its own did not induce calcium mobili-
zation in CHO/CRTH2 cells.
With increasing concentration of 10a, a dose-depend-
ent and parallel shift of the PGD2 dose response curve
in CHO/hCRTH2 cells was observed, thereby indicating
that the compound was indeed a competitive hCRTH2
antagonist (pA2 ) 7.48 ( 0.07). In addition, the antago-
nistic effect of the compound appeared to be hCRTH2
selective, since no inhibitory effect was seen with ATP-
stimulated Ca2+ flux. This important result led us to
prepare a series of analogues of 10a and to evaluate
them for selective binding to hCRTH2 over the DP
receptor. The test compounds were prepared according
to the conditions in Scheme 1.
The structure-activity relationship data presented in
Table 1 demonstrate the importance of a phenylsul-
fonamide group substituted at the para position with
an electron-withdrawing group to give optimal potency
and selectivity. Replacing the electron-withdrawing
group with a neutral (10d) or donating moiety (10h)
leads to reduced affinity at the hCRTH2 receptor. When
the electron-withdrawing group is present at the meta
position (10f), affinity for hCRTH2 is reduced. The
pendent phenyl group appears to be essential for activity
because replacement with a heterocyclic group (10i) or
an alkyl chain (10j) leads to a decrease in affinity. In
all cases the selectivity for hCRTH2 over the DP
a Standard error of the mean. b Carried out at 10 nM PGD2.
Table 2. Cellular Assay Data
eosinophil shape change
IC50 (nM)a
Th2 cell chemotaxis
IC50 (nM)
compd
10a
10c
908
74
not tested
67
a Forward scatter measured by fluorescence-activated cell sort-
ing carried out in the presence of 10% fetal calf serum.
receptor was high with >160-fold selectivity being
achieved for compound 10c. Interestingly, the dichloro
analogue (10g) demonstrated a small increase in affin-
ity for hCRTH2 despite lacking an electron-withdrawing
group at the para position; however, its functional
potency and selectivity over DP were not optimal.
The most potent and selective compounds (10a and
10c) were assessed in secondary assays to ensure that
the observed antagonism of hCRTH2 gave rise to
activity in primary human eosinophils and Th2 cells
that express endogenous CRTH2. The ability of the
compounds to inhibit PGD2 induced eosinophil or Th2
cell activation was assessed by shape change or chemo-
taxis; both compounds demonstrated an effect with 10c
showing particularly potent inhibition (Table 2). The
weaker effect of 10a on eosinophil shape change in the
presence of serum may be due to the poorer solubility
(0.05 mg/mL for 10a compared to 0.08 mg/mL for 10c)
and therefore putative higher protein binding for this
compound. The antagonistic effect of 10c appears to be
hCRTH2 selective, since no inhibitory effect was seen
when other chemoattractant compounds were used,
including eotaxin, 5-oxo-ETE, IL-5, C5a, and LTB4.12
The potency of 10c made it a candidate for further
evaluation, and so the potential for metabolic and toxic
liabilities was assessed. The data for these assays are
shown in Table 3. 10c showed no inhibition of the five
major cytochrome P450 (CYP450) isoforms and no

6176 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Letters
Table 3. Metabolic and Toxicity Assay Data for 10c
assay type
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Table 4. Pharmacokinetic Data for 10c in Rats
dose
Cmax
Tmax
AUCinf
Cl
t1/2
F
(mg/kg) (ng/mL) (h) (ng/(mL‚h)) ((mL/min)/kg) (h) (%)
1 (iv)
10 (po)
1801
332
619
3480
40
na
2.2
5.5 56
1.8
induction of three CYP450 compounds at 1 µM. No
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a human ether-a-go-go-related (hERG) potentiomentric
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additional information, and broad selectivity screening infor-
mation. This material is available free of charge via the
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Letters
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JM050519B