Discovery and optimization of CRTH2 and DP dual antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.09.052

A series of phenylacetic acid derivatives was discovered as CRTH2 antagonists. Modification of the series led to compounds that are also antagonists of DP. Since activation of CRTH2 and DP are believed to play key roles in mediating responses of asthma and other immune diseases, this series was optimized to increase the dual antagonistic activities and improve pharmacokinetic properties. These efforts led to selection of AMG 009 as a clinical candidate.

Full text of DOI:10.1016/j.bmcl.2009.09.052

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6419–6423
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of CRTH2 and DP dual antagonists
*
Jiwen Liu , Zice Fu, Yingcai Wang, Mike Schmitt, Alan Huang, Derek Marshall, George Tonn, Lisa Seitz,
Tim Sullivan, H. Lucy Tang, Tassie Collins, Julio Medina
Amgen Inc. 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2009
Revised 11 September 2009
Accepted 14 September 2009
Available online 17 September 2009
A series of phenylacetic acid derivatives was discovered as CRTH2 antagonists. Modification of the series
led to compounds that are also antagonists of DP. Since activation of CRTH2 and DP are believed to play
key roles in mediating responses of asthma and other immune diseases, this series was optimized to
increase the dual antagonistic activities and improve pharmacokinetic properties. These efforts led to
selection of AMG 009 as a clinical candidate.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
CRTH2
DP
Antagonists
GPCR
Asthma
PGD2
Prostaglandin D2 (PGD2) plays a key role in mediating allergic
reactions such as those seen in asthma, allergic rhinitis, atopic der-
matitis and allergic conjunctivitis.1 PGD2 is the major cyclooxygen-
ase product formed and secreted by activated mast cells and its
levels in bronchoalveolar lavage (BAL) fluid increase in response to
antigen provocation.2–6 In animals, including humans, PGD2 stimu-
lates several responses observed in asthma and other immune dis-
eases such as airway constriction, mucus secretion, increased
microvascular permeability and recruitment of eosinophils.7–12 In
addition, mice that overexpress PGD2 synthase, resulting in overpro-
duction of PGD2, experience increased levels of Th2 cytokines and
chemokines accompanied by enhanced accumulation of eosinophils
and lymphocytes in the lung following an allergic response to
ovalbumin.11 Thus, PGD2 is thought to be involved in the acute
and late phases of allergic reactions. PGD2 activates two receptors,
DP (prostanoid D receptor, DP1) and CRTH2 (chemoattractant recep-
tor-homologous molecule expressed on Th2 cells, DP2).
by sneezing, mucosal plasma exudation, and nasal blockage, as
well as late responses, such as mucosal plasma exudation and
eosinophil infiltration.16 Moreover, DP antagonism alleviated aller-
gen-induced plasma exudation in the conjunctiva in a guinea pig
allergic conjunctivitis model and antigen-induced eosinophil infil-
tration into the lung in a guinea pig asthma model.16 In addition,
human genetic data suggest that DP may play a role in asthma.17
These results demonstrate the importance of DP signaling for aller-
gic responses.
The second PGD2 receptor, CRTH2, is related to the N-formyl pep-
tide receptor (FPR) subfamily of chemoattractant receptors.18
CRTH2 is selectively expressed on Th2 cells, T cytotoxic type 2
(Tc2) cells, eosinophils, and basophils.19–21 CRTH2 activation in-
duces an increase in intracellular Ca2+ mobilization via Gi dependent
pathways, allowing CRTH2 to transmit promigratory signals in re-
sponse to PGD2.17,22,23 In leukocytes, PGD2 induces migration exclu-
sively via CRTH2.17,23–26 The ability of PGD2 to stimulate the
migration of inflammatory cells has led to the hypothesis that
CRTH2 may play a role in the recruitment of cellular components
of the allergic response into diseased tissues. Studies in rats using
agonists and antagonists selective for CRTH2 over DP lend support
to such a hypothesis. Selective activation of CRTH2 by intravenous
injection of 13,14-dihydro-15-keto (DK)-PGD2 into rats led to a
dose- and time-dependent increase in the number of eosinophils
in the peripheral blood. Pretreatment of the animals with Ramatro-
ban (BAY u3405, 17), a CRTH2/thromboxane A2 receptor dual
antagonist, completely abrogated DK-PGD2-induced eosinophilia.12
Furthermore, like DP, human genetic data also suggest that CRTH2
may play a role in asthma.27
DP was the first PGD2 receptor discovered.13 It belongs to the
prostanoid receptor family of GPCRs and is expressed on airway
epithelium, smooth muscle and platelets. Upon stimulation, DP
activates adenylate cyclase and increases the level of cAMP primar-
ily via Gs-dependent pathways.14 Genetic analysis of DP function
using DP deficient mice has shown that mice lacking DP do not
develop asthmatic responses in an ovalbumin-induced asthma
model.15 A selective DP antagonist in guinea pig allergic rhinitis
models dramatically inhibited early nasal responses, as assessed
* Corresponding author. Tel.: +1 650 244 2438; fax: +1 650 837 9369.
E-mail address: jiwenl@amgen.com (J. Liu).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.052

6420
J. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6419–6423
NO2
NO2
NO2
Several groups have reported their efforts of identifying selec-
O
Cl
Cl
O
OH
tive DP and CRTH2 inhibitors. Figures 1 and 2 show a few examples
of the DP and CRTH2 antagonists.28–32 Comprehensive reports on
DP and CRTH2 antagonists can be found in two review articles.33,34
Because the collective observations suggest that DP and CRTH2
play important and complementary roles in the physiological re-
sponses to PGD2, blockade of both receptors may prove more ben-
eficial in alleviating allergic diseases triggered by PGD2. In this
article, we report our efforts towards the identification of dual
DP and CRTH2 antagonists.
a
b
d
Cl
R1HN
R1HN
R3
R2
O
c
O
O
O
S
O
NH
NH2
O
O
O
OH
O
OH
R1HN
R1HN
R2
R2
O
O
1-12, 20-37
A phenylacetic acid derivative 1 (Table 1), discovered in a high
Scheme 1. Reagents and conditions: (a) amines, triethylamine, DCM, rt, 4 h, ꢀ90%;
(b) phenylacetic acids methyl ester, cesium carbonate, DMSO, 70 °C, 6 h, ꢀ85%; (c)
H2, Pd/C, EtOH, rt, 1 h, 100%; or SnCl2, EtOAc, 60 °C, 4 h, 80%; (d) sulfonyl chlorides,
pyridine, rt, 24 h, 70%.
throughput screen for CRTH2, inhibited the binding of 3H-PGD2 to
CRTH2 receptors on 293 cells with an IC50 of 0.010
ited the binding of 3H-PGD2 to DP receptors with an IC50 of only
8.3
M (Table 1).35 Compound 1 also inhibited CRTH2 mediated
lM, and inhib-
l
cell migrations in response to PGD2 with an IC50 of 0.0047
lM
NO2
NO2
using hCRTH2 stably transfected CEM cells.36
Cl
a
O
OH
O
b
Compound 1 and many its derivatives (2–12 and 20–37) were
synthesized according to Scheme 1. 4-Chloro-3-nitrobenzoyl chlo-
ride was reacted with amines to form the corresponding amides.
Displacement of the chlorine adjacent to the nitro group with
hydroxyphenylacetic acids gave the bis-aryl ethers in good yields.
Reduction of the nitro group followed by treatment with sulfonyl
chlorides in pyridine afforded the sulfonamides.
O
R
R
O
S
O
NH2
NH
O
OH
c
OH
O
R
O
R
R = C(O)Me, Cl, OMe
13, 15, 16
Compounds 13, 15 and 16 were synthesized in three steps
(Scheme 2), as described in Scheme 1, steps b, c and d.
Scheme 2. Reagents and conditions: (a) 3-hydroxyphenylacetic acid, cesium
carbonate, DMSO, 70 °C, 6 h, ꢀ85%; (b) SnCl2, EtOAc, 60 °C, 4 h, 80%; (c) benzene-
sulfonyl chlorides, pyridine, rt, 24 h, 70%.
S
The synthesis of compound 14 is shown in Scheme 3. 4-Chloro-
3-nitrobenzaldehyde was protected as the diethyl acetal, which
was carried into the same three steps as described in Scheme 2. Fi-
nally, the aldehyde was unmasked and converted into the ethyl
benzyl amine via reductive amination.
Compound 17 (Scheme 4) was also synthesized similarly in
three steps, as described in Scheme 2. Compounds 18 and 19 were
synthesized from compound 17. Reaction of propionyl chloride
O
F
COOH
Cl
NH
N
OH
COOH
O
S
Me
O
Laropiprant
S5751
Figure 1. DP antagonists.
O
O
O
NO2
NO2
O
NO2
S
S
MeN
HN
O
OH
Cl
a
b
Cl
EtO
O
EtO
O
OHC
c, d
F
F
OEt
OEt
N
N
O
COOH
O
S
O
O
NH
COOH
N
S
NH
Me
Ramatroban
e, f
O
OH
OH
EtHN
O
EtO
O
Me
N
F
14
OEt
Me
COOH
N
N
Scheme 3. Reagents and conditions: (a) EtOH, HCl, rt, 14 h, 100%; (b) 3-hydroxy-
phenylacetic acid methyl ester, cesium carbonate, DMSO, 70 °C, 6 h, ꢀ85%; (c) SnCl2,
EtOAc, 60 °C, 4 h, 80%; (d) benzenesulfonyl chlorides, pyridine, rt, 24 h, 70%; (e) TFA,
DCM/H2O, 0 °C, 2 h, 90%; (f) ethylamine, NaBH(OAc)3, DCE, rt, 3 h, 80%.
COOH
Figure 2. CRTH2 antagonists.
Table 1
NH2
NH2
Cl
a
O
OH
O
b, c
Cl
O
O
O
NH
O2N
O2N
OH
Cl
S
O
OH
O
O
S
O
O
O
Me
EtHN
Cl
d
S
Cl
NH
NH
OH
O
O
O
Cl
O
O
R
N
H
H2N
1
17
18, 19
a
a
Compd
CRTH2 IC50 in buffer (
0.010
lM)
DP IC50 in buffer (
8.3
lM)
Scheme 4. Reagents and conditions: (a) 3-hydroxyphenylacetic acid methyl ester,
cesium carbonate, DMSO, 70 °C, 6 h, ꢀ85%; (b) benzenesulfonyl chlorides, pyridine,
rt, 24 h, 70%; (c) SnCl2, EtOAc, 60 °C, 4 h, 80%; (d) for 18, propionyl chloride,
triethylamine, EtOAc, rt, 12 h, 80%; for 19, ethyl isocyanate, triethylamine, EtOAc, rt,
12 h, 80%.
1
a
Displacement of 3H-PGD2 from the CRTH2 or DP receptor expressed on 293
cells. Assay run in buffer containing 0.5% BSA. See Ref. 35 for assay protocol. Values
are means of three experiments, standard deviation is 30%.

J. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6419–6423
6421
with 17 yielded amide 18 and reaction of ethyl isocyanate with 17
afforded urea 19.
the primary or secondary amides (9 to 11), the reversed amide
(18) and the urea (19) derivatives.
The non-commercially available 3-hydroxy-5-chloro phenyla-
cetic acid was synthesized according to Scheme 5. Monochloro
displacement of 3,5-dichlorobenzoic acid by methoxide gave
3-chloro-5-methoxybenzoic acid. The carboxylic acid was then
converted into the acyl chloride, which was treated with trimeth-
ylsilyldiazomethane to afford the diazoketone. Wolff rearrange-
ment of the diazoketone provided the homologated methyl ester.
Finally, demethylation afforded 3-hydroxy-5-chloro phenylacetic
acid.
The optimization began with the examination of the effect of
substitutions on the sulfonamide benzene ring (Table 2). It was
found that the substitutions had little effect on the compounds’
affinity for CRTH2, as the unsubstituted compound 3 has similar
potency to other compounds in Table 2. However, in vitro meta-
bolic stability studies let us have a preference for the benzenesulf-
onamides with halogen substitutions, such as the 2,4-dichloro
benzenesulfonamide (2), because the stability studies using liver
microsomes indicated that the benzene ring of the benzenesulf-
onamides might be a vulnerable site of oxidative metabolism and
benzenesulfonamides with alkyl groups or other electron-donating
substitutions tend to have less optimal microsomal stability.
Next we investigated the role of the ethyl amide moiety. Ana-
logs devoid of the hydrogen bond donor NH, such as tertiary amide
12 and methyl ketone 13, has significant decreased activity. Like-
wise, analogs lacking the carbonyl group, such as compounds 14
(compared with 9) and 17 (compared with 18), also has signifi-
cantly decreased CRTH2 activity. This data suggests that a hydro-
gen bond donor and a hydrogen bond acceptor together is likely
to be beneficial for the activity on CRTH2, as demonstrated by
Evaluation of the phenylacetic acid moiety revealed that modi-
fications in this part of the molecule affect the selectivity of the
antagonists to the CRTH2 and DP receptors (Table 4). Compound
20 (Table 4) and compounds in Tables 2 and 3 that feature the ace-
tic acid moiety meta to the oxygen-linker are selective for CRTH2
over DP receptor. However, compounds that feature the acetic acid
moiety para to the oxygen-linker, such as 21, display CRTH2 and
DP dual inhibitory activity. Since we were interested in identifying
dual antagonists of CRTH2 and DP receptors, we decided to explore
the analogs of 21. Replacement of the acid moiety of 21 with a tet-
razole group (22) maintained the affinity for the DP receptor, but
the affinity for CRTH2 was greatly reduced. Likewise, addition of
a methyl group at the methylene next to the carboxylic acid (23)
resulted in a moderate enhancement of the affinity for DP, but a
significant loss of the CRTH2 affinity.
Table 3
Evaluation of the amide moiety
O
S
O
NH
R1
O
OH
O
R2
R2
a
Compd
R1
CRTH2 IC50 in buffer (lM)
9
10
11
12
13
14
15
16
17
18
19
H
H
Me
Me
H
H
H
H
CONHEt
CONH2
CONHEt
CONEt2
COMe
CH2NHEt
Cl
OMe
NH2
NHCOEt
NHCONHEt
0.013
0.047
0.007
1.10
0.38
>10
0.68
1.40
0.13
0.007
0.016
Cl
Cl
Cl
Cl
Cl
MeO
Cl
MeO
Cl
MeO
Cl
c
a
b
a
Displacement of 3H-PGD2 from the CRTH2 receptor expressed on 293 cells.
O
OH
O
OH
Cl
O
Cl
O
CHN2
Assay run in buffer containing 0.5% BSA. See Ref. 35 for assay protocol. Values are
means of three experiments, standard deviation is 30%.
HO
Cl
MeO
e
d
COOH
COOMe
Table 4
Scheme 5. Reagents and conditions: (a) NaOMe, HMPA, 120 °C, 2 days, 95%; (b)
SOCl2, reflux, 4 h, 100%; (c) TMSCHN2, triethylamine, ACN, THF, 0 °C, 3 h, 55%; (d)
silver benzoate, triethylamine, MeOH, -25 to 25 °C, 6 h, 70%; (e) 48% HBr, HOAc,
reflux, 22 h, 95%.
Exploration of the phenyl acetic acid moiety
Cl
O
S
O
NH
Cl
O
Ar
EtHN
Table 2
O
Evaluation of the sulfonamide benzene ring
a
a
O
R
Compd
Ar
CRTH2 IC50
in buffer (
DP IC50 in
O
NH
S
O
OH
lM)
buffer (lM)
O
OH
20
0.016
>10
EtHN
O
O
O
Cl
a
Compd
R
CRTH2 IC50 in buffer (lM)
21
22
23
0.004
2.37
0.12
OH
1
2
3
4
5
6
7
8
2,4-Cl-5-Me
2,4-Cl
0.010
0.012
0.003
0.005
0.003
0.004
0.004
0.028
N
N
N
H
4-Me
4-Cl
2-Cl
0.132
0.043
N
H
O
3-Cl-4-Me
2-Cl-4-CF3
0.107
OH
a
a
Displacement of 3H-PGD2 from the CRTH2 receptor expressed on 293 cells.
Displacement of 3H-PGD2 from the CRTH2 or DP receptor expressed on 293
Assay run in buffer containing 0.5% BSA. See Ref. 35 for assay protocol. Values are
means of three experiments, standard deviation is 30%.
cells. Assay run in buffer containing 0.5% BSA. See Ref. 35 for assay protocol. Values
are means of three experiments, standard deviation is 30%.

6422
J. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6419–6423
Table 6
Substitutions on the benzene ring of the phenyl acetic acid
Evaluation of the amide moiety
moiety were evaluated to study their effect on DP and CRTH2
selectivity (Table 5). Compound 21 has strong affinity for CRTH2,
but it only has moderate affinity for DP. Small substitutions such
as methoxy (24) and fluorine (25) next to the aryloxy group im-
prove the compounds’ affinity for the DP receptor (Table 5). How-
ever, ethoxy derivative 26 has decreased activity on DP compared
to methoxy analog 24, which suggests that the increased bulk may
be detrimental. Introduction of methoxy groups on both sides next
to the aryloxy group afforded a compound (27) with high affinity
for CRTH2 and dramatically decreased activity on DP.
Cl
O
O
NH
Cl
S
OMe
O
O
H
N
R
OH
O
a
a
Compd
R
CRTH2 IC50 in buffer (
l
M)
DP IC50 in buffer (lM)
24
30
31
32
33
34
35
36
37
Et
n-Pr
n-Bu
n-Pentyl
i-Pr
i-Bu
c-Pr
c-Bu
c-Pentyl
0.003
0.002
0.003
0.007
0.002
0.003
0.004
0.003
0.006
0.055
0.031
0.012
0.009
0.036
0.059
0.041
0.011
0.179
The linker between the carboxylic acid and the phenyl ring was
also studied (Table 5). A methylene linker (24) is preferred over an
ethylene linker (29) or direct attachment of carboxylic acid to the
phenyl ring (28).
Further study of the amide moiety (Table 6) provided additional
improvement on DP potency. Longer n-alkyl groups, such as n-pro-
pyl (30), n-butyl (31) and n-pentyl (32), improved the affinity for
DP, while maintaining the affinity for the CRTH2 receptor. Investi-
gation of branched alkyl groups, such as isopropyl (33) or isobutyl
(34), and cyclic alkyl groups, such as cyclopropyl (35) or cyclopen-
tyl (37), showed that these groups were well tolerated. However,
they did not lead to improvements on the affinity towards either
receptor. The cyclobutyl analog (36), on the other hand, displayed
increased affinity for the DP receptor while maintaining the affinity
for the CRTH2. All comparisons here are made to the parental ethyl
amide (24).
Because of its high affinity towards both receptors, compound
31 (AMG 009) was further investigated in DP and CRTH2 functional
assays. AMG 009 inhibited PGD2-induced down-modulation of
CRTH2 on CD16 negative granulocytes (eosinophils) in human
whole blood with a Ki of 1 nM.37 In addition, AMG 009 also inhib-
ited PGD2-induced cAMP response mediated by DP in platelets in
80% human whole blood with a Ki of 148 nM.38 The selectivity of
AMG 009 was studied in a commercially available GPCR panel
and AMG 009 demonstrated excellent selectivity, including its
selectivity against prostanoid receptors TP, EP2 and EP4.39 The
pharmacokinetic properties of AMG 009 were evaluated in several
species (Table 7). AMG 009 has low to moderate clearance across
species and good oral absorption.
Displacement of 3H-PGD2 from the CRTH2 or DP receptor expressed on 293
cells. Assay run in buffer containing 0.5% BSA. See Ref. 35 for assay protocol. Values
are means of three experiments, standard deviation is 30%.
a
Table 7
Pharmacokinetic properties of AMG 009
Cl
O
O
Cl
S
NH
OMe
O
O
H
N
OH
O
AMG 009
Species
Clearancea
MRT (h)
Vdss (L/kg)
Oral bioavailabilityb (%)
(L/h/kg)
Rat
Dog
Cyno
1.2
0.77
0.27
0.9
1.4
1.3
1.0
1.0
0.37
28
60
39
a
IV dose at 0.5 mg/kg.
Oral dose at 2 mg/kg.
b
Since guinea pigs have been shown to respond to aerosolized
PGD2 in a similar manner as humans,40 AMG 009 was investigated
in an acute guinea pig model of PGD2-induced airflow obstruction.
In this model, airway resistance is measured in response to in-
creased concentrations of PGD2 using a whole body plethysmogra-
phy.41 Groups of four Dunkin-Hartley guinea pigs were dosed once
with vehicle or AMG 009 at 3, 10 or 30 mg/kg by subcutaneous
administration 15 min prior to PGD2 challenges. The enhanced
pause (Penh), a measure of airway resistance, to inhaled PGD2
was measured over a 10-min span after the guinea pigs were sub-
jected to 1-min challenges with aerosolized saline or PGD2 in saline
of ascending concentrations at 0.063, 0.125, 0.25, 0.5 and 1.0 mg/
Table 5
Substitutions on the benzene ring of the phenyl acetic acid moiety
Cl
O
S
O
NH
Cl
R1
O
O
EtHN
R2
OH
n
O
R1
R2
n
CRTH2 IC50 in buffer (
l
M)
DP IC50 in buffer (
l
M)
a
a
Compd
21
24
25
26
H
OMe
F
OEt
OMe
OMe
OMe
H
H
H
H
OMe
H
H
1
1
1
1
1
0
2
0.004
0.003
0.002
0.002
0.002
>50
0.12
0.055
0.036
1.91
16.0
>10
27
28b
29b
0.74
2.07
a
Displacement of 3H-PGD2 from the CRTH2 or DP receptor expressed on 293 cells. Assay run in buffer containing 0.5% BSA. See Ref. 35 for assay protocol. Values are means
of three experiments, standard deviation is 30%.
b
Compounds are benzene sulfonamides instead of 2,4-dichlorobenzene sulfoamides.

J. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6419–6423
6423
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10 mg/kg
30 mg/kg
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Baseline
Saline
0.063
0.125
0.25
0.5
1.0
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Trottein, F. J. Immunol. 2003, 170, 4943.
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Figure 3. Dose–response of AMG 009 in the guinea pig model of PGD2-induced
airway constriction. AMG 009 was dosed SC 15 min before the challenges with
aerosolized PGD2; n = 4 guinea pigs/group. Data reported as mean standard error
of the mean (sem).
28. Sturino, C. F.; O’Neill, G.; Lachance, N.; Boyd, M.; Berthelette, C.; Labelle, M.; Li,
L.; Roy, B.; Scheigetz, J.; Tsou, N.; Aubin, Y.; Bateman, K. P.; Chauret, N.; Day, S.
H.; Lévesque, J.; Seto, C.; Silva, J. H.; Trimble, L. A.; Carriere, M.; Denis, D.; Greig,
G.; Kargman, S.; Lamontagne, S.; Mathieu, M.; Sawyer, N.; Slipetz, D.; Abraham,
W. M.; Jones, T.; McAuliffe, M.; Piechuta, H.; Griffith, D. A. N.; Wang, Z.;
Zamboni, R.; Young, R. N.; Metters, K. M. J. Med. Chem. 2007, 50, 794.
29. Mitsumori, S.; Tsuri, T.; Honma, T.; Hiramatsu, Y.; Okada, T.; Hashizume, H.;
Kida, S.; Inagaki, M.; Arimura, A.; Yasui, K.; Asanuma, F.; Kishino, J.; Ohtani, M.
J. Med. Chem. 2003, 46, 2446.
mL. Treatment with AMG 009 resulted in a dose dependent de-
crease in airway resistance provoked by PGD2 aerosol ( Fig. 3).
The in vivo effect of AMG 009 was most likely due to its DP antag-
onistic activity, because CRTH2 selective antagonist (27) did not re-
duce airway resistance in this guinea pig model.42
30. Ulven, T.; Kostenis, E. J. Med. Chem. 2005, 48, 897.
The ability of AMG 009 to block the guinea pig CRTH2 and DP
receptors was also evaluated in vitro using a 3H-PGD2 displace-
ment assay with cells transfected with the guinea pig CRTH2
receptors (IC50 = 3 nM)43 and a PGD2-induced cAMP response assay
with cells expressing the guinea pig DP receptors (Ki = 131 nM).44
In summary, we have discovered a novel series of CRTH2 antag-
onists and optimized their properties to also inhibit the DP recep-
tor. Based in part on the evaluation of CRTH2 and DP activities and
the outcomes of pharmacokinetic studies, we selected AMG 009
(31) as a candidate compound for evaluation in clinical studies.
This compound is a potent inhibitor of PGD2-induced CRTH2 recep-
tor down-modulation and DP mediated cAMP response in human
whole blood. This compound was also efficacious in a guinea pig
model of airway resistance.
31. Lovell, J.M. WO Patent 107772, 2007.
32. Royer, J. F.; Schratl, P.; Carrillo, J. J.; Jupp, R.; Barker, J.; Weyman-Jones, C.; Beri,
R.; Sargent, C.; Schmidt, J. A.; Lang-Loidolt, D.; Heinemann, A. Eur. J. Clin. Invest.
2008, 38, 663.
33. Medina, J. C.; Liu, J. Annu. Rep. Med. Chem. 2006, 41, 221.
34. Pettipher, R.; Hansel, T. T.; Armer, R. Nat. Rev. Drug Disc. 2007, 6, 313.
35. The CRTH2 or DP radioligand binding assay was performed on 293 cells stably
expressing human CRTH2 or DP. To measure binding, [3H]-PGD2 was incubated
together with 293(hCRTH2 or hDP) cells in the presence of increasing
concentrations of compounds. After washing, the amount of [3H]-PGD2 that
remained bound to the cells was measured by scintillation counting and the
concentration of compounds required to achieve a 50% inhibition of [3H]-PGD2
binding (the IC50) was determined. The binding buffer contains either 0.5% BSA
(buffer binding) or 50% human plasma (plasma binding).
36. CRTH2 mediated cell migration was analyzed in a transwell migration assay
using hCRTH2 stably transfected CEM cells (a T lymphoblast cell line). The cells
were incubated with increasing concentrations of compounds for 3 h in a 96-
well migration chamber on top of a transwell filter and the number of cells
migrating through the filter in response to 5 nM PGD2 in the lower chamber
was counted and the IC50 of the compounds determined. The migration buffer
contains 0.5% BSA.
References and notes
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Fluorochrome conjugated antibodies were used to label CRTH2 positive
granulocytes and CRTH2 receptor internalization was monitored by flow
cytometry. Ki was determined using Schild equation.
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38. Human whole blood was drawn into ACD vacutainer tubes, treated compounds
or DMSO and then stimulated with a dose response of PGD2. Cells were lysed
and cAMP was measured using a competitive ELISA. Comparison of the dose
response to PGD2 in samples containing DMSO only and samples containing
compounds were used in determining Ki using Schild equation.
39. AMG 009 has an IC50 of 30 l
M in a binding assay against 3H-labeled TP agonist
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aerosolized PGD2.
43. Guinea pig 3H-PGD2 displacement assay was done in the same way as in Ref. 35
using 293 cells stably expressing guinea pig CRTH2.
44. 293 cells stably-transfected with guinea pig DP receptors were treated
compounds or DMSO and then stimulated with a dose response of PGD2.
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Cells were lysed and cAMP was measured using
a competitive ELISA.
Comparison of the dose response to PGD2 in samples containing DMSO only
and samples containing compounds were used in determining Ki using Schild
equation.