Discovery and optimization of a biphenylacetic acid series of prostaglandin D2 receptor DP2 antagonists with efficacy in a murine model of allergic rhinitis

Article abstract of DOI:10.1016/j.bmcl.2011.01.024

Biphenylacetic acid (5) was identified through a library screen as an inhibitor of the prostaglandin D₂ receptor DP2 (CRTH2). Optimization for potency and pharmacokinetic properties led to a series of selective CRTH2 antagonists. Compounds demonstrated potency in a human DP2 binding assay and a human whole blood eosinophil shape change assay, as well as good oral bioavailability in rat and dog, and efficacy in a mouse model of allergic rhinitis following oral dosing.

Full text of DOI:10.1016/j.bmcl.2011.01.024

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6608–6612
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of a biphenylacetic acid series of prostaglandin
D2 receptor DP2 antagonists with efficacy in a murine model of allergic
rhinitis
⇑
Jill M. Scott , Christopher Baccei, Gretchen Bain, Alex Broadhead, Jilly F. Evans, Patrick Fagan,
John H. Hutchinson, Christopher King, Daniel S. Lorrain, Catherine Lee, Peppi Prasit, Pat Prodanovich,
Angelina Santini, Brian A. Stearns
Amira Pharmaceuticals, 9535 Waples Street, Suite 100, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Biphenylacetic acid (5) was identified through a library screen as an inhibitor of the prostaglandin D2
receptor DP2 (CRTH2). Optimization for potency and pharmacokinetic properties led to a series of selec-
tive CRTH2 antagonists. Compounds demonstrated potency in a human DP2 binding assay and a human
whole blood eosinophil shape change assay, as well as good oral bioavailability in rat and dog, and effi-
cacy in a mouse model of allergic rhinitis following oral dosing.
Received 10 November 2010
Revised 5 January 2011
Accepted 6 January 2011
Available online 11 January 2011
Ó 2011 Published by Elsevier Ltd.
Keywords:
CRTH2
DP2
Prostaglandin D2
Allergic rhinitis
Eosinophil shape change
Binding
Antagonist
Prostaglandin D2 (PGD2), a product of the arachidonic acid cas-
cade, is the major cyclooxygenase product formed and released
from activated mast cells, and is known to be involved in allergic
inflammatory response.1 The biological actions of PGD2 are medi-
ated by two G-protein coupled receptors (GPCRs) termed DP1
and DP2 (also known as CRTH2). While DP1 is expressed on airway
epithelium, smooth muscle, and platelets, DP2 is expressed on
eosinophils, basophils, and Th2 cells. It has been shown that the
PGD2-mediated activation and migration of these cells proceeds
selectively through the DP2 receptor. Therefore, the DP2 receptor
may have an important role in inflammatory diseases including
asthma, COPD, and allergic rhinitis.2 Towards that end, there have
been many recent publications detailing the discovery of small
molecule DP2 antagonists,3,4 as well as reports of their efficacy in
pre-clinical rodent models of various allergic diseases.4e,5
on cell membranes with an IC50 of 0.182 lM, and thus became a
lead for optimization. The molecule was divided into three sections
for a preliminary survey of the structure–activity relationship (see
structure, Table 1). All compounds were screened in a DP2 binding
assay6 both in the presence and absence of 0.2% human serum
albumin (HSA) to evaluate intrinsic potency and ascertain the de-
gree of protein shift associated with the compounds.
Compound 5 was synthesized according to Scheme 1, beginning
with the commercially available benzyl bromide 1 and bromophen-
ylacetic acid 3. (4S,5R)-4-Methyl-5-phenyl-oxazolidin-2-one was
alkylated with benzyl bromide 1 using sodium hydride as base. Bro-
mophenylacetic acid 3 was first converted to the ethyl ester then
treated with bis(pinacolato)diboron in the presence of tetrakis(tri-
phenylphosphine)palladium to generate aryl boronate 4. Palla-
dium-catalyzed Suzuki coupling with bromide 2 furnished the
biphenyl core, which was hydrolyzed to provide compound 5 (Table
1). Compounds 6–14 were synthesized in an analogous fashion.
Initial SAR studies began by exploring the substitution pattern
of the carboxylic acid function on the A ring (Table 1, compounds
5–11). The des-methoxy analog 6 was synthesized to use as a
benchmark in determining the correct chain length and placement
of the acid relative to the biphenyl ring. In the meta position, phen-
ylacetic acid proved to be the optimal linker length, as contracting
the chain to the benzoic acid 7 or extending to the phenyl-propi-
onic acid 8 led to decreased binding activity. Placing the acid in
We have previously described a novel tricyclic series of DP2
antagonists which were shown to have efficacy in a murine model
of allergic rhinitis.4e In this Letter, we describe the discovery and
exploration of a new series of small molecule DP2 antagonists.
Biphenylacetic acid 5, identified through a screen of existing
compounds, inhibited the binding of 3H-PGD2 to DP2 receptors
⇑
Corresponding author.
E-mail address: jill.scott@amirapharm.com (J.M. Scott).
0960-894X/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.01.024

J. M. Scott et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6608–6612
6609
Table 1
Exploration of phenyl acetic acid moiety and B ring substitution
R1
R2
R3
hDP2 binding IC50
(l
M)
hDP2 binding + 0.2% HSA IC50
(lM)
a
a
Compd
5
6
7
8
9
10
11
12
13
14
CF3
CF3
CF3
CF3
CF3
CF3
F
F
H
OMe
OMe
H
H
H
H
H
H
OMe
OMe
OMe
m-CH2CO2H
m-CH2CO2H
m-CO2H
m-CH2CH2CO2H
o-CH2CO2H
o-CH2CH2CO2H
p-CH2CO2H
m-CH2CO2H
m-CH2CO2H
m-CH2CO2H
0.18
0.34
27
9.8
41
4.5
3.0
91
75
62
11
38
0.36
0.36
0.88
1.3
1.8
1.3
2.0
6.1
a
Values are the mean of at least three experiments.
Scheme 2. Generic synthesis for compounds 17–25. Reagents and conditions: (a) 2-
bromo-5-(trifluoromethyl)benzaldehyde, K2CO3, Pd(PPh3)4, DME, H2O, 85 °C, 75%;
(b) RNH2, NaBH3CN, HOAc, MeOH; (c) ClCOOR, NEt3, CH2Cl2; (d) 1 M NaOH, EtOH,
THF (40% three steps).
Scheme 1. Representative synthesis for compounds 5–14. Reagents and conditions:
(a) oxazolidinone, NaH, DMF, 0 °C, 85–95%; (b) SOCl2, EtOH; (c) bis(pinacola-
to)diboron, KOAc, Pd(dppf)Cl2, 1,4-dioxane, 80 °C, 80% two steps; (d) K2CO3,
Pd(PPh3)4, DME, H2O, 85 °C, 60–80%; (e) 1 M NaOH, EtOH, THF, 70–80%.
lysis afforded compounds 17–25 (Table 2). In general, opening the
oxazolidinone at the C5–O1 bond provided highly potent com-
pounds with low serum shifts. Shortening the R1 chain length from
phenethyl 17 to benzyl 18 gave a twofold increase in potency in
the presence of HSA. While the secondary carbamate 19 showed
only a modest reduction in potency, deletion of the carboxyl group,
revealing a basic amine in 26 was not tolerated. Reversing the sub-
stituents such that the smaller alkyl group was attached directly to
the nitrogen and the phenyl chain was part of the carbamate also
gave potent compounds (20–24). There was some tolerance for
the size of the R1 alkyl group, as methyl, ethyl, cyclopropyl, and
cyclobutyl (20–23) were approximately equipotent across all as-
says. However, cyclopentyl 24 showed a decrease in both binding
and functional activity. Finally, compound 25 demonstrated that
the C group phenyl ring was not necessary to maintain good bind-
ing potency with a low protein shift.
the ortho position decreased potency regardless of chain length (9,
10). Compound 11 showed that para-phenylacetic acid was com-
parable to meta-phenylacetic acid.
The effect of substituents in the B ring was then explored while
maintaining the meta-phenylacetic acid in the A ring and reincor-
porating the ortho-methoxy group (Table 1, entries 12–14). Elec-
tron-deficient groups were preferred, as the increased potency of
trifluoromethyl 5 ꢀ fluoro 12 > hydrogen 13 > methoxy 14 tracked
with the electron withdrawing nature of the substituent. Changes
to the acid placement in the A ring and B ring substituent resulted
in no improvement in binding activity and minimal decrease in
protein shift. The focus was therefore shifted to the C group.
Since the oxazolidinone is a cyclic carbamate, the effect of open-
ing the oxazolidinone into its two parts, a carbamate and an alkyl
group, was explored. Compounds were synthesized as shown in
Scheme 2. Suzuki coupling between aryl boronate 4 and 2-bro-
mo-5-(trifluoromethyl)benzaldehyde provided aldehyde 15, which
was then subjected to reductive amination with the appropriate
amine to furnish the benzyl amines 16. Acylation and ester hydro-
Lastly, the phenyl ring of the carbamate of compound 21 was
elaborated. The halogenated compounds were synthesized as
shown in Scheme 3. Commercially available alcohols 28 were
treated with phosgene to generate chloroformates 29, which were
isolated by removal of solvent and reacted immediately with the

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J. M. Scott et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6608–6612
Table 2
Benzylamine substitution
R1
R2
hDP2 binding IC50 (nM)
hDP2 binding + 0.2% HSA IC50 (nM)
hESC IC50 (nM)
a
a
a
Compd
17
18
19
20
21
22
23
24
25
26
CH2CH2Ph
CH2Ph
H
Me
Et
cyPropyl
cyButyl
cyPentyl
Et
Me
Me
24
8
45
4
4
4
4
11
9
62
31
238
18
8
18
19
53
16
34
22
709
9
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
Me
5
10
14
53
8
CH2CH2Ph
—
3.4
l
M
11
l
M
nt
hESC = human whole blood eosinophil shape change assay.
a
Values are the mean of at least three experiments.
Table 3
Carbamate substitution
a
Compd
R
hDP2 binding hDP2 binding +
hESC IC50 (nM)
a
a
IC50 (nM)
0.2% HSA IC50 (nM)
30
31
32
33
34
35
2-Cl
3-Cl
4-Cl
3,5-Di-Cl
4-F
3
2
3
4
2
3
11
10
7
29
8
7
7
6
14
4
5
3,5-Di-F
11
a
Values are the mean of at least three experiments.
Scheme 3. Representative synthesis for compounds 30–35. Reagents and condi-
tions: (a) Phosgene, CH2Cl2; (b) iPr2NEt, CH2Cl2; (c) 1 M NaOH, EtOH, THF, 40% yield.
a pharmacological readout during in vivo pharmacology studies
or human clinical trials.
ethylamine intermediate 27 to furnish the substituted benzyl
carbamates. Hydrolysis to the acid provided compounds 30–35
(Table 3). In general, single halogens were well tolerated (30–32
and 34). The 3,5-difluoro derivative 35 was comparable to the
single halogen compounds, while the 3,5-dichloro derivative 33
was threefold less potent in the presence of HSA.
In order to assess functional activity, compounds were tested in
a human whole blood eosinophil shape change (hESC) assay.7
When incubated with purified eosinophils or blood, PGD2 activates
the DP2 receptor and its downstream intracellular signaling path-
ways, which results in eosinophil degranulation and changes in
eosinophil morphology. These effects can be analyzed by flow
cytometry due to a change in forward light scatter. As the data in
Tables 2 and 3 shows, compounds 20–22, 25, 30–32, and 34–35
were found to be quite potent (610 nM) in the hESC assay. Since
the hESC assay is performed in whole blood, the conditions of this
assay are more physiologically relevant than the binding assays.
More importantly, this assay could potentially be used to provide
Due to the structural similarity of ligands in the arachidonic
acid pathway, there exists the possibility for cross-reactivity of li-
gands with the various prostanoid receptors. Since small molecule
inhibitors also have the potential for cross-reactivity, a subset of
compounds was counterscreened against a select group of prosta-
noid receptors, including the other PGD2 receptor DP1, the human
thromboxane receptor (hTP), and the human prostacyclin receptor
(hIP) (Table 4). The compounds tested were highly selective for the
DP2 receptor. This subset was also profiled against the common
cytochrome P450 (CYP) isoforms to assess the potential for drug/
drug interactions8 as well as the ability to inhibit the hERG channel
utilizing a patch clamp assay.9 As the data in Table 5 show, these
compounds showed no CYP inhibitory concerns or activity at the
hERG channel.
The pharmacokinetic properties of the most potent compounds
in the series (25, 30–32, 34–35) were evaluated in rat (sodium salt,
PO 10 mpk in 0.5% Methocel). Compounds showed good exposure

J. M. Scott et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6608–6612
6611
Table 4
DP1, TP, IP counterscreen
32 mice received daily intranasal OVA (200
l
g in saline) challenge
to induce an allergic response consisting of sneezing and nasal
rubs. Compound 21 (10 mg/kg), dexamethasone (10 mg/kg) or
water vehicle was administered orally 1 h prior to OVA challenge
on days 31 and 32 only. The frequency of sneezing was recorded
during an 8 min session immediately following OVA challenge on
day 32. Compound 21 significantly reduced sneezing symptoms
in the sensitized mice with an effect similar to that of dexametha-
sone (Fig. 1).
In summary, we have discovered a new series of small molecule
DP2-selective antagonists. The biphenylacetic acid series has been
optimized to provide compounds such as 21 that are highly potent
in both binding and functional assays, and show good pharmacoki-
netic properties in rat and dog. Compound 21 was efficacious in a
murine model of allergic rhinitis following oral dosing. Further
investigation of compound 21 and others in this series is ongoing.
Compd
hDP1 binding
hTP binding
hIP binding
IC50 (lM)
a
a
IC50
(l
M)
IC50
(
lM)
5
18
21
25
32
31.3
12.6
4.0b
36.4
4.0
63.7b
62.4
23.0
67.4
11.3
nt
nt
56.2c
nt
33.0c
hDP1 binding performed using 3H-PGD2 and human platelet membranes. hTP
binding performed using human platelet membranes and 3H-SQ-29,548. hIP bind-
ing performed using hIP/293 membranes and 3H-iloprost.
a
Values are the mean of two experiments.
Values are the mean of at least three experiments.
Value from a single experiment (nt = not tested).
b
c
Table 5
Inhibition of human CYP isoforms and hERG
References and notes
Compd
CYP3A4
CYP2C9
CYP2D6
hERGa
inhibition
inhibition
inhibition
1. Pettipher, R.; Hansel, T. T.; Armer, R. Nat. Rev. Drug Disc. 2007, 6, 313.
2. Pettipher, R. Br. J. Pharmacol. 2008, 153, S191.
a
a
a
IC50
(l
M)
IC50
(l
M)
IC50
(l
M)
3. Reviews: (a) Ulven, T.; Kostenis, E. Expert Opin. Ther. Patents 2010, 11, 1505; (b)
Ulven, T.; Kostenis, E. Curr. Top. Med. Chem. 2006, 6, 1427; (c) Ly, T. W.; Bacon,
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5
18
21
25
32
>30
>30
18
>30
21
>30
>30
18
>30
18
>30
>30
>30
>30
>30
nt
1.2% at 10
IC50 >30
5.9% at 10
22% at 10
l
M
l
M
l
Mb
4. (a) Tumey, N. L.; Robarge, M. J.; Gleason, E.; Song, J.; Murphy, S. M.; Ekema, G.;
Doucette, C.; Hanniford, D.; Palmer, M.; Pawlowski, G.; Danzig, J.; Loftus, M.;
Hunady, K.; Sherf, B.; Mays, R. W.; Stricker-Krongrad, A.; Brunden, K. R.;
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L.; Medina, J. Bioorg. Med. Chem. Lett. 2010, 19, 6840; (e) Stearns, B. A.; Baccei,
C.; Bain, G.; Broadhead, A.; Clark, R. C.; Coate, H.; Evans, J. F.; Fagan, P.;
Hutchinson, J. H.; King, C.; Lee, C.; Lorrain, D. S.; Prasit, P.; Prodanovich, P.;
Santini, A.; Scott, J. M.; Stock, N. S.; Truong, Y. P. Bioorg. Med. Chem. Lett. 2009,
19, 4647; (f) Sandham, D. A.; Adcock, C.; Bala, K.; Barker, L.; Brown, Z.; Dubois,
G.; Budd, D.; Cox, B.; Fairhurst, R. A.; Furegati, M.; Leblanc, C.; Manini, J.; Profit,
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S.; Page, P.; Missotten, M.; Colovray, V.; Cleva, C.; Arrighi, J.-F.; Atherall, J.;
Macritchie, J.; Martin, T.; Humbert, Y.; Gaudet, M.; Pupowicz, D.; Maio, M.;
Pittet, P.-A.; Golzio, L.; Giachetti, C.; Rocha, C.; Bernardinelli, G.; Filinchuk, Y.;
Scheer, A.; Schwarz, M. K.; Chollet, A. J. Med. Chem. 2008, 51, 2227.
l
Mb
a
Values are the mean of means of at least two experiments.
Value from a single experiment (nt = not tested).
b
5. (a) He, R.; Oyoshi, M. K.; Wang, J. Y. T.; Hodge, M. R.; Jin, H.; Geha, R. S. J. Allergy
Clin. Immunol. 2010, 126, 784; (b) Stebbins, K. J.; Broadhead, A. R.; Correa, L. D.;
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Lorrain, D. S. Eur. J. Pharmacol. 2010, 638, 142; (c) Stebbins, K. J.; Broadhead, A.
R.; Baccei, C. S.; Scott, J. M.; Truong, Y. P.; Coate, H.; Stock, N. S.; Santini, A. M.;
Fagan, P.; Prodanovich, P.; Bain, G.; Stearns, B. A.; King, C. D.; Hutchinson, J. H.;
Prasit, P.; Evans, J. F.; Lorrain, D. S. J. Pharmacol. Exp. Ther. 2010, 332, 764; (d)
Carter, L. L.; Shiraishi, Y.; Shin, Y.; Burgess, L.; Eberhardt, C.; Wright, A. D.;
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Gelfand, E. W. J. Allergy Clin. Immunol. 2010, 125, AB124; (e) Boehme, S. A.;
Franz-Bacon, K.; Chen, E. P.; Sasik, R.; Sprague, L. J.; Ly, T. W.; Hardiman, G.;
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6. The DP2 radioligand binding assay was performed on membranes from 293
cells stably expressing human DP2. To measure binding, [3H]-PGD2 was
incubated together with 293(hDP2) membranes in the presence of increasing
concentrations of compounds. Membranes were harvested and washed using a
Brandel Harvester and the amount of [3H]-PGD2 that remained bound to the
cells was measured on a TopCount. The concentration of compounds required
to achieve a 50% inhibition of [3H]-PGD2 binding (the IC50) was determined. The
binding assay was carried out both in the absence and presence of 0.2% human
serum albumin (HSA) to evaluate the protein shift associated with the
compounds.
Figure 1. Mouse allergic rhinitis data for compound 21 (10 mg/kg) versus
dexamethasone (10 mg/kg) and vehicle. Bars represent means SEM of n = 4–10
mice per group. ⁄P <0.05 versus vehicle, Dunnett’s post hoc comparisons following
ANOVA. Average plasma concentration of compound 21 at 2 h post-dose was 1 lM.
following oral dosing. Compound 21 was tested further in rat (so-
dium salt, IV 2 mpk in water) and dog (sodium salt, IV 2 mpk in sal-
ine, PO 5 mpk in 0.5% Methocel). In both species, the compound
displayed a long half-life (8.7 h and 7.4 h, respectively), with oral
bioavailability of 77% in rat and 89% in dog.
Given the desirable PK profile observed, compound 21 was then
evaluated in an in vivo model of allergic disease. Allergic rhinitis
can be modeled in mice by first sensitizing animals to ovalbumin
(OVA) by intraperitoneal injection and subsequently performing
nasal challenge with OVA.10 The mice then develop symptoms
which are similar to those observed in human allergic rhinitis,
including nasal itch, sneezing, and nasal congestion. The effect of
drug treatment prior to challenge can be quantified by counting
the frequency of these behaviors and comparing to untreated
animals.
7. Human blood was drawn into EDTA tubes and used within 2 h of draw. 100 ll
aliquots of fresh blood were incubated for 15 min at 37 °C plus or minus test
compound in 50% DMSO/water. PGD2 (50 nM final concentration) or vehicle
was added from a 1
lM stock in PBS and incubations were continued for 5 min
at 37 °C. The reactions were placed on ice and 250
ll of ice-cold 1:4 diluted
Cytofix (BD Biosciences) in PBS immediately added. The reactions were
transferred to FACS tubes and lysed with ammonium chloride lysing solution
at room temperature for 15 min. Tubes were centrifuged and the cells washed
In this assay female BALB/c mice were primed by intraperito-
neal OVA on days 0 and 7 (10 lg in Alum). On days 21 through

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J. M. Scott et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6608–6612
once with 3 ml cold PBS before re-suspension in 200
Cytofix in PBS. Eosinophil shape change was analyzed on a FACScalibur by
analyzing forward light scatter of the autofluorescent cells.
l
l of ice-cold 1:4 diluted
9. hERG assays performed by ChanTest Corporation.
10. Nakaya, M.; Dohi, M.; Okunishi, K.; Nakagome, K.; Tanaka, R.; Imamura,
M.; Baba, S.; Takeuchi, N.; Yamamoto, K.; Kaga, K. Lab. Invest. 2006, 86,
917.
8. Walsky, R. L.; Obach, R. S. Drug Metab. Dispos. 2004, 32, 647.