Discovery of novel and potent CRTH2 antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.11.079

High throughput screening of our chemical library for CRTH2 antagonists provided a lead compound 1a. Initial optimization of the lead led to the discovery of a novel, potent and orally bioavailable CRTH2 antagonist 17.

Full text of DOI:10.1016/j.bmcl.2011.11.079

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1194–1197
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel and potent CRTH2 antagonists
Shinji Ito a, Tadashi Terasaka a, , Tatsuya Zenkoh a, Hiroshi Matsuda e, Hisashi Hayashida e,
⇑
Hiroshi Nagata e, Yoshimasa Imamura a, Miki Kobayashi d, Makoto Takeuchi c, Mitsuaki Ohta b
a Chemistry Research Labs, Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
b Research Planning & Administration, Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
c Research Management, Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
d Pharmacology Research Labs, Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
e Astellas Research Technologies Co., Ltd, 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2011
Revised 18 November 2011
Accepted 21 November 2011
Available online 28 November 2011
High throughput screening of our chemical library for CRTH2 antagonists provided a lead compound 1a.
Initial optimization of the lead led to the discovery of a novel, potent and orally bioavailable CRTH2
antagonist 17.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
CRTH2
DP2
PGD2
Asthma
Inflammation
Mast cells, which play important roles in allergic diseases, are
activated by immunoglobulin E (IgE). Activated mast cells produce
a variety of inflammation mediators, of which prostaglandin D2
(PGD2) is representative.1 It has been reported that antigen-
challenge-induced PGD2 production is promoted in the airway of
asthmatic patients2 and that overexpression of PGD synthase
enhances airway eosinophil infiltration and Th2 cytokine produc-
tion in an asthma model.3 These reports indicate that PGD2 is
closely related to the pathogenesis of allergic diseases, such as
asthma, allergic rhinitis, and atopic dermatitis.
Although PGD2 was initially considered to elicit its biological
actions through a classical PGD2 receptor (DP1), later findings sug-
gested that several PGD2-mediated actions of eosinophils arise via
DP2,4,5 which is also known as CRTH2 (chemoattractant receptor-
homologous molecule expressed on Th2 cells). CRTH2 is expressed
on inflammatory cells, such as Th2 cells,6 eosinophils and baso-
phils, and induces the chemotaxis of these cells.7 CRTH2 also plays
important roles in cytokine release by Th2 cells8 and in the degran-
ulation of eosinophils.9 CRTH2 antagonists are therefore expected
to be useful as anti-inflammatory agents in the treatment of
patients with allergic diseases.10
as a hit (Fig. 1), which inhibited the binding of 3H-labeled PGD2
to human and guinea pig CRTH2 receptors on HEK293 cells with
IC50 values of 42 and 256 nM, respectively.11 Moreover, 1a proved
to be selective over DP112 (IC50 > 1000 nM to human DP1) and dis-
played oral absorption in guinea pigs, with oral dosing of 1a at
10 mg/kg leading to a maximum plasma concentration (Cmax) of
O
N
X
HO2C
O
1a:
b:
X = CH
X = N
F
F
HN S
O
O
CO2H
N
N
High throughput screening (HTS) of our chemical library for
CRTH2 antagonists identified benzhydryl pyridone compound 1a
N
Me
CO2H
Ramatroban
2
⇑
Corresponding author. Tel.: +81 29 829 6201; fax: +81 29 852 5387.
E-mail address: tadashi.terasaka@astellas.com (T. Terasaka).
Figure 1. Chemical structures of HTS hit 1a, 1b and known CRTH2 antagonists.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.079

S. Ito et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1194–1197
1195
0.84
(AUC) of 5.95
l
g/mL and area under the blood concentration-time curve
The synthetic routes to 4,40-substituted benzhydryl derivatives
are shown in Scheme 1. 3-(3-Hydroxypropyl)phenol 3 was alkyl-
ated with ethyl bromoacetate in the presence of potassium carbon-
ate in acetonitrile. The resulting alcohol was converted to the
corresponding mesylate, followed by displacement with sodium
iodide in acetone to afford the alkyl iodide 4. p-Substituted diphe-
nylmethanols 5 were converted to diphenylmethylpyridones 6 in
the presence of sulfuric acid at 180–250 °C. Pyridones 6 were alkyl-
ated with the alkyl iodide 4 in the presence of lithium hydride in
N,N-dimethylformamide, followed by hydrolysis to give 7a–d.
The ether-linked compound 10 was synthesized by the routes
shown in Scheme 2. A nitrogen atom of pyridone 8 was alkylated
with 2-bromoethyl acetate and the resulting acetate was cleaved
with sodium hydroxide to afford alcohol 9. The alcohol was mesy-
lated with methanesulfonyl chloride, followed by alkylation and
hydrolysis of the ester to give 10.
The benzamide derivatives 13a–b were synthesized as shown
in Scheme 3. 3,3-Diphenylpropylamine 11 was alkylated with the
iodide 4 to give the secondary amine 12. Amine 12 was acylated
with benzoyl chloride or 4-methoxybenzoyl chloride, followed by
hydrolysis of the ester group to afford benzamides 13a–b.
The synthetic routes to convert the acetic acid moiety are out-
lined in Scheme 4. 3-(3-Hydroxypropyl)phenol 3 was converted
l
gꢀh/mL.13 In addition, a similar pyridazinone com-
pound 1b was discovered from further HTS. The pyridazinone 1b
also showed CRTH2 inhibitory activity and moderate oral absorp-
tion, that is, dosing of 1b (10 mg/kg) to guinea pigs showed a sim-
ilar Cmax (0.73
(2.40
lg/mL) to 1a and 2.5-fold decrease in AUC
gꢀh/mL). Although a number of CRTH2 antagonists have
l
been reported to date, including Ramatroban14 and indole acetic
acids exemplified with compound 215 (Fig. 1), pyridone or pyridaz-
inone scaffolds like 1a or 1b are not known as CRTH2 antagonists
and 1a was accordingly selected as a first lead compound. Given
that it was discovered by HTS, no information on the structure–
activity relationships (SAR) for this compound was available. We
therefore attempted to optimize compound 1a in order to obtain
SAR and to improve its activity and pharmacokinetic properties.
In this paper, we describe our initial optimization efforts and dis-
covery of novel analogs with higher potency for CRTH2.
While compound 1a had moderate inhibitory activity against
human CRTH2 receptor, its activity against guinea pig CRTH2
was relatively weak at only one-sixth that in humans. The common
use of a guinea pig hyperresponsiveness model to examine anti-
asthmatic activity in vivo mandated that we enhance inhibitory
activity against not only human but also guinea pig CRTH2.
I
OH
a, b, c
EtO2
C
O
OH
4
3
O
O
OH
HN
d
N
e, f
X
X
O
HO2C
X
X
X
X
5a: X = F
b: X = Cl
c: X = Me
6a-d
7a-d
d: X = OMe
Scheme 1. Reagents and conditions: (a) ethyl bromoacetate, K2CO3, MeCN, rt, 24 h; (b) methanesulfonyl chloride, Et3N, CH2Cl2, rt, 1 h; (c) NaI, acetone, rt, 18 h; (d) 2-
pyridone, H2SO4, 180–250 °C, 2 h; (e) 4, LiH, DMF, rt, overnight; (f) NaOH aq, MeOH, rt, 1 h.
O
O
O
O
HO
HN
N
a, b
N
c, d, e
HO2
C
O
9
10
8
Scheme 2. Reagents and conditions: (a) 2-bromoethyl acetate, NaH, DMF, rt, 24 h; (b) NaOH aq, EtOH, 5 °C, 1 h; (c) methanesulfonyl chloride, Et3N, CH2Cl2, 5 °C, 2 h; (d) ethyl
(3-hydroxyphenoxy)acetate, K2CO3, NaI, DMF, 80 °C, 2 h; (e) NaOH aq, EtOH, rt, 2 h.
O
NH
NH2
N
a
b, c
R
EtO2C
O
HO2C
O
11
12
13a: R = H
b: R = OMe
Scheme 3. Reagents and conditions: (a) 4, DMF, rt, 8 h; (b) BzCl or 4-MeO–BzCl, Py, CH2Cl2, rt, 8 h; (c) NaOH aq, MeOH, rt, 5 h.

1196
S. Ito et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1194–1197
O
(7b) and methyl (7c) groups led to a threefold or more loss. These
data suggest that only the hydrogen bond acceptor is tolerable to
guinea pig CRTH2 whereas a variety of substituents may be accept-
able to the human receptor, and accordingly that the introduction
of substituents at this position might enhance activity against hu-
man CRTH2 but would not improve the species difference between
humans and guinea pigs.
N
e
a, b, c, d
OH
OBn
OH
3
14
To facilitate synthesis and block the metabolically labile benzyl
position, we converted the methylene moiety of 1a into oxygen
(10). However, 10 showed 2–3-fold less potent inhibitory activity
both to human and guinea pig CRTH2. We did not perform further
optimization with this linker.
In consideration of planarity around the nitrogen atom, we con-
verted the pyridone scaffold of 1a to benzamides. The benzamides
13a and 13b showed highly potent (nM order) activity with IC50
values to human CRTH2 of 9.7 and 5.5 nM, respectively. In addi-
tion, these benzamides also displayed a greater than 10-fold
improvement in affinity for guinea pig CRTH2. These data suggest
that the lipophilicity of the scaffold may cause the enhancement of
the CRTH2 inhibitory activity.
O
O
N
f, g
N
OH
HO2C
O
Z
15
16a: Z = -CH2
-
b: Z = -CH(Me)-
c: Z = -CH(Et)-
d: Z = -(CH2)3-
Scheme 4. Reagents and conditions: (a) BnBr, K2CO3, MeCN, 60 °C, 2 h; (b) MsCl,
Et3N, CH2Cl2, rt, 30 min; (c) NaI, acetone, rt, 13 h; (d) 8, LiH, DMF, 50 °C, 3 h; (e) TFA,
1,2,3,4,5-pentamethylbenzene, rt, 12 h; (f) Br–Z–CO2Et, K2CO3, MeCN, 60 °C, 14 h;
(g) NaOH aq, MeOH, rt, 1 h.
Finally, we converted the terminal acetic acid moiety to explore
the capability (16a–d). Introduction of only one methyl group to
the
a-position of the carboxyl group enhanced CRTH2 inhibitory
activity (16a), whereas the activity was drastically diminished in
the case of the ethyl (16b) or dimethyl (16c) groups. Meanwhile,
elongation of the methylene chain contributed to the enhancement
of activity (16d). These data suggest that bulky substituents
around the acid moiety are not acceptable. Although the CRTH2
inhibitory activity of 16a and 16d are comparable, 16d is favorable
because it is free from the problem of chirality.
to pyridone 14 in four steps, followed by removal of the benzyl
group with trifluoroacetic acid in the presence of 1,2,3,4,5-pentam-
ethylbenzene16 to give phenol 15. The hydroxyl group of 15 was
alkylated with ethoxycarbonyl bromoalkane, followed by hydroly-
sis of ethyl ester to afford 16a–d. Compound 17 was prepared by
similar procedure to that of compound 7.
Given that 16d showed the most potent activity among the
pyridone series so far, we accordingly modified the pyridone scaf-
fold of 16d into pyridazinone 17. Although the inhibitory activity
of 17 against human CRTH2 was comparable to that of 16d, 17
showed twofold more potent activity in guinea pigs than 16d.
Compounds 13b, 16d and 17, with high potency compared to 1a
and 1b, were subjected to pharmacokinetic experiments in guinea
pigs as advanced candidates (Table 3). The resulting most potent
antagonist 13b showed poor oral availability. We consider that
the poor PK profile of 13b was due to greater metabolic lability
to in vitro clearance in liver microsomes in guinea pigs (766 mL/
The CRTH2 inhibitory activities of the synthesized compounds
are listed in Tables 1 and 2. At first we introduced halogen or other
substituents at the 4,40-position of phenyl rings in the benzhydryl
moiety in order to obtain SAR and to improve the metabolic stabil-
ity at this moiety (7a–d). It is well-known that introduction of hal-
ogen atom at the para-position of the phenyl ring can protect from
metabolism.17 All four p-substituted analogs 7a–d displayed 4–7-
fold more potent activity against human CRTH2 than 1a, but these
modifications did not contribute to enhancing activity against gui-
nea pig CRTH2. Specifically, the fluoro (7a) and methoxy (7d)
derivatives were comparable to 1a, but introduction of the chloro
Table 1
The CRTH2 inhibitory activities of the leads 1a, 1b and their derivatives
O
O
Y
N
N
N
HO2C
O
HO2
C
O
Z
Z
X
X
1a, 7, 10, 16
1b, 17
a
a
Compound
X
Y
Z
Human CRTH2 IC50 (nM)
Guinea pig CRTH2 IC50 (nM)
1a
1b
7a
7b
7c
7d
10
16a
16b
16c
16d
17
H
–(CH2)3–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH(Me)–
–CH(Et)–
–C(Me)2–
–(CH2)3–
–(CH2)3–
42
18
11
9.2
6.2
5.8
99
256
147
359
>1000
772
211
797
128
990
>1000
99
F
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–O(CH2)2–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
Cl
Me
OMe
H
H
H
17
502
NTb
11
H
H
13
52
a
See Ref. 11 for assay protocol. All values are mean of at least two experiments.
Not tested.
b

S. Ito et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1194–1197
1197
Table 2
References and notes
The CRTH2 inhibitory activities of the benzamide derivatives 13a–b
1. Peters, S. P.; MacGlashan, D. W., Jr.; Schulman, E. S.; Schleimer, R. P.; Hayes, E.
C.; Rokach, J.; Adkinson, N. F., Jr.; Lichtenstein, L. M. J. Immunol. 1984, 132, 1972.
2. Murray, J. J.; Tonnel, A. B.; Brash, A. R.; Roberts, L. J.; Gosset, P.; Workman, R.;
Capron, A.; Oates, J. A. N. Eng. J. Med. 1986, 315, 800.
3. Fujitani, Y.; Kanaoka, Y.; Aritake, K.; Uodome, N.; Okazaki-Hatake, K.; Urade, Y.
J. Immunol. 2002, 168, 443.
O
N
R
HO2
C
O
4. Woodward, D. F.; Hawley, S. B.; Williams, L. S.; Ralston, T. R.; Protzman, C. E.;
Spada, C. S.; Nieves, A. L. Invest. Ophthalmol. Vis. Sci. 1990, 31, 138.
5. Monneret, G.; Gravel, S.; Diamond, M.; Rokach, J.; Powell, W. S. Blood 2001, 98,
1942.
13a-b
a
a
6. Nagata, K.; Tanaka, K.; Ogawa, K.; Kemmotsu, K.; Imai, T.; Yoshie, O.; Abe, H.;
Tada, K.; Nakamura, M.; Sugamura, K.; Takano, S. J. Exp. Med. 1999, 162, 1278.
7. Hirai, H.; Tanaka, K.; Yoshie, O.; Ogawa, K.; Kenmotsu, K.; Takamori, Y.;
Ichimasa, M.; Sugamura, K.; Nakamura, M.; Takano, S.; Nagata, K. J. Exp. Med.
2001, 193, 255.
Compound
R
Human CRTH2 IC50
(nM)
Guinea pig CRTH2 IC50
(nM)
13a
13b
H
9.7
18
17
OMe 5.5
8. Xue, L.; Gyles, S. L.; Wettey, F. R.; Gazi, L.; Townsend, E.; Hunter, M. G.;
Pettipher, R. J. Immunol. 2005, 175, 6531.
a
See Ref. 11 for assay protocol. All values are mean of at least two experiments.
9. Gervais, F. G.; Cruz, R. P. G.; Chateauneuf, A.; Gale, S.; Sawyer, N.; Nantel, F.;
Metters, K. M.; O’Neill, G. P. J. Allergy Clin. Immunol. 2001, 108, 982.
10. Pettipher, R.; Hansel, T. T.; Armer, R. Nat. Rev. Drug Discov. 2007, 6, 313.
11. The human or guinea pig CRTH2 stable transfectants were generated by
transfecting the pcDNA3.1 expression vector containing human or guinea pig
CRTH2 gene into HEK293 cells (purchased from ATCC). They were suspended in
binding assay buffer (10 mM BES, 1 mM EDTA, 10 mM MnCl2, pH 7.0) and
Table 3
Pharmacokinetic parameters of CRTH2 antagonists after p.o. administration to guinea
pigs (10 mg/kg)
Compound
Pharmacokinetic parameters in guinea pigsa
disrupted by strong mixing using the injection needle. Cell suspension (10
protein/well for human CRTH2 assay or 20 g protein/well for guinea pig
CRTH2 assay) and [3H]PGD2 were mixed in
96-well round bottom
lg
l
Cmax
(lg/mL)
AUC (lgꢀh/mL)
a
1a
1b
13b
16d
17
0.84
0.73
0.04
0.50
2.86
5.95
2.40
0.27
1.02
4.85
polypropylene plate (Nunc) and incubated for 2 h at 4 °C in the absence or
the presence of increasing concentrations of the tested compounds. After
incubation, the cell suspension was transferred to a glass-filter plate (GF/B,
Perkin–Elmer). Scintillant was added to the filtration plate, and radioactivity
remaining on the filter was measured with a scintillation counter, TopCount
(Packard Bioscience). Nonspecific binding was determined in the presence of
a
10 lM DK-PGD2. The concentration of compounds causing a 50% decrease in
See Ref. 13 for assay protocol. All values are mean of n = 3 measurements.
the binding of [3H]PGD2 to the receptor was calculated as [IC50]value.
12. The human DP1 stable transfectants were generated by transfecting the
pcDNA3.1 expression vector containing human DP1 gene into HEK293 cells
(purchased from ATCC). They were suspended in binding assay buffer (10 mM
BES, 1 mM EDTA, 10 mM MnCl2, pH 7.0) and disrupted by strong mixing using
min/kg) than 1a (120 mL/min/kg). The chain elongated compound
16d showed poorer oral availability than the corresponding lead
1a. In contrast, pyridazinone 17 displayed excellent oral availabil-
ity, and dosing (10 mg/kg) to guinea pigs led to an approximately
the injection needle. Cell suspension (10 lg protein/well for human DP1 assay)
and [3H]PGD2 were mixed in a 96-well round bottom polypropylene plate
(Nunc) and incubated for 2 h at 4 °C in the absence or the presence of
increasing concentrations of the tested compounds. After incubation, the cell
threefold increase in Cmax (2.86
(4.85
gꢀh/mL) to 1a.
lg/mL) and comparable AUC
l
suspension was transferred to
Scintillant was added to the filtration plate, and radioactivity remaining on
the filter was measured with scintillation counter, TopCount (Packard
Bioscience). Nonspecific binding was determined in the presence of 10
a glass-filter plate (GF/B, Perkin–Elmer).
Evaluating the inhibitory activity of 17 against human DP1
proved that this compound was a selective CRTH2 antagonist
(IC50 = 5200 nM to human DP1). We next evaluated the in vivo
anti-asthmatic activity of compounds 1a and 17 in a guinea pig
model, and found that both showed in vivo efficacy orally in a gui-
nea pig model of airway hyperresponsiveness,18 with ED50 values
of 4.7 and 0.05 mg/kg u.i.d., respectively. The in vivo efficacy of
17 was thus drastically improved than that of 1a.
a
lM
PGD2. The concentration of compounds causing a 50% decrease in the binding
of [3H]PGD2 to the receptor was calculated as [IC50]value.
13. Male Hartley guinea pigs (6 weeks) were purchased from Japan SLC, Inc.,
(Shizuoka, Japan). Guinea Pigs were fasted overnight prior to dosing. Dosing
solution were prepared as
a 10 mg/mL suspending solution in 0.5% MC
(methylcellulose, COSMO BIO, Tokyo, Japan) in water. Guinea Pigs received
10 mg/kg dose by oral administration. Samples were analyzed in Astellas
Analysis & Pharmacokinetics Research Labs.
In summary, we discovered a novel and selective CRTH2 antag-
onist 1a from HTS of our chemical library. Initial optimization
based on 1a resulted in the discovery of the novel, potent and or-
ally bioavailable CRTH2 antagonist 17. We achieved not only a
improvement in in vitro CRTH2 antagonistic activity against both
human and guinea pig but also a drastic improvement in in vivo
efficacy compared to that of 1a. Further optimization of this series
aimed at improving activities and pharmacokinetic properties will
be reported later.
14. Robarge, M. J.; Bom, D. C.; Tumey, L. N.; Varga, N.; Gleason, E.; Silver, D.; Song,
J.; Murphy, S. M.; Ekema, G.; Doucette, C.; Hanniford, D.; Palmer, M.;
Pawlowski, G.; Danzig, J.; Loftus, M.; Hunady, K.; Sherf, B. A.; Mays, R. W.;
Stricker-Krongrad, A.; Brunden, K. R.; Harrington, J. J.; Bennani, Y. L. Bioorg.
Med. Chem. Lett. 2005, 15, 1749.
15. Middlemiss, D.; Ashton, M. R.; Boyd, E. A.; Brookfield, F. A. PCT Int. Appl. WO
044260, 2005.
16. Marriott, J. H.; Barber, A. M. M.; Hardcastle, R.; Rowlands, M. G.; Grimshaw, R.
M.; Neidle, S.; Jarman, M. J. Chem. Soc., Perkin Trans. 1 2000, 4265.
17. Smith, D. A.; Jones, B. C.; Walker, D. K. Med. Res. Rev. 1996, 16, 243.
18. Male Hartley guinea pigs were actively sensitized to ovalbumin (OVA) by i.p.
injection of 20 mg in 0.9% saline at day 0, and of 1 mg at day 2. Animals were
exposed for 10 min on days 14–21 to an aerosol of 0.5% OVA solution in 0.9%
saline or saline alone generated from a nebulizer. All animals were treated with
compound by po administration 60 min prior to the aerosol, and with 1 mg/kg
pyrilamine by ip injection 30 min prior to the aerosol. On day 22, animals were
anesthetized with urethane (1.5 g/kg). The trachea was cannulated and the
animal was mechanically ventilated (60 strokes/minutes; 1 mL/100 g body
weight) with a small animal respirator. Pulmonary inflation pressure (PIP) was
monitored with a pressure transducer. Doses of methacholine (0–14 mg/kg)
were administered sequentially at 3-minute intervals to each animal via the
right jugular vein. The area under the peak inflation pressure versus
methacholine dose curve (AUC) was calculated for each animal. The doses of
compounds causing 50% inhibition of the increase in the AUC were calculated
as [ED50] values.
Acknowledgments
The author would like to thank Mr. Yasuhiro Miyao for evaluat-
ing in vivo PK, Dr. David Barrett and Dr. Masaya Orita for their
valuable comments and help in the preparation of the manuscript,
and Mr. Mamoru Tasaki for his preparation of the manuscript con-
cerning the in vitro and in vivo assay method.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.079.