Isoquinoline derivatives as potent CRTH2 receptor antagonists: Synthesis and SAR

Article abstract of DOI:10.1016/j.bmcl.2012.03.009

Synthesis and structure-activity relationship of a novel series of isoquinoline CRTH2 receptor antagonists are described. One of the most potent compounds, TASP0376377 (6m), showed not only potent binding affinity (IC ₅₀ = 19 nM) but also excellent functional antagonist activity (IC₅₀ = 13 nM). TASP0376377 was tested for its ability of a chemotaxis assay to show the effectiveness (IC₅₀ = 23 nM), which was in good agreement with the CRTH2 antagonist potency. Furthermore, TASP0376377 showed sufficient selectivity for binding to CRTH2 over the DP1 prostanoid receptor (IC₅₀ >1 μM) and COX-1 and COX-2 enzymes (IC ₅₀ >10 μM).

Full text of DOI:10.1016/j.bmcl.2012.03.009

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3305–3310
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Isoquinoline derivatives as potent CRTH2 receptor antagonists: Synthesis
and SAR
Rie Nishikawa-Shimono a, , Yoshinori Sekiguchi a, Takeshi Koami a, Madoka Kawamura a,
⇑
Daisuke Wakasugi a, Kazuhito Watanabe b, Shunichi Wakahara b, Kayo Matsumoto b, Tetsuo Takayama a
a Medicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd, 1–403, Yoshino-Cho, Kita-Ku, Saitama-Shi 331-9530, Japan
b Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd, 1–403, Yoshino-Cho, Kita-Ku, Saitama-Shi 331-9530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and structure–activity relationship of a novel series of isoquinoline CRTH2 receptor antagonists
are described. One of the most potent compounds, TASP0376377 (6m), showed not only potent binding
affinity (IC50 = 19 nM) but also excellent functional antagonist activity (IC50 = 13 nM). TASP0376377 was
tested for its ability of a chemotaxis assay to show the effectiveness (IC50 = 23 nM), which was in good
agreement with the CRTH2 antagonist potency. Furthermore, TASP0376377 showed sufficient selectivity
Received 29 December 2011
Revised 1 March 2012
Accepted 3 March 2012
Available online 10 March 2012
for binding to CRTH2 over the DP1 prostanoid receptor (IC50 >1
(IC50 >10 M).
lM) and COX-1 and COX-2 enzymes
Keywords:
CRTH2 antagonist
PGD2
l
Ó 2012 Elsevier Ltd. All rights reserved.
Allergic diseases
Isoquinoline
Chemoattractant receptor–homologous molecule expressed on
Th2 lymphocytes (CRTH2) has been identified as a G protein-
coupled receptor, predominantly expressed on Th2 cells, and plays
a key role in allergic diseases, driving the IgE response, eosino-
philia, and release of proinflammatory cytokines.1–3 Activation of
CRTH2 promotes the release of histamine from basophils and
degranulation of eosinophiles.4–6 Thus, CRTH2 is involved in
complex inflammatory processes, and a CRTH2 antagonist might
have beneficial effects in a variety of inflammatory diseases.7–9
Ramatroban (1), a thromboxane A2 receptor (TP) antagonist
currently used for the treatment of allergic rhinitis, was shown
to be a potent CRTH2 antagonist. Indomethacin (2), a well-known
analgesic and cyclooxygenase 1 and 2 (COX-1 and COX-2) enzyme
inhibitor, was reported to show CRTH2 agonist potency. Since then,
a large number of CRTH2 antagonists including several series of in-
dole acids have been reported. These CRTH2 antagonists have a
variety of fused 6–5-membered ring chemotypes as a core struc-
ture such as indole (3),10–13 5-azaindole (4),14 7-azaindole (5),15
benzimidazole,16,17 indolizine,18 spiro–indoline;19 in addition, they
have an acetic acid moiety (Fig. 1).
date (3, 4, 5). Using a fused 6–6-membered ring system as a core
structure, we newly designed a 1,4-disubstituted isoquinoline lead
bearing a benzoyl group at the 1-position and an acetic acid moiety
essential to CRTH2 potency at the 4-position (Fig. 2). As a result, we
have identified a novel compound 6a showing moderate binding
affinity for the CRTH2 receptor in a radioligand binding assay
(3H-PGD2) using CHO cells stably transfected with the human
CRTH2 receptor (IC50 = 330 nM). In this paper, we describe the syn-
thesis and structure–activity relationship (SAR) of the new class of
isoquinoline derivatives.
Scheme 1 shows the synthesis of compounds 6a–m, 14, and 15
from commercially available 2-(4-aminophenyl)acetonitrile (7a).
Protection of the starting material 7a with di-t-butyl dicarbonate
afforded 8. The reaction of 8 with methyl 1-chloroisoquinoline-4-
carboxylate in the presence of sodium hexamethyldisilazane,
followed by oxidative decyanation under oxygen atmosphere
produced 920 in 83% yields. The carboxylic acid 10, prepared by
the basic hydrolysis of 9, was converted into the corresponding
acid chloride, and was treated with trimethylsilyldiazomethane
to afford the diazoketone intermediate. Wolff rearrangement of
the diazoketone intermediate followed by methylation of the
resulting carboxylic acid moiety with trimethylsilyldiazomethane
provided 11a21 in 69% yield from 9. Deprotection of the t-butoxy-
carbonyl group in 11a under an acidic condition (TFA), and the
subsequent acylation of the resulting aniline intermediate 12 with
various kinds of acid chlorides gave 13. Finally, hydrolysis of the
ester moiety afforded the target isoquinoline derivatives 6b–m,
14, and 15 in 22–96% yields. On the other hand, compound 6a
In the course of our program, which was aimed at developing
CRTH2 antagonists for the treatment of allergic diseases, we have
been pursuing a new class of potent and selective CRTH2 antago-
nist lead based on the structures of CRTH2 antagonists known to
⇑
Corresponding authors. Tel.: +81 48 669 3029; fax: +81 48 652 7254 (R.N.-S.).
E-mail address: rie.nishikawa@po.rd.taisho.co.jp (R. Nishikawa-Shimono).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.009

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R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3305–3310
O
MeO
N
F
H
N
O
N
N
Cl
O
Me
S
O
HO
O
HO
1
2
Ramatroban
Indomethacin
Me
Cl
O O
S
N
N
F
F
Me
S
N
N
O
O
Me
Me
N
O
Me
HO
HO
HO
3
4
5
Figure 1. Ligands for the CRTH2 receptor.
O
O
O
3 4 5
,
,
R
N
N
H
R1
CO2H
CO2H
6
Figure 2. Design of the novel isoquinoline chemotype for the CRTH2 receptor.
O
a
b
NC
NC
O
N
NH2
NHBoc
NHBoc
OMe
NHR
7a
8
9
O
O
c
d
f
N
O
N
NHBoc
OH
O
OMe
11a: R
= Boc
= H
10
e
12:
R
6a: R1 = Me
6i: R1 = 4-Cl-phenyl
6j: R1 = 2-Cl-phenyl
O
O
6b: R1 = phenyl
O
O
g
6c: R1 = cyclohexyl
6k: R1 = 4-CF3-phenyl
N
N
: R1 = 2-naphtyl
: R = 4-OMe-phenyl
1
N
H
R1
N
H
R1 6d
6l
6m
: R1 = 1H-indol-2-yl
: R = 3,4-di-Cl-phenyl
1
6e
O
OMe
O
OH
1
: R = 4-pyridyl
: R1 = 3,4-di-Cl-phenethyl
14
6f
13
6a-m, 14, 15
6g: R1 = 2-pyrimidyl
6h: R1 = 3-Cl-phenyl
15: R1 = (E)-2-(3,4-di-Cl-styryl)
Scheme 1. Reagents and conditions: (a) Boc2O, EtOH, rt, 57%; (b) methyl 1-chloroisoquinolinc-4-carboxylate, NaHMDS, THS. 0 °C, then, O2, rt, 83%; (c) 1 N NaOH aq, MeOH,
0 °C to rt, 94%; (d) (COCl)2 CHCl3, rt, TMSCMN2, THF/MeCN, rt, silver acetate, H2O/dioxane, 60 °C then TMSCHN2, MeOH, rt 69% (4 steps); (e) TFA, CHCl3, 0 °C to rt; (f) for 6b–m,
14, 15, RCOCl, pyridine, CHCl3, 0 °C to rt, 39–99%; for 6a, Av2O, pyridine, 70 °C, 97%; (g) 1 N NaOH aq, THF, 0 °C to rt, 22–96%.
was obtained by the treatment of 12 with acetic anhydride and the
subsequent basic hydrolysis.
Scheme 2 summarizes the synthesis of compounds 16–20. Com-
pound 16 was prepared by selective N-methylation of the amide of
6m. With regard to the synthesis of compound 17, the common
intermediate 12 was treated with 3,4-dichlorobenzylbromide in
the presence of potassium carbonate, and the ester moiety was
hydrolyzed by sodium hydroxide. Compound 18 was synthesized

R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3305–3310
3307
by the reaction of the aniline intermediate 12 with 3,4-dichloro-
benzene-1-sulfonyl chloride in the presence of pyridine followed
by basic hydrolysis of the ester moiety. Synthesis of compound
19 was achieved by demethylation of 11b by boron tribromide,
which was derived from methyl 1-chloroisoquinoline-4-carboxyl-
ate and 2-(4-methoxyphenyl)acetonitrile in a procedure similar
to the synthesis of 11a (Scheme 1), and the subsequent 3,4-dichlo-
robenzylation of the resulting phenol. Intermediate 11c was
prepared from methyl 1-chloroisoquinoline-4-carboxylate and
t-butyl 4-(cyanomethyl)benzoate, in a manner similar to the
synthesis of 11a as described in Scheme 1. By using 11c, compound
20 was prepared as follows: (1) acidic removal of the ester moiety
(TFA), (2) conversion into acyl chloride (oxalyl chloride), (3) con-
densation of the acyl chloride with 3,4-dichloroaniline.
Compounds 24, 25 and 33, in which the acetic acid part at the
4-position of the isoquinoline core was modified, were synthesized
as shown in Schemes 3 and 4. Geminal dimethylation of 11c by
sodium hydride and iodomethane produced 21 in a 58% yield.
Deprotection of the t-butyl ester moiety using TFA, followed by
condensation reaction of 22b with 4-chlorophenethylamine
(WSC-HCl, HOBT-H2O), gave 23b in a 44% yield. Finally, hydrolysis
of the ester moiety afforded the target derivative 25. In a similar
manner, compound 24 was prepared from 22a. Compound 33
was synthesized from commercially available 4-bromo-1-chloro-
O
b
c
17
a
6m
16
12
N
X
Cl
Cl
d
e
W
18
O
OH
O
f
i
g
h
l
6m
: W = NH, X = CO
19
11b
11c
NC
16: W = NMe, X = CO
17
: W = NH, X = CH2
18: W = NH, X = SO2
19
N
R
j, k
R
20
: W = O, X = CH2
20: W = CO, X = NH
O
OMe
11b: R = OMe
11c:
R = CO2Bu-t
7b: R = OMe
7c:
R = CO2Bu-t
Scheme 2. Reagents and conditions: (a) NaH, MeI, THF, rt, 31%; (b) 3,4-dichlorobenzylbromide, K2CO3, DMF, 90 °C, 35%; (c) 1 N NaOH aq, THF, rt, 58%; (d) 3,4-
dichlorobenzene-1-sulfonyl chloride, pyridine, 0 °C to rt, 39%; (e) 1 N NaOH aq, THF, rt, 12%; (f) BBr3, CHCI3, 0 °C to rt, 47%; (g) 3,4-dichlorobenzylbromide, K2CO3, DMF, 80 °C,
23%; (h) 1 N NaOH aq, THF, MeOH, rt, 48%; (i) TFA, CHC13, 0–50 °C, quant; (j) oxalyl chloride, DMF, CHCI3, rt; (k) 3,4-dichloroaniline, pyridine, CHCl3, 0 °C to rt, 35%; (l) 1 N
NaOH aq, MeOH, 0 °C, 87%.
O
O
b
R
R
N
O
N
OH
O
O
O
OMe
O
OMe
22a: R = H
11c
a
c
O
b
22b: R = Me
N
O
O
O
OMe
21
O
O
H
N
d
H
N
R
R
N
R
R
N
O
O
O
OMe
Cl
O
OH
Cl
23a: R = H
23b: R = Me
24: R = H
25: R = Me
Scheme 3. Reagents and conditions: (a) NaH, MeI, CHCl3, rt, 58%; (b) TFA, CHCl3, rt; (c) for 23a, oxalyl chloride, DMF, CHCl3, rt then 4-chlorophenethylamine, pyridine, CHCl3,
rt, 2 steps 45%; for 23b, 4-chlorophenethylamine, WSC-HCl, HOBT-H2O, CHCl3, rt, 2 steps 44%; (d) for 24, 1 N NaOH aq THF, rt, 78%; for 25, 1 N LiOH aq, dioxane, 100 °C, 70%.

3308
R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3305–3310
O
O
a
b
c
Cl
N
O
O
N
O
B
O
N
Br
Br
O
O
26
27
28
O
O
O
d
e
N
OH
N
O
O
O
N
O
HO
O
O
O
O
29
O
30
31
O
O
O
O
H
N
H
N
f
g
N
N
O
O
O
O
HO
O
Cl
Cl
32
33
O
O
Scheme 4. Reagents and conditions: (a) Methyl 4-(cyanomethyl)benzoate, NaHMDS, THF, 0 °C to rt, then O2, rt, quant; (b) bis(pinacolato)diboron, PdCl2(dppf), AcOK, 80 °C,
55%; (c) oxone, NaHCO3, acetone/H2O, 0 °C to rt, 9%; (d) t-butyl 2-bromoacetate, K2CO3, DMF, rt, 94%; (e) 0.2 N NaOH aq, THF, rt, 21%; (f) 4-chlorophenethylamine, WSC-HCl,
HOBT-H2O, CHCl3, rt, 59%; (g) 1 N NaOH aq, THF, rt, 39%.
isoquinoline (26), which was reacted with methyl 4-(cyanometh-
yl)benzoate in the presence of sodium hexamethyldisilazane. Sub-
sequent oxidative decyanation afforded 27. Compound 27 was
converted to 29 through the boronic acid ester 28 by the following
two-step reaction method: (1) treatment with bis(pinacolato)dibo-
ron in the presence of PdCl2(dppf) and potassium acetate, and (2)
oxidation of 28 using oxone. Transformation of 29 into the target
compound 33 was accomplished by the following conventional
method: (1) alkylation of the hydroxyl moiety in 29 with t-butyl
2-bromoacetate in the presence of potassium carbonate, (2) basic
hydrolysis of the methyl ester (NaOH), (3) condensation of the acid
with 4-chlorophenethylamine (WSC-HCl and HOBT-H2O), and (4)
hydrolysis of the t-butyl ester moiety.
As described above, we designed and synthesized a new 1,4-di-
substituted isoquinoline lead compound, and 6a was identified as a
ligand with moderate potency for the CRTH2 receptor. We initially
we examined the effects of the steric factor of R1 group on the
CRTH2 binding potency (Table 1). When the methyl group was
replaced with a bulky substituent such as a phenyl (6b), cyclohexyl
(6c), 2-naphthyl (6d), or 1H-indol-2-yl group (6e), the binding
affinity of the resulting compounds was dramatically enhanced
Next, we examined the rough SAR of the amide linker moiety of
6m (Table 2). Replacement of the amide linker with an propana-
mide linker was tolerated in CRTH2 binding affinity (14:
IC50 = 13 nM). When the propenamide linker was incorporated in
place of the amide linker, the binding affinity of the resulting com-
pound 15 (IC50 = 4.0 nM) increased by a factor of 4. By contrast, an
N-methylamide 16, which was devoid of the hydrogen bond donor
NH, showed a 15-fold decrease in binding affinity (IC50 = 210 nM).
These data suggested that the hydrogen bond donor (NH) played
an important role in maintaining the high binding affinity. Further-
more, replacement of the amide moiety of 6m with an aminometh-
ylene tether (17) maintained potent CRTH2 binding affinity (IC50
=
6.1 nM), while the sulfonamide linker (18) led to a significant
reduction in the binding (18: IC50 = 340 nM). Replacement with
Table 1
In vitro data of isoquinoline derivatives with R1 substitution modification
O
O
N
N
H
R1
(6b: IC50 = 7.9 nM, 6c: IC50 = 14 nM, 6d: IC50 = 4.0 nM, 6e: IC50
=
3.5 nM) as compared with the potency of 6a. By contrast, a
4-pyridyl (6f) or 2-pyrimidyl (6g) moiety led to little improvement
in potency. These results suggested that the steric factor of R1
group played a key role in increasing CRTH2 potency.
With these in vitro data in hand, further SAR studies of 6b were
conducted to examine the effects of substituents of the terminal
phenyl ring on CRTH2 binding affinity. Incorporation of a chlorine
atom into the meta or para position of the phenyl group led to an
increase in binding potency (6h: IC50 = 3.2 nM, 6i: IC50 = 3.4 nM),
while the installation of a chlorine atom into the ortho position
resulted in a 6-fold decrease in potency compared with 6b. With
regard to the para substituent, a trifluoromethyl group or a
methoxy group was tolerated, and these compounds exhibited
CO2H
a
Compund
R1
hCRTH2 binding IC50 (nM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
Methyl
Ph
330
7.9
14
4.0
3.2
83
290
3.2
3.4
45
Cyclohexyl
2-Naphthyl
1H-Indol-2-yl
4-Pyridyl
2-Pyrimidyl
3-Cl-Ph
4-Cl-Ph
2-Cl-Ph
6j
6k
6l
6m
4-CF3-Ph
4-OMe-Ph
3,4-Di-Cl-Ph
4.9
7.3
19
single digit nanomolar potency (6k: IC50 = 4.9 nM, 6l: IC50
=
a
7.3 nM). In addition, the meta- and para di-substituted phenyl
group was found to be well tolerated (6m: IC50 = 19 nM).
Mean values from at least two independent experiments. IC50 values were
determined from full 10-point, half-log concentration–response curves.

R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3305–3310
3309
an oxymethylene linker resulted in a slight loss of the potency (19:
IC50 = 38 nM). Interestingly, the inverse amide linker (20) retained
potent binding affinity (IC50 = 10 nM). These data supported the
importance of the hydrogen bond donor (NH) in the linker moiety
of the isoquinoline chemotype.
Subsequently, we examined the effects of substituents around
the carboxylic acid moiety on CRTH2 binding affinity (Table 3).
The carboxylic acid moiety is shared with the representative
CRTH2 antagonists and is essential for CRTH2 activity. Germinal
dimethylation of the methylene moiety next to the carboxylic acid
resulted in a 25-fold drop in potency (25: IC50 = 200 nM) compared
with the original compound 24. Insertion of an oxygen atom
between the carboxymethyl moiety and heteroaryl group resulted
in a slight loss in potency (33: IC50 = 25 nM). These data suggest
that the binding space of the CRTH2 receptor, where the acid moi-
ety of the antagonists interacted, is limited.
In addition, these isoquinoline derivatives were functionally
active and behaved as antagonists of PGD2 driven Ca2+ flux in
KB8 cells expressing human CRTH2.3,22 One of the most potent
antagonists, 6m (IC50 = 19 nM), was tested in a chemotaxis assay23
to determine its effectiveness (IC50 = 23 nM), which was in good
agreement with the CRTH2 antagonist potency. Furthermore, suffi-
cient level of selectivity was found for binding to CRTH2 over the
DP1 prostanoid receptor24 (IC50 >1
lM) and COX-1 and COX-2
enzymes25 (IC50 > 10
l
M).
In conclusion, we have identified the novel isoquinoline acetic
acid chemotype 6 as a potent CRTH2 antagonist. SAR of the scaffold
was explored, resulting in the identification of the compound 6m
(TASP0376377), which is a selective functional antagonist of
CRTH2. Studies are ongoing to explore the utility of these com-
pounds in inflammatory disease models and will be reported in
due course.
References and notes
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M.; Takanoa, S. FEBS Lett. 1999, 459, 195.
3. Pettipher, R.; Hansel, T. T.; Armer, R. Nat. Rev. Drug Disc. 2007, 6, 313.
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Ichimasa, M.; Sugamura, K.; Nakamura, M.; Takano, S.; Nagata, K. J. Exp. Med.
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Table 2
In vitro data of isoquinoline derivatives with linker modification
O
N
X
Cl
Cl
W
CO2H
a
Compound
W
X
hCRTH2 binding IC50 (nM)
19
7. Evi Kostenis, E.; Ulven, T. Trend Mol. Med. 2006, 12, 148.
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Chem. Lett. 2006, 16, 4287.
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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.;
Bennani, Y. L.; Harrington, J. J. Bioorg. Med. Chem. Lett. 2010, 20, 3287.
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Xue, L.; Pettipher, R. J. Med. Chem. 2005, 48, 6174.
6m
NH
CO
O
14
NH
13
∗
∗
∗
∗
O
15
NH
4.0
16
17
18
19
20
NMe
NH
NH
O
CO
210
6.1
340
38
CH2
SO2
CH2
NH
CO
10
a
Mean values from at least two indepentent experiments. IC50 values were
determined from full 10-point, half-log concentration–response curves.
13. Ulven, T.; Gallen, M. J.; Nielsen, M. C.; Merten, N.; Schmidt, C.; Mohr, K.;
Tränklec, C.; Kostenis, E. Bioorg. Med. Chem. Lett. 2007, 17, 5924.
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Cox, B.; Fairhurst, R. A.; Furegati, M.; Leblanc, C.; Manini, J.; Profit, R.; Reilly, J.;
Stringer, R.; Schmidt, A.; Turner, K. L.; Watson, S. J.; Willis, J.; Williams, G.;
Wilson, C. Bioorg. Med. Chem. Lett. 2009, 19, 4794.
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18. Hynd, G. WO 2006/136859; Chem. Abstr. 2006, 146, 100556.
19. Crosignani, 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.;
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Table 3
In vitro data of isoquinoline derivatives with R substitution modifications
O
H
N
N
R
O
Cl
a
Compound
R
hCRTH2 binding IC50 (nM)
∗
21. Kirmse, W. Eur. J. Org. Chem. 2002, 14, 2193.
22. Human CRTH2 transfectant KB8 cells (BML Co. Kawagoe, Japan) were
24
7.8
O
OH
∗
incubated with
1 lM Fluo-4 AM for 30 min at 37 °C in the dark. After
incubation, the cells were washed and suspended in HBSS containing 10 mM
HEPES, pH 7.3, 1 mM CaCl2 (2 Â 105cells/100
ll). The compound and 100 nM of
25
33
200
25
PGD2 were added, and the increase of intracellular Ca2+ concentration ([Ca2+]i)
was measured using the functional drug screening system FDSS6000
(Hamamatsu Photonics, Shizuoka, Japan). The IC50s of the representative
compounds (6b, 6g, 6k, 15, 19) were 42 nM, 550 nM, 15 nM, 17 nM, and
180 nM, respectively.
O
OH
O
∗
HO
23. Human Th2 cells were prepared as follows: the CD4+T lymphocytes separated
from PBMCs using anti-CD4 mAb were stimulated with anti-CD3 mAb and
O
anti-CD28 mAb in the presence of IL-4 and neutralizing anti-IFN
c mAb for
a
Mean values from at least two indepentent experiments. IC50 values were
determined from full 10-point, half-log concentration–response curves.
3 days and, then, expanded by IL-2 and IL-4 for 7 days. Th2 cells highly
expressing CRTH2 were separated with anti-CRTH2 mAb, and 2 days after the

3310
R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3305–3310
separation, these cells were used in the cell migration assay. Compound-
treated Th2 cells and 100 nM solution of PGD2 were applied to top and bottom
wells of the m-pore filter Chemo Tx-96 chamber (Neuroprobe,
Gaithersburg, MD, USA), respectively. After incubation at 37 °C for 1 h,
number of the cells in the bottom wells were counted by Burker–Turk
hemocytometer.
24. The binding assay for DP1 was run by Recerca Biosciences (Bothell, USA) using
the profiler service according to the manufacturer’s procedures.
25. The enzyme inhibition assays for COX-1 and COX-2 were run by Cerep
(Paris, France) using the profiler service according to the manufacturer’s
procedures.
5
l