Isoquinoline derivatives as potent CRTH2 antagonists: Design, synthesis and SAR

Article abstract of DOI:10.1016/j.bmc.2013.10.025

In this study, we describe the synthesis and structure-activity relationship (SAR) of a series of isoquinoline chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) antagonists. TASP0376377 (15-20), one of the most potent compounds, showed a potent binding affinity (IC₅₀ = 19 nM) in addition to the excellent functional antagonist activity (IC₅₀ = 13 nM). Moreover, the efficacy of this compound in a chemotaxis assay (IC₅₀ = 23 nM) was in good agreement with its potency as a CRTH2 antagonist. In addition, 15-20 exhibited greater selectivity in binding to CRTH2 than to the DP1 prostanoid receptor (IC ₅₀ >1 μM) or the enzymes COX-1 and COX-2 (IC₅₀ >10 μM).

Full text of DOI:10.1016/j.bmc.2013.10.025

Bioorganic & Medicinal Chemistry 21 (2013) 7674–7685
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Isoquinoline derivatives as potent CRTH2 antagonists: Design,
synthesis and SAR
a
a
a
Rie Nishikawa-Shimono a, , Yoshinori Sekiguchi , Takeshi Koami , Madoka Kawamura ,
Daisuke Wakasugi a, Kazuhito Watanabe b, Shunichi Wakahara b, Kayo Kimura b, Susumu Yamanobe a,
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:
In this study, we describe the synthesis and structure–activity relationship (SAR) of a series of
isoquinoline chemoattractant receptor–homologous molecule expressed on Th2 cells (CRTH2)
antagonists. TASP0376377 (15-20), one of the most potent compounds, showed a potent binding affinity
(IC50 = 19 nM) in addition to the excellent functional antagonist activity (IC50 = 13 nM). Moreover, the
efficacy of this compound in a chemotaxis assay (IC50 = 23 nM) was in good agreement with its potency
as a CRTH2 antagonist. In addition, 15-20 exhibited greater selectivity in binding to CRTH2 than to the
Received 5 September 2013
Revised 18 October 2013
Accepted 19 October 2013
Available online 30 October 2013
Keywords:
DP1 prostanoid receptor (IC50 >1
l
M) or the enzymes COX-1 and COX-2 (IC50 >10
lM).
CRTH2 antagonist
Allergic diseases
Isoquinoline
PGD2
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
In the course of our program, aimed at developing CRTH2 antag-
onists for the treatment of allergic diseases, we pursued a new class
of potent and selective CRTH2 antagonist. Figure 2 shows a superpo-
sition of four reported CRTH2 antagonists (2, 3, 618 and 719).
The alignment model was based on the molecular modeling soft-
ware MOE.20 The model shows three overlapping sites with similar
chemical features. P1 is a carboxylic acid site, P2 is an aromatic
ring, main scaffold including a monocyclic and/or fused-ring sys-
tem, and P3 consists of an aromatic ring with diverse functional
groups. Using a fused 6–6 membered ring system as a core struc-
ture, we newly designed a 1,4-disubstituted isoquinoline as a P2
scaffold. As a result, we have identified a novel compound 15-1
showing moderate binding affinity (IC50 = 330 nM) in a radioligand
binding assay (3H-PGD2) with the human CRTH2 receptor. A preli-
minary account of this work has been previously presented, and
herein we describe the synthesis and SAR of the new class of iso-
quinoline derivatives.
Prostaglandin D2 (PGD2), derived from arachidonic acid via the
cascade reactions of cyclooxygenase, is the major prostanoid re-
leased from activated mast cells. The biological actions of PGD2
are mediated by the G-protein-coupled receptors (GPCRs) termed
DP1 and CRTH2 (also known as DP2).1–3 The CRTH2 receptor is ex-
pressed on cells including eosinophils, basophils and Th2 cells and
is involved in the migration and activation of these cells.4–6 Partic-
ularly, the CRTH2 receptor may have an important role in inflam-
matory diseases including asthma, COPD, and allergic rhinitis.7,8
The emerging roles of the CRTH2 receptor in inflammatory diseases
led to extensive research aimed at identifying selective CRTH2
antagonists. The research into CRTH2 antagonists dramatically in-
creased after studies identifying ramatroban,
a thromboxane
receptor antagonist used in the treatment of allergic rhinitis, as a
CRTH2 antagonist, as well as reports demonstrating CRTH2 agon-
ism of the anti-inflammatory drug, indomethacin. Starting with
related indolacetic acids, such as 1,9 210 and 3,11 structure–activity
relationship (SAR) information around the indolacetic acids were
disclosed. Since then, a large number of CRTH2 antagonists includ-
ing several series of chemotypes including 5-azaindole (4),12
7-azaindole (5),13 benzimidazole,14,15 indolizine,16 spiro–indo-
line17 (Fig. 1) have been reported.
2. Chemistry
Scheme 1 shows the synthesis of compounds 15-1–15-33 from
commercially available 2-(4-aminophenyl)acetonitrile (8a).
Protection of the starting material 8a with di-t-butyl dicarbonate
afforded 9. The reaction of 9 with methyl 1-chloroisoquinoline-4-
carboxylate in the presence of sodium hexamethyldisilazane,
followed by oxidative decyanation under oxygen atmosphere
produced 10 in 58% yields.21 The carboxylic acid 11, prepared by
⇑
Corresponding author. Tel.: +81 48 669 3029; fax: +81 48 652 7254.
E-mail address: r-nishikawa@so.taisho.co.jp (R. Nishikawa-Shimono).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.025

R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. 21 (2013) 7674–7685
7675
Figure 1. Ligands for the CRTH2 receptor.
O
O
O
R
N
N
H
R
CO2H
CO2H
Figure 2. Design of the novel isoquinoline chemotype for the CRTH2 receptor.
Scheme 1. Reagents and conditions: (a) Boc2O, EtOH, rt, 40%; (b) methyl 1-chloroisoquinoline-4-carboxylate, NaHMDS, THF, 0 °C, then O2, rt, 58%; (c) 1 N NaOH aq., MeOH,
0 °C–rt, 84%; (d) (COCl)2, CHCl3, rt, TMSCHN2, THF/MeCN, rt, silver acetate, H2O/dioxane, 60 °C then TMSCHN2, MeOH, rt, 50% (steps); (e) TFA, CHCl3, 0 °C–rt; (f) for 15-2–15-
32 RCOCl, pyridine, CHCl3, 0 °C–rt, 28%-quant; for 15-1, Ac2O, pyridine, 70 °C, 97%; for 15-33, 1,2-dichloro-4-isocyanatobenzene, DMF, 0 °C-rt, 56%; (g) 1 N NaOH aq., THF,
0 °C–rt, 27%-quant.
the basic hydrolysis of 10, was converted to 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
12a in 50% yield from 11.22 Deprotection of the t-butoxycarbonyl
group in 12a under an acidic condition (TFA), and the subsequent
acylation of the resulting aniline intermediate 13 with a variety
of acid chlorides gave 14-1–14-33. Finally, hydrolysis of the ester
moiety afforded the target isoquinoline derivatives 15-1–15-33

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R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. 21 (2013) 7674–7685
Scheme 2. Reagents and conditions: (a) NaH, MeI, THF, rt, 31%; (b) phenyl carbonochloridate, CHCl3, 0–rt; (c) (3,4-dichlorophenyl)methanol, EtN(i-Pr2), THF, reflux, 2 steps
54%; (d) 1 N NaOH aq., THF, rt, 86%; (e) 3,4-dichlorobenzylbromide, K2CO3, DMF, 90 °C, 35%; (f) 1 N NaOH aq., THF, rt, 58%; (g) 3,4-dichlorobenzene-1-sulfonyl chloride,
pyridine, 0 °C–rt, 39%; (h) 1 N NaOH aq., THF, rt, 12%; (i) TFA, CHCl3, 0–50 °C, 96%; (j) oxalyl chloride, DMF, CHCl3, rt; (k) RNH2, pyridine, CHCl3, 0–rt, 35–78%; (l) 1 N NaOH aq.,
THF, MeOH, 0 °C, 74–87%; (m) BBr3, CHCl3, 0 °C–rt, 47%; (n) for 15-42, 3,4-dichlorobenzylbromide, K2CO3, DMF, 80 °C, 23%; for 15-43 and 15-44, ROH, n-Bu3P, TMAD, THF, rt,
29%, 34%; (o) 1 N NaOH aq., THF, MeOH, rt, 48%-quant.
Scheme 3. Reagents and conditions: (a) NaH, MeI, CHCl3, rt, 72%; (b) TFA, CHCl3, rt, quant; (c) 4-chlorophenethylamine, WSC–HCL, HOBT–H2O, CHCl3, rt, 44%; (d) 1 N LiOH
aq., dioxane, 100 °C, 70%.
in 27%-quant yields. Compound 15-1 was obtained by the treat-
ment of 14-1 with acetic anhydride and the subsequent basic
hydrolysis, and compound 15-33 was obtained by the treatment
of 14-33 with 1,2-dichloro-4-isocyanatobenzene followed by basic
hydrolysis.
Compounds 15-45 and 15-46, in which the acetic acid moiety
at the 4-position of the isoquinoline core was modified, were syn-
thesized as shown in Schemes 3 and 4. Geminal dimethylation of
12b by sodium hydride and iodomethane resulted in 16 with a
72% yield. Deprotection of the t-butyl ester moiety using TFA,
followed by the condensation reaction of 17 with 4-chlorophen-
ethylamine (WSCꢀHCl, HOBTꢀH2O), gave 18 in a 44% yield. Finally,
hydrolysis of the ester moiety afforded the target derivative
15-45. Compound 15-46 was synthesized from commercially
available 4-bromo-1-chloroisoquinoline (19), which was reacted
with methyl 4-(cyanomethyl)benzoate in the presence of sodium
hexamethyldisilazane. Subsequent oxidative decyanation afforded
20. Compound 20 was converted to 22 using the boronic acid ester
21 by the following two-step reaction method: (1) treatment with
bis(pinacolato)diboron in the presence of PdCl2(dppf) and potas-
sium acetate; and (2) oxidation of 21 using oxone. Transformation
of 22 into the target compound 15-46 was accomplished using the
following conventional method: (1) alkylation of the hydroxyl
moiety in 22 with t-butyl 2-bromoacetate in the presence of potas-
sium 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.
Scheme 2 summarizes the synthesis of compounds 15-34–15-
44. Compound 15-34 was prepared by selective N-methylation of
the amide of 15-20. The common intermediate 13 was reacted
with phenylcarbonochloridate. The addition of (3,4-dichloro-
phenyl)methanol followed by hydrolysis of the resulting ester moi-
ety generated 15-35. With regard to the synthesis of compounds
15-36, 13 was treated with 3,4-dichlorobenzylbromide in the pres-
ence of potassium carbonate, and the ester moiety was hydrolyzed
by sodium hydroxide. Compound 15-37 was synthesized by the
reaction of the aniline intermediate 13 with 3,4-dichloroben-
zene-1-sulfonyl chloride in the presence of pyridine followed by
basic hydrolysis of the ester moiety. Intermediate 12b was pre-
pared from methyl 1-chloroisoquinoline-4-carboxylate and t-butyl
4-(cyanomethyl)benzoate, in a manner similar to the synthesis of
12a as described in Scheme 1. Using 12b, compounds 15-38–15-
41 were prepared as follows: (1) acidic removal of the ester moiety
(TFA); (2) conversion to acyl chloride (oxalyl chloride); (3)
condensation of the acyl chloride with 3,4-dichloroaniline; and
(4) hydrolysis of the ester moiety. 15-42–15-44 were achieved
by demethylation of 12c by boron tribromide, which was
derived from methyl 1-chloroisoquinoline-4-carboxylate and
2-(4-methoxyphenyl)acetonitrile in a procedure similar to the
synthesis of 12a (Scheme 1), and the subsequent etherification of
the resulting phenol followed by hydrolysis.
3. Results and discussion
Compounds were tested for their binding affinity to human
CRTH2 in a radioligand binding assay (3H-PGD2) using CHO
cells stably transfected with human CRTH2. In addition, these

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7677
Scheme 4. Reagents and conditions: (a) Methyl 4-(cyanomethyl)benzoate, NaHMDS, THF, 0 °C–rt, then O2, rt, quant; (b) bis(pinacolato)diboron, PdCl2(dppf), AcOK, 80 °C,
55%; (c) oxone, NaHCO3, acetone/H2O, 0 °C–rt, 9%; (d) t-butyl 2-bromoacetate, K2CO3, DMF, rt, 94%; (e) 0.2 N NaOH aq., THF, rt, 48%; (f) 4-chlorophenethylamine, WSC–HCl,
HOBT–H2O, CHCl3, rt, 59%; (g) 1 N NaOH aq., THF, rt, 39%.
Table 1
In vitro data of isoquinoline derivatives with R1 substitution modification
R1
IC50 (nM)
Binding
Compound
R1
IC50 (nM)
Binding
a
a
Compound
Ca2+
Ca2+
15-1
15-2
15-3
15-4
15-5
15-6
15-7
15-8
Methyl
Cyclopropyl
Cyclohexyl
Cyclohexylmethyl
Adamantylmethyl
Tetrahydro-2H-yran-4-yl
Phenyl
330
50
14
20
21
400
7.9
4.0
NT
NT
15-9
Naphthalen-1-yl
1H-Indol-2-yl
Benzofuran-2-yl
Pyridin-2-yl
31
350
4.2
7.4
NT
>1000
>1000
550
15-10
15-11
15-12
15-13
15-14
15-15
15-16
3.5
3.8
48
270
220
97
>1000
42
Pyridin-4-yl
83
Pyridazine-3-yl
Pyrimidin-2-yl
Thiazole-4-yl
322
290
67
Naphthalen-2-yl
10
>1000
a
Mean value from at two or more independent experiments. IC50 values were determined from full ten-point, half-log concentration–response curves.
compounds were assessed for their functional activity in PGD2 dri-
ven Ca2+ flux in KB8 cells expressing human CRTH2.
15-11: IC50 = 3.8 nM) and the functional activity as well (15-8:
IC50 = 10 nM, 15-10: IC50 = 4.2 nM, 15-11: IC50 = 7.4 nM). In con-
trast, monocyclic heteroaromatic rings including pyridin-2-yl
(15-12), pyridin-4-yl (15-13), pyridazine-2-yl (15-14), pyrimidin-
2-yl (15-15), and thiazole-4-yl (15-30) had moderate binding
activity comparable to those containing methyl (15-1) and cyclo-
propyl (15-2) moieties. These results suggested that the lipophilic-
ity of R1 group played a key role in increasing CRTH2 binding and
functional potency.
We decided to further explore the SAR of the potent compound
15-7 by preparing a set of compounds with a substitution on the
terminal phenyl moiety (Table 2). At first, the compound 15-7
was modified by incorporating a chlorine atom into the ortho, meta,
and para positions of the phenyl ring. The meta-chlorophenyl
(15-18) and para-chlorophenyl (15-19) groups enhanced the bind-
ing affinity (15-18: IC50 = 3.2 nM, 15-19: IC50 = 3.4 nM) and the
functional antagonist activity (15-18: IC50 = 9.7 nM, 15-19:
IC50 = 9.1 nM). In contrast, the ortho-chlorophenyl (15-17) led to
a 4-fold reduction (IC50 = 45 nM) in the binding affinity, and
complete loss of the functional activity. Moreover, the meta- and
para-dichlorophenyl derivative (15-20) was well tolerated in the
binding and functional activity with CRTH2. For further SAR
development we focused on the modification of the para-substitu-
tion of the phenyl part of 15-7. Replacement of a methyl (15-21),
As described above, we designed a novel isoquinoline scaffold
and synthesized compound 15-1, based on the superimposition
of the critical P1, P2, and P3 sites of known CRTH2 antagonists
(Fig. 2). Inspection of the key interaction between the binding
pocket of CRTH2 and the P1 and P2 sites of the CRTH2 antagonists
identified a likely lipophilic side pocket on the P3 site. Considering
that the lipophilic side pocket could influence CRTH2 potency and
allow us to make a diverse library of derivatives, we decided there-
fore to first explore the acetyl moiety of the P3 site on 15-1
(Table 1). When the methyl group (R1) in the acyl moiety of 15-1
was replaced with bulkier cycloalkyl substituents such as a cyclo-
propyl (15-2), cyclohexyl (15-3), cyclohexylmethyl (15-4), and
1-adamantylmethyl (15-5), binding affinities were enhanced
(15-2: IC50 = 50 nM, 15-3: IC50 = 14 nM, 15-4: IC50 = 20 nM, 15-5:
IC50 = 21 nM) as compared with 15-1. However, these groups led
to submicromolar antagonist potency in the functional assay. In
addition, replacement of the methyl with a phenyl (15-7) resulted
in further improvement in the binding affinity (15-7: IC50 = 7.9 nM)
and in the functional antagonist affinity (15-7: IC50 = 42 nM). Other
aromatic rings such as naphthalen-2-yl (15-8), 1H-indol-2-yl
(15-10), and benzofuran-2-yl (15-11) led to greatly enhancing
the binding affinity (15-8: IC50 = 4.0 nM, 15-10: IC50 = 3.5 nM,

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Table 2
In vitro data of isoquinoline derivatives with R1 substitution modification
R1
IC50 (nM)
Compound
R1
IC50 (nM)
a
a
Compound
Binding
Ca2+
Binding
Ca2+
15-7
Phenyl
7.9
45
3.2
3.4
19
42
15-23
15-24
15-25
15-26
15-27
15-28
15-29
4-CF3-phenyl
4-OCF3-phenyl
4-CN-phenyl
4-NO2-phenyl
4-NMe2-phenyl
1,10-Biphenyl-4-yl
4-Phenoxyphenyl
4.9
11
7.8
9.9
6.2
18
15
15-17
15-18
15-19
15-20
15-21
15-22
2-Cl-Phenyl
3-Cl-Phenyl
4-Cl-Phenyl
3,4-Di-2-Cl-Phenyl
4-Me-Phenyl
4-OMe-Phenyl
>1000
9.7
9.1
13
18
17
8.7
>1000
28
13
14
5.2
7.3
14
120
a
Mean value from at two or more independent experiments. IC50 values were determined from full ten-point, half-log concentration–response curves.
Table 3
In vitro data of isoquinoline derivatives with linker modification
R1
IC50 (nM)
Binding
Compound
R1
IC50 (nM)
Binding
a
a
Compound
Ca2+
9.1
Ca2+
NT
15-19
3.4
15-37
340
15-20
15-30
15-31
15-32
15-33
15-34
19
13
30
18
17
36
NT
15-38
15-39
15-40
15-41
15-42
15-43
15-44
8.2
10
7.8
3.7
38
12
11
38
15
13
13
12
4.0
13
18
180
130
85
210
15-35
15-36
6.7
6.1
5.2
43
a
Mean value from at two or more independent experiments. IC50 values were determined from full ten-point, half-log concentration–response curves.
methoxy (15-22), trifluoromethyl (15-23), trifluoromethoxy
(15-24), nitro (15-26) and dimethylamino (15-27) groups resulted
in retention of the binding potency, simultaneously enhancing the
functional activity. Introduction of a cyano (15-25) maintained the
potent binding affinity (15-25: IC50 = 7.8 nM), while the functional
activity was greatly reduced (15-25: IC50 = >1000 nM). Additional
lipophilic substituents such as a phenyl (15-28) and phenoxy
(15-29) were tolerated with respect to the binding affinity
(15-28: IC50 = 18 nM, 15-29: IC50 = 14 nM).
Next, we examined the SAR of the amide linker moiety of 15-19
and 15-20 (Table 3). An N-methylamide 15-34, which was devoid
of the hydrogen bond donor (NH) showed a 15-fold reduction in
the binding affinity (15-34: IC50 = 210 nM), suggesting that the
hydrogen bond donor (NH) is likely to contribute to the greater
binding affinity. The amide derivatives 15-30–15-32, which differ
in the lengths of their linkers, displayed activity comparable to that
of the parent compound 15-20. It is interesting to note that incor-
poration of an aminocarbonyloxymethylene linker (15-35) led to a
2-fold improvement in the binding and functional potency. Com-
pounds (15-38 and 15-39) having the inverted amide linkers of
15-19 and 15-20, respectively, had acceptable binding affinity
(15-38: IC50 = 8.2 nM, 15-39: IC50 = 10 nM), and functional activity
(15-38: IC50 = 38 nM, 15-39: IC50 = 13 nM). In addition, an amido-
ethylene linker was also tolerable in the CRTH2 binding (15-40:
IC50 = 7.8 nM, 15-41: IC50 = 3.7 nM), and the functional potency
(15-40: IC50 = 12 nM, 15-41: IC50 = 18 nM). Next, we carried out

R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. 21 (2013) 7674–7685
7679
Table 4
5. Experimental
In vitro data of isoquinoline derivatives with R2 substitution modification
5.1. Chemistry
All starting materials and reagents were commercial products
that were used without further purification. The reaction
progresses were usually monitored by TLC using Merck silica gel
60 F254 plates or Fuji Silysia chromatorex NH plates. Column
chromatography was performed using silica gel Wako Pure
Chemical C-200 and NH-silica gel Fuji Silysia chromatorex
DM1020. Melting points were determined on a Mettler FP-61 or
a Yanaco MP-500D melting point apparatus and were uncorrected.
1H NMR spectra were recorded on a Varian Gemini-600 instrument
at 600 MHz. Chemical shifts were reported in parts per million as d
units relative to tetramethylsilane as an internal reference. Multi-
plicity was defined as s (singlet), d (doublet), t (triplet), q (quartet),
dd (double doublet), m (multiplet), or br s (broad singlet). Mass
spectra (MS) were recorded on a Shimadzu LCMS-2010EV mass
spectrometer with electrospray ionization (ESI)/atmospheric pres-
sure chemical ionization (APCI) dual source. Elemental analyses
were performed on a Yanaco MT-6 elemental analyzer, and the re-
sults were within 0.4% of the calculated values.
R3
IC50 (nM)
a
Compound
Binding
7.8
Ca2+
12
15-40
15-45
200
25
>1000
100
15-46
a
Mean value from two or more independent experiments. IC50 values were
determined from full ten-point, half-log concentration–response curves.
additional SAR study of the linker moiety. When an aminomethyl-
ene (15-36) or urea (15-33) tether was incorporated in place of the
amide linker, the resulting compounds maintained the potent
CRTH2 binding affinity, but it led to a 2- to 4-fold reduction in
functional activity compared to that of 15-19. In contrast, a
sulfonamide linker (15-37) led to a significant reduction in the
binding (15-37: IC50 = 340 nM). An oxymethylene linker resulted
in a slight loss of the binding affinity (15-42: IC50 = 38 nM), and a
substantial loss in the functional potency (15-42: IC50 = 180 nM).
The oxyethylene and oxypropylene linkers maintained the
potent binding affinity (15-43: IC50 = 12 nM, 15-44: IC50 = 11 nM),
while the functional activity of these compounds was weak
(15-43: IC50 = 130 nM, 15-44: IC50 = 85 nM).
5.1.1. 4-tert-Butoxycarbonylaminophenylacetonitrile (9)
To a solution of 4-aminophenylacetonitrile (1.41 g, 10.7 mmol)
in EtOH (5 ml) was added Boc2O (3.10 g, 14.2 mmol), and the mix-
ture was stirred at room temperature for 2 h. The resulting precip-
itate was collected by filtration, and dried to yield 9 (984 mg,
4.24 mmol, 40%) as a colorless powder: 1H NMR (CDCl3): d 1.52
(s, 9H), 3.69 (s, 2H), 6.49 (br s, 1H), 7.22–7.26 (m, 2H), 7.35–7.41
(m, 2H).
5.1.2. Methyl 1-{4-(tert-butoxycarbonylamino)phenylcarbonyl}-
isoquinolin-4-ylcarboxylate (10)
A 1.0 M of NaHMDS in THF (2.60 ml, 2.60 mmol) was added to a
solution of 9 (286 mg, 1.23 mmol) in THF (2 ml) at 0 °C, and the
mixture was stirred for 30 min. To the mixture was added a solu-
tion of methyl 1-chlorosioquinoline-4-carboxylate (300 mg,
1.35 mmol) in THF (3 ml) and the mixture was stirred at room tem-
perature for 2.5 h. Then, the reaction mixture was further stirred
for 17 h under oxygen atmosphere. The reaction was quenched
by adding a saturated ammonium chloride solution, and the mix-
ture was extracted with EtOAc. The organic layer was washed with
brine, dried over MgSO4, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(NH) eluting with 10-30% EtOAc/hexane to give 10 (289 mg, 58%)
as a yellow amorphous: 1H NMR (CDCl3): d 1.53 (s, 9H), 4.07
(s, 3H), 6.72 (br s, 1H), 7.43–7.49 (m, 2H), 7.62–7.66 (m, 1H),
7.83–7.89 (m, 3H),8.09–8.13 (m, 1H), 8.99–9.03 (m, 1H), 9.20 (s,
1H); MS (ESI/APCI Dual) m/z 407 (M+H)+.
We next examined the effects of substituents around the car-
boxylic acid moiety on CRTH2 binding (Table 4). The carboxylic
acid moiety shared by the representative CRTH2 antagonists and
is essential for the CRTH2 activity. Germinal dimethylation of the
methylene moiety next to the carboxylic acid resulted in a 25-fold
decrease in the binding potency (15-45) compared with that of the
original compound 15-40, and similarly, the functional activity of
15-45 was dramatically reduced. Insertion of an oxygen atom be-
tween the carboxymethyl moiety and the heteroaryl group re-
sulted in a slight loss in the binding and a significant decrease in
functional activity (15-46). These data suggest that the binding site
of the CRTH2 (P1), where the acid moiety of the antagonists inter-
acts, is strictly limited.
The SAR study of the isoquinoline scaffold demonstrated that
with a few exceptions, compounds with high binding affinities
generally exhibited highly potent functional activities. One of the
most potent antagonists, 15-20 (IC50 = 19 nM), was effective in a
chemotaxis assay (IC50 = 23 nM) and exhibited sufficient selectivity
in binding to CRTH2 over the DP1 prostanoid receptor23 (IC50
5.1.3. 1-{4-(tert-Butoxycarbonylamino)phenylcarbonyl}isoqui-
nolin-4-ylcarboxylic acid (11)
>1
l lM).
M) and the COX-1 and COX-2 enzymes24 (IC50 >10
To a solution of 10 (1.33 g, 3.28 mmol) in MeOH (30 ml) was
added 1 N NaOH (30 ml), and the mixture was stirred at room
temperature for 15 h, and then at 35 °C for 3 h. The mixture
was acidified with AcOH, and H2O was added to the mixture.
The resultant precipitate was collected by filtration, rinsed with
H2O, and dried to give 11 (1.08 g, 84%) as an orange powder:
1H NMR (CDCl3): d 1.53 (s, 9H), 6.90 (br s, 1H), 7.45–7.50
(m, 2H), 7.65–7.69 (m, 1H), 7.86–7.93 (m, 3H), 8.12–8.15
(m, 1H), 9.11–9.15 (m, 1H), 9.39 (s, 1H); MS (ESI/APCI Dual)
m/z 393 (M+H)+.
4. Conclusion
In conclusion, we have identified the novel isoquinoline acetic
acid chemotype 15-1 as a potent CRTH2 antagonist. SAR of the
scaffold was explored, resulting in the identification of compound
15-20 (TASP0376377), which is a selective functional antagonist of
CRTH2. Studies are ongoing to explore the utility of this class of
compounds in inflammatory disease models and will be reported
in due course.

7680
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5.1.4. 2-[1-[4-[(2-Methylpropan-2-yl)oxycarbonylamino]
benzoyl]isoquinolin-4-yl]acetic acid (12a)
under reduced pressure to give 15–20 (489 mg, 77%) as a colorless
powder: mp 124.5–126.5 °C; 1H NMR (DMSO-d6): d 4.17 (s, 2H),
7.70–7.74 (m, 1H), 7.83–7.93 (m, 4H), 7.94–7.99 (m, 3H),
8.02–8.06 (m, 1H), 8.13–8.16 (m, 1H), 8.24–8.26 (m, 1H), 8.54
(s, 1H), 10.78 (br s, 1H); MS (ESI/APCI Dual) m/z 479 (M+H)+.
To a suspension of 11 (1.08 g, 2.75 mmol) in CHCl3 (20 ml) was
added oxalyl chloride (0.709 ml, 8.26 mmol) at 0 °C, and the mix-
ture was stirred at room temperature for 3.5 h. After evaporation
of the volatile in the reaction mixture, the residue was dissolved
in a mixture of THF (10 ml) and CH3CN (10 ml). To this mixture
was added a 2.0 M of TMSCHN2 in CH2Cl2 (2.75 ml, 5.50 mmol)
at 0 °C, and the resulting mixture was stirred for 2 h. Then the
reaction mixture was concentrated under reduced pressure. To a
solution of the residue in a mixture of 1,4-dioxane (10 ml) and
H2O (10 ml) was added silver acetate (138 mg, 0.825 mmol), and
the mixture was stirred at 60 °C for 30 min. After cooling, the mix-
ture was extracted with EtOAc, and the organic layer was washed
with brine, dried over MgSO4 and concentrated under reduced
pressure. The resulting residue was dissolved in MeOH (20 ml),
and a 2.0 M of TMSCHN2 in CH2Cl2 (4.54 ml, 9.08 mmol) was added
to this mixture. After being stirred for 30 min, the reaction was
quenched by adding a few drops of AcOH. The mixture was diluted
with EtOAc and the organic layer was washed with brine, dried
over MgSO4 and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (60 N) eluting
with 10–35% EtOAc/hexane to give 12a (578 mg, 50% from 11) as
a yellowish powder: 1H NMR (CDCl3): d 1.51 (s, 9H), 3.72 (s, 3H),
4.10 (s, 2H), 6.72 (br s, 1H), 7.42–7.47 (m, 2H), 7.58–7.63 (m,
1H), 7.75–7.81 (m, 1H), 7.88–7.93 (m, 2H), 8.02–8.06 (m, 1H),
8.16–8.20 (m, 1H), 8.50 (s, 1H); MS (ESI/APCI Dual) m/z 421
(M+H)+.
5.1.8. 2-[1-(4-Acetamidobenzoyl)isoquinolin-4-yl]acetic acid
(15-1)
Pale yellow powder; mp 141.0–143.0 °C; 1H NMR (DMSO-d6): d
2.09 (s, 3H), 4.16 (s, 2H), 7.67–8.15 (m, 8H), 8.52 (s, 1H), 10.37 (s,
1H), 12.73 (br s, 1H); MS (ESI/APCI Dual) m/z 349 (M+H)+; Anal.
Calcd for C20H16N2O4ꢀ2H2O: C, 62.49; H, 5.24; N, 7.29. Found: C,
62.55; H, 5.06; N, 7.28.
5.1.9. 2-[1-[4-(Cyclopropanecarbonylamino)benzoyl]isoqui-
nolin-4-yl]acetic acid (15-2)
Pale yellow powder; mp 200.0–202.0 °C; 1H NMR (DMSO-d6): d
0.79–0.87 (m, 4H), 1.80–1.87 (m, 1H), 4.15 (s, 2H), 7.67–8.16 (m,
8H), 8.51 (s, 1H), 10.65 (s, 1H), 12.70 (br s, 1H); MS (ESI/APCI Dual)
m/z 375 (M+H)+; Anal. Calcd for C22H18N2O4ꢀ0.5H2O: C, 68.92; H,
4.99; N, 7.31. Found: C, 69.00; H, 4.96; N, 7.33.
5.1.10. [1-({4-[(Cyclohexylcarbonyl)amino]phenyl}carbonyl)-
isoquinolin-4-yl]acetic acid (15-3)
Colorless powder; mp 190.5–191.5 °C; 1H NMR (DMSO-d6): d
1.14–1.84 (m, 10H), 2.34–2.40 (m, 1H), 4.16 (s, 2H), 7.67–8.15
(m, 8H), 8.52 (s, 1H), 10.24 (br s, 1H), 12.67 (br s, 1H); MS (ESI/APCI
Dual) m/z 417 (M+H)+; Anal. Calcd for C25H24N2O4ꢀ0.1H2O: C,
71.79; H, 5.83; N, 6.70. Found: C, 71.61; H, 5.94; N, 6.50.
5.1.5. Methyl 1-[(4-(aminophenyl)carbonyl]isoquinolin-4-
ylacetate (13)
5.1.11. 2-[1-[4-[(2-Cyclohexylacetyl)amino]benzoyl]isoqui-
nolin-4-yl]acetic acid (15-4)
To a solution of 12a (100 mg, 0.238 mmol) in CHCl3 (1 ml) was
added TFA (1 ml) at 0 °C, and the mixture was stirred at room
temperature for 1.5 h. The reaction mixture was basified with a
saturated sodium bicarbonate solution and extracted with CHCl3.
The organic layer was dried over MgSO4 and concentrated under
reduced pressure to give 13 (91 mg, 100%) as a yellow oil. This
compound was used for the next reaction without further purifica-
tion: 1H NMR (CDCl3): d 3.73 (s, 3H), 4.09 (s, 2H), 6.62–6.66 (m,
2H), 7.58–7.62 (m, 1H), 7.75–7.82 (m, 3H), 8.01–8.04 (m, 1H),
8.15–8.18 (m, 1H), 8.50 (s, 1H); MS (ESI/APCI Dual) m/z 321
(M+H)+.
Pale yellow powder; mp 123.0–125.0 °C; 1H NMR (DMSO-d6): d
0.80–1.33 (m, 5H), 1.55–1.83 (m, 6H), 2.24 (d, J = 7.3 Hz, 2H), 4.16
(s, 2H), 7.65–8.16 (m, 8H), 8.52 (s, 1H), 10.29 (s, 1H), 12.73 (br s,
1H); MS (ESI/APCI Dual) m/z 431 (M+H)+; Anal. Calcd for C26H26N2-
O4ꢀ0.2H2O: C, 71.94; H, 6.13; N, 6.45. Found: C, 71.88; H, 6.29; N,
6.27.
5.1.12. 2-[1-[4-[[2-(1-Adamantyl)acetyl]amino]benzoyl]-
isoquinolin-4-yl]acetic acid (15-5)
Colorless powder; mp 173.0–173.5 °C; 1H NMR (DMSO-d6): d
1.56–1.69 (m, 12H), 1.90–1.96 (m, 3H), 2.11 (s, 2H), 4.16 (s, 2H),
7.67–8.15 (m, 8H), 8.52 (s, 1H), 10.19 (br s, 1H), 12.67 (br s, 1H);
MS (ESI/APCI Dual) m/z 483 (M+H)+; Anal. Calcd for C30H30N2O4-
ꢀ0.6H2O: C, 73.03; H, 6.37; N, 5.68. Found: C, 72.99; H, 6.25; N, 5.61.
5.1.6. Methyl 1-{4-(3,4-dichlorophenylcarbonylamino)phenyl-
carbonyl}isoquinolin-4-ylacetate (14–20)
To a solution of 13 (500 mg, 1.56 mmol) in pyridine (8 ml) was
added 3,4-dichlorobenzoyl chloride (490 mg, 2.34 mmol) at 0 °C,
and the mixture was stirred at room temperature for 13.5 h. The
reaction was quenched by adding H2O, and the mixture was ex-
tracted with EtOAc. The organic layer was washed with diluted
hydrochloric acid and brine, dried over MgSO4 and concentrated
under reduced pressure. The resulting residue was purified by sil-
ica gel column chromatography (60 N) eluting with 20-50% EtOAc/
hexane to give 14–20 (685 mg, 89%) as a pale yellow powder: 1H
NMR (CDCl3): d 3.74 (s, 3H), 4.12 (s, 2H), 7.55–7.83 (m, 7H),
7.97–8.08 (m, 4H), 8.21–8.24 (m, 1H), 8.52 (s, 1H); MS (ESI/APCI
Dual) m/z 493 (M+H)+.
5.1.13. 2-[1-[4-(Oxane-4-carbonylamino)benzoyl]isoquinolin-4-
yl]acetic acid (15-6)
Colorless powder; mp 157.0–159.0 °C; 1H NMR (DMSO-d6): d
1.61–1.75 (m, 4H), 2.60–2.67 (m, 1H), 3.32–3.37 (m, 2H), 3.88–
3.94 (m, 2H), 4.16 (s, 2H), 7.67–8.16 (m, 8H), 8.52 (s, 1H), 10.33
(s, 1H), 12.69 (br s, 1H); MS (ESI/APCI Dual) m/z 419 (M++H)+; Anal.
Calcd for C24H22N2O5ꢀ0.5H2O: C, 67.44; H, 5.42; N, 6.55. Found: C,
67.23; H, 5.44; N, 6.32.
5.1.14. 2-[1-(4-Benzamidobenzoyl)isoquinolin-4-yl]acetic acid
(15-7)
5.1.7. 2-[1-[4-[(3,4-Dichlorobenzoyl)amino]benzoyl]-
isoquinolin-4-yl]acetic acid (15-20)
Colorless powder; mp 195.0–197.0 °C; 1H NMR (DMSO-d6): d
4.18 (s, 2H), 7.52–8.16 (m, 13H), 8.54 (s, 1H), 10.65 (s, 1H), 12.72
(br s, 1H); MS (ESI/APCI Dual) m/z 411 (M+H)+; Anal. Calcd for
To a solution of 14–20 (650 mg, 1.32 mmol) in a mixture of THF
(10 ml) and MeOH (10 ml) was added 1 N NaOH (10 ml), and the
mixture was stirred for 30 min at room temperature. The mixture
was acidified with diluted hydrochloric acid and extracted with
CHCl3. The organic layer was dried over MgSO4 and concentrated
C25H18N2O4ꢀ0.2H2O: C, 72.52; H, 4.48; N, 6.77. Found: C, 72.46;
H, 4.46; N, 6.73.

R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. 21 (2013) 7674–7685
7681
5.1.15. 2-[1-[4-(Naphthalene-2-carbonylamino)
5.1.23. 2-[1-[4-(1,3-Thiazole-4-carbonylamino)benzoyl]isoqui-
benzoyl]isoquinolin-4-yl]acetic acid (15-8)
nolin-4-yl]acetic acid (15-16)
Colorless powder; mp 179.0–181.0 °C; 1H NMR (DMSO-d6): d
4.19 (s, 2H), 7.61–8.17 (m, 14H), 8.55 (s, 1H), 8.63 (s, 1H), 10.84
(s, 1H), 12.72 (br s, 1H); MS (ESI/APCI Dual) m/z 461 (M+H)+; Anal.
Calcd for C29H20N2O4ꢀ1.5H2O: C, 71.45; H, 4.76; N, 5.75. Found: C,
71.40; H, 4.39; N, 5.65.
Colorless powder; mp 194.5–196.5 °C; 1H NMR (DMSO-d6): d
4.17 (s, 2H), 7.69–8.59 (m, 10H), 9.28–9.30 (m, 1H), 10.74 (s, 1H),
12.69 (br s, 1H); MS (ESI/APCI Dual) m/z 418 (M+H)+; Anal. Calcd
for C22H15N3O4Sꢀ0.2H2O: C, 62.76; H, 3.69; N, 9.98. Found: C,
62.84; H, 3.68; N, 9.82.
5.1.24. 2-[1-[4-[(2-Chlorobenzoyl)amino]benzoyl]isoquinolin-
4-yl]acetic acid (15-17)
5.1.16. 2-[1-[4-(Naphthalene-1-carbonylamino)benzoyl]isoqu-
inolin-4-yl]acetic acid (15-9)
Colorless powder; mp 186.5–188.0 °C; 1H NMR (DMSO-d6): d
4.18 (s, 2H), 7.44–8.16 (m, 12H), 8.54 (s, 1H), 10.95 (br s, 1H),
12.67 (br s, 1H); MS (ESI/APCI Dual) m/z 445 (M+H)+; Anal. Calcd
for C25H17ClN2O4ꢀ0.2H2O: C, 66.95; H, 3.98; N, 6.14. Found: C,
67.14; H, 3.98; N, 6.14.
Colorless powder; mp 145.5–147.0 °C; 1H NMR (DMSO-d6): d
4.19 (s, 2H), 7.58–8.21 (m, 15H), 8.55 (s, 1H), 11.00 (br s, 1H),
12.67 (br s, 1H); MS (ESI/APCI Dual) m/z 461 (M+H)+; Anal. Calcd
for C29H20N2O4ꢀ1.0H2O: C, 72.79; H, 4.70; N, 5.80. Found: C,
72.98; H, 4.70; N, 5.80.
5.1.25. 2-[1-[4-[(3-Chlorobenzoyl)amino]benzoyl]isoquinolin-
4-yl]acetic acid (15-18)
5.1.17. 2-[1-[4-(1H-Indole-2-carbonylamino)benzoyl]isoqui-
nolin-4-yl]acetic acid (15-10)
Pale yellow powder; mp 181.0-182.0 °C; 1H NMR (DMSO-d6): d
4.18 (s, 2H), 7.57–8.16 (m, 12H), 8.54 (s, 1H), 10.73 (s, 1H), 12.72
(br s, 1H); MS (ESI/APCI Dual) m/z 445 (M+H)+; Anal. Calcd for
Pale yellow powder; mp 257.0–259.0 °C; 1H NMR (DMSO-d6): d
4.18 (s, 2H), 7.05–8.17 (m, 13H), 8.54 (s, 1H), 10.60 (s, 1H), 11.83
(br s, 1H), 12.73 (br s, 1H); MS (ESI/APCI Dual) m/z 450 (M+H)+;
Anal. Calcd for C27H19N3O4ꢀ0.6H2O: C, 70.46; H, 4.42; N, 9.13.
Found: C, 70.27; H, 4.35; N, 8.96.
C25H17ClN2O4ꢀ0.2H2O: C, 66.95; H, 3.98; N, 6.14. Found: C, 67.01;
H, 4.00; N, 6.11.
5.1.26. 2-[1-[4-[(4-Chlorobenzoyl)amino]benzoyl]isoquinolin-
4-yl]acetic acid (15-19)
5.1.18. 2-[1-[4-(1-Benzofuran-2-carbonylamino)benzoyl]isoqui-
nolin-4-yl]acetic acid (15-11)
Pale yellow powder; mp 180.0–182.0 °C; 1H NMR (DMSO-d6): d
4.17 (s, 2H), 7.61–8.16 (m, 12H), 8.54 (s, 1H), 10.71 (s, 1H); MS
(ESI/APCI Dual) m/z 445 (M+H)+; Anal. Calcd for C25H17ClN2O4: C,
67.50; H, 3.85; N, 6.30. Found: C, 67.29; H, 4.03; N, 6.17.
Pale yellow powder; mp 184.0–186.0 °C; 1H NMR (DMSO-d6): d
4.18 (s, 2H), 7.36–8.16 (m, 13H), 8.55 (s, 1H), 10.91 (s, 1H), 12.71
(br s, 1H); MS (ESI/APCI Dual) m/z 451 (M+H)+; Anal. Calcd for
C
2-7H18N2O5ꢀ0.2H2O: C, 71.42; H, 4.08; N, 6.17. Found: C, 71.52;
H, 4.34; N, 6.11.
5.1.27. 2-[1-[4-[(4-Methylbenzoyl)amino]benzoyl]isoquinolin-
4-yl]acetic acid (15-21)
5.1.19. 2-[1-[4-(Pyridine-2-carbonylamino)benzoyl]isoqui-
nolin-4-yl]acetic acid (15-12)
Colorless powder; mp 186.0–186.5 °C; 1H NMR (DMSO-d6): d
2.40 (s, 3H), 4.18 (s, 2H), 7.34–7.38 (m, 2H), 7.70–7.74 (m, 1H),
7.83–7.92 (m, 5H), 7.97–8.00 (m, 2H), 8.02 -8.05 (m, 1H),
8.12–8.15 (m, 1H), 8.54 (s, 1H), 10.56 (br s, 1H), 12.69 (br s, 1H);
MS (ESI/APCI Dual) m/z 425 (M+H)+; Anal. Calcd for C26H20N2O4ꢀ
0.2H2O: C, 72.95; H, 4.80; N, 6.54. Found: C, 72.90; H, 4.79; N, 6.52.
Colorless powder; mp 186.0–188.0 °C; 1H NMR (DMSO-d6): d
4.18 (s, 2H), 7.68–8.21 (m, 11H), 8.54 (s, 1H), 8.75–8.78 (m, 1H),
11.04 (s, 1H), 12.71 (br s, 1H); MS (ESI/APCI Dual) m/z 412
(M+H)+; Anal. Calcd for C27H17N3O4: C, 70.07; H, 4.16; N, 10.21.
Found: C, 70.01; H, 4.32; N, 10.14.
5.1.28. 2-[1-[4-[(4-Methoxybenzoyl)amino]benzoyl]isoqui-
nolin-4-yl]acetic acid (15-22)
5.1.20. 2-[1-[4-(Pyridine-4-carbonylamino)benzoyl]isoqui-
nolin-4-yl]acetic acid (15-13)
Colorless powder; mp 169.0–172.0 °C; 1H NMR (DMSO-d6): d
3.85 (s, 3H), 4.18 (s, 2H), 7.05–7.11 (m, 2H), 7.68–8.17 (m, 10H),
8.54 (s, 1H), 10.49 (s, 1H), 12.74 (br s, 1H); MS (ESI/APCI Dual)
m/z 441 (M+H)+; Anal. Calcd for C26H20N2O5ꢀ0.5H2O: C, 69.48; H,
4.71; N, 6.23. Found: C, 69.53; H, 4.80; N, 6.02.
Colorless powder; mp 200.0–202.0 °C; 1H NMR (DMSO-d6): d
4.17 (s, 2H), 7.69–8.16 (m, 10H), 8.54 (s, 1H), 8.79–8.82 (m, 2H),
10.88 (s, 1H); MS (ESI/APCI Dual) m/z 412 (M+H)+; Anal. Calcd
for C24H17N3O4ꢀ0.1H2O: C, 69.76; H, 4.20; N, 10.17. Found: C,
69.53; H, 4.23; N, 10.08.
5.1.29. 2-[1-[4-[[4-(Trifluoromethyl)benzoyl]amino]
5.1.21. 2-[1-[4-(Pyridazine-3-carbonylamino)benzoyl]isoqui-
nolin-4-yl]acetic acid (15-14)
benzoyl]isoquinolin-4-yl]acetic acid (15-23)
Colorless powder; mp 147.0–148.5 °C; 1H NMR (DMSO-d6): d
4.18 (s, 2H), 7.70–7.74 (m, 1H), 7.86–8.01 (m, 7H), 8.02–8.06 (m,
1H), 8.11–8.20 (m, 3H), 8.54 (s, 1H), 10.86 (br s, 1H), 12.70 (br s,
1H); MS (ESI/APCI Dual) m/z 479 (M+H)+ ; HRMS m/z Calcd for
Pale yellow powder; mp 190.5–192.0 °C; 1H NMR (DMSO-d6): d
4.18 (s, 2H), 7.70–8.36 (m, 10H), 8.55 (s, 1H), 9.48–9.51 (m, 1H),
11.46 (br s, 1H), 12.67 (br s, 1H); MS (ESI/APCI Dual) m/z 413
(M+H)+; Anal. Calcd for C23H16N4O4ꢀ0.2H2O: C, 66.41; H, 3.97; N,
13.47. Found: C, 66.51; H, 4.05; N, 13.15.
C
26H17F3N2O4 (M+Na)+, 501.1033. Found: 501.1014.
5.1.30. 2-[1-[4-[[4-(Trifluoromethoxy)benzoyl]amino]
benzoyl]isoquinolin-4-yl]acetic acid (15-24)
5.1.22. 2-[1-[4-(Pyrimidine-2-carbonylamino)benzoyl]isoqui-
nolin-4-yl]acetic acid (15-15)
Pale yellow powder; mp 165.0–168.0 °C; 1H NMR (DMSO-d6): d
4.17 (s, 2H), 7.51–8.17 (m, 12H), 8.54 (s, 1H), 10.75 (s, 1H), 12.79
(br s, 1H); MS (ESI/APCI Dual) m/z 495 (M+H)+; Anal. Calcd for
Pale yellow powder; mp 187.5–189.0 °C; 1H NMR (DMSO-d6): d
4.14 (s, 2H), 7.66–8.13 (m, 9H), 8.51 (s, 1H), 9.01–9.05 (m, 2H),
11.10 (br s, 1H), 12.64 (br s, 1H); MS (ESI/APCI Dual) m/z 413
(M+H)+; Anal. Calcd for C23H16N4O4ꢀ0.2H2O: C, 66.41; H, 3.97; N,
13.47. Found: C, 66.47; H, 4.02; N, 13.15.
C26H17F3N2O5: C, 63.16; H, 3.47; N, 5.67. Found: C, 62.90; H,
3.72; N, 5.52.

7682
R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. 21 (2013) 7674–7685
5.1.31. 2-[1-[4-[(4-Cyanobenzoyl)amino]benzoyl]isoquinolin-4-
yl]acetic acid (15-25)
5.1.38. 2-[1-[4-[[(E)-3-(3,4-Dichlorophenyl)prop-2-
enoyl]amino]benzoyl]isoquinolin-4-yl]acetic acid (15-32)
Colorless powder; mp 172.0–174.0 °C; 1H NMR (DMSO-d6): d
4.16 (s, 2H), 6.92 (d, J = 15.1 Hz, 1H), 7.58–8.17 (m, 12H), 8.53 (s,
1H), 10.68 (s, 1H); MS (ESI/APCI Dual) m/z 505 (M+H)+; Anal. Calcd
for C27H18Cl2N2O4ꢀ0.1H2O: C, 63.94; H, 3.62; N, 5.52. Found: C,
63.76; H, 3.70; N, 5.46.
Colorless powder; mp 192.0–193.5 °C; 1H NMR (DMSO-d6): d
4.17 (s, 2H), 7.70–7.74 (m, 1H), 7.86–7.92 (m, 3H), 7.96–8.00 (m,
2H), 8.02–8.07 (m, 3H), 8.11–8.16 (m, 3H), 8.54 (s, 1H), 10.88 (br
s, 1H); MS (ESI/APCI Dual) m/z 436 (M+H)+; HRMS m/z Calcd for
C
26H17N3O4 (M+Na)+, 458.1111. Found: 458.1098.
5.1.32. 2-[1-[4-[(4-Nitrobenzoyl)amino]benzoyl]isoquinolin-4-
yl]acetic acid (15-26)
5.1.39. 2-[1-[4-[(3,4-Dichlorophenyl)carbamoylamino]
benzoyl]isoquinolin-4-yl]acetic acid (15-33)
Colorless powder; mp 193.5–194.5 °C; 1H NMR (DMSO-d6): d
4.19 (s, 2H), 7.70–7.74 (m, 1H), 7.87–7.93 (m, 3H), 7.96–8.01 (m,
2H), 8.03–8.06 (m, 1H), 8.13–8.16 (m, 1H), 8.19–8.23 (m, 2H),
8.37–8.41 (m, 2H), 8.55 (s, 1H), 10.96 (br s, 1H), 12.70 (br s, 1H);
MS (ESI/APCI Dual) m/z 456 (M+H)+; Anal. Calcd for C25H17N3O6-
ꢀ0.3H2O: C, 65.16; H, 3.85; N, 9.12. Found: C, 65.07; H, 3.85; N, 8.93.
To a solution of 13 in DMF (3 ml) was added a solution of 3,4-
dichlorophenyl isocyanate (282 mg, 1.50 mmol) in DMF (2 ml) at
0 °C. The reaction mixture was stirred at room temperature for
1 h. Then the mixture was diluted with EtOAc, and the organic
layer was washed with brine, dried over MgSO4 and concentrated
under reduced pressure. The residue was purified by silica gel col-
umn chromatography (60 N) eluting with 0–70% EtOAc/CHCl3 to
give the target intermediate as a colorless powder (287 mg, 56%).
To a solution of this intermediate (287 mg, 0.564 mmol) in THF
(6 ml) was added 1 N NaOH (3 ml), and the mixture was stirred
for 18 h at room temperature. The mixture was acidified with di-
luted hydrochloric acid and extracted with CHCl3. The organic layer
was dried over MgSO4 and concentrated under reduced pressure to
give 15-33 (489 mg, 77%) as a yellowish powder: mp 157.0–
159.0 °C; 1H NMR (DMSO-d6): d 4.17 (s, 2H), 7.33–8.17 (m, 11H),
8.53 (s, 1H), 9.19 (br s, 1H), 9.42 (br s, 1H), 12.67 (br s, 1H) ; MS
(ESI/APCI Dual) m/z 494 (M+H)+; Anal. Calcd for C25H17Cl2N3O4-
ꢀ0.6H2O: C, 59.44; H, 3.63; N, 8.32. Found: C, 59.46; H, 3.72; N, 8.22.
5.1.33. 2-[1-[4-[[4-(Dimethylamino)benzoyl]amino]-
benzoyl]isoquinolin-4-yl]acetic acid (15-27)
Yellowish powder; mp 170.5–172.0 °C; 1H NMR (DMSO-d6): d
3.00 (s, 6H), 4.18 (s, 2H), 6.75–6.79 (m, 2H), 7.69–7.73 (m, 1H),
7.80–7.84 (m, 2H), 7.87–7.92 (m, 3H), 7.95 -7.99 (m, 2H), 8.00–
8.04 (m, 1H), 8.12–8.15 (m, 1H), 8.53 (s, 1H), 10.27 (br s, 1H),
12.69 (br s, 1H); MS (ESI/APCI Dual) m/z 454 (M+H)+; Anal. Calcd
for C27H23N3O4ꢀ0.3H2O: C, 70.67; H, 5.18; N, 9.16. Found: C,
70.62; H, 5.10; N, 9.06.
5.1.34. 2-[1-[4-[(4-Phenylbenzoyl)amino]benzoyl]isoquinolin-
4-yl]acetic acid (15-28)
Colorless powder; mp 213.5–215.0 °C; 1H NMR (DMSO-d6): d
4.19 (s, 2H), 7.42–7.46 (m, 1H), 7.50–7.54 (m, 2H), 7.70–7.74 (m,
1H), 7.75–7.79 (m, 2H), 7.84–7.93 (m, 5H), 7.99–8.06 (m, 3H),
8.07–8.11 (m, 2H), 8.13–8.16 (m, 1H), 8.55 (s, 1H), 10.70 (br s,
1H), 12.65 (br s, 1H); MS (ESI/APCI Dual) m/z 487 (M+H)+; Anal.
Calcd for C31H22N2O4ꢀ0.2H2O: C, 75.97; H, 4.61; N, 5.72. Found: C,
75.89; H, 4.66; N, 5.66.
5.1.40. 2-[1-[4-[(3,4-Dichlorobenzoyl)-methylamino]benzoyl]-
isoquinolin-4-yl]acetic acid (15-34)
To a suspension of sodium hydride (48 mg, 60% in oil, 1.2 mmol)
in THF (4 ml) was added 15-20 (190 mg, 0.396 mmol), and the
mixture was stirred at room temperature for 1 h. To the mixture
was added iodomethane (0.5 ml, 1.55 mmol), and the resulting
mixture was stirred at room temperature for 3 h. The reaction
was quenched with 2 N hydrochloric acid, and the mixture was ex-
tracted with EtOAc. The organic layer was washed with brine, dried
over MgSO4, and concentrated under reduced pressure. The crude
residue was crystallized from EtOH to yield 15-34 (60 mg, 31%)
as a colorless powder: mp 173.0–175.0 °C; 1H NMR (DMSO-d6): d
3.42 (s, 3H), 4.17 (s, 2H), 7.22–7.26 (m, 1H), 7.37–7.41 (m, 2H),
7.54–7.57 (m, 1H), 7.62–7.64 (m, 1H), 7.69–7.74 (m, 1H),
7.76–7.80 (m, 2H), 7.88–7.92 (m, 1H), 8.04–8.07 (m, 1H), 8.12–
8.15 (m, 1H), 8.52 (s, 1H), 12.68 (br s, 1H); MS (ESI/APCI Dual)
m/z 493 (M+H)+; Anal. Calcd for C26H18Cl2N2O4: C, 63.30; H, 3.68;
N, 5.68. Found: C, 63.01; H, 3.84; N, 5.61.
5.1.35. 2-[1-[4-[(4-Phenoxybenzoyl)amino]benzoyl]isoqui-
nolin-4-yl]acetic acid (15-29)
Colorless powder; mp 159.0–160.0 °C; 1H NMR (DMSO-d6): d
4.16 (s, 2H), 7.09–7.14 (m, 4H), 7.22–7.26 (m, 1H), 7.44–7.49 (m,
2H), 7.69–7.73 (m, 1H), 7.83–7.92 (m, 3H), 7.95–7.99 (m, 2H),
8.00–8.05 (m, 3H), 8.13–8.17 (m, 1H), 8.53 (s, 1H), 10.59 (br s,
1H); MS (ESI/APCI Dual) m/z 503 (M+H)+; HRMS m/z Calcd for
C
31H22N2O5 (M+Na)+, 525.1421. Found: 525.1386.
5.1.36. 2-[1-[4-[[2-(3,4-Dichlorophenyl)acetyl]amino]
benzoyl]isoquinolin-4-yl]acetic acid (15-30)
5.1.41. 2-[1-[4-[(3,4-Dichlorophenyl)methoxycarbonylamino]-
benzoyl]isoquinolin-4-yl]acetic acid (15-35)
Colorless powder; mp 194.0–197.0 °C; 1H NMR (DMSO-d6): d
3.76 (s, 2H), 4.16 (s, 2H), 7.31–7.34 (m, 1H), 7.58–7.62 (m, 2H),
7.68–7.72 (m, 1H), 7.73–7.77 (m, 2H), 7.80–7.83 (m, 2H),
7.87–7.91 (m, 1H), 7.99–8.02 (m, 1H), 8.11–8.14 (m, 1H), 8.52 (s,
1H), 10.62 (br s, 1H), 12.67 (br s, 1H); MS (ESI/APCI Dual) m/z
493 (M+H)+; Anal. Calcd for C26H18Cl2N2O4ꢀ0.4H2O: C, 62.39; H,
3.79; N, 5.60. Found: C, 62.35; H, 3.80; N, 5.54.
To a solution of 13 (320 mg, 1.00 mmol) in CHCl3 (5 ml) were
added pyridine (0.324 ml, 4.00 mmol) and phenyl chloroformate
(0.189 ml, 1.50 mmol) at 0 °C. The reaction mixture was stirred
at room temperature for 1 h. Then the mixture was concentrated
under reduced pressure. The crude residue was used in the next
step without further purification.
To a solution of the crude residue (440 mg) in THF (10 ml) were
added 3,4-dichlorobenzylalcohol (354 mg, 2.0 mmol) and
N,N-diisopropylethylamine (0.680 ml, 4.0 mmol), and the mixture
was stirred at reflux temperature for 8 h. To the mixture was added
3,4-dichlorobenzylalcohol (354 mg, 2.0 mmol), and the mixture
was stirred at reflux temperature for 8 h to complete the reaction.
After cooling to room temperature, the reaction mixture was diluted
with EtOAc, and the organic layer was washed with H2O and brine,
dried over MgSO4 and concentrated under reduce pressure. The res-
idue was purified by silica gel column chromatography (60 N)
5.1.37. 2-[1-[4-[3-(3,4-Dichlorophenyl)propanoylamino]
benzoyl]isoquinolin-4-yl]acetic acid (15-31)
Orange amorphous; 1H NMR (DMSO-d6): d 2.70 (t, J = 7.6 Hz,
2H), 2.92 (t, J = 7.6 Hz, 2H), 4.17 (s, 2H), 7.24–7.27 (m, 1H), 7.52–
7.56 (m, 2H), 7.68–7.75 (m, 3H), 7.78–7.82 (m, 2H), 7.87–7.91
(m, 1H), 7.99–8.02 (m, 1H), 8.11–8.15 (m, 1H), 8.52 (s, 1H), 10.36
(br s, 1H), 12.68 (br s, 1H); MS (ESI/APCI Dual) m/z 507 (M+H)+;
Anal. Calcd for C27H20Cl2N2O4ꢀ1.4H2O: C, 60.89; H, 4.31; N, 5.26.
Found: C, 60.89; H, 4.28; N, 5.01.

R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. 21 (2013) 7674–7685
7683
eluting with 0–50% EtOAc/hexane to give a target intermediate as a
pale yellow amorphous (280 mg, 0.535 mmol, 54%).
After cooling, the reaction mixture was diluted with H2O and ex-
tracted with CHCl3. The organic layer was dried over MgSO4 and
concentrated under reduced pressure to give the crude product
(3.39 g, 96%).
To a solution of the above intermediate (280 mg, 0.54 mmol) in
THF (6 ml) was added 1 N aqueous NaOH (3 ml), and the mixture
was stirred for 2 h at room temperature. The mixture was acidified
with diluted hydrochloric acid and extracted with CHCl3. The or-
ganic layer was dried over MgSO4 and concentrated under reduced
pressure to give 15-35 (235 mg, 86%) as a pale yellow powder: mp
159.0–161.0 °C; 1H NMR (DMSO-d6): d 4.16 (s, 2H), 5.19 (s, 2H),
7.41–8.17 (m, 11H), 8.52 (s, 1H), 10.35 (s, 1H), 12.73 (br s, 1H);
MS (ESI/APCI Dual) m/z 509 (M+H)+; Anal. Calcd for C26H18Cl2N2O5-
ꢀ0.8H2O: C, 59.62; H, 3.77; N, 5.35. Found: C, 59.68; H, 3.62; N, 5.34.
To a solution of the crude product (100 mg, 0.286 mmol) in
CHCl3 (3 ml) were added oxalylchloride (0.051 ml, 0.572 mmol)
and DMF (1 drop), and the mixture was stirred at room tempera-
ture for 1 h. Then the reaction mixture was concentrated under re-
duced pressure. To a solution of the residue in CHCl3 (3 ml) were
added 4-chloro-aniline (55 mg, 0.429 mmol) and pyridine
(0.060 ml, 0.429 mmol), and the mixture was stirred at room tem-
perature for 14 h. The reaction was quenched by adding a satu-
rated aqueous ammonium chloride solution, and the separated
organic layer was dried over MgSO4 and concentrated under re-
duced pressure. The residue was purified by silica gel column chro-
matography (OH) eluting with 30–60% EtOAc/hexane and
crystallized from EtOAc/hexane to give the target intermediate
(100 mg, 76%).
To a solution of the above intermediate (100 mg, 0.218 mmol)
in THF (3 ml) was added 1 N NaOH (1.5 ml), and the mixture
was stirred for 1 h at room temperature. The mixture was acidified
with diluted hydrochloric acid at 0 °C and extracted with EtOAc.
The organic layer was washed with brine, dried over MgSO4 and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (OH) eluting with 0–15%
MeOH/CHCl3 to give 15-38 (72 mg, 74%) as a colorless powder:
mp 184.0–187.0 °C; 1H NMR (DMSO-d6): d 4.19 (s, 2H), 7.41–
8.24 (m, 12H), 8.56 (s, 1H), 10.58 (s, 1H), 12.75 (br s, 1H); MS
(ESI/APCI Dual) m/z 445 (M+H)+; Anal. Calcd for C25H17ClN2O4-
ꢀ0.5H2O: C, 66.16; H, 4.00; N, 6.17. Found: C, 66.06; H, 3.96; N,
6.14.
5.1.42. 2-[1-[4-[(3,4-Dichlorophenyl)methylamino]benzoyl]-
isoquinolin-4-yl]acetic acid (15-36)
To a solution of 13 (180 mg, 0.562 mmol) and 3,4-dichloroben-
zyl bromide (162 mg, 0.674 mmol) in DMF (6 ml) was added potas-
sium carbonate (93 mg, 0.674 mmol), and the mixture was stirred
at reflux temperature for 5 h. To the mixture was added potassium
carbonate (162 mg, 1.17 mmol), and the mixture was further
stirred at the same temperature for 1 h. After cooling to room
temperature, the mixture was diluted with EtOAc. The organic
layer was washed with H2O and brine, dried over MgSO4 and con-
centrated under reduced pressure. The residue was purified by sil-
ica gel column chromatography (60 N) eluting with 0–50% EtOAc/
hexane to give the intermediate as a yellow amorphous (94 mg,
35%).
To a solution of the above intermediate (94 mg, 0.196 mmol) in
THF (2 ml) was added 1 N NaOH (1 ml), and the mixture was stir-
red for 2 h at room temperature. The mixture was acidified with di-
luted hydrochloric acid and extracted with CHCl3. The organic layer
was dried over MgSO4 and concentrated under reduced pressure to
give 15-36 (53 mg, 58%) as a yellowish powder: mp 115.0–
116.5 °C; 1H NMR (DMSO-d6): d 4.11 (s, 2H), 4.37–4.41 (m, 2H),
6.61–6.66 (m, 2H), 7.29–8.12 (m, 10H), 8.44–8.47 (m, 1H), 12.72
(br s, 1H); MS (ESI/APCI Dual) m/z 465 (M+H)+; Anal. Calcd for C25-
H18Cl2N2O3ꢀ0.5H2O: C, 63.30; H, 4.04; N, 5.91. Found: C, 63.24; H,
3.98; N, 5.82.
5.1.45. 2-[1-[4-[(3,4-Dichlorophenyl)carbamoyl]benzoyl]isoqui-
nolin-4-yl]acetic acid (15-39)
Pale yellow powder; mp 157.5–158.5 °C; 1H NMR (DMSO-d6): d
4.17 (br s, 2H), 7.62–8.56 (m, 12H), 10.72 (br s, 1H); MS (ESI/APCI
Dual) m/z 479 (M+H)+; Anal. Calcd for C25H16Cl2N2O4ꢀ0.3H2O: C,
61.95; H, 3.45; N, 5.78. Found: C, 61.89; H, 3.57; N, 5.74.
5.1.43. 2-[1-[4-[(3,4-Dichlorophenyl)sulfonylamino]benzoyl]-
isoquinolin-4-yl]acetic acid (15-37)
5.1.46. 2-[1-[4-[2-(4-Chlorophenyl)ethylcarbamoyl]benzoyl]
isoquinolin-4-yl]acetic acid (15-40)
To a solution of 13 (320 mg, 1.00 mmol) in pyridine (5 ml) was
added 3,4-dichlorobenzenesulfonyl chloride (368 mg, 1.50 mmol).
After being stirred at room temperature for 1 h, the mixture was
diluted with EtOAc, and the organic layer was washed with brine,
dried over MgSO4, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (60 N)
eluting with 0–40% EtOAc/CHCl3 to give the target intermediate
as a pale yellow amorphous (207 mg, 39%).
To a solution of the above intermediate (207 mg, 0.391 mmol)
in THF (4 ml) was added 1 N NaOH (2 ml), and the mixture was
stirred for 18 h at room temperature. The mixture was acidified
with diluted hydrochloric acid and extracted with CHCl3. The or-
ganic layer was dried over MgSO4 and concentrated under reduced
pressure to give 15-37 (24 mg, 12%) as a colorless powder: mp
125.5–127.5 °C; 1H NMR (DMSO-d6): d 4.16 (s, 2H), 7.23–7.27 (m,
2H), 7.66–8.14 (m, 9H), 8.49 (s, 1H), 11.16 (br s, 1H), 12.67 (br s,
1H); MS (ESI/APCI Dual) m/z 515 (M+H)+; Anal. Calcd for C24H16Cl2-
N2O5Sꢀ1.7H2O: C, 52.80; H, 3.58; N, 5.13. Found: C, 52.90; H, 3.30;
N, 4.96.
Colorless powder; mp 172.0–174.0 °C; 1H NMR (DMSO-d6): d
2.83–2.88 (m, 2H), 3.47–3.53 (m, 2H), 4.18 (s, 2H), 7.24–8.77 (m,
14H), 12.74 (br s, 1H); MS (ESI/APCI Dual) m/z 473 (M+H)+; Anal.
Calcd for C27H21ClN2O4: C, 68.57; H, 4.48; N, 5.92. Found: C,
68.49; H, 4.53; N, 5.88.
5.1.47. 2-[1-[4-[2-(3,4-Dichlorophenyl)ethylcarbamoyl]-
benzoyl]isoquinolin-4-yl]acetic acid (15-41)
Colorless powder; mp 178.5–179.5 °C; 1H NMR (DMSO-d6): d
2.85–2.90 (m, 2H), 3.50–3.55 (m, 2H), 4.18 (br s, 2H), 7.21–8.77
(m, 13H), 12.69 (br s, 1H); MS (ESI/APCI Dual) m/z 507 (M+H)+;
Anal. Calcd for C27H20Cl2N2O4: C, 63.92; H, 3.97; N, 5.52. Found:
C, 63.59; H, 4.05; N, 5.52.
5.1.48. 2-[1-[4-[(3,4-Dichlorophenyl)methoxy]
benzoyl]isoquinolin-4-yl]acetic acid (15-42)
To a solution of 12c (1.03 g, 3.07 mmol) in CHCl3 (30 ml) was
added a solution of boron tribromide (3.70 ml, 5.58 mmol) in CHCl3
(30 ml) at 0 °C and the mixture was stirred for 2 h. The reaction
was quenched by adding MeOH, and the organic layer was washed
with H2O, dried over MgSO4 and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatogra-
phy (60 N) eluting with 0–5% MeOH/CHCl3 to give the target
compound as a yellowish powder (463 mg, 47%).
5.1.44. 2-[1-[4-[(4-Chlorophenyl)carbamoyl]benzoyl]isoqui-
nolin-4-yl]acetic acid (15-38)
To a solution of 12b (4.09 g, 10.1 mmol) in CHCl3 (200 ml) was
added TFA (33 ml), and the mixture was stirred at 50 °C for 1 h.

7684
R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. 21 (2013) 7674–7685
To
a
solution of the above target compound (200 mg,
(DMSO-d6): 1.75 (s, 6H), 3.55 (s, 3H), 7.68–8.17 (m, 8H), 8.66 (s,
1H).
0.622 mmol) in DMF (5 ml) were added 3,4-dichlorobenzylamine
(0.111 ml, 0.747 mmol) and potassium carbonate (103 mg,
0.747 mmol), and the mixture was stirred at 80 °C for 4 h. After
cooling to room temperature, the reaction mixture was diluted
with EtOAc. The organic layer was washed with H2O and brine,
dried over MgSO4 and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (60 N)
eluting with 0–50% EtOAc/hexane to give a yellowish amorphous
(70 mg, 0.146 mmol, 23%).
To a solution of the above intermediate (70 mg, 0.146 mmol) in
a mixture of THF (2 ml) and MeOH (1 ml) was added 1 N NaOH
(1 ml), and the mixture was stirred for 2 h at room temperature.
The mixture was acidified with hydrochloric acid at 0 °C and ex-
tracted with EtOAc. The organic layer was washed with brine, dried
over MgSO4 and concentrated under reduced pressure. The residue
was purified by OH-silica gel plate eluting with MeOH/CHCl3
(10:90) to give 15-42 (33 mg, 48%) as a colorless amorphous: 1H
NMR (DMSO-d6): d 4.12–4.15 (m, 2H), 5.24 (s, 2H), 7.14–8.16 (m,
11H), 8.50 (s, 1H); MS (ESI/APCI Dual) m/z 466 (M+H)+; Anal. Calcd
for C25H17Cl2N2O4ꢀ1.1H2O: C, 61.77; H, 3.98; N, 2.88. Found: C,
61.50; H, 3.72; N, 2.81.
5.1.53. Methyl 2-[1-(4-{2-[(4-chlorophenyl)ethylamino]car-
bonyl}phenylcarbonyl)isoquinolin-4-yl]-2-methylpropinate
(18).
To a suspension of 17 (128 mg, 0.321 mmol), 2-(4-chlo-
rophenyl)ethylamine (0.054 ml, 0.385 mmol), and 1-hydroxyben-
zotriazole (74 mg, 0.482 mmol) in CHCl3 (1.5 ml) was added
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(92 mg, 0.482 mmol), and the mixture was stirred at room temper-
ature for 3 h. The mixture was washed with H2O, dried over MgSO4
and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (NH) eluting with
60–100% hexane/EtOAc to give 18 (73 mg, 44%) as a colorless
amorphous: 1H NMR (CDCl3): d 1.84 (s, 6H), 2.91–2.95 (m, 2H),
3.63 (s, 3H), 3.69–3.75 (m, 2H), 6.10–6.15 (m, 1H), 7.15–8.33 (m,
12H), 8.64 (s, 1H); MS (ESI/APCI Dual) m/z 515 (M+H)+.
5.1.54. 2-[1-[4-[2-(4-Chlorophenyl)ethylcarbamoyl]benzoyl]iso-
quinolin-4-yl]-2-methylpropanoic acid (15-45).
Pale yellow
powder; mp 105.0–106.0 °C; 1H NMR (DMSO-d6): d 1.76 (s, 6H),
2.83–2.89 (m, 2H), 3.47–3.53 (m, 2H), 7.24–8.77 (m, 14H), 12.80
(br s, 1H) ; MS (ESI/APCI Dual) m/z 501 (M++H)+; Anal. Calcd for C29-
H25ClN2O4ꢀ0.5H2O: C, 68.30; H, 5.14; N, 5.49. Found: C, 68.20; H,
5.42; N, 5.28.
5.1.49. 2-[1-[4-[2-(4-Chlorophenyl)ethoxy]benzoyl]isoquinolin-
4-yl]acetic acid (15-43)
Colorless amorphous; 1H NMR (DMSO-d6): d 3.06 (t, J = 6.6 Hz,
2H), 4.16 (s, 2H), 4.30 (t, J = 6.6 Hz, 2H), 7.04–8.14 (m, 12H), 8.51
(s, 1H), 12.69 (br s, 1H); MS (ESI/APCI Dual) m/z 446 (M+H)+; Anal.
Calcd for C26H20ClNO4ꢀ0.8H2O: C, 67.84; H, 4.73; N, 3.04. Found: C,
68.08; H, 4.80; N, 2.82.
5.1.55. 4-Bromo-1-{4-(methoxycarbonyl)phenylcarbonyl}iso-
quinoline (20).
This compound was prepared from 4-bro-
mo-1-chloroisoquinoline and methyl 4-(cyanomethyl)benzoate in
a similar manner as described for the synthesis of 10: 1H NMR
(CDCl3): d 3.96 (s, 3H), 7.71–7.75 (m, 1H), 7.87–7.91 (m, 1H),
7.99–8.02 (m, 2H), 8.12–8.15 (m, 2H), 8.30–8.34 (m, 2H), 8.78–
8.82 (m, 1H).
5.1.50. 2-[1-[4-[3-(4-Chlorophenyl)propoxy]benzoyl]isoqui-
nolin-4-yl]acetic acid (15-44)
Colorless amorphous; 1H NMR (DMSO-d6): d 2.00–2.07 (m, 2H),
2.72–2.76 (m, 2H), 4.07 (t, J = 6.4 Hz, 2H), 4.15 (s, 2H), 7.03–8.15
(m, 12H), 8.51 (s, 1H); MS (ESI/APCI Dual) m/z 460 (M+H)+; Anal.
Calcd for C27H22ClNO4ꢀ0.7H2O: C, 68.63; H, 4.99; N, 2.96. Found:
C, 68.73; H, 5.07; N, 2.672.
5.1.56. 1-{4-(Methoxycarbonyl)phenylcarbonyl}-4-(3,3,5,5,-tet-
ramethyl-2,5-dioxaborolidin-1-y1)isoquinoline (21).
To a
suspension of 20 (6.00 g, 16.2 mmol), bis(pinacolato)diboron
(41.1 g, 162 mmol), and potassium acetate (19.0 g, 194 mmol) in
DMF (120 ml) was added PdCl2(dppf) (1.32 g, 1.62 mmol), and
the mixture was stirred at 80 °C for 2 h. After the mixture being
cooled to room temperature, the mixture was diluted with H2O,
and extracted with EtOAc, The organic layer was washed with
brine, dried over MgSO4, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(OH) eluting 10–20% hexane/EtOAc to give 21 (3.72 g, 55%) as a
pale yellow powder: 1H NMR (CDCl3): d 1.46 (s, 12H), 3.95 (s,
3H), 7.58–7.63 (m, 1H), 7.77–7.81 (m, 1H), 7.96–8.01 (m, 2H),
8.09–8.13 (m, 2H), 8.16–8.20 (m, 1H), 8.78–8.82 (m, 1H), 9.03 (s,
1H).
5.1.51. tert-Butyl 4-[4-(2-methoxy-1,1-dimethyl-2-oxo-
ethyl)isoquinoline-1-carbonyl]benzoate (16)
To a solution of 12b (181 mg, 0.446 mmol) in DMF (3.6 ml)
was added sodium hydride (38 mg, 0.938 mmol) at 0 °C, and
the mixture was stirred for 10 min. To the mixture was added
iodomethane (0.058 ml, 0.938 mmol), and the reaction mixture
was stirred for 1.5 h. The reaction was quenched by adding sat-
urated ammonium chloride solution, and the mixture was ex-
tracted with EtOAc. The organic layer was dried over MgSO4
and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (OH) eluting
10–30% EtOAc/hexane to give 16 (139 mg, 0.321 mmol, 72%)
as colorless amorphous: 1H NMR (CDCl3): d 1.61 (s, 9H), 1.84
(s, 6H), 3.63 (s, 3H), 7.59–8.31 (m, 8H), 8.65 (s, 1H); MS (ESI/
APCI Dual) m/z 434 (M+H)+.
5.1.57. 4-Hydroxy-1-{4-(methoxycarbonyl)phenylcarbonyl}iso-
quinoline (22).
To a suspension of 21 (3.70 g, 8.87 mmol) and
sodium bicarbonate (5.22 g, 62.1 mmol) in a mixture of acetone
(30 ml) and H2O (30 ml) was added oxone (8.18 g, 13.3 mmol) at
0 °C. The reaction mixture was stirred at 0 °C for 1.5 h and then
at room temperature for 30 min. To the mixture was added 1 N
aqueous HCl, and the mixture was extracted with EtOAc. The
organic layer was washed with brine, dried over MgSO4 and con-
centrated under reduced pressure. The residue was purified by sil-
ica gel column chromatography (OH) eluting 50–100% hexane/
EtOAc and then 10% MeOH/CHCl3 to give 22 (260 mg, 9%) as a yel-
low solid: 1H NMR (DMSO-d6): d 3.91 (s, 3H), 7.75–7.83 (m, 2H),
7.93–7.96 (m, 2H), 8.06–8.09 (m, 2H), 8.16 (s, 1H), 8.27–8.30 (m,
1H), 8.52–8.55 (m, 1H); MS (ESI/APCI Dual) m/z 308 (M+H)+.
5.1.52. Methyl 2-[1-(4-carboxyphenylcarbonyl)isoquinolin-4-
yl]-2-methylpropinate (17)
To a solution of 16 (139 mg, 0.321 mmol) in CHCl3 (1.5 ml) was
added TFA (0.7 ml) at 0 °C, and the mixture was stirred at room
temperature for 17 h. The reaction mixture was concentrated un-
der reduced pressure, and the residue was neutralized by 1 N
NaOH, and extracted with EtOAc The organic layer was dried over
MgSO4 and concentrated under reduced pressure to give 17
(128 mg, 0.339 mmol, quant) as a colorless amorphous: 1H NMR

R. Nishikawa-Shimono et al. / Bioorg. Med. Chem. 21 (2013) 7674–7685
7685
5.1.58. tert-Butyl 1-[4-(methoxycarbonyl)phenylcarbonyl]iso-
quinolin-4-yloxyacetate (23). To a solution of 22 (254 mg,
ter Top Count NXT (Perkin Elmer, Boston, MA, USA) using a liquid
scintillation cocktail Micro-scinti-O (Packard, Meriden, CT, USA).
0.827 mmol) in DMF (5 ml) were added tert-butyl 2-bromoacetate
(242 mg, 1.24 mmol) and K2CO3 (343 mg, 2.48 mmol), and the mix-
ture was stirredat room temperaturefor 1.5 h. Then the mixture was
diluted with EtOAc, and the organic layer was washed with brine,
dried over MgSO4 and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (OH) elut-
ing with 10–20% hexane/EtOAc to give 23 (328 mg, 94%) as a pale
yellow powder: 1H NMR (CDCl3): d 1.53 (s, 9H), 3.96 (s, 3H), 4.85
(s, 2H), 7.69–7.80 (m, 2H), 7.99–8.02 (m, 2H), 8.03 (s, 1H), 8.12–
8.15 (m, 2H), 8.42–8.52 (m, 2H); MS (ESI/APCI Dual) m/z 422 (M+H)+.
5.2.2. Intracellular Ca2+ influx assay.
tant KB8 cells (BML Co. Kawagoe, Japan) were incubated with 1
Fluo-4 AM for 30 min at 37 °C in the dark. After incubation, the cells
HumanCRTH2transfec-
lM
were washed and suspended in HBSS containing 10 mM HEPES, pH
7.3, 1 mM CaCl2 (2 ꢁ 105cells/100
ll). The compound and 100 nM
of PGD2 were added, and the increase of intracellularCa2+ concentra-
tion ([Ca2+]i) was measured using the functional drug screening sys-
tem FDSS6000 (Hamamatsu Photonics, Shizuoka, Japan).
5.2.3. Th2 cells migration assay.
Human Th2 cells were pre-
5.1.59. tert-Butyl 1-[4-(carboxy)phenylcarbonyl]isoquinolin-4-
pared as follows: the CD4+T lymphocytes separated from PBMCs
using anti-CD4 mAb were stimulated with anti-CD3 mAb and
yloxyacetate (24).
To a solution of 23 (100 mg, 0.237 mmol)
in THF (6 ml) was added 0.2 M aqueous NaOH, and the mixture
was stirred at room temperature for 1.5 h. The mixture was neu-
tralized with saturated ammonium chloride solution, and the mix-
ture was extracted with EtOAc. The organic layer was dried over
MgSO4 and concentrated under reduced pressure to give 24
(40 mg, 48%), which was used in the next reaction without further
purification: 1H NMR (CDCl3): d 1.43 (s, 9H), 4.83–4.87 (m, 2H),
7.70–7.81 (m, 2H), 7.99–8.20 (m, 5H), 8.42–8.56 (m, 2H); MS
(ESI/APCI Dual) m/z 408 (M+H)+.
anti-CD28 mAb in the presence of IL-4 and neutralizing anti-IFNc
mAb for 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 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 5 lm-pore filter Chemo Tx-96 chamber (Neuroprobe, Gai-
thersburg, MD, USA), respectively. After incubation at 37 °C for 1
hr, number of the cells in the bottom wells were counted by Bur-
ker–Turk hemocytometer.
5.1.60. tert-Butyl 1-(4-{2-[(4-chlorophenyl)ethylamino]car-
bonyl}phenylcarbonyl)isoquinolin-4-yloxyacetate (25).
A
References and notes
mixture of 24 (19 mg, 0.047 mmol), 4-chlorophenethylamine
(15 mg, 0.094 mmol), WSC-HCl (18 mg, 0.094 mmol), and HOBT-
H2O (14 mg, 0.094 mmol) in CHCl3 (1 ml) was stirred at room
temperature for 1 h. Then the mixture was added H2O, extracted
with EtOAc, dried over MgSO4 and concentrated. The residue was
purified by silica gel column chromatography (OH, hexane/EtOAc
10–40%) to give 25 (15 mg, 59%) as a colorless powder: 1H NMR
(CDCl3): d 1.53 (s, 9H), 2.93 (t, J = 6.88 Hz, 2H), 3.69–3.74 (m,
2H), 4.85 (s, 2H), 6.11–6.15 (m, 1H), 7.15–7.19 (m, 2H), 7.28–
7.32 (m, 2H), 7.69–7.80 (m, 4H), 7.97–8.04 (m, 3H), 8.42–8.50
(m, 2H); MS (ESI/APCI Dual) m/z 545 (M+H)+.
1. Nagata, N.; Tanaka, K.; Ogawa, K.; Kemmotsu, K.; Imai, T.; Yoshie, O.; Abe, H.;
Tada, K.; Nakamura, M.; Sugamura, K.; Takano, K. J. Immunol. 1999, 162, 1278.
2. Nagata, K.; Hirai, H.; Tanaka, K.; Ogawa, K.; Aso, T.; Sugamura, K.; Nakamura,
M.; Takanoa, S. FEBS Lett. 1999, 459, 195.
3. Pettipher, R.; Hansel, T. T.; Armer, R. Nat. Rev. Drug Disc. 2007, 6, 313.
4. 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.
5. Monneret, G.; Gravel, S.; Diamond, M.; Rokach, J.; Powell, W. S. Blood 1942,
2001, 98.
6. Xue, L.; Gyles, S. L.; Wettey, F. R.; Gazi, L.; Townsend, E.; Hunter, M. G.;
Pettipher, R. J. Immunol. 2005, 175, 6531.
7. Woodward, D. F.; Jones, R. L.; Narumiya, S. Pharmacol. Rev. 2011, 63, 471.
8. Schuligoi, R.; Sturm, E.; Luschnig, P.; Konya, V.; Philipose, S.; Sedej, M.;
Waldhoer, M.; Peskar, B. A.; Heinemann, A. Pharmacology 2010, 85, 372.
9. Betancourt A. WO 2005/044260; Chem. Abstr. 2005, 142, 463599.
10. Birkinshaw, T. N.; Teague, S. J.; Beech, C.; Bonnert, R. V.; Hill, S.; Patel, A.;
Reakes, S.; Sanganee, H.; Dougall, I. G.; Phillips, T. T.; Salter, S.; Schmidt, J.;
Arrowsmith, E. C.; Carrillo, J. J.; Bell, F. M.; Paine, S. W.; Weaver, R. Bioorg. Med.
Chem. Lett. 2006, 16, 4287.
11. Bonnert R. WO 2004/106302; Chem. Abstr. 2012, 142, 23189.
12. Armer, R. E.; WO 2005/121141; Chem. Abstr. 2005, 144, 51458.
13. 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, 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.
14. Fretz, H. WO 2006/021418; Chem. Abstr. 2006, 144, 274271.
15. Bennani, Y. L. WO 2006/034418; Chem. Abstr. 2006, 144, 350544.
16. Hynd, G. WO 2006/136859; Chem. Abstr. 2006, 146, 100556.
17. 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.;
Filinchuk, Y.; Scheer, A.; Schwarz, M. K.; Chollet, A. J. Med. Chem. 2008, 51,
2227.
18. Trond, U.; Jean-Marie, R.; Marie, G.; Øystein, R.; Thomas, M. F.; Lars-Ole, G.;
Jesper, M. M.; Evi, K.; Lena, U.; Thomas, H. J. Med. Chem. 2006, 49, 6838.
19. Bonnert R. WO 2005/018529; Chem. Abstr. 2012, 142, 279943.
20. Molecular Operating Environment (MOE); Chemical Computing Group, Inc.
(CCG); Montreal, Quebec, Canada.
5.1.61. 2-[1-[4-[2-(4-Chlorophenyl)ethylcarbamoyl]benzoyl]iso-
quinolin-4-yl]oxyacetic acid (15-46).
To a solution of 25
(14 mg, 0.026 mmol) in THF (1 ml) was added 1 N aqueous NaOH
(0.5 ml), and the mixture was stirred for 16 h at room temperature.
The mixture was acidified with 1 N aqueous HCl and extracted
with EtOAc. The organic layer was dried over MgSO4 and concen-
trated under reduced pressure. The residue was crystallized to pro-
vide 15-46 (5 mg, 39%) as a colorless powder: 1H NMR (DMSO-d6):
d 2.86 (t, J = 7.11 Hz, 2H), 3.48–3.53 (m, 2H), 5.12 (s, 2H), 7.26–8.75
(m, 14H); MS (ESI/APCI Dual) m/z 489 (M+H)+.
5.2. In vitro pharmacological studies
5.2.1. Binding assay.
CRTH2 transfected cells were washed
and suspended in HBSS containing 10 mM HEPES, pH 7.3 (2 ꢁ 105-
cells/100 l
l). The cells were incubated with 5 nM 3H-PGD2 (GE
Healthcare, Little Chalfont, United Kingdom) for 60 min on ice.
The cells in suspension were rapidly filtered under vacuum
through a glass fiber filter plate GF/C (Whatman Inc., Clifton, NJ,
USA) using a cell harvester Filtermate (Packard, Meriden, CT,
USA). The filters were then washed four times with ice-cold phos-
phate buffered saline containing 0.1% bovine serum albumin.
Bound radio activity was measured with a liquid scintillation coun-
21. Kulp, S. S.; McGee, M. J. J. Org. Chem. 1983, 48, 4097.
22. Kirmse, W. Eur. J. Org. Chem. 2002, 14, 2193.
23. The binding assay for DP1 was run by Recerca Biosciences (Bothell, USA) using
the profiler service according to the manufacturer’s procedures.
24. 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.