Discovery of new chemical leads for prostaglandin D2 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.06.006

A series of Indomethacin analogs were synthesized and biologically evaluated. Among the compounds tested, N-(p-butoxy)benzoyl-2-methylindole-4- acetic acid 2 was discovered as a new chemical lead for a prostaglandin D ₂ (PGD₂) recept

Full text of DOI:10.1016/j.bmcl.2004.06.006

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4557–4562
Discovery of new chemical leads for prostaglandin D2
receptor antagonists
Kazuhiko Torisu,a,* Kaoru Kobayashi,a Maki Iwahashi,a Hiromu Egashira,a
Yoshihiko Nakai,a Yutaka Okada,a Fumio Nanbu,a Shuichi Ohuchida,b
Hisao Nakaia and Masaaki Todaa
aMinase Research Institute, Ono Pharmaceutical Co., Ltd, Shimamoto, Mishima, Osaka 618-8585, Japan
bFukui Research Institute, Ono Pharmaceutical Co., Ltd, Technoport, Yamagishi, Mikuni, Sakai, Fukui 913-8538, Japan
Received 24 May 2004; revised 4 June 2004; accepted 7 June 2004
Available online 2 July 2004
Abstract—A series of Indomethacin analogs were synthesized and biologically evaluated. Among the compounds tested, N-(p-
butoxy)benzoyl-2-methylindole-4-acetic acid 2 was discovered as a new chemical lead for a prostaglandin D2 (PGD2) receptor
antagonist. Structure–activity relationship data are also presented.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
role of the DP receptor remains unclear because of the
lack of potent and subtype-selective ligands. Here we
report on the discovery of a new chemical lead 2 for DP
receptor antagonists that were obtained from the
chemical modification of Indomethacin analogs 1a–c
(Fig. 1).
Coleman and co-workers proposed the existence of
specific receptors for thromboxane (TX), PGI, PGE,
PGF, and PGD namely the TP, IP, EP, FP, and DP
receptors, respectively.1 A number of specific ligands for
these receptors have already been described in the lit-
erature.2 Among them, the DP receptor is the newest
and the least characterized.3 PGD2 is the major
prostanoid released from mast cells after challenging
with IgE4 and it has also been shown to affect the sleep
cycle5 and body temperature.6 The discovery of a DP
selective receptor antagonist7 seems to offer significant
advantages, for investigating the role of this receptor in
the various pathologies described above. However, the
2. Chemistry
Synthesis of the compounds listed in Tables 1–4 is out-
lined in Schemes 1–3. Compounds 1a–c and 9a–b were
prepared as described in Scheme 1. p-Substituted phen-
ylhydrazines 11 were converted to the corresponding
hydrazones followed by N-acylation of the remaining
CO2H
CO2H
MeO
Me
Me
N
N
O
O
O
O
2
1a-c
Figure 1. Discovery of a new chemical lead for a DP receptor antagonist.
Keywords: Prostaglandin; DP receptor; Antagonist.
* Corresponding author. Tel.: +81-75-961-1151; fax: +81-75-962-9314; e-mail: torisu@ono.co.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.006

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K. Torisu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4557–4562
Table 1. Activity profile of Indomethacin and N-benzoyl-2-methyl-5-methoxyindole-3-acetic acids 2a–c
CO2H
MeO
Me
N
R
O
Compd
RBinding
Ki (lM)
mEP3
IC50 (lM)
mEP1
mEP2
mEP4
mDP
mDP
Indomethacin
>10
6.5
>10
>10
>10
>10
>10
>10
>10
>10
>10
NTa
1a
1b
1c
o- O
>10
>10
1.7
4.7
>10
>10
>10
>10
0.41
0.27
2.5
1.6
m- O
p- O
Using membrane fractions of CHO cells expressing the prostanoid receptors, the mouse (m) EP receptor or (m) DP receptor, Ki values were
determined by competitive binding assay, which was performed according to the method of Kiriyama et al.9 with some modifications.8 With regard
to the mDP receptor antagonist activity, IC50 values were determined based on the effects of the test compounds on the increase in intracellular
cAMP formation in the presence of BSA (bovine serum albumin) (0.1%) evoked by PGD2 in mDPreceptor expressing CHO cells.
a NT: not tested.
Table 2. Effect of a 2-substituent on the activity profiles
CO2H
MeO
R
N
O
O
Compd
RBinding
Ki (lM)
IC50 (lM)
mEP1
mEP2
mEP3
mEP4
mDP
mDP
3
H
>10
>10
3.8
8.9
4.7
2.9
>10
>10
>10
>10
>10
>10
2.2
NTa
1.6
NTa
1c
4
Me
Et
0.27
1.5
a NT: not tested.
Table 3. Effect of a 3-substituent on the activity profiles
R
MeO
Me
N
O
O
Compd
RBinding
Ki (lM)
IC50 (lM)
mEP1
>10
mEP2
>10
mEP3
>10
mEP4
mDP
mDP
4.0
CO2H
CO2H
5
>10
0.94
1c
>10
4.7
>10
>10
0.27
1.6
CO2H
6
7
>10
>10
5.7
2.7
>10
2.4
3.0
0.28
0.19
1.5
CO2H
1.5
4.2
0.91
CO2H
8
>10
5.2
>10
0.40
4.5
nitrogen to afford 12, which was cyclized using levulinic
acid to form the N-benzoyl indoles 1a–c and 9a–b.8
Compounds 3 and 10 were prepared as described in
Scheme 2. Demethylation of the 5-methoxy group of 13
gave 14, o-benzylation of which provided 15. N-Ben-
zoylation of 15 gave 16, catalytic hydrogenation of
which resulted in 10. Compound 3 was prepared from 17
according to the same procedures described for the
preparation of 10 from 13. Compounds 4–8 were pre-
pared from 18 according to the procedures described in

K. Torisu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4557–4562
Table 4. Effect of a 5-substituent on the activity profiles
4559
CO2H
4
3
R
2
5
6
Me
N
1
7
O
O
Compd
RBinding
Ki (lM)
IC50 (lM)
mEP1
mEP2
mEP3
mEP4
mDP
mDP
1c
9a
9b
10
OMe
H
>10
5.2
5.0
4.7
2.1
1.7
3.3
>10
>10
>10
>10
>10
>10
2.2
0.27
0.11
0.15
1.9
1.6
3.9
Me
OH
2.9
>10
>10
>10
CO2H
Me
R1
R1
R1
a, b
c, d
N
Me
NH2
N
N
N
R2
H
R2
O
O
11
1a-c and 9a-b
12
Scheme 1. Synthesis of compounds 1a–c and 9a–b. Reagents: (a) CH3CHO, toluene; (b) ArCOCl, pyridine, CH2Cl2; (c) 4 N HCl, 1,4-dioxane;
(d) levulinic acid, AcOH.
CO2R3
CO2R3
R2
1RO
1RO
c
R2
N
N
H
O
O
13 : R1 = Me, R2 = Me, R3 = H
14 : R1 = H, R2 = Me, R3 = H
15 : R1 = Bn, R2 = Me, R3 = Bn
16 : R1 = Bn, R2 = Me, R3 = Bn
10 : R1 = H, R2 = Me, R3 = H
3 : R1 = Me, R2 = H, R3 = H
a
b
d
17 : R1 = Me, R2 = H, R3 = H
Scheme 2. Synthesis of 3 and 10. Reagents: (a) HCl–pyridine, 180 °C; (b) benzyl bromide, K2CO3, DMF; (c) NaH, p-nbutoxybenzoyl chloride, DMF;
(d) H2, Pd–C, i-PrOH, EtOAc.
2 R2 OC
MeO
(CH )
2 n
MeO
NH2
a
R1
N
N
O
O
O
O
18
4 : n = 1, R1 = Et, R2 = H
6 : n = 2, R1 = Me, R2 = H
7 : n = 3, R1 = Me, R2 = H
8 : n = 4, R1 = Me, R2 = H
19 : n = 0, R1 = Me, R2 = allyl
5 : n = 0, R1 = Me, R2 = H
O
O
O
Et
CO2H
CO2H
Me
(CH )
2
20
b
21a : n = 1
21b : n = 2
21c : n = 3
O
Me
Oallyl
22
Scheme 3. Synthesis of 4–8. Reagents: (a) 20 or 21a–c or 22, AcOH; (b) morpholine, Pd(PPh3)4, THF.

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K. Torisu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4557–4562
R
OH
R
a
e
Me
Me
Me
N
N
N
H
H
23
O
O
24 : R = OTf
b
c
d
25 : R = CO2Me
26 : R = CO2H
27 : R = CO2Bn
28 : R = CO2Bn
29 : R = CO2H
f
g,h,i
30 : R =
2 : R1 =
CO2Bn
CO2H
f
Scheme 4. Synthesis of 2. Reagents: (a) Tf2O, 2,6-lutidine, CH2Cl2, )78 °C; (b) Pd(PPh3)4, CO, TEA, MeOH, DMF; (c) NaOH, MeOH, 1,4-dioxane;
(d) benzyl bromide, K2CO3, DMF; (e) NaH, p-nbutoxybenzoyl chloride, DMF; (f) Pd–C, H2, MeOH, EtOAc; (g) (COCl)2, DMF, toluene; (h)
TMSCHN2, THF, CH3CN; (i) 2,4,6-collidine, benzyl alcohol.
Scheme 3, using an appropriate ketoester or keto car-
boxylic acid selected from 20–22 instead of levulinic
acid. Compound 2 was prepared from 4-hydroxy-2-
methylindole 23 in ten steps as briefly described in
Scheme 4.
onist activity among the three isomers, was reconfirmed
as a chemical lead for further optimization.
As shown in Table 2, the effect of the 2-substituent of
the N-(p-butoxy)benzoyl-5-methoxyindole-3-acetic acid
skeleton on receptor affinity and mDP receptor anta-
gonist activity was evaluated.
3. Results and discussion
Removal of the methyl moiety of 1c led to 3, with nearly
10-fold lower mDP receptor affinity. Replacement of the
methyl moiety of 1c with an ethyl moiety provided 4,
which showed nearly 5.6-fold lower mDP receptor
affinity. Compound 4 also demonstrated weak affinity
for the mEP1 receptor in addition to the mEP2 and
mDP receptors, while 3 showed similar subtype selec-
tivity to 1c. As a result, the 2-substituent was optimized
as a methyl group. As shown in Table 3, the effect of the
3-substituent on the activity profile was investigated.
Removal of the methylene moiety from the acetic acid
moiety of 1c resulted in 5, with nearly 3-fold lower mDP
receptor affinity, 2.5-fold less potent antagonist activity,
and similar subtype selectivity. One carbon homologa-
tion of 1c gave 6, which showed retention of mDP
receptor affinity and antagonist activity.
As shown in Tables 1–5, the test compounds were bio-
logically evaluated for inhibition of the specific binding
of a radioligand, [3H]PGD2, to membrane fractions
prepared from cells stably expressing each prostanoid
receptor and for inhibition of cAMP formation
evoked by PGD2 in CHO cells9 in the presence of
BSA (bovine serum albumin) (0.1%). Test compounds
were also evaluated for binding to all subtypes of the
mouse PGE2 receptor (mEP1, mEP2, mEP3, and
mEP4).2
In the course of screening of our library focused on lipid
mediators, Indomethacin analog 1c was found to show
moderate affinity for the mouse DP (mDP) receptor,
while Indomethacin did not show any affinity at 10 lM
(Table 1). Indomethacin is a well-known orally active
anti-inflammatory drug that acts by inhibition of cyclo-
oxygenase in the arachidonic acid cascade. Thus, Indo-
methacin analog 1c was considered to be an excellent
chemical lead for an orally active DP receptor antagonist.
Based on the above information, structural optimization
was initiated with chemical modification of 1c. o-Isomer
1a and m-isomer 1b were synthesized and evaluated as
described in Table 1. m-Isomer 1b demonstrated slightly
lower receptor affinity and antagonist activity relative to
p-isomer 1c, while o-isomer 1a did not show any mDP
receptor affinity at 10 lM. Compounds 1a and 1b–c also
showed weak mEP1 receptor affinity and mEP2 receptor
affinity, respectively. As a result, p-isomer 1c, which
showed the strongest mDP receptor affinity and antag-
Compound 6 also demonstrated weak affinity for the
mEP2 and mEP4 receptors. Two carbon homologation
of 1c afforded 7, which had slightly increased mDP
receptor affinity and a slightly increased antagonist
activity, while it showed decreased subtype selectivity
because of an increase in mEP2, mEP3, and mEP4
receptor affinity. Three carbon homologation of 1c gave
8, with nearly 1.5-fold lower mDP receptor affinity and
2.8-fold less potent antagonist activity. It also showed
less subtype selectivity because of increased mEP4
receptor affinity. As a result, 1c was considered to be the
best chemical lead based on its high mDP receptor
affinity and the greatest subtype selectivity among the
compounds listed in Table 3.
Table 5. Effect of transformation of 2-methylindole-3-acetic acid to 2-methylindole-4-aceic acid on the activity profiles
Compd
Binding Ki (lM)
IC50 (lM)
mEP1
mEP2
mEP3
mEP4
mDP
mDP
1c
2
>10
>10
4.7
2.0
>10
3.3
>10
>10
0.27
0.010
1.6
0.30

K. Torisu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4557–4562
acetic acid moiety
4561
CO2H
Me
MeO
N
acetic acid moiety
O
CO2H
O
N-acyl moiety
1c
Me
N
CO2H
O
O
acidic part
N-acyl moiety
2
PGs
OH lipophilic part
PGs
Figure 2. Molecular design of 2-methyl-4-indole acetic acid 2.
The effect of a 5-substituent on the activity profile was
investigated as described in Table 4. Replacement of the
5-methoxy group of 1c with a hydrogen afforded 9a with
nearly 2.5-fold higher mDP receptor affinity, although it
showed 2.4-fold less potent antagonist activity.
template to an indole-4-acetic acid template such as 2.
As shown in Table 5, N-(p-butoxy)benzoyl-2-methyl-
idole-4-acetic acid 2 was synthesized and evaluated.
Compound 2 demonstrated 27-fold more potent mDP
receptor affinity and more than 5-fold stronger mDP
antagonist activity relative to 1c.
Replacement of the 5-methoxy group of 1c with a me-
thyl group provided 9b, with nearly 1.9-fold higher mDP
receptor affinity although it showed 1.8-fold less potent
antagonist activity. Compound 9b showed lower sub-
type selectivity relative to 1c because of increased mEP4
receptor affinity. Demethylation of the 5-methoxy group
of 1c provided the corresponding hydroxy analog 10,
with nearly 7-fold weaker mDP receptor affinity and no
antagonist activity at 10 lM. Compound 10 also showed
very weak mEP2 receptor affinity. Among the com-
pounds listed in Table 4, 1c again showed the most
promising activity profile with respect to both the
antagonist activity and subtype selectivity. On the basis
of the SARdescribed in Tables 1–4, further optimiza-
tion of N-acyl-2-methylindole-3-acetic acid analogs
seemed to be difficult. A breakthrough for further
chemical modification of this template was strongly
needed.
In summary, a series of Indomethacin analogs were syn-
thesized and evaluated as a new chemical lead for mDP
receptor antagonists. Their subtype selectivity was also
evaluated. As a result, N-(p-butoxy)benzoyl-2-methylin-
dole-4-acetic acid 2 was discovered as a new chemical lead
for DP receptor antagonists. Full details (including more
detailed chemistry) will be reported in due course.
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