Isosteric ramatroban analogs: Selective and potent CRTH-2 antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.12.055

The chemoattractant receptor-homologous molecule expressed on T _H2 cells (CRTH-2), also found on eosinophils and basophils, is a prostaglandin D₂ receptor involved in the recruitment of these cell types during an inflammatory response. In this report, we describe the synthesis and optimization of a ramatroban isostere that is a selective and potent antagonist of CRTH-2 which may be useful in the treatment of certain diseases.

Full text of DOI:10.1016/j.bmcl.2004.12.055

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1749–1753
Isosteric ramatroban analogs: selective and potent
CRTH-2 antagonists
Michael J. Robarge,* David C. Bom, L. Nathan Tumey, Norbert Varga,
Elizabeth Gleason, Daniel Silver, Jianping Song, Steven M. Murphy, George Ekema,
Chris Doucette, Doug Hanniford, Marc Palmer, Gary Pawlowski, Joel Danzig,
Margaret Loftus, Karen Hunady, Bruce A. Sherf, Robert W. Mays,
Alain Stricker-Krongrad, Kurt R. Brunden, John J. Harrington and Youssef L. Bennani
Athersys, Inc., Medicinal Chemistry, 3201 Carnegie Avenue, Cleveland, OH 441152634, USA
Received 6 August 2004; revised 17 December 2004; accepted 20 December 2004
Abstract—The chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH-2), also found on eosinophils and
basophils, is a prostaglandin D2 receptor involved in the recruitment of these cell types during an inflammatory response. In this
report, we describe the synthesis and optimization of a ramatroban isostere that is a selective and potent antagonist of CRTH-2
which may be useful in the treatment of certain diseases.
Ó 2004 Elsevier Ltd. All rights reserved.
Asthma is a chronic inflammatory disease of the air-
ways. It is characterized by bronchial hyperresponsive-
ness, chronic pulmonary eosinophilia, and increased
lung mucus production. The inflammation associated
with asthma is characterized by an infiltration of many
cell types, including but not limited to mast cells, eosino-
phils, T-lymphocytes, monocytes, and neutrophils.1
These cells, along with their mediators, form a complex
cascade of interactions, which ultimately result in
inflammation of the airways.2 The preferred treatment
of asthma includes the use of anti-inflammatory agents
(such as corticosteroids or leukotriene inhibitors) and
bronchodilators (b2 adrenoceptor agonists). The
increasing prevalence of asthma and the variable re-
sponse to these agents indicate the need for novel treat-
ments involving different mechanisms and approaches.
released by activated mast cells and is greatly increased
in the bronchiolar lavage fluid of asthmatics.6 PGD2
signals through two G-protein coupled receptors
(GPCRs) from distinct GPCR subfamilies: PGD2 recep-
tor (DP), a prostanoid receptor7 and chemoattractant
receptor-homologous molecule expressed on TH2 cells8,9
(CRTH-2), a receptor that is most closely related to
receptors for ÔclassicalÕ chemoattractants, such as N-for-
myl peptide (FMLP), C3a, and C5a.10 CRTH-2 is
expressed in several tissues but has been most carefully
studied on TH2 cells, eosinophils, and basophils where
it appears to mediate the chemoattractant effect of
PGD2 on each of these cell types.8 In addition, PGD2
can induce eosinophil degranulation via CRTH-2 stimu-
lation.11 Taken together, these findings suggest an
involvement of PGD2 and CRTH-2 in asthma and per-
haps other inflammatory diseases.
Leukotrienes, prostaglandins, and thromboxanes are a
series of arachidonic acid metabolites known to exert a
variety of physiological functions that are mediated
through their respective receptors.3–5 Prostaglandin D2
(PGD2) is one of the major inflammatory molecules
Ramatroban (BAY u3405, 1) has been marketed in
Japan for allergic rhinitis as a selective thromboxane-
type prostanoid receptor (TP) antagonist (Fig. 1). How-
ever, recently ramatroban was also identified as a
CRTH-2 antagonist.12 As an entry into SAR studies
based on diverse leads generated from an internal HTS
campaign, this finding prompted us to explore the
hCRTH-2 pharmacophore by simply reversing the ram-
atroban backbone (Fig. 1, 2).
Keywords: CRTH-2; Prostaglandin D2; Ramatroban; Asthma.
*
Corresponding author. Tel.: +1 2164319900; fax: +1 2163619596;
e-mail: mrobarge@athersys.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.055

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M. J. Robarge et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1749–1753
Table 1. Structures and activities of sulfonamide analogs of 2 at the
hCRTH-2 receptor
F
F
H
X
N
R
HN S
O
HN S
O
O
O
N
N
N
CO2H
COOH
COOH
Compd
X
R
Ki (lM)
2
1
2
5a
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
4-F-phenyl
3-F-phenyl
2-F-phenyl
Phenyl
0.25
1.3
Figure 1. Isostere of ramatroban—Ôreverse scaffoldÕ.
5b
5c
0.97
1.2
The synthesis of compound 2 began with tetrahydro-
carbazole 3 (Scheme 1).13 Michael addition to t-butyl
acrylate and reduction gave aniline 4. Sulfonylation
and deprotection gave the requisite sulfonamides 2 and
5a–q (Table 1). Treatment of aniline 4 with phenylisocya-
nate, benzoyl chloride, and benzaldehyde/NaCNBH3
gave compounds 6, 7, and 8, respectively.
5d
5e
4-Me-phenyl
3-Me-phenyl
2-Me-phenyl
2-Thienyl
3.2
4.2
5f
5g
>50
7.6
7.4
5h
5i
4-OH-phenyl
2-Naphthyl
4-OPh-phenyl
4-OMe-phenyl
4-Cl-phenyl
4-Ph-phenyl
4-F, 3-Cl-phenyl
2,4-Di-F-phenyl
3,4-Di-F-phenyl
n-Propyl
7.4
7.5
5j
5k
2.1
5l
5m
3
3.3
Comparison of the activities of compounds 2, 5a–q, and
6–9 in a hCRTH-2 binding assay, using membranes
from HEK293 cells stably expressing recombinant
hCRTH-2, illustrates the necessity for aryl sulfonamides
that are substituted in the para-position (Table 1).
Removal of the sulfonamide resulted in loss of activity
as shown for urea, 6, amide, 7, and amine, 9, while benz-
yl amine, 8 showed a significant drop in activity. Inter-
estingly, none of the substitutions attempted improved
upon the concept analog, compound 2, which incorpo-
rated a 4-fluorophenyl sulfonamide moiety.
5n
5o
1.7
3.6
5p
5q
4.2
>50
>50
>50
17
6
C(O)NH
C(O)
CH2
Phenyl
Phenyl
Phenyl
7
8
9
1, Ramatroban
—
H
N/A
>50
0.29
Having identified the optimal sulfonamide substitution,
we next investigated various carboxylic acid modifica-
tions. Propionic acid derivatives were generally synthe-
sized via a Michael addition to tetrahydrocarbazole 3
(Scheme 2). The use of stoichiometric base gave incom-
plete conversion to the Michael product. However, use
of a catalytic amount (10 mol%) of NaHMDS was
found to be effective at promoting this transformation.
In the case of ethylpropiolate, TBAF was found to be
a far more effective base than NaHMDS.
Scheme 4. The bromobenzoic ester was sufficiently reac-
tive for the synthesis of 23 and 24; however, in the case
of the ortho derivative, 25, the iodobenzoic ester was
necessary.
The ability of the carboxylic acid modified derivatives to
inhibit the binding of PGD2 to CRTH-2 is summarized
in Table 2. Substituted propionic acids 16 and 17 re-
sulted in complete loss of activity while acrylic acid ana-
log (15) gave an ꢀ75-fold decrease in potency indicating
the narrow structural and conformational requirement
for binding of the acid moiety. Attempts to change acid
from carboxylic to sulfonic (12) or phosphonic (13) re-
sulted in loss of potency. While the benzoic acid analogs
(23–25) were also found to be less potent than the con-
cept analog (2), the para-benzoic acid moiety was better
tolerated. Next, we explored the effects of lengthening
Lengthening and shortening of the carboxylic acid
spacer to give 18–22 was accomplished through alkyl-
ation with the appropriate bromoalkyl ester (Scheme
3). The carboxylic acid was also attached through an
aromatic linker via a recently reported modification of
the Ullmann coupling14 to give 23–25, as shown in
O
H
H2N
O
S N
O2N
R
a, b, c
N
d,e
N
N
H
4
3
5
CO2tBu
CO2H
Scheme 1. Reagents and conditions: (a) NaH, (b) t-butyl acrylate, (c) H2/Pd, (d) R–SO2Cl, Et3N, (e) TFA.

M. J. Robarge et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1749–1753
1751
F
H
O2N
N
S
O
O
N
c,d
e (for 12)
f (for 13)
N
X
X
10 X = SO2OPh
11 X = P(O)(OEt)2
12 X = SO3H
13 X = PO3H2
F
H
O2N
g
N
S
3
O
O
c, d, e
N
N
14
15
CO2Et
CO2H
F
O2N
H
N
S
c, d, e
O
O
N
R'
CO2Et
N
R
R'
R
CO2H
16 (R=Me, R'=H)
17 (R=H, R'=Me)
Scheme 2. Reagents and conditions: (a) Phenyl vinylsulfonate, NaHMDS (cat), DMF; (b) diethyl vinylphosphonate, NaHMDS (cat), DMF; (c) H2/
Pd; (d) 4-F–Ph–SO2Cl, Et3N; (e) NaOH, EtOH, heat; (f) TMS–Br, DCM; (g) ethyl propiolate, TBAF, THF; (h) RCH@CHR0CO2Et, NaHMDS
(cat), DMF, 60–70 °C.
F
H
Br
O2N
N
S
CO2R'
O
O
( )n
R
a, b, c
3
N
N
K2CO3, DMF
80-100O C
( )n
( )n
R
R
CO2R'
CO2H
18 (R=H, n=2)
19 (R=H, n=3)
20 (R=H, n=0)
21 (R=CH3, n=0)
22 (R=CH2CH3, n=0)
Scheme 3. Reagents and conditions: (a) H2/Pd; (b) 4-F–Ph–SO2Cl, Et3N; (c) NaOH, EtOH, heat or TFA.
F
O2N
H
N
S
O
Br (or I)
O
N
+
3
a
N
b, c, d
CO2Et
23 (ortho)
24 (meta)
25 (para)
CO2Et
CO2H
Scheme 4. Reagents and conditions: (a) CuI, NH(Me)CH2CH2NHMe, K2CO3, toluene, reflux; (b) H2/Pd; (c) 4-F–Ph–SO2Cl, Et3N; (d) NaOH,
EtOH, heat.

1752
M. J. Robarge et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1749–1753
Table 2. Inhibition constants of various carboxylic acid modifications
at the hCRTH-2 receptor
azole 3 were readily prepared via a Fischer indole syn-
thesis with an appropriate cyclic ketone (Table 3).
Standard transformations resulted in analogs 26–30.
The size of the ÔC-ringÕ was found to have a profound
effect on activity: the order of potency being 7-mem-
bered (30) P 6-membered (20) ꢁ 5-membered (29).
Lastly, the substitution effect on the 6-membered ÔC-
ringÕ series resulted in the finding that a methyl group
in the 3-position was optimal as typified by compound
26. A select group of the isosteric ramatroban analogs
were tested for their ability to inhibit PGD2-mediated
receptor activation in a fluorescence assay that measures
changes in intracellular calcium (Table 3). The IC50 val-
ues determined in this functional assay were very similar
to the inhibition constants determined in the CRTH-2
binding assay.
F
H
N
S
O
O
N
X
Compd
X
Ki (lM)
12
13
CH2CH2SO3H
CH2CH2PO3H2
CH@CH–CO2H
CH(Me)CH2CO2H
CH2CH(Me)CO2H
(CH2)3CO2H
(CH2)4CO2H
CH2CO2H
2.4
>50
19
15
16
>50
>50
8.3
17
18
Ramatroban (1) was initially developed as a thrombox-
ane A2 antagonist that was subsequently determined to
also be an inhibitor of CRTH-2. Since the compounds
reported herein were derived from ramatroban, select
analogs were counter-screened against the human
thromboxane receptor (hTP) using membranes from
HEK293 cells stably expressing recombinant hTP to
determine if any of them had dual activity. Interestingly,
ramatroban (1) is ꢀ16-fold selective for TP over CRTH-
2 while the concept compound (2) is ꢀ6-fold selective
for CRTH-2 over TP. This effect is amplified upon
examination of our lead compounds 20, 26, 27, and
30, which are all very selective, exhibiting >400-fold
preference for CRTH-2 over TP (Table 4).
19
20
4.6
0.030
4.4
21
22
CH(Me)CO2H
CH(Et)CO2H
ortho-Ph–CO2H
meta-Ph–CO2H
para-Ph–CO2H
(CH2)2CO2H
N/A
18
33
23
24
27
25
2
1, Ramatroban
6.4
0.25
0.29
and shortening the methylene-spacer between the acid
moiety and the indole. Increasing the chain length (com-
pounds 18 and 19) resulted in loss of potency while
decreasing the chain length resulted in an ꢀ10-fold
improvement in potency (compound 20). Finally, substi-
tuted acetic acids were explored. As in the case with
substituted propionic acids, substituted acetic acids also
showed a reduced potency, the magnitude of which was
proportional to the size of the substituent (compounds
21 and 22).
Table 4. Counter screening against hTP receptor
Compd
hTP % inhibition Ki (lM) hTP Ki (lM)
@ 50 lM
hCRTH-2
2
92
21
30
66
28
1.5
>20
0.25
20
26
27
30
0.030
0.013
0.050
0.020
0.29
>20
>20
>20
Next, we evaluated ÔC-ringÕ modified analogs incorpo-
rating the acetic acid moiety determined previously to
enhance potency. Various derivatives of tetrahydrocarb-
1, Ramatroban 98
0.018
Table 3. Synthesis and activity at the hCRTH-2 receptor of saturated ring variants
F
H
R
N
S
R
( )n
O2N
R
( )n
O
O
C
A
B
( )n
c, d,e
N
a, b
O
N
26-30
H
CO2H
Compd
R
n
Ki (lM)
IC50 (functional) (lM)
26
27
CH3
Phenyl
t-Butyl
H
1
1
1
0
2
0.013
0.050
0.15
0.0097
0.022
0.12
28
29
0.20
0.020
0.29
0.13
0.022
0.34
30
1, Ramatroban
H
N/A
Reagents and conditions: (a) 4-Nitrophenylhydrazine, EtOH; (b) HOAc / HCl (aq), 120 °C; (c) BrCH2CO2t-Bu, K2CO3, DMF; (d) H2/Pd; (e) 4-F–
Ph–SO2Cl, Et3N; (f) TFA.

M. J. Robarge et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1749–1753
1753
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In conclusion, we have designed and synthesized a series
of potent substituted indole acetic acids as CRTH-2
antagonists based on ramatroban as an initial foray into
defining the CRTH-2 antagonist pharmacophore.15
While ramatroban is moderately selective for TP over
CRTH-2, we have shown the compounds described
herein to be more than 400-fold selective for CRTH-2
over TP. Further studies have been undertaken to deter-
mine the potential clinical usefulness of these com-
pounds in allergic diseases.
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