Synthesis and in vitro evaluation of a selective antagonist and the corresponding radioligand for the prostaglandin D2 receptor CRTH2

Article abstract of DOI:10.1016/j.bmcl.2007.07.080

Synthesis and preliminary in vitro biological evaluation of a selective high-affinity CRTH2 antagonist is described. The stability of an N-benzyl group facilitated synthesis of the corresponding radioligand by tritiation of a brominated precursor. The com

Full text of DOI:10.1016/j.bmcl.2007.07.080

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5924–5927
Synthesis and in vitro evaluation of a selective antagonist
and the corresponding radioligand for the prostaglandin
D2 receptor CRTH2
Trond Ulven,a,* Michael J. Gallen,a Mads C. Nielsen,a Nicole Merten,b Carola Schmidt,b
Klaus Mohr,c Christian Tra¨nklec and Evi Kostenisb
aDepartment of Physics and Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
bDepartment of Molecular, Cellular and Pharmacobiology, Institute for Pharmaceutical Biology, University of Bonn,
Nussallee 6, D-53115 Bonn, Germany
cDepartment of Pharmacology and Toxicology, University of Bonn, Gerhard-Domagk-Strasse 3, D-53121 Bonn, Germany
Received 21 June 2007; revised 23 July 2007; accepted 24 July 2007
Available online 22 August 2007
Abstract—Synthesis and preliminary in vitro biological evaluation of a selective high-affinity CRTH2 antagonist is described. The
stability of an N-benzyl group facilitated synthesis of the corresponding radioligand by tritiation of a brominated precursor. The
compound [3H]TRQ11238 represents the first selective CRTH2 antagonist radioligand and exhibited a specific radioactivity of
52 Ci/mmol and a pKd of 9.0.
Ó 2007 Elsevier Ltd. All rights reserved.
It is well established that the arachidonic acid metabolite
prostaglandin D2 (PGD2) plays a central role as a mes-
senger molecule in inflammatory responses of allergy
and asthma. PGD2 is released in considerable amounts
by IgE-activated mast cells upon allergen exposure,
and one of its effects is to recruit immune cells to the site
of inflammation, which in turn produce the inflamma-
tory response. Two G protein-coupled 7-transmembrane
receptors, DP1 and CRTH2 (or DP2), have been identi-
fied for PGD2, and current evidence suggests that both
receptors play pivotal roles in mediating the effects of
PGD2 in allergic inflammation.1–3 The more recently
identified receptor CRTH2 is responsible for the chemo-
attractant effect of PGD2 on eosinophils, basophils, and
Th2-cells, supporting the view that this receptor pro-
vides the essential link between PGD2 and various as-
pects of initiation and perpetuation of allergic
inflammation.4,5 Findings that mutations in the CRTH2
gene segregated with asthma reinforced the conjecture
that the receptor might be an excellent drug target.6
The dual TP/CRTH2 antagonist ramatroban was indeed
observed to abrogate inflammatory response in a murine
model of contact hypersensitivity, an effect primarily as-
cribed to inhibition of CRTH2.7 Later, the selective
CRTH2 agonist DK-PGD2 was found to increase
pathology in both allergic asthma and atopic dermatitis
models,8 while two different selective CRTH2 antago-
nists relieved peribronchial eosinophilia and goblet cell
hyperplasia in sensitized mice.9,10
In parallel with the growing evidence suggesting a dom-
inant proinflammatory role of CRTH2, the receptor has
attracted increasing interest as a drug discovery target
for new anti-allergy and anti-asthma therapeutics, and
several series of CRTH2 antagonists have recently ap-
peared in the literature.9,11–13 Many of these originate
from the two first non-prostanoid CRTH2 ligands iden-
tified, the indole carboxylic acids indomethacin and
ramatroban (Fig. 1). Indomethacin is unusual in being
a potent CRTH2 agonist, but inhibiting binding of
[3H]PGD2 only at much higher concentrations.14 We
have previously described ramatroban analogs exhibit-
ing highly selective and potent CRTH2 antagonism.15
In further studies, these closely related compounds were
found to exhibit divergent pharmacology, in that the in-
dole-N-acetic acid analogs TM30089 and TM30463 be-
haved as insurmountable antagonists, whereas the
longer propionic acid analogs ramatroban and
Keywords: CRTH2; DP2; Prostaglandin D2; 7TM receptor; GPCR;
Antagonist; Radioligand.
*
Corresponding author. Tel.: +45 6550 2568; fax: +45 6650 8780;
e-mail: ulven@ifk.sdu.dk
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.080

T. Ulven et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5924–5927
5925
O
O
S
O
O
S
O
O
S
NH2
R
N
HN
Cl
a)
Cl
O
F
F
F
N
N
H
N
N
H
MeO
1
O
2
O
( )n
OH
OH
b) K2CO3, BnBr
Indomethacin
Ramatroban (R = H, n = 2)
TM30089 (R = Me, n = 1)
TM30463 (R = H, n = 1)
TM30462 (R = Me, n = 2)
O
O
S
O
O
S
N
c) NaH
O
N
Br
F
OEt
F
Figure 1.
N
O
N
H
3
4
TM30462 displayed classical competitive antagonism
(Fig. 1).16 Furthermore, we recently identified analogs
of indomethacin and ramatroban with divergent effects
on binding and function of PGD2: in radioligand bind-
ing assays using [3H]PGD2 as the tracer, both com-
pounds increased [3H]PGD2 binding reminiscent of
allosteric enhancers. However functional assays revealed
both compounds to exclusively inhibit PGD2-mediated
translocation of b-arrestin without interfering with G
protein-dependent signaling pathways of the receptor.17
The molecular mechanisms underlying these observa-
tions have not been dissected yet, but a selective antag-
onist radioligand, which does not discriminate between
discrete signaling-competent conformations of CRTH2,
could help clarify some of these questions. Although tri-
tiated ramatroban has been described, lacking selectivity
and a lower affinity than many of the currently known
selective CRTH2 ligands deter somewhat from its use-
fulness.18 At present, [3H]PGD2 is the only CRTH2
radioligand available, thus, providing complementary
tools is currently among our prioritized goals.
OEt
d) LiOH
O
O
S
N
e) H2, Pd/C
F
No reaction
N
O
5
OH
Scheme 1. Reagents and conditions: (a) 4-FC6H4SO2Cl, Et3N,
CH2Cl2, rt, 4 h, 96%; (b) K2CO3, BnBr, acetone, 50 °C, 86%; (c)
NaH, BrCH2CO2Et, DMF, rt, 12 h, 98%; (d) LiOH, H2O/THF (1:3),
12 h, 89%; (e) H2 (1 atm), Pd/C, MeOH, rt, 1 week.
to other methods. Nonetheless, realizing the opportu-
nity for easy access to a desired tool compound, we
decided to exploit the chemical inertness of the benzyl
group in construction of the first selective CRTH2
antagonist radioligand. Synthesis of precursor 8 by the
route described in Scheme 1 unexpectedly failed as the
3,5-dibromobenzylated compound decomposed under
the conditions used for N-alkylation of the indole.
Therefore, the compound was instead synthesized taking
advantage of the precursor 6,19 and the 3,5-dibromoben-
zyl moiety was introduced toward the end (Scheme 2).
Precursor 7 was radiolabeled by hydrogenation over
palladium on carbon under an atmosphere of tritium
gas. Purification by reverse phase HPLC provided the
compound [3H]TRQ11238 (8), exhibiting a specific
radioactivity of 52 Ci/mmol and a radiochemical purity
of 97.9%.21 Saturation binding experiments confirmed
The analogous structural features of indomethacin and
ramatroban suggest that they might share binding epi-
topes despite divergent functional activity, and the
structural class obtained by opening the aliphatic ring
of the ramatroban analogs may be viewed as bridging
toward the structural class of indomethacin (Fig. 1).
Being small and synthetically easily accessible, this sim-
plified structural class invited closer investigation. We
decided to enter this class by the way of 5, which was
readily synthesized from tryptamine by sulfonylation,
followed by benzylation of the sulfonamide, alkylation
of the indole nitrogen, and ester hydrolysis (Scheme
1). Compound 5, which has previously been disclosed
in a patent application by others,19 proved to be a high
affinity (pKi = 9.2 0.2, n = 8) CRTH2 antagonist
(pIC50 = 8.4 0.3, n = 3) in assays displacing
[3H]PGD2 binding or inhibiting PGD2-induced signal-
ing at hCRTH2-transfected cells, respectively. Further-
more and notably, the compound exhibited excellent
selectivity (>1000-fold) over relevant receptors such as
DP and TP.20
X
O
O
a) K2CO3,
Br
O
O
S
S
HN
Br
N
F
X
F
Br
b) NaOH
N
O
N
O
OMe
7
6
(X = Br)
OH
c) T2, Pd/C
8 (X = T)
Our original plan involved removal of the N-benzyl
group by hydrogenolysis and subsequent construction
of a library of analogs with diverse N-substituents.
Complete stability of the substrate to the reaction condi-
tions even after prolonged reaction times made us revert
Scheme 2. Reagents and conditions: (a) K2CO3, 3,5-dibromobenzyl
bromide, acetone, 50 °C, 12 h, 80%; (b) NaOH, H2O/THF/MeOH
(1:6:6), rt, 3 h, 98%; (c) T2, Pd/C, MeOH, rt, 2 h, 89% (yield from test
reaction using H2).

5926
T. Ulven et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5924–5927
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C.; Carrillo, J. J.; Bell, F. M.; Paine, S. W.; Weaver, R.
Bioorg. Med. Chem. Lett. 2006, 16, 4287.
Figure 2. Representative saturation analysis and Scatchard plot (inset)
of [3H]TRQ11238 (8) binding to cell-membrane preparations of
HEK293 cells expressing the human CRTH2 receptor. Data were
fitted best to a one-site binding model, and Kd and Bmax values were
determined. All data points are shown as mean values SE of one
individual experiment representative of four such experiments.
13. Sandham, D. A.; Aldcroft, C.; Baettig, U.; Barker, L.;
Beer, D.; Bhalay, G.; Brown, Z.; Dubois, G.; Budd, D.;
Bidlake, L.; Campbell, E.; Cox, B.; Everatt, B.; Harrison,
D.; Leblanc, C. J.; Manini, J.; Profit, R.; Stringer, R.;
Thompson, K. S.; Turner, K. L.; Tweed, M. F.; Walker,
C.; Watson, S. J.; Whitebread, S.; Willis, J.; Williams, G.;
Wilson, C. Bioorg. Med. Chem. Lett. 2007, 17, 4347.
14. Hirai, H.; Tanaka, K.; Takano, S.; Ichimasa, M.; Nakam-
ura, M.; Nagata, K. J. Immunol. 2002, 168, 981.
15. Ulven, T.; Kostenis, E. J. Med. Chem. 2005, 48, 897.
16. Mathiesen, J. M.; Christopoulos, A.; Ulven, T.; Royer, J.
F.; Campillo, M.; Heinemann, A.; Pardo, L.; Kostenis, E.
Mol. Pharmacol. 2006, 69, 1441.
high affinity of 8 for CRTH2, with binding parameters
of pKd = 9.0 0.1 and Bmax = 2790 360 fmol/mg pro-
tein as determined in membranes from stable hCRTH2
transfectants (Fig. 2).22 No binding was observed in
non-transfected cells.
In conclusion, we have described an expedient synthesis
of the indole-N-acetic acid TRQ11238 (5), a high-affin-
ity CRTH2 antagonist with appreciable selectivity over
relevant receptors. Furthermore, a route for tritiation
of the compound was developed, resulting in the first
selective antagonist CRTH2 radioligand. The radioli-
gand [3H]TRQ11238 (8) exhibited a pKd of 9.0 on
the human CRTH2 receptor, and we expect this new
tool to be of value in the continued discovery and char-
acterization of CRTH2 ligands as well as in studies of
the receptor.
17. Mathiesen, J. M.; Ulven, T.; Martini, L.; Gerlach, L. O.;
Heinemann, A.; Kostenis, E. Mol. Pharmacol. 2005, 68,
393.
18. Sugimoto, H.; Shichijo, M.; Okano, M.; Bacon, K. B. Eur.
J. Pharmacol. 2005, 524, 30.
19. Tanimoto, N.; Hiramatsu, Y.; Mitsumori, S.; Inagaki, M.
PCT Int. Appl. WO 2003/097598.
20. Affinity and functional activity of the antagonist were
determined essentially as described in Refs. 15,16. In brief,
antagonist affinity was derived from competition binding
analysis, using [3H]PGD2 as the tracer. Antagonist activity
was determined in inositolphosphate-assays (CRTH2 and
TP receptors) or in cyclic AMP accumulation assays (DP
receptor).
Acknowledgment
This work was supported by the Danish Research
Council.
21. Custom synthesis by Amersham Biosciences.
22. For saturation binding experiments, CRTH2-HEK293
cell membranes (15 lg of protein) were incubated with
0.7–70 nM [3H]TRQ11238 (52 Ci/mmol; Amersham Bio-
sciences) in a binding buffer consisting of HBSS (Invit-
rogen) and 100 mM Hepes (pH 7.4) under continuous
gentle shaking at 4 °C for 3 h. The exact concentration of
[3H]TRQ11238 used was determined from experiment to
experiment. Non-specific binding was defined in the
presence of 10 lM TM30089. The receptor bound
radioligand was filtered on a Tomtech 96-well Mach III
Harvester (Perkin-Elmer Life and Analytical Sciences
Wallac, Turku, Finland) using filters presoaked with
0.1% polyethylenimine (Filtermat A; Perkin-Elmer LAS
Wallac). Filtration was immediately followed by three
rinses with ice-cold 100 mM NaCl. Thereafter, scintilla-
tion wax (Meltilex A; Perkin-Elmer LAS Wallac) was
melted onto the dried filtermat. The filters were placed in
sample bags (Perkin-Elmer LAS Wallac), and filter-
bound radioactivity was measured using a Microbeta
Trilux-1450 scintillation counter (Perkin-Elmer LAS
Wallac). Determinations were made in triplicate in
References and notes
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5927
four-independent experiments. Analysis was performed
using Prism 4.02 (GraphPad Software Inc., San Diego,
CA). Data sets of saturation binding isotherms were
analyzed via non-linear regression applying a hyperbolic,
one-site binding model, and individual estimates for total
receptor number (Bmax) and the equilibrium radioligand
dissociation constant (Kd) of the radioligand were
calculated.