Novel 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetic acids: Discovery and hit-to-lead evolution of a selective CRTh2 receptor antagonist chemotype

Article abstract of DOI:10.1016/j.bmcl.2012.05.087

Hit-to-lead evolution of 2-(2-((2-(4-chlorophenoxy)ethyl)thio)-1H-benzo[d] imidazol-1-yl)acetic acid (1), discovered in a high-throughput screening campaign as a novel chemotype of CRTh2 receptor antagonist, is presented. SAR development as well as in vitro and in vivo DMPK properties of selected representatives of substituted 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetic acids are discussed.

Full text of DOI:10.1016/j.bmcl.2012.05.087

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4660–4664
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetic acids: Discovery
and hit-to-lead evolution of a selective CRTh2 receptor antagonist chemotype
Julien Pothier, Markus A. Riederer, Oliver Peter, Xavier Leroy, Anja Valdenaire, Carmela Gnerre,
⇑
Heinz Fretz
Actelion Pharmaceuticals Ltd, Gewerbestrasse, 16, CH-4123 Allschwil, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Hit-to-lead evolution of 2-(2-((2-(4-chlorophenoxy)ethyl)thio)-1H-benzo[d]imidazol-1-yl)acetic acid (1),
discovered in a high-throughput screening campaign as a novel chemotype of CRTh2 receptor antagonist,
is presented. SAR development as well as in vitro and in vivo DMPK properties of selected representatives
of substituted 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetic acids are discussed.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 11 May 2012
Accepted 23 May 2012
Available online 1 June 2012
Keywords:
CRTh2
DP2
Prostaglandin
Asthma
Rhinitis
Prostaglandin D2 (PGD2), a major metabolite of arachidonic
acid, is predominantly involved in the physiological response to al-
lergy. It is released at high concentration by immunoglobulin E
activated mast cells upon allergen challenge1 and was demon-
strated to activate two G-protein coupled receptors (GPCRs), the
classical DP1 receptor and the recently discovered chemoattractant
receptor-homologous molecule expressed on T-helper 2 cells
(CRTh2 also known as DP2) receptor.2 CRTh2 activation leads to
the recruitment of granulocytes and Th2 cells by chemotaxis3 to
the inflammation site and has been in almost all cases demon-
strated to be pro-inflammatory.4,5 Furthermore it is now well
established that antagonizing selectively the CRTh2 receptor could
be useful in the treatment of asthma and other inflammatory dis-
eases such as allergic rhinitis.6–10 Herein, we describe the discovery
and development of a novel CRTh2 antagonist chemotype series.
Our search for a selective CRTh2 receptor antagonist was initi-
ated by screening our GPCR biased in-house compound collection
by means of an in vitro fluorescent imaging plate reader (FLIPR™,
Molecular Devices) assay. The assay was performed in a 384-well
plate format. Human embryonic kidney cells (HEK-293) stably
expressing the human (h)CRTh2 receptor under the control of the
CMV promoter from a single insertion of the expression vector
pcDNA5 (Invitrogen), were challenged with PGD2 (10 nM concen-
tration) in the presence and absence of test compounds. In these
transformed cells, PGD2 induces release of calcium (Ca2+) from
intracellular Ca2+ stores. Changes of such intracellular Ca2+
concentrations were measured by monitoring changes in fluores-
cence emission at kex = 488 nm and kem = 540 nm of the added
cytoplasmic Ca2+ indicator Fluo-3 and Pluronic F-127 (Molecular
Probes) upon Ca2+ complexation. This screening effort revealed
hit compound 1 (Fig. 1) consisting of a 2-(2-((substituted)alkyl-
thio)-1H-benzo[d]imidazol-1-yl)acetic acid core. 1 antagonizes
PGD2 induced hCRTh2 activation with an IC50 = 6.6
more, we could demonstrate PGD2 competitive binding of 1 to
the hCRTh2 receptor with an IC50 = 1.5 M. The receptor binding
lM. Further-
l
assay was performed again with HEK-293 cells expressing hCRTh2
receptor employing the agonist radioligand [3H]PGD2 (2.5 nM,
169 Ci/mmol).
The carboxylic acid is a common functionality to all endoge-
nous prostanoids as well as many of the published prostanoid
receptor agonists and antagonists.11 Alike, the carboxylic acid of
1 was shown to be essential for hCRTh2 receptor interaction
and therefore was kept throughout the optimization process.
The focus of the first set of benzimidazole analogues was directed
towards the initial thiobenzimidazole hit core (X = S, R = H)
addressing part A of 2. As indicated (Fig. 1), part A represents a
(C1–C4)alkyl chain connected through a linker group Y to various
Z residues.
General synthetic pathways are outlined in Scheme 1(A) for
unsubstituted (R = H) and in 1(B) for unsymmetrically R-substituted
benzimidazole scaffold (R–H), respectively. Typically, starting
2-chlorobenzimidazole (3) was reacted with tert-butylbromoace-
tate and potassium carbonate in refluxing acetone to form tert-butyl
2-(2-chloro-benzimidazol-1-yl) acetate (4). Substitution of the
chlorine atom by means of thiourea in refluxing methanol yielded
⇑
Corresponding author. Tel.: +41 61 565 6208; fax: +41 61 565 8901.
E-mail address: heinz.fretz@actelion.com (H. Fretz).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.087

J. Pothier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4660–4664
4661
Part A
(CH2)n
R
4
5
N
N
N
N
O
S
X
Y
Z
6
Cl
7
COOH
COOH
1
2
μ
μ
M
Binding hCRTh2 IC50= 1.5
M
FLIPR Ca2+ flux hCRTh2 IC50= 6.6
Figure 1. Structure of initial high-throughput screening hit 1 and general formula 2, indicating the positions investigated during the hit-to-lead phase.
R
R
a)
N
N
N
(A)
(B)
Cl
Cl
N
H
b)
g)
COOtBu
R
R
R
4
H
N
3
4
c)
N
N
d)
N
N
(CH2)n
Y Z
(CH2)n
COOtBu
Y Z
5
6
S
S
S
R= H
N
7
COOH
COOtBu
R
R
R
5
NO2
NO2
F
NH2
NH
e)
6
2, 10-14
f)
NH
COOtBu
COOtBu
7
8
R ≠ H
9
Scheme 1. Reagent and conditions: (a) tert-butylbromoacetate, K2CO3, acetone, reflux (50–93%); (b) thiourea, methanol, reflux (45–98%); (c) Br-(CH2)n-Y–Z, K2CO3, acetone,
reflux; or HO-(CH2)n-Y–Z, (@NC(O)OtBu)2, Ph3P, THF; (d) TFA, DCM, rt; (e) NH2CH2CO2tBu hydrochloride, Na2CO3, DMSO, 50 °C (44–96%); (f) H2, 5% mol Pd/C, THF, rt; (g) 1,10-
thiocarbonyldiimidazole, THF, rt (41–73% over 2 steps).
tert-butyl 2-(2-thioxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)
acetate 5,12 which was then S-alkylated either with Br-(CH2)n -
Y–Z in the presence of potassium carbonate in refluxing acetone,
or in a Mitsunobu reaction with HO-(CH2)n -Y–Z in presence of
di-tert-butylazodicarboxylate and triphenylphosphine in THF
to give 6. Subsequent hydrolysis of tert-butyl acetate 6 with
TFA in dichloromethane yielded the expected thiobenzimidaz-
ole-1-acetic acid 2 and 10–12.
Depicted in Scheme 1(B) is a regioselective approach, devised to
provide benzimidazole analogue 2 with a substituent R other than
hydrogen at any of the positions C(4), C(5) or C(6) of the core,
avoiding cumbersome chromatographic separations of regioisom-
ers. Accordingly R substituted 1-fluoro-2-nitrobenzene (7) and
tert-butyl glycinate hydrochloride reacted in the presence of
sodium carbonate in DMSO at 50 °C to give the substituted 2-((2-
nitrophenyl)amino)acetate 8.13 Palladium-catalyzed hydrogenoly-
sis of the nitro group with H2 in THF under atmospheric pressure
yielded amine 9, which then was converted without purification
by means of 1,10-thiocarbonyl-diimidazole to the corresponding R
substituted tert-butyl 2-(2-thioxo-2,3-dihydro-1H-benzo[d]imida-
zol-1-yl)acetate 5. Subsequent S-alkylation and final deprotection
gave the desired products 13 and 14.
Following our screening cascade, the competitive radioligand
displacement assay described above was used as a test system to
establish the structure activity relationship (SAR) for all synthe-
sized potential hCRTh2 antagonists. The FLIPR assay was used as
a secondary assay in order to give evidence that the compounds
are truly antagonizing the effect of agonist PGD2 on the hCRTh2
receptor. The results from both assays are reported as IC50 values.
Selectivity of the antagonists towards the DP1 receptor was deter-
mined using a competitive receptor binding assay with CHO
(Chinese Hamster Ovary) cells expressing high levels of recombi-
nant DP1 receptor, employing [3H]PGD2 as ligand.14 Plasma as well
as chemical stabilities were assessed by incubating the antagonists
in rat plasma for up to 4 h, in simulated gastric fluid (SGF) for 1 h
and simulated intestinal fluid (SIF) for 4 h.15 Recovery of
unchanged parent was determined by LC-MS. Potent (binding
IC50 <100 nM), selective and stable antagonists were further
verified in a competitive radioligand displacement assay in the
presence of 0.5% human serum albumin (HSA) in order to identify
those antagonists that retained potency under conditions where
binding to blood proteins occurs. The positives were subsequently
tested in an eosinophil shape change (ESC) assay.16,17 In brief, hu-
man eosinophils were isolated from whole blood of healthy volun-
teers and were subsequently stimulated with 15 nM PGD2 in the
presence of 50% human plasma (HP) at 37 °C, for 5 min. Stimula-
tion was stopped by adding fixation solution and increase in light
scattering reflecting changes in cell shape was measured by flow
cytometry. A dose dependent inhibition of this shape change could
be demonstrated with potent antagonists by pre-incubating the
cells with the increasing concentrations of antagonist for 10 min
prior to stimulation with PGD2.
A preliminary SAR was established with compounds 2a–2r
(Table 1). The original hit 1 (Z = 4-chlorophenyl) was found to be
three times less potent than 2a with Z = phenyl. Also, 2b and 2c
containing the larger bicyclic substituents Z = naphth-2-yl and
naphth-1-yl respectively, were considerably less potent. No clear
correlation could be ascribed to the length of the n-alkyl chain
and the observed potency. The potency of the compounds was
dependant on both, the type of connecting group Y and the length
of the n-alkyl chain, for example Y = CH2, Z = phenyl and n = 0, was
found to be most potent (2h, IC50 = 388 nM) in this first series. An
additional CH2 (n = 1) led to more than a twofold drop of potency
(2g), whereas an almost complete regain was observed by further
elongating n-alkyl to n = 2, as shown with 2f. Optimal n-alkyl chain
length was observed if ethyl (n = 2) was combined with Y = O (2a),
or n-propyl (n = 3) with Y = NH (2i). An unexpected but interesting
discovery was made within the homologous alkanoate series. An
almost linear increase in potency was observed from ethyl propan-
oate up to ethyl hexanoate (2n–2q), 2q being the most potent

4662
J. Pothier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4660–4664
Table 1
Table 2
hCRTh2 receptor binding IC50 values of compounds 2a–2r
hCRTh2 receptor binding IC50 values of compounds 10a–12d
N
R
3
Z
2
1
N
N
S
nY
S
N
6
4
COOH
5
COOH
2a-r
10-12
a Values are the mean of two or more experiments.
(IC50 = 1010 nM). Absolutely no interaction with the receptor was
observed for the corresponding ethylamide 2r.
Subsequently, the sulfur atom of the thiobenzimidazole core (2,
X = S) was systematically replaced by X = O, CH2, and NH. All
compounds obtained were considerably less potent than the
corresponding thiobenzimidazole analogues (data not shown).
Oxidation of the sulfur atom to the corresponding 2-sulfinyl- or 2-
sulfonyl-1H-benzo[d]imidazole (X = SO, or SO2) analogues gave at
least a fivefold drop in potency (data not shown).
a Values are the mean of two or more experiments.
b Racemic.
With this information, we decided to focus on further exploring
2h. Subsequently, tolerability and effect of a substituent R at the
phenyl ring were investigated (10, Table 2). A positional scanning
was carried out comprising in parts the Topliss scheme for aro-
matic substitutents,18 first substituting the hydrogen atom at posi-
tion C(2), C(3) and C(4) with chloro, methoxy, methyl, bromo, and
methoxycarbonyl (Table 2). The latter was included at this stage
because of the discovery made with alkanoate homologues
2n–2q. However, an inconclusive SAR was obtained. The corre-
sponding 4-chloro, 4-methoxy and 4-methyl analogue 10a, 10d
and 10g, respectively, showed similar potencies and were found
to be approximately two times more potent than 2h. Bromine at
any of the three positions led to a minor loss of potency keeping
the same ranking order compared with respective chloro substitu-
tion. Clearly less tolerated was the 4-methoxycarbonyl (10m).
Furthermore, substituents at position 2 generally tended to result
in less potent compounds (10c, 10f, 10i, 10l) within a series. Com-
pared to 2h, a greater than five times lower IC50 value was obtained
with 3-methoxy substitution (10e).
Table 3
hCRTh2 receptor binding and FLIPR IC50 values of compounds 13a–13h
MeO
R
4
N
5
S
6
N
Me
7
O
COOH
13
The effect of a second substituent R at the phenyl ring was stud-
ied in a combinatorial approach. Only readily available building
blocks containing two substituents were selected and introduced
according to Scheme 1. Thereby building blocks were chosen
where a substituent like chloro, methoxy, methyl, bromo, or
methoxycarbonyl as in 10a–10o, respectively, was present in any
combination with a second substitutent such as chloro, methoxy,
methyl, or bromo, to give for example 11a to 11j (Table 2). In this
study, the 3,4-dichloro analogue 11a was found two times less
potent than the respective mono-substituted 4- or 3-chloro ana-
logue 10a and 10b.
a Values are the mean of two or more experiments.
Comparable values were measured for 2,5-dichloro analogue
11b and 2-chloro substituted 10c. Superimposing a phenyl ring
substituted with A at C(2) and a phenyl ring substituted with B at
C(3) implies a 2,3- or a 2,5-disubstitution pattern if both substitu-
ents A and B are combined on one phenyl ring. In our subsequent

J. Pothier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4660–4664
4663
Table 4
Table 6
hCRTh2 receptor binding and FLIPR IC50 values of compounds 14a–14k
Comparative in vivo pharmacokinetica profile of selected CRTH2 antagonists
R1
O
Compound AUC (ngxh/ml) CL (ml/min/kg) Vss (L/kg) T1/2 (h) F (%)
12b
13b
2360
919
20
36
1.1
1.2
2.5
0.9
27
19
F
N
N
a
S
Male Wistar rats (3 animals for each experiment), iv dose 1 mg/kg, po dose
R2
10 mg/kg as a solution.
O
COOH
14
Compound R1
R2
hCRTh2 binding
(Ca2+)i flux FLIPR
buffer IC50 (nM) hCRTh2 IC50a (nM)
a
(IC50 = 29 nM) so far. The positional scanning, whereupon hydrogen
was substituted with bromine at C(2), C(4) and C(5) as in 11g–11j,
afforded 11j with IC50 = 45 nM. That finding actually confirmed (a)
the positive ortho effect induced with this 2,5-disubstitution pat-
tern and (b) that this pattern is superior over all other investigated
combinations.
Replacing the initial 5-methoxycarbonyl with other groups,
for example more suitable alkoxycarbonyl groups such as 5-
isopropoxycarbonyl, (CO2iPr, 12a) caused a fivefold decrease in
potency. Further attempts to replace 5-methoxycarbonyl led to
the discovery of 12b with an IC50 = 8 nM, containing 5-methyl-
carbonyl instead. The importance of a carbonyl group at C(5)
in combination with a 2-methoxy substituent could be impres-
sively demonstrated, as 12b was found ꢀ30 times more potent
than 12c and >200 times more potent than 1-hydroxyethyl ana-
logue 12d.
14a
14b
14c
14d
14e
14f
14g
14h
14i
Et
nPr
nBu Me
Me
Me
4
5
9
1.7
16
16
59
5
9
12
4
15
15
94
130
725
91
4
Me
Me
Me
Me
Me
Me
Me
Me
Ph
NHEt
NHBu
NHBn
NEt2
72
NBnEt
Morpholino
Indolin-1-yl
495
107
3
14j
14k
a
Values are the mean of two or more experiments.
Table 5
Attaching a substituent R to the benzimidazole core 13 gener-
ated highly potent compounds (Table 3), binding to the hCRTh2
receptor with IC50 values in the single digit nM range, especially
if R represents a strong electron-withdrawing group at C(5) such
as fluorine, nitro, trifluoromethyl or formyl.
Comparative in vitro profiles of representative key antagonists
Assay
CRTh2 receptor antagonist
12b
13b
14f
14k
hCRTh2 Bdg in buffer IC50 (nM)
hCRTh2 FLIPR IC50 (nM)
hCRTh2 Bdg in HSAa IC50 (nM)
ESC in 50% plasma IC50 (nM)
hDP1Binding IC50 (nM)
hTP FLIPR IC50 (nM)
MW (Da)
9
25
2
16
2
16
3
Finally, a small library was produced exploiting in more detail
scope and limitations of the positions R1 and R2 of 14 (Table 4).
It was found that for R1 besides Me (11f, 12b, 13b) also the
C2-C44-alkyl homologues (14a, 14b and 14c) were tolerated. These
compounds were shown to inhibit PGD2 binding to the hCRTh2
receptor comparable to 13b with IC50 values <10 nM. Further mod-
ifications of R2 resulted in 14d displaying an IC50 = 1.7 nM. So far,
this was the most potent compound generated in this whole series.
However, more emphasis was put on replacing the metabolically
critical ketone or ester functionality. As illustrated with 14g–14j,
increasing bulkiness of the alkylamino part leads to decreasing
potency of the antagonists. Interestingly, regain of the full inhibi-
tory potential was observed by attaching the constrained bicyclic
indolin-1-yl residue as in 14k. hCRTh2 antagonism of selected
compounds was assessed in the aforementioned (Ca2+) flux FLIPR
assay (Tables 3 and 4). All compounds were confirmed as antago-
nists. Binding and FLIPR data correlate with comparable potencies
for the high affinity compounds, whereas a two- to fourfold shift is
observed for the weaker antagonists. In order to address protein
binding, the binding assay was performed in the presence of 0.5%
HSA prior to further profiling. Antagonists displaying no or only a
minor shift in potency in presence of HSA were tested for inhibi-
tory activity in the functional ESC assay, whereas antagonists dis-
playing a major shift under those conditions were not further
profiled.
5
15
4
14
81
8
5
41
51
>10000
>25000
370
ꢁ1.1
>10000
>25000
388
ꢁ0.8
>10000
ndb
445
0.1
>10000
nd
491
0.3
LogD7.4
Solubility (ng/ml)
0.1 M HCl
Phosphate buffer pH 7
Tris buffer pH9
575
602
583
484
399
583
670
719
>750
158
812
789
Chemical stability
SGF% parent after 1 h
SIF% parent after 4 h
92
100
95
100
94
78
100
100
Human plasma stability
% parent (4 h)
100
100
100
93
in vitro ADME profile
CLint (RLM) (
CLint (HLM) (
CLint (RHepa) (
l
l
l/min/mg protein)
l/min/mg protein)
l/min/106 cells)
17
0
60
13
60
7
91
2
l
10
>50
37
>50
24
20
>50
10
6
>50
4
12
>50
5
CYP2D6 IC50
(
lM)
CYP3A4 (Mid)c IC50
CYP3A4 (Test)c IC50
(
lM)
(lM)
15
>50
7
6
13
2
CYP2C9 IC50
(
lM)
a
b
c
0.5% Human serum albumin.
No data.
Substrates used: Mid = midazolam, Test = testosterone.
The properties of four selected representatives of this novel
chemotype lead series are summarized in Table 5. Comparable pro-
files were obtained concerning the activity on the hCRTh2 receptor
for 12b, 13b and 14k under various assay conditions, 14f per-
formed slightly weaker. High selectivity towards the hDP1 and
hTP receptor could be achieved. Low to medium molecular weight
(MW) ranging from 370 to 491 Da, and measured LogD7.4 values
ranging from ꢁ1.1 to +0.3 gave rise to highly soluble compounds
in aqueous buffer solutions. Chemical as well as plasma stability
studies we mainly focused on the 2,5-substitution pattern. For
example, unifying 2-methoxy of 10f (IC50 1100 nM) with 3-chloro
of 10b, or 3-methoxy of 10e, or 3-methyl of 10h, in one molecule
lead to a substantial loss of binding affinity as demonstrated for
11c, 11d, and 11e, respectively. However, to our surprise 2-meth-
oxy, as in 10f, combined with 3-methoxycarbonyl of 10n in a
2,5-disubstitution pattern yielded 11f the most potent compound

4664
J. Pothier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4660–4664
appeared not to be an issue, the selected compounds were found
stable under the various conditions, for example >90% of parent
was recovered after incubation in SGF for 1 h. Upon exposure to
SIF for 4 h, 14f turned out to be slightly less stable (78% recovery
of parent).
Acknowledgments
The authors thank A. Jonuzi, P. Risch, B. Butscha, J. Giller, B.
Lack, S. Brand for valuable technical support, B. Capeleto, R. Bravo,
S. Delahaye, H. Kletzl for providing DMPK data, K. Hilpert for
fruitful discussions and J. Williams for the proofreading of the
manuscript.
Compound 12b displayed low intrinsic clearance (Clint) in rat li-
ver microsomes (RLM) and suspended hepatocytes (RHepa) with
values of 17 ll/min/mg protein and 10 l
l/min/106 cells, respec-
tively. No turnover was measured in human liver micorosomes
(HLM). All three other analogues systematically showed higher
turnover in RLM and HLM. In RHepa, Clint values for 13b, 14f and
14k were within a factor of two compared to 12b.
All four selected representatives were weak inhibitors of the
cytochrome P450 isoform CYP2D6 with IC50 values higher than
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
05.087.
50
an IC50 of 6
with IC50 values of 24
l
M. Moderate inhibition of CYP2C9 was measured for 14f with
M, whereas 12b, 13b and 14k were weaker inhibitors
M, >50 M and 28 M, respectively. A sim-
l
References and notes
l
l
l
ilarly moderate inhibition pattern was observed on CYP3A4 with
12b displaying the weakest effect.
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In essence, the systematic optimization combined with seren-
dipitous findings during the evolution of the screening hit led to
the discovery of a structurally novel lead series comprising potent
and selective hCRTh2 receptor antagonists. Representative exam-
ples were shown to display encouraging in vitro and in vivo DMPK
properties. Therefore, this lead series was considered useful and
proposed for further lead optimization and development.
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