Novel selective thiazoleacetic acids as CRTH2 antagonists developed from in silico derived hits. Part 1

Article abstract of DOI:10.1016/j.bmcl.2009.12.008

Structure-activity relationships of three related series of 4-phenylthiazol-5-ylacetic acids, derived from two hits emanating from a focused library obtained by in silico screening, have been explored as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. Several compounds with double digit nanomolar binding affinity and full antagonistic efficacy for human CRTH2 receptor were obtained in all subclasses. The most potent compound was [2-(4-chloro-benzyl)-4-(4-phenoxy-phenyl)-thiazol-5-yl]acetic acid having an binding affinity of 3.7 nM and functional antagonistic effect of 66 nM in a BRET and 12 nM in a cAMP assay with no functional activity for the other PGD2 DP receptor (27 μM in cAMP).

Full text of DOI:10.1016/j.bmcl.2009.12.008

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1177–1180
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel selective thiazoleacetic acids as CRTH2 antagonists developed
from in silico derived hits. Part 1
Øystein Rist, Marie Grimstrup, Jean-Marie Receveur, Thomas M. Frimurer, Trond Ulven ,
à
*
Evi Kostenis , Thomas Högberg
7TM Pharma A/S, Fremtidsvej 3, DK-2970 Hørsholm, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure–activity relationships of three related series of 4-phenylthiazol-5-ylacetic acids, derived from
two hits emanating from a focused library obtained by in silico screening, have been explored as CRTH2
(chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. Several com-
pounds with double digit nanomolar binding affinity and full antagonistic efficacy for human CRTH2
receptor were obtained in all subclasses. The most potent compound was [2-(4-chloro-benzyl)-4-(4-
phenoxy-phenyl)-thiazol-5-yl]acetic acid having an binding affinity of 3.7 nM and functional antagonistic
effect of 66 nM in a BRET and 12 nM in a cAMP assay with no functional activity for the other PGD2 DP
Received 1 November 2009
Accepted 1 December 2009
Available online 4 December 2009
receptor (27 lM in cAMP).
Ó 2009 Elsevier Ltd. All rights reserved.
Prostaglandin D2 (PGD2) and one of its receptors CRTH2 (che-
moattractant receptor-homologous molecule expressed on Th2
cells) have been implicated in the pathogenesis of various inflam-
matory conditions.1 The CRTH2 receptor is expressed on eosino-
phils, basophils and Th2-type T lymphocytes, and mediates their
chemotaxis in response to PGD2.1,2 In addition to PGD2, a number
of other arachidonate metabolites activate the CRTH2 receptor,
CRTH2 receptor that was derived with input from the binding
pocket-related AT1 and AT2 receptors and associated ligands.11,12
The pharmacophore, containing a negatively charged site and three
hydrophobic regions, was used to extract about 600 compounds
from approximately 1 million publically available compounds from
vendors. In vitro binding of this library showed 10% of the com-
pounds to have IC50 values less than 10 lM. Some identified repre-
including 13,14-dihydro-15-keto-PGD2, PGJ2,
D
12PGJ2, 15-deoxy-
sentative compounds (1–2) are shown in Figure 1.11,12 The
phenoxyacetic acid derivative 2 provided input to the design of
potent and selective agents of the pyrazole-4-carbonyl type 3
displaying oral activity in allergic in vivo models.12
PGJ2 and 11-dehydro-TXB2.1,3 The Th2 cells are known as central
orchestrators of allergic asthma, driving IgE response and eosino-
philia. Hence, CRTH2 induces the production of proinflammatory
cytokines in Th2 cells,1,4 enhances the release of histamine from
basophils1,5 and mediates the respiratory burst and degranulation
of eosinophils.1,6 Recently, CRTH2 has also been implicated in
mediating an inhibitory effect of PGD2 on the apoptosis of human
Th2 cells induced by cytokine deprivation.7 Accordingly, CRTH2
antagonists are being developed for the treatment of asthma and
allergic disease.8,9 Some compounds have also advanced to clinical
trials directed towards asthma, allergic rhinoconjunctivitis and
chronic obstructive pulmonary disease.10
In this Letter we describe another set of compounds, that is, the
thiazoleacetic acids 4a and 4b shown in Figure 2 derived from the
same in silico screening campaign, and the optimization towards
more potent and selective compounds. Initially we expanded the
mining of public compound sources for additional compounds
with related structures to enrich the SAR. Using simple search
HO
O
We have earlier designed small target-specific libraries using a
physicogenetic approach to identify binding pocket-related 7TM
receptors associated with ligand information.11 As one example
we have described the generation of a pharmacophore for the
N
HN
S
N
HN
N
N
O
O
O
COOH
COOH
O
O
Cl
* Corresponding author. Tel.: +45 3925 7760; fax: +45 3925 7776.
E-mail address: th@7tm.com (T. Högberg).
Br
1
2
3
Br
Present address: Department of Physics and Chemistry, University of Southern
Denmark, Campusvej 55, DK-5230 Odense, Denmark.
Present address: Institute of Pharmaceutical Biology, University of Bonn, Nussal-
lee 6, D-53155 Bonn, Germany.
Figure 1. Some representative chemotypes identified after in silico screening, that
is, 1 (IC501.9
M)11 and 2 (IC50 0.044 M).12 The pyrazole 3 was developed as an
orally active potent antagonist with IC50 4 nM.12
à
l
l
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.008

1178
Ø. Rist et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1177–1180
Table 2
O
Et
Binding affinity and functional antagonism on hCRTH2 of 2,4-diphenylthiazoleacetic
acids
Cl
F
R4
R1
R3
N
N
N
N
N
S
S
H
H
HOOC
HOOC
S
4a
4b
Figure 2. Thiazoleacetic acid 4a (IC50 3.0
the initial in silico screening.
lM) and 4b (IC50 0.35 lM) identified in
COOH
IC50 Binda
No.
R1
R3
R4
(
lM)
IC50 BRETb
(lM)
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
H
H
H
H
H
H
F
Cl
OCH3
OPh
OPh
OPh
H
F
Cl
OCH3
Et
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.87
0.86
0.55
7.0
1.3
0.038
1.5
0.99
0.82
0.16
0.47
0.093
2.5
3.5
queries based on the hits 5a and 5b and the previous pharmaco-
phore led us to a set of thiazoleacetic acids 5a–5g with both phenyl
rings directly attached to the thiazole core (Table 1). The available
compounds did not display a great variation in potency, but one
may conclude that substituents are allowed in para (R1/R3) as well
as ortho (R2/R4) positions of both phenyl rings. Potency benefits
from having a larger halogen substituent in either para position
R1 or R3 of the two phenyl rings.
We decided to further explore this chemotype and synthesize
a more diverse range of structures. The 2,5-diphenylthiazoles 5h–5s
were conveniently obtained by condensing the appropriate
3-bromo-4-oxo-butyric acid derivatives II with thioamides IV by
microwave assisted heating in DMF. Standard Friedel–Crafts succi-
noylation afforded butyric acid I and subsequent treatment with bro-
mine in diethyl ether gave the monobromo compound II. The
thioamidesIVwereobtained by treating the nitriles III withhydrogen
sulfide in a mixture of pyridine and triethylamine. The compounds
5h–5s were characterized with respect to binding affinity (COS7 or
HEK385-7 cells with comparable affinities observed) and functional
antagonistic activity using a bioluminescence resonance energy
transfer (BRET) assay (Table 2).12–14
The introduction of a para halogen substituent in the western
(5i and 5j) or eastern (5n and 5o) phenyl ring gave compounds
equipotent to the parent compound 5h. Notably, introduction of
a para methoxy substituent in the western ring (5k) led to a 10-
fold drop in potency whereas the activity was retained by substi-
tuting the eastern phenyl ring (5p). A para ethyl substituent (5l)
in the western ring gave a functionally more active compound
but only with partial antagonistic activity. Better potency gains
were obtained by increasing lipophilicity by introduction of a phe-
nyl group in the western para position (5m) or phenoxy groups in
the eastern para position (5q–5s). The influence of electron donat-
ing (5r) or attracting (5s) substituents only had marginal effects on
the activity of the unsubstituted 5q.
0.96
5.0
0.35c
0.63
3.7
1.6
1.7
0.66
1.6
1.3
OCH3
Cl
5s
[3H]PGD2 equilibrium competition binding (COS7 or HEK385-7).
Antagonistic activity as inhibition of b-arrestin translocation measured in a
bioluminescence resonance energy transfer (BRET) assay in HEK385-7 cells. All
compounds displayed efficacy above 70% unless noted. All values are single or mean
of double determinations.
a
b
c
Compound having 50% antagonistic efficacy.
We expanded the SAR of the original 2-anilinothiazoleacetic
acids hits 4a and 4b by making a diverse set of compounds 4c–
4w using the same chemical route outlined in Scheme 1 starting
with thioureas IV. No significant activity differences by introducing
halogen, trifluoromethyl or methyl substituents (4d, 4e, 4l, 4n, 4p)
into the unsubstituted compound 4c were observed (Table 3). A
drop in antagonistic potency was however noted for the isopropyl
derivative 4o. Introduction of a methoxy group in western or east-
ern para positions (4f, 4q) or eastern ortho positions (4h, 4i) led to
comparable activities. A phenyl group in western para position (4g,
4j, 4m) did not improve potency. However, a more flexible phen-
oxy substituent gave rise to improved potency (4k, 4r, 4s) provided
R2
O
R3
A
+
O
III
IV
N
R1
R4
O
c
a
R3
O
R2
Table 1
Binding affinity on hCRTH2 of 2,4-diphenylthiazoleacetic acids extracted by ligand-
based searches from publicly available compound collections
OH
OH
H2N
A
I
R4
O
S
R3
R1
R4
N
S
b
R2
R3
Br
O
d
R1
II
R4
O
R1
R3
COOH
R4
No.
R1
R2
R3
R4
IC50 Binda
(lM)
N
S
5a
5b
5c
5d
5e
5f
H
H
OCH3
OCH3
NMe2
Cl
OCH3
OCH3
H
H
H
Et
Br
OCH3
Br
Br
H
H
H
H
H
H
CH3
1.4
0.35
4.1
0.39
0.46
0.21
0.99
A = NH
A = bond 5
A = CH2
4
A
R2
6
OH
O
H
H
OCH3
CH3
Scheme 1. Reagents and conditions: (a) AlCl3, <À10 °C with gradual warming to rt,
over night; (b) 1.1 equiv Br2 in Et2O, rt, 4 h; (c) H2S in pyridine/Et3N 5:1, rt, 3 days;
(d) equimolar amounts of II and IV, DMF, 100 °C, 10 min, microwave oven.
5g
Cl
[3H]PGD2 equilibrium competition binding in COS7 cells.
a

Ø. Rist et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1177–1180
1179
Table 3
Table 4
Binding affinity and functional antagonism on hCRTH2 of 2-anilino-4-phenylthiaz-
oleacetic acids
Binding affinity and functional antagonism on hCRTH2 of 2-benzyl-4-phenylthiaz-
oleacetic acids
R1
R1
R3
R3
N
S
N
S
R2
R2
N
H
COOH
COOH
R3
R1
R2
R3
IC50 Binda
(lM)
IC50 BRETb
(lM)
No.
R1
R2
IC50 Binda
(lM)
IC50 BRETb
(lM)
No.
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
H
H
H
H
H
H
Cl
H
Cl
OCH3
H
H
F
F
F
Cl
F
F
0.18
4.1
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
F
H
F
Cl
OCH3
Ph
F
0.35
0.65
0.10
1.9
0.66
1.5
1.5
1.3
1.3
2.4
1.2
2.1
0.73
1.9
Cl
Cl
Cl
CH3
OCH3
H
H
H
H
H
0.088
0.050
0.36
0.25
0.33
0.14
0.70
0.024
0.041
0.17
0.58
0.22
0.85
1.8
m-OCH3
H
Br
Ph
Ph
(p-CN)Ph
(m-CN)Ph
(p-OCH3)Ph
(m-OCH3)Ph
(o-OCH3)Ph
(m-OCF3)Ph
3.8
0.18c
>100
0.37
0.51
0.19c
>100
0.27
0.39
>100
>100
Cl
0.45
0.31
0.049
0.96
0.94
0.70
1.2
0.75
0.94
0.084
0.089
0.21
0.13
0.22
0.094
Ph
OPh
F
Ph
Cl
Cl
Cl
H
OPh
OPh
OPh
OPh
Br
H
0.48
1.9
Cl
Cl
CF3
iPr
Me
OCH3
H
F
H
H
3.8c
1.1
H
H
H
H
0.34
F
F
F
F
0.072
0.040
0.17
16
0.56
1.5
0.35
0.54
>100
>100
0.37
0.26c
H
0.16
O
6q
H
F
0.024
1.5
O
Cl
6r
6s
6t
H
Cl
OPh
H
2-Naphthyl
Cl
OPh
Cl
0.16
0.0037
0.041
>100
0.066
0.57
2-Naphthyl
2-Naphthyl
OPh
a
c
b
and as in Table 2.
Compounds having 20–30% antagonistic efficacies.
a
c
b
and as in Table 2.
Compounds having about 30% antagonistic efficacies.
no halogen substituent was present in the ortho position (4t, 4u)
which led to loss in functional antagonistic activity. By annelating
the eastern phenyl ring to a naphthyl substituent a high functional
antagonistic potency was achieved with 4v, whereas the very lipo-
philic 4w only behaved as a weak partial antagonist.
In summary, we described the structure–activity relationships
of a series of arylated thiazoleacetic acids derived from hits ob-
tained after in silico screening and their progression to potent
and selective compounds of potential use as anti-inflammatory
agents.
Subsequently, a larger set of analogous compounds 6a–6t hav-
ing the anilinic nitrogen replaced with a methylene linker was syn-
thesized according to Scheme 1 (Table 4). The mono and dichloro
compounds 6a–6c indicate that most potency enhancement is
delivered by the eastern para chloro group. However, introduction
of a western methoxy group reduces this enhancement (6d vs 6b
and 6c). Methyl (6e) or methoxy (6f) in the eastern para position
gives no improvement, which also is true for a fluoro substituent
in the western ring (6g, 6h). However, activity is gained when a
phenyl group is introduced in the ortho position (6i, 6j) (Table 4).
Additional functionalisations were investigated on this motif as
illustrated with the biphenyl derivatives 6k–6q carrying less lipo-
philic substituents to explore if this could be done with retained
potency. The nitrile derivatives 6k an 6l had poor functional activ-
ity. The para and meta methoxy (6m, 6n) and the methylenedioxy
(6q) derivatives had potencies comparable to the parent 6i
whereas the ortho compound 6o had inferior functional activity.
A trifluoromethoxy group in meta position was also less active than
the corresponding methoxy derivative (cf. 6p and 6n). The ortho
biphenyl ether 6r is also functionally inactive in contrast to the
corresponding biphenyl compound 6j. By bringing the additional
phenyl system even closer (2-naphthyl 6t) potency and functional
activity is regained. Notably, a phenoxy group in the western ring
(6s) gives rise to the most potent compound of these series exhib-
iting single digit binding affinity to the CRTH2 receptor (3.7 nM
n = 6). It displays full functional antagonistic effect at 66 nM
(n = 2) in BRET and 12 nM (n = 6) in a cAMP assay. Furthermore,
Acknowledgements
The authors thank Stina Hansen, Joan Gredal, Rokhsana Ander-
sen, Ann Christensen, and Helle Zancho Andresen, for excellent
technical assistance.
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lM

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14. Radioligand binding assay was conducted with stably transfected COS7 or
HEK385-7 cells, expressing human CRTH2 receptor, by competition binding
using[3H]PGD2. Total and nonspecific binding were determined in the
absence and presence of 10
l
M
PGD2. An improved functional
Bioluminescence Resonance Energy Transfer (BRET2) assay was performed
on CRTH2 transfected HEK385-7 cells. Antagonists were preincubated with
the cell suspension using a shaking table for 5 min. PGD2 was then added to
each well to elicit about 75–80% of the maximal agonist efficacy and cells
were further incubated for 5 min. After the incubation period, the 96-well
microplate was placed in the Mithras LB 940 instrument and DeepBlueC
coelenterazine was injected to one well at a time. Five seconds after the
injection, the light output from the well was measured sequentially at
400 nm and 515 nm. The BRET signal was calculated by the ratio of the
fluorescence emitted by GFP2-b-arr2 (515 nm) over the light emitted by the
receptor-Rluc (400 nm).
10. (a) Clinical studies: (a) AZD1981 from AstraZeneca in asthma and COPD; http://
www.clinicaltrials.gov; (b) OC000459 from Oxagen in asthma and allergic
rhinoconjunctivitis; http://www.clinicaltrials.gov; (c) ADC3680 reported from
Argenta.; (d) AM211 reported from Amira Pharmaceuticals.
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