Identification of novel pyrazole acid antagonists for the EP1 receptor

Article abstract of DOI:10.1016/j.bmcl.2006.06.086

The discovery, synthesis and structure-activity relationship (SAR) of a novel series of EP₁ receptor antagonists is described. Pyrazole acid 4, identified from a chemical array, had desirable physicochemical properties, an excellent in vitro microsomal inhibition and cytochrome P450 (CYP450) profile and good exposure levels in blood. This compound had an ED₅₀ of 1.3 mg/kg in a rat pain model. A range of more potent analogues in the in vitro assay was identified using efficient array chemistry. These EP₁ antagonists have potential as agents in the treatment of PGE₂ mediated pain.

Full text of DOI:10.1016/j.bmcl.2006.06.086

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4767–4771
Identification of novel pyrazole acid antagonists for the
EP1 receptor
Stephen C. McKeown,a,* Adrian Hall,b Gerard M. P. Giblin,b Olivier Lorthioir,a
Richard Blunt,a Xiao Q. Lewell,a Richard J. Wilson,a Susan H. Brown,b
Anita Chowdhury,b Tanya Coleman,b, Stephen P. Watson,a Iain P. Chessell,b
Adrian Pipe,a Nick Claytonb and Paul Goldsmithb
aDiscovery Research, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
bNeurology and Gastrointestinal Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park,
Third Avenue, Harlow, Essex CM19 5AW, UK
Received 25 May 2006; revised 29 June 2006; accepted 29 June 2006
Available online 14 July 2006
Abstract—The discovery, synthesis and structure–activity relationship (SAR) of a novel series of EP1 receptor antagonists is
described. Pyrazole acid 4, identified from a chemical array, had desirable physicochemical properties, an excellent in vitro micro-
somal inhibition and cytochrome P450 (CYP450) profile and good exposure levels in blood. This compound had an ED50 of 1.3 mg/
kg in a rat pain model. A range of more potent analogues in the in vitro assay was identified using efficient array chemistry. These
EP1 antagonists have potential as agents in the treatment of PGE2 mediated pain.
Ó 2006 Elsevier Ltd. All rights reserved.
Prostaglandin E2 (PGE2) is one of a number of prosta-
glandins generated through the metabolism of arachi-
donic acid and acts locally both in the periphery and
CNS. PGE2 mediates a multitude of pharmacological
actions and acts predominantly through four identified
GPCRs (EP1–4).1 EP1 receptor-deficient mice show
reduced pain sensitivity, similar to the effects of COX-2
inhibition with a NSAID (non-steroidal anti-inflamma-
tory drug). EP1 receptor antagonist ONO-8711 has
shown efficacy in numerous preclinical models of pain
in rats, including allodynia,2 neuropathic pain,3 and
postoperative pain.4 Furthermore, the EP1 receptor
antagonist ZD-6416 (1) has demonstrated human effica-
cy in visceral pain.5 Hence, selective EP1 receptor antag-
onists would be expected to be efficacious in the
treatment of pain and, in addition, have the potential
to circumvent the gastrointestinal (GI) side effects asso-
ciated with NSAIDS,6 and the cardiovascular side
effects associated with COX-2 inhibitors.7 Several small
molecule EP1 receptor antagonists have been identified
by various companies, including ZD-6416 (1)8,9 from
AstraZeneca, ONO-8711 and ONO-8713 (2)10 from
Ono and thiophene 311 from Merck Frosst, Figure 1.
In our efforts to discover new EP1 antagonists we initi-
ated a chemistry effort exploring azole acids as motifs.
We chose azoles because of their prevalence in drug mol-
ecules that have good general physico-chemical proper-
ties and good bioavailability.12 We report here the
discovery, SAR and synthetic accessibility of pyrazole
acids (e.g., compound 4, Fig. 2) as a new series of EP1
antagonists.
We chose to utilize the well-established lipophilic ortho-
benzyloxyphenyl portion of known EP1 antagonists
(e.g., 3) as a privileged fragment to attach to a variety
of heterocyclic acids using simple alkylation chemistry,
Scheme 1.
Alkylation of 54 azole esters with bromide 5 (detailed
chemistry shown in Scheme 2) gave, after ester hydroly-
sis, the final products as azole acids. In some cases
regioisomers were formed as a result of the azole alkyl-
ation chemistry, these were not separated but assayed as
Keywords: EP1 antagonist; Pain; Pyrazole; Prostanoid.
*
Corresponding author. Tel.: +44 1279 627558; fax: +44 1279 87
5414; e-mail: Steve.C.McKeown@gsk.com
Present address: AstraZeneca, Mereside, Alderley Park, Macclesfield,
Cheshire SK11 4TG, UK.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.086

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S. C. McKeown et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4767–4771
O
O
S
O O
S
OH
CO2H
Cl
N
H
F3C
O
O
N
Br
O
N
N
O
S
O
N
O
1, ZD-6416
2, ONO-8713
3, Merck Frosst
Figure 1. Selective ‘non-prostanoid’ EP1 antagonists.
Table 1. Physicochemical, in vitro metabolism and CYP450 profile of 4
O
O
N
Parameter
Value
N
EP1 pKi = 7.8
EP3 pKi = <5.7
OH
Solubility @ pH 7.4 (mg/mL)
CYP450,a IC50 (lM)
0.85
21 @ 1A2,
Br
4
>100 @ 2C9,>100 @ 2C19,
>100 @ 2D6, >100 @ 3A4
Figure 2.
Microsomal Cli (mL/min/g liver)
ClogP
logD @ pH 7.4
<0.5 rat; 0.6 human
5.1
1.15
401
mixtures. Our initial hit, 4, was identified as a potent
EP1 antagonist having a pKi of 7.8 against EP1 and
pKi < 5.7 against EP3 in our FLIPR assays.13 The EP1
potency was confirmed in a [3H]PGE2 displacement
binding assay (pIC50 8.2).14 Pyrazole 4 was immediately
attractive because of its relatively low molecular weight
(401 Da), good solubility and selectivity against EP3.
Molecular weight
a In vitro CYP450 assay results using Gentest protocol.
Table 2. In vivo PK data for compound 4
Parameter
Value
Cmax (lM)
Tmax (h)
5.69 1.41
0.5
Compound 4 showed a low level of inhibition of CYP450
isoforms routinely tested and showed excellent stability in
our microsomal clearance assay giving intrinsic clearanc-
es of <0.5 (rat) and 0.6 (human) mL/min/g liver (Table 1).
AUC (lM h)
AUC/dose (min kg/L)
16.26 6.49
124 51
Initially we investigated the substitution on the pyrazole
ring. Each of the compounds was prepared according to
Scheme 2. Phenol 7 was selectively alkylated and the
intermediate alcohol was then converted with PBr3 to
the bromide 5. A selection of pyrazole esters (commer-
cially available) were then alkylated with 5 under basic
conditions (KOtBu). Ester hydrolysis gave the final car-
boxylic acids. Any regioisomers formed from the alkyl-
ation step were separated by chromatography,
characterized by NOE and HMBC NMR experiments
and assayed individually.
The in vivo pharmacokinetic (PK) profile of 4 was as-
sessed in rat (Table 2) (dose = 3 mg/kg po). The data
show a high level of 4 in blood after 0.5 h with a Cmax
of 5.69 lM and an AUC/dose of 124 min kg/L suggest-
ing good bioavailability (absolute bioavailability for this
compound was not determined).
These data suggested that 4 and close analogues would
have a suitable profile for oral administration. A chem-
istry programme was then initiated to explore the SAR
around 4.
O
O
O
C
i) 54 Azole Esters, Base
ii) Ester Hydrolysis
Azole
OH
Br
B
Br
6
Br
5
Scheme 1. Array chemistry plan.
Br
Br
O
O
N
a,b
O
c,d
N
Br
OH
OH
OH
Br
7
4
5
Scheme 2. Reagents and conditions: (a) EtOH, NaOH, BnBr; (b) PBr3; (c) KOtBu, EtOH, azole ester, 60 °C; (d) LiOH, 40 °C, EtOH.

S. C. McKeown et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4767–4771
4769
Table 4. EP1 Potency of heterocycle replacement
No increase in potency was observed by varying either
the substituent at the 4- or the 5-position of the pyrazole
(Table 3). The des-methyl compound 8 showed similar
potency to the hit molecule 4. All analogues with sub-
stituents at the 4-position were less potent. Interestingly,
increasing the size of the group at the 5-position from
methyl to n-butyl (11) also led to a loss in potency.
Introduction of a larger, more polar acetamide substitu-
ent also led to a decrease in potency (10).
O
R
Br
Compound
R
Av EP1 pKi (FLIPR)13
N
8
7.26 0.29
N
N
CO2H
We then investigated the effect on potency of the hetero-
cycle and the importance of the regiochemistry of the
carboxylic acid using the chemistry described in Scheme
2 and employing commercially available heterocyclic
esters. Imidazole 18 and pyrazole 19 were inactive (Table
4) suggesting that the 1,3 relationship of the carboxylic
acid and the methylene linker was required for optimal
EP1 potency. It also appeared that the heteroatom in
the 2-position of the pyrazole was very important since
the analogues 20–22 were far less potent. We believe that
the 2-nitrogen between the carboxylic acid and methy-
lene linker plays a significant role in varying the affinity
to the receptor by influencing the acidity of the carbox-
ylic acid (either by electron density effects or by means
of an internal H-bond with the carboxylic acid) or by
forming an additional H-bond with the receptor.
CO2H
18
<5.7
N
CO2H
19
20
21
22
<5.7
<5.7
<5.7
<5.7
N
N
N
N
CO2H
CO2H
N
N
N
N
N
CO2H
We next examined the substitution around phenyl ring
C of the benzyl group. The synthesis of the analogues
is described in Scheme 3. The hydrazine 24 was formed
by reductive alkylation of tert-butyl carbazate with phe-
nol aldehyde 23. After TFA deprotection, the hydrazine
24 was treated with ethyl 2,4-dioxopentanoate (25) to
yield predominantly the desired regioisomer 26 which
was conveniently purified by preferential crystallization
from the reaction mixture upon cooling. An efficient
method16 was then developed whereby, in the same
pot, after alkylation of the phenol 26 (NaOH and alkyl-
ating agent), LiOH was added to affect ester hydrolysis.
Final purification was achieved using chromatography.
The results in Table 5 show that substitution at the
3-position of ring C was detrimental to potency (entries
35–41). However, substitution at the 2- and/or 4-posi-
tion led to analogues with up to 100-fold increase in
potency for EP1 with the 2,4-dichloro derivative (27)
giving the highest functional potency (pKi 9.2). All com-
pounds in Table 5 showed no inhibition in our EP3
FLIPR assay (pKi < 5.7).
Finally, we undertook a combinatorial approach to
investigate the effect of varying the bromine substituent
in 4 with either Cl, F, OMe, or H, together with varying
ring C with those substitutions that gave an increase in
potency relative to 4 in Table 5. The syntheses of the
four phenol intermediates were achieved by using the
conditions described in Scheme 3.16 Each of the four
phenols was then alkylated with 7 chosen alkylating
agents.
Table 3. EP1 Potency of pyrazole substitutions
O
O
N
N
OH
5
4
R
Br
Table 6 shows the potency of selected results from the
array with the bromo- and chloro-substituent, giving
highest potency on central ring B. Replacement of the
bromo with hydrogen (46) resulted in a 10-fold drop
in potency. Figure 3 shows the results of the complete
combinatorial array (and includes the original bromo
compounds for comparative purposes) and illustrates
that the SAR is consistent between the substituents on
ring C and B, with 2,4-dichloro consistently giving the
higher potency and the chloro substituent giving gener-
ally higher potency over bromo on ring B.
Compound
R
Av EP1 pKi
(FLIPR)13
Av EP1 pIC50
(binding)14
4
8
5-Me
5-H
7.82 0.38
7.26 0.29
6.21 0.14
<5.7
8.19 0.04
8.01 0.10
6.71 0.07
9
5-(2-)Thiophene
5-NHCOMe
5-nBu
10
11
12
13
14
15
16
17
311
6.01 0.18
6.16 0.32
5.87 0.16
<5.7
4-Fluoro
4-Me
4-Br
6.91 0.09
6.37 0.05
6.34 0.06
6.17 0.11
5.59 0.14
4-CH2OH
4-CF3
4-Cl
<5.7
<5.7
<5.7
8.90 0.6015
Each of the analogues 44–48 showed very low clearance
in the microsomal clearance assay and low inhibitory
n/a
8.45 0. 0415

4770
S. C. McKeown et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4767–4771
OH
OH
O
NH2
a-c
N
H
X
23: X=Br
24
X
O
O
OEt
d
O
25
O
OH
C
O
O
N
N
N
N
A
e,f
B
OH
OEt
Br
X
26
4
Scheme 3. Reagents and conditions: (a) AcOH, BocNHNH2; (b) Na(OAc3)BH; (c) TFA, CH2Cl2; (d) 25, AcOH, reflux, 1 h; (e) EtOH, NaOH, RBr,
70 °C, 16 h; (f) EtOH, LiOH, 40 °C, 3 h.
Table 5. EP1 Potency of benzyloxy analogues
10
9
O
C
O
N
N
R
A
B
3-Br
3-Cl
3-F
OH
8
Br
3-MeO
H
7
Compound
R
Av EP1 pKi
(FLIPR)13
Av EP1 pIC50
(binding)14
27
28
29
30
4
2,4-DiCl
2,4-DiF
4-Cl
9.31 0.20
8.58 0.41
8.06 0.33
7.93 0.34
7.82 0.38
7.54 0.19
7.16 0.19
7.01 0.11
6.98 0.15
6.89 0.23
6.37 0.15
6.28 0.09
6.47 0.54
5.96 0.07
5.95 0.15
<5.7
9.88 0.32
9.28 0.93
8.06 0.51
8.13 0.39
8.19 0.04
8.50 1.40
6
5
4-F
2,4-DiCl 2,4-DiF 2,6-DiF 2-Cl
4-Cl
4-F
H
H
2-Cl
Ring C Substitution (R1 )
31
32
33
34
35
36
37
38
39
40
41
42
43
2-MeO
4-CF3
2,6-DiF
3-Br
Figure 3. EP1 SAR around rings B (shapes) and C (x-axis).
7.45 0.50
7.83 0.55
7.75 0.12
6.94 0.66
6.90 0.53
(Freund’s Complete Adjuvant) in vivo rat model of
inflammatory pain.17 Pyrazole 4 was effective in this
model with an ED50 of 1.3 mg/kg. This and related com-
pounds are now the subject of further investigations.
3-Cl
3-Me
3-NO2
3-CF3
3,5-DiCl
6.51 0.61
In summary, we have described the discovery, synthesis
and structure–activity relationships (SARs) of a novel
series of selective EP1 antagonists. Pyrazole acid 4, iden-
tified from a chemical array, had desirable physicochem-
ical properties and was shown to attain high exposure
levels in blood after oral administration. A range of
potent analogues with potencies up to 9.3 (pKi) were
identified using efficient array chemistry. These EP1
antagonists show potential as useful agents in the treat-
ment of pain.
3-CF3,4-Cl
4-tBu
3,5-DiCF3
6.24 0.17
5.91 0.35
5.28 0.23
<5.7
<5.7
Table 6. EP1 Potency of examples from Scheme 3
7 x R
O
C
O
N
A
N
R
B
X
OH
X=Br,Cl, F, H,OMe
Compound
X
R
Av EP1 pKi
(FLIPR)13
Av EP3 pKi
(FLIPR)2
Acknowledgments
27
44
45
46
47
48
Br
Cl
Cl
H
F
2,4-DiCl
2,4-DiCl
2,4-DiF
2,4-DiCl
2,4-DiCl
2,4-DiF
9.31 0.20
9.26 0.14
8.95 0.24
8.08 0.01
7.69 1.14
8.10 0.16
<5.7
<5.7
We thank Pat McDonough and Klara Valko-Slegel for
carrying out the physical chemical measurements.
<5.7
<5.7
6.45 0.14
<5.7
F
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was added and the reaction mixture heated under nitrogen
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with ethanol (0.5 mL) and a 0.5 M solution of lithium
hydroxide in water (0.5 mL, 6.0 mg, 0.25 mmol) was added.
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assessed using
a Fluorimetric Image Plate Reader
(FLIPR). Values are means of at least three (EP1) or
two (EP3) experiments.