3-Urea-1-(phenylmethyl)-pyridones as novel, potent, and selective EP 3 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.08.137

A series of 3-urea-1-(phenylmethyl)-pyridones was discovered as novel EP₃ antagonists via high-throughput screening and subsequent optimization. The synthesis, structure-activity relationships, and optimization of the initial hit that resulted in potent and selective EP₃ receptor antagonists such as 11g are described.

Full text of DOI:10.1016/j.bmcl.2010.08.137

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6744–6747
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Urea-1-(phenylmethyl)-pyridones as novel, potent, and selective EP3
receptor antagonists
Yue H. Li a, , Pei-San Tseng c, Karen A. Evans a, Jon-Paul Jaworski b, Dwight M. Morrow b, Harvey E. Fries c,
⇑
Charlene W. Wu c, Richard M. Edwards c, Jian Jin c,
⇑
a Discovery Medicinal Chemistry, Discovery Research, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, PO Box 5089, Collegeville, PA 19426-0989, United States
b Screening & Compound Profiling, Discovery Research, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, PO Box 5089, Collegeville, PA 19426-0989, United States
c Cardiovascular and Urogenital Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-urea-1-(phenylmethyl)-pyridones was discovered as novel EP3 antagonists via high-
throughput screening and subsequent optimization. The synthesis, structure–activity relationships, and
optimization of the initial hit that resulted in potent and selective EP3 receptor antagonists such as
11g are described.
Received 26 July 2010
Revised 27 August 2010
Accepted 27 August 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
3-Urea-1-(phenylmethyl)-pyridones
EP3
Antagonists
Prostaglandins
Prostaglandins (PGs) are synthesized from arachidonic acid in
response to extracellular stimuli via the cyclooxygenase (COX1
and COX2) isozyme pathway. Once synthesized, PGs are immedi-
ately released from cells and act as hormones in the vicinity of
the cells to maintain local homeostasis.1 There are currently five
known naturally occurring PGs including PGD2, PGE2, PGF2, PGI2,
and thromboxane A2 (TXA2).2 Their ability to affect various physi-
ological and biological processes in the body depends solely on
their binding to the distinct prostanoid receptors. Prostanoid
receptors are G-protein coupled, rhodopsin type receptors with
seven transmembrane domains.3 The eight known prostanoid
receptors are EP1–4, DP, FP, IP, and TP. Prostaglandin E2 (PGE2), a
High-throughput screening (HTS) of the corporate compound
collection using a fluorometric imaging plate reader (FLIPR) assay
(which measures the inhibition of PGE2-induced [Ca2+]i-mobiliza-
tion in U2OS (human osteosarcoma) cells transfected with a vector
carrying full length human EP3 cDNA) led to the identification of
compound 1 as a hit with a functional pKi (fpKi) of 6.6 against
human EP3 receptor compound 1 also had good binding affinity
to the human EP3 receptor (Fig. 1).8 To improve potency and selec-
tivity, and establish tractable SAR, we focused on exploring the
highlighted four regions of compound 1: left-hand side (LHS) ben-
zyl, middle pyridone, urea linker (UL), and right-hand side (RHS)
phenyl moieties in Figure 1, and used the EP3 FLIPR assay as the
primary assay to support our SAR exploration.
SAR exploration of this chemical series was primarily achieved
through the general synthetic route outlined in Scheme 1. Benzyl
halides were used to alkylate 3-nitro-2(1H)-pyridinone under
basic conditions. Using KOH powder in DMSO, the reaction was
completed within 5 min to afford compound 4. Reduction of the
nitro group using tin(II) chloride in concentrated HCl gave the de-
sired 3-amino-2(1H)-pyridone 5. The urea was formed either by
pro-inflammatory mediator,4 binds preferentially to the four EP
5
receptors, EP1–4
.
Studies with EP3 knockout (KO) mice and EP3
agonists have implicated that activation of the EP3 receptor might
play a key role in fever generation, uterine contraction, gastric acid
secretion, smooth muscle contraction of GI tract, neurotransmitter
release, and sodium/water reabsorption in kidney.6
Several small molecule EP3 receptor antagonists have been
reported in the literature.7 Herein we describe the identification,
synthesis, structure–activity relationships (SAR), functional activ-
ity, and initial physicochemical properties of a novel series of
z3-urea-1-(phenylmethyl)-pyridones as potent and selective
human EP3 receptor antagonists.
reacting
5 with commercially available isocyanates or with
amines using triphosgene as the activating reagent. Intermediates
5 could also be reacted with sulfonyl chlorides, chloroformates,
acid chlorides, or carboxylic acids in a similar fashion to form sul-
fonamides, carbamates, and amides. Using this versatile three-
step synthetic route, multiple regions of this chemical series were
explored.
⇑
Corresponding authors. Tel.: +1 610 917 6999; fax: +1 610 917 7391.
E-mail address: yue.h.li@gsk.com (Y.H. Li).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.137

Y. H. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6744–6747
6745
Table 1
RHS
SAR of the left-hand side (LHS) benzyl moiety, compounds 1 and
7a–7o
LHS
H
N
UL
O
H
H
N
O
N
N
OMe
O
N
R1
O
OMe
N
H
Cl
Compound
R1
hEP3 FLIPRa (fpKi)
Substitution
1
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
2-Cl
H
2-F
2-Br
2-Me
3-F
3-Cl
3-Me
6.6
6.0
6.3
6.5
5.6
6.0
6.4
5.9
5.7
5.9
6.0
5.5
6.1
6.4
6.1
7.6
1
hEP3 binding: pKi = 7.8
hEP3 FLIPR: fpKi = 6.6
rEP3 FLIPR: fpKi = 6.3
Figure 1. HTS-hit compound 1.
3-MeO
4-F
4-Cl
We first explored the left-hand side (LHS) (R1) benzyl moiety of
the initial hit 1 while keeping the urea moiety and RHS constant. A
number of compounds were prepared and evaluated in the EP3
FLIPR assay (Table 1). It was observed that the position and nature
of the substituents on the benzylic phenyl ring was important for
EP3 potency. In general, electron-withdrawing substituents, as in
7b, 7c, 7e, 7f, were preferred over electron-donating ones, as in
7d, 7g, 7h. As for the position of substituents, it was found that
electron-withdrawing substituents in the ortho position had better
relative EP3 potency than their meta and para counter parts, as in 1
versus 7f, 7j, and 7b versus 7e, 7i. The best result was achieved by
installing both chloro and fluoro on the ortho positions (7o), which
improved the EP3 potency by more than 10-fold over the initial
hit 1.
4-Me
4-MeO
2-Cl-4-F
2-Cl-5-F
2-Cl-6-F
a
Mean of at least two determinations with standard deviation
of < 0.3.
Changes and requirements at the urea linkage (UL) was subse-
quently explored (Table 3). It was found that both hydrogen bond
donors on the urea moiety were critical to EP3 activity (1 vs 9a). In
addition, replacement of the urea with amides (9b, 9c) resulted in
the total loss of EP3 potency. Thus, the original N,N0-disubstituted
urea moiety was found to be optimal at this position.
Substitutions on the central pyridone ring were studied by
installing small substituents on the ring (Table 4). Unsubstituted
pyridone rings remained the most potent while compounds
10d–10g showed that small substituents at the 5- and 6-positions
retained some potency. Substitutions at the 4-position produced
inactive compounds (10a, 10b).
Having explored the four regions of defined in Figure 1 and
identified the potency-enhancing moieties, we then turned our
attentions to combining potency-enhancing moieties into single
molecules to further improve potency and prepared the com-
pounds listed in Table 5. We were pleased to find that, EP3 potency
improvement was indeed achieved when combining potency-
enhancing groups. For example, compound 11g with 2-chloro-6-
fluoro on the left-hand side benzyl ring (R1) and 3-ethyl on the
right-hand side phenyl ring (R2) showed an EP3 FLIPR fpKi of 8.2,
a more than 25-fold potency increase comparing to the initial hit.
We next investigated variation of the RHS phenyl moiety R2
while keeping other regions of 1 constant (Table 2). Repositioning
the methoxy group from the meta position (1) to the ortho and the
para position on the phenyl ring (8f, 8x) led to a decline in EP3
potency. It was found in general that substituents in the para posi-
tion resulted in the loss of activity. Variation of the ortho position
showed small substituents were tolerated, but no significant
improvement of activity was observed, as in 8a, 8b. Replacing
the methoxy group with other polar groups resulted in loss of po-
tency (8l, 8p, 8q, 8r) while replacement with electron-withdrawing
groups such as fluoro, chloro, bromo, iodo, and cyano resulted in
10-fold reduction of EP3 potency (8g, 8i, 8j, 8k). Improvement of
EP3 potency was achieved when the methoxy group was replaced
with smaller non-polar alkyl groups. In particular, both 3-ethyl
(8n) and 3-isopropyl (8o) analogs showed more than three-fold
improvement of EP3 potency and were the best substituents at this
RHS region.
O
H
R1
R1
NO2
b
N
O
+
a
N
Br
NO2
2
3
4
O
O
R2
R1
R1
H
N
H
N
NH2
c or d
N
N
O
5
6
Scheme 1. Reagents and conditions: (a) KOH (powder), DMSO, rt, 5 min; (b) SnCl2, HCl, rt; (c) R2NCO, DCM, rt; (d) Aryl–NH2, triphosgene, DIEA, DCM, rt.

6746
Y. H. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6744–6747
Table 2
Table 4
SAR of the right-hand side (RHS) phenyl moiety, compounds 1 and
8a–8x
SAR of substitutions on pyridone ring, compounds 1, 8n and 10a–10g
O
H
R2
O
N
N
O
H
R2
N
H
O
N
N
4
Cl
N
H
5
Cl
6
R3
R2
R3
hEP3 FLIPRa (fpKi)
Compound
R2
hEP3 FLIPRa (fpKi)
Compound
1
8a
8b
8c
8d
8e
8f
8g
8i
8j
3-OMe
2-F
2-Cl
2-I
2-Me
2-Et
2-OMe
3-F
3-Cl
6.8
6.8
6.9
5.3
6.1
5.4
6.2
5.8
6.0
1
8n
MeO
Et
Et
MeO
Et
Et
H
H
6.8
7.3
<4.6
5.9
6.7
6.3
5.9
6.9
10a
10b
10d
10e
10f
10g
4-Me
5-Me
5-Me
5-Cl
6-Me
6-Me
MeO
Et
a
Mean of at least two determinations with standard deviation of < 0.3.
3-Br
6.0
8k
8l
3-I
6.4
<4.6
6.6
7.3
7.2
<4.6
<4.6
<4.6
<4.6
<4.6
5.5
<4.6
<4.6
<4.6
3-CN
3-Me
3-Et
3-Isopropyl
3-Benzyl
3-CH2COOH
3-CH2COOCH3
3-COCH3
4-Cl
4-Me
4-Et
4-CF3
4-OMe
8m
8n
8o
8p
8q
8r
8s
8t
8u
8v
8w
8x
Table 5
hEP3 potency of some key combination compounds, compounds 1 and 11a–11g
R1
O
H
R2
O
N
N
N
H
Compound
R1
R2
hEP3 FLIPRa (fpKi)
a
1
11a
11b
11c
11d
11e
11f
2-Cl
3-MeO
3-Et
3-Et
3-Et
3-i-Pr
3-Et
6.8
6.7
6.9
7.0
7.2
7.3
7.6
8.2
Mean of at least two determinations with standard deviation
3-CF3
2-Cl, 4-F
2-F
2-Cl
2-Cl
of < 0.3.
2-Cl, 6-F
2-Cl, 6-F
3-MeO
3-Et
Table 3
11g
SAR of the urea moiety, compounds 1 and 9a–9c
a
Mean of at least two determinations with standard deviation of < 0.3.
Cl
O
R3
OMe
N
transporters, and ion channels (<23% inhibition at 1 lM against
all 50 targets in the panel) and showed at least 100-fold selectivity
versus a number of 7TM receptors including other prostanoid
receptors, such as hEP1, hEP2, hPE4, hDP, hFP, and hTP. It was found
that the potency of 8n at the rat receptors was comparable to that
for the human receptor with similar selectivity against rat EP1
receptor. Additionally, since prostanoid synthesis is dependent
upon the oxidative metabolism of arachidonic acid, COX1/2 activ-
ity was assayed. Compound 8n was inactive in COX1 and COX2
enzyme inhibitory assays. Moreover, no agonist activity was ob-
served for compounds in this series at the human EP3 receptor as
exemplified by compound 8n (Table 6).
R3
Compound
hEP3 FLIPRa (fpKi)
H
H
N
N
1
6.8
O
H
N
N
9a
<4.6
O
O
O
H
N
Compound 8n also had no hERG liability (dofetilide binding
pKi <5), though it showed some inhibition against P450 isozyme
1A2 (pKi = 5.7) and was clean against other major isozyme. Inter-
estingly, no 1A2 inhibition was observed in more potent analog
9b
9c
<4.6
<4.6
H
N
11f (IC50s: 1A2 > 25
25 M; 3A4 > 25 M).
The compounds in the series generally exhibited good artificial
lM; 2C19 = 4.5 lM, 2C9 = 3.7 lM; 2D6 >
l
l
a
Mean of at least two determinations with standard deviation
of < 0.3.
membrane permeability (e.g., 220 nm/s for 11f; 550 nm/s for 1),
but low aqueous solubility. In a rat pharmacokinetic (PK) study
(dosed at 1.2 mg/kg iv and 2.0 mg/kg po), compound 1 demon-
strated moderate clearance (Clb = 24 mL/min/kg) with a half life
of 1.3 h, volume of distribution (Vdss) of 3.4 L/kg and oral bioavail-
ability (F) of 27%. The initial developability data suggests that this
series constitutes a reasonable starting point for further lead
optimization.
Key compounds in the series were then evaluated for selectiv-
ity, cross-species activities, hERG and CYP450 inhibition, and
in vivo PK properties.
With regards to selectivity, compound 8n, for example, was
found to be highly selective in a CEREP screen of 50 receptors,

Y. H. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6744–6747
6747
Table 6
4. (a) Claveau, D.; Sirinyan, M.; Guay, J.; Gordon, R.; Chan, C.-C.; Bureau, Y.;
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Yamakawa, K.; Takegoshi, Y.; Mikami-Nakanishi, M.; Nakatani, Y.; Oh-ishi, O.;
Yasui, H.; Azuma, Y.; Hirasawa, N.; Ohuchi, K.; Kawaguchi, H.; Ishikawa, Y.; Ishii,
T.; Uematsu, S.; Akira, S.; Murakami, M.; Kudo, I. J. Biol. Chem. 2004, 27, 33684;
(c) Murakami, M.; Nakatani, Y.; Tanioka, T.; Kudo, I. Prostaglandins Other Lipid
Mediat. 2002, 68–69, 383; (d) Mabuchi, T.; Kojima, H.; Abe, T.; Takagi, K.;
Sakurai, M.; Ohmiya, Y.; Uematsu, S.; Akira, S.; Watanabe, K.; Ito, S. Neuroreports
2004, 1, 1395.
In vitro selectivity data for compound 8n9
O
H
Et
O
N
N
N
H
Cl
Selectivitya (pKi)
hEP1
<5
hTP
hEP2
<5
COX1
hEP4
<5
COX2
hDP
<5
Rat-EP3
hFP
<5
hEP3
EC50
<5
5. (a) Coleman, R. A.; Smith, W. L.; Narumiya, S. Pharmacol. Rev. 1994, 4, 205; (b)
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b
<5
<4
<4
6.8
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Gastroenterology 1988, 94, 1121; (c) Ushikubi, F.; Segi, E.; Sugimoto, Y.; Murata,
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a
Values are a mean of at least two determinations with a standard deviation of
< 0.3 log units.
b
Functional pKi.
As shown in Figure 1 and Table 6, the series have excellent
cross-species activities. For example, both the initial hit 1 and com-
pound 8n had fpKi of 6.3 and 6.8 against rat EP3 receptors.
In summary, exploration of a novel series of 3-urea-1-(phenyl-
methyl)-pyridones identified from high-throughput screening led
to the discovery of potent and selective human EP3 antagonists
exemplified by compound 11g (fpKi = 8.2). The series possesses
excellent functional activity, selectivity, and good initial developa-
bility properties.
Acknowledgments
The authors thank Walter Johnson for his assistance with the
LCMS spectra and Victoria Magaard for her assistance with com-
pound purification.
Supplementary data
8. (a) Functional activity was determined by measuring mobilization of
intracellular calcium on
a fluorometric imaging plate reader (FLIPR) as
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.137.
described in detail in Ref. 6f; (b) fpKi = IC50/(1+[A]/EC50), assuming slope = 1,
where [A] = ligand concentration, EC50 = ligand concentration at 50% activation;
IC50 = compd potency in antagonist mode.
References and notes
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COX1 and COX2 inhibition was measured using human whole blood in ELISA
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C.; Iacobelli, S.; Cipollone, F.; Ganci, A.; Creminon, C.; Maclouf, J.; Patrono, C. J.
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