Novel 3-oxazolidinedione-6-aryl-pyridinones as potent, selective, and orally active EP3 receptor antagonists

Article abstract of DOI:10.1021/ml100077x

High-throughput screening and subsequent optimization led to the discovery of novel 3-oxazolidinedione-6-aryl-pyridinones exemplified by compound 2 as potent and selective EP₃ antagonists with excellent pharmacokinetic properties. Compound 2 was orally active and showed robust in vivo activities in overactive bladder models. To address potential bioactivation liabilities of compound 2, further optimization resulted in compounds 9 and 10, which maintained excellent potency, selectivity, and pharmacokinetic properties and showed no bioactivation liability in glutathione trapping studies. These highly potent, selective, and orally active EP₃ antagonists are excellent tool compounds for investigating and validating potential therapeutic benefits from selectively inhibiting the EP₃ receptor.

Full text of DOI:10.1021/ml100077x

pubs.acs.org/acsmedchemlett
Novel 3-Oxazolidinedione-6-aryl-pyridinones as Potent,
Selective, and Orally Active EP3 Receptor Antagonists
,†
ꢀ
Jian Jin,* Angel Morales-Ramos, Patrick Eidam, John Mecom, Yue Li, Carl Brooks,
Mark Hilfiker, David Zhang, Ning Wang, Dongchuan Shi, Pei-San Tseng, Karen Wheless,
Brian Budzik, Karen Evans, Jon-Paul Jaworski, Jack Jugus, Lisa Leon, Charlene Wu,
Mark Pullen, Bhumika Karamshi, Parvathi Rao, Emma Ward, Nicholas Laping,
Christopher Evans, Colin Leach, Dennis Holt, Xin Su, Dwight Morrow, Harvey Fries,
Kevin Thorneloe, and Richard Edwards
GlaxoSmithKline, 709 Swedeland Road, King of Prussia, Pennsylvania 19406
ABSTRACT High-throughput screening and subsequent optimization led to the
discovery of novel 3-oxazolidinedione-6-aryl-pyridinones exemplified by com-
pound 2 as potent and selective EP3 antagonists with excellent pharmacokinetic
properties. Compound 2 was orally active and showed robust in vivo activities in
overactive bladder models. To address potential bioactivation liabilities of com-
pound 2, further optimization resulted in compounds 9 and 10, which maintained
excellent potency, selectivity, and pharmacokinetic properties and showed no
bioactivation liability in glutathione trapping studies. These highly potent, selec-
tive, and orally active EP3 antagonists are excellent tool compounds for investigat-
ing and validating potential therapeutic benefits from selectively inhibiting the EP3
receptor.
KEYWORDS EP3 receptor, novel, potent, selective, and orally active antagonists,
3-oxazolidinedione-6-aryl-pyridinones
rostanoids are members of a family of biologically
active lipids derived from arachidonic acid that include
prostaglandin E2 (PGE2), PGF2R, PGI2, PGD2, and throm-
response to bladder distension, nerve stimulation, inflam-
matory mediators, and mechanical trauma.6 Elevated urin-
ary PGE2 has been associated with OAB in humans.7,8 Third,
EP3 receptors are present in dorsal root ganglia and enhance
the excitability of dorsal root ganglia neurons.9 Finally, a
number of small clinical studies have shown that cycloox-
ygenase (COX) inhibitors that function to reduce production
of PGE2 improve OAB symptoms.10
A number of small molecule EP3 receptor antagonists
have been reported recently.11 -16 In this letter, we disclose
a novel series of 3-oxozolidinedione-6-aryl-pyridinones as
potent, selective, and orally active EP3 receptor antagonists,
which demonstrated robust in vivo activities in several OAB
animal models.
P
boxane A2. Each of the five prostanoids has a distinct receptor
class, named as EP, FP, IP, DP, and TP. Through their respective
receptors, prostanoids mediate a wide array of physiological
and pathophysiological processes including inflammation,
pain, smooth muscle function, platelet function, neuronal func-
tion, and kidney function.1,2 Among the prostanoids, PGE2 is the
most widely produced and has a number of diverse actions.1
Molecular cloning has revealed the existence of four different
receptors for PGE2, which accounts for the diverse effects of this
prostanoid. These receptors are designated as EP1, EP2, EP3,
and EP4 and are encoded by distinct genes.3
Overactive bladder (OAB) is characterized by symptoms
of increased voiding frequency and urgency. Although the
exact cause remains unknown, both bladder smooth muscle
excitability and increased bladder sensory nerve sensitivity
have been proposed.4 Several lines of evidence support that
EP3 receptor antagonism could provide a new and effective
treatment option for OAB. First, EP3 receptor knockout (KO)
mice demonstrated an enhanced bladder capacity as com-
pared to wild-type (WT)controls. Infusion of PGE2 or a selec-
tive EP3 receptor agonist, GR63799, into WT mice induced
bladder overactivity. These responses were absent in
KO mice.5 Second, PGE2 is synthesized in the bladder in
High-throughput screening (HTS) of the corporate com-
pound collection using a human EP3 fluorometric imaging
plate reader (FLIPR) assay17 led to the identification of 3-ox-
azolidinedione-6-aryl-pyridinone1 asan EP3 antagonist with a
functional pKi (fpKi) of 6.7 (Figure 1).18-20 Thecompoundwas
subsequently tested in human EP1 and rat EP3 FLIPR assays
and found to have good selectivity for EP3 over EP1 and good
cross-species activity.21 In a rat pharmacokinetic (PK) study,22
Received Date: April 15, 2010
Accepted Date: May 6, 2010
Published on Web Date: May 14, 2010
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Scheme 1. Synthesis of Pyridinones 1 and 2a
Figure 1. HTS hit 1.
a Reagents and conditions: (a) PXPd2, K2CO3, MeOH, 60 °C, 89%. (b) (i)
t-BuLi, THF, -78 °C; (ii) ethyl 3-methyl-2-oxobutyrate or ethyl 2-ox-
oacetate, THF, -78 °C to room temperature (4a, 30%; 4b, 62%). (c) (i)
Trichloroacetyl isocyanate, CH2Cl2, room temperature; (ii) 2 M K2CO3
aqueous solution, reflux (5a, 80%; 5b, 48%). (d) TMSCl, NaI or TMSI,
CH3CN, room temperature (1, 49%; 2, 58%).
Figure 2. Profiles of pyridinone 2.
2C9, 2D6, and 3A4) and in the hERG binding assay and had
good aqueous solubility (193 μM). The combination of high
potency with excellent cross-species activities, selectivity,
and rat PK properties makes pyridinone 2 an outstanding
tool compound for in vivo studies.
The synthesis of pyridinones 1 and 2 is outlined in Scheme 1.
Suzuki coupling of commercially available 2-bromo-6-methoxy-
pyridine with 2-naphthyl boronic acid yielded methoxypyridine
3, which was then deprotonated and added to R-keto esters to
afford R-hydroxy esters 4a and 4b. Oxozolidinediones 5a and
5b were prepared from R-hydroxy esters 4a and 4b via a one-
pot two-step sequence using trichloroacetyl isocyanate.25 De-
methylation of methoxypyridines 5a and 5b resulted in the
desired pyridinones 1 and 2 in good overall yields.
Pyridinone 2 was first evaluated in a GR63799-induced
OAB model in conscious, spontaneously hypertensive rats
(SHR).26 As shown in Figure 3, intraduodenal (id) pretreat-
ment with 2 at doses of 3 and 30 mg/kg (p = 0.04 and p =
0.01 vs GR63799 þ vehicle, two-way RM ANOVA) signifi-
cantly inhibited the GR63799-induced decrease in bladder
capacity (as measured by average voided volume), while
0.3 mg/kg of 2 was found to be ineffective. Compound 2 was
next tested in a PGE2-induced OAB model in the conscious
SHR.5 Intravesical infusion of PGE2 (120 μM) led to reduced
bladder capacity (Figure 4). Pretreatment with 2 (30 mg/kg,
id) (p = 0.006 vs PGE2 þ vehicle, two-way RM ANOVA)
significantly inhibited the bladder capacity decrease caused
by the PGE2 infusion. Compound 2 was also evaluated in an
acetic acid-induced OAB model in conscious SHR.5 Similar to
PGE2, intravesical infusion of acetic acid (0.25%) also re-
sulted in a bladder capacity decrease (Figure 5).5 This
bladder capacity decrease was significantly inhibited by id
pyridinone 1 had a moderate to high clearance, good half-life,
low dose-normalized area under curve (DNAUC) via oral
administration (po), and moderate oral bioavailability.
Optimization of this promising hit to improve potency and
PK properties led to the discovery of compound 2, which
possesses an isopropyl group at the 5-position of the oxazo-
lidinedione ring. Pyridinone 2 had excellent potency in
human EP3 FLIPR assay with an fpKi of 8.0, which is about
20-fold more potent as compared to compound 1 (Figure 2).
Compound 2 was also tested in a human EP3 binding assay23
and was found to have high binding affinity (pKi = 8.3),
consistent with its potency (fpKi = 8.0) in the FLIPR assay. In
addition, pyridinone 2 was very potent in rat (fpKi = 8.0)
and dog (fpKi = 7.7) EP3 FLIPR assays,21 demonstrating
excellent cross-species activities. Compound 2 had excellent
selectivity for EP3 over other EP and prostanoid receptors. It
was inactive in human EP1 FLIPR,21 EP2 LANCE,21 EP4
binding,23 DP binding,23 and FP FLIPR21 assays. Because
COX inhibitors function to reduce production of PGE2, this
promising lead was evaluated in human COX1 and COX2
enzyme inhibitory assays and rat COX1 and COX2 whole
blood assays to assess its selectivity for EP3 over COX1 and
COX2. We were pleased to find that pyridinone 2 was
inactive in the COX assays.24 Pyridinone 2 was next eval-
uated in a rat PK study22 and found to have a low clearance
(Cl = 2.6 mL/min/kg), good half-life (T1/2 = 4.0 h), and high
oral exposure as demonstrated by excellent oral DNAUC
(5.5 μg h/mL/mg/kg) and oral bioavailability (F = 83%). In
addition to excellent PK parameters, 2 had good develop-
ability properties. For example, 2 was inactive against all five
common cytochrome P450 (CYP450) isozymes (1A2, 2C19,
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Figure 6. Effect of iv administration of 2 on bladder rhythmic
contraction in anesthetized SD rats.
However, compound 2 may have potential bioactivation
liabilities as evidenced by the findings in glutathione (GSH)
trapping studies: NADPH-dependent GSH conjugates were
observed when rat liver S9 fractions were used, while no GSH
conjugates were observed when using human liver S9 frac-
tions (Table 1).21 It was hypothesized that the unsubstituted
naphthyl moiety of compound 2 could be oxidized to form
reactive metabolites. To overcome the potential bioactiva-
tion liabilities, compounds 8-10 were prepared using a new
synthetic strategy that introduces the right-hand side aryl
group closer to the end of the reaction sequence (Scheme 2).
Commercially available 2-chloro-6-methoxypyridine was
converted to R-hydroxy ester 6, which was subsequently
converted to oxozolidinedione 7 using chemistry similar to
that outlined in Scheme 1. Suzuki coupling of chloropyridine
7 with commercially available (1-methyl-1H-indol-5-yl)boro-
nic acid, 3,4-dimethylphenyl boronic acid, or boronic acid
12, followed by deprotection, afforded the desired pyridi-
nones 8, 9, and 10. Boronic acid 12 was prepared from
1-fluoro-naphth-7-ol 11,27 which was synthesized from com-
mercially available 8-amino- naphth-2-ol in three steps.
Compounds 8-10 had excellent potency in human and
rat EP3 FLIPR assays21 (Table 1). In particular, 8-fluoronaph-
thyl analog 10 was slightly more potent than unsubstituted
naphthyl compound 2. In rat PK studies,22 compounds 8-10
displayed excellent PK parameters-low clearance, good
half-life, and high oral exposure as demonstrated by excel-
lent oral DNAUC and oral bioavailability, similar to com-
pound 2. In addition, compounds 8-10 were inactive in EP1,
EP2, EP4, DP, TP, FP, COX1, and COX2 assays-demonstrat-
ing excellent selectivity for EP3 over other EPand prostanoid
receptors. In GSH trapping studies,21 no NADPH-dependent
GSH conjugates were detected for compounds 9 and 10
when using either rat or human liver S9 fractions. No GSH
conjugates were detected in the absence of NADPH as well.
Compound 8 was not metabolized by either rat or human
liver S9 fractions in the same experiment. These findings
indicated that the unsubstituted naphthyl moiety of com-
pound 2 was indeed responsible for the potential bioactiva-
tion liabilities, and such potential liabilities could be miti-
gated by replacing the unsubstituted naphthyl group with
other aryl groups (8 and 9) or a properly substituted naphthyl
group (10).
Figure 3. Effect of id administration of 2 on the GR63799-induced
decrease in conscious SHR bladder capacity.
Figure 4. Effect of id administration of 2 on the PGE2-induced
decrease in conscious SHR bladder capacity.
Figure 5. Effect of id administration of 2 on the acetic acid-
induced decrease in conscious SHR bladder capacity. Comparison
to the COX inhibitor ketoprofen.
pretreatment of 2 at 30 mg/kg (p = 0.002 vs acetic acid þ
vehicle, two-way RM ANOVA), an effect similar to that
observed with the COX inhibitor ketoprofen.
In addition, compound 2 was evaluated in a bladder
rhythmic contraction model in anesthetized Sprague-Daw-
ley (SD) rats.23 Intravenous (iv) administration of compound
2 at 3 and 10 mg/kg (p < 0.05 vs vehicle for both doses, two-
way ANOVA) significantly and dose dependently inhibited
the bladder rhythmic contraction induced by intravesical
infusion of saline (Figure 6). The robust in vivo activities in
several OAB models indicate that this novel and selective EP3
receptor antagonist series could potentially lead to effective
therapeutic agents for treating OAB.
In conclusion, a novel series of 3-oxozolidinedione-6-aryl-
pyridinones exemplified by compound 2 was discovered as
potent and selective EP3 receptor antagonists with excellent
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Table 1. Profile of 3-Oxazolidinedione-6-aryl-pyridinones 8-10
EP3 FLIPR fpKi rat PK parameters
NADPH-dependent GSH conjugates
T1/2
(h, iv)
Cl
DNAUC
(po) (μg h/mL/mg/kg)
oral F
(%)
human liver S9
fractions
rat liver S9
fractions
compound
human
rat
(mL/min/kg)
1
2
6.7
8.0
8.2
7.7
8.5
6.5
8.0
8.1
7.9
8.5
5.1
4.0
4.2
2.8
3.9
31
0.17
5.5
3.0
1.8
5.6
32
83
80
68
93
not tested
not tested
detected
2.6
4.5
6.3
2.8
not detected
8
no compound turnover
not detected
9
not detected
not detected
10
not detected
Scheme 2. Synthesis of Pyridinones 8-10a
SUPPORTING INFORMATION AVAILABLE Synthetic pro-
cedures and characterization data for all compounds; procedures
for human, rat, and dog EP3, EP1, EP2, and FP assays; and GSH
trapping studies. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Tel: 919-843-8459. Fax: 919-843-8465. E-mail: jianjin@
unc.edu.
Present Addresses: †Center for Integrative Chemical Biology
and Drug Discovery, Division of Medicinal Chemistry & Natural
Products, Eshelman School of Pharmacy, The University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
ACKNOWLEDGMENT We thank Minghui Wang for NMR, Bill
Leavens for HR-MS, and Jeff Guss, Mike Fisher, Kalindi Vaidya,
Elizabeth Davenport, Jim Fornwald, and Evanson Agyeman for
assay support.
ABBREVIATIONS PGE2, prostaglandin E2; OAB, overactive
bladder; KO, knockout; WT, wild-type; COX, cyclooxygen-
ase; HTS, high-throughput screening; FLIPR, fluorometric
imaging plate reader; fpKi, functional pKi; PK, pharmacoki-
netic; DNAUC, dose-normalized area under curve; CYP450,
cytochrome P450; SHR, spontaneously hypertensive rats;
GSH, glutathione.
a Conditions and reagents: (a) (i) t-BuLi, THF, -78 °C; (ii) ethyl 3-methyl-
2-oxobutyrate, THF, -78 °C to room temperature, 73%. (b) (i) Trichlor-
oacetyl isocyanate, CH2Cl2, room temperature; (ii) 2 M K2CO3 aqueous
solution, reflux, 94%. (c) Corresponding boronic acids, Suzuki coupling
conditions (see the Supporting Information). (d) TMSCl, NaI, or TMSI,
CH3CN, room temperature (8, 27%; 9, 48%; and 10, 64%). (e) NaH, MeI,
DMF, 0 °C to room temperature, 90%. (f) NaNO2, HCl, HBF4, H2O, 51%.
(g) BBr3,CH2Cl2,0°C to room temperature, 100%. (h) Tf2O, K3PO4,toluene,
H2O, 0 °C to room temperature, 91%. (i) PdCl2(dppf), dppf, KOAc, bis-
(pinacolato)diborane, dioxane, 80 °C, 70%. (j) NaIO4, HCl, THF, H2O, 77%.
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