Discovery of {4-[4,9-bis(ethyloxy)-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl]-2-fluorophenyl}acetic acid (GSK726701A), a novel EP4 receptor partial agonist for the treatment of pain

Article abstract of DOI:10.1016/j.bmcl.2018.03.091

A novel series of EP₄ agonists and antagonists have been identified, and then used to validate their potential in the treatment of inflammatory pain. This paper describes these novel ligands and their activity within a number of pre-clinical mo

Full text of DOI:10.1016/j.bmcl.2018.03.091

Accepted Manuscript
Discovery of {4-[4,9-bis(ethyloxy)-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-
yl]-2-fluorophenyl}acetic acid (GSK726701A), a novel EP4 receptor partial
agonist for the treatment of pain
Mark P. Healy, Amanda C. Allan, Kristin Bailey, Andy Billinton, Iain P.
Chessell, Nicholas M. Clayton, Gerard M. P. Giblin, Melanie A. Kay, Tarik
Khaznadar, Anton D. Michel, Alan Naylor, Helen Price, David J. Spalding,
David A. Stevens, Martin E. Swarbrick, Alexander W. Wilson
PII:
S0960-894X(18)30296-8
https://doi.org/10.1016/j.bmcl.2018.03.091
BMCL 25745
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 January 2018
29 March 2018
30 March 2018
Please cite this article as: Healy, M.P., Allan, A.C., Bailey, K., Billinton, A., Chessell, I.P., Clayton, N.M., M. P.
Giblin, G., Kay, M.A., Khaznadar, T., Michel, A.D., Naylor, A., Price, H., Spalding, D.J., Stevens, D.A., Swarbrick,
M.E., Wilson, A.W., Discovery of {4-[4,9-bis(ethyloxy)-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl]-2-
fluorophenyl}acetic acid (GSK726701A), a novel EP4 receptor partial agonist for the treatment of pain, Bioorganic
& Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.03.091
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Discovery of {4-[4,9-bis(ethyloxy)-1-oxo-1,3-dihydro-2H-
benzo[f]isoindol-2-yl]-2-fluorophenyl}acetic acid (GSK726701A),
a novel EP4 receptor partial agonist for the treatment of pain
Mark P. Healy,* Amanda C. Allan, Kristin Bailey,Andy Billinton, Iain P. Chessell,Nicholas M.
Clayton,Gerard M. P. Giblin,Melanie A. Kay,Tarik Khaznadar,Anton D. Michel,Alan Naylor,Helen
Price,David J. Spalding,David A. Stevens, Martin E. Swarbrick, Alexander W. Wilson
GlaxoSmithKline, Neurosciences Centre of Excellence for Drug Discovery; New Frontiers Science Park, Third Avenue,
Harlow, Essex CM19 5AW, UK
Declarations of interest: none
This is where the receipt/accepted dates will go; Received Month XX, 2018; Accepted Month XX, 2018 [BMCL RECEIPT]
Abstract— A novel series of EP4 agonists and antagonists have been identified, and then used to validate their potential in the treatment
of inflammatory pain. This paper describes these novel ligands and their activity within a number of pre-clinical models of pain,
ultimately leading to the identification of the EP4 partial agonist GSK726701A.
inflammatory resolution at later time points10 and these
Prostaglandin E2 (PGE2) 1, synthesised from arachidonic
effects may be mediated by different receptor sub-types.
Thus selectively targeting individual EP receptors offers
the opportunity to intervene in pathophysiology with
specificity and to potentially avoid toxicity.
acid (AA) by the cyclooxygenase enzymes, is a ubiquitous
mediator of mammalian physiology and pathophysiology.1
Physiologically, PGE2 contributes to the modulation of
bone formation2, gut homeostasis including pH control3,
renal function and blood pressure.4 In addition PGE2 is
associated with the growth of certain cancers5 and is a well
characterised inflammatory mediator contributing to pain
and inflammation.6 To date, pharmacological intervention
in the AA pathway has led to the discovery and
commercialisation of NSAIDS and COX2 inhibitors.7
However these drugs have a number of safety concerns
associated with gastro-intestinal bleeding and damage and
adverse cardiovascular events, respectively.8
As part of a wider investigation of the role of EP receptors
in pain and inflammation, we initiated a programme to
discover selective EP4 ligands. We were particularly
intrigued by seemingly conflicting roles of EP4 in pain and
inflammation. Studies have confirmed that PGE2 acting on
the EP4 receptor can either stimulate or inhibit
inflammation in concert with IFN.11 Furthermore, EP4
receptor knockout mice showed resistance to inflammation
in a collagen induced arthritis model12 and selective EP4
antagonists show robust anti-inflammatory effects in
vivo.13 However, EP4 agonists have also been reported to
attenuate levels of the pro-inflammatory mediator TNF in
rats14 and can potently suppress inflammation in an
adjuvant induced arthritis model.15
The biological effects of PGE2 are mediated by four G-
protein coupled receptors designated EP1-4.9 Each of these
receptors has characteristic distribution in various tissues
and this feature, together with the paracrine nature of
PGE2, allows PGE2 to play varied and sometimes opposing
roles in the mammalian system. For example, PGE2 is
thought to act as a pro-inflammatory agent in the early
stages of inflammation, while helping to promote
The PGE2 analogue ONO-4819CD (rivenprost) 2 has been
in clinical development and was reported to have an
 Corresponding author present address: Novartis Institutes for BioMedical Research, 22 Windsor St, Cambridge, MA,
02139, USA. e-mail: mark.healy@novartis.com (M.P.Healy).

improvement in histological scores in ulcerative colitis
patients.16 ONO-4819CD is the pro-drug of the EP4 agonist
ONO-AE1-437 3 (Figure 1). Previously we reported the
first non-prostanoid, selective EP4 agonist CCI17464 417 as
a result of an internal focussed screen. From the same
naphtholactam template an EP4 antagonist, GW627368X
518 was identified. The sulphonyl urea EP4 antagonist CJ-
023,423 (grapipant, RQ-007, AAT-007) 6,19 has been
reported as being effective for treating osteoarthritis (OA)
pain in a phase 2 study in humans,20 whilst recently been
approved to control pain and inflammation associated with
osteoarthritis in dogs. 21
Scheme 1. Reagents and conditions: i) Diethylsuccinate, NaOEt, EtOH;
ii) (a) R1I, NaOEt, EtOH then R2I, NaOEt, EtOH; (b) NaOH, EtOH; iii)
AcOH; iv) NaOH, EtOH; v) Zn, AcOH or i) NaBH4, then ii) Et3SiH,
TFA.
The synthesis of CCI17464 and analogues is shown in
Scheme 1. Diethyl phthalate 7 was treated under basic
conditions with diethyl succinate to give the naphthylene
diol 8. Diol 8 was then alkylated, followed by
saponification to yield the bis-acids 9. Condensation with
ethyl (4-amino phenyl) acetate 10 yielded the phthalimides
11, which were then hydrolysed to give carboxylic acids
13 and 15. Alternatively reduction of the imides 11 with
Zn/AcOH or a two-step protocol with NaBH4 followed by
Et3SiH/TFA,23 gave the lactam analogues 12, which were
then subsequently hydrolysed to give carboxylic acids 2,
14, and 16-19.
Figure 1 – Structures of selected EP4 receptor agonists
Table 1 summarises the early SAR in the series. The
phthalimide analogue 13, shows equipotency, but higher
intrinsic activity than the lactam partial agonist 2 in a
functional cAMP accumulation assay,24 which is a
consistent observation between these two functionalities.
Earlier studies have suggested the carboxylic acid
functionality on PGE2 forms a key interaction with a
conserved arginine residue within TMVII of all prostanoid
receptors.24 Indeed, the para-acetic acid is the optimum
position for EP4 agonist potency, and introduction of a
methyl group next to the carboxylic acid is detrimental to
agonist potency (compound 14) highlighting the sensitivity
of this part of the molecule for EP4 agonism.
Figure 2 – Structures of selected EP4 receptor antagonists
We were interested in exploring the biological profile of
EP4 ligands and as such set out to explore the SAR of the
EP4 partial agonist CCI17464. The reported potential for
partial agonists to induce less receptor desensitisation22 was
attractive to us if we could demonstrate human whole
blood activity and efficacy in preclinical models of pain.
Investigation into the size of the alkyl substituent on
oxygen atoms at the 4 and 9 positions of the naphthylene
ring showed the ethyl and iso-propyl groups to offer the
best potency (compounds 13 and 17). Increasing the size of
these groups beyond a butyl group leads to a loss in
potency (compound 19).
Removal of both oxygen atoms on the imide ring resulted
in a weak EP4 partial agonist (compound 20) suggesting
the carbonyl group has an important role in the agonist
mode of action.
Table 1 – SAR of compounds 2-20

p.o.; green bar 1); selective EP4 agonist CCI17464 2 (10mg/kg p.o.; red
bar 2); selective EP4 agonist CCI17464 2 (10mg/kg) pre-challenged with
selective EP4 antagonist GW627368X 5 (3mg/kg) (blue bar 3) on FCA-
induced hypersensitivity (weight bearing) versus vehicle (1%
DMSO/33% PEG400/66% distilled water p.o.; black bar 4).
Compound
R
X
Y
Z
cAMP
pEC50
IAb
pKic
a
Introduction of substitution onto the benzene ring of the
phthalimide was achieved by using the appropriately
substituted diethyl phthalate in the condensation with
diethyl succinate. The fluoro or methyl analogues (Table
3, compounds 21 and 22 respectively) showed good
agonist potency. Aza-analogue 23 showed no EP4 agonist
effects, further emphasising the sensitivity within the
template for EP4 agonism. Full agonist imide 22,
demonstrated a long half-life in rat (t1/2 = 31.7h) (as
observed with other imides e.g. 13) and was progressed to
the FCA model, however similar potency versus CCI17464
was observed (76% reversal of FCA induced
CCI17464, 2
Et
Et
Et
Me
nPr
iPr
nBu
nHex
Et
H,H
=O
H,H
=O
H,H
H,H
H,H
H,H
H,H
=O
=O
=O
=O
=O
=O
=O
=O
H,H
H
H
Me
H
H
H
H
H
H
7.7
7.6
<6
6.7
6.9
7.4
6.7
<6
61%
90%
-
69%
74%
87%
90%
-
7.1
6.8
6.7
NT
6.2
6.4
5.7
<5.2
6.2
13
14
15
16
17
18
19
20
6.6
43%
a functional cAMP accumulation assay, values are a mean of at least 2 experiments; bintrinsic
activity compared to the full agonist PGE2; c radioligand bindingassay, values are a mean of at
least 2 experiments; NT not tested
Selected compounds were assessed in human recombinant
CYP450 assays and it was found the analogues had low
potential for CYP450 inhibition with inhibitory activities
(IC50) typically in excess of 10 M (e.g. Compound 13
CYP1A2 IC50 65µM, CYP2C19 IC50 >100µM, CYP2C9
IC50 >100µM, CYP2D6 IC50 42µM, CYP3A4 (DEF) IC50
>100µM, CYP3A4 (7BQ) IC50 >100µM). Furthermore all
compounds tested had low intrinsic clearance in rat and
human microsomes (<0.5mL/min/g tissue). The rat in-vivo
pharmacokinetics were assessed for selected analogues by
intravenous administration and are summarised in Table 2.
hypersensitivity @10mg/kg), demonstrating there was no
apparent advantage for a compound with a full agonist
profile.
Table 3 – SAR of Phthalimide Ring.
Compound
R4
A
cAMP
pEC50
IAb
pKic
Table 2 – Rat iv pharmacokinetics (1mg/kg) for selected analogues
a
21
22
23
F
Me
H
C
C
N
7.1
7.7
<6
72%
112%
-
NT
6.8
7.5
Compound
CLb (mL/min/kg)
Vss
(L/kg)
0.30
0.28
t1/2 (h)
a functional cAMP accumulation assay, values are a mean of at least 2 experiments; bintrinsic
activity compared to the full agonist PGE2; c radioligand bindingassay, values are a mean of at
least 2 experiments; NT not tested
0.6#
0.2*
8.4
19.9*
CCI17464, 2
13
#dosed orally in 10% DMSO/water with pH adjustment to aid solubility; dosed orally in 5% (w/v)
glucose/2% DMSO (v/v)/water
It is interesting to note that in certain compounds that lost
EP4 agonist activity, binding affinity was sometimes
maintained (e.g. compounds 14 and 23 display pKi’s of 6.7
and 7.5 in a radioligand binding assay with 3H[PGE2])
demonstrating the sensitive structural requirements for
agonist activity.
Whereas compounds 2 and 13 had low clearance in rat, the
imide 13 showed considerably greater stability in-vivo and
hence had a much extended half-life. This was a common
feature of the imides within the series and few were
progressed further due to their long half lives. CCI17464 2
was progressed to the Freunds complete adjuvant (FCA)
acute rat model of inflammatory pain and shows full
reversal of the hypersensitivity,25 with an ED50 of 1.5mg/kg
(corresponding to a total blood concentration of ~5-11uM),
and we were keen to identify compounds with greater
potency in this model. Importantly, the anti-hyperalgesic
effect of CCI17464 2 is completely reversed when pre-
challenged with the selective EP4 antagonist GW627368X
5, confirming the anti-hyperalgesic effect is driven by the
selective EP4 agonist (Figure 3).
We next turned our attention to the phenylacetic acid ring.
Substituted phenyl acetic acids were condensed with the
bis-carboxylic acid 9. Table 4 summarises the SAR.
Substituents ortho to the lactam nitrogen generally gave an
increase in the in-vitro potency (Compounds 24-27 and
29). In contrast, only a F substituent was tolerated in the
ortho position to the acetic acid (see compounds 30-32).
Table 4 – Further Modifications to the Phenyl Ring
Compound
A
B
cAMP
pEC50
IAb
FCAc
reversal
a
5% @
3mg/kg
14% @
3mg/kg
47% @
10mg/kg
19% @
3mg/kg
NT
24
25
26
27
F
Cl
H
H
H
H
8.4
72%
73%
91%
78%
8.3
8.3
8.1
Br
Me
28
29
CF3
MeO
H
H
7.3
8.0
73%
68%
NT
80% @
3mg/kg
NT
30
31
H
H
F
7.4
<6
67%
-
Cl
Figure 3 – The effect of selective EP4 antagonist GW627368X 5 (3mg/kg

32
H
Me
<6
-
NT
a functional cAMP accumulation assay, values are a mean of at least 2 experiments; bintrinsic
activity compared to the full agonist PGE2; c % reversal of FCA induced hypersensitivity as
measured via weight bearing 1hr post dose. Compounds dosed orally in 1% methyl cellulose. NT
not tested.
Selected compounds were tested in the FCA model to
determine their in-vivo potencies. Surprisingly, the
compounds which had increased in-vitro potency
(Compounds 24-27) showed a poor response in-vivo,
whereas compound 30 showed an excellent reversal of the
FCA induced hypersensitivity at 3mg/kg. Compound 30,
GSK726701A was further investigated in this model and
was shown to have an ED50 of 0.2mg/kg (corresponding to
a total blood concentration at this dose of 0.6uM), which
compares well to the gold standard COX-2 inhibitor
celecoxib (full reversal of FCA induced hypersensitivity at
10mg/kg). This represents >10 fold increase in potency of
the lead compound CCI17474, 2.
Figure 5 – The effect of compound 30 (0.03-1mg/kg p.o. b.i.d. x5 days)
on FCA-induced chronic joint hypersensitivity (weight bearing) versus
vehicle (methylcellulose) and positive control (celecoxib, 30mg/kg p.o.).
GSK726701A was also examined in the chronic
constriction injury (CCI) model of neuropathic pain27 where
it was dosed twice daily for 8 days and demonstrated a
time-dependant, full reversal of CCI-induced mechanical
allodynia at 3mg/kg, equivalent to the clinical gold
standard gabapentin (30mg/kg).
GSK726701A has high selectivity (>100-fold) against a set
of other prostaglandin receptors (EP1-3, DP1, FP, IP, TP see
Table 5) and no significant activity against a wider panel
of targets. It demonstrates EP4 agonist activity with similar
potency and intrinsic activity (pEC50 8.2 (I.A. 68 %)) in a
human whole blood (HWB) assay on the inhibition of
LPS-mediated TNFαinduction (Figure 4), supporting the
potent anti-inflammatory potential of EP4 agonists.14,15
Table 5 – Profile of GSK726701A
EP4 pEC50 cAMP acc
EP4 pEC50 HWB
EP1 pIC50
7.4 (67%)
8.2 (68%)
<5
EP2 agonism
EP3 pIC50
14% activation @ 1uM
<5
DP1 pIC50
FP pIC50
<4.5
<5
IP agonism
IP antagonism
TP pIC50
11% activation at 1uM
31% inhibition at 1uM
<5
S9 stability (r, d, h)
P450 inhibition
<1ml/min/g liver
>100 M (CYP1A2, CYP2C9, CYP2C19,
CYP2D6, CYP3A4 (DEF), CYP3A4(7BQ))
1, 4, 4mL/min/kg
CLb (r,d,c)
t1/2(r,d,c)
6, 8, 5-10 h
FCA reversal of hypersensitivity ED50
Joint Pain reversal of hypersensitivity ED50
CCI reversal of mechanical allodynia
0.22mg/kg
0.23mg/kg
Full reversal @ 3mg/kg
In summary starting from the screening hit CCI17464 2,
the discovery of a highly selective and potent EP4 partial
agonist 30, {4-[4,9-bis(ethyloxy)-1-oxo-1,3-dihydro-2H-
benzo[f]isoindol-2-yl]-2-fluorophenyl}acetic acid
(GSK726701A), has been described which demonstrates
robust activity in a range of pre-clinical models of pain.
The data shows this mechanism has the potential to offer
an alternative therapeutic approach in the treatment of pain
and inflammatory conditions and supported the selection
of GSK726701A for further investigation.
Figure 4 – The effect of PGE2 (black), compound 2 (green) and
compound 30 (purple) on the inhibition of LPS-mediated TNF-alpha
induction in human whole blood.
GSK726701A has low human CYP inhibition potential
and low intrinsic clearance across species. In addition, it
displays good in-vivo pharmacokinetics with low blood
clearance and good oral bioavailability in rat (CLb 1
mL/min/kg, t1/2 6 h, Fpo 112 %), dog (CLb 4 mL/min/kg,
t1/2 8 h, Fpo 69 %) and cynomolgus monkey (CLb 4
mL/min/kg, t1/2 5-10 h, Fpo 50 %). GSK726701 has high
plasma protein binding across species (rat 99.8%, dog
99.1%, human 99.9%) and is non-CNS penetrant (rat
brain:blood ratio <0.05:1).
Acknowledgements
We would like to acknowledge Ann Walker, Sue
Roomans, Andy Mason and V Weller for the first synthesis
of selected compounds in Table 1, Anita Chowdhury and
Davina Mitchell for generation of EP1, EP3, and FP
selectivity data, and Simon Peace for assistance in
manuscript preparation.
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Graphical abstract:
Abstract— A novel series of EP4 agonists and antagonists have been identified, and then used to validate their potential in the treatment
of inflammatory pain. This paper describes these novel ligands and their activity within a number of pre-clinical models of pain,
ultimately leading to the identification of the EP4 partial agonist GSK726701A.
Keywords: EP4 receptor agonist, EP4 agonist, Prostaglandin E2, PGE2, Prostaglandin E2 receptor 4, EP4, pain, arthritis, inflammation,
anti-inflammatory, anti-hyperalgesic, neuropathic pain.
 Corresponding author present address: Novartis Institutes for BioMedical Research, 22 Windsor St, Cambridge, MA,
02139, USA. e-mail: mark.healy@novartis.com (M.P.Healy).

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A novel series of selective and potent EP4 receptor partial agonists identified
GSK726701A has good PK in rat, dog and monkey
Robust activity in a range of animal models of inflammatory and neuropathic pain