Discovery of 4-{1-[({1-[4-(trifluoromethyl)benzyl]-1H-indol-7-yl}carbonyl) amino]cyclopropyl}benzoic acid (MF-766), a highly potent and selective EP 4 antagonist for treating inflammatory pain

Article abstract of DOI:10.1016/j.bmcl.2010.04.065

The discovery of a highly potent and selective EP₄ antagonist MF-766 is discussed. This N-benzyl indole derivative exhibits good pharmacokinetic profile and unprecedented in vivo potency in the rat AIA model.

Full text of DOI:10.1016/j.bmcl.2010.04.065

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3760–3763
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 4-{1-[({1-[4-(trifluoromethyl)benzyl]-1H-indol-7-yl}car-
bonyl)amino]cyclopropyl}benzoic acid (MF-766), a highly potent and
selective EP4 antagonist for treating inflammatory pain
John Colucci a, Michael Boyd a, Carl Berthelette a, Jean-Francois Chiasson a, Zhaoyin Wang a,
Yves Ducharme a, Rick Friesen a, Mark Wrona b, Jean-Francois Levesque b, Danielle Denis c,
Marie-Claude Mathieu c, Rino Stocco c, Alex G. Therien c, Patsy Clarke d, Steve Rowland d,
Daigen Xu d, Yongxin Han a,
*
a Department of Medicinal Chemistry, Merck Frosst Canada Ltd, 16711 Trans-Canada Highway, Kirkland, Quebec, Canada H9H 3L1
b DMPK, Merck Frosst Canada Ltd, 16711 Trans-Canada Highway, Kirkland, Quebec, Canada H9H 3L1
c In Vitro Sciences, Merck Frosst Canada Ltd, 16711 Trans-Canada Highway, Kirkland, Quebec, Canada H9H 3L1
d In Vivo Sciences, Merck Frosst Canada Ltd, 16711 Trans-Canada Highway, Kirkland, Quebec, Canada H9H 3L1
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of a highly potent and selective EP4 antagonist MF-766 is discussed. This N-benzyl indole
derivative exhibits good pharmacokinetic profile and unprecedented in vivo potency in the rat AIA model.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 19 January 2010
Revised 14 April 2010
Accepted 16 April 2010
Available online 18 April 2010
Keywords:
EP4 antagonist
Inflammatory pain
Arthritis (OA and RA) is a chronic inflammatory condition lead-
ing to bone and cartilage destruction. Accumulated evidence in the
past decade suggests that PGE2 plays an important role in the path-
ogenesis of this disease such that inhibition of PGE2 production by
NSAIDs and more recently COX-2 inhibitors relieves arthritis symp-
toms.1 PGE2 is the ligand of four subtype EP receptors, EP1–4. In a
mouse model of collagen-antibody induced arthritis (CAIA), the
EP4À/À mice showed a remarkable resistance in both the incidences
and symptom scores (paw swelling/redness, ankylosis) compared
to the wild type control, while the EP1–3À/À mice had no effect,2 sug-
gesting that the effect of PGE2 in inflammation was mediated pre-
dominantly by the EP4 receptor. Lin et al. demonstrated that EP4,
not EP1–3, contributed to inflammatory pain hypersensitivity in
rats.3 Using highly selective EP1, EP3 and EP4 antagonists, we re-
cently demonstrated that EP4, not EP1 or EP3, was the primary
receptor involved in joint inflammation and pain in rodent models
of rheumatoid and osteoarthritis,4 further supporting EP4 antago-
nism as a valid strategy for treating inflammatory pain. In addition,
it is plausible that a highly selective EP4 antagonist can be a safer
alternative in relieving arthritis symptoms without causing poten-
tial cardiovascular side effects observed with NSAIDs and COX-2
inhibitors5 since it does not interfere with the biosynthesis of
important prostanoids such as prostacyclin (PGI2) and thrombox-
ane A2 (TxA2).6
We recently reported the discovery of our first generation acyl-
sulfonamide-based EP4 antagonists MF-4984 and MF-310.7 These
compounds, unfortunately, suffered from species dependent CYP
3A mediated hydrolysis of the acylsulfonamide moieties, resulting
in highly variable pharmacokinetic profile in preclinical species.
The corresponding sulfonamide from MF-310 also had the poten-
tial to accumulate in vivo due to its low clearance rate and exces-
sively long elimination half-life.8 SAR effort to prevent the
acylsulfonamide hydrolysis was unsuccessful, so we turned our
attention to non-acylsulfonamide analogs. This effort resulted in
the discovery of a highly potent and selective second generation
EP4 antagonist MF-766 (Fig. 1). We herein describe the SAR leading
to MF-766, its in vitro potency and selectivity, its pharmacokinetic
properties and its in vivo potency profile in the rat adjuvant-in-
duced-arthritis (AIA) model.
Non-acylsulfonamide EP4 antagonists such as CJ-042,794
(Fig. 1) were reported recently.9 Our efforts focused on compounds
bearing suitable heterocyclic templates in place of the central
phenyl core. By screening a number of monocyclic or bicyclic
heterocycles, we discovered that the indole core in 1 (Fig. 1) was
an excellent template, yielding highly potent and selective
* Corresponding author. Tel.: +1 514 428 3301; fax: +1 514 428 4900.
E-mail address: yongxin_han@merck.com (Y. Han).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.065

J. Colucci et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3760–3763
3761
O
methyl esters, gave the final desired compounds 6 and 9 in excel-
lent overall yield (>35% in four steps). The cyclopropylbenzamine
derivatives 8 was prepared by first submitting 1,4-dicyanobenzene
to the Szymoniak variation10 of the Kulinkovich reaction, in which
one of the cyano groups was modified to afford the cyclopropyl-
amine derivative 7 (Scheme 1). The other cyano group was then
hydrolyzed in boiling 6 N hydrochloric acid and the resultant car-
boxylic acid was esterified in MeOH in presence of catalytic
amount of concentrated HCl to give the methyl ester 8. The po-
tency and selectivity data of these compounds are summarized
in Table 1.
O
N
Cl
COOH
O
N
N
W
H
H
N
COOH
O
H
COOH
N
X
Ar
Y
CF3
F
1
CJ-042,794
MF-766
CF3
O
O
O
O
O
N
O
O
O
O
N
S NH
O O
S NH
O O O
N
N
O
As shown in Table 1, the m-Cl-benzyl analog 6a exhibited good
EP4 affinity (Ki = 2.7 nM) and functional activity (IC50 = 5.9 nM), but
was poorly selective against the DP1 receptor with a Ki of 16 nM.
Introduction of a methyl group at the 2-position (6b) was detri-
mental to the EP4 binding affinity (Ki = 56 nM). Incorporation of
substituents such as Cl (6c, Ki = 20 nM) and Me (6d, Ki = 39.5 nM)
at the 3-position was also detrimental to EP4 binding affinity.
Introduction of a larger group such as COMe at this position re-
sulted in an inactive compound (not shown). The 5-position of
the indole core, on the other hand, tolerated small groups such
CF3
MF-310
MF-498
Figure 1. Structures MF-766, CJ-042,794, MF-498, MF-310 and a suitable indole
template 1.
antagonists such as MF-766 with vastly improved overall profile
compared to previously reported compounds.
Synthesis of these indole analogs was easily accomplished as
shown in Scheme 1. Alkylation of commercially available methyl
1H-indole-7-carboxylates (2) with a variety of benzyl bromides 3
followed by hydrolysis gave the alkylated indole acids 4. Standard
amide coupling with commercially available (S)-methyl 4-(1-ami-
noethyl)benzoate (5) or cyclopropyl analog 8 in the presence of
HATU and Hunig’s base, followed by hydrolysis of the resultant
as Cl (6e, Ki = 0.81 nM, IC50 = 1.5 nM) and
F (6f, Ki = 2.4 nM,
IC50 = 3.3 nM). These two compounds also displayed marginal
DP1 selectivity but lose selectivity against the EP2 receptor (EP2
Ki = 110 and 504 nM, respectively). Benzyl groups bearing small
lipophilic moieties such as Cl or CF3 are preferred. The m-CF3
Br
H3N
Cl
O
O
N
O
COOMe
W
W
R
W
N
H
OH
OMe
5
c, b
3
COOH
N
X
NH
X
a, b
Y
Y
X
Y
R
R
2
4
6
c, b
H3N
Cl
e, f
O
d
COOMe
NC
W
H2N
N
H
8
CN
CN
COOH
N
X
7
Y
R
9
Scheme 1. Reagents and conditions: (a) NaH, DMF; (b) LiOH, MeOH; (c) HATU, Hunig’s base, DMF; (d) Ti(OiPr)4, EtMgBr, BF3ÁEt2O, CH2Cl2, rt; (e) 6 N HCl, reflux; (f) MeOH, cat.
concd HCl, reflux.
Table 1
Binding affinity, selectivity and functional potency of compounds 6 and 9
d
Compd
X, Y, W
R
Binding affinitya (nM)b
DP1
EP4 Functional IC50 (nM)
EP4
2.7
56.0
20.1
39.5
Othersc
6a
6b
6c
6d
6e
6f
6g
6h
H, H, H
Me, H, H
H, Cl, H
H, Me, H
H, H, Cl
H, H, F
H, H, H
H, H, H
H, H, H
H, H, Cl
H, H, H
H, H, Cl
m-Cl
m-Cl
m-Cl
m-Cl
m-Cl
m-Cl
m-CF3
3-Cl-5-OCF3
p-CF3
p-CF3
p-CF3
p-CF3
16
450
14
1205
63
404
885
49
1389
209
1648
561
P2500
nde
P930
nd
P110
P504
P785
P260
P580
P100
>6000
P1291
5.90
nd
nd
nd
0.81
1.50
3.30
11.8
1.40
2.70
1.12
1.30
1.26
2.40
4.80
0.38
0.62
0.44
0.23
0.31
6i
6j
9a (MF-766)
9b
a
b
c
For details on the binding assay for all PG receptors, see Ref. 4 and references cited therein.
Values are means from at least two independent experiments.
Other PG receptors refers to EP1, EP2, EP3, DP2, IP, TP and FP.
The EP4 functional assay measured the inhibition of PGE2-induced cAMP accumulation in HEK 293 cells, for details, see Ref. 4.
nd: not done.
d
e

3762
J. Colucci et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3760–3763
analog (6g, Ki = 4.8 nM, IC50 = 11.8 nM), for example, is only
slightly less potent than the m-Cl derivative 6a but is substantially
more selective against the DP1 receptor (Ki = 885 nM). The 3-Cl-5-
OCF3 analog 6h shows excellent EP4 affinity (Ki = 0.38 nM) and
functional potency (IC50 = 1.4 nM) but is again less selective
against DP1 (Ki = 49 nM). Moving the CF3 group from the meta-po-
sition in 6g to the para-position (6i) resulted in a significant
improvement in EP4 binding affinity (Ki = 0.62 nM), functional po-
tency (IC50 = 2.7 nM) and selectivity against DP1 (Ki = 1389 nM),
suggesting that the para-substitution was superior for both affinity
and selectivity. The corresponding 5-Cl analog 6j had similar affin-
ity (Ki = 0.44 nM) and functional potency (IC50 = 1.12 nM) but was
again significantly less selective against DP1 (Ki = 209 nM). Finally,
replacing the chiral (S)-Methyl 4-(1-aminoethyl)benzoate in 6i
with the achiral 4-(1-aminocyclopropyl)benzoate group gave the
title compound MF-766 (9a) with further improved affinity, func-
tional potency and selectivity. The corresponding 5-Cl analog 9b
had similar affinity and functional potency but was less selective.
The full in vitro profile of MF-766 is summarized in Table 2.
As shown, MF-766 exhibited excellent affinity at the EP4 recep-
tor (Ki = 0.23 nM), was not significantly shifted in presence of 10%
human serum (HS) (Ki = 0.34 nM) and had excellent selectivity
against other prostanoid receptors (>7000-fold). It behaved as a full
antagonist with an IC50 of 1.4 nM (shifted to 1.8 nM in the presence
of 10% HS) in the functional assay. In the human whole blood (HWB)
and receptors, and was found to have no appreciable activity at con-
centrations up to 10 M.
l
The in vitro metabolic stability of MF-766 was evaluated from
incubations in human, dog and rat hepatocytes. The major metab-
olite observed in all three species was the glucuronide conjugate of
the carboxylic acid. A minor taurine conjugate and several minor
oxidative metabolites were also observed in these species. MF-
766 was most stable in human, followed by rat and dog hepato-
cytes. The acyl glucuronide conjugate was also found to be the only
circulating metabolite in rat and dog plasma samples. Additional
in vitro incubations were performed in presence of fresh rat and
human hepatocytes using [3H]-MF-766 to assess potential protein
labeling due to acyl glucuronide migration or oxidative metabo-
lism, and acceptable levels of covalent binding were obtained
(rat: 61 pmol-equiv/mg @ 2 h; human: 50 pmol-equiv/mg @ 2 h).
No tritium loss was observed upon HPLC-radiometric detection
analysis of the in vitro samples.
The pharmacokinetic profile of MF-766 was evaluated in SD rats
and beagle dogs. The results are summarized in Table 3. As shown,
MF-766 exhibited desirable pharmacokinetic properties with high
oral bioavailability (F = 74–86%), low to moderate clearance rate
(CL = 3.7–6.3 mL/min/kg) and good elimination half-life (t1/2 = 2.6–
4.6 h), all of which contributed to the observed high systemic
exposures.
The excellent in vitro potency and PK profile of MF-766 trans-
lated into impressive in vivo potency and efficacy. When tested in
the rat AIA model,4,13,14 this compound was found to have unprec-
edented potency, with an ED50 of approximately 0.004 mg/kg/day
(Fig. 2). The minimum dose required for maximum inhibition
assay9c,11 measuring the blockade of inhibition of TNF -induced IP-
a
10 release by the specific EP4 agonist L-00090268812, MF-766
showed excellent activity with an IC50 of 9.5 nM. In addition, it
was evaluated in the MDS Pharma screen against >170 enzymes
Table 2
Full in vitro affinity, selectivity and functional potency profile of MF-766
Binding affinity Ki (nM)a
Functional potency IC50 (nM)b
EP1
EP2
EP3
EP4
EP4 + 10%HS
0.34
DP1
DP2
FP
IP
TP
HEK-293 cell
1.4
HEK-293 cell +10% HS
1.8
HWB
9.5
20,000
8000
7400
0.23
1800
>21,000
6400
>22,000
6700
a
Average of 3–11 independent experiments.
Average of 4–5 independent experiments.
b
Table 3
Pharmacokinetic parameters of MF-766
Species
Dose iv/poa (mg/kg)
Clp (mL/min/kg)
Vd (L/kg)
t1/2 (h)
F (%)
po AUC (lMÃh)0–24
Rat
Dog
5/20
1/4
3.7
6.3
1.0
0.8
4.6
2.6
86
74
150
15
a
iv: n = 2 (male), free acid in 60% PEG 200 at a dosing volume of 1 mL/kg; po: n = 4 in male rat, n = 2 in male dog, in 60% PEG 200 at a dosing volume of 5 mL/kg.
Secondary paw
Primary paw
**
125
100
75
125
100
75
**
*
50
50
*
25
25
0
0
-25
-25
treatments
treatments
Figure 2. In vivo potency and efficacy of MF-766 in the rat AIA model measuring inhibition of paw swelling at day 18, n = 3 for the non-AIA group and 6–7 for vehicle/drug
treated groups. Arthritis was induced by sub-plantar injection of complete Freund’s adjuvant in the primary paw at day 0, compound was dosed once daily in 0.5% methocel
starting from day 9. * P <0.005 and ** P <0.001 versus vehicle by one way ANOVA/Dunnett’s test.

J. Colucci et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3760–3763
3763
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8. Unpublished results: the elimination half-life of the corresponding
sulfonamide M1 from MF-310 in rat and dog was estimated to be >24 h.
When MF-310 was dosed orally at 20 mg/kg/day for 5 days in rats, M1 levels at
(ED100) of both the primary and the secondary paw swelling was
established at ꢀ0.025 mg/kg/day from a separate experiment, and
the corresponding drug levels at 2 h and 24 h post the final dose
were 28 and 3.1 nM, respectively. This potency was far superior
to that reported for CJ-042,749 (ED80 = 11.5 mg/kg).9b As a compar-
ison, the ED50s established for potent COX-2 inhibitors such as
refocoxib13 and MF-tricyclic14 from separate experiments under
similar paradigms were 0.7 and 0.5 mg/kg/day, respectively. Fur-
thermore, we observed that the maximum efficacy of MF-766 and
other highly selective EP4 antagonists in inhibiting swelling in the
primary paw (70%) and secondary paw (100%) in this model were
comparable to both COX-2 inhibitors and NSAIDs.
In conclusion, we have described the discovery and preclinical
characterization of the highly potent and selective EP4 antagonist
MF-766. In the rat AIA model of chronic inflammation, MF-766
exhibited comparable maximum efficacy to Coxibs and NSAIDs
but was >200-fold more potent than potent COX-2 inhibitors like
refocoxib and MF-tricyclic. Overall, MF-766 displayed the desired
potency, selectivity and pharmacokinetic profile for further devel-
opment, and would represent a potential safer alternative for the
treatment of pain and inflammation than traditional NSAIDs and
Coxibs.
24 h increased from 1.4
When it was dosed in dogs at 4 mg/kg/day for two weeks, M1 levels at 24 h
increased from 1.3 M after the first dose to 13.7 M after the last dose.
lM after the first dose to 4.6 lM after the last dose.
l
l
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