A novel series of potent and selective EP4 receptor ligands: Facile modulation of agonism and antagonism

Article abstract of DOI:10.1016/j.bmcl.2010.10.106

A novel series of EP₄ ligands, based on a benzyl indoline scaffold, has been discovered. It was found that agonism and antagonism in this series can be easily modulated by minor modifications on the benzyl group. The pharmacokinetic, metabolic

Full text of DOI:10.1016/j.bmcl.2010.10.106

Bioorganic & Medicinal Chemistry Letters 21 (2011) 484–487
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel series of potent and selective EP4 receptor ligands: Facile modulation
of agonism and antagonism
Michael J. Boyd a, , Carl Berthelette a, Jean-François Chiasson a, Patsy Clark d, John Colucci a, Danielle Denis c,
⇑
Yongxin Han a, Jean-Francois Lévesque b, Marie-Claude Mathieu c, Rino Stocco c, Alex Therien c,
Steve Rowland d, Mark Wrona b, Daigen Xu d
a Department of Medicinal Chemistry, Merck Frosst Canada Inc., 16711 Trans-Canada Highway, Kirkland, Quebec, Canada H9H 3L1
b Department of DMPK, Merck Frosst Canada Inc., 16711 Trans-Canada Highway, Kirkland, Quebec, Canada H9H 3L1
c Department of In Vitro Sciences, Merck Frosst Canada Inc., 16711 Trans-Canada Highway, Kirkland, Quebec, Canada H9H 3L1
d Department of In Vivo Sciences, Merck Frosst Canada Inc., 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:
A novel series of EP4 ligands, based on a benzyl indoline scaffold, has been discovered. It was found that
agonism and antagonism in this series can be easily modulated by minor modifications on the benzyl
group. The pharmacokinetic, metabolic and pharmacological profiles of these compounds was explored.
It was found that these compounds show good pharmacokinetics in rat and are efficacious in pre-clinical
models of pain and inflammation.
Received 8 September 2010
Revised 20 October 2010
Accepted 21 October 2010
Available online 28 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Prostaglandin E2
EP4
Inflammation
O
O
Arthritis is a chronic inflammatory condition leading to bone
and cartilage destruction. Accumulated evidence over past decades
suggests that PGE2 plays an important role in the pathogenesis of
this disease.1 Inhibition of prostaglandin E2 (PGE2) production by
NSAIDs and, more recently, Cox-2 inhibitors relieves arthritis
symptoms.
Cl
HO
HO
H
H
N
O
N
O
Ar
N
N
R
1
2
PGE2 is the ligand of four subtype of EP receptors: EP1-4. In a
mouse model of collagen-antibody induced arthritis (CAIA),
EP4ꢀ/ꢀ mice, unlike EP1–3ꢀ/ꢀ mice, showed remarkable symptom
resistance (paw swelling/redness, ankylosis). This suggests that the
effect of PGE2 in inflammation is mediated predominantly by the
EP4 receptor.2 To further support EP4 antagonism as a valid strat-
egy for treating inflammatory pain, we recently demonstrated,
using highly selective EP1, EP3 and EP4 antagonists, that EP4, not
EP1 or EP3, is the primary receptor involved in joint inflammation
and pain in rodent models of rheumatoid and osteoarthritis.3a
Moreover, since EP4 antagonism does not interfere with the bio-
synthesis of important prostanoids, such as prostacyclin (PGI2)
and thromboxane A2 (TxA2), it is plausible that a highly selective
EP4 antagonist would be a safer alternative for relieving arthritis
symptoms and thus lack the potential cardiovascular side effects
observed with NSAIDs and Cox-2 inhibitors.4
We previously disclosed the discovery of several series of
selective EP4 antagonists.3 We demonstrated that these com-
pounds exhibited excellent efficacy and GI tolerability. In this Let-
ter, we describe the SAR, pharmacokinetics, metabolism and
in vivo efficacy of a new series of EP4 ligands containing the indo-
line template shown in general structure 1.
In a previous communication,3d we described indole 2 as a po-
tent antagonist of EP4 with a Ki of 2.7 nM. It was highly selective
against the EP1, EP2, EP3, FP, IP, TP (>1000-fold) receptors but
was only sixfold selective against the DP receptor. Subsequently,
it was found that replacement of the indole core with an indoline
moiety (1a, Table 1) resulted in a sixfold increase of potency on
the EP4 receptor, as well as a substantial improvement in selectiv-
ity against DP.
The general synthetic route to the indolines is described in
Scheme 1. Commercially available N-Boc indoline (3) was treated
with s-BuLi, then quenched with CO2 to provide acid 4.5 Standard
HATU amide coupling conditions were used to couple 4 with
⇑
Corresponding author.
E-mail address: MICHAELJOHNBOYD@HOTMAIL.COM (M.J. Boyd).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.106

M. J. Boyd et al. / Bioorg. Med. Chem. Lett. 21 (2011) 484–487
485
Table 1
EP2 (from 241 and 338-fold, respectively, to >1000-fold). It
appeared that a variety of 4-substituents on the benzyl group (Cl,
F, CF3, OCF3, CN, Ph, COONH2, t-Bu and Me) were tolerated for
antagonist activity. An alternate way of producing antagonists
was to add a polar group at the 3 position of the benzyl group.
For example, 3-pyridyl (1n), 3-CONH2 (1r) and 3-CN (1s) behaved
as antagonists albeit with loss in potency. Incorporation of less
polar groups at the 3 position resulted in agonists (1a, 1f, 1j). Also
noteworthy, the 2- and 5-substitutions examined (1j, 1l, 1q) re-
sulted in a loss of selectivity. Interestingly, this agonist/antagonist
switch was not observed with the indole series3d (all the indole
derivatives prepared are antagonists).
With regards to potency, it appears that small non-polar
para-substituents (Cl, F, CF3 and CH3) are superior. With respect
to selectivity, 4-Cl, 4-CF3 and 4-CH3 are the most selective
(>1000-fold selectivity vs all other prostanoid receptors). Overall,
compound 1c (4-Cl) and 1e (4-CF3) have the best potency and
selectivity profile. Both are highly potent in both the binding
(Ki = 0.4 nM) and functional assays (IC50 = 1.0–2.0 nM), and are
>1000-fold selective versus all other prostanoid receptors.
Other SAR of interest is shown in Table 2. The tetrazole carbox-
ylic acid isostere 10 was prepared as shown in Scheme 2, and
binding data indicates that the tetrazole group is a suitable replace-
ment for the carboxylic acid. Also, substitution at the 5-position of
the indoline is tolerated, but loss of selectivity is observed against
EP2 and DP with groups larger than fluorine (1t–1v).
SAR of compounds of general structure 1 (R = H)
Ar
EP4
Ki,
nM
Func. assaya
EP2b/EP4
DPb/EP4
IC50(EC50
)
nM
2
2.7
6.0
>1000
595
338
>1000
488
>1000
>1000
687
603
139
151
>179
>1000
>1000
>127
>692
>1000
795
16
>1000
241
>1000
907
>1000
>1000
880
916
57
209
>73
114
>1000
>50
>281
>1000
88
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
1s
3-Cl
3,4-Cl
4-Cl
4-F
4-CF3
3-CF3
4-OCF3
4-CN
4-Ph
3,5-Br
4-COOH
2-Br,4-Cl
4-CONH2
3-Pyridyl
4-t-Bu
4-Me
2-Br,4-CF3
3-CONH2
3-CN
0.44
0.34
0.44
1.8
0.37
0.40
1.5
3.1
0.28
0.33
54
2.3
30
76
14
0.78
6.7
30
7.5
>10,000 (0.82)
1.1
0.92
6.2
2.1
>10,000 (0.20)
2.8
7.3
1.3
>10,000 (0.36)
—
8.3
73
258
114
5.8
4.7
72
>1000
582
461
>1000
212
a
Functional antagonist assay: inhibition of PGE2 evoked intracellular cAMP
elevation in HEK293 cells expressing EP4 receptors; agonist assay: potentiation of
cAMP elevation in the same cell line in the absence of PGE2
b
All compounds described in this Letter are greater than 1000-fold selective
versus EP1, EP3, FP, IP and TP.
The functional potency of 1c and 1e was measured in whole
blood. This functional cellular assay measures the blockade of inhi-
bition of TNFa
induced IP-10 release by a specific EP4 agonist.3 Both
Boc
Br
N
compounds are very potent in this assay with IC50s of 63 and
34 nM, respectively.
NH2
R
a
Pharmacokinetic experiments of select compounds were per-
formed in rats. Animals were dosed with the potassium salts at
20 mg/kg PO in 0.5% methocel and 5 mg/kg IV in 60% PEG 200.
Half-life, bioavailability and clearance data are shown in Table 3.
All compounds showed good to excellent bioavailability and low
to moderate clearance.
In order to investigate the metabolism of the series, rat hepato-
cyte incubation experiments were performed with compound 1e,
and the analysis of the incubation mixture was done by LC/MS.
After a 2 h incubation, 36% of parent compound was remaining.
Two major metabolites were observed and were identified as the
glucuronide and taurine conjugates of 1e. Indoles and metabolites
resulting from oxidation on the indoline core were among the
other minor metabolites observed.
f-h
3
O
OH
O
Boc
N
O
+
NH2
R
4
5
b
O
O
c-e
HO
O
H
H
N
O
N
O
Ar
Boc
N
N
R
R
1
6
Because of the high amount of metabolism on the acid moiety,
the tetrazole replacement of the carboxylic acid (10) could have
Scheme 1. Synthesis of compounds of general structure 2. Reagents: (a) s-BuLi,
TMEDA, CO2, THF; (b) HATU, DIPEA, ACN; (c) TFA, DCM; (d) NaH, ArCH2X, THF; (e)
NaOH, MeOH, THF; (f) Boc2O, TEA, MeOH; (g) MeLi, n-BuLi, CO2, THF; (h) TMSCl/
MeOH.
Table 2
R'
F
H
F
N
O
commercially available amine 5 to give 6. Afterwards, the amine
was deprotected and the indoline NH was benzylated with the
appropriate benzyl halide. Finally, hydrolysis of the methyl ester
provided compounds of general structure 1.
F
N
R
Interestingly, it was found that indoline 1a behaved as a full
agonist of EP4 with an EC50 of 0.82 nM (Table 1), and was inactive
(IC50 >10000 nM) in inhibiting PGE2 evoked cAMP elevation in
HEK293 cells over-expressing human EP4. Furthermore, we ob-
served that it was possible to switch from agonist to antagonist
by varying the aryl substitution pattern on the benzyl indoline.
For example, addition of a para-substituent such as Cl on the ben-
zyl group provided compound 1b as an antagonist (IC50 = 1.1 nM).
Omission of the 3-Cl (1c) still provided a full antagonist and this
modification also resulted in an increase in selectivity on DP and
R
R’
EP4
Ki,
nM
Func. assaya
EP2/EP4
DP/EP4
IC50
nM
,
1e
10
1t
1u
1v
H
H
Cl
CF3
F
COOH
Tetrazole
COOH
COOH
COOH
0.28
1.0
0.40
0.66
0.37
1.6
3.5
2.2
3.0
2.1
>1000
>1000
647
153
>1000
>1000
>1000
670
504
>1000
a
Functional assay: inhibition of PGE2 evoked intracellular cAMP elevation in
HEK293 cells expressing EP4 receptors.

486
M. J. Boyd et al. / Bioorg. Med. Chem. Lett. 21 (2011) 484–487
O
OH
120
100
Boc
Br
N
NH2
80
4
60
40
20
0
a-d
g-j
F
F
O
OH
N
F
N
Naïve 0.006
0.06
Dose (mg/kg)
0.2
0.6
2
6
NH2
+
Figure 1. Inhibition by 1e of secondary paw swelling in rats with AIA. Complete
Freund’s adjuvant was injected into left hind paw on day 0. Compound 1e was
administered starting on day 9, once daily for 9 days. Secondary paw volume was
measured at day 18. The percentage of inhibition was calculated from the difference
between a treated group (n = 7/group) and a vehicle control. Vertical bars show
SEM.
8
7
e
N
F
H
F
F
N
O
N
120
100
80
60
40
20
0
9
f
N
N
N
HN
F
H
N
F
F
O
Naïve Vehicle 0.03
0.1
1
3
10
N
Dose (mg/kg)
Figure 2. Inhibition by 1e of carrageenan-induced mechanical hyperalgesia.
Carrageenan is injected into the rat’s paw at t = 0 h and 1e was administered at
t = 2 h. The hyperalgesic response is measured at t = 3 h by applying mechanical
pressure to the paw and measuring the latency in paw withdrawal. **, p <0.01
versus vehicle control by one way ANOVA followed by Dunnett’s test. Vertical bars
show SEM (n = 5/group).
10
Scheme 2. Synthesis of tetrazole 10. Reagents: (a) CH2N2, MeOH; (b) TFA, DCM; (c)
NaH, 4-CF3ArCH2Br, THF; (d) KOH, MeOH, THF, ; (e) HATU, DIPEA, ACN; (f)
azidotributyltin, toluene,
N,N0-dimethylcyclohexane-1,2-diamine, dioxane,
D
D
, AcOH; (g) Boc2O, TEA, MeOH; (h) CuI, NaI, rac-trans-
D
; (i) CuI, NaCN, N,N0-dimethyl-
ethylenediamine, toluene,
D; (j) HCOOH, DCM.
The efficacy of compound 1e was evaluated in both the rat adju-
vant-induced arthritis model (AIA), and in the rat carrageenan-in-
duced mechanical hyperalgesia model. In the AIA, 1e dose
dependently reduced secondary paw swelling with NSAID/Coxib
like efficacy (Fig. 1) and completely inhibited secondary paw swell-
Table 3
Pharmacokinetics
t1/2 (h)
F (%)
Cl (mL/min/kg)
1a
1b
1c
1e
10
4.4
4.8
4.7
4.1
2.6
65
73
102
48
26
12
9
11
28
ing at
a dose of 0.6 mg/kg/day. In the carrageenan-induced
mechanical hyperalgesia model, 1e showed dose dependant inhibi-
tion of pain with a maximum of 64% inhibition relative to vehicle at
10 mg/kg (Fig. 2).
54
In conclusion, a novel benzyl indoline series of EP4 ligands was
discovered. Agonism and antagonism can be modulated by simple
modifications on the benzyl moiety. The compounds in this series
have good pharmacokinetic properties and are efficacious in
pre-clinical models of pain and inflammation.
potentially altered the pharmacokinetics; however, this modifica-
tion did not improve half-life or lower clearance in rats (Table 3).
In order to better understand the clearance pathway of these
compounds, a radiolabelled version of 1e was prepared from 1q
by palladium catalyzed tritiation. A rat bile cannulation study
was performed with 3H-1e, and LC/MS analysis of the bile indi-
cated that the compound is mostly excreted as glucuronide and
taurine conjugates, with very small amounts of parent. In total,
86% of the total radioactivity was recovered in the first 24 hours
from bile.
In order to predict the potential of toxicity resulting from cova-
lent protein labelling from acyl glucoronide migration or oxidative
metabolism,6 in vitro and in vivo covalent labelling studies were
performed with 3H-1e. It was found that the extent of covalent
protein labelling was relatively low in both rat and human hepato-
cytes (56 and 28 pmole-eq/mg @ 2 h, respectively). And an in vivo
covalent protein labelling study in rat also showed a relatively
small amount of labelling (5 pmol/mg protein).
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