Discovery of diphenyloxazole and Nδ-Z-ornithine derivatives as highly potent and selective human prostaglandin EP4 receptor antagonists

Article abstract of DOI:10.1021/jm050085k

Two novel classes of diphenyloxazole and Nδ-Z-ornithine derivatives as highly potent and selective EP₄ antagonists have been discovered. The optimized diphenyloxzole 8 and Nδ-Z-ornithine 11 effectively competed with [³H]-PGE₂

Full text of DOI:10.1021/jm050085k

J. Med. Chem. 2005, 48, 3103-3106
3103
Discovery of Diphenyloxazole and
Nδ-Z-Ornithine Derivatives as Highly
Potent and Selective Human
Prostaglandin EP4 Receptor Antagonists
Kouji Hattori,*,† Akira Tanaka,† Naoaki Fujii,†
Hisashi Takasugi,† Yoshiyuki Tenda,‡
Masayuki Tomita,§ Shoko Nakazato,§ Keiko Nakano,§
Yasuko Kato,§ Yutaka Kono,§ Hidetsugu Murai,| and
Kazuo Sakane†
Medicinal Chemistry Research Laboratories, Exploratory
Research Laboratories, Medicinal Biology Research
Laboratories, and Biopharmaceutical and Pharmacokinetic
Research Laboratories, Fujisawa Pharmaceutical Co., Ltd.,
2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan
Received January 29, 2005
Figure 1. Lead generation.
lymphocyte model of the cellular immune response,
Nataraj and colleagues have reported that EP2 and EP4
receptors can regulate the function of antigen-present-
ing cells.6 These finding may also be related to the
observation that homozygous deletion of the EP4 recep-
tor decreased the incidence and severity of collagen
antibody-induced arthritis by McCoy and co-workers.7
Gene knockout study has also been used to study the
potential involvement of the EP2 and EP4 receptors in
colon cancer. For example, Mutoh and co-workers have
reported homozygous deletion of the EP4 receptor
decreased the formation of aberrant crypt foci in ani-
mals treated with azoxymethane, a known colon car-
cinogen.8 On the other hand, there is little known about
selective ligands as EP2 and EP4 receptor antagonists.
Several PG congeners have been used as probes for the
EP2 and EP4 receptor antagonist ligands, although
subtype selectivity was not satisfaction.9 We disclose
herein two different structural scaffolds as high potent
and selective EP4 antagonists.10
Figure 1 shows two structural different series as a
seed/lead compound. One approach to find the lead
compound is to modify the previously reported diphen-
yloxazole series which was a potent and orally active
prostacyclin mimetic.11 1 was found to have affinity to
not only the IP receptor but also the EP4 receptor, with
Ki ) 54 nM for IP and Ki ) 1020 nM for EP4; therefore,
we modified 1 to generate a more selective EP4 ligand.
Initial SAR work on replacement of the acid moiety of
1 rapidly afforded a dramatic change in affinity and
selectivity. Phenylacetic acid type 2 was approximately
20-fold improved affinity for the EP4 receptor (Ki for EP4
) 50 nM and Ki for IP ) 170 nM, data not shown), and
the benzoic acid type 3, which we identified as a
promising lead, was improved 200-fold (Ki for EP4 ) 5.5
nM and Ki for IP ) 610 nM) and also showed excellent
bioavailability.
Abstract: Two novel classes of diphenyloxazole and Nδ-Z-
ornithine derivatives as highly potent and selective EP4
antagonists have been discovered. The optimized diphenylox-
zole 8 and Nδ-Z-ornithine 11 effectively competed with [3H]-
PGE2 binding to human recombinant EP4, with Ki values of
0.30 nM and 0.91 nM, respectively, and were selective for all
members of the human prostanoid receptor family. 8 was
shown to exhibit good pharmacokinetic properties in rats and
dogs and potent inhibitory activity toward in vitro PGE2-
promoted IgE synthesis.
The development of agents active at prostaglandin
(PG) E2 receptors has been an area of interest to the
pharmaceutical industry for the past three decades,
with both agonists and antagonists anticipated to have
therapeutic utility in treating diverse conditions includ-
ing asthma, inflammation, pain, ulcers, cancer, and
osteoporosis.1 However, success has been limited by the
problems of identifying suitably selective ligands. Many
of the problems encountered have been due to the
existence of multiple prostanoid receptor subtypes and
the lack of the selectivity of prostaglandin analogues.
PGE2 has been shown to be the preferred prostanoid
ligand for four of these receptor subtypes, the EP1, EP2,
EP3, and EP4 receptors: PGD2 for the DP receptor,
PGF2R for the FR receptor, PGI2 for the IP receptor, and
TXA2 for the TP receptor.2 The molecular characteriza-
tion of these receptors has resulted in renewed interest
in the field because the availability of cloned receptors
facilitates the identification of subtype-selective ligands
and in delineating the pathophysiological roles of each
of these receptor subtypes.3
EP2 and EP4 receptors coupled to an increase in
intracellular cAMP are smooth muscle relaxants.4 Fedyk
and Phipps have reported that EP2 and/or EP4 antago-
nists play an important role in diminishing allergic and
IgE-mediated asthmatic responses since EP2 and EP4
subtypes regulate activation and differentiation of
mouse B lymphocytes to IgE-secreting cells.5 In a mixed
The second approach to find a different scaffold was
to explore the Fujisawa sample collection. Random
screening identified the promising seed compound 4
with Ki for EP4 ) 80 nM, which had a unique dipeptide
structure containing N-R-indoylcarbonyl-N-δ-benzyloxy-
carbonyl ornithine and phenylalanine benzylmethyl-
amide. Transformation of the terminal benzylmethyla-
* To whom correspondence should be addressed. Phone: 81-6-6390-
1220. Fax: 81-6-6304-5435. E-mail address: kouji_hattori@
po.fujisawa.co.jp.
† Medicinal Chemistry Research Laboratories.
‡ Exploratory Research Laboratories.
§ Medicinal Biology Research Laboratories.
| Fujisawa Pharmaceutical Co.
10.1021/jm050085k CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/13/2005

3104 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Letters
Scheme 1a
a Reagents and conditions: (a) Tf2O, 2,6-lutidine, dichloromethane, 91%; (b) Pd(OAc)2, 1,3-bis(diphenylphosphino)propane, CO, Et3N,
DMF-MeOH, 83%; (c) 1 N NaOH, THF, MeOH, 92%; (d) BnONH2, WSC-HCl, HOBT, DMF, 95% for 7, BnSO2NH2, WSC-HCl, DMAPy,
DMF, 89% for 8.
Table 1. Profile of Eight Prostanoid Binding
human Ki (nM)
rat Ki (nm)
IP
DP
FP
TP
EP1
EP2
EP3
EP4
R-EP4
1
54
>1000
>3000
>1000
>3000
>1000
>10000
>1000
>3000
>1000
>3000
>1000
>10000
>1000
>3000
>1000
>3000
>1000
>10000
>1000
2500
>3000
>3000
>1000
>10000
>1000
>3000
>3000
>3000
>1000
>10000
>1000
>3000
>3000
>3000
>1000
>10000
1020
5.5 ( 0.23
0.68
0.30 ( 0.09
43
0.91 ( 0.10
3
610
7
>3000
>3000
>1000
>10000
8
0.74 ( 0.21
5.1 ( 0.2
5
11
a The results were presented as the mean ( SE of three independent experience for 3, 8, and 11 and as the average of two experiment
for 1, 7, and 5. Competitive binding assay based on the displacement of [3H]-Iloprost for human IP receptor, [3H]-PGD2 for human DP
receptors, [3H]-PGF2 for human FP receptors, [3H]-SQ29548 for human TP receptor, and [3H]-PGE2 for human EP1-4 receptors. Assays
were performed by the reported method; see in refs 2 and 4.
Table 2. Profile of Pharmacokinetica
rat: iv (n ) 2-3)
po (n ) 3)
dog: iv (n ) 2-3)
po (n ) 3)
t1/2 (h)
Cl (mL/min/kg) dose (mg/kg) Cmax (ng/mL) F(%)b
t1/2 (h)
Cl (mL/min/kg) dose (mg/kg) Cmax (ng/mL) F(%)
1
3
8
6.6 ( 0.33
4.0
4.4 ( 1.6
17.7 ( 0.33
11.2
10.2 ( 0.7
0.32
10
10
16.4 ( 0.88
9100 ( 1000
2100 ( 300
50
99
51
1
3
8
3.0 ( 1.4
5.5
5.5 ( 0.8
1.59 ( 0.29
0.52
0.52 ( 0.18
0.32
0.10
3.2
592 ( 196
57.3 ( 12.0
8200 ( 1400
72
25
97
a Results were shown as the mean ( SE. b F ) Bioavailability.
mide group of 4 to simple carboxylic acid led to the
identification of the 2-fold more potent compound 5 as
a second lead. We further optimize these two series of
diphenyloxazole-based lead and peptide-based lead to-
ward high EP4 subtype selectivity.
The diphenyloxazole derivatives in this study were
prepared as shown in Scheme 1. Key optically active
intermediate 6 was prepared by a simple and stereose-
lectve synthetic method.11 Optically active 6 was treated
with Tf2O, followed by carbonization with Pd(OAc)2, 1,3-
bis(diphenylphosphino)propane, and CO gas in a mix-
ture of DMF and MeOH and hydrolysis with 1 N NaOH
to give the desired compound 3 69% yield and >99% ee
from 6.
We focused our attention on further modification of
the benzoic acid part, which was transformed into amide
derivatives using various commercial available amines
to find a new pharmacophore for the EP4 receptor. We
discovered that aryl-substituted amide compounds formed
from the amines with two and three carbon atoms
between the nitrogen atom and the phenyl, such as 7,
had 10-fold improved potency toward the EP4. Phen-
oxymethylamide derivative 7 had high affinity for the
EP4 receptor with Ki ) 0.68 nM and good selectivity over
other EP receptors (Table 1). However, despite amide
derivatives showing high affinity and good selectivity,
compound 7 exhibited poor PK properties, hindering
further pharmacological evaluation. In consideration of
the PK profile of 1 and 3, acidic character of the
molecule would be essential for good bioavailability.
Combining both compounds character for potency and
bioavailability, we attempted to utilize acylsulfonamide
linked with benzyl substitution. The acyl sulfonamide
group is probably the most widely used replacement for
carboxylic acid and also recently has shown up in some
prostanoid directed ligands12 The new designed acyl-
sulfonamide analogue 8 showed good affinity for the EP4
receptor and excellent selectivity over other human
prostanoid receptors (each receptor selectivity >3000
nM). The acidic character of 8 was also displayed in the
good oral availability in rats and dogs, 50% and 90%,
respectively, in Table 2.13
Our next efforts on modifying another lead compound
5 started by exploring possible replacements for the
three fragments, as shown in Scheme 2. To optimize
rapidly and efficiently, we applied solid-phase synthesis
involving three major steps of three units: (i) coupling
of 2-chlorotrityl resin-bound to the first amino acid unit;
(ii) coupling of the second amino acid unit; (iii) capping
of the terminal amine with a substituted carboxylic acid.
This method produced the desired dipeptide-products
on a 1-10 mg scale in a total yield 80-90%.
We first determined the optimal side chain length of
the terminal carboxylic acid using building block X as
a carboxylic acid mimetic of the R-side chain of natural
PGE2. The homologue 9a was found to be same potency
while 9b was 50-fold more potent than lead 5 with 0.83
nM for the EP4 receptor. From this study, five-carbon

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3105
Scheme 2a
a Reagents and condition: (a) HO-AAx-NHFmoc, diisopropylethylamine, dichloromethane; (b) piperazine, DMF; (c) HO-AAy-NHFmoc,
TBTU, HOBT, diisopropylethyamine, DMF; (d) R-OH, 1,3-carbodiimide, diisopropylethyamine, dichloromethane; (e) TFA, dichloromethane.
chains were selected as the optimum length for further
optimization. Replacement of the Z-ornithine unit with
building block Y resulted in completely loss of affinity
for the EP4 receptor (Ki > 1000 nM) except Z-lysine.
L-Lysine 10a and D-lysine 10b showed different dis-
crimination for the EP4 receptor, 8.4 nM and 240 nM,
respectively. In the terminal capping unit R, 2-indoyl,
2-furanyl, 2-thiophenyl, and 2-naphthyl were the best
groups. Preliminary SAR work for the dipeptide series
indicated that substituted Z-ornithines 9b and 11
provide the best compound for potency and selectivity;
however, 9b and 11 showed problems in bioavailability.
The preliminary SAR results led to the generation of
11 are reported as Supporting Information and the
further optimization focused on improving bioavailabil-
ity will be published soon.
Next, we examined the in vitro antagonistic action of
8 and 11. It has already been confirmed that PGE2 is a
powerful immunomodulator that shifts the balance of
the cellular immune response away from Th1 and
toward Th2 and drives the humoral response toward
Figure 2. Effects of EP4 antagonists on in vitro PGE2-
IgE.14 Figure 2 shows EP4 antagonists have an inhibi-
promoted IgE synthesis in BDF1 mice B cells. Values were
*
presented as the mean ( SE, N ) 6. ##p < 0.01 vs base. p <
tory effect on PGE2-promoted IgE synthesis using BDF1
mice B cells.15 PGE2 were preincubated with small
dense B lymphocytes stimulated with IL-4 and LPS. IgE
were assayed in supernatants 6 days after addition of
IL-4 and LPS. PGE2 promotes IL-4, and LPS induced
IgE synthesis, which was significantly attenuated by
pretreatment with 8 at concentrations from 10-8 M to
10-6 M and 11 at concentrations from 10-7 M to 10-6
M. In this model, pretreatment of antagonists 8 and 11
without PGE2 had no effect on IgE level (data not
shown).
0.05 vs control. **p < 0.01 vs control.
We have discovered potent and selective EP4 antago-
nists according to two discovery approaches, from a PGI2
agonist and from random screening. Newly designed
EP4 antagonists 8 and 11 exhibited highly potent and
selective EP4 receptor activity. 8 has good pharmaco-
kinetic properties, and the inhibitory effect against
PGE2 promoted B lymphocyte Ig isotype switching to
IgE in vitro. These findings suggest 8 could be an

3106 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Letters
E receptor subtype EP4 in colon carcinogenesis. Cancer Res.
2002, 62, 28-32.
attractive therapeutic candidate for diminishing al-
lergic, asthmatic, and atopic disorders mediated by IgE.
(9) Ono-AE2-227 and EP4A have been reported as EP4 antagonists.
Ono-AE2-227: Ki values were found to be 2.7 nM for the mouse
EP4 receptor and 21 nM for mouse EP3 receptor, see in ref 8.
EP4A: Ki values were found to be 24 nM for the human EP4
receptor and 710 nM for human TP receptor, see in Machwate,
M.; Harada, S.; Leu, C. T.; Seedor, G.; Labelle, M.; Gallant, M.;
Hutchins, S.; Lachance, N.; Sawyer, N.; Slipetz, D.; Metters, K.
M.; Rodon, S. B.; Young, R.; Rodan, G. A. Prostaglandin receptor
EP4 mediates the bone anabolic effects of PGE2. Mol. Pharama-
col. 2001, 60, 36-41.
Acknowledgment. We express our thanks to Dr.
David Barrett for his critical reading of the manuscript.
Supporting Information Available: The experimental
procedures and compound characterization data. The prelimi-
nary SAR results of the dipeptide derivatives. This material
is available free of charge via the Internet at http://pubs.
acs.org.
(10) Saturation analysis was performed to determine the equilibrium
dissociation constants (Kd) and maximum number of binding
sites (Bmax) of radioligand [3H]PGE2 for EP4 receptors expressed
in COS cells: HumanEP4, Kd ) 0.28 ( 0.03 (nM), Bmax ) 3.1 (
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JM050085K