Synthesis and evaluation of novel pyrazolidinone analogs of PGE2 as EP2 and EP4 receptors agonists

Article abstract of DOI:10.1016/j.bmcl.2007.09.074

Replacement of the hydroxy cyclopentanone ring in PGE₂ with chemically more stable heterocyclic rings and substitution of the unsaturated α-alkenyl chain with a metabolically more stable phenethyl chain led to the development of potent and selective analogs of PGE₂. Compound 10f showed the highest potency and selectivity for EP₄ the receptor.

Full text of DOI:10.1016/j.bmcl.2007.09.074

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6572–6575
Synthesis and evaluation of novel pyrazolidinone analogs
of PGE2 as EP2 and EP4 receptors agonists
Zhong Zhao,a,*, Gian Luca Araldi,a Yufang Xiao,a,* Adulla P. Reddy,a Yihua Liao,a
Srinivasa Karra,a Nadia Brugger,a David Fischerb and Elizabeth Palmerb
aDepartment of Medicinal Chemistry, EMD-Serono Research Institute, Inc., Rockland, MA 02370, USA
bDepartment of Lead Discovery, EMD-Serono Research Institute, Inc., Rockland, MA 02370, USA
Received 23 August 2007; revised 18 September 2007; accepted 21 September 2007
Available online 26 September 2007
Abstract—Replacement of the hydroxy cyclopentanone ring in PGE2 with chemically more stable heterocyclic rings and substitution
of the unsaturated a-alkenyl chain with a metabolically more stable phenethyl chain led to the development of potent and selective
analogs of PGE2. Compound 10f showed the highest potency and selectivity for EP4 the receptor.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Prostaglandins are derived from arachidonic acid in a
two-step enzymatic reaction and act as autocrine and
paracrine lipid mediators.1 Prostaglandin E2 (PGE2) is
the most well known prostanoid derivative and exhibits
a broad range of biological actions in diverse tissues
through binding to specific receptors on the plasma
membrane. Four subtypes of the PGE receptor are
known (EP1, EP2, EP3 and EP4) and have been demon-
strated to belong to the G protein-coupled rhodopsin-
type receptor superfamily. Recent developments in the
molecular biology of the prostanoid receptors have en-
abled the investigation of roles specific to each receptor
by disruption of the respective gene.1 The EP2 and the
EP4 receptors are interesting pharmacological targets
because of their important regulatory roles in numerous
physiological processes, including bronchodilation, fer-
tility, bone resorption, and inflammation. Activation
of the EP2 and EP4 receptor increase the intracellular
cAMP level, which is linked to the treatment of preterm
labor by suppressing uterine contraction. Because EP2
receptor is induced in the cumulus in response to
gonadotropins and EP2 receptor system works as a po-
sitive-feedback loop to induce of oophorus maturation
required for fertilization.1b
PGE2, the natural ligand for these receptors, despite its
high potency, is not selective toward the individual EP
receptors. Also it is degraded quickly into inactive prod-
ucts under physiological conditions, with a half-life of
30 s to a few minutes.2 Natural prostaglandins are sus-
ceptible to three major modes of metabolic inactivation:
x-chain hydroxy oxidation, b-oxidation of the.a-chain,
and oxidation of the x-chain terminus.2 Furthermore,
PGE2 is chemically unstable since the hydroxyl group
on C-11 can easily undergo elimination, leading to en-
one derivatives like PGA2.2c In the last two decades, ef-
forts to improve the selectivity and chemical and
metabolic stability have been described3–6 (Fig. 1).
O
O
CO2Me
CO2H
OH
11
11
HO
HO
15
HO
butaprost
PGE2
CO2H
O
N
N
Keywords: Prostaglandin; EP2; EP4; Agonist.
*
Corresponding authors. Tel.: +1 781 681 2789; fax: +1 781 681 2939
(Y.X.); e-mail addresses: zhong.zhao@genzyme.com; yufang.xiao@
emdserono.com
R
HO
1
Present address: Genzyme Corporation, 153 Second Avenue, Walth-
man, MA 02451, USA.
Figure 1. PGE2 and pyrazolidinone derivative.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.074

Z. Zhao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6572–6575
6573
In order to improve the pharmacological properties of
PGE2, we have examined the replacement of the a-alke-
nyl side chain with the more stable phenethyl chain and
the substitution of the hydroxy cyclopentanone moiety
with different heterocyclic rings.3,4,6 This report de-
scribes the synthesis and structure-activity relationship
of a series of pyrazolidin-3-one derivatives of general
structure 1 bearing different R groups in the x-chain.
7 was carried out in EtOH at reflux for 2 h to furnish
8 in good to quantitative yields. The ketone intermediate
8 was transformed in 2 steps with excellent yield into the
final desired product 10 via reduction of 8 with NaBH4
in the presence of CeCl3 and saponification of the ester
group of 9.
Individual compounds were tested in vitro in the human
EP2/EP4 receptor binding assays and also in the human
EP2/EP4 functional assays.4 The data in Table 1 indicate
that the best results are obtained when a straight PGE2-
like chain is introduced at the x-position (compound
10a). Introduction of branching in the chain led to a
remarkable decrease in activity, especially for the EP4
receptor. Furthermore, it is interesting to note that com-
pound 10a showed 10-fold selectivity for the EP4 recep-
tor over the EP2 receptor.
Synthesis of the pyrazolidin-3-one derivatives is outlined
in Scheme 1. Introduction of the side chain in the a-po-
sition was achieved by alkylation of commercially avail-
able tert-butyl hydrazinecarboxylate with the phenethyl
bromide derivative 2 in the presence of a weak base. The
pyrazolidinone ring was then formed in one step by
reaction of chloropropanoyl chloride with the hydrazide
intermediate 3 under basic conditions.7 Deprotection of
the Boc group afforded the versatile intermediate 5
which was extensively used in our research effort for
the introduction of the x-chain.
The ONO’s researchers reported that the introduction of
a meta-position in phenyl group in the x-chain increased
selectivity and potency for EP4 receptor.6b Several 15-
hydroxy-16-aryl pyrazolidinone derivatives have been
synthesized and their in vitro data are summarized in
Table 2. As can be seen, the introduction of a simple
aromatic group as 10d led to a remarkable improvement
in EP4 affinity (100-fold over EP2) compared to the cor-
responding alkyl derivative 10a (10-fold over EP2).
Development of SAR for substituents at the meta posi-
tion of the aromatic ring led to the conclusion that the
best results are obtained with the introduction of halo-
gen atoms like iodo, bromo and chloro. Interesting re-
sults are also obtained with the introduction of bulky
groups (10m and 10n). There is still little known about
the structural determinants of the EP2 and EP4 receptors
that are required for the binding of PGE2, or the resi-
dues that dictate the selectivity of EP2 or EP4 receptors.
It is not at all clear that meta-position contributes signif-
icantly to binding or function at EP4 receptor.
Preparation of the pyrazolidinone derivatives bearing
the hydroxy group on C-15 of the x-chain was achieved
by Michael addition of the pyrazolidinone intermediate
5 to enone 7.8 As described in Scheme 1, the carboxylic
acid 6 was converted to the corresponding Weinreb
amide using EDCI and HOBt in DMF.9 Reaction of
the Weinreb amide with vinyl magnesium bromide pro-
vided enone derivative 7 that was used directly without
further purification.10 Michael addition of 5 to enone
CO2Me
CO2Me
a
HN
NH
Br
Boc
2
3
CO2Me
c
O
CO2Me
O
b
N
Due to the promising results in this series, we decided to
develop an enantioselective synthesis for these EP4 ago-
nists. As described in Scheme 2, the commercially avail-
able butadiene monooxide was converted to allylic
alcohol 12 by reaction with aryl magnesium bromide
11 in the presence of a catalytic amount of CuCN.
The allylic alcohol was then subjected to Sharpless epox-
idation condition with (+)-diethyl L-tartrate to furnish
the epoxide 13.11 Regioselective ring opening of epoxide
13 with Red-Al12 reduction afforded the diol derivative
14 (ee% >97% determined with chiral HPLC). Selective
protection of the secondary alcohol was achieved in a
two-step procedure by first using trimethyl orthoformate
and then reducing the cyclic ether intermediate with
N
N
N
H
Boc
4
5
CO2Me
O
R1
R
R1
g
d, e
N
R
CO2H
N
O
R
6
8
7
R1
O
CO2Me
h
CO2H
O
O
f
N
N
N
N
R
R
R1
R1
HO
HO
9
10
Table 1. 15-Hydroxy 16-alkyl pyrazolidinone derivatives
Scheme 1. Reagents and conditions: (a) tert-butyl hydrazinecarboxy-
late, NaHCO3, ACN, reflux, 15 h; (b) 3-chloropropanoyl chloride,
K2CO3, DMF, rt, 18 h; (c) TFA, DCM, RT, 1 h; (d) N,O-
dimethylhydroxylamine, EDCI, HOBt, Et3N, DMF, rt, 18 h; (e)
vinylmagnesium bromide, THF, 0 ꢁC, 1 h; (g) 5, EtOH, reflux, 2 h; (f)
NaBH4, CeCl3, EtOH, H2O, RT, 1 h; (h) NaOH, H2O, MeOH, THF,
rt, 18 h.
Compound CH2RR1
h-EP2
Ki (lM) EC50
(lM)
h-EP2 h-EP4 h-EP4
Ki
EC50
(lM) (lM)
10a
10b
10c
(CH2)4CH3
2.2
0.595 0.25
0.005
0.277
nd
CH2CH(Me)2 4.41
CH(Me)C3H7 2.76
15
5
2
6

6574
Z. Zhao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6572–6575
Table 2. 15-Hydroxy-16-aryl pyrazolidinone derivatives (R1@H)
Compound
R
h-EP2 Ki
(lM)
h-EP2
EC50 (lM)
h-EP4 Ki
(lM)
h-EP4 EC50 (lM)
10d
10e
10f
10g
10h
10i
Ph
m-I-Ph
6.2
0.068
0.027
0.013
0.059
0.036
0.057
0.018
0.33
2.36
5.45
2.83
6.29
6
m-Br-Ph
m-OCF3-Ph
m-F-Ph
0.0002
0.0002
m-CF3-Ph
m-Cl-Ph
10l
10m
5.69
8.34
m-(ethynylcyclopropyl)
phenyl
10n
m-(ethynylphenyl)
phenyl
0.56
0.044
PGE2
Butaprost
0.0049
0.11
0.00079
>10
R
Br
R
R
MgBr
R
OH
a
b
d
a, b, c
OH
O-TBS
18
12
11
17
R
c
OH
R
R
OH
CO2H
d, e
O
CO2Me
O
O
OH
14
N
N
e, f
N
N
13
R
CO2H
R
O
O-TBS
OH
f, g
20
19
O
N
N
Scheme 3. Reagents and conditions: (a) TBSCl, Imidazole, DMF, rt,
18 h; (b) (i) n-BuLi, THF, À78 ꢁC, 10 min, (ii) paraformaldehyde, rt,
2 h; (c) PPh3, CBr4, DCM, rt, 18 h; (d) 5, K2CO3, NaI, DMF, 50 ꢁC,
1 h; (e) H2 (1 atm), Pd/C 10%, MeOH, rt, 2 h; (f) (i) HCl 4 M, dioxane,
rt, 1 h; (ii) NaOH, H2O, MeOH, THF, rt, 18 h.
OMOM
R
15
OH
16
Scheme 2. Reagents and conditions: (a) 2-vinyloxirane, CuCN, THF,
À78 ꢁC, 2 h; (b) L-DET, Ti(O-i-Pr)4, t-BuO3H, molecular sieves,
DCM, À16 ꢁC, 4 h; (c) Red-Al, THF, 0 ꢁC, 15 h; (d) (i) trimethyl
orthoformate, CSA, DCM, RT, 2 h (ii) Dibal-H, À70 ꢁC, 2 h; (e) Dess-
Martin reagent, DCM, rt, 1 h; (f) Na(OAc)3BH, AcOH, DCE, rt, 2 h;
(g) (i) HCl (conc), MeOH, rt, 1 h; (ii) NaOH, H2O, MeOH, THF, rt,
18 h.
analogs bearing the hydroxyl group at C-16, similar to
the known EP2 selective agonist butaprost, which exhib-
ited a high selectivity (25000-fold) for EP2 receptor.13
Synthesis of the 16-hydroxyl pyrazolidinone derivatives
is outlined in Scheme 3. Protection of the propargyl
alcohol derivative 17 followed by formylation gave the
new propargyl alcohol in good yield. Conversion of
the alcohol into the bromide using triphenylphosphine
and carbon tetrabromide in DCM gave the correspond-
ing propargyl bromide 18 in quantitative yield, which
was used to alkylate pyrazolidinone 5. Hydrogenation
and deprotection of the TBS group and hydrolysis of
the methyl ester furnished the desired compounds of
general formula 20 in good yield.
DIBAL-H. Oxidation of the resulting alcohol intermedi-
ate using Dess-Martin periodine reagent afforded the
aldehyde intermediate 15 in quantitative yield, which
was used to alkylate the pyrazolidinone ring under stan-
dard procedure followed by saponification of the ester
groupto yield the desired chiral pyrazolidinone deriva-
tives 16. The in vitro activity of the compounds is sum-
marized in Table 3. The 16 with chiral center at C-15 did
not improve the binding affinity and potency (16a vs
10d, 16b vs 10f, 16c vs 10e). It indicates that the chiral
center at C-15 was not necessary to contribute the po-
tency and selectivity.
The in vitro activities are listed in Table 4. Moving the
hydroxy group from C-15 to C-16 decreased dramati-
cally in activity toward the EP4 receptor while maintain-
In order to improve the selectivity for the EP2 receptor,
we have investigated the synthesis of pyrazolidinone
Table 3. 15-Hydroxy-16-aryl pyrazolidinone derivatives
Table 4. 16-Hydroxy pyrazolidinone derivatives
Compound
R
h-EP2 Ki h-EP2 EC50 h-EP4 Ki h-EP4
(lM)
Compound
R
h-EP2 Ki h-EP2 EC50 h-EP4 Ki
(lM)
(lM)
(lM)
EC50
(lM)
(lM)
(lM)
20a
20b
(CH2)4CH3 2.44
1.3
2
16a
16b
16c
H
9.2
nd
0.134
0.013
0.026
nd
0.0001
nd
m-Br 3.07
m-I 2.03
>10
nd
0.75
0.393
>50

Z. Zhao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6572–6575
6575
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butaprost-like cyclobutyl group at C-17 (20b) increased
the EP2 activity to a certain degree, but showed a sharp
decrease in EP4 activity. However, 20b still showed less
selectivity and potency for EP2 receptor.
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In summary, for this series of pyrazolidinone deriva-
tives, compound 10f was found to be the most potent
and selective for the EP4 receptor than the EP2 receptor.
The rat pharmacokinetics data showed that compound
3. (a) Cameron, K. O.; Lefker, B. A.; Crawford, D. T.;
DaSilva-Jardine, P.; DeNinno, S. L.; Gilbert, S.; Grasser,
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E.; Reddy, A.; Xiao, Y.; Araldi, G. Abstracts of Papers,
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10f exhibited
a high intravenous distribute rate
(199.66 L/Kg), a moderate clearance rate (1.44 L/Kg/
h), a half-life time (4.25 h), and a low oral bioavailability
(6.7%). The low oral bioavailability might possibly be
caused by its low permeability.14 The low bioavailability
limited the assessment of these compounds in animal
model with per os route. The in vivo ovulation induction
of compound 10f in CD-1 adult female mice (10-week-
old) was evaluated. Preliminary results showed that a
3 mg/kg dose of 10f stimulated rupture of the follicle
and the induction and release of four oocytes into the
fallopian tube.11 oocytes were released when dosing
30 mg/kg at subcutaneous administration.15 10f also
exhibited good efficacy in a dose-dependent manner
(from 3 to 30 mg/kg).
Conclusions. In summary, we have found that analogs of
PGE2 wherein the hydroxyl cyclopentanone ring has
been replaced by a pyrazolidinone ring are potent EP4
receptor agonists. In particular, the pyrazolidin-2-one
derivatives having a phenethyl a-chain showed the best
in vitro profile among the different series explored. Mod-
ulation of the x-chain allowed us to find a potent and
selective EP4 agonist (for example 10f). Introduction
of a 16-hydroxy group on the x-chain (20a–b) did not
obviously improve the potency for the EP2, but dramat-
ically decreased EP4 receptor activity.
11. Rossiter, B. E.; Verhoeven, T. R.; Sharpless, K. B.
Tetrahedron Lett. 1979, 4733.
Acknowledgment
12. Wipf, P.; Lim, S. J. Am. Chem. Soc. 1995, 117, 558.
13. Gardiner, P. J. Br. J. Pharmac. 1986, 87, 45.
14. Compound 10f has low permeability with 9% passage, 10
papp · 10À6 cm/s. Rat plasma protein binding of
compound 10f (C = 10 mM, 2 h) was 61% unbound. After
1 h, live microsomes degradation was 7% in rat and 0%
in human, respectively. The solubility of compound
10f was measured to be 0.89 mg/mL in 2% DMSO in
HBSS or 0.84 mg/mL in 2%DMSO in saline. The result at
intravenous administration is not disclosed here.
15. We tested compounds in ovulation induction in two
routes: intravenous, subcutaneous. 2% DMSO + saline
was used to be vehicle control and FSH was a positive
control.
We are grateful to Dr. Lesley Liu-Bujalski for proof-
reading the manuscript.
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