Synthesis and evaluation of a γ-lactam as a highly selective EP2 and EP4 receptor agonist

Article abstract of DOI:10.1016/j.bmcl.2007.11.020

γ-Lactam analogs (2) of EP₄ receptor agonists were identified by substitution of the pyrazolidinone ring (1) with a pyrrolidinone ring. Several compounds (such as 2a, 2h) with high potency, selectivity and acceptable PK profiles were discovered. These were assessed in animal models of ovulation induction and bronchoconstriction.

Full text of DOI:10.1016/j.bmcl.2007.11.020

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 821–824
Synthesis and evaluation of a c-lactam as a highly selective
EP2 and EP4 receptor agonist
Yufang Xiao,a,* Gian Luca Araldi,a Zhong Zhao,a Adulla Reddy,a Srinivasa Karra,a
Nadia Brugger,a David Fischer,b Elizabeth Palmer,b Bagna Baob and Sean D. Mckennab
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 24 October 2007; revised 7 November 2007; accepted 8 November 2007
Available online 13 November 2007
Abstract—c-Lactam analogs (2) of EP4 receptor agonists were identified by substitution of the pyrazolidinone ring (1) with a pyr-
rolidinone ring. Several compounds (such as 2a, 2h) with high potency, selectivity and acceptable PK profiles were discovered. These
were assessed in animal models of ovulation induction and bronchoconstriction.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Prostaglandins are known to have a broad range of bio-
logical actions in diverse tissues through binding to spe-
cific receptors on the plasma membrane.1 Four subtypes
of the PGE2 receptor have been identified: EP1, EP2,
EP3, and EP4, which mediate a wide variety of biological
activities.1 Of these four receptors, three are involved in
the modulation of cAMP levels.2 Activation of the EP3
receptor results in a reduction of the intracellular cAMP
level. In contrast, activation of EP2 receptor and the EP4
receptor increases the intracellular cAMP level, which is
linked to the treatment of infertility. The EP1 receptor is
involved in regulating intracellular calcium levels. The
EP2 and the EP4 receptors are interesting pharmacolog-
ical targets because of their important regulatory roles
in numerous physiological processes, suggesting that
agonists may be useful in preventing and/or treating pre-
term labor, ovulation induction, asthma, fertility disor-
ders, undesired blood clotting, sexual dysfunction,
bone resorption, and inflammatory disorders, and other
diseases. Additional roles for EP receptors have been re-
ported, including smooth muscle relaxation in cat tra-
chea for EP2, vasodilation and anti-inflammatory
activity for EP4.3 EP2 and EP4 receptor agonists have
been proven to be beneficial for the treatment of preterm
labor by suppressing uterine contraction and inducing
oophorus maturation required for fertilization.1b
Several research groups have been investigating the
improvement of pharmacological properties of PGE2,
which shows non-selective binding to the EP receptors
(in-house binding data Ki = 9.1, 4.9, 0.33, and 0.79 nM
for h-EP1, h-EP2, h-EP3, and h-EP4, respectively) and
chemical and metabolic instability.4 Until now, efforts
to improve the selectivity and chemical stability of
PGE2 have been focused on only two general chemical
modifications;5 replacement of the a-alkenyl side chain
with the more chemically stable phenylethyl group and
substitution of heterocyclic rings for the 11-hydroxy
cyclopentanone moiety.5
CO2H
CO2H
O
O
N
N
N
R
R
HO
2
HO
1
O
O
CO2Me
CO2H
OH
HO
11
HO
15
HO
PGE2
Keywords: Prostaglandin; EP2 receptor; EP4 receptor.
*
butaprost
Figure 1. PGE2 and prostaglandin derivative.
Corresponding author. Tel.: +1 781 681 2789; fax: +1 781 681
2939; e-mail: yufang.xiao@emdserono.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.020

822
Y. Xiao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 821–824
In our previous communication, we described pyrazolid-
inone analogs (1 in Fig. 1) that were EP4 receptor ago-
nists.6 Poor oral bioavailability limited assessment of
these compounds in animal models. In order to over-
come this problem, we investigated pyrrolidinone (c-lac-
tam) derivatives (2 in Fig. 1). Derivatives (2 in Fig. 1)
showed good PK properties with high potency for the
EP4 receptor. In this report, we describe the synthesis,
the structure-activity relationship of this series of com-
pounds, as well as the results from in vivo animal model
studies.
reduced with NaBH4 and CeCl3 (for example R groups
in compounds 2f, 2g) or by chiral reduction using Cor-
ey’s procedure.8 Saponification of the esters with NaOH
in THF/MeOH/H2O, followed by preparative reverse
phase HPLC, furnished the desired target compound 2.
Individual compounds were tested in vitro in the human
EP2/EP4 receptor binding assays and also in the human
EP2/EP4 functional assays.5d 3H-PGE2/4 binding is eval-
uated by counting the plates on the top count using the
3H SPA dpm2 program. % Binding and Ki values for
inhibitors are calculated based on the one site competi-
tion parameter using the graphpad.? prism program.
EP2/4 EC50 was evaluated by measuring total cAMP
(intra- and extra- cellular) by using a cAMP-screen ELI-
SA System (Tropix, #CS1000). The binding and func-
tional data are summarized in Table 1. Compound 2a
with the PGE2x-side chain showed high affinity binding
against both EP2 and EP4 receptors (h-EP2
Ki = 120 nM, h-EP4 Ki = 2 nM). Interestingly, this com-
pound also showed very good in vitro potency against
the EP4 receptor with an EC50 of 0.2 nM, about 15-fold
more potent than PGE2 itself (EC50 = 3.0 nM). In vitro
activity of this compound displayed 60- or 750-fold
selectivity for the EP4 receptor versus the EP2 receptor.
Compounds with shorter alkyl groups (such as 2c, 2d,
The approach for the synthesis of c-lactams is outlined
in Scheme 1. Alcohol 9 was the key intermediate for
construction of the final c-lactam derivatives 2.The
(R)-stereochemistry of compound 9 (corresponding to
the natural PGE2 stereochemistry) was inherited from
the starting material H-D-Glu(O-t-Bu)-O-t-BuÆHCl (ee
> 95%). Reductive amination of aldehyde 5 with H-D-
Glu(O-t-Bu)-O-t-BuÆHCl smoothly provided triesters 6,
which underwent intramolecular cyclization to afford
lactam 7. The three-step conversion of 7 by hydrolysis,
anhydride formation, and then reduction with NaBH4
gave the key intermediate 9.7 Conversion of alcohol 9
into enone 11 was accomplished via Swern oxidation
followed by Wittig olefination. The enone 11 can be
CHO
CHO
a
CHOMe
b
MeOOC
MeOOC
CHO
HOOC
c
4
3
H
N
d
COO-t-Bu
MeOOC
COO-t-Bu
MeOOC
5
6
CO2Me
O
N
CO2Me
O
e
f
N
COO-t-Bu
CO2H
7
8
CO2Me
O
CO2Me
O
i
g, h
N
N
CH2OH
CHO
9
10
CO2H
CO2Me
m, n
O
O
N
l
N
R
O
R
11
2
HO
Scheme 1. Reagents and conditions: (a) SOCl2, MeOH, 91%; (b) ClPh3PCH2OMe, NaOMe, MeOH then benzene 73%; (c) aqueous H2SO4, THF,
94%; (d) i—H-D-Glu (O-t-Bu)-O-t-Bu.HCl, Et3N; ii—HOAc, MeOH, NaBH3CN; (e) xylene, reflux, 59%, 2 steps; (f) TFA, 0 ꢁC to rt; (g) N-
methylmorpholine, i-BuOC(O)Cl, THF; (h) NaBH4, THF/H2O, 54%, 3 steps; (i) (COCl)2, DMSO, Et3N, DCM, 92%; (l) i—NaH, THF, 0 ꢁC; ii—
(MeO)2P(O)CH2C(O)R, 0 ꢁC to rt, 90%; (m) NaBH4 and CeCl3, MeOH, H2O or (R)-2-methyl-CBS-oxazaborolidine, BH3ÆTHF, rt, 80%; (n) NaOH,
H2O/MeOH/THF, 100%.

Y. Xiao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 821–824
Table 1. 15-Hydroxy pyrrolidin-2-one derivatives
823
Compound
R
h-EP2
Ki (nM)
h-EP2
EC50 (nM)
h-EP4
Ki (nM)
h-EP4
EC50
2a
2b
2c
2d
2e
n-Pentyl
n-Hexyl
n-Butyl
n-Propyl
Et
120
54
15
80
2
0.02
1
425
25,000
>10,000
1
0.04
27
490
2f
141
21
19
3
9
2
0.3
0.3
2g
2h
2i
Bn
m-ClBn
2540
1000
4.9
434
250
0.05
0.02
0.03
0.03
PGE2
Butaprost
0.79
>10,000
110
2e) exhibited a dramatic decrease in EP2 and EP4 po-
tency (2a vs 2e). Butaprost was reported to have high
selectivity for the EP2 receptor over other EP subtypes.9
Compound 2g, with butaprost x-side chain, increased
remarkably the EP2 potency while still maintained great-
er affinity for the EP4 receptor.
tered. 5 IU PMSG in 200 UL PBS (i.p.) is injected
and then after 48 h hCG or hCG replacement (PGE2
or test compounds, sc, iv or ps routes) is injected. After
18 h, animals are sacrificed by CO2 asphyxiation and
abdominal cavities are opened using fine scissors and
forceps. Uterus, oviducts, and ovaries are collected
and placed in pre-labeled dishes containing phosphate-
buffered saline (PBS). The collected tissues are trans-
ferred to the laboratory and intact oviduct carefully dis-
sected out from uterus and ovary under the dissection
microscope. The dissected oviducts are placed on the
glass microscopic slide and covered with another slide.
Two slides are taped on two edges. The numbers of ovu-
lated ova in the oviducts are counted using upright
microscope with 4· objective and recorded.
Introduction of an aromatic moiety in the x-chain in-
creased by approximately 40-fold the EP4 affinity and
dramatically increased selectivity (from 60-fold for 2a
to 50,000-fold for 2h) over the EP2 receptor. Introduc-
tion of appropriate substituents at the meta position of
the phenyl ring (for example 2i) had similar selectivity
and potency for EP4 receptor. Similar results have been
reported by the ONo’s researchers.10
Rat pharmacokinetics data showed that compound 2a
exhibited high intravenous distribution rate with
42.44 L/kg, high area under the curve (AUC48,
18095.3 h*ng/mL for iv, 35175.6 h*ng/mL for sc,
14657.8 h*ng/mL for per os), a long half-life time
(14.30 h for iv, 31.61 for sc, 20.77 for per os), time to max-
imum (Tmax: 0.083 h for iv, 0.25 h for sc, 4.0 h for per os),
low clearance rate of 0.55 L/kg/h, and good bioavailabil-
ity (33% per os, 82% for subcutaneous) when dosing with
10 mg/kg. This compares favorably against the relatively
low oral bioavailability of 6.7% for the pyrazolidinone 1.6
For an evaluation of the oral activity of compound 2a,
two experiments were conducted. The first experiment
was conducted with non-fasted animals and the second
one was conducted in 24 h fasted animals (water pro-
vided). The results demonstrate that the compound
effectively induced ovulation when administered po
(ED50 = 21.97 mg/kg in non-fasted animals and
21.1 mg/kg in the fasted animals) and the fasting did
not affect in vivo activity.
Several compounds (Table 2) were tested in the same
model by different routes of administration (po and sc).
hCG and PGE2 were used to be reference samples. The re-
sultsin Table 2 demonstrate that EP4 agonistswereable to
stimulate ovulation induction in mature mice by both
routes of administration.12 Compound 2 h was the most
potent with an EC50 = 0.32 mg/kg (sc route) and 1 mg/
The in vivo ovulation induction activity of several com-
pounds in CD-1 adult female mice (10-week-old) was
evaluated.11 The general protocol for ovulation induc-
tion is described as follows. PMSG (pregnant mare ser-
um gonadotropin) (Calbiochem, cat #367222) and hCG
(Serono) are diluted in PBS. PGE2 (Cayman, Ann Ar-
bor MI) is dissolved in ethanol and diluted with
0.154 M NaHCO3 Buffer (pH 8.0) to final concentration
of ethanol of less than 3%. A test compound (based on
solubility) is pre-dissolved in ethanol, DMSO or other
reagent. Test compound is then diluted with saline or
other diluents such as PBS or NP3S (5% N-methyl-pyr-
rolidinone/30% PEG-400/25% PEG-200/20% Propylene
Glycol in saline). PMSG serves to stimulate follicle
growth and maturation. Mature follicles will ovulate
when an ovulation triggering dose of hCG or an hCG
replacement (PGE2 or the test compounds) is adminis-
Table 2. The ovulation induction (ED50, mg/kg)
Compound
Ovulation induction (ED50
mg/Kg)
,
sc
po
2a
2f
3.8
3
22
18
1
2i
2h
n.a.
0.32
n.a.
>30
1
2g
Butaprost
1
n.a.

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Y. Xiao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 821–824
(c) Regan, J. Life Sci. 2003, 74, 143; (d) Kobayashi, T.;
Narumiya, S. Prostaglandin Lipid Mediators 2002, 68-69,
557; (e) Sugimoto, Y.; Narumiya, S.; Ichikawa, A. Prog.
Lipid Res. 2000, 39, 289; (f) Boie, Y.; Stocco, R.; Sawyer,
N.; Slipetz, D.; Ungrin, M.; Neuschafer-Rube, F.;
Puschel, G.; Metters, K.; Abranovitz, M. Eur. J. Pharm.
1997, 340, 227.
kg (po route), respectively, which is consistent with its
high binding affinity (Ki = 0.05 nM) and potency
(EC50 = 0.03 nM) for EP4. In contrast, butaprost did
not show any activity when administered at up to
30 mg/kg (sc route) suggesting that EP4 might be more
important than EP2 in mediating this biological response.
2. Breyer, R. M.; Bagdassarian, C. K.; Myers, S. A.; Breyer,
M. D. Annu. Rev. Pharmacol. Toxicol. 2001, 41, 661.
3. Takayama, K.; Garcia-Cardena, G.; Sukhova, G. K.;
Comander, J.; Gimbrone, M. A., Jr.; Libby, P. J. Biol.
Chem. 2002, 277, 44147.
4. (a) Yamamoto, S. Comprehensive Nat. Prod. Chem. 1999,
1, 255; (b) Goto, M. P.; Goldman, A. S. Handbook Expr.
Pharmacol. 1997, 124, 433, Drug Toxicity in Embryonic
Development I; (c) Collins, P. W.; Djuric, S. W. Chem.
Rev. 1993, 93, 1533.
5. (a) Cameron, K. O.; Lefker, B. A.; Crawford, D. T.;
DaSilva-Jardine, P.; DeNinno, S. L.; Gilbert, S.; Grasser,
W. A.; Ke, H. Z.; Lu, B.; Owen, T. A.; Paralkar, V. M.;
Thompson, D. D.; Tjoa, C. M.; Zamistoski, M. P.;
Abstracts of Papers, 224th National Meeting of the
American Chemical Society, Boston, MA 2002; (b) Billot,
X.; Chateauneuf, A.; Chauret, N.; Denis, D.; Greig, G.;
Mathieu, M.-C.; Metters, K. M.; Slipetz, D. M.; Young,
R. N. Bioorg. Med.Chem. Lett. 2003, 13, 1129; (c) Zhao,
Z.; Bao, B.; Brugger, N.; Fischer, D.; Giachetti, C.;
Golzio, L.; Karra, S.; Marinelli, P.; McKenna, S.; Palmer,
E.; Reddy, A.; Xiao, Y.; Araldi, G. Abstracts of Papers,
228th National Meeting of the American Chemical Soci-
ety, Boston, MA, 2004; (d) Xiao, Y.; Araldi, G. L.; Zhao,
Z.; Brugger, N.; Karra, S.; Fischer, D.; Palmer, E. Bioorg.
Med. Chem. Lett. 2007, 17, 4323; (e) Tani, K.; Naganwa,
A.; Ishida, A.; Egashira, H.; Sagawa, K.; Harada, H.;
Ogawa, M.; Maruyama, T.; Ohuchida, S.; Nakai, H.;
Kondo, K.; Toda, M. Bioorg. Med. Chem. Lett. 2001, 11,
2025; (f) Elworthy, T. R.; Brill, E. R.; Chiou, F. C.;
Harris, J. R.; Hendricks, R. T.; Huang, J.; Kim, W.; Lach,
L. K.; Miradegan, T.; Yee, C.; Walker, K. A. M. J. Med.
Chem. 2004, 47, 6124.
The guinea pig pulmonary-cholinergic in vivo model is
generally used to test potential therapeutics for the treat-
ment of asthma in humans.13 In our studies, groups of 3
Duncan Hartley male or female guinea pigs, weighing
250 50 g, were anesthetized with sodium pentobarbital
(50 mg/kg ip) and succinylcholine chloride. The trachea
was cannulated and tracheal pressure was recorded
through a sidearm of the cannula connected to a
P23ID Statham transducer. Mean arterial pressure was
monitored from a cannulated carotid artery, and heart
rate was obtained from a chest-affixed electrode. The
jugular vein was cannulated for iv administration of
the test compounds in a volume of 1 mL/kg. Choliner-
gic-induced bronchoconstrictor responses, reflected as
increases in tracheal pressure (cm H2O), were elicited
by administration of methacholine hydrochloride
(10 lg/kg base weight iv). Compound 2a was injected
iv at doses 30 ng/kg to 0.3 mg/kg. Notably, significant
methacholine-induced bronchoconstriction (>50%) inhi-
bition was observed at doses >3 lg/kg. The calculated
effective dose (ED50) for Compound 2a was approxi-
mately 1.7 lg/kg, while not altering the blood pressure
or heart rate. This compound showed activity in dilation
of bronchiolar muscles, which resulted in inhibition of
methacholine-induced bronchomuscle constriction.
Conclusions. In summary, we have found that analogs of
PGE2 wherein the hydroxyl cyclopentanone ring has
been replaced by a pyrrolidin-2-one ring are potentially
useful EP4 receptor agonists. Compound 2a displayed
good PK parameters and showed activity in two
in vivo animal models. These compounds also exhibited
an anti-inflammatory activity and were a potent ana-
bolic agent for bone.11 More in vivo animal data will
be reported elsewhere.
6. Zhao, Z.; Araldi, G. L.; Reddy, P. A.; Xiao, Y.; Liao, Y.;
Karra, S.; Brugger, N.; Fischer, D.; Palmer, E. Bioorg.
Med. Chem. Lett. 2007, 17, 6572.
7. The ee of 9 was greater than 95% as determined by chiral
HPLC.
8. Corey, E. J.; Bakshi, R. K.; Shibata, S.; Chen, C.-P.;
Singh, V. K. J. Am. Chem. Soc. 1987, 109, 7925,
Diastereoisomers are separable with reverse phase HPLC
in the last purification.
9. Gardiner, P. J. Br. J. Pharmacol. 1986, 87, 45.
10. Maruyama, T.; kambe, T.; Maruyama, T.; Yoshida, H.;
Nishiura, A. WO 2003/009872 A1.
Acknowledgments
We gratefully acknowledge Dr. Ben Askew and Dr. Les-
ley Liu-Bujalski for proofreading the manuscript.
11. Araldi, G. L.; Reddy, A. P.; Zhao, Z.; Mckenna, S. D.;
Bao, B. WO 2003/10364 A2.
12. The compounds also showed ovulation induction when
delivered using the i.v. route (data were not disclosed here).
13. Fleisch, J. H.; Rinkema, L. E.; Haisch, K. D.; Swanson-
Bean, D.; Goodson, T.; Ho, P. P.; Marshall, W. S.
J. Pharmacol. Exp. Ther. 1985, 233, 148.
References and notes
1. (a) Funk, C. D. Science 2001, 294, 1871; (b) Narumiya, S.;
Sugimoto, Y.; Ushikubi, F. Pharm. Rev. 1999, 79, 1193;