Discovery of highly selective EP4 receptor agonists that stimulate new bone formation and restore bone mass in ovariectomized rats

Article abstract of DOI:10.1016/j.bmcl.2006.01.018

Heptanoic acid lactams, exemplified by 2, were identified as highly selective EP4 agonists via high throughput screening. Lead optimization led to the identification of lactams with a 30-fold increase in EP4 potency in vitro. Compounds demonstrated robust

Full text of DOI:10.1016/j.bmcl.2006.01.018

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1799–1802
Discovery of highly selective EP4 receptor agonists
that stimulate new bone formation and restore bone
mass in ovariectomized rats
Kimberly O. Cameron,* Bruce A. Lefker, Margaret Y. Chu-Moyer, David T. Crawford,
Paul DaSilva Jardine, Shari L. DeNinno, Sandra Gilbert, William A. Grasser, HuaZhu Ke,
Bihong Lu, Thomas A. Owen, Vishwas M. Paralkar, Hong Qi, Dennis O. Scott,
David D. Thompson, Christina M. Tjoa and Michael P. Zawistoski
Department of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton Laboratories,
Eastern Point Road, Groton, CT 06340, USA
Received 30 November 2005; revised 4 January 2006; accepted 5 January 2006
Available online 26 January 2006
Abstract—Heptanoic acid lactams, exemplified by 2, were identified as highly selective EP4 agonists via high throughput screening.
Lead optimization led to the identification of lactams with a 30-fold increase in EP4 potency in vitro. Compounds demonstrated
robust bone anabolic effects when administered in vivo in rat models of osteoporosis.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Prostaglandin E2 (PGE2, 1, Fig. 1) is an endogenous
ligand with high affinity to four receptor subtypes desig-
nated EP1–EP4. PGE2 is a local mediator of a number
of physiological responses including analgesia, smooth
muscle relaxation, and vasodilation.1 In addition,
administration of PGE2 in animals stimulates new bone
formation and increases bone mass and strength.2 There
Figure 1. Structures of 1 and 2a.
is significant interest in the discovery of a therapeutic
agent that can form new bone and therefore treat pa-
tients with established osteoporosis; however, side ef-
fects (diarrhea, hypotension) preclude the use of PGE2
for this purpose. Previous reports suggest that agonism
of the EP4 receptor is important for mediating PGE2’s
bone anabolic effects.3 We describe our efforts to identi-
fy potent and EP4-selective compounds in a lactam
series.4,5
ties of 2a were benchmarked in the Sprague–Dawley
rat. Lactam 2 exhibits low bioavailability (approxi-
mately 20%), high iv clearance (101 mL/min/kg),
and a short half-life (0.1 h). SAR goals were to im-
prove EP4 potency and oral bioavailability. Homo-
log 3 and truncated phenyl derivative 4 (Fig. 2)
had reduced EP4 potency (see Table 1). We there-
fore focused chemistry efforts on the synthesis of
benzyl analogs and explored phenyl substituent ef-
fects on EP4 potency. In addition, we hypothesized
that b-oxidation of the heptanoic acid side chain,
a significant clearance pathway for PGE2,6 may con-
tribute to the high clearance observed with 2a. A
survey of a variety of heptanoic acid replacements
led to the identification of propyl thiophene and
propyl benzene carboxylic acid analogs (9–23) as
potent, EP4-selective compounds. In vitro data,
Directed high throughput screening led to the iden-
tification of our lead heptanoic acid lactam 2a
(Fig. 1), a selective EP4 agonist with good in vitro
potency (see Table 1). The pharmacokinetic proper-
Keywords: Bone anabolic agent; EP4; EP2; Prostaglandin; Lactam;
Osteoporosis.
*
Corresponding author. Tel.: +1 860 441 3410; fax: +1 860 715 4706;
e-mail: kimberly.o.cameron@pfizer.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.018

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K. O. Cameron et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1799–1802
Table 1. In vitro results for selected lactam analogs
Compound
L
Ar
IC50 hEP1 (nM) IC50 rEP2 (nM) IC50 hEP3 (nM) IC50 rEP4 (nM) EC50 rEP4 nM
(% vs PGE2)
2a
2b
a
b
c
Phb
Pha
Phb
>3200
>3200
>3200
>3200
>3200
>3200
54 31
250 184
32.5 34 (114)
1.7 0.2 (88)
2c
3
690 156
>3200
>3200
21
>3200
4
a
a
c
4
9c
CH2Pha
2-Naphthylb
4-Fluorophenyla
4-Fluorophenyl
3-Fluorophenyla
3-Fluorophenylb
4-Chlorophenylb
3-Chlorophenyla
3-Chlorophenyla
3-Chlorophenyl
2-Biphenylb
2705 700
65 38
>3200
>3200
500 28 (73)
22 14 (70)
10b
10c
11b
11c
12c
13a
13b
13c
14c
15a
15b
15c
16a
16b
16c
17a
18a
19a
20a
21a
22a
22b
23c
b
c
>3200
>3200
2767 379
70 36
126 48
1205 64
>3200
b
c
640 71
86 44
>3200
17
80 64
22
7
2.4 2 (97)
40 24 (90)
8.8 6.7 (89)
425 35 (106)
0.6 0.7 (70)
c
>3200
>3200
>3200
>3200
a
b
c
3
>3200
42 35
1.1 1.1
1600 566
337 154
47 43
188 109
125 35
>3200
c
a
b
c
3-Biphenyl
3-Biphenyla
3-Biphenylb
195 106 (101)
440 260 (97)
203 138 (95)
13 13 (75)
>3200
18,550
135 49
>3200
>3200
20,000
50 11
21 11
a
b
c
3-Trifluoromethylphenyl
3-Trifluoromethylphenyl
3-Trifluoromethylphenyl
3-Cyanophenyl
70 27
1
105 62 (105)
0.5 0.7 (102)
96 19 (104)
170 28 (100)
18 25 (89)
483 237
>3200
>3200
6
a
a
a
a
a
a
b
c
388 134
1202 436
62 50
3-Hydroxyphenyl
3-Methoxymethylphenyl
3-Methoxyethylphenyl
3-Trifluoromethoxy-phenyl
3-Phenoxyphenylb
3-Phenoxyphenyla
4-Ethylphenylb
>3200
>3200
>3200
>3200
>2000
>3200
210 134
59 37
92 96 (82)
50 3 (78)
>3200
>3200
536 52
220 127
140 42
>3200
1200 283
275 49 (82)
Data are presented SD and are the means of at least two independent measurements.
a Mixture of isomers at C-12 and C-15.
b Mixture of isomers at C-15 alcohol.
Figure 2. Structures of 3 and 4.
pharmacokinetic properties as well as our in vivo re-
sults for selected analogs are presented.
Scheme 1. Reagents and conditions: (a) ArCH2MgX, CH2Cl2, 0 ꢁC;
(b) NaBH4, EtOH, 0 ꢁC; (c) TBSCl, imidazole, DMAP, DMF; (d)
NaHMDS, DMF, RX; (e) TBAF, THF; (f) NaOH, MeOH.
The synthesis of the requisite lactams as racemates is de-
scribed in Scheme 1. Addition of the appropriate Grig-
nard reagent into tetrahydro-6H-pyrrolizine-3,5-dione
(5) provided benzylic ketone 6.7 Reduction of the ke-
tone, followed by TBS protection of the resulting alco-
hol, generated lactam 7 as an approximate 1:1 mixture
of diastereomers. N-Alkylation, followed by saponifica-
tion, provided the desired lactam acids (2, 9–23).
(Scheme 2).8 Alkylation of intermediate 249 with either
methyl 7-bromoheptanoate or methyl 4-(3-bromopro-
pyl)benzoate10 followed by TBS deprotection provided
25a or 25b, respectively. Intermediate 25c was prepared
as described in Scheme 3. Conversion to the enone 26
was accomplished using modified Horner–Emmons con-
ditions. In some cases, 26 was hydrogenated, reduced
with sodium borohydride, and deprotected to generate
The chiral alcohols 25a, b, and c served as key interme-
diates in the enantioselective synthesis of lactam analogs

K. O. Cameron et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1799–1802
1801
with good EP4 binding potency were assessed for their
functional activity by measuring cAMP levels in HEK-
293 cells expressing the rEP4 receptor. Unless otherwise
stated, the lactam analogs were full agonists as com-
pared to PGE2 in the cAMP assay. In vitro data are
shown in Table 1.
The lactam analogs displayed excellent selectivity for the
rEP4 receptor versus EP1 and EP3. Typically, addition
of a small substituent on the meta position of the phenyl
ring maintained or enhanced EP4 potency, whereas para
substitution had a slight detrimental effect on potency.
The thiophene acid side chains (‘c’ compounds in Table
1) generally had reduced rEP4 to rEP2 binding selectiv-
ity as compared to the heptanoic acid and benzoic acid
side chains. However, enhanced EP4 functional potency
was achieved with the thiophene acid top chain.
Scheme 2. Reagents and condition: (a) NaHMDS, DMF, RBr; (b) 1 N
HCl, MeOH; (c) EDC, DMSO, pyridinium trifluoroacetate, benzene;
(d) NaH, ArCH2PO(OR)2; (e) H2, Pd/C; (f) NaBH4, EtOH; (g) NaOH,
MeOH; (h) (R)-2-methyl-CBS-oxazaborolidine, catecholborane,
CH2Cl2, À45 ꢁC.
A summary of pharmacokinetic parameters of selected
lactam derivatives in the Sprague–Dawley rat is shown
in Table 2. Compounds are generally characterized as
having high clearance, low volume of distribution, and
low oral bioavailability. Replacement of the heptanoic
acid with a propyl thiophene did little to reduce clear-
ance. The reduced oral exposure of 16c relative to 2a
and 16a is presumably due to decreased absorption.
Due to poor oral PK, analogs were tested in our in vivo
models using
administration.
a
subcutaneous (sc) route of
A sub-set of compounds was tested for bone anabolic
activity in an established osteopenic rat model.12 Female
rats (3.5–4 months) were ovariectomized (OVX) and, 5
weeks post-ovariectomy they were dosed daily (sc), with
compound for 28 days.13 No remarkable side effects,
such as GI effects, were observed with these subtype
selective agents at any dose. Peripheral distal femur
metaphysis showed that EP4 receptor selective agonists
can restore volumetric bone mineral content and density
in osteopenic rats. Selected bone efficacy parameters
are provided in Table 3. Administration of EP4
receptor agonists increased serum osteocalcin (a bone
formation marker) and bone formation rate (by bone
histomorphometric analysis). Compound 16c showed
Scheme 3. Reagents and condition: (a) DMF, NaHMDS, propargyl
bromide, 0 ꢁC; (b) Methyl 5-bromothiophene-2-carboxylate, Et3N,
Pd(PPh3)4, CuI, CH3CN; (c) H2, Pd/C.
a diastereomeric mixture of alcohols for in vitro testing.
Diastereo-enriched allylic alcohol was prepared via
reduction with (R)-2-methyl-CBS-oxazaborolidine and
catecholborane at À45 ꢁC.11 Subsequent hydrogenation
and saponification provided the desired lactams (2,
9–23).
Compounds were tested for binding at the EP receptors
in HEK-293 cells, which were stably expressed. Analogs
Table 2. Pharmacokinetics in Sprague–Dawley rat for selected lactam analogs
Compound
Cl (mL/min kg)
% F
Oral t1/2 (h)
iv t1/2 (h)
sc t1/2 (h)
Vd (L/kg)
2a
16a
16c
101
71
21
17
1
0.45
0.42
0.87
0.1
0.2
0.2
0.35
0.43
0.3
0.40
0.46
0.49
70
Table 3. Selected PQCT parameters from distal femoral metaphysis showing EP4 receptor selective agonists increased bone mineral content and
density
Compound
Dose (mg/kg)
Total content
Total density
Cortical area
Cortical thickness
Cortical content
8a
16a
16a
16c
16c
30
10
30
1
16.43
15.34
32.53
20.59
23.72
23.65
18.73
23.70
22.88
26.36
26.03
21.93
66.92
34.31
41.77
43.05
34.12
124.19
52.69
62.57
30.40
29.78
57.70
35.76
43.27
3
Values are given as percent increase (p < 0.01 for all values) compared with OVX rats treated with vehicle.

1802
K. O. Cameron et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1799–1802
Chemical Society: Washington, DC, 2003; MEDI-167.; (d)
Cameron, K. O.; Lefker, B. A.; Crawford, D. T.; DaSilva-
Jardine, P.; DeNinno, S. L.; Gilbert, S.; Grasser, W. A.;
Ke, H.; Lu, B.; Owen, T. A.; Paralkar, V. M.; Qi, H.;
Scott, D. O.; Thompson, D. D.; Tjoa, C. M.; Zawistoski,
M. P. Abstracts of Papers, 224th National Meeting of the
American Chemical Society, Boston, MA, August 18-22,
2002; American Chemical Society: Washington, DC, 2002;
MEDI-307.
comparable efficacy to our initial lead 2a at 1/30th the
dose. These data correlate well with the improved in vi-
tro functional potency of 16c. In addition, excellent effi-
cacy was achieved with QD dosing of short acting
compounds (sc t1/2 ꢀ 0.3 h). These data demonstrate
the importance of the EP4 receptor in bone formation
and in restoring bone lost due to estrogen deficiency.
In conclusion, we have shown that a selective EP4 ago-
nist 2a provides robust bone anabolism in an osteopenic
rat model. Chemical modification led to compound 16c,
which was about 30· more potent in vivo. Selective EP4
receptor agonists have potential as an anabolic therapy
for established osteoporotic patients.
5. After our initial disclosure additional reports on lactam
derivatives have been published: (a) Billot, X.; Chat-
eaunuf, A.; Chauret, N.; Denis, D.; Greig, G.; Mathieu,
M.; Metters, K. M.; Slipetz, D. M.; Young, R. N. Bioorg.
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E. R.; Lach, L. K.; McGee, D.; Yang, D. S.; Chiou, S.
Bioorg. Med. Chem. Lett. 2004, 14, 1655; (c) Elworthy, T.
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10. Prepared from methyl 4-iodobenzoate in three steps: a.
Sonagashira coupling with propargyl bromide; b. Hydro-
genation (Pd/C); c. Conversion to the bromide (CDI, allyl
bromide).
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37, 1986; (b) Corey, E. J.; Bakshi, R. K.; Shibata, S.;
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