Efficient synthesis of a novel m-phenylene derivative as a selective EP4 agonist inducing follicular growth and maturation in the ovary

Article abstract of DOI:10.1016/j.bmcl.2011.09.004

An efficient and straightforward synthesis of a novel m-phenylene derivative has been developed. The optically pure dibromo compound was selected as a starting material. Through a protocol involving the Prins reaction and two steps of the Horner-Wadsworth-Emmons reaction, the basic skeleton was constructed with appropriate alpha and omega side chains. The compound proved to be a highly selective EP₄ agonist and a possible drug candidate for maturation of the uterine cervix.

Full text of DOI:10.1016/j.bmcl.2011.09.004

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7505–7508
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Bioorganic & Medicinal Chemistry Letters
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Efficient synthesis of a novel m-phenylene derivative as a selective EP4
agonist inducing follicular growth and maturation in the ovary
⇑
Ryoji Hayashi , Yutaka Hosono, Koji Nakatsuji, Yoichiro Tanaka, Takeshi Mori, Takami Kanno,
Kei Makita, Mitsuhiro Moriwaki, Tomofumi Ohyama, Yasuo Ochi, Chifumi Inada, Masafumi Isogaya
Pharmaceutical Research Laboratories, Toray Industries, Inc., 6-10-1 Tebiro, Kamakura, Kanagawa 248-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient and straightforward synthesis of a novel m-phenylene derivative has been developed. The
optically pure dibromo compound was selected as a starting material. Through a protocol involving
the Prins reaction and two steps of the Horner–Wadsworth–Emmons reaction, the basic skeleton was
constructed with appropriate alpha and omega side chains. The compound proved to be a highly selective
EP4 agonist and a possible drug candidate for maturation of the uterine cervix.
Received 24 May 2011
Revised 31 August 2011
Accepted 1 September 2011
Available online 1 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Prostaglandin
EP4
m-Phenylene
Follicular growth
Maturation
Prostaglandin E2 (PGE2) exhibits a wide range of physiological
actions, including uterine constriction, suppression of gastric acid
secretion, protection of the gastric mucous membrane, stimulation
of digestive peristalsis, and induction of fever and diarrhea. In par-
ticular, it plays a crucial role in ovulation. PGE2 receptors can be
classified into four subtypes, namely, EP1, EP2, EP3, and EP4.1 It
has been revealed that the physiological actions of PGE2 are med-
iated by a specific receptor,2 and it has also been elucidated that
each receptor subtype mediates different physiological functions
of PGE2.3
The EP4 receptor is present in various organs including the
heart, kidney, liver, intestine, lung, and bones. EP4 receptor func-
tions include relaxation of smooth muscle, differentiation and pro-
liferation of lymphocytes, proliferation of mesangial cells, and
collagen production in fibroblasts. Therefore, EP4 receptor agonists
and antagonists can serve as preventives of or remedies for the
above pharmacological effects. For example, Ono showed that both
EP4 agonists and antagonists were useful drugs in the treatment of
bone diseases.4 Kanayama and co-workers revealed the following:
(1) localization of the EP4 receptor in the ovary plays a role in fol-
licular growth, (2) PGE2 induces ovarian follicular growth, and
development is mediated at least in part by the EP4 receptor,
(3) the action of an EP4 agonist is mediated through IL-8 up-regu-
lation, and (4) the new EP4 agonist could be a promising reagent for
various systems used to induce follicular maturation in clinical or
agricultural fields.5
Selectivity of EP4 agonism is necessary for developing a useful
drug without side effects, such as constriction of the uterus. Ono’s
strategy to develop a selective EP4 agonist could be regarded as a
modification of alpha and omega side chains in natural PGE1 or
PGE2. Although it seems to be a practical means to determine a
selective EP4 agonist starting with these natural products, Ono’s
EP4 agonists inevitably exhibited undesirable chemical instability,
which could not be corrected and thus proved a problem.6
Incontrast,wehavedevelopedachemicallystablePGI2analogcalled
‘Beraprost Sodium’ using the m-phenylene skeleton instead of the
unstable enol ether structure.7 During the course of the study, some
nonselective compounds that could be classified as EP4 agonists were
found. Therefore, we attempted to find a novel selective and chemically
stable EP4 agonist lead; we began by rescreening the m-phenylene li-
brary containing analogs with a variety of alpha and omega side chains.
Finally, we found a potent and selective EP4 agonist 3-((1R,2R,3aS,8bS)-
1-((S,E)-4-cyclohexyl-3-hydroxybut-1-enyl)-2-hydroxy-2,3,3a,8b-tet-
rahydro-1H-cyclopenta[b][1]benzofuran-5-propanoic acid, ‘APS-856’
(2), which was derivatized to a more potent and selective EP4 agonist,
(1R,2R,3aS,8bS)-5-(2-(1H-tetrazol-5-yl)ethyl)-1-((S,E)-4-cyclohexyl-3
-hydroxybut-1-enyl)-2,3,3a,8b-tetrahydro-1H-cyclopenta[b][1]benzo
f uran-2-ol, ‘APS-999’ (1)8 (Scheme 1).
Here we report the efficient synthesis and brief pharmacology
of the novel m-phenylene EP4 receptor agonist 1.
The first synthesis of compound 1 was conducted by functional
group transformation starting from methyl ester intermediate 3 of
⇑
Corresponding author. Tel.: +81 467 32 9643; fax: +81 467 32 2127.
E-mail address: Ryoji_Hayashi2@nts.toray.co.jp (R. Hayashi).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.004

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R. Hayashi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7505–7508
compound 17 was subjected to Prins reaction conditions and then
HN
N
N
COOH
hydrolyzed to afford diol 18. The two hydroxy groups of 18 were
then protected with TBDPS to afford the disilylether compound
19 (40% yield, 3 steps, after recrystallization). The next step was
a halogen-metal exchange reaction of the bromobenzene moiety
of disilylether 19 using i-PrMgCl; the product was then reacted
with dimethylformamide to give the corresponding aldehyde.
Without purification, the aldehyde was immediately condensed
with the corresponding Wadsworth reagent to afford a cis/trans
mixture of conjugated nitrile 20. After selective removal of the silyl
ether protecting group on the primary alcohol of 20, the resulting
compound was hydrogenated to afford the saturated nitrile 21
(53% yield, 3 steps). Simultaneously, unnecessary bromide on the
m-phenylene moiety was removed. The corresponding aldehyde,
which was obtained by Moffatt oxidation of 21, was converted to
enone 22 by Horner–Wadsworth–Emmons reaction in THF (90%
yield after recrystallization). Enone 22 was then stereoselectively
reduced by using the BH3-THF/(R)-5,5-Diphenyl-2-methyl-3,4-pro-
pano-1,3,2-oxazaborolidine system to yield an alcohol. Desilyla-
tion afforded the diols, which were a mixture of stereoisomers at
N
O
O
H
H
H
H
HO
OH
HO
OH
1
2
Scheme 1. Structure of APS-999 (1) and APS-856 (2).
our m-phenylene library compound 2, which was prepared by a
method described in a patent.9 Scheme 2 shows the synthetic route
to intermediate 3, which is a methyl ester prepared from optically
active cyclopenta[b][1]benzofuran derivative 4 over 13 steps.
Scheme 3 shows the sequence of transformations from interme-
diate 3 to target compound 1. This sequence started with silyl ether
protection of two hydroxy groups of methyl ester intermediate 3
(97% yield). A methyl ester group was hydrolyzed to be a carboxyl
group under alkaline conditions (95% yield). Oxalyl chloride and
ammonia saturated chloroform were used to obtain amide com-
pound 14 (63% yield), which was used as a substrate to yield nitrile
compound 15 by reaction with tosyl chloride in pyridine (53%
yield). After deprotection of the silyl ethers using tetrabutylammo-
nium fluoride (70% yield), the tetrazole ring, which would be a bio-
isostere of the carboxyl group of 2, was constructed by treatment
with sodium azide to yield 1 (57% yield).10 The target 1 was ob-
tained from 3 through six steps in 12.2% overall yield.
the allylic hydroxy group position (
Purification by column chromatography (silica gel) gave the pure
-alcohol 16 (75% yield, 2 steps). Conversion of the nitrile group
a-alcohol:b-alcohol = 94:6).
a
to a tetrazole group was performed by treatment with NaN3 to
yield compound 1 (70% yield, after recrystallization). Therefore, a
new and straightforward synthetic route with much fewer steps
(10 steps) than the corresponding conventional process (25 steps)
was developed. The overall yield of compound 1 from compound
17 was 10%.
The prostaglandin EP4 receptor agonistic activity and selectivity
of APS-856 (2) and APS-999 (1) were evaluated by the Magnus
method with appropriate isolated tissue (EP4: rabbit saphenous
vein; EP1+2: guinea pig ileum; EP3: guinea pig uterus).12 The results
are presented in Table 1. Converting the carboxylate group (2) to
its bioisosteric tetrazole moiety (1) improved EP4 activity and
especially EP4 selectivity.
Practically, the synthetic route described in Schemes 2 and 3 is
not straightforward, and can be simplified by introduction of an
appropriate alpha chain during the preparation of the m-phenylene
intermediates. Accordingly, we have attempted to develop a more
efficient method for the preparation of compound 1.
An improved method for the preparation of compound 1 is
shown in Scheme 4. The optically pure starting material 17 was
prepared by Nishiyama’s asymmetric synthesis.11 In the first step,
El-Nefiawy, Abdel-Hakim, and Kanayama have used compound
1 as a selective EP4 receptor agonist in vivo to explore whether this
compound has a positive impact on ovarian follicle growth in rats,
H3COOC
HOOC
O
H3COOC
OHC
HOOC
O
Br
Br
c
a, b
d, e, f
g
O
H
O
O
H
H
H
H
H
H
H
H
H
OH
OH
OTHP
OTHP
HO
HO
THPO
THPO
4
5
6
7
8
COOCH3
COOCH3
i, j
COOCH3
COOCH3
k
h
l, m
O
O
O
O
H
H
H
H
H
H
H
H
OTHP
OH
AcO
THPO
AcO
HO
O
OH
9
10
3
11
Scheme 2. Synthesis of starting material 3: reaction conditions a Trioxane, H2SO4, AcOH, 80 °C b NaOH, MeOH, reflux c H2, Pd/C, MeOH, rt - reflux d Dihydropyrane, p-
tolSO3H, THF, 35 °C e LAH, THF, -20 °C f MnO2, CH2Cl2, rt g H3COOCCH2=PPh3, THF, -10 °C h H2, Pd/C, MeOH, rt i p-tolSO3H, MeOH, 50 °C j Ph3CCl, Et3 N, THF, reflux then
Ac2O, Py, reflux then HCl/MeOH, rt k DMSO, DCC, CF3COOH, Py, THF, rt then NaH, WadsWorth reagent, THF, -20 °C l NaBH4, CeCl3-7H2O, MeOH, À20 °C m NaOMe, MeOH, rt
then silica gel column chromatography.

R. Hayashi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7505–7508
7507
COOCH3
COOCH3
COOH
CONH2
O
O
O
O
a
b
c, d
HN
H
H
H
H
H
H
H
H
HO
TBSO
TBSO
TBSO
OH
TBSO
TBSO
TBSO
3
12
13
14
N
N
CN
CN
N
O
H
O
O
g
e
f
H
H
H
H
H
HO
HO
TBSO
OH
16
OH
TBSO
1
15
Scheme 3. Functional group transformations of compound 3 to target compound 1: a TBSCl, Imidazole, DMF, 60 °C, 97% b 2 N NaOHaq, THF-MeOH, rt, 95% c (COCl)2, CH2Cl2,
rt d NH3, CHCl3, 0 °C, 63% e TsCl, pyridine, rt, 53% f TBAF, THF, rt, 70% g NaN3, NEt3, N-Me-2-pyrolidone, 150 °C, 57%.
CN
Br
Br
Br
Br
Br
Br
Br
a, b
c
d
O
O
O
O
H
H
H
H
H
H
H
H
OTBDPS
OH
OTBDPS
TBDPSO
HO
TBDPSO
18
19
17
20
HN
N
N
CN
CN
CN
N
e, f
O
O
g
h, i
O
j
O
H
H
H
H
H
H
H
H
OH
TBDPSO
TBDPSO
HO
HO
O
OH
OH
22
21
16
1
Scheme 4. An efficient and straightforward synthesis of compound 1. a Trioxane, H2SO4, AcOH, 70 °C b 3 N NaOHaq, THF, 50 °C c TBDPSCl, Imidazole, DMF, rt, 40% (3 steps,
after recrystallizaiton) d i-PrMgCl, THF then DMF, 60 °C then NaH, (EtO)2P(O)CH2CN, rt e TBAF, THF, À10 °C f H2, Pd/C, NaOAc, MeOH, rt, 53% (3 steps) g DCC, DMSO, TFA,
Pyridine, THF, rt then NaH, (MeO)2P(O)CH2C(O)CH2-c-Hex, rt, 90% (after recrystallization) h Oxazaborolidine, BH3-THF, THF, 0 °C i TBAF, THF, rt, 75% (2 steps) j NaN3, Et3 N
HCl, NMP, 140 °C, 70% (after recrystallization).
Table 1
In summary, we have developed an efficient and straightfor-
ward synthesis of an m-phenylene-type EP4 receptor agonist from
EP4 agonist activity and selectivity of synthesized compounds
an optically pure starting material. This simple approach gave
compound 1 in 10 steps. Compound 1 was subsequently proved
to be a selective EP4 receptor agonist and a possible drug candidate
for maturation of uterine cervix.
Compound Rabbit saphenous vein Guinea pig ileum Guinea pig uterus
(EP4)
(EP1+2
)
(EP3)
EC50 (nM)
EC50 (nM)
EC2000 (nM)
1
2
2.6
5.7
3400
150
2800
540
References and notes
1. Coleman, R. A.; Smith, W. L.; Narumiya, S. Pharmacol. Rev. 1994, 46, 205; (b)
Narumiya, S.; Sugimoto, Y.; Ushikubi, F. Physiol. Rev. 1999, 79, 1193.
2. Moncada, S.; Flower, R. J.; Vane, J. R. Prostaglandins, prostacyclin, thromboxane
A2, leukotriens. In The Pharmacological Basis of Therapeutics; Gilman, A. G.,
Good-man, L. S., Rall, T. W., Murad, F., Eds., 7th ed.; Macmillan Publishing: New
York, 1985; p 660.
while aiming to acquire a better understanding of the underlying
mechanism of action of PGE2.5 They concluded that the EP4 recep-
tor agonist could be a promising reagent for various systems used
to induce follicular maturation in clinical or agricultural fields.

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R. Hayashi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7505–7508
3. (a) Coleman, R. A.; Kennedy, I.; Humphrey, P. P. A.; Bunce, K.; Lumley, P.
Prostanoids and their receptors In Comprehensive Medicinal Chemistry; Hansch,
C., Sammes, P. G., Taylor, J. B., Emmett, J. C., Eds.; Pergaman: Oxford, 1990; Vol.
3, p 643; (b) Coleman, R. A.; Grix, S. P.; Head, S. A.; Louttit, J. B.; Mallett, A.;
Sheldrick, R. L. Prostaglandins 1994, 47, 151.
4. Hagino, H.; Kuraoka, M.; Kameyama, Y.; Okano, T.; Teshima, R. Bone 2005, 36,
444; T. Maruyama, S. Ouchida. Patent WO 003980, 2000.
5. El-Nefiawy, N.; Abdel-Hakim, K.; Kanayama, N. Eur. J. Endocrinol. 2005, 152,
315.
6. (a) Maruyama, T.; Asada, M.; Shiraishi, T.; Ishida, A.; Egashira, H.; Yoshida, H.;
Maruyama, T.; Ohuchida, S.; Nakai, H.; Kondo, K.; Toda, M. Bioorg. Med. Chem.
Lett. 2001, 11, 2029; (b) Maruyama, T.; Asada, M.; Shiraishi, T.; Sakata, K.; Seki,
A.; Yoshida, H.; Shinagawa, Y.; Maruyama, T.; Ohuchida, S.; Nakai, H.; Kondo,
K.; Toda, M. Bioorg. Med. Chem. Lett. 2001, 11, 2033; (c) Maruyama, T.; Asada,
M.; Shiraishi, T.; Yoshida, H.; Maruyama, T.; Ohuchida, S.; Nakai, H.; Kondo, K.;
Toda, M. Bioorg. Med. Chem. 2002, 10, 1743.
7. K. Ohno, H. Nagase, K. Matsumoto, S. Nishio, Patent EP 0084856, 1983.; (b)
Ohno, K.; Nishiyama, H.; Nagase, H.; Matsumoto, K.; Ishikawa, M. Tetrahedron
Lett. 1990, 31, 4489; (c) Nagase, H.; Matsumoto, K.; Yoshihara, H.; Tajima, A.;
Ohno, K. Tetrahedron Lett. 1990, 31, 4493; (d) Nishiyama, H.; Isaka, K.; Itoh, K.;
Ohno, K.; Nagase, H.; Matsumoto, K.; Yoshihara, H. J. Org. Chem. 1992, 57, 407.
8. H. Wakita, N. Yamada, H. Hatakeyama, G. Ishigaki, N. Hirano, T. Mori, Patent
WO 00/024727, 2000.
11. (a) Nishiyama, H.; Sakata, N.; Sugimoto, H.; Motoyama, Y.; Wakita, H.; Nagase,
H. Synlett 1998, 930; (b) Nishiyama, H.; Sakata, N.; Motoyama, Y.; Wakita, H.;
Nagase, H. Synlett 1997, 1147.
12. S.J. Lydford, K.C. McKechnie, I.G. Dougall, Br. J. Pharmacol. 1996, 117, 13.
Isolated preparation: All animal experiments were approved by our
institutional ethics review committee. EP4 receptor agonistic activity was
evaluated according to a previous method with slight modification. Male New
Zealand white rabbits (2–4 kg) were sacrificed by overdosing with the
anesthetic pentobarbital. Saphenous veins were excised and adherent fat and
connective tissue were removed. Vessel rings (4 mm wide) were cut and
suspended by stainless steel hooks under 1.0 g tension in 10 ml organ baths
containing Krebs solution (NaCl 118.1 mM, KCl 5.31 mM, CaCl2 3.52 mM,
MgSO4 1.01 mM, NaH2PO4 1.09 mM, NaHCO3 25 mM, glucose 9.99 mM; pH
7.4) at 37 °C and aerated with 95% O2/5% CO2. Isometric tension changes were
recorded with isometric transducers (Nihon-Kohden, Tokyo, Japan).
Experimental protocols: To reduce intertissue variation, relaxant responses
were measured by a paired curve experimental design. After an equilibration
period, tissues were initially exposed to 40 mM KCl to obtain
contraction. Subsequent tests were performed in the presence of TP
antagonist S-145 (1000 nM) to block TP receptor response. standard
concentration-response curve was obtained using PGE2, and following
washout period, testing curve was constructed in the presence of the
testing agonist. Data are expressed as percentage of the maximum
a stable
A
a
a
a
9. K. Ohno, T. Takahashi, A. Ohtake, H. Wakita, S. Nishio, Patent WO 89/003387,
1989.
contraction of 40 mM KCl. The EC50 value was calculated from each log
concentration-response curve as the concentration that caused a relaxation
response at 50% of its maximum value.
EP1 and EP2 receptor agonist activities were evaluated using guinea pig ileum
contracted with bethanechol (1000 nM) in the presence of TP antagonist S-145
(1000 nM).
EP3 receptor agonist activity was evaluated using guinea pig uterus.
Quantification of uterine contraction was expressed as uterine motility
index, which was calculated according to the following equation, where AUC
is area under the contraction curve.
Uterine Motility Index = (AUC for 15 min after test article treatment—AUC for
15 min before test article treatment)/contractile height induced with 60 mM
KCl.
10. Compound 16 (74 mg, 0.20 mmol) was dissolved in N-methyl-2-pyroridinone
(3.5 mL). To this solution, sodium azide (87 mg, 1.2 mmol) and triethylamine
(92 mg, 0.60 mmol) was added. After stirring for 56 h at 150 °C, sodium azide
(87 mg, 1.2 mmol) and triethylamine (92 mg, 0.6 mmol) was added to
complete substrate consumption following additional heating at 150 °C for
24 h. The reaction mixture was poured onto ice-cold 1 N hydrochloric acid
solution and extracted with ethyl acetate. Silica gel column chromatography
(cyclohexane/ethyl acetate: 1:3) was conducted to yield compound 1.
Analytical data: IR (neat, cmÀ1) 3386, 2922, 2850, 1655, 1560, 1509, 1450,
1257, 1194, 1064, 973, 863, 744. 1H NMR (CDCl3) d: 6.98 (1H, d, J = 6.9 Hz),
6.72–6.64 (2H, m), 5.59–5.57 (1H, m), 5.34–5.24 (1H, br s), 4.26–4.20 (2H, m),
4.08–3.42 (2H, m), 3.29–3.16 (2H, m), 2.80–2.72 (1H, m), 2.15–1.99 (3H, m),
1.79–1.68 (6H, m), 1.51–1.11 (6H, m), 0.97–0.90 (2H, m). Three protons (–OH, –
NH) were not observed. HRMS (EI) m/z calcd for C24H32N4O3 424.2427; found
424.2456.
The EP3 agonist potency is shown as EC2000 which is the concentration of test
article reached at number 2000 in Uterine Motility Index.