Synthesis and evaluation of a potent, well-balanced EP2/EP3 dual agonist

Article abstract of DOI:10.1016/j.bmc.2017.11.035

A highly potent and well-balanced dual agonist for the EP₂ and EP₃ receptors is described. Optimization of the lead compound was accomplished in consideration of the relative agonist activity against each EP subtype receptor and the pharmacokinetic profile. As the result, 2-[(2-{(1R,2R)-2-[(1E,4S)-5-cyclopentyl-4-hydroxy-4-methyl-1-penten-1-yl]-5-oxocyclopentyl}eth-yl)thio]-1,3-thiazole-4-carboxylic acid (10) showed excellent potency (human EC₅₀ EP₂ = 1.1 nM, EP₃ = 1.0 nM) with acceptable selectivity over the EP₁ and EP₄ subtypes (>2000-fold). Further fine-tuning of compound 10 led to identification of ONO-8055 as a clinical candidate. ONO-8055 was effective at an extremely low dose (0.01 mg/kg, po, bid) in rats, and dose-dependently improved voiding dysfunction in a monkey model of underactive bladder (UAB). ONO-8055 is expected to be a novel and highly promising drug for UAB.

Full text of DOI:10.1016/j.bmc.2017.11.035

Accepted Manuscript
Synthesis and Evaluation of a Potent, Well-balanced EP2/EP3 Dual Agonist
Akihiro Kinoshita, Masato Higashino, Koji Yoshida, Yoshiyuki Aratani, Akito
Kakuuchi, Keisuke Hanada, Hiroyuki Takeda, Atsushi Naganawa, Hidekazu
Matsuya, Kazuyuki Ohmoto
PII:
S0968-0896(17)31658-9
https://doi.org/10.1016/j.bmc.2017.11.035
BMC 14087
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 August 2017
19 November 2017
21 November 2017
Please cite this article as: Kinoshita, A., Higashino, M., Yoshida, K., Aratani, Y., Kakuuchi, A., Hanada, K., Takeda,
H., Naganawa, A., Matsuya, H., Ohmoto, K., Synthesis and Evaluation of a Potent, Well-balanced EP2/EP3 Dual
Agonist, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.11.035
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Bioorganic & Medicinal Chemistry
Synthesis and Evaluation of a Potent, Well-balanced EP2/EP3 Dual Agonist
Akihiro Kinoshita,a,* Masato Higashino,a Koji Yoshida,a Yoshiyuki Aratani,a Akito Kakuuchi,a Keisuke
Hanada,a Hiroyuki Takeda,a Atsushi Naganawa,a Hidekazu Matsuya,b and Kazuyuki Ohmotoa,*
aMedicinal Chemistry Research Laboratories, bDepartment of Biology & Pharmacology, Ono Pharmaceutical Co., Ltd., 3-1-1 Sakurai, Shimamoto-cho,
Mishima-gun, Osaka 618-8585, Japan.
* Corresponding Authors Tel.: +81-75-961-1151; fax: +81-75-962-9314.
E-mail addresses: ak.kinoshita@ono.co.jp (A. Kinoshita), k.ohmoto@ono.co.jp (K. Ohmoto)
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A highly potent and well-balanced dual agonist for the EP2 and EP3 receptors is described.
Optimization of the lead compound was accomplished in consideration of the relative agonist
activity against each EP subtype receptor and the pharmacokinetic profile. As the result, 2-[(2-
{(1R,2R)-2-[(1E,4S)-5-cyclopentyl-4-hydroxy-4-methyl-1-penten-1-yl]-5-oxocyclopentyl}eth-
yl)thio]-1,3-thiazole-4-carboxylic acid (10) showed excellent potency (human EC50 EP2 = 1.1
nM，EP3 = 1.0 nM) with acceptable selectivity over the EP1 and EP4 subtypes (>2000-fold).
Further fine-tuning of compound 10 led to identification of ONO-8055 as a clinical candidate.
ONO-8055 was effective at an extremely low dose (0.01 mg/kg, po, bid) in rats, and dose-
dependently improved voiding dysfunction in a monkey model of underactive bladder (UAB).
ONO-8055 is expected to be a novel and highly promising drug for UAB.
Available online
Keywords:
Prostaglandin
EP2 receptor
EP3 receptor
Dual agonist
2009 Elsevier Ltd. All rights reserved.
Underactive bladder
pharmacokinetics
however, the bioavailability of 1 in rats was quite low (Frat
2.5%) due to its low permeability. Compound 1 also showed
inadequate selectivity against the EP4 receptor (human EC50 EP2
= 2.9 nM，EP3 = 10 nM，EP4 = 195 nM). The EP4 receptor
subtype was also suggested to be involved in smooth muscle
relaxation.8 In terms of a manageable drug, EP4 agonist activity
must be removed because of a dramatic change in systemic blood
pressure by acting on vascular smooth muscle.9
=
1. Introduction
Prostanoid receptors are members of the G-protein coupled
receptor superfamily. Receptors for prostaglandin E2 (PGE2) can
be classified into four subtypes: EP1, EP2, EP3, and EP4.1 The
diverse biological activities of PGE2 are considered to be
expressed as a hybrid of the activities mediated by these four EP
receptor subtypes. Among them, the EP2 receptor subtype2,3
induces smooth muscle relaxation,4 while the EP3 receptor
subtype inhibits smooth muscle relaxation.5 Underactive bladder
(UAB) represents dysfunctional conditions of the bladder where
patients are unable to produce an effective voiding contraction.
The most common clinical signs are an elevation of post-void
residual urine volume and the lowering of urine flow rate. These
symptoms have a profoundly negative impact on quality of life.
PGE2 is considered to act on both bladder and urethral smooth
muscle. It has been reported that PGE2 prompts contraction of the
isolated bladder and relaxation of the isolated urethra.6
Furthermore, our pharmacological tests revealed that an EP3
selective agonist contracts the bladder and an EP2 selective
agonist relaxes the urethra (American Urology Association
2015).
In this article, well-balanced, a highly selective and potent
EP2/EP3 dual agonist is described. After extensive structural
optimization from previously reported compound 1, ONO-8055
was identified as a clinical candidate for treatment of UAB. In
European Phase I trials, ONO-8055 was well-tolerated at
multiple doses up to 700 g bid oral dosing.10
Table 1. Permeability and PK Profiles of Compound 1
CO2H
N
O
S
N
S
O
F
OH
1
We have already reported compound 1 as a highly potent EP2
and EP3 dual agonist.7 Compound 1 showed low clearance (CL =
9.5 mL/min/kg) as shown in Table 1. In spite of low clearance,
Rat PK profilea
cmpd Caco-2 × 10-6

(cm/s)
0.56
AUC
(μg  h/mL) (mL/min/kg)
0.041 9.5
CL
F
hydration. Therefore, we also tried to cut down on the number of
heteroatoms present in the compound. Removal of oxygen and
nitrogen atom from cyclic carbamate in 2 led to cyclopentanone 5,
which is closer structurally to natural prostaglandins. As shown
at Table 4, compound 5 showed a decreased topological polar
surface area (TPSA)12 in comparison to compound 2, with
concomitant balanced EP2 agonist activity and high permeability.
Unfortunately, cyclopentanone 5 exhibited inferior EP4 subtype
selectivity in comparison to cyclic carbamate 2.
(%)
1
2.5
aCompound 1 was administered at 0.01 mg/kg, i.v. and 1 mg/kg, p.o.
2. Results and discussion
2.1. Structure-activity and physical property relationships
At first, we focused on the improvement of EP4 subtype
selectivity. According to previous reports,11 the position of the
hydroxyl group at C-15 is likely to affect EP4 agonist activity
directly. C-15 is derived from the numbering for prostaglandin E2,
as shown in Figure 1. Displacement of hydroxyl group at C-15 in
lead compound 1 to next carbon (C-16) sharply reduced EP4
agonist activity (Table 2). The resulting homoallylic tertiary
alcohol 2 retained high EP2 and EP3 agonist activity, and showed
slightly improved permeability. Permeability is one of the most
important factors for an orally administered drug and it correlates
closely with the lipophilicity of the compound. In addition to
Caco-2 cell permeability studies, parallel artificial membrane
permeability assay (PAMPA) was used for a simple evaluation of
permeability.
Furthermore, we individually prepared and evaluated two
diastereomers at C-16 of compound 5. As shown in Table 5, the S
epimer (5S) turned out to be a highly active EP2 and EP3 dual
agonist. The absolute configuration was determined as described
below.
Table 3. Structure-Activity and Physical Property Relationship for Cyclic
Carbamate Compounds
CO2H
N
O
S
S
N
H3C OH
O
R2
R1
a
Functional assay EC50 (nM)
PAMPA × 10-6 Caco-2 × 10-6
cmpd R1
R2
hEP2
hEP3
hEP4
(cm/s) at pH=6.2
(cm/s)
cLogP
O
7
2
4
*
1
CH3
2
3
H
H
2.1
92
3.3
8.8
3380
2.9
9.6
1.0
4.1
4.7
6
8
5
3
 chain
 chain
9
CO2H
10
b
>10000
N.T.
N.T.
*
*
15
20
11
18
12
16
14
19
17
13
b
HO
4
Me
3400
36
>10000
19
5.2
OH
aAssay protocols are provided in Experiments section. EC50 values represent
the mean of more than two experiments.
bNot tested
Figure 1. Structure of Prostaglandin E2
Table 2. Structure Activity and Physical Property Relationship for Cyclic
carbamate Compounds
Table 4. Structure Activity and Physical Property Relationship for Cyclic
Carbamate Compound and Cyclopentanone Compound
CO2H
CO2H
N
S
N
S
O
O
S
S
N
N
O
H3C OH
O
CH3
CO2H
CO2H
F
N
S
N
S
OH
O
O
S
S
2
1
N
H3C OH
O
H3C OH
CH3
CH3
Caco-2 ×
10-6
2
5
Human Functional Assay, EC50
a
(nM)
cmpd
(cm/s)
hEP2
2.0
hEP3
10
hEP4
195
a
Functional assay EC50 (nM)
PAMPA × 10-6 Caco-2 × 10-6
0.56
1.0
1
2
cmpd
2
hEP2
2.1
hEP3
3.3
hEP4
(cm/s) at pH=6.2
(cm/s)
cLogP
4.1
TPSA
2.1
3.3
3380
aEC50 values respectively represent the mean of at least two experiments.
3380
91
2.9
1.0
100
87
5
0.48
2.7
40
4.1
4.6
In order to further improve permeability, we next tried to
increase lipophilicity of compound 2. As shown in Table 3, the
incorporation of cyclohexyl group into -side chain and a methyl
moiety onto the cyclic carbamate portion increased clog P, which
is an indicator for lipophilicity, and also increased PAMPA value,
as expected. However, EP2 agonist activity of both compounds 3
and 4 was significantly reduced, suggesting that EP2 agonist
activity might be incompatible with improvements to
permeability via introduction of hydrophobic groups.
Table 5. Structure Activity Relationship for Cyclopentanone Compound 5S
and 5R Isomer
C
O
H
C
O
H
2
N
S
2
N
S
O
O
S
3
S
3
H
C
O
H
H
C
O
H
C
H
C
H
3
3
5S
5R
Meanwhile, as the number of lone pair electrons increases,
permeability generally tends to decrease because of
a
corresponding increase in the polar surface area and degree of
cmpd
Human Functional Assay, EC50

a
6.8
16
2540
5R
(nM)
aEC50 values represent the mean of at least two experiments.
hEP2
hEP3
0.91
hEP4
146
0.086
5S
Table 6. Pharmacokinetic Profiles in Rats and Physical Properties of compound 1 and 5S
Oral dosea
Intravenous dosea
Dose
Cmax
T1/2
AUC
F
Dose
CL T1/2 AUCinf
Vss
cmpd
TPSA Clog P
(ng/mL) (hr) (ng h/mL) (%) (mg/kg) (mL/min/kg) (hr) (ng h/mL) (mL/kg)
(mg/kg)
1
3.7
-
41
2.5
17
0.01
0.1
9.5
34
4.4
11
17
51
728
100
87
3.7
4.6
1
0.1
0.96
4.1
8.9
107000
5S
a Each value is the mean of three animals in rat study.
of UAB, and motivated us to optimize compound 10 toward a
clinical candidate.
2.2. Further modification toward a clinical candidate
Further modification of the -side chain terminus in
compound 10 finally resulted in identification of our clinical
candidate, ONO-8055. As mentioned above, the bulkiness of the
-side chain terminus is a key factor for an EP2/EP3 dual agonist.
ONO-8055 was designed in consideration of the size of terminus
and the physical property as well. ONO-8055, which possessed S
stereochemistry at C-16, exhibits both well-balanced and highly
potent EP2 and EP3 agonist activity, in addition to a good
pharmacokinetic profile in both rats and cynomolgus monkeys
(for example, iv clearance of ONO-8055: <5 mL/min/kg). In the
rat UAB model, ONO-8055 demonstrated the efficacy with oral
dosing of 0.01 mg/kg .13
The pharmacokinetic (PK) profile of compound 5S was
evaluated and compared to the lead compound 1 (Table 6).
Compound 5S exhibited 17% bioavailability in rats due to its
higher permeability versus 1. According to this result, compound
5S could be considered to be a key compound toward a clinical
candidate. The concept behind an EP2/EP3 dual agonist is to
synergistically maximize drug potency while minimizing adverse
effects to the circulatory system. As mentioned earlier, our goal
is to identify well-balanced EP2 and EP3 agonist activity with
high selectivity over the EP4 subtype. Specifically, EP4 agonist
activity that drastically lowers blood pressure must be eliminated.
Finally, we focused on improving the EP4 subtype selectivity
of compound 5S while maintaining the other desired properties.
The structure-activity relationship of the resulting derivatives is
described in Table 7. The data for compounds 6-9 indicated that
the bulkiness of ω side chain reduced EP4 agonist activity, but
also tended to weaken EP2 agonist activity. No major change was
seen in EP3 agonist activity. This trend was especially noticeable
for compound 9, where the bulky tert-butyl group on the ω side
chain adequately improved EP4 subtype selectivity but also
resulted in a drastic loss of EP2 agonist activity even in
comparison with the trifluoromethylated 8. In terms of the
bulkiness of ω side chain, homology modeling based on publicly
disclosed data (GPCRdb, http://gpcrdb.org/alignment) supports
the difficulty in balancing EP2 and EP3 agonist activity.
According to the homology model, the binding pocket in the EP2
receptor seems to be narrower and less tolerant of bulkiness than
in the EP3 receptor, as shown in Figure 2. EP2 and EP3 receptors
respectively have Phe280 + Leu304 and Leu298 + Ala335
toward their binding pocket. This means there is larger bulge
protruding into the binding region of the EP2 receptor. Therefore,
we replaced the tert-butyl group of compound 9 with a flatter
cyclopentyl group. As expected, EP2 agonist activity of
cyclopentylated 10 was once again equal to EP3 agonist activity,
whereas selectivity against the EP4 subtype remained high. In this
case, the size of the cyclopentyl group turned out to be suitable to
bind to both EP2 and EP3 receptor and not to bind to the EP4
receptor. As an even more favorable characteristic, compound 10
Table 7. Structure Activity Relationship for Cyclopentanone Compounds
CO2H
N
O
S
S
H3C OH
R
a
Functional assay EC50 (nM)
EP4 selectivity
vs EP2 / vs EP3
Balance of
EP2 / EP3
cmpd
5S
R
hEP2
0.086
hEP3
0.91
hEP4
*
CH3
146
1698 / 160
0.095
CH3
*
6
7
0.068
0.11
0.80
3.0
26
382 / 33
0.085
0.037
*
578
5255 / 193
F
F
F
*
8
9
0.37
31
3.5
3.4
127
343 / 36
0.11
9.1
F
C
H
*
3
3
C
3
H
>10000
>323 / >2941
C
H
10
1.1
1.0
2636
2396 / 2636
1.1
*
aEC50 values represent the mean of at least two experiments.
also showed high selectivity against the EP1 subtype (EC50
=
Table 8. Structure Activity Relationship for Diastereomeric Isomers
2,000 nM). Regarding the configuration at C-16, we synthesized
the corresponding C-16(R) epimer 11 and reconfirmed that the S-
isomer 10 is more active (Table 8).
At this point, we tried to confirm whether an EP2/EP3 dual
agonist indicates the desired biological activity. When compound
10 was preliminarily evaluated by the use of a rat lumber spinal
canal stenosis derived UAB model, it reduced the residual
volume of urine with oral dosing of 0.03 mg/kg.13 This result
meant an EP2/EP3 dual agonist can be a novel drug for treatment

CO2H
N
S
O
S
Voided volume
220
200
180
160
140
120
100
80
*
R
*
*
a
Functional assay EC50 (nM)
EP4 selectivity
vs EP2 / vs EP3
Balance of
EP2 / EP3
cmpd
10
R
hEP2
hEP3
hEP4
H 3
C
O
O
H
1.1
1.0
2636
>10000
339
2396 / 2636
>5000 / >313
506 / 484
1.1
0.063
1.0
H 3
C
H
11
2.0
32
ONO-8055
0.67
0.7
aEC50 values represent the mean of at least two experiments.
EP2 Homology Model
EP2&EP3 Homology Model
60
NARROW SPACE
V
0.003 0.03
0.3
30
ONO-8055
(ng/kg/min)
Distigmine
(ng/kg/min)
V = vehicle
Leu304
Phe280
EP3 Homology Model
Figure 3. The effect of ONO-8055 and distigmine in monkey UAB
model
WIDE SPACE
3. Chemistry
Leu298 Ala335
The syntheses of 2 and 3 are described in Scheme 1.
Commercially available D-serine methyl ester hydrochloride 12
was treated with triphosgene to form the corresponding cyclic
carbamate. Subsequent reduction of the methyl ester afforded
alcohol 13. Protection of the hydroxyl group of 13 was followed
by N-alkylation of cyclic carbamate, reduction of the introduced
ethyl ester moiety, mesylation of the resulting alcohol 14 and
substitution by thioacetate to extend the α side chain. Treatment
of thioacetate 15 with ethyl 2-bromo-1,3-thiazole-4-carboxylate
and removal of TBS group provided alcohol 16. Oxidation of
alcohol 16, Julia olefination with sulfone 22 or 23, and removal
of the TMS protection by aqueous HCl were followed by
hydrolysis to provide 2 or 3, respectively. Regarding preparation
of sulfone 22 or 23, at first Reformatsky reaction between
ketones 18 or 19 and ethyl bromoacetate was performed,
followed by reduction of the ethyl ester moiety. The primary
alcohol of the resulting diol was tosylated and displaced with 1-
phenyl-1H-tetrazole-5-thiol to provide sulfide 20 or 21. Each
sulfide was oxidized with m-CPBA and protected at the tertiary
alcohol with TMS to furnish sulfone 22 or 23, respectively.
Figure 2. Homology models of EP2 and EP3 receptors
Regarding an expanded in vivo evaluation, ONO-8055
improved the lower urinary tract dysfunctions of neurogenic
UAB in a rat lumber spinal canal stenosis model with twice-daily
(bid) oral dosing of 0.01 mg/kg.14 It is worth noting that ONO-
8055 had little effect on blood pressure and heart rate at up to 0.3
mg/kg oral dosing. This strongly suggests that an appropriate
balance between EP2 and EP3 agonist activity alleviates
cardiovascular risks, as was predicted. Furthermore, ONO-8055
was compared with a cholinesterase inhibitor, distigmine, in a
monkey UAB model.15 Distigmine is one of the primary drugs
currently used for UAB. As shown in Figure 3, the voided
volume was dose-dependently increased by a 2-hour iv infusion
of 0.003, 0.03 and 0.3 ng/kg/min of ONO-8055. The voided
volume at 0.3 ng/kg/min was the same as that at 30 ng/kg/min of
distigmine. ONO-8055 demonstrated much higher in vivo
efficacy than the currently used drug for UAB.
The synthesis of 4 is described in Scheme 2. Compound 4 was
prepared by the use of D-threonine methyl ester hydrochloride 24
instead of D-serine methyl ester hydrochloride 12 as the starting
material and followed an otherwise identical route as shown in
Scheme 1.
Scheme 1. Syntheses of 2 and 3a

Cl
C
H
O
O
f), g)
As mentioned earlier, the S-configuration at C-16 is the more
active form. The isomers were separated and the more active
configuration was defined as follows: the syntheses of 5S and 5R
are described in Scheme 4. Compound 33 was at first protected at
the secondary alcohol with acetyl group, and then
macrolactonization employing the Yamaguchi conditions
afforded a mixture of two isomers (34S and 34R). Fortunately,
these two isomers had a distinct difference in polarity and were
separable by silica gel column chromatography. In addition,
when the cyclohexyl analog of 5 was prepared in parallel, the
more polar macrolactone 40 was obtained as crystals. Therefore,
the absolute configuration of 40 was determined by X-ray
crystallography as shown in Figure 4. According to this result,
the more polar macrolactone and the less active target compound
derived from it was determined to be the C-16(R) isomer. In
other words, the more active epimer is derived from the less polar
macrolactone and has the S-configuration at C-16. In terms of the
absolute configuration, it is also consistent with our knowledge in
a variety of prostaglandin analogs which we synthesized so far.
a), b)
c), d), e)
O
H
N
H
2
N
H
H
O
N
O
O
O
H
O
M
e
2
O
T
B
S
12
13
14
C O 2 E t
N
S
C
O
E
t
O
2
N
S
O
S
O
S
A
c
h), i)
j)
S
N
N
O
N
O
O
O
O
T
B
S
O
H
H
15
16
17
COH
2
N
S
O
S
k), l), m)
N
O
HC OH
3
R
*
CH3
2 : R =
3 : R =
*
C
H
3C H 3
N
N
N
Si
H3
C
OH
R
N
O
N
H 3
C
O
C H 3
N
N
n), o), p)
S
q), r)
N
S
R
H 3
C
R
O
O
*
*
*
*
*
*
CH3
CH3
CH3
18 : R =
19 : R =
20 : R =
21 : R =
22 : R =
23 : R =
aReagents and conditions: (a) K2CO3, water, triphosgene, toluene, 0 °C; (b)
NaBH4, EtOH, rt, 53% (for 2 steps); (c) TBSCl, imidazole, DMF, rt; (d) ethyl
bromoacetate, KOtBu, THF, rt; (e) NaBH4, THF-EtOH, rt, 86% (for 3 steps);
(f) MsCl, Et3N, CH2Cl2, 0 °C; (g) KSAc, DMF, rt, 100% (for 2 steps); (h)
ethyl 2-bromo-1,3-thiazole-4-carboxylate, tributylphosphine, K2CO3, EtOH,
50 °C; (i) TBAF, THF, rt, 78% (for 2 steps); (j) SO3-Py, Et3N, DMSO,
EtOAc, 10 °C; (k) KHMDS, 22 or 23, DME, -78 °C, 16-26% (for 2 steps); (l)
4N HCl(aq), EtOAc, 0 °C; (m) 2N NaOH(aq), EtOH, 0 °C, 33-65% (for 2
steps); (n) ethyl bromoacetate, zinc, iodine, dioxane, 0 °C; (o) LiAlH4, THF,
0 °C, 50-70% (for 2 steps); (p) 5N NaOH(aq), n-Bu4NBr, TsCl, toluene, 0 °C,
then 1-phenyl-1H-tetrazole-5-thiol, 60 °C, 80-86%; (q) m-CPBA, CH2Cl2, (r)
TMSCl, imidazole, CH2Cl2, 75-82% (for 2 steps).
After chromatographic separation of the two epimers, each
macrolactone (34S or 34R) was hydrolyzed to give the
corresponding ring-opened compound, followed by ethyl
esterification of carboxylic acid and oxidation of secondary
alcohol. The obtained ester (S or R-isomer) was hydrolyzed to
furnish the desired compound 5S or 5R, respectively.
The syntheses of 6-10 are described in Scheme 5. Compounds
6-10 all possess the more active S-configuration at C-16. The
requisite sulfones 37a-e were prepared in a similar way to that
shown in Scheme 1. Julia olefination between aldehyde 31 and
sulfones 37a-e gave the corresponding E-olefins, followed by
hydrolysis of the ethyl ester and deprotection of TMS group.
Subsequent macrolactonization of the resulting unmasked
carboxylic acids 38a-e and purification by silica gel column
chromatography were performed in a similar fashion. The desired
compounds 6-10 were derived from the corresponding less polar
macrolactones 39a-e. The R-isomer 11 in Table 8 was derived
from the more polar maclolactone produced in the process of
preparation of 10 as well.
Scheme 2. Synthesis of 4a
O
Cl
H
O
C
O
H
f), g)
N
H
2
N
H
a), b)
c), d), e)
H
O
N
O
O
3
O
H
C
O
M
e
O
T
B
S
2
H
C
O
C
3
H
C
3
H
24
25
26
Scheme 3. Synthesis of 5a
CO2H
C
O 2 E t
N
N
S
O
O
O
S
A
c
S
CO2Et
CO2Et
S
N
S
N
S
h), i)
O
HO
N
AcO
N
a), b), c), d)
J), k), l), m)
S
OH
e), f), g)
N
S
S
O
3
O
H3C
O
O
T
B
S
O
H
OTBDPS
OTBDPS
O
H
H3C
H 3
C
27
H
29
30
31
28
4
aReagents and conditions: (a) K2CO3, water, triphosgene, toluene, 0 °C; (b)
CO2
H
CO2
H
N
S
N
S
I
O
S
HO
S
H3
C
OH
h), i)
j), k)
Ph
NaBH4, EtOH, rt, 88% (for 2 steps); (c) TBSCl, imidazole, DMF, rt; (d) ethyl
bromoacetate, KOtBu, THF, rt; (e) NaBH4, THF-EtOH, rt, 94% (for 3 steps);
(f) MsCl, Et3N, CH2Cl2, 0 °C; (g) KSAc, DMF, rt, 100% (for 2 steps); (h)
ethyl 2-bromo-1,3-thiazole-4-carboxylate, tributylphosphine, K2CO3, EtOH,
50 °C; (i) TBAF, THF, rt, 83% (for 2 steps); (j) SO3-Py, Et3N, DMSO,
EtOAc, 10 °C; (k) KHMDS, 23, DME, -78 °C, 78% (for 2 steps); (l) 4N
HCl(aq), EtOAc, 0 °C; (m) 2N NaOH(aq), EtOH, 0 °C, 88% (for 2 steps);
Ph P+
Ph
CH3
H3
C
OH
H3
C
OH
CH3
CH3
32
33
5
aReagents and conditions: (a) LiAlH4, THF, 0 °C; (b) MsCl, DIPEA, THF,
-5 °C, then TMSCl, AcSK, K2CO3, DMF, 50 °C; (c) ethyl 2-bromo-1,3-
thiazole-4-carboxylate, tributylphosphine, K2CO3, EtOH, 50 °C; (d) 1N
HCl(aq), THF, 0 °C, 38% (for 4 steps); (e) Ac2O, Py, DMAP, rt; (f) TBAF,
THF, rt; (g) SO3-Py, Et3N, DMSO, EtOAc, 10 °C, 97% (for 3 steps); (h) n-
BuLi, 31, THF, -78 °C; (i) 2N NaOH(aq), MeOH, rt, 37% (for 2 steps); (j)
TBSCl, TEA, Toluene; (k) SO3-Py, DIPEA, DMSO, EtOAc, 0 °C, 62% (for 2
steps).
The synthesis of 5 is described in Scheme 3. Corey lactone 29
was first reduced with LiAlH4 to make the core structure. The
resulting diol was mesylated at the primary alcohol, protected at
the secondary alcohol with TMS, substituted by thioacetate and
treated with ethyl 2-bromo-1,3-thiazole-4-carboxylate to extend
the α side chain. Removal of TMS group furnished alcohol 30.
Subsequent acetylation of the hydroxyl group of 30, deprotection
of TBDPS group and oxidation of the resulting alcohol provided
aldehyde 31. E-selective Wittig olefination16 with phosphonium
salt 32 and aldehyde 31 followed by hydrolysis provided
carboxylic acid 33. After temporary protection of the carboxylic
acid with TBS and oxydation of the secondary hydroxyl group,
we obtained the desired compound 5.
Scheme 4. Syntheses of 5S,5Ra

CO2
H
S
N
S
N
N
S
good balance (human EC50 EP2 = 1.1 nM，EP3 = 1.0 nM) with
excellent EP1 and EP4 subtype selectivity (>2000-fold).
Moreover, ONO-8055, which is the derivative of compound 10,
dose-dependently increased voided volume in monkey UAB
model at a dose of 0.03 ng/kg, iv. In a European Phase I trial,
ONO-8055 proved to be well-tolerated at multiple doses up to
700 g bid oral dosing. ONO-8055 is under development as a
clinical candidate and it is expected to be novel and highly
promising drug for the treatment of UAB.
S
S
Ac O
HO
Ac O
S
a), b), c)
O
O
H3
C
O
H3
C
OH
H3
C
O
+
CH3
CH3
CH3
33
34S (less polar)
34R (more polar)
CO2
H
CO2
H
N
N
O
O
S
H3
S
d), e), f), g)
S
OH
S
OH
+
C
H3
C
CH3
CH3
5S
5R
aReagents and conditions: (a) Ac2O, pyridine, rt; (b) 2,4,6-trichlorobenzoyl
chloride, DMAP, toluene, 100 °C ; (c) separation by silica gel column
chromatography, 35% (for 34S), 25% (for 34R) (for 3 steps); (d) 2N
NaOH(aq), THF/MeOH, rt; (e) ethyl iodide, K2CO3, DMF ; (f) SO3-Py,
DIPEA, DMSO, EtOAc, 0 °C; (g) LiOH, DME/H2O, 80% (for 5S), 72% (for
5R) (for 4 steps).
5. Experiments
5.1. Chemistry
Scheme 5. Syntheses of 6-10a
Analytical samples were homogeneous as confirmed by TLC,
and afforded spectroscopic results consistent with the assigned
structures. NMR spectra were recorded as designated on either a
Varian Mercury 300 spectrometer or INOVA-500 spectrometer
using deuterated chloroform (CDCl3), deuterated methanol
(CD3OD) and deuterated dimethylsulfoxide (DMSO-d6) as the
solvent. Fast atom bombardment (FABMS, HRMS) and electron
ionization (EI) were obtained on a JEOL JMS-DX303HF
spectrometer. Atmospheric pressure chemical ionization (APCI)
mass spectra were determined by Hitachi M-1200H spectrometer.
IR spectra were measured on a JASCO FTIR-430 spectrometer.
Elemental analyzes were performed with a Perkin–Elmer PE2400
series II CHNS/O Analyzer and were only indicated as the
elements within ±0.4% of the theoretical values unless otherwise
noted. Purity analysis was carried out by the following HPLC or
LC/MS system. HPLC: Agilent Technologies 1200 Series with
Unison UK-C18 (4.6 x 150mm). LC/MS: Waters ACQUITY
UPLC system fitted by with Waters Micromass ZQ-2000
spectrometer. Column chromatography was carried out on silica
gel [Merck Silica Gel 60 (0.063–0.200 μm), Wako gel C-200, or
Fuji Silysia FL60D]. Thin layer chromatography was performed
on silica gel (Merck TLC or HPTLC plates, Silica Gel 60 F254).
The following abbreviations for solvents and reagents are used;
N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO),
ethanol (EtOH), ethyl acetate (EtOAc), methanol (MeOH),
tetrahydrofuran (THF), dichloromethane (CH2Cl2). tert-butyl
methyl ether (tBuOMe), diisopropyl ether (iPr2O), (acetonitrile
(CH3CN), triethylamine (Et3N), trifluoroacetic acid (TFA).
C
H
3C H 3
N
N
N
Si
H3C
OH
N
N
H 3
C
O
C H 3
O
N
a), b), c)
N
S
d), e)
R
N
S
R
O
O
H 3
C
R
35a-e
36a-e
37a-e
CO2 Et
S
N
N
S
CO2H
N
S
AcO
AcO
S
HO
S
h), i), j)
f), g)
S
OH
O
H3
C
O
H3
C
O
R
R
H
31
38a-e
39a-e
CO2
H
N
S
O
S
k), l), m), n)
H3
C
OH
R
6-10
35a-39a and 6:
35c-39c and 8:
35e-39e and 10:
F
CH3
*
*
R =
R =
R =
*
F
F
35b-39b and 7:
35d-39d and 9:
CH3
CH3
*
*
R =
R =
F
CH3
aReagents and conditions: (a) ethyl bromoacetate, zinc, iodine, dioxane, 0 °C;
(b) LiAlH4, THF, 0 °C, 50-70% (for 2 steps); (c) 5N NaOH(aq), n-Bu4NBr,
TsCl, toluene, 0 °C, then, 1-phenyl-1H-tetrazole-5-thiol, 60 °C, 80-86%; (d)
m-CPBA, CH2Cl2; (e) TMSCl, imidazole, CH2Cl2, 75-82% (for 2 steps) ; (f)
KHMDS, 37a-e, DME, -78 °C; (g) 2N NaOH(aq), EtOH, rt, then 1N
HCl(aq), 64-77% (for 2 steps); (h) Ac2O, Pyridine, rt; (i) 2,4,6-
trichlorobenzoyl chloride, DMAP, toluene, 100 °C, (j) separation by silica gel
column chromatography, 20-35% (for less polar lactone for 3 steps) (k) 2N
NaOH(aq), THF/MeOH, rt; (l) ethyl iodide, K2CO3, DMF; (m) SO3-Py,
DIPEA, DMSO, EtOAc, 0 °C; (n) LiOH, DME/H2O, 68-79% (for 4 steps).
5.1.1. (4S)-4-(hydroxymethyl)-1,3-oxazolidin-2-one (13)
Commercial available D-serine methyl ester hydrochloride
(5.76 g) was dissolved in water (52.0 mL), and stirred with
potassium bicarbonate (4.08 g) for 10 minutes. To the resulting
solution were added potassium bicarbonate (5.63 g) and a
solution of triphosgene (14.3 g) in toluene (26.0 mL) at 0 °C. The
reaction mixture was stirred at 0 °C for 2 hours, and then diluted
with ethyl acetate. The aqueous layer was removed and
concentrated in vacuo. The resulting residue was dispersed into
methylene chloride, dried over anhydrous sodium sulfate and
concentrated in vacuo. The ethanolic solution (73.0 mL) of the
obtained residue was treated with sodium borohydride (1.34 g)
and stirred for 3 hours at 0 °C. The reaction mixture was
quenched with saturated ammonium chloride at 0 °C and stirred
at room temperature for 30 minutes. The resulting mixture was
filtered and concentrated. The obtained residue was purified by
column chromatography on silica gel (ethyl acetate : methanol =
4 : 1) to give compound 13 (2.28 g, 53%).
S
S
3
A
c
O
N
O
H
C
O
40
Figure 4. X-ray crystallography of macrolactone 40
4. Conclusion
In summary, the optimization of compound 1 was carried out
to improve the PK profile and EP4 subtype selectivity while
maintaining the acceptable balance between EP2 and EP3 agonist
activity. As a result, 2-[(2-{(1R,2R)-2-[(1E,4S)-5-cyclopentyl-4-
hydroxy-4-methyl-1-penten-1-yl]-5-oxocyclopentyl}ethyl)thio]-
1,3-thiazole-4-carboxylic acid (10) showed excellent potency and
1H NMR (300 MHz, CDCl3) δ 3.62 (dd, J = 11.4, 5.7 Hz, 1H),
3.73 (m, 1H), 4.01 (m, 1H), 4.23 (m, 1H), 4.49 (t, J = 8.7 Hz, 1H),
5.99 (s, 1H).

with water and brine, dried over anhydrous magnesium sulfate
and concentrated. The crude product was purified by column
chromatography on silica gel (n-hexane : ethyl acetate = 2 : 3), to
give compound 16 (4.29 g, 78%).
1H NMR (300 MHz, CDCl3) δ 1.39 (m, 3H), 3.66 (m, 6H),
4.14 (m, 2H), 4.39 (m, 3H), 8.02 (s, 1H).
5.1.2. (4R)-4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-(2-
hydroxyethyl)-1,3-oxazolidin-2-one (14)
To a solution of alcohol 13 (2.28 g) in dimethylformamide
(20.0 mL) was added imidazole (1.60 g) and tert-
butyldimethylsilyl chloride (3.09 g). The resulting solution was
stirred at room temperature overnight, then poured into ice and
extracted with ethyl acetate. The organic layer was washed with
water and brine, dried over anhydrous sodium sulfate and
concentrated. The obtained residue was dissolved in anhydrous
tetrahydrofuran (45.0 mL), treated with potassium tert-butoxide
(2.41 g), when cooled in ice. After stirring for 10 minutes, the
reaction mixture was treated with 2-bromoethyl acetate (2.40
mL), and stirred at room temperature for 3 hours. The reaction
was quenched with saturated ammonium chloride, and extracted
with ethyl acetate. The organic layer was washed with brine,
dried over anhydrous sodium sulfate and concentrated. To a
solution of the obtained residue in tetrahydrofuran/ethanol (7:1)
(46.0 mL) was added sodium borohydride (2.22 g). The reaction
mixture was stirred at room temperature for 2 hours, then poured
into ice/saturated ammonium chloride and extracted with ethyl
acetate. The organic layer was washed with water and brine,
dried over anhydrous sodium sulfate and concentrated to give
compound 14 (4.62 g, 86%).
5.1.5. ethyl
yl]ethyl}thio)-1,3-thiazole-4-carboxylate (17)
To solution of the compound 16 (338 mg) in
2-({2-[(4R)-4-formyl-2-oxo-1,3-oxazolidin-3-
a
dimethylsulfoxide (3.40 mL)/ethyl acetate (6.80 mL) were added
triethylamine (1.00 mL) and sulfur trioxide-pyridine complex
(650 mg) at 10 °C. The reaction mixture was stirred at room
temperature for 1 hour, then treated with 1N hydrochloric acid,
and extracted with ethyl acetate. The organic layer was washed
water and brine, dried over anhydrous sodium sulfate and
concentrated to give compound 17 (336 mg, 100%).
1H NMR (300 MHz, CDCl3) δ 1.38 (t, J = 7.0 Hz, 3H), 3.84
(m, 9H), 8.02 (m, 1H), 9.81 (s, 1H).
5.1.6. 3-methyl-1-[(1-phenyl-1H-tetrazol-5-yl)thio]-3-octanol
(20)
To a solution of commercial available 2-heptanone (10.1 mL)
in 1,4-dioxane (150 mL) were added ethyl bromoacetate (9.6
mL), zinc (8.5 g) and iodine (3.7 g) at room temperature. The
reaction mixture was sonicated for 2 hours, then carefully treated
with 1N hydrochloric acid, and extracted with ethyl acetate. The
organic layer was washed with water and brine, dried over
anhydrous magnesium sulfate and concentrated. The residue was
purified by flush column chromatography (n-hexane : ethyl
acetate = 15 : 1). A solution of the obtained compound (13.86 g)
in tetrahydrofuran (100 mL) was added dropwise to a suspension
of lithium aluminum hydride (3.9 g) in tetrahydrofuran (100 mL)
at 0 °C. After 35 minutes, the reaction was diluted with ethyl
acetate at 0°C and quenched with 5N hydrochloric acid (20 mL)
until the evolution of gas ceased. The solution temperature was
raised to room temperature and the solution was stirred
overnight. The reaction solution was dried over anhydrous
magnesium sulfate, filtrated through Celite and concentrated. The
obtained residue was purified by column chromatography (n-
hexane : ethyl acetate = 1:1) to give diol compound (10.31 g). To
a solution of the diol compound (8.05 g) in toluene (98 mL) were
added tetrabutylammonium bromide (1.62 g) and 2N sodium
hydroxide (98 mL) at 0°C. The resulting mixture was treated
with a suspension of tosyl chloride (10.5 g) in toluene (40 mL).
The reaction mixture was warmed to room temperature and
stirred for 1 hour, followed by treatment with a solution of 1-
phenyl-1H-tetrazole-5-thiol (10.74 g) in toluene. The reaction
mixture was stirred at 60 °C for 3.5 hours, and extracted with
tert-butoxymethyl ether. The organic layer was washed with
brine, dried over anhydrous magnesium sulfate and concentrated.
The obtained residue was purified by silica gel column
chromatography (n-hexane : ethyl acetate = 81 : 19) to give
compound 20 (14.15 g, 86%).
1H NMR (300 MHz, CDCl3) δ 0.09 (s, 6H), 0.90 (s, 9H), 2.76
(s, 1H), 3.45 (m, 2H), 3.83 (m, 5H), 4.13 (dd, J = 8.6, 5.6 Hz,
1H), 4.38 (dd, J = 8.7, 8.6 Hz, 1H).
5.1.3. S-{2-[(4R)-4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-
oxo-1,3-oxazolidin-3-yl]ethyl} ethanethioate (15)
Methanesulfonyl chloride (1.43 mL) was added dropwise to a
solution of the compound 14 (4.58 g) and triethylamine (3.50
mL) in methylene chloride (32.0 mL) under cooling with ice. The
reaction mixture was stirred at 0 °C for 1 hour, then quenched
with water and extracted with ethyl acetate. The organic layer
was washed with 1N hydrochloric acid, water and brine, dried
over anhydrous magnesium sulfate and concentrated. To a
solution of the obtained residue in dimethylformamide (17.0 mL)
was added potassium thioacetate (1.91 g). The reaction mixture
was stirred at room temperature for 2 hours, then quenched with
water, and extracted with ethyl acetate. The organic layer was
washed with water and brine, dried over anhydrous sodium
sulfate and concentrated to give compound 15 (5.56 g, 100%).
1H NMR (300 MHz, CDCl3) δ 0.08 (s, 6H), 0.89 (s, 9H), 2.35
(s, 3H), 3.07 (m, 2H), 3.31 (m, 1H), 3.57 (m, 1H), 3.68 (dd,
J=10.8, 4.0 Hz, 1H), 3.79 (m, 1H), 3.93 (m, 1H), 4.12 (m, 1H),
4.33 (t, J=8.7 Hz, 1H).
5.1.4. ethyl
2-({2-[(4S)-4-(hydroxymethyl)-2-oxo-1,3-
oxazolidin-3-yl]ethyl}thio)-1,3-thiazol-4-carboxylate (16)
To a solution of the compound 15 (5.56 g), ethyl 2-bromo-1,3-
thiazole-4-carboxylate (4.34 g) and tributylphosphine (0.46 mL)
in ethanol (36.0 mL) was added potassium carbonate (3.70 g)
under cooling with ice. The reaction mixture was stirred at room
temperature for 1 hour and subsequently at 50 °C overnight. The
reaction was quenched with water, and extracted with ethyl
acetate. The organic layer was washed with water and brine,
dried over anhydrous magnesium sulfate and concentrated. To a
solution of the obtained residue in tetrahydrofuran (18.0 mL) was
1H NMR (300 MHz, CDCl3) δ 0.90 (t, J = 7.5 Hz, 3H), 1.25
(s, 3H), 1.42 (m, 6H), 1.57 (m, 2H), 1.65 (s, 1H), 2.04 (m, 2H),
3.57 (m, 2H), 7.73 (m, 5H).
5.1.7. 5-({3-methyl-3-[(trimethylsilyl)oxy]octyl}sulfonyl)-1-
phenyl-1H-tetrazole (22)
To a solution of the compound 20 (14.0 g) in methylene
chloride (200 mL) was added meta-chloroperbenzoic acid (27.3
g) at 0 °C. The reaction mixture was warmed to room
temperature and stirred overnight. The reaction was quenched
added
a solution of 1M tetrabutylammonium iodide in
tetrahydrofuran (18.4 mL) at 0 °C. The reaction mixture was
stirred at room temperature for 1 hour, then treated with water,
and extracted with ethyl acetate. The organic layer was washed

with an aqueous saturated sodium bicarbonate, evaporated to
remove methylene chloride and extracted with ethyl acetate. The
organic layer was washed with an aqueous saturated sodium
bicarbonate solution and brine, dried over anhydrous magnesium
sulfate and concentrated to give sulfone compound (15.4 g).
(79 mL). After stirring at –78 °C for 25 minutes, the reaction
mixture was warmed to 0 °C and stirred for 50 minutes. The
reaction was quenched with an aqueous saturated sodium
bicarbonate solution, and extracted with ethyl acetate. The
organic layer was washed with an aqueous saturated sodium
bicarbonate solution and brine, dried over anhydrous magnesium
sulfate, filtrated through Celite and concentrated under reduced
pressure. The obtained residue was purified by column
chromatography (n-hexane : ethyl acetate = 84 : 16) to give
olefin compound (6.62 g). To a solution of the olefin compound
(6.53 g) in ethyl acetate (114 mL) was slowly added dropwise 4N
hydrogen chloride/ethyl acetate solution (14 mL) at 0 °C and the
resulting solution was stirred for 10 minutes. An aqueous
saturated sodium bicarbonate solution was slowly added
dropwise to the reaction solution, followed by extraction with
ethyl acetate. The organic layer was washed with an aqueous
saturated sodium bicarbonate solution and brine, dried over
anhydrous magnesium sulfate and concentrated under reduced
pressure. The obtained residue was purified by column
chromatography (n-hexane : ethyl acetate = 40 : 60) to give
alcohol compound (3.81 g). To a solution of the alcohol
compound (3.7 g) in methanol (37 mL) was added dropwise 2N
aqueous sodium hydroxide solution (11 mL) at 0 °C. The
reaction mixture was warmed to room temperature and stirred for
45 minutes. After cooling to 0 °C, 2N hydrochloric acid was
added thereto and the solution was extracted with ethyl acetate.
The organic layer was washed with water and brine, dried over
anhydrous magnesium sulfate and concentrated. The obtained
residue was purified by column chromatography (n-hexane :
ethyl acetate = 1 : 1) to give compound 4 as colorless viscous oil
(2.95 g, 69%).
To a solution of the sulfone compound (15.4 g) in methylene
chloride (87 mL) were added imidazole (8.92 g) and
trimethylsilyl chloride (11.1 mL) at 0 °C. The reaction mixture
was stirred for 1 hour, and then treated with water, and extracted
with ethyl acetate. The organic layer was washed with brine,
dried over anhydrous magnesium sulfate and concentrated. The
obtained residue was purified by column chromatography (n-
hexane : ethyl acetate = 25 : 1), to give compound 22 (16.95 g,
82%).
1H NMR (300 MHz, CDCl3) δ 0.12 (s, 9 H), 0.90 (t, J = 6.0
Hz, 3H), 1.39 (m, 9H), 1.58 (m, 2H), 2.20 (m, 2H), 3.94 (m, 2H),
7.86 (m, 5H).
5.1.8. (4S,5S)-4-(hydroxymethyl)-5-methyl-1,3-oxazolidin-2-
one (25)
Starting from D-threonine methyl ester hydrochloride following
the same procedure as compound 13 provided compound 25.
1H NMR (300 MHz, CDCl3) δ 1.44 (d, J = 6.0 Hz, 3H), 3.62
(m, 3H), 4.51 (m, 1H), 6.20 (s, 1H).
5.1.9. (4S,5S)-4-({[dimethyl(2-methyl-2-
propanyl)silyl]oxy}methyl)-3-(2-hydroxyethyl)-5-methyl-1,3-
oxazolidin-2-one (26)
1H NMR (300 MHz, CDCl3) δ 1.86 (m, 19H), 2.76 (m, 4H),
3.73 (m, 4H), 3.93 (m, 1H), 4.28 (m, 1H), 5.36 (dd, J = 15.2, 9.0
Hz, 1H), 6.08 (m, 1H), 8.10 (s, 1H).
13C NMR (101 MHz, CDCl3) δ 18.66, 26.15, 26.41, 26.94,
27.33, 30.99, 31.04, 33.63, 35.21, 35.28, 35.34, 35.37, 41.41,
41.45, 45.35, 45.37, 49.37, 49.81, 67.06, 73.30, 73.35, 76.48,
127.44, 128.09, 129.02, 129.09, 129.45, 135.05, 135.14, 146.53,
157.69, 161.45, 161.49, 165.12, 165.14.
Starting from compound 25 following the same procedure as
compound 15 provided compound 26.
1H NMR (300 MHz, CDCl3) δ 0.09 (s, 6H), 0.90 (s, 9H), 1.44
(d, J = 6.0 Hz, 3H), 3.43 (m, 3H), 3.78 (m, 4H), 4.38 (m, 1H).
5.1.10. S-{2-[(4S,5S)-4-({[dimethyl(2-methyl-2-
propanyl)silyl]oxy}methyl)-5-methyl-2-oxo-1,3-oxazolidin-3-
yl]ethyl}ethanethioate (27)
HRMS (ESI, pos.) C22H33O5N2S2 (M + H) +, Obs. 469.1825,
Calc. 469.6378.
Starting from compound 26 following the same procedure as
compound 15 provided compound 27.
1H NMR (300 MHz, CDCl3) δ 0.08 (s, 6H), 0.89 (s, 9H), 1.42
(d, J = 6.0 Hz, 3H), 2.35 (s, 3H), 3.07 (m, 1H), 3.30 (m, 1H),
3.43 (m, 1H), 3.55 (m, 1H), 3.72 (m, 2H), 4.11 (m, 1H), 4.37 (m,
1H) .
5.1.13. 2-[(2-{(4S)-4-[(1E)-4-hydroxy-4-methyl-1-nonen-1-yl]-
2-oxo-1,3-oxazolidin-3-yl}ethyl)thio]-1,3-thiazole-4-carboxylic
acid (2)
Starting from compound 17 and compound 22 following the
same procedure as compound 4 provided compound 2 as pale yellow
5.1.11. ethyl
2-({2-[(4S,5S)-4-(hydroxymethyl)-5-methyl-2-
amorphous.
oxo-1,3-oxazolidin-3-yl]ethyl}thio)-1,3-thiazole-4-carboxylate
(28)
1H NMR (300 MHz, CDCl3) δ 1.02 (m, 3H), 1.64 (m, 11H),
2.47 (m, 2H), 5.07 (m, 9H), 5.57(m, 1H), 6.24 (m, 1H), 8.33 (m,
1H).
Starting from compound 27 following the same procedure as
compound 17 provided compound 28.
1H NMR (300 MHz, CDCl3) δ 1.39 (m, 6H), 3.60 (m, 6H),
3.84 (m, 2H), 4.40 (m, 2H), 8.02 (s, 1H).
13C NMR (101 MHz, CDCl3) δ 14.05, 18.70, 22.62, 22.63,
23.55, 26.68, 26.90, 30.93, 30.99, 32.28, 32.30, 41.42, 41.50,
41.84, 42.29, 44.40, 44.44, 67.05, 67.07, 72.84, 72.97, 76.55,
76.49, 127.51, 127.54, 128.99, 134.96, 134.98, 146.37, 157.63,
161.30, 161.44, 165.16, 165.20.
5.1.12. 2-[(2-{(4S,5S)-4-[(1E)-5-cyclohexyl-4-hydroxy-4-
methyl-1-penten-1-yl]-5-methyl-2-oxo-1,3-oxazolidin-3-
yl}ethyl)thio]-1,3-thiazole-4-carboxylic acid (4)
To a solution of the compound 23 (10.1 g) in dimethoxyethane
(79 mL) was slowly added dropwise potassium
hexamethyldisilazide (0.5M in toluene, 47.5 mL) at –78 °C. The
reaction mixture was stirred at –78 °C for 54 minutes, and treated
with a solution of the compound 28 (9.84 g) in dimethoxyethane
HRMS (ESI, pos.) C19H29O5N2S2 (M + H) +, Obs. 429.1512,
Calc. 429.5740.
5.1.14. 1-cyclohexyl-2-methyl-4-[(1-phenyl-1H-tetrazol-5-
yl)thio]-2-butanol (21)

diluted in ethyl acetate, washed with water and brine, dried over
anhydrous sodium sulfate and concentrated. The crude product
was purified by column chromatography on silica gel (n-hexane :
ethyl acetate = 4 : 1) to give compound 30 (624 mg, 38%).
1H NMR (300 MHz, CDCl3) δ 1.05 (s, 9H), 1.38 (t, J = 7.1 Hz,
3H), 1.77 (m, 8H), 2.82 (m, 1H), 3.20 (s, 1H), 3.58 (m, 3H), 4.41
(m, 3H), 7.41 (m, 6H), 7.65 (m, 4H), 7.96 (s, 1H).
Starting from commercial available 1-cyclohexylacetone
following the same procedure as compound 20 provided
compound 21.
1H NMR (300 MHz, CDCl3) δ 1.87 (m, 16H), 2.11 (m, 2H),
3.56 (m, 2H), 7.67 (m, 5H).
5.1.15. 5-({4-cyclohexyl-3-methyl-3-
[(trimethylsilyl)oxy]butyl}sulfonyl)-1-phenyl-1H-tetrazole
(23)
5.1.18. Ethyl
2-({2-[(1R,2S,5S)-2-(acetyloxy)-5-
Starting from compound 21 following the same procedure as
compound 22 provided compound 23.
formylcyclopentyl]ethyl}thio)-1,3-thiazole-4-carboxylate (31)
To a solution of compound 30 (42.2 g) in pyridine (75 mL)
were continuously added at 0 ºC acetic anhydride (13 mL) and 4-
N,N-dimethylaminopyridine (453 mg). The reaction mixture was
stirred at room temperature for 2 hours, and then diluted in ethyl
acetate. The organic layer was removed, washed with 1N
hydrochloric acid and brine, dried over anhydrous sodium sulfate
and concentrated under reduced pressure. The obtained residue
was dissolved in tetrahydrofuran (140 mL) and treated with 1M
tetrabutylammonium fluoride in tetrahydrofuran (110 mL) at 0 ºC.
After stirring at room temperature for 1 hour, the reaction was
quenched with water and extracted with ethyl acetate. The
organic layer was washed with water and brine, dried over
anhydrous magnesium sulfate, and concentrated under reduced
pressure. The obtained residue was purified by column
chromatography on silica gel (n-hexane : ethyl acetate = 7 : 3) to
give alcohol (27.0 g). The obtained alcohol (2.95 g) was
dissolved in dimethyl sulfoxide (20 mL) and ethyl acetate (30
mL), treated with triethylamine (7.8 mL) and sulfur trioxide-
pyridine complex (4.5 g) at 10 ºC, and stirred at room
temperature for 1 hour. To the reaction solution was added 1N
hydrochloric acid and the resulting mixture was extracted with
ethyl acetate. The organic layer was washed with water and
brine, dried over anhydrous sodium sulfate, and concentrated to
give compound 31 (2.93 g, 97%).
1H NMR (300 MHz, CDCl3) δ 0.13 (s, 9H), 1.81 (m, 16H),
2.15 (m, 2H), 3.88 (m, 2H), 7.76 (m, 5H).
5.1.16. 2-[(2-{(4S)-4-[(1E)-5-cyclohexyl-4-hydroxy-4-methyl-
1-penten-1-yl]-2-oxo-1,3-oxazolidin-3-yl}ethyl)thio]-1,3-
thiazole-4-carboxylic acid (3)
Starting from compound 17 and compound 22 following the
same procedure as compound 4 provided compound 3 as
colorless viscous oil.
1H NMR (300 MHz, CDCl3) δ 1.90 (m, 16H), 2.44 (m, 2H),
2.83 (s, 2H), 3.79 (m, 4H), 4.04 (m, 1H), 4.50 (m, 2H), 5.38 (dd,
J = 14.9, 8.6 Hz, 1H), 6.11 (m, 1H), 8.09 (s, 1H).
HRMS (ESI, pos.) C21H31O5N2S2 (M + H) +, Obs. 455.1669,
Calc. 455.6112.
5.1.17. Ethyl
2-({2-[(1R,2S,5S)-2-({[tert-
butyl(diphenyl)silyl]oxy}methyl)-5-
hydroxycyclopentyl]ethyl}thio)-1,3-thiazole-4-carboxylate
(30)
To
a
solution
of
(3aR,4S,6aS)-4-({[tert-
butyl(diphenyl)silyl]oxy}methyl)hexahydro-2H-
cyclopenta[b]furan-2-one (1.00 g) in anhydrous tetrahydrofuran
(9.00 mL) was added lithium aluminum hydride (97.0 mg) at 0
ºC. The reaction mixture was stirred at 0 ºC for 20 minutes, and
treated with water, and extracted with ethyl acetate. The organic
layer was washed with an aqueous saturated solution of sodium
tartrate and brine, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. A mixed solution of the
obtained residue and diisopropylethylamine (1.29 mL) in
1H NMR (300 MHz, CDCl3) δ 1.49 (m, 3H), 2.15 (m, 9H),
2.51 (m, 1H), 2.84 (m, 1H), 3.31 (m, 2H), 4.48 (m, 2H), 5.37 (m,
1H), 8.02 (s, 1H), 9.67 (d, J = 2.7 Hz, 1H).
5.1.19. 2-[(2-{(1R,2S,5R)-2-hydroxy-5-[(1E)-4-hydroxy-4-
methyl-1-nonen-1-yl]cyclopentyl}ethyl)thio]-1,3-thiazole-4-
carboxylic acid (33)
anhydrous tetrahydrofuran (9.00 mL) was added at –5 ºC to a
solution of methanesulfonyl chloride (0.23 mL) in anhydrous
tetrahydrofuran (5.00 mL). The reaction mixture was stirred for
20 minutes, and then quenched with anhydrous methanol (43.0
To
a
solution
of
(3-hydroxy-3-
methyloctyl)(triphenyl)phosphonium iodide 32 (800 mg) in
anhydrous tetrahydrofuran (9.00 mL) was added n-butyllithium
(1.60M in hexane, 1.90 mL) at room temperature. After stirring
for 1 hour, the resulting solution was treated with a solution of
the compound 31 (349 mg) in anhydrous tetrahydrofuran (6.00
mL) at –78 °C and then stirred for 2 hours. The reaction mixture
was warmed to room temperature, and poured into ice water,
followed by extraction with ethyl acetate. The organic layer was
washed with water and brine, dried over anhydrous sodium
sulfate and concentrated under reduced pressure. The obtained
residue was purified by column chromatography on silica gel (n-
hexane : ethyl acetate = 4 : 1). The obtained product was
dissolved in methanol (1.00 mL) and treated with 2N aqueous
sodium hydroxide solution (0.19 mL) at 0 °C. The resulting
solution was stirred at room temperature for 1 hour and acidified
with an aqueous solution of 2N hydrochloric acid (0.30 mL),
followed by extraction with ethyl acetate. The extract liquid was
washed with water and brine, dried over anhydrous sodium
sulfate and concentrated to give compound 33 (148 mg, 37%).
L) at –5 ºC for 15 minutes. The resulting mixture was treated
with trimethylsilyl chloride (0.49 mL) at –5 ºC, stirred at room
temperature for 10 minutes, subsequently treated with potassium
carbonate (1.10 g), potassium thioacetate (578 mg) and
anhydrous dimethylformamide (20.0 mL), and further stirred at
50 ºC for 5 hours. The reaction mixture was poured into iced
water, extracted with tert-butyl methyl ether, washed with water
and brine, dried over anhydrous sodium sulfate and concentrated.
To a solution of the obtained residue in ethanol (13.0 mL) were
added tri-n-butylphosphine (0.07 mL), ethyl 2-bromo-1,3-
thiazole-4-carboxylate (657 mg) and potassium carbonate (770
mg). The mixture was stirred at room temperature for 1 hour, and
subsequently stirred at 50 ºC overnight. The reaction mixture was
diluted in ethyl acetate, and washed with an aqueous saturated
solution of ammonium chloride, water and brine, dried over
anhydrous sodium sulfate and concentrated. The obtained residue
was dissolved in tetrahydrofuran (8.60 mL), cooled to 0 ºC, and
treated with 1N hydrochloric acid (1.86 mL). The resulting
mixture was stirred for 30 minutes at room temperature, then
1H NMR (300 MHz, CDCl3) δ 0.89 (t, J = 6.0 Hz, 3H), 1.13 (s,
3H), 1.30 (m, 12H), 1.79 (m, 2H), 2.02 (m, 3H), 2.40 (m, 1H),

2.88 (m, 1H), 3.56 (m, 1H), 4.50 (m, 1H), 5.32 (m, 1H), 5.45 (m,
1H), 8.02 (s, 1H).
13C NMR (101 MHz, CDCl3) δ 14.06, 22.65, 23.55, 26.54,
27.62, 28.25, 31.98, 32.33, 37.54, 41.95, 44.57, 46.35, 53.77,
72.95, 127.38, 127.44, 136.09, 146.41, 161.03, 166.10, 219.11.
HRMS (ESI, pos.) C21H32O4NS2 (M + H) +, Obs. 426.1767,
Calc. 426.6131.
5.1.20. 2-[(2-{(1R,2R)-2-[(1E)-4-hydroxy-4-methyl-1-
nonenyl]-5-oxocyclopentyl}ethyl)sulfanyl]-1,3-thiazole-4-
carboxylic acid (5)
To a solution of compound 33 (325 mg) in toluene (3.80 mL)
were added triethylamine (0.12 mL), t-butyl dimethyl silyl
chloride (130 mg) at 0 °C and the solution was stirred for 1 hour.
Ethyl acetate was added to the reaction solution, which was
filtered and concentrated under reduced pressure. The obtained
residue was dissolved in dimethylsulfoxide (1.20 mL)/ethyl
acetate (2.40 mL) and then treated with diisopropylamine (0.80
mL) and sulfur trioxide-pyridine complex (363 mg) at 0 °C,
followed by stirring for 30 min. The reaction mixture was diluted
with water, poured into 1N hydrochloric acid, and extracted with
ethyl acetate. The organic layer was washed with an aqueous
saturated ammonium chloride solution, water and brine, dried
over anhydrous sodium sulfate and concentrated under reduced
5.1.24. 2-[(2-{(1R,2R)-2-[(1E,4R)-4-hydroxy-4-methyl-1-
nonen-1-yl]-5-oxocyclopentyl}ethyl)thio]-1,3-thiazole-4-
carboxylic acid (5R)
Starting from compound 34R following the same procedure as
compound 10 provided compound 5R.
1H NMR (300 MHz, CDCl3) δ 0.89 (m, 3H), 2.58 (m, 21H),
3.67 (m, 4H), 5.53 (dd, J = 15.0, 8.2 Hz, 1H), 5.77 (m, 1H), 8.11
(s, 1H).
13C NMR (101 MHz, CDCl3) δ 14.07, 22.63, 23.55, 26.53,
27.62, 28.22, 31.98, 32.31, 37.55, 41.92, 44.57, 46.34, 53.77,
72.94, 127.38, 127.42, 136.09, 146.41, 161.01, 166.11, 219.11.
pressure.
The crude product was purified by column
HRMS (ESI, pos.) C21H32O4NS2 (M + H) +, Obs. 426.1767,
Calc. 426.6131.
chromatography on silica gel (n-hexane : ethyl acetate = 1 : 1) to
give compound 5 as yellow oil (199 mg, 62%).
1H NMR (300 MHz, CDCl3) δ 0.88 (t, J = 6.6 Hz, 3H), 2.59
(m, 21H), 3.75 (m, 4H), 5.51 (dd, J = 15.0, 9.0 Hz, 1H), 5.77 (m,
1H), 8.10 (s, 1H).
13C NMR (101 MHz, CDCl3) δ 14.06, 22.66, 23.56, 26.54,
27.62, 28.25, 31.98, 32.34, 37.54, 41.95, 44.57, 46.34, 53.77,
72.95, 127.39, 127.44, 136.08, 146.41, 161.03, 166.10, 219.11.
5.1.25. 3-methyl-1-[(1-phenyl-1H-tetrazol-5-yl)thio]-3-
heptanol (36a)
Starting from commercial available 2-hexanone following the
same procedure as compound 20 provided compound 36a.
1H NMR (300 MHz, CDCl3) δ 0.92 (t, J = 7.5 Hz, 3H), 1.25
(s, 3H), 1.34 (m, 4H), 1.52 (m, 2H), 2.03 (m, 2H), 3.47 (m, 2H),
7.57 (m, 5H).
HRMS (ESI, pos.) C21H32O4NS2 (M + H) +, Obs. 426.1767,
Calc. 426.6131.
5.1.26. 5-({3-methyl-3-[(trimethylsilyl)oxy]heptyl}sulfonyl)-1-
phenyl-1H-tetrazole (37a)
5.1.21. (5R,6S,9R,10E,13S)-13-methyl-15-oxo-13-pentyl-14-
oxa-2,18-dithia-19-azatricyclo[14.2.1.0~5,9~]nonadeca-
1(19),10,16-trien-6-yl acetate (34S)
Starting from compound 33 following the same procedure as
compound 39e provided compound 34S (less polar lactone).
1H NMR (300 MHz, CDCl3) δ 0.90 (t, J = 7.0 Hz, 3H), 1.46
(m, 13H), 2.16 (m, 8H), 2.39 (m, 1H), 2.68 (m, 1H), 2.88 (m, 2H),
3.32 (m, 1H), 5.31 (m, 1H), 5.43 (m, 1H), 5.62 (m, 1H), 7.94 (s,
1H).
Starting from compound 36a following the same procedure as
compound 22 provided compound 37a.
1H NMR (300 MHz, CDCl3) δ 0.12 (s, 9H), 0.92 (t, J = 6.0 Hz,
3H), 1.30 (m, 7H), 1.56 (m, 2H), 2.06 (m, 2H), 3.81 (m, 2H),
7.76 (m, 5H).
5.1.27. 7-fluoro-3-methyl-1-[(1-phenyl-1H-tetrazol-5-yl)thio]-
3-heptanol (36b)
Starting from commercial available 5-hexene-2-one following
the same procedure as compound 20 provided compound 36b.
1H NMR (300 MHz, CDCl3) δ 1.25 (s, 3H), 1.67 (m, 6H), 2.03
(m, 2H), 3.47 (m, 2H), 4.39 (dt, J = 57.0, 6.0 Hz, 2H), 7.57 (m,
5H).
5.1.22. (5R,6S,9R,10E,13R)-13-methyl-15-oxo-13-pentyl-14-
oxa-2,18-dithia-19-azatricyclo[14.2.1.0~5,9~]nonadeca-
1(19),10,16-trien-6-yl acetate (34R)
Starting from compound 33 following the same procedure as
compound 39e provided compound 34R (more polar lactone).
1H NMR (300 MHz, CDCl3) δ 0.90 (t, J = 7.0 Hz, 3H), 1.66
(m, 20H), 2.38 (m, 3H), 2.92 (m, 2H), 3.24 (m, 1H), 5.31 (m, 1H),
5.43 (m, 1H), 5.58 (m, 1H), 7.94 (s, 1H).
5.1.28. 5-({7-fluoro-3-methyl-3-
[(trimethylsilyl)oxy]heptyl}sulfonyl)-1-phenyl-1H-tetrazole
(37b)
Starting from compound 36b following the same procedure as
compound 22 provided compound 37b.
1H NMR (300 MHz, CDCl3) δ 0.12 (s, 9H), 1.54 (m, 7H), 1.70
(m, 2H), 2.06 (m, 2H), 3.81 (m, 2H), 4.39 (dt, J = 57.0, 6.0 Hz,
2H), 7.74 (m, 5H).
5.1.23. 2-[(2-{(1R,2R)-2-[(1E,4S)-4-hydroxy-4-methyl-1-
nonen-1-yl]-5-oxocyclopentyl}ethyl)thio]-1,3-thiazole-4-
carboxylic acid (5S)
Starting from compound 34S following the same procedure as
compound 10 provided compound 5S.
5.1.29. 6,6,6-trifluoro-3-methyl-1-[(1-phenyl-1H-tetrazol-5-
yl)thio]-3-hexanol (36c)
1H NMR (300 MHz, CDCl3) δ 0.88 (m, 3H), 2.59 (m, 21H),
3.75 (m, 4H), 5.52 (dd, J = 15.0, 7.8 Hz, 1H), 5.77 (m, 1H), 8.11
(s, 1H).
Starting from commercial available 5,5,5-trifluoropentane-2-
one following the same procedure as compound 20 provided
compound 36c.
1H NMR (300 MHz, CDCl3) δ 1.24 (s, 3H), 1.56 (m, 2H), 2.03
(m, 2H), 2.31 (m, 2H), 3.45 (m, 2H), 7.57 (m, 5H).

was diluted with water and extracted with ethyl acetate. The
organic layer was washed with brine, dried over anhydrous
sodium sulfate, and concentrated to obtain compound 38e (227
mg, 64%).
1H NMR (300 MHz, CDCl3) δ 1.15 (s, 3H), 1.49 (m, 8H), 1.88
(m, 5H), 2.08 (m, 2H), 2.20 (m, 2H), 2.48 (m, 4H), 2.93 (m, 1H),
3.55 (m, 1H), 4.52 (m, 1H), 5.37 (m, 1H), 5.48 (m, 1H), 8.07 (s,
1H).
5.1.30. 1-phenyl-5-({6,6,6-trifluoro-3-methyl-3-
[(trimethylsilyl)oxy]hexyl}sulfonyl)-1H-tetrazole (37c)
Starting from compound 36c following the same procedure as
compound 22 provided compound 37c.
1H NMR (300 MHz, CDCl3) δ 0.12 (s, 9H), 1.34 (m, 3H), 1.58
(m, 2H), 2.09 (m, 2H), 2.31(m, 2H), 3.80 (m, 2H), 7.75 (m, 5H)..
5.1.31. 3,6,6-trimethyl-1-[(1-phenyl-1H-tetrazol-5-yl)thio]-3-
heptanol (36d)
5.1.36. (5R,6S,9R,10E,13S)-13-(cyclopentylmethyl)-13-
methyl-15-oxo-14-oxa-2,18-dithia-19-
azatricyclo[14.2.1.0~5,9~]nonadeca-1(19),10,16-trien-6-yl
acetate (39e)
Acetic anhydride (0.06 mL) was added to a pyridine solution
(1.5 mL) of compound 38e (134 mg) under ice cooling, followed
by stirring at room temperature overnight. The reaction mixture
was poured into 1N hydrochloric acid and extracted with ethyl
acetate. The organic layer was washed with saturated brine, dried
over anhydrous sodium sulfate, and concentrated to furnish
acetate compound (131 mg).
Starting from commercial available 5,5-dimethylhexane-2-one
following the same procedure as compound 20 provided
compound 36d.
1H NMR (300 MHz, CDCl3) δ 0.89 (s, 9H), 1.23 (s, 3H), 1.33
(m, 2H), 1.54 (m, 2H), 2.04 (m, 2H), 3.46 (m, 2H), 7.57 (m, 5H).
5.1.32. 1-phenyl-5-({3,6,6-trimethyl-3-
[(trimethylsilyl)oxy]heptyl}sulfonyl)-1H-tetrazole (37d)
Starting from compound 36d following the same procedure as
compound 22 provided compound 37d.
1H NMR (300 MHz, CDCl3) δ 0.12 (s, 9H), 0.89 (s, 9H), 1.30
(m, 5H), 1.55 (m, 2H), 2.07 (m, 2H), 3.80 (m, 2H), 7.76 (m, 5H).
A solution of acetate compound (131 mg) in toluene (53 mL)
was treated with 4,4-dimethylaminopyridine (323 mg) at room
temperature and then heated to 100 ºC. 2,4,6-trichlorobenzoyl
chloride (0.25 mL) was added thereto. After stirring for 15 min at
100 ºC, the reaction mixture was cooled to room temperature,
then poured into a saturated aqueous solution of sodium
hydrogen carbonate, and extracted with ethyl acetate. The
organic layer was washed with water and brine, dried with
anhydrous sodium sulfate, and concentrated under reduced
pressure. The resulting crude product was purified by silica gel
column chromatography (n-hexane : ethyl acetate = 15 : 1) to
obtain compound 39e (less polar lactone, 49 mg, 35%).
5.1.33. 1-cyclopentyl-2-methyl-4-[(1-phenyl-1H-tetrazol-5-
yl)thio]-2-butanol (36e)
Starting from commercial available 4-cyclopentyl-2-butanone
following the same procedure as compound 20 provided
compound 36e.
1H NMR (300 MHz, CDCl3) δ 1.16 (m, 4H), 1.27 (s, 3H), 1.60
(m, 5H), 1.88 (m, 2H), 2.03 (m, 2H), 3.48 (m, 2H), 7.56 (m, 5H).
1H NMR (300 MHz, CDCl3) δ 1.20 (m, 4H), 1.66 (m, 8H),
1.89 (m, 5H), 2.22 (m, 6H), 2.37 (m, 2H), 2.82 (m, 2H), 3.34 (m,
2H), 5.31 (m, 1H), 5.45 (m, 1H), 5.62 (m, 1H), 7.95 (s, 1H).
5.1.34. 5-({4-cyclopentyl-3-methyl-3-
[(trimethylsilyl)oxy]butyl}sulfonyl)-1-phenyl-1H-tetrazole
(37e)
Starting from compound 36e following the same procedure as
compound 22 provided compound 37e.
1H NMR (300 MHz, CDCl3) δ 0.12 (s, 9H), 1.35 (m, 8H), 1.66
(m, 6H), 2.06 (m, 2H), 3.81 (m, 2H), 7.76 (m, 5H).
5.1.37. 2-[(2-{(1R,2R)-2-[(1E,4S)-5-cyclopentyl-4-hydroxy-4-
methyl-1-penten-1-yl]-5-oxocyclopentyl}ethyl)thio]-1,3-
thiazole-4-carboxylic acid (10)
Compound 39e (183 mg) was dissolved in a mixed solvent of
methanol (1.3 mL) and tetrahydrofuran (2.6 mL), and treated
with a 2N aqueous solution of sodium hydroxide (0.59 mL). The
reaction mixture was stirred at room temperature overnight,
subsequently poured into 1N hydrochloric acid and extracted
with ethyl acetate. The organic layer was washed with water and
brine, dried over anhydrous sodium sulfate, and concentrated to
give diol carboxylic acid compound (187 mg).
5.1.35. 2-[(2-{(1R,2R,5S)-2-[(1E)-5-cyclopentyl-4-hydroxy-4-
methyl-1-penten-1-yl]-5-hydroxycyclopentyl}ethyl)thio]-1,3-
thiazole-4-carboxylic acid (38e)
Potassium hexamethyldisilazide in toluene (0.5 M, 3.24 mL)
was slowly added dropwise to a solution of compound 37e (705
mg) in 1,2-dimethoxyethane (4.0 mL) at –78 ºC, followed by
stirring at the same temperature for 30 min. To the reaction
mixture, a solution of compound 31 (300 mg) in 1,2-
dimethoxyethane (3.0 mL) was slowly added dropwise at –78
ºC. After string at the same temperature for 30 min, the reaction
temperature was raised to 0 ºC. A saturated aqueous solution of
sodium hydrogen carbonate was added, followed by extraction
with ethyl acetate. The organic layer was washed with water and
saturated brine, dried over anhydrous sodium sulfate, and
concentrated. The resulting residue was purified by silica gel
column chromatography (n-hexane : ethyl acetate = 8 :1) to
obtain olefin compound (313 mg).
Potassium carbonate (218 mg) and ethyl iodide (0.064 mL)
were added to a dimethylformamide solution (2.0 mL) of diol
carboxylic acid compound (174 mg), followed by stirring at room
temperature overnight. The reaction solution was poured into
water and extracted with ethyl acetate. The organic layer was
washed with water and brine, dried over anhydrous sodium
sulfate, and concentrated to provide ethyl ester compound (173
mg).
Diisopropylethylamine (0.51 mL) and sulfur trioxide·pyridine
complex (286 mg) were added to a dimethyl sulfoxide (1.2
mL)/ethyl acetate (2.4 mL) solution of ethyl ester compound (173
mg) under ice cooling, followed by stirring for 15 min. The
reaction was quenched with water, and extracted with ethyl
acetate. The organic layer was washed with 1N hydrochloric
To an ethanol solution (3.0 mL) of olefin compound (313 mg),
2N aqueous sodium hydroxide solution (1.2 mL) was added
under ice cooling, followed by stirring at room temperature
overnight. The reaction mixture was acidified with 1N
hydrochloric acid. After stirring for 30 min, the reaction mixture

acid, water and brine, dried with anhydrous sodium sulfate, and
concentrated under reduced pressure. The resulting residue was
purified by silica gel column chromatography (n-hexane : ethyl
acetate = 3 : 2) to furnish keto ethyl ester (110 mg).
13C NMR (101 MHz, CDCl3) δ 14.12, 23.22, 26.08, 26.55,
27.60, 28.24, 32.00, 37.54, 41.71, 44.57, 46.34, 53.75, 72.92,
127.37, 127.43, 136.10, 146.44, 161.04, 166.08, 219.10.
HRMS (ESI, pos.) C20H30O4NS2 (M + H) +, Obs. 412.1611,
Calc. 412.5865.
The keto ethyl ester (110 mg) was dissolved in 1,2-
dimethoxyethane (2.0 mL)/water (1.0 mL), and treated with
lithium hydroxide (16.0 mg) under ice cooling, followed by
stirring at room temperature for 2 hours. The reaction mixture
was poured into a 5% aqueous solution of potassium hydrogen
sulfate and extracted with ethyl acetate. The organic layer was
washed with water and brine, dried with anhydrous sodium
sulfate, and concentrated in vacuo. The resulting residue was
purified by silica gel column chromatography (n-hexane : ethyl
acetate = 1 : 1 → methanol : ethyl acetate = 1 : 10) to provide
compound 10 as pale yellow viscous oil (90.5 mg, 72%).
1H NMR (300 MHz, CDCl3) δ 2.32(m, 22H), 2.59(m, 2H),
3.09(m, 2H), 3.36(t, J = 7.5 Hz, 2H), 5.51(dd, J = 15.0, 6.0 Hz,
1H), 5.70(dt, J = 15.0, 7.4 Hz, 1H), 8.10(s, 1H).
13C NMR (101 MHz, CDCl3) δ 25.00, 25.06, 27.09, 27.61,
28.24, 31.99, 34.50, 34.56, 36.00, 37.54, 45.37, 46.34, 48.04,
53.75, 73.33, 127.43, 127.50, 136.09, 146.40, 161.06, 166.11,
219.14.
5.1.41. 2-[(2-{(1R,2R,5S)-2-[(1E)-8-fluoro-4-hydroxy-4-
methyl-1-octen-1-yl]-5-hydroxycyclopentyl}ethyl)thio]-1,3-
thiazole-4-carboxylic acid (38b)
Starting from compound 37b instead of compound 37e
following the same procedure as compound 38e provided
compound 38b.
1H NMR (300 MHz, CDCl3) δ 1.15 (s, 3H), 1.42 (m, 12H),
2.03 (m, 3H), 2.42 (m, 1H), 3.11 (m, 1H), 3.55 (m, 1H), 4.36 (dt,
J = 47.4, 6.0 Hz, 2H), 4.50 (m, 1H), 5.32 (m, 1H), 5.44 (m, 1H)
8.08 (s, 1H).
5.1.42. (5R,6S,9R,10E,13S)-13-(4-fluorobutyl)-13-methyl-15-
oxo-14-oxa-2,18-dithia-19-
azatricyclo[14.2.1.0~5,9~]nonadeca-1(19),10,16-trien-6-yl
acetate (39b)
Starting from compound 38b instead of compound 38e
following the same procedure as compound 39e provided
compound 39b.
HRMS (ESI, pos.) C22H32O4NS2 (M + H) +, Obs. 438.1767,
Calc. 438.6238.
1H NMR (300 MHz, CDCl3) δ 1.34 (m, 11H), 2.04 (m, 7H),
2.39 (m, 1H), 2.64 (m, 1H), 2.85 (m, 2H), 3.30 (m, 2H), 4.34 (dt,
J = 47.4, 6.0 Hz, 2H), 5.30 (m, 1H), 5.46 (m, 1H), 5.61 (m, 1H),
7.94 (s, 1H).
Rotation []D : -34.56 (c = 1.05, CHCl3)
5.1.38. 2-[(2-{(1R,2S,5R)-2-hydroxy-5-[(1E)-4-hydroxy-4-
methyl-1-octen-1-yl]cyclopentyl}ethyl)thio]-1,3-thiazole-4-
carboxylic acid (38a)
5.1.43. 2-[(2-{(1R,2R)-2-[(1E,4S)-8-fluoro-4-hydroxy-4-
methyl-1-octen-1-yl]-5-oxocyclopentyl}ethyl)thio]-1,3-
thiazole-4-carboxylic acid (7)
Starting from compound 39b instead of compound 39e
following the same procedure as compound 10 provided
compound 7 as yellow viscous oil.
Starting from compound 37a instead of compound 37e
following the same procedure as compound 38e provided
compound 38a.
1H NMR (300 MHz, CDCl3) δ 0.91 (t, J = 7.0 Hz, 3H), 1.15
(s, 3H), 1.42 (m, 12H), 2.02 (m, 3H), 2.45 (m, 1H), 3.13 (m, 1H),
3.57 (m, 1H), 4.52 (m, 1H), 5.36 (m, 1H), 5.48 (m, 1H), 8.08 (s,
1H).
1H NMR (300 MHz, CDCl3) δ. 1.19(s, 3H), 2.60 (m, 16H),
3.44 (m, 2H), 4.46 (dt, J = 47.4, 6.0Hz, 2H), 5.53 (dd, J = 15.3,
8.1 Hz, 1H), 5.70 (dt, J = 15.3, 7.5 Hz, 1H), 8.11 (s, 1H).
13C NMR (101 MHz, CDCl3) δ 19.72, 26.52, 27.63, 28.22,
30.81, 31.99, 37.51, 41.45, 44.58, 46.38, 53.73, 72.75, 84.00,
127.10, 127.50, 136.38, 146.36, 161.03, 166.08, 219.03.
HRMS (ESI, pos.) C20H29O4NFS2 (M + H) +, Obs. 430.1517,
Calc. 430.5770.
5.1.39. (5R,6S,9R,10E,13S)-13-butyl-13-methyl-15-oxo-14-
oxa-2,18-dithia-19-azatricyclo[14.2.1.0~5,9~]nonadeca-
1(19),10,16-trien-6-yl acetate (39a)
Starting from compound 38a instead of compound 38e
following the same procedure as compound 39e provided
compound 39a.
5.1.44. 2-[(2-{(1R,2S,5R)-2-hydroxy-5-[(1E)-7,7,7-trifluoro-4-
hydroxy-4-methyl-1-hepten-1-yl]cyclopentyl}ethyl)thio]-1,3-
thiazole-4-carboxylic acid (38c)
1H NMR (300 MHz, CDCl3) δ 0.91 (t, J = 7.0 Hz, 3H), 1.36
(m, 11H), 2.06 (m, 7H), 2.38 (m, 1H), 2.66 (m, 1 H), 2.88 (m, 2
H), 3.32 (m, 2 H), 5.31 (m, 1 H), 5.43 (m, 1 H), 5.60 (m, 1 H),
7.94 (s, 1 H).
Starting from compound 37c instead of compound 37e
following the same procedure as compound 38e provided
compound 38c
1H NMR (300 MHz, CDCl3) δ 1.15 (s, 3H), 1.48 (m, 8H), 2.05
(m, 3H), 2.33 (m, 2H), 2.46 (m, 1H), 3.16 (m, 1H), 3.59 (m, 1H),
4.50 (m, 1H), 5.35 (m, 1H), 5.46 (m, 1H), 8.09 (s, 1H).
5.1.40. 2-[(2-{(1R,2R)-2-[(1E,4S)-4-hydroxy-4-methyl-1-
octen-1-yl]-5-oxocyclopentyl}ethyl)thio]-1,3-thiazole-4-
carboxylic acid (6)
Starting from compound 39a instead of compound 39e
following the same procedure as compound 10 provided
compound 6 as colorless oil.
5.1.45. (5R,6S,9R,10E,13S)-13-methyl-15-oxo-13-(3,3,3-
trifluoropropyl)-14-oxa-2,18-dithia-19-
azatricyclo[14.2.1.0~5,9~]nonadeca-1(19),10,16-trien-6-yl
acetate (39c)
Starting from compound 38c instead of compound 38e
following the same procedure as compound 39e provided
compound 39c.
1H NMR (300 MHz, CDCl3) δ 0.92 (t, J = 7.5 Hz, 3H), 1.16 (s,
3H), 1.37 (m, 4H), 1.53 (m, 2H), 1.77 (m, 1H), 2.27 (m, 7H),
2.59 (m, 2H), 3.51 (m, 2H), 5.50 (dd, J = 15.0, 8.0 Hz, 1H), 5.77
(m, 1H), 8.09 (s, 1H).

1H NMR (300 MHz, CDCl3) δ 1.36 (m, 7H), 2.06 (m, 7H),
2.38 (m, 3H), 2.68 (m, 1H), 2.85 (m, 2H), 3.31 (m, 2H), 5.34 (m,
1H), 5.45 (m, 1H), 5.63 (m, 1H), 7.95 (s, 1H).
Starting from more polar lactone derived from compound 38e
following the same procedure as compound 10 provided
compound 11 as colorless viscous oil.
1H NMR (300 MHz, CDCl3) δ 2.34 (m, 22H), 2.63 (m, 2H),
3.23 (m, 2H), 3.37 (t, J = 7.3 Hz, 2H), 5.51 (dd, J = 15.1, 6.0 Hz,
1H), 5.69 (dt, J = 15.1, 7.2 Hz, 1H), 8.10 (s, 1H).
5.1.46. 2-[(2-{(1R,5R)-2-oxo-5-[(1E,4S)-7,7,7-trifluoro-4-
hydroxy-4-methyl-1-hepten-1-yl]cyclopentyl}ethyl)thio]-1,3-
thiazole-4-carboxylic acid (8)
13C NMR (101 MHz, CDCl3) δ 24.96, 25.07, 27.14, 27.53,
28.24, 31.96, 34.47, 34.58, 36.07, 37.52, 45.46, 46.39, 47.78,
53.78, 73.36, 127.38, 127.48, 136.18, 146.41, 161.08, 166.12,
219.08.
Starting from compound 39c instead of compound 39e
following the same procedure as compound 10 provided
compound 8 as colorless viscous oil.
1H NMR (300 MHz, CDCl3) δ 1.19 (s, 3H), 1.81 (m, 3H), 2.31
(m, 9H), 2.57 (m, 2H), 3.37 (t, J = 6.7 Hz, 2H), 5.53 (dd, J =
15.1, 7.6 Hz, 1H), 5.75 (m, 1H), 8.10 (s, 1H).
HRMS (ESI, pos.) C22H32O4NS2 (M + H) +, Obs. 438.1767,
Calc. 438.6238.
Rotation []D : -36.15 (c = 1.15, CHCl3)
13C NMR (101 MHz, CDCl3) δ 26.39, 27.55, 28.12, 28.46,
31.83, 33.43, 33.45, 37.50, 44.97, 46.18, 53.62, 71.25, 126.36,
127.50, 136.89, 146.12, 161.04, 166.25, 218.98.
5.1.51. (5R,6S,9R,10E,13R)-13-(cyclohexylmethyl)-13-methyl-
15-oxo-14-oxa-2,18-dithia-19-
azatricyclo[14.2.1.0~5,9~]nonadeca-1(19),10,16-trien-6-yl
HRMS (ESI, pos.) C19H25O4NF3S2 (M + H) +, Obs. 452.1172,
Calc. 452.5313.
acetate (40)
5.1.47. 2-[(2-{(1R,2S,5R)-2-hydroxy-5-[(1E)-4-hydroxy-4,7,7-
trimethyl-1-octen-1-yl]cyclopentyl}ethyl)thio]-1,3-thiazole-4-
carboxylic acid (39c)
Starting from compound 23 and compound 31 following the
same procedure as compound 39e provided more polar
maclolactone compound 40.
Starting from compound 37d instead of compound 37e
following the same procedure as compound 38e provided
compound 38d.
1H NMR (300 MHz, CDCl3) δ 1.18 (m, 6H), 1.56 (m, 12H),
2.02 (m, 7H), 2.45 (m, 3H), 2.92 (m, 2H), 3.25 (m, 1H), 5.31 (m,
1H), 5.45 (m, 1H), 5.59 (m, 1H), 7.92 (s, 1H).
1H NMR (300 MHz, CDCl3) δ 0.88 (s, 9H), 1.15 (s, 3H), 1.24
(m, 2H), 1.39 (m, 6H), 1.88 (m, 2H), 2.10 (m, 3H), 2.44 (m, 1H),
3.13 (m, 1H), 3.57 (m, 1H), 4.52 (m, 1H), 5.37 (m, 1H), 5.46 (m,
1H), 8.06 (s, 1H).
5.2. Biology
To measure each EP agonistic activity, CHO cells expressing
human EP1, EP2, EP3, and EP4 receptors were used.
To measure EP1 and EP 3 agonistic activities, 100 μL loading
medium (medium containing 5 μmol/L fura-2-AM solution, 2.5
mmol/L probenecid, 20 μmol/L indomethacin, and 10 mmol/L
HEPES) was added, and the cells were incubated for about 60
minutes at 37 °C in a CO2 (5% CO2, 95% air) incubator to
incorporate the fura-2. An assay buffer [120 μL HBSS containing
1 w/v% albumin from bovine serum (Sigma-Aldrich Corp.), 2.5
mmol/L probenecid, 2 μmol/L indomethacin, and 20 mmol/L
HEPES] was added. The intracellular calcium level was
determined using a Fluorescence Drug Screening System (FDSS-
3000, Hamamatsu Photonics KK). Then, 2.45 min after the start
of measurement at 37°C, compound (0.03 to 300 nmol/L for EP3,
300 to 10,000 nmol/L for EP1) and PGE2 (0.03 to 300 nmol/L for
EP3, 0.1 to 1,000 nmol/L for EP1) were added. Measurements
were taken at approximately 3-sec intervals, and the indicator of
the change in intracellular calcium level was the change in the
fluorescence intensity ratio (f340/f380) at 500 nm during
irradiation with two alternating wavelengths of excitation light
(340 nm, 380 nm).
To measure EP2 and EP 4 agonistic activities, 500 μL of assay
medium A (MEM Alpha, Invitrogen Corp.) containing 2 μmol/L
diclofenac (Sigma-Aldrich Corp.) was added. Then, 450 μL of
assay medium B [MEM Alpha containing 2 μmol/L diclofenac, 1
mmol/L 3-isobutyl-1-methylxanthine (IBMX), and 1 w/v%
albumin from bovine serum (Sigma-Aldrich Corp.)] was added.
Next, compound (0.003 to 10 nmol/L for EP2, 0.3 to 10,000
nmol/L for EP4) and PGE2 (0.03 to 300 nmol/L for EP2, 0.01 to
3,000 nmol/L for EP1) were added. Finally, 500 μL of 10 w/v%
trichloroacetic acid solution (Wako Pure Chemical Industries)
was added to stop the reaction. Then, 300 μL of supernatant from
the centrifuged cell suspension was transferred to a polyethylene
tube and mixed with 300 μL of cAMP extraction solvent
[chloroform (Kishida Chemical Co., Ltd.) containing 0.45 mol/L
tri-n-octylamine (Tokyo Chemical Industry Co., Ltd.)].
5.1.48. (5R,6S,9R,10E,13S)-13-(3,3-dimethylbutyl)-13-methyl-
15-oxo-14-oxa-2,18-dithia-19-
azatricyclo[14.2.1.0~5,9~]nonadeca-1(19),10,16-trien-6-yl
acetate (39d)
Starting from compound 38d instead of compound 38e
following the same procedure as compound 39d provided
compound 39d.
1H NMR (300 MHz, CDCl3) δ 0.90 (s, 9H), 1.30 (m, 7H), 2.06
(m, 9H), 2.39 (m, 1H), 2.65 (m, 1H), 2.88 (m, 2H), 3.32 (m, 2H),
5.31 (m, 1H), 5.44 (m, 1H), 5.62 (m, 1H), 7.95 (s, 1H).
5.1.49. 2-[(2-{(1R,2R)-2-[(1E,4S)-4-hydroxy-4,7,7-trimethyl-1-
octen-1-yl]-5-oxocyclopentyl}ethyl)thio]-1,3-thiazole-4-
carboxylic acid (9)
Starting from compound 39d instead of compound 39e
following the same procedure as compound 10 provided
compound 9 as yellow viscous oil.
1H NMR (300 MHz, CDCl3) δ 0.89(s, 9H), 1.33(m, 5H),
1.53(m, 2H), 1.77(m, 1H), 2.30(m, 7H), 2.58(m, 2H), 3.35(t, J =
7.3 Hz, 2H), 5.54(dd, J = 15.7, 8.2 Hz, 1H), 5.76(m, 1H), 8.11(s,
1H).
13C NMR (101 MHz, CDCl3) δ 26.66, 27.52, 28.24, 29.35,
30.02, 31.97, 36.55, 37.56, 37.62, 44.38, 46.21, 53.72, 72.90,
127.32, 127.37, 136.06, 146.38, 160.87, 166.10, 219.15.
HRMS (ESI, pos.) C22H34O4NS2 (M + H) +, Obs. 440.1924,
Calc. 440.6397.
5.1.50. 2-[(2-{(1R,2R)-2-[(1E,4R)-5-cyclopentyl-4-hydroxy-4-
methyl-1-penten-1-yl]-5-oxocyclopentyl}ethyl)thio]-1,3-
thiazole-4-carboxylic acid (11)

Approximately 80 μL of the aqueous layer from the centrifuged
mixture was transferred to a polyethylene tube, and the level of
cAMP was assayed using a cAMP EIA kit [Amersham cAMP
Biotrak enzyme immunoassay (EIA) System (dual range), GE
Healthcare UK Ltd.].
The compound or control response rate (%) was used as an
evaluation indicator, where 100% represents the maximum
increase in intracellular calcium level of PGE2 in hEP3-CHO and
hEP1-CHO and 100% represents maximum cAMP production of
PGE2 in hEP2-CHO and hEP4-CHO.
humidified incubator (5% CO2, 37 C). Two h later, take aliquots
from the receiver chambers. Each determination was performed
in duplicate. The flux of co-dosed lucifer yellow was also
measured for each monolayer to ensure no damage was inflicted
to the cell monolayers during the flux period. Determine test
substance concentrations in the samples by LC/MS/MS.
The apparent permeability coefficient (Papp) is calculated
according to the following equation:
Papp = (dCr/dt)  Vr/(A  C0)
dCr/dt: slope of compound accumulation in the receiver
compartment over time (M/s)
Vr: volume of the receiver compartment (cm3)
A: area of the cell monolayer (1.13 cm2 for 12-well Transwell
plate)
5.3. Pharmacokinetics studies
The pharmacokinetics of compounds were studied following
single intravenous and oral administration to male rats. The IV
and PO solution formulations contained wellsolve/H2O = 1/9
(Celeste) and sodium hydroxide (two equivalents). All IV and PO
formulations were given as solution. Animals were fasted prior to
oral dosing in single-dose studies. Oral bioavailability was
estimated using non-cross-over study designs for the rat (n = 3).
Plasma samples were assayed for compounds using protein
precipitation by acetonitrile/ethanol (70/30, v/v) followed by
HPLC/MS/MS analysis employing negative-ion Turbo IonSpray
ionization. Plasma concentration-time data were analyzed by-
compartmental methods.
C0: initial normality of the buffer (M)
The involvement of efflux transporter(s) such as P-gp is
suspected if the ratio of the B-to-A Papp to the A-to-B Papp is 2
or more.
ACKNOWLEDGMENT
We thank Dr. Yoshiyuki Yamaura for measuring
pharmacokinetic profiles, Mr. Hiromu Egashira for homology
modeling of EP2 and EP3 receptor, Mr. Daisuke Wakamatsu for
performing the biological studies and animal models. We thank
Prof. Jason J. Chruma (Sichuan University) for helpful
manuscript revisions.
5.4. Parallel artificial membrane permeability assay
(PAMPA)
REFERENCES
1.
2.
3.
Coleman, R. A.; Smith, W.L.; Narumiya, S. Pharmacol. Rev. 1994,
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The PAMPA System (Pion) should be used for assay. Dilute
the System Solution Concentrate (Pion) with water, and add
DMSO to prepare a system solution (in 5% DMSO). Add 0.5
mol/L NaOH to the system solution using an automated buffer
preparation system to make three different buffers of pH 5.0, 6.2
and 7.4. Add 150-L of the buffers per well in a UV plate to
serve as blanks for determination of UV absorption (190-498
nm). Add 5 L of DMSO solution containing 10 mM the test
compound to 1000 L of the system solution, and filter by
suction through a 96-well filter plate (0.2 m PVDF hydrophilic
membrane, Corning) (final compound concentration of 50 M).
Add 150-L of the filtrate per well in the UV plate to serve as the
reference for determination of UV absorption (190-498 nm). Add
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use as the donor solution. Make a membrane by dropping 4 L of
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a 200-L aliquot of the acceptor solution (Acceptor Sink Buffer,
pH 7.4, Pion) per well in the acceptor plate. Place the acceptor
plate on the donor plate. After a 4-h incubation, transfer 150-L
aliquots from the donor and acceptor solutions to the UV plate
for determination of UV absorption (190-498 nm). The
membrane permeability coefficient should be calculated from the
observed values obtained as above using the PAMPA Evolution
Command Software.
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Grow Caco-2 cells on a 12-well Costar Transwell plate (with a
collagen-coated fine porous polycarbonate membrane) until a
confluent monolayer is formed. Prepare buffer for permeability
assay using 10 mM HEPES and Hank’s balanced salt solution
containing 15 mM glucose and0.2 mM Lucifer yellow with the
pH being adjusted at 7.4. Add the test substance solution to the
apical side (for the A-to-B permeability) or the basolateral side
(for the B-to-A permeability) of the Caco-2 cell monolayer at a
final concentration of 1 M, and then incubate the plate in a
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Graphical Abstract
Synthesis and Evaluation of a Potent, Well-
balanced EP2/EP3 Dual Agonist
Akihiro Kinoshita,a,* Masato Higashino,a Koji Yoshida,a Yoshiyuki Aratani,a Akito Kakuuchi,a
Keisuke Hanada,a Hiroyuki Takeda,a Atsushi Naganawa,a Hidekazu Matsuya,b and Kazuyuki
Ohmotoa,*
CO2H
CO2H
N
S
N
S
O
S
O
S
N
O
H3C OH
F
OH
10
1
human EC50 EP2 = 1.1 nM, EP3 = 1.0 nM,
EP1 = 2000 nM, EP4 = 2636 nM