Synthesis and evaluation of novel modified γ-lactam prostanoids as EP4 subtype-selective agonists

Article abstract of DOI:10.1016/j.bmc.2011.12.009

To identify chemically and metabolically stable subtype-selective EP4 agonists, design and synthesis of a series of modified γ-lactam prostanoids has been continued. Prostanoids bearing 2-oxo-1,3-oxazolidine, 2-oxo-1,3-thiazolidine and 5-thioxopyrrolidine as a surrogate for the γ-hydroxycyclopentanone without a troublesome 11-hydroxy group were identified as highly subtype-selective EP4 agonists. Among the tested, several representative compounds demonstrated in vivo efficacy after oral dosing in rats. Their pharmacokinetic and structure-activity relationship studies are presented.

Full text of DOI:10.1016/j.bmc.2011.12.009

Bioorganic & Medicinal Chemistry 20 (2012) 702–713
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of novel modified c-lactam prostanoids as EP4
subtype-selective agonists
⇑
Tohru Kambe , Toru Maruyama, Toshihiko Nagase, Seiji Ogawa, Chiaki Minamoto, Kiyoto Sakata,
Takayuki Maruyama, Hisao Nakai, Masaaki Toda
Minase Research Institute, Ono Pharmaceutical Co., Ltd, Shimamoto, Mishima, Osaka 618-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To identify chemically and metabolically stable subtype-selective EP4 agonists, design and synthesis of a
Received 7 November 2011
Revised 3 December 2011
Accepted 5 December 2011
Available online 13 December 2011
series of modified
2-oxo-1,3-thiazolidine and 5-thioxopyrrolidine as a surrogate for the
a troublesome 11-hydroxy group were identified as highly subtype-selective EP4 agonists. Among the
tested, several representative compounds demonstrated in vivo efficacy after oral dosing in rats. Their
pharmacokinetic and structure–activity relationship studies are presented.
Ó 2011 Elsevier Ltd. All rights reserved.
c
-lactam prostanoids has been continued. Prostanoids bearing 2-oxo-1,3-oxazolidine,
c-hydroxycyclopentanone without
Keywords:
Prostaglandin
EP4 receptor
Agonist
TNF-alpha
1. Introduction
structures have been reported as agonists for the EP2 and/or EP4
receptors with properties of increased chemical and metabolic
Prostaglandin E2 (PGE2) derived from arachidonic acid is one of
the most well-known prostanoids, and it exhibits a broad range of
biological actions in diverse tissues. These biological actions have
been known to mediate four subtypes of PGE2 receptor (EP1, EP2,
EP3 and EP4).1 Among them, the EP4 receptor subtype is of great
interest as a drug discovery target because of its important regula-
tory roles in numerous physiological processes, including broncho-
dilation, bone resorption, and inflammation.2 Activation of the EP4
receptor subtype increases the intracellular cAMP level, which is
linked to the treatment of diseases based on the above-described
mechanism of actions. Despite its high potency, PGE2, a natural
ligand for all of these receptor subtypes, shows strong nonselective
affinity to all the EP receptor subtypes.
stability.4–7 We also reported
c
-lactam prostanoids bearing the
16-(meta-substituted)phenyl
x-chain as a subtype-selective EP4
agonist and an EP2&EP4 dually selective agonist based on the
molecular design as schematically described in Figure 1.8–10 The
tertiary amide of the c-lactam moiety of II was assumed to have
a double bond character, which can retain the four atoms of the
7-, 8-, 9- and 10-positions (prostanoic acid numbering in Fig. 3)
in an identical plane, and was designed based on a hybrid structure
derived from an equilibrium mixture consisting of Ia and Ib. Based
O
S
Rα'
Natural PGs are susceptible to three major modes of metabolic
inactivation: oxidation of the 15-hydroxy group, b-oxidation of the
Rω
a-chain, and oxidation of the x-chain terminus. Furthermore, PGE2
HO
O
Rα
is chemically unstable since the 11-hydroxyl group can easily
undergo an elimination reaction, leading to PGA2.
Ia
N
X
Efforts to improve the chemical and/or metabolic instability of
PGE2 while maintaining the potency and the subtype selectivity
have been attempted. Previously, we reported 3,7-dithiaPGE ana-
logs as highly subtype-selective EP4 agonists, although they were
still susceptible to metabolism in addition to general chemical
OH
II
HO
S
Rα'
instability.3 Recently, many
c-lactam prostanoids and their related
Rω
HO
Ib
Figure 1. Reported template for EP4 receptor agonist.
⇑
Corresponding author. Tel.: +81 75 961 1151; fax: +81 75 962 9314.
E-mail addresses: kanbe@ono.co.jp, t-kam@iris.dti.ne.jp (T. Kambe).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.009

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 702–713
703
O
2. Chemistry
S
Rα
Rα
N
N
Synthesis of test compounds listed in Tables 2–4 is described in
Schemes 1–4. 2-Oxo-1,3-oxazolidine prostanoids 3–12 were syn-
thesized as outlined in Scheme 1a.
Y
X
X
OH
OH
Sodium borohydride reduction of an acid anhydride prepared
by the reaction of the commercially available 28 with isobutyl
chloroformate in the presence of N-methylmorpholine afforded
an alcohol 29 in good yield. Acidic deprotection of 29 with 4 N
hydrogen chloride in ethyl acetate followed by cyclization with
1,10-carbonyldiimidazole in the presence of triethylamine resulted
in a cyclic urethane 30. N-Alkylation of 30 with ethyl bromoacetate
in the presence of potassium tert-butoxide afforded 31. Sodium
borohydride reduction of 31 afforded an alcohol 32. O-Methane-
sulfonylation of 32 followed by the substitution reaction with
potassium thioacetate provided a thioacetate 33. Deacylation and
then S-alkylation of 33 with butyl 4-iodobutanoate followed by
acidic debenzylation with trifluoroacetic acid in the presence of
thioanisole produced 34, which was converted to prostanoids
3–12 by the following sequential reactions: (k) oxidation with
dimethyl sulfoxide in the presence of sulfur trioxide and N,N-diiso-
propylethylamine; (l) Horner–Emmons reaction with phosphonat-
es 35a–j in the presence of sodium hydride9; (m) stereoselective
reduction catalyzed with (R)-CBS11; and (n) alkaline hydrolysis.
Phosphonates 35h–i were prepared as described in Scheme 1b.
Cross-coupling reaction of the commercially available (3-bromo-
phenyl)acetic acid with phenylboronic acid in the presence of palla-
dium catalyst afforded 3-(phenyl)phenylacetic acid 36. Preparation
of the Weinreb amide from 36 followed by its reaction with the
anion prepared from dimethyl methylphosphonate in the presence
of n-butyl lithium afforded a phosphonate 35h. Another phospho-
nate 35i was prepared from (3-trifluoromethoxyphenyl)acetic acid
according to the same procedures as described above.
III
IV
a Y = O
b Y = S
Figure 2. New templates for EP4 receptor agonists.
O
X5
16
1CO2H
9 N8
10
Ar
11
OH
Figure 3. Numbering of the
c
-lactam prostanoid.
on the same concept as described above, 2-oxo-1,3-oxazolidine,
2-oxo-1,3-thiazolidine and 5-thioxopyrrolidine (Fig. 2) were also
considered to be surrogates for the
structures IIIa–b and IV were proposed as new templates for a sub-
type-selective EP4 agonist with increased chemical and/or meta-
bolic stability in addition to the
Herein, we report the synthesis and structure–activity relation-
ship (SAR) study of modified -lactam prostanoids IIIa–b and IV as
prostanoids bearing 2-oxo-1,3-oxazolidine, 2-oxo-1,3-thiazolidine
and 5-thioxopyrrolidine as surrogates for the -hydroxycyclopen-
tanone without a troublesome 11-hydroxy group as highly sub-
type-selective EP4 agonists. All of them were designed based on
the same synthetic concept as previously described for the
tam prostanoids since these modified -lactam prostanoids could
be synthesized as their optically active forms from commercially
available starting materials.
c-lactam moiety of II. Thus,
c-lactam prostanoids.
c
c
c-lac-
c
Synthesis of 2-oxo-1,3-thiazolidine prostanoids 13–18 were
synthesized as outlined in Scheme 2. Reaction of D-cysteine with tri-
phosgene in aqueous alkaline conditions followed by treatment
Table 1
Activity profiles of prostanoids bearing 16-(3-methoxymethyl)phenyl as a
x-chain moiety
X
CO2H
ring
Me
Binding assay (Ki, nM)
Functional assay EC50 (nM)
O
OH
Compound
ring
X
mEP1
>104
mEP2
>104
mEP3
>104
mEP4
10
rEP4
15
O
*
N
N
1
CH2
*
*
O
2
26
27
S
S
S
>104
>104
>104
8500
620
>104
8.0
0.7
2.4
1.3
1.4
1.1
*
O
*
*
56
HO
O
*
*
470
190

704
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 702–713
Table 2
Activity profiles of 2-oxo-1,3-oxazolidine prostanoids
O
S
CO2H
R
N
O
Binding assay (Ki, nM)
Functional assay EC50 (nM)
rEP4
OH
Compound
R
mEP1
mEP2
mEP3
mEP4
3
4
5
6
7
8
9
10
11
12
H
Me
Et
nPr
CF3
F
Cl
Ph
OCF3
CH2OMe
>104
4400
2400
>104
>104
1200
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
6400
2800
2700
1900
1600
4000
630
4.2
2.5
7.9
2.4
2.0
20
1.5
28
29
36
33
18
93
12
35
23
450
160
24
>104
1700
>104
7.2
Table 3
Activity profiles of 2-oxo-1,3-thiazolidine prostanoids
O
S
CO2H
R
N
S
Binding assay (Ki, nM)
Functional assay EC50 (nM)
rEP4
OH
Compound
R
mEP1
mEP2
mEP3
mEP4
13
14
15
16
17
18
H
Et
nPr
CF3
>104
4600
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
39
65
1200
280
1800
>104
0.65
0.88
0.98
0.97
1.5
4.8
1.9
6.1
4.3
5.7
2.4
F
CH2OMe
1.2
Table 4
Activity profiles of 5-thioxopyrrolidine prostanoids
S
CO2H
N
Binding assay (Ki, nM)
Functional assay EC50 (nM)
rEP4
R
OH
Compound
R
mEP1
mEP2
mEP3
mEP4
19
20
21
22
23
24
25
H
Et
nPr
CF3
>104
>104
>104
>104
>104
>104
2800
>104
1100
6800
>104
>104
1400
1700
2300
2300
2000
450
1700
6300
980
1.5
29
9.4
27
10
26
7.3
7.7
0.83
0.40
0.38
0.69
2.0
F
CH2OMe
Me (5-thia)
0.50
with diazomethane afforded 2-oxo-1,3-thiazolidine 37. Sodium
borohydride reduction of 37 provided an alcohol 38, which was con-
verted to 39 by the following sequential reactions: (d) O-protection
with tert-butyldimethylsilyl chloride (TBSCl); (e) N-alkylation with
ethyl bromoacetate in the presence of tert-butoxide; (f) sodium
borohydride reduction; (g) O-methanesulfonylation; and (h) substi-
tution reaction with potassium thioacetate. Deacetylation and then
S-alkylation of 39 with ethyl 4-bromobutanoate in the presence of
tert-butoxide followed by deprotection with tetrabutylammonium
fluoride (TBAF) afforded 40, which was converted to the prostanoids
13–18 according to the same procedure as described for the synthe-
sis of 3–12 from 34.
tion of which with TBAF provided 43. Conversion of 43 to their pro-
stanoid analogs was carried out according to the usual PG synthesis
resulting in 19–24.
The corresponding 5-thiaprostanoid analog 25 was synthesized
as shown in Scheme 4. Thiation of the amide oxygen of the
prostanoids 449 was carried out by using Lawesson’s reagent to
afford the corresponding thioamide 45, deprotection of which with
TBAF followed by alkaline hydrolysis resulted in 25.
3. Results and discussion
The binding assay was conducted according to the reported
method with minor modifications.12 The binding constants (Ki
values) for the mouse EP receptors (mEP1, mEP2, mEP3 and
mEP4) were determined by a competitive binding assay of the test
Synthesis of 5-thioxopyrrolidine prostanoids 19–24 is outlined
in Scheme 3. O-Protection of 418 with TBSCl followed by thiation
of the amide oxygen with Lawesson’s reagent afforded 42, deprotec-

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 702–713
705
(a)
O
NHBoc
OBn
e
NHBoc
OBn
c,d
a,b
NH
O
HO
OBn
HO2C
30
28
29
O
O
O
O
k, l, m, n
R
i, j
S
N
CH3
N
O
3 - 12
O
OBn
OH
34
31
R = CH2CO2Et
R = CH2CH2OH
R = CH2CH2SAc
f
32
33
g, h
Phosphonates:
f
a
H
F
X =
X
MeO
P
MeO
O
b
c
Me
Et
g
h
i
Cl
O
Ph
OCF3
35
d
nPr
e
CF3
j
CH2OMe
Scheme 1a. Synthesis of 3–12 reagents: (a) isobutyl chloroformate, N-methyl morpholine, DME; (b) NaBH4, H2O, 96% in two steps; (c) 4 N HCl, AcOEt, 79%;
(d) 1,10-carbonyldiimidazole, Et3N, THF, 66%; (e) ethyl bromoacetate, potassium tert-butoxide, DMF, 75%; (f) NaBH4, THF, MeOH, 94%; (g) MsCl, Et3N, THF; (h) potassium
thioacetate, K2CO3, DMF, 88%; (i) butyl 4-iodobutanoate, n-butanol, potassium tert-butoxide, DMF, 74%; (j) TFA, thioanisole, 52%; (k) SO3–Py, iPr2NEt, DMSO, AcOEt;
(l) phosphonates 35a–j, NaH, THF; (m) (R)-Me–CBS, BH3–THF, THF; (n) aq NaOH, MeOH, DME.
(b)
Br
b, c
a
MeO2C
35h
HO2C
36
b, c
OCF3
HO2C
35i
Scheme 1b. Preparation of phosphonates 35h–i. Reagents: (a) Phenylboronic acid, Pd(PPh3)4, Na2CO3, DME; (b) N,O-dimethylhydroxylamine hydrochloride, EDC–HCl, Et3N,
CH3CN; (c) dimethyl methylphosphonate, n-BuLi, toluene.
O
O
NH2
d, e, f, g, h
c
ClH
CO2H
a,b
NH
NH
HS
S
S
OH
CO2Me
38
37
O
O
O
CH3
S
i, j
S
N
N
O
S
13 - 18
S
O
OTBS
OH
40
39
Scheme 2. Synthesis of 13–18. Reagents: (a) Triphosgene, aq NaOH, dioxane; (b) CH2N2, AcOEt, 93% in two steps; (c) NaBH4, EtOH, 36%; (d) TBSCl, imidazole, DMF; (e) ethyl
bromoacetate, potassium tert-butoxide, THF; (f) NaBH4, THF, EtOH; (g) MsCl, Et3N, THF; (h) potassium thioacetate, K2CO3, DMF, 96% in five steps; (i) ethyl 4-bromobutanoate,
potassium tert-butoxide, THF; (j) TBAF, THF, 84% in two steps.
compounds using radiolabeled ligands such as [3H]PGE2. Prior to
the in vivo evaluation, the rat functional activity (EC50 values)
was conducted by a cell-based assay (EC50 for rEP4).
Results of the in vitro evaluation of the test compounds are
shown in Tables 1–4. As the standard compounds for SAR discus-
sion, activity profiles of c-lactam prostanoids 1, 2 and the cyclo-

706
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 702–713
O
O
O
S
O
S
c
a, b
CH3
N
O
CH3
N
N
O
O
OTBS
OH
OH
41
42
CH3
19 - 24
43
Scheme 3. Synthesis of 19–24. Reagents: (a) TBSCl, imidazole, DMF; (b) Lawesson’s reagent, toluene, 74%; (c) TBAF, THF, 80%.
O
O
S
O
c,d
S
S
a,b
CH3
N
O
CH3
N
O
25
CH3
CH3
OH
OTBS
44
45
Scheme 4. Synthesis of 25. Reagents: (a) TBSCl, imidazole, DMF, 79%; (b) Lawesson’s reagent, toluene; (c) TBAF, THF, 86% in two steps; (d) aq NaOH, MeOH, THF, 85%.
pentanone prostanoids 26–27 are described in Table 1. The
5-methylene -lactam analog 1 exhibited nearly equipotent activ-
rat functional activity of 6 relative to 3–5 and its relatively stronger
mouse receptor affinity was estimated to be due to the increased
lipophilicity of the 16-(3-propyl)phenyl moiety relative to those of
the 16-phenyl, 16-(3-methyl)phenyl and 16-(3-ethyl)phenyl moie-
ties since the higher lipophilicity causes more protein binding in the
cell-based assay. The 16-(3-fluoro)phenyl analog 8 did not show a
significant difference between the potency of the mouse EP4 binding
affinity and the rat functional activity. The significantly less potent
rat functional activities of 10 and 11 were considered to be due to
their weaker EP4 receptor affinities relative to those of the other
compounds.
c
ity and EP4 subtype selectivity with the corresponding 5-thia ana-
log 2 in the mouse binding assay, while 2 showed nearly 10-fold
more potency in the rat cell-based functional assay. As reported
previously,3 the 16-(3-methoxymethyl)phenyl moiety was the
most optimized 16-substituent (prostanoic acid numbering) in a
series of c-hydroxycyclopentanone prostanoids including 26. The
corresponding 11-deoxy analog 27 was also found to show almost
the same subtype selectivity and potency as 26 (in-house data).8
However, these two cyclopentanone analogs showed less subtype
selectivity relative to the
receptor affinity for EP2 and EP3, while they showed nearly the
same potency of the rat functional activity (EC50 values) as that
c
-lactam analogs owing to their increased
A series of 2-oxo-1,3-thiazolidine prostanoids were synthesized
also as their 5-thia analogs 13–18 and biologically evaluated. Results
are summarized in Table 3. It is of great interest to compare the
activity profiles of these 2-oxo-1,3-thiazolidine prostanoids with
their corresponding 2-oxo-1,3-oxazolidine prostanoids because of
their similarities in structures. The 16-phenyl analog 13 and
16-(3-ethyl)phenyl analog 14 showed very strong affinity for the
mouse EP4 receptor subtype while they showed increased affinity
for the mouse EP3 receptor subtype. The increased affinity of 13
and 14 seems to be one of the clear differences from the correspond-
of 2. Thus, both the c-lactam prostanoids 1 and 2 tended to show
better EP4 subtype selectivity than the cyclopentanone prosta-
noids 26–27, while the corresponding 5-thia analog 2 as well as
the cyclopentanone prostanoids 26–27 tended to show more po-
tency than the 5-methylene
EP4 functional activity.
c-lactam analog 1 in terms of rat
Keeping these results in mind, a series of 2-oxo-1,3-oxazolidine
prostanoids were synthesized as their 5-thia analogs and evaluated.
Results are shown in Table 2. The corresponding 16-substitutent
2-oxo-1,3-oxazolidine prostanoids was again optimized with
regard to this new template. As shown in Table 2, all the tested ana-
logs 3–12 exhibited good EP4 subtype selectivity and potent EP4
receptor affinity. Among them, 16-(3-fluoro)phenyl analog 8,
16-(3-phenyl)phenyl analog 10 and 16-(3-trifluoromethoxy)phenyl
analog 11 demonstrated significantly less potent receptor affinity
for the EP4 receptor subtype than 3–7, 9, and 12 because of
unknown reasons. Functional activities of these compounds in the
rat cell-based assay are also described in Table 2. Most of the com-
pounds showed 1.8–16-fold reduction in their potency of EC50 val-
ues in terms of the rat functional activity relative to the Ki values
of the mouse EP4 receptor affinity, while the 16-(3-propyl)phenyl
analog 6 showed remarkable reduction (38-fold) of the rat EC50
value relative to the Ki value of the mouse EP4 receptor affinity.
One of the plausible reasons for the remarkable reduction in the
ing 2-oxo-1,3-oxazolidine series
3 and 5, respectively. The
16-(3-propyl)phenyl analog 15 also exhibited very strong affinity
for the mouse EP4 receptor subtype with good subtype selectivity,
while its rat functional activity did not show such as remarkable
reduction as that of 6. The 16-(3-trifluoromethyl)phenyl analog 16
showed very strong affinity for the mouse EP4 receptor subtype with
slightly increased affinity for the mouse EP3 receptor subtype
relative to the corresponding 2-oxo-1,3-oxazolidine analog 7. The
16-(3-fluoro)phenyl analog 17 exhibited potent affinity for the
mouse EP4 receptor subtype with good subtype selectivity, while
it showed more increased mouse EP4 receptor affinity and rat func-
tional activity than 8. The 16-(3-methoxymethyl)phenyl analog 18
showed excellent activity profiles regarding both the mouse EP4
receptor affinity and the subtype selectivity, which are more potent
than the corresponding 2-oxo-1,3-oxazolidine analog 12. Activity
profiles of the most optimized structure in the mouse receptor sub-
type selectivity were found to be those of 18. All the analogs 13–18

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 702–713
707
Table 5
Biological evaluation and pharmacokinetic parameters of representative compounds
Compound
Rat TNF-
a
inhibition ED50 (mg/kg)
rEC50 (nM)
Pharmacokinetic parametersa
Cmax (ng/mL)
AUC (ng h/mL)
CLtot (mL/min/kg)
Vss (mL/kg)
B.A. (%)
1
4
7
13
14
15
16
18
24
25
1
>1
3
1
1
1
1
1
3
1
15
33
12
4.8
1.9
6.1
4.3
1.2
7.3
7.7
1.6b
5.4
3.8
6.4b
16.1
16.7
4.0
19.5
17.7
14.6
6.1
35
47
67
56
75
52
51
47
102
61
386
291
631
792
538
1280
512
325
530
1.2
4.5
6.7
3.5
8.6
6.3
5.5
2.0
12.3
9.8
2.7
10.4
11.4
5.3
2.6
6.5
20.6
29.9
3.4
1760
a
1 mg/kg, po and 0.1 mg/kg, iv.
Cmax and AUC were normalized to a dose of 1 mg/kg.
b
showed more potent rat functional activities relative to the corre-
sponding 2-oxo-1,3-oxazolidine analogs 3, 5–8 and 12, respectively.
dine prostanoids 24, 25 tended to show less in vivo potency. How-
ever, we could not find any clear SAR in their biological and PK
data. Further efforts to identify more optimized orally active EP4
selective agonists are being continued in our lab.
Replacement of the c-lactam carbonyl of 1 and 2 with a thiocar-
bonyl moiety afforded 5-thioxopyrrolidine analogs as illustrated
by 19–25 also with excellent affinity for the mouse EP4 receptor
subtype, good to excellent subtype selectivity and excellent rat
functional activity as shown in Table 4. This series of prostanoids
were first synthesized and evaluated as the 5-methylene analogs
since we wanted to avoid introduction of more than two sulfur
atoms into a compound because of the predicted oxidative metab-
olism. Also it was fortunate that all the synthesized 5-methylene
analogs 19–24 exhibited nearly equipotent mouse EP4 receptor
affinity and excellent subtype selectivity relative to the only syn-
5. Experimental
5.1. Chemistry
Analytical samples were homogeneous as confirmed by TLC,
and afforded spectroscopic results consistent with the assigned
structures. Proton nuclear magnetic resonance spectra (1H NMR)
were taken on a Varian Mercury 300 spectrometer, Varian GEM-
INI-200 or VXR-200s spectrometer using deuterated chloroform
(CDCl3), deuterated methanol (CD3OD) and deuterated dimethyl-
sulfoxide (DMSO-d6) as the solvent. Fast atom bombardment
(FAB-MS, HRMS) and electron ionization (EI) mass spectra were
obtained on a JEOL JMS-DX303HF spectrometer. Atmospheric pres-
sure chemical ionization (APCI) mass spectra were determined on a
HITACHI MI200H spectrometer. Infrared spectra (IR) were mea-
sured in a Perkin-Elmer FT-IR 1760X spectrometer. Melting points
and results of elemental analyses were uncorrected. Column chro-
matography was carried out on silica gel [Merck Silica Gel 60
thesized 5-thia analog 25 bearing a 16-(methyl)phenyl
x-chain.
All the tested compounds 19–25 tended to have less potent rat
functional activity relative to the corresponding 2-oxo-1,3-thiazol-
idine analogs.
As shown in Table 5, oral efficacy of the representative com-
pounds 1, 4, 7, 13–16, 18 and 24–25 selected based on their
in vitro activity profiles were evaluated for their inhibitory activity
of LPS-induced TNF-a production and pharmacokinetic (PK) param-
eters in rats. Rat functional assay results (rEC50 values) are again
listed for purposes of comparison. Their PK parameters are also
listed. Compounds 1, 13–18 and 25 had ED50 values of 1 mg/kg
and 4, 7 and 24 demonstrated less effective ED50 values after oral
dosing. We could not find any special SAR between their ED50 values
and their rat EC50 values. The parameters from the PK study
(1 mg/kg, po and 0.1 mg/kg, iv) are listed in Table 5. According to
the PK data described above, compounds 14 and 15 showed higher
Cmax values relative to the others. Compounds 4, 7, 14–16 and 24–25
showed higher oral exposure (AUC values). Relatively higher tissue
permeability (Vss values) and AUC values of 15 and 25 may suggest
that once compounds are distributed into the tissues, they circulate
systemically resulting in prolongation of their blood concentration.
Compound 24 exhibited the highest clearance (CLtot) while others
showed relatively lower CLtot values. Compounds 24 and 25 demon-
strated the best bioavailability (BA). However, we could not find any
clear SAR among the values of ED50, rEC50, Cmax, AUC, CLtot, Vss and
BA, as systemic effects were estimated to result in a hybrid of EC50
values and PK parameters.
(0.063–0.200 lm), 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; diethyl ether (Et2O), N,N-dimethyl-
formamide (DMF), dimethylsulfoxide (DMSO), ethanol (EtOH),
ethyl acetate (EtOAc), methanol (MeOH), tetrahydrofuran (THF),
methanol (MeOH), dichloromethane (CH2Cl2), chloroform (CHCl3),
dimethoxyethane (DME), acetonitrile (CH3CN), sulfur trioxide/pyr-
idine complex (SO3–Py), 4-(dimethylamino)pyridine (DMAP), tet-
rabutylammonium fluoride (TBAF), tert-butyldimethylsilyl (TBS)
chloride.
5.1.1. (R)-2-(tert-Butoxycarbonylamino)-3-benzyloxypropanol (29)
To a stirred solution of 28 (9.28 g, 31.7 mmol) and N-methyl
morpholine (3.7 mL, 33.7 mmol) in DME (70 mL) was added isobu-
tyl chloroformate (4.3 mL, 33.3 mmol) at ꢀ20 °C under argon atmo-
sphere. After being stirred for 30 min at 0 °C, the reaction mixture
was filtered to remove the resulting precipitates. To a cooled sus-
pension of sodium borohydride (2.5 g, 66.5 mmol) in water
(30 mL) was added the filtrate at 0 °C. Stirring was continued for
additional 30 min at the same temperature. The reaction mixture
was diluted with EtOAc, washed with hydrochloric acid, water,
brine, and dried over Na2SO4. The organic layer was removed by
evaporation to give an alcohol 29 as a colorless oil (8.47 g, 96%).
1H NMR (300 MHz, CDCl3): d 1.43 (s, 9H), 3.60–3.83 (m, 6H), 4.54
(s, 2H), 5.17 (br s, 1H), 7.40–7.28 (m, 5H).
4. Conclusion
In conclusion, the 2-oxo-1,3-oxazoline, 2-oxo-1,3-thiazolidine
and 5-thioxopyrrolidine were presented as candidates of the surro-
gate for the cyclopentanone and the c-lactam frameworks.
Among the tested compounds, all the optimized 2-oxo-1,3-thia-
zolidine prostanoids 13–18 were efficacious at 1 mg/kg (oral dosing)
while 2-oxo-1,3-oxazolidine prostanoids 4, 7 and 5-thioxopyrroli-

708
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 702–713
5.1.2. (4S)-4-[(Benzyloxy)methyl]-1,3-oxazolidin-2-one (30)
To a stirred solution of 29 (8.56 g, 30.3 mmol) in EtOAc (10 ml)
was added 4 N hydrogen chloride/EtOAc (30 mL) at 0 °C under
argon atmosphere, and the stirring was continued for 3 h. The
reaction mixture was evaporated. The remaining organic solvent
was further removed by repeated evaporation with toluene. To
the resulting residue was added EtOAc/hexane (1:5, 10 mL) and
the resulting mixture was stirred for 1 h. The resulting precipitates
were removed by filtration to afford an amino alcohol hydrochlo-
ride as a white powder (5.23 g, 79%). 1H NMR (300 MHz, DMSO-
d6): d 8.15 (m, 3H), 7.38–7.25 (m, 5H), 5.32 (m, 1H), 4.55 (s, 2H),
3.65–3.52 (m, 4H).
To a stirred solution of the above-described amino alcohol
hydrochloride (5.13 g, 23.6 mmol) and triethylamine (6.9 mL,
49.4 mmol) in THF (100 mL) was added 1,10-carbonyldiimidazole
(4.6 g, 28.3 mmol) at room temperature under argon atmosphere.
After being stirred for 2.5 h, the reaction mixture was diluted with
EtOAc, washed with water, brine, and dried over Na2SO4. The or-
ganic layer was evaporated, and the resulting residue was purified
by column chromatography on silica gel (EtOAc) to give 30 as a col-
orless oil (3.23 g, 66%). 1H NMR (300 MHz, CDCl3): d 3.44–3.48 (m,
2H), 4.04 (m, 1H), 4.14 (m, 1H), 4.55 (m, 1H), 4.56 (s, 2H), 5.88 (br s,
1H), 7.28–7.40 (m, 5H).
evaporated to afford a thioacetate 33 as a pale brown oil (2.93 g,
83% in two steps). 1H NMR (300 MHz, CDCl3): d 2.36 (s, 3H), 3.34
(m, 1H), 3.51–3.63 (m, 3H), 4.04 (m, 1H), 4.12 (m, 1H), 4.35 (m,
1H), 4.57 (s, 2H), 7.27–7.40 (m, 5H).
5.1.6. Butyl 4-({2-[(4S)-4-(hydroxymethyl)-2-oxo-1,3-
oxazolidin-3-yl]ethyl}thio)butanoate (34)
To a stirred solution of thioacetate 33 (2.92 g, 9.40 mmol) in
butanol (6 mL) and DMF (6 mL) was added potassium tert-butox-
ide (1.27 g, 11.3 mmol) and n-butyl 4-iodobutanoate (3.57 g,
13.2 mmol) at 0 °C under argon atmosphere. After being stirred
at room temperature for 2 h, the reaction was quenched with sat-
urated aq NH4Cl. The reaction mixture was extracted with EtOAc,
washed with H2O (ꢁ2), brine, dried over MgSO4 and evaporated
to give an ester as a brown oil (2.84 g). A solution of the above-de-
scribed ester in thioanisole (5 mL) was treated with trifluoroacetic
acid (5 mL) at 50 °C under argon atmosphere for 10 h. The reaction
mixture was evaporated. The resulting residue was purified by col-
umn chromatography on silica gel (EtOAc) to give a desilylated es-
ter 34 as a colorless oil (1.56 g, 52%). 1H NMR (300 MHz, CDCl3): d
0.95 (t, J = 7.4 Hz, 3H), 1.43–1.47 (m, 2H), 1.59–1.68 (m, 2H), 1.89–
2.00 (m, 2H), 2.43 (t, J = 7.1 Hz, 2H), 2.64 (t, J = 7.1 Hz, 2H), 2.74–
2.95 (m, 2H), 3.40 (m, 1H), 3.55 (m, 1H), 3.64 (m, 1H), 3.83 (m,
1H), 3.93 (m, 1H), 4.08 (t, J = 6.9 Hz, 2H), 4.25 (m, 1H), 4.38 (m, 1H).
5.1.3. Ethyl {(4S)-4-[(benzyloxy)methyl]-2-oxo-1,3-oxazolidin-3-
yl}acetate (31)
5.1.7. 4-[(2-{(4S)-4-[(1E,3S)-3-Hydroxy-4-phenylbut-1-enyl]-2-
oxo-1,3-oxazolidin-3-yl}ethyl)sulfanyl]butanoic acid (3)
To a stirred solution of 30 (3.22 g, 15.5 mmol) and ethyl bromo-
acetate (2.1 mL, 18.6 mmol) in DMF (35 mL) was added potassium
tert-butoxide (2.1 g, 18.6 mmol) at 0 °C under argon atmosphere.
After being stirred for 3 h at room temperature, the reaction was
quenched with saturated aq NH4Cl. The reaction mixture was ex-
tracted with EtOAc (ꢁ3). The combined organic layers were
washed with H2O (ꢁ2), brine, dried over MgSO4 and evaporated.
The resulting residue was purified by column chromatography on
silica gel (hexane/EtOAc, 1:2) to give an ester 31 as a colorless oil
(3.43 g, 75%). 1H NMR (300 MHz, CDCl3): d 1.23 (t, J = 7.2 Hz, 3H).
3.52 (m, 1H), 3.60 (m, 1H), 4.05 (m, 1H), 4.14 (m, 1H), 4.15 (q,
J = 7.2 Hz, 2H), 4.23 (m, 1H), 4.44 (m, 1H), 4.47 (m, 1H), 4.57 (s,
2H), 7.25–7.40 (m, 5H).
To a stirred solution of the alcohol 34 (500 mg, 1.57 mmol) in
EtOAc (3 mL) and N,N-diisopropylethylamine (0.98 mL, 9.36 mmol)
was added a solution of SO3–Py (750 mg, 4.71 mmol) in DMSO
(2.5 mL) at 0 °C under argon atmosphere. After being stirred at
the same temperature for 30 min, the reaction was quenched with
1 N hydrochloric acid. The reaction mixture was diluted with
EtOAc, washed with brine, dried over MgSO4 and evaporated to
yield the corresponding aldehyde as a yellow oil. To a stirred solu-
tion of a phosphonate 35a (455 mg, 1.88 mmol) in THF (15 mL)
was added sodium hydride (62.7% in mineral oil, 73.2 mg,
1.88 mmol) in several portions at 0 °C under argon atmosphere
and stirring was further continued at ambient temperature for
90 min. To the reaction mixture was added a solution of the
above-described aldehyde in THF (15 mL) at 0 °C and stirring was
continued at room temperature for 20 min. The reaction was
quenched with acetic acid. The reaction mixture was diluted with
EtOAc, washed with aq NaHCO3, water, brine, dried over MgSO4
and evaporated to give an enone as a pale yellow oil.
To a stirred solution of the above-described enone (285 mg) in
THF (5 mL) was added a solution of (R)-2-methyl-CBS-oxazaboroli-
dine (1.0 M in toluene, 0.24 mL, 0.24 mmol) at room temperature
under argon atmosphere. To the reaction mixture was added drop-
wise a solution of borane–THF complex (1.0 M in THF, 0.49 mL,
0.49 mmol) over 5 min. The resulting solution was stirred for 1 h,
and then treated with MeOH (0.3 mL). Stirring was continued for
additional 5 min. The reaction mixture was diluted with EtOAc,
washed with 1 N hydrochloric acid, water, aq NaHCO3, brine, and
dried over Na2SO4. After evaporation, the resulting residue was
purified by column chromatography on silica gel (EtOAc) to give
an allyl alcohol as a colorless oil.
5.1.4. (4S)-4-[(Benzyloxy)methyl]-3-(2-hydroxyethyl)-1,3-oxazo
lidin-2-one (32)
To a stirred solution of 31 (3.43 g, 11.7 mmol) in MeOH (10 mL)
and THF (50 mL) was added sodium borohydride (1.1 g,
29.2 mmol) at 0 °C under argon atmosphere. After being stirred
at room temperature for 3 h, the reaction was quenched with
H2O. The reaction mixture was extracted with EtOAc (ꢁ3). The
combined organic layers were washed with brine, and dried over
MgSO4. Removal of the solvent by evaporation gave an alcohol
32 as a colorless oil (2.75 mg, 94%). 1H NMR (300 MHz, CDCl3): d
3.36–3.63 (m, 4H), 3.72–3.88 (m, 2H), 4.06 (m, 1H), 4.12 (m, 1H),
4.38 (m, 1H), 4.57 (s, 2H), 7.28–7.40 (m, 5H).
5.1.5. S-(2-{(4S)-4-[(Benzyloxy)methyl]-2-oxo-1,3-oxazolidin-3-
yl}ethyl) ethanethioate (33)
To a stirred solution of 32 (2.72 g, 10.8 mmol) in THF (20 mL)
and triethylamine (2.1 mL, 15.2 mmol) was added methanesulfo-
nyl chloride (1.0 mL, 13.0 mL) at 0 °C under argon atmosphere.
Stirring was continued at room temperature for 30 min. To the
reaction mixture was added potassium carbonate (3.0 g,
A solution of the above-described allyl alcohol in EtOH (3 mL),
DME (1 mL) and 2 N sodium hydroxide (0.5 mL) was stirred at
ambient temperature for 16 h. After neutralization with 2 N hydro-
chloric acid (0.5 mL) under cooling, the reaction mixture was ex-
tracted with EtOAc three times, and the combined organic layers
were washed with brine, dried over Na2SO4 and evaporated. The
resulting residue was purified by column chromatography on silica
gel (CHCl3/MeOH, 50:1–20:1) to afford 3 as a white powder
21.6 mmol) and
a solution of potassium thioacetate (2.5 g,
21.6 mmol) in DMF (50 mL). Stirring was continued at 50 °C for
additional 90 min. After being cooled to room temperature, the
reaction was quenched with 1 N hydrochloric acid. The reaction
mixture was extracted with EtOAc twice. The combined organic
layers were washed with H2O twice, brine, dried over MgSO4 and

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 702–713
709
(154 mg, 24% in four steps): 1H NMR (300 MHz, CDCl3): d 1.88–1.92
(m, 2H), 2.60–2.64 (m, 8H), 3.06 (m, 1H), 3.45 (m, 1H), 3.89 (dd,
J = 8.1, 6.8 Hz, 1H), 4.30–4.39 (m, 3H), 5.55 (m, 1H), 5.89 (dd,
J = 15.6, 5.3 Hz, 1H), 7.24–7.30 (m, 5H); IR (film): 3454, 3026,
2911, 1729, 1704, 1474, 1437, 1217, 1141, 1095, 1021, 874, 764,
697, 484 cmꢀ1; FAB-MS (m/z): 380 (M+H)+; HRMS-FAB (m/z):
[M+H]+ calcd for C19H26NO5S, 380.1532; found, 380.1527.
(m, 1H), 3.90 (dd, J = 8.1, 6.6 Hz, 1H), 4.34–4.42 (m, 3H), 5.60 (m,
1H), 5.88 (m, 1H), 6.97 (m, 3H), 7.30 (m, 1 H); IR (film): 3410,
2925, 1729, 1631, 1602, 1501, 1436, 1292, 1153, 1047, 953 cmꢀ1
;
FAB-MS (m/z): 398 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for
19H25FNO5S, 398.1437; found, 398.1436.
C
5.1.13. 4-[(2-{(4S)-4-[(1E,3S)-4-(3-Chlorophenyl)-3-hydroxybut-
1-enyl]-2-oxo-1,3-oxazolidin-3-yl}ethyl)sulfanyl]butanoic acid
(9)
5.1.8. 4-[(2-{(4S)-4-[(1E,3S)-3-Hydroxy-4-(3-methylphenyl)but-1-
enyl]-2-oxo-1,3-oxazolidin-3-yl}ethyl)sulfanyl]butanoic acid (4)
Compound 4 was prepared from 34 according to the same pro-
cedure as described for the preparation of 3 from 34 as a pale yel-
low oil (43% in four steps): 1H NMR (300 MHz, CDCl3): d 1.85–1.96
(m, 2H), 2.36 (s, 3H), 2.44–2.89 (m, 8H), 3.11 (m, 1H), 3.45 (m, 1H),
3.91 (dd, J = 8.2, 8.0 Hz, 1H), 4.28–4.51 (m, 3H), 5.57 (ddd, J = 15.4,
8.8, 1.4 Hz, 1H), 5.90 (dd, J = 15.4, 5.2 Hz, 1H), 6.97–7.11 (m, 3H),
7.22 (t, J = 7.4 Hz, 1H); IR (film): 3410, 1732, 1420, 1235, 1028,
761, 704 cmꢀ1; FAB-MS (m/z): 394 (M+H)+; HRMS-FAB (m/z):
[M+H]+ calcd for C20H28NO5S, 394.1688; found, 394.1681.
Compound 9 was prepared from 34 according to the same pro-
cedure as described for the preparation of 3 from 34 as a pale yel-
low oil (11% in four steps): 1H NMR (300 MHz, CDCl3): d 1.87–1.98
(m, 2H) 2.44–2.77 (m, 6H), 2.80–2.84 (m, 2H), 3.10 (m, 1H), 3.46
(m, 1H), 3.89 (dd, J = 8.5, 8.2 Hz, 1H), 4.43 (dd, J = 8.5, 8.2 Hz, 1H),
4.29–4.50 (m, 2H), 5.56 (ddd, J = 15.4, 8.5, 1.4 Hz, 1H), 5.88 (dd,
J = 15.4, 5.2 Hz, 1H), 7.10 (m, 1H), 7.20–7.32 (m, 3H); IR (film):
2922, 1731, 1599, 1478, 1429, 1081, 784, 704 cmꢀ1; FAB-MS (m/
z): 414 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C19H25ClNO5S,
414.1142; found, 414.1158.
5.1.9. 4-[(2-{(4S)-4-[(1E,3S)-4-(3-Ethylphenyl)-3-hydroxybut-1-
enyl]-2-oxo-1,3-oxazolidin-3-yl}ethyl)sulfanyl]butanoic acid (5)
Compound 5 was prepared from 34 according to the same pro-
cedure as described for the preparation of 3 from 34 as a pale yel-
low oil (50% in four steps): 1H NMR (300 MHz, CDCl3): d 1.24 (t,
J = 7.5 Hz, 3H), 1.87–1.90 (m, 2H), 2.60–2.70 (m, 9H), 3.10 (m,
1H), 3.40–3.45 (m, 2H), 3.91 (m, 1H), 4.38–4.42 (m, 3H), 5.58
(ddd, J = 15.4, 8.5, 1.2 Hz, 1H), 5.90 (dd, J = 15.4, 5.3 Hz, 1H),
6.98–7.01 (m, 2H), 7.11 (m, 1H), 7.23 (m, 1H); IR (film): 3412,
2963, 2928, 2360, 1732, 1606, 1540, 1444, 1419, 1312, 1232,
1159, 1087, 1026, 976, 912, 797, 762, 704, 667, 517, 472, 456 cm
ꢀ1; FAB-MS (m/z): 408 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd
for C21H30NO5S, 408.1845; found, 408.1847.
5.1.14. 3-Biphenylacetic acid (36)
To a stirred solution of (3-bromo)phenylacetic acid (4.00 g,
18.26 mmol), Na2CO3 (2.13 g, 20.1 mmol) and phenylborinic acid
(2.45 g, 20.1 mmol) in DME (45 mL) was added tetrakis-triphenyl-
phosphine palladium (200 mg) at room temperature under argon
atmosphere. After being stirred at 100 °C for 16 h, the reaction
mixture was cooled to room temperature and filtered through Cel-
ite to remove the precipitates. The filtrate was diluted with EtOAc,
washed with 2 N hydrochloric acid, brine, dried over MgSO4 and
evaporated to afford 36 as a pale yellow powder (3.82 g, 99%). 1H
NMR (300 MHz, CDCl3): d 3.72 (s, 2H), 7.25–7.62 (m, 9H).
5.1.15. 3-(3-Biphenyl)-2-oxopropanephosphonate (35h)
To a stirred solution of N,O-dimethyl hydroxylamine hydrochlo-
ride (2.64 g, 27.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (4.49 g, 27.0 mmol) and triethylamine
(3.76 mL, 27.0 mmol) in CH3CN (30 mL) was added a solution of
36 (3.82 g, 18.0 mmol) in CH3CN (20 mL) at room temperature un-
der argon atmosphere. After being stirred for 1 h, the reaction was
quenched with water. The reaction mixture was diluted with
EtOAc, washed with 2 N hydrochloric acid, water, then brine, dried
over MgSO4 and evaporated to give a Weinreb amide as a colorless
oil. 1H NMR (300 MHz, CDCl3) d: 7.43–7.27 (m, 9H), 3.84 (s, 2H),
3.63 (s, 3H), 3.21 (s, 3H).
5.1.10. 4-[(2-{(4S)-4-[(1E,3S)-3-Hydroxy-4-(3-propylphenyl)but-1-
enyl]-2-oxo-1,3-oxazolidin-3-yl}ethyl)sulfanyl]butanoic acid (6)
Compound 6 was prepared from 34 according to the same proce-
dure as described for the preparation of 3 from 34 as a pale yellow oil
(22% in four steps): 1H NMR (300 MHz, CDCl3): d 0.94 (t, J = 7.3 Hz,
3H) 1.63–1.67 (m, 2H), 1.89–1.92 (m, 2H), 2.55–2.62 (m, 10H),
3.10 (m, 1H), 3.45 (m, 1H), 3.90 (dd, J = 8.1, 6.8 Hz, 1H), 4.30–4.39
(m, 3H), 5.58 (m, 1H), 5.90 (dd, J = 15.5, 5.2 Hz, 1H), 7.00–7.04 (m,
3H), 7.22 (m, 1H); IR (film): 3423, 3022, 2928, 2871, 1731, 1444,
1234, 1027 cmꢀ1; FAB-MS (m/z): 422 (M+H)+; HRMS-FAB (m/z):
[M+H]+ calcd for C22H32NO5S, 422.2001; found, 422.2001.
To a stirred solution of dimethyl methylphosphonate (2.32 g,
18.7 mmol) in toluene (40 mL) was added dropwise a solution of
n-BuLi (1.57 M in hexane, 11.9 mL, 18.7 mmol) at ꢀ78 °C under ar-
gon atmosphere, and stirring was continued for 1 h at the same
temperature. To the reaction mixture was added a solution of the
above-described Weinreb amide (18.0 mmol) in toluene (13 mL),
and stirring was continued for additional 2 h at the same temper-
ature. The reaction was quenched with acetic acid. The reaction
mixture was allowed to warm up to room temperature with stir-
ring, diluted with EtOAc, washed with water, then brine, dried over
MgSO4 and evaporated. The resulting residue was purified by col-
umn chromatography on silica gel (hexane/EtOAc, 2:1–0:1) to give
a phosphonate 35h as a colorless oil (3.12 g, 73% in two steps). 1H
NMR (300 MHz, CDCl3): d 3.14 (d, J = 22.8 Hz, 2H), 3.70 (s, 2H), 3.76
(s, 3H), 3.82 (s, 3H), 7.20–7.62 (m, 9H).
5.1.11. 4-{[2-((4S)-4-{(1E,3S)-3-Hydroxy-4-[3-(trifluoromethyl)
phenyl]but-1-enyl}-2-oxo-1,3-oxazolidin-3-yl)ethyl]sulfanyl}
butanoic acid (7)
Compound 7 was prepared from 34 according to the same pro-
cedure as described for the preparation of 3 from 34 as a pale yel-
low oil (10% in four steps). 1H NMR (300 MHz, CDCl3): d 1.88–1.90
(m, 2H), 2.50–2.57 (m, 6H), 2.89–2.92 (m, 2H), 3.10 (m, 1H), 3.46
(m, 1H), 3.88 (dd, J = 8.1, 6.4 Hz, 1H), 4.42 (m, 3H), 5.58 (m, 1H),
5.90 (dd, J = 15.4, 5.1 Hz, 1H), 7.45 (m, 4H); IR (film): 3407, 2924,
1732, 1469, 1447, 1435, 1330, 1190, 1121, 1074, 1026, 977, 801,
763, 705 cmꢀ1; FAB-MS (m/z): 448 (M+H)+; HRMS-FAB (m/z):
[M+H]+ calcd for C20H25F3NO5S, 448.1408; found, 448.1407.
5.1.12. 4-[(2-{(4S)-4-[(1E,3S)-4-(3-Fluorophenyl)-3-hydroxybut-
1-enyl]-2-oxo-1,3-oxazolidin-3-yl}ethyl)sulfanyl]butanoic acid
(8)
Compound 8 was prepared from 34 according to the same pro-
cedure as described for the preparation of 3 from 34 as a pale yel-
low oil (10% in four steps): 1H NMR (300 MHz, CDCl3): d 1.90–1.94
(m, 2 H), 2.55–2.68 (m, 6 H), 2.80–2.91 (m, 2H), 3.10 (m, 1H), 3.44
5.1.16. 4-[(2-{(4S)-4-[(1E,3S)-4-(1,10-Biphenyl-3-yl)-3-hydroxy
but-1-enyl]-2-oxo-1,3-oxazolidin-3-yl}ethyl)sulfanyl]butanoic
acid (10)
Compound 10 was prepared from 34 according to the same pro-
cedure as described for the preparation of 3 from 34 as a pale yel-

710
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 702–713
low oil (40% in four steps): 1H NMR (300 MHz, CDCl3): d 1.84–1.90
(m, 2H), 2.48–2.53 (m, 6H), 2.92–3.01 (m, 4H), 3.40 (m, 1H), 3.88
(m, 1H), 4.28–4.35 (m, 2H), 4.51 (m, 1H), 5.55 (dd, J = 15.4,
8.6 Hz, 1H), 5.90 (dd, J = 15.4, 5.5 Hz, 1H), 7.17 (m, 1H), 7.49 (m,
8H); IR (film): 3438, 3032, 2921, 1732, 1439, 1419, 1232, 1086,
1024, 976, 759, 729, 703 cmꢀ1; FAB-MS (m/z): 456 (M+H)+.
5.1.22. S-{2-[(4S)-4-(tert-Butylsilyloxymethyl)-2-oxo-1,3-thiaz
olidin-3-yl]ethyl} ethanethioate (39)
To a stirred solution of 37 (1.49 g, 11.2 mmol) was added TBS
chloride (1.85 g, 12.3 mmol) and imidazole (1.14 g, 16.8 mmol) in
DMF (15 mL) at 0 °C under argon atmosphere. After being stirred
at room temperature for 30 min, the reaction was quenched with
water. The reaction mixture was diluted with EtOAc. The organic
layer was washed with aq NaHCO3, brine, dried over MgSO4 and
evaporated to give a TBS ether as a colorless oil.
5.1.17. 3-(3-Trifluoromethoxyphenyl)-2-oxopropanephospho
nate (35i)
Compound 35i was prepared from (3-trifluoromethoxyphe-
nyl)acetic acid according to the same procedure as described for
the preparation of 35h from 36 as a colorless oil (58% in two steps).
1H NMR (300 MHz, CDCl3): d 3.13 (d, J = 21.0 Hz, 2H), 3.78 (s, 3H),
3.82 (s, 3H), 3.94 (s, 2H), 7.04–7.21 (m, 3H), 7.37 (m, 1H).
To a stirred solution of the above-described TBS ether in THF
(15 mL) was added potassium tert-butoxide (1.38 g, 12.3 mmol)
at 0 °C under argon atmosphere, and then stirring was continued
at 0 °C for 15 min. To the stirred suspension was added ethyl bro-
moacetate (2.05 g, 12.3 mmol) at 0 °C and stirring was continued at
room temperature for additional 1 h. The reaction was quenched
with aqueous NH4Cl. The reaction mixture was extracted with
EtOAc three times. The combined organic layers were washed with
H2O (ꢁ2), brine, dried over MgSO4 and evaporated to give an ester
as a colorless oil (3.97 g).
To a stirred solution of the above-described ester in MeOH
(5 mL) and THF (50 mL) was added sodium borohydride (509 mg,
13.4 mmol) at 0 °C under argon atmosphere. After being stirred
at room temperature for 3 h, the reaction was quenched with aq
NH4Cl. The reaction mixture was extracted with EtOAc (ꢁ3). The
combined organic layers were washed with brine, dried over
MgSO4 and evaporated to give an alcohol as a colorless oil
(3.28 g). 1H NMR (300 MHz, CDCl3): d 0.09 (s, 6H), 0.91 (s, 9H),
2.50 (m, 1H), 3.18 (m, 1H), 3.38–3.48 (m, 2H), 3.64 (m, 1 H),
3.71–3.84 (m, 4H), 3.92–4.01 (m, 1H).
To a stirred solution of the above-described alcohol in THF
(15 mL) and triethylamine (2.34 mL, 16.8 mmol) was added meth-
anesulfonyl chloride (0.91 mL, 11.8 mL) at 0 °C under argon atmo-
sphere. Stirring was continued at 0 °C for 1 h. To the reaction
mixture was added potassium carbonate (341 mg, 2.47 mmol)
and a solution of potassium thioacetate (1.35 g, 11.8 mmol) in
DMF (20 mL) and the stirring was continued at 50 °C for additional
16 h. After being cooled to room temperature, the reaction was
quenched with 1 N hydrochloric acid. The reaction mixture was ex-
tracted with EtOAc (ꢁ2). The combined organic layers were
washed with H2O (ꢁ2), brine, dried over MgSO4 and evaporated
to afford a thioacetate 39 as a pale yellow oil (3.78 g, 96% in five
steps). 1H NMR (300 MHz, CDCl3): d 0.09 (s, 6H), 0.90 (s, 9H),
2.36 (s, 3H), 3.02–3.09 (m, 2H), 3.14–3.32 (m, 2H), 3.38 (dd,
J = 11.1, 7.8 Hz, 1H), 3.66–3.84 (m, 3H), 3.93 (m, 1H).
5.1.18. 4-{[2-((4S)-4-{(1E,3S)-3-Hydroxy-4-[3-(trifluoromethoxy
)phenyl]but-1-enyl}-2-oxo-1,3-oxazolidin-3-yl)ethyl]sulfanyl}
butanoic acid (11)
Compound 11 was prepared from 34 according to the same pro-
cedure as described for the preparation of 3 from 34 as a pale yel-
low oil (27% in four steps): 1H NMR (300 MHz, CDCl3): d 1.88–1.91
(m, 2H), 2.55–2.59 (m, 6H), 2.85–2.89 (m, 2H), 3.10 (m, 1H), 3.46
(m, 1H), 3.89 (m, 1H), 4.37–4.44 (m, 3H), 5.59 (ddd, J = 15.4, 8.51,
1.4 Hz, 1H), 5.89 (dd, J = 15.4, 5.3 Hz, 1H), 7.11 (m, 3H), 7.36 (m,
1H); IR (film): 3734, 2925, 2360, 2342, 1869, 1733, 1654, 1614,
1589, 1488, 1446, 1420, 1259, 1217, 1161, 1087, 1026, 1003,
976, 763 cmꢀ1; FAB-MS (m/z): 464 (M+H)+.
5.1.19. 4-{[2-((4S)-4-{(1E,3S)-3-Hydroxy-4-[3-(methoxymethyl)
phenyl]but-1-enyl}-2-oxo-1,3-oxazolidin-3-yl)ethyl]sulfanyl}
butanoic acid (12)
Compound 12 was prepared from 34 according to the same pro-
cedure as described for the preparation of 3 from 34 as a pale yel-
low oil (18% in four steps). 1H NMR (300 MHz, CDCl3): d 1.88–1.92
(m, 2H), 2.40–2.65 (m, 8H), 3.20 (m, 1H), 3.39 (m, 1H), 3.43 (s, 3H),
3.92 (m, 1H), 4.41 (m, 3H), 4.47 (s, 2H), 5.63 (m, 1H), 5.92 (dd,
J = 15.4, 4.9 Hz, 1H), 7.24 (m, 4H); IR (film): 3842, 3411, 2924,
1732, 1421, 1384, 1311, 1233, 1158, 1087, 1026, 976, 892, 762,
704 cmꢀ1; FAB-MS (m/z): 424 (M+H)+; HRMS-FAB (m/z): [M+H]+
calcd for C21H30NO6S, 424.1794; found, 424.1798.
5.1.20. Methyl (4S)-2-oxo-1,3-thiazolidine-4-carboxylate (37)
To
a stirred solution of D-cysteine hydrochloride (5.26 g,
30.0 mmol) in 1 N sodium hydroxide (90 mL) was added a solution
of triphosgene (8.88 g, 30.0 mmol) in 1,4-dioxane (60 mL) at 0 °C
under argon atmosphere. After being stirred at the same tempera-
ture for 1 h, the reaction mixture was evaporated. To the resulting
residue was added warm acetonitrile. The resulting precipitates
were removed by filtration and the filtrate was evaporated. A solu-
tion of the resulting residue in EtOAc was treated with diazometh-
ane to give 37 (4.49 g, 93%) as a yellow oil. 1H NMR (300 MHz,
CDCl3): d 3.64 (dd, J = 11.4, 5.3 Hz, 1H), 3.68–3.81 (m, 1H), 3.83
(s, 3H), 4.45 (dd, J = 7.9, 5.3 Hz, 1H), 6.05 (m, 1H).
5.1.23. Ethyl 4-({2-[(4S)-4-(hydroxymethyl)-2-oxo-1,3-thiazoli
din-3-yl]ethyl}thio) butanoate (40)
To a stirred solution of thioacetate 39 (3.78 g, 10.8 mmol) in
EtOH (10 mL) and THF (10 mL) was added potassium tert-butoxide
(1.46 g, 13.0 mmol) and ethyl 4-bromobutyrate (2.54 g,
13.0 mmol) at 0 °C under argon atmosphere. After being stirred
at room temperature for 2 h, the reaction was quenched with aq
NH4Cl. The reaction mixture was extracted with EtOAc, washed
with water (ꢁ2), brine, dried over MgSO4 and evaporated to give
a sulfide as a yellow oil.
A solution of the above-described sulfide in THF (10 mL) was
treated with TBAF (16.2 mL, 1 M in THF, 16.2 mmol) at room tem-
perature under argon atmosphere for 1 h. After addition of brine,
the reaction mixture was extracted with EtOAc (ꢁ3). The combined
organic layers were dried over MgSO4 and evaporated. The result-
ing residue was purified by column chromatography on silica gel
(hexane/EtOAc, 1:1–0:1) to give an alcohol 40 as a colorless oil
(2.78 g, 84%). 1H NMR (300 MHz, CDCl3): d 1.27 (t, J = 7.1 Hz, 3H),
1.86–1.99 (m, 2H), 2.39–2.45 (m, 3H), 2.60 (t, J = 7.4 Hz, 2H), 2.79
(t, J = 6.6 Hz, 2H), 3.22–3.33 (m, 1H), 3.35–3.47 (m, 2H), 3.58–
3.80 (m, 2H), 3.82–4.00 (m, 2H), 4.13 (q, J = 7.1 Hz, 2H).
5.1.21. (4S)-4-(Hydroxymethyl)-1,3-thiazolidin-2-one (38)
To a stirred solution of 37 (5.30 g, 32.9 mmol) in EtOH (50 mL)
was added sodium borohydride (1.38 g, 36.2 mmol) at 0 °C under
argon atmosphere. After being stirred at room temperature for
3 h, the reaction was quenched with aq NH4Cl. The reaction mix-
ture was extracted with EtOAc three times. The combined organic
layers were washed with brine, dried over MgSO4 and evaporated.
The resulting residue was purified by column chromatography on
silica gel (EtOAc/MeOH, 1:0–10:1) to afford 38 as a white powder
(1.55 g, 36%). 1H NMR (300 MHz, CDCl3): d 3.19 (dd, J = 11.2,
5.1 Hz, 1H), 3.34–3.48 (m, 3H), 3.72 (m, 1H), 4.99 (t, J = 5.7 Hz,
1H), 8.03 (m, 1H).

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 702–713
711
5.1.24. 4-[(2-{(4S)-4-[(1E,3S)-3-Hydroxy-4-phenylbut-1-enyl]-2-
5.1.29. 4-{[2-((4S)-4-{(1E,3S)-3-Hydroxy-4-[3-(methoxymethyl)
oxo-1,3-thiazolidin-3-yl}ethyl)sulfanyl]butanoic acid (13)
Compound 13 was prepared from 40 according to the same pro-
cedure as described for the preparation of 3 from 34 as a yellow oil
(30% in four steps). 1H NMR (300 MHz, CDCl3): d 1.88–1.90 (m,
2H), 2.44–2.59 (m, 6H), 2.88–2.97 (m, 4H), 3.38 (m, 1H), 3.59 (m,
1H), 4.32 (m, 1H), 4.46 (m, 1H), 5.65 (dd, J = 15.4, 8.4 Hz, 1H), 5.86
(dd, J = 15.4, 5.3 Hz, 1H), 7.20–7.30 (m, 5H); IR (film): 3934, 3856,
3844, 3788, 3399, 3060, 3027, 2925, 1713, 1651, 1495, 1440, 1396,
1282, 1211, 1133, 1097, 1030, 977, 912, 841, 747, 702, 666, 607,
524, 470, 456 cmꢀ1; FAB-MS (m/z): 396 (M+H)+; HRMS-FAB (m/z):
[M+H]+ calcd for C19H26NO4S2, 396.1303; found, 396.1303.
phenyl]but-1-enyl}-2-oxo-1,3-thiazolidin-3-yl)ethyl]sulfanyl}
butanoic acid (18)
Compound 18 was prepared from 40 according to the same pro-
cedure as described for the preparation of 3 from 34 as a yellow
viscous oil (65% in four steps). 1H NMR (300 MHz, CDCl3): d
1.88–1.90 (m, 2H), 2.54–2.57 (m, 6H), 2.80–2.86 (m, 2H), 2.97
(dd, J = 11.2, 6.6 Hz, 1H), 3.07 (m, 1H), 3.39 (m, 1H), 3.42 (s, 3H),
3.57 (m, 1H), 4.32 (m, 1H), 4.45 (s, 2H), 4.46 (m, 1H), 5.65 (m,
2H), 5.67 (ddd, J = 15.3, 8.4, 1.3 Hz, 1H), 5.86 (dd, J = 15.3, 5.1 Hz,
1H), 7.13 (m, 1H), 7.20–7.24 (m, 2H), 7.29 (m, 1H); IR (film):
3409, 2926, 1725, 1720, 1654, 1649, 1440, 1386, 1311, 1281,
1210, 1093 cmꢀ1; FAB-MS (m/z): 440 (M+H)+; HRMS-FAB (m/z):
[M+H]+ calcd for C21H30NO5S2, 440.1565; found, 440.1564.
5.1.25. 4-[(2-{(4S)-4-[(1E,3S)-4-(3-Ethylphenyl)-3-hydroxybut-1-
enyl]-2-oxo-1,3-thiazolidin-3-yl}ethyl)sulfanyl]butanoic acid (14)
Compound 14 was prepared from 40 according to the same pro-
cedure as described for the preparation of 3 from 34 as a yellow oil
(29% in four steps). 1H NMR (300 MHz, CDCl3): d 1.23 (t, J = 7.5 Hz,
3H), 1.88–1.92 (m, 2H), 2.48–2.59 (m, 8H), 2.90–2.94 (m, 4H), 3.39
(m, 1H), 3.59 (m, 1H), 4.33 (m, 1H), 4.46 (m, 1H), 5.67 (dd, J = 15.4,
8.4 Hz, 1H), 5.87 (dd, J = 15.4, 5.3 Hz, 1H), 7.00–7.06 (m, 3H), 7.25
(m, 1H); IR (film): 3410, 3105, 3022, 2963, 2929, 2871, 1711,
1650, 1560, 1488, 1440, 1397, 1282, 1211, 1134, 1102, 1031,
976, 911, 849, 797, 736, 704, 663, 584, 504 cmꢀ1; FAB-MS (m/z):
424 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C21H30NO4S2,
424.1616; found, 424.1612.
5.1.30. Butyl 7-[(2R)-2-(tert-butyldimethylsilyloxymethyl)-5-thi
oxo-1-pyrrolidinyl]heptanoate (42)
To a stirred solution of 41 (3.28 g, 11.0 mmol) was added TBS
chloride (1.82 g, 12.1 mmol) and imidazole (1.12 g, 16.5 mmol) in
DMF (20 mL) at 0 °C under argon atmosphere. After being stirred
at room temperature for 30 min, the reaction was quenched with
H2O. The reaction mixture was diluted with EtOAc, washed with
aqueous NaHCO3, brine, dried over MgSO4 and evaporated to give
TBS ether as a colorless oil (5.25 g).
To a stirred solution of the above-described TBS ether in toluene
(12 mL) was added Lawesson’s reagent (2.67 g, 6.6 mmol) at room
temperature under argon atmosphere. After being stirred at 50 °C
for 2 h, the reaction was quenched with water. The reaction mix-
ture was diluted with EtOAc, washed with aqueous NaHCO3, brine,
and dried over MgSO4. The combined organic layers were dried
over MgSO4 and evaporated. The resulting residue was purified
by column chromatography on silica gel (hexane/EtOAc, 9:1–4:1)
to give 42 as a dark green oil (3.47 g, 74%). 1H NMR (300 MHz,
CDCl3): d 0.04 (s, 6H), 0.06 (s, 3H), 0.86–0.91 (m, 9H), 0.95 (t,
J = 7.2 Hz, 3H), 1.32–1.46 (m, 6H), 1.55–1.68 (m, 8H), 1.92 (m,
1H), 2.30 (t, J = 7.4 Hz, 2H), 2.95 (m, 1H), 3.30 (m, 1H), 3.62–3.83
(m, 2H), 3.96 (m, 1H), 4.09 (t, J = 7.2 Hz, 2H), 4.25 (m, 1H).
5.1.26. 4-[(2-{(4S)-4-[(1E,3S)-3-Hydroxy-4-(3-propylphenyl)but-1-
enyl]-2-oxo-1,3-thiazolidin-3-yl}ethyl)sulfanyl]butanoic acid (15)
Compound 15 was prepared from 40 according to the same pro-
cedure as described for the preparation of 3 from 34 as a yellow oil
(33% in four steps). 1H NMR (300 MHz, CDCl3): d 0.94 (t, J = 7.3 Hz,
3H), 1.62–1.65 (m, 2H), 1.89–1.93 (m, 2H), 2.56–2.59 (m, 7H),
2.90–2.96 (m, 5H), 3.38 (m, 1H), 3.60 (m, 1H), 4.32 (m, 1H), 4.46
(m, 1H), 5.68 (dd, J = 15.4, 8.4 Hz, 1H), 5.87 (dd, J = 15.4, 5.1 Hz,
1H), 7.00–7.05 (m, 3H), 7.24 (m, 1H); IR (film): 3390, 2956, 2928,
2870, 1709, 1649, 1441, 1397, 1282, 1211, 1134, 1103, 1032,
974, 786, 704 cmꢀ1; FAB-MS (m/z): 438 (M+H)+; HRMS-FAB (m/
z): [M+H]+ calcd for C22H32NO4S2, 438.1773; found, 438.1768.
5.1.31. Butyl 7-[(2R)-2-(hydroxymethyl)-5-thioxo-1-pyrrolidinyl
]heptanoate (43)
5.1.27. 4-{[2-((4S)-4-{(1E,3S)-3-Hydroxy-4-[3-
A solution of 42 (3.47 g, 8.09 mmol) in THF (8 mL) was treated
with TBAF (8.9 mL, 1 M in THF, 8.9 mmol) at room temperature un-
der argon atmosphere for 1 h. After addition of brine, the reaction
mixture was extracted with EtOAc (ꢁ2). The combined organic lay-
ers were dried over MgSO4 and evaporated. The resulting residue
was purified by column chromatography on silica gel (hexane/
EtOAc, 4:1–1:1) to give an alcohol 43 as a colorless oil (2.04 g,
80%). 1H NMR (300 MHz, CDCl3): d 0.94 (t, J = 7.2 Hz, 2H), 1.25–
1.47 (m, 6H), 1.54–1.91 (m, 8H), 1.95 (m, 1H), 2.13 (m, 1H), 2.30
(t, J = 7.4 Hz, 2H), 2.86–3.17 (m, 2H), 3.37 (m, 1H), 3.71 (m, 1H),
3.85 (m, 1H), 3.96–4.13 (m, 3H), 4.24 (m, 1H).
(trifluoromethyl)phenyl]but-1-enyl}-2-oxo-1,3-thiazolidin-3-
yl)ethyl]sulfanyl}butanoic acid (16)
Compound 16 was prepared from 40 according to the same pro-
cedure as described for the preparation of 3 from 34 as a yellow
viscous oil (15% in four steps). 1H NMR (300 MHz, CDCl3): d
1.88–1.90 (m, 2H), 2.73–2.76 (m, 10H), 3.39 (dd, J = 11.2, 7.5 Hz,
1H), 3.61 (m, 1H), 4.34 (m, 1H), 4.49 (m, 1H), 5.68 (m, 1H), 5.85
(m, 1H) 7.40–7.47 (m, 4H); IR (film): 3410, 2926, 2864, 1712,
1649, 1442, 1400, 1330, 1283, 1202, 1162, 1122, 1074, 1034,
977, 801, 704, 663 cmꢀ1; FAB-MS (m/z): 464 (M+H)+; HRMS-FAB
(m/z): [M+H]+ calcd for C20H25F3NO4S2, 464.1177; found, 464.1174.
5.1.32. 7-{(2R)-2-[(1E,3S)-3-Hydroxy-4-phenylbut-1-enyl]-5-thi
oxopyrrolidin-1-yl}heptanoic acid (19)
5.1.28. 4-[(2-{(4S)-4-[(1E,3S)-4-(3-Fluorophenyl)-3-hydroxybut-1-
enyl]-2-oxo-1,3-thiazolidin-3-yl}ethyl)sulfanyl]butanoic acid (17)
Compound 17 was prepared from 40 according to the same pro-
cedure as described for the preparation of 3 from 34 as a yellow
viscous oil (19% in four steps). 1H NMR (300 MHz, CDCl3): d
1.90–1.92 (m, 2H), 2.70–2.76 (m, 10H), 3.40 (dd, J = 11.1, 7.6 Hz,
1H), 3.61 (m, 1H), 4.34 (m, 1H), 4.47 (m, 1H), 5.66 (m, 1H), 5.85
(m, 1H), 6.94–6.98 (m, 3H), 7.28 (m, 1H); IR (film): 3407, 2926,
2872, 1718, 1648, 1587, 1488, 1444, 1399, 1281, 1246, 1212,
1140, 1033, 976, 944, 886, 786, 753, 692 cmꢀ1; FAB-MS (m/z):
414 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C19H25FNO4S2,
414.1209; found, 414.1202.
Compound 19 was prepared from 43 according to the same pro-
cedure as described for the preparation of 3 from 34 as a yellow
viscous oil (18% in four steps). 1H NMR (300 MHz, CDCl3): d
1.28–1.34 (m, 4H), 1.64–1.70 (m, 5H), 2.24 (m, 1H), 2.35 (t,
J = 7.3 Hz, 2H), 2.90–2.98 (m, 5H), 4.07 (m, 1H), 4.37–4.39 (m,
2H), 5.53 (m, 1H), 5.77 (m, 1H), 7.20–7.29 (m, 5H); IR (film):
3398, 2859, 1708, 1495, 1455, 1421, 1263, 1227, 1151, 1124,
1097, 1029, 975, 749, 684 cmꢀ1; FAB-MS (m/z): 376 (M+H)+;
HRMS-FAB (m/z): [M+H]+ calcd for C21H30NO3S, 376. 1946; found,
376. 1954.

712
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 702–713
5.1.33. 7-{(2R)-2-[(1E,3S)-4-(3-Ethylphenyl)-3-hydroxybut-1-enyl
]-5-thioxopyrrolidin-1-yl}heptanoic acid (20)
5.1.38. Butyl 4-[(2-{(2R)-2-[(1E,3S)-3-(tert-butyldimethylsilyl
oxy)-4-(3-methylphenyl)but-1-enyl]-5-thioxopyrrolidin-1-yl}
ethyl)sulfanyl]butanoate (45)
Compound 20 was prepared from 43 according to the same
procedure as described for the preparation of 3 from 34 as a yellow
viscous oil (18% in four steps). 1H NMR (300 MHz, CDCl3): d 1.24
(t, J = 7.7 Hz, 3H), 1.30–1.36 (m, 4H), 1.60–1.70 (m, 5H), 2.18–
2.23 (m, 2H), 2.34 (t, J = 7.2 Hz, 2H), 2.59–2.66 (m, 2H), 2.90–2.95
(m, 4H), 4.06 (m, 1H), 4.34–4.38 (m, 2H), 5.55 (dd, J = 15.9,
9.2 Hz, 1H), 5.79 (m, 1H), 7.15–7.20 (m, 3H), 7.24 (m, 1H); IR (film):
3377, 3023, 2932, 2859, 1709, 1490, 1457, 1421, 1374, 1321, 1263,
1227, 1153, 1100, 1030, 975 cmꢀ1; FAB-MS (m/z): 404 (M+H)+;
HRMS-FAB (m/z): [M+H]+ calcd for C23H34NO3S, 404.2259; found,
404.2255.
To a stirred solution of 44 (11.2 g, 25.0 mmol) was added TBS
chloride (4.13 g, 27.5 mmol) and imidazole (2.55 g, 37.5 mmol) in
DMF (70 mL) at 0 °C under argon atmosphere. After being stirred
at room temperature for 30 min, the reaction was quenched with
H2O. The reaction mixture was diluted with EtOAc, and washed
with aq NaHCO3, brine, and dried over MgSO4. The organic solvent
was removed by evaporation to give TBS ether as a colorless oil
(13.5 g).
To a stirred solution of the above-described TBS ether in toluene
(100 mL) was added Lawesson’s reagent (5.30 g, 13.1 mmol) at
room temperature under argon atmosphere. After being stirred at
50 °C for 1 h, the reaction was quenched with H2O. The reaction
mixture was diluted with EtOAc, washed with aq NaHCO3, brine,
dried over MgSO4. The combined organic layers were dried over
MgSO4 and evaporated. The resulting residue was purified by col-
umn chromatography on silica gel (hexane/EtOAc, 7:1–3:1) to give
45 as a colorless oil (10.5 g, 76%). 1H NMR (300 MHz, CDCl3): d
ꢀ0.15 (s, 3H), ꢀ0.07 (s, 3H), 0.86 (s, 9H), 0.94 (t, J = 7.2 Hz, 3H),
1.32–1.46 (m, 2H), 1.56–1.66 (m, 2H), 1.76 (m, 1H), 1.84–2.00
(m, 2H), 2.24 (m, 1H), 2.33 (s, 3H), 2.43 (t, J = 7.2 Hz, 2H), 2.56–
2.87 (m, 5H), 2.91–3.07 (m, 2H), 3.26 (m, 1H), 4.08 (s, 3H), 4.25–
4.50 (m, 2H), 5.40 (dd, J = 15.2, 6.6 Hz, 1H), 5.73 (dd, J = 15.2,
5.7 Hz, 1H), 6.88–7.07 (m, 3H), 7.16 (m, 1H).
5.1.34. 7-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(3-propylphenyl)but-1-
enyl]-5-thioxopyrrolidin-1-yl}heptanoic acid (21)
Compound 21 was prepared from 43 according to the same pro-
cedure as described for the preparation of 3 from 34 as a yellow
viscous oil (14% in four steps). 1H NMR (300 MHz, CDCl3): d 0.94
(t, J = 7.41 Hz, 3H), 1.32–1.35 (m, 4H), 1.60–1.70 (m, 7H), 2.21–
2.24 (m, 2H), 2.34 (t, J = 7.2 Hz, 2H), 2.54–2.57 (m, 2H), 2.95–2.96
(m, 4H), 4.06 (m, 1H), 4.36–4.38 (m, 2H), 5.55 (dd, J = 15.4,
8.6 Hz, 1H), 5.79 (m, 1H), 7.00–7.04 (m, 3H), 7.23 (m, 1H); IR (film):
3374, 3022, 2931, 2860, 1709, 1489, 1421, 1263, 1208, 1100, 1030,
974, 787, 733, 704 cmꢀ1; FAB-MS (m/z): 418 (M+H)+; HRMS-FAB
(m/z): [M+H]+ calcd for C24H36NO3S, 418.2416; found, 418.2413.
5.1.39. 4-[(2-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(3-methylphenyl)
but-1-enyl]-5-thioxopyrrolidin-1-yl}ethyl)sulfanyl]butanoic
acid (25)
A solution of 45 (10.5 g, 18.2 mmol) in THF (50 mL) was treated
with TBAF (50.0 mL, 1 M in THF, 50.0 mmol) at room temperature
under argon atmosphere for 15 h. After addition of water, the reac-
tion mixture was extracted with EtOAc (ꢁ3). The combined organic
layers were dried over MgSO4 and evaporated to give an alcohol as
a colorless oil (7.2 g).
A solution of the above-described allyl alcohol in EtOH (28 mL),
THF (28 mL) and 1 N sodium hydroxide (23 mL) was stirred at
ambient temperature for 18 h. After neutralization with 1 N hydro-
chloric acid (23 mL) under cooling, the reaction mixture was ex-
tracted with EtOAc (ꢁ3). The combined organic layers were
washed with brine, dried over Na2SO4 and evaporated. The result-
ing residue was purified by column chromatography on silica gel
(hexane/EtOAc, 1:1–0:1) to afford 25 as a white powder (5.7 g,
90% in two steps): 1H NMR (300 MHz, CDCl3): d 1.70–2.00 (m,
3H), 2.27 (m, 1H), 2.35 (s, 3H), 2.39–3.10 (m, 12H), 3.37 (m, 1H),
4.13 (m, 1H), 4.38–4.52 (m, 2H), 5.55 (ddd, J = 15.3, 8.7, 1.2 Hz,
1H), 5.82 (dd, J = 15.3, 5.1 Hz, 1H), 6.95–7.11 (m, 3H), 7.22 (dd,
J = 7.5, 7.5 Hz, 1H); IR (film): 2922, 1713, 1608, 1487, 1322, 1229,
1161, 1032, 976, 752, 701, 665, 440 cmꢀ1; FAB-MS (m/z): 408
(M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C21H30NO3S2,
408.1667; found, 408.1667.
5.1.35. 7-((2R)-2-{(1E,3S)-3-Hydroxy-4-[3-(trifluoromethyl)
phenyl]but-1-enyl}-5-thioxopyrrolidin-1-yl)heptanoic acid (22)
Compound 22 was prepared from 43 according to the same pro-
cedure as described for the preparation of 3 from 34 as a yellow
viscous oil (13% in four steps). 1H NMR (300 MHz, CDCl3): d
1.28–1.32 (m, 4H) 1.66–1.68 (m, 5H), 2.20–2.23 (m, 1H), 2.34 (t,
J = 7.2 Hz, 2H), 2.98–3.01 (m, 5H), 4.05 (m, 1H), 4.33 (m, 1H),
4.48 (m, 1H), 5.56 (m, 1H), 5.78 (m, 1H), 7.40–7.45 (m, 4H); IR
(film): 3046, 2935, 2861, 1710, 1494, 1451, 1422, 1329, 1264,
1227, 1200, 1163, 1122, 1074, 1033, 976, 800, 704 cmꢀ1; FAB-MS
(m/z): 444 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for
C22H29F3NO3S, 444.1820; found, 444.1817.
5.1.36. 7-{(2R)-2-[(1E,3S)-4-(3-Fuorophenyl)-3-hydroxybut-1-en
yl] -5-thioxopyrrolidin-1-yl}heptanoic acid (23)
Compound 23 was prepared from 43 according to the same pro-
cedure as described for the preparation of 3 from 34 as a yellow vis-
cous oil (11% in four steps). 1H NMR (300 MHz, CDCl3): d 1.33–1.36
(m, 4H), 1.68–1.71 (m, 5H), 2.26 (m, 1H), 2.35 (t, J = 7.3 Hz, 2H),
2.90–2.97 (m, 5H), 4.07 (m, 1H), 4.35 (m, 1H), 4.43 (m, 1H), 5.54
(m, 1H), 5.76 (m, 1H), 6.93–6.96 (m, 3H) 7.28 (m, 1H); IR (film):
3376, 3041, 2935, 2860, 1709, 1616, 1587, 1489, 1448, 1421,
1374, 1321, 1250, 1228, 1185, 1142, 1098, 1032, 976, 787, 753,
693 cmꢀ1; FAB-MS (m/z): 394 (M+H)+; HRMS-FAB (m/z): [M+H]+
calcd for C21H29FNO3S, 394.1852; found, 394.1858.
5.2. Biological assays
5.1.37. 7-((2R)-2-{(1E,3S)-3-Hydroxy-4-[3-(methoxymethyl)
phenyl]but-1-enyl}-5-thioxopyrrolidin-1-yl) heptanoic acid (24)
Compound 24 was prepared from 43 according to the same
procedure as described for the preparation of 3 from 34 as a brown
oil (64% in four steps). 1H NMR (200 MHz, CDCl3): d 1.20–1.90 (m,
10H), 2.33 (t, J = 7.2 Hz, 2H), 2.15–2.40 (m, 2H), 2.75–3.10 (m, 4H),
3.10–3.38 (m, 1H), 3.43 (s, 3H), 3.85–4.02 (m, 1H), 4.47 (s, 2H),
4.25–4.50 (m, 2H), 5.59 (dd, J = 15.4, 8.4 Hz, 1H), 5.82 (dd, J = 15.4,
5.0 Hz, 1H), 7.10–7.40 (m, 4H); IR (film): 3377, 2931, 2858, 1709,
1490, 1421, 1380, 1263, 1227, 1191, 1156, 1096, 1033, 975, 915,
793, 757, 733, 703 cmꢀ1; FAB-MS (m/z): 420 (M+H)+; HRMS-FAB
(m/z): [M+H]+ calcd for C23H34NO5S, 420.2209; found, 420.2208.
5.2.1. mEP1–4 receptor binding assay
Competitive binding studies were conducted using radiolabeled
ligands and membrane fractions prepared from Chinese hamster
ovary (CHO) cells, which stably express the prostanoid receptors
mEP1–4. Membranes from CHO cells expressing prostanoid recep-
tors were incubated with
a radiolabeled ligand (i.e. 2.5 nM
[3H]PGE2) and test compounds at various concentrations in an assay
buffer (i.e. 10 mM KH2PO4–KOH buffer containing 1 mM EDTA,
10 mM MgCl2 and 0.1 mM NaCl, pH 6.0). Incubation was carried
out at 25 °C for 60 min, with the exception of mEP1, which was incu-
bated for 20 min. Incubation was terminated via filtration through a

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 702–713
713
Whatman GF/B filter. The filter was subsequently washed with ice-
cold buffer (10 mM KH2PO4–KOH buffer containing 0.1 mM NaCl,
pH 6.0), and the radioactivity on the filter was measured in a 6 mL
liquid scintillation (ACSII) mixture with a liquid scintillation coun-
ter. Non-specific binding was achieved by adding excess amounts
of unlabeled PGE2 in the assay buffer. The concentration that causes
50% of inhibition (IC50 value) was estimated from the regression
curve. The Ki value (M) was calculated according to the following
equation: Ki = IC50/(1 + [L]/Kd), where [L] is the concentration of
radiolabeled ligand and Kd is the dissociation constant of radiola-
beled ligand for the prostanoid receptor of interest.
30 min, an intravenous administration of LPS (10
l
g/2 mL/kg)
was given, and 90 min after the LPS injection, blood samples were
withdrawn into heparinized syringes via aortic puncture. Follow-
ing centrifugation at 12,000 rpm for 3 min at 4 °C, plasma was
recovered and immediately frozen at 80 °C. Plasma TNF-a concen-
trations were determined via an ELISA kit (Biosource).
References and notes
1. Coleman, R. A.; Smith, W. L.; Narumiya, S. Pharmacol. Rev. 1994, 12, 205.
2. Narumiya, S.; Sugimoto, Y.; Ushikubi, F. Physiol. Rev. 1999, 79, 1193.
3. Maruyama, T.; Asada, M.; Shiraishi, T.; Ishida, A.; Yoshida, H.; Maruyama, T.;
Ohuchida, S.; Nakai, S.; Kondo, K.; Toda, M. Bioorg. Med. Chem. 2002, 10, 989.
4. Billot, X.; Chateauneuf, A.; Chauret, N.; Denis, D.; Greig, G.; Mathieu, M.-C.;
Metters, K. M.; Slipetz, D. M.; Young, R. N. Bioorg. Med. Chem. Lett. 2003, 13,
1129.
5. Elworthy, T. R.; Kertesz, D. J.; Kim, W.; Roepel, M. G.; Quattrocchio-Setti, L.;
Smith, D. B.; Tracy, J. L.; Chow, A.; Li, F.; Brill, E. R.; Lach, L. K.; McGee, D.; Yang,
D. S.; Chiou, S.-S. Bioorg. Med. Chem. Lett. 2004, 14, 1655.
6. Elworthy, T. R.; Brill, E. R.; Caires, C. C.; Kim, W.; Lach, L. K.; Tracy, J. L.; Chiou,
S.-S. Bioorg. Med. Chem. Lett. 2005, 15, 2523.
7. Cameron, K. O.; Lefker, B. A.; Chu-Moyer, M. Y.; Crawford, D. T.; Jardine, P. D.;
DeNinno, S. L.; Gilbert, S.; Grasser, W. A.; Ke, H.; Lu, B.; Owen, T. A.; Paralkar, V.
M.; Qi, H.; Scott, D. O.; Thompson, D. D.; Tjoa, C. M.; Zawistoski, M. P. Bioorg.
Med. Chem. Lett. 2006, 16, 1799.
5.2.2. Measurement of cAMP production
Chinese hamster ovary (CHO) cells expressing mouse or rat EP4
receptor were cultured in 24-well plates (1 ꢁ 105 cells/well). After
2 days, the media were removed and cells were washed with
500
10 min in 500
at 37 °C. After the removal of buffer via suction, cells were pre-
incubated in 450 L of assay medium (containing 1% of BSA) for
10 min at 37 °C. The reaction was started with the addition of each
test compound in 50 L of assay buffer. After incubation for 10 min
at 37 °C, the reaction was terminated by adding 500 L of ice-cold
l
L of Minimum Essential Medium (MEM) and incubated for
l
L of buffer (MEM containing 2 M of diclofenac)
l
l
l
8. Kambe, T.; Maruyama, T.; Naganawa, A.; Asada, M.; Seki, A.; Maruyama, T.;
Nakai, H.; Toda, M. Chem. Pharm. Bull. 2011, 59, 1494.
9. Kambe, T.; Maruyama, T.; Nakano, M.; Yoshida, T.; Yamaura, Y.; Shono, T.; Seki,
A.; Sakata, K.; Maruyama, T.; Nakai, H.; Toda, M. Chem. Pharm. Bull. 2011, 59,
1523.
l
10% trichloroacetic acid. cAMP production was determined via a
cAMP radioimmunoassay kit (Amersham).
10. Kambe, T.; Maruyama, T.; Nakano, M.; Nakai, Y.; Yoshida, T.; Matsunaga, N.;
Oida, H.; Konaka, A.; Maruyama, T.; Nakai, H.; Toda, M. Bioorg. Med. Chem. Lett.
in press.
11. Corey, E. J.; Bakshi, R. K.; Shibata, S. J. Am. Chem. Soc. 1987, 109, 5551.
12. Kiriyama, M.; Ushikubi, F.; Kobayashi, T.; Hirata, M.; Sugimoto, Y.; Narumiya, S.
Br. J. Pharmacol. 1997, 122, 217.
5.2.3. LPS-induced changes in plasma TNF-a levels in rats
LPS was dissolved in a sterile saline solution (1 mg/mL), and the
test compound was dissolved in a sterile saline solution containing
20% HP-b-CD. Then, the test compound was orally administered to
seven-week-old Sprague–Dawley rats (Charles River, Japan). After