Synthesis and evaluation of γ-lactam analogs of PGE2 as EP4 and EP2/EP4 agonists

Article abstract of DOI:10.1016/j.bmc.2012.04.008

To identify topically effective EP4 agonists and EP2/EP4 dual agonists with excellent subtype selectivity, further optimization of the 16-phenyl ω-chain moiety of the γ-lactam 5-thia prostaglandin E analog and the 2-mercaptothiazole-4-carboxylic acid anal

Full text of DOI:10.1016/j.bmc.2012.04.008

Bioorganic & Medicinal Chemistry 20 (2012) 3502–3522
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of c-lactam analogs of PGE2 as EP4
and EP2/EP4 agonists
⇑
Tohru Kambe , Toru Maruyama, Yoshihiko Nakai, Hiroji Oida, Takayuki Maruyama, Nobutaka Abe,
Akio Nishiura, 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:
Received 19 February 2012
Revised 1 April 2012
Accepted 4 April 2012
Available online 11 April 2012
To identify topically effective EP4 agonists and EP2/EP4 dual agonists with excellent subtype selectivity,
further optimization of the 16-phenyl
x-chain moiety of the c-lactam 5-thia prostaglandin E analog and
the 2-mercaptothiazole-4-carboxylic acid analog were undertaken. Rat in vivo evaluation of these newly
identified compounds as their poly (lactide-co-glycolide) microsphere formulation, from which sustained
release of the test compound is possible, led us to discover compounds that showed efficacy in a rat bone
fracture healing model after its topical administration without serious influence on blood pressure and
heart rate. A structure–activity relationship study is also presented.
Keywords:
Prostaglandin
EP4 receptor
EP2 receptor
Agonist
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
In a previous paper, we reported PGE analogs 1 and 2 (Fig. 1) as
the highly potent EP4 selective agonists.8 In the early stage of this
Prostaglandins (PGs) are a group of lipid mediators that are
produced from arachidonic acid in a variety of tissues under vari-
ous physiological and pathophysiological conditions and serve to
maintain local homeostasis.1 Among them, PGE2, which is the most
well-known prostanoid derivative and exhibits a broad range of
biological actions in diverse tissues through binding to specific
receptors (EP1, EP2, EP3 and EP4), potently induces bone resorp-
tion in bone organ cultures. Repeated injection of PGE2 also
induces bone formation in a variety of animals including humans.2
Despite its high potency, the use of PGE2 as a therapeutic agent in
the treatment of bone loss has been hindered by unwanted actions
because of its nonspecific affinity toward the individual EP recep-
tors. According to recent molecular biology developments, both
the EP2 and EP4 receptors, activation of which increases the intra-
cellular cAMP level, are interesting pharmacological targets
because of their important regulatory roles in numerous physio-
logical processes including bone metabolism.2,3
project, we tried to prepare the poly (lactide-co-glycolide) (PLGA)
microsphere formulation of the methyl ester 1 for its sustained
release during topical evaluation. This attempt was not successful
because of the low lipophilicity and instability of 1 as described
above for PGE2. In order to evaluate the in vivo efficacy in a bone
fracture healing model, the sustained release of a chemically and
metabolically more stable EP4 selective agonist was absolutely
needed. Previously, we reported the discovery of chemically and
metabolically stable c-lactam analogs of PGE as EP4 subtype-selec-
tive agonists and the EP2/EP4 dual agonists.9–11
In this report, we describe the synthesis and evaluation of two
series of
c-lactam PGE analogs 3 and 4 (Fig. 2) as an EP4
subtype-selective agonist and EP2/EP4 dual agonist, respectively,
and optimization of their sustained release from the PLGA formu-
lation. Some of the representative compounds were evaluated for
their in vivo efficacy towards bone fracture healing.
Natural PGE2 is susceptible to metabolic inactivation: 15-OH
oxidation, b-oxidation of the a-chain, and oxidation of the x-chain
O
S
CO2R
OMe
terminus. Furthermore, PGE2 is chemically unstable because of the
easy elimination of the 11-OH. Several research groups have inves-
tigated strategies for improving the pharmacological properties of
PGE2.4–7
HO
OH
1 R = Me
2 R = H active metabolite
⇑
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).
Figure 1. Structure of reported EP4 agonist.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.008

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
3503
CO2H
R
olefination followed by stereoselective reduction of the prochiral
enones and then alkaline hydrolysis. Thus, this section starts with
a description of the preparation of phosphonates (Fig. 3) for Horn-
er–Emmons olefination.
O
N
S
O
S
CO2H
R
S
N
N
OH
OH
Phosphonates 5a, 5c,d and 5g were prepared as shown in
Scheme 1a. Dehydrative condensation of 3-bromophenyl acetic
acid with N,O-dimethylhydroxylamine hydrochloride in the pres-
ence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (EDC-HCl) and triethylamine afforded 6, which was
converted to 5a by the reaction with lithium anion prepared from
dimethyl methylphosphonate and n-butyl lithium. The palladium-
catalyzed cross-coupling reaction of 6 with 4-methylphenylboron-
ic acid, 4-methoxyphenylboronic acid or indole-6-boronic acid
afforded 5c, 5d and 5g, respectively.
Phosphonates 5j and 5q,r were prepared as described in Scheme
1b. Compound 8a was prepared by the same method as described
above. The palladium-catalyzed cross-coupling reaction of the cor-
responding trifluoromethanesulfonate with benzofuran-2-boronic
acid afforded 8b, which was converted to the phosphonate 5j
according to the same procedure as described above. Compound
8a was converted to the phosphonate 5q by the following sequential
reactions: (1) trifluoromethanesulfonylation with trifluoroacetic
acid anhydride and pyridine; (2) palladium-catalyzed condensation
with pinacolborane; (3) palladium-catalyzed cross-coupling with 2-
bromopyridine; (4) reaction with diethyl methylphosphonate in the
presence of n-butyl lithium. O-silylation of 8a with tert-butyldi-
methylsilyl chloride in the presence of imidazole provided 8e,
which was converted to the phosphonate 5r by the same reaction
as described above.
3
4
a
i
R = CH3
a
i
R = CH3
R =
R =
*
*
O
O
N
CH3
CH3
m R =
*
*
O
N
n
R =
O
Figure 2. Molecular design of EP4 subtype-selective agonist 3 and EP2/EP4 dual
agonist 4 optimized for their PLGA microsphere formulation.
Since evaluation of a subtype-selective agonist in the bone frac-
ture healing model in rats requires 17 days, its sustained release
from the carrier after the topical injection was considered to be
an important factor necessary for good efficacy. For this reason,
evaluation of the in vitro release profile of representative test com-
pounds from PLGA microspheres was also carried out to estimate
their systemic sustained release after topical injection.
2. Chemistry
Test compounds listed in Tables 1 and 2 were synthesized as out-
lined in Schemes 1–5. Most of these compounds were synthesized
by the conventional synthetic method for PGs: Horner–Emmons
Preparation of phosphonates 5h, 5l–p, 5i and 5k is described
in Scheme 1c. Dehydrative condensation of commercially avail-
able aminophenols and 3-iodobenzoic acid in the presence of
Table 1
Effect of the
x-chain structure on the activity profiles of 8-aza-5-thia-16aryl PGE1 analogs
O
N
S
CO2H
R
Binding assay (Ki, nM)
Functional assay (EC50, nM)
rEP4
clogP
OH
Compound
R
mEP1
mEP2
mEP3
mEP4
3a
3b
CH3
Ph
>104
>104
>104
>104
5800
>104
1.8
1.1
5.7
52
2.2
3.6
3c
>104
2100
6100
>104
0.6
4.8
96
86
4.8
2.3
3d
>104
>104
N
3e
3f
7100
>104
>104
3500
>104
>104
4.4
28
85
3.5
3.6
N
H
N
0.28
N
N
3g
3h
3i
5000
>104
>104
1700
720
>104
>104
>104
0.2
13
3.1
3.8
4.4
O
S
0.36
0.1
3.2
7.0
2000
O
3j
>104
>104
>104
>104
>104
>104
>104
>104
>104
4.7
0.89
28
19
1.9
1.9
1.9
N
N
O
O
3k
3l
0.61
46
N
O

3504
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
Table 2
Effect of the
x
-chain structure on the activity profiles of ethyl-linked 2-mercaptothiazole-4-carboxylic acid analogs
CO2H
N
O
N
S
S
Binding assay (Ki, nM)
Functional assay (EC50, nM)
clogP
R
OH
Compound
R
mEP1
mEP2
mEP3
mEP4
rEP2
rEP4
4a
4b
CH3
Ph
>104
4200
9.3
3.0
540
4300
0.41
0.94
90
1800
0.79
8.0
3.0
4.4
CH3
Cl
4c
4d
4e
6100
6300
5400
2.0
1.7
9.2
>104
>104
>104
0.43
0.81
1.7
380
15
27
15
4.9
5.1
4.3
260
OMe
CH3
4600
4f
2400
23
>104
3.5
>104
160
5.1
CH3
CH3
CH3
4g
4h
4i
2500
>104
2700
1.5
22
>104
>104
1700
0.35
0.012
0.12
1400
>104
1500
21
5.3
5.6
5.1
480
5.3
2.1
O
O
S
N
N
4j
>104
8000
28
15
>104
>104
0.12
0.19
1600
2700
2.2
13
3.9
4.5
4k
Cl
4l
680
22
>104
0.25
>104
9.7
5.3
N
S
CH3
4m
4n
4o
1300
200
21
56
22
>104
>104
>104
0.21
0.16
0.21
>104
3200
8500
8.8
1.6
28
4.4
4.4
4.3
N
N
O
O
CH3
H3C
7700
N
N
O
O
CH3
4p
4q
3200
650
24
67
>104
>104
0.15
0.20
>104
>104
7.7
4.9
4.9
4.6
CH3
Cl
N
O
polyphosphoric acid (PPA) afforded oxazoles 9a–f, respectively,
which were converted to 10a–f, respectively, by the following
sequential reactions: (1) reaction with diethyl malonate in the
presence of sodium hydride and cuprous iodide; (2) alkaline
hydrolysis followed by decarboxylation; (3) condensation reac-
tion with N,O-dimethylhydroxylamine in the presence of EDC-
HCl and triethylamine. Compounds 10a–f were converted to 5h
and 5l–p, respectively, according to the same procedure as de-
scribed above. Dehydrative condensation of the commercially
available 2-aminothiophenols with 3-iodobenzoic acid in PPA
afforded 9g–h, respectively, which were converted to the
phosphonates 5i and 5k, respectively, according to the same
procedure as described above for the preparation of the corre-
sponding benzoxazoles.
Phosphonate 5f was prepared as shown in Scheme 1d. Esterifi-
cation of 3-aminophenylacetic acid with methanol in the presence
of thionyl chloride followed by N,N-dialkylation with 1,2-bis
(bromomethyl)benzene afforded 11, which was converted to the
phosphonate 5f according to the same procedure as described
above. Preparation of phosphonates 5b and 5e was described in
our preceding report.13
Synthesis of 3b–h is outlined in Scheme 2a. Horner–Emmons
olefination of the aldehyde 12 with the appropriate phosphonates
in the presence of sodium hydride followed by stereoselective (R)-

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
3505
CH3
N
b
Br
a
HO2C
Br
5a
MeO
O
6
c
b
CH3
N
5c-dand5g
R
MeO
O
7a-c
a
H
N
CH3
OMe
R =
*
*
*
b
c
Scheme 1a. Preparation of phosphonates 5a, 5c,d and 5g: (a) N,O-dimethylhydroxylamine hydrochloride, EDC-HCl, Et3N, CH3CN; (b) dimethyl methylphosphonate, n-BuLi,
toluene; (c) 4-methylphenylboronic acid or 4-methoxyphenylboronic acid, Pd(PPh3)4, or indole-6-boronic acid, Pd(OAc)2, (S)-BINAP, aq Na2CO3, DME.
CH3
N
OH
g
a
R
ence of sodium triacetoxyborohydride to afford 20, alkaline hydro-
lysis of which produced 3i.4
HO2C
MeO
5j and5q-r
O
Preparation of the aldehyde 23 is described in Scheme 3c.
Nucleophilic substitution of 2-bromoacetoaldehyde diethylacetal
with potassium thioacetate provided 20, reaction of which with
ethyl 2-bromothiazole-4-carboxylate in the presence of potassium
carbonate in ethanol afforded 23.
8a
8b
R = OH
R =
b,c
*
O
Synthesis of 4f,g is described in Scheme 4. Horner–Emmons
olefination of the aldehyde 16 with the phosphonate 5a in the
presence of sodium hydride followed by stereoselective CBS-
reduction afforded 24. Protection of the 15-hydroxy function of
24 as a TBS–ether provided 25. The palladium-catalyzed cross-cou-
pling reaction of 25 with 2,4-dimethylphenylboronic acid and 3,4-
dimethylphenylboronic acid afforded 26a and 26b, respectively.
Acidic deprotection of 26a,b followed by reductive amination with
the aldehyde 23 (Scheme 3c) afforded 27a,b accompanied by intra-
molecular cyclization. Alkaline hydrolysis of 27a,b yielded 4f,g,
respectively.
Synthesis of the additional 5-thia analogs 3j–l is outlined in
Scheme 5. Horner–Emmons olefination of the aldehyde 12 with
the phosphonate 5q followed by stereoselective CBS-reduction
afforded 28. Protection of the 15-hydroxy group of 28 as a tetrahy-
dropyranyl ether followed by deprotection of the TBS–ether with
tetrabutylammonium fluoride afforded 29. O-alkylation of 29 with
2-hydroxymethylpyridine, 3-hydroxymethylpyridine and 4-hydro-
xymethylpyridine under Mitsunobu conditions afforded 30a–c,
respectively. Acidic deprotection of 30a–c followed by alkaline
hydrolysis resulted in 3j–l, respectively.
8a
8c
R = OH
H3C
O
CH3
b, d
e
CH3
R =
*
B
CH3
O
R =
8d
8a
8e
N
*
R = OH
f
R = OTBS
Scheme 1b. Preparation of phosphonates 5j and 5q,r: (a) N,O-dimethylhydroxyl-
amine hydrochloride, EDC-HCl, Et3N, CH3CN; (b) trifluoromethanesulfonic anhy-
dride, pyridine, CH2Cl2; (c) benzofuran-2-boronic acid, PdCl2(PPh3)2, Et3N, DMF; (d)
pinacolborane, PdCl2(dppf), Et3N, CH3CN; (e) 2-bromopyridine, PdCl2(dppf), K3PO4,
DME; (f) TBSCl, imidazole, DMF; (g) dimethyl methylphosphonate or diethyl
methylphosphonate, n-BuLi, toluene.
CBS-reduction of the formed enones afforded 13a–g, respectively,
alkaline hydrolysis of which afforded 3b–h, respectively.10,14
Compounds 4c, 4e and 4j,k were synthesized as shown in Scheme
2b. Horner–Emmons olefination of the aldehyde 14 with the
appropriate phosphonates followed by stereoselective CBS-reduc-
tion provided 15a–e, respectively, alkaline hydrolysis of which
produced 4c, 4e and 4j,k, respectively.11,12
Synthesis of 4i and 4l–q is described in Scheme 3a. Horner–Em-
mons olefination of the aldehyde 16 with the appropriate phos-
phonates followed by stereoselective CBS-reduction afforded
17a–g.11,12 Acidic deprotection of 17a–g followed by reductive
amination with the aldehyde 23, which was prepared as shown
in Scheme 3c, in the presence of sodium triacetoxyborohydride
3. Results and discussion
Compounds listed in Tables 1 and 2 were evaluated for their
binding affinity using membrane fractions of CHO cells expressing
the mouse EP-receptor. Ki values were determined by a competi-
tive binding assay that was performed according to the method
of Kiriyama et al. with minor modifications.15
Prior to rat in vivo evaluation, the compounds listed in Tables 1
and 2 were tested for their agonist activity towards rat EP2 and EP4
receptors. Chemical modifications were conducted to concurrently
optimize the lipophilicity (clogP) and activity profiles so that
sustained release of the most active compounds from PLGA micro-
resulted in the c-lactam framework 18a–g accompanied by intra-
molecular cyclization, alkaline hydrolysis of which yielded 4i and
spheres could be achieved. Maintaining the optimal
a-chains, we
4l–q, respectively.
continued further chemical modifications of the -chain of 3 and
x
5-Thia-
c
-lactam analog 3i was synthesized as shown in Scheme
4 to investigate their SAR in more detail. Results are summarized
in Tables 1 and 2. As an index of lipophilicity, clogP values of test
compounds were calculated and are also summarized in the tables.
3b. Acidic deprotection of 17a was carried out as described above
followed by reductive amination with an aldehyde 19 in the pres-

3506
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
R2
R1
X
R1
CH3
N
R3
N
b, c, d
I
CO2H
a
R2
R3
H2N
R4
I
MeO
+
O
H
X
10a-h
9a-h
e
5h, 5l-p, 5i
and5k
H3C
CH3
N
CH3
N
N
N
R4 =
O
*
O
O
O
*
*
*
a
b
c
d
CH3
Cl
Cl
N
N
N
N
S
O
*
*
O
S
CH3
*
*
e
f
g
h
X = O
R1
H
R2
H
R3
H
H
H
CH3
CH3
H
CH3
H
H
H
Cl
H
CH3
H
CH3
H
X = S
R1
H
R2
H
R3
H
H
Cl
H
Scheme 1c. Preparation of phosphonates 5h, 5l–p, 5i and 5k: (a) 3-iodobenzoic acid, PPA; (b) diethyl malonate, NaH, CuI, DMI; (c) aq NaOH, DME then concd HCl; (d) N,O-
dimethylhydroxylamine hydrochloride, EDC-HCl, Et3N, CH3CN; (e) dimethyl methylphosphonate, n-BuLi, toluene.
NH2
c
a, b
N
HO2C
5f
MeO2C
11
Scheme 1d. Preparation of phosphonate 5f: (a) thionyl chloride, MeOH; (b) 1,2-dibenzyl bromide, K2CO3, DMPU; (c) dimethyl methylphosphonate, n-BuLi, toluene.
8-Aza-5-thia-16-(3-methyl)phenyl PGE1 analog 3a, which is
one of the optimized EP4 subtype-selective agonists, was subjected
to the above-described chemical modifications. Thus, the purpose
of chemically modifying this compound was to identify a structure
that shows a biological profile with higher mouse EP4 selectivity
and more potent rat EP4 agonist activity with higher lipophilicity.
Results are shown in Table 1. Replacement of the 3-methyl residue
of 3a with a phenyl or naphth-2-yl residue afforded 3b and 3c,
respectively, with reduced rat agonist activities despite increased
clogP values and the retention of the mouse EP4 subtype-selectiv-
ity. Replacement of the 3-methyl residue of 3a with a pyridine-2-yl
residue provided 3d without an increase in the clogP value.
Replacement of the 3-methyl residue of 3a with a dihydroisoin-
dol-2-yl moiety afforded 3e with reduced rat agonist activity and
an increased clogP value while it retained good mouse EP4 sub-
type-selectivity. Replacement of the 3-methyl residue of 3a with
an indol-5-yl residue afforded 3f with reduced rat agonist activity.
As described above, chemical optimization targeting profiles of
strong rat EP4 agonist activity with increased lipophilicity and
good EP4 subtype-selectivity was difficult. However, the desired
profiles were achieved with a series of fused 5–6 heterobiaryl
analogs, 3g, 3h and 3i. Replacement of the 3-methyl residue of
3a with a benzoxazol-2-yl, benzthiazol-2-yl or benzofuran-2-yl
residue afforded 3g, 3h and 3i, respectively. All of these three ana-
logs demonstrated excellent mouse EP4 subtype-selectivity and rat
agonist activity with increased clogP values. While the pyridyl-
methyl analogs 3j and 3l showed reductions in mouse EP4 affinity
and rat agonist activity, 2-pyridylmethyl analog 3k showed potent

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
3507
O
O
O
O
c
S
a, b
N
O
R
CH3
S
3b-h
N
O
CH3
CHO
OH
12
13a-g
R =
N
N
*
*
*
*
a
e
d
b
c
H
N
N
O
N
*
*
S
*
g
f
Scheme 2a. Synthesis of 3b–h. (a) the corresponding phosphonate, NaH, THF, (b) (R)-Me-CBS, BH3–THF, THF; (c) aq NaOH, MeOH, THF.
O
O
N
S
O
CH3
O
R
N
S
O
CH3
O
c
S
a, b
S
4c,4e
and4j-k
N
N
N
CHO
OH
15a-d
14
OMe
CH3
N
R =
S
O
*
*
*
*
a
b
c
d
Scheme 2b. Synthesis of 4c, 4e and 4j,k. (a) The corresponding phosphonate, NaH, THF, (b) (R)-Me-CBS, BH3–THF, THF; (c) aq NaOH, MeOH, THF.
CO2Et
N
S
O
NHBoc
R
e
c, d
S
4i and4l-q
N
EtO2C
a,b
R
OH
R = CHO
16
18a-g
R
*
R =
17a-g
OH
Cl
CH3
N
N
N
CH3
R =
O
*
S
O
O
*
*
*
a
b
c
d
Cl
CH3
H3C
N
N
N
O
O
*
*
O
CH3
*
e
f
g
Scheme 3a. Synthesis of 4i and 4l–q. (a) the corresponding phosphonate, NaH, THF; (b) (R)-Me-CBS, BH3–THF, THF; (c) HCl in dioxane, EtOH; (d) aldehyde 23, NaBH(OAc)3,
THF, DMF; (e) aq NaOH, MeOH, THF.
mouse EP4 subtype-selectivity and rat agonist activity. Despite low
clogP value of compound 3k, high entrapping efficiency and/or
extended release was expected due to the interaction between
the basic residue of 3k and acidic residue of PLGA micosphere. As
a result, 3g, 3h, and 3i, which bear the 5,6-fused heteroaromatic
Secondly, 2-mercaptothiazole-4-carboxylic acid analog 4a,
which was identified as one of the optimized EP2/EP4 dual ago-
nists, was also subjected to the above-described chemical modifi-
cations. Replacement of the 3-methyl residue of 4a with a phenyl
residue afforded 4b with retention of the excellent mouse EP2/
EP4 dual affinity. Based on the results described above, compounds
4c–4g were also synthesized and evaluated for their mouse EP2/
moiety as a
x-chain terminus, and 3k, which bear the basic resi-
due, were identified as the optimized compounds.

3508
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
OHC
S
CO2Et
O
19
S
CO2Et
c
a, b
N
3i
17a
O
OH
20
Scheme 3b. Synthesis of 3i. (a) HCl in dioxane, EtOH; (b) aldehyde 19, NaBH(OAc)3, THF; (c) aq NaOH, MeOH, THF.
OEt
CO2Et
exhibited significantly reduced rat EP2/EP4 agonist activity along
with its particularly increased mouse EP4 receptor affinity.12
Replacement of the methyl residue of 4a with benzofuran-2-yl,
benzoxazol-2-yl or benzthiazol-2-yl residues afforded 4i–4k,
respectively, with good EP2/EP4 dual selectivity, while they
showed good to excellent rat EP4 agonist activity with remarkably
reduced rat EP2 agonist activity. Introduction of a chloro or methyl
residue into the thiazol-2-yl and oxazol-2-yl residues afforded 4l
and 4m–q, respectively, with retention of mouse EP2/EP4 dual
affinity and good rat EP4 agonist activity with remarkably reduced
rat EP2 agonist activity. The clogP values of all the compounds 4b–
q were relatively higher than 4a. Thus, this series of compounds
exhibited EP2/EP4 dual subtype-selectivity in a mouse binding
assay while they tended to show rat EP4 selective agonist activity
because of the remarkably reduced rat EP2 agonist activity. These
results indicate that the rat EP2 agonist activity is much more sus-
ceptible to the species differences than the rat EP4 agonist activity
while the desired mouse EP2/EP4 dual selectivity may be repro-
duced in the human receptor assay and the functional assay.
c
N
S
OHC
S
X
EtO
23
X = Br
a
b
X = SAc
21
CO2Et
N
S
S
22 X =
*
Scheme 3c. Preparation of aldehyde 23: (a) potassium thioacetate, DMF, (b) ethyl
2-bromothiazole-4-carboxylate, K2CO3, EtOH; (c) 2 N HCl, CH3CN.
EP4 dual selectivity.12 All of these compounds showed excellent to
good mouse EP2/EP4 dual selectivity and increased clogP values,
while 2,4-dimethylphenyl analog 4f showed reduced affinity for
the mouse EP2/EP4 receptors and reduction of rat EP2/EP4 dual
agonist activity. All of the listed compounds tended to show
remarkable reduction of rat EP2 agonist activity. Compound 4h
CO2Et
N
O
e, f
S
g
NHBoc
N
4f-g
S
EtO2C
R
R
OH
16
24
R = CHO
27a-b
CH3
CH3
CH3
a,b
c
R =
Br
*
R =
R =
R =
*
*
OH
CH3
a
25
b
Br
*
OTBS
d
26a-b
R
*
OTBS
Scheme 4. Synthesis of 4f and g. (a) phosphonate 5a, NaH, THF; (b) (R)-Me-CBS, BH3–THF, THF; (c) tert-butyldimethylsilyl chloride, imidazole, DMF; (d) 2,4-
dimethylphenylboronic acid or 3,4-dimethylphenylboronic acid, Pd(OAc)2, (S)-BINAP, aq Na2CO3, DME; (e) HCl in dioxane, EtOH; (f) aldehyde 23, NaBH(OAc)3, THF; (g) aq
NaOH, MeOH, THF.
O
O
O
O
f, g
c, d
S
S
N
O
O
CH3
3j-k
N
O
CH3
R
R
OTHP
12
28
R = CHO
*
29
R = H
a, b
OTBS
e
30a-c
R =
N
N
*
OH
*
N
a
b
*
c
Scheme 5. Synthesis of 3j–l. (a) phosphonate 5q, NaH, THF; (b) (R)-Me-CBS, BH3–THF, THF; (c) DHP, p-TsOH, CH2Cl2; (d) tetrabutylammonium fluoride, THF; (e) 2-
hydroxymethylpyridine or 3-hydroxymethylpyridine or 4-hydroxymethylpyridine, DEAD, PPh3, THF; (f) 2 N HCl, MeOH; (g) aq NaOH, MeOH, THF.

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
3509
Phosphonates:
MeO
MeO
R1
P
O
O
5
CH3
OMe
Br
R1 =
*
*
*
*
*
a
e
b
c
d
H
N
N
N
N
O
*
*
S
*
*
*
g
h
i
f
Cl
CH3
N
N
N
CH3
O
S
O
*
O
*
*
j
m
k
l
H3C
CH3
Cl
N
N
N
OTBS
*
O
O
*
*
O
CH3
*
p
q
n
o
EtO
EtO
P
O
N
O
r
Figure 3. Phosphonates for the synthesis of analogs listed in Tables.
Table 3
Table 4
PLGA microsphere entrapping efficiency of representa-
tive compounds
In vitro release profile of representative compounds from PLGA microspheres
Compound
Remaining (%)
Compound
Entrapping efficiency (%)
Days
14
1
3i
3k
4i
4m
4n
22.0
77.3
17.2
59.7
70.2
60.6
0
7
21
28
3i
100
100
100
100
94.8
95.7
99.6
91.1
88.6
93.1
95
83.5
80.1
85.2
86.6
46.3
60.8
79.3
87.6
4i
4ma
4n
100
a
Containing 5% of methanol in the buffer system because of low solubility of 5m.
Prior to the rat in vivo evaluation, the compounds were also
investigated for their entrapping efficiency from PLGA micro-
spheres. As described in Table 3, compounds 3i, 4i, 4m and 4n,
which showed relatively higher clogP values, displayed better
entrapping efficacy relative to the others while 3k, which showed
a lower clogP value, displayed a lower entrapping effect. Expected
interaction between basic residue of 3k and acidic residue of PLGA
microsphere was not demonstrated.
They were also biologically evaluated for their effective dosage
for the rat bone fracture healing model. Results are summarized in
Table 5. Using the PLGA microsphere formulation described above,
the efficacy of 3i, 4i, 4m and 4n in the rat bone fracture healing
model was investigated. Table 5 shows the changes in mechanical
properties of the healing fibulae. Seventeen days after the topical
injection of the test compounds, the mechanical breaking strength
of the fractured fibulae increased in a dose-dependent manner. In
Compounds 3i, 4i, 4m and 4n were then evaluated for their
in vitro release profiles from PLGA microspheres. As shown in
Table 4, the in vitro sustained release of 3i, 4i, 4m and 4n from
PLGA microspheres over 28 days was observed by measuring their
remaining percentage in the PLGA microspheres. The remaining
percentage of 3i, 4i, 4m and 4n in PLGA microspheres was 46.3%,
60.8%, 79.3% and 87.6% after 28 days, respectively. These results
prompted us to investigate the rat in vivo study of these com-
pounds using their PLGA formulation. According to their remaining
percentages (%), a sufficient amount of test compounds remained
in the PLGA microspheres even after 28 days of incubation. Thus,
successful sustained release of these compounds from PLGA micro-
spheres was observed.
the fibulae treated with topical injection of 30, 100, 300 lg/kg of
3i/PLGA, the breaking strength reached 95.4%, 136%, 154% of non-
fractured fibulae, respectively, while it was 64.1% in the vehicle-
treated fibulae. Among the 2-mercaptothiazole-4-carboxylic acid
analogs, 4i started to show efficacy at 10
lg/kg of the PLGA formu-
lation. In the fibulae treated with topical injection of 1, 3, 10, 30
lg/
kg of 4i/PLGA, the breaking strength reached 76.1%, 83.3%, 104%,
116% of nonfractured fibulae, respectively, while it was 56.9% in
the vehicle-treated fibulae. Based on the results described above,
these subtype-selective agonists are expected to be promising drug
candidates for treating bone fracture healing.
To evaluate the safety of these subtype-selective agonists, the
effect of 3i/PLGA (300, 1000, 3000 lg/kg), one of the representative

3510
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
Table 5
Biological evaluation of representative compounds in a rat bone fracture healing model
Compound
Functional assay (EC50, nM)
Breaking strength (% intact) compound/PLGA (lg/kg)
rEP2
rEP4
Vehicle
1
3
10
30
100
300
3i/PLGA
4i/PLGA
4m/PLGA
4n/PLGA
NT
7.0
5.3
8.8
1.6
64.1 4.7
56.9 4.0
69.9 16.9
69.9 16.9
NT
76.1 8.2
NT
NT
NT
95.4 8.8
136.3 9.5*
NT
136.0 38.3
124.6 9.9
154 18*
NT
144.3 27.6
167.4 26.7
1500
>104
3200
83.3 6.3
77.7 12.0
84.4 14.5
104.2 7.3*
87.7 7.7
87.1 6.4
116.6 11.7*
98.1 5.4
NT
71.6 8.1
NT = not tested.
*
p <0.05 Dunnett0s test, n = 10.
ethyl acetate (EtOAc), methanol (MeOH), tetrahydrofuran (THF),
dichloromethane (CH2Cl2), chloroform (CHCl3), dimethoxyethane
(DME), acetonitrile (CH3CN), tert-butyl methyl ether (MTBE), 1,3-
dimethyl-2-imidazolidinone (DMI), N,N0-dimethylpropyleneurea
(DMPU), tetrabutylammonium fluoride (TBAF).
Table 6
Effect of compound 3i/PLGA on mean blood pressure and heart rate
Biological evaluation
% Change 3i/PLGA (lg/kg)
300
98.4
106.3
1000
3000
Mean blood pressure
Heart rate
98.2
104.6
97.3
111.4
5.1.2. N-Methoxy-N-methyl-2-(3-bromophenyl)acetamide (6)
To a stirred solution of N,O-dimethylhydroxylamine hydrochlo-
ride (6.81 g, 69.8 mmol), 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (11.6 g, 60.5 mmol) and triethylamine
(9.71 mL, 60.5 mmol) in CH3CN (150 mL) was added a solution of
(2-bromophenyl)acetic acid (10.0 g, 46.5 mmol) in CH3CN
(50 mL) at room temperature under argon atmosphere. After being
stirred for 2 h, the reaction was quenched with water. The reaction
mixture was diluted with EtOAc. The organic layer was washed
with 2 N HCl, water, brine, and dried over MgSO4. The organic sol-
vent was removed by evaporation to give a Weinreb amide 6 as a
colorless oil (8.16 g, 68%). 1H NMR (300 MHz, CDCl3): d 7.47–7.43
(m, 1H), 7.41–7.35 (m, 1H), 7.25–7.17 (m, 2H), 3.74 (s, 2H), 3.64
(s, 3H), 3.20 (s, 3H).
n = 4.
compounds of this series, on blood pressure and heart rate was
investigated. Results are summarized in Table 6. At the higher
dosages, 3i/PLGA did not influence blood pressure and heart rate
while it slightly increased heart rate at a dose of 3000 lg/kg.
4. Conclusion
In conclusion, two series of compounds were optimized in
terms of their PLGA microsphere formulation while retaining EP4
subtype-selectivity and EP2/EP4 dual selectivity with good
potency. Sustained release of the test compounds seemed to be
successful based on their in vitro release profile from PLGA micro-
spheres. Single topical injection of several representative com-
pounds as their PLGA microsphere formulation was found to be
5.1.3. Dimethyl 3-(3-bromophenyl)-2-oxopropanephosphonate
(5a)
To a stirred solution of dimethyl methylphosphonate (4.94 mL,
45.6 mmol) in toluene (100 mL) was added dropwise a solution of
n-BuLi (1.57 M in hexane, 47.3 mmol) at ꢀ78 °C under argon atmo-
sphere, and stirring was continued for 1 h at the same temperature.
To the reaction mixture was added a solution of 6 (32.6 mmol) in
toluene (20 mL), and stirring was continued for additional 2 h at
the same temperature. The reaction was quenched with acetic acid.
The reaction mixture was allowed to warm up to room tempera-
ture with stirring. The reaction mixture was diluted with EtOAc.
The organic layer was washed with water, brine, and dried over
MgSO4. The organic solvent was removed by evaporation and the
resulting residue was purified by column chromatography on silica
gel (hexane/EtOAc, 2:1–0:1) to give a phosphonate 5a as a yellow
oil (7.76 g, 74%). 1H NMR (300 MHz, CDCl3): d 7.43–7.38 (m, 2H),
7.25–7.13 (m, 2H), 3.83 (s, 2H), 3.82 (s, 3H), 3.78 (s, 3H), 3.12 (d,
J = 22.5 Hz, 2H).
effective in a rat bone fracture healing model. A series of c-lactam
5-thia PGE analogs and a series of 2-mercaptothiazole-4-carboxylic
acid PGE analogs showed EP4 subtype-selectivity and EP2/EP4 dual
selectivity in a mouse receptor assay while the series showed EP4
selectivity because of the reduced rat EP2 functional activity of the
2-mercaptothiazole-4-carboxylic acid PGE analogs. More details
will be reported in due course.
5. Experimental
5.1. Chemistry
5.1.1. General procedure
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 using deuter-
ated chloroform (CDCl3), deuterated methanol (CD3OD) and deu-
terated dimethylsulfoxide (DMSO-d6) as the solvent. Fast atom
bombardment (FABMS, HRMS) and electron ionization (EI) mass
spectra were obtained on a JEOL JMS-DX303HF spectrometer.
Atmospheric pressure chemical ionization (APCI) mass spectra
were determined on a HITACHI MI200H spectrometer. Infrared
spectra (IR) were measured in a Perkin-Elmer FT-IR 1760X spec-
trometer. Column chromatography was carried out on silica gel
5.1.4. N-Methoxy-N-methyl-2-(40-methyl-3-biphenylyl) acetamide
(7a)
To a stirred mixture consisting of a solution of 6 (1.20 g,
4.66 mmol) in DME (15 mL) and aqueous Na2CO3 (742 mg,
7.00 mmol) in H2O (12 mL) were added 4-methylphenylboronic
acid (951 mg, 7.00 mmol) and tetrakis(triphenylphosphine)palla-
dium (60 mg, 0.052 mmol) under argon atmosphere. After being
stirred at 90 °C for 2 h, the reaction mixture was cooled to room
temperature. The resulting mixture was filtered through a pad of
Celite. The filtrate was evaporated and the resulting residue puri-
fied by column chromatography on silica gel (hexane/EtOAc, 3:1–
1:1) to afford 7a as a pale brown solid (1.50 g, 100%). 1H NMR
(300 MHz, CDCl3): d 7.53–7.43 (m, 3H), 7.41–7.34 (m, 1H), 7.33–
7.19 (m, 4H), 3.83 (s, 2H), 3.62 (s, 3H), 3.21 (s, 3H), 2.38 (s, 3H).
[Merck Silica Gel 60 (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; N,N-dimethyl-
formamide (DMF), dimethylsulfoxide (DMSO), ethanol (EtOH),

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
3511
5.1.5. Dimethyl [3-(40-methyl-3-biphenylyl)-2-oxopropyl]
phosphonate (5c)
Compound 5c was prepared from 7a according to the same pro-
cedure as described for the preparation of 5a from 6 as a colorless
oil (1.17 g, 76%). 1H NMR (300 MHz, CDCl3): d 7.53–7.37 (m, 4H),
7.29–7.16 (m, 4H), 3.96 (s, 2H), 3.81 (s, 3H), 3.77 (s, 3H), 3.10
(d, J = 22.5 Hz, 2H), 2.36 (s, 3H).
To a stirred solution of the above-described trifluoromethane-
sulfonate (66 mmol) in DMF (70 mL) were added triethylamine
(36 mL, 260 mmol), benzofuran-2-boronic acid (21.0 g, 130 mmol),
bis(triphenylphosphine)palladium dichloride (4.2 g, 5.98 mmol)
under argon atmosphere. After being stirred at 60 °C for 15 h, the
reaction mixture was cooled to room temperature, and was
filtered through a pad of Celite. The filtrate was evaporated and
purified by column chromatography on silica gel (hexane/EtOAc,
2:1) to afford 8b as a pale brown oil (15.1 g, 77% in three steps).
1H NMR (300 MHz, CDCl3): d 7.84–7.71 (m, 3H), 7.69–7.61 (m,
1H), 7.61–7.50 (m, 1H), 7.46–7.35 (m, 1H), 7.34–7.19 (m, 2H),
7.05–7.00 (m, 1H), 3.84 (s, 2H), 3.65 (s, 3H), 3.23 (s, 3H).
5.1.6. N-Methoxy-N-methyl-2-(40-methoxy-3-biphenylyl)
acetamide (7b)
Compound 7b was prepared from 6 according to the same pro-
cedure as described for the preparation of 7a from 6 as a pale
yellow solid (1.58 g, 100%). 1H NMR (300 MHz, CDCl3): d 7.57–
7.32 (m, 4H), 7.2–7.26 (m, 2H), 6.90–7.01 (m, 2H), 3.85 (s, 3H),
3.83 (s, 2H), 3.63 (s, 3H), 3.21 (s, 3H).
5.1.12. Dimethyl {3-[3-(1-benzofuran-2-yl)phenyl]-2-oxopropyl}
phosphonate (5j)
Compound 5j was prepared from 8b according to the same pro-
cedure as described for the preparation of 5a from 6 (Scheme 1a) as
a yellow oil (13.9 g, 76%). 1H NMR (300 MHz, CDCl3): d 7.82–7.72
(m, 2H), 7.62–7.49 (m, 2H), 7.43 (t, J =7.69 Hz, 1H), 7.34–7.18 (m,
3H), 7.06–7.02 (m, 1H), 3.98 (s, 2H), 3.83 (s, 3H), 3.79 (s, 3H),
3.16 (d, J = 22.3 Hz, 2H).
5.1.7. Dimethyl [3-(40-methoxy-3-biphenylyl)-2-oxopropyl]
phosphonate (5d)
Compound 5d was prepared from 7b according to the same pro-
cedure as described for the preparation of 5a from 6 as a white
solid (1.02 g, 63%). 1H NMR (300 MHz, CDCl3): d 7.56–7.35 (m,
5H), 7.20–7.13 (m, 1H), 7.02–6.94 (m, 2H), 3.95 (s, 2H), 3.85 (s,
3H), 3.81 (s, 3H), 3.77 (s, 3H), 3.14 (d, J = 22.0 Hz, 2H).
5.1.13. N-Methoxy-N-methyl-2-[3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl]acetamide (8c)
5.1.8. N-Methoxy-N-methyl-2-(3-(1H-indol-5-yl)phenyl)
acetamide (7c)
To a stirred solution of 8a (4.87 mmol) in CH3CN (20 mL) and
triethylamine (2.04 mL, 14.6 mmol) were added pinacolborane
To a stirred solution of the above-described Weinreb amide 6
(500 mg, 1.94 mmol) in DME (11 mL) and 2 M aqueous sodium car-
bonate (2.90 mL, 5.80 mmol) were added indole-2-boronic acid
(623 mg, 3.87 mmol), (S)-2,20-bis(diphenylphosphino)-1,10-binaph-
thyl (BINAP) (240 mg, 0.386 mmol) and palladium acetate (44 mg,
0.194 mmol) at under argon atmosphere. After being stirred at
80 °C for 15 h, the reaction mixture was cooled to room tempera-
ture. The resulting mixture was filtered through a pad of Celite,
and the filtrate was evaporated and purified by column chromatog-
raphy on silica gel (hexane/EtOAc, 4:1–1:1) to afford 7c as a brown
oil (411 mg, 72%). 1H NMR (300 MHz, CDCl3): d 8.23 (br s, 1H), 7.86
(s, 1H), 7.61–7.49 (m, 2H), 7.46–7.44 (m, 2H), 7.38 (t, J = 7.7 Hz, 1H),
7.29–7.21 (m, 2H), 6.64–6.57 (m, 1H), 3.85 (s, 2H), 3.62 (s, 3H),3.21
(s, 3H).
(1.06 mL,
7.31 mmol)
and
dichloro[1,10-bis(diphenylphos-
phino)ferrocene]palladium CH2Cl2 complex (200 mg, 0.244 mmol)
at under argon atmosphere. After being stirred at 80 °C for 18 h,
the reaction mixture was cooled to room temperature. The result-
ing mixture was filtered through a pad of Celite, and the filtrate
was diluted with EtOAc. The organic layer was washed with 1 N
HCl, brine and dried over Na2SO4. The organic solvent was re-
moved by evaporation and the resulting residue was purified by
column chromatography on silica gel (hexane/EtOAc, 3:1–1:1)
to give 8c as
a
pale brown oil (491 mg, 33%). 1H NMR
(300 MHz, CDCl3): d 5.73 (dd, J = 15.9, 6.0 Hz, 1H), 5.52 (dd,
J = 15.9, 8.4 Hz, 1H), 4.21–4.02 (m, 4H), 3.65 (m, 1H), 3.10 (m,
1H), 2.78–2.50 (m, 4H), 2.45–2.20 (m, 5H), 1.98–1.20 (m, 6H),
0.93 (t, J = 6.9 Hz, 3H), 0.65 (m, 1H), 0.40 (m, 2H), ꢀ0.02 (m, 2H).
5.1.9. Dimethyl {3-[3-(1H-indol-5-yl)phenyl]-2-oxopropyl}
phosphonate (5g)
5.1.14. N-Methoxy-N-methyl-2-[3-(2-pyridinyl)phenyl]
acetamide (8d)
Compound 5g was prepared from 7c according to the same pro-
cedure as described for the preparation of 5a from 6 as a white so-
lid (137 mg, 28%). 1H NMR (300 MHz, CDCl3): d 8.28 (br s, 1H),
7.81–7.87 (m, 1H), 7.57 (d, J = 7.69 Hz, 1H), 7.37–7.52 (m, 4H),
7.21–7.28 (m, 1H), 7.17 (d, J = 7.42 Hz, 1H), 6.57–6.63 (m, 1H),
3.96 (s, 2H), 3.81 (s, 3H), 3.77 (s, 3H), 3.15 (d, J = 3.1 Hz, 2H).
To a stirred solution of the above-described Weinreb amide 8c
(491 mg, 1.61 mmol) in DME (5 mL) and potassium phosphate
(1.71 g, 8.05 mmol) were successively added 2-bromopyridine
(381 mg, 2.41 mmol) and dichloro[1,10-bis(diphenylphosphino)
ferrocene]palladium CH2Cl2 complex (132 mg, 0.161 mmol) under
argon atmosphere. After being stirred at 80 °C for 16 h, the reaction
mixture was cooled to room temperature. The resulting mixture
was filtered through a pad of Celite, and the filtrate was diluted
with EtOAc. The organic layer was washed with H2O, brine and
dried over Na2SO4. The solvent was removed by evaporation and
the resulting residue was purified by column chromatography on
silica gel (hexane/EtOAc, 2:1–1:1) to give 8d as a pale brown oil
(289 mg, 70%). 1H NMR (300 MHz, CDCl3): d 8.68 (m, 1H), 7.93
(m, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.74 (m, 2H), 7.44 (t, J = 7.8 Hz,
1H), 7.34 (m, 1H), 7.22 (m, 1H), 3.87 (s, 2H), 3.63 (s, 3H), 3.21 (s, 3H).
5.1.10. 2-(3-Hydroxyphenyl)-N-methoxy-N-methylacetamide
(8a)
Compound 8a was prepared from 3-hydroxyphenylacetic acid
according to the same procedure as described for the preparation
of 6 from 3-bromophenylacetic acid as a white solid (12.9 g, 100%).
5.1.11. 2-[3-(1-Benzofuran-2-yl)phenyl]-N-methoxy-N-
methylacetamide (8b)
To a stirred solution of 8a (12.9 g, 66.0 mmol) in CH2Cl2
(130 mL) and pyridine (6.4 mL, 79 mmol) was added trifluoro-
methanesulfonic anhydride (10.3 mL, 73 mmol) at 0 °C under ar-
gon atmosphere. After being stirred for 15 min, the reaction
mixture was poured into cold 1 N HCl, and extracted with EtOAc.
The organic layer was washed with brine, dried over MgSO4. The
organic solvent was removed by evaporation to give a trifluoro-
methanesulfonate as a pale brown oil.
5.1.15. Diethyl {2-oxo-3-[3-(2-pyridinyl)phenyl]propyl}
phosphonate (5r)
Compound 5r was prepared from 8d using diethylmethyl phos-
phonate instead of dimethylmethyl phosphonate according to the
same procedure as described for the preparation of 5a from 6
(Scheme 1a) as a colorless oil (276 mg, 70%). 1H NMR (300 MHz,

3512
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
CDCl3): d 8.70 (m, 1H), 7.90 (m, 2H), 7.76–7.70 (m, 2H), 7.46 (t,
J = 7.8 Hz, 1H), 7.32–7.10 (m, 2H), 4.20–4.03 (m, 4H), 4.00 (s, 2H),
3.13 (d, J = 22.8 Hz, 2H), 1.40–1.25 (m, 6H).
To a stirred solution of N,O-dimethylhydroxylamine hydrochlo-
ride (0.53 g, 5.45 mmol), 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (1.04 g, 5.45 mmol) and triethylamine
(1.52 mL, 10.9 mmol) in CH3CN (10 mL) was added a solution of
the above-described phenylacetic acid (1.15 g, 4.54 mmol) in
CH3CN (3 mL) at room temperature under argon atmosphere. After
being stirred for 6 h, the reaction was quenched with water. The
reaction mixture was diluted with EtOAc. The organic layer was
washed with 2 N HCl, water, brine, and dried over MgSO4. The
organic solvent was removed by evaporation to give a Weinreb
amide 10a as a pale brown solid (670 mg, 50%).
5.1.16. N-Methoxy-N-methyl-2-[3-(tert-butyldimethylsilyloxy)
phenyl]acetamide (8e)
To a stirred solution of 8a (12.8 g, 65.7 mmol) in DMF (60 mL)
and imidazole (4.47 g, 65.7 mmol) was added t-butyldimethylsilyl
chloride (9.90 g, 65.7 mmol) at room temperature under argon
atmosphere. After being stirred for 1 h, the reaction mixture was
diluted with EtOAc. The organic layer was washed with H2O twice,
brine and dried over MgSO4. The solvent was removed by evapora-
tion to give 8e as a pale brown oil (19.3 g, 95%). 1H NMR (300 MHz,
CDCl3): d 7.24–7.13 (m, 1H), 6.87–6.65 (m, 3H), 3.82 (s, 2H), 3.81 (s,
3H), 3.77 (s, 3H), 3.09 (d, J = 22.5 Hz, 2H), 0.98 (s, 9H), 0.18 (s, 6H).
5.1.20. Dimethyl {3-[3-(1,3-benzoxazol-2-yl)phenyl]-2-oxopropyl}
phosphonate (5h)
Compound 5h was prepared from 10a according to the same
procedure as described for the preparation of 5a from 6 as a yellow
oil (650 mg, 80%). 1H NMR (300 MHz, CDCl3): d 8.19 (d, J = 7.7 Hz,
1H), 8.15–8.10 (m, 1H), 7.83–7.72 (m, 1H), 7.64–7.56 (m, 1H),
7.55–7.48 (m, 1H), 7.44–7.32 (m, 3H), 4.03 (s, 2H), 3.84 (s, 3H),
3.81 (s, 3H), 3.18 (d, J = 22.5 Hz, 2H).
5.1.17. Dimethyl {3-[3-(tert-butyldimethylsilyloxy)phenyl]-2-
oxopropyl}phosphonate (5q)
Compound 5q was prepared from 8e according to the same pro-
cedure as described for the preparation of 5a from 6 (Scheme 1a) as
a colorless oil (16.3 g, 70%). 1H NMR (300 MHz, CDCl3): d 7.24–7.13
(m, 1H), 6.87–6.65 (m, 3H), 3.87–3.70 (m, 8H), 3.09 (d, J = 22.5 Hz,
2H), 0.98 (s, 9H), 0.24–0.12 (m, 6H).
5.1.21. 2-(3-Iodophenyl)-5-methyl-1,3-benzoxazole (9b)
To a stirred mixture of 3-iodobenzoic acid (5.00 g, 20.0 mmol)
and polyphosphoric acid (25.0 g) was added 2-amino-4-methyl-
phenol (2.49 g, 20.2 mmol) under argon atmosphere. After being
stirred at 200 °C for 5 h, the reaction mixture was cooled to room
temperature. The resulting mixture was diluted with EtOAc and
THF, and washed with H2O, saturated aqueous NaHCO3, brine
and dried over Na2SO4. The organic solvent was removed by evap-
oration and to give 9b as a beige powder (5.99 g, 88%). 1H NMR
(300 MHz, DMSO-d6): d 8.60 (m, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.85
(d, J = 8.0 Hz, 1H,) 7.55 (s, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.33–7.22
(m, 1H), 7.22–7.15 (m, 1H), 2.49 (s, 3H).
5.1.18. 2-(3-Iodophenyl)-1,3-benzoxazole (9a)
To a stirred mixture of 3-iodobenzoic acid (5.00 g, 20.0 mmol)
and polyphosphoric acid (25.0 g) was added 2-aminophenol
(2.20 g, 20.2 mmol) under argon atmosphere. After being stirred
at 200 °C for 5 h, the reaction mixture was cooled to room temper-
ature. The resulting mixture was diluted with EtOAc and THF and
washed with H2O, saturated aqueous NaHCO3, brine and dried over
Na2SO4. The organic solvent was removed by evaporation to give
9a as a pale brown powder (6.16 g, 96%). 1H NMR (300 MHz,
DMSO-d6): d 8.66–8.59 (m, 1H), 8.27–8.18 (m, 1H), 7.91–7.83 (m,
1H), 7.82–7.74 (m, 1H), 7.64–7.54 (m, 1H), 7.43–7.34 (m, 2H),
7.31–7.21 (m, 1H).
5.1.22. N-Methoxy-N-methyl-2-[3-(5-methyl-1,3-benzoxazol-2-
yl)phenyl]acetamide (10b)
To a stirred suspension of sodium hydride (63% dispersion in
mineral oil, 1.37 g, 35.7 mmol) in DMI (18 mL) was added dropwise
a solution of diethyl malonate (5.42 gm 35.7 mmol) at 0 °C under
argon atmosphere, and stirring was continued for 20 min at room
temperature. To the reaction mixture were added 9b (5.98 g,
17.8 mmol) and copper iodide (6.80 g, 35.7 mmol), and stirring
was continued for additional 4 h at 120 °C. After being cooled to
room temperature, the resulting mixture was poured into a mix-
ture of saturated aqueous NH4Cl and EtOAc under cooling. The
resulting mixture was filtered through a pad of Celite, and the fil-
trate was washed with H2O, brine, and dried over Na2SO4. The or-
ganic solvent was removed by evaporation and the resulting
residue was purified by column chromatography on silica gel (hex-
ane/EtOAc, 30:1–5:1) to give a diester as a yellow oil (6.27 g, 96%).
1H NMR (300 MHz, CDCl3): d 8.30–8.18 (m, 1H), 7.67–7.60 (m, 1H),
7.57–7.50 (m, 2H), 7.46 (d, J = 8.2 Hz, 1H), 7.36 (d, J = 1.1 Hz, 1H),
7.21–7.13 (m, 1H), 4.73 (s, 1H), 4.31–4.13 (m, 4H), 2.49 (s, 3H),
1.37–1.20 (m, 6H).
A solution of the above-described diester (6.27 g, 17.1 mmol) in
DME (30 mL) was treated with 2 N NaOH (36 mL, 72 mmol) at
60 °C for 4 h. The resulting reaction mixture was treated with conc.
HCl (8 mL), and stirred at 90 °C for additional 1 h. To the resulting
reaction mixture was added 5 N NaOH and diisopropyl ether at
0 °C with vigorous stirring. The resulting precipitates were col-
lected by filtration, dissolved in AcOEt and treated with 2 N HCl.
The aqueous layer of the filtrate was further extracted with AcOEt
after treating again with 2 N HCl. The combined organic layers
were washed with H2O, brine, and dried over Na2SO4. The organic
solvent was removed by evaporation and to give a phenylacetic
acid as a beige powder (2.53 g, 55%). 1H NMR (300 MHz, CDCl3):
5.1.19. N-Methoxy-N-methyl-2-[3-(1,3-benzoxazol-2-yl)phenyl]
acetamide (10a)
To a stirred suspension of sodium hydride (63% dispersion in
mineral oil, 1.37 g, 35.7 mmol) in DMI (18 mL) was added dropwise
a solution of diethyl malonate (5.42 g, 35.7 mmol) at 0 °C under ar-
gon atmosphere, and stirring was continued for 20 min at room
temperature. To the stirred mixture was successively added 9a
(6.16 g, 19.2 mmol) and copper iodide (6.80 g, 35.7 mmol), and
stirring was continued for additional 4 h at 120 °C. The reaction
mixture was cooled to room temperature and poured into a mix-
ture of cold saturated aqueous NH4Cl and EtOAc. The resulting
mixture was filtered through a pad of Celite and the filtrate was
washed with H2O, brine, and dried over Na2SO4. The organic sol-
vent was removed by evaporation and the resulting residue was
purified by column chromatography on silica gel (hexane/EtOAc,
30:1–5:1) to give a diester as a yellow oil (6.27 g, 11.9 mmol).
A solution of the above-described diester (6.27 g, 11.9 mmol) in
DME (15 mL) was treated with 2 N NaOH (24 mL, 120 mmol) at
60 °C for 2 h. The resulting reaction mixture was treated with conc.
HCl (12 mL) and stirred at 60 °C for additional 2 h. To the reaction
mixture was added 5 N NaOH and diisopropyl ether at 0 °C with
vigorous stirring. The resulting precipitates were collected by fil-
tration, dissolved in AcOEt and treated with 2 N HCl. The aqueous
layer of the filtrate was further extracted with AcOEt after treating
again with 2 N HCl. The combined organic layers were washed
with H2O, brine, and dried over Na2SO4. The organic solvent was
removed by evaporation to give a phenylacetic acid as a beige pow-
der (1.30 g, 43% in two steps).

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
3513
d 8.25–8.21 (m, 1H), 8.18–8.09 (m, 1H), 7.61–7.57 (m, 1H), 7.54–
7.40 (m, 3H), 7.21–7.13 (m, 1H), 3.78 (s, 2H), 2.48 (s, 3H).
To a stirred solution of N,O-dimethylhydroxylamine hydrochlo-
ride (1.31 g, 13.4 mmol), 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (2.22 g, 11.6 mmol) and triethylamine
(1.87 mL, 13.4 mmol) in CH3CN (20 mL) was added a solution of
the above-described phenylacetic acid (2.53 g, 9.48 mmol) in
CH3CN (3 mL) at room temperature under argon atmosphere. After
being stirred for 1 h, the reaction was quenched with water. The
resulting reaction mixture was diluted with EtOAc, washed with
2 N HCl, water, then brine, and dried over MgSO4. The organic sol-
vent was removed by evaporation to give a Weinreb amide 10b as
a light yellow powder(2.19 g, 75%). 1H NMR (300 MHz, CDCl3): d
8.20–8.16 (m, 1H), 8.16–8.10 (m, 1H), 7.55 (m, 1H), 7.52–7.41
(m, 3H), 7.16 (dd, J = 8.2, 1.1 Hz, 1H), 3.88 (s, 2H),3.67 (s,
3H),3.22 (s, 3H), 2.49 (s, 3H).
brown oil (3.86 g, 84% in two steps). 1H NMR (300 MHz, CDCl3): d
8.25–8.10 (m, 2H), 7.52–7.43 (m, 2H), 7.40 (d, J = 8.0 Hz, 1H),
7.25–7.19 (m, 1H), 7.17–7.10 (m, 1H), 3.89 (s, 2H), 3.67 (s, 3H),
3.23 (s, 3H), 2.67 (s, 3H).
5.1.29. Dimethyl {3-[3-(4-methyl-1,3-benzoxazol-2-yl)phenyl]-
2-oxopropyl}phosphonate (5n)
Compound 5n was prepared from 10d according to the same
procedure as described for the preparation of 5a from 6 (Scheme
1a) as a pale pink powder (2.9 g, 92%). 1H NMR (300 MHz, CDCl3):
d 8.23–8.10 (m, 2H), 7.56–7.46 (m, 1H), 7.44–7.35 (m, 2H), 7.30–
7.20 (m, 1H), 7.18–7.11 (m, 1H), 4.03 (s, 2H), 3.84 (s, 3H), 3.78 (s,
3H), 3.18 (d, J = 23.1 Hz, 2H), 2.67 (s, 3H).
5.1.30. 2-(3-Iodophenyl)-5,7-dimethyl-1,3-benzoxazole (9e)
Compound 9e was prepared from 3-iodobenzoic acid and 2-
amino-4,6-dimethylphenol according to the same procedure as de-
scribed for the preparation of 9a from 3-iodobenzoic acid and 2-
aminophenol as a reddish purple powder (5.67 g, 80%). 1H NMR
(300 MHz, DMSO-d6): d 8.49–8.44 (m, 1H), 8.22–8.14 (m, 1H),
8.02–7.94 (m, 1H), 7.44–7.35 (m, 2H), 7.07 (s, 1H), 2.51 (s, 3H),
2.39 (s, 3H).
5.1.23. Dimethyl {3-[3-(5-methyl-1,3-benzoxazol-2-yl)phenyl]-
2-oxopropyl}phosphonate (5l)
Compound 5l was prepared from 10b according to the same
procedure as described for the preparation of 5a from 6 (Scheme
1a) as a colorless oil (2.10 g, 81%). 1H NMR (300 MHz, CDCl3): d
8.20–8.14 (m, 1H), 8.13–8.08 (m, 1H), 7.59–7.37 (m, 4H), 7.21–
7.14 (m, 1H), 4.02 (s, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 3.18 (d,
J = 22.5 Hz, 2H), 2.49 (s, 3H).
5.1.31. N-Methoxy-N-methyl-2-[3-(5,7-dimethyl-1,3-
benzoxazol-2-yl)phenyl]acetamide (10e)
Compound 10e was prepared from 9e according to the same
procedure as described for the preparation of 10a from 9a as a
red oil (1.30 g, 49%). 1H NMR (300 MHz, CDCl3): d 8.22–8.10 (m,
2H), 7.52–7.44 (m, 2H), 7.37 (s, 1H), 6.97 (s, 1H), 3.89 (s, 2H),
3.67 (s, 3H), 3.22 (s, 3H), 2.55 (s, 3H), 2.44 (s, 3H).
5.1.24. 2-(3-Iodophenyl)-6-methyl-1,3-benzoxazole (9c)
Compound 9c was prepared from 3-iodobenzoic acid and 2-
amino-5-methylphenol according to the same procedure as de-
scribed for the preparation of 9a from 3-iodobenzoic acid and 2-
aminophenol as a gray powder. 1H NMR (300 MHz, DMSO-d6): d
8.49–8.41 (m, 1H), 8.20–8.14 (m, 1H), 8.02–7.93 (m, 1H), 7.68 (d,
J = 8.0 Hz, 1H), 7.62–7.5 (m, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.28–7.19
(m, 1H), 2.47 (s, 3H).
5.1.32. Dimethyl {3-[3-(5,7-dimethyl-1,3-benzoxazol-2-yl)phenyl]-
2-oxopropyl}phosphonate (5o)
Compound 5o was prepared from 10e according to the same
procedure as described for the preparation of 5a from 6 (Scheme
1a) as an orange oil (1.14 g, 74%). 1H NMR (300 MHz, CDCl3): d
8.21–8.15 (m, 1H), 8.13–8.08 (m, 1H), 7.56–7.46 (m, 1H), 7.42–
7.35 (m, 2H), 6.98 (s, 1H), 4.02 (s, 2H), 3.84 (s, 3H), 3.80 (s, 3H),
3.18 (d, J = 22.8 Hz, 2H), 2.55 (s, 3H), 2.45 (s, 3H).
5.1.25. N-Methoxy-N-methyl-2-[3-(6-methyl-1,3-benzoxazol-2-
yl)phenyl]acetamide (10c)
Compound 10c was prepared from 9c according to the same
procedure as described for the preparation of 10a from 9a as a
brown oil (2.74 g, 60% in two steps). 1H NMR (300 MHz, CDCl3): d
8.20–8.07 (m, 2H), 7.63 (d, J = 8.2 Hz, 1H), 7.51–7.44 (m, 2H),
7.40–7.35 (m, 1H), 7.20–7.13 (m, 1H), 3.88 (s, 2H), 3.67 (s, 3H),
3.22 (s, 3H), 2.51 (s, 3H).
5.1.33. 2-(3-Iodophenyl)-5-chloro-1,3-benzoxazole (9f)
Compound 9f was prepared from 3-iodobenzoic acid and
2-amino-4-chlorophenol according to the same procedure as
described for the preparation of 9a from 3-iodobenzoic acid and
2-aminophenol as brown powder (6.4 g).
5.1.26. Dimethyl {3-[3-(6-methyl-1,3-benzoxazol-2-yl)phenyl]-
2-oxopropyl}phosphonate (5m)
Compound 5m was prepared from 10c according to the same
procedure as described for the preparation of 5a from 6 (Scheme
1a) as a brown oil (2.74 g, 60%). 1H NMR (300 MHz, CDCl3): d
8.19–8.13 (m, 1H), 8.12–8.07 (m, 1H), 7.63 (d, J = 8.2 Hz, 1H),
7.55–7.46 (m, 1H), 7.42–7.35 (m, 2H), 7.21–7.13 (m, 1H), 4.02 (s,
2H), 3.84 (s, 3H), 3.80 (s, 3H), 3.18 (d, J = 23.1 Hz, 2H), 2.51 (s, 3H).
5.1.34. N-Methoxy-N-methyl-2-[3-(5-chloro-1,3-benzoxazol-2-yl)
phenyl]acetamide (10f)
Compound 10f was prepared from 9f according to the same
procedure as described for the preparation of 10a from 9a as a yel-
low oil (354 mg, 6%).
5.1.35. Dimethyl {3-[3-(5-chloro-1,3-benzoxazol-2-yl)phenyl]-
2-oxopropyl}phosphonate (5p)
Compound 5p was prepared from 10f according to the same
procedure as described for the preparation of 5a from 6 (in Scheme
1a) as a yellow oil (130 mg, 31%).
5.1.27. 2-(3-Iodophenyl)-4-methyl-1,3-benzoxazole (9d)
Compound 9d was prepared from 3-iodobenzoic acid and
2-amino-3-methylphenol according to the same procedure as
described for the preparation of 9a from 3-iodobenzoic acid and
2-aminophenol as a gray powder(5.0 g, 74%). 1H NMR (300 MHz,
DMSO-d6): d 8.51–8.47 (m, 1H), 8.23–8.17 (m, 1H), 8.03–7.96 (m,
1H), 7.64–7.55 (m, 1H), 7.47–7.29 (m, 2H), 7.26–7.21 (m, 1H),
2.59 (s, 3H).
5.1.36. 2-(3-Iodophenyl)-1,3-benzothiazole (9g)
Compound 9g was prepared from 3-iodobenzoic acid and 2-
aminothiophenol according to the same procedure as described
for the preparation of 9a from 3-iodobenzoic acid and 2-aminophe-
nol as a reddish purple powder (3.15 g, 93%). 1H NMR (300 MHz,
DMSO-d6): d 8.51–8.43 (m, 1H), 8.12–7.98 (m, 2H), 7.96–7.87 (m,
1H), 7.86–7.77 (m, 1H), 7.57–7.46 (m, 1H), 7.45–7.35 (m, 1H),
7.28–7.17 (m, 1H).
5.1.28. N-Methoxy-N-methyl-2-[3-(4-methyl-1,3-benzoxazol-2-
yl)phenyl]acetamide (10d)
Compound 10d was prepared from 9d according to the same
procedure as described for the preparation of 10a from 9a as a

3514
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
5.1.37. N-Methoxy-N-methyl-2-[3-(1,3-benzothiazol-2-yl)
phenyl]acetamide (10g)
5.1.43. Dimethyl {3-[3-(1,3-dihydro-2H-isoindol-2-yl)phenyl]-2-
oxopropyl}phosphonate (5f)
Compound 10g was prepared from 9g according to the same
procedure as described for the preparation of 10a from 9a as an or-
ange oil (791 mg, 43% in three steps). 1H NMR (300 MHz, CDCl3): d
8.10–8.02 (m, 1H), 8.00–7.94 (m, 1H), 7.93–7.87 (m, 1H), 7.53–7.48
(m, 1H), 7.48–7.43 (m, 2H), 7.42–7.34 (m, 1H), 7.26 (s, 1H), 3.88 (s,
2H), 3.67 (s, 3H), 3.22 (s, 3H).
Compound 5f was prepared from 11 according to the same
procedure as described for the preparation of 5a from 6 (Scheme
1a) as pale yellow oil (381 mg, 28%). 1H NMR (300 MHz, CDCl3):
d 7.40–7.24 (m, 5H), 6.68–6.47 (m, 3H), 4.65 (s, 4H), 3.87 (s, 2H),
3.82 (s, 3H), 3.78 (s, 3H), 3.13 (d, J = 22.5 Hz, 2H).
5.1.44. Butyl 4-({2-[(2R)-2-{(1E,3S)-4-[1,10-biphenyl-3-yl]-3-
hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl] ethyl} thio)
butanoate (13a)
To a stirred solution of phosphonate 5b (223 mg, 0.70 mmol) in
THF (3 mL) was added sodium hydride (62.3% in mineral oil,
23.1 mg, 0.60 mmol) at 0 °C under argon atmosphere and stirring
was continued at ambient temperature for 90 min. To the resulting
5.1.38. Dimethyl {3-[3-(1,3-benzothiaxazol-2-yl)phenyl]-2-
oxopropyl}phosphonate (5i)
Compound 5i was prepared from 10g according to the same
procedure as described for the preparation of 5a from 6 (Scheme
1a) as a yellowish-green oil (809 mg, 85%). 1H NMR (300 MHz,
CDCl3):
d
8.11–8.04 (m, 1H), 8.02–7.97 (m, 2H), 7.95–7.88
suspension was added
a solution of aldehyde 12 (158 mg,
(m, 1H), 7.56–7.45 (m, 2H), 7.43–7.33 (m, 2H), 4.02 (s, 2H), 3.84
0.50 mmol) in THF (1 mL) at 0 °C and stirring was continued for
2 h. The reaction was quenched with acetic acid, diluted with EtOAc,
washed with water, brine, and dried over MgSO4. The organic sol-
vent was removed by evaporation to give an enone as a pale yellow
oil. To a stirred solution of the above-described enone (0.50 mmol)
in THF (3.0 mL) was added a solution of (R)-2-methyl-CBS-oxaz-
aborolidine (1.0 M in toluene, 0.15 mL, 0.15 mmol) at room temper-
ature under argon atmosphere. To the resulting reaction mixture
was added dropwise a solution of borane–THF complex (1.0 M in
THF, 0.40 mL, 0.40 mmol) in 5 min. The resulting solution was stir-
red for 1 h, then treated with MeOH (0.1 mL) and stirring was con-
tinued for 5 min. The reaction mixture was diluted with EtOAc,
washed with 1 N HCl, water, saturated NaHCO3, brine, and dried
over Na2SO4. The organic solvent was removed by evaporation,
and the resulting residue was purified by column chromatography
on silica gel (EtOAc/hexane, 2:1–1:10) to give an allyl alcohol 13a
as a yellow viscous oil (180 mg, 72% in two steps). 1H NMR
(300 MHz, CDCl3): d 7.58 (d, J = 7.0 Hz, 2H), 7.54–7.30 (m, 6H),
7.20 (d, J = 7.3 Hz, 1H), 5.84–5.73 (m, 1H), 5.51 (dd, J = 15.4,
8.8 Hz, 1H), 4.39–4.53 (m, 1H), 4.16–4.01 (m, 3H), 3.67–3.51 (m,
1H), 2.98–2.84 (m, 3H), 2.69–2.10 (m, 10H), 1.97 (m, 1H), 1.93–
1.79 (m, 2H), 1.77–1.50 (m, 2H), 1.45–1.29 (m, 2H), 0.92 (t,
J = 7.5 Hz, 3H).
(s, 3H), 3.80 (s, 3H), 3.18 (d, J = 22.8 Hz, 2H).
5.1.39. 2-(3-Iodophenyl)-5-chloro-1,3-benzothiazole (9h)
Compound 9e was prepared from 3-iodobenzoic acid and
2-amino-4-chlorothiophenol according to the same procedure as
described for the preparation of 9a from 3-iodobenzoic acid and
2-aminophenol as a dark brown powder (8.60 g, 88%). 1H NMR
(300 MHz, DMSO-d6): d 8.43–8.38 (m, 1H), 8.24–8.14 (m, 2H),
8.10–8.03 (m, 1H), 7.99–7.92 (m, 1H), 7.57–7.49 (m, 1H), 7.38 (t,
J = 7.8 Hz, 1H).
5.1.40. N-Methoxy-N-methyl-2-[3-(5-chloro-1,3-benzothiazol-
2-yl)phenyl]acetamide (10h)
Compound 10h was prepared from 9h according to the same
procedure as described for the preparation of 10a from 9a as dark
brown oil (2.24 g, 34% in three steps). 1H NMR (300 MHz, CDCl3): d
8.05 (m, 1H), 8.01 (m, 1H), 7.97 (m, 1H), 7.81 (d, J = 8.5 Hz, 1H),
7.45 (m, 2H), 7.36 (dd, J = 8.5, 2.2 Hz, 1H), 3.88 (s, 2H), 3.69 (s,
3H), 3.23 (s, 3H).
5.1.41. Dimethyl {3-[3-(5-chloro-1,3-benzothiazol-2-yl)phenyl]-
2-oxopropyl}phosphonate (5k)
Compound 5k was prepared from 10h according to the same
procedure as described for the preparation of 5a from 6 (Scheme
1a) as an orange oil (2.02 g, 76%). 1H NMR (300 MHz, CDCl3): d
8.05 (m, 1H), 7.97 (m, 2H), 7.82 (dd, J = 8.5, 1.0 Hz, 1H), 7.50
(m, 1H), 7.37 (m, 2H), 4.03 (s, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 3.18
(d, J = 23.4 Hz, 2H).
5.1.45. 4-[(2-{(2R)-2-[(1E,3)-S4-(1,10-Biphenyl-3-yl)-3-hydroxybut-
1-enyl]-5-oxopyrrolidin-1-yl}ethyl)sulfanyl]butanoic acid (3b)
A solution of 13a (180 mg, 0.353 mmol) in MeOH (1.5 mL), THF
(1.5 mL) and 2 N NaOH (0.27 mL) was stirred at ambient tempera-
ture for 5 h. After neutralization with 2 N HCl (0.27 mL) under cool-
ing, the reaction mixture was extracted with EtOAc three times,
and the combined organic layers were washed with brine and
dried over Na2SO4. The organic solvent was removed by evapora-
tion. The resulting residue was purified by column chromatogra-
phy on silica gel (CHCl3/MeOH, 100:1–20:1) to afford 3b as a
colorless oil (91 mg, 57%). IR (film): 3735, 3386, 3031, 2923,
1725, 1658, 1479, 1454, 1419, 1362, 1217, 1159, 1101, 1026,
5.1.42. Methyl [3-(1,3-dihydro-2H-isoindol-2-yl)phenyl]acetate
(11)
To stirred MeOH (20 mL) was added dropwise thionyl chloride
(1.46 mL, 20 mmol) at 0 °C under argon atmosphere and stirring
was continued for 15 min. To the resulting mixture was added 3-
aminophenylacetic acid (3.02 g, 20 mmol). After being stirred at
room temperature for 3 h, the reaction mixture was evaporated
to give an ester as a beige powder. To a stirred mixture consisting
of a solution of the above-described ester (20 mmol) in N,N0-dim-
ethylpropyleneurea (DMPU) (40 mL) and potassium carbonate
(9.11 g, 66 mmol) was added 1,2-bis(bromomethyl)benzene
(5.81 g, 22 mmol) at room temperature under argon atmosphere.
After being stirred at 60 °C for 4 h, the reaction mixture was cooled
to room temperature. The resulting reaction mixture was diluted
with EtOAc, washed with H2O, brine, and dried over Na2SO4. The
organic layer was evaporated and the resulting residue was recrys-
tallized from MTBE to give 11 as a purple powder (3.14 g, 59% in
two steps). 1H NMR (300 MHz, CDCl3): d 7.43–7.19 (m, 4H), 6.76–
6.53 (m, 4H), 4.68 (s, 4H), 3.71 (s, 3H), 3.60 (s, 2H).
976, 903, 798, 759, 729, 702, 667, 616 cmꢀ1
;
1H NMR (300 MHz,
CDCl3): 7.59–7.55 (m, 2H), 7.49–7.33 (m, 6H), 7.17 (d,
d
J = 7.8 Hz, 1H), 5.76 (dd, J = 15.3, 6.0 Hz, 1H), 5.46 (dd, J = 15.3,
8.4 Hz, 1H), 4.45 (m, 1H), 4.09 (m, 1H), 3.57 (m, 1H), 2.98–2.82
(m, 3H), 2.61–2.26 (m, 8H), 2.18 (m, 1H), 1.92–1.78 (m, 2H), 1.63
(m, 1H); MS (FAB) m/z: 454 (M+H)+; HRMS-FAB (m/z): [M+H]+
calcd for C26H32NO4S, 454.2052; found, 454.2050.
5.1.46. Butyl 4-({2-[(2R)-2-{(1E,3S)-4-[3-(2-naphthyl)phenyl]-3-
hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]ethyl}thio)
butanoate (13b)
Compound 13b was prepared from 12 using the phosphonate 5e
instead of 5b according to the same procedure as described for the

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
3515
preparation of 13a from 12 as a pale yellow solid (74 mg, 42% in two
5.1.51. 4-{[2-((2R)-2-{(1E,3S)-4-[3-(1,3-Dihydro-2H-isoindol-2-
steps). 1H NMR (300 MHz, CDCl3): d 8.03 (m, 1H), 7.96–7.82 (m, 3H),
7.73 (m, J = 8.5, 1.6 Hz, 1H), 7.67–7.37 (m, 5H), 7.30–7.17 (m, 1H),
5.81 (dd, J = 15.5, 5.9 Hz, 1H), 5.61–5.43 (m, 1H), 4.54–4.43 (m,
1H), 4.19–3.99 (m, 4H), 3.68–3.42 (m, 4H), 3.00–2.84 (m, 3H),
2.63–2.43 (m, 4H), 2.40–2.11 (m, 5H), 2.00 (m, 1H), 1.92–1.76 (m,
1H), 1.44–1.27 (m, 2H), 0.91 (t, J = 7.40 Hz, 3H).
yl)phenyl]-3-hydroxybut-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]
sulfanyl}butanoic acid (3e)
Compound 3e was prepared from 13d according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (39 mg, 65%). IR (film): 3389, 3043, 2925, 2861,
1913, 1723, 1714, 1660, 1605, 1580, 1497, 1469, 1455, 1418,
1374, 1305, 1260, 1225, 1163, 1096, 1036, 976, 910, 850, 775,
733, 699, 664, 647, 571, 462 cmꢀ1 1H NMR (300 MHz, CDCl3): d
;
5.1.47. 4-{[2-((2R)-2-{(1E,3S)-3-Hydroxy-4-[3-(2-naphthyl)
phenyl]but-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]sulfanyl}
butanoic acid (3c)
7.33 (m, 6H), 6.57 (m, 2H), 5.81 (dd, J = 15.7, 5.5 Hz, 1H), 5.55
(dd, J = 15.7, 8.8 Hz, 1H), 4.78 (m, 4H), 4.49 (m, 1H), 4.12 (m,
1H), 3.59 (m, 1H), 2.72 (m, 13H), 1.79 (m, 3H); MS (FAB) m/z:
495 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C28H35N2O4S,
495.2318; found, 495.2330.
Compound 3c was prepared from 13b according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (45 mg, 65%). IR (film): 3422, 3054, 2922, 1728,
1658, 1488, 1444, 1418, 1370, 1268, 1238, 1158, 1099, 1030, 973,
891, 859, 822, 791, 749, 706, 661, 625, 607, 570 cmꢀ1 1H NMR
;
5.1.52. Butyl 4-({2-[(2R)-2-{(1E,3S)-4-[3-(1H-indol-5-yl)phenyl]-
3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]ethyl}thio)
butanoate (13e)
(300 MHz, CDCl3): d 8.03 (s, 1H), 7.94–7.82 (m, 3H), 7.73 (dd,
J = 8.7, 2.1 Hz, 1H), 7.64–7.57 (m, 1H), 7.57–7.40 (m, 4H), 7.21
(d, J = 7.5 Hz, 1H), 5.79 (dd, J = 15.3, 6.0 Hz, 1H), 5.49 (ddd,
J = 15.3, 8.7, 1.2 Hz, 1H), 4.54–4.44 (m, 1H), 4.14–4.04 (m, 1H),
3.66–3.52 (m, 1H), 3.00–2.85 (m, 3H), 2.60–2.10 (m, 9H), 1.90–
1.60 (m, 3H); MS (FAB) m/z: 504 (M+H)+; HRMS-FAB (m/z):
[M+H]+ calcd for C30H34NO4S, 504.2209; found, 504.2199.
Compound 13e was prepared from 12 using the phosphonate
5g instead of 5b according to the same procedure as described
for the preparation of 13a from 12 as a colorless oil (74 mg,
46% in two steps). 1H NMR (300 MHz, CDCl3): d 8.42–8.21 (m,
1H), 7.84 (s, 1H), 7.61–7.21 (m, 6H), 7.19–7.10 (m, 1H), 6.67–
6.52 (m, 1H), 5.78 (dd, J = 15.5, 5.9 Hz, 1H), 5.48 (dd, J = 15.4,
8.8 Hz, 1H), 4.53–4.37 (m, 1H), 4.20–3.99 (m, 3H), 3.65–3.47 (m,
1H), 3.01–2.77 (m, 3H), 2.66–2.07 (m, 9H), 2.02–1.19 (m, 8H),
0.92 (t, J = 7.3 Hz, 3H).
5.1.48. Butyl 4-({2-[(2R)-2-{(1E,3S)-4-(3-pyridin-2-ylphenyl)-3-
hydroxy-1-buten-1-yl}-5-oxo-1-
pyrrolidinyl]ethyl}thio)butanoate (13c)
Compound 13c was prepared from 12 using the phosphonate 5r
instead of 5b according to the same procedure as described for the
preparation of 13a from 12 as a pale yellow solid (105 mg, 48% in
two steps). 1H NMR (300 MHz, CDCl3): d 8.69 (d, J = 4.8 Hz, 1H),
7.92–7.70 (m, 4H), 7.41 (t, J = 7.5 Hz, 1H), 7.38–7.20 (m, 2H), 5.79
(dd, J = 15.0, 5.7 Hz, 1H), 5.51 (dd, J = 15.0, 8.4 Hz, 1H), 4.48 (m,
1H), 4.18–4.00 (m, 3H), 3.60 (m, 1H), 3.00–2.83 (m, 3H), 2.63–2.10
(m, 10H), 2.03 (br s, 1H), 1.87 (m, 2H), 1.78–1.50 (m, 3H), 1.38 (m,
2H), 0.92 (t, J = 7.5 Hz, 3H).
5.1.53. 4-{[2-((2R)-2-{(1E,3S)-3-Hydroxy-4-[3-(1H-indol-5-
yl)phenyl]but-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]sulfanyl}
butanoic acid (3f)
Compound 3f was prepared from 13e according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (40 mg, 60%). IR (film): 3410, 3032, 2925, 1714,
1647, 1604, 1458, 1419, 1348, 1310, 1247, 1161, 1097, 1066,
1042, 1026, 975, 894, 882, 792, 768, 732, 704, 671, 665, 643,
610, 600, 571, 564, 540 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.35
;
(br s, 1H), 7.84 (m, 1H), 7.46–7.40 (m, 4H), 7.25 (m, 2H), 7.14
(m, 1H), 6.60 (m, 1H), 5.78 (dd, J = 15.4, 5.8 Hz, 1H), 5.46 (ddd,
J = 15.4, 8.8, 1.1 Hz, 1H), 4.48 (m, 1H), 4.07 (m, 1H), 3.54 (m,
1H), 2.90 (m, 3H), 2.09–1.80 (m, 13H); MS (FAB) m/z: 493
(M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C28H33N2O4S,
493.2161; found, 493.2170.
5.1.49. 4-[(2-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(3-pyridin-2-
ylphenyl)but-1-enyl]-5-oxopyrrolidin-1-yl}ethyl)sulfanyl]
butanoic acid (3d)
Compound 3d was prepared from 13c according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (60 mg, 65%). IR (film): 3366, 2924, 1715, 1666,
1590, 1566, 1462, 1436, 1418, 1217, 1155, 1101,1033, 975, 914,
5.1.54. Butyl 4-({2-[(2R)-2-{(1E,3S)-4-[3-(1,3-benzoxazol-2-
yl)phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]
ethyl}thio)butanoate (13f)
754, 666 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.74 (m, 1H), 7.93
;
(s, 1H), 7.84 (dt, J = 1.8, 7.8 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.65
(d, J = 8.1 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.37–7.23 (m, 2H),
5.88 (dd, J = 15.0, 4.5 Hz, 1H), 5.64 (ddd, J = 15.0, 9.0, 1.5 Hz,
1H), 5.45 (br s, 2H), 4.58 (m, 1H), 4.10 (m, 1H), 3.40 (m, 1H),
3.21 (m, 1H), 3.02–2.80 (m, 2H), 2.78–2.10 (m, 9H), 1.99–1.82
(m, 2H), 1.73 (m, 1H); MS (FAB) m/z: 455 (M+H)+; HRMS-FAB
(m/z): [M+H]+ calcd for C25H31N2O4S, 455.1992; found, 455.2005.
Compound 13f was prepared from 12 using the phosphonate
5h instead of 5b according to the same procedure as described
for the preparation of 13a from 12 as a colorless oil (80 mg,
45% in two steps). 1H NMR (300 MHz, CDCl3): d 8.20–8.08 (m,
2H), 7.77 (m, 1H), 7.60 (m, 1H), 7.54–7.32 (m, 4H), 5.81 (dd,
J = 15.1, 6.6 Hz, 1H), 5.60–5.48 (m, 1H), 4.49 (br s, 1H), 4.17–
4.01 (m, 3H), 3.68–3.53 (m, 1H), 3.49 (s, 1H), 3.01–2.89 (m, 3H),
2.69–2.47 (m, 5H), 2.45–2.30 (m, 5H), 2.10–2.05 (m, 1H), 1.93–
1.78 (m, 3H), 1.12–1.05 (m, 1H), 0.91 (t, J = 7.2 Hz, 3H).
5.1.50. Butyl 4-({2-[(2R)-2-{(1E,3S)-4-[3-(1,3-Dihydro-2H-
isoindol-2-yl)phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-
pyrrolidinyl]ethyl}thio)butanoate (13d)
5.1.55. 4-{[2-((2R)-2-{(1E,3S)-4-[3-(1,3-Benzoxazol-2-yl)phenyl]
-3-hydroxybut-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]sulfanyl}
butanoic acid (3g)
Compound 3g was prepared from 13f according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (45 mg, 60%). IR (film): 3423, 3060, 2923,
2854, 1945, 1899, 1727, 1660, 1552, 1491, 1473, 1454, 1421,
1359, 1244, 1175, 1109, 1032, 1002, 977, 930, 879, 803, 793,
Compound 13d was prepared from 12 using the phosphonate
5f instead of 5b according to the same procedure as described for
the preparation of 13a from 12 as a colorless oil (86 mg, 53% in
two steps). 1H NMR (300 MHz, CDCl3): d 7.43–7.17 (m, 6H),
6.65–6.46 (m, 2H), 5.81 (dd, J = 15.4, 5.8 Hz, 1H), 5.55 (ddd,
J = 15.4, 8.7, 1.1 Hz, 1H), 4.66 (s, 4H), 4.50–4.36 (m, 1H), 4.19–
4.01 (m, 3H), 3.73–3.52 (m, 1H), 3.07–2.73 (m, 3H), 2.71–2.13
(m, 10H), 2.01–1.21 (m, 8H), 0.93 (t, J = 7.3 Hz, 3H).

3516
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
762, 747, 726, 698, 684 cmꢀ1; 1H NMR (300 MHz, CDCl3): d 8.31 (s,
1H), 8.08 (d, J = 7.8 Hz, 1H), 7.82–7.74 (m, 1H), 7.64–7.56 (m, 1H),
7.48 (t, J = 7.8 Hz, 1H), 7.44–7.34 (m, 3H), 5.89 (dd, J = 15.6, 4.5 Hz,
1H), 5.63 (dd, J = 15.6, 7.5 Hz, 1H), 4.65–4.55 (m, 1H), 4.20–4.05 (m,
1H), 3.55–3.40 (m, 1H), 3.30–3.10 (m, 1H), 3.30 (m, 1H), 2.89 (m,
1H), 2.75–2.15 (m, 9H), 1.95–1.85 (m, 2H), 1.80–1.60 (m, 1H);
MS (FAB) m/z: 495 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for
as described for the preparation of 13a from 12 as a colorless oil
(29 mg, 29% in two steps). 1H NMR (300 MHz, CDCl3): d 7.98 (s, 1
H), 7.58–7.28 (m, 5H), 7.15–7.05 (m, 1H), 7.02–6.89 (m, 2H), 5.84
(dd, J = 15.4, 5.8 Hz, 1H), 5.51 (dd, J = 15.4, 8.5 Hz, 1H), 4.48–4.08
(m, 4H), 3.85 (s, 3H), 3.74–3.59 (m, 1H), 3.33 (t, J = 6.6 Hz, 2H),
3.25–3.12 (m, 1H), 2.94–2.76 (m, 2H), 2.46–2.08 (m, 4H), 1.80–
1.62 (m, 3H), 1.49–1.34 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H).
C27H31N2O5S, 495.1954; found, 495.1954.
5.1.61. 2-[(2-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(40-methoxy-1,10-
biphenyl-3-yl)but-1-enyl]-5-oxopyrrolidin-1-yl}ethyl)sulfanyl]-
1,3-thiazole-4-carboxylic acid (4e)
5.1.56. Butyl 4-({2-[(2R)-2-{(1E,3S)-4-[3-(1,3-benzothiazol-2-yl)
phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]ethyl}
thio)butanoate (13g)
Compound 4e was prepared from 15b according to the same
procedure as described for the preparation of 3b from 13a as a
white amorphous powder (48 mg, 76%). IR (film): 3422, 3114,
2933, 2837, 1718, 1662, 1609, 1516, 1483, 1461, 1421, 1295,
1272, 1247, 1181, 1100, 1027, 975, 922, 835, 798, 788, 749, 710,
Compound 13g was prepared from 12 using the phosphonate 5i
instead of 5b according to the same procedure as described for the
preparation of 13a from 12 as a pale yellow oil (84 mg, 47% in two
steps).
664, 608, 572 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.07 (s, 1H),
;
5.1.57. 4-{[2-((2R)-2-{(1E,3S)-4-[3-(1,3-Benzothiazol-2-yl)phen
yl]-3-hydroxybut-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]sulfanyl}
butanoic acid (3h)
7.22 (m, 8H), 5.82 (dd, J = 15.4, 6.0 Hz, 1H), 5.51 (dd, J = 15.4,
8.8 Hz, 1H), 4.46 (m, 1H), 4.09 (m, 1H), 3.85 (s, 3H), 3.69 (m, 1H),
3.21 (m, 3H), 2.53 (m, 7H), 1.70 (m, 1H); MS (FAB) m/z: 525
(M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C27H29N2O5S2,
525.1518; found, 525.1520.
Compound 3h was prepared from 13g according to the same
procedure as described for the preparation of 3b from 13a as a pale
yellow amorphous powder (39 mg, 54%). IR (film): 3425, 3083,
3060, 3026, 2923, 2852, 1727, 1658, 1508, 1493, 1484, 1453,
1437, 1419, 1364, 1314, 1238, 1158, 1030, 976, 906, 760, 730,
5.1.62. Butyl 2-({2-[(2R)-2-{(1E,3S)-4-[3-(1,3-benzoxazol-2-yl)
phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]ethyl}
thio)-1,3-thiazole-4-carboxylate (15c)
697, 668, 615, 569, 560, 549, 522, 513 cmꢀ1 1H NMR (300 MHz,
;
CDCl3): d 8.14 (s, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.91 (d, J = 7.8 Hz,
1H), 7.84 (d, J = 7.8 Hz, 1H), 7.56–7.32 (m, 4H), 5.88 (dd, J = 15.0,
5.1 Hz, 1H), 5.61 (ddd, J = 15.0, 8.7, 1.5 Hz, 1H), 4.60–4.45 (m,
1H), 4.20–4.05 (m, 1H), 3.55–3.40 (m, 1H), 3.25–3.05 (m, 1H),
3.01 (dd, J = 13.8, 4.8 Hz, 1H), 2.88 (dd, J = 13.8, 8.7 Hz, 1H), 2.70–
2.10 (m, 9H), 1.96–1.82 (m, 2H), 1.80–1.60 (m, 1H); MS (FAB) m/
z: 511 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C27H31N2O4S2,
511.1725; found, 511.1738.
Compound 15c was prepared from the aldehyde 14 using the
phosphonate 5h instead of 5b according to the same procedure
as described for the preparation of 13a from 12 as a pale yellow
amorphous powder (69 mg, 28% in two steps). 1H NMR
(300 MHz, CDCl3): d 8.14–8.07 (m, 2H), 7.97 (s, 1H), 7.78 (m, 1H),
7.60 (m, 1H), 7.45 (m, 1H), 7.40–7.31 (m, 3H), 5.88 (dd, J = 15.5,
5.6 Hz, 1H), 5.59 (m, 1H), 4.49 (m, 1H), 4.33–4.16 (m, 3H), 3.66
(m, 1H), 3.42–3.23 (m, 3H), 2.97–2.87 (m, 2H), 2.45–2.11 (m,
4H), 1.77–1.62 (m, 3H), 1.48–1.33 (m, 2H), 0.94 (t, J = 7.7 Hz, 3H).
5.1.58. Butyl 2-({2-[(2R)-2-{(1E,3S)-4-(40-methyl-1,10-biphenyl-
3-yl)-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]
ethyl}thio)-1,3-thiazole-4-carboxylate (15a)
Compound 15a was prepared from aldehyde 14 using the phos-
phonate 5c according to the same procedure as described for the
preparation of 13a from 12 as a colorless oil (42 mg, 18% in two
steps). 1H NMR (300 MHz, CDCl3): d 8.02–7.95 (m, 1H), 7.54–7.18
(m, 7H), 7.16–7.05 (m, 1H), 5.84 (dd, J = 15.4, 5.8 Hz, 1H), 5.51 (dd,
J = 15.4, 8.8 Hz, 1H), 4.51–4.05 (m, 4H), 3.84–3.56 (m, 2H), 3.33 (t,
J = 6.6 Hz, 2H), 3.25–3.13 (m, 1H), 2.94–2.78 (m, 2H), 2.44–2.06 (m,
6H), 1.78–1.60 (m, 3H), 1.49–1.31 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H).
5.1.63. 2-{[2-((2R)-2-{(1E,3S)-4-[3-(1,3-Benzoxazol-2-yl)phenyl]
-3-hydroxybut-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]sulfanyl}-
1,3-thiazole-4-carboxylic acid (4j)
Compound 4j was prepared from 15c according to the same proce-
dure as described for the preparation of 3b from 13a as a white powder
(43 mg, 67%). IR (film): 3412, 3063, 2925, 1715, 1665, 1490, 1473,
1422, 1244, 1101, 1029, 1003, 977, 803, 793, 762 cmꢀ1 1H NMR
;
(300 MHz, CDCl3): d 8.32 (s, 1H), 8.12 (s, 1H), 8.06 (d, J = 7.5 Hz, 1H),
7.90–7.82 (m, 1H), 7.64–7.58 (m, 1H), 7.50–7.36 (m, 4H), 5.94 (dd,
J = 15.6, 4.5 Hz, 1H), 5.78 (dd, J = 15.6, 6.3 Hz, 1H), 4.70–4.50 (m, 1H),
4.15 (q, J = 7.2 Hz, 1H), 3.60–3.20 (m, 4H), 3.00 (dd, J = 14.4, 4.2 Hz,
1H), 2.85 (dd, J = 14.4, 9.0 Hz, 1H), 2.50–2.15 (m, 3H), 1.85–1.70 (m,
1H); MS (FAB) m/z: 536 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for
5.1.59. 2-[(2-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(40-methyl-1,10-
biphenyl-3-yl)but-1-enyl]-5-oxopyrrolidin-1-yl}ethyl)sulfanyl]-
1,3-thiazole-4-carboxylic acid (4c)
Compound 4c was prepared from 15a according to the same
procedure as described for the preparation of 3b from 13a as a
white amorphous powder (29 mg, 80%). IR (film): 3415, 3114,
3022, 2921, 1716, 1662, 1483, 1422, 1324, 1214, 1101, 1029,
976, 922, 824, 787, 718, 665, 561 cmꢀ1; 1H NMR (300 MHz, CDCl3):
d 8.01 (s, 1H), 7.39–7.29 (m, 8H), 5.82 (dd, J = 15.4, 5.8 Hz, 1H) 5.51
(dd, J = 15.4, 8.5 Hz, 1H), 4.47 (m, 1H), 4.10 (m, 1H), 3.69 (m, 1H),
3.23 (m, 3H), 2.92 (m, 2H), 2.38 (m, 8H), 1.71 (m, 1H); MS (FAB)
m/z: 509 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C27H29N2O4S2,
509.1569; found, 509.1562.
C27H26N3O5S2, 536.1314; found, 536.1303.
5.1.64. Butyl 2-({2-[(2R)-2-{(1E,3S)-4-[3-(1,3-benzothiazol-2-
yl)phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]
ethyl}thio)-1,3-thiazole-4-carboxylate (15d)
Compound 15d was prepared from aldehyde 14 using the
phosphonate 5i instead of 5b according to the same procedure as
described for the preparation of 13a from 12 as a pale yellow
amorphous powder (61 mg, 24% in two steps). 1H NMR
(300 MHz, CDCl3): d 8.05 (s, 1H), 8.00–7.86 (m, 4H), 7.56–7.28
(m, 4H), 5.90 (m, 1H), 5.60 (m, 1H), 4.48 (m, 1H), 4.35–4.15 (m,
3H), 3.75–3.59 (m, 2H), 3.44–3.32 (m, 2H), 3.26 (m, 1H), 2.97–
2.84 (m, 2H), 2.42–2.12 (m, 3H), 1.77–1.63 (m, 2H), 1.47–1.34
(m, 2H), 0.99–0.87 (m, 3H).
5.1.60. Butyl 2-({2-[(2R)-2-{(1E,3S)-4-(40-methoxy-1,10-
biphenyl-3-yl)-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]
ethyl}thio)-1,3-thiazole-4-carboxylate (15b)
Compound 15b was prepared from the aldehyde 14 using the
phosphonate 5d instead of 5b according to the same procedure

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
3517
5.1.65. 2-{[2-((2R)-2-{(1E,3S)-4-[3-(1,3-benzothiazol-2-yl)
phenyl]-3-hydroxybut-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]
sulfanyl}-1,3-thiazole-4-carboxylic acid (4k)
(M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C28H27N2O5S2,
535.1361; found, 535.1357.
Compound 4k was prepared from 15d according to the same
procedure as described for the preparation of 3b from 13a as a
white powder (36 mg, 65%). IR (film): 3059, 2921, 1946, 1716,
5.1.69. Ethyl (4R,5E,7S)-8-(3-(5-methyl-1,3-benzoxazol-2-yl) phenyl)-
7-hydroxy-4-({tert-butyoxycarbonyl}amino)-5-octenoate (17b)
Compound 17b was prepared from the aldehyde 16 using the
phosphonate 5l according to the same procedure as described for
the preparation of 13a from 12 as a colorless amorphous powder
(878 mg, 88% in two steps). 1H NMR (300 MHz, CDCl3): d 8.10
(m, 2H), 7.55 (s, 1H), 7.50–7.34 (m, 3H), 7.16 (dd, J = 8.2, 1.7 Hz,
1H), 5.73 (m, 1H), 5.58 (m, 1H), 4.62 (m, 1H), 4.44 (m, 1H), 4.09
(q, J = 7.1 Hz, 2H), 3.04–2.82 (m, 2H), 2.49 (s, 3H), 2.30 (t,
J = 7.4 Hz, 2H), 1.94–1.69 (m, 3H), 1.42 (s, 9H), 1.23 (t, J = 7.1 Hz,
3H).
1664, 1484, 1315, 1236, 1027, 976, 919, 760, 728 cmꢀ1 1H NMR
;
(300 MHz, CDCl3): d 8.16 (s, 1H) 7.95 (m, 4H), 7.44 (m, 4H), 5.79
(dd, J = 15.2, 6.8 Hz, 1H,) 5.40 (m, 1H), 4.40 (m, 1H), 4.24 (m, 1H),
3.58 (m, 1H), 3.25 (m, 2H), 2.91 (m, 3H), 2.22 (m, 3H), 1.67 (m,
1H); MS (FAB) m/z: 552 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd
for C27H26N3O4S3, 552.1085; found, 552.1073.
5.1.66. Ethyl (4R,5E,7S)-8-(3-(1-benzofuran-2-yl)phenyl)-7-
hydroxy-4-({tert-butyoxycarbonyl}amino)-5-octenoate (17a)
Compound 17a was prepared from the aldehyde 16 using the
phosphonate 5j according to the same procedure as described for
the preparation of 13a from 12 as a colorless amorphous powder
(4.27 g, 76% in two steps). 1H NMR (300 MHz, CDCl3): d 7.77–
7.69 (m, 2H), 7.59 (m, 1H), 7.55–7.49 (m, 1H), 7.40 (m, 1H),
7.33–7.16 (m, 3H), 7.04 (s, 1H), 5.76 (m, 1H), 5.60 (m, 1H), 4.55
(m, 1H), 4.42 (m, 1H) 4.18–4.06 (m, 3H), 2.99–2.80 (m, 2H), 2.31
(t, J = 6.9 Hz, 2H), 1.91–1.71 (m, 2H), 1.41 (s, 9H), 1.25 (t,
J = 7.7 Hz, 3H).
5.1.70. Ethyl 2-({2-[(2R)-2-{(1E,3S)-4-[3-(5-methyl-1,3-benzoxazol-
2-yl)phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]
ethyl}thio)-1,3-thiazole-4-carboxylate (18b)
Compound 18b was prepared from 17b according to the same
procedure as described for the preparation of 18a from 17a as a
pale yellow viscous oil (282 mg, 58%). 1H NMR (300 MHz, CDCl3):
d 8.15–8.04 (m, 2H), 7.99 (s, 1H), 7.54 (s, 1H), 7.48–7.24 (m, 3H),
7.16 (dd, J = 8.2, 1.5 Hz, 1H), 5.87 (dd, J = 15.4, 5.5 Hz, 1H), 5.56
(m, 1H), 4.47 (m, 1H), 4.42–4.28 (m, 2H), 4.22 (m, 1H), 3.67
(m, 1H), 3.38 (t, J = 6.6 Hz, 2H), 3.27 (m, 1H), 2.99–2.86 (m,
3H), 2.49 (s, 3H), 2.42–2.12 (m, 3H), 1.68 (m, 1H), 1.36 (t,
J = 7.1 Hz, 3H).
5.1.67. Ethyl 2-({2-[(2R)-2-{(1E,3S)-4-[3-(1-benzofuran-2-yl)phen
yl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl] ethyl} thio)-1,
3-thiazole-4-carboxylate (18a)
A solution of 17a (4.20 g, 8.50 mmol) in EtOH (10 mL) was trea-
ted with 4 N HCl in dioxane (3.2 mL, 25.5 mmol) at room temper-
ature under argon atmosphere. After being stirred for 6 h, the
reaction mixture was evaporated. The resulting residue was recrys-
tallized from MTBE and AcOEt to give an amine hydrochloride as a
white powder (2.81 g, 77%). To a stirred solution of aldehyde 23
(1.86 g, 8.07 mmol) in THF (25 mL) and DMF (3 mL) was added a
solution of the above-described amine hydrochloride (2.31 g,
5.38 mmol) in THF (3 mL) at room temperature under argon atmo-
sphere and stirring was continued for 1 h at 50 °C. To the stirred
reaction mixture was added sodium acetoxyborohydride (1.71 g,
8.07 mmol) at 0 °C under argon atmosphere. After being stirred
at room temperature for additional 15 h, the reaction mixture
was diluted with EtOAc, washed with water, saturated aqueous
NaHCO3, brine, and dried over Na2SO4. The organic layer was
evaporated and the resulting residue was purified by column chro-
matography on silica gel (hexane/EtOAc, 1:4–0:1) to give 18a as a
white amorphous (1.82 g, 60%). 1H NMR (300 MHz, CDCl3): d 7.99
(s, 1H), 7.72 (s, 2H), 7.58 (d, J = 7.1 Hz, 1H), 7.51 (d, J = 7.1 Hz,
1H), 7.36 (t, J = 8.0 Hz, 1H), 7.31–7.12 (m, 3H), 7.03 (s, 1H), 5.83
(m, 1H), 5.60 (m, 1H), 4.45 (m, 1H), 4.39–4.29 (m, 2H), 4.18 (m,
1H), 3.66 (m, 1H), 3.40–3.30 (m, 2H), 3.23 (m, 1H), 2.96–2.80 (m,
2H), 2.45–2.10 (m, 3H), 1.80–1.60 (m, 2H), 1.36 (t, J = 7.0 Hz, 3H).
5.1.71. 2-{[2-((2R)-2-{(1E,3S)-3-Hydroxy-4-[3-(5-methyl-1,3-benzox
azol-2-yl)phenyl]but-1-enyl}-5-oxopyrrolidin-1-yl) ethyl] sulfanyl}-1,
3-thiazole-4-carboxylic acid (4m)
Compound 4m was prepared from 18b according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (158 mg, 60%). IR (KBr): 3422, 2924, 1716,
1663, 1551, 1424, 1264, 1206, 1029, 797, 724, 567 cmꢀ1 1H
;
NMR (300 MHz, CDCl3): d 8.47 (s, 1H), 8.14 (s, 1H), 8.04 (d,
J = 7.8 Hz, 1H), 7.65 (s, 1H), 7.52–7.36 (m, 3H), 7.21 (dd, J = 8.4,
0.6 Hz, 1H), 5.94 (dd, J = 15.0, 4.5 Hz, 1H), 5.81 (m, 1H), 4.63 (m,
1H), 4.17 (m, 1H), 3.55–3.24 (m, 4H), 3.00 (dd, J = 14.7, 3.9 Hz,
1H), 2.84 (dd, J = 14.7, 9.0 Hz, 1H), 2.51 (s, 3H), 2.46–2.18 (m,
3H), 1.81 (m, 1H); MS (FAB) m/z: 550 (M+H)+; HRMS-FAB (m/z):
[M+H]+ calcd for C28H28N3O5S2, 550.1470; found, 550.1472.
5.1.72. Ethyl (4R,5E,7S)-8-(3-(6-methyl-1,3-benzoxazol-2-yl)
phenyl)-7-hydroxy-4-({tert-butyoxycarbonyl}amino)-5-octeno
ate (17c)
Compound 17c was prepared from the aldehyde 16 using the
phosphonate 5m instead of 5b according to the same procedure
as described for the preparation of 13a from 12 as a colorless amor-
phous powder (1.75 g, 89% in two steps).
5.1.68. 2-{[2-((2R)-2-{(1E,3S)-4-[3-(1-Benzofuran-2-yl)phenyl]-
3-hydroxybut-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]sulfanyl}-1,3-
thiazole-4-carboxylic acid (4i)
5.1.73. Ethyl 2-({2-[(2R)-2-{(1E,3S)-4-[3-(6-methyl-1,3-
benzoxazol-2-yl)phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-
pyrrolidinyl]ethyl}thio)-1,3-thiazole-4-carboxylate (18c)
Compound 18c was prepared from 17c according to the same
procedure as described for the preparation of 18a from 17a as a
pale yellow viscous oil (313 mg, 47% in two steps). 1H NMR
(300 MHz, CDCl3): d 8.15–8.02 (m, 2H), 7.99 (s, 1H), 7.63 (d,
J = 8.2 Hz, 1H), 7.49–7.30 (m, 3H), 7.17 (d, J = 7.7 Hz, 1H), 5.87
(dd, J = 15.4, 5.8 Hz, 1H), 5.56 (ddd, J = 15.4, 8.5, 1.1 Hz, 1H), 4.50
(m, 1H), 4.35 (q, J = 7.1 Hz, 2H), 4.25–4.14 (m, 1 H) 3.60 (m, 1H),
3.38 (m, 1H), 3.30 (m, 1H), 2.95–2.85 (m, 2H), 2.51 (s, 3H), 2.43–
2.09 (m, 3H), 1.69 (m, 1H), 1.35 (t, J = 7.1 Hz, 3H).
Compound 4i was prepared from 18a according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (1.62 g, 98%). IR (KBr): 3412, 3114, 2933, 1718,
1663, 1490, 1452, 1421, 1324, 1257, 1214, 1106, 1029, 976, 937,
795, 751, 720, 700 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.05 (s,
;
1H), 7.80–7.65 (m, 2H), 7.58 (d, J = 7.5 Hz, 1H), 7.51 (d, J = 7.5 Hz,
1H), 7.39 (t, J = 7.8 Hz, 1H), 7.35–7.15 (m, 3H), 7.03 (s, 1H), 5.82
(dd, J = 15.0, 5.7 Hz, 1H), 5.52 (d, J = 15.0, 8.7 Hz, 1H), 4.50 (m,
1H), 4.19–4.02 (m, 1H), 3.70 (m, 1H), 3.36–3.08 (m, 3H), 3.00–
2.82 (m, 2H), 2.50–2.10 (m, 3H), 1.72 (m, 1H); MS (FAB) m/z: 535

3518
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
5.1.74. 2-{[2-((2R)-2-{(1E,3S)-3-Hydroxy-4-[3-(6-methyl-1,3-
benzoxazol-2-yl)phenyl]but-1-enyl}-5-oxopyrrolidin-1-yl)
ethyl]sulfanyl}-1,3-thiazole-4-carboxylic acid (4n)
5.1.80. 2-{[2-((2R)-2-{(1E,3S)-4-[3-(5,7-Dimethyl-1,3-benzoxazol-
2-yl)phenyl]-3-hydroxybut-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]
sulfanyl}-1,3-thiazole-4-carboxylic acid (4p)
Compound 4n was prepared from 18c according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (245 mg, 82%). IR (film): 3060, 2941, 1723,
1683, 1551, 1474, 1418, 1381, 1277, 1149, 1032, 949, 892, 795,
Compound 4p was prepared from 18e according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (152 mg, 40%). IR (film): 3061, 2940, 2875,
1720, 1666, 1551, 1493, 1312, 1260, 1151, 1028, 942, 844, 718,
724, 696, 569 cmꢀ1
;
1H NMR (300 MHz, CDCl3): d 8.20 (s, 1H),
615 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.48 (s, 1H), 8.14 (s,
;
8.05 (m, 2H), 7.66 (d, J = 8.2 Hz, 1H), 7.42 (m, 3H), 7.20 (d,
J = 8.0 Hz, 1H), 5.92 (dd, J = 15.4, 4.9 Hz, 1H), 5.62 (ddd, J = 15.4,
8.8, 1.1 Hz, 1H), 4.49 (m, 1H), 4.19 (m, 1H), 3.60 (m, 1H), 3.34
(m, 3H), 2.90 (m, 2H), 2.33 (m, 7H), 1.75 (m, 1H); MS (FAB) m/z:
550 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C28H28N3O5S2,
550.1470; found, 550.1472.
1H), 8.06 (m, 1H), 7.42 (m, 3H), 7.01 (s, 1H), 5.81 (m, 1H), 4.63
(m, 1H), 4.15 (m, 1H), 3.39 (m, 4H), 3.01 (m, 1H), 2.83 (m, 1H),
2.39 (m, 11H), 1.81 (m, 1H); MS (FAB) m/z: 564 (M+H)+; HRMS-
FAB (m/z): [M+H]+ calcd for C29H30N3O5S2, 564.1627; found,
564.1631.
5.1.81. Ethyl (4R,5E,7S)-8-(3-(5-chloro-1,3-benzothiazol-2-yl)
phenyl)-7-hydroxy-4-({tert-butyoxycarbonyl}amino)-5-octenoate
(17g)
5.1.75. Ethyl (4R,5E,7S)-8-(3-(4-methyl-1,3-benzoxazol-2-yl)
phenyl)-7-hydroxy-4-({tert-butyoxycarbonyl}amino)-5-octeno
ate (17d)
Compound 17g was prepared from aldehyde 16 using phospho-
nate 5k instead of 5b according to the same procedure as described
for the preparation of 13a from 12 as a white powder (1.12 g, 51%
in two steps). 1H NMR (300 MHz, CDCl3): d 8.07 (m, 1H), 7.96 (s,
1H), 7.91 (m, 1H), 7.82 (m, 1H) 7.50–7.33 (m, 3H), 5.77 (m, 1H),
5.60 (m, 1H), 4.70 (m, 1H), 4.45 (m, 1H), 4.18–4.05 (m, 3H),
2.97–2.88 (m, 2H), 2.30 (t, J = 7.4 Hz, 2H), 1.91–1.72 (m, 2H), 1.43
(s, 9H), 1.24 (t, J = 7.1 Hz, 3H).
Compound 17d was prepared from the aldehyde 16 using the
phosphonate 5n according to the same procedure as described
for the preparation of 13a from 12 as a colorless amorphous pow-
der (1.75 g, 89% in two steps).
5.1.76. Ethyl 2-({2-[(2R)-2-{(1E,3S)-4-[3-(4-methyl-1,3-benzoxazol-
2-yl)phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]
ethyl} thio)-1,3-thiazole-4-carboxylate (18d)
Compound 18d was prepared from 17d according to the same
procedure as described for the preparation of 18a from 17a as a
pale yellow viscous oil (313 mg, 47% in two steps).
5.1.82. Ethyl 2-({2-[(2R)-2-{(1E,3S)-4-[3-(5-chloro-1,3-benzothiazol-2-yl)
phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]ethyl}thio)-1,3-
thiazole-4-carboxylate (18g)
Compound 18g was prepared from 17g according to the same
procedure as described for the preparation of 18a from 17a as a
pale brown oil (254 mg).
5.1.77. 2-{[2-((2R)-2-{(1E,3S)-3-Hydroxy-4-[3-(4-methyl-1,
3-benzoxazol-2-yl)phenyl]but-1-enyl}-5-oxopyrrolidin-1-yl)
ethyl]sulfanyl}-1,3-thiazole-4-carboxylic acid (4o)
Compound 4o was prepared from 18d according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (245 mg, 82%). IR (film): 3077, 2921, 1737,
1679, 1608, 1551, 1496, 1435, 1324, 1300, 1285, 1193, 1092,
5.1.83. 2-{[2-((2R)-2-{(1E,3S)-4-[3-(5-Chloro-1,3-benzothiazol-2-yl)
phenyl]-3-hydroxybut-1-enyl}-5-oxopyrrolidin-1-yl)ethyl] sulfanyl}-
1,3-thiazole-4-carboxylic acid (4l)
983, 886, 784, 713 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.16 (s,
;
Compound 4l was prepared from 18g according to the same
procedure as described for the preparation of 3b from 13a as a pale
yellow powder (140 mg, 29% in two steps). IR (film): 3076, 2987,
2871, 1749, 1675, 1548, 1465, 1415, 1324, 1295, 1180, 1099,
1H), 8.08 (m, 2H), 7.44 (m, 2H), 7.35 (d, J = 7.7 Hz, 1H), 7.27 (t,
J = 7.8 Hz, 1H), 7.17 (d, J = 7.4 Hz, 1H), 5.88 (dd, J = 15.3, 5.4 Hz,
1H), 5.53 (dd, J = 15.3, 9.0 Hz, 1H), 4.43 (m, 1H), 4.20 (m, 1H),
3.63 (m, 1H), 3.40 (m, 1H), 3.29 (m, 2H), 2.91 (d, J = 6.6 Hz, 2H),
2.68 (s, 3H), 2.37 (m, 2H), 2.24 (m, 1H), 1.74 (m, 1H); MS (FAB)
m/z: 550 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C28H28N3O5S2,
550.1470; found, 550.1465.
1071, 870, 845, 795, 735, 710, 695 cmꢀ1 1H NMR (300 MHz,
;
DMSO-d6): d 8.31 (s, 1H), 8.15 (m, 2H) 7.90 (m, 2H) 7.45 (m, 3H),
5.72 (dd, J = 15.4, 6.2 Hz, 1H), 5.33 (dd, J = 15.4, 8.8 Hz, 1H), 5.05
(m, 1H), 4.25 (m, 1H), 4.10 (m, 1H), 3.55 (m, 1H), 3.20 (m, 2H),
2.83 (m, 3H), 2.11 (m, 3H), 1.55 (m, 1H); MS (FAB) m/z: 586
(M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C27H25ClN3O4S3,
586.0696; found, 586.0692.
5.1.78. Ethyl (4R,5E,7S)-8-(3-(5,7-dimethyl-1,3-benzoxazol-2-yl) phenyl)-
7-hydroxy-4-({tert-butyoxycarbonyl}amino)-5-octenoate (17e)
Compound 17e was prepared from the aldehyde 16 using the
phosphonate 5o instead of 5b according to the same procedure
as described for the preparation of 13a from 12 as a colorless amor-
phous powder (347 mg, 27% in two steps). 1H NMR (300 MHz,
CDCl3): d 8.17–8.07 (m, 2H), 7.47 (m, 1H), 7.45–7.38 (m, 2H),
6.97 (s, 1H), 5.74 (m, 1H), 5.61 (m, 1H), 4.70 (m, 1H), 4.44 (m,
1H), 4.19–4.03 (m, 3H), 3.01–2.85 (m, 2H), 2.55 (s, 3H), 2.45 (s,
3H), 2.30 (t, J = 7.6 Hz, 2H), 1.91–1.69 (m, 2H), 1.43 (s, 9H), 1.25
(t, J = 7.4 Hz, 3H).
5.1.84. Ethyl (4R,5E,7S)-8-(3-(5-chloro-1,3-benzoxazol-2-yl)
phenyl)-7-hydroxy-4-({tert-butyoxycarbonyl}amino)-5-octenoate
(17f)
Compound 17f was prepared from aldehyde 16 using the phos-
phonate 5p instead of 5b according to the same procedure as
described for the preparation of 13a from 12 as a white powder
(185 mg, 84% in two steps).
5.1.85. Ethyl 2-({2-[(2R)-2-{(1E,3S)-4-[3-(5-chloro-1,3-
benzoxazol-2-yl)phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-
pyrrolidinyl]ethyl}thio)-1,3-thiazole-4-carboxylate (18f)
Compound 18f was prepared from 17f according to the same
procedure as described for the preparation of 18a from 17a as a
pale amorphous powder (75 mg, 35% in two steps).
5.1.79. Ethyl 2-({2-[(2R)-2-{(1E,3S)-4-[3-(5,7-dimethyl-1,3-
benzoxazol-2-yl)phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-
pyrrolidinyl]ethyl}thio)-1,3-thiazole-4-carboxylate (18e)
Compound 18e was prepared from 17e according to the same
procedure as described for the preparation of 18a from 17a as a
pale amorphous powder (258 mg, 64% in two steps).

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
3519
5.1.86. 2-{[2-((2R)-2-{(1E,3S)-4-[3-(5-Chloro-1,3-benzoxazol-2-
yl)phenyl]-3-hydroxybut-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]
sulfanyl}-1,3-thiazole-4-carboxylic acid (4q)
(m, 4H), 3.47 (d, J = 5.5 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H), 1.22 (t,
J = 7.1 Hz, 6H).
Compound 4q was prepared from 18f according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (46 mg, 65%). IR (film): 3093, 2922, 2686,
1717, 1666, 1587, 1490, 1330, 1257, 1208, 1179, 1028, 975, 860,
5.1.91. Ethyl 2-[(2-oxoethyl)thio]-1,3-thiazole-4-carboxylate
(23)
A stirred solution of 22 (16.8 g, 50 mmol) in CH3CN (150 mL)
was treated with 2 N HCl (15 mL) at room temperature under argon
atmosphere. After being stirred for 16 h, the reaction mixture was
evaporated up to about half of its volume, then poured into water
and extracted with AcOEt (five times). The organic solvent was re-
moved by evaporation, the resulting residue was recrystallized
from hexane to give 23 as pale brown powder (10.1 g, 87%). 1H
NMR (300 MHz, CDCl3): d 9.72 (t, J = 2.0 Hz, 1H), 8.05 (s, 1H), 4.39
(q, J = 7.1 Hz, 2H), 4.09 (d, J = 2.0 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H).
725, 685 cmꢀ1 1H NMR1H NMR (300 MHz, CDCl3): d 8.17 (s, 1H),
;
8.05 (m, 2H), 7.71 (m, 1H), 7.64 (m, 1H), 7.44 (m, 3H), 5.80 (dd,
J = 15.4, 7.0 Hz, 1H), 5.42 (dd, J = 15.4, 8.8 Hz, 1H), 4.40 (m, 1H),
4.25 (m, 1H), 3.59 (m, 1H), 3.23 (m, 1H), 2.92 (m, 3H), 2.24 (m,
3H), 1.67 (m, 1H); MS (FAB) m/z: 570 (M+H)+; HRMS-FAB (m/z):
[M+H]+ calcd for C27H25ClN3O5S2, 570.0924; found,570.0925.
5.1.87. Ethyl 4-({2-[(2R)-2-{(1E,3S)-4-[3-(1-benzofuran-2-
yl)phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]
ethyl}thio)butanoate (20)
5.1.92. Ethyl (4R,5E,7S)-8-(3-bromophenyl)-7-hydroxy-4-({tert-
butyoxycarbonyl}amino)-5-octenoate (24)
Compound 20 was prepared from 17a using aldehyde 19 in-
stead of 23 according to the same procedure as described for the
preparation of 18a from 17a as a beige amorphous powder
(290 mg, 56% in two steps). 1H NMR (300 MHz, CDCl3): d 7.79–
7.68 (m, 2H), 7.59 (d, J = 6.6 Hz, 1H), 7.50 (m, 1H), 7.40 (m, 1H),
7.33–7.16 (m, 3H), 7.04 (s, 1H), 5.80 (m, 1H), 5.53 (m, 1H), 4.49
(d, J = 6.0 Hz, 1H), 4.18–4.03 (m, 3H), 3.62 (m, 1H), 3.01–2.85 (m,
2H), 2.65–2.45 (m, 3H), 2.43–2.13 (m, 6H), 2.01 (br s, 1H), 1.91–
1.79 (m, 2H), 1.70 (m, 1H), 1.25 (t, J = 6.9 Hz, 3H).
Compound 24 was prepared from the aldehyde 16 using the
phosphonate 5a instead of 5b according to the same procedure
as described for the preparation of 13a from 12 as a colorless oil
(840 mg, 70% in two steps). 1H NMR (300 MHz, CDCl3): d 7.40–
7.33 (m, 2H), 7.21–7.09 (m, 2H), 5.68 (m, 1H), 5.55 (m, 1H), 4.50
(br s, 1H), 4.35 (m, 1H), 4.19–4.07 (m, 3H), 2.86–2.71 (m, 2H),
2.32 (t, J = 7.7 Hz, 2H), 1.91–1.66 (m, 3H), 1.45 (s, 9H), 1.26 (t,
J = 7.1 Hz, 3H).
5.1.93. Ethyl (4R,5E,7S)-8-(3-bromophenyl)-7-(tert-
5.1.88. 4-{[2-((2R)-2-{(1E,3S)-4-[3-(1-Benzofuran-2-yl)phenyl]-
3-hydroxybut-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]sulfanyl}
butanoic acid (3i)
butyldimethylsilyloxy)-4-({tert-butyoxycarbonyl}amino)-5-
octenoate (25)
To a stirred solution of 24 (590 mg, 1.29 mmol) in DMF (4.5 mL)
and imidazole (176 mg, 2.59 mmol) was added t-butyldiphenylsi-
lyl chloride (291 mg, 1.94 mmol) at room temperature under argon
atmosphere. After being stirred for 1 h, the reaction mixture was
poured into water and extracted with EtOAc. The organic layer
was washed with water (twice), brine, and dried over MgSO4.
The organic layer was evaporated to give a silyl ether 25 as a col-
orless oil (742 mg, 100%). 1H NMR (300 MHz, CDCl3): d 7.36–7.27
(m, 2H), 7.17–7.03 (m, 2H), 5.68–5.57 (m, 1H), 5.45 (m, 1H), 4.42
(br s, 1H), 4.23 (m, 1H), 4.17–4.05 (m, 3H), 2.75–2.60 (m, 2H),
2.30 (t, J = 7.6 Hz, 2H), 1.87–1.67 (m, 2H), 1.42 (s, 9H), 1.25 (t,
J = 6.6 Hz, 3H), 0.81 (s, 9H), ꢀ0.14 (s, 3H), ꢀ0.25 (s, 3H).
Compound 3i was prepared from 20 according to the same pro-
cedure as described for the preparation of 3b from 13a as colorless
amorphous (246 mg, 91%). IR (film): 3389, 2925, 1717, 1655, 1452,
1420, 1257, 751 cmꢀ1 1H NMR (300 MHz, CDCl3): d 7.76–7.70 (m,
;
2H), 7.59 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.40 (dd, J = 8.4,
8.4 Hz, 1H), 7.33–7.17 (m, 3H), 7.04 (s, 1H), 5.79 (dd, J = 15.3,
5.7 Hz, 1H), 5.51 (dd, J = 15.3, 8.4 Hz, 1H), 4.55–4.44 (m, 1H),
4.16–4.07 (m, 1H), 3.68–3.54 (m, 1H), 3.02–2.90 (m, 3H), 2.70–
2.10 (m, 9H), 1.92–1.78 (m, 2H), 1.78–1.62 (m, 1H); MS (APCI) m/
z: 492 (MꢀH)ꢀ.
5.1.89. S-(2,2-Diethoxyethyl) ethanethioate (21)
To a stirred solution of bromoacetaldehyde diethylacetal (29.6 g,
150 mmol) in DMF (70 mL) was added potassium thioacetate
(17.9 g, 157 mmol) at room temperature under argon atmosphere.
After being stirred at 50 °C for 4 h, the reaction mixture was allowed
to be cooled to room temperature. The reaction was quenched with
1 N HCl. The resulting reaction mixture was extracted with EtOAc
(twice). The combinedorganic layers were washed with H2O (twice),
brine, and dried over MgSO4. The organic solvent was removed by
evaporation to afford a thioacetate 21 as a brown oil (28.2 g). 1H
NMR (300 MHz, CDCl3): d 4.50 (t, J = 5.5 Hz, 1H), 3.76–3.47 (m,
4H), 3.12 (d, J = 5.5 Hz, 2H), 2.35 (s, 3H), 1.22 (t, J = 7.1 Hz, 6H).
5.1.94. Ethyl (4R,5E,7S)-8-(20,40-dimethyl-1,10-biphenyl-3-yl)-7-
(tert-butyldimethylsilyloxy)-4-({tert-butyoxycarbonyl}amino)-
5-octenoate (26a)
To a stirred solution of 25 (330 mg, 0.579 mmol) in DME (4 mL)
and 2 M aqueous sodium carbonate (0.87 mL, 1.74 mmol) were
added 2,4-dimethylphenylboronic acid (173 mg, 1.15 mmol), (S)-
2,20-bis(diphenylphosphino)-1,10-binaphthyl
(BINAP)
(72 mg,
0.116 mmol) and palladium acetate (13 mg, 0.058 mmol) under ar-
gon atmosphere. After being stirred at 80 °C for 15 h, the reaction
mixture was cooled to room temperature. The resulting mixture
was filtered through a pad of Celite, and the filtrate was evapo-
rated. The resulting residue was purified by column chromatogra-
phy on silica gel (hexane/EtOAc, 4:1–1:1) to afford 26a as a brown
oil (321 mg, 93%). 1H NMR (300 MHz, CDCl3): d 7.27 (s, 1H), 7.15–
7.00 (m, 6H), 5.65 (m, 1H), 5.46 (m, 1H), 4.43 (m, 1H), 4.30 (m, 1H),
4.17–4.02 (m, 3H), 2.80–2.71 (m, 2H), 2.35 (s, 3H), 2.33–2.22 (m,
2H), 2.23 (s, 3H), 1.86–1.68 (m, 2H), 1.41 (s, 9H), 1.25 (t,
J = 7.0 Hz, 3H), 0.79 (s, 9H), ꢀ0.13 (s, 3H), ꢀ0.24 (s, 3H).
5.1.90. Ethyl 2-[(2,2-diethoxyethyl)thio]-1,3-thiazole-4-
carboxylate (22)
To a stirred solution of thioacetate 21 (10.1 g, 52.5 mmol) in
EtOH (60 mL) and DMF (60 mL) was added potassium carbonate
(10.4 g, 75.0 mmol) and ethyl 2-bromothiazole-4-carboxylate
(11.8 g, 50.0 mmol) at room temperature under argon atmosphere.
After being stirred at room temperature for 15 h, the reaction was
quenched with saturated aqueous NH4Cl. The reaction mixture was
diluted with EtOAc, washed with H2O twice, brine, and dried over
MgSO4. The organic solvent was removed by evaporation to give a
sulfide 22 as a yellow oil (16.8 g). 1H NMR (300 MHz, CDCl3): d 8.02
(s, 1H), 4.77 (t, J = 5.4 Hz, 1H) 4.39 (q, J = 7.08 Hz, 2H), 3.81–3.52
5.1.95. Ethyl 2-({2-[(2R)-2-{(1E,3S)-4-(20,40-dimethyl-1,10-
biphenyl-3-yl)-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]
ethyl}thio)-1,3-thiazole-4-carboxylate (27a)
Compound 27a was prepared from 26a according to the same
procedure as described for the preparation of 18a from 17a as a

3520
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
pale amorphous powder (150 mg, 52%). 1H NMR (300 MHz, CDCl3):
d 8.00 (s, 1H), 7.30 (m, 1H), 7.18 (m, 1H), 7.14–7.01 (m, 5H), 5.82
(m, 1H), 5.55 (m, 1H), 4.38 (m, 1H), 4.25–4.07 (m, 3H), 3.67 (m,
1H), 3.37 (t, J = 6.6 Hz, 2H), 3.20 (m, 1H), 2.87–2.80 (m, 2H), 2.36
(s, 3H), 2.31 (m, 1H), 2.23 (s, 3H), 2.21–2.10 (m, 2H), 1.73 (m,
1H), 1.27 (t, J = 7.0 Hz, 3H).
5.76 (dd, J = 15.3, 5.4 Hz, 1H), 5.52 (dd, J = 15.3, 8.4 Hz, 1H), 4.45–
4.33 (m, 1H), 4.15–4.02 (m, 3H), 3.70–3.58 (m, 1H), 3.02–2.91
(m, 1H), 2.83–2.72 (m, 2H), 2.72–2.48 (m, 4H), 2.48–2.30 (m,
4H), 2.30–2.20 (m, 1H), 1.98–1.84 (m, 3H), 1.80–1.64 (m, 1H),
1.64–1.50 (m, 2H), 1.43–1.30 (m, 2H), 0.97 (s, 9H), 0.92 (t,
J = 7.2 Hz, 1H), 0.20 (s, 6H).
5.1.96. 2-[(2-{(2R)-2-[(1E,3S)-4-(20,40-Dimethyl-1,10-biphenyl-3-
yl)-3-hydroxybut-1-enyl]-5-oxopyrrolidin-1-yl}ethyl)sulfanyl]-
1,3-thiazole-4-carboxylic acid (4f)
5.1.101. Butyl 4-({2-[(2R)-2-{(1E,3S)-4-(3-hydroxyphenyl)-3-
(tetrahydro-2H-pyran-2-yloxy)-1-buten-1-yl}-5-oxo-1-
pyrrolidinyl]ethyl}thio)butanoate (29)
Compound 4f was prepared from 27a according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (142 mg, 100%). IR (film): 3407, 3112, 2924,
2858, 1717, 1663, 1649, 1639, 1561, 1506, 1479, 1458, 1421,
1326, 1214, 1098, 1028, 974, 921, 865, 822, 793, 714, 661, 594,
To a stirred solution of 28 (992 mg, 1.76 mmol) and 3,4-dihyd-
ropyran (0.32 mL, 3.52 mmol) in CH2Cl2 (10 mL) was added p-tolu-
enesulfonic acid monohydrate (3.3 mg, 0.018 mmol) at room
temperature under argon atmosphere After being stirred for
30 min, the reaction was quenched with several drops of triethyl-
amine and the solvent was evaporated to give crude tetrahydro-
pyranyl ether as a light yellow oil.
To a stirred solution of the above-described tetrahydropyranyl
ether in THF (5 mL) was added TBAF (1.0 M in THF, 2.6 mL,
2.6 mmol) at room temperature under argon atmosphere. The
solution was stirred for 1 h and then poured into saturated aque-
ous NH4Cl. The reaction mixture was extracted with EtOAc. The or-
ganic layer was washed with water, brine and dried over Na2SO4.
The organic solvent was evaporated and the residue was purified
by column chromatography on silica gel (hexane/EtOAc, 2:1–1:2)
to give 29 as a white solid. (873 mg, 93%) 1H NMR (300 MHz,
CDCl3): d 7.12 (t, J = 7.7 Hz, 1H), 6.80–6.57 (m, 3H), 6.14 (m, 1H),
5.61 (m, 1H), 5.35 (m, 2H), 4.60 (m, 1H), 4.30 (m, 1H), 4.16–3.97
(m, 3H), 3.88–3.33 (m, 3H), 3.06 (m, 1H), 2.80–2.39 (m, 7H),
2.37–2.28 (m, 2H), 2.15 (m, 1H), 1.98–1.30 (m, 13H), 0.93 (t,
J = 7.1 Hz, 3H).
583, 568 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.07 (s, 1H), 7.35 (t,
;
J = 8.0 Hz, 1H), 7.15 (m, 6H), 5.82 (m, 1H), 5.53 (m, 1H), 4.44 (m,
1H), 4.12 (m, 1H), 3.71 (m, 1H), 3.24 (m, 3H), 2.88 (d, J = 6.6 Hz,
2H), 2.36 (s, 3H), 2.33 (m, 3H), 2.23 (s, 3H), 1.74 (m, 1H); MS
(FAB) m/z: 523 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for
C28H31N2O4S2, 523.1725; found, 523.1720.
5.1.97. Ethyl (4R,5E,7S)-8-(30,40-Dimethyl-1,10-biphenyl-3-yl)-7-
(tert-butyldimethylsilyloxy)-4-({tert-butyoxycarbonyl}amino)-
5-octenoate (26b)
Compound 26b was prepared from 25 using 3,4-dim-
ethylphenylboronic acid instead of 2,4-dimethylphenylboronic
acid according to the same procedure as described for the prepara-
tion of 26a from 25 as a pale amorphous powder (306 mg, 87%). 1H
NMR (300 MHz, CDCl3): d7.45–7.27 (m, 4H), 7.19 (d, J = 8.1 Hz, 1H),
7.10 (d, J = 7.3 Hz, 1H), 6.97 (m, 1H), 5.76 (m, 1H), 5.51 (m, 1H),
4.44 (m, 1H), 4.31 (s, 1H), 4.18–4.06 (m, 3H), 2.83–2.74 (m, 3H),
2.33 (s, 3H), 2.30 (s, 3H), 1.78 (s, 2H), 1.42 (s, 9H), 1.30–1.20 (m,
3H), 0.81 (s, 9H), ꢀ0.12 (s, 3H), ꢀ0.24 (s, 3H).
5.1.102. Butyl 4-({2-[(2R)-2-{(1E,3S)-4-[3-(3-pyridinylmethoxy)
phenyl]-3-(tetrahydro-2H-pyran-2-yloxy)-1-buten-1-yl}-5-oxo-
1-pyrrolidinyl]ethyl}thio)butanoate (30a)
5.1.98. Ethyl 2-({2-[(2R)-2-{(1E,3S)-4-(30,40-Dimethyl-1,10-
biphenyl-3-yl)-3-hydroxy-1-buten-1-yl}-5-oxo-1-
pyrrolidinyl]ethyl}thio)-1,3-thiazole-4-carboxylate (27b)
Compound 27b was prepared from 26b according to the same
procedure as described for the preparation of 18a from 17a as a
pale amorphous powder (150 mg, 55%).
To a stirred solution of 29 (300 mg, 0.562 mmol), 3-hydroxy-
methylpyridine (73.5 mg, 0.674 mmol) and triphenylphosphine
(177 mg, 0.674 mmol) in THF (7 mL) was added diethyl azodicar-
boxylate (40% in toluene, 2.98 mL, 0.674 mmol) at 0 °C under argon
atmosphere. After stirring for 3 h at room temperature, the solu-
tion was diluted with EtOAc. The organic layer was washed with
water, brine and dried over Na2SO4. The organic solvent was re-
moved by evaporation and the resulting residue was purified by
column chromatography on silica gel (hexane/AcOEt, 1:2–0:1) to
give 30a as a colorless oil (168 mg, 48%).
5.1.99. 2-[(2-{(2R)-2-[(1E,3S)-4-(30,40-Dimethyl-1,10-biphenyl-3-
yl)-3-hydroxybut-1-enyl]-5-oxopyrrolidin-1-yl}ethyl)sulfanyl]-
1,3-thiazole-4-carboxylic acid (4g)
Compound 4g was prepared from 27b according to the same
procedure as described for the preparation of 3b from 13a as col-
orless amorphous (143 mg, 100%). IR (film): 3402, 3112, 2924,
2859, 1718, 1663, 1649, 1561, 1508, 1458, 1421, 1210, 1099,
5.1.103. 4-{[2-((2R)-2-{(1E,3S)-3-Hydroxy-4-[3-(pyridin-3-
ylmethoxy)phenyl]but-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]
sulfanyl}butanoic acid (3j)
To a stirred solution of 30a (167 mg, 0.268 mmol) in MeOH
(2 mL) was added 2 N HCl (1 mL) at room temperature under argon
atmosphere. The heterogeneous reaction mixture was stirred for
30 min and the resulting clear solution was treated with 2 N NaOH
(2 mL). After stirring for 20 min, the solution was diluted with
water and extracted with ether. The aqueous layer was neutralized
with 1 N HCl and then evaporated. The precipitating inorganic salt
was removed by filtration through a pad of Celite and washed with
EtOH and EtOAc repeatedly. The filtrate was evaporated and the
resulting residue was purified by column chromatography on silica
gel (CHCl3/MeOH, 1:0–100:1–50:1–30:1) to give 3j as a colorless
viscous oil (82 mg, 63%). IR (film): 3376, 2917, 1664, 1421, 1254,
1027, 881, 825, 790, 715, 610, 594 cmꢀ1 1H NMR (300 MHz,
;
CDCl3): d 8.06 (s, 1H), 7.45–7.35 (m, 5H), 7.20–7.12 (m, 2H), 5.82
(dd, J = 15.3, 6.0 Hz, 1H), 5.50 (dd, J = 15.3, 9.0 Hz, 1H), 4.46 (m,
1H), 4.10 (m, 1H), 3.68 (m, 1H), 3.24 (m, 2H), 3.13 (m, 1H), 2.91
(m, 2H), 2.33 (m, 3H), 2.31 (s, 3H), 2.30 (m, 3H), 1.70 (m, 1H);
MS (FAB) m/z: 523 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for
C28H31N2O4S2, 523.1725; found, 523.1724.
5.1.100. Butyl 4-({2-[(2R)-2-{(1E,3S)-4-[3-(tert-butyldimethyl-
silyloxy)phenyl]-3-hydroxy-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]
ethyl}thio)butanoate (28)
Compound 28 was prepared from the aldehyde 12 using the
phosphonate 5q instead of 5b according to the same procedure
as described for the preparation of 13a from 12 as a colorless oil
(992 mg, 65% in two steps). 1H NMR1H NMR (300 MHz, CDCl3): d
7.17 (t, J = 8.1 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 6.86–6.68 (m, 2H),
1157, 1036, 752 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.72 (s, 1H),
;
8.45 (d, J = 3.3 Hz, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.39 (dd, J = 7.8,
3.3 Hz, 1H), 7.16 (dd, J = 8.1, 8.1 Hz, 1H), 7.04 (s, 1H), 6.80–6.75
(m, 2H), 5.85 (dd, J = 15.3, 4.8 Hz, 1H), 5.62 (dd, J = 15.3, 8.7 Hz,
1H), 5.24 (d, J = 13.2 Hz, 1H), 5.17 (d, J = 13.2 Hz, 1H), 4.35–4.30

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
3521
(m, 1H), 4.20–4.10 (m, 1H), 3.53–3.40 (m, 1H), 3.30–3.16 (m, 1H),
2.81–2.38 (m, 10H), 2.35–2.12 (m, 1H), 1.95–1.80 (m, 2H), 1.81–
1.62 (m, 1H); MS (FAB) m/z: 485 (M+H)+; HRMS-FAB (m/z):
[M+H]+ calcd for C26H33N2O5S, 485.2110; found, 485.2116.
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 as-
say 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
incubated for 20 min. Incubation was terminated via filtration
through a Whatman GF/B filter. The filter was subsequently
washed with ice-cold buffer (10 mM KH2PO4–KOH buffer contain-
ing 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 counter. Nonspecific binding was achieved by
adding excess amounts of unlabeled PGE2 in the assay buffer. The
concentration that causes 50% of inhibition (IC50 value) was esti-
mated 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 disso-
ciation constant of radiolabeled ligand for the prostanoid receptor
of interest.
5.1.104. Butyl 4-({2-[(2R)-2-{(1E,3S)-4-[3-(2-pyridinylmethoxy)
phenyl]-3-(tetrahydro-2H-pyran-2-yloxy)-1-buten-1-yl}-5-oxo-
1-pyrrolidinyl]ethyl}thio)butanoate (30b)
Compound 30b was prepared from 29 according to the same
procedure as described for the preparation of 30a from 29 as a col-
orless oil (46 mg, 75%). 1H NMR (300 MHz, CDCl3): d 8.60 (d,
J = 4.8 Hz, 1H), 7.72 (m, 1H), 7.52 (d, J = 8.1 Hz, 1H),7.27–7.10 (m,
2H), 6.90–6.78 (m, 3H), 5.78–5.30 (m, 2H), 5.19 (s, 2H), 4.62–4.30
(m, 2H), 4.07 (t, J = 7.5 Hz, 2H), 3.83–3.35 (m, 4H), 3.03–2.16 (m,
3H), 1.95–1.30 (m, 7H), 0.93 (t, J = 7.5 Hz, 3H).
5.1.105. 4-{[2-((2R)-2-{(1E,3S)-3-Hydroxy-4-[3-(pyridin-2-
ylmethoxy)phenyl]but-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]
sulfanyl}butanoic acid (3k)
Compound 3k was prepared from 30b according to the same
procedure as described for the preparation of 3j from 30a as color-
less oil (25 mg, 62%). IR (film): 3383, 2924, 1715, 1666, 1598, 1488,
5.2.2. Measurement of cAMP production
1437, 1419, 1383, 1266, 1157, 1100, 1048, 975, 758, 697, 666 cm
;
Chinese hamster ovary (CHO) cells expressing mouse or rat EP2
or EP4-receptor were cultured in 24-well plates (1 ꢁ 105 cells/
well). After 2 days, the media were removed and cells were washed
ꢀ1
1H NMR (300 MHz, CDCl3): d 8.57 (d, J = 5.4 Hz, 1H), 7.80 (dt,
J = 1.5, 7.5 Hz, 1H), 7.60 (d, J = 7.5 Hz, 1H), 7.32 (m, 1H), 7.22 (t,
J = 7.8 Hz, 1H), 6.99–6.85 (m, 2H), 6.80 (d, J = 7.8 Hz, 1H), 5.85
(dd, J = 15.0, 4.8 Hz, 1H), 5.59 (ddd, J = 15.0, 8.7, 1.2 Hz, 1H), 5.32
(s, 2H), 4.43 (m, 1H), 4.11 (m, 1H), 3.43 (m, 1H), 3.18 (m, 1H),
2.88–2.18 (m, 13H), 1.97–1.83 (m, 2H), 1.72 (m, 1H); MS (FAB)
m/z: 485 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C26H33N2O5S,
485.2110; found, 485.2105.
with 500
for 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
lL of Minimum Essential Medium (MEM) and incubated
l
L of buffer (MEM containing 2 M of diclofenac)
l
l
l
l
5.1.106. Butyl 4-({2-[(2R)-2-{(1E,3S)-4-[3-(4-pyridinylmethoxy)
phenyl]-3-(tetrahydro-2H-pyran-2-yloxy)-1-buten-1-yl}-5-oxo-
1-pyrrolidinyl]ethyl}thio)butanoate (30c)
10% trichloroacetic acid. cAMP production was determined via a
cAMP radioimmunoassay kit (Amersham).
Compound 30c was prepared from 29 according to the same
procedure as described for the preparation of 30b from 29 as a
colorless oil (36 mg, 59%). 1H NMR (300 MHz, CDCl3): d 8.01
(d, J = 5.4 Hz, 2H), 7.35 (d, J = 5.4 Hz, 2H), 7.30–7.18 (m, 3H),
6.90–6.77 (m, 3H), 5.70 and 5.55–5.29 (m, 2H), 5.08 (s, 2H), 4.62,
4.50 and 4.40–4.28 (m, 2H), 4.07 (t, J = 6.6 Hz, 2H), 3.80 and
3.62–3.30 (m, 3H), 3.05–2.12 (m, 3H), 1.95–0.138 (m, 7H), 0.93
(t, J = 7.5 Hz, 3H).
5.2.3. Biological evaluation
Animals: Sprague–Dawley male rats (CrJ:CD(SD)IGS, 8-weeks
old, Charles River Laboratories (Atsugi, Japan), were maintained
in a controlled environment of 12 h light/12 h dark at 24 2 °C,
humidified at 55 15%. The animals were housed in polycarbonate
cage. All animal experiments in this study were performed in
accordance with ethical guidelines established by the Experimen-
tal Animal Care and Use Committee of ONO Pharmaceutical Co.,
Ltd.
5.1.107. 4-{[2-((2R)-2-{(1E,3S)-3-Hydroxy-4-[3-(pyridin-4-
ylmethoxy)phenyl]but-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]
sulfanyl}butanoic acid (3l)
Fracturemodels: Fibulae were fractured according to the method
of Kawaguchi et al. with minor modification.15 In brief, after anes-
thetizing the animals with sodium pentobarbital (30–50 mg/kg),
the midshaft of each left fibula was exposed and cut sharply with
bone cutter.
Determination of bone strength: Fibulae were removed from
anesthetized rats to estimate mechanical strength 17 days after
fracture. The three-point destructive bending test was performed
with a bone-testing machine (Instron 5544 model, Instron Japan).
Breaking strength was expressed as percentage of the mean value
of nonfractured fibulae harvested from intact rats.
Compound 3l was prepared from 30c according to the same
procedure as described for the preparation of 3j from 30a as color-
less oil (19 mg, 60%). IR (film): 3382, 2924, 1715, 1666, 1611, 1584,
1488, 1446, 1418, 1384, 1256, 1158, 1047, 975, 803, 755 cmꢀ1; 1H
NMR (300 MHz, CDCl3): d 8.58 (d, J = 6.0 Hz, 2H), 7.39 (d, J = 6.0 Hz,
2H), 7.22 (t, J = 7.8 Hz, 1H), 6.88–6.70 (m, 3H), 5.72 (dd, J = 15.3,
5.7 Hz, 1H), 5.45 (dd, J = 15.3, 8.1 Hz, 1H), 5.12 (s, 2H), 4.32 (m,
1H), 4.11 (m, 1H), 3.59 (m, 1H), 3.30 (m, 1H), 2.99 (m, 1H), 2.78
(m, 2H), 2.69–2.12 (m, 10H), 1.98–1.80 (m, 2H), 1.63 (m, 1H); MS
(FAB) m/z: 485 (M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for
5.2.4. Pharmacological evaluation of the hypotensive effects of
compound 3i/PLGA in rats
C26H33N2O5S, 485.2110; found, 485.2102.
Sprague–Dawley male rats (Crj:CD(SD)IGS, 7–9 weeks old
(Charles River Laboratories)) were anesthetized with urethane
(1.5 g/kg, sc). Blood pressure (BP) was measured directly from
the C3 arteria carotis tube catheter equipped with a pressure trans-
ducer. Heart rate was also calculated from the pulse wave of the
BP. Test compounds were injected into the femoral muscle.
5.2. Pharmacological evaluation
5.2.1. mEP1–4 receptor binding assay
Competitive binding studies were conducted using radiolabeled
ligands and membrane fractions prepared from Chinese hamster

3522
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 3502–3522
5.3. PLGA formulation
the pellet was dissolved with p-hydroxy benzoic acid n-nonyl ester
in acetonitrile. The concentration of compound in the solution was
analyzed by high performance liquid chromatography (HPLC).
The remaining percentage of compound was calculated as
follows:
5.3.1. Preparation of PLGA microspheres containing compound
3i, 4i, 4m,n
Microspheres were prepared by the emulsion–solvent evapora-
tion method.16,17 Compound (10 mg) and PLGA 75–65 (90 mg)
were dissolved in 1 mL of dichloromethane in the oil phase. The
oil phase was gradually added into an aqueous 0.1% PVA (polyvinyl
alcohol) solution stirred with a turbine-shaped mixer (Homo-
mixer) at 6000 rpm to obtain o/w emulsion. The emulsion was con-
tinuously stirred gently with a magnetic stirrer for 3 h to remove
CH2Cl2. After organic solvent evaporation, the PLGA microspheres
were suspended in the PVA solution. The suspension was centri-
fuged at 3000 rpm for 10 min to precipitate the microspheres
and remove any unreacted compound. The supernatant was then
discarded and replaced with fresh water or aqueous medium con-
taining 0.2% Tween 80. This washing procedure was performed
repeatedly. The washed microsphere precipitation was lyophilized
to remove residual organic solvent and water, and to recover com-
pound/ microspheres in solid form.
Rx ¼ Px=ðS0 þ P0Þ ꢁ 100
where, Rx = Remaining percentage of compound at x days (%),Px = A-
mount of compound in the pellet of 1 mL sample at x days
(l
g),S0 = Amount of compound in the supernatant of 1 mL sample
at 0 day ( g),P0 = Amount of compound in the pellet of 1 mL sample
at 0 day ( g).
l
l
References and notes
1. Narumiya, S.; Sugimoto, Y.; Ushikubi, F. Physiol. Rev. 1999, 79, 1193.
2. Yoshida, K.; Oida, H.; Kobayashi, T.; Maruyama, T.; Tanaka, M.; Katayama, T.;
Yamaguchi, K.; Segi, E.; Tsuboyama, T.; Matsushita, M.; Ito, K.; Ito, Y.; Sugimoto,
Y.; Ushikubi, F.; Ohuchida, S.; Kondo, K.; Nakamura, T.; Narumiya, S. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 4580.
3. Paralkar, V. M.; Borovecki, F.; Ke, H. Z.; Cameron, K. O.; Lefker, B.; Grasser, W.
A.; Owen, T. A.; Li, M.; DaSilva-Jardine, P.; Zhou, M.; Dunn, R. L.; Dumont, F.;
Korsmeyer, R.; Krasney, P.; Brown, T. A.; Plowchalk, D.; Vukicevic, S.;
Thompson, D. D. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6736.
4. Maruyama, T.; Asada, M.; Shiraishi, T.; Yoshida, H.; Maruyama, T.; Ohuchida, S.;
Nakai, H.; Kondo, K.; Toda, M. Bioorg. Med. Chem. 2002, 10, 1743.
5. Elworthy, T. R.; Brill, E. R.; Chiou, S.-S.; Chu, F.; Harris, J. R.; Hendricks, R. T.;
Huang, J.; Kim, W.; Lach, L. K.; Mirzadegan, T.; Yee, C.; Walker, K. A. M. J. Med.
Chem. 2004, 47, 6124.
5.3.2. Drug loading and encapsulation efficiency of compounds
3i, 4i, 4m,n PLGA microspheres
The appropriate quantity of compound compound-loaded PLGA
microspheres was dissolved with p-hydroxy benzoic acid n-nonyl
ester in acetonitrile. The concentration of compound in the solu-
tion was analyzed by high performance liquid chromatography
(HPLC). The drug loading and the encapsulation efficiency were
calculated as follows:
6. Xiao, Y.; Araldi, G. L.; Zhao, Z.; Brugger, N.; Karra, S.; Fischer, D.; Palmer, E.
Bioorg. Med. Chem. Lett. 2007, 17, 4323.
7. Xiao, Y.; Araldi, G. L.; Zhao, Z.; Reddy, A.; Karra, S.; Brugger, N.; Fischer, D.;
Palmer, E.; Bao, B.; Mckenna, S. D. Bioorg. Med. Chem. Lett. 2008, 18, 821.
8. 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.
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.
10. Kambe, T.; Maruyama, T.; Naganawa, A.; Asada, M.; Seki, A.; Maruyama, T.;
Nakai, H.; Toda, M. Chem. Pharm. Bull. 2011, 59, 1494.
11. 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.
2012, 22, 396.
12. Kambe, T.; Maruyama, T.; Nakai, Y.; Yoshida, H.; Oida, H.; Maruyama, T.; Abe,
N.; Nishiura, A.; Nakai, H.; Toda, M. Bioorg. Med. Chem. 2012, 20, 2235.
13. Kambe, T.; Maruyama, T.; Nagase, T.; Ogawa, S.; Minamoto, C.; Sakata, K.;
Maruyama, T.; Nakai, H.; Toda, M. Bioorg. Med. Chem. 2012, 20, 702.
14. Corey, E. J.; Bakshi, R. K.; Shibata, S. J. Am. Chem. Soc. 1987, 109, 5551.
15. Kiriyama, M.; Ushikubi, F.; Kobayashi, T.; Hirata, M.; Sugimoto, Y.; Narumiya, S.
Br. J. Pharmacol. 1997, 122, 217.
16. Okada, H.; Ogawa, Y.; Yashiki, T. US Patent, 4,652,441, 1987.
17. Kawagushi, H.; Kurokawa, T.; Hanada, K.; Hiyama, Y.; Tamura, M.; Ogata, E.;
Matsumoto, T. Endocrinology 1994, 135, 774.
Drug loading ð%; w=wÞ ¼ ðM=WÞ ꢁ 100
where,M = measured amount of compound in the compound-
loaded-PLGA microspheres (lg),W = weight of compound-loaded-
PLGA microspheres ( g),Encapsulation efficiency (%, w/w) = (Lm/
l
Lt) ꢁ 100, where Lm = measured drug loading (%, w/w) and Lt = the-
oretical drug loading (%, w/w).
5.3.3. In vitro release profile of compound 3i, 4i, 4m,n PLGA
microsphere
Compound-loaded PLGA microspheres were suspended in phos-
phate-buffered saline (PBS) containing 0.2% Tween 80 to adjust the
concentration of compound to 100 lg/mL. The microspheres were
then completely dispersed by vortex mixing and sonication. The
solution was divided into 1 mL samples and incubated at 37 °C.
At various time intervals, the aliquots were centrifuged for 5 min
at 12,000 rpm (n = 3 for each). The supernatant was removed and