Discovery of novel prostaglandin analogs as potent and selective EP2/EP4 dual agonists

Article abstract of DOI:10.1016/j.bmc.2012.02.018

To identify potent EP2/EP4 dual agonists with excellent subtype selectivity, a series of γ-lactam prostaglandin E analogs bearing a 16-phenyl ω-chain were synthesized and evaluated. Structural hybridization of 1 and 2, followed by more detailed chemical m

Full text of DOI:10.1016/j.bmc.2012.02.018

Bioorganic & Medicinal Chemistry 20 (2012) 2235–2251
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel prostaglandin analogs as potent and selective EP2/EP4
dual agonists
⇑
Tohru Kambe , Toru Maruyama, Yoshihiko Nakai, Hideyuki Yoshida, 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:
To identify potent EP2/EP4 dual agonists with excellent subtype selectivity, a series of c-lactam prosta-
Received 10 December 2011
Revised 4 February 2012
Accepted 6 February 2012
Available online 16 February 2012
glandin E analogs bearing a 16-phenyl
x-chain were synthesized and evaluated. Structural hybridization
of 1 and 2, followed by more detailed chemical modification of the benzoic acid moiety, led us to the dis-
covery of a 2-mercaptothiazole-4-carboxylic acid analog 3 as the optimal compound in the series. An iso-
mer of this compound, the 2-mercaptothiazole-5-carboxylic acid analog 13, showed 34-fold and 13-fold
less potent EP2 and EP4 receptor affinities, respectively. Structure activity relationship data from an
in vitro mouse receptor binding assay are presented. Continued evaluation in an in vivo rat model of
another 2-mercaptothiazole-4-carboxylic acid analog 17, optimized for sustained compound release from
PLGA microspheres, demonstrated its effectiveness in a rat bone fracture-healing model following topical
administration.
Keywords:
Prostaglandin
EP2 receptor
EP4 receptor
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Several research groups have investigated strategies for improv-
ing the pharmacological properties of PGE2.5–8 Efforts to improve its
Prostaglandin E2 (PGE2) is an oxidative metabolite produced by
the action of cyclooxygenase on arachidonic acid that is produced
in a multitude of significant physiological processes. Receptors for
PGE2 (EPs) can be classified into four subtypes (EP1, EP2, EP3 and
EP4), each of which mediates different effects in various tissues
and cells.1 The EP4 receptor is distributed in the thymus, lung,
heart, kidney, bone, womb and other organs, and mediates an in-
crease in intracellular cyclic AMP concentration. Various biological
actions of PGE2, such as cytoprotection, improvement of blood
flow, regulation of inflammatory cytokine production and bone
resorption/formation, are thought to be mediated by the EP4 sub-
type. In our previous study, we introduced the potent and orally
available EP4 agonist 1, which potently inhibited the production
subtype selectivity and chemical stability have focused mainly on
two general chemical modifications: replacement of the -alkenyl
side chain with a phenylethyl group; and replacement of the 11-hy-
a
droxy cyclopentanone moiety with 2-pyrrolidinone.
Because of the presumed therapeutic potential of EP2 and EP4
agonists for the treatment of bone diseases, our aim was to modify
PGE2 to produce analogs with high subtype selectivity and high
potency for both EP2 and EP4 receptor subtypes. In our previous
reports, we described a 2-pyrrolidinone prostanoid 1 (Fig. 1 and
Table 2) that was an EP4 receptor agonist with high selectivity
and potency.2 Compound 2 (Fig. 1 and Table 1) was also reported
as an EP4-selective agonist.5 We focused on the activity profiles
of 2 because it also displayed weak-to-moderate binding affinity
for the EP2 subtype (Ki = 340 nM for mEP2; in-house data in Table
1), in addition to potent affinity for EP4 and moderate affinity for
EP3. Based on the information described above, structural hybrid-
ization of 1 and 2, followed by more detailed chemical modifica-
tion of the benzoic acid moiety, was expected to generate a new
structure with the desired activity profiles: an EP2/EP4 dual ago-
nist with high selectivity and potency.
of TNF-
a
in LPS-induced rats.2 Current literature suggests that
EP4 agonists restore bone mass and strength normally lost in rats
subjected to ovariectomy or immobilization.3
Recently, EP2 receptor agonists have also been shown to have
an anabolic effect on bone formation in various animal models.4
These findings led us to develop a novel EP2/EP4 dual agonist that
would combine these actions on bone structure, and perhaps lead
to a putative novel therapeutic agent for bone diseases such as
osteoporosis and bone fracture healing in humans.
In this report, we describe the development of a new PGE ana-
log of structure 3 (Fig. 1 and Table 2) consisting of the
(beneficial for EP4 receptor affinity), a -lactam ring as a backbone
and a novel 2-mercaptothiazole-4-carboxylic acid substitution of
the -alkenyl side chain. In vivo assessment of 2-mercaptothiaz-
x-chain
c
a
ole-4-carboxylic acid analog 17 further optimized for sustained re-
lease is also presented.
⇑
Corresponding author. Tel.: +81 75 961 1151; fax: +81 75 962 9314.
E-mail addresses: kanbe@ono.co.jp, t-kam@iris.dti.ne.jp (T. Kambe).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.018

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T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
CO2H
The phosphonates 29a–e were prepared according to conven-
tional methodology.2,10,12 The preparation of phosphonates 29f–g
(Scheme 1c) is described in Scheme 1c. Condensation of (3-bromo-
phenyl)acetic acid with N,O-dimethylhydroxylamine hydrochlo-
ride in the presence of EDC–HCl produced the corresponding
Weinreb amide, palladium-catalyzed cross-coupling reaction of
which with 4-chlorophenylboronic acid and 2-naphthaleneboronic
acid gave 30a–b. Reaction of Weinreb amides 30a–b with the lith-
ium anion of dimethyl methylphosphonate provided 29f–g.
Synthesis of 7 is described in Scheme 2. O-Protection of the
O
O
S
CO2H
CH3
N
N
CH3
OH
OH
1
2
CO2H
R
N
S
O
S
N
commercially available
D-pyroglutaminol with methoxymethyl
OH
chloride, followed by palladium-catalyzed N-phenylation with
methyl 3-(3-bromophenyl)acrylate, provided 31. Sodium borohy-
dride reduction of 31 in the presence of nickel dichloride provided
saturated N-phenylpropionate 32. Compound 32 was converted to
3
R = CH3
Cl
17 R =
7 by the conventional procedure of
glandin (PG), as described above.
x-chain synthesis of prosta-
*
Figure 1. Molecular design of a
mercaptothiazole-4-caboxylic acid as its a-chain.
c-lactam PGE analogs bearing ethyl-linked 2-
Compounds 8 and 10b–c were synthesized as described in
Scheme 3. N-Alkylation of O-protected -pyroglutaminol 33 with
D
1,3-bis(bromomethyl)benzene in the presence of sodium hydride
provided 34a.10 Palladium-catalyzed insertion of carbon monoxide
into the benzyl bromide moiety of 34a provided 34b, which was
2. Chemistry
converted to 8 via 35 by the usual procedure of
x-chain synthesis
Synthesis of the test compounds listed in Tables 1–3 is described
in Schemes 1–9. Compounds 3 and 13–15 were synthesized as de-
scribed in Scheme 1a. Ethanolysis of 19, followed by S-arylation
with ethyl 2-bromothiazole-4-carboxylate in the presence of potas-
sium carbonate in ethanol, produced 20.10 Deprotection of 20 with
tetrabutylammonium fluoride (TBAF) provided an alcohol 21a. Oxi-
dation of 21a with dimethyl sulfoxide (DMSO) in the presence of
sulfur trioxide–pyridine complex (SO3–Py) and N,N-diisopropyleth-
ylamine produced an aldehyde 22a, which was converted to enones
23a–c by Horner–Emmons olefination using 29a–c (Scheme 1c) as
phosphonates. Stereoselective reduction of the 15-ketones of 23a–c
with (R)-Me-CBS produced 15(S)-alcohols 24a–c.11 Alkaline hydro-
lysis of 24a–c provided 3 and 14–15. Ethanolysis of 19, followed by
S-arylation with ethyl 2-bromothiazole-5-carboxylate, produced
25. Deprotection of 25 with TBAF yielded an alcohol 26. Oxidation
of 26 with DMSO in the presence of SO3–Py and N,N-diisopropyleth-
ylamine, followed by Horner–Emmons olefination, produced an en-
one 27 using 29a (Scheme 1c) as a phosphonate. Stereoselective
reduction of 27 with (R)-Me-CBS resulted in 15(S)-alcohol 28, alka-
line hydrolysis of which resulted in 13.
of PG, as described above. N-Alkylation of 33 with methyl 5-(3-bro-
mopropyl)thiophene-2-carboxylate in the presence of sodium hy-
dride produced 34c, deprotection of which with TBAF provided
36. Compound 36 was converted to 10b–c by the usual procedure
for prostaglandin synthesis.
To synthesize 9, we developed an alternative synthetic method
of 8-aza PGE analogs starting from N-Boc
scribed in Scheme 4. Oxidation of N-Boc
D
-glutaminol 37 as de-
-glutaminol 37 with
D
DMSO in the presence of oxallyl chloride and N,N-diisopropylethyl-
amine afforded an aldehyde 38.13 Horner–Emmons olefination of
38 with the phosphonate anion prepared from 29d followed by
the usual (R)-Me-CBS-reduction of the formed enone resulted in
an allyl alcohol 39 stereoselectively. Acidic deprotection of 39 with
trifluoroacetic acid afforded 40. Cyclization of 40 followed by N-
alkylation of the formed c-lactam with a bromide 42 prepared as
described below gave 41, alkaline hydrolysis of which afforded 9.
Synthesis of 10a is outlined in Scheme 5. Horner–Emmons olef-
ination of an aldehyde 38 with the phosphonate 29a in the pres-
ence of sodium hydride, followed by stereoselective reduction
with (R)-Me-CBS, produced an allyl alcohol 43. Acidic deprotection
of 43 provided 44. Reductive alkylation of the amine hydrochloride
44 with 3-(4-bromophenyl)propionaldehyde in the presence of
sodium triacetoxyborohydride, followed by intramolecular cycliza-
tion and then O-protection as a TBS–ether, produced 45. Carbonyl
insertion reaction into the phenylbromide 45 in ethanol provided
an ethyl ester 46. Acidic deprotection of 46 followed by alkaline
hydrolysis resulted in 10a.
Synthesis of 16–18 is outlined in Scheme 1b. Ester exchange
reaction of the above-described 20, followed by deprotection with
TBAF, provided an alcohol 21b. Oxidation of 21b with DMSO in the
presence of SO3–Py and N,N-diisopropylethylamine produced an
aldehyde 22b, Horner–Emmons olefination of which using 29e–g
(Scheme 1c) afforded enones 23d–f. Stereoselective reduction of
23d–f with (R)-Me-CBS afforded 24d–f. Alkaline hydrolysis of
24d–f afforded 16–18.
Table 1
Effect of the
a
-chain structure on the activity profiles of the
c
-lactam prostanoids bearing the natural x-chain
O
X
CO2H
N
A
CH3
OH
Binding assay (Ki, nM)
Compound
X
A
Functional assay (EC50, nM)
mEP1
>104
mEP2
mEP3
mEP4
1.7
rEP2
rEP4
2
Bond
340
86
1800
3.1
4
5
CH2
S
PGE2
–(CH2)3–
–(CH2)3–
>104
>104
6.0
>104
2500
22
39
26
5.0
9.2
2.0
3.1
>104
>104
19
8.4
2.0
3.5

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
2237
Table 2
Table 3
Effect of the
a
-chain structure on the activity profiles of the
c-lactam prostanoids
Effect of the
x
-chain structure on the activity profiles of the
c-lactam prostanoids
bearing the 16-(3-methylphenyl) or 16-(3-chlorophenyl)
x
-chains
bearing ethyl-linked 2-mercaptothiazole-4-carboxylic acid as the most highly
optimized -chain
a
O
R
N
CO2H
N
O
X
S
N
S
OH
R
Compound
X
R
Binding assay (Ki, nM)
OH
mEP1 mEP2 mEP3 mEP4
Compound
R
Binding assay (Ki, nM)
S
S
CO2H
CO2H
mEP1
3400
mEP2
3.0
mEP3
15
mEP4
0.5
1
6
CH3
Cl
>104
>104
>104
5800 1.8
*
*
14
15
–(CH2)3CH3
*
6900 >104
9.0
>104
19
770
1.5
7
8
9
Cl
>104
>104
6100 >104
3000
CO2H
CO2H
*
*
*
16
4200
3.0
4300
0.94
CH3
Cl
>104
>104
>104
78
53
Cl
5100 >104
CO2H
CO2H
17
18
6300
>104
1.7
22
>104
>104
0.81
*
*
*
10a
CH3
>104
410
>104
350
0.9
0.8
*
*
0.012
S
S
>104
>104
190
127
CO2H
CO2H
10b
10c
Cl
*
CH3
1600 0.6
S
S
>104
>104
>104
>104
>104
91
570
0.8
35
tive alkylation of 44 (Scheme 5) with the aldehyde 54 in the pres-
ence of sodium triacetoxyborohydride, followed by alkaline
hydrolysis, provided 12.
Compounds 2 and 5 were synthesized as outlined in Scheme 9.
Horner–Emmons olefination of 38 with the phosphonate 29b in
the presence of sodium hydride, followed by stereoselective reduc-
tion with (R)-Me-CBS, produced 55, acidic deprotection of which
provided 56. Reductive alkylation of 56 with 4-methoxycarbonyl
phenylacetaldehyde or 57, followed by alkaline hydrolysis,
resulted in 2 and 5, respectively.15
*
*
10d
11
12
3
CH3
CH3
CH3
CH3
CH3
CO2H
N
N
N
S
>104
>104
540
CO2H
CO2H
CO2H
CO2H
240
84
O
S
S
N
*
*
*
12
S
S
9.3
320
0.41
5.5
13
>104
3. Results and discussion
5-Mercaptothiophene-2-carboxylic acid analog 10d was syn-
thesized as outlined in Scheme 6. O-Methanesulfonylation of 47a
followed by substitution reaction with sodium iodide provided
47c, which was converted to 48 with sulfur (S8) and the anion pre-
pared from ethyl thiophene-2-carboxylate and lithium diisopro-
pylamine (LDA).10 Deprotection of 48 produced 49, which was
The compounds listed in Tables 1–3 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, which was performed according to the method
of Kiriyama et al. with minor modification.16
To investigate the effect of the
profiles of the compounds, -chain congeners 2, 4, and 5 (each
bearing a natural -chain) were compared with natural PGE2 for
a-chain structure on the activity
converted to 10d by the usual procedure for
x
-chain synthesis of
a
PG, as described above.
x
Compound 11 was synthesized as described in Scheme 7.
N-Acylation in the presence of EDC–HCl of DL-serine methyl ester
with 4-tert-butyldiphenylsilyl(TBDPS)oxybutyric acid, which was
prepared by O-protection of sodium 4-hydroxybutyrate with
TBDPS chloride in the presence of imidazole, provided 50. Treat-
ment of 50 with diethylaminosulfur trifluoride (DAST) in the pres-
ence of potassium carbonate, followed by treatment with
bromotrichloromethane and DBU, provided an oxazole 51, depro-
tection of which resulted in 52.14 Reductive alkylation in the pres-
ence of sodium triacetoxyborohydride of 44 (Scheme 5) with an
aldehyde, which was prepared by the DMSO oxidation of 52 in
the presence of SO3–Py and diisopropylethylamine, produced 53.
Alkaline hydrolysis of 53 produced 11.
their subtype selectivity and receptor affinity. The results are sum-
marized in Table 1. Compound 4, which possesses an N-heptanoic
acid moiety, showed moderate and potent binding affinity for EP3
and EP4 subtypes, respectively, but showed no affinity for the EP1
and EP2 subtypes at a concentration of up to 10 lM. The corre-
sponding 5-thia analog 5 showed moderate and highly potent
affinity for EP3 and EP4 subtypes, respectively, very weak affinity
for EP2 and no affinity for EP1 at a concentration of up to 10 lM.
Compared with congeners 4 and 5, our in-house data suggested
that compound 2 has increased affinity for EP2 and slightly more
reduced affinity for EP3, while retaining its potent EP4 receptor
affinity. As a result, all of these
show no affinity for the EP1 subtype at concentrations up to
10 M. Rat functional assay data for these chemical leads are also
c-lactam analogs were found to
Compound 12 was synthesized as described in Scheme 8. 4,4-
Dimethoxybutyronitrile was converted to 54 by the following
sequential reactions: (1) NaSH in the presence of MgCl2–6H2O in
DMF; (2) ethyl bromopyruvate in DMF; (3) 2 N HCl in DME. Reduc-
l
listed in Table 1. The potent mouse EP4 binding affinities of 2 and
4–5 were reflected in the rat EP4 agonist assay, while they tended
to show weaker EP2 functional activities than those expected from

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T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
X
N
S
O
O
a or b
g
S
SAc
N
3 and 13-15
N
Y
OTBS
R1
X = CO2Et, Y = H
19
20
R1 = CH2OTBS
c
21a
22a
R1 = CH2OH
R1 = CHO
d
e
*
*
R1 =
R1 =
23a-c
24a-c
R2
R2
f
O
OH
X = H, Y = CO2Et
25 R1 = CH2OTBS
c
R1 = CH2OH
26
27
d,e
CH3
CH3
*
R1 =
O
f
28
*
R1 =
OH
(a)
Synthesis of 3, and 13-15
O
N
O
O
CH3
g
h, i
S
N
20
S
16-18
R1
21b
22b
R1 = CH2OH
R1 = CHO
d
e
*
23d-f
24d-f
R1 =
R2
R2
f
O
R1 =
*
OH
(b) Synthesis of 16-18
Phosphonates:
R2 =
CH3
*
*
a
b
c
*
e
MeO
P
R2
MeO
Cl
CH3
O
O
*
f
*
*
29
Cl
g
*
d
CH3
N
Br
j, k
l
HO2C
R
29f-g
MeO
O
Cl
30a
R =
*
*
30b
R =
(c) Preparation of phosphonates 29f-g
Scheme 1. Synthesis of 3, 13–18 and preparation of phosphonates 29f–g. Reagents: (a) ethyl 2-bromothiazole-4-carboxylate, K2CO3, EtOH; (b) ethyl 2-bromothiazole-5-
carboxylate, K2CO3, EtOH; (c) TBAF, THF; (d) SO3–Py, i-Pr2NEt, DMSO, AcOEt; (e) 29a–c, 29e–g, NaH, THF; (f) (R)-Me-CBS, BH3–THF, THF; (g) aq NaOH, DME; (h) K2CO3, n-
BuOH; (i) TBAF, THF; (j) N,O-dimethylhydroxylamine hydrochloride, EDC–HCl, Et3N, CH3CN; (k) 4-chlorophenylboronic acid, Pd(PPh3)4, aq Na2CO3, DME or 2-
naphthaleneboronic acid, Pd(OAc)2, (S)-BINAP, aq Na2CO3, DME; (l) dimethyl methylphosphonate, n-BuLi, toluene.

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
2239
O
O
O
c, d
a, b
CO2Me
CO2Me
N
N
7
NH
O
OMe
OH
OH
32
31
Scheme 2. Synthesis of 7. Reagents and conditions: (a) MeOCH2Cl, trifluoromethanesulfonic acid, CH2Cl2, 80%; (b) methyl 3-(3-bromophenyl)acrylate, Pd(OAc)2, 1,1-
bis(diphenylphosphino)ferrocene, KOtBu, xylene, 49%; (c) NaBH4, NiCl2–6H2O, MeOH, THF; (d) 4 N HCl, dioxane, MeOH, 64% in two steps.
O
O
O
d
a or b
R2
R2
N
8, 10b-c
NH
N
OH
OTBS
OTBS
33
CO2Me
Br
*
*
*
34a
34b
R2 =
R2 =
R2 =
R2 =
35
36
c
S
CO2Me
*
CO2Me
S
R2 =
34c
CO2Me
*
Scheme 3. Synthesis of 8 and 10b–c. Reagents: (a) 1,3-bis(bromomethyl)benzene, NaH, DMF; (b) methyl 5-(3-bromopropyl)thiophene-2-carboxylate, NaH, DMF, 32%; (c) CO,
PdCl2(PPh3)2, K2CO3, THF, MeOH; (d) TBAF, THF.
O
(a)
CF CO H
f
NH
e
CO Me
3
2
2
2
NHBoc
R
d
N
Cl
9
EtOC
Cl
2
EtOC
2
OH
OH
41
40
OH
37
R2 =
*
*
a
38
39
R2 =
R2 =
CHO
b, c
Cl
*
OH
g, h, i
Br
(b)
CO Me
2
HO C
CO H
2
2
42
Scheme 4. Synthesis of 9. Reagents and conditions: (a) (COCl)2, i-Pr2NEt, DMSO, CH2Cl2; (b) phosphonate 29d, NaH, THF; (c) (R)-Me-CBS, BH3–THF, THF; (d) trifluoroacetic
acid, CH2Cl2; (e) 42, K2CO3, DMF; (f) 2 N NaOH, MeOH, THF; (g) TMSCHN2, MeOH; (h) BH3–SMe2, THF, 47%; (i) PBr3, CH2Cl2, 28%.
X
O
NHBoc
R
ClH
NH2
c
d, e
N
CH3
EtO2C
a,b
EtO2C
CH3
OH
44
OTBS
R = CHO
38
43
45
46
X = Br
X = CO Et
f
CH3
*
R =
2
OH
g, h
10a
Scheme 5. Synthesis of 10a. Reagents and conditions: (a) 29a, NaH, THF; (b) (R)-Me-CBS, BH3–THF, THF, 51% in two steps; (c) HCl in dioxane, EtOH, 100%; (d) 3-(4-
bromophenyl)propionaldehyde, NaBH(OAc)3, THF; (e) TBSCl, imidazole, DMF, 72% in two steps; (f) CO gas, PdCl2(PPh3)2, 1,1-bis(diphenylphosphino)ferrocene, Et3N, DMSO,
MeOH, 94%; (g) 2 N HCl, MeOH, DME; (h) 2 N NaOH, MeOH, DME, 76% in two steps.
their mouse binding affinities. Native PGE2 demonstrated non-spe-
cific affinities for all subtypes, but showed potent functional activ-
ities for both the rat EP2 and EP4 subtypes.
Based on the results described above, structural hybridization of
1 (Fig. 1 and Table 2) and 2 was considered a rational starting point
for the discovery of an EP2/EP4 dual agonist with high selectivity

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T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
CO2Et
CO2Et
S
S
O
O
O
d
c
X
S
S
10d
N
N
N
OTBS
OTBS
OH
48
49
47a X = OH
47b X = OMs
47c X = I
a
b
Scheme 6. Synthesis of 10d. Reagents and conditions: (a) MsCl, Et3N, THF; (b) NaI, CH3CN; (c) ethyl 2-thiophene carboxylate, S8, LDA, THF, 84%; (d) TBAF, THF, 83%.
a, b
H
N
c, d
e
N
CO2Me
OH
CO2Me
XO
TBDPSO
HO
CO2Na
O
O
51
52
X = TBDPS
X = H
50
CO2Me
CH3
O
N
O
h
f, g
N
11
OH
53
Scheme 7. Synthesis of 11. Reagents: (a) TBDPSCl, imidazole, DMF; (b) DL-serine methyl ester hydrochloride, EDC–HCl, Et3N, CH3CN, 32% in two steps; (c) diethylaminosulfur
trifluoride, K2CO3, CH2Cl2; (d) bromotrichloromethane, DBU, CH2Cl2, 54%; (e) TBAF, THF, 79%; (f) SO3–Py, i-Pr2NEt, DMSO, AcOEt; (g) 44, NaBH(OAc)3, THF, 74%; (h) aq NaOH,
MeOH, DME, 70%.
their
ylpropionic acid moiety is directly attached to the nitrogen of the
-lactam, showed weak receptor affinity for EP2 and EP4 subtypes,
with no affinity for EP1 and EP3 at concentrations up to 10 M.
Compound 8, in which the meta-substituted phenyl acetic acid
moiety is attached to the nitrogen of the -lactam through a meth-
a-chains. Compound 7, in which the meta-substituted phen-
OHC
a, b, c
OMe
d, e
12
N
MeO
c
CO2Et
S
CN
l
54
c
Scheme 8. Synthesis of 12. Reagents and conditions: (a) NaSH, MgCl2–6H2O, DMF;
(b) ethyl bromopyruvate, DMF; (c) 2 N HCl, DME, 12% in three steps; (d) 44,
NaBH(OAc)3, THF, 60%; (e) 2 N NaOH, DME, MeOH, 66%.
ylene moiety, displayed moderate affinity for the EP4 subtype
without showing any affinity for EP1, EP2 and EP3 subtypes at con-
centrations up to 10
tuted phenyl acetic acid moiety is attached to the nitrogen of the
-lactam through an ethylene moiety, retained the moderate EP4
lM. Compound 9, in which the meta-substi-
and potency. As shown in Table 2 and 5-thia analogs 1 and 6 bear-
c
ing a 16-(meta-substituted)aryl
x-chain were found to show
receptor affinity without losing its EP4 subtype selectivity. As a re-
highly selective and potent EP4 receptor affinity, without a con-
sult, compound 10a was found to show the most desired result for
comitant increase in the EP3 receptor affinity, relative to the corre-
EP2/EP4 dual selectivity and potency among the tested
lene analogs 7–10a. The corresponding five-membered heteroaryl
analogs 10b–c and 11–12 were synthesized as their N-propyl
a-pheny-
sponding natural
substituted)aryl
EP3 receptor affinity while retaining potent EP4 receptor affinity.
x
-chain analog 5 (Table 1).2 Thus, the 16-(meta-
x
-chain was found to be beneficial for reducing
linked analogs to adjust the length between the
and the carboxylic acid function. Replacement of the N-ethyl linked
benzoic acid -chain of 10a with N-propyl linked thiophene car-
c-lactam moiety
Our next focus was placed on the optimization of the
a-chain of
1 and 6 toward EP2/EP4 dual selectivity.
a
According to the reported data and our results described above,
we synthesized and evaluated the biological activity of compound
boxylic acid provided 10c, which exhibited EP2/EP4 selectivity
and slightly increased EP3 affinity. The corresponding 16-(3-chlo-
rophenyl) analog 10b exhibited potent affinity for the EP4 subtype,
and moderate affinity for the EP2 subtype with a tendency toward
increased EP3 affinity. Replacement of the N-propyl linker of 10c
with the N-ethylmercapto linker provided 10d, which showed po-
tent affinity for the EP4 subtype, and moderate affinity for the EP2
subtype together with weak affinity for EP3. Activity profiles of the
10a, which bears a benzoic acid moiety in its a
-chain.5 As shown in
Table 2, compound 10a exhibited very potent affinity for the EP4
receptor, and moderate affinity for EP2 receptor, without EP1 and
EP3 receptor affinities at concentrations up to 10 lM. On the basis
of the data described above, we synthesized and assessed com-
pounds 7–9, which bear a meta-substituted phenylene moiety in
d, e
ClH
NH2
NHBoc
R
c
CH3
2 and 5
EtO2C
EtO2C
OH
56
R = CHO
R =
OHC
S
CO2Me
38
55
a,b
57
CH3
OH
Scheme 9. Synthesis of
2 and 5. Reagents: (a) phosphonate 29b, NaH, THF; (b) (R)-Me-CBS, BH3–THF, THF; (c) HCl in dioxane, EtOH; (d) 4-methoxycarbonyl
phenylacetaldehyde or 57, NaBH(OAc)3, THF; (e) 2 N NaOH, MeOH, DME.

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
2241
thiophene analogs 10b–d indicated that they were not suitable as
highly selective EP2/EP4 dual agonists because of their insufficient
EP2 receptor affinity and increased affinity for the EP3 subtype.
Second, N-propyl linked oxazole-4-carboxylic acid analog 11
was synthesized and evaluated. Compound 11 showed a 1.7-fold
increase, and a 39-fold reduction, in EP2 and EP4 receptor affinity
relative to 10a, respectively. The corresponding thiazole-4-carbox-
ylic acid analog 12 showed increased affinity for both receptors
while retaining good EP2/EP4 dual selectivity. Replacement of
the N-propylene moiety attached to the 2-position of the thiazole
moiety of 12 with a N-ethylmercapto moiety was predicted to be
beneficial for our experimental aim. 2-Mercaptothiazole-4-carbox-
ylic acid analog 3, which was designed based on the concept de-
scribed above, resulted in excellent activity profiles. Its isomeric
compound 2-mercaptothiazole-5-carboxylic acid 13 demonstrated
34-fold less potent EP2 receptor affinity, and 13-fold less potent
EP4 receptor affinity, relative to 3, although its EP2/EP4 dual selec-
tivity was still maintained. The significant difference in the EP2/
EP4 dual receptor affinity between 3 and 13 was of great interest
because of their structural similarity. Compounds 11 and 12, both
15, were synthesized and evaluated by SAR studies. Compound 14
showed potent affinity for EP2, EP3 and EP4 subtypes, with negli-
gible affinity for the EP1 subtype. Replacement of the 16-alkyl moi-
eties of 14 with 16-phenyl moieties provided 15 with a clear
tendency of EP2/EP4 dual selectivity, or with clearly reduced affin-
ity for the EP3 receptor subtype. Analogous SAR was also observed
by the transformation of 2 to 10a. Thus, the improved activity pro-
file of 16-phenyl analog 15 relative to the natural
x-chain analog
14 was confirmed. In our previous report, introduction of a substi-
tuent into the meta-position of the 16-phenyl moiety was reported
to be acceptable for compound optimization.2,12 Based on this
information, we introduced phenyl, 4-chlorophenyl and naphth-
2-yl moieties into the meta-position of the 16-phenyl moiety of
15 to produce 16–18. Compounds 16 and 17 displayed excellent
activity profiles as EP2/EP4 dual agonists because of the increased
EP2 affinity and the reduced EP3 affinity relative to 15. Compound
18 also showed EP2/EP4 dual selectivity as a result of the retained
EP2 affinity, 18-fold more potent EP4 affinity and reduced EP3
affinity relative to 15.
Prior to in vivo evaluation in rats, the activities of compounds 3
and 16–18 in an EP2/EP4 functional assay, and their cLogP values,
were determined. The results are summarized in Table 4. Com-
pounds 3 and 16–18 showed, respectively, 1.9-fold, 8.5-fold, 33-
fold and 4 ꢀ 105-fold less potency relative to the corresponding
binding affinity (rEC50/mKi). Greater reduction of the agonist activ-
ity of 18 relative to the other compounds was considered mainly
due to its increased lipophilicity, which promotes strong protein
binding in the cell-based assay. The rat EP2 agonist activities, of
these compounds were reduced (9.7-fold, 600-fold, 153-fold and
>455-fold less potent rEC50 relative the mouse Ki value) compared
to the rat EP4 agonist activities. These results indicate that the rEP2
agonist activity was affected more than the rEP4 agonist activity in
the cell-based assay system. Compounds 16–18 showed higher
lipophilicity relative to 3, which is necessary for their sustained re-
lease from the PLGA microsphere. Among compounds 16–18, com-
pound 17 showed the most balanced profiles regarding dual
activity profiles in the rat functional assay and the cLogP value.
Based on the results described above, compound 17 was selected
as an EP2/EP4 dual agonist for in vivo evaluation because com-
pounds 16 and 18 demonstrated extremely reduced rEP2 and/or
rEP4 functional activities.
of which bear a nitrogen atom in the
a-position of the carboxylic
acid group, tended to show increased receptor affinity for the
EP2 subtype in comparison to 10a, which does not bear the corre-
sponding nitrogen atom. These a-nitrogen atoms of 11 and 12 may
be important for their interaction with the EP2 receptor through a
predicted hydrogen bond, whereas the corresponding interaction is
not expected in the thiazole-5-carboxylic acid derivative 13. Also
the nitrogen atom at the a-position of the carboxylic acid function
in 3 was estimated to strengthen the acidity of the carboxylic acid
function more than the isomer 13. Conversely, compounds 11 and
12 tended to show reduced EP4 receptor affinity relative to 10a
that was effectively recovered by the chemical modifications lead-
ing to the 2-mercaptothiazole-4-carboxylic acid derivative 3,
which displayed a remarkable increase in both EP2 and EP4 recep-
tor affinity. Thus, introduction of a sulfur atom into the 2-position
of the thiazole moiety was effective in increasing the EP2/EP4 dual
subtype selectivity.
The benzoic acid derivative 10a, oxazole carboxylic acid deriva-
tive 11 and thiazole carboxylic acid derivatives 3, 12 and 13, had
higher affinity for EP2/EP4 subtypes than 7–9. In compounds 3,
10a, and 11–13, but not in compounds 7–9, the carboxylic acid
group, and the carbon or sulfur atom directly attached to the aryl
moiety, occupy the same plane. This structural feature of 3, 10a
and 11–13 was considered to be a beneficial factor for EP2/EP4
dual selectivity, in addition to the above-described nitrogen atom
Because our aim was to identify a compound that exhibits effi-
cacy in the rat bone fracture-healing model following a single top-
ical injection, we initially investigated the sustained release of
compound 17 from PLGA microspheres in vitro. As shown in Table
5, the in vitro sustained release of 17 was observed by measuring
its remaining percentage in the PLGA microspheres after 28 days.
in the a-position of the carboxylic acid group and the 2-mercapto
moiety of the thiazole-4-carboxylic acid analog 3. Additionally, the
2-mercapto moiety of 3 may have some effects on the three dimen-
sional distance between the
acid function.9 Thus, compound 3, which bears N-ethyl linked 2-
mercaptothiazole-4-carboxylic acid as an -chain and 16-(3-
methyl)phenyl as a -chain, was found to have the most optimal
c-lactam nitrogen and the carboxylic
Table 4
a
Functional activities of compounds 3 and 16–18 on rat EP2/EP4 receptors
x
Compound
Functional assay (EC50, nM)
clogP
activity profiles among the tested compounds.
rEP2
90
rEP4
0.79
Because evaluation of the EP2/EP4 dual agonist in the bone frac-
ture-healing model in rats requires four weeks, its sustained re-
lease from the carrier after the topical injection is considered to
be another important determinant of efficacy. Further structural
optimization of compound 3 was performed to enable its sustained
release from the poly lactide-co-glycolide (PLGA) microsphere for-
mulation.17 Chemical modification was conducted to concurrently
optimize its lipophilicity (cLogP) and activity profiles so that sus-
tained release of the compound from PLGA microsphere could be
3
16
17
18
3.0
4.4
5.1
5.6
1800
260
8.0
27
480
>104
Table 5
In vitro release profile of compound 17 from PLGA microspheres
achieved. Maintaining the optimal
a
-chain, we continued further
Compound
% Remaining (days)
chemical modification of the -chain of 3 to investigate SAR in
x
0
7
21
28
more detail. The results are shown in Table 3. Compound 14,
substituted with 16-(n-butyl), and unsubstituted 16-phenyl analog
17
100
97.9
79.0
48.0

2242
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
Table 6
ethyl acetate (EtOAc), methanol (MeOH), tetrahydrofuran (THF),
methanol (MeOH), dichloromethane (CH2Cl2), chloroform (CHCl3),
dimethoxyethane (DME), acetonitrile (CH3CN), tert-butyl methyl
ether (MTBE), sulfur trioxide/pyridine complex (SO3–Py), tetrabu-
tylammonium fluoride (TBAF).
Biological evaluation of compound 17/PLGA in the rat bone fracture-healing model
Parameters
Vehicle
17/PLGA (lg/kg)
30
100
300
1000
Breaking strength (of % intact)
56.9
68.7
73.2
85.9
105.0
5.1.1. Ethyl 2-({2-[(2R)-2-({tert-butyldimethylsilyloxy}methyl)-
5-oxo-1-pyrrolidinyl]ethyl}thio)-1,3-thiazole-4-carboxylate
(20)
To a stirred solution of 19 (1.66 g, 5.00 mmol) in EtOH (25 mL)
were added potassium carbonate (897 mg, 6.50 mmol) and ethyl
2-bromothiazole-4-carboxylate (1.42 g, 6.00 mmol) at room tem-
perature under argon atmosphere. After being stirred at room
temperature for 16 h, the reaction mixture was diluted with EtOAc,
washed with H2O (ꢀ2), brine, and dried over MgSO4. The organic
layer was evaporated to give a sulfide 20 as a yellow oil.
The remaining percentage of 17 in PLGA microspheres was 97.9%
after 7 days and 48% after 28 days. These results gave us the confi-
dence to test 17 in PLGA microspheres (17/PLGA) in an in vivo rat
model of bone healing.
Using the 17/PLGA microsphere formulation described above,
the efficacy of 17 in the rat bone fracture-healing model was inves-
tigated. Table 6 shows the changes in the mechanical properties of
the healing fibulae. Seventeen days after the topical injection of 17
(30, 100, 300, 1000
fractured fibulae increased in a dose-dependent manner. In the fib-
ulae treated with a topical injection of 1000 g/kg of 17/PLGA, the
lg/kg), the mechanical breaking strength of the
5.1.2. Ethyl 2-({2-[(2R)-2-(hydroxymethyl)-5-oxo-1-pyrro
lidinyl]ethyl}thio)-1,3-thiazole-4-carboxylate (21a)
l
breaking strength reached 105% of nonfractured fibulae, while it
was 56.9% in the vehicle-treated fibulae. Based on the results de-
scribed above, compounds with EP2/EP4 dual agonist activity are
promising drug candidates for the treatment of bone fracture.
A solution of 20 (1.58 g, 3.56 mmol) in THF (3 mL) was treated
with a solution of TBAF (1.0 M in THF, 4.27 mL, 4.27 mmol) at room
temperature under argon atmosphere for 1 h. After addition of
brine, the reaction mixture was extracted with EtOAc repeatedly
(ꢀ5). The combined organic layers were dried over MgSO4, and
then evaporated. The resulting residue was purified by column
chromatography on silica gel (hexane/EtOAc, 1:1–0:1) to give
21a as a pale yellow oil (1.05 g, 63% in 2 steps). 1H NMR
(300 MHz, CDCl3): d 8.00 (s, 1H), 4.39 (q, J = 7.2 Hz, 2H), 3.93–
3.65 (m, 5H), 3.61–3.40 (m, 2H), 3.33 (m, 1H), 2.56–2.25 (m, 2H),
1.12 (m, 1H), 0.90 (m, 1H), 0.40 (t, J = 7.2 Hz, 3H).
4. Conclusion
In order to optimize EP2/EP4 subtype selectivity, we have gen-
erated ethyl-linked 2-mercaptothiazole-4-carboxylic acid analogs
3 and 15–18 bearing a 16-aryl moiety as the
x-chain. These com-
pounds were shown to be putative novel EP2/EP4 dual agonists
with excellent subtype selectivity and potency in a mouse receptor
assay. We have also discussed compound optimization by SAR,
which was initiated by structural hybridization of 5-thia c-lactam
PGE1 analog 1 and the benzoic acid prototype 2.
Further structural optimization was performed to enable the
sustained release of a test compound from PLGA microspheres.
Compound 17, which exhibited excellent in vitro activity profiles
as an EP2/EP4 dual agonist, displayed dose-dependent efficacy in
the rat bone fracture-healing model. Compound 17, and other
structurally related compounds, will now undergo full pharmaco-
logical and pharmacokinetic evaluation, the results of which will
be reported in due course.
5.1.3. Ethyl 2-({2-[(2R)-2-formyl-5-oxo-1-pyrrolidinyl]
ethyl}thio)-1,3-thiazole-4-carboxylate (22a)
To a stirred solution of the alcohol 21a (460 mg, 1.39 mmol) in
EtOAc (5 mL) and N,N-diisopropylethylamine (1.46 mL, 8.36 mmol)
was added a solution of SO3–Py (665 mg, 4.18 mmol) in DMSO
(2.5 mL) at 0 °C under argon atmosphere. After being stirred at
the same temperature for 20 min, the reaction was quenched with
1 N HCl. The reaction mixture was diluted with EtOAc, washed
with brine, and dried over MgSO4. The organic layer was evapo-
rated to yield an aldehyde 22a as a pale yellow oil.
5.1.4. Ethyl 2-[(2-{(2R)-2-[(1E)-4-(3-methylphenyl)-3-oxo-1-
buten-1-yl]-5-oxo-1-pyrrolidinyl}ethyl)thio]-1,3-thiazole-4-
carboxylate (23a)
5. Experimental
5.1. Chemistry
To
a stirred solution of dimethyl 3-(3-methylphenyl)-2-
oxopropanephosphonate 29a (166 mg, 0.649 mmol) in THF
(5 mL) was added sodium hydride (62.5% in mineral oil, 21.5 mg,
0.556 mmol) at 0 °C under argon atmosphere and stirring was fur-
ther continued at ambient temperature for 90 min. To the stirred
suspension was added a solution of the above-described aldehyde
22a in THF (2 mL) at 0 °C and stirring was continued at room tem-
perature for 1 h. The reaction was quenched with saturated aque-
ous NH4Cl. The reaction mixture was diluted with EtOAc, washed
with water, then brine, and dried over MgSO4. The organic layer
was evaporated to give an enone 23a as a pale yellow oil.
Analytical samples were homogeneous as confirmed by TLC,
and afforded spectroscopic results consistent with the assigned
structures. Proton nuclear magnetic resonance spectra (1H NMR)
were taken on a Varian Mercury 300 spectrometer, Varian GEM-
INI-200 or VXR-200s spectrometer using deuterated chloroform
(CDCl3), deuterated methanol (CD3OD) and deuterated dimethyl-
sulfoxide (DMSO-d6) as the solvent. Fast atom bombardment
(FAB-MS, HRMS) and electron ionization (EI) mass spectra were
obtained on a JEOL JMS-DX303HF spectrometer. Atmospheric pres-
sure chemical ionization (APCI) mass spectra were determined on a
HITACHI MI200H spectrometer. Infrared spectra (IR) were mea-
sured in a Perkin-Elmer FT-IR 1760X spectrometer. Melting points
and results of elemental analyses were uncorrected. Column chro-
matography was carried out on silica gel [Merck Silica Gel 60
5.1.5. Ethyl 2-[(2-{(2R)-2-[(1E,3S)-3-hydroxy-4-(3-methylphe
nyl)-1-buten-1-yl]-5-oxo-1-pyrrolidinyl}ethyl)thio]-1,3-thia
zole-4-carboxylate (24a)
To a stirred solution of 23a in THF (2.3 mL) was added a solution
of (R)-2-methyl-CBS-oxazaborolidine (1.0 M in toluene, 0.116 mL,
0.116 mmol) at room temperature under argon atmosphere. To
the reaction mixture was added dropwise a solution of borane–
THF complex (1.0 M in THF, 0.278 mL, 0.278 mmol) over 5 min.
The resulting solution was stirred for 20 min, then treated with
(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 were used; diethyl ether (Et2O), N,N-dimeth-
ylformamide (DMF), dimethylsulfoxide (DMSO), ethanol (EtOH),

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
2243
MeOH and stirring was continued for 5 min. The reaction mixture
was diluted with EtOAc, washed with 1 N HCl, water, saturated
aqueous NaHCO3, brine, and dried over Na2SO4. The organic layer
was evaporated and the resulting residue was purified by column
chromatography on silica gel (hexane/EtOAc, 1:2–0:1) to give an
alcohol 24a as a colorless oil (106 mg, 50% in 3 steps). 1H NMR
(300 MHz, CDCl3): d 8.01 (s, 1H), 7.18 (m, 1H), 7.08–6.93 (m, 3H),
5.81 (dd, J = 15.3, 5.7 Hz, 1H), 5.50 (ddd, J = 15.3, 8.7, 1.0 Hz, 1H),
4.43–4.30 (m, 3H), 4.20 (m, 1H), 3.72 (m, 1H), 3.40 (m, 2H), 3.20
(m, 1H), 2.80–2.73 (m, 2H), 2.45–2.10 (m, 7H), 1.75 (m, 1H), 1.38
(t, J = 7.2 Hz, 3H).
5.1.10. 2-[(2-{(2R)-2-[(1E,3S)-3-Hydroxy-4-phenylbut-1-enyl]-5-
oxopyrrolidin-1-yl}ethyl)sulfanyl]-1,3-thiazole-4-carboxylic
acid (15)
Compound 15 was prepared from 24c according to the same
procedure as described for the preparation of 3 from 24a as a col-
orless oil (80 mg, 85%). IR (film): 3398, 3005, 2923, 1715, 1664,
1496, 1421, 1325, 1216, 1099, 1029, 976, 750, 702 cmꢁ1 1H
;
NMR (300 MHz, CDCl3): d 8.09 (s, 1H), 7.38–7.14 (m, 5H), 5.80
(dd, J = 15.3, 6.0 Hz, 1H), 5.47 (dd, J = 15.3, 8.7 Hz, 1H), 4.40 (m,
1H), 4.21–3.61 (m, 4H), 3.38–3.16 (m, 3H), 2.97–2.79 (m, 2H),
2.52–2.18 (m, 3H), 1.76 (m, 1H); MS (APCI) m/z: 417 (MꢁH)ꢁ;
HRMS-FAB (m/z): [M+H]+ calcd for C20H23N2O4S2, 419.1099; found,
419.1105.
5.1.6. 2-[(2-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(3-methylphenyl)but-
1-enyl]-5-oxopyrrolidin-1-yl}ethyl)sulfanyl]-1,3-thiazole-4-car
boxylic acid (3)
5.1.11. Ethyl 2-({2-[(2R)-2-(hydroxymethyl)-5-oxo-1-pyrrolid
inyl]ethyl}thio)-1,3-thiazole-5-carboxylate (26)
To a stirred solution of 24a (105 mg, 0.229 mmol) in DME
(0.5 mL) was added 2 N NaOH (0.229 mL, 0.458 mmol) and stirring
was continued at ambient temperature for 2 h. After acidification
with 2 N HCl under cooling, the reaction mixture was extracted
with EtOAc (ꢀ3). The combined organic layers were washed with
brine, dried over Na2SO4 and evaporated. The resulting residue
was purified by column chromatography on silica gel (CHCl3/
MeOH, 100:1–9:1) to afford 3 as a colorless oil (79 mg, 80%). IR
(film): 3390, 3118, 3007, 2921, 1714, 1666, 1494, 1421, 1360,
1325, 1216, 1100, 1029, 975, 751 cmꢁ1; 1H NMR (300 MHz, CDCl3):
d 8.08 (s, 1H), 7.20 (m, 1H), 7.08–6.95 (m, 3H), 5.80 (dd, J = 15.3,
5.7 Hz, 1H), 5.50 (dd, J = 15.3, 8.7 Hz, 1H), 4.40 (m, 1H), 4.12 (m,
1H), 3.70 (m, 1H), 3.50–2.95 (m, 5H), 2.85–2.78 (m, 2H), 2.50–
2.19 (m, 6H), 1.77 (m, 1H); MS (APCI) m/z: 431 (MꢁH)ꢁ; HRMS-
FAB (m/z): [MꢁH]ꢁ calcd for C23H23N2O4S2, 431.1099; found,
431.1113.
Compound 26 was prepared from 19 according to the same pro-
cedure as described for the preparation of 21a from 19 as a pale
yellow oil (358 mg, 64% from 19). 1H NMR (300 MHz, CDCl3): d
8.19 (s, 1H), 4.35 (q, J = 7.2 Hz, 2H), 4.40–3.89 (m, 7H), 3.15–3.02
(br s, 1H), 2.58–2.33 (m, 2H), 2.19–2.05 (m, 1H), 1.90–1.78 (m,
1H), 1.38 (t, J = 7.2 Hz, 3H).
5.1.12. Ethyl 2-[(2-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(3-methylph
enyl)but-1-enyl]-5-oxopyrrolidin-1-yl}ethyl)sulfanyl]-1,3-thia
zole-5-carboxylate (28)
Compound 28 was prepared from 26 according to the same pro-
cedure as described for the preparation of 24a from 21a as a color-
less oil (248 mg, 50% in three steps). 1H NMR (300 MHz, CDCl3): d
8.18 (s, 1H), 7.20 (t, J = 8.0 Hz, 1H), 7.10–6.90 (m, 3H), 5.74 (dd,
J = 15, 6.0 Hz, 1H), 5.51 (dd, J = 15, 9.0 Hz, 1H), 4.41–4.33 (m, 1H),
4.36 (q, J = 7 Hz, 2H), 4.12 (q, J = 7 Hz, 1H), 3.75–3.6 (m, 1H), 3.40
(t, J = 7 Hz, 2H), 3.3–3.2 (m, 1H), 2.8–2.7 (m, 2H), 2.5–2.1 (m, 6H),
2.0–1.95 (br, 1H), 1.35 (t, J = 7 Hz, 3H).
5.1.7. Ethyl 2-[(2-{(2R)-2-[(1E,3S)-3-hydroxy-1-octen-1-yl]-5-
oxo-1-pyrrolidinyl}ethyl)thio]-1,3-thiazole-4-carboxylate (24b)
Compound 24b was prepared from 22a using a phosphonate
29b instead of 29a according to the same procedure as described
for the preparation of 24a from 22a as a colorless oil (99 mg, 51%
from 21a). 1H NMR (300 MHz, CDCl3): d 8.02 (s, 1H), 5.78 (dd,
J = 15.3, 5.7 Hz, 1H), 5.54 (dd, J = 15.3, 9.0 Hz, 1H), 4.39 (q,
J = 6.9 Hz, 2H), 4.21 (m, 1H), 4.10 (m, 1H), 3.79 (m, 1H), 3.50–
3.38 (m, 3H), 2.50–2.10 (m, 3H), 1.95 (br s, 1H), 1.77 (m, 1H),
1.66–1.20 (m, 11H), 0.87 (t, J = 7.2 Hz, 3H).
5.1.13. 2-[(2-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(3-methylphenyl)
but-1-enyl]-5-oxopyrrolidin-1-yl}ethyl)sulfanyl]-1,3-thiazole-
5-carboxylic acid (13)
Compound 13 was prepared from 28 according to the same pro-
cedure as described for the preparation of 3 from 24a as a colorless
viscous oil (44 mg, 93%). IR (film): 3387, 2922, 1701, 1520, 1418,
1359, 1241, 1158, 1097, 1035, 975, 755 cmꢁ1 1H NMR (300 MHz,
;
CDCl3): d 8.17 (s, 1H), 7.14 (t, J = 8 Hz, 1H), 7.0–6.9 (m, 3H), 5.68
(dd, J = 15, 7 Hz, 1H), 5.35 (dd, J = 15, 9 Hz, 1H), 4.31 (q, J = 7 Hz,
1H), 4.25–4.1 (m, 1H), 3.7–3.55 (m, 1H), 3.4–3.2 (m, 2H), 3.05–
2.9 (m, 1H), 2.88 (dd, J = 13, 6 Hz, 1H), 2.63 (dd, J = 13, 7 Hz, 1H),
2.4–2.25 (m, 5H), 2.25–2.1 (m, 1H), 1.75–1.6 (m, 1H); MS (APCI)
m/z: 431 (MꢁH)ꢁ; HRMS-FAB (m/z): [M+H]+ calcd for
5.1.8. 2-[(2-{(2R)-2-[(1E,3S)-3-Hydroxyoct-1-enyl]-5-oxopyrr
olidin-1-yl}ethyl)sulfanyl]-1,3-thiazole-4-carboxylic acid (14)
Compound 14 was prepared from 24b according to the same
procedure as described for the preparation of 3 from 24a as a color-
less oil (78 mg, 85%). IR (film): 3402, 3121, 2929, 2858, 1714, 1665,
1505, 1459, 1422, 1357, 1325, 1214, 1146, 1027, 975, 753 cmꢁ1; 1H
NMR (300 MHz, CDCl3): d 8.10 (s, 1H), 5.80 (dd, J = 15.6, 6.0 Hz, 1H),
5.55 (dd, J = 15.6, 8.7 Hz, 1H), 4.30–3.77 (m, 5H), 3.60–3.29 (m, 3H),
2.58–2.20 (m, 3H), 1.80 (m, 1H), 1.62–1.21 (m, 8H), 0.88 (t,
J = 7.5 Hz, 3H); MS (APCI) m/z: 397 (MꢁH)ꢁ; HRMS-FAB (m/z):
[M+H]+ calcd for C18H27N2O4S2, 399.1412; found, 399.1421.
C21H25N2O4S2, 433.1256; found, 433.1250.
5.1.14. Butyl 2-({2-[(2R)-2-(hydroxymethyl)-5-oxo-1-pyrroli
dinyl]ethyl}thio)-1,3-thiazole-4-carboxylate (21b)
To a stirred solution of 20 (1.71 g, 3.85 mmol) in n-butanol
(39 mL) was added potassium carbonate (53 mg, 0.385 mmol) un-
der argon atmosphere. After being stirred at 90 °C for 3 h, the reac-
tion mixture was cooled to room temperature, diluted with EtOAc,
washed with water, brine, and dried over Na2SO4. The organic layer
was evaporated to give an ester as a brown oil. A solution of the es-
ter in THF (3.9 mL) was treated with TBAF (1.0 M in THF, 3.85 mL,
3.85 mmol) at room temperature. After being stirred for 2 h, the
reaction mixture was diluted with EtOAc, washed with water,
brine, and dried over Na2SO4. The organic layer was evaporated
and the resulting residue was purified by column chromatography
on silica gel (EtOAc/MeOH, 100:0–20:1) to afford 21b as a brown-
ish orange oil (820 mg, 59% from 23). 1H NMR (300 MHz, CDCl3): d
7.99 (s, 1 H). 4.33 (t, J = 6.8 Hz, 2 H), 3.30–3.96 (m, 7H), 2.57–2.23
5.1.9. Ethyl 2-[(2-{(2R)-2-[(1E,3S)-3-hydroxy-4-phenylbut-1-
enyl]-5-oxo-1-pyrrolidinyl}ethyl)thio]-1,3-thiazole-4-carbox
ylate (24c)
Compound 24c was prepared from 22a using a phosphonate
29c instead of 29a according to the same procedure as described
for the preparation of 24a from 22a as a colorless oil (101 mg,
49% from 24a). 1H NMR (300 MHz, CDCl3): d 8.02 (s, 1H), 7.33–
7.12 (m, 5H), 5.80 (dd, J = 15.6, 6.0 Hz, 1H), 5.49 (ddd, J = 15.6,
8.4, 1.0 Hz, 1H), 4.42–4.30 (m, 3H), 4.20 (m, 1H), 3.70 (m, 1H),
3.39 (m, 2H), 3.20 (m, 1H), 2.85–2.78 (m, 2H), 2.45–2.08 (m, 4H),
1.72 (m, 1H), 1.38 (t, J = 7.2 Hz, 3H).

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T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
(m, 2H), 2.23–2.06 (m, 1H), 1.98–1.82 (m, 1H), 1.81–1.65 (m, 2H),
1.54–1.35 (m, 2H), 0.97 (t, J = 6.0 Hz, 3H), 0.75–0.35 (m, 1H).
J = 7.6 Hz, 2H), 7.18 (m, J = 7.4 Hz, 1H), 5.87 (dd, J = 15.4, 5.8 Hz,
1H), 5.53 (dd, J = 15.4, 8.8 Hz, 1H), 4.50–4.39 (m, 1H), 4.29 (t,
J = 7.1 Hz, 2H), 4.32–4.17 (m, 2H), 3.73–3.60 (m, 1H), 3.42–3.14
(m, 3H), 2.96–2.86 (m, 2H), 2.44–2.10 (m, 4H) 1.77–1.63 (m, 2H),
1.46–1.33 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H).
5.1.15. Butyl 2-[(2-{(2R)-2-[(1E,3S)-4-(3-biphenylyl)-3-hydroxy-
1-buten-1-yl]-5-oxo-1-pyrrolidinyl}ethyl)thio]-1,3-thiazole-4-
carboxylate (24d)
Compound 24d was prepared from 21b using a phosphonate
29e instead of 29a according to the same procedure as described
for the preparation of 24a from 21a as a colorless oil (67 mg, 29%
from 21b). 1H NMR (300 MHz, CDCl3): d 7.98 (s, 1H), 7.64–7.29
(m, 8H),7.19–7.08 (m, 1H), 5.85 (dd, J = 15.4, 5.8 Hz, 1H), 5.52
(dd, J = 15.4, 8.8 Hz, 1H), 4.49–4.03 (m, 4H), 3.84–3.57 (m, 2H),
3.33 (t, J = 6.6 Hz, 2H), 3.26–3.11 (m, 1H), 2.94–2.80 (m, 2H),
2.45–2.06 (m, 3H), 1.80–1.49 (m, 3H), 1.50–1.33 (m, 2H), 0.94 (t,
J = 7.2 Hz, 3H).
5.1.20. 2-{[2-((2R)-2-{(1E,3S)-3-Hydroxy-4-[3-(2-naphthyl)
phenyl]but-1-enyl}-5-oxopyrrolidin-1-yl)ethyl]sulfanyl}-1,3-
thiazole-4-carboxylic acid (18)
Compound 18 was prepared from 24f according to the same
procedure as described for the preparation of 3 from 24a as a col-
orless amorphous oil (49 mg, 83%). IR (film): 3114, 3053, 2923,
1717, 1663, 1507, 1490, 1456, 1421, 1324, 1213, 1101, 1028,
975, 891, 859, 821, 790 cmꢁ1 1H NMR (300 MHz, CDCl3): d 8.05–
;
8.00 (m, 2H), 7.93–7.82 (m, 3H), 7.71 (dd, J = 8.4, 1.8 Hz, 1H),
7.61 (d, J = 7.8 Hz, 1H), 7.56–7.47 (m, 4H), 7.42 (t, J = 7.8 Hz, 1H),
7.19 (d, J = 7.8 Hz, 1H), 5.84 (dd, J = 15.3, 5.7 Hz, 1H), 5.52 (dd,
J = 15.3, 8.7 Hz, 1H), 4.49 (q, J = 6.0 Hz, 1H), 4.15–4.05 (m, 1H),
3.75–3.65 (m, 1H), 3.35–3.05 (m, 3H), 2.95 (dd, J = 7.2, 3.3 Hz,
2H), 2.50–2.10 (m, 3H), 1.80–1.60 (m, 1H); FAB-MS (m/z): 545
(M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C30H29N2O4S2,
545.1569; found, 545.1573.
5.1.16. 2-[(2-{(2R)-2-[(1E,3S)-4-(1,1’-Biphenyl-3-yl)-3-hydroxy
but-1-enyl]-5-oxopyrrolidin-1-yl}ethyl)sulfanyl]-1,3-thiazole-
4-carboxylic acid (16)
Compound 16 was prepared from 24d according to the same
procedure as described for the preparation of 3 from 24a as a col-
orless amorphous oil (46 mg, 78%). IR (film): 3421, 2925, 1716,
1662, 1504, 1479, 1455, 1421, 1324, 1214, 1100, 1027, 975, 921,
853, 799, 758 cmꢁ1
;
1H NMR (300 MHz, CDCl3): 8.07 (s, 1H), 7.35
5.1.21. 2-(4’-Chloro-3-biphenylyl)-N-methoxy-N-methylace
tamide (30a)
(m, 9H), 5.82 (dd, J = 15.4, 5.8 Hz, 1H), 5.51 (dd, J = 15.4, 8.8 Hz,
1H), 4.47 (m, 1H), 4.11 (m, 1H), 3.68 (m, 1H), 3.06–2.96 (m, 7H),
2.30–2.15 (m, 3H), 1.72 (m, 1H); MS (APCI) m/z: 493 (MꢁH)ꢁ;
HRMS-FAB (m/z): [MꢁH]ꢁ calcd for C26H25N2O4S2, 493.1256;
found, 493.1230.
To a stirred solution of N,O-dimethylhydroxylamine hydrochlo-
ride (3.41 g, 35.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (5.81 g, 30.3 mmol) and triethylamine
(4.88 mL, 35.0 mmol) in CH3CN (40 mL) was added a solution of
(3-bromophenyl)acetic acid (5.00 g, 23.3 mmol) in CH3CN (17 mL)
at room temperature under argon atmosphere. After being stirred
for 1 h, the reaction was quenched with water. The reaction mixture
was diluted with EtOAc, washed with 2 N HCl, water, then brine,
and dried over MgSO4. The organic layer was evaporated to give a
Weinreb amide as a colorless oil.
To a stirred solution of above-described Weinreb amide (1.20 g,
4.66 mmol) in DME (15 mL) and H2O (10 mL) were added 4-chloro-
phenylboronic acid (1.09 g, 7.00 mmol), sodium carbonate (742 mg,
7.00 mmol) and tetrakis(triphenylphosphine)palladium (60 mg,
0.052 mmol) at under argon atmosphere. After being stirred at
90 °C for 5 h, the reaction mixture was cooled to room temperature.
The resulting mixture was filtered through a pad of Celite, and the
filtrate was evaporated and purified by column chromatography on
silica gel (hexane/EtOAc, 3:1–1:1) to afford 30a as a pale yellow so-
lid (1.58 g, 100%). 1H NMR (300 MHz, CDCl3): d 7.54–7.47 (m, 3H),
7.44–7.35 (m, 4H), 7.32–7.27 (m, 1H), 3.84 (s, 2H), 3.65 (s, 3H),
3.21 (s, 3H).
5.1.17. Butyl 2-[(2-{(2R)-2-[(1E,3S)-4-(4’-chloro-3-biphenylyl)-
3-hydroxy-1-buten-1-yl]-5-oxo-1-pyrrolidinyl}ethyl)thio]-1,3-
thiazole-4-carboxylate (24e)
Compound 24e was prepared from 21b using a phosphonate
29f instead of 29a according to the same procedure as described
for the preparation of 24a from 21a as a colorless oil (65 mg, 26%
from 21b). 1H NMR (300 MHz, CDCl3): d 7.99 (s, 1H), 7.55–7.30
(m, 7H), 7.21–7.12 (m, 1H), 5.85 (dd, J = 15.4, 5.8 Hz, 1H), 5.53
(dd, J = 15.4, 8.5 Hz, 1H), 4.48–4.07 (m, 4H), 3.79–3.58 (m, 1H),
3.35 (t, J = 6.9 Hz, 2H), 3.29–3.15 (m, 1H), 2.91–2.81 (m, 2H),
2.47–2.07 (m, 3H), 1.78–1.60 (m, 3H), 1.49–1.34 (m, 2H), 0.94 (t,
J = 7.4 Hz, 3H).
5.1.18. 2-[(2-{(2R)-2-[(1E,3S)-4-(4’-Chloro-1,1’-biphenyl-3-yl)-3-
hydroxybut-1-enyl]-5-oxopyrrolidin-1-yl}ethyl)sulfanyl]-1,3-
thiazole-4-carboxylic acid (17)
Compound 17 was prepared from 24e according to the same
procedure as described for the preparation of 3 from 24a as a col-
orless amorphous oil (49 mg, 83%). IR (film): 3115, 2923, 2691,
2581, 2510, 1716, 1661, 1498, 1478, 1421, 1396, 1372, 1323,
5.1.22. Dimethyl [3-(4’-chloro-3-biphenylyl)-2-oxopropyl]
phosphonate (29f)
1260, 1212, 1091, 1026, 1012, 974, 921, 833, 791, 748 cmꢁ1
;
1H
To a stirred solution of dimethyl methylphosphonate (0.76 mL,
7.00 mmol) in toluene (15 mL) was added dropwise a solution of n-
BuLi (1.57 M in hexane, 5.04 mL, 7.92 mmol) at ꢁ78 °C under argon
atmosphere, and stirring was continued for 1 h at the same tem-
perature. To the reaction mixture was added a solution of 30a
(1.58 g, 4.66 mmol) in toluene (4 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 temperature with stirring, diluted with EtOAc,
washed with water, then brine, and dried over MgSO4. The organic
layer was evaporated and the resulting residue was purified by col-
umn chromatography on silica gel (hexane/EtOAc, 2:1–0:1) to give
a phosphonate 29f as a colorless oil (1.07 g, 65%). 1H NMR
(300 MHz, CDCl3): d 7.57–7.32 (m, 7H), 7.22 (d, J = 7.1 Hz, 1H),
3.97 (s, 2H), 3.81 (s, 3H), 3.78 (s, 3H), 3.15 (d, J = 23.3 Hz, 2H).
NMR (300 MHz, CDCl3): d 8.07 (s, 1H), 7.35 (m, 8H) 5.83 (dd,
J = 15.4, 5.8 Hz, 1H), 5.52 (dd, J = 15.4, 8.8 Hz, 1H), 4.46 (m, 1H),
4.11 (m, 1H), 3.48–3.11 (m, 6H), 2.90–2.85 (m, 2H), 2.30–2.11
(m, 3 H), 1.69 (m, 1H); FAB-MS (m/z): 529 (M+H)+; HRMS-FAB (
m/z): [M+H]+ calcd for C26H26ClN2O4S2, 529.1023; found, 529.1025.
5.1.19. Butyl 2-({2-[(2R)-2-{(1E,3S)-3-hydroxy-4-[3-(2-naphthyl)
phenyl]-1-buten-1-yl}-5-oxo-1-pyrrolidinyl]ethyl}thio)-1,3-
thiazole-4-carboxylate (24f)
Compound 24f was prepared from 21b using a phosphonate
29g instead of 29a according to the same procedure as described
for the preparation of 24a from 21a as a colorless oil (102 mg,
40% from 21b). 1H NMR (300 MHz, CDCl3): d 8.02 (s, 1H), 7.95–
7.82 (m, 4H), 7.76–7.69 (m, 1H), 7.62–7.46 (m, 3H), 7.41 (t,

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
2245
5.1.23. N-Methoxy-N-methyl-2-[3-(2-naphthyl)phenyl]acetam
ide (30b)
3H), 7.05 (m, 1H), 4.56 (d, J = 6.3 Hz, 1H), 4.49 (d, J = 6.3 Hz, 1H),
4.35 (m, 1H), 3.67 (s, 3H), 3.56–3.50 (m, 2H), 3.26 (s, 3H), 2.95 (t,
J = 7.2 Hz, 2H), 2.82–2.16 (m, 6H).
To a stirred solution of the Weinreb amide (1.81 g, 7.00 mmol)
described in the experimental of 30a in DME (15 mL) and 2 M
aqueous sodium carbonate (10.5 mL, 21.0 mmol) were added
naphthalene-2-boronic acid (2.49 g, 14.0 mmol), (S)-BINAP
(871 mg, 1.4 mmol) and palladium acetate (157 mg, 0.7 mmol) at
under argon atmosphere. After being stirred at 80 °C for 12 h, the
reaction mixture was cooled to room temperature. The resulting
mixture was filtered through a pad of Celite. The filtrate was evap-
orated and purified by column chromatography on silica gel (hex-
ane/EtOAc, 4:1–1:1) to afford 30b as a pale yellow solid (1.61 g,
75%). 1H NMR (300 MHz, CDCl3): d 8.08–8.01 (m, 1 H) 7.94–7.83
(m, 3H), 7.75 (dd, J = 8.5, 1.9 Hz, 1H), 7.68–7.57 (m, 2H), 7.54–
7.41 (m, 3H), 7.35–7.29 (m, 1H), 3.88 (s, 2H), 3.65 (s, 3H), 3.23 (s,
3H).
A mixture of the above-described saturated ester (278 mg,
0.877 mmol) in MeOH (3.5 mL) and 4 N HCl in dioxane (0.88 mL)
was stirred at room temperature for 5 h. The reaction mixture
was poured into ice-water, extracted with EtOAc (ꢀ2), washed
with brine, dried over MgSO4 and evaporated. The resulting residue
was purified by column chromatography on silica gel (CHCl3/
MeOH, 170:–30:1) to give 32 as a brown viscous oil (156 mg,
64% in two steps). 1H NMR (300 MHz, CDCl3): d 7.38–7.20 (m,
3H), 7.07 (m, 1H), 4.28 (m, 1H), 3.76–3.54 (m, 2H), 3.67 (s, 3H),
2.96 (t, J = 7.5 Hz, 2H), 2.80–2.46 (m, 4H), 2.38–2.08 (m, 2H).
5.1.27. 3-(3-{(2R)-2-[(1E,3S)-4-(3-Chlorophenyl)-3-hydroxybut-
1-enyl]-5-oxopyrrolidin-1-yl}phenyl)propanoic acid (7)
Compound 7 was prepared from 32 according to the same pro-
cedure as described for the preparation of 3 from 21a as a colorless
oil (34 mg, 18% form 32). IR (film): 3404, 3013, 2360, 1681, 1600,
5.1.24. Dimethyl {3-[3-(2-naphthyl)phenyl]-2-oxopropyl}phos
phonate (29g)
Compound 29g was prepared from 30b according to the same
procedure as described for the preparation of 29f from 30a as a col-
orless oil (1.18 g, 61%). 1H NMR (300 MHz, CDCl3): d 8.03 (s, 1H),
7.95–7.83 (m, 3H), 7.73 (dd, J = 8.7, 1.8 Hz, 1H), 7.65 (d,
J = 8.7 Hz, 1H), 7.58 (s, 1H), 7.54–7.43 (m, 3H), 7.28–7.22 (m, 1H),
4.01 (s, 2H), 3.82 (s, 3H), 3.78 (s, 3H), 3.17 (d, J = 22.8 Hz, 2H).
1491, 1455, 1395, 1217, 1081, 1035, 971, 882, 756, 701 cmꢁ1 1H
;
NMR (300 MHz, CDCl3): d 7.60–6.80 (m, 8H), 5.70–5.50 (m, 2H),
5.20–4.00 (m, 2H), 4.61 (m, 1H), 4.33 (m, 1H), 3.04–2.20 (m, 8H),
1.84–1.60 (m, 2H).; MS (APCI) m/z: 412 (MꢁH)ꢁ; HRMS-FAB (m/
z): [M+H]+ calcd for C23H25ClNO4, 414.1472; found, 414.1470.
5.1.28. (5R)-1-[4-(Bromomethyl)benzyl]-5-({tert-butyldimethy
lsilyloxy}methyl)-2-pyrrolidinone (34a)
5.1.25. Methyl (2E)-3-(3-{(2R)-2-[(methoxymethoxy)methyl]-5-
oxo-1-pyrrolidinyl}phenyl)acrylate (31)
To a stirred solution of 33 (1.0 g, 4.40 mmol) in DMF (20 mL)
was added sodium hydride (62% in mineral oil, 200 mg,
5.17 mmol) at 0 °C under argon atmosphere. Stirring was contin-
ued at room temperature for 1 h and 50 °C for additional 1 h. To
the resulting suspension was added 1,4-bis(bromomethyl)benzene
(1.40 ml, 5.20 mmol) at room temperature, and stirring was con-
tinued at room temperature for 1.5 h. The resulting pale brown
solution was poured into saturated aqueous NH4Cl, extracted with
EtOAc, washed with H2O, brine, and dried over MgSO4. The organic
layer was evaporated and the resulting residue was purified by col-
umn chromatography on silica gel (hexane/EtOAc, 5:1–3:1) to give
34a as a colorless oil (499 mg, 27%). 1H NMR (300 MHz, CDCl3): d
7.38–7.34 (m, 2H), 7.24–7.20 (m, 2H), 4.96 (d, J = 15.4 Hz, 1H),
4.48 (d, J = 15.4 Hz, 1H), 3.65 (m, 1H), 3.57–3.50 (m, 2H), 2.56 (m,
1H), 2.48 (m, 1H), 2.07 (m, 1H), 1.89 (m, 1H), 0.89 (s, 9H), 0.04
(s, 6H).
To a stirred solution of (R)-5-(hydroxymethyl)-2-pyrrolidinone
(1.50 g, 13.0 mmol) in CH2Cl2 (12 mL) were added dimethoxyme-
thane (12 mL) and trifluoromethanesulfonic acid (1.0 mL) at room
temperature under argon atmosphere. After being stirred for 6 h,
the reaction mixture was poured into cold saturated aqueous
NH4Cl, and extracted with EtOAc (ꢀ6). The combined organic lay-
ers were washed with brine, dried over MgSO4, and evaporated to
give an methoxymethoxy(MOM) ether as a pale yellow oil (1.65 g,
80%). 1H NMR (300 MHz, CDCl3): d 6.07 (br s, 1H), 4.63 (s, 2H), 3.86
(m, 1H), 3.60 (dd, J = 9.9, 3.6 Hz, 1H), 3.38 (m, 1H), 3.37 (s, 3H),
2.44–2.14 (m, 3H), 1.75 (m, 1H).
To
a stirred solution of the above-described MOM ether
(300 mg, 1.89 mmol) in xylene (4.7 mL) were added methyl 3-(3-
bromophenyl)acrylate (682 mg, 2.83 mmol), palladium acetate
(32 mg, 0.141 mmol) and 1,1-bis(diphenylphosphino)ferrocene
(627 mg, 1.13 mmol) and potassium tert-butoxide (317 mg,
2.83 mmol) at room temperature under argon atmosphere. After
being stirred at 140 °C for 6 h, the reaction mixture was cooled
to room temperature and filtered through a pad of Celite. The fil-
trate was evaporated and purified by column chromatography on
silica gel (CHCl3/MeOH, 140:1–100:1) to afford 31 as a brown vis-
cous oil (296 mg, 49%). 1H NMR (300 MHz, CDCl3): d 7.74–7.32 (m,
5H), 6.44 (d, J = 16.2 Hz, 1H), 4.56 (d, J = 6.6 Hz, 1H), 4.49 (d,
J = 6.6 Hz, 1H), 4.39 (m, 1H), 3.81 (s, 3H), 3.64–3.50 (m, 2H), 3.25
(s, 3H), 2.75 (m, 1H), 2.54 (m, 1H), 2.35 (m, 1H), 2.16 (m, 1H).
5.1.29. Methyl (3-{[(2R)-2-({tert-butyldimethylsilyloxy}methyl)-
5-oxo-1-pyrrolidinyl]methyl}phenyl)acetate (34b)
To a stirred solution of 34a (491 mg, 1.19 mmol) in THF (3.0 mL)
and MeOH (1.5 mL) were added potassium carbonate (197 mg,
1.43 mmol) and bis(triphenylphosphine)palladium dichloride
(17 mg, 0.020 mmol) under argon atmosphere, and the argon gas
was replaced with carbon monooxide gas repeatedly. After being
stirred at room temperature for 20 h, the reaction mixture was
cooled to room temperature. The resulting mixture was evaporated
and purified by column chromatography on silica gel (hexane/
EtOAc, 5:1–1:1) to afford 34b as a colorless oil (291 mg, 62%). 1H
NMR (300 MHz, CDCl3): d 7.25–7.19 (m, 4H), 4.98 (d, J = 15.0 Hz,
1H), 4.02 (d, J = 15.0 Hz, 1H), 3.68 (s, 3H), 3.64 (m, 1H), 3.61 (s,
2H), 3.57–3.48 (m, 2H), 2.56 (m, 1H), 2.37 (m, 1H), 2.05 (m, 1H),
1.91 (m, 1H), 0.89 (s, 9H), 0.04 (s, 6H).
5.1.26. Methyl (2E)-3-{3-[(2R)-2-(hydroxymethyl)-5-oxo-1-pyr
rolidinyl]phenyl}acrylate (32)
To a stirred solution of 31 (280 mg, 0.877 mmol) in THF (4 mL)
and MeOH (4 mL) were added nickel chloride hexahydrate
(208 mg, 0.877 mmol), and then sodium borohydride (133 mg,
3.51 mmol) in several portions at 0 °C under argon atmosphere.
After being stirred for 1 h, the reaction mixture was diluted with
Et2O and filtered through a pad of Celite. The filtrate was poured
into cold aqueous NH4Cl, extracted with Et2O (ꢀ2). The combined
organic layers were washed with brine, dried over MgSO4 and
evaporated to give the corresponding saturated ester as a brown
viscous oil (278 mg). 1H NMR (300 MHz, CDCl3): d 7.36–7.16 (m,
5.1.30. Methyl (3-{[(2R)-2-(hydroxymethyl)-5-oxo-1-pyrroli
dinyl]methyl}phenyl)acetate (35)
A solution of 34b (288 mg, 0.740 mmol) in THF (1.5 mL) was
treated with a solution of TBAF (1.0 M in THF, 0.81 mL, 0.81 mmol)
at room temperature under argon atmosphere for 1 h. The reaction
mixture was diluted with EtOAc, washed with H2O, brine, and

2246
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
dried over Na2SO4. The organic layer was evaporated and the
resulting residue was purified by column chromatography on silica
gel (EtOAc/MeOH, 1:0–20:1) to give 35 as a colorless oil (158 mg,
77%). 1H NMR (300 MHz, CDCl3): d 7.25–7.19 (m, 4H), 4.82 (d,
J = 15.1 Hz, 1H), 4.24 (d, J = 15.1 Hz, 1H), 3.75 (m, 1H), 3.68 (s,
3H), 3.62 (s, 2H), 3.60–3.47 (m, 2H), 2.56 (m, 1H), 2.41 (m, 1H),
2.12–1.93 (m, 2H).
1H), 4.02 (m, 1H), 3.53 (m, 1H), 3.40 (br s, 1H), 2.90–2.70 (m,
5H), 2.50–2.10 (m, 3H), 1.90–1.60 (m, 3H); FAB-MS (m/z): 434
(M+H)+; HRMS-FAB (m/z): [M+H]+ calcd for C22H25ClNO4S,
434.1193; found, 434.1185.
5.1.35. 5-(3-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(3-methylphenyl)but-
1-enyl]-5-oxopyrrolidin-1-yl}propyl)thiophene-2-carboxylic
acid (10c)
5.1.31. [3-({(2R)-2-[(1E,3S)-3-Hydroxy-4-(3-methylphenyl)but-
1-enyl]-5-oxopyrrolidin-1-yl}methyl)phenyl]acetic acid (8)
Compound 8 was prepared from 35 according to the same pro-
cedure as described for the preparation of 3 from 21a as a colorless
oil (76 mg, 58% form 21a). IR (film): 3399, 2922, 1726, 1660, 1488,
Compound 10c was prepared from 34c using the phosphonate
29a according to the same procedure as described for the prepara-
tion of 3 from 21a as a colorless oil (60 mg, 59% from 21a). IR (KBr):
3391, 2924, 1666, 1540, 1463, 1421, 1378, 1264, 1097, 1033, 975,
820, 754, 701, 666 cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.68 (d,
;
1418, 1251, 1173, 1096, 1037, 974, 916, 784, 746, 702, 664, 609 cm
;
J = 3.8 Hz, 1H), 7.19 (t, J = 7.4 Hz, 1H), 7.07–6.96 (m, 3H), 6.83 (d,
J = 3.8 Hz, 1H), 5.75 (dd, J = 15.4, 6.0 Hz, 1H), 5.47 (ddd, J = 15.4,
8.8, 1.1 Hz, 1H), 4.38 (m, 1H), 4.02 (m, 1H), 3.53 (m, 1H), 2.90–
2.76 (m, 5H), 2.46–2.37 (m, 2H), 2.33 (s, 3H), 2.21 (m, 1H), 1.90–
1.65 (m, 3H). FAB-MS (m/z): 414 (M+H)+; HRMS-FAB (m/z):
[M+H]+ calcd for C23H28NO4S2, 414.1752; found, 414.1739.
ꢁ1
1H NMR (300 MHz, CDCl3): d 7.27–6.97 (m, 8H), 5.62 (dd,
J = 15.4, 5.8 Hz, 1H), 5.41 (ddd, J = 15.4, 8.8, 1.1 Hz, 1H), 4.74 (d,
J = 14.6 Hz, 1H), 4.36 (m, 1H), 3.87 (m, 1H), 3.81 (d, J = 14.6 Hz,
1H), 3.60 (s, 2H), 2.78 (d, J = 6.6 Hz, 2H), 2.55–2.35 (m, 2H), 2.32
(s, 3H), 2.15 (m, 1H), 1.69 (m, 1H); MS (APCI) m/z: 392 (MꢁH)ꢁ;
HRMS-FAB (m/z): [M+H]+ calcd for C24H28NO4, 394.2018; found,
394.2018.
5.1.36. Methyl [3-(2-bromoethyl)phenyl]acetate (42)
A solution of 1,3-phenylenediacetic acid (1.78 g, 9.20 mmol) in
MeOH (3 mL) and EtOAc (3 mL) was treated with a solution of
trimethylsilyldiazomethane (2.0 M in hexane, 4,6 mL, 9.2 mmol)
at 0 °C for 5 min. The resulting mixture was evaporated to give a
mixture of the corresponding half ester and diester. To a stirred
solution of a mixture of the half ester and the diester in THF
(9 mL) was added dropwise a solution of diborane–dimethylsulfide
complex (2.0 M in THF, 9.0 mL, 18 mmol) at 0 °C under argon
atmosphere. After being stirred at room temperature for 16 h, the
reaction was quenched with MeOH. The reaction mixture was di-
luted with EtOAc, washed with saturated aqueous NaHCO3, brine,
and dried over Na2SO4. The organic layer was evaporated, and
the resulting residue was purified by column chromatography on
silica gel (hexane/EtOAc, 1:1–1:2) to give the corresponding 1,3-
bisphenethyl alcohol as a colorless oil (834 mg, 47%). 1H NMR
(300 MHz, CDCl3): d 7.45–7.08 (m, 4H), 3.86 (t, J = 7.2 Hz, 2H),
3.70 (s, 3H), 3.62 (s, 2H), 2.86 (t, J = 7.2 Hz, 2H), 1.56–1.45 (br s,
1H).
To a stirred solution of the above-described 1,3-bispenethyl
alcohol (834 mg, 4.30 mmol) in CH2Cl2 (10 mL) was added phos-
phorous tribromide (0.75 mL, 5.20 mmol) at 0 °C under argon
atmosphere. After being stirred for 30 min, the reaction was
poured into ice-water. The resulting mixture was extracted with
EtOAc, washed with water, brine, and dried over Na2SO4. The or-
ganic layer was evaporated, and the resulting residue was purified
by column chromatography on silica gel (hexane/EtOAc, 4:1) to
give 42 as a pale brown oil (251 mg, 23%). 1H NMR (300 MHz,
CDCl3): d 7.45–7.12 (m, 4H), 3.70 (s,3H), 3.62 (s, 2H), 3.57 (t,
J = 8.0 Hz, 2H), 3.18 (t, J = 8.0 Hz, 2H).
5.1.32. Methyl 5-{3-[(2R)-2-({tert-butyldimethylsilyloxy}meth
yl)-5-oxo-1-pyrrolidinyl]propyl}-2-thiophenecarboxylate (34c)
To a stirred solution of the lactam 33 (922 mg, 4.03 mmol) in
DMF (10 mL) was added sodium hydride (62% in mineral oil,
183 mg, 4.80 mmol) at 0 °C under argon atmosphere. Stirring was
continued at room temperature for 1 h, and 60 °C for additional
1 h. To the resulting suspension was added methyl 5-(3-bromopro-
pyl)thiophene-2-carboxylate (1.05 g, 4.00 mmol) at 0 °C, and stir-
ring was continued at 80 °C for additional 1.5 h. The resulting
pale brown solution was poured into saturated aqueous NH4Cl, ex-
tracted with EtOAc, washed with H2O, brine, and dried over MgSO4.
The organic layer was evaporated, and the resulting residue was
purified by column chromatography on silica gel (hexane/EtOAc,
2:2–1:1) to give 34c as a colorless oil (530 mg, 32%). 1H NMR
(300 MHz, CDCl3): d 7.62 (d, J = 3.6 Hz, 1H), 6.81 (d, J = 3.6 Hz,
1H), 3.86 (s, 3H), 3.72–3.54 (m, 4H), 3.11 (m, 1H), 2.85 (t,
J = 7.5 Hz, 2H), 2.48–2.23 (m, 2H), 2.08–1.70 (m, 4H), 0.87 (s, 9H),
0.04 (s, 3H), 0.03 (s, 3H).
5.1.33. Methyl 5-{3-[(2R)-2-(hydroxymethyl)-5-oxo-1-pyrrolid
inyl]propyl}-2-thiophenecarboxylate (36)
A solution of 34c (530 mg, 1.29 mmol) in THF (5 mL) was trea-
ted with a solution of TBAF (1.0 M in THF, 2.60 mL, 2.60 mmol) at
room temperature under argon atmosphere for 1 h. After addition
of brine, the reaction mixture was extracted with EtOAc repeat-
edly. The combined organic layers were dried over MgSO4, and
evaporated. The resulting residue was purified by column chroma-
tography on silica gel (hexane/EtOAc, 1:1–0:1) to give 36 as a pale
yellow oil (394 mg, 100%). 1H NMR (300 MHz, CDCl3): d 7.62 (d,
J = 3.9 Hz, 1H), 6.81 (d, J = 3.9 Hz, 1H), 3.86 (s, 3H), 3.80–3.61 (m,
4H), 3.12 (m, 1H), 2.86 (t, J = 7.5 Hz, 2H), 2.48 (m, 2H), 2.32 (m,
1H), 2.15–1.86 (m, 4H), 1.74 (br s, 1H).
5.1.37. Ethyl (4R)-4-({2-tert-butoxycarbonyl}amino)-5-oxopent
anoate (38)
To a stirred solution of oxalyl chloride (0.49 mL, 5.64 mmol) in
CH2Cl2 (9 mL) was slowly added a solution of DMSO (0.50 mL,
7.05 mmol) in CH2Cl2 (3 mL) at ꢁ70 °C in 10 min. The resulting solu-
tion was stirred for an additional 5 min at the same temperature. To
the reaction mixture was added a solution of 37 (1.23 g, 4.70 mmol)
in CH2Cl2 (5 mL). The resulting suspension was allowed to warm up
to ꢁ40 °C over 30 min. After being stirred for an additional 10 min,
the reaction mixture was treated with N,N-diisopropylethylamine
(2.0 mL, 11.3 mmol) and the resulting suspension was allowed to
warm up to ꢁ5 °C over 30 min. The reaction was quenched with
water and then poured into ice-cold 1 N HCl. The reaction mixture
was extracted with EtOAc. The organic layer was washed with 1 N
HCl, water, brine, dried over MgSO4 and evaporated to give an
5.1.34. 5-(3-{(2R)-2-[(1E,3S)-4-(3-Chlorophenyl)-3-hydroxybut-
1-enyl]-5-oxopyrrolidin-1-yl}propyl)thiophene-2-carboxylic
acid (10b)
Compound 10b was prepared from 34c using a phosphonate
29d instead of 29a according to the same procedure as described
for the preparation of 3 from 21a as a colorless amorphous powder
(52 mg, 45% from 21a). IR (KBr): 3417, 2930, 2623, 1660, 1599,
1574, 1540, 1463, 1422, 1377, 1266, 1167, 1097, 1031, 975, 910,
819 cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.68 (d, J = 3.6 Hz, 1H),
;
7.23–7.18 (m, 3H), 7.08 (m, 1H), 6.83 (d, J = 3.6 Hz, 1H), 5.71 (dd,
J = 15.3, 6.0 Hz, 1H), 5.48 (ddd, J = 15.3, 8.7, 0.9 Hz, 1H), 4.39 (m,

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
2247
aldehyde 38 as a colorless oil. 1H NMR (300 MHz, CDCl3): d 9.60 (s,
1H), 5.25–5.20 (br s, 1H), 4.34–4.20 (m, 1H), 4.15 (q, J = 7.2 Hz,
2H), 2.42 (t, J = 7.2 Hz, 2H), 2.45–2.22 (m, 1H), 2.05–1.89 (m, 1H),
1.43 (s, 9H), 1.25 (t, J = 7.2 Hz, 3H).
3.50 (m, 7H), 3.04–2.77 (m, 5H), 2.46–2.22 (m, 2H), 2.21–2.00
(m, 1H), 1.89–1.70 (m, 1H).
5.1.41. [3-(2-{(2R)-2-[(1E,3S)-4-(3-Chlorophenyl)-3-hydroxybut-
1-enyl]-5-oxopyrrolidin-1-yl}ethyl)phenyl]acetic acid (9)
Compound 8 was prepared from 41 according to the same pro-
cedure as described for the preparation of 3 from 24a as a pale yel-
low amorphous (62 mg, 73%). IR (film): 3387, 2932, 1724, 1660,
5.1.38. Ethyl (4R,5E)-8-(3-chlorophenyl)-4-({[(2-methyl-2-
propanyl)oxy]carbonyl}amino)-7-oxo-5-octenoate (39)
To a stirred solution of the phoshonate 29d (1.56 g, 5.64 mmol)
in THF (50 mL) was added sodium hydride (63% in mineral oil,
197 mg, 5.17 mmol) in several portions at 0 °C under argon atmo-
sphere. Stirring was continued at ambient temperature for 30 min.
To the stirred suspension was added a solution of the above-de-
scribed aldehyde 38 in THF (10 mL) at room temperature and stir-
ring was continued for 2 h. The reaction was quenched with acetic
acid. The reaction mixture was diluted with EtOAc, washed with
water, then brine, dried over MgSO4. The organic layer was evapo-
rated. The resulting residue was purified by column chromatogra-
phy on silica gel (hexane/EtOAc, 5:1–3:1) to give an enone as a
white solid (1.40 g, 73% in 2 steps). 1H NMR (300 MHz, CDCl3): d
7.31–7.00 (m, 4H), 6.77 (dd, J = 15.6 , 6.0 Hz, 1H), 6.23 (d,
J = 15.6 Hz, 1H), 4.81-.466 (m, 1H), 4.47–4.20 (m, 1H), 4.14 (q,
J = 7.0 Hz, 1H), 3.82 (s, 2H), 2.38 (t, J = 7.2 Hz, 2H), 2.15–1.75 (m,
2H), 1.41 (s, 9H), 1.24 (t, J = 7.2 Hz, 3H).
To a stirred solution of the above-described enone (1.31 g,
3.20 mmol) in THF (10.0 mL) was added a solution of (R)-2-
methyl-CBS-oxazaborolidine (1.0 M in toluene, 0.90 mL, 0.90
mmol) at room temperature under argon atmosphere. To the reac-
tion mixture was added dropwise a solution of diborane–THF com-
plex (1.0 M in THF, 1.90 mL, 1.90 mmol) in 5 min. The resulting
solution was stirred for 1 h, then treated with MeOH and stirring
was continued 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 layer was evaporated. The resulting
residue was purified by column chromatography on silica gel (hex-
ane/EtOAc, 3:1–2:1) to give 39 as a pale yellow oil (1.13 g, 86%). 1H
NMR (300 MHz, CDCl3): d 7.25–7.22 (m, 3H), 7.15–7.05 (m, 1H),
5.69 (dd, J = 15.6, 6.0 Hz, 1H), 5.55 (d, J = 15.6, 9.0 Hz, 1H), 4.61–
4.55 (m, 1H), 4.45–4.32 (m, 1H), 4.22–4.10 (m, 3H), 2.95–2.74 (m,
2H), 2.32 (t, J = 7.2 Hz, 2H), 1.89–1.70 (m, 2H), 1,44 (s, 9H), 1.24
(t, J = 7.2 Hz, 3H).
1422, 1257, 1164, 1033, 976, 783, 757, 705 cmꢁ1
;
1H NMR
(300 MHz, CDCl3): d 7.25–7.0 (m, 8H), 5.51 (dd, J = 15, 6.0 Hz,
1H), 5.25 (dd, J = 15, 8 Hz, 1H), 4.4–4.3 (m, 1H), 3.75–3.65 (m,
1H), 3.62 (s, 2H), 3.65–3.55 (m, 1H), 3.3–2.4 (br), 3.0–2.7 (m, 5H),
2.4–2.2 (m, 2H), 2.1–1.95 (m, 1H), 1.65–1.5 (m, 1H); MS (APCI)
m/z: 428 (M+2ꢁH)ꢁ, 426 (MꢁH)ꢁ; HRMS-FAB (m/z): [M+H]+ calcd
for C24H27ClNO4, 428.1629; found, 428.1617.
5.1.42. Ethyl (4R,5E,7S)-8-(3-Methylphenyl)-7-hydroxy-4-({tert-
butyoxycarbonyl}amino)-5-octenoate (43)
Compound 43 was prepared from 38 according to the same pro-
cedure as described for the preparation of 39 from 38 as a colorless
oil (4.55 g, 84% from 38).
5.1.43. Ethyl (4R,5E,7S)-4-amino-7-hydroxy-8-(3-methylphenyl)
-5-octenoate hydrochloride (44)
A solution of 43 (3.82 g, 9.87 mmol) in EtOH (10 mL) was trea-
ted with 4 N HCl in dioxane (7.5 mL) at room temperature under
argon atmosphere. After being stirred for 8 h, the reaction mixture
was evaporated to give 44 as a brown oil (3.20 g, 100%). 1H NMR
(300 MHz, CD3OD): d 7.19–7.10 (m, 1H), 7.07–6.96 (m, 3H), 5.91
(dd, J = 15.7, 4.7 Hz, 1H), 5.54 (ddd, J = 15.5, 8.9, 1.5 Hz, 1H),
4.43–4.32 (m, 1H), 4.22–4.06 (m, 2H), 3.80–3.64 (m, 1H), 2.90–
2.67 (m, 2H), 2.39–2.19 (m, 5H), 2.16–1.92 (m, 2H), 1.88–1.70
(m, 1H), 1.26 (t, J = 7.1 Hz, 3H).
5.1.44. (5R)-1-[2-(4-Bromophenyl)ethyl]-5-[(1E,3S)-3-{tert-but
yldimethylsilyloxy}-4-(3-methylphenyl)-1-buten-1-yl]-2-pyrro
lidinone (45)
To a stirred solution of the amine hydrochloride 44 (200 mg,
0.61 mmol) in THF (5 mL) was added 3-(4-bromophenyl)propion-
aldehyde (134 mg, 0.670 mmol) at room temperature under argon
atmosphere. After being stirred for 30 min, to this reaction mixture
was added sodium acetoxyborohydride (194 mg, 0.92 mmol) at
0 °C. After being stirred at room temperature for additional 16 h,
the reaction mixture was diluted with EtOAc, washed with water,
then brine and dried over Na2SO4. The organic layer was evapo-
rated to afford a lactam alcohol as a yellow oil.
To a stirred solution of the above-described lactam alcohol in
DMF (3 mL) and imidazole (244 mg, 3.60 mmol) was added tert-
butyldimethylsilyl chloride (184 mg, 2.40 mmol) at room temper-
ature under argon atmosphere. After being stirred for 6 h at
50 °C, the reaction mixture was poured into water, and extracted
with EtOAc, washed with H2O twice, brine, and dried over Na2SO4.
The organic layer was evaporated. The resulting residue was puri-
fied by column chromatography on silica gel (hexane/EtOAc, 2:1)
to give 45 as a pale yellow oil (237 mg, 72% in steps). 1H NMR
(300 MHz, CDCl3): d 7.39 (d, J = 8.3 Hz, 2H), 7.18–7.08 (m, 1H),
7.06–6.89 (m, 5H), 5.57 (dd, J = 15.3, 5.9 Hz, 1H), 5.25 (dd,
J = 15.4, 8.5 Hz, 1H), 4.34–4.22 (m, 1H), 3.81–3.63 (m, 2H), 2.95–
2.60 (m, 5H), 2.43–2.18 (m, 5H)), 2.16–2.01 (m, 1H), 1.69–1.56
(m, 1H), 0.89–0.77 (m, 9H), ꢁ0.10 (s, 3H), ꢁ0.17 (s, 3H).
5.1.39. Ethyl (4R,5E,7S)-4-amino-8-(3-chlorophenyl)-7-hydroxy-
5-octenoate trifluoroacetate (salt) (40)
A solution of 39 (930 mg, 2.26 mmol) in CH2Cl2 (20 mL) was
treated with trifluoroacetic acid (2.3 mL) at 0 °C under argon atmo-
sphere. After being stirred for 90 min at room temperature, the tri-
fluoroacetic acid was removed by the repeated evaporation with
benzene to give 40 as a pale red oil. 1H NMR (300 MHz, CD3OD):
d
7.40–7.0 (m, 4H), 5.93 (dd, J = 15.3, 6.0 Hz, 1H), 5.56 (dd,
J = 15.3, 8.8 Hz, 1H), 4.49–4.37 (m, 1H), 4.21–4.10 (m, 2H), 3.87–
3.70 (m, 1H), 2.92–2.74 (m, 2H), 2.35–2.25 (m, 2H), 2.11–1.95
(m, 1H), 1.90–1.75 (m, 1H), 1.27 (t, J = 7.2 Hz, 3H).
5.1.40. Methyl [3-(2-{(2R)-2-[(1E,3S)-4-(3-chlorophenyl)-3-hyd
roxy-1-buten-1-yl]-5-oxo-1-pyrrolidinyl}ethyl)phenyl]acetate
(41)
To a stirred solution of 40 (255 mg, 0.60 mmol) in DMF (3 mL)
were added sodium bicarbonate (101 mg, 1.20 mmol) and the bro-
mide 42 (185 mg, 0.720 mmol) at room temperature under argon
atmosphere. After being stirred at 90 °C for 8 h, the reaction mix-
ture was cooled to room temperature, and diluted with EtOAc,
washed with water, then brine, and dried over Na2SO4. The organic
layer was evaporated. The resulting residue was purified by col-
umn chromatography on silica gel (EtOAc/MeOH, 30:1) to give
41 as a pale yellow oil (90 mg, 34%). 1H NMR (300 MHz, CDCl3): d
7.30–7.00 (m, 8H), 5.61–5.35 (m, 2H), 4.44–4.38 (m, 1H), 3.80–
5.1.45. Ethyl 4-(2-{(2R)-2-[(1E,3S)-3-(tert-butyldimethylsily
loxy)-4-(3-methylphenyl)-1-buten-1-yl]-5-oxo-1-pyrrolidinyl}
ethyl)benzoate (46)
To a stirred solution of 45 (237 mg, 0.437 mmol) in DMSO
(2.0 mL) and EtOH (3.0 mL) were added triethylamine (0.073 mL,

2248
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
0.52 mmol), palladium acetate (9.0 mg, 0.04 mmol) and 1,1-
bis(diphenylphosphino)ferrocene (32 mg, 0.08 mmol) under argon
atmosphere, and the argon gas was replaced with carbon monox-
ide gas. After being stirred at 80 °C for 8 h, the reaction mixture
was cooled to room temperature, and filtered through a pad of Cel-
ite. The filtrate was diluted with EtOAc, washed with water (ꢀ2),
then brine, and dried over Na2SO4. The organic layer was evapo-
rated. The resulting residue was purified by column chromatogra-
phy on silica gel (hexane/EtOAc, 2:1) to give 46 as a pale yellow oil
(220 mg, 94%). 1H NMR (300 MHz, CDCl3): d 7.96 (d, J = 8.0 Hz, 2H),
7.20 (d, J = 8.0 Hz, 2H), 7.15–7.10 (m, 2H), 7.04–6.87 (m, 2H), 5.56
(dd, J = 15.3, 5.9 Hz, 1H), 5.27 (d, J = 8.8 Hz, 1H), 4.37 (q, J = 7.1 Hz,
2H), 4.28 (m, J = 6.0 Hz, 1H), 3.83–3.65 (m, 3H), 3.00–2.61 (m, 5H),
2.38–2.18 (m, 6H), 1.67–1.60 (m, 1H), 1.40 (t, J = 7.0 Hz, 3H), 0.92–
0.77 (m, 9H), ꢁ0.10 (s, 3 H), ꢁ0.18 (s, 3H).
into saturated aqueous NH4Cl and extracted with EtOAc. The or-
ganic layer was washed with H2O, brine, dried over MgSO4 and
evaporated. The resulting residue was purified by column chroma-
tography on silica gel (hexane/EtOAc, 3:1–1:1) to give 48 as a
brown oil (1.86 g, 84%). 1H NMR (300 MHz, CDCl3): d 7.65 (d,
J = 3.9 Hz, 1H), 7.09 (d, J = 3.9 Hz, 1H), 4.32 (q, J = 7.5 Hz, 2H),
3.86–3.61 (m, 3H), 3.55 (m, 1H), 3.32 (m, 1H), 3.22–3.00 (m, 2H),
2.50–2.21 (m, 2H), 2.10 (m, 1H), 1.80 (m, 1H), 1.36 (t, J = 7.5 Hz,
3H), 0.86 (s, 9H), 0.03 (s, 3H), 0.02 (s, 3H).
5.1.50. Ethyl 5-({2-[(2R)-2-(hydroxymethyl)-5-oxo-1-pyrro
lidinyl]ethyl}thio)-2-thiophenecarboxylate (49)
Compound 49 was prepared from 48 according to the same pro-
cedure as described for the preparation of 21a from 20 as a color-
less oil (1.15 g, 83%). 1H NMR (300 MHz, CDCl3): d 7.64 (d,
J = 3.9 Hz, 1H), 7.10 (d, J = 3.9 Hz, 1H), 4.33 (q, J = 6.9 Hz, 2H),
3.80–3.68 (m, 3H), 3.60 (m, 1H), 3.40 (m, 1H), 3.17 (t, J = 7.0 Hz,
2H), 2.58–2.28 (m, 2H), 1.10 (m, 1H), 1.98–1.80 (m, 2H), 1.37 (t,
J = 7.2 Hz, 3H).
5.1.46. 4-(2-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(3-methylphenyl)but-
1-enyl]-5-oxopyrrolidin-1-yl}ethyl)benzoic acid (10a)
To a stirred solution of 46 (220 mg, 0.410 mmol) in DME (1 mL)
and MeOH (1 mL) was added 2 N HCl (0.5 mL) under argon atmo-
sphere. After being stirred at 50 °C for 8 h, the reaction mixture
was cooled to room temperature, and then treated with 2 N NaOH
(2 mL) for 2 h. The reaction mixture was acidified with 1 N HCl
and extracted with EtOAc. The organic layer was washed with
water, then brine, and dried over Na2SO4. The organic layer was
evaporated and the resulting residue was purified by column chro-
matography on silica gel (CHCl3/MeOH, 100:1–30:1) to give 10a as
a beige amorphous (122 mg, 76%). IR (film): 2927, 1666, 1261,
5.1.51. 5-[(2-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(3-methylphenyl)
but-1-enyl]-5-oxopyrrolidin-1-yl}ethyl)sulfanyl]thiophene-2-
carboxylic acid (10d)
Compound 10d was prepared from 49 according to the same
procedure as described for the preparation of 3 from 21a as a color-
less oil (67 mg, 20% from 49). IR (film): 3097, 3009, 2921, 1792,
1676, 1637, 1524, 1487, 1457, 1420, 1318, 1244, 1159, 1096,
1035, 975, 884, 820, 754 cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.69
;
755 cmꢁ1
;
1H NMR (300 MHz, CDCl3): d 8.02 (d, J = 8.1 Hz, 2H),
(d, J = 3.8 Hz, 1H), 7.19 (t, J = 7.3 Hz, 1H), 7.10–7.02 (m, 4H), 5.72
(m, 1H), 5.47 (m, 1H), 4.38 (m, 1H), 4.12 (m, 1H), 3.66 (m, 1H),
3.15–3.05 (m, 3H), 2.88–2.77 (m, 2H), 2.43–2.38 (m, 2H), 2.33 (s,
3H), 2.20 (m, 1H), 1.72 (m, 1H); FAB-MS (m/z): 432 (M+H)+;
HRMS-FAB (m/z): [M+H]+ calcd for C22H26NO4S2, 432.1303; found,
432.1299.
7.30–7.15 (m, 3H), 7.10–6.97 (m, 3H), 5.64 (dd, J = 15.6, 6.3 Hz,
1H), 5.37 (dd, J = 15.6, 8.7 Hz, 1H), 4.41–4.32 (m, 1H), 3.83–3.70
(m, 2H), 3.09–2.95 (m, 1H), 2.95–2.75 (m, 4H), 2.48–2.25 (m, 5H),
2.20–2.13 (m, 1H), 1.72–1.58 (m, 1H); MS (APCI) m/z: 392 (MꢁH)
ꢁ; HRMS-FAB (m/z): [M+H]+ calcd for C24H28NO4, 394.2018; found,
394.2023.
5.1.52. Methyl 3-hydroxy-2-[(4-{tert-butyldiphenylsilyloxy}
butanoyl)amino]propanoate (50)
5.1.47. 2-[(2R)-2-({tert-Butyldimethylsilyloxy}methyl)-5-oxo-1-
pyrrolidinyl]ethyl methanesulfonate (47b)
To a stirred solution of sodium 4-hydroxybutanonate (1.54 g,
12.2 mmol) in DMF (20 mL) and imidazole (1.25 g, 18.3 mmol)
was added tert-butyldiphenylsilyl chloride (4.03 g, 14.6 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 (ꢀ2), brine,
and dried over MgSO4. The organic layer was evaporated to give a
silyl ether as a colorless oil.
To a stirred solution of the alcohol 47a (11.8 g, 43.3 mmol) and
triethylamine (9.07 mL, 65.1 mmol) in THF (50 mL) was added
methanesulfonyl chloride (3.68 mL, 47.7 mmol) at 0 °C under ar-
gon atmosphere. After being stirred at the same temperature for
10 min, the reaction was quenched with H2O, diluted with EtOAc,
washed with brine, and dried over MgSO4. The organic layer was
evaporated to yield methanesulfonate 47b as a yellow oil.
To a stirred solution of DL-serine methylester hydrochloride
(2.85 g, 18.3 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide hydrochloride (EDC: 3.05 g, 15.9 mmol) and triethylamine
(2.55 mL, 18.3 mmol) in CH3CN (30 mL) was added a solution of
the above-described silyl ether in CH3CN (5 mL) at room tempera-
ture under argon atmosphere. After being stirred for 1 h, the reac-
tion was quenched with water. The reaction mixture was diluted
with EtOAc, washed with 1 N HCl, water, then brine, and dried over
MgSO4. The organic layer was evaporated and the resulting residue
was purified by column chromatography on silica gel (hexane/
EtOAc, 1:1–0:1) to give 50 as a colorless oil (1.73 g, 32% in two
steps). 1H NMR (300 MHz, CDCl3): d 7.65–7.50 (m, 4H), 7.41–7.30
(m, 6H), 6.41 (m, 1H), 4.67 (m, 1H), 4.05–3.97 (m, 2H), 3.79 (s,
3H), 3.85–3.72 (m, 2H), 2.42 (t, J = 7.2 Hz, 2H), 2.01–1.93 (m, 2H),
1.05 (s, 9H).
5.1.48. (5R)-5-({tert-Butyldimethylsilyloxy}methyl)-1-(2-
iodoethyl)-2-pyrrolidinone (47c)
To a stirred solution of the methanesulfonate 47b in CH3CN
(120 mL) was added sodium iodide (19.5 g, 130 mmol) at room
temperature under argon atmosphere, and stirring was continued
at 80 °C for 15 h. The resulting yellow solution was diluted with
EtOAc, and washed with water, then brine, and dried over MgSO4.
The organic layer was evaporated to afford 47c as a yellow oil
(11.3 g, 67%).
5.1.49. Ethyl 5-({2-[(2R)-2-({tert-butyldimethylsilyloxy}methyl)
-5-oxo-1-pyrrolidinyl]ethyl}thio)-2-thiophenecarboxylate (48)
To
a stirred suspension of ethyl thiophene-2-carboxylate
(936 mg, 6.0 mmol) and sulfur (S8, 240 mg, 7.5 mmol) in THF
(50 mL) was added dropwise lithium diisopropylamide (2.0 M in
THF/heptane/ethyl benzene, 4.0 mL: 8.0 mmol) at ꢁ78 °C under ar-
gon atmosphere. Stirring was continued for 35 min. To the stirred
suspension was added a solution of 47c (1.92 g, 5.0 mmol) in THF
(5.0 mL), and stirring was continued at room temperature for addi-
tional 1.5 h. The resulting mixture was diluted with MTBE, poured
5.1.53. Methyl 2-(3-{tert-butyldiphenylsilyloxy}propyl)-1,3-
oxazole-4-carboxylate (51)
To a stirred solution of 50 (1.72 g, 3.88 mmol) in CH2Cl2 (15 mL)
was
added
N,N-diethylaminosulfur
trifluoride
(0.56 mL,
4.27 mmol) at ꢁ78 °C under argon atmosphere and stirring was

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
2249
continued for 2 h. To the reaction mixture was added potassium
carbonate (1.60 g, 11.6 mmol), and the reaction mixture was al-
lowed to warm up to ꢁ20 °C under stirring. After being stirred for
additional 1 h, the reaction was quenched with water, extracted
with CH2Cl2, and dried over Na2SO4. The organic layer was evapo-
rated. To a stirred solution of the resulting residue in CH2Cl2
(19.4 mL) were added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU:
1.74 mL, 11.6 mmol) at 0 °C under argon atmosphere and then
powder (66 mg, 70% from 53). IR (KBr): 3422, 2933, 1727, 1656,
1586, 1459, 1421, 1280, 1161, 1110, 1036, 980, 747 cmꢁ1 1H
;
NMR (300 MHz, CDCl3): d 8.19 (s, 1H), 7.20 (m, 1H), 7.06–6.97
(m, 3H), 5.78 (dd, J = 15.3, 6.0 Hz, 1H), 5.50 (ddd, J = 15.3, 9.0,
1.2 Hz, 1H), 4.40 (m, 2H), 4.07 (m, 1H), 3.47 (m, 1H), 2.94 (m,
1H), 2.83–2.75 (m, 4H), 2.50–2.10 (m, 6H), 2.05–1.83 (m, 2H),
1.64 (m, 1H); MS (APCI) m/z: 397 (MꢁH)ꢁ; HRMS-FAB (m/z):
[M+H]+ calcd for C22H27N2O5, 399.1920; found, 399.1903.
dropwise
a
solution of bromotrichloromethane (0.80 mL,
8.14 mmol) in CH2Cl2 (4 mL). The reaction mixture was allowed
to warm up to room temperature under stirring. After being stirred
for 10 h, the reaction mixture was diluted with EtOAc, washed with
1 N HCl, water, saturated aqueous NaHCO3, brine, and dried over
Na2SO4. The organic layer was evaporated, and the resulting residue
was purified by column chromatography on silica gel (hexane/
EtOAc, 9:1–1:1) to give 51 as a colorless oil (886 mg, 54% from
53). 1H NMR (300 MHz, CDCl3): d 8.13 (s, 1H), 7.70–7.58 (m, 4H),
7.47–7.32 (m, 6H), 3.91 (s, 3H), 3.72 (t, J = 6.0 Hz, 2H), 3.01–2.90
(m, 2H), 2.11–1.98 (m, 2H), 1.08–0.99 (m, 9H).
5.1.57. Ethyl 2-(3-oxopropyl)-1,3-thiazole-4-carboxylate (54)
To
a stirred solution of 4,4-dimethoxybutyronitrile (5.0 g,
38.7 mmol) in DMF (50 mL) was added portionwise magnesium
chloride hexahydrate (10.2 g, 50.3 mmol) at 0 °C under argon
atmosphere. To the resulting suspension was successively added
sodium hydrosulfide (4.02 g, 50.3 mmol) at 0 °C under argon atmo-
sphere. After being stirred at room temperature for 16 h, the reac-
tion mixture was diluted with EtOAc, washed with H2O, and dried
over MgSO4. The organic layer was evaporated to give the corre-
sponding thioamide as a colorless oil.
To a stirred solution of the above-described thioamide (300 mg,
1.81 mmol) in DMF (2 mL) was added ethyl bromopyruvate
(422 mg, 2.17 mmol) at room temperature under argon atmo-
sphere. After being stirred for 2 h, the reaction was quenched with
H2O. The reaction mixture was diluted with EtOAc, washed with
saturated aqueous NaHCO3, brine and dried over MgSO4. The or-
ganic layer was evaporated to give ethyl 2-(3,3-dimethoxypro-
pyl)-1,3-thiazole-4-carboxylate as a brown oil.
A solution of the above-described dimethylacetal in DME (4 mL)
was treated with 2 N HCl at room temperature for 2 h. The homo-
geneous reaction mixture was diluted with EtOAc, washed with
H2O, and dried over Na2SO4. The organic layer was evaporated,
and the resulting residue was purified by column chromatography
on silica gel (hexane/EtOAc, 2:1–1:1) to give 54 as a brown oil
(225 mg, 12% in 3 steps). 1H NMR (300 MHz, CDCl3): d 9.85 (s,
1H), 8.06 (s, 1H), 4.41 (q, J = 6.9 Hz, 2H), 3.37 (t, J = 6.9 Hz, 2H),
3.08 (t, J = 6.9 Hz, 2H), 1.40 (t, J = 6.9 Hz, 3H).
5.1.54. Methyl 2-(3-hydroxypropyl)-1,3-oxazole-4-carboxylate
(52)
A solution of 51 (886 mg, 2.09 mmol) in THF (3 mL) was treated
with TBAF (1.0 M in THF, 4.27 mL, 4.27 mmol) at room tempera-
ture under argon atmosphere for 1 h and then with brine. The reac-
tion mixture was extracted with EtOAc repeatedly (ꢀ5). The
combined organic layers were dried over MgSO4, and evaporated.
The resulting residue was purified by column chromatography on
silica gel (hexane/EtOAc, 1:4–0:1) to give 52 as a pale yellow oil
(304 mg, 79%). 1H NMR (300 MHz, CDCl3): d 8.16 (s, 1H), 3.91 (s,
3H), 3.79–3.66 (m, 2H), 2.96 (t, J = 7.4 Hz, 2H), 2.14–1.97 (m, 2H),
1.76 (m, 1H).
5.1.55. Methyl 2-(3-{(2R)-2-[(1E,3S)-3-hydroxy-4-(3-methyl
phenyl)-1-buten-1-yl]-5-oxo-1-pyrrolidinyl}propyl)-1,3-oxa
zole-4-carboxylate (53)
To a stirred solution of the alcohol 52 (85 mg, 0.459 mmol) in
DMSO (2 mL) and N,N-diisopropylethylamine (0.48 mL, 2.75 mmol)
was added SO3–Py (219 mg, 1.38 mmol) at 0 °C under argon atmo-
sphere. After being stirred at room temperature for 20 min, the
reaction was quenched with 1 N HCl. The reaction mixture was di-
luted with EtOAc, washed with brine, and dried over MgSO4. The or-
ganic layer was evaporated to yield the corresponding aldehyde as a
pale yellow oil (98 mg).
To a stirred solution of the above-described aldehyde in THF
(0.5 mL) was added a solution of the amine hydrochloride 44
(100 mg, 0.306 mmol) in THF (0.5 mL) at room temperature under
argon atmosphere and stirring was continued for 1 h at 50 °C. To
the stirred reaction mixture was added sodium acetoxyborohy-
dride (117 mg, 0.550 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
chromatography on silica gel (hexane/EtOAc, 1:4–0:1) to give 53
as a pale yellow solid (93 mg, 74%). 1H NMR (300 MHz, CDCl3): d
8.14 (s, 1H), 7.24–7.14 (m, 1H), 7.10–6.93 (m, 3H), 5.75 (dd,
J = 15.1, 5.2 Hz, 1H), 5.50 (ddd, J = 15.1, 8.8, 1.1 Hz, 1H), 4.40 (m,
1H), 4.13–3.99 (m, 1H), 3.87 (s, 3H), 3.55–3.41 (m, 1H), 2.97–
2.67 (m, 5H), 2.47–2.11 (m, 7H), 2.03–1.85 (m, 2H), 1.72 (m, 1H).
5.1.58. 2-(3-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(3-methylphenyl)but-
1-enyl]-5-oxopyrrolidin-1-yl}propyl)-1,3-thiazole-4-carboxylic
acid (12)
Compound 12 was prepared from 54 using 44 according to the
same procedure as described for the preparation of 11 from 52 as a
white powder (66 mg, 70% form 54). IR (KBr): 3423, 2924, 1718,
1661, 1488, 1459, 1421, 1376, 1322, 1216, 1100, 1037, 975, 783,
748, 702 cmꢁ1 1H NMR (300 MHz, CDCl3): d 8.12 (s, 1H), 7.18
;
(m, 1H), 7.05–6.97 (m, 3H), 5.75 (dd, J = 15.3, 5.7 Hz, 1H), 5.60–
5.20 (m, 3H), 4.40 (m, 1H), 4.07 (m, 1H), 3.51 (m, 1H), 3.07–2.85
(m, 3H), 2.79 (d, J = 6.6 Hz, 2H), 2.50–2.12(m, 6H), 2.04–1.90 (m,
2H), 1.70 (m, 1H); MS (APCI) m/z: 413 (MꢁH)ꢁ; HRMS-FAB (m/z):
[M+H]+ calcd for C22H27N2O4, 415.1692; found, 415.1686.
5.1.59. Ethyl (4R,5E,7S)-7-hydroxy-4-({tert-butoxycarbonyl}
amino)-5-dodecenoate (55)
Compound 55 was prepared from 38 according to the same pro-
cedure as described for the preparation of 39 from 38 as a colorless
oil (2.97 g, 83% from 38). 1H NMR (300 MHz, CDCl3): d 5.71–5.47
(m, 2H), 4.51 (br s, 1H), 4.20–4.02 (m, 3H), 2.38 (t, J = 7.4 Hz, 2H),
1.95–1.73 (m, 2H), 1.54–1.49 (m, 2H), 1.46–1.30 (m, 11H), 1.29
(s, 9H), 0.89 (t, J = 6.6 Hz, 3H).
5.1.60. Ethyl (4R,5E,7S)-4-amino-7-hydroxy-5-dodecenoate
hydrochloride (56)
Compound 56 was prepared from 55 according to the same pro-
cedure as described for the preparation of 44 from 43 as a colorless
oil (2.49 g, 100%). 1H NMR (300 MHz, CD3OD): d 5.87 (dd, J = 15.4,
6.0 Hz, 1H), 5.57 (dd, J = 17.0, 9.6 Hz, 1H), 4.13 (m, 3H), 3.83–3.69
5.1.56. 2-(3-{(2R)-2-[(1E,3S)-3-Hydroxy-4-(3-methylphenyl)but-
1-enyl]-5-oxopyrrolidin-1-yl}propyl)-1,3-oxazole-4-carboxylic
acid (11)
Compound 11 was prepared from 53 according to the same pro-
cedure as described for the preparation of 10a from 46 as a white

2250
T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
w) = (Lm/Lt) ꢀ 100, where Lm = measured drug loading (%, w/w)
(m, 1H), 2.49–2.28 (m, 2H), 2.09 (m, 1H), 1.88 (m, 1H), 1.57–1.12
(m, 11H), 0.90 (t, J = 6.0 Hz, 3H).
and Lt = theoretical drug loading (%, w/w).
5.2.3. In vitro release profile of compound 17 PLGA microsphere
Compound 17-loaded PLGA microspheres were suspended in
phosphate-buffered saline (PBS) containing 0.2% Tween 80 to ad-
5.1.61. 4-(2-{(2R)-2-[(1E,3S)-3-Hydroxyoct-1-enyl]-5-oxopyrroli
din-1-yl}ethyl)benzoic acid (2)
Compound 2 was prepared from 56 using 4-methoxycarbonyl
phenylacetaldehyde according to the same procedure as described
for the preparation of 11 from 44 as a white powder (10 mg, 50%
from 56). IR (KBr): 3222, 2953, 2930, 2857, 1715, 1658, 1612,
1509, 1459, 1425, 1415, 1363, 1327, 1307, 1244, 1204, 1174,
just the concentration of compound 17 to 100 lg/ml. The micro-
spheres were then completely dispersed by vortex mixing and
sonication. The solution was divided into 1 ml samples and incu-
bated at 37 °C. At various time intervals, the aliquots were centri-
fuged for 5 min at 12,000 rpm (n = 3 for each). The supernatant was
removed and the pellet was dissolved with p-hydroxy benzoic acid
n-nonyl ester in acetonitrile. The concentration of compound 17 in
the solution was analyzed by high performance liquid chromatog-
raphy (HPLC).
1154, 1107, 1064, 1019, 977, 910, 867, 837, 749, 702.93, 612 cm
ꢁ1
;
1H NMR (300 MHz, DMSO-d6): d 7.84 (d, J = 8.4 Hz, 2H), 7.28
(d, J = 8.4 Hz, 2H), 5.62 (dd, J = 15.6, 6.3 Hz, 1H), 5.33 (dd, J = 15.6,
8.7 Hz, 1H), 4.71 (d, J = 4.8 Hz, 1H), 4.00–3.84 (m, 2H), 3.60 (m,
1H), 2.99 (m, 1H), 2.89–2.66 (m, 2H), 2.30–2.00 (m, 3H), 1.60 (m,
1H), 1.50–1.15 (m, 8H), 0.81 (t, J = 6.3 Hz, 3H); MS (APCI) m/z:
358 (MꢁH)ꢁ; HRMS-FAB (m/z): [M+H]+ calcd for C21H30NO4,
360.2175; found, 360.2173.
The remaining percentage of compound 17 was calculated as
follows:
Rx ¼ Px=ðS0 þ P0Þ ꢀ 100
where Rx = Remaining percentage of compound 17 at x days (%),
Px = Amount of compound 17 in the pellet of 1 mL sample at x days
5.1.62. 4-[(2-{(2R)-2-[(1E,3S)-3-Hydroxyoct-1-enyl]-5-oxopyrr
olidin-1-yl}ethyl)sulfanyl]butanoic acid (5)
(
l
g), S0 = Amount of compound 17 in the supernatant of 1 mL sam-
ple at 0 day ( g) and P0 = Amount of compound 17 in the pellet of
1 mL sample at 0 day ( g).
Compound 5 was prepared from 56 using aldehyde 57 accord-
ing to the same procedure as described for the preparation of 11
from 44 as a white powder (11 mg, 45% from 56). IR (KBr): 3214,
2928, 2857, 1730, 1660, 1460, 1369, 1320, 1269, 1210, 1173,
l
l
1150, 1022, 982, 910, 668, 570 cmꢁ1 1H NMR (300 MHz, CDCl3):
;
5.3. Biological evaluation
d 5.73 (dd, J = 15.3, 5.7 Hz, 1H), 5.53 (ddd, J = 15.3, 8.4, 1.2 Hz,
1H), 4.25–4.18 (m, 2H), 3.63 (m, 1H), 3.11 (m, 1H), 2.78–2.20 (m,
10H), 2.00–1.70 (m, 3H), 1.62–1.21 (m, 8H), 0.90 (t, J = 6.6 Hz,
3H); MS (APCI) m/z: 356 (MꢁH)ꢁ; HRMS-FAB (m/z): [M+H]+ calcd
for C18H32NO4S, 358.2052; found, 358.2051.
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 accor-
dance with ethical guidelines established by the Experimental Ani-
mal Care and Use Committee of ONO Pharmaceutical Co., Ltd.
Fracture models: Fibulae were fractured according to the meth-
od of Kawaguchi et al. with minor modification.18 In brief, after
anesthetizing 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.
5.2. PLGA formulation
5.2.1. Preparation of PLGA microspheres containing compound
17
Microspheres were prepared by the emulsion-solvent evapora-
tion method.17 Compound 17 (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 17. The supernatant was
then discarded and replaced with fresh water or aqueous medium
containing 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 17 microspheres in solid form.
References and notes
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Acad. Sci. U.S.A. 2002, 99, 4580.
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5.2.2. Drug loading and encapsulation efficiency of compound
17 PLGA microspheres
The appropriate quantity of compound 17-loaded PLGA micro-
spheres was dissolved with p-hydroxy benzoic acid n-nonyl ester
in acetonitrile. The concentration of compound 17 in the solution
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.
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Palmer, E.; Bao, B.; Mckenna, S. D. Bioorg. Med. Chem. Lett. 2008, 18, 821.
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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.
Drugloadingð%; w=wÞ ¼ ðM=WÞ ꢀ 100
where M = measured amount of compound 17 in the compound 17-
loaded-PLGA microspheres (
l
g), W = weight of compound 17-
loaded-PLGA microspheres (
l
g), Encapsulation efficiency (%, w/

T. Kambe et al. / Bioorg. Med. Chem. 20 (2012) 2235–2251
2251
9. Kambe, T.; Maruyama, T.; Nakano, M.; Nakai, Y.; Yoshida, T.; Matsunaga, N.;
Oida, H.; Akira Konaka, A.; Maruyama, T.; Nakai, H.; Toda, M. Bioorg. Med. Chem.
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Nakai, H.; Toda, M. Chem. Pharm. Bull. 2011, 59, 1494.
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