Discovery of potent and orally bioavailable 17β-hydroxysteroid dehydrogenase type 3 inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.03.052

We have previously reported the discovery of a new class of potent inhibitors of 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) derived from benzylidene oxazolidinedione and thiazolidinedione scaffolds. In this study, these analogs were designed, synthesized, and evaluated in a human cell-based assay. The detailed structure-activity relationship (SAR) surrounding this pharmacophore were developed, and consequently a number of compounds from this series demonstrated single-digit nanomolar 17β-HDS3 inhibitory activity in vitro. Subsequent optimization work in pursuit of the improvement of oral bioavailability demonstrated in vivo proof-of-concept by prodrug strategy based on phosphate esters for these 17β-HSD3 inhibitors. When a phosphate ester 16 was administered orally at a high dose of 100 mg/kg, 16 showed approximately two times more potent testosterone (T)-lowering effect against a positive control in the luteinizing hormone-releasing hormone (LH-RH)-induced T production assay. The T-lowering effect continued at ca 10% level of control over 4 h after administration. The nonsteroidal molecules based on this series have the potential to provide unique and effective clinical opportunities for treatment of prostate cancer.

Full text of DOI:10.1016/j.bmc.2012.03.052

Bioorganic & Medicinal Chemistry 20 (2012) 3242–3254
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of potent and orally bioavailable 17b-hydroxysteroid
dehydrogenase type 3 inhibitors
Koichiro Harada a, , Hideki Kubo a, Jun Abe a, Mari Haneta a, Arnel Conception b, Shinichi Inoue b,
⇑
Satoshi Okada b, Kazuhiko Nishioka c
a Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd, 1-98, Kasugadenaka 3-chome, Konohana-ku, Osaka 554-8558, Japan
b Organic Synthesis Division, Sumika Technoservice Corporation, 2-1, Takatsukasa 4-chome, Takarazuka, Hyogo 665-0051, Japan
c Crop Protection Division-International, Sumitomo Chemical Co., Ltd, 27-1, Shinkawa 2-chome, Chuo-ku, Tokyo 104-8260, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously reported the discovery of a new class of potent inhibitors of 17b-hydroxysteroid
dehydrogenase type 3 (17b-HSD3) derived from benzylidene oxazolidinedione and thiazolidinedione
scaffolds. In this study, these analogs were designed, synthesized, and evaluated in a human cell-based
assay. The detailed structure–activity relationship (SAR) surrounding this pharmacophore were devel-
oped, and consequently a number of compounds from this series demonstrated single-digit nanomolar
17b-HDS3 inhibitory activity in vitro. Subsequent optimization work in pursuit of the improvement of
oral bioavailability demonstrated in vivo proof-of-concept by prodrug strategy based on phosphate esters
for these 17b-HSD3 inhibitors. When a phosphate ester 16 was administered orally at a high dose of
100 mg/kg, 16 showed approximately two times more potent testosterone (T)-lowering effect against a
positive control in the luteinizing hormone-releasing hormone (LH–RH)-induced T production assay.
The T-lowering effect continued at ca 10% level of control over 4 h after administration. The nonsteroidal
molecules based on this series have the potential to provide unique and effective clinical opportunities
for treatment of prostate cancer.
Received 25 February 2012
Revised 23 March 2012
Accepted 23 March 2012
Available online 1 April 2012
Keywords:
17b-Hydroxysteroid dehydrogenase type 3
(17b-HSD3)
17b-HSD3 inhibitors
Prostate cancer
Oxazolidinedione
Thiazolidinedione
Phosphate ester prodrug
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
androgen biosynthesis or its action on the androgen receptor is also
central to the management of prostate cancer.2 Production of
Prostate cancer is the most frequently diagnosed cancer in men.
According to the American Cancer Society, an estimated 192,280
men were diagnosed with prostate cancer in the US during 2009.
With an estimated 27,360 deaths in 2009, prostate cancer is the
second-leading cause of cancer death in men.1 For advanced pros-
tate cancer, 10–60% of the patients experience biochemical recur-
rence associated with treatment failure, and no consensus exists
on the optimal therapy.
androgens is controlled at two levels within the central nervous
system; in addition, it is controlled locally in peripheral organs that
are targeted by the hormones.
The 17b-hydroxysteroid dehydrogenase (17b-HSD) family
mediates the activation and deactivation of sex steroids involving
redox reactions centered about the C17 area of the steroid back-
bone (Fig. 1).3 This family of enzymes has a direct impact on dis-
eases that have been shown to be hormone-dependent, such as
prostate and breast cancers. More specifically, both diseases de-
pend on the long-term exposure to potent androgens and estro-
gens, which are biosynthesized as a result of the action of
different types of 17b-HSD enzymes. 17b-Hydroxysteroid dehydro-
genase type 3 (17b-HSD3) catalyzes the final step in the steroid-
genesis of the potent androgen testosterone (T) by selectively
The human prostate is a hormone-sensitive organ that depends
on androgens for growth and development. The regulation of
Abbreviations: HSD, hydroxysteroid dehydrogenase; 17b-HSD3, 17b-hydroxy-
steroid dehydrogenase type 3; T, testosterone;
E1, estrone; E2, estradiol; AR, androgen receptor; ER
D
4-dione, 4-androstene-3,17-dione;
a, estrogen receptor alpha; GR,
reducing the C17 ketone of 4-androstene-3,17-dione (D
4-dione)
glucocorticoid receptor; LH–RH, luteinizing hormone–releasing hormone; NADP,
nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of NADP; PK,
pharmacokinetics; Cmax, maximum drug concentration; AUC, area under the plasma
concentration curve; HMBC, heteronuclear multiple bond connectivity; TBDMS,
tert-butyl dimethylsilyl; TMS, trimethylsilyl; MMC, magnesium methyl carbonate;
DMAP, N,N-dimethylaminopyridine; SAR, structure–activity relationship.
with NADPH as a cofactor, in testis and prostate tissue.4 17b-
HSD3 is almost exclusively located in testis, suggesting its poten-
tial involvement in gonadal T biosynthesis. 17b-HSD3 is also
responsible for pseudohermaphroditism in deficient man but is
asymptomatic in deficient women. Since 17b-HSD3 is not found
in the ovary, whereas 17b-HSD5 is, it is suggested that the latter
⇑
Corresponding author. Tel.: +81 6 6466 5321; fax: +81 6 6466 5442.
E-mail address: haradak@sc.sumitomo-chem.co.jp (K. Harada).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.052

K. Harada et al. / Bioorg. Med. Chem. 20 (2012) 3242–3254
3243
O
OH
O
17 -HSD3
17 -HSD5
R
S
O
S
N
a
b
R
N
S
R-NH2
Br
S
O
O
4-dione
1
T
3
4
OH
Scheme 1. Synthesis of rhodanine analogs 1. Reagents and conditions: (a)
S@C(SCH2CO2H)2, CDI, THF, reflux, or S@C(SCH2CO2H)2, Na2CO3, water, 60–70 °C,
then H2SO4, 50 °C, 35–60%; (b) 3-bromo-4-hydroxybenzaldehyde, ammonium
acetate or NaOAc, AcOH, reflux, 50–85%.
Aromatase
Aromatase
O
OH
17 -HSD1
17 -HSD5
Ar1
Ar1
17 -HSD2
HO
HO
NCO
d,e
NH2
a
7
5
E1
E2
c,e
Figure 1. The role of 17b-HSD family in human steroidogenesis.
is involved in the conversion of
D
4-dione to T in the ovary.5 In addi-
O
O
O
Ar1
Ar1
tion, the expression of 17b-HSD3 mRNA in cancerous prostate
biopsies was found to be 30-fold higher than in normal tissue.6
Therefore, the role of 17b-HSD3 in T biosynthesis makes this en-
zyme an attractive molecular target of a small-molecule inhibitor
for the treatment of prostate cancer.7
N
b
N
O
S
O
6
8
b
2. Chemistry
We recently described the identification of 3-aryl-5-benzyli-
dene rhodanine 1a and oxazolidione 2a as a novel series of 17b-
HSD3 inhibitors that were developed in a hit-to-lead optimization
effort starting from the hit compound identified in an in-house
screening (Fig. 2).8 Herein, we report the preliminary structure–
activity relationship (SAR) studies on these chemotypes and the
improvement upon the pharmacokinetic (PK) properties of these
compounds, which led to the discovery of orally bioavailable, po-
tent 17b-HSD3 inhibitors.
O
O
Ar1
Ar1
N
N
O
O
Ar2
Ar2
O
S
2 : Ar1=R-Ph, Ar =3-Br-4-OH-Ph
11 : Ar1=4-MeO-Ph, Ar2=R-Ph
13 : Ar1=4-MeO-Ph, Ar2=R-Py
9 : Ar1=R-Ph, Ar =3-Br-4-OH-Ph
10 : Ar1=R-Py, Ar2=3-Br-4-OH-Ph
12 : Ar1=4-MeO-Ph, Ar2=R-Ph
2
2
An efficient protocol was applied to the generation of 3-substi-
tuted 5-arylidene rhodanines 1 (Scheme 1). Various commercially
available amines (RNH2, 3) were used as building blocks to prepare
a collection of 3,5-disubstituted rhodanine derivatives 1 in a
sequential two-step process combining the Holmberg method,
which is based on the reaction between bis(carboxymethyl)trithio-
carbonate and primary amines, and the Knoevenagel condensation
with a benzaldehyde.9,10 In all cases, the products were isolated in
high purity and in moderate yields after a simple precipitation
from ethanol, and the thermodynamically more stable Z-isomers
predominated (Z/E ratio P98/2). The cis-trans isomers were as-
signed as previously reported in the literature.11
Scheme 2. Synthesis of 3-aryloxazolidinone analogs 2 and 9–13. Reagents and
conditions: (a) BnSC(S)OCH2CO2K, water, rt, then, Ac2O, 100 °C, 35–60%; (b)
Ar2CHO, ammonium acetate or b-alanine, AcOH, reflux, 45–95%; (c) HOCH2CO2Me,
CDI, Et3N, THF, rt 50 °C, crude; (d) HOCH2CO2Me, DMF, rt 80 °C, crude; (e) NaOMe,
toluene, 100 °C, 35–85%.
dithiocarbonate, followed by intramolecular cyclization with acetic
anhydride. Arylamines 5 or arylisocyanates 7 were treated with
glycolate ester to give the corresponding carbamates. Then, addi-
tion of catalytic sodium methoxide in toluene, followed by azeotro-
pic removal of the generated alcohol under reflux, provided the
cyclized 3-substituted 2,4-oxazolidinediones 8. Knoevenagel con-
densation of the oxazolidinones (6 and 8) and aryl aldehydes affor-
ded the corresponding 5-arylidene derivatives 2 and 9–13.
Substitution on the bridge part was achieved in a similar man-
ner to the unsubstituted analogs, using ketones instead of alde-
hydes (Scheme 3). The stereoisomers of compounds 14a,b were
determined to be E-isomers by heteronuclear multiple bond con-
nectivity (HMBC) experiment (see Supplementary data). Moreover,
benzylrhodanine 15, which is structurally more flexible than com-
pound 1a, was synthesized by a direct alkylation of compound 4a
with magnesium methyl carbonate (MMC) and a subsequent
deprotection of TBDMS in situ.12
The biaryl-substituted oxazolidione analogs were prepared by
the synthetic procedure outlined in Scheme 2. 2-Thioxo-oxazolid-
inones 6 were prepared by treatment of arylamines 5 with
MeO
O
N
X
S
Br
Finally, a phosphate ester 16 was prepared according to the
convenient synthetic route described in Scheme 4, to study bio-
precursors of this series for the purpose of an exploration of prac-
tical 17b-HSD3 inhibitors. We chose to synthesize a target phos-
OH
1a, X=S
2a, X=O
Figure 2. Structure of lead compounds 1a and 2a.

3244
K. Harada et al. / Bioorg. Med. Chem. 20 (2012) 3242–3254
Table 1
OH
MeO
MeO
MeO
Biological activity of rhodanine analogs
O
S
O
a
Br
O
N
R
S
N
N
S
S
R
S
S
b
Br
4a
14a : R=Me
14b : R=CF3
OH
a
Compound
R
HSD IC50 (nM)
O
1a
1b
1c
1d
1e
1f
1g
1h
1i
4-MeO-Ph
H
Me
allyl
14
N
nab
890
120
300
190
3
S
S
Br
propagyl
MeOCH2CH2
cyclohexyl
tetrahydropyran-4-yl
1-methylpiperidin-4-yl
4-MeO-PhCH2
15
OH
75
na
90
Scheme 3. Synthesis of 5-benzylidene and 5-benzyl rhodanes 14 and 15. Reagents
and conditions: (a) 3-Bromo-4-hydroxybenzaldehyde, b-alanine, AcOH, reflux, 50–
85%; (b) 3-bromo-4-(t-butyldimethylsilyloxy)benzaldehyde, MeOMgOCO2Me, DMF,
80 °C, 42%.
1j
a
Human cell-based assay. HeLa cells transiently transfected with 17b-HSD3. The
deviations were within 5%.
b
Not active.
phate-prodrug via a dibenzyl phosphate, as the benzyl group as a
protecting group is readily removed under mild conditions. A con-
venient phosphorylation of the phenols with dibenzyl phosphite
proceeded rapidly in one pot and in moderate yield, using dibenzyl
phosphate, only reagent quantities of CCl4, and catalytic amounts
of N,N-dimethylaminopyridine (DMAP).13 The benzyl groups on
the aryl phosphates were selectively removed with trim-
ethylsilylbromide (TMSBr), providing silyl esters that were subse-
quently hydrolyzed with H2O to afford the corresponding
phosphate ester 16.14
(1h and 1i). A benzyl analog 1j showed about 6-fold weaker inhib-
itory activity than the original 1a. As a result, introduction of steri-
cally bulky and hydrophobic moieties at the 3-position on the
rhodanine, which is practically perpendicular to the substituents,
enhanced the enzymatic inhibitory activity. The corresponding
hydrophobic binding pocket in the enzyme is observed to affect a
key interaction with this chemotype.
Next, acceptable diversity of substituents on the N-phenyl ring
was investigated. The results are summarized in Table 2. With few
exceptions, the 17b-HSD3 inhibitory activity was retained to a sat-
isfactory level in a cell-based assay. Concerning the regioisomers of
p-methoxyphenyl derivative 2a, o-methoxyphenyl derivative 2d
showed reduced activity to about one-fifth of that of compound
2a; on the other hand, m-methoxyphenyl analog 2c continued to
exhibit good potency, as did 3,4-methylenedioxy derivative 2e.
With compounds 2f–2i, bearing a halo, an alkyl, and a haloalkyl
substituent at the C40 of the aromatic ring, the activity increased
significantly compared with compound 2a. The assay result of an
unsubstituted phenyl (2b) and a C40 electro-withdrawing substitu-
ent (2j) instead of the electro-donating methoxy group (2a) sug-
gested that the electrostatic properties of the aromatic ring have
no effect against the activity. Replacement of the 2-thioxo group
in the hetroring system of compound 2a to 2-oxo group (9a) pro-
vided similar potency. In consideration of further investigation
concerning the aqueous solubility and the bioavailability, the 2,4-
oxazolidinedione derivatives were expected to possess more suit-
able physicochemical properties than the 2-thioxo analogs. There-
fore, water-soluble substituents at the C40 of the phenyl ring as the
3. Results and discussion
The analogs were evaluated in a cell based assay, which mea-
sured the ability of the compounds to inhibit 17b-HSD3. The IC50
values were calculated for the inhibition at each concentration
(1, 10, 100 nM, and 1
tration of T converted from
l
M) of each compound, by measured concen-
D
4-dione with HeLa cells expressing
the human recombinant 17b-HSD3 enzyme.
The initial result for the inhibition of 17b-HSD3 by our 5-arylid-
ene rhodanines suggested that the nature of the substituent at the
3-position significantly affects the potency in a cell-based assay
(Table 1). Replacement of the p-methoxyphenyl group with alkyl
chains resulted in reduction of the activity, as revealed by a com-
parison of biological activities between 1a and 1c–1f. Additionally,
N-unsubstituted rhodanine 1b led to lack of cellular activity. On
the other hand, cyclohexyl derivative 1g exhibited single-digit
nanomolar potency (IC50 = 3 nM). However, more extended incor-
poration of heteroatoms such as oxygen and nitrogen at the 4-po-
sition of the cyclohexyl group was found to decrease the potency
MeO
MeO
O
O
N
N
a,b
O
O
S
S
Cl
Cl
O
Cl
OH
Cl
O
P
OH
OH
11g
16
Scheme 4. Synthesis of a phosphate ester derivative 16. Reagents and conditions: (a) HP(O)(OBz)2, CCl4, DMAP, DIEA, DMF, ꢀ10 °C rt, 61%; (b) Me3SiBr, CHCl3, rt, then 1 N
HCl, rt, 43%.

K. Harada et al. / Bioorg. Med. Chem. 20 (2012) 3242–3254
3245
Table 2
Table 3
Biological activity of N-phenyl analogs
Biological activity of N-heteroaryl analogs
R
A
O
B
O
O
R
N
N
O
O
X
Br
Br
OH
OH
a
Compound
R
X
HSD IC50 (nM)
a
Compound
R
A
B
HSD IC50 (nM)
2a
2b
2c
2d
2e
2f
2g
2h
2i
4-OMe
H
3-OMe
2-OMe
3,4-OCH2O-
4-F
4-Cl
4-Me
4-CF3
4-CN
4-OMe
4-CO2H
4-CO2Me
4-C(O)NHMe
4-C(O)NMe2
4-C(O)-piperizine
4-C(O)-morphorine
4-NMe2
4-NHAc
4-NHCO-(c-hex)
4-SMe
4-SO2Me
4-SO2NH2
S
S
S
S
S
S
S
S
13
6
7
60
12
2
6
2
3
19
23
nab
24
56
65
42
300
25
57
21
90
900
na
9a
OMe
H
H
OMe
Me
F
Cl
NMe2
CH
CH
N
N
N
N
N
N
CH
N
23
74
22
4
67
21
20
4
10a
10b
10c
10d
10e
10f
10g
CH
CH
CH
CH
CH
CH
S
S
a
2j
Human cell-based assay. HeLa cells transiently transfected with 17b-HSD3. The
deviations were within 5%.
9a
9b
9c
9d
9e
9f
9g
9h
9i
O
O
O
O
O
O
O
O
O
O
O
O
O
Table 4
Biological activity of rhodanine analogs
MeO
O
9j
9k
9l
R
N
S
A
B
9m
S
Br
a
Human cell-based assay. HeLa cells transiently transfected with 17b-HSD3. The
deviations were within 5%.
b
OH
Not active.
Compound
A–B
R
HSD IC50 (nM)a
2,4-oxazolidinedione derivatives were sequentially evaluated for
the purpose of improvement of undesirable physical properties
in this series. Compounds bearing an ester (9c), amides (9d–9f),
an alkylamino (9h), retro-amides (9i and 9j), and a thioether (9k)
were found to show favorable biological activities. However, chan-
ged substituents such as the (4-morpholinyl)carbonyl group in 9g,
as well as the methylsulfonyl group in 9l, seemed not to be toler-
ated. In addition, introduction of acidic functionalities such as a
carboxyl (9b) and a sulfamoyl (9m) resulted in complete loss of
the 17b-HSD3 inhibitory activity.
With further modification on the N-aromatic ring, the pyridyl
analogs were studied as shown in Table 3. 30-Pyridyl derivative
10b showed more potent inhibitory activity than 20-pyridyl deriv-
ative 10a. When we introduced several substituents onto the pyr-
idine, a C60 methoxy and dimethylamino substituent of the 30-
pyridyl ring (10c and 10g) provided a substantial boost in potency,
possessing IC50 values of 4 nM for each. The compounds 10d–10f,
bearing a fluoro, a chloro, and a methyl substituent at the C60 of
the 30-pyridyl ring, showed similar activities compared with the
unsubstituted 10b. This SAR trend for the N-pyridyl series is differ-
ent from that observed for the N-phenyl series, where the presence
of a halo substituent and an alkyl group is likewise beneficial for
17b-HSD3 inhibitory activity.
1a
C@C
C@C
C@C
CH–CH
H
14
90
nab
90
14a
14b
15
Me
CF3
H
a
Human cell-based assay. HeLa cells transiently transfected with 17b-HSD3. The
deviations were within 5%.
b
Not active.
Finally, our efforts focused on the optimization of the phenol
moiety. As an initial result, the phenolic hydroxyl group at the
C40 was found to be essential, because replacement of the hydroxyl
group with various functional groups such as hydrogen bonding
donor/acceptor (an amino, a carboxyl, an amide, a cyano, and a flu-
oro substituent) resulted in a precipitous drop in enzymatic activ-
ity (data not shown). Next, substitution at in the C30 and C50
positions was evaluated. The results are summarized in Table 5.
Replacement of the bromine atom with a methyl group (11b)
was observed to keep the potency. The unsubstituted analog 11a
reduced the activity, and 30-methoxy substitution (11c) was less
tolerated. In contrast, the compounds 11d–11g with mono- or di-
halo substituents on the aromatic ring resulted in improvement
of the activity; in particular, 30-chloro-50-fluoro-40-hydroxylphenyl
derivative 11i demonstrated the best 17b-HSD3 inhibitory activity
in a cell-based assay, possessing an IC50 value of 1 nM. Surprisingly,
an abrupt drop of potency was observed in the case of X = O, 30,50-
dichloro derivative 12d compared with 11g. In further exploration,
introduction of substituents like a cyano in 12a, a carboxyl in 12b,
and (N,N-dimethylamino)methyl in 12c, were found not to be tol-
erated, and the inhibitory activities were lost. We speculated that
the optimal size and the lipophilic effect of R influenced the activ-
ity, and that the intermolecular H-bonding between the 40-hydro-
An influence of the bridge part on the activity was summarized
in Table 4. The introduction of a methyl and a trifluoromethyl
group at the benzylic position was unfavorable, giving consider-
ably decreased potency due to the predominance of the thermody-
namically more stable E-isomer (14a and 14b). The substitution at
the bridge part made the configuration convert from Z-isomer to E-
isomer, and even the size of the methyl group is critical to the
activity. In addition, more flexible benzyl analog 15 was found to
be a weak inhibitor, with an IC50 value of 90 nM.

3246
K. Harada et al. / Bioorg. Med. Chem. 20 (2012) 3242–3254
Table 5
Biological activity of oxazolidinone analogs
MeO
O
N
1
O
R
3
A
B
X
6
5
Compound
R
A
B
X
HSD IC50 (nM)a
2a
3-Br, 4-OH
4-OH
3-Me, 4-OH
3-OMe, 4-OH
3-F, 4-OH
3-Cl, 4-OH
2-Cl, 4-OH
3,5-DiCl, 4-OH
3,5DiF, 4-OH
3-Cl-5-F, 4-OH
3-CN, 4-OH
3-CO2H, 4-OH
3-CH2NMe2, 4-OH
3,5DiCl, 4-OH
4-OH
CH
CH
CH
CH
CH
CH
CH
CH
C
CH
CH
CH
CH
CH
N
C
C
C
C
C
C
C
C
CH
C
C
C
C
S
S
S
S
S
S
S
S
S
S
O
O
O
O
S
S
13
121
20
270
2
4
5
12
5
1
11a
11b
11c
11d
11e
11f
11g
11h
11i
12a
12b
12c
12d
13a
13b
nab
na
na
na
90
42
C
C
C
3-Br, 4-OH
N
a
Human cell-based assay. HeLa cells transiently transfected with 17b-HSD3. The
deviations were within 5%.
b
Not active.
xyl and the 30-substituent prevented interaction with a key amino
acid residue in the pore region of the enzyme and induced a signif-
icant loss of the activity. The 17b-HSD3 inhibitory activity of pyri-
dine derivative 13a was observed to be reduced to about one-third
of that of compound 2a (IC50 = 42 nM). In comparison between 13a
and 13b, this pyridyl analog was intuitively found to have a similar
tendency to the phenyl analog on SAR.
In general, these compounds did not show any undesired inhi-
bition of the oxidative isoenzyme 17b-HSD type 2 or 17b-HSD type
1, which convert the less potent estrogen estrone (E1) into the bio-
logically active estradiol (E2). In selectivity over nuclear receptors
Figure 4. (A) Plasma concentration of compound 2a and 2a-glucuronide when
dosed 2a, and compound 11g when dosed 11g after oral administration (10 mg/kg)
to male SD rats. (B) Plasma concentration of compound 11g when dosed 11 g or its
prodrug 16 after oral administration (10 mg/kg) to male SD rats. Each point with
vertical bar represents the mean standard error of the mean (SEM) (n = 3).
apparently not sufficient to enable detection of in vivo efficacy in
the LH–RH-induced testosterone production assay.15 Therefore,
we investigated the development of bio-precursor, chemically
modified versions of the pharmacologically active agent that must
undergo transformation in vivo to release the active drug.
Phosphate ester prodrugs, designed for hydroxyl and amine
functionalities of poorly water-soluble drugs with an aim to en-
hance their aqueous solubility to allow a more favorable oral or
parenteral administration, typically display excellent or adequate
chemical stability and rapid bioconversion back to the parent drug
by phosphatases present in the intestinal brush border or in the li-
ver.16 Therefore, we studied bioavailability of phosphate esters of
selected compounds. The strategy for enhanced bioavailability is
depicted in Figure 3. After compound 11g was converted to the
phosphate ester derivative 16, the oral bioavailability was exam-
ined by measuring the plasma concentration of the parent com-
pound 11g. As shown in Table 6, compound 16 demonstrated a
significantly improved oral bioavailability compared to the parent
such as androgen receptor (AR), estrogen receptor alpha (ERa), and
glucocorticoid receptor (GR), intersection with the nuclear recep-
tors was also not observed for these compounds. These chemo-
types showed excellent selectivity over 17b-HSD isoenzymes and
nuclear receptors (see previous report8).
Before examining androgen-lowering effect of the selected ori-
ginal 2a, we confirmed the oral bioavailability of this compound
(Fig. 4). Compound 2a was obviously not bioavailable in preclinical
species, owing to its ‘brickdust’ nature, with a solubility of less
than 1 lg per mL and a high clearance based on glucuronic acid
conjugation. Although for 30,50-dichloro analog (11g), improved
oral absorption to the systemic circulation without the glucuronic
acid conjugation was observed, the quantity in plasma was
O
R
N
O
O
O
O
R
R
N
N
Disubstitution (R, R')
Phosphate-esterification
O
S
R
O
S
S
Br
R
O
P
OH
R'
OH
OH
R'
OH
for protection against glucuronic acid conjugation
for improved aqueous solubility and oral absorption
Figure 3. Chemical modification-strategy for enhanced bioavailability.

K. Harada et al. / Bioorg. Med. Chem. 20 (2012) 3242–3254
3247
Table 6
levels in a dose-dependent manner (Fig. 5). When compound 16
was administered orally at a high dose of 100 mg/kg, 16 showed
approximately two times more potent T-lowering effect against
the positive control. The T-lowering effect continued at ca 10% le-
vel of control over 4 h after administration.
Phamacokinetic parameters of 11g and 16
Compound
11g
16
Tmax (h)
Cmax
AUC (
F (%)
1
1
5.25
18.8
35.1
(
l
l
g/mL)
0.40
4.0
5.5
g h/mL)
4. Conclusions
Tmax, Cmax, and AUC values were calculated from the plasma concentration after
10 mg/kg of 11g or 16 were administered orally to male SD rats. F values were
calculated by comparing the AUC in po study with the dose-corrected AUC values of
intravenously administered rat plasma at 0.1 mg/kg.
17b-HSD3. is an attractive biological target inhibitor for treating
and preventing hormone-dependent disorders such as prostate
cancer. In our previous work, several rhodanine- and oxazolidi-
none-based compounds were found to display not only potent
and selective inhibition but also reduced androgenic effects.7 An
efficient synthetic route was developed for the seed compound
and subsequently utilized in the synthesis of molecule libraries
based on rhodanine and oxazolidinone cores. The SAR studies on
the 3,5-disubstituted rhodanines and oxazolidinones culminated
in the discovery of novel, highly potent, and selective inhibitors
of 17b-HSD3. For example, 5-(3-chloro-5-fluoro-4-hydroxybenzy-
lidene)-3-(4-methoxyphenyl)-2-thioxo-1,3-oxazolidin-4-one 11i
is one of the most potent 17b-HSD3 inhibitors reported to date,
with an IC50 value of 1.0 nM (human cell-based assay). Although
representative compounds exhibited promising activity profiles,
the compounds displayed very low aqueous solubility and poor
PK profiles characterized by a lack of oral bioavailability. Therefore,
we investigated the development of phosphate ester prodrugs with
an aim to enhance their aqueous solubility to allow a more favor-
able oral administration. In this strategy, prodrug 16 demonstrated
a significantly improved oral bioavailability compared to the par-
ent 11g. When compound 16 was administered orally to male
Sprague–Dawley rats, 16 reduced plasma T levels in a dose-depen-
dent manner, and showed approximately two times more potent
T-lowering effect against the positive control. The T-lowering ef-
fect continued at ca 10% level of control over 4 h after administra-
tion. Preliminary biological evaluation in this study suggests that
the nonsteroidal molecules based on this series have the potential
to provide effective clinical opportunities for treatment of prostate
cancer.
5. Experimental section
NMR spectra were recorded on a JEOL EX-270 or Varian Unity
Inova 400 spectrometer. Chemical shifts are given in parts per mil-
lion (ppm) downfield from internal reference tetramethylsilane in
d units. Mass spectra were measured with an LCQ LC/MS (Thermo
Fisher Scientific) system for electro-spray ionization. Elemental
analysis was performed on a CE Instruments EA1110 elemental
analyzer. All solvents and reagents were obtained from commercial
sources and were used without further purification. Column chro-
matography was performed on Merck 230–400 mesh silica gel.
Analytical thin layer chromatography (TLC) was performed using
Merck 60-F-254 0.25 mm precoated silica gel plates.
Figure 5. (A) Effects of compound 16 (po, 20 and 100 mg/kg) and a positive control
CB7630 (po, 20 mg/kg) on the concentrations of testosterone (T) in plasma, after the
administration of LH–RH agonist to male SD rats. Each point with vertical bar
represents the mean standard error of the mean (SEM) (n = 4 or 5). (B) The effect
of each compound on the concentrations of T in plasma is represented as % of
control.
phenol 11g. The pharmacokinetic parameters revealed that the
phosphate ester was more druggable than the parent phenol, as
indicated by Cmax and area under the curve (AUC)0–24 h at 10 mg/
kg (16; Cmax = 5.25
l
g/mL, AUC0–24 h = 18.8
g h/mL).
lg h/mL versus 11g;
5.1. N-Substituted 2-thioxo-1,3-thiazolidin-4-ones 4: general
procedure 1
Cmax = 0.40 g/mL, AUC0–24 h = 4.0
l
l
Based on the results of pre-examination, we used the phosphate
ester 16 for in vivo evaluation to demonstrate in vivo proof-of-con-
cept for this series of 17b-HSD3 inhibitors. Compound 16 was eval-
uated in terms of inhibition of a LH–RH-induced testosterone
steroidogenesis. In this in vivo experiment, CB7630 (Abiraterone
A solution of trithiocarbonyl diglycolic acid (1.0 equiv) and 1,10-
carbonyldiimidazole (2.0 equiv) in THF (10 mL/mmol) was stirred
at room temperature for 1.5 h. An amine 3 (1.0 equiv) was then
added, and the mixture was refluxed for 4 h. A 2 M HCl solution
(30 mL/mmol) was added, and the mixture was extracted with
ethyl acetate. The combined organic layers were dried over anhy-
drous magnesium sulfate and concentrated in vacuo. The residue
was purified by silica gel column chromatography, eluted with
ethyl acetate/hexane to afford the title product (4).
acetate), which is a steroidal cytochrome P450 17a-hydroxylase-
17,20-lyase (CYP17) inhibitor approved by FDA as a drug for the
treatment of androgen-dependent prostate cancer, was used a po-
sitive control. Compared to CB7630 given orally at a dose of 20 mg/
kg to male Sprague–Dawley rats, compound 16 reduced plasma T

3248
K. Harada et al. / Bioorg. Med. Chem. 20 (2012) 3242–3254
5.1.1. Knoevenagel condensation: general procedure 2
To a solution of 3-substituted 2-thioxo-1,3-thiazolidin-4-one 4
(1.0 equiv) and 3-bromo-4-hydroxybenzaldehyde (1.0 equiv) in
acetic acid (4 mL/mmol) was added ammonium acetate (2.0 equiv)
or b-alanine (4.0 equiv). Then the mixture was stirred at 120 °C for
4 h. After cooling, the precipitate was collected by filtration and
washed with water. The solid was triturated with ethanol to afford
the title product (1).
5.1.1.7. 5-(3-Bromo-4-hydroxybenzylidene)-3-cyclohexyl-2-thi-
oxo-1,3-thiazolidin-4-one (1g). Compound 1g was prepared
following: general procedures 1 and 2. Yield 43%; 1H NMR
(270 MHz, CDCl3) d 1.24–1.92 (m, 10H), 4.96–5.05 (m, 1H), 5.91
(s, 1H), 7.11 (d, J = 8.6 Hz, 1H), 7.37 (dd, J = 2.2, 8.4 Hz, 1H), 7.59
(s, 1H), 7.61 (d, J = 2.4 Hz, 1H); MS (ESI) m/z 399 (MH), 401
(MH+2); Anal. Calcd for C16H16BrNO2S2: C, 48.25; H, 4.05; N,
3.52. Found: C, 48.20; H, 4.16; N, 3.51.
5.1.1.1.
5-(3-Bromo-4-hydroxybenzylidene)-3-(4-methoxy-
Compound 1a
5.1.1.8.
5-(3-Bromo-4-hydroxybenzylidene)-3-(tetrahydropy-
Compound
phenyl)-2-thioxo-1,3-thiazolidin-4-one (1a).
ran-4-yl)-2-thioxo-1,3-thiazolidin-4-one (1h).
was prepared following: general procedures 1 and 2. Yield 86%;
1H NMR (270 MHz, DMSO-d6) d 3.81 (s, 3H), 7.07 (d, J = 9.2 Hz,
2H), 7.13 (d, J = 8.6 Hz, 1H), 7.29 (d, J = 8.9 Hz, 2H), 7.51 (dd,
J = 2.2, 8.4 Hz, 1H), 7.72 (s, 1H), 7.87 (d, J = 2.2 Hz, 1H), 11.31 (s,
1H); MS (ESI) m/z 423 (MH), 425 (MH+2); Anal. Calcd for
1h was prepared following: general procedures 1 and 2. Yield
35%; 1H NMR (270 MHz, DMSO-d6) d 1.60 (d, J = 12.0 Hz, 2H),
2.60 (dd, J = 8.1, 16.5 Hz, 2H), 3.40 (d, J = 12.0 Hz, 2H), 3.98 (dd,
J = 4.2, 11.2 Hz, 2H), 5.10–5.20 (m, 1H), 7.12 (d, J = 8.6 Hz, 1H),
7.47 (dd, J = 2.2, 8.4 Hz, 1H), 7.67 (s, 1H), 7.85 (d, J = 2.2 Hz, 1H),
11.32 (s, 1H); MS (ESI) m/z 401 (MH), 403 (MH+2); Anal. Calcd
for C15H14BrNO3S2: C, 45.01; H, 3.53; N, 3.50. Found: C, 45.04; H,
3.70; N, 3.42.
C17H12BrNO3S2: C, 48.35; H, 2.87; N, 3.32. Found: C, 48.30; H,
2.98; N, 3.31.
5.1.1.2. 5-(3-Bromo-4-hydroxybenzylidene)-2-thioxo-1,3-thiaz-
olidin-4-one (1b).
Compound 1b was prepared from a com-
5.1.1.9. 5-(3-Bromo-4-hydroxybenzylidene)-3-(1-methylpiperi-
mercially available reagent, rhodanine 4b following: general
procedure 2. Yield 92%; 1H NMR (270 MHz, DMSO-d6) d 7.11 (d,
J = 8.4 Hz, 1H), 7.44 (dd, J = 2.2, 8.6 Hz, 1H), 7.56 (s, 1H), 7.80 (d,
J = 2.2 Hz, 1H), 11.31 (brs, 1H), 13.76 (brs, 1H); MS (ESI) m/z 317
(MH), 319 (MH+2); Anal. Calcd for C10H6BrNO2S2: C, 37.99; H,
1.91; N, 4.43. Found: C, 37.96; H, 1.91; N, 4.45.
din-4-yl)-2-thioxo-1,3-thiazolidin-4-one (1i).
Compound 4i
was prepared following: general procedures 1 and 2. Yield 12%;
1H NMR (270 MHz, DMSO-d6) d 1.85–1.92 (m, 2H), 2.46–2.56 (m,
4H), 2.57 (s, 3H), 2.67–2.78 (m, 2H), 4.09–4.19 (m, 1H), 6.60 (s,
1H), 6.88 (d, J = 8.6 Hz, 1H), 7.33 (d, J = 8.9 Hz, 1H), 7.47 (d,
J = 2.2 Hz, 1H); MS (ESI) m/z 414 (MH), 416 (MH+2); Anal. Calcd
for C16H17BrN2O2S2: C, 46.49; H, 4.15; N, 6.78. Found: C, 46.46;
H, 4.15; N, 6.80.
5.1.1.3. 5-(3-Bromo-4-hydroxybenzylidene)-3-methyl-2-thioxo-
1,3-thiazolidin-4-one (1c).
Compound 1c was prepared from
a commercially available reagent, 3-methyl-2-thioxo-1,3-thiazoli-
din-4-one 4c following: general procedure 2. Yield 91%; 1H NMR
(270 MHz, DMSO-d6) d 3.39 (s, 3H), 7.12 (d, J = 8.6 Hz, 1H), 7.48
(dd, J = 2.4, 8.6 Hz, 1H), 7.73 (s, 1H), 7.85 (d, J = 2.2 Hz, 1H), 11.31
(s, 1H); MS (ESI) m/z 331 (MH), 333 (MH+2); Anal. Calcd for
5.1.1.10. 5-(3-Bromo-4-hydroxybenzylidene)-3-(4-methoxyben-
zyl)-2-thioxo-1,3-thiazolidin-4-one (1j).
Compound 1j was
prepared following: general procedures 1 and 2. Yield 75%; 1H
NMR (270 MHz, CDCl3) d 3.72 (s, 3H), 5.16 (s, 2H), 6.88 (d,
J = 8.6 Hz, 2H), 7.12 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 8.6 Hz, 2H),
7.49 (dd, J = 1.6, 8.4 Hz, 1H), 7.76 (s, 1H), 7.86 (d, J = 1.6 Hz, 1 Hz),
11.35 (s, 1H); MS (ESI) m/z 437 (MH), 439 (MH+2); Anal. Calcd
for C18H14BrNO3S2: C, 49.55; H, 3.24; N, 3.21. Found: C, 49.54; H,
3.19; N, 3.33.
C11H8BrNO2S2: C, 40.01; H, 2.44; N, 4.24. Found: C, 40.21; H,
2.28; N, 3.96.
5.1.1.4.
3-Allyl-5-(3-bromo-4-hydroxybenzylidene)-2-thioxo-
1,3-thiazolidin-4-one (1d).
Compound 1d was prepared
from a commercially available reagent, 3-allyl-2-thioxo-1,3-thiaz-
olidin-4-one 4d following: general procedure 2. Yield 54%; 1H
NMR (270 MHz, DMSO-d6) d 4.64 (d, J = 5.1 Hz, 2H), 5.09–5.20
(m, 2H), 5.77–5.92 (m, 1H), 7.12 (d, J = 8.4 Hz, 1H), 7.50 (dd,
J = 2.3, 8.5 Hz, 1H), 7.75 (s, 1H), 7.87 (d, J = 2.2 Hz, 1H), 11.35 (s,
1H); MS (ESI) m/z 357 (MH), 359 (MH+2); Anal. (C13H10BrNO2S2):
Anal. Calcd for C13H10BrNO2S2: C, 43.83; H, 2.83; N, 3.93. Found:
C, 43.86; H, 3.00; N, 3.85.
5.1.1.11. N-Substituted 2-thioxo-1,3-oxazolidin-4-ones
general procedure 3. A solution of {[(benzylsulfanyl)carbo-
(6):
nothionyl]oxy} acetic acid potassium salt (1.0 equiv) and an aryl
amine 5 (1.0 equiv) in water (1 mL/mmol) was stirred at room
temperature for 5 h. The mixture was washed with benzyl methyl
ether, the aqueous layers acidified with 2 M HCl solution to pH 3.
The precipitate was collected by filtration. The solid was extracted
with chloroform, and the organic layers were washed with brine
and dried over anhydrous magnesium sulfate. After filtration, the
solvent was removed in vacuo, and then the residue was treated
with acetic anhydride (1.2 equiv) in acetic acid (1.0 mL/mmol) at
100 °C for 2 h. The mixture was concentrated in vacuo, and the res-
idue was purified by silica gel column chromatography, eluted
with ethyl acetate/hexane to afford the title product (6).
5.1.1.5. 5-(3-Bromo-4-hydroxybenzylidene)-3-propagyl-2-thi-
oxo-1,3-thiazolidin-4-one (1e).
Compound 1e was prepared
following: general procedures 1 and 2. Yield 30%; 1H NMR
(270 MHz, DMSO-d6) d 3.26 (t, J = 2.4 Hz, 1H), 4.76 (d, J = 2.4 Hz,
2H), 7.11 (d, J = 8.4 Hz, 1H), 7.49 (dd, J = 2.2, 8.6 Hz, 1H), 7.78 (s,
1H), 7.87 (d, J = 2.2 Hz, 1H), 11.36 (brs, 1H); MS (ESI) m/z 355
(MH), 357 (MH+2); Anal. Calcd for C13H8BrNO2S2: C, 44.08; H,
2.28; N, 3.96. Found: C, 44.27; H, 2.13; N, 3.71.
5.1.1.12. N-Substituted 1,3-oxazolidin-2,4-diones (8): general
procedure 4–1.
To a solution of ethyl glycolate (1.0 equiv)
and DMF (1 mL/mmol) was added an isocyanate (0.91 equiv) at
0 °C. The mixture was then stirred at 80 °C for 3 h, and the reaction
was quenched by adding water (5 mL/mmol). The precipitate was
collected by filtration. The solid was extracted with ethyl acetate,
and the organic layers were washed with brine and dried over
anhydrous magnesium sulfate. After filtration, the solvent was re-
moved in vacuo, and the residue was purified by silica gel column
chromatography, eluted with ethyl acetate/hexane to give ethyl
[(arylcarbamoyl)oxy]acetate. A mixture of the ethyl [(arylcarba-
5.1.1.6. 5-(3-Bromo-4-hydroxybenzylidene)-3-(methoxyethyl)-
2-thioxo-1,3-thiazolidin-4-one (1f).
Compound 1f was pre-
pared following: general procedures 1 and 2. Yield 31%; 1H NMR
(270 MHz, CDCl3) d 3.37 (s, 3H), 3.72 (t, J = 5.8 Hz, 2H), 4.36 (t,
J = 5.8 Hz, 2H), 5.98 (s, 1H), 7.11 (d, J = 8.6 Hz, 1H), 7.38 (dd,
J = 2.2, 8.6 Hz, 1H), 7.59 (s, 1H), 7.63 (d, J = 2.2 Hz, 1H); MS (ESI)
m/z 375 (MH), 377 (MH+2); Anal. Calcd for C13H12BrNO3S2: C,
41.72; H, 3.23; N, 3.74. Found: C, 41.74; H, 3.39; N, 3.65.

K. Harada et al. / Bioorg. Med. Chem. 20 (2012) 3242–3254
3249
moyl)oxy]acetate (1.0 equiv) and sodium methoxide (0.03 equiv)
in toluene (2 mL/mmol) was stirred under reflux for 1 h. After cool-
ing, the precipitate was collected by filtration and purified by silica
gel column chromatography, eluted with ethyl acetate/hexane to
afford the title product (8).
1H), 7.39–7.58 (m, 4H), 7.83 (dd, J = 2.2, 8.6 Hz, 1H), 8.11 (d,
J = 1.9 Hz, 1H), 11.22 (s, 1H); MS (ESI) m/z 395 (MH), 397
(MH+2); Anal. Calcd for C16H9BrFNO3S: C, 48.74; H, 2.30; N, 3.55.
Found: C, 48.81; H, 2.21; N, 3.51.
5.1.1.20.
5-(3-Bromo-4-hydroxybenzylidene)-3-(4-chloro-
Compound 2g
5.1.1.13. N-Substituted 1,3-oxazolidin-2,4-diones (8): general
phenyl)-2-thioxo-1,3-oxazolidin-4-one (2g).
procedure 4–2.
To a solution of ethyl glycolate (1.0 equiv) in
was prepared following: general procedures 2 and 3. Yield 46%;
1H NMR (270 MHz, DMSO-d6) d 6.98 (s, 1H), 7.13 (d, J = 8.6 Hz,
1H), 7.51–7.56 (m, 2H), 7.63–7.68 (m, 2H), 7.83 (dd, J = 2.2,
8.6 Hz, 1H), 8.11 (d, J = 1.9 Hz, 1H), 11.23 (s, 1H); MS (ESI) m/z
411 (MH), 413 (MH+2); Anal. Calcd for C16H9BrClNO3S: C, 46.79;
H, 2.21; N, 3.41. Found: C, 46.74; H, 2.32; N, 3.40.
THF (1 mL/mmol) was added 1,1’-carbonyldiimidazole (1.1 equiv)
at 0 °C, and the mixture was stirred at room temperature for
0.5 h. A solution of an aryl amine 5 (1.0 equiv) and triethylamine
(0.7 equiv) in THF (1 mL/mmol) was then added, and the mixture
was stirred under reflux for 5 h. After cooling, the mixture was con-
centrated in vacuo. The residue was purified by silica gel column
chromatography, eluted with ethyl acetate/hexane to afford the ti-
tle product (8).
5.1.1.21.
5-(3-Bromo-4-hydroxybenzylidene)-3-(4-methyl-
Compound 2h
phenyl)-2-thioxo-1,3-oxazolidin-4-one (2h).
was prepared following: general procedures 2 and 3. Yield 32%;
1H NMR (270 MHz, DMSO-d6) d 2.29 (s, 3H), 6.95 (s, 1H), 7.13 (d,
J = 8.6 Hz, 1H), 7.35 (brs, 4H), 7.82 (dd, J = 2.0, 8.5 Hz, 1H), 8.11
(d, J = 1.9 Hz, 1H), 11.20 (s, 1H); MS (ESI) m/z 391 (MH), 393
(MH+2); Anal. Calcd for C17H12BrNO3S: C, 52.31; H, 3.10; N, 3.59.
Found: C, 52.51; H, 2.94; N, 3.31.
5.1.1.14.
5-(3-Bromo-4-hydroxybenzylidene)-3-(4-methoxy-
Compound 2a
phenyl)-2-thioxo-1,3-oxazolidin-4-one (2a).
was prepared following: general procedures 2 and 3. Yield 70%;
1H NMR (270 MHz, DMSO-d6) d 3.82 (s, 3H), 6.94 (s, 1H), 7.08–
7.15 (m, 3H), 7.39 (d, J = 8.9 Hz, 2H), 7.82 (dd, J = 2.2, 8.6 Hz, 1H),
8.10 (d, J = 2.2 Hz, 1H), 11.20 (s, 1H); MS (ESI) m/z 407 (MH), 409
(MH+2); Anal. Calcd for C17H12BrNO4S: C, 50.25; H, 2.98; N, 3.45.
Found: C, 50.43; H, 2.96; N, 3.27.
5.1.1.22.
5-(3-Bromo-4-hydroxybenzylidene)-3-(4-trifluoro-
(2i). Com-
methylphenyl)-2-thioxo-1,3-oxazolidin-4-one
pound 2i was prepared following: general procedures 2 and 3.
Yield 25%; 1H NMR (270 MHz, DMSO-d6) d 7.01 (s, 1H), 7.15 (d,
J = 8.6 Hz, 1H), 7.83–8.05 (m, 5H), 8.12 (d, J = 2.2 Hz, 1H), 11.24
(s, 1H); MS (ESI) m/z 445 (MH), 447 (MH+2); Anal. Calcd for
5.1.1.15.
oxo-1,3-oxazolidin-4-one (2b).
5-(3-Bromo-4-hydroxybenzylidene)-3-phenyl-2-thi-
Compound 2b was prepared
following: general procedures 2 and 3. Yield 66%; 1H NMR
(270 MHz, DMSO-d6) d 6.97 (s, 1H), 7.13 (d, J = 8.6 Hz, 1H), 7.47–
7.61 (m, 5H), 7.83 (dd, J = 2.0, 8.5 Hz, 1H), 8.11 (d, J = 2.2 Hz, 1H);
MS (ESI) m/z 377 (MH), 379 (MH+2); Anal. Calcd for C16H10BrNO3S:
C, 51.07; H, 2.68; N, 3.72. Found: C, 51.11; H, 2.71; N, 3.61.
C17H9BrF3NO3S: C, 45.95; H, 2.04; N, 3.15. Found: C, 45.98; H,
2.21; N, 3.07.
5.1.1.23.
5-(3-Bromo-4-hydroxybenzylidene)-3-(4-cyano-
Compound 2j
phenyl)-2-thioxo-1,3-oxazolidin-4-one (2j).
5.1.1.16.
5-(3-Bromo-4-hydroxybenzylidene)-3-(3-methoxy-
Compound 2c
was prepared following: general procedures 2 and 3. Yield 63%;
1H NMR (270 MHz, DMSO-d6) d 7.01 (s, 1H), 7.14 (d, J = 8.4 Hz,
1H), 7.74 (d, J = 8.1 Hz, 2H), 7.84 (dd, J = 1.4, 8.4 Hz, 1H), 8.09 (d,
J = 8.4 Hz, 2H), 8.12 (d, J = 1.9 Hz, 1H); MS (ESI) m/z 402 (MH),
404 (MH+2); Anal. Calcd for C17H9BrN2O3S: C, 50.88; H, 2.26; N,
6.99. Found: C, 50.59; H, 2.21; N, 6.92.
phenyl)-2-thioxo-1,3-oxazolidin-4-one (2c).
was prepared following: general procedures 2 and 3. Yield 45%;
1H NMR (270 MHz, DMSO-d6) d 3.78 (s, 3H), 6.95 (s, 1H), 7.03–
7.14 (m, 4H), 7.47 (t, J = 8.5 Hz, 1H), 7.82 (dd, J = 2.2, 8.6 Hz, 1H),
8.11 (d, J = 2.2 Hz, 1H); MS (ESI) m/z 407 (MH), 409 (MH+2); Anal.
Calcd for C17H12BrNO4S: C, 50.25; H, 2.98; N, 3.45. Found: C, 50.45;
H, 2.82; N, 3.17.
5.1.1.24.
5-(3-Bromo-4-hydroxybenzylidene)-3-(4-methoxy-
Compound 9a was
phenyl)-1,3-oxazolidin-2,4-dione (9a).
5.1.1.17.
5-(3-Bromo-4-hydroxybenzylidene)-3-(2-methoxy-
Compound 2d
prepared following: general procedure 2 and 4–1. Yield 30%; 1H
NMR (270 MHz, DMSO-d6) d 3.81 (s, 3H), 6.89 (s, 1H), 7.03–7.12
(m, 3H), 7.41 (d, J = 9.2 Hz, 2H), 7.73 (dd, J = 2.2, 8.6 Hz, 1H), 8.02
(d, J = 2.2 Hz, 1H), 11.04 (s, 1H); MS (ESI) m/z 391 (MH), 393
(MH+2); Anal. Calcd for C17H12BrNO5: C, 52.31; H, 3.10; N, 3.59.
Found: C, 52.32; H, 2.96; N, 3.63.
phenyl)-2-thioxo-1,3-oxazolidin-4-one (2d).
was prepared following: general procedures 2 and 3. Yield 66%;
1H NMR (270 MHz, DMSO-d6) d 3.79 (s, 3H), 7.00 (s, 1H), 7.13 (d,
J = 8.6 Hz, 2H), 7.26 (d, J = 7.6 Hz, 2H), 7.43 (dd, J = 1.8, 7.7 Hz,
1H), 7.83 (dd, J = 2.2, 8.6 Hz, 1H), 8.11 (d, J = 2.2 Hz, 1H); MS (ESI)
m/z 407 (MH), 409 (MH+2); Anal. Calcd for C17H12BrNO4S: C,
50.25; H, 2.98; N, 3.45. Found: C, 50.17; H, 3.15; N, 3.48.
5.1.1.25. 5-(3-Bromo-4-hydroxybenzylidene)-3-(4-methoxycar-
bonylphenyl)-1,3-oxazolidin-2,4-dione (9c).
Compound 9c
5.1.1.18. 3-(1,3-Benzodioxol-5-yl)-5-(3-bromo-4-hydroxybenzy-
was prepared following: general procedure 2 and 4–1. Yield 42%;
1H NMR (270 MHz, DMSO-d6) d 3.90 (s, 3H), 6.95 (s, 1H), 7.10 (d,
J = 8.4 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.9 Hz, 1H),
8.04 (d, J = 1.7 Hz, 1H), 8.14 (d, J = 8.4 Hz, 2H), 11.09 (s, 1H); MS
(ESI) m/z 419 (MH), 421 (MH+2); Anal. (C18H12BrNO6): Anal. Calcd
for C17H12BrNO5: C, 52.31; H, 3.10; N, 3.59. Found: C, 52.32; H,
2.96; N, 3.63.
lidene)-2-thioxo-1,3-oxazolidin-4-one (2e).
Compound 2e
was prepared following: general procedures 2 and 3. Yield 79%;
1H NMR (270 MHz, DMSO-d6) d 6.14 (s, 2H), 6.95 (s, 1H), 6.93–
6.97 (m, 1H), 7.14 (t, J = 8.8 Hz, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.73
(d, J = 1.9, 12.4 Hz, 1H), 7.83 (dd, J = 2.4, 8.9 Hz, 1H), 8.28 (d,
J = 2.7 Hz, 1H); MS (ESI) m/z 421 (MH), 423 (MH+2); Anal. Calcd
for C17H10BrNO5S: C, 48.58; H, 2.40; N, 3.33. Found: C, 48.61; H,
2.41; N, 3.31.
5.1.1.26.
phenyl)-1,3-oxazolidin-2,4-dione (9b).
methoxycarbonylphenyl)-1,3-oxazolidin-2,4-dione
5-(3-Bromo-4-hydroxybenzylidene)-3-(4-carboxy-
A solution of 3-(4-
(1.38 g,
5.1.1.19.
5-(3-Bromo-4-hydroxybenzylidene)-3-(4-fluoro-
Compound 2f
phenyl)-2-thioxo-1,3-oxazolidin-4-one (2f).
5.20 mmol) and concd HCl–acetic acid (v/v = 1/1, 26 mL) was stir-
red at 120 °C for 3 h. After cooling to room temperature, the precip-
itate was filtered off, washed with water, and dried in vacuo to give
was prepared following: general procedures 2 and 3. Yield 44%;
1H NMR (270 MHz, DMSO-d6) d 6.98 (s, 1H), 7.13 (d, J = 8.6 Hz,

3250
K. Harada et al. / Bioorg. Med. Chem. 20 (2012) 3242–3254
3-(4-carboxyphenyl)-1,3-oxazolidin-2,4-dione (0.47 g, 38%); 1H
NMR (270 MHz, DMSO-d6) d 4.97 (s, 2H), 7.57 (d, J = 8.3 Hz, 2H),
8.09 (d, J = 8.6 Hz, 2H), 13.12 (s, 1H). Compound 9b was prepared
from the intermediate described above, following: general proce-
dure 2. Yield 20%; 1H NMR (270 MHz, DMSO-d6) d 6.95 (s, 1H),
7.09 (d, J = 8.3 Hz, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.75 (dd, J = 2.0,
8.7 Hz, 1H), 8.03 (d, J = 2.0 Hz, 1H), 8.10 (d, J = 8.6 Hz, 2H), 11.10
(brs, 1H), 13.16 (brs, 1H); MS (ESI) m/z 404 (M), 405 (M+2); Anal.
Calcd for C17H10BrNO6: C, 50.50; H, 2.49; N, 3.47. Found: C,
50.25; H, 2.41; N, 3.42.
41%; 1H NMR (270 MHz, DMSO-d6) d 3.00 (s, 6H), 6.88 (s, 1H),
7.03–7.14 (m, 3H), 7.35 (d, J = 8.1 Hz, 2H), 7.55 (d, J = 8.4 Hz, 1H),
8.64 (s, 1H); MS (ESI) m/z 404 (MH), 406 (MH+2); Anal. Calcd for
C18H15BrN2O4: C, 53.60; H, 3.75; N, 6.95. Found: C, 53.35; H,
3.67; N, 6.90.
5.1.1.32. 3-[4-(Acetylamino)phenyl]-5-(3-bromo-4-hydroxyben-
zylidene)-1,3-oxazolidin-2,4-dione (9i).
To a solution of 3-
(4-nitrophenyl)-1,3-oxazolidin-2,4-dione (1.0 g, 4.5 mmol) in
methanol (20 mL) was added 5% palladium on carbon (77 mg),
and the mixture was stirred under hydrogen atmosphere at room
temperature for 3 h. The reaction mixture was filtered through Cel-
ite, and the filtrate was concentrated. To a solution of the residue in
THF (30 mL) was added activated carbon (50 mg), and then the
mixture was stirred for 30 min. The mixture was filtered through
Celite, and the filtrate was concentrated. The residue was purified
by recrystallization with ethyl acetate/hexane (v/v = 1/1) to give 3-
(4-aminophenyl)-1,3-oxazolidin-2,4-dione (0.74 g, 86%); 1H NMR
5.1.1.27. 5-(3-Bromo-4-hydroxybenzylidene)-3-[4-(N,N-dimeth-
ylcarbamoyl)phenyl]-1,3-oxazolidin-2,4-dione (9e).
A solu-
tion of 3-(4-carboxyphenyl)-1,3-oxazolidin-2,4-dione (93 mg,
0.42 mmol), 50 wt% dimethylamine aqueous solution (48 mg,
1.3 equiv), N-methylmorpholine (85 lL, 1.8 equiv), 1-hydroxyben-
zotriazole (72 mg, 1.3 equiv), and 1-ethyl-3-(3’-dimethylamino-
propyl) carbodiimide hydrochloride (121 mg, 1.5 equiv) in DMF
(10 mL) was stirred at room temperature for 5 h. The mixture
was acidified to pH 4, by addition of 10 wt% HCl solution. The mix-
ture was extracted with ethyl acetate. The combined organic layers
were washed with saturated sodium hydrogen carbonate solution
and brine, dried over anhydrous magnesium sulfate, and concen-
trated in vacuo to give 4-[(N,N-dimethylcarbamoyl)phenyl]-1,3-
oxazolidin-2,4-dione (96 mg, 66%); 1H NMR (270 MHz, DMSO-d6)
d 2.89 (s, 3H), 3.00 (s, 3H), 4.96 (s, 2H), 7.46–7.58 (m, 4H). Com-
pound 9e was prepared from an intermediate described above, fol-
lowing: general procedure 2. Yield 34%; 1H NMR (270 MHz, DMSO-
d6) d 2.93 (s, 3H), 3.00 (s, 3H), 6.94 (s, 1H), 7.09 (d, J = 8.6 Hz, 1H),
7.57 (brs, 4H), 7.74 (dd, J = 2.0, 8.5 Hz, 1H), 8.03 (d, J = 2.1 Hz, 1H);
MS (ESI) m/z 432 (MH), 434 (MH+2); Anal. Calcd for C19H15BrN2O5:
C, 52.90; H, 3.51; N, 6.50. Found: C, 52.75; H, 3.14; N, 6.41.
(270 MHz, DMSO-d6)
d 4.90 (s, 2H), 5.38 (s, 2H), 6.61 (d,
J = 8.9 Hz, 2H), 6.97 (d, J = 8.6 Hz, 2H). To a solution of 3-(4-amino-
phenyl)-1,3-oxazolidin-2,4-dione (100 mg, 0.52 mmol) and trieth-
ylamine (110
lL, 1.5 equiv) in chloroform (4 mL) was added
acetyl chloride (41
lL, 1.1 equiv) at 5 °C. The mixture was stirred
at room temperature for 1.5 h. The reaction mixture was poured
into water, extracted with chloroform, and dried over anhydrous
magnesium sulfate. After filtration, the solvent was removed in va-
cuo, and the residue was purified by silica gel column chromatog-
raphy, eluted with ethyl acetate/hexane to give 3-(4-
acetylaminophenyl)-1,3-oxazolidin-2,4-dione (117 mg, 96%); 1H
NMR (270 MHz, DMSO-d6) d 2.34 (s, 3H), 4.94 (s, 2H), 7.31 (d,
J = 8.9 Hz, 2H), 7.67 (d, J = 8.9 Hz, 2H), 10.05 (s, 1H). Compound 9i
was prepared from an intermediate described above, following:
general procedure 2. Yield 40%; 1H NMR (270 MHz, DMSO-d6) d
2.36 (s, 3H), 6.90 (s, 1H), 7.08 (d, J = 8.5 Hz, 1H), 7.41 (d,
J = 8.1 Hz, 2H), 7.56 (d, J = 8.9 Hz, 1H), 7.64 (s, 1H), 7.73 (d,
J = 8.1 Hz, 2H), 10.08 (s, 1H); MS (ESI) m/z 418 (MH), 420
(MH+2); Anal. Calcd for C18H13BrN2O5: C, 51.80; H, 3.14; N, 6.72.
Found: C, 51.89; H, 3.16; N, 6.67.
5.1.1.28. 5-(3-Bromo-4-hydroxybenzylidene)-3-[4-(N-methylc-
arbamoyl)phenyl]-1,3-oxazolidin-2,4-dione
(9d).
Com-
pound 9d was prepared with methyl amine in a manner similar
to that described for compound 9e. Yield 54%; 1H NMR
(270 MHz, DMSO-d6) d 2.81 (d, J = 4.3 Hz, 3H), 6.94 (s, 1H), 7.09
(d, J = 8.7 Hz, 1H), 7.56–7.61 (m, 3H), 7.66 (d, J = 2.0 Hz, 1H), 7.96
(d, J = 8.3 Hz, 2H), 8.54 (d, J = 4.5 Hz, 1H), 10.68 (s, 1H); MS (ESI)
m/z 418 (MH), 420 (MH+2); Anal. Calcd for C18H13BrN2O5: C,
51.80; H, 3.14; N, 6.72. Found: C, 52.00; H, 2.96; N, 6.67.
5.1.1.33. 5-(3-Bromo-4-hydroxybenzylidene)-3-[4-(cyclohexan-
ecarbonyl)phenyl]-1,3-oxazolidin-2,4-dione
(9j).
Com-
pound 9j was prepared with cyclohexanecarbonyl chloride in a
manner similar to that described for compound 9i. Yield 36%; 1H
NMR (270 MHz, DMSO-d6) d 1.15–1.90 (m, 10H), 2.31–2.39 (m,
1H), 6.89 (s, 1H), 7.08 (d, J = 8.9 Hz, 1H), 7.39 (d, J = 8.9 Hz, 2H),
7.55 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 1.9 Hz, 1H), 7.74 (d, J = 8.9 Hz,
2H), 10.03 (s, 1H), 10.67 (brs, 1H); MS (ESI) m/z 486 (MH), 488
(MH+2); Anal. Calcd for C23H21BrN2O5: C, 56.90; H, 4.36; N, 5.77.
Found: C, 56.75; H, 3.99; N, 5.68.
5.1.1.29. 5-(3-Bromo-4-hydroxybenzylidene)-3-[4-(piperidin-1-
ylcarbonyl)phenyl]-1,3-oxazolidin-2,4-dione
(9f).
Com-
pound 9f was prepared with piperidine in a manner similar to that
described for compound 9e. Yield 23%; 1H NMR (270 MHz, DMSO-
d6) d 1.52–1.60 (m, 6H), 3.20–3.70 (m, 4H), 6.94 (s, 1H), 7.08 (d,
J = 8.6 Hz, 1H), 7.52–7.60 (m, 4H), 7.74 (dd, J = 2.1, 8.3 Hz, 1H),
8.03 (d, J = 1.9 Hz, 1H); MS (ESI) m/z 472 (MH), 474 (MH+2); Anal.
Calcd for C22H19BrN2O5: C, 56.05; H, 4.07; N, 5.95. Found: C, 55.76;
H, 4.02; N, 5.88.
5.1.1.34.
5-(3-Bromo-4-hydroxybenzylidene)-3-(4-methylthi-
Compound 9k was
ophenyl)-1,3-oxazolidin-2,4-dione (9k).
prepared following: general procedure 2 and 4–1. Yield 32%; 1H
NMR (270 MHz, DMSO-d6) d 3.87 (s, 3H), 6.92 (s, 1H), 7.06 (d,
J = 8.9 Hz, 2H), 7.23 (s, 1H), 7.32–7.38 (m, 3H), 8.12 (d, J = 2.4 Hz,
1H), 10.43 (s, 1H); MS (ESI) m/z 407 (MH), 409 (MH+2); Anal. Calcd
for C17H12BrNO4S: C, 50.25; H, 2.98; N, 3.45. Found: C, 50.27; H,
2.80; N, 3.40.
5.1.1.30. 5-(3-Bromo-4-hydroxybenzylidene)-3-[4-(morpholin-
4-ylcarbonyl)phenyl]-1,3-oxazolidin-2,4-dione (9g).
Com-
pound 9f was prepared with morpholine in a manner similar to
that described for compound 9e. Yield 14%; 1H NMR (270 MHz,
DMSO-d6) d 3.20–3.40 (m, 4H), 3.61–3.66 (m, 4H), 6.95 (s, 1H),
7.10 (d, J = 8.4 Hz, 1H), 7.60 (s, 4H), 7.75 (dd, J = 1.9, 8.5 Hz, 1H),
8.04 (d, J = 1.6 Hz, 1H), 11.07 (brs, 1H); MS (ESI) m/z 474 (MH),
476 (MH+2); Anal. Calcd for C21H17BrN2O6: C, 53.28; H, 3.62; N,
5.92. Found: C, 52.93; H, 3.25; N, 5.80.
5.1.1.35. 5-(3-Bromo-4-hydroxybenzylidene)-3-(4-methylsulfo-
nylphenyl)-1,3-oxazolidin-2,4-dione (9l).
To a solution of 3-
(4-methylthiophenyl)-1,3-oxazolidin-2,4-dione (0.45 g, 2.0 mmol)
in dichloromethane (20 mL) was added m-chloroperbenoic acid
(0.69 g, 2.0 equiv) at 5 °C. The mixture was stirred at room temper-
ature for 3 h. Saturated sodium thiosulfate solution and saturated
sodium hydrogen carbonate solution were added, and the mixture
5.1.1.31.
amino)phenyl]-1,3-oxazolidin-2,4-dione (9h).
9h was prepared following: general procedure 2 and 4–2. Yield
5-(3-Bromo-4-hydroxybenzylidene)-3-[4-(dimethyl-
Compound

K. Harada et al. / Bioorg. Med. Chem. 20 (2012) 3242–3254
3251
was extracted with chloroform. The combined organic layers were
washed with brine and dried over anhydrous magnesium sulfate.
After filtration, the solvent was removed in vacuo, and the residue
triturated with diethyl ether to give 3-(4-methylsulfonylphenyl)-
1,3-oxazolidin-2,4-dione (0.38 g, 75%); 1H NMR (270 MHz,
DMSO-d6) d 2.77 (s, 3H), 4.91 (s, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.81
(d, J = 8.4 Hz, 2H). Compound 9l was prepared from an intermedi-
ate described above, following: general procedure 2. Yield 38%; 1H
NMR (270 MHz, DMSO-d6) d 2.79 (s, 3H), 6.94 (s, 1H), 7.08 (d,
J = 8.9 Hz, 1H), 7.56 (dd, J = 1.9, 8.4 Hz, 1H), 7.64 (d, J = 1.9 Hz,
1H), 7.71 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H); MS (ESI) m/z
439 (MH), 441 (MH+2); Anal. Calcd for C17H12BrNO6S: C, 46.58;
H, 2.76; N, 3.20. Found: C, 46.29; H, 2.71; N, 3.33.
(MH), 381 (MH+2); Anal. Calcd for C15H8BrFN2O4: C, 47.50; H,
2.13; N, 7.39. Found: C, 47.25; H, 2.05; N, 7.34.
5.1.1.42. 5-(3-Bromo-4-hydroxybenzylidene)-3-(6-chloropyri-
dine-3-yl)-1,3-oxazolidin-2,4-dione (10f).
Compound 10f
was prepared following: general procedure 2 and 4–2. Yield 59%;
1H NMR (270 MHz, DMSO-d6) d 7.03 (s, 1H), 7.14 (d, J = 8.4 Hz,
1H), 7.81 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 1.9 Hz, 1H), 8.05 (dd,
J = 2.7, 8.4 Hz, 1H), 8.12 (d, J = 2.2 Hz, 1H), 8.57 (d, J = 2.2 Hz, 1H),
10.30 (s, 1H); MS (ESI) m/z 396 (MH), 398 (MH+2); Anal. Calcd
for C15H8BrClN2O4: C, 45.52; H, 2.04; N, 7.08. Found: C, 45.25; H,
1.96; N, 6.99.
5.1.1.43.
5-(3-Bromo-4-hydroxybenzylidene)-3-[6-(dimethyl-
Com-
5.1.1.36.
5-(3-Bromo-4-hydroxybenzylidene)-3-(4-sulfamoyl-
Compound 9m was
amino)pyridine-3-yl]-1,3-oxazolidin-2,4-dione (10g).
phenyl)-1,3-oxazolidin-2,4-dione (9m).
pound 10g was prepared following: general procedure 2 and 4–2.
Yield 48%; 1H NMR (270 MHz, DMSO-d6) d 3.07 (s, 6H), 6.75 (d,
J = 9.1 Hz, 1H), 6.89 (s, 1H), 7.08 (d, J = 8.8 Hz, 1H), 7.53–7.66 (m,
3H), 8.13 (d, J = 2.3 Hz, 1H), 10.64 (brs, 1H); MS (ESI) m/z 405
(MH), 407 (MH+2); Anal. Calcd for C17H14BrN3O4: C, 50.49; H,
3.49; N, 10.40. Found: C, 50.14; H, 3.12; N, 10.28.
prepared following: general procedure 2 and 4–1. Yield 49%; 1H
NMR (270 MHz, DMSO-d6) d 6.93 (s, 1H), 7.09 (d, J = 8.9 Hz, 1H),
7.55 (s, 2H), 7.59–7.72 (m, 4H), 7.98 (d, J = 8.4 Hz, 2H); MS (ESI)
m/z 440 (MH), 441 (MH+2); Anal. Calcd for C16H11BrN2O6S: C,
43.74; H, 2.53; N, 6.38. Found: C, 43.71; H, 2.22; N, 6.36.
5.1.1.37. 5-(3-Bromo-4-hydroxybenzylidene)-3-(pyridine-2-yl)-
5.1.1.44.
methoxyphenyl)-2-thioxo-1,3-thiazolidin-2,4-dione
(14a). Compound 14a was prepared with 1-(3-bromo-4-
hydroxyphenyl)ethanone in a manner similar to that described
for compound 1a. Yield 39%; 1H NMR (270 MHz, DMSO-d6) d 2.67
(s, 3H), 3.82 (s, 3H), 7.07 (d, J = 8.9 Hz, 2H), 7.17 (d, J = 8.6 Hz,
1H), 7.25 (d, J = 8.9 Hz, 2H), 7.39 (d, J = 1.6 Hz, 1H), 7.70 (d,
J = 2.2 Hz, 1H); MS (ESI) m/z 437 (MH), 439 (MH+2); Anal. Calcd
for C18H14BrNO3S2: C, 49.55; H, 3.24; N, 3.21. Found: C, 49.46; H,
3.26; N, 2.98.
5-[1-(3-Bromo-4-hydroxyphenyl)ethylidene]-3-(4-
1,3-oxazolidin-2,4-dione (10a).
Compound 10a was pre-
pared following: general procedure 2 and 4–2. Yield 48%; 1H
NMR (270 MHz, DMSO-d6) d 7.02 (s, 1H), 7.14 (d, J = 8.4 Hz, 1H),
7.61 (dd, J = 1.4, 6.2 Hz, 1H), 7.67 (d, J = 6.4 Hz, 1H), 7.84 (dd,
J = 2.0, 8.5 Hz, 1H), 8.02 (dt, J = 1.8, 7.8 Hz, 1H), 8.12 (d, J = 1.9 Hz,
1H), 8.69 (dd, J = 1.1, 4.9 Hz, 1H); MS (ESI) m/z 362 (MH), 364
(MH+2); Anal. Calcd for C15H9BrN2O4: C, 49.87; H, 2.51; N, 7.76.
Found: C, 49.52; H, 2.14; N, 7.64.
5.1.1.38. 5-(3-Bromo-4-hydroxybenzylidene)-3-(pyridine-3-yl)-
1,3-oxazolidin-2,4-dione (10b).
Compound 10b was pre-
5.1.1.45. 5-[1-(3-Bromo-4-hydroxyphenyl)-2,2,2-trifluoroethy-
lidene)-3-(4-methoxyphenyl)-2-thioxo-1,3-thiazolidin-2,4-
pared following: general procedure 2 and 4–2. Yield 32%; 1H
NMR (270 MHz, DMSO-d6) d 7.02 (s, 1H), 7.14 (d, J = 8.4 Hz, 1H),
7.74 (dt, J = 6.5, 3.5 Hz, 2H), 7.85 (d, J = 8.6 Hz, 1H), 7.97 (d,
J = 8.4 Hz, 1H), 8.12 (d, J = 1.4 Hz, 1H), 8.71 (s, 1H); MS (ESI) m/z
362 (MH), 364 (MH+2); Anal. Calcd for C15H9BrN2O4: C, 49.87; H,
2.51; N, 7.76. Found: C, 49.88; H, 2.37; N, 7.80.
dione (14b).
A mixture of 2,2,2-Trifluoro-1-(4-methoxy-
phenyl)ethanone (13.8 g, 67.6 mmol), silver oxide (II) (1.76 g,
0.12 equiv), concd sulfuric acid (6.5 mL), and carbon tetrachloride
(130 mL) was cooled to 5 °C. To the mixture was added bromine
(11.1 g, 1.03 equiv) at the temperature, and the reaction mixture
was stirred at 65 °C for 8 h. After cooling, the mixture was poured
into ice and extracted with chloroform. The combined organic lay-
ers were washed with saturated sodium hydrogen carbonate solu-
tion and brine, and dried over anhydrous magnesium sulfate. After
filtration, the solvent was removed in vacuo, and the residue was
purified by silica gel column chromatography, eluted with ethyl
acetate/hexane (v/v = 1/3) to give 1-(3-bromo-4-methoxyphenyl)-
2,2,2-trifluoroethanone (17.3 g, 90%); 1H NMR (270 MHz, CDCl3) d
4.03 (s, 3H), 7.00 (d, J = 8.9 Hz, 1H), 8.05 (dd, J = 1.1, 7.8 Hz, 1H),
8.29 (d, J = 1.1 Hz, 1H). To a solution of 1-(3-bromo-4-methoxy-
phenyl)-2,2,2-trifluoroethanone (7.52 g, 26.6 mmol) in DMF
(75 mL) was added lithium chloride (4.0 g, 3.6 equiv). Then the
mixture was stirred under reflux for 1 h. After cooling, the volatile
materials were removed in vacuo, the residue was dissolved in
methanol (100 mL). The mixture was acidified to pH 3, by addition
of 1 M HCl in methanol solution. The solvent was removed in va-
cuo, and the residue was purified by silica gel column chromatog-
raphy, eluted with ethyl acetate/hexane (v/v = 1/3) to give 1-(3-
bromo-4-hydroxyphenyl)-2,2,2-trifluoroethanone (4.68 g, 65%);
1H NMR (270 MHz, DMSO-d6) d 7.16 (d, J = 8.8 Hz, 1H), 7.93 (d,
J = 8.6 Hz, 1H), 8.11 (s, 1H). Compound 14b was prepared from
an intermediate described above, following: general procedure 2.
Yield 22%; 1H NMR (270 MHz, DMSO-d6) d 3.77 (s, 3H), 6.93 (d,
J = 8.4 Hz, 1H), 7.02 (d, J = 8.9 Hz, 2H), 7.20 (d, J = 8.9 Hz, 2H),
7.28 (d, J = 8.9 Hz, 1H), 7.53 (d, J = 1.9 Hz, 1H); MS (ESI) m/z 491
5.1.1.39. 5-(3-Bromo-4-hydroxybenzylidene)-3-(6-methoxypyr-
idine-3-yl)-1,3-oxazolidin-2,4-dione (10c).
Compound 10c
was prepared following: general procedure 2 and 4–2. Yield 78%;
1H NMR (270 MHz, DMSO-d6) d 3.92 (s, 3H), 7.00 (s, 1H), 7.03 (d,
J = 8.9 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.84 (dd, J = 1.5, 8.8 Hz,
2H), 8.12 (s, 1H), 8.28 (d, J = 2.4 Hz, 1H); MS (ESI) m/z 392 (MH),
394 (MH+2); Anal. Calcd for C16H11BrN2O5: C, 49.11; H, 2.84; N,
7.16. Found: C, 49.20; H, 2.86; N, 6.93.
5.1.1.40. 5-(3-Bromo-4-hydroxybenzylidene)-3-(6-methylpyri-
dine-3-yl)-1,3-oxazolidin-2,4-dione (10d).
Compound 10d
was prepared following: general procedure 2 and 4–2. Yield 67%;
1H NMR (270 MHz, DMSO-d6) d 2.56 (s, 1H), 7.15 (d, J = 8.4 Hz,
1H), 7.47 (d, J = 8.4 Hz, 1H), 7.55 (dd, J = 2.2, 8.9 Hz, 1H), 7.76–
7.80 (m, 2H), 7.86 (d, J = 2.2 Hz, 1H), 8.47 (d, J = 2.4 Hz, 1H); MS
(ESI) m/z 376 (MH), 378 (MH+2); Anal. Calcd for C16H11BrN2O4:
C, 51.20; H, 2.96; N, 7.47. Found: C, 51.22; H, 2.78; N, 7.88.
5.1.1.41. 5-(3-Bromo-4-hydroxybenzylidene)-3-(6-fluoropyri-
dine-3-yl)-1,3-oxazolidin-2,4-dione (10e).
was prepared following: general procedure 2 and 4–2. Yield 30%;
1H NMR (270 MHz, DMSO-d6) d 7.03 (s, 1H), 7.14 (d, J = 8.6 Hz,
1H), 7.47 (dd, J = 2.9, 8.9 Hz, 1H), 7.84 (dd, J = 2.9, 8.9 Hz, 1H),
8.12–8.24 (m, 2H), 8.41 (d, J = 1.9 Hz, 1H); MS (ESI) m/z 380
Compound 10e

3252
K. Harada et al. / Bioorg. Med. Chem. 20 (2012) 3242–3254
(MH), 492 (MH+2); Anal. Calcd for C18H11BrF3NO3S2: C, 44.09; H,
2.26; N, 2.86. Found: C, 43.69; H, 1.94; N, 2.79.
was prepared following: general procedure 2 and 3. Yield 40%;
1H NMR (270 MHz, DMSO-d6) d 3.82 (s, 3H), 6.92 (s, 1H), 6.99–
7.13 (m, 4H), 7.36–7.43 (m, 2H), 8.04 (d, J = 8.0 Hz, 1H), 10.81 (s,
1H); MS (ESI) m/z 362 (MH); Anal. Calcd for C17H12ClNO4S: C,
56.43; H, 3.35; N, 3.87. Found: C, 56.34; H, 3.37; N, 3.64.
5.1.1.46. 5-(3-Bromo-4-hydroxybenzyl)-3-(4-methoxyphenyl)-
2-thioxo-1,3-oxazolidin-4-one (15).
3-(Methoxyphenyl)-2-
thioxo-1,3-oxazolidin-4-one 4a (0.10 g, 0.48 mmol) and magne-
sium methyl carbonate (0.95 mL, 1.8 M in DMF solution) were
heated at 85 °C for 0.5 h. 3-Bromo-4-(t-butyldimethylsilyl)benzyl
chloride (0.18 g, 0.54 mmol) was added and the temperature spon-
taneously rose to 90 °C. After 3 h at 85–90 °C, the reaction mixture
was poured, with vigorous stirring, into 10 g of ice and 0.95 mL of
concd HCl. The precipitate was filtered off and recrystallized from
ethanol (6 mL) and water (10 mL). Yield 42%; 1H NMR (270 MHz,
CDCl3) d 3.18–3.37 (m, 2H), 3.81 (s, 1H), 5.14 (t, J = 4.5 Hz, 1H),
6.89–7.00 (m, 5H), 7.15 (dd, J = 2.0, 8.5 Hz, 1H), 7.41 (d, 2.2 Hz,
1H); MS (ESI) m/z 425 (MH), 427 (MH+2); Anal. Calcd for
5.1.1.53. 5-(3,5-Dichloro-4-hydroxybenzylidene)-3-(4-methoxy-
phenyl)-2-tioxo-1,3-oxazolidin-4-one (11g).
Compound 11g
was prepared following: general procedure 2 and 3. Yield 68%; 1H
NMR (270 MHz, DMSO-d6) d 3.82 (s, 3H), 6.96 (s, 1H), 7.10 (d,
J = 8.9 Hz, 2H), 7.38 (d, J = 8.9 Hz, 2H), 7.95 (s, 2H); MS (ESI) m/z
396 (MH); Anal. Calcd for C17H11Cl2NO4S: C, 51.52; H, 2.80; N,
3.54. Found: C, 51.54; H, 2.62; N, 3.49.
5.1.1.54. 5-(3,5-Difluoro-4-hydroxybenzylidene)-3-(4-methoxy-
phenyl)-2-tioxo-1,3-oxazolidin-4-one
(11h).
Compound
C
17H14BrNO3S2: C, 48.12; H, 3.33; N, 3.30. Found: C, 48.07; H,
11h was prepared following: general procedure 2 and 3. Yield
57%; 1H NMR (270 MHz, DMSO-d6) d 3.81 (s, 3H), 6.95 (s, 1H),
7.09 (d, J = 8.9 Hz, 2H), 7.37 (d, J = 8.9 Hz, 2H), 7.66 (d, J = 9.8 Hz,
2H); MS (ESI) m/z 364 (MH); Anal. Calcd for C17H11F2NO4S: C,
56.19; H, 3.05; N, 3.86. Found: C, 55.92; H, 2.98; N, 3.77.
3.15; N, 3.32.
5.1.1.47.
5-(4-Hydroxy-3-methylbenzylidene)-3-(4-methoxy-
phenyl)-2-tioxo-1,3-oxazolidin-4-one
(11a).
Compound
11a was prepared following: general procedure 2 and 3. Yield
50%; 1H NMR (270 MHz, DMSO-d6) d 3.82 (s, 3H), 6.93 (s, 1H),
6.94 (d, J = 8.9 Hz, 2H), 7.09 (d, J = 8.9 Hz, 2H), 7.39 (d, J = 8.9 Hz,
2H), 7.81 (d, J = 8.9 Hz, 2H), 11.33 (s, 1H); MS (ESI) m/z 328
(MH); Anal. Calcd for C17H13NO4S: C, 62.37; H, 4.01; N, 4.28. Found:
C, 62.14; H, 4.21; N, 4.19.
5.1.1.55.
5-(3-Chloro-5-fluoro-4-hydroxybenzylidene)-3-(4-
methoxyphenyl)-2-thioxo-1,3-oxazolidin-4-one (11i).
Compound 11i was prepared following: general procedure 2
and 3. Yield 78%; 1H NMR (270 MHz, DMSO-d6) d 3.81 (s, 3H),
6.95 (s, 1H), 7.09 (d, J = 8.9 Hz, 2H), 7.37 (d, J = 8.9 Hz, 2H), 7.75
(dd, J = 1.9, 10.8 Hz, 1H), 8.32 (d, J = 1.9 Hz, 1H); MS (ESI) m/z 380
(MH); Anal. Calcd for C17H11ClFNO4S: C, 53.76; H, 2.92; N, 3.69.
Found: C, 53.41; H, 2.61; N, 3.57.
5.1.1.48.
5-(4-Hydroxy-3-methylbenzylidene)-3-(4-methoxy-
phenyl)-2-tioxo-1,3-oxazolidin-4-one (11b).
Compound 11b was prepared following: general procedure 2
and 3. Yield 42%; 1H NMR (270 MHz, DMSO-d6) d 2.50 (s, 3H),
3.82 (s, 3H), 6.87 (s, 1H), 6.96 (d, J = 9.2 Hz, 1H), 7.09 (d, J = 8.9 Hz,
2H), 7.39 (d, J = 8.9 Hz, 2H), 7.66 (s, 1H), 7.68 (d, J = 9.2 Hz, 1H),
10.30 (brs, 1H); MS (ESI) m/z 342 (MH); Anal. Calcd for C18H15NO4S:
C, 63.33; H, 4.43; N, 4.11. Found: C, 63.08; H, 4.35; N, 4.06.
5.1.1.56.
5-(3-Cyano-4-hydroxybenzylidene)-3-(4-methoxy-
phenyl)-1,3-oxazolidin-2,4-dione (12a).
Compound 12a was prepared following: general procedure 2
and 4–1. Yield 56%; 1H NMR (400 MHz, CD3OD) d 3.85 (s, 3H),
6.82 (s, 1H), 7.04–7.08 (m, 3H), 7.41 (dt, J = 2.8, 9.1 Hz, 2H), 7.97
(dd, J = 2.3, 8.7 Hz, 1H), 8.03 (d, J = 2.3 Hz, 1H); MS (ESI) m/z 337
(MH); Anal. Calcd for C18H12N2O5: C, 64.27; H, 3.60; N, 8.33. Found:
C, 63.98; H, 3.55; N, 8.26.
5.1.1.49. 5-(4-Hydroxy-3-methoxybenzylidene)-3-(4-methoxy-
phenyl)-2-tioxo-1,3-oxazolidin-4-one (11c).
Compound 11c was prepared following: general procedure 2
and 3. Yield 96%; 1H NMR (270 MHz, DMSO-d6) d 2.50 (s, 3H),
3.82 (s, 3H), 6.92 (s, 1H), 6.96 (d, J = 8.4 Hz, 1H), 7.09 (d, J = 8.9 Hz,
2H), 7.39 (d, J = 8.9 Hz, 2H), 7.48 (d, J = 8.4 Hz, 1H), 7.53 (s, 1H),
9.96 (brs, 1H); MS (ESI) m/z 358 (MH); Anal. Calcd for C18H15NO5S:
C, 60.49; H, 4.23; N, 3.92. Found: C, 60.69; H, 4.07; N, 3.64.
5.1.1.57. 5-(3-Carboxy-4-hydroxybenzylidene)-3-(4-methoxy-
phenyl)-1,3-oxazolidin-2,4-dione(12b).
Compound 12b was prepared following: general procedure 2
and 4–1. Yield 40%; 1H NMR (400 MHz, DMSO-d6) d 3.81 (s, 3H),
6.98 (s, 1H), 7.08–7.12 (m, 3H), 7.42 (dt, J = 9.0, 2.2 Hz, 2H), 7.98
(dd, J = 8.8, 2.3 Hz, 1H), 8.37 (d, J = 2.3 Hz, 1H); MS (ESI) m/z 355
(M); Anal. Calcd for C18H13NO7: C, 60.83; H, 3.69; N, 3.94. Found:
C, 60.56; H, 3.62; N, 3.85.
5.1.1.50.
5-(3-Fluoro-4-hydroxybenzylidene)-3-(4-methoxy-
phenyl)-2-tioxo-1,3-oxazolidin-4-one (11d).
Compound 11d was prepared following: general procedure 2
and 3. Yield 75%; 1H NMR (270 MHz, DMSO-d6) d 3.82 (s, 3H),
6.95 (s, 1H), 7.07–7.13 (m, 3H), 7.36–7.43 (m, 2H), 7.64 (dd,
J = 2.2, 8.5 Hz, 1H), 7.73 (dd, J = 2.2, 12.6 Hz, 1H), 10.82 (s, 1H);
MS (ESI) m/z 346 (MH); Anal. Calcd for C17H12FNO4S: C, 59.12; H,
3.50; N, 4.06. Found: C, 58.84; H, 3.34; N, 4.26.
5.1.1.58. 5-[3-(N,N-dimethylaminomethyl)-4-hydroxybenzylid-
ene]-3-(4-methoxyphenyl)-1,3-oxazolidin-2,4-dione (12c).
To a mixture of p-hydroxybenzaldehyde (1.0 g, 8.2 mmol), N,N-
dimethylmethylene ammonium iodide (1.6 g, 1.0 equiv) in dichlo-
romethane (30 mL) was added anhydrous potassium carbonate
(1.7 g, 1.5 equiv). The mixture was stirred under reflux for 16 h.
After cooling, the mixture was extracted with chloroform, and
the organic layers was washed with brine and dried over anhy-
drous magnesium sulfate. After filtration, the solvent was removed
in vacuo, and the residue was purified by silica gel column chroma-
tography, eluted with chloroform/methanol (v/v = 30/1) to give 3-
(N,N-dimethylaminomethyl)-4-hydroxybenzaldehyde (1.0 g, 70%);
1H NMR (400 MHz, CDCl3) d 2.37 (s, 6H), 3.73 (s, 2H), 6.91 (d,
J = 8.3 Hz, 1H), 7.54–7.55 (m, 1H), 7.70 (dd, J = 2.1, 8.3 Hz, 1H),
8.00–9.50 (brs, 1H), 9.81 (s, 1H). Compound 12c was prepared from
an intermediate described above, following: general procedure 2.
Yield 44%; 1H NMR (400 MHz, CDCl3) d 2.37 (s, 6H), 3.73 (s, 2H),
5.1.1.51.
5-(3-Chloro-4-hydroxybenzylidene)-3-(4-methoxy-
(11e). Compound
phenyl)-2-tioxo-1,3-oxazolidin-4-one
11e was prepared following: general procedure 2 and 3. Yield
52%; 1H NMR (270 MHz, DMSO-d6) d 3.82 (s, 3H), 6.95 (s, 1H),
7.08–7.16 (m, 3H), 7.39 (d, J = 8.6 Hz, 2H), 7.78 (dd, J = 2.2, 8.6 Hz,
1H), 7.95 (d, J = 2.2 Hz, 1H), 11.13 (s, 1H); MS (ESI) m/z 362
(MH); Anal. Calcd for C17H12ClNO4S: C, 56.43; H, 3.35; N, 3.87.
Found: C, 56.46; H, 3.52; N, 3.79.
5.1.1.52.
5-(2-Chloro-4-hydroxybenzylidene)-3-(4-methoxy-
Compound 11f
phenyl)-2-tioxo-1,3-oxazolidin-4-one (11f).

K. Harada et al. / Bioorg. Med. Chem. 20 (2012) 3242–3254
3253
3.85 (s, 3H), 6.82 (s, 1H), 6.89 (d, J = 8.4 Hz, 1H), 7.02 (dt, J = 2.8,
9.1 Hz, 2H), 7.41 (dt, J = 2.8, 9.1 Hz, 2H), 7.53 (d, J = 2.2 Hz, 1H),
7.59 (dd, J = 2.2, 8.4 Hz, 1H); MS (ESI) m/z 369 (MH); Anal. Calcd
for C20H20N2O5: C, 65.19; H, 5.48; N, 7.61. Found: C, 64.90; H,
5.43; N, 7.54.
man 17b-HSD type 3 cDNA. HeLa cells expressing human 17b-HSD
type 3 were plated on a 96-well plate (1 ꢁ 104 cells, 100
lL per well)
in Dulbecco’s modified Eagle medium (DMEM) containing 5% char-
coal stripped fetal bovine serum (FCS), and incubated for 24 h at
37 °C in a 5% CO2 atmosphere. The cells were pretreated with the
compounds for 30 min, followed by incubation with 10 lL of
5.1.1.59. 5-(3,5-dichloro-4-hydroxybenzylidene)-3-(4-methoxy-
500 nM substrate (androstenedione) in DMEM media for 120 min
at 37 °C in a 5% CO2 atmosphere. The cell media were harvested,
and the testosterone concentration in the media was determined
by DELFIA Testosterone Reagents (PerkinElmer), for which measure-
ment was performed on an Ultra microplate detection system (TE-
CAN). Analytical conditions were as follows: Wavelength, 340 nm
phenyl)-1,3-oxazolidin-2,4-dione (12d).
Compound 12d
was prepared following: general procedure 2 and 4–1. Yield 70%;
1H NMR (270 MHz, DMSO-d6) d 3.81 (s, 3H), 6.95 (s, 1H), 7.11 (d,
J = 8.9 Hz, 2H), 7.39 (d, J = 8.9 Hz, 2H), 7.94 (s, 2H); MS (ESI) m/z
380 (MH); Anal. Calcd for C17H11Cl2NO5: C, 53.69; H, 2.92; N,
3.69. Found: C, 53.71; H, 2.74; N, 3.64.
(excitation) and 612 nm (fluorescence); integration time, 400
lag time, 400 s. The percent inhibition was calculated relative to
an uninhibited control and plotted against the compound concen-
trations (1, 10, 100 nM, and 1 M) to generate the IC50 results.
ls;
l
5.1.1.60.
5-[(5-Hydroxypyridin-2-yl)methylidene]-3-(4-
methoxyphenyl)-2-thioxo-1,3-oxazolidin-4-one (13a).
Compound 13a was prepared following: general procedure 2
and 3. Yield 34%; 1H NMR (270 MHz, DMSO-d6) d 3.82 (s, 3H),
6.77 (s, 1H), 7.10 (d, J = 8.9 Hz, 2H), 7.28–7.41 (m, 3H), 7.93 (d,
J = 8.6 Hz, 1H), 8.32 (d, J = 2.7 Hz, 1H); MS (ESI) m/z 313 (MH); Anal.
Calcd for C16H12N2O5: C, 61.52; H, 3.88; N, 8.97. Found: C, 61.37; H,
3.51; N, 8.88.
l
5.1.1.64. Inhibition of 17b-HSD type 1 and type 2 in a cell-based
assay. The assay was performed in a manner similar to that
described for 17b-HSD3 but estrone (E1) for 17b-HSD1 and estra-
diol (E2) for 17b-HSD2 as a tracer substrate.
5.1.1.65. Cotransfection assay.
Cotransfection assays using
5.1.1.61. 5-[(6-Bromo-5-hydroxypyridin-2-yl)methylidene]-3-
(4-methoxyphenyl)-2-thioxo-1,3-oxazolidin-4-one (13b).
Compound 13b was prepared following: general procedure 2
and 3. Yield 69%; 1H NMR (270 MHz, DMSO-d6) d 3.81 (s, 3H),
6.70 (s, 1H), 7.09 (d, J = 8.9 Hz, 2H), 7.38 (d, J = 8.9 Hz, 2H), 7.45
(d, J = 8.4 Hz, 1H), 7.91 (d, J = 8.6 Hz, 1H); MS (ESI) m/z 392 (MH),
394 (MH+2); Anal. Calcd for C16H11BrN2O5: C, 49.11; H, 2.84; N,
7.16. Found: C, 48.86; H, 2.76; N, 7.11.
human AR were performed in HeLa cells. HeLa cells were cultured
in the presence of DMEM supplemented with 10% FBS. Cells were
seeded 24 h prior to transfection on a 96-well plate. Cells were
transiently transfected by the calcium phosphate coprecipitation
procedure with the following plasmids: pRShAR, containing the
hAR cDNA under control of the RSV-LTR, a reporter plasmid, mouse
mammary tumor virus (MMTV)-luciferase (LUC), containing the
cDNA for firefly LUC under control of the MMTV long terminal re-
peat; a b-Gal expression plasmid, pRS-b-Gal, containing the cDNA
for bacterial b-galactosidase, and filler DNA. 6 h after transfection,
the medium was removed and cells were washed with phosphate-
buffered saline (PBS). After aspiration of PBS, medium containing
10% charcoal resin-stripped FBS containing seven incremental con-
centrations of compound ranging from 10ꢀ9 to 10ꢀ5 M was added
to the cells. For antagonist experiments, compounds were prepared
and diluted in medium containing an EC50 concentration of refer-
ence agonist (DHT). After 40 h, the medium was removed from
the cells, and the cells were lysed with a detergent containing buf-
fer. Luciferase and b-galactosidase activities were measured in cell
extracts to determine the level of transcriptional activation. The
normalized response was calculated as: luciferase response/b-gal
rate, where luciferase response = relative luciferase units (RLU)
and b-gal rate = b-gal O.D.415/b-gal incubation time in minutes.
For agonist experiments, the effective concentration that produced
50% of the maximum response (EC50) was determined for each
compound by interpolation between two concentrations spanning
the midpoint of the concentration–response curve. Agonist efficacy
for test compounds is calculated as a percent of normalized re-
sponse relative to the maximum normalized response by the refer-
ence agonist (DHT). For antagonist experiments, the concentration
of test compound that resulted in 50% of the maximum repression
observed (IC50) of the response induced by an EC50 of DHT was
determined for each compound by interpolation between two con-
centrations spanning the midpoint of the concentration–response
5.1.1.62.
5-[3,5-Dichloro-4-(phosphonoxy)benzylidene]-3-(4-
methoxyphenyl)-2-thioxo-1,3-oxazolidin-4-one (16).
Compound 11g (1.00 g, 2.44 mmol) was dissolved in DMF
(20 mL) and the solution was cooled to ꢀ10 °C. To the solution was
added carbon tetrachloride (1.80 g, 5.0 equiv), N,N-diisopropyleth-
ylamine (0.68 g, 2.1 equiv), N,N-dimethyl-4-aminopyridine
(31 mg, 0.1 equiv), and dibenzyl phosphite (1.00 g, 1.6 equiv) at
the temperature. The mixture was stirred at ꢀ10 °C for 0.5 h, and
then at room temperature for 1 h. A 0.5 M potassium dihydrogen
phosphate solution (13 mL) was added, extracted with chloroform,
and the organic layers were washed with brine and dried over anhy-
drous magnesium sulfate. After filtration, the solvent was removed
in vacuo, and the residue was recrystallized from ethyl acetate/
hexane (v/v = 1/5, 100 mL) to give 5-[3,5-dichloro-4-(phosphonoxy)
benzylidene]-3-(4-methoxyphenyl)-2-thioxo-1,3oxazolidin-4-one
dibenzyl ester (1.05 g, 61%); 1H NMR (270 MHz, CDCl3) d 3.86 (s, 3H),
5.25 (d, J = 8.1 Hz, 4H), 6.62 (s, 1H), 7.05 (d, J = 8.9 Hz, 2H), 7.27 (d,
J = 8.9 Hz, 2H), 7.35 (brs, 10H), 7.82 (s, 2H). To a solution of 5-[3,5-di-
chloro-4-(phosphonoxy)benzylidene]-3-(4-methoxyphenyl)-2-thi-
oxo-1,3oxazolidin-4-one dibenzyl ester (1.00 g, 1.41 mmol) in
chloroform (80 mL) was added bromotrimethylsilane (2.29 g,
10 equiv) at 0 °C, and the mixture was then stirred at room temper-
ature for 1.5 h. A 1 M HCl solution (20 mL) was added, and the mix-
ture was stirred for 0.5 h. The precipitate was collected by filtration
and washed with water. The solid was triturated with chloroform to
afford the title product (16). Yield 43%; 1H NMR (270 MHz, DMSO-
d6) d 3.82 (s, 3H), 7.00 (s, 1H), 7.10 (d, J = 8.6 Hz, 2H), 7.39 (d,
J = 8.6 Hz, 2H), 8.02 (s, 2H); MS (ESI) m/z 474 (M-1); Anal. Calcd for
curve. Cotransfection studies with ERa, and GR were carried out
in HeLa cells as described above to determine crossreactivity.
C17H12Cl2NO7PS: C, 42.86; H, 2.54; N, 2.94. Found: C, 42.51; H,
5.1.1.66. Rat pharmacokinetics.
Male Sprague–Dawley rats
2.17; N, 2.82.
were dosed orally with test compound in corn oil or 0.5% methyl-
cellulose (MC) solution, or intravenously with test compound dis-
solved in saline containing small amount of DMSO. Plasma samples
were measured using a selective HPLC/MS/MS method. The HPLC/
MS/MS system was operated in selected reaction monitoring (SRM)
5.1.1.63. Inhibition of 17b-HSD type
3
in
a
cell-based
assay.
Cellular activity of the compounds was evaluated in
HeLa cells, which were transiently transfected with full-length hu-

3254
K. Harada et al. / Bioorg. Med. Chem. 20 (2012) 3242–3254
mode under optimized conditions for detection of the selected
compounds using positive ions formed by atmospheric pressure
chemical ionization (APCI). Quantitation was determined using a
weighted regression analysis of peak area of analyte to define the
plasma concentration vs time profile.
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treated with aLH–RH agonist.
An LH–RH agonist, leuprorelin
(100 ng) was administered intramuscularly (im) to male Sprague–
Dawley rats that had been pretreated with two doses of the test
compound (20 and 100 mg/kg) and CB7630 (20 mg 1 h earlier.
Blood specimens were obtained 1, 2, and 4 h after LH–RH adminis-
tration in the rats. The plasma concentration of testosterone was
measured by gas chromatography-mass spectrometry (GC/MS) in
selected ion monitoring (SIM) mode, after conversion of testoster-
one to the heptafluorobutyric acid ester using heptafluorobutyric
acid anhydride (HFBA). The experimental protocol was approved
by the local ethics committee for animal studies.
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Acknowledgment
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The authors are grateful to Sumika Chemical Analysis Service,
Ltd for compound synthesis, characterization, and helpful
discussions.
Supplementary data
16. Jarkko, R.; Hanna, K.; Tycho, H.; Reza, O.; Dooman, O.; Tomi, J.; Junko, S. Nat.
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Supplementary data (NMR spectroscopic data for compounds
1a and 14a) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2012.03.052.